New compounds via Mannich reaction of cytosine, paraformaldehyde and cyclic secondary amines

Article abstract of DOI:10.1016/j.tetlet.2006.10.117

The Mannich reaction of cytosine, paraformaldehyde and cyclic secondary amines in the presence of acetic acid gives 5-(4′-morpholinyl)methylcytosine, 5-(1′-piperidinyl)methylcytosine, 5-(1′-pyrrolidinyl)methylcytosine, 5-(4′-methyl-1′-piperidinyl)methylcytosine, 5-(3′-methyl-1′-piperidinyl)methylcytosine and 5-(2′-methyl-1′-piperidinyl)methylcytosine. These products are quite different from those obtained via cytosine aminomethylation previously described in the literature.

Full text of DOI:10.1016/j.tetlet.2006.10.117

Tetrahedron Letters 47 (2006) 9045–9047
New compounds via Mannich reaction of
cytosine, paraformaldehyde and cyclic secondary amines
Dorota Prukała*
Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan´, Poland
Received 20 July 2006; revised 11 October 2006; accepted 19 October 2006
Abstract—The Mannich reaction of cytosine, paraformaldehyde and cyclic secondary amines in the presence of acetic acid gives
5-(40-morpholinyl)methylcytosine, 5-(10-piperidinyl)methylcytosine, 5-(10-pyrrolidinyl)methylcytosine, 5-(40-methyl-10-piperidinyl)-
methylcytosine, 5-(30-methyl-10-piperidinyl)methylcytosine and 5-(20-methyl-10-piperidinyl)methylcytosine. These products are quite
different from those obtained via cytosine aminomethylation previously described in the literature.
Ó 2006 Elsevier Ltd. All rights reserved.
Due to their antiviral and anticancer properties, 5-
substituted pyrimidine nucleosides, including 5-substi-
tuted derivatives of cytosine have been the subject of
increasing interest for many years.1,2 On the other hand,
there is continuing interest in the synthesis of nucleoside
analogues in which the carbon in the heterocyclic base is
linked to the nitrogen of the secondary cyclic amines.3 In
view of the above, we prepared a series of new 5-substi-
tuted derivatives of cytosine, which may have anticancer
and/or antivirus activity.
and in aqueous solutions decompose readily to the start-
ing components.
However, it is known that aminomethylation of uracil
gives 5-substituted products.5,6 Also 5-(40-morpholin-
yl)methyl-2-thiouracil was synthesized via the Mannich
reaction of 2-thiouracil, paraformaldehyde and mor-
pholine using ethanol as the solvent.7 The above method
with some modifications was adapted to prepare com-
pounds 1–6 (Fig. 1).8
This letter reports the synthesis of 5-(40-morpholin-
yl)methylcytosine 1, 5-(10-piperidinyl)methylcytosine
2, 5-(10-pyrrolidinyl)methylcytosine 3, 5-(40-methyl-10-
piperidinyl)methylcytosine 4, 5-(30-methyl-10-piperidin-
yl)methylcytosine 5 and 5-(20-methyl-10-piperidinyl)-
methylcytosine 6, in which the cyclic amines were intro-
duced through a methylene bridge at position-5 of cyto-
sine via a Mannich reaction.
Modification involved the use of glacial acetic acid in
combination with other reagents. The absence of acetic
acid in the reaction mixture resulted in only a small
amount of the desired product, even if the reaction
was refluxed over a long period of time (20 h). The use
of other acids (HCl, H2SO4) gave unsatisfactory results.
Addition of acetic acid, generally improved the solubil-
ity of cytosine in ethanol, which had a considerable
influence on the kinetics of the reaction. Secondly, acetic
acid can facilitate elimination of water from the interme-
diate formaldehyde-amine adduct. Thirdly, protonation
of the pyrimidine base under acidic conditions was
expected to make the pyrimidine ring more susceptible
towards the attack of the C-5 atom.9
Aminomethylated derivatives of cytosine have been syn-
thesized and isolated earlier.4 The Mannich reaction of
cytosine using 37% formaldehyde and morpholine or
diethylamine yielded bis-1,4 adducts. The reaction in
which all the substrates were stirred at room tempera-
ture using THF as the solvent resulted in bis-N4,1-(10-
piperidinyl)methylcytosine
and
bis-N4,1-(diethyl-
amino)cytosine. These compounds are rather unstable
The mechanism of this well known reaction (which can
occur in acidic solution through neutral to basic solu-
tions) involves electrophilic attack of the intermediate
on position-5 of the amino-oxo tautomer of cytosine.
This tautomer is the preferred one in the condensed
Keywords: Mannich reaction; Cytosine; Cyclic secondary amines;
Paraformaldehyde.
*
Tel.: +48 61 829 1435; e-mail: dprukala@amu.edu.pl
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.10.117

9046
D. Prukała / Tetrahedron Letters 47 (2006) 9045–9047
R1
N
R2
NH2
R1
R2
X
NH2
O
CH3COOH
ethanol
X
N
N
+
+
H
C
HN
H
O
N
H
O
N
H
1. R1 = H, R2 = H, X = O
3. R1 = H, R2 = H, X-null
2. R1 = H, R2 = H, X =CH2
R1 =H, R2 = H, X = CHCH
4.
3
5. R1 = H, R2 =CH3, X =CH2 6. R1 =CH3, R2 = H, X = CH2
Figure 1.
phase, in aqueous solution and even in acidic solution
(pH 66), in which the preferred protonation site of cyto-
sine is N3.10–13
were assigned to mas (NH2) and ms (NH2). In the 1600–
1700 cmꢀ1 range, intense amino group scissoring vibra-
tions a(NH2) were observed adjacent to the carbonyl
bond stretching vibrations m(C@O).
All reactions were monitored by TLC (CHCl3–CH3OH
5:1/SiO2) and also by electrospray ionisation mass spec-
trometry ESI MS.
The 13C NMR spectra were recorded in TFA-d solu-
tions, as the solubility of 1–6 in DMSO-d6 and in other
solvents was insufficient.8
To optimize the reaction conditions, ethanol and meth-
anol were used as solvents, with the same stoichiometry
of substrates and reaction time, however, when the reac-
tion was conducted in methanol a mixture of products
and a small amount of unreacted cytosine were isolated.
In all the reactions two equivalents of secondary amine
and paraformaldehyde were used and four equivalents
of acetic with respect to cytosine. An increase in the
equivalents of secondary amine and paraformaldehyde
resulted in a decrease of isolated products. It is worth
noting that after completion of the reaction and removal
of the solvent, the unreacted acetic acid should be care-
fully evaporated.
In conclusion it has been shown that reactions of cyto-
sine, paraformaldehyde and cyclic secondary amines in
the presence of acetic acid yielded 5-substituted deriva-
tives of cytosine.
This reaction indicates a new route for obtaining similar
compounds and calls for further developments. All the
obtained compounds are being tested for biological
activity.
References and notes
Finally, the resulting gummy oil was shaken with dry
benzene until a precipitated solid appeared. Dry benzene
was a unique solvent for this precipitation (for 1–5)
because no other solvent (diethyl ether, hexane, chloro-
form) worked nearly as well, if at all. After cooling in
a refrigerator for a few days while the precipitation
was in progress, the crude product was filtered to give
pure 1–6. The collected solids did not appear as salts;
hence addition of a base was not needed.
1. For example, (a) Prusoff, W. H.; Ward, D. C. Biochem.
Pharmacol. 1976, 25, 1233–1239; (b) Prusoff, W. H.; Chen,
M. S.; Fischer, P. H.; Lin, T. S.; Shiau, G. T.; Schinazi, R.
F.; Walker, J. Pharmacol. Ther. 1979, 7, 1–34; (c)
Shannon, W. M.; Schabel, F. M., Jr. Pharmacol. Ther.
1980, 11, 263–390; (d) Shealy, Y. F.; O’Dell, C. A.;
Shannon, W. M.; Arnett, G. J. Med. Chem. 1983, 26, 156–
161; (e) Shealy, Y. F.; O’Dell, C. A.; Shannon, W. M.;
Arnett, G. J. Med. Chem. 1986, 29, 79–84; (f) Motavia, M.
S.; Megied, A.; Pedersen, E. B.; Nielsen, C. M.; Ebbesen,
P. Acta Chem. Scand. 1992, 46, 77–81.
2. For example, (a) Greer, S.; Schildkraut, I. Ann. N.Y. Acad.
Sci. 1975, 255, 359–365; (b) Jones, A. S.; Serafinowski, P.
R.; Walker, T. Tetrahedron Lett. 1977, 28, 2459–2460; (c)
Ward, A. D.; Baker, B. R. J. Med. Chem. 1977, 20, 88–92.
3. Filichev, V. V.; Pedersen, E. B. Tetrahedron 2001, 57,
9163–9168, and references cited therein.
The structures of compounds 1–6 were determined by
comparison of their spectral data with those of 5-meth-
ylcytosine14,15 and cyclic amines.16–20
Data from IR, 1H NMR, 13C NMR, ESI MS spectra as
well as elemental analyses confirmed the identity of
products 1–6.
4. Sloan, K. B.; Siver, K. G. Tetrahedron 1984, 40, 3997–
4001.
5. Delia, T. J.; Scovill, J. P.; Munslow, W. D.; Burckhalter, J.
H. J. Med. Chem. 1976, 19, 344–346.
1
The H NMR spectra (in DMSO-d6 for 1, 3, 4 and in
TFA-d 1–6) for all the compounds showed a character-
istic singlet in the range 7.22–7.36 ppm (DMSO-d6) or in
the range 8.44–8.46 ppm (TFA-d) assigned to the proton
6. Motavia, M. S.; Jørgensen, P. T.; Lamkjær, A.; Pedersen,
E. B.; Nielsen, C. M. Monatsch. Chem. 1993, 124, 55–64.
7. Kamalakannan, P.; Vekappayya, D.; Balasubramanian,
T. J. Chem. Soc., Dalton Trans. 2002, 3381–3391.
8. General procedure for the syntheses of 1–6: Cytosine
(9 mmol), paraformaldehyde (18 mmol) and cyclic sec-
ondary amine (18 mmol) were suspended in ethanol
(99.8%, 20–25 ml). Glacial acetic acid (ꢁ36 mmol) was
added dropwise over 0.5 h to the boiling solution until the
cytosine dissolved. The resulting reaction solution was
1
at C-6 of the cytosine moiety. The H NMR spectra in
DMSO-d6 also exhibited two broadened signals at 7.10
and 10.41 ppm, assigned to the protons 4N–H2 and
1N–H, respectively.
The IR spectra of all compounds (KBr disks) revealed
two strong bands in the region 3430–3200 cmꢀ1, which

D. Prukała / Tetrahedron Letters 47 (2006) 9045–9047
9047
refluxed for 3.5–4 h. After the completion of the reaction,
as established by TLC (CHCl3–CH3OH, 5:1), the solvent
was evaporated under vacuum and the mixture was kept
under reduced pressure, on the same rotatory evaporator,
at 90 °C for 1 h. After cooling, the gummy oil was shaken
with benzene until a solid precipitated. Then the mixture
was left in a refrigerator for 2–3 days. The precipitated
solid was filtered off and crystallized from methanol. To
increase the amount of precipitate, the procedure was
repeated. Compound 6 was shaken with benzene and a
small amount of diethyl ether. The first precipitated
fraction was rejected and the procedure was repeated;
only subsequent fractions were suitable for further crys-
tallization. The spectral and analytical data of compounds
1–6 are given below.
3.14 (s, 2H). 1H NMR (300 MHz, TFA-d, ppm): d
piperidine moiety ꢀ1.59 (t, J = 12.3 Hz, 2H, H-3a,5a),
1.82 (m, 1H, H-4a), 2.10 (d, J = 14.0 Hz, 2H, H-3e,5e),
3.22 (t, J = 11.8 Hz, 2H, H-2a,6a), 3.85 (d, J = 10.8 Hz,
2H, H-2e,6e), 1.10 (d, J = 5.9 Hz, 3H, CH3); cytosine
moiety 8.44 (s, 1H, H-6); N–CH2–C5 4.51 (s, 2H). 13C
NMR (75 MHz, TFA-d, ppm): d piperidine moiety ꢀ28.5
(C4), 30.9 (C3,5), 53.9 (C2,6), 19.1 (CH3); cytosine moiety
ꢀ95.3 (C5), 148.1 (C2), 152.5 (C6), 159.9 (C4); N–CH2–C5
51.3. IR (KBr, cmꢀ1): 3422, 3374, 1671, 1627. ESI MS (m/z,
%int.) 223 (100) [M+H]+. Anal. Calcd for C11H18N4O: C,
59.46; H, 8.11; N, 25.23. Found: C, 59.26; H, 8.32; N, 25.14.
Compound
5
(isolated yield 39%, mp 263–268 °C
decomp.) white crystals. 1H NMR (300 MHz, TFA-d,
ppm): d piperidine moiety ꢀ1.16–1.35 (m, 1H, H-3a),
1.89–2.17 (m, 4H), 2.80 (t, J = 11.7 Hz, 1H, H-2a), 3.09
(t, J = 10.9 Hz, 1H, H-6a), 3.72 (d, J = 10.5 Hz, 1H, H-
2e), 3.83 (d, J = 10.9 Hz, 1H, H-6e), 1.09 (d, J = 6.3 Hz,
3H, CH3); cytosine moiety ꢀ8.44 (s, 1H, H-6); N–CH2–C5
4.51 (s, 2H). 13C NMR (75 MHz, TFA-d, ppm): d
piperidine moiety ꢀ22.7 (C5), 29.5 (C3), 30.0 (C4), 53.4
(C6), 59.6 (C2), 16.7 (CH3); cytosine moiety ꢀ95.5 (C5),
148.2 (C2), 152.6 (C6), 160.4 (C4); N–CH2–C5 51.6. IR
(KBr, cmꢀ1): 3295, 3123, 1668, 1628. ESI MS (m/z, %int.)
223 (100) [M+H]+. Anal. Calcd for C11H18N4O: C, 59.46;
H, 8.11; N, 25.23. Found: C, 59.36; H, 7.95; N, 25.36.
Compound
1
(isolated yield 65%, mp 275–280 °C
1
decomp.) white crystals. H NMR (300 MHz, DMSO-d6,
ppm): d morpholine moiety ꢀ2.31 (m, 4H, H-2,6), 3.56 (m,
4H, H-3,5); cytosine moiety ꢀ7.26 (s, 1H, H-6), 6.27 (br,
2H, NH2), 10.46 (br, 1H, 1NH); N–CH2–C5 3.15 (s, 2H).
1H NMR (300 MHz, TFA-d, ppm): d morpholine moiety
ꢀ3.58 (t, J = 11.5 Hz, 2H, H-2a,6a), 3.89 (d, J = 11.9 Hz,
2H, H-2e,6e), 4.09 (t, J = 12.5 Hz, 2H, H-3a,5a), 4.40 (d,
J = 12.3 Hz, 2H, H-3e,5e); cytosine moiety ꢀ8.46 (s, 1H,
H-6); N–CH2–C5 4.66 (s, 2H). 13C NMR (75 MHz, TFA-
d, ppm): d morpholine moiety ꢀ63.8 (C2,6), 51.9 (C3,5);
cytosine moiety ꢀ94.4 (C5), 148.1 (C2), 152.7 (C6), 159.9
(C4); N–CH2–C5 51.8. IR (KBr, cmꢀ1): 3428, 3290, 1664,
1654, 1617. ESI MS (m/z, %int.) 211 (100) [M+H]+. Anal.
Calcd for C9H14N4O2: C, 51.43; H, 6.67; N, 26.67. Found:
C, 51.13; H, 6.87; N, 26.46.
Compound
6
(isolated yield 25%, mp 205–209 °C
decomp.) white crystals. 1H NMR (300 MHz, TFA-d,
ppm): d piperidine moiety ꢀ1.35–2.20 (m, 6H), 3,16 (t,
J = 11.3 Hz, 1H, H-2a), 3.59 (m, 1H, H-6a), 3.72 (d,
J = 11.7 Hz, 1H, H-2e) 1.68 (d, J = 6.3 Hz, 3HCH3);
cytosine moiety 8.45 (s, 1H, H-6); N–CH2–C5 4.33 (s, 2H).
13C NMR (75 MHz, TFA-d, ppm): d piperidine moiety
ꢀ21.2 (C4), 22.6 (C5), 37.1 (C3), 47.9 (C6), 64.2 (C2), 16.7
(CH3); cytosine moiety ꢀ95.4 (C5), 148.1 (C2), 152.6 (C6),
160.2 (C4); N–CH2–C5 51.8. IR (KBr, cmꢀ1): 3342, 3125,
1670, 1632. ESI MS (m/z, %int.) 223 (100) [M+H]+. Anal.
Calcd for C11H18N4O: C, 59.46; H, 8.11; N, 25.23. Found:
C, 59.12; H, 8.09; N, 25.10.
Compound
2
(isolated yield 57%, mp 290–293 °C
1
decomp.) white crystals. H NMR (300 MHz, DMSO-d6,
ppm): d piperidine moiety ꢀ1.48 (m, 6H, H-3,4,5), 2.49 (s,
4H, H-2,6); cytosine moiety ꢀ7.22 (s, 1H, H-6), 7.10 (br,
2H, NH2), 10.33 (br, 1H, 1NH); N–CH2–C5 3.12 (s, 2H).
1H NMR (300 MHz, TFA-d, ppm): d piperidine moiety
ꢀ1.89–2.16 (m, 6H, H-3,4,5), 3.19 (t, J = 11.7 Hz, 2H, H-
2a,6a), 3.85 (d, J = 10.9 Hz, 2H, H-2e,6e); cytosine moiety
ꢀ8.44 (s, 1H, H-6); N–CH2–C5 4.51 (s, 2H). 13C NMR
(75 MHz, TFA-d, ppm): d piperidine moiety ꢀ20.6 (C4),
22.9 (C3,5), 54.1 (C2,6); cytosine moiety ꢀ95.2 (C5), 148.2
(C2), 152.5 (C6), 159.9 (C4); N–CH2–C5 51.5. IR (KBr,
cmꢀ1): 3363, 3132, 1672, 1629. ESI MS (m/z, %int.) 209
(100) [M+H]+. Anal. Calcd for C10H16N4O: C, 57.69; H,
7.69; N, 26.92. Found: C, 57.46; H, 7.91; N, 26.85.
9. Huang, Y.; Kentta¨maa, H. J. Am. Chem. Soc. 2003, 25,
9878–9889.
10. Steward, R.; Harris, M. G. Can. J .Chem. 1977, 55, 3807–
3814.
11. Colominas, C.; Luque, F. J.; Orozco, M. J. Am. Chem.
Soc. 1996, 118, 6811–6821.
12. Bruning, W.; Freisinger, E.; Sabat, M.; Sigel, R. K. O.;
¨
Compound
3
(isolated yield 47%, mp 283–285 °C
Lippert, B. Chem. Eur. J. 2002, 8, 4681–4692.
decomp.) white crystals. 1H NMR (300 MHz, TFA-d,
ppm): d pyrrolidine moiety ꢀ2.29 (s, 2H) and 2.38 (s, 2H):
H-3,4; 3.38 (s, 2H) and 3.98 (s, 2H): H-2,5; cytosine moiety
ꢀ8.44 (s, 1H, H-6); N–CH2–C5 4.62 (s, 2H). 13C NMR
(75 MHz, TFA-d, ppm): d pyrrolidine moiety ꢀ22.1
(C3,4), 54.9 (C2,5); cytosine moiety ꢀ96.8 (C5), 148.5
(2), 151.6 (C6), 159.6 (C4); N–CH2–C5 49.2. IR (KBr,
cmꢀ1): 3315, 3120, 1670, 1626. ESI MS (m/z, %int.) 195
(100) [M+H]+. Anal. Calcd for C9H14N4O; C, 55.67; H,
7.22; N, 28.87. Found: C, 55.63; H, 7.45; N, 28.57.
13. Florian, J.; Baumruk, V.; Leszczynski, J. J. Phys .Chem.
´
´
1996, 100, 5578–5589.
14. Shiragami, H.; Kudo, I.; Iida, S.; Hayatsu, H. Chem.
Pharm. Bull. 1975, 23, 3027.
15. Samijlenko, S. P.; Alexeeva, I. V.; Pachykirs’ka, L. H.;
Kondratyuk, I. V.; Stepanyugin, A. V.; Shalamay, A. S.;
Hovrun, D. M. J. Mol .Struct. 1999, 484, 31–38.
16. Abraham, R. J.; Medforth, C. J. Magn. Res. Chem. 1988,
26, 334–344.
17. Smith, W. B.; Shoulders, B. A. J. Phys. Chem. 1965, 69,
579–582.
18. Langner, R.; Zundel, G. J. Phys. Chem. A 1998, 102,
6635–6646.
19. Cannon, C. G.; Sutherland, G. B. B. M. Spectrochim. Acta
1951, 4, 373–395.
20. El-Gogary, T. M.; Soliman, M. S. Spectrochim. Acta A
2001, 57, 2647–2657.
Compound
4
(isolated yield 45%, mp 253–258 0C
1
decomp.) white crystals. H NMR (300 MHz, DMSO-d6,
ppm): d piperidine moiety ꢀ1.11 (t, J = 12.3 Hz, 2H,
H-3a,5a), 1.33 (m, 1H, H-4a), 1.59 (d, J = 12.0 Hz, 2H,
H-3e,5e), 1.82 (t, J = 11.3 Hz, 2H, H-2a,6a), 2.77 (d,
J = 11.3 Hz, 2H, H-2e,6e); cytosine moiety ꢀ7.23 (s, 1H,
H-6), 7.07 (br, 2H, NH2), 10.41 (br, 1H, 1NH) N–CH2–C5