5-arylaminouracil derivatives: New inhibitors of Mycobacterium tuberculosis

Article abstract of DOI:10.1111/cbdd.12603

Three series of 5-arylaminouracil derivatives, including 5-(phenylamino)uracils, 1-(4′-hydroxy-2′-cyclopenten-1′-yl)-5-(phenylamino)uracils, and 1,3-di-(4′-hydroxy-2′-cyclopenten-1′-yl)-5-(phenylamino)uracils, were synthesized and screened for potential antimicrobial activity. Most of compounds had a negative effect on the growth of the Mycobacterium tuberculosis H37Rv strain, with 100% inhibition observed at concentrations between 5 and 40 μg/mL. Of those, 1-(4′-hydroxy-2′-cyclopenten-1′-yl)-3-(4?-hydroxy-2?-cyclopenten-1?-yl)-5-(4″-butyloxyphenylamino)uracil proved to be the most active among tested compounds against the M. tuberculosis multidrug-resistant strain MS-115 (MIC₉₀ 5 μg/mL). In addition, the thymidylate kinase of M. tuberculosis was evaluated as a possible enzymatic target. Three series of 5-arylaminouracil derivatives were synthesized and screened for potential antimicrobial activity. Most of compounds had a negative effect on the growth of the M. tuberculosis H37Rv strain, with 100% inhibition observed at concentrations between 5 and 40 μg/mL. Of those, 1-(4′-hydroxy-2′-cyclopenten-1′-yl)-3-(4?-hydroxy-2?-cyclopenten-1?-yl)-5-(4″-butyloxyphenylamino)uracil proved to be the most active among tested compounds against the M. tuberculosis multidrug-resistant strain MS-115 (MIC₉₀ 5 μg/mL).

Full text of DOI:10.1111/cbdd.12603

Chem Biol Drug Des 2015; 86: 1387–1396
Research Article
5-Arylaminouracil Derivatives: New Inhibitors of
Mycobacterium tuberculosis
1
,†
2,†
Elena Matyugina , Mikhail Novikov , Denis
approximately 9 M new cases in 2013, 1.5 M deaths,
including 0.36 M coinfected with both HIV and TB. The
rapid emergence and spread of drug-resistant strains of
Mycobacterium tuberculosis requires new and more effica-
cious treatments and, in particular, new anti-TB agents. In
that regard, several pyrimidine derivatives have proved to
be highly effective as antiviral, anticancer, antifungal, and
pertinent to this work, antibacterial agents (1). These
reports have led to a new focus for exploring pyrimidine
heterocyclic inhibitors of M. tuberculosis.
2
2
Babkov , Alexander Ozerov , Larisa
Chernousova , Sofia Andreevskaya , Tatiana
Smirnova , Inna Karpenko , Alexander Chizhov ,
Pravin Murthu , Stefan Lutz , Sergei Kochetkov ,
Katherine L. Seley-Radtke * and Anastasia L.
Khandazhinskaya
3
3
3
1
4
5
5
1
6
,
1
1
Engelhardt Institute of Molecular Biology RAS, Vavilova
2, Moscow 119991, Russia
3
2
Department of Pharmaceutical & Toxicological Chemistry,
Volgograd State Medical University, Pavshikh Bortsov
Sq., 1, Volgograd 400131, Russia
Central Tuberculosis Research Institute RAMS, Yauzskaya
Alley 2, Moscow 107564, Russia
Zelinsky Institute of Organic Chemistry RAS, Leninsky
pr. 47, Moscow 119991, Russia
Department of Chemistry, Emory University, 1515 Dickey
Drive, Atlanta, GA 30322, USA
Department of Chemistry & Biochemistry, University of
Maryland, Baltimore, MD 21250, USA
Corresponding author: Katherine L. Seley-Radtke,
kseley@umbc.edu
Notably, TB utilizes a number of enzymes in the pyrimidine
salvage pathway which provide attractive targets for
rational drug design. This pathway is vital for all bacterial
cells and is composed of enzymes significantly different
from those present in humans (2). Moreover, the enzymes
involved in the pyrimidine salvage pathway have been pro-
posed to have an important role in the mycobacterial latent
state, as M. tuberculosis has to recycle heterobases and/
or nucleosides to survive in the hostile environment
imposed by the host (2). During last 10 years, a variety of
pyrimidine analogues have been reported to inhibit TB (3).
For example, bicyclic thymidine analogues I (Figure 1)
were shown to inhibit M. bovis (4), while derivatives of
3
4
5
6
*
†
Equal contribution.
Three series of 5-arylaminouracil derivatives, including
-(phenylamino)uracils, 1-(4 -hydroxy-2 -cyclopenten-
-yl)-5-(phenylamino)uracils, and 1,3-di-(4 -hydroxy-
-cyclopenten-1 -yl)-5-(phenylamino)uracils,
synthesized and screened for potential antimicrobial
activity. Most of compounds had a negative effect on
the growth of the Mycobacterium tuberculosis H37Rv
strain, with 100% inhibition observed at concentrations
between 5 and 40 lg/mL. Of those, 1-(4 -hydroxy-2 -cy-
clopenten-1 -yl)-3-(4‴-hydroxy-2‴-cyclopenten-1‴-yl)-5-
1
-benzyluracil II lacking the ribose moiety have been
0
0
5
1
2
patented as anti-TB compounds (5). The most active
0
0
0
0
0
compounds showed inhibitory activity with MIC50 s of
were
4
5–55 lg/mL (6).
Based on those results, the design and synthesis of a ser-
ies of 5-(phenylamino)uracils (III, Figure 1), which are
structural analogues of II, was undertaken in an effort to
pursue their potential anti-TB activity.
0
0
0
(4″-butyloxyphenylamino)uracil proved to be the most
active among tested compounds against the M. tuber-
culosis multidrug-resistant strain MS-115 (MIC90 5 lg/
mL). In addition, the thymidylate kinase of M. tuberculo-
sis was evaluated as a possible enzymatic target.
Methods and Materials
General
All reagents (highest grade available) were commercially
available and used without further purification unless other-
wise noted. Anhydrous DMF, isopropanol, and ethylene
glycol were purchased from Sigma-Aldrich Co (Madison,
WI, USA). NMR spectra were registered on a Bruker
Key words: carbocyclic nucleosides, Mycobacterium tuber-
culosis, uracil derivatives
Received 11 February 2015, revised
accepted for publication 30 May 2015
8 April 2015 and
1
Avance 400 spectrometer (400 MHz for H and 100 MHz
13
for C) in CDCl , DMSO-D , or CD CN with tetramethylsi-
3
6
3
Tuberculosis (TB) remains a global health issue in many
countries, with the World Health Organization reporting
lane as an internal standard. High-resolution mass spectra
were measured on Bruker micrOTOF II or maXis (Bruker,
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12603
1387

Matugina et al.
O
O
N
O
R
HN
HN
HN
NH
X
O
N
O
O
O
HN
O
S
R
CO H
NH
2
II
R = Me, Cl
III
X = CH, N
R = Alkyl, Alkoxy
I
Figure 1: Pyrimidine containing inhibitors of
M. tuberculosis.
+
Bremen, Germany) instruments using electrospray ioniza-
tion (HRESIMS). The measurements were taken in a posi-
tive ion mode (interface capillary voltage ꢀ4500 V) in a
mass range from m/z 50 to m/z 3000 Da; external or inter-
nal calibration was performed with Electrospray Calibrant
C
10
H
9
N
3
O
2
[M + H]
204.0768. Anal. calcd for
C H N O : C, 59.11; H, 4.46; N, 20.68; found: C, 59.18;
1
0 9 3 2
H, 4.41; N, 20.75.
TM
Solution (Fluka, Buchs, Switzerland). A syringe injection
5-[(3-Methylphenyl)amino]uracil (2)
was used for solutions in acetonitrile (flow rate 3 lL/min).
Nitrogen was applied as a dry gas, and it was produced by
Dominick Hunter LC-MS nitrogen generator; interface tem-
perature was set at 180 °C. TLC was performed on Merck
Yield 66%; mp: 324–326 °C. E1(k259.6) = 17 500,
1
E
2
3
(k328.2) = 6200. H-NMR (DMSO-d
6
1
): d 11.25 (1H, s,
N H), 10.62 (1H, d, J = 3.9 Hz, N H), 7.28 (1H, d,
J = 5.9 Hz, H-6), 6.98 (1H, t, J = 7.6 Hz, H-5 ), 6.47–6.53
0
0
0
0
13
(Kenilworth, NJ, USA) TLC Silica gel 60 F254 plates and
(4H, m, H-2 , H-4 , H-6 , 5-NH), 2.17 (3H, s, CH
3
). C-
developed with iodine or using UV lamp VL-6.LC (Vilber-
Lourmat Deutschland, Eberhardzell, Germany). Melting
points were determined using a MEL-TEMP 3.0 (Laboratory
Devices Inc., Placerville, CA, USA). Yields refer to spectro-
scopically ( H and C NMR) homogeneous materials. The
purities of the compounds were determined using elemental
analysis (C, H, N) on a CE1106 automatic analyzer (Italy)
and agreed with the theoretical values within 0.4%.
NMR (DMSO-d ): d 162.8, 150.8, 146.6, 138.3, 132.9,
6
129.0, 119.2, 116.2, 115.1, 111.9, 21.6. HRMS: found
+
m/z 218.0917; calcd for C H N O [M + H] 218.0924.
1
1 11 3 2
11 3 2
Anal. calcd for C11H N O : C, 60.82; H, 5.10; N, 19.34;
1
13
found: C, 60.80; H, 5.04; N, 19.31.
5-[(4-Methylphenyl)amino]uracil (3)
1
1
Yield 82%; mp: dec > 330 °C. E (k260.4) = 15 100. H-
3
1
NMR (DMSO-d ): d 11.21 (1H, s, N H), 10.51 (1H, s, N H),
6
General method for the synthesis of the
7.19 (1H, s, H-6), 6.94–6.92 (2H, m, H-3″, H-5″), 6.70–
13
5
6
-(phenylamino)uracils 1–8 and 5-(phenylamino)-
-azauracils 9–10
6.67 (3H, m, 5-NH, H-2″, H-6″), 2.17 (3H, s, CH3). C-
NMR (DMSO-d ): d 162.28, 150.27, 143.33, 129.94,
129.24 (C-3″, C5″), 126.84, 116.62, 114.91 (C-2″, C-6″),
20.10. HRMS: found m/z 218.0927; calcd for C11
6
A suspension of 5-bromo-6-azauracil (2.5 g, 13.09 mmol)
and aniline (40 mmol), 4-methylaniline (40 mmol), or 4-phe-
noxyaniline (40 mmol) was heated in 30 mL freshly distilled
ethylene glycol, until a homogeneous solution was observed.
The solution was then refluxed for 2 h, then cooled to room
temperature at which point the amination product precipi-
tated out. The reaction mixture then was poured into cold
water (150 mL), the solid filtered off, washed with water, and
reprecipitated from a 5% aqueous solution of NaOH. The
product was then recrystallized from DMF.
11 3 2
H N O
+
11 3 2
[M + H] 218.0924. Anal. calcd. for C11H N O : C, 60.82;
H, 5.10; N, 19.34; found: C, 60.98; H, 5.09; N, 19.43.
5-[(2,3-Dimethylphenyl)amino]uracil (4)
Yield 74%; mp: 306.5–308 °C.
E
1
(k253) = 13 600,
1
E (k320) = 5300. H-NMR (DMSO-d ): d 11.28 (1H, s,
2
6
3
1
N H), 10.53 (1H, d, J = 5.2 Hz, N H), 6.95 (1H, d,
0
J = 5.6 Hz, H-6), 6.88 (1H, t, J = 7.8 Hz, H-5 ), 6.64 (1H,
0
0
d, J = 7.3 Hz, H-6 ), 6.51 (1H, d, J = 8.0 Hz, H-4 ), 6.09
5
-(Phenylamino)uracil (1)
(1H, br. s, NH-Ar), 2.20 (3H, s, CH ), 2.05 (3H, s, CH ).
3
3
1
13
Yield 68%; mp: > 330 °C. E
1
3
(k260.0) = 12 400. H-NMR
6
C-NMR (DMSO-d ): d 162.5, 150.7, 143.6, 136.9,
129.8, 125.9, 124.2, 122.0, 118.0, 114.0, 20.6, 13.3.
1
(DMSO-d
6
): d 11.27 (1H, s, N H), 10.59 (1H, s, N H), 7.29
(
5
1H, s, H-6), 7.11–7.09 (2H, m, H-2″, H-6″), 6.83 (1H, s,
-NH), 6.75–6.72 (2H, m, H-3″, H-5″), 6.68–6.64 (1H, m,
HRMS: found m/z 231.1008; calcd for C12H N O [M]
13
3
2
231.1002, found m/z 232.1078; calcd for C12
H
13
N
3
O
2
1
3
+
H4″). C-NMR (DMSO-d ): d 162.38, 150.43, 146.26,
[M + H] 232.1081. Anal. calcd for C H N O : C,
6
12 13 3 2
1
32.45, 128.79 (C-3″, C5″), 117.90, 115.75, 114.26
62.33; H, 5.67; N, 18.34; found: C, 62.44; H, 5.68; N,
18.17.
(C-2″, C-6″). HRMS: found m/z 204.0771; calcd for
1
388
Chem Biol Drug Des 2015; 86: 1387–1396

5-Arylaminouracil Derivatives
3
1
5
-[(2,5-Dimethylphenyl)amino]uracil (5)
Yield 50%; mp: 269–270 °C. (k261.2) = 14 000,
): d 11.27 (1H, s,
N H), 11.28 (1H, s, N H), 8.61 (1H, s, 5-NH), 7.73 (2H, d,
0 0 0
J = 8.0 Hz, H-2 , H-6 ), 7.26 (2H, t, J = 8.4 Hz, H-3 , H-
E
1
1
0
0
13
E
2
(k322.5) = 4700. H-NMR (DMSO-d
6
1
5 ), 6.94 (1H, t, J = 7.2 Hz, H-4 ). C-NMR (DMSO-d
6
): d
3
00
00
N H), 10.60 (1H, d, J = 4.4 Hz, N H), 7.13 (1H, d,
J = 5.6 Hz, H-6), 6.91 (1H, d, J = 7.3 Hz, H-3 ), 6.49 (1H,
d, J = 7.6 Hz, H-4 ), 6.39 (1H, s, H-6 ), 6.10 (1H, br. s, 5-
154.6, 148.4, 139.7, 139.6, 128. 4(C-3 , C-5 )128.4,
121.5, 118.5 (C-2 , C-6 ). HRMS: found m/z 204.0647;
calcd for C H N O [M] 204.0642, found m/z 205.0725;
0
00
00
0
0
9
8 4 2
13
+
NH), 2.14 (3H, s, CH
DMSO-d ): d 162.6, 150.9, 144.0, 142.5, 135.7, 132.1,
30.4, 121.6, 119.9, 116.9, 114.9, 21.3, 17.5. HRMS:
found m/z 231.1005; calcd for C12 [M] 231.1002,
found m/z 232.1081; calcd for C12H N O [M + H]
3
), 2.10 (3H, s, CH
3
). C-NMR
calcd for C
9 8 4 2
H N O [M + H] 205.0720. Anal. calcd. for
: C, 52.94; H, 3.95; N, 27.44; found: C, 52.91;
(
1
6
9 8 4 2
C H N O
H, 3.95; N, 27.51.
13 3 2
H N O
+
13
3 2
2
32.1081. Anal. calcd for C12
H
13
N
3
O
2
: C, 62.33; H 5.67;
5-[(4-Butylphenyl)amino]-6-azauracil (10)
1
N, 18.21; found C, 62.54; H, 5.61; N, 18.17.
Yield 47%; mp: 306–308 °C. E(k2 ) = 15 150. H-MNR
60.2
3
(
DMSO-d
6
): d 11.96 (1H, br. s, N H), 11.23 (1H, br. s,
1
0
N H), 8.51 (1H, s, 5-NH), 7.61 (2H, d, J = 8.4 Hz, H-2 , H-
0
0
0
5
-[(4-Butylphenyl)amino]uracil (6)
6 ), 7.07 (2H, d, J = 8.8 Hz, H-3 , H-5 ), 2.50 (2H, m,
CH ), 1.52 (2H, m, CH ), 1.29 (2H, m, CH ), 0.88 (3H, t,
1
Yield 77%; mp: > 330 °C. E(k262) = 19200. H-NMR
2
2
2
3
1
13
(DMSO-d ): d 11.21 (1H, br. s, N H), 10.52 (1H, br. s, N H),
J = 7.4 Hz, CH3). C-NMR (DMSO-d ): d 154.7, 148.4,
6
6
0
0
7
6
.22 (1H, s, H-6), 6.94 (2H, d, J = 8.4 Hz, H-2 , H-6 ), 6.68–
.70 (3H, m, 5-NH, H-2″, H-6″), 2.44 (2H, t, J = 7.6 Hz,
139.7, 137.4, 135.5, 128.2, 118.6, 34.1, 33.2, 21.6, 13.7.
HRMS: found m/z 260.1270; calcd for C H N O [M]
13
16
4
2
CH
2
), 1.48 (2H, m, CH
2
), 1.29 (2H, m, CH
). C-NMR (DMSO-d ): d 162.3, 150.2,
43.6, 131.9, 130.2, 128.5, 116.5, 114.7, 34.0, 33.4, 21.6,
2
), 0.88 (3H, t,
260.1268, found m/z 261.1341; calcd for C13
H
16
N
4
O
2
13
+
J = 7.3 Hz, CH
3
6
16 4 2
[M + H] 261.1346. Anal. calcd. for C13H N O : C, 59.99;
1
1
H, 6.20; N, 21.52; found: C, 60.11; H, 6.21: N, 21.51.
3.7. HRMS: found m/z 259.1315; calcd for C14
H
17
N
3
O
2
[
[
M] 259.1315, found m/z 260.1387; calcd for C H N O
14 17 3
2
+
0
0
0
M + H] 260.1394. Anal. calcd for C14
H
17
N
3
O
2
: C, 64.85;
1-(4 -Hydroxy-2 -cyclopenten-1 -yl)-5-(phenylamino)
uracil (11)
H, 6.61; N, 16.20; found: C, 64.97; H, 6.68; N, 16.36.
To a solution of 1 (200 mg, 0.7 mmol) in dry DMF (50 mL)
epoxycyclopentene (63 mg, 0.77 mmol) in freshly distilled
THF (15 mL) and Pd(PPh3)4 (40 mg, 0.035 mmol) was
added. The reaction mixture was stirred for 4 h at rt. The
solvents were concentrated under reduced pressure, and
the residue purified by column chromatography on silica
5
-(4-Butyloxyphenylamino)uracil (7)
1
Yield 74%; mp: > 330 °C. E(k259.3) = 18 950. H-NMR
3
(DMSO-d ): d 11.21 (1H, br. s, N H), 10.42 (1H, br. s,
6
1
N H), 7.07 (1H, s, H-6), 6.76–6.78 (4H, m, aromatic H),
.54 (1H, s, 5-NH), 3.86 (2H, t, J = 6.5 Hz, CH O), 1.65
2H, m, CH ), 1.42 (2H, m, CH ), 0.92 (3H, t, J = 7.4 Hz,
). C-NMR (DMSO-d ): d 162.1, 152.0, 150.0, 138.6,
6
(
CH
2
3
eluting with CHCl :MeOH (98:2) to give 11 as an off-white
2
2
powder (56 mg, 0.16 mmol, 20%). Mp: decomposed at
1
3
1
3
6
1 2
214 °C. E (k263.4) = 21 500, E (k331.8) = 9700. H-NMR
1
26.6, 117.8, 117.1, 115.1, 67.4, 30.8, 18.7, 13.6. HRMS:
found m/z 275.1256; calcd for C14 [M] 275.1264.
Anal. calcd. for C H N O : C, 61.08; H, 6.22; N, 15.26;
(DMSO-d ): d 11.46 (1H, s, NH), 7.41 (1H, s, H-6), 7.12–
6
H
17
N
3
O
3
7.08 (2H, m, H-2″, H-6″), 7.01 (1H, s, 5-NH), 6.80–6.78
(2H, m, H-3″, H-5″), 6.69–6.67 (1H, m, H-4″), 6.14–6.12
14 17 3 3
0
found: C, 61.09; H, 6.31: N, 15.28.
(1H, d, J = 5.52 Hz, H-2 ), 5.85–5.83 (1H, d, J = 5.51 Hz,
0
0
H-3 ), 5.47–5.45 (1H, m, H-1 ), 5.25 (1H, s OH), 4.64–4.62
0
0
(
H
1H, m, H-4 ), 2.76–2.72 (1H, m, H -5 ), 1.46–1.43(1H, m,
0
-5 ). C-NMR (DMSO-d ): d 161.30, 149.63 (C-4, C-2),
b 6
145.48 (C-1″), 140, 130.97 (C-2 , C-3 ), 130.36 (C-6),
128.78 (C-3″, C-5″), 118.10 (C-5), 116.99 (C-4″), 114.41
(C-2″, C-6″), 73.13 (C-1 ), 58.16 (C-4 ), 40.91(C-5 ). HRMS:
found m/z 308.1001; calcd for C15
308.1006. Anal. calcd for C15 : C, 63.15; H, 5.30;
N, 14.73; found: C, 63.22; H, 5.38; N, 14.77.
a
13
5
-[(4-Phenoxyphenyl)amino]uracil (8)
1
0
0
Yield 59%; mp: 329–331 °C. E (k260.6) = 28 400. H-NMR
1
3
1
6
(DMSO-d ): d 11.23 (1H, s, N H), 10.58 (1H, s, N H), 7.33–
0
0
0
0 0 0
7.27 (3H, m, H-3 , H-5 , H-6), 7.04–7.00 (1H, m, H-4 ), 6.91–
0
0
+
6.77 (7H, m, 5-NH, H-2 , H-6 H-2″, H-3″, H-5″,
15 3 3
H N O [M + Na]
1
3
H-6″).
C-NMR (DMSO-d
6
): 162.34, 158.41, 150.39,
15 3 3
H N O
0 0
147.55, 142.70, 131.57, 129.73 (C-3 , C-5 ), 122.12
0
0
(
(
C-6), 120.52 (C-2 , C-6 ), 116.85 (C-3″, C5″), 116.26
C-2″, C-6″), 115.77. HRMS: found m/z 295.0941; calcd for
0
0
0
C
H N O
16 13 3 3
13 3 3
[M] 295.0951. Anal. calcd for C16H N O : C,
1-(4 -Hydroxy-2 -cyclopenten-1 -yl)-5-[(4″-
methylphenyl)amino]uracil (12)
65.08; H, 4.44; N, 14.23; found: C, 65.05; H, 4.44; N, 14.33.
In an analogous manner as was used to synthesize (11),
compound (12) was obtained as a pale yellow powder
5
-(Phenylamino)-6-azauracil (9)
(61 mg, 0.2 mmol, 21%); mp: 212–213 °C. E (k264.2)
1
1
Yield 68%; mp: > 330 °C.
E2(k325.8) = 11 900. H-NMR (DMSO-d ): d 11.99 (1H, s,
E
1
(k246.9) = 22600,
= 20 300, E
2
(k334.8) = 8400. H-NMR (DMSO-d
6
): d 11.44
1
(1H, s, NH), 7.35 (1H, s, H-6), 6.94–6.92 (2H, m, H-2″,
6
Chem Biol Drug Des 2015; 86: 1387–1396
1389

Matugina et al.
0
0
0
H-6″), 6.85 (1H, s, 5-NH), 6.75–6.73 (2H, m, H-3″, H-5″),
1-(4 -Hydroxy-2 -cyclopenten-1 -yl)-5-[(4″-
phenoxyphenyl)amino]uracil (15)
0
6
.13–6.11 (1H, d, J = 5.52 Hz, H-2 ), 5.83–5.81(1H, d,
0
0
J = 5.52 Hz, H-3 ), 5.47–5.44 (1H, m, H-1 ), 5.26 (1H, s
OH), 4.63–4.61 (1H, m, H-4 ), 2.75–2.67 (1H, m, H -5 ),
In an analogous manner as was used to synthesize (11),
compound (15) was obtained as a pale yellow powder
(45 mg, 0.12 mmol, 18%). Mp: decomposes at 200 °C.
E1(k265.3) = 26 750, E (k328.8) = 9320. H-NMR (DMSO-
6
d ): d 11.45 (1H, s, NH), 7.40 (1H, s, H-6), 7.32–7.30 (2H,
m, H-3 , H-5 ), 7.04 (2H, m, H-2 and H-6 ), 6.89–6.88
(2H, m, H-3
6.14–6.13 (1H, d, J = 5.50 Hz, H-2 ), 5.84–5.83(1H, d,
J = 5.51 Hz, H-3 ), 5.48–5.46 (1H, m, H-1 ), 5.28–5.27
(1H, m, H-4 ) 4.63–4.62 (1H, s OH), 2.71–2.67 (1H, m,
0 0
H -5 ), 1.44–1.41(1H, m, H -5 ). C-NMR (DMSO-d ): d
a b 6
161.24 (C-4), 158.24 (C-1Ph), 149.57 (C-2), 147.73 (C-1″),
41.85, 139.97 (C-2 , C-3 ), 131.01(C-6), 129.71 (C-3Ph,
0
0
a
0
13
2
.17 (3H, s, CH
3
), 1.45–1.39 (1H, m, H
b
-5 ). C-NMR
1
(
DMSO-d ): d 161.30, 149.63 (C-4, C-2), 145.48 (C-1″),
39.96, 131.00 (C-2 , C-3 ), 129.24 (C-3″, C5″), 127.67,
6
2
0
0
1
1
7
0
0
00
0
0
26.99 (C-6, C-5), 117.79 (C-4″), 115.02 (C-2″, C-6″),
0
0
0
0
0
00
00
00
3.13 (C-1 ) 58.12 (C-4 ), 41.11(C-5 ), 20.07 (CH
3
). HRMS:
[M + H]
,
H-5 ), 6.84 (4H, m, H-2 , H-4 , H-6 , 5-NH),
+
0
found m/z 300.1342; calcd for C16
H
17
N
3
O
3
0
0
3
00.1343; found m/z 322.1159; calcd for C H N O
6 17 3 3
1
+
0
[M + Na] 322.1162. Anal. calcd. for C16
H
17
N
3
O
3
: C,
13
6
1
4.20; H, 5.72; N, 14.07; found: C, 64.12; H, 5.78; N,
4.04.
0 0
1
C-5Ph), 129.51 (C-5), 122.17 (C-4Ph), 120.34 (C-2Ph,
0
0
0
1
-(4 -Hydroxy-2 -cyclopenten-1 -yl)-5-(4″-
C-6Ph), 117.46 (C-4″), 117.46(C-3″, C5″), 115.88 (C-2″,
0
0
0
butylphenylamino)uracil (13)
In an analogous manner as was used to synthesize (11), com-
C-6″), 73.15 (C-1 ) 58.17 (C-4 ), 41.21 (C-5 ). HRMS:
+
found m/z 400.1258; calcd for C21
19 3 4
H N O [M + Na]
pound (13) was obtained as off-white syrup (42 mg,
400.1268. Anal. calcd. for C H N O : C, 66.83; H, 5.07;
N, 11.13; found: C, 66.84; H, 5.08; N, 11.17.
2
1 19 3 4
1
0
.12 mmol, 17%). E(k261.4) = 22 400. H-NMR (DMSO-d
6
): d
11.45 (1H, s, NH), 7.37 (1H, s, H-6), 6.95–6.93 (2H, m, H-2″,
H-6″), 6.87 (1H, s, 5-NH), 6.76–6.74 (2H, m, H-3″, H-5″),
0
0
0
0
0
6
.14–6.12 (1H, m, H-2 ), 5.84–5.83 (1H, m, H-3 ), 5.48–5.45
1-(4 -Hydroxy-2 -cyclopenten-1 -yl)-3-(4‴-hydroxy-
2‴-cyclopent-en-1‴-yl)-5-(phenylamino)uracil (16)
The title compound was obtained as side product in the
synthesis of (11). The purification described for compound
(11) gave product (16) as a yellow syrup (140 mg,
0
0
(1H, m, H-1 ), 5.27 (1H, s OH), 4.63–4.62 (1H, m, H-4 ), 2.74–
0
2.70 (1H, m, H
a
-5 ), 2.46–2.42 (2H, m, CH
2
), 1.49–1.47 (3H,
0
3
m, H -5 , CH ), 1.31–1.29 (2H, m, CH ), 0.90–0.88 (3H, m,
CH
b
2
2
1
3
). C-NMR (DMSO-d
6
): d 161.22, 149.46 (C-4, C-2),
0
0
1
1
42.82 (C-4″), 139.96 (C-2 ), 132.08 (C-1″),130.97 (C-3 ),
28.55 (C-3″, C5″),128.28 (C-6), 117.61 (C-5), 114.74 (C-2″,
0.38 mmol, 39%). E
H-NMR (CDCl ): d 7.37 (1H, s, H-6), 7.24–7.21 (3H, m,
H-3″, H-5″, 5-NH), 6.95–6.94 (2H, m, H-2″
6.86 (1H, m, H-4″), 6.20–6.19 (1H, d, J = 5.51 Hz, H-2 ),
6.14–6.13 (1H, d, J = 5.50 Hz, H-2‴), 5.99–5.97 (2H, m,
H-1 , H-1‴), 5.82–5.81 (1H, d, J = 5.52 Hz, H-3 ), 5.77–
5.75 (1H, d, J = 5.49 Hz, H-3‴), 5.65–5.62 (1H, m, H-4 ),
4.85–4.83 (1H, m, H-4‴), 4.71 (1H, s, OH ), 4.24 (1H, s,
OH‴), 2.92–2.82 (2H, m, H -5 , H -5‴), 2.0 (1H, m, H -5 ),
1
(k264.7) = 15 360, E
2
(kk336.7) = 6000.
1
3
0
0
0
C-6″), 73.14 (C-1 ), 58.11 (C-4 ), 39.09 (C-5 ), 33.93 (CH
2
),
,
H-6″), 6.90–
0
3
3.33 (CH ), 21.62 (CH ), 13.70 (CH ). HRMS: found m/z
2
2
3
+
342.1805; calcd for C19
H
23
N
H
3
O
3
[M + H] 342.1812; found
+
0
0
m/z 364.1629; calcd for C19
Anal. calcd. for C19 : C, 66.84; H, 6.79; N, 12.31;
found: C, 66.74; H, 6.78; N, 12.34.
23
N O
3 3
[M + Na] 364.1632.
0
H N O
23 3 3
0
0
0
a
a
b
13
1
.96(1H, m, H
149.36 (C-4, C-2), 142.08 (C-1″), 139.42, 137.38 (C-2 ,
C-2‴), 132.23, 130.76 (C-3 , C-3‴), 129.54 (C-3″, C5″),
b
-5‴).
C-NMR (DMSO-d
6
): d 160.83,
0
0
0
0
1
-(4 -Hydroxy-2 -cyclopenten-1 -yl)-5-(4″-
0
butyloxyphenylamino)uracil (14)
In an analogous manner as was used to synthesize (11),
128.67 (C-4″), 121.20 (C-6), 118.76 (C-5), 117.20 (C-2″,
0
0
compound (14) was obtained as yellow syrup (54 mg,
C-6″), 76.31 (C-1 ), 74.71 (C-1‴), 60.38 (C-4 ), 56.94 (C-
1
0
0
.15 mmol, 22%). E(k262.2) = 21 100. H-NMR (DMSO-
4‴), 39.89 (C-5 ), 37.56 (C-5‴). HRMS: found m/z
+
d6): d 11.44 (1H, s, NH), 7.28 (1H, s, H-6), 6.84–6.82 (2H,
390.1417; calcd for C H N O [M + Na] 390.1424.
20
21 3 4
m, H-2″, H-6″), 6.75–6.72 (3H, m, 5-NH, H-3″, H-5″),
Anal. calcd. for C20
found: C, 65.35; H, 5.83; N, 11.43.
21 3 4
H N O : C, 65.38; H, 5.76; N, 11.44;
0
6
.13–6.11 (1H, d, J = 5.52 Hz, H-2 ), 5.83–5.80 (1H, d,
0
0
J = 5.51 Hz, H-3 ), 5.48–5.44 (1H, m, H-1 ), 5.26 (1H, s
OH), 4.63–4.61 (1H, m, H-4 ), 3.88–3.85 (2H, m, CH
0
2
),
0
0
0
0
2
.74–2.66 (1H, m, H
.41 (3H, m, CH
a
-5 ), 1.65–1.63 (2H, m, CH
2
), 1.44–
).
1-(4 -Hydroxy-2 -cyclopenten-1 -yl)-3-(4‴-hydroxy-
2‴-cyclopent-en-1‴-yl)-5-[(4″-methylphenyl)amino]
uracil (17)
0
1
2
, H
C-NMR (DMSO-d6):
b
-5 ), 0.94–0.90 (3H, m, CH
3
1
3
d
161.10, 151.97 (C-4, C-2),
0
0
1
1
7
49.29 (C-4″), 139.92 (C-2 ), 137.87 (C-1″), 131.05 (C-3 ),
24.99 (C-6), 116.96 (C-3″, C5″), 115.06 (C-2″, C-6″),
3.13, 73.03 (C-1 , C-4 ), 67.43 (C-5), 58.11 (C-5 ), 39.12
), 30.87 (CH ), 18.71 (CH ), 13.65 (CH ). HRMS:
found m/z 380.1570; calcd for C H N O [M + Na]
The title compound was obtained as side product in the
synthesis of (12). The purification described for compound
(12) gave product (17) as a yellow syrup (122 mg,
0
0
0
1
(CH
2
2
2
3
0.32 mmol, 35%). E
1
(k265.4) = 19 000. H-NMR (CDCl
3
): d
+
7.27 (1H, s, H-6), 7.05–7.03 (3H, m, H-3″, H-5″, 5-NH),
6.87–6.85 (2H, m, H-2″, H-6″), 6.19–6.17 (1H, d,
19 23 3 4
3
80.1581. Anal. calcd. for C19
23 3 4
H N O C, 63.85; H, 6.49;
0
N, 11.76; found: C, 63.84; H, 6.58; N, 11.77.
J = 5.52 Hz, H-2 ), 6.14–6.13 (1H, d, J = 5.51 Hz, H-2‴),
1
390
Chem Biol Drug Des 2015; 86: 1387–1396

5-Arylaminouracil Derivatives
0
6
(
.00–6.99 (1H, m, H-1 ), 5.97 (1H, m, H-1‴), 5.82–5.81
1.63–1.61 (1H, m, H
b
13
-5‴), 1.47–1.46 (2H, m, CH ), 0.97–
2
0
1H, d, J = 5.50 Hz, H-3 ), 5.77–5.75 (1H, d, J = 5.51 Hz,
H-3‴), 5.75–5.63 (1H, m, H-4 ), 4.85–4.84 (1H, m, H-4‴),
.71 (1H, s, OH ), 4.27 (1H, s, OH‴), 2.82–2.76 (2H, m,
-5‴), 2.25 (3H, s, CH
1H, m, H -5 ). C-NMR (DMSO-d ): d 160.78, 149.30
C-4, C-2), 139.29 (C-1″), 137.33 (C-2 , C-2‴), 132.28 (C-
, C-3‴), 130.79 (C-6), 130.05 (C-3″, C5″), 120.32 (C-4″),
0.94 (3H, m, CH
3
). C-NMR (CDCl
3
): d 160.80, 154.71
0
0
(C-4, C-2), 149.35 (C-4″), 139.32 (C-1″), 137.44 (C-2 ,
C-2‴), 134.81 (C-6), 132.33, 130.88 (C-3 , C-3‴), 121.60
0
0
4
H
(
0
0
a
-5 , H
a
3
), 2.0 (1H, m, H
b
-5 ), 1.65
(C-5), 120.64 (C-3″, C5″), 115.80, 115.70 (C-2″, C-6″),
0
00 13
0
0
0
76.41 (C-1 ), 74.85 (C-1‴), 68.32 (C-4 ), 60.57 (C-4‴),
b
6
0
(
56.95 (C-5 ), 39.96 (C-5‴), 37.67 (CH
2
), 31.57 (CH
2
),
0
3
1
1
2
19.41 (CH
calcd for C24
for C24
65.55; H, 6.63; N, 9.59.
2
), 14.00 (CH
[M + Na] 462.1999. Anal. calcd.
29 3 5
H N O : C, 65.59; H, 6.65; N, 9.56; found: C,
3
). HRMS: found m/z 462.1991;
0
+
17.87 (C-2″, C-6″), 117.24 (C-5), 76.31 (C-1 ), 74.76 (C-
‴), 60.42 (C-4 ), 56.90 (C-4‴), 39.90 (C-5 ), 37.72 (C-5‴),
29 3 5
H N O
0
0
0.67 (CH ). HRMS: found m/z 404.1570; calcd for
3
+
C
21
H
23
N
3
O
4
[M + Na]
404.1581. Anal. calcd. for
C H N O : C, 66.13; H, 6.08; N, 11.02; found: C,
2
1 23 3 4
0
0
0
66.09; H, 6.03; N, 11.03.
1-(4 -Hydroxy-2 -cyclopenten-1 -yl)-3-(4‴-hydroxy-
‴-cyclopent-en-1‴-yl)-5-[(4″-phenoxyphenyl)
2
amino]uracil (20)
0
0
0
1
2
-(4 -Hydroxy-2 -cyclopenten-1 -yl)-3-(4‴-hydroxy-
‴-cyclopent-en-1‴-yl)-5-(4″-butylphenylamino)
The title compound was obtained as side product in the
synthesis of (15). The purification described for compound
(15) gave product (20) as a yellow syrup (123 mg,
uracil (18)
The title compound was obtained as side product in the
synthesis of (13). The purification described for compound
0.27 mmol, 40%). E (k
H-NMR (CDCl ): d 7.29 (2H, m, H-3Ph, H-5Ph), 7.24 (1H,
3
) = 21 870, E (k333.0) = 6700.
1
265.0 2
1
(
3
13) gave product (18) as a yellow syrup (96 mg, 0.22 mmol,
s, H-6), 7.04–7.02 (1H, m, H-4Ph), 6.93 (7H, m, H-2Ph,
H-6Ph, H-2″, H-3″, H-5″, H-6″, 5-NH), 6.19–6.18 (1H, d,
1
1%). E(k336.2) = 8300. H-NMR (CDCl ): d 7.98 (1H, s, 5-
3
0
NH), 7.29 (1H, m, H-6), 7.06–7.04 (2H, m, H-3″, H-5″),
6
6
m, H-1 ), 5.82–5.81 (1H, m, H-3‴), 5.77–5.76 (1H, m, H-1‴),
5
s, OH ), 4.25 (1H, s, OH‴), 2.85–2.80 (2H, m, H
2
1
J = 5.52 Hz, H-2 ), 6.14–6.13 (1H, d, J = 5.51 Hz, H-2‴),
0
0
0
.89–6.87 (2H, m, H-2″ H-6″), 6.20–6.18 (1H, m, H-2 ),
6.00 (1H, m, H-1 ), 5.98 (1H, m, H-1‴), 5.83–5.81 (1H, d,
,
0
.15–6.14 (1H, m, H-2‴), 6.00–5.97 (1H, d, H-3 ), 5.94 (1H,
J = 5.50 Hz, H-3 ), 5.77–5.76 (1H, d, J = 5.51 Hz, H-3‴),
0
0
5.65–5.62 (1H, m, H-4 ), 4.84–4.83 (1H, m, H-4‴), 4.73
0
0
0
.64–5.62 (1H, m, H-4 ), 4.85–4.84 (1H, m, H-4‴), 4.72 (1H,
(1H, s, OH ), 4.28 (1H, s, OH‴), 2.83–2.79 (2H, m, H
a
-5 ,
0
0
0
a
-5 , H
a
-5‴),
H
a
0
-5‴), 2.01–1.97 (1H, m, H
5 ). C-NMR (DMSO-d ): d 160.78 (C-4), 158.09 (C-1Ph),
151.26 (C-2), 149.32 (C-4″), 139.33 (C-2 ), 137.80 (C-1″),
137.41 (C-2‴), 132.31 (C-3 ), 130.67 (C-3‴), 129.74 (C-
b
-5 ), 1.67–1.63 (1H, m, H
b
-
0
13
.54–2.50 (2H, m, CH ), 2.01–1.97 (1H, m, H -5 ), 1.68–
2
b
6
0
.67 (1H, m, H
b
-5‴), 1.55–1.53 (2H, m, CH
2
), 1.32–1.30
13
0
(2H, m, CH ), 0.92–0.91 (3H, m, CH ). C-NMR (CDCl ): d
2
3
3
0
0
1
1
1
1
4
60.89, 149.89 (C-4, C-2), 139.43, 137.46 (C-2 , C-2‴),
3Ph, C-5Ph), 122.84 (C-6), 120.61 (C-2Ph, C-6Ph),
120.34 (C-4Ph), 119.11 (C-3″, C5″), 118.12 (C-2″, C-6″),
36.13 (C-4″), 134.0 (C-1″), 132.34, 130.86 (C-3 , C-3‴),
0
0
29.50 (C-3″, C5″), 128.68 (C-6), 120.30 (C-5), 117.70,
117.24 (C-5), 76.32 (C-1 ), 74.72 (C-1‴), 60.42 (C-4 ),
0
0
17.56 (C-2″, C-6″), 76.41 (C-1 ), 74.86 (C-1‴), 60.53 (C-
56.95 (C-4‴), 39.79 (C-5 ), 37.58 (C-5‴). HRMS: found
0
0
+
), 57.0 (C-4‴), 40.0 (C-5 ), 37.68 (C-5‴), 36.61 (CH ), 35.0
m/z 482.1680; calcd for C H N O [M + Na] 482.1686.
2
26 25 3 5
(
CH
2
), 22.48 (CH
2
), 14.09 (CH
3
). HRMS: found m/z
25 3 5
Anal. calcd. for C26H N O : C, 67.96; H, 5.48; N, 9.14;
+
4
4
23.2141; calcd for C H N O [M] 423.2153; found m/z
found: C, 67.95; H, 5.43; N, 9.13.
2
4
29
29
3 4
+
46.2040; calcd for C24
: C, 68.06; H, 6.90; N, 9.92; found:
C, 68.15; H, 6.83; N, 9.93.
H
N
3
O
4
[M + Na] 446.2050. Anal.
29 3 4
calcd. for C24H N O
Cytotoxicity
Vero cells (green monkey kidney epithelial cells, ATCC No
CRL-1586), A549 cells (lung carcinoma cell line, ATCC No
CCL-185), and Huh7 cells (hepatocellular carcinoma cells)
were provided by Laboratory of cell cultures, Ivanovskii
Institute of virology (Moscow) and Engelhardt Institute of
Molecular Biology RAS (Moscow). The cell cultures were
grown in Dulbecco’s modified Eagle’s minimal essential
medium (DMEM) with 10% fetal calf serum (HyClone, GE
Healthcare Life Sciences, HyClone Laboratories, South
Logan, UT, USA) at 37 °C in a 96-well plate in the atmo-
0
0
0
1
2
-(4 -Hydroxy-2 -cyclopenten-1 -yl)-3-(4‴-hydroxy-
‴-cyclopenten-1‴-yl)-5-(4″-butyloxyphenylamino)
uracil (19)
The title compound was obtained as side product in the
synthesis of (14). The purification described for compound
14) gave product (19) as a yellow syrup (115 mg,
.26 mmol, 38%). E(k262.4) = 11 300. H-NMR (CDCl ): d
.98 (1H, s, 5-NH), 7.12 (1H, m, H-6), 6.93–6.91 (2H, m,
(
0
7
1
3
H-3″, H-5″), 6.82–6.79 (2H, m, H-2″ H-6″), 6.16–6.13
sphere of 5% CO . The tested compounds were dissolved
,
2
0
0
(
(
4
3
2H, m, H-2 , H-2‴), 6.00–5.98 (1H, d, H-3 ), 5.80–5.77
in DMSO at a concentration of 40 mg/mL (stock solu-
tions). Cytotoxicity (CD50) was estimated by MTT-assay in
the presence of tested compounds at the concentrations
of 0–100 lg/mL after 72 h of incubation with uninfected
cells and calculated as compound concentration, at which
0
0
3H, m, H-3‴, H-1 , H-1‴), 5.60–5.57 (1H, m, H-4 ), 4.83–
0
.81 (1H, m, H-4‴), 4.71 (1H, s, OH ), 4.30 (1H, s, OH‴),
0
.92–3.90 (2H, m, CH
2
), 2.80–2.79 (2H, m,
H
a
-5 ,
0
H -5‴), 2.0–1.97 (1H, m, H -5 ), 1.65–1.64 (2H, m, CH ),
a
b
2
Chem Biol Drug Des 2015; 86: 1387–1396
1391

Matugina et al.
0% cells died (7). DMSO (molecular biology grade; Sigma-
Aldrich) was additionally purified by double distillation. The
final concentration of DMSO in all tests was ≤ 1%. It has
been shown in control experiments that 1% of DMSO does
not affect the cell cultures we use in our experiments. At
the same time, DMSO at final concentration of 10% was
used as positive control (≥ 50% of cell died).
5
E. coli strain BL-21(DE3), under standard conditions
(0.2 mM IPTG, 20 °C for 16 h). The crude cell lysate was
washed over Ni-NTA agarose resin (Qiagen, Valencia, CA,
USA) and the target enzyme eluted with 250 mM imidazole
buffer. The purified enzyme was stored in buffer containing
50 mM Tris–HCl (pH 8), 300 mM NaCl, and 5 mM MgCl2.
Thymidylate kinase was > 95% pure as determined by
SDS–PAGE. Enzyme concentration was determined by UV
ꢀ1
ꢀ1
absorbance at 280 nm (e = 32 430 M cm ) (11).
Antituberculosis tests
Inhibition of thymidylate kinase activity was evaluated using
an established coupled spectroscopic assay (12). Initial
reaction rates were determined at 37 °C in reaction buffer
containing 50 mM Tris–HCl (pH 8), 100 mM KCl, 5 mM
Mycobacterial strains
The laboratory M. tuberculosis H37Rv strain susceptible to
anti-TB drugs and a M. tuberculosis MS-115 clinical strain
resistant to five-first line anti-TB drugs (rifampicin, isoniazid,
streptomycin, ethambutol, and pyrazinamide) were used.
The mycobacteria were transformed into a suspension of
single cells at the same growth phase and normalized by
CFU (8). Enriched Dubois medium (Difco, Leeuwarden, The
Netherlands) was used.
2
MgCl , 1 mM ATP, 0.2 mM PEP, and 0.2 mM NADH, as well
as five units of pyruvate kinase and lactate dehydrogenase,
respectively (Roche Biochemicals, Indianapolis, IN, USA).
Enzyme concentration was constant at 0.05 lM, while TMP
ranged from 0 to 128 lM and inhibitor was varied from 0 to
200 lM. Phosphorylation of TMP was monitored via NADH
ꢀ
1
ꢀ1
oxidation at 340 nm (e = 6220 M cm ). None of the
investigated inhibitors had significant absorbance at that
wavelength. All experiments were performed in the pres-
ence of 0.4% DMSO (v/v), maximizing inhibitor solubility
while minimizing detrimental effects on enzyme activity. Ini-
tial reaction rates at varying substrate/inhibitor concentra-
tions were fit to a competitive inhibition model.
Antibacterial activity
The influence of the tested compounds on the growth of the
bacterial strains was studied using the automated BACTEC
MGIT 960 system (BD, Becton Dickinson and Company,
Franklin Lakes, NJ, USA). The tested compounds were dis-
solved in DMSO at a concentration of 40 mg/mL (stock
solutions). Water and Tween-80 were added to the stock
solutions to give a 5: 0.5: 4.5 ratio of DMSO: Tween-80:
Results
2
H O. The mycobacterial suspension (500 lL) was inocu-
lated in the culture medium (7.9 mL) up to the final concen-
tration of 106 CFU/mL. Each sample including the control
samples lacking the tested compound were studied in tripli-
cate. It should be noted that this solvent mixture had no
effect on M. tuberculosis growth. The antibacterial activity
was evaluated by the proportion method with the TB Exist
software (9). The method is based on the comparison of the
bacterial growth in the samples containing the tested com-
pounds and the control samples diluted 1009 if compared
with the initially inoculated culture. The culture is susceptible
Synthesis of the 5-(phenylamino)uracils 1–10 was carried
out as previously described (13) by treating 5-bromouracil
with a threefold molar excess of the appropriate aniline in
boiling ethylene glycol, as shown in Scheme 1.
This procedure gave 5-(phenylamino)uracils 1–8 with yields
ranging between 50% and 82%. The moderate yields
observed are likely due to the tendency of anilines to
undergo oxidation. It should also be noted that the lowest
yields were observed for anilines possessing a methyl
group in the ortho-position (compounds 4 and 5).
(that is, the compound is active) if the optical density of the
control sample is 400 RFU (relative fluorescence units.) in
and that of the tested sample is < 100 RFU. In addition,
using the absolute concentrations, a delay in the bacterial
growth of the tested sample was measured in comparison
with the control samples, which reflected a negative effect
of the tested compounds at the concentrations lower than
MIC on the bacterial viability. The measurements were taken
automatically every 1 h and registered with the Epicenter
software (BD).
5-(Phenylamino)-6-azauracils 9 and 10 were prepared
under similar conditions by the condensation of 5-bromo-
6-azauracil (14) with a threefold molar excess of aniline or
4-n-butylaniline. The yield of compound 9 was high, while
compound 10, in which the butyl group is in the p-posi-
tion, was obtained in only a 47% yield.
Once in hand, the compounds were screened for their
inhibitory properties against TB. While several of the com-
pounds showed promising activity (see Table 1), most
proved to be too insoluble in the requisite medium (see
Table 1), thus a solution to this problem was sought.
Kinase assays
Compounds 6, 7 and 19 were tested as potential inhibi-
tors of thymidylate kinase from M. tuberculosis. The
enzyme was obtained by heterologous expression, using
pMCSG7-tmk (clone ID: APC105701.102; DNASU) (10) in
0
In that regard, we recently reported a series of 5 -nor carbo-
cyclic uracil derivatives, which displayed moderate anti-TB
1
392
Chem Biol Drug Des 2015; 86: 1387–1396

5-Arylaminouracil Derivatives
O
O
H
N
H N
OH
reflux,1 hr.
Br
2
HO
HN
+
HN
R
R
X
X
N
H
O
N
H
O
1
1
4
6
8
-8: X=CH; 9-10: X=N
: R=H; 2: R=3-Me; 3: R=4-Me
: R=2,3-diMe; 5:R=2,5-diMe;
: R=4-nBu; 7: R=4-nBuO;
: R=4-PhO; 9: R=H; 10: R=4-nBu
Scheme 1: Synthesis of 5-arylaminouracil derivatives.
Table 1: 100% inhibition of Mycobacterium tuberculosis growth,
strain H37Rv, and MIC99
Related to this, similar activity has been observed for a
series of 6-aryl purine analogues. These nucleoside displayed
selective antimycobacterial activity (19,20), with the flexible
versions exhibiting potent inhibitory activity against M. tuber-
culosis (20). Likewise, acyclic thymidine analogues were found
to inhibit M. tuberculosis monophosphate kinase (TMPKmt)
Concentration, lg/mL
a
Compound
40
20
10
5
1
MIC99
1
2
3
4
5
6
7
8
9
I
I
I
I
I
I
I
I
I
+
I
ꢀ
ꢀ
I
I
I
I
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
+
I
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
I
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
(21). Given the increased solubility generally observed for car-
ꢀ
20
10
ꢀ
bocyclic nucleosides, and the promising inhibitory properties
described above, combining the carbocyclic moiety with the
uracil scaffold was predicted to improve their solubility and
subsequently improve their biological activity.
+
ꢀ
ꢀ
I
I
I
ꢀ
10
5
+
I
ꢀ
In that regard, 5-phenylaminouracils 1, 3, 6–8 were con-
densed with epoxycyclopentene in the presence of a palla-
dium catalyst using Trost methodology (22). High regio-
and stereoselectivity of the reaction is achieved due to the
unique structure of the p-allyl intermediate complex formed
with the allylic acetate, which leads solely to cis-products
I
I
I
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
10
11
12
13
14
15
16
17
18
19
20
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
20
ꢀ
ꢀ
ꢀ
ꢀ
I
+
+
+
ꢀ
+
ꢀ
+
ꢀ
ꢀ
ꢀ
0
40
40
40
ꢀ
via retention of configuration at the 1 -position (23)
(Scheme 2). Due to their poor solubility in DMF, the heter-
ogeneous mixture containing the 5-substituted uracils was
vigorously stirred while heating to 50 °C. After isolation
20
ꢀ
1
and purification, the yields of racemic N -monosubstituted
products 11–15 were disappointingly low, ranging between
Rifampicin
1
1
3
1
8% and 25%, while the N , N -disubstituted uracil ana-
I: insoluble at this concentration.
Compound concentration in lg/mL at which growth is 99%
logues (16–20) were obtained in somewhat higher yields
(35–40%) as diastereoisomeric mixtures.
a
inhibited.
Compounds 1–20 were then screened for potential antimi-
crobial activity. The laboratory M. tuberculosis H37Rv
strain, which is a virulent strain susceptible to all antituber-
culosis drugs, was chosen for the initial screening. The
compounds were tested at concentrations of 1, 5, 10, 20
and 40 lg/mL. The minimal inhibitory concentrations
(MIC ) of compounds 1–20 were determined using the
0
activity (15). The 5 -nor carbocyclic nucleosides in general
possess several key advantages due to their increased sta-
bility, increased solubility, and lack of 5 -phosphorylation
16). This latter feature is responsible for a decrease in cyto-
0
(
toxicity that proved problematic for the triphosphates of
aristeromycin and neplanocin A, two naturally occurring car-
bocyclic nucleosides that exhibit potent therapeutic proper-
ties against viruses, parasites, and various diseases
including TB (17,18). The target 5-alkynyl uracil derivatives
were found to have an MIC99 of 10–40 lg/mL against the
H37Rv laboratory strain of M. tuberculosis with a corre-
sponding increase in activity noted as the length of the side
chain for the alkynyl derivatives increased (15).
99
proportion method in the BACTEC MGIT 960 system (9) in
the TB Exist mode (24).
Compounds 2, 3, 6, 7, 10, 15–17, and 19 all had a nega-
tive effect on the growth of mycobacterial cells, with 99%
inhibition observed at concentrations between 5 and
40 lg/mL. Compounds 2, 3, 6, 7, 10, 15–17, and 19
Chem Biol Drug Des 2015; 86: 1387–1396
1393

Matugina et al.
0
Scheme 2: Synthesis of 5 -norcarbocyclic 5-arylaminouracil derivatives.
Table 2: Mycobacterium tuberculosis (strain MS-115) % growth
inhibition
phate transfer in TMPKmt requires the transient binding of
a magnesium ion co-ordinating the phosphate acceptor.
Moreover, the unique configuration of the active site and
catalytic mechanism of TMPKmt renders it a promising tar-
get for the development of highly active and selective anti-
TB drugs (25,26). In addition, the crystallographic structure
of TMPKmt was published (25,27) therefore providing a
means for exploring potential inhibitors using computer-
mediated design (28).
Concentration, lg/mL
Compound
20
10
5
1
0.1
2
3
6
7
NA
I
I
I
NA
NA
NA
90%
100%
NA
NA
I
NA
NA
75%
75%
NA
NA
NA
NA
90%
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
I
1
1
1
1
1
0
5
6
7
9
NA
NA
NA
NA
99%
To evaluate TMPKmt as the possible target enzyme for the
compounds anti-TB activity, they were docked in the
active site of the enzyme using AUTODOCK VINA 1.1.2. (29) A
crystallographic model of thymidylate kinase in M. tubercu-
losis PDB: 1G3U, (27) previously used for modeling of
nucleoside inhibitors of the enzyme, was employed and
the results are shown below.
I: insoluble at this concentration; NA: not active.
were also tested at concentrations ranging between 0.1
and 20 lg/mL against M. tuberculosis multidrug-resistant
strain (MS-115), which is resistant to isoniazid, rifampicin,
streptomycin, ethambutol, and pyrazinamide. The results
are shown in Table 2.
Docking resulted in two distinctive conformations for com-
pounds studied. The first one represents mode of binding
that is analogous to previously described inhibitors of
TMPKmt (6) (Figure 2).
It is important for compounds that suppress bacterial or viral
replication to also be tested for toxicity, as, for example,
what appears to be an antibacterial effect against M. tuber-
culosis could, in fact, be the result of cell depletion. As a
result, the cytotoxicity of the compounds was evaluated in
Vero, A549, and Huh7 cells with none of the compounds dis-
playing cytotoxicity up to concentrations of 50 lg/mL.
The second, or ‘reversed’ binding pose, is shown in
2+
Figure 3. The Mg is displaced by the inhibitor, as is typi-
cal for these types of inhibitors, and the predicted bonding
interactions with the enzyme appeared encouraging. The
locations of the aromatic rings of 6, 7 and 19 were identical
to previously reported inhibitors and are likely due to p–p
stacking between the substituent at the 5th-position and
residues Phe70 and Tyr103, as well as the p-cation interac-
tion with the side chain of Arg95. The carbonyl function at
the 2nd-position of the pyrimidine ring formed a hydrogen
bond with the guanidine nitrogen Arg95, and analogous
interactions could be expected for inhibitors containing the
pyrrolidine-2-one moiety (21). Hence, a set of key features,
which are essential for inhibitory properties, was established
Discussion
Although the precise mechanism of action for these com-
pounds against TB is not known, speculation that it might
be a result of inhibition of the thymidylate kinase of
M. tuberculosis (TMPKmt, EC 2.7.4.9, ATP: phosphotrans-
ferase TMF) seemed most likely. TMPKmt belongs to the
family of nucleoside monophosphate kinases and cata-
0
(26,30). Finally, the 4 -hydroxyls of the cyclopentene appear
to interact with polar amino acids at the entrance to the
active site (the blue area shown in Figure 3B).
0
lyzes the reversible phosphorylation of 2 -deoxythymidine
0
phosphate to 2 -deoxythymidine diphosphate in the pres-
Faced with these conflicting results, we decided to evaluate
TMPKmt as the enzymatic target for compounds 6, 7 and
19. Heterologously expressed kinase, purified to homoge-
neity via its C-terminal polyHis tag, was used for these in vi-
ence of ATP (25,26). TMPKmt is required for DNA synthe-
sis, and, consequently, for the growth and reproduction of
mycobacteria. In contrast to other known TMPKs, phos-
1
394
Chem Biol Drug Des 2015; 86: 1387–1396

5-Arylaminouracil Derivatives
A
B
Figure 2: (A) dTMP in the active site of
TMPKmt. (B) Predicted binding mode for
compound 7—note the analogous with
dTMP ligand–protein interactions.
A
B
Figure 3: (A) Predicted ‘reversed’ binding
for compound 19. (B) Predicted ‘reversed’
binding modes for compounds 6, 7 and 19
overlaid with dTMP (shown in yellow) inside
the active center cavity. The blue surface
area indicates the entrance region, while the
green is the surface of the bottom of the
pocket.
tro experiments. Enzyme activity was initially tested with
native substrate (thymidine monophosphate; TMP), using a
spectrophotometric coupled-enzyme assay. The enzyme
showed activity and its steady-state parameters for TMP
were consistent with the literature data (30). Given the lim-
ited solubility of the inhibitors in buffer, the kinase’s toler-
ance for DMSO was tested. It was determined that the
catalytic activity was not significantly affected at concentra-
tions up to 0.4%. All inhibition studies were hence carried
out in reaction buffer (50 mM Tris–HCl pH 8, 100 mM KCl,
studies are needed. In addition, as solubility remains prob-
lematic, the development of prodrugs that could potentially
increase solubility and allow the use of lower inhibitor con-
centrations will now be considered.
Acknowledgments
The research was supported by the Russian Science
Foundation, project no. 14-50-00060.
5
2
mM MgCl , five units of PK and LDH, 1 mM ATP, 0.2 mM
PEP, and 0.2 mM NADH) supplemented with 0.4% DMSO
at 37 °C. Substrate phosphorylation was determined via the
coupled oxidation of NADH, and corresponding absorbance
References
ꢀ
1
ꢀ1
loss at 340 nm (reduced NADH: e340 = 6220 M cm ).
The three inhibitors were tested at concentrations from 0 to
1. Sharma V., Chitranshi N., Agarwal A.K. (2014) Signifi-
cance and biological importance of pyrimidine in the
microbial world. Int J Med Chem;2014:202784.
2. Villela A.D., Sanchez-Quitian A.Z., Ducati R.G., Santos
D.S., Basso L.A. (2011) Pyrimidine salvage pathway in
Mycobacterium tuberculosis. Curr Med Chem;18:1286–
1298.
200 lM in the presence of TMP at concentrations ranging
from 0 to 128 lM. Unfortunately, at higher inhibitor concen-
trations, solubility once again became problematic. Based
i
on the activity measurements, K values of > 200 lM must
be assumed for all three compounds; however, the low
affinity of these inhibitors is consistent with previous data
for the corresponding thymine analogues (6).
3. Shmalenyuk E.R., Kochetkov S.N., Alexandrova L.A.
(2013) New inhibitors of Mycobacterium tuberculosis
In summary, while the anti-TB activity observed is promis-
ing, the mechanism of action remains elusive and further
growth based on modified pyrimidine nucleosides and
their analogues. Russ Chem Rev;82:896–915.
Chem Biol Drug Des 2015; 86: 1387–1396
1395

Matugina et al.
4
. Van Daele I., Munier-Lehmann H., Hendrickx P.M.S.,
Marchal G., Chavarot P., Froeyen M., Qing L., Martins
J.C., Van Calenbergh S. (2006) Synthesis and biologi-
cal evaluation of bicyclic nucleosides as inhibitors of
18. Matyugina E., Khandazhinskaya A., Kochetkov S. (2012)
Carbocyclic nucleoside analogues: classification, target
enzymes, mechanisms of action and synthesis. Russ
Chem Rev;81:729–746.
M. tuberculosis
thymidylate
kinase.
ChemMed-
19. Read M.L., Braendvang M., Miranda P.O., Gundersen
L.L. (2010) Synthesis and biological evaluation of
pyrimidine analogs of antimycobacterial purines. Bioorg
Med Chem;18:3885–3897.
20. Braendvang M., Bakken V., Gundersen L.L. (2009)
Synthesis, structure, and antimycobacterial activity of
6-[1(3H)-isobenzofuranylidenemethyl]purines and ana-
logs. Bioorg Med Chem;17:6512–6516.
21. Familiar O., Munier-Lehmann H., Negri A., Gago F.,
Douguet D., Rigouts L., Hernandez A.I., Camarasa M.J.,
Perez-Perez M.J. (2008) Exploring acyclic nucleoside ana-
logues as inhibitors of Mycobacterium tuberculosis thymi-
dylate kinase. ChemMedChem;3:1083–1093.
Chem;1:1081–1090.
5
6
. Munier-Lehmann H., Douguet D., Labesse G., Pochet
S. (2006) Aryl pyrimidyl compounds, pharmaceutical
compositions comprising them, their use as antimicro-
bial agents. WO Patent 2006048336.
. Gasse C., Douguet D., Huteau V., Marchal G., Munier-
Lehmann H., Pochet S. (2008) Substituted benzyl-pyrimi-
dines targeting thymidine monophosphate kinase of
Mycobacterium tuberculosis: synthesis and in vitro anti-
mycobacterial activity. Bioorg Med Chem;16:6075–6085.
. Niks M., Otto M. (1990) Towards an optimized MTT
assay. J Immunol Methods;130:149–151.
7
8
. Andreevskaya S.N., Chernousova L.N., Smirnova T.G.,
Larionova E.E., Kuzmin A.V. (2006) Ex vivo study of
growth of different genotypic clusters of Mycobacte-
rium tuberculosis strains in macrophages. Probl Tuberc
Lung Dis;12:43–48.
22. Trost B.M., Kuo G.H., Benneche T. (1988) Transition-
metal-controlled synthesis of (ꢁ)-aristeromycin and
(ꢁ)-2,3-diepi-aristeromycin. An unusual directive effect
in hydroxylations. J Am Chem Soc;110:621–622.
23. Deardorff D.R., Linde R.G. II, Martin A.M., Shulman
M.J. (1989) Enantioselective preparation of functional-
ized cyclopentanoids via common chiral (p-allyl)palla-
dium complexJ. Org Chem;54:2759–2762.
9
. Siddiqi S.H., Rusch-Gerdes S. (2006) MGIT Procedure
Manual. Geneva, Switzerland: Foundation for Innovative
New Diagnostics (FIND).
1
0. Seiler C.Y., Park J.G., Sharma A., Hunter P., Surapaneni
P., Sedillo C., Field J., Alger R., Steel J., Throop A., Fiacco
M., LaBaer J. (2013) DNASU plasmid and PSI: biology-
materials repositories: resources to accelerate biological
research. Nucleic Acids Res;42:D1253–D1260.
1. Wilkins M.R., Gasteiger E., Bairoch A., Sanchez J.C.,
Williams K.L., Appel R.D., Hochstrasser D.F. (1999)
Protein identification and analysis tools in the ExPASy
server. Methods Mol Biol;112:531–552.
2. Schelling P., Folkers G., Scapozza L. (2001) Quater-
nary structure change as a mechanism for the regula-
tion of thymidine kinase 1-like enzymes. Anal
Biochem;295:82–87.
3. Gerns F.R., Perrotta A., Hitchings G.H. (1966) 5-Aryla-
minopyrimidines. J Med Chem;9:108–115.
24. Franzblau S.G., Witzig R.S., McLaughlin J.C., Torres
P., Madico G., Hernandez A., Degnan M.T., Cook
M.B., Quenzer V.K., Ferguson R.M., Gilman R.H.
(1998) Rapid, low-technology MIC determination with
clinical Mycobacterium tuberculosis isolates by using
the microplate alamar blue assay. J Clin Micro-
biol;36:362–366.
1
25. Fioravanti E., Adam V., Munier-Lehmann H., Bourgeois
D. (2005) The crystal structure of Mycobacterium tuber-
0
1
culosis thymidylate kinase in complex with 3 -azidode-
oxythymidine monophosphate suggests a mechanism
for competitive inhibition. Biochemistry;44:130–137.
26. Haouz A., Vanheusden V., Munier-Lehmann H., Fro-
eyen M., Herdewijn P., Van Calenbergh S., Delarue M.
(2003) Enzymatic and structural analysis of inhibitors
designed against Mycobacterium tuberculosis thymi-
dylate kinase. New insights into the phosphoryl transfer
mechanism. J Biol Chem;278:4963–4971.
1
1
4. Chang P., Ulbricht T.L.V. (1958) The reaction of chloral
hydrate with semicarbazides and the synthesis of
14
14
semicarbazide-C and 6-azauracil-2-C . J Am Chem
Soc;80:976–979.
27. Li de la Sierra I., Munier-Lehmann H., Gilles A.M.,
B a^ rzu O., Delarue M. (2001) X-ray structure of TMP
1
5. Matyugina E., Khandazhinskaya A., Chernousova L.,
Andreevskaya S., Smirnova T., Chizhov A., Karpenko
I., Kochetkov S., Alexandrova L. (2012) The synthesis
kinase from Mycobacterium tuberculosis complexed
with TMP at 1.95 A resolution. J Mol Biol;311:87–100.
28. Douguet D., Munier-Lehmann H., Labesse G., Pochet S.
(2005) LEA3D: a computer-aided ligand design for struc-
ture-based drug design. J Med Chem;48:2457–2468.
29. Trott O., Olson A.J. (2010) AutoDock Vina: improving
the speed and accuracy of docking. . . and multith-
reading. J Comput Chem;31:455–461.
30. Munier-Lehmann H., Chaffotte A., Pochet S., Labesse G.
(2001) Thymidylate kinase of Mycobacterium tuberculosis:
a chimera sharing properties common to eukaryotic and
bacterial enzymes. Protein Sci;10:1195–1205.
0
and antituberculosis activity of 5 -nor carbocyclic uracil
derivatives. Bioorg Med Chem;20:6680–6686.
0
1
1
6. Matyugina E., Khandazhinskaya A. (2014) 5 -Norcarbo-
cyclic
nucleoside
analogues.
Russian
Chem
Bull;5:1069–1080.
7. Patil S.D., Schneller S.W., Hosoya M., Snoeck R., Andrei
G., Balzarini J., De Clercq E. (1992) Synthesis and antiviral
0
properties of (ꢁ)-5 -noraristeromycin and related purine
carbocyclic nucleosides. A new lead for anti-human cyto-
megalovirus agent design. J Med Chem;35:3372–3377.
1
396
Chem Biol Drug Des 2015; 86: 1387–1396