Synthesis of 3-(2-hydroxy-1-phenylethyl)- and 3-(2-hydroxy-2-phenylethyl) adenine, DNA adducts derived from styrene

Article abstract of DOI:10.1002/jhet.5570450325

(Chemical Equation Presented) 3-(2-Hydroxy-2-phenylethyl)- and 3-(2-hydroxy-1-phenylethyl)adenine, DNA adducts derived from styrene, along with their 9-substituted analogues were prepared by alkylation of 8-bromoadenine with corresponding allyl-protected bromohydrins followed by a new deallylation procedure using tetrakis(triphenylphosphine)palladium catalyzed reductive cleavage by poly(methylhydrosiloxane) in the presence of p-toluenesulphonic acid. This novel procedure proved to be useful for purine derivatives, which were resistant to other deallylation protocols. Structure of positional isomers was assigned using 2D NMR experiments HMBC and HMQC.

Full text of DOI:10.1002/jhet.5570450325

May-Jun 2008
789
Synthesis of 3-(2-Hydroxy-1-phenylethyl)- and 3-(2-Hydroxy-2-
phenylethyl)adenine, DNA Adducts Derived from Styrene
Jan Krouželka, Igor Linhart* and Tomáš Tobrman
Department of Organic Chemistry, Institute of Chemical Technology, Prague, Technicka 1905, CZ-166 28
Prague, Czech Republic; E-mail: linharti@vscht.cz
Received August 27, 2007
NH2
N
NH2
N
H2N
N
N
N
N
Y
O
N
X
N
N
(i), (ii)
+
+
Br
N
Y
N
H
Br
OH
N
X
X
Y
OH
X = H, Y = Ph or
X = Ph, Y = H
(i) K2CO3/DMF; (ii) Pd(PPh3)4, TsOH, PMHS, DMF
3-(2-Hydroxy-2-phenylethyl)- and 3-(2-hydroxy-1-phenylethyl)adenine, DNA adducts derived from
styrene, along with their 9-substituted analogues were prepared by alkylation of 8-bromoadenine with
corresponding allyl-protected bromohydrins followed by new deallylation procedure using
a
tetrakis(triphenylphosphine)palladium catalyzed reductive cleavage by poly(methylhydrosiloxane) in the
presence of p-toluenesulphonic acid. This novel procedure proved to be useful for purine derivatives,
which were resistant to other deallylation protocols. Structure of positional isomers was assigned using 2D
NMR experiments HMBC and HMQC.
J. Heterocyclic Chem., 45, 789 (2008).
INTRODUCTION
In this study we describe a synthesis of both N3 and N9
substituted adenines derived from 1, the former ones as
biomarkers of DNA damage caused by styrene exposure,
the latter as suitable internal standards for the
development of analytical methods to determine adenine
adducts in urine. Because the primary aim was to obtain
pure samples of products to be used as analytical
standards we focused on the product purity rather than to
strive for maximum yields.
Alkyl-, aryl- and aralkyladenines play an important
role as bioactive compounds [1] and tools for the
detection and evaluation of DNA lesions [2,3]. Among
them, N3 substituted adenines are nucleobase DNA
adducts formed by covalent binding of electrophiles to
adenine in the DNA which are cleaved off
spontaneously from the DNA strand and finally
excreted in urine. Therefore, they can serve as urinary
biological markers of exposure to industrial and
environmental mutagens and genotoxic carcinogens for
early diagnosis of damage to DNA [2]. For styrene 7,8-
oxide (1), a reactive electrophilic metabolite of styrene,
N3 adenine adducts comprised 4 % of the total
alkylation of DNA when double stranded DNA was
alkylated at physiological pH, i.e., N3 adenine adducts
are the second most abundant DNA adducts derived
from styrene [4]. However, no N3 adenine adducts
were formed by the reaction of 1 with 2'-deoxy-
adenosine-3'-phosphate [4], adenosine [5] or 2'-deoxy-
adenosine [6]. Therefore, unlike other styrene derived
DNA adducts, N3 adenine adducts are not accessible
by direct reactions of styrene 7,8-oxide with
nucleosides or nucleotides.
RESULTS AND DISCUSSION
Styrene 7,8-oxide is a weak electrophile reacting with
nucleophiles non-selectively at both α- and β-position
giving rise to two isomeric products. In our previous work
we found that allyl and benzyl protected bromohydrins
2a,b and 3a,b can be used as suitable synthetic
equivalents of 1 to achieve selective alkylation (Scheme
1) [10,11]. In addition, benzyl-protected triflate 2-
benzyloxy-2-phenylethyl triflate (4) [12] was tested as a
more reactive alkylating reagent.
Although in the presence of a base, the alkylation of
adenine is directed mainly to the N9 position [7] a
selective alkylation at N3 was observed when the
alkylation of adenine by reactive allylic or benzylic
halides was performed in dimethylacetamide and no base
except adenine itself was employed [13]. However, our
attempts to alkylate adenine with allyl protected
bromohydrins 2a and 3a at the same reaction conditions
led to a mixture of at least four products as detected by
Adenine itself reacts with alkylating agents in the
presence of a base rather non-selectively to yield a
mixture of products alkylated at various nitrogens, N9
being usually the most prominent site of attack [7-9].

790
J. Krouželka, I. Linhart and T. Tobrman
Vol 45
3a and 3b each gave a mixture of alkylated adenines of
which corresponding protected 9-(2-hydroxy-1-phenyl-
ethyl)- and 7-(2-hydroxy-1-phenylethyl)adenines (6a,b
and 7a,b, respectively) were isolated.
To prepare both N3 and N9 substituted adenines we
decided to use 8-bromoadenine (8) as a precursor of
adenine, which after being alkylated can be converted
easily to the adenine moiety by catalytic reduction of
Scheme 1
OPG
Br
Br
OPG
Ph
Ph
2a, b
3a, b
(i)
(i)
bromine. In
a recent study, alkylation of 8 by
4-fluorobenzyl bromide gave N3- and N9-substituted
products in nearly 1:1 ratio [14]. The alkylations of 8 with
2a,b and 3a,b (Scheme 2) were carried out in DMF using
potassium carbonate as a base at temperatures between 50
and 100°C.
Nu
OH
+
OH
Nu
Ph
Ph
The results are summarized in the Table 1. The
alkylation proceeded at N3 and N9 position of the purine
moiety giving two products, the N3 substituted
derivatives being the major ones. Alkylation with 2a and
2b gave a mixture of two products, i.e., allyl- or benzyl-
protected 8-bromo-3-(2-hydroxy-2-phenylethyl)adenine
(9) and 8-bromo-9-(2-hydroxy-2-phenylethyl)adenine
(10).
NuH
Ph
O
1
PG = allyl or benzyl; (i) NuH followed by deprotection
Table 1
Alkylation of 8-Bromoadenine (8)
Scheme 2
NH2
Reagent
t [°C]
Reaction
time [h]
Product ratio
3N:9Na
Yield [%]
3N + 9N
Y
OPG
X
N
N
N
+
Br
2a
2a
70
70
4b
20
85 : 15
71 : 30
45
39
Br
N
2a, b
3a, b
8
2a
2b
2b
2b
3a
3a
3a
3b
3b
100
70
20
16
72
20
48
24
5
67 : 33
80 : 20
65 : 35
60 : 40
67 : 33
62 : 38
65 : 35
55 : 45
60 : 40
55
25
57
69
80
90
62
62
63
(i)
70
NH2
N
100
50
NH2
N
N
N
N
N
X
N
Br
+
70
Br
OPG
N
Y
90
50
18
7
X
Y
70
OPG
3b
4
90
r.t.
5
4
60 : 40
40 : 60
65
84
9a, b
11a, b
10a, b
12a, b
[a] Product ratios were determined by integration of the C(2)H signals in
the NMR spectra. [b] The ratio of the reactants 2a : 8 was 6 : 1 in this
experiment.
2, 9, 10 X=H, Y=Ph a, PG = allyl
3, 11, 12 X=Ph, Y=H b, PG = benzyl
(i) K2CO3/DMF, 50 - 100°C
The product ratio was temperature dependent, lower
temperatures and shorter reaction times gave a better
selectivity for the N3 alkylated products (Table 1). 2-
Benzyloxy-2-phenylethyl triflate (4) [12] showed a higher
reactivity than bromides but it gave predominantly the N9
substituted product 10b the ratio of 9b:10b being 2:3.
Protected bromohydrins 3a and 3b gave also
corresponding N3 and N9 substituted products, i.e., allyl-
or benzyl-protected 8-bromo-3-(2-hydroxy-1-phenyl-
ethyl)adenine (11a,b) and 8-bromo-9-(2-hydroxy-2-
phenyl-ethyl)adenine (12a,b). Unlike for the phenethyl
1
TLC and H-NMR. Adenine itself could be alkylated in
DMF at 125°C with non-protected 2-bromo-1-
phenylethanol (2) in the presence of 18-crown-6 and
potassium carbonate as a base yielding 29 % of 9-(2-
hydroxy-2-phenylethyl)adenine (5) as the main and only
isolated product. At least two by-products were detected
by TLC.
Isomeric bromohydrin, 2-bromo-2-phenylethanol (3)
did not react with adenine under the same reaction
conditions but its allyl- and benzyl-protected derivatives

May-Jun 2008
Hydroxy(phenyl)ethyladenines
791
bromides 2a and 2b alkylation product ratios for benzylic
bromides 3a and 3b were not dependent on the reaction
temperature in the range of 50 - 90°C (Table 1). When
pure phenethyl derivatives 9a and 9b were treated with 2a
and 2b, respectively, under the reaction conditions of
alkylation, i.e., heating in DMF for 24 h at 100°C in the
presence of potassium carbonate, no migration of the
substituent form N3 to N9 was observed. Therefore, the
temperature dependent selectivity of the alkylation cannot
be attributed to a reversible alkylation [15].
Debenzylation of 9b, 10b and 12b by hydrogenolysis
with 10% Pd on charcoal proceeded smoothly along with
the reduction of bromine in the position 8 but 11b was
cleaved to adenine at the same reaction conditions.
Therefore, allyl-protected derivatives 9a – 12a were used
preferentially to prepare the target compounds.
Allylic groups in bromoadenines 9a – 12a were
resistant to deallylation procedures such as cleavage with
NaBH4/I2 [16], SmI2 [17], DIBAL with [NiCl2(dppp)] as a
catalyst [18] and PhSiH3 with Pd(PPh3)4 as a catalyst [19].
Catalytic deallylation using Pd(PPh3)4, ZnCl2 and
poly(methyl)hydrosiloxane (PMHS) [20] which we
successfully applied previously to similar allyl-protected
guanine derivatives [10,11], gave poor yields (10 % at
most) with bromoadenines 9a – 12a. However, when
ZnCl2 was replaced by TsOH as a stronger acid,
deallylation proceeded smoothly along with reduction of
bromine in the position 8 (Scheme 3). Because isomeric
N3- and N9-bromoadenines were more difficult to
separate than the final deprotected product, mixtures of 9
and 10 or 11 and 12 were used as a starting material in
most deprotection experiments. Final products were then
separated by repeated column chromatography. So, pure
3-(2-hydroxy-2-phenylethyl)adenine (13) and 9-(2-4hy-
droxy-2-phenylethyl)adenine (5) were obtained by
deallylation of a 3:1 mixture of 9a and 10a followed by
separation of products. Similarly, deallylation of a 2:1
mixture of 11a and 12a yielded 3-(2-hydroxy-1-
phenylethyl)adenine (14) and 9-(2-hydroxy-1-phenyl-
Scheme 4
R
O
R
O
N
O
X
X
NH2
N
O
N
N
N
Y
HN
H2N
Y
N
N
N
HN
H2N
O
R
N
N
X
Y
7a
7
17a, 18a
15a, 16a
15, 16
(i)
(i)
(i)
17, 18
15, 17, X = H, Y = Ph, R = H
7, 16, 18, X = Ph, Y = H, R = H
a, R = allyl
(i) Pd(PPh2)4, TsOH, PMHS, DMF
ethyl)adenine (6). This new deallylation procedure was
also successfully applied to deprotection of N7-adenine
derivative 7a as well as of a series of guanine derivatives,
namely, 7-(2-allyloxy-2-phenylethyl)guanine (15a), 7-(2-
allyloxy-1-phenylethyl)guanine (16a), 9-(2-allyloxy-2-
phenylethyl)guanine (17a) and 9-(2-allyloxy-1-phenyl-
ethyl)guanine (18a) (Scheme 4). The yields of deallyl-
ation are listed in the Table 2.
For guanine derivatives they ranged from 74 - 96 % and
were slightly better than those previously reported (68-
90%) [11] with the catalytic system using ZnCl2 as an acid.
Table 2
Yields of Deallylation
Starting
Material
Product(s)
Yield [%]
Scheme 3
9a
11a
9a + 10a
11a + 12a
7a
13
14
13 + 5
14 + 15
7
16
17
18
19
55
45
60
59
60
96
74
90
76
NH2
N
NH2
N
N
N
N
N
N
X
Br
Br
N
Y
O
PG
16a
17a
18a
19a
10a, b, 12a, b
9a, 11a
X
Y
O
PG
(i), 9a, 11a
(ii), 9b
(i), PG = allyl
(ii), PG = benzyl
(ii), 11b
In the palladium complex catalyzed deallylation,
coordination of the allylic π-electrons to palladium is
essential to enable subsequent reductive cleavage of the
allyl group. Basic nitrogen atoms in purines may
coordinate to the transition metal and compete with the
allylic π-electrons. Addition of an acid to the catalytic
system seems to be necessary to prevent coordination of
purine nitrogens to palladium. In the case of guanine
derivatives ZnCl2 was sufficient to neutralize the purine
moiety, whereas adenine derivatives required TsOH as a
stronger acid to achieve the same effect.
NH2
N
NH2
N
NH2
N
N
N
X
N
N
N
N
N
N
H
N
OH
Y
Y
X
13, 14
5, 6
OH
5, 9, 10, 13, X=H, Y=Ph
6, 11, 12, 14, X=Ph, Y=H
a, PG = allyl
b, PG = benzyl
(i) Pd(PPh3)4, TsOH, PMHS, DMF; (ii) H2/Pd-C, MeOH

792
J. Krouželka, I. Linhart and T. Tobrman
Vol 45
Table 3
13C-NMR Chemical Shifts in ppm of Adenine Derivatives. Spectra were measured in CDClš or a)DMSO-d6.
Compound
C2
152.3
152.4
153.1
152.3
152.2
152.6
143.8
145.1
153.2
153.1
143.9
142.8
152.7
152.7
C4
149.6
149.6
150.5
151.8
151.3
151.7
150.7
150.4
151.7
151.6
151.3
151.3
151.8
151.9
C5
118.5
118.7
119.7
111.7
111.0
112.6
122.2
122.0
119.9
119.9
122.3
122.3
120.3
120.3
C6
155.9
156.0
155.1
160.2
159.8
160.1
153.2
154.3
154.2
154.1
152.8
152.8
154.6
154.3
C8
141.3
139.7
140.0
144.8
144.2
145.1
141.7
140.0
128.4
128.4
141.5
141.8
128.4
128.7
CH2
50.3
CH
70.7
aromatic CH and C
125.9, 127.5, 128.2, 142.4
127.0, 128.0, 128.6, 138.0
CH=CH2
(allyl)
---
5a
6a
69.9, 57.6
71.0
70.6, 58.2
73.6
116.8,
134.7
---
6b
127.3, 127.9, 128.1, 128.6, 129.0, 129.3,
136.9, 137.3
127.1, 128.5, 129.2, 138.6
7
63.3
62.2
---
7a
70.5, 59.3
71.0
71.2, 61.5
73.9
55.8, 78.0
70.0
55.0, 78.0
70.5
50.4, 78.7
69.9
50.5, 77.8
70.7
68.9, 62.1
72.7
68.8, 62.1
73.8
69.2, 62.4
72.2
69.0, 62.5
73.2
56.4
61.2
63.1
126.5, 128.2, 128.8, 137.8
117.0,
134.5
---
7b
126.9, 128.0, 128.5, 128.7, 129.7, 129.8,
135.5, 136,2
126.9, 129.0, 129.1, 137.6
9a
117.5,
133.8
---
9ba
10a
10b
11a
11b
12a
12b
127.5, 127.7, 128.1, 128.7, 129.3, 129.5,
138.3, 138.7
126.8, 128.8, 128.9, 138.3
116.9,
134.1
---
127.0, 127.6, 127.7, 128.3, 128.8,
129.0137.5, 138.2
128.5, 129.3, 129.4, 135.4
118.3,
133.7
---
128.0, 128.5, 128.7, 129.3, 129.4, 135.2,
137.1
127.8, 128.7, 129.0, 136.1
117.6,
134.2
---
127.8, 127.85, 127.9, 128.5, 128.7, 129.0,
136.1, 137.7
125.9, 127.5, 128.3, 142.1
128.1, 128.7, 129.1, 137.5
127.8, 128.7, 129.4, 138.9
13a
14a
15a
143.9
143.2
153.1
149.7
150.4
150.5
120.3
121.1
119.5
155.0
155.3
156.7
152.3
152.7
139.9
69.5
65.9
61.1
---
---
---
1
analytical or synthetic grade and were used as received. H and
13C NMR spectra were recorded with Bruker Avance DRX500
The position of the alkyl at the purine moiety was
determined unequivocally by 2D NMR experiments.
Parameters were set to show interactions J ≈ 7 Hz
corresponding to three-bond C-H coupling constants on
the heteroaromatic ring. Protons of NCH2 or NCH-CH2
showed cross-peaks with corresponding carbons of the
purine rings. To assign 2-H and 8-H protons in the
adenine derivatives unequivocally, their single bond C-H
correlation in HMQC was not sufficient because
corresponding carbon signals were very close. In HMBC,
2-H protons showed cross-peaks with both C-4 and C-6
and, similarly, 8-H protons correlated with C-4 and C-5.
In all cases the resonances of 2-H was found at lower field
values (8.14 - 8.56 ppm) than those of 8-H (7.70 - 8.10
ppm). This is in agreement with an earlier observation of
Kjellberg and Johansson [21].
1
(500 MHz for H) or with Varian Mercury 300 (300 MHz for
1H) Fourier transform NMR spectrometer. Mass spectra were
measured on a triple quadrupole HPLC-MS system Varian 1200
equipped with electrospray ionization (ESI) in the positive ion
mode.
Starting Materials. 8-Bromoadenine (8) was prepared by
bromination of adenine in aqueous solution [22]. Alkylating
reagents, i.e., 2-bromo-1-phenylethanol (2) [23], allyl(2-bromo-
1-phenylethyl) ether (2a) [11], allyl(2-bromo-2-phenylethyl)
ether (3a) [11], benzyl(2-bromo-1-phenylethyl) ether (2b) [11],
benzyl(2-bromo-2-phenylethyl) ether (3b) [24] and 2-benzyl-
oxy-2-phenylethyl triflate (4) [12] were prepared according to
published procedures.
Alkylation of adenine with 2-bromo-1-phenylethanol (2).
A mixture of 200 mg (1.48 mmol) of adenine, 205 mg (1.28
mmol) of potassium carbonate, 300 mg (1.14 mmol) of 18-
crown-6 and 607 mg (3 mmol) of bromohydrin 2 in 20 mL of
DMF was heated to 125°C for 20h under dry nitrogen. Three
products Rf = 0.49, 0.39 and 0.34 were detected by TLC
(CH2Cl2-MeOH 6:1). The reaction mixture was evaporated to
dryness and separated by repeated column chromatography on
silica gel.
EXPERIMENTAL
Column chromatography was performed on silica gel 60
purchased from Fluka, particle size 0.063-0.200 mm. For thin-
layer chromatography Merck Silica gel 60 F254 plates were used.
Dimethylformamide (DMF) was dried by vacuum distillation
from phosphorus pentoxide and stored over molecular sieves.
Other chemicals obtained from commercial sources were of
9-(2-Hydroxy-2-phenylethyl)adenine (5). This compound
was obtained by alkylation of adenine with 2 (109 mg, 29%) and
also by deallylation of a 3:1 mixture of 9a and 10a (150 mg, 0.4
mmol). Repeated column chromatography yielded 22 mg (87 %)

May-Jun 2008
Hydroxy(phenyl)ethyladenines
793
of a white powder, mp > 250°C; Rf (CH2Cl2-MeOH 6:1) = 0.49;
1H NMR (DMSO-d6): δ 4.26 (m, 2H, CH2N); 4.98 (m, 1H,
PhCH); 5.79 (d, 1H, J = 4.6 Hz, OH); 7.16 (br, 2H, NH2); 7.27
(m, 1H, Ph); 7,35 (m, 4H, Ph); 7.98 (s, 1H, 8-H); 8.14 (s, 1H, 2-
H); HMBC: Cross-peak of NCH2 at δ = 4.26 ppm with C-8 at
142.4 ppm and C-4 at 149.6 ppm; ESI-MS: m/z = 256 [M+H]+,
278 [M+Na]+. Anal. Calcd. for C13H13N5O: C, 61.2; H, 5.1; N,
27.4. Found: C, 60.9; H, 5.2; N, 27.3.
4.19 (dd, 1H, J = 10.5 and 8.2 Hz, NCHCH2); 4.24 (dd, 1H, J =
10.5 and 3.6 Hz, NCHCH2); 4.54 and 4.59 (d, 1+1H, J = 11.8
Hz, PhCH2O); 5.02 (br, 2H, NH2); 5.82 (dd, 1H, J = 8.2 and 3.6
Hz, CHN); 7.19 (m, 2H, Ph); 7.30 (m, 3H, Ph); 7. 41 (m, 5H,
Ph); 8.03 (s, 1H, 8-H); 8.46 (s, 1H, 2-H); ESI-MS: m/z 346
[M+H]+, 368 [M+Na]+; Anal. Calcd. for C20H19N5O: C, 69.5; H,
5.5; N, 20.3; Found: C, 69.2; H, 5.8; N, 20.3.
General Procedure for Alkylation of 8-Bromoadenine (8).
A mixture of 250 mg (1.17 mmol) of 8, 242 mg (1.75 mmol) of
potassium carbonate, 2.34 - 7.02 mmol of alkylating agent 2a,
2b, 3a or 3b and 10 mL of DMF was stirred under a nitrogen
atmosphere at 50 - 100°C and the course of reaction was
followed by TLC (Silica gel 60 F254, Merck, CHCl3-MeOH
8:1). After 4 - 72 h, when the spot of 8 at Rf = 0.16 disappeared,
the solvent was distilled off in vacuo, the residue was re-
suspended in 25 mL of dichloromethane - ethyl acetate 2:1,
poured onto a bed of 25 g silica gel (for flash chromatography)
and eluted subsequently with 100 mL of dichloromethane, 80
mL of ethyl acetate and 80 mL of ethanol. Crude products,
bromoadenines 9a,b - 12a,b, 2 for each alkylating reagent used,
were eluted in the ethyl acetate fraction. They were analyzed by
1H-NMR to determine the ratio of N3:N9 substituted product by
integration of corresponding 2-H signals and used directly in
further reaction steps. Pure samples of 9a,b - 12a,b were
obtained by column chromatography on silica gel followed by
crystallization or by semi-preparative HPLC.
Alkylation of Adenine with Allyl or Benzyl (2-Bromo-2-
phenylethyl) Ether (3a or 3b). Adenine (200 mg, 1.49 mmol),
NaH (36 mg, 1.4 mmol) and alkylating reagent 3a or 3b (2.2
mmol) were added to 15 mL of DMF. The reaction mixture was
stirred under a dry nitrogen atmosphere for two weeks at room
temperature, then it was diluted with 15 mL of chloroform, the
undissolved portion was filtered off, and the filtrate was
evaporated to dryness in vacuo and separated by repeated
column chromatography on silica gel using 10:1 and 6:1 CHCl3-
MeOH as an eluent.
9-(2-Allyloxy-1-phenylethyl)adenine (6a). A white powder
obtained by alkylation of adenine with 3a, crystallized from
chloroform – cyclohexane, 87 mg (20%), mp 158-160°C, Rf
(CHCl3 - MeOH 6:1) = 0.50; 1H-NMR (CDCl3): δ 4.00 (d, 2H, J
= 5.5 Hz, CH2O); 4.05 (dd, 1H, J = 10.5 and 4.4 Hz, CH2CHN),
4.13 (dd, 1H, J = 10.5 and 6.9 Hz, CH2CHN); 5.16 (d, 1H, J =
9.1 Hz, CH2=CH); 5. 18 (d, 1H, J = 14.6 Hz, CH2=CH); 5.94
(dd, 1H, J = 6.9 and 4.4 Hz, NCH); 6.2 (br, 2H, NH2); 7.30 (m,
5H, Ph); 8.00 (s, 1H, 8-H), 8.33 (s, 1H, 2-H); HMBC: Cross-
peaks of NCH at δ = 5.94 ppm with C-8 at 139.7 ppm and C-4 at
152.4 ppm were detected. ESI-MS: m/z 296 [M+H]+, 318
[M+Na]+ and 334 [M+K]+. Anal. Calcd. for C16H17N5O × 1/4
H2O: calc. C, 64.1; H, 5.9; N, 23.7; Found: C, 64.2; H, 5.8; N,
23.3.
When triflate 4 was used as an alkylation reagent instead of
protected bromohydrins, full conversion of bromoadenine 8 was
achieved in 4 h at room temperature.
8-Bromo-3-(2-allyloxy-2-phenylethyl)adenine (9a) and 8-
Bromo-9-(2-allyloxy-2-phenylethyl)adenine (10a). These
compounds were obtained by alkylation of
8 by 2a.
Crystallization of 350 mg of the crude product containing 7:2
mixture of 9a and 10a from dichloromethane - petroleum ether
afforded 32 mg (12%) of 9a. Remaining mixture of the two
isomers was separated by semi-preparative HPLC on a 250 x 8
mm Watrex C18 column (50 - 80 % MeOH, 27 min; 80%
MeOH for additional 5 min) affording analytical samples of pure
9a and 10a.
7-(2-Allyloxy-1-phenylethyl)adenine (7a). A minor product
of the alkylation of adenine with 3a obtained by repeated
column chromatography and crystallization from chloroform as
a white powder, 37 mg (8 %), mp 189-191°C, Rf (CHCl3
-
1
MeOH 6:1) = 0.39; H-NMR (CDCl3): δ 4.04 (m, 2H, CH2O);
4.19 (dd, 1H, J = 10.5 and 8 Hz, CH2CHN), 4.21 (dd, 1H, J =
10.5 and 3.9 Hz, CH2CHN); 5.19 (m, 2H, CH2=CH); 5.82 (dd,
1H, J = 7.7 and 3.9 Hz, NCH); 7.20 (m, 3H, Ph); 7.40 (m, 2H,
Ph); 8.02 (s, 1H, 8-H ); 8.43 (s, 1H, 2-H); HMBC: Cross-peaks
of NCH at δ = 5.82 ppm with C-8 at 144.2 ppm and C-5 at 111.0
ppm were detected. ESI-MS: m/z 296 [M+H]+, 318 [M+Na]+
and 334 [M+K]+; Anal. Calcd. for C16H17N5O: C, 65.1; H, 5.8; N
23.7; Found: C, 64.7; H, 5.7; N 23.3.
9a: White powder, mp 188-189°C; Rf (CHCl3-MeOH 10:1) =
1
044.; H-NMR (CDCl3): δ 3.64 (dd, 1H, J = 12.6 and 5.9 Hz,
OCH2); 3.90 (dd, 1H, J = 12.6 and 5.00Hz, OCH2); 4.22 (dd, 1H,
J = 9.5Hz and 13.6Hz, NCH2); 4.67 (dd, 1H J = 3.1Hz and
13.63Hz, NCH2); 4.85 (dd, 1H, J = 9.2 and 3.1Hz, PhCH); 5.05
(m, 2H CH=CH2); 5.63 (m, 1H, CH2=CH); 6.2-6.6 (br, 2H,
NH2); 7.25-7.38 (m, 5H, Ph); 7.99 (s, 1H, 2-H); HMBC: Cross-
peaks of NCH2 (δ = 4.22 and 4.67 ppm) with the signals of C-2
at 143.8 ppm and C-4 at 150.7 ppm as well as the cross-peak of
2-H (δ = 7.99 ppm) with the signal of CH2N at 55.8 ppm; ESI-
MS (m/z): 374 and 376 [M+H]+; Anal. Calcd. for
C16H16N5OBr.1/3 H2O: C, 50.5; H, 4.4; N, 18.4; Found: C, 50.2;
H, 4.0; N, 18.4.
9-(2-Benzyloxy-1-phenylethyl)adenine (7b). This compound
was obtained by alkylation of adenine with 3b. Repeated column
chromatography followed by crystallization from chloroform -
cyclohexane yielded 94 mg (18%) of a white powder, mp 197-
1
194°C, Rf (CHCl3 - MeOH 8:1) = 0.71; H-NMR (CDCl3): δ
4.09 (dd, 1H, J = 10.4 and 4.4 Hz, NCHCH2); 4.35 (dd, 1H, J =
10.4 and 6.9 Hz, NCHCH2); 4.53 and 4.58 (d, 1+1H, PhCH2O);
5.59 (br, 2H, NH2); 5.93 (dd, 1H, J = 6.9 and 4.4 Hz, CHN);
7.19 (m, 2H, Ph); 7.30 (m, 8H, Ph); 7.98 (s, 1H, 8-H); 8.32 (s,
1H, 2-H); ESI-MS: m/z 346 [M+H]+, 368 [M+Na]+; Anal.
Calcd. for C20H19N5O × 1/4 H2O: C, 68.7; H, 5.6; N, 20.0;
Found: C, 68.5; H, 5.8; N, 20.3.
10a: White powder, mp 151-153°C; Rf (CHCl3-MeOH 10:1)
1
= 0.48; H-NMR (CDCl3): δ 3.65 (m, 1H, OCH2); 3.92 (m, 1H,
OCH2); 4.28 (dd, 1H, J = 14.1 and 4.7Hz, NCH2); 4.49 (dd, 1H,
J = 14.1 and 8.8 Hz, NCH2); 4.87 (dd, 1H, J = 8.8 and 4.7Hz,
PhCH); 5.04 (m, 2H, CH=CH2); 5,61 (m, 1H, CH2=CH); 5.73
(br, 2H, NH2); 7.27-7.40 (m, 5H, Ph); 8.34 (s, 1H, 2-H); HMBC:
Cross-peaks of N-CH2 (δ = 4.28 and 4.49 ppm) with the signals
of C-8 at 128.4 ppm and C-4 at 151.6 ppm; ESI-MS: m/z 374
and 376 [M+H]+; Anal. Calcd. for C16H16N5OBr.1/3 H2O: C,
50.2; H, 4.2; N, 18.4; Found: C, 50.5; H, 4.4; N, 18.4.
7-(2-Benzyloxy-1-phenylethyl)adenine (7b).
product of the alkylation of adenine with 3b. Repeated column
chromatography and crystallization from chloroform
A
minor
-
cyclohexane yielded 59 mg (11.5 %) of a white powder, mp
211-215°C, Rf (CHCl3 - MeOH 8:1) = 0.61;1H-NMR (CDCl3): δ

794
J. Krouželka, I. Linhart and T. Tobrman
Vol 45
8-Bromo-3-(2-benzyloxy-2-phenylethyl)adenine (9b) and
with CHCl3 - Me2CO - MeOH 15:1:1 as an eluent yielded 40 mg
(8%) of 11b and 25 mg (5 %) of 12b.
11b: White solid, mp > 250°C; Rf (CHCl3-Me2CO-MeOH
8-Bromo-9-(2-benzyloxy-2-phenylethyl)adenine (10b). These
compounds were obtained by alkylation of 8 either with 2b or
with triflate 4. Pure analytical samples of 9b and 10b were
obtained by semi-preparative HPLC on a 250 × 8 mm Watrex
C18 column using a methanol - water gradient (63 - 90 %
MeOH, 25 min; 90% MeOH for additional 10 min).
1
15:1:1) = 0.21; H-NMR (CDCl3): δ 4.16 (dd, 1H, J = 10.8 and
4.3 Hz, CHCH2O); 4.40 (dd, 1H, J = 10.8 and 4.6 Hz,
CHCH2O); 4.53 (s, 2H, PhCH2O); 6.29 (t, 1H, J = 4.4 Hz,
NCH); 7.19 (m, 2H, Ph); 7.30 (m, 3H, Ph); 7.38 (m, 3H, Ph);
7.42 (m, 2H, Ph); 8.00 (s, 1H, 2-H); HMBC: Cross-peaks of
NCH (δ = 6.29 ppm) with the signals of C-2 at 143 ppm and C-4
at 151 ppm; ESI-MS: m/z 424 and 426 [M+H]+, 446 and 448
[M+Na]+, 462 and 464 [M+K]+; Anal. Calcd. for
C20H18N5OBr.1/4H20: C, 56.0; H, 4.2; N, 16.4; Found: C, 56.2;
H, 4.5; N, 16.6.
12b: White solid, mp > 250°C; Rf (CHCl3-Me2CO-MeOH
15:1:1) = 0.15; 1H-NMR (CDCl3): δ 4.15 (dd, 1H, J = 10 and 5
Hz, CHCH2O); 5.09 (t, 1H, J = 5.0 Hz, CHCH2O); 4.51 (d, 1H, J
= 12.1 Hz, PhCH2O); 4.55 (d, 1H, J = 12.1 Hz, PhCH2O); 5.7 (br
s, 2H, NH2); 5.89 (t, 1H, J = 5 Hz, NCH); 7.13 (m, 2H, Ph); 7.25
(m, 4H, Ph); 7.32 (m, 4H, Ph); 7.47 (m, 2H, Ph); 8.26 (s, 1H, 2-
H); HMBC: Cross-peaks of NCH (δ = 5.89 ppm) with the
signals of C-8 at 128.7 ppm and C-4 at 151.9 ppm; ESI-MS: m/z
424 and 426 [M+H]+, 446 and 448 [M+Na]+, 462 and 464
[M+K]+; Anal. Calcd. for C20H18N5OBr.1/4H20: C, 56.0; H, 4.2;
N, 16.4; Found C, 56.0; H, 4.1; N, 16.2.
General Procedure for Debenzylation. Palladium catalyst
(750 mg, 10% Pd-C, Degusa type, wet, containing approx. 50%
of water) was activated by heating to 100°C for 1 h in vacuo.
Argon was introduced into the reaction flask with the activated
catalyst, 400 mg (6.35 mmol) of ammonium formate and a
solution of 150 mg (0.353 mmol) of a bezyl protected compound
(9b, 10b, 12b, a mixture 9b and 10b or 11b and 12b) in absolute
MeOH (20 mL) was added. The reaction mixture was heated in
an oil bath to 60°C under an argon atmosphere. After 1h another
portion of 400 mg (6.35 mmol) of ammonium formate was
added and the reaction was continued for an additional 1h. The
catalyst was filtered off and washed with aqueous MeOH. The
solvents were evaporated in vacuo to yield crude products.
General Procedure for Deallylation. To a stirred solution of
150 mg (0.4 mmol) of an allyl-protected adenine derivative
(pure 9a, 11a or a mixture of either 9a + 11a or 10a + 12a) in
DMF (10 mL) under an argon atmosphere, 150 mg (0.8 mmol)
of TsOH monohydrate, 14 mg (0.012 mmol) of Pd(PPh3)4 and
100 µL of PMHS was added and the reaction mixture was
heated to 90°C. After 3 and 6 h additional portions of PMHS
were added (2 × 100 µL). When the reaction was complete (after
9 - 20 h), the solvent was evaporated in vacuo and the products
were isolated by repeated column chromatography. For
deallylation of guanine derivatives 15a – 18a (125 mg; 0.4
mmol) lower amounts of TsOH (75 mg; 0.4 mmol) and PMHS
(100 µL) were used. Full conversion of the starting material was
achieved in 3 h. Resulting alkylguanines were purified by
column chromatography on silica gel followed by anion
exchange on Dowex 1 to remove coeluting TsOH.
9b: White powder, mp 226-227°C; Rf (CHCl3-Me2CO-MeOH
1
15:1:1) = 0.47; H-NMR (DMSO-d6): δ 4.14 (d, 1H, J = 12.2
Hz, CH2O); 4.37 (d, 1H, J = 12.2 Hz, CH2O); 4.43 (dd, 1H, J =
13.6 and 3.8 Hz, CH2N); 4.50 (dd, 1H, J = 13.6 and 9.2 Hz,
CH2N); 4.92 (dd, 1H, J = 9.2 and 3.7 Hz, CHO); 6.97 (m, 2H,
Ph); 7.19 (m, 3H, Ph); 7.40 (m, 1H, Ph); 7.46 (m, 4H, Ph); 8.05
(br, 1H, NH) 8.22 (br, 1H, NH); 8.26 (s, 1H, 2-H); HMBC:
Cross-peaks of NCH2 (δ = 4.43 and 4.5 ppm) with the signals of
C-2 at 145 ppm, C-4 at 150 ppm and aromatic C at 138.7 ppm;
ESI-MS: m/z 424 and 426 [M+H]+, 446 and 448 [M+Na]+, 346
[M-Br]+; Anal. Calcd. for C20H18N5OBr.1/4H2O: C, 56.0; H, 4.2;
N, 16.4; Found C, 56.4; H, 3.8; N, 16.5.
10b: White powder, mp 146-147°C; Rf (CHCl3-Me2CO-
1
MeOH 15:1:1) = 0.43; H-NMR (CDCl3): δ 4.20 (d, 1H, J = 12
Hz, CH2O) 4.50 (d, 1H, J = 12 Hz, CH2O); 4.25 (dd, 1H, J =
14.2 and 4.1 Hz, CH2N); 4.50 (dd, 1H, J = 14.2 and 9.6 Hz,
CH2N); 4.87 (dd, J = 9.7 and 4.1 Hz, CHO); 5.6 (br, 2H, NH2);
6.92 (m, 2H, Ph); 7.14 (m, 3H, Ph); 7.42 (m, 5H, Ph); 8.27 (s,
1H, 2-H); HMBC: Cross-peaks of NCH2 (δ = 4.25 and 4.50
ppm) with the signals of C-8 at 128.4 ppm and C-4 at 151.6
ppm; ESI-MS: m/z 424 and 426 [M+H]+, 446 and 448 [M+Na]+,
346 [M-Br]+; Anal. Calcd. for C20H18N5OBr.1/4H2O: C, 56.0; H,
4.2; N, 16.4; Found: C, 56.2; H, 4.2; N, 16.3.
8-Bromo-3-(2-allyloxy-1-phenylethyl)adenine (11a) and 8-
Bromo-9-(2-allyloxy-1-phenylethyl)adenine (12a). These
compounds were obtained by alkylation of 8 with 3a. Separation
by semi-preparative HPLC on a 250 × 8 mm Watrex C18
column using a MeOH - H2O gradient (50 - 80 % MeOH, 27
min; 80% MeOH for additional 5 min) afforded analytical
samples of pure compounds 11a and 12a.
11a: White powder, mp 158-160°C; Rf (CHCl3-MeOH 8:1) =
1
0.53; H-NMR (CDCl3): δ 4.00 (d, 2H, J = 5.6 Hz, CH2=CH-
CH2); 4.10 (dd, 1H J = 10.9 and 4.4 Hz, NCHCH2O); 4.40 (dd,
1H, J = 10.9 and 4.7 Hz, NCHCH2O); 5.20 (m, 2H, CH2=CH);
5.80 (m, 1H, CH2=CH); 6.29 (t, 1H, J = 4.3 Hz, NCH). HMBC:
Cross-peaks of NCH (δ = 6.29 ppm) with the signals of C-2 at
144 ppm and C-4 at 151 ppm; ESI-MS: m/z 374 and 376
[M+H]+, 396 and 398 [M+Na]+, 412 and 414 [M+K]+; Anal.
Calcd. for C16H16N5OBr.H2O: C, 49.0; H, 4.6; N, 17.9; Found C,
48.9; H, 4.2; N, 17.7.
12a: White powder, mp > 250°C; Rf (CHCl3-MeOH 8:1) =
1
0.53; H-NMR (CDCl3): δ 4.00 (d, 2H, J = 5.9 Hz, CH2=CH-
CH2); 4.15 (dd, 1H, J = 10.2 and 5.1 Hz, NCHCH2O); 5.04 (t,
1H, J = 10.2 Hz, NCHCH2O); 5.11 (d, 1H, J = 10.9 Hz,
CH2=CH); 5.16 (dd, 1H, J = 18 and 1.5 Hz, CH2=CH); 5.76 (m,
1H, CH2=CH); 5.87 (dd, 1H, J = 10 and 5 Hz, NCH); ESI-MS:
m/z 374 and 376 [M+H]+, 396 and 398 [M+Na]+, 412 and 414
[M+K]+; Cross-peaks of NCH (δ = 5.87 ppm) with the signal of
C-4 at 152 ppm and with aromatic CH overlapping C-8 at 128
ppm were observed. Anal. Calcd. for C16H16N5OBr: C, 51.4; H,
4.3; N, 18.7; Found C, 51.5, H, 4.6; N, 18.3.
3-(2-Hydroxy-2-phenylethyl)adenine (13). Deallylation of
9a followed by crystallization from ethanol yielded 56 mg (55
%) of 13. Deallylation of a 3:1 mixture of 9a and 10a followed
by repeated column chromatography yielded 25 mg (24%) of 13
(the yield corrected to the content of 9a in the starting material).
Also obtained by debenzylation of 100 mg of 9b and
purification by column chromatography (CHCl3-MeOH 3:1) as a
white powder, 33 mg (55 %), mp > 250°C; Rf (CHCl3-MeOH
8-Bromo-3-(2-benzyloxy-1-phenylethyl)adenine (11b) and
8-Bromo-9-(2-benzyloxy-1-phenylethyl)adenine (12b). These
compounds were obtained by alkylation of 8 with 3b. Repeated
column chromatography of the crude product on silica gel (32 g)
1
8:1) = 0.15; H-NMR (DMSO-d6): δ 4.23 (dd, 1H, J = 13.5 and

May-Jun 2008
Hydroxy(phenyl)ethyladenines
795
9.4 Hz, CH2); 4.48 (dd, 1H, J = 13.5 and 3.4 Hz, CH2); 5.1 (m,
1H, CHOH): 5.9 (br, 1H, OH); 7.23-7.45 (m, 5H, Ph); 8.18 (s,
1H, 2-H); 7.78 (s, 1H, 8-H); HMQC: Cross-peaks of 2-H at δ =
8.18 ppm with C-2 at 144 ppm and 8-H at δ = 7.78 ppm with C-
8 at 152 ppm; HMBC: Cross-peaks of NCH2 (δ = 4.23 and 4.48
ppm) with C-2 at 144 ppm and with C-4 at 149.7 ppm; ESI-MS:
m/z 256 [M+H]+, 278 [M+Na]+; Anal. Calcd. for C13H13N5O: C,
61.2; H, 5.1; N, 27.4; Found C, 60.8; H, 5.1; N, 27.3.
(CH2O); 105.0 (C5); 127.5, 128.3 and 129.3 (Ar CH); 138.7 (C);
152.0 (C2); 154.4 (C4); 157.0 (C6); ESI-MS: m/z 272 [M+H]+,
294 [M+Na]+, 310 [M+K]+; Anal. Calcd. for C13H13N5O2: C,
57.6; H, 4.8; N, 25.8; Found: C, 57.4; H, 4.9; N, 25.5.
Acknowledgement. The financial support by grants No.
203/06/0888 from the Grant Agency of the Czech Republic and
grants MSM 604 613 73 01 and LC06070 from the Ministry of
Education of the Czech Republic is gratefully acknowledged.
3-(2-Hydroxy-1-phenylethyl)adenine (14). This compound
was obtained by deallylation of 12a (45%) and of a 2:1 mixture
of 11a and 12a (150mg, 0.4 mmol). Repeated column
chromatography followed by crystallization from hot aqueous
ethanol yielded 23 mg (33%) of 14 (the yield corrected to the
content of 11a in the starting material) as a white powder, mp >
REFERENCES
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450.
1
250°C; Rf (CHCl3-MeOH 6:1) = 0.29; H-NMR (DMSO-d6): δ
[3] Gluesenkamp, K.-H.; Krueger, K.; Eberle, G.; Drosdziok,
W.;E. Jaehde, E.; Gruendel, O.; Neuhaus, A.; Boese, R.; Stellberg P.;
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4.12 (dd, 1H, J = 11.3 and 3.5 Hz, CH2OH); 4.60 (m, 1H,
CH2OH); 5.92 (dd, 1H, J = 9.1 and 5 Hz, CHN); 5.45 (br, 1H,
OH); 7.30 (m, 3H, Ph); 7.45 (dd, 2H, J = 8.0 and 1.5 Hz, Ph);
7.90 (br, 2H, NH2); 7.70 (s, 1H, 8-H); 8.56 (s, 2-H); HMBC:
Cross-peaks of CHN (δ = 5.92 ppm) with C-2 at 143.2 and with
C-4 at 150.4; ESI-MS: m/z 256 [M+H]+, 278 [M+Na]+; Anal.
Calcd. for C13H13N5O: C, 61.2; H, 5.1; N. 27.4; Found: C, 60.9;
H, 5.1; N, 27.3.
[5] K. Savela, A. Hesso and K. Hemminki, Chem.-Biol. Interact.
1986, 60, 235.
[6] Qian, C.; Dipple, A Chem. Res. Toxicol. 1995, 8, 389.
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Tetrahedron 1987, 43, 2101.
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2003, 5, 637.
[11] Novák, J.; Linhart, I.; Dvořáková, H. Eur. J. Org. Chem.
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[12] Christoffers, J.; Roessler, U. J. Prakt. Chem. 2000, 342, 654.
[13a] Fujii, T.; Walker, G.C.; Leonard, N.J.; DeLong, D.C.;
Gerzon, K. J. Med. Chem. 1979, 22, 125. [b] Seley, K.L.; Mosley, S.L.;
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[14] Li, X.; Vince, R. Bioorg. Med. Chem. 2006, 14, 5742.
[15] Jones, J.B.; Young, J.M. Can. J. Chem. 1970, 48, 1566.
[16] Thomas, R.M.; Mohan, G.H.; Iyengar, D.S. Tetrahedron Lett.
1997, 38, 4721.
9-(2-Hydroxy-1-phenylethyl)adenine (6) [25]. This
compound was obtained by deallylation of a 2:1 mixture (150
mg; 0.4 mmol) of 11a and 12a. Separation of the crude product
by column chromatography yielded 19 mg (57%) of 15.
Alternatively, debenzylation of 150 mg (0.353 mmol) of a 1:1
mixture of 11a and 12a followed by separation on a silica gel
column using CHCl3 - MeOH 4:1 as an eluent yielded 15 mg
(62%) of adenine and 28 mg (62%) of 15 as a white powder, mp
1
> 250°C; Rf (CHCl3-MeOH 8:1) = 0.32; H-NMR (DMSO-d6):
δ 4.03 (m, 1H, CH2OH); 4.39 (m, 1H, CH2OH); 5.68 (dd, 1H, J
= 8.9 and 4.9 Hz, CH2N); 5.31 (t, 1H, J = 5.4 Hz, OH); 7.30 (m,
5H, Ph); 7.22 (br s, 2H, NH2); 8.10 (s, 1H, 8-H); 8.41 (s, 1H, 2-
H); ESI-MS: m/z 256 [M+H]+, 278 [M+Na]+; Anal. Calcd. for
C13H13N5O.1/4H2O: C, 60.1; H, 5.2; N, 27.0; Found: C, 59.9; H,
5.1; N, 27.0.
[17] Dahlén, A.; Sundgren, A.; Lahmann, M.; Oscarson S.;
Hilmersson, G.Org. Lett. 2003, 5, 4085.
9-(2-Hydroxy-1-phenylethyl)guanine (18). This compound
was obtained by deallylation of 18a (78 mg; 0.25 mmol).
Reaction mixture was evaporated to dryness in vacuo, the
residue was re-suspended in CHCl3 - MeOH 3:1 and poured onto
a silica gel bed (10 g). Elution with CHCl3 - MeOH 3:1 yielded a
product, which was contaminated with TsOH. Pure 18 was
obtained by crystallization from MeOH - CHCl3 (20 mg, 29%).
Another portion, 30 mg of the product was obtained by
separation on Dowex 1 anion exchange resin (3g). Total yield
was 50 mg (74%) of a white powder, mp > 250°C, Rf (CHCl3-
[18] Taniguchi, T.; Ogasawara, K. Angew. Chem. 1998, 110,
1137.
[19] Dessolin, M.; Guillerez, M; Thieriet, N.; Guibé F.; Loffet, A.
Tetreahedron Lett. 1995, 36, 5741.
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57, 3435.
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Metzler, M.R. Tetrahedron 1993, 49, 5635.
[25] Schaeffer, H.J.; Johnson, R.N.; Schwartz, M.A.; Schwender,
C.F. J. Med. Chem.1972, 15, 456.
1
MeOH) = 0.19; H-NMR (DMSO-d6): δ 3.98 (m, 1H, CH2O);
4.41 (m, 1H, CH2O); 5.25 (t, 1H, J = 5.4 Hz, OH); 5.44 (dd, 1H,
J = 8.7 and 5.1 Hz, CHN); 6.37 (br s, 2H, NH2); 7.28 (m, 5H,
Ph); 7.94 (s, 1H, 8-H); 13C-NMR (DMSO-d6): 60.6 (CHN); 63.2