Lens culinaris) seeds is well described in scientific literature, there is very little available data about secondary metabolites from lentil leaves and stems. Our research reveals that the aerial parts of lentil are a rich source of flavonoids. Six kaempferol and twelve quercetin glycosides were isolated, their structures were elucidated using NMR spectroscopy and chemical methods. This group includes 16 compounds which have not been previously described in the scientific literature: quercetin 3-O-β-D-glucopyranosyl(1→2)-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (1), kaempferol 3-O-β-D-glucopyranosyl(1→2)-β-D-galacto-pyranoside-7-O-β-D-glucuropyranoside (3), their derivatives 4–10, 12–15, 17, 18 acylated with caffeic, p-coumaric, ferulic, or 3,4,5-trihydroxycinnamic acid and kaempferol 3-O-{[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl(1→2)]-α-L-rhamnopyranosyl(1→6)}-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside (11). Their DPPH scavenging activity was also evaluated. This is probably the first detailed description of flavonoids from the aerial parts of lentil.While the phytochemical composition of lentil ( Lens culinaris Medik) whose cultivation started in the Near East in the Neolithic period, is one of the earliest domesticated plants. Nowadays, lentil is a crop of high importance in many countries of Northern Africa, Western and Southern Asia, as well as in Canada, which is the world leader in its production  and 2D  spectra  of these precursor ions revealed that they share the same fragmentation pattern. In the case of compounds 1 and 2, MS/MS spectra showed the presence of fragment ions of m/z 625 [(M−H)−176]−, suggesting the loss of a hexuronic acid, and m/z 300 [(M−H)−176−325]−, formed after the additional loss of the pair of hexoses and corresponding to the quercetin radical ion [Y0−H]−·. Similarly, compound 3 fragmented to ions of m/z 609 [(M−H)−176]− (the loss of an hexuronic acid), and m/z 285 [(M−H)−176−324]− , corresponding to the kaempferol ion Y0−. A precise structure elucidation was possible after 1D and 2D-NMR analyses of these compounds. The 13C-NMR spectrum of 1 showed 33 signals, sorted by 13C and DEPT-135 experiments into 2 CH2, 20 CH and 11 quaternary carbon atoms. The aromatic region of the 1H and COSY spectra of 1 exhibited the presence of two sets of aromatic protons, characteristic for the quercetin aglycone  and 6.50 , which were correlated in the HSQC spectrum with their aromatic carbon atoms at δC 95.8 and 100.7 ppm, respectively. The other set corresponded to 3,4-dihydroxyphenyl group at δH 7.80 , 7.62 , and 6.91 , in accordance with AMX system of ring B of the aglycone. The assignments of all carbons of the flavonol moiety were accomplished by interpretation of the HSQC and HMBC spectra. Observed heteronuclear multiple bond connectivity (HMBC) correlations from H-2' and H-6’ to C-2, 4J correlation from H-8 to C-4 and downfield shifted resonance at δC 159.4 for C-2, indicated that the compound 1 contained 3-O substituted quercetin. This was further supported by the result of UHPLC analysis of non-polar products of acid hydrolysis of 1. The carbohydrate region of 1H NMR spectrum showed the presence of the oxymethine protons in the range δ 3.36–4.14. Moreover, three anomeric proton signals at δH 5.35 , 4.79  and 5.20  were also observed, indicating the presence of three sugar units. Based on the values of coupling constants (J > 7 Hz), and the analysis of 1H-, 13C-NMR spectra, and 1D TOCSY, COSY, TOCSY, HSQC, HSQC-TOCSY and HMBC data, the three sugar units were elucidated as β-galactopyranoside δH/C 5.35 /101.6 , β-glucopyranoside δH/C 4.79 (H-1Glc)/105.1 (C-1Glc) and β-glucuropyranoside δH/C 5.20 (H-1GlcA)/101.4 (C-1GlcA)  . It was J coupled nuclei is of the same order as the coupling constant. It is said that this type of coupling shows dependence on solvent and field strength. Other factors influencing the complexity of the spectrum are both steric and electronic contributions. As it is shown later in this paper, other isolated compounds, substituted in either C-2GlcA or C-6Glc (or both) do not show the phenomenon of virtual coupling, but it is still visible in the GlcA of compound 3. The long range correlations observed in the HMBC spectrum between the anomeric proton of the glucose  and C-2 of galactose (δC 80.8) indicated the presence of interglycosidic linkage between these hexosyl units (1→2). This was further supported by the NOE effect detected in the rotating frame nuclear Overhauser effect spectroscopy spectrum (ROESY) between H-1Glc and H-2Gal. The 3-O glycosidation site was determined mainly by NOE effect in ROESY spectrum between the anomeric proton of galactopyranoside  and ring B  of the quercetin moiety. The other, indirect evidence, was the downfield shifted resonance at δC 159.4 for C-2, as mentioned earlier. The correlation observed in the HMBC spectrum from anomeric proton at δH 5.20 (H-1GlcA) to carbon C-7 (δC 164.3) and the NOE effect visible in the ROESY spectrum between H-1GlcA and H-6/8 indicated that the point of attachment of β-glucuropyranosyl unit to quercetin was at C-7 position. Therefore, the compound 1 was identified as quercetin 3-O-β-D-glucopyranosyl(1→2)-β-D-galactopyranoside-7-O-β-D-glucuropyranoside. Since the compound has not been reported before, we propose to name it lensoside A.Typically it occurs when the chemical shift difference between two 2, 11 and 16. Furthermore, on the basis of NMR spectra and the results of UHPLC analyses of non-polar products of acid hydrolysis, we established that the quercetin nucleus was also present in compounds 2, 4, 5, 7, 8, 12, 14, 15, 16, 17 and 18, which will be discussed later.This scheme of glycosidation, along with the type of sugar moieties, was observed in all isolated compounds, except for molecules 2 was identified, on the base of its fragmentation pattern and NMR spectrum, as quercetin 3-O-β-sophoroside-7-O-β-glucuronide, previously isolated from epidermis of onion (Allium cepa) − ions were detected during ESI-MS analyses of these flavonoids: m/z 979, 963, 947, 947, 977, 931 and 961, respectively. The precursor ion of compound 4 (m/z 979) gave fragment ions of m/z 803 [(M−H)−176]− after the loss of a hexuronic acid, m/z 625 [(M−H)−176−178]−, which could indicate the presence of trihydroxy-cinnamic acid, and m/z 300 [(M−H)−176−178−325]−, after the additional loss of dihexose, corresponding to the quercetin radical ion [Y0−H]−·.Compounds 5, 7, and 8 also seemed to be acylated quercetin derivatives, with the same fragmentation pattern , differing only in their putative phenolic acid moieties: caffeic (5), p-coumaric (7) and ferulic acid (8). The ESI-MS/MS analysis of the compounds 6, 9, and 10 showed that they were acylated derivatives of kaempferol. The precursor ion of compound 6 (m/z 947) fragmented into ions of m/z 771 [(M−H)−176]− (the loss of a hexuronic acid), m/z 609 [(M−H)−176−162]− (indicating a putative loss of caffeic acid), and m/z 284 [(M−H)−176−162−325]− , corresponding to the kaempferol radical ion [Y0−H]−·. Compounds 9 and 10 had the same fragmentation scheme, and differed only in their putative phenolic acid groups: p-coumaric and ferulic acid, respectively.Compounds 1–3. NMR analyses confirmed this conclusion. The 13C-NMR spectrum of the compound 4 showed 42 signals, sorted by 13C and DEPT-135 experiments into two CH2, 24 CH and 16 quaternary carbon atoms. Assignment of glucosidic protons systems and sites of glycosylation was achieved by analysis of 1D TOCSY, 1D ROESY, COSY, HSQC and HMBC experiments. The 1H-NMR spectrum of 4 contained resonances typical for the quercetin nucleus as expected  olefinic moiety and a downfield shifted aromatic singlet at δH 6.23 (2H) correlated in the HSQC spectrum with its aromatic carbon atom at δC 108.5. The long-range correlations observed in HMBC spectrum suggested that this is a E-3,4,5-trihydroxycinnamoyl moiety. For the Glc residue H-6 protons and C-6 carbon were downfield shifted to δH 4.41  and δC 64.8, espectively . Therefore, 4 was a monoacylated derivative of 1, established as quercetin 3-O-[-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside, named lensoside Aα.The UV and MS spectra, as well as the higher retention times of these flavonoids clearly suggested that they might be acylated forms of compounds expected , but alsectively . Additio5, 7 and 8, similarly to 4, were monoacylated with phenolic acids. All of them shared the same basic skeleton, identical with 1, which was confirmed with COSY, ROESY, HSQC and HMBC spectra along with selective experiments (1D TOCSY and 1D ROESY) used for the determination of nature of sugar moieties. The aromatic region of the 1H and COSY spectra of 5 contained sets of resonances characteristic for E- olefinic moiety and ABX system corresponding to a 3,4-dihydroxyphenyl group at δH 6.75 , 6.62 , and 6.59  and it was identified as E-caffeoyl group. Likewise, the aromatic region of the 1H and COSY spectra of 7 contained one pair of E-α-H and β-H doublets , but it also exhibited a AA’XX’ system corresponding to p-hydroxyphenyl group at δH 7.12  and 6.67 , which was interpreted as E-p-coumaroyl moiety in turn.Compounds 8 exhibited in the aromatic region of 1H and COSY spectra sets of resonances very similar to 5. One set of resonances was characteristic for E-  olefinic moiety and an ABX system corresponded to 3,4-dihydroxyphenyl group at δH 6.76 , 6.70  and 6.65 . The feruloyl nature of the acyl group in 8 was confirmed by a long-range correlation in HMBC between the 3-OCH3 group at δH 3.79  and an aromatic carbon C-3Fer that resonated at δC 148.9. The site of methylation in fthe eruloyl group of 8 was further confirmed by the NOE effect visible in the ROESY spectrum between protons of the CH3 group and H-2Fer. Protons H-6Glc in compounds 5, 7, 8 were downfield shifted to δH 4.43 , 4.44  and 4.49  as well as 4.44  and exhibited correlations in the HMBC spectra with carbonyl carbons resonated at δC 168.9, 168.9 and 168.9 corresponding with C-9Caf, C-9Cou and C-9Fer, respectively. Therefore, 5 was quercetin 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (named lensoside Aβ), 7 was quercetin 3-O-[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside(named lensoside Aγ) and compound 8 was quercetin 3-O-[(6-O-E-feruloyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (named lensoside Aδ).Compound 6, 9 and 10, similarly to 4, were monoacylated with phenolic acids. All of them shared the same basic skeleton, identical with 3, which was confirmed with COSY, ROESY, HSQC and HMBC spectra along with selective experiments (1D TOCSY and 1D ROESY) used for the determination of nature of sugar moieties. Compounds 6, 9 and 10 were kaempferol analogues of acylated quercetin glycosides 5, 7 and 8. The 1H-NMR spectra of 6, 9 and 10 contained resonances for the protons of kaempferol, and 1H- and 13C-NMR chemical shift values for the glycosyl moieties were similar to those of 5, 7 and 8, respectively. The biggest differences, apart from the aglycone part, were noticed in Gal anomeric protons upfield shifted to δH 5.06, 5.01 and 4.94 in 6, 9 and 10, comparing to δH 5.20, 5.19 and 5.11 in 5, 7 and 8, respectively. Thus 6 was kaempferol 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (named lensoside Bα), 9 was kaempferol 3-O-[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (named lensoside Bβ) and the compound 10 was kaempferol 3-O-[(6-O-E-feruloyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (named lensoside Bγ).Compounds 12–15, 17, and 18. Their UV spectra indicated that they were acylated with phenolic acids. [M−H]− ions of these compounds were as follows: m/z 1125, 1109, 1109, 1109, 1139, 1093. The fragmentation of the [M−H]− ion of flavonoid 12 comprised ions of m/z 963, indicating the loss of caffeic acid , m/z 787 (the loss of a hexuronic acid), and the further fragmentation path was the same as for 5. These facts indicate that 12 contains two caffeic acid moieties, and one of them is probably bound to the hexuronic acid. Similarly, the [M−H]− ion of compound 13 fragmented to the ions of m/z 947, which indicated the loss of coumaric acid, m/z 771 (the loss of an hexuronic acid), and other fragments were the same as for the flavonoid 6. Since the general pattern of fragmentation was similar for the remaining flavonoids of this group, it may be deduced that the compound 14 is a caffeoylated derivative of 7, 15—a coumaroylated derivative of 5, 17—a feruloylated derivative of 5, and 18 is a coumaroylated derivative of 7.The third group of structurally related flavonoids comprise compounds 13C-NMR spectrum of compound 12 showed 51 signals, sorted by 13C and DEPT-135 experiments into two CH2, 30 CH and 19 quaternary carbon atoms. Assignment of glucosidic protons systems and sites of glycosylation was achieved by analysis of 1D TOCSY, 1D ROESY, COSY, HSQC and HMBC experiments , and exhibited two separate AMX systems corresponded to 3,4-dihydroxyphenyl groups, which were interpreted as two E-caffeoyl moieties. Protons H-6Glc were downfield shifted at δH 4.42  and 4.38  and exhibited correlations in the HMBC spectra with carbonyl carbon resonated at δC 169.0 corresponding with C-9Caf. The H-2GlcA was downfield shifted to δH 5.16  and this resonance correlated to the carbonyl carbon C-9Caf of the second E-caffeoyl moiety at δC 168.3 in the HMBC spectrum. Therefore, 12 was in fact acylated form of the compound 5, quercetin 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-caffeoyl’)-β-D glucuropyranoside, named lensoside C.The eriments . The 1H-l with 1 . The aro13, like 12, was diacylated flavonol. It shared the same basic skeleton, identical with 3, which was confirmed with COSY, ROESY, HSQC and HMBC spectra along with selective experiments (1D TOCSY and 1D ROESY) used for the determination of nature of sugar moieties. The compound 13 was kaempferol analogue of the acylated quercetin glycoside 12. The 1H NMR spectrum of 13 contained resonances for the protons of kaempferol, and 1H and 13C-NMR chemical shift values for the glycosyl moieties were similar to those of 12. Thus, 13 was kaempferol 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-caffeoyl’)-β-D-glucuropyranoside, named lensoside D.Compound 15 and 17, were p-coumaroylated and feruloylated derivatives of compound 5, respectively. The 1H-NMR spectrum of 15 exhibited a AA’XX’ system corresponding to p-hydroxyphenyl group at δH 7.44  and 6.77 , which was interpreted as E-p-coumaroyl moiety. For the GlcA residue, H-2 was downfield shifted to δH 5.16 , as in 12, and this proton correlated to the carbonyl carbon C-9Cou resonated at δC 168.2 in the HMBC spectrum. Likewise, in 17, H-2GlcA was downfield shifted to δH 5.16  and this proton correlated to the carbonyl carbon C-9Fer resonated at δC 168.2 in the HMBC spectrum. Therefore, 15 was identified as quercetin 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-p-coumaroyl)-β-D-glucuropyranoside (named lensoside E) and 17 was quercetin 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-feruloyl)-β-D-glucuropyranoside (named lensoside F).Compounds 14 and 18, were caffeoylated and p-coumaroylated derivatives of compound 7, respectively. In the 1H-NMR spectrum of 14, H-2GlcA was downfield shifted to δH 5.16  and this proton correlated to the carbonyl carbon C-9Caf resonated at δC 168.2 in the HMBC spectrum. The aromatic region of the 1H and COSY spectra of 18 exhibited two sets of a AA’XX’ system corresponding to p-hydroxyphenyl group at δH 7.04  and 6.62  as well as δH 7.45  and 6.77 , which was interpreted as two E-p-coumaroyl moieties.Compounds H 5.16 , and this proton correlated to the carbonyl carbon C-9Cou’ resonated at δC 168.2 in the HMBC spectrum. Therefore, 14 was quercetin 3-O-[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-caffeoyl)-β-D-glucuropyranoside (named lensoside G) and 18 was identified as quercetin 3-O-[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-p--coumaroyl’)-β-D-glucuropyranoside, named lensoside H.For the GlcA residue, H-2 was downfield shifted to δ11 and 16 are significantly different from the above-described flavonoids. The MS/MS analysis of compound 11 gave a deprotonated molecule at m/z 1047, which fragmented into ions at m/z 901 [(M−H)−146]−, m/z 755 [(M−H)−146−146]−, indicating the loss of one and two deoxyhexose/coumaric acid units, respectively, and m/z 284 [(M−H)−146−146−471]−, formed after a putative loss of pair of hexoses bound to a deoxyhexose, and corresponding to kaempferol radical ion. Low value of UV absorption maximum for Band I (315 nm) and high retention time indicate the acylation of 11 with coumaric acid. The 13C-NMR spectrum of 11 showed 48 signals, sorted by 13C and DEPT-135 experiments into two CH3, two CH2, 32 CH and 12 quaternary carbon atoms. The aromatic region of the 1H and COSY spectra of 11 exhibited the presence of two sets of aromatic protons, characteristic for kaempferol aglycone  and 1.15 . Moreover, four anomeric proton signals at δH 5.53 , 4.99 , 4.71  and 4.48  were also observed, thus indicating the presence of four sugar units. Assignment of glucosidic protons system and sites of glycosylation was achieved by analysis of 1D TOCSY, 1D ROESY, COSY, HSQC and HMBC experiments (11 was kaempferol 3-O-{[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl(1→2)]-α-L-rhamnopyranosyl(1→6)}-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside.Compounds aglycone . The careriments . Therefo16 had a UV spectrum typical for quercetin 3-O-glycosides. The MS analysis of this flavonoid gave a deprotonated ion at m/z 447, as well as a dimeric ion [2M−H]− at m/z 895. The deprotonated ion fragmented to m/z 300 [(M−H)-147]−, corresponding to the quercetin radical ion, created after the loss of a deoxyhexose. On the base of its NMR spectra the compound 16 was identified as a widely occurring flavonoid, quercitrin, the quercetin 3-O-α-L-rhamnoside -7-O-α-L-rhamnopyranoside, a flavonoid occurring in lentil seeds, and found also in the legume Ateleia chicoasensis and the cactus Cephalocereus senilis − ; ESI-MS/MS (TQ), m/z: 801 [M−H]−, 625 [M−H−176]−, 300 [Y0−H]−· = [M−H−176−325 = quercetin-2H]−.Kaempferol 3-O-β-D-glucopyranosyl(1→2)-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (3): (6 mg), yellow solid, UV (UPLC-PDA) λmax (nm): 265, 346; HRESI-MS (Q-TOF), m/z: 785.1769 [M−H]− ; ESI-MS/MS (TQ), m/z: 785 [M−H]−, 609 [M−H−176]−, 285 [Y0]− = [M−H−176−324 = kaempferol−H]−.Quercetin 3-O-[-β-D-glucopyranosyl(1→2)]-β-D-galacto- pyranoside-7-O-β-D-glucuropyranoside (4): (1 mg), yellow solid, UV (UPLC-PDA) λmax (nm): 255, 335; HRESI-MS (Q-TOF), m/z: 1003.1959 [M+Na]+ ; ESI-MS/MS (TQ), m/z: 979 [M−H]−, 803 [M−H−176]−, 625 [M−H−176−178(triOHCin)]−, 300 [Y0−H]−· = [M−H−176−178−325 = quercetin−2H]−.Quercetin 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuro-pyranoside (5) (30.1 mg), yellow solid, UV (UPLC-PDA) λmax (nm): 253, 335; HRESI-MS (Q-TOF), m/z: 965.2171 [M+H]+ ; ESI-MS/MS (TQ), m/z: 963 [M−H]−, 787 [M−H−176]−, 625 [M−H−176−162]−, 300 [Y0−H]−· = [M−H−176−162−325 = quercetin−2H]−.Kaempferol 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (6): (14.6 mg), yellow solid, UV (UPLC-PDA) λmax (nm): 243, 266, 328; HRESI-MS (Q-TOF), m/z: 971.2070 [M+Na]+ ; ESI-MS/MS (TQ), m/z: 947 [M−H]−, 771 [M−H−176]−, 609 [M−H−176−162]−, 284 [Y0−H]−· = [M−H−176−162−325 = kaempferol−2H]−.Quercetin 3-O-[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl-(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (7): (28.5 mg), yellow solid, UV (UPLC-PDA) λmax (nm): 261sh, 268, 317; HRESI-MS (Q-TOF), m/z: 947.2078 [M−H]− ; ESI-MS/MS (TQ), m/z: 947 [M−H]−, 771 [M−H−176]−, 625 [M−H−176−146]−, 300 [Y0−H]−· = [M−H−176−146−325= quercetin−2H]−.Quercetin 3-O-[(6-O-E-feruloyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuro-pyranoside (8): (18.2 mg), yellow solid, UV (UPLC-PDA) λmax (nm): 253, 266sh, 332; HRESI-MS (Q-TOF), m/z: 977.200 [M−H]− ; ESI-MS/MS (TQ), m/z: 977 [M−H]−, 801 [M−H−176]−, 625 [M−H−176−176]−, 300 [Y0−H]−· = [M−H−176−176−325 = quercetin−2H]−.d-glucopyranosyl(1→2)]-β-d-galactopyranoside-7-O-β-D-glucuropyranosideKaempferol 3-O-− ; ESI-MS/MS (TQ), m/z: 931 [M−H]−, 755 [M−H-176]−, 609 [M−H−176−146]−, 284 [Y0−H]−· = [M−H−176−146−325= kaempferol−2H]−.Kaempferol 3-O-[(6-O-E-feruloyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (10): (17.1 mg), yellow solid, UV (UPLC-PDA) λmax (nm): 268, 327; HRESI-MS (Q-TOF), m/z: 961.2240 [M−H]− ; ESI-MS/MS (TQ), m/z: 961 [M−H]−, 785 [M−H−176]−, 609 [M−H−176−176]−, 284 [Y0−H]−· = [M−H−176−176−325= kaempferol−2H]−.d-galactopyranoside-7-O-α-L-rhamnopyranosideKaempferol 3-O-{[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl(1→2)]-α-L-rhamnopyranosyl(1→6)}-β- (11): (18.7 mg) yellow solid, UV (UPLC-PDA) λmax (nm): 269, 315; HRESI-MS (Q-TOF), m/z: 1049.3115 [M+H]+ ; ESI-MS/MS (TQ), m/z: 1047 [M−H]−, 901 [M−H−146]−, 755 [M−H−146−146]−, 284 [Y0−H]−· = [M−H−146−146−471= kaempferol−2H]−.Quercetin 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-caffeoyl')-β-D-glucuropyranoside (12): (14 mg) yellow solid, UV (UPLC-PDA) λmax (nm): 249, 266sh, 305sh, 332; HRESI-MS (Q-TOF), m/z: 1125.2423 [M−H]− ; ESI-MS/MS (TQ), m/z: 1125 [M−H]−, 963 [M−H−162]−, 787 [M−H−162−176]−, 625 [M−H−162−176−162]−, 300 [Y0−H]−· = [M−H−162−176−162−325= quercetin−2H]−.Kaempferol 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-caffeoyl')-β-D-glucuropyranoside (13): (9.2 mg) yellow solid, UV (UPLC-PDA) λmax (nm): 243, 268, 328; HRESI-MS (Q-TOF), m/z: 1109.2390 [M−H]− ; ESI-MS/MS (TQ), m/z: 1109 [M−H]−, 947 [M−H−162]−, 771 [M−H−162−176]−, 609 [M−H−162−176−162]−, 284 [Y0−H]−· = [M−H−162−176−162−325= kaempferol−2H]−.Quercetin 3-O-[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-caffeoyl)-β-D-glucuropyranoside (14): (4.6 mg) yellow solid, UV (UPLC-PDA) λmax (nm): 254, 269, 321; HRESI-MS (Q-TOF), m/z: 1111.2562 [M+H]+ ; ESI-MS/MS (TQ), m/z: 1109 [M−H]−, 947 [M−H−162]−, 771 [M−H−162−176]−, 625 [M−H−162−176−146]−, 300 [Y0−H]−· = [M−H−162−176−146−325= quercetin−2H]−.Quercetin 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-p-coumaroyl)-β-D-glucuropyranoside (15): (18 mg) yellow solid, UV (UPLC-PDA) λmax (nm): 254, 269, 320; HRESI-MS (Q-TOF), m/z: 1111.2557 [M+H]+ ; ESI-MS/MS (TQ), m/z: 1109 [M−H]−, 963 [M−H−146]−, 787 [M−H−146−176]−, 625 [M−H−146−176−162]−, 300 [Y0−H]−· = [M−H−146−176−162−325 = quercetin−2H]−.Quercetin 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-feruloyl)-β-D-glucuropyranoside (17): (6.4 mg) yellow solid, UV (UPLC-PDA) λmax (nm): 250, 266sh, 331; HRESI-MS (Q-TOF), m/z: 1141.2651 [M+H]+ ; ESI-MS/MS (TQ), m/z: 1139 [M−H]−, 963 [M−H−176]−, 787 [M−H−176−176]−, 625 [M−H−176−176−162]−, 300 [Y0−H]−· = [M−H−176−176−162−325 = quercetin−2H]−.Quercetin 3-O-[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl(1→2)]-β-D-galactopyranoside-7-O-(2-O-E-p-coumaroyl')-β-D-glucuropyranoside (18): (5.2 mg) yellow solid, UV (UPLC-PDA) λmax (nm): 268, 316; HRESI-MS (Q-TOF), m/z: 1095.2607 [M+H]+ ; ESI-MS/MS (TQ), m/z: 1093 [M−H]−, 947 [M−H−146]−, 771 [M−H−146−176]−, 625 [M−H−146−176−146]−, 300 [Y0−H]−· = [M−H−146−176−146−325 = quercetin−2H]−.O-β-D-glucopyranosyl(1→2)-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (1), its derivatives 4, 5, 7, 8, 12, 14, 15, 17, 18 acylated with 3,4,5-trihydroxycinnamic acid, kaempferol 3-O-β-D-glucopyranosyl(1→2)-β-D-galacto-pyranoside-7-O-β-D-glucuropyranoside (3), and its derivatives 6, 9, 10, 13 acylated with caffeic, p-coumaric, or ferulic acid, as well as kaempferol 3-O-{[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl(1→2)]-α-L-rhamno-pyranosyl(1→6)}-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside (11). Kaempferol and quercetin are known as potent antioxidants with diverse biological activity and health-promoting properties. Aerial parts of lentil may find use as a source of water-soluble flavonol glycosides, which can be applied, e.g., as nutraceuticals.As a concluding remark, it may be pointed out that aerial parts of lentil are a source of numerous flavonoids. In this study we presented the purification and structure elucidation of 18 acylated and non-acylated glycosides of kaempferol and quercetin, including 16 compounds which have not been reported previously in the scientific literature: quercetin 3-


The chiral Feist’s acids -3 and -3 were obtained in good isomeric purity by resolution of trans-(±)-3-methylene-cyclopropane-1,2-dicarboxylic acid from an 8:2 mixture of tert-butanol and water, using (R)-(+)-α-methylbenzyl amine as a chiral reagent. This process is reproducible on a large scale. All these new synthesized chiral ligands were characterized by 1H-NMR, 13C-NMR, IR, and mass spectrometry, as well as elemental analysis and their specific rotations were measured. These new classes of 2C symmetric chiral bisamide ligands could be of special interest in asymmetric transformations.The hemilabile chiral  Further recrystalization of compound -(+)-3a from tert-butanol and water (8:2) was carried out to obtain pure crystals of the (R)-(+)-α-methylbenzammonium salt of (+)-3-methylenecyclopropane--1,2-dicarboxylic acid -(+)-3a in approximately 92% yield , m.p. 219–222 °C, [α]observed + 0.60°, [α]20D + 120°, , [lit. [α]25D + 94.7° ]. The melting point and specific rotation values of compound -(+)-3a were in accordance with those reported in the literature 23.5), 25D terature . R,2R,1'R)-(+)-3a by using 1 N H2SO4 in ethyl acetate yielded -(+)-3-methylenecyclopropane-1,2-dicarboxylic acid {-3}, as an off white solid in 94% yield, m.p 200–203 °C, [lit. [α]observed + 0.65, [α]20D + 130, , lit. [α]20546 + 176° ]. Hydrolysis of the mother liquor -(+)-3b gave -(−)-3-methylenecyclopropane-1,2-dicarboxylic acid {-3} as an off-white solid in 90% yield, m.p. 201 203 °C, , lit. [α]25D − 131.3°; c, 0.81%, EtOH)], as shown in R,2R)-3 and -3 were in agreement with those reported in the literature 2054C, , lit. , [α]25D terature .2C Symmetric chiral bisamide ligands -a–d5 and -a–d6 have been synthesized in modest yield in two steps via the corresponding acid chloride intermediates, starting from the chiral Feist’s acids -3 and -3, respectively, as described in R,2R)-4 and -4 were carried out using two different procedures A and B, although the better yields of the ligands (up to 85%) were achieved by using the acid chloride obtained from procedure B, as described in the Experimental section. The formation of the acid chloride intermediates -4 and -4 was confirmed by IR spectroscopy, where the absence of a broad band around 3,452 cm–1 (due to OH stretching frequency) was observed, along with the shifting of the C=O stretching frequencies from 1,710 to 1,800 cm–1, indicating the formation of the acid chlorides. Furthermore, appearance of a sharp peak at 764 cm–1 was attributed to C–Cl stretching frequency.The syntheses of the acid chlorides -5a and -6a were synthesized by treating two equivalents of tert-butyl amine with one equivalent of the freshly prepared acid chlorides -4 and -4, respectively, in dry CH2Cl2 under inert conditions, as shown in R,2R)-5a and -6a were mainly deduced from their 1H-NMR spectra, where the presence of 18 protons at δ 1.24 and two protons at δ 7.87 as a singlet, corresponding to the 2×C(CH3)3 and 2×CONH groups, respectively, could be observed. These two ligands -5a and -6a were further confirmed by their IR spectra, where three characteristic sharp peaks were observed at 3,318 and 3,321, 1,641 and 1,642 and 1,551 and 1,549 cm−1 due to N–H(sec) stretching, C=O stretching and C–N stretching frequencies, respectively. The specific rotation values of the compounds -5a and -6a were found to be [α]20D + 150°  and [α]20D − 129 , respectively, in total agreement with their configuration.The chiral bisamide ligands -5b and -6b with a sec-butyl amide side chain, were obtained in good yield, in a similar fashion as described in the above paragraph  stretching, C=O stretching and C–N stretching vibrations, respectively. In case of their 1H-NMR spectra, the presence of a triplet at δ 0.81, a doublet at δ 1.02 and another doublet at δ 8.05, which were assigned to the protons of two sets of primary methyls, secondary methyls and two sets of amide groups, respectively. The splitting of the NH proton is mainly due to the presence of an adjacent tertiary proton in the sec-butyl chain. The calculated specific rotation of the ligands -5b and -6b were found to be [α]20D + 129  and [α]20D − 112 , respectively, which matching well with their configuration.The ligands -5c and -6c were obtained in very good yield, as described in the Experimental section  and [α]20D − 133 , respectively.The ligands with the isobutyl chain -5d and -6d with benzyl groups in amide side chain were prepared in very satisfactory yield by reaction of benzylamine (2 eq.) with the intermediates -4 and -4, respectively. These ligands were also characterized from the IR and 1H-NMR spectra. In the IR spectra of these two ligands, three characteristic sharp peaks at 3,289 and 3,297, 1,631 and 1,630 and 1,540 and 1,541 cm–1 were observed for the stretching frequencies of sec-N–H, C=O and C–N, respectively. The 1H-NMR of these ligands revealed characteristic peaks at δ 7.25–7.32 and 8.78 as a multiplet and triplet, which were assigned to the corresponding aromatic and amide protons, respectively. Again the splitting of the NH proton into a triplet was due to the presence of two benzylic protons in the amide chain. The configurations of these ligands were determined from their specific optical rotations. Thus the specific rotations for these two ligands were found to be [α]20D + 100  and [α]20D − 166 , respectively.Finally, the ligands -a–d5 and -a–d6, we found a shift of the NH proton’s δ values downfield to δ 7.87, 8.05, 8.21, and 8.78, and this downfield shift is due to the decreasing +I effect for the substituent groups in the amide chain in going from tert-butyl to a benzyl group.In 1H and 13C-NMR spectra were recorded on Jeol-400 spectrometer : using deuterated CHCl3 or DMSO as solvent. The chemical shifts (δ in ppm) for 1H- and 13C-NMR were referenced internally using residual non deuterated solvent resonance shift and reported to trimethylsilane (TMS). Coupling constants (J) are taken in Hertz (Hz). Specific rotations were measured by using ATAGO-POLAX-2L. Elemental analyses were performed on a Perkin Elmer 2400 Elemental Analyzer. IR spectra were recorded on a Model FTIR-800 Infrared FT-IR Spectrometer using KBr pellets for solids or neat for liquids. Mass spectrometric analysis was conducted by using ESI mode on AGILENT Technologies 6410-triple quad LC/MS instrument.All the moisture and air sensitive reactions were carried out under an inert atmosphere using an argon filled glove box and standard Schlenk-line techniques. All the chemicals were purchased from Aldrich, Sigma-Aldrich and Fluka and were used as received without purification, unless otherwise stated. TLC plates were used for monitoring the reactions. Flash chromatography was carried out with silica gel (100–200 mesh). Pyridine, triethylamine and diisopropylamine were dried over sodium hydroxide. Diethyl ether and tetrahydrofuran were distilled from sodium benzophenone ketyl. Hexane, heptane and pentane were distilled by using sodium/triglyme benzophenone ketyl. Chloroform, dichloromethane, benzene, toluene and dimethyformamide were dried using calcium hydride. Deuterated solvents were dried over calcium hydride and deoxygenated prior to use. Synthesis of the Ammonium Salts of trans-(±)-3-Methylenecyclopropane-1,2-dicarboxylic Acids [-(+)-3a&-(−)-3b]. The trans-(±)-3-methylenecyclopropane-1,2-dicarboxylic acid observed + 0.55°, [α]20D + 110° , .),  to afford fine crystals of the (R)-(+)-α-methylbenzammonium salt of -(+)-3-methylenecyclopropane-1,2-dicarboxylic acid (1:1) {-(+)-3a}; yield 17 g , based on the diastereomeric Feist’s acid salt), m.p. 219–222 °C, [α]observed + 0.60°, [α]20D + 120° , . A small sample was further recrystallized, but no change of specific rotation and melting point was observed. Elemental analyses were in accordance with the reported literature .C, , -(+)-3-Methylenecyclopropane-1,2-dicarboxylic Acid {-3: The (R)-(+)-α-methylbenzammonium salt of -(+)-3-methylenecyclopropane-(+)-1,2-dicarb-oxylic acid (1:1) {-(+)-3a} was dissolved in a 1:1 mixture of ethyl acetate and water (200 mL) and then acidified with 1 M H2SO4. The reaction mixture was stirred for 5 min then the organic phases were separated out. The aqueous phase was further extracted with 10% methanol in ethyl acetate (2 × 100 mL) and the combined organics were washed with brine (100 mL). Then the organics were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to afford -(+)-3-methylenecyclopropane-1,2-dicarboxylic acid {-3}, as an off white solid. Yield 8.5 g . 200–203 °C, , ; IR (cm–1): 3500–2500 , 1700 , 1470 (s), 1320 (s), 1300 (s), 1210 (s), 1100 (s), 980 (s), 920 (s), 790 (s), 660 (m); 1H-NMR (DMSO-d6): δ 2.85 , 5.9 , 12.9–13.2 ; 13C-NMR (DMSO-d6): δ 29.5 (CH), 110 (C=CH2), 133.5 (C=CH2), 174.0 (COOH); Anal. Calcd. for C6H6O4: C, 50.71; H, 4.26. Found: C, 50.52; H, 4.33; LC/MS (EI+): m/z = 141.02 [M−H+].C, , -3 or -3  was dissolved in benzene (20 mL) and 2 drops of DMF were added. Thionyl chloride  was added to the reaction mixture at room temperature. The reaction mixture was heated to 60 °C for two hours, a clear wine red colour solution formed. The benzene was then removed under reduced pressure to afford crude acid chlorides -4 and -4 with quantitative yield ~100%. IR (cm–1): 1,800 cm–1 (C=O str.), absence of OH str. Frequency from 3,452 cm–1. Procedure B: In a 100 mL round bottom flask equipped with a condenser and inert atmosphere, Compound -3 or -3  was suspended in dry dichloromethane (20 mL) and 2 drops of DMF were added. Then oxallyl chloride  was added dropwise to the reaction mixture at room temperature. The reaction mixture was stirred at ambient temperature for 1 h and it became the typical yellow colour of acid chloride solutions. The solvent was then removed under reduced pressure to afford crude acid chlorides -4 and -4 in very good yield . IR (cm–1): 1,800 cm–1 (C=O str.), absence of OH str. vibration at 3,452 cm–1.R,2R)-4 or -4  in CH2Cl2 (10 mL), was added slowly to the solution of amine a–d (2eq.) and diisopropyl amine (5 eq.) dissolved in CH2Cl2 (10 mL), at 0 to 5 °C. The reaction mixture was then allowed to stir at ambient temperature for 3 h. TLC showed (10% MeOH/CH2Cl2) complete consumption of starting material. The reaction mixture was then quenched with saturated solution of ammonium chloride (15 mL) and extracted with 10% methanol in chloroform (5 × 100 mL). The combine organic layers were washed with brine (100 mL) and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure to afford crude material which was washed with diethyl ether to afford pure amides -a–d5 and -a–d6.A solution of compound -N1,N2-Tert-butyl-3-methylenecyclopropane-1,2-dicarboxamide [-5a]: Compound -5a was obtained as a white solid by treating acid chloride -4 (3.52 mmol) with 2 eq. of tert-butylamine according to GP2. Yield 730 mg ; m.p. 235–238 °C, [α]observed + 0.45°, [α]20D + 150°, ; IR (cm–1): 3318 , 3073 (s), 2979 (s), 2961 (s), 1641 , 1551 , 1480 (s), 1360 (s), 1344 (s), 1249 (s), 1225 (s), 1108 (s), 890 (s), 652 (s); 1H-NMR (DMSO-d6): δ 1.24 3), 2.68 , 5.37 , 7.87 ; 13C-NMR (DMSO-d6): δ 29.1 (C(CH3)3), 39.43 (C(CH3)3), 50.9 (cyclopropyl CH), 103.6 (C=CH2), 133.4 (C=CH2), 167.9 (NHC=O); Anal. Calcd. for C14H24N2O2 (252.18); C, 66.63; H, 9.59; N, 11.10; Found: C, 66.52; H, 9.83; N, 10.72; LC/MS (EI+): m/z = 253.1 [M+H+].-N1,N2-sec-Butyl-3-methylenecyclopropane-1,2-dicarboxamide [-5b]: Compound -5b was obtained as an off white solid by treating acid chloride -4 (3.52 mmol) with 2 eq. of sec-butylamine according to GP2. Yield 677 mg ; m.p 235–238 °C, [α]observed + 0.4, [α]20D + 129, ; IR (cm–1): 3273 , 3080 (s), 2968 (s), 2929 (s), 1632 , 1558 , 1451 (s), 1361 (s),1219 (s), 1107 (s), 903 (s), 723, (s), 670 (s); 1H-NMR (DMSO-d6): δ 0.82 , 1.02 CH2), 1.37 , 2.69 , 3.64 CH2), 5.40 , 8.06 ; 13C-NMR (DMSO-d6): δ 11.0 (CH2CH3), 20.73 (CHCH3), 26.2 (CH2CH3), 29.5 (cyclopropyl CH), 46.6 (CHCH3), 103.9, (C=CH2), 133.16 (C=CH2), 167.8 (NHC=O); Anal. Calcd. for C14H24N2O2 (252.18); C, 66.63; H, 9.59; N, 11.10; Found: C, 65.99; H, 9.50; N, 10.11; LC/MS (EI+): m/z = 252.0 [M+].-N1,N2-Isobutyl-3-methylenecyclopropane-1,2-dicarboxamide [-5c]: Compound -5c was obtained as an off white solid by treating acid chloride -4 (3.52 mmol) with 2 eq. of iso-butylamine according to GP2. Yield 755 mg ; m.p. 225–229 °C, [α]observed + 0.2, [α]20D + 76, ; IR (cm–1): 3296 , 3084 (s), 2952 (s), 2913 (s), 1632 , 1558 , 1330 (s), 1212 (s), 1107 (s), 893 (s), 671 (s); 1H-NMR (DMSO-d6): δ 0.84 2), 1.66 2), 2.74 , 2.89 2), 5.41 , 8.21 ; 13C-NMR (DMSO-d6): δ 20.1(CH(CH3)2), 25.6 (CH(CH3)2), 28.0 (cyclopropyl CH), 104.89 (C=CH2), 132.5 (C=CH2), 167.9 (NHC=O); Anal. Calcd. for C14H24N2O2 (252.18); C, 66.63; H, 9.59; N, 11.10; Found: C, 66.50; H, 9.77; N, 10.69; LC/MS (ES+): m/z = 253.2 [M+H+].-N1,N2-Benzyl-3-methylenecyclopropane-1,2-dicarboxamide [-5d]: Compound -5d was obtained as an off white solid by treating acid chloride -4(3.52 mmol) with 2 eq. of benzylamine according to GP2. Yield 970 mg ; 231–235 °C, [α]observed + 0.4, [α]20D + 100, ; IR (cm–1): 3290 , 1631 , 1540 , 1325 (s), 732 (s), 696 (s); 1H-NMR (DMSO-d6): δ 2.82 , 4.29 , 5.48 , 7.24–7.33 , 8.77 ; 13C-NMR (DMSO-d6): δ 26.3 (cyclopropyl CH), 43.0 ((cyclopropyl CH), 104.6 (C=CH2), 127.4 (4C of aromatic), 127.9 (2C of aromatic), 128.9 (3C of aromatic), 132.6 (C=CH2), 139.7 (1C of aromatic ), 168.5 (NHC=O); Anal. Calcd. for C20H20N2O2 (320.15); C, 74.98; H, 6.29; N, 8.74; Found: C, 74.83; H, 6.47; N, 8.52; LC/MS (EI+): m/z = 321.15 [M+H+].-N1,N2-tert-Butyl-3-methylenecyclopropane-1,2-dicarboxamide [-6a]: Compound -6a was obtained as awhite solid by treating acid chloride -4 (3.52 mmol) with 2 eq. of tert-butylamine according to GP2. Yield 715 mg ; m.p. 234–236 °C, [α]observed − 0.40, [α]20D − 129, ; IR (cm–1): 3321 , 3071 (s), 2979 (s), 2960 (s), 1642 , 1549 , 1481 (s), 1362 (s), 1344 (s), 1249 (s), 1228 (s), 1108 (s), 890 (s), 654 (s); 1H-NMR (DMSO-d6): δ 1.24 3), 2.68 , 5.37 , 7.87 ; 13C-NMR (DMSO-d6): δ 29.09 (C(CH3)3), 39.4 (C(CH3)3), 50.9 (cyclopropyl CH), 103.6 (C=CH2), 133.38 (C=CH2), 167.9 (NHC=O); Anal. Calcd. for C14H24N2O2 (252.18); C, 66.63; H, 9.59; N, 11.10; Found: C, 66.62; H, 9.85; N, 10.91; LC/MS (EI+): m/z = 253.0 [M+H+].-N1,N2-sec-butyl-3-methylenecyclopropane-1,2-dicarboxamide [-6b]: Compound -6b was obtained as a white solid by treating acid chloride -4 (3.52 mmol) with 2 eq. of sec-butylamine according to GP2. Yield 690 mg ; m.p. 234–236 °C, [α]observed − 0.35, [α]20D − 112, ; IR (cm–1): 3269 , 3081 (s), 2968 (s), 2930 (s), 1630 , 1560 , 1450 (s), 1361 (s),1219 (s), 1109 (s), 903 (s), 723, (s), 672 (s); 1H-NMR (DMSO-d6): δ 0.82 , 1.02 CH2), 1.37 , 2.69 , 3.64 CH2), 5.40 , 8.06 ; 13C-NMR (DMSO-d6): δ 10.96 (CH2CH3), 20.73 (CHCH3), 26.18 (CH2CH3), 29.45 (cyclopropyl CH), 46.57 (CHCH3), 103.88 (C=CH2), 133.16 (C=CH2), 167.78 (NHC=O); Anal. Calcd. for C14H24N2O2 (252.18); C, 66.63; H, 9.59; N, 11.10; Found: C, 65.95; H, 9.51; N, 10.73; LC/MS (EI+): m/z = 252.2 [M+].-N1,N2-isobutyl-3-methylenecyclopropane-1,2-dicarboxamide [-6c]: Compound -6c was obtained as a white solid by treating acid chloride -4 (3.52 mmol) with 2 eq. of iso-butylamine according to GP2. Yield 740 mg ; 226–230 °C, [α]observed − 0.4, [α]20D − 133, ; IR (cm–1): 3288 , 3085 (s), 2950 (s), 2917 (s), 1634 , 1559 , 1330 (s), 1211 (s), 1107 (s), 893 (s), 670 (s); 1H-NMR (DMSO-d6): δ 0.84 2), 1.66 2), 2.74 , 2.89 2), 5.41 , 8.21 ; 13C-NMR (DMSO-d6): δ 20.1 (CH(CH3)2), 25.6 (CH(CH3)2), 28.0 (cyclopropyl CH), 104.9 (C=CH2), 132.47 (C=CH2), 167.9 (NHC=O); Anal. Calcd. for C14H24N2O2 (252.18); C, 66.63; H, 9.59; N, 11.10; Found: C, 66.55; H, 9.68; N, 10.93; LC/MS (EI+): m/z = 252.1 [M+].-N,2-benzyl-3-methylenecyclopropane-1N,2-dicarboxamide [-6d]: Compound -6d was obtained as an off white solid by treating acid chloride -4 (3.52 mmol) with 2 eq. of benzylamine according to GP2. Yield 944 mg ; m.p. 232–234 °C, [α]observed − 0.5, [α]20D − 166, ; IR (cm–1): 3297 , 1630 , 1541 , 1325 (s), 732 (s), 696 (s); 1H-NMR (DMSO-d6): δ 2.82 , 4.28 , 5.48 , 7.24–7.33 , 8.77 ; 13C-NMR (DMSO-d6): δ 26.30 (cyclopropyl CH), 43.01 (cyclopropyl CH), 104.47 (C=CH2), 127.4 , 127.84 , 128.91 , 132.60 (C=CH2), 139.73 , 168.53 (NHC=O); Anal. Calcd. for C20H20N2O2 (320.15); C, 74.98; H, 6.29; N, 8.74; Found: C, 74.74; H, 6.43; N, 8.87; LC/MS (EI+): m/z = 321.15 [M+H+].2C symmetric chiral bisamide ligands with high specific rotation values were carried out. All these ligands were derived from trans-(±)-3-methylene-cyclopropane-1,2-dicarboxylic acid (Feist’s acid), which served as the key precursor. In order to obtain the chiral backbone of the ligands, we have resolved trans-(±)-3-methylenecyclopropane-1,2-dicarboxylic acid in a new modified method, hence -(+)-3-methylenecyclopropane-1,2-dicarboxylic acid and -(−)-3-methylenecyclopropane-1,2-dicarboxylic acid were obtained with a high degree of enantioselectivity. The applications of these chiral ligands are under investigation and the results will be reported in the nearest future. On the basis of the chiral Feist’s acid, we are working to explore a large variety of novel classes of chiral 2C symmetrical bidentate, or tetradentate ligands, with bulky environments.In conclusion, the synthesis of the new class of


The structures were established as -20-O,22-O-(5′-hydroxymethyl)-furfurylidene-2β,3β,14α,25-tetrahydroxy-5β-ergost-7-en-6-one (1), -20-O,22-O-(5′-hydroxymethyl)-furfurylidene-2β,3β,25-trihydroxy-14β-methyl-18-nor-5β-cholesta-7,12-dien-6-one (2) and -20-O,22-O-(5′-hydroxymethyl)-furfurylidene-2β, 3β,5β,14α,26-pentahydroxycholest-7-en-6-one (3) by means of spectroscopic evidence.Three new phytoecdysteroid compounds, named niuxixinsterone A ( Achyranthes bidentata Bl., a member of Amaranthaceae, is an erect perennial herbaceous plant widely distributed and grown in hilly districts of India, China, Japan and Java. The roots of A. bidentata named “Niuxi” in Chinese, is an important medicinal herbal and documented in the Chinese Pharmacopeia. It is usually prescribed by practitioners of Traditional Chinese Medicine (TCM) as a tonic, emmenagogue, antiarthritic, diuretic, and antifertility agent to nourish the liver and kidneys, strengthen bones and muscles, and invigorate circulation + ion peak at m/z 625.3359 in the HRESIMS, corresponding to a molecular formula of C34H50O9. The UV spectrum was consistent with presence of a 7-en-6-one chromophore in an ecdysteroid, with a maximum value at 248 nm. By analyzing the 1H- and 13C-NMR spectroscopic data, it was determined that 1 was an ecdysone analog with a furan ring-containing substituent.Compound 1H-NMR spectrum of 1, five methyl singlets at δH 1.00 (×2), 1.53, 1.28 and 1.36 were attributed to CH3-18, CH3-19, CH3-21, CH3-26 and CH3-27, respectively. The CH3-28 signal appeared as a doublet at δH 1.19 (J = 6.8 Hz). In addition, signals for three olefin protons , two hydroxymethyl protons, and an acetal proton were observed in the spectrum.In the 13C-NMR spectrum of 1 had one carbonyl at δC 203.4 (C-6), three oxymethine at δC 68.2, 68.1 and 85.4 , three oxyquaternary carbons at δC 80.4, 85.5 and 72.1 , six olefinic carbons at δC 121.7, 165.4, 152.2, 110.1, 107.8 and 157.4 , together with an acetal carbon at δC 97.8. The full connectivity of 1 was deduced from the HSQC, 1H-1H-COSY and HMBC correlations  between Hβ-1 and H-2, a broad singlet of H-3, HMBC correlations between H3-19 and C-1, and NOESY correlation between H-2 and Hα-4. A NOESY correlation between Hα-12 and H-17 indicated that C-17 side-chain was β-oriented + ion peak at m/z 593.3091. The UV spectrum was consistent with presence of the ecdysteroid chromophore as above. Extensive NMR analysis of the parent nucleus indicated that compounds 2 and 1 were structurally different in terms of the presence of additional one olefin signal and the loss of one oxyquaternary carbon signal.Compound 1H- and 13C-NMR data from the spectra of 2 and -2β,3β,20,22,26-pentahydroxy-14β-methyl-18-nor-5β-cholesta-7,12-dien-6-one +) as C33H48O10 with a maximal UV absorption at 247 nm. Comparison of the 13C-NMR spectroscopic data of the tetracyclic ring system between 3 and 1 indicated that the major difference was a methine at δC 51.4 (C-5) in 1 being replaced by an oxyquaternary at δC 80.0 in 3. The HMBC correlations between H-7 and H3-19 with the signal at δC 80.0, respectively, which established the presence of an OH substituent at C-5. The 5β-OH configuration was indicated by the upfield resonance for the CH3-19 at δC 17.1 D20 +55 . IR (KBr) cm−1: 3460, 1660, 1454, 1380, 1062. UV (MeOH) λmax (log ε) nm: 226 (4.10), 248 (3.20), 323(1.15). HRESIMS (positive ion mode) m/z: 625.3359 [M+Na]+, . 1H-NMR and 13C-NMR data are shown in -20-O,22-O-(5′-Hydroxymethyl)-furfurylidene-2β,3β,25-trihydroxy-14β-methyl-18-nor-5β-cholesta-7,12-dien-6-one : white amorphous powder, mp: 230–231 °C. [α]D20 +46 . IR (KBr) cm−1: 3472, 1655, 1460, 1380, 1059. UV (MeOH) λmax (logε) nm: 224 (4.05), 248 (3.23), 321 (1.09). HRESIMS (positive ion mode) m/z: 593.3091 [M+Na]+, . 1H-NMR and 13C-NMR data are shown in -20-O,22-O-(5′-Hydroxymethyl)-furfurylidene-2β,3β,5β,14α,26-pentahydroxycholest-7-en-6-one : white amorphous powder, mp: 235–236 °C. [α]D20 +52 . IR (KBr) cm−1: 3480, 1656, 1456, 1380, 1062. UV (MeOH) λmax (logε) nm: 225 (4.09), 247 (3.51), 320 (1.07). HRESIMS (positive ion mode) m/z: 627.3150 [M+Na]+, . 1H-NMR and 13C-NMR data are shown in R,22R,24S)-20-O,22-O-(5′-hydroxymethyl)-furfurylidene-2β,3β,14α,25-tetrahydroxy-5β-ergost-7-en-6-one (1), -20-O,22-O-(5′-hydroxy-methyl)-furfurylidene-2β,3β,25-trihydroxy-14β-methyl-18-nor-5β-cholesta-7,12-dien-6-one (2) and -20-O,22-O-(5′-hydroxymethyl)-furfurylidene-2β,3β,5β,14α,26-pentahydroxy-cholest-7-en-6-one (3), were isolated from the EtOH extract of Achyranthes bidentata Bl. The discovery of compounds 1-3 is a further addition to the diverse phytoecdysteroids compounds.In conclusion, three new phytoecdysteroids, (20


Z)-4-((E)-3-(4-methoxyphenyl)allylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (4g) was characterized as an especially efficient UVA photoprotective agent with a UVA PF of 6.83 ± 0.05 and favorable photostability. Diethyl 2,2′-((Z)-4-(4-methoxybenzylidene)-2,5-dioxo- imidazolidine-1,3-diyl)diacetate (3b) was the most promising UVB-filter, with a SPFin vitro of 3.07 ± 0.04 and very good solubility and photostability. The main photodegradation products were geometric isomers of the parent compounds. These compounds were also shown to be non-cytotoxic at concentrations up to 50 µM when tested on three types of human skin cells and possess no estrogenic activity, according to the results of a MCF-7 breast cancer model.Effective protection from the harmful effects of UV radiation may be achieved by using sunscreens containing organic or inorganic UV filters. The number of currently available UV filters is limited and some of the allowed molecules possess limitations such as systemic absorption, endocrine disruption properties, contact and photocontact allergy induction, and low photostability. In the search for new organic UV filters we designed and synthesized a series consisting of 5-benzylidene and 5-(3-phenylprop-2-en-1-ylidene)imidazolidine-2,4-dione (hydantoin) derivatives. The photoprotective activity of the tested compounds was confirmed in methanol solutions and macrogol formulations. The most promising compounds possessed similar UV protection parameter values as selected commercially available UV filters. The compound diethyl 2,2′-(( Sunscreens are necessary to protect the skin against the acute and chronic consequences of ultraviolet radiation (UVR). The main active ingredients of sunscreens called UV-filters are inorganic blockers which reflect or scatter UVR or organic absorbers. Organic UV-filters act as chemical energy converters—after absorption of photons the molecule is excited to a higher energy state, then the absorbed energy is dissipated by the emission of photons or heat and molecule returns to the ground state ,2.Z-isomer has a lower extinction coefficient, which results in a significant reduction in its photoprotectiveness + calcd. for C10H8N2O2, 189.19, found, 189.01 100%. Physicochemical data for 1a were previously published by Martínez-López et al. + calcd. for C11H10N2O3, 219.21, found, 219.11 100%. Physicochemical data for 1b were previously published by Luo et al. + calcd. for C12H12N2O3, 233.24, found, 233.13 97.83%. Physicochemical data for 1c were previously published by Thenmozhiyal et al. + calcd. for C12H12N2O4, 249.24, found, 249.15 100%. Physicochemical data for 1d were previously published by Thenmozhiyal et al. + calcd. for C12H12N2O4, 249.42, found, 249.08 100%. Physicochemical data for 1e were previously published by Luo et al. + calcd. for C12H10N2O2, 215.12, found, 215.13 100%. Physicochemical data for 1f were previously published by Lamiri et al. + calcd. for C13H12N2O3, 245.25, found, 245.16 100%. CAS: 664353-78-0, CAS for Z,E isomer: none.(Z)-5-((E)-3-(2-Methoxyphenyl)allylidene)imidazolidine-2,4-dione (1h) was obtained as brown solid (yield 64%), M = 244.25, mp 260–263 °C, 1H-NMR (DMSO-d6) δ 11.05 , 10.65 , 7.55 , 7.33–7.16 , 7.10 , 7.05–6.90 , 6.24 , 3.82 , ESI-MS (m/z): [M + H]+ calcd. for C13H12N2O3, 245.25, found, 245.10 98.85%. CAS for E,E isomer: 1776969-29-9, CAS for Z,E isomer: none.Ethyl (Z)-2-acetate (2a) was obtained as white solid (yield 63%) M = 274.28, mp 169–171 °C, 1H-NMR (chloroform-d) δ 8.55 , 7.49–7.44 , 7.44–7.30 , 6.79 , 4.37 , 4.24 , 1.35–1.18 , ESI-MS (m/z): [M + H]+ calcd. for C14H14N2O4, 275.28, found, 275.20 96.84% CAS: 463317-70-6 CAS for Z isomer: 1416719-77-1.Ethyl (Z)-2-(4-(4-methoxybenzylidene)-2,5-dioxoimidazolidin-1-yl)acetate (2b) was obtained as white solid (yield 52%), M = 304.30, mp 178–179 °C, 1H-NMR (chloroform-d) δ 8.34 , 7.45–7.34 , 7.01–6.90 , 6.76 , 4.37 , 4.24 , 3.85 , 1.29  ESI-MS (m/z): [M + H]+ calcd. for C15H16N2O5, 305.30, found, 305.17 99,31%. CAS: 463317-69-3 CAS for Z isomer: 2247638-41-9.Ethyl (Z)-2-(4-(4-ethoxybenzylidene)-2,5-dioxoimidazolidin-1-yl)acetate (2c) was obtained as white solid (yield 61%), M = 318.33, mp 181–182 °C, 1H-NMR (chloroform-d) δ 8.66–8.60 , 7.48–7.34 , 6.98–6.87 , 6.75 , 4.37 , 4.23 , 4.07 , 1.43 , 1.36–1.19  ESI-MS (m/z): [M + H]+ calcd. for C16H18N2O5, 319.33, found, 319.20 99.02%.Ethyl (Z)-2--2,5-dioxoimidazolidin-1-yl)acetate (2d) was obtained as light yellow solid (yield 56%), M = 334.33, mp 197–198 °C, 1H-NMR (chloroform-d) δ 7.98 , 7.31–7.21 , 6.75–6.68 , 6.61–6.47 , 4.34 , 4.23 , 3.93 , 3.85 , 1.34–1.21  ESI-MS (m/z): [M + H]+ calcd. for C16H18N2O6, 335.33, found, 335.37 100%.Ethyl (Z)-2--2,5-dioxoimidazolidin-1-yl)acetate (2e) was obtained as white solid (yield 48%), M = 334.33, mp 198–200 °C, 1H-NMR (chloroform-d) δ 8.41 , 7.10–7.00 , 6.97–6.87 , 6.75 , 4.36 , 4.23 , 3.93 , 3.92 , 1.28 , ESI-MS (m/z): [M + H]+ calcd. for C16H18N2O6, 335.33, found, 335.15 100%. CAS: 463317-68-2 CAS for Z isomer: none.Diethyl 2,2′-((Z)-4-benzylidene-2,5-dioxoimidazolidine-1,3-diyl)diacetate (3a) was obtained as white solid (yield 81%), M = 360.37, mp 47–49 °C, 1H-NMR (chloroform-d) δ 7.39–7.30 , 7.26–7.19 , 7.01 , 4.37 , 4.23 , 4.20 , 3.95 , 1.28 , 1.09 . ESI-MS (m/z): [M + H]+ calcd. for C18H20N2O6, 361.37, found, 361.27 96.74%.Diethyl 2,2′-((Z)-4-(4-methoxybenzylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (3b) was obtained as white solid (yield 79%), M = 390.39, mp 74–76 °C, 1H-NMR (chloroform-d) δ 7.24–7.13 , 6.98 , 6.95–6.84 , 4.38 , 4.30–4.18 , 4.01 , 3.83 , 1.30 , 1.12 , ESI-MS (m/z): [M + H]+ calcd. for C19H22N2O7, 391.39, found, 391.17 100%.Diethyl 2,2′-((Z)-4-(4-ethoxybenzylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (3c) was obtained as light yellow solid (yield 84%), M = 404.42, mp 44–46 °C, 1H-NMR (chloroform-d) δ 7.22–7.11 , 6.97 , 6.95–6.82 , 4.37 , 4.30–4.15 , 4.02 , 1.42 , 1.34–1.21 , 1.17–1.04 , ESI-MS (m/z): [M + H]+ calcd. for C20H24N2O7, 405.42, found, 405.26 100%.Diethyl 2,2′-((Z)-4--2,5-dioxoimidazolidine-1,3-diyl)diacetate (3d) was obtained as white solid (yield 75%), M = 420.42, mp 88–90 °C, 1H-NMR (chloroform-d) δ 7.05 , 6.94 , 6.54–6.41 , 4.36 , 4.29–4.16 , 4.00 , 3.82 , 3.79 , 1.34–1.21 , 1.12 , ESI-MS (m/z): [M + H]+ calcd. for C20H24N2O8, 421.42, found, 421.22 100%.Diethyl 2,2′-((Z)-4--2,5-dioxoimidazolidine-1,3-diyl)diacetate (3e) was obtained as light yellow solid (yield 68%), M = 420.42, mp 80–82 °C, 1H-NMR (chloroform-d) δ 6.97 , 6.90–6.78 , 6.76 , 4.38 , 4.32–4.16 , 4.01 , 3.90 , 3.86 , 1.33–1.25 , 1.18–1.05 . ESI-MS (m/z): [M + H]+ calcd. for C20H24N2O8, 421.42, found, 421.22 100%.Diethyl 2,2′-((Z)-2,4-dioxo-5-((E)-3-phenylallylidene)imidazolidyne-1,3-diyl)diacetate (4f) was obtained as yellow solid (yield 84%), M = 386.40, mp 99–100 °C, 1H-NMR (DMSO-d6) δ 7.57–7.49 , 7.49–7.25 , 7.18–7.00 , 6.66–6.53 , 4.92 , 4.35 , 4.22–4.06 , 1.26–0.98 , ESI-MS (m/z): [M + H]+ calcd. for C20H22N2O6, 387.40, found, 387.19 100%.Diethyl 2,2′-((Z)-4-((E)-3-(4-methoxyphenyl)allylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (4g) was obtained as yellow solid (yield 89%), M = 416.43, mp 134–135 °C, 1H-NMR (DMSO-d6) δ 7.55–7.42 , 7.12–6.86 , 6.55 , 4.90 , 4.35 , 4.13 , 3.77 , 1.19 , 1.13 . ESI-MS (m/z): [M + H]+ calcd. for C21H24N2O7, 417.43, found,417.16 100%.Diethyl 2,2′-((Z)-4-((E)-3-(2-methoxyphenyl)allylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (4h) was obtained as yellow solid (yield 75%), M = 416.43, mp 134–135 °C, 1H-NMR (chloroform-d) δ 7.38 , 7.36–7.18 , 7.17–7.03 , 7.00–6.75 , 6.69 , 4.72 , 4.36 , 4.24 , 3.89 , 1.36–1.11 , ESI-MS (m/z): [M + H]+ calcd. for C21H24N2O7, 417.43, found, 417.29 97.93%.(Z)-2-acetic acid (5a) was obtained as white solid (yield 49%), M = 246.22, mp 215–217 °C, 1H-NMR (DMSO-d6) δ 10.91 , 7.69–7.59 , 7.47–7.28 , 6.58 , 4.19 ., ESI-MS (m/z): [M + H]+ calcd. for C12H10N2O4, 247.22, found, 247.16 100%. CAS: 857796-52-2 CAS for Z isomer: 850636-92-9.3b is disordered. The occupancy factors of these atoms after refinement are 0.797 and 0.203 for the major (A) and minor (B) components, respectively. For molecular graphics ORTEP  and R1 = 0.0417, wR2 = 0.0929 [all data].4g: C21H24N2O7, Mr = 416.42, crystal size = 0.17 x 0.23 x 0.79 mm3, monoclinic, space group I2/a, a = 17.7480(2) Å, b = 8.8899(1) Å, c = 27.1816(3) Å, β = 96.619(1)°, V = 4116.31(8) Å3, Z = 8, T = 130(2) K, 27541 reflections collected, 4942 unique reflections (Rint = 0.0202), R1 = 0.0435, wR2 = 0.1031 [I > 2σ(I)] and R1 = 0.0526, wR2 = 0.1094 [all data].www.ccdc.cam.ac.uk/data_request/cif.CCDC 1889259-1889260 contain the supplementary crystallographic data. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via max) of tested compounds was determined in methanol as the slope of the linear regression of absorbance vs. concentration of tested compound (from 10 to 50 μM or 2.5 to 30 μM).Spectra with a scan range of 290–400 nm were recorded in 50 or 25 μM methanol solutions, in 1 cm path length, 1.5 mL quartz cuvettes on a U-2800 double beam spectrophotometer  controlled by UV Solution version 2.2 software. The molar extinction coefficient at maximum absorption (ε3a–3e and 4f–4h and reference UV-filters were dissolved in a triacetin:Tween 20 (1:9) solvent system. For compounds 2a–2e it was necessary to use DMSO. The final composition of the macrogol formulations is presented in the photoprotective activity section.Tested compounds and commercial UV filters as reference standards were incorporated to a neutral macrogel formulation in the concentration of 2%. Prior to the incorporation, compounds 2 and 5 µm roughness value to simulate the skin surface . As a reference a PMMA plate treated with approximately 15 µL of glycerin was used. Measurements were carried out by reflectance spectrophotometry with SPF-290AS Analyser  equipped with integrating sphere controlled by WinSPF version X.X. software. For each sample two plates were prepared, absorbance measures were performed on 6 different positions of the plate from 290 to 400 nm with 1 nm steps. The results were expressed as an average of data from 12 scans. In vitro photoprotection study was performed according to EN ISO 24443:2012  with sli2 (cumulative dose of ultraviolet radiation 218 kJ/m2) with solar light simulator  equipped with an optical filter cutting off wavelengths shorter than 290 nm and IR-block filter to neutralize thermal effects. The UV absorption spectrum of the samples were analysed post-irradiation and compared with pre-irradiation results. UPLC-MS/MS analyses were also performed.The photostability evaluation of compounds was performed both in methanol solutions and in macrogol formulations. Irradiation of samples according to previous studies ,72 was cw/w) were tested on PMMA plates (1.3 mg/cm2). After post-irradiation measure of UV absorption, exposed and unexposed plates were washed with 5 mL of methanol, then mixtures were filtered and analysed by UPLC-MS/MS. All analyses were performed in duplicates.Methanol solutions were tested at concentration 25 or 50 µM in quartz cuvettes with the PTFE stopper to prevent evaporation or leaking of the solvent. Macrogol formulations  was added to each well. After 4 hours incubation , the medium was aspirated and the formazan produced in the cells appeared as dark crystals in the bottom of the wells. Next, DMSO (dissolving solution) was added to each wells. Then the optical density (OD) of solution was determined at 570 nm on micro plate reader . Viability (% of control) was determined by dividing A570 of experimental wells by of A570 of control wells × 100%.Primary Epidermal Melanocytes , human epidermal keratinocyte cell line HaCaT  and human skin fibroblast cell line BJ  were used in the study. Primary Epidermal Melanocytes were cultured in Dermal Cell Basal Medium (ATCC) supplemented with Adult Melanocyte Growth Kit Components (ATCC), HaCaT and BJ cells were cultured in Dulbecco’s modified Eagle’s medium  and supplemented with 10% fetal bovine serum . Cell maintenance was performed in a humidified incubator at 37 °C with 5% COFor hepatotoxicity and neurotoxicity assays a human neuroblastoma cell line—SH-SY5Y  and a human hepatocellular carcinoma cell line–HepG2, respectively have been used. Cells were seeded on 96-well plates at an initial density of 10,000 cells per well. Cells were cultured in standard conditions for 24 h and then study compounds were administrated at different concentrations (10–50 μM) for another 24 h. After incubation time cells viability was measured with MTT assay. For this purpose culture medium has been replaced for the fresh one and MTT reagent -2,5-diphenyltetrazolium bromide) has been added at the concentration 0.5 mg/mL. Cells were incubated with MTT reagent at 37 °C for 4 h. Then formazan crystals have been dissolved in DMSO solution and the absorbance has been measured at 570 nm. Experiments were conducted in triplicates. Values represent mean percent of viability (in the percent of control).570). Proliferation rate was determined by dividing the A570 of experimental wells by the A570 of control wells × 100%.To determine estrogenic activity of tested compounds their influence on estrogen-dependent MCF-7 breast cancer cell line proliferation was investigated. Cells were seeded into 24-wells plates in density of 40,000 per well. After 24 h incubation, cells were washed twice with PBS and fresh, estrogen- and phenol red-free medium  was added. Than cells were incubated with tested compounds or benzophenone-2 , which is a UV filter exhibiting estrogenic activity, to final concentration of 5 μM. After 120 h incubations, cells proliferation was determined with crystal violet assay. Briefly, cells were washed with PBS and fixed with 3,7% formaldehyde. Then, crystal violet solution was added for 10 minutes. After incubation, solution was removed and cells were washed five times with PBS. Crystal violate was extracted from cells using destaining solution  solution) and absorbance of the solution was determined at 570 nm  and the reference substance BP-2. Samples composed of test compound (20 µM), MLMs (0.8 mg/mL) and potassium phosphate buffer  were preincubated prior to addition of NADPH-regenerating system. NADPH-regenerating system contained NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase and potassium phosphate buffer . Control incubations were performed in the absence of the NADPH-regenerating system / protein in the incubation [mg]) × 0.693 / t1/2 . TA100, TA1535 and E. coli strains are used for the detection of base substitution mutations, whereas TA98 and TA1537 are utilized for the detection of frameshift mutations. S. typhimurium strains have GC base pairs, whereas E. coli strain has AT base pair at their primary reversion site and detect certain oxidizing mutagens, cross-linking agents and hydrazines.The following strains were used in the study: https://www.xenometrix.ch , –S9); 2-aminoanthracene (2-AA) at 0.5 µg/mL ; 2-AA at 1.25 µg/mL ; 9-AA at 2.5 µg/mL ; 2-aminofluorene (2-AF) at 400 µg/mL . Pure DMSO was used as the negative control.Positive controls used for the MPF protocol were 2-nitrofluorene (2-NF) at 2 µg/mL ; 4-nitroquinoline-N-oxide (4-NQO) at 0.1 µg/mL ; N4-aminocytidine (N4-ACT) at 100 µg/mL ; 9-aminoacridine (9-AAc) at 15 µg/mL ; 4-NQO at 2 µg/mL , and the dose dependency. The fold increase of revertants relative to the solvent control was determined by dividing the mean number of positive wells at each dose by the solvent control at baseline. The solvent control at baseline was defined as the mean number of positive wells in the solvent control plus one standard deviation (SD). When an increase of more than 2-fold relative to the baseline at more than one dose with a dose–response is observed the sample is classified as positive, whereas when no response >2 times the baseline and no dose–response is stated the sample is negative ,78,79,804f -2,4-dioxo-5-((E)-3-phenylallylidene)-imidazolidyne-1,3-diyl)diacetate) shows the best UVA photoprotective properties among the tested series with a UVA PF of 8.43 ± 1.35 but its photostability is unsatisfactory. Compound 2a (ethyl (Z)-2-acetate) shows the best UVB photoprotective properties within the tested series with a SPFin vitro of 4.79 ± 0.02, but it is only slightly soluble in the formulations used. Considering the other features of modern UV-filters such as good solubility in cosmetic solvents and high photostability, the most favorable UVA-filter seems to be compound 4g -4-((E)-3-(4-methoxyphenyl)allylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate) with a UVA PF of 6.83 ± 0.05 and better photostability than compound 4f. The most promising potential UVB-filter is compound 3b -4-(4-methoxybenzylidene)-2,5-dioxoimidazolidine-1,3-diyl)- diacetate) with SPFin vitro of 3.07 ± 0.04, characterized by very good solubility and photostability. The preliminary safety assessment of the compounds is also promising. Tested compounds at high concentrations (50 µM) are non-toxic toward human skin cells, hepatoma and neuroblastoma cells and possess no estrogenic activity. We may conclude that neither test compounds 3b and 4g nor their potential metabolites are base substitution or frameshift mutagens. Their metabolic stability is various and depends on substituents in phenyl and imidazolidine ring.In summary all tested compounds may be considered as potential UVA or UVB filters. Their absorption parameters are comparable or more favorable than those of selected commercially available UV-filters. Compound


Moderate to good enantiomeric excess values (ee ≥ 52%) were obtained when the CAL-B-catalyzed reactions were performed in t-BuOMe, at 60 ºC with 0.5 equiv. of added H2O as nucleophile.Enantiomerically enriched hydroxy-substituted  As it was later confirmed, the majority of the antipodes of unreacted β-amino acid ethyl ester enantiomers were consumed in side-reactions (polymerization). On the basis of our earlier results on the enantioselective hydrolyses of  solvent ,29,30,31)-(±)-10  with 0.5S*,2S*,5R*)-(±)-10 at 60 ºC , with better E and comparable reaction rate in toluene (entry 3) and with considerably higher E and higher reaction rate in t-BuOMe (entry 5) relative to iPr2O (entry 1). In order to enhance the observed enantioselectivity, several solvents were tested in the CAL-B-catalyzed hydrolysis of -(±)-4, -(±)-5, -(±)-6 and -(±)-10 were performed in t-BuOMe at 60 ºC in the presence of CAL-B (30 mg mL-1) with 0.5 equiv. of H2O. The results are summarized in β-amino acid enantiomers (11−14) are missing from the Table, as above observed, the β-amino acid ethyl esters -4, -5, -6 and -10 underwent polymerization (the side-product(s) were not characterized). As it was confirmed for the product obtained from -4, the mass spectrum shows two major peaks. The mass difference between these two peaks is 259.4 m/z. This corresponds to the mass of one Boc-protected saturated amino acid. Furthermore the MS/MS spectra of these two peaks show losses of Boc groups and Boc-protected saturated amino acid residues. These results suggest the polymerization of the amino acid derivative. A possible pathway is the enzyme catalyzed Michael addition. The NMR results support this view, because the signals of the protons of the double bonds and the OH groups are missing. Since CAL-B showed S-selectivity towards the earlier performed hydrolyses of cis and R-selectivity of trans carbocyclic β-amino esters . The ee values for -4, -5, -6, -11, -12 and -13, were determined by using GC equipped with Chromopack Chiralsil-Dex CB column (25 m) after derivatization with CH2N2 , . Racemic substrate (0.05 M solution) in an organic solvent (1 mL) was added to the lipase tested   for -(±)-1 or -(±)-7] in CH2Cl2 (200 mL), KI , NaHCO3 , H2O (200 mL) and I2  were added. After stirring for 12 h at room temperature, the mixture was taken up in CH2Cl2 (300 mL), and washed with saturated aqueous Na2SO3 solution. The organic layer was then dried (Na2SO4) and concentrated under reduced pressure and resulted -(±)-2  or -(±)-8 . To a solution of 1H-NMR  for -(±)-2: δ = 1.48 , 2.13–2.23 , 2.45–2.53 , 2.79–2.90 , 4.16–4.22 , 4.48–4.53 , 4.78 , 4.80–4.85 ; 13C-NMR : δ = 20.8, 28.7, 33.9, 36.9, 44.9, 46.1, 80.1, 82.0, 156.8, 172.0. 1H-NMR  for -(±)-8: δ = 1.50 , 2.23–2.30 , 2.33–2.43 , 2.87–3.07 , 4.22–4.25 , 4.38–4.43 , 4.88–4.93 , 5.38 ; 13C-NMR : δ = 20.1, 28.5, 29.1, 33.9, 43.0, 46.6, 79.4, 81.7, 177.0, 179.0. R*,2S*,4S*,5S*)-(±)-2 or -(±)-8] in THF (60 mL) DBU  was added and the mixture was stirred at 65 °C for 4 h. Then the solution was concentrated under reduced pressure and the residue was taken up in EtOAc (140 mL). The organic layer was washed with H2O (3 x 70 mL), dried (Na2SO4) and concentrated under vacuum. The residue was crystallized from n-hexane-EtOAc and resulted -(±)-3  or -(±)-9 . To a solution of iodolactone  in anhydrous EtOH (20 mL) NaOEt  was added at 0 °C and the reaction mixture was further stirred at the temperature and time indicated. Then the EtOH was removed under reduced pressure at 40 °C and the residue was taken up in EtOAc (50 mL). The organic layer was washed with H2O (3 × 25 mL), dried (Na2SO4) and concentrated under reduced pressure. The crude residue was crystallized from n-hexane-EtOAc to give the desired product: To a solution of unsaturated lactone D25 = +34 (c 0.19 EtOH); m.p. 69–71 °C;ee = 74%]. The filtered-off enzyme was washed with distilled H2O (3 x 15 mL), than with MeOH (3 x 15 mL) and the combined solvents was evaporated off, yielding the mixture of mainly polymeric products and amino acid. 1H-NMR  data for -4 were similar to those for -(±)-4. Analysis: calculated for C14H23NO5 (285.34): C, 58.93; H, 8.12; N, 4.91; found: C, 58.80; H, 8.08; N, 4.92. Compound -(±)-5  and H2O  in t-BuOMe (40 mL) on the presence of CAL-B  at 60 °C afforded after 10 days -5 D25 = +16 (c 0.165 EtOH); m.p. 100–102 °C;ee = 78%]. 1H-NMR  data for -5 were similar to those for -(±)-5. Analysis: calculated for C14H23NO5 (285.34): C, 58.93; H, 8.12; N, 4.91; found: C, 58.80; H, 8.08; N, 4.92. Following the procedure described above, the reaction of racemic -(±)-6  and H2O  in t-BuOMe (40 mL) on the presence of CAL-B  at 60 °C afforded after 10 days -6 D25 = -4 (c 0.24 EtOH); yellowish oil;ee = 54%]. 1H-NMR  data for -6 were similar to those for -(±)-6. Analysis: calculated for C14H25NO5 (287.35): C, 58.52; H, 8.77; N, 4.87; found: C, 58.66; H, 8.62; N, 4.85. Following the procedure described above, the reaction of racemic -(±)-10  and H2O  in t-BuOMe (40 mL) on the presence of CAL-B  at 60 °C afforded after 7 days -10 D25 = +100 (c 0.18 EtOH); m.p. 142–144 °C;ee = 90%]. 1H-NMR  data for -5 were similar to those for -(±)-5. 1H-NMR  data for -10 were similar to those for -(±)-10. Analysis: calculated for C14H23NO5 (285.34): C, 58.93; H, 8.12; N, 4.91; found: C, 58.91; H, 8.00; N, 4.97. Following the procedure described above, the reaction of racemic  were obtained for the unreacted amino esters when the reactions were performed with 0.5 equiv. of added H2O, in t-BuOMe, at 60 ºC. Due to polymerization, the supposed β-amino acid enantiomers practically could not be isolated. Four hydroxy-substituted


Malva verticillata , a leafy vegetable that has been popular in East Asia for a long time, has also been used in herbal teas and medicines. The aqueous fraction of the aerial parts of Malva verticillata, exhibiting a very high quantity of flavonoids compared to the EtOAc and n-BuOH fractions, exhibited significant recovery effects on pancreatic islets damaged by alloxan in zebrafish larvae. Thus, the bioactive components responsible for this anti-diabetic activity were investigated. A new flavonoid glucuronide (1) and five known flavonoids were isolated from the aqueous fraction. Based on several spectroscopic methods, compound 1 was identified to be nortangeretin-8-O-β-d-glucuronide, and was named malvaflavone A. The A-ring of compound 1 had a 5,6,7,8-tetrahydroxy moiety, which rarely occurs in plant systems. Also 8-O-glucuronide attached to the flavonoid moiety was rarely occurred in plant system. Compounds 1, 3, 4, and 6 significantly improved the pancreatic islet size in zebrafish at 0.1 μM, and compounds 1 and 6 were found to block β-cell K+ channels in experiments with diazoxide. In ABTS, ORAC, and SOD assays, compounds 1–5 exhibited high anti-oxidant activities compared with quercetin and BHA (positive controls), indicating that the 8-O-glucuronide attached to the flavonoid moiety is a key structure for the expression of anti-oxidant activity. This is the first report of the isolation of compounds 1–6 from M. verticillata as well evaluated for anti-diabetic and anti-oxidant ativities. ATP channels and voltage-gated Ca2+ channels +  in the positive ESI-QTof-MS. The IR spectrum suggested the presence of a hydroxyl group (3386 cm−1), a carbonyl group (1630 cm−1), a conjugated carbonyl group (1617 cm−1), and an aromatic double bond (1580 cm−1). The 1H-NMR spectrum contained four olefin methine proton signals , which were attributed to a paradisubstituted benzene ring B, and one olefin methine proton signal , which was attributed to H-3 of a flavone C-ring. In the oxygen region, one hemiacetal proton signal  and four oxygenated methine proton signals  were observed as the signals of a hexuronic acid moiety. The above-mentioned 1H-NMR spectrum indicated that Com 1 was a pentahydroxyflavone glucuronide. The 13C-NMR spectrum displayed 21 carbon signals, confirming that Com 1 was a pentahydroxyflavone with a hexuronic acid. One conjugated ketone carbon signal , nine olefin quaternary carbon signals , and five olefin methine carbon signals  in the down-field area were observed as the signals of the aglycone. Based on the Chem shift of the hexuronic acid carbon signals, such as a hemiacetal , four oxygenated methines , and one carboxyl δC 174.0 (C-6′′), the sugar was identified to be a β-glucuronic acid. From the coupling constant of the anomer proton signal (J = 7.2 Hz), the anomeric hydroxyl group was confirmed to be in the β-position. In the gradient heteronuclear multiple-bond connectivity (gHMBC) spectrum, the anomeric proton signal  exhibited 3J correlation with the olefin quaternary carbon signal , indicating that the β-d-glucuronopyranose was linked to C-8 of the pentahydroxyflavone. Therefore, the Chem structure of Com 1 was determined to be nortangeretin-8-O-β-d-glucuronide, and this Com was named malvaflavone A. The A-ring of malvaflavone A (1) contained 5,6,7,8-tetrahydroxy benzene, which rarely occurs in plant systems.Com n-BuOH, and aqueous Frs on zebrafish embryos, the survival rate related to EtOAc, n-BuOH, and aqueous Frs was investigated in zebrafish and LC50 value was calculated. Zebrafish were treated with EtOAc, n-BuOH, and aqueous Frs at ten different concentrations. The LC50 values of EtOAc, n-BuOH, and aqueous Frs were 91.5, 270.9 and 401.1 μg/mL, respectively  average in normal zebrafish was 1720 ± 300 μm2; furthermore, in AX- and glimepiride (GLM)-treated zebrafish, the average was 950 ± 200 μm2 and 1680 ± 220 μm2, respectively. PI size was 40.2% lower (p < 0.0001) in the AX treatment group than in the normal group amino]-2-deoxyglucose (2-NBDG), which is a fluorescent dye derived from glucose, modified with an amino group at the C-2 position  was subjected to ODS CC (φ 5 × 15 cm) and eluted with MeOH-H2O  to yield nine Frs (MVW-5-1 to MVW-5-9). Fr MVW-5-4  was subjected to SiO2 CC (φ 4.5 × 15 cm) and eluted with CHCl3-MeOH-H2O  to yield 17 Frs (MVW-5-4-1 to MVW-5-4-17). Fr MVW-5-4-13  was subjected to ODS CC (φ 3 × 5 cm) and eluted with acetone-H2O  to yield 7 Frs (MVW-5-4-13-1 to MVW-5-4-13-7). Fr MVW-5-4-13-3  was subjected to SiO2 CC (φ 2.5 × 5 cm) and eluted with CHCl3-MeOH-H2O  to yield 6 Frs (MVW-5-4-13-3-1 to MVW-5-4-13-3-6), which included two purified Coms, 4 and 5 . Fr MVW-5-4-13-4  was subjected to SiO2 CC (φ 2.5 × 15 cm) and eluted with EtOAc-n-BuOH-H2O  to yield nine Frs (MVW-5-4-13-4-1 to MVW-5-4-13-4-9), which included two purified Coms, 1 and 6 . Fr MVW-5-4-13-5  was subjected to ODS CC (φ 2.0 × 7 cm) and eluted with MeOH-H2O  to yield four Frs (MVW-5-4-13-5-1 to MVW-5-4-13-5-4), which included two purified Coms, 3 and 2 .Dried aerial parts of 1), yellow amorphous powder; [α]d–31.9° ; Mp decomposed >300 °C; IR  3386, 1630, 1617, 1580 cm−1; positive ESI-QTof-MS m/z 479.0828 [M + H]+ ; 1H-NMR  and 13C-NMR ; see Malvaflavone A , yellow amorphous powder; [α]D–11.2° ; Mp decomposed >300 °C; IR  3384, 1632, 1619, 1582 cm−1; positive ESI-QTof-MS m/z 463 [M + H]+; 1H-NMR  8.40 , 7.48 , 6.76 , 6.56 , 5.07 , 4.35 , 4.31 , 4.19 ; 13C-NMR  181.4 (C-4), 173.8 (C-6′′), 163.8 (C-2), 160.8 (C-4′), 156.5 , 148.8 (C-9), 128.5 , 125.3 (C-8), 120.7 (C-1′), 115.7 , 106.1 (C-1′′), 102.6 (C-10), 101.6 (C-3), 99.4 (C-6), 76.7 (C-3′′), 75.5 (C-5′′), 73.1 (C-2′′), 71.8 (C-4′′).Isoscutellarein 8-O-β-d-glucuronopyranoside , yellow amorphous powder; [α]D–7.1° ; Mp decomposed >300 °C; IR  3383, 1635, 1620, 1580 cm−1; positive ESI-QTof-MS m/z 479 [M + H]+; 1H-NMR  7.78 , 7.40 , 6.90 , 6.48 , 6.06 , 4.51 , 3.71 , 3.63 , 3.49 ; 13C-NMR  182.7 (C-4), 173.8 (C-6′′), 165.7 (C-2), 159.8 (C-7), 158.3 (C-5), 151.2 (C-4′), 150.4 (C-9), 146.9 (C-3′), 130.2 (C-8), 124.1 (C-1′), 120.3 (C-6′), 114.3 , 106.1 (C-1′′), 102.7 , 99.5 (C-6), 76.7 (C-3′′), 75.5 (C-5′′), 73.1 (C-2′′), 71.8 (C-4′′).Hypolaetin 8-O-β-d-glucuronopyranoside , yellow amorphous powder; [α]D–5.0° ; Mp decomposed >300 °C; IR  3380, 1633, 1622, 1579 cm−1; positive ESI-QTof-MS m/z 479 [M + H]+; 1H-NMR  8.74 , 7.31 , 6.50 , 4.89 , 4.16 , 4.05 , 3.90 ; 13C-NMR  181.5 (C-4), 171.2 (C-6′′), 159.4 (C-2), 159.2 (C-4′), 156.0 , 147.9 (C-9), 133.0 (C-3), 131.1 , 125.5 (C-8), 120.1 (C-1′), 114.6 , 105.7 (C-1′′), 102.1 (C-10), 99.1 (C-6), 76.4 (C-3′′), 75.7 (C-5′′), 74.1 (2′′), 71.5 (C-4′′).Herbacetin 8-O-β-d-glucopyranosyl-8-O-β-d-glucuronopyranoside , yellow amorphous powder; [α]D–5.7° ; Mp decomposed >300 °C; IR  3378, 1632, 1625, 1583 cm−1; positive ESI-QTof-MS m/z 641 [M + H]+; 1H-NMR  8.76 , 7.33 , 6.54 , 5.79 , 4.92 , 4.18 , 4.15 , 3.92–4.10 , 4.07 , 4.00 ; 13C-NMR  182.0 (C-4), 171.7 (C-6′′′), 159.9 (C-2), 159.8 (C-4′), 156.3 , 148.3 (C-9), 133.5 (C-3), 131.5 , 125.9 (C-8), 120.6 (C-1′), 115.0 , 106.1 (C-1′′′), 102.8 (C-1′′), 102.6 (C-10), 99.5 (C-6), 77.4 (C-3′′), 76.7 (C-3′′′), 76.1 (C-5′′′), 76.0 (C-5′′), 74.5 , 71.9 (C-4′′′), 69.8 (C-4′′), 60.9 (C-6′′).Herbacetin 3-O-β-d-glucopyranoside , yellow amorphous powder; [α]D–19.2° ; Mp decomposed >300 °C; IR  3477, 1616, 1579 cm−1; positive ESI-QTof-MS m/z 449 [M + H]+; 1H-NMR  8.40 , 7.48 , 6.91 , 6.71 , 5.56 , 4.21 , 3.85–4.10 , 3.68 ; 13C-NMR  182.0 (C-4), 163.8 (C-2), 161.9 (C-4′), 156.2 (C-7), 156.0 (C-5), 144.1 (C-9), 129.9 , 125.6 (C-8), 121.0 (C-1′), 115.8 , 104.7 (C-10), 102.5 (C-3), 101.2 (C-1′′), 98.7 (C-6), 77.2 (C-5′′), 75.8 (C-3′′), 73.1 (C-2′′), 69.6 (C-4′′), 60.9 (C-6′′).Isoscutellarein 7-Adult zebrafish were maintained in a zebrafish system S type (1500[W] × 400[D] × 2050[H] mm)  and a 14 h light/ 10 h dark cycle at 28.5 °C. Two pairs of adult zebrafish were placed in a spawning box overnight to obtain zebrafish larvae. The zebrafish spawned during a 30 min period of light. Zebrafish embryos were then collected at 3 h post-fertilization for incubation, and were maintained in a 0.03% sea salt solution for a 14/10 h, light/dark photocycle in an incubator at 28.5 °C. The fish were cared for in accordance with standard zebrafish protocols approved by the Animal Care and Use Committee of Kyung Hee University (KHUASP[SE]-15-10).n-BuOH, and aqueous Frs at the concentration 25, 50, 75, 100, 150, 200, 300, 400, 500 and 600 μg/mL, respectively. The zebrafish were observed under the microscope after 96 h treatment and dead zebrafish were recorded. LC50 values were calculated by non-linear regression using GraphPad Prism version 5.01 software.Twenty zebrafish embryos were used treatment for toxic test. Exposure of fish embryos was performed for 96 h as outlined in the OECD TG 236 for the Fish Embryo Acute Toxicity (FET) Test . 31 trea1–6. Wild-type zebrafish larvae (5 dpf) were placed into 24-well plates. The larvae were exposed to 600 μM AX for 3 h to induce PI damage. The AX-induced larvae were treated with 10 μg/mL Frs or 0.1 μM Coms for 12 h so that the recovery efficacy of the Frs and Coms could be determined. Then, the larvae were stained for 30 min with 40 μM 2-NBDG and rinsed with a 0.03% sea salt solution for 20 min. After the staining, the PIs were confirmed by fluorescence microscope and analyzed with Focus Lite software.The zebrafish larvae were divided into the normal group, AX-induced group (control group), and AX-induced groups treated with the different Frs and Coms 1, Com 1 + DZ, Com 3, Com 3 + DZ, Com 4, Com 4 + DZ, Com 6, and Com 6 + DZ. The Coms were applied at 0.1 μM each, and DZ was applied at 25 μM. The zebrafish larvae were treated with 600 μM AX for 3 h, after which the solution was rinsed with a 0.03% sea salt solution. The AX-induced zebrafish larvae were treated with the respective Coms and/or treatments for 12 h. Following treatment, the zebrafish larvae were stained with 40 μM 2-NBDG for 30 min and rinsed with a 0.03% sea salt solution for 20 min. After the staining, PI images were captured by fluorescence microscopy and analyzed with Focus Lite software.Wild-type zebrafish larvae (5 dpf) were placed in 24-well plates (10 zebrafish per well). The larvae were divided into the following 14 groups: normal, normal treated with DZ or AX, and AX-induced diabetic treated with DZ, GLM, GLM + DZ, Com p < 0.05. Statistical analysis was performed with GraphPad Prism (version 5). Data are expressed as the mean ± standard error of the mean (SEM) for three replicates. Significance was determined with repeated one-way ANOVA followed by Tukey’s test. The probability level for statistical significance was 1–6 (0.1 mL) were added to 2.9 mL of the methanolic DPPH• solution. The mixture was shaken vigorously and allowed to stand at 23 °C in the dark for 30 min. The decrease in absorbance of the resulting solution was monitored at 517 nm for 30 min. The control consisted of 0.1 mL of 70% aqueous methanol and 2.9 mL of DPPH• solution. Positive controls  were also subjected to the same procedure for comparison. The results were confirmed in triplicate analyses, and the DPPH RS activity was calculated with the following equation: According to the method of Brand-Williams , DPPH RS2HPO4/KH2PO4 buffer; 150 mM NaCl). The mixed solution was heated in a water bath at 68 °C. The resulting blue-green ABTS radical solution was adjusted to an absorbance of 0.650 ± 0.020 at 734 nm with additional PBS. Then, 20 µL of the sample was added to 980 µL of the ABTS radical solution. The mixture was incubated in a 37 °C water bath under restricted light for 10 min. A control  was run with each series of samples. Positive controls  were also subjected to the same procedure for comparison. The reduction of absorbance at 734 nm was measured 10 min later. The ABTS radical, exhibiting a characteristic blue-green color in its odd-electron state, loses color when its unpaired electron is paired with an electron from an anti-oxidant. The results were confirmed in triplicate analyses, and the ABTS RS activity was calculated with the following equation: The ABTS RS assay was carried out according to a published protocol . A radicThe ABTS RS assay was carried out according to a published protocol . ApproprSuperoxide RS activity was measured based on the capacity of the Coms to inhibit the photochemical production of superoxide in the riboflavin-light-NBT system. The reaction mixture for the measurement of superoxide RS activity contained 0.02 mM riboflavin, 3 mM methionine, and 0.18 mM NBT in 50 mM potassium phosphate buffer (pH 7.8). Samples in DMSO (10 µL) and 150 µL of the reaction mixture were dispensed into a 96-well plate. The reaction was started with fluorescent illumination (1000 lux) and allowed to proceed for 6 min at room temperature. The absorbance at 530 nm was measured on a microplate reader . The natural anti-oxidant quercetin and the synthetic anti-oxidant BHA were used as positive controls. The results were confirmed in triplicate analyses, and the superoxide scavenging activity was calculated with the following equation:


Natural products have a long history as a source of psychoactive agents and pharmacological tools for understanding the brain and its circuitry. In the last two decades, marine cyanobacteria have become a standard source of natural product ligands with cytotoxic properties. The study of cyanobacterial metabolites as CNS modulatory agents has remained largely untapped, despite the need for new molecules to treat and understand CNS disorders. We have generated a library of 301 fractions from 37 field collected cyanobacterial samples and screened these fractions against a panel of CNS receptors using radiolabeled ligand competitive-binding assays. Herein we present an analysis of the screening data collected to date, which show that cyanobacteria are prolific producers of compounds which bind to important CNS receptors, including those for 5-HT, DA, monoamine transporters, adrenergic, sigma, and cannabinoid receptors. In addition to the analysis of our screening efforts, we will also present the isolation of five compounds from the same cyanobacterial collection to illustrate how pre-fractionation followed by radioligand screening can lead to rapid identification of selective CNS agents. The systematic screening of natural products sources, specifically filamentous marine cyanobacteria, will yield a number of lead compounds for further development as pharmacological tools and therapeutics. Since the 1970s , marine 1 and σ2 receptors, which have been hypothesized to be targets for the treatment of neuropathic pain , 2.34 , 2.32 , 2.28, 2.13 , 2.01 , 1.41  together with a 12H signal at δ 1.27 for 6 defined the substituted diunsaturated alkyl chain.The C fraction, which had activity at 5-HTund A 2, . GCMS . The complete assignment of all protons and carbons of compound 2 was achieved through the construction of the substructures (3-18) and the allylic methylene at δ 2.01  ppm, respectively. This suggested the presence of a saturated hydrocarbon moiety, typical of fatty acid derivatives.The eriments , indicatructures  from 2D 2 hybridized carbon at C-8 (δ 125.2), together with correlations of H-19 to C-10 (δ 27.8) linked the saturated hydrocarbon moiety, comprised of C-10–C-18, to the trisubstituted alkene of the fatty acid. The spin system formed by H-2, H-3, H-4, H-5, H-6, H-7, H-8 was assigned by COSY and 2,3J HMBC correlations. The chemical shifts for carbon at C-3 and C-4 were consistent with resonances for alkenes, while the chemical shift of the carbon at C-7 indicated that it is attached to oxygen. The presence of the singlet signal at δ 3.23 corresponding to the hydrogens of the methyl group at C-19 (δ 55.5), and HMBC correlations from these hydrogens to the oxygen-bearing sp3 carbon at C-7, confirmed placement of the methoxy group at position 7. HMBC correlations from the α and β-methylene hydrogens (H-2 and H-3 respectively) to the carbonyl at δ173.6 (C-1) completed the assignment of substructure II. Long range C–H correlations between methylene hydrogens and the methine resonances at δ 5.45 , 4.99 , the hydrogens  of the methylene group at C-18 to C-1 allowed for the combination of substructures I and II and the characterization of compound 2.Long range HMBC connectivities from the methylene hydrogens at H-10 and the methyl hydrogens at H-19 (δ 1.64 s) to the sp3 was isolated as a colourless paste after purification by HPLC and was shown by 1H- and 13C-NMR data to be related to the C13 cyclopropyl fatty acid derivatives Compound2: colourless oil; 1H-, 13C- and 2D-NMR see m/z = 237, 194, 101, 55.Compound3: colourless oil; 1H-NMR  δ 4.18 , 2.39 , 1.56 , 1.35 , 1.31 , 1.28 , 1.23 , 1.14 , 0.90 , 0.46 , 0.46  0.22 ; 13C-NMR : δ 173.9 (C1), 60.3 (C14), 34.6 (C2), 34.2 (C7), 32.0 (C11), 29.6 (C3), 29.6 (C8-10), 22.7 (C12), 18.2 (C6), 18.0 (C4), 14.2 (C13), 14.2 (C15), 11.8 (C5); GCMS:11.3 min, m/z = 240, 194, 152.Propenediester (4): colourless oil; 1H-NMR  δ 7.25 , 5.10 , 4.78 , 4.74 , 4.75 , 2.45 , 2.09 , 2.09 , 2.01 , 1.83 , 1.43 , 1.28 ; 13C-NMR : δ 170.8 (C23), 170.1 (C1), 148.6 (C5), 137.0 (C20), 109.8 (C19), 107.5 (C21), 57.9 (C22), 35.8 (C6), 35.2 (C4), 33.3 (C2), 31.9 (C16), 29.7 (C8), 29.7–29.3 (C9-15), 27.8 (C7), 22.7 (C3), 22.7 (C7), 20.9 (C24), 14.1 (C18); GCMS: 20.5 min, m/z = 279, 110, 97.Grenadadiene (5): colourless oil; 1H-NMR  δ 7.32 , 7.00 , 5.75 , 4.86 , 2.55 , 2.16 , 1.61 , 1.34–1.28 , 1.28 , 1.27 , 1.24 , 1.15 , 0.90 , 2.55 , 0.48 , 0.48  0.25 ; 13C-NMR : δ 170.4 (C19), 169.9 (C1), 137.8 (C14), 124.5 (C16), 121.0 (C17), 109.0 (C15), 69.1 (C18), 34.3 (C2), 34.0 (C7), 31.8 (C11), 29.8 (C8–10), 29.2 (C3), 22.8 (C12), 20.9 (C20), 19.1 (C6), 18.2 (C4), 14.2 (C13), 11.8 (C5); GCMS:18.5 min, m/z = 414 (79Br), 416 (81Br), 293, 281, 220 (79Br), 222 (81Br).50 determinations/receptor binding profiles were generously provided by the National Institute of Mental Health’s Psychoactive Drug Screening Program, Contract # HHSN-271-2018-00023-C (NIMH PDSP). The NIMH PDSP is Directed by Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA. Detailed information regarding the assay protocol can be found at http://pdspdb.unc.edu/pdspWeb/.IC