Synthesis of modified uracil and cytosine nucleobases using a microwave-assisted method

Article abstract of DOI:10.1016/j.tetlet.2012.03.056

Modified nucleobases and nucleic acids have found many biological and pharmaceutical applications. Here we report a microwave-directed synthesis of a variety of modified uracil and cytosine nucleobases with high yields under solvent-free conditions. The reaction yields were further improved by addition of Lewis acid. The crystal structures of 5-isopropyl-6-methyluracil and 6-phenyluracil were also determined.

Full text of DOI:10.1016/j.tetlet.2012.03.056

Tetrahedron Letters 53 (2012) 2639–2642
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of modified uracil and cytosine nucleobases using a
microwave-assisted method
⇑
Laxmi Narayana Burgula, K. Radhakrishnan, Lal Mohan Kundu
Department of Chemistry, Indian Institute of Technology Guwahati, North Guwahati, Assam 781039, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Modified nucleobases and nucleic acids have found many biological and pharmaceutical applications.
Here we report a microwave-directed synthesis of a variety of modified uracil and cytosine nucleobases
with high yields under solvent-free conditions. The reaction yields were further improved by addition of
Lewis acid. The crystal structures of 5-isopropyl-6-methyluracil and 6-phenyluracil were also
determined.
Received 3 January 2012
Revised 14 March 2012
Accepted 18 March 2012
Available online 23 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Nucleobases
Pyrimidine
Lewis acid
Microwave
DNA damage
Unnatural nucleobases and oligonucleotides have been devel-
oped for many bio-molecular studies ranging from detection of
genomic mutations,1–4 disease diagnosis,5 gene silencing,6,7 and
as molecular probes.8–11 A number of nucleobase analogs, for
example, 5-fluorouracil, anilinouracils, and cyclopentenyl cyto-
sines are well-known antitumor and antibacterial drugs.12–14 In
the past decade a spectrum of non-natural nucleobases was also
developed to induce altered base-pairing properties into the DNA
duplex as well as for the expansion of genetic alphabets.15,16 Syn-
thesis of modified nucleobases, therefore, has drawn much interest
in bio-molecular studies as well as in pharmaceutical industries.
Synthesis of pyrimidine nucleobases is of particular interest to
study their propensity to undergo rapid photochemical lesion for-
mations under ultraviolet radiation, causing genomic damages.17,18
The conventional methods for the synthesis of pyrimidine nucleo-
bases require either multi-step reactions or harsh experimental
conditions and long reflux time.19,20 Several methods have been
developed, in the past, for the efficient synthesis of natural and
modified nucleobases, including metal catalyzed cross coupling
reactions.21 Microwave-directed methods were also used to syn-
thesize non-nucleobase pyrimidines, using N-vinyl and N-aryl
amides as substrates.22,23 Here we report a one-pot, efficient syn-
thesis of pyrimidine nucleobases with altered steric and electronic
properties at C-5 and C-6 positions. We found that the use of
microwave-assisted synthesis under solvent-free conditions led
to high yields of uracil and cytosine derivatives (Scheme 1). The
yield of the reactions as well as the rate of conversions were
further enhanced, significantly, by use of BF3ÁEt2O as Lewis acid.
We also demonstrate here the first crystal structure of 5-isopro-
pyl-6-methyluracil (5) and 6-phenyluracil (6) which clearly show
different packing and altered hydrogen bonding abilities in com-
parison to 6-propyl uracil (4).
A series of uracil and cytosine nucleobase derivatives were syn-
thesized in a single step, using solvent-free, microwave-assisted
synthesis. All reactions were carried out in a Anton Paar Synthos
3000 closed vessel microwave reactor at about 135–145 °C for var-
iable durations.27,28 Uracil nucleobases (1–10) were synthesized by
treatment of the respective b-ketoesters or b-aldehydo ester with
urea, whereas the cytosine derivatives (11, 12) were obtained from
benzoylacetonitrile or N,N-diethylamide precursors. The substrate
b-ketoesters (1a–8a, 10a), b-aldehydo ester (9a), benzoylacetonitri-
le, and N,N-diethylamide ( 11a, 12a) were either purchased or
O
O
R2
R1
O
R2
O
°
MW, 135
C
NH
OEt
H2N
H2N
NH2
NH2
+
solvent free
R1
N
H
NH2
O
O
R2
CN
R1
R2
R1
°
MW, 135
BF3.Et2O,
solvent free
C
+
N
O
N
H
O
⇑
Corresponding author. Tel.: +91 361 258 2326; fax: +91 361 258 2349.
E-mail addresses: lmkundu@iitg.ernet.in, kundu77@gmail.com (L.M. Kundu).
Scheme 1. Schematic presentation for the synthesis of pyrimidine nucleobases in a
microwave-directed method.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.03.056

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L. N. Burgula et al. / Tetrahedron Letters 53 (2012) 2639–2642
Table 1
Synthesis of a series of uracil and cytosine derivatives via Scheme 1
Entry No.
Substrate (1a–12a)
Microwave irradiation (min)
Product
% Yield
72
% Yield with BF3ÁEt2O (time)
COOEt
O
NH
1
9
1
83 (5 min)
O
N
H
O
COOEt
O
O
NH
2
8
70
72
70
48
80
67
55
75
25
70
65
85 (5 min)
86 (5 min)
83 (5 min)
60 (7 min)
92 (4 min)
76 (7 min)
75 (9 min)
87 (7 min)
55 (9 min)
80 (7 min)
78 (4 min)
2
3
N
H
O
O
COOEt
O
NH
3
8
N
H
O
COOEt
O
O
NH
4
10
12
8
4
5
nPr
N
H
O
O
COOEt
O
NH
5
N
H
O
O
COOEt
NH
6
6
O
Ph
Ph
N
O
H
O
COOEt
Ph
NH
7
9
7
O
Ph
Ph
N
H
O
COOEt
O
Ph
NH
8
12
9
8
9
O
O
O
N
H
O
O
COOEt
H
Ph
Ph
Ph
Ph
NH
9
N
H
O
O
COOEt
Ph
NH
10
11
12
15
9
10
Ph
N
H
O
CON(Et)2
NEt2
N
11
O
O
N
H
O
CN
Ph
NH2
N
12
6
Ph
N
H
O
The reactions were carried out at 600 W at around 135–145 °C.
synthesized following published procedures.24–26 Table 1 demon-
strates the synthesis of the nucleobases under microwave
conditions.27,28 Substrates were designed so that the product nucle-
obases were expected to have different stereo-electronic properties

L. N. Burgula et al. / Tetrahedron Letters 53 (2012) 2639–2642
2641
following reaction with urea gives an intermediate, characterized
by HRMS and NMR (Supplementary data). Finally, the intermediate
undergoes ring closure to yield the cyclized product (4, 9).
The crystal structure of 5-isopropyl-6-methyluracil (5) and that
of 6-phenyluracil (6) is demonstrated in Figure 2. The crystal struc-
ture of 5 shows a highly organized pattern with alternative hydro-
phobic and hydrophilic layers. The branched alkane chain forms
the hydrophobic layer whereas, the polar C(4)@O(2) accounts for
the hydrophilic interactions (Supplementary data). The crystal
structure of 6 also shows a self-assembled pattern through H-
BF3.Et2O
O
O
O
O
BF3.Et2O
R1
OEt
R2
R1
OEt
R2
4a. R1 = nPr, R2= H
9a. R1 = H, R2= Bn
- H2O NH2CONH2
O
R2
R1
HN
O
O
R2
- EtOH
NH
OEt
bonding (2.03 and 2.17 Å) as well as strong p–p interactions
(3.65 Å) between the phenyl and the heterocyclic ring.29
R1
N
H
O
NH2
4. R1 = nPr, R2= H
9. R1 = H, R2= Bn
Intermediate
Acknowledgments
We sincerely thank Professor R. C. Boruah, RRL Jorhat, India, for
providing the microwave reactor facility. We acknowledge the help
and cooperation of Dr. G. Das, IIT Guwahati, India, in analyzing the
crystal data. We also thank CSIR (New Delhi, India, Grant No.
02(0017)/11/EMR-II) for financial support.
Figure 1. Proposed mechanism of formation of nucleobases using Lewis acid.
at C-5 and C-6 positions. In order to test the robustness of the meth-
od, we have used b-aldehydo ester as substrate instead of a b-keto-
ester, which also shows rapid conversion (compound 9). The
versatility of the method was further demonstrated by the synthe-
sis of cytosine derivatives, 11 and 12. The optimized irradiation
times in Table 1 reflect completion of reactions, monitored by
checking TLC (thin layer chromatography) (see Fig. 1).
The yields of the cyclization reactions, in most cases, were sig-
nificantly increased by addition of BF3ÁEt2O as Lewis acid. This was
particularly evident for substrate (10a) which yielded a very low
amount of compound 10 in the absence of the Lewis acid. Addition
of BF3ÁEt2O also led to the reduction of the reaction time from
8–15 min to 4–7 min. The mechanism of the reaction is proposed
in Figure 2 where, in the first step, the carbonyl carbon of the
substrate b-ketoester (4a, 9a) is activated by the Lewis acid. The
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.03.
056.
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27. Representative procedure for microwave synthesis: Ethyl 3-oxohexanoate (4a,
2 mmol), taken in a reactor vessel, was mixed thoroughly for 1 min with urea
(156 mg, 2.6 mmol). The vessel was closed immediately and was subjected to
microwave irradiation for 10 min at about 135 °C. The completion of reaction
was monitored by checking TLC at regular time intervals. Compound (4) was
Figure 2. (a) Packing diagram of 5 showing organized layered structure. (b) Packing
diagram of 6 showing H-bonding (N–H. . .O@C, green dash) and p-stacking (purple
colored dummy atoms) interactions. Blue and red represents ‘N’ and ‘O’ atoms
respectively.

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L. N. Burgula et al. / Tetrahedron Letters 53 (2012) 2639–2642
further purified by column chromatography (Silica gel 60–120 mesh, 75% ethyl
acetate in hexane).
b = 105.156(12)°,
= 1.273 g cmÀ3
Crystal data for 6: CCDC # 844500; C10H8N2O2; M = 188.18, mp = 260–265 °C,
monoclinic; C-2/c, a = 9.5412(4) Å; b = 11.0300(4) Å, c = 16.1903(7) Å, = 90°,
b = 91.564(3)°, = 90°, V = 1703.22(12) Å, Z = 8,
= 0.105 mmÀ1
= 1.468 g cmÀ3, Mo K
radiation, R1 = 0.0453, wR2 = 0.0834, S = 1.355. The
crystallographic data for the complexes has been deposited with the
Cambridge Crystallographic Data Centre. Copies of this information may be
obtained free of charge from the Director, CCDC, 12, Union Road, Cambridge,
CB2 1EZ, UK (Fax: +44 1223336033; e-mail: deposit@ccdc.cam.ac.uk or http://
www.ccdc.cam.ac.uk).
c
= 90°,
V = 877.3(3) Å,
Z = 4,
l ,
= 0.093 mmÀ1
q
, Mo Ka radiation, R1 = 0.0489, wR2 = 0.0883, S = 1.102.
28. Representative procedure for microwave synthesis using Lewis acid: Ethyl 3-oxo-
hexanoate (4a, 2 mmol), taken in a reactor vessel with BF3 Et2O (339 mg,
2.4 mmol) was mixed thoroughly for 1 min with urea (156 mg, 2.6 mmol). The
vessel was closed immediately and was subjected to microwave irradiation at
135 °C for about 5 min. Reaction was complete within 5 min irradiation, which
was verified by TLC. Compound (4) was further purified by column
chromatography (Silica gel 60–120 mesh, 75% ethyl acetate in hexane).
29. Crystal data for 5: CCDC # 860110; C8H12N2O2; M = 168.2, mp = 285–287 °C,
a
c
l
,
q
a
monoclinic; P21/c, a = 11.0751(18) Å; b = 7.0759(12) Å, c = 11.598(2) Å,
a = 90°,