Aglais, Apatura, Araschnia, Argynnis, Brenthis, Boloria, Euphydryas, Inachis, Issoria, Libythea, Limenitis, Melitaea, Neptis, Nymphalis and Polygonia. Data published in a previous article add 101 specimens from the Vanessa genus.This paper complements the data published thus far about species of the nymphalid family with data collected in Romania’s eight regions between 1887 and 1984 and elsewhere in Europe , including the date and the site of original collection. For the first time, this research presents the collecting information of the species held in the entomological collection of the Museum of Natural History in Sibiu. It identifies the species of the nymphalid family in six of the museum’s lepidoptera collections. These collections are of extraordinary interest not least because they are associated with natural scientists of European renown, such as Daniel Czekelius, Eugen Worell, Viktor Weindel, Rolf Weirauch, Heinrich Hann von Hannenheim and Eckbert Schneider. The analysis, cataloguing, centralisation and updating of the nomenclature resulted in a number of 1,865 specimens from 49 species and fifteen genera (of the 90 referenced in Romania’s fauna): Polygoniaegea , Eugen Worrell collection and three species that are endemic to Romania: Melitaearetyezatica Diöszeghy, 1930, Argynnispandoradacica Hormuzaki, 1892, Daniel Czekelius collection and Boloriapales  carpathomeridionalis Crosson et Popescu-Gorj, 1963, both in the Viktor Weindel collection.Most species originate regionally from the nine counties of Transylvania followed by Oltenia and Moldova (three counties each), Banat and Dobrogea (two counties each), Crișana, Satu Mare and Muntenia (one county each) and the capital of Romania, Bucharest. The species presented in this paper also include the extinct taxon  The following natural scientists founded the collections studied in this paper and analysed and determined the preserved material, thus bringing an important contribution to lepidopterological research: Daniel Czekelius ; Arnold Müller ; Eugen Worell , Viktor Weindel ; Heinrich Hann von Hannenheim , Rolf Weirauch , Alexei Alexinschi ; Hans Rebel ; László Diószeghy ; Aurelian Popescu-Gorj  and Eckbert Schneider (1927)  . The NatLepidoptera collections are of central importance to the Natural History Museum’s patrimony. To date, partial collecting data of the species have been published for the Daniel Czekelius, Eugen Worell, Viktor Weindel, Rolf Weirauch, Heinrich Hann von Hannenheim and Eckbert Schneider collections , Eugen Worell (EW), Viktor Weindel (VW), Rolf Weirauch (RW), Heinrich Hann von Hannenheim (HH), Eckbert Schneider (ES).Romanian regions: Banat (BT), Crișana (CR), Oltenia (OT), Muntenia (MT), Moldova (MD), Dobrogea (DB), Satu Mare (SM), Transilvania (TR)Romania’s counties: Arad (AR), Alba (AB), Bistrița-Năsăud (BN), Brașov (BV), Caraș-Severin (CS), Cluj (CJ), Constanța (CT), Covasna (Cv), Galați (GL), Gorj (GJ), Harghita (HR), Hunedoara (HD), Ilfov (IF), Maramureș (MM), Mehedinți (MH), Mureș (MS), Neamț (NT), Satu Mare (SM), Sibiu (SB), Timiș (TM), Tulcea (TL), Vâlcea (VL), Vrancea (VN), București (B).European countries: Austria (AU), Croația (HR), Germany (GE), Republic of Moldova (RMO), Serbia (RS), Switzerland (CH).The conservation status: Extinct (EX), Critically endangered (CR), Endangered (EN), Vulnerable (VU), Near threatened (NT), Least concern (LC), Endemic (EN).712BE299-0967-567B-B3C7-268A247E89CAENROESBBC3CAF3-9FBC-5BF6-AC5E-25AA4214BB2FNTRO, AU, RMODC, EW, VW, ESF2784E02-C8CC-5642-A202-46D47F0CD613NTRO, AUEW6AEC3F5E-80FA-5698-B6A0-E02C8A0BAF61VURODC, EW, VW, RW, ESHormuzaki, 1892750E049F-AD99-5EE1-87DD-95D6BA671D07EndemicROEW140BAC9A-9D62-5CFC-957F-F39A3649987ALCRODC, EW, VW, RWBAA6E2A4-351F-50C9-B73D-425C551BD925NTRODC, EW, VWEsper 17892EE4629B-3152-5EE8-A0E3-A442460D6646NTROVW7F193551-7681-59C8-BA7B-D03E88C9714ANTRO, AUDC, EW, VW, ESBorkhausen, 17885FDFE764-3602-5637-A32B-77220338BE50NTRODC0039CA02-9EBC-57BC-AB05-376067FBD11EOUG 57/2007: 4B; IUCN: LC RODC, EW, VWAigner-Abafi, 19062D38C03E-89D4-5694-AE0E-79E0E1298A61LCRODC7B4BDEEB-64D4-5599-9163-2E016B8B593DLCRODC, VW, HH, ESRottemburg, 1775A4C7B511-3199-521A-8FCB-B3387DAE8490VURODC, VW18C952AB-FB4D-5CE4-8851-68F17921C48AVURODC, VW, ES3AEB1A85-E5E9-56DB-833D-9C63105853ECVUROVW, HH, ESF2708A60-936F-5346-A5F3-221F3F4705FCVURODC, VW, HH, ES535D70A7-6049-5AA0-B5B3-8FB55A29ED2DNTRODC, VW, HH, ESA87C6F0A-5484-5553-9E5F-F287DF1EAB58LCRODC, VW, HH, ES Crosson & Popescu-Gorj, 1963596F95A2-BAA9-5171-819C-73BEBC7C8814EndemicROVW, HH2F7E7ADA-889C-589A-86FD-7A97FC105FADLCRO, RMODC, EW, VW, HH, RW, ES57962061-12A0-555D-B630-BD063BD20278NTRO, RMODC, EW, VW, RW, ESA06CF910-C236-509F-BB36-010F3731A390NTRO, RMODC, EW, VW, HH, RW, ES9A2E99B8-F0D8-5A25-B65E-36405B617EF1EXHREWD3E722DC-9506-5D45-B832-B3BA4A070899NTRO, GEDC, EW, VW, HH, RW, ESOchsenheimer, 1807EE8A9D60-EC7D-51FB-B969-B201C6CFBC66NTRODCLinnaeus, 1758003B52E7-3ADC-5DF0-8750-30F9BFDFDF91NTRODC, EW, VWStichel, 19064D3D3472-7D09-5FFA-8018-720A521C4D89NTRODCB685CC7D-CBBB-5B0D-ADCC-A3B2FF4752E9VURODC, EW, VW, HH, RW, ES96DD0376-9A76-5D22-AB1F-F1AD1AB6F49EENRODC, EW, VW, RW, ES70ED3F29-66B6-5DFA-8D0F-D8E2A68C1712IUCN: LC RoDC, EW, VW, RW75410177-7E91-524C-81F4-B5E640E72577ONG 57/2007: 3, 4A, DH: II, IV; IUCN: LC RODC, VW, RW0DE8A529-1E85-5D37-9EA1-0879B8253924ONG 57/2007: 3, 4A, DH: II, IV, IUCN: DD RODC, EW, ESE5C9A171-0FEF-5EFE-BA4B-19F4B01C6ED6CRRORWC3FAFF5A-4A6B-5F92-8187-D211CB3092EFONG 57/2007: 3, 4A, DH: II RODC, EW, VW, ESA4794F46-BB83-5B37-8BC5-98E71579102ANTRODC, EW, VW, HH, RW, ES4047EC2F-01CE-5D1B-AB34-3240D7CCE67BNTRO, RMODC, EW, VW, RW, ESE9BF1AAA-3D0B-53EF-BEED-82B69164A82ENTRO, RMODC, EW, VW, ES4FF99F90-5309-564A-A5FC-C5B148FB9FB9LCRO, AUDC, EW, VW, HH, RWStaudinger, 1861C3117AD9-53C3-5082-A1E2-A837AEB17E0BVUROEWStaudinger, 18610F1979CF-8839-5093-B959-F24FA988A935VURORWStaudinger, 1870F37F8322-EE97-5FE3-A14F-210276674962VUROEWEversmann, 184719335340-B230-5B9B-90F9-911411A5C59CVURMOEWEsper, 177878990EA3-1A0B-510B-A4B9-BD003382B96AVURODC, EW, RWNickerl, 18500BA32BA6-F8A1-5CAD-BBCE-6AA1A2D73570VURO, RSDC, EW, VW, HH, RW, ES5A1FB6B2-732A-53DA-BAC7-F56B5ECC71DDNTRO, RMODC, EW, VW, HH, RW, ESGerhard, 18220D35EA98-BB3C-5AF0-BE6B-FAF59349C5FDNTROEW, RWAssmann, 1847E9E5050A-5B58-5B9E-8143-0C43A2183E13NTRORWFruhstorfer, 191743BC2528-DE7E-5814-9165-6E210E25A145NTRODCRehfons, 191018E62C71-ACCC-59FA-97EC-0556150D8A3BVURODCKeferstein, 1851575D35B8-580E-5917-A4B2-4BF62B0AF05DVURODC, EWMeyer-Dűr, 1851A10AEC62-5BF1-5CB6-A036-E4654D5CD9BDVUAUEWDiöszeghy, 19305D1B5CD2-247A-5AA6-A287-72994ED9E355EndemicRODCFreyer, 1828C5CF5101-3EAB-53B8-9DEE-3D03DB48E4DEVUROEWHormuzaki, 1898BB8C48BD-E921-5D9A-97B4-CB189EDC00BANTRODC968DE45F-B615-53D7-B484-BE4EF0883528VURODC, EW, VW, HH, RWEsper, 1800DD926DC0-8BBE-5EB5-AA46-6C35208A34DAVURODC, EW, VW, RW0B1A9DFD-51A4-5DB0-96F1-9A827068D13BVUROVW, RWLinnaeus, 176AB282179-EBC3-5D95-BD0A-C3CA032640CEVURO, CHDC, EW,Staudinger, 1901C100F37A-1141-5D78-A105-BB187627CA0FENRORWsensu Lhomme, 1924D6047C03-84A9-58DF-A18C-171FBE66DF71VURODC, HH8346C0BC-3B06-5392-8A63-7A35F7EF2691NTROEW, VW, RW, ESNordmann, 18515A4F5E37-A04C-58B7-AF74-76D5A2E791A3NTROVWPallas, 17719D61110B-586D-56B9-BACA-543C8A1DCC76VUROVW, RW, ES5283B693-74CF-5592-B7F2-64ADBF27C5CBNTRODC, EW, HH, RW, ES3A654A24-7B38-5944-9AA7-6A80D0EC4C62VURODC, EW, VW, RW0C9D4489-4625-5214-882A-CF730059A304VURODC, VW, RWRossi, 17942226EBCE-1F65-5F00-94EA-A62C68D784ADVURODC, EW, HH, ES9C7C8FE0-6D26-59DF-AFE5-ACA00C2969D8VURODC, EW, VW, HH, ESFreyer, 1829EA5D0E3F-A5DF-5910-B6D6-99E4F07533CCVUROEWVanessa genus are based on 101 specimens, 52 specimens of V.atalanta and 49 specimens of V.cardui  and Nymphalispolychloros  and were collected 135 years ago. The collectors specified on the labels are Daniel Czekelius, Béla Kiss, Arnold Müller, László Diószeghy, Friedrich Deubel, Paul Tiltscher, Miklos Tűtscher, Kimakowics, Adalbert Prall, Karl Alberti, Karl Petri and Vincenz Kollar.The analysis of the six collections of lepidoptera under scrutiny in this article focused on 1,865 specimens collected between 1887 and 1984 and identified 49 species and subspecies of the 90 mentioned in Romania’s fauna . EarlierV.cardui . The speV.cardui , HeinricTable 1Melitaearetyezatica Diöszeghy, 1930 from Melitaeaathalia , represented by twenty-six specimens collected by László Diószeghy in the Retezat Mountains (Southern Carpathians) between 1919 and 1922. The species in this collection represent the following conservation status: 18 taxa VU- 36.,74%, 18 taxa NT- 36.74%, six tax LC- 12.24%, four taxa EN-8.16%, two taxa CR-4.08% and one endemic taxon, 2.04%.Daniel Czekelius’s collection of lepidoptera is the oldest with specimens collected between 1887 and 1934 in the regions SM and TR, Sibiu County and nearby locations. This collection consists of 14 genera and 49 species represented by a number of 271 specimens  species and was collected in 1888. The collectors listed on the labels are Eugen Worell, Daniel Czekelius, László Diószeghy and Alexei Alexinschi. Considering their conservation status, these species are grouped as following: one species EX-2.38%, Polygoniaegea , from the site Pula, Istria, Croația, one species CR-2.38%, three species EN-7.15%, 19 species VU-45.23%, 15 species NT-35.71% and three species LC-7.15%  was collected in 1903. The collection also contains an endemic species, Boloriapales  carpathomeridionalis Crosson et Popescu-Gorj, 1963 that was collected in the Făgăraș Mountains. Viktor Weindel is the collector listed on the labels. The endangerment level of the specimens is the following: two species CR-5%, three species EN-7.5%, 15 species VU-37.5%, 15 species NT-37.5% and five species LC-12.5%  collected in 1928. The species were collected in BT, DB and TR and were determined by the collector himself whose name can be found on all labels. Conservation status is as follows: nine species VU-47.37%, six species NT-31.58% and four species LC-21.05%  sin. Euphydryasaurinia  collected in 1922. The information on the labels is unreadable. The species were collected in BT, DB, OT, MD, MT and TR from the Southern Carpathians, as well as in Austria. All species were determined by the collector whose name appears on all labels. The analysis of their endangerment level is as follows: three taxa CR-10.7%, two taxa EN-7.14%, 11 taxa VU-39.28%, 10 taxa NT-35.73% and two taxa LC-7.14%  collected from 1922 to 1978. The oldest specimen is Lybiteacelsis  species held by the Museum. The endangerment level of the collection’s specimens is as follows: three specimens EN-10%, 10 specimens VU-33.33%, 12 specimens NT-40% and five specimens LC-16.67% , we identified 157 sites where the butterflies of the nymphalid family were collected between 1887 and 1984. The species were collected in all of Romania’s eight regions, from 23 counties and the capital of Bucharest, i.e. on 56.1% of Romania’s territory. I designed a map based on these data that reflects the distribution of species in eight regions whose counties are listed by collector: (1-DC), seven counties, BN, BV, CJ, CV, HR, MS, SB, all in the TR and SM regions, (2-EW), nine counties, BV, CS, CT, GL, GJ, IF, MS, VL, SB, from five of Romania’s regions: BT, DB, OT, MD si TR; (3-VW), seven counties, AB, BN, BV, HR, NT, VL, SB, from three regions: MD, OT, and TR; (4-RW), 16 counties, BN, BV, CJ, CS, CT, CV, GL, GJ, HR, HD, IF, MH, VL, SB, TM and Bucharest, from six of Romania’s regions: BT, DB, MD, MT, OT, TR and Bucharest; (5-HH), six counties, AB, BV, CS, CJ, HR, SB, from two of Romania’s regions: BT and TR; (6-ES), 16 counties, AB, AR, BN, BV, CS, CJ, CT, GJ, HD, MH, MS, VL, SB, TM, TL, VN, from six of Romania’s regions: BT, CR, DB, OT, MD, TR  were also graphically represented in Fig. Conclusions2 that comprises a variety of geographic forms, from Alpine meadows to the Danube Delta’s hydrological profile. They are populated by a diverse fauna of lepidoptera containing over 200 diurnal butterfly species. Romania’s South, especially the South-East and South-West (BT and DB) is the region with the highest diversity of lepidoptera in the country  carpathomeridionalis Crosson et Popescu-Gorj, 1963. The Romanian Catalogue of Lepidoptera records the presence of 90 species of which the Nymphalids are distributed as follows: BT (78 species), CR (60 species), TR (78 species), SM (66 species), OT (73 species), MT (77 species), MD (68 species) and DB (62 species)  . I have species) , the preThe article illustrates the biodiversity of the nymphalid family and their historical evolution in Romania and Europe, thus bringing an important historical, scientific and biogeographic "


The synthetic pathway involved Steglich esterification of DHA with myristic acid; reduction of carbonyl group of 1,3-dimyristoylpropanone and esterification of 1,3-dimyristoylglicerol with stigmasterol chloroformate or stigmasterol hemisuccinate. The structure of the obtained hybrids was established by the spectroscopic methods . Obtained hybrid molecules were used to form new liposomes in the mixture with model phospholipid and their effect on their physicochemical properties was determined, including the polarity, fluidity, and main phase transition of liposomes using differential scanning calorimetry and fluorimetric methods. The results confirm the significant effect of both stigmasterol-containing acylglycerols on the hydrophilic and hydrophobic region of liposome membranes. They significantly increase the order in the polar heads of the lipid bilayer and increase the rigidity in the hydrophobic region. Moreover, the presence of both acylglycerols in the membranes shifts the temperature of the main phase transition towards higher temperatures. Our results indicate stabilization of the bilayer over a wide temperature range (above and below the phase transition temperature), which in addition to the beneficial effects of phytosterols on human health makes them more attractive components of novel lipid nanocarriers compared to cholesterol.New carriers of phytosterols; acylglycerols containing natural myristic acid at  Phytosterols are structural and functional analogues of cholesterol, synthesized by plants. These compounds are essential components of cell membranes that regulate their physicochemical properties. The most frequently referred plant sterols are 4-desmethyl sterols. Among them, one of the most common in the human diet is stigmasterol  . As otheDue to the low oil solubility and bioavailability of free stigmasterol, it is worth focusing on increasing its supply to the target tissues by its conjugation with other hydrophobic molecules. In this aspect, we paid our attention to acylglycerols, which, due to their natural presence both in food and the human body, can be an effective lipid delivery system and have been successively used as carriers of different bioactive ingredients , includiDue to their structure, acylglycerols are also an excellent base for their use as the components of liposomes, which may increase their stability and efficiency to deliver stigmasterol to the human body. Lipids in which two molecules of stigmasterol are covalently linked via succinate linker to glycerophosphocholine were used previously as the components of liposome formulations of doxorubicin and amphotericin B ,21. To tn-3 PUFA) and ceramide. It is the result of the myristoylation of N-terminal glycine residues of some enzymes, like NADH-cytochrome b5 reductase and dihydroceramide Δ4-desaturase  in 10% H2SO4 and heating the plates to 120–200 °C or 0.05% solution of primuline in acetone:water mixture  followed by observation of the spots under UV light (λ = 365 nm).Analytical Thin Layer Chromatography (TLC) was performed on 0.2 mm aluminum plates coated with silica gel 60 F® SX520 Plus Interchim system  gradient pump, UV detector, and fraction collector. The samples were dry loaded on a pre-column puriFlash® F0012 and the products were separated on puriFlash® SIHP 30 µm columns using gradient elution with mixtures of hexane and ethyl acetate as the mobile phase .Flash chromatography was carried out using puriFlash3 × MeOH complex solution according to the standard procedure +; found: 533.4179.White crystals, mp 72–75 °C (lit. 76°C ), Rf = 02, 1 g, 1.2 mmol) in 25 mL of THF placed in an ice bath with NaCl, few portions of NaBH4  were added. When substrate was not detected on TLC plate , THF was evaporated in vacuo, the residues was suspended in water and the product was extracted with CH2Cl2 (3 × 15 mL). The organic layer was washed with brine until neutral, dried over anhydrous MgSO4, filtered and concentrated in vacuo. After flash chromatography  0.56 g (yield 56%) of pure 1,3-dimyristoylglycerol (3) was isolated. Its physical and spectral data are given below:To the solution of 1,3-dimyristoylpropanone , 1H NMR : δ 0.87 (CH2)12C3H), 1.20–1.34 CH2CH2(C2H)10CH3), 1.62 CH2C2H(CH2)10CH3), 2.34 C2H(CH2)11CH3), 4.07 , 4.13 , 4.18 ; 13C NMR (100 MHz): δ 14.12 (2 × -OC(O)(CH2)12CH3), 22.68 (2 × -OC(O)(CH2)11CH2CH3), 24.86 (2 × -OC(O)CH2CH2(CH2)10CH3), 29.10, 29.23, 29.34, 29.43, 29.58, 29.63 and 29.66 (2 × -OC(O)CH2CH2(CH2)8CH2CH2CH3), 31.90 (2 × -OC(O)(CH2)10CH2CH2CH3), 34.07 (2 × -OC(O)CH2(CH2)11CH3), 65.00 (C-1 and C-3), 68.33 (C-2), 173.95 (2 × -OC(O)(CH2)12CH3); IR : 3484 (w), 1715 (s), 1732 (s), 1181 (s); HRMS (ESI): m/z calcd for C31H58O5: 535.4338 [M+Na]+; found: 535.4335.White crystals, mp 62–64 °C (lit. 64–66 °C ), Rf = 0rac-1,2-dimyristoylglycerol (3a) was isolated with the following physical and spectra data: As a minor product, 0.23 g (23% yield) of Rf = 0.16 , 1H NMR : δ 0.88 (CH2)12C3H), 1.20–1.34 CH2CH2(C2H)10CH3), 1.62 CH2C2H(CH2)10CH3), 2.32 and 2.34 C2H(CH2)11CH3), 3.71 , 3.74 , 4.23 , 4.32 , 5.08 ; 13C NMR (100 MHz): δ 14.12 (2 × -OC(O)(CH2)12CH3), 22.68 (2 × -OC(O)(CH2)11CH2CH3), 24.86 and 24.92 (2 × -OC(O)CH2CH2(CH2)10CH3), 29.07, 29.10, 29.26, 29.35, 29.46, 29.61, 29.64, 29.67 (2 × -OC(O)CH2CH2(CH2)8CH2CH2CH3), 31.90 (2 × -OC(O)(CH2)10CH2CH2CH3), 34.08 and 34.27 (2 × -OC(O)CH2(CH2)11CH3), 61.50 (C-3), 61.95 (C-1), 72.04 (C-2), 173.46 (>CHOC(O)(CH2)12CH3), 173.84 (-CH2OC(O)(CH2)12CH3); HRMS (ESI): m/z calcd for C31H58O5: 535.4338 [M+Na]+; found: 535.4336.White crystals, mp 54–56 °C, (lit. 56–58 °C ), Rf = 03, 0.1 g, 0.19 mmol), stigmasteryl chloroformate ) and DMAP  were dissolved in 50 mL of ethanol-free dry chloroform. The solution was stirred at room temperature for 48 h, and the solvent was evaporated in vacuo. Flash chromatography  afforded 0.17 g (92% yield) of 1,3-dimyristoyl-2-stigmasterylcarbonoylglycerol (4) with the following physical and spectral data:1,3-Dimyristoylglycerol , 1H NMR (600 MHz): δ 0.70 , 0.79 , 0.80 , 0.84 , 0.88 (CH2)12C3H), 0.95 , 1.00 , 1.01 , 1.02 , 1.06 ), 1.09–1.21 , H-17s, H-12s (α), one of CH2-28s), 1.22–1.33 CH2CH2(C2H)10CH3 and H-16s (β)), 1.38–1.67 , CH2-11s, H-2s (β), H-24s, H-25s, H-15s (α), 2 × -OC(O)CH2C2H(CH2)10CH3), 1.70 ), 1.89 ), 1.93–2.01 , H-7s (β), H-12 s (β)), 2.04 (H-20s), 2.31 and 2.32 C2H(CH2)11CH3), 2.36–2.44 , 4.18 and 4.19 , 4.32 and 4.33 , 4.48 , 5.02 , 5.10 , 5.15 , 5.39 ; 13C NMR (150 MHz): δ 12.02 (C-18s), 12.24 (C-29s), 14.11 (2 × -OC(O)(CH2)12CH3), 18.96 (C-27s), 19.23 (C-19s), 21.01 (C-11s), 21.08 (C-26s), 21.21 (C-21s), 22.69 (2 × -OC(O)(CH2)11CH2CH3), 24.33 (C-15s), 24.82 (2 × -OC(O)CH2CH2(CH2)10CH3), 25.40 (C-28s), 27.60 (C-2s), 28.89 (C-16s), 29.11, 29.27, 29.36, 29.48, 29.64 29.66 and 29.70 (2 × -OC(O)CH2CH2(CH2)8CH2CH2CH3), 31.82 (C-7s), 31.87 , 31.92 (2 × -OC(O)(CH2)10CH2CH2CH3), 34.03 (2 × -OC(O)CH2(CH2)11CH3), 36.53 (C-10s), 36.81 (C-1s), 37.92 (C-4s), 39.59 (C-12s), 40.48 (C-20s), 42.19 (C-13s), 49.99 (C-9s), 51.23 (C-24s), 55.92 (C-17s), 56.77 (C-14s), 61.99 and 62.00 , 72.68 (C-2), 78.46 (C-3s), 123.09 (C-6s), 129.29 (C-23s), 138.28 (C-22s), 139.16 (C-5s), 153.73 (Stig-OC(O)O-), 173.28 (2 × -OC(O)(CH2)12CH3); IR : 1739 (s), 1467 (s), 1278 (s), 1254 (s), 1083 (m), 787 (m), 723 (m); HRMS (ESI): m/z calcd for C61H106O7: 973.7836 [M+Na]+; found: 973.7831.White crystals, mp 43–45 °C, R3, 0.21 g, 0.41 mmol), DCC  and DMAP  in ethanol-free chloroform, stigmasteryl hemisuccinate  in chloroform was added dropwise. The reaction mixture was stirred at room temperature for 24 h and worked-up as described in paragraph 4.5.1. Flash chromatography  afforded 0.308 g (73% yield) of pure 1,3-dimyristoyl-2-stigmasterylsuccinoylglycerol (5) with physical and spectral data are given below:To the solution of 1,3-dimyristoylglycerol , 1H NMR (600 MHz): δ 0.69 , 0.79 , 0.80 , 0.84 , 0.88 (CH2)12C3H), 0.95 , 1.00 , 1.01 , 1.02 , 1.06 ), 1.10–1.21 , H-17s, H-12s (α), one of CH2-28s), 1.22–1.33 CH2CH2(C2H)10CH3 and H-16s (β)), 1.38–1.63 , CH2-11s, H-2s (β), H-24s, H-25s, H-15s (α), 2 × -OC(O)CH2C2H(CH2)10CH3), 1.70 ), 1.82–1.88 , H-2s (α)), 1.94–2.01 , H-12 s (β)), 2.03 (H-20s), 2.30–2.32 , 2.31 C2H(CH2)11CH3), 2.58–2.65 -CH2CH2-(O)CO-), 4.15 , 4.29 , 4.61 , 5.01 , 5.10 , 5.15 , 5.27 , 5.36 ; 13C NMR (150 MHz): δ 12.02 (C-18s), 12.23 (C-29s), 14.11 (2 × -OC(O)(CH2)12CH3), 18.96 (C-27s), 19.28 (C-19s), 21.00 (C-11s), 21.07 (C-26s), 21.20 (C-21s), 22.68 (2 × -OC(O)(CH2)11CH2CH3), 24.33 (C-15s), 24.83 (2 × -OC(O)CH2CH2(CH2)10CH3), 25.39 (C-28s), 27.72 (C-2s), 28.89 (C-16s), 29.12, 29.28, 29.30, 29.36, 29.49, 29.63, 29.66 and 29.69 (2 × -OC(O)CH2CH2(CH2)8CH2CH2CH3 and Stig-OC(O)CH2CH2(O)CO-), 31.82 (C-7s), 31.86 , 31.91 (2 × -OC(O)(CH2)10CH2CH2CH3), 34.00 (2 × -OC(O)CH2(CH2)11CH3), 36.57 (C-10s), 36.94 (C-1s), 38.04 (C-4s), 39.60 (C-12s), 40.49 (C-20s), 42.18 (C-13s), 50.02 (C-9s), 51.22 (C-24s), 55.91 (C-17s), 56.76 (C-14s), 61.94 , 69.44 (C-2), 74.40 (C-3s), 122.72 (C-6s), 129.27 (C-23s), 138.29 (C-22s), 139.50 (C-5s), 171.35 (Stig-OC(O)CH2CH2(O)CO-), 171.50 (Stig-OC(O)CH2CH2(O)CO-), 173.31 (2 × -OC(O)(CH2)12CH3); IR (cm−1): 1734 (s), 1467 (m), 1154 (s), 1091 (m), 721 (m); HRMS (ESI): m/z calcd for C64H110O8: 1029.8098 [M+Na]+; found: 1029.8085.White solid, mp 36–37 °C, R4,5) and dipalmitoylphosphatidylcholine (DPPC). The procedure was based on the hydration of a dry film and was described in previous papers [acylglycerol/nDPPC): 0.01, 0.02, 0.05, 0.1, and 0.2. The control liposomes contained only DPPC. The mixture of DPPC and the obtained acylglycerols were dissolved in chloroform. Next, the chloroform was slowly evaporated, first under nitrogen and then under vacuum pump for at least 2 h. Subsequently, the obtained dry film was hydrated with distilled water and shaken intermittently on a vortex at a temperature above the main phase transition of lipids (50 °C). We obtained the multilamellar vesicles (MLVs) when all lipids created a homogeneous milky suspension. Then, small unilamellar liposomes (SUVs) with a diameter of 120–140 nm were formed from MLVs using a Sonics VCX750 sonicator  for 15 min at 20 kHz. Liposomes were prepared using mixture of newly synthesized acylglycerols (s papers . Liposom1/2) were determined. The final concentration of the lipid in the sample was 25 mg/mL. The method was described precisely in our previous papers [Differential scanning calorimetry method was employed to evaluate the effect of hybrids of acylglycerols with stigmasterol on the thermotropic properties of MLVs liposomes. In particular, the temperature of pre- (Tp) and main phase transition (Tm), as well as the half width of the main peak (⊗Ts papers ,51. PrepThe fluorimetric method was used to determine the changes of polarity, fluidity, and main phase transition of SUVs liposomes composed of stigmasterol-containing acylglycerols and DPPC, prepared according to the procedure described in exc = 360 nm and λem = 425 nm; for Laurdan, λexc = 360 nm and the emission wavelengths were λem = 440 nm and λem = 490 nm. The details of this procedure was described in our previous papers [The excitation and emission wavelengths were as follows: for DPH, λs papers ,51.sn-1 and sn-3 positions and stigmasterol residue linked to sn-2 position by carbonate and succinate linker were designed, synthesized, and used to the formation of liposomes with DPPC as model phospholipid. The effect of newly synthesized lipids on the physicochemical properties of liposomes was determined, including the polarity, fluidity, and main phase transition using DSC and fluorimetric methods. In this work new acylglycerols containing natural myristic acid at Both stigmasterol-containing acylglycerols in molar ratios ranging from 0.2 to 0.05 significantly increase the order in the polar heads of the lipid bilayer and increase in the rigidity in the hydrophobic region of the membrane. Moreover, the presence of both acylglycerols in the membranes also shifts the temperature of the main phase transition towards higher temperatures. The trend of changes was similar for both compounds and the changes were proportional to the acylglycerols concentration. Taking into consideration the beneficial effects of stigmasterol and myristic acid on human health and the stabilization of liposome membranes containing synthesized compounds, the obtained hybrids of acylglycerols and stigmasterol are both good candidates for phytosterols delivery system and more attractive components of liposomes compared to cholesterol. The results of biophysical studies provide a basis for further research on the use of these compounds in the formation of novel lipid nanocarriers.


Chrysosporium sp. HSXSD-11-1, Cladosporium sp. HSXSD-12 and Acrostalagmus luteoalbus CH-6, were found to have the potential to produce antimicrobial compounds. Furthermore, the crude extracts of CH-6 displayed the strongest antimicrobial activities with 72.3–84.8% growth inhibition against C. albicans and Aeromonas salmonicida. The secondary metabolites of CH-6 were researched by bioactivity tracking combined with molecular networking and led to the isolation of two new α-pyrones, acrostalapyrones A (1) and B (2), along with one known analog (3), and three known indole diketopiperazines (4–6). The absolute configurations of 1 and 2 were identified through modified Mosher’s method. Compounds 4 and 6 showed strong antimicrobial activities. Remarkably, the antibacterial activity of 6 against A. salmonicida displayed two times higher than that of the positive drug Ciprofloxacin. This is the first report to discover α-pyrones from the genus Acrostalagmus, and the significant antimicrobial activities of 4 and 6 against C. albicans and A. salmonicida. This study further demonstrates the great potential of Antarctic fungi in the development of new compounds and antibiotics.With the increasingly serious antimicrobial resistance, discovering novel antibiotics has grown impendency. The Antarctic abundant microbial resources, especially fungi, can produce unique bioactive compounds for adapting to the hostile environment. In this study, three Antarctic fungi,  Antimicrobial drugs, including antibiotics, antivirals, antifungals, and antimalarials, are medicines that are active against various infections . HoweverPseudomonas aeurigenosa, Mycobacterium phlei, and MRCNS (methicillin-resistant coagulase-negative staphylococci) + , 275.1250 [M + Na]+.Acrostalapyrone A (2): amorphous powder; c 0.067, CH2Cl2); UV (CH2Cl2) λmax (log ε) 234 (4.67), 333 (4.48) nm; 1H and 13C NMR data, see m/z 253.1434 [M + H]+ , 275.1252 [M + Na]+.Acrostalapyrone B (S)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride ((S)-MTPA-Cl) (10 μL) was added to a stirred pyridine solution (500 μL) of 1 (1.0 mg) and 4-(dimethylamino)pyridine (2.0 mg). The mixture was stirred at rt for 10 h. The reaction mixture was evaporated to dryness under reduced pressure to get the reaction product, and then purified by HPLC eluted with 80% MeOH–H2O to give (S)-MTPA ester 1s. Treatment of 1 (1.0 mg) with (R)-MTPA-Cl (10 μL) as described above yielded the corresponding (R)-MTPA ester 1r. By the same procedure as for the preparation of the (S)- and (R)-MTPA esters of 1, (S)-MTPA ester (2s) and (R)-MTPA ester (2r) of 2 were obtained.(S)-MTPA ester of 1 (1s): 1H NMR  δ 7.53 , 7.43–7.37 , 6.34 , 6.11 , 5.10–5.05 , 3.90 , 3.57 , 2.79 , 1.94 , 1.84 , 1.32 , 0.95 ; HR-APCI-MS m/z 469.1828 [M + H]+ .(R)-MTPA ester of 1 (1r): 1H NMR  δ 7.52 , 7.44–7.38 , 6.40 , 6.12 , 5.08–5.04 , 3.90 , 3.52 , 2.84 , 1.95 , 1.89 , 1.25 , 1.06 ; HR-APCI-MS m/z 469.1826 [M + H]+ .-MTPA ester of 2 (2s): 1H NMR  δ 7.49–7.44 , 7.37–7.30 , 6.36 , 6.09 , 5.03 , 3.89 , 3.47 , 2.85 , 1.96 , 1.86 , 1.30 , 1.07 ; HR-APCI-MS m/z 469.1828 [M + H]+ .(R)-MTPA ester of 2 (2r): 1H NMR  δ 7.48 , 7.35–7.30 , 6.27 , 6.02 , 5.04 , 3.88 , 3.56 , 2.84 , 1.95 , 1.81 , 1.35 , 0.99 ; HR-APCI-MS m/z 469.1830 [M + H]+ . and B (2), and one known analog, multiforisin G (3), as well as three known indole diketopiperazines, luteoalbusin A (4), gliocladine C (5), and T988 C (6). Compounds 4 and 6 showed significant antimicrobial activities against C. albicans and A. salmonicida. In particular, the antibacterial activity against A. salmonicida of 6 displayed two times higher than that of the positive drug Ciprofloxacin. This is the first time to find α-pyrones in the fungal genus Acrostalagmus and the first report to discover significant antimicrobial activities of compounds 4 and 6 against C. albicans and A. salmonicida. This study further demonstrates the great potential of Antarctic fungi in the development of new compounds and antibiotics.In summary, three antimicrobial fungi,


Their structures were elucidated by extensive analysis of NMR and HRESIMS data. Their absolute configurations were determined by CD spectra, enzymatic hydrolysis, and GCMS analysis. Furthermore, all compounds were evaluated for anti-proliferative activities against various human cancer cellsin vitro. Compounds 2 and 3 exhibited weak inhibitorypotency against five human cancer cells.Five new furofurans lignans, Brasesquilignan A–E ( Selaginella braunii Baker is a perennial herb belonging to the genus Selaginella and mainly distributed south of the Yangtze River + . The 1H NMR spectrum of 1 exhibited signals for eight aromatic protons, indicating the existence of one set of 1, 3, 5-trisubstituted benzene system, 6.87 , and 6.78 ), one set of 1, 3, 4-trisubstituted aromatic proton signals , 6.78 , and 6.73 ), and one set of the 1, 3, 4, 5-tetra-substituted benzene system  and 6.75 ). Moreover, there was one anomeric proton signal at δH 4.25 , as well as three methoxyl proton signals at δH 3.79 , 3.76 , and 3.76 . The 13C NMR spectrum of 1 showed 30 carbon signals, of which δC 147.3, 119.2, 115.6, 111.0, 70.6, and 56.1 were overlapping signals. The 1H and 13C NMR -isoeucommin A 4‴-O-β-D-glucopyranoside +. The 1H NMR and 13C NMR data , 6.75 , and 6.72 ; a set of 1, 2, 4-trisubstituted aromatic proton signals, δH 7.06 , 6.97 , and 6.85 ; and a set of 1, 3, 4, 6-tetra-substituted aromatic proton signals, δH 6.87  and 6.86 . The location of the functional groups and NMR data assignments of 3 were determined by HMBC and HSQC spectroscopic analysis , enzymatic hydrolysis, and GC-MS analysis. Comparing coupling constants  and CD spectra of 3 with 1  -4, 2″- dihydroxy-3, 2′, 5″-trimethoxy-4″-O-β-D-glucopyranosyl-7, 9′: 7′, 9-diepoxy-4, 8″-oxy-8, 8′- sesquineolignan-7″, 9″-diol, named brasesquilignan C.Compound NMR data  of 3 weranalysis . The sug3 with 1 , the abs4 was obtained as a white amorphous powder, which had a molecular formula of C37H46O14 based on a protonated molecular ion peak at m/z 769.2638 [M + Na]+  in the HRESIMS data. The 1H NMR and 13C NMR data , 6.82 , 6.78 , and 6.70 ; a set of 1, 3, 5-trisubstituted aromatic proton signals, δH 6.82  and 6.70 ; and a 1, 2, 3, 4, 6-penta-substituted aromatic proton signal, δH 6.74 . In addition, compound 4 had one more methyl proton signal at δH 1.07 . In the 13C NMR spectrum of 4, the carbon signals δC 67.5, 52.9, 79.6,and 22.0 were significantly different from 1, and δC 79.6 is a quaternary carbon, suggesting that there is a methyl on the furan ring of 4. Further, the correlation between δH6.82  and δH6.78  in the 1H-1H COSY spectrum and the HMBC  to C-7′ (δC 82.3), H-3′  to C-8″ (δC 87.3),and 7-CH3  to C-8 (δC 67.5) indicated that the methyl was located at C-7  and C-4″ (δC132.4) indicated that the glycosyl was located at C-4″. The coupling constant of the anomeric protons (J = 7.8 Hz) prompted the existence of β-configuration. Moreover, the presence of D-glucose was confirmed by enzymatic hydrolysis and GC-MS analysiscompared with standard material. The coupling constants  showed that the relative configurations were both threo. The CD spectrum of 4  -5, 3″-dihydroxy-3, 2″, 6″-trimethoxy-4″-O-β-D-glucopyranosyl-7, 9′: 7′, 9-diepoxy-7-methyl-4, 8″-oxy-8, 8′-sesquineolignan-7″, 9″-diol, named brasesquilignan D.Compound NMR data  suggestethe HMBC  correlatd at C-7 . The HMBd at C-7  correlatrum of 4  showed a5 was obtained as a white amorphous powder. Its molecular formula was determined to be C31H36O9by HRESIMS at m/z 553.2416 [M + H]+ . Compound 5 had a similar 1H and 13C spectrum , 6.76 , 6.74 , and 6.59 ; one set of 1, 3, 4-trisubstituted aromatic proton signals, δH 6.65  and 6.60 ; and a set of 1, 3, 4, 5-tetrasubstituted aromatic proton signals, δH 6.73  and 6.71 . The 13C NMR spectrum showed18 aromatic carbon signals, δC 104.1~153.6; 4 methoxy carbon signals, δC 56.5 (5″-OCH3), 56.4 (4″-OCH3), 56.1 (3″-OCH3), and 56.0 (3′-OCH3); and 9 aliphatic carbon signals. In the HMBC spectrum, the correlation between H-5′  with C-8″ (δC 84.0) revealed that the connection of the two fragments is C-4′-O-C-8″. The location of the other functional groups and NMR data assignments of 5 were determined by HMBC and HSQC spectroscopic analysis  and ΔδH–9′(0.4) suggested thatH-7/H-8 and H-7′/H-8′ wereTrans + ,1H NMR (500 MHz in DMSO-d6) and 13C NMR (125 MHz in DMSO-d6). For data, see Brasesquilignan A (2): white amorphous powder, c 0.05, MeOH), HPLC-UV (ACN-H2O) λmax nm: 205, 225, 280, HRESIMS, m/z: 895.8730 [M+H]+ ,1H NMR (500 MHz in DMSO-d6) and 13C NMR (125 MHz in DMSO-d6).For data, see Brasesquilignan B (3): white amorphous powder, c 0.2, MeOH), HPLC-UV (ACN-H2O) λmax nm: 205, 225, 280, HRESIMS, m/z: 755.2473 [M+Na]+,1H NMR (500 MHz in DMSO-d6) and 13C NMR (125 MHz in DMSO-d6).For data, see Brasesquilignan C (4): white amorphous powder, 2O) λmax nm: 205, 225, 280, HRESIMS, m/z: 769.2638 [M+Na]+ ,1H NMR (500 MHz in DMSO-d6) and 13C NMR (125 MHz in DMSO-d6). For data, see Brasesquilignan D (5): white amorphous powder, c 0.05, MeOH), HPLC-UV (ACN-H2O) λmax nm: 205, 225, 280, HRESIMS, m/z: 553.2416 [M+H]+ , 1H NMR (500 MHz in DMSO-d6) and 13C NMR (125 MHz in DMSO-d6). For data, see Brasesquilignan E (1–5 (each 0.5 mg), cellulase (400 u/mg), and buffered saline solution  were added in centrifuge tube , and incubated for 96 h in 37 °C. After extraction with CHCl3, the aqueous layer of reaction mixture was concentrated and dried to obtain the monosaccharide fraction. The residue was dissolved in pyridine (0.6 mL) with 1.0 mg of L-cysteine methyl ester hydrochloride and heated . Then, trimethylsilylimidazole (0.6 mL) was added and heated . The reaction mixture was analyzed by GC-MS under the following conditions: Column, Rtx-5MS ; front inlet 300 °C, column 150–300 °C at 15 °C/min. The monosaccharides of compounds were confirmed by comparing the retention times with those of standard sugar (subjected to the same method).Compounds 2. MCF-7 and A549  were cultured by DMEM/HIGH GLUCOSE medium (Hyclone) supplemented with 10% FBS and 100 U/mL penicillin/streptomycin at 37 °C and 5% CO2. All human cancer cells were plated in 96-well plates . Then, each well was supplemented at various concentrations of test compounds in triplicate for 24 h, and MTT (20 μL 5 mg/mL) was added. After incubation for 4 h, the medium was removed, and DMSO (150 μL) was added before further incubation . The OD (570 nm) value of each well was measuredwitha microplate reader MD5 . The half-inhibitory concentration (IC50) values were calculated by SPSS 25. software.All compounds were tested with positive control . MDA-MB-231, SK-MEL-28, and A375  were cultured in DMEM/F12 (1:1) medium (Hyclone) supplemented with 10% FBS and 100 U/mL penicillin/streptomycin at 37 °C and 5% CO


Metasequoia glyptostroboides Hu & Cheng fruits (Cupressaceae) significantly reduced the aggregation of Aβ into oligomers and fibrils determined by Thioflavin T (ThT) assay. The solvent-partitioned ethyl acetate layer was further separated based on the bioassay-guided isolation method combined with the ThT assay. As a result, five compounds were isolated and elucidated as taxoquinone (1), sugiol (2), suginal (3), sandaracopimarinol (4), and sandaracopimaradien-19-ol (5) by comparing NMR data with references. All the compounds significantly reduced the aggregation of Aβ and enhanced the disaggregation of pre-formed Aβ aggregates in a dose-dependent manner. Furthermore, the inhibition of Aβ aggregation by the compounds protected PC12 cells from Aβ aggregate-induced toxicity. Among the five compounds, sandaracopimarinol (4) and sandaracopimaradien-19-ol (5) were the most effective. These results suggest that M. glyptostroboides and isolated five compounds have a potential for further study to be developed as anti-AD agents.Alzheimer’s disease (AD) is a serious neurodegenerative brain disease that interferes with daily life. The accumulation of beta-amyloid (Aβ), along with oxidative stress-inducing neurocellular apoptosis, has been considered one of the causes of AD. Thus, the purpose of this study is to find natural products that can reduce Aβ accumulation. The ethanol extract of  As the proportion of the elderly population increases, dementia, which exhibits various cognitive impairments, becomes an important social and economic problem. It is predicted that 1 out of 85 people will develop dementia by the year 2050 ,4. Among42, which consists of 42 amino acids, is known to be the most toxic. Aβ produced from APP accumulates extracellular compartments to form Aβ oligomers and fibrils, which cause the neurodegeneration, leading to the onset of AD symptoms [Aβ is a protein produced by the proteolytic cleavage of amyloid precursor protein (APP) by the proteases called β- and γ-secretases. The cleavage of APP by β-secretase produces the large N-terminal ectodomain of APP (sAPPβ) and the membrane-bound C-terminal fragment, C99. The subsequent cleavage of C99 by γ-secretase eventually produces Aβ, which consists of 36–43 amino acid residues ,8. Aβ42,symptoms .Metasequoia glyptostroboides Hu & Cheng fruits showed good inhibitory activity against Aβ aggregation. M. glyptostroboides is a deciduous coniferous tree belonging to the Cupressaceae family, distributed in many regions such as Europe, East Asia, and North America. It has feathery leaves and conical reddish-brown fruits [M. glyptostroboides such as anti-bacterial, antioxidant, and anti-fungal effects have been reported [M. glyptostroboides have not been studied for their effects on AD. Therefore, in this study, the ethanol extract of M. glyptostroboides fruits was tested for the potential inhibitory effects on Aβ aggregation with Thioflavin T (ThT) assay. In order to isolate active constituents that inhibit Aβ aggregation, a bioassay-guided isolation method using diverse column chromatographies (CCs) was employed. The structures of isolated compounds were elucidated by the comparison of NMR data with the references. The inhibitory effect of isolated compounds against Aβ aggregation and the protection of PC12 cells from Aβ aggregate-induced toxicity were determined by ThT and MTT assays, respectively.Despite the efforts to discover the mechanisms of AD pathology, the exact causes of AD are not elucidated, nor are there suitable treatments available. Therefore, the experts mentioned that the prevention of AD could be the best treatment ,12. Prevn fruits . Until nreported ,15. It hM. glyptostroboides extract on Aβ aggregation, the level of Aβ aggregation was measured by ThT assay. As shown in M. glyptostroboides significantly reduced the aggregation of Aβ in a dose-dependent manner. Particularly, 50 μg/mL of the extract inhibited the aggregation of Aβ close to the control level. Then, the ethanol extract was partitioned based on solvent polarity, and four fractions including n-hexane, dichloromethane, ethyl acetate, and water fractions were obtained. These four fractions (50 μg/mL) were also tested to measure the inhibitory effect on the aggregation of Aβ. As a result, n-hexane, dichloromethane, ethyl acetate, and water fractions at 50 µg/mL significantly reduced the aggregation of Aβ down to 45.9, 28.4, 8.8, and 55.7%, respectively, compared to the Aβ only control group. Among the four fractions, the ethyl acetate fraction was the most active and was used for further experiments.In order to determine the inhibitory effects of M. glyptostroboides. The structures of five compounds (compounds 1–5) were elucidated based on the 1H and 13C-NMR data, and comparison with relevant references [1), sugiol (2), suginal (3), sandaracopimarinol (4), and sandaracopimaradien-19-ol (5)  using silica gel as a stationary phase based on the bioassay-guided isolation method. As a result, five compounds were isolated from ferences ,18,19,209-ol (5) .1–5 was measured at various concentrations  to evaluate the antioxidant activity. As shown in 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging activity of compounds 1–5 isolated from M. glyptostroboides on the aggregation of Aβ, ThT assay was performed. As shown in 4 and 5 at 100 µg/mL reduced Aβ aggregation down to 26.9 and 25.4% compared to Aβ only group, which was close to the control group without Aβ. In addition, Aβ aggregations were reduced to 37.4 and 43.9% by the treatment with compounds 4 and 5 at 4 µg/mL. The inhibitory activities of compounds 4 and 5 at 4 µg/mL on Aβ aggregation were higher than compounds 1, 2, and 3 at 100 µg/mL which were 47.9, 66.8, and 54.8%, respectively.To determine the inhibitory effects of compounds 1–5, and then the level of Aβ aggregation was detected by ThT assay. All of the compounds exhibited a positive effect on Aβ disaggregation in a dose-dependent manner. In particular, compounds 4 and 5 efficiently disaggregated the pre-aggregated Aβ and the level of aggregated Aβ was down to 50.0 and 53.2%, respectively, at 100 µg/mL compared to Aβ only group  and andaracopimaradien-19-ol (5) efficiently decreased the aggregation of Aβ and enhanced the disaggregation of Aβ aggregates. Furthermore, sandaracopimarinol (4) and andaracopimaradien-19-ol (5) rescued the PC12 cells from Aβ aggregate-induced toxicity by inhibiting Aβ aggregation.Aβ, which is produced by proteolytic fragmentation of APP by β- and γ-secretases is considered one of the major causes of AD. In particular, over-produced Aβ aggregates into oligomers and fibrils, and Aβ aggregates are known to cause neurotoxicity and neuronal loss because of the increased mitochondrial dysfunction and ER stress ,5,8. In M. glyptostroboides is the only surviving large deciduous conifer known as a “living fossil” among the genus Metasequoia [M. glyptostroboides has been successfully cultivated in nearly 50 countries, including Asia, Africa, Europe, and the United States [M. glyptostroboides has been reported to have anti-bacterial, anti-fungal, antioxidant, and anti-inflammatory activities as well as neuroprotective effects [M. glyptostroboides includes the largest group of terpenoids. The most frequently identified terpenoid is a monoterpenoid and the second is a sesquiterpenoid [asequoia ,17,18. Md States . M. glyp effects ,15. It herpenoid . Dihydroerpenoid . In addierpenoid .M. glyptostroboides fruits was selected for further study because it efficiently inhibited Aβ aggregation determined by ThT assay. Then, the bioassay-guided isolation of ethanol extract of M. glyptostroboides fruits allowed to isolate five pure compounds. The structures of these compounds were elucidated based on 1H- and 13C-NMR data by comparison with references [1–3 were identified as taxoquinone, sugiol, and suginal, respectively, which are abietane-type diterpenoids. The aromatic abietane structure is the most abundant naturally occurring abietane-type diterpenoid. Aromatic abietane-type diterpenoids, such as ferruginol, sclareol, isorosmanol, and carnorol have been reported to possess various biological properties, including anti-bacterial, anti-cancer, anti-fungal, anti-tumor, anti-viral, and anti-inflammatory activities [The ethanol extract of ferences ,18,19,20tivities ,25,26,271) is known to be a natural anti-bacterial agent against a wide range of bacteria with little or no toxicity for centuries [1) has potent anti-diabetic and anti-melanin potential due to the inhibition of α-glucosidase and tyrosinase inhibition [2) is also reported to efficiently inhibit α-glucosidase and tyrosinase and have potential anti-diabetic and anti-menalin effects [3) has been reported to have anti-bacterial activity [Taxoquinone  and sandaracopimaradien-19-ol (5), pimarane-type diterpenoids. Pimarane-type diterpenoids are frequently produced from plants and fungi, but rarely produced from bacteria, insects, and marine organisms [4) is known to have strong anti-bacterial and antioxidant activity. Sandaracopimaradien-19-ol (5) is reported to have anti-bacterial activity [4) and sandaracopimaradien-19-ol (5) against Aβ aggregation were first reported in this study.In addition, compounds rganisms . Pimaranrganisms ,32,33. Sactivity . Even thM. glyptostroboides fruits and five diterpenoids isolated from M. glyptostroboides including taxoquinone (1), sugiol (2), suginal (3), sandaracopimarinol (4), and sandaracopimaradien-19-ol (5) significantly reduced the aggregation of Aβ and enhanced the disaggregation of Aβ aggregates from oligomers and fibrils to monomers. Furthermore, sandaracopimarinol (4), and sandaracopimaradien-19-ol (5) protected the neuronal cells from Aβ aggregate-induced toxicity by reducing Aβ aggregation. Therefore, the extract of M. glyptostroboides fruits and active constituents has the potential to prevent AD by inhibiting Aβ aggregation and protecting the neuronal cells.Taken together, these results suggest that the extract of 1-42 was purchased from GL Biochem . ThT and DPPH were purchased from Sigma . DMSO and 3--2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Biosesang . All the solvents for the extraction and CCs were purchased from Samchun Pure Chemical .Thin Layer Chromatography (TLC) plates were used including TLC silica gel 60 F254 . Stationary phases of open CC were Silica gel Si 60 . RPMI 1640 was purchased from Welgene . Fetal bovine serum (FBS) and horse serum were purchased from Gibco . AβM. glyptostroboides were collected from Dankook University  in January 2020. A voucher specimen has been deposited in the Pharmacognosy Laboratory of College of Pharmacy, Dankook University, Republic of Korea. The dried and pulverized M. glyptostroboids fruits (4 kg) were extracted three times with ethanol (30 L) under reflux for 24 h at room temperature. The ethanol filtrate was evaporated under vacuum to yield the ethanol extract (310 g). The extract was suspended in distilled water and then partitioned sequentially into n-hexane, dichloromethane, ethyl acetate, and water. Four fractions including n-hexane (115.2 g), dichloromethane (46.0 g), ethyl acetate (30.9 g), and water (115.8 g) fractions were obtained.The fruits of n-hexane and acetone (100:1), and 10 subfractions (MGE1~MGE10) were obtained. The subfraction 8 (MGE8) was further fractionated by open silica CC with solvent mixture of n-hexane and acetone (100:5), and compound 1 (89.5 mg) and compound 2 (222.5 mg) were obtained as pure compounds.The ethyl acetate fraction was fractionated by open CC using silica gel as stationary phase with solvent mixture of n-hexane and acetone (50:1). Among 9 subfractions, MGE3-9 was chromatographed by open silica gel CC with solvent mixture of n-hexane and acetone (10:1) and 6 (MGE3-9-1~MGE3-9-6) subfractions were obtained. The subfraction 3-9-5 (MGE3-9-5) was chromatographed by open silica gel CC with solvent mixture of chloroform and methanol (100:1), and compound 3 (76.0 mg) was obtained as a pure compound.The subfraction 3 (MGE3) was further fractionated by open CC using silica gel with solvent mixtures of 4 (55.0 mg).The subfractions 6 and 7  were combined and applied to open CC using silica gel with solvent mixture of dichloromethane and ethyl acetate (1:0~1:1), and 9 fractions  were obtained. The subfraction 6,7-4  was further chromatographed and purified by silica gel CC with solvent mixture of chloroform and methanol (1:0 ~ 1:1) to give compound n-hexane and acetone (20:1), and 6 fractions (MGE4-1~MGE4-6) were obtained. Subfractions 4-2 (MGE4-2) were chromatographed and purified by silica gel CC with solvent mixture of n-hexane and acetone (2:1), and compound 5 was obtained as a pure compound (16.7 mg).The subfraction 4 (MGE4) was chromatographed by MPLC  using silica gel with solvent mixture of 1 (Taxoquinone) 1H-NMR : δ 4.77 , 3.80 , 3.13 , 2.65 , 2.18 , 1.34–1.80 , 1.32 , 1.20 , 1.18 , 0.91 , 0.90 . 13C-NMR : δ 189.6 (C-14), 183.7 (C-11), 150.9 (C-12), 147.9 (C-9), 144.2 (C-8), 124.2 (C-13), 68.0 (C-7), 48.8 (C-5), 40.9 (C-3), 39.4 (C-10), 36.0 (C-1), 33.2 (C-18), 33.1 (C-4), 26.1 (C-6), 23.9 (C-15), 21.6 (C-19), 19.8 (C-16), 19.8 (C-17), 19.8 (C-20), 18.6 (C-2).Compound 2 (Sugiol) 1H-NMR : δ 7.61 , 6.75 , 3.13 , 2.49 , 2.13 , 1.75 , 1.34–1.80 , 1.13 , 1.12 , 1.11 . 0.90 , 0.85 . 13C-NMR : δ 197.0 (C-7), 160.6 (C-12), 156.3 (C-9), 133.0 (C-13), 125.5 (C-14), 123.1 (C-8), 109.8 (C-11), 49.6 (C-5), 41.4 (C-3), 38.0 (C-1), 38.0 (C-10), 36.1 (C-6), 33.4 (C-4), 32.8 (C-18), 26.6 (C-15), 23.6 (C-20), 22.9 (C-16), 22.7 (C-17), 21.7 (C-19), 19.0 (C-2).Compound 3  1H-NMR : δ 9.88 , 7.81 , 6.92 , 3.16 , 1.80–2.08 , 1.50–1.91 , 1.24–1.71 , 1.45–1.60 , 1.20 , 1.01 , 1.26 , 1.26 . 13C-NMR : δ 206.2 (C-18), 191.6 (C-7), 157.9 (C-9), 151.2 (C-12), 134.8 (C-13), 132.4 (C-14), 128.0 (C-8), 114.9 (C-11), 64.6 (C-5), 40.6 (C-4), 37.8 (C-1), 37.5 (C-10), 33.7 (C-6), 30.8 (C-3), 28.2 (C-15), 27.4 (C-19), 26.8 (C-20), 22.3 (C-16), 22.3 (C-17), 19.4 (C-2).Compound 4 (Sandaracopimarinol) 1H-NMR : δ 5.77 , 5.21 , 4.90 , 4.87 , 3.40 , 3.12 , 2.23 , 2.06 , 1.75 , 1.00–1.71 , 1.45–1.60 , 1.32–1.47 , 1.34 , 1.30–1.47 , 1.34–1.58 . 13C-NMR : δ 149.2 (C-15), 137.1 (C-8), 128.8 (C-14), 110.1 (C-16), 72.3 (C-18), 50.6 (C-9), 48.0 (C-5), 39.0 (C-1), 38.2 (C-10), 37.9 (C-4), 37.5 (C-13), 35.8 (C-7), 35.6 (C-3), 34.7 (C-12), 26.1 (C-17), 22.5 (C-6), 18.9 (C-11), 18.4 (C-2), 18.1 (C-19), 15.7 (C-20).Compound 5 (Sandaracopimaradien-19-ol) 1H-NMR : δ 5.70 , 4.90 , 4.95 , 5.12 , 3.82 , 3.42 , 2.31 , 2.02 , 1.83 , 1.75 , 1.68 , 1.66 , 1.48 , 1.44 , 1.33 , 1.27 , 0.97–1.27 , 1.03 , 1.22 , 1.03 , 0.98 , 0.98 , 0.97 . 13C-NMR : δ 149.1 (C-15), 136.8 (C-8), 128.9 (C-14), 110.1 (C-16), 73.2 (C-19), 50.7 (C-9), 48.8 (C-5), 38.9 (C-1), 38.2 (C-10), 37.5 (C-4), 36.7 (C-13), 36.1 (C-7), 35.8 (C-12), 34.7 (C-3), 26.0 (C-17), 22.6 (C-18), 21.1 (C-6), 18.9 (C-11), 18.3 (C-2), 15.7 (C-20).Compound 1-42 was dissolved in DMSO at 1 mg/mL concentration and test samples were also dissolved in DMSO. To monitor the effects of test samples on the Aβ aggregation, 20 µM of Aβ1-42 was incubated with various concentrations of test samples at 37 °C for 24 h. For the control group, DMSO was used instead of test samples. Then, 3 µM of ThT was added, and fluorescence was measured after 30 min using an Emax precision microplate reader  with excitation at 442 nm and emission at 485 nm. The Aβ only was used as a control, and each assay was repeated 3 times.To quantify the aggregation of Aβ to oligomers and fibrils, ThT assay was performed. The Aβ1-42 was pre-incubated at 37 °C for 24 h. After that, various concentrations of test samples or DMSO were added and incubated at 37 °C for additional 24 h. Then, 3 µM of ThT was added and fluorescence was measured after 30 min using an Emax precision microplate reader (Molecular Devices) with excitation at 442 nm and emission at 485 nm. The Aβ only was used as a control and each assay was performed in triplicate.To monitor the disaggregation effects of test samples on the pre-aggregated Aβ, the ThT assay was performed. Briefly, 20 µM of AβAcontrol − Atest)/Acontrol * 100.Antioxidant activity was measured using DPPH. The sample (10 µL) was mixed with 0.2 mM DPPH reagent (190 µL). The mixture was vortexed and kept in a dark place at 37 °C for 30 min. The change in absorbance was measured with an E-max precision microplate reader (Molecular Devices) at 540 nm. Ascorbic acid was used as a positive control. The inhibition rate was converted into a percentage of the difference between the absorbance values of a negative control treated with DMSO and the test samples. All experiments were repeated three times. Percent inhibition was calculated as (%) = (2 at 37 °C.PC12 cells (rat pheochromocytoma cells) were obtained from Korea Cell Line Bank  and grown in RPMI 1640 with 15% heat-inactivated horse serum and 5% heat-inactivated FBS. The cells were incubated in a humidified 5% CO4 cells per well. Plates were incubated at 37 °C for 3 h to allow the cells to attach to the plates. Test samples were diluted with DMSO and added to individual wells. DMSO was added in case of control group. The plates were then incubated for an additional 24 h at 37 °C. The cell viability was determined using an MTT toxicity assay by adding 10 µL of 5 mg/mL MTT to each well. After 3 h incubation at 37 °C, 70 µL of the medium was gently removed, and then 70 µL of DMSO was added to each well. Plates were incubated at room temperature for 30 min to dissolve the MTT formazan crystals, and then the absorbance was measured at 540 nm using an E-max precision microplate reader (Molecular Devices). An average from 3 replicate wells was used for each sample, and each assay was performed in triplicate.In order to determine the cytotoxicity of test samples, MTT assay was performed. PC12 cells were harvested from flasks and plated in 96-well plates with 6 × 101-42 (10 µM) were mixed together in 96-well plates and incubated at 37 °C for 24 h to inhibit the formation of Aβ aggregates. For the control group, DMSO was added instead of test samples. The mixture was added to PC12 cells and incubated at 37 °C for an additional 24 h. The cell viability was determined using MTT assay. An average from 3 replicate wells was used for each sample, and each assay was performed in triplicate.To investigate whether the inhibition of Aβ aggregation by test samples could rescue the cells from Aβ aggregate-induced toxicity, test samples  and Aβp-value smaller than 0.05 were considered statistically significant.All data in figures are expressed as mean ± SD of three different experiments. Data were analyzed using a two-tailed Student’s t-test in Microsoft Excel ver. 2016. Differences with a


The developed multistep method employed commercially available acid chlorides differing in alkyl chain length and terminal substituent, hydrazine hydrate, and phosphorus oxychloride. The intermediate bromine-containing 2,5-dialkyl-1,3,4-oxadiazoles were easily substituted with diisopropyl iminodiacetate, followed by hydrolysis in aqueous methanol solution giving the corresponding 1,3,4-oxadiazoles bearing carboxymethylamino substituents. The structure of all products was confirmed by conventional spectroscopic methods including  Oxadiazoles are five-membered heterocyclic compounds composed of two nitrogen atoms and one oxygen atom. Depending on the heteroatom position, oxadiazoles exist in the form of several isomers , includiN,N′-diacylhydrazines: polyphosphoric acid (PPA) [2SO4) [3) [2) [3SO2)2O) [2O5) [3∙OEt2) [N-acylhydrazones with oxidizing agents such as cerium ammonium nitrate (CAN) [2) [4) [2) [One of the most popular methods for 1,3,4-oxadiazole derivatives preparation include the cyclodehydration reaction. The following reagents are commonly employed in the cyclization of id (PPA) , sulfuri) [2SO4) , phosphoSO4) [3) , thionyl [3) [2) , trifluo3SO2)2O) , phosphoO) [2O5) , boron t[3∙OEt2) , and Bur[3∙OEt2) . Additiote (CAN) , bromineCAN) [2) , potassi [2) [4) , lead(IV [4) [2) , chloram [4) [2) , 2,3-dic [4) [2) , and hyp [4) [2) .N-(hydroxyethyl)ethylenediaminetriacetic acid (HEEDTA) [N,N′-bis(o-hydroxy-p-methylphenyl)acetic acid (EDDHMA) [1,3,4-Oxadiazoles containing alkyl chains at the 2 and 5 positions and substituted with carboxymethylamino groups are of particular interest. Generally, the presence of aminopolycarboxylic functionalities promote complexing properties in such derivatives, allowing for binding to metal cations. The literature shows a range of organic ligands of this type, some of which have been approved for use in medicine and agriculture. These include ethylenediaminetetraacetic acid (EDTA) , N-(hydr(HEEDTA) , ethylen(EDDHMA) , diethyl(EDDHMA) , nitrilo(EDDHMA) , glucohe(EDDHMA) , and cit(EDDHMA) ,43. Unfo(EDDHMA) . On the N,N′-diacylhydrazine derivatives with different alkyl chain lengths. In the second step, they were cyclized using POCl3, a known cyclodehydration reagent. Finally, the substitution reaction between bromine-containing 1,3,4-oxadiazole derivatives and iminodiacetic acid was studied.According to the above data, we assumed that the combination of the 1,3,4-oxadiazole core containing oxygen and two nitrogen atoms in the ring with aminopolycarboxylic groups allows for the development of a new family of organic ligands with potential applications as complexing agents in medicine and agriculture. Herein, we developed an effective method for the preparation of new symmetrically substituted 1,3,4-oxadiazole derivatives containing carboxymethylaminoalkyl groups at the 2 and 5 positions. Initially, a three-step transformation was conducted using commercially available reagents . In the N,N′-diacylhydrazine derivatives (2a–d) (1a–d) (1a) as the starting material. The optimization study consisted of examining the base (triethylamine (TEA) and sodium carbonate), solvent , and the influence of temperature. The best results were obtained at low temperature (0 °C) in diethyl ether using aqueous sodium carbonate as the base. The products were purified via recrystallization from either methanol or ethanol. The yields of the obtained hydrazine derivatives (2a–d) were 65–79%   using co) (1a–d)  differin3a–d)  were obtained in 40–76% yield and were used for the subsequent reactions without purification   involvedfication .4), which could directly react with the formed 2,5-dialkyl-1,3,4-oxadiazole derivatives (3a–d) containing bromine substituents -1,3,4-oxadiazoles (3a–d), followed by hydrolysis to restore the carboxyl groups. Hence, the esterification reactions were performed using ethanol (a) and isopropanol (b) as substrates, and in the presence of sulfuric acid as the catalyst  were obtained in good yields (37–52%).As part of the synthesis, we planned to use iminodiacetic acid , oxadiazole derivative (3a–d), and base in an aprotic solvent (3a) and diethyl iminodiacetate (5a) were first examined. When the reaction was conducted at room temperature, main product 6a was produced but in a low yield, at 23% (6a in a 91% yield (The next step was to perform the substitution reaction involving the appropriate ester ( solvent . In orde3b–d), and producing products (6a–e) in a 71–91% yield (6a–e) were purified by column chromatography on silica gel using ethyl acetate as the eluent.Among the solvents tested, acetonitrile gave the best result (6). Various literature methods were examined, including anhydrous lithium chloride [7b, 7d) at the side alkyl chains (7b) when the molar ratio between ester 6 and NaOH was 1:10. Methanol was found to be the best solvent owing to its ability to dissolve the substrate. The final products  were purified by recrystallization from methanol, providing the pure products in a 54–68% yield (The last step in the synthetic pathways was the restoration of the carboxyl groups from esters groups (chloride  and the chloride . It was l chains . The lit1H and 13C NMR spectroscopy (see 6a–e), and not described so far in the literature, characteristic 1H NMR signals included the doublet at 1.25 ppm and septet at 5.00 ppm corresponding to the isopropyl group. The singlet at 3.50 ppm was related to the iminodiacetate moiety. The remaining signals at 1.00–3.00 ppm corresponded to the alkyl side chain between the oxadiazole and diisopropyl ester. In the case of 13C NMR, the characteristic C2 and C5 signals of the heterocyclic 1,3,4-oxadiazole ring were found at 165.0 ppm. The remaining peaks from carbonyl groups were located at 170.0 ppm, while two signals from isopropyloxy group were found at 22.0 ppm (CH3) and 68.0 ppm (-CH-), respectively. Signals at 50.0–55.0 ppm corresponded to the iminodiacetate part of the molecule (-N(CH2)2<). Finally, carbons of the alkyl chain were in the range of 22.0–60.0 ppm. 1H and 13C spectra of the final products, containing carboxyl groups , showed no visible signals of the ester residue. High-resolution mass spectra further confirmed the structure of the obtained intermediates and final products.The structure of all obtained intermediates and final products was confirmed by 1H and 100 MHz for 13C, with CDCl3 or DMSO as solvent, and TMS as the internal standard. High-resolution mass spectra were acquired using a Waters ACQUITY UPLC/Xevo G2QT instrument . Thin-layer chromatography (TLC) was performed using silica gel 60 F254  thin-layer chromatography plates, with ethyl acetate, chloroform/ethyl acetate (5:1 v/v), or methanol/chloroform (4:1 v/v) as the mobile phases.All reagents were purchased from commercial sources and used without further purification. Melting points were measured using a Stuart SMP3 melting point apparatus . NMR spectra were recorded at 25 °C using an Agilent 400-NMR spectrometer  at 400 MHz for 1a–d, 0.06 mol) was dissolved in diethyl ether (20 mL) and added dropwise to the mixture. The temperature was carefully monitored, keeping it below 35 °C. Then, after 30 min, sodium carbonate  dissolved in water (40 mL) was added. After the evolution of carbon dioxide had ceased, acid chloride  dissolved in diethyl ether (10 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 h. The resulting precipitate was filtered, and dried products was recrystallized from methanol to obtain the desired pure products.Hydrazine hydrate  was dissolved in diethyl ether (50 mL), and the mixture was cooled to 0 °C. The appropriate amount of acid chloride (N′-(2-bromoacetyl)acetohydrazide (2a)2-Bromo-1H-NMR : δ 3.92 , 10.58 ; 13C-NMR : δ 26.9, 164.4.The product was obtained as white powder ; m.p. 174–176 °C. N′-(3-bromopropanoyl)propanehydrazide (2b)3-Bromo-1H-NMR : δ 2.78, 3.65 , 10.08 ;); 13C-NMR : δ 28.7, 36.3, 167.7.The product was obtained as white powder ; m.p. 182–183 °C. N′-(4-bromobutanoyl)butanehydrazide (2c)4-Bromo-1H-NMR : δ 2.04 , 2.28 , 3.55 , 9.75 ;); 13C-NMR : δ 28.3, 31.5, 34.2, 170.1.The product was obtained as white powder ; m.p. 159–160 °C. N′-(5-bromopentanoyl)pentanehydrazide (2d)5-Bromo-1H-NMR : δ 1.61 , 1.81 , 2.13 , 3.52 , 9.67 ; 13C-NMR : δ 23.6, 31.5, 32.1, 34.7, 170.7.The product was obtained as white powder ; m.p. 149–151 °C. N,N′-diacylhydrazine . The mixture was heated to reflux for 6–24 h. The progress of the reaction was monitored by TLC using methanol/chloroform (4:1 v/v) as the mobile phase. Excess phosphorus oxychloride was evaporated, and the residue in the flask was dissolved in diethyl ether (40 mL) and poured into water (100 mL). The mixture was neutralized using sodium carbonate, extracted with diethyl ether (40 mL), dried over anhydrous magnesium sulfate, and evaporated to dryness.Phosphorus oxychloride  was added to 3a)2,5-Bis(bromomethyl)-1,3,4-oxadiazole : δ 4.92 ; 13C-NMR : δ 17.6, 164.2. HRMS (ESI): m/z calcd for C4H4N2OBr2 + H+: 256.8748; found 256.8756.The product was obtained as yellow oil . 3b)2,5-Bis(2-bromoethyl)-1,3,4-oxadiazole : δ 2.96 , 3.56 ; 13C-NMR : δ 28.7, 36.3, 167.7. HRMS (ESI): m/z calcd for C6H8N2OBr2 + H+: 284.9061; found 284.9064.The product was obtained as yellow oil . 3c)2,5-Bis(3-bromopropyl)-1,3,4-oxadiazole : δ 2.21 , 2.96 , 3.63 ; 13C-NMR : δ 28.9, 31.5, 33.5, 165.5. HRMS (ESI): m/z calcd for C8H12N2OBr2 + H+: 312.9374; found 312.9386.The product was obtained as yellow oil . 3d)2,5-Bis(4-bromobutyl)-1,3,4-oxadiazole : δ 1.77 , 1.83 , 2.81 , 3.53 ; 13C-NMR : δ 23.6, 24.4, 31.4, 34.4, 166.1. HRMS (ESI): m/z calcd for C10H16N2OBr2 + H+: 340.9688; found 340.9692.The product was obtained as yellow oil . a) or isopropanol (b) (120 mL) and concentrated H2SO4 (7.5 mL) were added to iminodiacetic acid (4) . The reaction mixture was heated to reflux for 12 h. Excess alcohol was then evaporated using a rotary evaporator, and the mixture was neutralized with sodium bicarbonate solution. Then, the resulted solution was extracted with ethyl acetate (30 mL), dried over anhydrous magnesium sulfate, and evaporated to dryness.Ethanol (5a)Diethyl iminodiacetate : δ 1.24 , 3.42 , 4.15 ; 13C-NMR : δ 14.1, 50.1, 60.8, 171.6. HRMS (ESI): m/z calcd for C8H15NO4 + H+: 190.1079; found 190.1082 [The product was obtained as slightly yellow liquid . 190.1082 ,49.5b)Diisopropyl iminodiacetate : δ 1.25 , 3.42 , 5.07 ; 13C-NMR : δ 21.5, 50.3, 68.4, 171.2. HRMS (ESI): m/z calcd for C10H19NO4 + H+: 218.1392; found 218.1403.The product was obtained as slightly yellow liquid . 3a–d, 0.004 mol), iminodiacetic acid ester , and sodium carbonate  were dissolved in acetonitrile (50 mL). The reaction mixture was heated at 60 °C for 12 h. Water (20 mL) was added and extracted using ethyl acetate (50 mL). The organic phase was separated, dried over anhydrous magnesium sulfate, and evaporated to dryness. The crude product was purified using column chromatography with ethyl acetate as the mobile phase.2,5-Bis-1,3,4-oxadiazole (6a)Tetraethyl 2,2′,2″,2‴-bis(methylene))bis(azanetriyl))tetraacetate : δ 1.26 , 3.69 , 4.24 ; 13C-NMR : δ 14.1, 48.1, 54.5, 60.8, 164.3, 170.5. HRMS (ESI): m/z calcd for C20H32N4O9 + H+: 473.2248; found 473.2253.The product was obtained as yellow oil . 6b)Tetraisopropyl 2,2′,2″,2‴-bis(methylene))bis(azanetriyl))tetraacetate : δ 1.24 , 3.65 , 4.24 , 5.02 ; 13C-NMR : δ 21.8, 48.1, 54.8, 68.4, 164.4, 170.1. HRMS (ESI): m/z calcd for C24H40N4O9 + H+: 529.2874; found 529.2870.The product was obtained as yellow oil . 6c)Tetraisopropyl 2,2′,2″,2‴-bis)bis(azanetriyl))tetraacete : δ 1.25 , 3.34 , 3.43 , 3.90 , 5.07 ; 13C-NMR : δ 21.8, 29.0, 39.4, 50.3, 68.4, 164.1, 171.2. HRMS (ESI): m/z calcd for C26H44N4O9 + H+: 557.3185; found 557.3185.The product was obtained as yellow oil . 6d)Tetraisopropyl 2,2′,2″,2‴-bis)bis(azanetriyl))tetraacetate : δ 1.24 , 1.94 , 2.84 , 2.91 , 3.50 , 5.03 ; 13C-NMR : δ 21.9, 22.8, 24.7, 53.1, 55.2, 68.0, 166.8, 170.6. HRMS (ESI): m/z calcd for C28H48N4O9 + H+: 585.3500; found 585.3491.The product was obtained as yellow oil . %). 6e)Tetraisopropyl 2,2′,2″,2‴-bis)bis(azanetriyl))tetraacetate : δ 1.26 , 1.36 , 1.58 , 2.77 , 2.84 , 3.51 , 5.01 ; 13C-NMR : δ 21.7, 23.6, 23.9, 25.1, 49.1, 54.8, 68.4, 164.4, 170.1. HRMS (ESI): m/z calcd for C30H52N4O9 + H+: 613.3812; found 613.3810.The product was obtained as yellow oil . %). 6b, 6d) (0.18 mmol) were dissolved in methanol (24 mL), water (6 mL), and NaOH . The reaction mixture was heated at 50 °C for 1 h. The solution was then neutralized with 1 M HCl and evaporated to dryness. The crude product was purified by recrystallization from methanol.The ester derivatives of 2,5-dialkyl-1,3,4-oxadiazole (7b)2,2′,2″,2‴-bis(methylene))bis(azanetriyl))tetraacetic acid : δ 3.38 , 4.04 ; 13C-NMR : δ 47.4, 57.5, 164.5, 173.9. HRMS (ESI): m/z calcd for C12H16N4O9 + H+ + Na: 384.0893; found 384.0846.The product was obtained as white powder . 7d)2,2′,2″,2‴-bis)bis(azanetriyl))tetraacetic acid : δ 1.79 , 2.71 , 2.83 , 3.42 ; 13C-NMR : δ 22.1, 24.1, 52.8, 54.7, 166.3, 172.4. HRMS (ESI): m/z calcd for C16H24N4O9 + H+: 417.1621; found 417.1638.The product was obtained as white powder . An interesting methodological process was developed for the synthesis of extended 1,3,4-oxadiazole derivatives containing carboxymethylaminoalkyl groups at positions 2 and 5. The initial synthetic pathway, comprised of nucleophilic substitution of bromine-containing 2,5-dialkyl-1,3,4-oxadiazoles with iminodiacetic acid, was ineffective. However, the replacement of iminodiacetic acid with more reactive diisopropyl iminodiacetate led to the formation of the intermediate esters and final acids in satisfactory yields. The obtained final products constitute a new family of complexing agents with potential applications in various areas, such as agriculture, medicine, or pharmacy.