Syntheses of O6-alkyl- and arylguanine derivatives: Nucleobase adducts derived from styrene 7,8- and 3,4-oxides

Article abstract of DOI:10.1002/ejoc.200500477

A series of O⁶-alkyl and -arylguanine derivatives that may be formed in vivo after exposure to styrene has been prepared by reaction of 6-(4-aza-1-azoniabicyclo[2.2.2]octyl)-purine with alkoxides and aryloxides, respectively. The monoprotected diols 2-allyloxy- or 2-benzyloxy-1-phenyl- ethanol and 2-allyloxy- or 2-benzyloxy-2-phenylethanol were used as synthetic equivalents of styrene 7,8-oxide. 4-Vinylphenol, 2-(4-hydroxyphenyl)ethanol and 4-hydroxyphenylacetic acid were used as synthetic equivalents of arene oxide metabolites of styrene, i.e., styrene 3,4-oxide, 4-(2-hydroxyethyl)benzene 1,2-oxide and 4-carboxymethylbenzene 1,2-oxide, respectively. Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejoc.200500477

FULL PAPER
DOI: 10.1002/ejoc.200500477
Syntheses of O6-Alkyl- and Arylguanine Derivatives: Nucleobase Adducts
Derived from Styrene 7,8- and 3,4-Oxides
[a]
[a]
[a]
ˇ
Jan Novák, Zbynek Hasník, and Igor Linhart*
Keywords: Alkoxypurines / Aryloxypurines / DNA adducts / DNA damage / Heterocycles
A series of O6-alkyl and -arylguanine derivatives that may
be formed in vivo after exposure to styrene has been pre-
pared by reaction of 6-(4-aza-1-azoniabicyclo[2.2.2]octyl)-
purine with alkoxides and aryloxides, respectively. The
monoprotected diols 2-allyloxy- or 2-benzyloxy-1-phenyl-
ethanol and 2-allyloxy- or 2-benzyloxy-2-phenylethanol
were used as synthetic equivalents of styrene 7,8-oxide. 4-
Vinylphenol, 2-(4-hydroxyphenyl)ethanol and 4-hydroxy-
phenylacetic acid were used as synthetic equivalents of ar-
ene oxide metabolites of styrene, i.e., styrene 3,4-oxide, 4-
(2-hydroxyethyl)benzene 1,2-oxide and 4-carboxymeth-
ylbenzene 1,2-oxide, respectively.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
Introduction
O6-Alkylguanines are important DNA adducts formed
by alkylation with electrophilic mutagens, including some
cancer chemotherapeutic agents. Certain O6-alkylguanines
are potent inhibitors of alkylguanine-DNA transferase, an
enzyme responsible for repair of damage at the guanine O6
position in DNA. Inactivation of this enzyme leads to a
significant enhancement of the effect of chemotherapeutic
drugs whose mechanism of action involves alkylation at the
guanine O6 position.[1,2] Modified guanines are also used as
biomarkers of exposure to compounds that are capable of
DNA alkylation as they are indicators of exposure and can
also serve as indicators of damage to DNA, which is linked
to the risk of cancer.[3,4] The residues of modified DNA −
DNA adducts − can be determined in various tissues as
nucleotide adducts or in urine as modified nucleobases.[5,6]
In our previous work we described syntheses of 7-alkylgua-
nines derived from styrene 7,8-oxide (1), a carcinogenic me-
tabolite of styrene.[7] To extend the range of nucleobase ad-
ducts that can be used as markers of exposure to styrene, we
hereby present synthetic routes leading to O6-alkylguanines
derived from styrene 7,8-oxide (1) and O6-arylguanines de-
rived from arene oxide metabolites of styrene, such as sty-
rene 3,4-oxide (2), 4-(2-hydroxyethyl)benzene 1,2-oxide (3)
and 4-carboxymethylbenzene 1,2-oxide (4). A simplified
view of the possible formation of O6-guanine adducts from
styrene metabolites and metabolic intermediates is shown
in Scheme 1.
Scheme 1. O6-Alkyl- and -arylguanines derived from reactive meta-
bolic intermediates of styrene.
O6-Guanine adducts derived from 1 have been detected
in DNA samples treated with 1,[8,9] in experimental ani-
mals[10,11] and in humans exposed to styrene.[12,13] On the
other hand, no adducts derived from arene oxides 2–4 have
been reported as yet, although it is assumed that styrene
3,4-oxide (2) may contribute significantly to the cytogenetic
toxicity of styrene.[14,15]
O6-Alkylguanines (2-amino-6-alkoxypurines) can be pre-
pared by reaction of 2-amino-6-chloropurine (5) with so-
dium alkoxides. The reaction proceeds easily when the cor-
[a] Department of Organic Chemistry, Institute of Chemical Tech-
nology, Technická 1905,
166 28 Prague, Czech Republic
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responding alcohol is used as the solvent.[16] However, it is wave reactor. Also, the product ratio was affected by reac-
limited to liquid and relatively inexpensive alcohols. An- tion temperature and by microwave irradiation (Table 1).
other approach was therefore used to prepare alkoxypurines The products were separated by semi-preparative HPLC.
derived from solid or expensive alcohols. Position 6 in 5 was The two isomers differ in their stability in acidic aqueous
activated for nucleophilic attack by displacement of chlo- solutions. While isomer 8 is stable, 7 decomposes at pH 2
rine by trimethylamine[17] or 1,4-diazabicyclo[2.2.2]octane following first-order reaction kinetics with a rate constant,
(DABCO).[18,19] The ammonium salts formed react readily k, of 2.7×10–5 s (t1/2 = 42 min). A similar stability and reac-
with alkoxides and aryloxides in polar aprotic sol- tivity have been observed by Moschel et al. for correspond-
vents.[17–19] Moreover, they can be converted to 6-aryloxy- ing O6-guanosine derivatives.[24]
purinyl derivatives when 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) is used as a base.[20] We decided to use this synthetic
approach to prepare O6-alkylguanine derivatives derived
from styrene 7,8-oxide as well as O6-arylguanines derived
from styrene 3,4-oxide. The compounds synthesised will be
useful as analytical standards for the development of meth-
ods for the determination of nucleobase adducts in urine.
Unlike the corresponding guanine derivatives, O6-2Ј-de-
oxyguanosine and O6-2Ј-deoxyguanosine phosphate ad-
ducts have been described in the literature. In general, these
compounds are prepared by a non-selective alkylation of
nucleosides and nucleotides with 1. The reaction proceeds
with low conversion and is non-selective, affording a com-
plex mixture of products containing an excess of unreacted
nucleosides or nucleotides. Minor amounts of the adducts
can then be obtained by HPLC separation. O6-Substituted
2Ј-deoxyguanosines[13,21] and deoxyguanosine-3Ј-phos-
phates[22,23] derived from 1 have been obtained by this ana-
lytical approach. However, this approach is not applicable
to arene oxide adducts because arene oxides are not readily
available and are unstable in aqueous solutions. O6-Gua-
nosine adducts derived from 1 were prepared by reaction of
6-chloro-9-ribosylpurine with 1-phenylethane-1,2-diol (6) in
molten sodium. This reaction, which proceeds under rather
severe conditions, affords a mixture of two regioisomeric
adducts, i.e., 6-(2-hydroxy-1-phenylethyl)-9-ribosylpurine
and 6-(2-hydroxy-2-phenylethyl)-9-ribosylpurine, which
were separated by HPLC.[24] Similarly, a mixture of re-
gioisomeric adducts was obtained by Mitsunobu reaction
of protected 2Ј-deoxyguanosine-3Ј-phosphate with a mix-
ture of two isomeric 6 monoacetates.[25]
Scheme 2. Direct synthesis of O6-alkyklguanine adducts derived
from 1: (i) DABCO in DMF; (ii) 6 and NaH in DMF, 70 °C, 24 h
or MW, 50–90 °C, 2.5–3.5 h.
Table 1. The yields and isomer ratios obtained by the reaction of
DABCO-purine (9) with diol 6 induced thermally by conventional
heating or by microwaves.
Results
O6-(2-Hydroxy-1-phenylethyl)- (7) and O6-(2-Hydroxy-2-
Heating
Reaction time [h] Yield (7 + 8) Ratio 8/7[a]
phenylethyl)guanine (8)
Conventional, 70 °C
MW, 50 °C
MW, 70 °C
24
3.5
2.5
2.5
46%
39%
45%
43%
1.50
3.55
4.26
1.00
Reaction of chloropurine 5 with diol 6 proceeded only
after activation of the 6-position of the purine moiety by
DABCO and deprotonation of 6 with sodium hydride to
yield a mixture of 2-amino-6-(2-hydroxy-1-phenylethoxy)-
9H-purine [O6-(2-hydroxy-1-phenylethyl)guanine (7)] and 2-
MW, 90 °C
[a] Determined by HPLC with UV detection at 254 nm.
To prepare the adducts 7 or 8 selectively one of the hy-
amino-6-(2-hydroxy-2-phenylethoxy)-9H-purine [O6-(2-hy- droxy groups in diol 6 was protected. Allyl and benzyl pro-
droxy-2-phenylethyl)guanine (8)] (Scheme 2). The interme- tective groups were chosen, which meant that four mono-
diate in this reaction, 6-(4-aza-1-azoniabicyclo[2.2.2]octyl)- protected diols, i.e., 2-allyloxy-1-phenylethanol (10), 2-al-
purine [DABCO-purine (9)], can be prepared in situ or iso- lyloxy-2-phenylethanol (11), 2-benzyloxy-1-phenylethanol
lated and reacted in a subsequent step. Conventional heat- (12) and 2-benzyloxy-2-phenylethanol (13), were prepared.
ing to 70 °C for 24 h yielded 46% of products (7 + 8). Their structures are shown in Figure 1. Like with unprotec-
Shorter reaction times were achieved by heating in a micro- ted diol 6, it was necessary to activate chloropurine 5 with
508
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Nucleobase Adducts Derived from Styrene 7,8- and 3,4-Oxides
FULL PAPER
DABCO. The resulting DABCO-purine 9 reacted readily rather poor yields, even with a catalyst (10% Pd-C, De-
with the alkoxides derived from alcohols 10–13. Sodium hy- gussa-type containing nearly 50% of water), which was
dride was used for deprotonation of the alcohols. Allyl-pro- freshly activated by heating under vacuum to 110 °C. The
tected alcohols 10 and 11 gave O6-(2-allyloxy-1-phenyl- poor yields may be caused, at least in part, by a strong
ethoxy)guanine (14) and O6-(2-allyloxy-2-phenylethoxy)- adsorption on the catalyst and the low solubility of the
guanine (15), respectively. Similarly, benzyl-protected products. In fact, both 7 and 8 are poorly soluble even in
alcohols 12 and 13 gave O6-(2-benzyloxy-1-phenylethoxy)- dipolar aprotic solvents such as DMF and DMSO. Due to
guanine (16) and O6-(2-allyloxy-2-phenylethoxy)guanine its facile cleavage, allyl is a protective group of choice in
(17), respectively (Scheme 3). The yield of the reaction in- this reaction. The yields of both steps and overall yields are
creased with increasing amount of the base added, as summarised in Table 3.
shown in Table 2. The secondary alcohols 10 and 12 gave
lower yields than the primary ones (11 and 13). The differ-
ence between the reactivity of primary and secondary
Table 3. The reactions of DABCO-purine (9) with monoprotected
diols 10–13; the yields of the individual steps and the overall yield.
Reagent
Yield
Deprotection
alcohols can be explained by steric hindrance of the second-
ary hydroxy group.
Substitution
Overall yield
10
11
12
13
54% 14
94% 15
45% 16
79% 17
84% 7
80% 8
19% 7
21% 8
45% 7
75% 8
9% 7
17% 8
O6-Arylguanines
Figure 1. Structures of monoprotected diols used as synthetic
equivalents of styrene 7,8-oxide.
Four O6-arylguanines (aryloxypurines) derived from hy-
pothetical arene oxide metabolic intermediates of styrene
An efficient deprotection of the allyl-protected deriva- (2–4) were synthesised from 4-vinylphenol (18), 2-(4-hy-
tives 14 and 15 was achieved by tetrakis(triphenylphos- droxyphenyl)ethanol (19), 4-hydroxyphenylacetic acid (20)
phane)palladium-catalysed reductive cleavage according to and ethyl 4-hydroxyphenylacetate (21). The reaction of
Chandrasekhar et al.[26] On the other hand, debenzylation DABCO-purine 9 with 4-vinylphenol (18) in DMF with so-
of the analogous benzyl-protected products 16 and 17 gave dium hydride as base yielded a complex mixture of prod-
Scheme 3. Synthesis of O6-alkylguanines using monoprotected diols 10–13: (i) NaH in DMF, room temperature, 24 h; (ii) deallylation
with PMHS, [Pd(Ph3)4], ZnCl2 in DMF[19] for compounds 14 and 15; debenzylation with H2/Pd-C for compounds 16 and 17.
Table 2. The reactions of DABCO-purine (9) with monoprotected diols 10–13; product yields as influenced by the amount of base added.
Starting material
Product yield
2 equiv. of NaH[a]
2.5 equiv. of NaH[b]
3 equiv. of NaH[b]
9 + 10
9 + 11
9 + 12
9 + 13
48% 14
54% 15
29% 16
49% 17
52% 14
59% 15
32% 16
50% 17
54% 14
94% 15
45% 16
79% 17
[a] Three equivalents of the corresponding monoprotected diol were used. [b] Four equivalents of the corresponding monoprotected diol
were used.
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ucts. HPLC-MS analysis showed a number of peaks with
quasi-molecular ions at m/z = 254, 366, 374, 484, 494 and
606 corresponding to a purinyl or DABCO-purinyl moiety
with one to three vinylphenol molecules added. The ex-
pected product, 2-amino-6-(4-vinylphenyloxy)-9H-purine
[O6-vinylphenylguanine (22)], was detected as a minor peak
(Figure 2). This strongly suggests the formation of a series
of adducts. In these adducts, vinylphenol may be bound to
the purine either by its vinyl or its phenolic group. In the
latter case, Meisenheimer complexes can be formed (Fig-
ure 3).
Figure 3. Possible structures of Meisenheimer-type complexes cor-
responding to the main ionic species found by HPLC-ESI-MS of
the product mixture arising from the reaction of 9 with 18.
O6-Arylguanines derived from arene oxides 3 and 4,
namely O6-[4-(2-hydroxyethyl)phenyl]guanine (23) and O6-
(4-carboxymethylphenyl)guanine (24), respectively, were
obtained by the treatment of 9 with phenolic alcohol 19
and phenolic acid 20, respectively, as shown in Scheme 4.
Similarly, 21 gave O6-(4-ethoxycarbonylmethylphenyl)gua-
nine (25). Potassium carbonate, which was used as a base,
can deprotonate both carboxylic and phenolic groups whilst
leaving alcoholic hydroxy groups untouched. Nevertheless,
one equivalent of DBU was needed to obtain reasonable
yields of arylguanines 23–25. The yields of arylguanines 23,
24 and 25 were significantly better than that of arylguanine
22, amounting to 35%, 35% and 25%, respectively. A by-
product of arylguanine 23 was detected by HPLC/MS at
m/z = 384, which corresponds to a Meisenheimer adduct
containing phenol 19 and DABCO attached to the amino-
Figure 2. Products of the reaction of DABCO-purine (9) with vi-
nylphenol (18). HPLC-ESI-MS chromatogram.
Despite low abundance of arylguanine 22 in the reaction purine moiety. Unlike for the vinylphenylguanine 22 (Fig-
mixture, it could be isolated by column chromatography on ure 3), no by-products containing two or more phenolic
silica gel with an acidic eluent (CHCl3/MeOH/AcOH, 7:2:1) molecules attached to the purine moiety were detected. Pro-
followed by crystallisation. Under these separation condi- posed structures of Meisenheimer-type complexes 26 and
tions, at least some of the adducts were unstable and 22 was 27 derived from phenols 18 and 19, respectively, are shown
obtained in a 9% yield.
in Figure 3.
Scheme 4. Preparation of O6-arylguanines: (i) K2CO3 and DBU in DMF; 50 °C, 2 d for 21, 65 °C, 3 d for 22 and 23.
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Nucleobase Adducts Derived from Styrene 7,8- and 3,4-Oxides
FULL PAPER
dation of styrene and its metabolites (phenylethanol and
phenylacetic acid, respectively). Phenolic metabolites 18–20
themselves indicate that arene oxidation occurs during
biotransformation of styrene but do not indicate whether
or not the corresponding arene oxides are capable of
binding to DNA under physiological conditions. On the
other hand, arylguanines 22–24, if found in urine, would
indicate an arylation of nucleic acids by arene oxides 2–4 in
vivo.
Vinylphenol (18) is not commercially available and was
therefore prepared by alkaline hydrolysis of its acetate. This
reaction has been described previously by Corson et al.[31]
to proceed in aqueous KOH solution. After hydrolysis, the
phenolate obtained was acidified with carbon dioxide to
liberate phenol 18. Under these conditions, it was rather
difficult to control the pH of the solution and to prevent
the product from polymerising, which occurs at slightly
acidic pH. Therefore, we modified the reaction conditions
using KOH in aqueous ethanol for hydrolysis and an ad-
dition of toluene to the reaction mixture during acidifica-
tion. The product liberated from the phenolate was immedi-
ately extracted into the toluene layer and thereby protected
from polymerisation.
Discussion
1-Phenylethane-1,2-diol reacts non-selectively with
DABCO-purine in the presence of a base to give a mixture
of two O6-guanine adducts 7 and 8. Each of these products
can be obtained selectively by the reaction of monopro-
tected diols 10–13. In all cases an excess of sodium hydride
was required as a base. One equivalent of NaH is required
for neutralisation of the acidic NH at position 6 of
DABCO-purine 9, therefore more base is needed for depro-
tonation of the hydroxy group to obtain a nucleophilic alk-
oxide capable of the reaction with 9. For primary alcohols
11 and 13, the yield of the reaction increases significantly
as the amount of the base is increased from two to three
equivalents, giving high yields of 94 and 79% for 11 and
13, respectively. On the other hand, the secondary alcohols
10 and 12 gave rather low yields, even when three equiva-
lents of the base were used. This observation may be ex-
plained by a lower reactivity of the alkoxide formed due to
steric hindrance. This assumption is also supported by the
higher reactivity of allyl-protected diols 10 and 11 as com-
pared to their benzyl-protected analogues 12 and 13.
Monoprotected diols 10–13 react with DABCO-purine 9
exclusively at the unprotected hydroxy group. Thus, com-
Phenolates are much weaker nucleophiles than alkoxides.
pounds 10 and 12 give solely the α-isomers 14 and 16, It is therefore not surprising that they react much more
respectively, whereas compounds 11 and 13 give the β-iso- slowly and give lower yields than the alkoxides derived from
mers 15 and 16, respectively. The isomers were assigned un- 10–13. On the other hand, a combination of weaker bases
equivocally for allyl-protected derivatives 14 and 15 by (potassium carbonate and DBU instead of sodium hydride)
HMBC NMR experiments. Cross-peaks between purine C- is sufficient to deprotonate phenols and induce their reac-
6 signals and the CH or CH2 protons of the side-chain tion with DABCO-purine (9). The structures of the Mei-
clearly indicate which carbon of the side-chain is attached senheimer-type complexes shown in Figure 3 are based so-
to O6. Another distinctive feature of the NMR spectra of lely on ESI-MS data and should therefore be considered
the α- and β-isomers is a significant downfield proton shift tentative. Analogous complexes were to be expected when
of the CH and CH2 groups, respectively, directly attached reacting phenolic compounds 19 and 21 with 9. In fact,
to the O6 position. As a consequence, the difference be- in these reactions only hydroxyphenylethanol (19) gave a
tween the chemical shifts values [∆ = δH(CH) – δH(CH2)] compound that gives an ionic species in the ESI mass spec-
for α-isomers (2.6 0.1 ppm), is markedly greater than that trum that could be assigned as a Meisenheimer adduct of
for β-isomers (0.4 0.3 ppm). These data are summarised DABCO and phenol 19. The addition of DBU to the reac-
in Table 4.
tion mixture favoured the formation of O6-arylguanines 23–
While styrene 7,8-oxide (1) is a known metabolite of sty- 25. The ability of DBU to induce elimination reactions is
rene that is capable of DNA adduct formation,[27] the evi- well known, so thigher yields of arylguanines 23–25 are
dence of the formation of arene oxides 2–4 is indirect. It consistent with the formation of Meisenheimer complexes,
has been shown that several phenolic metabolites are which eliminate the corresponding phenols to give aryl-
formed during the biotransformation of styrene, mainly vi- guanines as the final products. On the other hand, DBU
nylphenol (18),[28] hydroxyphenylethanol (19) and hydroxy- did not improve the yield of vinylphenylguanine (22).
phenylacetic acid (20).[29] A general metabolic pathway Therefore, it is likely that the by-products detected by
from arenes to phenols proceeds via the corresponding ar- HPLC-MS at m/z = 374, 494 and 606 are adducts of the
ene oxides and an NIH shift.[30] Arylguanines 22–24 are aminopurinyl moiety to the vinyl group of 18 rather than
therefore expected DNA adducts arising from the arene oxi- Meisenheimer complexes.
Table 4. Comparison of the proton chemical shift values (in ppm) of the OCH(Ph)CH2O moiety for isomeric O6-guanine adducts.
α-Isomers
β-Isomers
7
14
16
mean
8
15
17
mean
δH(CH)
6.45
3.95
2.50
6.55
3.82
2.73
6.51
3.81
2.70
6.50 0.05
3.86 0.08
2.64 0.13
5.17
4.49
0.68
4.85
4.50
0.35
4.87
4.67
0.20
4.96 0.18
4.55 0.10
0.41 0.25
δH(CH2)[a]
∆ = δH(CH) – δH(CH2)
[a] Average value of δA and δB (AB system).
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J. Novák, Z. Hasník, I. Linhart
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mixture stirred at 70 °C for 24 h, then neutralised with acetic acid
(17.8 mg, 2.96 mmol) and the DMSO removed under vacuum. The
oily residue was purified by column chromatography on silica gel
using CHCl3/MeOH (6:1) as an eluent to obtain 182 mg (46%)
of a white powder of which a fraction was pure O6-(2-hydroxy-2-
phenylethyl)guanine (8; (O6-β-isomer, 18% yield) and the rest was
a 1:1 mixture of the O6-β-isomer 8 and O6-(2-hydroxy-1-phenylethyl)-
guanine (7; O6-α-isomer). The mixture was further separated by
HPLC using 0.02 m HCOONH4 and methanol at pH 6.0 as a mo-
bile phase.
O6-(2-Hydroxy-1-phenylethyl)guanine (O6-α-isomer, 7): M.p. 131–
135 °C. Rf (CHCl3/MeOH, 6:1) = 0.35. UV: λmax = 242 and 282 nm
(pH 6.5, 290 nm (pH 1); 286 nm (pH 12). 1H NMR (500 MHz,
[D7]DMF): δ = 3.85 (dd, J = 4.0 and 11.6 Hz, 1 H, CH2O), 4.05
(dd, J = 7.4 and 11.6 Hz, 1 H, CH2O), 6.45 (dd, J = 4.2 and 7.4
Hz, 1 H, CHO), 7.32 (m, 3 H, Ph), 7.51 (d, J = 7.2 Hz, 2 H, Ph),
8.02 (s, 1 H, 8-H) ppm. 13C NMR (125.8 MHz, [D7]DMF): δ =
66.0 (CH2O), 78.6 (CHO), 127.6, 128.2, 140.0 (Ph), 138.0 (C-8),
160.9 (C-6) ppm. ESI-MS: m/z = 294 [M + Na]+, 272 [M + H]+,
152 [M + H – PhCHOHCH2]+, 135 [M + H – PhCHOHCH2OH].
C13H13N5O2 (271.28): calcd. C 57.6, H 4.8, N 25.8; found C 57.4,
H 4.9, N 25.7.
Protection of the carboxylic group of phenol 20 as its
ethyl ester did not lead to any improvement of the reaction
yield. In fact, the reaction of 9 with ethyl ester 21 required
a higher reaction temperature, a prolonged reaction time
and gave a lower yield than that with unprotected carboxyl-
ate 20.
Unlike O6-alkylguanines 7 and 8 and their nucleoside
and nucleotide analogues, O6-arylguanine adducts 22–24
have not been prepared as yet and are hardly accessible by
the reaction of corresponding oxirane derivative (arene ox-
ide) with guanine
Experimental Section
General: Column chromatography was performed on silica gel 60
purchased from Fluka (particle size 0.063–0.200 mm). Merck Silica
gel 60 F254 plates were used for thin-layer chromatography. Di-
methyl sulfoxide (DMSO) was dried by distillation over calcium
hydride and stored over molecular sieves. Other chemicals obtained
from commercial sources were of analytical or synthetic grade and
were used as received. 1H and 13C NMR spectra were recorded
with a Bruker Avance DRX500 (500 MHz for 1H) or with a Varian
Mercury 300 (300 MHz for 1H) Fourier transform NMR spectrom-
O6-(2-Hydroxy-2-phenylethyl)guanine (O6-β-isomer, 8): M.p. 138–
141 °C. Rf (CHCl3/MeOH, 6:1) = 0.37. UV: λmax = 240 nm and
eter. In the HMBC experiments, the parameters were set to show 282 nm (pH 6.5); 288 nm (pH 1); 284 nm (pH 12). 1H NMR
cross-peaks for nuclei interacting with a JH,C coupling constant of
7 Hz. Mass spectra were measured with a triple quadrupole HPLC-
MS system Varian 1200 equipped with electrospray ionisation
(ESI). Positive ions were detected. Microwave-induced reactions
were performed in a Synthewave 402 reactor (Synthelabo, France)
operating in the controlled-temperature mode.
(500 MHz, [D7]DMF): δ = 4.42 (dd, J = 8.4 and 10.8 Hz, 1 H,
CH2O), 4.56 (dd, J = 3.6 and 10.8 Hz, 1 H, CH2O), 5.17 (dd, J =
3.4 and 8.1 Hz, 1 H, CHO), 7.39 (m, 3 H, Ph), 7.57 (d, J = 7.2 Hz,
2 H, Ph), 8.02 (s, 1 H, 8-H) ppm. 13C NMR (125.8 MHz, [D7]-
DMF): δ = 71.8 (CHO), 72.2 (CH2O), 113.2 (C-5), 127.0, 127.9,
128.7 and 143.1 (Ph), 138.0 (C-8), 138.6 (C-4), 142.1 (C-2), 160.9
(C-6) ppm. ESI-MS: m/z = 294 [M + Na]+, 272 [M + H]+, 152
[M + H – PhCHOHCH2]+, 135 [M + H – PhCHOHCH2OH]+.
C13H13N5O2 (271.28): calcd. C 57.6, H 4.8, N 25.8; found C 57.4,
H 4.9, N 25.7.
Starting Materials: 2-Allyloxy-1-phenylethanol (10) was prepared
from methyl mandelate as described in the literature.[32] 2-Ben-
zyloxy-1-phenylethanol (12) was prepared in an analogous way and
identified by comparing its NMR spectra with those published.[33]
Isomeric monoprotected diols, i.e., 2-allyloxy-2-phenylethanol (11)
and 2-benzyloxy-2-phenylethanol (13) were prepared by DDQ-cat-
alysed ring opening of 1 with allyl and benzyl alcohol, respec-
tively.[34] 2-Amino-6-(1-azonia-4-azabicyclo[2.2.2]oct-1-yl)purine
(2) (DABCO-purine) was obtained by reaction of 2-amino-6-chlo-
ropurine (5) with an excess of 1,4-diazabicyclo[2.2.2]octane
(DABCO).[18]
Reaction of 1-Phenyl-1,2-ethanediol with DABCO-Purine (MW-In-
duced): The reaction mixture was prepared as described for the ex-
periment with conventional heating and then irradiated by micro-
waves in a MW reactor with temperature set to 50, 70 or 90 °C.
The reaction time varied from 2.5 to 3.5 h. The products were iso-
lated by column chromatography on silica gel as described above.
The mixtures of 7 and 8 obtained were further separated by semi-
preparative HPLC to get samples of pure isomers. The yields and
ratios of the isomers are shown in Table 1.
4-Vinylphenol (18): 4-Vinylphenyl acetate (2 g, 12.3 mmol) was
added to a solution of potassium hydroxide (1.72 g, 32 mmol) in
18 mL of aqueous ethanol (1:1 mixture) and the reaction mixture
was stirred for 1.5 h under external cooling with crushed ice. Tolu-
ene (16 mL) was then added and the reaction mixture was neutral-
ised to pH 7–8 by introduction of carbon dioxide. The layers were
separated, the aqueous layer extracted with toluene (8 mL) and the
combined organic phases evaporated under vacuum to dryness.
Crystallisation of the residue from petroleum ether yielded 880 mg
Reaction of DABCO-Purine (9) with Monoprotected Diols 10–13: A
solution of the monoprotected diol (3.18 mmol) in DMF (7 mL)
was added to NaH (51 mg, 2.12 mmol) and the mixture was stirred
for 30 min at ambient temperature under dry nitrogen. DABCO-
purine (9; 0.300 g, 1.06 mmol) was added to the alkoxide formed
and the reaction mixture was allowed to react for 24 h at room
temperature. After neutralisation with acetic acid (121 µL, 127 mg,
(60%) of white crystals, m.p. 67–70 °C (ref.[31] 68–69 °C) which 2.12 mmol) and evaporation of DMF under vacuum the crude
were identified by comparing their NMR spectra with those pub-
product was purified by column chromatography on silica gel using
CHCl3/MeOH (10:1 or 15:1) as eluent for allyl- and benzyl-pro-
tected compounds, respectively. In another set of experiments,
4.24 mmol (4 equiv.) of the corresponding monoprotected diol was
used, while the amount of NaH was increased to 2.65 mmol
(2.5 equiv.) or 3.20 mmol (3 equiv.). The yields are shown in
Table 2.
O6-[2-(Allyloxy)-1-phenylethyl]guanine (14): A yellowish powder
(0.180 g, 54%) obtained by the reaction of alcohol 10 with 9.
Recrystallisation from H2O/EtOH (10:1) yielded a white powder,
lished.[35]
Reaction of 1-Phenylethane-1,2-diol (6) with DABCO-Purine (Con-
ventional Heating): The reaction conditions were analogous to
those of Lembitz et al.[19] A mixture of 2-amino-6-chloropurine (5;
0.25 g, 1.48 mmol) and DABCO (0.5 g, 4.45 mmol) was dissolved
in 4 mL of DMSO and left standing for 3 h to form a solution
of
2-amino-6-(1-azonia-4-azabicyclo[2.2.2]oct-1-yl)purine
(9;
DABCO-purine). The sodium salt of diol 6 was then added [1 g
(7.24 mmol) of 6 and 71 mg (2.96 mmol) of NaH] and the reaction
512
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 507–515

Nucleobase Adducts Derived from Styrene 7,8- and 3,4-Oxides
FULL PAPER
m.p. 160–162 °C. Rf (CHCl3/MeOH, 10:1) = 0.35. UV: λmax = 242
and 284 nm (pH 6.5); 290 nm (pH 1); 286 nm (pH 12). 1H NMR
(500 MHz, CDCl3): δ = 3.72 (dd, J = 3.5 and 10.7 Hz, 1 H,
OCHPhCH2), 3.91 (dd, J = 8.4 and 10.6 Hz, 1 H, OCHPhCH2),
4.00 (m, 2 H, OCH2CHCH2), 5.06 (s, 2 H, NH2), 5.17 (d, J =
10.6 Hz, 1 H, OCH2CHCH2), 5.21 (d, J = 17.3 Hz, 1 H,
OCH2CHCH2), 5.81 (m, 1 H, OCH2CHCH2), 6.55 (dd, J = 3.5 Hz
Deallylation: The reaction conditions were analogous to those de-
scribed by Chandrasekhar et al.[26] Poly(methylhydrosiloxane)
(PMHS; 115 mg), tetrakis(triphenylphosphane)palladium (10 mg)
and ZnCl2 were added to a stirred solution of the allyl-protected
purine derivative (0.300 g, 0.96 mmol) in 8 mL of DMF under ar-
gon. The reaction mixture was stirred at 60 °C for 7–10 h. After
evaporation of the solvent under vacuum, the crude product was
and 8.1 Hz, 1 H, OCHPhCH2), 7.25 (m, 3 H, Ph), 7.42 (d, J = purified by column chromatography on silica gel (chloroform/
7.1 Hz, 2 H, Ph), 7.68 (s, 1 H, 8-H) ppm. 13C NMR (125.8 MHz,
CDCl3): δ = 72.3 (OCHPhCH2), 73.2 (OCH2CHCH2), 76.5
(OCHPhCH2), 114.3 (C-5), 117.4 (OCH2CHCH2), 126.8, 128.1,
128.4 and 137.9 (Ph), 134.3 (OCH2CHCH2), 137.7 (C-8), 154.6 (C-
2), 159.1 (C-4), 160.3 (C-6) ppm. HMBC shows cross-peaks be-
tween C-6 (δC = 160.3 ppm) and CHO (δH = 6.55 ppm). ESI-MS:
m/z = 350 [M + K]+, 334 [M + Na]+, 312 [M + H]+. C16H17N5O2·
1/4H2O (315.85): calcd. C 60.8, H 5.6, N 22.2; found C 60.6, H
5.4, N 22.5.
methanol, 4:1) and crystallised from MeOH with the addition of
charcoal as a decolourising agent.
O6-(2-Hydroxy-1-phenylethyl)guanine (O6-α-isomer, 7): Obtained as
a greyish powder (0.220 g, 84%) by deallylation of 14. Crystallisa-
tion from MeOH with the addition of charcoal as a decolourising
agent yielded a white powder (0.084 g, 32%).
O6-(2-Hydroxy-2-phenylethyl)guanine (O6-β-isomer, ): Obtained as
a greyish powder (0.210 g, 80%) by deallylation of 15. Crystallisa-
tion from MeOH with the addition of charcoal as a decolourising
agent yielded a white powder (0.074 g, 28%).
O6-[2-(Allyloxy)-2-phenylethyl]guanine (15): A yellowish precipitate
(0.311 g; 94%) obtained from the reaction of alcohol 11 with purine
9. Recrystallisation from toluene/ethanol (10:1) gave a white pow-
Debenzylation: The reaction conditions were analogous to those
described by Bieg et al.[36] Palladium (10%) on carbon containing
nearly 50% of water, (Degussa type E101 NE/W) (1.484 g) was acti-
vated under vacuum at 110 °C. The benzyl-protected derivative
(0.300 g, 0.83 mmol) and ammonium formate (0.325 g) in 19 mL
of absolute MeOH was added to the catalyst and refluxed for 5–
7 h. The total amount of ammonium formate (1.3 g) was added
subsequently in four portions. Each portion was added after hydro-
gen had stopped forming. The catalyst was filtered off and washed
with hot aqueous methanol. Finally, the filtrate was evaporated
under vacuum.
der, m.p. 195–197 °C. Rf (CHCl3/MeOH, 10:1) = 0.40. UV: λmax
=
240 and 282 nm (pH 6.5); 288 nm (pH 1); 286 nm (pH 12). 1H
NMR (500 MHz, [D6]DMSO): δ = 3.95 (br. s, 2 H, OCH2CHCH2),
4.50 (d, J = 5.5 Hz, 2 H, OCH2CHPh), 4.85 (t, J = 5.5 Hz, 1 H,
OCH2CHPh), 5.11 (d, J = 10.5 Hz, 1 H, OCH2CHCH2), 5.27 (d,
J = 17.5 Hz, 1 H, OCH2CHCH2), 5.87 (m, 1 H, OCH2CHCH2),
6.25 (s, 2 H, NH2), 7.40 (m, 5 H, Ph), 7.81 (s, 1 H, 8-H) ppm. 13C
NMR (125.8 MHz, [D6]DMSO): δ = 69.1 (OCH2CHCH2), 69.2
(OCH2CHPh), 78.6 (OCH2CHPh), 113.4 (C-5), 116.3
(OCH2CHCH2), 127.0, 128.1, 128.5 and 138.8 (Ph), 135.0
(OCH2CHCH2), 137.8 (C-8), 155.2 (C-2), 159.6 (C-4), 159.9 (C-6)
ppm. HMBC shows cross-peaks between C-6 (δC = 159.9 ppm) and
OCH2CHPh (δH = 4.50 ppm). ESI-MS: m/z = 334 [M + Na]+, 312
[M + H]+, 152. C16H17N5O2·1/4H2O (315.85): calcd. C 60.8, H 5.6,
N 22.2; found C 60.5, H 5.4, N 22.5.
O6-(2-Hydroxy-1-phenylethyl)guanine (O6-α-isomer, 7): Obtained as
a white powder (0.043 g, 19%) by debenzylation of 16.
O6-(2-Hydroxy-2-phenylethyl)guanine (O6-β-isomer, 8): Obtained as
a white powder (0.045 g, 21%) by debenzylation of 17.
O6-[2-(Benzyloxy)-1-phenylethyl]guanine (16): A white precipitate
(0.173 g, 45%) obtained from the reaction of 12 with 9, m.p. 69–
73 °C. Rf (CHCl3/MeOH, 15:1) = 0.26. UV: λmax = 242 and 284 nm
(pH 6.5); 290 nm (pH 1); 286 nm (pH 12). 1H NMR (300 MHz,
CDCl3): δ = 3.71 (dd, J = 3.8 and 10.6 Hz, 1 H, OCHPhCH2), 3.92
(dd, J = 8.2 and 10.6 Hz, 1 H, OCHPhCH2), 4.47 (d, J = 12.0 Hz,
1 H, OCH2Ph), 4.54 (d, J = 12.0 Hz, 1 H, OCH2Ph), 4.83 (s, 2 H,
NH2), 6.51 (dd, J = 3.8 and 7.9 Hz, 1 H, OCHPhCH2), 7.19 (m, 8
H, Ph), 7.38 (d, J = 6.7 Hz, 2 H, Ph), 7.62 (s, 1 H, 8-H) ppm.
13C NMR (CDCl3): δ = 73.4, (OCHPhCH2), 73.5 (OCH2Ph), 76.6
(OCHPhCH2), 127.0, 127.8, 127.9, 128.3, 128.5 and 137.8, 138.0
(Ph), 159.2 (C-6) ppm. ESI-MS: m/z = 400 [M + K]+, 384 [M +
Na]+, 362 [M + H]+. C20H19N5O2·1/4H2O (365.91): calcd. C 65.6,
H 5.4, N 19.1; found C 65.8, H 5.5, N 18.9.
Preparation of O6-arylguanines Using NaH as a Base [Procedure (i)]:
DABCO-purine (9; 300 mg, 1.06 mmol) was added to a solution of
sodium phenolate in DMF prepared from the corresponding phe-
nol (3.18 mmol, threefold excess) and sodium hydride (76 mg,
3.17 mmol). The reaction mixture was heated at 60 °C under dry
nitrogen for 3 d. After cooling to room temperature, acetic acid
(180 µL, 189 mg, 3.15 mmol) was added for neutralisation and the
solvent was distilled off under vacuum. The residue was redissolved
in CHCl3/MeOH (8:1) and separated by column chromatography
on silica gel to obtain a crude product.
Preparation of O6-arylguanines Using K2CO3 and DBU [Procedure
(ii)]: A mixture of the phenol (3.28 mmol), potassium carbonate
(731 mg, 5.30 mmol) and DMF (5 mL) was stirred under dry nitro-
gen for 30 min. DABCO-purine (9; 300 mg, 1.06 mmol) was then
added and the reaction mixture was stirred for another 30 min at
ambient temperature. Thereafter, DBU (158 µL, 161 mg,
1.06 mmol) was added and the resulting mixture heated for 3 d at
65 °C. The solvent was evaporated under vacuum and the residue
separated by column chromatography on silica gel using CHCl3/
MeOH (8:1) to obtain a crude product.
O6-[2-(Benzyloxy)-2-phenylethyl]guanine (17): A white precipitate
(0.305 g, 79%) obtained from the reaction of 13 with 9, m.p. 73–
76 °C. Rf (CHCl3/MeOH, 15:1) = 0.23. UV: λmax = 240 and 282 nm
(pH 6.5); 290 nm (pH 1); 286 nm (pH 12). 1H NMR (500 MHz,
CDCl3): δ = 4.39 (d, J = 11.7 Hz, 1 H, OCH2Ph), 4.54 (d, J =
11.7 Hz, 1 H, OCH2Ph), 4.59 (dd, J = 3.3 and 11.3 Hz, 1 H,
OCH2CHPh), 4.75 (m, 1 H, OCH2CHPh), 4.87 (dd, J = 3.5 and
7.6 Hz, 1 H, OCH2CHPh), 5.33 (s, 2 H, NH2), 7.30 (m, 8 H, Ph), O6-(4-Vinylphenyl)guanine (22): Obtained by crystallisation of the
7.43 (d, J = 7.1 Hz, 2 H, Ph), 7.76 (s, 1 H, 8-H) ppm. 13C NMR crude product of the reaction of 9 with phenol 18 from CHCl3/
(CDCl3):
δ
=
70.1 (OCH2CHPh), 70.7 (OCH2Ph), 79.3
MeOH/AcOH. Procedures (i) and (ii) yielded 25 mg (9%) and
17 mg (6%), respectively, of a white powder identified as aryloxy-
(OCH2CHPh), 127.1, 127.5, 127.6, 128.2, 128.6 and 137.9, 138.3
(Ph), 138.5 (C-8), 159.2 (C-6) ppm. ESI-MS: m/z = 400 [M + K]+, purine 22, m.p. 212–217 °C. Rf (CHCl3/MeOH, 8:1) = 0.25. UV:
384 [M + Na]+, 362 [M + H]+. C20H19N5O2 (361.41): calcd. C λmax = 244 and 294 nm (pH 1); 246 and 286 nm (pH 6.5); 250 and
1
66.46, H 5.30, N 19.38; found C 66.2, H 5.1, N 19.3.
292 nm (pH 12). H NMR (500 MHz, [D6]DMSO): δ = 1.88 (s, 3
Eur. J. Org. Chem. 2006, 507–515
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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513

J. Novák, Z. Hasník, I. Linhart
FULL PAPER
H, CH3COO–), 5.23 (d, J = 10.7 Hz, 1 H, CH2), 5.80 (d, J =
17.6 Hz, 1 H, CH2), 6.75 (dd, J = 10.7, 17.6 Hz, 1 H, PhCH), 7.19
(d; 2 H; J = 8.3 Hz, Ph), 7.51 (d, J = 8 Hz, 2 H, 5 Hz, Ph), 7.91
(s, 1 H, C8-H) ppm. 13CNMR (75 MHz, [D6]DMSO): δ = 21.1
(CH3COO–), 114.0 (CH=CH2), 122.0 and 127.3 (aromatic CH),
134.1 and 152.4 (aromatic C), 135.9 (CH=CH2), 156.2 (C4),
Acknowledgments
Financial support though grants 310/03/0437 from the Grant
Agency of the Czech Republic and MSM 604 613 73 01 from the
Ministry of Education of the Czech Republic is gratefully acknowl-
edged.
159.7 (C6). ESI-MS: m/z
=
292 [M
+
K]+, 276
[M + Na]+, 254 [M + H]+. C13H11N5O·C2H4O2 (313.32): calcd. C
57.5, H 4.8, N 22.3; found C 57.8, H 5.0, N 22.8.
[1] A. E. Pegg, Cancer Res. 1990, 50, 6119–6129.
O6-[4-(2-Hydroxyethyl)phenyl]guanine (23): Obtained by the reac-
tion of 9 with phenol 19. The crude product was purified by col-
umn chromatography on silica gel using CHCl3/MeOH/AcOH,
30:3:1 as an eluent. Procedures (i) and (ii) gave 90 mg (35%) and
27 mg (9%), respectively, of a white powder which was identified
as arylguanine 23, m.p. 232–236 °C. Rf (CHCl3/MeOH/AcOH,
30:3:1) = 0.28. UV: λmax = 292 nm (pH 1); 286 nm (pH 6.5); 292 nm
(pH 12). 1H NMR (300 MHz, [D6]DMSO): δ = 2.72 (t, J = 6.9 Hz,
2 H, PhCH2), 3.62 (t, J = 6.9 Hz, 2 H, CH2O), 4.66 (s, 1 H,
CH2OH), 7.10 (d, J = 8.5 Hz, 2 H, Ph), 7.24 (d, J = 8.6 Hz, 2 H,
Ph), 7.91 (s, 1 H, C8-H) ppm. 13C NMR (75 MHz, [D6]DMSO): δ
= 38.0 (CH2CH2OH), 62.7 (CH2OH), 113.6 (C5), 122.0 and 130.6
(aromatic CH), 137.0 and 151.4 (aromatic C), 140.0 (C8), 157.4
(C4), 160.0 (C2), 160.4 (C6). ESI-MS: m/z = 310 [M + K]+, 294
[M + Na]+, 272 [M + H]+. C13H13N5O2·H2O (289.30): calcd. C
54.0, H 5.2, N 24.2; found C 53.6, H 4.8, N 22.9.
[2] C. Mi-Young, M. G. McDougall, M. E. Dolan, K. Swenn,
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O6-(4-Carboxymethylphenyl)guanine (24): Obtained by procedure
(ii) by reacting 9 with phenolic acid 20. The crude product was
purified by column chromatography on silica gel using CHCl3/
MeOH/AcOH (30:3:1) as eluent. Arylguanine 24 (105 mg, 35%)
was obtained as a white powder, m.p. Ͼ 300 °C, Rf (CHCl3/MeOH/
AcOH, 30:3:1) = 0.23. UV: λmax = 292 nm (pH 1); 286 nm (pH
6.5); 290 nm (pH 12). 1H NMR ([D6]DMSO): δ = 3.44 (s, 2 H,
OCH2CO2); 6.27 (s, 2 H, NH2), 7.10 (d, J = 8.5 Hz, 2 H, Ph), 7.27
(d, J = 8.5 Hz, 2 H, Ph), 7.9 (s, 1 H, C8-H) ppm. 13C NMR
(75 MHz, [D6]DMSO): δ = 43.0 (PhCH2), 113.6 (C5), 121.9 and
131.1 (aromatic CH), 134.3 and 151.5 (aromatic C), 157.5 (C4),
160.4 (C6), 175.0 (COOH). ESI-MS: m/z = 324 [M + K]+, 308 [M
+ Na]+, 286 [M + H]+. C13H11N5O3·H2O (303.28): calcd. C 51.5,
H 4.3, 23.1; found C 51.1, H 4.3, N 22.9.
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
O6-(4-Ethoxycarbonylmethylphenyl)guanine (25): Obtained by pro-
cedure (ii) by reacting 9 with phenol 21. Crystallisation of the crude
product from chloroform afforded 82 mg (25%) of arylguanine 25.
White powder, m.p. 215–217 °C, Rf (CHCl3/MeOH/AcOH, 30:3:1)
= 0.56. UV: λmax = 292 nm (pH 1); 286 nm (pH 6.5); 290 nm (pH
12). 1H NMR ([D6]DMSO): δ = 1.19 (t, J = 7.15 Hz, 3 H,
CH2CH3), 3.68 (s, 2 H, OCH2CO2), 4.85 (q, J = 7.15 Hz, 2 H,
CH2CH3), 6.26 (s, 2 H, NH2), 7.17 and 7.30 (d,, J = 8.4 Hz 2+2
H, Ph); 7.93 (s, 1 H, C8–H) ppm. 13C NMR (75 MHz, [D6]DMSO):
δ = 14.8 (CH2CH3), 52.4 (CH2COOH), 61.0 (OCH2CH3), 114.4
(C5), 122.3 and 131.2 (aromatic CH), 131.7 (aromatic C), 152.2
(aromatic COH), 156.8 (C4), 160.4 (C6), 172.0 (COOH). ESI-MS:
m/z = 352 [M + K]+, 334 [M + Na]+, 314 [M + H]+.
HPLC-MS Analyses: The crude products of the reaction of 9 with
phenols 18–20 were dissolved in aqueous methanol and analysed
by HPLC-MS on a Phenomenex Luna 2 C18 column (250×2 mm;
particle size: 5 µm). The column was eluted with 5 mm ammonium
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a rate of
[28]
[29]
[30]
O. M. Bakke, R. R. Scheline, Toxicol. Appl. Pharmacol. 1970,
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early up to 100% within 25 min and than kept unchanged for an
additional 5 min. The effluent was introduced into an ESI chamber
and the ions formed were detected in the mass range of m/z = 150–
700.
16, 691–700.
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2000, 13, 36–44.
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514
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 507–515

Nucleobase Adducts Derived from Styrene 7,8- and 3,4-Oxides
FULL PAPER
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Received: June 28, 2005
Published Online: November 9, 2005
Eur. J. Org. Chem. 2006, 507–515
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