An Efficient Synthesis and in vitro Cytostatic Activity of 5-Aminosulfonyl Uracil Derivatives

Article abstract of DOI:10.5562/cca3567

Efficient synthesis of 5-aminosulfonyl uracil derivatives 2?9 and results of their antiproliferative activity are provided. Sulfonylation of the amino group in 5-aminouracil 1 with selected arylsulfonyl chlorides occurs regioselectively when the reaction

Full text of DOI:10.5562/cca3567

O R I G I NA L S C I E N T I F I C P A P E R
Croat. Chem. Acta 2019, 92(2), 269–277
Published online: October 24, 2019
DOI: 10.5562/cca3567
An Efficient Synthesis and In vitro Cytostatic
Activity of 5-Aminosulfonyl Uracil Derivatives
Hamit Ismaili,1 Željka Ban,2 Josipa Matić,2 Dijana Saftić,2 Marijana Jukić,3
Ljubica Glavaš-Obrovac,3,* Biserka Žinić2,*
1
2
Faculty of Mathematics and Natural Sciences, University of Pristina, Agim Ramadani n.n. 10000, Pristina, Kosovo
Laboratory for Biomolecular Interactions and Spectroscopy, Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54,
HR-10000 Zagreb, Croatia
3
Department of Medicinal Chemistry, Biochemistry and Laboratory Medicine, Faculty of Medicine, Josip Juraj Strossmayer University of Osijek, Huttlerova 4,
HR-31000 Osijek, Croatia
*
Corresponding authors’ e-mail addresses: lgobrovac@mefos.hr, bzinic@irb.hr
RECEIVED: August 1, 2019
REVISED: September 18, 2019
ACCEPTED: September 19, 2019
TH
THIS PAPER IS DEDICATED TO PROF. KATA MLINARIĆ-MAJERSKI ON THE OCCASION OF HER 70 BIRTHDAY
Abstract: Efficient synthesis of 5-aminosulfonyl uracil derivatives 2−9 and results of their antiproliferative activity are provided. Sulfonylation of
the amino group in 5-aminouracil 1 with selected arylsulfonyl chlorides occurs regioselectively when the reaction is carried out in pyridine at
room temperature. Simple isolation of the products by recrystallization of the crude product mixture from aqueous methanol provides good
to excellent yields. The prepared 5-aminosulfonyl uracil derivatives 2−9 were tested for the antiproliferative activity on a panel of seven tumor
cell lines of different histological origin (HeLa, Caco-2, NCI-H358, Raji, HuT78, Jurkat, K562) and normal MDCK I cells. Derivatives 2−9 were
found more efficient to lymphoma and leukemia cells compared to solid tumor and normal cells.
Keywords: 5-aminouracil, arylsulfonyl chlorides, 5-aminosulfonyl uracil derivatives, in vitro, anticancer activity.
fluorescent uracil derivatives suitable for incorporation into
PNA's or nucleic acids.[24–29]
INTRODUCTION
HE pyrimidine derivatives are known as essential
constituents of nucleic acids and exhibit a variety of
Our group has a long-term experience in design,
synthesis, and characterization of biologically active nucleo-
side derivatives.[30–33] Previously, we have synthesized
N-sulfonylpyrimidine derivatives I (Figure 2) as a new type
of sulfonylcycloureas.[34–37] These compounds showed
strong antiproliferative activity on human tumor cell lines,
in in vitro[38–41] and in vivo[42–45] conditions.
T
biological activities[1] notable among which are the anti-
bacterial,[2–4] anticancer,[5–8] anti-inflammatory,[9,10] anti-
tubercular,[11] analgesic,[12] and antiviral[13–15] activities. The
pyrimidine base with modification at C5 position provides
the enhanced biological activity of the nucleobase and
nucleoside analogs.[16–18]
5-Fluorouracil[19,20] and the nucleoside floxuridine[21]
are frequently used for the treatment of various cancers.
Uramustine[22] is used orally in the treatment of leukemia
and thymine derivative HEPT is considered as a non-
nucleoside reverse transcriptase inhibitor in HIV-infection
therapy (Figure 1).[23] 5-Bromouracil and 5-aminouracil
could be incorporated into nucleic acids and could interfere
with their transcription or translation processes. They also
represent good starting material for the synthesis of the
O
Cl
H
O
F
O
N
N
O
O
O
H
O
CH3
S
N
H
O
F
H
O
N
N
N
Cl
N
HO
N
H
N
H
HO
O
OH
Floxuridine
5-FU
Uramustine
HEPT
Figure 1. Structures of some biologically active C-5
substituted pyrimidine derivatives.
This work is licensed under a Creative Commons Attribution 4.0 International License.

270
H. ISMAILI et al.: An Efficient Synthesis and In vitro Cytostatic Activity of …
derivatives
uracil
1-sulfonylpyrimidine
5-aminosulfonyl
derivatives
1
600.13 or 300.13 MHz for H nuclei using DMSO-d6 as the
R
3
N
R
O
O
H
3
internal standard. Elemental analyses were performed by
the Applied Laboratory Research Department at INA, d.d.
Research and Development Sector, Central Analytical
Laboratory.
H
O
H
O
Ar
NH2
5
H
O
H
O
N
N
N
N
S
5
1
O
O
N1
N
N
H
N
H
H
S
Ar
uracil
=
O
R
O
5-aminouracil
O
1
II
I
=
R
NH
cytosine
General Procedure for the Condensation of 5-aminouracil 1
with sulfonyl chlorides
Figure 2. Structures of 1-sulfonylpyrimidines I and 5-amino-
sulfonyl uracil derivatives II.
A suspension of 5-aminouracil 1 (1 mmol) in dry pyridine
(7.5 mL) was cooled to 0 °C and the appropriate sulfonyl
chloride (1 mmol) was added. The reaction mixture was
stirred at room temperature until the TLC showed the
reaction was completed (1.5−20 h). The solvent was
removed under reduced pressure and the crude product
was recrystallized from methanol or aqueous methanol to
afford the product.
The promising anticancer activity of 1-sulfonyl-
pyrimidines I inspired the design and synthesis of novel
series of 5-aminosulfonyl uracil derivatives of general
structure II (Figure 2; compounds 2−9, Scheme 1).
Sulfonylation of 5-aminouracil I appears particularly
interesting due to its enhanced hydrogen bonding ability by
5-amino hydrogens which enables the formation of
quadruplex and triplex structures.[46,47] By 5-amino
sulfonylation of I its hydrogen bonding potential is retained
and could be even enhanced due to increased acidity of
sufonylamide hydrogen.
For sulfonylation of 1, the aromatic sulfonyl
chlorides with different size and lipophilicity of aromatic
groups with electron-donating or electron-withdrawing
substituents on the aromatic system were selected. In this
way, the molecules with variable electronic and lipophilic
characteristics could be obtained, which may influence
their chemical stability as well as physical and biological
properties. Here we report the synthesis of 5-amino-
sulfonyl uracil derivatives 2−9 as well as their anti-
proliferative activity on normal MDCK I cells, and HeLa,
Caco-2, NCI-H358, Raji, HuT78, Jurkat, and K562 tumor cell
lines.
N-(2,4-Dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-4-methyl-
benzenesulfonamide (2)
Compound 2 was made in a different way than described in
Ref. [48]. According to the general procedure, 5-amino-
uracil 1 (0.5 g, 3.93 mmol) and tosyl chloride (0.749 g, 3.93
mmol) in pyridine (29 mL) were reacted for 19 h. Recrystal-
lization from aqueous methanol gave the analytically pure
product 2 as white crystals: 1.08 g (98 %; lit.[48] 49 %); Rf =
0.55 (CH2Cl2/MeOH 9:1); m.p. >300 °C; UV (MeOH):
λ
max/nm: 218 and 272; log ε/ dm3 mol–1 cm–1: 4.10 and 3.08;
IR (KBr) νmax/cm–1: 3330 (s), 3125 (m), 3042 (m), 2921 (w),
1689 (s), 1647 (s), 1414 (s), 1358 (m), 1222 (s), 1169 (s),
1
1092 (m); H NMR (DMSO-d6) δ/ppm: 11.18 (s, 1H, NH-3),
10.88 (brs, 1H, NH-1), 9.24 (brs, 1H, NH-SO2), 7.62 (d, J = 8.2
Hz, 2H, Ar), 7.32 (d, J = 8.2 Hz, 2H, Ar), 7.30 (brs, 1H, H-6),
2.35 (s, 3H, CH3); 13C NMR (DMSO-d6) δ/ppm: 161.0 (C-4),
150.3 (C-2), 142.9 (Cq, Ar), 138.6 (C-6), 137.5 (Cq, Ar), 129.3
(CH, Ar), 126.9 (CH, Ar), 110.2 (C-5), 21.0 (CH3); (see
Supporting Information Figures S1, S2). Anal. Calcd. mass
fractions of elements, w/%, for C11H11N3O4S (Mr = 281.29)
are: C, 46.97; H, 3.94; N, 14.94; S, 11.40; found: C, 47.02; H,
3.98; N, 15.00; S, 11.47.
EXPERIMENTAL
General
Chemicals and solvents were obtained from Sigma–Aldrich
Chemical Company (St. Louis, USA) and used without
further purification. Rf values refer to analytical TLC
performed using pre-coated silica gel 60 F254 plates
purchased from Sigma–Aldrich (Steinheim, Germany) and
developed in the solvent system indicated. Compounds
were visualized by use of UV light (254 and 365 nm).
Melting points were determined on a Kofler hot-stage
apparatus and were uncorrected. UV spectra were taken on
a Philips PU8700 UV/VIS spectrophotometer (Philips
Analytical, Cambridge, Great Britain). IR spectra were
obtained in KBr pellets on a Perkin-Elmer 297 spectro-
photometer (Perkin-Elmer, Waltham, MA, USA). NMR
spectra were recorded on AV600 and AV300 MHz
spectrometers (Bruker BioSpin GmbH, Rheinstetten,
Germany), operating at 150.92 or 75.47 MHz for 13C and
N-(2,4-Dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-2,4,6-
triisopropylbenzenesulfonamide (3)
According to the general procedure, 5-aminouracil 1 (0.5 g,
3.93 mmol) and 2,4,6-triisopropylbenzenesulfonyl chloride
(1.23 g, 3.93 mmol, 97 %) in pyridine (29 mL) were reacted
for 12 h. Recrystallization from methanol gave the
analytically pure product 3 as white crystals: 1.31 g (85 %);
Rf = 0.32 (CH2Cl2/MeOH, 20:1); m.p. >300 °C (decom.); UV
(MeOH): λmax/nm: 206, 208, and 278; log ε/ dm3 mol–1 cm–1:
4.61, 4.61, and 3.89; IR (KBr) νmax/cm–1: 3318 (m), 3133 (m),
2958 (m), 1721 (s), 1654 (s), 1490 (w), 1425 (m), 1248 (m),
1144 (s), 902 (w); 1H NMR (DMSO-d6) δ/ppm: 11.17 (s, 1H,
NH-3), 10.93 (d, 1H, J = 4.8 Hz, NH-1), 9.08 (s, 1H, NH-SO2),
7.29 (d, 1H, J = 6.1 Hz, H-6), 7.16 (s, 2H, Ar), 4.00−3.87 (m,
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DOI: 10.5562/cca3567

H. ISMAILI et al.: An Efficient Synthesis and In vitro Cytostatic Activity of …
271
2H, CH-Ar), 2.95−2.82 (m, 1H, CH-Ar), 1.20−1.14 (m, 18H,
CH-CH3); 13C NMR (DMSO-d6) δ/ppm: 161.3 (C-4), 151.9 (Cq,
Ar), 150.3 (C-2), 149.8 (Cq, Ar), 139.7 (C-6), 134.0 (Cq, Ar),
123.3 (CH, Ar), 109.2 (C-5), 33.1 (CH-CH3), 29.5 (CH-CH3),
24.5 (CH-CH3), 23.3 (CH-CH3); (see Supporting Information
Figures S3, S4). Anal. Calcd. mass fractions of elements,
w/%, for C19H27N3O4S (Mr = 393.50) are: C, 57.99; H, 6.92;
N, 10.68; S, 8.15; found: C, 58.03; H, 6.98; N, 10.70; S, 8.17.
124.2 (CH, Ar), 109.2 (C-5); (see Supporting Information
Figures S7, S8). Anal. Calcd. mass fractions of elements,
w/%, for C10H8N4O6S (Mr = 312.26) are: C, 38.46; H, 2.58; N,
17.94; S, 10.27; found: C, 38.51; H, 2.59; N, 17.96; S, 10.30.
N-(2,4-Dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-2-nitro-
benzenesulfonamide (6)
According to the general procedure, 5-aminouracil 1 (0.5 g,
3.93 mmol) and 2-nitrobenzenesulfonyl chloride (0.871 g,
3.93 mmol, 97 %) in pyridine (29 mL) were reacted for 20 h.
Recrystallization from methanol gave the analytically pure
product 6 as red crystals: 0.726 g (59 %); Rf = 0.22
(CH2Cl2/MeOH, 20:1); m.p. 280 °C (decom.); UV (MeOH):
4-Acetamido-N-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-
benzenesulfonamide (4)
Compound 4 was prepared by modification of the
described procedures.[49,50] According to the general
procedure, 5-aminouracil 1 (0.5 g, 3.93 mmol) and 4-acet-
amidobenzenesulfonyl chloride (0.937 g, 3.93 mmol, 98 %)
in pyridine (29 mL) were reacted for 20 h. Recrystallization
from methanol gave the analytically pure product 4 as light
brown crystals: 0.828 g (65 %): Rf = 0.22 (CH2Cl2/MeOH,
20:1); m.p. >290 °C (decom.), (lit.[50] m.p. >285−287 °C); UV
(MeOH): λmax/nm: 206 and 263; log ε/ dm3 mol–1 cm–1: 4.13
and 4.08; IR (KBr) νmax/cm–1: 3446 (w), 3321 (m), 3277 (m),
3035 (m), 2842 (m), 1735 (s), 1667 (s), 1586 (s), 1534 (s),
1494 (m), 1396 (m), 1313 (m), 1152 (s), 1011 (w), 846 (m);
1H NMR (300 MHz, DMSO-d6) δ/ppm: 11.17 (s, 1H, NH-3),
10.88 (brd, 1H, J = 5.0 Hz, NH-1), 10.28 (s, 1H, NH-C=O), 9.16
(s, 1H, NH-SO2), 7.70 (d, J = 8.9 Hz, 2H, Ar ), 7.65 (d, J = 9.0
Hz, 2H, Ar), 7.29 (d, 1H, J = 6.1 Hz, H-6), 2.07 (s, 3H, CH3);
13C NMR (DMSO-d6) δ/ppm: 168.9 (O=C-CH3) 161.0 (C-4),
150.3 (C-2), 143.0 (Cq, Ar), 138.5 (C-6), 133.8 (Cq, Ar), 128.0
(CH, Ar), 118.2 (CH, Ar), 110.1 (C-5), 24.1 (q, CH3); (see
Supporting Information Figures S5, S6). Anal. Calcd. mass
fractions of elements, w/%, for C12H12N4O5S (Mr = 324.31) )
are: C, 44.44; H, 3.73; N, 17.28; S, 9.89; found: C, 44.49; H,
3.74; N, 17.30; S, 9.91.
λ
max/nm: 268 and 326; log ε/ dm3 mol–1 cm–1: 3.99 and 2.92;
IR (KBr) νmax/cm–1: 3313 (m), 3108 (m), 3027 (m), 2830 (m),
1715 (s), 1679 (s), 1542 (s), 1421 (s), 1385 (s), 1325 (m),
1168 (s), 910 (m); 1H NMR (DMSO-d6) δ/ppm: 11.23 (s, 1H,
NH-3), 11.02 (brd, 1H, J = 5.4 Hz, NH-1), 9.70 (brs, 1H, NH-
SO2), 8.08−7.80 (m, 4H, Ph), 7.41 (d, 1H, J = 6.3 Hz, H-6); 13
C
NMR (DMSO-d6) δ/ppm: 161.4 (C-4), 150.5 (C-2), 147.6 (Cq,
Ar), 140.8 (C-6), 134.3 (CH, Ar), 132.6 (Cq, Ar), 132.2 (CH,
Ar), 130.4 (CH, Ar), 123.8 (CH, Ar), 109.2 (C-5); (see
Supporting Information Figures S9, S10). Anal. Calcd. mass
fractions of elements, w/%, for C10H8N4O6S (Mr = 312.26)
are: C, 38.46; H, 2.58; N, 17.94; S, 10.27; found: C, 38.49; H,
2.61; N, 17.99; S, 10.28.
4-Cyano-N-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-
benzenesulfonamide (7)
According to the general procedure, 5-aminouracil 1 (0.250 g,
1.97 mmol) and 4-cyanobenzenesulfonyl chloride (0.410 g,
1.97 mmol, 97 %) in pyridine (15 mL) were reacted for 1.5 h.
Recrystallization from methanol gave the analytically pure
product 7 as orange crystals: 0.403 g (70 %); Rf = 0.44
(CH2Cl2/MeOH, 20:1); m.p. > 300 °C decom.; UV (MeOH):
N-(2,4-Dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-4-nitro-
benzenesulfonamide (5)
λ
max/nm: 202, 231, and 271; log ε/ dm3 mol–1 cm–1: 4.03,
3.95, and 3.76; IR (KBr) νmax/cm–1: 3341 (s), 3112 (s), 2815
(m), 2236 (w), 1782 (w), 1690 (s), 1517 (w), 1416 (s), 1362
(s), 1221 (s), 1172 (s), 1089 (m); 1H NMR (DMSO-d6) δ/ppm:
11.19 (s, 1H, NH-3), 10.99 (d, 1H, J = 4.8 Hz, NH-1), 9.66 (s,
1H, NH-SO2), 8.02 (d, 2H, J = 8.4 Hz, Ar), 7.92 (d, 2H, J = 8.4
Hz, Ar), 7.43 (d, 1H, J = 6.2 Hz, H-6); 13C NMR (DMSO-d6)
δ/ppm: 161.1 (C-4), 150.4 (C-2), 144.6 (Cq, Ar), 140.4 (C-6),
133.0 (CH, Ar), 127.7 (CH, Ar), 117.8 (Cq, Ar), 114.9 (C≡N),
109.2 (C-5); (see Supporting Information Figures S11, S12).
Anal. Calcd. mass fractions of elements, w/%, for
C11H8N4O4S (Mr = 292.27) are: C, 45.20; H, 2.76; N, 19.17; S,
10.97; found C, 45.25; H, 2.80; N, 19.19; S, 11.01.
Compound 5 was prepared by modification of the
described procedure.[50] According to the general proced-
ure, 5-aminouracil 1 (0.5 g, 3.93 mmol) and 4-nitro-
benzenesulfonyl chloride (0.898 g, 3.93 mmol, 97 %) in
pyridine (29 mL) were reacted for 19 h. Recrystallization
from aqueous methanol gave the analytically pure product
5 as a light brown solid: 0.761 g (62 %); Rf = 0.52
(CH2Cl2/MeOH, 20:1); m.p. >300 °C (decom.), (lit.[50] m.p.
>300 °C); UV (MeOH): λmax/nm: 202, 204, and 266 log ε/
dm3 mol–1 cm–1: 4.03, 4.06, and 3.99; IR (KBr) νmax/cm–1:
3338 (s), 3113 (s), 3038 (m), 2923 (m), 1689 (s), 1648 (s),
1541 (s), 1416 (s), 1352 (s), 1313 (m), 1221 (s), 1174 (s),
1090 (m), 925 (m); 1H NMR (DMSO-d6) δ/ppm: 11.21 (s, 1H,
NH-3), 11.03 (brs, 1H, NH-1), 9.77 (brs, 1H, NH-SO2), 8.36
(d, 2H, J = 8.8 Hz, Ar), 8.01 (d, 2H, J = 8.8 Hz, Ar), 7.48 (s, 1H,
H-6); 13C NMR (DMSO-d6) δ/ppm: 161.1 (C-4), 150.4 (C-2),
149.6 (Cq, Ar), 146.1 (Cq, Ar), 140.5 (C-6), 128.5 (CH, Ar),
N-(2,4-Dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)naphtha-
lene-1-sulfonamide (8)
According to the general procedure, 5-aminouracil 1 (0.5 g,
3.93 mmol) and 1-naphtalenesulfonyl chloride (0.918 g,
3.93 mmol, 97 %) in pyridine (29 mL) were reacted for 6 h.
DOI: 10.5562/cca3567
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H. ISMAILI et al.: An Efficient Synthesis and In vitro Cytostatic Activity of …
Recrystallization from methanol gave the analytically pure
product 8 as yellow crystals: 0.723 g (58 %); Rf = 0.75
(CH2Cl2/MeOH, 20:1); m.p. 294−296 °C; UV (MeOH):
supplemented with 10 % heat-inactivated fetal bovine
serume FBS (Gibco, EU), 2 × 10–3 mol dm–3 glutamine
(Gibco, EU), 1 × 10–3 mol dm–3 sodium pyruvate (Gibco, EU),
1 × 10–2 mol dm–3 HEPES (Sigma-Aldrich, USA) and 100
U/0.1 mg penicillin/streptomycin. The MDCK I, HeLa and
Caco-2 cells were grown in Dulbecco’s Modified Eagle
Medium DMEM (Gibco, EU), supplemented with 10 % FBS,
2 × 10–3 mol dm–3 glutamine and 100 U/0.1 mg penicillin/
streptomycin; in tissue culture flasks and grown as mono-
layers. To detach them from the flask surface, cells were
trypsinized using a 0.25 % trypsin/EDTA solution. Cells were
cultured in a humidified atmosphere under the conditions
of 37 °C / 5 % of CO2 gas in a CO2 incubator (Shell Lab,
Sheldon Manufacturing, USA).
Tested compounds were dissolved in dimethyl
sulfoxide as a 1 × 10–2 mol dm–3 stock solution. Working
dilutions of derivatives 2−8 and 5FU were prepared in high
pure water at a concentration range 10–4−10–7 mol dm–3
and in the case of insoluble compound 9 at a concentration
range 10–5–10–7 mol dm–3.
For the MTT test, the adherent cells, MDCK I, HeLa,
and Caco-2, were seeded in 96 micro-well plates at
concentration of 2×104 cells/cm3 and allowed to attach
overnight in the CO2 incubator. After 72 hours of the expos-
ure to tested compounds, medium was replaced with
5 mg/cm3 MTT solution and the resulting formazane
crystals were dissolved in DMSO.
Leukemia cells (1 × 105 cells/cm3) were plated onto
96 micro-well plates and after 72 hours of incubation,
5 mg/cm3 MTT solution was added to each well and
incubated 4 hours in the CO2 incubator. To each well, 10 %
SDS with 0.01 mol dm–3 HCl was added to dissolve water-
insoluble MTT-formazane crystals. The microplate reader
(iMark, BIO RAD, Hercules, CA, USA) was used for measure-
ment of the absorbance at 595 nm.
λ
max/nm: 203, 220, and 279 log ε/ dm3 mol–1 cm–1, 4.81,
4.86, and 4.28; IR (KBr) νmax/cm–1: 3182 (m), 2923 (m), 1711
(s), 1663 (s), 1430 (m), 1354 (m), 1226 (m), 1164 (s), 1134
1
(s); H NMR (DMSO-d6) δ/ppm: 11.09 (s, 1H, NH-3), 10.86
(d, 1H, J = 5 Hz, NH-1), 9.61 (s, 1H, NH-SO2), 8.69 (d, 1H, J =
9.5 Hz, Ar), 8.21−8.04 (m, 3H, Ar), 7.71−7.56 (m, 3H, Ar),
7.23 (d, 1H, J = 6.1 Hz, H-6); 13C NMR (DMSO-d6) δ/ppm:
161.1 (C-4), 150.3 (C-2), 138.7 (C-6), 135.7 (Cq, Ar), 134.1
(CH, Ar), 133.8 (Cq, Ar), 128.8 (CH, Ar), 127.9 (Cq, Ar), 127.6
(CH, Ar), 126.7 (CH, Ar), 125.1 (CH, Ar), 124.3 (CH, Ar), 110.0
(C-5); (see Supporting Information Figures S13, S14). Anal.
Calcd for C14H11N3O4S (Mr = 317.32) are: C, 52.99; H, 3.49; N,
13.24; S, 10.10; found: C, 53.04; H, 3.51; N, 13.28; S, 10.12.
(E)-4-((4-(Dimethylamino)phenyl)diazenyl)-N-(2,4-dioxo-
1,2,3,4-tetrahydropyrimidin-5-yl)benzenesulfonamide (9)
According to the general procedure, 5-aminouracil 1 (0.098
g, 0.77 mmol) and 4-N,N-dimethylaminoazobenzene-4'-
sulfonyl chloride (0.256 g, 0.77 mmol, 97.5 %) in pyridine
(6 mL) were reacted for 6 h. Recrystallization from
methanol gave the analytically pure product 9 as a brown
solid: 0.268 g (84 %); Rf = 0.71 (CH3CH2CH2OH/NH3/H2O
7:1:2); m.p. >300 °C; UV (MeOH): λmax/nm: 204, 272, and
427; log ε/ dm3 mol–1 cm–1: 3.65, 3.38, and 3.62; IR (KBr)
ν
max/cm–1: 3320 (s), 3112 (s), 1691 (s), 1603 (s), 1519 (s),
1411 (s), 1361 (s), 1233 (s), 1145 (s), 1026 (s), 838 (s), 764
1
(s); H NMR (DMSO-d6) δ/ppm: 11.20 (s, 1H, NH-3), 10.96
(d, 1H, J = 5.9 Hz, NH-1), 9.43 (s, 1H, NH-SO2), 7.88–7.73 (m,
6H, Ar), 7.38 (d, J = 6.2 Hz, 1H, H-6), 6.86 (d, J = 9.1 Hz, 2H,
Ar), 3.08 (d, J = 5.2 Hz, 6H, NCH3); 13C NMR (DMSO-d6)
δ/ppm: 161.1 (C-4), 154.5 (Cq, Ar), 153.2 (Cq, Ar), 150.4 (C-
2), 142.6 (Cq, Ar), 140.0 (Cq, Ar), 139.3 (C-6), 128.3 (CH, Ar),
125.6 (CH, Ar), 121.9 (CH, Ar), 111.8 (CH, Ar), 109.9 (C-5),
39.9 (N-CH3); (see Supporting Information Figures S15,
S16). Anal. Calcd. mass fractions of elements, w/%, for
C18H18N6O4S (Mr = 414,44) are: C, 52.17; H, 4.38; N, 20.28;
S, 7.74; found: C, 52.21; H, 4.40; N, 20.31; S, 7.76.
All experiments were performed three times in
triplicates. The percentage of treated tumor cells growth
inhibition was calculated relative to the growth of
untreated (control) cells.
Selectivity index (SI) was calculated for each
compound using formula: SI = IC50 for normal cell line
MDCK I / IC50 for respective tumor cell line. Higher values of
SI indicate greater antitumor specificity and the SI > 1.00
identifies compounds with efficacy against tumor cells
greater than toxicity against normal cells.[52–54]
Cell Culturing and MTT Test[51]
5-Aminosulfonyl uracil derivatives 1−9 and 5-fluorouracil
(5-FU) as the positive control, were selected for preliminary
in vitro cytotoxicity testing against normal Madin-Darby
canine kidney (MDCK I) cells, and seven tumor cell lines of
different histological origin: cervix adenocarcinoma cells
(HeLa), human epithelial colorectal adenocarcinoma cells
(Caco-2), human caucasian bronchioalveolar carcinoma cells
(NCI-H358), human Burkitt lymphoma cells (Raji), human T
cell lymphoma cells (HuT78), human acute T cell leukemia
cells (Jurkat, and chronic myelogenous leukemia cells (K562).
The NCI-H358, Raji, HuT78, Jurkat, and K562
cells were grown in RPMI 1640 medium (Gibco, EU)
RESULTS AND DISCUSSION
Synthesis
Depending on the reaction conditions, alkylation of uracil
with alkyl halides may result in N-1 or N-3, alkylated and
N-1,N-3-dialkylated products. For example, in the reaction
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273
of uracil with a high excess of methyl iodide, in the presence
of alkali, 1,3-dimethyl uracil was formed,[55] while the
alkylation with a small excess alkyl halide in the presence of
a base in DMF yields N-1-alkylated products with a small
amount of N-1,N-3-dialkylated compounds.[56–57]
chlorides in pyridine.[34,36] The above procedures worked
well with aliphatic, aromatic, and heteroaromatic sulfonyl
chlorides.
For the synthesis of the 5-aminosulfonyl uracil
derivatives of general formula II (Figure 2), we used
5-aminouracil 1 and applied the method b) in pyridine for
the regioselective introduction of the aromatic sulfonyl
groups to the C5 amino group of uracil ring.
The substitution of the amino group in 1 proceeded
well when equimolar amounts of 5-aminouracil 1 and
p-toluenesulfonyl chloride were reacted in pyridine at room
temperature. After simple isolation by recrystallization of
the crude product mixture from aqueous methanol,
5-aminotosyl uracil 2 was isolated in 98 % yield (Scheme 1).
Employing the latter reaction conditions with
commercially available arylsulfonyl chlorides, which have
previously been associated with antitumor activity,[58–60]
the respective 5-aminosulfonyl uracil derivatives 3−9 were
obtained in good to excellent yields (Scheme 1).
The structures of the synthesized compounds were
confirmed by elemental analysis and by IR and NMR data.
The 1H NMR spectra of the products 2-9 confirm the
respective structures of 5-aminosulfonyl uracil derivatives.
Singlets, in the NMR spectra of 2-9 appearing in the range
of δ 11.09–11.23 ppm, are attributed to the uracil N3
protons. The uracil N1 protons appear as broad singlets (2
and 5 at 10.88 and 11.03 ppm, respectively) or doublets in
the range δ 10.86–11.02 ppm (J1,6 5−6 Hz), while the
On the other hand, 5-aminouracil 1 provides greater
substitution possibilities due to the presence of a basic
amino group at the C5 position of the ring. When potassium
salt of 5-aminouracil is allowed to react with methyl iodide
alkylation takes place in the N1 and N3 positions of the
pyrimidine ring with formation of 1,3-dimethyl-5-amino-
uracil.[48] Treatment of 5-aminouracil 1 with p-toluene-
sulfonyl chloride in aq. sodium hydroxide gave 5-amino-
tosyl uracil 2 in 49 % yield,[48] while 5-sulfanilamidouracil[49]
was synthesized by reacting 5-aminouracil 1 and N-acetyl-
sulfanilyl chloride in pyridine, followed by deprotection of
N-acetyl group in aqueous sodium hydroxide (52 % yield).
Pecorari et al.[50] showed that in the reaction of
4-acetamido and 4-nitrobenzenesulfonyl chloride with
5-aminouracil 1 in aq. sodium hydroxide, mono C5
aminosulfonyl products 4 and 5 are formed in a mixture
with different amounts of disubstituted products (N5,N5-
bis-sulfonyl), depending on pH.
We have previously described two methods for the
preparation of N-1-sulfonylpyrimidine derivatives of
general formula I (Figure 2): a) condensation of silylated
pyrimidine bases with different sulfonyl chlorides in
acetonitrile; b) reaction of pyrimidine bases with sulfonyl
N
N
N
O
H
H
O
N
CH3
N
S
O
H
O
H
N
O
O
H
O
N
H
O
N
H
N
S
N
N
S
9
O
O
O
O
N
H
N
H
2
84 %
8
58 %
98 %
O
CN
O
O
H
O
NH2
H
N
H
N
N
H
O
70 %
H
O
S
N
S
O
N
H
O
O
85 %
O
1
N
H
N
H
7
3
59 %
65 %
NO2
62 %
O2N
NHCOCH3
O
O
H
N
H
H
O
H
N
O
N
S
H
N
N
S
H
O
O
O
O
N
S
N
H
O
N
H
O
6
4
O
O
N
H
5
Scheme 1. Synthesis of 5-aminosulfonyl uracil derivatives 2−9 in pyridine at room temperature
(yields correspond to analytically pure products).
DOI: 10.5562/cca3567
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274
H. ISMAILI et al.: An Efficient Synthesis and In vitro Cytostatic Activity of …
singlets of NH protons of C5 sulfonamido groups are shifted
upfield and appear in the range δ 9.08–9.77 ppm. The
multiplet signals within the δ 6.86–8.69 ppm region are
assigned to the aromatic protons and H-6 protons of uracil
moiety.
In vitro Antiproliferative Screening
5-Aminosulfonyl uracil derivatives 2−9 were tested on in
vitro cytotoxicity against normal MDCK I cells, solid tumor
(HeLa, Caco-2, NCI-H358), lymphoma (Raji, HuT78), and
leukemia (Jurkat, K562) cell lines. 5-Fluorouracil (5-FU) was
used as the positive control. All cells were treated by
investigated compounds in the 10–4–10–7 mol dm–3 range of
concentrations (with exception of compound 9, which was
found insoluble at c = 10–4 mol dm–3) using 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
assay method.
Figure 3. Cytotoxic effects of 5-aminosulfonyl uracil
derivatives 2−9 on normal Madin-Darby canine kidney
(MDCK I) cell lines growth after 72 h of incubation in the final
concentration range (◼ 10–4, ◼ 10–5, ◼ 10–6 and ◼ 10–7
mol dm–3). Cytotoxicity was analyzed using the MTT survival
assay. Data are presented as the mean value ± SD of three
independent experiments done in triplicates.
Compounds 2−9 showed modest to negligible
effects on normal (MDCK I) and solid tumor (HeLa, Caco-2,
and NCI-H358) cells growth (Supporting Information
Figures S18-S20, Table S1). For example, at the highest
applied concentration (10–4 mol dm–3) 2,4,6-triisopropyl-
benzenesulfonamide 3, 4-nitrobenzenesulfonamide 5, and
2-nitrobenzenesulfonamide 6 derivatives showed low
(10−20 %) growth inhibition activities against MDCK I cell
lines (Figure 3; Supporting Information Figure S17). Similarly,
at 10–4 mol dm–3, compounds 2–4 and compound 9 at c =
10–5−10–6 mol dm–3, caused the reduction (10−20 %) of
HeLa cells growth (Supporting Information Figure S18);
compounds 2 and 9 inhibited growth of CaCo-2 cells
(Supporting Information Figure S19), while compounds 2–
4, 7, and 9 inhibited growth of NCI-H358 cells (Supporting
Information Figure S20). The lower concentrations of the
tested compounds 2−8 (c = 10–5−10–7 mol dm–3) had no
significant influence on the growth of normal and solid
tumor cells.
However, 5-aminosulfonyl uracil derivatives 2−9 were
more efficient to lymphoma and leukemia cells compared
to normal and solid tumor (HeLa, Caco-2, NCI-H358) cell
lines. Compounds 2−8 showed statistically significant
influence at the highest concentrations (c = 10–4 mol dm–3)
on the lymphoma (Raji, HuT78) and leukemia (Jurkat, K562)
cells (Supporting Information Figures S21–S24).
The strongest cytotoxic effect was found for 4-acet-
amidobenzenesulfonamide 4 (71.4 % of growth inhibition).
Also, strong inhibition effects were observed for tosyl-
sulfonamide 2 (64.8 %) and naphthalene-1-sulfonamide 8
(56.4 %). Other tested compounds showed only modest
cytotoxic activity on Raji cells (less than 50 % of inhibition). At
the lower range of concentrations (10–5−10–7 mol dm–3),
tested compounds exhibited insignificant effects on Raji cell
lines growth (Supporting Information Figure S21).
In comparison to effects on Raji cells, tested
compounds demonstrated similar results on HuT78 cells
(Supporting Information Figure S22). Compounds 2−8
applied at 10–4 mol dm–3 showed strong activity on HuT78
cells growth. The most potent effect showed 2-nitro
benzenesulfonamide 6, with 57 % of cell inhibition. Our
results indicate that only dabsyl-sulfonamide 9 applied at
concentration 10–5 mol dm–3 caused inhibition of 21 % of
human T cell lymphoma. Other compounds applied at
105−10–7 mol dm–3 concentration ranges have no or only
weak inhibition effect on the tested cell lines.
Applied at the highest concentration range,
compounds 2−6, and 8 inhibited more than 50 % of the
growth of Jurkat cells (Figure 4, Supporting Information
Figure S23). Also, 2,4,6-triisopropylbenzenesulfonamide 3
applied at c =10–5 mol dm–3, caused a 20 % reduction of
Jurkat cell growth. Interestingly, most of the tested
compounds applied at lower concentration range (10–6 and
10–7 mol dm–3) showed even a slight increase of Jurkat cell
growth.
The most interesting results were obtained for the
K562 cell lines (Figure 5; Supporting Information Figure
S24). Compounds 2, 3, 7, 8, and 9 showed significant
growth inhibitory effects in all applied range of con-
centrations. The most pronounced cytotoxic effect was
observed for 2,4,6-triisopropylbenzenesulfonamide 3 at
10–4 mol dm–3 (39.2 %). Also, at lower concentrations,
significant inhibitory effects was observed for 3 (65.6 %,
c = 105 mol dm–3) and dabsyl-sulfonamide 9 (63−74 %,
c = 105−10–7 mol dm–3). 4-Nitrobenzenesulfonamide 5 and
2-nitrobenzenesulfonamide 6 failed to show any K562
cytotoxicity at the applied concentration range of 10–5−
10–7 mol dm–3.
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275
Figure 5. Cytotoxic effects of 5-aminosulfonyl uracil
derivatives 2−9 on K562 cells growth after 72 h of
incubation in the final concentration range (◼ 10–4, ◼ 10–5,
◼ 10–6 and ◼ 10–7 mol dm–3). Cytotoxicity was analyzed
using the MTT survival assay. Data are presented as the
mean value ± SD of three independent experiments done in
triplicates.
Figure 4. Cytotoxic effects of 5-aminosulfonyl uracil
derivatives 2−9 on Jurkat cells growth after 72 h of
incubation in the final concentration range (◼ 10–4, ◼ 10–5,
◼ 10–6 and ◼ 10–7 mol dm–3). Cytotoxicity was analyzed
using the MTT survival assay. Data are presented as the
mean value ± SD of three independent experiments done in
triplicates.
Table 1. Inhibitory effects of 5-aminosulfonyl uracil derivatives 2−9 and 5-FU on the growth of leukemia, lymphoma, and normal cells.
IC50 (1×10–6 mol dm–3)
Normal cells
MDCK I
>100
Leukemia and lymphoma cells
Comp.
Raji
70.4 ± 0.6
>100
SI
HuT78
>100
SI
K562
>100
SI
Jurkat
80.6 ± 4.2
47.9 ± 4.3
79.9 ± 12.1
78.1 ± 8.2
63.3 ± 17.4
>100
SI
2
3
1.4
1.2
2.1
1.3
1.3
1.6
>100
>100
78.4 ± 28.2
>100
1.3
4
>100
57.2 ± 13.6
>100
1.7
1.1
>100
5
>100
>100
>100
6
>100
>100
86.0 ± 1.4
>100
1.2
>100
7
>100
>100
>100
8
>100
87.1 ± 4.5
>100
>100
>100
77.5 ± 8.7
>100
1.3
9
>100
>100
>100
5FU
55.0± 8.7
>100
>100
9.8±0.5
5.6
76.3± 11.4
0.7
IC50: the concentration that causes 50 % growth inhibition. Data represent the mean IC50 (1 × 10–6 mol dm–3) value of three independent experiments ± SD.
SI = IC50 for normal cell line MDCK I / IC50 for respective tumor cell line.
IC50 and SI values were also calculated for 5-amino-
sulfonyl uracil derivatives 2−9 for all the cell lines and
CONCLUSIONS
compared to those calculated for 5-FU, the common
chemotherapy drug (Supporting Information Table S1). IC50
values calculated for solid tumor (HeLa, Caco-2, NCI-H358)
cell lines along with the normal cell line, MDCK I, were more
than100×10–6 mol dm–3. Although compounds 2−8 showed
a statistically significant influence at the highest applied
concentrations (c = 10–4 mol dm–3) on lymphoma (Raji,
HuT78) and leukemia (Jurkat, K562) cells (Supporting
Information Figures S21–S24) calculated selectivity index
(SI) indicates that the new compounds generally lack
anticancer specificities (Table 1). An exception is Jurkat cells
where greater efficacy against tumor cells compared to
normal cells is observed. The SI value calculated for 5-FU
for Jurkat cell lines was low (0.7), indicating the superiority
of compounds 2-6 and 8 (SI 1.2−2.1) compared to 5-FU.
A small library of 5-aminosulfonyl uracil derivatives 2−9
with different aromatic substituents at C5-NH-SO2- of uracil
moiety was efficiently synthesized (yields 58−98 %). The
products were prepared using equimolar amounts of 5-
aminouracil 1 and selected aromatic sulfonyl chloride in
pyridine at room temperature.
The prepared compounds were tested for the
antiproliferative activity on normal MDCK I cells and tumor
HeLa, Caco-2, NCI-H358, Raji, HuT78, Jurkat, K562 cell lines.
All of the newly synthesized compounds 2−9 showed only
modest growth inhibition activity on normal (MDCK I) and
solid tumor (HeLa, Caco-2, NCI-H358) cells. However, they
were found more active on lymphoma (Raji, HuT78) and
leukemia (Jurkat, K562) cells, showing statistically
significant growth inhibition at the highest concentrations
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H. ISMAILI et al.: An Efficient Synthesis and In vitro Cytostatic Activity of …
(20−60 % growth inhibition at c =10–4 mol dm–3). It should
be noted that due to efficient synthetic protocol the
sulfonyl group substituents of 5-aminosulfonyl uracil
derivatives II could be easily varied providing the
opportunity for diverse structural variations which may
result with significant improvement of biological
properties.
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Acknowledgment. The authors gratefully acknowledge
financial support from the Ministry of Science, Education
and Sport of Croatia (Grants No098-0982914-2935, 219-
0982914-2176), Croatian Science Foundation (Grant HRZZ-
1477), and Faculty of Medicine, University of Osijek
supporting grant to Lj. Glavaš-Obrovac VIF2017-MEFOS-8.
Supplementary Information. Supporting information to the
paper is attached to the electronic version of the article at:
https://doi.org/10.5562/cca3567.
https://doi.org/10.1039/C8OB00253C
PDF files with attached documents are best viewed with Adobe Acrobat
Reader which is free and can be downloaded from Adobe's web site.
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