Symmetrically and unsymmetrically bridged methylenebis(allopurinols): Synthesis of dimeric potential anti-gout drugs

Article abstract of DOI:10.3390/12030563

Liquid-liquid phase transfer alkylation of 4-methoxy-pyrazolo[3,4-d]- pyrimidine (1a) with a dichloromethane/dibromomethane mixture (3:1, v/v) gave the regioisomeric methylenebis(heterocycles) 3a-5a. These were converted by dilute aqueous sodium hydroxide containing dimethylsulfoxide (DMSO) at concentrations between 0 and 60 vol-% into the methylenebis(allopurinols) 3b-5b by nucleophilic SNAr reactions at C(4). The effect of DMSO on the reaction kinetics was investigated.

Full text of DOI:10.3390/12030563

Molecules 2007, 12, 563-575
molecules
ISSN 1420-3049
http://www.mdpi.org
Full Paper
Symmetrically and Unsymmetrically Bridged
Methylenebis(allopurinols): Synthesis of Dimeric Potential
Anti-Gout Drugs
Helmut Rosemeyer 1,*, Martina Anders 1 and Frank Seela 1,2
1
Organische Chemie I – Bioorganische Chemie, Institut für Chemie, Fachbereich Biologie/Chemie,
Universität Osnabrück, Barbarastr. 7, D-49069 Osnabrück, Germany
2
Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
Heisenbergstr. 11, D-48149 Münster, Germany
*Author to whom correspondence should be addressed; E-mail: Helmut.Rosemeyer@uos.de
Received: 9 March 2007; in revised form: 19 March 2007 / Accepted: 19 March 2007 / Published:
21 March 2007
Abstract: Liquid-liquid phase transfer alkylation of 4-methoxy-pyrazolo[3,4-d]-
pyrimidine (1a) with a dichloromethane/dibromomethane mixture (3:1, v/v) gave the
regioisomeric methylenebis(heterocycles) 3a–5a. These were converted by dilute
aqueous sodium hydroxide containing dimethylsulfoxide (DMSO) at concentrations
between 0 and 60 vol-% into the methylenebis(allopurinols) 3b–5b by nucleophilic SNAr
reactions at C(4). The effect of DMSO on the reaction kinetics was investigated.
Keywords: Allopurinol, dimethylsulfoxide, dimeric drugs.
Introduction
1,5-Dihydro-4H-pyrazolo[3,4-d]pyrimidine = 8-aza-7-deazahypoxanthine (allopurinol, 1b, Figure
1; IUPAC numbering is used throughout the manuscript) [1,2] is a progressive inhibitor of xanthine
oxidase with alloxanthine – a 6-oxo derivative of 1b – being the actual inhibitor [3]. This has led to a
clinical application in the treatment of gout and related metabolic disorders [4]. The value of
allopurinol as well of its 4-amino analogue is augmented by their effects on pyrimidine and purine
biosynthesis [5,6].

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Molecules 2007, 12
We have previously reported on the inhibitor properties of pyrrolo[2,3-d]pyrimidines (i.e.
7-deazapurines) towards xanthine oxidase (from cow’s milk) [7]. In this context we now disclose that
2-methylthio-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (2a) [8,9] is also a moderately active
competitive inhibitor of xanthine oxidase, with an inhibition constant (Ki) of 120 µM. A corresponding
dimeric inhibitor was obtained when two 4-methoxy-2-methylthio-7H-pyrrolo[2,3-d]pyrimidine
molecules were linked by a methylene bridge via liquid-liquid phase-transfer-catalyzed alkylation of
the latter using dichloromethane, followed by hydrolysis of the methoxy groups with dilute
hydrochloric acid (→ 2b) [10]. The methylenebis(heterocycle) 2b was also tested as a xanthine
oxidase inhibitor and found to exhibit a Ki value of 6 µM, which is 20-fold more potent than the
corresponding Ki of the monomeric heterocycle 2a.
In this paper we now report the synthesis of methylenebis(allopurinols) 3b–5b applying
nucleophilic substitution reactions (SNAr) on the corresponding 4-methoxy precursors 3a–5a, as well
as the influence of dimethylsulfoxide on the reaction kinetics.
Figure 1. Chemical structures of novel methylenebis(heterocycles).

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Molecules 2007, 12
Results and Discussion
4-Methoxy-1H-pyrazolo[3,4-d]pyrimidine (1a) [1,11,12] has been proven as a versatile heterocycle
for the synthesis of alkylated [13] and glycosylated [14] pyrazolo[3,4-d]pyrimidines, while allopurinol
has not [15]. In phase-transfer-catalyzed reactions this lactam-protected chromophore can be alkylated
exclusively at N(1) and N(2), while reactions on the pyrimidine ring do not take place.
The methoxy group can subsequently undergo SNAr nucleophilic displacement reactions, which
are particularly advantageous for the synthesis of N(1)- and N(2)-glycosylated allopurinol nucleosides
[13,16,17]. Cleavage of the methoxy function with acid always bears the risk of concomitant N-
glycosylic bond cleavage. In a number of methylation and glycosylation reactions of pyrrolo[2,3-
d]pyrimidines under liquid-liquid phase-transfer catalysis conditions with dichloromethane as solvent
we observed a regioselective bridging of two chromophore moieties via N(7) by a methylene bridge
[10]. This reaction is then always favored when the alkyl halide is almost inert.
In contrast to this, the reaction of the heterocycle 1a with dichloromethane proceeded very slowly
with formation of three reaction products. However, when dibromomethane was added to the reaction
mixture compound 1a had disappeared completely with 60 min. Silica gel chromatography separated
1
three methylenebis(4-methoxy-pyrazolo[3,4-d]pyrimidines) in a 8:16:1 ratio. Comparison of their H-
and 13C-NMR spectra with those of 1a, as well as with those of its N(1)- and N(2)-methylated
derivatives, revealed that the fastest migrating product is the N(1)-N(1) bridged compound 3a, as only
one set of NMR signals could be observed. The second zone contained the unsymmetrically bridged
compound 4a. In this case, the NMR spectra showed two sets of signals – one for the N(1)- and a
second one for the N(2)-alkylated chromophore. The slowest migrating zone contained the N(2)-N(2)
bridged compound 5a in low yield. This may be due to the low reactivity of the 2-nitrogen compared
to N(1).
Table 1. [1H-13C] Coupling constants of compounds 3a–5a a.
Compound
C-6
3a
205.5
195.9
-
4a b
5a
204.1
197.6
3.0
H-C(6)
H-C(3)
CH2
204.1/205.6
C-3
195.9/196.2
C-3
3.0
C-3a
C-7a
C-7a
C-7a
CH2
H-C(3)
CH2
10.1
n. r.
n. r.
n. r.
155.4
-
7.2/10.4
8.0
-
-
H-C(6)
H-C(3)
CH2
-
12.8
6.8
-
157.3
-
156.0
~ 2
CH2
H-C(3)
a) Measured in DMSO-D6 at room temperature.
b) The first value refers to the N(2)-alkylated and the second to the N(1)-
alkylated chromophore of compound 4a.

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Molecules 2007, 12
For unequivocal structural proof proton-coupled 13C-NMR spectra of the three regioisomers 3a–5a
were obtained (Table 1). While C(7a) of compound 3a appears as a complex multiplet, due to three
3J(C,H) couplings with H-C(6), H-C(3) and CH2, the corresponding C(7a) of compound 5a shows only
3
two J(C,H) couplings with H-C(6) and H-C(3) (12.8 and 6.8 Hz, respectively). Correspondingly, the
CH2 groups of both isomers exhibit different coupling patterns: the multiplicity of CH2 of 3a is a
3
triplet (1J(C,H) = 155.4 Hz), while that of 5a shows a triplet of triplets due to an additional J(C,H)
coupling with both H-C(3) atoms. In addition, the C(3) signal of the isomer 5a exhibits an additional
fine splitting compared with that of 3a, due to a long range coupling (3J(C,H)) with its methylene
group. All these data confirmed the proposed structures of compounds 3a and 5a [18].
In the gated-decoupled 13C-NMR spectrum of the unsymmetrically bridged isomer 4a the coupling
patterns of both chromophore moieties behave additively. A positive indicator for the correct structure
is the fact that only the C(3) signal of the N(2)-alkylated moiety shows an additional fine splitting due
3
to a J(C,H) coupling with CH2 (3 Hz) while the corresponding C(3) signal of the N(1)-alkylated
chromophore moiety exhibits only a doublet (1J(C,H) = 196.2 Hz).
Nucleophilic displacement of the 4-methoxy groups of the methylenebis(4-methoxy-pyrazolo[3,4-
d]pyrimidines) 3a–5a by 1N NaOH/MeOH (1:1, v/v) gave the corresponding methylenebis-
1
(allopurinols) 3b–5b (Figure 1). Their structures were confirmed by UV- and H-NMR spectroscopy
as well as by elemental analyses. The different UV spectra of educts and products allow a simple
quantitative spectrophotometric monitoring and the determination of pseudo first-order kinetic
constants (half life τ and k) of the SNAr reactions (Table 2). As can be seen from Table 2 the reaction
rates of SNAr reactions of the methylenebis(4-methoxy-pyrazolo[3,4-d]pyrimidines) 3a–5a are
significantly faster than those of the N(1)- and N(2)-methylated compounds. In case of the N(2)-N(2)-
bridged derivative the half life is one order of magnitude higher than for the N(2)-methylated
chromophore.
Table 2. Pseudo first order kinetic data of nucleophilic OCH3 group displacement by
hydroxyl of 4-methoxy-pyrazolo[3,4-d]pyrimidine derivatives.
1N NaOH/MeOH
Reaction conditions
0.5 N NaOH
half life τ
(1:1, v/v)
half life τ
k
[min-1 x 103]
2.28
k
λ a)
[nm]
273
283
273
273
283
m b)
[min]
305
200
145
48
[min]
95
134
90
-
[min-1x103]
N(1)-methylated 1a
7.3
5.2
7.7
-
8.8
7.9
8.8
7.3
7.9
N(2)-methylated 1a
3.57
3a
4a
5a
4.81
14.4
20
34.6
-
-
a) Wavelength for spectrophotometric monitoring of the nucleophilic displacement reactions.
b) m = sensitivity of the substrates to the dimethylsulfoxide content in the varying solvent
compositions (1N NaOH/DMSO) according to equation 1 (correlation coefficients of linear
regression, r2, better than 0.99.

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Molecules 2007, 12
This leads to the assumption that a strong interaction exists between both chromophore moieties,
probably caused by an orbital overlap of the two heterocyclic π-electron systems separated by the non-
conjugating methylene group (homoconjugation). In this context it is interesting to see that for all three
regioisomeric methylenebis(allopurinols) 3D-optimized molecular models [19] display structures with
heterocyclic planes which are perpendicular to each other (Figures 2-4, Table 3); the heterocycles are
not stacked one upon the other, as it has been shown for ethylenebis- or higher alkyl analogues [20,21].
A mutual (-M) – effect of one chromophore on the other leads to an increase in electrophilicity at C(4),
compared with the methylated heterocycles and, therefore, to a higher reaction rate for displacement
reactions by hydroxyl. Even a weaker nucleophile such as ammonia (25 % aq. NH3) is able to
substitute the 4-methoxy group of compound 3a, yielding compound 3c. If the nucleophilic
displacement reaction of the methoxy group by hydroxyl on compound 5a was extended for more than
1 h, an opening of the pyrimidine rings at C(6) was observed, and small amounts of compound 6 were
formed [22]. If the nucleophilic displacement reactions of 3a–5a are performed in a methanol-free
medium (0.5 N NaOH), all reaction rates are enhanced (Table 3) which points to a weaker solvation of
the reaction intermediates by methanol-water mixtures compared to pure water.
Figure 2. 3D-Optimized structure of compound 3b [19].
Figure 3. 3D-Optimized structure of compound 4b [19].

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Molecules 2007, 12
Figure 4. 3D-Optimized structure of compound 5b [19].
Table 3. Selected torsion and bond angles as well as inter-moiety N-N distances (Å) of
compounds 3b–5b.
torsion and bond angles [deg];
Compound
inter-moiety N-N distances (Å)
3b [1,1’-methylenebis(allopurinol)]
4b [1,2’-methylenebis(allopurinol)]
5b [2,2’-methylenebis(allopurinol)]
N(2)-N(1)-C(8)-N(1’),
N(2’)-N(1’)-C(8)-N(1),
C(7a)-N(1)-C(8)-N(1’),
-79.3
-22.2
-90.4
C(7a’)-N(1’)-C(8)-N(1’), -41.4
N(1)-C(8)-N(1’).
N(2) - N(2’),
114.2
4.1
N(2)-N(1)-C(8)-N(2’),
N(1’)-N(2’)-C(8)-N(1),
-14.8
-59.2
C(7a)-N(1)-C(8)-N(2’), 165.8
C(3’)-N(2’)-C(8)-N(1),
N(1)-C(8)-N(1’),
122.0
115.9
3.2
N(2) – N(1’),
N(1)-N(2)-C(8)-N(2’),
N(1’)-N(2’)-C(8)-N(2),
C(3)-N(2)-C(8)-N(2’),
C(3’)-N(2’)-C(8)-N(2),
N(2)-C(8)-N(2),
177.8
94.1
-2.1
-86.4
112.4
4.5
N(1) – N(1’),
It has been postulated that any bimolecular reaction of a small anion passing through a larger
polarizable transition state will be accelerated considerably upon changing from protic to aprotic
solvents [23]. Based on this hypothesis, the rate of an ester saponification has been reported to be
drastically effected by the solvent composition, e.g. when aqueous alcohol is substituted by aqueous
dimethylsulfoxide [24-29].

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Molecules 2007, 12
Figure 5. Pseudo first-order rate constant k as well as ln k versus DMSO concentration
for the reaction of 3a → 3b.
This prompted us to measure the influence of the DMSO concentration on the reaction rates of
nucleophilic displacements on compounds 3a–5a in 1N NaOH/DMSO mixtures (Figure 5). This
figure using 3a as an example how the reaction rate (rate constant k for pseudo first-order reactions)
increases with the DMSO concentration. Analogous results were obtained for 4a and 5a (data not
shown). Two reasons have been reported [23] for such hydrolysis rate enhancements in water - DMSO
- sodium hydroxide mixtures: (i) with increasing DMSO concentrations the activity of the OH- ion is
enhanced; at a mole fraction xDMSO of 0.35 the thermodynamic excess functions are maximal, and with
increasing DMSO content the solvation of OH- is reduced. (ii) At lower DMSO concentrations the
solvent lowers the energy of the reaction intermediate by specific solvation. This leads consequently to
a rate enhancement. Because in our case the mole fraction of DMSO ranges between 0 and 0.3 (0 – 60
vol-%), the second effect should be predominant. For all reactions measured under pseudo first-order
conditions linear ln k versus xDMSO plots were obtained following the relation:
ln k = m xDMSO + ln k0
(1)

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Molecules 2007, 12
where m = sensitivity of the substrates to the dimethylsulfoxide content in the varying solvent
compositions; ln k = natural logarithm of the pseudo first-order rate constant; ln k0 = natural logarithm
of the pseudo first-order rate constant in a DMSO-free medium and xDMSO = mole fraction of
dimethylsulfoxide).
The sensitivity values, m, are listed in Table 2. As can be seen, all SNAr reactions performed show
almost the same sensitivity toward the dimethylsulfoxide concentration in the solvent composition (7.9
–8.8). There is no significant difference between the N(1)- and N(2)-alkylated heterocycles. This
becomes understandable under the assumption that in all cases similar reaction intermediates of the
Meisenheimer complex type are formed. This is likely because the OCH3 group is a rather weak
leaving group. Both intermediates can be formulated by three mesomeric Lewis structures which are
probably energetically equivalent as their negative charges are located on the nitrogen atoms (Scheme
1).
Scheme 1. Mesomeric structures of the Meisenheimer complexes formed as
intermediates during the nucleophilic displacement reactions of 3a and 5a with OH- and
its solvation by dimethylsulfoxide.

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Molecules 2007, 12
This implies that for the nucleophilic displacement reactions of the methoxy groups of the N(1)- as
well as for the N(2)-alkylated 4-methoxy-pyrazolo[3,4-d]pyrimidines the intermediates with a
tetrahedral C(4) are energetically lowered by the same amount through solvation by dimethylsulfoxide
(Scheme 1).
Conclusions
Liquid-liquid phase transfer alkylation of 4-methoxy-1H-pyrazolo[3,4-d]pyrimidine (1a) with a
mixture of dichloromethane/dibromomethane afforded the N(1)-N(1)-, N(1)-N(2)-, and N(2)-N(2)
methylene-bridged dimeric heterocycles 3a–5a which are not stacked, but rather form distorted
tetraedra at the central carbon. The three regioisomers were transformed into the corresponding
methylenebis(allopurinols) 3b–5b by nucleophilic SNAr reactions with dilute aqueous sodium
hydroxide containing dimethylsulfoxide at concentrations ranging between 0 and 60 vol-%. These
products represent a structural type of dimeric drug analogous to methylenebis(4-methoxy-2-
methylthio-7H-pyrrolo[2,3-d]pyrimidine) (2b) which is a 20-fold stronger competitive inhibitor of
xanthine oxidase than the corresponding monomeric heterocycle 2a. The various biomedical properties
of the methylenebis(allopurinols) will be investigated and the results published later.
Experimental Section
General
All chemicals were purchased from Aldrich, Sigma or Fluka (Sigma-Aldrich Chemie GmbH,
Deisenhofen, Germany). Solvents were of laboratory grade and were distilled before use. Thin layer
chromatography (TL): glass sheets, silica gel 60 F254, 0.2 mm layer (Macherey-Nagel & Co, Düren,
Germany). Column flash chromatography (FC): silica gel 60 (Merck, Germany) at 0.5 bar (4 x 104 Pa);
sample collection with an UltroRac II fraction collector (LKB Instrumnets, Bromma, Sweden).
Melting points (uncorrected): Linström apparatus (Wagner & Munz), Germany). UV spectra and
reaction kinetics were measured in MT-4 cuvettes (1 mL) on a SuperScan 3 (Varian, Australia) or a
Shimadzu U-210 (Shimadzu, Japan) spectrophotometers. The temperature was controlled with a Lauda
R20-K thermostat, connected to a R40/2 digital thermometer (MGW Lauda, Germany). NMR Spectra
were measured on AC-250 and AMX-500 spectrometers (Bruker, Karlsruhe, Germany) operating at
250.13/500.14 MHz (1H) and 62.896/125.700 MHz (13C), respectively. Chemical shifts (δ values) are
in parts per million relative to tetramethylsilane used as internal standard. Elemental analyses were
performed by Mikroanalytisches Laboratorium Beller (Göttingen, Germany). Inhibition constants, Ki,
were taken from Dixon plots, measured according to [7].
Methylenebis(4-methoxy-1H-pyrazolo[3,4-d]pyrimidines) 3a-c – General Procedure
Caution! Use a well ventilated hood. 4-Methoxy-1H-pyrazolo[3,4-d]pyrimidine (500 mg, 3.33
mmol), suspended in dichloromethane/dibromomethane (3:1, v/v, 20 mL), was added to a solution of
tetrabutylammonium hydrogen sulfate (700 mg, 2.5 mmol) in 50 % aq. sodium hydroxide (20 mL) and
agitated with a vibromixer for 1 h at room temperature. The biphasic mixture was then poured into

572
Molecules 2007, 12
chloroform (200 mL) containing glacial acetic acid (30 mL). After filtration of the precipitated sodium
acetate the organic layer was washed twice with water, dried (calcium chloride) and evaporated to
dryness. Crystallization from methanol gave a mixture of 3a–5a (305 mg, 61% yield). For isolation of
the three regioisomers the mixture (500 mg) was dissolved in chloroform/methanol (9:1, v/v, 10 mL),
applied to a silica gel column (silica gel 60H, 6 x 30 cm) and chromatographed with the same solvent
mixture as eluent. Three zones were separated.
1,1’-Methylenebis(4-methoxy-1H-pyrazolo[3,4-d]pyrimidine (3a). The fastest migrating zone afforded
compound 3a as colorless needles (155 mg, 32 % yield) with a melting point of 202-204 °C upon
evaporation of the solvent and crystallization from methanol. TLC (silica gel, chloroform/methanol,
1
9:1, v/v): Rf = 0.92. UV (chloroform/methanol, 1:1, v/v): λmax = 247 nm (ε = 16.600); H-NMR
((CD3)2SO) δ 8.68 (1H, s, H-C(6)); 8.03 (1H, s, H-C(3)); 6.99 (2H, s, CH2); 4.13 (3H, s, OCH3); 13C-
NMR ((CD3)2SO) δ 164.1 (C-4); 156.2 (C-6); 155.9 (C-7a); 133.2 (C-3); 103.0 (C-3a); 55.9 (CH2);
54.2 (OCH3); EI MS (70 eV): m/e 312 (52 %, M+), 163 (100 %, M+ - chromophore); Anal. Calcd. for
C13H12N8O2: C, 49.99; H, 3.87; N, 35.88. Found: C, 49.84; H, 4.01; N, 35.99.
4-Methoxy-1-[(4-methoxy-2H-pyrazolo[3,4-d]pyrimidin-2-yl)methyl]-1H-pyrazolo[3,4-d]pyrimidine
(4a). The second zone afforded compound 4a as colorless needles (309 mg, 64 % yield) with a melting
point of 211-214 °C upon evaporation of the solvent and crystallization from methanol. TLC (silica
gel, chloroform/methanol, 9:1, v/v): Rf = 0.68; UV (chloroform/methanol, 1:1, v/v): λmax = 251, 262
1
nm (ε = 15.100, 14.300); H-NMR ((CD3)2SO) δ 8.63 (1H, s, H-C(6), N(1)-alkylated moiety); 8.27
(1H, s, H-C(6), N(2)-alkylated moiety); 8.59 (1H, s, H-C(3), N(2)-alkylated moiety); 8.07 (1H, s, H-
C(3), N(1)-alkylated moiety); 6.89 (2H, s, CH2); 4.13 (3H, s, OCH3, N(1)-alkylated moiety); 4.08 (3H,
13
s, OCH3, N(2)-alkylated moiety); C-NMR ((CD3)2SO) δ 165.6 (C-4, N(2)-alkylated moiety); 164.2
(C-4, N(1)-alkylated moiety); 161.8 (C-7a, N(2)-alkylated moiety); 156.4 (C-6, N(2)-alkylated moety);
156.2 (C-6, N(1)-alkylated moiety); 156.0 (C-7a, N(1)-alkylated moiety); 133.8 (C-3, N(1)-alkylated
moiety); 123.7 (C-3, N(2)-alkylated moiety); 104.3 (C-3a, N(2)-alkylated moiety); 103.4 (C-3a, N(1)-
alkylated moiety); 62.2 (CH2); 54.3 (OCH3, N(1)-alkylated moiety); 54.0 (OCH3, N(2)-alkylated
moiety); EI MS (70 eV): m/e 312 (100 %, M+), 163 (57 %, M+ - chromophore); Anal. Calcd. for
C13H12N8O2: C, 49.99; H, 3.87; N, 35.88. Found: C, 50.10; H, 4.01; N, 35.92.
2,2'-Methylenebis(4-methoxy-2H-pyrazolo[3,4-d]pyrimidine) (5a). From the slowest migrating zone
compound 5a (18.6 mg, 4 % yield) was obtained as colorless needles upon evaporation of the solvent
and crystallization of the residue from 1,4-dioxane; melting point 202-204 °C. TLC (silica gel,
chloroform/methanol, 9:1, v/v): Rf = 0.45; UV (chloroform/methanol, 9:1, v/v): λmax = 262 nm (ε =
1
18.500); H-NMR ((CD3)2SO) δ 8.45 (1H, s, H-C(6)); 8.59 (1H, s, H-C(3); 6.79 (2H, s, CH2); 4.08
(3H, s, OCH3); 13C-NMR ((CD3)2SO) δ 165.9 (C-4); 161.2 (C-7a); 156.3 (C-6); 125.8 (C-3); 104.1 (C-
3a); 64.7 (CH2); 54.0 (OCH3); EI MS (70 eV) m/e 312 (45 %, M+), 163 (100 %, M+ - chromophore);
Anal. Calcd. for C13H12N8O2: C, 49.99; H, 3.87; N, 35.88. Found: C, 49.74; H, 3.97; N, 35.61.

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Molecules 2007, 12
Methylenebis(allopurinols) 3b–5b – General Procedure
Compounds 3a– 5a (3a, 4a: 500 mg, 1.65 mmol, each; 5a; 70 mg, 0.26 mmol) were each dissolved
in 2N sodium hydroxide/MeOH (1:1, v/v, 20 mL) and refluxed for 1 h. In the cases of compounds 3b
and 4b acidification of the reaction mixtures with 6N HCl afforded the corresponding
methylenebis(allopurinols) directly as colorless solids; in the case of 5b further chromatographic
purification proved to be necessary.
1,1'-Methylenebis(1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) (3b). Compound 3b (440 mg, 95
% yield); m.p. > 320 °C. TLC (silica gel, 0.25 M LiCl): Rf = 0.75; UV (1N sodium hydroxide): λmax
=
1
272 nm (ε = 19.500); H-NMR (CD3OD/NaOD): δ 6.71 (s, CH2); 8.02 (s, H-C(3)); 8.25 (s, H-C(6));
Anal. Calcd. for C11H8N8O2: C, 46.48; H, 2.84; N, 39.42. Found: C, 46.31; H, 2.98; N, 39.49.
1-[(4-Oxo-4,5-dihydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)methyl]-1,5-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-one (4b). Compound 4b (423 mg, 91 % yield); m.p. > 320 °C; TLC (silica gel, 0.25 M
LiCl): Rf = 0.70; UV (1N sodium hydroxide): λmax = 273, 281 (sh) nm (ε = 20.500, 18.700); 1H-NMR
(CD3OD/NaOD): δ 6.71 (2H, s, CH2); 8.08 (1H, s, H-C(3), N(1)-alkylated moiety); 8.14 (1H, s, H-
C(3), N(2)-alkylated moiety); 8.26 (1H, s, H-C(6), N(1)-alkylated moiety); 8.43 (1H, s, H-C(6), N(2)-
alkylated moiety); Anal. Calcd. for C11H8N8O2: C, 46.48; H, 2.84; N, 39.42. Found: 46.39; H, 2.90; N,
39.42.
2,2'-Methylenebis(2,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) (5b). The reaction mixture was
chromatographed on a Dowex® 1x2 ion exchange resin column (1.5 x 30 cm). After washing with
water (1 L) compound 5b (62 mg, 96 % yield) was eluted with aq. acetic acid (10 %). M.p. > 320 °C;
TLC (silica gel, 0.25 M LiCl): Rf = 0.77; UV (1N sodium hydroxide): λmax = 283 nm (ε = 19.500); 1H-
NMR (CD3OD/NaOD): δ 8.06 (2H, br. S, H-C(6) and H-C(3); 5.91 (2H, CH2); Anal. Calcd. for
C11H8N8O2: C, 46.48; H, 2.84; N, 39.42. Found: C, 46.33; H, 2.96; N, 39.31.
1,1'-Methylenebis(1H-pyrazolo[3,4-d]pyrimidin-4-amine) (3c)
1,1’-Methylenebis(4-methoxy-1H-pyrazolo[3,4-d]pyrimidine (3a, 100 mg, 0.33 mmol) was
dissolved in concentrated aq. ammonia (20 mL) and stirred for 60 min at room temperature. After
evaporation of the solvent the residue was taken up in water and chromatographed on Amberlite®
XAD, type 4 (Serva, Germany). Methanol/water (1:4, v/v) eluted one main peak from which
compound 3c (70 mg, 75 % yield) was isolated as colorless solid. M.p.: 88-89°C; TLC (silica gel,
chloroform/methanol, 9:1, v/v): Rf = 0.4; UV (chloroform/methanol, 1:1, v/v): λmax = 260, 276 nm (ε =
16.800, 18.700); 1H-NMR ((CD3)2SO) δ 8.25 (1H, s, H-C(6)); 8.09 (1H, s, H-C(3)); 7.77 and 7.26 (2H,
13
br, NH2); 6.69 (2H, s, CH2); C-NMR ((CD3)2SO) δ 54.8 (CH2); 99.9 (C-3a); 133.8 (C-3); 154.1 (C-
7a); 156.5 (C-6); 158.0 (C-4); Anal. Calcd. for C11H10N10: C, 46.80; H, 3.57; N, 49.62. Found: C,
46.72, H, 3.65; N, 49.50.

574
Molecules 2007, 12
References and Notes
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575
Molecules 2007, 12
13
1
13
18. Uzawa, J.; Uramoto, M. Assignment of Indirect C, H Couplings in the C- NMR Spectra of
1
Some Purine and Pyrimidine Nucleosides and Their Analogues by Long-Range Selective H
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1
22. Compound 6: Brownish solid. H-NMR ((CD3)2SO) δ 8.06 (2H, s, H-C(3)); 5.91 (2H, s, CH2); ~
13
5.5 (br, NH2); C-NMR ((CD3)2SO) δ 64.1 (CH2); 99.7 (C-4); 134.1 (C-3); 156.7 (C-5); 164.8
(C=O).
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