Synthesis and cytokinin activity of fluorescent 7- phenylethynylimidazo[4,5-b]pyridine and its riboside

Article abstract of DOI:10.1021/jf0000225

7-Phenylethynylimidazo[4,5-b]pyridine and its riboside have been newly developed as fluorescent carbon-substituted cytokinin analogues. Palladium- catalyzed coupling of 7-iodo-3-(tri-O-acetyl-β-D-ribofuranosyl)imidazo[4,5- b]pyridine with phenylacetylene followed by ammonolysis afforded the 7- phenylethynyl riboside via its tri-O-acetate. Acid hydrolysis of the riboside provided its free base, which showed a marked enhancement in fluorescence intensity in an aqueous alkaline solution. The free base and its riboside were more active than the corresponding 6-phenylethynylpurine and its riboside, respectively, in Amaranthus betacyanin and tobacco callus bioassays. Surprisingly, the imidazo[4,5-b]pyridine base exhibited strong cytokinin activity comparable to that of N⁶-benzyladenine in the tobacco callus bioassay. This compound would be useful for studying localization and transport of cytokinins in cells or tissues of plants.

Full text of DOI:10.1021/jf0000225

J. Agric. Food Chem. 2000, 48, 2559−2564
2559
Syn th esis a n d Cytok in in Activity of F lu or escen t
7-P h en yleth yn ylim id a zo[4,5-b]p yr id in e a n d Its Ribosid e
Shiro Nishikawa,* Masahiro Kurono, Kenji Shibayama, Sumiko Okuno, Minoru Inagaki, and
Naoki Kashimura
Department of Bioscience, Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, J apan
7-Phenylethynylimidazo[4,5-b]pyridine and its riboside have been newly developed as fluorescent
carbon-substituted cytokinin analogues. Palladium-catalyzed coupling of 7-iodo-3-(tri-O-acetyl-â-
D-ribofuranosyl)imidazo[4,5-b]pyridine with phenylacetylene followed by ammonolysis afforded the
7-phenylethynyl riboside via its tri-O-acetate. Acid hydrolysis of the riboside provided its free base,
which showed a marked enhancement in fluorescence intensity in an aqueous alkaline solution.
The free base and its riboside were more active than the corresponding 6-phenylethynylpurine and
its riboside, respectively, in Amaranthus betacyanin and tobacco callus bioassays. Surprisingly, the
imidazo[4,5-b]pyridine base exhibited strong cytokinin activity comparable to that of N6-benzyl-
adenine in the tobacco callus bioassay. This compound would be useful for studying localization
and transport of cytokinins in cells or tissues of plants.
Keyw or d s: 7-Alkynylimidazo[4,5-b]pyridine; 6-alkynyl-1-deazapurine; fluorescent cytokinin ana-
logue; tobacco callus growth; betacyanin biosynthesis
INTRODUCTION
N6-Adenines including N6-isopentenyladenine (2iP)
and N6-benzyladenine (BA, 1) markedly promote cell
division and regulate development of plants. The ad-
enines substantially show no fluorescence, although
they have a potent cytokinin activity. Therefore, fluo-
rescent cytokinin analogues with a potent activity have
been desired to be accessible, since they would provide
a useful tool to study the localization and the transport
of cytokinins in plant cells or tissues as well as in
binding experiments. In an early study of fluorescent
cytokinins, 2iP and BA analogues of benzoadenines, i.e.,
imidazo[4,5-g]- (2) and imidazo[ 4,5-f]quinazoline (3)
(Figure 1) were synthesized for assessing spatial pa-
rameters of the central moiety in cytokinins (Specker
et al., 1976). However, their cytokinin activities were
weak or negligible in a tobacco callus bioassay, although
they were fluorescent. The ring expansion of the purine
ring to make them fluorescent by inserting a phenyl
group resulted in a serious decrease in cytokinin activ-
ity. No attention has been paid to fluorescent cytokinins
thereafter.
On the other hand, 6-alkenyl- and 6-alkynylpurines
were also found to be cytokinin-active (Brathe et al.,
1999). We synthesized carbon-substituted analogues of
purine (Koyama et al., 1983; Nishikawa et al., 1994),
pyrimidine (Nishikawa et al., 1989), and pyridine (Nish-
ikawa et al., 1996) to identify the minimal structure as
well as active conformation responsible for the high
activity of cytokinins. Such analogues including â-sub-
stituted styryl derivatives were made conveniently from
the corresponding alkynylated azaheterocycles, among
which 6-phenylethynylpurine (5) exhibited moderate
activity, although its riboside 4 (Figure 1) was weak in
F igu r e 1. Structures of fluorescent cytokinin analogues and
related compounds.
an Amarathus bioassay (Koyama et al., 1982). We
noticed that most of the alkynylated azaheterocycles
showed fluorescence during the synthetic studies.
In recent years, purine-related 7-aminoimidazo[4,5-
b]pyridines and their ribosides have generated consider-
able interest in their biological activities involving
antitumor activity, inhibition of adenosine deaminase
(Cristalli et al., 1991), and antiviral activity against
human immunodeficiency virus (Cristalli et al., 1995).
The structural similarity of N6-adenines and 7-amino-
imidazo[4,5-b]pyridines suggested to us the development
of new carbon-substituted derivatives of the latter as
cytokinin analogues. However, such carbon-substituted
* Author to whom correspondence should be addressed
(telephone +81-59-231-9620; fax +81-59-231-9684; e-mail
nisikawa@bio.mie-u.ac.jp).
10.1021/jf0000225 CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/20/2000

2560 J. Agric. Food Chem., Vol. 48, No. 6, 2000
Nishikawa et al.
Sch em e 1. Syn th esis of Alk yn yl Der iva tives of Im id a zo[4,5-b]p yr id in esa
a
Conditions and reagents: (a) CH2I2, isoamyl nitrite/MeCN, hν, reflux; (b) PhCCH, (PPh3)2PdCl2, CuI, Et3N/MeCN, reflux; (c)
NH3/MeOH; (d) 0.1 N HCl/aqueous dioxane, reflux; (e) H2, Pd-C/MeOH.
derivatives have been rarely known until now. Early
studies of N6-alkyl (De Roos and Salemink, 1971;
Rogozinska et al., 1973; Kroon et al., 1974; Kitano et
al., 1975) and N6-acyl (Sugiyama et al., 1975; Matsubara
et al., 1978) derivatives of deazaadenines revealed that,
among the deazapurine isomers, only imidazo[4,5-b]-
pyridines retained high activity. Thus, in the develop-
ment of new fluorescent cytokinin analogues, we syn-
thesized 7-phenylethynylimizado[4,5-b]pyridine (11) and
its riboside 10 by a rational drug design in which they
could be endowed with both fluorescence and potent
cytokinin activity by transformation without ring ex-
pansion. Finally, we found that the free base 11 showed
strong fluorescence in an aqueous alkaline solution and
exhibited strong cytokinin activity comparable to that
of BA in a tobacco callus bioassay.
Thus, 7-amino-3-(2′,3′,5′-tri-O-acetyl-â-D-ribofurano-
syl)imidazo[4,5-b]pyridine (7) prepared from 2,3-di-
aminopyridine was subjected to radical iodination upon
irradiation (Nair et al., 1980) (Scheme 1). The reaction
of the amino derivative 7 with a mixture of CH2I2 and
isoamyl nitrite provided 7-iodide 8 in a moderate yield,
the structure being confirmed by LC-MS. The coupling
of 8 with phenylacetylene to make a conjugated system
ensures fluorescence of 10 and 11. The palladium-
catalyzed coupling of 8 with the terminal alkyne (So-
nogashira et al., 1975) proceeded smoothly in MeCN
(Koyama et al., 1982; Matsuda et al., 1985) to afford
7-alkynylated tri-O-acetate 9 in a high yield. Am-
monolysis of 9 provided 7-alkynyl riboside 10, which was
further hydrogenated to give phenylethyl compound 12.
The ribosyl group of 10 was cleaved in an aqueous
dioxane containing 0.1 N HCl to give its free base 11 in
a moderate yield without affecting the triple bond
(Koyama et al., 1982; Matsuda et al., 1992). The new
imidazo[4,5-b]pyridine 11 was resistant to hydrochloride
addition, in contrast to the corresponding purine base
5, which easily undergoes similar hydrogen halide
additions under electrophilic conditions to give â-sub-
stituted 6-styrylpurines (Koyama et al., 1983; Nish-
ikawa et al., 1985).
All the alkynyl derivatives synthesized were fluores-
cent, whereas the hydrogenated product 12 did not
fluoresce. Therefore, such a long conjugated system
comprised of the two aromatic rings connected with the
triple bond is necessary for fluorescence. Fluorescence
properties of imidazo[4,5-b]pyridines 10 and 11 were
compared with those of the purines 4 and 5 in aqueous
acidic, neutral and alkaline solutions. Compound 11
afforded simple fluorescence excitation and fluorescence
emission spectra under the different conditions (Figure
2). Excitation maxima of 11 appeared in a narrow range
of 310-325 nm, not far from the excitation maxima of
RESULTS AND DISCUSSION
Syn th esis. Practically, fluorescent cytokinins should
meet requirements for both high cytokinin activity and
strong fluorescence. Since cytokinins are small mol-
ecules, alteration in structure by the introduction of a
substituent or a fluorophore of even a relatively small
size may diminish or abort its activity, as verified by
quantitative structure-activity study of cytokinin-active
adenines (Iwamura et al., 1980) as well as our study on
the minimal structure of highly active cytokinins (Nish-
ikawa et al., 1996). Taking this into consideration, we
followed a different approach in which structural changes
in both the side chain and the central moiety were
minimized. In our synthesis, the ribosyl group intro-
duced at the 3-position of imidazo[4,5-b]pyridine not
only constitutes a ribose moiety of the riboside 10 but
also functions to increase the reactivity in the pal-
ladium-catalyzed coupling and is easily removed by acid
hydrolysis in the final step.

Fluorescent Imidazo[4,5-b]pyridine Cytokinins
J. Agric. Food Chem., Vol. 48, No. 6, 2000 2561
F igu r e 3. Effects of the alkynyl derivatives 4, 5, 10, and 11
on betacyanin biosynthesis of detached Amarathus seedlings:
BA (9); 4 (4); 5 (2); 10 (O); 11 (b).
F igu r e 2. Fluorescence excitation and emission spectra of 11
in aqueous neutral (- - -), 0.1 N HCl (s) and 0.1 N NaOH
(- - -) solutions. The relative fluorescence intensity is arbi-
trary for each solution.
Ta ble 1. F lu or escen t P r op er ties of th e Alk yn yl
Der iva tives of P u r in es 4 a n d 5 a n d
Im id a zo[4,5-b]p yr id in es 10 a n d 11a
H2O
0.1 N HCl
0.1 N NaOH
ex
em
(nm)
ex
(nm)
em
(nm)
ex
(nm)
em
(nm)
compd (nm)
4
5
10
11
328 416 (7.4)
329 430 (2.3) 328 423 (3.9)
330 397 (10.2) 332 447 (1.5) 327 499 (715)
310 373 (15.6) 322 406 (5.8) 322 390 (14.3)
309 363 (14.8) 319 425 (3.6) 324 395 (375)
F igu r e 4. Effects of the alkynyl derivatives 4, 5, 10, and 11
on tobacco callus growth: BA (9); kinetin; (0), 4 (4); 5 (2); 10
(O); 11 (b).
a
Numbers in parentheses indicate the relative fluorescence
intensities. Each compound (5 × 10-6 M) was excited at its
maximum at 25 °C. The fluorescence intensity of each control was
less than 0.04.
(Figure 4). The maximum callus yield of 11 was almost
the same as that of BA at the optimal concentration.
On the other hand, its riboside 10 was less active than
kinetin, but gave a maximum callus yield similar to that
of BA. The imidazo[4,5-b]pyridine base 11 induced shoot
formation in a high frequency at 40 µM, whereas its
ribose 10 promoted it in a low frequency at the same
concentration. The purines 4 and 5 were less active than
10 and 11, respectively. The former were toxic at 40 µM.
Cytokinin activities of BA, kinetin, 4, 5, 10, and 11 were
0.0063, 0.021, 4.3, 0.32, 0.061, and 0.0095 µM, respec-
tively. Thus, the order of the activity of the alkynyl
derivatives was 11 > 10 > 5 > 4 in the bioassay. A
minute structural change caused striking effects. The
displacement of the nitrogen atom at the 1-position of
5 with a methine group resulted in about 35-fold
enhancement in the activity, in accordance with the
results of N6-benzoyl-1-dezapurine (Matsubara et al.,
1978). More significantly, similar displacement of the
riboside 4 led to approximately a 70-fold increase in the
activity. Since most alkynyl derivatives of purine, pyr-
imidine, and pyridine were weak or inactive, it was
surprising to us that the alkynyl derivative 11 was
almost as active as BA.
tyrosine and tryptophan. The riboside 10 and its free
base 11 provided emission maxima at wavelength
approximately 35-45 nm shorter than those of 4 and
5, when excited at the excitation maximum of each
compound in water (Table 1). In 0.1 N HCl solution,
emission maxima of the four compounds shifted to
longer wavelength compared to those in water with the
decrease in fluorescence intensity. The most striking
was the change in fluorescence intensity in 0.1 N NaOH
solution. The free base 11 strengthened the intensity
25-fold compared to its intensity measured in water.
Similarly, the purine base 5 gave more significant
enhancement (70-fold). In contrast, the ribosides 4 and
10 gave slightly lower intensities than those measured
in water. Thus, deprotonation of the imidazo[4,5-b]-
pyridine or the purine ring under alkaline conditions
is critical for the enhancement.
Biologica l Activities. Cytokinin activities of the
imidazo[4,5-b]pyridines 10 and 11, and the purines 4
and 5 were compared in three different bioassays. In a
betacyanin bioassay, compounds 10 and 11 were strong-
er than 4 and 5, respectively (Figure 3). The order of
the activity was 11 > 10, 5 > 4. The imidazo[4,5-b]-
pyridine base 11 exhibited a strong activity, its equi-
active concentration (0.26 µM) being close to that of BA
(0.1 µM), a standard compound in this bioassay.
The free base 11 was more active than kinetin, and
slightly less active than BA in a tobacco callus bioassay
In a lettuce seed germination bioassay, however, 11
showed considerably lower activity compared to BA
(Figure 5). The activities of the four alkynyl derivatives
and BA in the lettuce seed germination bioassay were
not parallel to their activities in the tobacco callus

2562 J. Agric. Food Chem., Vol. 48, No. 6, 2000
Nishikawa et al.
Syn t h esis of Com p ou n d s. 7-Iodo-3-(2′,3′,5′-tri-O-acetyl-
â-D-ribofuranosyl)-3H-imidazo[4,5-b]pyridine (8). Method A. To
a mixture of 7 (500 mg, 1.27 mmol) in isoamyl nitrite (6.5 mL,
48.4 mmol) was added CH2I2 (6.0 mL, 74.5 mmol). The mixture
was degassed and stirred under nitrogen with illumination
with an unfrosted 200 W tungsten lamp at 90 °C for 2 h. It
was diluted with CH2Cl2, filtered, and concentrated in vacuo.
The residue was purified by column chromatography on silica
gel (EtOAc-benzene, 1:2) to give the iodide 8 as a yellow syrup
(485 mg, 76%): IR (KBr) 2930 (CsH), 1740 (CdO), 1554, 1486
1
(CdN, CdC) 1236, 1044 (CsOsC) cm-1; H NMR (CDCl3) δ
2.08, 2.12, and 2.15 (9H, s, Ac), 4.4-4.5 (3H, m, 4′-H, 5′-H),
5.70 (1H, m, 3′-H), 6.03 (1H, m, 2′-H), 6.24 (1H, d, 1′-H, J 1′,2′
) 5.5 Hz), 7.73 (1H, d, 6-H, J 5,6 ) 5.2), 8.04 (1H, d, 5-H, J 5,6
)
5.2), 8.29 (1H, s, 2-H); LC-MS m/z (rel intens) 503.9 ([M +
H]+, 100), C17H18IN3O7 requires 503.2.
Method B. To a mixture of 7 (1.48 g, 3.77 mmol) in isoamyl
nitrite (15 mL, 112 mmol) was added CH2I2 (12 mL, 149 mmol).
The mixture was reacted under the conditions described in
method A, except for illumination (Matsuda et al., 1992).
Similar workup and chromatographic separation on silica gel
as in method A gave the iodide (882 mg, 47%).
F igu r e 5. Effects of the alkynyl derivatives 4, 5, 10, and 11
on lettuce seed germination: BA (9); 4 (4); 5 (2); 10 (O); 11
(b).
7-Phenylethynyl-3-(2′,3′,5′-tri-O-acetyl-â-D-ribofuranosyl)-
3H-imidazo[4,5-b]pyridine (9). To a mixture of 8 (471 mg, 0.936
mmol), (PPh3)2PdCl2 (15 mg, 0.02 mmol), and CuI (8 mg, 0.04
mmol) in MeCN (10 mL) were added phenylacetylene (0.20
mL, 1.8 mmol) and triethylamine (0.10 mL, 0.72 mmol). The
mixture was degassed, and stirred at 90 °C under nitrogen
for 2 h. It was diluted with CH2Cl2, filtered, and concentrated
in vacuo. Purification of the residue by silica gel column
chromatography (EtOAc-benzene, 1:2) gave the alkynyl com-
pound 8 as a pale yellow syrup (417 mg, 93%): IR (KBr) 2923
(CsH), 2200 (CtC), 1740 (CdO), 1588, 1480 (CdN, CdC),
bioassay, although there was a good linear relationship
between the activities in the tobacco callus and the
betacyanin bioassays, when they were plotted against
each other (data not shown). The order of the activity,
11 > 5, 10 > 4, in the lettuce seed germination bioassay
was the same as that in the betacyanin bioassay.
The finding of a highly active imidazo[4,5-b]pyridine
derivative, 11, clearly showed that high cytokinin activ-
ity is maintained, even though the two N1- and N6-atoms
are replaced with carbon atoms. This suggests that the
exocyclic nitrogen atom does not function as a hydrogen
bond acceptor or a hydrogen donor, but orients the side
chain in a direction favorable for the activity (Nishikawa
et al., 1986). In addition, the nitrogen atom at the
1-position of purine may not play an essential role in
the receptor binding because it is replaceable with a
carbon atom, as shown in the previous and the present
studies. Otherwise, the nitrogen-carbon atom change
in purine ring may alter the basicity of the remaining
nitrogen atoms to intensify the activity.
1223 and 1044 (CsOsC), 756 and 690 (Ph CsH) cm-1 1H
;
NMR(CDCl3) δ 2.09, 2.13, and 2.16 (9H, s, Ac), 4.37-4.40 (1H,
m, 4′-H), 4.46-4.49 (2H, m, 5′-H), 5.72 (1H, m, 3′-H), 6.04 (1H,
m, 2′-H), 6.30 (1H, d, 1′-H, J 1′,2′ ) 5.5 Hz), 7.37-7.42 (3H, m,
H
m,p), 7.41 (1H, d, 6-H), 7.67-7.68 (2H, m, Ho), 8.28 (1H, s,
2-H), 8.39 (1H, d, 5-H, J 5,6 ) 4.9 Hz); LC-MS m/z (rel intens)
478.1 (100, [M + H]+), 220.3 (15), C25H23N3O7 requires 477.5.
7-Phenylethynyl-3-(â-D-ribofuranosyl)-3H-imidazo[4,5-b]-
pyridine(10). A solution of 9 (501 mg, 1.05 mmol) in MeOH
(50 mL) was saturated at 0 °C with ammonia and kept at 4
°C for 16 h. It was concentrated in vacuo to give the residue
which was purified by silica gel column chromatography
(EtOAc-MeOH, 20:1). Crystallization from EtOAc afforded the
riboside 10 (327 mg, 89%) as colorless crystals: mp 95-98 °C;
IR (KBr) 3380 (OH), 2920 (CsH), 2200 (CtC), 1592 and 1488
(CdN, CdC), 1195 and 1076 (CsOsC) 756 and 688 (Ph CsH)
cm-1; UV λmax (H2O) 321sh (ꢀ 20 200), 302 (ꢀ 27 400), λmin 253
(ꢀ 5850) nm; λmax (0.1 N HCl) 320sh (ꢀ 22 500), 301 (ꢀ 23 700),
λmin 255 (ꢀ 4920) nm; λmax (0.1 N NaOH) 322sh (ꢀ 21 600), 303
(ꢀ 27 100), λmin 254 (ꢀ 5 230) nm; 1H NMR (CD3OD) δ 3.8-3.9
(2H, m, 5′-H), 4.21 (1H, s, 4′-H), 4.38 (1H, t, 3′-H), 4.83 (1H, t,
2′-H), 6.14 (1H, d, 1′-H, J 1′,2′ ) 4.9), 7.4 (4H, m, 6-H Hm,p), 7.7
(2H, m, Ho), 8.34 (1H, d, 5-H, J 5,6 ) 4.9 Hz), 8.69 (1H, s, 2-H);
LC-MS m/z (rel intens) 352.1 ([M + H]+), 296.3 (39), 280 (20),
In this study, we synthesized a new cytokinin ana-
logue, 11, possessing both potent activity and strong
fluorescence. It will find use in the studies of membrane
transport and localization of cytokinins in plant cells
or tissues.
MATERIALS AND METHODS
1H NMR spectra were measured with a Hitachi R-90H (90
MHz) or a J EOL J NM-A500 (500 MHz) instrument at the Mie
University Cooperative Research Center, chemical shifts being
given in parts per million downfield from tetramethylsilane.
UV spectra were recorded on a Shimazu UV 300 spectrometer,
and IR spectra were taken with a Shimazu IR 470 spectrom-
eter. LC-MS analyses were performed with a Thermoquest
LCQ spectrometer by a APCI mode using methanol as solvent.
Microanalyses were carried out with a Yanagimoto MT-3 CHN
apparatus. Precoated silica gel plates 60 F254 and reversed-
phase plates RP-8 from Merck were used for TLC, and
Wakogel C-200 or Fuji Silisia BW-200 was used for column
chromatography. Fluorescence spectra were measured with a
Hitachi fluorospectrometer 650-60, and excitation and emis-
sion spectra were uncorrected.
7-Amino-3-(2′,3′,5′-tri-O-acetyl-â-D-ribofuranosyl)imidazo-
[4,5-b]pyridine was prepared as a syrup from 2,3-diamino-
pyridine by ribosylation of 7-nitroimidazo[4,5-b]pyridine (Delvin
et al., 1995; Wenzel and Seela, 1996), followed by palladium
reduction (Antonini et al., 1984; J ain et al., 1966; Cristalli et
al., 1987). Both 6-phenylethynylpurine and its riboside were
synthesized, as reported previously (Koyama et al., 1982).
220 (46), C19H17N3O4 requires 351.4. Anal. Calcd for C19H17
-
N3O4‚0.5H2O: C, 63.33; H, 5.03; N, 11.66. Found: C, 63.14;
H, 5.42; N, 9.99.
7-Phenylethynylimidazo[4,5-b]pyridine (11). The riboside 10
(100 mg, 0.285 mmol) was dissolved in a mixture of 0.2 N HCl
(5 mL) and dioxane (5 mL) and heated at reflux for 2 h. The
mixture was neutralized with saturated aqueous NaHCO3,
concentrated in vacuo, and purified by column chromatography
(EtOAc-MeOH, 20:1) to give 11 as a pale yellow syrup (46.5
mg, 74%). Recrystallization from EtOAc yielded a colorless
crystalline powder: mp 193-194 °C; IR (KBr) 2200 (CtC),
1593 (CdN, CdC), 755 and 690 (Ph CsH) cm-1; UV λmax (H2O)
322sh (ꢀ 20 700), 301 (ꢀ 28 300), λmin 252 (ꢀ 5230) nm; λmax (0.1
N HCl) 344sh (ꢀ 12 700), 318 (ꢀ 20 500), λmin 256 (ꢀ 3850) nm;
λmax (0.1 N NaOH) 315 (ꢀ 19 400), λmin 275 (ꢀ 7970) nm; 1H
NMR (CD3OD) δ 7.44 (4H, m, 5-H, Hm,p) 7.71 (2H, m, Ho), 8.39
(1H, d, 6-H, J 5,6 ) 4.9 Hz), 8.45 (1H, s, 2-H); LC-MS m/z (rel
intens) 220.2 (M + H, 100), C14H9N3 requires 219.24. Anal.

Fluorescent Imidazo[4,5-b]pyridine Cytokinins
J. Agric. Food Chem., Vol. 48, No. 6, 2000 2563
Calcd for C14H9N30.8 H2O: C, 71.97; H, 4.57; N, 17.98.
Found: C, 71.44; H, 4.29; N, 17.26.
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synthesis of base-modified nucleosides and alkylpurines:
6-Amino group replacement by H, Cl, Br and I. J . Org.
Chem. 1980, 45, 3969-3974.
Nishikawa, S.; Kumazawa, Z.; Kashimura, N.; Mizutani, H.;
Kondo, H. Synthesis of potent cytokinins from 6-alkynyl-
purines. Agric. Biol. Chem. 1985, 49, 3353-3354.
Nishikawa, S.; Kumazawa, Z.; Mizutani, H.; Kashimura, N.
Substituent-directing effect on cytokinin activity of the
R-double bond in the 6-substitutent of purine. Agric. Biol.
Chem. 1986a , 50, 1089-1091.
Nishikawa, S.; Kumazawa, Z.; Kashimura, N.; Nishikimi, Y.;
Uemura, S. Alternating dependency of cytokinin activity on
the number of methylene units in ω-phenylalkyl derivatives
of some purine cytokinins and 4-substituted pyrido[3,4-d]-
pyrimidine. Agric. Biol. Chem. 1986b, 50, 2243-2249.
Nishikawa, S.; Hayashi, E.; Kumazawa, Z.; Kashimura, N.
4-Styrylpyrimidines as a new class of cytokinins. Agric. Biol.
Chem. 1989, 53, 3387-3389.
Nishikawa, S.; Yamashita, F.; Kashimura, N.; Kumazawa, Z.;
Ohgami, N.; Mizuno, H. Synthesis and crystal structure and
cytokinin activities of â-substituted 6-styrylpurines. Phy-
tochemisty 1994, 37, 915-919.
Nishikawa, S.; Sato, M.; Kojima, H.; Suzuki, C.; Yamada, N.;
Inagaki, M.; Kashimura,; Mizuno, H. Convenient synthesis
and cytokinin activity of â-substituted 4-strylpyridines, the
simplest cytokinin analogs with a moderate cell division-
promoting activity. J . Agric. Food Chem. 1996, 44, 1337-
1342.
7-Phenylethyl-(3-â-D-ribofuranosyl)-3H-imidazo[4,5-b]pyri-
dine (12). To a solution of the riboside 10 (30.0 mg, 0.0854
mmol) in EtOH (10 mL) was added 10% Pd/C (15 mg), and
the mixture was stirred at room temperature under hydrogen
for 24 h. The catalyst was removed by filtration, and the
filtrate was concentrated in vacuo. The residue (23.9 mg, 79%)
was recrystallized from EtOAc to provide the phenylethyl
derivative 12 as colorless crystals: mp 171-173 °C; IR (KBr)
3380 (OH) 2915 (CsH), 1204, 1088 (CsOsC), 766 and 716
(Ph CsH), cm-1 1H NMR (CDCl3) δ 3.08 (2H, m, PhCsH),
;
3.33 (2H, m, PuCsH), 3.90 and 3.76 (2H, m, 5′-H), 4.20 (1H,
m, 4′-H), 4.35 (1H, t, 3′-H), 4.84 (1H, t, 2′-H), 6.08 (1H, d, 1′-
H, J 1,2 ) 6.1 Hz), 7.1-7.2 (6H, m, Ph-H, 6-H), 8.21 (1H, d,
5-H , J 5,6 ) 4.9 Hz), 8.58 (1H, s, 2-H). Anal. Calcd for
C
19H21N3O4: C, 64.21; H, 5.96; N, 11.81. Found: C, 63.47; H,
5.96; N, 11.67.
Beta cya n in Bioa ssa y. This bioassay was carried out in
two replications by using seedlings of Amaranthus caudatus
L., as reported previously (Nishikawa et al., 1986b). Briefly,
10 detached seedlings were incubated in a phosphate buffer
(pH 6.3) containing varying amounts of test sample and
L-tyrosine at 28 °C for 24 h in the dark, and the amount of
betacyanin was measured as the differential absorbance
between 542 and 620 nm. Cytokinin acivity was determined
in terms of C0.1 µM BA (µM). The averaged cytokinin activity was
calculated from the data in two experiments. The standard
error of the differential absorbance was 0.013.
Tob a cco Ca llu s Bioa ssa y. Callus tissues derived from
Nicotiana tabacum cv. Wisconsin No. 38 were grown on
Linsmaier and Skoog medium at 28 °C for 40 days in the dark,
as reported previously (Nishikawa et al., 1986b). Fresh weights
of the callus tissues obtained in four replications were aver-
aged, and cytokinin activity was determined as a defined
concentration, C1/2,max,K (µM). The averaged cytokinin activity
was calculated from the data in two experiments. The standard
error of the fresh weight was 0.20 g.
Lettu ce Seed Ger m in a tion Bioa ssa y. According to the
reported method (Nishikawa et al., 1986b), seeds of Lactuca
sativa cv. Great Lakes 366 were allowed to germinate on a
filter paper moistened with water solution containing sample
at 30.5 °C for 4 days in the dark. The germination of the control
was almost inhibited at the temperature due to thermo-
dormancy. The standard errors of the control and BA (0.1-10
µM) were 7.2% and 5.0%, respectively.
ACKNOWLEDGMENT
We are grateful to Mr. Eiji Yamazaki at The Mie
Prefectural Industrial Research Institute for taking the
LC-MS spectra.
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Recieved for review J anuary 3, 2000. Revised manuscript
received March 8, 2000. Accepted March 10, 2000.
J F0000225