K20, Novel Antifungal Aminoglycoside. Cheng-Wei Tom Chang Department of Chemistry and Biochemistry Utah State University January 29, 2013

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1 K20, ovel Antifungal Aminoglycoside Cheng-Wei Tom Chang Department of Chemistry and Biochemistry Utah State University January 29, 2013

2 Research verview Funding: I, DT, USTAR and companies ( 2 X) n Z Carbohydrates () n ( 2 ) n aminoglycosides Anticancer Antibacterial Antifungal SMA therapeutics bacteria bacteria Small molecule cyclopamine bacteria R 3 atural products C R R R heterocyclic Antibacterial Anticancer Me X R mice cancer cells C. elegans greenhouse 3 C pradimicin phycocyanobilin 3 C 3 C 3 C C 2 C 2 3 C C 3 3 C lignin Antiviral Antifungal C 3 Anti-inflammatory 2 Biofuel field testing

3 R Library of Synthesized Aminoglycosides 2 neomycin analogs with -1 AB (2) ( 2 ) n pyranmycin with -1 AB (5) kanamycin analogs with -1 AB (6) ( 2 ) n 2 R R' 2 neomycin analogs with various R (>30) ( 2 ) n Pyranmycin (>40) ( 2 ) n kanamycin analogs with 3',4'-dideoxygenation (8) ( 2 ) n kanamycin analogs (>40) ( ): number of aminoglycosides synthesized 3

4 istory: Six Years Ago Certain classical aminoglycosides like streptomycin, neomycin B, paromomycin inhibit important crop pathogenic fungal oomycetes 4

5 Research Model Synthesis Production, R&D Field testing FDA registration Marketing IP & Licensing Biology, plant pathology, R&D 5

6 Chemical Modification Can Change the Mode of Action ew method for reviving old drug Turning aminoglycoside from antibacterial to antifungal Kanamycin A Antibacterial but not antifungal III I Antifungal but not antibacterial II 4 FG08 C 3 C

7 FG08, ovel Antifungal Agent FG08 S. aureus E. coli S. cerevisiae Cladosporium KanB Fungi Diseases caused MIC ( µg/ml) Fusarium oxysporium Wilt disease 7.8 Ulocladium sp. uman allergen 7.8 Pythium irregularis Damping off Rhizopus stolonifer Bread mold 31.2 Cladosporium sp Tomato leaf mold Botrytis sp. Soft-rot of vegetables and fruits Fusarium graminearum Fusarium head blight (barley, wheat, etc) 15.6 Candida albicans uman pathogen 15.6 Phoma sp. uman allergen Chang, C.-W. T.; Fosso, M.; Kawasaki, Y.; Shrestha, S.; Bensaci, M. F.; Wang, J.; Evans, C. K.; Takemoto, J. Y. J. Antibiot. 2010, 63,

8 Aminoglycosides: Traditional Aminoglycoside Mode of Action ATP ADP Cell membrane Cell Death rra normal rra truncated DA rra Stage 1 Stage 2 8

9 ow Does FG08 Kill Fungi? Sytox Dye Application of Sytox Dye Fungi Membrane disrupting agents Green fluorescence indicates membrane disruption. 9

10 2 Effect of ctyl (C8) group III I II C 3 C 8 17 C. albicans F. graminearum FG08 Membrane-disrupting 2 daptomycin Antibacterial 10 Cl 2 C 2 syringomycin 2 Antifungal

11 Alkyl Mapping (C8) with FG08 Analog MIC (µg/ml) against Fusarium graminearum R = III I II C 3 C 8 17 (FG08) 7.8 R MIC (µg/ml) C 4 9 (FG01) 250 C (FG02 ) III I II (C8) 125 (C6) 11

12 Impact of Fungal Pathogens The annual cost of controlling black Sigatoka of banana caused by Mycosphaerella fijiensis in Costa Rica: $49 million Barrientos, E. et. al. Corporación Bananera acional, San José, Costa Rica, Economic losses from soybean rust caused by Phakopsora pachyrhizi in USA: $240 million to $2 billion Stokstad, E. Science 2004, 306, The annual economic losses from potato late blight caused by the fungus Phytophthora infestans worldwide: $3 billion McGraw, L. Article from Agriculture Research Service, USDA. Fungal pathogens destroy >125 million tons/yr of the top five food crops (rice, wheat, maize, potatoes and soybeans) that can feed more than 600 million people. Photo from Plant Research International Photo from Wikipedia Photo from Michigan Potato Diseases Fisher, M. C. et. al. ature, 2012, 484, Development of new fungicide: 12 close to priceless

13 From A Teaspoon To A Ton People don t really know what it takes to do a synthesis scale-up and produce material on large scale,.. Ahmed Abdel-Magid, a process chemist at Therachem Research Medilab. Without the ability to make a compound on a large scale, it s essentially no more than a laboratory curiosity and it s not going to be of any great benefit to the public. Jaan Pesti, a principal scientist in process chemistry at Bristol-Myers Squibb C & E ews September 6, 2010, 88,

14 Can FG08 Be Synthesized in Large Quantity? (a) IS, Tf, Et 2 :C 2 Cl 2 (3:1), 3 Ac Ac 3 3 R 4 R Bn C 2 Bn SPh 2a R = C 4 9, R 4 = 2b R = C 8 17, R 4 = 2c R = C 12 25, R 4 = 2d R = C 8 17, R 4 = Bn (b) ame, Me:TF (5:1) Bn Bn 3 3 C 2 R R 4 4a R = C 4 9, R 4 = 4b R = C 8 17, R 4 = 4c R = C 12 25, R 4 = 4d R = C 8 17, R 4 = Bn (a) PMe 3, TF, a, (b) 2, Pd() 2 /C, Ac/ 2 (1/3), (c) Dowex 1X8-200 (Cl - form) C 2 R R 4 FG01 R = C 4 9, R 4 = FG08 R = C 8 17, R 4 = FG02 R = C 12 25, R 4 = FG03 R = C 8 17, R 4 =

15 Kanamycin A Synthesis of FG08 analog Boc 2 Me, 2 Boc (2 nd Generation) Boc Boc Boc B4K C 8 17 S 2 Cl pyridine Synthesized in 3 steps (>80% yield) ver 1 Kg has been produced Boc Boc Boc Boc TFA C 2 Cl 2 S 2 C K S2 C 8 17 John blad 15 John Smitten

16 Minimum Inhibitory Concentration Microorganisms a MIC µg ml -1 FG08 FG03 K20 Bacteria Staphylococcus aureus nd >250 E. coli ATCC Filamentous Fungi Botrytis cinerea Curvularia brachyspora Pythium ultimum Verticillium spp nd Microdochium nivale 3.9 nd Fusarium graminearum Rhizopus stolonifer 31.3 b nd 62.5 Cladosporium cladosporioides 31.3 nd nd Fusarium oxysporum 7.8 nd nd Ulocladium spp. 7.8 nd nd Phoma spp nd nd Yeasts Candida albicans Rhodotorula pilliminae

17 Cytotoxicity against C cells (human melanoma cells) Percent Toxicity Triton X-100 FG08 FG03 K20 MTT assay 3-(4,5-dimethylthiazol-2- yl)-2,5- diphenyltetrazolium bromide (MTT) µg ml -1 In collaboration with Kawasaki, Y.; Shrestha, S; Takemoto, J. Y. 17

18 Cytotoxicity against I3T3 cells (mouse fibroblast) Percent Toxicity Triton X-100 FG08 FG03 K20 MTT assay 3-(4,5-dimethylthiazol-2- yl)-2,5- diphenyltetrazolium bromide (MTT) µg ml -1 In collaboration with Kawasaki, Y.; Shrestha, S; Takemoto, J. Y. 18

19 ovel Fungicide: K20 Specific against fungi US and international patent pending Fungi Low toxicity toward mammal cancer cells Effective in greenhouse and field greenhouse mice turf grass 19 untreated treated

20 K20 Is Active Against Cryptococcus neoformans In collaboration with Prof. icole Meissner, Montana State University 20

21 The Ergosterol Biosynthesis squalene-epoxidase epoxisqualene-cyclase C-14-demethylase Azoles (triazoles, diazoles and thiazoles) Azoles inhibit demethylation Sterol Biosynthesis Inhibitors or SBI Fungicides ergosterol a critical component of fungal cell membranes

22 Acknowledgement Collaborators Prof. Jon Takemoto, Utah State University Prof. icole Meissner, Montana State University Financial Support Graduate Jinhua Wang Marina Fusso Yukie Kawasaki Sanjib Shrestha Mekki F. Bensaci Intellectual Property Services Berry Treat Joe Christison David Clark 22