Synthesis and in vitro cancer cell growth inhibition evaluation of 11-amino-modified 5-Me-indolo[2,3-b]quinolines and their COMPARE analyses

Article abstract of DOI:10.1007/s00044-016-1508-z

A plant-derived neocryptolepine core, the 5-Me-indolo[2,3-b]quinoline skeleton, was emblazoned with substituents at C11 and C2 and then tested against various cancer cell lines to find potent anticancer agents. In the in vitro antiproliferative activity assay against the breast cancer MDA-MB-453 cell line, the attachment of alkylamino substituents at C11 of the 5-Me-indolo[2,3-b]quinoline induced improved activities. Specifically, 11-(3-aminopropylamino) and 11-(4-aminobutylamino) derivatives indicated the highest activity and selectivity against MDA-MB-453 (IC₅₀ = 0.3-0.5 μM) and also exhibited a higher cytotoxicity against the colon adenocarcinoma (WiDr) and ovarian cancer (SKOv3) cell lines. A synergistic effect by attachment of substituents at C2 was favorably observed with an electron-donating group, such as CH₃O, and unfavorably observed with an electron-withdrawing one, such as F and CF₃. Further modification of the terminal free amino group of the lariat attachment at C11 into the corresponding acylamides and 2,3-dihydrobenzo[e][1,3]thiazin-4-ones was not effective for the antiproliferative activity. The computer-assisted database analysis, COMPARE, suggested that 14e and 13b have a mode of action similar to actinomycin D and 13c has a mode of actions similar to vindesine sulfate or aclarubicin hydrochloride. However, the new compounds may have other unique mode of actions since the correlation coefficients (r) were in relatively low levels.

Full text of DOI:10.1007/s00044-016-1508-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1508-z
ORIGINAL RESEARCH
Synthesis and in vitro cancer cell growth inhibition evaluation
of 11-amino-modified 5-Me-indolo[2,3-b]quinolines and their
COMPARE analyses
Masashi Okada1 Zhen-Wu Mei1 Md. Imran Hossain1 Li Wang1 Taihei Tominaga1 Takeshi Takebayashi1
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Masaharu Murakami1 Mizuki Yasuda1 Tsukasa Shigehiro1 Tomonari Kasai1 Akifumi Mizutani1
Hiroshi Murakami1 Ibrahim El Tantawy El Sayed1,3 Shingo Dan2 Takao Yamori2 Masaharu Seno1
Tsutomu Inokuchi1
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Received: 7 June 2015 / Accepted: 6 January 2016
Ó Springer Science+Business Media New York 2016
Abstract A plant-derived neocryptolepine core, the
5-Me-indolo[2,3-b]quinoline skeleton, was emblazoned
with substituents at C11 and C2 and then tested against
various cancer cell lines to find potent anticancer agents. In
the in vitro antiproliferative activity assay against the
breast cancer MDA-MB-453 cell line, the attachment of
alkylamino substituents at C11 of the 5-Me-indolo[2,3-
b]quinoline induced improved activities. Specifically,
11-(3-aminopropylamino) and 11-(4-aminobutylamino)
derivatives indicated the highest activity and selectivity
against MDA-MB-453 (IC50 = 0.3–0.5 lM) and also
exhibited a higher cytotoxicity against the colon adeno-
carcinoma (WiDr) and ovarian cancer (SKOv3) cell lines.
A synergistic effect by attachment of substituents at C2
was favorably observed with an electron-donating group,
such as CH3O, and unfavorably observed with an electron-
withdrawing one, such as F and CF3. Further modification
of the terminal free amino group of the lariat attachment at
C11 into the corresponding acylamides and 2,3-dihy-
drobenzo[e][1,3]thiazin-4-ones was not effective for the
antiproliferative activity. The computer-assisted database
analysis, COMPARE, suggested that 14e and 13b have a
mode of action similar to actinomycin D and 13c has a
mode of actions similar to vindesine sulfate or aclarubicin
hydrochloride. However, the new compounds may have
other unique mode of actions since the correlation coeffi-
cients (r) were in relatively low levels.
Graphical Abstract
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-016-1508-z) contains supplementary
material, which is available to authorized users.
Keywords 5Me-indolo[2,3-b]quinoline Á
& Shingo Dan
sdan@jfcr.or.jp
Antiproliferative agent Á C11-alkylamino-substituted Á
MDA-MB-453 Á JFCR39 panel Á COMPARE study
& Masaharu Seno
mseno@okayama-u.ac.jp
& Tsutomu Inokuchi
inokuchi@cc.okayama-u.ac.jp
Introduction
1
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
Cancer is a leading cause of death worldwide, accounting
for approximately 14 million new cases and 8.2 million
deaths in 2012 (data from WHO) (World Cancer Report,
2014; Cancer Fact sheet No 297). A virtually promising
strategy for cancer prevention today is chemotherapy
(Neidle and Thurston, 2005). DNA has been a major target
for anticancer drugs (Gibson, 2002), because the
2
Division of Molecular Pharmacology, Cancer Chemotherapy
Center, Japanese Foundation for Cancer Research,
3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan
3
Chemistry Department, Faculty of Science,
El Menoufia University, Shebin El Koom, Egypt
123

Med Chem Res
interaction ability can interfere with transcription (gene
expression and protein synthesis) and DNA replication
(Yang and Wang, 1999; Denny, 1989).
screening is now predominant, cell-based screening is still
the main technique for revealing cytotoxic drugs. Thus, we
applied the synthesized agents to the different cell lines from
that used in our previous testing (Wang et al., 2012).
In the meantime, some of the 11-amino-modified 5-Me-
indolo[2,3-b]quinolines 13 and 14 were examined for their
antitumor effects across a panel of 39 human cancer cell
lines, termed JFCR39, which is comprised the subpanels of
human cancer cell lines derived from the breast, melanoma,
lung, colon, central nervous system (CNS), ovary, stomach,
kidney, and prostate. An antitumor spectrum of each
compound across JFCR39, or fingerprint, was calculated,
and a COMPARE analysis was carried out to predict the
molecular mode of action of a respective antitumor com-
pound by comparing the fingerprints with those of refer-
ence compounds possessing a known mechanism of action
previously determined (Yamori et al., 1999; Yamori,
2003).
Naturally occurring alkaloids are important chemical
compounds that serve as a rich reservoir of lead com-
pounds for drug discovery (Kittakoop et al., 2014; Leland,
2006). In a search for such lead compounds, the con-
stituents of the roots of the climbing shrub Cryptolepis
sanguinolenta, growing in West Africa, such as Ghana,
have attracted our attention, since an aqueous macerate or
decoction of this root is used for the treatment of endemic
disease, such as malaria fever, and various disorders of the
body (Alexandra et al., 2000; Cimanga et al., 1996a).
Quinoline–indole motif-containing alkaloids were the
main components identified in this plant (Cimanga et al.,
1996b). Among the known constituents, cryptolepine (i),
neocryptolepine (ii), and isocryptolepine (iii) are promising
scaffolds for developing more active derivatives in view of
their various biological properties (Fig. 1) (Bracca et al.,
2014).
The antiproliferative testing of the neocryptolepine
analogues indicated that the derivatives of 5-methylated ii
are usually more cytotoxic than their respective congeners
of 6-methylated iv (Wang et al., 2012). A synergistic effect
of the substituents at the C2 and/or C9 positions was also
observed (Kaczmarek et al., 1999).
Results and discussion
Synthesis
According to the reported procedure, the 11-chloro-
neocryptolepine cores with substituents at C2 were synthe-
sized starting from 1H-methyl indole-3-carboxylate (1) and
N-methylanilines 2 (Scheme 1) (Wang et al., 2014). Thus,
the intermediate 2-N-(N-methylanilino)indole-3-carboxy-
lates (3) were obtained by the chlorination of 1 with
N-chlorosuccinimide in the presence of 1,4-dimethylpiper-
azine followed by the addition of a mixture of 2 and tri-
chloroacetic acid. Cyclization of 3 via intramolecular
aromatic acylation was carried out by heating at 250 °C in
diphenyl ether to form tetracyclic ketones 4, which were
Intrigued by the importance of the substituents, such as
alkyl and alkylamino groups, on the indolo[2,3-b]quinoline
scaffolds ii and iv (Wang et al., 2012; Lu et al., 2013), we
attempted to synthesize the 5-Me-indolo[2,3-b]quinoline
derivatives 11–16 with various alkylamino and aminoalky-
lamino groups at the C11 positions and tested their antipro-
liferative activities against cancer cell lines, such as human
breast cancer (MDA-MB-453), colon adenocarcinoma
(WiDr), and ovarian cancer (SKOv3). Although target-based
Fig. 1 Representative
indoloquinolines from
Cryptolepis sanguinolenta and
congener
123

Med Chem Res
Scheme 1 Preparation of 11-chloroneocryptolepines 5–10 and their 11-amino derivatives 11–16. Reagents and conditions: (i) a. N-
chlorosuccinimide, 1,4-dimethylpiperazine, b. trichloroacetic acid. (ii) diphenyl ether, reflux. (iii) POCl3, toluene, reflux. (iv) appropriate amines
converted to 11-chloroneocryptolepines 5–10 by chlorina-
tion and dehydration with POCl3. Subsequently, amination
through the SNAr reaction of 5–10 was performed by heating
with the appropriate amines in DMF to afford the
11-aminoalkylamino-substituted neocryptolepine deriva-
tives 11–16.
The natural product neocryptolepine (ii) exhibited
comparable antiproliferative activities (IC50 7.48 lM)
against the MDA-MB-453 cell line with the reference
anticancer drug cisplatin (IC50 7.6 0.7 lM), but much
weaker than that of doxorubicin HCl (IC50
0.086 0.006 lM). Introduction of the Cl atom at the C11
of neocryptolepine induced a moderate improvement in the
activity (IC50 5.16 lM). Strikingly, introduction of an
amino group significantly increased the cytotoxic activity
compared with the non-substituted neocryptolepine.
Accordingly, various kinds of amino groups were then
evaluated to assess their antiproliferative activity.
Further modifications at the terminal amino function of
the lateral 3-aminopropylamino groups attached at C11 of
the indolo[2,3-b]quinoline 11e were carried out as outlined
in Scheme 2. First, the terminal amino group at C11 of 11e
was smoothly acylated with acetyl and 2-thienyl chlorides
to give the corresponding 11-(3-N-acylaminopropylamino)
derivatives 17. In order to connect the sulfur-containing
heterocyclic pendant at the C11 attachment, the thiazo-
lidin-4-one skeleton was constructed. Thus, the three-
component condensation of the 11-(3-aminopropylamino)-
substituted 11e, the substituted aldehydes, and 2-mercap-
tobenzoic acid was smoothly achieved with N,N-dicyclo-
hexylcarbodiimide (DCC) in toluene by heating in good
yields.
For the SAR study by varying the substituents at C11,
we applied three different kinds of amines as the sub-
stituents at C11, namely alkylamines, benzenamines, and
hydrazines, and the results are given in Table 1 and in the
supplementary Table. Among the three different kinds of
amines, the alkylamines showed a higher activity than the
benzenamines and are more favorable than hydrazines in
terms of their availability as structure-tunable materials.
Various alkylamines, which are classified into three cate-
gories, i.e., monoamines, 1,n-diamines, and polyamines, were
employed as the substituents. In our experiments, the diamines
and polyamines show higher activities compared with the
monoamines. The 1,x-diamino-substituted derivatives were
applied for screening under varying the size of carbon spacer
between two amino groups. Thus, the highest activities were
achieved with 4-aminobutylamino- and 3-aminopropylamino-
substituents, having LC50 values of 0.50 and 0.30 lM, respec-
tively. As shown in the supplementary Table, the hydrazine
derivatives show lower activities than that of the 1,3-diamino-
propane derivative, i.e., IC50 0.50 versus IC50 1.85–2.34.
We then focused our attention on the effect of the
substituent at C2 on the cytotoxicity. Thus, the substituents
at C2 were varied from H to Br, OCH3, CF3, F in the
In vitro antiproliferative activities
The results of the cytotoxic activity in vitro were expressed
as the IC50 concentration of the compound (in lM) that
inhibits proliferation 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 independent experiments.
The results on the antiproliferative activity of neocryp-
tolepine and its chemically modified analogues against the
human breast cancer MDA-MB-453 cell line are summa-
rized in Table 1, along with the data of the anticancer
drugs, cisplatin (Rakic et al., 2012) and doxorubicin HCl
(Sandhu et al., 2012).
123

Med Chem Res
Scheme 2 Synthesis of
indolo[2,3-b]quinolines by
further modifications of the
terminal amino group. Reagents
and conditions: (i) THF, rt; (ii)
THF, Et3N, rt
O
R3
NH
i
R3COCl
R3= methyl, 2-thienyl
HN
N
N
NH2
CH3
17
HN
N
ii
R4CHO
S
N
+
O
O
N
R4
CH3
11e
OH
SH
HN
N
N
CH3
18
presence of the (5-(diethylamino)pentan-2-yl)amino group
at C11 (including the supplementary Table). However, in
these cases, the substituent at C2 exerted little influence on
the activity. We observed that the strong electron-with-
drawing groups, such as CF3 and F, at C2 are not favorable
for the cytotoxicity and, in contrast, the electron-donating
group, such as OCH3, is fairly favorable. Indeed, com-
pound 14e bearing an OCH3 at C2 and a (3-amino-
propyl)amino group at C11 showed the improved IC50
value of 0.20 lM, the highest value of the evaluated data
compared with that of compound 11e bearing no sub-
stituent at C2 (0.50 lM).
Subsequently, these compounds, 11a–c, 11e, 11g, 11k,
11l, 13a, and 14e, which showed a potent antiproliferative
activity against the human breast cancer MDA-MB-453
cell line with IC50 \ 10 were then chosen for the next
stage, i.e., the antiproliferative activity against WiDr (colon
adenocarcinoma cell line), SKOV3 (ovarian cancer cell
line), along with the normal mice fibroblast BALB/3T3.
These results are summarized in Table 2. All the tested
compounds, except for 11l, were cytotoxic against the
WiDr and SKOv3 cancer cells, and their antiproliferative
activities against these cancer cells were much higher than
that of the control agent, cisplatin (Kuo et al., 2010; Ganta
et al., 2014). However, these tested compounds were ca. 5
times less active when compared to anticancer drug dox-
orubicin (Moon et al., 1999; Molphy et al., 2014)
(Table 2). Compounds 11e and 14e have much higher
antiproliferative activities against the cancer cell lines
WiDr and SKOv3, compared with other compounds, and
are less cytotoxic against normal mice fibroblast BALB/
3T3. Against the normal fibroblast BALB/3T3, the
antiproliferative activities of 11e and 14e were 1.4–2.6
times lower than that against the cancer cell lines. Com-
pounds 14e have a selective antiproliferative activity,
mostly against the SKOv3 cell line.
Based on these results, it is quite obvious that the
introduction of the proper alkylamino substituents into
biologically active derivatives can favorably influence their
activities and selectivity in DNA binding (Wang et al.,
2012; Kaczmarek et al., 1999).
As the additional SAR study, the C11 terminal amino
group modified compounds, such as the acylated 17 and
2,3-dihydrobenzo[e][1,3]thiazin-4-one derivatives 18, were
tested for their antiproliferative activity against the human
breast cancer MDA-MB-453 cell line, WiDr (colon ade-
nocarcinoma cell line), and SKOV3 (ovarian cancer cell
line). The biological results along with the cytotoxicity of
the anticancer drugs, cisplatin and doxorubicin HCl, are
summarized in Table 3. However, the chemically modified
analogues 17 and 18 showed slightly lower antiprolifera-
tive activities compared with the respective precursors
11–16 bearing a terminal free amino group. In the
series of modified compounds 18 bearing
a 2,3-
123

Med Chem Res
Table 1 Yields of 11-alkylaminated 5-methyl-indolo[2,3-b]quinolines and their antiproliferative activities against the human breast cancer
MDA-MB-453 cell line
Compound
R1
R2
Yield (%)
MDA-MB-453
IC50 (lM)
Neocryptolepine (ii)
H
H
F
H
7.48 4.42
5.16 2.74
3.78 0.12
6
Cl
Cl
75
95
10
11a
11b
H
H
99
94
1.53 1.00
4.41 3.37
11c
11e
11 g
H
H
H
77
96
96
5.12 0.43
0.50 0.24
1.76 1.08
11 k
11 l
H
H
78
92
5.41 1.58
5.72 1.31
11p
13a
14e
H
95
82
99
[10.4
Br
1.90 1.15
0.20 0.09
OCH3
Cisplatin
7.6 0.7a
Doxorubicin
0.086 0.006b
a
Rakic et al. (2012)
b
Sandhu et al. (2012)
123

Med Chem Res
Table 2 Antiproliferative activity of 11-alkylaminated 5-methyl-indolo[2,3-b]quinolines against normal mice fibroblast BALB/3T3 and against
cancer cell lines WiDr (colon adenocarcinoma) and SKOv3 (ovarian cancer)
Compound
BALB/3T3
IC50 (lM)
WiDr
IC50 (lM)
SKOv3
IC50 (lM)
11a
N.T.
4.18 0.09
5.12 4.11
5.25 0.47
0.64 0.13
2.81 0.55
7.20 1.20
[20
10.18 3.07
[20
11b
0.635 0.190
1.607 0.624
0.884 0.115
N.T.
11c
15.55 11.15
2.7 0.42
6.35 4.64
[20
11e
11 g
11 k
N.T.
11 l
N.T.
[20
13a
N.T.
4.23 0.62
0.37 0.07
8.84 0.52a
0.089 lMb
6.22 3.91
1.3 0.18
18.2 0.1c
0.52d
14e
0.978 0.021
8.700 0.970
1.078 0.033
Cisplatin
Doxorubicin
N.T. not tested
a
Kuo et al. (2010)
b
Moon et al. (1999)
c
Ganta et al. (2014)
d
Molphy et al. (2014)
dihydrobenzo[e][1,3]thiazin-4-one pendant, the aryl sub-
stituents at the C2 of the 1,3-thiazin-4-one unit revealed
that the electron-donating dimethylamino group is favor-
able, while an electron-withdrawing group, such as F, and
the pyridine motif are not favorable.
Compound 13b shows a mentionable cytotoxicity for the
CNS (SF-539, LC50 = 0.63 lM, SNB-75, LC50 = 0.93
lM), colon (HCC2998, LC50 = 0.69 lM), lung (NCI-
H522, IC50 = 0.57 lM, DMS273, LC50 = 0.61 lM), and
melanoma (LOX-IMVI, LC50 = 0.75 lM) cancer cell
lines. Compound 13c displayed a strong growth inhibitory
activity (GI50 \ 1 lM) for 28 panels in the JFCR39 panel
of the various cell lines. DMS273 (lung cancer) and NCI-
H522 (lung cancer) are the most sensitive cell lines of the
39 cell lines (GI50 = 0.42 lM for both and LC50 = 4.7
lM and 5.5 lM, respectively). Compound C also showed a
strong antitumor effect on LOX-IMVI (melanoma,
GI50 = 0.3 lM, LC50 = 7.6 lM).
Cytotoxic and growth inhibitory activities
across the JFCR39 cancer cell line panel
The anticancer screening of compounds 14e, 13b, and 13c
was performed in order to determine their cytotoxic and
growth inhibitory activities across the JFCR39 cancer cell
line panel. The compounds were evaluated at five con-
centration levels (100, 10, 1.0, 0.1, and 0.01 lM). The
results (the means of GI50, TGI, and LC50 values) are
summarized in Table 4.
COMPARE study
Table 4 shows that all of the compounds are highly
potent against the melanoma (LOX-IMVI) and lung (NCI-
H522, A549, and DMS273) cancer cell lines in the JFCR39
panels. Compounds 14e and 13b are more potent than
compound 13c toward the cancer cell lines in the JFCR39
panel. Compounds 14e and 13b show a similar growth
inhibitory activity (GI50 [ 0.01 lM) against the 31 and 28
cell lines, respectively, of the JFCR39 panel. Compound
14e shows a notable cytotoxicity against the breast (BSY-1,
LC50 = 0.82 lM), CNS (SF-539, LC50 = 0.60 lM, SNB-
75, LC50 = 0.91 lM), colon (HCC2998, LC50 = 0.74 lM),
lung (NCI-H522, LC50 = 0.71 lM, A549, LC50 = 0.85,
DMS273, LC50 = 0.70, DMS114, LC50 = 0.72), and
ovarian (VCAR-4, LC50 = 0.48 lM) cancer cell lines.
The COMPARE analysis assesses the correlation coefficient
between the fingerprints of the test compounds and those of
the various reference compounds (Kong and Yamori, 2012).
This system provides an information intensive approach to
identifying the molecular targets of new compounds. The
JFCR39 and COMPARE analysis-guided assay is a success-
ful means to find new anticancer drug candidates. The
COMPARE analysis is carried out by calculation of the
Pearson correlation coefficient (r value) between the finger-
prints of compounds X and Y. The r value is then used to
determine the degree of similarity, that is, the higher the
r value, the greater the similarity of X to Y. Generally, an
r value of 0.5\ r \ 0.75 between two agents suggests they
might have a similar mechanism of action (Fig. 2).
123

Med Chem Res
Table 3 Antiproliferative activity of terminal-modified 11-(3-aminopropylamino)-5-methyl-indolo[2,3-b]quinolines against MDA-MB-453,
WiDr, and SKOv3 cell lines
Compound
R2
MDA-MB-453
LC50 (lM)
WiDr
LC50 (lM)
SKOv3
LC50 (lM)
17a
2.06 1.00
3.27 1.18
2.12 1.09
3.99 0.41
1.41 1.00
4.25 1.18
8.43 4.16
13.60 3.96
17b
18a
18b
18c
18d
[7.8
[7.8
3.39 0.88
4.05 0.91
2.19 0.74
10.62 1.20
4.1 0.07
10.9 0.26
[20
13.1 0.18
Cisplatin
7.6 0.7a
0.086 0.006b
8.84 0.52c
0.089d
18.2 0.e
0.52f
Doxorubicin
a
Rakic et al., (2012)
b
Sandhu et al., (2012)
c
Kuo et al., (2010)
d
Moon et al., (1999)
e
Ganta et al., (2014)
f
Molphy et al., (2014)
123

Med Chem Res
The COMPARE analysis revealed that compounds 14e
and 13b have a very good match to actinomycin D (r = 0.7
for both). Similarly, compound 13b has a slight similarity
to paclitaxel (r = 0.64). Compound 13c shows some
resemblance to vindesine sulfate (r = 0.58) and aclaru-
bicin HCl (r = 0.57).
(3.0 mmol) were heated together at 135–155 °C for 1–4 h.
Monitoring by TLC was used to ensure the completion of
reaction. The resulting brown crude oil was purified by flash
chromatography using AcOEt-2Nammonia inMeOH (9:1) as
an eluent to yield pure 11–16 as yellowish-orange solids.
N1,N1-diethyl-N4-(5-methyl-5H-indolo[2,3-b]quinolin-11-
yl)pentane-1,4-diamine 11a Yield: 99 %; yellowish
solids; mp 70–73 °C; IR (KBr) mmax 3242, 3048, 2965,
2801, 2620, 1591, 1566, 1549, 1487, 1441, 1422, 1404,
Conclusion
1
1373, 1277, 1244, 1192, 1142, 1061, 748 cm-1; H NMR
Plant-derived 5-Me-indolo[2,3-b]quinolines with pendant
substituents at C11 and/or C2 were assayed for antiprolifera-
tive screening against several cancer cell lines. In particular,
the 11-(3-aminopropylamino)-substituted 11e and 14e and
11-(4-aminobutylamino)-substituted 11f recorded the highest
activity and selectivity against a breast cancer (MDA-MB-
453) cell line (IC50 = 0.3–0.5 lM). A synergistic effect bythe
additional attachment was observed with an electron-donating
group such as CH3O at C2, i.e., 14e (IC50 = 0.2 lM). On the
other hand, further modification of the terminal free amino
group of the lariat attachment at C11 into the corresponding
acylamides and 2,3-dihydrobenzo[e][1,3]thiazin-4-ones 18e
was not effective for the antiproliferative activity. The com-
puter-assisted database analysis, COMPARE, suggested that
14e and 13b have a mode of action similar to actinomycin D. It
also suggested that 13c has a mode of actions similar to vin-
desine sulfate or aclarubicin HCl. However, the new com-
pounds may have other unique mode of actions since the
correlation coefficients (r) were in relatively low levels, which
are interesting possibility to examine in further studies.
(CDCl3, 600 MHz): d = 0.92 (6H, t, J = 7.2 Hz), 1.36
(3H, d, J = 6.0 Hz), 1.50–1.60 (2H, m), 1.64–1.78 (2H,
m), 2.34 (2H, m), 2.41 (4H, q, J = 7.2 Hz), 4.29 (3H, s),
4.30 (1H, m), 4.91 (1H, d, J = 10.8 Hz), 7.21 (1H, td,
J = 7.8, 0.6 Hz), 7.38 (1H, t, J = 7.2 Hz), 7.46 (1H, t,
J = 7.8 Hz), 7.68–7.73 (2H, m), 7.77 (1H, d, J = 7.8 Hz),
7.91 (1H, d, J = 7.8 Hz), 8.16 (1H, d, J = 8.4 Hz); 13C
NMR (CDCl3, 150.8 MHz): d = 11.42 (2C), 22.35, 23.71,
32.74, 37.20, 46.73 (2C), 52.66, 54.34, 110.75, 114.77,
116.55, 117.50, 118.96, 120.60, 120.67, 124.09, 124.67,
126.32, 130.38, 138.15, 148.49, 153.15, 156.58; HRMS
(ESI) calcd for C25H31N4 [M - H]- exact mass: 387.2554,
found 387.2583.
3-((5-Methyl-5H-indolo[2,3-b]quinolin-11-yl)amino)pro-
pan-1-ol 11b Yield: 94 %, yellowish solids; mp
187–188 °C; IR (KBr) mmax 3391, 3080, 3042, 2922, 2859,
2359, 1912, 1618, 1591, 1570, 1514, 1443, 1416, 1341,
1
1292, 1248, 1225, 1074, 1063, 864, 748, 714 cm-1; H
NMR (CDCl3, 600 MHz): d = 1.95 (2H, quint,
J = 6.0 Hz), 3.96–4.02 (4H, m), 4.20 (3H, s), 6.20 (1H,
brs), 7.14 (1H, t, J = 7.2 Hz), 7.31 (1H, td, J = 7.8,
1.2 Hz), 7.37 (1H, t, J = 7.8 Hz), 7.60 (1H, d,
J = 8.4 Hz), 7.67 (1H, t, J = 7.2 Hz), 7.73 (1H, d,
J = 8.4 Hz), 7.91 (1H, d, J = 7.8 Hz), 8.11 (1H, d,
J = 7.8 Hz); 13C NMR (CDCl3, 150.8 MHz): d = 32.74,
32.87, 48.39, 62.20, 107.05, 114.63, 115.87, 117.08,
118.73, 120.50, 121.10, 124.00, 124.29, 125.58, 130.27,
138.06, 148.51, 152.30, 156.56; HRMS (ESI) calcd for
C19H18N3O [M - H]- exact mass: 304.1455, found
304.1485.
Experimental
Chemistry
General
The commercially obtained reagents were directly used
1
without further purification. The H-NMR and 13C-NMR
spectra were measured by a Varian INOVA-600 spec-
trometer with CDCl3 or DMSO-d6 as the solvent unless
otherwise indicated. High-resolution mass spectra were
obtained on a Bruker micrOTOF II-SKA spectrometer.
According to the reported procedure, the 11-chloro-
neocryptolepine cores with substituents at C2 were syn-
thesized starting from 1H-methyl indole-3-carboxylate (1)
and N-methylanilines 2 (Scheme 1) (Wang et al., 2014).
4-(5-Methyl-5H-indolo[2,3-b]quinolin-11-yl)morpholine 11c
Yield: 77 %, orange-colored solids; mp 205–207 °C; IR
(KBr) mmax 3046, 2951, 2849, 1734, 1614, 1599, 1564,
1520, 1489, 1437, 1416, 1387, 1366, 1292, 1269, 1242,
1
1194, 1171, 1105, 1063, 1009, 856, 746, 721 cm-1; H
NMR (CDCl3, 600 MHz): d = 3.58 (4H, t, J = 4.80 Hz),
4.09 (4H, t, J = 4.80 Hz), 4.36 (3H, s), 7.27 (1H, m), 7.46
(1H, m), 7.55 (1H, t, J = 7.20 Hz), 7.74–7.78 (3H, m),
8.36 (1H, d, J = 7.80 Hz), 8.57 (1H, d, J = 8.40 Hz); 13C
NMR (CDCl3, 150.8 MHz): d = 33.15, 49.34 (2C), 67.68
(2C), 114.41, 117.60, 119.49, 120.82, 121.38, 122.78,
General procedure for the synthesis of 11–16
11-Chloro-5-methyl-5H-indolo[2,3-b]quinolines 5–10 (0.3
mmol) and an excess of the appropriate aminoalkylamine
123

Med Chem Res
Table 4 Growth inhibitory (GI50) and cytotoxic (LC50) activities of compounds 14e, 13b, and 13c across the JFCR39 panel
Tissue of origin
Breast
Cell line
14e
13b
13c
GI (lM)
50
TGI (lM)
IC50 (lM)
GI (lM)
50
TGI
IC50 (lM)
GI50 (lM)
TGI (lM)
IC50 (lM)
HBC-4
0.06
0.11
0.06
0.04
0.08
0.04
0.05
0.10
0.05
0.13
0.12
0.05
0.06
0.04
0.72
0.04
0.06
0.08
0.04
0.05
0.06
0.04
0.08
0.03
0.06
0.04
0.05
0.05
0.13
0.27
0.27
0.06
0.09
0.04
0.05
0.05
0.05
0.1
0.80
0.30
0.33
0.24
0.33
0.47
0.59
0.81
0.20
0.34
0.78
0.22
0.64
0.54
6.9
9.5
0.82
3.4
17
0.08
0.12
0.09
0.03
0.07
0.05
0.06
0.11
0.07
0.15
0.20
0.07
0.09
0.06
1.0
0.61
0.35
0.41
0.31
0.34
0.55
0.75
0.55
0.23
0.38
0.98
0.23
0.59
0.64
3.7
6.1
3.5
4.1
3.1
3.4
7.9
4.7
2.9
0.63
0.93
5.2
0.69
4.6
28
1.3
4.3
5.2
10
19
20
53
44
44
58
76
49
22
42
62
8.0
64
46
49
15
98
44
5.5
20
8.3
4.7
18
7.6
82
19
32
100
51
18
51
44
14
69
100
100
100
22
56
BSY-1
2.0
HBC-5
0.86
0.87
0.50
0.7
MCF-7
7.9
4.1
13
MDA-MB-23
U251
6.9
17
CNS
SF-268
26
0.94
1.3
14
SF-295
14
14
SF-539
0.60
0.91
25
0.7
3.9
15
SNB-75
SNB-78
HCC2998
KM-12
3.3
2.9
20
Colon
0.74
23
1.0
2.9
7.0
5.6
5.8
2.3
4.8
4.8
1.8
2.6
2.5
1.6
3.7
1.5
4.6
2.7
3.1
3.4
7.4
3.0
7.1
4.9
3.6
8.0
12
0.72
0.56
0.58
0.43
1.2
HT-29
24
HCT-15
HCT-116
NCI-H23
NCI-H226
NCI-H522
NCI-H460
A549
50
100
5.1
2.5
6.2
0.57
1.7
1.9
0.61
1.10
0.75
42
0.57
0.28
0.49
0.20
0.27
0.23
0.18
0.26
0.11
0.28
0.15
0.35
0.33
1.0
22
0.04
0.07
0.09
0.04
0.05
0.05
0.05
0.10
0.03
0.05
0.04
0.05
0.05
0.13
0.19
0.25
0.12
0.11
0.06
0.05
0.05
0.06
0.09
0.16
0.80
0.36
0.63
0.18
0.23
0.26
0.18
0.33
0.11
0.30
0.16
0.26
0.43
1.2
Lung
1.8
25
0.74
0.42
0.45
0.68
0.42
1.1
0.71
3.4
0.85
0.70
0.72
1.1
8.0
0.48
9.5
23
DMS273
DMS114
LOX-IMVI
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
RXF-631L
ACHN
Melanoma
Ovarian
0.31
0.64
0.52
0.56
0.48
1.2
59
42
100
54
20
Renal
1.4
7.4
20
0.67
2.2
3.0
10
1.1
2.1
0.7
Stomach
St-4
0.43
0.38
0.34
0.9
3.8
12
0.60
0.47
0.48
1.1
3.7
3.1
36
0.72
1.1
MKN1
MKN7
23
0.9
MKN28
MKN45
MKN74
DU145
37
58
0.55
0.44
0.56
1.2
1.0
28
1.1
8.0
8.0
3.3
22
6.2
6.8
4.0
12
1.0
52
0.94
0.43
1.3
Prostate
0.34
1.3
17
PC-3
0.16
70
2.0
CNS central nervous system, GI50 50 % growth inhibition concentration (lM), TGI total growth inhibition concentration (lM), LC50 lethal concentration (lM)
123

Med Chem Res
Fig. 2 Growth inhibitory and
cytotoxic activities of
compounds 14e, 13b, and 13c
across a panel of the JFCR39
cell lines. The mean graph was
produced by computer
processing of the 50 % growth
inhibition (GI50) and the 50 %
lethal concentration (LC50
)
values. The logarithm of the
GI50 and the LC50 values for
each cell line is indicated. The
X axis shows the difference on a
logarithmic scale between the
mean of Log GI50/Log LC50
values for all 39-cell lines (MG-
MID, expressed as 0 in the
fingerprint) and the Log GI50
/
Log LC50 for each cell line in
the JFCR39 panel. Columns to
the right of 0 indicate the
sensitivity of the cell lines to a
given compound, and columns
to the left indicate their
resistance. The MG-MID mean
of the Log GI50/Log LC50
values for all 39 cell lines; delta
difference between the MG-
MID and the Log GI50/Log
LC50 value for the most
sensitive cell line; range
difference between the Log
GI50/Log LC50 values for the
most resistant cell line and the
most sensitive cell line
123

Med Chem Res
123.44, 124.91, 126.20, 128.50, 130.46, 138.03, 149.93,
154.55, 157.81; HRMS (ESI) calcd for C20H20N3O
[M ? H]? exact mass: 318.1601, found 318.1621.
N-(2-(1H-Indol-3-yl)ethyl)-5-methyl-5H-indolo[2,3-b]quino-
lin-11-amine 11l Yield: 92 %, yellow solids; mp
198–200 °C; IR (KBr) mmax 3410, 3053, 2918, 2862, 1717,
1622, 1593, 1568, 1499, 1441, 1418, 1343, 1290, 1248,
N1-(5-Methyl-5H-indolo[2,3-b]quinolin-11-yl)propane-1,3-
diamine 11e Yield: 96 %, yellowish solids; mp
69–71 °C; IR (KBr) mmax 3435, 2928, 2868, 2359, 2342,
1622, 1559, 1489, 1441, 1418, 1287, 1248, 1057, 750,
1
1109, 1071, 887, 745 cm-1; H NMR (CDCl3, 600 MHz):
d = 3.18 (2H, t, J = 6.6 Hz), 4.20–4.23 (2H, m), 4.22 (3H,
s), 5.44 (1H, brs), 7.04 (2H, m), 7.11 (1H, t, J = 7.8 Hz),
7.23 (1H, t, J = 8.4 Hz), 7.29 (1H, t, J = 7.8 Hz), 7.36
(1H, t, J = 7.8 Hz), 7.42 (1H, dd, J = 8.4, 0.6 Hz), 7.48
(1H, d, J = 8.4 Hz), 7.58–7.62 (2H, m), 7.68 (1H, t,
J = 7.8 Hz), 7.75 (1H, d, J = 8.4 Hz), 8.02 (1H, d,
J = 8.4 Hz), 8.54 (1H, brs); 13C NMR (CDCl3,
150.8 MHz): d = 26.62, 32.52, 48.61, 103.99, 110.81,
111.35, 115.18, 115.70, 115.96, 118.14, 118.25, 118.38.
120.87, 120.94, 122.02, 122.97 (2C), 123.62, 124.14,
124.73, 126.99, 130.84, 136.12, 137.26, 148.60, 155.31;
HRMS (ESI) calcd for C26H23N4 [M ? H]? exact mass:
391.1917, found 391.1943.
1
669 cm-1; H NMR (CDCl3, 600 MHz): d = 1.80 (2H,
quint, J = 6.0 Hz), 3.01 (2 H, t, J = 6.0 H), 4.01 (2H, t,
J = 6.0 Hz), 4.22 (3H, s), 7.17 (1H, t, J = 7.2 Hz), 7.18
(1H, brs), 7.31 (1H, t, J = 7.2 Hz), 7.41 (1H, t,
J = 7.2 Hz), 7.61 (1H, d, J = 9.0 Hz), 7.67 (1H, td,
J = 7.2, 1.2 Hz), 7.76 (1H, d, J = 7.8 Hz), 7.95 (1H, d,
J = 7.8 Hz), 8.13 (1H, d, J = 8.4 Hz); 13C NMR (CDCl3,
150.8 MHz): d = 32.64, 32.66, 41.26, 49.25, 105.61,
114.40, 115.86, 116.94, 118.49, 120.35, 121.37, 124.06,
124.12, 125.16, 130.08, 137.79, 148.52, 151.94, 156.55;
HRMS (ESI) calcd for C19H21N4 [M ? H]? exact mass:
305.1761, found 305.1765.
5-Methyl-N-phenyl-5H-indolo[2,3-b]quinolin-11-amine 11p
Yield: 95 %, yellowish solids; mp 187–188 °C; IR (KBr)
mmax 3296, 3053, 2943, 1620, 1589, 1568, 1524, 1497,
1485, 1439, 1416, 1402, 1317, 1261, 1242, 1171, 1142,
N1-(5-Methyl-5H-indolo[2,3-b]quinolin-11-yl)hexane-1,6-
diamine 11g Yield: 96 %, yellow solids; mp 54–56 °C;
IR (KBr) mmax 3350, 2928, 2855, 1622, 1593, 1568, 1489,
1
1099, 897, 858, 746, 721, 692 cm-1; H NMR (CDCl3,
1
1441, 1422, 1281, 1246, 1200, 1142, 882, 752 cm-1; H
600 MHz): d = 4.27 (3H, s), 6.89 (3H, m), 7.00 (2H, t,
J = 7.20 Hz), 7.19–7.25 (3H, m), 7.33 (1H, t,
J = 7.20 Hz), 7.43 (1H, t, J = 7.8 Hz), 7.68 (1H, m), 7.73
(2H, m), 8.05 (1H, d, J = 7.80 Hz); 13C NMR (CDCl3,
150.8 MHz): d = 32.99, 114.60, 114.88, 117.07, 117.16,
118.21 (2C), 119.32, 121.14, 122.25, 123.21, 123.45,
124.72, 127.44, 129.42 (2C), 130.54, 137.75, 140.05,
142.50, 153.30, 156.75; HRMS (ESI) calcd for C22H18N3
[M ? H]? exact mass: 324.1495, found 324.1509.
NMR (DMSO-d6, 600 MHz): d = 1.19 (5H, m), 1.36 (1H,
m), 1.68 (2H, m), 2.38 (1H, t, J = 6.6 Hz), 2.95 (1H, t,
J = 6.6 Hz), 3.83 (2H, m), 4.16 (3H, s), 6.99 (1H, brs),
7.07 (1H, t, J = 7.2 Hz), 7.28 (1H, t, J = 7.2 Hz), 7.41
(1H, t, J = 7.8 Hz), 7.50 (1H, d, J = 8.4 Hz), 7.79 (1H, t,
J = 7.8 Hz), 7.84 (1H, d, J = 7.8 Hz), 7.90 (1H, d,
J = 7.8 Hz), 8.53 (1H, d, J = 7.2 Hz); 13C NMR (DMSO-
d6, 150.8 MHz): d = 22.58, 26.24, 26.27, 30.98, 32.66,
41.20, 48.13, 104.68, 115.41, 115.82, 116.79, 118.47,
121.12, 122.22, 124.20 (2C), 124.98, 131.13, 137.61,
148.78, 152.10, 156.59; HRMS (ESI) calcd for C22H27N4
[M ? H]? exact mass: 347.2230, found 347.2235.
N4-(2-Bromo-5-methyl-5H-indolo[2,3-b]quinolin-11-yl)-N1,N1-
diethylpentane-1,4-diamine 13a Yield: 82 %, reddish
solids; mp 39 °C; IR (KBr) mmax 3296, 3050, 2967, 2934,
2799, 1622, 1559, 1489, 1443, 1418, 1379, 1273, 1242,
N1,N1-Bis(2-aminoethyl)-N2-(5-methyl-5H-indolo[2,3-b]quino-
lin-11-yl)ethane-1,2-diamine 11k Yield: 78 %, yellowish-
orange solids; mp 69 °C; IR (KBr) mmax 3354, 3048, 2934,
2855, 2357, 2189, 1616, 1591, 1564, 1495, 1489, 1418,
1188, 1107, 1059, 909, 802, 760, 741 cm-1 1H NMR
;
(CDCl3, 600 MHz): d = 0.93 (6H, t, J = 7.2 Hz), 1.35
(3H, d, J = 6.6 Hz), 1.58 (2H, m), 1.65–1.78 (2H, m), 2.37
(2H, m), 2.44 (4H, q, J = 7.2 Hz), 4.21 (1H, m), 4.25 (3H,
s), 4.87 (1H, d, J = 10.8 Hz), 7.22 (1H, td, J = 7.8,
1.2 Hz), 7.47 (1H, t, J = 7.8 Hz), 7.56 (1H, d,
J = 9.0 Hz), 7.73–7.80 (2H, m), 7.90 (1H, d, J = 7.2 Hz),
8.25 (1H, d, J = 2.4 Hz); 13C NMR (CDCl3, 150.8 MHz):
d = 11.38 (2C), 22.37, 23.65, 32.82, 37.14, 46.74 (2C),
52.57, 54.47, 111.65, 113.45, 116.40, 117.71, 118.13,
119.29, 120.83, 123.95, 126.79, 127.17, 132.98, 136.90,
147.18, 153.35, 156.30; HRMS (ESI) calcd for C25H32
BrN4 [M ? H]? exact mass: 467.1805, found 467.1807.
1
1279, 1246, 1142, 1103, 1024, 882, 853, 752 cm-1; H
NMR (CDCl3, 600 MHz): d = 2.41 (4H, brs), 2.61 (4H, t,
J = 6.6 Hz), 2.72 (2H, t, J = 6.0 Hz), 2.80 (4H, t,
J = 6.0 Hz), 3.94 (2H, t, J = 5.4 Hz), 4.23 (3H, s), 7.16
(1H, t, J = 7.2 Hz), 7.33 (1H, t, J = 7.2 Hz), 7.41 (1H, t,
J = 7.2 Hz), 7.63–7.69 (2H, m), 7.74 (1H, d, J = 7.8 Hz),
7.95 (1H, d, J = 7.2 Hz), 8.31 (1H, d, J = 8.4 Hz); 13C
NMR (CDCl3, 150.8 MHz): d = 32.73, 39.71 (2C), 45.66,
55.29, 56.56 (2C), 106.73, 114.68, 115.94, 117.18, 118.60,
120.45, 121.31, 123.99, 124.15, 125.57, 130.35, 137.97,
148.38, 152.37, 156.67; HRMS (ESI) calcd for C22H27N6
[M - H]- exact mass: 375.2303, found 375.2324.
N1-(2-Methoxy-5-methyl-5H-indolo[2,3-b]quinolin-11-yl)pro-
pane-1,3-diamine 14e Yield: 99 %, yellowish solids; mp
123

Med Chem Res
105–106 °C; IR (KBr) mmax 3381, 3268, 2934, 1614, 1593,
1568, 1489, 1445, 1424, 1348, 1288, 1246, 1184, 1140,
addition of thiosalicylic acid (3.0 equiv.) and DCC (1.2
equiv.). Then the reaction mixture was heated to reflux for
10–15 h. After cooling to room temperature, the mixture
was concentrated to dryness under reduced pressure, and
the residue was taken up in chloroform and washed with
aq. 5 % NaHCO3 and then finally with brine. The organic
layer was dried over MgSO4 and evaporated to get a crude
product, which was followed purification by flash chro-
matography using ethyl acetate (AcOEt)–2 N ammonia in
MeOH (9:1 v/v) as the eluent to yield pure products as
yellowish-orange solids (18a–d).
1038, 937, 810, 760 cm-1
;
1H NMR (DMSO-d6,
600 MHz): d = 1.72 (2H, quint, J = 6.6 Hz), 2.64 (2H, t,
J = 6.6 Hz), 3.91 (3H, s), 3.94 (2H, t, J = 6.6 Hz), 4.14
(3H, s), 7.04 (1H, t, J = 7.2 Hz), 7.26 (1H, t, J = 7.2 Hz),
7.43–7.47 (2H, m), 7.79 (1H, d, J = 9.6 Hz), 7.92 (2H, m);
13C NMR (DMSO-d6, 150.8 MHz): d = 32.23, 33.44,
39.82, 47.18, 55.80, 104.47, 105.88, 116.18, 116.28,
116.43, 117.47, 119.52, 122.15, 123.98, 124.43, 132.18,
147.73, 152.50, 153.55, 156.26; HRMS (ESI) calcd for
C20H23N4O [M ? H]? exact mass: 335.1866, found
335.1864.
2-(4-Chlorophenyl)-3-(3-((5-methyl-5H-indolo[2,3-b]quino-
lin-11-yl)amino)propyl)-2H-benzo[e][1,3]thiazin-4(3H)-one
18a Yield: 49.7 %, yellowish solids; mp 124–126 °C; IR
(KBr) mmax 3349, 3055, 2932, 1622, 1591, 1564, 1489, 1456,
1441, 1422, 1310, 1277, 1246, 1204, 1146, 1092, 1013, 841,
748 cm-1; 1H NMR (DMSO-d6, 600 MHz): d = 2.06 (1H,
m), 2.13 (1H, m), 3.23 (1H, dt, J = 15.0, 5.4 Hz), 3.82 (1H,
m), 4.22 (1H, m), 4.34 (3H, s), 4.51 (1H, m), 5.73 (1H, s),
7.11 (1H, d, J = 7.8 Hz), 7.15–7.22 (5H, m), 7.29 (1H, t,
J = 7.8 Hz), 7.32–7.36 (2H, m), 7.58 (1H, t, J = 7.2 Hz),
7.68 (1H, d, J = 9.0 Hz), 7.70 (1H, brs), 7.81 (1 H, t,
J = 7.8 Hz), 7.86 (1H, d, J = 8.4 Hz), 7.91 (1H, d,
J = 7.8 Hz), 8.16 (1H, dd, J = 7.8, 1.2 Hz), 8.63 (1H, d,
J = 8.4 Hz); 13C NMR (CDCl3, 150.8 MHz): d = 29.56,
35.64, 44.08, 45.23, 61.03, 102.48, 114.67, 115.41, 116.73,
120.94, 121.41, 121.77, 123.46, 124.67, 126.25, 126.71,
127.45 (2C), 127.79, 128.49, 128.82 (2C), 129.01, 129.95,
131.99, 132.53, 132.74, 134.51, 136.77, 136.97, 150.09,
151.09, 165.22; HRMS (ESI) calcd for C33H26ClN4OS
[M - H]- exact mass: 561.1516, found 561.1517.
General procedure for the preparation of compound 17
N1-(5-Methyl-5H-indolo[2,3-b]quinolin-11-yl)propane-1,3-
diamine (11e, 50 mg) was completely dissolved in dry
DMF (2 ml), then a mixture solution of R3COCl (1.2
equiv.) and dry DMF (1 ml) was added drop by drop under
stirring, finally 2.0 equiv of triethylamine was added, and
the reaction was carried out at room temperature for 2–4 h.
TLC monitoring was used to ensure the completion of
reaction. The crude product was purified by flash chro-
matography using AcOEt-2 N ammonia in MeOH (9:1 v/v)
as an eluent to yield pure products 17 as yellowish-orange
solids.
N-(3-((5-Methyl-5H-indolo[2,3-b]quinolin-11-yl)amino)propyl)
thiophene-2-carboxamide 17b Yield: 95 %, yellowish
solids; mp 204–205 °C; IR (KBr) mmax 3424, 3333, 3059,
2930, 1732, 1622, 1593, 1566, 1545, 1499, 1439, 1418,
1358, 1294, 1248, 1204, 1144, 1071, 862, 745, 719 cm-1
;
2-(4-Fluorophenyl)-3-(3-((5-methyl-5H-indolo[2,3-b]quino-
lin-11-yl)amino)propyl)-2H-benzo[e][1,3]thiazin-4(3H)-one
18b Yield: 69 %, yellowish solids; mp 104–108 °C; IR
(KBr) mmax 3349, 3055, 2934, 1622, 1593, 1564, 1506, 1456,
1H NMR (DMSO-d6, 600 MHz): d = 1.92 (2H, quint,
J = 7.2 Hz,), 3.26 (2H, q, J = 6.6 Hz,), 3.88 (2H, q,
J = 7.2 Hz), 4.16 (3H, s), 7.02–7.07 (2H, m), 7.10 (1H, dd,
J = 4.8, 2.4 Hz), 7.27 (1H, t, J = 7.2 Hz), 7.41 (1H, t,
J = 7.2 Hz), 7.49 (1H, d, J = 7.8 Hz), 7.64 (1H, dd,
J = 3.6, 1.2 Hz), 7.72 (1H, dd, J = 5.4, 1.2 Hz), 7.79 (1H,
td, J = 7.2 1.2 Hz), 7.85 (1H, d, J = 7.8 Hz), 7.94 (1H, d,
J = 7.2 Hz), 8.52 (2H, d, J = 6.6 Hz); 13C NMR (DMSO-
d6, 150.8 MHz): d = 30.92, 32.22, 36.69, 45.39, 105.08,
115.06, 115.65, 116.55, 118.00, 120.58, 122.08, 123.97,
124.02, 124.68, 127.81, 127.93, 130.64 (2C), 137.39,
139.84, 148.14, 152.38, 156.40, 161.33; HRMS (ESI) calcd
for C24H23N4OS [M ? H]? exact mass: 415.1587, found
415.1597.
1441, 1422, 1279, 1244, 1229, 1159, 1098, 845, 746 cm-1
;
1H NMR (CDCl3, 600 MHz): d = 2.13 (1H, m), 2.19 (1H,
m), 3.25 (1H, dt, J = 14.4, 4.8 Hz), 3.85 (1H, m), 4.22 (1H,
m), 4.35 (3H, s), 4.50 (1H, m), 5.82 (1H, m), 6.91 (2H, t,
J = 8.4 Hz), 7.10 (1H, d, J = 7.8 Hz), 7.16–7.33 (5H, m),
7.35 (1H, t, J = 7.2 Hz), 7.61 (1H, t, J = 7.8 Hz), 7.67 (1H,
d, J = 8.4 Hz), 7.82 (2H, t, J = 7.8 Hz), 7.93 (1H, d,
J = 7.8 Hz), 8.10 (1H, m), 8.14 (1H, d, J = 8.4 Hz), 8.68
(1H, d, J = 8.4 Hz); 13C NMR (CDCl3, 150.8 MHz):
d = 29.52, 36.51, 44.33, 45.07, 60.98, 100.67, 113.75,
115.63, 115.64 (d, J = 21.9 Hz, 2C), 116.77, 120.01,
121.72, 122.21, 124.23, 125.02, 126.31, 126.61, 127.78,
127.92 (d, J = 8.3 Hz, 2C), 128.43, 129.88, 132.50, 132.72,
132.74, 133.92, 136.68, 138.05, 147.76, 151.99, 161.75,
165.25; 19F NMR (CDCl3, 564 MHz): d = –112.96; HRMS
(ESI) calcd for C33H26FN4OS [M - H]- exact mass:
545.1817, found 545.1842.
General procedure for the synthesis of compound 18a–d
N1-(5-Methyl-5H-indolo[2,3-b]quinolin-11-yl)propane-
1,3-diamine (11e, 100 mg) and R4CHO (2.0 equiv.) was
stirred in dry toluene at 70–80 °C for 5 min, followed by
123

Med Chem Res
2-(4-(Dimethylamino)phenyl)-3-(3-((5-methyl-5H-indolo[2,3-
b]quinolin-11-yl)amino)propyl)-2H-benzo[e][1,3]thiazin-4(3H)-
one 18c Yield: 81 %, yellowish solids; mp 119–121 °C; IR
(KBr) mmax 3364, 3053, 2934, 1614, 1593, 1564, 1520, 1495,
1456, 1441, 1420, 1360, 1281, 1246, 1188, 1165, 1065, 947,
cancer-derived MDA-MB-453 cells and human colon can-
cer-derived WiDr cells were cultured in DMEM (Wako Pure
Chemicals, Osaka, Japan) supplemented with 10 % heat-
inactivated fetal bovine serum (FBS) and 100 lg/ml kana-
mycin at 37 °C under atmosphere of 5 % CO2/95 % air.
Human ovary cancer-derived SKOv3 cells and human leu-
kemia-derived K562 cells were cultured in medium for
RPMI-1640 (Wako Pure Chemicals, Osaka, Japan) supple-
mented with 10 % FBS and 100 lg/ml kanamycin at 37 °C
under atmosphere of 5 % CO2/95 % air.
1
748 cm-1; H NMR (CDCl3, 600 MHz): d = 1.82–1.94
(2H, m), 2.88 (6H, s), 3.32 (1H, m), 3.74 (1H, m), 4.11 (1H,
m), 4.28 (3H, s), 4.36 (1H, m), 5.61 (1H, s), 6.54 (2H, d,
J = 8.4 Hz), 7.01 (1H, brs), 7.09 (2H, d, J = 9.0 Hz), 7.14
(1H, d, J = 7.8 Hz), 7.20 (1H, t, J = 7.2 Hz), 7.28 (1H, t,
J = 7.2 Hz), 7.34 (1H, t, J = 7.2 Hz), 7.41 (1H, t,
J = 7.2 Hz), 7.46 (1H, t, J = 7.2 Hz), 7.65 (1H, m), 7.73
(1H, m), 7.81 (1H, d, J = 7.8 Hz), 7.95 (1H, d, J = 7.8 Hz),
Cytotoxic assay
8.25 (1H, dd, J = 7.8, 1.2 Hz), 8.53 (1H, d, J = 8.4 Hz); 13
C
Neocryptolepine derivatives were evaluated for anticancer
activity in vitro by MTT assay. Cells in logarithmic growth
phase were suspended at 100,000 cells/ml in the culture
medium. The cell suspension solutions in 100 ll were
dispensed into each well of 96-well plate. The plate was
incubated at 37 °C and 5 % CO2 overnight. Then sequen-
tially diluted solution of compounds was added to each
well. Then after 72 h, 20 ll of 5 mg/ml MTT dissolved in
PBS was added to each well. The plate was further incu-
bated at 37 °C for 4 h, and formazan crystal was dissolved
by 10 % SDS supplemented with 20 lM of hydrochloride.
Absorbance at 570 nm was observed followed by incuba-
tion overnight.
NMR (CDCl3, 150.8 MHz): d = 29.70, 33.42, 40.14 (2C),
43.71, 44.59, 62.10, 106.04, 111.85 (2C), 114.66, 116.48,
116.58, 119.29, 121.49, 121.95, 123.27, 124.04, 124.55,
125.68, 126.14, 127.25 (2C), 127.59, 128.69, 130.00,
130.64, 132.24, 133.82, 137.52, 149.05, 149.89, 150.40,
155.38, 165.34; HRMS (ESI) calcd for C35H32N5OS
[M - H]- exact mass: 570.2333, found 570.2346.
3-(3-((5-Methyl-5H-indolo[2,3-b]quinolin-11-yl)amino)propyl)-2-
(pyridin-3-yl)-2H-benzo[e][1,3]thiazin-4(3H)-one 18d Yield:
90 %, yellowish solids; mp 96–97 °C; IR (KBr) mmax 3418,
3053, 2934, 1622, 1591, 1564, 1495, 1456, 1441, 1418, 1283,
1
1246, 1202, 1153, 1024, 795, 748, 710 cm-1; H NMR
The results of the cytotoxic activity in vitro were
expressed as the IC50 concentration of the compound (in
lM) that inhibits proliferation 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 indepen-
dent experiments.
(CDCl3, 600 MHz): d = 1.92–2.02 (2H, m), 3.16 (1H, dt,
J = 14.4, 5.4 Hz), 3.75 (1H, m), 4.17 (1H, m), 4.25 (3H, s),
4.52 (1H, m), 5.63 (1H, s), 6.80 (1H, brs), 7.09–7.19 (3H, m),
7.29 (1H, t, J = 7.8 Hz), 7.33 (1H, t, J = 7.8 Hz), 7.38 (1H,
t, J = 7.8 Hz), 7.46 (1H, t, J = 7.2 Hz), 7.51 (1H, d,
J = 7.8 Hz), 7.65 (1H, d, J = 8.4 Hz), 7.73 (1H, t,
J = 7.2 Hz), 7.77 (1H, d, J = 7.8 Hz), 7.92 (1H, d,
J = 7.8 Hz), 8.20 (1H, d, J = 8.4 Hz), 8.44 (1H, d,
J = 4.2 Hz), 8.48 (1H, d, J = 7.8 Hz), 8.51 (1H, d,
J = 2.4 Hz); 13C NMR (CDCl3, 150.8 MHz): d = 29.66,
33.49, 43.81, 45.51, 59.65, 106.35, 114.78, 116.43, 116.49,
119.49, 121.65, 121.86, 123.07, 123.22, 124.06, 125.91,
126.87, 127.83, 128.66, 130.13, 130.80, 132.06, 132.69,
133.47, 134.25, 137.52, 147.49 (2C), 148.96, 149.67,
155.09, 164.85; HRMS (ESI) calcd for C32H26N5OS
[M - H]- exact mass: 528.1864, found 528.1870.
Acknowledgments We are grateful to Okayama University for its
support and to the Advanced Science Research Center for the NMR
experiments. We are thankful to Prof. X.-Q. Yu, Sichuan University,
for the HRMS analyses. This study was partially supported by the
Adaptable and Seamless Technology Transfer Program of JST, No.
AS242Z02199Q. Screening Committee of Anticancer Drugs sup-
ported by Grant-in-Aid for Scientific Research on Innovative Areas,
Scientific Support Programs for Cancer Research, from The Ministry
of Education, Culture, Sports, Science and Technology, Japan, is
gratefully acknowledged.
Pharmacology
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