Synthetic routes to N-9 alkylated 8-oxoguanines; Weak inhibitors of the human DNA glycosylase OGG1

Article abstract of DOI:10.3390/molecules200915944

The human 8-oxoguanine DNA glycosylase OGG1 is involved in base excision repair (BER), one of several DNA repair mechanisms that may counteract the effects of chemo- and radiation therapy for the treatment of cancer. We envisage that potent inhibitors of OGG1 may be found among the 9-alkyl-8-oxoguanines. Thus we explored synthetic routes to 8-oxoguanines and examined these as OGG1 inhibitors. The best reaction sequence started from 6-chloroguanine and involved N-9 alkylation, C-8 bromination, and finally simultaneous hydrolysis of both halides. Bromination before N-alkylation should only be considered when the N-substituent is not compatible with bromination conditions. The 8-oxoguanines were found to be weak inhibitors of OGG1. 6-Chloro-8-oxopurines, byproducts in the hydrolysis of 2,6-halopurines, turned out to be slightly better inhibitors than the corresponding 8-oxoguanines.

Full text of DOI:10.3390/molecules200915944

Molecules 2015, 20, 15944-15965; doi:10.3390/molecules200915944
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthetic Routes to N-9 Alkylated 8-Oxoguanines; Weak
Inhibitors of the Human DNA Glycosylase OGG1
Tushar R. Mahajan 1, Mari Eknes Ytre-Arne 2,3, Pernille Strøm-Andersen 3, Bjørn Dalhus 2,3
and Lise-Lotte Gundersen 1,*
1
Department of Chemistry, University of Oslo, P. O. Box 1033, Blindern, N-0315 Oslo, Norway;
E-Mail: t.r.mahajan@kjemi.uio.no
2
Department of Microbiology, Oslo University Hospital, P. O. Box 4950, Nydalen,
N-0424 Oslo, Norway; E-Mails: Mari.Ytre-Arne@rr-research.no (M.E.Y.-A.);
Bjorn.Dalhus@medisin.uio.no (B.D.)
3
Department of Medical Biochemistry, Institute of Clinical Medicine, University of Oslo,
P. O. Box 4950, Nydalen, N-0424 Oslo, Norway; E-Mail: pernille.strom-andersen@medisin.uio.no
* Author to whom correspondence should be addressed; E-Mail: l.l.gundersen@kjemi.uio.no;
Tel.: +47-228-570-19.
Academic Editor: Roman Dembinski
Received: 2 June 2015 / Accepted: 26 August 2015 / Published: 2 September 2015
Abstract: The human 8-oxoguanine DNA glycosylase OGG1 is involved in base excision
repair (BER), one of several DNA repair mechanisms that may counteract the effects of
chemo- and radiation therapy for the treatment of cancer. We envisage that potent inhibitors
of OGG1 may be found among the 9-alkyl-8-oxoguanines. Thus we explored synthetic routes
to 8-oxoguanines and examined these as OGG1 inhibitors. The best reaction sequence started
from 6-chloroguanine and involved N-9 alkylation, C-8 bromination, and finally simultaneous
hydrolysis of both halides. Bromination before N-alkylation should only be considered when
the N-substituent is not compatible with bromination conditions. The 8-oxoguanines were
found to be weak inhibitors of OGG1. 6-Chloro-8-oxopurines, byproducts in the hydrolysis of
2,6-halopurines, turned out to be slightly better inhibitors than the corresponding 8-oxoguanines.
Keywords: alkylation; cancer; DNA; enzyme inhibitors; guanine; halogenation

Molecules 2015, 20
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1. Introduction
Chemo- and radiotherapy are, in addition to surgery for removal of solid tumors, the two main treatment
protocols currently available to improve the outcome of cancer patients in general, but treatment-related
toxicity, the risk of secondary cancers, and the emergence of resistance limit their effectiveness [1].
Some chemotherapeutic drugs and radiotherapy work partly by imposing high concentrations of DNA
damage on the genome of cancer cells, beyond the repair capacity of those cells. The drug-exposed cancer
cells are heavily dependent on efficient DNA repair to survive. Consequently, inhibitors that reduce DNA
repair activities should sensitize cancer cells to chemo- and/or radiotherapy [2–5].
Several DNA repair mechanisms counteract exogenous and endogenous processes that destabilize or
directly damage genomes. The processes include, among others, base excision repair (BER), a mechanism
that depends on enzymes that recognize small modifications in the native bases in DNA, resulting
from alkylation, oxidation, deamination, or hydrolysis of the DNA bases. The pathway is initiated by a
damage-specific DNA glycosylase that removes the altered base [6]. Some of these enzymes mainly
remove oxidized bases, such as the human 8-oxoguanine DNA glycosylase (OGG1) that removes guanines
that have been oxidized at the C8-position. The 8-oxoguanine base in the DNA is flipped into a lesion
recognition pocket on the enzyme surface, exposing the Watson–Crick signature of guanine and the oxidized
C8 position (Figure 1).
Figure 1. Structural details of 8oxoG base flipped into the lesion recognition pocket of OGG1
(Protein Data Bank deposition 1EBM [7]). The protein backbone is shown as a blue ribbon/helix.
Selected amino acid side chains and the 8oxoG base are shown as ball-and-stick. Hydrogen
bonds between the protein and 8oxoG are shown as dashed lines. Asp268 is the catalytic
residue in OGG1. Symbols 5′ and 3′ indicate the position of the 5′ and 3′ phosphodiester links
in the DNA.
We envisage that potent inhibitors of OGG1 may be found among the 9-alkyl-8-oxoguanines.
The 8-oxo derivatives of guanosine or deoxyguanosine are probably not inhibitors of the glycosylases
since they themselves may be substrates for the enzymes that cleave N,O-acetals in nucleic acids. As a
continuance of our synthetic studies directed towards 9-substituted 8-oxoadenines [8,9], we herein present
strategies for the synthesis of N-9 substituted 8-oxoguanines. Previous routes include rather tedious
constructions of the guanine ring system [10–12], and hydrolysis of purine precursors; hydrolysis of

Molecules 2015, 20
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8-halopurines [13–16], or less conveniently hydrolysis of N-7 functionalizedpurines [11,17–19]. Results
regarding inhibitory activity against the human DNA glycosylase OGG1 are also presented.
2. Results and Discussion
2.1. Chemistry
We found it most convenient to start the synthesis of 9-alkyl-8-oxopurines from commercially available
purines, and in our opinion the best way to introduce the 8-oxo group would be by hydrolysis of an
8-halopurine. However, there still was the question of whether the halogen or the N-9 substituent should
be introduced first and which protection/activation groups should be employed in the synthesis. Ideally,
such groups should also be removed in the final hydrolysis step. Regioselectivity in N-alkylation of guanine
derivatives was also an issue [20–27]. We chose to start from two guanine precursors, commercially available
2-amino-6-chloropurine (1a) and the O-carbamoylguanine 1b, easily available from guanine [28,29].
The synthetic routes explored are all summarized in Scheme 1.
Reagents and conditions: (a) See Table 1; (b) See Table 2; (c) 1. Ac2O, NaOAc, AcOH, 2.
NaOH(aq), ∆; (d) 1. LDA, 2. (CCl2Br)2, THF, −78 °C; (e) Br2, CHCl3; (f) See [30]; (g)
HCl(aq), EtOH.
Scheme 1. Synthetic routes to 8-oxoguanines 5.

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Table 1. N-alkylation of guanine precursors 1a and 1b.
Reagents and
Entry
1
R2
Cl
Cl
R6
R
Ratio 2:3:1 a Yield (%) 2 b Yield (%) 3 b
Conditions
RBr, K2CO3,
NH2
NH2
CH2-c-hexyl
CH2-c-hexyl
80:20:0
93:7:0
67, 2a
76, 2a
45, 2e
70, 2e
10, 3a
DMF, rt, 72 h
ROH, DIAD, PPh3,
THF, 70 °C, 14 h
RBr, K2CO3,
2
5, 3a
7, 3e
3, 3e
–
3
OCONPh2 NHAc CH2-c-hexyl
OCONPh2 NHAc CH2-c-hexyl
81:19:0
82:18:0
15:0:85
DMF, rt, 72 h
ROH, DIAD, PPh3,
THF, 70 °C, 14 h
RI, K2CO3,
4
c
5
Cl
Cl
Cl
Cl
Cl
NH2
NH2
NH2
NH2
NH2
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-pentyl
c-pentyl
c-pentyl
c-pentyl
–
DMF, rt, 72 h
ROTs, K2CO3,
d
c
6
–
33, 2b
–
DMF, rt, 72 h
ROH, DIAD, PPh3,
THF, 70 °C, 14 h
ROH, DIAD, PPh3,
THF, ultrasound, 14 h
ROH, DIAD, PPh3,
DMF, 150 °C, μW, 2 h
ROTs, K2CO3,
c
c
7
8:4:88
27:0:73
41:8:51
–
–
8
20, 2b
–
c
c
9
–
–
d
c
10
11
12
13
14
15
16
17
18
19
OCONPh2 NHAc
OCONPh2 NHAc
OCONPh2 NHAc
–
30, 2f
–
THF, rt, 72 h
ROTs, K2CO3, DMF,
80 °C, 72 h
d,e
c
c
–
–
–
ROH, DIAD, PPh3,
THF, 70 °C, 14 h
RBr, K2CO3,
d
c
–
22, 2f
71, 2c
72, 2c
52, 2g
58, 2g
18, 2d
40, 2d
53, 2d
–
Cl
Cl
NH2
NH2
86:14:0
91:9:0
5, 3c
6, 3c
DMF, rt, 72 h
ROH, DIAD, PPh3,
THF, 70 °C, 14 h
RBr, K2CO3,
c
OCONPh2 NHAc
OCONPh2 NHAc
76:15:09
90:10:0
23:16:61
55:18:27
75:25:0
–
DMF, rt, 72 h
ROH, DIAD, PPh3,
THF, 70 °C, 14 h
RBr, K2CO3,
c
–
c
Cl
Cl
Cl
NH2 c-pent-2-enyl
NH2 c-pent-2-enyl
NH2 c-pent-2-enyl
–
DMF, rt, 24 h
ROH, DIAD, PPh3,
THF, 70 °C, 42 h
ROAc, Pd(PPh3)4,NaH,
DMSO, f 50 °C, 48 h
c
–
18, 3d
a From 1H-NMR spectra of the crude products, the signals from H-8 in compounds 1, 2, and 3 were integrated;
b Isolated yields; c Not isolated in pure form; d Difficult to determine due to overlapping signals in the 1H-NMR
spectra; e A complex mixture was formed; f Comparable results were obtained in DMF.

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Table 2. Synthesis of 8-bromopurines 4.
Entry Starting Material a
Reagents and Conditions
Br2, H2O
Yield (%) 4 a,b
79%, 4a
34%, 4a
56%, 4a
70%, 4b
81%, 4c
1
2
3
4
5
6
7
8
2a
10
10
2b
2c
2d
10
10
RBr, K2CO3, DMF
ROH, DIAD, PPh3, THF, 70 °C
Br2, H2O
Br2, H2O
1. LDA, 2. CCl2BrCCl2Br, THF, −78 °C
ROH, DEAD, PPh3, THF, 70 °C
ROAc, Pd(PPh3)4, NaH, DMF, 50 °C
32%, 4d
42%, 4d
29%, 4d
a The structures are shown in Scheme 1; b Isolated yields.
First we chose to N-alkylate the substrates 1 before C-8 halogenation and hydrolysis. Alkylations were
conducted by various methodologies in order to find the conditions that gave the desired N-9 alkylated
isomer 2 with high selectivity and in a good isolated yield (Scheme 1, Table 1). Relatively simple alkylating
agents were chosen for the model reactions and we focused on alkylation with alkyl halides in the presence
of base, Mitsunobu reactions, and Pd-catalyzed allylic alkylation.
The cyclohexylmethyl substituent could be introduced at N-9 either by reaction with alkyl bromide
in the presence of a base [31,32] (Table 1, Entries 1 and 3) or with cyclohexylmethanol under Mitsunobu
conditions (Table 1, Entries 2 and 4). The latter is often claimed to be more N-9 selective compared to
classical alkylations of purines [33–35]. In all cases a mixture of the N-9 and N-7 alkylated isomers
(2 and 3) was formed with good selectivity for the desired isomer 2. The isomers were identified from
HMQC and HMBC-NMR, as described before [31].
The guanine precursor 1b, carrying a bulky substituent at C-6 that may sterically block N-7, is reported
to react with high N-9 selectivity in other N-functionalization reactions [28,29,36–41]. Nevertheless, we
found the regioselectivity in N-alkylation of purine 1b equal or slightly poorer compared to 6-chloroguanine
1a in all reactions performed in this study. In the alkylation of compound 1b, minor amounts of other
relatively polar products were formed under both reaction conditions. These often made purification of
the N-7 alkylated isomer 3 difficult. The identity of the byproducts could not be determined, but they may
be formed as a result of cleavage of the O6-protecting group. Alkylation of N2, as observed by others [41],
was not seen.
Introduction of the cyclohexyl group at N-9 turned out to be quite difficult (Table 1, Entries 5–12).
Both starting materials (1a and 1b) did not react with cyclohexyl bromide (data not shown) and reacted
slowly with cyclohexyl iodide or the corresponding tosylate, but compounds 2b and 2f could be isolated
in modest yields (Table 1, Entries 5, 6, 10 and 11). It is, however, well known that cyclohexyl halides
or pseudo halides may react sluggishly in substitution reactions [42]. The results were not significantly
improved when the Mitsunobu reaction was employed (Table 1; Entries 7, 8, and 12), not even under
ultrasound (Table 1, Entry 8) or microwave conditions (Table 1, Entry 9).
The cyclopentyl group could easily be installed at N-9 on both starting materials 1a and 1b by reaction
with cyclopentyl bromide and base (Table 1, Entries 13 and 15) or by alkylation under Mitsunobu conditions
(Table 1, Entries 14 and 16). The selectivity for N-9 was higher in the Mitsunobu reactions, but the
isolated yields were comparable due to more tedious purification when Mitsunobu conditions, also
producing phosphine oxides and reduced azodicarboxylates, were employed.

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Finally we introduced the cyclopent-2-enyl group at N-9 (Table 1, Entries 17–19). These reactions were
only conducted at the guanine precursor 1a, since we so far had not observed any significant improvement
in regioselectivity when compound 1b was employed and we had observed problems with compounds
derived from purine 1b later in the planned synthetic sequence. In addition to alkylation with the halide
and Mitsunobu reaction with the alcohol, we also attempted palladium catalyzed alkylation with the allylic
acetate [43]. 3-Bromocyclopentene could only be generated as a 15% solution in CCl4 and the reagent
had a limited stability, probably partly due to traces of the radical initiator used in the synthesis left in the
solution [44], which may explain the low yield of product 2d (Table 1, Entry 17). The Mitsunobu reaction
between purine 1a and cyclopenten-2-ol was surprisingly slow, and full conversion was not achieved even
after several days. Furthermore, the N-9/N-7 selectivity was only ca. 4:1 (Table 1, Entry 18). Pd-catalyzed
allylic alkylation of purine 1a went to completion and gave the isomers 2d and 3d in a 4:1 ratio (Table 1,
Entry 19).
The 6-chloropurines 2a, 2b, and 2c were readily brominated on C-8 simply by treatment of bromine in
water (Scheme 1; Table 2; Entries 1, 4, and 5). For compound 2d, which has an alkene function, the bromide
was introduced by C-8 lithiation followed by trapping with CCl2BrCCl2Br (Table 2, Entry 6) [9,32,45,46].
However, the yield was surprisingly low and also another route to bromide 4d was examined (see
below). Finally hydrolysis of the dihalopurines 4, employing conditions used for hydrolysis of other
8-bromopurines [13–16,47], gave the 8-oxoguanines 5. Complete conversion was achieved in the
hydrolysis compound 4a, whereas small amounts of the partly hydrolyzed chlorides 6 where present after
hydrolysis of purines 4b–d even after prolonged reaction times.
Attempts to brominate the O-carbamoylguanine 2e failed (Scheme 1). Treatment with bromine or
lithiation followed by trapping with CCl2BrCCl2Br only resulted in cleavage of the carbamoyl protecting
group to give the guanine derivative 7. When compound 2e was treated with NBS, no reaction took place
at all. Thus, no attempts were made to brominate the carbamoyl protected guanines 2f and 2g.
Since bromination of the cyclopentenylpurine 2d turned out to be a challenge, we also examined the
possibility for introducing the 8-halo substituent before the N-9 alkyl group (Scheme 1). We chose to
brominate the THP protected compound 8 [30] and removed the protection group under mild acidic
condition, but direct bromination of purine 1a in a moderate yield has also been reported [48].
Alkylation of 8-bromo-6-chloropurin-2-amine (10) by bromomethylcyclohexane in the presence of
K2CO3/DMF (Table 2, Entry 2) occurred slowly compared to alkylation of 2-amino-6-chloropurine (1a)
under the same set of reaction conditions (for alkylation of compound 1a see Table 1). NMR analysis
showed that approximately 50% of the starting material was intact even after 96 h reaction time and the
desired product was isolated in a low yield. Also, ca. 4% of N-7 alkylated isomer was formed, as judged
by NMR. When compound 10 was reacted under Mitsunobu (Table 2, Entry 3) conditions, high conversion
(ca. 95%) and almost full selectivity towards the desired N-9 alkylated isomer 4a was achieved, as
judged by 1H-NMR. However, the product 4a was isolated only in 56% due to tedious separation from
reduced DIAD. Since compound 10 reacted slower (conventional alkylation) or comparably (Mitsunobu
alkylation) to compound 1a, it was concluded that there were no benefits associated with introducing the
bromide before the N-alkyl group for the synthesis of 8-bromopurines 4a–c.
Also, synthesis of the 9-cyclopentenylpurine 4d by N-alkylation of compound 10 was examined
(Table 2, Entries 7 and 8) since bromination of 2-amino-6-chloro-9-cyclopentenylpurine 2d turned out
to give only a low yield of the desired product. Again, isolation of the desired product from alkylation

Molecules 2015, 20
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under Mitsunobu conditions turned out to be troublesome. We tried this Mitsunobu alkylation using the
water-soluble azodicarboxylate DMEAD (di-2-methoxyethyl azodicarboxylate) as well as DIAD [49].
Purification of the product was less complicated, but the conversion was low and ca. 40% of starting
material 10 was recovered. Also, Pd-catalyzed allylation turned out to be a very slow reaction and even
after six days only 29% of the desired compound 4d could be isolated, together with 32% unconverted
starting material 10.
2.2. Biology
As previously mentioned, our hypothesis was that N-alkyl-8-oxoguanines may inhibit the human
8-oxoguanine DNA glycosylase (OGG1). Other substituents in the purine 8-position are probably
not tolerated, for instance 8-bromo- and 8-aminoguanines are reported to be enhancers for OGG1
activity [50]. Thus, the 8-oxoguanines 5 as well as the partly hydrolyzed 6-chloro-8-oxopurines 6 were
tested against human DNA glycosylases OGG1 and NTH1. A general structure of the tested compounds
is shown in Figure 2 and the results are presented in Tables 3 and 4, and Supplementary Figure S19.
Figure 2. General structure of the compounds shown in Table 3.
Table 3. % Activity of OGG1 in the presence of compounds 5 or 6 at 0.2 mM concentration.
Compound
X
R
% Activity
89 ± 5
5a
5b
6b
5c
6c
5d
6d
OH a CH2-c-hexyl
OH a
Cl
c-hexyl
c-hexyl
c-pentyl
c-pentyl
92 ± 2
70 ± 11
101 ± 12
72 ± 9
OH
Cl
OH c-pent-2-enyl
92 ± 7
Cl c-pent-2-enyl
84 ± 3
a The predominant 6-oxo tautomer of compounds 5 is shown in Scheme 1.
Table 4. % Activity of NTH1 in the presence of compounds 5 or 6 at 0.5 mM concentration.
Compound
X
R
% Activity
96 ± 3
5a
5b
6b
5c
6c
5d
6d
OH a CH2-c-hexyl
OH a
Cl
c-hexyl
c-hexyl
c-pentyl
c-pentyl
123 ± 20
73 ± 37
OH
Cl
102 ± 16
108 ± 18
104 ± 21
89 ± 13
OH c-pent-2-enyl
Cl c-pent-2-enyl
a The predominant 6-oxo tautomer of compounds 5 is shown in Scheme 1.

Molecules 2015, 20
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Compounds 6b and 6c inhibit the OGG1 enzyme by ca. 30%, followed by compounds 5a, 5b, and 6d
at ca. 10%–15%, all at 0.2 mM ligand concentration. Interestingly, the halogenated compounds seem in
general to be better inhibitors than their 6-oxo derivatives. To check enzyme specificity, we tested the same
seven compounds at the higher concentration of 0.5 mM against NTH1, a structural but not functional
homolog of OGG1. Both enzymes have a deep pocket for binding of oxidized bases; in general, OGG1
repairs oxidized purines while NTH1 is involved in repair of oxidized pyrimidines. Compound 6b
reduced the NTH1 activity by around 25% at 0.5 mM ligand concentration. An effect of varying the
N-9 substituent is not so evident from the few compounds examined.
3. Experimental Section
3.1. General Information
1H-NMR spectra were recorded at 300 MHz with a Bruker DPX 300, at 400 MHz with a Bruker
DPX 400 or at 600 with a Bruker AVI 600 instrument (Bruker BioSpin AG, Fällanden, Switzerland).
The 13C-NMR spectra were recorded at 75, 100, or 150 MHz with the Bruker instruments listed above.
1
13
1
13
Assignments of H and C resonances are inferred from 1D H-NMR, 1D C-NMR, DEPT, or APT,
and 2D NMR (HMQC, HMBC) spectroscopical data. 1H- and 13C-NMR spectra of all novel compounds
can be found in the Supplementary Material (Figures S1–S18). HRMS (EI) was performed with a
double-focusing magnetic sector VG Prospec Q instrument (Waters, Manchester, UK) and HRMS (ESI)
with a TOF quadrupole Micromass QTOF 2 W instrument (Waters). Melting points were determined with
a Büchi Melting point B-545 apparatus (Büchi Labortechnik AG, Flawil, Switzerland) and are uncorrected.
Dry DMF and THF were obtained from a solvent purification system, MB SPS-800 (MBraun, Garching,
Germany). Acetic anhydride and diisopropylamine were distilled over CaH2. DMSO was dried over
activated 3 Å molecular sieves for four days. Potassium carbonate was oven dried at 150 °C under
high vacuum for 12 h. A saturated aqueous solution of Br2 was prepared by stirring water (20 mL) with
Br2 (0.200 mL) in a closed container for 15 min at ambient temperature. Sodium hydride (ca. 60% in
mineral oil) was washed with dry pentane under inert atmosphere prior to use. All other reagents were
commercially available and used as received. The following compounds were available by literature
methods: Cyclohexyl tosylate [51], cyclopentenyl bromide [44], cyclopent-2-enol [52], cyclopentenyl
acetate [53], 1b [29], 8 [30].
3.2. Synthesis
3.2.1. 2-Amino-6-chloro-9-(cyclohexylmethyl)-9H-purine (2a) and 2-Amino-6-chloro-7-
(cyclohexylmethyl)-7H-purine (3a)
Method A: K2CO3 (1.63 g, 11.8 mmol) was added to a stirring solution of compound 1a (1.00 g,
5.90 mmol) in dry DMF (30 mL) at ambient temperature under N2. After 20 min bromomethylcyclohexane
(0.905 mL, 6.49 mmol) was added and the resulting mixture was stirred for 72 h, filtered. and evaporated.
The isomers were separated by flash chromatography on silica gel, eluting with MeOH–CH2Cl2 (1:9) to
yield 2a (1.05 g, 67%) and 3a (150 mg, 10%).

Molecules 2015, 20
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2a: colorless solid; mp 148–150 °C (lit. [54], 154–155 °C); 1H-NMR (DMSO-d6, 400 MHz) δ 8.10 (s, 1H,
H-8), 6.91 (s, 2H, NH2), 3.88 (d, J = 7.4 Hz, 2H, NCH2), 1.88–1.72 (m, 1H, H-1 in c-hex), 1.68–1.52
(m, 3H, c-hex), 1.51–1.42 (m, 2H, c-hex), 1.19–1.02 (m, 3H, c-hex) 1.00–0.85 (m, 2H, c-hex); 13C-NMR
(DMSO-d6, 100 MHz) δ 159.8 (C, C-2), 154.3 (C, C-4), 149.3 (C, C-6), 143.7 (CH, C-8), 123.3 (C,
C-5), 48.8 (CH2, NCH2), 37.1 (CH, C-1 in c-hex), 29.9 (CH2, C-3 and C-5 in c-hex), 25.8 (CH2, C-4 in
c-hex), 25.0 (CH2, C-2 and C-6 in c-hex); HREIMS m/z 265.1092 (calcd for C12H16ClN5, 265.1094).
Spectral data were in good agreement with those reported before [54].
1
3a: colorless solid mp 228–231 °C. H-NMR (DMSO-d6, 400 MHz) δ 8.32 (s, 1H, H-8), 6.62 (s, 2H,
NH2), 4.10 (d, J = 7.2 Hz, 2H, NCH2), 1.82–1.70 (m, 1H, H-1 in c-hex), 1.69–1.54 (m, 3H, c-hex),
1.50–1.41 (m, 2H, c-hex), 1.24–1.06 (m, 3H, c-hex), 1.03–0.89 (m, 2H, c-hex); 13C-NMR (DMSO-d6,
100 MHz) δ 164.2 (C, C-4), 159.9 (C, C-2), 149.8 (CH, C-8), 142.3 (C, C-6), 114.9 (C, C-5), 51.8
(CH2, NCH2), 38.6 (CH, C-1 in c-hex), 29.6 (CH2, C-3 and C-5 in c-hex), 25.8 (CH2, C-4 in c-hex), 25.1
(CH2, C-2 and C-3 in c-hex); HREIMS m/z 265.1096 (calcd for C12H16ClN5, 265.1094).
Method B: Compound 1a (200 mg, 1.18 mmol) was added to a solution of cyclohexylmethanol
(141 mg, 1.24 mmol) and PPh3 (325 mg, 1.24 mmol) in dry THF (10 mL) under N2. The resulting
suspension was treated with diisopropyl azodicarboxylate (DIAD) (0.244 mL, 1.24 mmol) and the
reaction mixture was stirred at 70 °C for 7 h before cyclohexylmethanol (141 mg, 1.24 mmol), PPh3
(325 mg, 1.24 mmol), and DIAD (0.244 mL, 1.24 mmol) were added. The mixture was stirred for another
7 h at 70 °C, cooled, treated with brine (10 mL), and extracted with CH2Cl2 (3 × 75 mL). The combined
organic layers were washed with water (50 mL), dried (Na2SO4) and evaporated in vacuo. The isomers
were separated by flash chromatography on silica gel eluting with EtOAc–Hexane (gradient; 70%–100%
EtOAc) followed by MeOH–EtOAc (1:9) to yield 2a (240 mg, 76%) and 3a (16 mg, 5%).
3.2.2. 2-Amino-6-chloro-9-(cyclohexyl)-9H-purine (2b)
Method A: The title compound was prepared from compound 1a (200 mg, 1.18 mmol), K2CO3 (326 mg,
2.36 mmol) and cyclohexyl tosylate (450 mg, 1.77 mmol) in DMF (15 mL) as described for the synthesis
of compounds 2a above. MeOH–EtOAc (1:19) was used for flash chromatography to yield 2b (98 mg,
33%). Colorless needles; mp 163–165 °C (lit. [55], 165 °C); 1H-NMR (DMSO-d6, 400 MHz) δ 8.23 (s, 1H,
H-8), 6.88 (s, 2H, NH2), 4.28–4.12 (m, 1H, H-1 in c-hex), 2.01–1.75 (m, 7H, c-hex), 1.45–1.17 (m, 3H,
c-hex); 13C-NMR (DMSO-d6, 100 MHz) δ 159.5 (C, C-6), 153.5 (C, C-4), 149.3 (C, C-2), 141.2 (CH,
C-8), 123.5 (C, C-5), 53.5 (CH, C-1 in c-hex), 31.9 (CH2, c-hex), 25.1 (CH2, c-hex), 24.7 (CH2, c-hex);
HREIMS m/z 251.0934 (calcd for C11H14ClN5, 251.0938). Spectral data were in good agreement with
those reported before [55].
Method B: The title compound was prepared from compound 1a (1.00 g, 5.90 mmol), cyclohexanol
[2 × (620 mg, 6.19 mmol)], PPh3 [2 × (1.62 g, 6.19 mmol)] and DIAD [2 × (1.22 mL, 6.19 mmol] in
THF (50 mL) as described for the synthesis of compounds 2a above. After each addition of cyclohexanol
the mixture was subjected to sonication for 20 min using a sonicator probe. EtOAc–Hexane (gradient;
30%–100% EtOAc) was used for flash chromatography to yield 2b (295 mg, 20%).

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3.2.3. 2-Amino-6-chloro-9-(cyclopentyl)-9H-purine (2c) and 2-Amino-6-chloro-7-(cyclopentyl)-7H-
purine (3c)
Method A: The title compounds were prepared from compound 1a (1.00 g, 5.90 mmol), K2CO3 (1.63 g,
11.8 mmol) and bromocyclopentane (0.696 mL, 6.49 mmol) in DMF (50 mL) as described for the
synthesis of compounds 2a and 3a above. MeOH–EtOAc (1:19) was used for flash chromatography to
yield 2c (994 mg, 71%) and 3c (75 mg, 5%).
2c: colorless solid; mp 137–140 °C (lit. [55], 142 °C); 1H-NMR (DMSO-d6, 400 MHz) δ 8.20 (s, 1H,
H-8), 6.86 (s, 2H, NH2), 4.77–4.65 (m, 1H, H-1 in c-pent), 2.16–2.02 (m, 2H, c-pent) 2.00–1.75 (m, 4H,
c-pent), 1.72–1.60 (m, 2H, c-pent); 13C-NMR (DMSO-d6, 100 MHz) δ 159.3 (C, C-2), 153.7 (C, C-4),
149.1 (C, C-6), 141.4 (CH, C-8), 123.5 (C, C-5), 55.1 (CH, C-1 in c-pent), 31.4 (CH2, C-3 and C-4 in
c-pent), 23.2 (CH2,C-2 and C-5 in c-pent); HREIMS m/z 237.0777 (calcd for C10H12ClN5, 237.0781).
Spectral data were in good agreement with those reported before [34,55,56].
3c: colorless solid; mp >230 °C (dec.); 1H-NMR (DMSO-d6, 400 MHz) δ 8.46 (s, 1H, H-8), 6.59 (s, 2H,
NH2), 5.11–5.01 (m, 1H, H-1 in c-pent), 2.24–2.10 (m, 2H, c-pent), 2.02–1.90 (m, 2H, c-pent),
13
1.86–1.62 (m, 4H, c-pent); C-NMR (DMSO-d6, 100 MHz) δ 164.3 (C, C-4), 159.7 (C, C-2), 146.6
(CH, C-8), 142.2 (C, C-6), 115.1 (C, C-5), 58.0 (CH, C-1 in c-pent), 32.6 (CH2, C-3 and C-4 in
c-pent), 23.1 (CH2, C-2 and C-5 in c-pent); HREIMS m/z 237.0776 (calcd for C10H12ClN5, 237.0781).
Spectral data were in good agreement with those reported before [34,55].
Method B: The title compounds were prepared from compound 1a (200 mg, 1.18 mmol), cyclopentanol
[2 × (107 mg, 1.24 mmol)], PPh3 [2 × (325 mg, 1.24 mmol)] and DIAD [2 × (244 µL, 1.24 mmol)] in
THF (10 mL) as described for the synthesis of compounds 2a and 3a above. EtOAc–hexane (gradient;
70%–100% EtOAc) followed by MeOH–EtOAc (1:9) were used for flash chromatography to 2c
(202 mg, 72%) and 3c (6 mg, 6%).
3.2.4. 2-Amino-6-chloro-9-(cyclopent-2-enyl)-9H-purine (2d) and 2-Amino-6-chloro-7-(cyclopent-2-
enyl)-7H-purine (3d)
Method A: The title compound 2d was prepared from compound 1a (200 mg, 1.18 mmol), K2CO3
(490 mg, 3.54 mmol) and 3-bromocyclopentene (0.29 mL, ca. 80% pure, ca. 2.4 mmol) in DMF (20 mL)
as described for the synthesis of compounds 2a and 3a above, except that the reaction time was 24 h.
EtOAc–hexane (gradient; 50%–100% EtOAc) followed by MeOH–EtOAc (1:9) were used for flash
chromatography to yield 2d (49 mg, 18%). Colorless solid; mp 154–154.5 °C (lit., [57] 166.0–166.7 °C);
1H-NMR (DMSO-d6, 400 MHz) δ 7.96 (s, 1H, H-8), 6.88 (s, 2H, NH2), 6.26–6.18 (m, 1H, c-pent),
5.93–5.84 (m, 1H, c-pent), 5.51–5.41 (m, 1H, c-pent), 2.73–2.61 (m, 1H, c-pent), 2.47–2.36 (m, 2H,
c-pent), 2.00–1.87 (m, 1H, c-pent); 13C-NMR (DMSO-d6, 100 MHz) δ 159.6 (C, C-6), 153.6 (C, C-4),
149.3 (C, C-2), 141.1 (CH, C-8), 137.3 (CH, C-2 in c-pent), 128.6 (CH, C-3 in c-pent), 123.6 (C, C-5),
59.4 (CH, C-1 in c-pent), 31.2 (CH2, C-5 in c-pent), 30.4 (CH2, C-4 in c-pent); HREIMS m/z 235.0624
(calcd for C10H10ClN5 235.0625). Spectral data were in good agreement with those reported before [57].

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Method B: The title compound 2d was prepared from compound 1a (340 mg, 2.01 mmol),
cyclopent-2-enol [2 × (0.180 mL, 2.03 mmol)], PPh3 [2 × (531 mg, 2.03 mmol)] and DIAD [2 × (0.442 mL,
2.03 mmol)] in THF (20 mL) as described for the synthesis of compounds 2a and 3a above.
EtOAc–Hexane (gradient; 70%–100% EtOAc) followed by MeOH–EtOAc (1:9) were used for flash
chromatography to yield 2d (187 mg, 40%).
Method C: A solution of compound 1a (100 mg, 0.590 mmol) and NaH (18 mg, 0.77 mmol) in dry
DMSO (5 mL) was stirred at room temperature for 20 min under Ar atmosphere. The mixture was added
to a solution of cyclopent-2-en-1-yl acetate (0.070 mL, 0.77 mmol) and Pd(PPh3)4 (103 mg, 0.0890 mmol)
in dry DMSO (5 mL) and the resulting mixture was stirred at 50 °C for 48 h under Ar and evaporated
in vacuo. The product was purified by flash chromatography as described in Method B to yield 2d
(73 mg, 53%) and 3d (25 mg, 18%).
3d: colorless solid; mp 155–157 °C (dec.); 1H-NMR (DMSO-d6, 400 MHz) δ 8.15 (s, 1H, H-8), 6.61 (s,
2H, NH2), 6.34–6.28 (m, 1H, c-pent), 6.03–5.96 (m, 1H, c-pent), 5.82–5.75 (m, 1H, c-pent) 2.61–2.34
(m, 3H, c-pent) 1.96–1.83 (m, 1H, c-pent); 13C-NMR (DMSO-d6, 100 MHz) δ 164.4 (C, C-4), 159.8 (C,
C-2), 146.2 (CH, C-8), 142.3 (C, C-6), 138.3 (CH, C-2 in c-pent), 127.9 (CH, C-3 in c-pent), 114.8 (C,
C-5), 62.5 (CH, C-1 in c-pent), 32.1 (CH2, C-5 in c-pent), 31.0 (CH2, C-4 in c-pent); HREIMS m/z
235.0631 (calcd for C10H10ClN5, 235.0625). Spectral data were in good agreement with those reported
before [57].
3.2.5. 2-Acetamido-9-(cyclohexylmethyl)-9H-purin-6-yl diphenylcarbamate (2e) and 2-Acetamido-7-
(cyclohexylmethyl)-7H-purin-6-yl diphenylcarbamate (3e)
Method A: The title compounds were prepared from compound 1b (200 mg, 0.515 mmol), K2CO3
(142 mg, 1.03 mmol) and bromomethylcyclohexane (0.144 mL, 1.03 mmol) in DMF (7 mL) as described
for the synthesis of compounds 2a and 3a above. MeOH–CH2Cl2 (1:9) followed by MeOH–CH2Cl2 (1:4)
were used for flash chromatography to yield 2e (111 mg, 45%) and 3e (18 mg, 7%).
1
2e: colorless solid; mp. 192–194 °C; H-NMR (CDCl3, 400 MHz) δ 7.97 (s, 1H, NH), 7.87 (s, 1H,
H-8), 7.47–7.24 (m, 10H, Ph), 3.99 (d, J = 7.0 Hz, 2H, NCH2), 2.58 (s, 3H, CH3), 1.85 (m, 1H, H-1 in
c-hex), 1.78–1.58 (m, 5H, c-hex), 1.30–1.07 (m, 3H, c-hex), 1.07–0.92 (m, 2H, c-hex); 13C-NMR
(CDCl3, 100 MHz) δ 171.1 (C, CONH), 156.2 (C, OCON), 155.4 (C, C-4), 152.2 (C, C-2), 150.6 (C,
C-6), 144.4 (CH, C-8), 141.9 (C, Ph), 129.3 (CH, Ph), 127.2 (br, 2 × CH2, Ph), 120.6 (C, C-5), 50.5
(CH2, NCH2), 38.4 (CH, C-1 in c-hex), 30.8 (CH2, C-3 and C-5 in c-hex), 26.1 (CH2, C-4 in c-hex), 25.6
(CH2, C-2 and C-3 in c-hex), 25.3 (CH3); HRESIMS m/z 485.2311 (calcd for C27H29N6O3 + 1, 485.2301).
3e: colorless oil; 1H-NMR (CDCl3, 400 MHz) δ 8.10 (s, 1H, NH), 7.96 (s, 1H, H-8), 7.42–7.36 (m, 8H,
Ph), 7.33–7.28 (m, 2H, Ph), 3.89 (d, J = 7.2 Hz, 2H, NCH2), 2.63 (s, 3H, CH3), 1.68–1.60 (m, 3H,
c-hex), 1.45–1.35 (m, 2H, c-hex), 1.13–0.98 (m, 3H, c-hex), 0.92–0.72 (m, 3H, c-hex); 13C-NMR
(CDCl3, 100 MHz) δ 172.0 (C, CONH), 164.9 (C-4), 152.2 (C, C-2), 151.9 (C, OCON), 149.5 (C,
C-6), 148.5 (CH, C-8), 141.5 (C, Ph), 129.6 (CH, Ph), 127.6 (br, 2 × CH, Ph), 111.9 (C, C-5), 53.8
(CH2, NCH2), 38.9 (CH, C-1 in c-hex), 30.3 (CH2, C-3 and C-5 in c-hex), 26.0 (CH2,C-4 in c-hex) 25.4
(CH2, C-2 and C-6 in c-hex ), 25.6 (CH3); HRESIMS m/z 485.2313 (calcd for C27H29N6O3 + 1, 485.2301).

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Method B: The title compounds were prepared from compound 1b (200 mg, 0.515 mmol),
cyclohexylmethanol [2 × (62 mg, 0.54 mmol)], PPh3 [2 × (142 mg, 0.540 mmol)] and DIAD [2 × (0.106 mL,
0.540 mmol)] in THF (10 mL) as described for the synthesis of compounds 2a and 3a above.
EtOAc–Hexane (gradient; 70%–100% EtOAc) followed by MeOH–EtOAc (1:9) were used for flash
chromatography to yield 2e (175 mg, 70%) and 3e (7 mg, 3%).
3.2.6. 2-Acetamido-9-(cyclohexyl)-9H-purin-6-yl diphenylcarbamate (2f)
Method A: The title compound was prepared from compound 1b (500 mg, 1.29 mmol), K2CO3 (329 mg,
2.38 mmol) and cyclohexyl tosylate (441 mg, 1.73 mmol) in THF (15 mL) as described for the synthesis
of compounds 2a above. MeOH–EtOAc (1:19) was used for flash chromatography to yield 2f (180 mg,
30%). Off-white solid; mp 189–190 °C; 1H-NMR (DMSO-d6, 300 MHz) δ 10.67 (s, 1H, NH), 8.55 (s,
1H, H-8), 7.54–7.38 (m, 8H, Ph), 7.37–7.25 (m, 2H), 4.46–4.28 (m, 1H, H-1 in c-hex), 2.20 (s, 3H, CH3),
2.06–1.80 (m, 6H, CH in c-hex), 1.76–1.65 (m, 1H, c-hex), 1.51–1.14 (m, 3H, c-hex); 13C-NMR (DMSO-d6,
75 MHz) δ 168.8 (C, CONH), 155.0 (C, OCON), 154.3 (C, C-4), 151.7 (C, C-2), 150.3 (C, C-6), 144.1
(CH, C-8), 141.6 (C, Ph), 129.4 (CH, Ph), 127.1 (CH, Ph), 120.1 (C, C-5), 54.1 (CH, C-1 in c-hex), 31.9
(CH2, C-3 and C-5 in c-hex), 25.1 (CH2, C-2 and C-6 in c-hex), 24.7 (CH2, C-4 in c-hex), 24.6 (CH3);
HREIMS m/z 470.2057 (calcd for C26H26N6O3, 470.2066).
Method B: The title compound was prepared from compound 1b (400 mg, 1.03 mmol), cyclohexanol
[2 × (108 mg, 1.08 mmol)], PPh3 [2 × (284 mg, 1.08 mmol)] and DIAD [2 × (0.213 mL, 1.08 mmol)]
in THF (10 mL) as described for the synthesis of compound 2a above. EtOAc–Hexane (gradient;
30%–100% EtOAc) was used for flash chromatography to yield 2f (107 mg, 22%) as an off-white solid.
3.2.7. 2-Acetamido-9-(cyclopentyl)-9H-purin-6-yl diphenylcarbamate (2g)
Method A: The title compound 2g was prepared from compound 1b (389 mg, 1.00 mmol), K2CO3
(277 mg, 2.00 mmol) and bromocyclopentane (0.120 mL, 1.10 mmol) in DMF (50 mL) as described for
the synthesis of compounds 2a above. MeOH–EtOAc (1:19) was used for flash chromatography to yield
1
2g (238 mg, 52%). Colorless solid; mp 137–140 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.62 (s, 1H,
NH), 8.51 (s, 1H, H-8), 7.53–7.40 (m, 8H, Ph), 7.36–7.27 (m, 2H, Ph), 4.79–4.62 (m, 1H, H-1 in c-pent),
2.21 (s, 3H, CH3), 2.19–2.11 (m, 2H, c-pent), 2.10–1.83 (m, 4H, c-pent), 1.77–1.62 (m, 2H, c-pent);
13C-NMR (DMSO-d6, 100 MHz) δ 168.8 (C, CONH), 155.0 (C, OCON), 154.6 (C, C-4), 151.7 (C,
C-2), 150.2 (C, C-6), 144.4 (CH, C-8), 141.6 (C, Ph), 129.4 (CH, Ph), 127.2 (CH, Ph), 120.3 (C,
C-5), 56.1 (CH, C-1 in c-pent), 31.7 (CH2, C-3 and C-4 in c-pent), 24.5 (CH3), 23.5 (CH2, C-2 and C-5
in c-pent), one Ph signal was hidden; HREIMS m/z 456.1903 (calcd for C25H24N6O3, 456.1910).
Method B: The title compound 2g was prepared from compound 1b (389 mg, 1.00 mmol), cyclopentanol
[2 × (91 mg, 1.1 mmol)], PPh3 [2 × (276 mg, 1.05 mmol)] and DIAD [2 × (0.207 mL, 1.05 mmol)] in
THF (10 mL) as described for the synthesis of compounds 2a above. EtOAc–Hexane (gradient;
70%–100% EtOAc) followed by MeOH–EtOAc (1:9) were used for flash chromatography to yield 2g
(264 mg, 58%).

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3.2.8. 2-Amino-8-bromo-6-chloro-9-(cyclohexylmethyl)-9H-purine (4a)
Method A: Sat. aq. Br2 (50 mL) was added dropwise to a rapidly stirred suspension of 2a (1.50 g,
5.64 mmol) in water (20 mL) over 10 min at ambient temperature. The flask was closed and the reaction
mixture was stirred for 74 h. The flask was left open in the hood until all Br2 was evaporated before the
water was removed in vacuo. The product was purified by flash chromatography on silica gel, eluting with
1
EtOAc–Hexane (1:1) to yield 4a (1.55 g, 79%). Yellow solid; mp 169–170 °C. H-NMR (DMSO-d6,
400 MHz) δ 7.07 (s, 2H, NH2), 3.86 (d, J = 8.0 Hz, 2H, NCH2), 1.96–1.81 (m, 1H, H-1 in c-hex), 1.72–1.44
(m, 5H, c-hex), 1.24–0.92 (m, 5H, c-hex); 13C-NMR (DMSO-d6, 100 MHz) δ 159.7 (C, C-2), 155.0 (C,
C-4), 148.0 (C, C-6), 129.5 (C, C-8), 123.3 (C, C-5), 49.6 (CH2, NCH2), 36.9 (CH, c-hex), 30.0 (CH2,
c-hex), 25.7 (CH2, c-hex), 25.1 (CH2, c-hex); HREIMS m/z 343.0198 (calcd for C12H15BrClN5, 343.0199).
Method B: K2CO3 (231 mg, 1.67 mmol) was added to a stirring solution of compound 10 (207 mg,
0.833 mmol) in dry DMF (15 mL) at ambient temperature under N2. After 20 min, bromomethylcyclohexane
(0.175 mL, 1.25 mmol) was added and the resulting mixture was stirred for 80 h before K2CO3 (115 mg,
0.835 mmol) and bromomethylcyclohexane (0.175 mL, 1.25 mmol) was added and the mixture was
stirred for additional 16 h and evaporated in vacuo. The product was purified by flash chromatography on
silica gel eluting with EtOAc–Hexane (2:3) to yield 4a (98 mg, 34%).
Method C: The title compound was prepared from compound 10 (175 mg, 0.704 mmol),
cyclohexylmethanol [2 × (0.091 mL, 0.74 mmol)], PPh3 [2 × (276 mg, 0.740 mmol)] and DIAD
[2 × (0.146 mL, 0.740 mmol)] in THF (10 mL) as described for the synthesis of compounds 2a
above. EtOAc–Hexane (gradient; 20%–100% EtOAc) was used for flash chromatography to yield 4a
(136 mg, 56%).
3.2.9. 2-Amino-8-bromo-6-chloro-9-(cyclohexyl)-9H-purine (4b)
The title compound was prepared from compound 2b (250 mg, 0.993 mmol) and saturated aqueous
Br2 (12 mL) in water (5 mL) as described for the synthesis of compound 4a above. EtOAc–Hexane (1:1)
was used for flash chromatography to yield 4b (229 mg, 70%). Yellow solid; mp 181–183 °C; 1H-NMR
(DMSO-d6, 600 MHz) δ 6.98 (br s, 2H, NH2), 4.35–4.22 (m, 1H, H-1 in c-hex), 2.46–2.27 (m, 2H, c-hex),
1.92–1.75 (m, 4H, c-hex), 1.74–1.62 (m, 1H, c-hex), 1.45–1.29 (m, 2H, c-hex), 1.28–1.12 (m, 1H,
c-hex); 13C-NMR (DMSO-d6, 150 MHz) δ 159.1 (C, C-2), 154.5 (C, C-4), 148.2 (C, C-6), 128.6 (C, C-8),
123.7 (C, C-5), 57.6 (CH, C-1 in c-hex), 29.7 (CH2, C-3 and C-5 in c-hex), 25.3 (CH2, C-2 and C-6 in
c-hex), 24.6 (CH2, C-4 in c-hex); HRESIMS m/z 330.0131 (calcd for C11H14BrClN5 + 1, 330.0121).
3.2.10. 2-Amino-8-bromo-6-chloro-9-(cyclopentyl)-9H-purine (4c)
The title compound was prepared from compound 2c (880 mg, 3.70 mmol) and sat. aq. Br2 (35 mL)
in water (10 mL) as described for the synthesis of compound 4a above. EtOAc–Hexane (1:1) was used
for flash chromatography to yield 4c (950 mg, 81%).Yellow solid; mp 172–174 °C; 1H-NMR (DMSO-d6,
600 MHz) δ 6.97 (s, 2H, NH2), 4.85–4.77 (m, 1H, H-1 in c-pent), 2.33–2.17 (m, 2H, c-pent), 2.11–1.87
(m, 4H, c-pent), 1.71–1.59 (m, 2H, c-pent); 13C-NMR (DMSO-d6, 150 MHz) δ 159.1 (C, C-2), 154.3 (C,
C-4), 148.2 (C, C-6), 129.3 (C, C-8), 123.9 (C, C-5), 57.7 (CH, C-1 in c-pent), 29.7 (CH2, C-3 and C-4 in
c-pent), 24.4 (CH2, C-2 and C-5 in c-pent); HREIMS m/z 314.9880 (calcd for C10H11BrClN5, 314.9886).

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3.2.11. 2-Amino-8-bromo-6-chloro-9-(cyclopent-2-enyl)-9H-purine (4d)
Method A: A solution of diisopropylamine (0.145 mL, 1.03 mmol) in dry THF (3 mL) was stirred at
−78 °C under Ar. n-BuLi (0.536 mL, 1.00 mmol, 1.87 M in hexane) was added dropwise. After stirring
for 30 min, a solution of compound 2d (118 mg, 0.500 mmol) in THF (1.5 mL) was added. After additional
stirring for 1 h at −78 °C, a solution of CBrCl2CBrCl2 (326 mg, 1.00 mmol) in THF (1.5 mL) was added
dropwise and the resulting mixture was stirred at −78 °C for 1 h, and then 10 min without cooling.
Saturated aqueous NH4Cl (5 mL) was added and the resulting mixture was extracted with EtOAc
(3 × 50 mL). The combined organic extracts were washed with brine (100 mL), dried (MgSO4), and
evaporated in vacuo. The product was purified by flash chromatography on silica gel eluting with
EtOAc–Hexane (1:1) to yield 4d (50 mg, 32%). Buff solid; mp 157–158 °C (dec.); 1H-NMR (DMSO-d6,
600 MHz) δ 6.95 (s, 2H, NH2), 6.15–6.13 (m, 1H, c-pent), 5.74–5.72 (m, 1H, c-pent), 5.69–5.60 (m, 1H,
13
c-pent) 2.90–2.79 (m, 1H, c-pent), 2.48–2.36 (m, 2H, c-pent), 2.22–2.14 (m, 1H, c-pent); C-NMR
(DMSO-d6, 150 MHz) δ 159.3 (C, C-2), 154.4 (C-4), 148.0 (C, C-6), 136.5 (CH, C-3 in c-pent), 128.0
(C, C-8), 127.7 (CH, C-2 in c-pent), 123.5 (C-5), 62.1 (CH, C-1 in c-pent), 32.0 (CH2, C-5 in c-pent),
27.9 (CH2, C-4 in c-pent); HREIMS m/z 312.9734 (calcd for C10H9BrClN5, 312.9730).
Method B: Compound 10 (64 mg, 0.26 mmol) was added to a cooled solution of cyclopent-2-en-1-ol
(44 mg, 0.51 mmol) and PPh3 (135 mg, 0.515 mmol) in anhydrous THF (5 mL) under Ar. The resulting
suspension was treated with diethyl azodicarboxylate (DEAD, 0.080 mL, 0.51 mmol) and the resulting
mixture was stirred at ambient temperature for 1 h and at 70 °C for 15 h. The mixture was cooled, treated
with brine (50 mL), and extracted with CH2Cl2 (3 × 50 mL). The combined organic layer was washed
with water (10 mL), dried (Na2SO4), and evaporated in vacuo. The product was purified by flash
chromatography on silica gel eluting with EtOAc–Hexane (3:7) to yield 4d (34 mg, 42%).
Method C: A solution of compound 10 (110 mg, 0.423 mmol) and NaH (16 mg, 0.67 mmol) in dry
DMF (10 mL) was stirred at ambient temperature for 20 min under Ar. Pd(PPh3)4 (77 mg, 0.067 mmol)
and cyclopent-2-en-1-yl acetate (84 mg, 0.66 mmol) were added, and the resulting mixture was stirred at
55 °C. After three days Pd(PPh3)4 (77 mg, 0.067 mmol) and cyclopent-2-en-1-yl acetate (84 mg, 0.66 mmol)
were added. The reaction mixture was stirred for three more days and evaporated under in vacuo.
The product was purified by flash chromatography on silica gel eluting with EtOAc–Hexane (gradient
50%–100% EtOAc) followed by MeOH–EtOAc (1:9) to yield 4d (41 mg, 29%).
3.2.12. 9-(Cyclohexylmethyl)-8-oxoguanine (5a)
A mixture of compound 4a (263 mg, 0.763 mmol), NaOAc (319 mg, 3.89 mmol), glacial AcOH (9 mL),
and Ac2O (1.5 mL, 17 mmol) was stirred at reflux under N2 for 16 h, before the mixture was cooled and
evaporated in vacuo. The residue was suspended in water (3 mL) and stirred at ambient temperature
while the pH was adjusted to 13 by dropwise addition of 10M NaOH (aq). The resulting solution was
refluxed for 20 min, cooled to 0 °C, and stirred while the pH was brought down to 7 by dropwise addition
of 6M HCl (aqueous). The precipitate was collected and dried in vacuo. The product was purified by
flash chromatography on silica gel eluting with MeOH–CH2Cl2 (1:4) to yield 5a (160 mg, 80%). Pinkish
solid; mp 297–300 °C; 1H-NMR (DMSO-d6, 400 MHz) δ 10.57 (s, 1H, NH), 10.48 (s, 1H, NH), 6.43 (s,
2H, NH2), 3.42 (d, J = 7.4 Hz, 2H, NCH2), 1.85–1.71 (m, 1H, H-1 in c-hex), 1.69–1.48 (m, 5H, c-hex),

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1.19–1.06 (m, 3H, c-hex), 0.98–0.85 (m, 2H, c-hex); 13C-NMR (DMSO-d6, 100 MHz) δ 153.4 (C, C-6),
152.6 (C, C-8), 150.9 (C, C-2), 148.2 (C, C-4), 98.0 (C, C-5), 44.9 (CH2, NCH2), 36.3 (CH, C-1 in c-hex),
30.1 (CH2, C-3 and C-5 in in c-hex), 25.9 (CH2, C-4 in c-hex), 25.1 (CH2, C-2 and C-6 in c-hex);
HREIMS m/z 263.1380 (calcd for C12H17N5O2, 263.1382).
3.2.13. 9-(Cyclohexyl)-8-oxoguanine (5b) and 2-Amino-6-chloro-9-cyclohexyl-7H-purin-8(9H)-one (6b)
The title compounds were prepared from compound 4b (186 mg, 0.563 mmol), NaOAc (231 mg,
2.81 mmol), glacial AcOH (7 mL), and Ac2O (1.20 mL, 12.7 mmol) as described for the synthesis of
compound 5a above, except that the reflux time with NaOH was 4 h. MeOH–EtOAc (1:9) was used for
flash chromatography to yield 5b (106 mg, 76%) and 6b (7 mg, 11%).
5b: colorless solid; mp 367–368 °C; 1H-NMR (DMSO-d6, 400 MHz) δ 10.57 (s, 1H, NH), 10.45 (s, 1H,
NH), 6.37 (s, 2H, NH2), 4.04–3.91 (m, 1H, H-1 in c-hex), 2.28–2.11 (m, 2H, c-hex), 1.86–1.71 (m, 2H,
c-hex), 1.69–1.52 (m, 3H, c-hex), 1.36–1.04 (m, 3H, c-hex); 13C-NMR (DMSO-d6, 100 MHz) δ 152.9
(C, C-6), 151.8 (C, C-8), 150.9 (C, C-2), 147.7 (C, C-4), 98.1 (C, C-5), 51.2 (CH, C-1 in c-hex), 29.5
(CH2, C-3 and C-5 in c-hex), 25.5 (CH2, C-2 and C-6 in c-hex), 24.8 (CH2, C-4 in c-hex); HREIMS m/z
249.1222 (calcd for C11H15N5O2, 249.1226).
6b: yellow solid mp 320–321 °C; 1H-NMR (DMSO-d6, 300 MHz) δ 11.20 (br s, 1H, NH), 6.54 (s, 2H,
NH2), 4.12–3.98 (m, 1H, H-1 in c-hex), 2.27–2.12 (m, 2H, c-hex), 1.88–1.75 (m, 2H, c-hex), 1.73–1.58
(m, 3H, c-hex), 1.39–1.09 (m, 3H, c-hex); 13C-NMR (DMSO-d6, 75 MHz) δ 158.3 (C, C-8), 152.5 (C,
C-4), 152.3 (C, C-2), 135.5 (C, C-6), 109.8 (C, C-5), 51.7 (CH, C-1 in c-hex), 29.1 (CH2, C-3 and C-5
in c-hex), 25.4 (CH2, C-2 and C-6 in c-hex), 24.8 (CH2,C-4 in c-hex); HREIMS m/z 267.0877 (calcd for
C11H14ClN5O, 267.0887).
3.2.14. 9-(Cyclopentyl)-8-oxoguanine (5c) and 2-Amino-6-chloro-9-cyclopentyl-7H-purin-8(9H)-one (6c)
The title compounds were prepared from compound 4c (250 mg, 0.790 mmol), NaOAc (325 mg,
3.96 mmol), glacial AcOH (10 mL), and Ac2O (3.00 mL, 31.6 mmol) as described for the synthesis of
compound 5a above, except that the refluxing time with NaOH was 6 h. MeOH–EtOAc (1:9) was used
for flash chromatography to yield 5c (130 mg, 70%) and 6c (12 mg, 6%).
5c: colorless solid; mp 309–310 °C; 1H-NMR (DMSO-d6, 400 MHz) δ 10.58 (s, 1H, NH), 10.47 (s, 1H,
NH), 6.35 (s, 2H, NH2), 4.46–4.42 (m, 1H, H-1 in c-pent), 2.17–2.01 (m, 2H, c-pent), 1.94–1.73 (m, 4H,
c-pent), 1.63–1.50 (m, 2H, c-pent); 13C-NMR (DMSO-d6, 100 MHz) δ 152.9 (C, C-6), 151.9 (C,
C-8), 150.9 (C, C-2), 147.8 (C, C-4), 98.2 (C, C-5), 51.8 (CH, C-1 in c-pent), 29.0 (CH2, C-2 and C-5 in
c-pent), 24.3 (CH2, C-3 and C-4 in c-pent); HREIMS m/z 235.1067 (calcd for C10H13N5O2, 235.1069).
6c: colorless solid; mp 321–322 °C (dec.); 1H-NMR (DMSO-d6, 400 MHz) δ 11.20 (s, 1H, NH), 6.52 (s,
2H, NH2), 4.70–4.46 (m, 1H, c-pent), 2.20–2.01 (m, 2H, c-pent), 1.98–1.74 (m, 4H, c-pent), 1.68–1.50
(m, 2H, c-pent); 13C-NMR (DMSO-d6, 100 MHz) δ 158.3 (C, C-4), 152.6 (C, C-8), 152.2 (C, C-6), 135.5
(C, C-2), 109.9 (C, C-5), 52.1 (CH, C-1 in c-pent), 28.7 (CH2, C-2 and C-5 in c-pent), 24.3 (CH2, C-3
and C-4 in c-pent); HREIMS m/z 253.0727 (calcd for C10H12ClN5O, 253.0734).

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3.2.15. 9-(Cyclopent-2-enyl)-8-oxoguanine (5d) and 2-Amino-6-chloro-9-(cyclopent-2-enyl)-7H-
purin-8(9H)-one (6d)
The title compounds were prepared from compound 4d (210 mg, 0.668 mmol), NaOAc (274 mg,
3.34 mmol), glacial AcOH (8 mL), and Ac2O (2.78 mL, 29.4 mmol) as described for the synthesis of
compound 5a above, except that the refluxing time with NaOH was 30 h and the heating bath was kept
at 160 °C in the first reaction step. Glacial AcOH was used for the final neutralization and EtOAc
followed by MeOH–EtOAc (1:9) were used for flash chromatography to yield 5d (119 mg, 71%) and
6d (9 mg, 5%).
5d: colorless solid; mp 322–325 °C (dec.); 1H-NMR (DMSO-d6, 400 MHz) δ 10.60 (s, 1H, NH), 10.49
(s, 1H, NH), 6.34 (s, 2H, NH2), 5.99–5.96 (m, 1H, H-3 in c-pent), 5.62–5.59 (m, 1H, H-2 in c-pent),
5.30–5.21 (m, 1H, H-1 in c-pent), 2.79–2.63 (m, 1H, H-5a in c-pent), 2.40–2.26 (m, 1H, H-5b in c-pent),
2.24–2.13 (m, 1H, H-4a in c-pent), 2.13–2.03 (m, 1H, H-4b in c-pent); 13C-NMR (DMSO-d6, 100 MHz)
δ 153.0 (C, C-6), 151.8 (C, C-8), 151.0 (C, C-2), 147.7 (C, C-4), 134.5 (CH, C-3 in c-pent), 129.0 (CH,
C-2 in c-pent), 98.3 (C, C-5), 56.8 (CH, C-1 in c-pent), 31.8 (CH2, C-4 in c-pent), 27.4 (CH2, C-5 in
c-pent); HREIMS m/z 233.0914 (calcd for C10H11N5O2, 233.0913).
6d: yellow solid; mp 310–310.5 °C; 1H-NMR (DMSO-d6, 300 MHz) δ 11.18 (s, 1H, NH), 6.48 (s, 2H,
NH2), 6.06–6.02 (m, 1H, H-3 in c-pent), 5.65–5.61 (m, 1H, H-2 in c-pent), 5.37–5.27 (m, 1H, H-1 in
13
c-pent), 2.84–2.69 (m, 1H, H-5a in c-pent), 2.43–2.05 (m, 3H, c-pent); C-NMR (DMSO-d6, 75 MHz)
δ 158.3 (C, C-8), 152.4 (C, C-4), 152.1 (C, C-2), 135.4 (C, C-6), 135.3 (CH, C-3 in c-pent), 128.1 (CH,
C-2 in c-pent), 109.9 (C, C-5), 57.2 (CH, C-1 in c-pent), 31.8 (CH2, C-5 in c-pent), 27.0 (CH2, C-4 in
c-pent); HREIMS m/z 251.0568 (calcd for C10H10ClN5O, 251.0574).
3.2.16. N-[9-(Cyclohexylmethyl)-6-oxo-6,9-dihydro-1H-purin-2-yl]acetamide (7)
Method A: Br2 (33 mg, 0.21 mmol) was added slowly to a stirred solution of compound 2e (20 mg,
0.41 mmol) in CHCl3 (4 mL) and the resulting mixture was stirred for 6 h at ambient temperature. The
reaction mixture was evaporated to dryness and the product was purified by flash chromatography on
silica gel eluting with MeOH–EtOAc (1:19) to yield 7 (10 mg, 84%). Off-white solid; mp 271–273 °C
(dec.); 1H-NMR (DMSO-d6, 400 MHz) δ 12.01 (s, 1H, N2H) 11.63 (s, 1H, NH), 7.95 (s, 1H, H-8), 3.90
(d, J = 7.4 Hz, 2H, NCH2), 2.17 (s, 3H, CH3), 1.88–1.74 (m, 1H, c-hex), 1.70–1.54 (m, 3H, c-hex),
1.53–1.44 (m, 2H, c-hex), 1.21–1.07 (m, 3H, c-hex), 1.01–0.87 (m, 2H, c-hex); 13C-NMR (DMSO-d6,
100 MHz) δ 173.5 (C, CON2), 154.9 (C, C-6), 148.8 (C, C-4), 147.6 (C, C-2), 140.2 (CH, C-8), 120.0
(C, C-5), 48.9 (CH2, NCH2), 37.4 (CH, C-1 in c-hex), 29.9 (CH2, C-3 and C-5 in c-hex), 25.8 (CH2,
C-4 in c-hex), 25.0 (CH2, C-2 and C-6 in c-hex), 23.8 (CH3); HREIMS m/z 289.1534 (calcd for
C14H19N5O2, 289.1539).
Method B: The title compound was prepared from compound 2e (20 mg, 0.41 mmol), diisopropylamine
(0.012 mL, 0.83 mmol), n-BuLi (0.060 mL, 0.83 mmol, 1.4 M in hexane) and CBrCl2CBrCl2 (27 mg,
0.83 mmol) in THF (tot. vol. 3 mL) as described for the synthesis of compound 4d above, except that
the reaction was stirred at −78 °C for 2 h after the addition of CBrCl2CBrCl2. The product was purified
by flash chromatography as described above to yield 7 (7 mg, 59%).

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3.2.17. 8-Bromo-6-chloro-N,9-bis(tetrahydro-2H-pyran-2-yl)-9H-purin-2-amine (9)
The title compound was prepared from compound 8 (1.00 g, 2.96 mmol), diisopropylamine (0.84 mL,
5.9 mmol), n-BuLi (4.23 mL, 5.20 mmol, 1.4 M in hexane), and CBrCl2CBrCl2 (1.93 g, 5.92 mmol) in
THF (tot. vol. 30 mL) as described for the synthesis of compound 4d above, except that the reaction was
stirred at −78 °C for 2 h after the addition of CBrCl2CBrCl2. EtOAc–Hexane (1:1) was used for flash
chromatography to yield 9 (987 mg, 80%). Colorless solid; mp 145–147 °C (dec.); 1H-NMR (DMSO-d6,
300 MHz) δ 8.23 (s, 1H, NH), 5.52 (dd, J = 11.0, 2.4 Hz, 1H, CH in THP), 5.13–5.02 (m, 1H, CH in THP),
4.10–3.97 (m, 1H, OCH2 in THP), 3.89–3.77 (m, 1H, OCH2 in THP), 3.69–3.56 (m, 1H, OCH2 in THP),
3.49–3.39 (m, 1H, OCH2 in THP), 3.14–2.90 (m, 1H, THP) 2.06–1.29 (m, 11H, CH2 in THP); 13C-NMR
(DMSO-d6, 75 MHz) δ 157.2 (C, C-2), 154.2 (C, C-8), 148.3 (C, C-6), 129.5 (C, C-4), 124.3 (C, C-5),
84.4 (CH, N9-THP), 80.2 (CH, THP), 68.0 (CH2, OCH2 in THP), 65.7 (CH2, OCH2 in THP), 30.1, 27.6,
24.9, 24.5, 22.6 and 22.5 (all CH2, THP); HREIMS m/z 415.0417 (calcd for C15H19BrClN5O2, 415.0411).
3.2.18. 2-Amino-8-bromo-6-chloro-1H-purine (10)
A mixture compound 9 (150 mg, 0.360 mmol), 96% EtOH (10 mL) and 9.6 M HCl (0.5 mL), was
stirred at ambient temperature for 30 min and neutralized by the addition of solid KHCO3. The resulting
mixture was evaporated in vacuo and the product was isolated by flash chromatography on silica gel
eluting with MeOH–CHCl3 (1:50:) to yield 10 (80 mg, 90%) as a yellow solid; mp >300 °C (dec.).
1H-NMR (DMSO-d6, 400 MHz) δ 13.65 (s, 1H, NH), 6.88 (s, 2H, NH2); 13C-NMR (DMSO-d6, 100 MHz)
δ 159.7, 156.2, 147.1, 126.5, 124.0; HREIMS m/z 246.9257 (calcd for C5H3BrClN5, 246.9260). Spectral
data were in good agreement with those reported before [48].
3.3. DNA Glycosylase Activity Assay
The enzyme OGG1 (residues12–327) was diluted to the desired concentration (60 pM) using a protein
dilution buffer (15% glycerol, 1 mM EDTA, 25 mM HEPES pH 7.9, 1 mM DTT, 0.1 μg/μL BSA).
Enzyme, compound 5 or 6 (0.2 mM), and 5′-32P end-labeled duplex DNA containing an 8-oxo-G/C base
pair were mixed in a 10 μL reaction volume of 50 mM MOPS pH 7.5, 1 mM EDTA, 5% glycerol, and
1 mM DTT. The sequence of the damaged strand in the DNA substrate used is 5′-GCATGCCTGCA
CGG-8oxoG-CATGGCCAGATCCCCGGGTACCGAG-3′, which was annealed with a complementary
strand containing a C opposite 8oxoG. The reactions were incubated for 10 min at 37 °C, followed by
addition of 2.5 μL 0.5 M NaOH and incubation for 20 min at 70 °C, in order to stop the reaction and
ensure complete strand cleavage. Then 0.5 M HCl/0.25 M MOPS pH 7.5 (2.5 μL) was added to each sample
to neutralize the pH. Formamide DNA loading buffer (15 μL) was added to the reaction mixtures and the
samples were incubated at 95 °C for 5 min to denature the DNA. The reaction products were analyzed
on 20% denaturing urea gels. The gels were transferred to 3M paper and dried at 80 °C for 45 min. The
dry gels were placed in a storage phosphor screen overnight, and subsequently scanned on a Typhoon
9410 Variable Mode Image. ImageQuant TL Version 2003.02 (Amersham Biosciences, Piscataway, NJ,
USA) was used to analyze the results. For human NTH1, the same procedure was followed, except that
the DNA substrate contained a 5-hydroxyuracil/G base pair instead of the 8oxoG/C pair in the OGG1

Molecules 2015, 20
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substrate. The concentration of NTH1 was 3 nM to make sure the activity in the assay was within the
linear range. Compounds were screened at 0.5 mM concentration.
4. Conclusions
Synthetic routes to 8-oxoguanines have been examined. The best reaction sequence from chloroguanine
1a to the target compounds was found to be N-9 alkylation, C-8 bromination, and finally simultaneous
hydrolysis of both halides. Bromination before N-alkylation should only be considered in cases where
the N-substituent is not compatible with bromination conditions, since a bromide in the purine 8-position
lowers the reactivity in N-alkylations. In most cases, alkylation with an alkyl halide in the presence of a
base compared favorably to reactions under Mitsunobu conditions. 2-Amino-6-chloropurine (1a) turned
out to be a superior guanine precursor compared to the O-carbamoylguanine 1b. The latter did not result
in improved N-9 selectivity in the alkylation and was not compatible with standard reaction conditions
for C-8 bromination.
Enzymatic assays show that partly hydrolyzed 6-chloro-8-oxopurines 6 are somewhat better OGG1
inhibitors than the 8-oxoguanines 5. However, an inhibitory effect was only observed when using at least
0.2 mM concentration of the compounds, suggesting that the R-group should be extended even further
to make more interactions with the enzyme’s substrate recognition pocket. Further, testing of the same
compounds at a 2.5-fold higher concentration against human NTH1, which is a structural homolog of
OGG1, showed that the synthesized compounds do not inhibit NTH1 at 0.5 mM, except possibly for
a weak effect for compound 6b. To develop these compounds into more potent inhibitors of OGG1,
one possibility is to try compounds with more ribose-like R-groups. In the present study, the R-group
contains a cyclic hydrocarbon only, and it would also be interesting to replace this with carbocyclic
2′-deoxyribose derivatives, as in antiviral drugs like abacavir and entecavir. In these nucleoside analog
drugs, the R-group is not particularly larger than the R-group in our study, but it contains 5′ and/or 3′
hydroxyl groups. Since the structure of the OGG1 enzyme is known [7], molecular modeling will be
included in the search for more potent OGG1 inhibitors in the future.
Supplementary Material
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/09/15944/s1.
Acknowledgments
The Molecular Life Science program at the University of Oslo MLSUiO is gratefully acknowledged
for a grant to Tushar R. Mahajan and the Research Council of Norway (RCN) for partial financing of
the Bruker Avance instruments used in this study. Bjørn Dalhus is supported by the South-Eastern
Norway Regional Health Authority (Grant No. 2014034) and Mari Eknes Ytre-Arne is supported by
RCN (project No. 228563). Support from the Anders Jahres Foundation for Medical Research to BD is
also acknowledged.

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Author Contributions
LLG and BD designed the research. TRM performed the synthetic organic chemistry, and MEYA
and PSA the biological experiments. All authors contributed to writing the paper and read and approved
the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Chabner, B.A.; Roberts, T.G., Jr. Chemotherapy and the war on cancer. Nat. Rev. Cancer 2005, 5,
65–72.
2. Madhusudan, S.; Middleton, M.R. The emerging role of DNA repair proteins as predictive,
prognostic and therapeutic targets in cancer. Cancer Treat. Rev. 2005, 31, 603–617.
3. Helleday, T.; Petermann, E.; Lundin, C.; Hodgson, B.; Sharma, R.A. DNA repair pathways as targets
for cancer therapy. Nat. Rev. Cancer 2008, 8, 193–204.
4. Plummer, R. Perspective on the pipeline of drugs being developed with modulation of DNA damage
as a target. Clin. Cancer Res. 2010, 16, 4527–4531.
5. Pallis, A.G.; Karamouzis, M.W. DNA repair pathways and their implication in cancer treatment.
Cancer Metast. Rev. 2010, 29, 677–685.
6. See for instance: Dalhus, B.; Lærdahl, J.K.; Backe, P.H.; Bjørås, M. DNA base repair–recognition
and initiation of catalysis. FEMS Microbiol. Rev. 2009, 33, 1044–1078.
7. Bruner, S.D.; Norman, D.P.G.; Verdine, G.L. Structural basis for recognition and repair of the
endogenous mutagen 8-oxoguanine in DNA. Nature 2000, 403, 859–866.
8. Siah, H.-S.M.; Gundersen, L.-L.; Gørbitz, C.H. NMR and X-ray structural studies on 3-benzyl-8-
bromoadenine. J. Heterocycl. Chem. 2011, 48, 1375–1378.
9. Siah, H.-S.M.; Gundersen, L.-L. Synthetic strategies to 9-substituted 8-oxoadenines. Synth. Commun.
2013, 43, 1469–1476.
10. Perini, F.; Tieckelmann, H. Conversion of ureidomalonates and 5-carbalkoxyhydantoins to
5-ureido-4,6-pyrimidinediones. J. Org. Chem. 1970, 35, 812–816.
11. Brown, R.; Joseph, M.; Leigh, T.; Swain, M.L. Synthesis and reactions of 7,8-dihydro-8-methylpterin
and 9-methylguanine 7-oxide. J. Chem. Soc. Perkin 1977, 1, 1003–1009.
12. Müller, H.; Carell, T.A. Carbocyclic analog of the oxidatively generated DNA lesion
spiroiminodihydantoin. Eur. J. Org. Chem. 2007, 1438–1445.
13. Robins, M.J.; Hatfield, P.W.; Balzarini, J.; de Clercq, E. Nucleic acid related compounds. 47. Synthesis
and biological activities of pyrimidine and purine “acyclic” nucleoside analogs. J. Med. Chem. 1984,
27, 1486–1492.
14. Michael, M.A.; Cottam, H.B.; Smee, D.F.; Robins, R.K.; Kini, G.D. Alkylpurines as immunopotentiating
agents. Synthesis and antiviral activity of certain alkylguanines. J. Med. Chem. 1993, 36, 3431–3437.

Molecules 2015, 20
15963
15. Zheng, Q.-H.; Wang, J.-Q.; Fei, X.; Hutchins, G.D. Synthesis of 8-methoxypenciclovir and
8-methoxyganciclovir through methyl triflate, a new potential approach to label penciclovir and
ganciclovir with carbon-11. Synthesis 2003, 2785–2794.
16. DeClue, M.S.; Monnard, P.-A.; Bailey, J.A.; Maurer, S.E.; Collins, G.E.; Ziock, H.-J.; Rasmussen, S.;
Boncella, J.M. Nucleobase mediated, photocatalytic vesicle formation from an ester precursor.
J. Am. Chem. Soc. 2009, 131, 931–933.
17. Kaiya, T.; Ohta, M.; Kohda, K. Electrophilic amination of imidazole moieties of 9-ethylguanine
and 1-methylbenzimidazole derivatives and reactivities of N-aminated products. Tetrahedron 1993,
49, 8795–8804.
18. Werbovetz, K.A.; Macdonald, T.L. On the mechanism of arylamine-DNA adduct formation.
Bioorg. Med. Chem. Lett. 1994, 4, 2323–2326.
19. Kaiya, T.; Fujiwara, T.; Kohda, K. Syntheses and properties of 1-methyl-3-phenylaminobenzimidazolium
salts, models of DNA adducts of N7-arylaminodeoxyguanosinium salt. Chem. Res. Toxicol. 2000,
13, 993–1001.
20. Ferenc, G.; Padar, P.; Szolomajer, J.; Kovacs, L. N-alkylated guanine derivatives. Curr. Org. Chem.
2009, 13, 1085–1135.
21. Kjellberg, J.; Liljenberg, M.; Johansson, N.G. Regioselective alkylation of 6-(β-methoxyethoxy)
guanine to give the 9-alkylguanine derivative. Tetrahedron Lett. 1986, 27, 877–880.
22. Kjellberg, J.; Hagberg, C.E.; Malm, A.; Noren, J.O.; Johansson, N.G. Studies on the Alkylation of
Guanine. 2. The synthesis of acyclic guanosine analogs via the precursor 7-methyl-10-oxo-9,10-
dihydropyrimido[1,2-a]purine. Acta Chem. Scand. B 1986, 40, 310–312.
23. Kjellberg, J.; Johansson, N.G. Regioselective alkylation of guanine via diacyloxyglyoxal-
N2-acetylguanine adduct to obtain 7-alkylguanine derivatives. Studies on alkylation of guanine I.
J. Heterocycl. Chem. 1986, 23, 625–627.
24. Kjellberg, J.; Johansson, N.G. Studies on the alkylation of derivatives of guanines.
Nucleosides Nucleotides 1989, 8, 225–256.
25. Geen, G.R.; Kincey, P.M.; Choudary, B.M. Regiospecific Michael additions with 2-aminopurines.
Tetrahedron Lett. 1992, 33, 4609–4612.
26. Bisacchi, G.S.; Singh, J.; Godfrey, J.D., Jr.; Kissick, T.P.; Mitt, T.; Malley, M.F.; di Marco, J.D.;
Gougoutas, J.Z.; Mueller, R.H.; Zahler, R. Regioselective coupling of tetraalkylammonium salts of
6-iodo-2-aminopurine to a cyclobutyl triflate: Efficient preparation of homochiral BMS-180,194, a
potent antiviral carbocyclic nucleoside. J. Org. Chem. 1995, 60, 2902–2905.
27. Geen, G.R.; Kincey, P.M.; Spoors, P.G. Regioselective alkylation of guanines using
2-acetoxytetrahydrofurans. Tetrahedron Lett. 2001, 42, 1781–1784.
28. Zou, R.; Robbins, M.J. High-yield regioselective synthesis of 9-glycosyl guanine nucleosides and
analogues via coupling with 2-N-acetyl-6-O-diphenylcarbamoylguanine. Can. J. Chem. 1987, 65,
1436–1437.
29. Dalpozzo, R.; de Nino, A.; Maiuolo, L.; Procopio, A.; de Munno, G.; Sindona, G. 9-Vinylguanine:
an easy access to aza-analogs of 2′,3′-dideoxyguanosine. Tetrahedron 2001, 57, 4035–4038.
30. Hocek, M.; Holy, A. A facile synthesis of 6-cyanopurine bases. Collect. Czech. Chem. Commun.
1995, 60, 1386–1389.

Molecules 2015, 20
15964
31. Langli, G.; Gundersen, L.-L.; Rise, F. Regiochemistry in Stille couplings of 2,6-dihalopurines.
Tetrahedron 1996, 52, 5625–5638.
32. Brændvang, M.; Gundersen, L.-L. Synthesis, biological activity and SAR of antimycobacterial 2-
and 8-substituted 6-(2-furyl)-9-(p-methoxybenzyl)purines. Bioorg. Med. Chem. 2007, 15, 7144–7165.
33. Toyota, A.; Katagiri, N.; Kaneko, C. Mitsunobu reactions for the synthesis of carbocyclic analogues
of nucleosides: Examination of the regioselectivity. Synth. Commun. 1993, 23, 1295–1305.
34. Toyota, A.; Katagiri, N.; Kaneko, C. Synthesis of nucleosides and related compounds. 31. The alkylation
of 2-amino-6-chloropurine with alcohols by Mitsunobu reaction for a synthesis of carbocyclic
guanosine analogs. Heterocycles 1993, 36, 1625–1630.
35. Maruyama, T.; Kozai, S.; Uchida, M. Synthesis of N-aryl uracils and hypoxanthines and their biological
properties. Nucleosides Nucleotides 1999, 18, 661–671.
36. Lu, W.; Sengupta, S.; Peterson, J.L.; Akhmedow, N.G.; Shi, X. Mitsunobu coupling of nucleobases
and alcohols: An efficient, practical synthesis for novel nonsugar carbon nucleosides. J. Org. Chem.
2007, 72, 5012–5015.
37. Trost, B.M.; Madsen, R.; Guile, S.G.; Brown, B. Palladium-catalyzed enantioselective synthesis of
carbanucleosides. J. Am. Chem. Soc. 2000, 122, 5947–5956.
38. Choo, H.; Chong, Y.; Chu, C.K. Solid phase synthesis of carbocyclic l-2′-deoxynucleosides.
Org. Lett. 2001, 3, 1471–1473.
39. Guillarme, S.; Legoupy, S.; Aubertin, A.-M.; Olicard, C.; Bourgougnon, N.; Huet, F. Rapid access
to acyclic nucleosides via conjugate addition. Tetrahedron 2003, 59, 2177–2184.
40. Velcicky, J.; Lanver, A.; Lex, J.; Prokop, A.; Wieder, T.; Schmalz, H.-G. Transition-metal-mediated
synthesis of novel carbocyclic nucleoside analogues with antitumoral activity. Chem. Eur. J. 2004,
10, 5087–5110.
41. Pautus, S.; Sehr, P.; Lewis, J.; Fortune, A.; Wolkerstorfer, A.; Szolar, O.; Guilligay, D.; Lunardi, T.;
Decout, J.-L.; Cusack, S. New 7-methylguanine derivatives targeting the influenza polymerase PB2
Cap-binding domain. J. Med. Chem. 2013, 56, 8915–8930.
42. Roberts, J.D.; Chambers, V.C. Small-ring compounds. VIII. Some nucleophilic displacement reactions
of cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl p-toluenesulfonates and halides. J. Am.
Chem. Soc. 1951, 73, 5034–5040.
43. For a review of Pd-catalyzed N-allylation of purines, see for instance: Gundersen, L.-L. Metal-mediated
C–C and C–N bond formation in the synthesis of bioactive purines. Targets Heterocycl. Syst. 2008,
12, 85–119.
44. Kobayashi, S.; Kawamoto, T.; Uehara, S.; Fukuyama, T.; Ryu, I. Black-light-induced radical/ionic
hydroxymethylation of alkyl iodides with atmospheric CO in the presence of tetrabutylammonium
borohydride. Org. Lett. 2010, 12, 1548–1551.
45. Gamadeku, T.; Gundersen, L.-L. Synthesis of 8-bromo-N-benzylpurines via 8-lithiated purines;
Scopes and Limitations. Synth. Commun. 2010, 40, 2723–2735.
46. Marzouk, V.H.R.; Hennum, M.; Gundersen, L.-L. Efficient synthesis of cytotoxic pyrido[1,2-e]
purines from purines employing direct C-allylation and RCM-oxidation as key-steps. Tetrahedron Lett.
2013, 54, 3437–3439.
47. Ikehara, M.; Tada, H.; Muneyama, K. Synthesis of 8-hydroxypurine nucleosides. Chem. Pharm. Bull.
1965, 13, 1140–1142.

Molecules 2015, 20
15965
48. Jang, M.-Y.; Lin, Y.; de Jonghe, S.; Gao, L.-J.; Vanderhoydonck, B.; Froeyen, M.; Rozenski, J.;
Herman, J.; Louat, T.; van Belle, K.; et al. Discovery of 7-N-piperazinylthiazolo[5,4-d]pyrimidine
analogues as a novel class of immunosuppressive agents with in vivo biological activity. J. Med. Chem.
2011, 54, 655–668.
49. Hagiya, K.; Muramoto, N.; Misaki, T.; Sugimura, T. DMEAD: A new dialkyl azodicarboxylate for
the Mitsunobu reaction. Tetrahedron 2009, 65, 6109–6114.
50. Fromme, J.C.; Bruner, S.D.; Yang, W.; Karplus, M.; Verdine, G.L. Product-assisted catalysis in
base-excision DNA repair. Nat. Struct. Biol. 2003, 10, 204–211.
51. Winstein, S.; Grunwald, E.; Buckles, R.E.; Hanso, C. The role of neighboring groups in replacement
reactions. XI. Some reactivities involving neighboring groups. J. Am. Chem. Soc. 1948, 70, 816–821.
52. Forkel, N.V.; Henderson, D.A.; Fuchter, M.J. Lanthanide replacement in organic synthesis: Luche-type
reduction of α,β-unsaturated ketones in the presence of calcium triflate. Green Chem. 2012, 14,
2129–2132.
53. Jacquet, O.; Bergholz, T.; Magnier-Bouvier, C.; Mellah, M.; Guillot, R.; Fiaud, J.-C. Palladium-catalyzed
and samarium-promoted coupling of stereochemically-biased allylic acetates with carbonyl compounds.
Tetrahedron 2010, 66, 222–226.
54. Kim, K.; McComas, W. Chemoselective high-throughput purification mediated by solid-supported
reagents: Its application to the first 6,9-disubstituted purine library synthesis. Comb. Chem. High
Throughput Screen. 2000, 3, 125–129.
55. Legraverend, M.; Ludwig, O.; Bisagni, E.; Leclerc, S.; Meijer, L.; Giocanti, N.; Sadri, R.; Favaudon, V.
Synthesis and in vitro evaluation of novel 2,6,9-trisubstituted purines acting as cyclin-dependent
kinase inhibitors. Bioorg. Med. Chem. 1999, 7, 1281–1293.
56. Lukin, K.A.; Yang, C.X.; Bellettini, J.R.; Narayanan, B.A. New purine derivatives as efficient
preparation of nucleoside analogs via alkylation. Nucleosides Nucleotides Nucleic Acids 2000, 19,
815–825.
57. Gültekin, Z. Palladium-catalyzed synthesis of 9-(2-cyclopentenyl)guanine. Asian J. Chem. 2006,
18, 1462–1466.
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