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1 2 ) United

S t a t e s Patent

Kawagishi

t a ] .

USOO8809328B2

US 8 , 8 0 9 , 3 2 8 B2

A u g .

1 9 , 2 0 1 4

1 0 ) Patent

N 0 . :

4 5 ) Date

o f

P a t e n t :

( 5 4 ) IMIDAZOLE DERIVATIVE

7 5 ) I n v e n t o r s :

H i r o k a z u

K a w a g i s h i , S h i z u o k a

J P ) ;

J a e - H o o n

C h o i , S h i z u o k a

J P )

7 3 ) A s s i g n e e : N a t i o n a l

U n i v e r s i t y

C o r p o r a t i o n

S h i z u o k a U n i v e r s i t y J P )

*

) N o t i c e : S u b j e c t t o a n y

d i s c l a i m e r ,

t h e t e r m o f h i s

p a t e n t

i s e x t e n d e d

o r a d j u s t e d

u n d e r 3 5

U . S . C .

1 5 4 ( b )

b y 0 d a y s .

2 1 ) A p p l . N 0 . : 1 4 / 1 1 3 , 7 3 0

2 2 ) PCT i l e d :

A p r . 2 4 , 2 0 1 2

( 8 6 ) PCT

N o . :

PCT/JP2012/060989

§

3 7 1

0 X 1 ) ’

2 ) , 4 ) D a t e : D e c .

1 9 ,

2 0 1 3

( 8 7 ) PCT

P u b .

N o . : WO2012/147750

PCT

P u b .

D a t e :

N o v . 1 , 2012

( 6 5 ) P r i o r P u b l i c a t i o n

Data

US 2 0 1 4 / 0 1 4 8 5 9 8

A1 May 2 9 , 2 0 1 4

3 0 ) F o r e i g n A p p l i c a t i o n P r i o r i t y

D a t a

Apr.

2 7 ,

201 1 ( J P ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2011-099456

Jul. 13,2011

JP) . . . . . . . .

.. 2011-154981

Feb. 28, 2 0 1 2 (JP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 0 12-0417 11

( 5 1 )

I n t . C l .

A 6 1 K 1 / 5 3 5

( 2 0 0 6 . 0 1 )

C07D 4 8

7 / 0 0

( 2 0 0 6 . 0 1 )

( 5 2 )

US.

C l .

USPC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514/230.5; 544/184

( 5 8 )

F i e l d

o f C l a s s i ? c a t io n S e a r c h

USPC

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544/184; 514/230.5

S e e

a p p l i c a t i on ? l e f o r

c o m p l e t e

s e a r c h

h i s t o r y .

( 5 6 )

R e f e r e n c e s C i t e d

FOREIGN PATENT

DOCUMENTS

JP 63-068570 3/1988

JP 63-104965 5 / 1 9 8 8

JP 04-210680 7/1992

JP

2009-001558

1 / 2 0 0 9

JP

4565018

8 / 2 0 1 0

W0 WO 011/010695

1 / 2 0 1 1

OTHER PUBLICATIONS

PCT/IB/ 3 8

N o t i ? c a t i o n o f

T r a n s m i t t a l

o f

C o p i e s o f T r a n s l a t i o n o f

t h e I n t e r n a t i o n a l P r e l i m i n a r y R e p o r t

o n

P a t e n t a b i l i t y

i n c l u d i n g PCT/

I B / 3 7 3 a n d PCT

ISN237

i n c o u n t e r p a r t

WO

a t e n t

A p p l i c a t i o n

N o .

P C T / 2 0 1 2 / 0 6 0 9 8 9 ) i n

E n g l i s h d a t e d N o v . 2 8 ,

2 0 1 3

5 p a g e s ) .

I n t e r n a t i o n a l

S e a r c h R e p o r t

m a i l e d J u n . 1 2 , 2 0 1 2 i n

c o u n t e r p a r t

A p p l i c a t i o n N o . P C T / J P 2 0 1 2 / 0 6 0 9 8 9 .

I v a n o v i c s , G e o r g e

A . ,

e t a l . , “ T h e

S y n t h e s i s

o f 2 - s u b s t i t u t e d d e r i v a

t i v e s

o f

5 - a m i n 0 - 1 - .

b e t a .

- D - r i b 0 f u r a n 0 s y l - i m i d a z o l e - 4

c a r b o x a m i d e . R i n g

o p e n i n g

e a c t i o n s

o f

- a z a p u r i n e

n u c l e o s i d e s ” ,

J .

O r g .

C h e m . ,

v 0 1 . 3 9 , 1 9 7 4 , p . 3 6 5 1 - p . 3 6 5 - 4 .

E l l i o t

Shaw

t a 1 . “ I m i d a z o - 1 , 2 , 3 - T r i a Z i n e s As S u b s t r a t e s

and

n h i b i

t o r s f o r X a n t h i n e O X i d a s e ” ,

J .

B i o l .

Chem. o l . 1 9 4 ,

1 9 5 2 , p . 6 4 1 - p .

6 5 4 .

Primary Examiner

*

e f f r e y H Murray

Assistant

Examiner luwafemi Masha

( 7 4 ) A t t o r n e y , A g e n t ,

o r

Firm *

s t r o l e n k Faber LLP

5 7 )

ABSTRACT

An b j e c t o f t h e p r e s e n t

i n v e n t i o n

i s t o p r o v i d e a compound

t h a t

c a n r e g u l a t e p l a n t g r o w t h . T h e

compound

s e l e c t e d f r o m

t h e g r o u p c o n s i s t i n g o f

( A )

a n d

B ) :

A )

3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i a Z i n - 4 , 6 5 H , 7 H ) - d i o n e ;

a n d

B )

3 - m e t h y l - 3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i a Z i n - 4 , 6 5 H , 7 H )

d i o n e ;

h a s a p l a n t g r o w t h r e g u l a t i n g

a c t i o n .

4 C l a i m s , 8

Drawing

S h e e t s

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F i g . 3

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o v mm mm mNo wm ?w ?tm ?m ?3m ?m ?;o w

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m m ?

_ = _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _

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w

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o wm

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0 0 ?

a :

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w ?

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o ?

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8 , 8 0 9 , 3 2 8

B2

1

IMIDAZOLE

DERIVATIVE

CROSS-REFERENCE TO RELATED

APPLICATIONS

The p r e s e n t a p p l i c a t i o n i s

a

3 5 U . S . C . § § 3 7 1

n a t i o n a l

p h a s e

c o n v e r s i o n

o f

P C T / J P 2 0 1 2 / 0 6 0 9 8 9 ,

? l e d A p r .

2 4 ,

2 0 1 2 , w h i c h

c l a i m s

p r i o r i t y t o J a p a n e s e P a t e n t A p p l i c a t i o n

N o s .

2 0 1 1 - 0 9 9 4 5 6 , ? l e d A p r . 2 7 , 2 0 1 1 , 2 0 1 1 - 1 5 4 9 8 1 , ? l e d

J u l .

1 3 , 2 0 1 1 ,

a n d 2 0 1 2 - 0 4 1 7 1 1 ,

? l e d

F e b .

2 8 , 2 0 1 2 , t h e

c o n t e n t s

o f l l o f

which

a r e i n c o r p o r a t e d h e r e i n

by

e f e r e n c e .

The PCT n t e r n a t i o n a l


was

u b l i s h e d

i n t h e a p a

n e s e

l a n g u a g e .

TECHNICAL FIELD

The p r e s e n t i n v e n t i o n r e l a t e s t o an i m i d a z o l e d e r i v a t i v e .

BACKGROUND

ART

A s c o m p o u n d s t o r e g u l a t e p l a n t g r o w t h , f o r e x a m p l e ,

p h y

tohormones a r e known. Phytohormones a r e

d e r i v e d

from

p l a n t s t h e m s e l v e s , b u t p h y t o h o r m o n e s a l s o c a n

b e

s y n t h e

s i z e d a s compounds and r e

u t i l i z e d a s a g r i c u l t u r a l

c h e m i c a l s

a n d v i t a l i z i n g

a g e n t s . As

s u c h

c o m p o u n d s t o

r e g u l a t e

p l a n t

g r o w t h ,

i m i d a z o l e d e r i v a t i v e s

a r e d e s c r i b e d

i n P a t e n t

L i t e r a

t u r e s 1

to

4 .

F o r

e x a m p l e ,

i n P a t e n t L i t e r a t u r e 4 , 7H-imidazo

[ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i a z i n - 4 3 H ) - o n e ( a n o t h e r

n a m e : 2 - a z a h y p o x

a n t h i n e , h e r e i n a f t e r , s o m e t i m e s r e f e r r e d t o a s “AHX”) i s

d e s c r i b e d , a n d t

i s

a l s o d e s c r i b e d t h a t

AHX

x h i b i t s g r o w t h

p r o m o t i o n o r g r o w t h s u p p r e s s i o n a c t i o n o n p l a n t s .

CITATION LIST

P a t e n t L i t e r a t u r e

P a t e n t

L i t e r a t u r e

1 : J a p a n e s e P a t e n t A p p l i c a t i o n

L a i d - O p e n

No. 63-104965

P a t e n t L i t e r a t u r e 2 : J a p a n e s e P a t e n t


L a i d - O p e n

No.

63-68570

P a t e n t

L i t e r a t u r e 3 :

J a p a n e s e P a t e n t A p p l i c a t i o n

L a i d - O p e n

No.

4-210680

P a t e n t L i t e r a t u r e 4 :

J a p a n e s e

P a t e n t No.

4565018

SUMMARY

OF

INVENTION

T e c h n i c a l Problem

A

compound,

a s

AH X ,

t h a t c a n r e g u l a t e p l a n t g r o w t h

a s

well

a s can be used a s an a g r i c u l t u r a l chemical o r v i t a l i z i n g

a g e n t

f o r a g r i c u l t u r e

a n d g a r d e n i n g

i s

r e q u i r e d .

T h u s , a n

o b j e c t o f h e p r e s e n t i n v e n t i o n s t o

p r o v i d e

a n o v e l

compound

t h a t

c a n

r e g u l a t e p l a n t g r o w t h .

S o l u t i o n

t o

Problem

I n t h e

c o u r s e o f AHX t u d y ,

t h e p r e s e n t

i n v e n t o r s h a v e

f o u n d a n

AHX e t a b o l i t e ,

3 H - i m i d a z o [ 4 , 5 - d ] 1

, 2 , 3 ] t r i a z i n

4 , 6 ( 5 H , 7 H ) - d i o n e

( a n o t h e r

n a m e : 2 - a z a - 8 - o x o h y p o x a n t h i n e ,

h e r e i n a f t e r , sometimes

r e f e r r e d t o

a s

“AOH”), t h a t

h a s a

p l a n t

g r o w t h

r e g u l a t i n g a c t i o n

l i k e AHX. A d d i t i o n a l l y ,

t h e i n v e n

t o r s

s y n t h e s i z e d 3 - m e t h y l - 3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i a z i n

4 , 6 ( 5 H , 7 H ) - d i o n e ( h e r e i n a f t e r ,

s o m e t i m e s r e f e r r e d t o a s

“ 3 - m e t h y l - A O H ” ) f r o m AOH n d h a v e

f o u n d t h a t

3 - m e t h y l

AOH l s o h a s a p l a n t g r o w t h r e g u l a t i n g a c t i o n . T h e r e f o r e , t h e

p r e s e n t i n v e n t i o n

p r o v i d e s a compound s e l e c t e d f r o m t h e

g r o u p

c o n s i s t i n g

o f

h e

f o l l o w i n g ( A )

a n d

B ) :

20

25

30

35

40

45

50

55

60

6 5

2

A ) 3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i a z i n - 4 , 6 5 H , 7 H ) - d i o n e ;

a n d

B )

3 - m e t h y l - 3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i a z i n - 4 , 6 5 H , 7 H )

d i o n e .

T h e p r e s e n t i n v e n t i o n

a l s o

p r o v i d e s

a p l a n t g r o w t h r e g u l a

t o r

c o m p r i s i n g a

compound s e l e c t e d

from t h e g r o u p c o n s i s t

i n g o f t h e a b o v e d e s c r i b e d

( A )

a n d

B ) .

T h e compound o f t h e


c a n b e u s e d

e f f e c t i v e l y

a s

a

p l a n t

g r o w t h

r e g u l a t o r b e c a u s e t h e

compound c a n g r e a t l y

p r o m o t e p l a n t

g r o w t h .

The p r e s e n t i n v e n t i o n

a l s o

p r o v i d e s a

method

f o r p r o d u c

i n g

AOH , c o m p r i s i n g a l l o w i n g x a n t h i n e o x i d a s e

t o

a c t o n

AHX

o

y i e l d

AOH.

W i t h t h i s p r o d u c t i o n

m e t h o d ,

i t

i s

p o s

s i b l e t o e a s i l y

p r o d u c e

AOH t a h i g h y i e l d .

The


a l s o p r o v i d e s a

method

f o r p r o d u c

i n g AOH o m p r i s i n g s t e p s o f :

e x t r a c t i n g

a p l a n t b o d y t o

p r e p a r e an e x t r a c t ; and i s o l a t i n g AOH rom t h e e x t r a c t . With

t h i s

p r o d u c t i o n m e t h o d , AOH a n b e y i e l d e d w i t h o u t

r e s o r t t o

enzyme r e a c t i o n .

A d v a n t a g e o u s E f f e c t s o f I n v e n t i o n

A c c o r d i n g

t o

t h e p r e s e n t

i n v e n t i o n ,

a

compound

t h a t

c a n

r e g u l a t e p l a n t g r o w t h s p r o v i d e d .

BRIEF

DESCRIPTION

OF DRAWINGS

F I G .

1

i s

a chromatogram

o b t a i n e d

from t h e c h r o m a t o g r a

phy o f a n e x t r a c t

o f

z o y s i a g r a s s c u l t i v a t e d i n s o i l added w i t h

AHX.

F I G . 2

i s

a chromatogram

o b t a i n e d

from t h e c h r o m a t o g r a

phy

o f an x t r a c t

o f i c e

c u l t i v a t e d

i n a c u l t u r e ? u i d

added i t h

AHX.

F I G . 3

i s

a

diagram showing

t h e c r y s t a l

s t r u c t u r e o f

a n

AHX m e t a b o l i t e d e t e r m i n e d w i t h X - r a y c r y s t a l s t r u c t u r e

a n a l y s i s .

F I G . 4 i s

a

g r a p h showing

h e

i n ? u e n c e o f a d d i t i o n o fAHX

or

AOH o a

medium

o n

r i c e

growth.

F I G .

5

i s a ) an

MS/MS

spectrum and ( b )

a

f u l l mas s

spectrum of s t a n d a r d

AOH.

F I G . 6 i s

a )

an

MS/MS

spectrum and ( b ) a f u l l mas s

spectrum of r i c e

r o o t

e x t r a c t .

F I G . 7 i s a ) an

MS/MS

spectrum and ( b ) a f u l l mas s

spectrum of tomato

r o o t

e x t r a c t .

F I G . 8 i s

a

g r a p h

showing h e

i n ? u e n c e o f

a d d i t i o n

o fAOH

o r 3-methyl-AOH

t o a

m e d i u m

on r i c e g r o w t h .

DESCRIPTION OF EMBODIMENTS

T h e p r e s e n t i n v e n ti o n p r o v i d e s

( A ) AOH, h a t i s , 3 H - i m i

d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r ia z i n- 4 , 6 ( 5 H , 7 H ) - d i o n e. AOH

s

a com

p o u n d

r e p r e s e n t e d b y

t h e

f o l l o w i n g f o r m u l a I ) .

AOH

I )

AOH an be

b t a i n e d

i n a c c o r d a n c e

with

h e

r e a c t i o n

of h e

f o l l o w i n g f o r m u l a

I I )

by a l l o w i n g x a n t h i n e o x i d a s e t o a c t o n

AHX,

h a t i s ,

7 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i a z i n - 4 3 H ) - o n e .

7/18/2019 Us 8809328


US

8 , 8 0 9 , 3 2 8

B2

3

H )

O o

N H

H N \ H N N

l

— '

>=O

\ N

g

N § N

g

AHX

AOH

X a n t h i n e o x i d a s e u s e d f o r

p r o d u c i n g AOH

r o m AHX

s

n o t p a r t i c u l a r l y l i m i t e d ,

a n d

x a n t h i n e o x i d a s e

d e r i v e d

f r o m ,

f o r e x a m p l e ,

b u t t e r

milk

c a n b e u s e d . To a l l o w

x a n t h i n e

o x i d a s e

t o

a c t on AHX, o r e x a m p l e ,

AHX

s d i s s o l v e d i n a

r e a c t i o n

s o l v e n t

t o make

a

r e a c t i o n m i x t u r e , and x a n t h i n e

o x i d a s e may be added o t h e r e a c t i o n m i x t u r e . The amount of

x a n t h i n e

o x i d a s e

t o

b e

added s , b u t n o t p a r t i c u l a r l y l i m i t e d

t o , p r e f e r a b l y 2 0 - 2 0 0

U ,

more r e f e r a b l y

5 0 - 2 0 0 U , a n d

more

p r e f e r a b l y 1 0 0 - 2 0 0 Upper g o f

AH X .

The r e a c t i o n c o n d i

t i o n s a r e n o t p a r t i c u l a r l y l i m i t ed , and t i s p o s s i b l e t o a l l o w


t o a c t ,

f o r e x a m p l e , w i t h i n

t h e

r a n g e o f i t s

optimum

r e a c t i o n c o n d i t i o n .

F o r e x a m p l e ,

t h e r e a c t i o n

tem

p e r a t u r e i s p r e f e r a b l y 2 0 t o 3 5 0 C . , more r e f e r a b l y

2 5

to

3 2 °

C . ,

and s t i l l more

p r e f e r a b l y

2 8

t o

3 0 ° C . F o r e x a m p l e ,

t h e

r e a c t i o n t i m e i s p r e f e r a b l y

6

t o

5 0 h o u r s ,

more r e f e r a b l y 2 4

to 50 h o u r s ,

and

t i l l more p r e f e r a b l y 48 t o 50 h o u r s .

As

t h e

r e a c t i o n

s o l v e n t , a l t h o u g h

n o t

p a r t i c u l a r l y l i m i t e d , w a t e r

based s o l v e n t s s uc h a s water

or

a buffer a r e p r e f e r r e d , and a s

t h e b u f f e r , f o r e x a m p l e , p h o s p h a t e b u f f e r s a l i n e , p h o s p h a t e

b u f f e r , and d i s t i l l e d water can

be

u s e d . Additives such a s a

r e a c t i o n a c t i v a t o r may e a d d e d t o t h e r e a c t i o n m i x t u r e .

I t i s

p o s s i b l e t o o b t a i n AOH o f

t h e


from t h e

r e a c t i o n

m i x t u r e b y

p e r f o r m i n g

o p e r a t i o n s

s u c h

s e x t r a c t i o n ,

c o n c e n t r a t io n , p u r i ? c a t i o n , a n d d r y i n g on t h e r e a c t i o n mix

t u r e a s a p p r o p r i a t e a f t e r t h e c o m p l e t i o n of t h e r e a c t i o n .

I t

i s a l s o

p o s s i b l e t o

p r o d u c e AOH r o m

a

p l a n t b o d y . The

p l a n t s a r e n o t p a r t i c u l a r l y l i m i t e d a s

l o n g

a s t h e y

p r o d u c e

AOH,

a n d

may

b e

s p e r m a t o p h y t e s , p t e r i d o p h y t e s , o r b r y o

p h y t e s . The s p e r m a t o p h y t e s may b e gymnosperms o r

a n g i o s p e r m s , a n d t h e a n g i o s p e r m s

may

m o n o c o t y l e d o n s o r

d i c o t y l e d o n s .

S u c h p l a n t s s p e c i ? c a l l y i n c l u d e p l a n t s o f t h e

f a m i l y

P o a c e a e ,

t h e f a m i l y S o l a n a c e a e , t h e f a m i l y T h e a c e a e , t h e

f a m i l y C o m p o s i t a e ,

t h e

f a m i l y

R o s a c e a e ,

a n d h e f a m i l y

L i l i

a c e a e . Amon g t h e s e , i n t h e v i e w p o i n t t h a t t h e p r o d u c t i o n

amount o f

AOH s

l a r g e , p l a n t s o f t h e

f a m i l y

P o a c e a e o r t h e

f a m i l y S o l a n a c e a e a r e p r e f e r a b l e . T h e p l a n t s o f t h e f a m i l y

P o a c e a e i n c l u d e t h e

g e n u s

P h y l l a s l a c h y s , t h e

g e n u s

H o r

d e u m , t h e g e n u s

T

i l i c u m , t h e g e n u s O r y z a , t h e g e n u s

A

r o s

l i s , t h e g e n u s Z o y s i a , t h e g e n u s S a c c h a ru m , a n d t h e g e n u s

Z e a . Amon g t h e s e , t h e g e n u s

O r y z a

i s p r e f e r r e d , a n d r i c e

( O r y z a s a l i v a ) i s

m o r e

p r e f e r r e d . T h e p l a n t s o f

t h e f a m i l y

S o l a n a c e a e i n c l u d e

t h e g e n u s

L y c o p e r s i c a n ,

t h e g e n u s

S o l a n u m , t h e

g e n u s

C a p s i c u m , t h e

g e n u s

N i c o l i a n a , t h e

g e n u s

D a l u r a ,

t h e g e n u s P h y s a l i s , and t h e g e n u s

P e t u n i a .

Among t h e s e ,

t h e

g e n u s

L y c o p e r s i c o n

i s p r e f e r r e d , a n d

t o m a t o

( S o l a n u m

l y c a p e r s i c u m ) i s more r e f e r r e d .

P l a n t s a p p l i e d w i t h

AHX may

b e

u s e d

a s p l a n t s f o r p r o

d u c i n g

AOH.

W h en MIX s a p p l i e d t o p l a n t s , AOH s p r o

duced

s anAHX e t a b o l i t e . T h i s AOH may

e i s o l a t e d from

t h e p l a n t b o d y . I f

AHX

s a p p l i e d

t o

p l a n t s , more AOH s

p r o d u c e d f r o m

t h e

p l a n t s

t h a n

t h a t u s u a l l y p r o d u c e d b y

t h e

p l a n t s .

As

a

method f o r p r o d u c i n g AOH

from a p l a n t b o d y , a

method

o m p r i s i n g s t e p s o f x t r a c t i n g a p l a n t

b o d y

o p r e p a r e

an e x t r a c t ;

and

i s o l a t i n g

AOH from

t h e e x t r a c t

can

be u s e d .

0 1

20

25

30

35

40

45

50

55

60

6 5

4

The method

f o r p r e p a r i n g a n e x t r a c t by e x t r a c ti n g a p l a n t

b o d y i n c l u d e s , b u t

n o t

p a r t i c u l a r l y l i m i t e d

t o ,

f o r e x a m p l e , a

method i n which a

p l a n t o r g a n

o f a

p l a n t

body c o n t a i n i n g

AOH r e h a r v e s t e d ,

t h e

p l a n t o r g a n

i s

p u l v e r i z e d , a n d

t h e

p u l v e r i z e d

o r g a n i s

e x t r a c t e d

w i t h an

o r g a n i c s o l v e n t

s u c h a s

ethanol

or

acetone

t o

prepare an

e x t r a c t . The met hod f o r

i s o l a t i n g

AOH

r o m

t h e

e x t r a c t

i s

n o t

a r t i c u l a r l y l i m i t e d ,

b u t ,

f o r e x a m p l e ,

i t i s p o s s i b l e t o i s o l a t e AOH o f t h e p r e s e n t

i n v e n t i o n b y p e r f o r m i n g o p e r a t i o n s s u c h a s

c o n c e n t r a t i o n ,

p u r i ? c a t i o n ,

and d r y i n g on t h e o b t a i n e d e x t r a c t a s a p p r o p r i

a t e .

The p l a n t o r g a n c o n t a i n i n g AOH ay e , b u t n o t p a r t i c u

l a r l y l i m i t e d

t o ,

a n

e n t i r e

p l a n t b o d y ,

o r a n y o f o o t s ,

s t e m s ,

l e a v e s , ? o w e r s , r e p r o d u c t i v e o r g a n s ,

a n d s e e d s , a n d f u r t h e r

m o r e ,

may e

c u l t u r e

c e l l s .

Among h e s e , i n t h e v i e w p o i n t

o f

c o n t a i n i n g m u c h

AOH ,

r o o t s a r e

p r e f e r r e d .

T h e p r e s e n t i n v e n t i o n a l s o

p r o v i d e s

B )

3 - m e t h y l - A O H ,

t h a t i s , 3 - m e t h y l - 3 H - i m i d a z o [ 4 , 5 - d ] [ l , 2 , 3 ] t r i a z i n - 4 , 6 5 H ,

7 H ) - d i o n e 3 - m e t h y l - A O H s a c o m p o u n d r e p r e s e n t e d

b y t h e

f o l l o w i n g

f o r m u l a I l l ) .

3 - m e t h y l - A O H c a n b e s y n t h e s i z e d b y m e t h y l a t i n g AOH,

a n d

3-methyl-AOH c a n b e

s y n t h e s i z e d

i n

a c c o r d a n c e w i t h ,

f o r

e x a m p l e , a method

d e s c r i b e d i n E x a m p l e s .

WithAOH r 3-methyl-AOH,

t becomes p o s s i b l e

t o r e g u

l a t e p l a n t g r o w t h .

T h e

l a n t g r o w t h i n t h e p r e s e n t i n v e n t i o n i s

n o t

p a r t i c u l a r l y l i m i t e d

a s

l o n g

a s

i t i s a phenomenon i n v o l v

i n g t h e u s u a l

d i f f e r e n t i a t i o n

o r

p r o l i f e r a t i o n

o f

l a n t

c e l l s ,

a n d

i n c l u d e s

n o t

o n l y

e x p a n s i o n

a n d

e n l a r g e m e n t o f t h e o r g a n s

c o n s t i t u t i n g

p l a n t

b o d i e s b u t a l s o g e r m i n a t i o n f r o m s e e d s ,

?ower

i n i t i a t i o n , a n d

s e e d f o r m a t i o n . AOH

a n d

3 - m e t h y l

AOH a n promote o r i n h i b i t

p l a n t

g r o w t h d e p e n d i n g on t y p e

o f p l a n t s to be t a r g e t e d ,

t h e

c o n c e n t r a t i o n

o f

AOH r 3-me

t h y l - A O H , a n d

t h e

p a r t

o f

l a n t s

o

b e

made o n t a c t withAOH

o r

3-methyl-AOH.

T h u s ,

i t

i s p o s s i b l e t o u s e a

p l a n t

g r o w t h

r e g u l a t o r

c o m p r i s i n g AOH o r 3-methyl-AOH a s

a

g r o w t h

p r o m o t e r

o r a

g r o w t h

i n h i b i t o r f o r v a r i o u s

o r g a n s o f

p l a n t s

d e p e n d i n g

on

a r g e t

p l a n t s a n d

p u r p o s e s .

The p l a n t s t o b e t a r g e t e d a r e n o t p a r t i c u l a r l y l i m i t ed , a s

l o n g a s

t h e y a r e p l a n t s

s u b j e c t e d o

a

g r o w t h

r e g u l a t i n g a c t i o n

by

AOH r

3 - m e t h y l - A O H .

The p l a n t s t o b e

t a r g e t e d

may b e

s p e r m a t o p h y t e s ,

p t e r i d o p h y t e s , o r

b r y o p h y t e s ,

t h e s p e r m a t o

p h y t e s

may b e gymnosperms o r a n g i o s p e r m s ,

and

t h e

a n g i o s p e r m s may

o n o c o t y l e d o n s o r d i c o t y l e d o n s .

S u c h p l a n t s s p e c i ? c a l l y i n c l u d e p l a n t s

o f t h e f a m i l y

P o a c e a e , t h e f a m i l y S o l a n a c e a e , t h e f a m i l y

T h e a c e a e , t h e

f a m i l y C o m p o s i t a e , t h e f a m i l y

R o s a c e a e , a n d

h e f a m i l y

L i l i

a c e a e . Amon g h e s e , i n t h e v i e w p o i n t t h a t t h e g r o w t h r e g u

l a t i n g a c t i o n b y AOH

s

s i g n i ? c a n t ,

i t

i s p r e f e r a b l e

o b e

l a n t s

o f t h e f a m i l y P o a c e a e . The

p l a n t s

o f t h e f a m i l y P o a c e a e

i n c l u d e t h e

g e n u s

P h y l l a s l a c h y s , t h e

g e n u s

Hordeum,

t h e

g e n u s

T r i l i c u m , t h e

g e n u s O r y z a ,

t h e

g e n u s

A g r a s l i s , t h e

g e n u s Z o y s i a , t h e g e n u s

S a c c h a r u m ,

and t h e g e n u s

Z e a .

Among t h e s e , t h e g e n u s O r y z a o r t h e g e n u s Z a y s i a i s p r e

f e r r e d .

The l a n t

o r g a n

t o b e t a r g e t e d

may

e, b u t n o t p a r t i c u l a r l y

l i m i t e d t o ,

a n y

o f

o o t s ,

s t e m s , l e a v e s , ? o w e r s , r e p r o d u c t i v e

I I I )

7/18/2019 Us 8809328


US

8 , 8 0 9 , 3 2 8

B2

5

o r g a n s , a n d s e e d s , a n d

f u r t h e r m o r e ,

may b e c u l t u r e

c e l l s .

Among t h e s e , i n

t h e

v i e w p o i n t t h a t

t h e

g r o w t h r e g u l a t i n g

a c t i o n b y AOH s s i g n i ? c a n t , i t i s p r e f e r a b l e t o b e r o o t s ,

s t e m s , l e a v e s ,

o r s e e d s .

The


and t h e c o n t a c t method

of

AOH o r

3-methyl-AOH t o be a p p l i e d on p l a n t s can be

s e l e c t e d

a s

a p p r o p r i a t e

d e p e n d i n g o n t y p e o f

p l a n t s

t o b e t a r g e t e d , t h e i r

o r g a n s ,

p u r p o s e s ,

and

t h e

l i k e .

For e x a m p l e ,

i n

a

c a s e

t h a t

t h e

t a r g e t p l a n t s

r i c e

( O r y z a s a l i v a ) , t h e t a r g e t o r g a n s t h e s t e m ,

and t h e

p u r p o s e i s

t o e x t e n d t h e l e n g t h

o f

t h e s t e m ,

i t i s

p r e f e r a b l e

t o c u l t i v a t e

t h e

p l a n t w i t h

a

c u l t u r e ? u i d i n

which

AOH s d i s s o l v e d t o

become

t o

2000

uM

n

a normal c u l t u r e

? u i d . I n t h i s c a s e , t h e


o f AOH

n t h e c u l t u r e

? u i d i s more

p r e f e r a b l y

1 0 0 t o

1500

uM,

and e t

more p r e f

e r a b l y 5 0 0 to

1 0 0 0

uM.

I n a c a s e

t h a t

t h e t a r g e t

p l a n t

i s O r y z a

s a l i v a ,

t h e

t a r g e t o r g a n s

t h e o o t ,

and

h e

p u r p o s e i s

t o e x t e n d

t h e

l e n g t h o f h e o o t ,

i t

i s

p r e f e r a b l e

o c u l t i v a t e

t h e p l a n t

w i t h

a

c u l t u r e

?uid

i n which

AOH s

d i s s o l v e d to

be come 50

to

2000

uM

n a normal c u l t u r e ? u i d .

I n

t h i s c a s e , t h e concen

t r a t i o n of AOH n t h e c u l t u r e ?uid s

more

p r e f e r a b l y 250 t o

1500 uM,

and

e t more r e f e r a b l y

500 t o

1000 uM. I n a c a s e

t h a t

t h e

t a r g e t p l a n t s O r y z a s a l i v a ,

t h e

t a r g e t o r g a n s s e e d s ,

and

t h e purpose s

to

i n c r e a s e t h e s i z e o r number of

s e e d s

t o

i n c r e a s e t h e y i e l d

o f s e e d s ,

i t

i s

p r e f e r a b l e

t o

c u l t i v a t e

t h e

p l a n t

w i t h

a

c u l t u r e ?uid i n which AOH s

d i s s o l v e d t o

become

t o

1000 uM

n

a normal

c u l t u r e

? u i d .

A l t e r n a t i v e l y ,

i n t h e c a s e t h a t t h e

p u r p o s e

i s t o

i n c r e a s e

t h e

y i e l d o f

t h e

s e e d

o f

O r y z a

s a l i v a ,

i t

i s

a l s o p r e f e r a b l e t o

c u l t i v a t e

O r y z a s a l i v a

i n

s o i l

w i t h

a p p l y i n g

a c u l t u r e ? u i d i n w h i c h A O H s d i s s o l v e d

to

become 5 t o

500

uM.

F o r 3-methyl-AOH, f o r

e x a m p l e ,

i n

a

c a s e t h a t

t h e

t a r g e t

p l a n t

i s O r y z a

s a l i v a , t h e t a r g e t o r g a n i s t h e

r o o t , a n d

t h e

p u r p o s e i s

t o

e x t e n d t h e l e n g t h o f t h e

r o o t , i t

i s

p r e f e r a b l e t o

c u l t i v a t e t h e p l a n t w i t h a

c u l t u r e

? u i d

i n

which 3 - m e t h y l

AOH s d i s s o l v e d t o become

50 t o

1000 uM n a

normal

c u l t u r e

? u i d . I n h i s

c a s e ,

t h e

c o n c e n t r a t i o n o f 3-methyl-AOH

i n t h e

c u l t u r e

? u i d i s

more p r e f e r a b l y 200 to 500

uM.

T h e

p l a n t

g r o w t h r e g u l a t o r c o m p r i s i n g

AOH

r 3 - m e t h y l

AOH

f

t h e p r e s e n t i n v e n t i o n

may

o n t a i n

b a c t e r i c i d e s ,

a n t i

mold a g e n t s , i n s e c t i c i d e s , o r compounds

h a v i n g

a p l a n t

g r o w t h r e g u l a t i n g a c t i o n o t h e r t h a n AOH r 3 - m e t h y l - A O H ,

i n

a d d i t i o n

t o

AOH

r 3-methyl-AOH. F u r t h e r m o r e , t h e r e g u

l a t o r

m a y c o n t a i n kno wn a d d i t iv e s f o r f o r m u l a t i o n .

As s u c h

a d d i t i v e s f o r

f o r m u l a t i o n , t i s p o s s i b l e o

u s e ,

b u t n o t

p a r t i c u

l a r l y l i m i t e d

t o ,

f o r e x a m p l e , e x c i p i e n t s , e m u l s i ? e r s ,

a n d

h u m e c t a n t s . The t y p e s o f

t h e

f o r m o f

t h e

p l a n t g r o w t h r e g u

l a t o r o f

t h e p r e s e n t

i n v e n t i o n

c a n b e ,

b u t n o t p a r t i c u l a r l y

l i m i t e d t o , f o r e x a m p l e , e m u l s i o n s , w e t t a b l e p o w d e r s , w a t er

s o l u b l e p o w d e r s ,

s o l u t i o n s ,

g r a n u l e s , p o w d e r s ,

m i c r o c a p

s u l e s , f u m i g a n t s ,

s m o k i n g

a g e n t s ,

a e r o s o l s , ? o w a b l e

a g e n t s ,

p a s t e s , t a b l e t s , c o a t i n g a g e n t s , u l t r a - l o w - v o l u m e s p r a y i n g

a g e n t s , o i l a g e n t s , and complex

f e r t i l i z e r s ,

and u s e r s c a n

s e l e c t a s a p p r o p r i a t e

d e p e n d i n g

on t y p e o f p l a n t s

t o

be

t a r

g e t e d ,

t h e i r

o r g a n s , p u r p o s e s , a n d t h e

l i k e .

P l a n t g r o w t h r e g u

l a t o r i n t h e s e f o r m s can b e

produced

w i t h kno wn e t h o d s .

EXAMPLES

H e r e i n b e l o w ,


i s d e s c r i b e d more

s p e

c i ? c a l l y i n r e f e r e n c e

o E x a m p l e s ,

b u t t h e p r e s e n t i n v e n t i o n

s

n o t i n t e n d e d t o be l i m i t e d

to t h e s e Examples.

E x a m p l e 1

D e t e c t i o n o f an AHX e t a b o l i t e o f Z o y s i a g r a s s

Z o y s i a g r a s s ( A g r o s l i s

s l a l o n i f e r a ) wa s c u l t i v a t e d

i n a

con

t r o l s e c t i o n

o f

n o r m a l

a g a r medium 1 0 0 mL) and n anAHX

20

25

30

35

40

45

50

55

60

6 5

6

s e c t i o n

o f

an

a g a r medium

added i t h 1 mM

HX

1 0 0

mL)

f o r 3 0

d a y s . E x t e n s i o n

o f

s h o o t s o f h e z o y s i a g r a s s i n t h e MIX

s e c t i o n was l e a r l y p r o m o t e d c o m p a r e d o

t h a t

o f

t h e

z o y s i a

g r a s s

i n t h e

c o n t r o l s e c t i o n . The

s h o o t s and r o o t s o f t h e

c u l t i v a t e d z o y s i a g r a s s were h a r v e s t e d , e x t r a c t e d

w i t h

e t h a

n o l , and h e e x t r a c t of

h o o t s

and

h e e x t r a c t o f t h e r o o t s were

s u b j e c t e d

t o

r e v e r s e d - p h a s e

h i g h

p e r f o r m a n c e

i q u i d

c h r o m a

t o g r a p h y

r e v e r s e d - p h a s e H P L C ) . T h e

a n a l y s i s

c o n d i t i o n s

were

a s

f o l l o w s . Column:

D e v e l o s i l

C30-UG-5

column s i z e

4 . 6 > < 2 5 0 mm),

? o w

a t e : 0 . 5 m L / m i n u t e , m o b i l e p h a s e : g r a

d i e n t e l u t i o n o f 2 methanol i n 0.05 t r i ? u o r o ac e t i c a c i d

( l i q u i d A )

f o r

1 2

m i n u t e s ;

2-100 e t h a n o l i n t h e

l i q u i d A o r

1 2 0 m i n u t e s ;

100 m e t h a n o l

f o r 2 0 m i n u t e s , d e t e c t i o n :

absorbance measurement a t

UV 254

nm.

A

chromatogram

o b t a i n e d w i t h c h r o m a t o g r a p h y

s

shown n F I G . 1 . No e a k o f

AHX a s

d e t e c t e d b u t

a

peak

of AHX e t a b o l i t e w a s

d e t e c t e d

from t h e

e l u a t e

o f

t h e s h o o t

e x t r a c t o f

t h e

AHX

s e c t i o n

( F I G .

1 b ) ) . P e a k s o f AHX n d t h e AHX e t a b o l i t e

were d e t e c t e d from

t h e

e l u a t e

of h e r o o t e x t r a c t of

h e AHX

s e c t i o n ( F I G . 1 d ) ) . I t i s b e l i e v e d t h a t t h i s i s b e c a u s e , o t h e r

t h a n

t h e

AHX

e t a b o l i t e i n t h e r o o t s , t h e

AHX

n t h e medium

i s

contaminated

i n t o

t h e

e l u a t e

of t h e r o o t e x t r a c t . N e i t h e r a

peak of

AHX or

a peak

o f t h e AHX

e t a b o l i t e

wa s

d e t e c t e d

from h e e l u a t e s of h e s h o o t e x t r a c t and h e r o o t e x t r a c t of h e

c o n t r o l s e c t i o n ( F I G S . 1 a ) a n d 0 ) ) .

E x a m p l e 2

D e t e c t i o n of

an AHX

e t a b o l i t e of Rice

Rice (Oryza s a l i v a ) wa s c u l t i v a t e d i n a c o n t r o l s e c t i o n o f a

normal

c u l t u r e ?uid and i n

an

MIX e c t i o n of c u l t u r e ?uid

i n

which AHX

as

added

o

be

0 . 2 mM n t h e

normal

c u l t u r e

? u i d

f o r

1 4

d a y s . E x t e n s i o n

o f

s h o o t s o f t h e r i c e i n t h e AHX

s e c t i o n

was

c l e a r l y promoted

compared o

t h a t o f

t h e r i c e

i n

t h e c o n t r o l s e c t i o n . The s h o o t s and o o t s o f t h e c u l t i v a t e d r ic e

w e r e h a r v e s t e d ,

e x t r a c t e d

a n d a n a l y z e d

w i t h r e v e r s e d - p h a s e

HPLC

s i n

Example

A

h romatog ram

o b t a i n e d with h r o

m a t o g r a p h y

i s shown i n F I G . 2 . I n t h e

MIX e c t i o n , no p e a k

of AHX w a s

d e t e c t e d

n e i t h e r from t h e e l u a t e of t h e s h o o t

e x t r a c t

nor from h e e l u a t e o f h e r o o t

e x t r a c t ,

b u t a

peak

of h e

AHX e t a b o l i t e was d e t e c t e d ( F I G S .

2 ( b )

and d ) ) .

N e i t h e r

a

peak of

AHX

or a peak o f t h e

AHX

e t a b o l i t e wa s d e t e c t e d

from h e

e l u a t e s

of h e s h o o t e x t r a c t and h e r o o t

e x t r a c t

of h e

c o n t r o l s e c t i o n

( F I G S .

2 a ) a n d 0 ) ) .

E x a m p l e 3

I s o l a t i o n o f t h e AHX e t a b o l i t e

Rice

(Oryza s a l i v a ) wa s

c u l t i v a t e d i n a c o n t r o l s e c t i o n

o f

a

normal c u l t u r e

?uid

and i n an AHX

e c t i o n

of

a

c u l t u r e

?uid

i n which AHX

as

added t o be 1 mM n t h e normal

c u l t u r e

? u i d

f o r 2 0

d a y s .

S h o o t s ( 3 6 0

g ) o f t h e c u l t i v a t e d r i c e were

h a r v e s t e d

and

e x t r a c t e d

w i t h

e t h a n o l .

An

e t h a n o l - s o l u b l e

f r a c t i o n

w a s c o n c e n t r a t e d under reduced p r e s s u r e

and

e x t r a c t e d

w i t h

d i c h l o r o m e t h a n e .

A

d i c h l o r o m e t h a n e - i n

s o l u b l e f r a c t i o n w a s e x t r a c t e d

with

e t h a n o l t o y i e l d an e t h a

n o l - s o l u b l e

f r a c t i o n

( 9 . 8 g ) .

The

e t h a n o l - s o l u b l e

f r a c t i o n

was

s u b j e c t e d t o s i l i c a g e l

c h r o m a t o g r a p h y

? l l e r :

3 5 0 g o f

i l i c a

g e l

6 0 N , c o l u m n s i z e : 4 x 6 0 c m ) a n d

e l u t e d s e q u e n t i al l y

w i t h

m i x t u r e s

o f

d i c h l o r o m e t h a n e : m e t h a n o l : 9 :

, 7 : 3 , 5 : 5

t o y i e l d

e i g h t

f r a c t i o n s .

Amon g

h e s e , t h e

f r a c t i o n 3

( 2 8 8 mg)

w a s

p u r i ? e d s e q u e n t i a l l y w i t h

HPLC ( c o l u m n :

D e v e l o s i l

C30

UG-15/30

c o l u m n ,

s i z e :

5 0 > < 5 0 0

m m , ? o w a t e :

2 5

mL/min,

m o b i l e p h a s e :

5

m e t h a n o l ,

d e t e c t i o n : UV

1 0

nm) a n d

HPLC ( c o l u m n : D e v e l o s i l C30-UG-5 c o l u m n , s i z e :

2 0 > < 2 5 0

7/18/2019 Us 8809328


US

8 , 8 0 9 , 3 2 8

B2

7

m m ,

? o w

a t e :

5

m L / m i n u t e ,

m o b i l e p h a s e :

1 0

m e t h a n o l ,

d e t e c t i o n : UV 10 n m) t o

? n a l l y

i s o l a t e 1 0 . 5

mg

f an AHX

m e t a b o l i t e .

E x a m p l e

4

D e t e r m i n a t i o n o f

t h e S t r u c t u r e

w i t h

X-Ray

C r y s t a l

S t r u c t u r e

A n a l y s i s

X - r a y c r y s t a l

s t r u c t u r e

a n a l y s i s

was

p e r f o r m e d on t h e

i s o

l a t e d

AHX

e t a b o l i t e a s

f o l l o w s . S i n g l e - c r y s t a l X - r a y d i f

f r a c t i o n m e a s u r e m e n t w a s

p e r f o r m e d

u s i n g

S P r i n g - 8 s i n g l e

c r y s t a l s t r u c t u r e a n a l y s i s

b e a m l i n e

BL02B1).

The m e a s u r e

ment c o n d i t i o n s

were

a s

f o l l o w s .

W a v e l e n g t h :

0 . 8 2 6 0

4 )

A ,

beam

s i z e : l e n g t h

l 4 0 > < w i d t h 1 5 9 u m z ,

P h o t o n

F l u x :

1 . 8 1 > <

1 0 8 p h o t o n s / e c ,

a n d P h o t o n F l u x D e n s i t y : 8 . 1 3 > < 1 0 3

p h o t o n s /

s e c / u m z .

F I G .

3 i s a d i a g r a m s h o w i n g t h e

c r y s t a l

s t r u c t u r e

o f

t h e AHX e t a b o l i t e o b t a i n e d

w i t h

X - r a y c r y s t a l l o g r a p h y .

From

X - r a y c r y s t a l l o g r a p h y , i t

was con?rmed

t h a t t h e

i s o

l a t e d AHX e t a b o l i t e wa s 3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i a z i n

4 , 6 ( 5 H , 7 H ) - d i o n e ( a n o t h e r n a m e :

2 - a z a - 8 - o x o h y p o x a n t h i n e ,

“AOH”) h a v i n g t h e s t r u c t u r e o f f o r m u l a I ) .

E x a m p l e

5

P r o d u c t i o n o f AOH

AOH

as

produced by a l l o w i n g x a n t h i ne o x i d a s e t o a c t on

AHX s f o l l o w s .

I n

1

L

o f p h o s p h a t e b u f f e r s a l i n e

( 1 0

mM,

p H

7 . 4 ) , 1 3 7

mg f AHX was d i s s o l v e d

t h e r e t o , 2 5

mg

f

x a n t h i n e o x i d a s e ( d e r i v e d f r o m b u t t e r m i l k , 0 . 2 8 U/mg)

was

a d d e d ,

and

h e

r e s u l t a n t m i x t u r e

was

e f t

t o s t a n d t

3 0 °

C . To

t h e m i x t u r e ,

2 5 mg f

t h e x a n t h i n e o x i d a s e w a s

added

t h r e e

t i m e s e v e r y

2 4

h o u r s . A f t e r t h e

l a s t

a d d i t i o n , t h e m i x t u r e w a s

l e f t

t o

s t a n d f o r f u r t h e r 24 h o u r s . T h a t i s , i n t h e t o t a l a m o u n t ,

100

mg

f xanthine

oxidase w a s

allowed t o

a c t on

137 mg f

AHX f o r

9 6

h o u r s .

C o n s e q u e n t l y , i t

wa s

con?rmed

w i t h

a n a l y s i s

o f

HPLC

( D e v e l o s i l

C30-UG-5

c o l u m n

s i z e

4 . 6 > <

2 5 0

mm),

?ow a t e : 0 . 5 m L / m i n u t e , m o b i l e p h a s e : g r a d i e n t

e l u t i o n

o f

2 e t h a n o l i n

0.05

t r i ? u o r o a c e t i c a c i d ( l i q u i d

A) f o r 1 2 m i n u t es ; 2-100 m e t h a n o l i n t h e l i q u i d A o r 1 2 0

m i n u t e s ;

100

m e t h a n o l f o r 1 2 0 m i n u t e s ; d e t e c t i o n : a b s o r

bance t UV 54 n m)

t h a t

AHX as c o m p l e t e l y c o n v e r t e d t o

AOH.

The p r o d u c t was p u r i ? e d w i t h ODS

e l ? a s h

chroma

t o g r a p h y ? l l e r :

3 5 0

g o f

O D S g e l ,

c o l u m n s i z e :

4 > < 6 0

c m ,

m o b i l e

p h a s e :

w a t e r , w a t e r / m e t h a n o l : 9 : 1 )

t o

i s o l a t e 1 2 0 m g

o f

AOH y i e l d 7 8 . 4 % ) . I t was con?rmed by t h e HPLC

e t e n

t i o n

t i m e ,

t h e a b s o r b a n c e w a v e l e n g t h ,

a n d

mass s p e c t r o m e t r y

t h a t

t h e i s o l a t e d s u b s t a n c e w a s AOH.

E x a m p l e 6

The

In?uence ofAOH

n

Rice

S t e r i l i z e d

s e e d s o f

r i c e

( N i p p o n b a r e ) ( O r y z a s a l i v a L . c v .

N i p p o n b a r e ) were a l l o w e d t o

s p r o u t

a t 2 8 ° C . i n

t h r e e d a y s .

The

s p r o u t e d s e e d s ( f o u r s e e d s p e r

t e s t

t u b e ) were

c u l t i v a t e d

i n a t e s t t u b e ,

i n

w h i c h a

c o n t r o l c u l t u r e

?uid

o r

a

c u l t u r e

?uid

t o

w h i c h

AHX

rAOH a s added

t o

d i f f e r e n t c o n c e n t r a t i o n s

and

w a s l a c e d ,

a t

2 8 0 C .

f o r

a week. AOH

h a t w a s produced

a c c o r d i n g

t o

t h e

method o f Example 5 was

u s e d .

The o n t r o l

c u l t u r e

? u i d

c o m p r i s e s 0 . 5 mM

f

NH4NO3, 0 . 3 mM

f

Na2HPO4, 0 . 1 5 mM f K 2 S O 4 , 0 . 2 mM f M g C l Z , 0 . 1 mM f

C a C l Z , 2 3 u M o f F e - e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( F e

E D T A ) , 2 5 uM f H 3 B O 3 ,

4 . 5

uM f MnSO4,

0 . 1 5

uM f

C u S O 4 , 0 . 3 5

uM

nnSO4,

a n d

0 . 0 5

uM

f N a 2 M o O 4 . As

o r

t h e

c u l t u r e

? u i d s

c o n t a i n i n g AHX r

AO H ,

AHX r AOH s

20

25

30

35

40

45

50

55

60

6 5

8

added

t o t h e above d e s c r i b e d c o n t r o l

c u l t u r e

? u i d s

s u c h

t h a t

t h e

? n a l c o n c e n t r a t i o n

becomes 5 0 uM, 200

uM r 1 0 0 0

uM.

The c u l t u r e ?uid w a s r e p l a c e d with new c u l t u r e ?uid e v e r y

o t h e r

d a y .

A f t e r u l t i v a t i o n , t h e l e n g t h o f s h o o t s

a n d

r o o t s

was

m e a s u r e d .

The measured e x t e n s i o n

o f t h e s h o o t s and r o o t s i s

shown

i n F I G .

4 .

I t was

con?rmed t h a t AO H , s i m i l a r l y

t o

AH X ,

r o m o t e d

e x t e n s i o n

o f

s h o o t s

a n d

r o o t s c o n c e n t r a t i o n

d e p e n d e n t l y

and t h a t AOH as a n Ala e t a b o l i t e i n

F I G .

4 ,

“Con” e p r e s e n t s c o n t r o l . “*” i n d i c a t e s t h e P

a l u e

< 0 . 0 5 and

“**” i n d i c a t e s

t h e

P

v a l u e

< 0 . 0 1 . n : 1 1 . ) .

E x a m p l e

7

I s o l a t i o n o f AOH rom R i c e

R i c e

( O r y z a s a l i v a ) was h y d r o p o n i c a l l y c u l t i v a t e d w i t h a

n o r m a l c u l t u r e ? u i d f o r r i c e f o r t w o m o n t h s , a n d r o o t s

( 5 2

g )

were

a r v e s t e d . A f t e r t h e r o o t s

were

u l v e r i z e d

with

a m i x e r ,

t h e p u l v e r i z e d r o o t s were e x t r a c t e d

w i t h

e t h a n o l and a c e t o n e ,

and t h e e x t r a c t wa s t h e n

c o n c e n t r a t e d

t o

d r y n e s s t h e r e b y

t o

o b t a i n

r o o t e x t r a c t

(340 mg).

T h i s e x t r a c t w a s

d i s s o l v e d

i n a

s o l v e n t

(95

c e t o n i t r i l e

a n d 0.05

f o r m i c

a c i d )

t o

a d j u s t t o

a


o f 1 0

m g / mL,

and

t h e

r e s u l t a n t

s o l u t i o n

w a s

a n a l y z e d

w i t h

a l i q u i d c h r o m a t o g r a p h y - t a n d e m mass

s p e c

t r o m e t e r (LC-MS/MS). The r e f e r e n c e s t a n d a r d

AOH

a s a

peak t

a

r e t e n t i o n t i m e of

. 5

m i n u t e s w i t h LC,

a

peak of

m o l e c u l a r w e ig h t o f 152.0[M—H]— was

d e t e c t e d

from

a

f u l l

mas s

s p e c t r u m t t h i s

r e t e n t i o n

t i m e ,

and

peak f

a

f r a g m e n t

i o n o f molecular weight of 9 7 wa s d e t e c t e d from

an MS/MS

s p e c t r u m . S i m i l a r l y

t o

t h e

r e f e r e n c e s t an d a r d AOH,

t h e r i c e

r o o t e x t r a c t h a s a peak at a

r e t e n t i o n

time of

5

minutes

with

LC,

a

p e a k

o f a m o l e c u l a r w e i g h t o f 152.0[M—H]— was

d e t e c t e d

from a f u l l m a s s spectrum t t h i s r e t e n t i o n t i m e , and

a

peak

o f

f r a g m e n t

i o n o f a

m o l e c u l a r

w e i g h t

o f

9 7 w a s

d e t e c t e d from

an MS MS p e c t r u m .

S i n c e t h e same

molecular

w e i g h t peak a s t h a t

o f

t h e

r e f e r e n c e s t a n d a r d

AOH

w a s

d e t e c t e d from t h e e x t r a c t

of

h e

r i c e , i t

wa s

con?rmed

h a t t h e

r i c e

r o o t s

c o n t a i n e d

e n d o g e n o u s

AOH.

The

MS/

MS

p e c t r u m

a ) a n d

t h e f u l l mass s p e c t r u m b ) o f t h e r e f e r e n c e s t a n d a r d

AOH

r e s h o w n i n

F I G .

5 . The MS/

MS

p e c t r u m a ) and t h e

f u l l m a s s

spectrum

( b )

o f t h e r i c e r o o t

e x t r a c t

a r e

a l s o

s h o w n

i n

F I G .

6 . The amount

o f AOH

o n t a i n e d i n t h e

r i c e r o o t s ,

w h i c h w a s a l c u l a t e d

on h e

b a s i s

of h e

c a l i b r a t i o n

curve and

t h e peak a r e a o f AO H , wa s a b o u t 2 . 5

ng

p e r

52 g of

h e

r i c e

r o o t s . The LC-MS/MS

a n a l y s i s

c o n d i t i o n s and t h e

a p p a r a

tuses

u s ed

are as follows.

<The LC-MS/MS n a l y s i s C o n d i t i o n s

a n d

t h e A p p a r a t u s >

( L C

p a r t )

P u m p :

LC-20AD

(SHIMADZU

CORPORATION)

Column: P C

HILIC s i z e :

2 . 0

mm><100 m m , S h i s e i d o

C o . ,

L t d . )

F l o w a t e :

0 . 2

mL/minute

I n j e c t i o n a m o u n t : 1 0 uL

( M S / MS a r t )

M a s s s p e c t r o m e t e r : LTQ ORBITRAP D ISCOVERY

( I o n

t r a p t y p e , N e g a t i v e m o d e , THERMO S C I E N T I F I C )

E x a m p l e 8

I s o l a t i o n

o f

AOH

rom

Tomato

T o m a t o ( S o l a n u m

l y c o p e r s i c u m ) w a s h y d r o p o n i c a l l y c u l

t i v a t e d with a normal

c u l t u r e

?uid f o r tomatoes f o r

two

m o n t h s ,

and o o t s ( 1 7 g ) were a r v e s t e d . A f t e r t h e r o o t s were

p u l v e r i z e d

w i t h

a

m i x e r ,

t h e p u l v e r i z e d

r o o t s

were e x t r a c t e d

with

t h a n o l and c e t o n e , t h e e x t r a c t w a s h e n

c o n c e n t r a t e d

t o

d r y n e s s ,

a n d were

s t r i p p e d o f t h e d i c h l o r o m e t h a n e - s o l u b l e

7/18/2019 Us 8809328


US

8 , 8 0 9 , 3 2 8

B2

9

p a r t t o o b t a i n r o o t e x t r a c t ( 4 . 7

m g ) .

T h i s e x t r a c t wa s

i s s o l v e d

i n a s o l v e n t (95

c e t o n i t r i l e , 0.05 f o r m i c

a c i d ) t o a d j u s t to

a

c o n c e n t r a t i o n o f 1 0 m g / mL,

and t h e r e s u l t a n t s o l u t i o n

wa s

a n a l y z e d w i t h a n

LC-MS/MS as

i n Example

.

The

a n a l y s i s

c o n d i t i o n s and

p p a r a t u s e s

used o r a n a l y s i s were h e same a s

i n Example

7 .

C o n s e q u e n t l y , t h e t o m a t o r o o t e x t r a c t , s i m i

l a r l y

to

t h e r e f e r e n c e s t a n d a r d

AO H ,

h a s a

peak

t

r e t e n t i o n

t i m e

3 . 5 m i n u t e s w i t h LC,

a

p e a k

o f m o l e c u l a r w e i g h t 1 5 2 . 0

[M—H] w a s

detected

f r o m a

f u l l

m a s s

s p ectr u m

t t h i s reten

t i o n

t i m e , a n d a

p e a k o f f r a g m e n t i o n o f o l e c u l a r w e i g h t 9 7

w a s d e t e c t e d

from an MS/MS spectrum. Thus, i t w a s con

?rmed h a t t h e t o m a t o r o o t s a l s o c o n t a i n e d

endogenous

AO H .

The MS/MS

s p e c t r u m

a )

a n d t h e f u l l mass s p e c t r u m b ) o f

t h e

tomato

r o o t e x t r a c t

a r e s h o w n

i n

FIG. 7 . The

amount

of

AOH ontained i n t h e tomato r o o t s , w h i c h w a s c a l c u l a t e d on

t h e b a s i s

o f

t h e c a l i b r a t i o n c u r v e and t h e peak a r e a

o f

AO H ,

w a s

about

0 . 1 ng o r l e s s

per

1 7

g of

h e

tomato

r o o t s ,

which

w a s lower

t h a n

t h a t i n

t h e

r i c e .

E x a m p l e 9

The In?uence ofAOH n S o i l C u l t i v a t i o n of Rice

P o t C u l t i v a t i o n )

R i c e ( N i p p o n b a r e) ( O r y z a s a l i v a L . c v .

N i p p o n b a r e )

was

sown

on

A p r .

2 9 , 2 0 1 1 ,

and

e a c h

o f grown s e e d l i n g s w a s

t r a n s p l a n t e d t o a p o t

( 1 / 5 0 0 0 a

p o t) ? l l e d w i t h s o i l which

c o n t a i n s a f e r t i l i z e r

c o m p r i s i n g N

1 4 4 0 m g ) , P 2 0 5 ( 1 2 m g ) ,

K20 ( 7 6 0

mg)

a n d CaO ( 8 0 6

mg)

o n J u n e

7 t h , a n d w a s

s o i l - c u l t i v a t e d

i n

a

g r e e n h o u s e

( 2 8 ° C . )

u n d e r a n y one o f

s e v e n t y p e s

o f

c u l t i v a t i o n c o n d i t i o n s

o f t h e

f o l l o w i n g

1 ) to

7 )

u n t i l

S e p t e m b e r

2 4 t h . S u p p l y o f

w a t e r w a s

p e r f o r m e d

b y

p r o v i d i n g

two l i t e r of a p w a t e r o r t a p w a t e r t o which AOH

was

a d d e d e v e r y

week

( s e e

b e l o w ) .

20

25

30

35

1 0

C u l t i v a t i o n

C o n d i t i o n s

1 ) T a p w a t e r w a s p r o v i d e d

d u r i n g

p o t

c u l t i v a t i o n .

( 2 ) Tap

w a t e r to

which

AOH

as

added s u c h

t h a t t h e ? n a l

c o n c e n t r a t i o n became 50 uM was p r o v i d e d f o r two

weeks

( f r o m J u n e

7 t h t o

J u n e 2 0 t h )

i n

t h e p l a n t i n g s t a g e .

T a p

w a t e r

was

p r o v i d e d i n

t h e o t h e r

p e r i o d .

( 3 ) Tap w a t e r to which AOH

as

added s u c h t h a t t h e ? n a l


became

50

uM was

p r o v i d e d

f o r

two

weeks

( f r o m

J u l y 4 t h

t o

J u l y

1 7 t h ) i n

t h e t i l l e r i n g

s t a g e . T a p

w a t e r

was p r o v i d e d i n t h e o t h e r p e r i o d .

( 4 ) Tap w a t e r added

t o which

AOH

as

added s u c h

t h a t t h e

? n a l

c o n c e n t r a t i o n became 50

uM

was

p r o v i d e d

f o r two

w e e k s

( f r o m

u l y

2 5 t h o A u g u s t 7 t h ) i n

t h e t o p d r e s s i n g

s t a g e

f o r

p a n i c l e f o r m a t i on . T a p

w a t e r

was

p r o v i d e d

i n t h e o t h e r

p e r i o d .

( 5 ) Tap w a t e r added

t o which

AOH

as

added s u c h

t h a t t h e

? n a l

c o n c e n t r a t i o n became 50

uM

was

p r o v i d e d

f o r two

w e e k s ( f r o m

A u g u s t 1 5 t h

t o

A u g u s t 2 8 t h )

i n


s t a g e

f o r

r i p e n i n g .

T a p w a t e r

was

r o v i d e d n t h e

o t h e r p e r i o d .

( 6 ) Tap

w a t e r

to which

AOH

as

added s u c h


c o n c e n t r a t i o n became uM as p r o v i d e d d u r i n g p o t c u l t i v a

t i o n .

( 7 ) Tap w a t e r to

which

AOH

as

added s u c h



became

5 0

uM

as

p r o v i d e d d u r i n g

p o t

c u l t i

v a t i o n .

Brown r i c e and p l a n t b o d i e s

s o i l - c u l t i v a t e d were

d r i e d a t

3 0 ° C .

f o r 1 5 d a y s , a n d brown i c e w e i g h t

( b o t h

o f e i g h t

p e r

p l a n t a n d

w e i g h t p e r 1 0 0

b r o w n

r i c e g r a i n s ) ,

p a n i c l e

l e n g t h ,

c u l m

e n g t h ,

number f a n i c l e s , number f i l l e r s , a n d

e r i a l

p a r t w e i g h t were m e a s u r e d .

The

e s u l t s a r e

shown

n T a b l e 1 .

By

p p l y i n g AOH l l

t h e t i m e , t h e number f a n i c l e s a n d t h e

a e r i a l p a r t weight were i n c r e a s e d

more h a n t h e c o n t r o l . Addi

t i o n a l l y ,

b y a p p l y i n g AOH n t h e

t o p d r e s s i n g

s t a g e f o r p a n i c l e

f o r m a t i o n s t a g e

o r

l a t e r , t h e

brown

r i c e w e i g h t

p e r

p l a n t

was

i n c r e a s e d

more t h a n

t h e

c o n t r o l

( I n

T a b l e 1 ,

n u m e r i c a l v a l u e s

i n d i c a t e t h e a v e r a g e 1 s t a n d a r d d e v i a t i o n . “ I n c r e a s i n g r a t e ”

i n d i c a t e s an

i n c r e a s i n g r a t e ( ) r e l a t i v e

t o

t h e c o n t r o l . “*”

i n d i c a t e s

t h e P v a l u e

< 0 . 0 5 . Number o f

s a m p l e s p o t s ) : 6 . ) .

TABLE

5 0 pM AOH

T o p d r e s s i n g

5 0

uM AOH

5 0 pM AOH

s t a g e

f o r

P l a n t i n g T i l l e r i n g p a n i c l e

C o n t r o l s t a g e s t a g e f o r m a t i o n

Brown r i c e

B r o w n

r i c e

weight 41.7

1 1 0 .9

46.6 1 9 . 4 3 42.3 1 1 2 . 2 5 3 . 7 1 3.59*

( g / P m m )

I n c r e a s i n g

r a t e ( % ) 1 2 2 9

B r o w n

r i c e weight 2 . 1 9 10.06 2 . 1 1 1 0 0 7 2 . 1 2 1 0 . 1 2 2 . 1 2 10.07

( @ 1 0 0

g r a i n s )

P l a n t b o d y

Number f t i l l e r s

31.3

1

3.30 34.6

1

2.70 30.9

1

3.67 31.6

1

3.60

P a n i c l e l e n g t h ( c m )

2 5 . 3 11.91

2 3 . 5

11.61

2 6 . 2 1 1 . 0 3

2 4 . 5 11.61

C u l m l e n g t h

(cm)

9 5 . 3

1

7 . 4 5 9 7 . 1

112.1

9 4 . 5

1

4 . 6 3 9 0 . 2

1

6 . 1 5

n u m b e r

ofpanicles

26.5 1

4.76

27.8 1 4.26 23.0 1 5.44 30.5 1

4.50

I n c r e a s i n g

r a t e ( % )

A e r i a l p a r t

g )

1 1 6

123.8 1 2 5 121.2 1 1 2 1 3 0 0 1 3 4 1 1 6 5

I n c r e a s i n g

r a t e ( % )

5 0 pMAOH

T o p d r e s s i n g

s t a g e

5 pMAOH

5 0

pMAOH

f o r r i p e n i n g A l l t h e

t i m e

A l l

t h e t i m e

Brown

r i c e

B r o w n

r i c e weight

5 3 . 2 1

2.61*

5 9 . 3 1

2.27*

5 8 . 2 1

3.76*

g / p l a n t )

7/18/2019 Us 8809328


US

8 , 8 0 9 , 3 2 8

B2

1 1 1 2

TABLE - c o n t i n u e d

I n c r e a s i n g r a t e ( % )

2 8

42

4 0

Brown i c e weight 2.18 r 0.08 2 . 1 3 r 0.09 2 . 1 5 r

0.16

g / 1 0 0 g r a i n s )

P l a n t b o d y

Number f t i l l e r s

31.4

r 3 . 9 1 37.9 r

3.71*

35.7 r

4.15 *

P a n i c l e

l e n g t h

(c m) 2 4 . 0

r

2 . 0 7

2 4 . 5

r

2 . 2 4 2 4 . 4

r

1 . 9 6

Culrn l e n g t h

(cm) 9 2 . 8 r 3 . 9 8

8 9 . 7

r

6 . 4 0 9 2 . 3

r

5.64

number

ofpanicles

28.8 r 4.67 32.8 r 2 48 * 32.7 r

4.03*

I n c r e a s i n g r a t e

( % ) 2 4

2 3

A e r i a l

p a r t ( g ) 131 r 1 3 . 3 151 r

6 1 9 *

147 r 9 6 3 *

I n c r e a s i n g

r a t e

( % )

3 0 2 7

Example 1 0 1 5 TABLE

. . . .

0.5

mM

OH

The I n ? u e n c e o f AOH n

8 0 1 1 ' C u l t 1 v a t 1 o n

o f

R i c e

D u r i n g 1 0

mM OH

F l e l d c u n l v a n o n ) s e e d l i n g

D u r i n g

s e e d l i n g

P e r 1 0

p l a n t s

C o n t r o l r a i s i n g r a i s i n g

Rlce

( N l p p _ 0 n b a r e _ )

(On/Z” s a l i v a

L '

C V '

N l p p c f n b e l r e )

w a s

2 0

T 0 1 3 1 w e i g h t

g )

6 0 4 1 5 2 . 8

6 3 0

1 7 1 . 4

6 3 8

1 5 9 . 6 *

u s e d and cult1vated 1n a ? e ld as follows.

As

cult1vat1on

con-

H un

e i g h t

g ) 2 6 0

I

2 8 3 , 2 8 3

I

350* 284

I

244*

d i t i o n s , ? v e sections

of 1 )

t o ( 5 )

w e r e

s e t .

N o n — s i e v e d

brown

i c e

2 0 8

1

2 4 . 2 2 2 7

1

2 8 . 9 * 2 2 8

1

201*

~ ~ ~ ~

weight (g)

RIC? (Nlpponbare)

(On/2a “mm

' C V '

Nlppopbare)

. W a s S i e v e d b r o w n r i c e g ) 2 0 1 1

2 4 . 6

2 1 8 1 2 9 . 0 * 2 2 1

1 1 9 . 5 *

s o w n 1 n a

nursery

box on p r . 2 9 , 201 , and p ro w d ed

W1th

a I n m a s i n g rm

% ) 89 1 0 2

t o t a l of

15 l i t e r s

o f t a p w a t e r

or tap

w a t e r t o whichAOH as 2 5 Thousand k e r n e l 2 3 . 6 1

0 . 5 2 3 . 0

1 0 . 5 * 2 3 . 5 1 0 . 3

a d d e d f o r

t w o weeks f r o m May

4 t h w h i l e r a i s i n g

s e e d l i n g

i n

W e l g h t

g )

t h e n u r s e r y b o x .

On

J u n e

7 t h ,

e a c h o f

grown s e e d l i n g s was

0 5 mM

OH

t r a n s p l a n t e d

t o

t h e ? e l d . The p l a n t 1 n g d e n 5 1 t y

w a s

s e t

t o

t h e

0 . 5 T n M AO H T o p d r e s s i n g

a t

p a n i c l e

i n t e r r o w

space of

30

cm and

t h e p l a n t i n g d i s t a n c e

of

1 5

cm 3 0

P M 1 0 P l a n t s B a s a l f e l ? l i z e r f o r m a t i o n

( t h r e e

r e p r o d u c t i o n s

of 3x33 m e r one s e c t i o n ) . As b a s a l T o t a l w ? i g h t g ) 6 4 4 1 4 9 5 * 6 4 3 I 5 4 1 *

f e r t 1 l 1 z e r ,

50

L

of t a p water or t a p water t o w h 1 c h AOH a s Hun e i g h t

g )

2 8 8 1 239* 2 7 9 1 2 4 . 9 *

added

per s e c t i o n w a s

s u p p l i e d

on

June 7 t h .

The c u l t i v a t i o n

N o u n - i i i ?

b r o w n r i m

2 3 0

I 1 9 7 * 2 2 4 I

2 0 9 *

_ . . - we1g

t g

w a s o n t 1 n u e d , and s topdressmg f o r p a n 1 c l e f o r m a t 1 0 n , 50

L S i m / 6 d

b r o w n mg g )

2 2 2

I 1 9 8 * 2 1 5 I

2 0 7 *

of

a p water or a p water o w h 1 c h AOH a s added

per s e c t 1 o n

3 5

I n c r e a s i n g

rm % )

1 0 5 7 _ 2

was supplied o n July 25th.

Plant

bodies w e r e harvested o n T h o u s a n d

k ? r n e l

w e i g h t

g ) 2 3 - 5 I 0 - 6 2 3 - 4 I 0 - 5

O c t o b e r

1 2 t h .

C u l t i v a t i o n C o n d i t i o n s

1 ) T a p w a t e r was u s e d a s t h e c u l t u r e

? u i d d u r i n g

s e e d l i n g Exam 1 e 1 1

r a i s i n g , t h e b a s a l

f e r t i l i z e r ,

a n d


f o r

p a n i c l e 4 0 p

f o r m a t i o n .

2 ) Tap w a t e r t o which AOH as added s u c h t h a t t h e

? n a l P r o d u c t i o n o f

3-Methyl-AOH

c o n c e n t r a t i o n became 0 . 5 mM

as

provided

a s t h e c u l t u r e

? u 1 d d u r 1 n g s e e d h n g r a l s 1 n g . T a p

w a t e r

w a s s e d .

a s

t h e b a s a l A c c o r d i n g

t o

t h e

f o l l o w i n g

m e t h o d , 3 _ m e t h y 1 _ A O H w as

f g r n r l i z e r a r t i d t h e t o l p d i e s i g i l g l f o r p a n ; n g O I ‘ I I I I I a E I O I t L t h ? 1 4 5 p r o d u c e d f r o m A O H

a

waeroW1c

wasa e suc a e

na . .

) p To 5 mL of DMSO a n h y d r o u s d 1 m e t h y l s u l f o x 1 d e ) , 1 5 3

concentratlon

became 1 . 0 mM

as

prowded a s

the culture

0

?

mg of

AOH was d 1 s s o l v e d at 50 C., 0.075 mL

of

u1d during

s eedh ng

r a l s 1 n g .

Tap water w a s

u sed

a s

t h e basal

f 1odomethane

was added

thereto, and t he r e s u lt an t m1xture

e r t 1 1 1 z e r

and

t h e topdressmg f o r p a n 1 c l e f o r m a t 1 o n . t

d

4

h

A t . b t

d

t .

( 4 ) Tap water t o w h i c h

AOH

as added s u c h t h a t

the

?nal 5 0

w a s

reac

e

or o u r s '

r a c

l o ne ame usmg repara we

c o n c e n t r a t i o n became

0 . 5 mM as

provided

a s

t h e

b a s a l

f e r t i l i z e r .

Tap

w a t e r

wa s

used

a s t h e c u l t u r e

?uid

d u r i n g

s e e d l i n g

r a i s i n g

a n d

t h e t o p d r e s s i n g f o r

p a n i c l e f o r m a t i o n .

( 5 ) Tap w a t e r to which AOH

as

added s u c h


c o n c e n t r a t i o n became

0 . 5 mM as p r o v i d e d a s t h e t o p d r e s s

i n g f o r p a n i c l e f o r m a t i o n .

T a p

w a t e r was

u s e d

a s t h e

c u l t u r e

? u i d

d u r i n g s e e d l i n g

r a i s i n g a n d

t h e b a s a l

f e r t i l i z e r .

Brown r i c e was d r i e d , and t o t a l brown

r i c e

w e i g h t ,

h u l l

w e i g h t , n o n - s i e v e d

brown

i c e

w e i g h t a n d

s i e v e d

brown i c e ,

p e r 1 0

p l a n t b o d i e s ,

and t h o u s a n d

k e r n e l

w e i g h t were mea

s u r e d . The e s u l t s a r e shown n

T a b l e

2 . By applyingAOH, t h e

t o t a l

brown

i c e w e i g h t , t h e h u l l w e i g h t , t h e

n o n -

s i e v e d

brown

r i c e

w e i g h t , and

t h e

s i e v e d brown r i c e were i n c r e a s e d more

t h a n t h e

c o n t r o l

I n T a b l e

2 , n u m e r i c a l

v a l u e s

i n d i c a t e

t h e

55

60

a v e r a g e z s t a n d a r d

d e v i a t i o n . “ I n c r e a s i n g r a t e ” i n d i c a t e s

an

6 5

i n c r e a s i n g

r a t e

( )

r e l a t i v e t o

t h e c o n t r o l .

“ * ”

i n d i c a t e s t h e P

v a l u e < 0 . 0 5 . ) .

t h i n - l a y e r c h r o m a to g r ap h y ( T L C ,

m o b i l e p h a s e

C H 2 C 1 2 :

m e t h a n o l : 9 : 1 )

was

f u r t h e r s u b j e c t e d t o

H PLC ( D e v e l o s i l

C30-UG-5

column s i z e 20x250 m m ,

? o w

a t e :

5

m L / m i n u t e ,

m o b i l e

p h a s e :

1 0

m e t h a n o l

i n

0.05

t r i ? u o r o a c e t i c

a c i d ,

d e t e c t i o n :

UV

1 0 nm) t o y i e l d 1 0 . 2 m g y i e l d

6 . 1 1 % )

o f

3 - m e t h y l -AOH.

I t w a s

con?rmed by

h e

measurement e s u l t s

of

a s s

s p e c

t r o m e t r y , 1H-NMR

a n d

l3C-NMR t h a t t h e

p r o d u c e d

s u b

s t a n c e w a s

3-methyl-AOH, and

t h e

d e t a i l s were a s

f o l l o w s .

When h e

sample w a s measured

with a

m a s s

s p e c t r o m e t e r

J MS-T100LC

a s s

s p e c t r o m e t e r ) i n

t h e p o s i t i v e m o d e , m / z

168[M+H]+ a n d m/z

190[M+Na]+

w e r e i n d i c a t e d .

A d d i t i o n a l l y , w i t h

1H-NMR a n d l 3 C - N M R , t h e s a m p l e

showed t h e f o l l o w i n g v a l u e s .

lH-NMR 5 0 0

MHz)

3 . 8 8

13c-NMR

1 2 5 MHz)

3 7 . 9 ,

1 1 2 . 8 , 1 4 2 . 1 , 1 4 8 . 0 ,

1 5 2 . 7

7/18/2019 Us 8809328


US

8 , 8 0 9 , 3 2 8

B2

1 3

E x a m p l e

1 2

The In?uence

o f AOH

nd 3-Methyl-AOH on

R i c e

S t e r i l i z e d s e e d s

o f

r i c e ( N i p p o n b a r e ) ( O r y z a s a l i v a L . c v .

N i p p o n b a r e ) were a l l o w e d t o

s p r o u t

a t 2 8 ° C . i n

t h r e e d a y s .

The

s p r o u t e d

s e e d s

( f o u r

s e e d s

p e r t e s t

t u b e )

were c u l t i v a t e d

i n

a

test

t u b e , i n

w h i c h

a

c o n t r o l c u l t u r e

? u i d , a

c u l t u r e

?uid

o

w h i c h

. 2

mM

fAOH as a d d e d , o r

a

c u l t u r e ?uid o w h i c h

0 . 2 mM f 3-methyl-AOH was added and wa s p l a c e d ,

at

2 8 0

C .

f o r a week. The o n t r o l

c u l t u r e

? u i d c o m p r i s e s 0 . 5 mM f

NH4NO3, . 3 mM fNazHPO4,

0 . 1 5

mM

f K Z S O 4 , 0 . 2

mM

o f M g C l 2 ,

0 . 1

mM f

C a C l Z ,

2 3

uM

f

F e - e t h y l e n e d i a m i n e

t e t r a a c e t i c

a c i d ( F e - E D T A ) , 2 5

uM

f H 3 B O 3 , 4 . 5

uM

f

M n S O 4 ,

0 . 1 5 u M

f C u S O 4 ,

0 . 3 5 u M onnSO4, a n d . 0 5 u M

o f

Na2MoO4.

The u l t u r e

? u i d w a s e p l a c e d

w i t h

new u l t u r e

? u i d e v e r y o t h e r d a y . A f t e r

c u l t i v a t i o n ,

t h e l e n g t h o f s h o o t s

and o o t s w a s

measured.

The measured

e x t e n s i o n of h e

r o o t s

i s shown i n F I G .

8 .

I t wa s

con?rmed

t h a t

3-methyl-AOH,

s i m i l a r l y

t o AOH, a d

e x t e n s i o n c t i v i t y f o r

r o o t s . I n

F I G .

8 ,

“*” i n d i c a t e s P a l u e < 0 . 0 5 , and “**” i n d i c a t e s P a l u e < 0 . 0 1 .

n : 1 6 . ) .

I n c o n t r a s t , 3-methyl-AOH g a v e no i n ? u e n c e

on

e x t e n s i o n o f t h e s h o o t s .

INDUSTRIAL APPLICABILITY

S i n c e h a v i n g a

g r o w t h

r e g u l a t i n g

a c t i o n , AOH n d 3-me

thyl-AOH

o f


c a n

be e f f e c t i v e l y

u s e d

a s

1 0

20

25

1 4

p l a n t g r o w t h e g u l a t o r s . S u c h p l a n t g r o w t h r e g u l a t o r s c a n b e

w i d e l y a p p l i e d t o

a g r i c u l t u r e

a n d g a r d e n i n g .

The

i n v e n t i o n

c l a i m e d

i s :

1 . A ompound s e l e c t e d f r o m t h e g r o u p c o n s i s t i n g o f t h e

f o l l o w i n g A ) a n d B ) :

A ) 3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i a Z i n - 4 , 6 5 H , 7 H ) - d i o n e ;

and

B ) 3 - m e t h y l - 3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i a Z i n - 4 , 6 5 H ,

7 H ) - d i o n e .

2 . A l a n t

g r o w t h

r e g u l a t o r c o m p r i s i n g t h e

c o m p o u n d

a c c o r d i n g t o c l a i m 1 .

3 .

A

e t h o d

f o r p r o d u c i n g

3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i

a Z i n - 4 , 6 5 H , 7 H ) - d i o n e , c o m p r i s i n g :

a l l o w i n g


t o a c t

on 7 H - i m i d a z o [ 4 , 5 - d ] 1 ,

2 , 3 ] t r i a Z i n - 4 3 H ) - o n e

t o y i e l d 3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 ,

3 ] t r i a Z i n - 4 , 6 5 H , 7 H ) - d i o n e .

4 .

A

e t h o d

f o r p r o d u c i n g 3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i

a Z i n - 4 , 6 ( 5 H , 7 H ) - d i o n e c o m p r i s i n g s t e p s o f :

e x t r a c t i n g a p l a n t body to

p r e p a r e

a n e x t r a c t ; and

i s o l a t i n g 3 H - i m i d a z o [ 4 , 5 - d ] [ 1 , 2 , 3 ] t r i a Z i n - 4 , 6 5 H , 7 H ) - d i

one from

t h e e x t r a c t .

* * * * *

us 8809328

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