Antimalarial activity of 2,6-dibenzylidenecyclohexanone derivatives

Article abstract of DOI:10.1016/j.bmcl.2021.128216

Malaria remains one of the deadliest infectious diseases worldwide and continues to infect hundreds of millions of individuals each year. Here we report the discovery and derivatization of a series of 2,6-dibenzylidenecyclohexanones targeting the chloroquine-sensitive 3D7 strain of Plasmodium falciparum. While the initial lead compound displayed significant toxicity in a human cell proliferation assay, we were able to identify a derivative with no detectable toxicity and sub-micromolar potency.

Full text of DOI:10.1016/j.bmcl.2021.128216

Bioorg. Med. Chem. Lett. 47 (2021) 128216
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antimalarial activity of 2,6-dibenzylidenecyclohexanone derivatives
,
Scott Eagona *, Jared T. Hammillb, Kasey Fitzsimmonsa, Natalie Sienkoa, Brandon Nguyena,
Jarvis Lawa, Aashrita Manjunatha, Steven P. Wilkinsona, Kara Thompsona,
Julia Elizabeth Gliddena, Amy L. Riceb, Mofolusho O. Faladeb, Joshua J. Kimballc,
Celine DiBernardoa, R. Kiplin Guyb
a Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, CA 93407, USA
b Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40508, USA
c Promega Biosciences, 277 Granada Drive, San Luis Obispo, CA 93401, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Malaria remains one of the deadliest infectious diseases worldwide and continues to infect hundreds of millions
of individuals each year. Here we report the discovery and derivatization of a series of 2,6-dibenzylidenecyclo-
hexanones targeting the chloroquine-sensitive 3D7 strain of Plasmodium falciparum . While the initial lead
compound displayed significant toxicity in a human cell proliferation assay, we were able to identify a derivative
with no detectable toxicity and sub-micromolar potency.
Malaria
Plasmodium falciparum
2,6-Dibenzylidenecyclohexanone
Structure activity relationship
Michael system
Malaria remains one of the deadliest diseases on the planet, infecting
an estimated 229 million individuals in 2019 with >400,000 associated
deaths, primarily in young children and pregnant women.1 Malaria is
caused by Plasmodium parasite infection of the liver and blood, with
P. falciparum accounting for the vast majority of deaths.2 The complexity
of the Plasmodium life cycle3 coupled with mounting resistance to cur-
rent treatments4 underscores the need for new treatment options. As a
part of this discovery effort, we recently identified a number of active
compounds in a high-throughput screen designed to identify molecules
that arrest the growth of P. falciparum in co-culture with human eryth-
rocytes.5 One compound displaying notable activity was compound 1, a
2,6-dibenzylidenecyclohexanone shown in Fig. 1.
main areas of substitution: modification of meta-CF3 groups, different
substitutions at the 4-position of the cyclohexanone core, and manipu-
lation of the Michael system. We also decided to focus on symmetrical
aryl substitutions for this study in order to minimize the synthetic effort
required to construct the compounds until we could determine if these
aryl groups have any effect on the SAR and toxicity.
To begin our synthesis of derivatives of 1, we first prepared the 4,4-
disubstitued cyclohexanone core according to a previously published
protocol11 then used a simple acid promoted double aldol reaction to
furnish a selection of symmetrical compounds with variations about the
pendant aromatic rings (Scheme 1). Our primary aim was to observe the
effects of moving the meta-CF3 to a different position or replacing it
entirely with a fluorine or hydrogen atom. As a part of this group of
compounds we also resynthesized the original hit compound 1 as a
positive control.
While this is the first report of 2,6-dibenzylidenecyclohexanones
displaying antimalarial activity, similar compounds have been previ-
ously reported as having antitubercular,6 anti-inflammatory7 and anti-
neoplastic8 activity, as well as being inhibitors of the STAT3 pathway9
and HIV-1 integrase.10 Given the promising activity of 1 against the
clinically susceptible P. falciparum 3D7 strain and the drug resistant V1S
and K1 strains, we decided to make a library of related compounds to
probe the structure activity relationship (SAR) of the scaffold. Fig. 1
moreover, we wished to see if potency against P. falciparum could be
maintained while minimizing the antiproliferative activity observed
against a human cell line (BJ). Since nothing is currently known about
the mechanism of action of this compound, we decided to focus on three
While we were able to synthesize a handful of derivatives in a
straightforward manner, we were unable to isolate an ortho-CF3 deriv-
ative, as all tested aldol conditions failed to yield any product.
We next chose to investigate the effects of changing the substitutions
about the 4-position of the cyclohexanone ring by using commercially
available cyclohexanones (Scheme 2).
We also prepared heteroaromatic derivatives of 1 by utilizing 4-oxo-
tetrahydropyran and 4-oxotetrahydrothiopyran according to Scheme 3.
Finally, we modified the Michael system present in compound 1 by
* Corresponding author.
E-mail address: seagon@calpoly.edu (S. Eagon).
https://doi.org/10.1016/j.bmcl.2021.128216
Received 14 May 2021; Received in revised form 8 June 2021; Accepted 16 June 2021
Available online 19 June 2021
0960-894X/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

S. Eagon et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128216
Fig. 1. Bioactivity of 2,6-dibenzylidenecyclohexanone 1 against P. falciparum.
Scheme 2. Synthesis of 4-substititued cyclohexanone derivatives of com-
pound 1.
reducing the alkenes with palladium on carbon and the carbonyl with
sodium borohydride to provide 12 and 13, respectively.
With our compounds in hand, we next tested their proliferation
inhibitory activity against the chloroquine-sensitive P. falciparum 3D7
strain (MRA-102) in human O-positive erythrocytes as well as the sol-
ubility of the compounds in a PBS buffer. We also tested for growth
inhibition of a human fibroblast cell line (BJ) as a marker for toxicity
(Table 1).
Modification about the phenyl rings (1–4) did not improve potency
of the compounds, and toxicity in the form of antiproliferative activity
against the BJ cell line remained an issue. Changing the substitutions
about the 4-position of the cyclohexanone core (5–8) revealed that
observed toxicity could be alleviated by removal of the aromatic ring,
with the 4-tert-butyl substituent (8) presenting no detectable toxicity
while maintaining sub-micromolar potency. Replacement of the cyclo-
hexanone core with either 4-oxotetrahydropyran (9) or 4-oxotetrahy-
drothiopyran (10) likewise improved the toxicity profile, although
potency was reduced. In contrast, however, the sulfone derivative (11)
was more active against the human cell line than P. falciparum. Reduc-
tion of either the alkene or the ketone functional groups to remove the
Michael system (12 and 13) also resulted in compounds with no
detected toxicity, although potency against P. falciparum fell by
approximately one order of magnitude. Solubility in a PBS buffer was
low for all compounds, which is not surprising given that most were
highly conjugated aromatic systems with few polar groups.
In conclusion, our in vitro results demonstrate that the anti-
proliferative activity of 2,6-dibenzylidenecyclohexanone derivatives
against a human cell line can be resolved by changing the substitutions
about the 4-position of the cyclohexanone core or by reducing the
Michael system, with compound 8 maintaining sub-micromolar potency
against the 3D7 strain of P. falciparum. The ease of construction of these
compounds makes them attractive targets for further modification, and
future studies will explore additional derivatives designed to improve
both potency and solubility.
Scheme 3. Preparation of heterocyclic cyclohexanone derivatives of 1.
Scheme 1. Synthesis of 4-phenyl-4-cyanocyclohexanone derivatives of compound 1.
2

S. Eagon et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128216
Table 1
Antimalarial activity, cytotoxicity, therapeutic index (T.I.) and solubility of all synthesized compounds.
Compound
Structure
EC50 3D7a
(μM)
EC50 BJb
(μM)
T.I.
2
Solubility (
n.d.
μ
M)c
1
0.331 ± 0.185
0.207 ± 0.031
1.13 ± 0.320
1.36 ± 0.410
0.570 ± 0.099
0.406 ± 0.048
2.27 ± 0.084
5.85 ± 1.62
2
3
4
5
2
2
< 1
< 1
< 1
< 1
4
2.29 ± 0.289
0.491 ± 0.188
1.10 ± 0.268
23.5 ± 1.02
1.53 ± 0.225
20.1 ± 7.8
10
6
7
3
1.1 ± 0.7
< 1
18
8
0.641 ± 0.285
> 24
> 37
< 1
9
1.16 ± 0.422
0.820 ± 0.033
0.393 ± 0.107
1.03 ± 0.171
3.21 ± 0.344
23.7 ± 0.539
23.2 ± 0.181
0.120 ± 0.016
> 24
20
28
1.4 ± 0.7
1.2 ± 0.9
< 1
10
11
12
< 1
> 23
> 7
2.3 ± 2.1
13
> 24
2.5 ± 1.7
a
EC50 values are reported as the mean ± SD of four measurements. Assay endpoints were normalized from 0% (DMSO only) to 100% inhibition (16
μM Mefloquine).
bEC50 values are reported as the mean ± SD of four measurements. Assay endpoints were normalized from 0% (DMSO only) to 100% inhibition (58
μM Idarubicin).
cSolubility in PBS buffer, pH 7.4, with DMSO used as the carrier vehicle. The detection limit of the solubility assay was 100
μM. Albendazole (<1 μM) and carba-
mazepine (>100 μM) were used as poorly soluble and highly soluble standards, respectively. Values are reported as the mean ± SD of six measurements.
3

S. Eagon et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128216
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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This work was supported by the Bill and Linda Frost Fund at Cali-
fornia Polytechnic State University (no associated grant number).
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