Developing ciprofloxacin analogues against plant DNA gyrase: A novel herbicide mode of action

Article abstract of DOI:10.1039/c7cc09518j

Ciprofloxacin has been shown to exhibit potent herbicidal activity through action against plant DNA gyrase, presenting a novel mode of action. Analogues of ciprofloxacin have been prepared with increased herbicidal activity and diminished antibacterial activity, compared to ciprofloxacin, as demonstrated using model systems.

Full text of DOI:10.1039/c7cc09518j

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Received 00th January
20xx,
Developing ciprofloxacin analogues against plant DNA gyrase: A
novel herbicide mode of action
a
b
ac
ad
Michael D. Wallace, Nidda F. Waraich, Aleksandra W. Debowski, Maxime G. Corral, Anthony
b
ad
a
Maxwell, Joshua S. Mylne* and Keith A. Stubbs*
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Ciprofloxacin has been shown to exhibit potent herbicidal activity
through action against plant DNA gyrase, presenting a novel mode
of action. Analogues of ciprofloxacin have been prepared with
increased herbicidal activity and diminished antibacterial activity,
compared to ciprofloxacin, as demonstrated using model systems.
The rapid emergence of herbicide resistance is reducing the
efficacy of herbicides, the most important tool in the control of
1
,2
weeds. Currently, 252 weed species have been reported to
have resistance to one or more herbicide chemistries
Figure 1.
Post-emergent herbicidal activity of ciprofloxacin 1.
(
A
)
Ciprofloxacin with sites of modification highlighted. (B)
(www.weedscience.org). More importantly 38 species are
8
-10
9
reported to have developed resistance to glyphosate, which is
considered the most important herbicide in global weed
in plants
and shown to be essential for proper growth.
Interestingly the fluoroquinolone ciprofloxacin
9
has herbicidal activity (Figure 1B) and it has been shown that
1
(Figure 1A)
3
control. Therefore, there is a desperate need for the
1
1
introduction of herbicides with novel modes of action,
4
something which has not been achieved in over 20 years.
the mode of action of
This presents a potential new herbicidal mode of action.
Despite having the benefits of high potency towards
plant DNA gyrase,
1 is through inhibition of DNA gyrase.
DNA topoisomerases are enzymes that control the
unwinding of DNA during DNA replication, and are classified
based on whether the enzymatic reaction they catalyse
proceed via a transient single- (type I) or double-stranded
1
9
,11
it lacks selectivity relative to bacterial
DNA gyrases, hindering its potential as a herbicide. Here in an
effort to increase selectivity towards a herbicidal mode of
5
break (type II) in DNA. DNA gyrase is an essential type II
6
topoisomerase, first found in the bacterium Escherichia coli,
action we present a synthesis for preparing analogues of
1
where the fluoroquinolone scaffold is retained, but is
elaborated at the N-1 and C-7 positions with various chemical
moieties varying in size, shape and hydrophobicity (Figure 1A).
Using the model plant Arabidopsis thaliana, (a widely used
model plant in plant biology as its genome is known and
sequenced and since it is small, it can be grown under
and has been demonstrated to have the unique ability to relax
positively supercoiled DNA by introducing negative supercoils
using ATP hydrolysis or relaxing negative supercoils
5
independently of ATP. As DNA gyrase is absent in humans it is
an appealing antibacterial target with fluoroquinolones
7
developed as a successful class of antibiotics. Recently, DNA
1
2
laboratory conditions ), as well as bacterial strains and in vitro
assays, against both plant and bacterial DNA gyrase, we
evaluated these molecules for potency and selectivity.
gyrase and its activity has been characterized
To realise a synthetic pathway for preparing analogues
a.
School of Molecular Sciences, University of Western Australia, Crawley, WA 6009, bearing the fluoroquinolone core we felt that the ester
Australia. E-mail: keith.stubbs@uwa.edu.au, joshua.mylne@uwa.edu.au
2
would serve as a useful intermediate. Starting with the aniline
application and adaptation of the known synthetic
b.
c.
Department of Biological Chemistry, John Innes Centre, Norwich Research Park,
Norwich NR4 7UH, United Kingdom.
3
13
School of Biomedical Sciences, University of Western Australia, Nedlands, WA procedures of Koga et al. (Figure 2A) gave the desired
009, Australia.
6
d.
fluoroquinolone substructure 2. With this compound in hand,
ARC Centre of Excellence in Plant Energy Biology, University of Western Australia,
Crawley, WA 6009, Australia.
attention focussed on modifying the N-1 position. Recently an
interactive database containing physiochemical data of 334
Electronic
DOI: 10.1039/x0xx00000x
Supplementary
Information
(ESI)
available:
See
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and benzyl derivative 33 retained modest activity and the i-
propyl derivative 28 also retained some activity, compared to
1. The smaller methyl group of 29 and the longer alkyl groups
of 31 and 32 as well as the introduction of H-bond acceptors as
in 34 and 35 resulted in a loss of activity.
Consequently, attention turned to analogues with
modification at the C-7 position. As ciprofloxacin
1 and 30
(norfloxacin.HCl) were the best compounds in the first round
of herbicidal assays we decided to retain the cyclopropyl and
ethyl groups at the N-1 position for the second round of
analogues. Eighteen analogues were selected using the same
methodology as for the preparation of 28
use of the interactive database (Supporting Figure 1).
Treatment of the chloride 36 or 14 with the appropriate amine
-35, specifically via
1
4
1
8
using S
N
Ar reaction conditions as described by Grohe et al.
43 and 44 50 respectively in
gave the desired compounds 37
-
-
good yields (Table 1). In addition, the (2-aminoethyl)amino
analogues 51 and 52 were prepared via a similar methodology
with 36 and 14, respectively but using an intermediate Boc-
protected moiety to aid purification. (Supporting Scheme 1).
Furthermore, the N-methyl derivative of ciprofloxacin
1,
namely 53, was prepared via an Eschweiler-Clarke methylation
of
commercially.
Assays against A. thaliana with 37
1
(Supporting Scheme 2) and 54
,
was obtained
-
54 found that
substitution of the secondary amine nitrogen to a methylene
or oxygen atom 38 retained herbicidal activity, with an
increase in potency for 38 (Figure 3). A change in ring size as
40 46 and 47 resulted in a reduction of activity
and 30 respectively. The
50 had differing activity,
3
7
Figure 2. (A) a) R-X (X = I, Br or Cl), K
2 3
CO
, DMF; b) 2M aq. NaOH, EtOH; c) i. piperazine, for 39
,
,
DMSO; ii. Boc
2
O, 1M aq. NaOH, THF; d) 3M aq. HCl, EtOH. (B) Representative compared to the parent compounds
1
screening assay of ciprofloxacin 1 and analogues 28-35 with modification at N-1. A.
open chained moieties 41-43 and 48-
thaliana Col-0 was grown on soil and treated with the relevant compound after three
and six days of growth, with 2% DMSO as a control. Images were taken at day 16.
Table 1. Synthesis of compounds based on ciprofloxacin 1 and 30 with modification at
C-7.
1
commercial herbicides was prepared and has been employed
4
1
5-17
for herbicide discovery
and so we used this database
tochoose a simple set of analogues that varied in size, shape
and hydrophobicity, but retained favourable physiochemical
properties for herbicides (Supporting Figure 1). The synthesis
of the esters
position with the relevant alkyl halide in good yields (73-93%),
except for the i-propyl derivative , where a low yield of 17%
was obtained, which was consistent with the alkylation
reaction involving being unfavourable toward secondary
alkyl halides. Subsequent base-mediated hydrolysis of the
esters 11 gave the acids 12 19 in good yields. Nucleophilic
aromatic substitution of the chlorides 12 19 with piperazine
4-11 was achieved via alkylation of the N-1
5
Entry
1
Amine
Product
Yield (%)
61
Product Yield (%)
2
37
44
37
1
8
4-
-
2
3
38
39
85
58
45
46
59
40
-
1
8
using a modified procedure followed by the Boc-protection
of the resultant amine using a modified procedure by
1
9
Dhaneshwar et al., in a one-pot reaction, produced the Boc-
protected fluoroquinolones 20 27. Removal of the Boc-
protecting group from 20 27 furnished the desired compounds
35 with acceptable overall yields.
The compounds 28 35 were evaluated for their
post-emergent herbicidal activity against A. thaliana
Figure 2B). Overall, all the modified compounds showed a loss
in activity compared to ciprofloxacin . The ethyl derivative 30
4
5
6
40
41
42
58
41
32
47
48
49
43
44
35
-
-
28-
-
(
7
43
32
50
28
1
2
| J. Name., 2012, 00, 1-3
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Figure 3. Representative screening assay of analogues of ciprofloxacin 1 and 30, which have been modified at C-7. The screening was conducted as described in Figure 2B and
images were taken at day 20 of growth.
with the smaller dimethyl moieties 41 and 48 being the most compounds were more active against recombinant E. coli
potent and with 41 being similar to 1. Compounds 51 and 52 gyrase compared to A. thaliana gyrase. From these results it is
bearing a single linear chain had very little herbicidal activity. clear that the overall differences in potency observed against
Finally, compound 53 retained herbicidal activity compared to organismal models is not manifested at the level of enzyme
1
, and 54 retained modest herbicidal activity compared to 30
.
inhibition, but rather the availability of the compound through
To assess if there was any selectivity gained between plant and/or bacterial uptake. This result is important in terms
plants and bacteria, either due to a difference in the of potential future development of the scaffolds for use as
compounds ability to penetrate the organism or its affinity for herbicides for not only ciprofloxacin-based analogues, but
plant and bacterial gyrase, antibiotic susceptibility tests (MIC) fluoroquinolones in general. Typically compounds are first
against Escherichia coli (Table 2), Pseudomonas aeruginosa evaluated against recombinantly expressed enzymes for
and Staphylococcus aureus (Supporting Table 2) were potency and selectivity (where needed) and only then
2
0
conducted, as well as DNA gyrase supercoiling assays against progressed to more complex systems. Our data suggest
recombinant A. thaliana and E. coli DNA gyrase (Table 2, evaluation should be conducted at the level of the
Supporting Figure 2A and Supporting Table 1).
Specifically for E. coli, the synthesised analogues all had compounds may be excluded if a protein-first evaluation is
MIC values greater than ciprofloxacin , except for 53. Not used.
surprisingly the selectivity of the known antibiotics 30 and 54 Analogues that demonstrated reasonable herbicidal
remained poor along with the parent compound , towards a activity namely 30 37 39 41 44 and 45, were assayed against
plant/bacterium to evaluate potency and selectivity as
1
1
,
-
,
,
herbicidal mode of action. On the other hand some
compounds saw a considerable improvement in selectivity
Table 2. Overview of compounds displaying herbicidal potency against A. thaliana with
an IC50 value less than 25 mg/L. Minimum inhibitory concentrations (MICs) against
E. coli B. IC50 values of compounds against recombinant A. thaliana and E. coli gyrase in
towards a herbicidal mode of action. Analogues 37 and 38
,
which have similar herbicidal activities compared to
1
, showed supercoiling assays. Errors represent standard error of the mean.
a significant increase in MIC against E. coli and P. aeruginosa,
and modest changes against S. aureus (See Supporting Table
a
a
b
c
IC50
MIC
Selectivity
DNA Gyrase IC50
A. thaliana
10.2 ± 0.4
19.3 ± 2.8
6.1 ± 1.0
E. coli
A. thaliana
0.68 ± 0.06
3.15 ± 0.15
4.70 ± 0.70
E. coli
2). Of note is 44 which, despite having a slight reduction in
-4
1
0.0078
7 x 10
0.56 ± 0.07
1.35 ± 0.25
1.90 ± 0.50
herbicidal potency compared to ciprofloxacin 1, has >600-fold
-3
30
0.0625
3 x 10
increase in selectivity toward a herbicidal mode of action
based on testing against the organismal models. Evaluation of
the potency of all the compounds against the recombinant
DNA gyrases of A. thaliana and E. coli in supercoiling assays
also produced interesting results. Overall the potency of the
prepared analogues was less to that observed for ciprofloxacin
37
1
0.25
2
0.16
0.06
0.09
0.02
0.45
0.08
38
39
41
4.0 ± 0.3
5.85 ± 0.65 0.77 ± 0.095
21.4 ± 2.6
24.0 ± 3.2
17.8 ± 1.6
12.0 ± 1.0
2.80 ± 0.60
8.13 ± 1.76
26.9 ± 3.3
28.4 ± 3.6
0.25 ± 0.04
1.90 ± 0.40
2.65 ± 0.05
2.45 ± 0.45
0.5
8
44
45
1
a
c
µ
b
mg/L. selectivity towards a herbicidal mode of action (defined as MIC/IC50).
M
1
with respect to both enzymes however like 1 each of the
This journal is © The Royal Society of Chemistry 20xx
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Microbiology Culture Collection. The authors also thank the
Centre for Microscopy, Characterisation and Analysis at The
University of Western Australia, which is supported by
University, State and Federal Government funding. KAS and
JSM also thank the Australian Research Council for funding
(
FT100100291 and FT120100013 respectively). MDW thanks
the University of Western Australia for a UWA Alumni SWANS
Rural Scholarship and Research Training Program
a
Scholarship. MGC is supported by a University Postgraduate
Award for International Students and a Scholarship for
International Research by the University of Western Australia.
AWD thanks the National Health and Medical Research Council
for funding (APP1073250). AM is supported by the
Biotechnology and Biological Sciences Research Council UK
(BB/P012523/1) and the John Innes Foundation. NFW is
supported by a CASE studentship from the BBSRC and
Inspiralis. This work was also funded in part by a Bayer AG
division Crop Science Grants4Targets grant 2016-01-55 to JSM,
KAS and AM.
Figure 4. Representative testing assay of analogues which showed herbicidal activity
Conflicts of Interest
(
IC50 <25 mg/L), against wild-type (WT) and gyra-3 mutant A. thaliana. Testing was
conducted as described in Figure 2B and images were taken at day 20 of growth.
There are no conflicts to declare.
1
1
a ciprofloxacin resistant A. thaliana mutant (gyra-3), which
Notes and references
has a point mutation that causes an amino acid substitution in
its encoded ATGYRA, to confirm these compounds were on 1. S. B. Powles and Q. Yu, Annu. Rev. Plant Biol., 2010, 61, 317.
target, affecting only the DNA gyrase protein in vivo (Figure 4). 2. L. P. Gianessi, Pest Manag. Sci., 2013, 69, 1099.
3
4
5
6
.
.
.
.
S. O. Duke and S. B. Powles, Pest Manag. Sci., 2008, 64, 319.
S. O. Duke, Pest Manag. Sci., 2012, 68, 505.
N. Bush, K. Evans-Roberts and A. Maxwell, in EcoSal Plus, 2015.
M. Gellert, K. Mizuuchi, M. H. O'Dea and H. A. Nash, Proc. Natl.
Acad. Sci. USA, 1976, 73, 3872.
The results are highly suggestive that DNA gyrase is the sole
target as the mutants demonstrate an increased resistance to
not only ciprofloxacin
1, but also to the analogues. To further
confirm that these compounds act in a similar manner to
ciprofloxacin
1
,
DNA cleavage assays were conducted. 7. G. A. Jacoby and D. C. Hooper, in Antibiotic Discovery and
Development, eds. T. J. Dougherty and M. J. Pucci, Springer US,
Fluoroquinolones, such as 1, inhibit DNA gyrase by stabilising
Boston, MA, 2012, pp. 119.
the covalent complex that forms between the enzyme and
6
DNA and such complexes can be detected as a linear band in
8
.
H. S. Cho, S. S. Lee, K. D. Kim, I. Hwang, J. Lim, Y. Park and H. Pai,
Plant Cell, 2004, 16, 2665.
agarose gel electrophoresis. The most herbicidal compounds 9. M. K. Wall, L. A. Mitchenall and A. Maxwell, Proc. Natl. Acad.
were assayed (Supporting Figure 2B) and found to be able to
Sci. USA, 2004, 101, 7821.
1
1
0. C. V. Morgante, R. A. O. Rodrigues, P. A. S. Marbach, C. M.
Borgonovi, D. S. Moura and M. C. Silva-Filho, Mol. Genet.
Genomics, 2009, 281, 525.
1. K. M. Evans-Roberts, L. A. Mitchenall, M. K. Wall, J. Leroux, J. S.
Mylne and A. Maxwell, J. Biol. Chem., 2016, 291, 3136.
stabilise the gyrase cleavage complex with both E. coli and A.
thaliana gyrase, similar to ciprofloxacin 1, suggesting that they
all act by a similar mechanism.
In conclusion, herbicide resistance is a growing concern
and there is a pressing need for new modes of action. Previous 12. M. Koornneef and D. Meinke, Plant J., 2010, 61, 909.
1
3. H. Koga, A. Itoh, S. Murayama, S. Suzue and T. Irikura, J. Med.
Chem., 1980, 23, 1358.
4. M. N. Gandy, M. G. Corral, J. S. Mylne and K. A. Stubbs, Org.
Biomol. Chem., 2015, 13, 5586.
research has shown that DNA gyrase presents a potential new
target for herbicide development. We have demonstrated
here using a known chemical inhibitor scaffold of DNA gyrase,
1
fluoroquinolones, that tuning of molecules for selectivity 15. M. Corral, J. Leroux, K. A. Stubbs and J. S. Mylne, Sci. Rep., 2017,
towards a herbicidal rather than an antibiotic mode of action is
possible at the organism level. However, the gyra-3 mutant
highlights that, as for all herbicidal targets, resistance can be
selected for so practices that delay the evolution of resistance
7, 45871.
1
1
6. S. Funar-Timofei, A. Borota and L. Crisan, Mol Divers., 2017, 21,
4
37.
7. M. G. Corral, J. Leroux, S. Tresch, T. Newton, K. A. Stubbs and J.
S. Mylne, Angew. Chem. Int. Ed., 2017, 56, 9881.
will also need to be considered. Overall this work presents a 18. K. Grohe and H. Heitzer, Liebigs Ann. Chem., 1987, 29.
2
1
platform for further research into this enzyme, both at the 19. S. Dhaneshwar, K. Tewari, S. Joshi, D. Godbole and P. Ghosh,
Chem. Phys. Lipids, 2011, 164, 307.
0. J. P. Hughes, S. Rees, S. B. Kalindjian and K. L. Philpott, Br. J.
Pharmacol., 2011, 162, 1239.
1. J. A. Evans, P. J. Tranel, A. G. Hager, B. Schutte, C. Wu, L. A.
Chatham and A. S. Davis, Pest. Manag. Sci, 2015, 72, 74.
chemical, biological and structural level, as a possible new
2
2
mode of action for herbicide development.
The authors wish to thank Melissa Andrade and Marina
Silich-Carrara for providing bacterial strains from the UWA
4
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The development of ciprofloxacin analogues against plant DNA gyrase, a novel herbicidal
target, with increased herbicidal activity and diminished antibacterial activity is described.