Synthesis and Cytokinin Activity of New Zeatin Derivatives

Article abstract of DOI:10.1021/jf970752l

The analogue of zeatin bearing a vinylic fluorine atom and its geometrical isomer were synthesized. The fluorine atom exerts a favorable influence on cytokinin activity in the fluoro analogue of ciszeatin, but not in the fluoro analogue of zeatin itself. Another series of zeatin derivatives in which the methyl group was replaced by alkyl (ethyl, propyl, and isopropyl), phenyl, and benzyl groups were also obtained. The ethyl analogue was found to be more active than zeatin, while the others were inactive or slightly active.

Full text of DOI:10.1021/jf970752l

J. Agric. Food Chem. 1998, 46, 1577−1582
1577
Syn th esis a n d Cytok in in Activity of New Zea tin Der iva tives
M’Barek Haidoune,†,‡ Isabelle Raynaud,† Norval O’Connor,† Pascal Richomme,§
Rene´ Mornet,*,† and Michel Laloue|
Faculte´ des Sciences, Laboratoire d’Inge´nierie Mole´culaire et Mate´riaux Organiques, 2 Boulevard Lavoisier,
49045 Angers Cedex, France, SCRMN, 16 Boulevard Daviers, 49045 Angers Cedex, France, and
Laboratoire de Biologie Cellulaire, INRA, Route de Saint Cyr, 78026 Versailles Cedex, France
The analogue of zeatin bearing a vinylic fluorine atom and its geometrical isomer were synthesized.
The fluorine atom exerts a favorable influence on cytokinin activity in the fluoro analogue of cis-
zeatin, but not in the fluoro analogue of zeatin itself. Another series of zeatin derivatives in which
the methyl group was replaced by alkyl (ethyl, propyl, and isopropyl), phenyl, and benzyl groups
were also obtained. The ethyl analogue was found to be more active than zeatin, while the others
were inactive or slightly active.
Keyw or d s: Cytokinins; fluoro compounds; synthesis; biological activity
INTRODUCTION
hydrogen atom in 2 was substituted for fluorine. Thus,
an exceptionnally high cytokinin activity was expected
for fluorozeatin (5), since zeatin (1) has been found to
be more active than N6-isopentenyladenine (2) in vari-
ous bioassays (Matsubara, 1990). We describe here the
synthesis of this new zeatin analogue and of its geo-
metrical isomer 6 related to cis-zeatin (7) and the results
of their cytokinin bioassays.
Zeatin (1) and N6-isopentenyladenine (2) are the most
We previously described a method of synthesis of
zeatin (1) (Mornet and Gouin, 1977) which could be used
for zeatin analogues where the methyl group is substi-
tuted for various other groups. To enrich structure-
activity relationships in the zeatin series, we have also
synthesized compounds 8a -e to evaluate their cytoki-
nin activity.
widespread natural plant hormones of the cytokinin
family. Except for a series of N,N′-disubstituted ureas,
the most active cytokinins are N6-substituted adenines.
In this latter class of compounds, studies of structure-
activity relationships have demonstrated that optimal
cytokinin activity was observed when the N6-chain
contains five or six carbon atoms and bears a double
bond in the 2,3-position (Matsubara, 1990). Analogues
of zeatin (1) have been studied, corresponding to various
chain isomerisms (Schmitz et al., 1972), hydroxylation
or dihydroxylation (Leonard et al., 1968; Letham, 1973;
Van Staden and Drewes, 1982) and hydrogenation (Fujii
and Ogawa, 1972) of the double bond, and methylation
at the C-1 of the chain (Fujii et al., 1989). However, in
zeatin (1), neither the influence on the activity of the
replacement of the methyl group by other substituents
nor that of the replacement of the vinylic proton by
halogens has been yet investigated.
EXPERIMENTAL PROCEDURES
Melting points were measured with a Reichert Thermovar
hotstage apparatus. 1H NMR spectra were recorded with TMS
as an internal standard, at 270 MHz on a J EOL GSX 270 WB
spectrometer or at 60 MHz on a Varian EM 360 apparatus.
13C NMR spectra were obtained from the J EOL spectrometer
at 67.9 MHz, and 19F spectra from the same apparatus at 254.2
MHz, using TMS and CFCl3 as internal standards, respec-
tively. EI mass spectra were recorded with a VG Autospec
spectrometer. Microanalyses were obtained from the Service
d’Analyses du CNRS (Lyon, France).
We recently synthesized two new derivatives (3 and
4) of N6-isopentenyladenine (2), the activities of which
were found to be significantly higher than the activity
of 2 (Clemenceau et al., 1996). In particular, a near 10-
fold increase in activity was observed when the vinylic
* Author to whom correspondence should be addressed.
E-mail: Rene.Mornet@univ-angers.fr.
† Laboratoire d’Inge´nierie Mole´culaire et Mate´riaux Orga-
niques.
2-Meth yl-1-(tr ityloxy)-2-p r op en e (9). To a solution of
2-methyl-2-propen-1-ol (11.0 g, 153 mmol), triethylamine (21.6
mL, 153 mmol), and 4-(dimethylamino)pyridine (0.50 g, 4.1
mmol) in dimethylformamide (200 mL) was added trityl
chloride (43.4 g, 156 mmol). The reaction mixture was stirred
in a nitrogen atmosphere for 24 h. The solution was then
diluted with water (350 mL). The white precipitate which was
obtained was filtered and extracted with hot cyclohexane (300
‡ On leave from the University of Casablanca (Casablanca,
Morocco) from 1993 to 1996.
§ SCRMN.
| Laboratoire de Biologie Cellulaire.
S0021-8561(97)00752-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/03/1998

1578 J. Agric. Food Chem., Vol. 46, No. 4, 1998
Haidoune et al.
3
mL). The white crystals obtained on cooling were filtered and
dried to give 25.0 g (52%) of compound 9: 1H NMR (270 MHz,
CDCl3) δ 1.51 (s, 3H, CH3), 3.30 (s, 2H, CH2O), 4.72 and 5.04
(2 s, 2 × 1H, dCH2), 7.01-7.15 and 7.25-7.30 (2 m, 15H,
H-phenyl).
4J HF ) 1.9 Hz, CH2O), 4.56 (d, 2H, J HF ) 22.0 Hz, CH2N),
7.73 and 7.84 (2 m, 4H, H-phthalimide); 13C NMR (CDCl3) δ
3
2
12.3 (d, J CF ) 7.3 Hz, CH3), 36.5 (d, J CF ) 31.2 Hz, CH2N),
3
63.9 (d, J CF ) 8.3 Hz, CH2O), 119.7 (d, J ) 13.2 Hz, CdCF),
1
152.0 (d, J CF ) 251.3 Hz, dCF).
1-Br om o-1-flu or o-2-m eth yl-2-[(tr ityloxy)m eth yl]cyclo-
p r op a n e (10). To a mixture of the ethylenic compound 9 (20.0
g, 0.064 mol), benzyltriethylammonium chloride (0.4 g, 0.88
mmol), sodium hydroxide (25 mL of a 50% aqueous solution),
and dichloromethane (250 mL) was added dibromofluo-
romethane (19.2 g, 0.1 mol) dropwise while the solution was
stirred. The mixture was heated to reflux with vigorous
stirring for 36 h. After cooling, water (1 L) was added, and
the solution was extracted with dichloromethane (3 × 100 mL).
The combined organic layers were washed with water and
dried over magnesium sulfate. After evaporation of the solvent
under low pressure, the residue was chromatographed through
a silica gel column with hexane/dichloromethane (8:2) as the
eluent. The appropriate fractions were combined and evapo-
rated to leave 19 g of a mixture containing 13.7 g (50%) of 10,
as a mixture of diastereomers, and 5.3 g of the starting
compound 9 (according to 1H NMR determination). 10 was
(Z)-2-Flu or o-4-h ydr oxy-3-m eth ylbu t-2-en ylam in e (13Z).
A solution of the phthalimide 12Z (996 mg, 4 mmol) and 0.25
mL of hydrazine hydrate in methanol (20 mL) was heated to
reflux with stirring for 2 h. After cooling, the white precipitate
obtained was filtered and washed with water (5 mL). The
combined filtrates were evaporated under reduced pressure,
and the residue was chromatographed through a silica gel
column with dichloromethane/ethanol (95:5) as the eluent. 13Z
was obtained as a yellowish oil and was not further purified
(305 mg, 64%): 1H NMR (270 MHz, CDCl3) δ 1.70 (d, 3H, 4J HF
3
) 2.8 Hz, CH3), 3.4 (d, 2H, J HF ) 21.6 Hz, CH2N), 4.13 (d,
4
3
2H, J HF ) 3.0 Hz, CH2O); 13C NMR (CDCl3) δ 13.1 (d, J CF
)
)
2
3
4.2 Hz, CH3), 39.2 (d, J CF ) 31.1 Hz, CH2N), 59.1 (d, J CF
2
11.4 Hz, CH2O), 112.7 (d, J CF ) 14.5 Hz, CdCF), 155.4 (d,
1J CF ) 253.5 Hz, dCF).
(E)-2-Flu or o-4-h ydr oxy-3-m eth ylbu t-2-en ylam in e (13E).
This compound was obtained with the same procedure used
for 13Z, from impure 12E (240 mg, 1 mmol). The product was
isolated as a yellowish oil (83 mg, 70%) and was contaminated
(<10%) by 13Z: 1H NMR (270 MHz, CDCl3) δ 1.73 (d, 3H, 4J HF
not further purified: 1H NMR (270 MHz, CDCl3) δ 0.90-1.10
4
and 1.20-1.40 (2 m, 2 × 1H, CH2-ring), 1.34 (d, 3H, J HF
)
2.1 Hz, CH3), 3.06-3.14 (m, 2H, CH2O), 7.10-7.30 and 7.41-
7.47 (2 m, 15H, H-phenyl).
3
) 3.3 Hz, CH3), 3.46 (d, 2H, J HF ) 22.0 Hz, CH2N), 4.02 (d,
4
3
N-[2-F lu or o-3-m et h yl-4-(t r it yloxy)-2-b u t en yl]p h t h a l-
im id es (11Z a n d 11E). Crude cyclopropane 10 (15 g contain-
ing 10.79 g of 10, 26 mmol) and potassium phthalimide (9 g,
48.6 mmol) were dissolved in dimethylformamide (100 mL).
The mixture was heated to 110 °C for 72 h. After cooling,
water (200 mL) was added, and the mixture was extracted with
dichloromethane (3 × 100 mL). The combined organic extracts
were washed with water (100 mL) and dried over magnesium
sulfate. After evaporation of the solvent, the residue was
chromatographed through a silica gel column with hexane/
ethyl acetate (8:2) as the eluent. Two fractions were isolated
which contained respectively the isomers 11Z and 11E as
major products. After evaporation of the solvents from these
fractions, the residues were recrystallized from pentane. Pure
11Z was obtained as a white powder (6.0 g, 47%): mp 175 °C;
1H NMR (270 MHz, CDCl3) δ 1.96 (d, 3H, 4J HF ) 2.8 Hz, CH3),
2H, J HF ) 1.9 Hz, OCH2); 13C NMR (CDCl3) δ 12.3 (d, J CF
)
)
2
3
7.3 Hz, CH3), 39.1 (d, J CF ) 30.1 Hz, CH2N), 61.7 (d, J CF
9.3 Hz, CH2O), 114.3 (d, 2J CF ) 14.5 Hz, CdCF), 156.6 (d, 1J CF
) 250.2 Hz, dCF).
6-[(Z)-2-F lu or o-4-h yd r oxy-3-m eth ylbu t-2-en yla m in o]-
p u r in e (5). The amino alcohol 13Z (250 mg, 2.1 mmol),
6-chloropurine (320 mg, 2.07 mmol), and triethylamine (0.65
mL) were dissolved in ethanol (20 mL). The solution was
heated at 80 °C for 24 h. The solvent was evaporated under
reduced pressure, and the residue was recrystallized from
water. After filtration and drying at 100 °C under reduced
pressure, 5 was obtained as a white powder (350 mg, 74%):
1
mp 223-224 °C; H NMR (270 MHz, DMSO-d6) δ 1.78 (3H,
3
4J HF ) 2.6 Hz, CH3), 3.96 (s, 2H, CH2O), 4.41 (d, 2H, J HF
)
21.0 Hz, CH2N), 4.67 (br s, 1H, OH), 7.80 (br s, 1H, NH), 8.10
and 8.20 (2 s, 2 × 1H, 2 and 8H-purine); 19F NMR (pyridine-
d5) δ -115.9 (unresolved peak). Elemental analysis found: C,
50.50; H, 5.10; N, 29.03. Calcd for C10H12FN5O: C, 50.63; H,
5.09; N, 29.05.
4
3
3.73 (d, 2H, J HF ) 3.3 Hz, CH2O), 4.44 (d, 2H, J HF ) 20.2
Hz, CH2N), 7.10-7.35 and 7.40-7.50 (2 m, 15H, H-triphenyl),
7.72 and 7.83 (2 m, 2 × 2H, H-phthalimide). Elemental
analysis found: C, 77.94; H, 5.20; F, 3.74; N, 2.81. Calcd for
6-[(E)-2-F lu or o-4-h yd r oxy-3-m eth ylbu t-2-en yla m in o]-
p u r in e (6). It was prepared from the impure amino alcohol
13E (59.5 mg, 0.5 mmol), with the same procedure used for 5.
The crude product 6 obtained was purified by reversed phase
C
32H26FNO3: C, 78.19; H, 5.33; F, 3.86; N, 2.85. 11E was
obtained as a white powder containing about 10% of its isomer
11Z (3.0 g, 23.5%): 1H NMR (270 MHz, CDCl3) δ 1.70 (d, 3H,
4
4J HF ) 3.5 Hz, CH3), 3.79 (d, 2H, J HF ) 1.7 Hz, CH2O), 4.20
chromatography through a Lichroprep (40-63 µm) glass
(d, 2H, 3J HF ) 19.7 Hz, CH2N), 7.10-7.30 and 7.40 (2 m, 15H,
H-triphenyl), 7.63 and 7.75 (2 m, 2 × 2H, H-phthalimide).
N-[(Z)-2-F lu or o-4-h yd r oxy-3-m eth yl-2-bu ten yl]p h th a l-
im id e (12Z). The phthalimide 11Z (3.58 g, 7.3 mmol) was
dissolved in methanol (100 mL), and p-toluenesulfonic acid (1.5
g, 7.5 mmol) was added. The solution was stirred for 48 h at
room temperature. The solvent was evaporated, and the
residue was chromatographed through a silica gel column with
dichloromethane/ethanol (95:5) as the eluent. Evaporation of
the appropriate fraction left pure 12Z as a white powder (1.20
g, 66%): mp 111 °C; 1H NMR (270 MHz, CDCl3) δ 1.96 (d, 3H,
column (310 × 25 mm), using water/methanol (70:30) as the
eluent. After evaporation of the solvents from the appropriate
fractions of the eluent, pure 6 (59 mg, 50%) was obtained as a
1
white powder: mp 220 °C; H NMR (270 MHz, DMSO-d6) δ
1.63 (d, 3H, 4J HF ) 3.20 Hz, CH3), 4.08 (d, 2H, 4J HF ) 4.23 Hz,
CH2O), 4.40 (d, 2H, 3J HF ) 20.3 Hz, CH2N), 4.8 (br s, 1H, OH),
7.7 (br s, 1H, NH), 8.12 and 8.19 (2 s, 2 × 1H, 2 and 8
H-purine); 19F NMR (pyridine-d5) δ -113.0 (unresolved peak).
Elemental analysis found: C, 50.47; H, 5.11; N, 28.82. Calcd
for C10H12FN5O: C, 50.63; H, 5.09; N, 29.05.
Gen er a l Meth od of P r ep a r a tion of th e 2-Su bstitu ted
(E)-1-Acetoxy-4-(N-p h th a lim id o)bu t-2-en es (15a -e). Po-
tassium phthalimide (11.1 g, 60 mmol) and a chloroacetate 14
(50 mmol) were stirred overnight in dimethylformamide (100
mL). Water (100 mL) was then added, and the mixture was
extracted with diethyl ether (3 × 100 mL). The combined
organic layers were dried over sodium sulfate, and the solvent
was evaporated. The residue was recrystallized from petro-
leum ether or petroleum ether/toluene, leaving white crystals.
15a (2-ethyl) (13.3 g, 93%): mp 56.5 °C (petroleum ether); 1H
NMR (60 MHz, CCl4) δ 1.10 (t, 3H, J ) 7.5 Hz, CH3CH2), 2.02
(s, 3H, CH3CdO), 2.28 (q, 2H, J ) 7.5 Hz, CH2CH3), 4.25 (d,
2H, J ) 7.5 Hz, CH2N), 4.47 (s, 2H, CH2O), 5.48 (t, 1H, J )
7.5 Hz, HCd), 7.60-8.00 (m, 4H, H-phenyl). Elemental
4
4J HF ) 2.8 Hz, CH3), 4.22 (d, 2H, J HF ) 3.1 Hz, CH2O), 4.57
3
(d, 2H, J HF ) 20.0 Hz, CH2N), 7.70 and 7.80 (2 m, 2 × 2H,
3
H-phthalimide); 13C NMR (CDCl3) δ 13.3 (d, J CF ) 4.2 Hz,
2
3
CH3), 35.2 (d, J CF ) 31.2 Hz, CH2N), 59.7 (d, J CF ) 10.4 Hz,
CH2O), 116.7 (d, 2J CF ) 11.4 Hz, CdCF), 149.0 (d, 1J CF ) 250.0
Hz, dCF). Elemental analysis found: C, 62.59; H, 4.72; F,
7.54; N, 5.64. Calcd for C13H12FNO3: C, 62.65; H, 4.85; F, 7.62;
N, 5.62.
N-[(E)-2-F lu or o-4-h yd r oxy-3-m eth yl-2-bu ten yl]p h th a l-
im id e (12E ). This compound was obtained (300 mg, 52%)
with the same procedure used for 12Z from the impure
phthalimide 11E (1.139 g, 2.32 mmol). It was contaminated
by a small amount (<10%) of its isomer 12Z: 1H NMR (270
4
MHz, CDCl3) δ 1.76 (d, 3H, J HF ) 3.3 Hz, CH3), 4.26 (d, 2H,
analysis found: C, 66.87; H, 5.92; N, 4.63. Calcd for C16H17-

Cytokinin Activity of New Zeatin Derivatives
J. Agric. Food Chem., Vol. 46, No. 4, 1998 1579
NO4: C, 66.89; H, 5.96; N, 4.88. 15b (2-propyl) (13.4 g, 89%):
mp 72 °C (petroleum ether); H NMR (60 MHz, CCl4) δ 1.00
Sch em e 1. Syn th esis of th e Tetr a su bstitu ted F lu or o-
vin ylic Com p ou n d s 11 fr om 2-Meth yl-2-p r op en -1-ol
1
(t, 3H, J ) 7.0 Hz, CH3CH2), 1.20-1.90 (m, 2H, CH2CH2CH3),
2.02 (s, 3H, CH3CdO), 2.30 (t, 2H, J ) 7.0 Hz, CH2CH2CH3),
4.27 (d, 2H, J ) 7.0 Hz, CH2N), 4.43 (s, 2H, CH2O), 5.53 (t,
1H, J ) 7.0 Hz, HCd), 7.50-7.90 (m, 4H, H-phenyl). Elemen-
tal analysis found: C, 67.67; H, 6.32; N, 4.54. Calcd for C17H19
-
NO4: C, 67.76; H, 6.36; N, 4.65. 15c (2-isopropyl) (12.9 g,
86%): mp 56.5 °C (petroleum ether); 1H NMR (60 MHz, CCl4)
δ 1.15 [d, 6H, J ) 7 Hz, (H3C)2CH], 2.02 (s, 3H, CH3CdO),
3.20 [h, 1H, J ) 7.0 Hz, CH(CH3)2], 4.30 (d, 2H, J ) 7.0 Hz,
CH2N), 4.50 (s, 2H, CH2O), 5.47 (t, 1H, J ) 7.0 Hz, HCd),
7.60-7.90 (m, 4H, H-phenyl). Elemental analysis found: C,
67.59; H, 6.25; N, 4.72. Calcd for C17H19NO4: C, 67.76; H, 6.36;
N, 4.65. 15d (2-phenyl) (13.2 g, 79%): mp 87-88 °C (petro-
1
leum ether/toluene); H NMR (60 MHz, CCl4) δ 1.97 (s, 3H,
CH3CdO), 4.20 (d, 2H, J ) 7.0 Hz, CH2N), 4.70 (s, 2H, CH2O),
5.72 (t, 1H, J ) 7.0 Hz, HCd), 7.33 (br s, 5H, H-phenyl), 7.60-
7.90 (m, 4H, H-phthalimide). Elemental analysis found: C,
71.59; H, 5.02; N, 4.22. Calcd for C20H17NO4: C, 71.63; H, 5.11;
N, 4.18. 15e (2-benzyl) (14.3 g, 82%): mp 103-104 °C
MHz, DMSO-d6) δ 3.40 (s, 2H, CH2-benzyl), 3.72 (br s, 2H,
CH2O), 4.28 (br s, 2H, CH2N), 4.78 (br s, 1H, OH), 5.70 (t, 1H,
J ) 7.0 Hz, dCH), 7.20-7.40 (m, 5H, H-phenyl), 7.80 (br s,
1H, NH), 8.08 and 8.18 (2 s, 2 × 1H, 2 and 8 H-purine); HRMS
found 295.14177 (M+), calcd for C16H17N5O 295.14331.
Biologica l Assa y. Compounds 5, 6, and 8a -e were
assayed according to the procedure previously described
(Clemenceau et al., 1996; Nogue et al., 1995), using zeatin (1)
and its geometrical isomer cis-zeatin (7) as reference active
compounds.
1
(petroleum ether/toluene); H NMR (60 MHz, CCl4) δ 1.95 (s,
3H, CH3CdO), 3.67 (s, 2H, CH2-benzyl), 4.42 (d, 2H, J ) 7.0
Hz, CH2N), 4.42 (s, 2H, CH2O), 5.73 (t, 1H, J ) 7.0 Hz, HCd),
7.23 (br s, 5H, H-phenyl), 7.60-7.90 (m, 4H, H-phthalimide).
Elemental analysis found: C, 72.25; H, 5.40; N, 4.16. Calcd
for C21H19NO4: C, 72.19; H, 5.48; N, 4.01.
Gen er a l Meth od of P r ep a r a tion of th e (E)-3-Alk yl(or
p h en yl)-4-h yd r oxybu t-2-en yla m in es (16a -e). A suspen-
sion of a phthalimide/ester 15 (10 mmol) and of barium
hydroxide octahydrate (9.45 g, 30 mmol) in water (15 mL) was
heated to reflux for 3 h. After cooling, the solution was
acidified to pH 3. After centrifugation of the mixture, the
supernatant was evaporated to dryness under reduced pres-
sure. Recrystallization of the residue in ethanol left a gummy
product which was used without further purification, except
for 15c (3-isopropyl), which was obtained as white plates (1.226
g, 69%): mp 70-80 °C dec. Elemental analysis found: C,
47.26; H, 9.14; N, 7.93. Calcd for C14H32N2O6S: C, 47.17; H,
9.05; N, 7.86.
RESULTS AND DISCUSSION
Ch em istr y. Fluorozeatins 5 and 6. Most of the
methods used for obtaining tetrasubstituted fluorovi-
nylic compounds were initially investigated for the
synthesis of the trisubstituted analogues. These include
condensation of diethyl fluoromalonate with ketones
(Kitazume and Ishikawa, 1981), reaction of trifluorovi-
nyllithium with ketones (Poulter et al., 1977; Normant
et al., 1975), Grignard reaction of 1-acyl-1,2-difluoro-2-
phenylsulfanylethylenes (Nakayama et al., 1985), alkyl-
ation by alkyl halides of ethyl phenylsulfinylfluoroac-
etate (Allmendiger, 1991), and Horner-Wadworth-
Emmons reaction of a ketone with a 2-(diethylphos-
phono)-2-fluoroacetate (Massoudi et al., 1983; Etemad-
Moghadam and Seyden-Penne, 1985; Machleidt and
Weissendorf, 1964), with the corresponding acid (Cou-
trot and Grison, 1987) or with the corresponding nitrile
(Xu and Desmarteau, 1992). The addition of bromof-
luorocarbene to a double bond, followed by the ring
opening of the cyclopropane with the use of a nucleo-
phile (Chau and Schlosser, 1974; Schlosser et al., 1975;
Schlosser and Chau, 1975; Mu¨ller et al., 1977), was also
an efficient method. The nucleophiles used included the
diethyl malonate anion, the acetate anion, or toluene.
The addition of the carbene was not stereoselective, and
a mixture of the two fluoroethylenic compounds was
generally obtained at the final step.
Gen er a l Meth od of P r ep a r a tion of th e 6-[(E)-3-Alk yl-
(or p h en yl)-4-h yd r oxybu t-2-en yla m in o]p u r in es (8a -e).
6-Chloropurine (154.5 mg, 1 mmol), an amine 16 as the
ammonium sulfate (0.75 mmol), and triethylamine (1 mL) were
heated in butanol to reflux for 2 h. After cooling, the solvent
was evaporated to dryness under reduced pressure, and the
residue was recrystallized from water with decolorizing on
Darco carbon, leaving white crystals. 8a (3-ethyl) (82 mg,
35%): mp 174-176 °C; 1H NMR (270 MHz, DMSO-d6) δ 0.99
(t, 3H, J ) 7.0 Hz, CH3), 2.13 (q, 2H, J ) 7.0 Hz, CH2CH3),
3.84 (br s, 2H, CH2O), 4.15 (br s, 2H, CH2N), 4.70 (br s, 1H,
OH), 5.50 (t, 1H, J ) 7.0 Hz, dCH), 7.81 (br s, 1H, NH), 8.08-
8.17 (2 s, 2 × 1H, 2 and 8-H purine). Elemental analysis
found: C, 56.54; H, 6.66; N, 30.12. Calcd for C11H15N5O: C,
56.64; H, 6.48; N, 30.02. 8b (3-propyl) (148 mg, 60%): mp
1
129 °C; H NMR (270 MHz, DMSO-d6) δ 0.89 (t, 3H, J ) 6.5
Hz, CH3), 1.40 (m, 2H, CH2CH3), 2.08 (t, 2H, J ) 7.5 Hz, CH2-
CH2CH3), 3.83 (br s, 2H, CH2O), 4.15 (br s, 2H, CH2N), 4.70
(m, 1H, OH), 5.54 (t, 1H, J ) 7.0 Hz, dCH), 7.66 (br s, 1H,
NH), 8.09-8.18 (2 s, 2 × 1H, 2 and 8-H purine). Elemental
analysis found: C, 58.09; H, 6.86; N, 28.27. Calcd for
C
12H17N5O: C, 58.28; H, 6.93; N, 28.32. 8c (3-isopropyl) (128
1
We selected this last method as a common source of
the two geometrical isomers, for building the skeleton
of the aliphatic chain in fluorozeatins 5 and 6 (Scheme
1). We started from 1-hydroxy-2-methyl-2-propene by
protecting its alcohol function with the trityl group. The
obtained ethylenic compound 9 reacted with the carbene
generated from dibromofluoromethane and sodium hy-
droxide in a phase-transfer conditions reaction. At this
step, the cyclopropane 10 which was a mixture of
diastereomers, considering the complexity of the NMR
signals corresponding to the ring methylene protons,
could not be completely separated from the unreacted
mg, 52%): mp 226 °C; H NMR (270 MHz, DMSO-d6) δ 1.03
(d, 6H, J ) 7.1 Hz, 2 CH3), 2.94 [h, 1H, J ) 7.0 Hz, CH(CH3)2],
3.91 (br s, 2H, CH2O), 4.18 (br s, 2H, CH2N), 4.59 (br s, 1H,
OH), 5.50 (t, 1H, J ) 7.0 Hz, dCH), 7.73 (br s, 1H, NH), 8.08
and 8.17 (2 s, 2 × 1H, 2 and 8-H purine). Elemental analysis
found: C, 57.97; H, 7.15; N, 28.23. Calcd for C12H17N5O: C,
58.28; H, 6.93; N, 28.32. 8d (3-phenyl) (70 mg, 25%): mp 130
°C; 1H NMR (270 MHz, DMSO-d6) δ 4.10 (br s, 2H, CH2N),
4.15 (br s, 2H, CH2O), 5.0 (br s, 1H, OH), 5.85 (t, 1H, J ) 7.0
Hz, dCH), 7.25-7.50 (m, 5H, H-phenyl), 8.07 and 8.16 (2 s, 2
× 1H, 2 and 8-H purine). Elemental analysis found: C, 64.31;
H, 5.29; H, 25.01. Calcd for C15H16N5O: C, 64.04; H, 5.37; N,
24.89. 8e (3-benzyl) (207 mg, 70%): mp 217 °C; 1H NMR (270

1580 J. Agric. Food Chem., Vol. 46, No. 4, 1998
Haidoune et al.
Sch em e 2. Syn th esis of F lu or ozea tin 5
F igu r e 1. Significant nOe enhancements (percentage) ob-
served for 11Z and 12E.
starting material 9. The Rf of this compound in thin-
layer chromatography was indeed very close to that of
10 under various conditions of elution. The crude
product 10 reacted with potassium phthalimide as a
nucleophile, leaving a mixture of the two geometrical
isomers 11Z and 11E. Pure fluoroethylenic phthalimide
11Z was isolated by chromatography on silica gel and
further recrystallization, but after the same treatment,
its isomer 11E remained contaminated by 11Z.
The route from the phthalimide 11Z to the fluo-
rozeatin 5 is described in Scheme 2. The trityl group
was first eliminated by methanolysis in the presence of
p-toluenesulfonic acid, leading to the phthalimide alco-
hol 12Z which further reacted with hydrazine to give
the amino alcohol 13Z. Reaction of this amine with
6-chloropurine in the last step gave finally the expected
fluorozeatin 5. A similar pathway from the impure
compound 11E gave the fluorozeatin 6, which was
separated from a small amount of its isomer 5 by
reversed phase chromatography.
F igu r e 2. Cytokinin activity assay. Fresh weight of mutant
plantlets of N. plumbaginifolia cultured with compounds 5 and
6 at various concentrations. The response is compared to that
of zeatin (1) and cis-zeatin (7). Bars indicate standard devia-
tions.
Sch em e 3. Syn th eses of Zea tin An a logu es 8a -e (for
8a , R ) Et; for 8b, R ) P r ; for 8c, R ) iP r ; for 8d , R )
P h ; a n d for 8e, R ) Bz)
The configurations of the ethylenic isomers were
checked by NMR spectroscopy. Examination of 1H NMR
data found in the literature for tetrasubstituted ethyl-
enic compounds revealed that the allylic coupling con-
4
stants J HF between the vinylic fluorine nucleus and
methyl protons followed the rule J trans < J cis (Normant
et al., 1975; Poulter et al., 1977; Lovey and Pauson,
1982; Coutrot et al., 1987; Abraham and Ellison, 1987).
This feature was also found for compounds 5, 6, and
11-13. However, 13C NMR data, including CF coupling
constants, were not available for tetrasubstituted fluo-
3
3
rovinylic compounds. A correlation J cis < J trans was
found in trisubstituted fluoroethylenes (Bosch et al.,
1987), but we did not found a similar sequence with
tetrasubstituted compounds 12 and 13. The 13C NMR
zeatin (1) (Mornet and Gouin, 1977a) from the allylic
chlorides 14a -e (Mornet and Gouin, 1977b) (Scheme
3). Phthalimides 15a -e were obtained by reaction of
these chlorides with potassium phthalimide in dimeth-
ylformamide. Hydrolysis of both the phthalimide and
the acetate was carried out by heating compounds
15a -e in an aqueous solution of barium hydroxide.
Amino alcohols 16a -e were isolated as the ammonium
sulfates and further reacted with 6-chloropurine to give
the expected N6-substituted adenines 8a -e.
Biologica l Activity. The biological properties of
zeatin derivatives 5, 6, and 8a -e were evaluated with
the same bioassay that was used for the previously
studied fluorocytokinins (Clemenceau et al., 1996), using
mutant plantlets of Nicotiana plumbaginifolia (Nogue
et al., 1995), which exhibit a characteristic hypertrophy
specifically evoked by cytokinins.
3
spectra of these compounds showed that J transCH F (4.2
3
3
Hz) was smaller than J cisCH F (7.3 Hz). Thus, the
configurations of the fluoroeth3ylenic compounds were
definitely assigned from {1H}-1H nuclear Overhauser
effect studies (NOEDIFF) on final compounds 5 and 6.
Presaturation of the CH2O protons led to an enhance-
ment of 7% in 5 and 8% in 6 for the methyl proton
signal. However, a 3% enhancement of the CH2N signal
was observed in 6, while the expected negative relayed
nOe (-3%) appeared for 5 for the same protons. The
configurations of the phthalimide ether 11E and of the
phthalimide alcohol 12Z have been also assigned from
nOe studies. The signal enhancements, consistent with
the proposed structures, are shown in Figure 1.
The variations of the fresh weight of plantlets versus
concentration of fluorozeatins 5 and 6, and of reference
compounds zeatin (1) and cis-zeatin (7), are represented
Zeatin Analogues 8a -e. Zeatin analogues 8a -e were
synthesized using a pathway similar to that used for

Cytokinin Activity of New Zeatin Derivatives
J. Agric. Food Chem., Vol. 46, No. 4, 1998 1581
zeatin (5). The particular behavior of this compound
might be attributed to the existence of an internal
hydrogen bond between the fluorine and the OH hy-
drogen atoms. Examination of a molecular model shows
that the loss of side chain planarity which could be
detrimental to activity (Hecht et al., 1970) is not
observed. Moreover, the model allows the three atoms
(F‚‚‚H-O) to be almost aligned, with a F-O distance
(2.26 Å) that is compatible with the existence of a weak
but true hydrogen bond (Howard et al., 1996). In this
case, the molecule might be forced to adopt a conforma-
tion unfavorable to the binding at the cytokinin recep-
tors. However, this hypothesis appears to be unprob-
able if we consider the weak energy expected for such a
hydrogen bond. As a comparison, a hydrogen bond
energy as weak as 1.48 kcal mol-1 has been calculated
recently for fluoroethylene in water (Howard et al.,
1996). One will be able to verify this hypothesis only
when receptors to cytokinins are discovered.
Except for the ethyl analogue 8a (Figure 3A,B) which
was as active as zeatin (1), zeatin analogues 8a -e
exhibit weaker activity than zeatin (1), 8d and -e being
completely inactive, as seen with the absence of root
growth inhibition. In this respect, at the lowest con-
centrations assayed (i.e. 0.03-0.3 µM), 8a was slightly
more active than zeatin in term of root growth inhibition
(Figure 3A). These observations let us suppose that
zeatin (1) is not the optimal ligand for the cytokinin
receptors. In particular, the bulk and hydrophobicity
of the chain may be slightly increased in the surround-
ings of the methyl group for a better fitting. This
information may be valuable for further studies of three-
dimensional quantitative structure-activity relation-
ships (QSAR).
F igu r e 3. Cytokinin activity assay. (A) Phenotype of mutant
plantlets of N. plumbaginifolia cultured on compounds 8a -e
at various concentrations. (B) Fresh weight of plantlets
cultured on compounds 8a -c at various concentrations. Bars
indicate standard deviations. In both cases, the response is
compared to that of zeatin (1).
LITERATURE CITED
Abraham, R. J .; Ellison, S. L. R.; Barfield, M.; Thomas, W. A.
An experimental and theoretical investigation of orienta-
tional dependence of H-F couplings in fluoro-olefins. J .
Chem. Soc., Perkin Trans. 2 1987, 977-985.
Allmendiger, T. Ethyl phenylsulfinyl fluoroacetate, a new and
versatile reagent for the preparation of R-fluoro-R,â-
unsaturated carboxylic acid esters. Tetrahedron 1991, 47,
4905-4914.
Bosch, M. P.; Camps, F.; Fabria`s, G.; Guerrero, A. Determi-
nation of the stereochemistry of disubstituted fluoroethyl-
enes by 13C NMR spectroscopy. Magn. Reson. Chem. 1987,
25, 347-351.
Chau, L. V.; Schlosser, M. Silberion-induzierte ringo¨ffnung von
chlorofluorocyclopropanen (Silver ion induced ring opening
of chlorofluorocyclopropanes). Synthesis 1974, 115-116.
Clemenceau, D.; Cousseau, J .; Martin, V.; Molines, H.; Wak-
selman, C.; Mornet, R.; Nogue, F.; Laloue, M. Synthesis and
cytokinin activity of two fluoro derivatives of N6-isopente-
nyladenine. J . Agric. Food Chem. 1996, 44, 320-323.
Coutrot, P.; Grison, C.; Sauveˆtre, R. 2-Diethoxyphosphoryl
alcanoic acid dianions (lithium-R-lithiocarboxylates) [Ste-
reoselective synthesis of (E)-2-fluoro-2,3-ethylenic esters by
Wittig-Horner reaction of methyl (diethylphosphono)-2-
fluoroacetate. Comparative study with methyl (diphe-
nylphosphono)-2-fluoroacetate]. J . Organomet. Chem. 1987,
332, 1-8.
Etemad-Moghadam, G.; Seyden-Penne, J . Synthe`se ste´re´ose´-
lective d’esters R,â-ethyle´niques R-fluore´s E par re´action de
Wittig-Horner a` partir du die´thyl phosphono R-fluoroac-
e´tate de me´thyle. Etude comparative avec le diphe´nyl
phosphonoxy R-fluoroace´tate de me´thyle. Bull. Soc. Chim.
Fr. 1985, 448-454.
in Figure 2. As observed with other bioassays (Schmitz
et al., 1972), cis-zeatin was less active than zeatin.
Maximal hypertrophy of the plantlets was obtained in
the presence of 30 µM cis-zeatin. Such a stimulation
was already attained in the presence of 3 µM zeatin.
On the contrary, the fluoro analogue (6) of cis-zeatin
was more active than zeatin at intermediate concentra-
tions (1 and 3 µM) and at high concentrations (10 and
30 µM), for which a relative inhibition was observed due
to a supraoptimal effect. Thus, in this range of concen-
tration, 6 was at least 10-fold more active than cis-zeatin
(7) itself, as previously observed in the case of N6-
isopentenyladenine and of its fluoro analogue (Clem-
enceau et al., 1996). However, and unexpectedly, such
an increase of cytokinin activity was not observed in the
case of the fluoro analogue (5) of zeatin which appeared
to be about 10-fold less active than the parent compound
(1) over the whole range of concentrations.
Thus, the beneficial effect on cytokinin activity at-
tributed to a vinylic fluorine atom, which we observed
in the fluoro analogue of N6-isopentenyladenine (3)
(Clemenceau et al., 1996), is confirmed only in the
analogue of cis-zeatin (7). We proposed earlier that the
presence of the vinylic fluorine atom in these molecules
would render them less sensitive to oxidative cleavage
of the aliphatic chain by cytokinin oxidases. However,
this would not be the case for the fluoro analogue of
Fujii, T.; Ogawa, N. Absolute configuration of (-)-dihy-
drozeatin. Tetrahedron Lett. 1972, 3075-3078.

1582 J. Agric. Food Chem., Vol. 46, No. 4, 1998
Haidoune et al.
Fujii, T.; Itaya, T.; Matsubara, S. Purines. XXXIII. Syntheses
and cytokinin activities of both enantiomers of 1′-meth-
ylzeatin and their 9-â-D-ribofuranosides. Chem. Pharm.
Bull. 1989, 37, 1758-1763.
Nakayama, Y.; Kitazume, T.; Ishikawa, N. Synthesis and
useful building blocks for monofluorinated compounds de-
rived from trifluoroethene. J . Fluorine Chem. 1985, 29, 445-
458.
Hecht, S. M.; Leonard, N. J .; Schmitz, R. Y.; Skoog, F.
Cytokinins: influence of side-chain planarity of N6-substi-
tuted adenines and adenosines on their activity in promoting
cell growth. Phytochemistry 1970, 9, 1907-1913.
Howard, J . A. K.; Hoy, V. J .; O’Hagan, D.; Smith, G. T. How
good is fluorine as a hydrogen bond acceptor? Tetrahedron
1996, 52, 12613-12622.
Kitazume, T.; Ishikawa, N. One-pot synthesis of R-fluoro-R,â-
unsaturated esters from chloromalonic ester and carbonyl
compounds using “spray-dried” potassium fluoride. Chem.
Lett. 1981, 1259-1260.
Leonard, N. J .; Hecht, S. M.; Skoog, F.; Schmitz, R. Y.
Cytokinins: Synthesis and biological activities of related
derivatives of 2iP, 3iP, and their ribosides. Isr. J . Chem.
1968, 6, 539-550.
Letham, D. S. Cytokinins from Zea Mays. Phytochemistry
1973, 12, 2445-2455.
Lovey, A. J .; Pawson B. A. Fluorinated retinoic acids and their
analogues. 3. Synthesis and biological activity of aromatic
6-fluoro analogues. J . Med. Chem. 1982, 25, 71-75.
Machleidt, H.; Wessendorf, R. Organische Fluorverbindungen,
IV Carbonyl-fluorolefinierungen (Fluoroorganic compounds,
IV. Carbonyl fluoroolefination). Liebigs Ann. Chem. 1964,
674, 1-10.
Massoudi, H. H.; Cantacuze`ne, D.; Wakselman, C.; Bouthier
de la Tour, C. Synthesis of the cis- and trans-isomers of
homoaconitic and fluorohomoaconitic acid. Synthesis 1983,
1010-1012.
Matsubara, S. Structure-activity relationships of cytokinins.
Crit. Rev. Plant Sci. 1990, 9, 17-57.
Mornet, R.; Gouin, L. Synthe`se de l’ace´tate du chloro-4
me´thyl-2 bute`ne-2-ol-1 (E). Nouvelle voie d’acce`s a` la trans-
ze´atine [Synthesis of (E)-4-chloro-2-methyl-2-butenyl ac-
etate. New access to trans-zeatin]. Tetrahedron Lett. 1977a ,
167-168.
Mornet, R.; Gouin, L. The carbonochloridate cleavage of
tertiary amines; a simple step in the preparation of di- and
trisubstituted olefinic (allylic) synthons. Synthesis 1977b,
786-787.
Mu¨ller, C.; Stier, F.; Weyerstahl, P. Darstellung, ringo¨ffnung-
sreaktionen und halogen/lithium-austauch von 1-brom-1-
fluorcyclopropanen (Synthesis, ring cleavage, and halogen/
lithium exchange of 1-bromo-1-fluorocyclopropanes). Chem.
Ber. 1977, 110, 124-137.
Nogue, F.; J ullien, M.; Mornet, R.; Laloue, M. The response of
a cytokinin resistant mutant is highly specific and permits
a new bioassay. Plant Growth Regul. 1995, 17, 87-94.
Normant, J . F.; Foulon, J . P.; Masure, D.; Sauveˆtre, R.;
Villie´ras, J . A new preparation of trifluorovinyllithium.
Applications to the syntheses of trifluorovinylalcohols and
of R-fluoro-R-ethylenic acids and derivatives. Synthesis 1975,
122-125.
Poulter, C. D.; Argyle, J . C.; Mash, E. A. Prenyltransferase.
New evidence for an ionization-condensation-elimination
mechanism with 2-fluorogeranyl pyrophosphate. J . Am.
Chem. Soc. 1977, 99, 957-959.
Schlosser, M.; Chau, L. V. Fluororganische synthesen. V.
Darstellung und abwandlung von chlorfluorcyclopropanen.
Ein gezielter zugang zu fluorallylalkoholen (Fluoroorganic
syntheses, V. Preparation and transformations of chloro-
fluorocyclopropanes. A selective approach to fluoroallylic
alcohols). Helv. Chim. Acta 1975, 58, 2595-2603.
Schlosser, M.; Spahic, B.; Tarchini, C.; Chau, L. V. Insertion
of a carbon atom into olefinic double bonds: synthesis of
fluorodienes and fluoroalkenes. Angew. Chem., Int. Ed. Engl.
1975, 14, 365-366.
Schmitz, R. Y.; Skoog, F.; Playtis, A. J .; Leonard, N. J .
Cytokinins: Synthesis and biological activity of geometric
and position isomers of zeatin. Plant Physiol. 1972, 50, 702-
705.
Van Staden, J .; Drewes, S. E. 6-(2,3,4-Trihydroxy-3-methyl-
butylamino)purine, a cytokinin with high biological activity.
Phytochemistry 1982, 21, 1783-1784.
Xu, Z. Q.; DesMarteau, D. D. A convenient one-pot synthesis
of R-fluoro-R,â-unsaturated nitriles from diethyl cyanofluo-
romethanephosphonate. J . Chem. Soc., Perkin Trans. 1
1992, 313-315.
Received for review September 2, 1997. Revised manuscript
received February 4, 1998. Accepted February 11, 1998.
J F970752L