Convenient synthesis and cytokinin activity of β-substituted 4-styrylpyridines, the simplest cytokinin analogs with a moderate cell division-promoting activity

Article abstract of DOI:10.1021/jf9502313

Designed synthesis of Z- and E-isomers of β-substituted 4-styrylpyridines as cytokinin analogs was conveniently achieved by nucleophilic and electrophilic addition of ethanol, methyl mercaptan, and hydrogen halides (HCl, HBr, and HI) to 4-(phenylethynyl)pyridine prepared by palladium-catalyzed coupling. They easily underwent E-Z photoisomerization under monochromatic UV light or sunlight. A tobacco callus assay revealed that the Z-isomers were more active than their E-isomers and that the Z-ethoxy derivative, which showed the highest activity among the β-substituted 4-styrylpyridines, was one-fifth as potent as kinetin. The Z-ethoxy derivative also promoted betacyanin biosynthesis of Amaranthus seedlings at 4-100 μM.

Full text of DOI:10.1021/jf9502313

J. Agric. Food Chem. 1996, 44, 1337
−1342
1337
Con ven ien t Syn th esis a n d Cytok in in Activity of â-Su bstitu ted
4-Styr ylp yr id in es, th e Sim p lest Cytok in in An a logs w ith a Mod er a te
Cell Division -P r om otin g Activity
Shiro Nishikawa,* Masakazu Sato, Hisaki Kojima,† Chi-e Suzuki, Nobuko Yamada,
Minoru Inagaki, Naoki Kashimura, and Hiroshi Mizuno‡
Laboratory of Biofunctional Chemistry, Department of Bioscience, Faculty of Bioresources,
Mie University, Tsu, Mie 514, J apan, and National Institute of Agrobiological Resources,
2-1-2 Kannondai, Tsukuba, Ibaraki 305, J apan
Designed synthesis of Z- and E-isomers of â-substituted 4-styrylpyridines as cytokinin analogs was
conveniently achieved by nucleophilic and electrophilic addition of ethanol, methyl mercaptan, and
hydrogen halides (HCl, HBr, and HI) to 4-(phenylethynyl)pyridine prepared by palladium-catalyzed
coupling. They easily underwent E-Z photoisomerization under monochromatic UV light or
sunlight. A tobacco callus assay revealed that the Z-isomers were more active than their E-isomers
and that the Z-ethoxy derivative, which showed the highest activity among the â-substituted
4-styrylpyridines, was one-fifth as potent as kinetin. The Z-ethoxy derivative also promoted
betacyanin biosynthesis of Amaranthus seedlings at 4-100 µM.
Keyw or d s: Synthetic cytokinin; 4-styrylpyridines; palladium-catalyzed coupling; addition reaction;
photoisomerization; tobacco callus bioassay; betacyanin bioassay
INTRODUCTION
Cytokinins promote cell division of plant tissues.
Recent studies on their analogs revealed that the purine
ring is not necessarily required for strong cytokinin
activity and is replaceable with other nitrogen-contain-
ing heterocycles. For example, modification of N6-
benzyladenine (BA, 1) on the basis of analysis of
structural correlation led to the finding of N-(2-chloro-
4-pyridyl)-N′-phenylurea (3), the strongest synthetic
cytokinin ever known, via pyrimidine derivative 2
(Figure 1; Okamoto et al., 1981; Shudo, 1994). Most
natural and synthetic cytokinins are N-alkyl and N-acyl
derivatives. In contrast, there are few carbon-substi-
tuted cytokinin analogs, although they give insight into
the mode of the cytokinin-receptor interaction that has
not been fully studied. For these N-alkyl and N-acyl
derivatives, analogous modification is also available. In
fact, 6-trans-styrylpurine derivative 4 (Nishikawa et al.,
1985, 1994) and 4-styrylpyrimidine derivative 5 (Nish-
ikawa et al., 1989) promoted significantly the growth
F igu r e 1. Structural relationships of N- and carbon-substi-
of tobacco callus tissues as well as the betacyanin
biosynthesis of Amaranthus seedlings. These results
indicate that the exocyclic nitrogen atoms of N6-
substituted adenines can be replaced with a sp2 carbon
atom without lowering their cytokinin activity. On the
other hand, however, little has been known about the
function of nitrogen atoms of the heterocyclic rings,
particularly the purine ring of N6-substituted adenines
on cytokinin action at the molecular level, in spite of
the fact that there have been some systematic studies
on deazaadenines (Rogozinska et al., 1973; Sugiyama
et al., 1975).
tuted cytokinin analogs.
Our main concern is to specify that the nitrogen atom
of the purine ring participated in cytokinin-receptor
binding and to disclose the role of other nitrogen atoms
in the binding by comparative studies on purine and
non-purine cytokinins. Pyridine derivatives which have
a minimal structure required for the activity are ideal
for this purpose. Our success in the development of the
carbon-substituted derivatives 4 and 5 prompted us to
develop 4-styrylpyridine analogs 8 and 9 as a logical
extension (Scheme 1), although (E)-4-styrylpyridine,
known as stilbazole, was completely inactive in a
betacyanin bioassay (Nishikawa et al., 1989). For the
synthesis of â-substituted 4-styrylpyridines, 4-(phenyl-
ethynyl)pyridine (7) is a key intermediate. Palladium-
catalyzed coupling using CuI as cocatalyst (Sonogashira
et al., 1975) has been widely used to introduce an
alkynyl group into pyridine (Kanesho Co. Ltd., 1981;
Sakamoto et al., 1986) because of its convenience and
* Author to whom correspondence should be ad-
dressed (e-mail nisikawa@bio.mie-u.ac.jp).
† Present address: Tsukuba Research Institute, Banyu
Pharmaceutical Co., Ltd., Tsukuba Techno Park O-ho,
3 Ohkubo, Tsukuba, Ibaraki 300-03, J apan.
‡ National Institute of Agrobiological Resources.
S0021-8561(95)00231-7 CCC: $12.00
© 1996 American Chemical Society

1338 J. Agric. Food Chem., Vol. 44, No. 5, 1996
Nishikawa et al.
Sch em e 1. Syn th esis of â-Su bstitu ted 4-Styr ylp yr i-
CuI (70 mg, 0.36 mmol) in triethylamine (18 mL) in acetonitrile
(50 mL) was stirred under a nitrogen atmosphere at room
temperature for 44 h. The resultant crystals of triethylamine
hydrochloride were filtered off, and the filtrate was evaporated
under reduced pressure to give the residue. Purification of
the residue by column chromatography (n-hexane-ethyl ac-
etate, 3:1, v/v) afforded 7 as yellow crystals (5.50 g, 85%), mp
87-91 °C. Recrystallization from n-hexane-ethyl acetate
yielded plates of mp 93 °C (lit. mp 95-95.5 °C, Smith, J r., et
al., 1948; lit. mp 102-104 °C, Lukes and Ernest, 1949; lit. mp
92-93 °C, Beccalli et al., 1985).
d in esa
(Z)- (8a ) and (E)-4-[2-(methylthio)-2-phenylethenyl]pyridine
(9a ). A mixture of alkyne 7 (897 mg, 5.00 mmol) and 15%
aqueous methyl mercaptan sodium salt (5.0 mL, 8.6 mmol) in
EtOH (10 mL) was refluxed for 1 h. TLC analysis showed
completion of the reaction. Without neutralization, the mix-
ture was dried under reduced pressure and chromatographed
on silica gel to give the Z-isomer 8a (853 mg, 75%), mp 44-51
°C. Recrystallization from n-hexane-ethyl acetate furnished
yellow needles, mp 52-54 °C: UV (MeOH) λmax 319 nm (7820),
λmin 269 nm (4890); IR (KBr) νmax 3050 (aromatic CH), 2900
a
(i) PhCtCH, (PPh3)2PdCl2, CuI, Et3N/MeCN; (ii) HX or
Me3SiX (X ) Cl, Br, I)/CHCl3, NaOEt/EtOH or MeSNa/EtOH;
(iii) hν/AcOEt.
(CH3), 1580 (CdC), 1530, 1480, 1400, 760, 700 (phenyl) cm-1
;
1H NMR (CDCl3) δ 2.01 (3H, s, SCH3), 6.60 (1H, s, CHdC),
7.2-7.6 (7H, m, 3-H, 5-H, phenyl), 8.60 (2H, d, J ) 5.1 Hz,
2-H, 6-H). Anal. Calcd for C14H13NS: H, 5.76; C, 73.97; N,
6.16. Found: H, 5.77; C, 74.00; N, 6.10. Picrate recrystallized
from EtOH was of mp 174-179 °C.
high efficiency. Simple addition of small molecules
including hydrogen halide, alcohol, and mercaptan to 7
together with E-Z-photoisomerization would provide
â-substituted (Z)- and (E)-4-styrylpyridines, as in the
cases of 6-styrylpurines (Nishikawa et al., 1985, 1994)
and 4-styrylpyrimidines (Nishikawa et al., 1989). Thus
we have synthesized some â-substituted 4-styrylpy-
ridines conveniently from commercially available 4-bro-
mopyridine (6) via alkynyl compound 7 prepared by the
palladium-catalyzed coupling and tested their cytokinin
activity in both betacyanin and tobacco callus bioassays.
Eventually, we found Z-â-ethoxy derivative 8b which
exhibited activity at the same order of concentration as
kinetin in the tobacco callus bioassay. To our knowl-
edge, there has been no report on cytokinin activity of
carbon-substituted pyridine derivatives with a moderate
cytokinin activity.
Further elution with the same solvent mixture followed by
evaporation gave the E-isomer 9a as a yellow oil (220 mg,
19%): UV (MeOH) λmax 313 nm (7660), λmin 270 nm (3000); IR
(KBr) νmax 3050 (aromatic CH), 2900 (CH3), 1670 (CHdC), 1580
(CdC), 1530, 1480, 1430, 1400, 760, 710, 700 (phenyl) cm-1
;
1H NMR (CDCl3) δ 2.31 (3H, s, SCH3), 6.28 (1H, s, CHdC),
6.75 (2H, d, J ) 5.7 Hz, 3-H, 5-H), 7.33 (5H, s, phenyl), 8.23
(2H, d, J ) 5.7 Hz, 2-H, 6-H). Picrate, mp 156-161 °C (with
sublimation), was similarly prepared. Anal. Calcd for C20H16
-
N4O7S: H, 3.53; C, 52.63; N, 12.27. Found: H, 3.43; C, 51.63;
N, 12.06.
(Z)- (8b) and (E)-4-(2-Ethoxy-2-phenylethenyl)pyridine (9b).
After sodium (162 mg, 7.0 mg atom) dissolved completely in
dry EtOH, 7 (896 mg, 5.00 mmol) was added to the solution
and refluxed for 4 days. The mixture was dried under reduced
pressure and submitted to column chromatography. Elution
with n-hexane-ethyl acetate (1:1, v/v) afforded the Z-isomer
8b as a colorless oil (552 mg, 49%) after evaporating the
solvents: UV (MeOH) λmax 298 nm (27 700), λmin 244 nm (2230);
IR (KBr) νmax 3050 (aromatic CH), 2950 (CH3), 1620, 1580
This paper describes convenient synthesis of the
â-substituted 4-styrylpyridines and their cytokinin ac-
tivity in both bioassays. In addition, a possible role of
nitrogen atoms of N6-substituted adenines is discussed
based on their structural similarity to the new cytokinin
analogs.
(CdC), 1530, 1480, 1400, 760, 700 (phenyl) cm-1 1H NMR
;
(CDCl3) δ 1.26 (3H, t, J ) 7.0 Hz, CH2CH3), 5.82 (1H, s,
CHdC), 7.2-7.5 (7H, m, 3-H, 5-H, phenyl), 8.46 (2H, d, J )
5.9 Hz, 2-H, 6-H). Picrate, mp 165-170 °C (with sublimation),
MATERIALS AND METHODS
was prepared as described above. Anal. Calcd for C21H18
-
Melting points were measured with a Yanagimoto hot stage
apparatus and are uncorrected. UV and IR spectra were
recorded on a Hitachi UV 200-10 or Shimazu UV 300 spec-
trometer and a Shimazu IR 470 spectrophotometer, respec-
tively. EI and HR mass spectra were taken with a Hitachi
M80-B and a J EOL J MS-DX-303 spectrometer, respectively.
N4O8: H, 3.99; C, 55.51; N, 12.33. Found: H, 3.94; C, 54.36;
N, 11.98.
Further elution with the same solvents separated the
E-isomer 9b from 8b. Evaporating the solvents yielded 9b as
colorless needles (161 mg, 14%), mp 82.5-87 °C, which were
recrystallized from n-hexane-ethyl acetate to afford pure
product of the same melting point: UV (MeOH) λmax 292 nm
(10 800), λmin 257 nm (4710); IR (KBr) νmax 3050 (aromatic CH),
2950 (CH3), 1620, 1580 (CdC), 1530, 1480, 1400, 1260, 760,
700 (phenyl) cm-1; 1H NMR (CDCl3) δ 1.43 (3H, t, J ) 7.0 Hz,
CH2CH3), 4.03 (2H, q, J ) 7.0 Hz, CH2CH3), 5.68 (1H, s,
CHdC), 6.75 (2H, d, J ) 6.2 Hz, 3-H, 5-H), 7.34 (5H, s, phenyl),
8.25 (2H, d, J ) 6.2 Hz, 2-H, 6-H). Picrate, mp 146-147 °C.
Anal. Calcd for C21H18N4O8: H, 3.99; C, 55.51; N, 12.33.
Found: H, 3.91; C, 54.24; N, 11.97.
(Z)-4-(2-Chloro-2-phenylethenyl)pyridine (8c). HCl gas, gen-
erated from NaCl and H2SO4, was passed through a solution
of 7 (537 mg, 2.99 mmol) in dry CHCl3 (20 mL) for 40 min,
and the mixture was refluxed. After reflux for 2 days, the
mixture was saturated with HCl again and refluxed for an
additional 2 days. The mixture was diluted with MeOH,
neutralized with 28% ammonia, dried under reduced pressure,
and purified by column chromatography (n-hexane-ethyl
acetate, 1:1, v/v) to afford colored crystalline powders (169 mg,
1H NMR spectra were measured with
a Hitachi R-90H
spectrometer (90 MHz) using tetramethylsilane as an internal
standard. For measurement of HMBC spectra, a J EOL J NM-
A500 spectrometer (500 MHz) of the Mie University Coopera-
tive Research Center was used. Microanalyses were performed
with a Yanagimoto MT-3 CHN apparatus. For photoisomer-
ization at a given wavelength, a J ASCO CT-10s monochrom-
eter equipped with a 150 W xenon lamp was used. For TLC
and column chromatography, Merck precoated silica gel plates
60F254 and Wakogel C-200 or Fuji Silisia BW-200 were used,
respectively. Unless otherwise noted, all operations including
syntheses and bioassays were carried out with shielding from
light or under light as weak as possible because of lability to
light of the â-substituted 4-styrylpyridines synthesized.
Syn th esis of Com p ou n d s. 6-(Phenylethynyl)pyridine (7).
In a manner similar to the reported methods (Sonogashira et
al, 1975; Kanesho Co., 1981), a mixture of 4-bromopyridine
hydrochloride (6; 7.00 g, 36.0 mmol; Aldrich), phenylacetylene
(4.04 g, 39.6 mmol), (PPh3)2PdCl2 (253 mg, 0.36 mmol), and

Cytokinin Analogs of 4-Styrylpyridines
J. Agric. Food Chem., Vol. 44, No. 5, 1996 1339
26%): mp 56-59 °C; UV (MeOH) λmax 287 nm (18 800), λmin
240 nm (6130); IR (KBr) νmax 3000 (aromatic CH), 1575 (CdC),
760, 680 (phenyl) cm-1; 1H NMR (CDCl3) δ 6.98 (1H, s, CHdC),
7.4-7.7 (7H, m, 3-H, 5-H, phenyl), 8.64 (2H, d, J ) 5.9 Hz,
2-H, 6-H). Anal. Calcd for C13H10ClN: H, 4.67; C, 72.40; N,
6.49. Found: H, 4.60; C, 71.07; N, 6.61.
(Z)-4-(2-Bromo-2-phenylethenyl)pyridine (8d ). Similarly, 7
(538 mg, 3.00 mmol) was dissolved in CHCl3 (20 mL) saturated
with HBr and refluxed for 8 days, during which HBr gas was
passed through the solution three times after 8 h, 2 days, and
3 days. The mixture contained unreacted 7 (by TLC). It was
dried under reduced pressure and purified by column chro-
matography (n-hexane-ethyl acetate, 1:1, v/v) to yield 8d as
a colorless crystalline mass (289 mg, 26%): mp 54-57 °C; UV
(MeOH) λmax 284 nm (19 600), λmin 241 nm (5240); IR (KBr)
P r ep a r a tion of E-Meth ylth io Der iva tive 9a by P h o-
toisom er iza tion . A mixture of 8a (252 mg, 1.11 mmol)
dissolved in ethyl acetate (50 mL) in a 100 mL Pyrex flask
was exposed directly to sunlight for 3.5 h on a fine day. The
Z/E ratio of the mixture was 57/43 (by 1H NMR). Evaporation
of the solvent and subsequent chromatographic separation (n-
hexane-ethyl acetate, 3:1, v/v) yielded the E-isomer 9a as a
yellow oil (57 mg, 23%), which in all respects was identical
with the product obtained by chemical synthesis described
above.
P r ep a r a tion of E-Br om id e 9c by P h otoisom er iza tion .
The Z-isomer 8d (119 mg) in ethyl acetate was similarly
photoisomerized indoors with indirect exposure to sunlight for
6 h to avoid decomposition. Purification by column chroma-
tography (n-hexane-ethyl acetate, 1:1, v/v) followed by evapo-
ration afforded a brown crystalline solid (41 mg, 35%), mp 25-
30 °C, positive to Beilstein’s test: UV (MeOH) λmax 273 nm
(10 600), λmin 248 nm (7700); IR (KBr) νmax 3030, 3000
(aromatic CH), 2970 (aliphatic CH), 1620 (CHdC), 1590 (CdC),
1410, 710, 690 (phenyl) cm-1; 1H NMR (CDCl3) δ 6.83 (2H, m,
3-H, 5-H), 7.20 (1H, s, CHdC), 7.34 (5H, m, phenyl), 8.38 (2H,
d, J ) 5.5 Hz, 2-H, 6-H); EIMS (70 eV) m/ z (rel intensity) 259
(M+, 24), 217 (37), 215 (79), 180 (55), 152 (60), 77 (61). Anal.
Calcd for C13H10BrN: H, 3.87; C, 60.02; N, 5.38. Found: H,
4.27; C, 63.41; N, 5.65.
Tobacco Callu s Assay. Callus tissues of Nicotiana tabacum
var. Wisconsin No. 38 grown on agar medium (Linsmaier and
Skoog, 1965) for 40 days were weighed, as reported previously
(Nishikawa et al., 1986). The assay was done in four replicas
for each concentration, and fresh weights of callus tissues were
averaged. Cytokinin activity of each compound was deter-
mined from its concentration-response curve and expressed
in terms of a concentration at which it gives the half-maximum
yield (fresh weight) induced by kinetin.
Beta cya n in Assa y. As reported (Biddington and Thomas,
1973; Nishikawa et al., 1986), 10 derooted seedlings of Ama-
ranthus caudatus L. were incubated in a phosphate buffer (pH
6.3) containing varying amount of test compound at 28 °C for
24 h in the dark, and the amount of betacyanin was measured
photometrically as differential absorbance between 542 and
620 nm.
ν
max 3000 (aromatic CH), 1610 (CHdC), 1590 (CdC), 1410, 760,
690 (phenyl), 570 cm-1; 1H NMR (CDCl3) δ 7.14 (1H, s, CHdC),
7.3-7.7 (7H, m, 3-H, 5-H, phenyl), 8.65 (2H, d, J ) 5.9 Hz,
2-H, 6-H). Anal. Calcd for C13H10BrN: H, 3.87; C, 60.02; N,
5.38. Found: H, 4.27; C, 57.37; N, 5.07.
(Z)-4-(2-Iodo-2-phenylethenyl)pyridine (8e). A mixture of 7
(176 mg, 1.00 mmol) and iodotrimethylsilane (0.80 mL, 4.9
mmol) in distilled CHCl3 (15 mL) was refluxed for 24 h. TLC
analysis showed completion of the reaction. Similar chro-
matographic purification (n-hexane-ethyl acetate, 1:1, v/v)
provided 8e as a colorless crystalline powder (176 mg, 57%):
mp 49-54 °C; UV (MeOH) λmax 277 nm (12 200), λmin 242 nm
(8400); IR (KBr) νmax 3050, 3000 (aromatic CH), 1580 (CdC),
1
1430, 1395, 755, 690 (phenyl) cm-1; H NMR (CDCl3) δ 6.98
(1H, s, CHdC), 7.3-7.7 (7H, m, 3-H, 5-H, phenyl), 8.65 (2H,
d, J ) 5.9 Hz, 2-H, 6-H). Anal. Calcd for C13H10IN: H, 3.28;
C, 50.84; N, 4.56. Found: H, 3.26; C, 50.41; N, 4.47.
(Z)-4-[1-Chloro-2-(methylthio)-2-phenylethenyl]pyridine (8f).
Alkyne 7 (180 mg, 1.00 mmol) was reacted with chlorotri-
methylsilane (1.7 mL, 13 mmol) in DMSO (4.0 mL) at 100 °C
for 4 h. After completion of the reaction (by TLC), the mixture
was concentrated under reduced pressure below 40 °C and
submitted to column chromatography. Elution with n-hex-
ane-ethyl acetate (1:1, v/v) and then with MeOH removed the
DMSO from the reaction mixture. The eluates containing 8f
were concentrated, neutralized with 28% ammonia, dried
under reduced pressure, and purified by column chromatog-
raphy (n-hexane-ethyl acetate, 1:1, v/v) to yield colorless
needles (190 mg, 73%): mp 84-89 °C; IR (KBr) νmax 2900
(CH3), 1620 (CHdC), 1580 (CdC), 1395, 1300, 1260, 750, 700
(phenyl) cm-1; 1H NMR (CDCl3) δ 1.84 (3H, s, SCH3), 7.4-7.6
(7H, m, 3-H, 5-H, phenyl), 8.71 (2H, d, J ) 6.0 Hz, 2-H, 6-H);
EIMS (70 eV) m/ z (rel intensity) 261 (M+, 100), 211 (70), 208
(14), 152 (19), 77 (76); HRMS found m/ z 261.0244, calcd for
C14H12ClNS 261.0351.
X-r a y An a lysis. X-ray diffraction data were measured on
a Rigaku AFC-5R automated four-circle diffractometer equipped
with Cu KR radiation (λ ) 1.5418 Å) at 20 °C. A total of 1648
independent reflections (2θ e 122°, θ - 2θ scan technique)
were observed (|Fo| g 3σ(Fo)). The structure was solved by
the program MULTAN (Main et al., 1984) and refined in the
usual way (final R ) 0.046, Rw ) 0.049, residual electron
density 0.29 eÅ-3). The crystal structure model was drawn
by ORTEP (J ohnson, 1976).
NMR spectrum of its E-isomer in a mixture obtained in
photoisomerization by using a monochrometer described later
is as follows: 1H NMR (CDCl3) δ 1.94 (3H, s, SCH3), 7.4-7.6
(7H, m, 3-H, 5-H, phenyl), 8.54 (2H, d, J ) 6.0 Hz, 2-H, 6-H).
Crystal data of 8f are as follows: formula, C14H12ClNS;
crystal morphology, prismatic; crystal size, 0.1 × 0.1 × 0.04
mm; Z, 8; Dc, 1.367; space group, orthorhombic, Pbca; cell
dimensions, a ) 12.447(1), b ) 17.011(1), c ) 12.064(1) with
estimated standard deviations in parentheses.
(Z)-4-(2-Ethoxy-2-phenylethenyl)pyridine N-Oxide (8g).
A
mixture of compound 8b (224 mg, 1.00 mmol) and m-chloro-
peroxybenzoic acid (70% purity, 259 mg, 1.05 mmol; Nacalai
Tesque) in distilled CHCl3 (10 mL) was stirred at room
temperature overnight. The mixture was dried and chromato-
graphed on silica gel (n-hexane-ethyl acetate, 1:1, v/v) to give
8g as a colorless oil (158 mg, 65%): IR (KBr) νmax 3060
(aromatic CH), 2920, 2890 (aliphatic CH), 1630 (CHdC), 1480,
1240 (N-oxide), 770, 700 (phenyl) cm-1; 1H NMR (CDCl3-CD3-
OD) δ 1.33 (3H, t, J ) 7.0 Hz, CH2CH3), 3.89 (2H, q, J ) 7.0
Hz, CH2CH3), 5.83 (1H, s, CHdC), 7.2-7.5 (5H, m, phen-
yl),7.61 (2H, dd, J ) 5.2, 2.1 Hz, 3-H, 5-H), 8.12 (2H, dd, J )
5.2, 2.1 Hz, 2-H, 6-H). Picrate, mp 126-130 °C. Anal. Calcd
for C21H18N4O9: H, 3.86; C, 53.62; N, 11.91. Found: H, 3.78;
C, 52.87; N, 11.96.
P h otoisom er iza tion Usin g a Mon och r om eter . Com-
pound 8a dissolved in CDCl3 (10 mg/mL, 0.50 mL) in a Pyrex
NMR tube was kept at a distance of 11 cm from the shutter of
a monochrometer and irradiated at 280, 320, or 380 nm. NMR
spectra were taken at a given time to determine the Z/E ratio
by integrating the signals of 2-H and 6-H protons of 8a and
its isomer 9a .
RESULTS AND DISCUSSION
Syn th esis of 4-Styr ylpyr idin es. Compared to R-sub-
stituted 4-styrylpyridines, there have been few â-sub-
stituted 4-styrylpyridines, some of which were synthe-
sized by the condensation of substituted acetophenone
and 4-methylpyridine (Terada et al., 1989) or 4-pyridi-
necarboxaldehyde and substituted benzylphosphonate
(Koeppel et al., 1975; Terada et al., 1986). Palladium-
catalyzed alkynylation by using (PPh3)2PdCl2-CuI (So-
nogashira et al., 1975) is a method of choice to obtain
4-(phenylethynyl)pyridine (7) (Kanesho Co., Ltd., 1981),
a key intermediate for the synthesis of â-substituted
4-styrylpyridines 8 and 9 (Scheme 1). Thus 4-bromopy-
ridine (6) was reacted with phenylacetylene in the
presence of the catalysts in a solvent mixture of MeCN-
Et3N at room temperature to yield alkyne 7 in a high
yield. While electrophilic addition of HCl to 7 using

1340 J. Agric. Food Chem., Vol. 44, No. 5, 1996
Nishikawa et al.
chlorotrimethylsilane in boiling MeOH did not proceed
at all, the reaction with HCl in CHCl3 at reflux afforded
Z-chloride 8c in a low yield (26%). The position of the
chlorine atom was determined by HMBC NMR. The
1
compound gave a cross-peak due to a long range H-
13C J coupling between its olefinic proton of singlet and
the carbons at the 3- and 5-positions of the pyridine ring,
not the ortho-carbons of the phenyl ring. This indicated
that the chlorine atom is attached at the â-position.
Compound 8c was assumed to have Z-configuration by
comparing chemical shifts in 1H NMR between a pair
of geometrical isomers which were interchangable by
photoisomerization, as described later.
Similar reaction of 7 with bromotrimethylsilane in the
presence of t-BuOH in CHCl3 gave Z-bromide 8d in 14%
yield after a prolonged reflux (16 days). Alternatively
the reaction with HBr in boiling CHCl3 improved the
yield of 8d to 26%. HCl and HBr additions by refluxing
for a long period accompanied serious degradation that
led to the low yields of 8c,d which were isolated as a
sole product. HI generated from iodotrimethylsilane in
CHCl3 was added to 7, giving Z-iodide 8e in a better
yield (57%).
The reaction of 7 with chlorotrimethylsilane in DMSO
to obtain 8c proceeded smoothly to provide an unex-
pected R,â-disubstituted product in a satisfactory yield.
Its molecular weight determined by HRMS coincided
with that of MeSCl adduct 8f. Similar to other Z-â-
F igu r e 2. Crystal structure and atomic numbering system
of E-methanesulfenyl chloride adduct 8f.
was determined on the basis of different chemical shifts
in
1H NMR of the olefinic protons, phenyl protons, and
1
those of the pyridine ring. The E-isomers showed
signals of the protons of the pyridine ring at higher field
than the Z-isomers, as expected from the shielding effect
of the phenyl ring of the former.
In case of the nucleophilic additions, the formation
of the E-isomers diminished stereoselectivity in the
reactions, but regioselectivity was still high. Compound
8b was oxidized with m-chloroperoxybenzoic acid to its
N-oxide 8g in order to assess the effect of N-oxidation
on cytokinin activity.
substituted 4-styrylpyridines 8, the adduct showed H
NMR signals (CDCl3) of 2-H and 6-H protons at lower
field (8.71 ppm) than those of its photoisomerized one
(8.54 ppm), suggesting the E-configuration. In addition,
methylthio group and chlorine atom were assumed to
be at R- and â-positions of the vinyl group, respectively,
on the basis of the following mechanistic consideration.
Methanesulfenyl chloride generated via chlorosulfonium
ion (Knipe, 1981) in the reaction of DMSO with chlo-
rotrimethylsilane as a source of hydrogen chloride
probably attacks the triple bond of 7 by AdE2 mecha-
nism (Capozzi et al., 1977). Similar methylthiation
using DMSO and chloromethyl methyl ether or acetyl
chloride (Hocker et al., 1975; Anzai, 1979) has been
reported. Thus, we assigned the structure of 8f to the
adduct. The geometry of 8f was finally confirmed by
X-ray crystallographic study as the E-configuration
(Figure 2). In crystals, 8f exists in a conformation in
which the phenyl and pyridine rings are largely slant
against the vinyl group due to bulky R- and â-substit-
uents, and the resultant interplanar angle between the
two rings is 71.0°.
P h otoisom er ization . 4-Styrylpyridines undergo E-Z
photoisomerization under UV and visible light (Galiazzo
et al., 1969). In order to study the effect of wavelength
on the Z/E ratio at a photostationary state, the Z-
methylthio derivative 8a , which photoisomerized to its
E-isomer 9a without byproduct formation, was em-
ployed for an experiment by using a monochrometer.
Absorption maxima of 8a and 9a in MeOH were 319
and 313 nm, respectively. Irradiation of 8a in CDCl3
at 280 nm for 3 h and at 320 nm for 1 h afforded a
mixture of almost the same Z/E ratio of 57/43 that was
1
determined by H NMR (Figure 3). Photoequilibrium
Electrophilic additions of HI and MeSCl under acidic
conditions afforded only the trans-isomers 8e,f, respec-
tively, with high regio- and stereoselectivity, whereas
similar additions of HCl and HBr were not selective due
to byproduct formation.
was achieved more rapidly by irradiation at 320 nm
than by irradiation at 280 nm. On the other hand,
exposure of 8a to UV light at 380 nm for 3 h gave a
mixture of the Z/E ratio of 69/31 in a new photoequi-
librium in which the amount of 8a increased. This
indicates that photostationary Z/E ratio of â-substituted
4-styrylpyridines can be altered in order to enrich one
of the geometrical isomers by irradiating at different
wavelength, as observed for R-substituted 4-styrylpy-
ridines (Galiazzo et al., 1969). Further irradiation of
the mixture at 320 nm for 1 h led to the former
photoequilibrium. These results confirm that the E-Z
isomerization under the conditions is reversible. For
other â-substituted styrylpyridines, particularly â-ha-
logenated derivatives, degradation hampered quanti-
On the other hand, nucleophilic addition of 7 under
basic conditions yielded a mixture of Z- and E-isomers.
Treatment of 7 with methyl mercaptan sodium salt in
EtOH at reflux proceeded easily to yield a mixture of
Z-8a and its E-isomer 9a , the ratio of Z/E being 86/14
(based on isolated yield). In contrast, similar addition
of EtOH in the presence of NaOEt required reflux for a
longer period to provide a mixture of Z-8b and E-isomer
9b with the Z/E ratio of 78/22. Similarly a cross-peak
arising from the coupling between the olefinic proton
and the 3- and 5-carbons in HMBC NMR spectra proved
that the methylthio and ethoxy groups of these isomers
are at the â-position. In addition, their configuration
1
tation by H NMR.
For preparative purpose, similar photoisomerization
was carried out by exposing directly or indirectly to

Cytokinin Analogs of 4-Styrylpyridines
J. Agric. Food Chem., Vol. 44, No. 5, 1996 1341
Ta ble 1. Cytok in in Activity of â-Su bstitu ted
4-Styr ylp yr id in es for th e Gr ow th of Toba cco Ca llu s of
N. ta ba cu m Va r . Wiscon sin No. 38a
compound
cytokinin activity
R1
R2
config
(µM)
8a
8b
8c
8d
8e
8f
8g
9a
H
H
H
H
H
SMe
H
H
H
SMe
OEt
Cl
Br
I
Z
Z
Z
Z
Z
E
E
E
E
E
E
0.29
0.052
3.6
0.53
1.8
ND
0.38
inactive
1.6
ND
Cl
OEt
SMe
OEt
Br
9b
9c
H
H
stilbazole
kinetin
H
inactive
0.011
a
ND, not determined. Tobacco callus assay was done in four
replicas for each concentration, and the fresh weights of callus
tissues after ca. 40 days incubation were averaged. Cytokinin
activity was determined in terms of a defined concentration
(C1/2maxK) from the dose-response curve of each compound, as
described in Materials and Methods.
F igu r e 3. Cis-trans photoisomerization of Z-methylthio
derivative 8a to its E-isomer 9a at different wavelengths.
Compound 8a in CDCl3 in a Pyrex NMR tube was irradiated
at 280 (b), 320 (9), and 380 (4) nm, and Z/E ratios were
1
determined by H NMR at a given time. Arrows indicate the
time at which the wavelength was changed.
the more sensitive tobacco callus assay was used to
assess their substituent effects on the activity. The
4-styrylpyridines except E-methylthio derivative 9a ,
which was inactive at 100 µM, were weakly or moder-
ately active, whereas E-4-styrylpyridine, stilbazole, was
inactive (Table 1). The most active among the deriva-
tives was the Z-ethoxy derivative 8b, the activity of
which was approximately one-fifth that of kinetin.
Ethoxy group was the most favorable â-substituent, as
in the case of 4-styrylpyrimidines (Nishikawa et al.,
1989). Its N-oxide 8g showed lower activity than the
parent 8b. Thus, there was no enhancement in the
activity due to N-oxidation of the pyridine ring, in
contrast to the results of N-(2-chloro-4-pyridryl)-N′-
phenylurea N-oxides (Henrie et al., 1988). Additionally,
Z-bromide 8d was as active as Z-methylthio derivative
8a . Among the Z-halides, the bromide 8d exceeded
iodide 8e and chloride 8c in activity. Thus, the observed
activity order of the Z-isomers was EtO > MeS, Br >
Cl, I . H (inactive). Since stilbazole was completely
inactive, the presence of â-substituents is essential for
the activity. We cannot explain the order in terms of
electronic or steric parameters of the â-substituent at
present. Restriction of their conformation by the â-sub-
stituent might be involved.
It is important that the activity of 8b became close
to that of kinetin in the tobacco callus bioassay because
the pyridine derivative is the simplest cytokinin-active
analog ever developed. The compound is composed of
two planar rings attached to a vinyl moiety and has only
one nitrogen atom and no hydrogen donor, different
from BA and N-(2-chloro-4-pyridryl)-N′-phenylurea.
Structural similarity of the heteroaromatic rings among
6-styrylpurines, 4-styrylpyrimidines, 4-styrylpyridines,
and BA implies that the 3-nitrogen atom of the N6-
substituted adenines plays a crucial role in cytokinin-
receptor interaction. This is supported by the fact that
N6-substituted 1-deazaadenines retained high activity
in contrast to the corresponding 3-deazaadenines
(Rogozinska et al., 1973; Sugiyama et al., 1975). It is
reasonably conceivable that the exocyclic nitrogen atom
at the 6-position acts as a substituent-directing linkage
atom (Nishikawa et al., 1986) due to its sp2 nature
(Soriano-Garcia and Parthasarathy, 1975), when we
take into consideration that the nitrogen atom is
replaceable with a sp2 carbon.
F igu r e 4. Effects of â-substituted 4-styrylpyridines and N6-
benzyladenine on betacyanin biosynthesis by Amaranthus
seedlings: Z-methylthio 8a (b), Z-ethoxy (2), Z-ethoxy N-oxide
8g (0), E-methylthio 9a (O), E-ethoxy (4), and N6-benzylad-
enine (9).
sunlight. Direct exposure of 8a in AcOEt to sunlight
for 3.5 h yielded a mixture of 8a and its E-isomer 9a
1
with the Z/E ratio of 57/43 (by H NMR); the value was
identical with that obtained in the photoisomerization
at 280 and 320 nm. Chromatographic separation gave
9a in 23% yield. Similarly, indirect exposure of Z-bromo
derivative 8d to sunlight for 6 h yielded its E-isomer
9c in 35% yield. However, the photoisomerization was
accompanied with serious degradation, even under weak
sunlight conditions.
Biologica l Activity. In an Amaranthus betacyanin
assay, Z-ethoxy derivative 8b promoted betacyanin
biosynthesis at 4-100 µM and produced almost the
same amount of betacyanin as that induced by 0.1 µM
BA at 10 µM; thus it was about 100 times weaker than
BA (Figure 4). Z-Methylthio derivative 8a was less
active than 8b, and the activity of N-oxide 8g was
intermediate between them. E-Isomers 9a ,b were
weaker than their Z-isomers 8a ,b, respectively, and
exhibited weak or no activity at concentrations above
40 µM.
Since cytokinin activities of the 4-styrylpyridines
detected in the Amaranthus assay were relatively low,

1342 J. Agric. Food Chem., Vol. 44, No. 5, 1996
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ACKNOWLEDGMENT
We thank Munetaka Ishida, Central Research Labo-
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