Here we present a checklist of the bee species found on the C. Hart Merriam elevation gradient along the San Francisco Peaks in northern Arizona. Elevational gradients can serve as natural proxies for climate change, replacing time with space as they span multiple vegetation zones over a short geographic distance. Describing the distribution of bee species along this elevation gradient will help predict how bee communities might respond to changing climate. To address this, we initiated an inventory associated with ecological studies on pollinators that documented bees on the San Francisco Peaks. Sample sites spanned six life zones (vegetation zones) on the San Francisco Peaks from 2009 to 2019. We also include occurrence data from other studies, gathered by querying the Symbiota Collection of Arthropods Network (SCAN) portal covering the San Francisco Peaks region (hereafter referred to as “the Peaks”).Our checklist reports 359 bee species and morphospecies spanning five families and 46 genera that have been collected in the Peaks region. Prior to our concerted sampling effort there were records for 155 bee species, yet there has not been a complete list of bee species inhabiting the Peaks published to date. Over a 10-year period, we documented an additional 204 bee species inhabiting the Peaks. Our study documents range expansions to northern Arizona for 15 species. The majority of these are range expansions from either southern Arizona, southern Utah, or the Rocky Mountain region of Colorado. Nine species are new records for Arizona, four of which are the southernmost record for that species. An additional 15 species are likely undescribed. The North American Southwest has one of the highest biodiversity of bee species worldwide , with ArIn northern Arizona, the San Francisco Peaks region (hereafter referred to as “the Peaks”) is one of the northern most sky islands and is characterized by the C. Hart Merriam elevational gradient, ranging from 785 to 3,850 meters . This raThere have been multiple checklists published within the last year summarizing the bee species found in various regions of North America, including areas in the southern and western US . HoweverResearch was conducted on the San Francisco Peaks in northern Arizona Fig. . A totalCup Sampling: 2009–2012 : Pollinators were sampled from 2009-2012 at five life zones ranging from desert shrub to mixed conifer, with one site established at each life zone. At each site we placed one pollinator cup array, which consisted of 30 pollinator cups (i.e. elevated pan traps). Each cup was filled with 50/50 water/propylene glycol about 2/3 of the way full. The pollinator cups were 12 oz. plastic stadium cups . White, yellow and blue colors accounted for all of the major flora colors in this area : Bees were sampled using pollinator cups at three life zones on the Peaks: ponderosa pine, mixed conifer and spruce-fir. We sampled at three unique sites at each life zone and set up pollinator arrays in two distinct locations per site: one array was placed in a meadow habitat and one was placed in a forest habitat. An array consisted of nine pollinator cups . Details on our method of pollinator cup trapping is described above. Each year pollinator cups were set up during two seasons: dry pre-monsoon (June) and monsoon (August). During the monsoon season of 2013, 50% of the pre-monsoon cups were lost to animal damage at our Peaks sites at the spruce-fir elevation.Cup Sampling: 2015 : Cup sampling methods were identical to those used in 2013-2014, however we added an additional three sites at both ponderosa pine and spruce-fir . In addition, we established pollinator cup arrays on Kendrick Mountain, a neighboring mountain within the Peaks region, where we sampled at three life zones: ponderosa pine, mixed conifer, and spruce-fir, with three sites at each life zone . Cup sampling methods and array design were identical to that used for the cup sampling on the Peaks. Each year, for both mountains, pollinator cups were set up during two seasons: dry pre-monsoon (June) and monsoon (August).Cup Sampling: 2016 : Cup sampling methods were identical to those used in 2013-2015, however there were differences in the sampling sites. Some sites were reused from previous years . An additional five sites were established at each of the three higher life zones , and seven new sites were established at the pinyon juniper ife zone (PJ1-PJ7). This led to a total of 32 sites, with eight sites per life zone.Flower Sampling: 2016–2018 : In 2016, transect plots were established at four life zones: pinyon-juniper, ponderosa pine, mixed conifer, and spruce-fir. Eight sites were established at each life zone that were at least 1 km apart, with each site containing three 60-meter × 1-meter transects. Five sites were re-used from previous sampling years . Using modified hand vacuums  as well as near Snowbowl Ski Resort (considered mixed conifer and spruce-fir life zones). A few additional specimens were collected at sites used in previous years . Latitude and longitude decimal points for all 2019 sampling locations are provided , Jason Gibbs (Lasioglossum) and Karen Wright (Melissodes). Vouchers were deposited in the Colorado Plateau Museum of Arthropod Biodiversity, ARS Pollinating Insect Research Unit, Wallis Roughley Museum of Entomology, and Texas A&M University Insect Collection.All bees collected in samples were curated and initially identified in the Northern Arizona University (NAU) pollinator ecology lab. Bees were identified using DiscoverLife.org and published identification guides. Classification for species of For those genera or subgenera where taxonomic information was lacking, we classified bees with similar morphological distinctions into morphospecies. Each morphospecies is classified by the genus (and subgenus if determined) followed by a unique three-digit number. Male and female specimens of the same morphospecies were combined. Species that were morphologically different were treated as unique morphospecies. All morphospecies listed are all potentially undescribed taxa.We established a reference collection of bee species that is currently stored in the Colorado Plateau Museum of Arthropod Biodiversity at NAU. All specimens were digitally cataloged in the Symbiota Collections of Arthropods Network (SCAN) online data portal. Identification of the 65 species that were not collected by the NAU lab and confirmed by NAU, the Logan Bee Lab, Jason Gibbs or Karen Wright need further consideration, especially in instances/localities where they have not been collected for 20+ years. These 65 taxa are noted with the year that they were last collected on the Peaks. Further, one-third of these taxa (20 species) were not assigned to a life zone due to a lack of precision in the latitude and longitude coordinates. These 20 species were removed from further analysis , GBIF , and DiscoverLife. Species were deemed a new record for Arizona if there were not any previous records documented within the Arizona state boundaries on any of the four data portals mentioned above. We examined published literature to verify that these species were not previously recorded within the Arizona state boundary . SpeciesSpecies were assigned "notes" if 1) they had not been recorded in our study range prior to our 10-year NAU study or 2) they were not collected in our 10-year NAU study but were collected in previous years from other sampling events (followed by the year that the species was last collected). Records obtained through SCAN, GBIF and iDigBio databases provided this information.LaBerge, 1980EE553594-CFF1-5C83-B19B-73804BDE5E40Last collected on the Peaks in 1952Smith, 185310FF2589-57E7-565C-958C-49FAF484AADFLast collected on the Peaks in 1986FCEDFE2B-0D94-5206-8C18-20B51588540FB4BB85C9-7692-5FB4-B8B0-0226E2C544FFCockerell, 1913056650F2-725E-5D9B-A65D-A50C61DC4E66LaBerge, 19671E322496-9272-5707-A468-CEADE384DD51Last collected on the Peaks in 1976LaBerge, 1967D57674BA-0323-597A-B835-F09BE76A2FBFLaBerge, 197899932799-3006-5D38-8B1E-981DE30DFE90LaBerge, 19673994EE18-CC22-5AC2-8FA6-C6EC2AD6ADE5Smith, 18794330DD37-635D-5EC9-857A-283B5478CAD0LaBerge, 19673C77101A-900E-5B83-8AF1-B59BB96CBC69Cockerell, 1897B5BBC7A3-8950-5173-9ACB-5ECC0CC367D4Cockerell, 1914E3FEF0B8-6F65-5D56-B34D-E394731B8B9DSmith, 185368E728AC-8DD3-577D-BA1F-D71661CC6E7BFabricius, 177567C51B3F-834B-54CC-BCB5-13F84759CFD7Smith, 1853568DF721-899F-50FE-8F0C-2274C5F2DC6BDE667109-A24C-5A68-B100-411B8960BF9BOur record is the first documentation of this species in northern Arizona. Species occurs in neighboring areas.Viereck & Cockerell, 19142EA72269-F9CE-50D6-9DE4-6430570D3D22Last collected on the Peaks in 1902Smith, 1879700CE654-CF4D-5C48-B5F5-605F5E77D1BFBouseman & LaBerge, 19790C07EB11-8C64-5C6A-A3DD-72CFCCABD514Cockerell, 19300797F720-36D2-55E1-B241-6BDCAC7C1D9DCockerell, 18968F3E4AAE-633B-599F-9FD7-9CE4927DBD74Robertson, 1893FF2B235A-AEAC-5B35-926A-40A91CD1B1EECockerell, 1896ED0E9277-5D85-56BB-90F2-50ECD178A79DDCBB3B1E-36ED-5D22-B457-5A9C24BCDA36Cockerell, 1902841FFBB1-E793-5E7C-BA22-A88C5B63770BViereck 19046D40E315-9042-5FBF-9FAF-0EDF2071BC249DAB65D5-0AE3-521B-A5C4-0D4C2E34F489Our records are the first documentation of this species in Arizona and the southernmost extension of its range. Species occurs in neighboring areas.Cockerell, 19069E133523-1B84-57BF-B7F4-C90F9BE4350827D16DB7-9C8C-5A6C-9330-F8D09371DD8FOur record is the first documentation of this species in Arizona. Species occurs in neighboring areas.Smith, 18794478B4E8-75B2-5204-863D-03007914CB8F9D5EB134-49BA-5959-B60B-47F61EBAB150Robertson, 19027A325DBC-2149-53AC-B8ED-35F8F3EBD1ACFabricius, 1775EFA2D9EE-87F0-5E8E-B207-EC8EF0C3ACCBFabricius, 1775EF3FCD8B-548D-574D-843C-EC20245A629FFabricius, 1775BC10DF13-E9F1-5A81-98B2-3EE659988B9DFabricius, 17758ABE8CA3-E2C0-5698-BDF7-2F93EDBF16EAFabricius, 1775E58F4BC1-E6A5-525C-A649-49B4B0525389Cockerell, 18996F5938D7-FE8E-5166-9BB4-85C1BC4AB355Shinn, 1965DB5A2656-696A-5BFB-A0EF-59C68319E760Last collected on the Peaks in 1951Cockerell, 18997DCA38EA-0BDD-59BA-AC76-4318DEFBA20DLast collected on the Peaks in 196152F4D3BE-C47B-5743-9DEF-979B5D2AC36372A91B0C-7F35-58D1-8AC7-196ED20C5DD577C27628-DB02-5C8D-A13B-F815B99BD568FF62439F-9B05-594B-8625-8628AC9C3227Smith, 18992E8A5D47-E8A7-5615-9000-8C72A195CE6B4B53C0FE-5DDF-5AF9-BB57-C2E8159FFBFBTimberlake, 1954D3BA903F-89E5-585B-9E76-F380D5DA309CLast collected on the Peaks in 1964Cockerell, 18967B31D55E-3C61-57B0-B0CF-9075E8DA1E88Last collected on the Peaks in 1965Cockerell, 18963BAE7D80-66A4-5A50-8A4F-6A7D1E602EA7Last collected on the Peaks in 1967Cresson, 187805A7D981-951F-5CDE-836D-80F7E0A99041Last collected on the Peaks in 1952Smith, 18538B97B4A9-C73F-52B2-8E9D-42F39C2BB14CSmith, 1853FAA139D9-5498-5423-97DE-EBF99F502AABSmith, 1853C995971D-99E1-5005-9D55-D74BF7E532CASmith, 185341551D8F-A56B-5278-8E74-F31832755B7ESmith, 1853233873F0-F640-5EE1-9156-E2A5888E83F0Smith, 1853A78FA619-8E98-5C8C-8B6C-05EE14BFDAC7Smith, 185321EFA0A9-DF26-507B-B6CD-30F0F277C08BSmith, 185316D25AEA-6330-5F24-A9C2-2C02E5402005Smith, 185352F34359-2036-5282-8C20-FEEB9827493FSmith, 185302B1A8EA-F937-5287-A410-0E4E341B7A4DCockerell, 1906DDF4C8B3-0AED-535B-8B5A-520194FC088DLast collected on the Peaks in 1958Robertson, 1904CC35BF5D-643A-579E-BE92-6AB414294EAARobertson, 190424FC68C2-808C-5A65-BB42-62C54D43197ARobertson, 1904D8DD6A0E-56A8-51C4-8054-8FEA79E9D944Robertson, 1904A6CEBAB5-0724-531B-A230-DBE70ECE6668Robertson, 1904520675AE-04BA-5260-B208-911B76741DEE1EA30B11-24D3-50DC-812A-FDF10A80B5BFLast collected on the Peaks in 1934F579F21A-1A0C-552D-B793-634C0467A70CLast collected on the Peaks in 193413BA9562-77B9-5197-B666-B1F3C054FC0ALast collected on the Peaks in 193459525654-F81D-55B8-AF97-5B63C060D0BDLast collected on the Peaks in 1934C3E5BA68-B867-534B-AD98-715A29ECFEDDLast collected on the Peaks in 1934881812EB-7BB2-5C04-9507-5B42A70E9E6657C635A3-CF90-54C9-B181-7AA2C19FF86FLast collected on the Peaks in 19506C716627-8356-5614-A048-44D41F3A6FCDCresson, 1878BBFF9861-254C-5843-AC1B-7104CA3287D9Timberlake, 19511774824C-F765-511F-9156-1967A0D36ABECockerell, 1900231EC23F-EAB4-53EA-A453-1503D2B54C71Cresson, 1869BB4690FF-4BAB-508A-AFE7-24BFC6D1FF71Cresson, 187981FBFCCA-3DB9-5745-85D3-E25E1C7DA2EATimberlake, 19370993D304-79EA-5126-BB2F-F789B1304A33Cockerell, 190564CED378-58B9-53EF-985B-6CCBA9D0D55DED9C59A1-A894-5117-95EE-4E8BEBA156CC09E57354-58E3-5150-AB6A-1BF1091800DF85002FA7-FCD2-595E-912C-AF6C54A7744FTimberlake, 19518F15967C-4BF3-58F2-B8E0-6E4A33C0BAE6Linnaeus, 1758AD05F484-EB86-5DF2-A3E9-035AD77BECE4Cresson, 187469BE6BA7-1C79-5C68-8FD8-D85E93B968E9Greene, 185897480866-BB49-5C87-8C41-1185424A51C3Cresson, 1878BB8FF8B3-5928-582A-9602-C11A43A1DB5661002286-D32C-5673-B728-E30210ECB5D7C5BAB641-7876-5B79-B54F-B08B5A39F10A27B4DBCD-6EBE-5B6A-B840-F88C3E85D691Last collected on the Peaks in 1934Cresson, 187819CF1D60-686C-5314-9F82-AB1E993596AACresson, 1864FF42A1CB-F814-5282-B79E-E8E4C6B293F1Cresson, 1863843CD184-8F64-5042-9561-D04A8471A5C5Greene, 1860215807B2-E8A0-51C3-8285-033FEC1666BFNylander, 1848B855CDCC-AB96-57BB-9267-B18AA5963101Kirby, 1837ACDF91D2-2723-56E7-88AC-1B4583074676Our record is the first documentation of this species in northern Arizona. Species occurs in neighboring areas.Cresson, 1878D985F9EF-BB41-59EB-B16D-C2761C86F2BESmith 1854BE798AAC-77C0-5027-A225-9291AAA655C7DDD82638-7E4F-5449-BC21-177FC6D37871Cockerell, 18973C816D2B-A855-517D-B9D1-B2ACE5F910F3Last collected on the Peaks in 1936Cockerell, 189856DB7CEB-494C-5DDD-A475-7C13B04E0797Daly, 19733F3B35EE-A673-557F-9B24-44D4361F1800Cockerell, 18975A22010D-B8B0-505C-AAE7-ACEA87698113Cockerell, 19018D28E922-E1D5-5F3B-B1B8-C17EAE7174F2H.S. Smith, 1907198583E5-7DEB-5303-A8C0-B772E7375990Latreille, 18027CEC19A9-8C9F-5700-AAEA-08293162C8BA9421FB6C-7C0E-5D32-9759-FE7B6413CE088A1F6FE9-F8E8-5C70-9DD6-C0CD0F9E58F7(Patton)2249A62A-2664-520E-BFDB-91843641A887Cockerell, 1897E3E69466-60F7-5F86-8F58-B32379C018ED9C20E895-676A-5ABF-9A60-123F1D1584CECresson, 187801911AC1-9778-540E-9551-D9E2F59C93814C562B3C-7A54-5487-B63A-C07F13BD1B63Last collected on the Peaks in 1961Robertson, 190056B652D0-78A0-5539-BE2A-8115569BB02BCresson, 18644F4054C0-185B-58AC-A352-939D8A501110D65AA518-D59D-5D34-9300-7526D032B7E0Last collected on the Peaks in 19363D7D2BBA-6C95-5A54-9B55-F68E07E1E41BOur records are the first documentation of this species in Arizona. Species occurs in neighboring areas.1DE722EF-E89E-5133-A129-CECCD75522F0Our records are the first documentation of this species in Arizona and the southernmost extension of its range. Species occurs in neighboring areas.1451C664-8283-5895-AA5F-2E7D47A4B8217BBBBCFA-A70D-564A-B0B7-F282476556D2Our records are the first documentation of this species in Arizona and the southernmost extension of its range. Species occurs in neighboring areas.77973694-8E46-5163-82EB-F1B23BBBC93FPatton5064B76A-3934-57E0-8BC3-62A66846AD5F3B719BF3-D987-5373-8118-A4BE188205DE07B13FE4-7890-5063-A1BC-BD9B59F48D02Last collected on the Peaks in 1934CF0AE348-1E9E-5354-84B4-61517A87961ALast collected on the Peaks in 1952Scopoli, 17706B47BF03-61A4-5D4F-8E25-E65ACD53122BScopoli, 17704D91EE8B-D621-5268-84E8-CA09479A6EDDScopoli, 177090276146-B226-5EFF-9945-7932D8309A1FCockerell, 1896975FD7ED-63E7-5D03-A4D8-903B79585AEBCockerell, 1898B62F14B8-F410-598D-BFFF-1E2682EEDD0C83D3B5F6-1C41-5E2E-8D1D-C75499900DCDLinsley, 193977C50B6B-14C0-578E-9DC9-41C53A37784009E87481-7C7E-561F-8653-B337F73D9259Cockerell, 190507C8BD2B-5184-562B-B2CB-0B1CADA85496Cresson, 187862C38DEA-3206-5D55-86D4-D9EB89D62387538577C4-918F-5C49-9FFE-2ED001C8692BLaBerge, 196131F704B2-DBE8-5D62-A316-D51B6F8B81496DA62249-6A10-51B1-B95E-A6C05F05B8FFCockerell, 190502CECEEF-BE99-53BF-B834-3B8927A608DCLaBerge, 1961A8A65DC4-5EBD-5FA9-A5AA-2D512008E498Cockerell, 1908A1BC510E-B1A8-52E5-804E-49D5C263BFBFOur records are the first documentation of this species in northern Arizona. Species occurs in neighboring areas.Cockerell,1905365E6DF7-1BDC-5ABC-A421-02319C597693Cockerell, 1894423D4BFA-89C0-5371-BD4E-26CA0EDFDBDFCockerell, 1905FD8E71F3-2B71-5F24-ADC7-5AE6069BA948Cresson, 187213550B29-21F9-5E7A-BD88-791ABFB26FFFCresson, 187894B687AF-53B9-5F2A-B225-0358B1545C69Cockerell, 1896DE431962-A538-5F9A-9D91-DA9B394760EFCresson, 187811AC8B2B-59D1-581E-873D-50E2652D6F18Last collected on the Peaks in 1938Tucker, 1909DE709306-02C6-52AF-BDD4-FABC1A2E0786Last collected on the Peaks in 1950Cresson, 1878719DC6A9-024E-5245-810E-47A7074090DELast collected on the Peaks in 1966LaBerge, 1961CEE5160B-0247-5905-913E-30278C796640Last collected on the Peaks in 2002Robertson, 19052A4A4B8C-4472-5513-AC92-9AEA2918EF99Last collected on the Peaks in 1934Cockerell, 1898BAEE7A38-FB9E-5CF0-9077-DDA4AB033909Last collected on the Peaks in 1964Cresson, 1868F5B95FE5-509E-5EC8-BBBD-64F766F4C036Last collected on the Peaks in 1939Cockerell,1905F59C46D8-30FF-5431-AE0E-2C8297384434Last collected on the Peaks in 193653CC35DE-C07A-5EAD-A7DE-9B7DFA517DC4Last collected on the Peaks in 1952Moalif, 198867B88550-DE12-5065-8C0F-87146F814D08Last collected on the Peaks in 1951Cresson, 18784679D53A-6335-52C6-A36B-1A83E1C64E55Last collected on the Peaks in 19559670619C-F80F-5F25-96A5-DC19B8E93599Robertson, 19014D740556-8DCE-589A-B4DF-B45C2A6FB8EDRobertson, 19019F0ACE0A-A3C4-52C1-8504-12F3D581D68DCockerell, 1921574308C0-060C-50D7-A408-12AA7B168E407CF1B193-1F5A-5A78-BB24-2DB58B84A81BCresson, 1864A3779B64-DDE3-5E2F-9EAD-856F3271974FTimberlake, 195189A18588-76FD-5387-BCBA-E0D2DE6C7A11Cresson, 1868095260D2-E700-5B46-B10B-FC4A4535D13ARobertson, 189199466871-F606-5596-AB78-E2594FA12E6FLast collected on the Peaks in 1952Cockerell, 189773436295-6217-524B-883A-B4E46002BEC5Cockerell, 1898EF41FE6E-DAAE-5F20-BC94-D6258023DFEFViereck, 190352831829-81C4-577B-856F-F98A76B3AD12Swenk, 19086A901250-C3FA-53C8-9710-A6304661DCE7Cresson, 1868363F7B1F-E1D6-5A26-9544-86AA505611FBLast collected on the Peaks in 1939Timberlake, 1943E6353080-AD56-59BB-9D55-0F1B73D155C5Cockerell, 1897CF5525AE-0A6E-5924-BA68-2C396C09C39BLast collected on the Peaks in 1950Latreille, 180219D320CB-40A0-50C1-9968-F5ECB5CF68F0Latreille, 1802309A114F-900F-5C95-90E7-0BBD0FCDAE03Latreille, 18020FA55442-D987-5009-B1B2-DFADE29E0F17Latreille, 18022D8C9376-57BB-557B-8C68-8ACD253CDC1ELatreille, 1802F3C82C88-4447-5B3F-A2A5-7862A09D715254ACD16F-8F03-526F-B6DF-A5912CAACD3E3D6BC026-4750-53C0-97BB-BDAB456BB5B33A6E9F36-C2B9-56F2-A436-E8E294FAC2569EF85B97-E096-5380-8C4D-F097AF97D68FC4CFBB53-8F71-57D6-A367-1949EB65E74DSnelling, 19668D54DDCC-DAB2-5C38-8B03-7D172B0286AALast collected on the Peaks in 1950Cockerell, 1924A61A09B3-8018-5011-A577-65F1D780E0FD55381D3E-CA54-509E-AC82-601E4B1EC4DECresson, 1872F081C65F-8D69-5BC0-A3F6-562FBABF67EBCresson, 1868EB5F8CEA-152E-5647-BCEE-3AEE21FAD5449A508195-04C3-5F5B-8F0D-E74AC60A8AF8ACB6F9E6-ECD9-524F-9875-6D3F4C05B247BB2C1310-0DC0-523D-9808-24DD3FEC6848779E5604-A561-55DE-9C99-95436040049F51976C15-6CB7-5AFB-818E-8C8947286AFBOur record is the first documentation of this species in Arizona. Species occurs in neighboring areas.131867BE-FE75-5D0F-B0CD-BF34B50D66D26B9F643F-C2BB-5843-81F1-1F6D3E15E8943EA4C936-805F-5776-8DDB-3FC61AEF2D066126A584-0A67-575B-A841-A7E3FB28C67240F66CC3-4640-5ED6-9684-162D328D637C61E3D8F6-9F67-5266-BA2F-0DFE16AE48F9FCAD2544-FA92-5432-ADA4-6D88A40450FCAFEDFE4B-9D7C-59AD-916C-5916D8C1AC970ED293BE-79C2-5CF3-B12F-0037C4347CD412C98648-4BE5-5E0B-9B34-05E7A155B7A8EC04E4E0-92BE-5D84-A776-37A8A89CC0D55E6E032A-0E15-545B-AB7C-CACF11945C97B212D413-2750-5BD2-AFE5-76C74F87371FBE39684D-D5BE-5690-AF4A-364A2965B6593F96DEE1-890A-5B53-A23E-F94615533EEEA5AB3A91-8526-578A-A285-8CCE0DD8F745E6496AF4-65AD-58B4-9072-508A299027FECF1D1E27-A717-5C25-A06F-FAABDD9487A9AF4B7621-C065-5139-9BCB-87E347C6BE9E9CCFA909-7A6F-5596-94E8-8EC89A729073CFA20CB8-BD7D-58CE-B619-515AA8B7BA73Curtis, 1833A123E3A3-F5B3-59FD-AC90-4426D333A7BDA91D4F2E-C135-5FBD-A4DE-2EC674350ED96C505270-36E5-566E-BA24-F5317EDF4882B600CEED-6991-5089-BC28-2F2232BA2985BB4F1AB0-40AD-5DCB-82AE-E7AEC40F6A5C8D08A081-A556-5290-A02D-47EC4105EB20C8C91C4F-4854-5F9B-8ECE-15E2739594FDDalla Torre, 1896E420AEB6-29EF-5086-BA48-1920649D519BLast collected on the Peaks in 1952Cockerell, 1910377F7BBE-C739-5274-B365-ED5BB0A36625Last collected on the Peaks in 1936Bohart and Griswold, 19971C66F359-655F-5550-AA0D-C37E8CD6413B020AC94A-577D-5276-B5ED-EFA3012A8218Latreille, 1804C02DE143-777D-50F0-903D-12FAF3776917Latreille, 1804BB499844-23A4-538C-9A10-F0312628F90CLatreille, 1804C2222EDD-CAE1-54AF-BA73-4CB82F865207Latreille, 1804B9CF8DEF-302F-5610-801E-C234003C0C46Latreille, 1809BAE6A4CA-C990-570A-93D0-44A8554A33E9Last collected on the Peaks in 1986Cresson, 1879A53509B1-34B9-5A11-9CC1-0AAF642445E0Swenk, 19143C4145DC-12A1-5CC9-B4AF-38E641DB88B2Schwarz, 192888C5F24F-058B-5AAA-B070-CECCF1249F0DCockerell, 1937B5CC4AB9-A72E-50BF-9883-080D5F5897D1Gonzalez and Griswold, 201393B1174A-5EBD-5BE4-BF2B-7DE58671B0F404D69FB2-D395-5C1D-B82D-1F15A3AF99A1ACAC14C8-EF0B-593C-BA13-12C97EE918F2Smith, 1854125BAE85-62A9-5F75-A19E-F4943C67F057Cresson, 187868A36BC9-833C-50D1-AE95-B8056720933CCresson, 187862FA6188-87FC-5E04-B993-1756158EEF24Cockerell, 19040BE6422D-910E-5624-8E0C-AD3E96E16D83Cockerell, 1900FDA5046E-05EF-5B8A-A177-1124D76A49FFGonzalez and Griswold, 201303113CE2-58FA-5BF3-87E2-81CE6C718C25Michener, 19369CF8445F-BDA8-547C-BD81-8F34035951C8Our records are the first documentation of this species in Arizona. Species occurs in neighboring areas.Cockerell, 1910C9200FFA-D0C1-5579-A0C1-2857501133488B69962F-2C49-50DE-9DAE-66393E8866EDA7785634-EC5B-532B-AAE9-AC4809FDED78FA1FDF0A-E5C9-525E-8AE5-5C6B6D8F2299Titus, 190421124E45-8257-5766-8076-4C766A5738957546446C-5771-53AF-94B1-FEF0A6CAC973CE127362-721B-528D-A2C7-1F692299BF80Michener, 193985B8E1EE-C86E-5FF0-A106-D478A5005BF6Michener, 1949B102C47A-9B80-5CF0-9352-5EC3FD0B7BBDOur record is the first documentation of this species in northern Arizona. Species occurs in neighboring areas.Michener, 193912C4C393-B89A-5BBA-ADC6-3EE608E1DB5E7C0BAE73-8CAE-5976-9863-9FA3BD8A7580Cresson, 18644D72E702-1E52-5293-8583-CA9871F37AC3Say, 1824BB9894C9-A340-5DD8-9605-D2DB2363FAB1Last collected on the Peaks in 1950Cockerell, 19000A4D28B0-FE00-566C-BDB7-02E4F5B47581Last collected on the Peaks in 1971Smith, 18547679E5FE-BD0E-51CC-9898-5641C9DBD3A4Cresson, 18784BD08A37-08A0-5B43-972E-B773633733B0Last collected on the Peaks in 1971Cockerell, 189800F26440-024B-5D85-A354-2F31B746D29FCockerell, 1900C6FFEE43-6CD3-5816-8F7F-0667F6E4AE4ALast collected on the Peaks in 1961Cockerell, 1912BB0A1D90-10A0-5375-8C3A-FD5990CC0325Last collected on the Peaks in 1971Latrielle, 18091C3226BB-605C-5440-B92A-EFA651385ACDLatrielle, 180922478A7D-2496-55C3-9BE7-EBF5D7598E18Latrielle, 18093DB0592C-A6AB-5C98-97D6-3C5732F643AE641219AC-313A-531A-A03C-960307F35BBCLast collected on the Peaks in 1967749C398C-2064-54D1-A717-E0DDB1267E1E34510EE2-2457-514D-BB22-B0EC5680241416FF1507-3BCB-5F1F-881D-46B401E7C7973BDE7590-D51F-5614-84A7-7F3BE2AF024ETimberlake, 194359913C69-A703-5B3B-AA07-8BAD43EDCB93Schwarz, 194001BFA83D-8AFB-5506-9057-65816C2A59603D666D51-0826-5D19-9A5D-F298E5A442CCCockerell, 1923C14A7BBC-CD8F-5229-9ADB-23868158000CC2862546-7B1B-57CC-88B8-4594587C8AEB178C47DF-663B-5509-A200-658FCB63684DSwenk, 191492AB344E-1C0D-5004-8837-D0F4A97BCB16A2B8AD88-A359-59B3-BF44-E681CBE84E2BMichener, 193837047D7D-09E9-5EF8-AEF3-E0810D125318Cockerell, 1897B1234A4A-CFA6-5858-BEB0-CE4CA20B64C7Michener, 19545EBCD303-52B6-5C46-9BFA-5DD6CDD7F13FLast collected on the Peaks in 1934Michener, 1938C7337709-9CB4-5D9E-88A6-05708BF1758ELast collected on the Peaks in 1947Michener, 1938D00098F2-786C-539F-B1B8-169EDA4EE637Spinola, 1808C72F32D2-0B53-5708-9712-FC1C0863C1EC53435A45-9C43-5AB9-9A96-EAA39425EEBFLast collected on the Peaks in 1986Michener, 1947749ECFED-338B-5C0F-8D82-B82D404B65F30578FF96-A121-5C09-ADF8-C93602AA8BF8Last collected on the Peaks in 1961DF505F9C-E4C7-5176-A9F4-54AC1D1CED57Our records are the first documentation of this species in northern Arizona. Species occurs in neighboring areas.Klug, 1916FD64131E-8E42-5123-8486-08573F841B9B2156F194-8D81-5D6E-9201-F354CEE9FFADF3A31A76-2213-5CC4-935A-BB5CCAC8B79AMitchell, 1934179F0CFA-95FC-5BC0-A359-BD62177D10AD3C43EC5B-F658-57F0-BC4F-46751F8866EFCF0C95BD-28E7-527A-96FC-193F406227015342E8CB-C6A2-5DBD-8D0D-9C565120D9DE8730FAF1-ABCE-589B-BE68-E9581454F768Say, 1837CA4F5EE1-4A73-547D-98C5-64415D02861ALast collected on the Peaks in 1967Cockerell, 190055414545-4385-55D5-9299-6EE344FD1B5ACockerell, 1908941697B0-CA32-52B5-A0DC-B402200573BEMitchell, 1927D4F4E31E-3156-5ECF-A9AE-58E223EEB0D9Last collected on the Peaks in 1950Cresson, 18782387BFEC-058E-5148-9CF0-42E574E7C2A5Last collected on the Peaks in 1950Thomson 18729FA9D24F-6A7D-5D55-A4D5-D1EC1072427DOur records are the first documentation of this species in Arizona and the southernmost extension of its range. Species occurs in neighboring areas.2FCA13AB-9A03-5210-A7A6-3000C023CD9ACresson, 18782C3B94E6-982A-561F-839E-6E4ACC8BFD7ECresson, 18786B59B132-1445-5F2B-9ADA-4FA2FEF799B1Last collected on the Peaks in 1951Cockerell, 190896819045-E65A-5130-876C-577AFB575762Mitchell, 1927D6CB5597-7329-5DA5-BF45-FD83AA057913Cockerell, 19055AAEAB31-8BFD-55D0-94A5-A86929783648Cresson, 1872C61B21B7-4B75-5933-BE5F-FD2F8DA36AB7Cockerell, 18975C144971-E02C-57E1-B0A5-6C6F8B10E945Cresson, 187866BE7DC1-2EDA-5C7A-8AC3-81B53830936DCresson, 1872AF20374E-5A07-5254-BA36-0B6611F19F86Cresson, 1878DF589A16-C83F-5DFB-BE2E-464600EBA21FSay, 18311E259CB3-F534-52FB-9C34-09F5E0EF97FBSay, 1837212CB3E5-B659-5307-A02C-36BC70D69FF5Cresson, 18725EBC9F90-67A2-5634-9393-129081BD135CSmith, 18532E0F4BA3-06C7-5C10-AD6B-AF61D5F953F339896233-4AFC-56AA-B96A-40C70881FEA0Our records are the first documentation of this species in northern Arizona. Species occurs in neighboring areas.Smith, 18531764E39A-99E4-5389-8EDD-80D88D136266Cresson, 18780604FF57-8863-5796-9E22-D676E87A8985Cockerell, 189838B75DF5-11FF-594C-AF7C-AA530A4D22C4Last collected on the Peaks in 1990Latreille321D89DD-E224-51F5-ABAB-93385134CC91Latreille6FD35A59-95EB-5845-967E-2669C5444753Cresson, 1864E90B9B6E-38A2-518F-94C0-1A58CCDAEFEBCockerell, 1900A9E9D523-92A3-5440-B2C3-886AC1D80423Cresson, 187886860BB2-A34B-51B1-AD48-9243CE96AB38Sladen, 191696246B5E-A63C-5756-BB30-86CFAF0E030BSladen, 19162A4483DB-CF2F-5CAE-9A3E-983BCC505C69Cresson, 187207126906-5E9E-576C-8B4C-E9FE989FC67CCockerell, 1906373F363D-1B20-54E1-9E2A-13C6FD0F2B11Last collected on the Peaks in 1971Cresson, 186433BE49FA-2176-5C4B-8E93-DCB38B86F6F325C53F95-FE5E-5E04-B860-989E67A94812Our records are the first documentation of this species in Arizona. Species occurs in neighboring areas.Cresson, 186461C17325-8EF8-5719-A018-74391E60094CLast collected on the Peaks in 1971Cresson, 1864E555D374-0A3D-5030-889F-7B3FFC09CF51Cockerell, 19334D10EF63-60D3-5F8E-949B-A01C4924E6D6Cockerell, 19068A438904-9830-5276-A73F-E21A74B5D7EELast collected on the Peaks in 1950BC6961F1-01AB-5590-B17C-5C53BE9273F081B70320-D52E-5394-ABCD-4094129EC875Michener, 1937CBC3DB17-0FFD-5433-B435-E279AC72B8E0Last collected on the Peaks in 1964Schmiedeknecht, 1885E7C3157A-FE80-5D23-B0DF-8574F9B6D2F4Schmiedeknecht, 18859C4357D9-1796-5603-8189-05855C9BE9DA2BA137A5-FAEF-5E24-B51F-2F1E50F10D57Last collected on the Peaks in 1971Panzer, 18063A5AD2B3-49E4-5848-BBE6-80EFFB447658Panzer, 180697B4F1DC-12AE-5C19-8436-9876D63A6D5DPanzer, 1806FAA4FEE6-14A5-5A38-8C4D-BC886910D7BFPanzer, 18064DE2B128-4594-5228-B1BA-FBDD00F17F70Panzer, 1806C84FD703-DCEB-5006-887B-3482E01BC39APanzer, 1806658A6E39-C2FB-512D-9CC1-640CE7A8FE87Panzer, 180615C80295-02F9-5D8E-8AF0-28BABDEFC05DPanzer, 1806A8EEB544-FC55-57FA-B343-3F5A69609100Panzer, 18060A77F222-76E2-5D28-A33E-8C015AAFD1E03C5FA2D8-F26D-5421-95DD-7B81A08B3056036E2376-060D-5AD5-BCC0-C7188E7980A9C58148E8-6B1D-585B-B304-CF4FB5E56192Perditazebrata and Coelioxysporterae are of note because they were not collected in our 10-year study but were collected in high abundance in previous years. P.zebratahad 150+ records documented in the pinyon-juniper life zone but has not been recorded on the Peaks since 1971. C.porterae had 100+ records documented throughout the Peaks since 1952 but was not collected in our 10-year study.Prior to the start of our study, past collection events had documented 155 bees on the Peaks between 1908 and 2009. Records that were not identified to species, outside of our collection, were not considered in our checklist. Two collection events in particular significantly advanced the known number of bee species on the San Francisco Peaks  with Megachilini and Osmiini , and they were also the most species rich family in the Peaks region. An exception is at the mixed conifer life zone where Andrenidae was the most dominant family . Further, all but one of our reported Andrena species were found to inhabit mixed conifer, and 41% of Andrenidaeonly occurred at mixed conifer. Andrenids are typically ground nesters that prefer the sandy soils found at lower elevations  is seen at desert grassland and there is little diversity in Halictidae at the higher elevations. This trend is consistent with the idea that lower elevation habitats may provide greater nesting resources for halictids due to the warm, dry environment  but do have representative species at every life zone.In general, the percentage of species composed by a particular family at each life zone seemed to positively correlate with the overall diversity of that family found on the Peaks. For example, evations  and suchironment . ColletiAnthophoramontana, Diadasiadiminuta, Melissodestristis, Agapostemonangelicus, Agapostemontexanus, and Lasioglossumsisymbri. These six species only come from two families  and all six are ground nesters. Evidence suggests that with changing climate or other anthropogenic disturbances, higher species loss may occur with species that encompass smaller geographic ranges or specialized habitats (Apidae) are also likely susceptible to climate change. Additionally, lower elevation habitats will get increasingly warmer and are likely to experience more drought events. This could potentially limit the already scarce floral resources available in these desert environments. It is therefore important to document ranges and habitat requirements of the bee species found on the Peaks to predict future shifts in local distribution.Our results indicate a high degree of habitat specialization along the elevational gradient of the Peaks, with 49% of the total bee species found in only one life zone (177 species) Fig. . Convershabitats . Bees inhabitats . If broahabitats  most megBombusoccidentalis, has published accounts showing population trends (Insect species richness and abundance is reported to be declining globally, pointing to the importance of regularly monitoring populations worldwide . Howevern trends . Checklin trends  could se85A74899-1530-5E38-A8D4-ACBA285C578210.3897/BDJ.8.e49285.suppl1Supplementary material 1Localities for 2019 qualitative samplingData typedecimal latitude/longitude localitiesFile: oo_392852.xlsxhttps://binary.pensoft.net/file/392852McCabe et alF1DB3B69-7782-5FE9-93C7-C22AA707ADDB10.3897/BDJ.8.e49285.suppl2Supplementary material 2Occurrence Records removed from analysis Data typeoccurrencesFile: oo_392854.xlsxhttps://binary.pensoft.net/file/392854McCabe et alCDCE64C2-007D-57EA-BD04-267EF5BA868F10.3897/BDJ.8.e49285.suppl3Supplementary material 3DwC archive file of the bee records on the San Fransico PeaksData typeoccurrencesFile: oo_362433.csvhttps://binary.pensoft.net/file/362433McCabe et al


Cardiocrinum cordatum (Thunb.) Makino (Family: Liliaceae), commonly known as ‘Ubayuri’, is native to Japan and some islands in the Russian Far East. It has high value as food, medicinal, and ornamental species. The aim of this study was to isolate and characterize the main chemical constituents of the leaves of C. cordatum. A total of 19 compounds, namely caffeic acid (1), caffeic acid methyl ester (2), caffeic acid β-glucopyranosyl ester (3), caffeic acid 4-O-β-glucopyranoside (4), ferulic acid (5), isoferulic acid (6), protocatechuic acid (7), syringic acid (8), 2,6-dimethoxy-p-hydroquinone 1-O-β-glucopyranoside (9), esculetin (10), taxifolin (11), quercetin 3-O-(6-O-α-rhamnopyranosyl)β-glucopyranoside-7-O-β-rhamnopyranoside (12), 2,7-dimethyl-2,4-diene-deca-α,ω-diacid β-glucopyranoside (13), 4-[formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]butanoic acid (14), (3Z)-3-hexenyl β-glucopyranoside (15), tryptophan (16), adenine (17), adenosine (18), and 2-deoxyadenosine (19) were isolated using various chromatographic methods. The structures of isolated compounds were elucidated on the basis of their NMR spectroscopic data. All these compounds were isolated for the first time from the genus Cardiocrinum. Phenolic acid derivatives and flavonoids can be considered as chemotaxonomic markers in the leaves of Cardiocrinum species. Cardiocrinum (Liliaceae) comprises of three perennial bulbiferous herb species, C. cathayanum (E. H. Wilson) Stearn, C. cordatum (Thunb.) Makino., and C. giganteum  Makino butanoic acid (14)  butanoic acid (14), (3Z)-3-hexenyl β-glucopyranoside (15), amino acid (16), and nucleic acid derivatives (17–19) were isolated and identified for the first time from the genus Cardiocrinum. The genus Cardiocrinum comprises only three species, C. cathayanum, C. cordatum, and C. giganteum butanoic acid (14), a pyrrole alkaloid derivative, was previously reported from Lycium chinense Mill. (Solanaceae) as a hepatoprotective compound 004) has been deposited at the Department of Medicinal Botany, School of Pharmacy, Kumamoto University, Kumamoto, Japan.The young leaves of C. cordatum (960 g) were extracted twice with MeOH (15 L each time). The extracts were then combined and evaporated under reduced pressure to give 202.0 g extract. The extract was then subjected on MCI gel CHP20P column chromatography (CC) and eluted successively with water, 20%, 40%, 60%, 80%, 100% MeOH and CHCl3:MeOH (1:1) to give 10 fractions (1–10). Fraction 3  was subjected on Sephadex LH-20 CC (50% MeOH) to give four subfractions (3-1–3-4). Fraction 3-4 was subjected on ODS CC (5–100% MeOH) to afford compound 7 (9.7 mg). Fraction 4  was subjected on Sephadex LH-20 CC (50% MeOH) to give seven subfractions (4-1–4-7). Subfraction 4-3 (311.0 mg) was subjected to repeated column chromatography on silica gel CC (CH2Cl2:MeOH:H2O = 8:2:0.1) to afford compound 9 (6.9 mg). Subfraction 4-4 (589.0 mg) was subjected to silica gel CC (CH2Cl2:MeOH:H2O = 8:2:0.1) followed by ODS CC (10% MeOH) to afford compounds 17 (6.1 mg) and 18 (1.4 mg) and 19 (4.2 mg). Subfraction 4-6 (138.3 mg) was ODS CC (20–40% MeOH) followed by repeated CC using Sephadex LH-20 (50% MeOH) to afford compounds 1 (4.7 mg), 3 (14.2 mg), 4 (11.0 mg), and 16 (23.0 mg). Fraction 5  was subjected to Sephadex LH-20 CC (50% MeOH) and then to repeated CC on silica gel (CH2Cl2:MeOH:H2O = 8:2:0.1) to afford compound 10 (2.4 mg). Fraction 6  was subjected to Sephadex LH-20 CC (50% MeOH) to obtain 4 subfractions (6-1–6-4). These subfractions were further purified using silica gel, Sephadex LH-20, and ODS CC to afford compounds 12 (15.9 mg), 13 (4.3 mg), 14 (11.0 mg) and 15 (4.6 mg). Fraction 7  was subjected to silica gel CC (CH2Cl2:MeOH:H2O = 8:2:0.1) to obtain 5 subfractions (7-1–6-5). These subfractions were further purified using silica gel, Sephadex LH-20 and ODS CC to afford compounds 2 (1.8 mg), 5 (4.1 mg), 6 (1.1 mg), 8 (5.0 mg), and 11 (11.5 mg).The shade dried young leaves of 1H-NMR (in CD3OD) δH 7.84 , 7.34 , 7.04 , 6.21 . 13C-NMR (in CD3OD) δC 163.7 (C-2), 153.4 (C-7), 152.7 (C-6), 146.1 (C-4), 144.5 (C-9), 116.8 (C-5), 113.2 (C-3), 112.9 (C-10), 104.4 (C-8).1H-NMR (in CD3OD) δH 7.17 , 6.47 , 6.11 , 4.33 , 4.02 , 3.83–3.65 , 3.45–3.15 , 2.64–2.54 , 2.47–2.16 , 1.95 , 0.95 . 13C-NMR (in CD3OD) δC 176.6 (C-10), 172.3 (C-1), 142.6 (C-5), 140.1 (C-3), 129.0 (C-4), 126.5 (C-2), 104.8 (Glc-1), 81.9 (C-8), 78.2 (Glc-3), 77.8 (Glc-5), 75.4 (Glc-2), 71.6 (Glc-4), 62.9 (Glc-6), 39.4 (C-9), 38.7 (C-7), 37.0 (C-6), 15.6 (C-11), 12.7 (C-12).1H-NMR (in CD3OD) δH 9.45 , 6.98 , 6.28 , 4.49 , 4.37 , 3.35 , 2.32 , 2.01 . 13C-NMR (in CD3OD) δC 181.1 (CHO), 176.6 (COOH), 141.1 (C-5), 133.9 (C-2), 125.9 (C-3), 112.9 (C-4), 66.4 (C-6), 58.3 (OCH3), 45.9 (C-1′), 31.8 (C-3′), 27.7 (C-2′).1H-NMR (in CD3OD) δH 5.47 , 5.37 , 4.33 , 3.86 , 3.68–3.18 , 2.37 , 2.05 , 0.94 ; 13C-NMR (in CD3OD) δC 135.3 (C-3), 125.8 (C-4), 104.1 (Glc-1), 77.7 , 74.9 (Glc-2), 71.4 (Glc-4), 71.0 (C-1), 62.5 (Glc-6), 28.7 (C-2), 21.6 (C-5), 14.8 (C-6).Cardiocrinum. From the results of current study and with the previous reports, it can be assumed that the flavonoids and other phenolic compounds can be considered as main components in the aerial parts including leaves, stems, and seeds, which may serve as the chemotaxonomic markers of these species. Future studies are necessary to isolate and identify chemical constituents from the aerial parts of other two species and comparison of interspecific variation. On the other hand, bioactivity evaluations of the extracts and isolated compounds is necessary to explore the potential of C. cordatum for use in pharmaceutical, cosmetic, and functional food industries.A total of 19 compounds belonging to different chemical classes such as phenylpropanoids, benzoic acid derivatives, flavonoids, coumarins, etc., were isolated and identified for the first time from the genus


A novel series of pyrazoline 3a–e, phenylpyrazoline 4a–e, isoxazoline 5a–e and pyrazoline carbothioamide derivatives 6a–e were synthesized and screened for in vitro antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO) and superoxide radical scavenging assay as well as 15-lipoxygenase (15-LOX) inhibition activity. Compounds 3a, 4e, 5b, 5c, 6a, 6c, and 6e showed excellent radical scavenging activity in all three methods in comparison with ascorbic acid and 15-LOX inhibition potency using quercetin as standard then were subjected to in vivo study. Catalase (CAT) activity, glutathione (GSH) and malondialdehyde (MDA) levels were assayed in liver of treated rats. Compounds 5b, 5c, and 6e showed significant in vivo antioxidant potentials compared to control group at dose of 100 mg/kg B.W. Molecular docking of compound 6a endorsed its proper binding at the active site pocket of the human 15-LOX which explains its potent antioxidant activity in comparison with standard ascorbic acid.Oxidative stress is one of the main causes of significant severe diseases. The discovery of new potent antioxidants with high efficiency and low toxicity is a great demand in the field of medicinal chemistry. Herein, we report the design, synthesis molecular modelling and biological evaluation of novel hybrids containing pyrazole, naphthalene and pyrazoline/isoxazoline moiety. Chalcones   The balance between the production and neutralization of ROS by antioxidants is very delicate.OH), and other species inducing oxidative stress.OH, hydroperoxyl radical (HO2.), superoxide anion (.O2−), nitric oxide (NO), singlet oxygen (O) and H2O2 as well as nitrogen reactive species (RNS) and reactive sulphur species (RSS). These species are generated either internally from normal metabolic activities or external factors, such as smoking, environmental pollutants and radiation, that promote the production of free radicals. The main human body targets of ROS, RNS and RSS are sugars, proteins, lipids, DNA and RNA moleculesOxidative stress is one of the main causes of significant severe diseases, i.e. cancer, aging, atherosclerosis, hypertension, inflammation, renal disorders, liver disorders, rheumatoid arthritis, neurological disorders, cardiovascular, autoimmune diseases and neurodegenerative disorders such as Alzheimer’s, Huntington’s diseases and Parkinson’s diseasesI as anti-inflammatoryII as anticancerIII as anticoagulantIV as anticancer, antibioticV as antidepressantPyrazole ring is an important scaffold in medicinal chemistry. Pyrazole is a five-membered heterocyclic ring that consists of three carbons and two adjacent nitrogen atoms. Pyrazole derivatives have received considerable attention due to their remarkable broad spectrum of medicinal and pharmacological activities i.e. anticancerVI  has antioxidant activity and can prevent oxidative stress by increasing antioxidant enzymes, such as GPx, and diminishing the lipid peroxidation process. Examples for the pharmacological effects of 1,2-diazole or its related drugs. 1,2-Diazole was found to be effective in preventing nephrotoxicity caused by the anti-neoplastic drug cisplatinVI  is a novVII showed potent radical scavenging activity (RSA) against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicalVIII showed good DPPH RSAIX has shown potent RSA as well. The antioxidant activity of pyrazole is attributed to the presence of NH proton of the pyrazole moietyX showed excellent RSA using DPPH, .OH, .O2− and NO anion assays, compared to butylated hydroxy toluene (BHT7)XI showed good scavenging activity  in the DPPH assay at 10−4 M concentrationXII showed good RSA using DPPH, NO and H2O2 methods in comparison with ascorbic ascidXIII possessed potent DPPH RSAXIV exhibited good antioxidant activity at low concentrations (0.25 mg/mL) in DPPH methodRecently, the study of pyrazole as a pharmacophore for the development of potential antioxidants has led to the synthesis of several compounds containing pyrazole core in their structures. Among the reported synthetic pyrazoles: 3-(Pyridin-4-yl)-1H-pyrazole-5-carboxamide chalcones ,,A) was found to work as a potent rabbit 15-LOX-1 inhibitorB) showed novel 15- LOX inhibition with potent activity against human 15-LOX-1 in both a cellular and an in vivo model of strokeThe mechanism of action of antioxidants can be through various pathways such as free radical scavengers (preventive oxidants) and as lipoxygenase inhibitors (pro-oxidative enzymes)H-pyrazole  and lipid peroxidation (MDA) assays. The results of in vitro antioxidant activity of the newly designed hybrids and their 15-LOX inhibitory activity would identify the required antioxidant parameters that are most reliable in the design of 15-LOX inhibitors for the future studies. The structure–activity relationship (SAR) and possible mechanisms of action of these derivatives were also investigated.In this study, we report the design, synthesis and biological evaluation of a hybrid scaffold in which 3-naphthyl pyrazole is substituted with pyrazoline/isoxazoline ring at position 3 to generate novel and new derivatives of 3-(2-naphthyl)-1-phenyl-1pyrazole . These n2.2.1.1HNMR) and carbon-13 nuclear magnetic resonance (13C-NMR) spectra were recorded in dimethyl sulfoxide-d6 (DMSO-d6) on a Varian Mercury (300 MHz) spectrometer (Varian UK) using TMS as internal standard and chemical shifts were given as ppm . Mass spectra were carried out using 70 eV EI Ms-QP 1000 EX , Faculty of Science, Cairo University, and Cairo, Egypt. Microanalyses were performed on Vario, Elementar apparatus , Organic Microanalysis Unit, Faculty of Science, Cairo University, Cairo, Egypt and the results were within the accepted range (0.40) of the calculated values. Column Chromatography was performed on (Merck) Silica gel 60 (particle size 0.06–0.20 mm).Melting points were determined with Electro-thermal IA 9100 apparatus  and the values given were uncorrected. Fourier-transform infrared spectroscopy (FT-IR) spectra were recorded as KBr pellets on a Perkin-Elmer 1650 spectrophotometer (USA), Faculty of Science, Cairo University, Cairo, Egypt. Proton nuclear magnetic resonance (2.2.3-(2-Naphthyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (1). The titled compound 1 was synthesized according to the literature procedure,3) with the phenyl hydrazone (0.01 mol) in DMF (3 mL). The reaction mixture was stirred at 70–80 °C for 5–6 h. The progress of reaction was monitored by TLC using hexane and ethanol (90:10). The reaction was cooled to room temperature, then poured into cold water and a saturated solution of sodium bicarbonate was added to neutralise the mixture. The white solid obtained was filtered followed by washing with water.3--1-aryl propenone (2a–e). A mixture of 4-substituted acetophenone (0.03 mol) and the aldehyde 1 (0.03 mol) in 25 mL 50% alcoholic NaOH solution were stirred at room temperature for 24 h, then the solution was cooled, poured on ice/water acidified with dil. HCl. The produced solid was filtered off, dried and crystallized from ethanol to give compounds 2a–e.3--1-phenyl propenone (2a).Yellow solid, yield 81%, m.p.158–159 °C. IR (KBr) vmax (cm−1): 3150 (CH–Ar), 1695 (C=O), 1604 (C=N). 1H NMR  δ: 6.5 , 6.6 , 6.8 , 7.1–7.9 .13C NMR  δ: 105.0 (pyrazole-C4), 126.0 (pyrazole-C5), 114.7–140.1 (aromatic Cs), 129. 26, 142.8 (CH=CH), 160.0 (pyrazole-C3), 187.0 (C=O). MS (EI): m/z: 400 [M+] (20%). Anal. Calcd for C28H20N2O (400.471): C, 83.98; H, 5.03; N, 7.00; Found: C,83. 77; H, 5.15; N, 6.93.1-(4-Methoxyphenyl)-3--propenone (2b). Brown solid, yield 85%, m.p.187–188 °C. IR (KBr) vmax (cm−1): 2970 (CH-sp3), 3157 (CH–Ar), 1691 (C=O), 1605 (C=N). 1H NMR  δ: 3.3 , 7.0 , 7.4 , 6.6 , 7.5-8.55 . 13C NMR  δ: 55.87 (OCH3), 105.21 (pyrazole-C4), 126.66 (pyrazole-C5), 113.33–145.0 (aromatic carbons), 129.30, 148.40 (CH=CH), 161.0 (pyrazole-C3), 183.0 (C=O). MS (EI): m/z: 430 [M+] (20%). Anal. Calcd for C29H22N2O2 (430.497): C,80.91; H, 5.15; N, 6.51. Found: C,80.78; H,5.17; N,6.72.3--1-p-tolyl propenone (2c). Yellow solid, yield 80%, m.p.146–147 °C. IR (KBr) vmax (cm−1): 2975 (CH-sp3), 3160 (CH–Ar), 1696 (C=O), 1605 (C=N). 1H NMR  δ: 2.3 , 6.5 , 6.7 , 6.8 , 7.0–7.9 . 13C NMR  δ: 20.7 (CH3), 105.0 (pyrazole-C4), 126.50 (pyrazole-C5), 112.7–142.1 (aromatic carbons), 126.26, 140.8 (CH=CH), 163.0 (pyrazole-C3), 187.0 (C=O). MS (EI): m/z: 414 [M+] (17.7%). Anal. Calcd for C29H22 N2O (414.49): C,84.03; H, 5.35; N, 6.76; Found: C,84.19; H,5.27; N,6.67.1-(4-chlorophenyl)-3-propenone (2d). Yellow solid, yield 77%, m.p.161–162 °C. IR (KBr) vmax (cm−1): 3157 (CH–Ar), 1692 (C=O), 1655 (C=N). 1H NMR  δ: 6.4 , 6.8 , 6.9 , 7.1–7.8 . 13C NMR  δ:105.5 (pyrazole-C4), 126.2 (pyrazole-C5), 115.7–145.1 (aromatic carbons), 126.2,141.1 (CH=CH), 160.3 (pyrazole-C3), 187.0 (C=O). MS (EI): m/z: 434 [M+] (20.1%), 436 . Anal. Calcd for C28H19ClN2O: (434.916): C, 77.33; H, 4.40; N, 6.44; Found: C,77.29; H, 4.45; N,6.47.1--3-propenone (2e). Yellow solid, yield 79%, m.p.168-169 °C. IR (KBr) vmax (cm−1): 3156 (CH–Ar), 1691 (C=O), 1603 (C=N). 1H NMR  δ: 6.2 , 6.4 , 6.7 , 7.0–7.9 . 13 C NMR  δ: 105.0 (pyrazole-C4), 126.0 (pyrazole-C5), 113.3–140.4 (aromatic carbons), 126.2,142.2 (CH=CH), 160.2 (pyrazole-C3), 187.0 (C=O). MS(EI): m/z: 469 [M+] (15.3%), 471 . Anal. Calcd for C28H18Cl2N2O (469.36): C,71.65; H, 3.87; N, 5.97; Found: C,71.55; H, 3.85; N,5.83.3-Naphthalen-2-yl-5-aryl,1’-phenyl-3,4-dihydro-2H,1H’- bipyrazole (3a–e). A solution of (2a–e) (1.0 mmol) and hydrazine hydrate 99% (1.0 mmol) in absolute ethanol (15 mL) was refluxed for 6-8 h. The resulting solution was concentrated, cooled, the solid obtained was filtered off and recrystallized from ethanol to give compounds 3a–e.3′-Naphthalen-2-yl-5,1′-diphenyl-3,4-dihydro-2H,1’H-bipyrazole (3a). Yellow solid, yield 61%, m.p.172–173 °C. IR (KBr) vmax (cm−1): 2960 (CH-sp3), 3052 (CH–Ar), 3439 (NH), 1593 (C=N). 1H NMR  δ: 3.10–3.88 , 5.26 , 6.8 ,7.0–8.0 , 8.3 . 13C NMR  δ: 39.79 (pyrazoline-C4), 55.87 (pyrazoline-C5), 105.0 (pyrazole-C4), 125.99 (pyrazole-C5), 118.68–145.05 (aromatic carbons), 150.71 (pyrazoline-C3), 158.01 (pyrazole-C3). MS (EI): m/z:414 [M+] (13.7%). Anal. Calcd for C28H22N4 (414.50): C,81.13; H, 5.35; N, 13.52; Found: C,81.17; H,5.23; N,13.43.5-(4-Methoxyphenyl)-3′-naphthalen-2-yl-1′-phenyl-3,4-dihydro-2H,1’H- bipyrazole (3b). Brown solid, yield 67%, m.p.177–178 °C. IR (KBr) vmax (cm−1): 2965 (CH-sp3), 3163 (CH–Ar), 3356 (NH), 1605 (C=N). 1H NMR  δ: 3.22–3.40 , 3.9 , 5.20 , 6.7, 6.9–7.9 , 8.0 . 13C NMR  δ: 38.30 (pyrazoline-C4), 55.60 (pyrazoline-C5), 56.0(OCH3), 105.0 (pyrazole-C4), 126.0 (pyrazole-C5), 112.2–141.1 (aromatic carbons), 148.54 (pyrazoline-C3), 159.0 (pyrazole-C3). MS (EI): m/z: 444 [M+] (14.3%). Anal. Calcd for C29H24N4O: (444.53):C,78.36; H, 5.44; N, 12.60; Found: C,78. 33; H,5.43; N,12.70.3’-Naphthalen-2-yl-1’-phenyl-5-p-tolyl-3,4-dihydro-2H, 1’H -bipyrazole (3c). Yellow crystals, yield 66%, m.p.187-188 °C. IR (KBr) vmax (cm−1): 2963 (CH-sp3), 3164 (CH–Ar), 3357 (NH), 1605 (C=N). 1H NMR  δ: 2.5 , 3.20–3.51 , 5.3 , 6.6 , 7.0-7.9 , 8.0 . 13C NMR  δ: 20.7 (CH3), 36.30 (pyrazoline-C4), 57.50 (pyrazoline-C5), 105.0 (pyrazole-C4), 125.0 (pyrazole-C5), 115.5-145.1 (aromatic carbons), 148.54 (pyrazoline-C3) 160.0 (pyrazole-C3). MS (EI): m/z: 428 [M+] (9.8%). Anal. Calcd for C29H24N4 (428.53): C,81. 28; H, 5.65; N, 13.07; Found: C,81.27; H,5.58; N,13.03.5-(4-Chlorophenyl)-3′-naphthalen-2-yl-1′-phenyl-3,4-dihydro-2H,1’H- bipyrazole (3d). Yellow crystals, yield 75%, m.p.190–191 °C. IR (KBr) vmax (cm−1): 2979 (CH-sp3), 3173 (CH–Ar), 3354 (NH), 1605 (C=N). 1H NMR  δ: 3.2-3.5 , 5.20 , 6.8 , 7-7.8 , 8.01 . 13C NMR  δ: 34.30 (pyrazoline-C4), 56.57 (pyrazoline-C5), 105.0 (pyrazole-C4), 127.0 (pyrazole-C5), 117.7–147.1 (aromatic carbons), 148.50 (pyrazoline-C3), 161.0 (pyrazole-C3). MS (EI): m/z: 448 [M+] (11.8%),450. Anal. Calcd for C28H21ClN4 (448.95):C,74.91; H,4.71; N,12.48; Found: C,74.87; H, 4.80; N,12. 53.5--3′-naphthalen-2-yl-1′-phenyl-3,4-dihydro-2H,1’H--bipyrazole (3e). Yellow solid, yield 77%, m.p.195–196 °C. IR (KBr) vmax (cm−1): 2973 (CH-sp3), 3175 (CH–Ar), 3351 (NH), 1608 (C=N). 1H NMR  δ: 3.4 , 3.2 , 5.23 , 6.8 , 7.2–7.8 , 8.0 . 13C NMR  δ: 38.30 (pyrazoline-C4), 58.50 (pyrazoline-C5), 108.0 (pyrazole-C4), 128.0 (pyrazole-C5), 118.7–140.1 (aromatic carbons), 148.54 (pyrazoline-C3), 158.5 (pyrazole-C3). MS (EI): m/z: 482 [M+] (14.5%), 484 . Anal. Calcd for C28H20Cl2N4 (483.39): C,69.57; H, 4.17; N, 11.59; found: C,69.77; H,4.10; N,11.60.3-Naphthalen-2-yl-5-aryl,2,1’-diphenyl-3,4-dihydro-2H,1H’- bipyrazole (4a–e). A solution of (2a–e) (1.0 mmol) and phenyl hydrazine (1.0 mmol) in 25 mL ethanol containing 0.5 mL piperidine was refluxed for 6–8 h. The mixture was cooled, filtered off and recrystallized from ethanol to give compounds 4a–e.3′-Naphthalen-2-yl-2,5,1′-triphenyl-3,4-dihydro-2H,1’H-bipyrazole (4a). Yellow solid, yield 68%, m.p.187–188 °C. IR (KBr) vmax (cm−1): 2963 (CH-sp3), 3186 (CH–Ar), 1607 (C=N). 1H NMR  δ: 3.0–3.4 , 5.21 , 6.8 , 6.9-7.7 . 13C NMR  δ: 35.2 (pyrazoline-C4), 57.50 (pyrazoline-C5), 106.2 (pyrazole-C4), 129.0 (pyrazole-C5), 116.0-140.0 (aromatic carbons), 148.8 (pyrazoline-C3), 158.8 (pyrazole-C3). MS (EI): m/z: 490 [M+] (9.7%). Anal.Calcd for C34H26N4 (490.6): C, 83.24; H, 5.34; N, 11.42; Found: C,83.47; H,5.33; N,11.49.5-(4-Methoxyphenyl)-3′-naphthalen-2-yl-2,1′-diphenyl-3,4-dihydro-2H,1’H- bipyrazole (4b). Brown solid, yield 60%, m.p.156–157 °C. IR (KBr) vmax (cm−1): 2967 (CH-sp3), 3187 (CH–Ar), 1600 (C=N). 1H NMR  δ: 3.1–3.3 , 3.8 , 5.3 , 6.6 , 6.8-7.6 . 13C NMR  δ: 35.8 (pyrazoline-C4), 57.6 (pyrazoline-C5), 56.0 (OCH3), 106.8 (pyrazole-C4), 129.2 (pyrazole-C5), 116.0 − 140.0 (aromatic carbons), 149.0 (pyrazoline-C3), 159.0 (pyrazole-C3). MS (EI): m/z: 520 [M+] (11.3%). Anal. Calcd for C35H28N4O (520.62): C,80.74; H, 5.42; N, 10.76. Found: C,80.73; H,5.43; N,10.70.3′-Naphthalen-2-yl-2,1′-diphenyl-5-p-tolyl-3,4-dihydro-2H, 1’H -bipyrazole (4c). Yellow solid, yield 77%, m.p.198–199 °C. IR (KBr) vmax (cm−1): 2923 (CH-sp3), 3052 (CH–Ar), 1595 (C=N). 1H NMR  δ: 2.5 , 3.3–3.6 , 5.28 , 6.6 , 6.9–8.2 . 13C NMR  δ: 20.3 (CH3), 39.5 (pyrazoline-C4), 55.8 (pyrazoline-C5), 107.1 (pyrazole-C4), 129.5 (pyrazole-C5), 118.6-140.6 (aromatic carbons), 150.0 (pyrazoline-C3), 159.0 (pyrazole-C3). MS(EI): m/z: 504 [M+] (13.5%). Anal. Calcd for C35H28N4(504.62): C,83.30; H, 5.59; N, 11.10; Found: C,83.37; H, 6.00; N,11.01.5-(4-Chlorophenyl)-3′-naphthalen-2-yl-2,1′-diphenyl-3,4-dihydro-2H,1'H- bipyrazole (4d). Yellow solid, yield 71%, m.p.192-193 °C. IR (KBr) vmax (cm−1): 2978 (CH-sp3), 3174 (CH–Ar), 1605 (C=N). 1H NMR  δ: 3.3–3.5 , 5.27 , 6.7 , 7-7.8 . 13C NMR  δ:35.9 (pyrazoline-C4), 57.8 (pyrazoline-C5), 106.9 (pyrazole-C4), 129.4 (pyrazole-C5), 116.3–141.1 (aromatic carbons), 149.6 (pyrazoline-C3), 159.6 (pyrazole-C3). MS (EI): m/z: 524 [M+] (10.8%), 426 . Anal. Calcd for C34H25ClN4(525.04): C,77.78; H, 4.80; N, 10.67; Found: C,77.87; H, 4.82; N,10.53.5--3′-naphthalen-2-yl-2,1′-diphenyl-3,4-dihydro-2H,1’H--bipyrazole (4e). Brown solid, yield 75%, m.p.172–173 °C. IR (KBr) vmax (cm−1): 2974 (CH-sp3), 3176 (CH–Ar), 1605 (C=N). 1H NMR  δ: 3.5 , 3.3 , 5.26 , 6.8 , 7–7.8 . 13C NMR  δ: 38.80 (pyrazoline-C4), 58.80 (pyrazoline-C5), 108.8 (pyrazole-C4), 128.2(pyrazole-C5), 118.8–140.4 (aromatic carbons), 148.58 (pyrazoline-C3), 158.8 (pyrazole-C3). MS(EI): m/z: 558 [M+] (16.5%), 560 . Anal. Calcd for C34H24Cl2N4 (559.48): C,72.99; H, 4.32; N, 10.01; Found: C,72.96; H,4.30; N,10.10.5--3-aryl-4,5-dihydro-isoxazole (5a–e). A solution (2a–e) (1.0 mmol), and hydroxylamine HCl 99% (1.0 mmol) in absolute ethanol (15 mL) with 0.5 mL piperidine was refluxed for 8–10 h. The resulting solution was concentrated, cooled, the solid obtained was filtered off and recrystallized from ethanol to give compounds 5a–e.5--3-phenyl-4,5-dihydro-isoxazole (5a).White solid, yield 81%, m.p.190–191 °C. IR (KBr) vmax (cm−1): 2967 (CH-sp3), 3167 (CH–Ar), 1608 (C=N). 1H NMR  δ: 3.60-3.80 , 5.50 , 6.5 , 6.8–7.6 . 13C NMR  δ: 37.30 (isoxazoline-C4), 56.50 (isoxazoline-C5), 114.0 (pyrazole-C4), 125.0 (pyrazole-C5), 118.0–140.1 (aromatic carbons), 157.0 (pyrazole-C3), 160. 0 (isoxazoline-C3). MS (EI): m/z: 415 [M+] (17.7%). Anal. Calcd for C28H21N3O (415.48): C,80.94; H, 5.09; N, 10.11; Found: C,80.97; H, 5.13; N,10.23.3-(4-Methoxyphenyl)-5--4,5-dihydro isoxazole (5b). Yellow solid, yield 85%, m.p.197–198 °C. IR (KBr) vmax (cm−1): 2966 (CH-sp3), 3169 (CH–Ar), 1605 (C=N). 1H NMR  δ: 3.61-3.90 , 3.5 , 5.40 , 6.6 , 7.1-7.9 . 13C NMR  δ: 33.30 (isoxazoline-C4), 56.1 (OCH3), 60.0 (isoxazoline-C5), 115.0 (pyrazole-C4), 127.0 (pyrazole-C5), 119.0–141.0 (aromatic carbons), 158.0 (pyrazole-C3), 162.0 (isoxazoline-C3). MS (EI): m/z: 445 [M+] (18.9%). Anal. Calcd for C29H23N3O2: (445.51): C,78.18; H, 5.20; N, 9.43; Found: C,78.27; H,5.14; N,9.33.5--3-p-tolyl-4,5-dihydroisoxazole (5c). Yellowish brown solid, yield 77%, m.p.181–182 °C. IR (KBr) vmax (cm−1): 2967 (CH-sp3), 3170 (CH–Ar), 1600 (C=N). 1H NMR  δ: 2.5 , 3.66-3.82 , 5.55 , 6.7 , 7.0-7.9 . 13C NMR  δ: 20.5 (CH3), 32.30 (isoxazoline-C4), 60.6 (isoxazoline-C5), 115.5 (pyrazole-C4), 127.2 (pyrazole-C5), 118.0-144.0 (aromatic carbons), 157.5 (pyrazole-C3), 160.6 (isoxazoline C3). MS (EI): m/z: 429 [M+] (11.8%). Anal. Calcd for C29H23N3O (429.51): C,81.09; H, 5.40; N, 9.78; Found: C,81.17; H, 5.43; N,9.76.3-(4-Chlorophenyl)-5--4,5-dihydro isoxazole (5d). White solid, yield 79%, m.p.185–186 °C. IR (KBr) vmax (cm−1): 2916 (CH-sp3), 3046 (CH–Ar), 1594 (C=N). 1H NMR  δ: 3.31–3.50 , 5.56 , 6.60,7-8 . 13C NMR  δ:32.5 (isoxazoline-C4), 60.8 (isoxazoline-C5), 115.6 (pyrazole-C4), 127.1 (pyrazole-C5), 117.6-140.2 (aromatic carbons), 159.7 (pyrazole-C3), 162.8(isoxazoline-C3). MS (EI): m/z: 449 [M+] (12.6%), 451 . Anal. Calcd for C28H20ClN3 (449.93): C,74.74; H, 4,48; N, 9.34; Found: C,74.77; H,4.43; N,9.36.3--5--4,5-dihydro isoxazole (5e). White solid, yield 79%, m.p.187–189 °C. IR (KBr) vmax (cm−1): 2963 (CH-sp3), 3166 (CH–Ar), 1604 (C=N). 1H NMR  δ: 3.69 , 3.82 , 6.06, 6.6 , 7.0-7.8 . 13C NMR  δ: 32.5 (isoxazoline-C4), 60.3 (isoxazoline-C5), 118.0 (pyrazole-C4), 128.8 (pyrazole-C5), 119.9-143.0 (aromatic carbons), 159.5 (pyrazole-C3), 162.0 (isoxazoline-C3). MS (EI): m/z: 484 [M+] (20.7%),486 . Anal. Calcd for C28H19Cl2N3O (484.3): C,69.43; H,3,95; N,8.68. Found:C,69. 47; H,3.93; N,8.66.3’-Naphthalen-2-yl-5-aryl,1’-phenyl-3,4-dihydro-1’H-bipyrazolyl-2-carbo thioic acid amide (6a–e). To a solution of chalcones 2a–e (1.6 mmol) in absolute ethanol (25 mL), semicarbazide hydrochloride (3.66 mmol) and piperidine (0.5 mL) were added and the solution was refluxed for 9–12 h. The reaction mixture was poured on ice water. The obtained solid was filtered off and recrystallized from ethanol to give compounds 6 a–e.3′-Naphthalen-2-yl-5,1′-diphenyl-3,4-dihydro-1’H-bipyrazolyl-2-carbothioic acid amide (6a). Yellow solid, yield 65%, m.p.198–199 °C. IR (KBr) vmax (cm−1): 2966 (CH-sp3), 3166 (CH–Ar), 3350 (NH2), 1603 (C=N). 1H NMR  δ: 2.9–3.3 , 5.40 , 6.7, 7–7.7 , 10.1 . 13C NMR  δ: 45.1 (pyrazoline-C4), 55.5 (pyrazoline-C5), 117.1 (pyrazole-C4), 125.1 (pyrazole-C5), 118.5–145.0 (aromatic carbons), 155.0 (pyrazoline-C3), 157.0 (pyrazole-C3), 180.0 (C=S). MS (EI): m/z:473 [M+] (16.7%). Anal. Calcd for C29 H23N5S (473.59): C,73.55; H, 4.90; N, 14.79. Found: C,73.47; H,4.87; N,14.73.5-(4-Methoxyphenyl)-3′-naphthalen-2-yl-1′-phenyl-3,4-dihydro-1’H- bipyrazolyl-2-carbothioic acid (6b). Yellow solid, yield 67%, m.p.193-194 °C. IR (KBr) vmax (cm−1): 2956 (CH-sp3), 3049 (CH–Ar), 3255 (NH2), 1597 (C=N). 1H NMR  δ: 2.8-3.03 , 3.36 , 5.66 , 6.0 , 6.7–7.6 , 9.66 . 13C NMR  δ:40.0 (pyrazoline-C4), 55.0 (pyrazoline-C5), 56.0 (OCH3), 113.9 (pyrazole-C4), 126.9 (pyrazole-C5), 118.6–148.4 (aromatic carbons), 155.6 (pyrazoline-C3), 158.3 (pyrazole-C3), 180.0 (C=S). MS(EI): m/z: 503 [M+] (12.8%). Anal. Calcd for C30H25N5OS: (503.62): C,71.55; H, 5.00; N, 13.91. Found: C,71.47; H,4.97; N,13.90.3′-Naphthalen-2-yl-1′-phenyl-5-p-tolyl-3,4-dihydro-1’H-bipyrazolyl-2-carbo thioic acid amide (6c). Yellow solid, yield 63%, m.p.184–185 °C. IR (KBr) vmax (cm−1): 2968 (CH-sp3), 3161 (CH–Ar), 3307 (NH2), 1606 (C=N). 1H NMR  δ: 2.51, 3.25–3.51 , 5.43 , 6.7, 6.9–7.9, 10.5 . 13C NMR  δ: 20.6 (CH3), 45.5 (pyrazoline-C4), 56.6 (pyrazoline-C5), 117.5 (pyrazole-C4), 125.5 (pyrazole-C5), 118.7–144.0 (aromatic carbons), 155.6 (pyrazoline-C3), 157.50 (pyrazole-C3), 180.2 (C=S). MS(EI): m/z: 487 [M+] (16.3%). Anal. Calcd for C30H25N5S (487.62): C,73.89; H, 5.17; N, 14.36. Found: C,73.87; H,5.13; N,14.43.5-(4-Chlorophenyl)-3′-naphthalen-2-yl-1′-phenyl-3,4-dihydro-1’H- bipyrazo lyl-2-carbothioic acid (6d). Yellow solid, yield 69%, m.p.186–187 °C. IR (KBr) vmax (cm−1): 2963 (CH-sp3), 3162 (CH–Ar), 3310 (NH2), 1606 (C=N). 1H NMR  δ: 2.90–3.37 , 5.62 , 6.6 , 7.0–7.8 , 10.1 . 13C NMR  δ: 45.1 (pyrazoline-C4), 56.6 (pyrazoline-C5), 117.6.0 (pyrazole-C4), 125.3 (pyrazole-C5), 118.9–143.0 (aromatic carbons), 157.7 (pyrazoline-C3), 160.0 (pyrazole-C3), 180.0 (C=S). MS (EI): m/z: 507 [M+] (20%), 509 . Anal. Calcd for C29H22ClN5S (508.3): C,68.56; H, 4.36; N, 13.79. Found: C, 68.57; H,4.33; N,13.63.5--3′-naphthalen-2-yl-1′-phenyl-3,4-dihydro-1’H-bi pyrazolyl-2-carbothioic acid (6e). Yellow solid, yield 69%, m.p. 180–181 °C. IR (KBr) vmax (cm−1): 2967 (CH–sp3), 3163 (CH–Ar), 3317 (NH2), 1600 (C=N). 1H NMR  δ: 3.01 , 3.6 , 5.25 , 6.8 , 7.0–7.8 , 10.2 . 13C NMR  δ: 45.3 (pyrazoline-C4), 56.3 (pyrazoline-C5), 117.3.0 (pyrazole-C4), 125.4 (pyrazole-C5), 118.1–142.0 (aromatic carbons), 155.3 (pyrazoline-C3), 157.1 (pyrazole-C3), 180.0 (C=S). MS (EI): m/z: 541 [M+] (22.1%), 543 . Anal. Calcd for C29H21Cl2N5S (542.48):C,64.21; H, 3.90; N,12.91. Found: C,64.27; H,3.83; N,12.83.2.3.Chemicals: All chemicals required for all assays were used as analytical grade, and were purchased from Sigma-Aldrich Chemicals Co., St. Louis, MO, USA.2.3.1.517 nm.Acontrol is the absorbance of the control reaction , and Asample is the absorbance of the test sample. Linear regression analysis was performed to calculate drug concentration showing 50% free radical inhibition activity (IC50). All tests were performed in triplicates.The DPPH scavenging activity of all synthesized compounds was measured as previously described by Nahar et al.2.3.2.3PO3) was added to each well to measure the nitrite content. The absorbance of the formed pink-coloured chromophore was measured at λ 546 nm. DMSO and ascorbic acid were used as the negative and positive control, respectively. All tests were performed in triplicate. The percentage of NO scavenging activity was calculated according to the following equation:Acontrol is the absorbance of the control reaction , and Asample is the absorbance of the test sample. Linear regression analysis was performed to calculate drug concentration showing 50% free radical inhibition activity (IC50).The NO scavenging activity of all synthesized compounds was measured as previously described by Ho et al.2.3.3.2−. RSA of all synthesized compounds as previously described2EDTA. Next, 50 µL of 60-mM pyrogallol in1mM HCl had been thoroughly mixed with the mixture. The absorbance of the reaction mixture was measured at A325 nm every 30 s for 5 min. O2− RSA was expressed by the oxidation degree of a test group in comparison to that of the control. The absorbance at 325 nm was measured against the Tris-HCl buffer every 30 s for 5 min. The percentage of scavenging effect was calculated using the following equation:A325nm, control is the increase in A325 nm of the reaction mixture without the sample and ΔA325 nm, sample is that for the mixture with the sample; T=5 min. The experiments were performed in triplicate. The IC50 value was defined as the concentration for 50% superoxide free radical inhibition and was calculated by linear regression.The improved pyrogallol autoxidation method was used to determine O2.4.Ainhibitor sample) is the absorbance of the test sample. Dose–response curve was plotted between % inhibition and the drug concentration. The non-linear dose–response curve was used for calculating drug concentration showing 50% enzyme inhibition.The assay was performed using Cayman’s lipoxygenase inhibitor screening assay kit  according to the manufacturer’s instructions. Briefly, 90 µL of 15-LOX was pipetted into a 96-well plate. Next, 10 µL of test compound at concentrations  dissolved in DMSO were added to each well. The reaction was initiated by adding 10 µL substrate (AA) and the plate was placed on a shaker for at least 5 minutes. Finally, 100 µL of chromogen (prepared according to manufacturer’s instructions) was added to each well to stop enzyme catalysis and develop the reaction. 100 µL of Assay buffer  was used in blank wells. Quercetin and DMSO were used as the positive control and 100% initial activity, respectively. The absorbance of the solution was measured at λ 490–500 nm. The percentage inhibition was calculated according to the following equation:2.5.in vivo antioxidant potentials of the test compounds which showed promising in vitro antioxidant activities, CAT activity, GSH and MDA levels were assayed in liver of treated animals.To determine 2.5.1.ad libitum and maintained in a controlled environment under standard conditions of temperature and humidity with an alternating 12 h light and dark cycle for about a week for acclimatization. The protocol of the study was approved by the Animal Ethics Committee of the Faculty of Pharmacy, Helwan University . The study was conducted in accordance with the EC, directive 86/609/EEC for animal experiments.The complete progress of the experiment was conducted using male Wistar albino rats (200–250 g), delivered by the Institutional Breeding House, Egypt, reared and maintained in the animal house of the institution. The animals had free access to food and water 2.5.2.in vivo antioxidant study was 100 mg/kg B.W.The acute toxicity study of the selected compounds was performed on albino rats according Organization for Economic Co-operation and development guidelines-4252.5.3.n=6) were divided into nine different groups. Group I served as a control group and treated with the same volume vehicle only. Group II treated with 100 mg/kg of Ascorbic acid as standard antioxidant drug. Groups (III–IX) orally administered 100 mg/kg of compounds 3a, 4e, 5b, 5c, 6a, 6c, 6e, respectively, for 3 days. The animals were sacrificed by cervical dislocation 24 h after the last dose. Sacrificing is carried out at the same time of the day, to avoid the circadian variation in the level of tissue GSH®, Cat no.099C K6424, TERRE HAUTE, USA).Rats are weighed at the beginning and at the end of experiment. Fifty-four male albino rats  in liver homogenates were assayed by the deproteinization of tissue homogenate2.5.3.3.Lipid peroxidation level in the liver homogenates was determined as thiobarbituric acid reactive substances (TBARS) by measuring malondialdehyde ‘MDA’ level according to Mihara and Uchiyama2.6.post hoc Scheffe’s test. A value of p < 0.05 was considered statistically significant.The data were represented as mean ± SEM. Significant differences between groups were tested by using GraphPad InStat software version 3.05 . Appropriate graphs were plotted when needed using GraphPad Prism version 5 for Windows . The results were analysed using one-way analysis of variance (ANOVA) with 2.7.www.rcsb.org). The hydrogen atoms were then added to both the small molecule and the 15-LOX enzyme structure. The atom and bond types as well as the protonation state for the small molecule and the binding site were checked and corrected when needed. Water molecules were deleted. This was followed by minimising the complex with the DS force field using the default parameters. The resulted binding mode of the designed compound in bound to catalytic active site of 15-LOX will be discussed later.The modelling experiment described in this study was performed by using the Discovery Studio (DS) version 4.5  software3.3.1.(2-6) was depicted in 1) was prepared via Vilsmeier-Haack reaction3. Claisen-Schmidt condensation1) with different aromatic ketones such as 1-phenylethanone, 1-(4-methoxyphenyl)ethanone, 1-(4-methylphenyl)ethanone, 1-(4-chlorophenyl)ethanone and 1-ethanone was performed in 30% ethanolic sodium hydroxide solution at room temperature to afford the corresponding chalcones (2a–e), respectively, as outlined in ((2a–e) was used as key intermediates for synthesizing the target pyrazole-pyrazolines (3) and (4) through 1,4-addition of hydrazine hydrate or phenyl hydrazine to the α, β-unsaturated carbonyl system of the precursor chalcones 2a–e, followed by dehydration and rearrangement. Cyclocondensation of the chalcones (2a–e) with hydrazine hydrate or phenyl hydrazine(3a–e) and phenylpyrazolines (4a–e), respectively ((2a–e) into the corresponding isoxazolines (5a–e) was conducted by condensation of the chalcones with hydroxylamine hydrochloride(6a–e) were prepared by base-catalysed cyclization of chalcones 2a–e through reaction with semicarbazide HCl2 to carbonyl carbon which is stabilized by the proton transfer and further dehydration leads to the formation of pyrazoline (The synthesis of the target compounds lined in  . The foectively . Cyclizarazoline .2-6) were confirmed by IR, 13C NMR, mass spectroscopy and microanalysis. The IR spectrum of compounds 2a–e exhibited characteristic bands at around 1690 cm−1 for C=O. 1H NMR spectra showed two doublets signals at δ 6.2–7.4 ppm for CH=CH protons of chalcones. The 1H NMR spectra of compounds 3-6 showed signal doublet of doublet at δ 2.80–3.90 ppm and triplet at δ 5.20-5.66 ppm corresponding to the protons at C-4 and C-5 of the pyrazoline ring in addition to the signals of pyrazole and other protons  . Moreover, the 1H NMR spectra of compounds 3 and 6 showed singlet exchangeable signal at around δ 8 ppm or δ 10 ppm corresponding to (NH) of the pyrazoline or (NH2) of the carboxamide, respectively. 13C NMR showed the characteristic signals at d δ 32.1-38.3 and 55.1-60.6 ppm corresponding to C-4 and C-5 carbon of the pyrazoline ring, respectively. In addition to the other signals for the carbons of the target compounds, The 13C NMR spectra of compounds 2 and 6 showed the presence of signals corresponding to C=O around at δ 187 ppm and C=S at around δ 180 ppm, respectively .The formed new compounds (3.2.2–6 (a–e) were investigated for their RSA by DPPH, NO and superoxide assays while Ascorbic Acid (AA) was used as antioxidant reference standard. The in vitro antioxidant activities of tested compounds were expressed as IC50 values  was determined using DPPH radical scavenging assay in comparison with ascorbic acid (AA) as control treatment. The mechanism of radical scavenging is based on acidic H-atom transfer from the compound to the DPPH to form DPPH-H. The results are presented in (3a-d) and pyrazoline carbothioamides (6a-d) have potent antioxidant activities while pyrazolyl isoxazolines (5a-d) have demonstrated moderate RSA. The most active compounds were 3b, 3c, 3e, 6b and 6e . They exhibited potent RSA than ascorbic acid (IC50= 13.67 ± 0.97 μg/mL). Suggesting that, the presence of free NH of pyrazoline enhances the antioxidant activity by increasing their hydrogen donor capacity. Moreover, good radical scavenger property of S atom C=S and free NH2 that act as hydrogen donor in pyrazoline carbothioamides possessed potent DPPH RSA. Compounds 3a, 6a, and 6c  displayed good RSA but lower than ascorbic acid. In addition, N-phenyl pyrazolyl pyrazolines 4b (IC50=21.28 ± 1.14 μg/mL), 4c (IC50=24.42 ± 0.9 μg/mL) and 4e (IC50= 19.56 ± 1.06 μg/mL) showed good activity in comparison to standard treatment AA. The lower DPPH RSA of N-phenyl pyrazolyl pyrazolines (4a–e) and pyrazolyl isoxazolines (5a–e) prove the significant role of NH of pyrazoline in antioxidant activity. All compounds (3–6) showed higher antioxidant RSA than their precursors, chalcones (2a–e) thus indicating that pyrazoline and isoxazoline rings enhance RSA of these compounds. SAR studies showed that antioxidant activity of compounds tested by DPPH assay depends not only on the type of heterocyclic pharmacophore but also on substituents R on the aromatic ring of pyrazoline/isooxazoline since antioxidant activity is related to electron or hydrogen donation capacity to DPPH.Radicals (3e > 6e> 4e> 5e). Pyrazolines 3 and pyrazoline carbothioamides 6 have higher FRSA than N-phenyl pyrazolines 4 and isoxazolines 5. For compounds 3e and 6e their potent antioxidant activity is due to the presence of pyrazoline and carbothioamide moiety,5. Concerning substitution patterns of pyrazolineAQ4/isoxazoline. On the other hand, the order of antioxidant activity of pyrazoline and isoxazoline compounds was found to be: 3,4-(Cl) 2(3e) > 4-OCH3(3b) > 4-CH3(3c) > H (3a) > 4-Cl (3d), in descending order.Currently, antioxidants showing DPPH scavenging activity are receiving attention due to their role as anticancer, anti-inflammatory and antiaging agentsRadicals . Regardi3e and 6e show the most potent antioxidant activity, both having 3,4 (di-Cl) substituents on phenyl ring, which is in accordance with the reported results3 and CH3 are more beneficial than unsubstituted or mono chloro-substituted phenyl ring, which may be due to + I and mesomeric effectsThe di-halogenated compounds exhibited significant DPPH RSA than the corresponding chloro-substituted compounds. Pyrazole derivative 3.2.2.(2–6) to NO radical. The results are presented in 2e, 6c, 3a and 6a showed NO scavenging activity through competing with oxygen to scavenge for the nitrite radical; higher than that of ascorbic acid . Pyrazole 2e was the most potent antioxidant derivative via reducing nitrite production with 3.4 folds that of ascorbic acid, Pyrazolyl isoxazolines 5a, 5c  displayed comparable potency to ascorbic acid. Moreover, N-phenyl pyrazolyl pyrazoline 4e displayed good antioxidant activity (IC50=57.01 ± 1.29 μg/mL). SAR studies showed that pyrazoline carbothioamide 6c and pyrazolyl pyrazoline 3a exhibited higher NO scavenging activity than pyrazolyl isoxazoline 5a and N-phenyl pyrazolyl pyrazoline 4e. This confirms that pyrazoline and carbothioamide rings were favourable substitutions over isoxazoline and N-phenyl pyrazoline for the antioxidant activity of the tested compounds against NO assay.NO assay was used to determine the scavenging power of the target compounds 3.2.3.(2–6) were evaluated for their antioxidant activity via superoxide scavenging assay to estimate their capability to scavenge O2−. and so, preventing the formation of elemental oxygen. The resulted IC50 values were measured in μg/mL as shown in 5c, 3a, 4b, 6e and 5b  possessed pronouncing superoxide scavenging activity than ascorbic acid (IC50=124.99 ± 1.32 μg/mL). Pyrazolyl isoxazoline 5c and pyrazolyl pyrazoline 3a were the most potent derivatives with 2.8 and 2.5 folds of ascorbic acid, respectively. In addition, pyrazolyl pyrazolines 3b and 3e , pyrazoline carbothioamide 6a (IC50= 127.25 ± 1.47 μg/mL) displayed comparable O2−. scavenging potency to ascorbic acid. Moreover, pyrazoline carbothioamide 6c (IC50 = 140.17 ± 1.52 μg/mL), N-phenylpyrazoline 4c, 4e  showed good superoxide scavenging activity. SAR studies showed that most of synthesized compounds revealed higher activity than the parent chalcones in superoxide scavenging assay. Pyrazoline 3a, isoxazoline 5c, pyrazoline carbothioamide 6e and N-phenylpyrazoline 4b rings showed a significant RSA towards superoxide radical anion. Di-halogenated compounds displayed good superoxide RSA while, mono halogenated ones didn’t.All target compounds 3.3.2–6) were tested against Soybean 15-LOX enzyme. The results are expressed as IC50 values (µM) as shown in 2b, 2c, 3a, 3e, 4a, 4c, 5e, 6a, 6b, 6c and 6d showed potential 15-LOX inhibition activity when compared to quercetin (IC50=3.34 µM) as reference inhibitor. Carbothioamides 6a, 6e in which the pyrazoline ring is substituted with phenyl moiety and 3,4-di-Cl phenyl, were the most potent compounds  with 2.2 and 2.1 folds that of quercetin, respectively. Pyrazoline 3c and N-phenyl pyrazoline 4e showed comparable potency to that of quercetin . Moreover, pyrazole 2e (IC50=4.63 ± 0.09 µM), pyrazoline 3b (IC50=4.60 ± 0.06 µM), isoxazolines 5a, 5b, and 5c  and N-phenyl pyrazoline 4b (IC50=4.00 ± 0.06 µM) displayed good 15-LOX inhibitory activity but lower than quercetin. The results of the tested compounds (2–6) as 15-LOX inhibitors emphasized the important role of 3-naphthylpyarazole in this enzymatic assay regardless the derivative was either α, β-unsaturated ketone 2c, pyrazoline 3a,N-phenylpyrazoline 4c, isoxazoline 5e or pyrazoline carbothioamide counterpart 6a. Those derivatives were superior to quercetin in 15-LOX inhibition. 15-LOX inhibition appeared to be (6a> 4c >3a> 2c> 5e) to confirm the excel pyrazoline carbothioamide over pyrazoline, N-phenyl pyrazoline and isoxazoline for antioxidant activity of the tested compounds against Soybean 15-LOX enzyme. It was also noticed that di-halogenated derivatives showed significant 15-LOX inhibition activity. This might be due to better fitting of derivative into the catalytic pocket of 15-LOX enzyme. In summary, compounds 3a, 4e, 5b, 5c, 6a, 6c and 6e showed significant RSA in all three methods in comparison with ascorbic acid and 15-LOX inhibition potency using quercetin as standard. This suggests an important influence of EDGs  and di-halogen (di-Cl) in benzene ring. Regarding heterocyclic pharmacophore, pyrazoline carbothioamide and pyrazoline showed higher RSA and 15-LOX inhibition potency than N-phenyl pyrazoline and isoxazoline and these observations should be regarded in the future on the designed LOX inhibitors.All target compounds  leading to cell injury and death2O2 and lipid peroxides. Oxidative stress readily oxidises GSH to glutathione disulphide by free radicals and ROS causing depletion of GSH level2O2 and protects the tissues from highly reactive stabilization that may be produced from H2O2. Pyrazole derivatives  that showed promising in vitro antioxidant activities were subjected to in vivo study. In the acute toxicity study, the orally administered compounds did not show toxic effects in doses up to 100 mg/kg B.W. Oral administration of test compounds for 3 days increased CAT activity and GSH level and decreased the MDA concentration in the liver, which indicated that they could enhance the antioxidant status as presented in in vitro antioxidant activity shown by these compounds. However, only compounds  showed significant potent antioxidant activity compared to control group at dose of 100 mg/kg. This may be attributed to the short treatment period of the animals. The results of our study showed that treatment of animals by those compounds significantly increased the level of CAT enzyme by about 101, 89, and 141%, respectively, compared to control group. The increased level of CAT leads to break down of H2O2 and prevent further generation of free radicals. The increase in intracellular thiol-based antioxidant GSH was by about 52, 55, 51%, shown by compounds . The antioxidant activity may be due to potent radical-scavenging activity of isoxazoline and carbothioamide pyrazoline.Measuring the 3.5.6a into the active binding site pocket of the human 15-LOX (PDB: 4NRE)www.rcsb.org). The hydrogen atoms were then added to both the small molecule and the protein. The atom and bond types as well as the protonation state for the small molecule and the binding site were checked and corrected when needed. Water molecules were deleted. This was followed by minimizing the complex with the DS force field by using the default parameters. Analysis of the proposed binding of inhibitor 6a  with the carbonyl of the Ile 676 amino acid residue.To investigate the orientation of the most potent compound bitor 6a  into the4.in vitro antioxidant activity using DPPH, NO and Superoxide radical scavenging assays as well as 15-LOX inhibition activity. One important pathway for antioxidant agents is through inhibiting lipid peroxidation that is mainly catalyzed by 15-LOX. The activity of the compound 6a was assed towards both antioxidant and anti-LOX activities. It was found that compound 6a showed NO scavenging activity higher than that of ascorbic acid. In addition, it showed potent anti-LOX activity by 2.2 folds compared to that of quercetin. Furthermore, compounds 5a and 5c showed comparable potency to ascorbic acid. Moreover, compound 4e displayed good antioxidant activity. SAR studies showed that pyrazoline carbothioamide 6c and pyrazolyl pyrazoline 3a exhibited higher NO scavenging activity than pyrazolyl isoxazoline 5a and N-phenyl pyrazolyl pyrazoline 4e. In addition, compounds 2c, 3a, 3e, 4a, 4c, 5e, 6a, 6b, 6c, 6d, and 6e showed potential 15-LOX inhibition activity which was almost aligned with DPPH assay results only but was conflicted for some compounds such as 2c, 3e, 4a, 4c, 5e, 6b, and 6d with the other antioxidant assays. Interestingly, carbothioamides 6a and 6e were the most potent compounds with 2.2 and 2.1 folds that of quercetin, respectively, and almost showed similar potential antioxidant activity in all three assays. Furthermore, compounds 3a, 4e, 5b, 5c, 6a, 6c, and 6e showed significant RSA in all three in vitro assays in comparison with ascorbic acid along with 15-LOX inhibition potency using quercetin as standard suggesting an important influence of EDGs  and di-halogen (di-Cl) on the benzene ring. Regarding heterocyclic pharmacophore, pyrazoline carbothioamide and pyrazoline showed higher RSA and 15-LOX inhibition potency than N-phenyl pyrazoline and isoxazoline and these observations should be taken in consideration for future designed LOX inhibitors. Furthermore, the in vivo results supported the in vitro data, i.e. compounds 5b, 5c and 6e at dose of 100 mg/kg B.W showed significant in vivo antioxidant activity through increased CAT activity, GSH level and decreased lipid peroxidation in the treated rat liver compared to control treatment. This indicates their role in enhancing the antioxidant status. These data validate the potent in vitro antioxidant activity shown by those derivatives. Docking study of the most potent candidate 6a revealed that stabilization of the ligand inhibitor through hydrophobic-hydrophobic interactions. In addition, π–cationic interactions with the catalytic Fe3+ of the active site as well as π–π interactions with the His378 amino acid residue might be required for the potential activity of 15-LOX inhibitor. In conclusion, the obtained results suggest that these potent compounds may serve as lead candidates for 15-LOX inhibitors. Furthermore, the designed pyrazole hybrid scaffold is an interesting antioxidant pharmacophore and considered as novel lead scaffold for any future optimization.In summary, novel hybrids containing pyrazole, naphthalene and pyrazoline/isoxazoline pharmacophore were synthesized and investigated for their


N-phenacylated. N-Phenacyl derivatives of 4,6-dimethyl-5-nitropyridin-2-one under the action of base gave 5-hydroxy-8-nitroindolizine and under acidic conditions gave 5-methyl-6-nitrooxazolepyridinium salt, which underwent recycization with MeONa to 5-methoxy-8-nitroindolizine.Reaction between the derivatives of 6-methyl-beta-nitropyridin-2-one and phenacyl bromides was studied, and the yields observed were extremely low. The pyridones were converted via chloropyridines to methoxyderivatives, which were  A, NH reaction of 8-nitroindolizines at this position pyridinium perchlorate (6). A mixture of 0.1 g (0.3 mmol) of phenacylpyridone 5a and 1 mL of concentrated sulfuric acid was maintained at 22 °C for 18 h. Then, 0.2 mL of 70% HCiO4 was added to the mixture, incubated for 1 h, poured into 100 mL of absolute ether and the precipitate formed was filtered off. The yield of perchlorate is 92%; m.p. 295–297 °C (decomp.). 1H-NMR Spectrum (DMSO-6d): 9.72 ; 8.60 ; 8.07 ; 7.82 ; 2.89 ; 2.67 . 13C-NMR (DMSO-6d): 15.4; 18.5; 110.6; 112.1; 122.6; 127.5; 130.1; 137.1; 137.8; 144.8; 145.8; 152.2; 152.4. X-Ray data see 2-(p-Chlorophenyl)-5,7-dimethyl-6-nitrooxazolo pyridinium salt 6, which underwent recycization with MeONa to 5-methoxy-8-nitroindolizine 7.In conclusion, the reaction between the derivatives of 6-methyl-beta-nitropyridin-2-one (


Despite the development of targeted therapies in cancer, the problem of multidrug resistance (MDR) is still unsolved. Most patients with metastatic cancer die from MDR. Transmembrane efflux pumps as the main cause of MDR have been addressed by developed inhibitors, but early inhibitors of the most prominent and longest known efflux pump P-glycoprotein (P-gp) were disappointing. Those inhibitors have been used without knowledge about the expression of P-gp by the treated tumor. Therefore the use of inhibitors of transmembrane efflux pumps in clinical settings is reconsidered as a promising strategy in the case of the respective efflux pump expression. We discovered novel symmetric inhibitors of the symmetric efflux pump MRP4 encoded by the ABCC4 gene. MRP4 is involved in many kinds of cancer with resistance to anticancer drugs. All compounds showed better activities than the best known MRP4 inhibitor MK571 in an MRP4-overexpressing cell line assay, and the activities could be related to the various substitution patterns of aromatic residues within the symmetric molecular framework. One of the best compounds was demonstrated to overcome the MRP4-mediated resistance in the cell line model to restore the anticancer drug sensitivity as a proof of concept. Cancer is an ongoing burden for humanity with increasing costs for therapy of patients ,2,3. OldBesides the various resistance mechanisms that occur toward single drugs, multidrug resistance (MDR) is the main obstacle in cancer therapy ,12. TranN-benzyl substituted compounds as potential efflux pump inhibitors with a symmetric molecular scaffold to inhibit the symmetric efflux pump MRP4 encoded by the ABCC4 gene. As only a few inhibitors of MRP4 are known [We discovered symmetric 1,4-dihydropyridines as novel inhibitors of the symmetric efflux pump MRP4 that plays a role in various types of solid tumors . Early 1re known ,37, we d1, methyl propiolate 2, and a benzylamine compound 3 as shown in 4-21 own no substituents in the 2- and 6-positions where those formed with the dicarbonyl compound own alkyl substituents at the respective 1,4-dihydropyridine positions.Known inhibitors of transmembrane efflux pumps as natural or synthetic compounds like cyclosporine or valspodar own complicated molecular structures that are partly difficult to synthesize. Our 1,4-dihydropyridines as novel MRP4 inhibitors are accessible in a simple one-pot reaction of three components, namely an aromatic aldehyde 1H NMR spectra.After finishing the reaction that was followed by thin-layer chromatography (TLC), the solvent was removed. The compound purification and final isolation led to solid products that were spectroscopically characterized to confirm the given structures with proton signals for the hydrogen atoms at the 2- and 6-positions at about 7.45 ppm and for the 4-hydrogen atom at about 4.75 ppm in the However, due to the recent promotion of MRP4 to an anticancer target, there is a certain lack of MRP4 inhibitors. From those few inhibitors with low potency, the most prominent one is the cysteinyl leukotriene receptor 1 antagonist MK571 that shows additional pharmacological activities as a phosphodiesterase inhibitor ,45. ThusMRP4 has a symmetric molecular framework built of two transmembrane domains (TMDs) that each consist of six α-helical subunits surrounding the inner channel for outward transport of the MRP4 substrates ,47. The FAR). The FAR values are shown in FAR values.In our assay system, we determined the uptake of the fluorescent MRP4 substrate calcein in the pancreatic colo357 and the colo357 MRP4-overexpressing cell lines both with and without our 1,4-dihydropyridines as potential inhibitors. In the case of inhibition of MRP4, an increase of the uptake of the fluorescent MRP4 substrate calcein was observed in the MRP4-overexpressing cell line. The fluorescence amount of the MRP4 substrate was measured for the inhibited cell lines where it increased only in the MRP4-overexpressing cell line due to the inhibition while in the non-inhibited cells fluorescence remained unchanged. A difference in fluorescence was calculated from these values for each cell line. The given values were divided to result in a fluorescence activity ratio  were measured using tetramethylsilane as an internal standard. Thin-layer chromatography (TLC) was performed on E. Merck 5554 silica gel plates. The high-resolution mass spectra were recorded on a Finnigan LCQ Classic mass spectrometer .Commercial reagents were used without further purification. The 1, two equivalents of the methyl propiolate 2, and one equivalent of the benzylamine 3 were dissolved in 2 mL of freshly distilled acetic acid and heated under reflux under stirring at 80 °C. The reaction procedure was followed by TLC and mass spectrometry. After finishing, a brine solution was added, and the mixture was extracted with chloroform several times. The unified organic layer was reduced in volume, and residual acetic acid was removed with toluene. Then methanol or a mixture of diethyl ether and ethanol was added under cooling to give the final product under crystallization. In the case of non-crystallizing, the reaction mixture had to be purified by column chromatography using silica gel and eluent mixtures of cyclohexane and ethyl acetate. The unified compound-containing fractions were evaporated, and again methanol or a mixture of diethyl ether and methanol was added and the purified compound crystallized.One equivalent of the aromatic aldehyde 4). Yield 15%, yellow powder; mp 99–101 °C; 1H NMR (DMSO-d6) δ = 3.54 , 4.80 , 4.92 , 7.29 , 7.37–7.44 , 7.46–7.50 , 7.62 , 7.637.68 , 7.68–7.73 , 7.76 ; m/z (ESI) 522.48 (M+Na+).Dimethyl N-(3-trifluoromethylbenzyl)-4-(3-trifluoromethylphenyl)-1,4-dihydropyridine-3,5-carboxylate (5). Yield 8%, yellow-red powder; mp 104–105 °C; 1H NMR (DMSO-d6) δ = 3.53 , 3.74 , 4.77 , 4.79 , 6.87–6.95 , 7.31 , 7.35 , 7.39–7.45 , 7.46–7.50 , 7.54 ; m/z (ESI) 484.49 (M + Na+).Dimethyl N-(3-methoxybenzyl)-4-(3-trifluoromethylphenyl)-1,4-dihydropyridine-3,5-carboxy-late (6). Yield 15%, yellow crystals; mp 101–103 °C; 1H NMR (DMSO-d6) δ = 3.53 , 3.74 , 4.73 , 4.78 , 6.94 , 7.29 , 7.32 , 7.36–7.50 , 7.52 ; m/z (ESI) 484.52 (M + Na+).Dimethyl N-(4-methoxybenzyl)-4-(3-trifluoromethylphenyl)-1,4-dihydropyridine-3,5-carboxy-late (7). Yield 24%, yellow solid; mp 131–133 °C; 1H NMR (DMSO-d6) δ = 3.53 , 3.73, 3.74 , 4.70 , 4.79 , 6.88 , 6.95 , 7.00 , 7.35 , 7.37-7.44 , 7.43–7.51 , 7.53  514.63 (M + Na+).Dimethyl N--4-(3-trifluoromethylphenyl)-1,4-dihydropyridine-3,5-carboxylate (8). Yield 10%, yellow-orange solid; mp 116–117 °C; 1H NMR (DMSO-d6) δ = 3.53 , 3.74 , 4.77 , 4.79 , 6.87–6.94 , 7.31 , 7.32–7.36 , 7.52 , 7.54 ; m/z (ESI) 484.74 (M + Na+).Dimethyl N-(3-methoxybenzyl)-4-(4-trifluoromethylphenyl)-1,4-dihydropyridine-3,5-carboxylate (9). Yield 26%, yellow-white solid; mp 129–130 °C; 1H NMR (DMSO-d6) δ = 3.52 , 3.75 , 4.72 , 4.77 , 6.96–6.99 , 7.25–7.33 , 7.29 , 7.51 , 7.53 ; m/z (ESI) 461.40 (M+).Dimethyl N-(4-methoxybenzyl)-4-(4-trifluoromethylphenyl)-1,4-dihydropyridine-3,5-carboxylate (10). Yield 22%, yellow-orange powder; mp 131–132 °C; 1H NMR (DMSO-d6) δ = 3.53 , 3.73, 3.74 , 4.70 , 4.79 , 6.88 , 6.97 , 6.99 , 7.00 , 7.31 , 7.51 , 7.54 ; m/z (ESI) 514.49 (M + Na+).Dimethyl N--4-(4-trifluoromethylphenyl)-1,4-dihydropyridine-3,5-carboxylate (11). Yield 22%, yellow powder; mp 99–100 °C; 1H NMR (DMSO-d6) δ = 3.54 , 3.74 , 4.70 , 4.76 , 6.86–6.93 , 6.93–7.01 , 7.18 7.27, 7.29–7.35 , 7.50 ; m/z (ESI) 452.63 (M + Na+).Dimethyl 4--N-(3-methoxybenzyl)-1,4-dihydropyridine-3,5-carboxylate (12). Yield 46%, yellow crystals; mp 129–130 °C; mp 129–130 °C; 1H NMR (DMSO-d6) δ = 3.54 , 3.74 , 4.68 , 4.71 , 6.86–6.70 , 7.17–7.33 , 7.49 ; m/z (ESI) 452.46 (M + Na+).Dimethyl 4--N-(4-methoxybenzyl)-1,4-dihydropyridine-3,5-carboxylate (13). Yield 49%, white-yellow solid; mp 164–165 °C; 1H NMR (DMSO-d6) δ = 3.54 , 3.73, 3.74 , 4.69 , 4.70 , 6.88 , 6.92–7.01 , 7.24 , 7.49 ; m/z (ESI) 460.48 (M + H+).Dimethyl 4--N--1,4-dihydropyridine-3,5-carboxylate (14). Yield 29%, yellow crystals; mp 135–136 °C; 1H NMR (DMSO-d6) δ = 3.54 , 3.73, 3.74 , 4.69 , 4.72 , 6.80 , 6.88 , 6.88–7.00 , 7.16–7.25 , 7.49 ; m/z (ESI) 464.76 (M + H+).Dimethyl N--4-(3-fluorophenyl)-1,4-dihydropyridine-3,5-carboxylate (15). Yield 22%, yellow-orange powder; mp 159–161 °C; 1H NMR (DMSO-d6) δ = 3.53 , 3.73, 3.74 , 4.69 , 4.70 , 6.87 , 6.97 , 6.97 , 6.99 , 7.11 , 7.12 , 7.46 ; m/z (ESI) 464.76 (M + H+).Dimethyl N--4-(4-fluorophenyl)-1,4-dihydropyridine-3,5-carboxylate (16). Yield 49%, yellow solid; mp 155–156 °C; 1H NMR (DMSO-d6) δ = 3.54 , 3.81 , 4.71 , 4.90 , 7.06 , 7.18 , 7.34 , 7.58 , 7.62-7.68 , 7.70–7.77 ; m/z (ESI) 552.54 (M + Na+).Dimethyl 4-(4-methoxy-3-trifluoromethylphenyl)-N-(3-trifluoromethylbenzyl)-1,4-dihydro-pyridine-3,5-carboxylate (17). Yield 45%, yellow-white solid; mp 142–144 °C; 1H NMR (DMSO-d6) δ = 3.54 , 3.74, 3.82 , 4.71 , 4.76 , 6.85-6.95 , 7.08 , 7.26 , 7.28–7.33 , 7.35 , 7.50 ; m/z (ESI) 514.79 (M + Na+).Dimethyl N-(3-methoxybenzyl)-4-(4-methoxy-3-trifluoromethylphenyl)-1,4-dihydropyridine-3,5-carboxylate (18). Yield 41%, yellow-white crystals; mp 152–153 °C; 1H NMR (DMSO-d6) δ = 3.53 , 3.74, 3.81 , 4.68 , 4.71 , 6.94 , 7.07 , 7.22 , 7.28 , 7.32 , 7.49 ; m/z (ESI) 514.76 (M + Na+).Dimethyl N-(4-methoxybenzyl)-4-(4-methoxy-3-trifluoromethylphenyl)-1,4-dihydropyridine-3,5-carboxylate (19). Yield 32%, yellow solid; mp 142–143 °C; 1H NMR (DMSO-d6) δ = 3.54 , 3.82, 3.83, 3.88 , 4.77 , 4.86 , 6.96–7.01 , 7.04 , 7.07 , 7.18 , 7.43 , 7.47 ; m/z (ESI) 544.54 (M + Na+).Dimethyl N--4-(4-methoxy-3-trifluoromethylphenyl)-1,4-dihydro-pyridine-3,5-carboxylate (20). Yield 55%, white-yellow solid; mp 161–163 °C; 1H NMR (DMSO-d6) δ = 3.50 , 3.53, 3.66 , 4.64 , 4.89 , 6.58 , 6.60 , 6.71 , 7.54 , 7.62–7.73 , 7.77 ; m/z (ESI) 514.12 (M + Na+).Dimethyl 4--N-(3-trifluoromethylbenzyl)-1,4-dihydropyridine-3,5-carboxylate (21). Yield 6%, yellow solid; mp 97–98 °C; 1H NMR (DMSO-d6) δ = 3.57 , 4.81 , 4.99 , 5.02, 5.08 , 6.76 , 6.88 , 6.99 , 7.25–7.41 , 7.44–7.49 , 7.42 , 7.62–77.78 ; m/z (ESI) 666.71 (M + Na+).Dimethyl 4--N-(3-trifluoromethylbenzyl)-1,4-dihydropyridine-3,5-carboxylate . The measurement was conducted three times each for inhibitor-treated and untreated cells of both cell lines. The MRP4 50 values were determined from the resulting sigmoid curves.10.000 cells of each cell line were cultured in wells of a 96-well plate at 37 °C under a carbon dioxide atmosphere (5%) for 24 h. Increasing concentrations of the MRP4 substrate 6-mercaptopurine from 0.0001 µM to finally 100 µM were added after the test compound and MK571, each at a concentration of 10 µM, had been added. The plate was incubated for 48 h under the original culture conditions. Then the MTT reagent was added to each well (10 µL of a stock solution of 5 mg/mL in PBS) and incubation continued for 4 h. Then 100 µL DMSO was added to each well to solve the formazan reduction product. The plate was shaken for 30 min on a plate shaker and, finally, the formazan absorption was measured. The described method was repeated three times. The ICMDR is the main problem in anticancer treatment, especially in the case of metastatic cancer that generally cannot be treated as it would afford the effectiveness of various anticancer drugs. As novel anticancer drugs are specific to single cancer drug targets, their use is limited to certain kinds of cancer but not for the treatment of MDR. Moreover, these novel drugs also become affected by the MDR phenomenon. The aim to block the MDR by the use of effective drugs is still a challenge. If a respective tumor expresses a transmembrane efflux pump as the main cause of the MDR phenomenon, the use of an inhibitor will treat the MDR and resensitize the tumor toward the anticancer drug treatment.meta substituted N-benzyl ring residue at the symmetric molecular scaffold. All compounds showed a mainly better activity than the used MRP4 inhibitor MK571. In order to further profile our compound class, the best inhibitor was investigated to restore the MRP4-mediated anticancer drug toxicity in the used MRP4-overexpressing cell line model. The use of that compound almost reached the anticancer drug toxicity determined in the non-MRP4-expressing cell line. Thus our novel compound class is promising for further studies to perspectively combat MRP4-mediated anticancer drug resistance.We developed a novel class of MRP4 inhibitors that was evaluated to block the MRP4 activity toward the fluorescent MRP4 substrate calcein. Certain substituent-dependent effects were observed with best activities for a disubstituted 4-phenyl residue and a