CHA KUIN TAITEIT US009770431B2VAN A TANO UN TRAT TO (12 ) United States Patent ( 10 ) Patent No. : US 9 ,770 ,431 B2 Reddell et al. (45 ) Date of Patent: Sep . 26 , 2017 ( 54 ) TIGLIEN - 3 -ONE DERIVATIVES OTHER PUBLICATIONS @(71 ) Applicant: QBiotics Limited , Yungaburra , Mackenzie ; Expert Review of Anti- infective Therapy ; 11( 6 ) , 539 541, 2013 . * Queensland ( AU ) Silver ; Clinical Microbiology Reviews, Jan . 2011 , 71 - 109 . * Malcolm ; Current Opinion in Pharmacology ; 2014 , 18 ;91 -97 . * @(72 ) Inventors: Paul Warren Reddell , Yungaburra Brynes et al. , “ Plasminogen Activator Induction and Platelet Aggre (AU ) ; Victoria Anne Gordon , gation by Phorbol and Some of Its Derivatives : Correlation with Yungaburra ( AU ) Skin Irritancy and Tumor- promoting Activity , ” J. Cancer Res . Clin . Oncology 97 : 257 - 266 , 1980 . Assignee : QBiotics Limited , Yungaburra (AU ) CA Abstract No . 101 : 147806 , taken from Adolf et al. , “ On the active ( 73 ) principles of the spurge family , X . Skin irritants , cocarcinogens, and @ cryptic cocarcinogens from the latex of the manchineel tree , ” ( * ) Notice : Subject to any disclaimer , the term of this Journal of Natural Products 47 ( 3 ) : 482 - 496 , 1984 . ( 2 pages ) . patent is extended or adjusted under 35 CA Abstract No . 115 :273076 , taken from Roeser et al ., U . S . C . 154 (b ) by 0 days. “ Toxicokinetics of tumor promoters of mouse skin . II . Metabolism of the tumor promoter 12 - 0 - tetradecanoylphorbol 13 - acetate in @(21 ) Appl. No. : 15 /041 , 960 mouse skin and biological activities of metabolites , " Carcinogenesis 12 ( 9 ) : 1563 - 1570 , 1991 . ( 2 pages ) . CA Abstract No . 69 : 10570 , taken from Crombie et al ., " Chemistry ( 22 ) Filed : Feb . 11 , 2016 and structure of phorbol, the diterpene parent of the cocarcinogens of croton oil, ” Journal of the Chemical Society C : Organic 11 :1347 (65 ) Prior Publication Data 1362 , 1968 . ( 1 page ) . US 2016 /0158187 A1 Jun . 9 , 2016 CA Abstract No . 70 :47622 , taken from Schairer et al. , " Chemistry of phorbol . IV . Polybenzoates and polyacertates of phorbol and Related U . S . Application Data 3 - phorbolol and functional derivatives of the allyl group of phorbol, ” Zeitschrift fuer Naturforschung , Teil B : Anorganische (62 ) Division of application No . 14 /084 ,949 , filed on Nov. Chemi, Organische Chemie , Biochemie , Biophysik , Biologie 20 , 2013 , now Pat . No . 9 , 289, 410 , which is a division 23 ( 11 ) : 1430 - 1443 , 1968 . ( 2 pages ) . of application No . 12 / 158 , 461, filed as application CA Abstract No. 88 :37991 , taken from Snatzke et al. , " Chemistry of No . PCT /AU2006 / 002001 on Dec . 22 , 2006 , now Pat . phorbol. XIX . On circular dichroism . LXX . Chiroptical properties No. 8 ,598 ,229 . of phorbol - 12, 13 , 20 - triacetate and some other phorbol derivatives, " Israel Journal of Chemistry 15 ( 1 - 2 ): 46 -56 , 1977 . ( 4 pages ). Foreign Application Priority Data CA Abstract No. 89 : 192165 , taken from Brune et al . , “ Inflamma ( 30 ) tory , tumor initiating and promoting activities of polycyclic aro matic hydrocarbons and diterpene esters in mouse skin as compared Dec . 23 , 2005 (AU ) .. 2005907278 with their prostaglandin releasing potency in vitro , ” Cancer Letters 4 (6 ) : 333 - 342 , 1978 . ( 2 pages ) . (51 ) Int. Ci. CA Abstract No . 89 : 3208 , taken from Ogura et al. , “ Potential A61K 31 / 352 ( 2006 .01 ) anticancer agents . VIII . Constituents of Baliospermum montanum A61K 31/ 336 ( 2006 . 01 ) ( Euphorbiaceae ) ,” Planta Medica 33 ( 2 ) : 128 - 143 , 1978 . ( 2 pages ) . AOIN 43 / 20 ( 2006 . 01 ) CA Abstract No. 92: 141462 , taken from Freeman et al. , “ The AOIN 43 /90 ( 2006 .01 ) constituents of Australian Pimelea species I. The isolation and AOIN 45 / 00 ( 2006 . 01 ) structure of the toxin of Pimelea simplex and P . trichostachya Form C07D 301 /32 ( 2006 . 01 ) B responsible for St. George disease of cattle ," Australian Journal C07D 303 / 14 ( 2006 .01 ) of Chemistry 32 ( 11 ) : 2495 - 2506 , 1979 . ( 1 page ) . CA Abstract No . 93 : 232311 , taken from Goerttler et al. , " Two - stage C07D 303 /32 ( 2006 .01 ) carcinogenesis in NMRI mice : intravaginal application of 7 , 12 CO7D 493 /04 ( 2006 . 01 ) dimethylbenz [ a ] anthracene as initiator followed by the phorbol AOIN 53 / 00 ( 2006 .01 ) ester 12 - 0 - tetradecanoylphorobol - 13 -acetate as promoter, " CO7D 303 / 44 ( 2006 . 01 ) Carcinogenesis 1 ( 8 ) : 707 -713 , 1980 . ( 2 pages ) . CO7D 303 / 17 ( 2006 . 01) CA Abstract No. 97 : 176609, taken from Zayed et al. , “ On the active (52 ) U . S . CI. principles of the Thymelaeaceae . I . The irritants and cocarcinogens CPC . .. .. .. .. A61K 31/ 336 (2013 .01 ) ; AOIN 43 /20 of Pimelea prostrata , ” Planta Medica 45 ( 2 ) : 67- 77 , 1982 . ( 2 pages ) . ( 2013 .01 ) ; A01N 43 / 90 ( 2013 .01 ) ; AOIN (Continued ) 45 /00 (2013 . 01 ) ; A01N 53 / 00 ( 2013 .01 ) ; CO7D 301 /32 (2013 .01 ) ; C07D 303 / 14 Primary Examiner — Pancham Bakshi ( 2013 .01 ) ; C07D 303 / 17 ( 2013 . 01) ; C07D ( 74 ) Attorney , Agent , or Firm — Seed IP Law Group LLP 303 /32 ( 2013 .01 ) ; C07D 303 /44 ( 2013 .01 ) ; C07D 493/ 04 ( 2013 .01 ) (57 ) ABSTRACT (58 ) Field of Classification Search The present invention relates to tiglien - 3 - one compounds USPC .. 514 /229 , 530 , 475, 715 and their use in methods of treating or preventing protozoal See application file for complete search history . infections, bacterial infections, parasitic infections and cell ( 5656 ) References Cited proliferative disorders . The tiglien - 3 -one compounds are also used in methods of controlling pests in humans , ani U . S . PATENT DOCUMENTS mals , plants and the environment. 6 ,268 ,395 B1 * 7 / 2001 Hattori .. .. .. A61K 31 /215 514 /530 16 Claims, 7 Drawing Sheets US 9 , 770 ,431 B2 Page 2 ( 56 ) References Cited OTHER PUBLICATIONS Challacombe et al. , “ Neutrophils Are a Key Component of the Antitumor Efficacy of Topical Chemotherapy with Ingenol - 3 Angelate , " The Journal of Immunology 177 ( 11 ) : 8123 -8132 , Dec . 2006 . Cozzi et al. , “ Induction of Senescence in Diterpene Ester - Treated Melanoma Cells via Protein Kinase C - Dependent Hyperactivation of the Mitogen -Activated Protein Kinase Pathway, ” Cancer Research 66 ( 20 ) : 10083 - 10091 , Oct. 2006 . Hafez et al. , “ Active Principles of the Thymelaeaceae, ” Planta Medica 49 : 3 - 8 , 1983 . Hampson et al. , “ PEP005 , a selective small -molecule activator of protein kinase C , has potent antileukemic activity mediated via the delta isoform of PKC , ” Blood 106 ( 4 ) : 1362- 1368, Aug . 2005 . Tyler et al ., " Synthesis and Structure -Activity Studies of Potential Antitumour Agents Derived from Esters of Phorbol, ” Australian Journal of Chemistry 40 ( 1 ) : 193 - 200 , 2002 . * cited by examiner U . S . Patent Sep . 26 , 2017 Sheet 1 of 7 US 9 , 770 , 431 B2 BiomassBiomass Methanol Extraction Extract 1 Extract 2 HPLC HPLC Rotary - Evaporation Aqueous Concentrate FIG . 1 U . S . Patent Sep . 26 , 2017 Sheet 2 of 7 US 9 , 770 , 431 B2 Aqueous Concentrate Partition EtOAC1 H201 HPLC HPLC ETOAC2 H202 HPLC HPLC EtOAC3 H203 ThereHPLC HPLC FIG . 2A atent Sep . 26 , 2017 Sheet 3 of 7 US 9 , 770 , 431 B2 EtOAC Residue 10 : 9 : 1 Hex :MeOH :Water Hexane Methanol: Water HPLC HPLC FIG . 2B U . S . Patent Sep . 26 , 2017 Sheet 4 of 7 US 9 ,770 ,431 B2 Active C18che Fractionsera cons Preparative HPLC 100 Fractions analytical HPLC Pool related fractions Pool 1 Pool 2 Pool 3 Pool 4 Pool N weigh , assay, HPLC?? weigh , assay, HPLC? weigh , assay, HPLC? -weigh , assay , HPLC? weigh , assay , HPLC FIG . 3 U . S . Patent Sep . 26 , 2017 Sheet 5 of 7 US 9 , 770 , 431 B2 180 .00 - - - - B16 - - MM96 160. 00 * * * * * * * * * * * VAN nesmenas -- - * NFF 140. 00 RENSA www -- - -- MCF7 C180 1 100 .00 WA wwwwm ann * * * 35 CellGrowth(%control) mwenen sunDU145 80 .00 wwwwwwwwwwwwwwwwwwwwwwww w - -- - K562 425 * * * 60. 00 ASSSS * * * * * 141 www bmw HL60 wwerraneanfermercaderienciaerrorriceperetetrelercorrederier KA 40 .00 20 .00 * ** * * * * * * * * * * * * * * * * * * manu * * * * * * * * * * * * * * w wwwwwwwwwwww wwwwwwwwwwwwwwwwwwwwwwwwww www * * * * ** * * ** 0 .00 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * r enews * 0 .01 0 . 1 - 20 . 00 EBI- 46 Concentration (ug /ml ) wwwwwwwwwwwwwwwwwwwwwwwwwwwwwww FIG . 4 užg Tumorvolume(cubicmm) Controls ãö EB1- 46 ? 15 ? Days after injecting tumor cells FIG . 5 atent Sep . 26 , 2017 Sheet 6 of 7 US 9 , 770 , 431 B2 ão?ñã?eo Tumorvolume(cubicmm) - Control * EBI- 46 10 15 20 25 30 Days after tumor cell injection FIG . 6 : e Controls sa EBI46 low dose ? - EB146 high dose Tumorvol.(cubicmm) § § § 1 51051 Days after injecting tumor cells FIG . 7 atent Sep . 26 , 2017 Sheet 7 of 7 US 9 , 770 , 431 B2 Tumorvolume(cubicmm) Control EB548 - 29 oog + EB?- 46 0 5 10 15 20 25 30 Days after tumor cell injection FIG . 8 US 9 ,770 ,431 B2 TIGLIEN - 3 -ONE DERIVATIVES one or more of R ! (or R ? ) is connected to R3 ( or R4) , and / or R3 ( or R4 ) is connected to RS ( or R ' ), and / or RS ( or FIELD OF THE INVENTION R ) is connected to R², and/ or R7 is connected to R8 ( or R ) , and /or R8 (or R ) is connected to R10 ( or Rll ) , and / or R10 ( or This invention relates to bioactive molecules. More par- 5 Ril ) is connected to R12 , and /or R12 is connected to R13 ( or ticularly , this invention relates to tiglien - 3 -one derivatives of R14 ), and / or R13 connected to R14 , and / or R13 ( or R14 ) is potential therapeutic benefit