Antioxidant activity of synthetic cytokinin analogues: 6-alkynyl- and 6-alkenylpurines as novel 15-Lipoxygenase inhibitors.

Article abstract of DOI:10.1016/S0968-0896(01)00427-8

Synthetic cytokinin analogues as well as the well known CKs 6-benzylaminopurine (BAP), kinetin and trans-zeatin were examined for antioxidant activity. The compounds were tested as potential diphenylpicrylhydrazyl (DPPH) scavengers and as inhibitors of 15

Full text of DOI:10.1016/S0968-0896(01)00427-8

Bioorganic & Medicinal Chemistry 10 (2002) 1581–1586
Antioxidant Activity of Synthetic Cytokinin Analogues:
6-Alkynyl- and 6-Alkenylpurines as Novel
15-Lipoxygenase Inhibitors
Anders Brathe,a Geir Andresen,a Lise-Lotte Gundersen,a,*
Karl E. Malterudb and Frode Risea
aDepartment of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Norway
bSchool of Pharmacy, University of Oslo, PO Box 1068, Blindern, N-0316 Oslo, Norway
Received 4 October 2001; accepted 29 November 2001
Abstract—Synthetic cytokinin analogues as well as the well known CKs 6-benzylaminopurine (BAP), kinetin and trans-zeatin were
examined for antioxidant activity. The compounds were tested as potential diphenylpicrylhydrazyl (DPPH) scavengers and as
inhibitors of 15-lipoxygenase (15-LO). The natural plant hormones were essentially inactive in both assays, but several synthetic
analogues have a profound inhibiting effect on 15-lipoxygenase from soybeans. The same compounds were only weak DPPH sca-
vengers and they may therefore be regarded as so-called non antioxidant inhibitors of 15-LO. # 2002 Elsevier Science Ltd. All
rights reserved.
Introduction
Kinetin is reported to reduce lipoxygenase activity in
Lipoxygenase (LO) was first reported from soybeans
more than 50 years ago, and has since then been found
in a large number of higher plants.1 In spite of the wide-
spread occurrence of these enzymes in the plant king-
dom, their physiological role is still disputed. At pre-
sent, it is generally believed that lipoxygenases play a
role in the response of plants to wounding,2 possibly
due to the toxicity of LO metabolites towards invading
fungi and bacteria. Other activities of lipoxygenase
metabolites have been suggested as well, such as plant
hormonal activity,2,3 growth regulation and senescence
induction,4 plant maturation5 and decreasing oxygen
tension in seeds.6
detached pea leaves and induce chlorophyll retention,
and some years ago it was proposed that CKs may act
as radical scavengers and react directly with superoxide
anion radical (O.2ꢀ).7 The only support for this theory
was, however, formation of amides when some aryl
amines (not CKs) were reacted with superoxide. In fact,
CKs does not react fast with the superoxide anion radi-
cal in aqueous solution.8 Antioxidant effects mediated
by CKs even in mammalian cells are also reported.
trans-Zeatin and derivatives inhibits lipoperoxidation in
rat kidney homogenates. These results indicate that
CKs may have a potential as preventors of lipid perox-
idation in living animal cells.9 However, there are also
studies that does not support cytokinin mediated lowering
of lipoxygenase activity,10 and clearly the relationships
between cytokinins and antioxidant effects are largely
unexplored.
Cytokinins (CK) are plant growth hormones which
among other things, promote cell division and cell
growth and are involved in retardation of senescence
and protection against abiotic oxidative stress. The
structures of the cytokinins kinetin, N6-benzylamino-
purine (BAP) and trans-zeatin are shown in Figure 1.
*Corresponding author. Tel.:+47-22-85-7019; fax:+47-22-85-5507;
e-mail: 1.1.gundersen@kjemi.uio.no
Figure 1. Structure of the cytokinins kinetin, BAP and trans-zeatin.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00427-8

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A. Brathe et al. / Bioorg. Med. Chem. 10 (2002) 1581–1586
1582
We have prepared a number of cytokinin analogues
where the –NHCH2R substituent found in the purine 6-
position in naturally occurring CKs are replaced with a
–CꢁCR, –CH¼CHR or –CH2CH2R group. These
compounds were believed to be less susceptible to
degradation by cytokinin oxidase11 and several of the
compounds promoted cell growth within the same range
as naturally occurring CKs.12,13 We have now examined
the potential antioxidant abilities of the same CK
analogues and we herein report their ability to scav-
enge diphenylpicrylhydrazyl (DPPH) and to inhibit
15-lipoxygenase.
for scavenging ability. Only alkynes 2 and 5 and trans
alkenes 3 and 6 displayed any activity. The most active
compounds were 6-(3-thienylethenyl)purine 3m with
18% scavenging after 15 min and the furanes 3 h and 3i.
These were actually more active that the phenols 3d and
3e (entries 11 and 12). We found no correlation between
DPPH scavenging and the plant growth stimulation we
previously reported.12,13
The CKs and analogues were also tested as potential
inhibitors of 15-lipoxygenase (15-LO) from soybeans.
Again the well known CKs BAP, and trans-zeatin were
completely inactive and kinetin showed only a weak
inhibitory activity. However, several synthetic CK ana-
logues were profound enzyme inhibitors. All purines
were tested for enzyme inhibition at 167 mM concn. IC50
values were determined for compounds showing more
than 50% inhibition in the initial screening (Table 1).
There were no correlations between the ability to med-
iate antioxidant activity and to stimulate plant growth.
Furthermore, there were no direct correlations between
enzyme inhibition and DPPH scavenging activity, indi-
cating that the purines mediate their effect by binding to
the enzyme and not by scavenging radicals formed
from 15-LO oxidation of fatty acids. A –CꢁCR or
trans –CH¼CHR groups in the 6-position were required
for high activity. Highest activity (IC50 values between
90 and 110 mM) were found for the alkynes and alkenes
2, 3a, 3b and 3f, which can be regarded as BAP analogues,
and the trans-zeatin analogues 5a, 5d, 6a and 6b. Com-
pounds 4 and 7 with a –CH2CH2R group in the purine 6-
position or a phenyl group bound directly to the purine
ring (1) were only weakly active (5–25% inhibition at
Results and Discussion
Our synthetic cytokinin analogues as well as the well
known CKs BAP, kinetin and trans-zeatin were exam-
ined as potential DPPH scavengers. All compounds
examined can formally be represented as the general
structures shown in Figure 2, all though the oxopurines
(X=OH) exist mainly as lactam tautomers.
The results from the reaction with DPPH are shown in
Table 1. The 6-aminopurines BAP, kinetin and trans-
zeatin were not at all able to scavenge DPPH at 667 mM
concn. None of the synthetic CK analogues was highly
active DPPH scavengers, but several compounds were
significantly more active than BAP, kinetin and trans-
zeatin. Generally 2-oxopurines were somewhat more
active than the corresponding purines without any sub-
stituent in the 2-position. No conclusion can be drawn
at this point as to whether a 9-substituent is beneficial
Figure 2. Structures of the cytokinin analogues 1–7.

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A. Brathe et al. / Bioorg. Med. Chem. 10 (2002) 1581–1586
1583
167 mM concn). The substituents in the 2- and 9-posi-
tion have no consistent effect on the enzyme inhibiting
ability.
connected to the purine mediated enzyme inhibition
described herein.
The results presented herein demonstrates that several
6-alkenyl and 6-alkynylpurines have a profound inhibit-
ing effect on 15-lipoxygenase from soybeans. The same
compounds were only weak DPPH scavengers and they
may therefore be regarded as so-called non antioxidant
inhibitors of 15-LO. We used 15-lipoxygenase from soy-
beans, but it has been shown that there is a good correla-
tion for inhibitory activity for the soybean and
mammalian 15-LO enzyme18,19 even though these two are
not identical. 15-Lipoxygenase has been implicated in
oxidation of low density lipoproteins (LDL). This process
is believed to be important for the development of ather-
osclerosis20,21 and non antioxidant 15-LO inhibitors may
have an anti-atherosclerotic effect in vivo.22ꢀ24 The current
lack of lipoxygenase inhibitors limits further investigation
of the biological role of these enzymes.25 If continued
research on the synthetic cytokinin analogues reported
herein reveals that they also are inhibitors of human
lipoxygenases, the purines may have a potential as drugs
against atherosclerosis and maybe even against other
major diseases as for instance cancer, Parkinson’s disease,
Alzheimer’s disease, stroke, heart infarction and rheuma-
toid arthritis which are also linked to free radicals.26ꢀ28
Most of the 15-LO inhibiting compounds reported
herein have IC50 values between 90 and 120 mM. This
compares well with previously reported 15-LO inhibi-
tors of plant origin, such as quercetin (98 mM), sinense-
tin (114 mM) and tetramethylscutellarein (110 mM),14
and also with synthetic inhibitors, for example dia-
lkylphtalates (IC50 values between 93 and 108 mM).15
We can see no obvious structural relationship between
the purines examined in this study and known LO inhi-
bitors. To the best of our knowledge, this is the first
report on lipoxygenase inhibitors with purine structure,
except for the possible role of cytokinins as inhibitors of
lipoxygenase activity. The only other relationship
between purines and lipoxygenases we know of, is the
fact that calcium dependent 5-lipoxygenase activity is
stimulated by the addition of adenosine triphosphate
(ATP)16 and that it has recently been reported that 15-
LO from soybeans catalyze hydrolysis of ATP and the
subsequent release of inorganic phosphate. However,
the latter reaction was not affected by the addition of
the lipoxygenase inhibitor phenidone17 and may not be
Table 1. Scavenging of DPPH and inhibition of 15-lipoxygenase
Compd
% DPPH scavenging after 5 min
Inhibition of 15-Lipoxygenase
a
667 mM concna
% Inhibition at 167 mMa
IC50
BAP
Kinetin
t-Zeatin
1
2a
2b
2c
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
4a
4b
4c
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
ꢀ0.1Æ0.4
0.1Æ0.29.2
ꢀ0.2Æ0.1
1.1Æ0.3
0.2Æ0.3
0.6Æ0.1
3.2Æ0.1
0.4Æ0.0
ꢀ0.2Æ0.3
1.0Æ0.4b
5.9Æ0.5
3.5Æ0.3
0.2Æ0.3
0.8Æ0.1
9.7Æ0.7
8.6Æ0.4
1.4Æ0.1
1.1Æ0.1
2.6Æ0.9
18.0Æ0.9
0.0Æ0.3
ꢀ0.1Æ0.24.8
1.2Æ0.3c
1.6Æ0.3
3.6Æ0.5
4.2Æ0.6
1.9Æ0.1
4.0Æ0.4
2.9Æ0.6
7.0Æ1.239.3
0.8Æ0.1
1.1Æ0.1
0.5Æ0.1
ꢀ6.1Æ7.6
Æ2.6
n.d.
n.d.
n.d.
n.d.
102Æ11
102Æ3
107Æ6
104Æ6
96Æ5
n.d.
ꢀ1.3Æ10.4
17.1Æ4.0
76.2Æ4.7
85.4Æ1.1
83.8Æ2.9
84.9Æ1.5
89.6Æ3
27.1Æ6.5d
73.4Æ2.2
61.7Æ3.5
82.4Æ1.295
79.8Æ1.2
54.1Æ4.9
45.0Æ4.4
39.8Æ5.1
45.9Æ5.3
59.8Æ2.1
63.8Æ4.1
24.5Æ4.5
Æ8
119Æ11
138Æ10
Æ5
>166
148Æ22
n.d.
n.d.
n.d.
141Æ8
136Æ11
n.d.
n.d.
n.d.
15.9Æ7.7
77.2Æ4.9
61.2Æ4.3
90.8Æ1.5
74.5Æ1.5
76.9Æ1.6
83.5Æ2.5
Æ5.1
108Æ10
130Æ17
116Æ7
105Æ6
106Æ5
92Æ5
n.d.
63.6Æ4.8
5.3Æ5.5
15.7Æ6.4
131Æ12
n.d.
n.d.
aData are shownÆSD.
b83.5 mM concn.
c333 mM concn.
d83.5 mM concn.

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A. Brathe et al. / Bioorg. Med. Chem. 10 (2002) 1581–1586
1584
Experimental
and ethanol (20 mL, 96%) was stirred at ambient tem-
perature for 1 h 45 min before neutralizing with solid
NaHCO3. A small amount of silica gel was added, and
the volatiles were evaporated in vacuo. The crude pro-
duct was purified by flash chromatography on silica gel
eluting with 0–10% ethanol in ethyl acetate. Yield 85
mg (89%) off-white powder, mp 206–207 ꢂC (lit.32 198–
199 ꢂC). 1H NMR (200 MHz, CD3OD) d 8.90 (s, 1H, H-
2), 8.60 (s, 1H, H-8), 7.80–7.75 (m, 2H, Ph), 7.52–7.41
(m, 3H, Ph). MS (EI): 220 (M+, 100), 219 (5), 193 (6),
166 (7), 139 (12), 114 (4), 113 (5), 87 (1), 66 (3).
6-Benzylaminopurine, kinetin and trans-zeatin were pur-
chased from Fluka (Buchs, Switzerland) and diphenyl-
picrylhydrazyl (DPPH) and 15-lipoxygenase (Type 1)
from Sigma (St. Louis, MO, USA). Silica gel for flash
chromatography was available from Merck (Darmstadt,
Germany) (Merck No. 9385) or Fluka (Fluka No.
60752). DMF, pyrrolidine, and diisopropylethylamine
were distilled from CaH2, and THF from Na/benzo-
phenone. Tetramethylammonium fluoride trihydrate
was dried by azeotropic distillation with abs ethanol.
Compounds 2c,13 3a–b,12 3c,13 3d–3m,12 4c,13 5a,12 5b,13
6a–b12 6c,13 6d,12 and 7a–b13 were prepared as we have
described before. 6-Iodopurine,29 6-iodo-9-(tetrahydro-
2H-pyran-2-yl)-9H-purine30 and 6-phenyl-9-(tetrahydro-
2H-pyran-2-yl)-9H-purine 131 were available by litera-
6-(2-Phenylethyl)- 9- (tetrahydro -2H -pyran -2-yl) -9H -
purine (4a). 6-Iodo-9-(tetrahydro-2H-pyran-2-yl)-9H-
purine (660 mg, 2.0 mmol), tris(dibenzylideneacetone)-
dipalladium chloroform adduct (52mg, 0.05 mmol) and
triphenylphosphine (105 mg, 0.4 mmol) were dissolved
in dry THF (5 mL) and stirred under N2 atmosphere
until the mixture turned yellow. Phenylethylzinc chlo-
ride (8 mL, 4 mmol, 0.5M in THF) was added and the
mixture was stirred at 50 ꢂC for 24 h and poured into
NH4Cl (satd, 50 mL). The resulting mixture was
extracted with EtOAc (3Â40 mL), and the combined
extracts were dried (MgSO4) and evaporated in vacuo.
The crude product was purified by flash chroma-
tography on silica gel eluting with 0–70% EtOAc in
hexane. Yield 411 mg (67%) off-white solid, mp 113–
1
ture methods. The H NMR spectra were recorded at
500 MHz with a Bruker Avance DRX 500 instrument,
at 300 MHz with a Bruker Avance DPX 300 instrument
or at 200 MHz with a Bruker Avance DPX 200 instru-
ment. The 13C NMR spectra were recorded at 125, 75 or
50 MHz using instruments mentioned above. Unless
otherwise stated, the spectra are recorded at ambient
temperature. Chemical shifts (d) are given in ppm
downfield from tetramethylsilane. Mass spectra were
recorded with a VG Prospec instrument at 70 eV ionizing
voltage unless otherwise stated, and are presented as m/z
(% rel. int.). Melting points are uncorrected. All mea-
surements of DPPH scavenging and 15-lipoxygenase
activity were carried out in a Shimadzu UV-160A spec-
trophotometer (Shimadzu, Kyoto, Japan).
114 ꢂC. H NMR (200 MHz, CDCl3) d 8.85 (s, 1H, H-
1
2), 8.20 (s, 1H, H-8), 7.26–7.05 (m, 5H, Ph), 5.71 (m,
1H, THP), 4.05 (m, 1H, THP), 3.68 (m, 1H, THP), 3.45
(m, 2H, CH2), 3.18 (m, 2H, CH2), 2.04–1.95 (m, 3H,
THP), 1.67–1.56 (m, 3H, THP). 13C NMR (50 MHz,
CDCl3) d 161.3, 152.0, 149.7, 141.3, 140.8, 132.3, 128.0,
127.98, 125.6, 81.5, 68.4, 34.5, 33.8, 31.3, 24.5, 22.4. MS
(EI): 308 (M+, 6), 225 (15), 224 (100), 223 (60), 208 (9),
147 (30), 120 (5), 91 (33), 84 (6), 65 (13). HRMS: found:
308.1636, calcd for C18H20N4O: 308.1637.
6-(Phenylethynyl)-9-(tetrahydro-2H-pyran -2-yl)-9H-
purine (2a). 6-Iodo-9-(tetrahydro-2H-pyran-2-yl)-9H-
purine (500 mg, 1.51 mmol), copper(I) iodide (29 mg,
0.15 mmol) and bis(triphenylphosphine)palladium(II)
chloride (53 mg, 0.076 mmol) was dissolved in dry
DMF (10 mL) under Ar atmosphere. Phenylethyne
(3.35 mL, 3.03 mmol) and ethyldiisopropylamine (6.00
mL, 4.54 mmol) were added, and the mixture was
heated at 60 ꢂC for 17 h. After cooling to ambient tem-
perature, a small amount of silica gel was added, and
the volatiles were evaporated in vacuo. The crude pro-
duct was purified by flash chromatography on silica gel
eluting with pure hexane to pure ethyl acetate. Yield 386
mg (84%), mp 124–126ꢂC. 1H NMR (200MHz, CDCl3)
d 8.92(s, 1H, H-2), 8.32(s, 1H, H-8), 7.75–7.68 (m, 2H,
Ph), 7.41–7.33 (m, 3H, Ph), 5.81–5.75 (m, 1H, H-2in
THP), 4.20–4.13 (m, 1H, THP), 3.83–3.70 (m, 1H, THP),
2.17–2.01 (m, 3H, THP), 1.85–1.66 (m, 3H, THP). 13C
NMR (50 MHz, CDCl3) d 152.6, 150.8, 143.0, 141.8,
134.3, 132.6, 129.9, 128.4, 121.4, 98.4, 84.1, 82.1, 68.8,
31.8, 24.8, 22.7, 19.0. MS (EI): 304 (M+, 42), 276 (16),
247 (7), 222 (12), 221 (79), 220 (100), 219 (9), 193 (10), 166
(10), 165 (7), 140 (8), 139 (17), 128 (6), 113 (8), 85 (31).
HRMS: found: 304.1330, calcd for C18H26N4O: 304.1324.
6-(2-Phenylethyl)-1H - purine (4b). A mixture of 6 - (2-
phenylethyl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine 4a
(228 mg, 0.74 mmol), ethanol (15 mL, 96%) and
hydrochloric acid (10 mL, 1.0 M) was stirred at ambient
temperature for 4 h before neutralizing with solid
NaHCO3. A small amount of silica gel was added, and
the volatiles were evaporated in vacuo. The crude pro-
duct was purified by flash chromatography on silica gel
eluting with 0–20% ethanol in EtOAc. Yield 124 mg
(74%) colorless powdery crystals, mp 138–139 ꢂC (lit.32
135–137 ꢂC). 1H NMR (500MHz, CD3OD) d 8.81, (s, 1H,
H-2), 8.44 (s, 1H, H-8), 7.19 (m, 4H, Ph), 7.11 (m, 1H, Ph),
3.42(m, H2 , CH 2), 3.14 (m, 2H, CH2). 13C NMR
(125MHz, CDCl3, 50ꢂC) d 160.6, 150.1, 153.2, 145.7,
142.2, 130.6, 129.4, 129.3, 127.1, 35.9, 35.2. MS (EI): 224
(M+, 100), 223 (70), 209 (4), 208 (9), 207 (3), 197 (2), 196
(3), 147 (36), 120 (5), 101 (7), 91 (42), 77 (5), 65 (16).
6-(5-Carbomethoxy-4-methyl-3-penten-1-yn-1-yl)-1H-
purine (5c). To a solution of 2-methyl-pent-2-en-4-ynoic
acid (435 mg) in methanol (30 mL) was added concd
sulfuric acid (10 drops) and the resulting mixture was
refluxed for 24 h, cooled to ambient temperature and
poured into aqueous potassium carbonate (10%, 50 mL).
The mixture was extracted with chloroform (4Â50 mL).
6-(Phenylethynyl)-1H-purine (2b). A mixture of 6-(phe-
nylethynyl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine 2a
(150 mg, 0.493 mmol), hydrochloric acid (10 mL, 1.0M)

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A. Brathe et al. / Bioorg. Med. Chem. 10 (2002) 1581–1586
1585
After drying (Na2SO4) and evaporation, the product
was purified by Kugelrohr distillation (17 mmHg,
150 ꢂC) to give methyl 2-methylpent-2-en-4-ynoate 178
517 nm of a methanolic DPPH solution (A517=1.0, 2.95
mL) over a 5-min period after addition of 50 mL of a
DMSO solution of the test substance.33 Appropriate
corrections were made for dilution and for absorbance
of the reaction product, reduced DPPH. Calculation of
radical scavenging activity was carried out as previously
described.34
1
mg (36%) as a pale yellow oil. H NMR (200 MHz,
CDCl3) d 6.57 (m, 1H, H-3), 3.75 (s, 3H, OCH3), 3.51
(m, 1H, H-5), 2.06 (m, 3H, CH3). 6-Iodopurine (295 mg,
1.20 mmol), bis(triphenylphosphine)palladium(II) di-
chloride (42mg, 0.060 mmol) and CuI (32 mg, 0.12
mmol) were dissolved in dry DMF (6 mL) under nitro-
gen atmosphere. Ethyldiisopropylamine (620 mL, 3.60
mmol) was added via syringe, before methyl 2-methyl-
pent-2-en-4-ynoate (178 mg, 1.43 mmol) was added and
the mixture was stirred at 70 ꢂC for 18 h. The volatiles
were removed in vacuo and the crude product was purified
using flash chromatography on silica gel eluting with
ethyl acetate. Yield 121 mg (42%) colorless powdery
Inhibition of 15-lipoxygenase
Lipoxygenase activity was measured in borate buffer
solutions (0.2M, pH 9.00) as previously described 14,33
by the increase in absorbance at 234 nm from 30 to 90 s
after addition of the enzyme, using linoleic acid (134
mM) as substrate. The final enzyme concentration was
167 U/mL. Test substances were added as DMSO solu-
tions (final DMSO concn 1.6%); DMSO alone was
added in uninhibited control experiments. Six or more
parallels of controls and three or more parallels for each
test substance solution were measured. To ensure con-
stant enzyme activity throughout the experiment, the
enzyme solution was kept on ice, and controls were
measured at regular intervals.
crystals, mp 177–181 ꢂC. H NMR (500 MHz, CD3OD)
1
d0 8.90 (s, 1H, H-2), 8.59 (s, 1H, H-8), 6.95 (m, 1H, H-
3 ), 3.82(s, 3H, CO 3CH3), 2.26 (m, 3H, CH3). 13C NMR
(125 MHz, CDCl3, 50 ꢂC) d 168.2, 153.5, 147.7, 144.6,
133.7, 133.2, 130.0, 118.2, 94.8, 94.5, 53.5, 16.1. MS
(EI): 242 (M+, 100), 211 (5), 184 (10), 183 (37), 182
(52), 156 (4), 129 (4), 128 (1), 102 (2), 76 (1). HRMS:
found: 242.0830, calcd for C12H10N4O2: 242.0804.
Calculation of enzyme activity was carried out as pre-
viously described14 and IC50 values were determined by
linear interpolation between the measuring points clo-
sest to 50% activity. Values are expressed as means
ÆSD. Student’s t-test was employed for determination
of statistical significance.
6-(4-Methyl-3-penten-1-yn-1-yl)-1H-purine (5d). A mix-
ture of tris(dibenzylideneacetone)dipalladium chloro-
form adduct (129 mg, 0.125 mmol), triphenylphosphine
(262 mg, 1.00 mmol) and copper(I) iodide (95 mg, 0.50
mmol) in dry pyrrolidine (10 mL) was stirred at ambient
temperature under N2-atm. 1-Bromo-2-methylpropene
(675 mg, 5.0 mmol) and ethynyl(trimethyl)silane (1.40
mL, 10 mmol) were added and the resulting mixture was
stirred at 40 ꢂC for 14 h. Satd aq NH4Cl (40 mL) was
added and the mixture was extracted with diethyl ether
(4Â20 mL). After drying (Na2SO4) and evaporation, the
product was purified by Kugelrohr distillation (12
mmHg, 70–80 ꢂC) to give 4-methyl-1-trimethylsilyl-3-
penten-1-yne 484 mg (64%) as a colorless liquid. 1H
NMR (CDCl3, 300 MHz): d 5.27 (m, 1H, H-3), 1.89 (d,
J 0.5 Hz, 3H, CH3), 1.78 (d, J 1.2Hz, 3H, CH 3), 0.15 (s,
9H, TMS); MS (EI) 152(25, M +), 137 (100). 4-Methyl-
1-trimethylsilyl-3-penten-1-yne (1.34 g, 8.8 mmol) and
ethyldiisopropylamine (0.51 mL, 3.0 mmol) were added
to a mixture of 6-iodo-1H-purine (246 mg, 1.0 mmol),
bis(triphenylphosphine)palladium(II) chloride (35 mg,
0.05 mmol), copper(I) iodide (19 mg, 0.1 mmol) and dry
tetramethylammonium fluoride (10 mmol) in dry DMF
(6 mL) and the resulting mixture was stirred for 24 h at
60 ꢂC. The mixture was evaporated and the product
isolated after flash chromatography on SiO2 eluting
with EtOAc followed by EtOAc–EtOH (95:5). Yield 69 mg
Acknowledgements
The Norwegian Research Council is greatly acknowl-
edged for partial financing of the 200, 300 and 500 MHz
Bruker Avance NMR instruments used in this study.
References and Notes
1. Hertel, H. Biol. Rundsch. 1986, 24, 257.
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(35%) yellow crystalline solid, mp 189–191 ꢂC. H NMR
1
(CD3OD, 300MHz) d 8.81 (s, 1H, H-2), 8.52 (s, 1H, H-8),
5.69 (m, 1H, H-30), 2.11 (s, 3H, CH3), 1.95 (s, 3H, CH3).
ꢂ
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