Dimeric cyclohexane-1,3-dione oximes inhibit wheat acetyl-CoA carboxylase and show anti-malarial activity

Article abstract of DOI:10.1016/j.bmcl.2010.06.007

A series of dimeric 1,3-cyclohexanedione oxime ethers were synthesized and found to have significant antiplasmodial activity with IC₅₀'s in the range 3-12 μM. The most active dimer was tested in the Plasmodium berghei mouse model of malaria and

Full text of DOI:10.1016/j.bmcl.2010.06.007

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4611–4613
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dimeric cyclohexane-1,3-dione oximes inhibit wheat acetyl-CoA carboxylase
and show anti-malarial activity
Theola Louie a,b, C. Dean Goodman c, Georgina A. Holloway a, Geoffrey I. McFadden c, Vanessa Mollard c,
Keith G. Watson a,
*
a The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Victoria, Australia
b Department of Medical Biology, The University of Melbourne, Parkville 3010, Victoria, Australia
c School of Botany, University of Melbourne, Parkville 3010, Victoria, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dimeric 1,3-cyclohexanedione oxime ethers were synthesized and found to have significant
antiplasmodial activity with IC50’s in the range 3–12 lM. The most active dimer was tested in the Plas-
modium berghei mouse model of malaria and at a dose of 48 mg/kg gave a 45% reduction in parasitaemia.
Several commercial herbicides, all known to be inhibitors of maize acetyl-CoA carboxylase, were also
tested for antimalarial activity, but were essentially inactive with the exception of butroxydim which
Received 23 April 2010
Accepted 1 June 2010
Available online 8 June 2010
Keywords:
Acetyl-CoA carboxylase
Antimalarial
gave an IC50 of 10 lM.
Ó 2010 Elsevier Ltd. All rights reserved.
1,3-Cyclohexanedione
Acetyl-CoA carboxylase (ACC) performs the first and generally
rate-limiting step in fatty acid biosynthesis and ACC’s are found
in most living organisms including bacteria, fungi, plants, animals
and humans.1 ACC catalyses the production of malonyl-CoA which
is used in the biosynthesis of long chain fatty acids vital for many
cellular functions including the cell membranes. The ACC catalysed
formation of malonyl CoA from acetyl-CoA and CO2 takes place in
two stages. Thus, the biotin carboxylase (BC) sub-unit of ACC catal-
yses the ATP-dependent carboxylation of biotin and then the carb-
oxyltransferase (CT) sub-unit catalyses the transfer of this
activated carboxyl group to the acceptor acetyl-CoA. As the sup-
plier of malonyl-CoA and the control point for fatty acid synthesis
(FAS) ACC is an excellent target for inhibitor studies and drug dis-
covery.2 Until recently it was believed that the malaria parasite,
Plasmodium sp., relies entirely on the host erythrocyte and serum
for its fatty acid requirements, but recent studies have shown that
Plasmodium falciparum and other apicomplexan parasites possess a
plastid known as the apicoplast, a similar organelle to plant chlo-
roplasts, which includes all the biochemical machinery to carry
out FAS.3 Thus the P. falciparum genome contains FAS genes and
a single copy of ACC which is closely related to the ACC from the
plastid of many grasses. This indicates that malaria uses the type
II FAS pathway common to plant chloroplasts and bacteria, but dis-
tinct from the type I FAS pathway of animals including humans.4 It
also suggests that inhibitors of ACC or enzymes in the type II FAS
pathway would be good candidates for testing as antimalarials.5
Two major classes of herbicide, the cyclohexane-1,3-dione oxi-
mes (dims, e.g., tralkoxydim 1) and the aryloxyphenoxypropionic
acids (fops, e.g., quizalofop 2), which were both discovered in the
early 1970s are important to agriculture because of their selective
activity against grass weeds in broad-leaf crops (Fig. 1).6 The mode
of action of the dims and fops was a mystery for many years, but in
1987-88 several laboratories independently reported that both
types of herbicide are selective inhibitors of ACC’s from grass
plants.7 Waller et al. have reported that the dim herbicide tralk-
oxydim 1, and also the fops diclofop and fenoxaprop, show weak
activity (IC50’s of 181, 210 and 144 lM, respectively) against P. fal-
ciparum,4 so we decided to test a wider range of these compound
classes for antimalarial activity and herein report our findings.
As a preliminary set of compounds for testing on P. falciparum
we selected three of the most active commercially available dim
herbicides clethodim 3, tepraloxydim 4 and butroxydim 5, plus
the cyclohexane-1,3-dione oximes 6, 7 and 8 which are also known
Me
Me
OH
O
O
N
N
N
O
Me
Me
OH
Cl
O
O
Quizalofop, 2
Tralkoxydim, 1
* Corresponding author. Tel.: +61 3 93452113; fax: +61 3 93452211.
E-mail address: kwatson@wehi.edu.au (K.G. Watson).
Figure 1. Structures of a representative dim 1 and fop 2 herbicide.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.007

4612
T. Louie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4611–4613
O
OH
O
OH
OH
O
O
Cl
O
Cl
N
N
O
N
O
S
O
3
4
5
O
HO
O
OH
O
OH
OH
O
O
O
N
O
N
O
N
N
Fe
O
8
6
7
Figure 2. Structures of cyclohexanedione herbicides tested on P. falciparum.
to have high herbicidal activity (Fig. 2).8 In addition we selected for
testing quizalofop 2 which is one of the most active and widely
used fop herbicides. Since it is well known that fop herbicides
show better activity when applied to plants as alkyl esters, which
are much more cell-permeable and the esters are readily hydroly-
sed in vivo to the active acid form,7 we therefore also tested the
quizalofop ethyl ester 2a.
The compounds were tested for antiplasmodial activity using an
in vitro SyberGreen stain method for determining P. falciparum cell
viability after exposure to the compound for 48 h.9 Compounds
were tested against the laboratory strain D10 using chloroquine
as a control and the results are summarized in Table 1. Included
in Table 1 are representative reported IC50 values for the inhibition
of maize ACC for most of the compounds. The results reveal that
reduces the activity, but a larger R1 (n-Pr) is tolerated, with com-
pound 10 showing similar activity to compound 8. Altering the
central phenyl ring substitution from the 1,4 arrangement in com-
pound 8 to the isomeric 1,3 orientation in compound 11 signifi-
cantly improved the activity with 11 having an IC50 of 3 lM. The
results for variation of the oxime ether group clearly show that lar-
ger R2 groups are not tolerated with the benzyl oxime ether 13
being less active and the diphenyl-methyl oxime ether 14 only
weakly active. On the other hand compound 12 with an allyl oxime
ether group shows similar activity to 11. In general terms the re-
sults from variation of the R1 and R2 groups in this set of dimeric
compounds are consistent with what would be predicted from
the known herbicidal structure–activity studies with cyclohex-
ane-1,3-dione oximes, with optimal R1 being ethyl or propyl and
optimal R2 being ethyl or allyl.15
compounds 1–4 are essentially inactive (IC50 >100 lM) and
compounds 6–7 are only weak inhibitors of P. falciparum. The
two compounds showing the best activity were butroxydim 5
To test the importance of the length of the central spacing group
in the dimeric cyclohexane-1,3-diones, two extra dimers 16 and 17
(Fig. 4) were prepared starting from the appropriate di-aldehydes
and following a similar reaction sequence to that shown in
Figure 3. In the in vitro antiplasmodium assay dimers 16 and 17
(IC50 = 10
lM) and the dimeric cyclohexane-1,3-dione 8 (IC50 =
6
lM). These two cyclohexanediones 5 and 8 were also tested for
activity against the resistant strain W2mef and an alternative
strain 3D7 with both compounds showing very similar IC50’s to
that found with the D10 strain. Compounds 5 and 8 were also
tested for inhibition of wheat ACC and, as expected, both com-
showed IC50’s of 12 and 5 lM, respectively, suggesting that the
length of the spacer group is not critical to the activity.
Butroxydim 5 plus three of the most active dimeric compounds
8, 10 and 11 were tested for in vivo antimalarial activity using the
Plasmodium berghei-mouse model. Mice were inoculated with
1.5 Â 107 parasites followed by four daily subcutaneous injections
of placebo, chloroquine or the test compounds. The daily dosages
received were 10 mg/kg of chloroquine; 160 mg/kg of butroxydim
5; 20 mg/kg of dimer 8; 20 mg/kg of dimer 10 and 48 mg/kg of 11.
pounds showed complete inhibition of ACC activity at 20 lM.
To try to improve the activity of the promising dimeric com-
pound 8 a series of dimers 8–15 (Table 2) was prepared in which
the acyl grouping R1, the oxime ether substituent R2 and the orien-
tation of the cyclohexane-dione ring substitution on the central
phenyl group were varied. The synthesis of the dimers was carried
out using the simple general synthetic method shown in Figure 3
for the 1,4-di-substituted compounds 8–10.
All the dimers 8–15 were tested using the in vitro antiplasmo-
dium assay and the results are summarized in Table 2. The data
shows that a smaller acyl group, as in compound 9 (R1 = Me),
Table 2
Antiplasmodial activity of the dimeric cyclohexane-1,3-diones 8–15
R2
OH
N O
R2
HO
O
4
O N
R1
R1
Table 1
O
2
3
Antiplasmodial and ACC activity of 1–8
Compound no.
IC50
(lM)
IC50 (lM)
Compound
no.
Acyl
Oxime
Substitution
IC50 (lM)
P. falciparum
maize ACC
group R1 subst. R2
of the phenyl ring P. falciparum*
1
2
2a
3
4
5
6
7
>100
>100
>100
>250
>100
10
>10 < 100
>10 < 100
6
0.5210
0.01611
>111
8
9
10
11
12
13
14
15
Et
Et
Et
Et
Et
1,4
1,4
1,4
1,3
1,3
1,3
6
12
5
3
2.5–5.0
12.5
ꢀ25
4
Me
n-Pr
Et
n-Pr
n-Pr
n-Pr
n-Pr
—
0.0212
0.7*,13
0.314
ND
Allyl
Benzyl
(Phenyl)2CH 1,3
Et
—
ND
ND
ND
1,3
—
8
Chloroquine
0.015
Chloroquine
0.015
*
*
All assays were run in triplicate and the results shown are the average value.
IC50 for Giant foxtail ACC; ND not determined.

T. Louie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4611–4613
4613
1. Na/EtOH
HO
OH
acetone
CH2(CO2Et)2
O
O
OHC
CHO
HO
2. aq NaOH
3. HCl
aq. NaOH
O
O
(R1CO)2O
DMAP
Toluene reflux
OH
O
HO
O
OH
O
R1
O
R
R2
N O
R1
2O N
R1
O
R2ONH2
R1
O
8-10
Figure 3. Synthetic method for the dimeric cyclohexane-1,3-diones.
O
Et
N
HO
Et
O
OH
Et
HO
O
Et
HO
Et
N O
O N
Et
O N
Et
(CH2)4
O
O
O
Et
O
O
16
17
Figure 4. Dimeric cyclohexane-1,3-diones with longer central spacer groups.
Parasitaemia was monitored by blood smears on day 4. Four repli-
cate mice were used for each treatment and the results averaged
and standard deviations and t-tests performed. In summary it
was found that treatment with butroxydim 5 gave a 35% reduction
in parasitaemia at the 160 mg/kg dose and dimer 11 gave a 45%
reduction of parasitaemia with the 48 mg/kg dose (both results
were statistically significant with p <0.01). The chloroquine treated
mice were completely cleared of all parasites whereas dimers 8
and 10 were essentially inactive albeit at lower doses than used
for 5 and 11.
The high resolution crystal structure of tepraloxydim 4 in com-
plex with the CT domain of yeast ACC was recently solved and re-
veals that the small molecule inhibitor is bound in the active site at
the interface of a dimer of the enzyme.16 We were intrigued that
the most active compounds from our exploration are dimeric
cyclohexanedione oximes with each end of the molecules poten-
tially able to bind into an ACC-CT active site. However, a cursory
look at the ACC-CT X-ray structure in complex with 4 shows that
the oxime ether moiety is close to the interface of the protein di-
mer and thus the inhibitor is the wrong way around to allow
simultaneous binding of both ends of dimers such as 8–17.
The fact that the aryloxyphenoxy propionic acid 2 and many of
the cyclohexanedione herbicides do not show significant activity
on P. falciparum in the in vitro assay suggests that neither class
of molecule are good inhibitors of malaria ACC. However, recent re-
ports indicate that cultured P. falciparum parasites can survive in
the absence of type II FAS and that fatty acid synthesis is only
essential during the liver stage of the life cycle in the mouse malar-
ia model P. berghei.17 Thus, it appears that ACC inhibitors will only
show full activity in liver stage testing. Alternatively, it is possible
that the antiplasmodial activity of butroxydim 5 and dimers such
as 11 may be due to activity on some other as yet unidentified
target.
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