Study on condensation of N-aryl thioureas with 3-bromo-acetylacetone: Synthesis of aminothiazoles and iminodihydrothiazoles, and their in vitro antiproliferative activity on human cervical cancer cells

Article abstract of DOI:10.1002/jhet.602

The condensation of N-aryl thioureas with 3-bromo-acetylacetone in neutral solvent acetone not only led to 5-acetyl-4-methyl-2-(substituted anilino) thiazoles 3 but also 2-imino-3-(substituted phenyl)-4-methyl-5-acetyl-2,3- dihydrothiazoles 4. Further study found that different reaction solvents displayed an important role toward the ratio of aminothiazoles 3 and iminodihydrothiazoles 4, and the reaction scope was extended. A plausible mechanism involving solvent effect and in situ hydrobromic acid catalyzation was proposed. Some selected isomers exhibited moderate in vitro antiproliferative activity on human cervical cancer cell lines (Hela, Siha).

Full text of DOI:10.1002/jhet.602

September 2011
Study on Condensation of N-Aryl Thioureas with 3-Bromo-
acetylacetone: Synthesis of Aminothiazoles and
Iminodihydrothiazoles, and Their In Vitro Antiproliferative
Activity on Human Cervical Cancer Cells
1061
Hai-Bo Shi,a,b Shi-Jie Zhang,a Yan-Fang Lin,c Wei-Xiao Hu,a,*
and Chao-Ming Caib
aCollege of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032,
People’s Republic of China
bZhejiang Pharmaceutical College, Ningbo 315100, People’s Republic of China
cABON Biopharm (Hangzhou) Co., Ltd., Hangzhou 310018, People’s Republic of China
*E-mail: huyang@mail.hz.zj.cn
Received April 29, 2010
DOI 10.1002/jhet.602
Published online 18 May 2011 in Wiley Online Library (wileyonlinelibrary.com).
The condensation of N-aryl thioureas with 3-bromo-acetylacetone in neutral solvent acetone not only
led to 5-acetyl-4-methyl-2-(substituted anilino) thiazoles 3 but also 2-imino-3-(substituted phenyl)-4-
methyl-5-acetyl-2,3-dihydrothiazoles 4. Further study found that different reaction solvents displayed an
important role toward the ratio of aminothiazoles 3 and iminodihydrothiazoles 4, and the reaction scope
was extended. A plausible mechanism involving solvent effect and in situ hydrobromic acid catalyzation
was proposed. Some selected isomers exhibited moderate in vitro antiproliferative activity on human
cervical cancer cell lines (Hela, Siha).
J. Heterocyclic Chem., 48, 1061 (2011).
INTRODUCTON
[23]. Reaction under DMSO with in situ formed a-halo-
ketones in the presence of hydrochloric acid or hydrobro-
mic acid also afforded both aminothiazoles and iminodi-
hydrothiazoles [24].
Thiazoles are an important class of heterocyclic com-
pounds with biological activities and as versatile synthetic
building blocks [1]. Thiazole derivatives exhibit a wide
range of pharmacological activities such as antibacteria
and fungi [2,3], anti-inflammation [4], antihypertension
[5], anticonvulsion [6], anti-Parkinson’s disease [7], anti-
Alzheimer’s disease [8], anti-HIV [9], and anticancer [10–
14]. The synthesis of thiazole derivatives has drawn con-
siderable attention. The most straight forward procedure
reported by Hantzsch and Weber in 1887 involved a one-
pot condensation of a-haloketones and thiourea deriva-
tives in refluxing alcohol with long reaction time [15].
Modified procedures or novel approaches leading to thia-
zoles have long been explored [16–21]. As far as we
known, Traumann in 1888 first observed an isomer
formed by condensation of chloroacetone with N-methyl-
thiourea, which was 2-imino-3,4-dimethyl-2,3-dihydrothia-
zole [22]. Not until a century later did Meakins and co-
workers give an explanation that the acidic reaction condi-
tions in the Hantzsch thiazole synthesis promoted the for-
mation of iminodihydrothiazoles, while in neutral solvents,
without exception, only aminothiazoles were obtained
We have carefully reexamined our work on a condensa-
tion of substituted arylthioureas with 3-bromo-acetylace-
tone in refluxing acetone [25] and found some desired 5-
acetyl-4-methyl-2-(substituted anilino) thiazoles were not
pure in that trace of 2-imino-3-(substituted phenyl)-4-
methyl-5-acetyl-2,3-dihydrothiazoles were existed as indi-
1
cated from H-NMR spectra. This result encouraged us to
undertake a study on the synthesis of aminothiazoles 3 and
iminodihydrothiazoles 4 in neutral solvents (Scheme 1).
RESULTS AND DISCUSSION
In continuation of our study on the synthesis of thia-
zoles, we found that the reaction of p-nitrophenyl thiourea
1a with 3-bromo-acetylacetone 2 under refluxing acetone
was largely converted to 5-acetyl-4-methyl-2-(4-nitrophe-
nylamino)-thiazole 3a along with a very small amount of
a second product that was identified as 2-imino-3-(4-nitro-
phenyl)-4-methyl-5-acetyl-2,3-dihydrothiazole 4a and
C
V
2011 HeteroCorporation

1062
H.-B. Shi, S.-J. Zhang, Y.-F. Lin, W.-X. Hu, and C.-M. Cai
Vol 48
Scheme 1. Synthetic route to thiazoles 3 and 4.
confirmed by X-ray diffraction [26]. The molecular struc-
ture is shown in Figure 1. In planar thiazole ring, the
In addition, we found that the salts of iminodihydro-
thiazoles 4 were generally less soluble in acetone than
those of aminothiazoles 3, so the salts of 4 could be
obtained conveniently by filtration after cooling the
reaction mixture before neutralization. This method
avoided chromatographic separation to get pure 4, but
the yields still on the lower level than that resulted from
carbon tetrachloride.
˚
S(1)–C(2) [1.756 (2) A], S(1)–C(5) [1.762 (2) A], C(2)–
˚
˚
˚
N(3) [1.404 (2) A], and N(3)–C(4) [1.383 (2) A] bond
lengths corresponded to single bonds, and C(4)–C(5) was
typical for double bond. Also, the C(2)–N(6) [1.265 (2)
˚
A] indicated a double bond.
As we had confirmed the crystal structure of 4a, the
reaction conditions were studied to amplify the ratio of
4a as a major product as shown in Table 1. It could be
found that different solvents significantly affected the ra-
tio of 3a and 4a (entries 3–8) and seemed to have the
trend that the amount of 4a increased in nonpolar sol-
vents and 3a was predominant in polar solvents.
Besides, the reaction temperature did not fundamentally
change the ratio between 3a and 4a neither in acetone
(entries 1–3) nor in carbon tetrachloride (entries 8–10);
however, reflux temperature led to higher isolated yields
and greatly shortened the reaction time. In addition, we
tried to use 3-chloro-acetylacetone in refluxing acetone,
the ratio (3a:4a ¼ 99:1) was almost the same as with 3-
bromo-acetylacetone (entry 3).
The UV absorption spectra of some aminothiazoles 3,
iminodihydrothiazoles 4, and their hydrochlorides in
absolute ethanol at 10ꢀ4M concentration are profiled in
Figure 2. In general, aminothiazoles 3 had longer wave-
lengths of their absorption maxima than that of iminodi-
hydrothiazoles 4, showing compounds 3 possessed stron-
ger aromatic conjugated systems. Moreover, from the
similar wavelengths of absorption maxima between 3
and their hydrochlorides, it could be concluded that the
conjugated systems were not destroyed after forming
hydrochlorides. However, the hydrochlorides of iminodi-
hydrothiazoles 4 strikingly led to blue shifts, indicating
protonation of heterocyclic nitrogen atom with a lone
pair of electrons impaired conjugation and gave rise to
shorter wavelengths of the absorption maxima.
Considering the reaction time and isolated yields, we
extended the reaction scope in refluxing solvents with
different substituted arylthioureas as shown in Table 2.
It could be found that acetone was not an efficient regio-
selective solvent except for p-nitrophenyl thiourea 1a.
For other substituted arylthioureas, it was not obvious
that compounds 3 were favorable in refluxing acetone;
however, compounds 4 seemed to have overwhelming
yields in refluxing carbon tetrachloride.
A plausible mechanism was proposed as illustrated in
Scheme 2. Similar to the Hantzsch thiazole synthesis,
the reactions were proposed to involve a sequence of
three stages from nucleophilic displacement of halogen
Table 1
The effects of solvents and temperature in the synthesis of
3a and 4a.
Ratioa
Temperature
(ꢁC)
Reaction
time (h)
Entry
Solvent
3a
4a
1
2
Acetone
Acetone
Acetone
THF
0
25
22
4
85
84
99
98
85
47
18
12
7
15
16
1
3
4
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
0
1
1
2
5
6
EtOH
1
1
15
53
82
88
93
93
Benzene
CHCl3
CCl4
7
8
1
1
9
10
CCl4
CCl4
24
12
25
7
Figure 1. ORTEP view of iminodihydrothiazole 4a (thermal ellipsoids
at 30% probability).
a The ratio was determined by HPLC.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2011
Study on Condensation of N-Aryl Thioureas with 3-Bromo-acetylacetone
1063
Table 2
Synthesis of thiazoles 3 and 4.
c
c
Yieldb tR
(%) (min)
Yieldb tR
(%) (min)
Entry R1
Solventa
3
4
1
p-NO2 Acetone 3a 90.3
p-EtO Acetone 3b 37.7
o-Cl Acetone 3c 31.4
m-Cl Acetone 3d 44.3
p-Cl Acetone 3e 31.5
3,4-Cl Acetone 3f 45.8 16.44 4f 19.2
o-CH3 Acetone 3g 45.5
p-CH3 Acetone 3h 46.3
7.35 4a
2.0
1.91
1.90
1.64
2.24
1.94
2.04
1.79
2.45
1.99
1.91
1.64
1.94
1.99
2
3
6.50 4b 37.7
5.13 4c 52.6
9.32 4d 35.3
9.05 4e 45.6
4
5
6
7
8
5.41 4g 40.7
9.20 4h 23.9
5.75 4i 17.1
7.35 4a 80.3
5.13 4c 78.6
9.05 4e 76.7
5.75 4i 72.3
9
H
Acetone 3i 41.6
10
11
12
13
p-NO2 CCl4 3a
9.5
4.1
Figure 2. UV comparison of compounds 3, 4, and their
hydrochlorides.
o-Cl
p-Cl
H
CCl4 3c
CCl4 3e 15.5
CCl4 3i 14.5
and no 4c was detected by HPLC with triethylamine. In
addition, from our results discussed above, it could also
be speculated that nonpolar solvents might enhance the
yields of iminodihydrothiazoles 4.
a Under reflux.
b Isolated yield.
c Retention time determined by HPLC.
The in vitro antiproliferative activity of some selected
isomers on human cervical cancer cell lines (Hela and
Siha) was evaluated as shown in Table 3. The antiprolifer-
ative activity of all compounds was weaker than that of
cisplatin (DDP), but some still exhibited moderate anticer-
vical activity. It seemed that isomers with p-substituted
phenyl moieties were less effective against cervical cancer
cells. The selectivity between aminothiazoles 3 and imino-
dihydrothiazoles 4 on cell lines (Hela or Siha) was not
obvious. Further work is being done to illustrate the
detailed structure–activity relationships with more amino-
thiazoles 3 and iminodihydrothiazoles 4.
by sulfur together with nitrogen attacking carbonyl
group, cyclization with proton transfer, and dehydration
[23]. The final ratio of aminothiazoles 3 and iminodihy-
drothiazoles 4 was depended on the equilibrium of 6
and 9, which might probably affected by the nature of
solvents, and in situ released hydrobromic acid might
accelerate the formation of compounds 4 as it was
reported that in neutral medium entirely aminothiazoles
3 were exclusively obtained and only under acidic con-
ditions could compounds 4 be obtained [23]. It was
observed from HPLC detection that the formation of 4
could be greatly inhibited when we added equivalent
pyridine or triethylamine into the reaction in advance as
hydrobromic acid capturers. Our study with o-chloro-
phenyl thiourea 1c as an example showed that the final
ratio of 3c:4c in acetone reached 99.4:0.6 with pyridine,
EXPERIMENTAL
Chemistry. Melting points were taken on an XRC-1 appara-
tus and are uncorrected. Infrared spectra were obtained on a
Scheme 2. Plausible mechanism for thiazoles 3 and 4.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1064
H.-B. Shi, S.-J. Zhang, Y.-F. Lin, W.-X. Hu, and C.-M. Cai
Vol 48
Table 3
262(15), 230(20), 163(76), 117(55), 90(7), 76(30); HRMS
(APCI): calcd for C12H11N3O3SþH 278.0599. Found
Antiproliferative activity of some selected isomers on human cervical
cancer cell lines.
˚
278.0600. Crystal data: Monoclinic, P21/c, a ¼ 11.446 (2) A,
ꢁ
˚
˚
b ¼ 11.1475 (19) A, c ¼ 10.5155 (18) A, b ¼ 103.108 (2) , V
¼ 1306.8 (4) A , Z ¼ 4, Dx ¼ 1.409 mg mꢀ3, Mo Ka radia-
3
˚
Human cervical cancer cell lines
IC50 (lM)
tion, l ¼ 0.26 mmꢀ1, T ¼ 296 (2) K, 8104 measured reflec-
tions, 2957 independent reflections, Rint ¼ 0.020, fine R1
0.038, wR(F2) ¼ 0.110.
¼
Compd.
Hela
Siha
CCDC 653947 contains the supplementary crystallographic
data for 4a. They can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.a-
c.uk/data_request.cif.
3a
4a
3b
4b
3d
4d
3f
>100
>100
>100
>100
>100
76.1
>100
>100
>100
>100
>100
97.3
5-Acetyl-4-methyl-2-(p-ethoxyphenylamino)-thiazole (3b). Pale
yellow crystal, mp: 172–174ꢁC (lit. [27]: 176ꢁC); IR mmax
(KBr)/cmꢀ1: 3273, 3132, 2977, 1609, 1554, 1510, 1484, 1372,
56.5
49.5
37.1
42.0
31.3
66.1
5.53
84.4
29.8
13.3
52.3
21.3
33.4
5.57
1
4f
1326; H-NMR (CDCl3, 500 MHz) d: 7.25 (d, J ¼ 8.5 Hz, 2H,
3g
4g
3i
4i
DDP
Ar-H), 6.93 (d, J ¼ 8.5 Hz, 2H, Ar-H), 4.04 (q, J ¼ 7.0 Hz,
2H, CH3CH2), 2.55 (s, 3H, CH3), 2.41 (s, 3H, CH3CO), 1.43 (t,
J ¼ 7.0 Hz, 3H, CH2CH3); EIMS m/z (%): 276 [Mþ](83),
261(13), 247(100), 233(14), 205(7), 178(4), 150(3), 134(7).
5-Acetyl-4-methyl-2-imino-3-(p-ethoxyphenyl)-2,3-dihydro-
thiazole (4b). Gray white crystal, mp: 113–115ꢁC; IR mmax
(KBr)/cmꢀ1: 3295, 2973, 1583, 1562, 1511, 1359, 1322, 1251;
1H-NMR (CDCl3, 500 MHz) d: 7.15 (d, J ¼ 9.0 Hz, 2H, Ar-
H), 7.03 (d, J ¼ 9.0 Hz, 2H, Ar-H), 4.07 (q, J ¼ 7.0 Hz, 2H,
CH3CH2), 2.34 (s, 3H, CH3), 2.21 (s, 3H, CH3CO), 1.45 (t, J
¼ 7.0 Hz, 3H, CH2CH3); EIMS m/z (%): 276 [Mþ](100),
275(57), 261(7), 247(48), 233(7), 205(5), 162(52), 134(19);
HRMS (APCI): calcd for C14H16N2O2SþH 277.1011. Found
277.1013.
1
Thermo Nicolet Avatar 370 FTIR spectrophotometer. H-NMR
spectra were recorded on a Brucker AC 400 spectrometer
operating at 400 MHz or a Bruker AVANCE III spectrometer
at 500 MHz using TMS as the internal standard. MS spectra
were run on an HP5989B instrument or a Waters GCT Premier
with EI source. HRMS spectra were recorded on Agilent 6210
TOF LC/MS. HPLC were performed on a Shimadzu LC-10AT
Liquid Chromatograph (Diamonsil C18, 250 mm ꢂ 4.6 mm)
with MeOH/H2O (8:2) as an eluent, Tf ¼ 1.0 mL/min, k ¼
254 nm. UV spectra were recorded on a Shimadzu UV-
2400PC spectrophotometer. All the chemicals and solvents
were of analytical reagent and used as received. Arylthioureas
1 and 3-bromo-acetylacetone 2 were prepared according to lit-
erature methods [25].
General procedure for aminothiazoles 3 and iminodihy-
drothiazoles 4 in acetone. To a stirred mixture of arylthiourea
1 (10 mmol) in acetone (40 mL) was added a solution of 3-
bromo-acetylacetone 2 (1.8 g, 10 mmol) in acetone (5 mL),
keeping the reaction temperature below 25ꢁC throughout the
addition. Then, the reaction was kept to reflux temperature for
about 1 h (completion by TLC). After cooling, the mixture
was neutralized with 5% aqueous K2CO3 to pH 9, and the
resulting precipitate was filtered and purified by preparative
layer chromatography, eluting with petroleum ether/ethyl ace-
tate (2:1) to afford 3 and 4, respectively. The isolated yields
are presented in Table 2.
5-Acetyl-4-methyl-2-(p-nitrophenylamino)-thiazole (3a). Yellow
crystal, mp: 208–209ꢁC (lit. [27]: 205ꢁC); IR mmax (KBr)/
cmꢀ1: 3282, 3104, 1611, 1597, 1577, 1478, 1320, 1253; 1H-
NMR (CDCl3, 400 MHz) d: 8.32 (d, J ¼ 9.2 Hz, 2H, Ar-H),
7.57 (d, J ¼ 9.2 Hz, 2H, Ar-H), 2.71 (s, 3H, CH3), 2.55 (s,
3H, CH3CO); EIMS m/z (%): 277 [Mþ](90), 262(100), 247(5),
234(7), 216(20), 86(13), 71(16), 50(7).
5-Acetyl-4-methyl-2-imino-3-(p-nitrophenyl)-2,3-dihydrothia-
zole (4a). Yelow crystal, mp: 168–169ꢁC; IR mmax (KBr)/
cmꢀ1: 3287, 3075, 1607, 1568, 1523, 1350, 1324, 1103; 1H-
NMR (CDCl3, 400 MHz) d: 8.42 (d, J ¼ 8.8 Hz, 2H, Ar-H),
7.51 (d, J ¼ 9.2 Hz, 2H, Ar-H), 2.36 (s, 3H, CH3), 2.25 (s,
3H, CH3CO); EIMS m/z (%): 277 [Mþ](100), 276(100),
5-Acetyl-4-methyl-2-(o-chlorophenylamino)-thiazole (3c). White
crystal, mp: 163–165ꢁC; IR mmax (KBr)/cmꢀ1: 3235, 3000,
1633, 1543, 1480, 1439, 1384, 1314; 1H-NMR (CDCl3, 400
MHz) d: 7.94 (d, J ¼ 8.0 Hz, 1H, Ar-H), 7.44 (d, J ¼ 8.0 Hz,
1H, Ar-H), 7.34 (t, J ¼ 7.8 Hz, 1H, Ar-H), 7.07 (t, J ¼ 7.8
Hz, 1H, Ar-H), 2.64 (s, 3H, CH3), 2.48 (s, 3H, CH3CO);
EIMS m/z (%): 266 [Mþ](73), 251(82), 231(100), 223(10),
189(15), 138(20), 86(15), 71(25).
5-Acetyl-4-methyl-2-imino-3-(o-chlorophenyl)-2,3-dihydro-
thiazole (4c). White crystal, mp: 90–92ꢁC; IR mmax (KBr)/
cmꢀ1: 3252, 1622, 1382, 1476, 1435, 1383, 1363, 1326; 1H-
NMR (CDCl3, 400 MHz) d: 7.62 (d, J ¼ 9.2 Hz, 1H, Ar-H),
7.51–7.46 (m, 2H, Ar-H), 7.37 (d, J ¼ 9.2 Hz, 1H, Ar-H),
2.37 (s, 3H, CH3), 2.20 (s, 3H, CH3CO); EIMS m/z (%): 266
[Mþ](32), 231(99), 189(17), 152(60), 111(17), 75(20); HRMS
(APCI): calcd for C12H11ClN2OSþH 267.0359. Found
267.0365.
5-Acetyl-4-methyl-2-(m-chlorophenylamino)-thiazole (3d). Pale
yellow crystal, mp: 191–193ꢁC; IR mmax (KBr)/cmꢀ1: 3289,
3131, 1613, 1594, 1553, 1490, 1371, 1319; H-NMR (CDCl3,
1
500 MHz) d: 7.41 (s, 1H, Ar-H), 7.33 (t, J ¼ 8.0 Hz, 1H, Ar-
H), 7.24 (d, J ¼ 7.8 Hz, 1H, Ar-H), 7.14 (d, J ¼ 7.5 Hz, 1H,
Ar-H), 2.61 (s, 3H, CH3), 2.48 (s, 3H, CH3CO); EIMS m/z
(%): 266 [Mþ](84), 251(100), 223(10), 182(10), 138(9),
111(10), 86(13), 71(22); HRMS (APCI): calcd for C12H11
ClN2OSþH 267.0359. Found 267.0359.
5-Acetyl-4-methyl-2-imino-3-(m-chlorophenyl)-2,3-dihydro-
thiazole (4d). White crystal, mp: 156–158ꢁC; IR mmax (KBr)/
cmꢀ1: 3330, 3063, 1578, 1472, 1424, 1384, 1358, 1323; 1H-
NMR (CDCl3, 500 MHz) d: 7.52–7.48 (m, 2H, Ar-H), 7.29 (s,
1H, Ar-H), 7.19–7.17 (m, 1H, Ar-H), 2.35 (s, 3H, CH3), 2.23
(s, 3H, CH3CO); EIMS m/z (%): 266 [Mþ](72), 265(100),
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Study on Condensation of N-Aryl Thioureas with 3-Bromo-acetylacetone
1065
251(13), 223(8), 152(64), 111(32), 85(6), 75(16); HRMS
(APCI): calcd for C12H11ClN2OSþH 267.0359. Found
267.0341.
5-Acetyl-4-methyl-2-(p-chlorophenylamino)-thiazole (3e). Gray
white crystal, mp: 195–197ꢁC (lit. [27]: 185ꢁC); IR mmax
(KBr)/cmꢀ1: 3276, 3127, 1608, 1554, 1492, 1373, 1319, 1237;
1H-NMR (CDCl3, 400 MHz) d: 7.37 (d, J ¼ 8.8 Hz, 2H, Ar-
H), 7.32 (d, J ¼ 8.8 Hz, 2H, Ar-H), 2.60 (s, 3H, CH3), 2.47
(s, 3H, CH3CO); EIMS m/z (%): 266 [Mþ](90), 251(100),
223(10), 182(10), 152(20), 138(15), 111(12), 86(17).
1H-NMR (CDCl3, 400 MHz) d: 7.34 (d, J ¼ 8.0 Hz, 2H, Ar-
H), 7.12 (d, J ¼ 8.4 Hz, 2H, Ar-H), 2.42 (s, 3H, CH3), 2.34
(s, 3H, CH3CO), 2.21 (s, 3H, Ar-CH3); EIMS m/z (%): 246
[Mþ](100), 231(21), 203(10), 132(93), 91(63), 65(30); HRMS
(APCI): calcd for C13H14N2OSþH 247.0905. Found 247.0913.
5-Acetyl-4-methyl-2-phenylamino-thiazole (3i). Pale yellow
crystal, mp: 154–155ꢁC (lit. [27]: 147ꢁC); IR mmax (KBr)/
cmꢀ1: 3280, 3203, 3140, 3096, 1617, 1558, 1483, 1332; 1H-
NMR (CDCl3, 400 MHz) d: 7.42 (t, J ¼ 8.0 Hz, 2H, Ar-H),
7.34 (d, J ¼ 8.0 Hz, 2H, Ar-H), 7.19 (t, J ¼ 7.2 Hz, 1H, Ar-
H), 2.60 (s, 3H, CH3), 2.46 (s, 3H, CH3CO); EIMS m/z (%):
232 [Mþ](100), 217(35), 189(47), 148(57), 118(36), 104(77),
77(90), 51(15).
5-Acetyl-4-methyl-2-imino-3-phenyl-2,3-dihydrothiazole (4i). Pale
yellow crystal, mp: 128–129ꢁC (lit. [29]: 128–129ꢁC; IR mmax
(KBr)/cmꢀ1: 3325, 3284, 3054, 1586, 1496, 1327, 1093, 1022;
1H-NMR (CDCl3, 400 MHz) d: 7.56 (t, J ¼ 7.4 Hz, 2H, Ar-
H), 7.50 (t, J ¼ 7.4 Hz, 1H, Ar-H), 7.26 (d, J ¼ 7.2 Hz, 2H,
Ar-H), 2.35 (s, 3H, CH3), 2.21 (s, 3H, CH3CO); EIMS m/z
(%): 232 [Mþ](92), 231(100), 217(12), 189(13), 130(7),
118(94), 91(7), 77(89); HRMS (APCI): calcd for
C12H12N2OSþH 233.0749. Found 233.0760.
5-Acetyl-4-methyl-2-imino-3-(p-chlorophenyl)-2,3-dihydro-
thiazole (4e). Gray white crystal, mp: 121–123ꢁC; IR mmax
(KBr)/cmꢀ1: 3207, 3014, 1652, 1633, 1587, 1489, 1383, 1315;
1H-NMR (CDCl3, 400 MHz) d: 7.53 (d, J ¼ 8.4 Hz, 2H, Ar-H),
7.21 (d, J ¼ 8.4 Hz, 2H, Ar-H), 2.35 (s, 3H, CH3), 2.22 (s, 3H,
CH3CO); EIMS m/z (%): 266 [Mþ](90), 251(20), 152(100),
129(15), 111(57), 97(15), 83(20), 75(30); HRMS (APCI): calcd
for C12H11ClN2OSþH 267.0359. Found 247.0347.
5-Acetyl-4-methyl-2-(3,4-dichlorophenylamino)-thiazole (3f). Pale
yellow crystal, mp: 220–221ꢁC; IR mmax (KBr)/cmꢀ1: 3270,
3070, 1604, 1587, 1546, 1472, 1362, 1318; H-NMR (CDCl3,
1
500 MHz) d: 7.56 (d, J ¼ 2.0 Hz, 1H, Ar-H), 7.47 (d, J ¼ 8.5
Hz, 1H, Ar-H), 7.24 (dd, J1 ¼ 2.0 Hz, J2 ¼ 8.5 Hz, 1H, Ar-
H), 2.65 (s, 3H, CH3), 2.50 (s, 3H, CH3CO); EIMS m/z (%):
300 [Mþ](80), 285(100), 257(12), 218(7), 186(6), 172(7),
86(13), 71(9).
Biological section. In vitro cervical cancer cell antipro-
liferation assay. The human cervical cancer cell lines (Hela
and Siha derived from Shanghai Institutes for Biological Sci-
ence, Chinese Academy of Sciences) were cultivated at 37ꢁC,
5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM,
purchased from Gibco) supplemented with 800 (U/v) penicil-
lin, 0.1% (w/v) streptomycin, and 10% (v/v) fetal bovine se-
rum for 3–5 days. Human cervical cancer cells, treated with
trypsin-EDTA solution, were seeded into 96-well flat bottom
plates at 106 cells per well and incubated in a 5% CO2 incuba-
tor at 37ꢁC for 24 h. Cultures were treated with compounds
prepared in different concentrations. Mitochondrial metabolism
was measured as a marker for cell growth by adding 10 micro-
liters per well MTT (5 mg/mL in medium, Sigma) with 3 h of
incubation at 37ꢁC. Crystals formed were dissolved in 150 lL
of DMSO. The absorbance was determined using a microplate
reader at 490 nm. The absorbance data were converted into a
cell proliferation percentage, compared to DMSO-treated cells,
to determine the IC50s.
5-Acetyl-4-methyl-2-imino-3-(3,4-dichlorophenyl)-2,3-dihydro-
thiazole (4f). Pale yellow crystal, mp: 195–196ꢁC; IR mmax
(KBr)/cmꢀ1: 3306, 3057, 1647, 1604, 1549, 1472, 1305, 1091;
1H-NMR (CDCl3, 500 MHz) d: 7.63 (d, J ¼ 8.5 Hz, 1H, Ar-
H), 7.41 (d, J ¼ 2.0 Hz, 1H, Ar-H), 7.15 (dd, J1 ¼ 2.0 Hz, J2
¼ 8.5 Hz, 1H, Ar-H), 2.35 (s, 3H, CH3), 2.24 (s, 3H,
CH3CO); EIMS m/z (%): 300 [Mþ](88), 299(100), 285(20),
257(10), 186(73), 145(30), 109(15), 75(7); HRMS (APCI):
calcd for C12H10Cl2N2OSþH 300.9969. Found 300.9945.
5-Acetyl-4-methyl-2-(o-methylphenylamino)-thiazole (3g). White
crystal, mp: 153–154ꢁC (lit. [28]: 169–170ꢁC); IR mmax (KBr)/
cmꢀ1: 3165, 3062, 2930, 1598, 1541, 1500, 1431, 1328; 1H-
NMR (CDCl3, 500 MHz) d: 7.49 (d, J ¼ 8.0 Hz, 1H, Ar-H),
7.31–7.26 (m, 2H, Ar-H), 7.21 (d, J ¼ 7.5 Hz, 1H, Ar-H),
2.51 (s, 3H, CH3), 2.40 (s, 3H, CH3CO), 2.33 (s, 3H, Ar-
CH3); EIMS m/z (%): 246 [Mþ](100), 231(57), 213(16),
203(26), 170(27), 118(27), 91(19), 71(22).
5-Acetyl-4-methyl-2-imino-3-(o-methylphenyl)-2,3-dihydro-
thiazole (4g). Yellow crystal, mp: 102–104ꢁC; IR mmax (KBr)/
cmꢀ1: 3613, 3018, 2976, 1627, 1571, 1487, 1359, 1325; 1H-
NMR (CDCl3, 500 MHz) d: 7.46–7.38 (m, 3H, Ar-H), 7.18 (d,
J ¼ 7.6 Hz, 1H, Ar-H), 2.38 (s, 3H, CH3), 2.17 (s, 6H,
CH3CO þ Ar-CH3); EIMS m/z (%): 246 [Mþ](100), 231(75),
203(27), 170(28), 132(68), 118(40), 91(63), 71(59); HRMS
(APCI): calcd for C13H14N2OSþH 247.0905. Found 247.0884.
5-Acetyl-4-methyl-2-(p-methylphenylamino)-thiazole (3h). White
crystal, mp: 190–192ꢁC (lit. [27]: 189ꢁC); IR mmax (KBr)/
cmꢀ1: 3281, 3131, 1610, 1483, 1372, 1323, 1309, 1239; 1H-
NMR (CDCl3, 400 MHz) d: 7.22–7.05 (m, 4H, Ar-H), 2.58 (s,
3H, CH3), 2.44 (s, 3H, CH3CO), 2.36 (s, 3H, Ar-CH3); EIMS
m/z (%): 246 [Mþ](100), 231(96), 203(11), 162(10), 132(13),
118(18), 91(15), 71(10).
Acknowledgments. The authors thank Dr. Hai-Bo Li, Nantong
Center for Disease Control and Prevention, Nantong, People’s
Republic of China, for conducting anticancer evaluation. The
authors are also very grateful to the Natural Science Foundation
of Ningbo City (grant No. 2009A610185) for financial support.
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