Synthesis, anticancer activity and DNA-binding properties of novel 4-pyrazolyl-1,8-naphthalimide derivatives

Article abstract of DOI:10.1016/j.bmcl.2013.12.014

A novel series of 4-pyrazolyl-1,8-naphthalimide derivatives have been designed and facilely synthesized. For anticancer activity in vitro, most of the compounds were found to be more toxic against human mammary cancer cells (MCF-7) than human cervical car

Full text of DOI:10.1016/j.bmcl.2013.12.014

Bioorganic & Medicinal Chemistry Letters 24 (2014) 586–590
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, anticancer activity and DNA-binding properties of novel
4-pyrazolyl-1,8-naphthalimide derivatives
Shenghui Li a,b, , Shengjie Xu , Yonghe Tang , Shan Ding , Jinchao Zhang
⇑
a
a
a
a,b,
⇑
, Shuxiang Wang a,b
,
a
b
a,b
Guoqiang Zhou , Chuanqi Zhou , Xiaoliu Li
a
Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Hebei University, Baoding 071002, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 4-pyrazolyl-1,8-naphthalimide derivatives have been designed and facilely synthesized.
For anticancer activity in vitro, most of the compounds were found to be more toxic against human mam-
mary cancer cells (MCF-7) than human cervical carcinoma cells (Hela) and human lung cancer cells
Received 10 September 2013
Revised 1 November 2013
Accepted 3 December 2013
Available online 14 December 2013
(
A549). Compounds 4i, 4h, 4b and 4a showed improved cytotoxic activity against MCF-7 cells over
amonafide, in particular compounds 4i and 4h, the IC50 values of which against cell lines of MCF-7 were
.51 M and 0.79 M, respectively. The DNA-binding properties of 4i were investigated by UV–vis, fluo-
0
l
l
Keywords:
Naphthalimides
Pyrazole
Anticancer
DNA-binding
rescence, and Circular Dichroism (CD) spectroscopies and thermal denaturation. The results indicated
that compound 4i as the DNA-intercalating agent exhibited middle binding affinity with CT-DNA.
Ó 2013 Elsevier Ltd. All rights reserved.
Cancer is well-known as one of the leading causes of death
worldwide, and it is estimated that about 10 million new cancer
cases are diagnosed every year which represents a real crisis for
public health in the world.1 Therefore, the discovery of selective,
efficient, and safe drugs for cancer chemotherapy remains an ur-
gency and high priority for medicinal research.
O
N
O
O
N
O
H
N
H
N
N
Elinafide
O
N
O
NO
2
O
O
N
O
Now DNA is still an important target for cancer chemotherapy.
Generally, DNA interactive drugs include DNA-alkylating agents,
H
N
2
N
O
DNA groove binders and DNA intercalators. Naphthalimide deriv-
N
H
NH
2
atives, known as DNA intercalators, showed high anticancer activ-
Amonafide
3
ities against a broad spectrum of cell lines. Some compounds, such
O
2
N
Bisnafide
as amonafide, elinafide and bisnafide (Fig. 1) have reached clinical
trials. However, heretofore no naphthalimide derivative has ever
entered the antineoplastic market because of dose-limiting
toxicity.4 To improve anticancer activities and/or decrease side
effects, great efforts have been made to fuse aromatic rings, such
as benzene, imidazole, pyrazine, furan or thiophene, and others,
to naphthalene nucleus, some of the obtained compounds showed
significant improvement in cellular cytotoxic activity over
Figure 1. Representative naphthalimide derivatives in clinical trial.
to 4- or 3-position of naphthalimide skeleton and found that the
majority of these compounds showed better cytotoxic activity
7,8
against MCF-7 cells than amonafide.
Pyrazolesareconsideredasextremelyversatilebuildingblocksin
3
,5
amonafide. Another series of naphthalimides, where an unfused
benzene or furan ring was introduced, were reported by Brana and
co-workers exhibited extraordinary cytotoxicity for cancer cell
9,10
organicchemistry,
whichis alsoan importantclassof compounds
11
in medicinal chemistry. Pyrazole derivatives possess a broad spec-
12
trum of pharmacological activities such as anti-inflammatory,
6
lines. More recently, Qian and co-workers reported a series of
13
14
15,16
antibacterial,
antifungal,
hypoglycemic,
modified naphthalimide derivatives by linking 1,2,3-triazole ring
17
anti-hyperlipidemic, etc. A variety of pyrazole derivatives have
also been tested for their antitumor activity in vivo, which often
result in promising lead compounds.
1
8–22
This gave us a great
⇑
Corresponding authors. Tel.: +86 312 507 9359; fax: +86 312 507 9005.
E-mail addresses: lish@hbu.edu.cn, shenghui2299@126.com (S.
jczhang@126.com (J. Zhang).
impetus to the search for potential pharmacologically active drugs
carrying pyrazole substituent. Herein, we described the design,
Li),
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.014

S. Li et al. / Bioorg. Med. Chem. Lett. 24 (2014) 586–590
587
synthesis, anticancer activity and DNA-binding properties of novel
-pyrazolyl-1,8-naphthalimide derivatives, expecting the incorpo-
Table 1
The cytotoxicity of compounds against MCF-7, Hela and A549 cells
4
ration of a pyrazole heterocyclic unit into naphthalimide could im-
prove the biological activities while eliminating the N-acetyl
transferase-2 (NAT2) acetylation of amonafide.23
Compound
Cytotoxicity (IC50
, lM)
MCF-7
Hela
A549
8.52
24.02
17.16
>50
5.09
7.69
11.95
11.84
5.14
25.03
25.36
4
4
a
b
1.58
1.56
4.24
10.66
17.01
2.62
1.99
0.79
0.51
4.26
3.39
2.72
1.68
6.50
3.10
10.64
3.47
4.60
4.99
14.60
3.50
3.09
13.32
16.60
9.47
As shown in Scheme 1, 4-pyrazolyl-1,8-naphthalimide deriva-
tives 4a–4l were synthesized by two steps starting from 4-bro-
mo-1,8-naphthalic anhydride through condensation with N,N-
dimethylethylenediamine in ethanol, followed by aromatic substi-
4c
4d
4e
2
4
4f
tution reaction with various 4-aryl-1H-pyrazoles or 1H-pyrazole.
4
4
g
h
1
Structures of all the final products were confirmed by using H and
1
3
C NMR, IR, MS and elemental analysis.25
4i
4j
The anticancer activity of the compounds 4a–4l, was evaluated
4
4
k
l
in vitro against MCF-7, Hela and A549 cells by MTT tetrazolium
>50
13.00
2
6
assay. The IC50 values were listed in Table 1 and the activities
of amonafide were as a control. The preliminary results showed
that most of the obtained compounds were more toxic against
MCF-7 than Hela and A549 cells. Toward MCF-7 and Hela cells,
compounds 4b, 4h and 4i were found to be more toxic than
amonafide. Importantly, compound 4i, bearing 3,4,5-trimethoxy
group on its phenyl ring, was the most effective one, its inhibition
abilities was 3.3-fold and 2.2-fold higher than that of amonafide
under the same experimental conditions. Toward A549 cells, com-
pounds 4a, 4e–4i exhibited higher activity than amonafide, espe-
cially, compound 4e, decorating with a 4-methoxy group on its
phenyl ring, was the most effective, its inhibition ability was 2.6-
fold higher than that of amonafide.
Amonafide
6.71
cytotoxic SAR of the number of methoxy groups substituted on
the phenyl ring (3 > 2 > 1). Secondly, the position of electron-donat-
ing group seems to have great effect on cytotoxic activity. Compar-
ing compounds 4f, 4g and 4h, it could be concluded that the
substituted C-3 position is essential for cytotoxicity against MCF-7
cell line. Thirdly, the nature of the electron-donating group seems
to be essential too. Compound 4e, bearing a weakly electron-donat-
ing methyl group in C-4 position, is weakly cytotoxic against MCF-7
cells than 4f with a relative strongly electron-donating methoxy
group. For Hela and A549 cells, exactly different observation was
obtained and this may be related to the tumor cell species. These re-
sults will be very helpful for designing new anticancer drugs in the
future.
For these novel naphthalimide derivatives functionalized with
pyrazole moiety, the structure–activity relationships (SAR) are
summarized as follows: (1) Phenyl pyrazole-substitution makes
the obtained naphthalimide derivatives more toxic than simple pyr-
azole-substitution. Compound 4a exhibited better activity than
compound 4l against MCF-7, Hela and A549 cells. (2) The introduc-
tion of different substituent on the phenyl pyrazole unit could obvi-
ously affect the cytotoxicity against A549 cells. Generally, electron-
donating group on the position-4 of the phenyl ring is essential for
optimal anti-tumor activity. For example, compounds 4e and 4f
with electron-donating groups on the benzene ring displayed high-
er cytotoxic activity than compounds 4b, 4c, 4d and 4j with elec-
tron-withdrawing groups. (3) The number, position and nature of
electron-donating groups on substituted phenyl ring also played a
profound role on cytotoxic activity against MCF-7 and Hela cells.
Firstly, the cytotoxicity increases with the number of electron-do-
nor group present on phenyl ring. A comparison between the cyto-
toxic activities of compounds 4f, 4g, 4h and 4i illustrated the
In order to understand the cytotoxicity of the amonafide-ana-
logues, the DNA-binding properties of the most active 4i with calf
thymus DNA (CT-DNA) were investigated by UV–vis absorption
spectra, fluorescent spectra, Circular Dichroism spectra (CD), and
thermal denaturation experiment.
It is widely accepted that if the compound can intercalate DNA,
the UV–vis curve of their complex will be induced bathochromic
2
7
shift and hypochromicity. The UV–vis spectra of 4i, the efficient
cytotoxic among compounds, was measured (Fig. 2). As DNA con-
centration was increased, the curve showed significant
hypochromicities and obvious bathochromic shifts. The spectral
characteristic implies that compound 4i can insert into the base
2
8
pairs of DNA. Its binding constant was calculated with the fol-
lowing equation:
2
9
N
N
HN
Ar
N
N
O
O
O
O
NH2
N
O
N
O
N
O
3
i
ii
N
Br
Br
Ar
1
2
4a-4l
4
4
4
a: Ar = Ph; 4b: Ar = 4-FC H ; 4c: Ar = 4-ClC H ; 4d: Ar = 4-BrC H ; 4e: Ar = 4-CH C H ;
6
4
6
4
6
4
3 6 4
f: Ar = 4-CH OC H ; 4g: Ar = 3-CH OC H ; 4h: Ar = 3,4-(CH O) C H ;
3
6
4
3
6
4
3
2 6 3
i: Ar = 3,4,5-(CH O) C H ; 4j: Ar = 4-CF C H ; 4k: Ar = 3,5-(CF ) C H ; 4l: Ar = H
3
3
6
2
3
6
4
3 2 6 3
Scheme 1. Synthesis of 4-pyrazolyl-1, 8-naphthalimide derivatives. Reagents and conditions: (i) ethanol, reflux, 2 h; (ii) t-BuOK, DMSO, 72 °C, 1.5 h.

5
88
S. Li et al. / Bioorg. Med. Chem. Lett. 24 (2014) 586–590
4
ꢁ1
0
0
0
0
0
0
.25
.20
.15
.10
.05
.00
to be 1.04 ꢂ 10 M . As shown in Figure 3, the emission intensi-
ties of compound 4i decreased with increasing the amount of
CT-DNA and the wavelength showed slightly blue shift (Fig. 3).
The observed quenching may be due to the binding of compound
a
l
4
i with CT DNA, while the blue shift is probably consistent with
3
0
intercalation.
Circular Dichroism (CD) is a valuable technique for examining
the structure of DNA. From the changes in DNA’s CD and the In-
duced Circular Dichroism (ICD) of compounds, the binding mode
and the mechanism of interaction can be deduced. Compound 4i
was chosen to illustrate the CD spectra at different ratio (Fig. 4).
Both the changes of DNA and ICD of 4i were obtained. The CD spec-
trum of free CT-DNA shows bands at 279 nm (positive) and 247 nm
negative) typical of the B-form. When R(4i/DNA) 6 0.1 (Fig. 4A),
the increase of positive band and the decrease of the negative band
were observed, and without significant wavelength change, which
was consistent with the B to A-like conformational change.32 This
may be explained by the intercalation of 4i into stacked DNA base
pairs, which resulted in the separation of the base pairs and
31
(
3
00
350
400
450
500
Wavelength(nm)
7
distortion of the sugarphosphate backbone. Additionally, signifi-
Figure 2. UV–vis spectral of compound 4i at the concentration of 20
addition of CT-DNA ((a) 0 M, (b) 10 M, (c) 20 M, (d) 30 M, (e) 40
g) 70 M, (h) 80 M, (i) 110 M, (j) 120 M, (k) 130 M, (l) 140
CH COONa–CH COOH buffer (pH 4.0).
l
M upon
cant Induced Circular Dichroisms (ICD) signals was observed in
the region of the characteristic absorption of the naphthalimides
(325–475 nm). The weak and negative signal at low R values
l
l
l
l
l
M, (f) 60
lM,
(
l
l
l
l
l
lM) in 0.1 M
3
3
(
0.1 < R(4i/DNA) 6 0.5, Fig. 4A) further indicated that 4i intercalated
33,34
DNA with a vertical orientation in the intercalation pocket.
½
DNAꢀ=ð
ꢀ
a
ꢁ
ꢀ
f
Þ ¼ ½DNAꢀ=ð
ꢀ
b
ꢁ
ꢀ
f
Þ þ 1=K
b
ð
ꢀ
b
ꢁ
ꢀ
f
Þ
where [DNA] is the concentration of DNA in base pairs,
a f b
e , e and e
1
0
8
6
4
2
0
2
4
correspond to average molar extinction coefficient of the solution
[compound 4i]), molar extinction coefficient of free 4i, and molar
extinction coefficient of totally binding 4i, respectively, and K is the
intrinsic binding constant. A linear fit of the plot of [DNA]/(
versus [DNA] gives a slope of 1/( ) and an intercept of 1/K
= 1.01 ꢂ 10 M was determined by this meth-
od, however, it is lower than that of amonafide (K
1
.0
(A
obsd
/
0.8
0.6
b
0
.4
c
e
a
ꢁ
e
f
)
0.2
0.0
0.2
e
b
ꢁ
e
f
b b-
(e
-
4
ꢁ1
ꢁe
f
). A value of K
b
-0.4
-0.6
a
b
,
-
0.8
5
ꢁ1
28
1
4
.05 ꢂ 10 M in 30 mM Tris–HCl, pH 7.5), which implies that
-1.0
3
40 360 380 400 420 440 460
i bind to DNA relatively less strongly. The decreased intercalation
Wavelength/nm
of 4i compared with amonafide may be due to the incorporation of a
pyrazole heterocyclic unit into naphthalimide.
To further investigate the interactions of compound 4i with
CT-DNA, the fluorescence quenching technique was employed to
c
-
-
b
measure the Scatchard binding constants (K ) for compound 4i
a
and the apparent binding constant (K
b
) to CT-DNA was determined
A
250
300
350
400
450
500
550
600
Wavelength/nm
1000
1
5
a
i
1
0
0
.0
.5
.0
8
6
4
2
00
00
00
00
0
10
5
g
d
g
d
-
0.5
3
20 340 360 380 400 420 440 460 480
Wavelength/nm
0
B
-5
4
00
450
500
550
300
400
Wavelength/nm
500
600
Wavelength/nm
Figure 4. Titration CD spectra of CT-DNA by 4i at different R ratio in 0.1 M
CH COONa–CH COOH buffer (pH 4.0). The CT-DNA concentration is 60 M. The
value of R (4i/DNA) is (a) 0 (b) 0.02 (c) 0.1 (d) 0.5 (e) 1.0 (f) 2.0 (g) 4.0.
Figure 3. Fluorescence spectral of compound 4i at the concentration of 20
addition of CT-DNA((a) 0 M, (b) 10 M, (c) 20 M, (d) 30 M, (e) 50
g) 90 M, (h) 110 M, (i) 130 M) in 0.1 M CH COONa–CH COOH buffer (pH 4.0).
l
M upon
l
l
l
3
l
lM, (f) 70 lM,
3
3
l
(
l
l
l
3

S. Li et al. / Bioorg. Med. Chem. Lett. 24 (2014) 586–590
589
When 0.5 < R(4i/DNA) < 2.0 (Fig. 4B), change trends of CD spectra
Table 2
Average T
m
and
D
T
m
for CT-DNA in the absence and presence of 4i
Tm (°C)
were continued, but the negative ICD signals disappeared, and a
new positive ICD signals appeared. The ICD behaviour at high R
Compound
D
Tm (°C)
3
3,34
values is presumably a consequence of external stacking.
CT-DNA
59.7
61.5
0
1.8
When R(4i/DNA) P 2.0, the positive band reached the highest peak,
which suggested that the DNA had been saturated with 4i.
Thermal denaturation studies of CT-DNA are useful in deter-
mining the ability of the present compound to stabilize the double
stranded DNA. The intercalation of small molecules into the double
CT-DNA+4i
showed that compound 4i as the DNA intercalator exhibited mid-
dle binding affinity with CT-DNA. These results will be helpful for
designing naphthalimide-based lead compounds with improved
anticancer activity. Further structural optimization and detailed
biological studies on the mechanism of action about the designed
naphthalimide derivatives were under way.
m
helix was known to increase the DNA melting temperature (T ),
which characterizes the transition of DNA from double-stranded
to single standard nucleic acid.35 The melting curve of CT-DNA in
the absence and presence of 4i are illustrated in Figure 5 and
Table 2, respectively. The T
Upon addition of 4i, obvious changes in the DNA melting temper-
ature were observed. The T value increased to 61.5 °C. The level of
the increased melting temperature ( ) induced by DNA-com-
m
value for the free CT-DNA is 59.7 °C.
Acknowledgments
m
D
T
m
This work was supported by the National Basic Research 973
Program (Grant No. 2010CB534913), Hebei Provincial Natural Sci-
ence Foundation of China-Shijiazhuang Pharmaceutical Group
(CSPC) Foundation (B2011201174, B2012201044), Hebei Province
Nature Science Fund for Distinguished Young Scholars
(B2011201164), the Nature Science Fund of Hebei Province
(B2011201135), the Key Basic Research Special Foundation of Sci-
ence Technology Ministry of Hebei Province (11966412D,
12966418D) and Open Fund of Key Laboratory of Chemical Biology
of Hebei Province (No. 09265631D-7).
pound interactions is 1.8 °C. Compound 4i possessed lower DNA
melting temperature than some other amonafide analogues re-
ported in related literatures. These results illuminated that com-
pounds 4i, as the DNA intercalator, exhibited middle affinity with
CT-DNA, this is consistent with the results from the UV–Vis titra-
3
6
tion data.
The present work describes the design, synthesis and biological
activity evaluation of the 4-pyrazolyl-1,8-naphthalimide deriva-
tives as DNA-intercalating agents. The convenient method that
introduce pyrazole ring to the 4 site of naphthalimide was offered.
This reconstruction improved the cytotoxicity of the compounds
over the lead compound amonafide. Based on the testing of anti-
tumor activity in vitro, most of the obtained compounds were
found to be more cytotoxic against MCF-7 than Hela and A549
cells. In particular, compound 4i, bearing 3,4,5-trimethoxy group
on its phenyl ring, with the values of IC50 against three cell lines
were 3.3-fold, 2.2-fold, and 2.5-fold lower than that of amonafide.
Compound 4e, decorating with a 4-methoxy group on its phenyl
ring, with the cytotoxicity against A549 cells was 2.6-fold higher
than that of amonafide. The preliminary SAR revealed that 1) by
linking a pyrazole ring onto the naphthalene core can significantly
improve cytotoxic activity and 2) activity would be enhanced if the
phenyl pyrazole moiety possessed electron-donating groups. The
DNA binding properties were investigated by UV–vis, fluorescence
and CD spectra and thermal denaturation experiment. The results
Supplementary data
Supplementary data (such as synthetic procedure, cytotoxicity
analysis, and DNA binding experiment) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl. 2013.12.014.
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Figure 5. DNA melting curves for CT-DNA (50
l
M) in the absence and presence of
4
i with concentration of 5 M in 0.1 M CH COONa–CH
l
3
3
COOH buffer (pH 4.0).

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Sanchez, A.; Cobo, J. Bioorg. Med. Chem. 2010, 18, 4965.
0. Labbozzetta, M.; Baruchello, R.; Marchetti, P.; Gueli, M. C.; Poma, P.;
Notarbartolo, M.; Simoni, D.; D’Alessandro, N. Chem. Biol. Interact 2009, 181, 29.
1. Perchellet, E. M.; Ward, M. M.; Skaltsounis; Alexios, L.; Kostakis, I. K.; Pouli, N.;
Marakos, P.; Perchellet, J. H. Anticancer Res. 2006, 26, 2791.
2. Pevarello, P.; Brasca, M. G.; Amici, R.; Orsini, P.; Traquandi, G.; Corti, L.; Piutti,
C.; Sansonna, P.; Villa, M.; Pierce, B. S.; Pulici, M.; Giordano, P.; Martina, K.;
Fritzen, E. L.; Nugent, R. A.; Casale, E.; Cameron, A.; Ciomei, M.; Roletto, F.;
Isacchi, A.; Fogliatto, G. P.; Pesenti, E.; Pastori, W.; Marsiglio, A.; Leach, K. L.;
Clare, P. M.; Fiorentini, F.; Varasi, M.; Vulpetti, A.; Warpehoski, M. A. J. Med.
Chem. 2004, 47, 3367.
8.10 (s, 1H), 8.16 (s, 1H), 8.69 (m, 3H). 13C NMR (CDCl
, 150 MHz) d (ppm):
3
38.33, 45.74, 55.37, 56.96, 114.55, 122.09, 122.36, 122.86, 123.94, 125.22,
126.33, 127.06, 127.34, 127.74, 129.46, 130.82, 131.10, 131.92, 140.07, 141.86,
159.02, 163.49, 164.02. Anal. Calcd for C26
12.46. Found: C, 69.51; H, 5.37; N, 12.29. ESI-MS m/z: 441.5 [M+H] . Compound
H
24
N
4
O
3
ꢃ0.5H
2
O: C, 69.47; H, 5.61; N,
+
ꢁ
1
4g (84%), light yellow solid, mp 145.2–145.8 °C. IR (KBr, cm ): 3112, 2944,
2777, 1697, 1658, 1519, 1473, 1396, 1240, 1176, 937, 779; 1H NMR (CDCl
600 MHz) d (ppm): 2.56 (s, 6H), 2.93(t, J = 7.2 Hz, 2H), 3.90(s, 3H), 4.46 (t,
J = 7.2 Hz, 2H), 6.90 (dd, J = 8.4 Hz, J = 1.8 Hz, 1H), 7.16 (s, 1H), 7.22 (d,
J = 7.8 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.84 (m, 2H), 8.18 (s, 1H), 8.20 (s, 1H),
8.67 (d, J = 8.4 Hz, 1H), 8.70 (d, J = 7.8 Hz, 2H). 13C NMR (CDCl
, 150 MHz) d
,
3
1
2
3
(ppm): 38.37, 45.78, 55.33, 57.00, 111.83, 112.47, 118.37, 122.29, 122.48,
122.90, 125.33, 126.38, 127.79, 128.14, 129.45, 130.15, 130.67, 131.04, 131.93,
132.74, 140.26, 141.75, 160.21, 163.44, 163.96. Anal. Calcd for C26H N O : C,
24 4 3
2
3. Norton, J. T.; Witschi, M. A.; Luong, L.; Kawamura, A.; Ghosh, S.; Stack, M. S.;
Sim, E.; Avram, M. J.; Appella, D. H.; Huang, S. Anti-Cancer Drugs 2008, 19, 23.
4. Yang, P.; Yang, Q.; Qian, X. Tetrahedron 2005, 61, 11895.
5. To a solution of 4-aryl-1H-pyrazole or 1H-pyrazole (1.25 mmol) in dimethyl
sulfoxide (DMSO) (1.5 mL), solid potassium tert-butoxide (1.38 mmol) was
added, the mixture was stirred at room temperature for 15 min, then a solution
70.89; H, 5.49; N, 12.72. Found: C, 70.68; H, 5.40; N, 12.51. ESI-MS m/z: 441.7
+
[M+H] . Compound 4h (88%), light yellow solid, mp 171.2–172.1 °C. IR (KBr,
ꢁ
1
2
2
cm ): 3116, 2948, 2763, 1695, 1652, 1583, 1508, 1475, 1398, 1247, 1025, 970,
782; 1H NMR (CDCl
, 600 MHz) d (ppm): 2.79 (s, 6H), 3.24(t, J = 7.2 Hz, 2H),
3.96 (s, 3H), 3.99 (s, 3H), 4.57 (t, J = 7.2 Hz, 2H), 6.97 (d, J = 7.8 Hz, 1H), 7.11 (d,
J = 1.8 Hz, 1H), 7.19 (dd, J = 8.4 Hz, J = 1.8 Hz, 1H), 7.85 (m, 2H), 8.14 (s, 1H),
8.16 (s, 1H), 8.70 (m, 3H). C NMR (CDCl , 150 MHz) d (ppm): 38.35, 45.76,
3
1
2
13
of N-(2-(dimethylamino)ethyl)-4-bromo-naphthalimide
DMSO (0.5 mL) was added through a syringe. The mixture was heated to
2 °C and kept at this temperature for 1.5 h. Then the mixture was cooled to
2
(1.31 mmol) in
3
56.03, 56.99, 109.48, 111.89, 118.40, 122.15, 122.36, 122.87, 124.34, 125.41,
126.34, 127.51, 127.74, 129.45, 130.76, 131.04, 131.90, 140.11, 141.80, 148.63,
7
room temperature and quenched with ice water (10 mL), the precipitate was
collected by filtration and oven-dried in vacuum. The crude product was
further purified by recrystallization from ethanol to give pure products 4a–4l
in excellent yields. Compound 4a (92%), light yellow solid, mp 138.3–139.0 °C.
149.51, 163.44, 163.96. Anal. Calcd for C27
N, 11.79. Found: C, 68.06; H, 5.32; N, 11.64. ESI-MS m/z: 471.5 [M+H] .
Compound 4i (91%), golden yellow solid, mp 184.6–186.8 °C. IR (KBr, cm ):
H
26
N
4
O
4
ꢃ0.25H
2
O: C, 68.27; H, 5.62;
+
ꢁ
1
2937, 2817, 2767, 1698, 1664, 1587, 1506, 1457, 1398, 1365, 1243, 1029, 998,
ꢁ
1
971, 836, 782. 1H NMR (CDCl
IR (KBr, cm ): 3100, 2958, 2763, 1697, 1652, 1585, 1425, 1398, 1376, 1344,
3
, 600 MHz) d (ppm): 2.72 (s, 6H), 3.15 (t,
1
1
242, 1118, 952, 758, 694; H NMR (CDCl
3
, 600 MHz) d (ppm): 2.39 (s, 6H),
J = 7.2 Hz, 2H), 3.92 (s, 3H), 3.97 (s, 6H), 4.54 (t, J = 7.2 Hz, 2H), 6.82 (s, 2H), 7.85
(m, 2H), 8.16 (s, 1H), 8.17 (s, 1H), 8.67 (d, J = 7.8 Hz, 2H), 8.71 (d, J = 7.8 Hz, 2H).
2
.71(t, J = 7.2 Hz, 2H), 4.38 (t, J = 7.2 Hz, 2H), 7.35 (t, J = 7.8 Hz, 1H), 7.47 (t,
13
J = 7.8 Hz, 2H), 7.64 (d, J = 7.2 Hz, 2H), 7.86 (m, 3H), 8.18 (s, 1H), 8.22 (s, 1H),
3
C NMR (CDCl , 150 MHz) d (ppm): 38.39, 45.78, 56.32, 57.01, 61.00, 103.46,
1
3
8
.67 (d, J = 8.4 Hz, 1H), 8.71 (d, J = 7.8 Hz, 2H). C NMR (CDCl
3
, 150 MHz) d
122.31, 122.48, 122.90, 125.59, 126.41, 127.15, 127.82, 127.97, 129.45, 130.68,
131.03, 131.95, 137.77, 140.19, 141.73, 153.85, 163.44, 163.96. Anal. Calcd for
(
ppm): 38.37, 45.74, 55.77, 56.99, 122.47, 122.89, 125.45, 125.86, 126.37,
1
1
27.32, 127.79, 127.99, 129.10, 129.44, 130.68, 131.05, 131.39, 131.92, 140.19,
41.77, 163.43, 163.96. Anal. Calcd for C25 O: C, 72.36; H, 5.47;
O2ꢃ0.25H
C H N O : C, 67.19; H, 5.64; N, 11.19. Found: C, 66.76; H, 5.47; N, 11.00. ESI-
28 28 4 5
+
H
22
N
4
2
MS m/z: 501.5 [M+H] . Compound 4j (91%), yellow solid, mp 190.7–192.1 °C. IR
+
ꢁ1
N, 13.50. Found: C, 72.49; H, 5.44; N, 13.35. ESI-MS m/z: 411.4 [M+H] ;
Compound 4b (93%). Golden yellow solid, mp 155.1–155.6 °C. IR (KBr, cm ):
(KBr, cm ): 3118, 2950, 2777, 1697, 1650, 1585, 1519, 1471, 1396, 1328,
ꢁ
1
1
1166, 1124, 1066, 838, 779. H NMR (CDCl
3
, 600 MHz) d (ppm): 2.55 (s, 6H),
3
8
124, 2958, 1700, 1654, 1583, 1513, 1473, 1427, 1398, 1367, 1232, 1168, 970,
2.92 (t, J = 7.2 Hz, 2H), 4.46 (t, J = 7.2 Hz, 2H), 7.73 (m, 4H), 7.86 (m, 2H), 8.25 (s,
1H), 8.26 (s, 1H), 8.63 (d, J = 8.4 Hz, 2H), 8.71 (d, J = 7.8 Hz, 2H). C NMR
27, 781, 750. 1
H
NMR (CDCl 600 MHz) (ppm): 2.39 (s, 6H), 2.71(t,
,
d
13
3
J = 7.2 Hz, 2H), 4.38 (t, J = 7.2 Hz, 2H), 7.16 (t, J = 8.4 Hz, 2H), 7.59 (td,
3
(CDCl , 150 MHz) d (ppm): 38.41, 45.78, 57.00, 122.67, 122.97, 123.23, 124.08,
J1 = 7.2 Hz, J1 = 3.6 Hz, 2H), 7.85 (m, 2H), 8.13 (s, 1H), 8.17 (s, 1H), 8.66 (d,
125.03, 125.94, 126.09, 126.43, 127.95, 128.63, 129.16, 129.43, 130.42, 130.99,
132.01, 135.06, 140.12, 141.49, 163.38, 163.90. Anal. Calcd for
1
3
J = 8.4 Hz, 1H), 8.71 (d, J = 7.8 Hz, 2H). C NMR (CDCl
3
, 150 MHz) d (ppm):
8.38, 45.77, 57.00, 115.98 116.12, 122.35, 122.48, 122.92, 124.55, 126.38,
27.45 127.50, 127.57 127.59, 127.82 127.85, 129.45, 130.59, 131.03, 131.94,
40.04, 141.68, 161.32, 162.95, 163.41, 163.94. Anal. Calcd for C25 F: C,
0.08; H, 4.94; N, 13.08. Found: C, 69.70; H, 5.02; N, 12.84. ESI-MS m/z: 429.4
3
1
1
7
C
26
H
21
N
4
O
2
F
3
ꢃ0.25H
2
O: C, 64.66; H, 4.49; N, 11.60. Found: C, 64.70; H, 4.30;
+
N, 11.49. ESI-MS m/z: 479.4 [M+H] . Compound 4k (86%), white solid, mp
ꢁ
1
H
21
N
4
O
2
180.5–181.1 °C. IR (KBr, cm ): 3102, 2962, 2796, 1695, 1656, 1587, 1382,
1351, 1276, 1176, 1122, 941, 781, 703. 1H NMR (CDCl
3
, 600 MHz) d (ppm): 2.58
+
[
M+H] ; Compound 4c (95%), light yellow solid, mp 174.8–175.4 °C. IR (KBr,
(s, 6H), 2.96 (t, J = 7.2 Hz, 2H), 4.47 (t, J = 7.2 Hz, 2H), 7.29 (s, 1H), 7.87 (m, 2H),
ꢁ
1
cm ): 3108, 2971, 1700, 1658, 1583, 1471, 1432, 1396, 1240, 1097, 971, 856,
8.04 (s, 2H), 8.29 (s, 1H), 8.32 (s, 1H), 8.58 (d, J = 8.4 Hz, 2H), 8.72 (d, J = 7.8 Hz,
86, 754; 1H NMR (CDCl
H), 4.38 (t, J = 7.2 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.85
, 600 MHz) d (ppm): 2.39 (s, 6H), 2.71(t, J = 7.2 Hz,
13
7
2
3
3
2H). C NMR (CDCl , 150 MHz) d (ppm): 38.39, 45.75, 56.99, 120.71, 122.33,
122.86, 123.02, 124.14, 125.69, 126.46, 128.11, 128.98, 129.41, 130.19, 130.92,
132.08, 132.46, 132.68, 133.83, 139.87, 141.22, 163.34, 163.86. Anal. Calcd for
(
m, 2H), 8.17 (s, 1H), 8.19 (s, 1H), 8.64 (d, J = 8.4 Hz, 1H), 8.71 (d, J = 7.8 Hz, 2H).
1
3
C NMR (CDCl
3
, 150 MHz) d (ppm): 38.39, 45.77, 57.00, 122.43, 122.53,
C
27
H
20
N
4
O
2
F
6
ꢃ0.25H
2
O: C, 58.38; H, 3.81; N, 10.09. Found: C, 58.62; H, 3.50; N,
+
1
1
C
22.94, 124.34, 126.38, 127.06, 127.85, 128.05, 129.27, 129.45, 129.94, 130.53,
31.00, 131.95, 133.04, 140.01, 141.61, 163.39, 163.91. Anal. Calcd for
9.97. ESI-MS m/z: 547.4 [M+H] . Compound 4l (96%), light yellow solid, mp
ꢁ
1
141.1–142.0 °C. IR (KBr, cm ):3118, 2971, 2771, 1697, 1654, 1587, 1515,
1396, 1371, 1238, 1043, 790, 755; 1H NMR (d
-CDCl , 600 MHz) d (ppm): 2.37
(s, 6H), 2.68 (t, J = 7.2 Hz, 2H), 4.35 (t, J = 7.2 Hz, 2H), 6.64 (t, J = 1.8 Hz, 1H),
7.78 (m, 2H), 7.94 (s, 2H), 8.55 (d, J = 8.4 Hz, 2H), 8.66 (d, J = 7.8 Hz, 2H).
NMR (CDCl , 150 MHz) d (ppm): 38.35, 45.76, 56.98, 108.05, 122.21, 122.64,
122.85, 126.55, 127.73, 129.40, 130.70, 131.06, 131.52, 131.88, 141.99, 142.37,
163.47, 163.99. C19 O: C, 67.34; H, 5.50; N, 16.53. Found: C,
25
H
21
N
4
O
2
Clꢃ0.25H
2
O: C, 66.81; H, 4.82; N, 12.47. Found: C, 66.97 H, 4.92;
6
3
+
N, 12.29. ESI-MS m/z: 445.6 [M+H] ; 4d (96%), light yellow solid, mp 177.8–
ꢁ
1
13
1
1
3
80.3 °C. IR (KBr, cm ): 3106, 2960, 1702, 1658, 1583, 1471, 1432, 1396, 1365,
240, 1062, 971, 856, 786, 754. H NMR (CDCl
.28 (t, J = 7.2 Hz, 2H), 4.59 (t, J = 7.2 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.59 (d,
C
1
3
, 600 MHz) d (ppm): 2.82 (s, 6H),
3
J = 8.4 Hz, 2H), 7.85 (m, 2H), 8.18 (s, 1H), 8.19 (s, 1H), 8.65 (d, J = 7.8 Hz, 1H),
H
18
N
4
O
2
ꢃ0.25H
2
1
3
+
8
1
1
.71 (d, J = 7.2 Hz, 2H). C NMR (CDCl
3
, 150 MHz) d (ppm): 38.38, 45.79, 56.99,
67.65; H, 5.28; N, 16.26. ESI-MS m/z: 335.3 [M+H] .
21.04, 122.43, 122.56, 122.92, 124.35, 126.37, 127.37, 127.89, 128.05, 129.44,
30.38, 130.56, 131.03, 131.99, 132.22, 139.99, 141.60, 163.60, 163.95. Anal.
26. Mosmann, T. J. Immun. Meth. 1983, 65, 55.
27. Selvi, P. T.; Palaniandavar, M. Inorg. Chim. Acta 2002, 337, 420.
28. Xie, L.; Xu, Y.; Wang, F.; Liu, J.; Qian, X.; Cui, J. Bioorg. Med. Chem. 2009, 17, 804.
29. Pyle, A. M.; Rehmann, J. P.; Meshoyrer, R.; Kumar, C. V.; Turro, N. J.; Barton, J. K.
J. Am. Chem. Soc. 1989, 111, 3051.
30. (a) Suh, D.; Chaires, J. B. Bioorg. Med. Chem. 1995, 3, 723; (b) Yang, X.; Liu, W.;
Jin, W.; Shen, G.; Yu, R. Spectrochim. Acta. A 1999, 55, 2719; (c) Banerjee, S.;
Kitchen, J. A.; Gunnlaugsson, T.; Kelly, J. M. Org. Biomol. Chem. 2012, 10, 3033;
(d) Banerjee, S.; Kitchen, J. A.; Gunnlaugsson, T.; Kelly, J. M. Org. Biomol. Chem.
2013, 11, 5642.
31. Jiang, X.; Shang, L.; Wang, Z.; Dong, S. Biophys. Chem. 2005, 118, 42.
32. Maheswari, P. U.; Palaniandavar, M. J. Inorg. Biochem. 2004, 98, 219.
33. Norden, B.; Kurucsev, T. J. Mol. Recognit. 1994, 7, 141.
34. Schipper, P. E.; Norden, B.; Tjerneld, F. Chem. Phys. Lett. 1980, 70, 17.
35. Cao, Y.; He, X. Spectrochim. Acta Part A 1998, 54, 883.
Calcd for C25
H
21
N
4
O
2
Brꢃ0.25H
2
O: C, 60.80; H, 4.39; N, 11.34. Found: C, 60.79;
+
H, 4.40; N, 11.19. ESI-MS m/z: 491.1 [M+H] . Compound 4e (95%), light yellow
solid, mp 180.5–181.3 °C. IR (KBr, cm ): 3112, 2973, 2821, 1698, 1654, 1396,
ꢁ
1
1
1
2
2
241, 1174, 1118, 975, 779; H NMR (CDCl
.42(s, 3H), 2.71 (t, J = 7.2 Hz, 2H), 4.38 (t, J = 7.2 Hz, 2H), 7.27 (d, J = 8.4 Hz,
H), 7.53 (d, J = 8.4 Hz, 2H), 7.85 (m, 2H), 8.15 (s, 1H), 8.20 (s, 1H), 8.69 (d,
3
, 600 MHz) d (ppm): 2.39 (s, 6H),
1
3
J = 7.8 Hz, 1H), 8.71 (d, J = 7.8 Hz, 2H). C NMR (CDCl
3
, 150 MHz) d (ppm):
1.18, 38.36, 45.77, 56.99, 122.16, 122.39, 122.88, 125.45, 125.76, 126.35,
27.69, 127.75, 128.46, 129.46, 129.77, 130.77, 131.07, 131.91, 137.12, 140.18,
41.84, 163.46, 163.99. Anal. Calcd for C26 O: C, 72.04; H, 5.81; N,
ꢃ0.5H
2.92. Found: C, 72.18; H, 5.59; N, 12.70. ESI-MS m/z: 425.5 [M+H] . Compound
2
1
1
1
4
2
H
24
N
4
O
2
2
+
ꢁ
1
f (90%); light yellow solid, mp 156.7–157.8 °C. IR (KBr, cm ): 3108, 2964,
765, 1698, 1660, 1583, 1508, 1471, 1243, 1180, 1027, 833, 779. 1H NMR
(
CDCl
3
, 600 MHz) d (ppm): 2.39 (s, 6H), 2.71(t, J = 7.2 Hz, 2H), 3.88 (s, 3H), 4.38
36. Wang, K.; Wang, Y.; Yan, X.; Chen, H.; Ma, G.; Zhang, P.; Li, J.; Li, X.; Zhang, J.
Bioorg. Med. Chem. Lett. 2012, 22, 937.
(
t, J = 7.2 Hz, 2H), 7.00 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.84 (m, 2H),