Synthesis and biological evaluation of novel quinoline-piperidine scaffolds as antiplasmodium agents

Journal Pre-proof

Synthesis and biological evaluation of novel quinoline-piperidine scaffolds as

antiplasmodium agents

Tim Van de Walle, Maya Boone, Julie Van Puyvelde, Jill Combrinck, Peter J. Smith,

Kelly Chibale, Sven Mangelinckx, Matthias D'hooghe

PII:

S0223-5234(20)30299-3

DOI:

https://doi.org/10.1016/j.ejmech.2020.112330

EJMECH 112330

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 24 March 2020

Revised Date: 10 April 2020

Accepted Date: 10 April 2020

Please cite this article as: T. Van de Walle, M. Boone, J. Van Puyvelde, J. Combrinck, P.J. Smith, K.

Chibale, S. Mangelinckx, M. D'hooghe, Synthesis and biological evaluation of novel quinoline-piperidine

scaffolds as antiplasmodium agents, European Journal of Medicinal Chemistry (2020), doi: https://

doi.org/10.1016/j.ejmech.2020.112330.

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Graphical Abstract

Synthesis and biological evaluation of novel quinoline-piperidine

scaffolds as antiplasmodium agents

Tim Van de Walle,^aMaya Boone,^aJulie Van Puyvelde,^aJill Combrinck,^{b, c}Peter J.

Smith,^bKelly Chibale,^dSven Mangelinckx,^aand Matthias D’hooghe*^a

^aSynBioC Research Group, Department of Green Chemistry and Technology, Faculty of Bioscience Engineering,

Ghent University, Coupure Links 653, B-9000 Ghent, Belgium

^bDivision of Clinical Pharmacology, Department of Medicine, Medical School, University of Cape Town, K45,

OMB, Groote Schuur Hospital, Observatory, 7925, South Africa

^cWellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular

Medicine, South Africa

^dSouth African Medical Research Council Drug Discovery and Development Research Unit, Department of

Chemistry and Institute of Infectious Disease & Molecular Medicine, University of Cape Town, Rondebosch

7701, South Africa

* Corresponding author. E-mail: Matthias.Dhooghe@UGent.be

Abstract

The parasitic disease malaria places almost half of the world’s population at risk of infection and is responsible

for more than 400,000 deaths each year. The first-line treatment, artemisinin combination therapies (ACT)

regimen, is under threat due to emerging resistance of Plasmodium falciparum strains in e.g. the Mekong delta.

Therefore, the development of new antimalarial agents is crucial in order to circumvent the growing resistance.

Chloroquine, the long-established antimalarial drug, still serves as model compound for the design of new

quinoline analogues, resulting in numerous new active derivatives against chloroquine-resistant P. falciparum

strains over the past twenty years. In this work, a set of functionalized quinoline analogues, decorated with a

modified piperidine-containing side chain, was synthesized. Both amino- and (aminomethyl)quinolines were

prepared, resulting in a total of 18 novel quinoline-piperidine conjugates representing four different chemical

series. Evaluation of their in vitro antiplasmodium activity against a CQ-sensitive (NF54) and a CQ-resistant (K1)

strain of P. falciparum unveiled highly potent activities in the nanomolar range against both strains for five 4-

aminoquinoline derivatives. Moreover, no cytotoxicity was observed for all active compounds at the maximum

concentration tested. These five new aminoquinoline hit structures are therefore of considerable value for

antimalarial research and have the potency to be transformed into novel antimalarial agents upon further hit-

to-lead optimization studies.

Keywords: Quinolines, Piperidines, Malaria, Chloroquine, Plasmodium

1. Introduction

Malaria refers to a fatal infection of red blood cells, caused by a protozoan parasite of the genus

Plasmodium and transmitted to humans by Anopheles mosquitoes. Out of the five Plasmodium

species that can cause malaria, Plasmodium falciparum and Plasmodium vivax are the most

prevalent, with P. falciparum being the most lethal [1]. Despite numerous endeavours to reduce

malaria incidence over the last decades, this tropical disease remains far from being vanguished.

According to the latest WHO report, malaria gave rise to 228 million new cases and 405,000 deaths

worldwide in 2018, mostly in the African region (93% of all new cases and 94% of all deaths) and in

the age group of children under five years (67% of all deaths) [2]. These numbers confirm that

malaria remains the most lethal parasitic infection worldwide. Current malaria control relies on three

pillars: mosquito vector control with insecticides, treatment of infected people using antimalarial

1

agents and, most recently, vaccination [3]. Although the employment of insecticides and vaccination

are important prevention tools, the cure of infected people completely depends on antimalarial

medicines. In the past, all antimalarial agents that have been deployed for the treatment of malaria,

e.g. quinine, chloroquine, amodiaquine, sulfadoxine-pyrimethamine, mefloquine, piperaquine and

halofantrine, shared the same fate [1,4–6]. Plasmodium parasites developed clinical resistance to

these antiplasmodials in one area and then spread to other parts of the world [6,7]. Currently,

artemisinin derivatives (artesunate, artemether and dihydroartemisinin (DHA)) are the first-line

drugs to cure malaria, on the one hand as monotherapy for severe malaria cases, but mostly as part

of the artemisinin combination therapies (ACT) regimen for uncomplicated malaria, whereby

artemisinin derivatives are co-administered with a longer half-life antimalarial drug such as

amodiaquine, piperaquine, mefloquine or sulfadoxine-pyrimethamine [1,6,8]. However, a reduced in

vivo susceptibility of P. falciparum to artemisinins was already noticed more than ten years ago in the

Cambodia-Thailand border area, which were the first signs of resistance development [9]. At present,

an emerging multidrug resistance and growing ability to withstand ACT is the sobering situation [3,6].

A spread of this ACT-resistant P. falciparum from the Cambodia-Thailand area to other parts of the

world, which has occurred in the past for both chloroquine and sulfadoxine-pyrimethamine, would

result in a public health disaster, certainly when there is no plan B in terms of the availability of new

potent antimalarial agents [1,3,6,10].

Throughout the history of antimalarial chemotherapy, the quinoline scaffold has always played a

leading role, as can be seen from the numerous quinoline-containing antimalarial drugs such as

quinine 1, chloroquine 2, mefloquine 3, amodiaquine 4, primaquine 5 and piperaquine 6 (Figure 1).

Although the Plasmodium parasite (especially P. falciparum) has developed resistance against all

these quinolines, they are still of value as antimalarial medicines against more sensitive Plasmodium

parasites or in combination with artemisinins [1,8,11]. Quinolines, in particular 4-aminoquinolines,

based on the reference antimalarial chloroquine 2, are still of high interest in the quest for novel

antiplasmodium compounds, resulting in various highly potent quinolines over the last twenty years

[3,4,12–14]. It is generally believed that quinine 1, chloroquine 2, mefloquine 3, amodiaquine 4 (and

by extension all 4-substituted quinolines) rely on the same mode of action against Plasmodium [15].

Human hemoglobin is digested in the digestive vacuole of the parasite to produce amino acids, which

are needed for its growth. Heme, a toxic side product formed during this degradation, is normally

bio-crystallized by the Plasmodium parasite into hemozoin, a non-toxic crystal form [16]. When

administered to infected people, chloroquine 2 and other quinolines enter the acidic digestive

vacuole (pH 5) of the Plasmodium parasite through diffusion, where they are protonated. These

protonated forms are unable to diffuse back across the membrane and are thereby accumulated in

high concentrations in this digestive vacuole [17–19]. They subsequently inhibit the crystallization of

heme into hemozoin by forming a heme-quinoline complex, resulting in a build-up of toxic heme

molecules and consequently death of the protozoans [4,13,20]. The Plasmodium parasite can gain

resistance against established antimalarial drugs via two different mechanisms: by means of

mutations in the drug target or via mutations in drug transporters [21,22]. For chloroquine 2 and

mefloquine 3, and presumably also other quinolines having the same mode of action, the latter

mechanism is responsible for the developed resistance [6]. Although not fully understood, there is

much evidence that multiple point mutations in the PfCRT (P. falciparum chloroquine resistance

transporter) gene in case of chloroquine resistance and an increased copy-number of the pfmdr1 (P.

falciparum multidrug resistance) gene in case of mefloquine resistance are involved [4,6,23–25].

Either way, these mutated transporters, located in the membrane of the parasite’s digestive vacuole,

impair the accumulation of chloroquine 2 or mefloquine 3 inside the digestive vacuole, probably via

an increased efflux mechanism. In that way, they prevent the inhibitory activity of these antimalarial

2

drugs, simply because they cannot reach their target [6,17,21,25–27]. However, the target activity

itself, i.e. binding to heme units, is not altered in these resistant Plasmodium strains and therefore

remains very interesting to tackle, as this is a biological process that is unique to the malarial

parasite, resulting in limited host toxicity [28]. Examining the potent chloroquine derivatives that

have been designed over the past twenty years, a variety of side chain modifications, including the

introduction of different heterocyclic moieties, has been applied and well tolerated without being

detrimental to biological activity, while the pharmacophoric quinoline moiety is always maintained.

Authors frequently refer to the term hybrid structures, i.e. the implementation of a second

pharmacophore group as side chain onto the quinoline nucleus, with the rationale of obtaining a

compound with a dual mode of action [3,4,12,14]. It is believed that these side chain modifications

can also hinder binding and/or transport by mutated PfCRT in chloroquine-resistant (CQ-resistant)

strains, due to steric hindrance [17,29]. Even though many of these so called hybrid structures

possess very good antiplasmodium properties against CQ-resistant P. falciparum, there is not always

experimental proof that this improved activity can truly be attributed to a dual mode of action, or

solely to an excellent chloroquine-type mode of action that has surpassed resistance by

circumventing the mutated transporter mechanisms. Regardless of which mechanism is responsible

for their superior activity, the abundance of potent examples proves that the development of new

chloroquine analogues with a functionalized side chain still constitutes a promising strategy in the

quest for new antimalarial agents.

Figure 1. Examples of clinically used antimalarials containing a quinoline scaffold: quinine 1, chloroquine 2,

mefloquine 3, amodiaquine 4, primaquine 5 and piperaquine 6.

Within that framework, and inspired by the structural properties of quinine 1 and mefloquine 3, we

aimed to synthesize functionalized quinoline derivatives bearing a piperidine moiety in their side

chain. In our opinion, the introduction of piperidine as heterocyclic structure in antimalarial quinoline

analogues is underinvestigated compared to other heterocycles such as triazine, triazole and

pyrimidine [3,4,12]. Furthermore, the introduction of a piperidine moiety will deliver a weakly basic

side chain, which is presumed to be essential for uptake and accumulation of the antimalarial drug

via pH trapping in the acidic digestive vacuole [17,30,31]. In analogy with quinine 1 and mefloquine 3,

we selected one bridged and one monocyclic functionalized piperidine scaffold, suitable for linking

with different quinoline cores. This paper presents the synthesis of four different series of novel

quinoline derivatives modified with a piperidine side chain and the assessment of their

antiplasmodium activity against a CQ-sensitive strain (NF54) of P. falciparum. The most promising

analogues were further evaluated for their potency in a CQ-resistant strain (K1) of P. falciparum, and

for their cytotoxicity in a mammalian Chinese hamster ovary (CHO) cell line.

3

2. Results and discussion

2.1. Synthesis

In order to have convenient access to more decorated piperidine side chains, we decided to

construct our contemplated quinoline-piperidine analogues from appropriate piperidine scaffolds,

instead of commencing with the modification of a quinoline nucleus, as is most commonly done in

the literature. To that end, two different piperidine compounds were selected, both bearing a

functional group (an N-4-methoxybenzyl and a nitrile moiety, respectively) that can be converted

into a free amino group, since this was the preferred route towards reaction with various quinolines.

As a first heterocyclic scaffold, a bridged piperidine, i.e. 2-[N-(4-methoxybenzyl)aminomethyl]-4-

phenyl-1-azabicyclo[2.2.1]heptane 7 was chosen on the basis of its similarity to the side chain

present in quinine 1 and based on our in-house knowledge pertaining to the synthesis of this class of

compounds. A series of 1-azabicyclo[2.2.1]heptanes had previously been synthesized in our group

and evaluated for in vitro antiplasmodium activity [32]. Based on in silico docking studies of the most

active compound, the moderate antiplasmodium activity observed was hypothesized to (partially) be

attributed to inhibition of plasmepsin II, an enzyme essential for hemoglobin digestion in the parasite

[32]. Therefore, it seemed reasonable to explore whether or not the observed moderate

antiplasmodium activity could contribute to a potential dual mode of action in our target analogues.

For the synthesis of the contemplated bridged piperidine-quinoline analogues, 1-

azabicyclo[2.2.1]heptane 7, bearing a p-methoxybenzyl (PMB) protecting group, was used as a

starting point because of the inability to transform the other known 1-azabicyclo[2.2.1]heptanes

(with N-benzyl, N-4-(chlorobenzyl) and N-(4-methylbenzyl)) [32] into the corresponding primary

amine via common debenzylation methods. As described in the literature, bridged piperidine 7 was

prepared by treatment of the corresponding 2-(bromomethyl)aziridine [33–35] with α-lithiated

phenylacetonitrile in THF, followed by deprotonation with LDA and subsequent quenching with 1-

bromo-2-chloroethane,[36] and finally a one-step LiAlH₄-mediated ring closure to afford the

constrained piperidine (Synthesis and Experimental Data, see Supporting Information) [32]. It should

be noted that the separation of both diastereomers of 1-azabicyclo[2.2.1]heptane 7 is possible via

selective crystallization in ethanol,[36] but for practical reasons in view of the reaction scale,

constrained piperidine 7 was used as mixture of endo- and exo-isomers (dr 55/45) for further

synthetic elaboration. Treatment of 1-azabicyclo[2.2.1]heptane 7 with cerium ammonium nitrate

(CAN) in an acetonitrile/water (1/4) mixture resulted in primary amine 8 (Scheme 1). Purification

using both normal phase or reversed phase column chromatography led to product degradation. For

this reason, after acid-base work-up to remove the 4-methoxybenzaldehyde byproduct, the crude 2-

aminomethyl-4-phenyl-1-azabicyclo[2.2.1]heptane 8 was used as such in the next step. To chemically

link this constrained piperidine scaffold with a quinoline nucleus, two different coupling reactions

were deployed, resulting in two different sets of quinoline-piperidines (Scheme 1). First, reaction

with 4,7-dichloroquinoline 9a and 4-chloroquinoline 9b under microwave irradiation, without the

addition of solvent (‘neat’), produced 4-aminoquinolines 11a and 11b. Remarkably, performing the

synthesis in the high-boiling solvent phenol, commonly applied for this type of amine-halide

exchange reactions,[37,38] resulted in a considerable side reaction of 4-chloroquinolines 9 with

phenol, which rendered this procedure not useful. Purification by means of automated reversed

phase column chromatography yielded 4-aminoquinolines 11a-b in pure form (purity >95%, based on

¹H NMR and LC-MS analysis), as a mixture of diastereomers. As can be seen in Scheme 1, the endo

and exo forms eventually displayed the same reactivity, since the diastereomeric ratio did not change

significantly after reaction (dr determined before purification). In order to expand our library of

quinoline-piperidine analogues,

a

second series of new chloroquine analogues, i.e.

4

(aminomethyl)quinolines (inspired by quinine 1 and mefloquine 3), having an extra methylene linker

between the quinoline core and the amino group, was developed, as this part of the antimalarial

drug space is mostly untouched. Moreover, a recently reported 3-(aminomethyl)quinoline with

promising activity against both a CQ-sensitive (IC₅₀= 4.73 µM) and CQ-resistant (IC₅₀= 15.25 µM)

strain of P. falciparum,[39] prompted us to combine our 1-azabicyclo[2.2.1]heptane with quinolines

in this fashion. To that end, free amine 8 was subjected to reductive amination conditions using

different commercially available quinoline carboxaldehydes 10a-e and NaBH₄in methanol. After

purification using automated reversed phase column chromatography, five novel

(aminomethyl)quinolines 12a-e, bearing a bridged piperidine side chain at either the 2-, 3-, or 4-

position of the quinoline core (Scheme 1), were obtained in pure form (purity >95%, based on

¹H NMR and LC-MS analysis). Also here, the reaction conditions applied did not affect the

diastereomeric ratio (dr determined before purification). It must be emphasized that the reported

yields in Scheme 1 for both 4-aminoquinolines 11 and (aminomethyl)quinolines 12 are calculated

over two steps, starting from the N-protected 1-azabicylo[2.2.1]heptane 7. The low yields are mainly

attributed to the elaborate work-up procedure after CAN deprotection, which, after intensive

extraction procedures, only afforded roughly 50% of crude primary amine 8. The extended

purification en route to analytically pure samples for bio-evaluation also contributed to the low

yields.

Scheme 1. Synthetic route towards 4-aminoquinolines 11a-b and (aminomethyl)quinolines 12a-e

functionalized with a 1-azabicyclo[2.2.1]heptane scaffold.

As a second heterocyclic structure, we proposed the introduction of a monocyclic piperidine ring in

our desired set of quinoline-piperidine analogues, based on the structure of mefloquine 3. Since our

synthetic strategy required the presence of a functional group that could be converted into a primary

amine, the cis-1-allyl-5-benzyloxy-3,3-dimethylpiperidine-4-carbonitrile 13 available in-house was

selected as suitable and easily accessible monocyclic piperidine (Scheme 2). This class of piperidine-4-

5

carbonitriles has not been previously screened for antiplasmodium activity, which allows for an

interesting comparison with the 1-azabicyclo[2.2.1]heptane-quinolines 11 and 12. The synthesis of

piperidine-4-carbonitrile 13 is based on a 5-step literature procedure, involving the preparation of 4-

(2-bromoalkyl)-β-lactams via the Staudinger reaction,[40] their subsequent reduction to the

corresponding azetidines and finally a ring transformation with potassium cyanide to obtain the

desired piperidine-4-carbonitrile scaffold (Synthesis, see Supporting Information) [41]. Based on

¹H NMR analysis of the intermediate cis-β-lactams, it was confirmed that piperidine-4-carbonitrile 13

also displayed the cis-configuration [41]. The introduction of an N-allyl group was envisaged to

provide a handle for further synthetic modification later on, if desired, although other substituents

(e.g. benzyl or tBu) can also be easily incorporated. Reduction of the nitrile functionality with LiAlH₄

yielded the corresponding 4-(aminomethyl)piperidine 14, but also gave rise to a substantial amount

of side product 15, in which the benzyloxy moiety was cleaved off (Scheme 2). Although the removal

of a methoxy group from a piperidine ring with LiAlH₄is known in the literature,[42,43] this is the

first reported example of a simultaneous nitrile reduction and benzyloxy group removal through the

use of LiAlH₄. Modification of the reaction parameters (adaptation of numbers of equivalents of

LiAlH₄, temperature and time) always resulted in a mixture with at least 50% of 5-unsubstituted

piperidine 15. Separation of the primary amines by means of reversed phase column

chromatography only succeeded partially due to their tailing behaviour and was also accompanied by

some product degradation. Therefore, we decided to use the mixture of primary amines 14 and 15 as

such to react with an appropriate quinoline scaffold in the next step. To calculate the correct amount

of reagents, an average molecular weight of the reaction mixture was used according to the ratio of

1

both primary amines, which was approximately 1/4 (14/15) based on H NMR analysis. In order to

compare the antiplasmodium activity of these monocyclic piperidine-quinoline series with the

previously synthesized 1-azabicyclo[2.2.1]heptane-quinolines, the monocyclic piperidines were

reacted with the same quinolines as in Scheme 1. Thus, 4-chloroquinolines 9a-b were reacted with

the mixture of primary amines 14 and 15 under microwave irradiation (‘neat’), which afforded a

mixture of the corresponding 4-chloroquinolines 16 (Scheme 2). Only 0.8 equivalents of 4,7-

dichloroquinoline 9a or 4-chloroquinoline 9b were used, since upon addition of an equimolar

quantity, a significant amount of 4-chloroquinolines 9a-b remained unreacted. By means of

purification using automated reversed phase column chromatography, both piperidine-quinoline

analogues 16a and 16b could be isolated in high purity in the case of R¹= Cl, whereas for R¹= H, only

the analogue without the benzyloxy group (16c) was separated from the reaction mixture in high

1

purity (purity >95%, based on H NMR and LC-MS analysis). Next to 4-aminoquinolines, a series of

(aminomethyl)quinolines decorated with a monocyclic piperidine side chain was synthesized as well

via reductive amination using four different quinoline carboxaldehydes 10a,c-e (Scheme 2). In case

no complete conversion was achieved after addition of 1.5 equivalents of NaBH₄, 0.5 extra

equivalents were added to reach a conversion of 100%. Table 1 gives an overview of

1

(aminomethyl)quinolines 17a-g that were isolated in a purity of >95% (based on H NMR and LC-MS

analysis) after automated reversed phase column chromatography. Only derivative 17c had a purity

of >85% (based on ¹H NMR and LC-MS analysis), but could still be characterized. In addition to these

seven monocyclic piperidine-quinoline analogues 17a-g, (aminomethyl)quinoline 17h (see Figure 2),

containing a 5-methoxy-substituted piperidine structure, was synthesized in a similar fashion in a

yield of 6%, by reaction of cis-1-allyl-5-methoxy-3,3-dimethylpiperidine-4-carbonitrile and quinoline-

4-carboxaldehyde 10a. Since the intermediate mixture of primary amines 14 and 15 was not purified,

the yields for all novel 4-aminoquinolines 16a-c and aminomethylquinolines 17a-h, functionalized

with a monocyclic piperidine side chain, were calculated over two steps, starting from the

corresponding piperidine-4-carbonitrile 13. Also, novel analogues bearing a methoxy or benzyloxy

6

group (16a, 17a, 17c, 17f, 17h) featured the cis-configuration, since no diastereomerisation was

observed via ¹H NMR analysis during all reactions.

Scheme 2. Synthetic route towards 4-aminoquinolines 16a-c and (aminomethyl)quinolines 17a-g functionalized

with a monocyclic piperidine side chain.

Table 1. Specific substitution pattern and yields of all piperidine-(aminomethyl)quinolines 17a-g, functionalized

with a monocyclic piperidine side chain, that were obtained in pure form.

Compound

OBn?

Aldehyde 10

Equiv. NaBH₄

Yield (%)

17a

17b

17c

17d

17e

17f

Yes

No

Yes

No

Yes

No

10a

10c

10d

10e

1.5

16

11

5^a

26

12

15

9

1.5 + 0.5

1.5

1.5 + 0.5

17g

^a: Purity >85%, based on ¹H NMR and LC-MS analysis.

2.2. Antiplasmodium activity assessments

An antiplasmodium assay was performed on this set of 18 novel quinoline-piperidine analogues to

establish their potential as antimalarial agents. Figure 2 shows an overview of the different

compounds subjected to biological screening. All compounds were screened for in vitro

antiplasmodium activity against a CQ-sensitive strain of P. falciparum (NF54). Subsequently,

compounds showing promising antiplasmodium activity were also tested against a CQ-resistant

strain of P. falciparum (K1) and analysed for in vitro cytotoxicity against a mammalian cell line,

Chinese

hamster

ovarian

(CHO)

cells,

using

the

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazoliumbromide (MTT)-assay. Continuous in vitro cultures of asexual erythrocyte stages

7

of P. falciparum were maintained using a modified method of Trager and Jensen,[44] and

quantitative assessment of antiplasmodium activity in vitro was determined via the parasite lactate

dehydrogenase assay using a modified method described by Makler [45]. The test samples were

tested in triplicate on one occasion. The MTT-assay was used as a colorimetric assay for cellular

growth and survival, and compares well with other available assays [46,47]. The tetrazolium salt MTT

was used to measure all growth and chemosensitivity. Again, samples were tested in triplicate on

one occasion. Chloroquine was used as reference compound in both antiplasmodium screenings,

while emetine was used as known antiprotozoal agent in the MTT-assay.

N

NH

N

NH

N

Cl

11a

11b

12a

12b

12c

N

O

N

HO

Cl

NH

N

NH

N

Cl

12d

12e

16a

16b

16c

N

O

N

O

OH

NH

N

NH

N

17e

17a

17b

17c

17d

N

OMe

NH

O

N

Cl

NH

N

OMe

17f

17g

7

17h

Figure 2. Overview of novel 4-aminoquinoline-piperidines 11 and 16, novel (aminomethyl)quinoline-piperidines

12 and 17, and 1-azabicyclo[2.2.1]heptane 7, which were assayed on their antiplasmodium activity.

Table 2 summarizes the results of the biological tests. The IC₅₀values against NF54, K1 and CHO,

accompanied by, if applicable, selectivity (SI) and resistance indices (RI) are shown. Since the

compounds were not analysed all together but in three separate subsets, three IC₅₀values for

chloroquine and emetine are reported.

8

Table 2. IC₅₀values of 4-aminoquinolines (11 and 16), (aminomethyl)quinolines (12 and 17) and

1-azabicyclo[2.2.1]heptane 7 tested for their in vitro antiplasmodium activity against a CQ-sensitive (NF54) and

a CQ-resistant (K1) P. falciparum strain and their cytotoxicity. Data are expressed as mean ± SD values of three

independent experiments.

Compound

NF54 IC₅₀(µM)

K1 IC₅₀(µM)

CHO IC₅₀(µM)

SI^a

RI^b

11a^d

11b^e

12a^c

12b^c

12c^c

12d^c

12e^c

16a^d

16b^d

16c^e

17a^d

17b^d

17c^d

17d^d

17e^d

17f^d

17g^d

17h^d

7^c

0.021 ± 0.0050

0.045 ± 0.007

6.77 ± 0.25

4.07 ± 0.74

1.27 ± 0.04

0.72 ± 0.03

3.60 ± 0.31

0.012 ± 0.0010

0.015 ± 0.0017

0.236 ± 0.032

2.37 ± 1.05

4.94± 1.18

1.57 ± 0.17

3.66 ± 0.82

2.89 ± 1.06

1.28 ± 0.33

7.75 ± 1.44

17.99 ± 2.44

1.55 ± 0.13

0.033 ± 0.0062

0.069 ± 0.0113

-

26.0 ± 2.9

11.2 ± 2.1

-

1266

250

-

1.6

1.5

-

6.4

-

2.2

1.7

0.3

1.5

-

4.64 ± 0.13

-

0.026 ± 0.0057

0.025 ± 0.0089

0.0685 ± 0.0078

3.66 ± 0.43

-

102.65 ± 19.67

-

121.8 ± 41.4

29.9 ± 1.2

27.0 ± 3.2

139.4 ± 14.1

-

210.6 ± 31.4

-

142

-

10149

1991

114

59

-

134

-

2.82 ± 0.14

-

1.95 ± 0.078

-

1.8

-

1.5

-

14.7

17.8

8.5

-

199.0 ± 23.3

156

-

Chloroquine 0.011 ± 0.001^c

0.010 ± 0.0004^d

0.167 ± 0.028^c

0.169 ± 0.047^d

0.169 ± 0.047^e

-

0.020 ± 0.001^e

-

Emetine

0.04 ± 0.01^c

0.14 ± 0.04^d

0.03 ± 0.01^e

-

^a: SI (selectivity index) = IC₅₀CHO/IC₅₀NF54.

^b: RI (resistance index) = IC₅₀K1/IC₅₀NF54.

^c: Reference values: CQ: IC₅₀(NF54) 11.3 ± 1.0 nM, IC₅₀(K1) 166.5 ± 27.9 nM; emetine: IC₅₀0.04 ± 0.01 µM.

^d: Reference values: CQ: IC₅₀(NF54) 9.5 ± 0.4 nM, IC₅₀(K1) 169.0 ± 46.7 nM; emetine: IC₅₀0.14 ± 0.04 µM.

^e: Reference values: CQ: IC₅₀(NF54) 20.0 ± 1.4 nM, IC₅₀(K1) 169.0 ± 46.7 nM; emetine: IC₅₀0.03 ± 0.01 µM.

-: not tested.

The data obtained reveals that all piperidine-(aminomethyl)quinoline analogues 12 and 17 possess

moderate activity against the CQ-sensitive strain of P. falciparum (NF54) with IC₅₀values below 8 µM,

except for 17h. However, the 4-aminoquinoline-piperidines 11 and 16 clearly stand out, exhibiting

antiplasmodium activities in the low nanomolar range, comparable to chloroquine itself, with the

exception of 16c. Results in the CQ-resistant P. falciparum strain (K1) are in line with those observed

in NF54. All 4-aminoquinoline-piperidines 11 and 16 demonstrate excellent inhibitory activity with

nanomolar IC₅₀values in K1 (25 – 69 nM), 2.5- to 7-fold more potent than chloroquine. Their RI

values are approximately 10 times lower compared to chloroquine. Peculiarly, 4-aminoquinoline-

piperidine 16c has a RI value below 1 (RI = 0.3). Although not unheard of, RI values lower than 1 are

quite unusual, and cannot be explained straightforwardly. Nevertheless, these five 4-aminoquinoline-

piperidines can be considered very promising potential novel antimalarial hit structures, showing the

same intrinsic inhibitory activity as chloroquine (except for 16c) in NF54 data, but able to circumvent

the chloroquine resistance mechanisms. Additionally, data from the MTT-assay indicate no

cytotoxicity issues for all 4-aminoquinoline-piperidines 11 and 16, with SI values above 100.

9

A selective screening of the best performing (aminomethyl)quinolines (12d, 17a, 17c and 17f) shows

moderate antiplasmodium activity (IC₅₀1.95 – 4.64 µM) in K1, comparable with that in NF54. These

IC₅₀values in K1 are still higher than those of chloroquine, suggesting that this class of novel

(aminomethyl)quinolines 12 and 17 exert their medium activity either much less effectively than

chloroquine, or via another mechanism that does not significantly contribute to the antiplasmodium

effect. Nevertheless, it is clear from these results that insertion of an extra methylene unit between

the quinoline moiety and the amino functionality, as in (aminomethyl)quinoline-piperidines 12 and

17, is detrimental to the antiplasmodium activity. Analysing the structures of the best performing

compounds 11 and 16, it seems that the presence of a chlorine atom on the 7-position of the

quinoline nucleus delivers the best activity (analogues 11a and 16a-b). Conversely, omitting this

chlorine atom (analogues 11b and 16c) lowers the activity only 2 to 3 times in the CQ-resistant strain,

while its influence on activity in the CQ-sensitive strain varies, but still leads to nanomolar IC₅₀values

in its absence. Thus, it appears that, with respect to the 4-amino-7-chloroquinoline nucleus present

in chloroquine 2, the 4-aminoquinoline moiety is the principal pharmacophore required for good

activity, while the 7-chloro substitution only provides an additional effect. Furthermore, no

substantial difference in antiplasmodium activity can be observed between bridged piperidine-

quinolines 11 and monocyclic piperidine-quinolines 16. In contrast, 1-azabicyclo[2.2.1]heptane 7

possesses moderate antiplasmodium activity, comparable to previous reported activities for this

class,[32] while their monocyclic counterparts were also tested and, as expected, displayed no

antiplasmodium activity at all (data not shown). Thus, the antiplasmodium activity of the introduced

piperidine side chain itself seems to exert little influence on the final activity of the 4-

aminoquinoline-piperidines. From these findings, it seems unlikely that 1-azabicyclo[2.2.1]heptane-

piperidines 11 are hybrid structures with a dual mode of action. Therefore, we assume that our novel

4-aminoquinoline-piperidines 11 and 16 bypass the chloroquine resistance mechanism and are not

recognized by the mutated chloroquine transporters, or at least to a much lesser extent, and

therefore can still exert their antiplasmodium activity by inhibition of heme crystallization. However,

elaborate biological follow-up studies are necessary to confirm this hypothesis.

3. Conclusion

In this work, a library of 18 novel quinoline-piperidine conjugates was synthesized with the aim to

investigate their antimalarial potential. To that end, two different piperidine moieties, a bicyclic

1-azabicyclo[2.2.1]heptane scaffold and a 4-substituted monocyclic piperidine, were selected and

coupled with different quinoline cores either via an addition-elimination reaction or a reductive

amination, affording in total four different series of quinoline-piperidines. Antiplasmodium

evaluation against a CQ-sensitive (NF54) and a CQ-resistant (K1) strain of P. falciparum revealed a

promising activity for almost all compounds. Amongst them, five novel analogues belonging to the

class of 4-aminoquinoline-piperidines (11a-b and 16a-c) demonstrated excellent in vitro

antiplasmodium activity, with nanomolar IC₅₀values against both NF54 (12 – 236 nM) and K1 (25 – 69

nM) parasite strains. Furthermore, biological assessment in mammalian CHO cells did not indicate

any cytotoxicity issues. Given the urgent need for novel antimalarial agents in the battle against this

lethal parasitic disease and the emerging resistance to artemisinins, we propose our small library of

five novel 4-aminoquinoline-piperidines 11a-b and 16a-c as promising hit structures for further hit-

to-lead optimization towards new potent antimalaria medicines.

10

4. Experimental section

4.1. Synthesis and characterization

4.1.1. General

All reagents were purchased from commercial suppliers and were used as received without any

further purification. Dry solvents (THF, toluene, dichloromethane and diethyl ether) were obtained

using the MBraun SPS-800 solvent purification system. Dry methanol was purchased from Acros

Organics. Other solvents were purchased at the highest quality possible and used as supplied. Thin

layer chromatography (TLC) analysis of crude reaction mixtures or pure samples was performed using

glass-backed 0.25 mm Merck silica gel 60 F₂₅₄TLC plates, and visualised under UV light (254 nm) or by

using a KMnO₄or ninhydrin stain. Purification by means of column chromatography was executed on

chromatographic silica gel (particle size 35 – 70 µM, pore diameter 6 nm). Automated flash

chromatography was carried out on a Grace Reveleris® X1 flash chromatography system (reversed

phase) or Büchi Reveleris® X2 flash chromatography system (normal phase), using prepacked

Reveleris® silica or Reveleris® C18 cartridges. ¹H and ¹³C NMR spectra were recorded at 400 MHz and

100.6 MHz, respectively, on a Bruker Avance III, equipped with ¹H/BB z-gradient probe (BBO, 5 mm).

CDCl₃was used as solvent, and TMS was used as an internal chemical shift standard. All spectra were

processed using TOPSPIN 3.6.2 and were acquired through the standard sequences available in the

Bruker pulse program library. 2D spectra (COSY, HSQC, HMBC) were recorded as additive spectra for

complete structure elucidation. IR spectra were obtained from samples in neat form with a Quest

ATR (Attenuated Total Reflectance) accessory with diamond crystal puck using a Shimadzu

IRAFFINITY-1S Fourier Transform Infrared Spectrophotometer (FTIR). Melting points of solid

compounds were determined using a Kofler Bench, type WME Heizbank of Wagner & Munz. For

HPLC analyses, an Agilent 1200 Series HPLC equipped with a Supelco Ascentic Express C18 column (3

cm × 4.6 mm, 2.7 µm fused-core particles, 90 Å), a Phenomenex Guard column (SecurityGuard

Standard) and a UV-DAD detector was used. The HPLC is coupled to an Agilent 1100 Series MS with

electrospray ionisation (70 eV) with a single quadrupole detector for HPLC-MS analyses. The latter

one was also used for low resolution mass spectra (LRMS). High resolution electron spray (ES-TOF)

mass spectra were obtained with an Agilent Technologies 6210 Series Time of Flight. Microwave

reactions were performed in a CEM FocusedTM Microwave Synthesis System, Model Discover in 10

ml vials.

4.1.2. Procedure for the synthesis of (4-phenyl-1-azabicyclo[2.2.1]heptan-2-yl)methanamine

8

To a mixture of 10 ml acetonitrile and 40 ml water, 2-[N-(4-methoxybenzyl)aminomethyl]-4-phenyl-

1-azabicyclo[2.2.1]heptane 7 [32] (322 mg, 1 mmol) and CAN (2.74 g, 5 mmol) were added to achieve

a concentration of 0.1 M CAN in an acetonitrile/water mixture with a ratio of 1/4. The reaction

mixture was stirred for 24 hours at room temperature and checked afterwards for having reached

acidic conditions (litmus paper). It was poured in 50 ml water and extracted with 50 ml

dichloromethane. The dichloromethane phase was separated, while the emulsion-like aqueous

phase was extracted in a second separation funnel containing 50 ml dichloromethane. After

separation of the dichloromethane phase, the emulsion-like aqueous phase was extracted a last time

in a third separation funnel with 50 ml dichloromethane, in order to remove as much as possible of

the 4-methoxybenzaldehyde byproduct in the dichloromethane phase. Subsequently, the aqueous

phase was basified with 1 N NaOH (litmus paper), and filtrated over celite to remove the solids

present. After filtration, the aqueous phase was extracted with dichloromethane in three different

11

separation funnels (3 × 50 ml), like previously described. The combined organic phases were washed

with 50 ml brine, dried over MgSO₄, filtrated and evaporated under reduced pressure. The obtained

crude (4-phenyl-1-azabicyclo[2.2.1]heptan-2-yl)methanamine 8 (94.2 mg) was used as such without

purification in the next reactions.

4.1.3. Procedure for the synthesis of a mixture of cis-1-allyl-4-aminomethyl-5-benzyloxy-3,3-

dimethylpiperidine 14 and 1-allyl-4-aminomethyl-3,3-dimethylpiperidine 15

A solution of cis-1-allyl-5-benzyloxy-3,3-dimethylpiperidine-4-carbonitrile 13 [41] (284 mg, 1 mmol) in

15 ml dry THF was cooled to 0 °C. A 1.0 M LiAlH₄solution in THF (5 ml, 5 mmol) was carefully added

at 0 °C under argon atmosphere. Subsequently, the reaction was heated to reflux temperature and

stirred for 15 hours. Afterwards, the reaction mixture was cooled down to 0 °C again, after which the

excess of LiAlH₄was quenched by carefully adding dropwise 5 ml water, followed by 5 ml 2 N NaOH.

The salts in the mixture were removed via filtration, after which the solvent was evaporated. Then,

10 ml water was added and it was extracted with 3 × 20 ml dichloromethane, whereafter the

combined organic phases were dried over MgSO₄and filtrated. Removal of the solvent under

reduced pressure

afforded

a

mixture of

cis-1-allyl-4-aminomethyl-5-benzyloxy-3,3-

dimethylpiperidine 14 and 1-allyl-4-aminomethyl-3,3-dimethylpiperidine 15 (228.6 mg), which was

used without further purification in the next steps.

4.1.4. Representative procedure for the synthesis of N-[(4-phenyl-1-azabicyclo[2.2.1]heptan-

2-yl)methyl]quinolin-4-amines 11a-b

For ease of calculations, it was assumed that the amount of crude starting material equals 1

equivalent. Primary amine (4-phenyl-1-azabicyclo[2.2.1]heptan-2-yl)methanamine 8 (94.2 mg, 0.47

mmol) and 4,7-dichloroquinoline 9a (93 mg, 0.47 mmol) were put into a microwave vessel of 10 ml.

The mixture was stirred under microwave irradiation for two hours at 130 °C. After confirmation of

full conversion via LC-MS analysis, the reaction product was dissolved in 50 ml dichloromethane, and

if necessary 5 ml methanol was used to dissolve all precipitates. 5 ml 2 N NaOH and 10 ml water

were added, followed by an extraction with dichloromethane (2 × 20 ml). The combined organic

phases were washed with water (10 ml), dried over MgSO₄, filtrated and evaporated under reduced

pressure. Purification by means of automated column chromatography (C18, gradient MeOH/H₂O

50/50 to 100/0) resulted in 45.2 mg 7-chloro-N-[(4-phenyl-1-azabicyclo[2.2.1]heptan-2-

yl)methyl]quinolin-4-amine 11a (0.124 mmol) in a purity of >95%, corresponding with a yield of 12%

over two steps. The purification conditions for derivative 11b are specified in the characterization.

4.1.4.1. 7-Chloro-N-[(4-phenyl-1-azabicyclo[2.2.1]heptan-2-yl)methyl]quinolin-4-amine 11a

Spectral data derived from the mixture of diastereomers (dr = 55/45).

¹H NMR (400 MHz, CDCl₃): δ 1.29 (1H, ddd, J = 11.7, 6.2, 1.4 Hz), 1.56 (1H, ddd, J = 11.9, 5.1, 3.6 Hz),

1.60-1.64 (1H, m), 1.72-1.78 (1H, m), 1.91-2.03 (3H, m), 2.19 (1H, ddd, J = 11.6, 10.6, 3.5 Hz), 2.66

(1H, dt, J = 9.8, 1.6 Hz), 2.79-2.87 (2H, m), 2.93-3.02 (5H, m), 3.13-3.32 (4H, m), 3.51-3.57 (1H, m),

3.72 (1H, hept, J = 5.4 Hz), 5.71 (1H, d, J = 4.1 Hz, NH_minor), 5.97 (1H, d, J = 7.4 Hz, NH_major), 6.37 (1H, d,

J = 5.3 Hz), 6.41 (1H, d, J = 5.3 Hz), 7.24-7.39 (12H, m), 7.796 (1H, d, J = 9.0 Hz), 7.801 (1H, d, J = 8.9

Hz), 7.95 (1H, d, J = 2.1 Hz), 7.97 (1H, d, J = 2.1 Hz), 8.53 (1H, d, J = 5.3 Hz), 8.56 (1H, d, J = 5.3 Hz).

¹³C NMR (100 MHz, CDCl₃): δ 37.6, 38.7, 42.3, 42.9, 44.5, 46.6, 47.1, 53.9, 55.0, 56.4, 59.8, 62.5,

64.87, 64.90, 99.1, 99.3, 117.1, 117.4, 121.4, 121.6, 125.2, 125.4, 126.6, 126.7, 126.8, 128.5, 128.76,

128.79, 134.8, 135.0, 141.8, 142.1, 149.2, 149.3, 149.4, 149.6, 152.1. IR (cm^-1) ν_NH= 3294, ν_max= 2922,

2870, 1608, 1578, 1447, 1136, 806, 698. MS (70 eV): m/z (%) = 364/366 ([M+H]⁺, 100). HRMS (ESI)

12

calcd for C₂₂H₂₂ClN₃[M+H]⁺364.1575, found 364.1577. Light yellow oil. Yield after automated column

chromatography (C18, MeOH/H₂O: 50/50 to 100/0) = 12%.

4.1.4.2. N-[(4-Phenyl-1-azabicyclo[2.2.1]heptan-2-yl)methyl]quinolin-4-amine 11b

Spectral data derived from the mixture of diastereomers (dr = 60/40).

¹H NMR (400 MHz, CDCl₃): δ = 1.33 (1H, dd, J = 11.7, 5.4 Hz), 1.57 (1H, ddd, J = 11.9, 4.5, 4.5 Hz),

1.62-1.67 (1H, m), 1.72-1.79 (1H, m), 1.93-2.04 (3H, m), 2.22 (1H, ddd, J = 11.7, 11.2, 2.9 Hz), 2.67

(1H, d, J = 9.5 Hz), 2.81-2.89 (2H, m), 2.97-3.06 (5H, m), 3.16-3.35 (4H, m), 3.54-3.60 (1H, m), 3.74-

3.82 (1H, m), 5.79 (1H, br s, NH_minor), 6.00 (1H, d, J = 6.8 Hz, NH_major), 6.40 (1H, d, J = 5.3 Hz), 6.43 (1H,

d, J = 5.2 Hz), 7.24-7.37 (10H, m), 7.45 (2H, dd, J = 7.6, 7.5 Hz), 7.64 (2H, dd, J = 8.0, 7.5 Hz), 7.88 (1H,

d, J = 7.6 Hz), 7.89 (1H, d, J = 7.6 Hz), 7.98 (1H, d, J = 8.0 Hz), 7.99 (1H, d, J = 8.0 Hz), 8.56 (1H, d, J =

5.3 Hz), 8.59 (1H, d, J = 5.2 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 37.6, 38.6, 42.3, 43.0, 44.6, 46.5, 47.2,

53.8, 54.8, 56.4, 59.6, 62.4, 64.8, 64.9, 98.8, 99.0, 118.7, 119.0, 119.88, 119.94, 124.6, 124.7, 126.58,

126.63, 126.7, 126.8, 128.51, 128.55, 129.06, 129.12, 129.7, 129.8, 141.8, 142.0, 148.37, 148.42,

149.5, 149.7, 151.0. IR (cm^-1) ν_max= 2951, 2172, 1580, 1572, 1128, 758, 725, 698. MS (70 eV): m/z (%)

= 330 ([M+H]⁺, 100). Mp = 60 °C. Light yellow powder. Yield after automated column

chromatography (C18, MeOH/H₂O: 50/50 to 100/0) = 13%.

4.1.5. Representative procedure for the synthesis of N-[(4-phenyl-1-azabicyclo[2.2.1]heptan-

2-yl)methyl]-1-quinolinylmethanamines 12a-e

For ease of calculations, it was assumed that the amount of crude starting material equals 1

equivalent. Crude (4-phenyl-1-azabicyclo[2.2.1]heptan-2-yl)methanamine 8 (94.2 mg, 0.47 mmol)

was dissolved in 10 ml dry methanol and stirred under argon atmosphere. To this mixture, quinoline-

4-carboxaldehyde 10a (81.3 mg, 0.52 mmol) was added and subsequently stirred overnight at room

temperature. After complete conversion, determined via LC-MS analysis, NaBH₄(26.7 mg, 0.71

mmol) was added portionwise and the reaction mixture was stirred for an additional three hours at

room temperature. When the reaction reached complete conversion, methanol was evaporated. The

reaction mixture was resolved in 20 ml chloroform and 20 ml brine and extracted with chloroform (3

× 20 ml), after which the combined organic phases were dried over MgSO₄, filtered and evaporated

under reduced pressure. The resulting crude product was purified with automated column

chromatography (C18, gradient MeOH/H₂O 0/100 to 100/0). Finally, 20.5 mg N-[(4-phenyl-1-

azabicyclo[2.2.1]heptan-2-yl)methyl]-1-(quinolin-4-yl)methanamine 12a (0.060 mmol) was obtained

in a purity of >95%, in a yield of 6% over two steps.

4.1.5.1. N-[(4-Phenyl-1-azabicyclo[2.2.1]heptan-2-yl)methyl]-1-(quinolin-4-yl)methanamine 12a

Spectral data derived from the mixture of diastereomers (dr = 54/46).

¹H NMR (400 MHz, CDCl₃): δ 1.12 (1H, ddd, J = 11.4, 5.8, 2.0 Hz), 1.39 (1H, ddd, J = 11.7, 5.5, 3.6 Hz),

1.48-1.54 (1H, m), 1.64-1.71 (1H, m), 1.83-1.92 (3H, m), 2.07 (1H, ddd, J = 11.4, 10.7, 3.5 Hz), 2.32 (2

× 1H, br s, 2 × NH), 2.57-2.62 (2H, m), 2.70 (1H, dd, J = 11.8, 10.0 Hz), 2.75-2.81 (2H, m), 2.86-2.93

(6H, m), 3.01-3.07 (1H, m), 3.13 (1H, ddd, J = 11.8, 11.2, 5.9 Hz), 3.50-3.58 (1H, m), 4.26-4.37 (4H, m),

7.19-7.34 (10H, m), 7.51 (2H, d, J = 4.4 Hz), 7.55-7.59 (2H, m), 7.69-7.73 (2H, m), 8.09-8.14 (4H, m),

8.87 (2H, d, J = 4.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 37.5, 38.7, 42.6, 43.2, 46.8, 50.0, 50.2, 51.4,

53.5, 54.6, 54.9, 56.6, 60.1, 63.8, 65.0, 66.0, 119.7, 119.9, 123.3, 126.4, 126.47, 126.53, 126.7, 126.8,

127.0, 127.1, 128.39, 128.41, 129.0, 129.1, 130.18, 130.22, 142.3, 142.6, 145.5, 145.8, 148.2, 148.3,

150.37, 150.40. IR (cm^-1) ν_NH= 3306, ν_max= 2949, 2884, 1593, 1570, 1508, 1497, 754, 698. MS (70 eV):

m/z (%) = 344 ([M+H]⁺, 100). HRMS (ESI) calcd for C₂₃H₂₅N₃[M+H]⁺344.2121, found 344.2112. White-

13

yellow oil. Yield after automated column chromatography (C18, gradient MeOH/H₂O 0/100 to

100/0): 6%.

4.1.5.2. N-[(4-Phenyl-1-azabicyclo[2.2.1]heptan-2-yl)methyl]-1-(quinolin-3-yl)methanamine 12b

Spectral data derived from the mixture of diastereomers (dr = 53/47).

¹H NMR (400 MHz, CDCl₃): δ 1.10 (1H, ddd, J = 11.6, 6.0, 1.6 Hz), 1.36 (1H, ddd, J = 11.7, 5.5, 3.6 Hz),

1.45-1.52 (1H, m), 1.63-1.69 (1H, m), 1.80-1.91 (3H, m), 2.05 (1H, ddd, J = 11.6, 10.6, 3.7 Hz), 2.13 (2

× 1H, br s, 2 × NH), 2.52 (1H, dd, J = 11.9, 4.3 Hz), 2.58-2.65 (2H, m), 2.73-2.94 (8H, m), 2.97-3.04 (1H,

m), 3.12 (1H, ddd, J = 11.9, 11.1, 5.9 Hz), 3.46-3.54 (1H, m), 3.98-4.08 (4H, m), 7.19-7.33 (10H, m),

7.51-7.56 (2H, m), 7.66-7.71 (2H, m), 7.80 (2H, dd, J = 7.8, 3.6 Hz), 8.09-8.14 (4H, m), 8.92 (2H, dd, J =

5.0, 2.1 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 37.5, 38.8, 42.7, 43.2, 46.9, 51.0, 51.51, 51.55, 53.4, 54.5,

54.7, 56.6, 60.0, 63.8, 65.1, 66.1, 126.31, 126.33, 126.6, 126.68, 126.71, 126.8, 127.6, 128.03, 128.04,

128.4, 128.95, 129.01, 129.23, 129.25, 133.01, 133.08, 134.4, 134.5, 142.3, 142.7, 147.48, 147.53,

151.5, 151.6. IR (cm^-1) ν_NH= 3285, ν_max= 2949, 2868, 1601, 1570, 1495, 1125, 756, 700. MS (70 eV):

m/z (%) = 344 ([M+H]⁺, 100). HRMS (ESI) calcd for C₂₃H₂₅N₃[M+H]⁺344.2121, found 344.2126.

Brown-yellow oil. Yield after automated column chromatography (C18, gradient MeOH/H₂O 0/100 to

100/0): 6%.

4.1.5.3. N-[(4-Phenyl-1-azabicyclo[2.2.1]heptan-2-yl)methyl]-1-(quinolin-2-yl)methanamine 12c

Spectral data derived from the mixture of diastereomers (dr = 55/45).

¹H NMR (400 MHz, CDCl₃): δ 1.13 (1H, ddd, J = 11.6, 6.0, 1.9 Hz), 1.40 (1H, ddd, J = 11.8, 5.1, 3.6 Hz),

1.45-1.52 (1H, m), 1.62-1.69 (1H, m), 1.80-1.91 (3H, m), 2.07 (1H, ddd, J = 11.6, 10.4, 3.7 Hz), 2.29 (2

× 1H, br s, 2 × NH), 2.53-2.61 (2H, m), 2.68-2.97 (8H, m), 3.02-3.10 (2H, m), 3.11-3.17 (1H, m), 3.47-

3.57 (1H, m), 4.13-4.22 (4H, m), 7.19-7.33 (10H, m), 7.49-7.54 (4H, m), 7.66-7.72 (2H, m), 7.78-7.81

(2H, m), 8.07 (2H, d, J = 8.4 Hz), 8.12 (2H, d, J = 8.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 37.5, 38.8, 42.8,

43.2, 47.0, 47.8, 51.4, 53.5, 54.7, 55.96, 56.04, 56.6, 60.1, 64.0, 65.2, 66.1, 120.4, 120.5, 126.00,

126.04, 126.3, 126.7, 126.8, 127.3, 127.6, 128.4, 129.0, 129.37, 129.44, 136.4, 142.9, 147.7, 160.5,

160.6. IR (cm^-1) ν_NH= 3323, ν_max= 2951, 2870, 1599, 1425, 1111, 829, 756, 698. MS (70 eV): m/z (%) =

344 ([M+H]⁺, 100). HRMS (ESI) calcd for C₂₃H₂₅N₃[M+H]⁺344.2121, found 344.2109. Brown-yellow

oil. Yield after automated column chromatography (C18, gradient MeOH/H₂O 0/100 to 100/0): 4%.

4.1.5.4. 2-{N-[(4-Phenyl-1-azabicyclo[2.2.1]heptan-2-yl)methyl]aminomethyl}quinolin-8-ol 12d

Spectral data derived from the mixture of diastereomers (dr = 54/46).

¹H NMR (400 MHz, CDCl₃): δ 1.10 (1H, dd, J = 11.4, 6.0 Hz), 1.34 (1H, ddd, J = 11.6, 5.5, 3.6 Hz), 1.46-

1.52 (1H, m), 1.66-1.72 (1H, m), 1.81-1.93 (3H, m), 2.05 (1H, ddd, J = 11.4, 10.6, 3.5 Hz), 2.53-3.11

(12H, m), 3.17-3.24 (1H, m), 3.52-3.59 (1H, m), 4.05-4.22 (4H, m), 7.16-7.47 (18H, m), 8.08-8.11 (2H,

m). ¹³C NMR (100 MHz, CDCl₃): δ 37.5, 38.8, 42.6, 43.3, 46.8, 51.5, 53.1, 54.4, 54.6, 54.7, 55.3, 56.4,

60.1, 64.0, 65.0, 66.4, 110.5, 110.6, 117.5, 117.6, 120.9, 121.0, 126.3, 126.7, 126.8, 127.1, 127.2,

127.5, 127.6, 128.3, 128.4, 136.4, 136.5, 137.7, 142.3, 142.7, 152.4, 152.7, 157.7, 158.2. IR (cm^-1) ν_NH

= 3304, ν_max= 2932, 2870, 1599, 1505, 1466, 1242, 754, 698. MS (70 eV): m/z (%) = 360 ([M+H⁺],

100). HRMS (ESI) calcd for C₂₃H₂₅N₃O [M+H]⁺360.2070, found 360.2057. Brown oil. Yield after

automated column chromatography (C18, gradient MeOH/H₂O 0/100 to 100/0): 3%.

4.1.5.5.

N-[(4-Phenyl-1-azabicyclo[2.2.1]heptan-2-yl)methyl]-1-(2-chloroquinolin-3-

yl)methanamine 12e

Spectral data derived from the mixture of diastereomers (dr = 51/49).

14

¹H NMR (400 MHz, CDCl₃): δ 1.13 (1H, ddd, J = 11.5, 6.0, 1.7 Hz), 1.39 (1H, ddd, J = 11.6, 5.2, 3.7 Hz),

1.48-1.54 (1H, m), 1.65-1.71 (1H, m), 1.83-1.91 (3H, m), 2.08 (1H, ddd, J = 11.5, 10.5, 3.5 Hz), 2.29 (2

× 1H, br s, 2 × NH), 2.54-2.62 (2H, m), 2.66-2.71 (1H, m), 2.75-2.82 (2H, m), 2.85-2.98 (6H, m), 3.02-

3.09 (1H, m), 3.15 (1H, ddd, J = 12.1, 11.2, 5.7 Hz), 3.51-3.59 (1H, m), 4.05-4.08 (4H, m), 7.20-7.33

(10H, m), 7.52-7.56 (2H, m), 7.66-7.71 (2H, m), 7.82 (2H, d, J = 8.0 Hz), 8.01 (2H, d, J = 8.4 Hz), 8.27

(1H, s), 8.28 (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ 37.5, 38.8, 42.6, 43.2, 46.8, 50.7, 50.9, 51.1, 53.5,

54.5, 54.6, 56.6, 60.0, 63.9, 65.1, 66.0, 126.4, 126.7, 126.8, 127.0, 127.1, 127.4, 128.2, 128.4, 129.9,

130.0, 131.8, 132.0, 137.0, 137.2, 142.3, 142.6, 146.7, 146.8, 150.5, 150.6. IR (cm^-1) ν_NH= 3316, ν_max

=

2936, 2870, 1593, 1491, 1327, 1032, 756, 700. MS (70 eV): m/z (%) = 378/380 ([M+H]⁺, 100). HRMS

(ESI) calcd for C₂₃H₂₄ClN₃[M+H]⁺378.1732, found 378.1724. Brown-yellow oil. Yield after automated

column chromatography (C18, gradient MeOH/H₂O 0/100 to 100/0): 5%.

4.1.6. Representative procedure for the synthesis of N-[(1-allyl-3,3-dimethylpiperidin-4-

yl)methyl]quinolin-4-amines 16a-c

The crude mixture of cis-1-allyl-4-aminomethyl-5-benzyloxy-3,3-dimethylpiperidine 14 and 1-allyl-4-

aminomethyl-3,3-dimethylpiperidine 15 (101.8 mg, 0.50 mmol)^awas brought into a microwave vessel

of 10 ml, and 4,7-dichloroquinoline 9a (79.2 mg, 0.40 mmol) was added. The reaction mixture was

stirred for 2 hours at 130 °C under microwave irradiation. In case no complete conversion was

reached, the product first had to be dissolved in methanol and evaporated again prior to further

microwave irradiation, in order to avoid product degradation. After complete conversion, the

reaction product was dissolved in 50 ml dichloromethane, and if necessary 5 ml methanol was used

to dissolve all precipitates. 5 ml 2 N NaOH and 10 ml water were added, followed by an extraction

with dichloromethane (2 × 20 ml). The combined organic phases were washed with water (10 ml),

dried over MgSO₄, filtrated and evaporated under reduced pressure. Purification by means of

automated column chromatography (C18, gradient acetonitrile/H₂O 30/70 to 100/0 with slow

gradient of 1.4% per CV between 50 and 100% acetonitrile) afforded 3.2 mg N-[(cis-1-allyl-5-

benzyloxy-3,3-dimethylpiperidin-4-yl)methyl]-7-chloroquinolin-4-amine 16a (0.007 mmol) and 12.8

mg N-[(1-allyl-3,3-dimethylpiperidin-4-yl)methyl]-7-chloroquinolin-4-amine 16b (0.037 mmol) in a

purity of >95%, in a yield over two steps of 2% and 10% respectively. The purification conditions for

N-[(1-allyl-3,3-dimethylpiperidin-4-yl)methyl]quinolin-4-amine

16c

is

specified

in

the

characterization.

^a: The ratio of mixture 14/15 was 1/4, so an average MW of 203.5 g/mol was used for calculations.

4.1.6.1. N-[(cis-1-Allyl-5-benzyloxy-3,3-dimethylpiperidin-4-yl)methyl]-7-chloroquinolin-4-amine

16a

¹H NMR (400 MHz, CDCl₃): δ 1.11 (3H, s), 1.18 (3H, s), 1.60-1.77 (1H, m), 1.96 (1H, d, J = 11.5 Hz),

2.13-2.32 (1H, m), 2.38 (1H, d, J = 11.5 Hz), 2.89-3.06 (1H, m), 3.03 (2H, d, J = 6.3 Hz), 3.42-3.48 (1H,

m), 3.54-3.63 (1H, m), 3.76-3.85 (1H, m), 4.34 (1H, d, J_AX= 11.8 Hz), 4.79 (1H, d, J_AX= 11.8 Hz), 5.13-

5.25 (2H, m), 5.84-5.95 (1H, m), 6.33 (1H, d, J = 5.4 Hz), 6.77 (1H, d, J = 9.0 Hz), 7.02 (1H, dd, J = 9.0,

1.8 Hz), 7.40-7.42 (5H, m), 7.87 (1H, d, J = 1.8 Hz), 8.45 (1H, d, J = 5.4 Hz). ¹³C NMR (100 MHz, CDCl₃):

δ 24.2, 28.4, 33.8, 39.8, 46.0, 54.0, 61.6, 71.0, 74.6, 98.3, 117.2, 117.6, 121.1, 124.9, 128.1, 128.3,

128.5, 128.8, 134.6, 135.3, 138.6, 142.5, 149.7, 151.9. IR (cm^-1): ν_max= 2953, 1582, 1368, 914, 743. MS

(70 eV): m/z (%) = 450/452 ([M+H]⁺, 100). HRMS (ESI) calcd for C₂₇H₃₂ClN₃O [M+H]⁺450.2307, found

450.2324. Yellow oil. Yield after automated column chromatography (C18, gradient ACN/H₂O 30/70

to 100/0 with slow gradient of 1.4% per CV between 50 and 100% acetonitrile): 2%.

*Remark: The signal for C_quatCH₂N is not visible on ¹³C NMR.

15

4.1.6.2. N-[(1-Allyl-3,3-dimethylpiperidin-4-yl)methyl]-7-chloroquinolin-4-amine 16b

¹H NMR (400 MHz, CDCl₃): δ 1.04 (3H, s), 1.05 (3H, s), 1.43-1.51 (1H, m), 1.52-1.63 (1H, m), 1.69 (1H,

d, J = 11.3 Hz), 1.73-1.79 (1H, m), 1.88 (1H, ddd, J = 11.6, 11.6, 2.8 Hz), 2.50 (1H, dd, J = 11.3, 1.5 Hz),

2.85 (1H, dd, J = 13.5, 6.8 Hz), 2.90-3.05 (3H, m), 3.50-3.57 (1H, m), 4.98 (1H, br s, NH), 5.10-5.21 (2H,

m), 5.78-5.89 (1H, m), 6.42 (1H, d, J = 5.3 Hz), 7.36 (1H, dd, J = 8.9, 1.9 Hz), 7.64 (1H, d, J = 8.9 Hz),

7.96 (1H, d, J = 1.9 Hz), 8.54 (1H, d, J = 5.3 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 20.3, 27.4, 27.5, 33.4,

44.34, 44.35, 54.5, 61.9, 67.4, 99.0, 117.19, 117.23, 120.7, 125.3, 129.0, 134.9, 135.6, 149.1, 149.8,

152.0. IR (cm^-1): ν_max= 2951, 2868, 1611, 1452, 1368, 1136, 918. MS (70 eV): m/z (%) = 344/346

([M+H]⁺, 100). HRMS (ESI) calcd for C₂₀H₂₆ClN₃[M+H]⁺344.1888, found 344.1891. Mp = 154 °C.

White-yellow powder. Yield after automated column chromatography (C18, gradient ACN/H₂O 30/70

to 100/0 with slow gradient of 1.4% per CV between 50 and 100% acetonitrile): 10%.

4.1.6.3. N-[(1-Allyl-3,3-dimethylpiperidin-4-yl)methyl]quinolin-4-amine 16c

¹H NMR (400 MHz, CDCl₃): δ 1.04 (3H, s), 1.06 (3H, s), 1.44-1.52 (1H, m), 1.53-1.63 (1H, m), 1.69 (1H,

d, J = 11.3 Hz), 1.77 (1H, dd, J = 12.9, 2.3 Hz), 1.88 (1H, ddd, J = 11.6, 11.5, 2.3 Hz), 2.50 (1H, d, J =

11.3 Hz), 2.84 (1H, dd, J = 13.4, 6.6 Hz), 2.93 (1H, br d, J = 11.5 Hz), 2.97-3.04 (2H, m), 3.55 (1H, dt, J =

12.7, 4.3 Hz), 4.99 (1H, br t, J = 4.3 Hz, NH), 5.11-5.19 (2H, m), 5.79-5.89 (1H, m), 6.43 (1H, d, J = 5.3

Hz), 7.42 (1H, dd, J = 7.9, 7.6 Hz), 7.63 (1H, dd, J = 8.1, 7.6 Hz), 7.71 (1H, d, J = 7.9 Hz), 7.98 (1H, d, J =

8.1 Hz), 8.56 (1H, d, J = 5.3 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 20.3, 27.4, 27.5, 33.4, 44.31, 44.34, 54.5,

61.8, 67.5, 98.7, 117.1, 118.8, 119.0, 124.6, 129.0, 130.2, 135.7, 148.5, 149.7, 151.1. IR (cm^-1): ν_NH

=

3325, ν_max= 2934, 1574, 1545, 1436, 1375, 764. MS (70 eV): m/z (%) = 310 ([M+H]⁺, 100), 156 (30).

Mp = 134 °C. White-yellow powder. Yield after automated column chromatography (C18, gradient

ACN/H₂O 40/60 to 100/0 with slow gradient of 1.4% per CV): 8%.

4.1.7. Representative procedure for the synthesis of N-[(1-allyl-3,3-dimethylpiperidin-4-

yl)methyl]-1-quinolinylmethanamines 17a-h

The crude mixture of cis-1-allyl-4-aminomethyl-5-benzyloxy-3,3-dimethylpiperidine 14 and 1-allyl-4-

aminomethyl-3,3-dimethylpiperidine 15 (50.9 mg, 0.25 mmol)^a,bwas dissolved in 10 ml dry methanol.

Subsequently, 8-hydroxyquinoline-2-carboxaldehyde 10d (47.6 mg, 0.275 mmol) was added under

argon atmosphere and stirred for 15 hours at room temperature. After confirmation of complete

conversion by LC-MS analysis, NaBH₄(14.2 mg, 0.375 mmol) was added portionwise and stirring was

continued for three hours at room temperature. In case no complete conversion was reached after

three hours, an extra 0.5 equivalents of NaBH₄(4.7 mg, 0.125 mmol) were added. Methanol was

evaporated, and the crude product was resolved in 10 ml dichloromethane, to which 10 ml brine was

added. After a first extraction, the aqueous phase was extracted three times more with 10 ml

dichloromethane. The combined organic phases were dried over MgSO₄, filtrated and evaporated

under reduced pressure. The obtained mixture of two N-[(piperidin-4-yl)methyl]-1-

quinolinylmethanamines was purified by means of automated column chromatography (C18,

gradient MeOH/H₂O 30/70 to 100/0 with slow gradient of 0.625% per CV between 70 and 100%

methanol),

which

resulted

in

9.3

mg

2-{N-[(1-allyl-3,3-dimethylpiperidin-4-

yl)methyl]aminomethyl}quinolin-8-ol 17e (0.028 mmol) in a purity of >95%, and in a yield of 12%

over two steps. The purification conditions for the other N-[(1-allyl-3,3-dimethylpiperidin-4-

yl)methyl]-1-quinolinylmethanamines 17a-d,f-h are specified in the characterization.

^a: The ratio of mixture 14/15 was 1/4, so an average MW of 203.5 g/mol was used for calculations.

^b: For derivative 17h, a mixture in a ratio of 1/2 was used, with an average MW of 192.3 g/mol.

16

4.1.7.1. N-[(cis-1-Allyl-5-benzyloxy-3,3-dimethylpiperidin-4-yl)methyl]-1-(quinolin-4-

yl)methanamine 17a

¹H NMR (400 MHz, CDCl₃): δ 0.94 (3H, s), 1.09 (3H, s), 1.42-1.49 (1H, m), 1.84 (1H, d, J = 11.1 Hz),

1.98-2.10 (1H, m), 2.36 (1H, d, J = 11.1 Hz), 2.75 (1H, dd, J = 11.4, 3.4 Hz), 2.94-3.01 (1H, m), 2.98 (2H,

dd, J = 6.3, 1.0 Hz), 3.05 (1H, dd, J = 11.4, 9.8 Hz), 3.79-3.83 (1H, m), 4.13 (1H, d, J_AB= 14.9 Hz), 4.19

(1H, d, J_AB= 14.9 Hz), 4.35 (1H, d, J_AX= 12.2 Hz), 4.68 (1H, d, J_AX= 12.2 Hz), 5.10-5.22 (2H, m), 5.82-

5.93 (1H, m), 7.15-7.20 (1H, m), 7.23-7.27 (2H, m), 7.29-7.33 (3H, m), 7.49 (1H, ddd, J = 8.4, 7.0, 1.2

Hz), 7.68 (1H, ddd, J = 8.5, 7.0, 1.2 Hz), 7.99 (1H, dd, J = 8.4, 1.2 Hz), 8.10 (1H, dd, J = 8.5, 1.2 Hz), 8.73

(1H, d, J = 4.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 23.7, 28.5, 33.5, 45.8, 48.4, 50.4, 50.9, 55.0, 61.7,

70.9, 73.8, 117.3, 119.5, 123.2, 126.4, 127.0, 127.38, 127.41, 128.3, 129.0, 130.2, 135.6, 139.0, 145.4,

148.1, 150.4. IR (cm^-1): ν_max= 2951, 2868, 2749, 1595, 1462, 1357, 1113, 1069, 756. MS (70 eV): m/z

(%) = 430 ([M+H]⁺, 100). HRMS (ESI) calcd for C₂₈H₃₅N₃O [M+H]⁺430.2853, found 430.2855. Yellow

oil. Yield after automated column chromatography (C18, gradient MeOH/H₂O 30/70 to 100/0 with

slow gradient of 1.25% per CV between 70 and 100% MeOH): 16%.

*Remark: One of the C_arom,quatsignals is missing in ¹³C NMR.

4.1.7.2. N-[(1-Allyl-3,3-dimethylpiperidin-4-yl)methyl]-1-(quinolin-4-yl)methanamine 17b

¹H NMR (400 MHz, CDCl₃): δ 0.905 (3H, s), 0.909 (3H,s), 1.20-1.28 (1H, m), 1.37-1.58 (1H, m), 1.63

(1H, d, J = 11.2 Hz), 1.80-1.91 (2H, m), 2.38-2.46 (2H, m), 2.82 (1H, dd, J = 13.6, 6.7 Hz), 2.89-2.95 (2H,

m), 3.00 (1H, dd, J = 13.6, 5.7 Hz), 4.24 (1H, d, J_AB= 12.8 Hz), 4.29 (1H, d, J_AB= 12.8 Hz), 5.08-5.19 (2H,

m), 5.78-5.89 (1H, m), 7.46 (1H, d, J = 4.4 Hz), 7.57 (1H, ddd, J = 8.4, 6.9, 1.5 Hz), 7.71 (1H, ddd, J =

8.2, 6.9, 1.4 Hz), 8.08 (1H, d, J = 8.4 Hz), 8.13 (1H, d, J = 8.2 Hz), 8.87 (1H, d, J = 4.4 Hz). ¹³C NMR (100

MHz, CDCl₃): δ 20.4, 27.4, 27.6, 33.2, 45.5, 50.5, 50.9, 54.8, 62.0, 67.7, 117.0, 119.6, 123.3, 126.4,

127.1, 129.1, 130.2, 135.9, 145.9, 148.1, 150.4. IR (cm^-1): ν_max= 2947, 2743, 1510, 1462, 916, 754. MS

(70 eV): m/z (%) = 324 ([M+H]⁺, 100). HRMS (ESI) calcd for C₂₁H₂₉N₃[M+H]⁺324.2434, found

324.2434. Yellow oil. Yield after automated column chromatography (C18, gradient MeOH/H₂O

30/70 to 100/0 with slow gradient of 1.25% per CV between 70 and 100% MeOH): 11%.

4.1.7.3. N-[(cis-1-Allyl-5-benzyloxy-3,3-dimethylpiperidin-4-yl)methyl]-1-(quinolin-2-

yl)methanamine 17c

Purity > 85% (based on ¹H NMR analysis).

¹H NMR (400 MHz, CDCl₃): δ 0.91 (3H, s), 1.06 (3H, s), 1.41-1.50 (1H, m), 1.81 (1H, d, J = 11.2 Hz),

1.96-2.06 (1H, m), 2.32-2.45 (1H, m), 2.72 (1H, dd, J = 11.6, 3.4 Hz), 2.77-3.07 (4H, m), 3.82-3.86 (1H,

m), 3.99 (1H, d, J_AB= 14.6 Hz), 4.06 (1H, d, J_AB= 14.6 Hz), 4.43 (1H, d, J_AB= 12.2 Hz), 4.68 (1H, d, J_AB

=

12.2 Hz), 5.07-5.21 (2H, m), 5.79-5.92 (1H, m), 7.15-7.20 (1H, m), 7.23-7.28 (2H, m), 7.31-7.35 (2H,

m), 7.38 (1H, d, J = 8.4 Hz), 7.49 (1H, ddd, J = 8.0, 7.0, 1.0 Hz), 7.67 (1H, ddd, J = 8.3, 7.0, 1.1 Hz), 7.76

(1H, dd, J = 8.0, 1.1 Hz), 8.00 (1H, d, J = 8.3 Hz), 8.02 (1H, d, J = 8.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ

23.8, 28.4, 33.4, 45.4, 48.5, 50.6, 55.3, 56.2, 61.8, 71.0, 73.8, 117.1, 120.3, 120.5, 125.9, 127.2, 127.3,

127.4, 127.5, 128.2, 129.0, 129.3, 135.8, 136.3, 139.3, 147.7. IR (cm^-1): ν_max= 2949, 2868, 1508, 1358,

1112. MS (70 eV): m/z (%) = 430 ([M+H]⁺, 100). Yellow oil. Yield after automated column

chromatography (C18, gradient MeOH/H₂O 30/70 to 100/0 with slow gradient of 0.625% per CV

between 70 and 100% MeOH): 5%.

4.1.7.4. N-[(1-Allyl-3,3-dimethylpiperidin-4-yl)methyl]-1-(quinolin-2-yl)methanamine 17d

¹H NMR (400 MHz, CDCl₃): δ 0.88 (3H, s), 0.89 (3H, s), 1.23-1.31 (1H, m), 1.41-1.53 (1H, m), 1.62 (1H,

d, J = 11.2 Hz), 1.84-1.92 (2H, m), 2.38 (1H, dd, J = 11.4, 9.9 Hz), 2.43 (1H, dd, J = 11.2, 1.7 Hz), 2.81

(1H, dd, J = 13.7, 6.8 Hz), 2.86-2.94 (2H, m), 3.00 (1H, dd, J = 13.7, 5.8 Hz), 4.06 (1H, d, J_AB= 14.5 Hz),

17

4.12 (1H, d, J_AB= 14.5 Hz), 5.08-5.19 (2H, m), 5.78-5.89 (1H, m), 7.46 (1H, d, J = 8.4 Hz), 7.51 (1H, ddd,

J = 8.1, 7.0, 1.1 Hz), 7.69 (1H, ddd, J = 8.4, 7.0, 1.1 Hz), 7.80 (1H, dd, J = 8.1, 1.1 Hz), 8.05 (1H, d, J = 8.4

Hz), 8.11 (1H, d, J = 8.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 20.4, 27.4, 27.5, 33.2, 45.4, 50.6, 54.9, 56.2,

62.1, 67.7, 116.9, 120.5, 126.0, 127.3, 127.6, 129.0, 129.4, 136.0, 136.4, 147.8, 160.6. IR (cm¹): ν_max

=

2947, 2772, 2745, 1601, 1503, 1425, 1126, 916. MS (70 eV): m/z (%) = 324 ([M+H]⁺, 100). HRMS (ESI)

calcd for C₂₁H₂₉N₃[M+H]⁺324.2434, found 324.2430. Yellow oil. Yield after automated column

chromatography (C18, gradient MeOH/H₂O 30/70 to 100/0 with slow gradient of 0.625% per CV

between 70 and 100% MeOH): 26%.

4.1.7.5. 2-{N-[(1-Allyl-3,3-dimethylpiperidin-4-yl)methyl]aminomethyl}quinolin-8-ol 17e

¹H NMR (400 MHz, CDCl₃): δ 0.88 (3H, s), 0.89 (3H, s), 1.23-1.32 (1H, m), 1.42-1.55 (1H, m), 1.64 (1H,

d, J = 11.2 Hz), 1.84-1.92 (2H, m), 2.35-2.47 (2H, m), 2.83 (1H, dd, J = 13.6, 6.8 Hz), 2.86-2.96 (2H, m),

3.01 (1H, dd, J = 13.6, 5.6 Hz), 4.08 (1H, d, J_AB= 15.1 Hz), 4.12 (1H, d, J_AB= 15.1 Hz), 5.08-5.19 (2H, m),

5.79-5.89 (1H, m), 7.16 (1H, d, J = 7.4 Hz), 7.31 (1H, d, J = 7.9 Hz), 7.39-7.44 (1H, m), 7.46 (1H, d, J =

8.4 Hz), 8.11 (1H, d, J = 8.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 20.4, 27.3, 27.5, 33.2, 45.4, 50.4, 54.8,

55.6, 62.0, 67.6, 110.1, 117.1, 117.7, 121.2, 127.1, 127.5, 135.8, 136.6, 137.5, 151.9, 158.1. IR (cm^-1):

ν_max= 2949, 2791, 2745, 1504, 1329, 1246, 833, 750. MS (70 eV): m/z (%) = 340 ([M+H]⁺, 100). HRMS

(ESI) calcd for C₂₁H₂₉N₃O [M+H]⁺340.2383, found 340.2381. Yellow oil. Yield after automated column

chromatography (C18, gradient MeOH/H₂O 30/70 to 100/0 with slow gradient of 0.625% per CV

between 70 and 100% MeOH): 12%.

4.1.7.6. N-[(cis-1-Allyl-5-benzyloxy-3,3-dimethylpiperidin-4-yl)methyl]-1-(2-chloroquinolin-3-

yl)methanamine 17f

¹H NMR (400 MHz, CDCl₃): δ 0.93 (3H, s), 1.08 (3H, s), 1.43-1.51 (1H, m), 1.81-1.87 (1H, m), 1.96-2.15

(1H, m), 2.31-2.47 (1H, m), 2.68 (1H, dd, J = 11.6, 3.4 Hz), 2.94-3.05 (4H, m), 3.79-3.83 (1H, m), 3.87

(1H, d, J_AB= 15.3 Hz), 3.92 (1H, d, J_AB= 15.3 Hz), 4.40 (1H, d, J = 12.2 Hz), 4.70 (1H, d, J = 12.2 Hz),

5.09-5.23 (2H, m), 5.82-5.93 (1H, m), 7.10-7.15 (1H, m), 7.21-7.25 (2H, m), 7.31-7.35 (2H, m), 7.50-

7.54 (1H, m), 7.66-7.71 (2H, m), 7.98-8.00 (1H, m), 8.01 (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ 23.7,

28.5, 33.5, 45.2, 48.4, 50.9, 51.1, 55.1, 61.8, 70.9, 73.5, 117.2, 127.0, 127.35, 127.41, 127.5, 127.8,

128.19, 128.24, 129.8, 132.1, 135.7, 136.9, 139.1, 146.7, 150.6. IR (cm^-1): ν_max= 2951, 2868, 1460,

1327, 1030, 914. MS (70 eV): m/z (%) = 464/466 ([M+H]⁺, 100). Yellow oil. Yield after automated

column chromatography (C18, gradient MeOH/H₂O 30/70 to 100/0 with slow gradient of 0.625% per

CV between 70 and 100% MeOH): 15%.

4.1.7.7. N-[(1-Allyl-3,3-dimethylpiperidin-4-yl)methyl]-1-(2-chloroquinolin-3-yl)methanamine 17g

¹H NMR (400 MHz, CDCl₃): δ 0.89 (3H, s), 0.90 (3H, s), 1.21-1.30 (1H, m), 1.42-1.54 (1H, m), 1.64 (1H,

d, J = 11.2 Hz), 1.82-1.93 (2H, m), 2.37 (1H, dd, J = 10.3, 10.1 Hz), 2.44 (1H, dd, J = 11.2, 1.6 Hz), 2.83

(1H, dd, J = 13.9, 7.6 Hz), 2.85-2.96 (2H, m), 3.01 (1H, dd, J = 13.9, 5.4 Hz), 3.98 (1H, d, J_AB= 15.2 Hz),

4.04 (1H, d, J_AB= 15.2 Hz), 5.08-5.19 (2H, m), 5.78-5.89 (1H, m), 7.56 (1H, ddd, J = 8.0, 7.0, 1.1 Hz),

7.71 (1H, ddd, J = 8.4, 7.0, 1.4 Hz), 7.82 (1H, d, J = 8.0 Hz), 8.01 (1H, d, J = 8.4 Hz), 8.20 (1H, s).

¹³C NMR (100 MHz, CDCl₃): δ 20.4, 27.4, 27.5, 33.2, 45.5, 50.3, 51.2, 54.8, 62.0, 67.7, 117.0, 127.1,

127.36, 127.44, 128.2, 130.0, 132.1, 135.9, 137.2, 146.8, 150.6. IR (cm^-1): ν_max= 2945, 2778, 1329,

1134, 1034, 914, 752. MS (70 eV): m/z (%): 358/360 ([M+H]⁺, 100), 446 (30). HRMS (ESI) calcd for

C₂₁H₂₈ClN₃[M+H]⁺358.2045, found 358.2049. Mp = 50 °C. White-yellow powder. Yield after

automated column chromatography (C18, gradient MeOH/H₂O 30/70 to 100/0 with slow gradient of

0.625% per CV between 70 and 100% MeOH): 9%.

18

4.1.7.8. N-[(cis-1-Allyl-5-methoxy-3,3-dimethylpiperidin-4-yl)methyl]-1-(quinolin-4-

yl)methanamine 17h

¹H NMR (400 MHz, CDCl₃): δ 0.92 (3H, s), 1.01 (3H, s), 1.38-1.42 (1H, m), 1.78 (1H, d, J = 9.6 Hz), 1.97-

2.03 (1H, m), 2.34 (1H, d, J = 9.6 Hz), 2.78 (1H, dd, J = 11.3, 4.1 Hz), 2.91-3.05 (4H, m), 3.31 (3H, s),

3.58 (1H, q, J = 3.6 Hz), 4.26 (1H, d, J_AB= 14.9 Hz), 4.28 (1H, d, J_AB= 14.9 Hz), 5.11-5.20 (2H, m), 5.84-

5.94 (1H, m), 7.47 (1H, d, J = 4.4 Hz), 7.56 (1H, ddd, J = 8.7, 7.0, 1.0 Hz), 7.71 (1H, ddd, J = 8.4, 7.0, 1.1

Hz), 8.10 (1H, dd, J = 8.7, 1.1 Hz), 8.13 (1H, dd, J = 8.4, 1.0 Hz), 8.87 (1H, d, J = 4.4 Hz). ¹³C NMR (100

MHz, CDCl₃): δ 23.6, 28.5, 33.4, 45.8, 48.4, 50.6, 54.7, 57.3, 61.9, 66.3, 76.1, 117.4, 119.8, 123.3,

126.4, 127.1, 129.1, 130.2, 135.5, 146.0, 148.2, 150.4. IR (cm^-1): ν_max= 2928, 2868, 1593, 1508, 1462,

1364, 1111, 1082, 754. MS (70 eV): m/z (%) = 354 ([M+H]⁺, 100). HRMS (ESI) calcd for C₂₂H₃₁N₃O

[M+H]⁺354.2540, found 354.2538. Yellow oil. Yield after automated column chromatography (C18,

gradient ACN/H₂O 30/70 to 100/0): 6%.

4.2. Biological evaluation

4.2.1. In vitro antiplasmodium assay

The test samples were prepared to a 20 mg/ml stock solution in 100% DMSO. Stock solutions were

stored at -20 °C. Further dilutions were prepared in complete medium on the day of the experiment.

Chloroquine (CQ) was used as the reference drug. A full dose-response was performed to determine

the concentration inhibiting 50% of parasite growth (IC₅₀value). Test samples were tested at a

starting concentration of 10 µg/ml (or 1 µg/ml), which was then serially diluted 2-fold in complete

medium to give 10 concentrations, with the lowest concentration being 20 ng/ml (or 2 ng/ml). The

same dilution technique was used for all samples. The highest concentration of solvent to which the

parasites were exposed to had no measurable effect on the parasite viability (data not shown). The

50% inhibitory concentration (IC₅₀) values were obtained from full dose-response curves, using a non-

linear dose-response curve fitting analysis via GraphPad Prism v.4 software.

4.2.2. In vitro assay for the evaluation of cytotoxic activity

The same stock solutions prepared for antiplasmodium testing were used for cytotoxicity testing.

Test compounds were stored at -20 °C until use. Dilutions were prepared on the day of the

experiment. Emetine was used as the reference drug in all experiments. The initial concentration of

emetine was 100 µg/ml, which was serially diluted in complete medium with 10-fold dilutions to give

6 concentrations, the lowest being 0.001 µg/ml. The same dilution technique was applied to all the

test samples. The highest concentration of solvent to which the cells were exposed to had no

measurable effect on the cell viability (data not shown). The 50% inhibitory concentration (IC₅₀)

values were obtained from full dose-response curves, using a non-linear dose-response curve fitting

analysis via GraphPad Prism v.4 software.

Transparency declarations

The authors declare no conflict of interest.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships

that could have appeared to influence the work reported in this paper.

Acknowledgements

19

The authors are indebted to the Special Research Fund (BOF) of Ghent University for financial

support. The University of Cape Town, South African Medical Research Council, and South African

Research Chairs Initiative of the Department of Science and Technology, administered through the

South African National Research Foundation are gratefully acknowledged for support (K.C.).

Appendix A. Supplementary data

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23

Highlights

1. Four different series of new quinoline analogues decorated with a (monocyclic or bridged)

piperidine-containing side chain were synthesized

2. Antiplasmodium assessments in NF54 (CQ-sensitive) and K1 (CQ-resistant) strains of P.

falciparum and cytotoxic evaluation in CHO cells were performed

3. Five 4-aminoquinoline-piperidine compounds displayed nanomolar activities against both NF54

and K1 and no CHO toxicity, and were thus identified as promising hits

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships

that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered

as potential competing interests:

Article Doi

Synthesis and biological evaluation of novel quinoline-piperidine scaffolds as antiplasmodium agents

DOI: 10.1016/j.ejmech.2020.112330

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