Adenine derivatives and their use as a plant growth regulator

A denine derivatives represented by the following formula: ##STR1## (wherein n is 1 or 2 and R and R' represent hydrogen or a methyl, ethyl, allyl, or propargyl group) and agriculturally acceptable acid addition salts thereof, which have cytokinin activity and besides, excellent rooting action, is applied to as plant growth regulators.

The present invention relates to adenine derivatives represented by the 
following formula (1): 
##STR2## 
(wherein n represents 1 or 2 and R and R' each represents a hydrogen atom 
or a methyl, ethyl, allyl or propargyl group). 
The adenine derivatives of the present invention show cytokinin activity 
and are useful for acceleration of plant physiological actions such as 
acceleration of plant cell division, promotion of growth of lateral buds 
and fruits, stimulation of seed germination and bud formation, 
acceleration of blooming and fruit bearing, senescence retardation and 
promotion of substance accumulation in storage organs. Moreover, the 
adenine derivatives of the present invention have a stimulating activity 
of root formation in addition to cytokinin activity. 
Hitherto, many compounds have been proposed for regulation of vegetation of 
plants, especially cereals, fruits and vegetables. Among them, compounds 
generically called cytokinins are known to have many physiological 
activities and typical examples thereof are zeatin, kinetin and 
benzyladenine(6-benzylaminopurin). These cytokinins are known to have 
plant physiological activities such acceleration of plant cell division, 
promotion of growth of lateral buds and fruits, stimulation of seed 
germination and bud formation and acceleration of blooming and fruit 
bearing, senescence retardation and promotion of substance accumulation in 
storage organs. It is also known that rooting can be further stimulated by 
using auxins, but it is well known that there are many plants which cannot 
be or are hardly stimulated by such method. Furthermore, compounds having 
cytokinin activity which have hitherto been found have substantially no 
action of stimulating root formation. 
Benzyladenines represented by the following formula (2) have been known as 
cytokinin compounds utilized in the field of agriculture, but are not wide 
in their applicability in practical use and more effective compounds have 
been demanded. Besides, more effective rooting stimulators have also been 
desired. 
##STR3## 
(wherein Ph represents a phenyl group). 
An object of the present invention is to provide novel substances which 
have cytokinin activity and rooting action superior to those of known 
adenine derivatives and can be utilized in a wide variety of uses, namely, 
to provide excellent plant growth regulators. 
The inventors have conducted intensive research on synthesis of compounds 
having various substituents on N.sup.6 nitrogen atom of adenines and 
screening of biological activity thereof and as a result they have found 
that the novel adenine derivatives represented by the following formula 
(1) have both the excellent cytokinin activity and the action of 
stimulating root formation. Thus, the present invention has been 
accomplished. 
##STR4## 
(wherein n represents 1 or 2 and R and R' each represents hydrogen or a 
methyl, ethyl, allyl or propargyl group.). 
That is, the present invention relates to adenine derivatives represented 
by the above formula (1). 
The adenine derivatives represented by the above formula can be obtained, 
for example, by the following process. 
6-Chloropurine and an amine compound having the structure represented by 
the following formula (3) are heated in organic solvents such as alcohols 
in the presence of trialkylamines such as ethyldiisopropylamine to allow 
6-chloropurine and the amine compound to react with each other. 
##STR5## 
(wherein n represents 1 or 2 and R and R' each represents hydrogen or a 
methyl, ethyl, allyl or propargyl group). 
The amine compounds represented by the formula (3) can be obtained, for 
example, by deprotecting phthalimides represented by the formula (5) which 
are obtained by allowing N-(oxoalkyl)phthalimides represented by the 
formula (4) to react with hydroxylamine or O-substituted hydroxylamines. 
The deprotection can be carried out by reaction with acids such as 
hydrochloric acid and hydrobromic acid or hydrazine. 
##STR6## 
(wherein n represents 1 or 2 and R and R' each represents hydrogen or a 
methyl, ethyl, allyl or propargyl group). 
Typical examples of the adenine derivatives represented by the formula (1) 
are as follows. 
N.sup.6 -[2-(N-methoxyimino)ethyl]adenine represented by the following 
formula (6): 
##STR7## 
N.sup.6 -[2-(N-methoxyimino)propyl]adenine represented by the following 
formula (7): 
##STR8## 
N.sup.6 -[3-(N-hydroxyimino)butyl]adenine represented by the following 
formula (8): 
##STR9## 
N.sup.6 -[3-(N-methoxyimino)butyl]adenine represented by the following 
formula (9): 
##STR10## 
N.sup.6 -[2-(N-ethoxyimino)propyl]adenine represented by the following 
formula (10): 
##STR11## 
N.sup.6 -[2-(N-allyloxyimino)propyl]adenine represented by the following 
formula (11): 
##STR12## 
N.sup.6 -[2-(N-propaglyoxyimino)propyl]adenine represented by the following 
formula (12): 
##STR13## 
The adenine derivatives of the present invention can be easily converted to 
salts of mineral acids such as hydrochloric acid, hydrobromic acid, 
sulfuric acid, nitric acid and phosphoric acid or salts of organic acids 
such as formic acid and acetic acid which are represented by the following 
formula (13) by conventional process 
##STR14## 
(wherein n represents 1 or 2, R and R' each represents hydrogen or a 
methyl, ethyl, allyl or propargyl group and Z represents an equivalent 
amount of an acid such as hydrochloric acid, hydrobromic acid, sulfuric 
acid, nitric acid, phosphoric acid, formic acid or acetic acid). 
Physical properties of the adenine derivatives represented by the formula 
(1) are shown in Table 1. 
TABLE 1 
______________________________________ 
Physical properties of the adenine derivatives 
Com- 
pound 
No. Formula n R R' mp(.degree.C.) 
______________________________________ 
A (6) 1 H CH.sub.3 239-246 
decomposition 
B (7) 1 CH.sub.3 
CH.sub.3 220-227 
decomposition 
C (8) 2 CH.sub.3 
H 216-238 
D (9) 2 CH.sub.3 
CH.sub.3 189-201 
E (10) 1 CH.sub.3 
C.sub.2 H.sub.5 
226-228 
F (11) 1 CH.sub.3 
CH.sub.2 CH.dbd.CH.sub.2 
206-208 
G (12) 1 CH.sub.3 
CH.sub.2 C.tbd.CH 
194-196 
______________________________________ 
The adenine derivatives represented by the formula (1) and salts thereof 
represented by the formula (13) have plant hormone activity and cytokinin 
activity, and stimulate root formation, and therefore are expected to be 
able to be used as plant growth regulators in the fields or as medium 
components for culturing of plant tissue. 
These compounds per se can be used alone or as the active ingredient in a 
composition for plant growth regulator. Typically, they are used in 
combination with agriculturally acceptable liquid or solid carriers and, 
optionally, with surface active agents as emulsifiers, dispersants, 
spreaders, and stickers such as those which are generally used in the 
field of formulation of agrochemicals. 
Examples of formulations within the scope of the invention are emulsifiable 
concentrates, wettable powders, dusts and liquids. 
As diluents for preparation of a liquid formulation, mention may be made of 
polar solvents such as alcohols, e.g., methanol and ethanol, water, 
dimethylformadmide and dimethylsulfoxide. 
As carriers for preparation of a wettable powder, granule or dust, there 
may be used talc, clay, bentonite, kaolin, montmorilonite, diatomaceous 
earth, phenol resin and white carbon. 
Typical examples of surface-active agents which may be usefully employed in 
the practice of the invention are anionic surface-active agents such as 
sodium alkylbenzene-sulfonates and sodium laurylsulfate, cationic 
surface-active agents such as stearyltrimethylammonium chloride and 
nonionic surface-active agents such as polyoxyethylenealkylphenyl ether. 
As dispersants, there may be used, in addition to the above surface-active 
agents, for example, sodium ligninsulfonate, methyl cellulose, and sulfite 
waste liquor. 
As stickers i.e. materials to adhere the active agents to the plants, there 
may be used, for example, casein lime, glue, carboxymethyl cellulose, 
sodium alginate and polyvinyl alcohol. 
When the composition of the present invention is used as rooting 
stimulators, there may be employed the method of immersing a cutting of 
plant in an aqueous solution of the compound and then planting the cutting 
in nursery or the method of coating or covering a cut section of a cutting 
with the composition. Furthermore, other rooting stimulators such as 
indoleacetic acid, indolebutyric acid, naphthylacetic acid and 
2,4-dichlorophenoxyacetic acid which are plant hormones, and fungicides 
and fertilizers may be used in combination with the composition of the 
present invention and further enhancement of the effects and labor-saving 
can be attained thereby. 
The composition of the present invention can be applied, as plant growth 
regulators, to fruit trees such as grapevine, mandarin, pear tree, and 
peach tree, woods such as oak, pine, and Japanese cypress, and flowering 
plants such as camellia, rhododendron, azalea, chrysanthemum, carnation, 
rose and poinsettia. 
Synthesis of the adenine derivatives of the present invention and 
physiological activities thereof will be illustrated by the following 
examples and test examples.

EXAMPLE 1 
Synthesis of N.sup.6 -[2-(N-methoxyimino)ethyl]adenine (compound A) 
A branched chain amine compound was first synthesized and then, this was 
allowed to react with 6-chloropurine in the following manner. 
(1) Synthesis of N-[2-(N-methoxyimino)ethyl]phthalimide 
0.560 g (2.96 mmol) of N-(2-oxoethyl)phthalimide was dissolved in 20 ml of 
ethanol and to the solution was added 0.330 g (3.95 mmol) of 
O-methylhydroxylamine hydrochloride. To this suspension was added 10 ml of 
0.35M aqueous sodium carbonate solution, followed by being stirred at room 
temperature for 3 days. The reaction mixture was poured into 100 ml of 
water and extracted with chloroform (20 ml.times.3), and the combined 
organic layer was dried over magnesium sulfate. Chloroform was distilled 
off and the residual solids were washed with petroleum ether to give 0.291 
g (yield 45%) of the titled compound as colorless crystals. 
(2) Synthesis of N.sup.6 -[2-(N-methoxyimino)ethyl]adenine (compound A) 
0.290 g (1.33 mmol) of N-[(2-(N-methoxyimino)ethyl]phthalimide was 
dissolved in 8 ml of ethanol and to the solution was added 77.7 .mu.l 
(1.60 mmol) of hydrazine hydrate, followed by being refluxed for 5 hours. 
The reaction mixture was cooled to room temperature and then, 15 ml of 
ether was added thereto and the mixture was left to stand at 0.degree. C. 
for 1 hour to sufficiently precipitate solids. The solids were removed by 
suction filtration and the filtrate was dried over sodium sulfate and 
concentrated to 1 ml by an avaporator. This concentrated filtrate was 
dissolved in 5 ml of isopropanol and to the solution were added 77.7 mg 
(0.503 mmol) of 6-chloropurine and 88 .mu.l of ethyldiisopropylamine, 
followed by being refluxed with heating on an oil bath for 5 hours. The 
precipitated crystals were collected by filtration and thereafter, were 
purified by a thin layer chromatography (silica gel, developing agent: 10% 
ethanolchloroform) to give 5.2 mg (yield 2%) of the titled compound as a 
white powder. 
.sup.1 H-NMR spectrum (DMSO-d.sub.6, TMS as internal standard); 
.delta.=3.77(s, 3H.times.0.5), 3.88(s, 3H.times.0.5), 4.37(m, 2H), 6.86(t, 
J=3.7 Hz, 1H.times.0.5), 7.53(t, J=5.4 Hz, 1H.times.0.5), 8.17(s, 1H), 
8.23(s, 1H), 12.90(br, 1H)ppm, 
IR spectrum (KBr); .nu.max=2900.sup.s, 2700.sup.vs, 2660.sup.s, 1570.sup.s, 
1435.sup.m, 1390.sup.m, 1295.sup.m, 1240.sup.m, 1045.sup.w, 1010.sup.w, 
885.sup.m, 855.sup.m, 825.sup.m, 725.sup.w, 630.sup.w cm.sup.-1, 
UV spectrum: .lambda.max(H.sub.2 O)=208(24,600), 266(20,800)nm; 
.lambda.max(0.1 NHCl)=207(20,600)nm; .lambda.max(0.1 NNaOH)=272(18,400), 
280.sup.sh (13,800)nm, 
Elemental analysis; C.sub.8 H.sub.10 N.sub.6 O; Calcd. C:46.60% H:4.89% 
N:10.76%, Found C:46.95% H:5.08% N:40.33%. 
EXAMPLE 2 
Synthesis of N.sup.6 -[2-(N-methoxyimino)propyl]adenine (compound B) 
A branched chain amine compound was first synthesized and then, this was 
allowed to react with 6-chloropurine in the following manner. 
(1) Synthesis of N-[2-(N-methoxyimino)propyl]-phthalimide 
1.33 g (6.55 mmol) of N-(2-oxopropyl)phthalimide was dissolved in 30 ml of 
ethanol and to the solution was added 1.64 g (19.7 mmol) of 
O-methylhydroxylamine hydrochloride. To this suspension was added 20 ml of 
2N aqueous sodium hydroxide solution, followed by being stirred at room 
temperature for 12 hours. The reaction mixture was poured into 100 ml of 
water and extracted with chloroform (30 ml.times.3), and the combined 
organic layer was dried over magnesium sulfate. Chloroform was distilled 
off and the residual solids were washed with hexane to give 1.03 g (yield 
68%) of the titled compound as colorless crystals. 
Melting point 114.degree.-131.degree. C., 
.sup.1 H-NMR spectrum (DMSO-d.sub.6, TMS as internal standard); 
.delta.=1.83(s, 3H), 3.73(s, 3H), 4.35(m, 2H), 7.82(s, 4H)ppm, 
IR spectrum (KBr); .nu.max=2870.sup.w, 1680.sup.s, 1360.sup.s, 1315.sup.s, 
1035.sup.s, 900.sup.w, 825.sup.m, 695.sup.m cm.sup.-1, 
(2) Synthesis of N.sup.6 -[2-(N-methoxyimino)propyl]adenine (compound B) 
0.232 g (1.00 mmol) of N-[2-(N-methoxyimino)propyl]phthalimide was 
dissolved in 6 ml of methanol and to the solution was added 60 .mu.l (1.2 
mmol) of hydrazine hydrate, followed by being refluxed for 5 hours. The 
reaction mixture was cooled to room temperature and then, 20 ml of ether 
was added thereto and the mixture was left to stand at 0.degree. C. for 1 
hour to sufficiently precipitate solids. The solids were removed by 
suction filtration and the filtrate was dried over sodium sulfate and 
concentrated to 1 ml by an evaporator. This concentrated filtrate was 
dissolved in 5 ml of isopropanol and to the solution were added 105 mg 
(0.680 mmol) of 6-chloropurine and 101 .mu.l of ethyldiisopropylamine, 
followed by being refluxed with heating on an oil bath for 5 hours. The 
reaction mixture was poured into 20 ml of water and extracted with 
chloroform (15 ml.times.5). The extract was dried over magnesium sulfate 
and the solvent was distilled off. The residue was purified by a thin 
layer chromatography (silica gel, developing agent: 15% 
ethanol-chloroform) to give 32.0 mg (yield 15%) of the titled compound as 
white solids. 
.sup.1 H-NMR spectrum (DMSO-d.sub.6, TMS as internal standard); 
.delta.=1.72(s, 3H.times.0.14), 1.79(s, 3H.times.0.86), 3.74(s, 3H), 
4.28(d, J=6 Hz, 2H), 7.63(t-like, J=6 Hz, 1H), 8.07(s, 1H), 8.17(s, 1H), 
12.70(br, 1H)ppm, 
IR spectrum (KBr); .nu.max=2910.sup.s, 2770.sup.br, 1585.sup.vs, 
1245.sup.m, 1130.sup.w, 1035.sup.m, 880.sup.m, 625.sup.w cm.sup.-1. 
UV spectrum: .lambda.max(H.sub.2 O)=208(30,800), 266(24,000)nm; 
.lambda.max(0.1NHCl)=275(15,700)nm; .lambda.max(0.1NNaOH)=273(15,900), 
280.sup.sn (12,400)nm. 
Elemental analysis; C.sub.9 H.sub.12 N.sub.6 O; Calcd. C:49.08% H:5.49% 
N:38.16%, Found C:49.29% H:5.77% N:38.50%. 
EXAMPLE 3 
Synthesis of N.sup.6 -[3-(N-hydroxyimino)butyl]adenine (compound C) 
A branched chain amine compound was first synthesized and then, this was 
allowed to react with 6-chloropurine in the following manner. 
(1) Synthesis of N-[3-(N-hydroxyimino)butyl]-phthalimide 
1.08 g (5.00 mmol) of N-(3-oxobutyl)phthalimide was dissolved in 10 ml of 
methanol and to the solution was added 0.695 g (10.0 mmol) of 
hydroxylamine hydrochloride. To this suspension was added 2 ml of 5N 
aqueous sodium hydroxide solution, followed by being stirred at room 
temperature for 16 hours. The reaction mixture was poured into 100 ml of 
saturated aqueous sodium hydrogencarbonate and extracted with chloroform 
(30 ml.times.3), and the combined organic layer was dried over magnesium 
sulfate. Chloroform was distilled off and the residual solids were washed 
with hexane to give 0.98 g (yield 84%) of the titled compound as colorless 
crystals. 
Melting point 151.degree.-172.degree. C., 
.sup.1 H-NMR spectrum (DMSO-d.sub.6, TMS as internal standard); 
.delta.=1.81(s, 3H.times.0.75), 1.83(s, 3H.times.0.25), 2.3-2.6(m, 2H), 
3.78(t, J=6 Hz, 2H.times.0.75), 3.91(t, J=6 Hz, 2H.times.0.25), 7.85(s, 
4H), 10.15(bs, 1H.times.0.75), 10.34(s, 1H.times.0.25)ppm, 
(2) Synthesis of N.sup.6 -[3-(N-hydroxyimino)butyl]adenine (compound C) 
0.232 g (1.00 mmol) of N-[3-(N-hydroxyimino)butyl]phthalimide was dissolved 
in 6 ml of methanol and to the solution was added 60 .mu.l (1.2 mmol) of 
hydrazine hydrate, followed by being refluxed for 5 hours. The reaction 
mixture was cooled to room temperature and then, 20 ml of ether was added 
thereto and the mixture was left to stand at 0.degree. C. for 1 hour to 
sufficiently precipitate solids. The solids were removed by suction 
filtration and the filtrate was dried over sodium sulfate and concentrated 
to 1 ml by an evaporator. This concentrated filtrate was dissolved in 5 ml 
of isopropanol and to the solution were added 75 mg (0.50 mmol) of 
6-chloropurine and 101 .mu.l of ethyldiisopropylamine, followed by being 
refluxed on an oil bath for 5 hours. The reaction mixture was poured into 
20 ml of water and extracted with chloroform (15 ml.times.5). The extract 
was dried over magnesium sulfate and the solvent was distilled off. The 
residue was purified by a thin layer chromatography (silica gel, 
developing agent: 15% ethanol-chloroform) to give 45.1 mg (yield 22%) of 
the titled compound as white solids. 
.sup.1 H-NMR spectrum (DMSO-d.sub.6, TMS as internal standard); 
.delta.=1.80(s, 3H), 3.30(m, 2H), 3.69(m, 2H), 7.45(m, 1H), 8.08(s, 1H), 
8.19(s, 1H), 10.29(br, 1H), 12.70(br, 1H)ppm, 
IR spectrum (KBr); .nu.max=2900.sup.br,vs, 2810.sup.s, 1590.sup.s, 
1405.sup.m, 1300.sup.m, 1255.sup.s, 1160.sup.m, 930.sup.m 885.sup.m, 
635.sup.m cm.sup.-1, 
UV spectrum: .lambda.max(H.sub.2 O)=208(26,100), 267(19,100)nm; 
.lambda.max(0.1NHCl)=269(20,100)nm; .lambda.max(0.1NNaOH)=273(20,900), 
282.sup.sn (15,800)nm, 
Elemental analysis; C.sub.9 H.sub.12 N.sub.6 O; Calcd. C:49.08% H:5.49% 
N:38.16%, Found C:49.32% H:5.78% N:37.88%, 
EXAMPLE 4 
Synthesis of N.sup.6 -[3-(N-methoxyimino)butyl]adenine (compound D) 
A branched chain amine compound was first synthesized and then, this was 
allowed to react with 6-chloropurine in the following manner. 
(1) Synthesis of N-[3-(N-methoxyimino)butyl]-phthalimide 
1.08 g (5.00 mmol) of N-(3-oxobutyl)phthalimide was dissolved in 10 ml of 
methanol and to the solution was added 0.835 g (10.0 mmol) of 
O-methylhydroxylamine hydrochloride. To this suspension was added 2 ml of 
5N aqueous sodium hydroxide solution, followed by being stirred at room 
temperature for 16 hours. The reaction mixture was poured into 100 ml of 
saturated aqueous sodium hydrogencarbonate solution and extracted with 
chloroform (30 ml.times.3), and the combined organic layer was dried over 
magnesium sulfate. Chloroform was distilled off and the residual solids 
were washed with hexane to give 1.23 g (yield 100%) of the titled compound 
as white crystals. 
Melting point 55.degree.-64.degree. C., 
.sup.1 H-NMR spectrum (DMSO-d.sub.6, TMS as internal standard); 
.delta.=1.86(s, 3H.times.0.66), 1.91(s, 3H.times.0.34), 2.5(t, J=6 Hz, 
2H.times.0.66), 2.66(t, J=6 Hz, 2H.times.0.34), 3.48(s, 3H.times.0.34), 
3.65(s, 3H.times.0.66), 3.84(t, J=6 Hz, 2H.times.0.66), 3.87(t, J=6 Hz, 
2H.times.0.034), 7.85(s, 4H)ppm, 
(2) Synthesis of N.sup.6 -[3-(N-methoxyimino)butyl]adenine (compound D) 
0.282 g (1.00 mmol) of N-[3-(N-methoxyimino)butyl]phthalimide was dissolved 
in 6 ml of methanol and to the solution was added 60 .mu.l (1.2 mmol) of 
hydrazine hydrate, followed by being refluxed for 7 hours. The reaction 
mixture was cooled to room temperature and then, 20 ml of ether was added 
thereto and the mixture was left to stand at 0.degree. C. for 1 hour to 
sufficiently precipitate solids. The solids were removed by suction 
filtration and the filtrate was dried over sodium sulfate and concentrated 
to 1 ml by an evaporator. This concentrated filtrate was dissolved in 5 ml 
of isopropanol and to the solution were added 105 mg (0.680 mmol) of 
6-chloropurine and 101 .mu.l of ethyldiisopropylamine, followed by being 
refluxed on an oil bath for 5 hours. The reaction mixture was poured into 
20 ml of water and extracted with chloroform (15 ml.times.5). The extract 
was dried over magnesium sulfate and the solvent was distilled off. The 
residue was purified by a thin layer chromatography (alumina, developing 
agent: 15% ethanol-chloroform) to give 85 mg (yield 35%) of the titled 
compound as white solids. 
.sup.1 H-NMR spectrum (DMSO-d.sub.6, TMS as internal standard); 
.delta.=1.84(s, 3H), 2.47(t, J=6.5 Hz, 2H), 3.55(m, 2H), 3.70(s, 
3H.times.0.7), 3.74(s, 3H.times.0.3), 7.44(br, 1H), 8.05(s, 1H), 8.17(s, 
1H), 12.60(br, 1H)ppm, 
IR spectrum (KBr); .nu.max=3160.sup.sn,m, 3050.sup.m, 2900.sup.s, 
2770.sup.s, 1580.sup.v, 1280.sup.m, 1240.sup.m, 1125.sup.w, 1020.sup.m, 
915.sup.w, 870.sup.m, 780.sup.w, 625.sup.w cm.sup.-1, 
UV spectrum: .lambda.max(H.sub.2 O)=206(26,200), 268(15,000)nm; 
.lambda.max(0.1NHCl)=274(14,100)nm; .lambda.max(0.1NNaOH)=274(14,500), 
282.sup.sn (11,300)nm, 
Elemental analysis; C.sub.10 H.sub.14 N.sub.6 O: Calcd. C:51.27% H:6.02% 
N:35.87%, Found C:51.49% H:6.39% N:35.51%, 
Test Example 1 Cytokinin activity test based on the chlorophyll retention 
effect 
Rice seeds (var.: Nankin No. 11) were sown in the cultivation soil filled 
in a seed box and raised for about 1 month in a greenhouse (25.degree. C. 
in the daytime/15.degree. C. in the nighttime). 
Leaf sections of 1 cm long were cut off from the central portion of the 
fourth leaves of seedlings in the sixth leaf stage. A group of five leaf 
sections cut off from the seedlings were floated on 2 ml of test solution 
containing a given concentration of the test compound in a glass tubular 
bottle having an inner diameter of 32 mm. Three bottles each containing 
five leaf sections and 2 ml of the test solution were used for each 
concentration tested. These bottles were placed in the dark at 30.degree. 
C. for 3 days and thereafter, the leaf sections were put in a test tube 
containing 10 ml of 80% ethanol and this test tube was dipped in a water 
bath of 80.degree. C. for 20 minutes to extract chlorophyll. 
After cooling, 80% ethanol was added to make up 10 ml and absorbance of the 
solution was measured with a wavelength of 665 nm. 
Senescence retardation rate based on the degree of chlorophyll retention 
effect caused by test compounds was obtained by the following formula. 
The results are shown in Table 2. 
##EQU1## 
a: Absorbance of the treated group after lapse of 3 days. b: Absorbance of 
the untreated group after lapse of 3 days. 
c: Absorbance of the leaves before treated. 
TABLE 2 
______________________________________ 
Cytokinin activity test based on 
chlorophyll retention effect 
Senescence retardation rate (%) 
Concentration (mg/l) 
Compound 0.1 1 10 
______________________________________ 
B 92 96 98 
Benzyladenine 
82 95 96 
______________________________________ 
Test Example 2 Cytokinin activity test based on soybean hypocotyl section 
growth 
Soybean seeds (var.: Enrei) were disinfected with antiformin containing 1% 
effective chlorine for 12 minutes and washed with sterile water six times. 
15 ml of 1.6% agar was put in a test tube of 2.5 cm in diameter and a 
groove was made on the surface by a forceps. Then, one seed disinfected 
above was placed therein and left to stand in the dark at 30.degree. C. 
for 5 days in the dark. In a glass tubular bottle of 26 mm in inner 
diameter was put 10 ml of Miller's culture medium containing a given 
concentration of the test compound, benzyladenine or trans Zeatine, and 
2,4-D. The central portion of the hypocotyl budding out of the seed was 
cut at a thickness of 1 mm and the four sections as one group were 
transplanted onto the Miller's culture medium. Three bottles each 
containing four leaf sections and 10 ml of the Miller's culture medium 
were used for each concentration tested. They were placed in the dark at 
30.degree. C. for 3 weeks and thereafter, fresh weight of the callus was 
measured. 
The results are shown in Tables 3 and 4. 
TABLE 3 
______________________________________ 
Cytokinin activity test based on 
acceleration of cell division of soybean 
callus 
Fresh weight of soybean callus 
(mg/flask) 
Concentration (mg/1) 
Compound 0 1 10 
______________________________________ 
B -- 874 741 
Benzyladenine 
-- 809 636 
Untreated 86 
______________________________________ 
2,4-D concentration: 2 mg/l 
TABLE 4 
______________________________________ 
Cytokinin activity test based on 
acceleration of cell division of soybean 
callus 
Fresh weight of soybean callus 
(mg/flask) 
Concentration (.mu.M) 
Compound 0 0.1 1 10 
______________________________________ 
B -- 661 986 1167 
E -- 585 715 1062 
trans Zeatine 
-- 502 730 1003 
Untreated 132 
______________________________________ 
2,4-D concentration: 2 .mu.M 
Test Example 3 Tests on absorption and translocation in rice; Chlorophyll 
retention effects by penetration of compound B and benzyladenine into rice 
leaves 
Rice seeds (var.: Nankin No. 11) were sown in cultivation soil in a seed 
box and raised for about 1 month in a greenhouse (25.degree. C. in the 
daytime/15.degree. C. in the nighttime). 
Leaf sections of 30 mm in length were cut off from the central portion of 
the fifth leaves of seedlings in seventh leaf stage. The bottom of a petri 
dish was covered with one circular filter paper, which was wetted with 2.5 
ml of distilled water. A slide glass was put on the filter paper and the 
five leaf sections were arranged on the slide glass with the back of the 
leaves facing upward. 10 .mu.l of a test solution prepared by dissolving, 
at a given concentration, the test compound in 0.1% aqueous solution of 
Tween 20 was put on the central portion of the respective leaf sections. 
Three dishes each containing five leaf sections were used for each 
concentration tested. These petri dishes were covered and then left in the 
dark at 30.degree. C. for 5 days. Length of the green portion which 
remained due to the chlorophyll-retaining effect of the test solution was 
measured. 
The results are shown in Table 5. 
Comparison of the results on the test solutions of the same concentration 
revealed that compound B gave the larger green portion than benzyladenine. 
This indicates that compound B is superior in absorbability and 
translocation. 
TABLE 5 
______________________________________ 
Test on absorbability and translocation in rice 
Length of green portion (mm) 
______________________________________ 
Benzyladenine 1 (mg/l) 18.3 
10 (mg/l) 18.1 
Compound B 1 (mg/l) 20.9 
10 (mg/l) 24.3 
______________________________________ 
Test Example 4 
Shoots of Oak subjected to aseptic subcultivation were planted in a WP 
culture medium (0.3% "gelrite", 1% sucrose) containing a given 
concentration of N.sup.6 -[2-(N-methoxyimino)propyl]adenine compound B and 
were cultivated for 8 weeks with 16 hours in the light of 4,000 lux and 8 
hours in the dark per one day at 25.degree..+-.1.degree. C. Benzyladenine 
having known cytokinin activity and indolebutyric acid known as a rooting 
stimulator were used for control test. The results are shown in Table 6. 
TABLE 6 
______________________________________ 
Rooting stimulator effect of N.sup.6 -{2-(N- 
methoxyimino)propyl}adenine(compound B)on 
shoots of oak. 
Concent- Number of Number of 
Rooting 
Test ration planted rooted rate 
compounds (mg/l) shoots shoots (%) 
______________________________________ 
Compound B 
0.02 81 51 63 
0.05 92 53 58 
0.1 94 48 51 
BA 0.1 88 1 1 
IBA 0.1 75 25 33 
______________________________________ 
BA: Benzyladenine, IBA: Indolebutyric acid 
Rooting rate = (Number of rooted shoots/number of planted shoots) .times. 
100