Polymethylene derivatives of nucleic bases with ω-functional groups. III.1 N-[7-(2-oxocyclohexyl)-7-oxoheptyl]-substituted pyrimidines

Article abstract of DOI:10.1023/B:RUBI.0000043786.12780.8a

New polymethylene derivatives of nucleic bases containing a β-dioxo function at the ω-position were synthesized by alkylation of uracil, thymine, and cytosine with 1-(7-chloroheptanoyl)cyclohexan-2-one, and their physicochemical properties were studied.

Full text of DOI:10.1023/B:RUBI.0000043786.12780.8a

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 5, 2004, pp. 436–440. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 5, 2004, pp. 487–492.
Original Russian Text Copyright © 2004 by Kritzyn, Komissarov.
Polymethylene Derivatives of Nucleic Bases with w-Functional
Groups. III.1 N-[7-(2-Oxocyclohexyl)-7-oxoheptyl]-Substituted
Pyrimidines
A. M. Kritzyn*, 2 and V. V. Komissarov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
Received July 2, 2003; in final form, October 17, 2003
Abstract—New polymethylene derivatives of nucleic bases containing a β-dioxo function at the ω-position
were synthesized by alkylation of uracil, thymine, and cytosine with 1-(7-chloroheptanoyl)cyclohexan-2-one,
and their physicochemical properties were studied.
Key words: alkylation, nucleosides, polymethylene analogues
21
INTRODUCTION
obvious. In this work, we describe the synthesis of the
first pyrimidine-derived compounds of this type.
We have previously shown that polymethylene
derivatives of nucleic bases with various functional
groups at the ω-position of their carbon chains (more
than four carbon atoms in length) are convenient and
promising tools for studying the enzymes of nucleic
RESULTS AND DISCUSSION
We chose the alkylation of a nucleic base or its pro-
tected derivative with 2-(7-chloroheptanoyl)cyclohex-
anone (I) for the preparation of new polymethylene
derivatives of nucleic bases bearing a β-diketone func-
tion in the ω-position.
3
exchange [1, 2]. For example, we found that such
thymine and uracil derivatives can effectively interact
with the reverse transcriptase site responsible for the
recognition of the tRNALys anticodon and activate this
enzyme. The study of interaction with DNA topo-
isomerase I demonstrated that these derivatives only
slightly inhibit the enzyme, the activity of the uracil
derivatives being higher than that of the thymine deriv-
atives.
The alkylating reagent (I) was prepared, using the
procedure described by Nesmeyanov et al. [7], by the
acylation of cyclohexanone morpholine enamine [8]
with 7-chloroheptanoyl chloride under mild conditions.
The subsequent acidic hydrolysis of the resulting acy-
lated enamine led to the target 1,3-diketone (I). Note
that a considerable amount of side product with a sub-
stantially lower boiling point arose in this reaction; its
structure was not determined.
It has recently been demonstrated that 4-aryl-2,4-
dioxobutanoic acids are the effective inhibitors of HIV-1
integrase. The mechanism of action and the peculiari-
ties of binding to the HIV-1 integrase are still not clear,
but the presence of β-dioxo fragment was found to be
one of the factors determining the activity of these com-
pounds [6]. The keto–enol tautomerism inherent in
β-diketones might contribute to the interaction with the
corresponding enzymes or receptors, since the pH val-
ues around the enzyme active sites could substantially
differ from the physiological values.
O
O
Cl
(I)
As we shown in [2], the alkylation of 2,4-dihydroxy-
pyrimidines with alkyl halides in DMF in the presence
of DBU results in moderate yields of N1-substituted
derivatives (method A). The corresponding and N1-[7-
(2-oxocyclohexyl)-7-oxoheptyl] derivatives of uracil
(II) and thymine (IV) were obtained by this method in
30–40% yields. Silica gel column chromatography
allowed the separation of target derivatives (II) and
(IV) from N1,N3-bisalkylated products (III) and (V),
respectively; the admixtures of them were ~5%.
No compounds containing β-diketone fragments in
the ω-position of carbon chain have yet been described
among a few polymethylene derivatives of nucleic
bases derivatives with functional groups. Moreover,
simple synthetic schemes for their preparation are not
1
For communication II, see [1].
Corresponding author: fax: +7 (095) 135-1405; e-mail:
amk@eimb.ru
2
3
Abbreviations: DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene.
1068-1620/04/3005-0436 © 2004 MAIK “Nauka /Interperiodica”

POLYMETHYLENE DERIVATIVES OF NUCLEIC BASES
437
the purification of product on a Dowex 50 × 8 (H+ form)
column led to the target diketone (VI‡) in a total yield
of 27%.
Another way involved the alkylation of cytosine
sodium salt, prepared with sodium hydride in DMF
(method B). This allowed us both to avoid the protec-
tion of exocyclic amino group (as in method A) and to
increase the yield of the target cytosine derivative (VI‡)
The alkylation of cytosine with chloride (I) by
method A led to a multicomponent mixture, and we
succeeded in the isolation of the target (VI‡) only in 6%
yield. Therefore, other synthetic ways for the prepara-
tion of this product were tried. In particular, cytosine
protected at the exo amino group with a benzoyl residue
was alkylated with chloride (I) by method A to give
(VIb). The removal of the N4-benzoyl group using 5 M
ammonia in methanol (24 h at 20°ë) [9] followed by to 43%.
O
O
O
O
R
O
R
O
CH3
CH3
HN
N
N
HN
O
N
R
N
R
N
R
O
N
R
(V)
(II)
(III)
(IV)
X
NH
O
O
1'
3'
5'
R =
8'
N
10'
11'
9'
7'
2'
4'
6'
13'
N
R
O
12'
(VI‡), (VIb) ‡: X = H; b: X = Bz.
The structures of the alkylated pyrimidines (II)– the ionization method. NMR spectra were registered on
(VI) were confirmed by NMR and UV spectroscopy
and mass spectrometry (see Tables 1, 2 and the Experi-
mental section).
a Bruker AMXIII-400 spectrometer (Germany) with
the working frequency of 400 MHz for H1 and 100 MHz
Note that, according to UV spectra, the β-diketone
fragments in (II)–(VI) are only slightly enolized in acid
and neutral aqueous solutions. A pH increase causes an
increase in the portion of enol form, which manifests
itself in the appearance of absorption maximum at
315 nm. The UV spectra of thymine derivative (IV)
exemplify this dependence (figure). On the other hand,
according to NMR spectra, the enolization of the
β-diketone fragments is nearly complete (more than
90%) in anhydrous media (DMSO or ëDCl3).
A
1.4
1.2
1.0
0.8
0.6
0.4
EXPERIMENTAL
Uracil, thymine, and cytosine were from Sigma
(United States); DBU was from Aldrich (United
States); sodium hydride (80% suspension in mineral
oil) was from Fluka (Switzerland); ion-exchange resin
Dowex 50 × 8 was from Serva (Germany).
Solvents were purified and dried using standard pro-
tocols [10]. UV spectra were recorded on a Cary 50
spectrophotometer (Varian, Australia) at pH 1, 7, and
14. The values of λmax are given in nm (ε, å–1 cm–1).
Mass spectra were registered on an MS-30 mass spec-
trometer (Kratos, Japan); electron impact was used as
0.2
0
220
240
260
280
300
320
340
λ, nm
1
UV spectrum of N -[7-(2-oxocyclohexyl)-7-oxohep-
tyl]uracil (II); pH 1 (- -); pH 7 ( ); pH 14 (—).
…
·
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004

438
KRITZYN, KOMISSAROV
1
Table 1. H NMR spectral data of the synthesized compounds (δ, ppm; J, Hz)
Protons
H3
(II)
(III)
(IV)
(V)
(VIa)
(VI·)
8.99 (1 H, s)
5.68 (1 H, d,
5,6 7.8)
–
7.99 (1 H, c)
–
–
–
–
–
H5
5.64 (1 H, d,
J5,6 7.8)
5.6 (1 H, d,
J5,6 7.7)
5.63 (1 H, d,
J5,6 7.8)
J
H6
7.14 (1 H, d,
J5,6 7.8)
7.1 (1 H, d,
J5,6 7.8)
6.95 (1 H, s)
6.92 (1 H, s)
7.9 (1 H, d,
J5,6 7.7)
6.88 (1 H, d,
J5,6 7.8)
5-Me
–
–
–
–
–
–
–
–
1.9 (3 H, c)
1.87 (3 H, c)
–
–
4-NH2
4-NHBz
PhCO-
–
–
–
–
–
–
6.75 (2 H, s)
–
–
–
7.24 (1 H, s)
o–7.89 (2 H, d,
Jo, m 7.5)
m–7.5 (2 H, m)
p–7.59 (1 H, t,
Jp, m 7.8)
H1'
3.72 (2 H, t,
J1',2' 7.2)
3.86 (2 H, t,
J1',2' 7.5)
3.67 (2 H, t,
J1',2' 7.5)
3.88 (2 H, t,
J1',2' 7.8)
3.65 (2 H, t,
J1',2' 7.2)
3.89 (2 H, t,
J1',2' 7.2)
H1''
–
3.66 (2 H, t,
J1'',2'' 7.2)
–
3.65 (2 H, t,
J1'',2'' 7.2)
–
–
H2'-H5'
H2''-H5''
H6'
1.36 (8 H, m)
–
1.36 (8 H, m)
–
1.36 (8 H, Ï)
–
1.36 (8 H, m)
–
1.36 (16 H, m)
2.34 (4 H, m)
1.36 (16 H, m)
2.35 (4 H, m)
2.41 (2 H, t,
J6',5' 7.5)
2.4 (2 H, t,
J6',5' 7.5)
2.41 (2 H, t,
J6',5' 7.5)
2.41 (2 H, t,
J6',5' 7.5)
H6''
–
–
–
–
9'-OH
15.9 (1 H, s)
15.9 (1 H, s)
15.95 (1 H, s)
15.9 (1 H, s)
15.92 (1 H, s)
15.96 (1 H, s)
15.9 (1 H, s)
15.9 (1 H, s)
9''-OH
–
–
–
–
H10', H13'
H10'', H13''
H11', H12'
H11'', H12''
2.3 (4 H, m)
2.3 (4 H, m)
2.3 (4 H, m)
2.3 (4 H, m)
2.3 (8 H, m)
2.3 (8 H, m)
–
–
–
–
1.38 (4 H, m)
–
1.38 (4 H, m)
–
1.38 (4 H, m)
–
1.38 (4 H, m)
–
1.38 (8 H, m)
1.38 (8 H, m)
for C13 at 300K in DMSO-d6 (if not stated otherwise);
tetramethylsilane was used as an internal reference;
distilled in a vacuum to give 29.7 g (60.7%) of (I); bp
147–149°ë/1 mmHg; 1H NMR (CDCl3): 1.02 (2 H, m,
chemical shifts are given in ppm and coupling constants H4'), 1.08 (2 H, m, H5'), 1.24 (2 H, m, H6'), 1.30 (4 H,
in Hz. TLC was performed on Kieselgel 60 F254 plates m, H4 and H5), 1.40 (2 H, m, H3'), 1.94 (4 H, m, H6,
H3), 2.05 (2 H, t, J2',3' 7.0, H2'), 3.16 (2 H, t, J7',6' 6.5,
(Merck, Germany); elution in chloroform (systemA) or
19 : 1 chloroform–ethanol (system B). Column chro-
matography was carried out on silica gel L40/100 (Che-
mapol, Czech Republic).
13
H7'), and 15.6 (1 H, s, 1-OH); C NMR: 22.75 (C4),
22.99 (C5), 23.99 (C3'), 25.82 (C3), 27.65 (C5'), 29.96
(C4'), 31.58 (C6'), 35.81 (C2'), 44.0(C6'), 44.98 (C7'),
105.59 (C2), 179.8 (C1), and 200.58 (C1'); MS, m/z:
244.8 [M]+; calc. for C13H21ClO2: 244.8.
7-Chloroheptanoylcyclohexanone-2 (I). A solu-
tion of 7-chloroheptanoyl chloride (36.6 g, 0.2 mol) in
dry chloroform (100 ml) was added dropwise under
stirring to a solution of N-(cyclohexen-1-yl)morpholine
[8] (37 g, 0.22 mol) and triethylamine (35.5 ml,
0.25 mol) in chloroform (250 ml), and the mixture was
kept at 20°ë for 14 h. A solution of concentrate H2SO4
(60 ml) in water (80 ml) was added dropwise under vig-
orous stirring, and the mixture was refluxed under stir-
ring for 4 h and cooled. The organic layer was sepa-
rated, the aqueous fraction was extracted with chloro-
form (2 × 100 ml), the organic extracts were combined,
dried with Na2SO4, and evaporated. The residue was
Alkylation of pyrimidine bases with 7-chlorohep-
tanoylcyclohexanone-2 using DBU as a base
(method A). Alkylating reagent (I) (3.7 g, 15 mmol)
and DBU (2.3 g, 15 mmol) were added to a suspension
of pyrimidine base or its protected derivative (10 mmol)
in anhydrous DMF (25 ml). The reaction mixture was
kept at 80–100°ë for 20 h (TLC monitoring). The mix-
ture was cooled and evaporated to dryness. The residue
was suspended in chloroform and chromatographed on
a silica gel column (5 × 28 cm) eluted with a gradient of
ethanol (0
10%) in chloroform. The target fractions
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004

POLYMETHYLENE DERIVATIVES OF NUCLEIC BASES
Table 2. 13C NMR spectral data of the synthesized compounds
439
Compound, δ, ppm
Carbon atoms
(II)
(III)
(IV)
(V)
(VIa)
(VI·)
166.2
C2
150.9
163.75
102.25
144.5
–
151.3
162.95
101.5
142.0
–
150.8
164.1
107.8
140.5
12.4
–
151.3
163.65
109.6
138.2
12.9
156.4
165.5
93.8
141.4
–
C4
170.0
93.5
148.9
–
C5
C6
5-Me
PhCO-
–
–
–
–
213.0 (CO),
127.7 (m),
129.2 (o),
133.2 (p)
C1'
48.9
–
49.6
41.0
48.6
–
49.3
41.3
50.1
–
51.1
–
C1''
C2'
31.25
–
32.7
31.25
–
31.09
31.03
28.97
28.87
26.7
31.25
–
31.25
–
C2''
C3'
32.3
28.9
–
28.76
28.73
27.73
26.67
31.08
31.02
36.7
28.9
–
28.9
–
28.9
–
C3''
C4'
26.4
–
26.4
–
26.5
–
26.5
–
C4''
C5'
26.3
29
29.2
–
28.8
29.0
–
29.0
–
C5''
C6'
–
27.4
36.8
–
36.8
–
36.7
36.8
–
36.8
–
C6''
C7'
36.6
36.6
201.4
–
201.3
201.1
201.4
–
201.3
201.2
201.4
–
201.4
–
C7''
C8'
106.9
–
106.9
–
106.9
–
106.9
–
106.6*
106.7*
C8''
C9'
181.8
–
181.62
181.56
28.7
181.8
–
181.64
181.57
28.9
181.8
–
181.8
–
C9''
C10'
C10''
C11'
C11''
C12'
C12''
C13'
C13''
28.9
–
28.8
–
28.9
–
28.7
–
28.8
28.7
21.8
–
21.61
21.57
24.07
23.83
21.8
–
23.61
21.58
22.81
22.78
21.8
–
21.8
–
23.0
–
23.0
–
23.0
–
23.0
–
24.0
–
24.0
–
24.3
–
24.2
–
23.71**
23.7**
* C8' and C8'' resonances are overlapped.
** C13' and C13'' resonances are overlapped.
were evaporated, and the residue was recrystallized After 30-min stirring at 20°ë, the alkylating reagent (I)
from water–ethanol mixtures or ethanol.
(2.9 g, 12 mmol) was added, and the reaction mixture
was kept at 80–100°ë for 20 h (TLC monitoring). The
solvent was evaporated, and the residue was shaken in
a mixture of water (20 ml) and chloroform (50 ml). The
Alkylation of sodium salt of cytosine with 7-chlo-
roheptanoylcyclohexanone-2 (method B). Sodium
hydride (0.33 g, 11 mmol) was added to a suspension of
cytosine (1.2 g, 10 mmol) in anhydrous DMF (25 ml). organic layer was separated, the aqueous phase was
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004

440
KRITZYN, KOMISSAROV
extracted with chloroform (5 × 70 ml), and the com- icochemical characteristics were identical to those of
bined extracts were dried with anhydrous sodium sul- the derivative obtained by the methods A and B.
fate. The evaporated residue was chromatographed on a
silica gel column as described above. The target frac-
ACKNOWLEDGMENTS
tions were evaporated, and the residue was recrystal-
lized from ethanol.
The authors are grateful to Dr. A.R. Khomutov for
fruitful discussion of the results. The work was sup-
ported by the Russian Foundation for Basic Research,
project 03-04-49224.
N1-[7-(2-Oxocyclohexyl)-7-oxoheptyl]uracil (II)
was obtained by method A in a yield of 35%; Rf 0.59
(A); mp 79–81°ë (1 : 3 water–ethanol); UV: pH 1, 270
(17700); pH 7, 270 (18 800); pH 14, 269 (13 200) and
315 (24100); MS, m/z 320 [M+],calc. for C17H24N2O4:
320.
REFERENCES
1. Makinsky, A.A., Kritzyn, A.M., Ul’yanova, E.A.,
Zakharova, O.D., Bugreev, D.V., and Nevinsky, G.A.,
Bioorg. Khim., 2001, vol. 27, pp. 191–196.
2. Makinsky, A.A., Kritzyn, A.M., Ul’yanova, E.A.,
Zakharova, O.D., and Nevinsky, G.A., Bioorg. Khim.,
2000, vol. 26, pp. 735–742.
N1,N3-Bis[7-(2-Oxocyclohexyl)-7-oxoheptyl]uracil
(III) was obtained by method A in a yield of 3%; Rf
0.23 (B). UV: pH 1, 286 (11400); pH 7, 286 (12300);
pH 14, 280 (9600) and 313 (22 400); MS, m/z: 529
[M]+; calc. for C30H44N2O6: 529.
N1-[7-(2-Oxocyclohexyl)-7-oxoheptyl]thymine (IV)
was obtained by method A in a yield of 39%; Rf 0.645
(A); mp 119–120°ë (1 : 2 water–ethanol); UV: pH 1,
275 (14500); pH 7, 276 (16200); pH 14, 278 (12000)
and 315 (19 800); MS, m/z: 334 [M]+; calc. for
C18H26N2O4: 334.
N1,N3-Bis[7-(2-Oxocyclohexyl)-7-oxoheptyl]thym-
ine (V) was obtained by method A in a yield of 4%; Rf
0.31 (A). UV: pH 1, 290 (17900); pH 7, 290 (19000);
pH 14, 285 (17700), 313 (25200); MS m/z: 543 [M]+;
calc. for C31H46N2O6: 543.
N1-[7-(2-Oxocyclohexyl)-7-oxoheptyl]cytosine (VIa)
was obtained by method A in a yield of 6% and by
method B in a yield of 43%; Rf 0.07 (A); mp 157–159°ë
(ethanol). UV: pH 1, 286 (15800); pH 7, 277 (11400);
pH 14, 281 (11400) and 315 (16400); MS, m/z: 319
[M]+; calc. for C17H25N3O3: 319.
3. Hazuda, D.J., Felock, P., Witmer, M., Wolfe, A., Still-
mock, K., Grobler, J.A., Espeseth, A., Gabryelski, L.,
Schleif, W., Blau, C., and Miller, M.D., Science, 2000,
vol. 287, pp. 646–650.
4. Espeseth, A., Felock, P., Wolfe, A., Witmer, M., Grob-
ler, J., Anthony, N., Egbertson, M., Melamed, J.Y.,
Young, S., Hamill, T., Cole, J.L., and Hazuda, D.J., Proc.
Natl. Acad. Sci. USA, 2000, vol. 97, pp. 11 244–11 249.
5. Wai, J.S., Egbertson, M.S., Payne, L.S., Fisher, T.E.,
Embrey, M.W., Tran, L.O., Melamed, J.Y., Lang-
ford, H.M., Guare, J.P., Zhuang, L., Grey, V.E.,
Vacca, J.P., Holloway, M.K., Naylor-Olsen, A.M.,
Hazuda, D.J., Felock, P.J., Wolfe, A.L., Stillmock, K.A.,
Schleif, W.A., Gabryelski, L.J., andYoung, S.D., J. Med.
Chem., 2000, vol. 43, pp. 4923–4926.
6. Grobler, J.A., Stillmock, K., Hu, B., Witmer, M.,
Felock, P., Espeseth, A.S., Wolfe, A., Egbertson, M.,
Bourgeois, M., Melamed, J., Wai, J.S., Young, S.,
Vacca, J., and Hazuda, D.J., Proc. Natl. Acad. Sci. USA,
2002, vol. 99, pp. 6661–6666.
7. Nesmeyanov, A.N., Zakharkin, L.I., Kost, T.A., Fre-
idlina, R.Kh., and Nesmeyanov, A.N., Izbrannye trudy
(Selected Works), M.: Acad. Sci. USSR, 1959, vol. 3,
pp. 466–472.
8. Fizer, L.F. and Fizer, M., Reagents for Organic Synthe-
sis, New York: John Wiley and Sons, 1968. Translated
under the title Reagenty dlya organicheskogo sinteza,
Moscow: Mir, 1970, vol. 2, p. 314.
9. Zorbach, W.W. and Tipson, R.S., Synthetic Procedures
in Nucleic Acid Chemistry, New York: John Wiley and
Sons, 1968, vol. 1, p. 291.
10. Tietze, L.F. and Eicher, T., Reaktionen und Synthesen im
organische-chemischen Praktikum und Forschungslab-
oratorium, New York: Gerg Thieme, 1991. Translated
under the title Preparativnaya organicheskaya khimiya,
Moscow: Mir, 1999.
N1-[7-(2-Oxocyclohexyl)-7-oxoheptyl]-N4-benzo-
ylcytosine (VIb) was obtained by method A in a yield
of 30.5%; Rf 0.67 (A); mp 132–134°ë (4 : 5 water–eth-
anol); MS, m/z: 423.5 [M]+; calc. for C24H29N3O4:
423.5.
Removal of benzoyl protective group. Cytosine
derivative (VIb) (0.25 g, 0.6 mmol) was kept in 5 M
ammonia solution in methanol at room temperature.
After 24 h (TLC monitoring), the reaction mixture was
evaporated, the residue was dissolved in ethanol
(15 ml), and applied onto a Dowex 50 × 8 (H+ form)
column. The column was washed with water and alco-
hol. The product was eluted with a 4 : 1 ethanol–conc.
aqueous ammonia mixture (150 ml). The eluate was
evaporated to give 0.17 g (89%) of (VIa), whose phys-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004