Therefore, the adenosine A2B receptor is currently in focus as a novel target for cancer therapy as well as for noninvasive molecular imaging via positron emission tomography (PET). Aiming at the development of a radiotracer labeled with the PET radionuclide fluorine-18 for imaging the adenosine A2B receptor in brain tumors, one of the most potent and selective antagonists, the xanthine derivative PSB-603, was selected as a lead compound. As initial biodistribution studies in mice revealed a negligible brain uptake of [3H]PSB-603 (SUV3min: 0.2), structural modifications were performed to optimize the physicochemical properties regarding blood–brain barrier penetration. Two novel fluorinated derivatives bearing a 2-fluoropyridine (5) moiety and a 4-fluoro-piperidine (6) moiety were synthesized, and their affinity towards the four adenosine receptor subtypes was determined in competition binding assays. Both compounds showed high affinity towards the adenosine A2B receptor (Ki (5) = 9.97 ± 0.86 nM; Ki (6) = 12.3 ± 3.6 nM) with moderate selectivity versus the other adenosine receptor subtypes.The G protein-coupled adenosine A Th. Th2B reN-alkylation at the nitrogen atoms N1 and N3  or 4-bromophenyl (PSB-1901) piperazine moiety, respectively  of 0.2 at 3 min p.i.  at 3 min p.i. of [3H]PSB-603 was determined, indicating a low uptake of the radioligand. For mouse brain, an SUV of 0.21 ± 0.07  and human embryonic kidney (HEK) cells, respectively, expressing the corresponding human receptor subtype  = 9.97 ± 0.86 nM; Ki (6) = 12.3 ± 3.6 nM), which was slightly increased compared to the nonfluorinated derivatives 7 and 8 . Animal experiments were performed with female CD-1 mice, 10–12 weeks old, obtained from the Experimental Centre of the Faculty of Medicine (MEZ) at the Leipzig University, Leipzig, Germany.3H]PSB-603  Ltd, Cardiff, UK; the precursor was synthesized at the University of Bonn, Germany  to calculate the standardized uptake value (SUV) for each tissue.- 443.09433, found 443.09483, calculated for C38H33O8N12F2S2- [2M-H]- 887.19593, found 887.19637.R4, 690 mg  4-fluoropiperidine TFA salt, and 466 µL  triethylamine (TEA) were mixed and heated for 2.5 h at 125 °C. After the addition of 44 µL  TEA and heating at 125 °C for 4 h, the reaction mixture was quenched with 250 mL 1M aq. HCl and extracted with 4 × 50 mL ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude material was prepurified by column chromatography  to yield 61 mg  of 6.In 3 mL DMF, 150 mg  6 was further purified via semipreparative HPLC ).For binding studies, f = 0.66 . 1H NMR  δ = 0.86 , 1.52–1.59 , 1.76–1.93 , 2.91–3.12 , 3.80 , 4.72 , 7.85 , 8.31 , 11.99  ppm. 13C NMR  δ = 11.2, 21.0, 30.1 , 41.5, 42.2 , 86.7 , 108.7, 127.0 (2 C), 128.1 (2 C), 133.1, 136.1, 147.5, 148.0, 151.0, 155.0 ppm. 19F NMR  δ = −181.83 ppm. HR-MS m/z calculated for C19H23O4N5FS+ [M+H]+ 436.14493, found 436.14542; HR-MS m/z calculated for C19H22O4N5FSNa+ [M+Na]+ 458.12633, found 458.12774.R1, A2A, A2B, and A3 receptors were performed according to Alnouri et al. [3H]2-chloro-N6-cyclopentyladenosine  3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine  8-(4-[4-(4-chlorophenyl)piperazine-1-sulfonyl]phenyl)-1-propyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione  (R)-8-ethyl-4-methyl-2-(phenyl)1,4,7,8-tetrahydro-5H-imidazopurin-5-one  PSB-603 resulted in a low brain uptake with an SUV of 0.2. Therefore, the structure of the lead compound PSB-603 was modified to achieve better BBB penetration by reducing the molecular size and the lipophilicity. Furthermore, fluorine was introduced into the molecule to allow the possibility for future radiolabeling with fluorine-18. Both novel fluorinated compounds 5 and 6 showed high binding affinity towards the A2B receptor  and moderate selectivity towards the other adenosine receptor subtypes . These findings provide a good basis for further modifications of the ligand structure to enhance selectivity towards the adenosine receptor subtypes with respect to future brain PET imaging of the adenosine A2B receptor.In conclusion, a biodistribution study with [


Two decades have passed since the publishing of the last checklist of the millipedes of Slovakia. During this time, several new faunistic records have been added and taxonomic revisions have occurred. The present updated checklist summarises data on all millipede species recorded in Slovakia, including altogether 93 species.Geoglomerissubterranea Verhoeff, 1908, Brachyiuluslusitanus Verhoeff, 1898, Cylindroiulusbritannicus , C.parisiorum  and Polydesmusburzenlandicus Verhoeff, 1925, as well as to C.arborum Verhoeff, 1928, the species newly confirmed for Slovakia after more than 70 years.For each species, general habitat characteristics, ecological classification and distributional pattern are provided. Ecological classification is presented for the first time for the millipede species occurring in Slovakia and is proposed as a tool for ecological studies and for the nature protection purposes. Special remarks are given to the species newly found for Slovakia,  The studies on the millipedes in Slovakia have a long history, with the first papers published at the end of the 19 century . One of th century was done by other authors, including Jawlowski  , based on material and manuscripts from his estate. Besides this, Gulička's unpublished compendia  . Gulička25–2009) , which w25–2009) . This laompendia , which wSince 2001, the number of species has changed significantly, thus warranting a new update. The aim of this report is to update the list of millipede species and their distribution in Slovakia, to summarise published, as well as unpublished data and records, respecting recent taxonomic changes and to provide a useful tool in the form of ecological and geographical classification of species for other studies.Diplopoda used for this checklist follows the systematic arrangement by Brachydesmus and Polydesmus into other genera.The classification for We do not include the subspecific categories as subspecies, nor all the relevant literature since that information has been included by the author of the last published checklist . ReferenRelic species (R) - stenotopic species inhabiting exclusively undisturbed habitats with low impact of human activities, for example, the nature-closest forests, remnants of steppes, caves, stony debris, mountain habitats.Adaptable species (A) - species inhabiting nature-close habitats (various types of forests or meadows), able to penetrate artificial and man-made habitats .Eurytopic species (E) - species with the widest ecological valence, frequently found in forested and non-forested biotopes and in man-made plots .Synanthropic species (S) - species inhabiting a wide spectrum of man-made habitats, missing in natural biotopes, species significantly synanthropic, although occasionally found in natural, mainly altered, sites.Exotic species (X) – this category includes mostly tropical species, non-native or introduced species, found exclusively in hothouses.Ecological classification of each species  is provi(c) - corticolous(e) - epigeic(ed) - edaphic(h) - hygrophilous(m) - mountainous(n) - nidicolous(tb) - troglobitic(tp) - troglophilous(x) - xerophilous(?) next to the classification refers to problematic data on the occurrence of the species.We would like to note that we did not adopt this habitat evaluation scheme mechanically, but we evaluate each species, based on the knowledge about its ecology in Slovakia. Question mark In addition, a general geographical category that outlines the distribution of each species is included in the list .Records from Slovakia compiled from the published literature and additional collected material are summarised. The actually sampled material is in the collection at the workplace of the authors' team. Part of the data was obtained by preliminary revision of the collection by J. Gulička, including his unpublished compendia. A large portion of Gulička's complete written legacy is lost. However, his views on the taxonomy, biogeography and ecology of individual species can be found in Gulička's additionally handwritten notes in the monographs of other authors e.g.  etc..51B5879F-5FC7-5A53-8809-BE8BA4E6EFD41474AEEA-6B12-5591-ACB8-91EDF280965C828426A7-1F69-533A-8D81-0CB37BEC7213West Palearctic and NearcticA, c, nBFC7F491-9FAA-5D78-ADF4-E7B530A4DD4EGlomerida, according to published data, is represented by three genera in Slovakia – Trachysphaera, Glomeris and Geoglomeris  is characterised by a south-western occurrence in Europe Nowicki, 1870694103EA-A3AF-533E-A057-1E6D5868BA6AWest CarpathianR, eGlomerismnischechi is considered the only endemic species of the order Glomerida north of the Alps. According to G.mnischechi 8EAAF47F-F7BE-5D57-AD24-C8CA0B7A8FC5South and Central EuropeanA, e, cBrandt, 1833D64F6E1F-0B9A-5880-9006-DBADB01E7E7CCentral and East EuropeanA, eGlomerisconnexa C. L. Koch, 1847 in Slovakia. This species is characteristic for its south-western European distribution . However, he did not comment on the finding and attached only a picture taken from other literature  in the water of three wells in Northern Bavaria, Germany. The author explained its occurrence as random, presuming the animals entered the well through crevices in the wall.This species was described by terature . Since tterature . In the E2F129A8-8841-5B7A-8884-F673C64DA7E8CarpathianR, ed, tp, hB05A6FD8-BBE1-5C39-87F4-27EE0DDCB1E5East and Central EuropeanR, ed, mC8D3F0D0-4F6A-570C-A64D-2544106EAECDAlpine and West CarpathianR, ed0D81710B-775D-5A5F-B443-9D6023099906621915C8-EAFC-5396-9714-FE20D7462F70Verhoeff, 1907F24C4010-6F24-56CD-9527-A5CFB0D89E06East Alpine and West CarpathianR, eBrandt, 18370FF2874E-FB0B-579D-AE75-912804D3A386EuropeanA, e, hVerhoeff, 18986E9952B6-3AB8-57ED-927F-53563C7C8C6CEast CarpathianR, h85AF59DC-241D-5259-BB9E-A2B5FD794658EFAA8300-886A-5590-B1A3-EAECD325EF463481208A-BB4B-5F5A-8FB5-5089E9D8B446West, North, Central and East EuropeanR, ed, tpReference: Mock et al. (2015)B89DF54C-253C-5767-887F-BF4C2CF3B1A6EuropeanS, edAntić, Mock & Enghoff, 20156BD50117-1D65-52DF-B16F-788C6FE3D833West CarpathianR, ed, tp58F43795-2A1E-5F3A-917B-9BBF992DCF83EuropeanS, edReference: Mock (2001b)82D904AB-2C93-5895-9895-696840D9AD3BEuro-CaucasianS, ed90CD54F7-E609-5EEA-A47D-CA7C9BFB183FEuropeanE, c, n17D6B204-1121-5909-8A04-30BB23FCB962C. L. Koch, 1847E2AE1783-D0ED-5389-99E3-EA3FDCEA4048EuropeanR, c, nA0FD6BA1-3FBD-5217-908B-7229ADD0FA3B8A120E9F-8E37-5E28-A5AE-266719F586D5South and Central EuropeanE, e, n, xVerhoeff, 1898DA264670-66AC-5322-B965-09E7A9B62558Type status:Other material. Occurrence: individualCount: 1; sex: male; lifeStage: adult; Taxon: scientificName: Brachyiuluslusitanus Verhoeff, 1898; class: Diplopoda; family: Julidae; genus: Brachyiulus; specificEpithet: lusitanus; taxonRank: species; Location: country: Slovakia; locality: Košice Basin, Košice – a city park (Mestský park) at the railway station; verbatimElevation: 210 m a.s.l.; locationRemarks: Acer platanoides, Aesculus hippocastaneum)leaf litter  at the railway station; verbatimElevation: 210 m a.s.l.; locationRemarks: Acer platanoides, Aesculus hippocastaneum)leaf litter 4B2E539A-C953-5C56-A2A3-B021375BA5F8South and Central EuropeanR, eVerhoeff 1928B89DFBDB-1AE3-5FF3-9F8F-FC29FCCBABC9Type status:Other material. Occurrence: individualCount: 1; sex: male; lifeStage: adult; Taxon: scientificName: Cylindroiulusarborum Verhoeff, 1928; class: Diplopoda; family: Julidae; genus: Cylindroiulus; specificEpithet: arborum; taxonRank: species; Location: country: Slovakia; locality: Burda Mts.; verbatimElevation: 190 m a.s.l.; locationRemarks: Quercus spp., Carpinusbetulus, Fraxinusexcelsior), decomposed wood from an oak-tree cavityforest , decomposed wood from an oak-tree cavityforest , decomposed wood from an oak-tree cavityforest 00B4D62F-CAB4-5AC1-A501-206AD0322D4CType status:Other material. Occurrence: individualCount: 5; sex: male; lifeStage: adult; Taxon: scientificName: Cylindroiulusbritannicus ; class: Diplopoda; family: Julidae; genus: Cylindroiulus; specificEpithet: britannicus; taxonRank: species; Location: country: Slovakia; locality: Košice Basin, Košice, Public cemetery; verbatimElevation: 225 m a.s.l.; locationRemarks: Fraxinusexcelsior, Acer platanoidesTilia cordata, )leaf litter 59F9CDA8-596B-5293-B1F4-B134097D301FCosmopolitanS, eReference: Mock (2006)015DCC44-B303-577D-8F68-8D8FD23D8EBDAtlantic, North-western and Central EuropeanS, eReference: Mock (2001b)2169FE95-512D-5B5C-9687-EE993EA34BAFType status:Other material. Occurrence: individualCount: 21; sex: male; lifeStage: adult; Taxon: scientificName: Cylindroiulusparisiorum ; class: Diplopoda; family: Julidae; genus: Cylindroiulus; specificEpithet: parisiorum; taxonRank: species; Location: country: Slovakia; locality: Košice Basin, Košice – a city park (Mestský park) at the railway station; verbatimElevation: 210 m a.s.l.; locationRemarks: Acer platanoides, Aesculus hippocastaneum)leaf litter ; class: Diplopoda; family: Julidae; genus: Cylindroiulus; specificEpithet: parisiorum; taxonRank: species; Location: country: Slovakia; locality: Košice Basin, Košice – a city park (Mestský park) at the railway station; verbatimElevation: 210 m a.s.l.; locationRemarks: Acer platanoides, Aesculus hippocastaneum)leaf litter ; class: Diplopoda; family: Julidae; genus: Cylindroiulus; specificEpithet: parisiorum; taxonRank: species; Location: country: Slovakia; locality: Košice Basin, Košice – a city park (Mestský park) at the railway station; verbatimElevation: 210 m a.s.l.; locationRemarks: Acer platanoides, Aesculus hippocastaneum)leaf litter 3F2C90F6-E45B-5522-BEE0-7729C555013FWest and Central EuropeanX, e, hReference: Mock (2001b)6A9BDF51-A050-5A30-8414-E0B0559A7BD7EuropeanA, e, hA4829F1E-DA65-527C-B7B1-D37F201BA8C6West CarpathianR, e, mFC523E99-BEB5-5322-BB58-4FFED80B2107East CarpathianR, eReference: Gulička et al. (2014)Verhoeff, 1899C4854698-B0A2-5FEA-88FC-10B1D892E657CarpathianR, eLatzel, 1884838E1EA4-4893-559E-AC0B-12AF67C88986Central European and ScandinavianA, e, nLohmander, 1925ACE7AF66-74D7-5242-A32D-D26CEF6D988ECentral EuropeanA, e, hLinnaeus, 1758991BA89E-2910-5CC7-A987-3ADC15B2DB20North, Central, South and East EuropeanR, e, hF1365525-3B19-500B-BE44-03B9C771BE26East EuropeanA, e, x15D295F6-7BF1-5386-A52E-66E7A0C596D2East and Central EuropeanR, e08E79850-A0D8-5202-90FB-1DCFEE256CFFNorth and Central European, BalticR, e, h, n37631AAA-BF6F-5888-9930-B3D690E6A46DWest CarpathianR, e, mVerhoeff, 19138C2CE56F-DB40-5F25-A980-976BB5F8386ESudetico-CarpathianR, e, mGulička, 1952528708CF-BDA4-5421-8648-F5613BD4F96AWest CarpathianR, e, tp5D4CD2CC-5E74-584E-B453-8BD294C5FA8DNorth, Central and East EuropeanA, eGulička, 19565CFD59DC-D3C9-51A1-A7EC-982F005C6A4BWest CarpathianR, e, mE70CBA85-A434-5CCF-8054-9F30CB9DE647Central and East EuropeanA, e10EE350B-3B20-5539-A3F8-F06E5320DA26West CarpathianR, e, mVerhoeff, 18942F4ADCC2-0CAE-5197-96DE-987A6C1EE653Central and East EuropeanA, e18575707-9CD0-58B8-AC98-F6C577D56B10East and Central EuropeanR, e, m82675623-7D8A-587F-9DA2-ECC464C45BD1South and Central European, BalkanE, e, xF3581CAB-3DDD-5006-9F26-42C2AB926E38EuropeanE, e, x002768BF-03DC-5C20-AF68-E1D1D6813B89EuropeanE, e968A4371-833F-5CF1-8DEB-6403DC30EB83EuropeanE, e, h590A2C07-04D4-5B08-82B9-6E7AB7E2C297Central EuropeanA, e2AC4FCC1-001A-5F77-88D6-0BF22F3560D6North CarpathianR, e, m9DECA411-8B99-5EBF-BFCC-9236A24D3D0BEast and Central EuropeanR, e, h, nReference: Tajovský et al. (2001)0E3D0A19-D96F-53D6-B9B2-04BDD47C8879F11EB5B5-46CA-5729-AA43-7CDCC80AFBAA8779CE31-7CCF-5ED2-844B-F748C93AF4F1Central European, BalkanS, eReference: Mock and Tajovský (2002)160BA32D-8C74-58FD-A7F6-B98739996D13East CarpathianA, eReference: Mock and Tajovský (2002)A36ACD63-2589-50F5-BCEE-180E889D5584North and Central EuropeanS, eReference: Mock and Tajovský (2002)331205F4-1713-5FBA-8F29-22CB684628EACD723F79-E5EA-5909-A6A2-ABF03F0F0BACWest EuropeanR, tpReference: Kováč et al. (2005)BCF46DAF-6878-5C36-8C31-E0AFFA337A96Loksa, 1960FF9DAFFD-2F98-5440-B74F-938C13BBFF45West CarpathianR, edReference: Mock et al. (2019)E6E1CFB1-CB6A-50D7-BF31-8C5C8BFE1358Verhoeff, 192810E8DB47-2C13-54BF-97D3-62954C7C3AB6West CarpathianR, e, hOchogonacervina , recently pointed out by O.cervina and our knowledge of H.bokori must be reviewed. In addition, in order to justify the name of the family Hungarosomatidae and its position in the Chordeumatida system, the decision as to which genus the species belongs: Ochogona, Octeicosisoma, Triakontizona or Ceratosoma, has to be resolved 0A4EF9DD-BE59-578D-AE90-9AF360667BBF5465705D-4E49-593D-8E26-4B084410442CEast CarpathianR, e, mReference: Gulička et al. (2014)Leach, 181418001744-0F61-565E-9304-C5DC381DEE11West, Central and South EuropeanA, eVerhoeff, 1897C034B994-8521-5B2A-BF62-86B943A61F9CSouth and Eastern EuropeanR, e9791AA65-998E-5E80-8ACA-379C1C6853A6West CarpathianR, e, mF85EE3C3-6D8D-598A-800C-A0E921089325Central EuropeanR, eReference: Gulička et al. (2014)8465A86A-22AF-58EA-A29D-62CCEF45F63CC84E98DF-B346-5CFA-997A-1DBA43A3F8F5South and Central EuropeanA, eA999896F-BD64-58F6-A12B-B3C7541A1B9FWest CarpathianR, tbVerhoeff, 1899140D933F-8B82-55A7-98C8-2C3B87E733ADNorth CarpathianR, e, mE7D5C923-C05B-5F7F-9308-8679655690C8A238419E-E84D-5864-94E8-30FCFEB8B7DAEast CarpathianR, eReference: Gulička et al. (2014)65C7370E-1D2E-5072-9B36-54FF3C108D9C2A92FCE4-9022-5340-833A-B55860611281West CarpathianR, tpMock & Tajovský, 20088E374C6F-3304-51BB-B71F-3023B348134DWest CarpathianR, tpF3341C42-CE6E-5A79-97D3-5A0080CB7F459CADC961-AE1B-5FD5-9F57-4B13B5EA3CCFCentral EuropeanR, e10F158A8-343B-5272-98A4-A5C356723B03Central and East EuropeanE, eVerhoeff, 1899304B8977-AF62-58F9-BDF5-7CCD99B24798West CarpathianR, e, m725C200A-8CB2-5B3F-899B-73FEFDE655573B6E25C9-4384-59EA-8A19-7E147787968DAlpineS, eReference: Haľková and Mock (2018)04B3F154-EEB0-589A-AAB6-EA53CFF83B6FCE25DD76-98C6-5791-BFB1-E37E5C39CA55Verhoeff, 1895533BBE4D-9FCB-563B-B251-E8258224F4C2South, Central and East EuropeanA, e, hLatzel, 1884694DE3E8-C9E8-52CC-A576-03D8521719E2EuropeanS, e, hVerhoeff, 19253F6D92C3-5485-51AF-A747-5E482E314050South-eastern CarpathianR, e, cPolydesmusburzenlandicus is a small hygrophilous representative of the order Polydesmida, widespread mainly in the south-eastern Carpathians 2F776C36-2364-557E-9339-FA5E7F1A6C08North, Central, South and East EuropeanE, e, hC. L. Koch, 1847DFBCDACE-498E-578F-9B2B-6666B24578C4EuropeanE, eLatzel, 188441C36D84-A0C7-5A99-A3F6-40C3FF568B9BEuropeanS, e, hReference: Mock (2004)Ložek & Gulička, 19627A252CA8-152E-5F89-9304-7D099E79DD1BEast CarpathianR, e, mDaday, 1889AED868E6-77C3-5328-A02B-3A599AD52E30East CarpathianR, e, c, mLatzel, 18847602842C-F4AB-568C-A312-4A9572FEE2B7East CarpathianR, e, mLatzel, 18848387BDA8-3A08-5193-B8A5-708A271BC0ABSouth CarpathianR, e, tp, ?P.transylvanicus Daday, 1889 in nearby locations within the Eastern Slovak Plain CA7A8050-3949-55B0-8481-75FBA30951DF00AF0722-FD46-50DD-A656-DB0CEE6CB79ECosmopolitanX, e58E29EC2-4A4D-5FF3-8A8E-6095FD2EB639East and Central European, BalticA, e, tp, h39B0E373-F2B2-5294-A509-D0FC388031668C690F2D-6B47-56FC-B104-FF46490EB680North, West and Central EuropeanX, e, c, hReference: Mock (2001b)5A793D16-C55A-5C43-9D9B-5CC8B6D5FECAFC24AFCE-C982-58AC-A70A-8C2F9A09C482East and Central EuropeanX, e, c, hReference: Mock (2001b)The increase in faunistic research over the past two decades has resulted in significant expansion of knowledge of the Slovak millipede fauna. Since the last published review of millipede species from the territory of Slovakia , 23 specMycogonagermanica, Allaiulusnitidus or Blaniulusguttulatus and Cylindroiuluslatestriatus (as Aneuloboiulusfrisius). From the faunistic point of view, the article is very inaccurate and should be approached with great caution.Most of the unclear and questionable data that were preliminarily excluded from the list are from the older literature. One of the most recent studies, creating confusion due to imprecision in accurate and reliable determination, was published by Leptoiulustatricus, Mecogonopodiumcarpathicum and Hylebainosomagulickai). The presence of the Western Carpathians has an undeniable influence on the composition of Slovak millipedes. Almost 20% of the species are represented by rare mountainous fauna and more than 10% are characteristic for their preference of cave habitat.The millipede fauna of Slovakia can be characterised as a combination of European, Alpine-Atlantic and Carpathian elements, with the occasional influence of Caucasian and Balkan fauna . From thAccording to ecological classification, 50% of Slovak millipede fauna are represented by stenotopic species inhabiting exclusively undisturbed habitats with a low impact of human activities. A total of 20% can be classified as adaptable and only 7% can be classified as eurytopic. While the original classification, proposed by Typhloiulus sp. from the Domica Cave (Trichopolydesmidae (Despite the increased interest in faunistic research, the millipede fauna of Slovakia has not yet been completely investigated. Several findings are still to be determined at the species level. Such findings include subadult and female individuals of ica Cave  and a redesmidae . Both ta


The relative and absolute stereochemistry of 1 was completely established by a combination of spectroscopic data and chemical reactions. The structures of the isolated compounds were elucidated by analysis of HRMS and NMR data. Notably, compounds 1–3 had a β-resorcylic acid harboring a long unesterified aliphatic side chain, whereas the long aliphatic chains were esterified to form macrolactones in 4–9. Among the isolated compounds, monocillin I and radicicol showed potent antifungal activities against Cryptococcus neoformans, comparable to clinically available antifungal agents and radicicol showed weak antifungal activity against Candida albicans. These findings provide insight into the chemical diversity of fungal RAL-type compounds and their pharmacological potential.Six new β-resorcylic acid derivatives ( A variety of bioactive metabolites have been reported from plant-associated microorganisms . NotablyMonocillium nordinii in 1953 and found to be an excellent Hsp20 inhibitor  corresponding to C-10 to C-18, and we confirmed its connection with the β-resorcylic acid moiety according to the HMBC spectrum  and oxymethines , as shown in δH 6.31) of the aliphatic chain with C-2 (δC 99.6) and C-6 (δC 103.9) of the β-resorcylic acid moiety indicated that the long-chain moiety was attached to C-7. Additionally, we considered the possibility that the long aliphatic chain of 1 was macrolactonylated through an ester bond between C-1 and C-17, as many RAL-type compounds have previously been reported in fungi. However, it was more probable to have a free hydroxyl group at C-17 rather than to be esterified to form a macrolactone ring due to the chemical resonance of H-17 (δH 4.0) in the long chain. Evidently, no HMBC correlation from H-17 to the carbonyl carbon C-1 supported the presence of free hydroxyl groups. Although a number of RAL-type compounds have been reported from fungi, to our knowledge, this is a rare case to isolate β-resorcylic acid with an unesterified long aliphatic side chain. We then confirmed the presence of β-resorcylic acid with a non-esterified aliphatic chain by a simple chemical reaction. Compound 1 was acetylated to confirm the number of exchangeables in the side chain. Acetylation of 1 caused downfield shifts of three oxymethine protons  in the aliphatic side chain by ~1 ppm (compared with those in compound 1), thus completing the planar structure of compound 1  data . The 1H spectrum . Althougmpound 1 . Compounmula C18HO8 based 2 (colletogloeic acid B) was a yellowish amorphous solid and determined to be chlorinated according to the characteristic isotopic pattern in its MS spectrum. Its molecular formula was found to be C18H25ClO9 based on (+)HRESIMS (observed [M + Na − H2O]+ at m/z 425.1010) and spectroscopic data. This chlorinated compound had one aromatic proton, one olefinic proton, and four oxymethine proton signals. The C-6 of the β-resorcylic acid moiety was confirmed to be chlorinated based on HMBCs of the aromatic proton H-4 (δH 6.42) and the olefinic proton H-8 (δH 6.72) with C-6 (δC 108.3). Detailed analysis of 1H-1H COSY revealed the presence of a 1,3,4,6-tetraol system in the aliphatic chain instead of the 1,2,4-triol system shown in 1. The position of the additional carbinol group  was confirmed to be C-12 by HMBCs of H2-10 (δH 2.75 and 2.67) with C-8 (δC 101.9) and C-12 (δC 70.4), suggesting its planar structure (Compound tructure .3 (colletogloeic acid C) was a yellowish amorphous solid with the molecular formula C18H24O7 according to (+)HRESIMS . Although the NMR spectral data of 3 were similar to those of 1, an additional double bond in trans at δH 6.54  and 6.16  and the lack of the oxygenated methine group revealed a distinct structural difference between 1 and 3. The additional double bond was positioned at C-10 and C-11 based on the HMBC correlations of H-10 (δH 6.16) with C-8 (δC 106.0) and C-12 (δC 33.8), as well as H-11 (δH 6.54) with C-9 (δC 153.5) and C-13 (δC 26.1). Additionally, a spin system CH(O)-CH2-CH(O)-CH3 corresponding to C15(O)-C16-C17(O)-C18 obtained by 1H-1H COSY indicated hydroxylation at C-15 and C-17, which was also supported by HMBCs.Compound 4 (Colletoresorcylic lactone) was a yellowish amorphous solid and its molecular formula was found to be C18H24O7 based on the HSQC NMR and (+)HRESIMS (observed [M + Na]+ at m/z 375.1419). Compound 4 showed different UV profiles from compounds 1–3. While 1–3 had a strong UV absorption at 244 nm and a medium absorption at 320 nm, 4 showed three UV absorptions at 211, 262, and 298 nm, suggestive of a lactonized resorcylic acid. The 1H, 13C, and 1H-1H COSY NMR data of 4 were similar to those of 1 in the presence of the 2,4-dihydroxybenzoic acid moiety and a 1,2,4-triol group, CH3-CH(O)-CH2-CH(O)-CH(O)-CH2, in the aliphatic chain. A methylene group at δH 4.48 and δH 3.82 and a ketone carbon (δC 212.5) in 4 instead of the enol group indicated that 4 had a keto group. Notably, compound 4 had an oxymethine proton signal at δH 4.95 unlike previous compounds 1–3, suggesting that 4 had an ester group. An HMBC correlation of the oxymethine proton δH 4.95 with the carboxylic carbon (C-1) at δC 171.9 in addition to two oxymethine carbons (C-14 and C-17) indicated the presence of the 12-membered lactone ring. Additionally, HMBCs of the methylene protons H2-8 (δH 4.48 and 3.82) with C-2 (δC 107.5), C-6 (δC 113.9), and C-9 (δC 212.5) confirmed the presence of the keto group at C-9.Compound 5 had the molecular formula C18H23ClO7 according to (+)HRESIMS data and it exhibited a similar UV pattern to 4, characteristic for RAL-type compounds. Moreover, this was supported by an HMBC of H-17 (δH 5.43) in a long chain with the carboxylic carbon C-1 (δC 172.5). Chlorination at C-6 of the β-resorcylic acid moiety was assigned based on its HMBCs of H-4 and H2-8 with C-6 along with typical isotopic mass fragmentation. Compound 5 was structurally similar to the known compound nordinonediol (6), except for the presence of chlorine at C-6 of the RAL moiety.Compound 7 was a yellowish amorphous solid with a molecular formula of C23H28O12 based on (+)HRESIMS data. Additionally, we determined 7 to be a RAL-type compound based on its characteristic UV absorption, which was also supported by NMR resonances for an oxymethine group  representative of a lactone. Unlike previous RAL-type compounds 5 and 6, 7 had a conjugated double bond, which was confirmed by proton resonances and their coupling constants , 7.59 , 6.22 , and 5.77 ) together with 1H-1H COSY , δC 56.6 (C-14); 3.08 , 56.9 (C-15)) than the diol group were indicative of epoxide functionality. Moreover, the degrees of unsaturation suggested by the molecular formula were supportive of the epoxide ring. The conjugated double bond was then connected to the epoxide group according to 1H-1H COSY and HMBC correlations of the epoxide protons, H-14 and H-15 with C-13. The geometries of the double bonds  were determined to be E, Z, and Z configurations, respectively, based on their coupling constants. Additionally, 1H and 13C resonances downfield  suggested the presence of a pentose, which was confirmed to be ribose by comparing its NMR resonances with those of the references + .Colletogloeic acid A (2): yellowish amorphous solid; [α]D25 = −9.9 ; IR νmax 3300, 1640, 1020 cm−1; UV (MeOH) λmax 244, 326 nm; for 1H and 13C NMR data (500 MHz in CD3OD), see 3OD, H-#→C-#) H-4→C-1, C-2, C-3, C-5, and C-6; H-8→C-2, C-6, C-9, and C-10; H-10a and H-10b→C-8, C-9, C-11, and C-12; H-11a→C-10; H-11b→C-12; H-13a→C-11, C-12, and C-15; H-16a→C-14, C-17, and C-18; H-16b→C-17 and C-18; H-17→C-15 and C-16; H3-18→C-16 and C-17; (+)HRESIMS m/z 425.1010 [M + Na − H2O]+ .Colletogloeic acid B (3): yellowish amorphous solid; IR νmax 3320, 2940, 2820, 1020 cm−1; UV (MeOH) λmax (log ε) 244 (4.00), 328 (3.21) nm; for 1H and 13C NMR data (500 MHz in CD3OD), see 3OD, H-#→C-#) H-4→C-2, C-5, and C-6; H-6→C-2, C-4, and C-8; H-8→C-2, C-6, C-9, and C-10; H-10→C-8, C-9, C-11, and C-12; H-11→C-9, C-12, and C-13; H-12→C-10, C-11, C-13, and C-14; H-16a→C-14, C-15, and C-17; H3-18→C-16 and C-17; (+)HRESIMS m/z 357.1312 [M + Na − H2O]+ .Colletogloeic acid C (4): yellowish amorphous solid; [α]D25 = −5.1 ; IR νmax 3380, 1680, 1620 cm−1; UV (MeOH) λmax 211, 262, 298 nm; for 1H and 13C NMR data (500 MHz in CD3OD), see 3OD, H-#→C-#) H-4→C-2, C-3, C-5, and C-6; H-6→C-2, C-4, C-5, and C-8; H-8a and H-8b→C-2, C-6, C-7, and C-9; H-10a and H-10b→C-9, C-11, and C-12; H-12→C-13; H-13a→C-11, C-12, C-14, and C-16; H-13b→C-11, C-12, and C-14; H-14→C-12, C-13, C-15, and C-16; H-15→C-1, C-14, and C-17; H-16a→C-14, C-17, and C-18; H-16b→C-14, C-15, and C-18; H3-18→C-16 and C-17; (+)HRESIMS m/z 375.1419 [M + Na]+ .Colletoresorcylic lactone (5): yellowish amorphous solid; [α]D25 = −21.7 ; IR νmax 3300, 1640, 1240 cm−1; UV (MeOH) λmax (log ε) 220 (3.85), 260 (3.74), 310 (3.34) nm; for 1H and 13C NMR data (500 MHz in CD3OD), see 3OD, H-#→C-#) H-4→C-1, C-2, C-3, and C-5; H-8a and H-8b→C-2, C-6, and C-9; H-10a→C-11; H-10b→C-9; H-14→C-15 and C-16; H-15→C-14, C-16, and C-17; H-16a→C-17 and C-18; H-16b→C-15 and C-18; H-17→C-1, C-16, and C-18; H3-18→C-16 and C-17; (+)HRESIMS m/z 409.1041 [M + Na]+ .Nordinonediol chloride (7): yellowish amorphous solid; [α]D25 = +4.5 ; IR νmax 3300, 1650, 1010 cm−1; UV (MeOH) λmax (log ε) 220 (3.70), 258 (3.52), 308 (3.25) nm; for 1H and 13C NMR data (500 MHz in CD3OD), see 3OD, H-#→C-#) H-4→C-2, C-3, C-5, and C-6; H-8a and H-8b→C-2, C-7, and C-9; H-10→C-12; H-12→C-10; H-17→C-1, C-16, and C-15; H3-18→C-16 and C-17; H-1′→C-5, C-2′, C-3′, and C-4′; (+)HRESIMS m/z 519.1033 [M + Na]+ .Hydroxymonocillin I glycoside (1 (4.5 mg) was dissolved in anhydrous solution  and treated with 2,2-dimethoxypropane (4.5 mL) and pyridinium p-toluenesulfonate (9 mg) in a reaction vial. After reaction on a stirrer at room temperature for 6 h, saturated NaHCO3 (20 μL) was added to quench the reaction. The mixture was purified by reversed-phase HPLC (Luna C18 (2), 5 µm, 250 × 10.0 mm; Phenomenex; HPLC conditions: room temperature, 2.0 mL/min, and UV 210 and 254 nm) using gradient elution from 35% to 55% in ACN/H2O for 30 min. The final product  was eluted at a retention time of 29 min under HPLC conditions.The thoroughly dried compound 1a): 1H NMR  δH 6.34 , 6.30 , 4.30 , 4.09 , 3.92 , 2.54 , 1.74 , 1.61 , 1.55 , 1.50 , 1.46 , 1.42 , 1.38 , 1.31 , 1.19 ; 13C NMR  δC 168.1 (C-1), 167.6 (C-3), 165.0 (C-5), 158.9 (C-9), 141.5 (C-7), 108.8 (C-19), 105.4 (C-8), 103.8 (C-4), 102.7 (C-6), 99.8 (C-2), 79.2 (C-14), 76.2 (C-15), 65.7 (C-17), 40.2 (C-16), 34.1 (C-10), 30.8 (C-13), 29.1 (C-20), 28.0 (C-11), 26.8 (C-12), 26.3 (C-21), 24.8 (C-18); HMBCs  H-4→C-2, C-3, C-5, and C-6; H-6→C-4, C-5, and C-8; H-8→C-2, C-7, C-9, and C-10; H-10→C-8, C-9, C-11, and C-12; H-11→C-9, C-10, C-12, and C-13; H-12a→C-13; H-13→C-12 and C-14; H-14→C-12, C-16, and C-20; H-15→C-13, C-17, and C-20; H-16a→C-14, C-15, C-17, and C-18; H-16b→C-17 and C-18; H-17→C-15 and C-16; H3-18→C-16 and C-17; H3-20→C-19 and C-21; H3-21→C-19 and C-20; ESIMS m/z 415.4 [M + Na − H2O]+Colletogloeic acid A acetonide derivative (2a): 1H NMR  δH 6.70 , 6.42 , 4.02 , 3.94 , 3.73 , 2.67 , 1.85 , 1.80 , 1.71 , 1.68 , 1.43, 1.41, 1.31, and 1.30 , 1.19 , 1.18 , 1.08 ; 13C NMR  δC 167.5 (C-1), 165.4 (C-3), 163.3 (C-5), 160.1 (C-9), 137.9 (C-7), 107.4 (C-6), 103.2 (C-4), 102.1 (C-8), 100.1 , 100.0 (C-2), 73.5 (C-15), 73.4 (C-14), 69.2 (C-12), 66.6 (C-17), 36.2 (C-16), 34.7 (C-11), 34.5 (C-13), 30.6 (C-10), 30.5, 30.4, 20.23, and 20.17 (four acetonide methyl), 22.7 (C-18); HMBCs  H-4→C-2, C-3, and C-6; H-8→C-2, C-9, and C-10; H-10→C-8, C-9, C-11, and C-12; H-11a and H-11b→C-9, C-10, C-12, and C-13; H-14→C-15 and C-16; H-15→C-14 and C-16; H3-18→C-16 and C-17; acetonide methyl protons (δH 1.43/1.41)→acetonide ketal and acetonide methyl carbon (δC 30.4/30.5); acetonide methyl protons (δH 1.31/1.30)→acetonide ketal and acetonide methyl carbon (δC 20.23/20.17); H3-21→C-15, C-17, C-19, and C-20; H3-23→C-22 and C-24; H3-24→C-12, C-14, C-22, and C-23; ESIMS m/z 505.4 [M + Na − H2O]+.Colletogloeic acid B diacetonide derivative (1 (1a) was prepared in two vials and evaporated to dryness under a high vacuum for 24 h. Each sample was dissolved in anhydrous pyridine (600 μL), sealed completely, and allowed to stand at room temperature for 5 min, followed by the addition of 30 μL of (R)- and (S)-MTPA chloride (MTPA-Cl) to the reaction vials. After reaction for 18.5 h under argon, to quench the reaction, anhydrous MeOH (50 μL) was added. The mixture was purified by reversed-phase HPLC (Luna C18 (2), 5 µm, 250 × 10.0 mm; Phenomenex; HPLC conditions: room temperature, 2.0 mL/min, and UV 210 and 254 nm) using gradient elution (from 10% to 100%) in ACN/H2O for 70 min. Under the HPLC conditions described above, the S-MTPA (1b) and R-MTPA (1c) esters eluted at retention times of 62 min and 61 min, respectively. The molecular formula was confirmed as C51H51F9O14 using (+) ESI-MS ([M + Na − H2O]+m/z at 1063.6).The acetonide product of S-MTPA ester (1b): 1H NMR  δH 7.75-7.39 , 7.38 , 6.83 , 6.59 , 5.28 , 3.86 , 3.77 , 3.75 , 3.70 , 3.54 , 2.57 , 1.73 , 1.65 , 1.51 , 1.46 , 1.33 , 1.29 , 1.38 , 1.37 , 1.24 .R-MTPA ester (1c): 1H NMR  δH 7.75-7.40 , 7.39 , 6.83 , 6.60 , 5.26 , 4.10 , 3.77 , 3.70 , 3.50 , 2.59 , 1.76 , 1.69 , 1.40 , 1.31 , 1.29 .C. albicans (SC5314) and C. neoformans (H99) strains were cultured overnight at 30 °C in YPD liquid medium, washed twice with sterilized H2O, and then resuspended in sterilized H2O. We then added 100 µL of the cell suspension, in which the cell concentration was adjusted to an OD600 of 1.0, to 10 mL of 3-(N-morpholino)propanesulfonic acid (MOPS)-buffered Roswell Park Memorial Institute (RPMI)-1640 medium  and loaded onto 96-well plates containing two-fold serially-diluted compounds. The final concentration of the tested compounds ranged from 1.56 µM to 200 µM. The 96-well plates were incubated at 35 °C for 2 days, and the cell density in each well was measured at OD595 [We determined the minimum inhibitory concentration (MIC) of the selected compounds according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines using the E.Def 7.1 methodology. C. gloeosporioides JS0419. While most RALs reported to date feature a 14-membered macrocyclic ring fused to β-resorcylic acid, compounds 1–3 harbored β-resorcylic acid with a long unesterified aliphatic side chain and compound 4 had a 12-membered macrocyclic ring fused to the β-resorcylic acid in this chemical investigation. Although several ring-opened β-resorcylic acids have been reported to date [4 was lactonized with the ester linkage between C-15 and C-1 to form a 12-membered lactone ring. Although many RALs had 12-membered rings, to our knowledge, this is the first report of RAL with not methyl but propyl group at C-15. Although many RAL-type compounds have been evaluated for antifungal activities against several plant pathogenic fungi, to our knowledge, there is no report on their activity against C. albicans and C. neoformans causing fatal candidiasis and cryptococcosis, respectively. The known compounds, radicicol, and monocillin I, exhibit potent in vitro antifungal activities, suggesting that an epoxide group, non-sugar moiety, and lactonization of the long polyketide chain could be important for antifungal activities. Optimization of the polyketides through structure–activity relationship studies may further increase their antifungal activity and allow the examination of their in vivo efficacy for treating systemic fungal infections.In this study, we isolated nine β-resorcylic acid derivatives, represented by RALs, from cultures of  to date ,37, they


Their structures were elucidated by spectroscopic analyses, including 1D- and 2D-NMR experiments with those of known analogues, and on the basis of HR-EI-MS mass spectrometry, their antimycobacterial activities were also evaluated.The cultivation of one actinobacteria strain, Herbidospora yilanensis, was isolated from sediment samples collected from Yilan County City in Taiwan, resulting in the isolation of five previously undescribed compounds: herbidosporayilanensins A–E ( H. yilanensis isolated from the sediments of northern rivers and lakes is very novel and worthy of in-depth research and discussion.Actinobacteria have the ability to produce a variety of physiologically active products, so they play a very important role in the food industry, pharmaceutical industry, and environmental protection. Our team has also separated and collected actinobacteria resources from all over Taiwan and various environments over the years, except for common chains. In addition to molds, there are many rare genera. Based on the concept of “new species and new compounds”, it is hoped that special compounds can be found from these new species. In recent years, studies have also found that these new species of actinobacteria can produce many active secondary metabolites. In order to further explore the efficacy of different strains of actinobacteria and expand the application range of actinobacteria, this study focused on a strain from central Taiwan, and the new strain T, which was isolated from a sediment sample collected from the Yilan County of Taiwan with a unique morphology + ion peak at m/z 410.2667 , consistent with a molecular formula of C23H38O6. The IR absorption bands suggested the presence of a COOH and OH (2500–3400 cm−1), a terminal double bond , and a CO (1698 cm−1) group. The 13C-NMR (DEPT) spectrum of 1 (2 (one being oxygenated and one exocyclic double bond), and four CH groups (two being oxygenated), as well as five quaternary C-atoms, including a C=O function (δC 182.6).Compound rum of 1  exhibiteFive indices of hydrogen deficiency (IHD) were determined from the molecular formula. After subtracting one double bonds and carboxyl groups, the remaining unsaturation degree is 3, and then the three typical methyl groups at δH 1.25 (s), 1.22 (s), and 0.59 (s) in 1H-NMR, it was deduced that compound 1 is a diterpenoid containing two six-membered labdane-type structures, and the basic structure of labdane is a C20 compound, so it was deduced that it contains a three-carbon derivative functional groups, which contain the two methyl signals δH 1.39 (s) and 1.35 (s).13C-NMR spectrum at δC 108.4, a signal was found and confirmed by DEPT as a quaternary carbon. Furthermore, the HMBC spectrum showed that H-21/H-23 were correlated with C-22, along with no COSY correlations between H-21 and H-23, and established that two methyl groups are located at C-22 (C 108.4) had such a low field that it must be a conjugated double bond, but this quaternary carbon did not have other conjugated double bonds and was connected to two methyl groups. Therefore, it was deduced that this quaternary carbon is an acetonide functional group with two oxygens. In combination with the HMBC correlations of H-15, H-21, and H-23 to C-22, this led to the establishment of acetonide being located on C-14 and C-15 instead of the C-12/C-13 or C-13/C-14 position. From the HSQC spectrum, it can be seen that the proton signal corresponding to δC 74.4 is δH 3.56 , and it has an HMBC correlation with C-9, C-11, and C-13. It was inferred that δC 74.4 is C-12, and that C-12 is connected to a hydroxyl group (-OH). In addition, δC 73.9 has an HMBC correlation with H-12, H-14, and H-16 and from the IR, it was found that there was an OH signal at 3418 cm−1, so it was inferred that δC 73.9 is C-13, which is connected to a hydroxyl group (-OH), and δH 1.25 (s) is correlated to C-12, C-13, and C-14, so this methyl group was deduced as being located on C-13. From the NOESY results, H-11 is correlated to H-20, it can be confirmed that C-20 is in the axial. The presence of NOESY correlations between H-11 and H-20, between H-18 and H-3/5, and the absence between H-18 and H-20 indicated that Me-18 was α-orientated at C-4 in equatorial  is determined, the 1H-NMR chemical shift of one of the protons on C-17 will be close to about δH 4.50 . In compound 1, one of the hydrogens at the C-17 position was δH 4.53 (br s) and is similar to the reference analog, (12R)-Hydroxylabda-8(17),13(Z)-dien-12,19-dioic acid ; in an opposite reference compound (12S)-hydroxylabda-8(17),13(Z)-dien-12,19-dioic acid, its H-17a is about δH 4.71 )  in 3, instead of a Me group (δH 0.86 (Me-19); δC 72.5 (C-19)) at C-4 (δC 33.6) in labda-8(17),13(E)-diene-15-oic acid. The 1H-NMR, 13C-NMR (E) -triene-15-oic acid, named herbidosporayilanensin C.Compound  13C-NMR , COSY, N 13C-NMR , HSQC, a 13C-NMR  data con4 was isolated as colorless oil with c 0.011, MeOH). The HR-EI-MS data determined the molecular formula to be C20H26O3 ). The UV absorption of 4 at 280 nm suggested the presence of a benzenoid nucleus. The IR spectrum showed absorption bands for a hydroxyl group at 3400 cm−1 and an aromatic ring at 1597 and 1505 cm−1, respectively.Compound 1H-NMR and 13C-NMR spectra (4 were similar to those of sugiol [C 209.9 (C-2)] of 4 replaced a methylene group [δC 19.4 (C-2)] at C-2 of sugiol. The 1H- and 13C-NMR , corresponding to seven degrees of unsaturation. The IR spectrum of 5 showed absorption bands at 3410; 1696 cm−1 ascribable to hydroxyl, and C=O groups. The 1H and 13C NMR spectral data of 5 showed signals for a terminal vinyl group, one acetyl, a hydroxyl-substituted methine, one carboxylic acid, and two singlet methyl groups. A careful comparison of the 13C NMR data of 5 , 1.57/2.00 , 2.56 , 2.19 , 5.65 ] in 5 replaced a but-3-en-2-ol moiety , 5.19 , 5.33 , 5.91) in the C-12 position in 12,13-dihydroxylabda-8(17),14-dien-19-oic acid. A 4-acetylcyclohex-1-en-1-yl group was located at C-12 further confirmed by the HMBC correlations from H–12 ((δH 3.99) to C-14 ((δC 119.4), H-14 ((δH 5.65) to C-12 (δC 73.9). The 1H- and 13C-NMR (5 as 5-(2-(4-acetylcyclohex-1-en-1-yl)-2-hydroxyethyl)-1,4a-dimethyl-6-methylenedecahydronaphthalene-1-carboxylic acid, named herbidosporayilanensin E.A major difference was that a 4-acetylcyclohex-1-en-1-yl group at C-12 +, and the molecular formula C12H10O5 of 6 was resolved using HR-EI-MS. The IR (KBr) spectrum of 6 showed absorption bands at 1770; 1668 cm−1 ascribable to γ-lactone, and conjugated C=O groups. The 1H-NMR spectrum of 6 displayed signals for a ABX-pattern splits benzene ring , 6.88  and 7.40 ), two methylene groups , 3.00 , 4.43 , 4.57 , one methine ), and one methylenedioxy at δH 6.07 . From the 13C-NMR, there was a benzene rings at δC 108.1 (C-2), 148.8 (C-3), 152.8 (C-4), 108.3 (C-5), 124.9 (C-6), 129.8 (C-1), with two carbonyl groups, one of which was δC 194.1 (C-7), which is a conjugated carbonyl group. UV absorption at 231 nm showed that it should be adjacent to the benzene ring. The carbon signal at δC 175.29 (C-11) is a characteristic signal of the γ-lactone, which forms a five-ring lactone with the remaining signals at δC 69.19 (C-9), 31.06 (C-10), and 41.97 (C-8). This is the framework of C6-C5 type. Compound 6 showed dextrorotatory optical activity with c 0.02, CHCl3). By comparing the reference to the R-configuration of (R)-(+)-4-(3-Methoxyphenyl)methylbutyrolactone + ion peak at m/z 236.1044 in the HREIMS. The UV spectrum of 8 showed maximal absorptions between 229–280 nm, indicating the presence of a benzenoid moiety  and the CH2 [δH 2.70 ] of 7 replaced a OCH2O group [δH 6.07 ] at C-3 and 4 and a C=O at C-7 [δC 194.1 (C-7)] of 6. The dextrorotatory optical activity {c 0.18, CHCl3} once again indicated the stereochemistry of C-8 as 8R , and the HMBC spectrum  and 7-Me (ca. δH 1.34) of 8 were also similar to those of related analogues. The NOESY correlations were observed for H-4/H-5, indicating that they were on the same side of the molecular plane, assumed to have an R-orientation. Therefore, the absolute configuration of C-4 and C-5 of 8 was deduced to be the  configuration   ,15. No N1–7) in compounds from fermented broth were filtered to separate the mycelium and culture broth. The culture broth was repeatedly extracted three times with EtOAc. The EtOAc-soluble fraction of the fermented broth of the actinobacteria H. yilanensis was tested in vitro against M. tuberculosis strain H37Rv. The antimycobacterial activity data are shown in 1), herbidosporayilanensin A (2), and herbidosporayilanensin F (6) exhibited more potent antimycobacterial activities against M. tuberculosis strain H37Rv in vitro, showing MIC values of 16.6, 19.2, and 18.2 μM, respectively, than did the clinical drug, ethambutol (MIC 6.25 μg/mL). The results showed moderate antimycobacterial activity, indicating that 3 and 4 had MIC values of 40.8 and 50.6 μg/mL, respectively. Compounds 5 and 7 showed no antimycobacterial activities. When comparing the two benzenoids (6–7), the presence of ketone groups on the 7 position of the side chain in 6 was seen to play an important role in antimycobacterial activity.The biological activities of the isolates present in sufficient amounts (254 precoated plates (Merck); for column chromatography (CC), silica gel 60  and Spherical C18 100A Reversed Phase Silica Gel (RP-18) (particle size: 20–40 μm) (Silicycle). For HPLC, we used spherical C18 column  (Waters) and LDC-Analytical-III apparatus. For the UV spectra, we used a Jasco UV-240 spectrophotometer, λmax (log ε) in nm. For the optical rotation, we used Jasco DIP-370 polarimeter, in CHCl3. For the IR spectra, we used a Perkin-Elmer-2000 FT-IR spectrophotometer; ν in cm−1. For the 1H-, 13C- and 2D-NMR spectra, we used Varian-Mercury-500 and Varian-Unity-Plus-400 spectrometers; δ in ppm rel. to Me4Si, J in Hz. For ESI and HRESIMS, we used a Bruker APEX-II mass spectrometer, in m/z.For TLC, we used silica gel 60 FHerbidospora yilanensis (0351M-12T), was isolated from sediment collected from the northern area of Taiwan using HVY agar and was then incubated at 45 °C for 7 days. This actinobacteria was identified by Mrs. Min Tseng, and the specimens (0351M-12T) were deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI). The strain was maintained on oatmeal agar, and the spores or mycelia suspension were harvested with 20% (v/v) glycerol and stored at −20 °C. A mature slant culture of strain 0351M-12T was inoculated into a 500 mL flask containing 100 mL of the seed medium consisting of 0.4% glucose, 0.4% yeast extract, and 1% malt extract (pH 7.3). After growing at 30 °C for 4 d on a rotary shaker (200 rpm), the aliquots (2 mL) of the seed culture were transferred into a 500 mL flask containing 200 mL of production medium . After 18 days cultivation at 30 °C temperature on a rotary shaker (200 rpm), the culture filtrates were obtained by filtering through filter paper.The actinobacteria, Fermented broth (3 l) was filtered to separate the mycelium and culture broth. The culture broth was repeatedly extracted three times with EtOAc. The EtOAc layers were combined and dried to give fraction-soluble EtOAc (25.2 g).n-hexane/acetone to give 10 fractions (1–10). Fraction 2 (1237 mg) was applied to a silica gel , eluting with a gradient of n-hexane/acetone to give eight fractions (2-1–2-8). Fraction 2-1 (179 mg) was chromatographed on a Sephadex LH-20 column (MeOH) to give five fractions (2-2-1–2-2-5). Fr. 2-2-3 (19 mg) was applied to an RP-C18 column (10 g), eluting with acetonitrile/H2O (2.5:1) to obtain 1 (1.9 mg) and 2 (1.8 mg). Fr. 2-2-4 (89.4 mg) was chromatographed on a silica gel column  eluting with CHCl3/MeOH (20:1) to give seven fractions (2-2-4-1~2-2-4-7). Fr. 2-2-4-3 was applied to an RP-C18 column, eluting with acetonitrile/H2O (3:1) to obtain 7 (2.8 mg) and 3 (1.1 mg). Fraction 5 (13.2 mg) was further purified by preparative RP-18 TLC  to afford 6 (3.8 mg) and 9 (0.53 mg). Fraction 7 (3.20 g) was applied to a silica gel column , eluting with CH2Cl2 to obtain seven fractions (7-1–7-7). Fraction 7-3 (85 mg) was chromatographed on an RP-C18 column (1 g), eluting with MeOH/H2O (2.5:1) to afford 5 (3.1 mg). Fraction 8 (2.25 g) was chromatographed on a silica gel column , eluting with a gradient of n-hexane/acetone, to give 10 fractions (8-1–8-10). Fr. 8-7 (188.6 mg) was chromatographed on an RP-18 column (2 g), eluting with  to afford 4 (5.1 mg) and 9 (0.41 mg).The EtOAc fraction (25.2 g) was applied to silica gel column , eluting with a gradient of 1): oil; c 0.01, CHCl3); IR (Neat): 3418(-OH), 2500–3400 (COOH), 1698 (COOH), 1644, 858 (C=CH2) cm−1; 1H NMR : see 13C NMR : see m/z (%):410 , 392 (5), 370 (44), 321 (34); HREIMS m/z 410.2667 [M]+ .Herbidosporayilanensin A (2): oil; c 0.01, CHCl3); UV (MeOH): 224 (4.11) nm; IR (Neat): 3462 (OH), 2500–3400 (COOH), 1693 (COOH), 1664, 905 (C=CH2) cm−1; 1H NMR : see 13C NMR : see m/z (%): 306 , 275 (4); HREIMS m/z 306.1835 [M]+ .Herbidosporayilanensin B (3): oil; c 0.012, CHCl3); UV (MeOH): 220 (4.00) nm; IR (Neat): 3444 (OH), 2500–3400 (COOH), 1682 (COOH), 1645, 1660, 894 (C=CH2) cm−1; 1H NMR : see 13C NMR : see m/z (%):332 , 314 (74), 145 (23), 105 (30); HREIMS m/z 332.1960 [M]+ .Herbidosporayilanensin C (4): oil; c 0.01, CHCl3); UV (MeOH): 294 (4.31), 280 (4.23), 228 (4.70) nm; IR (Neat): 3400 (OH), 1651 (conjugated C=O), 1597, 1505 (aromatic ring) cm−1; 1H NMR : see 13C NMR : see m/z (%): 330 , 312 (9), 285 (12), 121 (98), 55 (100); HREIMS m/z 314.1884 [M]+ .Herbidosporayilanensin D (5): oil; c 0.01, CHCl3); IR (Neat): 2500~3400 (COOH), 3403 (OH), 1696 (COOH), 1645, 890 (C=CH2) cm−1; 1H NMR : see 13C NMR : see m/z (%):387, 333 (61), 321 (100), 313 (38), 123 (32); HREIMS m/z 388.2615 [M]+ .Herbidosporayilanensin E (6): amorphous solid.; c 0.01, CHCl3); UV (MeOH): 231 (4.20), 276 (3.78), 312 (3.85) nm; IR (KBr): 1770 (OCO), 1668 (CO), 1605,1506, 1419 (aromatic ring) cm−1; 1H NMR : see 13C NMR : see m/z (%): 234 , 149 (100), 121 (13); HREIMS m/z 238.0538 [M]+ .Herbidosporayilanensin F (7): amorphous solid.; c 0.01, CHCl3); UV (MeOH): 230 (4.02), 280 (3.59) nm; IR (KBr): 1779 (OCO), 1594, 1517, 1463 (aromatic ring) cm−1; 1H NMR : see 13C NMR : see m/z (%): 234 , 149 (100), 121 (13); HREIMS m/z 236.1044 [M]+ .Herbidosporayilanensin G (8): oil.; c 0.01, CHCl3); IR (Neat): 3422 (OH), 1769 (γ-lactone ring) cm−1; 1H NMR : δH 1.05 , 1.75 , 1.88 , 2.53 , 2.79 , 4.28 , 4.49 ; 13C NMR : δC 9.9 (C-7), 21.5 (C-6), 39.5 (C-3), 68.8 (C-4), 85.9 (C-5), 175.4 (C-2).; EIMS (70 eV) m/z (%): 131 , 72 (100); HREIMS m/z 131.0721 [M+H]+ .Herbidosporayilanensin H (9): oil.; c 0.022, CHCl3); IR (Neat): 3424 (OH), 1772 (γ-lactone ring) cm−1; 1H NMR : δH 1.01 , 1.67 , 2.50 , 2.81 , 4.27–4.31 ; 13C NMR : δC 9.5 (C-7), 26.1 (C-6), 37.7 (C-3), 71.2 (C-4), 89.1 (C-5), 175.3 (C-2).; EIMS (70 eV) m/z (%): 131 , 72 (10); HREIMS m/z 131.0721 [M+H]+ .Herbidosporayilanensin I  is the lowest concentration of test compounds that completely inhibits the growth of the test isolate of M. tuberculosis, as detected by the unaided eye.The in vitro antitubercular activity of each tested compound (1–7) was evaluated using n method . BrieflyHerbidospora yilanensis is a new strain isolated from the sediment of rivers in northern Taiwan. It is very novel and worthy of further study. This strain has been published in the International Journal of Systematic and Evolutionary Microbiology (IJSEM). Based on the concept of “new species and new compounds”, it is expected that special compounds will be found from these new strains. The current study was conducted on the biologically active metabolites of the EtOAc soluble fraction of by the actinomycetes H. yilanensis. This led to the isolation of five previously undescribed compounds, namely, herbidosporayilanensin A-E (1–5), and four compounds isolated from nature for the first time, namely, herbidosporayilanensins F–I (6–9) (H. yilanensis fermentation broth was tested in vitro against Mycobacterium tuberculosis strain H37Rv. See 1), B (2), and F (6) showed more effective resistance to Mycobacterium tuberculosis strain H37Rv in vitro with MIC values of 16.6, 19.2, and 18.2 μM, respectively, than did the clinical drug, ethambutol (MIC 6.25 μg/mL). Compounds 5 and 7 did not show anti-mycobacterial activity. When comparing the two benzene analogues (6 and 7), it was observed that the presence of the ketone group at position 7 of the side chain in 6 played an important role in the anti-mycobacterial activity.–I (6–9) . The strMycobacterium tuberculosis in vitro. Therefore, it is still worth continuing to study the active substance against different Mycobacterium tuberculosis strains.In previous surveys, there have been many reports on the activity of actinomycete metabolites in the literature, but there are some reports of active natural products against


Rubus chingii Hu , a number of highly oxygenated terpenoids were isolated and characterized. These included nine ursane-type , five oleanane-type , and six cucurbitane-type (15–20) triterpenoids, together with five ent-kaurane-type diterpenoids (21–25). Among them, -2,19α,23-trihydroxy-3-oxo-urs-1,12-dien-28-oic acid , -2α,19α,24-trihydroxy-3-oxo-urs-12-en-28-oic acid , -2,19α-dihydroxy-olean-1,12-dien-28-oic acid , and -3α,16α,17-trihydroxy-ent-kaur-2-one  were previously undescribed. Their chemical structures and absolute configurations were elucidated on the basis of spectroscopic data and electronic circular dichroism (ECD) analyses. Compounds 1 and 3 are rare naturally occurring pentacyclic triterpenoids featuring a special α,β-unsaturated keto-enol (diosphenol) unit in ring A. Cucurbitacin B (15), cucurbitacin D (16), and 3α,16α,20(R),25-tetrahydroxy-cucurbita-5,23- dien-2,11,22-trione (17) were found to have remarkable inhibitory effects against NF-κB, with IC50 values of 0.08, 0.61, and 1.60 μM, respectively.During a phytochemical investigation of the unripe fruits of  Rubus genus (family Rosaceae) is large and diverse (with about 700 species distributed worldwide), and Flora of China lists 139 species as endemic to China D21 14.0 ; UV (MeOH) λmax (log ε) 265 (2.31) nm; ECD  λmax (Δε): 217 (−16.3), 274 (+13.9), 330 (−3.0) nm; IR (KBr) vmax 3576, 3446, 2970, 2937, 2870, 1721, 1703, 1690, 1644, 1464, 1402, 1379, 1270, 1242, 1157, 1058, 931, 863, 754 cm−1; 1H and 13C NMR data, see m/z 499 [M − H]−; HRESIMS m/z 499.3067 [M − H]− .-2α,19α,24-Trihydroxy-3-oxo-urs-12-en-28-oic acid . White powder; [α]D21 11.7 ; UV (MeOH) λmax (log ε) 202 (2.14) nm; ECD  λmax (Δε): 217 (−5.3) nm; IR (KBr) vmax 3446, 2975, 2927, 2848, 1703, 1691, 1459, 1387, 1210, 1185, 1140, 1053, 1028, 975, 868 cm−1; 1H and 13C NMR data, see m/z 501 [M − H]−, 525 [M + Na]+; HRESIMS m/z 525.3184 [M + Na]+ .-2,19α-Dihydroxy-olean-1,12-dien-28-oic acid . White powder; [α]D21 16.3 ; UV (MeOH) λmax (log ε) 266 (1.53) nm; ECD  λmax (Δε): 215 (−20.5), 274 (+12.8), 333 (−4.0) nm; IR (KBr) vmax 3571, 3446, 2972, 2935, 2868, 1706, 1695, 1634, 1462, 1407, 1384, 1237, 1212, 1157, 1050, 936, 756, 649 cm−1; 1H and 13C NMR data, see m/z 485 [M + H]+ and 507 [M + Na]+; HRESIMS m/z 485.3254 [M + H]+ .-3α,16α,17-Trihydroxy-ent-kaur-2-one . White powder; [α]D21 −30.2 ; UV (MeOH) λmax (log ε) 202 (1.53) nm; ECD  λmax (Δε): 285 (−8.7) nm; IR (KBr) vmax 3379, 2935, 2865, 1701, 1619, 1514, 1449, 1379, 1317, 1290, 1207, 1182, 1142, 1043, 916, 871 cm−1; 1H and 13C NMR data, see m/z 337 [M + H]+, 359 [M + Na]+; HRESIMS m/z 337.2389 [M + H]+ ., based on a sigmoidal dose response curve (variable slope) and computed using Graphpad Prism 5 (Graphpad Software). Bortezomib  was used as the positive control [HEK293 with a stable NF-se assay ,12,47. C control ,12,43.Candida albicans (the resistant strain 901 or the sensitive strain 5314) was chosen from Sabouraud’s agar and then inoculated in yeast extract peptone dextrose medium (YEPD) for activation by shake bottled cultivation . After 16 h, fungi in the late-exponential growth phase were obtained, followed by being adjusted to 1 × 103–5 × 103 CFU/mL with RPMI 1640 medium. The density of the fungi in 96-well plates was 103 CFU/mL, while the final concentrations of the test compounds ranged from 64 to 0.125 μg/mL in triplicate. The assay volume in each well was 100 μL with MIC80 determined following 48 h incubation at 37 °C. Optical density was measured with a microplate reader  at 630 nm. MIC80 was determined as the lowest concentration of the drugs that inhibited growth by 80% compared with the positive control. Fluconazole was used as the positive control [An anti-fungal assay was carried out on the basis of the Clinical and Laboratory Standards Institute (CLSI) method M27-A3 ,48. A si control ,46.R. chingii were extensively reviewed [R. chingii [R. chingii. Three highly oxygenated triterpenoids  and one ent-kaurane-type diterpenoid  were reported that were hitherto unknown. Compounds 1 and 3 possess a special diosphenol unit in ring A. Diosphenols are α-diketones, in which one of the carbonyls is enolized; they are part of the structure of several products of natural and synthetic origin, but are quite rare in naturally occurring ursane- and oleanane-type pentacyclic triterpenoids. To our knowledge, only a few plant-originated oleanane-/ursane-type [15–20) were obtained from the unripe fruits of R. chingii for the first time. Regarding the bioactivity evaluations, three isolates (15–17) showed potent inhibitory effects against NF-κB. It is worth noting that some cucurbitane-type tetracyclic triterpenoid glycosides from Momordica charantia [12), might be the principle in the anti-fungal activity of 70% ethanol Fructus Rubi [R. chingii and could provide useful clues for the discovery and development of new therapeutic or preventive agents for the treatment of NF-κB related diseases.Previous phytochemical studies on the fruits and leaves of reviewed ,5. Aroun chingii ,5,49. Inane-type ,51,52,53ane-type  triterpeharantia  have alstus Rubi . In gene


N,N-dialkylamino)-2,5-dihydrioimidazol-1-oxyl, with functional groups suitable for specific binding, were synthesized. The dependence of the parameters of their EPR spectra on pH was studied. Several showed a pKa close to 7.4, following the pH changes in a normal physiological range, and some demonstrated a monotonous change of the hyperfine coupling constant by 0.14 mT upon pH variation by four units.Local acidity and electrostatic interactions are associated both with catalytic properties and the adsorption activity of various materials, and with the vital functions of biomolecules. The observation of acid–base equilibria in stable free radicals using EPR spectroscopy represents a convenient method for monitoring pH changes and the investigation of surface electrostatics, the advantages of which are especially evident in opaque and turbid samples and in porous materials such as xerogels. Imidazoline nitroxides are the most commonly used pH-sensitive spin probes and labels due to the high sensitivity of the parameters of the EPR spectra to pH changes, their small size, and their well-developed chemistry. In this work, several new derivatives of 4-( Interfacial phenomena and local protonation effects play an important role in biophysics, biochemistry, and in the chemistry of heterogeneous systems . CatalytA-tensor and g-factor matrix, as well as the rotational dynamics of the nitroxide molecule in the proximity of charged surfaces, and this is reflected in the EPR spectra [2-SiO2 xerogels [EPR spectroscopy of ionizable nitroxides is a convenient method for the investigation of the above-mentioned phenomena ,4,5, and spectra . An analxerogels .N,N-dialkylamino)-2,5-dihydrioimidazol-1-oxyls from 4H-imidazole-3-oxides [a values showing high sensitivity in a broad range of pH [1, and pH-sensitive alkylating spin, labels 2a–c [3, for site-directed labeling of proteins and lipids [4, capable of binding to silica or alumina surfaces [Imidazoline nitroxides are the most commonly used pH-sensitive spin probes and labels due to the high sensitivity of the parameters of the EPR spectra to pH changes, their small size, and their well-developed chemistry. A large number of pH-sensitive nitroxides of the imidazoline series have been prepared ,4,9. Som3-oxides  allowed ge of pH ,12, e.g.els 2a–c ,14,15, wels 2a–c ,14, thiod lipids ,17, and 2 in nucleophilic substitution reactions offers easy access to new functional derivatives. Expectedly, 2b readily reacts with sodium azide to produce 5 with a nearly quantitative yield. Nitroxide azides can be used for the spin labeling of acetylene-modified molecules via Huisgen 1,3-dipolar cycloaddition [5 with tetraisopropyl but-3-yne-1,1-diyldiphosphonate 6 in the presence of Cu(II) salt and ascorbic acid after subsequent re-oxidation produced 7  produced spin label 15.Unless the attachment of a pH-sensitive nitroxide to a primary amino group is successfully performed via alkylation with 2b , the acyrocedure   to give 20a,b. To demonstrate the feasibility of the carbonylimidazole pH-sensitive spin labels for binding to primary amino groups, 20b was allowed to react with N,N-diethyl-1,3-diaminopropane.To prepare 2-functionalized nitroxides with a pKwas used . The tretroxides . The rea21 was isolated with a 60% yield. The oxidative cleavage of the terminal double carbon–carbon bond in 18b with osmium tetraoxide–oxone system yielded carboxylic acid 22.The nitroxide 17 is another convenient way to create 2-functionalized pH-sensitive spin labels. The reaction produced nitroxide 23 with a high yield. The dioxolane protection group in 23 was readily removed under relatively mild conditions to give the corresponding aldehyde 24, which can be either oxidized to carboxylic acid 25 using the Lindgren-Kraus-Pinnick procedure, or reduced with sodium borohydride to 19a. The sequence 17 → 23 → 24 → 19a gives a remarkably higher yield of the target nitroxide than the addition of allylmagnesium bromide with subsequent hydroboration. A titration of the nitroxides 20, 22, and 25 showed that they may be valuable spin labels and probes with high sensitivities to changes of pH within the physiological range  ethylmagnesium bromide to ange see .a nitroxide 1, which was successfully used in numerous studies [1 with a functional group in a substituent at the exocyclic nitrogen atom of the amidine moiety.An investigation of the surfaces of many inorganic and organo-inorganic materials  requires nitroxides with a high sensitivity to acidity changes within a broad range of pH. A good example of such a spin probe is two-pK studies ,29,30,31N-benzyl)-N-methylamine 26 was prepared in two steps from tereftaldicarboxaldehyde 27  corresponds to a smaller change in the hyperfine constant (0.05–0.063 mT), which may correspond to pyridine moiety protonation. Moreover, the basic pKa showed minor dependence on the nature of the substituent at the exocyclic nitrogen, while the acidic pKa varies from 2.58 for 34 to 2.19 for 35. Meanwhile, a comparison of 34 and 35 shows that an increase in the electron-withdrawing character of the substituent at the exocyclic nitrogen leads to an increase of ΔaN in the more acidic region, and a decrease of that correspondent to higher pKa. A comparison of the titration data for 1 and 36 [a values for 4-amino-2,5-dihydroimidazol-1-oxyls are strongly dependent on substituents at exocyclic nitrogen and can go below four [31–35 have similar basicity and the contribution of different monoprotonated forms is varying depending on the electronic effect of the substituents at the exocyclic nitrogen.In accordance with the general concept of basicity, the pK1 and 36  gives silow four ,33. ThusIn this paper feasibility of the approach to synthesis of pH-sensitive spin labels of 4-amino-2,5-dihydrioimidazol-1-oxyl series was once again demonstrated. We have showed that various functional groups can be easily placed in the substituents both in position 2 and to exocyclic nitrogen to make the spin probe suitable for a specific purpose. The potential of this synthetic scheme is still far from exhaustion.H-imidazol-3-oxides 2b, 8, 17 and 29 were prepared according to the literature protocols [The nitroxides and 4rotocols ,11,14. 1−4 M). NMR spectra 1H and 13C were recorded on a Bruker AV-300 (300.132 and 75.467 MHz), AV-400 (400.134 and 100.614 MHz). 1H and 13C chemical shifts (δ) were internally referenced to the residual solvent peak. The nitroxides were reduced to diamagnetic compounds with PhSH [2D4 [3COOH [4Cl/D2O [1H NMR spectra. HRMS analyses were performed with High Resolution Mass Spectrometer DFS . Reactions were monitored by TLC carried out using UV light 254 nm or 1% aqueous permanganate. Column chromatography was performed on silica gel 60 (70−230 mesh).The IR spectra were recorded on a Bruker Vector 22 FT-IR spectrometer  in KBr pellets (1:150 ratio) or in neat samples (for oily compounds). UV spectra were acquired on a HP Agilent 8453 spectrometer  in ethanol solutions ). The details are shown in The X-ray diffraction experiment was carried out on a Bruker KAPPA APEX II  diffractometer (graphite-monochromated Mo Kα radiation). Reflection intensities were corrected for absorption by SADABS-2016 program . The str program  and refiLXL-2018 . The pos2-(4-(Azidomethyl)phenyl)-2,5,5-triethyl-4-pyrrolidino-2,5-dihydro-1H-imidazol-1-oxyl (5)2b hydrochloride [3  was added, and the mixture was stirred at 60 °C for 10 h. The mixture was diluted with water (20 mL) and saturated solution of NaCl (50 mL) and extracted with diethyl ether. The extract was washed with saturated solution of NaCl and dried with Na2CO3, concentrated in vacuum, and the residue was separated using column chromatography on silica gel, eluent chloroform, to give 5, yield 290 mg (95%), yellow crystals, m.p. 63–65 °C (hexane). Elemental analysis, found: C, 65.33; H, 8.01; N, 22.47; calcd. for C20H29N6O: C, 65.01; H, 7.91; N, 22.75%. IR (KBr) νmax: 2091 (N3), 1597 and 1574 .A mixture of chloride  2CO3 and concentrated in vacuum. The residue was separated using column chromatography on silica gel, eluent chloroform, to give 6, yield 1.4 g (25%), colorless liquid. Elemental analysis, found: C, 50.35; H, 8.49; P, 16.10; calcd. for C16H32O6P2: C, 50.26; H, 8.44; P, 16.20%; IR (neat) νmax (cm−1): 2122 (C≡C). 1H NMR : 1.25 , 1.93 , 2.43 , 2.64 , 4.7 ; 13C{1H} NMR : 15.87 , 23.70 , 24.05 , 37.87 , 69.66 , 71.36 , 81.60 . Tetrais2-ethyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)-2,5,5-triethyl-4-pyrrolidino-2,5-dihydro-1H-imidazol-1-oxyl (7)5 , 6 , EtOH (1.5 mL), H2O (1.5 mL), and saturated solution of CuSO4 in water (0.15 mL). The mixture was stirred for 2 h, then PbO2  was added, the mixture was stirred for 1 h, then the precipitate was filtered off and washed with ethanol. The combined solutions were evaporated in vacuum and the residue was separated using column chromatography on silica gel, eluent chloroform, to give 7, yield 320 mg (65%), yellow oil. Elemental analysis, found: C, 57.23; H, 8.30; N, 10.98; P, 8.35; calcd. for C36H61N6O7P2: C, 57.51; H, 8.18; N, 11.18; P, 8.24%; IR (neat) νmax (cm−1): 1595, 1576 . 1H NMR : 0.76 , 0.90 , 1.04 , 1.26 2), 1.45 , 1.83 , 2.12 , 2.86 (1H (partly exchanged), tt, PJ 23, JH 6, P–CH–P), 3.26 , 3.7 , 4.69 , 5.51 , 7.32 , 7.39 , 7.52 , 7.69 ; 1H NMR : 0.85 , 0.89 , 1.04 , 1.19 2), 1.27 2), 1.33 2), 1.49 , 2.02 , 2.99 (1H (partly exchanged), tt, PJ 24, HJ 6, P−CH-P), 3.26 , 3.7 , 4.75 , 5.56 , 7.37  and 7.60 , 7.75 ; 31P NMR : 20.44, 20.47.Ascorbic acid  was added to a mixture of 2,5,5-Triethyl-2-(4-formylphenyl)-4-pyrrolidino-2,5-dihydro-1H-imidazol-1-oxyl (9)Method A8  in methanol (50 mL). The mixture was stirred for 4 h, manganese oxides were filtered off through celite 281, the solvent was distilled off in vacuum and the residue was separated using column chromatography on silica gel, eluent chloroform, to give 9, yield 340 mg (85%), yellow crystals, m.p. 84–85 °C dec. (chroloform-hexane). Elemental analysis, found: C, 70.20; H, 8.15; N, 12.31; calcd. for C20H28N3O2: C, 70.14; H, 8.24; N, 12.27%. IR (KBr) νmax (cm−1): 1697 (C=O); 1593, 1570 ; UV (EtOH) λmax (log ε): 229 (4.22), 253 (4.26).Activated manganese dioxide  was added to a stirred solution of Method B8  in chloroform (10 mL) and the solution was stirred for 2 h at room temperature. The solvent was distilled off in vacuum, the residue was separated using column chromatography on silica gel, eluent chloroform, to give 9, yield 440 mg (85%).A powder of 2,2,6,6-tetramethylpiperidinium chloride  was added to a solution of 2,5,5-Triethyl-2-(4-ethynylphenyl)-4-pyrrolidino-2,5-dihydro-1H-imidazol-1-oxyl (11)8 , freshly annealed K2CO3  and anhydrous methanol (5 mL). The mixture was stirred overnight, methanol was distilled off in vacuum, the residue was triturated with ethyl acetate, the precipitate was filtered off and washed with ethyl acetate, the combined solution was concentrated in vacuum and separated by column chromatography on silica gel, eluent diethyl ether–hexane 1:1 to give 11, yield 82 mg (84%), orange crystals, m.p. 156–158 °C (hexane-ethyl acetate). Elemental analysis, found: C, 74.81; H, 7.97; N, 12.50; calcd. for C21H28N3O: C, 74.52; H, 8.34; N, 12.41%. IR (KBr) νmax (cm−1): 3151 (≡C–H), 2094 (C≡C); 1587, 1554 .A solution of dimethyl (1-diazo-2-oxopropyl)phosphonate in acetonitrile  was added to a mixture of 2-(4-Carboxyphenyl)-2,5,5-triethyl-4-pyrrolidino-2,5-dihydro-1H-imidazol-1-oxyl (12)8  in CHCl3 (10 mL) was cooled to 0 °C and 2,2,6,6-tetramethyloxopiperidinium chloride (10)  was added in one portion. The mixture was stirred for 2 h at 0 °C. Then 2-methylbut-2-ene  was added to reaction mixture followed by addition of a solution of NaClO2  and KH2PO4  in H2O (44 mL). The mixture was stirred for 2 h, the organic layer was separated, washed with saturated aqueous solution of Na2CO3 (3 × 20 mL) and concentrated in vacuum. The residue was separated by column chromatography on silica gel using CHCl3–EtOH mixture (100:16) as an eluent to give light-yellow crystals of 12, yield 463 mg (89%), m.p. 205–207 °C (AcOEt—i-PrOH 10:1). Elemental analysis, found: C, 66.85; H, 7.87; N, 11.71; calcd. for C20H28N3O3: C, 67.01; H, 7.87; N, 11.72%. IR (KBr) νmax (cm−1): 2974 (C–H), 1693 (C=O), 1591 (C=N), 1571 (C=C). UV (EtOH) λmax (log ε): 232 (4.41). 1H NMR : 0.78 , 0.85 , 0.95 , 1.00–1.12 , 1.36, 1.75 , 1.85–1.99 , 2.00 , 3.50–3.55 , 7.66 , 8.01 .A solution of 2-(4-(Ethoxycarbonyl)phenyl)-2,5,5-triethyl-4-pyrrolidino-2,5-dihydro-1H-imidazol-1-oxyl (14)12  acid in dry CHCl3 (10 mL). The resulting solution was stirred at 0 °C, and SOCl2  was added dropwise. The stirring continued for 3 h, then ethanol  was added in one portion. The mixture was stirred for 2 h, the solvent was removed in vacuum, and the residue was separated using column chromatography on silica gel, eluent CHCl3–EtOH 200:1, to give 14, yield 352 mg (65%), yellow crystals, m.p. 85–90 °C (hexane). Elemental analysis, found: C, 68.60; H, 8.10; N, 10.80; calcd. for C23H32N3O3: C, 68.27; H, 8.35; N, 10.87). IR (KBr) νmax (cm−1): 2970 (C–H), 1718 (C=O), 1593 (C=N), 1571 (C=C). UV (EtOH) λmax (log ε): 231 (4.45). 1H NMR : 0.62 , 0.68 , 0.81 , 0.88–1.12 , 1.17 , 1.2, 1.60–1.84 , 1.89 , 3.44 , 4.15 , 7.44 , 7.80 .Pyridine  was added to a suspension of 2-carbonyl)phenyl)-2,5,5-triethyl-4-pyrrolidino-2,5-dihydro-1H-imidazol-1-oxyl (15)12  in dry CHCl3 (5 mL), the resulting solution was stirred at 0 °C, and SOCl2  was added dropwise. The reaction mixture was stirred for 3 h, then N-hydroxysuccinimide  was added in one portion. The mixture was stirred for 1 h, the solvent was removed in vacuum, and residue was separated using column chromatography on silica gel, eluent CHCl3–EtOH (100:1), to give 15, yield 102 mg (58%), yellow crystals, m.p. 107–108 °C (Et2O—hexane 1:2). Elemental analysis, found: C, 63.09; H, 6.86; N, 11.92; calcd. for C24H31N4O5: C, 63.28; H, 6.86; N, 12.30%. IR (KBr) νmax (cm−1): 2974 (C–H), 1774, 1743 (C=O), 1591 (C=N), 1571 (C=C). UV (EtOH) λmax (log ε): 235 (4.41).Pyridine  was added to a suspension of 2-(4-((3-Carboxypropanoyloxy)methyl)phenyl)-2,5,5-triethyl-4-pyrrolidino-2,5-dihydro-1H-imidazol-1-oxyl (16)8  in CHCl3 (10 mL) and the reaction mixture was heated to reflux for 2 h. The resulting solution was washed with H2O (10 mL), dried with Na2SO4, and the solvent was removed in vacuum. The solid residue was triturated with ether, the crystalline precipitate of 16 was filtered off and washed with diethyl ether, yield 213 mg (80%), yellow crystals, m.p. 168–169 °C dec. (Et2O). Elemental analysis, found: C, 64.37; H, 7.52; N, 9.48; calcd. for C24H34N3O5: C, 64.24; H, 7.71; N, 9.45%. IR (KBr) νmax (cm−1): 2969 (C-H), 1731 (C=O ester), 1587 (C=N), 1569 (C=O carboxy). UV (EtOH) λmax (log ε): 220 (4.03). 1H NMR : 0.66 , 0.81 , 1.00, 1.24 , 1.56–1.81 , 1.89 , 2.44 , 3.44 , 4.9 , 7.14 , 7.31 .Succinic anhydride  was added to a solution of 2-Allyl-2-ethyl-5,5-dimethyl-4-(pyrrolidino)-2,5-dihydroimidazol-1-oxyl (18a)17  in THF (15 mL). The reaction mixture was stirred for 1 h, then water (30 mL) was added dropwise under vigorous stirring. Then manganese dioxide  was added and the reaction mixture was stirred for 1 h. The manganese oxides were filtered off and the precipitate was washed with tert-butylmethyl ether. The organic layer was separated, the water solution was saturated with NaCl and extracted with tert-butylmethyl ether. The combined organic extracts were concentrated in vacuum and the residue was separated using column chromatography on Al2O3, eluent tert-butylmethyl ether–hexane (1:1) to give 18a, yield 630 mg (63%), yellow crystals, m.p. 55–57 °C (hexane). Elemental analysis, found: C, 67.22; H, 10.23; N, 16.88; calcd. for C14H24N3O: C, 67.16; H, 9.66; N, 16.78%. IR (KBr) νmax (cm−1): 2975 (C–H), 1645 (C=C), 1590 (C=N). UV (EtOH) λmax (log ε): 225 (4.17). 1H NMR : 0.91 , 1.47 , 1.60–1.83 , 2.01 ), 2.39–2.63 , 3.56 , 5.08 , 5.97 .A solution of allylmagnesium bromide prepared from allyl bromide  and Mg  in diethyl ether (15 mL) under argon was added dropwise to a stirred solution of nitrone 2-Ethyl-5,5-dimethyl-2-(pent-4-enyl)-4-(pyrrolidino)-2,5-dihydroimidazol-1-oxyl (18b)17  in THF (20 mL). The reaction mixture was stirred overnight, then water (4 mL) was added dropwise under vigorous stirring. The reaction mixture was vigorously stirred in air for 1 h, then organic layer was separated, and the aqueous layer was extracted with Et2O–EtOH (100:1). The combined organic extracts were dried with Na2SO4, solvents were distilled off in vacuum, and the residue was separated by column chromatography on Al2O3 using hexane–CHCl3 mixture (2:1) as an eluent to give 18b. Yield 931 mg (70%), yellow oil. Elemental analysis, found: C, 68.93; H, 9.80; N, 15.00; calcd. for C16H28N3O: C, 69.02; H, 10.14; N, 15.09%. IR (KBr) νmax (cm−1): 2973 (C–H), 1639 (C=C), 1594 (C=N). UV (EtOH) λmax (log ε): 225 (4.19). 1H NMR : 0.65 , 1.16 , 1.23 , 1.32 , 1.36–1.64 , 1.77 , 1.85 , 3.23, 3.43 , 4.60–4.76 , 5.48 .A solution of pent-4-enylmagnesium bromide was prepared from 5-bromopentene  and Mg  in THF (20 mL) under argon. This solution was added dropwise to a stirred solution of 2-Ethyl-2-(3-hydroxypropyl)-5,5-dimethyl-4-(pyrrolidino)-2,5-dihydroimidazol-1-oxyl (19a)Method A18a  in THF (10 mL) under argon. The reaction mixture was vigorously stirred for 4 h, then cooled to 0 °C and cold (0 °C) aqueous NaOH  and cold (0 °C) H2O2  were added dropwise successively. The mixture was allowed to warm to room temperature upon stirring (ca. 2 h), organic layer was separated, dried with Na2CO3, and the solvent was distilled off in vacuum. The residue was dissolved in CHCl3 (25 mL), anhydrous Na2CO3 (1 g) was added, and mixture was allowed to stand overnight in air. The solution was concentrated in vacuum and separated by column chromatography on silica gel using CHCl3–EtOH mixture (100:4) as an eluent to give 19a. Yield 150 mg (35%), yellow oil. Elemental analysis, found: C, 62.53; H, 9.49; N, 15.45; calcd. for C14H26N3O2: C, 62.65; H, 9.76; N, 15.66%. IR (KBr) νmax (cm−1): 3386 , 1592 (C=N). UV (EtOH) λmax (log ε): 225 (4.07). 1H NMR : 0.94 , 1.43 , 1.53–1.92 , 1.98 , 3.52 , 3.57 . 2-Ethyl-2-(5-hydroxypentyl)-5,5-dimethyl-4-(pyrrolidino)-2,5-dihydroimidazol-1-oxyl (19b) was prepared similarly from 18b. Yield 42%, yellow crystals, m.p. 68–73 °C (Et2O). Elemental analysis found: C, 65.17; H, 10.56; N, 14.08; calcd. for C16H30N3O2: C, 64.83; H, 10.20; N, 14.18%. IR (KBr) νmax (cm−1): 3261 , 1593 (C=N). UV (EtOH) λmax (log ε): 225 (4.1). 1H NMR : 0.62 , 1.06 2OH), 1.21–1.28 , 1.40 , 1.57 –CH2), 1.79 , 3.20, 3.44 , 3.26 .A solution of 9-BBN in THF  was added dropwise to a stirred solution of Method B24  in EtOH (10 mL) at 0 °C. The reaction was controlled with TLC, Silufol UV-254, eluent CHCl3–EtOH (25:1). Inorganic residue was filtered off, the solution was distilled off in vacuum, and the residue separated by column chromatography as described above to give 19a. Yield 309 mg (72%).Sodium borohydride  was added portionwise to a stirred solution of 2-(3-(1H-Imidazole-1-carbonyloxy)propyl)-2-ethyl-5,5-dimethyl-4-(pyrrolidino)-2,5-dihydro-1H-imidazol-1-oxyl (20a)19a  in dry CHCl3 (5 mL) and the mixture was allowed to stand for 24 h. The solution was washed with brine, dried with Na2SO4, and concentrated in vacuum. The residue was separated by column chromatography on silica gel using CHCl3–EtOH mixture (100:2) as an eluent, producing 20a as yellow oil. Yield 139 mg (90%). Elemental analysis, found: C, 59.69; H, 7.72; N, 19.45; calcd. for C18H28N5O3: C, 59.65; H, 7.79; N, 19.32%. IR (KBr) νmax (cm−1): 1760 (C=O), 1592 (C=N). UV (EtOH) λmax (log ε): 223 (4.17). 2-(5-(1H-Imidazole-1-carbonyloxy)pentyl)-2-ethyl-5,5-dimethyl-4-(pyrrolidino)-2,5-dihydro-1H-imidazol-1-oxyl (20b) was prepared similarly, yield 80%, yellow oil. Elemental analysis, found: C, 61.30; H, 8.26; N, 17.70; cacld. for C20H32N5O3: C, 61.51; H, 8.26; N, 17.93%. IR (KBr) νmax (cm−1): 1762 (C=O), 1593 (C=N). UV (EtOH) λmax (log ε): 226 (3.99).Carbonyldiimidazole  was added to a solution of alcohol 2-(3-(3-(Diethylamino)propylcarbamoyloxy)propyl)-2-ethyl-5,5-dimethyl-4-(pyrrolidino)-2,5-dihydro-1H-imidazol-1-oxyl (21)N,N-Diethyl-1,3-diaminopropane  was added to a solution of 20a  in dry Et2O (5 mL), and mixture was allowed to stay for 24 h. The solution was concentrated in vacuum, and the residue was separated by column chromatography on Al2O3 using CHCl3 as an eluent to give 21  νmax (cm−1): 1718 (C=O), 1593 (C=N). UV (EtOH) λmax (log ε): 225 (4.19). 1H NMR : 0.75 , 1.11  1.33 , 1.40 , 1.43–1.79 , 1.90 , 2.87–3.03 , 3.36, 3.52 , 3.84 . give 21 . Yield 82-(3-Carboxypropyl)-2-ethyl-5,5-dimethyl-4-(pyrrolidino)-2,5-dihydro-1H-imidazol-1-oxyl (22)18b  in DMF (20 mL) and the mixture was stirred for 3 h. A powder of Na2SO3  was added in one portion. Inorganic precipitate was filtered off and washed with EtOH, the combined solution was evaporated to dryness in vacuum and the residue was separated by column chromatography on silica gel using EtOH as an eluent to give 22, yield 90 mg (21%), yellow oil. M+ (cacld./found) 296.1969/296.1972. IR (neat) νmax (cm−1): 2977 (C-H), 1664 (C=O), 1592 (C=N). λmax (EtOH)/nm: 225 (lgε 4.19).Osmium tetroxide  and oxone  were added successively to a solution of 2-ethyl)-2-ethyl-5,5-dimethyl-4-(pyrrolidin-1-yl)-2,5-dihydro-1H-imidazol-1-oxyl (23)17  in 20 mL Et2O and 6 mL THF. The reaction mixture was stirred overnight, then water (5 mL) was added dropwise under vigorous stirring. The reaction mixture was allowed to air for 1 h, then organic layer was separated, inorganic residue was quenched with Et2O–EtOH (100:1). An isolated organic layer was dried over Na2SO4, solvents were removed in vacuum. The residue was separated using column chromatography on Al2O3 using CHCl3 as an eluent to give 23, yield 1.13 g (90%), yellow oil. Elemental analysis, found: C, 62.08; H, 9.21; N, 13.43; calcd for C16H28N3O3: C, 61.91; H, 9.09; N, 13.54%. IR (KBr) νmax (cm−1): 2972 (C-H), 1593 (C=N), 1143 (C–O). λmax (EtOH)/nm: 225 (lgε 3.90).A solution of 2-ethylmagnesium bromide was prepared from 2-(2-bromoethyl)-1,3-dioxolan  and Mg  in 20 mL THF under a stream of argon. This solution was added dropwise to a stirred solution of nitrone 2-Ethyl-5,5-dimethyl-2-(3-oxopropyl)-4-(pyrrolidin-1-yl)-2,5-dihydro-1H-imidazol-1-oxyl (24)3 (10 mL) was added to a residue. The product was extracted with CHCl3–i-PrOH mixture (50:1) (3 × 15 mL). An isolated organic layer was dried over Na2SO4, the solvents were removed in vacuum, the residue was separated using column chromatography on silica gel using CHCl3-EtOH mixture (50:1) as an eluent to give 24, yield 161 mg (75%), yellow oil. Elemental analysis, found: %: C, 63.08; H, 9.18; N, 15.63; calcd. for C14H24N3O2: C, 63.13; H, 9.08; N, 15.78. IR (neat) νmax (cm−1): 2972 (C-H), 1720 (C=O), 1593 (C=N). λmax (EtOH)/nm: 225 (lgε 4.16).A solution of oxalic acid  in water (6 mL) was added to a solution of nitroxide 23  in EtOH (4 mL). The reaction mixture was stirred for 3 h under reflux, then ethanol was removed in vacuum, saturated aqueous KHCO2-(2-Carboxyethyl)-2-ethyl-5,5-dimethyl-4-(pyrrolidin-1-yl)-2,5-dihydro-1H-imidazol-1-oxyl (25)24  in 10 mL CH3CN followed by addition of a solution of NaClO2  and KH2PO4  in H2O (20 mL). Progress of the reaction was monitored by TLC , developing with 1% aq KMnO4). CH3CN was removed in vacuum, the product was extracted from water by CHCl3–i-PrOH mixture (100:1) (5 × 15 mL). An isolated organic layer was dried over Na2SO4, the solvents were removed in vacuum, the residue was separated using column chromatography on silica gel using CHCl3–EtOH mixture (5:2) as an eluent to give 25, yield 121 mg (57%), yellow oil, M+  282.1812/282.1811. IR (neat) νmax (cm−1): 2973 (C–H), 1729 (C=O), 1591 (C=N). λmax (EtOH)/nm: 223 (lgε 4.04). 1H NMR  0.75 , 0.98–1.14 , 1.34, 1.40 , 1.47–1.61 , 1.91 , 2.14–2.27 , 3.36, 3.55 .Trimethylethylene  was added to a cooled (0 °C) solution of aldehyde 1-phenyl)-N-methylmethanamine (26)27  in 175 mL PhCH3. Water was distilled off with Dean-Stark tube. The reaction mixture was then quenched with aqueous NaHCO3, dried over Na2CO3, the solvent was remove in vacuum, and residue was dissolved in methanol saturated with methylamine (20 mL). The resulting solution was added to the previously maintained under vigorous stirring for 10 min in a solution of Ti(Oi-Pr)4  in methanol saturated with methylamine (30 mL). The mixture was stirred for 5 h, then NaBH4  was added portionwise, and mixture was stirred for 2 h. Water (7 mL) was added dropwise, solvents were removed in vacuum, brine was added to a residue, and the product was extracted by ether. Organic layer was dried over NaOH. Residue was separated using column chromatography on silica gel using Et2O–EtOH mixture (10:1) as an eluent, yield 12.24 g (85%), colorless oil. Elemental analysis, found: C, 67.84; H, 7.91; N, 6.94; calcd for C11H15NO2: C, 68.37; H, 7.82; N, 7.25%. 1H NMR : 2.38 , 3.70 , 3.95–4.09 , 5.75 , 7.29, 7.38 ), 13C NMR : 35.77 (N–CH3), 55.56 (N–CH2), 65.06 (O–CH2–CH2–O), 103.44 (O–CH–O), 126.31 (CH–C–CH2NHCH3), 127.89 (CH–C–CH), 136.32 (C–CH2NHCH3), 141.10 (C–CH). IR (neat) νmax (cm−1): 3325 (N–H), 1082 (O–C–O). λmax (EtOH)/nm: 210 (logε 3.94), 260 (logε 2.36).p-Toluene sulfonic acid monohydrate  was added to a solution of terephthalic aldehyde 5-benzyl)(methyl)amino)-4,4-dimethyl-2-(pyridin-4-yl)-4H-imidazole 3-oxide (30)N-methylmethanamine 26  was added to a solution of 5-cyano-4,4-dimethyl-2-(pyridin-4-yl)-4H-imidazole 3-oxide 29  in THF (25 mL) and the mixture was allowed to stand at r.t. for 24 h. The solvent was removed in vacuum, residue was triturated with ether and crystallization from CH3CN to give 30, yield 3.97 g (75%), dirty-yellow crystals, m.p. 160 °C (dec.). Elemental analysis, found: C, 65.81; H, 6.25; N, 14.41; calcd for C21H24N4O3: C, 66.30; H, 6.36; N, 14.73%. 1H NMR  1.66 , 3.06 , 3.86–4.16 , 4.77 , 5.75 , 7.26, 7.46 ), 8.46, 8.70 . 13C NMR  21.50 (2 × Me), 35.22 (N–CH3), 53.11 (N–CH2), 65.00 (O–CH2–CH2–O), 75.73 (Me2C), 102.82 (O–CH–O), 121.00 , 126.77 ), 133.86 , 136.36 (C–CH2NCH3), 137.47 (C–CH), 144.67 (C=N→O), 149.82 , 172.33 (C=N). IR (KBr) νmax (cm−1): 1597 (C=N), 1082 (O–C–O). λmax (EtOH)/nm: 263 (logε 4.30), 389 (logε 3.78).1-phenyl)-4-benzyl)(methyl)amino)-2-ethyl-5,5-dimethyl-2-(pyridin-4-yl)-2,5-dihydro-1H-imidazol-1-oxyl (31)2O under a stream of argon. This solution was added dropwise to a stirred solution of nitrone 27  in 15 mL THF. The reaction mixture was allowed to stand for 1 h. Then water (3 mL) was added dropwise under vigorous stirring followed by MnO2  addition. Progress of the reaction was monitored by TLC , developing with 1% aq. KMnO4). The mixture was stirred vigorously for 2 h, the oxidant was filtered off and the residue was washed by CHCl3 and MeOH. The solvent from filtrate was removed in vacuum and the residue was separated by column chromatography on silica gel using CHCl3-EtOH (100:3) as an eluent. The product 31 was isolated as a hydrochloride. Yield 797 mg (68%), yellow oil. Elemental analysis, found: C, 62.18; H, 6.83; N, 12.48; Cl, 6.70; calcd. for C23H30ClN4O3: C, 61.94; H, 6.78; N, 12.56; Cl, 6.95%. IR (neat) νmax (cm−1): 1593 (C=N), 1082 (O–C–O). λmax (EtOH)/nm: 216 (logε 4.34).A solution of ethylmagnesium bromide was prepared from ethyl bromide  and Mg  in 35 mL Et2-Ethyl-4-((4-formylbenzyl)(methyl)amino)-5,5-dimethyl-2-(pyridin-4-yl)-2,5-dihydro-1H-imidazol-1-oxyl (32)30  in 15 mL 0.5 M aq. HCl was refluxed for 4 h, then Na2CO3 added to the end of gas evolution. A product was extracted by mixture of 20 mL CHCl3 + 1 mL i-PrOH three times, organic layer was dried over Na2CO3, the solvents were removed in vacuum, and the nitroxide 29 was isolated from the residue by column chromatography on silica gelusing CHCl3 as an eluent. Yield 1.14 g (71%), yellow oil. Elemental analysis, found: C, 68.73; H, 6.88; N, 14.92; calcd. for C21H25N4O2: C, 69.02; H, 6.90; N, 15.33%. IR (KBr) νmax (cm−1): 1701 (C=O), 1593 (C=N). λmax (EtOH)/nm: 252 (logε 4.29).A solution of nitroxide 4-((4-Carboxybenzyl)(methyl)amino)-2-ethyl-5,5-dimethyl-2-(pyridin-4-yl)-2,5-dihydro-1H-imidazol-1-oxyl (33)29  in 20 mL CHCl3 followed by addition of a solution of NaClO2  and KH2PO4  in H2O (50 mL). Progress of the reaction was monitored by TLC , developing with 1% aq. KMnO4). The organic layer was separated, the product was extracted from water by CHCl3—i-PrOH mixture (20:1) (2 × 20 mL). A combined organic extracts were washed with brine, dried over Na2SO4, the solvents were removed in vacuum, the residue was separated using column chromatography on silica gel using AcOEt–EtOH mixture (10:1) as an eluent. Yield 285 mg (47%), yellow crystals, compound 33 was isolated as a crystal solvate 3 (33) × 2 EtOH (ether–EtOH 100:2), m.p. 204 °C (dec.). Elemental analysis, found: C, 65.12; H, 6.42; N, 13.12; calcd. for C67H87N12O11: C, 65.08; H, 7.09; N, 13.59%. IR (KBr) νmax (cm−1): 2474 (O–H), 1708 (C=O), 1597 (C=N), λmax (EtOH)/nm: 242 (logε 4.20).Trimethylethylene  was added to a cooled (0 °C) solution of aldehyde 2-Ethyl-4-((4-(hydroxymethyl)benzyl)(methyl)amino)-5,5-dimethyl-2-(pyridin-4-yl)-2,5-dihydro-1H-imidazol-1-oxyl (34)4  was added portionwise to a cooled (0 °C) solution of aldehyde 32  in EtOH (20 mL). The reaction mixture was stirred until the reaction was complete . The solvent was removed in vacuum, the residue was separated using column chromatography on silica gel using AcOEt as an eluent. Yield 308 mg (60%), yellow crystals, compound 34 was isolated as a crystal solvate 2 (34) × 3 H2O (ether), m.p. 147–148 °C. Elemental analysis, found: C, 66.31; H, 7.12; N, 14.55; calcd. for C63H85N12O8: C, 66.47; H, 7.53; N, 14.76%. IR (KBr) νmax (cm−1): 3178 (O-H), 1595 (C=N). λmax (EtOH)/nm: 220 (logε 4.30). 1H NMR : 1.03 , 1.29 , 1.79 , 1.90 , 3.20 , 4.65 , 4.97 , 7.26 , 7.46 , 7.94 , 8.73 NaBH4-oxy)carbonyl)benzyl)(methyl)amino)-2-ethyl-5,5-dimethyl-2-(pyridin-4-yl)-2,5-dihydro-1H-imidazol-1-oxyl (35)33  in 10 mL of dry CHCl3 followed by addition of SOCl2 . The reaction mixture was vigorously stirred for 3 h, then N-hydroxysuccinimide  was added and the mixture was allowed to stand for 24 h. The solvents were then removed in vacuum, residue was separated using column chromatography on silica gel using CHCl3–EtOH mixture (100:2) as an eluent to give 35, yield 123 mg (40%), yellow crystals, compound 35 was isolated as a hydrochloride (hexane), m.p. 58 °C (dec.). Elemental analysis, found: C, 58.42; H, 5.47; N, 13.25; Cl, 6.56; calcd for C25H29ClN5O5: C, 58.31; H, 5.68; N, 13.60; Cl, 6.88%. IR (KBr) νmax (cm−1): 2976 (C-H), 1770 (O=C-N-C=O), 1741 (C=O), 1593 (C=N). λmax (EtOH)/nm: 239 (logε 4.25)Pyridine  was added to a cooled (0 °C) suspension of acid EPR experiments were performed on X-band EPR (9.8 GHz) spectrometer Bruker ER-200D. Titrations of the radicals (~0.2 mM) were performed in a buffer mixture of acetate-phosphate-borate (0.5 mM of each) in a pH range of 2–10 starting from the acidic value. Small aliquots of NaOH solution were used for titration to a higher pH. The observed hfi constants were measured as a distance between low field and central lines of nitroxide triplet spectra.a titration curve function:Single proton exchange:a titration curve function:Double proton exchange implies the serial protonation,EPR settings. Microwave power, 5 mW; modulation amplitude, 0.08–0.12 mT.a, 0.05; for hfc, 0.005 mT.SD for pK