K20, Novel Antifungal Aminoglycoside

Cheng-Wei Tom Chang

Department of Chemistry and Biochemistry

Utah State University

January 29, 2013

2

O

N

HO

H

HH

H

H

NN

NO

(HO)n

O

(H2N)n

ONH2

HOHO H2N

O NH2

NH2HO

O

Research Overview

Anticancer

Funding: NIH, DOT, USTAR and companies

O

(H2X)n Z

SMA therapeutics

O

O

Carbohydrates

Small molecule

R

O

O

NN

NR

N

O

O

RNH

O

O

R

O

O

NN

NMe

X

Antibacterial Anticancer

Natural products

NH

H3C

NH

N

H3CCH3

NH

O

H3CH3C

CO2HCO2H H3C

O

Anti-inflammatory Antiviral Antifungal Biofuel

bacteria

cancer cells

bacteria bacteria

mice

C. elegans

field testing

greenhouse

aminoglycosides

RN3

cyclopamine

heterocyclic

OOH

H3CO

O NH

HOO

O

OH

OHO

HONHCH3

O

COOH

O

OH

HOHO

lignin pradimicin phycocyanobilin

Antibacterial Antifungal

3

Library of Synthesized Aminoglycosides

OHO

O OHR

O

NH2

HOHO

H2NNH2

OOH

HNO

OH

ONH2

neomycin analogs with N-1 AHB (2)

O

NH2

HOHO

H2NNH2

OOH

HNO

OH

ONH2

O(H2N)n

pyranmycin with N-1 AHB (5)

O

NH2

HOHO

H2NNH2

HOO

HNO

OH

ONH2

O (NH2)n

OR'

O OHR

O

NH2

HOHO

H2NNH2

OOH

NH2O

O

NH2

HOHO

H2NNH2

OOH

NH2OO(H2N)n

O

NH2

H2NNH2

HOO

NH2O

O (NH2)n

O

NH2

HOHO

H2NNH2

HOO

NH2O

O (NH2)n

kanamycin analogs with N-1 AHB (6)

neomycin analogs with various R’ (>30)

Pyranmycin (>40) kanamycin analogs with 3',4'-dideoxygenation (8)

kanamycin analogs (>40)

( ): number of aminoglycosides synthesized

4

History: Six Years Ago

“Certain classical aminoglycosides like streptomycin, neomycin B, paromomycin inhibit important crop pathogenic fungal oomycetes…”

5

Research Model

Synthesis Production, R&D

Field testing FDA registration Marketing

IP & Licensing

Biology, plant pathology, R&D

6

Chemical Modification Can Change the Mode of Action

Turning aminoglycoside from antibacterial to antifungal

New method for reviving old drug

ONH2

HOHO

HOO NH2

NH2O

HO

O

H2NHO

OHOH

Kanamycin A

ONH2

HOHO

H2N O NH2

NH2O

HO

O

HOHO O

CH3

C8H17

FG08

III

III

3” 4” 6” 4”

Antibacterial but not antifungal

Antifungal but not antibacterial

7

FG08, Novel Antifungal Agent S. aureus E. coli S. cerevisiae

FG08

KanB

Cladosporium

Fungi Diseases caused MIC ( µg/mL)

Fusarium oxysporium Wilt disease 7.8

Ulocladium sp. Human allergen 7.8

Pythium irregularis Damping off 15.12

Rhizopus stolonifer Bread mold 31.2

Cladosporium sp Tomato leaf mold 31.25

Botrytis sp. Soft-rot of vegetables and fruits 31.25

Fusarium graminearum Fusarium head blight (barley, wheat, etc) 15.6

Candida albicans Human pathogen 15.6

Phoma sp. Human allergen 31.25 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, 667-672.

8

Aminoglycosides: Traditional Mode of Action

ATP

rRNA

Aminoglycoside

Stage 1 Stage 2

Cell Death

rRNA DNA rRNA

normal

truncated

Cell membrane

ADP

9

How Does FG08 Kill Fungi? Application of Sytox Dye

Fungi

Sytox Dye

Membrane disrupting agents

Green fluorescence indicates membrane disruption.

10

Effect of Octyl (C8) group

ONH2

HOHO

H2N O NH2

NH2O

HO

O

HOHO O

CH3

C8H17

FG08

III

III

F. graminearum

C. albicans

Membrane-disrupting

HN

OO

OO

HN

HN

O

ClOH

ONH

OH

HN

NH

HOO

NH2OHHO2C O

O

HN

OHN

NH

HN

O

NH2

NH2

NHHN

NH

HN

NH

HN

HN

NH

O

O

O

O

O

O

OH

OH

O

OO

O

NH

O

NH

NH

OHN

O

OH

O

NH

H

NHO

OH

O

O

NHNH

OHO

O

HN

O

OHO

H

daptomycin syringomycin Antibacterial Antifungal

11

Alkyl Mapping (C8) with FG08 Analog

ONH2

HOHO

H2N O NH2

NH2O

HO

O

HOHO O

CH3

R

III

III

C8H17 (FG08) 7.8

OH

ONH2

HOHO

H2N O NH2

NH2O

HO

O

HOHO OH

III

III

MIC (µg/mL) against Fusarium graminearum

R = MIC (µg/mL)

C4H9 (FG01) ≥ 250 C12H25 (FG02 ) ≥ 250

≥ 500

≥ 500

≥ 500

20 62.5

31.3 (C8) 125 (C6)

12

Impact of Fungal Pathogens

Development of new fungicide:

The annual cost of controlling black Sigatoka of banana caused by Mycosphaerella fijiensis in Costa Rica: $49 million

Economic losses from soybean rust caused by Phakopsora pachyrhizi in USA: $240 million to $2 billion

The annual economic losses from potato late blight caused by the fungus Phytophthora infestans worldwide: $3 billion

Barrientos, E. et. al. Corporación Bananera Nacional, San José, Costa Rica, 1995.

Stokstad, E. Science 2004, 306, 1672-1673.

McGraw, L. Article from Agriculture Research Service, USDA.

close to priceless

Photo from Plant Research International

Photo from Wikipedia

Photo from Michigan Potato Diseases 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.

Fisher, M. C. et. al. Nature, 2012, 484, 186-194.

http://www.pri.wur.nl/NR/rdonlyres/B4153B95-3D8D-44B7-BB46-C108E02B9DC6/64353/DSC00125602.jpghttp://en.wikipedia.org/wiki/File:Soybean_rust.jpg

“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 & EN News September 6, 2010, 88, 17-28.

“From A Teaspoon To A Ton”

14

Can FG08 Be Synthesized in Large Quantity?

OCH2

BnORO

OBn

SPh

(a) NIS, TfOH, Et2O:CH2Cl2 (3:1),

(b) NaOMe, MeOH:THF (5:1).

ON3

AcOAcO N3 O N3

N3HO

HO

(a) PMe3, THF, NaOH, (b) H2, Pd(OH)2/C, HOAc/H2O (1/3), (c) Dowex 1X8-200 (Cl- form).

ONH2

HOHO H2N

O NH2

NH2HO

O

O

ORHOHO CH2

ON3

HOHO N3 O N3

N3HO

O

O

ORBnOBnO

CH22a R = C4H9, R4 = H

3

2b R = C8H17, R4 = H2c R = C12H25, R4 = H

R4

R4

2d R = C8H17, R4 = OBn4a R = C4H9, R4 = H4b R = C8H17, R4 = H4c R = C12H25, R4 = H4d R = C8H17, R4 = OBn

FG01 R = C4H9, R4 = HFG08 R = C8H17, R4 = HFG02 R = C12H25, R4 = HFG03 R = C8H17, R4 = OH

R4

15

Synthesis of FG08 analog (2nd Generation)

K20

Synthesized in 3 steps (>80% yield) Over 1 Kg has been produced

Kanamycin A

ONH

HOHO

O NHHN

HO

O

O

OHHNHO

HO

OH

Boc

Boc

Boc

Boc

C8H17SO2Clpyridine

ONH

HOHO

O NHHN

HO

O

O

OHHNHO

HO

O

Boc

Boc

Boc

BocSO2C8H17

TFACH2Cl2

ONH3

HOHO

O NH3NH3

HO

O

O

OHH3NHO

HO

OSO2C8H17

B4K

ONH2

HOHO

O NH2NH2

HO

O

O

OHH2NHO

HO

OH

Boc2OMeOH, H2O

John Oblad John Smitten

16

Microorganisms aMIC µg mL-1

FG08 FG03 K20 Bacteria

Staphylococcus aureus 25923 62.5 nd >250 E. coli ATCC25922 250 500 250

Filamentous Fungi Botrytis cinerea 31.3 31.3 31.3 Curvularia brachyspora 31.3 31.3 62.5 Pythium ultimum 15.6 31.3 62.5 Verticillium spp. 15.2 15.2 nd Microdochium nivale 3.9 nd 3.9-7.8 Fusarium graminearum 7.8 7.8 7.8 Rhizopus stolonifer 31.3 bnd 62.5 Cladosporium cladosporioides 31.3 nd nd Fusarium oxysporum 7.8 nd nd Ulocladium spp. 7.8 nd nd Phoma spp. 31.3 nd nd

Yeasts Candida albicans 31.3 62.5 31.3 Rhodotorula pilliminae 7.8 62.5 31.3

Minimum Inhibitory Concentration

17

Cytotoxicity against C8161.9 cells

0

20

40

60

80

100

10 20 50 100 250

Pe

rce

nt T

ox

icit

y

µg mL-1

Triton X-100

FG08

FG03

K20

(human melanoma cells)

MTT assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

In collaboration with Kawasaki, Y.; Shrestha, S; Takemoto, J. Y.

18

Cytotoxicity against NIH3T3 cells

-10

10

30

50

70

90

10 20 50 100 250

Per

cen

t To

xici

ty

µg mL-1

Triton X-100

FG08

FG03

K20

(mouse fibroblast)

In collaboration with Kawasaki, Y.; Shrestha, S; Takemoto, J. Y.

MTT assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

19

Novel Fungicide: K20

Specific against fungi

Low toxicity toward mammal Fungi

mice

cancer cells

Effective in greenhouse and field greenhouse

turf grass

US and international patent pending

treated untreated

20

K20 Is Active Against Cryptococcus neoformans

In collaboration with Prof. Nicole Meissner, Montana State University

ergosterol

C-14-demethylase

squalene-epoxidase

epoxisqualene-cyclase

The Ergosterol Biosynthesis

Azoles (triazoles, diazoles and thiazoles)

HO

HO

HO

a critical component of fungal cell membranes

Azoles inhibit demethylation Sterol Biosynthesis Inhibitors or SBI Fungicides

22

Acknowledgement

Graduate Collaborators Prof. Jon Takemoto, Utah State University Prof. Nicole Meissner, Montana State University

Jinhua Wang Marina Fusso Yukie Kawasaki Sanjib Shrestha Mekki F. Bensaci

Intellectual Property Services

Berry Treat Joe Christison David Clark

Financial Support

K20, Novel Antifungal AminoglycosideSlide Number 2Library of Synthesized AminoglycosidesSlide Number 4Research Model Chemical Modification Can Change the Mode of ActionFG08, Novel Antifungal AgentSlide Number 8Slide Number 9Effect of Octyl (C8) groupAlkyl Mapping (C8) with FG08 AnalogImpact of Fungal Pathogens“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. Can FG08 Be Synthesized in Large Quantity?Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Acknowledgement