Syntheses and electrochemical properties of novel aminopyrimidinone derivatives as a new class of abasic-site binders

Article abstract of DOI:10.1007/s11164-012-0641-7

We describe syntheses and electrochemical properties of a new class of aminopyrimidinone derivatives: 2-amino-4-(3',4'-dimethoxyphenyl)-6-pyrimidinone, 2-amino-4-(3',4'-dihydroxyphenyl)-6-pyrimidinone, 2,4-diamino-5-(3',4'- dimethoxyphenyl)-6-pyrimidinone, 2,4-diamino-5-(3',4'-dihydroxyphenyl)-6- pyrimidinone, and 4-amino-5-(3',4'-dihydroxyphenyl)-2,6-pyrimidinione, three of which possess a catechol unit as an oxidation-active moiety for developing electrochemically detectable abasic-site binders. These compounds were synthesized via a pyrimidine-ring forming reaction with guanidine. Cyclic voltammetry measurements revealed that the catechol-bearing derivatives showed oxidation potentials lower than that of DNA, indicating that they satisfied the requirements for the purpose. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11164-012-0641-7

Res Chem Intermed (2013) 39:177–183
DOI 10.1007/s11164-012-0641-7
Syntheses and electrochemical properties of novel
aminopyrimidinone derivatives as a new class
of abasic-site binders
•
Junya Chiba Yasuhiro Doi Masahiko Inouye
•
Received: 3 October 2011 / Accepted: 7 January 2012 / Published online: 22 June 2012
Ó Springer Science+Business Media B.V. 2012
Abstract We describe syntheses and electrochemical properties of a new class of
aminopyrimidinone derivatives: 2-amino-4-(30,40-dimethoxyphenyl)-6-pyrimidinone,
2-amino-4-(30,40-dihydroxyphenyl)-6-pyrimidinone, 2,4-diamino-5-(30,40-dimethoxyphenyl)-
6-pyrimidinone, 2,4-diamino-5-(30,40-dihydroxyphenyl)-6-pyrimidinone, and 4-amino-5-
(30,40-dihydroxyphenyl)-2,6-pyrimidinione, three of which possess a catechol unit as an
oxidation-active moiety for developing electrochemically detectable abasic-site binders.
These compounds were synthesized via a pyrimidine-ring forming reaction with guani-
dine. Cyclic voltammetry measurements revealed that the catechol-bearing derivatives
showed oxidation potentials lower than that of DNA, indicating that they satisfied the
requirements for the purpose.
Keywords Electrochemistry ꢀ Abasic site ꢀ Aminopyrimidinone ꢀ Catechol
Introduction
Abasic sites, also known as AP (apurinic/apyrimidinic) sites, are locations in DNA
duplexes that have neither a purine nor a pyrimidine base. Abasic sites are naturally
occurring and one of the well-known forms of DNA damage [1–3]. Various types of
small organic molecules have been studied as ‘‘abasic-site binders’’ that are able to
bind selectively to nucleobases opposite an abasic site in DNA duplexes, attempting
to develop anti-cancer agents, to modulate gene expression and to detect SNPs
(single nucleotide polymorphisms) [4–8]. In addition, abasic-site binders with an
J. Chiba (&) ꢀ Y. Doi ꢀ M. Inouye (&)
Graduate School of Pharmaceutical Sciences, University of Toyama, Sugitani 2630,
Toyama 930-0194, Japan
e-mail: chiba@pha.u-toyama.ac.jp
M. Inouye
e-mail: inouye@pha.u-toyama.ac.jp
123

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J. Chiba et al.
cytosine
cytosine
cytosine
thymine
Me
adenine
A
B
C
D
E
N
N
N
N
N
N
H
H
H
N
O
N
N
H
O
N
N
O
N
H
O
O
N
N
H
H
H
N
N
H
H
N
H
H
N
H
N
H
N
H
N1
H
N
H
N3
O
H
N1
O
N3
O
H
H
H
H
H
N3
N1
N3
H
N1
O
N1
O
H
H
N3
H
O
N
O
H
H
2-AP
2,4-DAP
4-APDO
N
2,4-DAP
H
4-APDO
Chart 1 Possible hydrogen-bonding structures of a 2-amino-6-pyrimidinone (2-AP) with cytosine,
b 2,4-diamino-6-pyrimidinone (2,4-DAP) with cytosine, c 4-amino-2,6-pyrimidindione (4-APDO) with
cytosine, d 2,4-DAP with thymine and e 4-APDO with adenine
information-signaling ability will be useful reporter molecules for detecting an
abasic site in a DNA duplex.
We previously reported an aminopyrimidinone derivative that serves as a
hydrogen-bonding array in an acceptor–donor–donor (ADD, A: acceptor; D: donor)
fashion and selectively binds with a cytosine (DAA) derivative (Chart 1a–c) [9]. We
also reported the duplex formation of artificial oligonucleotides bearing 2-amino-
pyrimidinones with native deoxycytidine homooligomers even in an aqueous buffer
solution [10]. Moreover, it is well known that diaminopyrimidine (DAD) and
pyrimidindione (ADA) skeletons can selectively interact with thymine (Chart 1d)
and adenine (Chart 1e), respectively [11]. With these in mind, we thought that these
three frameworks would be useful as hydrogen-bonding sites for designing new
abasic-site binders. Thus, we planned to develop some abasic-site binders as an
electrochemically detectable reporter molecule. For this purpose, we introduced
catechol-derived units into the abasic-site binders described above. In particular,
1,2-dihydroxyphenyl groups will serve as an oxidation active moiety because of
the reversible redox combination of catechol/o-quinone [12, 13]. In this paper, we
report the syntheses and electrochemical properties of a new class of aminopyri-
midinone derivatives; 2-amino-4-(30,40-dimethoxyphenyl)-6-pyrimidinone (1),
2-amino-4-(30,40-dihydroxyphenyl)-6-pyrimidinone (2), 2,4-diamino-5-(30,40-dime-
thoxyphenyl)-6-pyrimidinone (3), 2,4-diamino-5-(30,40-dihydroxyphenyl)-6-pyri-
midinone (4), and 4-amino-5-(30,40-dihydroxyphenyl)-2,6-pyrimidindione (5)
(Scheme 1).
Experimental
General
1H and 13C NMR spectra were obtained at 500 and 125 MHz, respectively, on a
Varian Unity 500 spectrometer. IR spectra were measured on a JASCO FT/IR-460
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Syntheses and electrochemical properties
179
O
O
O
HN
N
HN
HN
R2
HN
R1
H2N
O
d
OH
N
R1
NH2
R2
H2N
NH
NH2
NH2
OH
1: R1 = OMe (47%)
2: R1 = OH (72%)
5 (50%)
H2N
3: R2 = OMe (92%)
4: R2 = OH (42%)
a
b
c
c
aKey: (a) NaOH, ethyl 3-(3',4'-dimethoxyphenyl)-3-oxopropanoate (6), EtOH; (b) EtONa, methyl
2-(3',4'-dimethoxyphenyl)-2-cyanoethanoate (7), EtOH; (c) Pyridine•HCl; (d) 47% HBr, AcOH.
Scheme 1 Synthetic routes for aminopyrimidinones 1–5
plus spectrometer. ESI-HRMS analyses were carried out on a JEOL JMS-T100LC
mass spectrometer. Melting points were determined with Yanako MP-500D and not
corrected.
Materials
Ethyl 3-(30,40-dimethoxyphenyl)-3-oxopropanoate (6) [14, 15] and methyl 2-(30,40-
dimethoxyphenyl)-2-cyanoethanoate (7) [16] were prepared as previously
described. All other materials used were commercially available.
2-Amino-4-(30,40-dimethoxyphenyl)-6-pyrimidinone (1)
An EtOH (50 mL) solution of guanidine carbonate (25.0 g, 139 mmol) and NaOH
(2.78 g, 69.4 mmol) was added to a toluene (100 mL) solution of 6 (17.5 g,
69.4 mmol), then the mixture was refluxed for 5 h. After removal of the solvent, the
residue was resolved in 1 M NaOH and neutralized with AcOH (pH 6–7). The
resulting precipitate was then filtered and washed with H2O, acetone and Et2O to
give crude 1 (8.03 g, 47 %). Recrystallization from MeOH gave pure 1 as a
colorless solid. Mp 271–274 °C; IR (KBr) 3,410, 3,170, 3,101, 1,654, 1,264 cm-1
;
1H NMR (DMSO-d6) d 10.72 (brs, 1 H), 7.55 (dd, J = 9.0, 2.0 Hz, 1 H), 7.51 (d,
J = 2.0 Hz, 1 H), 7.00 (d, J = 9.0 Hz, 1 H), 6.56 (brs, 2 H), 6.09 (s, 1 H), 3.81 (s, 3
H), 3.80 (s, 3 H) ppm; 13C NMR (DMSO-d6) d 163.4, 162.5, 155.6, 150.6, 148.5,
129.9, 119.8, 111.3, 110.1, 96.6, 55.7, 55.6 ppm; HRMS calculated for MH?,
C12H14N3O3: 248.1035; found 248.1026.
2-Amino-4-(30,40-dihydroxyphenyl)-6-pyrimidinone (2)
A mixture of 1 (1.00 g, 4.05 mmol) and pyridine hydrochloride (10.0 g, 86.5 mmol)
was heated at 200 °C for 1 h. After being cooled to room temperature, the mixture
was suspended in H2O (100 mL) and neutralized with 1 M NaOH (pH 6–7). After
removal of the solvent, H2O (100 mL) was added to the residue. The resulting
precipitate was filtered and then washed with H2O, EtOH, and Et2O to give 2
(638 mg, 72 %). Recrystallization from 1 M HCl gave pure 2 (hydrochloride salt)
as a pale yellow powder. Mp 262–264 °C; IR (KBr) 3,282, 3,114, 1,700, 1,637,
1
1,523 cm-1; H NMR (DMSO-d6) d 10.62 (brs, 1 H), 9.28 (brs, 1 H), 9.01 (brs, 1
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J. Chiba et al.
H), 7.36 (d, J = 2.0 Hz, 1 H), 7.25 (dd, J = 8.0, 2.0 Hz, 1 H), 6.75 (d, J = 8.0 Hz,
1 H), 6.45 (brs, 2 H), 5.86 (s, 1 H) ppm; 13C NMR (DMSO-d6) d 162.5, 156.9,
154.6, 148.8, 145.6, 124.5, 118.9, 115.8, 114.3, 97.1 ppm; HRMS calculated for
MH?, C10H10N3O3: 220.0722; found 220.0730.
2,4-Diamino-5-(30,40-dimethoxyphenyl)-6-pyrimidinone (3)
Guanidine hydrochloride (16.3 g, 170 mmol) was added to an EtOH (200 mL)
solution of NaOEt (11.6 g, 170 mmol), then the mixture was stirred for 5 min at
room temperature. After filtration, 7 (10.0 g, 42.5 mmol) was added to the filtrate.
The resulting mixture was stirred for 16 h at ambient temperature and evaporated
under atmospheric pressure to remove the EtOH. The residue was solidified with
water (300 mL), and the resulting precipitate was filtered, washed with EtOH and
Et2O, and air-dried to give crude 3 (9.80 g, 92 %). Recrystallization from H2O gave
pure 3 as a colorless solid. Mp [ 313 °C (decomp.); IR (KBr) 3,437, 3,175, 1,639,
1
1,600, 1,239 cm-1; H NMR (DMSO-d6) d 9.93 (brs, 1 H), 6.90 (d, J = 8.5 Hz, 1
H), 6.80 (d, J = 2.0 Hz, 1 H), 6.76 (dd, J = 8.5, 2.0 Hz, 1 H), 6.15 (brs, 2 H), 5.46
(brs, 2 H), 3.74 (s, 3 H), 3.71 (s, 3 H) ppm; 13C NMR (DMSO-d6) d 161.4, 153.7,
148.4, 146.9, 127.8, 123.1, 114.9, 111.9, 90.3, 55.6, 55.4 ppm; HRMS calculated for
MH?, C12H15N4O3: 263.1144; found 263.1141.
2,4-Diamino-5-(30,40-dihydroxyphenyl)-6-pyrimidinone (4)
A mixture of 3 (1.10 g, 4.21 mmol) and pyridine hydrochloride (10.0 g, 86.5 mmol)
was heated at 170 °C for 30 min. The mixture was cooled to room temperature,
dissolved in H2O (70 mL) and neutralized with 1 M NaOH (pH 6–7). After removal
of the solvent, H2O (50 mL) was added to the residue. The resulting precipitate was
filtered and then washed with H2O, EtOH and Et2O to give 4 (412 mg, 42 %).
Recrystallization from 1 M HCl gave pure 4 (hydrochloride salt) as a pale yellow
powder. Mp 253–255 °C; IR (KBr) 3,185, 2,733, 1,694, 1,657, 1,595, 1,535,
1,431 cm-1; 1H NMR (DMSO-d6) d 9.85 (brs, 1 H), 8.69 (brs, 1 H), 8.60 (brs, 1 H),
6.67 (d, J = 7.5 Hz, 1 H), 6.64 (d, J = 2.0 Hz, 1 H), 6.47 (dd, J = 7.5, 2.0 Hz, 1
H), 6.09 (brs, 2 H), 5.29 (brs, 2 H) ppm; 13C NMR (DMSO-d6) d 152.1, 151.2,
145.3, 144.7, 121.9, 121.6, 118.5, 115.9, 90.4 ppm; HRMS calculated for MH?,
C10H11N4O3: 235.0831; found 235.0839.
4-Amino-5-(30,40-dihydroxyphenyl)-2,6-pyrimidindione (5)
A mixture of 47 % HBr (5 mL), AcOH (2.5 mL) and 3 (540 mg, 2.06 mmol) was
heated at 140 °C for 10 h. After being cooled to room temperature, the mixture was
treated with H2O (15 mL). The resulting precipitate was filtered and then washed
with H2O, EtOH and Et2O to give 5 (243 mg, 50 %). Recrystallization from H2O
gave pure 5 as a colorless solid. Mp [ 307 °C (decomp.); IR (KBr) 3,384, 3,039,
2,711, 1,693, 1,609, 1,534, 1,423, 1,291 cm-1; 1H NMR (DMSO-d6) d 10.38 (brs, 2
H), 8.48 (brs, 1 H), 8.41 (brs, 1 H), 6.93 (d, J = 1.5 Hz, 1 H), 6.74 (dd, J = 8.0,
1.5 Hz, 1 H), 6.56 (d, J = 8.0 Hz, 1 H), 6.50 (brs, 2 H) ppm; 13C NMR (DMSO-d6)
123

Syntheses and electrochemical properties
181
d 163.3, 152.0, 143.8, 142.5, 125.8, 121.9, 118.4, 118.3, 114.5, 91.7 ppm; HRMS
calculated for MNa?, C10H9N3O4Na: 258.0491; found 258.0479.
Electrochemical measurements [17]
Cyclic voltammetry (CV) was performed using a Bioanalytical Systems (BAS)
Model 620A electrochemical analyzer with a normal three-electrode configuration
consisting of a gold working electrode (/ = 1.6 mm, BASi), saturated Ag/AgCl
reference electrode (BAS) and platinum wire auxiliary electrode. Measurements for
2, 4, and 5 were performed with a scanning rate (v) of 0.1 V/s at 25.0 0.1 °C in
65 mM phosphate buffer (pH 7.0) that had been thoroughly degassed with N2.
Redox potentials of dimethoxy derivatives 1 and 3 (0.5 mM) were measured in N2
bubbled acetonitrile containing 100 mM tetrabutylammonium perchlorate (condi-
tions: v = 0.1 V/s, 25.0 0.1 °C).
Results and discussion
Syntheses of the new aminopyrimidinones
The new aminopyrimidinones were synthesized via a pyrimidine-ring forming
reaction with commercially available guanidine carbonate and guanidine hydro-
chloride (Scheme 1). Base-catalyzed cyclocondensation of guanidine carbonate and
B
+4
+6
A
+4
+2
0
+2
0
-2
-4
-2
-4
-0.2
0
+0.2
+0.4
+0.6
-0.2
0
+0.2
+0.4
+0.6
Potential (V vs. Ag/AgCl)
Potential (V vs. Ag/AgCl)
C
+4
+2
0
-2
-4
-0.4
-0.2
0
+0.2
+0.4
Potential (V vs. Ag/AgCl)
Fig. 1 Cyclic voltamograms of a 2, b 4, and c 5 (0.5 mM)
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J. Chiba et al.
oxopropanoate 6 afforded 2-aminopyrimidinone 1 in moderate yield. Next, a
deprotection reaction of the methyl group with pyridine hydrochloride at 200 °C
proceeded for 1 h to give the water-soluble dihydroxy compound 2 in good yield.
The condensation of cyanoethanoate 7 and guanidine hydrochloride under basic
conditions gave 2,4-diaminopyrimidinone 3 in 92 % yield. Subsequent treatment of
3 with pyridine hydrochloride at 180 °C for 30 min provided the corresponding
dihydroxy derivative 4. It is important to regulate the reaction time for increasing
the yield of the deprotection reaction. A longer reaction time gave a mixture of 4
and 2,6-pyrimidindione 5, the hydrolyzed compound of 4. The by-product 5 could
be mainly obtained from 3 by treatment with 47 % HBr and AcOH at 140 °C for
10 h.
Electrochemical properties
Redox potentials of aminopyrimidinones 1–5 were measured by CV. Dimethoxy
compounds 1 and 3 exhibited no electrochemical response within a scanning range
of –0.4 to ?0.6 V versus Ag/AgCl. On the other hand, the dihydroxy derivatives 2,
4 and 5 showed reversible cyclic voltammograms, in which the oxidation potentials
were ?0.25, ?0.21, and ?0.10 V versus Ag/AgCl, respectively (Fig. 1). These
results suggested that the catechol derivatives 2, 4, and 5 showed clear
electrochemical responses owing to the catechol/o-quinone redox activity. On the
other hand, the methyl-protected 1 and 3 were hardly oxidized within the potential
range. The oxidation potentials of 2, 4, and 5 to give the corresponding o-quinone
derivatives were similar to that of catechol previously reported (ca. ?0.2 V vs. Ag/
AgCl in aqueous media [12]). In addition, these values are favorable for a reporter
molecule in abasic sites because DNA is electrochemically silent in the potential
range lower than ca. ?0.8 V [18, 19].
Conclusion
In conclusion, we synthesized novel aminopyrimidinones possessing a catechol
moiety for providing oxidation activity. Because all derivatives with the dihydroxy
form of the catechol unit showed low oxidation potentials to give the corresponding
o-quinone derivatives, they will be appropriate for using as abasic-site binders with
a function of electrochemically detectable reporter. Development of a simple
electrochemical detection system for nucleobases at abasic sites using these
compounds is now underway.
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