(+)-(R)- and (?)-(S)-Perhexiline maleate: Enantioselective synthesis and functional studies on Schistosoma mansoni larval and adult stages

Article abstract of DOI:10.1016/j.bioorg.2020.104067

Schistosomiasis is a neglected tropical disease mainly affecting the poorest tropical and subtropical areas of the world with the impressive number of roughly 200 million infections per year. Schistosomes are blood trematode flukes of the genus Schistosoma causing symptoms in humans and animals. Organ morbidity is caused by the accumulation of parasite eggs and subsequent development of fibrosis. If left untreated, schistosomiasis can result in substantial morbidity and even mortality. Praziquantel (PZQ) is the most effective and widely used compound for the treatment of the disease, in prevention and control programs in the last 30 years. Unfortunately, it has no effect on juvenile immature schistosomes and cannot prevent reinfection or interfere with the schistosome life cycle; moreover drug-resistance represents a serious threat. The search for an alternative or complementary treatment is urgent and drug repurposing could accelerate a solution. The anti-anginal drug perhexiline maleate (PHX) has been previously shown to be effective on larval, juvenile, and adult stages of S. mansoni and to impact egg production in vitro. Since PHX is a racemic mixture of R-(+)- and S-(?)-enantiomers, we designed and realized a stereoselective synthesis of both PHX enantiomers and developed an analytical procedure for the direct quantification of the enantiomeric excess also suitable for semipreparative separation of PHX enantiomers. We next investigated the impact of each enantiomer on viability of newly transformed schistosomula (NTS) and worm pairs of S. mansoni as well as on egg production and vitellarium morphology by in vitro studies. Our results indicate that the R-(+)-PHX is mainly driving the anti-schistosomal activity but that also the S-(?)-PHX possesses a significant activity towards S. mansoni in vitro.

Full text of DOI:10.1016/j.bioorg.2020.104067

Journal Pre-proofs
(+)-(R)- and (-)-(S)-Perhexiline maleate: enantioselective synthesis and func-
tional studies on Schistosoma mansoni larval and adult stages
Alessandra Guidi, A Prasanth Saraswati, Nicola Relitti, Roberto Gimmelli,
Fulvio Saccoccia, Carmina Sirignano, Orazio Taglialatela-Scafati, Giuseppe
Campiani, Giovina Ruberti, Sandra Gemma
PII:
S0045-2068(20)31364-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.104067
YBIOO 104067
Reference:
To appear in:
Bioorganic Chemistry
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Accepted Date:
26 May 2020
28 June 2020
Please cite this article as: A. Guidi, A. Prasanth Saraswati, N. Relitti, R. Gimmelli, F. Saccoccia, C. Sirignano, O.
Taglialatela-Scafati, G. Campiani, G. Ruberti, S. Gemma, (+)-(R)- and (-)-(S)-Perhexiline maleate:
enantioselective synthesis and functional studies on Schistosoma mansoni larval and adult stages, Bioorganic
Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104067
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(+)-(R)- and (-)-(S)-Perhexiline maleate: enantioselective synthesis and functional studies on
Schistosoma mansoni larval and adult stages
Alessandra Guidi,a,1 A Prasanth Saraswati,b,1 Nicola Relitti,b Roberto Gimmelli,a Fulvio Saccoccia,a Carmina
Sirignano,c Orazio Taglialatela-Scafati,c Giuseppe Campiani,b.* Giovina Ruberti,a,** and Sandra Gemmab,***
aInstitute of Biochemistry and Cell Biology, National Research Council, Campus A. Buzzati-Traverso via E.
Ramarini, 32 00015 Monterotondo (Rome) Italy,
bDepartment of Biotechnology, Chemistry and Pharmacy, University of Siena, via Aldo Moro 2, 53100,
Siena, Italy
cDepartment of Pharmacy, School of Medicine and Surgery, University of Naples Federico II, Via D.
Montesano 49, 80131, Naples, Italy
1Equal contribution: Both authors contributed equally to this work
*Corresponding Authors: E-mail addresses: G.C. campiani@unisi.it; G.R. giovina.ruberti@cnr.it; S.G.
gemma@unisi.it
Abstract
Schistosomiasis is a neglected tropical disease mainly affecting the poorest tropical and subtropical areas of
the world with the impressive number of roughly 200 million infections per year. Schistosomes are blood
trematode flukes of the genus Schistosoma causing symptoms in humans and animals. Organ morbidity is
caused by the accumulation of parasite eggs and subsequent development of fibrosis. If left untreated,
schistosomiasis can result in substantial morbidity and even mortality. Praziquantel (PZQ) is the most effective
and widely used compound for the treatment of the disease and in prevention control programs in the last 30
years. Unfortunately, it has no effect on juvenile immature schistosomes and cannot prevent re-infection or
interfere with the schistosome life cycle; moreover drug-resistance represents a serious threat. The search for
an alternative or complementary treatment is urgent and drug repurposing could accelerate a solution. The
anti-anginal drug perhexiline maleate (PHX) has been previously shown to be effective on larval, juvenile, and
adult stages of S. mansoni and to impact egg production in vitro. Since PHX is a racemic mixture of R-(+)- and
S-(-)-enantiomers, we designed and realized a stereoselective synthesis of both PHX enantiomers and
developed an analytical procedure for the direct quantification of the enantiomeric excess also suitable for
semipreparative separation of PHX enantiomers. We next investigated the impact of each enantiomer on
viability of newly transformed schistosomula (NTS) and worm pairs of S. mansoni as well as on egg production
and vitellarium morphology by in vitro studies. Our results indicate that the R-(+)-PHX is mainly driving the
anti-schistosomal activity but that also the S-(-)-PHX possesses a significant activity towards S. mansoni in
vitro.
KEYWORDS: PHX maleate enantiomers; Stereoselective synthesis; Michael initiated ring closure reactions;
Schistosoma mansoni

1. Introduction
Schistosomiasis is a neglected tropical parasitic disease affecting more than 200 million people in the poorest
regions of the world, mainly in sub-Saharan Africa (85%) [1]. Schistosomiasis is caused by a parasitic
trematode of the genus Schistosoma with different species accounting for the majority of human infections in
different world regions, namely S. haematobium in Africa and the Middle East, S. mansoni in Africa and South
America, and S. japonicum in China and the Philippines; S. intercalatum in Africa and S. mekongi in Southeast
Asia are less common [2]. Recent outbreaks revealed the re-emergence of urogenital schistosomiasis in
southern Europe [3]. Importantly, S. haematobium is a biological carcinogen as classified by the International
Agency for Research in Cancer of the World Health Organization (WHO) [4].
A vaccine is unavailable to date; in addition, the treatment and most control initiatives rely on a single drug,
praziquantel (PZQ) that has high efficacy, few and transient side effects, competitive cost. However, while very
active on adult parasites, PZQ is poorly effective against juvenile worms both in vitro and in vivo. Therefore, in
endemic areas, where patients are likely to harbor juvenile and adult parasites concurrently and reinfection is
common, a complete cure is not reliably achieved with a single dose of PZQ [5]. Moreover, the extensive use
of PZQ has brought concern about the emergence of PZQ-resistance/tolerance by schistosomes, also for the
isolation of field and laboratory isolates less responsive to treatment, thus reaffirming an urge for the
development of new alternative treatments [5].
In the search for novel schistosomicidal multi-stage compounds, and taking into account drug-repositioning as
a key aspect of drug discovery trajectories [6], we have identified perhexiline maleate (PHX, 1, Figure 1) as a
novel promising multi-stage lead compound [7]. This drug, introduced to clinical use over 30 years ago as an
efficacious monotherapy for the treatment of angina [8], showed activity against schistosomula (NTS), juvenile,
and adult worms in vitro [7]. In addition, at sub-lethal doses, PHX impairs egg-laying in vitro, impact the
morphology of the parasite reproductive systems, and increases lipids accumulation in the vitellarium [7]. The
in vivo study in S. mansoni-infected mice showed variability in worm burdens and an overall minimal effect
upon PHX treatment. The short half-life in mice, due to very high plasma clearance [9] together with a very
high plasma protein binding, could be a possible explanation for the modest efficacy of PHX in a
schistosomiasis murine model [7]. In mammalian cells, PHX appears to act on several molecular targets [10],
with carnitine plamitoyltransferase 1 (CPT-1) and 2 (CPT-2), enzymes involved in the metabolism of fatty acids,
being among the most investigated [10-14]. On the contrary, the molecular mechanisms of action of PHX in

Schistosoma is still unknown and it should be considered that the parasite lacks CPT-1 and CPT-2 orthologues
and that fatty acid oxidation still remains controversial in schistosomes [15-18].
PHX is formulated for therapeutic use as a racemic mixture of the (+) and (−) enantiomers ((+)-(R)-2 and (-)-
(S)-2, Figure 1). Reports of severe side effects upon chronic treatment, including peripheral neuropathy and
fatal hepatotoxicity, firstly prompted the restriction of its use and then the worldwide withdraw from the market,
with the exception of Australia and New Zealand where is still used for the treatment of refractory angina [11].
The major determinant of the oral clearance of PHX is hepatic metabolism mediated by the cytochrome P450
2D6 (CYP2D6) [19] and polymorphism of CYP2D6 is the primary contributor to the highly variable clinical
pharmacokinetics of racemic PHX, thus limiting its therapeutic application [20-23]. Interestingly, it has been
reported that in human liver microsomes, the intrinsic clearance of (-)-(S)-2 is greater than that of the (+)-(R)-
2 [22], while differential toxicity of each enantiomer in rat has been observed [21].
In order to understand whether the anti-schistosomal activity of PHX is mediated by a single enantiomer or
both are involved in the observed phenotypic effects, we developed a straightforward stereoselective synthesis
of this piperidine-based drug. In particular, we exploited a stereoselective Michael initiated ring closing reaction
as the key step for the construction of the chiral piperidine ring (Figure 1) [24]. The enantiomeric purity of the
single PHX enantiomers was established on the basis of a newly developed chiral HPLC method that can be
extended to the semi-preparative scale. The in vitro activities of (+)-(R)-2 and (-)-(S)-2, both prepared as
maleate salts, and the racemic PHX were then comparatively assessed against NTS and adult worm pairs.
Drug effects were evaluated by using viability assays and confocal microscopy studies on adult parasites.
Grignard
reaction
N
H
N
N
CO2H
H
H
HO2C
(-)-(S)-2
(+)-(R)-2
(+/-)-Perhexiline maleate, 1
(PHX)
Retrosynthetic analysis for
the synthesis of (+)- and (-)-2
CO2R
N
O
Xc
Michael initiated
ring closure
3
Figure 1. Structure of PHX maleate (1), of enantiomers (+)-(R)-2 and (-)-(S)-2, and retrosynthetic analysis for
their stereoselective synthesis.

2. Results and discussion
2.1. Stereoselective synthetic approach and chiral HPLC
In the previous years, several attempts have been made to separate or synthesize both enantiomers of 1 in
high enantiomeric excess. Davies et al. [25] employed fractional crystallization processes to obtain optically
enriched perhexiline by resolving 1,1’-binaphthyl-2,2’-diyl(hydrogen)phosphate (BNPA) salts of PHX. In this
process, the racemic PHX was treated with (S)-(+)-BNPA to obtain (S)-(-)-2, followed by crystallization using
methanol-acetone mixture. Consequently, (R)-(+)-2 was obtained by the resolution with (R)-(-)-BNPA. Even
though both the enantiomers obtained were of high optical purity, the tedious crystallization operation involving
the diastereomeric salts limits the availability of the enantiomeric forms. To this end, Tseng et al. [26] recently
reported the synthesis and absolute configuration of both (S)-(-)-2 and (R)-(+)-2 using a protocol that parallels
the synthesis of racemic 1. The limit of this strategy is highlighted by the reduction step: in fact the catalytic
hydrogenation of a pyridine ring appropriately functionalized at C2 and bearing an Evans chiral auxiliary at C5,
afforded both enantiomers with enantiomeric excess of 93% for the (S)-(-)-2 and 88% for the (R)-(+)-2. In our
pursuit to improve upon the enantiomeric purity of the PHX enantiomers, and to develop a more versatile
synthetic approach also suitable for structure-activity relationship investigations, we prepared the
stereochemically defined 2-piperidineacetate reported in Figure 1 as the key intermediate, whose ester group
could be used for the introduction of a variety of substituents beside the dicyclohexylmethyl moiety of PHX.
Among the described procedures for the synthesis of this key building block, we applied a stereoselective
Michael initiated ring closing reaction as described in Scheme 1. Accordingly, 3,4-dihydropyran (3) was
converted to the unsaturated iodide (4) through a described 3-step protocol [27]. The key precursors 5 (major
product) and 6 were obtained by the reaction of intermediate 4 with (R)--methylbenzylamine in the presence
of triethylamine as a base in ethanol [28].
Due to the limited 6:5 diastereomeric ratio obtained, we investigated if an improvement of the diastereomeric
ratio of this reaction could be achieved by isolating intermediate 11 (Scheme 1) and submitting it to an n-
butyllithium-promoted cyclization reaction. Accordingly, dihyropyran 3 was converted into the corresponding
-hydroxyaldehyde by treatment with HCl, followed by conversion to aminoalcohol 7 through a reductive
amination protocol using NaBH3CN as the reducing agent [29]. The free secondary amine 7 was then protected
as the corresponding N-tert-butoxycarbonyl-derivative 8 in presence of triethylamine and this latter
intermediated was then converted to the corresponding aldehyde 9 using Dess-Martin Periodinane. Aldehyde
9 was subjected to the Horner-Wadsworth-Emmons olefination reaction to obtain the α,β-unsaturated ethyl

ester 10 employing triethylphosphonoacetate (TEPA) and sodium hydride as the base. Compound 10 was
deprotected using trifluoroacetic acid and the resulting amine was treated with n-butyllithium to obtain
diastereomers 12 and 13, respectively. As expected, 12 was the major product of the reaction but no
improvement of the overall diastereomeric ratio was achieved. Interestingly, it was observed that the
deprotected amine was able to spontaneously cyclize intramolecularly to obtain low amounts of piperidine
products at room temperature before the cyclization step with n-butyllithium.
Scheme 1.a
aReagents and conditions: (a) (R)-α-methylbenzylamine, Et3N, EtOH, reflux, 16 h (b) 1 N HCl, 0 to 25 °C, 30
mins (c) (R)-α-methylbenzylamine, acetic acid, NaCNBH3, EtOH, 1 h, 25 °C (d) (Boc)2O, Et3N, THF, 12 h (e)
Dess-Martin Periodinane, DCM, 25 °C, 4 h (f) TEPA, NaH, THF, 25 °C, 30 min (g) Trifluoroacetic acid, DCM,
(1:1), 25 °C (h) n-butyl lithium, -78 °C, 2 h.
The final steps for the synthesis of S-(-)-2 are described in Scheme 2. The key precursor (12) was subjected
to a Grignard-reaction using 2 M cyclohexylmagnesium chloride in THF to afford the alcohol intermediate 14.
Subsequent treatment with thionyl chloride afforded the chlorination/elimination product 15 which on catalytic
hydrogenation with Pd(OH)2 in ethanol yielded the debenzylated intermediate which was further reduced using
catalytic Pd/C in H2 atmosphere to afford the desired enantiomer. (R)-(+)-2 was prepared using the similar
procedure as discussed above, starting from (S)-α-methylbenzylamine and dihydropyran 3. The obtained
enantiomers were converted to their maleate salts using maleic acid.

Scheme 2.a
aReagents and conditions: (a) Cyclohexylmagnesiumchloride (2 M), THF, reflux, 3 h (b) SOCl2, THF, reflux, 5
h (c) Pd(OH)2, ammonium formate, EtOH, reflux, 4 h (d) H2, Pd/C, MeOH, 25 °C, 8 h.
The chiral purity of synthesized piperidines was assessed through the development of a chiral HPLC
methodology. After considerable experimentation on several stationary phases, better results were obtained
on
a
Chirex® (S)-Val/(R)-NEA column (stationary phase including S-valine and R-1-(alpha-
naphthyl)ethylamine), with mobile phase a mixture of n-hexane 85%/isopropanol 10%/methanol 5%. A
representative chromatogram is reported in Figure 2.
Figure 2. Representative Chiral HPLC chromatogram of PHX racemic mixture. Optical rotation was used as
simple tool to assign the separated enantiomers. Thus, peak eluting at T = 2,74 min was identified as (S)-
(-)-2 and peak eluting at T = 3,06 min was identified as (R)-(+)-2.
2.2. Antischistosomal properties of PHX and enantiomers (S)-(-)-2 and (R)-(+)-2
Each PHX maleate enantiomer, together with the racemic mixture, was tested in vitro for its relative
schistosomicidal properties. The in vitro assay on NTS, was based on ATP quantitation by a luminescence
assay at 24 h, as previously described [30]. DMSO (vehicle) and gambogic acid (positive control) that induces
complete death of NTS, were respectively used as 100% and 0% viability reference values. A dose-response
curve ranging from 0.78 µM up to 100 µM was performed, and the LC50 value was estimated for each
compound. In this setting, (R)-(+)-2 appears to be more effective (LC50 = 9 µM) than the (S)-(-) enantiomer

(LC50 =16 µM) and overall comparable to the LC50 value of the racemic PHX preparation (LC50 = 8 µM) (Figure
2).
Figure 3. Effects of PHX maleate, (R)-(+) , and (S)-(-) PHX maleate enantiomers on NTS. Dose-response
curves of racemic PHX (A), (R)-(+) PHX (B), and (S)-(-) PHX (C) on NTS. For each compound the LC50 value
was estimated 24 h upon incubation by means of the % ATP reduction (death of NTS) plotted on the y-axis
and normalized with 50 μM GA-treated control (0% survival) and DMSO treated-schistosomula (100% survival).
The PHX enantiomers and the racemic PHX maleate were then tested against adult worm pairs in vitro.
Viability was recorded by assigning a multi-parametric score as previously reported [7]. In these experiments
the concentration of racemic PHX was doubled in order to match that of the two enantiomers, under the
assumption that: (i) only one of the two enantiomers might be active and (ii) that the racemate would contain
only 50% of active substance, as already proposed for the anti-schistosomal drug oxamniquine (OXA)
enantiomer studies [31].
For the treatment 3 different concentrations (5, 10, and 20 M) of racemic PHX together with the equivalent
halved dose of each PHX enantiomers were tested (Table 1).
Our results indicate that on adult worm pairs, the (R)-(+)-2 enantiomer was markedly more active than the (S)-
(-)-enantiomer (Table 1). In particular the dose range of 10 M of (R)-(+)-2 reduced worm vitality to 20% at 72
h, while (S)-(-)-2 had only a modest effect, lowering worm vitality to 80% at 72 h and 50% at day 7. The racemic
mixture used at 20 M showed a strong reduction on worm’s vitality with about 10% survival 72 h after
treatment. Thus, under these conditions the (R)-(+)-2 enantiomer and the racemic mixture had a comparable
effect on adult parasites as observed for NTS. On the other hand, the (S)-(-)-enantiomer resulted to be about
four time less active than the (R)-(+)-2 enantiomer. When lower concentrations of both enantiomers were
tested (2,5 and 5 M) the overall activity of both compounds appeared to be quite similar with the (R)-(+)-2
resulting always slightly more active than (S)-(-)-2. It is worth to point out that in these conditions the racemic

mixture used at 5 and 10 M showed a strong reduction in worm’s vitality to 50% and 10% respectively upon
72 h incubation (Table 1).
When higher drug concentrations (20 M) of both enantiomers was tested, the effect on worms was very
pronounced for both enantiomers, and even (S)-(-)-2 led to a complete worm death. These results suggest
that the bulk of anti-schistosomal activity is mainly exerted by (R)-(+)-2, with (S)-(-)-2 retaining a considerable
activity when used at higher concentration.
Table 1 Effects of PHX and PHX enantiomers on Schistosoma mansoni adult pairs.
Treatment (μM)
% Viability ± SEM
3d
% Pairing
% Relative egg
reduction
% Viability ± SEM
7d
DMSO
PHX 5
R-(+)-PHX 2.5
S-(-)-PHX 2.5
100±0
47±7
97±3
100
60-100
100
100
20±3 ***
89±12
100±0
52±10
92±8
100±0
100
132±28
100±0
DMSO
PHX 10
100±0
18±2
100
0
100
/
100±0
/
R-(+)-PHX 5
S-(-)-PHX 5
79±6
100±0
100
100
60±9 *
86±10
83±3
97±3
DMSO
PHX 20
R-(+)-PHX 10
S-(-)-PHX 10
98±2
11±6
20±3
75±8
100
/
12±3 ****
47±5 **
98±2
/
13±2
50±3
0
80-100
100
DMSO
R-(+)-PHX 20
S-(-)-PHX 20
98±2
0±0
16±1
100
0
0
100
/
/
98±2
/
/
Viability and egg reduction are reported as residual percentage with respect to the DMSO-treated samples.
Each point represents the average ± SEM of three independent experiments. *P <0.05, **P <0.01 or ****P
<0.0001, Student's t test.
Due to the high relevance of eggs for both disease transmission and pathology, the effect of the PHX
enantiomers was also investigated for their impact on egg-laying in vitro (Table 1). Eggs released by worm
pairs treated with 2,5, 5 and 10 M of each compound were counted after 72 h from treatment and normalized
to the number of couples. At both concentrations (R)-(+)-2 had a significant effect in lowering eggs laid by
treated female parasites while the (S)-(-)-2 had a substantial effect only when used at 10 M. No significant
differences were observed at 2.5 M for both compounds.
Adult worm couples treated with 10 μM of PHX enantiomers for 72 h, the condition at which differences were
observed for both viability and egg production, were considered for morphological analysis by means of
carmine red-staining, followed by confocal laser scanning microscopy (CLSM). In particular, vitellaria of female
parasites treated with (R)-(+)-2, showed high levels of damage with numerous unstained, dark, hole-like areas,
representing lipid droplets. Also, the loss of gut epithelial cells in 4 of 6 male worms was observed. (S)-(-)-2

had a less severe effect on the overall tissue morphology: only a small number of dark, hole-like areas were
present in the vitellocytes and subtle damage of gut endothelium was observed only in 1 out of 8 worms
analyzed.
Figure 4. Confocal microscopy of carmine red-stained adult worm couples. Confocal microscopy of
carmine red-stained worm couples treated with DMSO and PHX enantiomers (10 μM). Images represent
female vitellarium and male gut. Abbreviations: vt, vitellarium; g, gut; white arrows indicate gut epithelium,
white triangles indicate black areas round shaped in vitellocytes. Scale bar 50 μm.
Data presented here show that the (R)-(+)-2 enantiomer accounts for the majority of the anti-schistosomal
activity in particular on adult parasites at 10 M, while the (S)-(-)-2 is capable of exerting activity only at higher
concentration (20 M). This suggests that when schistosomes are exposed to the racemic PHX mixture, the
(R)-(+)-2 component is likely to be the more active, though (S)-(-)-2 is not devoid of any activity. How the two
enantiomers behave remains to be unveiled.
3. Conclusions
We here developed a strategy for the synthesis and enantiomeric analysis of PHX. The synthetic strategy here
presented is versatile and will be useful for the exploration of the structure-activity relationships at the pendant
aliphatic chain on the single enantiomers. The separated enantiomers and the racemic mixture have been
comparatively tested on various life cycle stages of S. mansoni with the aim of assessing the relative
contribution of each enantiomer to the anti-schistosomal activity. Due to the reported difference in
pharmacology, toxicology, pharmacokinetics, and metabolism of PHX enantiomers, our studies are important
in the frame of designing novel PHX derivatives with improved pharmacokinetics properties for in vivo efficacy
studies as well as for the identification of PHX targets in schistosomes.

4. Experimental Section
4.1. Chemistry.
Reactions were carried out under nitrogen atmosphere in oven-dried (120 °C) glassware. All reagents and
chemicals were purchased from commercial suppliers and used without further purification. Anhydrous
solvents were obtained as follows: dichloromethane from calcium hydride diethyl ether and tetrahydrofuran
from sodium benzophenone. All purification procedures were carried out with reagent grade solvents
(purchased form Fisher Scientific) in air. TLC analysis was conducted using aluminium foil supported thin-layer
silica gel chromatography plates (F254 indicator). Column chromatography was performed using 230–400
mesh, 60 Å pore diameter silica gel. Chiral chromatography was performed on a Chirex® (S)-Val/(R)-NEA 5
µm column (Phenomenex, Torrance, CA, USA) using a mxiture n-hexane 85%/isopropanol 10%/methanol 5%.
as eluent at a flow rate of 0.5 mL/min. Samples were solubilized in the mobile phase. 1H and 13C NMR spectra
were recorded at room temperature on a Varian 300 or a Bruker ARX-400. Chemical shifts (d values) are
reported in parts per million and are referenced to the deuterated residual solvent peak. NMR data are reported
as: d value (chemical shift, J-value (Hz), integration, where s = singlet, d = doublet, t = triplet, q = quartet, br =
broad peak). Low-resolution mass analyses were performed on an Agilent 1100 spectrometer.
4.1.1.(E)-Methyl 7-iodohept-2-enoate (4).
To a solution of Ph3P (160 mg, 0.60 mmol) in anhydrous DCM (3 mL) were added imidazole (41 mg, 0.60
mmol), iodine (154 mg, 0.60 mmol), and a solution of the (E)-methyl 7-hydroxyhept-2-enoate (80 mg, 0.50
mmol) in anhydrous DCM (2 mL). The mixture was stirred at room temperature for 30 min, concentrated in
vacuo, and chromatographed on silica gel (PE : EtOAc 20:1) to afford the title compound 4 as a clear oil (85
mg, 63%). 1H NMR (300 MHz, CDCl3) δ 7.00 – 6.86 (m, 1H), 5.83 (d, J = 15.7,Hz, 1H), 3.72 (s, 3H), 3.17 (t, J
= 6.9, 2H), 2.22 (q, J = 7.2, 2H), 1.93 – 1.75 (m, 2H), 1.66 – 1.45 (m, 2H). ESI MS: 269 [M+H]+
4.1.2.Methyl 2-((S)-1-((R)-1-phenylethyl)piperidin-2-yl)acetate (5).
(R)-α-Methylbenzylamine (40 µL, 0.30 mmol) was added dropwise to a stirred solution of 4 (75 mg, 0.28 mmol)
and triethylamine (80 µL, 0.56 mmol) in EtOH (4 mL) at 25 °C under nitrogen. The resulting solution was
heated at reflux for 16 h. After cooling to 25 °C, the solvent was evaporated under reduced pressure. Water
(5 mL) was added and the mixture was extracted with Et2O (2  10 mL). The combined organic extracts were
washed with water (5 mL), 5% aqueous sodium thiosulfate solution (5 mL) and brine (5 mL), then dried over
Na2SO4 and evaporated under reduced pressure. The crude product was purified by flash column

chromatography on silica gel (PE: EtOAc: Et3N 30:10:0.1) affording the amino ester 5 (31 mg, 42%) as a pale-
yellow oil. 1H NMR (300 MHz, CDCl3) δ 7.41 – 7.10 (m, 5H), 3.76 – 3.59 (m, 4H), 3.53 – 3.41 (m, J = 9.1, 4.6
Hz, 1H), 2.64 – 2.45 (m, 2H), 2.38 – 2.10 (m, 2H), 1.83 – 1.66 (m, 1H), 1.62 – 1.32 (m, 5H), 1.29 (d, J = 6.7
Hz, 3H). ESI MS: 262 [M+H]+
4.1.3.Methyl 2-((R)-1-((R)-1-phenylethyl)piperidin-2-yl)acetate (6).
Pale-yellow oil (17 mg, 23%) 1H NMR (300 MHz, CDCl3) δ 7.39 – 7.08 (m, 5H), 3.82 (q, J = 6.7 Hz, 1H), 3.59
(s, 3H), 3.24 – 3.06 (m, 1H), 2.69 (dd, J = 14.0, 4.9 Hz, 1H), 2.61 – 2.53 (m, 2H), 2.53 – 2.33 (m, 1H), 1.76 –
1.36 (m, 6H), 1.32 (d, J = 6.7 Hz, 3H). ESI MS: 262 [M+H]+..
4.1.4.(R)-5-((1-Phenylethyl)amino)pentan-1-ol (7).
A suspension of 3,4-dihydropyran (1000 mg, 1084 µL, 11.88 mmol) in 1 M aqueous solution of HCl (3 mL) was
heated to reflux for 1 h. Successively, the reaction was cooled to 25 °C, neutralized with a 0.5 M solution of
NaOH and extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL),
dried over Na2SO4 and evaporated in vacuo. The residue was dissolved in a mixture of ethanol (4 mL) and
acetic acid (30 µL), then (R)-α-methylbenzylamine (484 mg, 363 µL, 4.00 mmol) was added. The mixture was
stirred at 25 °C for 1 h then sodium cyanoborohydride (251 mg, 4.00 mmol) was added and the reaction was
stirred at 25 °C for 1 h. After this time a 1 M solution of NaOH in water was added and the mixture was extracted
with diethyl ether (3 x 10 mL), the combined organic layers were dried over Na2SO4 and evaporated in vacuo,
affording 7 (788 mg, 32%). as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.40 – 7.13 (m, 5H), 3.74 (q, J =
6.6 Hz, 1H), 3.61 (t, J = 6.4, 2H), 2.60 – 2.30 (m, 2H), 1.69 (brs, 2H), 1.58 – 1.44 (m, 4H), 1.41 – 1.30 (m, 5H).
ESI MS: 208 [M+H]+
4.1.5.(R)-tert-Butyl (5-hydroxypentyl)(1-phenylethyl)carbamate (8).
To a stirred solution of 7 (310 mg, 1.49 mmol) in anhydrous THF (5 mL) cooled to 0 °C, triethylamine (625 µL,
4.48 mmol) and Boc2O (652 mg, 2.99 mmol) were added. The reaction was stirred for 12 h at 25 °C, then it
was diluted with DCM (10 mL) and extracted 3 times with a saturated solution of NaHCO3 (10 mL). The
combined organic layers were dried by over anhydrous Na2SO4 and the solvent was removed in vacuo. The
crude mixture was purified by flash column chromatography on silica gel (3:1 PE : EtOAc) to obtain the 8 (288
mg, 63%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.39 – 7.11 (m, 5H), 5.40 (brs, 1H), 3.54 (t, J = 6.5
Hz, 2H), 3.13 – 2.70 (m, 2H), 1.53 – 1.31 (m, 18H). ESI MS: 308 [M+H]+

4.1.6.(R)-tert-Butyl (5-oxopentyl)(1-phenylethyl)carbamate (9).
To a stirred solution of 8 (115 mg, 0.37 mmol) in anhydrous DCM (4 mL) cooled to 0 °C, Dess-Martin
Periodinane (364 mg, 0.86 mmol) was added. The reaction was allowed to reach 25 °C and it was stirred until
complete consumption of the starting material monitored by TLC. On completion, the reaction was neutralized
with a saturated solution of NaHCO3 and extracted with DCM (3 x 5 mL). The combined organic layers were
dried over anhydrous Na2SO4 and evaporated in vacuo. The crude mixture was purified by flash column
chromatography on silica gel (4:1 PE : EtOAc) to obtain the ntermediate 9 (65 mg, 57%) as a colorless oil. 1H
NMR (300 MHz, CDCl3) δ 9.67 (s, 1H), 7.39 – 7.13 (m, 5H), 5.41 (brs, 1H), 3.23 – 2.66 (m, 2H), 2.31 (s, 2H),
1.57 – 1.45 (m, 16H). ESI MS: 306 [M+H]+
4.1.7.(R,E)-Ethyl 7-((tert-butoxycarbonyl)(1-phenylethyl)amino)hept-2-enoate (10).
To a stirred suspension of NaH (60% dispersion in mineral oil, 42 mg, 1.06 mmol) in anhydrous THF (3 mL)
cooled to 0 °C, was added TEPA (210 µL, 1.06 mmol) dropwise. After 30 min, a solution 9 (65 mg, 0.21 mmol)
in anhydrous THF (2 mL) was added dropwise. The reaction was allowed to reach 25°C and stirred for 2 h
until the complete consumption of the starting material monitored by TLC. On completion, water (5 mL) was
added to the reaction mixture and the mixture was extracted with EtOAc (3 x 5 mL). The combined organic
layers were dried over anhydrous Na2SO4 and the solvent was removed in vacuo. The crude mixture was
purified by flash column chromatography on silica gel (4:1 PE : EtOAc) to obtain intermediate 10 (56 mg, 70%)
as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.40 – 7.15 (m, 5H), 6.92 - 6.79 (m, 1H), 5.74 (d, J = 15.6, 1H),
5.40 (br, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.12 – 2.77 (m 2H), 2.18 – 1.95 (m, 2H), 1.57 – 1.39 (m, 16H), 1.28 (t,
J = 7.1 Hz, 3H). ESI MS: 376 [M+H]+.
4.1.8.(R,E)-Ethyl 7-((1-phenylethyl)amino)hept-2-enoate (11).
Intermediate 10 (56 mg, 0.15 mmol) was dissolved in anhydrous DCM (3 ml) and cooled to 0 °C. To this cold
solution, TFA (610 µL) was added dropwise and the reaction was allowed to stir for 2 h until the complete
consumption of the starting material monitored by TLC. The volatiles were removed in vacuo and the crude
was diluted with DCM (5 mL) and extracted 3 times with a saturated solution of NaHCO3 (5 mL). The combined
organic layers were dried by passage through anhydrous Na2SO4 and the solvent was removed in vacuo to
obtain the pure product as a pale-yellow oil (38 mg, 93%). 1H NMR (300 MHz, CDCl3) δ 7.43 – 7.16 (m, 5H),
7.02 – 6.83 (m 1H), 5.78 (d, J = 15.6, 1H), 4.17 (q, J = 7.1 Hz, 2H), 3.77 (q, J = 6.6 Hz, 1H), 2.78 – 2.27 (m,
3H), 2.28 – 1.92 (m, 2H), 1.48 (m, 4H), 1.37 (d, J = 6.5 Hz, 3H), 1.29 (t, J = 7.1, 3H). ESI MS: 276 [M+H]+

4.1.9.Ethyl 2-((S)-1-((R)-1-phenylethyl)piperidin-2-yl)acetate (12).
n-Butyllithium (49 µL, 0.12 mmol) was added dropwise to a solution of 11 (34 mg, 0.12 mmol) in anhydrous
THF (4 mL) at -78 °C and the resultant mixture was stirred for 2 h at the same temperature. A saturated solution
of NH4Cl was then added and the aqueous layer was extracted with EtOAc (3 x 5 mL). The combined organic
layers were dried over anhydrous Na2SO4 and the solvent was removed in vacuo. The resulting mixture was
purified by flash column chromatography on silica gel (50:1:0.1 PE : EtOAc : Et3N) to obtain 12 (11 mg, 32%)
as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.42 – 7.11 (m, 5H), 4.25 – 4.00 (m, 2H), 3.68 (q, J = 6.6 Hz,
1H), 3.58-3.40 (m, 1H), 2.64 – 2.41 (m, 2H), 2.38 – 2.11 (m, 2H), 1.85 – 1.68 (m, 1H), 1.63 – 1.32 (m, 5H),
1.32 – 1.21 (m, 6H). ESI MS: 276 [M+H]+
4.1.10. Ethyl 2-((R)-1-((R)-1-phenylethyl)piperidin-2-yl)acetate (13).
Colorless oil (7 mg, 21%). 1H NMR (300 MHz, CDCl3) δ 7.32 – 7.16 (m, 5H), 4.06 (q, J = 7.1 Hz, 2H), 3.84 (q,
J = 6.7 Hz, 1H), 3.21 – 3.12 (m, 1H), 2.74 – 2.36 (m, 4H), 1.78 – 1.37 (m, 5H), 1.34 (d, J = 3.1 Hz, 3H), 1.29
– 1.00 (m, 4H). ESI MS: 276 [M+H]+
4.1.11. 1,1-Dicyclohexyl-2-((S)-1-((R)-1-phenylethyl)piperidin-2-yl)ethanol (14).
To a stirred 2.0 M solution of cyclohexylmagnesium chloride (306 µL, 0.61 mmol) in anhydrous THF (3 mL)
intermediate 12 (40 mg, 0.15 mmol) dissolved in anhydrous THF was added. The reaction was heated at reflux
for 2 h until the complete consumption of the starting material monitored using TLC. A saturated solution of
NH4Cl (5 mL)) was added to the reaction mixture that was successively extracted with EtOAC (3 x 5 mL). The
combined organic extracts were collected, dried over anhydrous Na2SO4 and concentrated in vacuo. The crude
was purified by flash column chromatography on silica gel (PE: EtOAc 5:1) obtaining intermediate 14 (38 mg,
62%) as a pale-yellow oil. 1H NMR (300 MHz, CDCl3) δ 7.32 – 7.23 (m, 5H), 4.09 (q, J = 6.6 Hz, 1H), 3.37 –
2.85 (m, 3H), 2.38 - 2.23 (m 1H), 2.21 – 1.89 (m, 2H), 1.85 – 1.48 (m, 10H), 1.49 – 0.59 (m, 20H). ESI MS:
398 [M+H]+
4.1.12. (S)-2-(2,2-Dicyclohexylvinyl)-1-((R)-1-phenylethyl)piperidine (15).
To a stirred solution of 10 (30 mg, 0.08 mmol) in anhydrous DCM (3 mL) cooled at 0 °C, SOCl2 (18 µL, 0.15
mmol) was added dropwise. The reaction was heated at reflux for 2 h until the complete disappearance of the
starting material monitored using TLC. Then a saturated solution of NaHCO3 (5 mL) was added to the reaction
mixture and extracted with DCM (3 x 5 mL). The combined organic extracts were collected, dried over
anhydrous Na2SO4 and concentrated in vacuo. The crude was purified by flash column chromatography on
silica gel (PE: EtOAc 6:1) obtaining intermediate 15 (22 mg, 77%) as a colorless oil. 1H NMR (300 MHz, CDCl3)

δ 7.51 – 7.39 (m, 2H), 7.35 – 7.11 (m, 3H), 5.17 (d, J = 9.2 Hz, 1H), 4.19 (q, J = 6.8 Hz, 1H), 3.38 (td, J = 9.3,
3.1 Hz, 1H), 2.65 – 2.50 (m, 1H), 2.49 – 2.37 (m, 1H), 2.12 (td, J = 11.0, 2.9 Hz, 1H), 1.92 – 1.04 (m, 30H).
ESI MS: 398 [M+H]+
4.1.13. (S)-2-(2,2-dicyclohexylethyl)piperidine (S-(-)-Perhexiline, 2).
To a stirred solution of 15 (15 mg, 0.04 mmol) in EtOH (2 mL) ammonium formate (8 mg, 0.12 mmol) and a
catalytic amount of Pd(OH)2 oncarbon were added. The reaction mixture was refluxed at 85 °C under nitrogen
for 2 h. Reaction was monitored by TLC for the complete consumption of starting material, upon which it was
filtered through a pad of CeliteTM. The filtrate was concentrated in vacuo and the crude obtained (debenzylated
product) (7 mg, 64%) was used in the next step without further purification. ESI MS: 276 [M+H]+. To a stirred
solution of the above mentioned intermediate (10 mg, 0.04 mmol) dissolved in MeOH (2 mL), a catalytic
amount of Pd/C was added under nitrogen atmosphere. The nitrogen was replaced by hydrogen and the
reaction was stirred for 8 h until complete disappearance of the starting material was observed. The reaction
mixture was filtered through a pad of CeliteTM and the filtrate was concentrated. The crude was purified by
flash column chromatography on alumina (DCM: MeOH 150:1) to obtain the desired product (5 mg, 50%) as
a pale-yellow oil. 1H NMR (300 MHz, Chloroform-d) δ 3.09 – 3.00 (m, 1H), 2.59 (td, J = 11.7, 2.9 Hz, 1H), 2.43
– 2.32 (m, 1H), 1.84 – 0.77 (m, 32H). 13C NMR (75 MHz, CDCl3) δ 57.0, 47.4, 45.3, 40.1, 39.9, 36.6, 33.2,
31.9 (2C), 30.0, 29.8, 27.1(2C), 26.9, 26.8, 26.7, 26.6, 25.1. ESI MS: 278 [M+H]+
4.1.14. (R)-2-(2,2-dicyclohexylethyl)piperidine (R-(+)-Perhexiline, 2).
The title compound was prepared by the same method as described above for preparing S-(-)-Perhexiline, 2,
but by using (R)-2-(2,2-dicyclohexylvinyl)piperidine (11 mg, 0.04 mmol), to obtain a pale-yellow oil (6 mg, 55%).
The analytical and spectroscopic data were identical to those for S-(-)-Perhexiline.
4.2. Antischistosomal activity
4.2.1.Reagents
Dimethyl sulfoxide (DMSO), percoll, foetal bovine serum (FBS), gambogic acid, perhexiline maleate (1:1
racemic mixture of (R) and (S) enantiomers (PHX), carmine-red, and Canada balsam were purchased from
Sigma-Aldrich (Saint Louis, USA); CellTiter-Glo (CTG) reagent from Promega (Madison, USA); Dulbecco-
Modified Eagle’s Medium (DMEM) with or without phenol red, Hepes, L-glutamine from Lonza (Basel,
Switzerland); antibiotic-antimycotic reagent (100×) from Thermo Fisher Scientific (Waltham, USA); Mowiol 4–
88 from Calbiochem was used to prepare Calbiochem mounting medium using a standard protocol.

4.2.2.Ethical statement
The experimental protocols involving the use of animals were reviewed and approved by the Public Veterinary
Health Department of the Italian Ministry of Health (Authorization N. 25/2014-PR and no. 336/2018-PR). All
experiments were conducted in respect to the 3R rules according to the ethical and safety rules and guidelines
for the use of animals in biomedical research provided by the relevant Italian law and European Union Directive
(Italian Legislative Decree 26/2014 and 2010/63/EU) and the international guiding principles for biomedical
research involving animals (Council for the International Organizations of Medical Sciences, Geneva,
Switzerland).
4.2.3.Parasite maintenance
A Puerto Rican strain of S. mansoni, that has been maintained in the laboratory for several decades, was used
throughout this study. An albino strain of Biomphalaria glabrata served as the intermediate host, while ICR
(CD-1) outbred female mice (Envigo, Italy) were used for the mammalian stage. Mice were infected by tail
immersion with 200-300 double sex cercariae and adult worms recovered after 7 weeks by perfusion as
previously described [30].
4.2.4.Schistosomes’ viability assay and egg count
Schistosomula were obtained by mechanical transformation of cercariae and percoll gradient as previously
described [30]. The viability assay of NTS was performed in 96-well black plates with 150-200 NTS/well
cultured in Dulbecco modified Minimum Eagle’s Medium (DMEM) (w/o phenol red) complete medium,
containing 4500 mg/L glucose, 1 mM Hepes pH 6.98 –7.30, 2 mM L-glutamine, 1x antibiotic-antimycotic
reagent and 10% heat inactivated FBS, by CellTiterGlo as previously described [30]. Sample luminescence
levels (proportional to ATP levels) was quantified as RLU (Relative Luminescence Unit) and ATP signal
percentage normalization (% live parasites) was calculated as previously described [30].
Worm pairs, isolated from infected mice, were used for drug assay as previously described [7]. Briefly, 5 worm
pairs were distributed in tissue culture dishes (3.5 cm) in DMEM complete medium (with phenol red). After 24
h parasites were exposed to racemic PHX maleate or the single enantiomers, and cultured for 7 days at 37°C
in an atmosphere of 5% CO2. Vitality was assessed daily under a Leica MZ12.5 stereomicroscope, as
previously described [7, 32] by using the following phenotypical score: 3, plate-attached, good movements,
clear; 2, slower or diminished movements, darkening, minor tegumental damage; 1, movements heavily
lowered, darkening, tegument heavily damaged; 0, dead, lack of any movement. For each sample the scores
of all worms divided by the number of worms present in the dish represented the average scores. For each

compound, three experiments were carried out and compounds were added only once without medium
addition and/or replacement. DMSO (vehicle) treated worms were used as control.
For egg count, the number of eggs laid in vitro by worm couples was assessed three days upon compound
treatment using an inverted Leica DM IL microscope (Leica Microsystems, Wetzlar, Germany) with a gridded-
plate as supporting tool [7, 32].
4.2.5.Carmine red staining and Laser scanning confocal microscopy analysis.
Carmine red staining was performed as previously described [32]. Images were acquired on an Olympus
FV1200 confocal laser scanning microscope using a UplanFLN 40x immersion oil lens (NA = 1.30) with optical
pinhole at 1 AU and a multiline argon laser focused. Excitation was at 488 nm and fluorescence was recovered
in the range 500 nm to 700 nm. Images were collected as single stack. Data analysis was performed using
GraphPad Prism v7.0 software
Acknowledgments
This work was supported by Ministero dell’Istruzione, dell’ Università e della Ricerca (MIUR) (PRIN Project
20154JRJPP to G.C., O.T.S., and G.R.), by the CNR (National Research Council)-CNCCS (Collezione
Nazionale di Composti Chimici e Centro di screening) “Rare, Neglected and Poverty Related Diseases -
Schistodiscovery Project” (DSB.AD011.001.003) to G.R. A special thanks to Stefania Colantoni for mouse
husbandry and Pierluigi Palozzo for dishwashing lab technical support.
Abbreviations
World Health Organization (WHO); praziquantel (PZQ); schistosomula (NTS);carnitine plamitoyltransferase 1
(CPT-1); carnitine plamitoyltransferase 2 (CPT-2); 1,1’-binaphthyl-2,2’-diyl(hydrogen)phosphate (BNPA);
triethylphosphonoacetate (TEPA).
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Highlights
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Both enantiomers of perhexiline were prepared through an enantioselective synthesis
Their antischistosomal activity was assessed in vitro in comparison to the racemate
(R)-(+)-Perhexiline accounts for the majority of the antischistosomal activity