Photochemical reactions of 5-fluoropyrimidine bases with selected alkylamines

Article abstract of DOI:10.1016/S0040-4039(02)02654-0

Novel photochemical reactions between 5-fluoropyrimidine bases and primary alkylamines are described. Photoreaction of 5-fluorouracil with alkylamines in aqueous solution (pH>8) leads to 5-alkylaminouracils as the main photoproducts. Similarly, photoreact

Full text of DOI:10.1016/S0040-4039(02)02654-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 761–763
Photochemical reactions of 5-fluoropyrimidine bases with
selected alkylamines
Anna Kanciurzewska, Mateusz Raczkowski, Krzysztof Ciszewski and Lech Celewicz*
Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland
Received 8 October 2002; revised 15 November 2002; accepted 22 November 2002
Abstract—Novel photochemical reactions between 5-fluoropyrimidine bases and primary alkylamines are described. Photoreaction
of 5-fluorouracil with alkylamines in aqueous solution (pH>8) leads to 5-alkylaminouracils as the main photoproducts. Similarly,
photoreaction of 5-fluorocytosine with alkylamines yields 5-alkylaminocytosines. © 2003 Elsevier Science Ltd. All rights reserved.
The photochemistry of 5-halogenopyrimidine bases has
been extensively studied because of the enhanced pho-
toreactivity of 5-halogenopyrimidine-substituted nucleic
acids as compared to native nucleic acids and the poten-
tial use of photochemical cross-linking of such nucleic
acids with proteins to investigate contact positions
located in nucleoprotein complexes.1 Despite the fact
that there is ample literature on photocross-linking of
proteins to 5-halogenopyrimidine substituted nucleic
acids, detailed information on the chemical structures of
the conjugates and the reaction mechanisms involved in
their formation is often lacking.2,3 In this study, we have
focused on the amino group functionality which is
present in the side-chain of the amino acid lysine and N-
terminal amino acid residue of proteins. We report here
our results on the isolation and characterization of prod-
ucts obtained from photochemical reactions of 5-fluorou-
racil 1 and 5-fluorocytosine 8 with primary alkylamines.
Irradiation of 5-fluorouracil 1 and the appropriate alky-
lamine with 254 nm ultraviolet light in aqueous solution
(pH>8) yields 5-alkylaminouracils 2a–c and by-product
5-fluoro-6-hydroxy-5,6-dihydrouracil 3 which partially
undergoes conversion to barbituric acid 4 by elimination
of hydrogen fluoride (Scheme 1).4 Formation of small
amounts of some uncharacterized minor products was
also detected by HPLC.
The structures of the products 2a–c were established on
the basis of spectroscopic data (UV, NMR and MS)5 as
well as by independent syntheses from 5-bromouracil and
alkylamines (thermal reactions).6 In addition, we have
also synthesized 6-alkylaminouracils7 by thermal reac-
tion of 6-chlorouracil and alkylamines and used them in
identification procedures in order to exclude the possibil-
ity that these compounds are the products of our pho-
toreactions.
Scheme 1.
Keywords: 5-fluorouracil; 5-fluorocytosine; alkylamines; photochemistry.
* Corresponding author. Tel.: +48-61 829-1370; fax: +48-61 865-8008; e-mail: celewicz@amu.edu.pl
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02654-0

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A. Kanciurzewska et al. / Tetrahedron Letters 44 (2003) 761–763
bamoyl)-3-amino-2-bromoacrylamides.11 In the first
case, cleavage of C(5)ꢁBr bond takes place (which is
much weaker than C(5)ꢁF bond), in the second, nucle-
ophilic attack of an amine on the C-2 position of the
pyrimidine ring of 5-bromouracil causes the splitting of
the C(2)ꢁN(1) bond and formation of the ring opened
product. The photoreactions of non-halogenated
pyrimidine bases (uracil,12 thymine,13 cytosine12,14 and
5-methylcytosine14) with alkylamines yield mainly ring
opened products, which subsequently undergo thermal
ring closure to 1-alkylpyrimidine bases.
The photoreactions were monitored by high perfor-
mance liquid chromatography (HPLC) using a photodi-
ode array detector (PAD) and
a mass detector
(electrospray ionisation mass spectrometry, ESI MS).8
We have detected the major product 2 by HPLC during
the early stages of the photoreaction. It was not possi-
ble to observe postulated precursors of 2 (Scheme 2),
which should have UV spectra lacking an absorption
band with a maximum at about 270 nm, which is
characteristic of the pyrimidine chromophore with a
saturated double bond at C(5)ꢀC(6). This suggests that
the transformation of 5-fluoro-6-alkylamino-5,6-dihy-
drouracil 5 (Scheme 2) is a process occurring on a faster
time scale than that of HPLC experiments designed to
detect it.
Analogously, irradiation of 5-fluorocytosine 8 and the
appropriate alkylamine with 254 nm ultraviolet light in
aqueous solution (pH>8) produces 5-alkylaminocytosi-
nes 9a–c and by-product 5-fluoro-6-hydroxy-5,6-dihy-
drocytosine 10 which undergoes partial transformation
to 4-amino-2,6-dihydroxypyrimidine 11 by elimination
of hydrogen fluoride (Scheme 3).4 Formation of some
unidentified minor products was also detected by
HPLC.
Treatment of pure photohydrate 3 with an alkylamine
does not produce 2. This experiment allowed us to
exclude the possibility that product 2 is formed in a
dark reaction via 3. Irradiation of 3 and an alkylamine
with 254 nm ultraviolet light in aqueous solution like-
wise does not yield 2 (on the other hand 3 does not
absorb light significantly at 254 nm); this eliminates the
possibility that 2 is formed by a photochemical reaction
from 3. It is reasonable to postulate that the first step of
the reaction involves nucleophilic attack of an amine on
the C-6 position of excited 5-fluorouracil to give the
intermediate product 5. It can be noted that, other
nucleophiles (e.g. water9 and alcohols10) attack the C-6
position of excited 5-fluoropyrimidine bases. Subse-
quently, the intermediate 5 could undergo nucleophilic
displacement of the fluorine substituent at the 5 posi-
tion either by an amine acting as external nucleophile
to yield 7 or intramolecularly with the aid the of
6-alkylamino group (neighboring group effect) to pro-
duce the aziridine intermediate 6.
The identity of products 9a–c was established by com-
parison of their spectral data15 with those obtained
from thermal reactions of 5-bromocytosine and alkyl-
amines at 90–100°C. The photochemical reactions of
5-fluorocytosine with alkylamines presumably proceed
by a similar mechanism to that proposed in Scheme 2
above for 5-fluorouracil and alkylamines. Because the
photoreactions of 5-fluorouracil and 5-fluorocytosine
with alkylamines are not photosensitized by acetone
and as the presence of oxygen has no significant effect
on product formation, it can be concluded that they
proceed with involvement of a singlet excited state of
5-fluoropyrimidine bases.
In conclusion, we have shown that photochemical reac-
tion of 5-fluoropyrimidine bases with alkylamines yields
5-alkylaminopyrimidines as the major products. This
suggests possible modes of photoinduced cross-linking
between the amine side chain of the amino acid lysine
It is interesting to note that photoreactions of 5-bro-
mouracil with alkylamines in aqueous solution yield
mainly uracil (debromination reaction) and, in addi-
tion, ring opened products namely N-(N%-alkylcar-
Scheme 2.

A. Kanciurzewska et al. / Tetrahedron Letters 44 (2003) 761–763
763
Scheme 3.
and the N-terminal amino acid residue contained in
proteins and 5-fluoropyrimidine-substituted nucleic
acids.
2.76 (t, J=7.3 Hz, 2H, CH2), 3.95 (s, 1H, C5-NH), 6.30
(s, 1H, H-6), 7.89 (s, 1H, N-1), 10.39 (s, 1H, N-3). 13C
NMR (300 MHz, DMSO-d6) l: 21.4, 40.1, 45.2, 112.1,
123.8, 149.1, 161.1.
6. (a) Philips, A. P. J. Am. Chem. Soc. 1951, 73, 1061–1062;
(b) Benitez, A.; Ross, L. O.; Goodman, L.; Baker, B. R.
J. Am. Chem. Soc. 1960, 82, 4585–4591.
7. (a) Pfleiderer, W.; Nubel, G. Liebigs Ann. Chem. 1960,
631, 168–174; (b) Focher, F.; Ubiali, D.; Pregnolato, M.;
Zhi, C.; Gambino, J.; Wright, G. E.; Spadari, S. J. Med.
Chem. 2000, 43, 2601–2607.
Acknowledgements
The authors are grateful to Professor Martin D. Shetlar
of the Department of Pharmaceutical Chemistry, Uni-
versity of California-San Francisco, USA, for helpful
discussions.
8. Analytical HPLC was carried out on a XTerra RP18
reverse phase column (4.6×150 mm, 5 mm) using water–20
mM potassium dihydrogen phosphate pH 2.5 (90:10 v/v)
as eluent or water–methanol for HPLC-MS.
9. Fikus, M.; Wierzchowski, K. L.; Shugar, D. Photochem.
Photobiol. 1965, 4, 521–536.
References
1. Meisenheimer, K. M.; Koch, T. H. Crit. Rev. Biochem.
Mol. Biol. 1997, 32, 101–140.
2. Shetlar, M. D. The Spectrum 1994, 7, 12–19.
10. Urjasz, W.; Maciejewski, A.; Celewicz, L. Tetrahedron
3. Norris, C. L.; Meisenheimer, K. M.; Koch, T. H. Pho-
Lett. 1999, 40, 3243–3246.
11. Shetlar, M. D.; Rose, R. B.; Hom, K.; Shaw, A. A.
Photochem. Photobiol. 1991, 53, 595–609.
tochem. Photobiol. 1997, 65, 201–207.
4. General procedure for irradiations: An aqueous solution
of 5-fluoropyrimidine base (0.2 mmol) and appropriate
alkylamine (200 mmol) at pH>8 (pH was usually adjusted
to 9 with hydrochloric acid) was irradiated for 4 h in a
cylindrical reactor using an immersed, water-cooled, 15
W low pressure mercury lamp (Heraeus, Germany) with
a cylindrical Vycor light filter (2 mm thick). After
removal of solvent in vacuo, the products 2a–c and 9a–c
were isolated by preparative HPLC on XTerra Prep RP18
reverse phase column (19×100 mm, 5 mm) using water–
methanol (90:10 v/v) as eluent in yields 36–40%.
5. Compound 2a: UV (in 20 mM aqueous KH2PO4 pH 2.5):
umax=262 nm. EIMS (high resolution): calcd for
C5H7N3O2 141.05302, found 141.05383. 1H NMR (300
MHz, DMSO-d6) l: 2.25 (s, 3H, CH3), 4.23 (s, 1H,
C5-NH), 6.19 (s, 1H, H-6), 10.15 (s, 1H, N-1), 11.10 (s,
1H, N-3). 13C NMR (300 MHz, DMSO-d6) l: 39.5,
111.3, 125.2, 149.2, 161.0. Compound 2b: UV (in 20 mM
aqueous KH2PO4 pH 2.5): umax=261 nm. EIMS (high
resolution) calcd for C6H9N3O2 155.07054, found
155.06947. 1H NMR (300 MHz, DMSO-d6) l: 1.10 (t,
J=7.3 Hz, 3H, CH3), 2.80 (q, J=7.3 Hz, 2H, CH2), 4.10
(s, 1H, C5-NH), 6.15 (s, 1H, H-6), 10.16 (s, 1H, N-1),
11.13 (s, 1H, N-3). 13C NMR (300 MHz, DMSO-d6) l:
13.9, 40.3, 112.4, 132.9, 149.4, 161.3. Compound 2c: UV
(in 20 mM aqueous KH2PO4 pH 2.5): umax=252 nm.
EIMS (high resolution) calcd for C7H11N3O2 169.08418,
found 169.08513. 1H NMR (300 MHz, DMSO-d6) l: 0.91
(t, J=7.3 Hz, 3H, CH3), 1.56 (m, J=7.3 Hz, 2H, CH2),
12. Hom, K.; Strahan, G.; Shetlar, M. D. Photochem. Photo-
biol. 2000, 71, 243–253.
13. Saito, I.; Sugiyama, H.; Matsuura, T. J. Am. Chem. Soc.
1983, 105, 956–962.
14. Shetlar, M. D.; Hom, K.; Distefano, S.; Ekpenyong, K.;
Yang, J. Photochem. Photobiol. 1988, 47, 779–786.
15. Compound 9a: UV (in 20 mM aqueous KH2PO4 pH 2.5):
umax 1=219 nm, umax 2=276 nm. EIMS (high resolution)
calcd for C5H8N4O 140.07091, found 140.06981. 1H
NMR (300 MHz, DMSO-d6) l: 2.25 (s, 3H, CH3), 3.30
(s, 1H, C5-NH), 4.90 (s, 1H, H-6), 5.23 (s, 2H, NH2), 6.70
(s, 1H, N-1). 13C NMR (300 MHz, DMSO-d6) l: 30.7,
39.8, 119, 153.3, 159.6. Compound 9b: UV (in 20 mM
aqueous KH2PO4 pH 2.5): umax 1=205 nm, umax 2=270
nm. EIMS (high resolution) calcd for C6H10N4O
154.08551, found 154.08546. 1H NMR (300 MHz,
DMSO-d6) l: 1.15 (t, J=7.2 Hz, 3H, CH3), 2.81 (q,
J=7.2 Hz, 2H, CH2), 6.52 (s, 1H, C5-NH), 7.89 (s, 2H,
NH2), 10.12 (s, 1H, N-1). 13C NMR (300 MHz, DMSO-
d6) l: 14.2, 39.2, 117.3, 120.4, 155.2, 161.0. Compound
9c: UV (in 20 mM aqueous KH2PO4 pH 2.5): umax=276
nm. EIMS (high resolution) calcd for C7H12N4O
168.10239, found 168.10110. 1H NMR (300 MHz,
DMSO-d6) l: 0.91 (t, J=7.2 Hz, 3H, CH3), 1.30 (m,
J=7.2 Hz, 2H, CH2), 2.80 (t, J=7.2 Hz, 2H, CH2), 4.12
(s, 1H, C5-NH), 6.40 (s, 1H, H-6), 7.70 (s, 1H, NH2),
10.22 (s, 1H, N-1). 13C NMR (300 MHz, DMSO-d6) l:
21.8, 39.2, 46.2, 117.2, 120.2, 155.0, 161.0.