Synthesis of neocryptolepines and carbocycle-fused quinolines and evaluation of their anticancer and antiplasmodial activities

Article abstract of DOI:10.1016/j.bioorg.2020.103732

This study reported the discovery of novel compounds containing five-membered ring fused quinoline core structures as anticancer and antimalarial agents. Two libraries containing these core structures, neocryptolepines and carbocycle-fused quinolines, were prepared and evaluated. Compound 3h was found to be much more potent than other analogs against cancer cell lines with high selectivity. Meanwhile, carbocycle-fused quinolines 5h and 5s showed moderate anticancer properties but much less cytotoxicity to normal cell than doxorubicin. In addition, compound 3h also showed much lower cytotoxic against human normal kidney cell line compared to doxorubicin standard. However, only compounds 3s and 3p provided acceptable results for antimalarial activities.

Full text of DOI:10.1016/j.bioorg.2020.103732

Journal Pre-proofs
Synthesis of Neocryptolepines and Carbocycle-Fused Quinolines and Evalua-
tion of Their Anticancer and Antiplasmodial Activities
Bhornrawin Akkachairin, Warabhorn Rodphon, Onrapak Reamtong, Mathirut
Mungthin, Jumreang Tummatorn, Charnsak Thongsornkleeb, Somsak
Ruchirawat
PII:
S0045-2068(20)30190-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103732
YBIOO 103732
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
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24 January 2020
20 February 2020
6 March 2020
Please cite this article as: B. Akkachairin, W. Rodphon, O. Reamtong, M. Mungthin, J. Tummatorn, C.
Thongsornkleeb, S. Ruchirawat, Synthesis of Neocryptolepines and Carbocycle-Fused Quinolines and Evaluation
of Their Anticancer and Antiplasmodial Activities, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/
j.bioorg.2020.103732
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Synthesis of Neocryptolepines and Carbocycle-Fused Quinolines and Evaluation of
Their Anticancer and Antiplasmodial Activities
Bhornrawin Akkachairin,a Warabhorn Rodphon,a Onrapak Reamtong,d Mathirut Mungthin,b
Jumreang Tummatorn,a,c,* Charnsak Thongsornkleeb,a,c and Somsak Ruchirawata,c
aProgram on Chemical Biology, Chulabhorn Graduate Institute, Center of Excellence on Environmental Health and
Toxicology, CHE, Ministry of Education, 54 Kamphaeng Phet 6, Laksi, Bangkok 10210, Thailand
bDepartment of Parasitology, Phramongkutklao College of Medicine, Ratchawithi Road, Bangkok 10400, Thailand
cChulabhorn Research Institute, 54 Kamphaeng Phet 6, Laksi, Bangkok 10210, Thailand
dDepartment of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University,
Bangkok, Thailand
Keywords:
Neocryptolepine
Antiplasmodial
Anticancer
Indoloquinoline
Azide
Carbocycle-fused quinolines
Abstract
This study reported the discovery of novel compounds containing five-membered ring fused
quinoline core structures as anticancer and antimalarial agents. Two libraries containing these core
structures, neocryptolepines and carbocycle-fused quinolines, were prepared and evaluated.
Compound 3h was found to be much more potent than other analogs against cancer cell lines with
high selectivity. Meanwhile, carbocycle-fused quinolines 5h and 5s showed moderate anticancer
properties but much less cytotoxicity to normal cell than doxorubicin. In addition, compound 3h
also showed much lower cytotoxic against human normal kidney cell line compared to doxorubicin
standard. However, only compounds 3s and 3p provided acceptable results for antimalarial
activities.
1. Introduction
According to World Health Organization (WHO), the emergence and spread of cancer
and malaria are major threats to the world public health concern. Cancer is non-communicable
disease which is one of major disease concerns related to human mortality. The International
Agency for Research on Cancer (IARC) reported that the estimated cancer cases around the
world have been rising up to 18.1 million new cases causing 9.6 million deaths in 2018. In
addition, nearly one-half of the estimated cases and over one-half of cancer deaths worldwide are
found in Asia for both sexes due to the high contribution to global population.1 Another concern
is malaria disease which is one of global infectious diseases caused by Plasmodium parasites and
transmitted by female Anopheles mosquitos. There are four common species of Plasmodium that

cause human infection including P. falciparum, P. malariae, P. ovale and P. vivax. The P.
falciparum is the most threatening contributing strain to the highest prevalence of malaria.2-3 The
2018 World Malaria Report described that the number of malaria cases in 2017 was significantly
lower than in 2010 by approximately 20 million cases. However, there was no significant
decrease from 2015 to 2017. The estimated number of infections was up to 219 million cases in
90 countries and closely a million deaths were reported in 2017.3 The resistance of P. falciparum
strain against currently available anti-malarial drugs such as chloroquine and artemisinin is
growing. The incidence of malarial infection is rising in both developing and industrialized
countries, leading to an urgent need in the exploration of new therapeutic agents.4-6 Therefore,
the development of therapeutic treatment for both diseases is still in need and catches the interest
in the pharmaceutical field.
One of the most important core structures in drug discovery and development for treatment
of cancer and malaria is quinoline as this core structure plays an important role in increasing
potency of biologically active compounds. Nowadays, several anticancer and antimalarial drugs
in global markets contain quinoline moiety in the molecules.7-9 The examples of quinoline
antimalarial drugs include chloroquine, quinine, mefloquine, amodiaquine, and
hydroxychloroquine.9-11 In addition, quinoline core structure also has high impact on the
development of anticancer therapeutic agents.12 For examples, bosulif is a synthetic chronic
myeloid leukemia drug which was reported as one of the best-selling anticancer drugs.13 Therefore,
several libraries of quinoline analogs were designed, synthesized and evaluated for their anticancer
properties leading to the discovery of new lead anticancer compounds. In addition, polycyclic
quinoline is a privileged core structure used as a structure-based drug design to improve biological
activities of compounds. This core structure also is also present in the structure of well-known
anticancer drugs, topotecan and irinotecan.9,14 These two synthetic analogs of polycyclic quinoline
were originally developed from camptothecin natural product and used for treatment of ovarian
cancer, and metastatic colon and rectal cancer, respectively.15-18
Moreover, plants are rich sources of five-membered ring fused quinoline core structure.
Cryptolepine (1), neocryptolepine (2) and isocryptolepine (3) are examples of polycyclic
quinoline natural products which were isolated from the West and Central African plant,
Cryptolepis sanguinolenta.19-21This plant has been used in traditional medicine to treat various
health disorders including malaria, inflammation, urinary tract infections, and other diseases.22-25
These natural quinoline alkaloids have revealed potent antiplasmodial and anticancer properties
which show the potential to be developed as antimalarial and anticancer agents.26-28 They also
display biological functions including intercalation of DNA, inhibition of topoisomerease II
enzyme, cytotoxicity, antibacterial activity, and antifungal activity.26,29-30 However, due to their
ability to intercalate DNA and inhibit topoisomerease II, these indoloquinoline alkaloids can
cause toxicity to human. Cryptolepine shows stronger DNA-intercalation and inhibition of
human topoisomerase II compared to neocryptolepine and isocryptolepine.31-33

Me
N
N
Me
Me
N
O
HO
O
O
N
N
O
N
N
O
O
O
O
Irinotecan
Topotecan
OH
Me
OH
Me
N
Y
N
X
N
N
N
N
Me
Me
Me
1
Cryptolepine ( )
3
Neocryptolepine ( )
2
Isocryptolepine (X, Y = H:
Isocryptolepine (X = F, Y = Br:
)
2a
)
Scheme 1. Natural indoloquinoline and five-membered N-heterocyclic-fused quinoline
alkaloids.
Previously, our group reported the synthesis and structure–activity relationship (SAR)
studies of isocryptolepine derivatives as anticancer and antimalarial agents.34 The novel
isocryptolepines were conveniently synthesized in four steps from simple substrates, arylmethyl
azides and N-phenylsulfonyl indoles, via azide rearrangement chemistry.35 The results
demonstrated that halogen-substituted isocryptolepines (2a) showed high activities against and
selectivities for four Plasmodium falciparum strains and four primary cancer cell lines.34 As
numerous compounds containing five-membered nitrogen ring fused with quinoline core
structure showed a broad spectrum of biological activities including anticancer and antimalarial
properties; this inspired us to study the structure-activity relationship of neocryptolepine and
carbocycle-fused quinolines, as anticancer and antimalarial agents.
2. Results and Discussion
2.1. Chemistry
Recently, our research group developed synthetic methodology for the construction of 6H-
indolo[2,3-b]quinoline and carbocycle fused-quinoline derivatives using the N2-extrusion of azido
complexes of alkynylarylketone derivatives as shown in Scheme 2.36 Neocryptolepine derivatives
could be synthesized from regioselective methylation of indoloquinoline precursors which were
prepared using our developed method. A wide variety of indoloquinoline skeletons with broad range
of substituents were prepared from domino N2-extrusion–cyclization of alkynylarylketone starting
material which could be obtained by simple and convenient procedure via Sonogashira coupling of
2-iodobenzophenone derivatives with terminal alkynes. The reactions proceeded through double N2-
extrusion and double aryl migration of ortho-alkynylarylketones to generate carbodiimidium ion
intermediate (Scheme 2a) to provide indoloquinoline products. In this work, this useful synthetic
method of indoloquinolines was used to create a library of neocryptolepine analogs via
regioselective methylation of these indoloquinolines. The results showed that neocryptolepine
derivatives could be prepared from a broad range of substrates to give the desired products in low to
excellent yields. All neocryptolepines were further used for evaluation of biological activities. In

addition, our research group has reported the development of a synthetic method to construct five-
membered ring-fused quinoline using azide rearrangement chemistry.36 This method could be used
to create a novel library of carbocycle–fused quinolines with a broad range of substituents.
Alkynylarylketones were employed as the starting material which were treated with TMSN3 and
TfOH to generate the nitrilium ion, followed by double cyclization to give the desired products
(Scheme 2b). In this study, twenty-three neocryptolepines and twenty-five carbocycle-fused
quinolines were prepared for biological evaluation. In this study, R1, R2 and R3 were varied as shown
in Table 1.
a) Synthesis of neocryptolepine derivatives
R3
CH3
H
N
O
N
N
N
TMSN3, TfOH
DCM
MeI, 85 C
R1
R1
R1
R2
N-Methylation
R2
R2
R3
R3
Cyclization
Carbodiimidium ion generated in situ
H
N
R1
R2
R1
N
H
R1
C
R1
R2
C
N
R2
N
N
N
C
C
R2
H
N
N
+
H
+
R3
O
R3
O
R3 OH
R3 OH
b) Synthesis of cabocycle-fused quinoline derivatives
N
R2
B
R1
O
TMSN3, TfOH
DCM
R1
A
R2
N
Ph
N
N
N
N
-H2O
OH
N
Ph
Ph
Ph
H
NH
N
Ph
Ph
Ph
N
Ph
Scheme 2. Synthetic methods for the synthesis of neocryptolepines and carbocycle-fused
quinolines

Table 1. Neocryptolepine derivatives for biological studies.
Me
N
Me
N
Me
N
Me
N
Me
N
N
N
N
N
N
OMe
Bu
: 33%
Bu
3a
3e
3b
3d
: 91%
: 42%
: 68%
3c
:68%
Me
Me
Me
N
Me
N
Me
Me
N
Me
N
N
N
N
N
N
N
OMe
OMe
OMe
Bu
: 33%
Bu
MeO
Me
MeO
3i
: 97%
3j
: 29%
3h
: 85%
3f
3g
: 59%
Me
Me
Me
Me
Me
N
Me
N
Me
Me
N
N
N
N
N
N
N
N
OMe
F
MeO
3k
: 25%
F
3l
F
: 67%
F
MeO
3m
3n
3o
: 40%
: 75%
: 45%
Me
Me
N
Me
Me
N
Me
N
N
N
N
N
N
N
N
OMe
OMe
F
Bu
: 25%
MeO
F
3q
3t
: 92%
: 99%
3r
: 96%
3s
: 99%
3p
F
F
F
Me
N
Me
N
Me
N
N
N
N
Me
3u
MeO
Cl
3w
: 76%
3v
: 80%
: 89%
2.2. Biological evaluation
2.2.1. In vitro anticancer assays
Neocryptolepine (3a-3w) and carbocycle-fused quinoline (5a-5y) libraries were employed to
evaluate the biological activities including, in vitro antiplasmodial activity against chloroquine-
sensitive 3D7 (CQ-S) and chloroquine-resistant K1 (CQ-R) Plasmodium falciparum strains and
antiproliferative activity against four primary cancer cell lines; HepG2 (human liver carcinoma),
HuCCA-1 (human cholangiocarcinoma), MOLT-3 (human acute lymphoblastic leukemia), and
A549 (human lung adenocarcinoma). The cytotoxicity against normal human embryonic kidney cell
line-293 (HEK-293) was also investigated to determine the selectivity indices (SIs). The MTT assay
was employed to measure cytotoxicity of HepG2, HuCCA-1, A549, and HEK-293 cell lines while
XTT method was applied to MOLT-3 cell line. In this study, doxorubicin or etoposide or both were
used as the positive controls for these assays. In this studies, cytotoxic activities of neocryptolepine
analogs 3a-3w were investigated with the results shown in Table 3.

Table 2. Carbocycle-fused quinoline analogsa
N
N
N
N
N
5a
5b
5c
5d
5e
F
Cl
Br
CF3
N
Me
N
N
F
N
N
F
5g
5j
5f
5h
5i
OMe
NO2
N
N
Cl
N
N
N
Cl
MeO
F
Cl
5m
5l
5n
5k
5o
MeO
N
N
BnO
N
N
HO
N
MeO
MeO
OBn
5p
5r
5t
5q
5s
N
N
MeO
MeO
N
N
N
F3CO
MeO
5x
5u
5w
5v
5y
Cl
aAll compounds were prepared using our reported method.36
The results from cytotoxicity evaluation against HEK-293 showed that compound 3h, 3k and
3m were much less toxic compared to other derivatives of neocryptolepines and at least 35 times
less toxic than doxorubicin over 35 times. For the cytotoxicity against HuCCA-1, compound 3g, 3h,
3i, 3p, and 3s were all found to have good activity among other analogs, especially compound 3s
containing both methoxy group and fluorine atom showed highest activity, although, the selectivity
index of compound 3h was highest in this cell lines. In case of A549 cancer cell line, compounds
3e, 3f, 3g, 3h, 3i and 3s displayed good anticancer activity. Among these compounds, compound 3i
provided highest activity while compound 3h showed highest selectivity. The structures of both 3h
and 3i contain methoxy and isopentyl groups in the molecules with the methoxy group located at
different positions. The results from cytotoxicity evaluation against HepG2 cell line showed similar

range of IC50 to A549 cancer cell line (13-16 µM). Compounds 3e, 3f, 3g, 3h, 3i and 3s also provided
good activity and compound 3h also showed highest selectivity. Interestingly, compound 3h has
greater selectivity than doxorubicin standard and also provided better IC50 value than etoposide
standard in this cell line. In case of MOLT-3, compounds 3a, 3c, 3e, 3f, 3g, 3h, and 3i gave good
biological activity against this cancer cell line with good IC50 values. Compound 3f containing
methoxy and butyl groups has the best activity (IC50 2.54 µM) a of all analogs in the series among
this library while compound 3h again provided highest selectivity with IC50 of 4.90 µM and SI of
68.13. In this series of neocryptolepine anologs, compound 3h illustrated promising medicinal
property both for its cytotoxicity and selectivity in all cancer cell lines. Based on several studies of
cryptolepine and its analogues, including isocryptolepine and neocryptolepine, topoisomerase II has
been shown as target of inhibition by these compounds in several cancer cell lines via DNA
intercalation mechanism.37-39 Therefore, the mode of action of compound 3h was suspected to
involve the inhibition of topoisomerase II in these cancer cell lines as well.
Table 3 In vitro cytotoxic activities of neocryptolepine analogs 3a-3w against four primary cancer
cell lines and one normal lung cell line.
HEK-293
(µM)
HuCCA-1 (µM)
IC50 (µM)
A549 (µM)
IC50 (µM)
HepG2 (µM)
IC50 (µM)
MOLT-3 (µM)
IC50 (µM)
Comp.
IC50 (µM)
SI
SI
SI
SI
3a
3b
3c
3d
3e
3f
129.87±28.44
160.70±38.47
152.13±37.61
105.86±47.88
13.04±20.43
37.75±6.93
31.68 ± 0.17
123.26 ± 0.20
30.19 ± 0.56
58.04 ± 1.12
17.21 ± 3.24
14.07 ± 0.93
13.13 ± 1.82
13.39 ± 1.68
13.63 ± 0.00
30.13 ± 1.46
26.13 ± 0.26
108.13 ± 5.60
Inactive
4.10
1.30
5.04
1.82
0.76
2.68
10.81
24.93
7.00
7.61
11.86
2.21
-
27.12 ± 0.95
103.60 ± 6.56
26.85 ± 2.48
58.90 ± 2.86
15.58 ± 0.78
13.63 ± 1.57
14.89 ± 0.46
15.31 ± 0.57
10.80 ± 1.57
30.89 ± 1.63
27.03 ± 0.35
103.41 ± 4.98
Inactive
4.79
1.55
5.67
1.80
0.84
2.77
9.54
21.80
8.83
7.42
11.46
2.31
-
19.18 ± 0.62
76.66 ± 4.86
17.62 ± 0.40
72.00 ± 3.66
15.51 ± 1.00
15.36 ± 0.79
14.57 ± 0.75
15.58 ± 0.42
13.72 ± 0.69
17.04 ± 0.81
15.53 ± 1.16
56.81 ± 10.63
79.36 ± 2.30
21.38 ± 2.72
26.46 ± 0.34
24.88 ± 4.19
40.60 ± 1.72
73.81 ± 6.68
15.88 ±1.32
19.39 ± 1.09
25.77 ± 3.16
21.07 ± 3.96
85.87 ± 28.35
6.77
2.10
8.63
1.47
0.84
2.46
9.75
21.43
6.95
13.45
19.95
4.21
5.92
7.52
5.73
8.89
3.52
1.08
4.31
2.60
1.92
4.00
1.48
5.31 ± 0.38
18.55 ± 2.69
7.14 ± 2.08
19.85 ± 2.48
4.49 ± 0.44
2.54 ± 0.19
3.74 ± 0.69
4.90 ± 0.45
4.18 ± 0.75
13.47 ± 2.00
9.29 ± 1.34
17.62 ± 3.55
16.58 ± 1.84
8.47 ± 1.85
35.19 ± 39.56
67.92 ± 58.92
23.10 ± 1.96
20.25 ± 6.46
10.19 ± 1.01
10.35 ± 1.91
11.66 ± 2.17
9.28 ± 1.42
Inactive
24.46
8.66
21.31
5.33
2.90
14.86
37.97
68.13
22.82
17.02
33.35
13.57
28.32
18.99
4.31
3g
3h
3i
142.00±4.88
333.83±27.75
95.39±26.62
229.23±10.10
309.79±42.30
239.03±36.89
469.59±64.78
160.84±60.87
151.66±39.14
221.11±10.39
142.85±44.93
79.64±25.91
68.44±4.33
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
Inactive
-
102.55 ± 8.77
44.11 ± 0.49
24.85 ± 1.17
Inactive
1.57
3.44
8.90
-
51.43 ± 0.55
13.53 ± 0.42
Inactive
2.95
16.34
-
3.26
6.18
141.19 ± 3.99
9.65 ± 1.57
29.88 ± 1.33
36.29 ± 2.32
49.17 ± 2.69
Inactive
0.56
7.09
1.69
1.36
1.72
4.10
110.92 ± 3.20
14.00 ± 1.37
28.68 ± 0.30
51.33 ± 1.223
50.26 ± 7.50
Inactive
0.72
4.89
1.76
0.96
1.68
4.79
3.93
6.72
3t
50.48±8.87
4.88
3u
3v
3w
49.50±6.45
4.25
84.35±13.59
127.00±5.45
9.09
-

8.62±8.36
-
0.79 ± 0.08
-
10.91
-
0.34 ± 0.01
-
25.23
-
0.53 ± 0.09
54.17 ± 1.67
16.26
-
0.01 ± 0.00
0.05 ± 0.01
862
-
Doxorubicin
Etoposide
Table 4. In vitro cytotoxic activities of carbocycle-fused quinoline analogs 5a-5y against four
primary cancer cell lines.
HepG2
A549
HuCCA-1
MOLT-3
Comp.
IC50 (µM)
104.56±19.48
91.56±12.95
88.04±7.83
87.56±6.51
90.58±12.45
inactive
IC50 (µM)
147.27±9.71
133.83±18.10
115.45±8.59
87.29±10.03
127.50±2.26
inactive
IC50 (µM)
157.95±2.71
140.97±15.46
125.53±4.45
114.52±5.00
118.92±11.46
inactive
IC50 (µM)
63.75±7.21
45.84±6.04
49.76±5.33
45.09±3.27
38.94±5.65
105.50±14.12
42.75±8.57
inactive
5a
5b
5c
5d
5e
5f
5g
inactive
Inactive
inactive
5h
62.27±8.79
90.73±8.96
107.25±17.93
106.49±9.34
91.36±3.15
124.14±14.62
90.61±1.61
94.68±1.60
121.95±19.43
90.76±3.05
73.69±3.56
72.27±0.88
inactive
inactive
inactive
5i
137.29±5.40
178.49±5.37
181.68±3.79
160.85±10.11
166.00±7.35
154.70±16.96
139.46±15.66
inactive
156.96±1.77
180.85±4.15
183.43±6.80
145.69±7.93
178.29±0.43
156.95±4.63
145.23±8.73
inactive
53.81±0.20
57.19±0.22
56.78±0.22
39.68±0.14
50.47±0.18
54.37±0.19
28.87±0.10
46.63±0.17
27.64±0.10
30.42±0.09
20.71±0.06
67.92±0.26
25.57±0.08
32.55±0.11
28.00±0.09
54.83±0.21
inactive
5j
5k
5l
5m
5n
5o
5p
5q
122.21±6.49
inactive
143.56±6.85
inactive
5r
5s
70.79±9.27
inactive
70.42±3.48
inactive
5t
5u
77.37±5.83
81.08±3.80
77.54±1.18
122.00±2.78
87.97±9.04
50.21±1.78
0.50±0.05
111.78±17.45
115.46±15.33
116.32±4.88
inactive
113.75±12.61
119.79±5.32
121.65±4.88
inactive
5v
5w
5x
5y
inactive
inactive
Etoposide
Doxorubicin
0.34±0.00
-
0.05±0.00
0.34±0.01
0.83±0.05
0.01±0.00
The results in Table 4 displayed cytotoxic activities of carbocycle-fused quinoline analogs.
Compound 5h showed highest cytotoxicity against HepG2 cell line which was slightly less cytotoxic

than the etoposide standard. It is important to note that compound 5h selectively inhibited only
HepG2 cell line while it was inactive towards A549, HuCCA-1, and MOLT-3 cancer cell lines.
Compound 5s was found to have good cytotoxicity against all four cell lines. However, the general
potency of carbocycle-fused quinoline was less than those of neocryptolepine analogs. In addition,
the selectivity index of compounds 5h and 5s were also evaluated against human normal HEK-293
cell line. The results showed that compounds 5h and 5s demonstrated lower selectivities than
doxorubicin standard while the cytotoxicity of compounds 5h and 5s are much lower than
doxorubicin as shown in Table 5. Therefore, these results provided a room for improvement of their
cytotoxicity against cancer cell lines while maintaining or lowering toxicity against normal cell line
in the future investigation.
Table 5. Cytotoxicities and selectivity indices of carbocycle-fused quinoline analogs 5h and 5s.
HEK-293 (µM)
IC50 (µM)
HuCCA-1 (µM)
A549 (µM)
IC50 (µM)
HepG2 (µM)
IC50 (µM)
MOLT-3 (µM)
Comp.
IC50 (µM)
SI
SI
SI
IC50 (µM)
SI
5h
5s
135.39±28.41
359.70±49.21
8.62±8.36
inactive
-
inactive
-
62.27±8.79
72.27±0.88
0.53 ± 0.09
inactive
-
70.42±3.48
0.79 ± 0.08
5.11
10.91
70.79±9.27
0.34 ± 0.01
5.08
25.23
4.98
20.71±0.06
17.39
862
16.26 0.01 ± 0.00
Doxorubicin
2.2.2.
In vitro antiplasmodial assays
In this assay, twenty-two neocryptolepine analogs (3a-3w) were evaluated for their
antiplasmodial activity using two standard laboratory P. falciparum isolates, chloroquine-sensitive
(CQ-S) clone 3D7 and chloroquine-resistant (CQ-R) clone K1. In these studies, chloroquine (CQ)
and artesunate (ARS) were employed as positive controls for the assays. In addition, the selectivity
index was also determined from the IC50 values of pure compounds towards normal HEK-293 cell
line. However, the results showed that only compounds 3e, 3h, 3o, and 3p gave positive results in
both strains as shown in Table 6. In a recent study, we found that an isocryptolepine analogue could
disrupt several biological processes of P. falciparum 3D7, including ribosomal, proteasomal, and
metabolism pathways.40 Therefore, we suspected that neocryptolepine analogues could also inhibit
P.falciparum in a similar manner.
Table 6. In vitro antiplasmodial activities of neocryptolepine analogs
HEK-293
IC50 (nM)
3D7
K1
Comp.
IC50 (nM)
SI
IC50 (nM)
SI
3e
3h
3o
13037.6±20427
333831.7±27749
151661.1±39142
1479.1 ± 596.4
2028.6 ± 252.1
1225.7 ± 11.9
8.81
1326.3 ± 24.9
1575.1± 9.1
9.83
164.6
123.7
211.9
145.6
1041.7 ± 157.2

3p
CQ
221109.6±10386
1233.1 ± 176.5
19.1 ± 1.5
179.3
1361.3 ± 6.4
82.2 ± 8.9
2.8 ± 0.1
162.4
-
-
-
-
-
-
ARS
4.6 ± 0.1
3. Conclusion
In summary, we investigated new neocryptolepine (3a-3w) and cyclopentyl-fused quinoline
(5a-5y) analogs for their anticancer and antiplasmodial activities. Through these studies, we found
that compound 3h exhibited good anticancer activities in four primary cancer cell lines; HepG2,
HuCCA-1, MOLT-3, and A549 and also showed higher selectivity in HepG2 and HuCCA-1 cell
line than doxorubicin while its selectivity in A549 cell line was comparable to this standard and was
lower than the standard MOLT-3 cancer cell line. Moreover, the results also showed that
neocryptplepine derivatives had higher potency than carbocycle-fused quinoline analogs. In case of
antiplasmodial activities, compound 3h also showed the best activity among these analogs against
K1 strain with good selectivity while compound 3p showed good activity and selectivity against
3D7 strain. Based on these results and on our previous results,34 isocryptolepine analogs had superior
activities in inhibiting cancer cell lines and plasmodium activities than both neocryptolepines and
carbocycle-fused quinolines and warrant further investigation.
Acknowledgments
This research work was supported by Chulabhorn Research Institute, Mahidol University,
and the Center of Excellence on Environmental Health and Toxicology, Science & Technology
Postgraduate Education and Research Development Office (PERDO), Ministry of Education
Education, and Thailand Research Fund through the Royal Golden Jubilee (RGJ) Ph.D. Program
(Grant number PHD/0181/2559) and Thailand Research Fund (RSA6080085).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http:// to be inserted.
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Graphical abstract
Me
N
N
N
R
R
R2
R1
R1
 25 examples of carbocycle-fused quinoline analogs
 anticancer activities
 23 examples of neocryptolepine analogs
 anticancer activities
 antiplasmodial activities
Me
N
N
N
OBn
OMe
5s
3h
Me
high anticancer activity
high antiplasmodial activity
high selectivity
high anticancer activity
Me

Highlights
 Twenty-three neocryptolepine and twenty-five carbocycle-fused quinoline analogues were
conveniently and rapidly synthesized.
 The synthetic procedures allowed for structure-activity relationship studies of these
compounds against cancer cell lines and P. falciparum strains.
 Some of these analogues were identified to possess high activities against four primary
cancer cell lines and two P. falciparum strains.
 These novel analogues also showed high selectivities when tested in normal human kidney
cell.
 These compounds demonstrated potential for future development as medicinal agents against
cancer and malaria.