New (1-deaza)purine derivatives via efficient C-2 nitration of the (1-deaza)purine ring

Article abstract of DOI:10.1081/NCN-200027566

Nitration of substituted (1-deaza)purines using a mixture of tetrabutylammonium nitrate (TBAN) and trifluoracetic acid anhydride (TFAA) was applied to prepare nitrosubstituted (1-deaza)purines at low temperature. The nitro group influences the system twofold: 1) it activates other substituents towards nucleophilic aromatic substitution and 2) it can be substituted itself leading to a variety of di-substituted (1-deaza)purines, also via solid phase syntheses. Several of the molecules obtained were studied for their antiprotozoal activity and for interactions with the different human adenosine receptors.

Full text of DOI:10.1081/NCN-200027566

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New (1‐Deaza)Purine Derivatives via Efficient C‐2
Nitration of the (1‐Deaza)Purine Ring
M. J. Wanner a , B. Rodenko a , M. Koch a & G. J. Koomen a
a Bioorganic Chemistry, Institute of Molecular Chemistry, University of Amsterdam, Nieuwe
Achtergracht 129, Amsterdam, 1018 WS, The Netherlands
Published online: 17 Aug 2006.
To cite this article: M. J. Wanner , B. Rodenko , M. Koch & G. J. Koomen (2004) New (1‐Deaza)Purine Derivatives via Efficient
C‐2 Nitration of the (1‐Deaza)Purine Ring, Nucleosides, Nucleotides and Nucleic Acids, 23:8-9, 1313-1320, DOI: 10.1081/
NCN-200027566
To link to this article: http://dx.doi.org/10.1081/NCN-200027566
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, Nos. 8 & 9, pp. 1313–1320, 2004
New (1-Deaza)Purine Derivatives via Efficient C-2 Nitration
of the (1-Deaza)Purine Ring
*
M. J. Wanner, B. Rodenko, M. Koch, and G. J. Koomen
Bioorganic Chemistry, Institute of Molecular Chemistry, University of Amsterdam,
Amsterdam, The Netherlands
ABSTRACT
Nitration of substituted (1-deaza)purines using a mixture of tetrabutylammonium
nitrate (TBAN) and trifluoracetic acid anhydride (TFAA) was applied to prepare
nitrosubstituted (1-deaza)purines at low temperature. The nitro group influences the
system twofold: 1) it activates other substituents towards nucleophilic aromatic
substitution and 2) it can be substituted itself leading to a variety of di-substituted
(1-deaza)purines, also via solid phase syntheses. Several of the molecules obtained
were studied for their antiprotozoal activity and for interactions with the different
human adenosine receptors.
Key Words: Antiprotozoal activity; Temozolomide.
INTRODUCTION
Nucleoside and nucleotide analogues have been of interest since many years.
Initially mainly as antiviral and anticancer agents, which has resulted in development of
many clinically useful drugs. In view of the instability of adenosine and its many
*
Correspondence: G. J. Koomen, Bioorganic Chemistry, Institute of Molecular Chemistry,
University of Amsterdam, Nieuwe Achtergracht 129, 1018 WS Amsterdam, The Netherlands;
E-mail: gjk@science.uva.nl.
1313
DOI: 10.1081/NCN-200027566
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

1314
Wanner et al.
Scheme 1. Nitration of subsituted purines.
biological functions, interest in inhibitors of the metabolising enzyme adenosine
deaminase is also still very lively. In addition to that, the increasing knowledge of
describing processes in the CNS in molecular terms has stimulated the research in the
field of the different adenosine receptors. For the development of selective agonists and
(anta)gonists of the human adenosine receptors and for basic receptor studies, many
more adenosine analogues with well defined structures are required and consequently
also new synthetic approaches, in particular at solid phase should be developed.
A couple of years ago, a new selective nitration reaction of substituted purines and
deazapurines at the 2-position was developed in our lab.[1,2] The reaction, using a
mixture of tetrabutyl ammonium nitrate (TBAN) and trifluoroacetic acid anhydride
(TFAA) is simple and proceeds at low temperature if the purine system is substituted at
the six position (purine numbering) with a functional group without acidic protons and
containing a free electron pair (Scheme 1).
This nitration reaction turned out to be a combination of an electrophilic and a
1
radical process, as could be established using H, 13C and 15N NMR spectra.
For the studies towards the structure elucidation of possible intermediates of the
reaction via 15N NMR, the corresponding fully labeled 15N TBAN was prepared from
TBACl and Na15NO3. Recently, the proposed mechanism was unequivocally estab-
lished in our lab by observing CIDNP effects during the reaction at low temperature
(Scheme 2).
After initial nitration at N-7 of N-9 protected 6-chloropurine, addition of
trifluoroacetate at C-8 takes place, leading to an intermediate which can be observed
in NMR at low temperature. Slowly warming up triggers a radical rearrangement of the
nitro group to carbon-2 under restoration of aromaticity via elimination of trifluoro-
acetic acid.
The reaction could also be carried out with 6-nitro-1-deazapurine[3] leading to 2,6-
dinitro-1-deazapurine, which could also be easily substituted to difunctionalised
1-deaza systems (Scheme 3). The nitration of 1-deaza purines was improved con-
siderably via protection of N-9 with a BOC group.
The resulting nitro groups introduced in the (deaza) purine systems could be easily
substituted with different nucleophiles in aromatic nucleophilic substitution reactions.
Scheme 2. Intermediate in the radical nitration.

New (1-Deaza)Purine Derivatives
1315
Scheme 3. Synthesis of 1-deazapurines.
In the purine series even aromatic amines showed sufficient nucleophilicity. An
additional advantage of introducing the nitro group is the enhancement of the reactivity
of other functional groups toward nucleophilic substitution. For instance, a 6-chloro
group in a purine ring system can be replaced in presence of a 2-nitro substituent at
temperatures below 0°C.
This nitration reaction could also be carried out on solid support at room
temperature (Scheme 4). The protected ribonucleosides were coupled to carboxystyr-
ene. As expected from a mechanistic point of view, nitration of the polystyrene support
Scheme 4. Solid phase synthesis of disubstituted adenosine analogues.

1316
Wanner et al.
Scheme 5. Cyclophane type analogues.
did not take place under the reaction conditions. After cleavage, the products were
purified via semi preparative HPLC. The procedure gave access to a variety of
disubstituted purine analogues.[4]
Thus, both in solution as well as on solid support, replacement of both
6-substituents and 2-nitro groups resulted in the preparation of a wide variety of
Table 1. Antiprotozoal activity-IC50’s in mM.

New (1-Deaza)Purine Derivatives
1317
disubstituted purine and 1-deazapurine systems both for biological screening and for
studies of the interactions with adenosine receptors.
To block the syn/anti rotations around the glycosidic bond, syntheses of 5’-2
cyclonucleosides were also realised (Scheme 5).
Since Plasmodium Falciparum, the causative parasite of Malaria is not capable to
carry out its own de novo purine syntheses, it is strongly dependent on supply of
nucleosides and nucleobases from its host. Via its transportmechanism, uncommon
and reactive nucleoside analogues can be administered to the parasite. Many of our
di- and trisubstituted analogues were tested in cooperation with R. Brun, Tropical
Institute, Basel on Plasmodium falciparum (P.f.) and Trypanosoma Brucei (T.b.r.) (see
Table 1).
Reduction of the nitro group in 2-nitroadenosine with H2/Pd, followed by oxidation
of the resulting hydroxyaminogroup produced nitrosoadenosine, which could also be
used for a variety of structural modifications, partly via hetero Diels-Alder reactions.
The possibility of reductive ringopening of the N–O bridge led to a new series of
substituted adenosine derivatives[5] (Scheme 6).
The synthetic approach developed for the anticancer agent temozolomide[6] could
be used for the synthesis of a series of substituted carbamoyl triazenes (Scheme 7).
In cooperation with the group of IJzerman (University of Leiden) these compounds
were tested for their activity on different Adenosine receptors. Some of the analogues,
like TCPA showed remarkable selective agonist activity on the human A1 receptor[7]
(see Table 2).
The clinically active cytostatic Temozolomide exerts is activity via in vivo
production of the methyldiazonium ion (protonated diazomethane), a powerful
DNA alkylator, which alkylates guanine bases in DNA at the O-6 position.
Scheme 6. Nitroso adenosine, a versatile synthon.

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Wanner et al.
Scheme 7. Preparation of biologically active triazenes.
Resistance of cancer cells against temozolomide is developed by the production
of the repair enzyme O6-alkylguanine-DNA-alkyltransferase (AGT). By com-
bining the drug with O6-benzylguanine the level of the repair protein is strong-
ly diminished.
Our synthetic approach allowed for the synthesis of a series of molecules in which
both the alkylating agent and the enzyme inhibitor are formed at the same time in vivo.
Table 2. Activity of Triazene derivatives on the different human Adenosine receptors.
Ki S.E.M. in nM (n = 3) or % displacement at 10 mM (n = 2)
R1
R2
A1
A2A
A3
A2B
H
Ethyl
H
H
H
H
H
H
H
H
H
H
H
H
H
140 34
45 17
5.8 1.8
6.1 1.3
15 11
88 12
19 10
209 62
113 21
63 17
9%
15%
6%
Cyclohexyl
Phenyl
25
25
3
3
110 20
39
54%
21%
64%
30%
15%
18%
4-Cl-Phenyl
3-Cl-Phenyl
2-Cl-Phenyl
4-MeO-Phenyl
2-MeO-Phenyl
3-CF3-Phenyl
Benzyl
4.4%
29.9%
45 19
6
15
23
4
8
59 25
179 33
73 11
20 10
20
25
1
3
125 49
insoluble
25 11
38.9%
81 13
insoluble
42 15
43%
insoluble
147 18
577 138
610 340
175 57
90 12
insoluble
9%
6%
O-Phenyl*
Thiazol
24
1
25
1
79%
10%
9%
H
Phenyl
Phenyl
Phenyl
204 136
430 132
2.8 0.8
17.7%
24.3%
Phenyl (TCPA)
Phenyl
CCPA
Cyclopentyl
Ph2CHCH2
210 20
980 110
639 55
60 10
600 230
140 20
281 56
11 0.8
10%
10%
3.1 0.9
6.4 1.8
12 (9.6–15)
NECA
2200 600

New (1-Deaza)Purine Derivatives
Table 3. Stability of the precursors of diazomethane and O6-benzylguanine.
1319
Introduction of different substituents could be used for the finetuning of the half lives
of the molecules (Table 3).
Biological studies are currently in progress in cooperation with the group of G.J.
Peters, VU University Medical Center, Amsterdam.
CONCLUSIONS
Nitration of substituted (deaza)purines using a mixture of tetrabutylammonium
nitrate and trifluoracetic acid anhydride is a powerful synthetic instrument to prepare
nitrosubstituted heteroyclic systems at low temperature. The nitro group influences the
system twofold: 1) it activates other substituents towards nucleophilic aromatic
substitution and 2) it can be substituted with different nucleophiles leading to a variety
of di-substituted (deaza)purines, also via solid phase syntheses.
Several of the molecules obtained are of interest for different biological reasons,
like antiprotozoal activity and for interaction studies with the different human
adenosine receptors.

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Wanner et al.
REFERENCES
1. Deghati, P.Y.F.; Wanner, M.J.; Koomen, G.J. Regioselective nitration of purine
nucleosides: synthesis of 2-nitroadenosine and 2-nitroinosine. Tetrahedron Lett.
2000, 41, 1291–1295.
2. Deghati, P.Y.F.; Wanner, M.J.; Biera¨ugel, H.; Koomen, G.J. Mild and regioelective
nitration of 1-deazapurine nucleosides using TBAN/TFAA. Tetrahedron Lett. 2000,
41, 569–573.
3. Cristalli, G.; Franchetti, P.; Grifantini, M.; Vittori, S.; Klotz, K.-N.; Lohse, M.J.
Adenosine receptor agonists: synthesis and biological evaluation of 1-deaza
analogues of adenosine derivatives. J. Med. Chem. 1988, 31, 1179–1183.
4. Rodenko, B.; Wanner, M.J.; Koomen, G.J. Solid phase synthesis of C-2,
N6-disubstituted adenosine analogues. J. Chem. Soc., Perkin I 2002, 1245–1252.
5. Wanner, M.J.; Koomen, G.J. Syntheses and properties of 2-nitrosoadenosine.
J. Chem. Soc., Perkin I 2001, 1908–1915.
6. Wanner, M.J.; Koomen, G.J. A new syntheses of temozolomide. J. Chem. Soc.,
Perkin I 2002, 1877–1880.
7. Beukers, M.W.; Wanner, M.J.; Von Frijtag Drabbe Ku¨nzel, J.K.; Klaasse, E.C.;
IJzerman, A.P.; Koomen, G.J. N6-Cyclopentyl-2-(3-phenylaminocarbonyl-triazene-
1-yl)adenosine (TCPA) a very selective agonist with high affinity for the human
adenosine A1 receptor. J. Med. Chem. 2003, 46, 1492–1503.