Journal of Applied Pharmaceutical Science Vol.

9(02), pp 012-019, February, 2019

Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2019.90202
ISSN 2231-3354

Synthesis and anticonvulsant activity of 6-methyl-2-thioxo-2,

3-dihydropyrimidin-4(1H)-one acetamides

Hanna Severina1*, Olha Skupa2, Andrei Khairulin3, Natalya Voloshchuk2, Victoria Georgiyants1
1
National University of Pharmacy, Department of Pharmaceutical Chemistry, Kharkiv, Ukraine.
2
National Pirogov Memorial Medical University, Department of Pharmacology, Department of Pharmaceutical Chemistry, Vinnytsya, Ukraine.
3
Institute of Organic Chemistry NAS of Ukraine, Department of Sulfur Chemistry, Laboratory of Condensed Heterocyclic Compounds, Kyiv, Ukraine.

ARTICLE INFO ABSTRACT

Received on: 30/10/2018 This research aimed at synthesizing new potential anticonvulsants in the series of 2-(4-methyl-6-oxo-1,6-dihydropyrimidin-
Accepted on: 19/01/2019 2-yl)thio-acetamides. An initial intermediate 6-methyl-2-thioxo-2,3-dihydro-pyrimidin-4(1Н)-one was obtained by the
Available online: 28/02/2019 reaction of thiourea with an acetoacetic ester in the presence of sodium methoxide. The target 2-(4-methyl-6-oxo-1,6-
dihydropyrimidin-2-yl) thioacetamides were synthesized by alkylation of initial 6-methyl-2-thiopyrimidin-4-one with
corresponding 2-chloroacetamides in Dimethylformamide (DMF) in the presence of potassium carbonate. The structure
Key words:
of compounds was confirmed by 1H Nuclear magnetic resonance (NMR)-spectroscopy, LCMS, and elemental analysis.
2-thiopyrimidine, alkylation,
A screening of anticonvulsant activity of synthesized compounds was carried out using the pentylenetetrazole- and
acetamides, anticonvulsant
maximal electroshock-induced seizures models. In these studies, the highest anticonvulsant activity demonstrated a
activity.
compound 5.5 2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-bromophenyl)-acetamide, which decreased the
lethality, the number and the severity of seizures, and increased their latent period. For these compound parameters of
ЕD50, acute (LD50) and neurotoxicity (TD50), as well as therapeutic (TI) and protective (PI) indexes were determined.
Logical structure analysis of anticonvulsant activity screening revealed some patterns of “structure–activity” relationship.

INTRODUCTION an antiplatelet activity (Crepaldi et al., 2009), anticancer (Gorneva

The medical use of 2-thiopyrimide derivatives began et al., 2005) and antibacterial agents (Basavaraja et al., 2010)
many decades ago. Thiobarbiturates exhibit psychotropic which are 2-thiopyrimidine derivatives were also created.
activity and are used as medicines for anesthesia (Russo and High psychotropic activity of pyrimidines has attracted
Bressolle, 1998). Propylthiouracil is used as an antithyroid the interest of scientists, whose focus is on developing new
drug for the treatment of hyperthyroidism (Azizi et al., 2017; pyrimidine containing drugs that affect the central nervous system.
Cooper, 2005). Recently, the range of pharmacological activity A successful strategy for the synthesis of new antiepileptic drugs
of 2-thiopyrimidines was significantly expanded. Thus, among was the introduction of the exocyclic sulfur atoms into molecules
them were found the reverse transcriptase inhibitors of human of compounds (Levin et al., 1986; Matias et al., 2017).
immunodeficiency virus type 1 (HIV-1) of non-nucleoside The positive experience of our own research concerning
structure, and the effective concentration of which in vitro was in the synthesis of new anticonvulsants in the series of pyrimidin-
the nanomolar range (Mai et al., 2001; Nawrozkij et al., 2008). 4(3H)-one and 4-thione derivatives (Severina et al., 2013; 2015)
The inhibitors of herpes simplex virus (HSV) of type 1 and 2 and determination of the thioacetamide fragment (Saidov et al.,
(Shigeta et al., 2003), antagonists of P2Y12 receptors that possess 2014) effect on increase of antiepileptic activity prompted us to
search for the new highly potent anticonvulsants among thioalkyl
pyrimidine derivatives.
Therefore, this research aimed at synthesizing new
Corresponding Author
*
potential anticonvulsants—S-alkylated derivatives of 6-methyl-2-
Hanna Severina, National University of Pharmacy, Kharkiv, Ukraine. thioxo-2,3 ethyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one.
E-mail: severina.ai @ ukr.net

© 2019 Hanna Severina et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License
(https://creativecommons.org/licenses/by/4.0/).
 Severina et al. / Journal of Applied Pharmaceutical Science 9 (02); 2019: 012-019 013

MATERIALS AND METHODS (CH3), 20.77 (CH3). Found, m/z: 290.09 [M+H]+. The Anal. Calcd.
was for C14H15N3O2S: C, 58.11; N,14.52; S, 11,08; we found: C,
Chemistry 58.01; N, 14.57; and S, 11.05.
All of the solvents and reagents were obtained from
the commercial sources. The progress of the reactions was 2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(3-
monitored by thin layer chromatography (TLC) using aluminum methoxyphenyl)acetamide, 5.3
silica gel plates. The melting points (°C) were determined in a Yield: 76%, mp 192°C–4°C; 1H NMR (300 MHz,
capillary using an electrothermal IA9100X1 (Bibby Scientific DMSO-d6, δ (ppm)): 12.52 (1H, br. s, NH-3), 9.99 (1H, s,
Limited, Staffordshire, UK) digital melting point apparatus. 1H NHCO), 7.28 (1H, s, H-2′), 7.12 (1H, t, J = 8.2, H-5′), 7.02 (1H, d,
Nuclear magnetic resonance (NMR) spectra were recorded on a J = 7.5, H-6′), 6.52 (1H, d, J = 7.5, H-4′), 5.99 (1H, s, CH-5),
Varian Mercury-400 (Varian Inc., Palo Alto, CA) spectrometer 4.00 (2H, s. SCH2), 3.75(3H, s, OCH3), 2.19 (3H, s, OCH3). 13C
(300 MHz) in hexadeuterodimethyl sulfoxide (DMSO-d6) using NMR (126 MHz, DMSO-d6): δ 166.46 (C=O, amide), 166.21
tetramethylsilane (TMS) as an internal standard (chemical shifts (6-C=O), 165.46, 164.12 (C-S), 159.92, 140.39, 129.89, 111.87,
are in ppm). 13C NMR spectra were recorded at Bruker Avance 400 109.21, 105.44, 107.13 (5-CH, br), 55.33 (OCH3,) 35.62 (CH2),
(100.6 MHz). Chemical shifts were reported in ppm downfield 23.74 (CH3). Found, m/z: 306.08 [M+H]+. The Anal. Calcd. was for
from TMS as internal standards. The elemental analysis was C14H15N3O3S: C, 55.07; N, 13.76; S, 10.04; we found: C, 55.25; N,
performed on a Euro Vector EA-3000 (Eurovector SPA, Redavalle, 13.71; and S, 9.99.
Italy) microanalyzer. Elemental analyses were within ±0.4% of
the theoretical values. LCMS was recorded with РЕ SCIEX API 2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-
150EX chromatograph. chlorophenyl)acetamide, 5.4
6-Methyl-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one 3 Yield: 76%, mp >282°C–4°C; 1H NMR (300 MHz,
was obtained according to the method reported in the literature DMSO-d6, δ (ppm)): 12.48 (1H, br. s, NH-3), 10.22 (1H, s, NHCO),
(Novikov et al., 2005). 7.56 (2H, d J = 8, H-3′,5′), 7.32 (2H, d, J = 8, H-2′,6′), 5.99 (1H, s,
CH-5), 4.09 (2H, s. SCH2), 2.45 (3H, s, CH3). 13C NMR (126 MHz,
General procedure of the synthesis of S-alkylated derivatives of DMSO-d6): δ 166.61 (C=O, amide), 166.43 (6-C=O), 165.54,
6-Methyl-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one, 5.1-15 164.09 (C-S), 138.28, 129.09 (2C), 127.38, 121.25 (2C), 107.48
A mixture of 7, 34 mmol of 2-thiouracil (3) and 10, 85 (5-CH, br), 35.59 (CH2), 23.32 (CH3). Found, m/z: 309.03 [M+H]+.
mmol of potassium carbonate in 10 ml of Dimethylformamide The Anal. Calcd. was for C13H12ClN3O2S: C, 50.40; N, 13.56; S,
(DMF) was stirred at 70°C–80°C for 1 hour, and the reaction 10.35; we found: C, 50.32; N, 13.61; and S, 10.31.
mixture was cooled to room temperature; a solution of 7, 34 mmol
of appropriate chloroacetanilide (4) in 10 ml of DMF was added, 2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-
stirred for 5 hours, and left for 12 hours afterward. The reaction bromophenyl)acetamide, 5.5
mixture was filtered and the filtrate evaporated in a vacuum; Yield: 79%, mp >259°C –61°C; 1H NMR (300 MHz,
the residue was treated with 100 ml of cold water. The formed DMSO-d 6, δ (ppm)): 12.48 (1H, br. s, NH-3), 10.22 (1H, s,
precipitate was filtered, air dried, and recrystallized from an NHCO), 7.61 (2H, d, J = 7.9, H-3′,5′), 7.42 (2H, d, J = 7.9, H-2′,6′),
acetone-DMF mixture. 5.98 (1H, s, CH-5), 4.05 (2H, s. SCH2), 2.15 (3H, s, CH3). Found,
m/z: 353.99 [M+H]+. The Anal. Calcd. was for C13H12BrN3O2S: C,
2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N- 44.08; N, 11.86; S, 9.05; we found: C, 43.95; N, 11.90; and S, 9.02.
phenylacetamide, 5.1
Yield: 82%, melting point (mp) 241°C –3°C; 1H NMR 2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(2,3-
(300 MHz, DMSO-d6, δ (ppm)): 12.52 (1H, br. s, NH-3), 10.16 (1H, dichlorophenyl)acetamide, 5.6
s, NHCO), 7.59–7.27 (4H, m, H-2′,3′,5′,6′), 7.05 (1H, t, J = 7.5, Yield: 80%, mp 230°C–2°C; 1H NMR (300 MHz,
H-4′), 5.99 (1H, s, CH-5), 4.07 (2H, s, SCH2), 2.13 (3H, s, CH3). DMSO-d6, δ (ppm)): 12.50 (1H, br. s, NH-3), 10.10 (1H, s, NHCO),
13
C NMR (126 MHz, DMSO-d6): δ 169.82 (C=O, amide), 166.39 7.82 (1H, d, J = 8,2, H-4′), 7.41-7.28 (2H, m, H-5′, 6′), 6.01 (1H, s,
(6-C=O), 165.22, 164.12 (C-S), 139.3, 129.14 (2C), 123.72, 119.54 CH-5), 4.12 (2H, s. SCH2), 2.19 (3H, s, CH3). Found, m/z: 344.21
(2C), 107.66 (5-CH, br), 35.61 (CH2), 23.84 (CH3). Found, m/z: [M+H]+. The Anal. Calcd. was for C13H11Cl2N3O2S: C, 45.36; N,
276.32 [M+H]+. The Anal. Calcd. was for C13H13N3O2S: C, 56.71; 12.21; S, 9.32; we found: C, 45.29; N, 12.23; and S, 9.30.
N, 15.26; S, 11.65%; we found: C, 56.79; N, 15.31; and S, 11.68%.
2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(2,5-
2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4- dimethylphenyl)acetamide, 5.7
methylphenyl)acetamide, 5.2 Yield: 72%, mp >248°C –50°C; 1H NMR (300 MHz,
Yield: 79%, mp 253°C –5°C; H NMR (300 MHz,
1
DMSO-d6, δ (ppm)): 12.42 (1H, br. s, NH-3), 9.52 (1H, s,
DMSO-d6, δ (ppm)): 12.52 (1H, br. s, NH-3), 10.16 (1H, s, NHCO), NHCO), 7.22 (1H, s, H-6'), 7.12 (1H, d, J = 8, H-4′), 6.89 (1H, d,
7.45 (2H, d, J = 8.4, H-2′,6′), 7.10 (2H, d, J = 8.4, H-3′,5′), 5.99 J = 8, H-3′), 6.01 (1H, s, CH-5), 4.05 (2H, s, SCH2), 2.29 (3H, s,
(1H, s, CH-5), 4.05 (2H, s, SCH2), 2.24 (3H, s, CH3), 2.13 (3H, s, CH3), 2.15 (3H, s, CH3); 2.09 (3H, s, CH3). 13C NMR (126 MHz,
CH3). 13C NMR (126 MHz, DMSO-d6): δ 169.78 (C=O, amide), DMSO-d6): δ 166.37 (C=O, amide), 166.21 (6-C=O), 165.34,
167.38 (6-C=O), 166.46 (C-S), 165.33, 143.36, 136.82, 132.61, 164.12 (C-S), 136.35, 135.2, 130.46, 128.87, 126.39, 125.62,
129.48, 119.67 (2C, Ar), 107.13 (5-CH, br), 35.45 (CH2), 23.75 107.48 (5-CH, br), 34.75 (CH2), 23.52 (CH3-4), 20.95 (CH3),
014 Severina et al. / Journal of Applied Pharmaceutical Science 9 (02); 2019: 012-019

17.75 (CH3). Found, m/z: 304.11 [M+H]+. The Anal. Calcd. was 2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-
for C15H17N3O2S: C, 59.38; N, 13.85; S, 10.57; we found: C, cyclohexlyl-acetamide, 5.13
59.19; N, 13.89; and S, 10.55. Yield: 65%, mp 245°C–7°C; 1H NMR (300 MHz,
DMSO-d6, δ (ppm)): 12.51 (1H, br. s, NH-3), 7.88 (1H, s, NHCO),
2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(2,5-
5.98 (1H, s, CH-5), 4.00 (2H, s. SCH2), 2.21 (3H, s, CH3) 1.90–
dimethoxyphenyl)acetamide, 5.8
1.18 (м, 11H, С6H11). 13C NMR (126 MHz, DMSO-d6): δ 167.58
Yield: 68%, mp 188°C–90°C; 1H NMR (300 MHz, (C=O, amide), 166.4 (6-C=O), 166.09, 165.43 (C-S), 107.65 (5-
DMSO-d6, δ (ppm)): 12.43 (1H, br. s, NH-3), 9.39 (1H, s, NHCO), CH, br), 48.42, 36.66, 34.6, 32.74, 31.99, 25.55, 24.66, 23.53
7.86 (1H, s, H-6′), 6.90 (1H, d, J = 7.9, H-4′), 6.56 (1H, d, J = 7.9, (CH3). Found, m/z: 282.19 [M+H]+. The Anal. Calcd. was for
H-3′), 6.05 (1H, s, CH-5), 4.01 (2H, s, SCH2), 3.75 (6H, s, 2CH3); C13H19N3O2S: C, 55.49; N, 10.68; S, 11.40; we found: C, 55.36;
2.23 (3H, s, CH3). Found, m/z: 336.11 [M+H]+. The Anal. Calcd. N, 10.65; and S, 11.36.
was for C15H17N3O4S: C, 53.72; N, 12.53; S, 9.56; we found: C,
53.60; N, 12.58; and S, 9.53. Ethyl 2-[2-(4-methyl-6-oxo-1,6-dihydropyrimidin-2-ylthio)-
acetylamino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-
2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(2,4,6- carboxylate, 5.14
trimethylphenyl)acetamide, 5.9
Yield: 55%, mp 230°C –2°C; 1H NMR (300 MHz,
Yield: 69%, mp >271°C–3°C; 1H NMR (300 MHz, DMSO-d6,, δ (ppm)): 12.61 (1H, br. s, NH-3), 11.29 (1H, s,
DMSO-d6, δ (ppm)): 12.33 (1H, br. s, NH-3), 9.39 (1H, s, NHCO), 6.03 (1H, s, CH-5), 4.35 (2H, q, OCH2CH3), 4.10 (2H,
NHCO), 6.85 (2H, s, H-3′,5′), 6.00 (1H, s, CH-5), 4.02 (2H, s, s. SCH2), 2.90-2.70 (2H, m, CH2), 2.52–2.31 (4H, m, 2CH2), 2.12
SCH2), 2.22 (6H, s, 2CH3); 2.11 (6H, s, 2CH3). 13C NMR (126 (3H, s, CH3) 1.20 (3H, t, J = 5.2, OCH2CH3). 13C NMR (126 MHz,
MHz, DMSO-d6): δ 166.43 (C=O, amide), 166.09 (6-C=O), DMSO-d6): δ 167.66 (C=O amide), 166.10 (6-C=O), 166.05,
165.43, 164.12 (C-S), 135.82, 135.26 (2C), 132.67, 128.66 (2C), 166.01, 164.12, 150.52, 141.37, 132.21, 108.54, 107.65 (5-CH,
107.84 (5-CH, br), 34.32 (CH2), 23.7 (CH3 -4), 20.92 (CH3), 18.27 br), 60.81, 33.99 (CH2), 30.37, 28.86, 27.7, 23.69 (CH3) 14.42
(2CH3). Found, m/z: 318.12 [M+H]+. The Anal. Calcd. was for (CH3). Found, m/z: 393.48 [M+H]+. The Anal. Calcd. was for
C16H19N3O2S: C, 60.54; N, 13.24; S, 10.10; we found: C, 60.42; C17H19N3O4S2: C, 55.49; N, 10.68; S, 11.40; we found: C, 55.36;
N, 13.19; and S, 10.05. N, 10.72; and S, 11.35.
2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(2,4,6- 2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-
trichlorophenyl)acetamide, 5.10 phenoxy-phenyl)-acetamide, 5.15
Yield: 76%, m.p. >258°C–60°C; 1H NMR (300 MHz, Yield: 60%, mp 224°C–6°C; 1H NMR (300 MHz,
DMSO-d6, δ (ppm)): 12.50 (1H, br. s, NH-3), 10.01 (1H, s, DMSO-d6,, δ (ppm)): 12.45 (1H, br. s, NH-3), 10.08 (1H, s,
NHCO), 7.69–7.51 (2H, m, Ar-H), 6.03 (1H, s, CH-5), 4.10 (2H, s, NHCO), 7.75–7.55 (2H, m, Ar-H), 7.40–7.29 (2H, m, Ar-H), 7.10–
SCH2), 2.22 (3H, s, CH3). Found, m/z: 379.66 [M+H]+. The Anal. 6.91 (5H, m, Ar-H), 5.98 (1H, s, CH-5), 4.08 (2H, s. SCH2), 2.21
Calcd. was for C13H10Cl3N3O2S: C, 41.23; N, 11.10; S, 8.47; we (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 166.43 (C=O,
found: C, 41.19; N, 11.06; and S, 8.44. amide), 166.09 (6-C=O), 165.43, 164.12 (C-S), 157.78, 152.44
2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(2-methyl- (2C), 135.25, 130.43(2C), 123.49 (2C), 121.37, 119.94 (2C),
3-chlorophenyl) acetamide, 5.11 118.39, 107.83 (5-CH, br), 35.54 (CH2), 23.91 (CH3). Found, m/z:
368.43 [M+H]+. The Anal. Calcd. was for C19H17N3O3S: C, 62.11;
Yield: 75%, mp >262°C; 1H NMR (300 MHz, DMSO-d6, N, 11.44; S, 8.73; we found: C, 62.02; N, 11.40; and S, 8.69.
δ (ppm)): 12.50 (1H, br. s, NH-3), 10.01 (1H, s, NHCO), 7.69–7.51
(3H, m, Ar-H), 6.01 (1H, s, CH-5), 4.09 (2H, s, SCH2), 2.17 (3H, Anticonvulsant activity
s, CH3). Found, m/z: 324.05 [M+H]+. The Anal. Calcd. was for
Animals
C14H14ClN3O2S: C, 51.93; N, 12.98; S, 9.90; we found: C, 51.79;
N, 12.93; and S, 9.87. The experiments were carried out on 95 adult male
rats (130–150 g) and adult random-bred albino mice of either
2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-benzyl- sex weighing 22–28 g. The animals were bred in the vivarium
acetamide, 5.12 of National Pirogov Memorial Medical University, Vinnytsya
Yield: 66%, mp 196°C –8°C; 1H NMR (300 MHz, housed in cages under standard conditions with a temperature of
DMSO-d6, δ (ppm)): 12.50 (1H, br. s, NH-3), 10.01 (1H, t, J = 5.1, (22°C ± 1°C), relative humidity of (55% ± 15%) with free access
CH2NHCO), 7.60–7.27 (4H, m, H-2′,3′,5′,6′), 7.05 (1H, t, J = 7.5, to food and water and a 12-hour light/darkness cycle (8.00–20.00),
H-4'), 6.05 (1H, s, CH-5), 4.11 (2H, s. SCH2), 4.01 (2Н, d, J = 5.3, respectively. All of the experiments were conducted in accordance
NHCH2Ph), 2.18 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): with “Directive 2010/63/EU of the European Parliament and of the
δ 167.58 (C=O, amide), 166.43 (6-C=O), 166.09, 165.43 Council of 22 September 2010 on the protection of animals used
(C-S), 139.61, 128.7 (2C), 127.59 (2C), 127.27, 107.84 (5- for scientific purposes,” with the procedures and requirements
CH, br), 42.98 (CH2), 34.27 (CH2, amide), 23.83 (CH3). Found, of the State Expert Center of the Ministry of Health of Ukraine
m/z: 290.09 [M+H]+. The Anal. Calcd. was for C14H15N3O2S: and with the rules of European Convention for the Protection of
C, 58.11; N, 14.52; S, 11.08; we found: C, 58.01; N, 14.48; Vertebrate Animals used for Experimental and Other Scientific
and S, 11.04. Purposes (Strasbourg, 1986), resolution of the First National
 Severina et al. / Journal of Applied Pharmaceutical Science 9 (02); 2019: 012-019 015

Congress on Bioethics (Kyiv, 2001), with the Law of Ukraine After estimation of the most active substance, its acute
National Congress on Bioethics (Kyiv, 2001) and with the Law toxicity (LD50), neurotoxicity (TD50), and protective index (PI =
of Ukraine №3447-IV “On Protection of Animals from Cruel LD50/TD50) were determined.
Treatment” dated 02.21.2006.
Animals were randomly assigned to the experimental Neurotoxic activity
and control groups. The substances tested were dissolved in 1% The possible neurotoxic effects in mice were quantified
starch gel and administered by oral gavage cannula at a volume using a rotarod test. The test compound was administered
of 0.5 ml/100 g body weight in rats and 0.2 ml/10 g body weight intraperitoneally to animals at a dose rate of 10–200 mg/kg and
in mice 1 hour before seizure induction. Screening dose for the control mice received an equivalent amount of solvent. Mice
testing compounds was 80 and 50 mg/kg. Referent compounds were placed on a rotating knurled rod (12 rpm). Neurotoxic
(phenobarbital, lamotrigine, and carbamazepine) were given the action was manifested in the form of violations of coordination of
same manner in their median anticonvulsant doses 20, 20, and motion. Mice were considered impaired if they fell off the rotarod
15 mg/kg body weight, respectively (Metcalf et al., 2017). The three times during the 1-minute observation period performed
determination of time for the experiment was based on the data immediately prior to stimulation (rotarod failure). The mice of the
concerning the peak of the anticonvulsant activity of the drug, control group were kept on the rods for several minutes. The value
described in the literature (Fisher, 1989; Löscher, 2011). Control of TD50 was calculated by the probit analysis method.
rats received equivolume amounts of solvents. All seizures [both
chemical (PTZ) and electrical (MES)] were induced between Acute toxicity
9:00 and 11:00 to minimize possible inconsistencies arising from During this experiment, we used the white nonlinear
circadian rhythms (Löscher, 1996). mice (24 ± 3g), which were divided into five groups. Animals
of the experimental groups received a substance 5.5, ranging in
Pentylenetetrazole-induced seizures doses from 100 mg/kg to 400 mg/kg. The drug was administered
Seizures in animals were modeled by a single in appropriate doses intragastrically, dissolving it in the required
subcutaneous pentylentetrazole injection (Sigma) at a dose of amount of 1% starch gel. Animal observations were carried out
80 mg/kg. The animals were administered intragastrically with within 14 days; then, the number of dead animals in each group was
a freshly prepared suspension of experimental compounds and noted and using the method implemented by Prozorovsky (2007),
reference drugs phenobarbital and lamotrigine (20 mg/kg). The acute toxicity (LD50 and its confidence interval) was evaluated.
anticonvulsant activity was assessed by the dynamic of the latent We recorded the behavior and body weight of mice, the clinical
period, the intensity and duration of seizures in minutes, and the signs of intoxication, the general condition of animals, the nature
lethality rate of mice. of motor activity, the characteristics of breathing, the condition of
The intensity of seizures was evaluated using a 5-point hair and skin, the presence of a vessel, the consumption of food
scale, taking as a basis the following criteria (including the number and water, and also we noted the number of dead animals in each
of animals that died) (Gerald, 1973): 0—no seizure activity; 1— group and the value of LD50 was defined.
hyperkinesia; 2—trembling, twitching; 3—clonic seizures of
upper limbs with the rise on their lower limbs; 4—pronounced Statistical analysis
tonic-clonic seizures, the animal’s fall to the side, and the available All values were expressed as the means ± S.E.M. The
phase of tonic extension; and 5—repeated tonic-clonic seizures, data were analyzed by ANOVA (analysis of variance) followed
loss of posture, and death. by Dunnett’s test (Statistical package for social sciences, SPSS
Anticonvulsant effect was considered an animal 16.0). An X2 analysis was performed to compare the neurotoxicity
protection based on the clonic and tonic seizures and the lethality. differences (number of failures per test). p values ≤ 0.05 was
considered as significant.
Maximal electroshock seizure testing in mice (MES)
The research was conducted on the non-linear mice of RESULTS AND DISCUSSION
both sexes, weighing 25–28 grams. The animals were divided Chemistry
into four groups with 10 animals in each group. Group one served
To establish the prospects of synthesis and optimization
as the control group. Animals of other groups were receiving
of further pharmacological screening, we performed a prediction
intragastrically compounds (5.1-15) (50 mg/kg), lamotrigine
of the biological activity that was planned for the synthesis
(20 mg/kg), or carbamazepine (15 mg/kg). The investigation
of compounds using a PASS computer program (http://www.
of anticonvulsant activity was conducted 1 hour later after the
pharmaexpert.ru/passonline/). 6-Methyl-2-thioxo-2,3-dihydro-
administration of the experimental compounds. The MES test
1H-pyrimidin-4-one acetamides were selected for synthesis.
consisted of electrical stimulation of the cornea using 60 Hz of
High psychotropic activities such as antiepileptic, anxiolytic,
alternating current (150 mA), with a stimulus duration of 0.2
antidepressant, antineurotic, and anticonvulsant activities (Pa ≥
second, using a custom-build MES stimulator. The electrodes
0.50) were predicted for these compounds.
were soaked with a 0.9% sodium chloride solution. A number of
The synthesis of initial 6-methyl-2-thioxo-2,3-dihydro-
animals with hind limb flexion-extension and mortality rate were
1H-pyrimidin-4-one 3 consisted of condensation of the ethyl
estimated.
acetoacetate 1 with a 2.5-fold molar excess of thiourea 2 (Fig. 1).
The reaction occurred successfully in refluxing absolute methanol
016 Severina et al. / Journal of Applied Pharmaceutical Science 9 (02); 2019: 012-019

Figure 1. The synthesis of 2-[(1,6-dihydro-6-oxo-4-methyl-2-pyrimidinyl)thio]-N-acetamides.

with a 2.6–2.8-fold molar excess of sodium methylate as reported to relate all other signals to any specific carbon atom (especially
in known methods used (Novikov et al., 2005). in the aromatic region of the spectrum) without additional
Alkylation of the resulting 6-methyl-2-thiopymidin-4- two-dimensional experiments. However, they also give useful
one 3 was carried out by an equimolar amount of N-arylsubstituted information—at least, concerning the number of carbon atoms in
2-chloroacetamides, 2-chloro-N-benzylacetamide, and ethyl the molecule. 13C NMR spectra for compounds 5.5, 5.6, 5.8, and
2-[(chloroacetyl)amino]-5,6-dihydro-4H-cyclopenta[b]thiophene- 5.10 were not recorded due to insufficient solubility.
3-carboxylate according to known procedure of alkylation of
thiopyrimidines (Gagnon et al., 2007; Rakhimov and Titova, Anticonvulsant activity
2007). The reaction was conducted in dimethylformamide solution In the control group, pentylentetrazole administration
in the presence of an excess of potassium carbonate and led to the led to the development of seizures in all animals. The duration
formation of only 2-[(1,6-dihydro-6-oxo-4-methyl-2-pyrimidinyl) of the latent period averaged 4.7 minutes and the duration of
thio]-N-acetamides 5.1-15. The yield of reaction products was seizures —9.7 minutes (Table 1). The seizures which developed
55%–82%. in this group of rats were accompanied by severe periodically
Because the sulfur atom in the molecule of 4-methyl- repeated tonic-clonic convulsions. There was a clear phase of
2-thiopyrimidin-4-one 3 is the most nucleophilic, the alkylation tonic extension (epistotonus). The lethality in this group of rats
reaction on this direction was expected and most likely. was 100%. Phenobarbital prevented the development of the
In the literature (Danel, 1998), it was noted that the seizures in all animals. At the same time, after administration of
usage of primary alkyl halides as alkylating agents leads to the lamotrigine in rats, pentylentetrazole induced some manifestations
formation of a complex mixture consisting of S-mono, SN1- and of the seizures (convulsive twitching, jumps, and contractions
SN3-disubstituted alkylation products. TLC, LCMS, and 1H NMR of the upper limbs), but the duration of the latent period was
spectroscopy data confirmed the formation of only S-derivatives statistically reliably lengthened (in 5.8 times), the degree of the
which probably can be explained by using sizable alkylating seizure severity, and their total duration were significantly lower
agents that shielded Nitrogen atoms. than in the control group. Lamotrigine prevented the lethality in
The 1H NMR spectra of synthesized compounds 5.1–5.15 80% of animals.
contain a wide singlet proton signal of the NH group in the third All investigated compounds reduced the development
position of the pyrimidine cycle located at δ 12.52–12.43 ppm, a of seizures in the chemoconvulsive seizure model (Table 1).
singlet signal of NHCO proton group of acetamide residue at δ After their administration, the extension of the latent period was
11.23–9.39 ppm, a signal of a methine proton at 5 position of the observed in relation to the control group; however, compounds
pyrimidine cycle at δ 6.05–5.98 ppm, and a singlet of a methylene 5.4, 5.10, 5.12, 5.14, and 5.15 slightly increased it (in 0.2–1.5
SCH2-group within a range 4.12–4.00 ppm. Protons of the phenyl times).
radical resonate at δ 7.92–6.52 ppm and their multiplicity and The extension of the latent period of seizures, reduction
intensity correspond to the nature and location of substituents. of their intensity, and duration of seizures were most observed
13
C NMR spectra of all 2-[(1,6-dihydro-6-oxo-4- while administering the compounds 5.2, 5.5, 5.8, 5.13, and 5.11.
methyl-2-pyrimidinyl)thio]-N-acetamides 5.1-15 allow for Compounds 5.5, 5.7, 5.8, 5.11, and 5.13 prevented the
reliably identifying the signals of only some carbon atoms: C=O lethality in 100% of rats similarly to phenobarbital. Compounds 5.1,
amide, 6-C=O, C-S, 5-CH, 6-CH3, CH2, and CH3. It is incorrect 5.2, 5.3, and 5.10 and lamotrigine reduced lethality, respectively,
 Severina et al. / Journal of Applied Pharmaceutical Science 9 (02); 2019: 012-019 017

Table 1. The effect of the synthesized compounds 5.1–5.15, lamotrigine, and phenobarbital on seizures caused by administration of pentylentetrazole in rats.

Number of rats Duration of the latent Lethality abs. units Intensity of seizures,
Groups of animals Dose, mg/kg Duration of seizures, min
in the group period, min (%) (points)
Control n =10 - 4.7 ± 0.30#/& 9.70±0.90#/& 10 (100%) 4.96
5.1 n=5 80 11.2 ± 2.0*/#/& 5.6 ± 1.0*/#/& 2 (40%) 3.6
5.2 n=5 80 14.4 ± 4.0*/#/& 7.6 ± 2.0#/& 2 (40%) 3.2
5.3 n=5 80 1.8 ± 2.1*/#/& 15.0 ± 2.4*/#/& 2 (40%) 3.2
5.4 n=5 80 5.0 ± 0.4#/& 9.2 ± 1.6#/& 5 (100%) 4.8
5.5 n=5 80 27.0 ± 3.0* 1.0 ± 1.0*/# 0* 0.4
5.6 n=5 80 7.6 ± 1.2*/#/& 14.8 ± 2.1*/#/& 3 (60%) 3.8
5.7 n=5 80 8.4 ± 1.7*/#/& 11.4 ± 3.5#/& 0* 3.4
5.8 n=5 80 16.2 ± 3.6*/#/& 4.4 ± 1.3*/# 0* 1.0
5.9 n=5 80 13.8 ±2.2*/#/& 13.4 ± 1.8#/& 3 (60%) 2.6
5.10 n=5 80 5.8 ± 0.9#/& 8.8 ± 2.2#/& 2 (40%) 3.2
5.11 n=5 80 18.0 ± 5.1*/# 2.8 ± 1.2*/# 0* 1.8
5.12 n=5 80 7.0 ± 0.3*/#/& 9.40 ±0.7*/#/& 5 (100%) 4.4
5.13 n=5 80 12.6±1.4*/#/& 12.0 ±3.3#/& 0* 2.8
5.14 n=5 80 11.8± 4.6#/& 7.2 ± 2.1#/& 3 (60%) 2.8
5.15 n=5 80 5.0 ± 0.8#/& 10.4± 0.5#/& 5 (100%) 4.6
Phenobarbital n=5 20 30.0 ± 0.0* 0* 0* 0
Lamotrigine n=5 20 27.6 ± 0.8* 2.40±0.40*/# 1 (20%) 2.20

n—a number of animals in the group; *—statistically significant differences of obtained results in the experimental group (p < 0.05) compared with a control; #—statistically significant
differences of obtained results in the experimental group (p < 0.05) compared with phenobarbital. &—statistically significant differences of obtained results in the experimental group (p <
0.05) compared with lamotrigine.

Table 2. The effect of the synthesized compounds (5.5, 5.7, 5.8, 5.11, and Structure-activity relationship (SAR) studies
5.13), carbamazepine, and lamotrigine on seizures caused by maximal
electroshock in mice. Analysis of the results of screening studies of
anticonvulsant activity of thiopyrimidine derivatives 5.1–5.15 on
Duration of the model of pentylentetrazole seizures in rats allowed making
Number of Number of electroshock
Experiment
animals in
Dose,
mice with seizures (clonic some general conclusions about the influence of structural
conditions mg/kg
group seizures seizures + tonic fragments on anticonvulsant activity of synthesized compounds.
extension), sec
It can be assumed that the introduction of the N-phenyl
Control 10 - 9 47.3 ± 0.48 radical into the structure of compounds contributes to the increase
5.5 10 50 1 5.4 ± 0.31*# of anticonvulsant activity, since the replacement of the N-phenyl
(-88.5%)
radical with benzyl, 4-phenoxyphenyl, and cyclopentathiophene
5.7 10 50 7 30.2±0.25*#& (5.12, 5.14, and 5.15) leads to reduction of the latent period,
5.8 10 50 5 10.1±1.36*# prolongation of the duration of seizures, and an increase the
5.11 10 50 5 12.4±2.15*# percentage of lethality of the experimental animals (100%, 100%,
5.13 10 50 6 18.2±0.18*#& and 60%, respectively).
Lamotrigine 10 20 3 8.4 ± 0.39* Substituents in the phenyl radical also have a significant
(−82.3%) role in an anticonvulsant activity. 4-, 2,3- and 2,4,6-chloro-
Carbamazepine 10 15 2 6.2 ± 0.56* substituted derivatives (5.4, 5.6, and 5.10) minimally extended
(−86.9%) the latent period, did not reduce the duration and severity of the
*—statistically significant differences of obtained results in the experimental group seizures and did not prevent the lethality of animals, whereas, Me-,
(p < 0.05) with a control; #—statistically significant differences of obtained results in the MeO- and, in particular, 4-Br-substituted derivatives improved
experimental group (p < 0.05) compared with carbamazepine; &—statistically significant
differences of obtained results in the experimental group (p < 0.05) compared with this data.
lamotrigine. The following pattern was observed: with the increase
of the number of chlorine atoms, the lethality of the experimental
in 80% and 60% of the experimental rats. 100% lethality of the animals decreased: 5.4, 5.6, and 5.10 = 4-Cl, 2,3-diCl, 2,4,6-triCl
experimental animals, similar to the control group, was observed = 100%, 60%, 40%. Such results were unexpected as there is
while administering the compounds 5.4, 5.12, and 5.15. a significant amount of literary data describing an increase of
According to our research, the most pronounced anticonvulsant activity when introducing chlorine atoms.
anticonvulsant activity was shown by compound 5.5, which The next step in our research was focused on investigating
contained 4-bromophenyl radical. This compound reduced the the effects of the synthesize substances on the models of primary
duration of seizures in 2.4 times and the seizure severity in 5.5 generalized seizures caused by maximal electroshock which is
times. essential for identifying potential anticonvulsants.
018 Severina et al. / Journal of Applied Pharmaceutical Science 9 (02); 2019: 012-019

Table 3. Characteristic of anticonvulsant activity of 2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)

thio]-N-(4-bromophenyl)acetamide (5.5) in intragastric administration.

LD50, Pentylenetetrazole (rats) МЕS (mice)

Compound ТD50, mg/kg
mg/kg ЕD50, mg/kg ТІ РІ ЕD50, mg/kg ТІ РІ
5.5 258.0± 105.9 ± 11.7 53.0± 0.33 9.96 1.98 12.5± 0.14 20.6 8.47
(23.3)

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