Synthesis and in vitro antiproliferative activity of new 11-aminoalkylamino-substituted 5H- and 6H-indolo[2,3-b]quinolines; Structure-activity relationships of neocryptolepines and 6-methyl congeners

Article abstract of DOI:10.1016/j.bmc.2012.05.054

The present report describes the synthesis and antiproliferative evaluation of certain 11-aminoalkylamino-substituted 5H- and 6H-indolo[2,3-b]quinolines and their methylated derivatives. These 5-Me- and 6-Me-indolo[2,3-b]quinoline derivatives 10-14, 20 were prepared by amination at the C-11 position of the 11-chloro-5-methyl-5H- and 11-chloro-6-methyl-6H-indolo[2,3-b]quinolines with different substituents on the quinoline ring. The 11-aminoalkylaminomethylated 23, the homologue of 11, was prepared from the same intermediate for a further SAR study. These intermediates are accessible from 4-substituted anilines or their N-methylated analogues and methyl indole-3-carboxylate as a counterpart. The in vitro antiproliferative assay indicated that the 5-methylated derivatives 10-14 are more cytotoxic than their respective 6-methylated 6H-indolo[2,3-b]quinoline derivatives 20. Among them, N-(3-aminopropyl)-2-bromo- 5-methyl-5H-indolo[2,3-b]quinolin-11-amine 12f was the most cytotoxic with a mean IC₅₀ value of 0.12 μM against human leukemia MV4-11 cell line, and also exhibited selective cytotoxicities against A549 (lung cancer), HCT116 (colon cancer) cell lines and normal fibroblast BALB/3T3 with IC ₅₀ values of 0.543, 0.274 and 0.869 μM, respectively. The binding constant of products 12f and 20f to salmon fish sperm DNA were also evaluated using UV-vis absorption spectroscopy, indicating intercalation binding with a constant of 2.93 × 10⁵ and 3.28 × 10⁵ L mol^-1, respectively.

Full text of DOI:10.1016/j.bmc.2012.05.054

Bioorganic & Medicinal Chemistry 20 (2012) 4820–4829
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro antiproliferative activity of new
11-aminoalkylamino-substituted 5H- and 6H-indolo[2,3-b]quinolines;
structure–activity relationships of neocryptolepines and 6-methyl congeners
a
b
a
a
a
a
´
Li Wang , Marta Switalska , Zhen-Wu Mei , Wen-Jie Lu , Yoshito Takahara , Xing-Wen Feng ,
Ibrahim El-Tantawy El-Sayed a,c, Joanna Wietrzyk b, , Tsutomu Inokuchi
a,
⇑
⇑
a Division of Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
b Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12, R. Weigl Street, 53-114 Wroclaw, Poland
c Chemistry Department, Faculty of Science, El Menoufeia University, Shebin El Koom, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The present report describes the synthesis and antiproliferative evaluation of certain 11-aminoalkylami-
no-substituted 5H- and 6H-indolo[2,3-b]quinolines and their methylated derivatives. These 5-Me- and
6-Me-indolo[2,3-b]quinoline derivatives 10–14, 20 were prepared by amination at the C-11 position of the
11-chloro-5-methyl-5H- and 11-chloro-6-methyl-6H-indolo[2,3-b]quinolines with different substituents
on the quinoline ring. The 11-aminoalkylaminomethylated 23, the homologue of 11, was prepared from
the same intermediate for a further SAR study. These intermediates are accessible from 4-substituted ani-
lines or their N-methylated analogues and methyl indole-3-carboxylate as a counterpart. The in vitro
antiproliferative assay indicated that the 5-methylated derivatives 10–14 are more cytotoxic than their
respective 6-methylated 6H-indolo[2,3-b]quinoline derivatives 20. Among them, N-(3-aminopropyl)-2-
bromo-5-methyl-5H-indolo[2,3-b]quinolin-11-amine 12f was the most cytotoxic with a mean IC50 value
Received 1 May 2012
Revised 24 May 2012
Accepted 27 May 2012
Available online 12 June 2012
Keywords:
5H-Indolo[2,3-b]quinolines
6H-Indolo[2,3-b]quinolines
Anticancer agents
Antiproliferative acivity
of 0.12
A549 (lung cancer), HCT116 (colon cancer) cell lines and normal fibroblast BALB/3T3 with IC50 values of
0.543, 0.274 and 0.869 M, respectively. The binding constant of products 12f and 20f to salmon fish
lM against human leukemia MV4-11 cell line, and also exhibited selective cytotoxicities against
l
sperm DNA were also evaluated using UV–vis absorption spectroscopy, indicating intercalation binding
with a constant of 2.93 Â 105 and 3.28 Â 105 L molÀ1, respectively.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Nature is a treasured source to find and develop new therapeu-
tic agents.1 Indoloquinolines are unique natural alkaloids found
almost exclusively in the West African climbing shrub Cryptolepis
sanguinolenta which are used in traditional medicine for the
treatment of infectious diseases, including malaria.2 Among the
various isolated products with indoloquinoline structures,3 cry-
ptolepine i (5-methylindolo[3,2-b]quinoline), and neocryptolepine
ii (5-methylindolo[2,3-b]quinoline) (Fig. 1), a minor alkaloid, have
been intensively studied for their biological activities, namely
antibacterial,4a–c antifungal,4a–c and antimalarial.4d–f
N
N
N
N
CH3
CH3
ii
i
Cryptolepine
Neocryptolepine
CH3
Due to the linearly arranged tetracyclic plane structure, cryptol-
epine i is a DNA intercalating agent and inhibits topoisomerase II,
showing a high level of cytotoxicity.5 On the other hand, neocry-
ptolepine ii, a regioisomer of i in the orientation of indole moiety
to the quinoline ring, can also intercalate into DNA, but its affinity
N
N
H
N
N
CH3
CH3
iv
iii
6-Methylindolo[2,3-b]quinoline
Ellipticine
⇑
Corresponding authors.
E-mail addresses: wietrzyk@iitd.pan.wroc.pl (J. Wietrzyk), inokuchi@cc.okaya-
Figure 1. Structures of cryptolepine i, neocryptolepine ii, 6-methylindolo[2,3-
b]quinoline iii, and ellipticine iv.
ma-u.ac.jp (T. Inokuchi).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.054

L. Wang et al. / Bioorg. Med. Chem. 20 (2012) 4820–4829
4821
is slightly weaker compared with i, and its capability to inhibit
2. Chemistry
topoisomerase II is reduced.5c
Regarding the structurally related anticancer drug ellipticine iv
(Fig. 1),6 a tetracyclic 2-aza analogue with the same indole core, a
series of 5,11-dimethylindolo[2,3-b]quinolines derivatives have
been tested for their chemotherapeutic activities,7 showing a cyto-
toxicity against several human cancer cell lines, with IC50 value
The preparations of the 11-chloro-5-methyl-5H-indolo[2,3-
b]quinolines 5–9, the scaffold for the synthesis of the 11-amino
derivatives 10À14 are shown in Scheme 1.11 Thus, the N-methyl-
anilines 1 and methyl indole-3-carboxylate 2 were oxidatively
combined by the action of N-chlorosuccinimide (NCS), giving 3,
which was converted into the tetracyclic ketones 4 upon heating
at 250 °C in diphenyl ether. The treatment of 4 with POCl3 afforded
the desired chlorides 5À9.11c
ranging from 0.6 to 1.4 l
M.7c Furthermore, the 6,11-dimethylindo-
lo[2,3-b]quinolines,8 the 6H congener of neocryptolepine, showed
a cytotoxicity against KB cell lines with IC50 value ranging from
2.1 to 9 l
M.8b In these studies, the substituents, such as the Me
On the other hand, the 11-chloro-6-methyl congeners 19 were
prepared starting from anilines 15. Thus, the treatment of 16, pre-
pared from 15 and 2 by oxidation with NCS, with NaH–MeI in THF
afforded the 2-arylamino-1-methylindoles-3-carboxylates 17 as a
result of methylation at the nitrogen of the indole ring.11c The
cyclization of 17 to 18 was smoothly achieved by heating at reflux
in diphenyl ether, and the subsequent dehydroxy chlorination of
the resulting 18 with POCl3 afforded the desired 19.
As shown in Schemes 1 and 2, these 11-chloro-5-methyl- and
11-chloro-6-methylindolo[2,3-b]quinolines 5–9 and 19 were con-
verted into the corresponding 11-amino derivatives 10–14 and
20, respectively. The results shown in Table 1 and 3 indicate that,
in general, the 11-chloroneocryptolepines 5–9 are prone to under-
go addition-elimination reactions of the amine nucleophiles of the
linear and cyclic primary amines at 70–130 °C, giving 10–14 in
good to excellent yields. On the other hand, the amination of the
6-methyl congeners 19 with linear and cyclic primary amines were
performed in DMF at 120 °C for a longer reaction time, giving the
corresponding amine adducts 20 in slightly lower yields.
and MeO groups, introduced into the indolo[2,3-b]quinoline core
were shown to have highly infectious activities. Later, the 11-ani-
linoindolo[2,3-b]quinolines were synthesized and their anticancer
activity as bioisosteric compounds of acridine derivatives were
evaluated,9 which have been extensively studied as potential che-
motherapeutic agents due to their capability of intercalating DNA
that leading to the inhibition of mammalian topoisomerase II.
However, until now, the types of examined substituents on the
11-aminoindolo[2,3-b]quinolines have been limited, and the effect
of halogens, such as Cl and Br, were scarcely discussed. Recently,
aminoalkylamino-substituted neocryptolepine was shown to be
1500-fold efficacious in comparison to the natural product itself
against the chloroquine-sensitive Plasmodium falciparum Ghana
strain.10 Based on these facts, we examined the introduction of
the alkylamino and aminoalkylamino groups at the C-11 positions
of indolo[2,3-b]quinoline skeleton with varying the kinds of
substituents at the C-2 positions. In this study, we investigated
the effects of substituents types at the C-2 and C-11 positions on
antiproliferative activity in vitro in the 5-methyl-ii and 6-methy-
lindolo[2,3-b]quinoline series iii.
In addition to these 11-aminoalkylamino derivatives 10–14 and
20, we prepared the 11-aminoalkylaminomethylated 23, the
R1
O
MeO2C
R1
R1
MeO2C
i
ii
N
H
N
N
N
N
H
2
H
NHMe
1
Me
3
Me
4
R2
Cl
iii R1
R1
iv
D
5, 10
R
1= H
C
N
A
B
N
6, 11 R1= Cl
7, 12 R1= Br
8, 13 R1= F
9, 14 R1= OMe
N
N
Me
Me
10-14
5-9
Scheme 1. Preparation of 11-chloroneocryptolepines 5–9 and their 11-amino derivatives 10–14. Reagents and conditions: (i) (a) N-chlorosuccinimide, 1, 4-dimethylpi-
perazine. (b) trichloroacetic acid; (ii) diphenyl ether, reflux; (iii) POCl3, toluene, reflux; (iv) appropriate amines.
R1
MeO2C
R1
MeO2C
R1
i
v
ii
2
N
N
N
N
H
H
Me
NH2
H
17
15
16
R2
N
Cl
O
iii R1
R1
R1
iv
D
R1 =Cl
C
N
A
B
N
N
N
N
H
Me
Me
Me
19
20
18
Scheme 2. Preparation of 11-chloro-6-methylindolo[2,3-b]quinolines 19 and their 11-amino derivatives 20. Reagents and conditions: (i) (a) N-chlorosuccinimide, 1, 4-
dimethylpiperazine; (b) trichloroacetic acid; (ii) diphenyl ether, reflux; (iii) POCl3, toluene, reflux; (iv) appropriate amines; (v) NaH, MeI, THF.

4822
L. Wang et al. / Bioorg. Med. Chem. 20 (2012) 4820–4829
Table 1
Yields of 11-alkylaminated 5-methyl-indolo[2,3-b]quinolines and their antiproliferative activity against human leukemia MV4-11 cell line
Compound
R1
R2
Yield (%)
MV4-11
IC50 M)
(
l
Cisplatin
Doksorubicin HCl
5
7
9
2.820 0.450
0.006 0.002
1.312 0.262
0.810 0.145
1.206 0.182
H
Br
OMe
Cl
Cl
Cl
10a
H
N
98
0.721 0.154
HN
NH
10b
10c
H
H
90
94
0.871 0.256
0.170 0.010
HN
HN
OH
N
10d
H
94
7.171 0.987
N
NH2
HN
HN
10e
10f
H
H
96
45
0.100 0.035
0.066 0.023
NH2
O
10g
11a
H
92
55
0.164 0.047
0.780 0.142
N
Cl
N
HN
NH
11b
11c
Cl
Cl
67
27
0.447 0.212
0.530 0.118
HN
HN
OH
N
11d
Cl
48
0.106 0.053
N
NH2
HN
HN
11e
11f
Cl
Cl
86
97
0.680 0.028
0.068 0.018
NH2
12a
12c
Br
Br
N
64
70
0.578 0.043
0.703 0.088
HN
HN
OH
N
12d
Br
82
0.737 0.208
N
NH2
HN
HN
12e
12f
13e
14c
14e
14f
Br
96
63
94
66
79
80
0.091 0.015
0.012 0.002
0.095 0.012
0.135 0.052
0.132 0.017
0.102 0.021
NH2
NH2
Br
HN
HN
F
OH
NH2
OMe
OMe
OMe
HN
HN
NH2

L. Wang et al. / Bioorg. Med. Chem. 20 (2012) 4820–4829
4823
Table 2
3. Biological results and discussion
Antiproliferative activity of 11-alkylaminated 5-methyl-indolo[2,3-b]quinolines
against normal mice fibroblast BALB/3T3 and against cancer cell lines A549 and
HCT116
The results of the cytotoxic activity in vitro were expressed as
IC50-the concentration of the compound (in M) that inhibits pro-
liferation of the cells by 50% as compared to the untreated control
cells. The IC values were separately calculated for each experiment
and the mean values SD were calculated from at least 3–5 inde-
pendent experiments.
The results of the studies on the antiproliferative activity of the
neocryptolepine analogues against human leukemia MV4-11 cells
are summarized in Table 1, along with the results of anticancer
drugs, cisplatin and doksorubicin HCl. All tested compounds, ex-
cept the 10d, were cytotoxic against the MV4-11 leukemia cells
l
Compound
BALB/3T3
IC50 M)
A549
IC50
HCT116
IC50 M)
(l
(lM)
(l
Cisplatin
Doksorubicin HCl
8.700 0.970
1.078 0.033
0.635 0.190
0.936 0.069
0.884 0.115
1.607 0.624
6.413 2.278
0.401 0.015
1.197 0.807
7.600 0.260
0.742 0.091
0.869 0.018
0.853 0.104
0.807 0.054
1.622 0.537
0.978 0.021
9.870 2.400
0.329 0.097
0.973 0.239
0.911 0.113
0.205 0.079
1.509 0.410
7.390 3.880
0.761 0.169
1.587 0.729
4.535 2.031
0.579 0.204
0.543 0.256
0.785 0.169
1.010 0.069
0.674 0.189
0.407 0.117
8.500 0.540
0.390 0.098
0.714 0.085
0.669 0.229
0.302 0.056
1.795 0.277
1.750 0.335
0.195 0.044
0.989 0.234
1.160 0.097
0.209 0.111
0.274 0.050
0.178 0.048
0.278 0.160
0.657 0.382
0.155 0.042
10c
10e
10f
10g
11d
11f
12c
12d
12e
12f
13e
14c
14e
14f
(IC50 below 0.9
than the activity of the known anticancer drug, cisplatin (IC50
2.82 M). The most potent of all the tested compounds was 12f
(IC50 0.012 M) with a 3-aminopropylamino group at the 11-posi-
lM), and their antiproliferative activity is higher
l
l
tion. A very high antiproliferative activity was also revealed for
compounds 10c, 10e–g, 11b, 11d, 11f, 12e, 13 and 14 of IC50
between 0.06–0.45 lM. A halogen substituent at the 2-position
can influence the antiproliferative activity. In some cases, the
halogen substitution can increase the antiproliferative activity.
Thus, The 11-aminopropylamino-2-bromo-indolo[2,3-b]quinoline
12f was about 4 times more active than the corresponding 11-
aminopropylamino-2H-indolo[2,3-b]quinoline 10f against MV4-
11 cells. The same trend was also observed for compounds 10b
and 11b. On the contrary, for the 3-hydroxypropylamino-substi-
tuted agents 10c, 11c, 12c and 14c, the antiproliferative activity
decreased about 4 times when the H at the C2 was changed by
Cl or Br. Good antiproliferative activity against MV4-11 cells was
also observed when an electron-donating group –MeO was intro-
duced as a substituent at the C-2 position.
Table 3
Yields of 11-alkylaminated 6-methyl-indolo[2,3-b]quinolines and their antiprolifer-
ative activity against human leukemia MV4-11 cell line
Compound
R1 R2
Yield (%) MV4-11
IC50 M)
(l
Cisplatin
Doksorubicin HCl
2.820 0.450
0.006 0.002
20a
Cl
N
27
5.248 0.804
HN
NH
Accordingly, these compounds, 10c, 10e–g, 11d, 11f, 12c–f, 13
and 14, were then chosen for next study: the antiproliferative activ-
ity against human lung (A549) and colon (HCT116) cancer cell lines
and normal murine fibroblasts (BALB/3T3). These results are sum-
marized in Table 2. All the tested compounds were cytotoxic against
the A549 and HCT116 cancer cells, and their antiproliferative activ-
ities against the cancer cells were much higher than the activity of
cisplatin used as control agent (Table 2). Compound 10f, 12e, 12f
and 14f have high antiproliferative activities against cancer cell
lines A549 and also HCT116, but a lower cytotoxicity. Against the
normal fibroblast BALB/3T3, their antiproliferative activities were
3–4 times lower than that against cancer cells. Compound 11d,
11f, 13e and 14c have a selective antiproliferative activity, mostly
against the HCT116 cell line. Against the A549 cancer cell line and
normal fibroblast BALB/3T3 cell line, the activities were about 4
times lower. It is quite obvious that the introduction of proper alkyl-
amino substituents into biologically active derivatives can favorably
influence their activities and selectivities in DNA binding.
An SAR study of indolo[2,3-b]quinoline from series 5H- and 6H-
series showed that the 6,11-dimethyl-6H-indolo[2,3-b]quinoline is
an inactive isomer of 5,11-dimethyl-5H-indolo[2,3-b]quinoline
(DIMIQ).7c Therefore, we hoped that the 6H-series might acquire
an activity when proper substituent was introduced.
Similarly, the antiproliferative activity of the 6-methylated
congeners 20 were tested and summarized in Tables 3 and 4. Gen-
erally, the 6-methylated derivatives were still less cytotoxic than
their respective 5-methylated analogues. The activities of 6,11-di-
methyl-6H-indolo[2,3-b]quinoline series were improved when
proper amino groups were introduced at the C-11. Only com-
pounds 20b and 20g were not cytotoxic against MV4-11 leukemia
cells. Compounds 20d-f, and 20i showed the IC50 value between
20b
20c
Cl
Cl
28
70
18.827 3.106
6.209 0.824
HN
HN
OH
N
20d
Cl
35
0.773 0.346
N
NH2
HN
HN
20e
20f
Cl
Cl
26
61
0.669 0.312
0.800 0.159
NH2
O
20g
Cl
27
>25
N
N
20h
20i
Cl
Cl
44
50
2.834 0.876
0.456 0.123
HN
NH2
HN
N
23d
23j
Cl
Cl
36
56
3.436 0.585
3.465 0.814
N
HN
N
homologue of 11, from 6 for a SAR study (Scheme 3). Thus, the
reaction of 6 with sodium nitromethylate afforded the adduct of
nitromethane, 21, which was treated with KMnO4 to give the
C-11 formylated 22. The reductive amination of 22 with the appro-
priate amines using NaBH4 as a hydride donor afforded 23.
0.46–0.80
lM against MV4-11 leukemia cells, which were higher
than the known anticancer drug cisplatin.

4824
L. Wang et al. / Bioorg. Med. Chem. 20 (2012) 4820–4829
R2
NO2
CHO
Cl
Cl
Cl
iii
i
ii
6
N
N
N
N
N
N
Me
Me
Me
23
22
21
Scheme 3. Preparation of 11-aminoalkylaminomethylated neocryptlepine 23. Reagents and conditions: (i) CH3NO2, NaH; (ii) KMnO4; (iii) appropriate amines, NaBH3CN.
compared to the 11-aminated neocryptolepines or their congeners
(11d, 20d and 23d).
Table 4
Antiproliferative activity of 11-alkylaminated 6-methylindolo[2,3-b]quinolines
against normal mice fibroblast BALB/3T3 and against cancer cell lines A549 and
HCT116
Indeed, according to literature data, the inactive isomer of 6,11-
dimethyl-6H-indolo[2,3-b]quinoline (DIMIQ) can acquire activity
after introduction of substituents.9 On the other hand, introduction
of proper aminoalkyl substituents into biologically active deriva-
tives can favor its activity and selectivity in DNA binding.8b How-
ever, the cytotoxicity against normal in relation to cancer cells of
these compounds was not described. Therefore, obtained new
derivatives of indolo[2,3-b]quinoline possessing a high antiprolif-
erative activity against cancer cells accompanied by a low cytotox-
icity against normal fibroblasts can offer a new valuable group of
potential anticancer compounds.
Compound
BALB/3T3 IC50
M)
A549 IC50
M)
HCT116 IC50
M)
(l
(l
(l
Cisplatin
Doksorubicin
HCl
20d
20e
8.700 0.970
1.078 0.033
9.870 2.400
0.329 0.097
8.500 0.540
0.390 0.098
>25
11.24 0.950
6.093 0.595
7.378 0.974
8.282 0.585
8.000 0.369
5.498 2.182
4.486 1.417
6.989 1.416
9.012 1.304
9.798 0.354
10.437 0.400
20f
20i
Further studies are needed to explain the mechanism of action
of the obtained derivatives with selective activity against cancer
cells.
From the results summarized in Table 4, we can see that the
antiproliferative activities of these four compounds against human
lung (A549) and colon (HCT116) cancer cell lines are comparable
with the cisplatin. Against normal fibroblast BALB/3T3, compound
20d showed selectivity, which was not cytotoxic (IC50 higher than
4. Spectroscopic characterization of neocryptolepine derivative
12f and 20f interacting with salmon fish sperm DNA
25
lM).
The 11-aminoalkylaminomethylated neocryptolepine deriva-
The DNA binding studies of compounds 12f or 20f was per-
formed using UV–vis absorption spectroscopy with salmon fish
sperm DNA in phosphate buffer of pH 7.0 at 20 °C. The red shift
and hypochromic effect were observed in the absorption spectra
while the DNA solution was gradually added to the solution of
tives 23, the homologs of 20, were also tested, the results of which
are listed in Table 3. Though the 11-aminoalkylaminomethylated
neocryptolepine derivatives 23 were also cytotoxic against MV4-
11 leukemia cells, the activity was significantly decreased when
(a)
(b)
(c)
(d)
Figure 2. (a) UV–vis absorption spectra of compound 12f and 12f-DNA at 20 °C. C12f = 50 lmol/L, CDNA = 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 lmol/L for curve 1–11 in
pH 7.0 phosphate buffer solution; (b) the plot of 1/
D
A vis 1/[DNA] for 12f-DNA; (c) UV–vis absorption spectra of compound 20f and 20f-DNA at room temperature.
C20f = 50 mol/L, CDNA = 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0
l
l
mol/L for curve 1–11 in pH 7.0 phosphate buffer solution; (d) the plot of 1/ A vis 1/[DNA] for 20f-DNA.
D

L. Wang et al. / Bioorg. Med. Chem. 20 (2012) 4820–4829
4825
the compound 12f or 20f. The results in Figure 2 showed that the
absorption band at 349 nm for the 12f (a) decreased while increas-
ing the DNA concentration. The maximum absorption shifted from
349 nm to a longer wavelength at 356 nm. It illustrated that the
mode of 12f binding to DNA was intercalation. Then the binding
constant of 12f-DNA and 20f-DNA was calculated as 2.93 Â 105
and 3.28 Â 105 L molÀ1, respectively, according to double-recipro-
cal equation.13
J = 1.17 Hz, 1H), 7.51 (d, J = 7.92 Hz, 1H), 7.80 (m, 1H), 7.86 (t,
J = 6.90 Hz, 2H), 8.58 (dd, J = 8.36, 1.03 Hz, 1H); 13C NMR
(150 MHz, DMSO-d6) d ppm 11.5, 21.6, 23.4, 32.2, 36.2, 46.0,
51.9, 52.6, 106.1, 115.0, 116.0, 116.6, 117.9, 120.6, 121.7, 124.1,
124.2, 124.9, 130.6, 137.4, 148.3, 152.5, 156.4; HRMS (ESI) Calcd
for C25H31N4 [MÀH]À 387.2554. Found 387.2522.
6.1.2.2.
b]quinolin-11-amine (10b).
201–203 °C; 1H NMR (600 MHz, DMSO-d6)
N-(2-(1H-Indol-3-yl)ethyl)-5-methyl-5H-indolo[2,3-
Yield: 90%, yellow solid, Mp:
ppm 3.12 (t,
d
5. Conclusion
J = 7.63 Hz, 2H), 4.09–4.15 (m, 2H), 4.16 (s, 3H), 6.91 (ddd,
J = 7.92, 7.04, 0.88 Hz, 1H), 6.98–7.05 (m, 2H), 7.08 (d, J = 2.35 Hz,
1H), 7.12 (t, J = 5.87 Hz, 1H), 7.25–7.28 (m, 1H), 7.28–7.31 (m,
1H), 7.41 (ddd, J = 8.22, 7.04, 1.17 Hz, 1H), 7.47 (d, J = 7.92 Hz,
1H), 7.50 (d, J = 7.92 Hz, 1H), 7.77–7.81 (m, 1H), 7.82–7.85 (m,
1H), 7.87 (d, J = 7.63 Hz, 1H), 8.54 (dd, J = 8.36, 1.03 Hz, 1H),
10.80 (br s, 1H); 13C NMR (150 MHz, DMSO-d6) d ppm 26.7, 32.2,
48.6, 104.7, 110.9, 111.4, 115.0, 115.6, 116.5, 117.9, 118.2, 118.3,
120.5, 121.0, 121.9, 122.9, 124.0, 124.1, 124.6, 127.0, 130.6,
136.1, 137.4, 148.1, 152.3, 156.4; HRMS (ESI) Calcd for C26H21N4
[MÀH]À 389.1772. Found 389.1760.
We have described the synthesis of a series of 11-amino-substi-
tuted 5H- and 6H-indolo[2,3-b]quinolines, whose antiproliferative
activities were evaluated using the MV4-11 (human leukemia),
A549 (human lung cancer), HCT116 (human colon cancer), and
normal mice fibroblast (BALB/3T3) cell lines. A few of the tested
compounds showed very high antiproliferative activities against
the MV4-11 leukemia cell line (IC50: 0.012–0.450 lM). Some drug
candidate compounds showed selective antiproliferative activity
against cancer cell lines A549 or HCT116, but have lower cytotox-
icities against the normal fibroblast BALB/3T3. These results indi-
cated that the 11-amino group is important for their activity,
especially the 3-aminopropylamino group, which could increase
the activity against MV4-11 about 67 times compared to its pre-
cursor 7. The antiproliferaive test indicated that the 5-methylated
derivatives are usually more cytotoxic than their respective 6-
methylated congeners.
6.1.2.3. 3-(5-Methyl-5H-indolo[2,3-b]quinolin-11-ylamino)pro-
pan-1-ol (10c).
Yield: 94%, yellow solid, Mp: 183–186 °C; 1H
NMR (600 MHz, DMSO-d6) d ppm 1.85 (quin, J = 6.38 Hz, 2H),
3.46 (t, J = 5.43 Hz, 2H), 3.90–3.98 (m, 2H), 4.16 (s, 3H), 4.60 (br
s, 1H), 7.04 (t, J = 5.58 Hz, 1H), 7.06–7.10 (m, 1H), 7.26–7.31 (m,
1H), 7.41 (ddd, J = 8.14, 6.97, 1.03 Hz, 1H), 7.50 (d, J = 7.63 Hz,
1H), 7.76–7.81 (m, 1H), 7.82–7.87 (m, 1H), 7.97 (d, J = 7.63 Hz,
1H), 8.48 (dd, J = 8.22, 1.17 Hz, 1H); 13C NMR (150 MHz, DMSO-
d6) d ppm 32.2, 33.5, 46.0, 58.5, 104.5, 115.0, 115.5, 116.4, 117.9,
120.5, 121.9, 124.0, 124.1, 124.5, 130.6, 137.4, 148.3, 152.0,
156.2; HRMS (ESI) Calcd for C19H18N3O [MÀH]À 304.1455. Found
304.1461.
6. Experimental
6.1. Chemistry
6.1.1. General
The commercially obtained reagents were used without further
purification. The 1H NMR, 13C NMR and 19F NMR spectra were mea-
sured on the Varian INOVA-600 or Varian INOVA-400 spectrome-
ter. High resolution mass spectra were obtained on a Bruker
micrOTOF II-SKA spectrometer. Melting points were determined
on a J-Science RFS-10 hot stage microscope. The salmon fish sperm
DNA was received as a gift from Maruha-nichiro company, and it
was used directly without further purification. The UV spectra
were recorded with Hitachi U-2910 spectrophotometer in a 1 cm
path length quartz cuvette at indicated wavelength, and the sam-
ple was dissolved in phosphate buffer with 2.5% DMSO. The scaf-
folds 5À9 and 19 were prepared by the method we previously
mentioned.11c
6.1.2.4.
lo[2,3-b]quinoline (10d).
5-Methyl-11-(4-methyl-1,4-diazepan-1-yl)-5H-indo-
Yield: 94%, orange solid, Mp: 106–
107 °C; 1H NMR (400 MHz, CDCl3) d ppm 2.12–2.20 (m, 2H), 2.53
(s, 3H), 2.82–2.88 (m, 2H), 2.92–2.97 (t, J = 5.60 Hz, 2H), 3.70 (t,
J = 6.06 Hz, 2H), 3.74–3.79 (m, 2H), 4.31 (s, 3H), 7.21–7.27 (m,
1H), 7.42 (ddd, J = 8.17, 5.14, 2.93 Hz, 1H), 7.48–7.55 (m, 1H),
7.65–7.80 (m, 3H), 8.19 (dd, J = 7.63, 0.59 Hz, 1H), 8.45–8.53 (d,
J = 8.40 Hz, 1H); 13C NMR (100 MHz, CDCl3) d ppm 30.0, 32.9,
47.5, 52.0, 53.0, 58.3, 60.9, 114.3, 117.3, 119.3, 120.8, 121.2,
122.3, 122.9, 123.8, 126.6, 128.0, 130.1, 138.2, 151.4, 154.1, 157.8.
6.1.2.5. N-(4-Aminobutyl)-5-methyl-5H-indolo[2,3-b]quinolin-
11-amine (10e).
NMR (600 MHz, CDCl3)
Yield: 96%, yellow solid, Mp: 69–70 °C; 1H
6.1.2. General procedure for the synthesis of 11-amino-2-
chloro-5-methyl-5H-indolo[2,3-b]quinoline (10–14) and 11-
amino-2-chloro-6-methyl-6H-indolo[2,3-b]quinoline 20
11-Chloro-indolo[2,3-b]quinoline and a large excess of the
appropriate amine were heated together at 80À120 °C for 1–8 h.
The reaction was monitored by TLC. Then the mixture was washed
with water and extracted with AcOEt. The organic phase was dried
over MgSO4 and concentrated under vacuo. The crude product was
purified by chromatography using eluent changed from AcOEt to
AcOEt-2 N ammonia in MeOH (20: 1) to give final product.
d
ppm 1.44–1.49 (m, 2H), 1.70 (dt,
J = 14.75, 7.15 Hz, 2H), 2.66 (t, J = 6.75 Hz, 2H), 3.73 (t, J = 6.90 Hz,
2H), 4.15 (s, 3H), 5.53 (br s, 1H), 7.15–7.19 (m, 1H), 7.24–7.28
(m, 1H), 7.40–7.44 (m, 1H), 7.53 (d, J = 8.22 Hz, 1H), 7.61 (ddd,
J = 8.44, 6.97, 1.32 Hz, 1H), 7.76 (d, J = 7.92 Hz, 1H), 7.83 (d,
J = 7.63 Hz, 1H), 8.04 (dd, J = 8.22, 1.17 Hz, 1H); 13C NMR
(150 MHz, CDCl3) d ppm 29.1, 30.5, 32.5, 41.4, 48.9, 107.3, 114.5,
115.6, 117.1, 118.6, 120.3, 120.8, 123.9, 124.1, 125.6, 130.1,
137.8, 148.4, 152.4, 156.4; HRMS (ESI) Calcd for C20H21N4 [MÀH]À
317.1772. Found 317.1769.
6.1.2.1.
N-(5-(Diethylamino)pentan-2-yl)-5-methyl-5H-indo-
Yield: 98%, yellow solid,
6.1.2.6. N-(3-Aminopropyl)-5-methyl-5H-indolo[2,3-b]quinolin-
11-amine (10f).
Yield: 45%, yellow solid, Mp: 95–97 °C; 1H
lo[2,3-b]quinolin-11-amine (10a).
Mp: 89–91 °C; 1H NMR (600 MHz, DMSO-d6) d ppm 0.70 (t,
J = 7.19 Hz, 6H), 1.13–1.27 (m, 2H), 1.44 (d, J = 6.46 Hz, 3H), 1.57–
1.65 (m, 1H), 1.65–1.74 (m, 1H), 2.01–2.12 (m, 2H), 2.16 (q,
J = 5.87 Hz, 4H), 4.18 (s, 3H), 4.29–4.39 (m, 1H), 6.57 (d,
J = 10.27 Hz, 1H), 7.09 (m, 1H), 7.30 (m Hz, 1H), 7.43 (t,
NMR (600 MHz, CDCl3) d ppm 1.77–1.80 (m, 2H), 2.97–3.01 (t,
J = 6.16 Hz, 2H), 3.99 (t, J = 6.16 Hz, 2H), 4.20 (s, 3H), 7.16 (td,
J = 7.48, 1.17 Hz, 1H), 7.29 (ddd, J = 8.22, 7.04, 1.17 Hz, 1H), 7.41
(td, J = 7.48, 1.17 Hz, 1H), 7.56–7.61 (m, 1H), 7.65 (ddd, J = 8.51,
7.04, 1.47 Hz, 1H), 7.75 (d, J = 7.63 Hz, 1H), 7.95 (d, J = 7.63 Hz,

4826
L. Wang et al. / Bioorg. Med. Chem. 20 (2012) 4820–4829
1H), 8.11 (dd, J = 8.36, 1.32 Hz, 1H); 13C NMR (150 MHz, CDCl3) d
ppm 32.6, 32.7, 41.3, 49.3, 105.8, 114.4, 115.9, 117.0, 118.5,
120.3, 121.4, 124.1, 124.2, 125.2, 130.1, 137.9, 148.5, 152.2, 156.7.
1.66 (dt, J = 14.23, 7.07 Hz, 2H), 2.66 (t, J = 6.65 Hz, 2H), 3.64 (t,
J = 6.75 Hz, 2H), 4.00–4.07 (s, 3H), 5.59–5.91 (br s, 1H), 7.09–7.17
(m, 1H), 7.32–7.42 (m, 2H), 7.43–7.52 (m, 1H), 7.68 (d,
J = 7.83 Hz, 1H), 7.76 (d, J = 7.63 Hz, 1H), 7.99 (d, J = 2.15 Hz, 1H);
13C NMR (100 MHz, CDCl3) d ppm 28.9, 30.3, 32.6, 41.3, 48.8,
107.4, 115.7, 116.6, 117.1, 118.8, 121.1, 123.5, 123.8 125.6, 125.8,
129.9, 136.1, 147.2, 152.4, 156.2.
6.1.2.7.
(10g).
5-Methyl-11-morpholino-5H-indolo[2,3-b]quinoline
Yield: 92%, red solid, Mp: 212–215 °C; 1H NMR
(600 MHz, CDCl3) d ppm 3.50–3.55 (m, 4H) 4.03–4.08 (m, 4H)
4.30 (s, 3H) 7.23–7.28 (m, 1H) 7.43 (ddd, J = 8.16, 6.84, 1.10 Hz,
1H) 7.52 (td, J = 7.61, 1.10 Hz, 1H) 7.68–7.76 (m, 3H) 8.33 (d,
J = 7.94 Hz, 1H) 8.53 (dd, J = 8.27, 1.21 Hz, 1H); 13C NMR
(150 MHz, CDCl3) d ppm 33.0, 49.2, 67.6, 114.3, 117.5, 119.4,
120.6, 121.2, 122.7, 123.2, 124.8, 126.1, 128.3, 130.3, 137.9,
149.8, 154.5, 157.6.
6.1.2.13. N-(3-aminopropyl)-2-chloro-5-methyl-5H-indolo[2,3-
b]quinolin-11-amine (11f).
Yield: 97%, yellow solid, Mp:
123–125 °C; 1H NMR (600 MHz, DMSO-d6) d ppm 1.73–1.77 (m,
2H), 2.61 (t, J = 6.46 Hz, 2H), 3.91 (t, J = 6.75 Hz, 2H), 4.13–4.16 (s,
3H), 7.06–7.10 (m, 1H), 7.27–7.31 (m, 1H), 7.50 (d, J = 7.63 Hz,
1H), 7.78–7.82 (m, 1H), 7.85–7.88 (m, 1H), 7.92 (d, J = 7.92 Hz,
1H), 8.60 (d, J = 2.35 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) d
ppm 32.4, 33.1, 40.0, 46.6, 104.7, 116.7, 116.8, 117.1, 118.2,
122.4, 123.1, 124.1, 124.8, 125.0, 130.2, 136.0, 147.0, 152.4, 156.3.
6.1.2.8.
2-Chloro-N-(5-(diethylamino)pentan-2-yl)-5-methyl-
(11a). Yield: 55%,
5H-indolo[2,3-b]quinolin-11-amine
brown solid, Mp: 76–78 °C; 1H NMR (600 MHz, DMSO-d6) d ppm
0.71 (t, J = 6.60 Hz, 6H), 1.11–1.26 (m, 2H), 1.44 (d, J = 6.46 Hz,
3H), 1.55–1.64 (m, 1H), 1.64–1.73 (m, 1H), 2.01–2.13 (m, 2H),
2.13–2.23 (m, 4H), 4.16 (s, 3H), 4.29–4.38 (m, 1H), 6.73 (d,
J = 10.27 Hz, 1H), 7.11 (t, J = 7.48 Hz, 1H), 7.32 (t, J = 7.80 Hz, 1H),
7.52 (d, J = 7.92 Hz, 1H), 7.77–7.83 (m, 2H), 7.88 (dd, J = 9.10,
0.88 Hz, 1H), 8.74 (d, J = 2.05 Hz, 1H); 13C NMR (150 MHz, DMSO-
d6) d ppm 11.4, 21.4, 23.4, 32.5, 36.1, 46.0, 51.8, 52.4, 106.5,
116.8, 117.1, 117.3, 118.2, 122.0, 123.2, 124.0, 125.2, 125.3,
130.3, 136.0, 147.1, 152.6, 156.4; HRMS (ESI) Calcd for C25H30ClN4
[MÀH]À 421.2164. Found 421.2153.
6.1.2.14. 2-Bromo-N-(5-(diethylamino)pentan-2-yl)-5-methyl-
5H-indolo[2,3-b]quinolin-11-amine (12a).
Yield: 64%, yel-
low solid, Mp: 77–79 °C; 1H NMR (600 MHz, DMSO-d6) d ppm
0.72 (t, J = 6.90 Hz, 6H), 1.12–1.27 (m, 2H), 1.44 (d, J = 6.46 Hz,
3H), 1.55–1.64 (m, 1H), 1.64–1.74 (m, 1H), 2.08–2.19 (m, 6H),
4.16 (s, 3H), 4.27–4.39 (m, 1H), 6.72–6.79 (m, 1H), 7.11 (t,
J = 7.20 Hz, 1H), 7.32 (t, J = 7.80 Hz, 1H), 7.52 (d, J = 7.92 Hz, 1H),
7.79 (d, J = 7.63 Hz, 1H), 7.82 (d, J = 9.10 Hz, 1H), 7.92 (dd,
J = 9.10, 2.05 Hz, 1H), 8.86 (d, J = 2.35 Hz, 1H); 13C NMR
(150 MHz, DMSO-d6) d ppm 11.4, 21.4, 23.3, 32.4, 36.0, 46.0,
51.8, 52.4, 106.4, 113.1, 116.8, 117.4, 117.8, 118.2, 122.0, 124.0,
125.2, 126.1, 133.0, 136.3, 147.0, 152.6, 156.3; HRMS (ESI) Calcd
for C25H30BrN4 [MÀH]À 465.1659. Found 465.1667.
6.1.2.9. N-(2-(1H-Indol-3-yl)ethyl)-2-chloro-5-methyl-5H-indo-
lo[2,3-b]quinolin-11-amine (11b).
Yield: 67%, yellow solid,
Mp: 173–175 °C; 1H NMR (400 MHz, CDCl3) d ppm 3.19 (t,
J = 6.40 Hz, 2H), 4.18 (t, J = 6.40 Hz, 2H), 4.22 (s, 3H), 5.25 (brs,
3H), 7.03–7.07 (m, 2H), 7.12 (t, J = 7.20 Hz, 6H), 7.22–7.26 (m,
2H), 7.38–7.43 (m, 2H), 7.46 (d, J = 12.00 Hz, 1H), 7.55–7.64 (m,
3H), 7.75 (d, J = 7.60 Hz, 1H), 7.96 (d, J = 2.00 Hz, 1H), 8.29 (brs,
1H); 13C NMR (100 MHz, CDCl3) d ppm 26.5, 32.50, 48.6, 104.9,
110.9, 111.4, 116.5, 116.9, 117.0, 118.2, 118.3, 118.3, 120.9,
122.4, 122.9, 123.1, 123.8, 125.0, 125.2, 127.0, 130.3, 135.9,
136.1, 147.1, 151.7, 155.9; HRMS (ESI) Calcd for C26H20ClN4
[MÀH]À 423.1382. Found 423.1393.
6.1.2.15.
ylamino)propan-1-ol (12c).
3-(2-Bromo-5-methyl-5H-indolo[2,3-b]quinolin-11-
Yield: 70%, yellow solid, Mp:
234–236 °C; 1H NMR (600 MHz, DMSO-d6) d ppm 1.84 (s, 2H),
3.42 (d, J = 4.99 Hz, 2H), 3.90 (d, J = 6.16 Hz, 2H), 4.14 (s, 3H),
4.54 (s, 1H), 7.05–7.10 (m, 1H), 7.11–7.16 (m, 1H), 7.29 (s, 1H),
7.50 (s, 1H), 7.80 (d, J = 9.10 Hz, 1H), 7.88–7.94 (m, 2H), 8.73 (d,
J = 2.05 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) d ppm 32.3, 33.5,
45.7, 58.3, 104.9, 112.8, 116.7, 117.3, 117.3, 118.2, 122.4, 124.1,
124.8, 125.9, 132.9, 136.3, 147.0, 152.4, 156.3; HRMS (ESI) Calcd
for C19H17BrN3O [MÀH]À 382.0560. Found 382.0574.
6.1.2.10.
ylamino)propan-1-ol (11c).
3-(2-Chloro-5-methyl-5H-indolo[2,3-b]quinolin-11-
Yield: 27%, yellowish green so-
lid, Mp: 223–226 °C; 1H NMR (600 MHz, DMSO-d6) d ppm 1.87–
1.89 (m, 2H), 3.45–3.48 (q, J = 4.80 Hz, 2H), 3.95 (q, J = 6.00 Hz,
2H), 4.19 (s, 3H), 4.58 (t, J = 4.80 Hz, 1H), 7.11–7.16 (m, 2H), 7.33
(t, J = 7.80 Hz, 1H), 7.55 (d, J = 7.80 Hz, 1H), 7.84–7.86 (m, 1H),
7.92 (d, J = 9.00 Hz, 1H), 7.97 (d, J = 7.80 Hz, 1H), 8.66 (d,
J = 2.40 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) d ppm 32.4, 33.5,
45.7, 58.3, 105.0, 116.7, 116.8, 117.1, 118.2, 122.3, 123.0, 124.1,
124.8, 125.0, 130.2, 136.0, 147.1, 152.4, 156.3; HRMS (ESI) Calcd
for C19H18ClN3O [MÀH]À 338.1066. Found 338.1036.
6.1.2.16. 2-Bromo-5-methyl-11-(4-methyl-1,4-diazepan-1-yl)-
5H-indolo[2,3-b]quinoline (12d).
Yield: 82%, yellow solid,
Mp: 134–137 °C; 1H NMR (400 MHz, CDCl3) d ppm 2.10–2.19 (m,
2H), 2.55–2.61 (s, 3H), 2.86–2.92 (t, J = 4.80 Hz, 2H), 2.93–2.99 (t,
J = 5.20 Hz, 2H), 3.70 (t, J = 5.97 Hz, 2H), 3.73–3.78 (t, J = 4.80 Hz,
2H), 4.29 (s, 3H), 7.22–7.28 (m, 1H), 7.49–7.55 (t, J = 7.20 Hz, 1H),
7.58 (d, J = 9.00 Hz, 1H), 7.71–7.75 (d, J = 8.00 Hz, 1H), 7.75–7.81
(dd, J = 8.80, 2.00 Hz, 1H), 8.12 (d, J = 7.63 Hz, 1H), 8.78 (d,
J = 2.15 Hz, 1H); 13C NMR (100 MHz, CDCl3) d ppm 30.0, 33.2,
47.5, 51.9, 52.8, 58.4, 60.6, 114.4, 116.1, 117.5, 119.8, 122.5,
122.7, 122.9, 123.9, 128.5, 129.2, 132.8, 137.2, 149.9, 154.0, 157.4.
6.1.2.11. 2-Chloro-5-methyl-11-(4-methyl-1,4-diazepan-1-yl)-
5H-indolo[2,3-b]quinoline (11d).
Yield: 48%, orange solid,
Mp: 103–105 °C; 1H NMR (300 MHz, CDCl3) d ppm 2.13–2.16 (m,
2H), 2.57 (s, 3H), 2.89 (t, J = 5.10 Hz, 2H), 2.95 (t, J = 5.40 Hz, 2H),
3.69–3.77 (m, 4H), 4.31 (s, 3H), 7.23 (t, J = 9.90 Hz, 1H), 7.53 (t,
J = 7.20 Hz, 1H), 7.66 (d, J = 1.20 Hz, 2H), 7.75 (d, J = 8.10 Hz, 1H),
8.15 (d, J = 7.20 Hz, 1H), 8.61 (s, 1H); 13C NMR (75 MHz, CDCl3) d
ppm 30.3, 33.2, 47.7, 52.1, 53.0, 58.6, 60.7, 115.9, 117.6, 119,8,
122,2, 122.9, 123.2, 124.1, 126.1, 127.0, 128.6, 130.2, 136.8,
150.1, 154.5, 157.7.
6.1.2.17. N-(4-Aminobutyl)-2-bromo-5-methyl-5H-indolo[2,3-
b]quinolin-11-amine (12e).
Yield: 96%, yellow solid, Mp:
81–82 °C; 1H NMR (600 MHz, CDCl3) d ppm 1.45–1.50 (m, 2H),
1.65–1.69 (m, 2H), 2.68 (t, J = 6.60 Hz, 2H), 3.64 (d, J = 4.11 Hz,
2H), 4.05 (s, 3H), 5.72 (br., 1H), 7.17 (t, J = 7.34 Hz, 1H), 7.29 (d,
J = 8.80 Hz, 1H), 7.39–7.44 (m, 1H), 7.58 (dd, J = 9.10, 1.17 Hz,
1H), 7.73 (d, J = 7.92 Hz, 1H), 7.78 (d, J = 7.92 Hz, 1H), 8.09 (s,
1H); 13C NMR (150 MHz, CDCl3) d ppm 29.0, 30.5, 32.6, 41.4,
48.9, 107.6, 112.9, 116.0, 117.1, 117.3, 118.8, 121.1, 123.9, 125.9,
126.6, 132.5, 136.5, 147.0, 152.7, 156.2; HRMS (ESI) Calcd for
6.1.2.12. N-(4-Aminobutyl)-2-chloro-5-methyl-5H-indolo[2,3-
b]quinolin-11-amine (11e).
Yield: 86%, yellow solid, Mp:
208–210 °C; 1H NMR (400 MHz, CDCl3) d ppm 1.40–1.50 (m, 2H),
C
20H20BrN4 [MÀH]À 395.0877. Found 395.0862.

L. Wang et al. / Bioorg. Med. Chem. 20 (2012) 4820–4829
4827
6.1.2.18. 2-Bromo-5-methyl-11-(4-methyl-1,4-diazepan-1-yl)-
5H-indolo[2,3-b]quinoline (12f). Yield: 63%, yellow solid,
4.13 (m, 1H), 4.52 (d, J = 10.76 Hz, 2H), 7.30 (td, J = 7.63, 0.98 Hz,
2H), 7.38 (d, J = 8.02 Hz, 2H), 7.49–7.55 (m, 2H), 7.57 (dd, J = 9.00,
2.35 Hz, 1H), 7.97 (d, J = 9.00 Hz, 1H), 8.00 (d, J = 7.63 Hz, 2H),
8.09 (d, J = 2.15 Hz, 1H); 13C NMR (100 MHz, CDCl3) d ppm 11.1,
22.1, 23.3, 27.6, 36.9, 46.6, 52.5, 54.1, 106.3, 108.4, 119.4, 119.8,
120.3, 121.9, 121.9, 126.4, 126.7, 129.3, 129.5, 141.6, 146.3,
147.4, 153.8; HRMS (ESI) Calcd for C25H30ClN4 [MÀH]À 421.2164.
Found 421.2155.
Mp: 143–145 °C; 1H NMR (600 MHz, DMSO-d6) d ppm 1.71–1.77
(m, 2H), 2.59 (t, J = 6.46 Hz, 2H), 3.91 (t, J = 6.75 Hz, 2H), 4.11–
4.16 (m, 3H), 7.08 (ddd, J = 7.92, 7.04, 0.88 Hz, 1H), 7.29 (td,
J = 7.63, 1.17 Hz, 1H), 7.50 (dd, J = 7.92, 0.59 Hz, 1H), 7.79 (d,
J = 9.10 Hz, 1H), 7.90 (dd, J = 9.10, 2.05 Hz, 1H), 7.92 (d,
J = 7.63 Hz, 1H), 8.71 (d, J = 2.35 Hz, 1H); 13C NMR (150 MHz,
DMSO-d6) d ppm 32.3, 33.5, 39.5, 46.7, 104.5, 112.8, 116.7, 117.3,
117.3, 118.1, 122.4, 124.1, 124.7, 126.0, 132.9, 136.3, 147.0,
152.4, 156.3; HRMS (ESI) Calcd for C19H18BrN4 [MÀH]À 381.0720.
Found 381.0714.
6.1.2.24.
N-(2-(1H-Indol-3-yl)ethyl)-2-chloro-6-methyl-6H-
Yield: 28%, grey so-
indolo[2,3-b]quinolin-11-amine (20b).
lid, Mp: 207–209 °C; 1H NMR (600 MHz, CDCl3) d ppm 3.15 (t,
J = 6.46 Hz, 2H), 3.87 (s, 3H), 4.07 (d, J = 5.58 Hz, 2H), 4.85 (br s,
1H), 7.04 (s, 1H), 7.05–7.09 (t, J = 7.80 Hz, 1H), 7.16 (t, J = 7.48 Hz,
1H), 7.26 (t, J = 9.60 Hz, 1H), 7.30 (d, J = 7.92 Hz, 1H), 7.40–7.46
(m, 2H), 7.49–7.56 (m, 2H), 7.65 (d, J = 7.92 Hz, 1H), 7.94–8.00
(m, 2H), 8.43 (br s, 1H); 13C NMR (150 MHz, CDCl3) d ppm 27.1,
27.7, 48.8, 104.9, 108.2, 111.4, 111.8, 118.5, 118.7, 119.7, 119.7,
120.0, 121.9, 122.0, 122.4, 122.9, 126.1, 126.4, 127.1, 129.1,
129.2, 136.5, 141.2, 146.1, 147.8, 153.7; HRMS (ESI) Calcd for
6.1.2.19. N-(4-Aminobutyl)-2-fluoro-5-methyl-5H-indolo[2,3-
b]quinolin-11-amine (13e).
Yield: 94%, yellow solid, Mp:
78–79 °C; 1H NMR (400 MHz, CDCl3) d ppm 1.47–1.53 (m, 2H),
1.65–1.75 (m, 2H), 2.69 (t, J = 6.65 Hz, 2H), 3.62–3.72 (m, 2H),
4.12 (s, 3H), 5.57 (br s, 1H), 7.12–7.20 (t, J = 7.60 Hz, 1H), 7.31–
7.38 (m, 1H), 7.39–7.50 (m, 2H), 7.70–7.77 (m, 2H), 7.80 (d,
J = 7.83 Hz, 1H); 13C NMR (100 MHz, CDCl3) d ppm 29.1, 30.5,
32.8, 41.4, 48.8, 108.2, 109.3, 109.6, 115.9, 116.0, 116.4, 116.4,
117.2, 118.0, 118.2, 118.7, 121.0, 123.6, 126.0, 134.5, 147.5,
152.8, 155.4, 156.4, 157.7; 19F NMR (376 MHz, CDCl3) d ppm
121.33; HRMS (ESI) Calcd for C20H20FN4 [MÀH]À 335.1677. Found
335.1650.
C
26H20ClN4 [MÀH]À 423.1441. Found 423.1422.
6.1.2.25.
ylamino)propan-1-ol (20c).
3-(2-Chloro-6-methyl-6H-indolo[2,3-b]quinolin-11-
Yield: 74%, grey solid, Mp:
133–135 °C; 1H NMR (400 MHz, DMSO-d6) d ppm 1.81 (m, 2H),
3.39–3.49 (m, 2H), 3.76 (q, J = 6.52 Hz, 2H), 3.86 (s, 3H), 4.52 (br
s, 1H), 6.58 (br s, 1H), 7.24–7.32 (m, 1H), 7.49 (t, J = 8.00 Hz, 1H),
7.57 (d, J = 8.00 Hz, 1H), 7.64 (dd, J = 9.00, 2.15 Hz, 1H), 7.88 (d,
J = 9.00 Hz, 1H), 8.09 (d, J = 7.63 Hz, 1H), 8.58 (d, J = 2.15 Hz, 1H);
13C NMR (100 MHz, CDCl3) d ppm (ppm) 27.6, 33.8, 46.0, 58.6,
102.7, 108.6, 118.5, 119.8, 119.9, 122.2, 122.9, 125.4, 125.8,
129.0, 140.7, 145.3, 148.2, 153.7; HRMS (ESI) Calcd for
6.1.2.20. 3-(2-Methoxy-5-methyl-5H-indolo[2,3-b]quinolin-11-
ylamino)propan-1-ol (14c).
Yield: 66%, yellow solid, Mp:
206–208 °C; 1H NMR (600 MHz, DMSO-d6) d ppm 1.82 (m, 2H),
3.42–3.47 (q, J = 4.20 Hz, 2H), 3.89–3.91 (m, 2H), 3.92 (s, 3H),
4.15 (s, 3H), 4.60 (t, J = 4.55 Hz, 1H), 7.00 (t, J = 5.43 Hz, 1H),
7.03–7.07 (m, 1H), 7.28 (td, J = 7.56, 1.03 Hz, 1H), 7.44–7.49 (m,
2H), 7.81 (d, J = 9.10 Hz, 1H), 7.91–7.95 (m, 2H); 13C NMR
(150 MHz, DMSO-d6) d ppm 32.3, 33.7, 45.9, 55.9, 58.6, 105.2,
106.0, 116.2, 116.3, 116.5, 117.6, 119.5, 122.1, 123.9, 124.6,
132.2, 147.8, 152.3, 153.6, 156.1; HRMS (ESI) Calcd for
C
19H17ClN3O [MÀH]À 338.1066. Found 338.1062.
6.1.2.26. 2-Chloro-6-methyl-11-(4-methyl-1,4-diazepan-1-yl)-
6H-indolo[2,3-b]quinoline (20d). Yield: 35%, off white solid,
C
20H20N3O2 [MÀH]À 334.1556. Found 334.1537.
Mp: 183–184 °C; 1H NMR (400 MHz, CDCl3) d ppm 2.21 (m, 2H),
2.58 (s, 3H), 2.86–2.94 (t, J = 2.40 Hz, 2H), 3.00 (t, J = 5.20 Hz, 2H),
3.68 (t, J = 6.06 Hz, 2H), 3.75 (t, J = 4.80 Hz, 2H), 3.94 (s, 3H), 7.34
(t, J = 7.53 Hz, 1H), 7.39 (d, J = 8.02 Hz, 1H), 7.55–7.62 (m, 2H),
8.02 (d, J = 9.00 Hz, 1H), 8.38 (d, J = 7.83 Hz, 1H), 8.43 (d,
J = 2.35 Hz, 1H); 13C NMR (100 MHz, CDCl3) d ppm 27.7, 29.9,
47.4, 52.1, 52.9, 58.3, 61.1, 108.3, 114.9, 119.3, 120.0, 123.7,
124.0, 124.5, 127.6, 127.7, 129.1, 129.5, 142.3, 146.7, 150.6, 154.4.
6.1.2.21.
N-(4-Aminobutyl)-2-methoxy-5-methyl-5H-indo-
Yield: 79%, gel; 1H NMR
lo[2,3-b]quinolin-11-amine (14e).
(600 MHz, CDCl3) d ppm 1.41–1.49 (m, 2H), 1.67–1.72 (m, 2H),
2.62–2.67 (m, 2H), 3.64–3.72 (m, 2H), 3.85 (s, 3H), 4.11 (s, 3H),
5.49 (br s, 1H), 7.13 (t, J = 7.20 Hz, 1H), 7.23 (dd, J = 6.00, 3.00 Hz,
1H), 7.38 (t, J = 8.40 Hz, H), 7.42–7.47 (m, 2H), 7.70 (d,
J = 7.92 Hz, 1H), 7.81 (d, J = 7.63 Hz, 1H); 13C NMR (150 MHz,
CDCl3) d ppm 29.1, 30.5, 32.6, 41.4, 48.8, 55.6, 106.4, 108.2,
115.7, 116.4, 116.8, 118.3, 118.9, 120.9, 123.7, 125.7, 132.7,
148.0, 152.6, 153.4, 156.1; HRMS (ESI) Calcd for C21H23N4O
[MÀH]À 347.1877. Found 347.1857.
6.1.2.27. N-(4-Aminobutyl)-2-chloro-6-methyl-6H-indolo[2,3-
b]quinolin-11-amine (20e).
Yield: 26%, light grey solid, Mp:
222–224 °C; 1H NMR (600 MHz, CDCl3) d ppm 1.53 (m, 2H), 1.77
(m, 2H), 2.71 (t, J = 7.20 Hz, 2H), 3.68 (t, J = 7.20 Hz, 2H), 3.88 (s,
3H), 4.92 (br s, 1H), 7.25–7.29 (m, 1H), 7.35 (d, J = 8.40 Hz, 1H),
7.46–7.51 (m, 1H), 7.55 (dd, J = 8.80, 2.40 Hz, 1H), 7.91–7.98 (m,
2H), 8.09 (d., J = 2.40 Hz, 1H); 13C NMR (150 MHz, CDCl3) d ppm
27.6, 29.3, 30.9, 41.7, 49.2, 104.6, 108.3, 118.4, 119.7, 120.2,
121.8, 121.8, 126.1, 126.4, 129.2, 129.4, 141.3, 146.3, 147.9,
153.9; HRMS (ESI) Calcd for C20H20ClN4 [MÀH]À 351.1382. Found
351.1366.
6.1.2.22.
N-(3-Aminopropyl)-2-methoxy-5-methyl-5H-indo-
Yield: 80%, yellow solid,
lo[2,3-b]quinolin-11-amine (14f).
Mp: 73–74 °C; 1H NMR (600 MHz, CDCl3) d ppm 1.68–1.71 (m,
2H), 2.92 (t, J = 5.87 Hz, 2H), 3.82 (s, 3H), 3.90 (t, J = 5.40 Hz, 2H),
4.12 (s, 3H), 7.05 (br s, 1H), 7.14 (t, J = 7.19 Hz, 1H), 7.21 (dd,
J = 9.24, 2.79 Hz, 1H), 7.35–7.44 (m, 3H), 7.72 (d, J = 7.92 Hz, 1H),
7.93 (d, J = 7.63 Hz, 1H); 13C NMR (150 MHz, CDCl3) d ppm 32.5,
32.5, 41.4, 49.2, 55.6, 106.0, 106.4, 115.5, 116.4, 116.8 118.0,
118.8, 121.5, 124.0, 125.2, 132.6, 147.8, 152.7, 153.4, 156.6; HRMS
(ESI) Calcd for C20H21N4O [MÀH]À 333.1721. Found 333.1684.
6.1.2.28. N-(3-Aminopropyl)-2-chloro-6-methyl-6H-indolo[2,3-
b]quinolin-11-amine (20f).
Yield: 61%, light grey solid, Mp:
78–79 °C; 1H NMR (400 MHz, CDCl3) d ppm 1.84–1.90 (m, 2H),
2.99 (t, J = 6.40 Hz, 2H), 3.85–3.86 (m, 2H), 3.92 (s, 3H), 6.03 (br
s, 1H), 7.28 (t, J = 8.00 Hz, 1H), 7.38 (d, J = 7.60 Hz, 1H), 7.50 (t,
J = 7.20 Hz, 1H), 7.57 (dd, J = 8.80, 2.40 Hz, 1H), 7.97 (s,
J = 9.20 Hz, 1H), 8.08 (d, J = 8.00 Hz, 1H), 8.19 (d, J = 2.40 Hz, 1H);
13C NMR (100 MHz, CDCl3) d ppm 27.7, 33.7, 41.0, 48.9, 104.1,
108.2, 118.5, 119.7, 120.7, 122.1, 122.2, 126.0, 126.3, 129.2,
6.1.2.23. 2-Chloro-N-(5-(diethylamino)pentan-2-yl)-6-methyl-
6H-indolo[2,3-b]quinolin-11-amine
(20a).
Yield:
27%,
brown gel; 1H NMR (400 MHz, CDCl3) d ppm 0.95 (t, J = 7.14 Hz,
6H), 1.31 (d, J = 6.26 Hz, 3H), 1.52–1.66 (m, 3H), 1.66–1.76 (m,
1H), 2.39 (t, J = 7.34 Hz, 2H), 2.46 (q, J = 7.11 Hz, 4H), 3.92 (s, 3H),

4828
L. Wang et al. / Bioorg. Med. Chem. 20 (2012) 4820–4829
129.4, 141.3, 146.4, 148.2, 154.2; HRMS (ESI) Calcd for C19H18ClN4
of celit. The filtrate was extracted with CH2Cl2 and the organic
phase was dried over anhydrous MgSO4. Further purification was
achieved by flash chromatography on silica gel. Yield: 27%, red so-
lid, Mp: 261–263 °C; 1H NMR (600 MHz, CDCl3) d ppm 4.15 (s, 3H),
7.08 (ddd, J = 7.78, 6.90, 1.17 Hz, 1H), 7.45–7.49 (m, 2H), 7.50–7.52
(m, 1H), 7.57 (dd, J = 9.10, 2.35 Hz, 1H), 7.84 (d, J = 7.63 Hz, 1H),
8.72 (d, J = 2.35 Hz, 1H), 11.03 (s, 1H); 13C NMR (150 MHz, CDCl3)
d ppm 33.7, 115.4, 117.1, 118.3, 120.9, 121.9, 125.9, 126.0, 128.6,
130.1, 130.6, 131.7, 131.8, 134.6, 155.8, 156.7, 190.6.
[MÀH]À 337.122. Found 337.1205.
6.1.2.29.
b]quinoline (20g).
2-Chloro-6-methyl-11-morpholino-6H-indolo[2,3-
Yield: 27%, yellow solid, Mp: 245–
247 °C; 1H NMR (600 MHz, CDCl3) d ppm 3.54 (m, 4H), 3.95 (s,
3H), 4.09–4.14 (t, J = 4.80 Hz, 4H), 7.36 (t, J = 7.63 Hz, 1H), 7.41
(d, J = 8.22 Hz, 1H), 7.58–7.63 (m, 2H), 8.04 (d, J = 8.80 Hz, 1H),
8.50 (d, J = 2.35 Hz, 1H), 8.54 (d, J = 7.63 Hz, 1H); 13C NMR
(150 MHz, CDCl3) d ppm (ppm) 27.8,, 49.4, 67.8, 108.6, 115.6,
119.2, 20.0, 123.4, 124.0, 125.5, 127.9, 128.0, 129.4, 129.5, 142.6,
146.5, 149.1, 154.1.
6.1.5. Preparation of 11-aminoalkylaminomethylated
neocryptolepine derivatives 23
The aldehyde 22 (0.1 mmol) and appropriate amine (0.5 mmol)
was dissolved in MeOH. After stirring overnight at room tempera-
ture, NaBH4 (0.5 mmol) was added. Then the mixture was stirred
for an additional 2 h. The reaction was quenched with saturated
NH4Cl, extracted with CH2Cl2, and the organic phase was dried
over anhydrous MgSO4. Further purification was achieved with
chromatography on silica gel.
6.1.2.30.
2-Chloro-N-(2-(dimethylamino)ethyl)-6-methyl-6H-
Yield: 44%, brown
indolo[2,3-b]quinolin-11-amine (20h).
solid, Mp: 152–153oC; 1H NMR (400 MHz, CDCl3) d ppm 2.44 (s,
6H), 2.61 (t, J = 5.58 Hz, 2H), 3.82 (q, J = 5.22 Hz, 2H), 3.95 (s, 3H),
6.19 (br s, 1H), 7.28–7.34 (t, J = 7.20 Hz, 1H), 7.39 (d, J = 8.02 Hz,
1H), 7.49–7.54 (m, 1H), 7.57 (dd, J = 9.00, 2.35 Hz, 1H), 7.98 (d,
J = 9.00 Hz, 1H), 8.22 (d, J = 7.83 Hz, 1H), 8.28 (d, J = 2.15 Hz, 1H);
13C NMR (100 MHz, CDCl3) d ppm 27.7, 44.9, 46.0, 58.9, 104.6,
108.3, 118.6, 119.9, 120.4, 121.8, 122.8, 126.0, 126.2, 129.2,
129.3, 141.2, 146.5, 148.5, 153.7; HRMS (ESI) Calcd for C20H20ClN4
[MÀH]À 351.1382. Found 351.1380.
6.1.5.1.
2-Chloro-5-methyl-11-((4-methyl-1,4-diazepan-1-
Yield: 36%, or-
yl)methyl)-5H-indolo[2,3-b]quinoline (23d).
ange solid, Mp: 164–166 °C; 1H NMR (600 MHz, CDCl3) d ppm
1.83–1.90 (m, 2H), 2.34 (s, 3H), 2.54 (dt, J = 4.92, 2.38 Hz, 2H),
2.63–2.72 (m, 2H), 2.88–2.93 (m, 2H), 2.96 (t, J = 6.16 Hz, 2H),
4.32 (s, 3H), 4.49 (s, 2H), 7.20–7.25 (m, 1H), 7.54 (td, J = 7.63,
1.17 Hz, 1H), 7.63–7.70 (m, 2H), 7.72 (d, J = 7.92 Hz, 1H), 8.28 (d,
J = 7.63 Hz, 1H), 8.60 (d, J = 2.05 Hz, 1H); 13C NMR (150 MHz,
CDCl3) d ppm 27.3, 33.2, 46.6, 54.1, 55.1, 56.3, 58.1, 115.4, 117.9,
119.9, 122.1, 124.1, 124.3, 126.9, 126.9, 128.3, 129.3, 130.1,
135.2, 139.1, 155.7, 155.9; HRMS (ESI) Calcd for C23H24ClN4
[MÀH]À 391.1689. Found 391.1750.
6.1.2.31. N-(2-Aminoethyl)-2-chloro-6-methyl-6H-indolo[2,3-
b]quinolin-11-amine (20i).
Yield: 50%, light grey solid, Mp:
138–141 °C; 1H NMR (600 MHz, CDCl3) d ppm 2.92 (t, J = 5.40 Hz,
2H), 3.65 (q, J = 5.28 Hz, 2H), 3.84 (s, 3H), 5.83 (t, J = 5.28 Hz, 1H),
7.24 (t, J = 7.48 Hz, 1H), 7.29 (d, J = 7.92 Hz, 1H), 7.45 (t,
J = 7.80 Hz, 1H), 7.51 (dd, J = 9.10, 2.35 Hz, 1H), 7.90 (d,
J = 9.10 Hz, 1H), 8.12 (d, J = 7.63 Hz, 1H), 8.15 (d, J = 2.35 Hz, 1H);
13C NMR (150 MHz, CDCl3) d ppm 27.5, 42.0, 50.4, 104.5, 108.0,
118.4, 119.6, 120.1, 121.8, 122.2, 125.9, 126.0, 129.0, 129.1,
141.0, 146.3148.0, 153.6; HRMS (ESI) Calcd for C18H16ClN4 [MÀH]À
323.1069. Found 323.1039.
6.1.5.2. N1-((2-Chloro-5-methyl-5H-indolo[2,3-b]quinolin-11-
yl)methyl)-N3,N3-dimethylpropane-1,3-diamine
(23j).
Yield: 56%, red solid, Mp: 150–153 °C; 1H NMR
(600 MHz, CDCl3) d ppm 1.68–1.74 (m, 3H), 2.15–2.19 (m, 10H),
2.29–2.34 (m, 4H), 2.90 (t, J = 6.75 Hz, 3H), 4.25 (s, 5H), 4.53 (s,
3H), 7.21 (td, J = 7.48, 1.17 Hz, 2H), 7.50–7.54 (m, 2H), 7.57–7.60
(m, 2H), 7.62–7.65 (m, 2H), 7.69 (d, J = 7.92 Hz, 2H), 8.05 (d,
J = 7.63 Hz, 2H), 8.26 (d, J = 2.05 Hz, 2H); 13C NMR (150 MHz,
CDCl3) d ppm 27.9, 33.3, 45.6, 45.7, 46.6, 49.1, 58.1, 115.8, 118.1,
120.3, 121.6, 123.3, 123.8, 125.5, 127.3, 127.5, 129.4, 130.2,
135.4, 139.6, 155.7, 155.7.
6.1.3. Preparation of 2-chloro-5-methyl-11-(nitromethyl)-5H-
indolo[2,3-b]quinoline 21
The solution of nitromethane (4 mmol) in DMSO (1 mL) was
added to the suspension of NaH in dry DMSO (5 mL) with stirring.
When the babbling subsided, the 6 (1 mmol) in DMSO was added.
The mixture was heated at 80 °C for 4 h. After cooled down to room
temperature, 20 mL water was added, then the mixture was
washed with ethyl acetate for two times to remove the byproducts.
The aquous phase was acidified with saturated NH4Cl, and the pre-
cipitation was filtrated. The filtration 21 was washed with water
and dried. Yield: 96%, brown solid; 1H NMR (600 MHz, DMSO-d6)
d ppm 4.36 (s, 3H), 6.89 (s, 2H), 7.23 (t, J = 7.48 Hz, 1H), 7.54–
7.59 (m, 1H), 7.61–7.65 (m, 1H), 7.94 (dd, J = 9.10, 2.35 Hz, 1H),
8.11 (d, J = 9.10 Hz, 1H), 8.18 (d, J = 7.92 Hz, 1H), 8.60 (d,
J = 2.35 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) d ppm 33.5, 72.4,
117.8, 117.8, 120.0, 120.5, 122.6, 123.9, 124.7, 126.8, 128.4,
129.0, 130.2, 130.7, 135.3, 154.9, 156.0; HRMS (ESI) Calcd for
6.2. Bioactivity study
6.2.1. Cell lines
Established in vitro, human cancer cell lines: MV4-11 (bipheno-
typic B myelomonocytic leukemia), A549 (non-small cell lung car-
cinoma), HCT116 (colorectal carcinoma) and normal mice
fibroblasts BALB/3T3 was used.
All lines were obtained from American Type Culture Collection
(Rockville, Maryland, USA). All the cell lines were maintained in
the Institute of Immunology and Experimental Therapy, Wroclaw,
Poland.
C
17H11ClN3O2 [MÀH]À 324.0545. Found 324.0539.
6.1.4. Preparation of 2-chloro-5-methyl-5H-indolo[2,3-
b]quinoline-11-carbaldehyde 22
MV4-11 cells were cultured in RPMI 1640 medium (Gibco, Scot-
To the stirred suspension of 21 (0.5 mmol) in methanol (5 mL)
at 0 °C, a freshly prepared solution of KOH (1.5 mmol) in methanol
(10 mL) was added dropwise. After being stirred for 30 min, a solu-
tion of KMnO4 (0.335 mmol) and MgSO4 (1.5 mmol) in water
(30 mL) was added dropwise with vigorous stirring. When the
reaction was complete, the mixture was filtered over a thin layer
land, UK) supplemented with 2 mM L-glutamine and 1.0 mM so-
dium pyruvate, 10% fetal bovine serum (all from Sigma–Aldrich
Chemie GmbH, Steinheim, Germany), 100 units/mL penicillin, and
100 lg/mL streptomycin (both from Polfa, Tarchomin S.A., Poland).
The cell line was grown at 37 °C with 5% CO2 humidified
atmosphere.

L. Wang et al. / Bioorg. Med. Chem. 20 (2012) 4820–4829
4829
HCT116 and A549 cells were cultured in RPMI 1640+OptiMEM
(50:50) medium (Gibco, Scotland, UK) supplemented with 2 mM
-glutamine and 5% fetal bovine serum (all from Sigma–Aldrich
Chemie GmbH, Steinheim, Germany). BALB/3T3 cells were cultured
in Dulbecco’s medium (IIET, Wroclaw, Poland) supplemented with
2 mM L-glutamine and 1.0 mM sodium pyruvate, 10% fetal bovine
serum (all from Sigma–Aldrich Chemie GmbH, Steinheim, Ger-
many). All culture mediums were supplemented with 100 units/
Acknowledgements
L
We are grateful to Okayama University for support and to the
Advanced Science Research Center for the NMR experiments. We
are thankful to Prof. S. Nakashima for HRMS analyses and Prof. J.
Futami for UV analyses. We thank the Ministry of Education, Cul-
ture, Sports, Science and Technology (MEXT) for the scholarship
to L.W. and Nichiro, Inc. for the generous gift of salmon fish sperm
DNA.
mL penicillin and 100 lg/mL streptomycin (both from Polfa,
Tarchomin S.A., Poland). The cells were grown at 37 °C with 5%
CO2 humidified atmosphere.
Supplementary data
6.2.2. Anti-proliferative assay in vitro
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.054.
Test solutions of the compounds tested (1 mg/mL) were pre-
pared by dissolving the substances in 100
drich, Germany) completed with 900
l
l DMSO (Sigma–Al-
l
l of tissue culture
References and notes
medium. Afterwards, the tested compounds were diluted in cul-
1. (a)Functional Molecules from Natural Sources; Wrigley, S. K., Thomas, R.,
Nicholson, N., Bedford, C. T., Nicholso, N., Eds.; RSC Publishing, 2011;
(b)Biodiversity: New Leads for the Pharmaceutical and Agrochemical Industries;
Chrystal, E. J. T., Wrigley, S. K., Thomas, T., Nicholson, N., Hayes, M., Eds.; Royal
Society of Chemistry, 2000; (c) Butler, M. S. J. Nat. Prod. 2004, 67, 2141.
2. Reviews: (a) Lavrado, J.; Moreira, R.; Paulo, A. Curr. Med. Chem. 2010, 17, 2348;
(b) Suresh, K.; Eyunni, V. K.; Etukala, J. R.; Ablordeppey, S. Y. Mini-Rev. Med.
Chem. 2008, 8, 538.
ture medium to reach the final concentrations of 10, 1, 0.1, 0.01
and 0.001 lg/mL.
Twenty-four hours before addition of the tested agents, the cells
were plated in 96-well plates (Sarstedt, Germany) at a density of
1 Â 104 cells per well. The assay was performed after 72 h of expo-
sure to varying concentrations (from 0.01 to 10 lg/mL) of the
tested agents. The in vitro cytotoxic effect of all agents against
HCT116, A549 and BALB/3T3 were examined using the SRB assay
and the in vitro cytotoxic effect of all agents against MV4-11 was
examined using the MTT assay to avoid rinsing out of the cells dur-
ing washing. Both assays were recommended by NCI as giving sim-
ilar results.12 Each compound in given concentration was tested in
triplicates in each experiment, which was repeated 3–5 times.
3. Cimanga, K.; De Bruyne, T.; Pieters, L.; Claeys, M.; Vlietinck, A. J. Tetrahedron
Lett. 1996, 37, 1703.
4. (a) Cimanga, K.; De Bruyne, T.; Pieters, L.; Totte, J.; Tona, L.; Kambu, K.; Gerghe,
D. V.; Vlietinck, A. J. Phytomed. 1998, 5, 209; (b) Cimanga, K.; De Bruyne, T.;
Pieters, L.; Vlietinck, A. J. J. Nat. Prod. 1997, 60, 688; (c) Cimanga, K.; De Bruyne,
T.; Lasure, A.; Van Poel, B.; Pieters, L.; Claeys, M.; Berghe, D. V.; Kambu, K.; Tona,
L.; Vlietinck, A. Planta Med. 1996, 62, 22; (d) Grellier, P.; Frappier, F.; Trigalo, F.;
Ramiaramanana, L.; Millerioux, V.; Deharo, E.; Schrével, J.; Bodo, B.; Pousset, J.
L. Phytother. Res. 1996, 10, 317; (e) Wright, C. W.; Phillipson, J. D.; Awe, S. O.;
Kirby, G. C.; Warhurst, D. C.; Quetin-Leclercq, J.; Angenot, L. Phytother. Res.
1996, 10, 361; (f) Kirby, G. C.; Paine, A.; Warhurst, D. C.; Noamese, B. K.;
Phillipson, J. D. Phytother. Res. 1995, 9, 359.
5. (a) Guittat, L.; Alberti, P.; Rosu, F.; Van Miert, S.; Thetiot, E.; Pieters, L.; Gabelica,
V.; De Pauw, E.; Ottaviani, A.; Riou, J. F.; Mergny, J. L. Biochimie 2003, 85, 535;
(b) Jonckers, T. H. M.; Van Miert, S.; Cimanga, K.; Bailly, C.; Colson, P.; De Pauw-
Gillet, M. C.; Van Den Heuvel, H.; Claeys, M.; Lemière, F.; Esmans, E. L.;
Rozenski, J.; Quirijnen, L.; Maes, L.; Dommisse, R.; Lemière, G. L. F.; Vlietinck, A.;
Pieters, L. J. Med. Chem. 2002, 45, 3497; (c) Bailly, C.; Laine, W.; Baldeyrou, B.;
De Pauw-Gillet, M. C.; Colson, P.; Houssier, C.; Cimanga, K.; Van Miert, S.;
Vlietinck, A. J.; Pieters, L. Anti-Cancer Drug Des. 2000, 15, 191; (d) Dassonneville,
L.; Lansiaux, A.; Wattelet, A.; Wattez, N.; Mahieu, C.; Van Miert, S.; Pieters, L.;
Bailly, C. Eur. J. Pharmacol. 2000, 409, 9.
6.2.3. MTT assay
For the last 3–4 h of incubation 20 ll of MTT solution (MTT: 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;
stock solution: 5 mg/mL, Sigma–Aldrich, Germany) were added
to each well. The mitochondria of viable cells reduce the pale yel-
low MTT to a navy blue formazan: the more viable cells are present
in well, the more MTT will be reduced to formazan. When incuba-
tion time was completed, 80 ll of the lysing mixture were added to
each well (lysing mixture: 225 mL dimethylformamide, POCh,
Gliwice, Poland, 67.5 g sodium dodecyl sulphate, Sigma–Aldrich,
Germany, and 275 mL of distilled water). After 24 h, when forma-
zan crystals had been dissolved, the optical densities of the sam-
ples were read on a Multiskan RC photometer (Labsystems,
Helsinki, Finland) at 570 nm wavelength. The background optical
density was measured in the wells filled with culture medium,
without the cells.
6. (a) Potier, P. Chem. Soc. Rev. 1992, 21, 113; (b) Stiborová, M.; Sejbal, J.; Borek-
Dohalská, L.; Aimová, D.; Poljaková, J.; Forsterová, K.; Rupertová, M.; Wiesner,
J.; Hudecek, J.; Wiessler, M.; Frei, E. Cancer Res. 2004, 64, 8374.
7. (a) Peczynska-Czoch, W.; Pognan, F.; Kaczmarek, L.; Boratynski, J. J. Med. Chem.
1994, 37, 3503; (b) Kaczmarek, L.; Peczynska-Czoch, W.; Osiadacz, J.;
Mordarski, M.; Sokalski, W. A.; Marcinkowska, E.; Glazman-Kusnierczyk, H.;
Radzikowski, C. Bio. Med. Chem. 1999, 7, 2457; (c) Osiadacz, J.; Majka, J.;
Czarnecki, K.; Kaczmarek, L.; Sokalski, W. A. Bio. Med. Chem. 2000, 8, 937; (d)
Humeniuk, R.; Kaczmarek, L.; Peczynska-Czoch, W.; Marcinkowska, E. Oncol.
Res. 2003, 13, 269; (e) Jaromin, A.; Kozubek, A.; Suchoszek-Lukaniuk, K.;
Malicka-Blaszkiewicz, M.; Peczynska-Czoch, W.; Kaczmarek, L. Drug Deliv.
2008, 15, 49.
6.2.4. SRB assay
8. (a) Kaczmarek, L.; Luniewski, W.; Zagrodzki, B.; Godlewska, J.; Osiadacz, J.;
Wietrzyk, J.; Opolski, A.; Peczynska-Czoch, W. Acta. Pol. Pharm. 2002, 59, 199;
(b) Godlewska, J.; Luniewski, W.; Zagrodzki, B.; Kaczmarek, L.; Bielawska-Pohl,
A.; Dus, D.; Wietrzyk, J.; Opolski, A.; Siwko, M.; Jaromin, A.; Jakubiak, A.;
Kozubek, A.; Peczynska-Czoch, W. Anticancer Res. 2005, 25, 2857.
9. Chen, Y. L.; Hung, H. M.; Lu, C. M.; Li, K. C.; Tzeng, C. C. Bio. Med. Chem. 2004, 12,
6539.
10. El Sayed, I.; Van der Veken, P.; Steert, K.; Dhooghe, L.; Hostyn, S.; Van Baelen,
G.; Lemière, G.; Maes, B. U. W.; Cos, P.; Maes, L.; Joossens, J.; Haemers, A.;
Pieters, L.; Augustyns, K. J. Med. Chem. 2009, 52, 2979.
11. (a) Bergman, J.; Engqvist, R.; Stålhandske, C.; Wallberg, H. Tetrahedron 2003, 59,
1033; (b) Parvatkar, P. T.; Parameswaran, P. S.; Tilve, S. G. Curr. Org. Chem. 2011,
15, 1036; (c) Wang, L.; Lu, W. J.; Odawara, T.; Mei, Z. W.; Peng, W.; El-Tantawy
El-Sayed, I.; Inokuchi, T. J. Heterocycl. Chem., in press.
12. Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.; Tosini, S.; Skehan,
P.; Scudiero, D. A.; Monks, A.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113.
13. Purcell, M.; Neault, J. F.; Tajmir-Riahi, H. A. Biochem. Biophys. Acta 2000, 1478,
61.
The cells attached to the plastic were fixed by gently layering
cold 50% TCA (trichloroacetic acid, Sigma–Aldrich, Germany) on
the top of the culture medium in each well. The plates were incu-
bated at 4 °C for 1 h and then washed five times with tap water.
The cellular material fixed with TCA was stained with 0.4% SRB sul-
forhodamine B (Sulforhodamine B, Sigma–Aldrich, Germany) dis-
solved in 1% acetic acid (POCh, Gliwice, Poland) for 30 min.
Unbound dye was removed by rinsing (4Â) with 1% acetic acid.
The protein-bound dye was extracted with 10 mM unbuffered Tris
base (Sigma–Aldrich, Germany) for determination of optical den-
sity (at 540 nm) in a computer-interfaced, 96-well microtiter plate
reader Multiskan RC photometer (Labsystems, Helsinki, Finland).
The background optical density was measured in the wells filled
with culture medium, without the cells.