Synthesis and bioactivity of 2-cyanoacrylates containing a trifluoromethylphenyl moiety

Article abstract of DOI:10.1016/j.jfluchem.2004.10.041

A series of 2-cyanoacrylates containing a trifluoromethylphenyl moiety were synthesized in three steps: ethyl 2-cyano-3,3-dimethylthioacrylate was prepared from ethyl cyanoacetate with carbon disulfide and dimethyl sulfate, then its reaction with 4-trifluoromethylaniline yielded ethyl 2-cyano-3-(4- trifluoromethylphenylamino)-3-methylthioacrylate, and then reacted with an alkylamine. The new structures were verified by elemental analysis, IR, ¹H NMR and mass spectra. In the MTT test, these new compounds were found to possess high antitumor activities against PC3 and A431 cells.

Full text of DOI:10.1016/j.jfluchem.2004.10.041

Journal of Fluorine Chemistry 126 (2005) 87–92
www.elsevier.com/locate/fluor
Synthesis and bioactivity of 2-cyanoacrylates containing
a trifluoromethylphenyl moiety
*
Baoan Song , Song Yang, Huimin Zhong, Linhong Jin, Deyu Hu, Gang Liu
Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education,
Center for Research and Development of Fine Chemicals, Guizhou University, Huaxi District, Guiyang 550025, PR China
Received 1 September 2004; received in revised form 19 October 2004; accepted 23 October 2004
Available online 15 December 2004
Abstract
A series of 2-cyanoacrylates containing a trifluoromethylphenyl moiety were synthesized in three steps: ethyl 2-cyano-3,3-dimethylthioa-
crylate was prepared from ethyl cyanoacetate with carbon disulfide and dimethyl sulfate, then its reaction with 4-trifluoromethylaniline
yielded ethyl 2-cyano-3-(4-trifluoromethylphenylamino)-3-methylthioacrylate, and then reacted with an alkylamine. The new structures were
verified by elemental analysis, IR, 1H NMR and mass spectra. In the MTT test, these new compounds were found to possess high antitumor
activities against PC3 and A431 cells.
# 2004 Elsevier B.V. All rights reserved.
Keywords: Trifluoromethylphenylcyanoacrylate; Synthesis; Antitumor bioactivity
1. Introduction
often possess unexpected biological activity [8–9]. Indeed,
the substitution of a main group by a trifluoromethyl group
in a bioactive compound might be expected to induce great
changes in molecular properties, such as the hydrophobicity,
solubility, special biomimetic effect and electronegativity
[10]. Hence, in a search for new anticancer drug, we thought
that the replacement of the methylthio moiety by 4-
trifluoromethylphenyl-amino group in some 2-cyano-3,3-
dimethylthioacrylate may change the bioactivity. Therefore,
several trifluoromethyl-substituted cyanoacrylates were
synthesized and their anticancer bioactivities were evalu-
ated. The reaction route is shown in Scheme 1.
2-Cyanoacrylates are of considerable importance
because of their versatile biological activity and the
possibility for application in agrochemistry, e.g. herbicides
that disrupt photosynthetic electron transportation at a
common binding domain on the 32 kDa polypeptide of the
photosystem II (PSII) reaction center [1–2]. Among these
cyanoacrylates, ethoxyethyl (Z)-3-(4-chlorophenylmethyla-
mino)-2-cyano-3-isopropyl-acrylate (CPNPE) exhibits the
highest Hill inhibitory activity yet reported [3–7].
On the other hand, fluorinated compounds, in general, are
the focus of much interest in modern medicinal chemistry
and are ideal for use in drug design because of the good
biological activity and low toxicity of molecules containing
the trifluoromethyl moiety plus the ease of substitution of a
conventional phenyl or heteroaromatic group with trifluoro-
methyl. Moreover, trifluoromethyl-containing compounds
2. Results and discussion
Ethyl 2-cyano-3,3-dimethylthioacrylate (1) was prepared
in 56.2% yield from ethyl cyanoacetate, carbon disulfide,
dimethyl sulfate with sodium methoxide as the base. It was
found that the dropwise adding of sodium methoxide was
better than pouring in at one time since, many side products
were observed if the sodium methoxide was added at once.
* Corresponding author. Tel.: +86 851 3620521; fax: +86 851 3622211.
E-mail addresses: songbaoan22@yahoo.com,
gzgdjh@tmail.gzu.edu.cn (B. Song).
0022-1139/$ – see front matter # 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2004.10.041

88
B. Song et al. / Journal of Fluorine Chemistry 126 (2005) 87–92
Scheme 1.
Compound 1 was then reacted with 4-trifluoromethylani-
From the yields given below, it could be seen that the
yields of the title compounds decreased with the order:
4a > 4e > 4f > 4b > 4d > 4c.
line to yield ethyl (E)-2-cyano-3-(4-trifluoromethylpheny-
lamino)-3-methylthioacrylate (2). In this reaction step,
sodium hydride was used as deprotonator for the aryl amine
so that the nucleophilicity of the amine would be enhanced
(no reaction was observed even after refluxing the reaction
mixture for 10 h, if sodium hydride was not used). Through
using the solvent mixture DMF and toluene, the mineral oil
coated on the sodium hydride powder is dissolved in toluene,
so the reaction system could be more homogenized. When
the reaction was completed and the mixture was treated with
water, the organic impurity is dissolved by toluene.
When the amine attacks the double bond to form a
transition state, the orientation of 4-trifluoromethylaniline
and ester carbonyl should be of a cis configuration due to the
presence of hydrogen bond, as shown in Scheme 2. Also, the
configuration of the target compound was kept due to the
loss of a methylthio group, which resulted in an (E)
configuration of the compound and this was confirmed by
the X-ray single crystal analysis of 2.
Compound 2 was treated with the aliphatic amine (3) to
obtain 3-aliphatic amino-2-cyano-3-(4-trifluoromethylphe-
nylamino) acrylate (4). In our experiment, we found the
nucleophilicity of the aliphatic amine attached to the a,b-
unsaturated double bond of S,N-kentene acetals was
reduced. When amine (3) was treated with 2, the reaction
was relatively slow, probably due to the steric hindrance of
the alkyl group, inhibiting the reactivity of nucleophilic
substitution. Therefore, the order of reactivity of aliphatic
amine is probably as follows:
The structures of all of the products were confirmed by 1H
NMR, 13C NMR, elemental analysis, IR and mass spectro-
scopy. From the spectra data of 4, it can be seen that the IR
spectra of 4 exhibited bands at about 3204.7–3287.9 cmꢀ1
,
indicating the presence of NH. The signals at 1627.9–
1660.4 cmꢀ1 were assigned to C=O vibrations. The strong
absorptions C=C appeared at 1531–1548 cmꢀ1. A set of
peaks at 2193.0–2204.6 cmꢀ1 was due to C=N vibrations.
The signals at 1112.9–1165.0(s) were assigned to C–F
1
vibrations. In H NMR data, all phenyl protons showed
multiplet at d, 7.12–7.74. While chemical shifts of CH2 of
ester were at about 4.06 and 4.15 ppm, respectively,
exhibiting doublet. The NH proton of 4 was at 9.52–
11.07 ppm in a singlet probably due to the existence of
hydrogen bonding between ester carbonyl and NH of 4-
trifluoromethylphenylamino group, which leads to its
chemical shift moving to lower field. As for the 13C
NMR data of 4, all of the carbon atoms were identified and
the numbers of protons calculated from the integration curve
were tallied with what was expected from the molecular
formula. The MS spectra revealed that the molecular ion and
fragmentation peaks were in accordance with the proposed
structure of compound 4.
It could be seen from the X-ray single crystal analysis that
compound 2 maintains a planar structure (see Fig. 1). It can
˚
be seen that the bond length of C(1)–C(2) (1.386 A) is longer
˚
˚
than normal C=C (1.34 A), C(3)–O(2) (1.331 A) is shorter
˚
than normal single C–O (1.44 A), C(2)–C(3) (1.453 A) and
˚
NH3 > NH2ꢀCH2Ph > NHðCH3Þ2 > NH2ꢀPr
ꢀ n > NH2ꢀPr ꢀ i > NH2ꢀBu ꢀ n
˚
C(2)–C(6) are shorter than normal C–C (1.54 A), C(3)–O(1)
˚
˚
(1.213A) is longer than normal C=O (1.34 A), C(1)–S(1)
˚
(1.754 A) is shorter than the normal covalent single S–C
˚
bond (1.801–1.825 A), C(1)–N(1) (1.333 A) bond is shorter
˚
˚
than the normal C–N single bond (1.49 A) and C=N is longer
than the normal C=N, which suggest that the electron
density is localized among C(8)–N(1)–C(1)–C(2)–C(3)–
O(1). From the view of Fig. 2 and Table 1, it is known that
the two neighboring molecules are linked by hydrogen
bonds, in the form of N(1)–H(1)ꢁꢁꢁO(1). The donor and
Scheme 2.

B. Song et al. / Journal of Fluorine Chemistry 126 (2005) 87–92
89
Fig. 1. Molecular structure of compound 2.
˚
acceptor distance is N(1)–O(1) 2.644(5) A and the bond
angle is 135.3(7)8. The crystal packing is stabilized by these
extensive hydrogen bonds.
IC50 values for compounds, 4a–f, indicated that PC3 cells
were more sensitive to compound 4 than A431 cells were.
The results presented in Table 2 indicate that these newly
synthesized compounds exhibited promising anticancer
activities against two cancer cells in vitro. The compounds
4e and b have most potent antitumor activity than 4a, c, d
and f do. The bioassay indicated that the nature of the R
group affected anticancer activity. For example, when R is
2.1. Anticancer bioactivity assay
The inhibitive effects of cyanoacrylates, 4a–f, against cell
proliferation of PC3 and A431 cell lines were evaluated. The
Fig. 2. Packing diagram of the unit cell of compound 2.

90
B. Song et al. / Journal of Fluorine Chemistry 126 (2005) 87–92
Table 1
room temperature. The samples were applied in methanol
and the chromatograms were developed in the following
eluting systems: (a) benzene–methanol (10:1) and (b)
ethyl acetate–tetrahydrofuran–methanol (12:3:1). The
spots were visualized with UV light and with iodine
vapor. All solvents were dried, deoxygenated and redis-
tilled before use.
˚
Hydrogen bonds for compound 2 (A and degree (8))
D–HꢁꢁꢁA
N(1)–H(1)ꢁꢁꢁO(1)
0.86
d(D–H)
d(HꢁꢁꢁA)
d(DꢁꢁꢁA)
<(DHA)
1.96
2.644(5)
135.3
Table 2
4.2. Synthesis
Bioassay of cyanoacrylates with a trifluoromethylphenyl moiety against
PC3 and A431 cell lines
4.2.1. Ethyl 2-cyano-3,3-dimethylthioacrylate (I)
Compounds
R
IC50 (mM)
To a mixture of anhydrous ethanol (400 ml), ethyl
cyanoacetate (34.0 g, 0.301 mol) and carbon disulfide
(23.0 g, 0.303 mol) under 20 8C was added dropwise a
solution of sodium methoxide (38.48 g, 0.610 mol) in
anhydrous methanol (360 ml) for 1 h. After adding the
solution, the mixture was stirred for 8 h at room temperature.
Then, dimethyl sulfate was added dropwise during 0.5 h and
the reaction mixture was heated under reflux for 2.5 h. The
solvent was removed under reduced pressure and then water
(40 ml) was added and filtered. The residue was dried and
recrystallized twice from a mixture of n-hexane/diethyl
ether, 1/1 (v/v) to give a white solid (36.7 g): yield, 56.2%;
m.p., 53.5–54.5 8C (lit. ref. [11], m.p., 53–54 8C).
PC3
A431
4a
4b
4c
4d
4e
4f
–H
32.1 ꢂ 2.1
18.2 ꢂ 0.3
23.1 ꢂ 1.1
21.1 ꢂ 2.2
12.1 ꢂ 1.6
42.1 ꢂ 2.1
36.1 ꢂ 1.1
22.1 ꢂ 2.5
32.1 ꢂ 1.7
26.1 ꢂ 2.6
12.1 ꢂ 2.4
39.2 ꢂ 4.3
–Pr-n
–Bu-n
–Pr-i
–CH2Ph
(CH3)2
benzyl, compound 4e has relatively higher activity to PC3
and A431 cells.
3. Conclusion
4.2.2. Ethyl 2-cyano-3-(4-trifluoromethylphenylamino)-3-
(methylthio)acrylate (2)
A series of 3-aliphatic amino-2-cyano-3-(4-trifluoro-
methylphenylamino) acrylate (4) containing a trifluoro-
methylphenyl moiety were synthesized in three steps. Their
structures were verified by spectroscopic method. In the
MTT bioassay, these new compounds were found to possess
high antitumor activities against PC3 and A431 cells. As
far as we know, it is the first report that the substituted
2-cyanoacrylates have anti-cancer bioactivity.
To an oven-dried three-necked 100-ml round-bottom
flask fitted with a magnetic stirring bar and charged with
dry N2 was added intermediates 1 (2.17 g, 0.01 mol),
4-trifluoromethylaniline (1.61 g, 0.01 mol), 60% sodium
hydride (0.80 g, 0.02 mol), DMF (20 ml) and toluene
(20 ml). The resulting mixture was then stirred at 10–
20 8C for 40 h. The extent of the reaction was monitored by
TLC. The mixture was poured into ice water (100 ml)
and separated. The aqueous phase was acidified with 10%
HCl to pH 6–7, and filtered. The residue was dried and
recrystallized from anhydrous ethanol to give a white solid,
yield 63.5%, m.p., 78–79 8C. IR (KBr): 3005.1, 2208.4,
1653.0, 1593.2, 1537.2, 1396.4, 1377.1, 1313.5, 1307.7,
1269.1, 1172.7, 1124.5, 1118.7, 1024.2, 881.4, 810.1, and
4. Experimental
4.1. Instruments
The melting points of the products were determined on
a XT-4 binocular microscope (Beijing Tech Instrument
Co., China) and are not corrected. The IR spectra were
recorded on a Bruker VECTOR22 spectrometer in KBr
1
675.0 cmꢀ1. H NMR (CDCl3): d 11.40(s, 1H, NH), 7.82–
7.69(m, 4H, Ar–H), 4.24(q, 2H, CH2), 2.05(s, 3H, SCH3),
and 1.27(t, 3H, CH3–C). MS m/z: 330, 311, 283, 270, 255,
237, 212, 198, 184, 160, 145, 125, 111, 95, and 75. Anal.
Calc. for C14H13F3N2O2S: C, 50.90; H, 3.97; N, 8.48.
Found: C, 50.73; H, 3.81; N, 8.30.
1
disks. H NMR (solvent CDCl3) and 13C (solvent CDCl3)
NMR spectra were performed on a Varian–Inova 500 MHz
spectrometer at room temperature using TMS as internal
standard. D2O exchange was applied to confirm the
assignment of the signals of NH protons. 19F NMR spectra
were obtained on a Varian EM-360A Spectrometer using
CF3COOH as an external standard, positive for downfield
shift. The mass spectra were taken on an HP5988A
spectrometer. Elemental analysis was performed by an
Elementar Vario-III CHN analyzer. The reagents were all
analytical reagent-grade or chemically pure. The thin layer
chromatography was conducted on GF254 plastic sheets at
4.2.3. General procedure for the preparation of
products 4a–f
A solution of ethyl 2-cyano-3-(4-trifluoromethylpheny-
lamino)-3-methylthio-acrylate (2) (0.75 mmol) in EtOH
(40 ml) was stirred, followed by the addition of amine (3)
(0.82 mmol). The mixture was then heated under reflux for
8–12 h. The extent of the reaction was monitored by TLC.
The solvent was removed under reduced pressure. The

B. Song et al. / Journal of Fluorine Chemistry 126 (2005) 87–92
91
product was purified by column chromatography on a silica
gel (eluent: ethyl acetate/petroleum ether, 2/8, v/v) to give
the title compounds.
1348.2, 1303.8, 1273.0, 1253.7, 1170.7, 1157.2, 1026.1, and
701.1 cmꢀ1. 1H NMR (CDCl3/TMS): d 9.56(s, 1H, NH–Ar),
8.87–8.89(d, J = 7.4 Hz, 1H, NH–C), 7.20–7.67(m, 4H, Ar–
H), 4.04–4.10(q, 2H, OCH2), 3.82–3.87(m, 1H, NCH), and
1.04–1.19(m, 9H, 3 ꢃ CH3). 19F NMR (CDCl3/TFA): d
ꢀ11.4. MS m/z: 341, 332, 322, 312, 296, 282, 273, 264, 254,
237, 226, 210, 199, 187, 172, 161, 145, 134, 125, 111, 102,
93, 83, and 68. Anal. Calc. for C16H18F3N3O2: C, 56.30; H,
5.32; N, 12.31. Found: C, 56.36; H, 5.11; N, 12.39.
4.2.3.1. Ethyl 3-amino-2-cyano-3-(4-trifluoromethylpheny-
lamino)acrylate (4a). White crystal; yield: 72.6%; m.p.,
193.5–194.5 8C. IR (KBr): 3204.7, 2976.1, 2200.7, 1635.6,
1620.2, 1608.6, 1585.4, 1531.4, 1458.1, 1446.6, 1492.2,
1386.8, 1369.4, 1298.0, 1269.1, 1120.6, 1109.0, 1085.9, and
1
779.2 cmꢀ1; H NMR (CDCl3/TMS): d 11.07(s, 1H, NH–
Ar), 9.21(s, 2H, NH2), 7.26–7.74(m, 4H, Ar–H), 4.18–
4.25(q, 2H, –OCH2), and 1.29–1.65(m, 3H, CH3). 13C NMR
(CDCl3/TMS): 169.36, 161.83, 161.26, 138.40, 127.48,
127.44, 127.40, 125.18, 118.82, 60.32, and 14.47. 19F NMR
(CDCl3/TFA): d ꢀ11.9. Anal. Calc. for C13H12F3N3O2: C,
52.18; H, 4.04; N, 14.04. Found: C, 52.13; H, 4.00; N, 13.98.
4.2.3.5. Ethyl 3-benzylamino-2-cyano-3-(4-trifluoromethyl-
phenylamino)acrylate (4e). White solid; yield: 71.5%;
m.p., 139–141 8C. IR (KBr): 3287.9, 3012.8, 2997.3,
2980.0, 2937.5, 2204.6, 1668.4, 1558.5, 1531.4, 1479.4,
1436.9, 1408.0, 1369.4, 1348.2, 1303.8, 1273.0, 1253.7,
1
1186.2, 1165.0, 1157.2, 1026.1, and 781.1 cmꢀ1. H NMR
(CDCl3/TMS): d 9.52(s, 1H, NH–C–Ar), 9.32(s, 1H, NH–C–
Ph), 7.13–7.63(m, 9H, Ar–H), 4.45(d, J = 8.2 Hz, 2H, O–
CH2), 4.07(dd, J = 10.2, 7.1 Hz, 2H, NCH2), and 1.16(t,
J = 7.2 Hz, 3H, CH3). 13C NMR (CDCl3/TMS): 169.14,
161.17, 137.35, 136.90, 134.38, 129.29, 128.96, 128.66,
127.63, 127.45, 125.84, 125.80, 123.96, 117.18, 59.40,
57.34, 46.22, and 14.36. 19F NMR (CDCl3/TFA): d ꢀ11.0.
MS m/z: 389, 370, 358, 344, 328, 316, 299, 299, 288, 276,
264, 251, 235, 223, 210, 187, 172, 155, 142, 128, 106, 91, 77,
and 65. Anal. Calc. for C20H18F3N3O2: C, 61.69; H, 4.66; N,
10.79. Found: C, 61.52; H, 4.52; N, 10.62.
4.2.3.2. Ethyl 2-cyano-3-(4-trifluoromethylphenylamino)-
3-(propylamino)acrylate (4b). White crystal; yield:
58.6%; m.p., 95.5–96.5 8C. IR (KBr): 3213.4, 2995.4,
2970.3, 2200.7, 1651.0, 1587.4, 1537.2, 1444.6, 1427.3,
1384.8, 1369.4, 1294.2, 1274.9, 1244.0, 1226.7, 1188.1,
1165.0, 1107.1, 1089.7, 1002.9, 873.7, 775.3, 734.8, and
1
698.2 cmꢀ1. H NMR (CDCl3/TMS): d 10.70(s, 1H, NH–
Ar), 9.25(s, 1H, NH–C), 7.13–7.62(m, 4H, Ar–H), 4.06–
4.26(q, 2H, O–CH2), 2.86(d, J = 7.3 Hz, 2H, NCH2), 1.54–
1.68(m, 2H, CH2), 1.31–1.35(m, 3H, CH3), and 0.87–
0.91(m, 3H, CH3). 13C NMR (CDCl3/TMS): 169.33, 162.15,
126.71, 122.89, 120.42, 60.37, 47.37, 22.83, 14.44, and
11.11. 19F NMR (CDCl3/TFA): d ꢀ11.3. MS m/z: 341, 326,
313, 298, 287, 273, 254, 237, 226, 210, 199, 187, 174, 161,
145, 135 125, 111, 95, 85, and 69. Anal. Calc. for
C16H18F3N3O2: C, 56.30; H, 5.32; N, 12.31. Found: C,
56.17; H, 5.20; N, 12.30.
4.2.3.6. Ethyl 2-cyano-3-(dimethylamino)-3-(4-trifluoro-
methylphenylamino)acrylate (4f). White solid; yield:
63.1%; m.p., 126–128 8C. IR (KBr): 3263.5, 2987.7,
2927.9, 2193.0, 1662.6, 1614.4, 1537.2, 1431.1, 1388.7,
1332.1, 1265.1, 1161.7, 1118.7, 1066.6, 1060.8,1014.5, and
839.0 cmꢀ1. 1H NMR (CDCl3/TMS): d 9.52(s, 1H, NH–Ar),
7.01–7.62(m, 4H, Ar–H), 4.23–4.18(q, 2H, O–CH2), 2.96(s,
6H, 2CH3), and 1.31(t, J = 7.4 Hz, 3H, CH3–C). 13C NMR
(CDCl3/TMS): 169.17, 164.34, 142.71, 126.88, 126.85,
126.82, 126.58, 120.93, 119.48, 77.32, 77.00, 76.68, 63.43,
60.59, 41.40, and 14.36. 19F NMR (CDCl3/TFA): d ꢀ11.6.
Anal. Calc. for C15H16F3N3O2: C, 55.04; H, 4.93; N, 12.84.
Found: C, 55.06; H, 4.89; N, 12.84.
4.2.3.3. Ethyl 3-butylamino-2-cyano-3-(4-trifluoromethyl-
phenylamino)acrylate (4c). White solid; yield: 45.0%;
m.p., 105–106 8C. IR (KBr): 3304.1, 3053.3, 2975.9,
2200.8, 1627.9, 1589.3, 1548.8, 1473.6, 1438.9, 1417.6,
1375.2, 1294.2, 1112.9, 1085.9, 858.3, 819.7, 655.8, and
1
526.5 cmꢀ1; H NMR (CDCl3/TMS): d 10.70(s, 1H, NH–
Ar), 9.24(s, 1H, NH–C), 7.12–7.62(m, 4H, Ar–H), 4.22(t,
J = 8.1 Hz, 2H, O–CH2), 2.89–3.94(m, 2H, NCH2), and
0.83–1.62(m, 10H, CH2–CH2–CH3+CH3). 13C NMR
(CDCl3/TMS): 169.33, 162.16, 142.37, 126.78, 122.86,
120.51, 60.40, 45.32, 31.45, 19.65, 14.45, and 13.46. 19F
NMR (CDCl3/TFA): d ꢀ11.2. MS m/z: 355, 332, 322, 312,
296, 282, 273, 264, 254, 237, 226, 210, 199, 187, 172, 161,
145, 134, 125, 111, 102, 93, 83, and 68. Anal. Calc. for
C17H20F3N3O2: C, 57.46; H, 5.67; N, 11.82. Found: C,
57.59; H, 5.71; N, 11.74.
4.3. Crystal structure determination
For the determination of structure, using a single crystal,
X-ray intensity data were recorded on a Rigaku Raxis-IV
diffraction meter using graphite monochromated Mo Ka
˚
radiation (l = 0.71073 A). In the range 1.768 ꢄ u ꢄ 25.018,
2739 independent reflections were obtained. Intensities
were corrected for Lorentz and polarization effects and
empirical absorption, and all data were corrected using
SADABS [12] program. The structure was solved by direct
methods SHELXS-97 program [13]. All the non-hydrogen
atoms were refined on F2 anisotropically by the full-matrix
least squares method. The hydrogen atoms were located
from the difference Fourier map, but their positions were not
4.2.3.4. Ethyl 2-cyano-3-isopropylamino-3-(4-trifluoro-
methylphenylamino)acrylate (4d). White solid; yield:
56.1%; m.p., 100.5–102 8C. IR (KBr): 3219.9, 2204.6,
1668.4, 1583.5, 1531.4, 1479.4, 1440.8, 1408.0, 1369.4,

92
B. Song et al. / Journal of Fluorine Chemistry 126 (2005) 87–92
refined. The contributions of these hydrogen atoms were
included in structure–factor calculations. The final least-
squares cycle gave wR = 0.1793, R = 0.1107 for 7835
reflection with I > 2s(I); the weighting scheme, w = 1/
[s2(Fo ) + (0.0940P)2 + 1.0028P], where P = (Fo2 + 2Fc )/
3. The maximum and minimum difference peaks and holes
are 0.346 and ꢀ0.227 e Aꢀ3, respectively, s = 1.034 and
(D/s)max = 0.0100(16). Atomic scattering factors and
anomalous dispersion corrections were taken from Inter-
national Table for X-ray Crystallography [14]. Crystal-
lographic data (excluding structure factors) for the structure
have been deposited to the Cambridge Crystallographic Data
Center as supplementary publication no. CCDC-245102.
L-glutamine. Tissue culture reagents were obtained from
Gibco Co.
2
2
Acknowledgements
The authors wish to thank the National Key Project for
Basic Research (Grant no. 2003CB114404), High-Tech
Research and Development Program of China (Contract no.
2002AA217131, 2002AA649190) and the National Natural
Science Foundation of China (Grant no. 20362004) for
financial support.
4.4. MTT assay against cell proliferation
Reference
All compounds tested were dissolved in DMSO (1–
100 mM solutions) and subsequently diluted in the culture
medium before treatment of the cultured cells. Tested cells
were plated in 96-well plates at a density of 3 ꢃ 103 cells/
well/100 ml of the proper culture medium and treated with
the compounds at concentration of 1–100 mM for 48 h. In
parallel, the cells were treated with 0.1% of DMSO as
control. A MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphe-
nyltetrazolium bromide] assay (Roche Molecular Bio-
chemicals) was performed according to the instructions
provided by Roche. This assay is based on the cellular
cleavage of the tetrazolium salt, MTT, into a formazan that
is insoluble in the cell culture medium and is measured at
550 nm directly in 96-well assay plates. Absorbance is
directly proportional to the number of living cells in
culture. Two types of cells were used in these studies, PC3
(prostate cancer) and A431 (uterus cancer) cell lines
(provided by Cell Bank of Committee on Type Culture
Collection of Chinese Academy of Science) were
cultivated in F-12 medium (for PC3) or RPMI 1640
medium (for A431) supplemented with 10% fetal bovine
serum (provided by TBD & HY Bio. Co.) and 2 mM of
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