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IMIDAZOLE DERIVATIVE
Notice:
JP
JP
JP
JP
JP
W0
3/1988
5/1988
7/1992
1/2009
8/2010
1/2011
(86)
PCT No.:
PCT/JP2012/060989
371 (0X1)
(2), (4) Date:
US 2014/0148598 A1
63-068570
63-104965
04-210680
2009-001558
4565018
WO 2011/010695
(65)
References Cited
OTHER PUBLICATIONS
14/113,730
(87)
US 8,809,328 B2
654.
May 29,2014
Primary Examiner * Jeffrey H Murray
(30)
.....
. . . ..
2011-154981
Int. Cl.
A61K 31/535
C07D 48 7/00
(52) US. Cl.
(58)
(2006.01)
(2006.01)
ABSTRACT
(51)
USPC
(57)
(B) 3-methyl-3H-imidazo[4,5-d][1,2,3]triaZin-4,6(5H,7H)
dione;
has a plant growth regulating action.
US. Patent
Fig.3
Sheet 3 0f8
US 8,809,328 B2
US. Patent
Sheet 4 0f8
'
0
o
US 8,809,328 B2
(HM)550120000
AOH
200
AHX
a:
Z]
50
'
o
o
I
(D
Con
13
Fig.4
(W9)NOISNEIJ_X3
US. Patent
Sheet 6 0f 8
US 8,809,328 B2
__
1._____.__.-
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cm ow ow ON
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_=0.
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US. Patent
US 8,809,328 B2
Sheet 7 0f 8
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US 8,809,328 B2
1
IMIDAZOLE DERIVATIVE
(B) 3-methyl-3H-imidazo[4,5-d][1,2,3]triazin-4,6(5H,7H)
dione.
CROSS-REFERENCE TO RELATED
APPLICATIONS
ing of the above described (A) and (B). The compound of the
growth.
The present invention also provides a method for produc
ing AOH, comprising allowing xanthine oxidase to act on
AHX to yield AOH. With this production method, it is pos
sible to easily produce AOH at a high yield.
The present invention also provides a method for produc
BACKGROUND ART
20
25
30
CITATION LIST
Patent Literature
35
analysis.
No. 63-104965
No. 63-68570
45
Technical Problem
(I)
60
AOH
65
US 8,809,328 B2
4
3
(H)
O
o
N
HN
\N
AHX
'
HN
NN
01
>=O
AOH
The plant organ containing AOH may be, but not particu
20
25
30
40
dicotyledons.
Such plants speci?cally include plants of the family
Poaceae, the family Solanaceae, the family Theaceae, the
family Compositae, the family Rosaceae, and the family Lili
45
50
60
plants.
As a method for producing AOH from a plant body, a
65
US 8,809,328 B2
5
organs, purposes, and the like. For example, in a case that the
target plant is rice (Oryza saliva), the target organ is the stem,
and the purpose is to extend the length of the stem, it is
preferable to cultivate the plant with a culture ?uid in which
AOH is dissolved to become 5 to 2000 uM in a normal culture
?uid. In this case, the concentration of AOH in the culture
?uid is more preferably 100 to 1500 uM, and yet more pref
erably 500 to 1000 uM. In a case that the target plant is Oryza
saliva, the target organ is the root, and the purpose is to extend
that the target plant is Oryza saliva, the target organ is seeds,
than the AHX metabolite in the roots, the AHX in the medium
25
Example 2
plant is Oryza saliva, the target organ is the root, and the
purpose is to extend the length of the root, it is preferable to
cultivate the plant with a culture ?uid in which 3-methyl
AOH is dissolved to become 50 to 1000 uM in a normal
35
45
60
65
EXAMPLES
Example 1
Detection of an AHX Metabolite of Zoysiagrass
Zoysiagrass (Agroslis slalonifera) was cultivated in a con
trol section of a normal agar medium (100 mL) and in anAHX
US 8,809,328 B2
7
Example 4
Determination of the Structure with X-Ray Crystal
Structure Analysis
Example 7
Example 5
Production of AOH
30
40
AOH. The product was puri?ed with ODS gel ?ash chroma
tography (?ller: 350 g of ODSgel, column size: 4><60 cm,
mobile phase: water, water/methanol:9:1) to isolate 120 mg
of AOH (yield 78.4%). It was con?rmed by the HPLC reten
tion time, the ab sorbance wavelength, and mass spectrometry
the peak area of AOH, was about 2.5 ng per 52 g of the rice
roots. The LC-MS/MS analysis conditions and the appara
45
(LC part)
Pump: LC-20AD (SHIMADZU CORPORATION)
Example 6
Ltd.)
Flow rate: 0.2 mL/minute
Injection amount: 10 uL
(MS/ MS part)
Sterilized seeds of rice (Nipponbare) (Oryza saliva L. cv.
Nipponbare) were allowed to sprout at 28 C. in three days.
The sprouted seeds (four seeds per test tube) were cultivated
55
and was placed, at 280 C. for a week. AOH that was produced
60
65
months, and roots (17 g) were harvested. After the roots were
pulverized with a mixer, the pulverized roots were extracted
with ethanol and acetone, the extract was then concentrated to
US 8,809,328 B2
9
10
Cultivation Conditions
part to obtain root extract (4.7 mg). This extract was dissolved
(2) Tap water to which AOH was added such that the ?nal
concentration became 50 uM was provided for two weeks
(from June 7th to June 20th) in the planting stage. Tap water
was provided in the other period.
(3) Tap water to which AOH was added such that the ?nal
concentration became 50 uM was provided for two weeks
(from July 4th to July 17th) in the tillering stage. Tap water
(4) Tap water added to which AOH was added such that the
?nal concentration became 50 uM was provided for two
The MS/MS spectrum (a) and the full mass spectrum (b) of
period.
(5) Tap water added to which AOH was added such that the
?nal concentration became 50 uM was provided for two
the basis of the calibration curve and the peak area of AOH,
(6) Tap water to which AOH was added such that the ?nal
20
Example 9
tion.
(7) Tap water to which AOH was added such that the ?nal
vation.
Brown rice and plant bodies soil-cultivated were dried at
(Pot Cultivation)
plant and weight per 100 brown rice grains), panicle length,
culm length, number of panicles, number of tillers, and aerial
part weight were measured. The results are shown in Table 1.
30
By applying AOH all the time, the number of panicles and the
formation stage or later, the brown rice weight per plant was
increased more than the control (In Table 1, numerical values
aerial part weight were increased more than the control. Addi
35
Topdressing
50 uM AOH
50 pM AOH
Planting
Tillering
panicle
stage
stage
formation
42.3 1 12.2
53.7 1 3.59*
2.12 1 0.12
2.12 10.07
Control
stage for
Brown rice
Brown rice weight
41.7 110.9
46.6 1 9.43
2.19 10.06
2.111007
(g/Pmm)
Increasing rate (%)
Brown rice weight
12
29
(@100 grains)
Plant body
Number oftillers
31.3 1 3.30
34.6 1 2.70
30.9 1 3.67
31.6 1 3.60
25.3 11.91
23.5 11.61
26.2 1 1.03
24.5 11.61
95.3 1 7.45
97.1 112.1
94.5 1 4.63
90.2 1 6.15
number ofpanicles
26.5 1 4.76
27.8 1 4.26
23.0 1 5.44
30.5 1 4.50
116 123.8
125 121.2
1121300
1341165
5 pM AOH
50 pM AOH
for ripening
59.3 1 2.27*
58.2 1 3.76*
Brown rice
Brown rice weight
(g/plant)
53.2 1 2.61*
US 8,809,328 B2
11
12
TABLE 1-continued
Increasing rate (%)
28
42
40
2.18 r 0.08
2.13 r 0.09
2.15 r 0.16
(g/100 grains)
Plant body
Number oftillers
31.4 r 3.91
37.9 r 3.71*
35.7 r 4.15*
24.0 r 2.07
92.8 r 3.98
28.8 r 4.67
24.5 r 2.24
89.7 r 6.40
32.8 r 248*
24.4 r 1.96
92.3 r 5.64
32.7 r 4.03*
24
23
131 r 13.3
151 r 619*
147 r 963*
30
27
Example 10
15
TABLE 2
0.5 mM AOH
During
10 mM AOH
(Fleld cunlvanon)
seedling
During seedling
Per 10 plants
Control
raising
raising
604 1 52.8
630 1 71.4
638 1 59.6*
260 I 283,
208 1 24.2
283 I 350*
227 1 28.9*
284 I 244*
228 1 201*
201 1 24.6
218 1 29.0*
89
221 119.5*
102
23.6 1 0.5
23.0 1 0.5*
23.5 1 0.3
Rlce (Nlpp_0nbare_) (On/Z saliva L' CV' Nlppcfnbelre) was 20 T0131 weight (g)
used and cult1vated 1n a ?eld as follows. As cult1vat1on conditions, ?ve sections of (1) to (5) were set.
~
weight (g)
total of 15 liters oftap water or tap water to which AOH was 25 Thousand kernel
added for two weeks from May 24th while raising seedling in
the nursery box. On June 7th, each of grown seedlings was
Welght (g)
05 mM AOH
Topdressing at panicle
formation
Totalw?ight (g)
644 1495*
643 I 541*
288 1 239*
230 I 197*
279 1 24.9*
224 I 209*
222 I 198*
215 I 207*
we1g
of tap water or tap water to wh1ch AOH was added per sect1on 35 Increasing rm (%)
was supplied on July 25th. Plant bodies were harvested on
105
October 12th.
Cultivation Conditions
(1) Tap water was used as the culture ?uid during seedling
7_2
23-5 I 0-6
23-4 I 0-5
Exam 1e 11
formation.
(2) Tap water to which AOH was added such that the ?nal
Production of 3-Methyl-AOH
fgrnrlizer, artid the tolpdiesigilgl for pan;ng fOIIIIIIaEIOItLth ? 1 45 produced from AOH
( )
waeroW1c
wasa
suc
na
? -
- -
mg
u1d during seedhng rals1ng. Tap water was used as the basal
- -
of AOH
t df
d1ssolved at
50
C.,
0.075
mL
of
or
4h
Af
ours'
t.
rac lone
bt .
t.
was
(4) Tap water to which AOH was added such that the ?nal 50 was reac e
(5) Tap water to which AOH was added such that the ?nal
concentration became 0.5 mM was provided as the topdress
ing for panicle formation. Tap water was used as the culture
55
Brown rice was dried, and total brown rice weight, hull
value <0.05.).
US 8,809,328 B2
13
14
Example 12
(A) 3H-imidazo[4,5-d][1,2,3]triaZin-4,6(5H,7H)-dione;
and
10
aZin-4,6(5H,7H)-dione, comprising:
20
INDUSTRIAL APPLICABILITY
25
3]triaZin-4,6(5H,7H)-dione.
4. A method for producing 3H-imidazo[4,5-d][1,2,3]tri
(B) 3-methyl-3H-imidazo[4,5-d][1,2,3]triaZin-4,6(5H,
7H)-dione.
isolating 3H-imidazo[4,5-d][1,2,3]triaZin-4,6(5H,7H)-di
one from the extract.
*