Reynoutria multiflora Thunb. Moldenke  has been widely used in TCM clinical practice for centuries. The raw R. multiflora (RRM) should be processed before use, in order to reduce toxicity and increase efficiency. However, the content of trans-2, 3, 5, 4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside (trans-THSG), which is considered to be the main medicinal ingredient, decreases in this process. In order to understand the changes of stilbene glycosides raw R. multiflora (RRM) and processed R. multiflora (PRM), a simple and effective method was developed by ultra high performance liquid chromatography tandem quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive plus orbitrap MS/MS). The content and quantity of stilbene glycosideshave undergone tremendous changes during the process. Seven parent nucleus of stilbene glycosides and 55 substituents, including 5-HMF and a series of derivatives, were identified in PM. 146 stilbene glycosides were detected in RRM, The number of detected compounds increased from 198 to 219 as the processing time increased from 4 to 32 h. Among the detected compounds, 102 stilbene glycosides may be potential new compounds. And the changing trend of the compounds can be summarized in 3 forms: gradually increased, gradually decreased, first increased and then decreased or decreased first. The content of trans-THSG was indeed decreased during processing, as it was converted into a series of derivatives through the esterification reaction with small molecular compounds. The clarification of secondary metabolite group can provide a basis for the follow-up study on the mechanism of pharmacodynamics and toxicity of PM, and for screening of relevant quality markers.The root of  Traditional Chinese medicine processing is a unique pharmaceutical technology derived from the theory of traditional Chinese medicine. It has played a prominent role in the clinical practice of traditional Chinese medicine for thousands of years, ensuring the safety and effectiveness of treatment. After processing with different temperatures, durations, solvents or excipients, the components of traditional Chinese medicine have undergone different changes. Ingredients will be dissolved, decomposed or transformed into new components, resulting in increasing or decreasing of the compounds. All these changes are closely related to the property and efficacy of traditional Chinese medicine. Therefore, it is of great significance to study the changes of chemical components before and after processing of traditional Chinese medicine.Reynoutria multiflora Thunb. (Polygonum multiflorum Thunb.), well known as He-shou-wu in China, has been widely used in TCM clinical practice for centuries , cis-THSG and polydatin were purchased from yuanye Bio-Technology Co., Ltd. Acetonitrile . acetonitrile and methanol (HPLC grade), were supplied by E. Merck , formic acid (HPLC grade) was purchased from fisher (United States), ultra-pure water was prepared by a Mili-Q water purification system .All the samples were prepared using following method: 1 g sample powder was ultrasonicated for 30 min with 25 ml of 70% ethanol, followed by filtration and then evaporated the filtrate. 5 ml of ultrapure water were added to dissolve the residue and then extracted twice with 15 ml of ethyl acetate. The resulting mixture was combined with an ethyl acetate solution and evaporated over a water bath; after that, 1 ml of methanol was added to dissolve the residue and centrifugation  for 10 min by a 1.5 ml centrifuge tube. Finally, the supernatant of the treatment samples was injected into the UPLC-Q-Exactive plus orbitrap MS/MS system.All the samples were analysed using an Ultimate 3000 UPLC system  that was controlled with Thermo Xcalibur software . The samples were separated using a Kinetex UPLC C18 column  . The mobile phase consisted of solvent A (0.1% formic acid) and solvent B (acetonitrile). A gradient elution was applied using the following optimized gradient program: 8-8% B at 0–3 min, 8–28% B at 3–25 min, 28–40% B at 25–26 min, 40–50% B at 26–28 min, 50–70% B at 28–30 min, 70–90% B at 30–32 min, and 90–90% B at 32–35 min. The flow rate was kept at 0.4 ml/min, the sample injection volume was 1 μL, and the column temperature was maintained at 25°C.m/z 100–1,200. The parameter settings were as follows: a full scan and fragment spectral resolution of 70,000 FWHM and 17, 50 FWHM, respectively; capillary temperature was 350°C; auxiliary gas heater temperature was 350°C; spray voltage was −3.2 KV; sheath gas flow rate was 40 Arb; auxiliary gas flow rate was 15 Arb; and S-lens RF level was set at 50. The acquisition mode of stepped NCE  was using with settings of 30, 50, and 70 eV. The accumulated resultant fragment ions were injected into the Orbitrap mass analyzer for single-scan detection.Mass spectrometry was performed on a Q-Exactive Plus™ quadrupole-Orbitrap mass spectrometer  in negative ion mode. The scan mass range was set at Considering the possible elemental composition of the RM components, the types and quantities of expected atoms were set as follows: carbon ≤50, hydrogen ≤200, oxygen ≤20, nitrogen ≤3. The accuracy error threshold was fixed at 5 ppm.The chemical profiles of RRM and processing PRMs were analyzed by UHPLC-Q-Exactive plus orbitrap MS/MS, the representative base peak chromatograms of RRM and processing PRM (24 h) are shown in A3-5, tR = 11.18 min) and cis-THSG  had a [M-H]− ion at m/z 405.1187 with only a dominant ion at m/z 243.0654 (C14H11O4) in MS2 spectrum. These two ions could be used as a diagnostic ion for identify stilbene glycosides. Compound A3-1, A3-3 and A3-4  also had an [M-H]− ion at m/z 405.1187 (C20H21O9), and showed a fragment ion at m/z 243.0654 in their MS2 spectrum, indicating that they are isomers of THSG. A3-5 was identified as trans-THSG and A3-2 was cis-THSG, and A3-1 should be isomer of cis-THSG, A3-3 and A3-4 should be isomers of trans-THSG. , 5-hydroxymethyl furfural (5-HMF) and its derivatives . Stilbenm/z 405.1192(C20H21O9) and 243.0654 (C14H11O4). These were used for rapidly extracting and analyzing unknown stilbene glycosides. According to the structural characteristics of THSG, the linking points of stilbene derivatives with other compounds contain glycosyl hydroxyl moiety and phenyl hydroxyl moiety. According to the cleaved fragments, it can be inferred as follows: 1. if there is a fragment from loss of C6H10O5 by parent ion, the linking point should be phenyl hydroxyl moiety; 2. The cleavage fragment contains the ion at m/z 405.1187 of THSG and the ion at m/z (hexoside + substituent), and there is no fragment to loss of C6H10O5, so the linking point should be glycosyl hydroxyl moiety; 3. if the fragment is only the ion at m/z 243.0654, most of the glycosyl hydroxyl moiety may be linked, but there is also a probability that the hexoside of THSG and the substituent on the phenyl hydroxyl moiety will split at the same time, so the linking point cannot be determined in this case. We use tetrahydroxystilbene-O- (substituent)-hexoside to name them.Most stilbene glycosides in RM showed common fragmentation pathways and two diagnostic fragment ions at A1-1 and A1-2 displayed a [M-H]− ion at m/z 375.1081 (C19H19O8) and the product ion at m/z 243.0654 derived from the loss of a pentose (mostly arabinose). By comparing with literature, Compounds A1-1 and A1-2 were tentatively identified as tetrahydroxystilbene-O-pentose.Compounds A2-1, A2-2, and A2-3 gave a [M-H]− ion at m/z 389.1242 (C20H21O8) and the product ion at m/z 243.0654 derived from the loss of a deoxyhexose (mostly rhamnose), indicated that it was a THSG derivative. Compounds A2-1, A2-2, and A2-3 were tentatively characterized as tetrahydroxystilbene-O-deoxyhexoside.Compounds A4-1 and A4-2 displayed a high resolution [M-H]− ion at m/z 423.1295 and gave element composition of C20H23O10. The MS2 spectra gave identical ions at m/z 261.0764 (C14H13O5) and 243.0654 (C14H11O4), respectively. The loss of C6H10O5 (hexoside) and H2O to produce the deprotonated moiety ion at m/z 243.0655, indicated can be identified as stilbene derivatives, but the specific structure is not yet determined.Compounds A5-1 ∼ A5-4 showed the same [M-H]− ion at m/z 433.1136 (C21H21O10) and the MS2 spectra gave ions at m/z 271.0608 (C15H11O5) and 243.0654 (C14H11O4). Without further information, compounds A5-1 ∼ A5-4 were tentatively characterized as tetrahydroxystilbene-O-hexoside-O-formic acid acyl (phenolic hydroxyl moiety).Compounds A6-1 and A6-2 showed the same [M-H]− ion at m/z 437.1450 (C21H25O10) and the MS2 spectra gave ions at m/z 275.0922 (C15H15O5) and m/z 243.0655 (C14H11O4), the loss of C6H10O5 (hexoside) and CH4O to produce the deprotonated moiety ion at m/z 243.0655, allowed us to infer that they were tetrahydroxystilbene derivative, but the specific structure is not yet determined.Compounds A7-1 ∼ A7-5 gave a [M-H]− ion at m/z 447.1300 (C22H23O10) and loss 204 Da to produce ion at m/z 243.0656 in the MS2 spectrum, which indicated that the presence of a hexose group and an acetyl. Thus, compounds A7-1∼ A7-5 were preliminarily characterized as tetrahydroxystilbene-O-(acetyl)-hexoside.Compounds A8-1 and A8-2 showed the same [M-H]− ion at m/z 449.1086 (C21H21O11) and the MS2 spectra gave ions at m/z 287.0554 (C15H11O6) and 243.0654 (C14H11O4) form continuous loss of C6H10O5 and CO2. Thus, the carbonate acyl substituted THSG was detected and compounds A8-1 and A8-2 were identified as tetrahydroxystilbene-O-hexoside-O-carbonate acyl (phenolic hydroxyl moiety).Compounds A9 displayed a high resolution [M-H]− ion at m/z 457.1116 and gave element composition of C23H21O10, the product ion at m/z 243.0654 originated from the loss of C9H10O6 (hexoside + hydroxycyclopropenon). By investigating literature, compound A9 was preliminarily identified as tetrahydroxystilbene-O-(hydroxycyclopropenon)-hexoside. Similarly, compounds A10 and A11 were tentatively identified as tetrahydroxystilbene-O-(acrylic acid acyl)-hexoside and tetrahydroxystilbene-O-(propionyl)-hexoside, since the loss of C9H12O6 (hexoside + acrylic acid) and C9H14O6 (hexoside + propionic acid) were detected.Compound A12-1 and A12-2 showed the same [M-H]− ion at m/z 463.1244 (C22H23O11) and the MS2 spectra gave ion at m/z 243.0654 (C14H11O4) form loss of C8H12O7 (C6H10O5 and C2H2O2). Thus, the glycolic acid substituted THSG was detected and compounds A12-1 and A12-2 were identified as tetrahydroxystilbene-O- (glycolic acid acyl)-hexoside.Compounds A13-1, A13-2, and A13-3 showed the same [M-H]− ion at m/z 477.1396 (C23H25O11) and the MS2 spectra gave ions at m/z 405.1184 (C20H2109), 315.0859 (C17H15O6), 297.0763 (C17H13O5) and 243.0655 (C14H11O4). The ion at m/z 477.1396 loss of C3H4O2 produce the ion at m/z 405.1184, By comparing literature, compounds A13-1, A13-2, and A13-3 were tentatively identified as tetrahydroxystilbene-O-hexoside-O-lactic acid acyl (phenolic hydroxyl moiety).Compounds A14-1 ∼ A14-4 showed the same [M-H]− ion at m/z 489.1759 (C25H29O10) and the MS2 spectra gave identical ions at m/z 405.1176 (C20H2109), 327.1222 (C19H19O5) and 243.0656 (C14H11O4). The loss of C5H8O to produce the deprotonated THSG moiety ion at m/z 405.1176, Furthermore, the ion at m/z 327.1222 assigned as loss of C6H10O5 form the m/z 489.1759. By investigating literatures, compounds A14-1 ∼ A14-4 were identified as tetrahydroxystilbene-O-hexoside-O-valerate acyl (phenolic hydroxyl moiety).Compounds A15 displayed a high resolution [M-H]− ion at m/z 499.1241 and gave element composition of C25H23O11, the product ions at m/z 337.0704 (C19H13O6), 293.0812 (C18H13O4) and 243.0654 (C14H11O4) originated from the consecutive loss of C6H10O5 (hexoside), CO2 and C4H2 . By investigating literature, compound A15 was preliminarily identified as tetrahydroxystilbene-O-hexoside-O-5-hydroxyfuran-2-carbaldehyde (phenolic hydroxyl moiety).Compound A16-1 and A16-2 showed the same [M-H]− ion at m/z 501.1393 (C25H25O11) and the MS2 spectra gave ions at m/z 339.0858, 321.0756 and 243.0654 form continuous loss of C6H10O5 (hexoside), CO2 and C4H4 (A16-1 and A16-2 were identified as tetrahydroxystilbene-O-hexoside-O-4-hydroxymethyl-5H-furan-2-one (phenolic hydroxyl moiety).Compounds and C4H4 . By inveA17 gave a [M-H]− ion at m/z 503.1553 (C25H27O11) and the product ions at m/z 341.1019 (C19H17O6), 297.1125 (C18H17O4) and 243.0656 (C14H11O4). By comparing literature, compound A17 was tentatively characterized as tetrahydroxystilbene-O-hexoside-O- 5-hydroxymethyl-4, 5-dihydrofuranone (phenolic hydroxyl moiety).Compound A18-1 ∼ A18-5 showed the same [M-H]− ion at m/z 505.1346 (C24H25O12) and in A18-1 and A18-2 MS2 spectra, gave ions at m/z 405.1178 and m/z 243.0655, in A18-3 ∼ A18-5 MS2 spectra, gave ions at m/z 343.0799 and m/z 243.0655. By comparing literature, compounds A18-1 and A18-2 were preliminarily characterized as tetrahydroxystilbene-O-(succinic acid acyl)-hexoside, and compounds A18-3 ∼ A18-5 were identified as tetrahydroxystilbene-O-hexoside-O-succinic acid acyl (phenolic hydroxyl moiety).Compounds A19-1 and A19-2 were eluted at 11.85 and 12.00 min, and the molecular formula was C24H27O12 (m/z 507.1500). The MS2 spectra gave identical ions at m/z 345.0966 (C18H17O7), m/z 313.0709 (C17H13O6), m/z 285.0763 (C16H13O5), m/z 255.0656 (C15H11O4) and m/z 243.0654 (C14H11O4). By comparing literature, compounds A19-1 and A19-2 were identified as tetrahydroxystilbene-O-hexoside-O-dihydroxybutyrate (phenolic hydroxyl moiety).Compounds A20 gave a [M-H]− ion at m/z 511.1603 (C27H27O10) and the product ions at m/z 349.1068 (C21H17O5) and 243.0655 (C14H11O4) form continuous loss of C6H10O5 (hexoside) and C7H6O . By comparing literature, compound A20 was tentatively characterized as tetrahydroxystilbene-O-hexoside-O-salicyloyl (phenolic hydroxyl moiety).Compound A21-1 and A21-2 showed the same [M-H]− ion at m/z 512.1555 (C26H26O10N) and the MS2 spectra gave ions at m/z 405.1175 (C20H21O9) and 243.0655 (C14H11O4). By comparing literature, compounds A21-1 and A21-2 were tentatively identified as tetrahydroxystilbene-O-(aminocatecholoyl)-hexosides. Similarly, compound A25 was tentatively identified as tetrahydroxystilbene-O-(pyroglutamyl)-hexoside.Compounds A22-1 ∼ A22-4 showed the same [M-H]− ion at m/z 513.1497 (C26H25O11), compounds A22-1, A22-2 and A22-3 loss 162 Da to produce ion at m/z 351.0862, and then loss 108 Da (C6H4O2) to produce ion at m/z 243.0656 in the MS2 spectrum. And in compound A22-4 MS2 spectra, there was a fragment ion at m/z 243.0655, the proposal fragmentation pathway shown in A22-1, A22-2, and A22-3 were identified as tetrahydroxystilbene-O-hexoside-O-5-HMF (phenolic hydroxyl moiety), A22-4 was identified as tetrahydroxystilbene-O-(5-HMF)-hexoside.Compounds A23-1 and A23-2 were eluted at 15.29 and 15.69 min, and the molecular formula was C26H27O11 (m/z 515.1555). In addition, the product ions at m/z 353.1021 (C20H17O6) and m/z 243.0654 (C14H11O4) originated from the consecutive loss of hexoside (C6H10O5) and C6H6O2 . By comparing literature, compounds A23-1 and A23-2 identified as tetrahydroxystilbene-O-hexoside-O-2, 5-bis-hydroxymethyl furan (phenolic hydroxyl moiety). And compound A24 displayed a high resolution [M-H]− ion at m/z 515.1179 and gave element composition of C25H23O12, the loss of C5H2O3 and C6H10O5 to produce the deprotonated moiety ion at m/z 243.0655. By investigating literatures, compound A24 was tentatively characterized as tetrahydroxystilbene-O-(5-hydroxyfuran-2-carboxylic acid)-hexoside.Compounds A26-1 ∼ A26-4 showed the same [M-H]− ion at m/z 519.1495 (C25H27O12). In A26-1 MS2 spectra, the fragment ion at m/z 243.0655, in A26-2, A26-3 and A26-4 MS2 spectra, the fragment ions at m/z 405.1167 (C20H21O9), 357.0967 (C19H17O7), 339.0855 (C19H15O6), 297.0760 (C17H13O5) and 243.0655 (C14H11O4), by investigating literatures, compound A26-1 was identified as tetrahydroxystilbene-O-(glutaryl)-hexoside, A26-2, A26-3 and A26-4 were identified as tetrahydroxystilbene-O-hexoside-O-glutaryl (phenolic hydroxyl moiety).Compounds A27-1 (tR = 12.84 min), A27-2 (tR = 13.07 min) and A27-3 (tR = 13.82 min) showed the same [M-H]− ion at m/z 521.1294 (C24H25O13) and the MS2 spectra gave identical ions at m/z 405.1177 (C20H21O9), 359.1115 (C19H19O7) and 243.0754 (C14H11O4). The loss of C4H4O4 to produce the deprotonated THSG moiety ion at m/z 405.1177, thus, compounds A27-1, A27-2, and A27-3 identified as tetrahydroxystilbene-O-hexoside-O-malic acid acyl (phenolic hydroxyl moiety).Compounds A28-1 ∼ A28-5 showed the same [M-H]− ion at m/z 525.1398 (C27H25O11), the MS2 of A28-1 ∼ A28-3 spectra gave ions at m/z 525.1398, 405.1179, 363.0883, 243.0858 and 137.0228. The product ions at m/z 363.0883 originated from the loss of hexoside (C8H10O5). Thus, the salicylic acid acyl substituted THSG was detected and compounds A28-1 ∼ A28-3 identified as tetrahydroxystilbene-O-hexoside-O-salicylic acid acyl (phenolic hydroxyl moiety). The MS2 of A28-4 and A28-5 spectra gave ions 405.1179, 243.0858 and 137.0228, but there were no 363.0833 fragment ion. Thus, compounds A28-4 and A28-5 were identified as tetrahydroxystilbene-O- -hexoside.Compounds A29-1, A29-2 and A29-3 gave a [M-H]− ion at m/z 527.1190 (C26H23O12) and the MS2 spectra showed identical ions at m/z 365.0652 (C20H13O7) and 243.0659 (C14H11O4). The MS2 spectrum showed losses of C6H10O5 and C6H2O3, respectively, to produce characteristic aglycone ion at m/z 243.0659. By comparing literature, compounds A29-1, A29-2, and A29-3 were tentatively identified as tetrahydroxystilbene-O-hexoside-O-5-formylfuran-2-carboxylyl (phenolic hydroxyl moiety).Compounds A30-1 ∼ A30 ∼ 7 showed the same [M-H]− ion at m/z 529.1345 (C26H25O12), the MS2 spectra of A30-1, A30-2 and A30-6 gave ions at m/z 367.0870 (C20H15O7), 323.0914 (C19H15O5) and 243.0651 (C14H11O4). The product ions originated from the consecutive loss of hexoside (C6H10O5), CO2 and C5H4O. In A30-3 and A30-4 spectra, gave ions at m/z 367.0807 (C20H15O7), 243.0656 (C14H11O4) and 123.0071 (C6H3O3), and in A30-5 and A30-7 spectra, gave ions at m/z 405.1176 (C20H21O9), 243.0656 (C14H11O4) and 123.0071 (C6H3O3). By investigating literatures, the substituent group of the compound was 5-hydroxymethyl-furfural. And according to the fragmentation fragments, it can be inferred that the binding sites are different -O-5-hydroxymethylfuran-2-carboxylyl-hexosides (hydroxyl moiety), A30-3 and A30-4 were identified as tetrahydroxystilbene (phenolic hydroxyl moiety)-O-5-hydroxymethylfuran- 2-carboxylyl-hexoside (carboxyl moiety), A30-5 and A30-7 were identified as tetrahydroxystilbene-O-(5-hydroxymethylfuran-2-carboxylyl)-hexoside.Compounds ifferent . A30-1, A31-1 ∼ A31-10 were eluted at 2.58, 2.75, 3.58, 4.66, 5.00 min 5.64, 12.14, 15.02, 15.36, 15.74 min, they both showed an accurate [M-H]− ion at m/z 531.1511 (C26H27O12), which were 128 Da higher than that of THSG. In their MS2 spectra, the [M-H]− ion showed fragment ions at m/z 405.1180 (C20H21O9), 369.0966 (C20H17O7), 351.0863 (C20H15O6), 319.0796 (C18H13O5), 295.0609 (C17H11O5), and 243.0655 (C14H11O4). All the MS2 of the compounds have molecular fragment m/z 369.0966, indicated that the compounds are formed by dehydration of substituents and phenolic hydroxyl groups of stilbene glycosides. By investigating literature (A31-1 ∼ A31-10 were tentatively characterized as tetrahydroxystilbene-O-hexoside-DDMP (phenolic hydroxyl moiety).Compounds terature , compounA32-1 ∼ A32-5 showed the same [M-H]− ion at m/z 533.1658 (C26H29O12) and the MS2 spectra gave identical ions at m/z 371.1124 (C20H18O7), 327.0863 (C18H16O6) and 243.0657 (C14H11O4). The MS2 showed losses of C6H3O3 and C6H10O5 due to substituent and hexose moiety, respectively, to produce characteristic aglycone ion at m/z 243.0657 (C14H11O4). By comparing literature, compounds A32-1 ∼ A32-5 were tentatively characterized as tetrahydroxystilbene-O-hexoside-adipic acid acyl (phenolic hydroxyl moiety).Compounds A33-1 ad A33-2 gave a [M-H]− ion at m/z 537.1609 (C25H29O13) and the product ion at m/z 243.0655 (C14H11O4) form loss of C11H18O9 (C6H10O5 and C5H8O4). By comparing literature, compound A33-1 and A33-2 were tentatively characterized as tetrahydroxystilbene-O-(arabinoyl)-hexoside.Compound A34-1 ∼ A34-4 showed a [M-H]− ion at m/z 541.1353 (C27H25O12) and the MS2 spectra of A34-1 and A34-2, showed fragment ions at m/z 405.1175 (C20H21O9), 297.0610 (C13H13O8), 243.0657 (C14H11O4), 153.0179 (C7H5O4), respectively, indicated that they were THSG derivatives. The loss of C7H5O4 to produce the deprotonated THSG moiety ion at m/z 405.1175, allowed us to infer that they were protocatechuic acid substituted THSG products. And the ion at m/z 297.0610 assigned as protocatechuic acid acyl-hexoside moiety, produced protocatechuic acid ion at m/z 153.0179. A34-3 and A34-4 were eluted at 21.70 and 22.07 min, the [M-H]− ions showed fragment ions at m/z 379.0793 (C21H15O7) and 243.0657 (C14H11O4), respectively, originated from the consecutive loss of hexoside (C6H10O5) and protocatechuic acid (C7H5O4). There results indicate that protocatechuic acid substituted to the phenolic hydroxyl moiety in compounds A34-3 and A34-4. Therefore, compounds A34-1 and A34-2 were identified as tetrahydroxystilbene-O-hexoside-O-protocatechuic acid acyl (glycosyl hydroxyl moiety), and A34-3 and A34-4 were identified as tetrahydroxystilbene-O-hexoside-O-protocatechuic acid acyl (phenolic hydroxyl moiety).Compounds A35 gave a [M-H]− ion at m/z 543.1121 (C26H33O13) and the product ions at m/z 405.1196 (C20H21O9) and 243.0655 (C14H11O4) form consecutive loss of C6H12O4 and C6H10O5. By comparing literatures, compound A35 was tentatively identified as tetrahydroxystilbene-O- (furan-dicarboxylic acid acyl)-hexoside. And compound A36 gave a [M-H]− ion at m/z 543.1501 (C27H27O12) and the product ion at m/z 381.0963 (C21H17O7), 337.1069 (C20H17O5) and 243.0655 (C14H11O4) form consecutive loss of C6H10O5, CO2 and C6H6O. By investigating literatures, A36 was tentatively identified as tetrahydroxystilbene-O-hexoside-methoxymethyl-furancarboxylic acid acyl (phenolic hydroxyl moiety).Compound A37-1 and A37-2 showed the same [M-H]− ion at m/z 547.1453 (C26H27O13) and the MS2 spectra gave ions at m/z 385.0914 (C20H17O8) and 243.0655 (C14H11O4) from consecutive loss of C6H10O5 and C6H6O4. By comparing literature, compounds A37-1 and A37-2 were tentatively identified as tetrahydroxystilbene-O-hexoside-oxoadipic acid acyl (phenolic hydroxyl moiety). Similarly, compounds A38-1, A38-2 and A38-3 were tentatively identified as tetrahydroxystilbene-O-hexoside-hydroxyadipic acid acyl (phenolic hydroxyl moiety), since the ions at m/z 387.1066 (C20H19O8) and 243.0655 (C14H11O4) from consecutive loss of C6H10O5 and C6H8O4.Compounds A39-1 ∼ A39-5 displayed a high resolution [M-H]− ion at m/z 551.1556 and gave element composition of C29H27O11. In A39-2 ∼ A39-5 MS2 spectra, the [M-H]− showed fragment ions at m/z 405.1180 (C20H21O9), 243.0656 (C14H11O4), 163.0397(C9H7O3) and 145.0280 (C9H5O2). The product ions at m/z 405.1180 and 234.0656 originated from the consecutive loss of p-hydroxycinnamoyl (C9H6O2) and hexoside (C6H10O5), Thus, p-hydroxycinnamoyl substituted THSG was detected and compounds A39-2 ∼ A39-5 were identified as tetrahydroxystilbene-O-(p-hydroxycinnamoyl)-hexoside. In A39-1 MS2 spectra, the [M-H]− showed fragment ions at 399.1018 , 243.0655, 163.0396 and 145.0279, indicated p-hydroxycinnamoyl was linked to phenolic hydroxyl moiety. Thus, compound A39-1 was identified as tetrahydroxystilbene-O-hexoside-p-hydroxycinnamoyl (phenolic hydroxyl moiety).Compounds A40-1 ∼ A40-8 showed the same [M-H]− ion at m/z 557.1295 (C27H25O13) and the MS2 spectra gave identical ions at m/z 405.1179 (C20H21O9), 313.0555 (C13H13O9), 243.0654 (C14H11O4) and 169.0127 (C7H5O5). The loss of C6H10O5 and C7H4O4 to produce the deprotonated resveratrol moiety ion at m/z 243.0654, a galloyl group was present, the ion at 313  appeared as base peak in the MS2 spectra. It could further fragment ion m/z 169 . Therefore, compounds A40-1 ∼ A40-8 were identified as tetrahydroxystilbene-O-hexoside-O-galloyl (glycosyl hydroxyl moiety) (Compounds  moiety) .41-1 and 41-2 showed the same [M-H]− ion at m/z 561.1609 (C27H29O13) and the MS2 spectra gave identical ions at m/z 405.1174 (C20H21O9) and 243.0654 (C14H11O4). By comparing literatures, compounds 41-1 and 41-2 were identified as tetrahydroxystilbene-O- (gabosine C)-hexoside.Compounds A 42-1 ∼ A 42-13 gave precursor ion [M-H]− at m/z 567.1712 (C26H31O14), in A42-2 ∼ A42-4 and A42-7 ∼ A42-13 MS2 spectra, the [M-H]− ion showed fragment ion at m/z 243.0654 (C14H11O4), indicated that the consecutive neutral loss of hexoside, they were disaccharide THSGs. By comparing literature, they were tentatively characterized as tetrahydroxystilbene-O-di-hexosides , 447.1278 (C22H23O10), 315.0857 (C17H15O6), 285.0761 (C16H13O5), and 243.0655 (C14H11O4), respectively. The fragment ions 477.1401 −, 447.1278 − are diagnostic the last two neutral loss fragments suggested that a C-glycoside was connected with the stilbene glycoside. Therefore, A42-1, A42-5 and A42-6 were determined as tetrahydroxystilbene-O-hexoside-C-glycoside.Compounds exosides . But in A42-14 (tR = 18.29 min), A42-15 (tR = 19.50 min) and A42-16 (tR = 21.64 min) showed the molecular formula were C29H27H12 (m/z = 567.1504), which was 162 Da heavier than that of THSG, and different from A42-1 ∼ A42-13. In their MS2 spectra, the [M-H]− ion showed fragment ions at m/z 405.1203 (C20H21O9), 243.0654 (C14H11O4), 161.0229 (C9H5O3), respectively. Indicated that the consecutive loss of caffeoyl (C9H6O3) and hexoside (C6H10O5). Thus, the caffeoyl substituted THSG was detected and compound A42-14, A42-15 and A42-16 identified as tetrahydroxystilbene-O-(caffeoyl)-hexoside.A43-1 ∼ A43-6 showed the same [M-H]− ion at m/z 573.1251 (C27H25O14) and the MS2 spectra gave identical ions at m/z 243.0657 (C14H11O4), 166.9971 (C7H3O5) and 123.0071 (C6H3O3). By comparing literature, Compounds A43-1 ∼ A43-6 were tentatively identified as tetrahydroxystilbene-O- (tetrahydroxybenzoic acid acyl)-hexoside.Compounds A44 gave a [M-H]− ion at m/z 575.1402 (C27H27O14) and the product ions at m/z 337.0707 (C19H13O6) and 244.0655 (C14H11O4). By comparing literatures, compound A44 was tentatively identified as tetrahydroxystilbene-O- (dioxoheptane-dicarboxylic acid acyl)-hexoside.Compound A45-1 ∼ A45-5 showed the same [M-H]− ion at m/z 581.1655 (C30H29O12), the MS2 spectra gave ions at m/z 405.1177 (C20H21O9), 337.0921(C16H17O8), 243.0655(C14H11O4), 193.0493 (C10H9O4) and 175.0387 (C10H7O3). Its MS2 spectrum gave characteristic ions at m/z 337.0921 ([feruoylglucose-H]−), m/z 193.0493 ([ferulic acid-H]−) and m/z 175.0387, therefore, compounds A45-1 ∼ A45-5 were identified as tetrahydroxystilbene-O-hexoside-O-feruloyl (glycosyl hydroxyl moiety).Compounds A46 gave a [M-H]− ion at m/z 591.2075 (C29H35O13) and the product ions at m/z 405.1203 (C20H21O9), 243.0655 (C14H11O4) and 185.0806 (C9H13O4). By comparing literature, compound A46 was tentatively identified as tetrahydroxystilbene-O- (hydroxynonanedioic acid acyl)-hexoside.Compound A47 gave a [M-H]− ion at m/z 613.1769 (C27H33O16) and the product ions at m/z 405.1203 (C20H21O9) and 243.0655 (C14H11O4). By comparing literatures, compound A47 was tentatively identified as tetrahydroxystilbene-O-(Glucoheptanoyl)-hexoside.Compound A48-1, A48-2 and A48-3 showed the same [M-H]− ion at m/z 719.1825 (C33H35O18) and the MS2 spectra gave identical ions at m/z 557.1287 (C27H25O13), 405.1174 (C20H21O9), 313.0557 (C13H13O9), 243.0655 (C14H11O4) and 169.0126 (C7H5O5), the loss of C6H10O5 to produce compound A39 ion at m/z 557.1287, and the fragmentation ions were the same. Therefore, A48-1, A48-2 and A48-3 were identified as tetrahydroxystilbene-O-dihexoside -galloyl (glycosyl hydroxyl moiety).Compounds A49-1 ∼ A49-6 displayed a high resolution [M-H]− ion at m/z 827.2399 and gave element composition of C40H43O19. The MS2 spectra gave ions at 405.1165 (C20H21O9), 259.0607 (C14H11O5) and 243.0656 (C14H11O4), forming the ions 405.1165 and 243.0656 indicated that they should be THSG derivatives. By comparing literature, compounds A49-1 ∼ A49-6 were tentatively identified as polygonumoside C/D.Compounds A50-1 ∼ A50-8 displayed a high resolution [M-H]− ion at m/z 837.2601 and gave element composition of C42H45O18. The MS2 spectra gave ions at m/z 675.2068 (C36H35O13), 513.1556 (C30H25O8), 431.1342 (C22H23O9), 405.1165 (C20H21O9), 269.0813 (C16H13O4) and 243.0656 (C14H11O4). The consecutive neutral loss of hexoside, forming ions at m/z 675.2068 and 513.1556, and forming the ions 405.1165, 243.0656 and 431.1342, 269.0813 indicated that was cleavage into two glycosides. By comparing literature, compounds A50-1 ∼ A50-8 were identified as polygonumnolide D , which was 16 Da (O) higher than that of A50, and the MS2 spectra gave ions at m/z 447.1300 (C22H23O10), 405.1175 (C20H21O9), 285.0765 (C16H13O5) and 243.0656 (C14H11O4).Compounds nolide D . SimilarA51-1 ∼ A51-5 showed the same [M-H]− ion at m/z 841.2551 (C41H45O19), which was 14 Da (CH2) higher than that of A49. The MS2 spectra gave ions at m/z 405.1182 (C20H21O9), 273.0764(C15H13O5) and 243.0655(C14H11O4), and the ion m/z 273.0764 was 14 Da (CH2) higher than that of A49 ion m/z 259.0607. therefore, compounds A51-1 ∼ A51-5 were identified as methylation polygonumoside C/D. Similarly, compounds A53-1 ∼ A53-4 were identified as hydroxylation methylation polygonumoside C/D. since the [M-H]− ions at m/z 857.2502 (C41H45O20), which was 14 Da (CH2) higher than that of A51, and the MS2 spectra gave ions at m/z 405.1172 (C20H21O9), 289.0709 (C15H13O6) and 243.0656 (C14H11O4).Compounds B1-1, B1-2 and B1-3 gave a [M-H]− ion at m/z 389.1240 (C20H21O8) and the product ion at m/z 227.0702 (C14H11O3). The loss of C6H10O5 was confirmed by MS2 spectra and indicated a hexose neutral loss. Compared with the control substance, B1-2 was identified as polydatin, compounds B1-1 and B1-3 were identified as isomer polydatin.Compounds B2-1 ∼ B2-4 showed the same [M-H]− ion at m/z 541.1345 (C27H25O12) and the MS2 spectra gave ions at m/z 313.0559 (C13H13O9), 227.0702 (C14H11O3), 169.0128 (C7H5O5). Similar with compounds A40, compounds B2-1 ∼ B2-4 were identified as trihydroxystilbene-O-hexoside-O-galloyl (glycosyl hydroxyl moiety).Compounds B3-1 and B3-2 showed the same [M-H]− ion at m/z 457.1136 (C23H21O10) and the MS2 spectra gave ions at m/z 295.0605 (C17H11O5) and 227.0702 (C14H11O3). By comparing literature, compounds B3-1 and B3-2 were identified as trihydroxystilbene-O-hexoside-O-acid deltique acyl (phenolic hydroxyl moiety).Compounds B4 gave a [M-H]− ion at m/z 535.1816 (C26H31O12) and the product ion at m/z 227.0702 (C14H11O3) derived from the loss of C6H10O5 (hexoside) and C6H10O4 . By investigating literatures, compound B4 was identified as trihydroxystilbene-(deoxyhexose)-O-hexoside.Compound B5 displayed a high resolution [M-H]− ion at m/z 359.1132 and gave element composition of C19H19O7, the product ions at m/z 359.1129 (C19H19O7) and 227.0701 (C14H11O3). The product ion at m/z 227.0701 originated from the loss of pentose (mostly arabinose). Therefore, compound B5 was identified as trihydroxystilbene-O-pentose.Compound C1-1 ∼ C1-7 displayed a high resolution [M-H]− ion at m/z 421.1138, and gave element composition of C20H21O10, which was 14 Da (CH2) higher than that of THSG, the MS2 spectra gave ion at 259.0609 (C14H11O5). By comparing literature, compounds C1-1 ∼ C1-7 were tentatively identified as pentahydroxystilbene glycosides.Compounds C2 gave a [M-H]− ion at m/z 545.1291 (C26H25O13) and the product ions at m/z 421.1128 (C20H21O10), 259 (C14H11O5) and 123.0070 (C6H3O3) derived from the loss of C6H5O5 (5-HMF) and C16H10O5 (hexoside). By investigating literatures, compound C2 was identified as pentahydroxystilbene-(5-HMF)-O-hexoside.Compound D1-1 and D1-2 gave a [M-H]− ion at m/z 403.1030 (C20H19O9) and prominent fragment ion at m/z 241.0497 (C14H9O4) in MS2 spectrum, which were showed 2 Da less than that of THSG. It can be inferred that they were dehydrogenated product of THSG. By comparing literatures  and the MS2 spectra gave ions at m/z 387.1072 (C20H19O8), 297.0760 (C17H13O5) and 241.0497 (C14H9O4). Similarly compounds A38, compounds D2-1 ∼ D2-8 were identified as tetrahydroxy-phenanthrene-O-hexoside-O-p-hydroxycinnamoyl (phenolic hydroxyl moiety).Compounds E1 gave a [M-H]− ion at m/z 407.1343 (C20H23O9) and prominent fragment ion at m/z 245.0811 (C14H13O4) in MS2 spectrum, which were showed 2 Da higher than that of THSG. It can be inferred that they were dihydrogenated product of THSG. By comparing literatures, compounds E1 was identified as dihydrotetrahydroxystilbene-O-hexoside.Compound E2-1 and E2-2 showed the same [M-H]− ion at m/z 527.1552 (C27H27O11) and the MS2 spectra gave ions at m/z 365.1017 (C21H17O6), 335.0918 (C20H15O5) and 245.0814 (C14H13O4). Similarly compounds A27, compounds E2-1 and E2-2 were identified as dihydrotetrahydroxystilbene-O-hexoside-salicylic acid acyl (phenolic hydroxyl moiety).Compounds E3 displayed a high resolution [M-H]− ion at m/z 539.1766, and gave element composition of C25H31O13. The MS2 spectra gave ion at 245.0811 (C14H13O4) derived from the loss of C6H10O5 (hexoside) and C5H8O4 . Compound E3 was identified as dihydrotetrahydroxystilbene-O-(pentose)-hexoside.Compound F1-1 and F1-2 showed the same [M-H]− ion at m/z 419.0980 (C20H19O10) and the MS2 spectra gave ion at m/z 257.0542 (C14H9O5), which were showed 16 Da higher than that of compounds D1. Therefore, compounds F1-1 and F1-2 were identified as pentahydroxy-phenanthrene-O-hexosides.Compounds G1 gave a [M-H]− ion at m/z 373.1286 (C20H21O7) and prominent fragment ion at m/z 211.0751 (C14H13O2) in MS2 spectrum, which were showed 32 Da less than that of THSG. It can be inferred that they were dedihydroxylation product of THSG. Therefore, compounds G1 was identified as dihydroxystilbene-O-hexoside.Compound G2-1 and G2-2 showed the same [M-H]− ion at m/z 525.1396 (C27H25O11) and the fragment ions at m/z 525.1392, 313.0558 (C13H13O9), 211.0751 (C14H13O2), 169.0128 (C7H5O5) and 151.0020 (C7H3O4) in MS2 spectra. Similar with compounds A39 and compounds B2, compounds G2-1 and G2-2 were identified as dihydroxystilbene-O-hexoside-O-galloyl (glycosyl hydroxyl moiety).Compounds A3) (B1-2) was glycosylated form resveratrol. Pentahydroxystilben-O-hexoside (compounds C1) was synthesized by rehydroxylation of tetrahydroxystilbene and then glycosylation. The phenanthrene moiety was formed by cyclization of 6-H and 2′-H positions of tetrahydroxystilbene, which can stabilize the cyclization of aglycone, and then glycosylated to form tetrahydroxy-phenanthrene-O-hexoside (compounds D1). The tetrahydroxy-phenanthrene was hydroxylated to form pentahydroxy-phenanthrene, then glycosylated to form pentahydroxy-phenanthrene-O-hexoside (compounds F1). Dihydrotetrahydroxystilbene-O-hexoside (compounds E1) was synthesized from tetrahydroxystilbene by double bond opening and then glycosylated. The resveratrol lost a hydroxyl group in the plant, and dihydroxystilbene-O-hexoside (G1) was obtained by the glycosylation of dihydroxystilbene  . Polydatstilbene .p-hydroxybenzoic acid. Maillard reaction products, mainly includes 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4H-pyranone  and 5-hydromethylfurfura  and its derivatives from glucose or glycine were also observed as stilbene glycosides substituents (A30) was produced, then methylation and oxidation produced methoxymethyl-furancarboxylic acid  and furan-dicarboxylic acid . 2, 5-bis-(hydroxymethyl) furan  was synthesized by the reduction of 5-HMF. 5-formylfuran-2-carboxyl acid  was synthesized by the oxidation of the hydroxyl end of 5-HMF. Then decarboxylation and reoxidation were carried out to form 5-hydroxyfuran-2-carbaldehyde , then, 5-hydroxyfuran-2-carboxylic acid  was formed by oxidation of aldehyde end. 4-hydroxymethyl-5h-furan-2-one  was synthesized from 2, 4-dihydroxy-5- (hydroxymethyl) oxolane-2-carbaldehyde by dehydration and CH2O removal, then hydrogenation reaction was reacted to produce 5-hydroxymethyl-4,5-dihydrofuranone , and the change path of 5-HMF is shown in Compounds containing malonyl, acetyl, caffeoyl and other aromatic acyls are very common in various plants . And galtituents . The aldThe peak areas of all compounds were based on the extracted ion chromatographic peaks. The mean and SD values were calculated and the column diagram of each compound was drawn . A totalA3-4 (isomer trans-THSG), gradually increase during the processing time. The third model, such as compound A45-1 (tetrahydroxystilbene-O-hexoside-feruloyl (glycosyl hydroxyl moiety), gradually decreased with processing time.The change trend of the highest content compound trans-THSG and its cis-THSG slightly increased before 8 h, then decreased gradually, and was lower than that of RRM at 24 h. This is a model of content change, but the peak time may be 4, 8, 12 or 18 h. The second model, such as compound in vivo, enhancing the efficacy of THSG.The results showed that the content and quantity of stilbene glycoside compounds have undergone tremendous changes during the processing process. Although the content of THSG in PRM is indeed lower than that in RRM, a large number of stilbene glycoside derivatives are produced in the processing process, so the total content of stilbene glycoside compounds in the PRM will not be reduced. Conventional understanding, RRM after processing can enhance efficiency and reduce toxicity, and the content of THSG also decreases with the processing time, is THSG toxic? After this experimental study, it can be proved that THSG should not be a toxic component, because its derivatives will metabolize into compounds similar to THSG m/z 405.1087 and 243.0656, accurate mass measurements and fragmentation pathways. Based on the proposed strategy, the metabolic process of 7 stilbene glycosides in plants was identified, and 55 substituent and Maillard reaction process were identified. Finally, 219 stilbene glycosides derivatives were identified, of which 102 compounds may be potential new compounds. The 55 substituents include monosaccharide, disaccharide, organic acid and Maillard reaction products  and so on. The quality and quantity of stilbene glycosides changed during the processing of RM. 73 compounds were not found in RRM, 21 compounds were not found in 4 h PRM, 9 compounds were not found in 8 h PRM and 1 compound was not found in 12 h PRM, and the change trend of the compounds can be summarized into 3 models: gradually increased, gradually decreased, first increased and then decreased. 181 trans-THSG derivative products were obtained through the hydrolysis and dehydration reaction between trans-THSG and small molecules compounds, after this experimental study, it can be proved that THSG should not be a toxic component, because its derivatives will metabolize into compounds similar to THSG in vivo, enhancing the efficacy of THSG.In the present study, a simple and effective method was developed for characterization of stilbene compounds in the roots of RRM and PRMs by UHPLC-Q-Exactive plus orbitrap MS/MS. Stilbene glycosides were distinguished by diagnostic fragment ions at


Callicarpa nudiflora. The structures of the new compounds were established by 1D-, 2D-NMR, and HR-ESI-MS spectral analyses. Compounds 1–3 showed inhibitory activities on lipopolysaccharide-induced nitric oxide (NO) production in RAW264.7 cells, and new compounds 1–2 exhibited more potent inhibitory activity than compound 3. The cytotoxicity of compounds 1–3 against human hepatocellular carcinoma HepG2 cells and human gastric carcinoma SGC-7901 cells were evaluated, while all of them exhibited no cytotoxicity.Two new seco-labdane diterpenoids, nudiflopene N (1) and nudiflopene O (2), and four known compounds were isolated from the leaves of  Callicarpa comprises about 190 species and is widely dispersed across tropical and subtropical Asia and Oceania. Callicarpa nudiflora Hook. & Arn. (Verbenaceae) is a shrub or small tree mainly found in Guangdong, Guangxi, Hainan, China, with the medicinal materials in Wuzhishan, Hainan Province representing the best specimens + at 345.2058  in the HR–ESI–MS. This molecular was consistent with the 1H and 13C NMR data , four aliphatic methylenes at δ 2.44 , 2.36 , 2.27 , 2.16 , 1.51 , 1.50 , 1.40 , 1.24 , six olefinic protons at δ 6.20 , 5.64 , 4.88 , 4.81 , 4.76 , 4.53 , and one methoxy singlet at δ 3.50 . The 13C NMR spectrum (see δ 51.7 (C-21), two carbonyl carbon at δ 173.9 (C-3), 169.1 (C-15), and eight olefinic carbons at δ 156.2 (C-13), 151.3 (C-12), 148.1 (C-8), 146.8 (C-4), 116.3 (C-14), 114.4 (C-18), 110.6 (C-11) and 109.6 (C-17). With the aid of DEPT and HSQC spectra, the remaining ten aliphatic carbons were classified into three methyls at δ 23.8 (C-19), 17.1 (C-20), and 11.9 (C-16), four methylenes at δ 23.8 (C-19), 17.1 (C-20), and 11.9 (C-16), two methines at δ 49.4 (C-5), 47.2 (C-9), and one quaternary carbon at δ 41.6 (C-10). According to these spectroscopic data, compound 1 was inferred to be a diterpenoid carrying a methoxy group. The diterpenoid scaffold was elucidated by 1H-1H COSY and HMBC spectra  and an isopropenyl group attached at C-5. The residual moiety was extrapolated to form a five-membered unsaturated lactone ring with a methyl group attached at C-13, which was confirmed by the HMBC couplings of H3-16 to C-12, C-13, and C-14, and H-14 to C-12, C-13, C-15, and C-16. This five-membered unsaturated lactone ring was linked to C-9 of the six-membered ring via the olefinic carbon C-11 (δ 110.6), confirmed by the HMBC correlations of H-11 to C-8, C-9, C-10, C-12, and C-13. All of the above analysis permitted the planar structure of 1 to be elucidated. The configuration of compound 1 was elucidated based on the NOESY spectrum and compared the optical rotation and electronic circular dichroism (ECD) spectrum  in the HR–ESI–MS spectrum. This molecular was consistent with the 1H and 13C NMR data . To prove that compounds 1 and 2 are not artifacts, we extracted the medicinal materials again with ethyl acetate and analyzed its constituents with HPLC-DAD. The results showed that compounds 1 and 2 can be found in the HPLC chromatogram of the ethyl acetate extract based on the retention time and UV spectrum , 1H-1H Correlated Spectroscopy (1H-1H COSY), Heteronuclear Multiple Quantum Correlation (HMQC), and Heteronuclear Multiple Bond Correlation (HMBC) experiments were performed on a Bruker Avance Neo 400 MHz NMR spectrometer ; TMS was used as international standard. Mass spectra were obtained on a Bruker micro TOF mass spectrometer (ESI-MS) . High-performance liquid chromatography (HPLC) was performed using an Agilent 1260 Series HPLC system  equipped with a four-pump with an in-line degasser, autosampler, oven, and Diode-array detector (DAD). The semi-preparative HPLC was performed using a YMC ODS-A chromatographic column .Optical rotations were recorded on a Bellingham-Stanley ADP 440+ polarimeter . ECD spectra were obtained on a JASCO J-715 CD spectrometer . Callicarpa nudiflora were collected from Wuzhishan of Hainan Province, China in October 2015 and identified by Prof. Xi-Feng Sheng . A voucher specimen (No. LHZZ-2015) has been deposited in the Department of Pharmacy, School of Medicine, Hunan Normal University.The air-dried leaves of C. nudiflora (8.5 kg) were extracted twice with EtOH-H2O , and the concentrated liquid was dispersed in water and using petroleum ether, ethyl acetate, and n-butanol to extract twice to obtain three portions of petroleum ether (170 g), ethyl acetate (258 g), and n-butanol. The ethyl acetate portion (230 g) was subjected on a silica gel column with a gradient elution of CH2Cl2-MeOH (100:0-0:100) to obtain 9 fractions (A–I). Based on TLC analysis, the fractions B-D were combined and subjected on a silica gel column chromatography using CH2Cl2-MeOH (100:0-0:100) as a gradient elution to obtain 200 fractions. Fr. 47–51 was purified by semi-preparative HPLC  with ACN–H2O  to obtain compound 6 . Fr. 20–40 were combined and chromatographed on an ODS column chromatography to obtain 200 fractions by a gradient elution of MeOH-H2O (40:60–100:0). Fr. 27–31 was purified by semi-preparative HPLC  with ACN–H2O  to obtain compound 5 . Fr. 79–120 was purified by semi-preparative HPLC  with ACN–H2O  to obtain compound 1 . The petroleum ether portion (161 g) was subjected on a silica gel column with a gradient elution of PE-EA (10:0.6-0:1) to obtain 226 fractions. Fr. 47–51 was purified by semi-preparative HPLC  with ACN–H2O  to obtain compound 2 . Fr. 111–112 was purified by semi-preparative HPLC  with ACN–H2O  to obtain compound 4 . Fr. 120–123 was purified by semi-preparative HPLC  with ACN–H2O  to obtain compound 3 .The air-dried leaves of 1): Colorless oil. [α]ε-2.93) nm; UV (MeOH) λmax: 274 nm; 1H-NMR and 13C-NMR (DMSO-d6) see 21H29O4 [M + H]+ 345.2066; found 345.2058.Nudiflopene N (2): Colorless oil. [α]ε -2.17) nm; UV (MeOH) λmax: 274 nm. 1H-NMR and 13C-NMR (DMSO-d6) see 22H31O4 [M + H]+ 359.2222; found 359.2224.Nudiflopene O (3): Colorless oil. [α]1H NMR : δH 6.18 , 5.64 , 4.87 , 4.79 , 4.73 , 4.50 , 3.21 , 2.37 , 2.37 , 2.26 , 2.23 , 2.14 , 2.06 , 1.74 , 1.73 , 1.50 , 1.47 , 1.32 , 0.87 . 13C NMR : δC 169.2 (C-3), 169.1 (C-15), 156.1 (C-13), 151.2 (C-12), 148.4 (C-8), 147.0 (C-4), 116.3 (C-14), 114.1 (C-18), 111.0 (C-11), 109.4 (C-17), 49.3 (C-5), 47.5 (C-9), 41.4 (C-10), 35.9 (C-7), 34.9 (C-1), 29.2 ,24.4 (C-19), 17.1 (C-20), 12.0 (C-16).Nudiflopene H (4): White amorphous powder. UV λmax: 196, 226 nm. 1H NMR : δH 6.90 , 6.12 , 4.88 , 4.80 , 4.72 , 4.42 , 2.84 , 2.31 , 2.26 , 2.15 , 2.08 , 1.73 , 1.49 , 1.27 , 0.86 . 13C NMR : δC 198.5 (C-13), 175.9 (C-3), 148.9 (C-8), 146.3 , 133.9 (C-12), 114.2 (C-16), 109.5 (C-15), 52.9 (C-9), 49.1 (C-5), 40.9 (C-10), 35.9 (C-2), 34.3 (C-1), 29.1 (C-6), 28.3 (C-7), 27.3 (C-14), 24.3 (C-17), 17.4 (C-18).Latisilinoid (5): Yellow amorphous powder. UV λmax: 240 nm. 1H NMR : δH 7.02 , 6.45 , 5.96 , 2.63 , 2.27 , 2.33 , 1.92 , 1.08 , 1.04 . 13C NMR : δC 199.4 (C-9), 199.0 (C-3), 163.2 (C-5), 146.9 (C-7), 130.4 (C-8), 126.6 (C-4), 78.5 (C-6), 49.1 (C-2), 41.2 (C-1), 26.2 (C-10), 23.3 (C-12), 22.1 (C-11), 17.7 (C-13).(+)-dehydrovomifoliol (6): Yellow amorphous powder. UV λmax: 196, 242 nm. 1H NMR : δH 5.79 , 5.69 , 5.67 , 4.19 , 2.37 , 2.06 , 1.81 , 1.12 , 0.93 , 0.92 . 13C NMR : δC 197.8 (C-3), 164.8 (C-5), 136.3 (C-7), 128.3 (C-8), 125.9 (C-4), 78.2 (C-6), 66.5 (C-9), 49.8 (C-2), 41.3 (C-1), 24.5 (C-12), 24.4 (C-10), 23.5 (C-11), 19.4 (C-13).Vomifoliol  and allowed to adhere for 24 h at 37 °C. At the same time, a blank control group and a drug group were set up and cultured 2 h in a cell temperature incubator. 10 μg/mL of LPS  per well were added to induce inflammation, and the culture was continued in the incubator for 24 h. As a parameter of NO synthesis, the nitrite concentration was measured by the Griess reaction using the supernatant of the RAW 264.7 cells. 50 μL of cell supernatant per well, then Griess reagent I and Griess reagent II, respectively, were added. The absorbance was read with a microplate reader  at 550 nm. The experiment was performed three times. SPSS 16.0 and GraphPad Prism 6.01 software were used for statistical analysis.NO is an important inflammatory factor, and the NO inhibitory effects of compounds 2, respectively. The cells of the logarithmic growth phase were seeded into 96-well plates with a density of 4000 cells/well in 200 µL medium, respectively. The cells were treated with the tested compounds at various concentrations , with sorafenib and cisplatin as positive control. Each of three parallel holes were located, then incubated for 24 h. Subsequently, the 96-well plate was taken out, and 10 μL of CCK-8 was added to the experimental and control wells; at the same time, two other individual wells were taken as blank controls, and only 10 μL of CCK-8 in 0.1 mL of DMEM was added to each well. Then incubation under the same conditions was conducted for 4 h. The optical density (OD) was measured at 450 nm using a Bio-Tek Synergy . The experiment was repeated 3 times. Finally, the impact of drugs on cell growth inhibition rate and IC50 values was calculated.The cytotoxicity assay was carried out using the CCK-8 method. HepG2 and SGC-7901 cells were cultured in a cell temperature incubator and a DMEM at 37 °C in 5% COCallicarpa nudiflora Hook. & Arn. The structures of the new compounds were elucidated by spectroscopic analysis. Compounds 1–3 from this plant were evaluated for their NO inhibitory activity and cytotoxicity against human hepatocellular carcinoma HepG2 cells and human gastric carcinoma SGC-7901 cells. All of them showed good inhibitory activity on the LPS-induced NO production in RAW 264.7 cells, and compounds 1–2 showed more potent activity than compound 3.In this study, two new seco-labdane diterpenoids, nudiflopene N (1) and nudiflopene O (2), along with four known compounds, were isolated from the leaves of


Burkholderia cenocepacia that binds  fucosides at the N-terminal domain and  mannosides at the C-terminal domain. This double carbohydrate specificity allows the lectin to crosslink host cells and bacterial cells. We have recently reported the design and generation of the first glycomimetic antagonists of BC2L-C, β-C- or β-N-fucosides that target the fucose-specific N-terminal domain (BC2L-C-Nt). The low water solubility of the designed N-fucosides prevented a full examination of this promising series of ligands. In this work, we describe the synthesis and biophysical evaluation of new L-fucosyl and L-galactosyl amides, designed to be water soluble and to interact with BC2L-C-Nt. The protein–ligand interaction was investigated by Saturation Transfer Difference NMR, Isothermal Titration Calorimetry and crystallographic studies. STD-NMR experiments showed that both fucosyl and galactosyl amides compete with α-methyl fucoside for lectin binding. A new hit compound was identified with good water solubility and an affinity for BC2L-C-Nt of 159 μM (ITC), which represents a one order of magnitude gain over α-methyl fucoside. The x-ray structure of its complex with BC2L-C-Nt was solved at 1.55 Å resolution.The inhibition of carbohydrate-lectin interactions is being explored as an efficient approach to anti adhesion therapy and biofilm destabilization, two alternative antimicrobial strategies that are being explored against resistant pathogens. BC2L-C is a new type of lectin from  Lectins are carbohydrate-binding proteins, widely distributed in Nature. They read out the signals associated with glycans and thus mediate numerous cellular recognition processes. In particular, microorganisms often use lectins to bind specific carbohydrates expressed on the outer membrane of host cells in a process that leads to cell adhesion . AdhesioBurkholderia cenocepacia BC2L-C lectin, a candidate target for AAT against this pathogen, which is associated with fatal pulmonary infections of cystic fibrosis patients + m/z: 354.13; found: 354.21. 1H NMR  δ 7.69–7.64 , 7.38 , 7.30–7.21 , 6.74 , 4.90 , 4.38–4.33 , 3.91 , 1.47 . 13C chemical shifts extrapolated from HSQC exp.: δ 129.2 (Ph-C-4), 128.3 (Ph-C-5), 124.1 , 120.4 (Fur-C-3), 107.1 (Fur-C-4), 51.7 (-OCH3), 28.4 (tBu).To a solution of methyl 5-bromofuran-2-carboxylate tert-butoxycarbonyl)aminomethyl)phenyl)furan-2-carboxylate 11  in a ternary mixture of tetrahydrofuran/methanol/water , LiOH.H2O was added and the reaction mixture was stirred for 2 h at room temperature. The organic solvents were removed under reduced pressure. The aqueous phase was diluted with water (2 mL) and washed with ethyl acetate (4 mL), then it was acidified with 1M aqueous solution of HCl until pH 2. The acid aqueous layer was extracted with ethyl acetate (2 × 5 mL). The organic phase was washed with brine (4 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure, to give the desired product as a white solid. Y = 77%. Rf (n-Hex/AcOEt 1:1 + 0.1% formic acid) = 0.46. MS (ESI) calcd for C17H18NO5 [M + Na]+ m/z: 340.12; found: 340.20. 1H NMR  δ 7.73–7.67 , 7.42–7.34 , 7.30 , 6.78 , 4.96 , 4.39–4.32 , 1.48 . 13C chemical shifts extrapolated from HSQC exp.: 128.6 , 123.8 , 121.3 (Fur-C-3), 106.8 (Fur-C-4), 44.2 (-CH2), 27.8 (tBu). To a stirred solution of methyl 5-(3-((tert-butoxycarbonyl)aminomethyl)phenyl)furan-2-carboxylic acid 12  was suspended in anhydrous dichloromethane  and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide  was added, together with 1-hydroxy-7-azabenzotriazole  and N,N-diisopropylethylamine . The resulting mixture was stirred at room temperature for 1 h under N2.  azide 5  or 2,3,4,6-tetra-O-acetyl-β-L-galactopyranosyl azide 6 were dissolved in anhydrous dichloromethane  and cooled to 0 °C under N2. Trimethylphosphine  was slowly added and the reaction mixture was stirred at room temperature for 20 min. The activated acid mixture was combined to the reduced azide one and the whole reaction was stirred overnight at room temperature. It was diluted with dichloromethane (2 mL) and washed with sat. solution of NH4Cl (2 × 3 mL), sat. solution of NaHCO3 (3 mL) and brine (3 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to dryness. The product was purified by automatic chromatography, using n-Hex/AcOEt as eluent. 5-(3-((n-Hex/AcOEt 6:4) to give the desired product as yellow oil. Y = 53%. Rf (CH2Cl2/MeOH 98:2) = 0.41. MS (ESI) calcd for C29H36N2O11 [M + Na]+ m/z: 611.22; found: 611.39.1H NMR  δ 7.64 , 7.59 , 7.38–7.26 , 7.23 , 7.16 , 6.67 , 5.33–5.18 , 5.14 , 4.98 , 4.32 , 3.96 , 2.14 , 1.98 , 1.95 , 1.40 , 1.16 . 13C chemical shifts extrapolated from HSQC exp.: 129.4 (Ph-C-5), 128.2 (Ph-C-4), 123.7 , 117.6 (Fur-C-3), 107.4 (Fur-C-4), 78.7 (C-1), 70.6 , 68.5 (C-4), 28.7 (tBu), 20.8 (OAc), 16.3 (C-6). The product was purified by automatic chromatography  to give the desired product as yellow oil. Y = 40%. Rf (CH2Cl2/MeOH 98:2) = 0.65. MS (ESI) calcd for C31H38N2O13 [M + Na]+ m/z: 669.23; found: 611.40.1H NMR  δ 7.71 , 7.65 , 7.45–7.32 , 7.29 , 7.23 , 6.74 , 5.49 , 5.48–5.32 , 5.30–5.27 , 5.21 , 5.04 , 4.40–4.36 , 4.18–4.07 , 2.17 , 2.08–1.97 , 1.46 . 13C chemical shifts extrapolated from HSQC exp.: 129.5 (Ph-C-5), 128.3 (Ph-C-4), 123.8 , 117.7 (Fur-C-3), 107.7 (Fur-C-4), 78.7(C-1), 72.6 (C-5), 70.8 (C-3), 68.3 (C-2), 67.2 (C-4), 28.4 (tBu), 20.9 (OAc).The product was purified by automatic chromatography  to give the desired product as yellow oil. Y = 95%. Rf (n-Hex/AcOEt 4:6) = 0.43. Characterization data in agreement with those reported in literature [17H21NO9 [M + Na]+ m/z: 406.11; found: 406.66. 1H NMR  1H NMR  δ 7.42 , 7.10–7.05 , 6.44 , 5.32–5.22 , 5.21–5.07 , 3.93 , 2.12 , 1.95 , 1.14 . 13C chemical shifts extrapolated from HSQC exp.: δ = 144.9 (Ar-C-5), 115.8 (Ar-C-3), 112.2 (Ar-C-4), 78.2 (C-1), 71.4 (C-3), 70.4 , 68.1 (H-2), 20.6 (OAc), 16.2 (C-6). The product was purified by automatic chromatography  were dissolved in anhydrous methanol  under N2, and a 0.1 M solution of sodium methoxide in anhydrous methanol (0.05 mL) was added . The resulting mixture was stirred at room temperature until full conversion (~2 h). The reaction was quenched by adding Amberlite IRA 120 H+ until pH 7, then the resin was filtered out and the solvent was removed under vacuum to give the desired product; this was used without further purification for Boc removal.Here, 2, and trifluoroacetic acid (1.2 mL) was added. The reaction mixture was stirred at room temperature until TLC showed the full conversion (1 h). The solvent was removed under reduced pressure and co-evaporated with methanol. The final compound was purified by HPLC using acetonitrile/water (+ 0.1% formic acid) as eluent. The deacetylation crude  was dissolved in anhydrous dichloromethane (11 mL) under N13 was subjected to standard deacetylation and the crude was submitted to the following step. (5-(3-((tert-butoxycarbonyl)aminomethyl)phenyl)furan-2-carboxamido)-β-L-fucopyranose: Colourless oil. Y = 98%. Rf (CH2Cl2/MeOH 93:7) = 0.22. MS (ESI) calcd for C23H30N2O8 [M + Na]+ m/z: 485.19; found: 485.40.1H NMR  δ 7.80–7.75 , 7.41 , 7.33–7.25 , 6.93 , 5.08 , 4.29 , 3.82–3.72 , 3.69 , 3.59 , 1.47 , 1.27 . 13C chemical shifts extrapolated from HSQC exp.: 129.1 (Ph-C-5), 127.4 (Ph-C-4), 123.4 , 117.2 (Fur-C-3), 107.1 (Fur-C-4), 80.4 (C-1), 74.9 (C-3), 72.0 , 69.8 (C-2), 27.6 (tBu), 15.7 (C-6).An analytical sample was used for the characterization of the intermediate 3 as a white foam. Y = 95%. Rf (CH2Cl2/MeOH 8:2) = 0.24. The product was further purified by semi-preparative RP-HPLC (H2O + 0.1% formic acid/CH3CN) with gradient: 0–2 min: 0%, 2–17 min: 0–20%. tr = 13.06 min. [α]D18 = 5.5 . MS (ESI) calcd for C18H22N2O6 [M + H]+ m/z: 363.16; found: 363.27. HRMS (ESI) calculated for C18H22N2O6 [M + Na]+. m/z: 385.1376; found 385.1376.1H NMR  δ 7.95 , 7.90 , 7.51 , 7.44 , 7.28 , 6.96 , 5.09 , 4.18 , 3.83–3.75 , 3.70 , 3.61 , 1.26 . 13C NMR  δ 159.7 (-CONH), 155.6 (Fur quat), 146.4 (Fur quat), 133.9 (Ar quat), 130.4 (Ar quat), 129.8 (Ph-C-5), 128.8 (Ph-C-4), 124.9 (Ph-C-2), 124.8 (Ph-C-6), 117.0 (Fur-C-3), 107.6 (Fur-C-4), 80.1 (C-1), 74.7 (C-3), 72.8 (C-2), 72.0 (C-4), 69. (C-5), 42.8 (-CH2), 15.6 (C-6). [α]D17.6 = 5.5 (MeOH)Boc deprotection of the deacetylation crude afforded 14 was subjected to standard deacetylation and the crude was submitted to the following step. (5-(3-((tert-butoxycarbonyl)aminomethyl)phenyl)furan-2-carboxamido)-β-L-galactopyranose: Colourless oil. Y = 85%. Rf (CH2Cl2/MeOH 93:7) = 0.22. MS (ESI) calcd for C23H30N2O9 [M + Na]+ m/z: 501.18; found: 501.31.1H NMR  δ 7.79–7.74 , 7.40 , 7.30–7.27 , 6.92 , 5.10 , 4.27 , 3.93 , 3.84–3.63 , 3.59 , 1.45 . 13C chemical shifts extrapolated from HSQC exp.: 132.8 (Ph-C-5), 131.5 (Ph-C-4), 127.4 , 121.2 (Fur-C-3), 110.9 (Fur-C-4), 84.4 (C-1), 81.2 (C-2), 78.5 (C-3), 73.3 , 31.7 (tBu).An analytical sample was used for the characterization of the intermediate 4 as a white foam. Y = quant.. Rf (CH2Cl2/MeOH 7:3) = 0.20. The product was further purified by semi-preparative RP-HPLC (H2O + 0.1% FA/CH3CN) with gradient: 0–2 min: 0%, 2–12 min: 0–10%. tr = 12.12 min. [α]D18 = 7.3 . MS (ESI) calcd for C18H22N2O7 [M + H]+ m/z: 379.15; found: 379.50. HRMS (ESI) calculated for C18H22N2O7 [M + H]+. m/z: 379.1505; found 379.1507. 1H NMR  δ 8.56 , 8.01 , 7.95 , 7.55 , 7.47 , 7.34 , 7.02 , 5.14 , 4.20 , 3.97 , 3.85–3.68 , 3.62 . 13C NMR  δ 159.8 (-CONH), 155.6 (Fur quat), 146.4 (Fur quat), 133.9 (Ar quat), 130.4 (Ar quat), 129.5 (Ph-C-5), 129.0 (Ph-C-4), 124.9 (Ph-C-2), 124.8 (Ph-C-6), 117.0 (Fur-C-3), 107.6 (Fur-C-4), 80.3 (C-1), 77.1 (C-2), 74.6 (C-3), 69.8 (C-5), 69.4 (C-4), 61.3 , 42.8 (-CH2). Boc deprotection of the deacetylation crude afforded 16 was subjected to standard deacetylation to afford 17 as a white foam. Y = 90%. Rf (nHex:EtOAc=95:5) 0.1. Characterization data in agreement with those reported in the literature [11H15NO6 [M + Na]+ m/z: 280.08; found: 280.35. 1H NMR  δ 7.59 , 7.11 , 6.50 , 4.94 , 3.67 , 3.63–3.56 , 3.49 , 1.16 . 13C chemical shifts extrapolated from HSQC exp.: δ = 145.2 (C-5), 114.7 (C-3), 111.5 (C-4), 80.0 (C-1), 74.5 (C-3), 72.6 (C-5), 71.8 (C-4), 69.6 (C-2), 15.4 (C-6). terature . MS  were calculated by division through integrals over the respective signals in STD reference spectra. The different signal intensities of the individual protons are best analyzed from the integral values in the reference and STD spectra, respectively.0 is the intensity of one signal in the off-resonance or reference NMR spectrum, Isat is the intensity of a signal in the on-resonance NMR spectrum, and I0 − Isat represents the intensity of the STD spectrum.In order to map the epitope of the analyzed molecules to BC2L-C-Nt, the relative STD percentages were also calculated by comparing the STD intensities to the strongest signal, which was given an arbitrary value of 100%. The relative STD percentages are shown on the group epitope map using the following color code: purple circles are used to highlight the strongest signal (100% relative STD), green circles are for protons between 40 and 80%, and light blue circles are for signals under 40% relative to the strongest STD signal. 3 and 4), with intervals of 100 s while stirring at 850 rpm. For compound 17, the protein concentration was between 139 and 187 µM, and the ligand concentration was 50 mM. The supplied software MicroCal PEAQ-ITC  was used to fit the experimental data to a theoretical titration curve using one set of site models; the stoichiometry was fixed to 1 as all measurements were performed in an excess of ligand. We were able to determine the affinity  and the binding enthalpy (ΔH). The quality of the protein batches was controlled by titrating with 3mM H-type 1 tetrasaccharide . A KD of 56 ± 4.5 µM was obtained and was equivalent to that described previously. We estimated from the stoichiometry that 80% of the protein was actively binding and, therefore, protein concentration was corrected accordingly. . The protein and the different ligands were dissolved in a buffer composed of 20 mM Tris HCl pH 7.0 and 100 mM NaCl. The 200 μL sample cell containing the protein at a concentration ranging from 119 to 127 μM was subjected to 39 injections of 1 μL of 30 mM ligand solution  and flash cooled in liquid nitrogen. Data were collected on Proxima 1 beamline at the synchrotron SOLEIL, Saint Aubin, France using an Eiger 16M detector (see statistics in 3 was solved by molecular replacement using the protein coordinates of PDB-ID 6TID as search model in PHASER [3. The coordinates were deposited in the Protein Data Bank (PDB) under code 8BRO.Cocrystals of BC2L-C-Nt in complex with H-type 1 trisaccharide were obtained as described previously . Then, 1n PHASER . Iteraten PHASER ,26. The B. cenocepacia lectin BC2L-C-Nt. In order to obtain these data, we had to overcome the low water solubility of initial hits, such as 2, that were found to interact in the targeted binding site by X-ray crystallography, but whose affinity could not be properly assessed. This is a common issue for glycomimetics, which are often designed by exploiting the lipophilic interaction potential of aromatic substructures, together with the lectin-targeting ability of specific monosaccharides. Thus, the approach we followed here can be of general interest for the design of viable glycomimetic ligands. Water solubility was pursued by a) reducing the “flatness” of the structure by turning an aniline into a benzylamine moiety, and b) replacing L-fucose with L-galactose. While the latter move reduced the affinity of the ligand by a factor of 2.5, as measured by ITC, it still produced a ligand, compound 4, which stands among the most active glycomimetics described so far against BC2L-C-Nt. STD-NMR data showed that 4 competes for binding with α-methyl fucoside, and thus suggests that the lectin can tolerate a hydroxyl group in the binding region used by fucose methyl group. This is a useful indication because, beside increasing solubility, the additional hydroxyl group of L-galactose can provide a handle for the conjugation of the ligand, for instance, to polyvalent scaffolds. The L-fucosylamide 3, with a KD 159 µM, is one of the most potent glycomimetic ligands of BC2L-C-Nt described so far, and presents a great potential for structure activity relationship (SAR) studies, given the optimal synthetic accessibility of the chemotype. All the available data indicate that, as previously observed in the β-fucosylalkyne series, represented by 1, lipophilic interactions of the aromatic fragment within the targeted crevice at the monomer interface of the lectin are paramount in establishing the ligand affinity. The terminal amino group helps by interacting with the side chain of Asp70, at the opposite end of the crevice from the sugar. This interaction occurs near the surface of the protein and, as shown by the available X-ray structures, it cannot be described as the charge/charge interaction predicted by docking calculations, but rather as a water mediated contact, that can extend to other elements of the protein surface. All together, these studies are amassing a series of information and SAR that will be instrumental for the design of high affinity ligands to be tested in the disruption of B. cenocepacia biofilm.The results discussed above represent the first quantitative evaluation of the β-fucosylamide chemotype as a ligand of the


Baccharis genus Asteraceae is widely used in traditional treatment against fever, headache, hepatobiliary disorders, skin ulcers, diabetes, and rheumatism, as well as an antispasmodic and diuretic. Its phytochemistry mainly shows the presence of flavonoids and terpenoids such as monoterpenes, sesquiterpenes, diterpenes, and triterpenes. Some of them have been evaluated for biological activities presenting allelopathic, antimicrobial, cytotoxic, and anti-inflammatory properties. In this paper, our research group reported the isolation, characterization, and antifungal evaluation of several molecules isolated from the dichloromethane extract from Baccharis prunifolia, Baccharis trinervis, and Baccharis zumbadorensis against the phytopathogen fungus Botrytis cinerea. The isolated compounds have not previously been tested against Botrytis, revealing an important source of antifungals in the genus Baccharis. Six known flavones were isolated from B. prunifolia. The dichloromethane extracts of B. trinervis and B. zumbadorensis were subjected to a bio-guided isolation, obtaining three known flavones, an α-hydroxidihydrochalcone mixture, one labdane, one triterpene, and two norbisabolenes from the most active fractions. The compounds 4′-methoxy-α-hydroxydihydrochalcone (7A), 3β,15-dihydroxylabdan-7-en-17-al (8), and 13-nor-11,12-dihydroxybisabol-2-enone (11) are novel. The most active compounds were the Salvigenin (5) and 1,2-dihydrosenedigital-2-one (10) with an IC50 of 13.5 and 3.1 μg/mL, respectively. Asteraceae constitutes a defined family among flowering plants distributed primarily in the tropical areas of South America − , exact mass calcd for C16H16O6, 304.0947. 1H-NMR ; δ 2.81 , 3.11 , 3.84 , 5.37 , 5.98 , 5.99 , 6.96 , 6.96 , 7.38 , 7.38 , 12.05 .13C-NMR ; δ 43.14 (CH2), 55.37 (OCH3), 79.02 (CH), 95.32 (CH), 96.65 (CH), 103.26 (C), 114.23 (CH), 114.23 (CH), 127.73 (CH), 127.73 (CH), 130.27 (C), 160.07 (C), 163.25 (C), 164.13 (C), 164.36 (C), 196.01 (CO). R,6S,8aS)-6-Hydroxy-1-(5-hydroxy-3-methylpentyl)-5,5,8a-trimethyl-1,4,4a,5,6,7,8,8a-octahydronaphthalen-2-yl)ethan-1-one or 3β,15-Dihydroxylabdan-7-en-17-al (8). (+)-HRESIMS m/z 323.2559 [M + H]+ , 2930 (=C-H), 2868 (C-H), 1682 (C=O) cm−1. 1H-NMR ; δ 0.80 , 0.91 , 0.91 , 1.01 , 1.20 , 1.48 , 1.52 , 1.65 , 1.95 , 2.35 , 3.27 , 3.67 , 6.79 , 9.39 . 13C-NMR ; δ 14.3 (CH3), 15.2 (CH3), 19.6 (CH3), 23.9 (CH2), 24.9 (CH2), 27.2 (CH2), 27.9 (CH3), 30.3 (CH), 36.6 (C), 37.1 (CH2), 38.5 (CH2), 38.5 (CH2), 38.6 (C), 50.4 (CH), 61.3 (CH2), 78.8 (C-OH), 144.4 (C), 151.7 (C), 194.6 (CHO).1- was chromatographed on silica gel column with hexane-acetate mixtures in increasing polarity obtaining 15 fractions. Fraction E5 was submitted to CC on silica gel using hexane:dichloromethane mixtures, yielding 87 mg of flavonoid 10. Fraction I (24.234 g) was chromatographed on silica gel CC with hexane–acetate mixtures in increasing polarity, obtaining 15 fractions. The I12 fraction was submitted to a CC obtaining a pure compound 13-nor-11,12-dihydroxybisabol-2-enone (11) (60 mg).From R)-6-((6R)-6,7-Dihydroxyheptan-2-yl)-3-methylcyclohex-2-en-1-one (11).(6 (+)-HRESIMS m/z 263.1611 [M + Na]+ , 2932 (C-H), 1656 (C=O), 909 (C=CH2) cm−1. 1H-NMR ; δ 0.80 , 1.30 , 1.31 , 1.44 , 1.49 , 1.78 , 1.92 , 1.93 , 2.12 , 2.29 , 2.30 , 3.43 , 3.64 , 3.70 , 5.85 . 13C-NMR ; δ 15.8 , 22.6 , 23.4 , 24.1 , 30.4 , 30.7 , 33.2 , 34.5 , 49.8 , 66.8 , 72.2 , 127.0 , 161.4 , 201.3 . Botrytis cinerea UCA992 using different methodologies according to the characteristics of the samples . Th. Th5 andThe data were analyzed using an ANOVA test with PRISM © statistical analysis software (version 5.01). Dose–response analysis was performed to estimate the IC50 values with 95% confidence ranges.Baccharis species ) were identified as Nevadensin (1) D25: −24.4. The analysis of the high-resolution mass spectrum (HRESIMS (+) [M + Na]+: 263.1623, found: 263.1611) confirms the molecular formula C14H24O3. NMR spectroscopic data of 11 showed similarities with those published from the known compound 10 . The 1H-NMR spectrum (δH-14: 0.80), a proton next to an alkene system (δH-4: 2.29), and a methyl group on sp2-carbon (δH-15: 1.93). Furthermore, three oxygenated groups (δH-11: 3.70 and δH-12: 3.64 and 3.63) and an olefinic group (δH-2: 5.85) were present. The 13C-NMR spectrum (δC: 201.3 (C-1), an olefinic group δC: 161.4 (C-3), 127.0 (C-2), and two oxygenated carbons, δC: 72.2 (C-11) and 66.8 (C-12). The 1H-13C HMBC correlation observed between H-6 and C-5, H-6 and C-14, H-7 and C-5, H-7 and C-14, and between H-8 and C-14 supports the idea that the di-alcohol moiety was connected to C-6 -6-((6R)-6,7-dihydroxyheptan-2-yl)-3-methylcyclohex-2-en-1-one  (IC50 of 41.1 μM) isolated from B. prunifolia and B. trinervis, and 6(R)-6-((R)-hept-6-en-2-yl)-3-methylcyclohex-2-en-1-one (10) (IC50 15.2 μM) isolated from B. Zumbadorensis showed the highest activity  had the lowest activity (IC50 of 57.0 ppm), 4′, 7-dimethoxyapigenin (2); Genkwanin (3) and Cirsimaritin (4) had a similar activity ; all of them have a hydroxyl group in C-5. On the other hand, Genkwanin (3) has a similar structure to 2 with the main difference being the free hydroxyl group presented in C-4′ (3 (IC50 of 35.9 ppm)  see . 5) and Galangustin (6) showed similar activities. In both cases, there is a hydroxyl group in C-5 and a methoxyl group in C-4′ (compound 5) and C-8 (compound 6). Salvigenin (5) showed the highest activity of all flavonoid compounds tested. A structural comparison with compounds 3 and 6 shows the importance of the free hydroxyl group in C-4′ as seen in the previous case (compounds 2 and 3) and the presence of a methoxyl group in C-7. In conclusion, for this family of compounds, the IC data confirms the importance of presenting at least a free hydroxyl group to increase the polarity, which increases the solubility in polar media (a hydroxyl group in C-5). A hydroxyl group in C-5 and a methoxyl group in C-8 (compound 6) or C-4′ (compound 5) could be related to the more efficient structural requirements for a high activity against B. cinerea. Compound 8, a novel compound identified from B. trinervis, showed a lower activity with an IC50 of 70.04 ppm.Salvigenin ; this data is the first report on the fungicide activity of these structures. Compound 10, with an alkene group in C-11-C-12 showed higher activity than the oxidized analog compound 11  were also tested by the poisoned food technique [The most active compounds  were studied in order to isolate new compounds with antifungal activity against the phytopathogen fungus B. cinerea UCA992. This is the first report of biological assays against the phytopathogen Botrytis cinerea which tests these isolated compounds.Three species from the 7A), (8), and (11). All compounds were tested against Botrytis cinerea UCA 992. The most active compounds were Salvigenin (5) with an IC50 of 13.5 ppm and 1,2-dihydrosenedigital-2-one (10) with an IC50 of 3.1 ppm.For this purpose, the extracts and the fractions from an initial chromatographic analysis were submitted to bio-guided isolation. Phytochemical investigation of the most active fractions of DCM extracts allowed for the identification of twelve compounds. Three of them reported here for the first time: (


Their chemical structures are confirmed by NMR, IR and MS spectra analyses. Density–functional theory (DFT) studies are performed using (DFT/B3LYP) with the 6-311G basis sets to recognize different chemical and physical features of the target compounds.The cyclic anhydrides are broadly employed in several fields, such as the chemical, plastic, agrochemical, and pharmaceutical industries. This study describes the chemical reactivity of 4,5-dichlorophthalic anhydride towards several nucleophiles, including thiosemicarbazide and different amines, to produce the carboxylic acid derivatives resulting from anhydride’s opening, namely, phthalimide and dicarboxylic acid ( Synthesis of new chemical compounds and evaluation of their biological properties are the main tasks in organic medicinal chemistry research. A literature survey revealed that several heterocyclic compounds originating from cyclic acid anhydrides have made a great contribution in a number of interesting areas ,4,5,6,7.1–12 starting with 4,5-dichlorophthalic as starting material. The chemical structures of the synthesized compounds 1–12 were confirmed using different spectroscopic analytical tools, i.e., MS, IR, and NMR. Density function theory (DFT) calculations were carried out based on set (B3LYP) with the 6-311G level of theory. To clarify the chemical and physical characteristics of the target compounds 1–12, various descriptors such as ionization potential (IP), the electron affinity (EA), chemical potential (μ), electronegativity (χ), hardness (η), softness (S), electrophilicity index (ω), and nucleophilicity index (N) were calculated.Furthermore, phthalic anhydrides are characterized by dominant saturated dibasic acids with a high chemical affinity towards different nucleophiles , affording the carboxylic acid derivatives arising from anhydride opening, namely, phthalimides, dicarboxylic acids and carboxylic acid esters. Considering both the above facts and the continuing work in chemical research dealing with cyclic anhydrides, the present work aimed to synthesize compounds 1–12 using 4,5-dichlorophthalic anhydride as key intermediate. The 4,5-dichlorophthalic anhydride was dissolved in boiling glacial acetic acid and treated with thiosemicarbazide under reflux condition to obtain the parent compound 1, with a good yield [1 was fully characterized via interpretation of its IR, MS, and NMR spectral data. The IR spectrum of compound 1 revealed stretching absorption bands at 1663 and 1702 cm−1 for carbonyl groups and weak absorption bands in the range of 3243–3439 cm−1 for amino groups , which corresponds to a molecular formula of C9H7Cl2N3O3S for 1.Many reactions are aimed at, as indicated by od yield ,16. The oups see . The EI-1H and 13C NMR spectral data. In the aromatic region of the 1H NMR spectrum, compound 1 showed two singlets at δ ppm 8.22 (H-6) and 8.07 (H-3) for the ortho 4,5-dichlorophenyl moiety , whereas the 13C NMR spectrum exhibited a thioxo carbon resonance at 182.7 ppm  at 8.50 ppm and then from the 13C, which exhibited its 13C-resonance at 127.2 (C-4/7). Further, a characteristic resonance was recorded at 163.3 ppm, assignable to the two equivalent dione carbons (C-1/3). The N-acetyl group was proven from its 1H-singlet at 2.43  and 13C-signals at 170.0 and 24.7, corresponding to the carbonyl and methyl carbons, respectively  in boiling DMF produced compounds 3–5. Their IR spectra revealed absorption peaks in the ranges of 1689–1703 and 3367–3405 cm−1, indicative of the carbonyl and amino functional groups, respectively. Products 3–5 were established on the basis of the characteristic signals of 4,5-dichlorobenzoic acid and corresponding aldehyde together with the two most downfield singlets at about 11.42 and 10.20, assignable to the –NH protons in -CO.NH.NH.CS- group. 1H NMR spectra revealed the two singlets typical of H-6 and H-3 at almost 8.21 and 8.16 of the 4,5-dichlorobenzoic acid moiety (4), alongside the intrinsic –N=CH- singlet at about 8.33 ppm. Three further informative 1H-signals that described the carbaldehyde moieties were unambiguously assigned . The most characteristic 13C-resonances in case of such products (3–5) were assigned at the ranges of 178.0–179.0 and 155.0–156.0 ppm, interpretable as C=S and –N=CH- carbons, respectively. Two other confirmative 13C-signals for all 4,5-dichloro derivatives were assigned at about 138.0 and 135.0 for the two chlorinated carbons C-4 and C-5. In regard to the NMR spectrum of compound 5, it revealed a singlet at 2.22 ppm, assignable to the methyl group and its characteristic 13C-resonance at 20.45 ppm  was established by IR, NMR, and HRMS analytical techniques. As in the case of products 3–5, both 6 and 8 showed the two characteristic singlets in the range of 8.1–8.0 ppm in their 1H NMR spectra, indicative of H-6 and H-3. In addition, the spectrum of 6 exhibited the characteristic broad singlets at 7.23 (H-5’) and 7.13 (H-4’) together with the ethyl ester resonances at 4.29  and 1.29   together with a singlet at 3.83 for the 3CH-ester. It is important to note that the three carbonyl-carbons of –COOH, -CONH- and –COOEt or COOMe appeared in the range of 163.0–167.0 ppm together with the ethyl carbons at 61.2 (-COO 2CHCH3), 14.6 (-COOCH23CH), and methyl ester-carbon at 52.6 , because of their symmetric structures. This was further documented via the disappearance of the 13C-signals of carbonyl-carboxyl and carbonyl-imide and appearance of a dione of two equivalent carbons (C-1/3) instead, observed at about 161.1, and 161.65 ppm confirming a symmetrical dione structure in both 7 and 9 (The formation of the two coupling products (CH3) see . However52.6 see . Refluxi 7 and 9 . In the  7 and 9 . 7 showed strong absorption peaks at 1688, 1702 and 1780 cm−1, consistent with the stretching frequencies of the carbonyl groups  and 13C-resonance at δ163.0 (C-1/3) for the two equivalent dione carbonyls. Moreover, the presence of an ethylene diamine group was concluded from its two 1H-triplets at 3.80  and 2.92  along with their corresponding 13C-resonances at 43.3 and 36.5 (-N2CH-2CHNH2). When 4,5-dichlorophthalic anhydride was allowed to react with 2-amino-5-methyl-benzoic acid or 3-amino-2-naphthoic acid in glacial acetic acid under reflux condition, the resulting products 11 and 12 were obtained, and their structures established on the basis of IR, HRMS, and NMR spectral data. Concerning both coupling products , they showed more or less the same 1H- and 13C-resonances as 6 and 8, except for the interpretation of the resonances corresponding to aminobenzoic or aminonaphthoic moieties instead of the characteristic aminothiophene ethyl or methyl esters. The 1H NMR spectrum of 11 displayed a methyl-Ar singlet at δ 2.45 ppm and its 13C-resonance at 21.1 ppm together with the carboxyl-carbonyl carbon at 166.5 ppm, while the 13C spectrum of 12 showed the carboxyl-carbonyl carbon of the naphthoic acid moiety at 166.6 ppm.The IR spectrum of cyclic oups see . Treatme product . The che1–12 via the density functional theory (DFT) method using the Gaussian 09 suite of programs. All compounds were calculated at the B3LYP level with 6–311G basis sets. For this study, we combined the predicted simulation and practical verification in order to carry out a profound study. We optimized the structures of IP), electron affinity (EA), softness (S) chemical potential (μ), electrophilicity index (ω), and hardness (η) were calculated at the same levels; the results are provided in 5 appeared to have the lowest η value; thus, it can be easily excited. On the other hand, compound 2 had the highest chemical η value; thus, it is the hardest molecule and is more stable. Compound 5 is considered an electron donor, and is good electron acceptor as well due to its lowest IP and highest EA characteristics, respectively. Compound 2 possesses a higher χ value than all of the other compounds; thus, it seems to be the best electron acceptor. In this study, ionization potential (1–5 is mainly on the (Ar−NH) and (NH− CS−NH−NH−) moieties (the electron transfer zones), as shown by their HOMO in 6–9, it is mostly distributed on the thiophene moiety. The LUMO spatial distributions of 6–12 is located on the carboxylic and benzene, groups (the electron acceptor zones), while they are mostly located on −NH−Ar and (NH− CS−NH−) in 1–5. The CO−N−NH, −N−NH−CS−NH−, Ar−OH, COOH, and Cl-Ar groups thus likely represent the most plausible reaction sites for nucleophilic and electrophilic attacks.The density distribution of compounds f  +f−f0 q(N) is the charge on the kth atom for a neutral molecule and q(N + 1) and q(N − 1) are the same for its anionic and cationic species, respectively. Using Hirshfeld, the descriptors’ values were calculated at the B3LYP/6-31G  level. At the DFT level, the most susceptible site to a nucleophilic attack for compound 2 is located on oxygen (O16), while for electrophilic attack the most reactive sites are on O19 and O20. The chloro atoms Cl9 and Cl10 are the most reactive sites for a free radical attack , therefore, this is a useful region for nucleophilic activity. In addition, the lowest electron density was found in compound 5 at 8.610 × 10−2 (a.u.) with the highest intensity blue colour shown for the hydrogen atom in O-H, making it an appropriate site for electrophilic attraction.The various electrostatic potential values at the surface are represented by different colors: red for electronegative, blue for positive electrostatic potential, and green for zero potential . The inc2 was selected; its experimentally detected and theoretically determined harmonic vibrational frequencies and its correlations are presented in From the experimental and calculated FT–IR spectra see , a sligh1H NMR and 13C NMR spectra of the synthesized compounds are shown in the 1H and 13C chemical shifts are shown in 1H and 13C NMR chemical shift correlation coefficients (R2) were found to be 0.978203291 and 0.97954627 ) for compounds 2 and 5, respectively. The observed correlation coefficient (R2) value shows that the practical and calculated chemical shift values were very close to each other [The ch other ,22.1–12 were successfully synthesized and their structures established on the basis of their NMR, IR, and MS spectral data. Practical and theoretical vibrational analysis of compound 2 was performed and showed fairly good agreement. The experimental data were correlated with DFT theoretical calculations employing (DFT/B3LYP) with the 6-311G basis sets. Different electronic and reactivity descriptors of the target compounds 1–12 were calculated in order to clarify their physical and chemical features. Various 4,5-dichlorophthalic anhydride derivatives were submitted for biological evaluation and will be discussed in a full study in forthcoming research. The target compounds d6 on a Bruker AMX 700 spectrometer operated at 700 MHz for 1H NMR and 175 MHz for 13C NMR. High-resolution mass spectra (EI-HRMS) were measured on a JEOL MStation JMS-700 system. A Brucker Alpha II FTIR-ATR spectrometer was used to record the IR spectra . Melting points (uncorrected) were measured using a STUART SMP 10 melting point apparatus. Monitoring the reactions and checking compound purity were carried out by TLC on a DC Mikrokarten polygram SIL G/UV254  with a thickness of 0.25 mm.NMR spectra were measured in DMSO-1H NMR : δ 14.11 , 10.48 , 9.47 , 8.22 , 8.07 , 7.85, 7.43 ; 13C NMR : δ 182.7 (C=S),166.5 (C-7), 166.0 (−CONH−), 136.8 (C-4), 135.4 (C-5), 133.3 (C-2), 131.6 (C-1), 130.9 (C-6),130.7 (C-3); IR : 1663 and 1702 cm−1 (C=O), 3243–3439 cm−1 (NH &NH2); HRMS (EI), m/z calcd. for C9H7Cl2N3O3S (M)•+ 306.9556, found 306.9503.4,5-Dichlorophthalic anhydride (1 mmol) was dissolved in boiling glacial acetic acid (15 mL), then thiosemicarbazide (1.5 mmol) was added and the reaction mixture was refluxed for 6 hrs. The obtained solid was filtrated and washed with water and ether. Yield (75%); mp 220 °C; 1 (1 mmol) was refluxed with acetic anhydride (10 mL) for 7 hrs. The reaction mixture was cooled and the resulting solid was collected. Yield (60%); mp 190 °C; 1H NMR : δ 8.50 , 2.43 ; 13C NMR : δ 170.0 ((−NCOCH3), 163.3 (C-1/3), 139.5 (C-5/6), 129.6 , 127.2 (C-4/7), 24.7 (−NCOCH3); IR : 1727 and 1796 cm−1 (C=O), HRMS (EI), m/z calcd. for C10H5Cl2NO3 (M)•+ 256.9646, found 256.9598.Compound 1 (1.1 mmol) was refluxed in DMF (15 mL) in the presence of few drops of glacial acetic acid for 5–9 hrs. After cooling, the precipitate was filtered off, washed with water, and dried to obtain the pure compounds 3–5.A mixture of aldehyde (1.1 mmol) and compound 1H NMR : δ 11.42 , 10.24 , 8.30 , 8.21 , 8.17 , 8.15 , 7.33 , 6.82 ; 13C NMR : δ 178.3 (C=S), 166.4 (C-7), 166.0 (−CONH−), 155.7 (−N=CH−), 149.8 C-2’), 137.8 (C-4), 135.3 (C-5), 133.5 ,130.8 (C-6), 128.8 (C-3), 123.4 (C-3’), 118.5 (C-5’), 111.6 (C-4’); IR : 1693 cm−1 (C=O), 3405 cm−1 (-NH-); HRMS (EI), m/z calcd. for C14H9Cl2N3O3S2 (M)•+ 400.9462, found 400.9411.Yield (43%); mp 187 °C; 1H NMR : δ 11.42 , 10.98 , 10.20 , 8.33 , 8.21 , 8.16 , 7.72 , 7.33 , 6.81 ; 13C NMR : δ 179.0 (C=S), 166.4 (C-7), 166.0 (-CONH-), 160.0 (C-2’), 156.0 (−N=CH-7’), 138.9 (C-4’), 137.5 (C-4), 134.5 (C-5), 133.3 (C-2/1), 131.1 (C-6/6’), 128.0 (C-3), 120.4 (C-1’), 118.0 (C-3’), 111.4 (C-5’); IR : 1689 cm−1 (C=O), 3367cm−1 (-NH-); HRMS (EI), m/z calcd. for C16H10BrCl2N3O4S (M)•+ 488.8952, found 488.9001.Yield (47%); mp 203 °C; 1H NMR : δ 11.35 , 9.63 , 8.34 , 8.11 , 7.92 , 7.75 , 7.03 , 6.76 , 2.22 ; 13C NMR : δ 178.1 (C=S), 166.4 (C-7), 166.0 (−CONH−), 162.7 (C-2’), 154.6 (−N=CH-7’), 140.3 (C-4’), 137.6 (C-4), 134.5 (C-5), 133.3 (C-2/4’/1), 132.1 (C-6/5’), 128.8 (C-3), 127.1 (C-6’), 120.6 (C-1’), 116.3 (C-3’), 20.45 (Ar-CH3); IR : 1703 cm−1 (C=O), 3400 cm−1 (-NH-); HRMS (EI), m/z calcd. for C17H13Cl2N3O4S (M)•+ 425.0003, found 425.0045.Yield (45%); mp 239 °C; 6 and 8 as the final products. Compounds 6 or 8 (1 mmol) was refluxed in acetic anhydride (10 mL) for 8 h. After cooling, the obtained solid compound (7 or 9) was collected and dried (the reaction for preparation 8 proceeded in trimethylamine and used DMF as solvent). A mixture of 4,5-dichloro-phthalic anhydride (1 mmol) and 2-amino-thiophene-3-carboxylic acid ethyl ester (1.3 mmol) or methyl-4-minothiophene-3-carboxylate hydrochloride (1.3 mmol) was stirred under reflux conditions in glacial acetic acid (15 mL) or DMF (15 mL) for 7 h. After cooling, the solid was filtrated and washed with water to produce compounds 1H NMR : δ 11.22 , 8.38 , 8.09 , 8.04 , 7.23 , 7.13 , 4.29 , 1.29 ; 13C NMR : δ 167.0 (C-2’), 166.0 (C-7), 165.7 (−CONH−), 164.6 (−COOCH2CH3), 138.5 (C-4), 136.5 (C-5), 133.2 (C-2), 132.2 (C-1), 130.6 (C-6/3), 124.3 (C-4’), 117.3 (C-5’), 114.3 (C-3’), 61.2 (−COO2CHCH3), 14.6 (−COOCH23CH); IR :1730, 1662 cm−1 (C=O), 3270, 3376 cm−1 (-NH-); HRMS (EI), m/z calcd. for C15H11Cl2NO5S (M)•+ 386.9735, found 386.9694.Yield (76%); mp 170 °C; 1H NMR : δ 8.43 , 7.79 , 7.51 , 4.10 , 1.02 ; 13C NMR : δ164.9 (−COOCH2CH3), 161.1 (C-1/3), 139.5 (C-5/6), 136.8 (C-2’), 131.7 (C-4’), 129.8 , 127.7 (C-5’), 127.6 (C-3’), 126.3 (C-4/7), 61.1 (−COO2CHCH3), 13.9 (−COOCH23CH); IR : 1688, 1702 and 1780 cm−1 (C=O); HRMS (EI), m/z calcd. for C15H9Cl2NO4S (M)•+ 368.9628, found 368.9674.Yield (52%); mp 140 °C; 1H NMR : δ 10.25 , 8.41 , 8.06 , 8.00 , 7.99 , 3.83 ; 13C NMR : δ 166.0 (C-7), 164.1 (−CONH−), 162.8 (−COOCH3), 137.7 (C-4), 135.7 (C-5), 135.0 (C-4’), 133.0 (C-2), 132.0 (C-2’), 131.6 (C-1), 130.1 (C-6/3), 122.2 (C-3’),112.9 (C-5’), 114.3 (C-3’), 52.6 (−COOCH3); IR :1723, 1656 cm−1 (C=O), 3282, 3400cm−1 (-NH-); HRMS (EI), m/z calcd. for C14H9Cl2NO5S (M)•+ 372.9578, found 372.9612.Yield (63%); mp 195 °C; 1H NMR : δ 8.54 , 8.32 , 7.90 , 3.67 ; 13C NMR : δ 165.5 (−COOCH3), 161.9 (C-1/3), 138.7 (C-5/6), 135.7 (C-4’), 132.2 (C-2’), 129.6 , 127.9 (C-3’), 126.4 , 52.5 (−COOCH3); HRMS (EI), m/z calcd. for C14H7Cl2NO4S (M)•+ 354.9473, found 354.9433.Yield (52%); mp 165 °C; 1H NMR : δ 8.22 , 7.95, 7.73 , 3.80 , 2.92 ; 13C NMR : δ 163.0 (C-1/3), 137.8 (C-5/6), 126.0 , 125.7 (C-4/7), 43.3, 36.5 (-NCH2-CH2NH2); HRMS (EI), m/z calcd. for C10H8Cl2N2O2 (M)•+ 257.9963, found 258.0001.A mixture of 4,5-dichlorophthalic anhydride (1 mmol) and ethylene diamine (4 mmol) was stirred under reflux in glacial acetic acid (10 mL) for 8 hrs. After cooling, the precipitate was filtrated and washed with water to give the final product. Yield (65%); mp 231 °C; 1H NMR : δ 8.70 , 8.21 , 8.06 , 7.75, 7.55 , 3.73 , 2.74 ; 13C NMR : δ 166.7 (C-7), 166.5 (-CONH-),137.1 (C-4), 134.3 (C-5), 133.2 (C-2), 130.9 (C-1), 130.8 (C-6), 129.6 (C-3), 31.0 (-NHCH2-CH2NH2).A mixture of 4,5-dichlorophthalic anhydride (1 mmol) and 2-amino-5-methyl-benzoic acid (1.2 mmol) or 3-amino-2-naphthaoic acid (1.2 mmol) was refluxed in glacial acetic acid for 6–8 hrs. After cooling, the obtained solid was collected and washed with water to obtain the final products.1H NMR : δ 14.15, 13.35 , 8.33 , 8.06 , 8.01 , 7.89 , 7.59 , 7.42 , 2.45 ; 13C NMR : δ 166.5 (C-7’), 166.2 (C-7), 165.9 (-CONH-), 140.0 (C-1’), 138.3 (C-4), 135.2 (C-5), 134.1 (C-2), 132.1 (C-1/5’), 131.6 (C-3’), 130.8 (C-6), 130.0 (C-3), 129.0 (C-4’), 126.2 (C-2’/6’), 21.1 (Ar-CH3); HRMS (EI), m/z calcd. for C16H11Cl2NO5 (M)•+ 367.0014, found 367.0059.Yield (66%); mp 234 °C; 1H NMR : δ 13.25 , 8.74 , 8.37 , 8.06 , 8.01 , 8.24 , 8.12 , 8.09 , 7.77 , 7.74 ; 13C NMR : δ 166.6 (C-7’), 166.2 (C-7/-CONH-), 138.4 (C-4/3’), 134.7 (C-5), 133.3 (C-2), 132.4 (C-4’a), 132.1 (C-1/1’), 130.6 (C-6), 129.8 (C-3), 129.6 (C-8’), 128.6 (C-6’), 128.2 (C-8’a), 127.7 (C-5’), 126.3 (C-2’/4’/7’); HRMS (EI), m/z calcd. for C19H11Cl2NO5 (M)•+ 403.0014, found 403.0048.Yield (56%); mp 217 °C; 1–12 in the ground state was performed by density functional theory (DFT/B3LYP) using the 6-311G basis [Optimization of the molecular structures of compounds p) basis ,24. The p) basis . The Gaup) basis . Calculap) basis . The symp) basis ,33,34. Fp) basis .


Synthetic access is challenging and the few reports found in the literature suffer from the requirement of hazardous intermediates and harsh reaction conditions. Here, we report a new six-step synthesis for c1G base, starting from 6-iodo-1-deazapurine. The key transformations are copper catalyzed C–O-bond formation followed by site-specific nitration. A further strength of our route is divergency, additionally enabling the synthesis of 1-deazahypoxanthine (c1I base).Imidazopyridines and pyrrolopyrimidines are an important class of compounds in medicinal chemistry. They can also be considered as deaza-modified purine nucleobases, and as such have attracted a lot of interest recently in the context of RNA atomic mutagenesis. In particular, for 1-deazaguanine (c After complete reaction (TLC reaction control), the suspension was allowed to cool to room temperature and the catalyst was filtered through celite and washed with dichloromethane containing 1% triethylamine. The filtrate was concentrated to dryness and the crude product was purified via silica gel chromatography using 20 to 40% ethyl acetate in cyclohexane (containing 1% triethylamine) as gradient. Yield: 1.18 g (73%) of compound 18 as a brownish solid. TLC (cyclohexane/ethyl acetate 1:1): Rf 0.23; 1H NMR  δ 1.57 -pyran), 1.74 -C(4)-pyran), 1.95 -C(3)-pyran & H(a)-C(4)-pyran), 2.29 -C(3)-pyran), 3.69 -C(6)-pyran), 4.01 -C(6)-pyran), 5.53 ), 5.76 -pyran), 6.96 ), 7.34–7.51 ), 8.21 ), 8.50 ); 13C NMR  δ 22.58 (H-C(3)-pyran), 24.57 (H-C(4)-pyran), 30.10 (H-C(5)-pyran), 67.69 (H-C(6)-pyran), 70.51 (H2C-benzyl), 80.77 (H-C(2)-pyran), 103.74 (H-C(1)), 124.73 (H-C(5)), 127.96, 128.14, 128.49 (HC-benzyl), 136.39 (C-quart.-benzyl), 140.79 (H-C(8)), 145.59 (H-C(2)), 148.17 (H-C(4)), 156.27 (H-C(6)); ESIMS (m/z): [M + H]+ calcd for 310.16; found, 310.16.Compound 18  was dissolved in methanol (20 mL), then 5% hydrochloric acid (5 mL) was added and stirred for 2 hours at 50 °C. After complete deprotection (TLC reaction control!), the solvent and all volatiles were removed under reduced pressure and the crude residue was suspended in dichloromethane (6.5 mL). Afterwards, di-tert-butyl dicarbonate , 4-(dimethylamino)pyridine  and triethylamine  were added and the clear solution was stirred for one hour at room temperature. The mixture was subsequently quenched with saturated ammonium chloride solution, filtrated and concentrated to dryness. The crude product was purified via silica gel chromatography using 0 to 3 % methanol in dichloromethane as gradient. Yield: 730 mg (77%) of compound 19 as a brownish solid. TLC: (6% methanol in dichloromethane): Rf 0.70; 1H NMR  δ 1.69 3-Boc), 5.51 ), 6.82 ), 7.31–7.49 ), 8.35 ), 8.36 ); 13C NMR  δ 28.14 (C(CH3)3-Boc), 71.60 (H2C-benzyl), 86.18 (C(CH3)3-Boc), 105.44 (H-C(1)), 126.46 (H-C(5)), 127.71, 128.48, 128.81 (HC-benzyl), 135.89 (C-quart.-benzyl), 140.80 (H-C(8)), 146.50 , 148.08 (H-C(2) & H-C(4)), 157.22 (H-C(6)). ESIMS (m/z) [M + H]+ calcd for 326.15; found, 326.15.Compound 19  was dissolved in dry dichloromethane (6 mL) and cooled to 0 °C under argon atmosphere. The nitration mixture was transferred with a syringe and added over a period of 10 minutes to the dissolved substrate. The resulting reaction mixture was stirred for 1.5 hours at 0 °C under an argon atmosphere. Afterwards, the reaction was quenched by adding the whole reaction mixture into a separatory funnel containing saturated sodium bicarbonate solution, followed by vigorously shaking (do not use a stopper!). The organic layer was washed with saturated sodium bicarbonate solution and brine and was finally dried over Na2SO4, filtered and concentrated to dryness. The crude product was purified via silica gel chromatography using 0 to 4% methanol in dichloromethane as gradient. Yield: 395 mg of a mixture containing the 2-nitro-compound 20 and 21 (for NMR spectra see Rf 0.34 (compound 20), Rf 0.05 (compound 21).Nitration: Tetrabutylammonium nitrate  was dissolved in 6 mL dry dichloromethane under argon atmosphere, then, trifluoroacetic anhydride  was added and the mixture was stirred at 0 °C for 10 minutes. Meanwhile, compound N,N-diisopropylethylamine  was added. A solution containing dry dichloromethane (4 mL) and trichlorosilane  was prepared at 0 °C under an argon atmosphere, and then added via syringe to the substrate. The mixture was stirred at 0 °C for one hour. Subsequent quenching was achieved by the addition of saturated bicarbonate solution (10 mL) and further stirring for one hour. Afterwards, two spoons of silica were added and the whole suspension was concentrated to dryness and dried under high vacuum until a fine powder remains. The silica adsorbed with the product was loaded onto a short silica column and the product was eluted using 0 to 20% methanol in dichloromethane as gradient. The brownish solid was dissolved in boiling water, filtered and cooled to room temperature to precipitate a white solid. Yield: 110 mg  of compound 22 as a white solid. TLC: (15% methanol in dichloromethane): Rf 0.44; 1H NMR  δ 5.34 ), 6.10 ), 7.36–7.52 ), 7.93 ); 13C NMR  δ 71.03 (H2C-benzyl), 88.06 (H-C(1)), 110.84 (H-C(5)), 128.30, 128.38, 128.71 (HC-benzyl), 135.11 (C-quart.-benzyl), 140.84 (H-C(4)), 145.69 (H-C(8)), 155.63 (H-C(2)), 157.47 (H-C(6)); ESIMS (m/z): [M + H]+ calcd for 241.11; found, 241.11.Reduction: The mixture from the above procedure was suspended in dry dichloromethane (8 mL) at 0 °C, and 22  and palladium on carbon 10%  were suspended in H2O/methanol 1:1 (7 mL). A rubber septum was applied and hydrogen gas  was bubbled through the solution for 10 minutes. The mixture was stirred under hydrogen atmosphere for further four hours at room temperature. The catalyst was filtered through celite and the filtrate was concentrated to dryness. The crude product was purified via silica gel chromatography using 0 to 25% methanol in dichloromethane as gradient. Yield: After recrystallization from water 48 mg (70%) of compound 11 as a white solid. TLC: (25% methanol in dichloromethane) Rf 0.19; 1H NMR  δ 5.43 ), 5.76 , 7.78 ), 11.69 ; 13C NMR  δ 89.87 (H-C(1)), 113.28 (H-C(5)), 139.82 (H-C(8)), 145.69 (H-C(4)), 154.08 (H-C(2)), 162.52 (H-C(6)); ESIMS (m/z): [M + H]+ calcd for 151.06; found, 151.06.Compound 17 , copper(I) iodide , 1,10-phenanthroline , caesium carbonate  and benzyl alcohol  were suspended in 0.76 mL of toluene (0.5 mL per 1 mmol compound 17) and stirred for 4 hours at 110 °C under atmospheric conditions. After complete reaction (TLC reaction control), the suspension was allowed to cool to room temperature and the catalyst was filtered through celite and washed with dichloromethane containing 1% triethylamine. The filtrate was concentrated to dryness and the remaining solid was dissolved in methanol (20 mL) and 5% hydrochloric acid (5 mL). This solution was stirred for two hours at 50 °C and concentrated to dryness. The crude compound was purified via silica gel chromatography using 0 to 10% methanol in dichloromethane as gradient. Yield: 250 mg (73%) of compound 31 as a white solid. TLC: (10% methanol in dichloromethane): Rf 0.44; 1H NMR  δ 5.62 ), 5.76 -pyran), 7.39–7.62  & H-C(1)), 8.63 ), 8.91 ); 13C NMR  δ 71.89 (H2C-benzyl), 103.56 (H-C(1)), 118.33 (H-C(5)), 128.29, 128.66 (HC-benzyl), 134.85 (C-quart.-benzyl), 140.65 (H-C(2)), 145.81 (H-C(8)), 148.62 (H-C(4)), 157.10 (H-C(6)); ESIMS (m/z): [M + H]+ calcd for 226.10; found, 226.10.Compound 31  was dissolved in methanol (15 mL), then palladium on charcoal 10%  was added and hydrogen  was bubbled through the suspension for 10 minutes. Subsequently, the mixture was vigorously stirred under hydrogen atmosphere for 4 hours at room temperature. Afterwards, the suspension was filtered through celite, and the filtrate was concentrated to dryness. The remaining crude product was purified by recrystallization in water. Yield: 83 mg (60%) of compound 30 as a white solid. 1H NMR  δ 6.96 ), 8.26 ), 8.58 ); 13C NMR  δ 107.00 (H-C(1)), 118.88 (H-C(5)), 137.87 (H-C(2)), 145.78 (H-C(8)), 149.08 (H-C(4)), 160.86 (H-C(6)); ESIMS (m/z): [M − H]− calcd for 134.04; found, 134.03.Compound File 117 to 22, 11, 31 and 30.NMR spectra of compounds