Inhibitory effect of some new uracil and thiouracil derivatives on cercarial penetration enzymes

Article abstract of DOI:10.1007/s11164-012-0748-x

Some uracil- and thiouracil-5-sulfonohydrazide derivatives have been synthesized to be evaluated as antischistosomal agents. N-[2-(1,5-Dimethyl-3- oxo-2-phenylpyrazolin-4-yl)-4-oxo-1,3-thiazolidin-3-yl]-4-oxo-2-thioxo-1,2,3, 4-tetrahydropyrimidine-5-sulfonamide (3c) was formulated in jojoba oil and used to paint mice tails before infection with Schistosoma mansoni cercariae. Using Boc-Val-Leu-Gly-Arg-_PNA, a specific substrate for trypsin-like serine proteinases, compound 3c inhibited cercarial serine protease activity with 50 % inhibition concentration (IC₅₀) of 160 μg. Upon topical application on mice tails before infection with S. mansoni cercariae, it caused a 20 % reduction in worm burden compared with untreated infected mice. Using soluble crude cercarial antigen in enzyme-linked immunosorbent assay (ELISA), no significant changes were observed in the levels of immunoglobulin M (IgM) and IgG in sera from treated infected mice at 2, 4, and 6 weeks postinfection (WPI) compared with the level in sera from infected untreated mice. At 4 WPI, sera from treated infected mice showed significantly low (P < 0.05) IgM reactivity to crude soluble worm antigen compared with infected nontreated ones. IgG levels in sera from treated infected mice at 2 and 4 WPI were significantly lower (P < 0.05) than in sera from infected nontreated mice. At 6 WPI, the IgG response showed no significant differences in sera from both mice groups. Sera from treated infected mice at 2, 4, and 6 WPI had generally lower IgM reactivity to soluble egg antigen when compared with the level in sera from nontreated infected mice. At all time points postinfection, sera collected from treated infected mice showed significantly low IgG reactivity (P < 0.05) compared with infected nontreated mice.

Full text of DOI:10.1007/s11164-012-0748-x

Res Chem Intermed
DOI 10.1007/s11164-012-0748-x
Inhibitory effect of some new uracil and thiouracil
derivatives on cercarial penetration enzymes
•
O. A. Fathalla M. E. Haiba M. M. Anwar
•
•
•
Maha S. Almutairi A. S. Maghraby M. M. Bahgat
•
Received: 28 May 2012 / Accepted: 25 July 2012
Ó Springer Science+Business Media B.V. 2012
Abstract Some uracil- and thiouracil-5-sulfonohydrazide derivatives have
been synthesized to be evaluated as antischistosomal agents. N-[2-(1,5-Dimethyl-3-
oxo-2-phenylpyrazolin-4-yl)-4-oxo-1,3-thiazolidin-3-yl]-4-oxo-2-thioxo-1,2,3,4-tet-
rahydropyrimidine-5-sulfonamide (3c) was formulated in jojoba oil and used to
paint mice tails before infection with Schistosoma mansoni cercariae. Using Boc-
Val-Leu-Gly-Arg-PNA, a specific substrate for trypsin-like serine proteinases,
compound 3c inhibited cercarial serine protease activity with 50 % inhibition
concentration (IC50) of 160 lg. Upon topical application on mice tails before
infection with S. mansoni cercariae, it caused a 20 % reduction in worm burden
compared with untreated infected mice. Using soluble crude cercarial antigen in
enzyme-linked immunosorbent assay (ELISA), no significant changes were
observed in the levels of immunoglobulin M (IgM) and IgG in sera from treated
infected mice at 2, 4, and 6 weeks postinfection (WPI) compared with the level in
sera from infected untreated mice. At 4 WPI, sera from treated infected mice
showed significantly low (P \ 0.05) IgM reactivity to crude soluble worm antigen
compared with infected nontreated ones. IgG levels in sera from treated infected
mice at 2 and 4 WPI were significantly lower (P \ 0.05) than in sera from infected
nontreated mice. At 6 WPI, the IgG response showed no significant differences in
sera from both mice groups. Sera from treated infected mice at 2, 4, and 6 WPI had
O. A. Fathalla ꢀ M. E. Haiba ꢀ M. M. Anwar (&) ꢀ A. S. Maghraby ꢀ M. M. Bahgat
Department of Therapeutical Chemistry, National Research Centre, Dokki, Cairo, Egypt
e-mail: Manal.hasan52@yahoo.com
M. E. Haiba ꢀ M. S. Almutairi
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia
A. S. Maghraby ꢀ M. M. Bahgat
Laboratory of Immunology and Infectious Diseases, the Center of Excellence for Advanced
Sciences, National Research Center, Dokki, Cairo, Egypt
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O. A. Fathalla et al.
generally lower IgM reactivity to soluble egg antigen when compared with the level
in sera from nontreated infected mice. At all time points postinfection, sera col-
lected from treated infected mice showed significantly low IgG reactivity
(P \ 0.05) compared with infected nontreated mice.
Keywords Cercariae ꢀ Cercarial serine protease ꢀ IgM ꢀ IgG ꢀ 4-Oxo-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-sulfonohydrazide ꢀ Schistosoma mansoni
Introduction
Schistosomiasis (formerly called bilharziasis or bilharziosis) is a parasitic disease
produced by a flatworm of the class trematoda, relatively common in developing
countries, especially in Africa, called schistosoma (or esquistosoma). More than 207
million individuals are infected worldwide, and in addition, 700 million are at risk
of infection [1]. Around 90 % of individuals who need preventive chemotherapy for
schistosomiasis are in Africa, where the disease is endemic in 42 countries [1].
Chemotherapy is an important control strategy against this parasitic disease [2];
however, it has not reduced endemicity [3], and rapid reinfection demands frequent
treatment [4]. Therefore, it is considered that an effective vaccine combined with
chemotherapy would be an efficient control mechanism [5]. The initial step in the
infection of humans by schistosomal parasites is penetration of cercariae, the
infective stage of the parasite, into the host skin [6, 7]. Attempts to develop a topical
formulation to prevent this step date back to Pellegrino [8]. Niclosamide was tested
as a cercaricidal compound in the field for its ability to prevent such infections [9,
10]. Some efficacy of niclosamide in preventing infection by Schistosoma mansoni
but not by Schistosoma haematobium was reported, but this was insufficient to
warrant a recommendation for its widespread use. Penetration of cercariae into the
host skin is facilitated by secretion of a potent cercarial serine protease from the
preacetabular glands of cercariae [11–13]. Several uracil and thiouracil derivatives
constitute an interesting group of compounds, many of which possess widespread
pharmacological activities especially as antiviral [14], antibacterial [15], antituber-
cular [16], inhibition of serine protease of S. mansoni [12], antihypertensive [17],
analgesic and anti-inflammatory [18], anticonvulsant [19], and anticancer agents
[20]. As a continuation of our previous studies on uracil and thiouracil chemistry
[12, 21–23], the present work deals with synthesis of new compounds carrying these
important nuclei in order to study their inhibitory effects on cercarial penetration
enzymes to block penetration of S. mansoni cercariae into mouse skin.
Experimental
All melting points were uncorrected and determined in capillary tubes by melting
point microscope (Boetius; Great Britain Stuart Scientific Co. Ltd.). Infrared (IR)
spectra were recorded on an infrared spectrophotometer (PU9712; Beckman,
Madison, WI, USA) with KBr discs. 1H and 13C nuclear magnetic resonance (NMR)
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Inhibitory effect of some new uracil and thiouracil derivatives
spectra were measured on a Joel EX500-MHZ (Japan) spectrometer using
tetramethylsilane (TMS) as internal standard. Mass spectra were recorded by mass
spectrometer (SSQ7000; California, USA) at 70 eV. Microanalyses were carried out
at the Microanalytical Center, Cairo University. All reactions were followed and
checked by thin-layer chromatography (TLC) using chloroform/methanol (3:1), and
spots were examined by UV analysis lamp k 254/366 nm.
Synthesis of the starting compound (1)
The starting material thiouracil-5-sulfonylhdrazide (1) was prepared by reaction of
thiouracil-5-sulfonylchloride with hydrazine hydrate 99 % [12].
General procedure for synthesis of the Schiff bases (2a–d)
A mixture of thiouracil-5-sulfonylhdrazide (1, 2.3 g, 10 mmol) and the appropriate
aldehyde, namely 4-bromobenzaldehyde, 5-methyl-2-furfural, 4-antipyrinecarbox-
aldehyde or salicylaldehyde (10 mmol), in absolute ethanol (25 ml) was stirred at
room temperature for 2 days. The reaction mixture was cooled, and the formed
precipitate was then filtered off and dried. The obtained product was crystallized
from the proper solvent to give the title compounds 2a–d, respectively.
N0-(4-Bromobenzylidin)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
sulfonylhydrazide (2a)
Yield: 64 %; m.p.: 251–253 °C; crystallized from dimethylformamide (DMF)/
water; IR (KBr, cm-1): 3325, 3225, 3122 (3NH), 1680 (CO of thiouracil), 1322,
1
1125 (SO2NH); H-NMR (dimethyl sulfoxide, DMSO-d6, d ppm): 7.30, 7.71 (d, d,
4H, aromatic H, J = 6.5 Hz), 8.42 (s, 1H, thiouracil-H6), 8.90 (s, 1H, –N=CH),
11.10, 11.33, 11.80 (3s, 3H, 3NH exchangeable with D2O); 13C-NMR (DMSO-d6, d
ppm): 178.3 (C=S), 164.0 (C=O), 103.1, 125.1, 131.2, 132.4, 133.0, 143.7, 150.1
(aromatic-C; CH=N); MS m/z: 388 (M?) (30 %), 390 (M? ? 2) (29 %); Anal.
calcd. for C11H9BrN4O3S2 (389.25): C, 33.94; H, 2.33; N, 14.39. Found: C, 33.82;
H, 2.31; N, 14.34.
N0-[(5-Methyl-2-furyl)methylene]-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
sulfonylhydrazide (2b)
Yield: 72 %; m.p.: 263–265 °C; crystallized from DMF/water; IR (KBr, cm-1):
3318, 3217, 3198 (3NH), 1680 (CO of thiouracil), 1327, 1162 (SO2NH); H-NMR
1
(DMSO-d6, d ppm): 2.33 (s, 3H, CH3), 6.31, 7.20 (d,d, 2H, furan ring, J = 7.4 Hz),
8.34 (s, 1H, thiouracil-H6), 8.72 (s, 1H, –N=CH), 10.32, 11.41, 11.72 (s, 3H, 3NH
exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 178.1 (C=S), 164.6 (C=O),
103.2, 106.7, 110.2, 134.7, 147.3, 150, 152.5 (aromatic-C; CH=N), 15.1 (CH3); MS
m/z: 314 (M?) (34 %); Anal. calcd. for C10H10N4O4S2 (314.36): C, 38.20; H, 3.21;
N, 17.82. Found: C, 38.17; H, 3.16; N, 17.78.
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O. A. Fathalla et al.
N0-[(1,5-Dimethyl–3-oxo-2-phenylpyrazolidin-4-yl)methylene]-4-oxo-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-sulfonylhydrazide (2c)
Yield: 66 %; m.p.: 275–277 °C; crystallized from DMF/water; IR (KBr, cm-1):
3325, 3225, 3215 (3NH), 3080 (C–H aromatic), 1712 (CO of pyrazoline ring), 1683
(CO of thiouracil), 1345, 1127 (SO2NH); 1H-NMR (DMSO-d6, d ppm): 2.50 (s, 3H,
CH3), 3.71 (s, 3H, –N–CH3), 6.43–7.33 (m, 5H, aromatic-H), 8.2 (s, 1H, thiouracil-
H6), 9.10 (s, 1H, –N=CH), 10.11, 11.01, 11.42 (3s, 3H, 3NH exchangeable with
D2O); 13C-NMR (DMSO-d6, d ppm): 176.1 (C=S), 165.2, 164.1 (2C=O), 103.2,
102.7, 113.2, 119.2, 129.1, 136.7, 147.5, 150, 154.2 (aromatic-C; CH=N), 15.3, 15.5
(2CH3); MS m/z: 420 (M?) (6 %); Anal. calcd. for C16H16N6O4S2 (420.48): C,
45.70; H, 3.83; N 19.98; Found: C, 45.43; H, 3.71; N, 19.95.
N0-(2-Hydroxybenzylidin)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
sulfonylhydrazide (2d)
Yield: 71 %; m.p.: 281–283 °C; crystallized from DMF/water; IR (KBr, cm-1):
3450 (OH), 3315, 3225, 3115 (3NH), 1692 (CO of thiouracil), 1343, 1125 (SO2NH);
1H-NMR (DMSO-d6, d ppm): 7.31–7.80 (m, 4H, aromatic-H), 8.32 (s, 1H,
thiouracil-H6), 8.74 (s, 1H, –N=CH), 10.32, 10.99, 11.62, 12.21 (4s, 4H, 3NH, OH
exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 178.1 (C=S), 164.0 (C=O),
103.1, 116.7, 118.2, 130.3, 121.0, 143.2, 150.5, 161.4 (aromatic-C; CH=N); MS m/
z: 326 (M?) (17 %); Anal. calcd. for C11H10N4 O4S2 (326.38): C, 40.48; H, 3.09; N
17.17. Found: C, 40.39; H, 3.04; N, 17.11.
General procedure for preparation of thiazolidinone derivatives 3a–d
A solution of thioglycolic acid (1 g, 10 mmol) in dry benzene (15 ml) was added to
a solution of the compound 2a–d (10 mmol) in dry benzene (20 ml), then the
mixture was refluxed on a water bath for 8–12 h. The formed precipitate was filtered
off and crystallized from the proper solvent to give 3a–d.
N-[2-(4-Bromophenyl)-4-oxo-1,3-thiazolidin-3-yl]-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-sulfonamide (3a)
Yield: 70 %; m.p.: 271–273 °C; recrystallized from ethanol; IR (KBr, cm-1): 3410,
3321, 3225 (3NH), 1725 (CO of thiazolidine), 1672 (CO of thiouracil), 1320, 1135
1
(SO2NH); H-NMR (DMSO-d6, d ppm): 4.30 (s, 2H, CH2), 4.52 (s, 1H, –N–CH),
6.3, 7.4 (d,d 4H, aromatic-H, J = 8.4 Hz), 8.23 (s, 1H, thiouracil-H6), 10.32, 10.52,
11.33 (3s, 3H, 3NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 178.1
(C=S), 170.1, 164.0 (2C=O), 103.2, 121.2, 131.1, 132.0, 138.4 150.9 (aromatic-C),
51.5, 55.3 (thiazolidinone-C); MS m/z: 462 (M?.) (11 %), 464 (M? ? 2) (10 %);
Anal. calcd. for C13H11 BrN4O4S3 (463.36): C, 33.69; H, 2.39; N, 12.09. Found: C,
33.56; H, 2.16; N, 12.05.
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Inhibitory effect of some new uracil and thiouracil derivatives
N-[2-(5-Methyl-2-furyl)-4-oxo-1,3-thiazolidin-3-yl]-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-sulfonamide (3b)
Yield: 72 %; m.p.: 280–282 °C; crystallized from ethanol; IR (KBr, cm-1): 3340,
3225, 3210 (3NH), 1715 (CO of thiazolidine), 1695 (CO of thiouracil), 1323, 1120
(SO2NH); 1H-NMR (DMSO-d6, d ppm): 2.43 (s, 3H, CH3), 4.10 (s, 2H, CH2), 4.41
(s, 1H, –N–CH, thiazolidine), 7.11, 7.59 (d, d, 2H, furan-H, J = 7.0 Hz), 8.10 (s,
1H, thiouracil-H6), 10.41, 11.23, 11.52 (3s, 3H, 3NH exchangeable with D2O); 13C-
NMR (DMSO-d6, d ppm): 178.0 (C=S), 164.2, 169.3 (2C=O), 103.2, 107.2, 149.2,
150.0, 151.0, 152.5 (aromatic-C), 34.1, 58.2 (thiazolidinone-C), 15.4 (CH3); MS m/
z: 388 (M?) (12 %); Anal. calcd. for C12H12N4O5S3 (388.45): C, 37.10; H, 3.11; N,
14.42; Found: C, 37.03; H, 3.08; N, 14.39.
N-[2-(1,5-Dimethyl-3-oxo-2-phenylpyrazolin-4–yl)-4-oxo-1,3-thiazolidin-3-yl]-4-
oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (3c)
Yield: 65 %; m.p.: 290–292 °C; recrystallized from ethanol; IR (KBr, cm-1): 3330,
3321, 3225 (3NH), 1721 (CO of thiazolidine), 1695 (CO of thiouracil), 1323, 1122
1
(SO2NH); H-NMR (DMSO-d6, d ppm): 2.62 (s, 3H, CH3), 3.80 (s, 3H, –N–CH3),
4.52 (s, 2H, CH2), 4.72 (s, 1H, –N–CH), 6.60–7.31 (m, 5H, aromatic-H), 8.22 (s,
1H, thiouracil-H6), 10.21, 10.40, 11.62 (3s, 3H, 3NH exchangeable with D2O); 13C-
NMR (DMSO-d6, d ppm): 178.0 (C=S), 164.2, 169.3, 166.2 (3C=O), 103.2, 104.2,
121.5, 124.1, 129.1, 134.1, 150.1, 154.0 (aromatic-C), 33.5, 70.9 (thiazolidinone-C),
15.2, 15.8 (2CH3); MS m/z: 494 (M?) (4 %); Anal. calcd. for C18H18N6O5S3
(494.58): C, 43.71; H, 3.66; N, 16.99; Found: C, 43.65; H, 3.44; N, 16.76.
N-[2-(2-Hydroxyphenyl)-4-oxo-1,3-thiazolidin-3-yl]-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-sulfonamide (3d)
Yield: 70 %; m.p.: [300 °C; recrystallized from ethanol; IR (KBr, cm-1): 3343,
3225, 3219 (3NH), 1724 (CO of thiazolidine), 1675 (CO of thiouracil), 1325, 1120
1
(SO2NH); H-NMR (DMSO-d6, d ppm): 4.23 (s, 2H, CH2), 4.62 (s, 1H, –N–CH),
7.31–7.80 (m, 4H, aromatic-H), 8.12 (s, 1H, thiouracil-H6), 10.32, 11.23, 11.6,
12.01 (4s, 4H, 3NH, OH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm):
178.0 (C=S), 164.2, 169.3 (2C=O), 103.5, 114.2, 158.2, 130.1, 150.5, 154.0
(aromatic-C), 35.5, 56.9 (thiazolidinone-C), MS m/z: 400 (M?) (13 %); Anal. calcd.
for C13H12N4O5S3 (400.46): C, 38.99; H, 3.01; N, 13.98; Found: C, 38.65; H, 3.24;
N, 13.76.
General procedure for synthesis of Mannich bases 4a, b, 5b, c, and 6c, d
A mixture of paraformaldehyde (0.5 g, 5 mmol) and the appropriate amine, namely
N-methyl piperazine, diethylamine or morpholine (52 mmol), in ethanol (25 ml)
was refluxed for 0.5 h till complete solubility of paraformaldehyde. A warmed
solution of thiazolidinone derivative 3a–d (20 mmol) in absolute ethanol (30 ml)
was then added to the reaction mixture. The whole mixture was refluxed for 6–9 h
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O. A. Fathalla et al.
and then left at room temperature for 3 days. The volatile material was evaporated
under vacuum, and the dry residue was extracted with chloroform to give 4a, b, 5b,
c, and/or 6c, d, respectively.
4-Oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonic acid [5-(4-methyl-
piperazine-1-ylmethyl)-4-oxo-2-(5-methyl-2-furyl)-thiazolidin-3-yl]amide (4a)
Yield: 65 %; m.p.:[300 °C; crystallized from DMF/water; IR (KBr, cm-1): 3335,
3230, 3210 (3NH), 1723 (CO of thiazolidine), 1697 (CO of thiouracil), 1322, 1125
(SO2NH); 1H-NMR (DMSO-d6, d ppm): 3.11–3.50 (m, 7H, N–CH3, CH2–N–CH2),
3.62–3.82 (m, 4H, CH2–N–CH2), 4.11 (d, 2H, CH2, J = 6.2 Hz), 4.44 (s, 1H –N–
CH), 6.11 (t, 1H, J = 6.6 Hz, S–CH), 6.53–7.40 (d,d, 4H, aromatic-H J = 7.2 Hz),
8.32 (s, 1H, thiouracil-H6), 10.5, 10.7, 11.2 (3s, 3H, 3NH exchangeable with D2O);
13C-NMR (DMSO-d6, d ppm): 179.1 (C=S), 171.1, 165.2 (2C=O), 103.1, 121.1,
131.0, 131.9, 138.1, 150.3 (aromatic-C), 52.3, 54.1, 56.3, 52.0, 55.2, 43.3
(thizolidinone-C; –CH2–N); MS m/z: 574 (M?) (7 %), 576 (M? ? 2) (6.5 %);
Anal. calcd. for C19H23BrN6O4S3 (575.53): C, 39.65; H, 4.02; N, 14.60; Found: C,
39.61; H, 4.11; N, 14.56.
4-Oxo-2-thioxo-1,2,3,4 3-tetrahydropyrimidine-5-sulfonic acid [5-(4-methyl-
piprazin-1-ylmethyl)-4-oxo-2-(5-methyl-2-furyl)-thiazolidin-3-yl]amide (4b)
Yield: 71 %; m.p.:[300 °C; crystallized from DMF/water; IR (KBr, cm-1): 3335,
3240, 3220 (3NH), 1723 (CO of thiazolidine), 1692 (CO of thiouracil), 1322, 1125
1
(SO2NH); H-NMR (DMSO-d6, d ppm): 2.31 (s, 3H, CH3), 3.13-3.5 (m, 7H, –N–
CH3, CH2–N–CH2), 3.60–3.81 (m, 4H, CH2–N–CH2), 4.12 (d, 2H, CH2,
J = 6.5 Hz), 4.43 (s, 1H, –N–CH), 6.14 (t, 1H, J = 6.5 Hz, S–CH), 7.20–7.41
(d, d, 2H, furan ring, J = 7.4 Hz), 8.42 (s, 1H, thiouracil-H6), 10.24, 10.91, 11.31
(3s, 3H, 3NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 179.1 (C=S),
171.1, 165.2 (2C=O), 103.1, 107.2, 149.4, 150.1, 150.3, (aromatic-C), 52.3, 54.1,
56.3, 52.0 (thizolidinone-C; –CH2–N), 43.2, 15.2 (2CH3); MS m/z: 500 (M?) (3 %);
Anal. calcd. for C18H24N6O5S3 (500.63): C, 43.18; H, 4.83; N, 16.78; Found: C,
43.15; H, 4.93; N, 16.63.
4-Oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonic acid (5-diethyl-amino-
methyl-4-oxo-(5-methyl-2-furyl)–thiazolidin-3-yl)amide (5b)
Yield: 67 %; m.p.:[300 °C; crystallized from DMF/water; IR (KBr, cm-1): 3335,
3246, 3229 (3NH), 1723 (CO of thiazolidine), 1692 (CO of thiouracil), 1322, 1130
(SO2NH); 1H-NMR (DMSO-d6, d ppm): 1.11, 1.30 (2t, 6H, 2CH3 of ethyl gp), 2.31
(s, 3H, CH3)), 4.22 (q, 4H, J = 7.9 Hz, 2CH2),), 4.47 (d, 2H, CH2, J = 5.9 Hz),
4.56 (s, 1H, –N–CH), 6.10 (t, 1H, J = 6.9 Hz, S–CH), 7.12, 7.52 (d,d, 2H, furan
ring, J = 6.8 Hz), 8.41 (s, 1H, thiouracil-H6), 10.22, 11.00, 11.53 (3s, 3H, 3NH
exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 178.0 (C=S), 164.2, 169.3
(2C=O), 103.1, 109.0, 143.2, 143.4, 150.1 (aromatic-C), 48.5, 50.2, 54.1, 55.6,
(thiazolidinone-C; 3CH2), 13.4, 14.3 (3CH3); MS m/z: 473 (M?) (30 %). Anal.
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Inhibitory effect of some new uracil and thiouracil derivatives
calcd. for C17H23N5O5S3 (473.60): C, 43.11; H, 4.89; N, 14.78; Found: C, 43.05; H,
4.83; N, 14.63.
4-Oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonic acid (5-diethyl-
aminomethyl-2-(1,5-dimethyl-3-oxo-2-phenylpyrazolidin-4-yl)-4-oxo-(thiazolidin-3-
yl)amide (5c)
Yield: 65 %; m.p.: [300 °C; recrystallized from DMF/water. IR (KBr, cm-1):
3325, 3219, 3210 (3NH), 1723 (CO of thiazolidine), 1687 (CO of thiouracil), 1322,
1115 (SO2NH); 1H-NMR (DMSO-d6, d ppm): 1.11, 1.31 (2t, 6H, J = 6.7 Hz,
2CH3), 2.42 (s, 3H, CH3), 3.80 (s, 3H, –N–CH3), 4.20 (q, 4H, J = 7.7 Hz, 2CH2 of
ethyl gp), 4.45 (d, 2H, J = 5.7 Hz, CH2), 4.58 (s, 1H, –N–CH), 6.13 (t, 1H,
J = 6.7 Hz, S–CH), 7.10–7.5 (m, 5H, aromatic-H), 8.30 (s, 1H, thiouracil-H6),
10.32, 11.01, 11.23 (3s, 3H, 3NH exchangeable with D2O); 13C-NMR (DMSO-d6, d
ppm): 178.0 (C=S), 163.3, 164.2, 169.3 (3C=O), 103.1, 147.2, 119.1, 113.1, 129.4,
136.1, 150.1 (aromatic-C), 48.5, 50.2, 54.1, 55.6, (thiazolidinone-C; 3CH2), 13.4,
14.3 (3CH3); MS m/z: 480 (M?) (52 %); Anal. calcd. for C23H29N7O5S3 (579.73):
C, 47.65; H, 5.04; N, 16.91; Found: C, 47.52; H, 5.31; N, 16.81.
4-Oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonic acid (1,5-dimethyl-
3-oxo-2-phenylpyrazolin-4-yl)-5-[(morpholin-4-yl-)methyl]-4-oxo–(thiazolidin-3-
yl)amide (6c)
Yield: 61 %; m.p.: [300 °C; recrystallized from DMF/water; IR (KBr, cm-1):
3350, 3220, 3210 (3NH), 1710 (CO of thiazolidine), 1691 (CO of thiouracil), 1320,
1118 (SO2NH); 1H-NMR (DMSO-d6, d ppm): 2.21 (s, 3H,CH3), 2.41, 3.31 (m, 4H,
CH2–N–CH2), 3.82 (s, 3H, N–CH3), 3.71, 4.21 (m, 4H, CH2–O–CH2), 4.63 (s, 1H, –
N–CH), 4.82 (d, 2H, J = 6. 1 Hz, CH2), 6.22 (t, 1H, J = 6.1 Hz, S–CH), 6.69–7.50
(m, 5H, aromatic-H), 8.32 (s, 1H, thiouracil-H6), 10.32, 10.61, 11.30 (3s, 3H,3NH
exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 176.0 (C=S), 164.2, 165.2,
169.5 (3C=O), 103.1, 112.3, 115.2, 130.2, 131.1, 147.2, 150.2, 156.6 (aromatic-C),
52.3, 53.2, 66.8, 54.1, 56.3 (thiazolidinone-C; morpholine-C; –CH2–N), 15.4
(2CH3); MS m/z: 593 (M?) (5 %); Anal. calcd. for C23H27N7O6S3 (593.71): C,
46.52; H, 4.58; N, 16.51; Found: C, 46.48; H, 4.52; N, 16.47.
4-Oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonic acid -5[(morpholin-4-
yl)methyl]-4-oxo-2-[(2-hydroxyphenyl)-thiazolidine-3-yl]amide (6d)
Yield: 55 %; m.p.:[300 °C; recrystallized from DMF/water; IR (KBr, cm-1): 3332,
3240, 3215 (3NH), 1723 (CO of thiazolidine), 1697 (CO of thiouracil), 1322, 1125
(SO2NH); 1H-NMR (DMSO-d6, d ppm): 2.32, 3.12 (m. 4H, CH2–N–CH2), 3.92, 4.10
(m, 4H, CH2–O–CH2), 4.62 (s, 1H, –N–CH), 4.81 (d, 2H, J = 6.2 Hz CH2), 6.21 (t,
1H, J = 6.7 Hz, –S–CH), 7.27–7.90 (m, 4H, aromatic-H), 8.13 (s, 1H, thiouracil),
10.30, 10.51, 11.60, 12.01 (4s, 4H, 3NH, OH exchangeable with D2O); 13C-NMR
(DMSO-d6, d ppm): 176.0 (C=S), 165.2, 169.5 (2C=O), 103.1, 115.2, 130.2, 131.1,
150.2, 156.6 (aromatic-C), 52.3, 53.2, 66.8, 54.1, 56.3 9 (thiazolidinone-C;
123

O. A. Fathalla et al.
morpholine-C; –CH2–N); Ms m/z: 499 (M?) (40 %); Anal. calcd. for C18H21N5O6S3
(499.59): C, 43.27; H, 4.23; N, 14.02; Found: C, 43.21; H, 4.41; N, 14.12.
General procedure for synthesis of N-[4-(6-amino or oxo-5-cyano-4-substituted
phenyl)-1,2-dihydropyridin-2-yl)-phenyl]-2,4-dioxo or 4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-sulfonamides 8a–c, 9a–c, 10a–c, and 11a–c
A mixture of the compound 7a, b (3 mmol), the appropriate aldehyde, namely
2-chloro-5-nitrobenzaldehyde, 2,5-dimethoxybenzaldehyde and 3,5-dimethoxybenz-
aldehyde (3 mmol), malononitrile or ethyl cyanoacetate (21 mmol), and ammonium
acetate (24 mmol) in n-butanol (50 ml) was refluxed for 8–10 h. The reaction mixture
was concentrated to half volume, then cooled and left overnight. The formed
precipitate was filtered off, dried, then crystallized from the proper solvent.
N-[4-(6-Amino-5-cyano-4-(2-chloro-5-nitrophenyl)-1,2-dihydropyridin-2-
yl)phenyl]-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (8a)
Yield: 65 %; m.p.:[300 °C; recrystallized from DMF/water; IR (KBr, cm-1): 3467
(NH2), 3442, 3320, 3220 (3NH), 2212 (CN), 1735, 1691 (2 CO of uracil), 1563,
1
1350 (NO2), 1327, 1129 (SO2NH); H-NMR (DMSO-d6 d ppm): 4.92 (s, 2H, NH2
exchangeable with D2O), 6.52 (s, 1H, pyridine-H3), 7.21–7.69 (m, 7H, aromatic-H),
8.40 (s, 1H, uracil-H6), 10.21, 10.80, 11.22 (3s, 3H, 3NH exchangeable with D2O);
13C-NMR (DMSO-d6, d ppm): 150.5, 164.2 (2C=O), 162.0 (C–NH2), 150.0 (C–
NO2), 100.2, 107.2, 111.1, 116.8, 119.1, 120.2, 124.3, 129.3, 126.3, 128.4, 133.2,
136.4, 142.1, 145.2 158.2 (aromatic-C), 117.2 (CN); MS m/z: 540 (M?) (40 %), 542
(M?.?2) (13 %); Anal. calcd. for C22H14ClN7O6S (539.92): C, 48.94; H, 2.61; N,
18.16; Found: C, 48.92; H, 2.59; N, 18.11.
N-[4-(6-Amino-5-cyano-4-(2,5-dimethoxyphenyl)-1,2-dihydropyridin-2-yl)phenyl]-
2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (8b)
Yield: 68 %; m.p.: [300 °C; crystallized from DMF/water; IR (KBr, cm-1): 3553
(NH2), 3440, 3321, 3210 (3NH), 2209 (CN), 1722, 1685 (2CO of uracil), 1327, 1129
1
(SO2NH); H-NMR (DMSO-d6, d ppm): 3.62, 3.71 (2s, 6H, 2OCH3), 4.72 (s, 2H,
NH2), 6.62 (s, 1H, pyridine-H3), 7.41–7.82 (m, 7H, aromatic-H), 8.52 (s, 1H, uracil-
H6), 10,00, 10.62, 11.21, (3s, 3H, 3NH exchangeable with D2O); 13C-NMR (DMSO-
d6, d ppm): 153.2, 164.2 (2CO), 162.0 (C–NH2), 103.1, 118.3, 112.0, 124.2, 127.9,
127.4, 128.2, 129.3, 138.2, 139.2, 150.0, 152.3, 155.2, 159.1 (aromatic-C), 117.0
(CN), 55.9 (2OCH3); MS m/z: 520 (M?) (40 %); Anal. calcd. for C24H20N6O6S
(520.53): C, 55.37; H, 3.87; N, 16.14; Found: C, 55.46; H, 3.85; N 16.44.
N-[4-(6-Amino-5-cyano-4-(3,5-dimethoxyphenyl)-1,2-dihydropyridin-2-yl)phenyl]-
2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (8c)
Yield: 71 %; m.p.: 290–292 °C; crystallized from DMF/water; IR (KBr, cm-1):
3540 (NH2), 3435, 3430, 3220 (3NH), 2211 (CN), 1720, 1689 (2CO of uracil),
123

Inhibitory effect of some new uracil and thiouracil derivatives
1
1325, 1220 (SO2NH); H-NMR (DMSO-d6, d ppm): 3.59, 3.80 (2s, 6H, 2OCH3),
4.82 (s, 2H, NH2), 6.71 (s, 1H, pyridine-H3), 7.32–7.72 (m, 7H, aromatic-H), 8.32
(s, 1H, uracil-H6), 10,21, 10.52, 11.30 (3s, 3H, 3NH exchangeable with D2O); 13C-
NMR (DMSO-d6, d ppm): 150.5, 164.2 (2C=O), 162.0 (C–NH2), 100.2, 100.9,
103.3, 107.2, 111.1, 116.8, 120.2, 126.3, 128.4, 136.4, 140.2, 142.1, 158.2, 162.2
(aromatic-C), 117.3 (CN), 55.9 (2OCH3) MS m/z: 520 (M?) (40 %); Anal. calcd. for
C24H20N6O6S (520.52): C, 55.37; H; 3.87; N, 16.14; Found: C, 55.35, H, 3.94; N,
16.00.
N-[4-(6-Amino-5-cyano-4-(2-chloro,5-nitrophenyl)-1,2-dihydropyridin-2-
yl)phenyl]-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (9a)
Yield: 66 %; m.p.: 295–297 °C; crystallized from DMF/water; IR (KBr, cm-1):
3520 (NH2), 3420, 3360, 3240 (3NH), 2212 (CN), 1720 (CO of thiouracil), 1560,
1
1347 (NO2), 1327, 1129 (SO2NH); H-NMR (DMSO-d6, d ppm): 4.90 (s, 2H, NH2
exchangeable with D2O), 6.51 (s, 1H, pyridine-H3), 7.21, 7.61 (m, 7H, aromatic-H),
8.41 (s, 1H, uracil-H6), 10.21, 11.21, 11.63 (3s, 3H, 3NH exchangeable with D2O);
13C-NMR (DMSO-d6, d ppm): 178.9 (C=S), 164.2 (C=O), 162.4 (C–NH2), 151.2
(C–NO2), 101.2, 107.2, 111.2, 116.8, 119.1, 120.2, 124.3, 129.3, 126.3, 128.4,
133.2, 136.4, 142.1, 145.2, 158.2 (aromatic-C), 117.3 (CN); MS m/z: 540 (M?)
(40 %), 542 (M? ? 2) (13 %); Anal. calcd. for C22H14ClN7O5S2 (555.97): C,
47.94; H, 2.54; N, 17.64; Found: C, 48.22; H, 2.39; N, 18.01; MS m/z: 555 (M?)
(27 %), 557 (M? ? 2).
N-[4-(6-Amino-5-cyano-4-(2,5-dimethoxyphenyl)-1,2-dihydropyridin-2-
yl)phenyl]4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (9b)
Yield: 68 %; m.p.: 288–290 °C; crystallized from DMF/water; IR (KBr, cm-1):
3510 (NH2), 3439, 3346, 3250 (3NH), 2212 (CN), 1720 (CO of thiouracil), 1329,
1120 (SO2NH); 1H-NMR (DMSO-d6, d ppm): 3.61, 3.80 (2s, 6H, 2OCH3), 4.90 (s,
2H, NH2 exchangeable with D2O), 6.51 (s, 1H, pyridine-H3), 7.21–7.62 (m, 7H,
aromatic-H), 8.41 (s, 1H, thiouracil-H6), 10.21, 10.83, 11.24, (3s, 3H, 3NH
exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 178.3 (C=S), 164.2 (CO),
162.2 (C–NH2), 103.5, 118.3, 112.2, 124.2, 127.9, 127.4, 128.2, 129.3, 138.2, 139.2,
150.0, 152.3, 155.2, 156.1 (aromatic-C), 117.0 (CN), 54.8 (2OCH3); MS m/z: 536
(M?) (15 %); Anal. calcd. for C24H20N6O5S2 (536.59): C, 53.72; H, 3.75; N, 15.66;
Found: C, 53.70; H, 3.70; N, 15.59.
N-[4-(6-Amino-5-cyano-4-(3,5-dimethoxyphenyl)-1,2-dihydropyridin-2-yl)phenyl]-
4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (9c)
Yield: 71 %; m.p.: 287–289 °C; crystallized from DMF/water; IR (KBr, cm-1):
3520 (NH2), 3435, 3340, 3210 (3NH), 2212 (CN), 1720 (CO of thiouracil), 1327,
1129 (SO2NH); 1H-NMR (DMSO-d6, d ppm): 3.61, 3.80 (2s, 6H, 2OCH3), 4.91 (s,
2H, NH2 exchangeable with D2O), 6.52 (s, 1H, pyridine-H3), 7.22–7.63 (m, 7H,
aromatic-H), 8.42 (s, 1H, thiouracil-H6), 10.21, 10.83, 11.25 (3s, 3H, 3NH
123

O. A. Fathalla et al.
exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 179.2 (C=S), 164.2 (C=O),
162.0 (C–NH2), 100.2, 100.9, 103.3, 107.2, 111.1, 116.8, 120.2, 126.3, 128.4,
136.4, 140.2, 142.1, 158.2, 162.2 (aromatic-C), 117.2 (CN), 55.9 (2OCH3); MS m/
z 536 (M?) (17 %); Anal. calcd. for C24H20N6O5S2 (536.59): C, 53.72; H, 3.75; N,
15.66; Found: C, 53.67; H, 3.65; N, 15.52.
N-{4-[4-(2-Chloro-5-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyridin-2-yl]phenyl}-
2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (10a)
Yield: 68 %; m.p.:[300 °C; crystallized from DMF/water; IR (KBr, cm-1): 3532,
3219, 3160, 3157 (4NH), 2209 (CN), 1720, 1697, 1689 (3CO), 1550, 1347 (NO2),
1
1322, 1127 (SO2NH); H-NMR (DMSO-d6, d ppm): 6.42 (s, 1H, pyridone-H3),
7.11–7.80 (m, 7H, aromatic-H), 8.32 (s, 1H, uracil-H6), 9.82, 10.31, 11.23, 12.23
(4s, 4H, 4NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 150.5, 162.0,
164.2 (3C=O), 150.3 (C–NO2), 101.2, 108.2, 111.1, 116.8, 119.1, 121.2, 124.3,
129.3, 126.3, 128.4, 133.2, 136.4, 142.1, 145.2 158.2 (aromatic-C), 117.2 (CN); MS
m/z 540.5 (M?) (30 %), 542.5 (M?.?2) (10 %); Anal. calcd. for C22H13ClN6O7S
(540.90): C, 48.85; H, 2.42; N, 15.53; Found: C, 48.83; H, 2.40; N, 15.32.
N-{4-[5-Cyano-4-(3,5-dimethoxyphenyl)-6-oxo-1,6-dihydropyridin-2-yl]phenyl}-
2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (10b)
Yield: 62 %; m.p.:[300 °C; crystallized from DMF/water; IR (KBr, cm-1): 3442,
3260, 3160, 3157 (4NH), 2209 (CN), 1715, 1689, 1679 (3CO), 1322, 1126
1
(SO2NH); H-NMR (DMSO-d, ppm): 3.71, 3.96 (2s, 6H, 2OCH3), 6.42 (s, 1H,
pyridone-H3), 7.33–7.90 (m, 7H, aromatic-H), 8.32 (s, 1H, uracil-H6), 9.81–10.31,
11.20, 12.25 (4s, 4H, 4NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm):
153.2, 162.3, 164.2 (3CO), 103.1, 118.3, 112.0, 124.2, 127.9, 127.4, 128.2, 129.3,
138.2, 139.2, 150.0, 152.3, 155.2, 159.1 (aromatic-C), 117.0 (CN), 55.9 (2OCH3);
MS m/z 521 (M?) (25 %); Anal. calcd. for C24H19N5O7S (521.51): C, 55.27; H,
3.67; N, 13.42; Found: C, 55.55; H, 3.85; N, 13.49.
N-{4-[5-Cyano-4-(2,5-dimethoxyphenyl)-6-oxo-1,6-dihydropyridin-2-yl]phenyl}-
2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (10c)
Yield: 65 %; m.p.:[300 °C; crystallized from DMF/water; IR (KBr, cm-1): 3442,
3360, 3264, 3157 (4NH), 2209 (CN), 1725, 1685, 1676 (3CO), 1321, 1122
1
(SO2NH); H-NMR (DMSO-d6, d ppm): 3.62, 3.90 (2s, 6H, 2OCH3), 6.64 (s, 1H,
pyridone-H3), 7.21–8.13 (m, 7H, aromatic-H), 8.41 (s, 1H, thiouracil-H6), 9.92,
10.82, 11.52, 12.32 (4s, 4H, 4NH exchangeable with D2O); 13C-NMR (DMSO-d6, d
ppm): 150.5, 163.0, 164.2 (3C=O), 100.2, 100.9, 103.3, 107.2, 111.1, 116.8, 120.2,
126.3, 128.4, 136.4, 140.2, 142.1, 158.2, 162.2 (aromatic-C), 117.2 (CN), 54.9
(2OCH3); MS m/z 521.5 (M?) (23 %); Anal. calcd. for C24H19N5O7S (521.50): C,
55.27; H, 3.67; N, 13.42; Found: C, 55.34; H, 3.64; N, 13.60.
123

Inhibitory effect of some new uracil and thiouracil derivatives
N-{4-[4-(2-Chloro-5-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyridin-2-yl]phenyl}-
4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (11a)
Yield: 67 %; m.p.: 294–296 °C; crystallized from DMF/water; IR (KBr, cm-1):
3510, 3442, 3264, 3157 (4NH), 2205 (CN), 1720, 1685 (2CO), 1560, 1350 (NO2),
1
1322, 1126 (SO2NH); H-NMR (DMSO-d6, d ppm): 6.41 (s, 1H, pyridone-H3),
7.32–8.22 (m, 7H, aromatic-H), 8.52 (s, 1H, thiouracil-H6), 9.82, 10.51, 11.22, 11.52
(4s, 4H, 4NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 178.9 (C=S),
164.4, 165.2 (2C=O), 150.1 (C–NO2), 101.2, 107.2, 111.2, 116.8, 119.1, 120.2, 124.3,
129.3, 126.3, 128.4, 133.2, 136.4, 142.1, 145.2, 155.2 (aromatic-C), 117.1 (CN); MS
m/z 556 (M?) (27 %), 558 (M? ? 2) (9 %); Anal. calcd. for C22H13ClN6O6S2
(556.96): C, 47.44; H, 2.35; N, 15.08; Found: C, 47.64; H, 2.41; N, 15.34.
N-{4-[5-Cyano-4-(3,5-dimethoxyphenyl)-6-oxo-1,6-dihydropyridin-2-yl]phenyl}-4-
oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (11b)
Yield: 71 %; m.p.: 286–288 °C; crystallized from DMF/water; IR (KBr, cm-1):
3490, 3360, 3265, 3224 (4NH), 2208 (CN), 1718, 1680 (2CO), 1320, 1120
1
(SO2NH); H-NMR (DMSO-d6, d ppm): 3.71, 3.92 (2s, 6H, 2OCH3), 6.52 (s, 1H,
pyridone-H3), 7.11–8.32 (m, 7H, aromatic-H), 8.42 (s, 1H, thiouracil-H6), 9.82,
10.51, 11.23, 11.53 (4s, 4H, 4 NH exchangeable with D2O); 13C-NMR (DMSO-d6, d
ppm): 178.3 (C=S), 162.2, 164.2 (2CO), 103.5, 119.3, 111.2, 125.2, 127.4, 127.9,
128.2, 129.3, 138.2, 139.2, 151.0, 152.3, 155.2, 156.1 (aromatic-C), 117.2 (CN),
55.8 (2OCH3); MS m/z 537.5 (M?) (22 %); Anal. calcd. for C24H19N5O6S2
(537.57): C, 53.62; H, 3.56; N, 13.02; Found: C, 53.40, H, 3.54; N, 13.31.
N-{4-[5-Cyano-4-(2,5-dimethoxyphenyl)-6-oxo-1,6-dihydropyridin-2-yl]phenyl}-4-
oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (11c)
Yield: 68 %; m.p.:[300 °C; crystallized from DMF/water; IR (KBr, cm-1): 3490,
3357, 3265, 3125 (4NH), 2208 (CN), 1718, 1680 (2CO), 1320, 1120 (SO2NH); H
1
NMR (DMSO-d6, d ppm): 3.61, 3.82 (2s, 6H, 2OCH3), 6.32 (s, 1H, pyridone-H3),
7.21–8.42 (m, 7H, aromatic-H), 8.32 (s, 1H, thiouracil-H6), 9.82, 10.51, 11.02,
11.51 (4s, 4H, 4NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 179.2
(C=S), 163.0, 164.2 (C=O), 100.2, 102.9, 103.3, 107.2, 111.1, 117.8, 120.2, 126.3,
128.4, 136.4, 140.2, 142.1, 158.2, 162.2 (aromatic-C), 117.2 (CN), 55.7 (2OCH3);
MS m/z 537.5 (M?) (21 %); Anal. calcd. for C24H19N5O6S2 (537.57): C, 53.62, H,
3.56; N, 13.02; Found: C, 53.59; H, 3.54; N, 13.22.
General procedure for synthesis of chalcones 12a–c and 13a–c
A mixture of 7a, b (3 mol) and the appropriate aromatic aldehyde, namely 2-chloro-
5-nitrobenzaldehyde, 2,5-dimethoxybenzaldehyde, and 3,5-dimethoxybenzaldehyde
(3 mol), in 10 % ethanolic sodium hydroxide solution (50 ml) was stirred at room
temperature for 24 h, then refluxed for 1 h. After reaction completion, the mixture
was cooled and poured onto ice-cold water. The precipitate that appeared after
123

O. A. Fathalla et al.
neutralization with diluted HCl was filtered off and crystallized from acetic acid to
give the desired products.
N-{4-[(2Z)-3-(2-Chloro-5-nitrophenyl)prop-2-enoyl]phenyl}-2,4-dioxo-1,2,3,4-
tetrahydropyrimidine-5-sulfonamide (12a)
Yield: 63 %; m.p.: [300 °C; crystallized from DMF/water; IR (KBr, cm1): 3349,
3340, 3215 (3NH), 1724, 1715, 1685 (3CO), 1557, 1350 (NO2), 1322, 1130
(SO2NH); 1H-NMR (DMSO-d6, d ppm): 6.51, 6.80 (d,d, 2H, J = 7.2 Hz, –
CH=CH–), 7.30–8.22 (m, 7H, aromatic-H), 8.32 (s, 1H, uracil-H6), 9.82, 10.61,
11.00 (3s, 3H, 3NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 150.1,
164.2, 170.2 (3C=O), 100.1, 116.8, 119.2, 121.4, 123.3, 127.1, 128.7, 130.2, 132.4,
139.5, 142.2, 143.3, 145.2, 149.3(aromatic-C; HC=CH); MS m/z 476.5 (M?)
(15 %), 478 (M? ? 2) (5 %); Anal. calcd. for C19H13ClN4O7S (476.86): C, 47.85;
H, 2.74; N, 11.74; Found: C, 47.44; H, 2.73; N, 11.53.
N-{4-[(2Z)-3-(2,5-Dimethoxyphenyl)prop-2-enoyl]phenyl}-2,4-dioxo-1,2,3,4-
tetrahydropyrimidine-5-sulfonamide (12b)
Yield: 65 %, m.p.:[300 °C; crystallized from DMF/water; IR (KBr, cm-1): 3349,
3219, 3160 (3NH), 1720, 1710, 1683 (3CO), 1322, 1128 (SO2NH); 1H-NMR
(DMSO-d6, d ppm): 3.62, 3.81 (2s, 6H, 2 OCH3), 6.32, 6.61 (d,d, 2H, J = 7.2 Hz –
CH=CH–), 7.21–8.51 (m, 7H, aromatic-H), 8.43 (s, 1H, uracil-H6), 9.52, 10.42,
11.22 (s, 3H, 3NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 150.1,
164.4, 170.6 (3C=O), 100.1, 101.5, 106.4, 107.3, 116.8, 121.4, 127.1, 128.4, 130.2,
142.2, 143.3, 145.2, 158.3, 158.5 (aromatic-C; HC=CH), 55.9 (2OCH3); MS m/
z 457.5 (M?) (17 %); Anal. calcd. for C21H19N3O7S (457.45): C, 55.13; H, 4.18; N,
9.18; Found: C, 55.12; H, 4.17; N, 9.07.
N-{4-[(2Z)-3-(3,5-Dimethoxyphenyl)prop-2-enoyl]phenyl}-2,4-dioxo-1,2,3,4-
tetrahydropyrimidine-5-sulfonamide (12c)
Yield:68 %, m.p.:[300 °C; crystallized fromDMF/water;IR(KBr, cm-1) 3421, 3232,
3119 (3NH), 1715, 1697, 1685 (3CO), 1322, 1130 (SO2NH); 1H-NMR (DMSO-d6, d
ppm): 3.56, 3.81 (2s, 6H, 2OCH3), 6.22, 6.52 (d,d, 2H, J = 7.4 Hz –CH=CH–),
7.22–8.11 (m, 7H, aromatic-H), 8.32 (s, 1H, uracil-H6), 9.62, 10.22, 11.32 (3s, 3H, 3NH
exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 150.1, 164.4, 170.6 (3C=O),
100.1, 101.5, 106.5, 107.3, 117.8, 121.4, 127.7, 128.4, 131.2, 142.2, 143.3, 145.2, 158.1,
158.8 (aromatic-C; HC=CH), 56.2 (2OCH3); MS m/z 457.5 (M?) (13 %); Anal. calcd.
forC21H19N3O7S(457.54):C, 55.13;H, 4.18; N, 9.18;Found:C, 55.11,H, 4.00;N, 9.36.
N-{4-[(2Z)-3-(2-Chloro-5-nitrophenyl)prop-2-enoyl]phenyl}-4-oxo-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-sulfonamide (13a)
Yield: 66 %; m.p.:[300 °C; crystallized from DMF/water; IR (KBr, cm-1): 3232,
3219, 3157 (3NH), 1720, 1684 (2CO), 1560, 1346 (NO2), 1321, 1127 (SO2NH); 1H-
123

Inhibitory effect of some new uracil and thiouracil derivatives
NMR (DMSO-d6, d ppm): 6.52, 6.80 (d,d, 2H, J = 7.2 Hz, –CH=CH–), 7.32–8.21
(m, 7H, aromatic-H), 8.32 (s, 1H, thiouracil-H6), 9.82, 10.62, 11.00 (3s, 3H, 3NH
exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 178.4 (C=S), 164.2, 170.2
(2C=O), 100.5, 117.8, 119.2, 121.4, 124.3, 127.1, 128.7, 130.2, 132.4, 138.5, 142.2,
143.3, 145.2, 149.3 (aromatic-C; HC=CH); MS m/z 492 (M?) (15 %), 494
(M? ? 2) (5 %); Anal. calcd. for C19H13ClN4O6S2 (492.92): C, 46.29; H, 2.65; N
11.36; Found: C, 46.26; H, 2.44; N, 11.15.
N-{4-[(2Z)-3-(2,5-Dimethoxyphenyl)prop-2-enoyl]phenyl}-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-sulfonamide (13b)
Yield: 69 %; m.p. 292–294 °C; crystallized from DMF/water; IR (KBr, cm-1):
3352, 3220, 3150 (3NH), 1723, 1687 (2CO), 1325, 1127 (SO2NH); 1H-NMR
(DMSO-d6, d ppm): 3.51, 3.83 (3s, 6H, 2OCH3), 6.21, 6.54 (d, d, 2H, J = 6.9 Hz –
CH=CH–), 7.22–8.11 (m, 7H, aromatic-H), 8.32 (s, 1H, thiouracil-H6), 9.62, 10.22,
11.32 (3s, 3H, 3NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 178.4
(C=S), 164.4, 170.6 (2C=O), 100.1, 101.5, 106.4, 107.3, 116.8, 121.4, 127.1, 128.4,
130.2, 142.2, 143.3, 145.2, 158.3, 158.5 (aromatic-C; HC=CH), 55.9 (2OCH3); MS
m/z 473 (M?) (21 %); Anal. calcd. for C21H19N3O6S2 (473.52): C, 53.26; H, 4.04;
N, 8.87; Found: C, 53.05; H, 4.00; N, 8.65.
N-{4-[(2Z)-3-(3,5-Dimethoxyphenyl)prop-2-enoyl]phenyl}-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-sulfonamide (13c)
Yield: 63 %; m.p.: 286–288 °C; crystallized from DMF/water; IR (KBr, cm-1):
3232, 3220, 3119 (3NH), 1723, 1697 (2CO), 1322, 1215 (SO2NH); 1H-NMR
(DMSO-d6, d ppm): 3.61, 3.90 (2s, 6H, 2OCH3), 6.11, 6.42 (d,d, 2H, J = 7.4 Hz, –
CH=CH–), 7.00–8.32 (m, 7H, aromatic-C), 8.32 (s, 1H, thiouracil-H6), 9.62, 10.50,
11.42 (3s, 3H, 3NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 178.4
(C=S), 164.4, 170.6 (2C=O), 100.4, 101.5, 106.5, 107.3, 118.8, 121.4, 127.7, 129.4,
130.2, 142.2, 143.3, 144.2, 158.1, 158.8 (aromatic-C; HC=CH), 55.8 (2OCH3); MS
m/z 473.5 (M?) (12 %); Anal. calcd. for C21H19N3O6S2 (473.52): C, 53.26; H, 4.04;
N, 8.87; Found: C, 53.24; H, 4.00; N, 8.64.
(2Z)-2-[1-(4-{[(4-Oxo-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-
yl)sulfonyl]amino}phenyl)ethylidene]hydrazinecarbothioamide (14)
A mixture of 7b (3.25 g, 10 mmol) and thiosemicarbazide (0.91 g, 10 mmol) in
absolute ethanol (20 ml) containing a few drops of glacial acetic acid was refluxed
for 3 h. The formed precipitate was filtered off and crystallized from DMF/water to
give compound 14. Yield: 68 %; m.p.: [300 °C; IR (KBr, cm-1): 3490 (NH2),
1
3330, 3265, 3220, 3215 (4NH), 1718 (CO of thiouracil), 1320, 1210 (SO2NH); H-
NMR (DMSO-d6, d ppm): 2.24 (s, 3H, CH3), 6.52 (s, 2H, NH2), 7.13–7.69 (m,4H,
aromatic-H), 8.30 (s, 1H, thiouracil-H6), 9.81, 10.52, 11.21, 11.59 (4s, 4H, 4 NH
exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 178.1, 180.1 (2C=S), 168
(C=N), 164.2(C=O), 103.2, 116.3, 124.9, 130.2, 140.2, 150.1(aromatic-C), 15.1
123

O. A. Fathalla et al.
(CH3); MS m/z 398 (M?) (15 %); Anal. calcd. for C13H14N6O3S3 (398.49): C,
39.18; H, 3.54; N, 21.08; Found: C, 39.16; H, 3.51; N, 21.27.
(2Z)-2-[1-(4-{[(4-Oxo-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)sulfonyl]-
amino}phenyl)ethylidene]-N-phenylhydrazinecarbothioamide (15)
A mixture of 7b (3.25 g, 10 mmol) and phenylthiosemicarbazide (1.70 g, 10 mmol)
in absolute ethanol (20 ml) containing a few drops of hydrochloric acid was
refluxed for 3 h. The formed precipitate was filtered off and crystallized from DMF/
water to give the desired compound. Yield: 68 %; m.p.:[300 °C; IR (KBr, cm-1):
3410, 3360, 3265, 3224, 3215 (5NH), 1718 (CO of thiouracil), 1320, 1130
1
(SO2NH); H-NMR (DMSO-d6, d ppm): 2.23 (s, 3H, CH3), 7.22–7.93 (m, 9H,
aromatic-H), 8.32 (s, 1H, thiouracil-H6), 9.82, 10.50, 11.22, 11.51, 12.00 (5s, 5H,
5NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 178.1, 180.1 (2C=S),
168 (C=N), 164.2 (C=O), 103.2, 116.3, 124.3, 124.9, 126.5, 129.1, 130.2, 137.2,
140.2, 150.1 (aromatic-C), 15.1 (CH3); MS m/z 474 (M?) (21 %); Anal. calcd. for
C19H18N6O3S3 (474.59): C, 48.08; H, 3.82; N, 17.71; Found: C, 48.27; H, 3.80; N,
17.59.
N-{4-[2-(Morpholin-4-yl)-2-thioxoethyl]phenyl}-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-sulfonamide (16)
A mixture of 7b (3.25 g, 10 mmol), morpholine (0.86 g, 10 mmol), and sulfur
(0.32 g, 10 mmol) was refluxed gently till the evolution of H2S subsided, then more
vigorously for a total period of 14 h. The hot reaction was poured onto warm
ethanol (10 ml) and left to crystallize. The obtained crystals were filtered off and
crystallized from ethanol. Yield: 68 %; m.p.: [300 °C; IR (KBr, cm-1): 3490,
3350, 3222 (3NH), 1718 (CO of thiouracil), 1320, 1120 (SO2NH), 1200 (SO2);
1H-NMR (DMSO-d6, d ppm): 3.00 (m, 4H, N(CH2)2), 3.61 (m, 4H, O(CH2)2), 4.32
(s, 2H, CH2), 7.20–7.94 (m, 4H, aromatic-H), 8.32 (s, 1H, thiouracil-H6), 9.81,
10.52, 11.21 (3s, 3H, 3NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm):
181.2, 178.3 (2C=S), 164.6 (C=O), 128.2, 129.3, 139.2, 148.2, 150.9 (aromatic-C),
46.2, 69.0 (morpholine-C), 37.4 (CH2–CS); MS m/z 426 (M?) (21 %); Anal. calcd.
for C16H18N4O4S3 (426.55): C, 45.05; H 4.25; N 13.14; Found: C, 45.23; H, 4.23;
N, 13.32.
(4-{[(4-Oxo-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-
yl)sulfonyl]amino}phenyl)acetic acid (17)
A mixture of 16 (4.26 g, 10 mmol), glacial acetic acid (5 ml), conc. H2SO4 (1 ml),
and water (2 ml) was refluxed for 5 h. Then the mixture was poured onto water
(50 ml) and kept overnight. The formed solid was filtered off and washed with cold
water and digested with a solution of 5 % NaOH. Then the solution was filtered off
and the filtrate was acidified with HCl. The formed precipitate was filtered off and
crystallized from DMF/water to give the desired product. Yield: 68 %; m.p.
[300 °C; IR (KBr, cm-1): 3543 (OH), 3460, 3353, 3222 (3NH), 1718, 1710 (2CO),
123

Inhibitory effect of some new uracil and thiouracil derivatives
1320, 1130 (SO2NH), 1200 (SO2); 1H-NMR (DMSO-d6, d ppm): 4.53 (s, 2H, CH2),
7.20–7.81 (m, 4H, aromatic-H), 8.31 (s, 1H, thiouracil-H6), 9.81, 10.50, 11.21,
11.52 (4s, 4H, 3NH and OH exchangeable with D2O); 13C-NMR (DMSO-d6, d
ppm): 179.2 (C=S), 164.1, 176.1 (2C=O), 128.2, 129.3, 139.2, 148.2, 150.9
(aromatic-C), 48.2 (CH2); MS m/z 341.63 (M?) (30 %); Anal. calcd. for
C12H11N3O5S2 (341.63): C, 42.22; H, 3.24; N, 12.30; Found: C, 42.29; H, 3.53;
N, 12.50.
General procedure for synthesis of compounds 18a–f
A mixture of uracil-5-sulfonylhydrazide (2.0 gm, 0.02 mol) and the appropriate
sulfonyl chloride, namely benzenesulfonyl chloride, 4-bromobenzene sulfonyl
chloride, 4-methylbenzenesulfonyl chloride, 2-nitrobenzenesulfonyl chloride, ura-
cil-5-sulfonyl chloride, 2-thiouracil-5-sulfonyl chloride (0.02 mol), in methanol
(30 ml) containing a few drops of pyridine was refluxed for 8–12 h. The precipitate
obtained after cooling was filtered off, dried, and crystallized to give compounds
18a–f, respectively.
N-[(2,4-Dioxo-1,2,3,4-tetrahydropyrimidine-5-
yl)sulfonodithioyl]benzenesulfonylhydrazide (18a)
Yield: 85 %; m.p.: 230–232 °C; crystallized from DMF/water; IR (KBr, cm-1):
3426–3257 (4NH), 1710, 1685 (2 CO of uracil), 1320, 1123 (SO2NH); H-NMR
1
(DMSO-d6, d ppm): 7.11–7.90 (m, 5H, Ar–H), 8.11 (s, 1H, uracil-H6) and 10.43,
10.62, 10.91, 11.45 (4s, 4H, 4 NH exchangeable with D2O); 13C-NMR (DMSO-d6, d
ppm): 150.1, 164.2 (2C=O), 100.1, 128.6, 129.7, 130.2, 142.2 (aromatic-C); MS m/
z 346 (M?) (4 %); Anal. calcd. for C10H10N4O6S2 (346.35): C, 34.67; H, 2.91; N,
16.18; Found: C, 34.43, H, 2.87; N, 16.24.
4-Bromo-N-[(2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-
yl)sulfonodithioyl]benzenesulfonylhydrazide (18b)
Yield: 82 %; m.p.: 262–264 °C; IR (KBr, cm-1): 3430–3208 (4NH), 1713, 1682
1
(2CO of uracil), 1322, 1213 (SO2NH); H-NMR (DMSO-d6, d ppm): 7.41, 7.62
(d,d, 4H, J = 7.6 Hz, Ar–H), 8.42 (s, 1H, uracil-H6) and 10.23, 10.51, 10.75, 11.32
(4s, 4H, 4NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 150.3, 164.7
(2C=O), 100.1, 123.4, 128.6, 131.7, 132.2, 142.2 (aromatic-C); MS m/z 424 (M?)
(16 %), 426 (M? ? 2) (14 %); Anal. calc. for C10H9BrN4O6S2 (425.24): C, 28.24;
H, 2.13; N, 13.17; Found: C, 28.41; H, 2.09; N, 13.00.
N-[(4-Methylphenyl)sulfonyl]-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-
sulfonylhdrazide (18c)
Yield: 80 %; m.p.: 233–235 °C; IR (KBr, cm-1): 3520–3278 (4NH), 1731, 1679
1
(2CO of uracil), 1324, 1124 (SO2NH); H-NMR (DMSO-d6, d ppm): 2.71 (s, 3H,
CH3), 7.23, 7.80 (d,d, 4H, J = 7.4 Hz, Ar–H), 8.2 (s, 1H, uracil-H6) and 10.00,
123

O. A. Fathalla et al.
10.48, 10.91, 11.43 (4s, 4H, 4NH exchangeable with D2O); 13C-NMR (DMSO-d6, d
ppm): 150.1, 164.7 (2C=O);, 101.2, 128.0, 129.1, 135.2, 135.6, 143.3 (aromatic-C),
24.2 (CH3); MS m/z 360 (M?) (8 %); Anal. calc. for C11H12N4O6S2 (360.37): C,
36.66; H, 3.36; N, 15.54; Found: C, 36.41; H, 3.02; N, 15.50.
N-[(2-Nitrophenyl)sulfonyl]-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-
sulfonylhydrazide (18d)
Yield: 76 %; m.p.: 245–247 °C; IR (KBr, cm-1): 3480–3259 (4NH), 1722, 1684
(2CO of uracil), 1320, 1123 (SO2NH); 1H-NMR(DMSO-d6, d ppm): 7.01, 7.53 (d,d,
4H, J = 8.0 Hz, Ar–H), 8.42 (s, 1H, uracil-H6), 10.41, 10.72, 10.92, 11.45 (4s, 4H,
4NH exchangeable with D2O); 13C-NMR (DMSO-d6, d ppm): 150.1, 164.2 (2C=O),
100.1, 121.4, 124.2, 129.6, 131.7, 134.2, 142.3, 149.2 (aromatic-C); MS
m/z 391(M?) (6 %); Anal. calcd. for C10H9N5O8S2 (391.34): C, 30.69; H, 2.32;
N, 17.90; Found: C, 30.63; H 2.20; N, 17.63.
N-[(2,4-Dioxo-1,2,3,4-tetrahydropyrimidine-5-yl)sulfonoyl]2-4-dioxo-1,2,3,4-
tetrahydropyramidine-5-sulfonylhydrazide (18e)
Yield: 80 %; m.p.:[300 °C; IR (KBr, cm-1): 3450–3108 (6NH), 1720, 1681 (4CO
of 2 uracil rings), 1317, 1213 (SO2NH); 1H-NMR (DMSO-d6, d ppm): 8.41, 8.62 (s,
2H, 2 uracil-H6), 10.21–11.75 (6s, 6H, 6NH exchangeable with D2O); 13C-NMR
(DMSO-d6, d ppm): 150.1, 164.5 (4C=O), 100.1, 142.5 (aromatic-C); MS m/z 380
(M?) (9 %); Anal. calc. for C8H8N6O8S2 (380.32): C, 25.26; H, 2.11; N, 22.09;
Found: C, 25.03; H, 2.28; N, 22.27.
2,4-Dioxo-N-[(4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-yl)sulfonyl]-1,2,3,4-
tetrahydropyrimidine-5-sulfonylhydrazide (18f)
Yield: 85 %; m.p.:[300 °C; IR (KBr, cm-1): 3470–3110 (6NH), 1709, 1681 (3CO
of uracil), 1321, 1119 (SO2NH); 1H-NMR (DMSO-d6, d ppm): 8.11, 840 (2s, 2H, 2
uracil-H6) and 10.43–11.89 (6s, 6H, 6NH exchangeable with D2O); 13C-NMR
(DMSO-d6, d ppm): 150.1, 164.5 (3C=O), 173.2 (C=S), 100.1, 114.2, 138.1, 142.5
(aromatic-C); MS m/z 396 (M?) (11 %); Anal. calc. for C8H8N6O7S3 (396.39): C,
24.24; H, 2.03; N 21.20; found: C, 24.11; H, 2.31; N, 21.15.
Treatment and infection
Inhibitory effect of the different pyrimidine derivatives (prepared in the present
work) on S. mansoni cercarial serine protease activity was investigated.
Pyrimidine derivative 3c, which showed in vitro inhibitory effect on cercarial
penetration enzymes, was further tested at its most potent inhibitory concentration
activity to block invasion of mice skin by cercariae of S. mansoni. Compound 3c
was dissolved in DMSO/ethanol (1/1), mixed with jojoba oil, and used to paint the
tails of five mice. A control group of five mice was included, whose tails were
painted with the oil mixed with the solvents without the tested compound. Each
123

Inhibitory effect of some new uracil and thiouracil derivatives
mouse was infected with 80 cercariae of S. mansoni strain by tail immersion method
[12].
Separation of mouse sera
Sera were collected from the treated infected and untreated infected mice at 2, 4,
and 6 weeks postinfection and from uninfected mice to be used as negative control.
For different mice groups (treated infected, control infected, and control uninfected)
available sera for IgG and IgM measurements at the three time points were from at
least three individual mice. Sera missing at one or more time points were excluded
for either being hemolyzed or insufficient to be included or the bleeding was not
successful at one or more time point because of some technical problem.
Assessments of worm recovery
Seven weeks postinfection, treated and untreated infected mice were perfused.
Recovery of adult worms was performed through hepatic portal [24]. Total worm
counts in intestines and livers were determined. Protection was assessed as percentage
reduction in worm counts according to the following formula: P = C - T/C 9 100,
where P is the percentage reduction in worm counts in liver and intestine, C is the mean
worm count in control infected mice (tail painted with oil containing the solvent
without compound 3c), and T is the mean worm count in pre-tail painted mice with the
oil containing compound 3c before infection with S. mansoni cercariae.
Enzyme-linked immunosorbent assay (ELISA)
The assay was performed according to the method of Bahgat et al. [11] to determine
levels of IgG and IgM in sera of different mice groups against antigens derived from
early and late stages of the S. mansoni parasite. Plates were coated with 100 ll/well of
three different schistosome antigens: cercarial antigenic preparation (CAP, early-
stage antigen) at concentration 25 lg/ml, soluble worm antigen preparation (SWAP,
late-stage antigen) at concentration 50 lg/ml, and soluble egg antigen (SEA, late-
stage antigen) at concentration 25 lg/ml; then plates were incubated at room
temperature overnight. Plates were washed three times using phosphate-buffered
saline (PBS) containing 0.05 % 20 (PBST), and antigen-free sites were blocked
against nonspecific binding using 200 ll/well PBST containing 1 % bovine serum
albumin (BSA) (PBST-BSA), then left for 1 h at 37 °C. After three washes, 100 ll/
well diluted sera (1:100 in PBST-BSA) was added and incubated at 37 °C for 2 h.
Peroxidase-labeled anti-mouse IgG or IgM conjugates were added at dilution of
1:500 and 1:5,000, respectively, in PBST-BSA and incubated for 1 h at 37 °C.
O-Phenylenediamine-dihydrochloride (OPD) substrate was used as substrate for
visualization of the antigen antibody binding in the presence of H2O2. To avoid the
increase in the background of the enzyme substrate reaction, HCl (2 N) was used as
stopper, and changes in optical densities were measured at kmax of 490 nm using a
microwell plate ELISA reader (Tecan; Sunrise Remote Control, Groedig, Austria).
123

O. A. Fathalla et al.
Preparation of cercarial secretions (CSs)
The secretions of schistosome cercariae were prepared following the previously
described procedure [11] and were used as source for the cercarial serine protease
activity. To obtain cercariae, infected snails were exposed to light for 1 h and were
then gently poured into Petri dishes (15 cm diameter) that were previously painted
with linoleic acid (0.9 g/ml) and air dried. A total of 10,000 cercariae were used per
dish. The plates were incubated at 37 °C for 30 min. During this incubation,
cercariae release the contents of acetabular glands while trying to penetrate into the
linoleic acid and transforming into schistosomula. All the water containing cercarial
materials was collected in 15-ml Falcon tubes, and larvae were sedimented on ice.
The supernatant containing CSs was spun down for 2 min at 3,0009g to eliminate
cercarial debris, then concentrated by lyophilization and reconstituted with water to
one-tenth of its original volume. Protein content was measured by bicinchoninic
acid (BCA) test.
Quantitative assay of serine proteinase activity using chromogenic substrate
The serine protease activity in CSs was quantitatively assessed by using Boc-Val-
Leu-Gly-Arg-PNA, a specific substrate for trypsin-like serine proteinases [25]. The
release of yellow color was quantified by measuring the absorbance at 405 nm using
microtiter plate reader (Tecan, Sunrise Remote Control, Groedig, Austria).
The intensity of the yellow color is directly proportional to the enzyme activity.
Stock solution of the substrate at concentration of 10 mg/ml was prepared in
dimethyl sulfoxide (DMSO) and was then diluted with substrate buffer (30 mM Tris
containing 60 mM NaCl and 0.05 % NaN3) to the final concentration of optimal pH.
Inhibition of cercarial serine protease activity
Inhibitory effect of the different pyrimidine derivatives (prepared in the present
work) on S. mansoni cercarial serine protease activity was investigated.
All derivatives were found to be completely soluble in DMSO/ethanol mixture
(1/1). Stock solutions of the pyrimidine derivatives of interest (10 mg/ml) were
prepared. For performing inhibition assays, 50 ll CSs were preincubated with 10 ll
of each of the serial dilutions of different pyrimidine derivatives for 10 min before
adding the substrate L-1195. Similar volumes of the solvents (DMSO/ethanol) were
mixed with the CSs as negative controls.
Results and discussion
Chemical studies
The key starting material thiouracil-5-sulfonylhydrazide prepared according to
the reported method [12] was allowed to react with different aromatic aldehydes,
namely 4-bromobenzaldehyde, 5-methylfurfural, 4-antipyrinecarboxaldehyde,
123

Inhibitory effect of some new uracil and thiouracil derivatives
and/or 2-salicylaldehyde, to give the corresponding Schiff bases 2a–d, respectively.
The structures of the obtained derivatives were confirmed by microanalyses and
spectral data. IR spectra exhibited different absorption bands at the region
3,325–3,122 cm-1 corresponding to NH groups, in addition to the presence of
absorption bands at the region 1,692–1,680 cm-1 due to C=O of thiouracil ring. IR
spectrum of 2c showed another band at 1,712 cm-1 due to C=O of pyrazoline ring.
1H NMR spectra of the compounds confirmed their chemical structures; e.g., the
spectrum of 2b showed a singlet signal at d 2.33 ppm due the methyl group of furan
ring and two doublets at d 6.31, 7.20 ppm due to the two protons of the furan
moiety, in addition to the other expected protons of the compound. Also, the
spectrum of 2c exhibited two singlet signals at d 2.50, 3.71 ppm referring to the two
methyl groups of the pyrazoline ring, in addition to the other aromatic protons and
those of NH groups appearing in their expected regions. MS spectra of the
compounds showed the molecular ion peaks of the compounds. Since Schiff bases
are important intermediate precursors for synthesis of different heterocyclic ring
systems, the derivatives 2a–d were allowed to react with thioglycolic acid in dry
benzene to furnish the new thiazolidinone derivatives 3a–d, and their structures
were confirmed using microanalytical and spectral data. IR spectra revealed the
appearance of an absorption band at the range 1,715–1,725 cm-1 corresponding to
C=O of thiazolidinone ring in addition to the absorption band at 1,680–1,692 cm-1
1
that is relevant to the carbonyl group of thiouracil ring. H NMR spectra of 3a–
d exhibited the presence of two singlets at the range d 4.10–4.50 and 4.51–4.61 ppm
due to the protons of SCH2 and N–CH–S groups of thiazolidinone ring. MS spectra
showed the molecular ion peaks of the compounds. The Mannich bases 4a, b, 5b, c,
and 6c, d were obtained by reaction of the thiazolidinone derivatives 3a–d with
different secondary amines, namely N-methylpiperazine, diethylamine, and/or
morpholine in refluxing ethanol in presence of paraformaldehyde (Scheme 1). The
structures of the novel derivatives were established by microanalytical and spectral
data. IR spectra appeared in agreement with their structures. 1H NMR spectra of the
compounds exhibited a doublet signal at the range d 4.10–4.89 ppm corresponding
to the two protons of CH2 bridge that connects the amine moiety with the
thiazolidinone ring. Also, a triplet signal appeared at the range d 6.11–6.14 ppm due
to the methine proton of S–CH of thiazolidinone ring, in addition to other signals
corresponding to the expected protons of each derivative. MS spectra showed their
molecular ion peaks.
The respective iminopyridine and pyridone derivatives 8a–c, 9a–c, 10a–c, and
11a–c were synthesized by one-pot reaction of the compounds 7a, b [15] with the
appropriate aldehydes, namely 2-chloro-5-nitrobenzaldehyde, 2,5-dimethoxybenz-
aldehyde and 3,5-dimethoxybenzaldehyde, malononitrile, and/or ethyl cyanoacetate
and ammonium acetate in refluxing n-butanol for 8–10 h (Scheme 2). Microanal-
yses and spectral data confirmed the chemical structures of the novel derivatives. IR
spectra of the compounds displayed the characteristic absorption bands of CN
groups at the range 2,205–2,212 cm-1, and those of CO groups of the pyrimidine
rings appeared at the range 1,685–1,720 cm-1. Compounds 10a–c and 11a–
c showed additional bands around the same range corresponding to the pyridinyl CO
group. 1H NMR spectra of the compounds appeared in agreement with their
123

O. A. Fathalla et al.
O
O
SO2NHN=CHR
SO2NHNH2
HN
HN
i
S
N
H
S
N
H
2a-d
1
R
S
O
N
N
N
SO2NH
HN
O
4a,b
S
N
H
i
i
i
R
R
O
S
S
O
N(C2H5)2
N
SO2NH
N
SO2NH
HN
iv
HN
O
O
S
N
H
5b,c
S
N
H
3a-d
v
R
S
O
O
N
N
SO2NH
HN
O
S
N
H
6c,d
a
c
b
d
R
Br
N
O
O
N
HO
Reaction conditions:i: appropriate aldehyde, absolute ethanol, stirr at rm. temp., two days;
ii:thioglyclic acid, dry benzene, reflux, water bath, 8-12 h; iii, iv, v: paraformaldehyde,
appropriate amine, absolute ethanol, reflux, 6-9 h.
Scheme 1 Preparation of Schiff bases 2a–d, thiazolidinone derivatives 3a–d, and Mannich bases 4a, b,
5b, c, and 6c, d starting from thiouracil-5-sulfonylhydrazide (1)
expected chemical structures. The molecular ion peaks of the compounds are
exhibited in their MS spectra.
Treatment of compounds 7a, b with the above-mentioned aldehydes in 10 %
ethanolic NaOH solution furnished the chalcones 12a–c and 13a–c. IR spectra of the
compounds showed the absorption bands of the chalcone CO groups in addition to
1
those of the pyrimidine rings at the range 1,680–1,720 cm-1. H NMR spectra
123

Inhibitory effect of some new uracil and thiouracil derivatives
R
CN
O
SO2NH
R
N
NH2
HN
CN
O
O
X
N
H
SO2NH
8a-c, 9a-c
N
H
HN
X
N
H
10a-c, 11a-c
O
O
SO
2NH
COCH=CHR
HN
SO2NH
COCH3
iii
HN
X
N
H
N
H
X
12a-c, 13a-c
7a,b
O
CH3
S
CNHR
7a; X=O
7b; X=S
SO2NH
C=NNH
1
HN
v
S
N
H
O
14, 15
SO2NH
CH2C=S
HN
N
16
S
N
H
O
O
CH2COOH
SO2NH
HN
S
17
N
H
c
14
15
a
Cl
b
OCH3
OCH3
H
R1
R
OCH3
NO2
OCH3
X
O, S
Reaction conditions: i: Ar-CHO, malononitrile, ammonium acetate, reflux for 8-10h; ii:Ar-CHO,
ethylcyanoactate, ammonium acetate, 8-10h; iii: Ar-CHO, ethanolic NaOH 10%, stirr
at rm.temp.; iv: substituted thiosemicarbazide, ethanol, reflux for 3h; v: S, morpholine, reflux for 14h; vi:
glacial acetic acid, conc. H2SO4, reflux for 5h.
Scheme 2 Preparation of 6-iminopyridines 8a–c, 9a–c, 6-oxopyridines 10a–c, 11a–c, chalcone
derivatives 12a–c, 13a–c, hydrazinecarbothioamide derivatives 14, 15, thiomorpholide 16, and acetic
acid derivative 17 starting from compounds 7a, b
exhibited two doublet signals at d 6.10–6.81 ppm referring to the two protons of
–CH=CH– group. MS spectra exhibited the molecular ion peaks of the compounds
(Scheme 2).
123

O. A. Fathalla et al.
Hydrazinecarbothioamide derivatives 14, 15 were gained upon reaction of
compounds 7a, b with (phenyl)thiosemicarbazide in acidified ethanol. Microanal-
1
yses and spectral data confirmed the chemical structures of the compounds. H
NMR spectra exhibited a singlet signal around d 2.23 ppm corresponding to the
ethylidene CH3 group.
Willgerodt reaction [26] was carried out by the reaction of 7b with morpholine
and sulfur to get thiomorpholide derivative 16, which was converted easily to the
corresponding acetic acid derivative 17 (Scheme 2). Microanalyses and spectral
data confirmed the chemical structures of the derivatives. IR spectrum of 16
revealed absorption bands at 1,718–1,710 cm-1 referring to 2CO of thiouracil ring
1
and the carboxylic group. H NMR spectrum of 16 presented two multiplets at d
3.00 and 3.61 ppm assigned to the 8H of morpholine ring, in addition to a singlet at
1
d 4.31 ppm due to 2H of thioxoethyl group. H NMR spectrum of 17 showed the
disappearance of the multiplets of morpholine ring and the presence of a singlet due
to 2H of acetic acid group at d 4.53 ppm. The other expected protons of the
derivatives were apparent in the spectra. MS spectra showed the molecular ion
peaks of the compounds.
Further reactions were carried out by refluxing uracil-5-sulfonylhydrazide with
equimolar amounts of different substituted benzenesulfonyl chloride in alkaline
methanol to give benzenesulfonohydrazide derivatives 18a–f (Scheme 3). Micro-
analytical and spectral data complied with the chemical structures of the novel
derivatives.
Pharmacological studies
In this report, we describe a new serine protease inhibitor. We employed a cercarial
serine protease that could be inhibited by known serine protease inhibitors [25]. The
protease inhibitory effect of our new pyrimidine derivative is not surprising in the
light of previous reports demonstrating that previously characterized pyrimidine
derivatives can block several serine proteases, such as nifedipine, alkyl [sulfo-
nyl(oxy)] uracils, 1,2-dihydrouracil, and hexahydroimidazo[1,2-c]pyridine deriva-
tive that could block leukocyte enzyme [27], and (5-amino-6-oxo-2-phenyl-1,6-
dihydro-1-pyrimidinyl)-acetyl-Phe-CF3 and pyrimidinone derivatives that showed
chymase inhibitory effect [28]. Although the serine protease inhibitor described in
this report and its effect to block cercarial penetration are new, the approach is a
continuation of earlier trials carried out to develop protease inhibitory formulations
that can successfully block cercarial penetration both in vitro and in vivo [29].
When our compound 3c was formulated in jojoba oil, it slightly blocked cercarial
penetration as indicated by 20 % reduction in worm burden. Although this reduction
is not a high degree of lowering the worm burden, it may contribute to decreasing
the pathology of the disease since reduction of worm count will certainly lead to
reduction of egg count, which is the major cause of the severe pathology.
Cruthers et al. [30] reported that topical application of a 2-hydroxybenzoic
anilide provided prophylactic protection against penetration by infective cercariae
of the parasitic worms. Salafsky et al. found that praziquantel (PZQ) was
moderately effective in blocking cercarial penetration when used as a topical paint.
123

Inhibitory effect of some new uracil and thiouracil derivatives
O
SO2NHNH
SO2
HN
O
N
H
R
18a-d
O
SO2NHNH2
HN
O
N
H
O
O
SO2
SO2NHNH
HN
NH
O
N
H
N
H
X
18e,f
a
b
d
e
f
c
R
H
4-Br 4-CH3 2-NO2
O
S
X
Reaction conditions: i, ii: appropriate sulfonylchloride, methanol, few drops of pyridine, reflux for 8-12h
Scheme 3 Preparation of benzenesulfonohydrazide derivatives 18a–f starting from uracil-5-
sulfonylhydrazide
Cremophor EL, 50 % propylene glycol, vegetable oil, and cod liver oil were used as
PZQ vehicles. When Cremophor EL (ethoxylated castor oil) was used to administer
PZQ, a 93 % reduction in cercarial penetration was seen at 0 h and a
98 %? reduction rate was seen from 4 to 24 h postexposure. However, Cremo-
phor EL alone had an essentially equivalent effect on cercarial penetration from 8 to
92 h after administration. Castor oil gave protection rate ranging from 90 to almost
100 %. They suggested that chemoprophylaxis may be possible by dietary
supplementation with lipids having antipenetration activity or by molecules that
resemble these lipids [31].
Cooper et al. [32] showed a dimethicone formulation to be effective at
preventing S. cercariae from infecting skin. Current treatment depends on
praziquantel, raising concerns of emergence of resistant parasites. Oxadiazole-2-
123

O. A. Fathalla et al.
oxide was identified as an inhibitor of thioredoxin glutathione reductase (TGR), a
selenocysteine-containing flavoenzyme required by the parasite to maintain proper
cellular redox balance.
Bahgat et al. [12] found that N0-(4-methylbenzylidine)-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-sulfonylhydrazide has potent inhibitory effect against
S. mansoni cercariae; upon its use as a paint on mice tails before infection with
S. mansoni cercariae, the compound formulated in jojoba oil caused a significant
reduction (93 %) in worm burden. Rai et al. [33] studied the utility of oxadiazole-2-
oxides as novel inhibitors and as efficacious antischistosomal agents.
The humoral, both primary and secondary, immune responses against several
antigens derived from different stages of S. mansoni lifecycles were measured in
sera from treated then infected mice, infected untreated mice, and uninfected
untreated control mice. The antigens used were soluble cercarial antigenic
preparation (CAP) that represents larval early-stage antigen and, as late-stage
derived antigens, SWAP and SEA.
Comparing the IgM reactivity to CAP in sera collected from treated infected
mice at regular time intervals postinfection with its reactivity in sera collected from
infected untreated mice at the same time points showed that sera collected from
treated infected mice at two weeks treated infection (2WTI) had 0.59-fold lower
IgM reactivity when compared with sera collected from treated infected mice at two
weeks treated infection (2WI). This could be due to the fact that treatment retarded
penetration of cercariae and as a result lowered the primary immune response,
particularly at such an early phase postinfection.
At 4 weeks postinfection, treated infected mice (4 WTI) showed no change to
CAP compared with infected untreated (4 WI) ones. This could be due to the
beginning of an enhanced primary immune response due to chemical attenuation of
some of the cercariae by treatment. Those cercariae may have penetrated the mice
skin and stimulated the increase in the IgM immune response among the treated
infected mice when compared with the untreated infected ones.
At 6 weeks, the IgM response to CAP in sera from treated infected mice was
0.47-fold lower than in those from untreated mice. This may reflect that, although
treatment caused attenuation of some cercariae and may have caused enhancement
of the IgM response at 4 weeks postinfection, this effect was very transient and the
duration of this IgM response was so short that it was lower at 6 weeks in the treated
infected mice than in the untreated infected mice. The IgG level in the sera collected
from treated infected mice at 2 and 4 weeks postinfection generally showed lower
IgG reactivity when compared with the IgG level in sera collected from infected
untreated mice at the same time point. This confirms our conclusion that treatment
with our new compound caused attenuation of some of the penetrating cercariae.
Comparison of both IgM and IgG responses to SWAP in sera collected from treated
infected mice at regular time intervals postinfection versus the reactivity in sera
collected from infected untreated mice at the same time points revealed that sera
collected from treated infected mice had lower IgM level (0.84-fold) at 2 and 4 WTI
and IgG at 2, 4, and 6 WTI than IgM level in sera from the infected untreated ones at
the same time points. This could be due to the fact that attenuation of the cercariae
caused by skin treatment may have caused retardation in the development of
123

Inhibitory effect of some new uracil and thiouracil derivatives
Fig. 1 Inhibition of serine protease activity of Schistosoma mansoni cercariae by compound 3c
cercariae into schistosomula and juvenile worms and as a result caused retardation
of the IgM and IgG responses developed to the adult worm antigens among sera
from treated mice when compared with those of untreated ones.
Both IgM and IgG responses to soluble egg antigen were generally lower in
sera collected at different time intervals from treated infected mice when compared
with in sera collected from infected untreated mice at the same time points. We
concluded that compound 3c has an immunosuppressive response, and our results are
in agreement with Bahgat et al. [34], who reported evaluation of a cyclohexanecarb-
oxamide derivative, N-phenyl-N-[1-(piperidine-1-carbonyl)cyclohexyl]benzamide,
for its inhibitory effects on S. mansoni cercarial serine protease activity and cercarial
penetration. In addition, the IgM and IgG responses to crude S. mansoni cercarial,
worm, and egg antigens were generally lower in sera from treated infected mice than
untreated infected mice.
Inhibition of cercarial serine protease activity of S. mansoni cercariae
The prepared pyrimidine derivatives were tested in vitro for their inhibitory effects.
Compound 3c exerted an inhibitory effect on cercarial serine protease when tested
at serial concentrations (Fig. 1). The IC50 (inhibitor concentration that abolishes
50 % of enzyme activity) of the compound was observed to be 160 lg.
Antischistosomal activity
The perfusion results of mice treated with compound 3c are presented in Fig. 2. It
caused a reduction (20 %) in the worm burden when compared with control infected
mice, although the worm burden results of treated, tail-painted, mice did not differ
significantly (P = 0.2920) from the control mice.
123

O. A. Fathalla et al.
Mean and Standard Deviation
16
14
12
10
8
6
4
2
0
A
B
Column
Fig. 2 Reduction in worm burden in mice treated with 3c (column B) when compared with untreated
control mice (column A). The results indicate 20 % reduction in worms recovered from treated mice
compared with control mice
Detection of IgM and IgG levels posttreatment and infection with S. mansoni
Measurements of both IgM and IgG levels were carried out in sera collected at 2, 4,
and 6 weeks postinfection from both treated and untreated mice.
Using CAP as an antigen in ELISA, the level of IgM or IgG showed
nonsignificant change in sera from treated infected as compared with infected
untreated mice at different time points (Table 1).
IgM reactivity to SWAP level in sera collected from treated infected mice at 2
(2 WTI) and (6 WTI) weeks postinfection showed nonsignificant differences when
compared with sera collected from infected treated mice at the same time point
(2 WI and 6 WI, respectively). At 4 weeks postinfection, the treated infected mice
(4 WTI) showed significant decrease (P = 0.004) in IgM reactivity to SWAP as
compared with infected untreated (4 WI) ones (Table 1).
The same comparison for IgG level is demonstrated in Table 1. The level of IgG
in sera from treated infected mice at 2 and 4 weeks postinfection (2 WTI, 4 WTI)
against SWAP showed significant decrease (P = 0.04 and 0.004, respectively)
when compared with sera collected from infected treated mice at the same time
point (2 WI and 4 WI, respectively). At 6 weeks, the IgG response showed
nonsignificant differences in sera from treated infected mice compared with those
from untreated mice (Table 1).
The IgM reactivity to SEA in sera collected from treated infected mice at 2, 4,
and 6 WTI weeks postinfection showed nonsignificant differences when compared
with the IgM level in sera from infected treated mice at the same time point (2, 4,
and 6 WI). The IgG reactivity in sera from treated infected mice at 2, 4, and 6 WTI
showed significant decrease (P \ 0.05) when compared with the IgG level in sera
from infected treated mice at the same time point (2, 4, and 6 WI) (Table 1).
123

Inhibitory effect of some new uracil and thiouracil derivatives
123

O. A. Fathalla et al.
Conclusions
A new series of uracil- and thiouracil-5-sulfonohydrazide derivatives were
synthesized to test their antischistosomal activity. It was found that compound 3c,
N-[2-(1,5-dimethyl-3-oxo-2-phenylpyrazolin-4-yl)-4-oxo-1,3-thiazolidin-3-yl]-4-oxo-
2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide, inhibited cercarial serine pro-
tease activity with IC50 = 160 lg. Upon topical application on mice tails before
infection with S. mansoni cercariae, it caused a reduction in worm burden when
compared with untreated infected mice. Thus, further modifications must be
performed on the synthesized derivatives to obtain more potent cercarial serine
protease inhibition, greater reduction of cercarial penetration, and more potent
antischistosomal efficacy.
Acknowledgments This research project was supported by a grant from the research center of the
center for female scientific and medical colleagues in King Saud University.
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