Cytotoxic activity and DNA binding of naphthalimide derivatives with amino acid and dichloroacetamide functionalizations

Article abstract of DOI:10.1016/j.cclet.2014.04.020

A series of novel naphthalimide derivatives modified by amino acids and their dichloroacetamide derivatives at the 3-position have been synthesized. Their cytotoxic activities were preliminarily evaluated against Hela, A549 and K562 cells, which showed that the length of the side chains of the amino acids influenced the cytotoxic activities. Moreover, compound 7d showed a very good cytotoxic activity against A549 cells with an IC₅₀ value of 4.78 μmol L^-1. Furthermore, the UV-vis, fluorescence, and circular dichroism (CD) spectroscopies and thermal denaturation experiment indicated that compounds 6a, 6d and 7a, 7d, as DNA intercalators, exhibited binding affinities with calf-thymus DNA (Ct-DNA).

Full text of DOI:10.1016/j.cclet.2014.04.020

G Model
CCLET 2964 1–7
Chinese Chemical Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
1
2
Original article
3
4
Cytotoxic activity and DNA binding of naphthalimide derivatives with
amino acid and dichloroacetamide functionalizations
a
c
a
c
*
5
6
Q1 Ke-Rang Wang a,b, , Feng Qian , Xiao-Man Wang , Guan-Hai Tan , Rui-Xue Rong ,
Zhi-Ran Cao c, Hua Chen a,b, Ping-Zhu Zhang a,b, Xiao-Liu Li a,b,
*
7
8
9
a Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China
b Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002, China
c Department of Immunology, School of Basic Medical Science, Hebei University, Baoding 071002, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A series of novel naphthalimide derivatives modified by amino acids and their dichloroacetamide
derivatives at the 3-position have been synthesized. Their cytotoxic activities were preliminarily
evaluated against Hela, A549 and K562 cells, which showed that the length of the side chains of the
amino acids influenced the cytotoxic activities. Moreover, compound 7d showed a very good cytotoxic
Received 5 March 2014
Received in revised form 7 April 2014
Accepted 9 April 2014
Available online xxx
activity against A549 cells with an IC50 value of 4.78
m
mol LÀ1. Furthermore, the UV–vis, fluorescence,
and circular dichroism (CD) spectroscopies and thermal denaturation experiment indicated that
compounds 6a, 6d and 7a, 7d, as DNA intercalators, exhibited binding affinities with calf-thymus DNA
(Ct-DNA).
Keywords:
Naphthalimide
Dichloroacetamide
Cytotoxic activity
DNA binding
ß 2014 Ke-Rang Wang and Xiao-Liu Li. Published by Elsevier B.V. on behalf of Chinese Chemical
Society. All rights reserved.
10
11
1. Introduction
On the other hand, sodium dichloroacetate (DCA), as a 29
targeting agent of the tumor metabolic process, has reached 30
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Naphthalimide derivatives, known to be DNA intercalators,
have been widely investigated as anticancer agents for many years,
and some compounds (Fig. 1) have reached clinical trials
(Mitonafide, Amonafide, UNBS5162, Elinafide and Bisnafide) [1–
3]. Amonafide (Fig. 1) as the first compound reaching the clinical
trial stage exhibited potent anticancer activity against solid tumors
[1–3]. However, due to unexpected central neurotoxicity, hema-
totoxicity and limited efficiency, the further application of
Amonafide in the clinical trial was limited [1–3]. In order to
improve its selectivity, efficiency, and safety, a broad variety of
novel naphthalimide derivatives have been developed [1–8]. For
example, UNBS5162, as a novel anticancer agent in a Phase I trial,
showed potent anticancer activity and lower toxic side effects,
which decreased CXCL chemokine expression in advanced solid
tumors or lymphoma [4,9]. Moreover, the synthesis of novel
naphthalimide derivatives as potent anticancer agents is of
considerable interest.
Phase II trials [10–13]. Recently, the multifunctional drugs with 31
DCA functionalization attracted special interest for their en- 32
hanced anticancer and lower toxic side effects [14]. For example, 33
the multifunctional, hybrid, platinum (IV) prodrug with DCA 34
functionalization [15–17], targeted to the metabolic process of 35
the mitochondria and the DNA cross-linking process, showed 36
much higher cytotoxicity toward the cancer cells, and paved a 37
pathway for developing selective anticancer agents. In the 38
interim, Cheng and coworkers reported a series of N-phenyl 39
dichloroacetamide derivatives [18–20], which exhibited potent 40
anticancer activity to cancer cells, and have valuable potential for 41
drug development.
42
Inspired by the above results, novel naphthalimide derivatives, 43
modified by dichloroacetamide conjugated with amino acid of 44
different lengths (Scheme 1), were designed and synthesized in 45
this paper. Their cytotoxic activities against human lung 46
adenocarcinoma epithelial cells (A549), henrietta lacks strain of 47
cancer cells (Hela) and human myelogenous leukemia cells (K562) 48
were evaluated. Furthermore, their binding properties with calf- 49
thymus DNA (Ct-DNA) were studied by UV–vis, fluorescence and 50
circular dichroism spectroscopies and thermal denaturation 51
*
Corresponding authors at: Key Laboratory of Chemical Biology of Hebei
Q2
Province, College of Chemistry and Environmental Science, Hebei University,
Baoding 071002, China.
experiment.
52
E-mail addresses: kerangwang@hbu.edu.cn (K.-R. Wang), lixl@hbu.cn (X.-L. Li).
http://dx.doi.org/10.1016/j.cclet.2014.04.020
1001-8417/ß 2014 Ke-Rang Wang and Xiao-Liu Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Please cite this article in press as: K.-R. Wang, et al., Cytotoxic activity and DNA binding of naphthalimide derivatives with amino acid
and dichloroacetamide functionalizations, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.020

G Model
CCLET 2964 1–7
2
K.-R. Wang et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
Fig. 1. The structures of naphthalimide derivatives in clinical trials.
53
54
2. Experimental
determined by its absorption intensity at 260 nm with a known
molar absorption coefficient value of 6600 molÀ1 L cmÀ1
73
74
.
2.1. Instruments
2.3. Cytotoxicity analysis
75
55
56
57
58
59
60
61
Melting points were measured with a SGW X-4 micro-melting
point apparatus and were uncorrected. The 1H NMR, 13C NMR
spectra were recorded with RT-NMR Bruker AVANCE 600 NMR
spectrometers. Chemical shifts were reported in ppm with TMS as
the internal standard. High-resolution mass spectra (HRMS) were
recorded with a Bruker Apex Ultra FTMS in the electrospray
ionization (ESI) mode.
The compounds 6a–d and 7a–d were dissolved in phosphate
buffered saline(PBS) or PBS buffer containing10% DMSO, and diluted
to the required concentration with culture medium. The cytotoxicity
was evaluated by MTT assay. Briefly, cells were plated in 96-well
microassay culture plates (104 cells per well) and grown overnight at
37 8C in a 5% CO2 incubator. The compounds 6a–d and 7a–d were
then added to the wells to achieve final concentrations ranging from
10À7 to 10À4 mol LÀ1. The content of DMSO in the final solution of
compounds 7a–d is less than 0.1%. Wells containing culture medium
without cells were used as control blanks; wells containing culture
medium and cisplatin were used as positive control. The plates were
incubated at 37 8C ina 5% CO2 incubator for 48 h. Upon completion of
the incubation, stock 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
62
2.2. DNA-binding studies
63
64
65
66
67
68
69
70
71
72
The UV–vis spectra and thermal denaturation studies were
recorded on a Shimadzu UV-3600 spectrophotometer equipped
with S-1700 temperature controller and Tm analysis soft. The
fluorescent spectra were measured on a Hitachi F-7000 lumines-
cence spectrophotometer. CD spectra were recorded on a Bio-Logic
MOS-450/AF-CD. Ct-DNA was purchased from the Sigma–Aldrich
tetrazolium bromide (MTT) (Sigma) dye solution (20
mL, 5 mg/mL)
was added to each well. After 4 h incubation, 2-propanol (100
mL)
Company. Solution of Ct-DNA in phosphate buffer (10 mmol LÀ1
,
was added to solubilize the MTT formazan. The optical density of
each well was then measured on a microplate spectrophotometer at
a wavelength of 570 nm. The IC50 value was determined from the
plot of % viability against dose of complexes added.
pH 7.4) containing 50 mmol LÀ1 NaCl gave a ratio of UV absorbance
at 260 and 280 nm of 1.8–1.9:1, indicating that the DNA was
sufficiently free from protein. The concentration of Ct-DNA was
Scheme 1. Reagents and conditions: (a) H2SO4, HNO3, 5–20 8C, 82%; (b) N,N-dimethylethylenediamine, EtOH, reflux, 82%; (c) 10% Pd/C, H2, EtOH, 86%; (d) HATU, DIPEA, DMF;
(e) CF3COOH, CH2Cl2; (f) dichloroacetyl chloride, TEA, CH2Cl2.
Please cite this article in press as: K.-R. Wang, et al., Cytotoxic activity and DNA binding of naphthalimide derivatives with amino acid
and dichloroacetamide functionalizations, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.020

G Model
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K.-R. Wang et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
3
95
2.4. Synthesis
solvent was removed in vacuo. The yields of 6a–d were 90%, 91%, 159
64% and 73%, respectively.
160
96
2.4.1. General synthesis of 5a–d
Compound 6a: Mp 118.5–119.9 8C; 1H NMR (600 MHz, CD3OD): 161
d 3.06 (s, 6H, –CH3), 3.58 (t, 2H, J = 5.4 Hz, –CH2), 4.03 (s, 2H, –CH2), 162
97
98
99
Boc-protected amino acids (0.80 mmol) and 2-(7-aza-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoropho-
sphate (HATU) (241 mg) were dissolved in 5 mL of dimethylfor-
mamide (DMF); the mixture was stirred at room temperature for
30 min, and then compound 4 (150 mg, 0.53 mmol) and N,N-
diisopropylethylamine (DIPEA) (0.46 mL) were added. After that,
the reaction mixture was stirred at room temperature for 18 h and
then evaporated. The residue was dissolved with 100 mL of 5%
hydrochloric acid solution. The solution was dried by Na2SO4, and
evaporated in vacuo. The residues were further purified by silica-
gel column chromatography using CH2Cl2/MeOH (10:1, v/v), as the
eluent, to give the product in the yields of 80% (5a), 60% (5b), 80%
(5c) and 86% (5d), respectively.
4.51(t, 2H, J = 4.8 Hz, –CH2), 7.70 (t, 1H, J = 6.6 Hz, Ar–H), 8.11 (d, 163
1H, J = 7.8 Hz, Ar–H), 8.34 (d, 1H, J = 6.6 Hz, Ar–H), 8.44 (s, 1H, Ar– 164
H), 8.50 (s, 1H, Ar–H); 13C NMR (150 MHz, CD3OD):
d 35.16, 41.05, 165
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
42.74, 42.79, 55.96, 121.56, 122.13, 122.47, 123.82, 124.78, 127.35, 166
129.89, 132.26, 133.92, 136.74, 164.05, 164.37, 165.00; HRMS (m/ 167
z): calcd. for C18H21N4O3+: 341.1613; found 341.1604.
168
Compound 6b: Mp 286.7–287.8 8C (hydrochloride); 1H NMR 169
(600 MHz, CD3OD): 2.38 (s, 6H, –CH3), 2.70 (m, 4H, –CH2), 3.10 (t, 170
d
2H, J = 6.6 Hz, –CH2), 4.33 (t, 2H, J = 6.6 Hz, –CH2), 7.76 (t, 1H, 171
J = 7.8 Hz, Ar–H), 8.23 (d, 1H, J = 7.8 Hz, Ar–H), 8.44 (d, 1H, 172
J = 7.2 Hz, Ar–H), 8.60 (d, 1H, J = 1.8 Hz, Ar–H), 8.71 (d, 1H, 173
J = 1.2 Hz, Ar–H); 13C NMR (150 MHz, CD3OD):
d 33.66, 35.10, 174
Compound 5a: Mp 203.3–204.1 8C; 1H NMR (600 MHz, CDCl3):
d
35.70, 43.22, 46.05, 55.31, 121.60, 122.42, 123.25, 124.36, 124.72, 175
128.21, 129.76, 132.44, 134.35, 138.01, 164.25, 164.50, 169.65; 176
1.53 (s, 9H, –CH3), 2.44 (s, 6H, –CH3), 2.76 (s, 2H, –CH2), 4.07 (d, 2H,
J = 5.4 Hz, –CH2), 4.34 (t, 2H, J = 6.6 Hz, –CH2), 5.58 (br, 1H, –NH),
7.66 (t, 1H, J = 7.2 Hz, Ar–H), 8.03 (d, 1H, J = 7.8 Hz, Ar–H), 8.21 (s,
1H, Ar–H), 8.41 (d, 1H, J = 6.6 Hz, Ar–H), 8.69 (s, 1H, Ar–H), 9.07 (br,
HRMS (m/z): calcd. for C19H23N4O3+: 355.1770; found 355.1762.
177
Compound 6c: Mp 72.6–73.5 8C; 1H NMR (600 MHz, DMSO-d6): 178
2.81 (t, 2H, J = 6.6 Hz, ÀCH2), 2.90 (s, 6H, –CH3), 3.15 (m, 2H, – 179
d
1H, CONH–Ar); 13C NMR (150 MHz, CDCl3):
d
28.36, 37.96, 45.67,
CH2), 3.32 (s, 1H), 3.45 (t, 2H, J = 5.4 Hz, –CH2), 4.38 (t, 2H, 180
J = 5.4 Hz, –CH2), 7.85 (t, 1H, J = 8.4 Hz, Ar–H), 8.40 (m, 2H, Ar–H), 181
8.71 (s, 2H, Ar–H), the protons of another CH2 maybe hidden in the 182
57.23, 80.94, 121.96, 122.14, 123.05, 123.82, 124.88, 127.44,
129.88, 132.31, 133.75, 136.37, 163.69, 164.15, 168.63; HRMS (m/
z): calcd. for C23H29N4O5+: 441.2138; found: 441.2126.
solvent peaks; 13C NMR (150 MHz, CD3OD):
d 22.74, 33.12, 35.11, 183
Compound 5b: Mp 205.7–206.9 8C; 1H NMR (600 MHz, CDCl3):
d
38.99, 42.79, 55.99, 121.37, 121.68, 122.03, 123.82, 124.40, 127.17, 184
1.46 (s, 9H, –CH3), 2.47 (s, 6H, –CH3), 2.72 (t, 2H, J = 4.8 Hz, –CH2),
2.84 (br, 2H, –CH2), 3.57 (m, 2H), 4.34 (t, 2H, J = 6.0 Hz, –CH2), 5.30
(br, 1H, –NH), 7.62 (t, 1H, J = 7.8 Hz, Ar–H), 7.91 (d, 1H, J = 6.6 Hz,
Ar–H), 8.06 (s, 1H, Ar–H), 8.33 (d, 1H, J = 7.2 Hz, Ar–H), 8.57 (s, 1H,
129.54, 132.12, 133.83, 137.38, 164.08, 164.35, 171.92; HRMS (m/ 185
z): calcd. for C20H25N4O3+: 369.1927; found 369.1918.
186
Compound 6d: Mp 57.8–58.9 8C; 1H NMR (600 MHz, DMSO-d6): 187
d
1.55 (m, 2H, –CH2), 1.77 (m, 2H, –CH2), 1.83 (m, 2H, –CH2), 2.55 (t, 188
Ar–H), 8.95 (s, 1H, CONH–); 13C NMR (150 MHz, CDCl3):
d
28.45,
2H, J = 7.2 Hz, –CH2), 2.98 (t, 2H, J = 7.8 Hz, –CH2), 3.04 (s, 6H, – 189
CH3), 3.55 (t, 2H, J = 6.0 Hz, –CH2), 4.56 (t, 2H, J = 6.0 Hz, –CH2), 7.82 190
(t, 1H, J = 8.4 Hz, Ar–H), 8.31 (d, 1H, J = 8.4 Hz, Ar–H), 8.52 (d, 1H, 191
J = 7.2 Hz, Ar–H), 8.68 (d, 1H, J = 1.8 Hz, Ar–H), 8.78 (d, 1H, 192
36.42, 37.47, 37.79, 46.06, 58.08, 79.65, 121.08, 121.97, 122.78,
123.16, 124.37, 127.37, 129.54, 132.15, 133.64, 136.79, 156.32,
163.56, 163.56, 164.33, 170.70; HRMS (m/z): calcd. for
C24H31N4O5+: 455.2294; found: 455.2287.
J = 1.8 Hz, Ar–H); 13C NMR (150 MHz, CD3OD):
d 24.58, 25.64, 193
Compound 5c: Mp 170.2–171.7 8C; 1H NMR (600 MHz, CDCl3):
d
26.96, 35.24, 36.13, 39.20, 42.84, 56.13, 115.81, 117.75, 121.65, 194
122.04, 124.32, 127.21, 129.60, 132.40, 133.89, 137.62, 161.52, 195
161.75, 164.25, 164.52, 173.32; HRMS (m/z): calcd. for 196
0.86 (m, 2H), 1.45 (s, 6H, –CH3), 1.48 (s, 12H, –CH3), 1.96 (t, 2H,
J = 6.0 Hz, –CH2), 2.53 (m, 2H, –CH2), 2.89 (d, 2H, J = 5.4 Hz, –CH2),
3.31 (d, 2H, J = 5.4 Hz, –CH2), 5.12 (s, 1H, –NH), 7.64 (t, 1H,
J = 7.8 Hz, Ar–H), 8.04 (d, 1H, J = 6.6 Hz, Ar–H), 8.32 (s, 1H, Ar–H),
8.36 (d, 1H, J = 6.6 Hz, Ar–H), 8.80 (s, 1H, Ar–H), 9.80 (s, 1H, CONH–
C
22H29N4O3+: 397.2240; found 397.2229.
197
2.4.3. General synthesis of 7a–d
198
); 15C NMR (150 MHz, CDCl3):
d
27.00, 28.46, 34.42, 38.01, 39.34,
To a solution of compounds 6a–d (1.0 mmol) and dichloroacetyl 199
chloride (0.12 mL, 1.3 mmol) in CH2Cl2 (10 mL) were added 200
triethylamine (TEA) (0.3 mL). The reaction mixture was stirred 201
at room temperature for 10 h and then evaporated. The residue 202
was dissolved with 10 mL CH2Cl2, and washed with water. The 203
solution was dried over Na2SO4, and evaporated in vacuo. The 204
solids obtained were purified by column chromatography using 205
CH2Cl2/MeOH (20:1, v/v) as the eluent to afford pure 7a (48%), 7b 206
45.90, 57.51, 79.97, 121.58, 122.12, 122.92, 123.97, 124.64, 127.25,
129.52, 132.37, 133.70, 137.25, 163.74, 164.30, 171.99; HRMS (m/
z): calcd. for C25H33N4O5+: 469.2451; found 469.2443.
Compound 5d: Mp 187.1–188.7 8C; 1H NMR (600 MHz, CD3OD):
d
1.43 (s, 9H, –CH3), 1.47 (m, 2H, –CH2), 1.57 (m, 2H. –CH2), 1.81 (m,
2H, –CH2), 2.43 (s, 6H, –CH3), 2.49 (t, 2H, J = 7.8 Hz, –CH2), 2.76 (t,
2H, J = 7.2 Hz, –CH2), 3.09 (t, 2H, J = 6.6 Hz, –CH2), 4.29 (t, 2H,
J = 6.6 Hz, –CH2), 7.69 (t, 1H, J = 7.8 Hz, Ar–H), 8.12 (d, 1H,
J = 8.4 Hz, Ar–H), 8.33 (d, 1H, J = 7.2 Hz, Ar–H), 8.46 (d, 1H,
(38%), 7c (47%) and 7d (45%), respectively.
Compound 7a: Mp 186.2–189.3 8C; 1H NMR (600 MHz, DMSO- 208
d6): 2.21 (s, 6H, –CH3), 2.52 (br, 2H, –CH2), 4.11 (s, 2H, –CH2), 4.15 209
207
J = 1.8 Hz, Ar–H), 8.57 (s, 1H, Ar–H); 13C NMR (150 MHz, CD3OD):
d
d
25.01, 26.13, 27.40, 29.36, 36.55, 37.15, 39.83, 44.24, 56.31, 78.42,
121.56, 121.84, 122.59, 123.95, 124.46, 127.05, 129.11, 132.26,
133.44, 137.51, 157.17, 163.69, 164.04, 173.46; HRMS (m/z): calcd.
for C27H37N4O5+: 497.2764; found 497.2756.
(t, 2H, J = 7.2 Hz, –CH2), 6.65 (s, 1H, –CH), 7.82 (t, 1H, J = 7.8 Hz, Ar– 210
H), 8.38 (t, 2H, J = 7.2 Hz, Ar–H), 8.63 (d, 1H, J = 1.8 Hz, Ar–H), 8.74 211
(d, 1H, J = 1.8 Hz, Ar–H), 9.02 (br, 1H, CONH–), 10.79 (br, 1H, 212
CONH–); 13C NMR (150 MHz, DMSO-d6):
d 35.69, 43.22, 43.70, 213
45.94, 55.20, 55.25, 66.99, 121.59, 122.49, 123.35, 124.32, 124.75, 214
128.15, 129.67, 132.53, 134.37, 138.08, 164.49, 164.43, 164.70, 215
167.91; HRMS (m/z): calcd. for C20H21Cl2N4O4+: 451.0940; found 216
150
151
152
153
154
155
156
157
158
2.4.2. General synthesis of 6a–d
In a 25-mL flask, 1.0 mmol each of compounds 5a–d was
dissolved in 10 mL CH2Cl2. The solution was cooled to 0 8C, and
10 mL of CF3COOH was carefully added under stirring. The mixture
was allowed to warm to room temperature and stirred for 1 h.
Then, the solvent was removed under reduced pressure. The
residue was dissolved with 20 mL of 5% aqueous sodium
carbonate. The organic phase was separated, washed with water,
and dried over sodium sulfate. The product was obtained after the
451.0936.
Compound 7b: Mp 169.1–165.7 8C; 1H NMR (600 MHz, DMSO- 218
d6): 2.70 (t, 2H, J = 6.6 Hz, –CH2), 2.81 (s, 3H, –CH3), 3.48 (m, 2H, – 219
217
d
CH2), 4.36 (t, 2H, J = 5.4 Hz, –CH2), 6.56 (s, 1H, –CH), 7.81 (t, 1H, 220
J = 7.8 Hz, Ar–H), 8.37 (t, 2H, J = 7.8 Hz, Ar–H), 8.72 (s, 1H, Ar–H), 221
8.77 (s, 1H, Ar–H), 8.96 (s, 1H, CONH–), the protons of another CH2 222
maybe hidden in the solvent peaks; 13C NMR (150 MHz, CD3OD):
d
223
Please cite this article in press as: K.-R. Wang, et al., Cytotoxic activity and DNA binding of naphthalimide derivatives with amino acid
and dichloroacetamide functionalizations, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.020

G Model
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K.-R. Wang et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
Table 1
The cytotoxic activities of compounds 6a–d and 7a–d.
Cell lines
Cytotoxicity (IC50, m )
mol LÀ1
6a
6b
6c
6d
7a
7b
7c
7d
Amonafide
Hela
A549
K562
21.50 Æ 1.50
11.42 Æ 0.40
12.17 Æ 2.10
47.45 Æ 8.56
20.62 Æ 1.97
60.51 Æ 21.05
29.78 Æ 1.70
15.36 Æ 1.34
50.72 Æ 10.03
34.40 Æ 4.11
13.28 Æ 0.86
8.52 Æ 1.18
41.22 Æ 1.45
11.29 Æ 0.53
23.11 Æ 5.20
73.37 Æ 13.27
26.48 Æ 4.45
34.92 Æ 13.23
47.44 Æ 7.79
28.10 Æ 3.70
16.17 Æ 1.47
22.86 Æ 1.77
4.78Æ 0.50
17.08 Æ 6.90
12.09 Æ 1.33
14.3 Æ 1.0
1.94 Æ 0.31
+
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
36.05, 36.24, 41.70, 43.18, 55.20, 67.21, 121.45, 122.44, 123.19,
124.50, 124.66, 128.02, 129.49, 132.48, 134.27, 138.41, 164.18,
164.41, 170.46; HRMS (m/z): calcd. for C21H23Cl2N4O4+: 465.1096;
found 465.1087.
164.23, 134.46, 172.59; HRMS (m/z): calcd. for C24H29Cl2N4O4
507.1565; found 507.1559.
:
246
247
3. Results and discussion
248
Compound 7c: Mp 174.5–176.3 8C; 1H NMR (600 MHz, CD3OD):
d
1.83 (m, 2H, –CH2), 2.48 (m, 2H, –CH2), 2.87 (s, 6H, –CH3), 3.23 (m,
The target compounds 7a–d were synthesized by six steps
(Scheme 1). Commercially available 1,8-naphthalic anhydride 1
was reacted under concentrated H2SO4 and HNO3 conditions to
afford compound 3-nitro-1,8-naphthalic anhydride 2 [21]. Then,
compound 2 was condensed with N,N-dimethylethylenediamine
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265
2H, –CH2), 3.42 (s, 2H, –CH2), 4.39 (t, 2H, J = 5.4 Hz, –CH2), 6.59 (s,
1H, –CHCl2), 7.82 (t, 1H, J = 7.8 Hz, Ar–H), 8.37 (t, 2H, J = 8.4 Hz, Ar–
H), 8.72 (s, 1H, Ar–H), 8.79 (s, 1H, Ar–H), 8.96 (s, 1H, CONH–), 9.88
(s, 1H, CONH–); 13C NMR (150 MHz, CD3OD):
d 24.99, 34.06, 35.60,
43.10, 55.06, 67.38, 121.29, 122.44, 123.17, 124.47, 124.58, 127.98,
129.38, 132.52, 134.29, 138.57, 164.13, 134.19, 134.43, 172.07;
HRMS (m/z): calcd. for C22H25Cl2N4O4+: 479.1252; found 479.1244.
Compound 7d: Mp 158.4–159.5 8C; 1H NMR (600 MHz, DMSO-
to yield compound
3 [6], which was reduced by catalytic
hydrogenation to obtain compound 4. Condensation of compound
4 with various Boc-protected amino acids yielded compounds 5a–
d. Subsequent removal of the Boc protecting groups under TFA
conditions, gave compounds 6a–d, which were directly condensed
with dichloroacetyl chloride to form the corresponding target
compounds 7a–d.
The cytotoxic activities of the naphthalimide derivatives 6a–d
and 7a–d against cancer cell lines (Hela, A549 and K562) in vitro
were measured with comparison to the control drug, Amonafide.
As shown in Table 1, the length of the side chains of the amino acid
influenced the cytotoxic activities. Compounds 6a with a glycine
d6):
d 1.35 (m, 2H, –CH2), 1.49 (m, 2H, –CH2), 1.67 (m, 2H, –CH2),
2.43 (t, 2H, J = 7.8 Hz, –CH2), 3.13 (dd, 2H, J = 6.6 Hz, 12.6 Hz, –CH2),
3.42 (br, 2H, –CH2), 4.38 (t, 2H, J = 6.0 Hz, –CH2) 6.51 (s, 1H, –CH),
7.82 (t, 1H, J = 7.8 Hz, Ar–H), 8.37 (t, 2H, J = 8.4 Hz, Ar–H), 8.70 (d,
1H, J = 1.8 Hz, Ar–H), 8.75 (br, 1H, CONH–), 8.79 (d, 1H, J = 1.8 Hz,
Ar–H), 9.82 (br, 1H, CONH–); 13C NMR (150 MHz, DMSO-d6):
d
25.12, 26.33, 28.76, 34.56, 36.79, 43.14, 45.88, 55.14, 67.41, 121.32,
122.44, 124.58, 127.99, 129.40, 132.55, 134.29, 138.63, 164.00,
0.20
0.20
a
b
0.15
0.10
0.05
0.00
0.15
0.10
0.05
0.00
320
360
400
440
320
360
400
440
Wavelength/nm
Wavelength/nm
0.12
0.10
0.08
0.06
0.04
0.02
0.00
d
c
0.15
0.10
0.05
0.00
320
360
400
440
480
320
360
400
440
480
Wavelength/nm
Wavelength/nm
Fig. 2. UV–vis spectral changes of compounds 6a and 6d (2.0 Â 10À5 mol LÀ1, a and b), 7a and 7d (2.0 Â 10À5 mol LÀ1, c and d) upon addition of Ct-DNA (from 0 mol LÀ1 to
2 Â 10À4 mol LÀ1) in phosphate buffer (10 mmol LÀ1, pH 7.4, 50 mmol LÀ1 NaCl) or in phosphate buffer (10 mmol LÀ1, pH 7.4, 50 mmol LÀ1 NaCl) containing 5% DMSO at 25 8C.
Please cite this article in press as: K.-R. Wang, et al., Cytotoxic activity and DNA binding of naphthalimide derivatives with amino acid
and dichloroacetamide functionalizations, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.020

G Model
CCLET 2964 1–7
K.-R. Wang et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
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conjugate and 6d with an aminocaproic acid conjugate, and their
dichloroacetamide derivatives 7a and 7d exhibited better cytotox-
using the nonlinear least-squares curve-fitting method [25] by 297
fitting the experimentaldata of the maximum fluorescence changes. 298
Circular dichroism (CD) is a powerful and reliable technique to 299
investigate the conformational changes in DNA morphology during 300
small molecules–DNA interactions. As shown in Fig. 4, the CD 301
spectrum of free Ct-DNA showed a negative band at 246 nm due to 302
the polynucleotide helicity, and a positive band at 276 nm due to 303
the base staking, which conformed that the Ct-DNA existed in the 304
right-band B form [26,27]. Upon addition of compounds 6a, 6d and 305
7a, 7d, the CD signals of Ct-DNA underwent obvious changes in 306
both the negative and positive bands without significant wave- 307
length change. These results indicated that compounds 6a, 6d and 308
7a, 7d intercalated into the base pairs of DNA, causing the changes 309
of the degree of rotation between successive base pairs [28,29]. 310
Additionally, weak, positive induced circular dichroism (ICD) 311
signals were observed in the region of the characteristic absorption 312
of the naphthalimides (300–380 nm), which indicated that 6a, 6d 313
and 7a, 7d intercalated DNA with a vertical orientation in the 314
ic activity than 6b, 6c with
b-alanine and 4-aminobutyric acid
conjugates and their derivatives 7b and 7d. In addition, 6a, 6d, 7a
and 7d had more toxicity against A549 than against Hela and K562
cells compared with the control drug (Amonafide), especially 7d
which had an IC50 value of 4.78
m .
mol LÀ1
Furthermore, the DNA binding properties of compounds 6a, 6d
and 7a, 7d with Ct-DNA were investigated by using UV–vis,
fluorescence and CD spectroscopies and DNA thermal denaturation
experiment.
The UV–vis spectra of compounds 6a and 6d with Ct-DNA in PBS
buffer (10 mmol LÀ1, pH 7.4, 50 mmol LÀ1 NaCl) at 25 8C are shown
in Fig. 2. The absorption intensity of compounds 6a and 6d
decreased with the increase of Ct-DNA concentrations, and the
maximum absorption peak showed a bathochromic shift of about
5 nm. This spectral characteristic implied that compounds 6a and
6d were inserted into the base pairs of DNA [22–24]. On the other
hand, the DNA binding properties of the dichloroacetamide
derivatives 7a and 7d with Ct-DNA showed hypochromicities
upon addition of Ct-DNA, but the maximum absorption peak
showed no obvious change. These results indicated that 7a and 7d
exhibited weaker binding interactions with Ct-DNA than com-
intercalation pocket [30].
315
Moreover, the thermal denaturation experiments of the Ct-DNA 316
in the absence, and presence, of 6a, 6d and 7a, 7d (Fig. 5) showed an 317
obvious change in the melting temperatures (Tm), which could 318
provide useful information on the conformational change and the 319
pounds 6a and 6d, as
a
result of the dichloroacetamide
interaction strength between the DNA and the compounds [31].
320
functionalization.
The melting curves of Ct-DNA are illustrated in Fig. 5 and 321
Table 2, respectively. The Tm value for the free Ct-DNA is 72.9 8C in 322
PBS buffer. Upon addition of 6a, 6d, obvious changes in the DNA 323
melting temperature were observed and the Tm values increased to 324
77.4 8C and 76.1 8C, respectively. The increases of the melting 325
The fluorescencespectra of compounds 6a, 6d and 7a, 7d withCt-
DNA are given in Fig. 3. Upon addition of Ct-DNA, the fluorescence
of 6a, 6d and 7a, 7d was quenched. Furthermore, the binding
constants between compounds 6a, 6d and 7a, 7d with Ct-DNA were
obtained as 1.29 Â 105 molÀ1 L (6a), 1.15 Â 105 molÀ1 L (6d),
5.1 Â 103 molÀ1 L (7a), and 5.1 Â 103 molÀ1 L (7d), respectively,
temperature (DTm) induced by DNA–compound interactions were 326
4.5 8C and 3.2 8C, respectively, indicating that compound 6d 327
1200
a
1000
b
1000
800
600
400
200
0
800
600
400
200
0
400
450
500
550
600
400
450
500
550
600
Wavelength/nm
Wavelength/nm
d
c
1600
1400
1200
1000
800
600
400
200
0
1500
1200
900
600
300
0
400
450
500
550
600
400
450
500
550
600
Wavelength/nm
Wavelength/nm
Fig. 3. Fluorescence spectral changes of compound 6a and 6d (2.0 Â 10À5 mol LÀ1, a and b), 7a and 7d (2.0 Â 10À5 mol LÀ1, c and d) upon addition of Ct-DNA (from 0 mol LÀ1 to
2 Â 10À4 mol LÀ1) in phosphate buffer (10 mmol LÀ1, pH 7.4, 50 mmol LÀ1 NaCl) or in phosphate buffer (10 mmol LÀ1, pH 7.4, 50 mmol LÀ1 NaCl) containing 5% DMSO at 25 8C.
Inset: the fitted plots of the binding constants at the maximum emission band.
Please cite this article in press as: K.-R. Wang, et al., Cytotoxic activity and DNA binding of naphthalimide derivatives with amino acid
and dichloroacetamide functionalizations, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.020

G Model
CCLET 2964 1–7
6
K.-R. Wang et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
18
a
b
15
12
9
Ct-DNA
Ct-DNA+7a
Ct-DNA+7d
Ct-DNA
Ct-DNA+6a
Ct-DNA+6d
15
12
9
6
6
3
3
0
0
-3
-6
-9
-3
-6
250
300
350
400
450
250
300
350
400
450
Wavelength/nm
Wavelength/nm
Fig. 4. CD spectra of Ct-DNA (6.0 Â 10À5 mol LÀ1) upon addition of 6a and 6d (1.0 Â 10À4 mol LÀ1) in phosphate buffer (10 mmol LÀ1, pH 7.4, containing 50 mmol LÀ1 NaCl) (a),
and CD spectra of Ct-DNA (8.0 Â 10À5 mol LÀ1) upon addition of 7a and 7d (1 Â 10À4 mol LÀ1) in phosphate buffer (10 mmol LÀ1, pH 7.4, containing 50 mmol LÀ1 NaCl)
containing 5% DMSO at 25 8C (b).
a
b
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
Ct-DNA
Ct-DNA+7a
Ct-DNA+7d
Ct-DNA
Ct-DNA+6a
Ct-DNA+6d
50
60
70
80
50
60
70
80
Temperature/oC
Temperature/oC
Fig. 5. DNA melting curves for Ct-DNA (5.0 Â 10À5 mol LÀ1) in the absence and presence of 6a and 6d (5.0 Â 10À6 mol LÀ1) in phosphate buffer (1 mmol LÀ1, pH 7.4,
5 mmol LÀ1 NaCl) (a), 7a and 7d (5.0 Â 10À6 mol LÀ1) in phosphate buffer (1 mmol LÀ1, pH 7.4, 5 mmol LÀ1 NaCl) containing 5% DMSO (b).
328
329
330
331
332
333
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339
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341
possessed a lower DNA melting temperature than 6a. On the other
hand, the Tm value for the free Ct-DNA in PBS buffer containing 5%
DMSO was 67.9 8C. Upon addition of compounds 7a, and 7d, the
cytotoxic activity and the binding property with DNA of the amino
acids modified naphthalimide derivatives and their dichloroace-
tamide functionalized derivatives showed a similar trend with the
side chains.
342
343
344
345
increases in the melting temperature (DTm) induced by DNA–
compound interactions were 4.3 8C, and 1.9 8C, respectively, which
suggested that compound 7d had a lower DNA melting tempera-
ture than 7a. These results illustrated that glycine conjugated
naphthalimide derivatives 6a and 7a showed the stronger binding
interactions with the Ct-DNA than the aminocaproic acid linked
derivatives 6d and 7d, which was consistent with the results from
the fluorescence titration data.
Considering the above results, we could conclude that the novel
naphthalimides derivatives 6a–d and 7a–d, as the DNA inter-
calators, possessed similar binding properties with DNA. The
4. Conclusion
346
A series of novel naphthalimide derivatives modified by amino
acids and dichloroacetamide at 3-position have been synthesized.
Their cytotoxic activities were preliminarily evaluated against
Hela, A549 and K562 cells and their DNA binding properties were
investigated by UV–vis, fluorescence, and circular dichroism (CD)
spectroscopies and thermal denaturation experiment. The results
revealed that the lengths of the side chain of the amino acids
influenced the cytotoxic activities and the binding properties. The
glycine and aminocaproic acid modified naphthalimide derivatives
6a (7a) and 6d (7d) exhibited better cytotoxic activity. Especially,
347
348
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350
351
352
353
354
355
356
357
358
359
Table 2
Average Tm and
DTm for Ct-DNA in the absence and presence of 6a, 6d and 7a, 7d.
compound 7d with an IC50 value of 4.78
m
mol LÀ1 against A549
Compounds
Tm (8C)
D
Tm (8C)
cells would dictate further investigation to develop a potential
anticancer agent.
Ct-DNAa
6a
72.9
77.4
76.1
67.9
72.2
69.8
0
4.5
3.2
0
6d
Ct-DNAb
7a
Acknowledgments
360
4.3
1.9
7d
This work was supported by the National Natural Science Q3 361
a
The Tm value for the free Ct-DNA was determined in phosphate buffer
Foundation of China (Nos. 21372059 and 21172051), the Hebei
Natural Science Foundation (No. B2012201041) and the Founda-
tion of Hebei Education Department (No. YQ2013006).
362
363
364
(1 mmol LÀ1, pH 7.4, 5 mmol LÀ1 NaCl).
b
The Tm value for the free Ct-DNA was determined in phosphate buffer
(1 mmol LÀ1, pH 7.4, 5 mmol LÀ1 NaCl) containing 5% DMSO.
Please cite this article in press as: K.-R. Wang, et al., Cytotoxic activity and DNA binding of naphthalimide derivatives with amino acid
and dichloroacetamide functionalizations, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.020

G Model
CCLET 2964 1–7
K.-R. Wang et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
7
365
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Please cite this article in press as: K.-R. Wang, et al., Cytotoxic activity and DNA binding of naphthalimide derivatives with amino acid
and dichloroacetamide functionalizations, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.020