Design, synthesis and anthelmintic activity of 7-keto-sempervirol analogues

Article abstract of DOI:10.1016/j.ejmech.2018.04.032

The plant-derived, diterpenoid 7-keto-sempervirol was recently reported to display moderate activity against larval stages of Schistosoma mansoni (IC₅₀ = 19.1 μM) and Fasciola hepatica (IC₅₀ = 17.7 μM), two related parasitic blood and liver flukes responsible for the neglected tropical diseases schistosomiasis and fascioliasis, respectively. Here, we aimed to increase the potency of 7-keto-sempervirol by total synthesis of 30 structural analogues. Subsequent screening of these new diterpenoids against juvenile and adult lifecycle stages of both parasites as well as the human HepG2 liver cell line and the bovine MDBK kidney cell line revealed structure-activity relationship trends. The most active analogue, 7d, displayed improved dual anthelmintic activity over 7-keto-sempervirol (IC₅₀ ≈ 6 μM for larval blood flukes; IC₅₀ ≈ 3 μM for juvenile liver flukes) and moderate selectivity (SI ≈ 4–5 for blood flukes, 8–13 for liver flukes compared to HepG2 and MDBK cells, respectively). Phenotypic studies using scanning electron microscopy revealed substantial tegumental alterations in both helminth species, supporting the hypothesis that the parasite surface is one of the main targets of this family of molecules. Further modifications of 7d could lead to greater potency and selectivity metrics resulting in a new class of broad-spectrum anthelmintic.

Full text of DOI:10.1016/j.ejmech.2018.04.032

European Journal of Medicinal Chemistry 152 (2018) 87e100
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and anthelmintic activity of 7-keto-sempervirol
analogues
,
Alessandra Crusco a b, Cinzia Bordoni b, Anand Chakroborty a, Kezia C.L. Whatley a,
,
, *
Helen Whiteland a, Andrew D. Westwell b **, Karl F. Hoffmann a
a Institute of Biological, Environmental and Rural Sciences (IBERS), Penglais Campus, Aberystwyth University, Aberystwyth SY23 3DA, United Kingdom
b School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff CF10 3NB, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The plant-derived, diterpenoid 7-keto-sempervirol was recently reported to display moderate activity
Received 20 December 2017
Received in revised form
8 April 2018
Accepted 15 April 2018
Available online 17 April 2018
against larval stages of Schistosoma mansoni (IC50 ¼ 19.1
m
M) and Fasciola hepatica (IC50 ¼ 17.7
mM), two
related parasitic blood and liver flukes responsible for the neglected tropical diseases schistosomiasis and
fascioliasis, respectively. Here, we aimed to increase the potency of 7-keto-sempervirol by total synthesis
of 30 structural analogues. Subsequent screening of these new diterpenoids against juvenile and adult
lifecycle stages of both parasites as well as the human HepG2 liver cell line and the bovine MDBK kidney
cell line revealed structure-activity relationship trends. The most active analogue, 7d, displayed
Keywords:
improved dual anthelmintic activity over 7-keto-sempervirol (IC50 z 6
mM for larval blood flukes;
Natural products
Diterpenoids
Schistosomiasis
Fascioliasis
Antiparasitic
Anthelmintic
IC50 z 3 M for juvenile liver flukes) and moderate selectivity (SI z 4e5 for blood flukes, 8e13 for liver
m
flukes compared to HepG2 and MDBK cells, respectively). Phenotypic studies using scanning electron
microscopy revealed substantial tegumental alterations in both helminth species, supporting the hy-
pothesis that the parasite surface is one of the main targets of this family of molecules. Further modi-
fications of 7d could lead to greater potency and selectivity metrics resulting in a new class of broad-
spectrum anthelmintic.
© 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-
ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
against the immature forms, leading easily to reinfections [5];
moreover, due to its large scale administration, concerns about
Schistosomiasis, caused by infection with blood fluke schisto-
somes, is the most devastating human parasitic disease after ma-
laria considering the number of people currently infected and at
risk of infection [1]. It is a chronic disease of poverty characterized
by pain and disability that, collectively, exacerbates the already
compromised healthcare situation of developing tropical countries
[2]. The number of people infected is approximately 600 million [3]
and, along with 300 thousand deaths per year, schistosomiasis is
considered as the most deadly neglected tropical disease (NTD) [4].
No vaccines are available to prevent infection and, therefore,
treatment of infected individuals is predominantly facilitated by
chemotherapy with praziquantel (PZQ) (Fig. 1A). PZQ is active
against adult worms of all Schistosoma species, but less effective
drug resistance are increasing [5,6].
Another NTD predominantly controlled by a single drug, tricla-
bendazole, is fascioliasis. Ingestion of liver fluke parasites initiates
fascioliasis and leads to up to 17 million human [7] and numerous
livestock animal infections per annum, causing enormous economic
losses in the global beef, lamb and milk industries [8]. In the United
Kingdom alone, an estimated £23 million is lost annually due to
fascioliasis [9]. However, recent reports suggest that this figure
could rapidly increase due to changes in global climate and exten-
sive animal movement [10,11]. Triclabendazole (Fig. 1B) is the only
commercially available drug that is active against both juvenile and
adult liver fluke lifecycle stages. Unfortunately, triclabendazole
resistant Fasciola parasites have been described throughout Asia,
Africa, South America, North America, Australia and Europe [12],
making the discovery of new drugs for combating this human and
animal disease an urgent healthcare priority.
* Corresponding author.
** Corresponding author.
E-mail addresses: WestwellA@cf.ac.uk (A.D. Westwell), krh@aber.ac.uk
(K.F. Hoffmann).
For these reasons, research into the discovery of new anti-
flukicidal drugs is increasing with natural products being an
https://doi.org/10.1016/j.ejmech.2018.04.032
0223-5234/© 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

88
A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
Fig. 1. Anthelmintic compound structures discussed in this study. Illustrated are the structures used for treating schistosomiasis, Praziquantel (A), and for fascioliasis, Tricla-
bendazole (B). The anthelmintic diterpenoid 7-keto-sempervirol and the scaffold numbering system are also shown (C).
exceptionally attractive resource for beginning such a process [13].
Indeed, plant-derived natural products are a well-recognised
source of anti-infective compounds due to natural selection
shaping the production of more toxic and protective compounds to
facilitate survival in a microorganisms-rich environment [14]. One
example of a plant-derived compound isolated from Lycium chi-
nense with bioactivity against both Schistosoma mansoni and Fas-
ciola hepatica is the diterpenoid 7-keto-sempervirol (Fig. 1C) [15].
While this diterpenoid was only moderately potent, it was effective
in killing juvenile and adult forms of both fluke species. Consid-
ering the fact that some endemic areas for schistosomiasis are co-
endemic for fascioliasis and that, in these areas, it is common to
find people with both infections [16e18], a compound with potent
dual anthelmintic activity against multiple lifecycle stages would
be highly desirable.
Fig. 2. Description of the 7-keto-sempervirol analogues. The scheme shows the
basic scaffold of 7-keto-sempervirol (A) and the six families (I-VI) of analogues (B)
designed from it. Each family differs for modification of the scaffold (in red) and in-
cludes several analogues differing for the R group.
In this study, 7-keto-sempervirol was subjected to medicinal
chemistry optimisation, with the aim of exploring whether
different substitutions could increase the dual anthelmintic po-
tency of this natural product. To achieve this, structural related
analogues were obtained by total synthesis using the different
synthetic pathways reported here. The compounds were subse-
quently screened against larval (schistosomula) and adult
S. mansoni blood flukes as well as against newly excysted juvenile
(NEJs), immature and adult F. hepatica liver flukes. The newly
synthesised compounds were also screened against HepG2 liver
human cells and MDBK kidney bovine cells to assess potency and
selectivity as well as to deduce preliminary structure activity re-
lationships (SARs). The results of compound synthesis and bio-
activities are presented and discussed here.
obtained by oxidation in the presence of tetrapropylammonium
perruthenate (TPAP) and N-methylmorpholine N-oxide (NMO)
(Scheme 1).
From the synthesised keto-analogues, two different cyclization
methods were performed to obtain the tricyclic diterpenoid scaf-
fold with a keto-group in position six (Scheme 2). The first method
(Scheme 2A) used the Lewis acid BBr3 to facilitate the synthesis of
the trans diastereoisomers (3a-c) as described by Huang et al. [19],
while the second method (Scheme 2B) used RuCl3 and AgOTf to
obtain the cis diastereoisomer (4a-c), according to the method of
Youn et al. [20]. However, this set of compounds showed instability
when exposed to oxygen at room temperature and, therefore, their
biological activity was not measured. Indeed, changes in colour and
composition of the 6-keto compounds were observed as quickly as
two (and up to seven) days post synthesis (See Supplementary
information). Analysis of the resulting mixtures revealed high
levels of autoxidation (cis > trans) that, upon subsequent purifica-
tion and characterisation, revealed the presence of a keto group in
positions 6 and 7 (Scheme 2C). These 6,7-diketo derivatives (5a-c)
were more stable than the original 6-keto compounds and, for this
reason, are considered a new family of analogues. Literature pre-
cedent for spontaneous oxidation in terpene-based systems,
including mechanistic studies, is known [21].
2. Results and discussion
2.1. Chemistry
Based on the previously reported anthelmintic activity of the
diterpenoid 7-keto-sempervirol, we investigated the impact of
chemical structure modification on activity to obtain more potent
and selective analogues. To achieve this, six families of compounds
containing the basic scaffold of 7-keto-sempervirol were syn-
thesised with each family containing distinct aromatic sub-
stitutions (Fig. 2).
Diverse synthetic strategies were adopted for each family. Non-
cyclised analogues lacking bond C9-C10 were obtained from the
coupling between commercially available beta-cyclocitral and
substituted benzyl chlorides, as described by Huang et al. [19], to
generate the first set of analogues as secondary alcohols (1a-f)
(Scheme 1). Corresponding keto-analogues (2a-f) were then
The other three families of compounds were obtained from one
synthetic pathway according to the method described by Surendra
and Corey [22] (Scheme 3). Substituted Grignard compounds were
added to geraniol acetate in the presence of Li2CuCl4 (Scheme 3A)
and the products obtained (6a-c) were then cyclised through an
enantioselective reaction catalysed by the complex (R)-BINOL-
SbCl5; the resulting hydrophobic compounds (7a-d) lacked a keto

A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
89
Scheme 1. Synthesis of diterpenoid analogues 1a-f and 2a-f. The scheme shows the synthesis of the non-cyclised analogues 1a-f and 2a-f from beta-cyclocitral using lithiation
and oxidation chemistry.
Scheme 2. Synthesis of diterpenoid analogues 3a-c and 5a-c. The scheme shows the synthesis of 6-keto (3a-c) and 6,7-diketo (5a-c) analogues based on diastereoselective ring
closure and subsequent autoxidation of the cis-isomer.
group (Scheme 3B). Compounds 7a-d were then oxidized by CrO3
to generate the corresponding 7-keto analogues 8a-d (Scheme 3C).
For our studies we made use of the commercially available chiral
ligand (R)-BINOL, as opposed to the o,o’-dichloro-BINOL employed
by Surendra and Corey in their reported enantioselective synthesis
[22]. Preliminary polarimeter analysis of our final compounds 8a-

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A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
Scheme 3. Synthesis of diterpenoid analogues 6a-c, 7a-d, 8a-d. The scheme shows the synthesis of no-keto (7a-d), 7-keto analogues (8a-d) and their synthetic intermediates (6a-
c) via addition of benzylic Grignard agent to geraniol acetate followed by enantioselective antimony-promoted cyclization and chemical oxidation. Compounds 7d and 8d were
obtained by demethylation of 7b and 8b, respectively, by BBr3 in DCM.
d indicated enantiomeric excesses (ee's) in the range of 20e30%
(data not shown). For the purpose of this screening analysis, these
compounds should be treated as racemic (as is the case of the
standard of care drug praziquantel [5]). Enantiomeric synthesis
and/or enantiomer resolution to study the contributory biological
activity of single enantiomers will form the basis of future studies.
control wells) included smaller eggs lacking both lateral spines and
regular autofluorescence (Fig. 4B and C). While the significance of
abnormally produced eggs is currently unknown, the substantial
‘anti-fecundity’ activity is noteworthy as schistosome eggs are
responsible for transmission in endemic areas and are the major
cause of schistosomiasis-attributable chronic disease in infected
humans [6].
Egg images obtained by fluorescence microscopy are also shown
(under natural light in the left panels and using a GFP filter in the
right panels). Eggs collected from control wells (B) are oval, contain
a lateral spine and exhibit a homogeneous auto-fluorescent surface.
Eggs collected from treatment wells (6.25 mM 7d) are abnormally
shaped (C), smaller and are characterized by loss of the lateral spine
and irregular auto-fluorescence.
The six most active compounds on schistosomula (2c, 2d, 5a, 5b,
7d and 8c) were also screened against the related trematode,
F. hepatica (Fig. 5). Initial screening on F. hepatica newly excysted
juveniles (NEJs) was performed at 25 and 12.5 mM and demon-
strated that, similarly to schistosomula, the most active anti-NEJ
compounds were 7d and 8c (Fig. 5A and B).
Subsequently, titrations of these two compounds (and of 7ks as
reference compound) allowed the calculation of the respective
IC50s, with 7d exhibiting values of 3.2 and 2.5 mM for phenotype
and motility (Table 2). Both 7d and 8c were next screened on
immature and mature liver fluke lifecycle stages (Fig. 6; Supple-
mentary Information, S2), with 7d again showing the most potent
2.2. Biological activities
All stable compounds (1a-f, 2a-f, 5a-c, 6a-c, 7a-d, 8a-d) were
first screened for anthelmintic activity on the larval stage (schis-
tosomula) of S. mansoni using an integrated high-throughput, high
content image (HCI) analysis platform called Roboworm [23,24].
Here, compounds are quantified according to their ability to affect
both phenotype and motility of the treated larva. Any compound
that leads to a score threshold that is equal to or falls below the
values of ꢀ0.15 and ꢀ0.35 for phenotype and motility, respectively,
in ꢁ70% of the assayed larva is considered a hit [25]. As all com-
pounds tested were hits at 50
schistosomula screens were performed at 25 and 10
The gold thiolate anti-parasitic compound Auranofin was used
as a positive control [27]. At 25 M, six compounds (2c, 2d, 5a, 5b,
7d and 8c) were considered hits and at 10 M, only compound 7d
was still active. This compound was later screened at 1 M but
showed no anti-schistosomula activity (data not shown). The
estimated IC50 values for 7d phenotype and motility were 6.8 and
6.3 mM, respectively and represent the most potent values when
m
M (data not shown), subsequent
m
M (Fig. 3).
m
m
m
activity (IC50 values of 10.4 and 28.4 mM, respectively (Table 2)).
Surrogate selectivity was assessed by screening all synthesised
compounds on HepG2 liver human cells using the MTT assay. Here,
all diterpenoids showed moderate selectivity, being on average 3e5
times more toxic on blood or liver flukes than human cells (Tables 1
and 2). Due to the veterinary importance of these parasitic diseases
[9,17,28], compounds were also screened against MDBK kidney
bovine cells and showed a toxicity comparable or lower than that
observed for HepG2 cells (Tables 1 and 2).
Looking at the difference in the anti-schistosomula activity of
the analogues (where the most complete dataset has been
collected), some trends in structure-activity relationship were
found. Considering the non-cyclised analogues (1a-f and 2a-f), the
alcohol series 1a-f was generally less active on schistosomula than
the correspondent ketone series 2a-f. With regards to the aromatic
portion, methoxy, iso-propyl and tert-butyl substitutions in posi-
tion 12 (diterpenoid numeration from Fig. 1) increased the
anthelmintic activity when compared to the non-substituted
compared to all other analogues (Table 1).
A general observation was that most of the diterpenoids
screened had a greater effect on schistosomula motility rather than
phenotype. The lead compound 7-keto-sempervirol (7ks) was
additionally screened by Roboworm and the anthelmintic activity,
previously quantified by a different method [15], was comparable.
The six compounds active against schistosomula at 25 mM were
subsequently screened on adult male and female worm pairs
(Table 1). Amongst these six, the most active analogue was again
7d, able to kill all parasites at 50
mM and to significantly affect their
motility until 12.5 M (Fig. 4). While there was no significant
m
variation in the ability of 7d to differentially affect male and female
worm motility, the compound inhibited egg production even at the
lowest concentration tested, 6.25 mM (Fig. 4A; for adult worm
screening of the other compounds, see Supplementary Information,
S1). At this concentration, only eggs with abnormal phenotypes
were found; these phenotypes (when compared to eggs from

A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
91
Fig. 3. Diterpenoid screening on S. mansoni schistosomula. Schistosome (n ¼ 120)/diterpenoid co-cultures were incubated for 72 h at 37 ꢂC in a humidified atmosphere con-
taining 5% CO2. The effect that each of these diterpenoids had on schistosomula phenotype and motility at 25 mM (A) and 10 mM (B) is illustrated and compared with the negative
(DMSO) and the positive controls (Auranofin). Compounds are scored by the high throughput platform Roboworm according to their effect on schistosomula phenotype and motility
(indicated by change in red outlines on figures). Hit compounds (within the hit threshold) affect ꢁ 70% of the larvae. Each point represents the average score of two replicates. Z0-
scores [26] were calculated for these anti-schistosomula screens and included values corresponding to 0.69159 for phenotype and 0.48074 for motility. Images of the parasites after
treatment with the controls and the six hits at 25 mM are also shown (C). *Result from a separate assay where the anthelmintic activity of 7-keto-sempervirol (7ks) was assessed.
analogues or to the ones with the methoxy group in 11 or 13. As a
result, the most active compounds were 2c and 2d. This relation-
ship was confirmed in the 7-keto series 8a-d with the most active
compound being 8c. However, in the di-keto series 5a-c, the most
active compound was 5b, which contained a methoxy group in
position 13. This structure activity relationship, supported by
different schistosomula phenotypes (Fig. 3), could perhaps indicate
a different mechanism of action. The strongest anti-schistosomal
(both schistosomula and adult worms) activity was associated
with a hydroxy group in position 12 as demonstrated by the most
potent compound 7d. However, when other substituents were
added along with the hydroxy group (like those found in 7ks and
8d), the anti-schistosomal activity was reduced. Similar findings
were observed for F. hepatica NEJs, with the best activity against
this liver fluke being associated with 7d and 8c.
To begin developing hypotheses regarding 7d's mechanism(s) of
action, Scanning Electron Microscopy (SEM) was used. Here, sur-
face integrity of parasites treated with a sublethal concentration of
7d (25
mM for S. mansoni adults, 40 mM for F. hepatica adults,
13.3 M for F. hepatica immatures) was specifically examined
m
(Fig. 7). In the case of adult blood fluke parasites, S. mansoni males
treated with 7d displayed substantial tegumental disruption
throughout the whole body, with the spinous tubercles losing their
natural shape when compared to the control (Fig. 7A and B). A
similar phenotype, but less severe, was found in 7d-treated female
schistosomes (not shown). Compound 7d's apparent gender biased

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A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
Table 1
Diterpenoid anti-schistosomal activity and cell cytotoxicity summary.
Compounds IC50 on schistosomula IC50 on IC50 on adult
CC50 on HepG2 CC50 on MDBK Selectivity Index HepG2 cells Selectivity Index MDBK cells
phenotype (
mM)
schistosomula
worms (
mM)
cells (
m
M)
cells (
m
M)
(Phenotype/Motility)
(Phenotype/Motility)
motility (mM)
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
5a
5b
5c
6a
6b
6c
7b
7c
7d
8a
8b
8c
8d
7ks
43.6
39.9
42.5
39.9
40.2
43.8
43.8
41.4
25.3
25.4
28.0
43.7
25.0
22.9
43.7
43.8
43.9
39.4
43.4
44.1
6.8
21.5
23.5
26.4
27.3
25.6
43.6
26.0
28.1
19.1
18.2
18.9
27.2
13.0
6.4
26.5
43.8
27.1
40.1
27.9
26.6
6.3
NA
NA
NA
NA
NA
NA
NA
NA
>50
>50
NA
NA
>50
>50
NA
NA
NA
NA
NA
NA
19.9
NA
NA
>50
NA
NA
>100
>100
>100
>100
>100
>100
>100
>100
>100
71.7
>100
>100
>100
84.9
>100
>100
>100
>100
89.0
>100
>100
>100
>100
>100
>100
>100
>100
91.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
32.5
NA
NA
NA
NA
NA
NA
NA
NA
NA
2.8/3.9
NA
NA
NA
3.7/13.2
NA
NA
NA
NA
2.1/3.3
NA
3.8/4.1
2.1/5.7
NA
2.3/3.2
2.3/3.0
3.7/3.1
NA
NA
NA
NA
NA
NA
NA
NA
3.6/4.8
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
4.8/5.2
NA
NA
5.0/6.9
NA
>100
25.6
84.2
>100
33.1
65.1
39.5
28.6
14.2
28.0
21.9
14.7
17.6
10.3
21.9
26.2
>100
>100
70.6
>100
63.8
80.0
2.9/2.4
Estimated IC50s calculated from diterpenoid dose response curves for S. mansoni schistosomula phenotype and motility and S. mansoni adult worm motility. Estimated CC50
s
calculated from diterpenoid dose response curves for HepG2 and MDBK cells and selectivity index of activity on schistosomula when compared to both cell lines. The values
are results of experiments in duplicate (for helminths) or triplicate (for cells).
effect (with regards to surface architecture) is in agreement with
other studies and could be associated with females having a less
absorptive surface [29]. In the case of liver flukes, 7d also caused
noticeable damage to hermaphroditic adult F. hepatica, with the
ventral surface decorated by swollen tegumental ridges and
smaller spines (Fig. 7C and D).
Swelling of the tegument is likely due to an osmotic effect after
changes in the permeability of the membrane surface and/or in the
osmoregulatory system, often linked to unbalanced ion distribution
[30]. Even more pronounced was the damage of 7d to F. hepatica
immature flukes, where a complete loss of spines on the ventral
surface was observed together with sloughing of the tegument and
exposure of the basal lamina (Fig. 7E and F).
These findings, together with other data reported in the litera-
ture [2,31,32], support the hypothesis that one of the main targets
of terpenoid like molecules is the tegument, a vital structure for
anti-parasitic drugs because of its barrier function in host in-
teractions [29]. However, the decreased motility and the inhibition
of phenotypically normal egg production could also indicate other
possible mechanisms of action as hypothesised for 7ks [15].
F. hepatica NEJs, with IC50 of 2.5e3.2 mM (phenotype and motility,
respectively) and selectivity index of 8e10.2 when compared with
HepG2 cells and 10.2e13.0 with MDBK cells. This compound
additionally displayed activity against immature and adult liver
fluke parasites, with estimated IC50 values of 10.4 mM and 28.4 mM.
SEM studies of parasites treated with 7d, revealed tegumental al-
terations that could be the basis for the mechanism of action.
The results of this study showed the potential of this class of
diterpenoids as promising new anthelmintic compounds and how a
medicinal chemistry optimisation approach can lead to the iden-
tification of more potent molecules. Further studies deciphering
mechanisms of action and improving the physicochemical prop-
erties should be pursued to help develop this promising class of
molecules.
4. Experimental section
4.1. Chemistry
Commercial reagents and solvents were purchased from Sigma
Aldrich or Fisher Scientific and used without further purification. 7-
keto-sempervirol was obtained as previously described [15]. The
synthesised compounds were characterized by high resolution
mass HRMS, 1H, 13C and two-dimensional nuclear Magnetic Reso-
nance (NMR) spectroscopy. NMR spectra were recorded on a Bruker
Avance 500 MHz NMR spectrometer in CDCl3 solution referenced to
the solvent residual peak. The reactions were monitored by Thin
Layer Chromatography (TLC) on pre-coated TLC aluminium sheets
of silica gel. The compounds were purified by column chromatog-
raphy on silica gel (35e70 mesh) using the eluents indicated. Mass
spectrometry was performed on a Bruker Daltonics microTof-LC
system or on an Orbitrap Fusion Thermo Scientific with a Dionex
UltiMate 3000 UHPLC system.
3. Conclusions
In conclusion, 30 analogues of the diterpenoid 7-keto-semper-
virol were synthesised and 25 of them initially screened for
anthelmintic activity on the larval stage of the parasite S. mansoni.
Amongst these 25 compounds, the best was 7d having an IC50 of
6.8e6.3 mM (phenotype and motility, respectively) and selectivity
index of 3.8e4.1 when compared with HepG2 liver human cells and
4.8e5.2 when compared with MDBK kidney bovine cells. This
compound was also active against adult schistosomes
(IC50 ¼ 19.9
mM) and inhibited egg production at the lowest test
concentration. More potent and selective activity was displayed on

A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
93
Fig. 4. Anthelmintic screening of compound 7d on adult S. mansoni worm pairs. (A) Adult schistosome worm pairs (3 pairs/well) were co-cultivated with 7d (50
m
M - 6.25
m
M)
for 72 h at 37 ꢂC in a humidified atmosphere containing 5% CO2. The effect that 7d had on schistosome motility and egg production was quantified according to the methodologies.
Each bar represents the mean motility score SD of 12 adult worms (6 worms x 2 independent experiments) when treated with different concentrations and compared to the
negative (DMSO) and positive control (Auranofin). The overlying line graph represents the mean egg count SD. * Significant difference compared to DMSO controls (p < 0.05).
For each family of molecules, the general synthetic method is
reported together with an example of compound characterisation.
All the compound characterisation can be found in the Supple-
mentary Information.
4.1.1.1. 2-Phenyl-1-(2,6,6-trimethylcyclohex-1-en-1-yl)ethan-1-ol
(1a). Colourless oil, yield: 60%. 1H NMR:
ArH), 4.48 (1H, dd, J ¼ 10.4, 3.15 Hz, CHOH), 3.17 (1H, dd, J ¼ 14.0,
10.6 Hz, CH2Ph), 2.89 (1H, dd, J ¼ 14 Hz, 3.2 Hz, CH2Ph), 2.05e2.00
(2H, m, CH2), 2.01 (3H, s, CH3C¼C), 1.63e1.61 (2H, m, CH2),
1.50e1.48 (2H, m, CH2), 1.14 (3H, s, CH3), 1.05 (3H, s, CH3); 13C NMR:
d
7.37e7.25 (5H, m, 5 ꢃ
d
139.92 (C), 139.14 (C), 131.87 (C), 129.33 (2 ꢃ ArCH), 128.60 (2 ꢃ
4.1.1. General method for synthesis of compounds 1a-f [19]
ArCH), 126.44 (ArCH), 72.37 (CHOH), 43.23 (CH2), 39.95 (CH2), 34.86
(C(CH3)2), 34.12 (CH2), 28.67 (CH3), 28.08 (CH3), 21.30 (CH3), 19.34
(CH2); HRMS-ESI m/z: [M þ Na]þ calcd for C17H24ONa 267.1719,
found 267.1720.
Under nitrogen, a mixture of naphthalene (3.75 eq) and lithium
(3.75 eq) in anhydrous THF was stirred at room temperature for 2 h.
Then, a solution of beta-cyclocitral (1.1 eq) and the substituted
benzyl chloride (1 eq) was added dropwise via syringe at 0 ꢂC. The
reaction mixture was stirred at room temperature for 2 h, then
diluted with diethyl ether and treated with saturated NH4Cl. The
organic phase was separated and the aqueous phase extracted with
ethyl acetate. The combined organic layers were washed with
brine, dried over Na2SO4 and concentrated under reduced pressure.
The crude product was purified by silica gel column chromatog-
raphy (4e10% ethyl acetate in petroleum ether).
4.1.2. General method for synthesis of compounds 2a-f
The previously obtained alcohol (1 eq) and N-methylmorpho-
line N-oxide (NMO) (1.5 eq) were dissolved in anhydrous
dichloromethane under nitrogen. After stirring for 15 min, tetra-
propylammonium perruthenate (TPAP) (0.05 eq) dissolved in
dichloromethane was added and the mixture stirred overnight. The
reaction mixture was then diluted with dichloromethane, and

94
A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
Fig. 5. Anthelmintic screening of diterpenoids on F. hepatica newly excysted juveniles (NEJs). NEJs (n ¼ 25)/diterpenoid co-cultures were incubated for 72 h at 37 ꢂC in a hu-
midified atmosphere containing 5% CO2. The effect that each of these diterpenoids had on NEJ phenotype and motility at 25 mM (A) and 12.5 mM (B) is illustrated and compared with
the negative (DMSO) and the positive controls (Triclabendazole). Each bar graphs represents the average score SD (n ¼ 25) for phenotype (green) and motility (blue). Compounds
7d and 8c showed the greatest significant activity at the lowest test concentration. Images of the parasites after treatment with the controls and the 2 hits at 12.5
(C). * Significant difference with DMSO control (p < 0.05).
mM are also shown
Table 2
Diterpenoid anti-Fasciola activity and selectivity summary.
Compounds IC50 on NEJs
phenotype (
IC50 on NEJs
IC50 on
immatures (
IC50 on adults Selectivity Index HepG2 cells
Selectivity Index MDBK cells
(Phenotype/Motility)
mM)
motility (
m
M)
mM)
(
m
M)
(Phenotype/Motility)
7d
8c
7ks
3.2
9.9
16.8
2.5
8.4
13.1
10.4
14.6
NA
28.4
39.9
NA
8/10.2
3.3/3.9
4.8/6.1
10.2/13.0
7.1/8.3
3.8/4.9
Estimated IC50s calculated from diterpenoid dose response curves for F. hepatica NEJ phenotype and motility, as well as motility for both F. hepatica immature and adult flukes.
Selectivity index when compared to CC50s on HepG2 and MDBK cells is also illustrated.
washed with sodium sulphite solution and brine. The organic layer
was dried over Na2SO4 and concentrated under reduced pressure.
The crude product was purified by silica gel column chromatog-
raphy (4e10% ethyl acetate in petroleum ether).
4.1.3. General method for synthesis of compounds 3a-c [19]
To a solution of the previously obtained ketone (1 eq) in anhy-
drous dichloromethane was added a solution of BBr3 1 M (6 eq) in
dichloromethane dropwise at ꢀ78 ꢂC, under nitrogen. The resulting
mixture was stirred for 30 min and then allowed to reach 0 ꢂC
before continued stirring for 3 h. The reaction was quenched
carefully with NaHCO3 solution and the aqueous phase was
extracted with ethyl acetate. Organic layers were washed with
brine and dried over Na2SO4. The crude product was purified by
silica gel column chromatography (5e30% ethyl acetate in petro-
leum ether).
4.1.2.1. 2-Phenyl-1-(2,6,6-trimethylcyclohex-1-en-1-yl)ethan-1-one
(2a). Colourless oil, yield: 72%.1H NMR:
d
7.25e7.22 (2H, m, 2 ꢃ ArH),
7.17e7.13 (3H, m, 3 ꢃ ArH), 3.77 (2H, s, CH2Ph), 1.90 (2H, t, J ¼ 6.3 Hz,
CH2),1.62e1.57 (2H, m, CH2),1.51 (3H, s, CH3, C¼C),1.39e1.37 (2H, m,
CH2), 1.01 (6H, s, 2 ꢃ CH3); 13C NMR:
d
208.29 (C¼O), 143.31 (C),
134.03 (C), 129.98 (2 ꢃ ArCH), 129.62 (C), 128.42 (2 ꢃ ArCH), 126.86
(ArCH), 52.16 (CH2Ph), 38.96 (CH2), 33.46 (C(CH3)2), 31.23 (CH2),
28.87 (2 ꢃ CH3), 21.18 (CH3), 18.93 (CH2); HRMS-ESI m/z: [M þ Na]þ
calcd for C17H22ONa 265.1563, found 265.1564.
4.1.3.1. Trans 6-oxopodocarpa-8,11,13-triene (3a). Colourless oil,
yield: 82%. 1H NMR:
d
7.35 (1H, d, J ¼ 7.7 Hz, ArH), 7.26 (1H, td,

A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
95
Fig. 6. Anthelmintic screening of compound 7d on F. hepatica immature and adult parasites. Immature (3/well) and adult (2/well) parasites were co-cultivated with 7d (40
m
M -
4.4 m
M) for 72 h at 37 ꢂC in a humidified atmosphere containing 5% CO2. The effect that the compounds had on the immature (A) and adult (B) parasites was quantified according to
the methodologies. Each bar represents the mean motility score SD of the worms when treated with different concentrations and compared to the negative (DMSO) and positive
control (Triclabendazole). * Significant difference compared to DMSO controls (p < 0.05).
J ¼ 7.7, 0.9 Hz, ArH), 7.20 (1H, td, J ¼ 7.4 Hz,1.3 Hz, ArH), 7.07 (1H, dd,
J ¼ 7.4 Hz, 0.9 Hz, ArH), 3.64 (2H, s, CH2-7), 2.41 (1H, s, CH-5),
2.41e2.36 (1H, m, CH2), 1.80e1.67 (4H, m, 2 ꢃ CH2), 1.46e1.43 (1H,
ArH), 2.68 (1H, s, CH-5), 2.62e2.59 (1H, m, CH2), 1.63e1.52 (2H, m,
CH2), 1.45e1.37 (1H, m, CH2), 1.37e1.32 (2H, m, CH2), 1.23 (3H, s,
CH3), 0.97 (3H, s, CH3), 0.38 (3H, s, CH3); 13C NMR:
d
198.94 (C¼O),
m, CH2), 1.34 (3H, s, CH3C¼C), 1.19 (3H, s, CH3), 1.12 (3H, s, CH3); 13
C
181.48 (C¼O), 150.00 (ArC), 135.73 (ArCH), 133.84 (ArC), 130.36
(ArCH), 127.55 (ArCH), 124.82 (ArCH), 68.97 (CH-5), 42.05 (CH2),
39.83 (C), 38.83 (CH3), 36.18 (CH2), 35.62 (C), 31.45 (CH3), 24.20
(CH3), 18.92 (CH2); HRMS-ESI m/z: [M þ Na]þ calcd for C17H20O2Na
279.1356, found 279.1357.
NMR:
d
209.47 (C¼O), 149.00 (ArC), 132.44 (ArC), 128.47 (ArCH),
126.95 (ArCH), 126.45 (ArCH), 123.68 (ArCH), 62.55 (CH-5), 45.12
(CH2), 42.91 (CH2), 40.58 (CH), 38.68 (CH2), 33.08 (CH3), 32.76 (C),
24.62 (CH3), 21.65 (CH3), 18.76 (CH2); HRMS(ESI) m/z: [M þ Na]þ
calcd for C17H22ONa 265.1563, found 265.1565.
4.1.6. General method for synthesis of compounds 6a-c [22]
To a solution of geraniol acetate (1 eq) in THF at 0 ꢂC, 0.1 M
Li2CuCl4 solution in THF (0.1 eq) was added dropwise and then a
solution of Grignard reagent (2 eq) was added dropwise at 0 ꢂC.
After 2 h at 0 ꢂC, the mixture was quenched with saturated aqueous
NH4Cl solution and the aqueous layer extracted with diethyl ether.
Combined organic layers were washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (0e1% ethyl acetate
in petroleum ether) to afford the coupling product.
4.1.4. General method for synthesis of compounds 4a-c [20]
A mixture of AgOTf (0.4 eq) and RuCl3 x H2O (0.2 eq) in dry
dichloroethane was stirred for 1 h. The previously obtained ketone
(1 eq) was then added and the resulting solution was stirred
overnight at 80 ꢂC. The solvent was evaporated and crude product
purified by silica gel column chromatography using dichloro-
methane and petroleum ether (5e10% ethyl acetate in petroleum
ether).
4.1.4.1. Cis 6-oxopodocarpa-8,11,13-triene (4a). Colourless oil, yield:
51%. 1H NMR:
d
7.34 (1H, d, J ¼ 7.8 Hz, ArH), 7.26 (1H, t, J ¼ 7.4 Hz,
4.1.6.1. Trans
(4,8-Dimethylnona-3,7-dien-1-yl)benzene
(6a).
Colourless oil, yield: 90%. 1H NMR:
d
7.32 (2H, t, J ¼ 7.5 Hz, 2 ꢃ ArH),
ArH), 7.19 (1H, t, J ¼ 7.4 Hz, ArH), 7.07 (1H, d, J ¼ 7.4 Hz, ArH),
3.74e3.51 (2H, m, CH2-7), 2.56e2.53 (1H, m, CH2), 2.12 (1H, s, CH-
5), 1.55e1.53 (1H, m, CH2), 1.33e1.25 (4H, m, 2 ꢃ CH2), 1.08 (3H, s,
7.25e7.21 (3H, m, 3 ꢃ ArH), 5.24 (1H, t, J ¼ 7.0 Hz, CH ¼ ), 5.15 (1H, t,
J ¼ 7.0 Hz, CH ¼ ), 2.69 (2H, t, J ¼ 8.0 Hz, CH2), 2.38e2.34 (2H, m,
CH2), 2.14e2.10 (2H, m, CH2), 2.05e2.02 (2H, m, CH2), 1.74 (3H, s,
CH3), 0.95 (3H, s, CH3), 0.31 (3H, s, CH3); 13C NMR:
d
212.38 (C¼O),
CH3), 1.66 (3H, s, CH3), 1.61 (3H, s, CH3); 13C NMR:
d 142.54 (C),
141.65 (ArC), 134.22 (ArC), 128.69 (ArCH), 127.24 (ArCH), 126.50
(ArCH), 124.05 (ArCH), 66.55 (CH-5), 44.17 (CH2), 42.32 (CH2), 38.90
(C), 36.13 (CH2), 34.40 (C), 33.42 (CH3), 32.28 (CH3), 22.58 (CH3),
18.94 (CH2); HRMS(ESI) m/z: [M þ Na]þ calcd for C17H22ONa
265.1563, found 265.1565.
135.87 (C), 131.42 (C), 128.61 (2 ꢃ CH), 128.33 (2 ꢃ CH), 125.78 (CH),
124.50 (CH), 123.75 (CH), 39.86 (CH2), 36.29 (CH2), 30.11 (CH2),
26.87 (CH2), 25.84 (CH3), 17.83 (CH3), 16.09 (CH3); HRMS-ESI m/z:
[M þ Na]þ calcd for C17H22O2 258.1620, found 258.1625.
4.1.5. General method for synthesis of compounds 5a-c
After exposure to air and light at room temperature for 2e7
days, changes in colour, from colourless to yellow, were noticed on
the cis-6-keto compounds. The new di-keto compounds generated
were isolated after purification by column chromatography (8e15%
ethyl acetate in petroleum ether). The oxidation procedure was
repeated using the procedure followed for compounds 8a-c (see
below).
4.1.7. General method for synthesis of compounds 7a-c [22]
A solution of R-BINOL (0.5 eq) in dry dichloromethane was
cooled to ꢀ78 ꢂC and then 1 M SbCl5 solution in dichloromethane
(0.5 eq) was added and the mixture was stirred for
15 min at ꢀ78 ꢂC. A precooled solution of the previously obtained
alkene (1 eq) in dry dichloromethane was added at ꢀ78 ꢂC and the
reaction mixture was stirred at the same temperature for 5 h. The
reaction mixture was quenched with saturated NaHCO3 solution
and the aqueous layer was extracted with diethyl ether. Combined
organic layers were washed with brine, dried over Na2SO4 and
concentrated under reduced pressure. The crude product was pu-
rified by silica gel chromatography (1e2% ethyl acetate in
4.1.5.1. Cis 6,7-dioxopodocarpa-8,11,13-triene (5a). Yellow solid,
yield: 95%. 1H NMR:
(1H, m, ArH), 7.48 (1H, d, J ¼ 7.9 Hz, ArH), 7.41 (td, J ¼ 7.8, 1.0 Hz,
d
8.13 (1H, dd, J ¼ 7.8 Hz,1.3 Hz, ArH), 7.68e7.65

96
A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
Fig. 7. Scanning electron microscopy (SEM) images of S. mansoni and F. hepatica worms co-cultivated with 7d. SEM images of the adult blood flukes after treatment with
control DMSO (A) and with compound 7d at 25 M (B). The tubercles of the treated parasite appear damaged when compared to the control. Images of adult liver flukes after
treatment with control DMSO (C) and 7d at 40 M (D) are also shown. The ventral area of the parasite contains smaller spines within the swollen tegument. Finally, comparison of
control (E) and compound 7d at 13.3 M (F) on immature liver flukes showed more substantial loss of spines and sloughing of the tegument on the ventral area of treated parasites.
m
m
m

A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
97
petroleum ether) to afford the cyclised product.
4.2. Biological evaluation
4.2.1. Compound handling and storage
4.1.7.1. Trans
13-methoxypodocarpa-8,11,13-triene
(7b). White
solid, yield: 63%. 1H NMR:
d
7.18 (1H, d, J ¼ 8.7 Hz, ArH), 6.71 (1H,
In preparation for biological assays conducted, all compounds
were solubilised in DMSO (Fisher Scientific, Loughborough, UK) and
stored at ꢀ20 ꢂC at a stock concentration of 16 mM.
dd, J ¼ 8.7, 2.8 Hz, ArH), 6.58 (1H, d, J ¼ 2.7 Hz, ArH), 3.77 (3H, s,
OCH3), 2.95e2.82 (2H, m, CH2), 2.28e2.26 (1H, m, CH2), 1.90e1.86
(1H, m, CH2), 1.74e1.67 (2H, m, CH2), 1.62e1.58 (1H, m, CH2),
1.50e1.48 (1H, m, CH2), 1.41e1.37 (1H, m, CH2), 1.35e1.32 (1H, m,
CH), 1.26e1.23 (1H, m, CH2), 1.18 (3H, s, CH3), 0.96 (3H, s, CH3), 0.94
4.2.2. Schistosoma mansoni schistosomula culture and compound
screening
(3H, s, CH3); 13C NMR:
d 157.08 (ArC), 142.86 (ArC), 136.70 (ArC),
S. mansoni (Puerto Rican Strain, Naval Medical Research Institute
- NMRI) cercariae were collected from infected Biomphalaria glab-
rata (NMRI) snails after exposure to 2 h of light at 26 ꢂC and then
mechanically transformed into schistosomula as described [33].
Newly transformed schistosomula were prepared for 72 h high
throughput screening (HTS) in 384-well black-sided microtiter
plates (Perkin Elmer, MA, USA) as described in Nur-e-Alam [24],
with a final DMSO concentration of 0.625%. The effect of com-
pounds on 72 h cultured schistosomula was deduced by analysing
the effect on both motility and phenotype of treated schistosomula
using the image analysis model described by Paveley [25].
125.60 (ArCH), 113.31 (ArCH), 111.95 (ArCH), 55.24 (OCH3), 50.72
(CH), 41.84 (CH2), 39.16 (CH2), 37.39 (C), 33.53 (C), 33.48 (CH3),
30.83 (CH2), 25.09 (CH3), 21.73 (CH3), 19.47 (CH2), 19.17 (CH2);
HRMS-ESI m/z: [2 M þ Na]þ calcd for C36H52O2Na 539.3860, found
539.3863.
4.1.8. General method for synthesis of compounds 8a-c
The previously obtained no-keto diterpenoid 7a-c was added
dropwise to a solution of CrO3 (1 eq) in acetic acid at room tem-
perature. After overnight stirring, the reaction mixture was
neutralized with NaHCO3 and the aqueous phase extracted with
diethyl ether. The organic layer was washed with brine, dried over
Na2SO4 and concentrated under reduced pressure. The crude
product was purified by silica gel column chromatography (2e5%
ethyl acetate in petroleum ether).
4.2.3. Schistosoma mansoni adult worms culture and compound
screening
MF-1 mice (Harlan, UK) were infected by percutaneous expo-
sure to 200 cercariae. Mature adult parasites were recovered from
hepatic portal veins by perfusion as described by Smithers and
Terry [34] seven weeks post infection. Three adult male and three
adult female worms (i.e. three worm pairs) were cultured per well
in a 48-well tissue culture plate (Fisher Scientific, Loughborough,
UK) containing 1 ml of modified DMEM (Gibco, Paisley, UK) media
(containing 10% v/v Hepes (Sigma-Aldrich, Gillingham, UK), 10% v/v
4.1.8.1. Trans 7-oxopodocarpa-8,11,13-triene (8a). Colourless oil,
yield: 20% (2 steps). 1H NMR:
d
8.01 (1H, dd, J ¼ 7.8, 1.6 Hz, ArH),
7.52 (1H, dd, J ¼ 8.0, 1.6 Hz, ArH), 7.38 (1H, d, J ¼ 7.9 Hz, ArH),
7.30e7.26 (1H, m, ArH), 2.76e2.62 (2H, m, CH2), 2.36e2.34 (1H, m,
CH2), 1.90 (1H, dd, J ¼ 13.8, 4.2 Hz, CH), 1.79 (1H, tt, J ¼ 13.7, 3.3 Hz,
CH2), 1.72e1.68 (1H, m, CH2), 1.58e1.55 (2H, m, CH2), 1.30e1.25 (1H,
m, CH2), 1.25 (3H, s, CH3), 1.01 (3H, s, CH3), 0.95 (3H, s, CH3); 13C
Foetal Calf Serum (Gibco, Paisley, UK), 0.7% v/v 200 mM L-Gluta-
mine (Gibco, Paisley, UK), 1% v/v Antibiotic/antimycotic (Gibco,
Paisley, UK). Worms were incubated for 1 h at 37 ꢂC in a humidified
atmosphere containing 5% CO2 before being dosed with test com-
NMR:
d
199.74 (C¼O), 156.29 (ArC), 134.17 (ArCH), 131.00 (ArC),
127.46 (ArCH), 126.24 (ArCH), 123.82 (ArCH), 49.42 (CH), 41.49
(CH2), 38.30 (C), 38.06 (CH2), 36.36 (CH2), 33.46 (C), 32.69 (CH3),
23.58 (CH3), 21.48 (CH3), 19.03 (CH2); HRMS(ESI) m/z: [M þ Na]þ
calcd for C17H22ONa 265.1563, found 265.1565.
pounds obtaining final concentrations of 50, 25, 12.5 and 6.25 mM
(0.3% DMSO final concentration). While all worms were scored
manually after 24hr, 48 h and 72hr using microscopic methods
described in the literature [35], only motility metrics at 72hr are
reported. At 72hr, eggs were also collected and counted from each
well. After enumeration, eggs were finally subjected to fluorescence
microscopy as described below.
4.1.9. General method for synthesis of compounds 7d and 8d
To a solution of the previously obtained methoxy-diterpenoids
(7 b, 8 b), a solution of 1 M BBr3 (6 eq) in dichloromethane
at ꢀ78 ꢂC was added dropwise under nitrogen. After stirring for
30 min, the reaction mixture was warmed to 0 ꢂC and stirred for 3 h.
The mixture was then neutralized with aqueous NaHCO3 and the
aqueous phase extracted with ethyl acetate. The organic layer was
washed with brine, dried over Na2SO4 and concentrated under
reduced pressure. The crude product was purified by silica gel
column chromatography (10e15% ethyl acetate in petroleum ether)
to afford the hydroxy-diterpenoids.
4.2.4. Fasciola hepatica newly excysted juvenile (NEJ) culture and
screening
F. hepatica (Italian strain) metacercariae were obtained from
Ridgeway Research, Gloucestershire, UK. During our experiments,
two procedures were used to generate newly excysted juveniles
(NEJs) from metacercariae. In the first procedure, NEJs were pro-
duced according to Dixon et al. [36] and Wilson et al. [37] with
minor modifications. These modifications included overnight in-
cubation of cysts in distilled water before treating them in 5 ml
solution containing 1% w/v pepsin (Sigma-Aldrich, St Louis, USA)
and 0.4% v/v 1 M HCl for 1 h at 37 ꢂC. After pepsin treatment, the
cysts were washed with distilled water before suspending them in
5 ml Na2S2O4 solution (0.035 g Na2S2O4 (Fisher Scientific,
UK) þ 0.1 g NaHCO3 (Acros Organics, USA) þ 0.08 g of NaCl (Acros
Organics, USA) and 1% v/v 1 M HCl) for 2 h at 37 ꢂC. Subsequently,
the parasites were washed with distilled water and DMEM con-
taining 1% v/v Antibiotic/antimycotic solution, respectively. Finally,
the cysts were incubated in 5 ml DMEM solution containing 0.02 g
of Sodium Tauroglycocholate (Fisher Scientific, UK) for 1 h at 37 ꢂC
to facilitate parasite excystment. The second method used to pre-
pare NEJs from metacercariae involved initial rupturing of cyst
outer walls with a dissecting needle instead of pepsin digestion.
4.1.9.1. Trans podocarpa-8,11,13-trien-13-ol (7d). White solid, yield:
59%. 1H NMR:
d
7.12 (1H, d, J ¼ 8.6 Hz, ArH), 6.61 (1H, dd, J ¼ 8.5,
2.8 Hz, ArH), 6.50 (1H, d, J ¼ 2.8 Hz, ArH), 4.56 (3H, br s, OH),
2.90e2.77 (2H, m, CH2), 2.26e2.23 (1H, m, CH2), 1.88e1.85 (1H, m,
CH2), 1.76e1.67 (2H, m, CH2), 1.61e1.58 (1H, m, CH2), 1.49e1.47 (1H,
m, CH2), 1.37 (1H, td, J ¼ 13.2, 3.5 Hz, CH2), 1.31 (1H, dd, J ¼ 12.5,
2.3 Hz, CH), 1.22 (1H, td, J ¼ 13.5, 4.0 Hz, CH2), 1.16 (3H, s, CH3), 0.95
(3H, s, CH3), 0.93 (3H, s, CH3); 13C NMR:
d 152.92 (ArC), 143.08 (ArC),
137.05 (ArC), 125.81 (ArCH), 115.00 (ArCH), 113.01 (ArCH), 50.71
(CH), 41.86 (CH2), 39.19 (CH2), 37.43 (C), 33.54 (C), 33.46 (CH3),
30.60 (CH2), 25.11 (CH3), 21.74 (CH3), 19.47 (CH2), 19.12 (CH2);
HRMS-ESI m/z: [2 M þ Na]þ calcd for C34H48O2Na 511.3547, found
511.3547.

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A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
Following cyst rupturing, excystment of NEJs was performed
essentially as described above.
Post-excystment, the NEJs were distributed into 48-well tissue
culture plates at a density of 25 parasites per well in RPMI-1640
(Gibco, Paisley, UK) containing 1% v/v Foetal Calf Serum. Test
4.2.7. Scanning electron microscopy and fluorescence microscopy
Following 72 h co-culture with compounds at sub-lethal con-
centrations, parasites were processed for SEM analysis alongside
those parasites derived from positive (Auranofin) and negative
(DMSO) control wells. To separate male and female schistosomes
for fixing and further SEM processing, several steps from Collins
et al. were adapted [40]. Briefly, schistosomes were collected
following treatment and washed in 1 ml of anaesthetic (1% ethyl 3-
aminobenzoate methane (Sigma-Aldrich, Gillingham, UK) dis-
solved in DMEM) to relax and separate male and female worms.
Gender separated schistosome worms were then further relaxed
and killed in a solution of 1 mL of 0.6 mM MgCl2 (Fisher Scientific,
Loughborough, UK) and then briefly washed in PBS before being
placed in SEM fixative (0.1 M sodium cacodylate, 2.5% v/v glutar-
aldehyde (Agar Scientific, Stansted, UK) in ultra-pure water).
Following fixation, adult schistosomes were stored at 4 ꢂC until
ready for SEM analysis. The same fixation procedure was adopted
for all F. hepatica immature and adult hermaphroditic worms.
In preparation for SEM analysis, all blood and liver fluke samples
were then exposed to a number of wash, staining and dehydration
steps before mounting for SEM. Initially, worms were washed twice
in 0.1 M sodium cacodylate for 30 min, and then stained in 1%
osmium tetroxide solution (Agar Scientific, Stansted, UK) for 2 h.
Following staining, worms were then washed in 0.1 M sodium
cacodylate for 1 h and then ultra-pure water for a further 30 min.
Worms were then dehydrated using a 30, 50, 70, 95, 100% ethanol
series (Fisher Scientific, Loughborough, UK) for 30 min each and
then dried using hexamethyldisilazane critical drying point agent
(Agar Scientific, Stansted, UK). Initially, worms were left in the
drying agent for 3 h. This was then removed and replaced with
further drying agent, which was then allowed to evaporate off over
night.
compounds were initially screened at 25
DMSO) before a dose response titration of the most active ones was
performed (10 M, 5 M, 2.5 M, 1.25 M and 0.625 M; all in 0.16%
DMSO). Control wells included parasites incubated with 0.16%
DMSO (negative) and 10 M Triclabendazole (positive, Sigma-
mM and 12.5 mM (0.16%
m
m
m
m
m
m
Aldrich, UK). All parasite/compound co-cultures were performed
in a total volume of 1 ml. Parasites were scored for both motility
and phenotype at 24, 48 and 72 h as previously described [38], but
only results at 72hr are reported. Motility was scored from 1 to 5,
with 1 signifying normal movement and 5 no movement; pheno-
type was scored from 1 to 6, with 1 representing a normal
phenotype and 6 a dissolved parasite [15].
4.2.5. Fasciola hepatica adult and immature culture and screening
F. hepatica adult (8 weeks post-infection) and immature (4e5
weeks post-infection) parasites were collected from infected livers
of cattle and springer lambs (Randall Parker Foods, Llanidloes,
Wales) and washed three times in phosphate buffered saline (PBS).
Over the next 24 h, parasites were subsequently washed in RPMI
1640 containing 1% v/v Antibiotic solution and 10% v/v Foetal
Bovine Serum (Gibco, Paisley, UK) (changing the wash media every
hour for the first 3 h and then every 6 h). After washing, adult
parasites were moved into 50 ml falcon tubes (2 parasites/tube)
containing 6 ml of media (RPMI 1640, 2.5% v/v HEPES,1% Antibiotic/
antimycotic, 1% v/v Foetal Bovine Serum) whereas immature par-
asites (3 parasites/well) were moved into 6-well tissue culture
plates (Thermo Scientific, Denmark) containing 3 ml of the same
media. All parasites were placed at 37 ꢂC in a humidified environ-
ment containing 5% CO2. Test compounds were added to parasite
Once worm samples were fully dried, they were carefully
mounted on self-adhesive conductive carbon tabs, on aluminium
specimen stubs (both Agar Scientific, Stansted, UK) and then coated
with gold using a Polaron E5000 SEM Coating Unit. Coated worms
could then be stored in a desiccation jar until imaging. SEM imaging
was conducted using a Hitachi S-4700 FESEM microscope (Ultra
High Resolution, an accelerating voltage of 5.0 kV with a working
distance of 5.0 mm). Images were captured at 2560 ꢃ 1920
resolution.
cultures at 40, 13.3 and 4.4
DMSO). Control wells included parasites incubated with 0.25%
DMSO (negative) and 10 M Triclabendazole (positive, Sigma-
mM final concentrations (in 0.25%
m
Aldrich, UK). Both mature and immature parasites were then
scored for motility at 24, 48 and 72 h, but only results at 72 h
showed For adult worms, motility was scored from 1 to 6 where 1
equates to good movement (curled, sticking on wall, movement on
petri plate or conical flask), 2 equates to moderate movement (less
vigour but more than 10 s pulses or peristaltic waves), 3 equates to
resting (less than 10 s pulses in head and body), 4 equates to
lethargy (less than 2 s pulses in head and body), 5 equates to faint
movement of suckers (movement of oral or ventral suckers only,
whole body paralysed) and 6 equates to no movement at all (or
dead). The movement of immature parasites was scored from 1 to 5
(1 good or normal, 2 moderate, 3 low, 4 very little and 5 none).
After collection of schistosome worms for SEM analyses, media
left in the wells were collected in separated Eppendorf tubes, spun
down (200 ꢃ g for 2 min) and then carefully removed leaving a thin
pellet of eggs at the bottom. Eggs were then fixed with 500 ml of 10%
formaldehyde in PBS and stored at 3e4 ꢂC until use. Eggs were then
transferred onto glass slides and visualised using a Leica LMD 6000
Laser Microdissection Microscope under natural light and GFP filter
conditions.
4.2.6. HepG2 and MDBK cell culture and MTT assay
4.2.8. Statistics
Cells were grown tơ 80% confluency in culture media (BME with
phenol red for HepG2 cells or EMEM for MDBK cells, 10% v/v Foetal
Bovine Serum, 1% v/v MEM non-essential amino acid solution, 1% v/
Multiple t-tests and Bonferroni post hoc test were used to
determine any significant difference between each compound
treatment and the DMSO control. Statistical tests and IC50 calcula-
tions were performed using GraphPad Prism 7.02.
v 200 mM L-Glutamine, 1% v/v antibiotic/antimycotic). Confluent
cells were prepared for cytotoxicity assays in the same manner as
stated by Nur-e-Alam [24]. Briefly, 2.5 ꢃ 104 were seeded in a black
walled 96-well microtiter plate (Fisher Scientific, Loughborough,
UK) and incubated for 24 h at 37 ꢂC in a humidified atmosphere
containing 5% CO2. Test compounds were then added at final con-
4.2.9. Ethics statement
All procedures performed on mice adhered to the United
Kingdom Home Office Animals (Scientific Procedures) Act of 1986
(project license PPL 40/3700) as well as the European Union Ani-
mals Directive 2010/63/EU and were approved by Aberystwyth
University's (AU) Animal Welfare and Ethical Review Body
(AWERB).
centrations of 100, 75, 50, 25, 10, 1, 0.1, 0.01 mM (1.25 final % DMSO)
in parallel to negative (DMSO; 1.25%) and positive (1% v/v Triton X-
100) [39] controls. Following a further incubation for 24 h, the MTT
assay was performed as previously described [24].

A. Crusco et al. / European Journal of Medicinal Chemistry 152 (2018) 87e100
99
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anthelmintic activity against both Schistosoma mansoni and Fasciola hepatica,
Author contribution
Conceived and designed the experiments: AC, ADW, KFH. Per-
formed the experiments: AC (compound synthesis), AC, CB (com-
pound characterisation), AC, KCLW, HW (S. mansoni screening), AC,
ACh, KCLW (F. hepatica screening), AC (cell screening). Manuscript
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Acknowledgment
ꢀ
[18] S.J. Krauth, C. Musard, S.I. Traore, J. Zinsstag, L.Y. Achi, E.K. N'Goran, J. Utzinger,
We thank the Welsh Government, Life Sciences Research
Network Wales scheme for financial support to AC. We thank Dr.
Iain Chalmers, Ms. Holly Craven, Dr. Josephine Ford-Thomas, Mr.
Tom Gasan, Dr. Kathrin Geyer, Ms. Julie Hurst, and Ms. Gilda
Padalino for help in maintaining the S. mansoni life cycle. We also
thank Dr. Robert J. Nash (Phytoquest Ltd) for providing 7-keto-
sempervirol as well as Dr. Manfred Beckmann (IBERS, AU) and the
analytical services unit (School of Chemistry, Cardiff University) for
provision of accurate mass spectrometry. We finally thank Dr.
Russell Morphew (IBERS, AU) for helpful discussions regarding
F. hepatica excystment protocols and acknowledge that IBERS re-
ceives strategic funding from BBSRC.
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fascioliasis co-endemic area of northern Cote d'Ivoire, Acta Trop. 149 (2015)
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