The synthesis and antituberculosis activity of 5-alkynyl uracil derivatives

Article abstract of DOI:10.1016/j.bmcl.2020.127351

A series of new 5-alkynyl-substituted uracil and uridine derivatives were synthesised via palladium-catalysed Sonogashira cross-coupling reaction of 5-bromo-pyrimidine base with terminal acetylenes with good yields in DMF at room temperature. All obtained compounds were tested for antimycobacterial activity against Mycobacetrium bovis and Mycobacterium tuberculosis (H37Ra) at concentrations of 1–100 μg/ml using MABA test. Obtained results revealed that most of tested uracil derivatives exhibited high antimycobacterial activity (MIC₅₀ = 1.1–19.2 μg/ml) in comparison with therapeutic agents such as rifampicin, isoniazid and D-cycloserine, excluding compounds having alkyl substituent at triple alkyne bond.

Full text of DOI:10.1016/j.bmcl.2020.127351

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The synthesis and antituberculosis activity of 5-alkynyl uracil derivatives
Yana B. Platonova, Alexander N. Volov, Larisa G. Tomilova
PII:
S0960-894X(20)30462-5
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127351
BMCL 127351
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Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
11 May 2020
10 June 2020
12 June 2020
Please cite this article as: Platonova, Y.B., Volov, A.N., Tomilova, L.G., The synthesis and antituberculosis
activity of 5-alkynyl uracil derivatives, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/
10.1016/j.bmcl.2020.127351
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The synthesis and antituberculosis activity of 5-alkynyl uracil
derivatives
Yana B. Platonova,a,b Alexander N. Volov,a,* and Larisa G. Tomilovaa,b
a Department of Chemistry, Lomonosov Moscow State University, 1 Leninskie Gory, Moscow 119991,
Russian Federation
b Institute of Physiologically Active Compounds, Russian Academy of Sciences, 1 Severny proezd,
Chernogolovka, Moscow Region 142432, Russian Federation
*Corresponding author E-mail: volovalexander@gmail.com
ABSTRACT:
A series of new 5-alkynyl-substituted uracil and uridine derivatives were synthesised via
palladium-catalysed Sonogashira cross-coupling reaction of 5-bromo-pyrimidine base with
terminal acetylenes with good yields in DMF at room temperature. All obtained compounds
were tested for antimycobacterial activity against Mycobacetrium bovis and Mycobacterium
tuberculosis (H37Ra) at concentrations of 1-100 µg/ml using MABA test. Obtained results
revealed that most of tested uracil derivatives exhibited high antimycobacterial activity
(MIC50=1.1-19.2 µg/ml) in comparison with therapeutic agents such as rifampicin, isoniazid and
D-cycloserine, excluding compounds having alkyl substituent at triple alkyne bond.
Derivatives of uracil have found significant and wide application in the construction of
pharmacologically valuable compounds.1-3 Due to strong binding to the various biological
targets, uracil derivatives can enhance the physiological effect or give a new type of activity to
the organic molecules. It is known that 5-substituted uracil derivatives are good recommended as
selective HIV-1 reverse transcriptase inhibitors,4 antagonists of AMPA5 and kainate receptors6,
and compounds with a potent antimycobacterial activity against Mycobacterium tuberculosis7.
According to the World Health Organization (WHO), tuberculosis is an extremely socially
dangerous disease, killing millions of people around the world.8 It should be noted that
appearance of new drug-resistant tuberculosis strains, which are responsible for infection and
development of tuberculosis in biological objects (Mycobacterium tuberculosis and
Mycobacterium avium), encourages to creation of novel therapeutic agents9,10. In this regard, the
creation of new effective inhibitors of mycobacteria responsible for the onset and development
of tuberculosis is extremely urgent. An analysis of the literature data of the currently existing
compounds with antimycobacterial activity against Mycobacterium tuberculosis and
Mycobacterium avium showed that effective inhibitors of the aforementioned mycobacteria are
uracil derivatives and nucleosides based on it.11,12 In a number of studies, it was found that the
presence of an alkynyl fragment at the C-5 position of pyrimidine nucleosides plays a major role

in their antimycobacterial properties.12-14 Unfortunately, the mechanisms of their action and
target enzymes were unclear.7 In this reason, we selected 5-alkynyl-substituted derivatives of
uracil and uridine as the objects of study - potential inhibitors of mycobacteria, the structure of
which is shown in Figure 1.
R
O
NH
R
O
N
O
HO
NH
O
N
H
O
OH OH
Figure 1. The structures of C-5-substituted derivatives of uracil and uridine
RESULTS AND DISCUSSION
To introduce the alkynyl fragment in the C-5 position of the pyrimidine heterocycle, we
have chosen the palladium-catalyzed Sonogashira cross-coupling reaction since it allows direct
C-alkynylation without the formation of a large number of by-products (Scheme 1). The starting
5-bromouracil was synthesized by a regioselective C-5 bromination reaction using
N-bromosuccinimide as a brominating agent in 92% yield. It should be noted that reaction in
absolute dichloromethane with using of NBS significantly increased the conversion of the
product in comparison with other brominating agents and allow avoiding the formation of
by-products. Unlike 5-bromouracil, the uridine bromination reaction was carried out using
N-bromosuccinimide and sodium azide, since a halogenation at the C-5 position of the
pyrimidine ring of the nucleoside requires the presence of a substituent and at the same time a
well-leaving group at the C-6 position.
It should be noted that we have chosen palladium(II) tetra-tert-butyl-substituted phthalocyanine
as catalyst for Sonogashira cross-coupling reaction because this Pd complex has shown excellent
catalytic activity in reaction of phenylacetylene with p-substituted aryl bromides and chlorides
with it’s low loading (0.5 mol%) at room temperature.15 During the selection and optimization of
the reaction conditions of 5-bromouracil with phenylacetylene, it was found that the optimal
solvent for the reaction is N,N-dimethylformamide (DMF) and cesium carbonate (Table 1). It
should be noted that the use of polar solvents (DMF, N,N-dimethylacetamide (DMA),
acetonitrile) is associated exclusively with the low solubility of the initial substrate due to the
formation of intermolecular hydrogen bonds.

N
R2
N
N
N
N
O
NBS, DCM, RT (R1=H)
or NBS, NaN3, DME, RT,
(R1=riboside)
O
O
R2
Pd
tBuPcPd =
N
N
Br
NH
tBuPcPd (0.5 mol%)
NH
NH
CuI (1mol%)
Cs2CO3 (3 eq.)
N
N
R1
O
N
O
N
R1
O
R1
DMF, RT
1-15
73-89%
R1=H, riboside; R2=Ph, SiMe3, Me, Et,
n-Bu, n-C6H14, c-C3H7, c-C6H14,
C6H5-CH(OH)
O
O
Me3Si
O
O
Me
O
NH
NH
NH
NH
NH
N
O
5
80%
N
H
O
H
N
H
O
N
H
O
N
O
H
1
89%
4
86%
2
87%
3
84%
O
N
OH
O
NH
O
O
O
NH
NH
O
NH
NH
HO
N
O
N
H
O
O
H
N
H
O
N
H
82%
O
7
82%
6
83%
OH OH
10
8
9
84%
73%
OH
O
N
O
N
O
N
O
O
NH
NH
NH
NH
NH
O
O
O
HO
HO
O
N
O
N
O
HO
HO
O
HO
O
O
O
OH OH
11
OH OH
12
OH OH
13
OH OH
15
OH OH
14
76%
81%
77%
77%
79%
Scheme 1. Synthesis of C-5-substituted derivatives of uracil and uridine via Sonogashira
coupling reaction
The use of amines as base, such as triethylamine (Et3N), N,N-diisopropyl-ethylamine (i-Pr2NEt),
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), led to a decrease in the conversion of the target
product due to the formation of by-products. The reaction progress was monitored by
chromatography-mass spectrometry (GC/MS) and the conversion of the starting substrate was
determined by the peak area in the chromatogram. To exclude a spontaneous reaction without
using a catalyst, a blank experiment was conducted between phenylacetylene and 5-bromouracil,
and a complete absence of any conversion of the starting substrates was found. Comparison of
the catalytic activity of the system based on the palladium complex of phthalocyanine with
traditional and commonly used catalysts (Pd(PPh3)2Cl2, Pd(PPh3)4, Pd(OAc)2) showed the high
efficiency of the complex which we have developed. The obtained data can be explained by the
agglomeration of palladium particles and the formation of the so-called “metallic black”, which
leads to a loss of the catalytic activity of the system due to the release of the palladium complex
from the reaction medium.

Table 1. Optimisation of Sonogashira cross-coupling reaction of phenylacetylene with
5-bromouracil
O
O
tBuPcPd (0.5 mol%)
Br
NH
NH
CuI (1 mol%)
Base (3 eq.)
Solvent
250C, 4h
N
H
O
N
H
O
1
Entry
Solvent
Base
Yieldа (%)
Yielda (%) with
Pd(PPh3)4
1
2
CH3CN
CH3CN
CH3CN
CH3CN
DMA
DMA
DMA
DMA
DMF
Et3N
i-Pr2NEt
Cs2CO3
DBU
26
32
77
41
21
30
68
52
32
44
89
74
Trace
5
3
7
4
11
14
9
5
Et3N
6
i-Pr2NEt
Cs2CO3
DBU
7
21
17
20
14
15
24
8
9
Et3N
10
11
12
DMF
i-Pr2NEt
Cs2CO3
DBU
DMF
DMF
aYield was determined by GC/MS without of product isolation
After establishing the optimal reaction conditions of Sonogashira cross-coupling reaction
between 5-bromouracil and phenylacetylene a series of 5-alkynyl-substituted derivatives of
uracil and uridine was successfully synthesized, varying the nature of the substituents at the
terminal triple -C≡C bond of the acetylene fragment to establish structure-activity relationship.
It was also found that the use of a sufficiently small amount of both a catalyst and a co-catalyst
of copper (I) iodide made it possible to avoid both the formation of bicyclic furano[2,3-
d]pyrimidine and the product of oxidative dimerization of the starting acetylene proceeding
according to the Glaser reaction mechanism.
The structure of the obtained compounds was unequivocally confirmed by NMR and high
resolution mass spectrometry. The purity of the obtained 5-alkynyl-substituted derivatives of
uracil and uridine was determined using HPLC/MS and elemental analysis. It was established
that the values of chemical shifts in the 13C NMR spectra in the aromatic region of

5-alkynyluridines were very different from furano[2,3-d]pyrimidines. In high-resolution mass
spectra the m/z values are fully correspond to the calculated ones and the obtained
fragmentations also confirm the formation of target compounds.
The obtained 5-alkynyl-substituted derivatives of uracil 1-9 and uridine 10-15 were tested for
cytotoxicity and antimycobacterial activity. Cytotoxicity was determined by measuring 50%
inhibition of cell growth (CC50, average concentration of cytotoxicity) using the MTT test
(calorimetric test using MTT dye (3-4,5-dimethylthiazol-2-yl-2,5-diphenylterazole)). The MTT
cytotoxicity test data showed (Table 2) that all of the above compounds have a fairly low
cytotoxicity with respect to the studied cell lines. Only 5-phenylethinyluridine 10 and
5-(3-hydroxy-3-phenylprop-1-yn-1-yl)uridine 15 showed a rather high toxicity on the MRC-5,
A-549 and Vero cell lines.
Table 2. Cytotoxicity of C-5-alkynyl-substituted derivatives of uracil and uridine
Compound
Cell line (СС50, µМ)
A-549
MRC-5
75.1±0.5
124.0±0.8
117.2±0.9
119.6±0.8
112.8±0.7
94.9±0.5
101.2±0.6
62.8±0.5
38.4±0.4
1.2±0.1
Vero
1
2
248.3±3.6
268.2±4.4
256.1±3.2
252.6±2.9
250.4±2.6
248.7±2.8
251.4±2.9
244.8±2.5
236.7±2.3
42.5±0.4
254.5±4.2
269.7±4.6
262.5±4.4
263.4±4.1
253.4±3.8
249.8±3.6
252.4±3.9
247.4±3.4
242.1±3.3
78.4±0.6
3
4
5
6
7
8
9
10
11
12
13
14
15
11.6±0.3
15.7±0.4
24.2±0.3
36.8±0.4
0.9±0.1
83.2±0.7
102.6±1.5
111.4±1.2
132.7±1.4
148.3±1.1
65.4±0.5
87.2±0.6
114.6±0.9
126.7±0.8
34.2±0.4
CC50 (50% cytotoxic concentration), concentration that caused a 50% decrease in cell viability
by the standard formazan method (MTT). The data are expressed as CC50±SD (µM).
The target compounds 1-15 were also tested for antimycobacterial activity against
Mycobacetrium bovis and Mycobacterium tuberculosis (H37Ra) using a quantitative test with
alamar blue reagent (Microplate Alamar Blue Assay test) in concentrations from 1 to 100 μg/ml.

Rifampicin, isoniazid and D-cycloserine were used as substances in the control group, since they
are known and effective drugs used in the treatment of tuberculosis.
Table 3. Antimycobaceterial activity of C-5-alkynyl-substituted derivatives of uracil 1-9 and
uridine 10-15
Compound
M. Bovis (BCG)
MIC50[µg/ml]
M. tuberculosis (H37Ra)
MIC50[µg/ml]
% inhibition
% inhibition
(concentration, µg/ml)
(concentration, µg/ml)
1
2
3
4
5
6
90(50,100), 40(10)
17.4±0.4
90(100,50), 40(10)
18.2±0.5
50(100)
100
50(100)
100
0
0
0
0
-
-
-
-
0
-
-
-
-
0
0
0
90(100), 70(50), 60(10),
50(5)
7
80(100), 60(50,10)
7.8±0.5
5.2±0.2
8
90(100), 75(50,10), 50(1-5)
90(100,50), 80(10), 50(1-5)
90(100,50), 75(10), 50(1-5)
90(100), 50(50), 0(10)
90(100), 60(50), 0(10)
90(100), 80(10), 50(1-5)
100(50), 60(25), 10(1-5)
100(1-5)
2.5±0.3
3.1±0.3
2.7±0.2
50
90(100), 75(50,10), 50(1-5)
90(100,50), 75(10), 50(1-5)
90(100,50), 70(10), 50(1-5)
90(100), 50(50), 10(10)
90(100), 70(50), 0(10)
90(100), 70(10), 50(1-5)
100(50), 55(25), 10(1-5)
100(1-5)
2.8±0.2
2.9±0.4
3.2±0.3
50
9
10
11
12
13
<50
<50
2.4±0.2
19.2±0.5
1.4±0.1
0.8±0.1
0.9±0.1
2.7±0.4
18.9±0.4
1.1±0.1
0.7±0.2
0.8±0.1
14
15
Rifampicin
Isoniazid
D-
100(1)
100(1)
100(1)
100(1)
71(10), 50(5)
5.1±0.3
67(10), 50(5)
5.2±0.2
cycloserine
The data of the MABA test for antimycobacterial activity showed (Table 3) that most of the
synthesized derivatives of 5-alkynyl-substituted derivatives of uracil and uridine have a fairly
high inhibition efficiency of the studied cell lines, with the exception of compounds having an
alkyl substituent at the acetylene fragment. As in the case of cytotoxicity studies on the MRC-5,
A-549 and Vero cell lines, 5-phenylethynyluridine 10 and 5-(3-hydroxy-3-phenylprop-1-yn-1-
yl)uridine 15 showed high activity, which, as we believe, is associated with the presence of a
riboside moiety and effects of substituent at C≡C bond. Based on the data of in vitro biological
studies, it can be concluded that it is possible to use the synthesized 5-alkynyl-substituted
derivatives of uracil and nucleosides based on it as chemotherapeutic agents for the treatment of
tuberculosis.

ACKNOWLEDGEMENTS
The work was supported by a grant from the Russian Science Foundation (RSF Grant No. 18-73-
00216).
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Declaration of interests
x The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

N
O
R
R
O
N
N
N
N
Br
tBuPcPd (0.5 mol%)
tBuPcPd =
NH
Pd
N
N
NH
CuI (1mol%)
Cs2CO3 (3 eq.)
N
O
N
O
N
R'
DMF, RT
R'
1-15
73-89%
Antimycobacterial activity against Mycobacetrium bovis and Mycobacterium tuberculosis, MIC50=1.1-100 µg/ml
R=Ph, SiMe3, Me, Et, n-Bu, n-C6H14,
c-C3H7, c-C6H14, C6H5-CH(OH);
R'=H, riboside
Highlights
 A series of new 5-alkynyl-substituted uracil derivatives were synthesised via
Sonogashira cross-coupling reaction.
 New palladium catalyst has shown excellent catalytic activity to yield C-5-
alkynyl-substituted uracils with good yields.
 Obtained target compounds were characterized by NMR, FT-IR
spectroscopy, HRMS and elemental analysis.
 Target compounds were tested on antimycobacterial activity against
Mycobacetrium bovis and Mycobacterium tuberculosis.

 Most of tested uracil derivatives exhibited high antimycobaceterial activity
against mycobacteria (MIC50=1.1-19.2 µg/ml).