The uses of non-steroidal anti-inflammatory drugs (NSAIDs) are limited by a variety of side effects. So research on preparing new analgesic agents is important. According to some reports about the analgesic activity of hydrazide and hydrazine derivatives a new series of these compounds were synthesized in order to obtain new analgesic compounds. The final compounds 10a-10e and 15a-15d were prepared by condensation of corresponding hydrazides 7,8 and 11-14 with different aldehydes 9a-9e. The structures of all synthesized compounds were confirmed by means of FT-IR, 1H-NMR and Mass spectra. All compounds were evaluated for their analgesic activities by abdominal constriction test (writhing test). Most of the synthesized compounds induced significant reduction in the writhing response when compared to control and compound 15 was more potent than mefenamic acid in the writhing test.  Non-steroidal anti-inflammatory drugs (NSAIDs) are extremely used in the treatment of pain and inflammation. These compounds non selectively inhibit the two isoforms of the cyclooxygenase (COX-1 and COX-2) and so prevent the metabolism of cellular arachidonic acid (AA) and the upregulation of prostaglandin formation, which in other respects lead to an increase of vascular permeability, edema, hyperalgesia, pyrexia and inflammation . Additionally, Leukotrienes, particularly LTB4 which are products of 5-LO together with decrease in prostaglandin formation are incriminated in the acute ulceration induced by NSAID’s. Accordingly, compounds that obtain dual inhibition of enzymes COX and 5-LO can show safer profile of activity and enhanced efficacy in the battle of pain in inflammatory diseases. Some reports suggest that the hydrazone moiety present in phenylhydrazone derivative 1  is a pha2-phenoxyphenyl ring system and it’s analogues has attracted some interests in medicinal chemistry and this scaffold have demonstrated a wide range of biological effects which include anticonvulsant, antimycobacterial, analgesic and anti-inflammatory activities - 11.According to the above and on the basis of molecular hybridization approach which is based on the combination of different pharmacophores to produce a new hybrid molecule with improved activity, we aimed at the synthesis of new hybrid compounds containing both of hydrazone and 2-phenoxyphenyl structures in the hope of obtaining new potent analgesic agents with potential dual COX/5-LO inhibitory activity and lower side effects -15. Chemistry 2-phenoxybenzoic acid hydrazide 8 which used in the preparation of compounds 10b-10e was synthesized according to our previously described method  3). Tar. Tar3). Chemicals were purchased from Merck Chemical company  and ACROS company . The melting points were determined in open capillary tubes and presented uncorrected. 1H-NMR spectra were obtained using a Bruker FT-400 spectrometer . Tetramethylsilane was used as an internal standard. Mass Spectra were obtained using a Finnigan Mat TSQ-70 spectrometer at 70 eV . The IR spectra were obtained using a Nicolet FT-IR Magna 550 Spectrographs (KBr disks) . The target compounds were synthesized according to the General procedure for the synthesis of hydrazides (10b-10e) and hydrazines (15a-15d) A mixture of hydrazides 7-8 or hydrazines 11-14 (2mmol) and corresponding aldehydes 9a-9d (2.1mmol) in absolute ethanol (20ml) were stirred at room temperature for 1-5 hr in the presence of two drops of hydrochloric acid as a catalyst. The end of the reaction was observed by TLC, and then neutralized by 10% aqueous solution of sodium bicarbonate. The resulting precipitate was filtered, washed with 20 ml water and dried and crystallized by ethanol.Spectral data of selected compounds N’-(2-phenoxybenzylidene)benzohydrazide (10a) -1, 3310 (NH), 1682 (C=O), 1643(C=N). Mass (m/z): 316, 121(100), 105 (64), 93 (55), 77 (95); Anal. Calcd. for C20H16N2O2: C, 75.93; H, 5.10; N, 8.86. Found: C, 75.64; H, 5.32; N, 8.72. 1H-NMR  : 11.93 , 8.78 , 7.90 , 7.58-7.16 , 7.01, 6.96. IR (KBr) : ν cmN ’ - ( 2 - p h e n o x y b e n z y l i d e n e ) - 2 - phenoxybenzohydrazide (10b) + ,100), 197(74), 169(38), 93(61), 77(84); Anal. Calcd. for C26H20N2O3: C, 76.45; H, 4.94; N, 6.86. Found: C, 76.20; H, 5.22; N, 6.69.1H-NMR : 11.94 , 8.58 , 7.99, 7.85 , 7.66-6.84 . IR (KBr): ν cm-1, 3290(NH), 1678(C=O), 1638(C=N). Mass (m/z): 408(MN’-(3-phenoxybenzylidene)-2-phenoxybenzohydrazide (10c)-1, 3320(NH), 1680 (C=O), 1640 (C=N). Mass (m/z): 408, 213(81), 197(80), 169(51), 93 (78), 77(85); Anal. Calcd. for C26H20N2O3: C, 76.45; H, 4.94; N, 6.86. Found: C, 76.38; H, 5.17; N, 6.73.1H-NMR : 11.72 , 8.27 , 7.95, 7.69-6.94 . IR (KBr): ν cmN’-(4-(methylthio)benzylidene)-2-phenoxybenzohydrazide (10d)-1, 3295(NH), 1685(C=O), 1646(C=N). Mass (m/z): 362, 197(88), 120(49), 93(70), 77(65); Anal. Calcd. for C21H18N2O2S: C, 69.59; H, 5.01; N, 7.73. Found: C, 69.72; H, 5.24; N, 7.41.1H-NMR : 11.81, 7.93, 7.22, 7.60 , 7.15-6.93, 2.51. IR (KBr): ν cmN’-((1H-indol-2-yl)methylene)-2-phenoxybenzohydrazide(10e)-1, 3375, 3324(NH), 1685(C=O), 1645(C=N). Mass (m/z): 355, 135(100), 120(87), 104(65), 77(82); Anal. Calcd. for C22H17N3O2: C, 74.35; H, 4.82; N, 11.82. Found: C, 74.01; H, 4.99; N, 11.51.1H-NMR  : 13.46, 12.1, 8.21, 7.90, 7.71-7.37, 7.19-6.90, IR (KBr) : ν cm2-(2-phenoxybenzylidene)-1-phenylhydrazine (15a)-1, 3335(NH), 1635(C=N). Mass (m/z): 288, 93(79), 92(68), 77(95).; Anal. Calcd. for C19H16N2O: C, 79.14; H, 5.59; N, 9.72. Found: C, 79.43; H, 5.39; N, 9.88.1H-NMR : 11.20, 8.15, 7.70-7.20, 7.15-6.91. IR (KBr): ν cm2-(2-phenoxybenzylidene)-1-(2-nitrophenyl) hydrazine (15b) 1H-NMR : 11.33, 8.79, 8.10, 7.97, 7.65-6.92.-1, 3322(NH), 1630(C=N), 1359, 1548(NO2). Mass (m/z): 333, 137(48), 93(80), 77(53); Anal. Calcd. for C19H15N3O3: C, 68.46; H, 4.54; N, 12.61. Found: C, 68.39; H, 4.31; N, 12.69.IR (KBr): ν cm2-(2-phenoxybenzylidene)-1-(4-nitrophenyl) hydrazine (15c) -1, 3342(NH), 1639(C=N), 1352, 1543(NO2). Mass (m/z): 333, 137(57), 93(74), 77(61); Anal. Calcd. for C19H15N3O3: C, 68.46; H, 4.54; N, 12.61. Found: C, 68.37; H, 4.542; N, 12.08.1H-NMR : 11.42, 8.71, 8.15, 7.79-7.18, 7.11-6.95. IR (KBr): ν cm2-(2-phenoxybenzylidene)-1-hydrazine (15d) -1, 3322(NH), 1634(C=N), 1359, 1548(NO2). Mass (m/z): 378, 182(91), 167(50), 77(94); Anal. Calcd. for C19H14N4O5: C, 60.32; H, 3.73; N, 14.81. Found: C, 60.16; H, 3.38; N, 14.56.1H-NMR  : 11.54, 9.10, 8.78, 8.63, 7.92, 7.77-7.20, 7.15-6.93. IR (KBr): ν cmPharmacological evaluationMale NMRI mice weighting 20-25 g  were used for abdominal constriction test (writhing test). The animal were housed in colony cages and conditions of constant temperature (22 ± 2°C) and a 12 h hlight/dark schedule and allowed free access to standard diet and tap water except during the experiment. The animals were allowed to habituate to the laboratory environment for 2h before the experiments were initiated. All ethical manners for use of laboratory animals were considered carefully and the protocol of study was approved by TUMS ethical committee. The compounds were administered intraperitoneally (IP)  as a suspension in saline and tween 80 (4%w/ν). Mefenamic acid   was used as standard drug under the same conditions. The control group received vehicle  alone Analgesic activityThe analgesic activity was determined in vivo by the abdominal constriction test induced by acetic acid in mice . An acetStatistical analysisStstistical analysis was done by one-way analysis of variance (ANOVA) and followed by Tukey multiple comparison test. Differences with p < 0.05 between experimental groups were considered statistically significant.In this study, a new series of hydrazide and hydrazine derivatives were synthesized and evaluated for analgesic activity using the acetic acid induced mice abdominal constriction test and the results are shown in As shown in So withdrawing of carbonyl moiety doesn’t have a deleterious effect on the analgesic potency. It has been observed that attachment of phenoxy group in both 2 and 3 positions of phenyl ring can result good compounds. Since compounds 10a, 10d and 10e showed weak activity, we can conclude that in hydrazide derivatives existence of phenoxy moiety in both side of the molecule is essential. Attachment of electronwithrawing groups like Nitro substitution on ortho or para position of phenyl ring in hydrazines can improve the activity but substitution of both positions by these groups has a decrescent effect on analgesic activity. In conclusion several synthesized compounds showed comparable or better analgesic activity than the reference drug.New hydrazide and hydrazone derivatives were synthesized and their structures were confirmed by spectral data. Their analgesic activity was tested by writhing test and most of them were active analgesic agents. Since in vivo activity depend on highly complex physiological interactions, therefore the SAR’s which were presented earlier are just probable and their validity can’t be claimed absolutely.


In continuation of our earlier research, we aimed at the synthesis and development of various potent antihypertensive 1,2,4-triazoloquinazoline derivatives.Several quinazoline and triazole derivatives are reported to possess a wide-range of interesting pharmacological effects. Although various triazoloquinazoline subclasses having been synthesized and studied, the preparation of 1,2,4-triazoloquinazoline derivatives have been synthesized. Their structures were mainly established by spectroscopic methods of analysis . Their in vivo antihypertensive activity was evaluated by tail cuff method using Muromachi Blood Pressure Monitor (Model MK 2000) for rats and mice. Some of the tested compounds were found to exhibit valuable effects in terms of heart rate and blood pressure. According to the biological results, some of tested derivatives have abolished completely the tachycardia of the parent compounds and may be studied and modified as potential adrenoblockers and cardiac stimulant.A new series of 1,2,4-triazoloquinazolines were synthesized by convenient methodology from four key molecules, whereby their structures were established by advanced spectroscopic analyses. Some lead compounds have abolished completely the tachycardia of the parent compounds, that may be examined as potent adrenoblockers and some other compounds seem to be a cardiac stimulant or may be modified to enhance their hypotensive activity.New series of fifteen 1,2,4-triazolo[1,5- High blood pressure has been identified as the most powerful one among the major risk factors for arterial diseases . Now-a-d(1–4) according to the literature triazoloquinazolin-5-ones (5–10) resulted in 71-87% yield. The products 5–10 were obtained as coloress solid and their IR spectra displayed a strong (C = O) absorption band in the range of 1675–1689 cm-1. When equimolar amounts of triazoloquinazolin-5-one 1 and phosphorus pentasulfide were allowed to react in dry pyridine under reflux for 3 h, the desired triazoloquinazolin-5-thione (15) could be isolated as yellow solid in excellent yield of 92% triazoloquinazolin-5-ones  into 5-chloro-triazoloquinazolines (11–13) have been successfully achieved by phosphorus oxychloride in boiling benzene for 2.5 h, followed by treatment with a saturated aqueous solution of potassium carbonate quinazolin-5-one (4) was obtained in good yield. IR spectrum of compound 4 is characterized by a strong (C = O) absorption band at 1699 cm-1. Analogous to the reaction of 2 with hydrogen peroxide, the corresponding compound 13 was obtained from 12. The MS spectra of 4 and 13 showed molecular ion peaks at m/z 263 and 282 , corresponding to their molecular formulae. As well as the structures of 4 and 13 were characterized by NMR, MS, IR spectra, and have been unambiguously proven by X-ray crystallography . In the tricyclic nucleus, the benzofused moiety was deduced from its own four one proton 1H-signals as two dd (or br d) resonances with Jortho (7.5-8.5 Hz) and Jmeta (1–2 Hz) assignable for H-9 and H-6 and two td (or br t) resonances with Jortho and Jmeta for H-8 and H-7, respectively. The exchangeable NH-proton was interpreted in 1H NMR of 3 and 4 at δ > 13 ppm and were absent in all spectra of N-alkyl or 5-chloro-triazoloquinazolines. 13C NMR spectra proved the main tricyclic moiety through characteristic nine resonances including the most downfield key signal of C-2 assigned at ≈ 165 ppm in 2-phenoxy derivatives  that was observed relatively upfield at ≈ 160 in case of 2-methylsulfonyl function  due to the stronger –R and –I (deshielding) effect of O-phenoxy than -SO2. Another key 13C-signal was C-5 that interpreted at δ ≈ 158–160 ppm in 5-ones (3–10), ≈ 167 in 5-Cl derivatives  and ≈ 185 in case of 5-thione (15). The 2-phenoxy function was easily deduced from its intrinsic three resonances at ≈ 7.46 , 7.33  and 7.26  for H-3′/5′, H-2′/6′ and H-4′  and confirmed through its four 13C resonances at δ ppm ≈ 154, 130, 124 and 119 interpretable for C-1′, 3′/5′, 4′, and 2′/6′, respectively. Methylsulfonyl function was concluded from its own 1H and 13C resonances of O2S-CH3 at ≈ 3.45 (s) and 42 ppm, respectively. N-alkylation with p-nitrobenzyl was unambiguously confirmed by its A2M2 spin coupling system of the two ortho-doublets at about 8.2 and 7.7 (J = 8.5) for H-3″/5″ and H-2″/6″ together with the downfield located CH2-singlet at ≈ 5.4 and its 13C-signal at 46 ppm by the deshielding effect of tertiary N-atom . Whereas, alkylation with allyl moiety was proved by its four characteristic 1H-resonances with intrinsic splitting pattern at about 5.9 (m), 5.25 , 5.20  and 4.72  assignable for olefinic methine and methylene and CH2-saturated types . The corresponding three 13C-resonances were interpreted at δ ppm 131.2, 117.5 and 45.2 for C-2, C-3 and C-1 of allyl group, respectively. In compound 18, 5-S-allyl protons were interpreted with the same splitting pattern but highly deshielded at 6.04, 5.42, 5.20 and 4.09 by higher electronegativity of S-atom and their 13C-signals were reported at 129.6 (C-2″), 115.1 (C-3″), and 32.1 (C-1″). Moreover, N-ethyl function was observed in the form of a typical A2X3 spin coupling system as a quartet (CH2) and triplet (CH3) at about 4.2  and 1.3  and confirmed by their corresponding 13C-resonances at δ ppm 38.5 and 12.4 . In 17 and 19 S-ethyl and S-phenyl functions showed the same splitting pattern of N-ethyl and O-phenyl but with slightly different δ-values in both 1H and 13C NMR spectra. All other 1H- and 13C-resonances of all structures were finally assigned on the basis of comparison with literature data of structure related compounds and according to application of substitution additive rules of 13C NMR.Generally, uncorrected melting points of all compounds were recorded and their chemical structures primarily consistent with their IR and MS data  and confirmed by In vivo antihypertensive activity study of the title compounds 1–19 was performed by tail cuff method using Muromachi Blood Pressure Monitor for rats and mice (Model MK 2000). The obtained results, in Table 1–4 may have had a considerable impact on the heart rate and blood pressure in all synthesized derivatives (1–19).1 was found to increase the heart rate, however it has not demonstrated any effect on blood pressure. Replacement of phenoxy group on position 2 by a sulfanyl group in case of compound 2 has been abolished effect on the heart rate. On the other hand, presence of sulfonyl group in 4 was found to induce the bradycardia, however no effect on blood pressure was noticed. Introduction of methyl group on the lead compound 1 to afford 3 did offer advantageous effect on reduction of blood pressure accomplished by suppressing effect on heart rate in regard to 3. Alkylation of lactam moiety in 1 furnished N-alkylated products 5–7, that did demonstrate remarkable effect. For instance, compound 5 was induced bradycardia, however great increasing in the heart rate showed by 6, whereas abolished in the tachycardia term and slightly induction on bradycardia was demonstrated by 7. Similarly, transformation of 4 into 9 has emerged more bradycardia effect accomplished by moderate decreasing on blood pressure. However the product 8 showed induction on bradycardia and 10 exhibited only bradycardia effect. Further transformation of 1 into 11 does not seem to offer any advantages effect on heart rate but a very slightly decrease in blood pressure was observed. Whereas, compound 12 showed more bradycardia and completely abolished in tachycardia effects together with a slightly increase on blood pressure. Moreover, decreasing on tachycardia profile was exhibited by compound 13. The replacement of the chlorine atom in 11 by ethoxide group gave 14, which has shown decrease on blood pressure and tachycardia effects. Thionation of 1 into 15 was accomplished by abolishing the tachycardia profile and induce on bradycardia term. The same behavior on tachycardia effect was observed by compound 16 but no influence on blood pressure profile has been recorded. Moreover, the transformation of 15 into 17 demonstrated almost the same behavior in the terms of the effects on heart rate and blood pressure. Whereas, compound 18 was exerted complete abolishing in tachycardia and slight induction on bradycardia profiles. However, induction on tachycardia and decreasing on blood pressure terms was demonstrated by compound 19.Basically, the parent 1–4 have afforded derivatives with different effects on heart rate and blood pressure profiles. Variation in the substituted alkyl groups has demonstrated slightly remarkable activity in regards to the parents, such as increasing on heart rate emerged by 6, induction in bradycardia proved by 5 and slightly decreasing in blood pressure occurred with 9. This could be attributed to the characteristic features of their alkyl groups. Introduction of lipophilic group on position 5 of the parent was appeared to offer slightly decrease in blood pressure in case of compounds 11 and 12. Nevertheless, the slight effect on blood pressure of 11 and 12 bearing at the 5-position a chlorine atom indicates that, not only chloro lipophilic factor, but also the sterric effect are important for decrease on blood pressure as shown by 14. Thionation targets 15 and 16 showed remarkable attenuated on profiles activity, despite these compounds possess enhanced lipophilicity comparable to that parent compounds. However thioether products have almost emerged the same behavior on blood pressure and heart rate as in 17, and decreasing on blood pressure by 19. The actual explanation for these changes regarding SAR will await the elucidation of the mechanism(s) of action of the compounds.Structure modifications on the lead compounds -1) spectra were recorded on a Perkin Elmer FT-IR Spectrum BX system. NMR spectra were recorded on a Bruker AMX 500 spectrometer in DMSO-d6 and reported as δ ppm values relative to TMS at 500 and 125 MHz for 1H- and 13C NMR, respectively. Mass spectra were measured on an Agilent 6410 TSQ system connected to Agilent 1200 HPLC interface (samples were infused in MeOH). Follow up of the reactions and checking the purity of compounds was made by TLC on DC-Mikrokarten polygram SIL G/UV254, from the Macherey-Nagel Firm, Duren Thickness: 0.25 mm. Column chromatography was conducted on silica gel .Melting points were determined on open glass capillaries using a Mettler FP 62 apparatus and are uncorrected. The IR  in EtOH (20 mL) at 0°C. Afterwards, triethylamine (30 mmol) was added drop-wise over a period of 30 min. After the addition was complete, the reaction mixture was left to stirr overnight at room temperature. Acidification of the mixture was performed by conc. HCl under ice cooling followed by refluxing for 1–3 h. After cooling, the mixture was poured into ice/water, the resulting solid was filtered, washed with water and dried. Recrystallization from THF gave analytically pure colored as white solid, yield: 77%, m.p. 218–220°C; IR (KBr): v/cm-1 1692, (C = O), 3219 (NH); 1H NMR (DMSO-d6): δ = 13.04 , 7.98 , 7.72 , 7.45 , 7.32 , 7.25 , 2.45 ; 13C NMR (DMSO-d6): δ = 165.7 (C-2), 159.4 (C-5), 154.2 (C-1′), 147.1 (C-9a), 136.2 (C-8), 135.3 (C-3a), 133.6 (C-5a), 129.7 (C-3′/5′), 127.8 (C-6), 124.9 (C-4′), 119.4 (C-2′/6′), 116.6 (C-7), 114.1 (C-9), 20.5 (Ar-CH3); MS (ESI) negative mode: m/z 291.1 (M- - 1) for MW = 292.2-Hydrazino-5-methyl benzoic acid (10 mmol) was added portion wise to a stirred solution of diphenyl-2O2 (10 mL), was added drop-wise over a period of 5 min. while heating. After the addition was complete, the mixture was poured into hot water and left at room temperature, the resulting solid was filtered, washed with water and dried. Recrystallization from toluene gave analytically pure colored as white solid, yield: 50%, m.p. 261–263°C; IR (KBr): v/cm-1 1690 (C = O) 3240 (NH); 1H NMR (DMSO-d6): δ = 13.39 , 8.22 , 8.04 , 7.97 , 7.65 , 3.35 ; 13C NMR (DMSO-d6): δ = 160.9 (C-2), 160.2 (C-5), 149.9 (C-9a), 135.4 (C-8), 135.3 (C-3a), 128.4 (C-6), 127.2 (C-5a), 118.0 (C-7), 114.8 (C-9), 42.0 (O2S-CH3); MS (ESI) (negative mode): m/z 263.1 (M- - 1), (positive mode): m/z 265.0 (M+ + H), 287.1 (M+ + Na) for MW = 264.An amount of 2-methylsulfanyl-4H-triazoloquinazolin-5-one (1 mmol) was dissolved in bioling glacial acetic acid (10 mL), afterward H1 or 4 (I mmol) in DMF (5 mL) was added potassium carbonate (1.2 mmol) portion wise over a period of 10 min at room temperature. After stirring for 20 min, the appropriate alkyl halide (1.5 mmol) was added drop wise, and the reaction mixture was stirred for 18 h at room temperature. The mixture was poured into ice/water, the precipitate was filtered off, washed with water and dried. Analytically pure products 5–10 were obtained after recrystallization from THF.To a solution of v/cm-1 1677 (C = O); 1H NMR (DMSO-d6): δ = 8.25 , 8.19 , 7.95 , 7.87 , 7.71 , 7.59 , 7.46 , 7.33 , 7.26 , 5.43 ; 13C NMR (DMSO-d6): δ = 165.2 (C-2), 158.7 (C-5), 154.1 (C-1′), 148.4 (C-9a), 146.9 (C-4″), 143.4 (C-1″), 135.8 (C-8), 135.3 (C-3a), 129.8 (C-3′/5′), 128.8 (C-2″/6″), 128.6 (C-6), 126.0 (C-5a), 124.9 (C-4′), 121.5 (C-3″/5″), 119.0 (C-2′/6′), 116.2 (C-7), 114.2 (C-9), 46.1 (-CH2-Ar); MS (EI): m/z (%) 413  for MW = 413.White solid, yield: 78%, m.p. 190–192°C; IR (KBr): v/cm-1 1675 (C = O ); 1H NMR (DMSO-d6): δ = 8.23 , 7.92 , 7.83 , 7.56 , 7.46 , 7.35 , 7.27 , 4.15 , 1.29 ; 13C NMR (DMSO-d6): δ = 165.4 (C-2), 158.3 (C-5), 154.2 (C-1′), 148.2 (C-9a), 135.4 (C-8), 135.0 (C-3a), 129.8 (C-3′/5′), 128.4 (C-6), 125.8 (C-5a), 124.9 (C-4′), 119.1 (C-2′/6′), 116.3 (C-7), 114.1 (C-9), 38.5 (-CH2-CH3), 12.4 (-CH2-CH3); MS (EI): m/z (%) 306  for MW = 306.White solid, yield: 73%, m.p. 165–167°C; IR (KBr): v/cm-1 1684 (C = O); 1H NMR (DMSO-d6): δ = 8.23 , 7.92 , 7.83 , 7.57 , 7.47 , 7.35 , 7.27 , 5.97 , 5.24 , 5.19 , 4.72 ; 13C NMR (DMSO-d6): δ = 165.4 (C-2), 158.2 (C-5), 154.2 (C-1′), 148.2 (C-9a), 135.5 (C-8), 135.1 (C-3a), 131.2 (C-2″), 129.8 (C-3′/5′), 128.5 (C-6), 125.9 (C-5a), 124.9 (C-4′), 119.1 (C-2′/6′), 117.5 (C-3″), 116.1 (C-7), 114.1 (C-9), 45.2 (C-1″); MS (EI): m/z (%) 318  for MW = 318.White solid, yield: 85%, m.p. 133–135°C; IR (KBr): v/cm-1 1675 (C = O); 1H NMR (DMSO-d6): δ = 8.27 , 8.07 , 8.00 , 7.69 , 4.22 , 3.49 , 1.34 ; 13C NMR (DMSO-d6): δ = 160.8 (C-2), 158.3 (C-5), 149.8 (C-9a), 135.5 (C-8), 134.5 (C-3a), 128.6 (C-6), 127.6 (C-5a), 117.4 (C-7), 114.8 (C-9), 42.1 (O2S -CH3), 38.9 (-CH2-CH3), 12.4 (-CH2–CH3); MS (EI): m/z (%) 292  for MW = 292.White solid, yield: 71%, m.p. 187–189°C; IR (KBr): v/cm-1 1684 (C = O); 1H NMR (DMSO-d6): δ = 8.27 , 8.10 , 8.03 , 7.70 , 6.00 , 5.31 , 5.21 , 4.80 , 3.48 ; 13C NMR (DMSO-d6): δ = 160.7 (C-2), 158.3 (C-5), 149.9 (C-9a), 135.7 (C-8), 134.8 (C-3a), 131.0 (C-2′), 128.7 (C-6), 127.7 (C-5a), 117.6 (C-3′), 117.2 (C-7), 114.9 (C-9), 45.5 (C-1′), 42.0 (O2S -CH3); MS (EI): m/z (%) 304  for MW = 304.White solid, yield: 87%, m.p. 152–154°C; IR (KBr): v/cm-1 1678 (C = O); 1H NMR (DMSO-d6): δ = 8.29 , 8.19 , 8.14 , 8.05 , 7.75 , 7.72 , 5.51 , 3.47 ; 13C NMR (DMSO-d6): δ = 160.6 (C-2), 158.8 (C-5), 150.2 (C-9a), 146.9 (C-4′), 143.2 (C-1′), 135.9 (C-8), 135.0 (C-3a), 128.7 (C-2′/6′), 128.7 (C-6), 127.7 (C-5a), 123.5 (C-3′/5′), 117.3 (C-7), 114.9 (C-9), 46.5 (-CH2-Ar), 42.0 (O2S –CH3); MS (EI): m/z (%) 399  for MW = 399.White solid, yield: 72%, m.p. 199–201°C; IR (KBr): 1 (1 mmol) was refluxed with Phosphorous oxychloride (1 mL) in benzene (7 mL) for 2.5 h. The solvent was evaporated and the residue was treated with saturated solution of potassium carbonate. The solid was filtered, washed thoroughly with water, dried and recrystallized from THF to give pure compound as white solid, yield: 82%, m.p. 167–169°C; 1H NMR (DMSO-d6): δ = 8.07 , 7.64 , 7.40 , 7.30 , 7.25 , 7.18 ; 13C NMR (DMSO-d6): δ = 167.6 (C-2), 166.3 (C-5), 157.0 (C-9a), 155.0 (C-1′), 136.3 (C-3a), 131.7 (C-8), 129.4 (C-3′/5′), 128.0 (C-7), 123.8 (C-4′), 123.0 (C-6), 119.0 (C-2′/6′), 118.6 (C-5a), 112.9 (C-9). MS (EI): m/z (%) 296  for MW = 296.Compound a]quinazoline was dissolved in bioling glacial acetic acid (10 mL), afterward H2O2 (10 mL) was added drop-wise over a period of 5 min. while heating. After the addition was complete, the mixture was poured into hot water and lef at room temperature, the resulting solid was filtered, washed with water and dried. Recrystallization from Toluene gave analytically pure colored as white solid, yield: 62%, m.p. 196–198°C; 1H NMR (DMSO-d6): δ = 8.21 , 7.99 , 7.66 , 3.54 ; 13C NMR (DMSO-d6): δ = 162.9 (C-5), 160.9 (C-2), 158.6 (C-9a), 137.3 (C-3a), 135.5 (C-8), 128.8 (C-7), 127.4 (C-6), 118.5 (C-5a), 114.7 (C-9), 42.1 (O2S-CH3); MS (EI): m/z (%) 282  for MW = 282.1 mmol of 2-methylsulfanyl-5-chloro-triazolo[1,5-11 (1 mmol) by stirring at room temperature for 30 min. Afterwards the solid was collected by filtration, air dried, and recrystallized from THF to give product as white solid, yield: 56%, m.p. 197–199°C; IR (KBr): v/cm-1 1602 (C = N); 1H NMR (DMSO-d6): δ = 8.15 , 7.71 , 7.65 , 7.49 , 7.43 , 7.36 , 7.04 , 3.72 , 1.57 ; 13C NMR (DMSO-d6): δ = 167.3 (C-2), 155.5, (C-5), 154.2 (C-9a), 150.7 (C-1′), 135.6 (C-3a), 133.5 (C-8), 129.8 (C-3′/5′), 127.9 (C-7), 123.8 (C-4′), 123.0 (C-6), 119.6 (C-2′/6′), 118.9 (C-5a), 112.9 (C-9), 24.1 (-CH2CH3), 13.9 (-CH2CH3); MS (EI): m/z (%) 306  for MW = 306.A freshly prepared sodium ethoxide solution from sodium (150 mg) and absolut ethanol (35 mL) was reacted with compound 1 (1 mmol) was refluxed with phosphorous pentasulfide (1 mmol) in absolute pyridine (5 mL) for 3 h. Afterwards the reaction mixture was cooled and poured into ice/water, the yellow precipitate was separated by filtration and washed thoroughly with water. Recrystallization from aqueous dimethylformamide furnished analytically pure as yellow solid, yield: 92%, m.p. 209–211°C; IR (KBr): v/cm-1 1197 (C = S); 1H NMR (DMSO-d6): δ = 8.64 , 7.95 , 7.89 , 7.56 , 7.47 , 7.35 , 7.27 ; 13C NMR (DMSO-d6): δ = 185.3 (C-5), 165.0 (C-2), 154.2 (C-1′), 149.3 (C-9a), 135.9 (C-8),132.4 (C-3a), 131.8 (C-5a), 129.8 (C-3′/5′), 126.3 (C-6), 125.0 (C-4′), 123.9 (C-7), 119.4 (C-2′/6′), 114.5 (C-9); MS (EI): m/z (%) 294  for MW = 294.Compounds 15 (1 mmol) was dissolved in 2 M sodium hydroxide solution (10 mL), alkyl halide (1.5 mmol) was added dropwise over a period 2 min, the mixture was left to stir for I h at room temperature, and the obtained solid was separated by filtration, washed thoroughly with water and dried. Recrystallization of the crude products from THF afforded 17–19 as colored pure solid.Compound 1H NMR (DMSO-d6): δ = 8.26 , 7.75 , 7.77 , 7.63 , 7.51 , 7.41 , 7.11 , 4.39 , 1.41 ; 13C NMR (DMSO-d6): δ = 167.9 (C-2), 156.7, (C-5), 154.0 (C-9a), 150.9 (C-1′), 136.1 (C-3a),134.8 (C-8), 130.1 (C-3′/5′), 128.3 (C-7), 125.2 (C-4′), 123.9 (C-6), 120.3 (C-2′/6′), 119.1 (C-5a), 112.1 (C-9), 62.1 (-CH2CH3), 13.9 (-CH2CH3); MS (EI): m/z (%) 322  for MW = 322.White solid, yield: 68%, m.p. 185–187°C; 1H NMR (DMSO-d6): δ = 8.08 , 7.72 , 7.66 , 7.37 , 7.33 , 7.25 , 7.17 , 6.04 , 5.42 , 5.20 , 4.09 ; 13C NMR (DMSO-d6): δ = 167.4 (C-2), 166.6 (C-5), 154.9 (C-1′), 154.2 (C-5a), 151.0 (C-9a), 136.2 (C-3a), 135.7 (C-8), 129.6 (C-2″), 129.5 (C-3′/5′), 127.9 (C-6), 125.0 (C-7), 123.9 (C-4′), 119.0 (C-2′/6′), 115.1 (C-3″), 112.9 (C-9), 32.1 (C-1″); MS (EI): m/z (%) 334  for MW = 334.Yellow solid, yield: 75%, m.p. 109–111°C; 1H NMR (DMSO-d6): δ = 8.81 , 7.78 , 7.71 , 7.66 , 7.43 , 7.38, 7.31 , 7.27 , 7.21 , 7.17 ; 13C NMR (DMSO-d6): δ = 166.8 (C-2), 155.0 (C-5), 154.8 (C-1′), 152.4 (C-3a), 132.9 (C-9a), 132.3 (C-8), 132.1 (C-3″/5″), 129.5 , 129.4 (C-4″), 127.9 (C-5a), 124.2 , 123.8 (C-6), (C-3′/5′), 119.1 (C-2′/6′), 119.0 (C-7), 113.5 (C-9); MS (EI): m/z (%) 370  for MW = 370.Yellow solid, yield: 54%, m.p. 172–174°C; In vivo antihypertensive activity study of the title compounds was performed by tail cuff method using Blood Pressure Monitor for rats and mice (Model MK 2000- Muromachi kikkai Co. ltd. Japan). The samples of the investigated compounds were administered orally at a dose of 5 mg/kg as suspension in 1% sodium carboxy methyl cellulose. Measurements (blood pressure and heart rate) were recorded before and after the treatment of tested compounds at the intervals of 1 h for 5 h.4, 8, 9, 12, and 15 have abolished completely the tachycardia of the parent compounds, they may be studied as potential adrenoblockers. Compounds 9 and 14 may be modified to enhance their hypotensive activity. Furthermore, compound 6 seem to be a cardiac stimulant and it will be studied further for this concern. Finally, the structure–activity relationship (SAR) study of the compounds gave us some useful insights about the characteristic requirements, which may be taken into consideration in the design of new antihypertensive agents.Since compounds The authors declare that they have no competing interests.RA and NL have made a substantial contribution to experimental design. RA and MM made a significant contribution to acquisition of data, analysis, manuscript preparation. KE designed and performed the biological study and manuscript revision. IA and MH read, revised and approved the final manuscript. All authors read and approved the final manuscript.


Z,6E)-1-N-methyl-3-benzylidene-6-(2S-methyl-3-hydroxypropylidene)piperazine-2,5-dione (1), -1-N-methyl-3-benzylidene-6-(2R-methyl-3-hydroxypropylidene)piperazine-2,5-dione (2), -3-(4-hydroxybenzylidene)-6-isobutylidenepiperazine-2,5-dione (3), -3-((1H-imidazol-5-yl)-methylene)-6-isobutylidenepiperazine-2,5-dione (4), and -3-benzylidene-6-(2S-but-2-yl)piperazine-2,5-dione (5), were isolated from the marine-derived actinomycete Streptomyces sp. FXJ7.328. The structures of 1–5 were determined by spectroscopic analysis, CD exciton chirality, the modified Mosher’s, Marfey’s and the C3 Marfey’s methods. Compound 3 showed modest antivirus activity against influenza A (H1N1) virus with an IC50 value of 41.5 ± 4.5 μM. In addition, compound 6 and 7 displayed potent anti-H1N1 activity with IC50 value of 28.9 ± 2.2 and 6.8 ± 1.5 μM, respectively. Due to the lack of corresponding data in the literature, the 13C NMR data of -3-benzylidene-6-isobutylpiperazine-2,5-dione (6) were also reported here for the first time. Five new diketopiperazine derivatives, (3 Micromonospora sp. L-13-ACM2-092 + . The similarity of the 1D NMR spectra to those of albonoursin (7) + . 1D NMR  to C-13 (δC 119.8) and C-12 (δC 137.0), of H-13 (δH 7.52) to C-15 (δC 137.1), and of H-11 (δH 6.60) to C-2 (δC 157.8) and C-12 suggested a dehydro-histidine (deh-His) unit. The Z-configurations of both Δ3,11 and Δ6 double bonds were deduced from the relative downfield shifts of H-11 and H-7 consistent with those of -3-((1H-imidazol-5-yl)methylene)-6-isopropylpiperazine-2,5-dione (δH 6.51) + , indicating an isomer of 6. The 1D NMR  derivatives of the acid hydrolysates of 5 and four authentic isoleucine samples  were prepared. HPLC analysis over ODS column (5 displayed the same retention time (tR 23.06 min) as the authentic l-Ile (tR 23.06 min) and l-allo-Ile (tR 23.06 min) but were different from d-Ile (tR 28.11 min) and d-allo-Ile (tR 28.11 min). The FDAA derivatives of the acid hydrolysates of 5 and the authentic l-Ile and l-allo-Ile were further analyzed by C3 HPLC column (5 was the same as for the authentic l-Ile (tR 38.50 min), but different from the authentic l-allo-Ile (tR 37.49 min). Thus, the isoleucine moiety in 5 was unambiguously identified as l-Ile, and the structure of compound 5 was elucidated as -3-benzylidene-6-(2S-but-2-yl)piperazine-2,5-dione.The molecular formula of Ce 1D NMR  spectra H-7/H-10  and the s method . The 1-fS column  revealedC column . The ret1–10 were postulated to be produced biogenetically from the amino acid pathway (cyclo(Phe-Leu) that underwent dehydrogenation in Phe moiety to form compounds 6 and 7. The N-methylation of 6 produced an un-isolated intermediate (a) that further underwent another dehydrogenation in the Leu moiety to form compound 8. The oxidation of 8 at the same homoallylic positions produced compounds 1 and 2. By a similar biosynthetic pathway, compounds 3–5 and 9 were produced from the cyclic condensation and successive dehydration of Tyr with Leu, His with Leu, Phe with Ile, and Phe with Val, respectively. The dehydrogenation is favored to form Z-products because of the steric hindrance of the carbonyl oxygen, while the dehydrogenation of Leu moiety in the N-Me substituted compounds only produced the E-products due to the strong steric hindrance by N-Me. So, compound 10 could be an artifact produced from the photo-isomerization of compound 8 under light during the extraction and the subsequent isolation steps. Compounds  pathway . Cyclic 1 and 3–10 were tested for antivirus effects on H1N1 by the CPE inhibition assay 25D −28 , IR (KBr) νmax 3496, 3208, 3073, 3022, 1675, 1617, 1494, 1454, 1376, 1033, 997, 763, 688 cm−1; 1H and 13C NMR data, Compound2: Yellow solid; UV (MeOH) λmax (log ε): 238 (4.10), 323 (4.51) nm; [α]25D +28 , IR (KBr) νmax 3496, 3208, 3073, 3022, 1675, 1617, 1494, 1454, 1376, 1033, 997, 763, 688 cm−1; 1H and 13C NMR data, Compound3: Yellow amorphous solid; UV (MeOH) λmax (log ε): 248 (3.75), 347 (4.10) nm; IR (KBr) νmax 3183, 3073, 2960, 2867, 1681, 1639, 1606, 1541, 1421, 1359, 1275, 1173, 1024, 998, 829, 761 cm−1; 1H and 13C NMR data, m/z 273.1231 [M + H]+ .Compound4: Yellow solid; UV (MeOH) λmax (log ε): 250 (3.71), 347 (4.25) nm; IR (KBr) νmax 2973, 2937, 2879, 1708, 1638, 1577, 1459, 1379, 1273, 1122, 1092, 1025, 960 cm−1; 1H and 13C NMR data, m/z 247.1189 [M + H]+ .Compound5: White solid; UV (MeOH) λmax (log ε): 229 (4.05), 302 (4.17) nm; [α]25D −36 , IR (KBr) νmax 3168, 3077, 3042, 2961, 2867, 1680, 1646, 1546, 1426, 1392, 1357, 1092, 933, 800, 760, 619 cm−1; 1H and 13C NMR data, m/z 259.1439 [M + H]+ .Z,6S)-3-Benzylidene-6-isobutylpiperazine-2,5-dione (6); 1H NMR : δ 8.58 , 9.98 , 3.94 , 1.58 , 1.80 , 0.89 , 0.88 , 6.67 , 7.48 , 7.39 , 7.30 . 13C NMR : δ 161.0 , 127.3 , 168.1 , 54.2 , 44.2 , 24.1 , 23.3 , 22.6 , 114.9 , 133.9 , 129.2 × 2 , 129.8 × 2 , 128.5 . HRESIMS m/z 259.1456 [M + H]+ .1  was dissolved in 1 mL of CH2Cl2, and triethylamine (10 μL) and p-bromobenzoyl chloride  were added. The mixture was stirred for 7 h at room temperature. Then 2 mL of H2O were added and the solution was extracted three times with CH2Cl2 (5 mL each). The CH2Cl2 solutions were combined and evaporated under reduced pressure to give a gum. p-Bromobenzoate 1c  was obtained by HPLC purification eluting with 85% (MeOH-H2O).Compound p-Bromobenzoate (1c): Yellow solid; [α]25D −50 ; CD  λext (Δε) 313 (−5.2), 254 (+4.78), 208 (−1.73). 1H NMR : δ 3.18 , 5.57 , 4.09 , 4.20 , 4.28 , 1.14 , 6.71 , 7.53 , 7.38 , 7.30 , 7.86 , 7.67 . 13C NMR : δ 31.3 , 158.5 , 126.0 , 158.8 , 131.1 , 130.4 , 31.4 , 69.3 , 17.5 , 116.6 , 133.5 , 129.7 × 2 , 129.1 × 2 , 128.0 , 165.6 , 129.4 , 132.3 × 2 , 131.5 × 2 , 127.9 . ESIMS m/z 469.1 and 471.1; HRESIMS m/z 469.0755 [M + H]+ .1  was dissolved in 500 μL of anhydrous pyridine and 4-dimethyl-aminopyridine  and (R)-MTPACl (10 μL) were added. The reaction was stirred for 12 h at room temperature. Then 1 mL of H2O was added, and the solution was extracted three times with CH2Cl2 (5 mL each). After removal of CH2Cl2 under reduced pressure, the residue was purified by semipreparative HPLC (70% MeOH-H2O) to yield (S)-MTPA ester 1a . By the same procedure, (R)-MTPA ester 1b , (S)-MTPA ester 2a  and (R)-MTPA ester 2b  were obtained from the reaction of 1 and 2  with (S)-MTPACl, (R)-MTPACl and (S)-MTPACl (10 μL each), respectively. Compound S)-MTPA ester 1a: δ 3.07 , 3.99 , 1.06 , 5.51 , 6.76 , 4.36 , 4.28 . 7.51 , 7.41 , 7.32 . HRESIMS m/z 525.1612 [M + Na]+ .R)-MTPA ester 1b: δ 3.12 , 3.99 , 1.07 , 5.51 , 6.76 , 4.33 , 7.50 , 7.41 , 7.32 . HRESIMS m/z 525.1613 [M + Na]+ . S)-MTPA : δ 3.12 , 3.99 , 1.07 , 5.51 , 6.76 , 4.33 , 7.50 , 7.41 , 7.32 . HRESIMS m/z 525.1607 [M + Na]+ .R)-MTPA ester 2b: δ 3.07 , 3.98 , 1.06 , 5.51 , 6.76 , 4.35 , 4.27 , 7.50 , 7.41 , 7.32 . HRESIMS m/z 525.1606 [M + Na]+ . 5  was dissolved in 6 M HCl (1 mL) in a sealed tube and the mixture was heated at 105 °C for 17 h. Then the solution was cooled and evaporated to dryness. The residue, l-Ile, l-allo-Ile and d-Ile, d-allo-Ile, was dissolved in H2O (250 μL each), respectively. 50 μL of each solution was treated with 200 μL of 1% FDAA in acetone followed by 1.0 M NaHCO3 (40 μL). The reaction was maintained 1 h at 45 °C and then quenched by addition of 2.0 M HCl (10 μL). The corresponding FDAA derivatives of hydrolysate of 5, l-Ile, l-allo-Ile, d-Ile and d-allo-Ile were analyzed by ODS HPLC column maintained at 30 °C using the following programs: solvent A, H2O + 0.2% TFA; solvent B, MeCN; linear gradient, 0 min 25% B (75% A), 40 min 60% B (40% A), 45 min 100% B ; UV detection at 340 nm. The retention times for the FDAA derivatives of the hydrolysate of 5, l-Ile, l-allo-Ile, d-Ile and d-allo-Ile were 23.06, 23.06, 23.06, 28.11 and 28.11 min, respectively  over 55 min with UV detection at 340 nm. The retention times for the FDAA derivatives of the hydrolysate of 5, standard l-Ile, and l-allo-Ile, were 38.50, 38.50, and 37.49 min, respectively  was then added to each well and incubated for 4 h. Old medium containing MTT (150 μL) was then gently replaced by DMSO and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at 570 nm. In the SRB assay, 200 μL of the A549 cell suspension was plated in 96-well plates at density of 2 × 105 cell mL−1. Then, 2 μL of the test solutions (in MeOH) were added to each well and the culture was further incubated for 24 h. The cells were fixed with 12% trichloroacetic acid and the cell layer stained with 0.4% SRB. The absorbance of the SRB solution was measured at 515 nm. Adriamycin was used as positive control .Cytotoxicity was assayed by the MTT  and SRB 50 was calculated as the compound concentration required inhibiting influenza virus yield at 48 h post-infection by 50%. Ribavirin was used as the positive control with an IC50 value of 38.8 μM.The antiviral activity against H1N1 was evaluated by the CPE inhibition assay ,22. Conf5 cells/well) were placed in a 24-well plate. The cells were then stably transfected with pNF-κB-Luc expression plasmid (0.5 μg/well). Transfections were performed using lipofectamine 2000 in accordance with the instructions of the manufacturer . Stably transfected cells were pretreated 2 h with the test compounds and stimulated with 0.1 μg/mL of LPS for an additional 4 h. The luciferase assay was performed with the aid of a Steady-Glo Luciferase assay system in accordance with the instructions of the manufacturer .The anti-inflammatory effects of compounds were assayed by inhibition of lipopolysaccharide (LPS)-mediated NF-κB transcriptional activity in RAW 264.7 cells . RAW 264E. coli, E. aerogenes, P. aeruginosa, B. subtilis, S. aureus, and C. albicans were evaluated by 2-fold dilution method [C. albicans at 37 °C. The test compounds were dissolved in DMSO at different concentrations from 100 to 0.78 μg/mL (from 6.25 to 0.025 μg/mL for the positive controls) by the continuous 2-fold dilution methods. The minimum inhibitory concentrations (MICs) were determined in 96-well plates, and each well contains 100 μL of contents composed of 20 μL of inoculums (5 × 105 CFU/mL), test compounds and LB or YPD media. The microtiter plates were incubated at 35 °C for 24 h and were examined for microbes’ growth by turbidity in daylight. The MICs were defined as the lowest concentration at which no visible growth of microbes could be observed. Ciprofloxacin lactate and ketoconazole were used as positive controls for E. coli, E. aerogenes, P. aeruginosa, B. subtilis, S. aureus, and C. albicans with MIC values of 0.05, 0.19, 0.1, 0.39, 3.12 and 0.025 μg/mL, respectively. The antimicrobial activities against n method . The tes1–5 were isolated and their structures including absolute configurations were determined. The new compound 3 displayed modest activity and the known analogs 6 and 7 displayed potent activity against H1N1 virus with IC50 values of 41.5 ± 4.5, 28.9 ± 2.2 and 6.8 ± 1.5 μM, respectively. The results showed that both Z-deh-Phe and Leu or Z-deh-Leu substitutions significantly increase the anti-H1N1 activity of DKPs, while E-isomerization and hydroxylation of both deh-Phe and deh-Leu moieties, and N-methylation reduce the activity. The substitutions of both deh-Phe or deh-Tyr with deh-His and Leu or deh-Leu with iso-Leu also reduce the anti-H1N1 activity. In addition, the 13C NMR data of -3-benzylidene-6-isobutylpiperazine-2,5-dione (6) was reported here for the first time. Five new vinylidene substituted diketopiperazines (DKPs)


The structures of these compounds were established on the basis of extensive spectroscopic analysis. The absolute configurations of compounds 1–5, were determined by CD spectroscopic analysis and comparison with literature data. All isolated compounds (1–5) were evaluated for their cytotoxic activities against MCF-7, NCI-H460, and SF-268 tumor cell lines by the 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) method. Five new oxygenated pimarane diterpenes, named scopararanes C–G ( Eutypella scoparia FS26 from marine sediment.In recent years, the rich and unprecedented chemical diversity of natural products from marine-derived fungi and their pronounced biological activities continue to attract the interest of natural product chemists, ecologists and pharmacologists alike ,2,3,4,5.Eutypella species are present in a diverse array of habitats, including soil in Antarctica and tropical forests of Australia and Thailand. It was also found in the marine pulmonate mollusc Onchidium sp.  , an olefinic proton at [δH 7.04 ], and four tertiary methyl groups at  , two sp2 methines, and eight quaternary carbons . The 1H and 13C NMR spectra of 1 were similar to those reported earlier for pimarane-type diterpene libertellenone A (6)  in 6 was replaced by the methylene at  in 1, and the chemical shift of C-2 (δC 17.7) in 1 was upfield shifted by ∆δ 11.5 ppm compared with that of C-2 (δC 29.2) in 6  according to proton coupling constants and NOESY spectrum results  showed the characteristics of geminal and axial-axial relationships, which indicated the pseudoaxial position of H-11β. H-11β showed NOE correlations with H-17 and H-20, and H-20 showed NOE correlation with H-19, demonstrating that C-17, C-19, and C-20 are in axial configurations on the top face of the molecule. The hydroxyl group at C-9 was established as beta-axial based upon the NOE correlation between H-11β and H-20.Scopararane C  ,14, exce.2) in 6 . 1H,1H-Cand H-16 . The obsand H-16 , further results . The cou2) was obtained as colorless crystals. Its molecular formula, C20H28O4, was determined by HR-EI-MS  indicating the presence of seven degrees of unsaturation. Comparison of the 1H and 13C NMR data with those of the scopararane C (1) revealed that the signals were similar, except one methylene signal at  in 1 was absent, and one new hydroxyl-bearing methine was present in 2 at , which suggested that scopararane D (2) was the 3-hydroxyl derivative of scopararane C (1). The HMBC correlations observed between the proton at δH 3.42 (H-3) and the 13C signals at δC 40.7 (C-4), 28.8 (C-1), 24.9 (C-18), and 18.1 (C-19), further confirmed the assignment of new hydroxyl group to the 3-position. The entire sequence of H-atoms and the C-skeleton of 2 was established by 1H, 1H-COSY and HMBC. The relative configuration of 2 was identical to that in libertellenone A (6) and scopararane C (1) according to proton coupling constants and NOESY spectrum data. The proton coupling constants of H-11β , H-1α , and H-3α , indicated the axial orientations of H-11β, H-1α, and H-3α. H-11β showed NOE correlations with H-17 and H-20, and H-20 showed NOE correlation with H-19, indicated the beta-axial positions of Me-17, Me-19, and Me-20. H-3α showed NOE correlations with H-1α and H-18, in agreement with the beta-equatorial position of 3-OH.Scopararane D (3) was obtained as colorless crystals, which were analyzed for the molecular formula C20H28O5  by HR-EI-MS . The 1H NMR spectrum of 3 was similar to that of scopararane D (2), except the methylene proton signal at  in 2 was replaced by the oxymethine proton resonance in 3 at [δH 4.23 ]. Comparison of the 13C NMR data with those of 2 showed analogy of the chemical shifts except for C-11 and C-12. The above evidence suggested that scopararane E (3) was the 11-hydroxyl derivative of scopararane D (2). The HMBC correlations observed between the proton at δH 4.23 (H-11) and the 13C signals at δC 39.9 (C-12), and 46.7 (C-10), further confirmed the assignment of new hydroxyl group to the 11-position. Analyses of 1H, 1H COSY and HMBC spectral data allowed complete assignment of protons and carbons in 3 , scopararane C (1), and scopararane D (2) according to proton coupling constants and NOESY spectrum data.Scopararane E  was obtained as colorless crystals, having a molecular formula of C20H30O5 , determined by HR-EI-MS . The 1H, 13C, DEPT135 and HSQC NMR spectra of 4 showed 20 carbon signals and 26 protons for three singlet methyls, seven methylenes , three methines , and seven quaternary carbons . Comparison of the 1H and 13C NMR data of 4 with those of scopararane B (7) , and the α,β-unsaturated ketone C-7 (δC 194.6) in 7 were absent, and one new methylene at , and a hydroxyl-bearing singlet methine signal at  were present in 4. The new methylene protons and singlet methine signal were then attributed to H2-11 and H-7 respectively. HMBC cross-peaks of H-7 with C-6, C-8, and C-9, and H-11 with C-8, C-9, C-12, and C-13, further confirmed the 7-position of the methine and 11-position of the methylene respectively. Analyses of 1H, 1H COSY and HMBC spectral data allowed complete assignment of protons and carbons in 4 ] with H-12, indicated the axial location of H-11β (δH 1.92). H-11β showed NOE correlations with H-17 and H-20b [δH 3.40 ], and H-20a [δH 4.13 ] showed NOE correlation with H-19, indicating the beta-axial orientation of Me-17, Me-19, and H-20. H-14 showed NOE correlations with H-7 and H-17, in agreement with alpha-axial positions of 7-OH and 14-OH. The relative configurations of C-7 and C-14 were identical to that in sphaeropsidin E  in 4, the presence of a singlet methyl at  in 5, and the replacement from the oxygenated quaternary C-6 at (δC 107.2) in 4 to the carbonyl resonance at (δC 210.9) in 5. Based on these spectral data, scopararane G (5) was assigned as the ketone derivative of scopararane F (4). HMBC cross-peaks of H-20 with C-1, C-5, and C-9, and H-7 with C-6, C-8, and C-9, further confirmed the 20-position of the methyl and 6-position of the carbonyl respectively. The entire sequence of H-atoms and the C-skeleton of 5 was established by 1H, 1H COSY and HMBC , MCF-7 (human breast adenocarcinoma cell line) and NCI-H460  by the MTT method. The results of the cytotoxicity tests suggested that all the analogues demonstrated quite different cytotoxic activities (9) exhibited strong cytotoxicities against SF-268, MCF-7 and NCI-H460 cell growth with IC50 values ranging from 4.4–9.9 μM. Libertellenone A (6) displayed varying cytotoxic activities against the three cell lines with IC50 values in the range of 12.0–40.2 μM. Scopararanes C (1) and D (2) showed moderate cytotoxic activities against the tumor cell line MCF-7 with IC50 values of 35.9 μM and 25.6 μM, respectively. The other compounds displayed no appreciable activity, except that compounds 3, 5, and 7 showed weak cytotoxic activities against the MCF-7 cell line with IC50 values of 74.1 μM, 85.5 μM, and 60.1 μM, respectively.Compounds tivities . Diaport9) with diaporthein A (8), scopararane B (7), and libertellenone A (6), we found that compound 9 displayed strong analogies with compounds 8 and 7, differing only in the presence of a ketone moiety at C-7 replacing a hydroxy group, and a ∆8, 14 olefinic bond instead of a ∆8, 9 olefinic bond, respectively; compound 9 exhibited high structural similarity with compound 6 and the main structural difference between them is at position C-20. It also should be noted that the only structural differences between libertellenone A (6) and scopararanes C–E (1–3) are the number and positions of hydroxyl groups. All the above suggested that the carbonyl at position C-7, ∆8,14 olefinic bond, whether the C-20 methyl forms a furan ring with C-6, C-5, and C-10, and the number and positions of hydroxyl groups, might be the important structural factors leading to the distinction of their cytotoxicities. Diaporthein B and the 11-dehydroxy derivative of diaporthein B (scopararane A) were reported to show significant cytotoxic activities against human cancer cell lines  and nonmalignant Vero cells (African green monkey kidney fibroblasts) 25D −160.40 ; UV (MeOH) λmax (log ε) 219 (3.75), 263 (3.68), 325 (3.92) nm; CD (CH3CN) (∆ε) 352 (−32.4), 308 (+7.3); IR (KBr) vmax 3528, 3412, 3011, 2954, 2924, 2866, 1652, 1622, 1590, 1462, 1394, 1369, 1287, 1211, 1122, 1085, 1046, 997, 842, 806, 780, 679, 545 cm−1; 1H and 13C NMR, see m/z 316.2032 [M]+.Scopararane C (2): colorless crystals; mp 197.4 °C; [α]25D −200.36 ; UV (MeOH) λmax (log ε) 218 (3.94), 260 (3.81), 325 (4.02) nm; CD (CH3CN) (∆ε) 351 (−37.7), 310 (+9.1); IR (KBr) vmax 3522, 3410, 3089, 2968, 2877, 1650, 1620, 1591, 1465, 1392, 1369, 1289, 1231, 1206, 1142, 1085, 1044, 1012, 957, 919, 878, 806, 780, 679, 548 cm−1; 1H and 13C NMR, see m/z 332.1983 [M]+ .Scopararane D (3): colorless crystals; colorless crystals; mp 100.0 °C; [α]25D −92.30 ; UV (MeOH) λmax (log ε) 218 (4.02), 259 (3.90), 324 (4.12) nm; CD (CH3CN) (∆ε) 350 (−36.4), 312 (+15.1); IR (KBr) vmax 3417, 3087, 2970, 2941, 2874, 1653, 1622, 1592, 1464, 1396, 1374, 1333, 1289, 1230, 1153, 1064, 1041, 1017, 1002, 968, 916, 877, 835, 778, 731, 678, 545 cm−1; 1H and 13C NMR, see m/z 371.1833 [M + Na]+ .Scopararane E (4): colorless crystals; mp 170.0 °C; [α]25D −94.70 ; UV (MeOH) λmax (log ε) 207 (3.41) nm; CD (CH3CN) (∆ε) 207 (−103.4); IR (KBr) vmax 3382, 3086, 2932, 1641, 1460, 1393, 1302, 1247, 1212, 1180, 1125, 1001, 949, 914, 877, 845, 804, 709 cm−1; 1H and 13C NMR, see m/z 373.1984 [M + Na]+ .Scopararane F (5): a colorless oil; [α]25D +1.43 ; UV (MeOH) λmax (log ε) 206 (3.67), 232 (3.45), 278 (3.21) nm; CD (CH3CN) (∆ε) 342 (−0.8), 277 (+0.4), 250 (−1.1), 227 (+1.8); IR (KBr) vmax 3436, 2960, 2928, 2874, 1725, 1461, 1379, 1286, 1125, 1074, 1043, 844, 805, 745 cm−1; 1H and 13C NMR, see m/z 334.2138 [M]+ .Scopararane G (6): colorless crystals; CD (CH3CN) (∆ε) 353 (−46.3), 311 (+13.8).Libertellenone A (1–5) were assayed against three tumor cell lines MCF-7, NCI-H460 and SF-268. Assays were performed by the MTT method [Cytotoxicities of compounds . Cytotoxic tests for the five novel compounds (1–5) and their related six analogues [8, 14 olefinic bond, whether the C-20 methyl forms a furan ring with C-6, C-5, and C-10, and the number and positions of hydroxyl groups might be the important structural factors leading to the distinction of their cytotoxicities.Investigation of biologically active secondary metabolites from the marine-derived fungus nalogues  displaye


O-β-d-glucopyranosyloxy-2α,3α,24-trihydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (1) and 2α,3β,3,30-tetrahydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (2) were isolated from roots of Actinidia valvata Dunn. Their structures were elucidated by means of extensive spectroscopic studies. Both these two new compounds showed moderate cytotoxic activity in vitro against BEL-7402 and SMMC-7721 tumor cell line.Two new triterpenoids, 30- Actinidia valvata Dunn is a shrub mainly growing in eastern China (Zhejiang and Jiangxi province) D25 = 5.89 , and had the molecular formula C42H66O16, with ten degrees of unsaturation, as determined according to a pseudo-molecular-ion peaks at 849.4240  in the HR-ESI-MS.Compound 13C-NMR (DEPT) spectra of compound 1 revealed 42 carbon signals, including five CH3, two C=C bonds  and one C=O group. Assuming compound 1 has skeleton of urs-triterpenoid, the assignment of the two C=C bonds should be highly concerned. In 1H-NMR spectra of compound 1, five singlets at δ(H) 0.80–1.89 consisting of five CH3, and proton signal at δ(H) 5.53 (br) was assigned to 12-position. In HMBC spectra, clear correlation of 19–CH3 with two quaternary C-atoms at δ(C) 126.17 (s) and δ(C) 136.92 (s) was observed, and the correlation signals of 12-H with these two quaternary C-atoms were also observed. Thus, the two C=C bonds can be rightly assigned to 18-, 19-position, respectively. The HMBC correlation signal of 20-H with C-atom at δ(C) 69.68(t), was observed, then the oxygenation of 30-C may be deduced successfully. In NOSEY spectra, as the correlations of 2-H with 3-H, 2-H with 10-Me, 8-Me with 10-Me were observed, and by comparing with reference data .1H-, 13C-NMR spectra .Basing on above analysis, in combination with the  spectra , HMQC, H spectra , establi2 was obtained as white amorphous powder, displayed positive Liebermann–Burchard test, was optically active with [α]D25 = 7.35 , and had the molecular formula C36H56O11 with nine degrees of unsaturation, as determined according to a pseudo-molecular-ion peaks at 687.3717  in the HR-ESI-MS.The compound 1H-NMR and 13C-NMR .Comparing the  13C-NMR  spectra,nce data ,7. In co spectra , HMQC, H spectra , establi1 and 2 showed moderate in vitro cytotoxic activity against BEL-7402  and SMMC-7721 , as determined by classical MTT -2,5-diphenyl-2H-tetrazolium bromide) colorimetric assay.Compounds −1. 1H-, 13C-, 2D-NMR spectra: BRUKER AVANCE 600 spectrometer; chemical shifts δ in ppm rel. to (CH3)4Si, coupling constant J in Hz. ESI-MS: Finnigan LCQ mass spectrometer; in m/z. HR-ESI-MS: Q-Tof micro YA019 mass spectrometer.Silica-gel plates  were used for TLC analysis. mp: WRS-1A micro-melting-point apparatus; uncorrected. Optical rotations: JASCO P-1300 spectropolarimeter. IR: Spectra: BRUKER VECTOR-22 spectrophotometer; in cmActinidia valvata Dunn were collected in Changshan County, Zhejiang Province, China, in October 2006, and identified by Zheng Han-Chen, Department of pharmacognosy, School of pharmacy, Second military medical university. A voucher specimen (No. 20061005) was deposited at Department of pharmacognosy, School of pharmacy, Second military medical university.The roots of Actinidia valvata Dunn 30 kg was refluxed with 8 times of 80% EtOH solution for 3 times, 1.5 h each time. The extract was concentrated under reduced pressure to brown syrup, which was partitioned between H2O and petroleum ether (PE), AcOEt, and BuOH, successively. The n-BuOH soluble fraction (280.6 g) was subjected to column chromatography (CC) on silica gel (SiO2), eluting with CHCl3/MeOH/H2O (10:1:0.1 to 2:1 0.1) to afford 9 fraction 1–9. Fraction 5 were repeatedly subjected to CC (Pharmadex LH-20 and RP C-18) to yield compound 1 (10.4 mg) and 2 (14.6 mg).The powdered plant material of roots of O-β-d-glucopyranosyloxy-2α,3α,24-trihydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (1): white amorphous powder, mp 140°–142°, [α]D25 = 5.89 . IR (KBr): 3432, 2920, 2852, 1641, 1380, 1038. 1H-NMR  and 13C-NMR : M + Na]+), HR-ESI-MS: 849.4240 .30-O-β-d-glucopyranosyl ester (2): white amorphous powder. mp 220° (carbonification), [α]D25 = 7.35 . IR (KBr): 3448, 2963, 1681, 1644, 1381, 1278, 1080. 1H-NMR  and 13C-NMR : M + Na]+), HR-ESI-MS: 687.3717 .2α,3β,23,30-tetrahydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyloxy-2α,3α,24-trihydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester and 2α,3β,23,30-tetrahydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester were isolated from roots of Actinidia valvata Dunn, and their structures were elucidated by means of extensive spectroscopic studies. Both these new compounds showed moderate cytotoxic activity in vitro against BEL-7402 and SMMC-7721 tumor cell line.In the present research, two new triterpenoids, 30-