Anticancer activities of some arylcarbamoylalkyltriphenylphosphonium chlorides

Article abstract of DOI:10.1007/s00044-007-9050-7

A series of novel arylcarbamoylalkyltriphenylphosphonium chlorides were synthesized. The newly synthesized compounds, [R-(p-C₆H ₄)-NH-CO-R₁P^⊕(C₆H ₄)₃]Cl^θ, R = H (2), CH₃ (4), NO₂ (6), R₁ = -CH₂- (b), CH₃CH< (a), -CH₂CH₂- (c), -CH₂CH₂CH ₂ (d), the analogs of an anticancer drug, were characterized by infrared (IR), ¹H nuclear magnetic resonance (NMR), ¹³C-NMR, ³¹P-NMR, mass spectrometry (MS), thermogravimetry (TG), and conductivity measurements. The anticancer activities of the obtained compounds were measured by MTT. The preliminary results indicated that some compounds showed potent anticancer activities against HCT-8, Bel-7402, A549, and S180.

Full text of DOI:10.1007/s00044-007-9050-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2007) 16:380–391
DOI 10.1007/s00044-007-9050-7
ORIGINAL RESEARCH
Anticancer activities of some
arylcarbamoylalkyltriphenylphosphonium chlorides
Lou Zhaowen Æ Zhou Li Æ Xiao Chunfen Æ Ye Yong Æ
Zeng Fanbo Æ Huang Kaixun
Received: 27 June 2007 / Accepted: 5 November 2007 / Published online: 7 December 2007
¨
Ó Birkhauser Boston 2007
Abstract A series of novel arylcarbamoylalkyltriphenylphosphonium chlorides
were synthesized. The newly synthesized compounds, [R-(p-C6H4)-NH-CO-
R1PÈ(C6H4)3]Clh, R = H (2), CH3 (4), NO2 (6), R1 = –CH2- (b), CH3CH\ (a), –
CH2CH2- (c), –CH2CH2CH2 (d), the analogs of an anticancer drug, were charac-
1
terized by infrared (IR), H nuclear magnetic resonance (NMR), 13C-NMR, 31P-
NMR, mass spectrometry (MS), thermogravimetry (TG), and conductivity mea-
surements. The anticancer activities of the obtained compounds were measured by
MTT. The preliminary results indicated that some compounds showed potent
anticancer activities against HCT-8, Bel-7402, A549, and S180.
Keywords Phosphonium Á Synthesis Á Characterization Á Anticancer activity
Introduction
Phosphonium, as a bactericide, has been used in the overseas market (Fu et al.,
2003), and its anticancer activity has been studied. Since Dubois and Lin (1978)
found that phosphonium bromide showed activity on P-388 of lymphocyte
leukemia, there have been many studies of the synthesis and anticancer activity
of this kind of compounds. The results of these studies have showed that these
compounds demonstrate certain activity against colonic cancer (Patel, et al., 1994;
Lou and Zhang, 2000), ovarian cancer (Manetta et al., 1996), liver cancer (Lou and
L. Zhaowen (&) Á Z. Fanbo Á H. Kaixun
Department of chemistry, Hua Zhong University of Science and Technology, Wuhan, Hubei
430074, China
e-mail: hdlouwen@public.wh.hb.cn
L. Zhaowen Á Z. Li Á X. Chunfen Á Y. Yong
Key Laboratory of Synthesis and Application of Organic Functional Molecules of the Education
Ministry, Hubei University, Wuhan, Hubei 430062, China

Med Chem Res (2007) 16:380–391
381
Shang, 2000), oropharyngeal cancer (Lou and Shang, 2000), human breast cancer
(Delikatny et al., 1996), and so on. The highly negative plasma membrane
potentials characteristic of neoplastic cells were believed to account for the selective
accumulation and toxicity of phosphonium and other cationic lipophilic compounds
against malignant cells (Rideout et al., 1989; Delikatny et al., 2002; Rhys et al.,
1995). In order to further study the anticancer activities, to select useful anticancer
compounds, and to study their anticancer mechanisms, we synthesized 12
phosphonium compounds based on these facts. Firstly, we chose the compounds
containing amino groups as original sources to react with chloroalkyl chlorides, then
the products reacted with triphenyl phosphates to produce the following phospho-
nium compounds: [R-(p-C6H4)-NH-CO-R1PÈ(C6H4)3]Clh, R = H (2), CH3 (4), NO2
(6),R1 = –CH2- (b),CH3CH\ (a),–CH2CH2- (c),–CH2CH2CH2 (d). The antican-
cer activities of the compounds were studied by MTT. The general synthesis route
of the target compounds is shown in Fig. 1.
Results and discussion
Chemistry
1H nuclear magnetic resonance (NMR), 13C NMR, 31P NMR, and infrared (IR)
analyses were used to characterize all the synthesized arylcarbamoylalkyltriphe-
nylphosphonium chlorides. The formation of the product was further confirmed by
mass spectrometry (MS) analyses. Because the target compounds partially
decomposed while measuring the melting point, thermogravimetry analyses were
conducted (Table 1). The conductivity results are given in Table 2, indicating that
all of the products existed as ion in ethanol.
Anticancer activities
The influence of these compounds on the HCT-8, A549, and Bel-7402 cell lines was
studied (Table 3). The preliminary bioassays indicated that 4a and 4d showed
O
H
N
Cl
H
N
R2
R2
R2
Cl
PPh3
NH2
Cl
PPh3
R1
R1
R1
Cl
O
O
1, 3, 5
2, 4, 6
1, 2: R1=H; 3, 4: R1=Me; 5, 6: R1=NO2
a: R2=CH2; b: R2=CH3CH; c: R2=CH2CH2; d: R2=CH2CH2CH2
Fig. 1 Synthesis route for the target compounds

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Med Chem Res (2007) 16:380–391
Table 1 The thermogravimetry
data of target compounds
Compounds Start losing Start melting
T/°C
T/°C
2a
2b
2c
2d
4a
4b
4c
4d
6a
6b
6c
6d
225
250
230
287
228
233
230
281
235
224
244
267
235
253
224
278
240
234
235
252
223
213
234
224
T = 378°C, lost 88%
T = 34°C , lost 96%
T = 312°C, lost 99%
T = 362°C, lost 94%
T = 340°C, lost 96%
T = 392°C, lost 90%
T = 314°C, lost 99%
T = 377°C, lost 96%
T = 395°C, lost 89%
T = 381°C, lost 91%
T = 396°C, lost 78%
T = 395°C, lost 89%
Table 2 The molar
conductivity of the products
dissolved in ethanol
Compounds Concentration Conductivity
(mmolÁL-1
Molar conductivity
(sÁcm2Ámol-1
)
(9102wsÁcm-1
)
)
2a
4.44
4.22
4.35
4.44
4.00
4.04
4.05
4.01
4.00
4.03
4.00
4.00
0.72
16.1
18.8
22.9
21.0
16.9
20.4
21.6
20.3
13.6
15.2
19.1
19.9
2b
0.80
2c
1.00
2d
0.94
4a
0.68
4b
0.83
4c
0.88
4d
0.82
6a
0.55
6b
0.62
6c
0.77
6d
0.80
Ethanol
0.55 ls/cm
promising anticancer activity against A549 and Bel-7402, respectively, while 4b
showed promising anticancer activity against HCT-8, A549, and Bel-7402.
We have reported (Lou and Shang, 2000) that benzyltriphenylphosphonium
chloride displayed activities against BEL-7420, HCT-8, and KB, at a density of 5
lg/mL, with inhibitions of 57.82%, 19.1%, and 62.8%, respectively. Table 3
indicates that compounds 4a, 4b, and 4d showed higher activities than benzyltri-
phenylphosphonium chloride.
The inhibition of these compounds on the S180 cell line was measured by MTT,
with 5-fluorouracil as a reference (Table 4). The results indicated that the
investigated compounds showed certain activities against S180 cell lines at
different concentrations, and that most of these compounds displayed better activity

Med Chem Res (2007) 16:380–391
383
Table 3 Antitumor activities
of triphenylphosphonium
compounds (5 lg/mL,
inhibitory rate)
Compounds
Inhibitory rate (%)
HCT-8
Bel-7420
A549
2a
2b
2c
2d
4a
4b
4c
4d
6a
6b
6c
6d
-35.61
-18.63
-23.95
-20.75
-25.53
63.34
25.36
0.33
5.31
21.94
19.76
17.07
12.73
70.28
20.47
61.82
26.70
27.84
20.62
3.47
3.32
-3.54
73.98
45.04
-4.39
13.14
13.94
-1.19
-6.14
-3.64
-6.19
43.98
14.47
-9.03
-2.50
-11.69
Table 4 The inhibitory rate of compounds on S180 [5-fluorouracil (5-Fu) as reference]
Compound
1 mg/ml
100 lg/ml
10 lg/ml
1 lg/ml
Activity range (lg/ml)
31P NMR
5-Fu
2a
2b
2c
72.77%
58.80
46.20
53.69
61.01
72.75
61.44
46.20
35.91
–
34.92%
88.66
76.95
86.54
87.42
103.55
82.62
81.04
67.21
57.82
139.20
97.71
56.93
28.3%
89.05
74.84
89.51
90.57
83.14
76.78
85.30
62.37
159.51
162.54
80.95
69.89
21.92% [ 100
102.24
97.45
87.27
92.11
77.69
84.17
143.70
43.80
102.55
176.55
127.69
63.82
\ 1
\ 1
10
26.703
21.317
23.289
22.841
26.721
22.113
23.414
22.841
26.443
21.173
23.197
22.608
2d
4a
4b
4c
\ 1
100
\ 1
\ 1
100
10
4d
6a
6b
6c
81.52
53.44
29.10
\\ 1
\ 1
10
6d
than 5-fluorouracil at low concentrations. In addition, the inhibition of 5-fluorouracil
increased with increasing concentration, while the inhibitory rate of the phospho-
nium compounds obviously increased with decreasing concentration, suggesting
that different mechanisms are involved. The mechanism of 5-fluorouracil is
inversion, corresponding to nucleotide and restrained pedantic chest glycoside
synzyme, followed by inhibited synthesis of DNA, so the inhibition of 5-Fu
increased with increasing concentration. On the other hand, the mechanism of action
of the phosphonium compounds is selective accumulation of cationic lipophilic
phosphonium salts (CLPS) in mitochondria neoplastic cells and inhibition of
mitochondrial function. Thus, the excess charge of mitochondria in tumor cells is

384
Med Chem Res (2007) 16:380–391
limited. Consequently, the absorptive ability of tumor cells does not increase with
increasing concentration. Furthermore, compounds 6a–c displayed higher activities
than the other compounds, while compounds 4a–d displayed lower activity.
Compound 6d, however, did not display any obvious activity. Thus, the relationship
between the structure of the compounds and their activity was analyzed. The effect
of compound 6d disappeared because of the butyl group. Compounds 6a, 6b, and 6c
include strong electron-withdrawing groups, and compounds 4a–d include electron-
releasing groups. This clearly shows that the positive charge of the phosphate atom
influenced the activity of the compounds. The 31P NMR results also supported this
explanation. Although the difference in the structures was small, the difference in
the 31P NMR delta values of the compounds was large. Regarding the specific
similar structure of the P atom, the positive charge density was higher, and the P
spectrum displacement delta value was smaller. Therefore, compounds 6a–d,
including electron-withdrawing nitryl groups, displayed the best antitumor activity,
while compounds 4a–d, including electron-releasing groups, displayed the weakest
activity. The order of positive charge density and compound P spectrum
displacement were exactly opposite, but were consistent with the rate of tumor
cell inhibition, in which both the positive charge density of the compound and the
inhibitory rate were higher.
Surface-enhanced Raman spectroscopy
We carried out surface-enhanced Raman spectroscopy (SERS) on the above
compounds. The results, shown in Figs. 2–4, indicate that the phosphonium
compounds and DNA did not display obviously mutual effects. Figure 4 (SERS of
the mixture of DNA and 4d) may be regarded as the superimposition of Figs. 2 and
3. The corresponding Raman displacement and relative strength did not obviously
change, so we can predict that the phosphonium compounds have no toxicity
737
DNA
1325
1655
1467
1028
960
200 400 600 800 1000 1200 1400 1600 1800 2000 2200
cm-1
Fig. 2 SERS of DNA

Med Chem Res (2007) 16:380–391
385
1592
M-2d
1000
1176
1370
1028
1108
851
200
400
600
800 1000 1200 1400 1600 1800 2000 2200
cm-1
Fig. 3 SERS of compound 4d
1325
M-2d-DNA
737
1592
1467
1000
1028
960
200
400
600
800 1000 1200 1400 1600 1800 2000 2200
cm-1
Fig. 4 SERS of a mixture of DNA and compound 4d
towards DNA. These results prove that the antitumor activity of the phosphonium is
mainly concerned with the absorption of tumor cell surface. Fig. 4
Conclusion
In conclusion, arylcarbamoylalkyltriphenylphosphonium chloride derivatives were
synthesized and their anticancer activities were evaluated. Compounds 4a, 4b, and
4d showed better activities than benzyltriphenylphosphonium chloride and most of
these compounds displayed better activity than 5-fluorouracil at low concentration.
The analysis of their anticancer mechanism proved that the antitumor activity of
phosphonium is mainly concerned with absorption at the tumor cell surface. Further
research in this area is in progress in our laboratory.

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Med Chem Res (2007) 16:380–391
Experimental
Chemistry
The solvents and reagents were used as received or were dried prior to use as
needed. Melting points were determined with a Tech X-6 micro melting point
apparatus made in Beijing. Infrared spectra were recorded on a Perkin-Elmer PE-
SPECTRUM ONE apparatus with the t values recorded in wave numbers (cm-1).
Solid compounds were pressed with KBr to form disks. 1H NMR, 13C NMR, and 31
P
NMR spectra were obtained on a Varian Unity INOVA-600 MHz spectrometer
1
(600 MHz, H; 150 MHz, 13C) in CDCl3 with tetramethylsilane as the internal
reference. The mass spectra were obtained on a Finnigen trace mass spectrometer
(70 eV). Thermogravimetry was carried out on a Perkin–Elmer TG Thermal System
TGS2 in a flow of nitrogen gas and a heating rate of 10°C/min from 20°C to 400°C
in a platinum crucible. The conductivities of the products were all measured at the
same condition by dissolving them in ethanol. The molar conductivity was
calculated by the formula k = (jsolu–jsolv)/c.
Synthesis of N-phenyl-2-chloroacetamide (1a)
To a stirred solution of aniline (9.3 ml, 0.10 mol) in acetone (20 ml), 2-chloroacetyl
chloride (4.1 ml, 0.05 mol) at 0–5°C was added dropwise and maintained with an
ice bath. The reaction mixture was stirred at room temperature for an additional four
hours. Then, HCl (40 ml, 10%) was added to the mixture. A white solid was formed
thereafter. The precipitate was separated by filtration and washed successively with
HCl (10%) and water. The product was dried under vacuum. Yield, 96.3%; m.p. 89–
90°C IR (t/cm-1): 3260, 1671.
The method for synthesis of 1b–d was the same as that of 1a. Data for 1b: yield,
93.2%; m.p. 140–142°C. IR (t/cm-1): 3267, 1673. Data for 1c: yield, 94.8%; m.p.
118–120°C. IR (t/cm-1): 3302, 1660. Data for 1d: yield, 93.5%; m.p. 69–71°C . IR
(t/cm-1): 3322, 1669.
Synthesis of phenylcarbamoyl-methyltriphenylphosphonium chloride (2a)
To N-phenyl-2-chloroacetamide (1.57 g, 9.3 mmol) in benzene (20 ml), triphenyl
phosphate (2.44 g, 9.3 mmol) was added and the mixture was refluxed for six hours.
The solvent, when cooled to room temperature, was removed from the crystalline
reaction product by filtration and the crystals were washed successively with
benzene and ethyl acetate to provide 1.5 g of 2a. The crude product was
recrystallized by ethanol. Yield, 37.5%; IR (t/cm-1): 3394, 1679, 1600, 1553, 1439,
1
1333, 1111, 738, 689, 509; H-NMR d: 11.89 (1H, s, NH), 7.84*7.88 (6H, m,
ArH), 7.75*7.78 (3H, m, ArH), 7.60*7.66 (8H, m, ArH), 7.20*7.22 (2H, t, ArH),
7.03*7.05 (1H, t, ArH), 5.26 (2H, d, CH2); 13C-NMR d: 160.60, 160.56, 137.77,

Med Chem Res (2007) 16:380–391
387
134.75, 134.73, 133.85, 133.78, 129.93, 129.84, 128.25, 124.12, 120.01, 118.34,
117; 31P-NMR d: 26.703; MS (ESI) m/z: 396.16 (M+-Cl).
Synthesis of 1-(phenylcarbamoyl)-ethyltriphenylphosphonium chloride (2b)
To N-phenyl-2-chloropropanamide (1.51 g, 8.2 mmol) in xylene (20 ml), triphenyl
phosphate (2.25 g, 8.6 mmol) was added and the mixture was refluxed for 60 hours.
The solvent, when cooled to room temperature, was removed from the crystalline
reaction product by filtration and the crystals were washed with ethyl acetate to
provide 0.89 g of 2b. The crude product was dissolved in ethanol and the solution
was then subjected to column chromatography (using silica gel), eluting succes-
sively with ethyl acetate and ethanol. The elution was analyzed by AgNO3 (5%).
Yield, 24.3%; IR (t/cm-1): 3435, 1678, 1596, 1542, 1495, 1437, 1447, 1109, 749,
1
778, 757, 724, 697, 686; H-NMR d: 12.03 (1H, s, NH), 7.93*7.96 (6H, q, ArH),
7.74*7.76 (3H, t, ArH), 7.62*7.66 (8H, m, ArH), 7.21*7.23 (2H, t, ArH),
7.03*7.05 (1H, t, ArH), 6.59*6.65 (1H, m, CH3), 1.76 (3H, q, CH3); 13C-NMR d:
166.70, 138.37, 135.25, 135.23, 134.87, 134.80, 130.60, 130.52, 129.03, 124.93,
120.90, 118.63, 118.07, 38.12, 37.78, 15.59; 31P-NMR d: 21.317; MS (ESI) m/z:
410.18 (M+-Cl).
Synthesis of 2-(phenylcarbamoyl)-ethyltriphenylphosphonium chloride (2c)
To N-phenyl-3-chloropropanamide (1.56 g, 8.5 mmol) in DMF (15 ml) was added
triphenyl phosphate (2.33 g, 8.9 mmol) and the mixture was refluxed for 70 hours.
The solvent was evaporated under reduced pressure and the solid obtained was
dissolved in ethanol and the solution was then subjected to column chromatography
(using silica gel), eluting successively with ethyl acetate and ethanol. The elution
was analyzed by AgNO3 (5%). Yield, 51.8%; IR (t/cm-1): 3430, 1684, 1595, 1543,
1494, 1441, 1112, 756, 743, 722, 698, 690; 1H-NMR d: 11.31(1H, s, NH),
7.74*7.80 (11H, m, ArH), 7.67*7.70 (6H, m, ArH), 7.21*7.24 (2H, t, ArH),
7.00*7.02 (1H, t, ArH), 3.85 (2H, m, CH2), 3.20 (2H, m, CH2); 13C-NMR d:
168.13, 168.04, 139.12, 135.44, 135.42, 133.85, 133.78, 133.31, 130.76, 130.67,
130.56, 128.50, 123.59, 120.16, 118.05, 117.47, 29.96, 20.28, 19.91; 31P-NMR d:
23.289; MS (ESI) m/z: 410.26 (M+-Cl).
Data for 2d
Yield, 29.4%; IR (t/cm-1): 3422, 1675, 1601, 1542, 1439, 1113, 752, 723, 691; 1H-
NMR (600 MHz, CDCl3, TMS) d: 10.91(1H, s, NH), 7.88*7.89 (3H, d, ArH),
7.68*7.81(14H, m, ArH), 7.24*7.27 (2H, t, ArH), 7.02 (1H, s, ArH), 3.68 (2H, s,
CH2), 3.04 (2H, t, CH2), 2.07 (2H, d, CH3); 13C-NMR d: 171.43, 139.61, 135.66,
133.94, 131.04, 128.98, 123.91, 120.57, 118.80, 118.23, 37.42, 20.08; 31P-NMR d:
22.841; MS (ESI) m/z: 424.25 (M+-Cl).

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Med Chem Res (2007) 16:380–391
Synthesis of N-(p-tolyl)-2-chloroacetamide (3a)
To a stirred solution of p-methylanline (3.42 g, 32 mmol) in toluene (15 ml), a
solution of sodium carbonate (2.76 g, 26 mmol) in water (20 ml) was added. To this
mixture dropwise 2-chloroacetyl chloride (3.72 ml, 38 mmol) was added at 0*5°C
maintained with an ice bath. The reaction mixture was stirred at room temperature
for additional 2.5 h and the precipitate formed was separated by filtration and
washed successively with water and toluene. The product was dried under vacuum.
The yield was 91.2; %. m.p. 164–165.4°C. IR (t/cm-1): 3274, 1669.
The method for the synthesis of 3b–d was the same as that of 3a. Data for 3b:
yield, 89.9%; m.p.: 124–125.5°C. IR (t/cm-1): 3252, 1663. Data for 3c: yield,
85.5%; m.p. 123–124°. IR (t/cm-1): 3294, 1655. Data for 3d: yield, 68.9%; m.p.
89.6–92°C. IR (t/cm-1): 3321, 1661.
Synthesis of 1-(p-tolylcarbamoyl)-methyltriphenylphosphonium chloride (4a)
Triphenyl phosphate (2.64 g, 10 mmol) was added to N-(p-tolyl)-2-chloroacet-
amide (1.84 g, 10 mmol) in xylene (20 ml) and the mixture was refluxed for
48 hours. The solvent, when cooled to room temperature, was removed from the
crystalline reaction product by filtration and the crystals were washed with ethyl
acetate to provide 4.05 g of 4a. The crude product was dissolved in ethanol and the
solution was then subjected to column chromatography (using silica gel), eluting
successively with ethyl acetate and ethanol. The elution was analyzed by AgNO3
(5%). Yield, 90.7%; IR (t/cm-1): 3422, 3031, 1676, 1607, 1542, 1514, 1438, 1111,
1
820, 747, 727, 689; H-NMR d: 11.76 (1H, s, NH), 7.48*7.87 (6H, q, ArH),
7.75*7.77 (3H, t, ArH), 7.63*7.66 (6H, m, ArH), 7.48*7.49 (2H, d, ArH),
7.00*7.02 (2H, d, ArH), 5.24(2H, d, CH2), 2.25 (3H, s, CH3); 13C-NMR d: 160.48,
135.45, 134.79, 133.99, 133.92, 133.72, 130.01, 129.92, 128.85, 120.13, 118.67,
118.08, 33.36, 20.69; 31P-NMR d: 22.113; MS (ESI) m/z: 410.19 (M+-Cl).
Data for 4b
Yield, 35.5%; IR (t/cm-1): 3421, 1677, 1609, 1544, 1514, 1438, 1110, 821, 752,
726, 690; 1H-NMR d: 11.92 (1H, s, NH), 7.93*7.96 (6H, q, ArH), 7.32*7.56 (3H,
t, ArH), 7.64 (6H, s, ArH), 7.50*7.51 (2H, d, ArH), 7.01*7.02 (2H, d, ArH), 6.59
(1H, s, CH), 2.25 (3H, s, CH3), 1.75 (3H, m, CH3); 13C-NMR d:166.39, 135.80,
135.19, 134.85, 134.78, 134.41, 130.56, 130.47, 129.48, 120.80, 118.63, 118.07,
38.03, 21.33, 15.56; 31P-NMR d: 26.721; MS(ESI) m/z: 424.22(M+-Cl).
Data for 4c
Yield, 83.7%; IR (t/cm-1): 3420, 1675, 1605, 1541, 1514, 1438, 1113, 822, 743,
724, 690; 1H-NMR d: 11.15 (1H, s, NH), 7.75*7.78 (9H, q, ArH), 7.63*7.69 (8H,

Med Chem Res (2007) 16:380–391
389
m, ArH), 7.00*7.01 (2H, d, ArH), 3.83 (2H, m, CH2), 3.17 (2H, m, CH2, 2.26 (3H,
s, CH3); 13C-NMR d: 168.18, 168.09, 136.85, 135.71, 134.16, 133.30, 130.98,
129.31, 120.52, 120.41, 118.50, 117.93, 30.36, 21.32,20.06; 31P-NMR d: 23.414;
MS (ESI) m/z: 424.31 (M+-Cl).
Synthesis of 3-(p-tolylcarbamoyl)-propyltriphenylphosphonium chloride (4d)
A mixture of N-(p-tolyl)-4-chlorobutyramide (1.81 g, 8.6 mmol) and triphenyl
phosphate (2.25 g, 8.6 mmol) was heated to 90? for eight hours. To this mixture
ethyl acetate (20 ml) was added and heated to reflux for several minutes. The white
solid formed was separated by filtration from the solution and washed with ethyl
acetate to provide 2.34g of 4d. The crude product was dissolved in ethanol and the
solution was then subjected to column chromatography (using silica gel), eluting
successively with ethyl acetate and ethanol. The elution was analyzed by AgNO3
(5%). Yield, 57.8%; IR (t/cm-1): 3420, 1674, 1602, 1536, 1514, 1438, 1112, 822,
743, 723, 691; 1H-NMR d: 10.75 (1H, s, NH), 7.68*7.81 (17H, m, ArH),
7.05*7.06 (2H, d, ArH), 3.67 (2H, t, CH2), 3.01 (2H, t, CH2), 2.27 (3H, s, CH3),
2.05 (2H, d, CH2); 13C-NMR d: 171.19, 137.03, 135.63, 133.97, 133.32, 131.08,
131.01, 129.47, 120.53, 118.83, 118.26, 37.31, 37.19, 22.29, 21.33, 20.04; 31P-NMR
d: 22.841; MS (ESI) m/z: 438.24 (M+-Cl).
Synthesis of N-(4-nitro-phenyl)-2-chloroacetamide (5a)
To a stirred solution of 4-nitroaniline (1.58 g, 11.4 mmol) in dichloromethane
(50 ml), sodium carbonate (0.96 g, 9.1 mmol) was added. To this mixture a solution
of 2-chloroacetyl chloride (1.2 ml, 15.1 mmol) in dichloromethane (3 ml) was
added dropwise at room temperature. The reaction mixture was stirred at room
temperature for additional four hours. Water (15 ml) was added to the mixture. The
mixture was separated by separation funnel and the organic layer was dried with
anhydrous Na2SO4. The organic solvent was evaporated and yellow crystal
obtained. The crude product was dissolved in minimum ethanol and deposited by
adding water. Yield, 87.2%; m.p. 189–191°C. IR (t/cm-1): 3278, 1687.
Data for 5b: yield, 92.3%; m.p. 139–141°C. IR (t/cm-1): 3278, 1680. Data for
5c: yield, 89.8%; m.p. 172–174°C. IR (t/cm-1): 3351, 1705. Data for 5d: yield,
93.4%; m.p. 102–104°C. IR (t/cm-1): 3356, 1702.
Synthesis of 1-(4-nitro-phenylcarbamoyl)-methyl triphenylphosphonium chloride
(6a)
A mixture of N-(4-nitro-phenyl)-2-chloroacetamide (0.91 g, 4.2 mmol) and
triphenyl phosphate (1.15 g, 4.4 mmol) was heated to 100°C for 11 h. To the
mixture was added ethyl acetate (20 ml). The mixture gave 1.85 g yellow solid that
was separated by filtration. The crude product was recrystallized with ethanol.

390
Med Chem Res (2007) 16:380–391
Yield, 91.5%; IR (t/cm-1): 3422, 1690, 1617, 1596, 1563, 1509, 1334, 1111, 855,
750, 719, 689, 513; 1H-NMR d: 12.63(1H, s, NH), 8.08*8.09 (2H, d, ArH),
7.78*7.87 (11H, m, ArH), 7.65*7.68 (6H, m, ArH), 5.26 (2H, d, CH2); 13C-NMR
d: 162.33, 162.30, 144.35, 143.92, 135.55, 134.41, 134.33, 130.63, 130.55, 124.84,
120.05, 118.62, 118.03, 34.07, 33.70; 31P-NMR d: 21.173; MS (ESI) m/z: 441.20
(M+-Cl).
Data for 6b
Yield, 57.8%; IR (t/cm-1): 3433, 1687, 1619, 1595, 1565, 1509, 1437, 1337, 1111,
1
863, 752, 724, 698; H-NMR d: 12.82 (1H, s, -NH-), 8.06*8.08 (2H, d, ArH),
7.94*7.97 (8H, m, ArH), 7.80*7.83 (3H, t, ArH), 7.69*7.72 (6H, m, ArH), 6.48
(1H, t, CH), 1.82 (3H, q, CH3); 13C-NMR d: 167.34, 167.32, 144.15, 143.62,
135.28, 135.19, 134.42, 134.32, 134.25, 130.45, 130.36, 130.27,124.64, 124.53,
119.98, 119.88, 117.68, 117.11,38.19, 38.13, 37.79, 15.12 ; 31P-NMRd: 26.443; MS
(ESI) m/z: 455.20 (M+-Cl).
Data for 6c
Yield, 91.5%; IR (t/cm-1): 3421, 1701, 1614, 1595, 1563, 1499, 1333, 1111, 856,
743, 724, 691; 1H-NMR d: 12.04 (1H, s, NH), 8.09*8.11 (2H, d, ArH), 8.00*8.01
(2H, d, ArH), 7.69*7.82 (15H, m, ArH), 3.82 (3H, m, CH2), 3.25 (2H, m, CH2);
13C-NMR d: 169.05, 168.97, 145.23, 143.07, 135.63, 135.61, 133.87, 133.80,
130.86, 130.77, 124.65, 119.67, 117.86, 117.28, 30.18, 20.13, 19.78; 31P-NMR d:
23.197; MS (ESI) m/z: 455.24 (M+-Cl).
Data for 6d
Yield, 39.8%; IR (t/cm-1): 3409, 1683, 1612, 1595, 1555, 1507, 1438, 1336, 1112,
1
856, 744, 723, 690; H-NMR d: 11.72 (1H, s, NH), 8.10*8.14 (4H, q, ArH),
7.82*7.83 (3H, q, ArH), 7.72*7.75 (12H, m, ArH), 3.59 (2H, s, CH2), 3.09 (2H, s,
CH2), 2.08 (3H, d, CH2); 13C-NMR d: 171.53, 145.19, 142.39, 135.10, 133.23,
133.16, 130.44, 130.36, 124.28, 119.19, 117.81, 117.23 36.74, 36.62, 21.92, 18.80;
31P-NMR d: 22.608; MS (ESI) m/z: 469.22(M+-Cl).
Biology
Cell growth inhibition assays
Four human cancer cell lines, HCT-8, A549, Bel-7402, and S180 were used in this
study. The cells were added to 96-well plates in 100 lL of growth medium. After
treatment with the drug and supplementation with 10% fetal calf serum, the cells

Med Chem Res (2007) 16:380–391
391
were cultured at 37°C in an atmosphere of 5% CO2. Then, 12 lL of 5 mg/ml MTT
was added to each well. After incubation at 37°C for four hours, the supernatant was
removed, and 100 lL DMSO was added to dissolve the water-insoluble
phosphonium salt. The absorbancies at OD570 nm was measured with an ELISA
micro-plate reader (Bio-Rad, Hercules, CA, USA). Drug resistance was represented
as the percentage of viable cells suspension culture after exposure to idarubicin,
compared to the untreated control group.
Surface-enhanced Raman spectroscopy (SERS)
The sample was added to a capillary vessel with an inner diameter of 1 mm. An Ar+
laser with an output of 514.5 nm was used as a light source. The Raman spectrum
was collected by the scattering disposition at 90°. SERS of DNA and phosphonium
compounds were carried out separately. SERS of a mixture of DNA and
phosphonium compounds was tested in 30 min.
Acknowledgments Hu Juanjuan and Du Guanhua of Institute of Materia Medica, Chinese Academy of
Medical Sciences, are greatly acknowledged for determining the data in Table 2.
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