Synthesis and evaluation of the tetracyclic ring-system of isocryptolepine and regioiso-mers for antimalarial, antiproliferative and antimicrobial activities

Article abstract of DOI:10.3390/molecules26113268

A series of novel quinoline-based tetracyclic ring-systems were synthesized and evaluated in vitro for their antiplasmodial, antiproliferative and antimicrobial activities. The novel hydroiodide salts 10 and 21 showed the most promising antiplasmodial inhibition, with compound 10 displaying higher selectivity than the employed standards. The antiproliferative assay revealed novel pyridophenanthridine 4b to be significantly more active against human prostate cancer (IC₅₀ = 24 nM) than Puromycin (IC₅₀ = 270 nM) and Doxorubicin (IC₅₀ = 830 nM), which are used for clinical treatment. Pyridocarbazoles 9 was also moderately effective against all the employed cancer cell lines and moreover showed excellent biofilm inhibition (9a: MBIC = 100 μM; 9b: MBIC = 100 μM).

Full text of DOI:10.3390/molecules26113268

molecules
Article
Synthesis and Evaluation of the Tetracyclic Ring-System of
Isocryptolepine and Regioisomers for Antimalarial,
Antiproliferative and Antimicrobial Activities
Katja S. Håheim 1, Emil Lindbäck 1, Kah Ni Tan 2 , Marte Albrigtsen 3, Ida T. Urdal Helgeland 1,
Clémence Lauga 1, Théodora Matringe 1 , Emily K. Kennedy 2, Jeanette H. Andersen 3 , Vicky M. Avery 2,4,5
and Magne O. Sydnes 1,
*
1
2
3
Department of Chemistry, Bioscience and Environmental Engineering, University of Stavanger,
NO-4036 Stavanger, Norway; katja.s.haheim@uis.no (K.S.H.); emil.lindback@uis.no (E.L.);
ida.t.helgeland@uis.no (I.T.U.H.); clemence.lauga08@gmail.com (C.L.); theodora.matringe@gmail.com (T.M.)
Discovery Biology, Griffith Institute for Drug Discovery, Griffith University, Don Young Road,
Nathan, QLD 4111, Australia; kahni.tan@griffith.edu.au (K.N.T.); emily.kennedy@griffith.edu.au (E.K.K.);
v.avery@griffith.edu.au (V.M.A.)
The Norwegian College of Fishery Science, Marbio, UiT—The Arctic University of Norway, Breivika,
NO-9037 Tromsø, Norway; marte.albrigtsen@uit.no (M.A.); jeanette.h.andersen@uit.no (J.H.A.)
CRC for Cancer Therapeutics, Griffith University, Nathan, QLD 4111, Australia
School of Environment & Science, Nathan, Griffith University, Nathan, QLD 4111, Australia
Correspondence: magne.o.sydnes@uis.no
4
5
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
*
Citation: Håheim, K.S.; Lindbäck, E.;
Tan, K.N.; Albrigtsen, M.; Urdal
Helgeland, I.T.; Lauga, C.; Matringe,
T.; Kennedy, E.K.; Andersen, J.H.;
Avery, V.M.; et al. Synthesis and
Evaluation of the Tetracyclic
Abstract: A series of novel quinoline-based tetracyclic ring-systems were synthesized and evaluated
in vitro for their antiplasmodial, antiproliferative and antimicrobial activities. The novel hydroiodide
salts 10 and 21 showed the most promising antiplasmodial inhibition, with compound 10 displaying
higher selectivity than the employed standards. The antiproliferative assay revealed novel pyri-
dophenanthridine 4b to be significantly more active against human prostate cancer (IC50 = 24 nM)
than Puromycin (IC50 = 270 nM) and Doxorubicin (IC50 = 830 nM), which are used for clinical
Ring-System of Isocryptolepine and
Regioiso-Mers for Antimalarial,
Antiproliferative and Antimicrobial
Activities. Molecules 2021, 26, 3268.
https://doi.org/10.3390/
treatment. Pyridocarbazoles
9 was also moderately effective against all the employed cancer cell
lines and moreover showed excellent biofilm inhibition (9a: MBIC = 100 µM; 9b: MBIC = 100 µM).
molecules26113268
Keywords: indoloquinoline; antiplasmodial activity; antiproliferative activity; antimicrobial activity;
biofilm inhibition
Academic Editor: Valeria
Patricia Sülsen
Received: 28 April 2021
Accepted: 26 May 2021
Published: 30 May 2021
1. Introduction
Malaria and cancer are two major health issues affecting millions of lives annually.
Malaria is a parasitic blood disease caused by protozoans of the Plasmodium genus. Al-
though five Plasmodium strains are known to infect humans, namely P. falciparum, P. vivax,
P. ovale, P. knowlesi and P. malariae, infections by P. falciparum are responsible for the ma-
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
jority of malaria-related deaths [
the number of malaria cases to be 229 million in 2019, claiming approximately 409,000
lives [ ], despite considerable global efforts to combat the disease. A major obstacle in
the battle against malaria has been the rapid appearance and spread of resistant strains
across endemic areas [ ]. An excess of 90% of all malaria incidents occur in sub-Saharan
Africa [ ], a region sorely dependent on the availability of affordable treatments. Originally,
1,2]. The World Health Organization (WHO) estimated
1
3
Copyright:
© 2021 by the authors.
1
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
malaria-endemic regions were primarily limited to the immediate areas surrounding the
tropics. The increasing surface air temperatures as a consequence of global warming is
predicted to change this, leaving also temperate climates susceptible to the disease, and
with it, a larger part of the human population [
4]. Following the widespread appearance
of chloroquine (CQ)-resistant strains of P. falciparum, artemisinin-based therapies have
been the gold standard of malaria treatment [
5]. However, in 2008, the first reports of
Molecules 2021, 26, 3268. https://doi.org/10.3390/molecules26113268
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3268
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artemisinin-based resistance were observed in Cambodia [
6
] and ten years later, over
30 independent cases had been documented throughout southeast Asia [7]. Therefore, the
development of novel and affordable treatments remains of paramount importance.
Contrary to malaria, which is an infectious disease, cancer is a noncommunicable
disease, ranking as the second leading cause of death globally, responsible for approx-
imately 1 in 6 deaths. Estimates from WHO put the number of cancer cases in 2018 at
18.1 million, accompanied by 9.6 million fatalities [8]. The five most diagnosed cancers
are lung, breast, colorectal, prostate and stomach. A variety of anti-cancer therapies are
currently available, however, those treated suffer from the unwanted side effect of being
highly immunosuppressed. Patients suffering from a compromised immune system follow-
ing cancer treatments are therefore more likely to contract nosocomial infections [9], such
as infection caused by drug-resistant Staphylococcus aureus, increasing the overall burden of
nosocomial infectious diseases [10]. This is further complicated by the increased likelihood
of formations of multidrug-resistant biofilms, which are notoriously hard to treat [11
Bacterial infections are also known to be a cause of cancer on their own, and according to
the WHO, roughly 13% of all cancers globally occur as a result of chronic infections [ ].
Additionally, research in recent years has started to uncover a direct link between the
formation of microbial biofilms in the body and the growth of certain cancers [13 15]. The
,12].
8
–
availability of anticancer drugs with the dual capability of inhibiting biofilm growth is
severely limited, making the development of such drugs greatly needed.
Natural products have proven to be an invaluable source of lead compounds for
medicinal research in the past and present due to their wide array of structural
diversity [16–18]. As of 2020, roughly 40% of all Food and Drugs Administration (FDA)
approved drugs have natural origins [19], further demonstrating the importance of nat-
ural products in drug discovery. Accordingly, discovery and characterization of natural
products and their semi-synthetic derivatives remain pivotal in the search for novel drug
candidates [20]. The quinoline core represents a versatile structural motif, possessing
applications in the fields of material science, the dye industry and moreover constitute an
important building block in the design of pharmaceutical compounds [21]. In particular,
due to the presence of the quinoline skeleton in numerous natural products displaying a
vast array of biological activities, quinoline-based natural products and their derivatives
are attractive medicinal targets [22–25].
Almost exclusively found in the West African climbing shrub Cryptolepis sanguino-
lenta [26,27], the indoloquinoline natural products cryptolepine (1), neocryptolepine (2),
and isocryptolepine (3a) (Figure 1) represent a unique class of bioactive compounds. These
alkaloids are characterized by a fused quinoline and indole moiety [28] and long before the
constituents of C. sanguinolenta were identified, the extracts were used in herbal remedies
to treat malarial fevers among other ailments [29]. The major bioactive component of the
shrub was eventually determined to be cryptolepine (
the most attention in the literature of the three regioisomeric indoloquinolines
3a. A host of biological properties have been observed in cryptolepine ( ) assays, such as
antiplasmodial, antimalarial [30 35], anti-inflammatory [33], antifungal [36 38], antimi-
crobial [39 42], antiproliferative [43 46] and antiviral [40]. The linearly arranged planar
structure of cryptolepine ( ) is believed to be related to its high level of undesired cytotoxic-
ity, resulting in its ability to non-specifically intercalate into DNA, inhibiting topoisomerase
II [44 47 49]. Neocryptolepine ( ) and isocryptolepine (3a) have also been demonstrated
to possess similar biological profiles, although inferior to cryptolepine ( ) [28 50]. Despite
the lower potency, both neocryptolepine ( ) and isocryptolepine (3a) were revealed to
be significantly less cytotoxic than cryptolepine ( ), allowing for the possibility of their
derivatives to be developed into new lead compounds [49,51].
1), which has subsequently received
1, 2, and
1
–
–
–
–
1
,
–
2
1
,
2
1

Molecules 2021, 26, 3268
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Figure 1. Structures of the major bioactive compounds isolated from Cryptolepis Sanguinolenta;
cryptolepine (1), neocryptolepine (2) and isocryptolepine (3a).
The biological activities for the core structures of C. sanguinolenta have been extensively
studied while their regioisomers have been largely undescribed. In particular, the novel
pyridophenanthridine scaffold 4a (Figure 2) unveiled in our previous study [52], represents
an interesting target for biological evaluation. The pyridophenanthridine skeleton may be
regarded as a regioisomer to the pyridoacridines (the core structure of which is illustrated
in compound
for exhibiting potent antiproliferative qualities [53
all naturally occurring pyridoacridines have been shown to act as DNA intercalating agents,
resulting in cytotoxic effects in cultured tumor cells [54 55 58]. They also possess the ability
to inhibit topoisomerase II [53 58] and further contain biological profiles such as antibacte-
rial, antifungal, antiviral, antiparasitic and insecticidal [53 56 57 59 61]. Consequently, it
5
in Figure 2), a well-studied class of marine alkaloids most notably known
–
57]. Similarly, to cryptolepine ( ), nearly
1
,
,
,
,
, , –
is postulated that the pyridoacridines and their synthetic derivatives are pivotal for the
future generation of medicinal compounds [58].
Figure 2. Structures of pyridophenanthridines 4a and 4aa along with pyridoacridine 5.
Recently, we described the preparation of several isocryptolepine regioisomers and
certain chemoisomers [52]. In this paper, we present modifications to our previous synthetic
strategies which allowed for the realization of novel tetracyclic ring-systems (compounds
4b
,
8
, and
9
) along with the N-alkylation of several compounds to furnish new analogues
3c 10, and 21). Moreover, the newly synthesized compounds, along with
(compounds 3b
,
,
our existing library of natural products and analogues, were evaluated for their in vitro
antiplasmodial activity against Plasmodium falciparum 3D7 parasites; cytotoxicity against
normal mammalian cell line (HEK293), and three cancer cell lines (HCT116, MDA-MB-231
and PC-3). The compounds were also evaluated as antimicrobial agents against common
pathogenic bacteria as well as their ability to inhibit biofilm growth.

Molecules 2021, 26, 3268
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2. Results and Discussion
2.1. Chemistry
We recently reported a concise synthesis of isocryptolepine (3a) and some regioisomers
in which the two key synthetic steps were a Suzuki-Miyaura cross-coupling reaction
followed by a palladium-catalyzed intramolecular cyclization [52,62]. The most unexpected
result of our previous endeavor was the formation of a pyridophenanthridine scaffold 4a,
when biaryl 7a was treated with palladium under our intramolecular cyclization conditions
(Path A, Scheme 1). Shortly after our report, Kumar and co-workers reported the formation
of compound 4a by a palladium-catalyzed arylation technique utilizing diaryliodinium
salts [63]. To the best of our knowledge, these two preparations of pyridophenanthridine
4a remain the only descriptions in the literature. However, Beauchard and coworkers
describe the accidental synthesis of the functionalized pyridophenanthridine 4aa in 2006
(Figure 2) [64]. This was the result of attempting to synthesize isocryptolepine analogues
by a microwave-induced thermal decomposition of a benzotriazole-coupled quinoline.
Scheme 1. Synthesis of pyridophenanthridines
(i) coupling partner
8
and pyridocarbazoles
9
from a common starting material. Conditions:
◦
6
, Cs2CO3, Pd(PPh3)4 (5 mol%), DME/H2O, 80 C; Path A: (ii) PdCl2 (dppf) (20 mol%), IMes (5 mol%),
◦
◦
H2O2 (35 wt%, 29 mol%), AcOH, 118 C, MW; (iii) CH3I, CH3CN, reflux; Path B: (iv) 1) HCl (37%), NaNO2 (0.4 M), 0 C,
◦
◦
1.5 h; 2) NaN3/NaOAc, 0 C, 1 h; (v) 1,2-dichlorobenzene, 180 C, 3 h; (vi) CH3I, CH3CN, reflux.
Intrigued by these results, we decided to investigate further and wondered if the
regioselectivity would be the same utilizing a different synthetic strategy. Drawing inspira-
tion from Timári et al. [65] in the synthesis of isocryptolepine and further expanded on by
Hostyn et al. [66] for the synthesis of isoneocryptolepine, a Suzuki-Miyaura cross-coupling
reaction and nitrene insertion approach was undertaken. Standard azidation of biaryl
7a via installation of a diazonium salt yielded the aryl azide 7I, which upon thermal de-
composition in refluxing 1,2-dichlorobenzene interestingly gave pyridocarbazole 9a as the
only product without any traces of its regioisomer 4a (Scheme 1, Path B). Thereby, it was
concluded that 4a and 9a can be achieved from a common starting material by following
reaction pathway A and B, respectively, in Scheme 1. A fluoro-substituted analogue of
compound 9a, namely 9b, was further possible to construct starting from boronic acid 6b
.
To conclude the synthetic pathways, compounds and 9a were finally regioselectively
4
N-methylated using excess iodomethane in refluxing acetonitrile [49] to realize tetracycles
8 and 10.
In Timári et al.’s original synthesis of isocryptolepine (3a) by means of a thermally
induced nitrene insertion, only one regioisomeric product was observed, namely isocryp-
tolepine precursor 14 [65]. Following the same conditions in our laboratories, the approach
primarily resulted in the construction of indoloquinoline 14 but its regioisomer 13 was
also formed in minor quantities (Scheme 2). Applying the nitrene insertion approach to

Molecules 2021, 26, 3268
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biaryls 15
,
17, and 19, we were able to significantly improve the yields of tetracycles 18
and 20 compared to our previous endeavors (Scheme 3, previous yields in brackets) [52].
Following a literature procedure, neocryptolepine (2) was obtained in good yield starting
from its precursor 13 (Scheme 4) [67].
Modification of our previously reported conditions for the N-methylation of tetracycle
14 to furnish isocryptolepine (3a) [62], allowed the formation of two novel isocryptolepine
analogues 3b and 3c, albeit in lower yields than the parent alkaloid (Scheme 4). Of the re-
maining tetracycles, namely compounds 16, 18, and 20, only compound 16 was successfully
N-methylated using the same conditions as reported in our previous work [62]. Efforts to
explain the failure of tetracycles 18 and 20 to undergo N-alkylation at the most reactive
ring-nitrogen, presumably the quinoline moiety, is currently under way in our laboratories.
Scheme 2. Synthesis of 6H-indolo[2,3-b] quinoline (13) and 11H-indolo[3,2-c] quinoline (14) via a Suzuki-Miyaura cross-
coupling and thermally induced nitrene insertion approach. Conditions: (i) boronic acid 6a, K2CO3, PdCl2 (dppf)
(5 mol%), EtOH/H2O (5:1), 60 ◦C [52
(iii) 1,2-dichlorobenzene, 180 C, 3 h.
,
62]; (ii) 1) HCl (37%), NaNO2 (0.4 M), 0 ◦C, 1.5 h; 2) NaN3/NaOAc, 0 ◦C, 1 h;
◦
Scheme 3. Synthesis of 11H-pyrido[3,2-a] carbazole (16), 11H-pyrido[2,3-c] carbazole (18) and 7H-
pyrido[3,2-c] carbazole (20) using a diazotization-azidation-nitrene insertion approach. In brackets:
◦
yields from our previous endeavors [52]. Conditions: (i) HCl (37%), NaNO2 (0.4 M), 0 C, 1.5 h;
◦
(ii) 1,2-dichlorobenzene, 180 C, 3 h. For the synthesis of compounds 15
,
17, and 19, refer to our
previous work [52].

Molecules 2021, 26, 3268
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Scheme 4. Regioselective N-alkylations to synthesize neocryptolepine (2), isocryptolepine (3a), N-
alkyl isocryptolepine derivatives (3b and 3c) and 4-methyl-11H-pyrido[3,2-a]carbazolium iodide (21).
Conditions: (i) CH3CN, THF, reflux, 24 h [67]; (ii) a: CH3I, PhMe, reflux, 3 h [62]; b: CH3CH2I, PhMe,
reflux, 3 h; c: CH2=CHCH2Br, PhMe, reflux, 22 h; (iii) CH3I, CH3CN, reflux, 20 h.
2.2. Antiplasmodial Assay
The prepared natural products and their derivatives were evaluated for their in vitro
antiplasmodial activities against the Plasmodium falciparum 3D7 strain. The compounds
were further tested for their in vitro cytotoxicity against HEK293 cells (human embryonic
kidney cells) for the determination of their selectivity indices. Furthermore, to serve
as positive controls for our analyses, chloroquine (CQ), dihydroartemisinin (DHA) and
puromycin were employed. Results from these studies are summarized in Table 1.
The tested compounds were found to possess diverse activities against the Pf3D7
cell line. Albeit being well documented to have antiplasmodial activity in the literature,
the parent alkaloid neocryptolepine (2) has thus far not been evaluated for in vitro an-
timalarial activity against Pf3D7 (IC50 = 7249 nM), showing a lower potency compared
to isocryptolepine (3a) (IC50 = 1211 nM). Out of the two novel isocryptolepine deriva-
tives, allyl variant 3c showed a marginal improvement compared to the natural product
(IC50 = 1198 nM), while ethyl variant 3b showed a lower activity (IC50 = 1318 nM). Both
derivatives were revealed to be notably more cytotoxic than the parent alkaloid 3a.
The neocryptolepine precursor 13 was revealed to display no antiplasmodial inhibi-
tion, which is in accordance with a previous study conducted by Jonckers et al., where
they highlighted the importance of the N-5 methyl group for activity in certain halogen-
substituted indolo[3,2-b]quinolines [68]. The regioisomer 15 was also shown to be inactive
against Pf3D7. Contrary to this, the isocryptolepine precursor 14 displayed more potent
antimalarial activities (IC50 = 977 nM) compared to the parent alkaloid 3a. For the isocryp-
tolepine precursor 14, it has been shown through previous work that by introduction of
certain basic side chains at C-9, the in vitro antimalarial activity against the K-1 strain of
P. falciparum was dramatically increased compared to isocryptolepine (3a). The authors

Molecules 2021, 26, 3268
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argued that these observations could be attributed to the basic properties allowing the
compound to experience a lower degree of hydrophobicity [69], a quality also observed for
CQ [28].
Table 1. In vitro antiplasmodial activities of tetracyclic ring-systems
2–3, 4, 8–10, 13–14, 16, 18, 20, and 21 against the 3D7
P. falciparium strain, cytotxicity against HEK293 cells and selectivity indices.
Entry
Compound
3D7 IC50 (nM)
Cytotoxicity IC50 (nM)
SI a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Neocryptolepine (2)
Isocryptolepine (3a)
7249 ± 6
1211 ± 84
1318 ± 5
1198 ± 32
548 ± 3
866 ± 2
1698 ± 5
1546 ± 27
6825 ± 61
NT b
>20,000
2074 ± 70
3078 ± 49
3152 ± 40
2834 ± 92
3657 ± 2
7410 ± 207
5057 ± 45
>80,000
NT b
2.8
1.7
2.3
2.6
5.2
4.2
4.4
3.3
11.7
-
3b
3c
4a
4b
8a
8b
9a
9b
10
13
14
16
18
20
21
128 ± 2
NA c
213.9
-
18.9
-
NA c
NA c
977 ± 11
NA c
18460 ± 183
NA c
NA c
NA c
-
2414 ± 42
380 ± 0.5
24 ± 1
NA c
16.6
105.4
165
74
NA c
Chloroquine
DHA
Puromycin
>4000
1 ± 0.07
93 ± 2
NA c
3 ± 3
0.03
Data are presented as the mean
±
standard deviation from two separate experiments. IC50 values were calculated using non-linear dose-
response curves in GraphPad Prism. SI = selectivity index = cytotoxicity in HEK293/activity in 3D7; b NT = not tested; c NA = not active.
a
Pyridophenanthridines
4 (a: IC50 = 548 nM; b: IC50 = 866 nM) outperformed both
neocryptolepine ( ) and isocryptolepine (3a) in terms of activity and selectivity; however, it
2
displayed an unfavorable increase in cytotoxicity. Keeping in mind the effects observed by
Jonckers et al. [68] for the functionalization of the isocryptolepine precursor 14, addition of
appropriate substituents to pyridophenanthridine 4a could potentially result in increased
antiplasmodial activity. Evidently, the presence of the methoxy substituent in compound
4b negatively impacted both the antiplasmodial activity and cytotoxicity compared to
the naked pyridophenanthridine 4a. Interestingly, the addition of an N-methyl group to
pyridophenanthridines 4 to furnish compounds 8 (a: IC50 = 1698 nM; b: IC50 = 1546 nM)
negatively impacts the antiplasmodial activity. For the indoloquinoline natural products,
the N-methyl group is considered an instrumental aspect for their parasitic inhibition [28],
this is evidently not the case for the pyridophenanthridines, possibly suggesting the
presence of a novel mode of action against the parasitic life cycle. As this represents the
first case in the literature of the antiplasmodial evaluation of a pyridophenanthridine, other
functionalizations of the core scaffold should nonetheless be further researched.
The two most prominent results of our studies were the novel hydroiodide salts 10
(IC50 = 128 nM) and 21 (IC50 = 380 nM), where the latter showed improved selectivity
compared to the standards. Their precursors 9a and 15 showed little to no activity, high-
lighting the importance of the N-methyl functionality. These results are possibly aided
by the fact that the salt structure likely promotes increased solubility in aqueous media,
further increasing the biological availability of the compounds, a fact which should be
carefully considered when exploring new lead compounds.
2.3. Antiproliferative Assay
All prepared samples were evaluated in vitro against a panel of three cancer cell lines,
including HCT116 (human colon cancer), MDA-MB-231 (human breast adenocarcinoma)

Molecules 2021, 26, 3268
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and PC-3 (human prostate cancer) using a resazurin assay. Puromycin and Doxorubicin
were employed as positive controls for the obtained IC50 results, which are summarized in
Table 2.
Table 2. In vitro antiproliferative activities and cytotoxicities of tetracyclic ring-systems
against three cancer cell lines.
2–3, 4, 8–10, 13–14, 16, 18, 20 and 21
Entry
Compound
HCT116 IC50 (nM)
MDA-MB-231 IC50 (nM)
PC-3 IC50 (nM)
13
14
15
16
1
2
3
4
5
6
Neocryptolepine (2)
Isocryptolepine (3a)
6218 ± 90
667 ± 45
742 ± 11
1243 ± 80
721 ± 27
166 ± 16 a
444 ± 52
871 ± 172
20,015 ± 1665
NT c
10,435 ± 375
695 ± 130
998 ± 300
3064 ± 467
594 ± 140
1002 ± 297
360 ± 51
814 ± 162
21,540 ± 2480
NT c
27% at 80 µM
1821 ± 7
2440 ± 94
1296 ± 51
1630 ± 173
24 ± 3 b
3b
3c
4a
4b
8a
8b
9a
9b
2571 ± 114
4539 ± 361
17,790 ± 1640
NT c
7
8
9
10
11
12
17
18
19
10
13
14
16
18
20
21
38% at 40 µM
24% at 40 µM
36% at 40 µM
NA d
NA d
NA d
36% at 80 µM e
80% at 80 µM
NT c
30% at 80 µM f
NA d
3573 ± 309
82% at 80 µM
NT c
NT c
17,030 g
NA d
16,415 ± 2305
47% at 40 µM
NA d
NA d
Puromycin
Doxorubicin
85
150
300
590
270
830
Data are presented as the mean
±
sem (standard error of the mean) from two separate experiments. IC50 values were calculated using
a
non-linear dose-response curves in GraphPad Prism. 89% metabolic activity at 40 nM; b 45% metabolic activity at 40 nM; c NT = not
tested; d NA = not active; e 55% metabolic activity at 40 µM; f 50% metabolic activity at 40 µM; g 12% metabolic activity at 40 µM.
Both parent alkaloids neocryptolepine (
against the HCT116 cell line ( : 6218 nM; 3a: 667 nM) (Table 2). It is evident that isocryp-
tolepine (3a) had an overall better performance against the tested cancer cell lines than
neocryptolepine ( ). The same was observed for the isocryptolepine derivatives 3b and
2) and isocryptolepine (3a) performed best
2
2
3c; however, the potency was less than for the parent isocryptolepine (3a). Derivatives
3b and 3c were revealed to become less potent with increasing alkyl chain length for the
human colon cancer (3b: IC50 = 742 nM; 3c: 1243 nM) and human breast adenocarcinoma
(
3b: IC50 = 998 nM; 3c: 3064 nM) cell lines. Interestingly, for the human prostate cancer cell
line, a different trend was observed (3b: IC50 = 2440 nM; 3c: 1296 nM). The N-allyl group
outperformed both the methyl and ethyl groups in terms of activity, suggesting that the
alkene functionality is somehow important to the mechanism of cell growth inhibition. It
is believed that the indoloquinolines inhibit cell growth by direct interactions with DNA,
although the exact mechanism(s) remain uncertain [28,44,47,50,70].
Several of the tested compounds were found to display no activity against the panel
of cancer cell lines, including novel compounds 10 and 21. Another compound which was
observed to be inactive was neocryptolepine precursor 13, being inactive against all three
cell lines. The isocryptolepine precursor 14 showed poor activity against all cancer cell
lines and further highlights the necessity of the N-methyl group for cell growth inhibition.
The importance of incorporating an N-methyl is further demonstrated in compounds
4a and its corresponding N-methylated product 8a, showing an increase in activity against
the HCT116 and MDA-MB-231 cell lines, favoring the inclusion of an N-methyl group. In
the PC-3 cell lines, the pyridophenanthridines
4
showed a decrease in activity with the
addition of an N-methyl substituent to give the corresponding compound
8. However, the
assay revealed the methoxy pyridophenanthridine 4b to contain potent anticarcinogenic
properties (IC50 = 24 nM) against the PC-3 cell line. Compound 4b showed a 10-fold and
35-fold increase in activity compared to the positive controls Puromycin (IC50 = 270 nM)

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and Doxorubicin (IC50 = 830 nM), respectively. The positioning of the methoxy substituent
at C-6 of the pyridophenanthridine scaffold appears to be key to the observed increase in
activity, as the naked pyridophenanthridine 4a showed only modest activity against the
PC-3 cell line (IC50 = 1630 nM). A previous study by Lu and coworkers demonstrated the
potential of the strategic installation of appropriate ring-substituents to obtain increased
antiproliferative activity in various indolo[3,2-b]quinolines [71]. Similar to the observations
made in this work, Lu et al. noted the potency of C-9 ester substituted indoloquinolines
in their screening of several cancer cell lines [71], despite the parent neocryptolepine (2)
displaying only minor inhibition of cell growth. The N-methylated pyridophenanthridine
8a evaluated in this work was further shown to be more potent against the MDA-MB-
231 (IC50 = 360 nM) cell line than Doxorubicin (IC50 = 590 nM). Being novel compounds,
the mode of action of the pyridophenanthridines against proliferative cancer is naturally
unknown. Thus, proceeding studies have the potential to unveil a new mode of action.
The discovery of new modes of action is regarded as highly important in the field of drug
discovery [72], further illustrating the potential for the novel pyridophenanthridine scaffold
as a lead for subsequent development into a new anticancer therapy.
2.4. Antimicrobial and Biofilm Iinhibition Assay
The prepared samples were tested for in vitro antimicrobial activity against E. faecalis,
E. coli, P. aeruginosa, S. aureus, Streptococcus agalactiae and S. epidermis using gentamycin
as a reference compound. The compounds were tested at 100, 75, 50, 25, 12.5, 10, 6.3,
3.1 and 1.6 µM and the obtained minimal inhibitory concentrations (MIC) and minimal
bacterial inhibition concentrations (MBIC) can be seen in Table 3. Several of the screened
compounds contained no antibacterial properties against the tested panel of bacteria,
including tetracycles 8a, 10, 13-14, 16, 18, and 20-21, while compounds 3b, 3c, and 21 were
not tested.
Table 3. In vitro antimicrobial activities of tetracyclic ring-systems 2–3, 4, and 8–9 against five bacterial cell lines and
inhibition of biofilm formation.
Tested Strain
MIC (µM)
2
3a
4a a
4b a
8a a
9a
9b
Gentamycin
NA b
NA b
NA b
NA b
100
NA b
50
NA b
NA b
NA b
NA b
100
NA b
NA b
NA b
NA b
NA b
E. faecalis (ATCC 29122)
E. coli (ATCC 259233)
P. aeruginosa (ATCC 27853)
S. aureus (ATCC 25923)
100
100
100
NA b
NA b
100
75
8
NA b
NA b
75
0.13
0.25
0.06
4
NA b
100
NA b
NA b
75
NA b
NA b
Streptococcus agalactiae (ATCC 12386)
100
MBIC (µM)
NA b
NA b
NA b
NA b
S. epidermis (ATCC 35984)
100
100
100
NT c
a
Also tested at concentrations of 100, 75, 50, 25, 12.5, 10, 6.3, 3.1 and 1.6 µM. Compounds 8b, 10, 13–14, 16, 18, and 20 showed no activity
against any of the tested strains; b NA = not active; c NT = not tested. Compounds 3b, 3c, and 21 were not tested.
Neocryptolepine (
(MIC = 100 M), while its precursor 13 was inactive against all bacterial strains. It has been
shown previously that neocryptolepine ( ) only possesses bacteriostatic properties against
Gram-positive bacteria and displays no activity whatsoever against Gram-negative bacte-
ria [40 72 74], which fits well with our observations. With the exception of P. aeruginosa,
2) showed only modest activity against Streptococcus agalactiae
µ
2
,
–
isocryptolepine (3a) contained modest activity against all the tested strains and excellent
inhibition of biofilm growth.
The novel pyridophenanthridines 4a and 8a were both effective against the Gram-
positive bacteria E. faecalis (4a: MIC = 100
µM; 8a: MIC = 75 µM) and S. aureus (4a:
MIC = 100 M; 8a: MIC = 75 M) but were inactive against the rest. These results are
µ
µ
comparable to previous observations for the indolo[2,3-b]quinolines (i.e., neocryptolepines),
showing that the presence of an N-methyl substituent is essential for antimicrobial inhi-

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bition [73]. Methoxy substituted pyridophenanthridine 4b was proven to be the most
successful in the evaluated series, being moderately effective against E. coli (MIC = 50
µM)
and S. aureus (MIC = 75 M). Interestingly, addition of the N-methyl functionality to
µ
produce pyridophenanthridine 8b, resulted in a complete loss of activity. Representing
unknown scaffolds, the mode of action of the pyridophenanthridines are naturally not
known; however, these data indicate that the methoxy substituted 4b and 8b could differ
from their non-functionalized counterparts 4a and 8a.
Novel pyridocarbazoles
MBIC = 100 M; 9b: MIC = 100
cus agalactiae (MBIC = 100 M). The incorporation of a fluorine into a molecule is usually
associated with a significant increase in biological activity [75], which is not the case for
compound , having the non-fluorinated 9a performing better overall. In general, pyrido-
9
showed excellent biofilm formation inhibition (9a
:
µ
µM) and variant 9a was also active against Streptococ-
µ
9
carbazoles have been primarily studied for their antiproliferative qualities in the past, with
natural products such as the ellipticines containing potent anticancer properties [76]. The
ellipticines are currently employed clinically as antiproliferative agents, though little is
known about the inherent antimicrobial potential of such motifs. Although the antimicro-
bial activities observed for compound 9 were not particularly significant, this structural
motif should be explored in greater detail in future research to uncover its full potential as
a dual antimicrobial and antiproliferative agent.
3. Materials and Methods
3.1. Chemistry
3.1.1. General
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AscendTM
1
400 series (Billerica, MA, USA), operating at 400.13 MHz for H, 376.49 MHz for 19F
and 100.61 MHz for 13C, respectively. Chemical shifts (
δ) are expressed in ppm relative
to residual chloroform-d (1H, 7.26 ppm; 13C, 77.16 ppm), DMSO-d6 (1H, 2.50 ppm; 13C,
39.52 ppm), methanol-d4 (1H, 3.31 ppm; 13C, 49.00 ppm), acetone-d6 (1H, 2.09 ppm; 13C,
30.60 ppm), dichloromethane-d2 (1H, 5.32 ppm; 13C, 53.84 ppm) or
α
,α
,α
-trifluorotoluene
(19F,
−
62.61 ppm) [77] as an external reference. The assignment of signals in various NMR
spectra were often assisted by conducting correlation spectroscopy (COSY), heteronuclear
single-quantum correlation spectroscopy (HSQC), heteronuclear multiple bond correlation
spectroscopy (HMBC) and nuclear Overhauser effect spectroscopy (NOESY).
Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm
silica gel F254 coated aluminum sheets using UV light as a visualizing agent. Silica gel 60
(particle size 40–63 µm) was used for flash chromatography.
In addition to TLC, low resolution mass spectrometry (LRMS) was routinely used
to monitor and identify the various components of reaction mixtures. The LRMS spectra
were obtained using an Advion expressions CMS mass spectrometer operating at 3.5 kV in
electrospray ionization (ESI) mode.
Infrared spectroscopy (IR) was performed on a Agilent Technologies Cary 360 FTIR
spectrophotometer (Santa Clara, CA, USA). Solids were dissolved in CHCl3 or CH2Cl2 and
adsorbed on a NaCl plate, or by placing the sample directly onto the crystal of an attenuated
total reflectance (ATR) module. Melting points were measured using a Stuart Scientific
SMP3 melting point apparatus and are uncorrected. High resolution mass spectrometry
(HRMS) were conducted externally at the University of Bergen (UiB) or the University
of Tromsø, using ESI mode. The microwave-assisted experiments were performed in a
CEM Focused MicrowaveTM Synthesis System (Charlotte, NC, USA), model type Discover,
◦
operating at 0–300 W, a pressure of 0–290 psi, at a temperature of 118 C, using reactor vial
volumes of either 10 or 35 mL. Commercially available chemicals were used as delivered
from the supplier unless otherwise noted.
Detailed experimental procedures and full characterizations for compounds 3a
, 4, 5b,
7, 15, 17, and 19 are available through our previous works [52,62].

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3.1.2. 4-Fluoro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (6b)
To a mixture of 2-bromo-4-fluoroaniline (1000.0 mg, 5.26 mmol), anhydrous Et3N
(2.93 m, 21.04 mmol), PdCl2(PPh3)2 (369.2 mg, 0.53 mmol, 10 mol%) in 20 mL anhydrous
dioxane, was added 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.30 mL, 15.79 mmol) drop-
wise. The resulting mixture was refluxed for 22 h and then allowed to cool to rt before
being quenched by addition of suitable amounts of sat. aq. NH4Cl. The crude was sub-
sequently extracted using CH2Cl2 (3
×
20 mL) and the combined organic phases were
20 mL), dried (MgSO4), filtered and concen-
washed with water (1 20 mL), brine (1
×
×
trated in vacuo. The concentrate was then evaporated onto celite and purification by silica
gel column chromatography (pet. ether/EtOAc, 9:1 v/v) and concentration of the relevant
fractions [Rf = 0.33 (pet. ether/EtOAc, 9:1 v/v)] gave the target compound 6b as a red
solid (975.7 mg, 78%), mp 49–50 ◦C (lit. [78] 50–52 ◦C); IR (ATR):
ν
3481, 3388, 2978,
max
2931, 1621, 1431, 1137, 854 cm−1; 1H NMR (400 MHz, CDCl3):
1H), 6.92 (ddd, J = 8.6 Hz, 8.3 Hz, 3.1 Hz, 1H), 6.53 (dd, J = 8.8 Hz, 4.3 Hz, 1H), 4.55 (bs,
2H), 1.34 (s, 12H); 13C NMR (100 MHz, CDCl3):
155.3 (d, JCF = 235.0 Hz), 149.9, 121.6 (d,
δ 7.28 (dd, J = 9.1 Hz, 3.1 Hz,
δ
J
CF = 20.3 Hz), 119.8 (d, JCF = 23.0 Hz), 116.1 (d, JCF = 6.9 Hz), 83.9, 25.0 (one carbon was
obscured or overlapping); 19F NMR (376 MHz, CDCl3): δ −129.0. The spectroscopic data
are in accordance with previously reported data [78].
3.1.3. 4-Fluoro-2-(quinolin-5-yl)aniline (7c)
To a solution of 5-bromoquinoline (5a) (512.3 mg, 2.46 mmol) in 25 mL
1,2-dimethoxyethane (DME) under an argon atmosphere was added 4-fluoro-2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (6b) (875.7 mg, 3.69 mmol), an aqueous solution
of Cs2CO3 (2805.3 mg, 8.61 mmol in 5 mL H2O) and Pd(PPh3)4 (142.1 mg, 0.12 mmol).
The resulting mixture was stirred at 80 ◦C for 17 h before being allowed to cool to rt. The
volatiles were then removed under reduced pressure and the concentrate was evaporated
onto celite. Purification by silica gel column chromatography (pet. ether/EtOAc, 1:1 v/v)
and concentration of the relevant fractions [Rf = 0.10 (pet. ether/EtOAc, 1:1 v/v)] gave the
target compound 7c as an orange solid (419.5 mg, 72%), mp 196–197 ◦C; IR (ATR):
ν
max
3041, 2921, 2852, 1635, 1490, 1192, 900, 792 cm−1; 1H NMR (400 MHz, CD2Cl2):
δ 8.90 (dd,
J = 4.1 Hz, 1.7 Hz, 1H), 8.14–8.12 (m, 1H), 7.96 (ddd, J = 8.5 Hz, 1.6 Hz, 0.8 Hz, 1H), 7.79 (dd,
J = 8.5 Hz, 7.1 Hz, 1H), 7.51 (dd, J = 7.0 Hz, 1.1 Hz, 1H), 7.36 (dd, J = 8.5 Hz, 4.2 Hz, 1H),
7.00 (td, J = 8.6 Hz, 3.0 Hz, 1H), 6.88 (dd, J = 9.0 Hz, 3.0 Hz, 1H), 6.79 (dd, J = 8.8 Hz, 4.8 Hz,
1H), 3.42 (bs, 2H) (Figure S3.1); 13C NMR (100 MHz, CD2Cl2):
δ 156.4 (d, JCF = 235.8 Hz),
151.0, 149.1, 141.4, 136.8, 134.5, 130.1, 129.6, 128.3, 127.0, 125.7 (d, JCF = 7.2 Hz), 121.8, 117.7
(d, JCF = 22.1 Hz), 116.6 (d, JCF = 8.0 Hz), 115.9 (d, JCF = 22.1 Hz) (Figure S3.2); 19F NMR
(376 MHz, CD2Cl2):
δ
-128.0 (Figure S3.3); HRMS (ESI): calcd. for C15H11FN2 [M + H+]
239.0979, found 239.0988.
3.1.4. Intramolecular Cyclization to Form Tetracycles 9, 13, 14, 18 and 20
General Procedures
Method 1—palladium-catalyzed intramolecular C-H activation/C-N bond formation:
The appropriate biaryl (1 equiv.) in a suitable amount of glacial acetic acid was added to a
premixed solution of PdCl2(dppf) (10 mol%), 1,3-bis(2,4,6-trimethylphenyl)imidazolium
(IMes) (5 mol%), H2O2 (35 wt%, 29 mol%) and a suitable amount of glacial acetic acid.
The reaction mixture was then placed in a sealed reactor tube and immersed into the
cavity of a microwave oven and heated at 118 ◦C until completion as indicated by TLC
analysis. The reaction mixture was then transferred to a round-bottom flask with the aid
of EtOAc/CHCl3 and the volatiles were removed under reduced pressure. The reaction
mixture was finally evaporated onto celite and purified by column chromatography with
the eluents as indicated in order to give the target compounds.
Method 2—diazotization-azidation-nitrene insertion: The appropriate biaryl (1 equiv.)
was dissolved in a suitable amount of aq. HCl (37%) and the mixture was cooled to
◦
0 C using an ice bath. Next, to ice-cooled aq. NaNO2 (0.4 M) was added the solution

Molecules 2021, 26, 3268
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◦
dropwise and the resulting mixture was stirred at 0 C for 1.5 h. An ice-cooled aq. solution
of NaN3/NaOAc (2.1 equiv./14 equiv. in an appropriate amount of H2O) was added
◦
dropwise and the mixture stirred for 1 h while keeping the temperature at 0 C. The
reaction mixture was quenched by addition of appropriate amounts of sat. aq. K2CO3
and subsequently extracted with CH2Cl2 (3
×
20 mL). The combined organic phases were
washed with water (1 20 mL), brine (1 20 mL), dried (MgSO4), filtered and concentrated
×
×
in vacuo. The obtained residue was dissolved in a suitable amount of 1,2-dichlorobenzene
◦
and flushed with argon. The resulting mixture was stirred at 180 C for 3 h under an argon
atmosphere before being cooled to rt. The solvent was removed under reduced pressure
and the concentrate was evaporated onto celite and purified by column chromatography
using the eluents as indicated in order to give the target compounds.
7H-Pyrido[2,3-c]carbazole (9a)
Method 2: Following the general procedure, the title compound was prepared from
2-(quinolin-5-yl)aniline (7a) (100.0 mg, 0.45 mmol), HCl (37%, 3 mL), NaNO2 (82.9 mg,
1.20 mmol in 3 mL H2O), NaN3 (61.4 mg, 0.94 mmol) and NaOAc (516.8 mg, 6.30 mmol
in 5 mL H2O). After formation of the azide was confirmed by IR, the cyclization was
carried out using 3 mL of 1,2-dichlorobenzene. The crude was purified by silica gel
column chromatography (CH2Cl2/EtOAc, 95:5
fractions [Rf = 0.22 (CH2Cl2/EtOAc, 95:5 v/v)] gave the target compound 9a as a light
brown solid (78.8 mg, 80%), mp 204–205 ◦C; IR (ATR):
3045, 2919, 2842, 1523, 1274,
→
9:1 v/v) and concentration of the relevant
ν
max
956, 804, 728 cm−1; 1H NMR (400 MHz, DMSO-d6):
δ 11.92 (bs, 1H), 9.17 (dd, J = 8.4 Hz,
1.4 Hz, 1H), 8.84 (dd, J = 4.1 Hz, 1.4 Hz, 1H), 8.60 (d, J = 8.0 Hz, 1H), 8.03–7.98 (m, 2H),
7.69–7.65 (m, 2H), 7.47–7.44 (m, 1H), 7.34.7.31 (m, 1H) (Figure S6.1, S6.3, and S6.4); 13C
NMR (100 MHz, DMSO-d6):
δ 146.4, 144.3, 139.0, 136.9, 130.8, 127.7, 124.5, 124.3, 122.9,
121.6, 121.5, 119.8, 116.8, 113.6, 111.9 (Figure S6.2, S6.5, and S6.6); HRMS (ESI): calcd. for
C15H10N2 [M + H+] 219.0917, found 219.0927.
10-Fluoro-7H-pyrido[2,3-c]carbazole (9b)
Method 2: Following the general procedure, the title compound was prepared from
4-fluoro-2-(quinolin-5-yl)aniline (7c) (419.5 mg, 1.76 mmol), HCl (37%, 8 mL), NaNO2
(137.9 mg, 2.00 mmol), NaN3 (240.5 mg, 3.70 mmol) and NaOAc (2021.2 mg, 24.64 mmol
in 15 mL H2O). The crude was essentially pure by 1H NMR and 100.0 mg of the azide
was dissolved in 2 mL 1,2-dichlorobenzene and reacted without any further purification.
The cyclization yielded a reaction crude which was also pure by NMR and the target
◦
compound 9b was obtained as a dark green solid (87.3 mg, 97%), mp 256–257 C; IR (ATR):
1
ν
3137, 2974, 2746, 1460, 1149, 789 cm−1; H NMR (400 MHz, CD3OD):
δ
9.07 (ddd,
max
J = 8.4 Hz, 1.6 Hz, 0.8 Hz, 1H), 8.74 (dd, J = 4.4 Hz, 1.6 Hz, 1H), 8.18–8.15 (m, 1H), 7.98
(dd, J = 9.1 Hz, 0.7 Hz, 1H), 7.91 (d, J = 9.1 Hz, 1H), 7.67 (dd, J = 8.4 HZ, 4.4 HZ, 1H), 7.58
(ddd, J = 8.8 Hz, 4.5 Hz, 0.5 Hz, 1H), 7.25-7.20 (m, 1H) (Figure S7.1, S7.4, and S7.5); 13
C
NMR (100 MHz, CD3OD):
δ 159.2 (d, JCF = 234.5 Hz), 146.9, 145.1, 139.8, 137.3, 132.9, 127.9,
126.4, 124.7 (d, JCF = 9.5 Hz), 122.7, 118.3, 115.1 (d, JCF = 5.3 Hz), 113.7 (d, JCF = 24.0 Hz),
113.5 (d, JCF = 7.2 Hz), 107.6 (d, JCF = 24.8 Hz) (Figure S7.2, S7.6, and S7.7); 19F NMR (376
MHz, CD3OD):
found 237.0830.
δ
-123.6 (Figure S7.3); HRMS (ESI): calcd. for C15H9FN2 [M + H+] 237.0823,
6H-Indolo[2,3-b]quinoline (13) and 11H-indolo[3,2-c]quinoline (14)
Method 2: Following the general procedure, the title compounds were prepared
starting from 2-(quinolin-3-yl)aniline (12) (100.0 mg, 0.45 mmol), HCl (37%, 3 mL), NaNO2
(82.8 mg, 1.20 mmol in 3 mL H2O), NaN3 (61.4 mg, 0.94 mmol) and NaOAc (516.8 mg,
6.30 mmol in 5 mL H2O). After formation of the azide was confirmed by IR, the cyclization
was carried out using 3 mL 1,2-dichlorobenzene. The crude was purified by silica gel
column chromatography (CH2Cl2/EtOAc, 8:2
→
0:1 v/v) and concentration of the relevant

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fractions [Rf = 0.56 (CH2Cl2/EtOAc, 2:8 v/v)] gave compound 13 as off-white crystals
(4.2 mg, 4%) along with compound 14 as an off-white solid (86.4 mg, 88%).
Characterization of Compound 13
mp 341–342 ◦C (lit. [79] 342–346 ◦C); IR (ATR):
ν
3139, 2923, 2849, 1402, 725 cm−1
;
max
1H NMR (400 MHz, DMSO-d6):
8.11 (dd, J = 8.1 Hz, 1.3 Hz, 1H), 7.99–7.97 (m, 1H), 7.75–7.70 (m, 1H), 7.55–7.46 (m, 3H),
7.29–7.25 (m, 1H); 13C NMR (100 MHz, DMSO-d6):
152.7, 146.1, 141.4, 128.7, 128.6, 128.2,
δ 11.72 (bs, 1H), 9.06 (s, 1H), 8.26 (d, J = 7.7 Hz, 1H),
δ
127.7, 126.8, 123.6, 122.8, 121.8, 120.3, 119.7, 118.0, 110.9. The spectroscopic data are in
accordance with previously reported data [79].
Characterization of Compound 14
◦
◦
mp 333-335 C (lit. [62] 340–341 C); IR (NaCl):
1493 cm−1; 1H NMR (400 MHz, DMSO-d6):
12.74 (bs, 1H), 9.60 (s, 1H), 8.52 (dd, J = 7.8 Hz,
1.1 Hz, 1H), 8.33–8.31 (m, 1H), 8.14 (dd, J = 8.4 Hz, 1.1 Hz, 1H), 7.77–7.68 (m, 3H), 7.52–7.48
(m, 1H), 7.37–7.33 (m, 1H); 13C NMR (100 MHz, DMSO-d6):
145.3, 144.7, 139.8, 138.8,
νmax 3060, 2958, 2854, 1682, 1582, 1515,
δ
δ
129.4, 128.1, 125.7, 125.6, 122.1, 121.9, 120.6, 120.1, 117.1, 114.3, 111.9. The spectroscopic
data are in accordance with previously reported data [62].
11H-Pyrido[2,3-a]carbazole (18)
Method 2: Following the general procedure, the title compound was prepared from
2-(quinolin-7-yl)aniline (17) (100.0 mg, 0.45 mmol), HCl (37%, 3 mL), NaNO2 (82.8 mg,
1.20 mmol in 3 mL H2O), NaN3 (61.4 mg, 0.94 mmol) and NaOAc (516.8 mg, 6.30 mmol
in 3 mL H2O). After formation of the azide was confirmed by IR, the cyclization was
carried out using 3 mL 1,2-dichlorobenzene. The crude was purified by silica gel column
chromatography (CH2Cl2/EtOAc, 9:1 v/v) and concentration of the relevant fractions
[Rf = 0.36 (CH2Cl2/EtOAc, 9:1 v/v)] gave◦the target compound 18 as off-white crystals
◦
(40.0 mg, 41%), mp 164 C (lit. [52] 165–167 C); IR (ATR):
ν
max 3263, 3043, 2923, 2854, 1523,
1
1369, 820, 734 cm−1; H NMR (400 MHz, CDCl3):
δ 10.20 (bs, 1H), 8.92 (dd, J = 4.4 Hz,
1.5 Hz, 1H), 8.35 (dd, J = 8.3 Hz, 1.5 Hz, 1H), 8.24 (d, J = 8.5 Hz, 1H), 8.19–8.17 (m, 1H),
7.62–7.60 (m, 2H), 7.51–7.47 (m, 2H), 7.35–7.31 (m, 1H); 13C NMR (100 MHz, CDCl3):
δ
147.8, 139.2, 137.4, 136.8, 134.9, 127.3, 125.9, 123.8, 121.7, 120.8, 120.5, 120.4, 120.2, 118.8,
111.8. The spectroscopic data are in accordance with previously reported data [52].
7H-Pyrido[3,2-c]carbazole (20)
Method 2: Following the general procedure, the title compound was prepared from
2-(quinolin-8-yl)aniline (19) (450.0 mg, 2.04 mmol), HCl (37%, 10 mL), NaNO2 (137.9 mg,
2.00 mmol in 5 mL H2O), NaN3 (278.5 mg, 4.28 mmol) and NaOAc (2342.8 mg, 28.56 mmol
in 10 mL H2O). After formation of the azide was confirmed by IR, the cyclization was
carried out using 5 mL 1,2-dichlorobenzene. The crude was purified by silica gel column
chromatography (pet. ether/EtOAc, 1:1 v/v) and concentration of the relevant fractions
[Rf = 0.85 (pet. ether/EtOAc, 1:1 v/v)] gave the target compound 20 as a dark red oil
(195.9 mg, 44%). IR (ATR):
ν
3207, 2976, 2919, 2850, 2740, 2605, 2499 cm−1; 1H NMR
max
(400 MHz, DMSO-d6): δ 11.92 (bs, 1H), 9.02 (dd, J = 4.4 Hz, 1.8 Hz, 1H), 8.90–8.88 (m, 1H),
8.46 (dd, J = 8.1 Hz, 1.4 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.66–7.64
(m, 1H), 7.49 (dd, J = 8.0 Hz, 4.3 Hz, 1H), 7.46–7.42 (m, 1H), 7.33-7.29 (m, 1H); 13C NMR
(100 MHz, DMSO-d6):
δ 149.8, 145.3, 139.6, 138.5, 136.5, 126.0, 124.5, 123.1, 122.9, 122.8,
119.7, 118.3, 115.3, 114.2, 111.4. The spectroscopic data are in accordance with previously
reported data [52].
3.1.5. Neocryptolepine (2)
To a solution of 6H-indolo[2,3-b]quinoline (13) (23.0 mg, 0.10 mmol) in 2 mL THF,
iodomethane (0.66 mL, 10.0 mmol) was added and the resulting mixture refluxed for
24 h. The volatiles were then removed under reduced pressure and the concentrate was

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evaporated onto celite. Purification by silica gel column chromatography (CH2Cl2/MeOH,
95:5 v/v) and concentration of the relevant fractions [Rf = 0.18 (CH2Cl2/MeOH, 95:5 v/v)]
gave the hydroiodide salt of neocryptolepine. To obtain the free base, the hydroiodide
salt was dissolved in a 20 mL 1:1 mixture of CH2Cl2 and NH3(aq) (20%) and stirred at rt
for 30 min. The organic layer was then separated and the aqueous layers were extracted
with CH2Cl2 (3
(1 10 mL), brine (1
neocryptolepine ( ) as a dark yellow solid (19.5 mg, 84%), mp 85–86 C (lit. [67] 104–105 C);
IR (ATR):
max 3051, 2961, 2923, 2852, 1494, 1012, 741 cm−1; 1H NMR (400 MHz, CD3OD):
8.67 (s, 1H), 8.05-8.02 (m, 2H), 7.90 (d, J = 8.6 Hz, 1H), 7.83–7.78 (m, 1H), 7.59–7.57 (m,
1H), 7.50–7.45 (m, 2H), 7.19 (td, J = 7.5 Hz, 1.0 Hz, 1H), 4.23 (s, 3H); 13C NMR (100 MHz,
CD3OD): 156.9, 155.4, 138.1, 132.0, 131.2, 130.4, 130.1, 128.4, 124.9, 123.6, 122.4, 122.2,
×
10 mL) and the combined organic layers were washed with water
×
×
10 mL), dried (MgSO4), filtered and concentrated in vacuo to give
◦
◦
2
ν
δ
δ
121.2, 117.7, 115.7, 33.7. The spectroscopic data are in accordance with previously reported
data [67].
3.1.6. 5-Ethyl-5H-indolo[3,2-c]quinoline (3b)
To a solution of 11H-indolo[3,2-c]quinoline (14) (15.0 mg, 0.068 mmol) in 3 mL toluene,
ethyl iodide (1.1 mL, 13.68 mmol) was added and the resulting mixture was refluxed for
3 h. The volatiles were then removed under reduced pressure and the concentrate was
evaporated onto celite. Purification by silica gel column chromatography (CHCl3/MeOH,
9:1 v/v) and concentration of the relevant fractions [Rf = 0.21 (CHCl3/MeOH, 9:1 v/v)]
gave the hydroiodide salt of compound 3b. To obtain the free base, the hydroiodide salt
was dissolved in a 40 mL 1:1 mixture of CH2Cl2 and NH3 (aq) (20%) and stirred at rt for
5 min. The organic layer was then separated and the aqueous layers were extracted with
CH2Cl2 (2
dried (MgSO4), filtered and concentrated in vacuo to give the target compound 3b as a
yellow solid (11.0 mg, 64%), mp 198 ◦C. IR (NaCl):
3371, 3049, 2960, 2927, 2856, 1731,
1640, 1598, 1455, 1392, 1353 cm−1; 1H NMR (400 MHz, DMSO-d6):
×
10 mL) and the combined organic layers were washed with brine (1
×
10 mL),
ν
max
δ
9.59 (s, 1H), 8.84 (dd,
J = 8.1 Hz, 1.3 Hz, 1H), 8.23 (d, J = 8.7 Hz, 1H), 8.20–8.18 (m, 1H), 7.93–7.89 (m, 1H), 7.81 (d,
J = 8.1 Hz, 1H), 7.79–7.75 (m, 1H), 7.51–7.47 (m, 1H), 7.34–7.30 (m, 1H), 4.81 (q, J = 7.1 Hz,
2H), 1.56 (t, J = 7.1 Hz, 3H) (Figure S1.1, S1.3, and S1.4); 13C NMR (100 MHz, DMSO-d6):
δ
151.2, 150.5, 138.6, 134.3, 130.1, 126.1, 125.8, 124.7, 124.4, 120.5, 120.2, 119.9, 117.8, 117.2,
115.9, 49.7, 15.0 (Figure S1.2, S1.5, and S1.6); HRMS (ESI): calcd. for C17H14N2 [M + H+]
247.1235, found 247.1238.
3.1.7. 5-Allyl-5H-indolo[3,2-c]quinoline (3c)
To a solution of 11H-indolo[3,2-c]quinoline (14) (30.0 mg, 0.14 mmol) in 5 mL toluene,
allyl bromide (1.14 mL, 13.76 mmol) was added and the resulting mixture was refluxed for
22 h. The volatiles were then removed under reduced pressure and the concentrate was
evaporated onto celite. Purification by silica gel column chromatography (CHCl3/MeOH,
95:5
→
9:1 v/v) and concentration of the relevant fractions [Rf = 0.18 (CHCl3/MeOH, 9:1
v/v)] gave the hydroiodide salt of compound 3c. To obtain the free base, the hydroiodide
salt was dissolved in a 20 mL 1:1 mixture of CH2Cl2 and NH3 (aq) (20%) and stirred at rt for
45 min. The organic layer was then separated and the aqueous layers were extracted with
CH2Cl2 (3
brine (1
compound 3c as a yellow viscous oil (15.5 mg, 43%). IR (ATR):
×
10 mL) and the combined organic layers were washed with water (1
10 mL), dried (MgSO4), filtered and concentrated in vacuo to give the target
2924, 2723, 1596, 1349,
×
10 mL),
×
ν
max
1204, 927, 743 cm−1; 1H NMR (400 MHz, DMSO-d6):
δ 9.67 (s, 1H), 8.84 (dd, J = 8.0 Hz,
1.3 Hz, 1H), 8.22–8.20 (m, 1H), 8.15 (d, J = 8.7 Hz, 1H), 7.92–7.88 (m, 1H), 7.84–7.77 (m,
2H), 7.55–7.51 (m, 1H), 7.37–7.34 (m, 1H), 6.26–6.17 (m, 1H), 5.46–5.44 (m, 2H), 5.31–5.28
(m, 1H), 5.15–5.11 (m, 1H) (Figure S2.1, S2.3, and S2.4); 13C NMR (100 MHz, DMSO-d6):
δ
150.0, 149.9, 139.6, 134.9, 132.8, 130.3, 126.5, 126.1, 124.4, 124.3, 121.0, 120.1, 119.7, 118.5,
118.2, 116.8, 115.7, 56.5 (Figure S2.2 and S2.5); HRMS (ESI): calcd. for C18H14N2 [M + H+]
259.1230, found 259.1232.

Molecules 2021, 26, 3268
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3.1.8. 4-Methyl-4H-pyrido[4,3,2-gh]phenanthridine (8a)
To a solution of 7H-pyrido[4,3,2-gh]phenanthridine (4a) (70.0 mg, 0.32 mmol) in
2 mL acetonitrile, iodomethane (2.0 mL, 32.0 mmol) was added and the resulting mixture
was refluxed for 2 h. The volatiles were then removed under reduced pressure and the
concentrate was evaporated onto celite. Purification by silica gel column chromatography
(CHCl3/MeOH, 95:5 + 0.3% NH3 (aq) v/v) and concentration of the relevant fractions [Rf
= 0.33 (CHCl3/MeOH, 95:5 + 0.3% NH3(aq) v/v)] gave the hydroiodide salt of compound
8a. To obtain the free base, the hydroiodide salt was dissolved in a 20 mL 1:1 mixture of
CH2Cl2 and NH3 (aq) (20%) and stirred at rt for 20 min. The organic layer was separated
and the aqueous layers were extracted with Et2O (2
layers were washed with water (1 10 mL), brine (1
and concentrated in vacuo to give the target compound 8a as dark yellow crystals (52.8 mg,
×
20 mL) and the combined organic
×
×
10 mL), dried (MgSO4), filtered
◦
−1
;
71%), mp 182–183 C; IR (ATR):
ν
max 3485, 3051, 2922, 2851, 2574, 1601, 1327, 820, 748 cm
1H NMR (400 MHz, DMSO-d6): δ 8.29 (dd, J = 8.1 Hz, 1.1 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H),
7.71 (t, J = 8.1 Hz, 1H), 7.56 (dd, J = 8.2 Hz, 1.2 Hz, 1H), 7.51–7.47 (m, 2H), 7.30–7.26 (m, 1H),
7.05 (d, J = 8.1 Hz, 1H), 6.18 (d, J = 7.6 Hz, 1H), 3.45 (s, 3H) (Figure S4.1, S4.3, and S4.4); 13
C
NMR (100 MHz, DMSO-d6): "δ" 152.9, 145.9, 141.0, 140.9, 133.8, 131.6, 129.2, 127.0, 123.2,
122.9, 121.3, 119.6, 112.2, 108.8, 106.2, 39.6 (Figure S4.2, S4.5, and S4.6); HRMS (ESI): calcd.
for C16H12N2 [M + H+] 233.1073, found 233.1073.
3.1.9. 6-Methoxy-4-methyl-4H-pyrido[4,3,2-gh]phenanthridine (8b)
To a solution of 6-methoxy-7H-pyrido[4,3,2-gh]phenanthridine (4b) (90.0 mg,
0.36 mmol) in 10 mL acetonitrile, iodomethane (2.25 mL, 36.3 mmol) was added and
the resulting mixture refluxed for 2 h. The volatiles were then removed under reduced
pressure and the concentrate was evaporated onto celite. Purification by silica gel column
chromatography (EtOH + 0.1
→
5% NH3 (aq) v/v) and concentration of the relevant frac-
tions [Rf = 0.23 (EtOH)] gave the hydroiodide salt of compound 8b. To obtain the free
base, the hydroiodide salt was dissolved in a 20 mL 1:1 mixture of CH2Cl2 and NH3 (aq)
(20%) and stirred at rt for 20 min. The organic layer was separated and the aqueous layers
were extracted with CH2Cl2 (4
×
20 mL) and the combined organic layers were washed
20 mL), dried (MgSO4), filtered and concentrated in
with water (1 20 mL), brine (1
×
×
vacuo to give the target compound 8b as a dark yellow gel (55.1 mg, 58%). IR (ATR):
1
ν
2918, 2850, 1600, 1255, 1059, 745 cm−1; H NMR (400 MHz, CD2Cl2):
δ
8.19 (dd,
max
J = 8.2 Hz, 1.3 Hz, 1H), 7.79 (dd, J = 8.2 Hz, 1.0 Hz, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.57–7.51
(m, 2H), 7.34–7.30 (m, 1H), 6.74–6.72 (m, 2H), 3.85 (s, 3H), 3.34 (s, 3H) (Figure S5.1, S5.3 and
S5.4); 13C NMR (100 MHz, CD2Cl2):
δ 148.1, 146.3, 140.8, 139.9, 134.8, 130.9, 129.4, 128.6,
124.1, 122.7, 122.5, 119.9, 110.6, 107.7, 57.1, 40.4 (one carbon was obscured or overlapping)
(Figure S5.2, S5.5 and S5.6); HRMS (ESI): calcd. for C17H14N2O 263.1179, found 263.1188.
3.1.10. 4-Methyl-7H-pyrido[2,3-c]carbazolium Iodide (10)
To a solution of 7H-pyrido[2,3-c]carbazole (9a) (40.7 mg, 0.19 mmol) in 5 mL ace-
tonitrile, iodomethane (1.20 mL, 19.6 mmol) was added and the resulting mixture re-
fluxed for 20 h. The volatiles were then removed under reduced pressure and the con-
centrate was evaporated onto celite. Purification by alumina gel column chromatogra-
phy (CHCl3/MeOH, 9:1 v/v + 1% NH3 (aq)) and concentration of the relevant fractions
[Rf = 0.12 (CHCl3/MeOH, 9:1 v/v + 2% NH3 (aq))] gave the target compound 10 as a
bright yellow solid (20.9 mg, 47%), mp 284–286; IR (ATR):
ν
max 3353, 3043, 3006, 2960, 2921,
2853, 1556, 1370, 1326, 741 cm−1; 1H NMR (400 MHz, DMSO-d6):
δ
12.84 (bs, 1H), 9.99 (d,
J = 8.4 Hz, 1H), 9.39 (d, J = 5.6 Hz, 1H), 8.76 (d, J = 8.1 Hz, 1H), 8.50 (d, J = 9.3 Hz, 1H),
8.43 (d, J = 9.4 Hz, 1H), 8.22 (dd, J = 8.5 Hz, 5.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.64-7.60
(m, 1H), 7.48–7.44 (m, 1H), 4.74 (s, 3H) (Figure S8.1, S8.3, and S8.4); 13C NMR (100 MHz,
DMSO-d6):
δ 145.0, 140.8, 139.8, 137.5, 134.5, 126.6, 125.8, 122.4, 122.1, 121.9, 121.6, 121.1,
116.0, 114.3, 112.8, 46.3 (Figure S8.2, S8.5 and S8.6); HRMS (ESI): calcd. for C16H13N2I [M –
I-] 233.1073, found 233.1073.

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3.1.11. 4-Methyl-11H-pyrido[3,2-a]carbazolium Iodide (21)
To a solution of 11H-pyrido[3,2-a]carbazole (15) (32.1 mg, 0.15 mmol) in 5 mL acetoni-
trile, iodomethane (0.92 mL, 14.72 mmol) was added and the resulting mixture stirred at
reflux for 20 h. The volatiles were then removed under reduced pressure and the obtained
yellow crystals were thoroughly washed with n-hexanes and dried in vacuo to give the
◦
target compound 21 as a dark orange crystalline solid (47.6 mg, quant.), mp 279–280 C; IR
(ATR):
CD3OD):
(m, 1H), 8.15–8.10 (m, 2H), 7.79–7.77 (m, 1H), 7.65–7.61 (m, 1H), 7.46–7.42 (m, 1H), 4.76 (s,
3H) (Figure S9.1, S9.3, and S9.4); 13C NMR (100 MHz, CD3OD):
148.2, 141.7, 141.6, 139.1,
ν
max 3416, 3165, 3077, 2997, 2905, 1599, 1452, 1371, 740 cm−1; 1H NMR (400 MHz,
δ
9.64 (d, J = 8.6 Hz, 1H), 9.28 (d, J = 5.7 Hz, 1H), 8.99 (d, J = 9.1 Hz, 1H), 8.35–8.33
δ
136.1, 131.1, 128.4, 123.6, 122.4, 122.3, 121.6, 121.1, 119.9, 113.1, 109.0, 46.9 (Figure S9.2, S9.5
and S9.6); HRMS (ESI): calcd. for C16H13N2I [M – I-] 233.1073, found 233.1075.
3.2. Biological Testing Assay
3.2.1. General
All compounds for antimicrobial testing were diluted to a final assay concentration
of 40 µL, 0.4% DMSO, and tested in full dose-response using three concentrations per log
dose (16 points with a concentration range of 0.33 nM–40 µM, for reference compounds:
21 points with a concentration of 0.01 nM–40 µM).
All compounds for antiplasmodial testing were diluted to a final assay concentration
L, 0.4% DMSO, and tested in full dose-response using three concentrations per log
dose (16 points with a concentration range of 0.4 nM–40 M, for reference compounds:
16 points with a concentration rage of 0.4 nM–40 M for chloroquine and puromycin:
0.001 nM–0.1 M for artemisinin). Compounds tested in the antiproliferative assays were
of 40
µ
µ
µ
µ
tested in 11 dilution points (0.02 µM–40 µM or 0.04 µM–80 µM).
Antiplasmodial Imaging Assay
Antiplasmodial activity was determined as previously described by Duffy and Av-
ery [80]. Briefly, compounds were incubated with 2% parasitemia in 0.3% hematocrit, in
an assay volume of 50 µ
L for 72 h at 37 ◦C and 5% CO2 in CellCarrier Ultra 384-well PDL-
imaging plates. After incubation, plates were stained with 4’,6-diamidino-2-phenylindole
(DAPI) in a permeabilization buffer for 5 h at rt in the dark. Plates were imaged on the
Opera confocal microplate image reader (PerkinElmer). Parasite inhibition was calculated
using the minimum (0.4% DMSO) and maximum (5
values determined using GraphPad Prism software.
µM puromycin) controls, and IC50
Cytotoxicity Assay
The cytotoxicity of compounds was determined using a resazurin-based viability assay
in HEK293 (ATCC®, CRL-1573), as described by Fletcher and Avery [81]. Compounds
were added to TC-treated 384-well plates (Greiner, Kremsmünster, AT) containing 2500
HEK293 cells per well, total assay volume of 50
CO2. After incubation, media was removed, replaced with 44
µ
L and incubated for 72 h at 37 ◦C, 5%
M resazurin and plates
µ
incubated 6 h under the same experimental conditions. Fluorescence was measured using
an EnSight plate reader (PerkinElmer, Waltham, MA, USA). Cell viability was calculated
using positive (45 µM puromycin) and negative (0.4% DMSO) controls, and the IC50 values
determined using GraphPad Prism software.
Antiproliferative Assay
Antiproliferative activity was assessed in HCT116 (ATCC® CCL-247; 1000 cells/well),
MDA-MB-231 (ATCC® HTB-26; 2000 cells/well) and PC-3 (ATCC® CRL-1435; 1000 cells/well)
cells. HCT116 cells were maintained in McCoy’s 5A media (Life Technologies, CA, USA),
MDA-MB-231 cells were cultured in DMEM media (Life Technologies) with 10 mM HEPES
(Life Technologies), whilst PC-3 cells were maintained in RPMI media (Life Technologies).
All media were supplemented with 10% heat-inactivated fetal bovine serum (Australian

Molecules 2021, 26, 3268
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◦
source; Corning, CA, USA) and all cells were incubated at 37 C in a humidified incubator
with 5% CO2.
Cells were seeded in 384-well plates (Greiner Bio-One, NC, USA) using the respective
complete media. After 24 h cell seeding, compounds were added and antiproliferative
activity was determined using the resazurin assay after 72 h compound incubation. Briefly,
◦
cells were incubated with 60
µ
M resazurin (Cayman, MI, USA) for 6 h at 37 C and
fluorescence signals were measured using a microplate reader (EnSight, PerkinElmer,
Waltham, MA, USA). Fluorescence signals were normalized to 0.4% DMSO and 50
µM
puromycin and IC50 values were calculated from non-linear dose-response curves using
GraphPad Prism 7 software (La Jolla, CA, USA).
3.2.2. Growth Inhibition Assay
To determine and quantify antimicrobial activity, a bacteria growth inhibition assay
in liquid media was executed. Compounds 2–3, 4, 8–10, 13–14, 16, 18, and 20 were tested
against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 259233), Enterococcus
faecialis (ATCC 29122), Pseudomonas aeruginosa (ATCC 27853) and Streptococcus agalactiae
(ATCC 12386); all strains from LGC Standards (Teddington, UK). S. aureus, E. coli, and
P. aeruginosa were grown in Muller Hinton broth (275730, Becton, Franklin Lakes, NJ, USA).
E. faecalis and S. agalactiae were cultured in brain hearth infusion broth (53286, Sigma, St
Louise, MO, USA). Fresh bacterial colonies were transferred in the respective medium
and incubated at 37 ◦C overnight. The bacterial cultures were diluted to a culture density
representing the log phase and µL/well were pipetted into a 96-well microtiter plate
(734–2097, NunclonTM, Thermo Scientific, Waltham, MA, USA). The final cell density was
1500–15.000 colony forming units/well. The compound was diluted in 2% (v/v) DMSO
in ddH2O, providing a final assay concentration of 50% of the prepared sample, since
50
µ
L of sample in DMSO/water were added to 50
µ
L bacterial culture. After adding
◦
the samples to the plates, they were incubated overnight at 37 C and the growth was
determined by measuring the optical density at
λ = 600 nm (OD600) with a 1420 Multilabel
Counter VICTOR3TM (Perkin Elmer, Waltham, MA, USA). A water sample was used as
a reference control, growth medium without bacteria was used as a negative control and
dilution series of gentamycin (A2712, Merck, Darmstadt, DE) from 32 to 0.01 µg/mL was
used as positive control and visually inspected for bacterial growth. The positive control
was used as a system suitability test and the results of the antimicrobial assay were only
considered valid when positive control was passed. The final concentration of DMSO in
the assays was
was processed using GraphPad Prism 8.
≤
2% (v/v) and was known to have no effect in the tested bacteria. The data
3.2.3. Biofilm Formation Inhibition Assay
For testing the inhibition of biofilm formation, the biofilm-producing Staphylococcus
epidermidis (ATCC 35984) was grown in Tryptic Soy Broth (TSB, 105459, Merck, Kenilworth,
NJ, USA) overnight at 37 ◦C. The overnight culture was diluted in fresh medium with 1%
glucose (D9434, Sigma) before being transferred to a 96-well microtiter plate; 50
µL/well
were incubated overnight with 50 L of the test compound dissolved in 2% (v/v) DMSO aq.
µ
added in duplicates. The bacterial culture was removed from the plate and the plate was
◦
washed with tap water. The biofilm was fixed at 65 C for 1 h before 70
µL 0.1% crystal violet
(115940, Millipore, Burlington, MA, USA) was added to the wells for 10 min of incubation
and 70 L of 70% ethanol was then added to each well and the plate incubated on a shaker
for 5–10 min. Biofilm formation inhibition was assessed by the presence of violet color
and measured at 60 nm absorbance using a 1420 Multilabel Counter VICTOR3TM; 50
of a non-biofilm forming Staphylococcus haemolyticus (clinical isolate 8-7A, University
Hospital of North Norway Tromsø, Norway) mixed in 50 L autoclaved Milli-Q water was
used as a control; 50 L S. epidermidis mixed in 50 L autoclaved Milli-Q water was used
L TSB with 50 L autoclaved Milli-Q water
µ
µL
µ
µ
µ
as the control for biofilm formation; and 50
was used as a medium blank control.
µ
µ

Molecules 2021, 26, 3268
18 of 22
4. Conclusions
In conclusion, a series of quinoline-based tetracyclic ring-systems were prepared and
evaluated for their in vitro antiplasmodial, antiproliferative and antimicrobial activities
against selected strains. Through these studies, it was determined that the ionic pyridocar-
bazoles 10 and 21 showed the best antiplasmodial activity against the Plasmodium falciparum
3D7 strains (10: IC50 = 128 nM; 21: IC50 = 380 nM) of the evaluated compounds. The
antiproliferative assay revealed that the novel pyridophenanthridine scaffold
4 was the
most active. In particular, compound 4b showed excellent potency against the PC-3 cell line
(IC50 = 24 nM), significantly outperforming Puromycin (IC50 = 270 nM) and Doxorubicin
(IC50 = 830 nM). The pyridophenanthridines
Gram-positive and Gram-negative bacteria, with compound 4b being moderately active
against E. coli (MIC = 50 M) and Streptococcus agalactiae (MIC = 75 M). The antimicro-
bial studies further demonstrated pyridocarbazoles to be highly potent against biofilm
growth (9a: MBIC = 100 M; 9b: MBIC = 100 M). Overall, this study has highlighted the
potential for the novel pyridophenanthridine motif and the studied pyridocarbazoles
4 were also active against certain strains of
µ
µ
9
µ
µ
4
9
to be developed into future drug candidates, with emphasis on the formulation of a dual
antimicrobial and antiproliferative agent.
Supplementary Materials: Figure S1.1: 1H NMR of 5-ethyl-5H-indolo[3,2-c]quinoline (3b),
Figure S1.2: 13C NMR of 5-ethyl-5H-indolo[3,2-c]quinoline (3b), Figure S1.3: COSY of 5-ethyl-5H-
indolo[3,2-c]quinoline (3b), Figure S1.4: NOESY of 5-ethyl-5H-indolo[3,2-c]quinoline (3b),
Figure S1.5: HSQC of 5-ethyl-5H-indolo[3,2-c]quinoline (3b), Figure S1.6: HMBC of 5-ethyl-5H-
indolo[3,2-c]quinoline (3b), Figure S2.1: 1H NMR of 5-allyl-5H-indolo[3,2-c]quinoline (3c),
Figure S2.2: 13C NMR of 5-allyl-5H-indolo[3,2-c]quinoline (3c), Figure S2.3: COSY of 5-allyl-5H-
indolo[3,2-c]quinoline (3c), Figure S2.4: NOESY of 5-allyl-5H-indolo[3,2-c]quinoline (3c),
Figure S2.5: HMBC of 5-allyl-5H-indolo[3,2-c]quinoline (3c), Figure S3.1: 1H NMR of 4-fluoro-2-
(quinolin-5-yl)aniline (7c), Figure S3.2: 13C NMR of 4-fluoro-2-(quinolin-5-yl)aniline (7c), Figure S3.3:
1
19F NMR of 4-fluoro-2-(quinolin-5-yl)aniline (7c), Figure S4.1: H NMR of 4-methyl-4H-pyrido[4,3,2-
gh]phenanthridine (8a), Figure S4.2: 13C NMR of 4-methyl-4H-pyrido[4,3,2-gh]phenanthridine (8a),
Figure S4.3: COSY of 4-methyl-4H-pyrido[4,3,2-gh]phenanthridine (8a), Figure S4.4: NOESY of
4-methyl-4H-pyrido[4,3,2-gh]phenanthridine (8a), Figure S4.5: HSQC of 4-methyl-4H-pyrido[4,3,2-
gh]phenanthridine (8a), Figure S4.6: HMBC of 4-methyl-4H-pyrido[4,3,2-gh]phenanthridine (8a),
1
Figure S5.1: H NMR of 6-methoxy-4-methyl-4H-pyrido[4,3,2-gh]phenanthridine (8b), Figure S5.2:
13C NMR of 6-methoxy-4-methyl-4H-pyrido[4,3,2-gh]phenanthridine (8b), Figure S5.3: COSY of
6-methoxy-4-methyl-4H-pyrido[4,3,2-gh]phenanthridine (8b), Figure S5.4: NOESY of 6-methoxy-
4-methyl-4H-pyrido[4,3,2-gh]phenanthridine (8b), Figure S5.5: HSQC of 6-methoxy-4-methyl-4H-
pyrido[4,3,2-gh]phenanthridine (8b), Figure S5.6: HMBC of 6-methoxy-4-methyl-4H-pyrido[4,3,2-
gh]phenanthridine (8b), Figure S6.1: 1H NMR of 7H-pyrido[2,3-c]carbazole (9a), Figure S6.2: 13C
NMR of 7H-pyrido[2,3-c]carbazole (9a), Figure S6.3: COSY of 7H-pyrido[2,3-c]carbazole (9a),
Figure S6.4: NOESY of 7H-pyrido[2,3-c]carbazole (9a), Figure S6.5: HSQC of 7H-pyrido[2,3-c]carbazole
(
9a), Figure S6.6: COSY of 7H-pyrido[2,3-c]carbazole (9a), Figure S7.1: 1H NMR of 10-fluoro-7H-
pyrido[2,3-c]carbazole (9b), Figure S7.2: 13C NMR of 10-fluoro-7H-pyrido[2,3-c]carbazole (9b),
Figure S7.3: 19F NMR of 10-fluoro-7H-pyrido[2,3-c]carbazole (9b), Figure S7.4: COSY of 10-fluoro-
7H-pyrido[2,3-c]carbazole (9b), Figure S7.5: NOESY of 10-fluoro-7H-pyrido[2,3-c]carbazole (9b),
Figure S7.6: HS of 10-fluoro-7H-pyrido[2,3-c]carbazole (9b), Figure S7.7: HMBC of 10-fluoro-7H-
1
pyrido[2,3-c]carbazole (9b), Figure S8.1: H NMR of 4-methyl-7H-pyrido[2,3-c]carbazolium iodide
(
10), Figure S8.2: 13C NMR of 4-methyl-7H-pyrido[2,3-c]carbazolium iodide (10), Figure S8.3: COSY
of 4-methyl-7H-pyrido[2,3-c]carbazolium iodide (10), Figure S8.4: NOESY of 4-methyl-7H-pyrido[2,3-
c]carbazolium iodide (10), Figure S8.5: HSQC of 4-methyl-7H-pyrido[2,3-c]carbazolium iodide (10),
Figure S8.6: HMBC of 4-methyl-7H-pyrido[2,3-c]carbazolium iodide (10), Figure S9.1: 1H NMR
of 4-methyl-11H-pyrido[3,2-a]carbazolium iodide (21), Figure S9.2: 13C NMR of 4-methyl-11H-
pyrido[3,2-a]carbazolium iodide (21), Figure S9.3: COSY of 4-methyl-11H-pyrido[3,2-a]carbazolium
iodide (21), Figure S9.4: NOESY of 4-methyl-11H-pyrido[3,2-a]carbazolium iodide (21), Figure S9.5:
HSQC of 4-methyl-11H-pyrido[3,2-a]carbazolium iodide (21), Figure S9.6: HMBC of 4-methyl-11H-
pyrido[3,2-a]carbazolium iodide (21).

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Author Contributions: Conceptualization, M.O.S.; methodology, K.S.H., E.L., J.H.A., V.M.A. and
M.O.S.; synthesis, K.S.H., I.T.U.H., C.L. and T.M.; antiplasmodial imaging assay, K.N.T., E.K.K. and
V.M.A.; cytotoxicity assay, K.N.T., E.K.K. and V.M.A.; antiproliferative assay, K.N.T., E.K.K. and
V.M.A.; growth inhibition assay, M.A. and J.H.A.; biofilm formation inhibition assay, M.A. and J.H.A.;
writing—original draft preparation, K.S.H.; writing—review and editing, K.S.H., E.L., M.A., J.H.A.,
V.M.A. and M.O.S.; supervision, J.H.A., V.M.A. and M.O.S.; project administration, M.O.S.; funding
acquisition, J.H.A., V.M.A. and M.O.S. All authors have read and agreed to the published version of
the manuscript.
Funding: The authors acknowledge the ToppForsk program at the University of Stavanger for
financial support and the provision of a PhD fellowship for K.S.H. (PR-10550). K.N.T is funded by a
Griffith University Postdoctoral Fellowship.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are contained within the article and
Supplementary Material.
Acknowledgments: B Holmelid, University of Bergen is thanked for recording HRMS analysis.
K. B. Jørgensen is sincerely thanked for his help operating and maintaining the NMR instrument.
The authors would like to acknowledge the Australian Red Cross Blood Bank for providing fresh
human erythrocytes.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
References
1.
World Health Organization. World Malaria Report. 2020. Available online: https://www.who.int/publications/i/item/978924
0015791 (accessed on 11 January 2021).
2.
3.
Collins, F.H.; Paskewitz, S.M. Malaria: Current and future prospects for control. Annu. Rev. Entomol. 1995, 40, 195–219. [CrossRef]
Achan, J.; Talisuna, A.O.; Erhart, A.; Yeka, A.; Tibenderana, J.K.; Baliraine, F.N.; Rosenthal, P.J.; D’Alessandro, U. Quinine, an old
anti-malarial drug in a modern world: Role in the treatment of malaria. Malar. J. 2011, 10, 1–12. [CrossRef]
Van Baelen, G.; Hostyn, S.; Dhooghe, L.; Tapolcsányi, P.; Mátyus, P.; Lemière, G.; Dommisse, R.; Kaiser, M.; Brun, R.; Cos, P.; et al.
Structure-activity relationship of antiparasitic and cytotoxic indoloquinoline alkaloids, and their tricyclic and bicyclic analogues.
Bioorg. Med. Chem. 2009, 17, 7209–7217. [CrossRef] [PubMed]
4.
5.
6.
7.
8.
9.
Tse, E.G.; Korsik, M.; Todd, M.H. The past, present and future of anti-malarial medicines. Malar. J. 2019, 18, 1–21. [CrossRef]
[PubMed]
Noedl, H.; Se, Y.; Scaecher, K.; Smith, B.L.; Socheat, D.; Fukuda, M.M. Evidence of artemisinin-resistant malaria in western
Cambodia. N. Engl. J. Med. 2008, 359, 2619–2620. [CrossRef]
Amato, R.; Pearson, R.D.; Almagro-Garcia, J.; Amaratunga, C.; Lim, P. Origins of the current outbreak of multidrug-resistant
malaria in southeast Asia: A retrospective genetic study. Lancet Infect. Dis. 2018, 18, 337–345. [CrossRef]
World Health Organization. World Cancer Report 2020: Cancer Research for Cancer Prevention. Available online: www.iarc.fr/
cards_page/world-cancer-report/ (accessed on 24 August 2020).
´
Sidoryk, K.; Jaromin, A.; Edward, J.A.; Switalska, M.; Stefanska, J.; Cmoch, P.; Zagrodzka, J.; Szczepek, W.; Peczynska-Czoch, W.;
Wietrzyk, J.; et al. Searching for new derivatives of neocryptolepine: A synthesis, antiproliferative, antimicrobial and antifungal
activities. Eur. J. Med. Chem. 2014, 78, 304–313. [CrossRef]
10. Stamm, A.M.; Long, M.N.; Belcher, B. Higher overall nosocomial infection rate because of increased attack rate of methicillin-
resistant Staphylococcus aureus. Am. J. Infect. Control 1993, 21, 70–74. [CrossRef]
11. Drenkard, E. Antimicrobial resistance of Preudomonas aeruginosa biofilms. Microb. Infect. 2003, 5, 1213–1219. [CrossRef]
12. Teng, C.P.; Zhou, T.; Ye, E.; Liu, S.; Koh, L.D.; Low, M.; Loh, X.J.; Win, Y.; Zhang, L.; Han, M.-Y. Effective Targeted Photothermal
Ablation of Multidrug Resistant Bacteria and Their Biofilms with NIR-Absorbing Gold Nanocrosses. Adv. Healthcare Mater. 2016
5, 2122–2130. [CrossRef]
,
13. Rizzato, C.; Torres, J.; Kasamatsu, E.; Carmorlinga-Ponce, M.; Bravo, M.M.; Canzian, F.; Kato, I. Potential role of biofilm formation
in the development of digestive tract cancer with special reference to Heliobacter pylori infection. Front Microbiol. 2019, 10, 1–21.
[CrossRef] [PubMed]
14. Johnson, C.H.; Dejea, C.M.; Edler, D.; Hoang, L.T.; Santidrian, A.F.; Felding, B.H.; Ivanisevic, J.; Cho, K.; Wick, E.C.; Hechen-
bleikner, E.M.; et al. Metabolism links bacterial biofilms and colon carcinogenesis. Cell Metab. 2015, 21, 891–897. [CrossRef]

Molecules 2021, 26, 3268
20 of 22
15. Drewes, J.L.; White, J.R.; Dejea, C.M.; Fathi, P.; Iyadorai, T.; Vadivelu, J.; Roslani, A.C.; Wick, E.C.; Mongodin, E.F.; Loke, M.F.; et al.
High-resolution bacterial 16S rRNA gene profile meta-analysis and biofilm status reveal common colorectal cancer consortia.
NPJ Biofilms Microbiomes 2017, 3, 1–34. [CrossRef] [PubMed]
16. Lee, K.H. Discovery and development of natural product-derived chemotherapeutic agents based on a medicinal chemistry
approach. J. Nat. Prod. 2010, 73, 500–516. [CrossRef] [PubMed]
17. Newman, D.J.; Cragg, G.M. Natural products as sources of new drugs over the last 25 years. J. Nat. Prod. 2007, 70, 461–477.
[CrossRef] [PubMed]
18. Vuorela, P.; Leinonen, M.; Saikku, P.; Tammela, P.; Rauhad, J.-P.; Wennberg, T.; Vuorela, H. Natural products in the process of
finding new drug candidates. Curr. Med. Chem. 2004, 11, 1375–1389. [CrossRef]
19. Newman, D.J.; Cragg, G.M. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019.
J. Nat. Prod. 2020, 83, 770–803. [CrossRef] [PubMed]
20. Bracca, A.B.J.; Heredia, D.A.; Larghi, E.L.; Kaufman, T.S. Neocryptolepine (cryprotackieine), a unique bioactive natural product:
Isolation, synthesis, and profile of its biological activity. Eur. J. Med. Chem. 2014, 2014, 7979–8003. [CrossRef]
21. Madapa, S.; Tusi, Z.; Batra, S. Advances in the syntheses of quinoline and quinoline-annulated ring systems. Curr. Org. Chem.
2008, 12, 1116–1183. [CrossRef]
22. Sydnes, M.O. Recent progress in the synthesis of antimalarial indoloquinoline natural products and analogues. In Studies in
Natural Products Chemistry: Bioactive Natural Products; Rahman, A., Ed.; Elsevier: Karachi, Pakistan, 2020; pp. 59–84.
23. Sydnes, M.O. Synthetic Strategies for the Synthesis of Indoloquinoline Natural Products. In Targets in Heterocyclic Systems;
Attanasi, O.A., Merino, P., Spinelli, D., Eds.; Italian Society of Chemistry: Rome, Italy, 2019; Volume 23, pp. 201–219.
24. Wang, N.; Wicht, K.J.; Imai, K.; Wang, M.-Q.; Ngoc, T.A.; Kiguchi, R.; Kaiser, M.; Egan, T.J.; Inokuchi, T. Synthesis, β-haematin
inhibition, and in vitro antimalarial testing of isocryptolepine analogues: SAR study of indolo[3,2-c]quinolines with various
substituents at C2, C6 and N11. Bioorg. Med. Chem. 2014, 22, 2629–2642. [CrossRef]
25. Aroonkit, P.; Thongsornkleeb, C.; Tummatorn, J.; Krajangsri, S.; Mungthin, M.; Ruchirawat, S. Synthesis of isocryptolepine
analogues and their structure-activity relationship studies as antiplasmodial and antiproliferative agents. Eur. J. Med. Chem. 2015
,
94, 56–62. [CrossRef] [PubMed]
26. Pousset, J.L.; Martin, M.T.; Jossang, A.; Bodo, A. Isocryptolepine from Cryptolepis sanguinolenta. Phytochemistry 1995, 39, 735–736.
[CrossRef]
27. Sharaf, M.H.H.; Schiff, P.L.; Tackie, J.A.N.; Phoebe, C.H.; Johnson, J.R.L.; Minick, D.; Andrews, C.W.; Crouch, R.C.; Martin, G.E.
The isolation and structure determination of cryptomisrine, a novel indolo[3,2-b]quinoline dimeric alkaloid from cryptolepis
sanguinolenta. J. Heterocycl. Chem. 1996, 33, 789–797. [CrossRef]
28. Lavrado, J.; Moreira, R.; Paulo, A. Indoloquinolines as scaffolds for drug discovery. Curr. Med. Chem. 2010, 17, 2348–2370.
[CrossRef] [PubMed]
29. Cimanga, K.; De Bruyne, T.; Pieters, L.; Vlietnck, A.J. In Vitro and in Vivo Antiplasmodial Activity of Cryptolepine and Related
Alkaloids from Cryptolepis sanguinolenta. J. Nat. Prod. 1997, 60, 688–691. [CrossRef] [PubMed]
30. Sofowora, A. Medicinal Plants and Traditional Medicine in Africa; John Wiley & Sons: Chichester, UK, 1982; pp. 183–256.
31. Kirby, G.C.; Paine, A.; Warhurst, D.C.; Noamese, B.K.; Phillipson, J.D. In vitro and in vivo antimalarial activity of cryptolepine, a
plant-derived indoloquinoline. Phytother. Res. 1995, 9, 359–363. [CrossRef]
32. Grellier, P.; Ramiaramanana, L.; Millerioux, V.; Deharo, E.; Shrével, J.; Frappier, F.; Trigalo, F.; Bodo, B.; Pousset, J.L. Antimalarial
Activity of Cryptolepine and Isocryptolepine, Alkaloids Isolated from Cryptolepis sanguinolenta. Phytother. Res. 1996, 10,
317–321. [CrossRef]
33. Olajide, O.A.; Heiss, E.H.; Schachner, D.; Wright, C.W.; Vollmar, A.M.; Dirsch, V.M. Synthetic cryptolepine inhibits DNA binding
of NF-κB. Bioorg. Med. Chem. 2007, 15, 43–49. [CrossRef]
34. Bierer, D.E.; Fort, D.M.; Mendez, C.D.; Luo, J.; Imbach, P.A.; Dubenko, L.G.; Jolad, S.D.; Gerber, R.E.; Litvak, J.; Lu, Q.;
et al. Ethnobotanical-Directed Discovery of the Antihyperglycemic Properties of Cryptolepine: Its Isolation from Cryptolepis
sanguinolenta, Synthesis, and in Vitro and in Vivo Activities. J. Med. Chem. 1998, 41, 894–901. [CrossRef]
35. Rauwald, H.W.; Kober, M.; Mutschler, E.; Lambrecht, G. Cryptolepis sanguinolenta: Antimuscarinic properties of cryptolepine
and the alkaloid fraction at M1, M2 and M3 receptors. Planta Med. 1992, 58, 486–488. [CrossRef] [PubMed]
36. Chen, Y.-J.; Liu, H.; Zhang, S.-Y.; Li, H.; Ma, K.-Y.; Liu, Y.-Q.; Yin, X.-D.; Zhou, R.; Yan, Y.-F.; Wang, R.-X.; et al. Design, Synthesis,
and Antifungal Evaluation of Cryptolepine Derivatives against Phytopathogenic Fungi. J. Agric. Food Chem. 2021, 69, 1259–1271.
[CrossRef] [PubMed]
37. Ablordeppey, S.Y.; Fan, P.; Li, S.; Clark, A.M.; Hufford, C.D. Substituted indoloquinolines as new antifungal agents. Bioorg. Med.
Chem. 2002, 10, 1337–1346. [CrossRef]
38. Singh, M.; Singh, M.P.; Ablordeppey, S. In vitro studies with liposomal cryptolepine. Drug Dev. Ind. Pharm. 1996, 22, 377–381.
[CrossRef]
39. Paulo, A.; Duarte, A.; Gomes, E.T. In vitro antibacerial screening of Cryptolepis sanguinolenta alkaloids. J. Ethnopharmacol. 1994
44, 127–130. [CrossRef]
,
40. Cimanga, K.; De Bruyne, T.; Lasure, A.; Van Poel, B.; Pieters, L.; Claeys, M.; Berghe, D.V.; Kambu, K.; Tona, L.; Vlietinch, A.
In vitro biological activities of alkaloids from Cryptolepis sanguinolenta. Planta Med. 1996, 62, 22–27. [CrossRef]

Molecules 2021, 26, 3268
21 of 22
41. Zhao, M.; Kamada, T.; Takeuchi, A.; Nishioka, H.; Kuroda, T.; Takeuchi, Y. Structure-activity relationship of indoloquinoline
analogs anti-MRSA. Bioorg. Med. Chem. 2015, 25, 5551–5554. [CrossRef]
42. Karou, D.; Savadogo, A.; Canini, A.; Yameogo, S.; Montesano, C.; Simpore, J.; Colizzi, V.; Traore, A.S. African ethnopharmacology
and new drug discovery. Afr. J. Biotechnol. 2007, 5, 195–200.
43. Lu, C.-M.; Chen, Y.-L.; Chen, H.-L.; Chen, C.-A.; Lu, P.-J.; Yang, C.-N.; Tzeng, C.-C. Synthesis and antiproliferative evaluation of
certain indolo[3,2-c]quinoline derivatives. Bioorg. Med. Chem. 2010, 18, 1948–1957. [CrossRef]
44. Dassonneville, L.; Lansiaux, A.; Wattelet, A.; Watterz, N.; Mahieu, C.; Van Miert, S.; Pieters, L.; Bailly, C. Cytotoxicity and cell
cycle effects of the plant alkaloids cryptolepine and neocryptolepine: Relation to drug-induced apoptosis. Eur. J. Pharmacol. 2000
,
409, 9–18. [CrossRef]
45. Zhu, H.; Gooderham, N.J. Mechanisms of induction of cell cycle arrest and cell death by cryptolepine in human lung adenocarci-
noma A549 cells. Toxicol. Sci. 2006, 91, 132–139. [CrossRef]
46. Matsui, T.-A.; Sowa, Y.; Murata, H.; Takagi, K.; Nakanishi, R.; Aoki, S.; Yoshikawa, M.; Kobayashi, M.; Sakabe, T.; Kubo, T.; et al.
The plant alkaloid cryptolepine induced p21WAF1/CIP1 and cell cycle arrest in a human osteosarcoma cell line. Int. J. Oncol.
2007, 31, 915–922. [PubMed]
47. Bonjean, K.; De Pauw-Gillet, M.C.; Defresne, M.P.; Colson, P.; Houssier, C.; Dassonneville, L.; Bailly, C.; Greimers, R.; Wright, C.W.;
Quetin-Leclercq, J.; et al. The DNA intercalating alkaloid cryptolepine interferes with topoisomerase II and inhibits primarily
DNA synthesis in B16 melanoma cells. Biochemistry 1998, 37, 5136–5146. [CrossRef]
48. Dassonneville, L.; Bonjean, K.; De Pauw-Gillet, M.C.; Colson, P.; Houssier, C.; Quetin-Leclercq, J.; Angenot, L.; Bailly, C.
Stimulation of topoisomerase II-mediated DNA cleavage by three DNA-intercalating plant alkaloids: Cryptolepine, matadine
and serpentine. Biochemistry 1999, 38, 7719–7726. [CrossRef]
49. Whittel, L.R.; Batty, K.T.; Wong, R.P.M.; Bolitho, E.M.; Fox, S.A.; Davis, T.M.E.; Murray, P.E. Synthesis and antimalarial evaluation
of novel isocryptolepine derivatives. Bioorg. Med. Chem. 2011, 19, 7519–7525. [CrossRef] [PubMed]
50. Van Miert, S.; Hostyn, S.; Maes, B.U.W.; Cimanga, K.; Brun, R.; Kaiser, M.; Mátyus, P.; Dommisse, R.A.; Lemière, G.; Vlietinck,
A.; et al. Isoneocryptolepine, a synthetic indoloquinoline alkaloid, as an antiplasmodial lead compound. J. Nat. Prod. 2005, 68,
674–677. [CrossRef] [PubMed]
51. 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. DNA intercalation, topoisomerase II inhibition and cytotoxic activity of the plant alkaloid neocryptolepine. Anti-Cancer
Drug Des. 2000, 15, 191–201.
52. Håheim, K.S.; Helgeland, I.T.U.; Lindbäck, E.; Sydnes, M.O. Mapping the reactivity of the quinoline ring-systems—Synthesis of
the tetracyclic ring-system of isocryptolepine and regioisomers. Tetrahedron 2019, 75, 2924–2957. [CrossRef]
53. Schmitz, F.J.; Deguzman, F.S.; Hossain, M.B.; Vanderhelm, D. Cytotoxic aromatic alkaloids from the ascidian Amphicarpa
meridiana and Leptoclinides sp.: Meridine and 11-hydroxyascididemin. J. Org. Chem. 1991, 56, 804–808. [CrossRef]
54. Gunawardana, G.P.; Koehn, F.E.; Lee, A.Y.; Clardy, J.; Hee, H.Y.; Faulkner, D.J. Pyridoacridine alkaloids from deep-water
marine sponges of the family Pacchastrellidae: Structure revision of dercitin and related compounds and correlation with the
kuanoniamines. J. Org. Chem. 1992, 57, 1523–1526. [CrossRef]
55. Molinski, T.F. Marine pyridoacridine alkaloids: Structure, synthesis, and biological chemistry. Chem. Rev. 1993, 93, 1825–1838.
[CrossRef]
56. Eder, C.; Schupp, P.; Proksch, P.; Wray, V.; Steube, K.; Müller, C.E.; Frobenius, W.; Herderich, M.; van Soest, R.W.M. Bioactive
Pyridoacridine Alkaloids from the Micronesian Sponge Oceanapia sp. J. Nat. Prod. 1998, 61, 301–305. [CrossRef]
57. Marshall, K.M.; Barrows, L.R. Biological activities of pyridoacridines. Nat. Prod. Rep. 2004, 61, 731–751. [CrossRef] [PubMed]
58. Delfourne, E.; Bastide, J. Marine pyridoacridine alkaloids and synthetic analogues as antitumor agents. Med. Res. Rev. 2003, 23,
234–252. [CrossRef] [PubMed]
59. De Guzman, F.S.; Carte, B.; Troupe, N.; Faulkner, D.J.; Harper, M.K.; Concepcion, G.P.; Mangalindan, G.C.; Matsumoto, S.S.;
Barrows, L.R.; Ireland, C.M. Neoamphimedine: A new pyridoacridine topoisomerase II inhibitor which catenates DNA. J. Org.
Chem. 1999, 64, 1400–1402. [CrossRef]
60. Feng, Y.R.; Davis, A.; Sykes, M.L.; Avery, V.M.; Carroll, A.R.; Camp, D.; Quinn, R.J. Antitrypanosomal pyridoacridine alkaloids
from the Australian ascidian Polysyncraton echinatum. Tetrahedron Lett. 2010, 51, 2477–2479. [CrossRef]
61. Fuente, J.A.D.L.; Martin, M.J.; Blanco, M.D.M.; Alfonso, E.P.; Avendano, C.; Mendez, J.C. A C-Ring regioisomer of the marine
alkaloid meridine exhibits selective in vitro cytotoxicity for solid tumors. Bioorg. Med. Chem. 2001, 9, 1807–1814. [CrossRef]
62. Helgeland, I.T.U.; Sydnes, M.O. A Concise Synthesis of Isocryptolepine by C-C Cross-Coupling Followed by a Tandem C-H
Activation and C-N Bond Formation. SynOpen 2017, 1, 41–44. [CrossRef]
63. Mehra, M.K.; Sharma, S.; Rangan, K.; Kumar, D. Substrate of Solvent-Controlled PdII-Catalyzed Regioselective Arylation of
Quinolin-4(1H)-ones Using Diaryliodonium Salts: Facile Access to Benzoxocine and Aaptamine Analogues. Eur. J. Org. Chem.
2020, 2020, 2409–2413. [CrossRef]
64. Beauchard, A.; Chabane, H.; Sinbandhit, S.; Guenot, P.; Thiéry, V.; Besson, T. Synthesis of original thiazoloindolo[3,2-c]quinoline
and novel 8-N-substituted-11H-indolo[3,2-c]quinoline derivatives from benzotriazoles. Part I. Tetrahedron 2006, 62, 1895–1903.
[CrossRef]
65. Timári, G.; Soós, T.; Hajós, G. A convenient synthesis of two new indoloquinoline alkaloids. Synlett 1997, 1997, 1067–1068.
[CrossRef]

Molecules 2021, 26, 3268
22 of 22
66. Hostyn, S.; Maes, B.U.W.; Pieters, L.; Lemière, G.L.F.; Mátyus, P.; Hajós, G.; Dommisse, R.A. Synthesis of the benzo-
β-carboline
isoneocryptolepine: The missing indoloquinoline isomer in the alkaloid series cryptolepine, neocryptolepine and isocryptolepine.
Tetrahedron 2005, 61, 1571–1577. [CrossRef]
67. Miller, M.; Vogel, J.C.; Tsang, W.; Merrit, A.; Procter, D.J. Formation of N-heterocycles by the reaction of thiols with glyoxamides:
Exploring a connective Pummerer-type cyclisation. Org. Biomol. Chem. 2009, 7, 589–597. [CrossRef]
68. 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.; et al. Synthesis, cytotoxicity, and antiplasmodial and antitrypanosomal activity of new neocryptolepine
derivatives. J. Med. Chem. 2002, 45, 3497–3508. [CrossRef]
69. Go, M.L.; Koh, H.L.; Ngiam, T.L.; Phillipson, J.D.; Kirby, G.C.; Oneill, M.J.; Warhurst, D.C. Synthesis and in vitro antimalarial
activity of some indolo[3,2-c]quinolines. Eur. J. Med. Chem. 1992, 27, 391–394. [CrossRef]
70. Ansah, C.; Gooderham, N.J. The Popular Herbal Antimalarial, Extract of Cryptolepis sanguinolenta, Is Potently Cytotoxic.
Toxicol. Sci. 2002, 70, 245–251. [CrossRef] [PubMed]
71. Lu, W.-J.; Switalska, M.; Wang, L.; Yonezawa, M.; El-Sayed, I.E.-T.; Wietrzyk, J.; Inokuchi, T. In vitro antiproliferative activity
of 11-aminoalkylamino-substituted 5H-indolo[2,3-b]quinolines; improving activity of neocryptolepines by installation of ester
substituent. Med. Chem. Res. 2013, 22, 4492–4504. [CrossRef]
72. Iorio, F.; Bosotti, R.; Scacheri, E.; Belcastro, V.; Mithbaokar, P.; Ferriro, R.; Murino, L.; Tagliaferri, R.; Brunetti-Pierri, N.; Isacchi, A.;
et al. Discovery of drug mode of action and drug repositioning from transcriptional responses. Proc. Natl. Acad. Sci. USA 2012
107, 14621–14626. [CrossRef]
,
73. Peczynska-Czoch, W.; Pognan, F.; Kaczmarek, L.; Boratynski, J. Synthesis and structure-activity relationship of methyl-substituted
indolo[2,3-b]quinolines: Novel cytotoxic, DNA topoisomerase II inhibitors. J. Med. Chem. 1994, 37, 3503–3510. [CrossRef]
74. Cimanga, K.; De Bruyne, T.; Pieters, L.; Totte, J.; Tona, L.; Kambu, K.; Vanden Berghe, D.; Vlietinck, A.J. Antibacterial and
antifungal activiies of neocryptolepine, biscryptolepine and cryptoquindoline, alkaloids isolated from Cryptolepis sanguinolenta.
Phytomedicine 1998, 5, 209–214. [CrossRef]
75. Shah, P.; Westwell, A.D. The role of fluorine in medicinal chemistry. J. Enzyme Inhib. Med. Chem. 2007, 22, 527–540. [CrossRef]
76. Miller, C.M.; McCarthy, F.O. Isolation, biological activity and synthesis of the natural product ellipticine and related pyridocar-
bazoles. RSC Adv. 2012, 2, 8883–8918. [CrossRef]
77. Rosenau, C.P.; Jelier, B.J.; Gossert, A.D.; Togni, A. Exposing the origins of irreproducibility in fluorine NMR spectroscopy. Angew.
Chem. Int. Ed. 2018, 57, 9528–9533. [CrossRef] [PubMed]
78. Zhou, F.; Driver, T.G. Efficient Synthesis of 3H-Indoles Enables by the Lead-Mediated α-Arylation of β-Ketoesters or γ-Lactams
Using Aryl Azides. Org. Lett. 2014, 16, 2916–2919. [CrossRef]
79. Alajarin, M.; Molina, P.; Vidal, A. Formal total synthesis of the alkaloid cryptotackieine (neocryptolepine). J. Nat. Prod. 1997, 60,
747–748. [CrossRef]
80. Duffy, S.A.; Avery, V.M. Development and optimization of a novel 384-well anti-malarial imaging assay validated for high-
throughput screening. Am. J. Trop. Med. Hyg. 2012, 86, 84–92. [CrossRef]
81. Fletcher, S.; Avery, V.M. A novel approach for the discovery of chemically diverse anti-malarial compounds targeting the
Plasmodium falciparum Coenzyme A synthesis pathway. Malar. J. 2014, 13, 343. [CrossRef]