Synthetic kavalactone analogues with increased potency and selective anthelmintic activity against larvae of haemonchus contortus in vitro

Article abstract of DOI:10.3390/molecules25082004

Kava extract, an aqueous rhizome emulsion of the plant Piper methysticum, has been used for centuries by Pacific Islanders as a ceremonial beverage, and has been sold as an anxiolytic agent for some decades. Kavalactones are a major constituent of kava extract. In a previous investigation, we had identified three kavalactones that inhibit larval development of Haemonchus contortus in an in vitro-bioassay. In the present study, we synthesized two kavalactones, desmethoxyyangonin and yangonin, as well as 17 analogues thereof, and evaluated their anthelmintic activities using the same bioassay as employed previously. Structure activity relationship (SAR) studies showed that a 4-substituent on the pendant aryl ring was required for activity. In particular, compounds with 4-trifluoromethoxy, 4-difluoromethoxy, 4-phenoxy, and 4-N-morpholine substitutions had anthelmintic activities (IC₅₀ values in the range of 1.9 to 8.9 μM) that were greater than either of the parent natural products-desmethoxyyangonin (IC₅₀ of 37.1 μM) and yangonin (IC₅₀ of 15.0 μM). The synthesized analogues did not exhibit toxicity on HepG2 human hepatoma cells in vitro at concentrations of up to 40 μM. These findings confirm the previously-identified kavalactone scaffold as a promising chemotype for new anthelmintics and provide a basis for a detailed SAR investigation focused on developing a novel anthelmintic agent.

Full text of DOI:10.3390/molecules25082004

molecules
Article
Synthetic Kavalactone Analogues with Increased
Potency and Selective Anthelmintic Activity against
Larvae of Haemonchus contortus In Vitro
H.M.P. Dilrukshi Herath 1,†, Aya C. Taki 1,† , Nghi Nguyen 2,3, José Garcia-Bustos 1,
Andreas Hofmann 1 , Tao Wang 1, Guangxu Ma 1 , Bill C.H. Chang 1, Abdul Jabbar 1,
Brad E. Sleebs 2,3,* and Robin B. Gasser 1,
*
1
Faculty of Veterinary and Agricultural Sciences, The University of Melbourne,
Parkville, Victoria 3010, Australia; dili.herath@unimelb.edu.au (H.M.P.D.H.);
aya.taki@unimelb.edu.au (A.C.T.); Jose.GarciaB@unimelb.edu.au (J.G.-B.);
a.hofmann@structuralchemistry.org (A.H.); tao.wang1@unimelb.edu.au (T.W.);
guangxu.ma@unimelb.edu.au (G.M.); bill.chang@yourgene-health.com (B.C.H.C.);
jabbara@unimelb.edu.au (A.J.)
Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia;
nguyen.n@wehi.edu.au
Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne,
Parkville, Victoria 3010, Australia
2
3
*
†
Correspondence: robinbg@unimelb.edu.au (R.B.G.); sleebs@wehi.edu.au (B.E.S.)
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 31 March 2020; Accepted: 22 April 2020; Published: 24 April 2020
Abstract: Kava extract, an aqueous rhizome emulsion of the plant Piper methysticum, has been used
for centuries by Pacific Islanders as a ceremonial beverage, and has been sold as an anxiolytic agent
for some decades. Kavalactones are a major constituent of kava extract. In a previous investigation,
we had identified three kavalactones that inhibit larval development of Haemonchus contortus in
an in vitro-bioassay. In the present study, we synthesized two kavalactones, desmethoxyyangonin
and yangonin, as well as 17 analogues thereof, and evaluated their anthelmintic activities using
the same bioassay as employed previously. Structure activity relationship (SAR) studies showed
that a 4-substituent on the pendant aryl ring was required for activity. In particular, compounds
with 4-trifluoromethoxy, 4-difluoromethoxy, 4-phenoxy, and 4-N-morpholine substitutions had
anthelmintic activities (IC50 values in the range of 1.9 to 8.9 µM) that were greater than either of the
parent natural products—desmethoxyyangonin (IC50 of 37.1
The synthesized analogues did not exhibit toxicity on HepG2 human hepatoma cells in vitro at
concentrations of up to 40 M. These findings confirm the previously-identified kavalactone scaffold
µM) and yangonin (IC50 of 15.0 µM).
µ
as a promising chemotype for new anthelmintics and provide a basis for a detailed SAR investigation
focused on developing a novel anthelmintic agent.
Keywords: kavalactone; synthetic analogues; anthelmintic; in vitro-activity; Haemonchus contortus
1. Introduction
Kava extract is an aqueous emulsion that is prepared from the plant Piper methysticum. It has been
used for centuries by Pacific Islanders as an inebriant beverage in ritual and cultural ceremonies [1,2].
Since the 1990s, the commercial extract has been ‘popularized’ as an anxiolytic agent in countries
including the USA, Australia, New Zealand, and Germany, although its use has been restricted in some
countries due to concerns regarding possible, rare hepatotoxicity associated with consumption [3].
Molecules 2020, 25, 2004; doi:10.3390/molecules25082004
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2004
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Kavalactones, also called kavapyrones, are lipophilic pyrones, which usually concentrate in the
roots of the plant and are responsible for the anxiolytic activity of the kava extract [ ]. To date,
4
18 such kavalactones have been identified, and six of them (i.e., kavain, dihydrokavain, methysticin,
dihydromethysticin, desmethoxyyangonin, and yangonin) have been reported to be responsible for
~96% of the pharmacological activity of this extract [5,6].
In a recent effort to discover anthelmintic candidates from natural sources [7], we identified, for
the first time, an enriched root extract from P. methysticum, collected from Papua New Guinea, with
activity against one of the most important parasitic nematodes of livestock animals—Haemonchus
contortus. Subsequent bioassay-guided fractionation of this extract yielded three kavalactones (i.e.,
dihydrokavain, desmethoxyyangonin, and yangonin) that inhibited the development of exsheathed
third-stage larvae (xL3s) to fourth-stage larvae (L4s) [
at inhibiting L4 development (IC50 of 207.6 M), both desmethoxyyangonin (IC50 of 31.7
yangonin (IC50 of 23.7 M) were moderately potent inhibitors of larval development. Interestingly,
each of these compounds induced a lethal ‘evisceration’ (Evi) phenotype in treated larvae [ ]. Using one
7]. While dihydrokavain had a limited potency
µ
µM) and
µ
7
representative from the identified kavalactone panel, desmethoxyyangonin, we also showed that
exposure to the compound for 30 h was sufficient to induce the Evi phenotype in H. contortus
(see [
toxicity on MCF10A, non-tumorigenic breast epithelial cells in an in vitro-cell proliferation assay [
Together with knowledge of the drug-like features of kavalactones, these findings suggested that
the kavalactone ( -pyrone) scaffold has potential for ‘lead’ identification in the quest to develop
7]). All three kavalactones (i.e., dihydrokavain, desmethoxyyangonin, and yangonin) had limited
7].
α
new nematocides. Therefore, we undertook a structure-activity relationship (SAR) investigation of
this natural product scaffold, with the aim of synthesizing analogues with improved efficacy and
pharmacokinetic profiles, and with a view toward identifying potential ‘lead’ candidates for future
anthelmintic drug development.
2. Results and Discussion
Initially, desmethoxyyangonin and yangonin were synthesized in-house and evaluated for their
anthelmintic activity. These kavalactones had IC50 values (for larval development-inhibition) in a
similar range to those purchased and evaluated in a previous study [
the synthesized compounds. The IC50 values obtained with desmethoxyyangonin
(purchased) and 37.1 M (synthesized) for larval development-inhibition, and the values for yangonin
were 23.7 M (purchased) and 15.0 M (synthesised) (Table 1; Figure 1). The methoxy-substitution at
7], confirming the activity of
1
were 31.7
µM
µ
2
µ
µ
the 4-position of the pendant phenyl ring is the only difference between desmethoxyyangonin and
yangonin, which might be the reason for the observed differences in the IC50 values.

Molecules 2020, 25, 2004
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Table 1. The activities of synthesized kavalactone analogues against Haemonchus contortus in the
larval development assay and HepG2 human hepatoma cells in vitro. A comparison of half maximum
inhibitory concentration (IC50) values of the synthesized compounds with those of monepantel and
moxidectin, expressed as mean IC50 ± standard error of the mean (SEM).
Inhibition of Larval
HepG2
Compound
R2
R3
R4
Development in H.
EC50 (µM)
Contortus IC50 (µM) ± SEM
1 (desmethoxyyangonin)
H
H
H
H
H
OCH3
OCF3
37.1 ± 3.1
15.0 ± 3.0
1.9 ± 0.1
8.9 ± 0.3
5.2 ± 2.2
12.4 ± 3.5
12.6 ± 2.1
10.3 ± 1.2
> 50
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
>40
> 40
> 40
>40
> 40
> 40
> 40
> 40
> 40
nd
2 (yangonin)
3 (WEHI-408)
H
H
4
H
H
OCHF2
OPh
5
H
H
6
H
H
Cl
7
H
H
Me
8
H
H
CF3
9
H
H
N(CH3)2
N-morpholine
N-piperazine
CO2Me
CN
10
H
H
6.4 ± 0.2
> 100
11
H
H
12
H
H
> 100
13
H
H
> 100
14
OCH3
H
H
H
> 100
15
OCH3
H
61.9 ± 7.3
> 100
16
H
-OCH2O-
17
18
H
CN
CO2Me
[N]
H
H
> 100
H
> 100
19
H
OCH3
> 100
Monepantel
Moxidectin
0.07 ± 0.01
0.02 ± 0.00
nd
nd = not determined.
Figure 1. Structures of the natural products, yangonin and desmethoxyyangonin, and their activities
against Haemonchus contortus in the larval development assay (see [7]).
To investigate the SAR of the kavalactone framework, we synthesized 17 analogues to explore the
effect of the pendant aryl ring-substitution on anthelmintic activity. Seven of the synthesized analogues
had IC50 values of < 20 µM for L4 development-inhibition; four of them had IC50 values of < 10 µM.

Molecules 2020, 25, 2004
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Specifically, the potencies of the analogues
3
(WEHI-408),
4,
5, and 10, with IC50 values ranging from 1.9
to 8.9
µM, were greater than those of their parent kavalactones desmethoxyyangonin 1 and yangonin 2
(Table 1; Figure 2).
7 days
120
1 (desmethoxyyangonin)
2 (yangonin)
3 (WEHI-408)
80
40
0
4
5
10
Monepantel
Moxidectin
02
3
4
5
Log (concentration; nM)
Figure 2. The activity of desmethoxyyangonin, yangonin and selected analogues (
against Haemonchus contortus in the larval development assay (conducted over 7 days). Dose-response
curves for the two parent kavalactones ( and ) and four analogues ( and 10) for developmental
inhibition in comparison to monepantel or moxidectin. Information on compounds is given in Table 1.
3, 4, 5, and 10)
1
2
3, 4, 5
The SAR derived from the bioassay data (Table 1) showed that analogues 14 and 15, with 2-methoxy
or 3-methoxy substitution on the phenyl ring, did not possess significant anthelmintic effects (IC50
values of 61.9
substitution, the data showed that methoxy substitutions at the 2- or 3-position were not tolerated.
Analogues 16 18 with 3,4-methylenedioxy, 3-cyano, or 3-carboxylate substitutions on the phenyl ring
µM and > 100 µM, respectively). In contrast to the activity of 2, bearing a 4-methoxy
–
were also inactive, further confirming that substitution at the 3-position is not tolerated.
A set of analogues with varying substitutions at the 4-position of the phenyl ring showed that
analogues
increased anthelmintic activity (IC50 values of 1.9, 8.9, and 5.2
4-methoxy parent compound (Table 1). Analogues with 4-chloro, 4-methyl, and 4-trifluoromethyl
substitutions all had comparable activities (IC50 values of 12.4, 12.6, and 10.3 M, respectively) to
yangonin , suggesting that non-polar functionalities with electron neutral or withdrawing properties
and with less steric volume than a methoxy group were tolerated. In contrast, analogues 12, and
3–
5
with 4-(trifluoromethoxy), 4-(difluoromethoxy) and 4-phenoxy substituents significantly
µM, respectively) compared with the
2
6–8
µ
2
9,
13 with the polar groups dimethylamino, cyano, and carboxylate at the 4-position were inactive at
the highest concentration tested. Interestingly, the analogue 10 with N-morpholine in the 4-position
exhibited an IC50 of 6.4
balance between the polarity and lipophilicity of functionalities tolerated at the 4-position. Notably,
the activity of analogues and 10 with phenoxy or morpholine groups in the 4-position suggests that
µM, but the 4-N-piperazine analogue 11 was inactive, suggesting that there is a
5
these groups with a large steric volume could be orientated towards solvent space, or alternatively, be
accommodated in a suitable cavity within the presumed protein-target of the kavalactones. The addition
of a morpholine group to the 4-position had the added benefit of likely enhancing the aqueous solubility
of the kavalactones. Additional solubility improvements could accrue from the addition of an endocyclic
nitrogen; however, the only synthetically tractable position on the pendant aryl ring is the 3-position,
and analogue 19 was shown to be inactive at the highest concentration tested.

Molecules 2020, 25, 2004
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Summarizing the activity data, the anthelmintic potency of the parent kavalactones
1
and
2
was
significantly increased by minor substitutions on the 4-position of the phenyl-ring, without modifying
the -pyrone scaffold (Table 1). Importantly, all analogues with a significant inhibitory effect induced
an Evi phenotype (cf. [ ]), suggesting that these analogues have a mode of action that is the same or
α
7
similar to their respective parent kavalactone.
All analogues assessed for inhibitory activity against H. contortus were also tested against human
HepG2 hepatoma cells in vitro (Table 1), using CellTiter-Glo® to measure ATP concentration as a
metabolic marker for cell numbers. The data showed that none of the compounds inhibited cell growth
at the highest concentration tested (IC50 > 40
selectivity index (> 20-fold) against H. contortus. Overall, this investigation showed that four of
17 analogues synthesized (i.e., 5, and 10) had enhanced potency against and selectivity for H.
contortus, without evidence of significant in vitro-toxicity on hepatoma cells. Clearly, this increased
anthelmintic activity and lack of apparent in vitro-toxicity of analogues 5, and 10, all bearing
µM), with analogue 3 (WEHI-408) having the highest
3, 4,
3, 4,
relatively minor structural modifications, indicate a pathway for future work on the pyrone scaffold
toward novel anthelmintic leads.
Given that the development of new anthelmintics is critical to counteract widespread resistance
problems in nematodes of livestock animals, the current investigation—enabled by our previous
study of kavalactones [
7
]—offers guidance for developing the
α
-pyrone scaffold into an anthelmintic.
11],
Although different synthetic kavalactones and analogues have been reported in the literature [
8–
none of them had been tested against a parasitic nematode of animals. The target/s of natural
kavalactones or synthetic analogues has/have not yet been identified in nematodes, but some targets,
including
γ
-aminobutyric acid (GABA) receptors, voltage-gated ion channels, monoamine oxidase,
19]. Orthologues of these proteins
and the arachidonate cascade, have been proposed for mammals [12
–
in H. contortus and related parasites should be investigated as possible targets responsible for the
anthelmintic activity of the kavalactones reported here. Available genomic, transcriptomic, and
proteomic data sets (see [20–23]) would provide a foundation for such an extension.
3. Materials and Methods
3.1. Chemical Synthesis of Kavalactones and Analogues
Nineteen compounds were synthesized. In brief, desmethoxyyangonin
1, yangonin 2, and all the
4-methoxypyrone derivatives ( 19) were synthesized by condensation of the appropriate aryl aldehyde
3
–
with 4-methoxy-6-methyl-2H-pyran-2-one under basic conditions, following a previously published
method [24] (Supplementary file 1, Scheme S1). Synthesis protocols and compound characterization
are given in the Supplementary file 1.
3.2. Procurement of H. contortus
The Haecon-5 strain of H. contortus was produced in experimental sheep [21,25], according to
animal ethics guidelines of the University of Melbourne, Australia (permit no.1413429). The feces were
collected from the infected sheep and incubated at 27 ◦C for 1 week, allowing first-stage larvae to
emerge and develop to third-stage larvae (L3s) [25]. L3s were collected and stored at 10 ◦C until used
in experiments; xL3s were prepared by treating L3s with 0.15% v/v sodium hypochlorite (NaClO) and
incubated for 20 min at 38 ◦C, and subsequently washed five times with sterile physiological saline (pH
7.0). These xL3s were immediately incubated at 38 ◦C in sterile Luria Bertani (LB) broth supplemented
with 100 IU/ml of penicillin, 100 µg/ml of streptomycin, and 0.25 mg/ml of amphotericin B (fungizone,
antibiotic—antimycotic; cat. no. 15240-062; Gibco, Thermo Fisher Scientific, Waltham, MA, USA); this
supplemented medium was designated as LB*.

Molecules 2020, 25, 2004
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3.3. Assessment of Potency of Synthesized Compounds at Inhibiting Larval Development of H. contortus
In our previous study [7], larval development-inhibition results guided the fractionation of
the active extract from P. methysticum, and the findings were consistent with subsequent potency
assessments of the purified kavalactones. Therefore, the developmental inhibition assay for H. contortus
was used to determine the activity/potency of the kavalactone analogues synthesized in the present
study. In brief, all synthesized compounds as well as monepantel (Zolvix, Elanco Animal Health,
Australia) and moxidectin (Cydectin, Virbac, France) (two positive controls) were individually tested for
inhibition of larval development in H. contortus using an 18-point, two-fold dilution series, commencing
at 100
to 96-well flat bottom plates to achieve the desired final concentrations in a final volume of 100
and xL3s (as described in Section 3.2) were dispensed into individual wells at a density of 300 worms
µM. Compound solutions (in dimethyl sulfoxide [DMSO] plus LB*) were prepared and added
µl,
per well (in 50
µ
l of LB*). Matched concentrations of DMSO (0.25% in LB*) were used as negative
◦
(untreated) controls. The plates were incubated (10% v/v CO2) at 38 C for 7 days [25]. Larvae in
each well were fixed with 50
µl of 1% iodine at 7 days, and the number of xL3s that developed to
L4s in each well was counted by light microscopic examination at 20
×
magnification [25]. L4s were
differentiated from xL3s by their well-developed mouth and pharynx characteristics [26]. The assay
was repeated three times on different days using three replicates in each assay. The half-maximum
inhibitory concentration (IC50) of each compound was determined by fitting the log10-transformed
concentration to a variable slope four-parameter model using the GraphPad Prism (v. 8.3.1) software
(GraphPad Software Inc., San Diego, CA, USA).
3.4. Assessment of Cytotoxicity and Selectivity of Synthesized Compounds
Synthesized compounds were tested for their toxicity on HepG2 human hepatoma cells, as
described previously [27]. In brief, compounds were tested in a 10-point, two-fold dilution series,
commencing at 40
supplemented with 10% fetal bovine serum (FBS) and incubated at 37 ◦C with 5% v/v CO2. The cells
were seeded into 384-well plates at a density of 1
103 per well and exposed to compounds for 48 h.
µM. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
×
The toxicity was determined by CellTiter-Glo® cell viability assay (Promega, USA), which assessed the
number of viable cells based on the quantity of ATP present. Matched percentages of etoposide and
DMSO were used as positive and negative (untreated) controls, respectively. The EC50 values were
determined as described in Section 3.3, and the selectivity index was calculated by dividing the EC50
value for HepG2 cells by the IC50 value for H. contortus. Bortezomib, which has an EC50 of 0.01 µM,
was used as the reference control compound.
Supplementary Materials: The following are available online, Chemistry methods, Scheme S1. General synthetic
pathway for kavalactone analogues. References [9,24,28,29] are cited in the supplementary materials
Author Contributions: Conceptualization, R.B.G., B.E.S., and J.G.-B.; methodology, R.B.G., A.C.T., N.N., and
B.E.S.; validation, H.M.P.D.H., A.C.T., R.B.G., and B.E.S.; formal analysis, H.M.P.D.H. and A.C.T.; investigation,
H.M.P.D.H., A.C.T., and B.E.S.; resources, A.C.T., B.E.S., and R.B.G.; data curation, H.M.P.D.H., A.C.T., N.N., and
B.E.S.; writing—original draft preparation, H.M.P.D.H., A.C.T., B.E.S., and R.B.G.; writing—review and editing,
H.M.P.D.H., A.C.T., N.N., J.G.-B., A.H., T.W., G.M., B.C.H.C., A.J., B.E.S., and R.B.G.; visualization, A.C.T. and
B.E.S.; supervision, R.B.G.; project administration, R.B.G.; funding acquisition, R.B.G. and B.E.S. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by the Australian Research Council, Yourgene Health Singapore, and The
University of Melbourne. B.E.S. is a Corin Centenary Fellow.
Acknowledgments: The authors thank NatureBank (www.griffith.edu.au/institute-drug-discovery/unique-
resources/naturebank) for previous access to the natural product extract library and raw plant materials.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

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Sample Availability: Samples of the compounds 1 to 5 and 10 are available from the authors.
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