Synthesis, characterization and in vitro evaluation of novel enantiomerically-pure sulphonamide antimalarials

Article abstract of DOI:10.1039/c5ob01479d

Malaria parasites are currently gaining drug-resistance rapidly, across countries and continents. Hence, the discovery and development of novel chemical scaffolds, with superior antimalarial activity remain an important priority, for the developing world. Our report describes the development, characterization and evaluation of novel bepotastine-based sulphonamide antimalarials inhibiting asexual stage development of Plasmodium falciparum parasites in vitro. The screening results showed potent inhibitory activity of a number of novel sulphonamides against P. falciparum at low micromolar concentrations, in particular in late-stage parasite development. Based on computational studies we hypothesize N-myristoyltransferase as the target of the compounds developed here. Our results demonstrate the value of novel bepotastine-based sulphonamide compounds for targeting the asexual developmental stages of P. falciparum.

Full text of DOI:10.1039/c5ob01479d

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H. Ximei, P. R. Preiser and S. NanjundaSwamy, Org. Biomol. Chem., 2015, DOI: 10.1039/C5OB01479D.
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Page 1 of 27
Organic & Biomolecular Chemistry
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DOI: 10.1039/C5OB01479D
Synthesis, characterization and in vitro evaluation of novel enantiomerically-pure
sulphonamide antimalarials
1$
2$
1
2
3
Sebastian Anusha , Ameya Sinha , Babu Rajeev C.P , Trang TT Chu , Jessin Mathai , Huang
4
5,6
7
5
4
Ximei , Julian E Fuchs , NanjundaSwamy Shivananju, Andreas Bender , Peter Rainer Preiser ,
7
1
2
Kanchugarakoppal S. Rangappa , Basappa *, Rajesh Chandramohanadas *
1
2
Laboratory of Chemical Biology, Department of Chemistry, Bangalore
University, Bangalore, India
Pillar of Engineering Product Development, Singapore University of Technology & Design,
Singapore.
3
4
5
Gulf Medical University, Ajman, United Arab Emirates
School of Biological Sciences, Nanyang Technological University, Singapore.
Centre for Molecular Science Informatics, Department of Chemistry, University of Cambridge,
Lensfield Road, CB2 1EW, Cambridge, United Kingdom.
6
7
Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innrain 82,
6
020 Innsbruck, Austria
Department of Studies in Chemistry, University of Mysore, Mysore, India
$
*
These authors contributed equally to this work.
Correspondence: Basappa*(salundibasappa@gmail.com); Rajesh Chandramohanadas*(rajesh@sutd.edu.sg)

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Abstract
Malaria parasites currently gain resistance rapidly, across countries and continents. Hence, the discovery
and development of novel chemical compounds, as well as scaffolds, with superior antimalarial activity
remains an important priority, not only for the developing world. Our report describes the development,
characterization and evaluation of novel bepotastineꢀbased sulphonamide antimalarials inhibiting asexual
stage development of Plasmodium (P.) falciparum parasites in vitro. Screening results showed potent
inhibitory activity of a number of novel sulphonamides against P. falciparum at low micromolar
concentrations, in particular in lateꢀstage parasite development, as well as selectivity over mammalian cells.
Based on computational studies we hypothesize Nꢀmyristoyltransferase as the target of the compounds
developed here. Our results demonstrate the value of novel bepotastineꢀbased sulphonamide compounds for
targeting th asexual developmental stages of P. falciparum.
Keywords: Plasmodium falciparum, Antiꢀmalarials, in vitro screening, Drug Discovery, Giemsaꢀstaining,
Microscopy, Flow cytometry,

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Background
Malaria caused by parasitic protozoans of the family Plasmodium continues to be a major threat to millions
of people across mainly Africa, India, and South East Asia. Based on recent reports, at least 700,000
people, the majority of which are children under the age of 8, die every year due to malariaꢀassociated
pathology [1]. There are 4 major classes of plasmodia that cause human malaria: Plasmodium (P.)
falciparum, P. vivax, P. ovale and P. malariae. Among these, P. falciparum and P. vivax are the most
commonly found parasitic variants in Africa and Asia, respectively [2]. Current treatment for malaria relies
on the use of a series of chemotypes that operate through distinct mechanisms impairing parasitic survival
within human hosts [3-4], primarily by targeting pathways directly related to hemoglobin degradation.
However, drug resistant strains for all of the common antimalarials including artemisinin are currently
emerging [5-6]. Of late, given the emergence of multidrugꢀresistant malaria, even combination therapy
shows to be ineffective in many situations as well [2]. Thus, the identification of new chemical entities
targeting specific stages of parasite development is of extreme importance to global human health.
Classical antiꢀmalarial drugs include chloroquine, artemisinin and pyrimethamine. In combination with
pyrimethamine, folic acid antagonists such as sulfadoxine were and are widely used for malaria treatment;
however resistance against this combination has been reported extensively [7]. Histamineꢀ1 receptor
antagonists like bepotastine were shown to be effective against the proliferation of Plasmodia both in vitro
and in vivo [8]. Thus, we in this work designed and evaluated novel chemical scaffolds, which combine
structural features of both sulphonamides and bepotastine, for their efficacy against the asexual
development stages of P. falciparum. Our results revealed efficient inhibition of parasite development and
replication by these compounds at low micromolar concentrations, interfering at the final stages of intraꢀ
erythrocytic life cycle progression. These molecules showed low toxicity to mammalian cells (>10 fold
selectivity), and therefore represent potential lead scaffolds for developing novel antiꢀmalarials.
Compound Synthesis and Characterization
We previously developed many new molecules of diverse structure that targets cancer pathways [9-14].
Here, to synthesize the reported compounds, we used commercially available 2ꢀbromopyridine and 4ꢀ
chlorobenzaldehyde to yield racemic (4ꢀchlorophenyl)methyl)(pyridineꢀ2ꢀyl)methanol (1) via Grignard

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reaction [15], which upon reacting with 1ꢀbenzylꢀ4ꢀchloropiperidine (2) in DMF using potassium
carbonate as base under reflux condition resulted in the racemic compound 2ꢀ((1ꢀbenzylpiperidinꢀ4ꢀ
yloxy)(4ꢀchlorophenyl)methyl)pyridine (3). Compound 3 was then subjected to debenzylation followed by
resolution using R(ꢀ)/S(+) mandelic acid in methanol to yield 2ꢀ((4ꢀchlorophenyl)(piperidinꢀ4ꢀyloxy)
methyl)pyridine (4a-b). Other reported procedures for the synthesis of 2ꢀ((4ꢀchlorophenyl)(piperidinꢀ4ꢀ
yloxy) methyl)pyridine in literature mostly involve the use of organolithium reagents and other extreme
conditions, such as longer reaction times and low temperature [16]. The resolution using mandelic acid
was less laborious compared to other resolution methods eg. where repeated crystallizations were involved
in achieving the required chiral purity [17]. The reaction of chirally pure (4a-b) with different
commercially available benzene sulfonyl chlorides (5a-i) in the presence of triethylamine and
dichloromethane at room temperature gave sulphonamides (6a-r) in 82ꢀ90% yield (Scheme 1). The
1
13
compounds (6a-r) were characterized by melting point, LCꢀMS, HRMS, H NMR, C NMR, and chiral
HPLC analysis (Supplementary Information, S1).
In vitro screening against Plasmodium falciparum parasites
To identify active molecules among the newly synthesized sulphonamides, we initially chose to conduct a
preꢀscreening against laboratory strains (3D7) of P. falciparum. Three different concentrations of the
compounds dissolved in DMSO were added to actively metabolizing parasites (at trophozoite stage, ~30
hours post invasion (hpi)). DMSOꢀtreated parasites were included as negative controls. 48h postꢀtreatment,
parasitemia was scored (after staining with Hoechst 33342) using a standard flow cytometric method [18].
Giemsaꢀstained smears prepared in parallel were examined microscopically to validate the flow cytometry
results. From this, we were able to identify ten molecules (6b, 6c, 6e, 6f, 6g, 6k, 6l, 6n, 6p & 6r) with
significant inhibitory activity at 20 ꢁM against P. falciparum (Supplementary Figure S2), which were
chosen for subsequent experiments.
In order to accurately determine inhibitory potential of the compounds, trophozoite stage parasites (3D7)
were treated at concentrations ranging from 1.6 ꢁM to 200 ꢁM. DMSOꢀtreated and chloroquineꢀtreated
parasites served as negative and positive controls respectively. After 48h of incubation with the inhibitors,
parasites were fixed with gluteraldehyde and stained with Hoechst for flow cytometry. The sensitivity
profiles obtained against 3D7 and chloroquineꢀresistant (K1) strains are provided in Figures 1A and 1B,

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and corresponding IC50 values are summarized in Table 1. Based on these results, three of the tested
compounds (6e, 6k, 6n) showed inhibitory activity in the low micromolar range (<4ꢁM), while all the other
compounds (6b, 6c, 6f, 6g, 6l, 6p, 6r) were effective only below 15ꢁM. Apart from 6k, all other compounds
showed comparable inhibitory effect on both parasites (Table 1). Cytotoxicity assays (MTT) [19] were
performed for all ten selected compounds using a MadinꢀDarby Canine Kidney (MDCK) cell line. For all
compounds except 6c, a selectivity (IC50 MDCK/IC50 3D7) of larger than 10ꢀfold was observed (see Table
1
and Supplementary Figure S3). Compound 6n turned out to be most selective, with a selectivity index
of 92.
To further examine putative biological mechanisms that these molecules are interfering with, we performed
experiments probing their stageꢀspecific effects. In order to do this, parasites were continuously treated
with each compound (6b, 6c, 6e, 6f, 6g, 6k, 6l, 6n, 6p, 6r) at various lifeꢀstages (ring, trophozoite and
schizonts). In brief, parasites were treated at 4*IC , 2*IC , IC and 0.25*IC (as determined from Table
50
50
50
50
1
) and monitored using Giemsaꢀstained images and flow cytometry counting. The results are displayed in
Figures 2A, 2B and 2C for the different development stages. It can be seen that most of the compounds
showed an effect on schizont to ring transition at concentrations at IC50 and at 2* IC . This means that
50
these molecules were primarily interfering with the late stages of merozoite development and merozoite
rupture.
Giemsaꢀstained smears from each experiment were examined for specific phenotypes in order to
understand the lead compound (6n) action further. A representative panel is shown in Supplementary
Figure S4. Uninfected RBCs looked morphologically indistinct even after addition of the compound.
Controlꢀtreated (DMSO) parasites developed as anticipated over 46 hours post invasion to form
multinucleated schizonts. However, parasites treated with the compounds presented here appear to become
trapped in a unique phenotype whereby they fail to segment out into multiple schizonts. At 52 hours post
invasion, all DMSOꢀtreated parasites had egressed and formed ringꢀstage parasites, whereas inhibitorꢀ
treated parasites appear to remain lockedꢀin, thereby resulting in parasite death.
Based on the screening results, most of the R and S forms of 2ꢀ((4ꢀchlorophenyl)(piperidinꢀ4ꢀ
yloxy)methyl)pyridine (CPMP) attached to the substitutedꢀphenyl sulphonyls showed significant inhibition
of malaria growth. Molecules with 2,5ꢀdichloro, 4ꢀnitro, 2,4,6ꢀtrymethyl, and 2ꢀnitroꢀsubstituted phenyl

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sulphonyl groups attached to CPMP inhibited parasite growth with IC50 values of 2.1, 3.2, 7.4, and 13.33
M, respectively. The IC50 value for the positive control sulfadoxine is 1 ꢁM, and it is hence comparable in
ꢁ
activity to the newer scaffolds presented here. On the other hand, the R forms of 4ꢀmethoxyꢀ as well as the
S forms of 4ꢀmethylꢀsubstituted phenylsulphonyl groups were found to have slightly higher IC values, of
50
6
.8 and 8.8ꢁM respectively. Additionally, 3ꢀnitro, and 3ꢀtrifluoromethylꢀsubstituted phenyl sulphonyls
attached to CPMP significantly reduced antimalarial properties. To evaluate potential in vivo efficacy of
the most effective compound 6n, BALB/c mice infected with P. yoelii, were given a daily dosage of 10
mg/kg body weight or 50mg/kg body weight via i.p. injection. The compound only had mild inhibitory
effect on parasite replication (see Supplementary Figure S5).
In silico mode-of-action analysis
Aiming to rationalize antimalarial activity of the bepotastineꢀbased sulfonamides, we searched the Protein
Data Bank [20] for coꢀcrystal structures of aromatic sulfonamide ligands bound to proteins of plasmodial
origin and retrieved two hits. Based on ligand similarity beyond the sulfonamide functionality we decided
to investigate Nꢀmyristoyltransferase. Further, given that it has recently been validated as a novel
antimalarian drug target in Plasmodium vivax by Wright et al. [21]. In this work it was shown on a
structural basis that aromatic sulfonamides are capable of inhibiting Nꢀmyristoyltransferase, and hence we
chose the published coꢀcrystal structure of enzyme and sulfonamide inhibitor as the basis for our in silico
analysis (PDB 2YND). Induced fit docking of our series of sulfonamides to P. vivax Nꢀmyristoyltransferase
yielded consistent binding modes, in contrast to static docking runs that often led to severely distorted
ligand conformations. Additionally, flexible docking enabled successful redocking of the cognate ligand
with an RMSD <0.6Å.
In silico docking predicted our compounds to occupy a very similar region of the binding pocket (see
Figure 3). The inverted sulfonamide functionality allows accommodation of the substituted benzene in the
region occupied by the pyrazole ring of the cocrystallized ligand (Figure 3A). As this region is particularly
involved in πꢀπ interactions with Pheꢀ103 and Pheꢀ105, the steric and electronic structure of the substituted
benzenes is deemed crucial for optimal molecular interactions [22]. The Yꢀshaped tail group of the ligands
is predicted to overlap with the long hydrophobic stretch of the coꢀcrystallized ligand, thereby filling major

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parts of the enzyme's binding cavity and forming multiple additional hydrophobic contacts (Figures 3B
and 3C). Moreover, we find polar contacts to Serꢀ319 as well as contacts to the Cꢀterminal Leuꢀ410 for
some ligands. Several structural water molecules are involved in anchoring the polar interaction sites in the
binding site. Docking scores of our ligands were found in similar range as the cognate ligand, thus pointing
towards similar interaction energies.
Summary
In this work, we report the synthesis, characterization and in vitro testing of a novel library of
sulphonamides for their antimalarial activity. To this end, number of bepotastineꢀbased sulphonamides
were synthesized, as both of these compound classes are known to efficiently inhibit parasitic development
[
7, 8]. Subsequently, these compounds were tested to establish their inhibitory potential on asexual stages
of human malaria parasite. To prioritize potential antimalarial scaffolds within the synthesized library, we
conducted preliminary screening experiments, based on which the ten most active molecules were selected
for detailed analyses. It was found in doseꢀresponse experiments that the majority of the selected
compounds killed malaria parasites at lower micromolar concentrations, were in most (but not all) cases
comparably effective on regular and drugꢀresistant parasitic variants, and demonstrated selectivity over
mammalian cells (>10ꢀfold, and up to 92ꢀfold). The compounds reported here in general appeared to
primarily manipulate later stages of asexual development, based on preliminary phenotypic screening
results reported. These phenotypes were, however, not entirely comparable to typical egressꢀimpaired
phenotypes induced by serine/cysteine protease inhibitors [23]. Based on computational work we
hypothesize the potential target for the compounds reported here to be Nꢀmyristoyltransferase. Further
work is needed to accurately document the precise molecular mechanism, and to ensure that these
molecules are indeed interfering with this molecular target.
Materials and Methods
1
All solvents used were of analytical grade and reagents used were purchased from SigmaꢀAldrich. H and
1
3
C NMR spectra were recorded on a Bruker WHꢀ200 (400 mHz or 300 mHz) spectrometer in CDCl or
3
DMSOꢀd as solvent, using TMS as an internal standard and chemical shifts are expressed as ppm. High
6
resolution mass spectra were determined on a Bruker Daltonics instrument. SOR was recorded using

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Rudolph autopol IV polarimeter. The progress of the reaction was monitored by TLC preꢀcoated silica gel
G plates.
Synthesis & Characterization
Typical procedure for the synthesis of (4-chlorophenyl)methyl)(pyridine-2-yl)methanol (1): In a
3
00mL round bottomꢀ3ꢀneck flask fitted with a mechanical stirrer, a reflux condenser, and an immersion
thermometer were charged32 mL of tisopropyl magnesium bromide (1M in THF, 0.03 mol) under nitrogen
0
atmosphere. The reaction mass was cooled to 10 C and 2ꢀbromopyridine 5g (0.03 mol) was added slowly
0
maintaining the temperature below 20 C. Then the reaction mass was brought to room temperature and
stirred for 1hr. 4ꢀchlorobenzaldehyde 4.4g (0.03 mol) was added drop wise and the reaction mixture was
stirred for 4h until completion of the reaction was observed by TLC. The reaction was quenched with
ammonium chloride and the organic layer was separated, dried over sodium sulphate and concentrated
under reduced pressure. The obtained crude product was purified by flash column chromatography in
1
hexane/ethyl acetate.Yield (6g, 86%); H NMR (300 MHz, CDCl ) 8.58 (d, 1H), 8.57ꢀ7.20(m, 7H), 5.75
3
+
(
s,1H), 1.60(s, 1H); LCMS (MM:ES+APCI) 220.3(M+H) ; C H ClNO: C, 65.61; H, 4.59; N, 6.38;
12
10
Found: C, 65.78; H, 4.69; N, 6.32.
Typical
procedure
for
the
synthesis
of
2-((1-benzylpiperidin-4-yloxy)(4-
chlorophenyl)methyl)pyridine (3): 5g (22.7mmol) of 1 and 5.7g (27.3mmol) of 1ꢀbenzylꢀ4ꢀ
chloropiperidine (2) was dissolved in 30mL of DMF. Dried Potassium carbonate 12.56g (91.04mmol) was
charged to the above reaction mass in a round bottom flask equipped with a magnetic stir bar. The reaction
mass was heated to reflux for 20h. Completion of the reaction was monitored by TLC. The reaction mass
was quenched to water extracted with ethyl acetate 80mL three times. The combined organic phase was
subjected to brine wash to remove DMF, dried over anhydrous sodium sulphate and concentrated under
vacuum. The crude product (3) was flash chromatographed on a short silica gel column in hexane/ethyl
1
acetate. Yield (8.1g, 90.8%).%) H NMR (300 MHz, CDCl ) 8.48ꢀ7.25 (m, 13H), 5.69(s, 1H), 3.97 (s,2H),
3
+
3
.64(m, 1H), 3.14(m, 2H), 2.92(m, 2H), 1.96(m, 2H), 1.74(m, 2H); LCMS (MM:ES+APCI) 394.4(M+H) ;
Anal.Calcd for C H ClN O: C 73.36; H 6.41; N 7.13; Found: C, 73.26; H, 6.45; N, 7.16.
2
4
25
2

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Typical procedure for the synthesis of 2-((4-chlorophenyl)(piperidin-4-yloxy) methyl)pyridine (4):
The compound 3 (7g) obtained in the above process was diluted in 200 ml methanol and transferred
under nitrogen atmosphere into an autoclave, charged with 5% Pd/C catalyst (0.8g). The mixture was
pressurized with hydrogen gas at 40psi maintaining the reaction temperature below 30°C. Hydrogen
uptake initially was ~40 psi per 30 min for about 4h. The uptake reduced to ~40 psi per 2 to 3 h. The
reaction was continued for 16 h until the hydrogen uptake stopped. TLC analyses were performed to check
for completion of the reaction. The reaction mixture was cooled, and the catalyst was filtered off. The
methanolic solution was dried over sodium sulphate and concentrated to dryness.
1
2
-((4-chlorophenyl)(piperidin-4-yloxy)methyl)pyridine (4): Yield (5g, 94%) H NMR (300 MHz,
CDCl3) 8.50ꢀ7.12 (m, 8H), 5.64(s, 1H), 3.53 (m,1H), 3.09(m, 2H), 2.59ꢀ2.46 (m,3H,ꢀCH ,ꢀ
2
1
3
NH) 1.9(m, 2H), 1.6(m, 2H); C NMR (75 MHz, CDCl ) 162.04, 148.82, 140.37, 136.94, 133.20,
3
1
28.48, 128.21, 122.46, 120.64, 80.59, 73.76, 44.17, 32.94, 32.91, 29.66; DEPT attached; LCMS (MM:
+
ES+APCI) 303 (M+H) ; Chiral HPLC (% ee) 49.91: 50.09.
Typical procedure for the separation of the R and S forms of 4: 5 g of the syrupy racemic 2ꢀ((4ꢀ
0
chlorophenyl)(piperidinꢀ4ꢀyloxy)methyl)pyridine (4) was taken in 15ml methanol and heated to 60 C, a hot
solution of S(+) mandelic acid (2eq) in 10ml methanol was added and entire reaction mass was refluxed
for 2h. The reaction mixture was concentrated to half the volume and the S(ꢀ)ꢀ2ꢀ((4ꢀ
chlorophenyl)(piperidinꢀ4ꢀyloxy)methyl)pyridine (4b) was precipitated out by adding 2ꢀpropanol. The
maximum chiral purity of the S isomer was achieved by recrystallization from methanol/isopropyl alcohol
mixture. The mother liquor collected after the filtration of the S isomer was concentrated to dryness and the
crude mass was treated in the same mannr as above with R(ꢀ)mandelic acid to separate the R(+)ꢀ2ꢀ((4ꢀ
chlorophenyl)(piperidinꢀ4ꢀyloxy)methyl)pyridine (4a).
Characterization of (4a): Yield (2g, 40%). Anal.Calcd for C H ClN O: C 67.43; H 6.32; N 9.25; Found:
17
19
2
C, 67.49; H, 6.28; N, 9.29; Chiral HPLC (%ee) 98.4; [α] = +11(C 0.05, MeOH);
D
Characterization of (4b): Yield 2.1g, 42%); Anal.Calcd for C H ClN O: C 67.43; H 6.32; N 9.25; Found:
17
19
2
C, 67.38; H, 6.37; N, 9.24; Chiral HPLC (%ee) 100; [α] = ꢀ11(C 0.05, MeOH).
D

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General procedure for the synthesis of 6(a-r): The salt forms of the compounds (4a-b) were made free
bases as follows: The salt was taken in dichloromethane (DCM) in a beaker, cooled in an ice bath and
basified using aqueous sodium hydroxide (NaOH) solution (6M) until the pH reached ~12. The free amine
was extracted two times using DCM, the combined organic phase was washed with water, followed by
brine solution. The entire organic phase was dried over anhydrous sodium sulphate and concentrated under
vacuum .200mg (0.6mmol) of free amine was dissolved in 5 mL DCM. Triethylamine (0.5 mL) was added
to the above reaction mass in a round bottom flask equipped with a magnetic stir bar. Sulphonyl chloride
0
(
5a-i) (0.65mmol) were added to the reaction mass and stirred at 30 C for 14 h. Completion of the reaction
was monitored by TLC analysis. The reaction mass was quenched with water and then extracted with 10 ml
DCM three times. The combined organic phase was subjected to saturated sodium bicarbonate wash, brine
wash, dried over anhydrous sodium sulphate, and concentrated under vacuum. The crude product (6a-r)
was flash chromatographed on a short silica gel column in hexane/ethyl acetate.
Characterization
of
(R)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((3ꢀ
nitrophenyl)sulfonyl)piperidinyl)oxy)methyl)pyridine (6a): Yield (270mg, 84.3%); Tan colored solid;
0
mp: 78 C; 1HꢀNMR (400 MHz, DMSOꢀd ) 8.55ꢀ 8.53(d,J=8.0Hz,1H), 8.43ꢀ 8.42(d, J=4.0Hz,1H), 8.35
6
(
s,1H),
8.17ꢀ
8.15
(d,J=8.0Hz,1H),
7.95ꢀ
7.91(t,J=8.0Hz,1H),7.75ꢀ7.71(m,1H),
7.42ꢀ
7
1
1
3
4
.40(d,J=8.0Hz,1H),7.31ꢀ7.21(m, 5H), 5.59(s, 1H), 3.45ꢀ3.41(m, 1H), 3.25ꢀ3.23(m, 2H), 2.83ꢀ 2.78(m, 2H)
.89ꢀ 1.88 (m, 2H) 1.67ꢀ 1.60 (m, 2H); 13CꢀNMR ( DMSOꢀd );161.53, 149.29, 148.53, 140.98, 137.58,
6
37.51, 133.83, 132.45, 131.97, 129.11, 128.64, 128.18, 123.10, 122.43, 120.72, 80.18, 71.11, 43.74,
+
0.53, 30.38; HRMS Calcd 510.086; Found: 510.086(M+Na ); Anal.Calcd for C H ClN O S : C 56.61; H
23
22
3
5
.54; N 8.67; Found: C, 56.63; H, 4.57; N, 8.65; Chiral HPLC (%ee) 99.9.
of
Characterization
(R)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((4ꢀ
nitrophenyl)sulfonyl)piperidinꢀ4ꢀyl)oxy)methyl)pyridine (6b): Yield (287mg, 89.6%); Tan coloured solid;
0
mp: 110 C; 1HꢀNMR (400 MHz, DMSOꢀd ) 8.45ꢀ 8.44 (d,J=4Hz,1H), 7.81ꢀ 7.73(m,3H), 7.45ꢀ 7.35
6
(
m,7H), 7.25ꢀ 7.22(d,J=8.0Hz,1H), 5.59(s, 1H), 3.47ꢀ 3.39(m, 1H), 3.04ꢀ 3.02(m, 2H), 2.78ꢀ 2.76(m, 2H),
1
1
.86ꢀ 1.84 (m, 2H), 1.66ꢀ 1.62 (m, 2H); 13CꢀNMR ( DMSOꢀd );161.67, 149.11, 148.63, 140.38, 137.58,
6
37.45, 133.13, 132.45, 132.09, 129.11, 128.64, 128.13, 123.39, 122.43, 120.12, 80.09, 71.01, 43.74,

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+
3
0.50, 30.22; HRMS Calcd 510.086; Found: 510.086 (M+Na ); Anal.Calcd for C H ClN O S : C 56.61;
23
22
3
5
H 4.54; N 8.67; Found: C, 56.64; H, 4.53; N, 8.64; [α] = ꢀ37.6(C 0.01, MeOH);
D
Characterization
of
(R)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((2ꢀ
nitrophenyl)sulfonyl)piperidinꢀ4ꢀyl)oxy)methyl)pyridine (6c): Yield (267mg, 83.4%); Tan coloured solid;
0
mp: 85 C; 1HꢀNMR (400 MHz, DMSOꢀd ) 8.43ꢀ 8.41 (d,J=8Hz,1H), 7.73ꢀ 7.70(m,1H), 7.60ꢀ 7.58
6
(
d,J=8.0Hz, 2H), 7.43ꢀ 7.32(m, 3H),7.30ꢀ 7.21 (m, 5H), 5.57(s, 1H), 3.35(m, 1H), 3.11ꢀ 3.07(m, 2H), 2.73ꢀ
1
3
2
1
7
.69(m, 2H), 1.84ꢀ 1.83 (m, 2H), 1.64ꢀ 1.61 (m, 2H); C NMR ( DMSOꢀd );161.55, 149.29, 148.53,
6
40.98, 137.18, 137.11, 133.83, 132.48, 131.97, 129.11, 128.64, 128.18, 123.10, 122.43, 120.72, 79.99,
+
1.74, 43.84,
30.54, 30.28; HRMS Calcd 510.086; Found: 510.086(M+Na ); Anal.Calcd for
C H ClN O S : C 56.61; H 4.54; N 8.67; Found: C, 56.64; H, 4.55; N, 8.69; Chiral HPLC (%ee) 96.0
23
22
3
5
Characterization of
(R)ꢀ2ꢀ((chlorophenyl)((1ꢀ((3ꢀ (trifluoromethyl)phenyl)sulfonyl)piperidinꢀ4ꢀ
0
1
yl)oxy)methyl)pyridine
(6d): Yield (290mg, 86.5%); Offꢀwhite solid; mp: 108 C; HꢀNMR (400 MHz,
DMSOꢀd ) 8.43ꢀ 8.42(d, J=4Hz,1H), 8.14ꢀ 8.12(d, J=8.0Hz, 1H), 8.07ꢀ 8.05(d, J=8.0Hz, 1H), 7.94ꢀ 7.90(m,
6
2
2
1
1
5
5
H),7.72ꢀ 7.70 (m, 1H),7.40ꢀ 7.38(d, J=8Hz,1H),7.30ꢀ 7.21(m,5H), 5.59(s, 1H), 3.47(m, 1H), 3.19ꢀ 3.16(m,
H), 2.84ꢀ 2.82(m, 2H), 1.88ꢀ 1.86 (m, 2H), 1.64ꢀ 1.62 (m, 2H); 13CꢀNMR ( DMSOꢀd );161.56, 149.29,
6
41.02, 137.46, 137.23, 132.43, 132.00, 131.56, 130.64, 130.38, 130.35, 130.31, 129.07, 128.61, 125.18,
24.15, 124.11, 123.08, 122.47, 120.63, 80.12, 70.96, 43.60, 30.46, 30.31; HRMS Calcd 511.107; Found:
11.1067(M+H) Anal.Calcd for C H ClFN O S : C 56.42; H 4.34; N 5.48; Found: C, 56.39; H, 4.37; N,
24
22
3
3
.47; [α] = ꢀ39.7(C 0.01, MeOH);
D
Characterization
of
(R)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((2,5ꢀ
dichlorophenyl)sulfonyl)piperidinꢀ4ꢀ
yl)oxy)methyl)pyridine (6e): Yield (296 mg, 88.3%); Brown low melting solid; 1HꢀNMR (400 MHz,
DMSOꢀd ) 8.44ꢀ 8.42(d, J=8.0Hz,1H), 7.73ꢀ 7.70(m,1H), 7.60ꢀ 7.58 (d, J=8.0Hz, 1H), 7.43ꢀ 7.32(m,
6
3
H),7.30ꢀ 7.21 (m, 5H), 5.57(s, 1H), 3.36(m, 1H), 3.11ꢀ 3.07(m, 2H), 2.74ꢀ 2.69(m, 2H), 1.84ꢀ 1.83 (m,
2
H), 1.63ꢀ 1.62 (m, 2H); 13CꢀNMR ( DMSOꢀd );160.59, 151.29, 150.53, 139.96, 137.48, 137.11, 133.83,
6
1
32.48, 131.43, 129.18, 128.83, 128.19, 123.04, 122.38, 80.18, 73.64, 43.81, 30.78, 30.57; HRMS Calcd
11.0417; Found: 511.0411(M+H); Anal.Calcd for C H Cl N O S : C 53.97; H 4.14; N 5.47; Found:
5
23
21
3
2
3
C,53.94 ; H, 4.19; N, 5.50; Chiral HPLC (%ee) 98.7.

Organic & Biomolecular Chemistry
Page 12 of 27
View Article Online
DOI: 10.1039/C5OB01479D
Characterization
of
(R)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((4ꢀmethoxyphenyl)sulfonyl)piperidinꢀ4ꢀ
0
1
yl)oxy)methyl)pyridine (6f): Yield (260 mg, 83.8%); Offꢀwhite solid; mp: 114 C; HꢀNMR (400 MHz,
DMSOꢀd ) 8.44ꢀ8.43 (d, J=4Hz, 1H), 7.74ꢀ7.70 (m,1H), 7.65ꢀ 7.63 (d, J=8.0Hz, 2H), 7.39ꢀ7.37 (d,
6
J=8.0Hz, 1H), 7.32ꢀ7.22 (m, 5H), 7.14ꢀ7.12 (d, J= 8.0 Hz, 1H), 5.56 (s, 1H), 3.84 (s, 3H), 3.44 (m, 1H),
3
1
7
.07 (m, 2H), 2.73ꢀ 2.69(m, 2H), 1.84ꢀ 1.83 (m, 2H), 1.64ꢀ 1.62 (m, 2H); 13CꢀNMR ( DMSOꢀd ); 162.96,
6
61.06, 148.99, 141.00, 137.40, 132.41, 130.05, 129.09, 128.61, 126.90, 123.17, 120.72, 115.00, 79.88,
+
1.16, 55.99, 43.52, 30.42, 30.21; HRMS Calcd 495.112; Found: 495.112 (M+Na ); Anal.Calcd for
C H ClN O S : C 60.94; H 5.33; N 5.92; Found: C, 60.98; H, 5.31; N, 5.96; [α] = ꢀ40.5(C 0.01, MeOH).
24
25
2
4
D
Characterization of (R)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ(mesitylsulfonyl)piperidinꢀ4ꢀ yl)oxy)methyl)pyridine (6g):
0
1
Yield (280mg, 88%); White solid; mp: 106 C; HꢀNMR (400 MHz, DMSOꢀd ) 8.46ꢀ 8.45 (d, J=4Hz, 1H),
6
7
5
1
1
.81ꢀ 7.77(m, 1H), 7.53ꢀ 7.51 (d, J= 8.0Hz, 1H), 7.39ꢀ 7.34 (m, 4H),7.27ꢀ 7.24 (m, 1H),7.05 (s, 2H),
.64(s, 1H), 3.56 (m, 1H), 3.32 (s, 6H),3.24(m, 2H), 2.93ꢀ 2.88 (m, 2H), 2.26 (s, 3H), 1.83ꢀ1.80 (m, 2H),
13
.60ꢀ 1.57 (m, 2H); CꢀNMR ( DMSOꢀd ) 161.71, 149.32, 142.85, 141.17, 140.03, 137.60, 132.45, 132.31,
6
32.14, 129.16, 128.70, 120.80, 80.34, 71.79, 41.63, 30.68, 30.59, 22.76, 20.90; HRMS Calcd 507.148;
+
Found: 507.148 (M+Na ); Anal.Calcd for C H ClN O S : C 64.38; H 6.03; N 5.78; Found: C, 64.41; H,
26
29
2
3
6
.01; N, 5.79;Chiral HPLC (%ee) 96.2.
Characterization
of
(R)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((4ꢀ
fluorophenyl)sulfonyl)piperidinꢀ4ꢀ
0
1
yl)oxy)methyl)pyridine (6h): Yield (268mg, 88.7%);Tan coloured solid; mp: 106 C; HꢀNMR (400 MHz,
DMSOꢀd ): 8.44ꢀ 8.43 (d, J= 4Hz, 1H), 7.81ꢀ 7.73 (m,3H), 7.49ꢀ 7.22 (m, 8H), 5.59 (s, 1H), 3.46 (m, 1H),
6
1
3
3
1
1
.14ꢀ 3.10 (m, 2H), 2.78ꢀ 2.76 (m, 2H), 1.85ꢀ 1.84 (m, 2H), 1.66ꢀ1.63 (m, 2H); CꢀNMR (DMSOꢀd6):
66.29, 163.80, 161.56, 149.30, 141.02, 137.48, 132.43, 132.16, 132.13, 131.03, 130.94, 129.09, 128.63,
23.10, 120.68, 117.10, 116.88, 80.11, 70.99, 43.57, 30.41, 30.29; HRMS Calcd 483.092; Found: 483.092
+
(
M+Na ); Anal.Calcd for C H ClFN O S : C 59.93; H 4.81; N 6.08; Found: C, 59.89; H, 4.77; N, 6.12;
2
3
22
2
3
Chiral HPLC (%ee) 97.2.
Characterization of (R)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀtosylpiperidinꢀ4ꢀ yl)oxy)methyl)pyridine (6i): Yield
0
1
(
254mg, 84.3%); Tan coloured solid; mp: 98 C; HꢀNMR (400 MHz, DMSOꢀd ): 8.39ꢀ 8.37 (d, J=8Hz,
6
1
3
H), 7.72ꢀ7.70 (m, 1H), 7.59ꢀ7.57 (d, J=8.0 Hz, 2H), 7.43ꢀ7.38 (m, 3H), 7.32ꢀ7.21 (m, 5H), 5.56 (s, 1H),
.34 (m, 1H), 3.11ꢀ3.07 (m, 2H), 2.73ꢀ2.69 (m, 2H), 2.40 (s, 3H), 1.85ꢀ1.83 (m, 2H), 1.64ꢀ1.62 (m, 2H);

Page 13 of 27
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB01479D
1
3
CꢀNMR ( DMSOꢀd ): 160.96, 149.30, 143.43, 141.94, 137.49, 132.42, 132.21, 130.13, 128.96, 128.63,
6
1
27.96, 122.94, 120.71, 79.99, 71.11, 43.29, 30.51, 30.32, 21.57; HRMS Calcd 479.117; Found: 479.117
+
(
M+Na ); Anal.Calcd for C H ClN O S : C 63.08; H 5.51; N 6.13; Found: C, 63.06; H, 5.48; N, 6.14;
2
4
25
2
3
Chiral HPLC (%ee) 99.
Characterization
of
(S)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((3ꢀ
nitrophenyl)sulfonyl)piperidinꢀ4ꢀyl)oxy)methyl)pyridine (6j): Yield (265mg, 82%); Tan colored solid;
0
1
mp: 78 C; HꢀNMR (400 MHz, DMSOꢀd ): 8.55ꢀ 8.53 (d, J=8.0 Hz, 1H), 8.42ꢀ 8.41(d, J=4.0 Hz, 1H),
6
8
.35 (s, 1H), 8.19ꢀ8.17 (d, J=8.0Hz, 1H), 7.94ꢀ7.90 (t, J=8.0Hz, 1H), 7.75ꢀ7.71 (m, 1H), 7.42ꢀ7.40 (d,
J=8.0Hz, 1H), 7.32ꢀ7.21 (m, 5H), 5.63(s, 1H), 3.45ꢀ3.41(m, 1H), 3.25ꢀ3.23(m, 2H), 2.83ꢀ 2.77(m, 2H)
1
3
1
1
3
4
.89ꢀ 1.87 (m, 2H) 1.67ꢀ 1.61 (m, 2H); CꢀNMR (DMSOꢀd ); 162.53, 149.59, 148.03, 140.88, 137.88,
6
37.11, 133.93, 132.54, 132.07, 130.07, 128.64, 128.10, 123.13, 122.43, 120.72, 80.96, 71.99, 43.70,
+
0.55, 30.36; HRMS Calcd 510.086; Found: 510.086(M+Na ); Anal.Calcd for C H ClN O S : C 56.61; H
23
22
3
5
.54; N 8.67; Found: C, 56.58; H, 4.57; N, 8.67; Chiral HPLC (%ee) 97.2.
of
nitrophenyl)sulfonyl)piperidinꢀ4ꢀyl)oxy)methyl)pyridine (6k): Yield (283mg, 88%); Tan coloured solid;
Characterization
(S)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((4ꢀ
0
1
mp: 110 C; HꢀNMR (400 MHz, DMSOꢀd ): 8.45ꢀ8.44 (d,J=4Hz, 1H), 7.81ꢀ7.73 (m, 3H), 7.45ꢀ7.35
6
(
m,7H), 7.25ꢀ 7.22(d,J=8.0Hz,1H), 5.61(s, 1H), 3.47ꢀ 3.39(m, 1H), 3.04ꢀ 3.03(m, 2H), 2.78ꢀ 2.76(m, 2H),
1
1
3
4
.86ꢀ 1.84 (m, 2H), 1.66ꢀ1.62 (m, 2H); 13CꢀNMR ( DMSOꢀd );161.99, 148.99, 148.63, 140.93, 137.57,
6
37.47, 133.11, 132.45, 132.09, 129.11, 128.64, 128.13, 123.33, 122.44, 120.11, 80.99, 71.70, 43.75,
+
0.50, 30.22; HRMS Calcd 510.086; Found: 510.086(M+Na ); Anal.Calcd for C H ClN O S : C 56.61; H
23
22
3
5
.54; N 8.67; Found: C, 56.68; H, 4.53; N, 8.69; [α] = +37.6(C 0.01, MeOH).
D
Characterization
of
(S)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((2ꢀ
nitrophenyl)sulfonyl)piperidinꢀ4ꢀyl)oxy)methyl)pyridine (6l): Yield (273mg, 85%); Tan coloured solid;
0
1
mp: 85 C; HꢀNMR (400 MHz, DMSOꢀd ): 8.43ꢀ8.41 (d,J=8Hz,1H), 7.73ꢀ7.70 (m,1H), 7.60ꢀ7.58 (d,
6
J=8.0Hz, 2H), 7.43ꢀ7.32 (m, 3H),7.30ꢀ7.21 (m, 5H), 5.61 (s, 1H), 3.35 (m, 1H), 3.11ꢀ 3.07 (m, 2H), 2.73ꢀ
1
3
2
1
.69 (m, 2H), 1.84ꢀ1.83 (m, 2H), 1.64ꢀ1.61 (m, 2H); CꢀNMR (DMSOꢀd ): 162.05, 149.29, 148.53,
6
40.98, 137.12, 137.11, 133.83, 132.42, 130.07, 129.11, 128.64, 128.18, 123.10, 122.43, 120.79, 80.89,

Organic & Biomolecular Chemistry
Page 14 of 27
View Article Online
DOI: 10.1039/C5OB01479D
+
7
1.98, 43.84,
30.54, 30.29; HRMS Calcd 510.086; Found: 510.086(M+Na ); Anal.Calcd for
C H ClN O S : C 56.61; H 4.54; N 8.67; Found: C, 56.56; H, 4.59; N, 8.71; Chiral HPLC (%ee) 97.6.
23
22
3
5
Characterization
yl)oxy)methyl)pyridine
DMSOꢀd ) 8.44ꢀ 8.43(d, J=4Hz,1H), 8.14ꢀ 8.12(d, J=8.0Hz, 1H), 8.07ꢀ 8.05(d, J=8.0Hz, 1H), 7.94ꢀ 7.90(m,
of
(S)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((3ꢀ(trifluoromethyl)phenyl)sulfonyl
piperidinꢀ4ꢀ
0
1
(6m): Yield (288 mg, 86%); Offꢀwhite solid; mp: 108 C; HꢀNMR (400 MHz,
6
2
3
1
1
H),7.72ꢀ 7.70 (m, 1H),7.40ꢀ 7.38(d, J=8.0Hz,1H),7.30ꢀ 7.21(m,5H), 5.60(s, 1H), 3.47(m, 1H), 3.19ꢀ
13
.16(m, 2H), 2.84ꢀ 2.82(m, 2H), 1.88ꢀ 1.86 (m, 2H), 1.66ꢀ 1.63 (m, 2H); CꢀNMR ( DMSOꢀd );162.00,
6
49.26, 141.04, 137.46, 137.23, 132.43, 132.08, 131.55, 130.64, 130.38, 130.35, 130.31, 129.03, 128.62,
25.18, 124.15, 124.11, 123.00, 122.44, 120.63, 80.99, 71.00, 43.60, 30.48, 30.32; ; HRMS Calcd 511.107;
Found: 511.1067(M+H) ; Anal.Calcd for C H ClFN O S : C 56.42; H 4.34; N 5.48; Found: C, 56.47; H,
24
22
3
3
4
.33; N, 5.52; [α] = +39.7(C 0.01, MeOH).
D
Characterization
of
(S)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((2,5ꢀ
dichlorophenyl)sulfonyl)piperidinꢀ4ꢀ
1
yl)oxy)methyl)pyridine (6n): Yield (291mg, 87%); Brown low melting solid; HꢀNMR (400 MHz, DMSOꢀ
d6) 8.43ꢀ 8.42(d, J=8.0Hz,1H), 7.73ꢀ 7.70(m,1H), 7.60ꢀ 7.58 (d, J=8.0Hz, 1H), 7.43ꢀ 7.32(m, 3H),7.30ꢀ 7.21
(
(
m, 5H), 5.59(s, 1H), 3.35(m, 1H), 3.11ꢀ 3.07(m, 2H), 2.73ꢀ 2.69(m, 2H), 1.84ꢀ 1.83 (m, 2H), 1.64ꢀ 1.61
m, 2H); 13CꢀNMR ( DMSOꢀd );160.99, 151.99, 150.93, 139.98, 137.48, 137.11, 133.83, 132.48, 131.43,
6
1
5
5
29.18, 128.83, 128.11, 123.01, 122.33, 80.99, 73.98, 43.81, 30.78, 30.91; HRMS Calcd 511.0417; Found:
11.0411(M+H); Anal.Calcd for C H Cl N O S : C 53.97; H 4.14; N 5.47; Found: C,53.93 ; H, 4.16; N,
23
21
3
2
3
.56; Chiral HPLC (%ee) 98.
of
Characterization
(S)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((4ꢀ
methoxyphenyl)sulfonyl)piperidinꢀ4ꢀ
0
1
yl)oxy)methyl)pyridine (6o): Yield (266mg, 86%); Offꢀwhite solid; mp: 114 C; HꢀNMR (400 MHz,
DMSOꢀd ): 8.43ꢀ 8.42 (d, J=4Hz,1H), 7.74ꢀ7.70 (m,1H), 7.65ꢀ 7.63 (d, J=8.0Hz, 2H), 7.39ꢀ7.37 (d,
6
J=8.0Hz, 1H), 7.32ꢀ 7.22 (m, 5H), 7.14ꢀ 7.12 (d, J=8.0Hz, 1H), 5.57 (s, 1H), 3.84 (s,3H), 3.44(m, 1H),
3
1
7
.07(m, 2H), 2.73ꢀ 2.69(m, 2H), 1.84ꢀ 1.83 (m, 2H), 1.64ꢀ 1.62 (m, 2H); 13CꢀNMR ( DMSOꢀd );163.13,
6
61.56, 149.29, 141.04, 137.49, 132.41, 130.15, 129.09, 128.62, 127.22, 123.11, 120.72, 114.90, 80.08,
+
1.05, 56.16, 43.56, 30.36, 30.30; HRMS Calcd 495.112; Found: 495.112(M+Na ); Anal.Calcd for
C H ClN O S : C 60.94; H 5.33; N 5.92; Found: C, 60.91; H, 5.37; N, 5.95; [α] = +40.5(C 0.01,
24
25
2
4
D
MeOH).

Page 15 of 27
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB01479D
Characterization of (S)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ(mesitylsulfonyl)piperidinꢀ4ꢀ yl)oxy)methyl)pyridine (6p) :
0
Yield (281mg, 88.5%); White solid; mp: 106 C;
1HꢀNMR (400 MHz, DMSOꢀd ) 8.46ꢀ
6
8
1
1
1
.45(d,J=4Hz,1H), 7.81ꢀ 7.77(m,1H), 7.53ꢀ 7.51 (d,J=8.0Hz, 1H), 7.39ꢀ 7.34(m, 4H),7.27ꢀ 7.24 (m,
H),7.05 (s, 2H), 5.67(s, 1H), 3.56(m, 1H), 3.32(s, 6H),3.24(m, 2H), 2.93ꢀ 2.88(m, 2H), 2.26 (s,3H), 1.83ꢀ
.80 (m, 2H), 1.60ꢀ 1.57 (m, 2H); 13CꢀNMR ( DMSOꢀd ) 160.03, 149.85, 142.14, 141.17, 140.03, 137.17,
6
32.45, 132.31, 132.16, 129.16, 128.60, 120.80, 80.99, 71.99, 41.63, 30.62, 30.53, 22.75, 20.96; HRMS
+
Calcd 507.148; Found: 507.148 (M+Na ); Anal.Calcd for C H ClN O S : C 64.38; H 6.03; N 5.78;
26
29
2
3
Found: C, 64.42; H, 5.99; N, 5.81; Chiral HPLC (%ee) 97.1.
Characterization of (S)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀ((4ꢀ
fluorophenyl)sulfonyl)piperidinꢀ4ꢀ
0
1
yl)oxy)methyl)pyridine (6q): Yield (259mg, 86%); Tan coloured solid; mp: 98 C; HꢀNMR (400 MHz,
DMSOꢀd ) 8.44ꢀ 8.43(d,J=4Hz,1H), 7.81ꢀ 7.73(m,3H), 7.49ꢀ 7.22 (m, 8H), 5.61(s, 1H), 3.46(m, 1H), 3.14ꢀ
6
1
3
3
1
1
.10(m, 2H), 2.78ꢀ 2.76(m, 2H),1.85ꢀ 1.84 (m, 2H)1.66 1.63(m, 2H); CꢀNMR ( DMSOꢀd )166.39,
6
63.83, 161.54, 149.34, 141.01, 137.48, 132.43, 132.16, 132.13, 131.03, 130.94, 129.04, 128.61, 123.10,
20.48, 117.40, 116.18, 80.94, 71.03, 43.57, 30.46, 30.31; HRMS Calcd 483.092; Found: 483.092
+
(
M+Na ); Anal.Calcd for C H ClFN O S : C 59.93; H 4.81; N 6.08; Found: C, 59.85; H, 4.87; N, 6.11;
2
3
22
2
3
Chiral HPLC (%ee) 98.9.
Characterization of (S)ꢀ2ꢀ((4ꢀchlorophenyl)((1ꢀtosylpiperidinꢀ4ꢀ yl)oxy)methyl)pyridine (6r): Yield
0
1
(
263mg, 87.3%); Tan coloured solid; mp: 98 C; HꢀNMR (400 MHz, DMSOꢀd ) 8.43ꢀ 8.41(d,J=8Hz,1H),
6
7
1
.73ꢀ 7.70(m,1H), 7.60ꢀ 7.58 (d,J=8.0Hz, 2H), 7.43ꢀ 7.38(m, 3H),7.32ꢀ 7.21 (m, 5H), 5.57(s, 1H), 3.35(m,
13
H), 3.11ꢀ 3.07(m, 2H), 2.73ꢀ 2.69(m, 2H), 2.40(s,3H), 1.84ꢀ 1.83(m, 2H), 1.64ꢀ 1.61 (m, 2H); CꢀNMR
(
DMSOꢀd )161.56, 149.30, 143.93, 141.04, 137.49, 132.82, 132.41, 130.23, 129.10, 128.63, 127.96,
6
+
1
23.10, 120.70, 80.11, 71.09, 43.29, 30.41, 30.32, 21.48; HRMS Calcd 479.117; Found: 479.117(M+Na );
Anal.Calcd for C H ClN O S : C 63.08; H 5.51; N 6.13; Found: C, 63.11; H, 5.55; N, 6.09; Chiral HPLC
2
4
25
2
3
(
%ee) 97.5.
Blood Collection and malaria experiments
Fresh blood was collected from healthy donors with their informed consent in EDTA tubes (VACUETTE®
EDTA Tubes, Greiner BioꢀOne). The blood collection procedure was verified and approved by the

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Institutional Review Board (IRB) of the National University of Singapore (NUS). Blood was washed three
times by centrifugation (600g for 10 minutes) using RPMI 1640 (R8758 SigmaꢀAldrich) to remove the
buffy coat. Washed RBCs were stored at 50% hematocrit in RPMI 1640. P. falciparum (3D7 clone of the
NF54 strain) was cultured in 2.5% haematocrit, RPMIꢀHEPES medium at pH 7.4 supplemented with
hypoxanthine 50 ꢁg/mL, NaHCO 25 mM, gentamicin 2.5 ꢁg/mL, and Albumax II (Gibco, Singapore)
3
0
.5% wt/vol in an atmosphere containing 5% CO as routinely followed [24].
2,
Determination of parasitemia by flow cytometry
For the determination of parasitemia, 100 ꢁL aliquots of the culture from the respective wells were
collected and fixed overnight with 0.1% glutaraldehyde (SigmaꢀAldrich) at 4°C. Fixed cells were washed
twice with PBS (400xg, 5 min), permeablised with 0.25% Triton Xꢀ100 (SigmaꢀAldrich) for 10min at RT
and then washed again with PBS. Samples were then incubated with 25ꢁg/ml Hoechst 3342 for 30 minutes
and washed twice with PBS and then analyzed by flow cytometry using an LSRII (BD) flow cytometer as
reported elsewhere [25-27]. The samples were excited using a 355nm UV laser for detection and 75,000
RBCs were analyzed for each sample. Data acquisition and analysis was carried out using FlowJo (Tree
Star, Inc., Ashland, OR).
Phenotypic Analysis through microscopy
Thin blood smears were prepared on glass slides and fixed with 100% methanol. Smears were stained with
filtered 1/10 dilution of Giemsa (Sigma) solution in phosphate buffered saline (PBS), incubated for 10 min
at room temperature, washed with distilled water, dried, and observed under an oil immersion lens (100x).
Cytotoxicity Assay
To determine cell viability the colorimetric MTT metabolic activity assay was used. MDCK cells were
4
seeded in a 96ꢀwell plate at a density of 10 cells/well at 37°C, 5% CO in medium comprising of DMEM
2
(
SigmaꢀAldrich), 10% FBS (Sigma) and PenꢀStrepꢀGlut and left to adhere overnight. This medium was
removed after 24 h and medium containing varying concentrations of compound was added and incubated
overnight. Cells treated with medium only served as a negative control group. The supernatant of each well

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was then aspirated and the wells were washed with PBS. This was replaced with medium containing
.5mg/ml MTT dye (Sigma) and the plates were left to incubate at 37°C for 4 h. The supernatant was
0
carefully aspirated and the resultant formazan crystals were dissolved in dimethyl sulfoxide (100 µL) and
the absorbance intensity measured using a microplate reader at 495 nm with a reference wavelength of 620
nm. All experiments were performed in duplicates, and the relative cell viability was expressed as a
percentage relative to the untreated control cells.
In vivo efficacy testing in rodent malaria parasites
Male BALB/c 6ꢀ8weeks old mice were obtained from InVivos Singapore, and subsequently bred under
specific pathogen free (SPF) condition at Nanyang Technological University Animal Holding Unit.
Genetically modified Plasmodium yoelii (P.yoelii) lethal strain YM, which is expressing mcherry (YMꢀ
mcherry) throughout the life cycle, ws used for infection. When parasitemia reached around 1% mice were
subjected to drug treatment by intraperitoneal (i.p.) injection for 4 consecutive days. Positive control groups
were treated with Chloroquine (CQ, dissolved in distilled water) of 10mg/kg body weight or 50mg/kg body
weight, and negative control mice were treated with DMSO, while experimental groups received compound
6
n (dissolved in DMSO) of 10mg/kg body weight or 50mg/kg body weight. Parasitemia was monitored
daily 4 hours after drug treatment by flow cytometry. All animal experiments were carried out in
accordance with institutional guidelines of Nanyang Technological University.
Molecular Docking analysis
Transferability of in silico docking results from P. vivax to P. falciparum Nꢀmyristoyltransferase was
ensured by performing a sequence alignment of sequences using BLASTP as provided by UniProt [28]. We
found 80% overall sequence identity and 93% sequence similarity. As all amino acids in a radius of 4.5Å
around the ligand binding site are identical and second sphere amino acid exchanges are highly
conservative, we expect validity of our in silico analysis for P. falciparum. We prepared chain A of the
crystal structure for docking using protonate3d in MOE [29-31]. Since several rigid docking attempts did
not yield consistent binding modes for our compound series, we performed induced fit docking runs for all
compounds using MOE's default parameters (Molecular Operating Environment 2014.0901, Chemical

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Computing Group, Montreal, Canada). Thereby, we allowed for conformational flexibility of the protein
sideꢀchains in addition to the ligand during energy minimization [32]. Furthermore, we introduced
pharmacophore filters to reduce search space by enforcing a hydrogen bond acceptor function at the
position of the sulfonamide and a hydrophobic group at the pyrazole position (the tolerance radius for both
features was set to 2.5Å).
Figure & Table Legends
Scheme 1: Schematic representation for the synthesis of the novel sulphonamides reported here.
Figure 1: Dose response curve for the most effective 10 compounds identified from the library screen.
Shortlisted compounds were screened against common laboratory strains of malaria parasites, 3D7 (Fig.
1
A) and chloroquine resistant parasite variant, K1 (Fig. 1B). Screening was performed at concentrations
ranging from 0ꢁM to 100ꢁM. Both parasite strains showed comparable sensitivity to most inhibitors (all
except 6k).
Figure 2: Effect of the selected compounds on distinct stages of parasite development. To document
stageꢀspecificity of the compounds tested, experiments were carried out using parasites at early stage
during ringꢀtrophozoite transition (Fig. 2A), trophozoiteꢀschizont transition (Fig. 2B) and schizontꢀring
transition (Fig. 2C), at four different compound concentrations (4*IC , 2*IC , IC and 0.25*IC ). It can
50
50
50
50
be seen that the majority of compounds has an effect during the later stages of parasite development.
Figure 3: Predicted protein-ligand interactions of the sulphonamides with N-myristoyltransferase.
Induced fit docking of the bepotastineꢀderived sulfonamide series to Plasmodium vivax Nꢀ
myristoyltransferase yielded a binding mode in agreement with an Xꢀray structure of another sulfonamide
(
PDB: 2YND). Crystal structure of the enzyme (green cartoon) with the template ligand (atomic coloring
with carbon in white). Sideꢀchains of Pheꢀ103 and Pheꢀ105 are shown to indicate πꢀπ stacking in this region
Fig. 3A). Predicted binding poses for compounds 6l and 6i that showed similar occupation of the binding
(
pocket (Fig. 3B & 3C). The substituted benzene rings of the title compounds forming πꢀπ interactions of
variable strength, which occupies the similar positions of the pyrazole ring of the coꢀcrystallized ligand.
Table 1: Overall susceptibility to each molecules of Plasmodium falciparum strains 3D7 and K1.
Sensitivity of parasites (3D7 and K1) within a 0 ꢁM to 100 ꢁM compound concentration range was

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evaluated. Further, cytotoxicity of the same compounds was tested on the MDCK cell line, and the
resulting selectivity is evaluated as 3D7 IC /MDCK IC .
50
50
Table 2: Stage-specific effects of the compounds presented here on Plasmodium falciparum. ‘ꢀ’
represents no effect; ‘+’ indicates effect at 4*IC ; ‘++’ indicates effect at 4*IC and 2*IC ; ‘+++’
50
50
50
indicates effect at 4*IC , 2*IC and IC . It can be seen that most compound are active on the later stages
50
50
50
of parasite development.
Acknowledgments
This research was supported by University Grants Commission (41ꢀ257ꢀ2012ꢀSR), Department of Science
and Technology (NO. SR/FT/LSꢀ142/2012) to Basappa. RC, AS and TC acknowledge the following grants:
SRLS13049, SUTDꢀZJU/RES/02/2013 PRP acknowledges the following research support from the
Ministry of Education, Singapore: MOE2012ꢀT2ꢀ2ꢀ093. RC acknowledges laboratory/instrument support
provided by the SingaporeꢀMIT Alliance for Research & Technology (SMART) Center funded by the
National Research Foundation, Singapore.
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Table 1
Comp IC50
95%
Confidence
Interval
IC50
K1
(µM) Interval
95%
Confidence
Cytotoxicity Selectivit
ound
3D7
µM)
IC MDCK
y
5
0
(
(µM)
Index
6
6
6
6
6
6
6
6
6
6
b
c
e
f
g
k
l
n
p
r
8.85
8.086 to 9.687
14.42 to 16.90
2.677 to 3.160
5.938 to 7.816
9.937 to 13.10
2.623 to 4.079
11.67 to 15.22
1.876 to 2.506
6.856 to 8.018
6.622 to 11.80
7.345 6.444 to 8.371
9.231 8.589 to 9.921
4.187 3.386 to 5.178
6.952 6.060 to 7.976
9.777 8.703 to 10.98
7.219 6.218 to 8.380
13.22 12.27 to 14.24
2.379 2.171 to 2.606
6.342 5.936 to 6.776
9.111 7.820 to 10.62
>100
>100
>200
>100
>200
>200
>200
>200
>200
>200
>11.3
>6.4
15.61
2.908
6.812
11.41
3.271
13.33
2.168
7.414
8.84
>68.8
>14.7
>17.5
>61.1
>15.0
>92.3
>27.0
>22.60

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Table 2
Ring to
Trophozoite
Trophozoites
to Schizont
Schizont to
Ring
Compounds
6
b
ꢀ
ꢀ
ꢀ
ꢀ
++
+++
++
+
6
6
c
e
ꢀ
ꢀ
6
f
ꢀ
ꢀ
6
g
ꢀ
+
ꢀ
+
6
k
ꢀ
+
6
l
+
ꢀ
+
ꢀ
++
+++
+++
++
ꢀ
6
6
n
p
+
+
ꢀ
ꢀ
6
r
ꢀ
Control
ꢀ

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9
4x48mm (300 x 300 DPI)

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85x309mm (300 x 300 DPI)

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75x260mm (300 x 300 DPI)

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99x237mm (300 x 300 DPI)