Synthesis and Antimycobacterial Activity of 5-(Arylmethylene)Hexahydropyrimidine-2,4,6-Triones

Article abstract of DOI:10.1007/s11094-016-1376-3

A series of 5-(arylmethylene)hexahydropyrimidine-2,4,6-triones were synthesized. Their antimycobacterial activity and acute daily toxicity with respect to M. lufu were investigated.

Full text of DOI:10.1007/s11094-016-1376-3

DOI 10.1007/s11094-016-1376-3
Pharmaceutical Chemistry Journal, Vol. 49, No. 12, March, 2016 (Russian Original Vol. 49, No. 12, December, 2015)
SYNTHESIS AND ANTIMYCOBACTERIAL ACTIVITY
OF 5-(ARYLMETHYLENE)HEXAHYDROPYRIMIDINE-2,4,6-TRIONES
1
S. A. Luzhnova, A. G. Tyrkov, N. M. Gabitova, and E. A. Yurtaeva
2
1
1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 49, No. 12, pp. 12 – 14, December, 2015.
Original article submitted December 29, 2014.
A series of 5-(arylmethylene)hexahydropyrimidine-2,4,6-triones were synthesized. Their antimycobacterial
activity and acute daily toxicity with respect to M. lufu were investigated.
Keywords: synthesis, 5-(arylmethylene)hexahydropyrimidine-2,4,6-triones, antimycobacterial activity, min-
imum inhibiting and bactericidal concentrations, acute toxicity.
Several well-known antileprosy drugs are currently
widely used in medical practice. However, their high toxicity
1, 2] and the development of resistance to them have shifted
on condensation of 2,4,6-pyrimidinetrione (I) with an excess
of the aromatic aldehydes (II – VII). The reaction was car-
ried out in EtOH. The reaction mixture was refluxed for
45 min.
[
the first priority to the synthesis of novel antimycobacterial
drugs. In our previous studies, the daily toxicity and anti-
mycobacterial activity of substituted 2-nitro-1-(4-toluene-
sulfonyl)-2-[3-methyl(phenyl)-1,2,4-oxaliazol-5-yl]ethanes
against M. lufu and M. tuberculosis were investigated [3].
In continuation of research and development in this area
and in order to discover the least toxic compounds, we syn-
thesized a series of 5-(arylmethylene)hexahydropyrimidine-
The structures of the compounds were established using
1
3
IR and electronic spectroscopy, PMR and C NMR spectra,
and mass spectrometry. The compositions were determined
by elemental analyses. IR spectra displayed a new absorption
band that was missing in the starting materials for an ethyl-
–
ene bond at 1625 cm . A methine singlet at 8.20 – 8.36 ppm
1
in the PMR spectrum and a methine resonance for C at
7
1
50 ppm in the C NMR appeared characteristically. Elec-
3
1
2
,4,6-triones (VIII – XIII) and investigated their antimyco-
tronic spectra showed two absorption bands with clearly re-
bacterial activity.
O
O
solved maxima at 260 nm (local p -electron excitation) and
350 – 450 nm (intramolecular charge transfer characteristic
CH
4 6
H C R
HN
HN
+
OHC H C R
4 6
of conjugated ethylene [4]). Mass spectra of the synthesized
compounds contained both very strong peaks for the molecu-
lar ions that allowed their molecular masses to be estimated
and peaks for fragments from primary and secondary disso-
ciative ionization. The hexahydropyrimidinetriones were
high melting colorless or colored compounds that were solu-
EtOH
O
NH
I
O
O
NH
O
II – VII
VIII – XIII
R = H (II, VIII);
R = 4-CH O (III, IX);
3
R = 4-(CH ) N (IV, X);
2
3
ble in most organic solvents and poorly soluble in H O.
R = 4-Cl (V, XI);
2
R = 3-NO (VI, XII);
2
R = -OH(VII, XIII)
EXPERIMENTAL CHEMICAL PART
The method for preparing the target compounds
The target compounds were synthesized from I and aro-
matic aldehydes (II – VII) (Aldrich, USA). Their physical
constants agreed with the literature data. IR spectra of the
synthesized compounds in KBr pellets were recorded in the
(
VIII – XIII), the yields of which were 82 – 92%, was based
1
Leprosy Research Institute, RF Ministry of Health, Astrakhan, 414057
Russia.
2
–
range 4000 – 400 cm on an InfraLUM FT-02 spectrophoto-
1
Astrakhan State University, Astrakhan, 414956 Russia.
810
0
091-150X/15/4912-0810 © 2015 Springer Science+Business Media New York

Synthesis and Antimycobacterial Activity
811
1
meter. PMR and C NMR spectra were recorded in
3
5-[(4-Chlorophenyl)methylene]-2,4,6-pyrimidinetrio-
–
1
DMSO-d with HMDS internal standard on a Bruker DRX
ne (XI). Yield 90%, (dec.) 298 °C. IR spectrum, n max, cm :
3550 (NH), 1770, 1750 (C=O), 1625 (C=C). PMR spectrum,
d , ppm: 11.45 (br.s, 1H, NH); 11.30 (br.s, 1H, NH);
6
5
00 SF at operating frequency 500 and 300 MHz, respec-
tively. Electronic spectra (0.3 mg/mL in EtOH) were taken
on a Cary-50 spectrophotometer. Mass spectra were obtained
on a Finnigan SSQ 7000 mass spectrometer with direct sam-
ple introduction into the ion source at ionizing potential
1
3
8
.15 – 7.55 (m, 4Harom, C H ); 8.32 (s, 1H, CH). C NMR
6 4
spectrum, d , ppm: 163.3 (C ), 162.9 (C ), 155.2 (C ), 150.6
4
6
2
(
C ), 135.7 (C ), 133.2 (C ), 129.7 (C ), 129.3 (C ), 118.1
7 11 8 10 9
7
0 eV, sample temperature 150°C, and accelerating potential
000 V (resolution 5000). The course of reactions and purity
(
C ). UV spectrum, l , nm: 260 (lge 3.6), 350 (lge 3.3).
+
5
max
5
+
Mass spectrum, m/z (I , %): 250 [M] (100), 249 [M – 1]
rel
of products were monitored by TLC on Silufol
+
+
(42), 215 [M-Cl]
(10), 207 [M-CHNO]
(30).
UV-254 plates using Me CO:hexane (2:3) and detection by
2
C H N O Cl. M 250.62.
11
7
2
3
I2 vapor [5]. Elemental analyses were performed on a Euro
5-[(3-Nitrophenyl)methylene]-2,4,6-pyrimidinetrione
Vector automated Euro EA-3000 CHNS analyzer and agreed
– 1
(
XII). Yield 82%, (dec.) 245 °C. IR spectrum, n max, cm :
with those calculated.
3
550 (NH), 1770, 1750 (C=O), 1625 (C=C), 1540, 1365
5
-(Arylmethylene)hexahydropyrimidine-2,4,6-triones
VIII – XIII). A solution consisting of compound I
10 mmol) and II – VII (11 mmol) in EtOH (25 mL) was
(
NO ). PMR spectrum, d , ppm: 11.50 (br.s, 1H, NH); 11.35
2
(
(
br.s, 1H, NH); 8.91 – 7.75 (m, 4Harom, C H ); 8.36 (s, 1H,
1
6
4
(
3
CH). C NMR spectrum, d , ppm: 162.8 (C ), 161.5 (C ),
refluxed for 45 min and cooled to room temperature. The
solid was filtered off, rinsed twice with cold EtOH (15 mL
each), dried in air, and recrystallized from MeOH.
4
6
1
51.2 (C ), 150.2 (C ), 147.2 (C ), 138.4 (C ), 134.5 (C ),
2 7 12 13 10
1
29.4 (C ), 126.1 (C ), 125.5 (C ), 121.6 (C ). UV spec-
8 9 11 5
trum, l max, nm: 260 (lge 3.7), 350 (lge 3.3). Mass spectrum,
+
m/z (I , %): 261 [M] (65), 260 [M – 1] (47), 244 [M-NH]
5
-(Phenylmethylene)-2,4,6-pyrimidinetrione (VIII).
+
+
–
1
Yield 85%, (dec.) 295 °C. IR spectrum, n max, cm : 3550
NH.), 1770, 1750 (C=O), 1625 (C=C). PMR spectrum, d ,
ppm: 11.35 (br.s, 1H, NH); 11.20 (br.s, 1H, NH); 8.10 – 7.45
rel
+
100), 214 [M-NO2] (69), 172 [M-NO -CHNO] (21).
+
(
2
(
C H N O . M 261.16.
11
7
3
5
5-[(2-Hydroxyphenyl)methylene]-2,4,6-pyrimidine-
13
(
m, 5Harom, C H ); 8.30 (s, 1H, CH). C NMR spectrum, d ,
ppm: 163.4 (C ), 161.6 (C ), 154.7 (C ), 150.2 (C ), 133.1
6 5
trione (XIII). Yield 85%, (dec.) 290 °C. IR spectrum, n max,
–
cm : 3550 (NH), 3250 (OH), 1770, 1750 (C=O), 1625
1
4
6
2
7
(
C ), 132.7 (C ), 132.2 (C ), 128.0 (C ), 119.1 (C ). UV
9 8 10 11 5
(
C=C). PMR spectrum, d , ppm: 11.20 (br.s, 1H, NH); 11.05
spectrum, l max, nm: 260 (lge 3.5), 350 (lg e 3.1). Mass spec-
(
br.s, 1H, NH); 9.45 (br.s, 1H, OH), 8.05 – 7.05 (m, 4Harom
,
+
trum, m/z (I , %): 216 [M] (70), 215 [M – 1] (100), 172
+
rel
13
C H ); 8.22 (s, 1H, CH). C NMR spectrum, d , ppm: 163.4
6 4
+
M-CHNO] (51), 102 [C H ] (15). C H N O . M 216.18.
+
[
8 6 11 8 2 3
(
C ), 161.9 (C ), 159.1 (C ), 155.1 (C ), 150.2 (C ), 134.8
4 6 9 2 7
5
-[(4-Methoxyphenyl)methylene]-2,4,6-pyrimidine-
trione (IX). Yield 87%, (dec.) 270 °C. IR spectrum, n max.,
–
1
cm : 3550 (NH.), 1770, 1750 (C=O), 1625 (C=C). PMR
spectrum, d , ppm: 11.25 (br.s, 1H, NH); 11.10 (br.s, 1H,
NH); 8.35 – 7.10 (m, 4Harom, C H ); 8.25 (s, 1H, CH); 3.87
TABLE 1. Acute Daily Toxicity (LD ) and Antimycobacterial
5
0
Activity of 5-(Arylmethylene)hexahydropyrimidine-2,4,6-triones
6
4
1
3
(VIII – XIII)
(
s, 3H, CH O). C NMR spectrum, d , ppm: 163.9 (C4),
3
1
63.4 (C ), 162.2 (C ), 155.0 (C ), 150.2 (C ), 137.5 (C ),
Antimycobacterial activity
6
11
2
7
9
LD50,
Compound
1
25.2 (C ), 115.5 (C ), 113.9 (C ), 55.7 (CH O). UV spec-
mg/kg
8
5
10
3
MIC, m g/mL
MBC, m g/mL
8.1 ± 1.9*
trum, l max., nm: 260 (lge 3.6), 380 (lge 3.2). Mass spectrum,
+
m/z (I , %): 246 [M] (100), 245 [M – 1] (66), 215
*
**
VIII
> 1500
> 1500
1500
2
.5 ± 0.09
+
rel
+
+
IX
42.8 ± 3.4**
64.0 ± 12.3**
220.0 ± 19.8
7.65 ± 1.65*
428.5 ± 28.3**
560.0 ± 26.4**
8.3 ± 1.56
[
M-CH O]
(10),
202
[M-CHNO]
(38),
172
3
+
M-CHNO-CH O] (5),. C H N O . M 246.20.
X
128.0 ± 11.6**
[
3
12 10
2
4
5
-[(4-Dimethylaminophenyl)methylene]-2,4,6-pyrimi-
XI
> 1500
1500
2
.3 ± 0.11**
dinetrione (X). Yield 92%, (dec.) 277 °C. IR spectrum, n max,
***
XII
256.0 ± 16.2
256.0 ± 18.9
4.0 ± 0.7
–
1
cm : 3550 (NH.), 1770, 1750 (C=O), 1625 (C=C). PMR
spectrum, d , ppm: 11.20 (br.s, 1H, NH); 11.05 (br.s, 1H,
NH); 9.60 – 8.30 (m, 4Harom, C H ); 8.20 (s, 1H, CH); 2.95
*
**
XIII
Dapsone
1500
600
6
4
1
3
(
s, 6H, CH N). C NMR spectrum, d , ppm: 163.4 (C4),
*
3
Statistically significant differences relative to dapsone
statistically significant differences relative to dapsone
1
1
61.6 (C ), 155.1 (C ), 151.1 (C ), 150.2 (C ), 130.7 (C ),
6 2 11 7 9
(p £ 0.05);
**
21.9 (C ) 112.3 (C ), 40.1 (CH N). UV spectrum, l max,
8
10
3
nm: 260 (lge 3.7), 450 (lge 3.3). Mass spectrum, m/z (I , %):
2
(p £ 0.01);
***
rel
+
59 [M] (100), 258 [M – 1] (53), 216 [M-CHNO] (34),
+
+
statistically significant differences relative to dapsone
+
15 [M-(CH ) N] (15). C H N O . M 259.26.
(p £ 0.001).
2
3
2
13 13
3
3

8
12
S. A. Luzhnova et al.
(
C ), 132.9 (C ), 121.2 (C ), 120.1 (C ), 118.3 (C12), 115.5
An analysis of the results in Table 1 showed that
VIII – XIII possessed low toxicity (³ 1500 mg/kg). The
antimycobacterial activity of VIII – XIII against M. lufu
varied widely. Compounds VIII and XI possessed the great-
est antimycobacterial activity (MBA). Their activity was
similar to that of dapsone. The differences between them
were statistically insignificant (p £ 0.05). The activity of
compound XI [MIC (2.3 ± 0.11) m g/mL] was statistically
significantly (p £ 0.01) different from that of dapsone [MIC
(4.0 ± 0.7) m g/mL] whereas that of VIII [MIC
11
13
5
8
(
C ). UV spectrum, l , nm: 260 (lge 3.5), 350 (lge 3.2).
10 max
+ +
Mass spectrum, m/z (I , %): 232 [M] (100), 231 [M – 1]
rel
+
41), 215 [M-OH] (18), 139 [M-C H O] (54). C H N O .
+
(
6 5 11 8 2 4
M 232.17.
EXPERIMENTAL BIOLOGICAL PART
Compounds VIII – XIII were screened microbiologi-
cally for antibacterial activity against M. lufu as a test culture
for determining the in vitro activity of antileprosy drugs [6].
The procedure was based on the serial dilution method that
was used to study antibacterial activity against M. tuberculo-
sis [7]. M. lufu were cultivated in Lowenstein—Jensen dense
medium for cultivation of mycobacteria [7]. The antimyco-
bacterial activity of the compounds was studied in
Shkol’nikova medium [7]. A two-week culture of M. lufu
(
2.5 ± 0.09) m g/mL] was statistically significantly greater
p £ 0.001). The other compounds were also active with re-
(
spect to M. lufu. However, their MIC values were statistically
significantly greater than that of dapsone (p £ 0.01;
p £ 0.001).
5
was seeded at a dose of 10 microbes per mL of medium.
Thus, the investigation showed that the compounds had
low toxicity. The compounds exhibited antimycobacterial ac-
tivity of varying degrees compared to that of dapsone. The
results allowed VIII and XI to be considered promising for
further studies of their antimycobacterial activity.
Cultures were synchronized in the cold (4 ± 1)°C for 72 h be-
fore seeding in order to obtain the most consistent results.
The reference drug was dapsone (Novartis, Switzerland).
Seeded samples were incubated with the compounds in
Shkol’nikova medium for 10 d at (31 ± 1)°C. After this, sus-
pension (0.05 mL) was transferred from each tube into tubes
with slanted Lowenstein—Jensen medium in order to deter-
mine the viability of M. lufu. The remaining contents of the
tubes were centrifuged. Smears were prepared from the pre-
cipitate and tested with Ziehl—Neelsen acid-fast stain [7].
Twenty fields-of-view were examined in the smears. The to-
tal number of mycobacteria was counted. Their morphology
and the presence of acid-fast and mutated forms were as-
sessed. Seedings in Lowenstein—Jensen medium were incu-
bated for 10 d at (31 ± 1)°C. Colonies growing on dense me-
dium were counted. The minimum inhibiting concentration
ACKNOWLEDGMENTS
The work was supported financially by the program “De-
velopment of innovative infrastructure at Russian Higher Ed-
ucational Institutions” (Grant No. 13.637.31.0038) and used
scientific equipment of the CCU “Biotechnology for creating
original drug substances.”
REFERENCES
1
2
3
. J. S. Blanchard, Annu. Rev. Biochem., 65, 215 – 239 (1996).
. T. Beardsley, V Mire Nauki, 1, 78 – 79 (1993).
(
MIC) of the compounds was the lowest concentration at
which the number of M. lufu colonies growing on the me-
dium was less than 50% of the control value. The minimum
bactericidal concentration (MBC) of the compounds was that
concentration at which colonies of M. lufu were not observed
after incubation in the medium. The investigation used five
series of repeated experiments. The results were processed
statistically using the Student t-criterion.
. A. G. Tyrkov, N. G. Urlyapova, and A. D. Daudova, Khim.-farm.
Zh., 40, 30 – 31 (2006); Pharm. Chem. J., 40(7), 377 – 379
(
2006).
4
. E. Pretsch, P. Buhlmann, and C. Affolter, Structure Determina-
tion of Organic Compounds, Springer, Berlin, New York (2000)
[Russian translation, (2006), pp. 393 – 395].
5. J. G. Kirchner, Techniques of Chemistry, Vol. 14: Thin-Layer
Chromatography, 2nd Ed., Wiley-Interscience, New York
1978), 1137 pp [Russian translation, (1981), pp. 129, 218].
Acute toxicity (LD ) was determined using male CBA
50
(
mice (25 – 28 g) with seven animals per group. Compounds
were injected into the stomach as an aqueous suspension at
doses of 5 – 50 and 1500 mg/kg. LD50 values were calcu-
lated using the Miller and Tainter method and that of
Gaddam [8]. All animals were synchronized with respect to
housing and feeding and were observed for 10 d.
6
. O. A. Irtuganova and N. A. Urlyapova, Critical Questions in
Leprology [in Russian], Astrakhan (1984), pp. 147 – 150.
. P. Gerhardt, Manual of Methods for General Bacteriology, Amer-
ican Society for Microbiology, Washington DC (1981) [Russian
translation, (1983), p. 29].
7
8
. O. N. Elizarova, Determination of Threshold Oral Doses of In-
dustrial Poisons [in Russian], Meditsina, Moscow (1971),
pp. 240, 207.
Table 1 presents the results for the antimycobacterial ac-
tivity of VIII – XIII.