Polymethylene derivatives of nucleic bases bearing ω-functional groups: IX—An unusual reaction of methyl 2-thiocyano-5-chloropentanoate with uracyl and thymine

Article abstract of DOI:10.1134/S1068162017060061

Although the search of inhibitors of the key enzymes of HIV replication has remained a topical and intensively studied topic over the last several decades, potential inhibitors of the enzymes bearing thiocyano groups have been little studied. In this work, we tried to synthesize polymethylene derivatives of nucleic bases bearing thiocyano groups at the ω-position of the polymethylene chain. The reaction of new alkylating reagents, methyl α-thiocyano-ω-chloroalkanoates, with nucleic bases led to a complicated and barely separated product mixture. The only exceptions were the reactions of uracil or thymine with methyl 2-thiocyano- 2-chloropentanoate, in which 2-(N¹-uracilyl or -thyminyl)tetrahydrothiophene-2-carboxylates were isolated in 45–50% yields. The potential mechanism of the reaction is discussed.

Full text of DOI:10.1134/S1068162017060061

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 6, pp. 669–672. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © V.V. Komissarov, A.M. Kritzyn, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 6, pp. 650–654.
Polymethylene Derivatives of Nucleic Bases Bearing ω-Functional
Groups: IX—An Unusual Reaction of Methyl 2-Thiocyano-5-
1
Chloropentanoate with Uracyl and Thymine
2
V. V. Komissarov and A. M. Kritzyn
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia
Received March 31, 2017; in final form, April 21, 2017
Abstract⎯ Although the search of inhibitors of the key enzymes of HIV replication has remained a topical and
intensively studied topic over the last several decades, potential inhibitors of the enzymes bearing thiocyano
groups have been little studied. In this work, we tried to synthesize polymethylene derivatives of nucleic bases
bearing thiocyano groups at the ω-position of the polymethylene chain. The reaction of new alkylating
reagents, methyl α-thiocyano-ω-chloroalkanoates, with nucleic bases led to a complicated and barely sepa-
rated product mixture. The only exceptions were the reactions of uracil or thymine with methyl 2-thiocyano-
1
2
-chloropentanoate, in which 2-(N -uracilyl or -thyminyl)tetrahydrothiophene-2-carboxylates were isolated
in 45–50% yields. The potential mechanism of the reaction is discussed.
Keywords: alkylation, nucleosides, polymethylene analogues, thiocyanates
DOI: 10.1134/S1068162017060061
INTRODUCTION
effectively inhibiting HIV RT, the only one derivative con-
taining a thiocyanate group was studied [5, 6].
Drug products inhibiting HIV replication play an
important role in the therapy of AIDS. The products
referred to this group are inhibitors of the virus sorp-
tion on cell membranes, fusion inhibitors of the viral
particle and the cell, protease inhibitors, integrase
inhibitors, and nucleoside- or nonnucleoside-based
reverse transcriptase inhibitors.
The capacity of HIV to rapidly produce mutations,
thereby developing resistance to drugs, is the reason for
the search for new inhibitors of the viral replication.
Polymethylene derivatives of nucleic bases with
various functional groups at the end of the hydropho-
bic hydrocarbon chain are convenient tools for the
evaluation of the structure–function relationship
upon the interaction of these derivatives with various
proteins including HIV enzymes. Previously, we stud-
ied polymethylene ω-carboxy-, carbalkoxy-, and
hydroxyl derivatives of pyrimidine [2] and purine [3]
bases as potential HIV RT inhibitors, as well as DNA
polymerase I inhibitors. We also searched for HIV
integrase inhibitors within 5-(4-halogenophenyl)-5-
oxopentyl derivatives of nucleic bases [4].
Polymethylene derivatives of nucleic bases with
thiocyanate groups have not yet been described.
RESULTS AND DISCUSSION
3'-Thiocyano-3'-deoxythymidine, a potential anti-
HIV agent, was synthesized in [5] in a yield of 21%.
The target compound was synthesized by nucleophilic
substitution of a mesyloxy group of 1-(2-deoxy-3-О-
mesyl-5-О-trityl-β-D-threo-pentofuranosyl)thymine
by a thiocyanate residue followed by the removal of the
protective group with acetic acid. Later, this com-
pound was obtained by opening the cyclic anhydride
5
'-О-trityl-2,3'-anhydrothymidine in the presence of
lithium cyanate [6].
For the preparation of polymethylene derivatives of
nucleic bases bearing a ω-carboxy group and a thiocy-
anate group located at the α-position relative to it, we
planned to use alkylation of a nucleic base with α-
thiocyano-ω-chlorocarboxylates in an aprotic solvent
in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU).
Among a tremendous number of nucleoside analogues
bearing various modifications of the ribose residue and
The starting compounds for the preparation of
alkylating agents were ω-chloropentanoic, ω-chloro-
hexanoic, and ω-chlorononanoic acids. α-Bromo-ω-
chloropentanoate, -hexanoate, and -nonanoate were
prepared by bromination of the proper ω-chloroacids
1
2
For Part VIII, see [1].
Corresponding author: fax: +7(499) 135-1405; e-mail:
amk@eimb.ru.
669

6
70
KOMISSAROV, KRITZYN
Table 1
α-Thiocyanocarboxylates*
OR
OR
X
n
O
X
n
O
Br
SCN
Ia)−(Id)
(
IR, (SCN),
Compound
n
X
R
Solvent
Molecular formula
C H NO S
M
Yield,%
–1
сm
(Ia)
(Ib)
(Ic)
(Id)
0
1
2
5
H
Сl
Cl
Cl
Et MeOH
173.3
207.6
221.7
263.8
90.7
98.0
91.4
97.6
2159
2155
2157
2166
7
11
2
Me Butanone-2
Me DMF
C H ClNO S
7 10 2
C H ClNO S
8
12
2
Me Butanone -2
C H ClNO S
11 18 2
*
For the NMR spectral data, see the Experimental part.
using the Hell–Volhard–Zelinsky reaction followed but used column chromatography. According to the
by esterification.
NMR spectral data the purity of the isolated com-
13
pounds achieved 92 to 96%. In C NMR spectra, a
chemical shift at 110 ppm was observed, which is char-
acteristic for the SCN carbon atom; in IR spectra, a
With ethyl α-bromobutanoate as an example, we
developed a standard procedure of α-thiocyanocarboxyl-
ate synthesis. 2-Thiocyanobutanoate (Ia) was obtained in
–1
a yield of 91% by refluxing a suspension of a small excess of clear band at 2130–2160 cm characteristic for a C≡N
potassium rodanide with ethyl α-bromobutanoate in group was seen (cf. [8]).
methanol for 4 h (Table 1). The reaction can also be per-
Alkylation of nucleic bases with α-thiocyano-ω-chlo-
formed in butanol-2 or dimethylformamide.
rocarboxylates was performed as described in [9]. Under
Due to a considerable difference in the reactivity of
the conditions used, a starting nucleic base was recovered
the bromine atom in the α-position and the terminal
in a nearly quantitative yield. We did not analyze a compli-
chlorine atom of the carboxylic acid [7], we prepared
cated product mixture formed in the reaction of α-thiocy-
new methyl α-thiocyano-ω-chloropentanoate, -hexa-
ano-ω-chlorocarboxylates with DBU.
noate, and -nonanoate in nearly quantitative yields
(
Ib–d) (Table 1).
Interactions of methyl α-thiocyano-ω-chloropen-
tanoate with uracil and thymine were the only excep-
ates were viscous liquids. Due to their inclination to tions. The expected α-thiocyano-ω-pyrimidinopenta-
isomerization we did not purify them by distillation noates (II) and (III) were not found either (Scheme 1).
The synthesized α-thiocyano-ω-chlorocarboxyl-
O
R
NH
O
N
O
(
II) R = H
O
(III) R = CH3
SCN
O
O
SCN
O
R
NH
R
Cl
O
+
NH
i
N
H
O
N
O
(IV) R = H
COOMe
(Ib)
i: DBU, DMF, 90°C
(V) R = CH3
S
Scheme 1.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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POLYMETHYLENE DERIVATIVES
671
In the IR spectral patterns of the isolated com- phen-2-carboxylate (IV) and methyl 2-(5-methyl-
pounds, absorption bands of a thiocyano group 2,4-dioxo-3,4-dihydro-1(2Н)-pyrimidinyl)tetrahydro-
were lacking. In the NMR spectra, a signal of the
nitrile group was not found. The data of element
analysis, NMR spectroscopy, and mass spectra of
the compounds synthesized in 40–50% yields
allowed their structures as methyl 2-(2,4-dioxo-
thiphen-2-carboxylate (V). Characteristic proton
resonances of a tetrahydrothiophene cycle at 1.88,
1
2
8
.33, and 3.02 ppm in H and 30.0, 34.4, and
13
1.8 ppm in C NMR spectra confirmed these
3
,4-dihydro-1(2Н)-pyrimidinyl)tetrahydrothio-
structures.
O
O
O
.
DBU HCN
−
DBU
Cl
O
N
Cl
O
N
Cl
O
S
S
S
(
Ib)
(1)
(2)
O
R
NH
N
H
O
DBU
O
O
N
O
R
NH
R
R
NH
NH
N
O
−
(IV) R = H
V) R = CH3
O
N
O
(
O
O
S
S
−
O
O
Cl
(3)
Scheme 2.
We proposed the potential reaction mechanism performed on silica gel 60 (0.040–0.063 mm) (Merck,
(
Scheme 2). The key step may be the formation of Germany). Mass spectra were recorded on an MS-30
methyl 5-chloro-2-thiooxopentanoate (2). In this mass spectrometer (Kratos, Japan) using electron
case, the nitrile group at the sulfur atom of the thiocy- impact ionization. NMR spectra (δ, ppm; J, Hz) were
ano group served a leaving group. A similar behavior of recorded in CDCl on a Bruker AMXIII-400 spec-
3
the nitrile group within a thiocyano substituent was trometer (Germany) with the working frequency
1
13
observed in [8].
4
00 MHz for H and 100 MHz for C NMR spectra at
Similar cyclic products were not found in the reac- 300 К.
tions of compounds (Ic) and (Id) with nucleic bases,
IR spectra were recorded on a FT-IR Nicolet
most probably due to the enormous benefits of the
five-membered cycle formation, which was only pos-
sible for the interaction with compound (Ib).
IR200 spectrometer (United States) in fine film. Sam-
ples were suspended in Vaseline oil, the suspension
was placed between ZnSe plates, and the spectra were
–1
recorded. The resolution was 4 cm ; the number of
scans, 32.
EXPERIMENTAL
In this work, we used adenine, uracil, thymine, and
General procedure for the preparation of α-thiocy-
cytosine (Sigma, United States). DBU and sodium ano-ω-chlorocarboxylates
hydride, 80% suspension in mineral oil were from
A suspension of potassium thiocyanate (33 mmol)
Fluka (Switzerland); CDCl and DMSO-d , from
3
6
and α-bromo-ω-chlorocarboxylate (30 mmol) in buta-
none-2 (50 mL) was refluxed under stirring for 5 h. The
mixture was kept overnight at room temperature. The
precipitate was filtered off, and the filtrate was evapo-
Acros Organics (Belgium).
Purification and dehydration of solvents were per-
formed by standard procedures.
TLC was carried out on Kieselgel 60 F2 plates rated to dryness. The residue was dissolved in dry
54
(
Merck, Germany) eluting with 19 : 1 chloroform– methylene chloride (75 mL). The precipitate was fil-
ethanol. The compounds were visualized by UV tered off, the filtrate was evaporated in vacuum, dried
absorption at 254 nm. Column chromatography was by distillation with benzene, and analyzed (Table 1).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 6 2017

6
72
KOMISSAROV, KRITZYN
Ethyl 2-thiocyanobutanoate (Ia) (methanol as a sol- ethyl acetate to give the product (49.5%, 1.26 g); mp
1
+
vent). H NMR: 1.06 (3 H, t, J 7.4, CH CH CH- 162–3°C (EtOAc). Mass: m/z 256.05 [M] , calc.
3
2
1
(
SCN)COOEt); 1.30 (3 H, t, J 7.2, COOCH CH ), 256.05 (C H N O S). H-NMR: 1.88, 2.33, 3.07
2 3
10 12 2 4
1
(
.96–2.17 (2 H, m, CH CH(SCN)COOEt); 3.78 (6 H, 3 m, CH CH CH ); 3.75 (3 H, s, COOCH );
2
2 2 2 3
1 H, t, J 6.8, CH(SCN)COOEt); 4.25 (2 H, q, J 7.2, 5.71 (1 H, d, J 8.3, H5); 8.35 (1 H, d, J 8.3, H6); 9.21
13
13
COOCH CH ). C NMR: 10.80 (CH CH CH- (1 H, s, H3). C NMR: 29.97 (С4'); 34.42 (C5'); 40.76
2
3
3
2
(
(
SCN)COOEt); 14.10 (COOCH CH ); 25.56 (C3'); 53.54 (COOCH
3
); 82.18 (C2'); 100.94 (C5);
2
3
CH CH CH(SCN)COOEt); 51.52 (CH(SCN)- 141.72 (C6); 150.74 (C2); 163.64 (C4); 169.02
3
2
(
COOMe). Found: C 47.06, H 4.88, N 10.87, S 12.29
C H N O S. Calc.: C 46.87, H 4.72, N 10.93, S 12.15.
COOEt); 62.62 (COOCH CH ); 110.15 (SCN);
2
3
1
68.82 (COOEt).
10 12
2
4
Methyl 2-(5-methyl-2,4-dioxo-3,4-dihydro-1(2Н)-
pyrimidinyl)tetrahydrothiophen-2-carboxylate (V) was
prepared similarly to (IV). Yield 1.22 g (45.2%).
Methyl 2-thiocyano-5-chloropentanoate (Ib) (buta-
1
none-2 as a solvent). H NMR: 1.94 (2 H, m,
ClCH CH ); 2.11-2.32 (2 H, m, ClCH CH CH ),
2
2
2
2
2
R 0.72, mp 178–9°C (EtOAc). Mass: m/z 270.07
f
3
.57 (2 H, t, J 6.3, ClCH ); 3.82 (3 H, s, COOCH );
2
3
+
1
[
M] , calc. 270.07 (C H N O S). H NMR: 1.9 (3 H,
1
3
11 14 2 4
3
.84 (1 H, t, J 6.8, CH(SCN)COOMe). C NMR:
s, 5-CH ) 1.88, 2.25, 3.02 (6 H, 3 m, CH CH CH ); 3.69
3
2
2
2
2
9.25 (ClCH CH ); 29.47 (ClCH CH CH ); 43.59
2
2
2
2
2
(
3 H, s, COOCH ); 7.28 (1 H, s, H6); 9.14 (1 H, s, H3).
3
(
(
ClCH );
48.97
(CH(SCN)COOMe);
53.50
2
1
3
C NMR: 12,74 (5-CH ); 30.03 (С4'); 34.44 (C5'); 40.78
COOCH ); 109.55 (SCN); 169.02 (COOMe).
3
3
(
1
C3'); 53.45 (COOCH ); 81.84 (C2'); 109.32 (C5);
3
Methyl 2-thiocyano-6-chlorohexanoate (Ic) (DMF
37.48 (C6); 150.74 (C2); 164.13 (C4); 169.27 (COOMe).
1
as a solvent, reaction temperature 100°С). H NMR:
Found: C 48.72, H 5.27, N 10.43, S 12.16. C H N O S.
11
14
2
4
1
.61 (2 H, m, ClCH CH ); 1.81 (2 H, m, ClCH -
2
2
2
Calc.: C 48.88, H 5.22, N 10.36, S 11.86.
CH CH ); 1.92–2.15 (2 H, m, ClCH CH CH CH ),
2
2
2
2
2
2
3
3
2
.52 (2 H, t, J 6.4, ClCH ); 3.80 (3 H, s, COOCH );
2
3
13
ACKNOWLEDGMENTS
.80 (1 H, t, J 6.8, CH(SCN)COOMe). C NMR:
3.94 (ClCH CH CH ); 31.41 (ClCH CH ); 31.82
The authors are grateful to Prof. J. Vepsalainen and
2
2
2
2
2
(
(
(
CH CH(SCN)COOMe); 44.32 (ClCH ); 49.45 J. Vaisel (Department of Pharmacy, Biocenter of
2
2
CH(SCN)COOMe); 53.51 (COOCH ); 109.85 Kuopio, University of Eastern Finland, Kuopio, Fin-
3
SCN); 169.33 (COOMe).
land) for fruitful discussions and ascription of the
structures of the compounds synthesized.
Methyl 2-thiocyano-9-chlorononanoate (Id) (buta-
1
none-2 as a solvent). H NMR: 1.20–1.45 (8 H, m,
ClCH CH CH CH CH CH CH ); 1.70 (2 H, m,
2
2
2
2
2
2
2
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2
2
2
2
2
2
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2
2
2
2
2
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SCN)COOMe); 32.51 (CH CH(SCN)COOMe);
2
4
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2
4
(
COOCH ); 109.95 (SCN); 169.47 (COOMe).
3
Alkylations of nucleic bases with methyl α-thiocy-
ano-ω-chloropentanoate, -hexanoate, and -nona-
5
noate (Ib–d) were performed as described in [9].
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idinyl)tetrahydrothiophen-2-carboxylate (IV). DBU
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stirring for 15 min. The reaction mixture was stirred for
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methylene chloride (~15 mL), loaded on a silica gel
column (200 g), and chromatographed eluting in a
gradient of ethanol (0 → 10 mL) in methylene chloride
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(
100 mL). The fractions with R 0.63 were combined
f
and evaporated. The residue was recrystallized from
Translated by E. Shirokova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 43
No. 6
2017