Gram-Positive and Gram-Negative Antibiotic Activity of Asymmetric and Monomeric Robenidine Analogues

Article abstract of DOI:10.1002/cmdc.201800463

Desymmetrisation of robenidine (1: N′,2-bis((E)-4-chlorobenzylidene)hydrazine-1-carboximidhydrazide) and the introduction of imine alkyl substituents gave good antibiotic activity. Of note was the increased potency of two analogues against vancomycin-resistant Enterococci (VRE), one of which returned a MIC of 0.5 μg mL^?1. Five analogues were found to be equipotent or more potent than the lead 1. Introduction of an indole moiety resulted in the most active robenidine analogue against methicillin-resistant S. aureus (MRSA), with a MIC of 1.0 μg mL^?1. Imine C=NH isosteres (C=O/C=S) were inactive. Monomeric analogues were 16–64 μg mL^?1 active against MRSA and VRE. An analogue that lacks the terminal hydrazide NH moiety showed modest Gram-negative activity at 64 μg mL^?1. A 4-tert-butyl analogue was shown to be active against both Gram-positive and -negative strains at 16–64 μg mL^?1. In general, additional modifications with aromatic moieties was poorly tolerated, except with concomitant introduction of an imine C-alkyl group. The activity of these analogues against MRSA and VRE ranged from 8 μg mL^?1 to inactive (MIC>128 μg mL^?1) with the naphthyl and indole analogues. Gram-negative activity was most promising with two compounds at 16 μg mL^?1 against E. coli. Against P. aeruginosa, the highest activity observed was with MIC values of 32 μg mL^?1 with another two analogues. Combined, these findings support the further development of the (E)-2-benzylidenehydrazine-1-carboximidamide scaffold as a promising scaffold for the development of antibiotics against Gram-positive and Gram-negative strains.

Full text of DOI:10.1002/cmdc.201800463

Accepted Article
Title: Gram-positive and Gram-negative Antibiotic Activity of
Asymmetric and Monomeric Robenidine Analogues
Authors: Cecilia C Russell, Andrew Stevens, Hongfei Pi, Manouchehr
Khazandi, Abiodun Ogunniyi, Kelly A Young, Jennifer R
Baker, Siobhann Niamh McCluskey, Stephen W Page, Darren
J Trott, and Adam McCluskey
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201800463
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800463
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10.1002/cmdc.201800463
ChemMedChem
FULL PAPER
Gram-positive and Gram-negative Antibiotic Activity of
Asymmetric and Monomeric Robenidine Analogues
Cecilia C Russell,[a] Andrew Stevens,[a] Hongfei Pi,[b] Manouchehr Khazandi,[b] Abiodun Ogunniyi,[b]
Kelly A Young,[a] Jennifer R Baker,[a] Siobhann N McCluskey,[a] Stephen W Page,[c] Darren J Trott,[b]
and Adam McCluskey *[a]
[a]
Dr CC Russell (0000-0001-8401-5856), Dr A Stevens (0000-0002-5114-9134), Ms KA Young (0000-0002-7252-4693), Ms JR Baker (0000-0002-9560-
301X), Ms SN McCluskey (0000-0003-3609-9316), Prof A McCluskey (0000-0001-7125-863X)
Chemistry, School of Environmental & Life Sciences
The University of Newcastle
University Drive, Callaghan NSW 2308 Australia
E-mail: Adam.McCluskey@newcastle.edu.au
[b]
Mr H Pi (0000-0002-5758-6044), Dr M Khazandi (0000-0002-9806-9959); Dr A Ogunniyi (0000-0001-9308-5629), Prof DJ Trott (0000-0002-8297-5770)
Australian Centre for Antimicrobial Resistance Ecology
School of Animal and Veterinary Sciences, University of Adelaide
Roseworthy Campus, Mudla Wirra Road, Roseworthy, 5371 SA, Australia.
Dr SW Page (0000-0001-5312-2149),
[c]
Neoculi Pty Ltd
Burwood, 3125 VIC, Australia.
Supporting information for this article is given via a link at the end of the document.
Abstract: Desymmetrisation of robenidine 1, N',2-bis((E)-4-
chlorobenzylidene)hydrazine-1-carboximidhydrazide, and the
Control and Prevention in 2013 estimated the US deaths from
bacterial infections at >23,000 [2]. In Australia, there are an
estimated 165,000 hospital acquired bacterial infections [3]. By
2050 The Review on Antimicrobial Resistance estimates bacterial
infection will result in 10 million deaths globally [4].
introduction of imine alkyl substituents gave good antibiotic activity.
Of note was the increased potency of 17 and 20 against VRE with 20
the most active, MIC of 0.5 µg mL-1. Analogues 2, 14, 17, 19 and 20
were equipotent or more potent than the lead 1. Introduction of an
indole moiety, 30, resulted in the most MRSA active robenidine
analogue, MIC of 1.0 µg mL-1. Imine C=NH isosteres (C=O / C=S)
were inactive. Monomeric analogues, 33–35 were 16 – 64 µg mL-1
active against MRSA and VRE. Analogue 36, lacking the terminal
hydrazide NH moiety showed modest Gram-negative activity at 64 µg
mL-1. 4-t-Butyl 45 was Gram -positive and -negative active at of 16 –
64 µg mL-1. Typically additional aromatic moiety modification was
poorly tolerated, except with concomitant introduction of an imine C-
alkyl moiety. The activity of these analogues against MRSA and VRE
ranged from 8 µg mL-1 with 64 and 68, to inactive (MIC > 128 µg mL-
1) with the naphthyl 69 and 70 and the indole 73. Gram-negative
activity was most promising with 62 and 68 at 16 µg mL-1 against E.
coli. Against Ps. aeruginosa, the highest activity observed was with
MIC values of 32 µg mL-1 with 62 and 64. Combined, these findings
support the further development of the (E)-2-benzylidenehydrazine-1-
carboximidamide scaffold as a promising Gram-positive and Gram-
negative antibiotic development.
Effective treatment of bacterial infections is becoming
increasingly difficult with the emergence of resistance to multiple
classes of antimicrobial drug. In particular, sepsis due to the
multidrug-resistant (MDR) ESKAPE pathogens (Enterococcus
faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and E.
coli/Enterobacter species) contribute significantly to morbidity and
mortality associated with antimicrobial resistance (700,000
deaths annually) worldwide [5-10]. Therefore, the discovery and
development of new broad-spectrum antimicrobials with activity
against pan-resistant resistant ESKAPE pathogens causing
sepsis is one of the highest global priorities [11-13]. In 2010, the
Infectious Diseases Society of America (IDSA) made an
ambitious call for 10 new antimicrobial agents to be registered by
2020 [14]. However, since that call, only a single agent with a new
mode of action (the narrow-spectrum drug fidaxomicin for
Clostridium difficile infections) has been registered. The
remainder represent modifications to existing drug classes
targeting
dalbavancin, tedizolid, oritavancin) or Gram-negative organisms
(ceftolozane-tazobactam, ceftazidime-avibactam) [15-18].
Reiterating the call for novel agents with new modes of action, the
World Health Organization (WHO) recently published a priority list
of bacteria for which new antibiotics are urgently needed, in which
MDR ESKAPE pathogens are prominent [19]. As of March 2017,
there were 41 new antibiotics in clinical development within the
US, but it is expected that less than 1 in 5 of these agents will
either
Gram-positive
organisms
(telavancin,
Introduction
Pathogenic bacteria that are resistant to multiple classes of
antimicrobials account for massive morbidity and mortality in
humans worldwide, and poses both a significant public health
concern and ever increasing health-care costs in many countries.
A 2016 joint report from the European Center for Disease
Prevention and Control and the European Medicines, based on
2007 data estimated that there were >25,000 bacterial infection
associated deaths in Europe [1]. The US Centre for Disease
enter
the
clinic.
Baxdela
(delafloxacin)
and
Mereopenen+Vaborbactam have NDAs submitted are potential
treatments for the ESKAPE pathogens [20].
1
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10.1002/cmdc.201800463
ChemMedChem
FULL PAPER
We recently looked to repurpose the anti-coccidial agent
robenidine in this space (1; NCL812). Our preliminary reports
show promising levels of bactericidal activity [21, 22]. Of these, 2
exhibits bactericidal activity against both S. pneumoniae
(minimum inhibitory concentration (MIC) range 2-8 µg mL-1) and
S. aureus (MIC range 1-2 µg ml-1) via disruption of the cell
membrane potential. In the presence of polymyxin B nonapeptide,
2 also showed Gram-negative activity against E. coli and P.
aeruginosa at 10 and 5 µM concentrations, respectively (Figure
1). Importantly, our preliminary analogues were less cytotoxic to
a variety of mammalian cell lines than the parent compound, and
were typically non-toxic to erythrocytes at the highest
concentration tested (128 µg mL-1). This suggests specificity for
the prokaryotic cell membrane, which can be engineered by
further modification of this scaffold, whilst identifying analogues
with improved solubility, pharmacokinetics and potency against
ESKAPE pathogens.
R2
R2
R2
H
H
H
N
H
N
N
i
H2N
NH2
N
O
N
N
NH
HCl
NH
R1
R1
R1
ii
R2
R2
R3
H
H
H
N
H
iii
N
N
N
N
NH2
N
N
NH
NH
R4
R1
R1
Scheme 1. Reagents and Conditions: i) 2 equiv. ‘C=O’ (see Table 1 for detail),
1,3-diaminoguanidine hydrochloride, EtOH reflux; ii) 1 equiv. ‘C=O’ (see Table
1 for detail), 1,3-diaminoguanidine hydrochloride, EtOH reflux; and iii) as per (ii)
with different ‘C=O’ as detailed in Table 1.
Our initial efforts examined the modification of one of the aromatic
rings of the lead 1, followed by the introduction of imine C-alkyl
substituents. However, we note that the yields of this ‘two-step’
process were typically far lower than the equivalent one pot
procedure. In the presence of the second equivalent of the C=O
entity we typically observed a product distribution that related to
an imine equilibrium in the resulting reaction mixture. Flash
chromatography to isolate the mono adduct was possible, but on
introduction of the mono-imine adduct to the reaction mixture, the
H
H
H
H
N
N
N
N
N
N
N
N
NH
NH
Cl
Cl
Cl
Cl
1
NCL812
2
Figure 1. Chemical structures of lead Robenidine analogues 1 (NCL812) and
2.
initial equilibrium mixture was isolated.
The outcome of
In our initial report on the modifications of robenidine (1), our
investigation was restricted to the SAR of symmetrical dimeric
analogues and simple monomers. These preliminary SAR
studies demonstrated a key role for the ring C2’-/C4’- halogens;
that imine C-alkylation gives enhanced hydrolytic stability (t1/2 >
240 min in hepatic microsomal preparations); imine reduced
analogues are inactive [21]; and N to O bioisosteric modifications
abrogated activity. Herein we report on efforts to expand the
known pharmacophore of robenidine analogues as potential
antibiotic agents.
subsequent antibiotic screening against methicillin resistant
Staphylococcus aureus (MRSA), vancomycin resistant
Enterococci (VRE), E. coli and Ps. aeruginosa is presented in
Table 1. Lead 1, displayed good levels of activity against both
MRSA and VRE with MIC values of 1-4 µg mL-1. Reposition of
one of the 4-Cl moieties to the asymmetrically substituted 2-Cl 3
resulted in a 2-4 fold reduction in activity (8 µg mL-1), with the
analogous 3-Cl 4 inactive (MIC >128 µg ml-1).
Isosteric
replacement of a 4-Cl with a 4-CH3 5 or a 4-CF3 6 moiety saw a
complete loss of activity. The 2-F 7 and 4-F 8 analogues were
active against MRSA and VRE at 2-4 µg mL-1. However the loss
of activity with the introduction of a 2- or 3- or 4- CN (9-11)
suggests that this is not purely a function of electronegativity at
this position. The introduction of a second halogen to one
aromatic ring saw a modest reduction in potency with 12 at 2-16
µg mL-1, while the combination of a 2-F, 4-CF3 and a 2-F, 4-Cl
moieties with 13 and 14 saw excellent MIC values of 2-4 µg mL-1.
The symmetrically substituted 2-F, 4-Cl 15 saw a modest
reduction in activity relative to 14. Replacement of the 2-F of 15
with the H-bonding capable 2-NH2, 16, resulted in a 4-fold loss of
activity suggesting that a hydrogen bonding (acceptor or donor)
group in this position was not favoured.
Results and Discussion
Access to robenidine analogues is rapidly accomplished via a
condensation of an aldehyde or phenone with 1,3-
diaminoguanidine hydrochloride. The use of two equivalents of
the carbonyl entity affords symmetrically substituted analogues,
while careful control of the reaction stoichiometry (one equivalent
of C=O entity) allows isolation of the mono-adduct, which can be
further reacted with a second equivalent of the same or a different
aldehyde or phenone (Scheme 1).
2
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Table 1. Inhibition of two isolates of MRSA, VRE, E. coli, and Ps. aeruginosa growth by 1,3-aminoguanidine Schiff Base analogues possessing mono- and di-
substituted aromatic rings (1− 25).
R2
R3
H
H
N
N
N
N
R1
R4
NH
MIC mode (µg mL-1)
R1
R2
H
R3
H
R4
MRSA
VRE
E. coli
Ps. aeruginosa
b
4-Cl
4-Cl
2
2
8
-
2
2
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
4-Cl
Me
H
Me
H
4-Cl
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
4-Cl
2-Cl
3
4-Cl
H
H
3-Cl
4
4-Cl
H
H
4-Me
-
-
5
4-Cl
H
H
4-CF3
-
-
6
4-Cl
H
H
2-F
8
4
-
4
4
-
7
4-Cl
H
H
4-F
8
4-Cl
H
H
2-CN
9
4-Cl
H
H
3-CN
-
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
4-Cl
H
H
4-CN
-
-
4-Cl
H
H
2,4-di-Cl
2-F, 4-CF3
2-F, 4-Cl
2-F, 4-Cl
2-NH2, 4-Cl
4-Cl
16
4
2
8
32
4
-
2
4
2
4
32
2
-
4-Cl
H
H
4-Cl
H
H
2-F, 4-Cl
2-NH2, 4-Cl
4-Cl
Me
H
Me
H
H
Me
H
4-Cl
Me
H
4-CF3
4-Cl
Me
Me
H
4-CF3
4
4
-
2
0.5
-
4-Cl
Me
Me
Me
Me
Et
4-CF3
4-CF3
2-NH2, 4-Cl
2-OH, 4-Cl
2-OH, 4-Cl
2-OH, 4-Cl
4-CF3
Me
Me
Et
2-NH2, 4-Cl
2-OH, 4-Cl
2-OH, 4-Cl
2-OH, 4-Cl
-
-
16
4
64
32
4
128
c-Pent
c-Pent
a compounds isolated as the HCl salts; b inactive at 128 µg mL-1
The introduction of a CH3 moiety at each of the imine carbons
resulted, with 2, in a moderate potency enhancement against
MRSA and VRE, and as previously reported an in vitro stability
enhancement [16]. Selective removal of one the imine carbon
CH3 moieties resulted in a modest decrease in MRSA but not VRE
activity with 17, but combined with the introduction of a 4-CF3
moiety, abolished activity with 18. With the 4-CF3 analogues 19
and 20, the loss of a CH3 moiety was well tolerated with retention
of the activity noted at 2-4 µg mL-1, but with 20, which retained
both CH3 moieties, activity was retained against MRSA with a
concurrent 2-fold increase in potency against VRE (0.5 – 1 µg
mL-1). Bis-4-CF3 21 is inactive. This suggests that the position,
and potentially the steric bulk, of the C-alkyl moiety, plays a key
role in the observed antibiotic activity in combination with the
nature of the aromatic substituent.
3
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Given the retention of activity noted with 1 and 2 on
introduction of the imine C-CH3 moieties we looked to apply the
same modification to the 2-NH2, 4-Cl substituted 16, however this
removed all antibiotic activity (MIC > 128 µg mL-1; 22). The
corresponding 2-OH 23 is more potent than either 16 or 22 with
MIC values 4-32 µg mL-1. Increasing the imine C-alkyl moiety to
ethyl 24 gave a further 4-fold potency enhancement. However,
the c-pentyl analogue 25 is only active against one of MRSA and
one VRE strains examined. The 4-Cl, CH2OH analogue 22
displays MIC values of 8 µg mL-1 against MRSA and VRE. None
of these analogues show efficacy against the two exemplar Gram-
negative bacterial strains examined, i.e. E. coli and Ps.
auruginosa.
Phenyl ring modifications were investigated via the synthesis
of the 2-pyridyl 26, 3-pyridyl 27 and 28, as well as the indole 29
and 30. Subsequent screening of these analogues revealed that
the pyridyl analogues resulted in a significant loss of activity with
only 4-Cl-3-pyridyl 27 displaying modest activity against VRE.
The 2-indole 29 displayed good activity against Gram-positive
MRSA and VRE, 4 and 8 µg mL-1respectively. The corresponding
5-bromoindole 30 was 4-fold more MRSA active while retaining
the same 8 µg mL-1 activity against VRE.
Table 2. Inhibition of two isolates of MRSA, VRE, E. coli and Ps. aeruginosa growth by 1,3-aminoguanidine Schiff Base analogues possessing heterocyclic aromatic
rings (26 − 30).
MIC mode (µg mL-1)
Compound
Structure
MRSA
VRE
E. coli
Ps. aeruginosa
H
N
H
N
b
26a
27
128
-
-
-
N
N
N
NH
N
H
N
H
N
N
N
128
128
-
-
NH
Cl
N
N
Cl
H
N
H
N
N
N
28
29
-
-
-
-
NH
N
NH2
H2N
N
4
8
-
-
Br
Br
H
N
H
30
1
4
-
-
N
N
N
NH
HN
NH
a compounds isolated as the HCl salts; b in active at 128 µg mL-1
Isosteric modification of the central imine C=NH to a C=O
removed activity for all analogues excepting the 3-CF3-substituted
31 (Figure 2), which retained modest potency against one of the
VRE lines examined (32 µg mL-1). In a similar manner a C=NH to
C=S modification only afforded a single analogue, the 3-Cl
substituted 32 (Figure 2), which retained activity against one of
of these findings we examined the activity of three simple
monomeric compounds, the 4-Cl 33, 34 and 4CF3 35 guanidine
analogues, and the effect of removal of the terminal amino moiety
of the guanidine hydrazine in the synthesis of 4-Cl 36 (Figure 3).
H
H
H
H
N
N
N
N
the VRE strains examined with
(Supplementary Data).
a
MIC of 32 µg mL-1
N
NH2
N
NH2
NH
NH
Cl
Cl
33
34
H
N
H
N
F3C
CF3
H
H
H
N
N
N
N
N
N
N
NH2
NH
N
NH2
N
O
NH
31
F3C
Cl
35
36
H
N
H
N
Cl
Cl
Figure 3. Chemical structures of monomeric analogues 33-36.
O
Both 33 and 34 showed reduced Gram-positive antibiotic
activity, with 33 active at 32 and 64 µg/mL against MRSA and
VRE respectively, 34 showed >64 µg mL-1 activity against VRE
only, the 4-Cl to 4-CF3 modification with 35 saw and increase in
VRE activity at 16 µg mL-1, but similarly no activity against MRSA.
Interestingly, 36 lacking one of the guanidine NH moieties showed
32
Figure 2. Chemical structure of 31 and 32.
As noted above, the presence of asymmetrically di-
substituted analogues retained, and in some cases enhanced
activity against the Gram-positive strains examined. As a result
4
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10.1002/cmdc.201800463
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no Gram-positive activity (MRSA and VRE MIC values >128 µg
mL-1), but did show promising and unexpected levels of Gram-
negative activity with a 64 µg mL-1 MIC value for both E. coli and
Pseudomonas.
Emboldened by the identification of Gram-negative activity of
36, we explored the development of an additional focused
compound library that saw further modification to the aromatic
scaffold, but lacking one of the guanidine NH moieties. These
synthesises were accomplished using a slight modification of the
procedure described in Scheme 1. A total of 39 monomeric
analogues 37-73, and in many cases the corresponding C=S and
C=O isosteres were generated, but only the parent analogues
Supplementary Data). In most cases the removal of the second
aromatic moiety removed all vestiges of antibiotic activity for
analogues lacking an imine C-alkyl moiety, viz halogens 37-41,
small alkyl 42-44, CN 46-48, OCH3 49 and N(CH3)2 50 moieties
were inactive. However 4-t-Bu 45 afforded activity across Gram-
positive (16 and 64 µg mL-1) and Gram-negative bacteria (64 and
64 µg mL-1). Of the trifluoromethyl substituted analogues 51 – 53
only the 4-CF3 53 was active. The 2-F,4-Cl 54 was very weakly
active, as was the corresponding 2-F,4-CF3 55, but this analogue
showed low levels of Gram-negative activity as well.
returned any noteworthy antibiotic activity (Table
3 and
Table 3. Inhibition of two isolates of MRSA, VRE, E. coli, and Ps. aeruginosa growth by monomeric 1,3-Aminoguanidine Schiff Base analogues (37−73).
R2
H
N
NH2
NH
MIC mode (µg ml-1)
N
R1
R1
R2
H
MRSA
VRE
E. coli
Ps. aeruginosa
b
37a
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
2-Cl
-
-
-
-
3-Cl
H
-
-
-
-
2-F
H
-
-
-
-
3-F
H
-
-
-
-
4-F
H
-
-
-
-
2-CH3
3-CH3
4-CH3
4-t-Bu
2-CN
3-CN
4-CN
2-OCH3
4-N(CH3)2
2-CF3
3-CF3
4-CF3
2-F,4-Cl
2-F, 4-CF3
4-Cl
H
-
-
-
-
H
-
-
-
-
H
-
-
-
-
H
16
-
64
-
64
64
H
-
-
H
-
-
-
-
H
-
-
-
-
H
-
-
-
-
H
-
-
-
-
H
-
-
-
-
H
-
-
-
-
H
64
128
64
>64
16
32
64
32
32
8
64
128
64
>64
32
32
64
32
32
8
-
-
H
-
-
H
128
>64
32
>128
128
64
64
16
8
128
>64
64
128
Me
Me
Me
Et
Pr
n-Bu
n-pent
n-hept
4-t-Bu
2-F,4-Cl,
4-Cl
a
-
4-Cl
128
>64
32
-
4-Cl
4-Cl
4-Cl
2
2
5
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64
65
66
67
68
4-Cl
CH-cyclohexane
(CH2)2-cyclopentane
CH2NHNH2
2
8
16
32
-
4-Cl
4
4
16
4-Cl
32
>64
8
64
>64
8
>64
>64
16
>64
>64
-
H
COOH
2-OH,4-Cl
CH-cyclohexane
H
N
NH2
NH
69
70
64
64
128
64
128
128
128
N
H
N
NH2
NH
-
N
OH
H
N
NH2
NH
N
71
72
32
32
64
64
128
128
128
H
N
NH2
NH
N
H
NH2
NH
N
N
73
-
-
-
-
N
H
a compounds isolated as the HCl salts; b inactive at 128 µg mL-1
The parent imine C-alkyl analogue in this series 56 was
inactive at 64 µg mL-1, with the corresponding 4-t-Bu 57 16-64 µg
mL-1 active across all bacteria screened (Table 3). Introduction of
the imine C-methyl to 53 gave 58, which showed a 4-fold
(MRSA, n=20), Escherichia coli (n=20), Enterobacter aerogenes
(n=16), Enterobacter cloacae (n=19), Klebsiella pneumoniae
(n=20) and Klebsiella oxytoca (n=16). Both analogues (64 and 63)
were bactericidal against all of the strains tested with MBC/MIC90
improved Gram-positive activity and some Gram-negative activity. ratios ≤ 2 (Table 4). The results demonstrated potent activity
Conducting a systematic evaluation of the effect of the C-alkyl
moiety we examined the activity of a series of 4-Cl analogues
through an ethyl 59, propyl 60, butyl 61, pentyl 62, CH-
cyclohexane 64 and (CH2)2-cyclopentane 65. Over this series,
the antibiotic activity increased from methyl 64 to pentyl 70 with
the latter analogue returning MIC values of 8 µg mL-1 against
MRSA and VRE, and 16 and 32 µg µL-1 against E. coli and Ps.
aeruginosa respectively. Further increasing in the size of the alkyl
moiety was detrimental to activity.
Our findings within this compound series, that a reduction in
molecular weight combined with an increase in polarity affords
promising levels of Gram-negative activity, is in keeping with
recent reports [23]. This is in part a consequence of reduced
efflux liability [24], combined with the poor passive permeability
through the outer membrane of gram-negative bacteria of
hydrophobic molecules [25-27].
against major target pathogens in human isolates. Time−kill curve
evaluation of 64 and 63 at 1x 2x and 4x their MIC against one
susceptible S. aureus isolate (ATCC 25923), one clinical MRSA
isolate (USA300) and one clinical E. coli isolate and one clinical
K. pneumoniae ATCC 4352 confirmed their bactericidal activity,
resulting in a >5-log reduction in bacterial cell number over an 0.5
h period across both analogue doses evaluated (Figure 4). After
24 h, no bacteria regrowth was observed with both compounds at
all concentrations tested. Introduction of polar C-substituents was
detrimental to activity, with CH2NHNH2 66 and COOH 67 showing
reduced activity. However introduction of a hydrogen bonding
moiety to the aromatic ring with the 2-OH,4-Cl 68 was well
tolerated with MIC values of 8-16 µg mL-1 observed. Increasing
the size of the aromatic moiety to naphthyl with 69 – 71, extension
of the conjugation between the aromatic moiety and the amino
warhead with 72, or via an indole 73 all resulted in a loss of activity
suggesting that the phenyl moiety maybe the optimal aromatic
moiety in this compound series.
Having established the antibacterial activity of this class of
compounds, we examined the activity of 64 and 63 against Gram-
positive and Gram-negative isolates obtained from clinical cases
of infection including, methicillin resistant Staphylococcus aureus
Table 4. MIC range, MIC50, MIC90, MBC values (μM) and MBC/MIC for 64 and 63 against methicillin resistant staphylococcus aureus (MRSA, n=20), Escherichia
coli (n=20), Enterobacter aerogenes (n=16), Enterobacter cloacae (n=19), Klebsiella pneumoniae (n=20) and Klebsiella oxytoca (n=16) isolated from human clinical
cases.
MBCd range
(µM)
MBC/
MIC
MBC range
(µM)
MBC/
MIC
MICa values (µM) for 64
MIC values (µM）for 63
b
c
MIC range
MIC50
6.8
MIC90
6.8
MIC range
MIC50
3.4
MIC90
MRSA (n=20)
6.8
6.8
1
3.4-6.8
3.4
3.4
1 (90%)
(85%)
1
E. coli (n=20)
27.3-218.6
109.3
109.3
27.3-437.2
6.8-54.3
27.1
27.1
6.8-108.5
1 (85%)
(95%)
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E. aerogenes
(n=16)
E. cloacae (n=19)
27.3-218.6
27.3-218.6
54.6-218.6
54.6-218.6
109.3
109.3
109.3
109.3
109.3
218.6
218.6
109.3
27.3-437.2
27.3-437.2
54.6-437.2
54.6-437.2
1
(88%)
1
(89%)
1
6.8-54.3
6.8-108.5
6.8-108.5
13.6-108.5
27.1
27.1
27.1
27.1
27.1
108.5
54.3
6.8-108.5
6.8-217
6.8-217
6.8-217
1 (94%)
1 (89%)
1 (80%)
1 (94%)
K. pneumoniae
(n=20)
K. oxytoca (n=16)
(95%)
1
54.3
(87%)
a MIC = drug concentration required to inhibit bacterial growth, b MIC50 = drug concentration required to inhibit 50% of the bacteria isolates tested. c MIC90 = drug
concentration required to inhibit 90% of the bacteria isolates tested, d MBC = drug concentration required to kill 99.9% bacteria.
A
B
● control; ■ MIC; ▲ 2 x MIC; ▼ 4 x MIC
Figure 4. Time−kill assay of (A) 64 and (B) 63 against methicillin-sensitive S. aureus, methicillin-resistant S. aureus USA300, E. coli ATCC 25922 and K. pneumoniae
ATCC 4352. Cultures were inoculated with 106 CFU/mL bacteria and exposed to increasing concentrations of compounds for 24 h. Samples were taken at 0.5, 1,
2, 4 and 24 h to determine viable cell numbers. After 30 min, all MIC data shows zero CFU, with all data points overlapping.
The robenidine core has proved to be amenable to multiple
structural modifications that allowed retention, and enhancement,
Conclusions
of antibiotic activity. In particular the introduction of asymmetry
and the inclusion of imine C-alkyl substituents were well tolerated.
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CAMHB. Plates were incubated for 20 - 24 hours at 37°C before
determination of the MIC. Similarly, the MIC50 and MIC90 for 63 and 64 were
determined as described above.
Of particular note was the increased potency of 17 and 20 against
VRE with 20 the most active with an MIC of 0.5 µg mL-1. In this
initial library analogues 2, 14, 17, 19 and 20 were equipotent or
more potent than the lead 1. The introduction of an indole moiety
with 30 resulted in the most MRSA robenidine based analogue to
date with an MIC of 0.5 µg mL-1. In all instances imine C=NH
isosteres were poorly tolerates; the C=O and C=S isosteres were
inactive.
Simple monomeric analogues, e.g. 33 – 35 returned low levels
of Gram-positive activity at ca. 16 – 64 µg mL-1. Surprisingly
removal of an NH moiety from the terminal hydrazide of 33 in the
synthesis of 36 gave rise to modest Gram-negative activity at 64
µg mL-1. Building on this finding, screening of a focused library
revealed the 4-t-butyl 45 with broad spectrum Gram-positive and
Gram-negative activity with MICs of 16 – 64 µg mL-1. Other
modifications to the aromatic substituent were poorly tolerated,
but modifications in conjunction with the introduction of an imine
C-alkyl moiety gave rise to more promising levels of both Gram-
positive and Gram-negative antibiotic activity. Gram-positive MIC
values ranged from 8 µg mL-1 with 64 and 68, to inactive (MIC >
128 µg mL-1) with the naphthyl 69 and 70 and the indole 73 based
analogues. Gram-negative activity was most promising with 62
and 68 at 16 µg mL-1 against E. coli. Against Ps. aeruginosa, the
highest activity observed was with MIC values of 32 µg mL-1 with
Minimum bactericidal concentration
The minimum bactericidal concentration (MBC) was determined according
to the CLSI guidelines.26 Briefly, the MIC assay was extended and after
determination of the MIC 10 µL aliquots were taken from all wells above
the MIC and spotted onto sheep blood agar. The plates were incubated for
24 and 48 hours before determination of the MBC as the lowest
concentration where 99.9% of the final inoculum is killed.
Time kill assays
Time kill assays were performed in 20mL samples at 1×, 2× and 4× the
MIC90 of each compound in glass flasks according to CLSI guidelines with
LB broth replacing the CAMHB.28 The compounds were serially diluted in
100% DMSO at 100x the final desired concentration and 200 µL of
appropriate concentrations added to each flask. Cultures were incubated
at 37 °C with samples taken at 0, 0.5, 1, 2, 4 and 24 hours.
Chemistry - General Methods
All reagents were purchased from Sigma-Aldrich, AK Scientific, Matrix
Scientific or Lancaster Synthesis and were used without purification. All
solvents were re-distilled from glass prior to use.
1H, 13C and 19F (decoupled) NMR spectra were recorded on a Bruker
Advance™ AMX 400 at 400.13, 100.62 and 376 MHz, respectively and
Advance™ AMX 600 at 600.21 and 150.92 MHz, respectively. Chemical
shifts (δ) are reported in parts per million (ppm) measured relative to the
internal standards. Coupling constants (J) are expressed in hertz (Hz).
Mass spectra were recorded on a Shimadzu LCMS 2010 EV and Agilent
6100 series single quadrupole LCMS using a mobile phase of 1 : 1
acetonitrile :H2O with 0.1% formic acid. The University of Wollongong,
62 and 64.
As a result, we believe that the (E)-2-
benzylidenehydrazine-1-carboximidamide scaffold is a promising
development candidate targeting both Gram-positive and Gram-
negative bacteria.
Acknowledgements
Australia, Mass Spectrometry User resource
& Research Facility
This research was supported by a Linkage Project grant from
the Australian Research Council in collaboration with Neoculi Pty.
Ltd (ARC LP110200770).
(MSURRF) analysed samples for High Resolution Mass Spectrometry
HRMS Analytical HPLC traces were obtained using a Shimadzu system
possessing a SIL-20A auto-sampler, dual LC-20AP pumps, CBM-20A bus
module, CTO-20A column heater, and a SPD-20A UV/vis detector. This
system was fitted with an Alltima™ C18 5 µm 150 mm × 4.6 mm column
with solvent A: 0.06% trifluoroacetic acid (TFA) in water and solvent B:
0.06% TFA in CH3CN–H2O (90 : 10). In each case HPLC traces were
acquired at a flow rate of 2.0 mL min-1, gradient 10–100 (%B), over 15.0
min, with detection at 220 nm and 254 nm. All samples returned
satisfactory analyses. Compound purity was confirmed by a combination
of LC-MS (HPLC), micro and/or high resolution mass spectrometry and
NMR analysis. All analogues are ≥ 95% purity.
Experimental
Microbiology
Bacterial isolates
A total of 20 MRSA clinical isolates, details listed in the Supplementary
Data, were kindly provided by Professor Geoffrey Coombs (Department of
Microbiology, Pathwest Laboratory Medicine WA). A total of Escherichia
coli (n=20), Enterobacter aerogenes (n=16), Enterobacter cloacae (n=19),
Klebsiella oxytoca (n=16) and Klebsiella pneumoniae (n=20) clinical
isolates were kindly provided by Mrs Jan Bell (SA Pathology, Institute of
Medical and Veterinary Science, South Australia). E. coli ATCC 25922, P.
aeruginosa ATCC 27853, MSSA strains ATCC 25922 and 25923 were
obtained from the American Type Culture Collection. Isolates used in initial
screening assay were sourced as follows: SCCmec type IV MRSA and
VRE, multidrug resistant clinical isolates for initial screen of NCL812
analogues were kindly provided by Professor Mary Barton (University of
South Australia).
Melting points were recorded on a Büchi Melting Point M-565 instrument.
IR spectra were recorded on a PerkinElmer Spectrum Two™ FTIR
Spectrometer with the UATR accessories. Thin layer chromatography
(TLC) was performed on Merck 60 F254 pre-coated aluminium plates with
a thickness of 0.2 mm. Column chromatography was performed under
‘flash’ conditions on Merck silica gel 60 (230–400 mesh).
Compounds 3, 4, 8, 9-11, 15-17, 21-23, 69, 71, S1, S3, and S5-S7, S36,
S37 were synthesized by an external contractor, EpiChem Pty Ltd
(Melbourne, Australia).
Susceptibility testing
General Method A
The MIC of all analogues was determined using a slightly modified
microdilution method described in CLSI guidelines,26 as follows: All
analogues were dissolved in 100% DMSO to a concentration of 12.8
µg.mL-1. Luria Bertani (LB) broth was used instead of CAMHB as it has
been previously shown that 1 can chelate calcium ions.27 In addition the
antimicrobial dilutions of all analogues were completed in 100% DMSO,
with 1 µL added to each well, as the compounds are hydrophobic. The
assay was performed in a total volume of 100 µL with test concentration
increasing 2-fold from 0.25 µg.mL-1 to 128 µg.mL in 96 well plates. MIC
tests involving ampicillin were performed according to CLSI guidelines in
A
suspension of aldehyde or phenone (2.2 equiv) and 1,3-
diaminoguanidine hydrochloride (1.0 equiv) in EtOH (5 mL) was subjected
to microwave irradiation (150 W) at 100 °C for 10 min. Most of the solvent
was then removed in vacuo, Et2O (5 mL) was added, and the flask was
chilled to effect crystallization. The resulting precipitate was collected and
washed with Et2O (5 mL) to afford the carbonimidic dihydrazide.
General Method B
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A
suspension of 1,3-diaminoguanidine hydrochloride (3.0 equiv) in
7
8
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ESKAPE pathogens. Expert Rev Anti Infect Ther. 2013;11:297-308.
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water/EtOH (50/50 v/v, 20 mL) was heated at 70°C. A solution of aldehyde
or phenone (1.0 equiv) in Et2O (2 mL) was added to the flask and heating
was continued for 4 hr. The reaction mixture was filtered hot and the filtrate
concentrated to near dryness. The residue was dissolved in Et2O and the
organic was washed with water and dried. The resulting solid was
recrystallized from EtOH/water (50/50 v/v) to afford the mono-adduct.
10
11
12
Arzanlou M, Chai WC, Venter H. Intrinsic, adaptive and acquired
antimicrobial resistance in Gram-negative bacteria. Essays Biochem.
2017;61:49-59.
A solution of the mono-adduct (1.0 equiv) and second aldehyde or
phenone (1.2 equiv) in anhydrous DMSO (3 mL) was stirred at ambient
temperature for 16 hr. The reaction mixture was taken up in CH2Cl2 and
the organic washed with NaHCO3(aq). The organic was concentrated and
the crude material was dissolved in Et2O (~5 mL). Hydrochloric acid (2M
in ether, 3 mL) was added and the resulting precipitate was collected,
washed with Et2O and dried to afford the unsymmetrical carbonimidic
dihydrazide.
Bush K. Investigational Agents for the Treatment of Gram-Negative
Bacterial Infections:
A Reality Check. ACS Infectious Diseases.
2015;1:509-511.
Perez F, El Chakhtoura NG, Papp-Wallace K, Wilson BM, Bonomo RA.
Treatment Options for Infections Caused by Carbapenem-resistant
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2016;DOI:
10.1517/14656566.2016.1145658.
General Method C
13
14
Poirel L, Kieffer N, Liassine N, Thanh D, Nordmann P. Plasmid-mediated
carbapenem and colistin resistance in a clinical isolate of Escherichia coli.
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pipelines. Int J Antimicrob Agents. 2015;45 (Supplement 2), S10.
Butler MS, Blaskovich MA, Cooper MA. Antibiotics in the clinical pipeline
in 2013. J Antibiot. 2013;66:571-91.
A suspension of the appropriate aldehyde or acetophenone (2.2 equiv) and
N,N-diaminoguanidine hydrochloride (1.0 equiv) in EtOH was heated at
reflux for 16 h. The mixture was concentrated and Et2O added to effect
crystallization. The resulting precipitate was collected and washed with
Et2O to afford the carbonimidic dihydrazide.
15
16
17
18
General Method D
A suspension of the appropriate aldehyde or acetophenone (1.0 equiv) and
aminoguanidine hydrochloride (1.0 equiv) in EtOH was heated at reflux for
16 h. The mixture was concentrated and Et2O added to effect
crystallization. The resulting precipitate was collected and washed with
Et2O to afford the carbonimidic hydrazide.
Eliopoulos G. New antimicrobial agents for Gram-positive pathogens in
pipelines. Int J Antimicrob Agents. 2015;45 (Supplement 2), S5.
Silver LL. New Targets for Antibacterial Compounds. Antibiotics: Current
Innovations and Future Trends. S. Sánchez and A. L. Demain. Portland,
Oregon, Caister Academic Press. 2015.
General Method E
Chlorobenzene (2.0 equiv) and aluminium chloride (1.0 equiv) were stirred
together before the slow addition of the appropriate acid chloride (1.0
equiv), the reaction was stirred at room temperature overnight. The
reaction mixture was added to ice and diluted with 10% HCl, and then
extracted with 2 x CHCl3. The combined organic phases were washed with
brine, saturated sodium bicarbonate and then dried. The solvent was then
removed to afford the desired substituted phenylketone.
19
WHO. World Health Organization Media Centre; WHO publishes list of
bacteria for which new antibiotics are urgently needed. 27 FEBRUARY
2017. 2017.
20
21
http://www.pewtrusts.org/~/media/assets/2017/05/antibiotics-currently-
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Abraham RJ, Stevens AJ, Young KA, Russell C, Qvist A, Khazandi M,
Wong HS, Abraham S, Ogunniyi AD, Page SW, O’Handley R, McCluskey
A, Trott DJ. Robenidine analogues as Gram positive antibacterial agents.
J. Med. Chem. 2016;59;2126-2138
22
Ogunniyi AD, Khazandi M, Stevens AJ, Sims SK, Page SW, Garg S,
Venter H, Powell A, White K, Petrovski KR, Laven-Law G, Tótoli EG,
Salgado HR, Pi H, Coombs GW, Shinabarger DL, Paton JC, McCluskey
A, Trott DJ. Evaluation of robenidine analogue NCL195 as a scaffold for
development of a novel class of broad-spectrum antibacterial agents.
PLos ONE, 2017, 12(9), e0183457.
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Entry for the Table of Contents
Insert graphic for Table of Contents here. ((Please ensure your graphic is in one of following formats))
G +ve
G +ve; lead
G -ve
Repurposing Robenidine leads to novel Gram-positive and Gram-negative agents with activity against MRSA, VRE, E. coli and Ps.
Aeruginosa. The most promising analogues return MIC values from 0.5-16 µg mL-1 against Gram-positive MRSA and VRE; and
against the Gram-negative E. coli and Ps. Aeruginosa MIC values of 16 and 32 µg mL-1 were noted.
11
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