Facile synthesis of some condensed 1,3-thiazines and thiazoles under conventional conditions: antitumor activity

Article abstract of DOI:10.1007/s11164-017-2990-8

1,3-Thiazine 3 was obtained from cinnamoyl thiourea derivative 2 as the kinetic control product. Refluxing of 2 with sodium ethoxide afforded pyrimidine derivative 4. Moreover, stirring of 2 with bromine/acetic acid gave thiazole 5 that was condensed with o-phenylene diamine forming benzimidazole 6. Heating of arylthiourea 8 with maleic anhydride or phenacyl chloride afforded thiazole derivatives 9 and 10, respectively. Condensation of compound 10 with o-phenylene diamine gave benzimidazole 11. Reaction of p-amino benzoic acid with chloro acetyl isothiocyanate, acetylacetone and ethylacetoacetate produced imidazole 14, enaminone 15 and crotonate 16 derivatives, respectively. Stirring a mixture of benzoyl isothiocyanate with 15 and/or 16 resulted in pyridine-2-thione 17. The yields of the prepared compounds were 41–93%. The experimental section is simple and easy. The detailed synthesis, spectroscopic data, IC₅₀ and antitumor activity of the synthesized compounds were reported. The cytotoxicity of the newly synthesized products showed that compound 4 is the most active compound towards the cancer cell line at which its reactivity is higher than that of the standard doxorubicin (anticancer reference drug).

Full text of DOI:10.1007/s11164-017-2990-8

Res Chem Intermed
DOI 10.1007/s11164-017-2990-8
Facile synthesis of some condensed 1,3-thiazines
and thiazoles under conventional conditions: antitumor
activity
Reda A. Haggam1,2 Mohamed G. Assy2 Mohamed
•
•
H. Sherif2 Mohamed M. Galahom3
•
Received: 30 October 2016 / Accepted: 23 May 2017
Ó Springer Science+Business Media Dordrecht 2017
Abstract 1,3-Thiazine 3 was obtained from cinnamoyl thiourea derivative 2 as the
kinetic control product. Refluxing of 2 with sodium ethoxide afforded pyrimidine
derivative 4. Moreover, stirring of 2 with bromine/acetic acid gave thiazole 5 that
was condensed with o-phenylene diamine forming benzimidazole 6. Heating of
arylthiourea 8 with maleic anhydride or phenacyl chloride afforded thiazole
derivatives 9 and 10, respectively. Condensation of compound 10 with o-phenylene
diamine gave benzimidazole 11. Reaction of p-amino benzoic acid with chloro
acetyl isothiocyanate, acetylacetone and ethylacetoacetate produced imidazole 14,
enaminone 15 and crotonate 16 derivatives, respectively. Stirring a mixture of
benzoyl isothiocyanate with 15 and/or 16 resulted in pyridine-2-thione 17. The
yields of the prepared compounds were 41–93%. The experimental section is simple
and easy. The detailed synthesis, spectroscopic data, IC50 and antitumor activity of
the synthesized compounds were reported. The cytotoxicity of the newly synthe-
sized products showed that compound 4 is the most active compound towards the
cancer cell line at which its reactivity is higher than that of the standard doxorubicin
(anticancer reference drug).
Keywords 1,3-Thiazines Á Benzothiazoles Á Isothiocyanate Á Imidazoles Á
Pyrimidines and antitumor activity
&
Reda A. Haggam
rhaggan99@hotmail.com
1
Department of Chemistry, Faculty of Science, Islamic University in Almadinah Almonawara,
Almadinah Almonawara, Saudi Arabia
2
3
Department of Chemistry, Faculty of Science, Zagazig University, Zagazig 44511, Egypt
Department of Chemistry, Faculty of Science, Jazan University, Jazan 2097, Saudi Arabia
123

R. A. Haggam et al.
Introduction
Synthetic heterocyclic compounds especially containing heteroatoms N, S, O have
enormous potential primarily as agrochemicals and drugs. 1,3-Thiazines with N–C–
S linkage have promising pharmacological activities which have drawn the attention
of scientists. 1,3-Thiazines and subsequently their derivatives have been used as
antitubercular, antiradiation [1] and antitumor [2] agents. Further, they are used in
various organic synthesis and transformations as reaction intermediates [1, 3].
Moreover, the core moiety of 1,3-thiazines is present in the fused form with a b-
lactam ring in major class of antibiotics like cephalosporins (Fig. 1) [4, 5].
Benzothiazoles are widely found in bioorganic and medicinal chemistry with
applications in drug discovery and are of great scientific interest. Benzothiazole
moities are a part of compounds showing numerous biological activities such as
anticancer [6–9], antihelmintic [10] and antidiabetic [11] activities. Several
benzimidazoles have been reported as antiviral [12], anticoagulant [13] and
anticancer agents [14, 15]. As a result, our research project was directed towards the
synthesis of some new fused azoles and azines of expected antitumor activity. As a
part of our programme, we published a paper on azoles and azines derivatives of
expected biological activity [16].
Experimental
Melting points were measured using an Electro thermal IA 9100 apparatus with
open capillary tubes and are uncorrected. All experiments were carried out using
drying solvents. Thin-layer chromatography (TLC) was performed on a Merck
Silica Gel 60F254 with detection by UV light. Products were purified by
crystallization. The IR spectra (KBr disc) were recorded on a Pye Unicam Sp-3-
1
300 or a Shimadzu FTIR 8101 PC infrared spectrophotometer. The H/(13C) NMR
spectra were recorded at a Varian Mercury VX-300 NMR 300 (75.4) MHz
spectrometer using DMSO-d6 as a solvent. All chemical shifts were expressed on
the d (ppm) scale using TMS as an internal standard reference. The coupling
constant (J) values are given in Hz. Mass spectrometery and analytical data were
obtained from the Microanalysis Center at Cairo University, Giza, Egypt.
(E)-4-(3-cinnamoylthioureido)benzoic acid (2) [21] A mixture of 4-aminobenzoic
acid (1) (24.0 mmol) and cinnamoyl isothiocyanate (24.0 mmol) in dry acetone
Fig. 1 Structure of
cephalosporins
O
H
S
R2
N
H
O
N
R1
COOH
R1 = -CH2COOMe, -Me, -CH2Cl
R2 = -CH2CN, p-(NH2)C6H4, p-(OH)C6H4
123

Facile synthesis of some condensed 1,3-thiazines and…
(50 mL) was stirred at room temperaure for 1 h. The reaction mixture was poured
into water. The formed product was filtered off, washed with water, dried and
crystallized from methanol to give a yellow powder in 82% yield. mp 260–261 °C;
mmax (KBr) 3454, 3163, 3151, 1286 cm-1; dH (300 MHz, DMSO-d6) 12.90 (1H,
D2O exchangeable, s, OH), 11.68 (1H, D2O exchangeable, s, NH), 11.65 (1H, D2O
exchangeable, s, NH), 6.93–7.95 (11H, m, ArH’s ? vinylic protone); m/z (EI,
70 eV) 326(98), 293(15), 179(10), 131(98), 103(65), 77(40); Elem. Anal. Calcd for
C17H14N2O3S (326.37): C, 62.56; H, 4.32; N, 8.58. Found: C, 62.43; H, 4.18; N,
8.42.
4-(4-oxo-6-phenyl-1,3-thiazinan-2-ylideneamino)benzoic acid (3) A mixture of
(E)-4-(3-cinnamoylthioureido) benzoic acid (2) (3.00 mmol) and sodium ethoxide
(3.00 mmol) in ethanol (20 mL) was stirred overnight at room temperature. The
reaction mixture was neutralized by HCl. The formed product was filtered off, dried
and crystallized from ethanol to give a yellow powder in 58% yield. mp
232–233 °C; mmax (KBr) 3720, 3450, 1710, 1648 cm-1; dH (300 MHz, DMSO-d6)
12.89 (1H, br s, COOH), 10.52 (1H, br s, NH), 7.24–8.22 (9H, m, ArH’s), 4.89 (2H,
d, J = 2.4 Hz, CH2), 2.93 (1H, t, J = 2.4 Hz, CH); m/z (EI, 70 eV) 326(20),
147(25), 131(98), 103(70), 91(80), 77(50); Elem. Anal. Calcd for C17H14N2O3S
(326.37): C, 62.56; H, 4.32; N, 8.58. Found: C, 62.33; H, 4.21; N, 8.36.
4-(4-oxo-6-phenyl-2-thioxotetrahydropyrimidin-1(2H)-yl)benzoic acid (4) A mix-
ture of (E)-4-(3-cinnamoylthioureido) benzoic acid (2) (3.00 mmol) and sodium
ethoxide (3.00 mmol) in ethanol (20 mL) was heated under reflux for 3 h. The
reaction mixture was cooled and neutralized by HCl. The formed product was
filtered off, washed with water, dried and crystallized from ethanol to give yellow
crystals in 41% yield. mp 319–320 °C; mmax (KBr) 3429, 3266, 1712, 1240 cm-1
;
dH (300 MHz, DMSO-d6) 12.53 (1H, br, D2O exchangeable, COOH), 11.33 (1H, s,
D2O exchangeable, NH), 7.19–7.90 (9H, m, ArH’s), 4.73 (2H, d, J = 2.4 Hz, CH2),
2.98 (1H, t, J = 2.4 Hz, CH); m/z (EI, 70 eV) 326(15), 131(30), 103(15), 91(98),
80(15), 65(20); Elem. Anal. Calcd for C17H14N2O3S (326.37): C, 62.56; H, 4.32; N,
8.58. Found: C, 62.44; H, 4.16; N, 8.45.
2-cinnamamidobenzo[d]thiazole-6-carboxylic acid (5) A mixture of (E)-4-(3-
cinnamoylthioureido) benzoic acid (2) (3.00 mmol) and bromine (3.00 mmol) in
acetic acid (20 mL) was stirred overnight at room temperature. The reaction mixture
was poured into cold water. The formed product was filtered off, dried and
crystallized from acetic acid to give white crystals in 65% yield. mp[ 360 °C; mmax
(KBr) 3443, 3233, 1710 and 1679 cm-1; dH (300 MHz, DMSO-d6) 12.76 (1H, br,
COOH), 11.06 (1H, s, NH), 6.83–6.87 (2H, dd, J = 14.8 Hz, vinylic protons),
7.44–7.90 (8H, m, ArH’s),; m/z (EI, 70 eV) 324(15), 146(98), 131(98), 103(85),
77(65), 65(30); Elem. Anal. Calcd for C17H12N2O3S (324.35): C, 62.95; H, 3.73; N,
8.64. Found: C, 62.74; H, 3.56; N, 8.48.
N-(6-(1H-benzo[d]imidazol-2-yl)benzo[d]thiazol-2-yl)cinnamamide (6) A mixture
of thiazole carboxylic acid 5 (1.00 mmol) and o-phenylene diamine (1.00 mmol) in
ethanol (10 mL) was heated under reflux for 6 h. The reaction mixture was cooled
123

R. A. Haggam et al.
and poured into ice-cold water. The formed product was filtered off, dried and
crystallized from ethanol to give a green powder in 62% yield. mp 220–221 °C;
mmax (KBr) 3414, 1684 cm-1; dH (300 MHz, DMSO-d6) 10.98 (1H, s, D2O
exchangeable, NH), 8.04 (1H, s, D2O exchangeable, NH), 7.25–7.85 (12H, m,
ArH’s), 6.83–6.87 (2H, dd, J = 14.8 Hz, vinylic protons); m/z (EI, 70 eV) 397(10),
146(30), 131(20), 108(85), 80(98), 64(35); Elem. Anal. Calcd for C23H16N4OS
(396.56): C, 69.68; H, 4.07; N, 14.13. Found: C, 69.57; H, 3.98; N, 14.01.
4-(3-benzoylthioureido)benzoic acid (7) [22] A mixture of 4-aminobenzoic acid
(1) (71.0 mmol) and benzoyl isothiocyanate (71.0 mmol) in dry acetone (50 mL)
was stirred at room temperature for 1 h. The reaction mixture was poured into
water. The formed product was filtered off, washed with water, dried and
crystallized from methanol to give yellow crystals in 73% yield. mp 229–230 °C;
mmax (KBr) 3766, 3373, 3256, 1690, 1592, 1261 cm-1; dH (300 MHz, DMSO-d6)
12.93 (1H, s, COOH), 9.14 (1H, s, NH), 7.25–7.95 (9H, m, ArH’s), 3.48 (1H, s,
NH); m/z (EI, 70 eV) 300(60), 179(25), 137(25), 120(25), 105(98), 77(60); Elem.
Anal. Calcd for C15H12N2O3S (300.33): C, 59.99; H, 4.03; N, 9.33. Found: C, 59.82;
H, 3.85; N, 9.22.
4-thioureidobenzoic acid (8) [23] A mixture of 4-(3-benzoylthioureido) benzoic
acid (7) (10.0 mmol) and 10% sodium hydroxide (10.0 mmol) in water (20 mL)
was heated under reflux for 30 min. The reaction mixture was cooled and
neutralized by HCl. The formed product was filtered off, washed with water, dried
and crystallized from butanol to give a white powder in 93% yield. mp [ 300 °C;
mmax (KBr) 3440, 3339, 3279, 3170, 1684, 1291 cm-1; dH (300 MHz, DMSO-d6)
12.72 (1H, br s, COOH), 9.96 (1H, s, NH), 7.54–7.90 (4H, m, ArH’s), 3.37 (1H, s,
NH2); m/z (EI, 70 eV) 196(10), 179(98), 162(98), 134(75), 120(95), 92(35); Elem.
Anal. Calcd for C8H8N2O2S (196.23): C, 48.97; H, 4.11; N, 14.28. Found: C, 48.84;
H, 4.07; N, 14.24.
4-(5-(carboxymethyl)thiazol-2-ylamino)benzoic acid (9) A mixture of 4-thiourei-
dobenzoic acid (8) (5.00 mmol) and maleic anhydride (5.00 mmol) in dry pyridine
(10 mL) was heated under reflux for 6 h. The reaction mixture was cooled and
poured into ice-cold water. The formed product was filtered off, washed with water,
dried and crystallized from ethanol to give brown crystals in 47% yield. mp
279–280 °C; mmax (KBr) 3323, 3194, 3173, 1690 and 1713 cm-1; dH (300 MHz,
DMSO-d6) 12.72 (1H, s, COOH), 10.52 (1H, s, COOH), 9.19 (1H, s, NH),
7.53–7.92 (5H, m, ArH’s), 1.95 (2H, s, CH2); m/z (EI, 70 eV) 278(15), 241(98),
186(55), 146(65), 105(98), 77(55); Elem. Anal. Calcd for C12H10N2O4S (278.28):
C, 51.79; H, 3.62; N, 10.07. Found: C, 51.65; H, 3.41; N, 10.01.
4-(4-phenylthiazol-2-ylamino) benzoic acid (10) A mixture of 4-thioureidobenzoic
acid (8) (5.00 mmol) and phenacyl chloride (5.00 mmol) in dry pyridine (10 mL)
was heated under reflux for 6 h. The reaction mixture was cooled and poured into
ice-cold water. The formed product was filtered off, washed with water, dried and
crystallized from ethanol to give brown crystals in 78% yield. mp. 249–250 °C;
mmax (KBr) 3395, 3263, 1675, cm-1; dH (300 MHz, DMSO-d6) 12.58 (1H, br s,
123

Facile synthesis of some condensed 1,3-thiazines and…
COOH), 10.67 (1H, s, NH), 7.29–7.92 (10H, m, ArH’s); m/z (EI, 70 eV) 296(98),
134(55), 104(25), 90(25), 77(25), 65(20); Elem. Anal. Calcd for C16H12N2O2S
(296.34): C, 64.85; H, 4.08; N, 9.45. Found: C, 64.67; H, 3.95; N, 9.23.
N-(4-(1H-benzo[d]imidazol-2-yl) phenyl)-4-phenylthiazol-2-amine (11) A mixture
of 4-(4-phenylthiazol-2-ylamino) benzoic acid (10) (1.00 mmol) and o-phenylene
diamine (1.00 mmol) in ethanol (10 mL) was heated under reflux for 6 h. The
reaction mixture was cooled and poured into ice-cold water. The formed product
was filtered off, dried and crystallized from ethanol to give yellow crystals in 66%
yield. mp 170–171 °C; mmax (KBr) 3440, 3315 cm-1; dH (300 MHz, DMSO-d6)
10.72 (1H, s, D2O exchangeable, NH), 7.41–7.93 (14H, m, ArH’s ? SCH =), 4.22
(1H, s, D2O exchangeable, NH); m/z (EI, 70 eV) 368(98), 324(98), 295(25),
279(25), 134(45), 90(30); Elem. Anal. Calcd for C22H16N4S (368.45): C, 71.71; H,
4.38; N, 15.21. Found: C, 71.57; H, 4.31; N, 15.07.
4-(2-chloroacetamido) benzoic acid (12) [24] To a mixture of 4-aminobenzoic
acid (1) (15.0 mmol) and pyridine (15.0 mmol) in benzene (20 mL) chloroacetyl
chloride (15.0 mmol) was added dropwise. The formed product was filtered off,
washed with water, dried and crystallized from ethanol to give a white powder in
77% yield. mp 274–275 °C; mmax (KBr) 3401, 3279, 1688, 1603 cm-1; dH
(300 MHz, DMSO-d6) 12.73 (1H, br, COOH), 10.58 (1H, s, NH), 7.66–7.88 (4H, m,
ArH’s), 4.39 (2H, s, CH2); m/z (EI, 70 eV) 213(35), 164(25), 137(98), 120 (65),
77(20), 65(35); Elem. Anal. Calcd for C9H8ClNO3 (213.62): C, 50.60; H, 3.77; N,
6.56. Found: C, 50.47; H, 3.60; N, 6.41.
4-(2-imino-4-oxothiazolidin-3-yl) benzoic acid (13) A mixture of 4-(2-chloroac-
etamido) benzoic acid (11) (5.00 mmol), ammonium thiocyanate (5.00 mmol) and
sodium ethoxide (5.00 mmol) in ethanol (20 mL) was heated under reflux for 3 h.
The reaction mixture was cooled and neutralized by HCl. The formed product was
filtered off, washed with water, dried and crystallized from ethanol to give a white
powder in 51% yield. mp 288–289 °C; mmax (KBr) 3411, 3346, 1708, 1674 cm-1
;
dH (300 MHz, DMSO-d6) 12.64 (1H, br s, COOH), 10.69 (1H, s, NH), 7.64–7.89
(4H, m, ArH’s), 4.16 (2H, s, CH2); m/z (EI, 70 eV) 236(55), 163(35), 137(90),
120(98), 90(40), 65(70); Elem. Anal. Calcd for C10H8N2O3S (236.25): C, 50.84; H,
3.41; N, 11.86. Found: C, 50.72; H, 3.27; N, 11.76.
4-(4-oxo-2-thioxoimidazolidin-1-yl) benzoic acid (14) A mixture of 4-aminoben-
zoic acid (1) (29.0 mmol) and chloroacetylisothiocyanate (29.0 mmol) in dry
acetone (20 mL) was heated under reflux for 1 h. The reaction mixture was cooled
and poured into cold water. The formed product was filtered off, washed with water,
dried and crystallized from ethanol to give yellow crystals in 48% yield. mp
228–229 °C; mmax (KBr) 3400, 3278, 1689, 1602, 1251, cm-1; dH (300 MHz,
DMSO-d6) 12.75 (1H, br s, COOH), 10.59 (1H, s, NH), 7.66–7.89 (4H, m, ArH’s),
4.26 (2H, s, CH2); m/z (EI, 70 eV) 236(15), 202(15), 162(10), 100(10), 80(20),
56(25); Elem. Anal. Calcd for C10H8N2O3S (236.25): C, 50.84; H, 3.41; N, 11.86.
Found: C, 50.68; H, 3.28; N, 11.63.
123

R. A. Haggam et al.
4-((4-oxopent-2-en-2-yl) amino) benzoic acid (15) [25] A mixture of 4-aminoben-
zoic acid (1) (29.0 mmol), acetylacetone (29.0 mmol) and 3 drops of acetic acid in
ethanol (10 mL) was heated under reflux for 2 h. The reaction mixture was cooled
and poured into ice-cold water. The formed product was filtered off, washed with
water, dried and crystallized from ethanol to give yellow crystals in 55% yield. mp
172–173 °C; mmax (KBr) 3450, 3384, 1696, 1683 cm-1; dH (300 MHz, DMSO-d6)
12.56 (1H, s, COOH), 8.90 (1H, s, NH), 7.22–7.88 (4H, m, ArH’s), 5.27 (1H,
s, = CH), 2.47 (3H, s, CH3), 2.09 (3H, s, CH3); m/z (EI, 70 eV) 219(75), 204(98),
160(65), 132(40), 117(30), 65(70); Elem. Anal. Calcd for C12H13NO3 (219.24): C,
65.74; H, 5.98; N, 6.39. Found: C, 65.55; H, 5.81; N, 6.24.
4-(4-ethoxy-4-oxobut-2-en-2-yl) amino) benzoic acid (16)
A mixture of
4-aminobenzoic acid (1) (29.0 mmol), ethylacetoacetate (29.0 mmol) and 3 drops
of acetic acid in ethanol (10 mL) was heated under reflux for 2 h. The reaction
mixture was cooled and poured into ice-cold water. The formed product was filtered
off, washed with water, dried and crystallized from ethanol to give white crystals in
59% yield. mp 146–148 °C; mmax (KBr) 3465, 3383, 1705 and 1683 cm-1; dH
(300 MHz, DMSO-d6) d 12.10 (1H, s, COOH), 10.74 (1H, s, NH), 6.64–8.04 (4H,
m, ArH’s), 4.79 (1H, s, = CH), 4.14 (2H, q, J = 2.2 Hz, CH2) 2.26 (3H, s, CH3),
1.27 (3H, t, J = 2.2 Hz, CH3); m/z (EI, 70 eV) 249(60), 203(30), 176(40), 162(98),
130(45), 65(50); Elem. Anal. Calcd for C13H15NO4 (249.26): C, 62.64; H, 6.07; N,
5.62. Found: C, 62.49; H, 6.00; N, 5.46.
4-((2-mercapto-6-phenylpyridin-4-yl) amino) benzoic acid (17) A mixture of
compounds 15 or 16 (5.00 mmol), benzoyl isothiocyanate (5.00 mmol) and and few
drops of triethylamine in dry acetone (50 mL) was stirred at room temperature for
30 min. The reaction mixture was poured into water. The formed product was
filtered off, washed with water, dried and crystallized from ethanol/acetic acid (1:3)
to give yellow crystals in 55% and 85% yield, respectively. mp 226–228 °C; mmax
(KBr) 3380, 3257, 1697 cm-1; dH (300 MHz, DMSO-d6) 12.74 (1H, s, D2O
exchangeable, NH), 11.65 (1H, s, D2O exchangeable, NH), 7.51–7.95 (11H, m,
ArH’s); m/z (EI, 70 eV) 323(5), 300(25), 179(20), 137 (20), 105(98), 77(55); Elem.
Anal. Calcd for C18H14N2O2S (322.38): C, 67.06; H, 4.38; N, 8.69. Found: C, 66.91;
H, 4.31; N, 8.55.
Antitumor activity tests
Reagents
Fetal bovine serum (FBS) and L-glutamine, were from Gibco Invitrogen (UK).
RPMI-1640 medium was from Cambrex (NJ, USA). Dimethyl sulfoxide (DMSO),
doxorubicin, penicillin, streptomycin and sulforhodamine B (SRB) were from
Sigma Chemical (Saint Louis, USA).
123

Facile synthesis of some condensed 1,3-thiazines and…
Cell cultures
Human tumor cell line MCF-7 (breast adenocarcinoma) was kindly provided by the
National Cancer Institute (NCI, Cairo, Egypt). They grow as a monolayer and were
routinely maintained in RPMI-1640 medium supplemented with 5% heat-inacti-
vated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin
100 mg/mL), at 37 °C in a humidified atmosphere containing 5% CO2. Exponen-
tially growing cells were obtained by plating 1.5*105 cells/mL for MCF-7 followed
by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of
these cell line was evaluated in all the experiments by exposing untreated control
cells to the maximum concentration (0.5%) of DMSO used in each assay.
Tumor cell growth assay
The effects of compounds 3, 4, 10, 13 and 14 on the in vitro growth of the human
tumor cell line were evaluated according to the procedure adopted by the National
Cancer Institute (NCI, USA) in the ‘In vitro Anticancer Drug Discovery Screen’
which uses the protein-binding dye sulforhodamine B to assess cell growth [26].
Briefly, exponentially, cells growing in 96-well plates were then exposed for 48 h to
five serial concentrations of each compound, starting from a maximum concentra-
tion of 150 lM. Following this exposure period, adherent cells were fixed, washed,
and stained. The bound stain was solubilized and the absorbance was measured at
492 nm in a plate reader (Power wave XS; Bio-Tek Instruments, Wincoski, USA).
For each test compound and cell line, a dose–response curve was obtained and the
growth inhibition of 50% (GI50), and IC50 (minimum inhibitory concentration)
corresponding to the concentration of the compounds that inhibited 50% of the net
cell growth was calculated, as described elsewhere [27]. Doxorubicin was used as a
positive control and tested in the same manner.
Results and discussion
This report presents the synthesis of some novel fused azoles and azines via
isothiocyanates that are versatile reagents and have been used as synthetic
intermediates to prepare biologically active heterocyclic compounds [17, 18].
Initially, the addition of p-aminobenzoic acid (1) to cinnamoyl isothiocyanate [19]
in acetone with stirring at room temperature for 1 h gave cinnamoyl thiourea
derivative 2 (Scheme 1). Base-induced intramolecular ring closing of 2 led to 1,3-
thiazine cyclization 3 as the kinetic control product. On the other hand, refluxing of
2 with sodium ethoxide afforded pyrimidine derivative 4 as a thermodynamic
stable product via the addition of nucleophilic nitrogen to the electrophilic
cinnamoyl carbon. Stirring of cinnamoyl derivative 2 with bromine/acetic acid
resulted in thiazole 5 in 65% yield through oxidative cyclization. Condensation of
the carboxylic function 5 with o-phenylene diamine produced the polycyclic-
bearing benzimidazole ring system 6 in 62% yield (Scheme 1).
123

R. A. Haggam et al.
O
HN
S
HOOC
N
Ph
3
stirring/r.t/ over night
NaOEt/EtOH
58%
H
S
COOH
AcOH/Br2
stirring/r.t/
over night
HOOC
O
S
stirring/r.t/ 1 h
acetone
82%
HOOC
N
H
NH
S
C
N
Ph
N
H
65%
N
Ph
O
O
Ph
NH2
1
2
5
NH2
NaOEt/EtOH
3 h reflux
41%
EtOH
6 h reflux
62%
NH2
S
NH
N
O
S
N
HOOC
N
NH
N
H
Ph
O
Ph
4
6
Scheme 1 Reaction of cinnamoyl thiourea 2 with ethoxide or bromine/acetic acid
The chemical structures of the synthesized compounds were elucidated by
analysis of their spectroscopic data. In the IR spectrum of compound 2, there are
absorption bands at 3454, 3163, 3151 and 1286 cm-1 for OH, 2NH and C=S,
1
respectively. Its H NMR displayed three broad singlets at d = 12.90, 11.68 and
11.65 ppm for COOH and 2NH protons, respectively. In addition, a doublet of
doublet is in between d = 6.93–7.95 ppm for vinylic protons (Scheme 1). The IR
spectrum of compound 3 exhibited absorption bands at 3720, 3450, 1710 and
1
1684 cm-1 for OH, NH, acidic C=O and amidic carbonyl, respectively. The H
NMR spectrum of 3 showed two singlets at d = 12.89 and 10.52 ppm for COOH
and NH protons, respectively. Moreover, there is a doublet at d = 4.89 ppm for
COCH2 and a triplet at d = 2.93 ppm for the CHPh group. (Scheme 1). The IR
spectrum of 4 showed absorption bands at 3429, 3266, 1710, 1682 and 1240 cm-1
1
for OH, NH, 2C=O and C=S, respectively. Its H NMR explained two singlets at
d = 12.53 and 11.33 ppm for COOH and NH protons, respectively. There is a
doublet at d = 4.73 ppm for COCH2 and a triplet at d = 2.98 ppm for the CHPh
proton with coupling constant J = 2.4 Hz. The IR spectrum of 5 showed absorption
bands at 3443, 3233, 1710 and 1679 cm-1 for OH, NH, acidic C=O and amidic
1
carbonyl, respectively, while its H NMR spectrum revealed two sharp singlets at
d = 12.76 and 11.06 ppm for COOH and NH protons, respectively. In addition,
doublet of doublet is in between d = 6.83–6.87 ppm for vinylic protons with
coupling constant J = 14.8 Hz. The structure of compound 6 was elucidated from
its IR spectrum which showed that the acidic carbonyl band disappeared at
1710 cm-1. However, there are absorption bands at 3414 and 1684 cm-1 for 2NH
and amidic C=O, respectively. Its 1H NMR spectrum displayed two broad singlets at
d = 10.98 ppm and d = 8.05 ppm for 2NH protons (Fig. 2).
123

Facile synthesis of some condensed 1,3-thiazines and…
Fig. 2 The 1H NMR spectrum of 6
Fig. 3 The D2O spectrum of 6
123

R. A. Haggam et al.
The disappearance of the two peaks at d = 10.98 and 8.05 ppm in the D2O
spectrum of 6 (Fig. 3) confirmed that the 2NH protons were exchangable with D2O
and this is evidence for the suggested structure.
Moreover, the mass spectrometry of compound 6 is in agreement with its
structure as well as its molecular weight (Fig. 4).
Upon addition of p-aminobenzoic acid (1) to benzoyl isothiocyanate [20] in
acetone with stirring at room temperature for 1 h led to the formation of benzoyl
thiourea 7 in 73% yield which in turn underwent basic hydrolysis by 10% NaOH
producing arylthiourea 8 in 93% yield (Scheme 2). Heating of compound 8 with
maleic anhydride in the presence of pyridine for 6 h produced a cycloaddition
reaction to form thiazole derivative 9 in 47% yield via a thiourea derivative
intermediate. Arylthiourea 8 was reacted with phenacyl chloride in pyridine for 6 h
to afford the thiazole ring 10 in 78% yield. Condensation of compound 10 with o-
phenylene diamine in ethanol gave benzimidazole derivative 11 in 66% yield
(Scheme 2).
The chemical structure of compound 9 was elucidated by analysis of its IR and
1H NMR spectra. Its IR spectrum showed absorption bands at 3323, 3194, 3173,
1690 and 1713 cm-1 for 2OH, NH, and 2C=O, respectively. The 1H NMR spectrum
of 9 gave three singlet signals at d = 12.72, 10.52 and 9.19 ppm for 2COOH and
NH protons, respectively. Moreover, there is another singlet at d = 1.95 ppm for
CH2CO protons. The structure of compound 11 was confirmed by its IR spectrum
which exhibited the disappearance of the acidic carbonyl band at 1710 cm-1. This
indicates that the carboxylic group was condensed with the amino group to form an
Fig. 4 The MS spectrum of 6
123

Facile synthesis of some condensed 1,3-thiazines and…
COOH
stirring/r.t/ 1 h
acetone
73%
S
C
O
C
H
N
H
N
S
C
N
Ph
HOOC
HOOC
Ph
O
NH2
7
1
O
10%NaOH
30 min reflux
93%
O
O
C
S
H
Ph
CH2Cl
O
N
C NH2
pyridine
6 h reflux
78%
pyridine
6 h reflux
47%
8
O
HOOC
NH
N
S
O
O
HOOC
HOOC
NH
NH
S
S
Ph
10
NH2
NH2
NH
N
EtOH
6 h reflux
66%
H
H
N
COOH
COOH
NH
S
N
OH
N
Ph
11
HOOC
NH
S
N
9
Scheme 2 Synthesis of thiazole 9 and benzimidazole derivatives 11
imidazole ring. In addition, there were two bands at 3440 and 3315 cm-1 for the
2NH groups. The 1H NMR spectrum of 11 showed a singlet signal at
d = 10.72 ppm for NH which was exchangable with D2O. There was an inferred
multiplet at d = 7.41–7.93 ppm for the aromatic protons and SCH = group
(Fig. 5).
Acetylation of p-amino benzoic acid (1) with chloroacetyl chloride resulted in
arylchloroacetyl chloride 12 in 77% yield. Reflux of compound 12 with ammonium
thiocyanate in the presence of sodium ethoxide produced thiazole derivative 13 in
51% yield (Scheme 3). Refluxing of 1 with chloro acetyl isothiocyanate in dry
acetone for 1 h gave imidazole derivative 14 in 48% yield. Condensation of
compound 1 with acetylacetone and/or ethylacetoacetate in the presence of ethanol
and acetic acid produced enaminone 15 and crotonate 16 derivatives in 55 and 59%
yields, respectively (Scheme 3).
The IR spectrum of compound 13 showed absorption bands at 3411, 3346, 1674
1
and 1708 cm-1 for OH, NH, amidic and acidic C=O, respectively. Its H NMR
spectrum exhibited three singlet signals at d = 12.64, 10.69 and 4.16 ppm for
COOH, NH and CH2 protons, respectively. The aromatic protons appeared as a
multiplet at d = 7.64–7.89 ppm. The structures of compounds 14 and 15 are in
agreement with their analysis data. The IR spectrum of 16 showed absorption bands
123

R. A. Haggam et al.
Fig. 5 The 1H NMR spectrum of 11
COOH
O
HOOC
NH C CH2Cl
l
O
C
e
H
3
n
C
i
12
C
O
d
i
r
NH
C
O
C
H 2
y
p
C
/
C
NaOEt/EtOH
3 h reflux
e
H3C
CH
OEt
n
%
l
CH
2
E
e
7
C
z
7
t
O
2
n
O
O
e
b
H/
NH4SCN
h
16
COOH
r
A
x
e
C
c
f
5
51%
O
H
l
9
u
%
E
O
t
HOOC
N
H 3
S
NH2
C
O
C
l
C
C
COOH
NH
1
HN
a
H
2
c
H 2
et
13
O
H
C
O
C
o
O
x
n
c
C
e
A
C
/
/
1 h
u
4
H
l
N
f
8
%
O
e
t
%
r
5
H 3
E
r
C
h
e
5
S
f
2
l
u
S
x
O
NH
HOOC
N
H3C
C CH C CH3
O
15
14
Scheme 3 Acetylation and condensation of p-amino benzoic acid 1 with different reagents
1
at 3465, 3383, 1705 and 1683 cm-1 for OH, NH and 2C=O, respectively. In its H
NMR spectrum, there is no signal for the NH2 group but a singlet signal
d = 10.74 ppm for NH. This emphasizes that the reaction occurred at the amino
123

Facile synthesis of some condensed 1,3-thiazines and…
group not the carboxylic one. Moreover, there was another singlet at d = 4.79 ppm
for the =CH proton (Scheme 3).
Stirring a mixture of benzoyl isothiocyanate and enamine 15 and/or crotonate
derivatives 16 in acetone and a few drops of TEA for 30 min, pyridine-2-thione
derivative 17 was obtained in 55 and 85% yields, respectively (Scheme 4).
The IR spectrum of compound 17 showed absorption bands at 3380, 3257 and
1
1697 cm-1 for OH, NH, SH and C=O, respectively. The H NMR spectrum of 17
displayed two broad singlets at d = 12.74 and 11.65 for 2NH protons which were
also exchangable with D2O (Fig. 6). There was a multiplet at d = 7.51–7.95 ppm
for aromatic protons.
Moreover, the mass spectrometry of 17 is in agreement with its structure as well
as with its molecular weight (Fig. 7).
The proposed mechanism for the building of pyridine-2-thione derivative 17
showed the formation of an acyclic thioamide intermediate. Subsequently,
dehydration followed by deacetylation took place (Scheme 5).
Pharmacological studies
Antitumor activity tests
The effect of the newly synthesized compounds 3, 4, 10, 13 and 14 was evaluated on
the in vitro growth of a human tumor cell line, namely breast adenocarcinoma
(MCF-7), after a continuous exposure for 48 h. The results are introduced in
Table 1 and Fig. 8.
The results are given in concentrations that were able to cause 50% of cell growth
inhibition (GI50) after a continuous exposure of 48 h and show mean SEM of
three-independent experiments performed in duplicate.
All the examined compounds were able to inhibit the growth of the tested human
tumor cell line in a dose-dependent manner. The results indicated in Table 1 and
Fig. 8 reveal that compound 4 showed the highest inhibitory effect against the
MCF-7 cell line, and that such activity is higher than the reference doxorubicin.
Meanwhile, compound 14 showed high inhibitory effects against the MCF-7 cell
line, which is less than the corresponding reference doxorubicin. Compound 13
COOH
COOH
COOH
O
C
O
C
Ph
N
C
S
Ph
N
C
S
NH
acetone/TEA
stirring/r.t 30 min
acetone/TEA
NH
O
C
NH
O
C
stirring/r.t 30 min
85%
H3C
C
CH
CH3
H3C
C
CH
16
OEt
%
5
5
15
Ph
N
H
S
17
Scheme 4 Synthesis of pyridine-2-thione derivative 17
123

R. A. Haggam et al.
Fig. 6 1H NMR spectrum of compound 17
Fig. 7 MS spectrum of 17
123

Facile synthesis of some condensed 1,3-thiazines and…
COOH
COOH
COOH
COOH
O
- H2O
Ph
C
N
C
S
NH
C
O
C
N
O
NH
C
O
NH
H
O
H3C
CH
CH3
H2C
H
H3C
Ph
C
C
S
CH3
C
C
C
S
CH3
CH3
15
Ph
HO
NH
Ph
NH
N
H
S
O
O
COOH
COOH
COOH
COOH
N
OH
-AcOH
H
H
N
O
NH
N
O
CH3
CH3
Ph
OH
S
N
H
S
Ph
Ph
Ph
N
H
S
N
H
N
H
S
17
Scheme 5 Suggested mechanism for the building of pyridine-2-thione derivative 17
Table 1 Effect of the obtained
compounds on the growth of
MCF-7 cell line
Compound
MCF-7 GI50 (lg L-1
)
3
20.4 6.8
0.01 0.001
22.22 4.12
30.7 8.60
2.6 1.14
4
10
13
14
Doxorubicin
0.04 0.008
Fig. 8 The inhibitory activities
against the MCF-7 cell line
MCF-7
35
30
25
20
15
10
5
0
3
4
10
compound
13
14 Doxorubicin
showed the lowest inhibitory effect, while the compounds 3, 10 exhibited a
moderate growth inhibitory effect.
The cytotoxic and antitumor activities of compounds 3, 4, 10, 13 and 14 were
tested against MCF-7 cell line. The inhibitory activities were detected by using
different concentrations of the tested compounds (0.10, 1.00 and 10.0 lg) and the
123

R. A. Haggam et al.
Table 2 Evaluation of cytotoxicity of the obtained compounds against MCF-7 cell line
Compound
Viability rate (%)
IC50 (mg/mL)
0.1 lg/mL
1 lg/mL
10 lg/mL
3
72.56 2.44
52.32 4.69
78.26 8.22
78.26 6.29
93.26 4.22
70.12 4.60
56.99 6.50
77.44 4.29
72.21 3.98
90.20 3.66
63.31 4.22
52.29 3.94
70.18 4.68
70.23 5.56
82.09 6.41
20.32 3.55
0.01 0.006
22.50 2.35
30.29 5.44
2.59 0.03
4
10
13
14
MCF-7
100
90
80
70
60
50
40
30
20
10
0
3
4
10
13
14
0.1
1
10
concentraꢀon μg
Fig. 9 The cytotoxic activities against the MCF-7 cell line
viability cells (%) were determined by colorimetric method. Also, the IC50 was
calculated (Table 2; Fig. 9).
The results presented in Table 2 and Fig. 9 reveal that compounds 4 and 14 have
very strong cytotoxic antitumor activity, compounds 3 and 10 have strong cytotoxic
antitumor activity and compound 13 has moderate cytotoxic antitumor activity
against the MCF-7 cell line.
Conclusion
In this study, we succeeded in synthesizing some novel condensed azoles and azines
via the addition of p-aminobenzoic acid to a heteroallene such as isothiocyanate.
Some of the compounds exhibited very good antitumor activity in the MCF-7 cell
line. The cytotoxicity of compound 4 is the most active one towards the cancer cell
line where its reactivity is more than that of the standard doxorubicin (anticancer
reference drug). The obtained results are considered as a promotive force to
encourage us for further work on such a ring system.
123

Facile synthesis of some condensed 1,3-thiazines and…
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