Robenidine Analogues as Gram-Positive Antibacterial Agents

Article abstract of DOI:10.1021/acs.jmedchem.5b01797

Robenidine, 1 (2,2′-bis[(4-chlorophenyl)methylene]carbonimidic dihydrazide), was active against MRSA and VRE with MIC's of 8.1 and 4.7 μM, respectively. SAR revealed tolerance for 4-Cl isosteres with 4-F (8), 3-F (9), 3-CH₃ (22), and 4-C(CH₃)₃ (27) (23.7-71 μM) and with 3-Cl (3), 4-CH₃ (21), and 4-CH(CH₃)₂ (26) (8.1-13.0 μM). Imine carbon alkylation identified a methyl/ethyl binding pocket that also accommodated a CH₂OH moiety (75; 2,2′-bis[1-(4-chlorophenyl)-2-hydroxyethylidene]carbonimidic dihydrazide). Analogues 1, 27 (2,2′-bis{[4-(1,1-dimethylethyl)phenyl]methylene}carbonimidic dihydrazide), and 69 (2,2′-bis[1-(4-chlorophenyl)ethylidene]carbonimidic dihydrazide hydrochloride) were active against 24 clinical MRSA and MSSA isolates. No dose-limiting cytotoxicity at ≥2× MIC or hemolysis at ≥8× MIC was observed. Polymyxin B addition engendered Escherichia coli and Pseudomonas aeruginosa Gram-negative activity MIC's of 4.2-21.6 μM. 1 and 75 displayed excellent microsomal stability, intrinsic clearance, and hepatic extraction ratios with T_1/2 > 247 min, CL_int < 7 μL/min/mg protein, and E_H < 0.22 in both human and mouse liposomes for 1 and in human liposomes for 75.

Full text of DOI:10.1021/acs.jmedchem.5b01797

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Article
Robenidine analogues as Gram positive antibacterial agents
Rebecca Abraham, Andrew Stevens, Kelly Anne Young, Cecilia Russell, Anastasia
Qvist, Manouchehr Khazandi, Hui San Wong, Sam Abraham, Abiodun David
Ogunniyi, Stephen W Page, Ryan M O'Handley, Adam McCluskey, and Darren Trott
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01797 • Publication Date (Web): 14 Jan 2016
Downloaded from http://pubs.acs.org on January 15, 2016
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Robenidine analogues as Gramꢀpositive antibacterial
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Rebecca J Abraham†,¶, Andrew J Stevens‡,¶, Kelly A Young‡, Cecilia Russell‡, Anastasia Qvist‡,
Manouchehr Khazandi †, Hui San Wong †, Sam Abraham †,#, Abiodun D Ogunniyi †, Stephen W
Page §, Ryan O’Handley †, Adam McCluskey ,‡,* and Darren Trott†,*
† School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, Mudla
Wirra Rd, Roseworthy, SA, Australia, 5371
‡ Chemistry, Centre for Chemical Biology, School of Environmental and Life Sciences, The
University of Newcastle, University Drive, Callaghan, NSW, Australia, 2308
# School of Veterinary and Life Sciences, Murdoch University, 90 South Street, Murdoch, WA,
Australia, 6150
§ Neoculi Pty Ltd, Burwood, Vic, Australia, 3125
Keywords: Robenidine, antibacterial, Staphylococci, aminoguanidine Schiff bases
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Abstract
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Robenidine, 1 (2,2'ꢀbis[(4ꢀchlorophenyl)methylene]carbonimidic dihydrazide), was active
against MRSA and VRE with MIC’s of 8.1 and 4.7 ꢀM respectively. SAR revealed tolerance for 4ꢀ
Cl isosteres with 4ꢀF (8), 3ꢀF (9), 3ꢀCH3 (22), 4ꢀC(CH3)3 (27) (23.7ꢀ71 ꢀM) and 3ꢀCl (3), 4ꢀCH3
(21), 4ꢀCH(CH3)2 (26) (8.1ꢀ13.0 ꢀM). Imine carbon alkylation identified a methyl/ethyl binding
pocket which also accommodated a CH2OH moiety (75; 2,2'ꢀbis[1ꢀ(4ꢀchlorophenyl)ꢀ2ꢀ
hydroxyethylidene]carbonimidic dihydrazide). Analogues 1, 27 (2,2'ꢀbis{[4ꢀ(1,1ꢀ
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dimethylethyl)phenyl]methylene}carbonimidic dihydrazide) and 69 (2,2'ꢀbis[1ꢀ(4ꢀ
chlorophenyl)ethylidene]carbonimidic dihydrazide hydrochloride), were active against 24 clinical
MRSA and MSSA isolates. No dose limiting cytotoxicity at ≥ 2× MIC or haemolysis at ≥ 8× MIC
was observed. Polymyxin B addition engendered E. coli and P. aeruginosa Gramꢀnegative activity
MIC’s of 4.2ꢀ21.6 ꢀM. 1 and 75 displayed excellent microsomal stability, intrinsic clearance and
hepatic extraction ratios with T1/2 > 247 min, CLint <7 ꢀL/min/mg protein and EH <0.22 in both
human and mouse liposomes for 1, and human liposomes with 75.
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Introduction
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The management of bacterial infections has become increasingly difficult with the emergence of
antimicrobial resistance. In particular the ESKAPE pathogens (Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa, and Enterobacter species), are causing significant problems worldwide.1 Methicillinꢀ
resistant S. aureus (MRSA) is a major pathogen causing both hospital and communityꢀacquired
infections. In addition to methicillin resistance, S. aureus isolates resistant to the last line
antimicrobials vancomycin, linezolid, daptomycin, antiꢀMRSA cephalosporins (ceftobiprole and
ceftaroline) and mupirocin have been isolated from clinical cases.2ꢀ6
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Concurrent with the increasing prevalence of antimicrobial resistance, there has been a large gap
in the development of new antimicrobials to treat these pathogens and as time from initial
identification to release of a product onto the market can take ten years, alternatives for treatment
need to be rapidly identified to alleviate the problem in the short term. One attractive approach is
the repurposing of food and drug administration (FDA) approved drugs in clinical use as potential
antimicrobials. Another under evaluated area is reꢀexamination of compounds previously
developed for use to support animal health for antimicrobial activity as candidates for further
structural modification for possible use in human medicine. These approaches offer known safety
and development pathways with associated time and cost savings.7
Within the animal medicine sphere, Robenidine (1, 2,2'ꢀbis[(4ꢀchlorophenyl)methylene]ꢀ
carbonimidic dihydrazide hydrochloride; Figure 1) is a wellꢀknown antiꢀcoccidial agent which has
been used worldwide since the early 1970s in the prevention of coccidian infections in commercial
poultry and rabbit production.8,9 Robenidine 1 belongs to the aminoguanidine compound class, a
diverse group of bioactive compounds used in the treatment of a broad range of diseases from
bacterial infections through to cancer and diabetes and many are already approved for human
use.10,11 Excellent activity against Babesia microti infection has been reported in mouse models
with 100% inhibition noted at 25 and 50 mg/kg.12
Despite 1 having a long history of use in animals there have been no inꢀdepth evaluations as a
potential antibacterial agent, although early work demonstrated robenidine had activity against
unidentified Gramꢀpositive soil bacteria.13 Herein we report on the in vitro antibacterial activity of
1 and the development of preliminary structure activity relationship (SAR) data around this
scaffold. The efficacy of selected analogues against clinical isolates of MRSA is also examined.
Results and Discussion
Access to a focused library of Robenidine analogues was realized by the condensation of
selected monoꢀsubstituted benzaldehydes with 1,3ꢀdiaminoguanidine hydrochloride.14 Under
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ethanol reflux conditions low (18%) to excellent (93%) yields of the desired analogues (1 – 31)
were obtained (Scheme 1). The minimum inhibitory concentration (MIC50; the drug concentration
resulting in the growth inhibition of the upper 50 percentile of a bacteria population) of these
analogues was determined against two strains of MRSA and VRE, and one strain each of E. coli
and P. aeruginosa with the data presented in Table 1.
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Ampicillin was used as an internal control and showed both Gramꢀnegative (E. coli, 45.8 ꢀM; P.
aeruginosa, 366 ꢀM) and Gramꢀpositive (MRSA, 183 ꢀM; VRE 11.5 ꢀM) activity. However none
of the Robenidine analogues, 1 – 31, showed Gramꢀnegative activity (Table 1). Robenidine, 1
inhibited the growth of MRSA and VRE with MIC50 values of 8.1 and 4.7 ꢀM respectively.
Removal of the 4ꢀCl moiety with the synthesis of 2 resulted in a complete loss of activity. A
halogen scan revealed a preference for the 4ꢀCl moiety, with a twoꢀfold loss in VRE activity with
the 3ꢀCl (3) and no activity with 2ꢀCl (4) analogues. Other halogen substituents were poorly
tolerated with only 3ꢀBr (6), 4ꢀF (8) and 3ꢀF (9) displaying modest levels of activity (MIC50 < 75
ꢀM). The 3ꢀBr (6) displayed a near 10ꢀfold VRE vs. MRSA selectivity. Of the additional electron
withdrawing groups examined: CN, NO2 and CF3, only the 4ꢀCF3 (11) displayed moderate levels of
activity with MIC50 values of 36.5 and 11.4 ꢀM against MRSA and VRE respectively (Table 1 and
supplementary data). These data suggest that an electronꢀwithdrawing group is poorly tolerated and
that the effect of the 4ꢀCl moieties in 1 may be steric.
Hydrogen bond donors, e.g. 12ꢀ14, were poorly tolerated with the 4ꢀOH (12) analogue ~25ꢀfold
less MRSA and ~60ꢀfold less VRE active than 1. Methylation saw a modest activity improvement,
but only with the 3ꢀOCH3 (16). The more electronegative 4ꢀOCF3 (18) retained VRE activity but
was twoꢀfold less active against MRSA. The corresponding 4ꢀSCH3 (19) was more active,
particularly against VRE with a MIC50 of 12.7 ꢀM. The 4ꢀSCF3 (20) showed reduced activity,
again suggesting a poor tolerance for electronegative substituents (Table 1).
In general, alkyl substituents were well tolerated with the 4ꢀCH3 (21) displaying comparable
MRSA (MIC50 9.1 ꢀM) activity to 1. The equivalent 3ꢀ and 2ꢀCH3 (22 and 23) were an order of
magnitude less active, as were the 4ꢀpropyl and butyl analogues (24 and 25). The 4ꢀCH(CH3)2 (26)
with MIC50 values of 9.1 and 13.0 ꢀM against MRSA and VRE respectively, displayed activity
comparable with 21 and 1. However, the isoꢀpropyl moiety is known to be metabolized via highly
reactive intermediates,13 which resulted in our examination of replacement with both 4ꢀC(CH3)3
(27) and 4ꢀN(CH3)2 (28), but both were less active than 26.15 A 2ꢀ or 4ꢀ Ph moiety (29 and 30) was
poorly tolerated, but the 4ꢀethynyl (31) showed moderate (MIC50 17.2 ꢀM) and selective activity
against VRE. Polar substituents such as 4ꢀCH=CHCOOH, 4ꢀNHCOCH3 and 3ꢀCOOH were
inactive (MIC50 > 400 ꢀM; supplementary data).
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With an understanding of the SAR of the simple phenyl rings, the effects of multiple moieties
were next explored. These analogues were synthesized as described in Scheme 1 from the
corresponding aldehydes and the outcome of screening is presented in Table 2.
The inclusion of two halogens was initially investigated. Interestingly, despite the parent 2ꢀF
(10) and the 2,5ꢀdiꢀF (32) analogues being inactive, combination of the 2ꢀF with a 4ꢀCl moiety (33)
gave MRSA and VRE activity comparable to 1. The 3,4ꢀdiꢀF (34) saw a reduction in MRSA
activity but retention of VRE activity. A 4ꢀCl moiety in combination with a 2ꢀNH2 (38), 2ꢀ
NHCH(OH)CH3 (39), and 2ꢀOH (42) afforded good to modest MRSA and VRE activity. These
data suggest that the 2ꢀF of 33 may be acting as a hydrogen bond isostere. In an attempt to improve
aqueous solubility, the 4ꢀCl was replaced with 4ꢀN(CH3) 2 with 2ꢀOH retention with the synthesis of
43, but this resulted in a loss of activity. Combining the 2ꢀOH with 3ꢀCH3 (41), both individually
active substituents (Table 1, 14 and 22), resulted in no activity, possibly due to the proximity of the
substituents interfering with their respective interactions. Of the catechol bearing analogues (44 –
46), only modest activity was noted with 44. The introduction of a third OH moiety (50 – 52) saw
both the reintroduction of low levels of activity against MRSA and VRE, and with 52 also a low
level of activity against P. aeruginosa (MIC50 322 ꢀM). These catechol analogues were not
investigated further, nor were the variations that saw methylation or OH to Br exchange or
introduction of pentafluoro moiety examined as these analogues, 53 – 57, were inactive across all
bacterial strains examined.
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The introduction of phenyl ring isosteres, fused aromatics, heterocyclic and methylene spaced
analogues (58ꢀ67) was accomplished as per Scheme 1.14,16 These analogues were screened for
MRSA and VRE activity (Table 3). In virtually all instances the phenyl ring isosteric modification
resulted in a significant loss of both MRSA and VRE activity relative to 1. In multiple instances
selectivity towards MRSA (63 and 64) and VRE (67) was observed. With the pyridyl analogues
only the 4ꢀCl (60) was active, suggesting again a key role for a 4ꢀCl substituent in this class of
compounds. Other fused aromatics, 2ꢀhydroxynaphthyl, 3ꢀquinolinyl, 2ꢀquinoxalinyl and chromene
analogues were inactive (data not shown).
Installation of a range of isocyanates and chloroformates to afford the respective carbamate and
urea analogues of 1, only resulted in a complete loss of activity (Scheme 2; Supplementary Data).
Potential imine carbon based modifications were explored through coupling of phenones with
1,3ꢀdiaminoguanidine hydrochloride to afford alkyl substituted analogues (68ꢀ80) (Scheme 3; Table
4). While the compounds were evaluated against the Gramꢀnegative pathogens, no activity was
observed (data not shown).
Introduction of a methyl substituent at the imine carbon imparted activity to compounds whose
methine counterpart bore no activity, such as 68 and 70 whereas their desmethylated analogues 2
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and 5, respectively were MRSA and VRE inactive (Table 1). In a similar manner ‘Cꢀmethylation’
of 1 to afford 69 provided a marginal improvement in activity (Table 4). However, this ‘Cꢀ
alkylation’ strategy was not universally applicable, as it appears to be incompatible with the CF3
substitution of 71, abolishing MRSA activity with retention of VRE inhibition.
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Sequential probing of this space with straight chain aliphatics (72 ꢀ 74) suggested the presence of
a Cꢀmethyl/ethyl binding pocket with the Cꢀethyl (72); the largest Cꢀalkyl substituent to retain
antibacterial activity. Further probing of this region with hydrogen bond donor/acceptor atoms,
bearing either neutral or ionic qualities, showed that the pocket would tolerate a neutral hydrogen
bond donor/acceptor (CH2OH; 75), however introduction of ionic species in 76 (CH2NH2) and 77
(COOH) resulted in abolition of potency (Table 4).
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Coupling of the favorable aromatic substituents, the 4ꢀCH3 (21) and 4ꢀC(CH3)3 (27), moieties
from the parent phenyl analogues with the C-methyl substituents from this series of analogues gave
rise to 78 and 79, which were amongst the most potent analogues developed herein with MIC
values in 4.5 – 36.2 ꢀM. Attempts to enhance compound solubility through the introduction of a 4ꢀ
piperazinꢀ1ꢀyl moiety (80) were unsuccessful with a significant loss in potency noted (Table 4).
Having established the antibacterial activity of this class of compounds, we examined the
activity of 1, 27 and 69 against twenty clinically obtained isolates of MRSA and four methicillinꢀ
sensitive S. aureus (MSSA) isolates obtained from the ATCC. All three analogues (1, 27 and 69)
were bactericidal against all the S. aureus strains tested with MBC:MIC90 ratios of ≤2 (Table 5).
The lead, 1, was uniformly active across all clinical isolates with a 5.4 ꢀM MIC90 value, 27
displayed a broader range of activity from 4.8 – 38.6 ꢀM and 69 from 1.3 – 5.0 ꢀM. A profound
and negative impact on the MIC values with a 4ꢀfold decrease with 1 and 27 at 2% and no activity
at 50% serum was observed. The effect with 69 was less stark with the MIC90 values dropping to
10 ꢀM at 2% serum. This suggests a high level of protein binding of these analogues, but does not
necessarily render these analogues ineffective in vivo.17
As anticipated analogues 1, 27 and 69 failed to display any Gramꢀnegative activity against E.
coli or P. aeruginosa (Table 6). However in the presence of subꢀinhibitory concentrations of
polymyxin B nonapeptide (PMBN; 4.1 ꢀM), a polymyxin derivative with almost absent
antimicrobial activity but known to disrupt the outer membrane of Gram negative bacteria, 1 and 69
showed at least a 10ꢀfold MIC90.19 Administration of the same subꢀinhibitory concentration (4.1
ꢀM) of PMBM alone had no antibiotic activity (data not shown). These data are consistent with the
target of these analogues being present in both Gramꢀpositive and Gramꢀnegative bacteria and that
improvement in activity noted on coꢀadministration of PMBM suggests that the lack of Gramꢀ
negative antibacterial activity for this compound was a consequence of lack of compound
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penetration of the bacterial outer membrane.20 With 1 and 69, MIC90 values were comparable with
those observed with Clofazimine (Table 6).18
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Time kill curve evaluation of 1, 27 and 69 at 1, 2, 4 and 8 times their MIC90 against one
susceptible S. aureus isolate (ATCC 29213) and one clinical MRSA isolate (USA300) confirmed
their bactericidal activity resulting in a ≥ 4ꢀlog reduction in bacterial cell numbers over an 8 hour
period across all the analogue doses evaluated (Figure 2). After 24 hours some bacteria regrowth
was observed with all compounds at all concentrations tested (data not shown).
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Of the analogues evaluated, only 1 showed statistically significant activity against S. aureus
biofilms (based on a oneꢀway ANOVA analysis). Significant biofilm retardation was only noted at
concentrations > 43.2 ꢀM, and resulted in a ≤3ꢀlog reduction in cells irrespective of analogue
concentration.
Hemolysis was evident with 1 at ≥ 173 ꢀM while no hemolysis was observed with 27 or 69 at
concentrations as high as 600 ꢀM. Low to moderate levels of cytotoxicity against a normal human
lung fibroblast cell line (HEL 299) with 1, 27 and 69 were observed with growth inhibition (GI50)
values of 17.7, 28.8 and 6.7 ꢀM respectively; and against a human hepatocellular carcinoma cell
line (Hep G2) returned GI50 values of 25.4, 26.3 and 13.8 ꢀM respectively. These values are at
least twice the observed MIC50 values for these three analogues. The observed cytotoxicity of these
compounds is considered acceptable at this stage of compound development especially as extensive
in vivo studies have been performed previously with 1 which determined the oral LD50 to be ~3000
mg/kg in rats and 1500 mg/kg and 2900 mg/kg per day in male and female rabbits respectively with
no toxicity concerns reported.21 Robenidine, 1 has been used safely since the early 1970’s as an
oral antiꢀcoccidial agent in poultry and rabbits.8 Furthermore no adverse effects were reported after
dosing mice with 50 mg/kg p.o. of 69.22
Analogues 1, 27 and 69 were considered for preliminary in vivo stability and degradation studies,
however we were concerned that the Cꢀmethyl imine moiety might be highly susceptible to
oxidation, and as such examined the stability and degradation of the related CH2OH substituted 75,
which also afforded the possibility of enhanced solubility as a consequence of the additional polar
moiety (cf. 69). We examined the physicochemical and metabolism parameters of 1, 27 and 75 in
human and mouse liposomes and this data is presented in Table 7. As anticipated these analogues
conformed with Lipinski’s rules, and presented moderately favorable LogD values at both pH 3.0
and 7.4, although the solubility was modest, but highest with 1 and 75.23 Even 75 which was
selected to best mimic a first pass metabolism of 69 was within the Lipinski criteria for an orally
active drug (Table 7).23 Of the three analogues evaluated, 27 displayed the lowest T1/2 and
correspondingly higher intrinsic elimination and hepatic extraction ratios, CLint and EH respectively
(Table 7). Both CLint and EH with 1 and 75 were considerably more favorable with T1/2 > 247 min,
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CLint <7 ꢀL/min/mg protein and EH <0.22 in both human and mouse liposomes for 1, and human
liposomes with 75. The metabolic parameters were slightly poorer with 75 in mouse liposomes, but
still acceptable at 131 min, 13 ꢀL/min/mg protein and 0.22 for T1/2, CLint and EH respectively.24,25
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Our screening confirmed Robenidine, 1; a widely used poultry industry antiꢀcoccidial agent was
active against both MRSA and VRE. Removal of the 4ꢀCl moiety resulted in loss of activity,
however a wide array of isosteric and other monoꢀsubstituents were moderately (4ꢀF (8), 3ꢀF (9), 3ꢀ
CH3 (22), 4ꢀC(CH3)3 (27)) to well tolerated (3ꢀCl (3), 4ꢀCH3 (21), 4ꢀCH(CH3)2 (26). Bulky
substituents were poorly tolerated. The introduction of diꢀsubstituted phenyl moieties was well
tolerated with 2ꢀF,4ꢀCl (33) and 2ꢀNH2,4ꢀCl (38). No noteworthy potency was observed with only
the indole (66) and benzothiophene (67) showing modest levels of activity. Similarly no activity
was noted with the development of carbamate and urea analogues. Modifications at the imine
carbon revealed the presence of a methyl/ethyl binding pocket with good activity with 69ꢀ70 and
the CH2OH analogue 75.
Analogues 1, 27 and 69, had rapid and potent bactericidal activity against MRSA in vitro.
Robenidine 1 displayed promising activity against S. aureus biofilms in vitro. These promising
results were further validated with excellent in vitro antibacterial profiles against clinical S. aureus
(MRSA and MSSA) isolates. Of particular note was the limited effect of 2% serum on the MIC
values of 69, where both 1 and 27 were rendered essentially inactive. Moreover none of these
analogues displayed dose limiting cytotoxicity against the two cell lines examined, returning GI50
values at least twoꢀfold higher than the compound MIC50, no significant hemolysis was observed
until compound concentrations reached at least 8ꢀtimes the MIC value. Efficacy against Gramꢀ
negative E. coli and P. aeruginosa was only observed in the presence of polymyxin B nonapeptide,
with activities comparable with Clofazimine.18
Preliminary evaluation of physicochemical and metabolic parameters in human and mouse
liposomes further supports the development of these analogues as potential clinical antibacterial
leads with both 1 and 75 displaying excellent stability, intrinsic clearance rates (CLint) and hepatic
extraction (EH) ratios. Both CLint and EH with 1 and 75 were highly promising for future
development with T1/2 > 247 min, CLint <7 ꢀL/min/mg protein and EH <0.22 in both human and
mouse liposomes for 1, and human liposomes with 75.24,25
Experimental
Chemistry - General Methods
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All reactions were performed using standard laboratory equipment and glassware. Solvents and
reagents were purchased from Sigma Aldrich, Lancaster International or TCI and used as received.
Organic solvents were bulk quality, and were distilled from glass prior to use. Organic solvent
extracts were dried with magnesium sulfate (MgSO4), and dried under reduced pressure with either
Büchi or Heidolph rotary evaporators. Melting points were recorded in open capillaries on a Stuart
SMP11 Melting Point Apparatus. Temperatures are expressed in degrees Celsius (°C). Where
available, literature values are provided and appropriately referenced. Electrospray mass spectra
were recorded using 10% DMSO/H2O or HPLCꢀgrade methanol or acetonitrile as carrier solvents
on a Shimadzu LCꢀMS spectrometer. All compounds were ≥ 95% purity as determined by HPLC and
LCMS analysis.
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Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker Avance 300 MHz
spectrometer, where proton NMR (1HꢀNMR) spectra and carbon NMR (13CꢀNMR) spectra were
acquired at 300 and 75 MHz respectively, or a Bruker Avance III 400 MHz spectrometer, where
1HꢀNMR and 13CꢀNMR were acquired at 400 and 100 MHz respectively. All spectra were recorded
in deuterated dimethyl sulfoxide (DMSOꢀd6), obtained from Sigma Aldrich or Cambridge Isotope
Laboratories Inc., unless otherwise stated, with the residual solvent peaks used as the internal
reference (δ 2.49 (quintet) and δ 39.7 (septet) for 1HꢀNMR and 13CꢀNMR respectively). Chemical
shifts (δ) were measured in parts per million (ppm) and referenced against the internal reference
peaks. Coupling constants (J) were measured in Hertz (Hz).
NMR assignments were determined through the interpretation of oneꢀ and twoꢀdimensional
spectra, specifically gradient heteronuclear single quantum correlation (gHSQC), gradient
heteronuclear multiple bond correlation (gHMBC) and distortionless enhancement by polarization
transfer quaternary (DEPTQ) spectroscopy. Multiplicities are denoted as singlet (s), broad singlet
(br s), doublet (d), doublet of doublets (dd), doublet of doublet of doublets (ddd), triplet (t), triplet
of doublets (td), doublet of triplets (dt), quartet (q), quintet (quin) and multiplet (m). Peaks are
listed in increasing chemical shift in the following format: chemical shift (integration (1H),
multiplicity (1H), coupling constant (1H), ascribed assignment).
Microbiology
Antimicrobial agents
Robenidine (1, NCL812) was provided by Neoculi Pty. Ltd. Ampicillin and Clofazimine used in
this study were sourced from Sigma Aldrich.
Bacterial isolates
A total of 20 MRSA clinical isolates, details listed in Table S1 (supplementary material), were
kindly provided by Associate Professor Geoffrey Coombs (Department of Microbiology, Pathwest
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Laboratory Medicine WA). MSSA strains ATCC 25923, 6538, 49775 and 29213 were obtained
from the American Type Culture Collection together with E. coli ATCC 25922 and P. aeruginosa
ATCC 27853. Isolates used in initial screening assay were sourced as follows: SCCmec type IV
MRSA, VRE, multidrugꢀresistant E. coli and P. aeruginosa clinical isolates were kindly provided
by Prof Mary Barton, University of South Australia.
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Susceptibility testing
The MIC of all analogues was determined according to CLSI guidelines in cationꢀadjusted
MuellerꢀHinton broth (CAMHB) at a total volume of 100 ꢀL with test concentration increasing 2ꢀ
fold from 0.25 ꢀg.mLꢀ1 to 128 ꢀg.mLꢀ1.26 All analogues were dissolved in 100% DMSO to a
concentration of 12.8 mg.mLꢀ1 prior to dilution in broth.
The MIC50 and MIC90 for 1, 27 and 69 was determined using a modified MIC assay based on
CLSI guidelines.26 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 1, 27 and 69 were
completed in 100% DMSO, with 2 ꢀL added to each well, as the compounds are hydrophobic. The
assay was performed in a total volume of 200 ꢀL in 96 well plates. Antimicrobials were tested in
final concentrations as follows; 1: 0.7 – 345.3 ꢀM, 27: 0.6 – 309.2 ꢀM and 69: 0.6 – 321 ꢀM. MIC
tests involving ampicillin were performed according to CLSI guidelines in CAMHB. Plates were
incubated for 20 ꢀ 24 hours at 37°C before determination of the MIC. For Gramꢀnegative organisms
the MIC was determined as above with and without supplementation of media with polymyxin B
nonapeptide (PMBN) (4.1 ꢀM). Clofazimine was used as a control in PMBN studies and prepared
as the test compounds.18
The effect of serum on the MIC90 of the compounds was also determined. MIC90 assays were
performed as described above, however the media was supplemented with 2, 10 or 50% heat
inactivated adult bovine serum.
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 ꢀL
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 20 ꢀL samples at 1×, 2×, 4× and 8× the MIC90 of each
compound in glass flasks according to CLSI guidelines with LB broth replacing the CAMHB.28
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The compounds were serially diluted in 100% DMSO at 100x the final desired concentration and
200 ꢁL of appropriate concentrations added to each flask. Cultures were incubated at 37 °C with
samples taken at 0, 4, 8 and 24 hours.
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Biofilm Assays
Biofilm activity was determined using the MBEC assay system as previously outlined.29 Briefly,
biofilms were allowed to grow for 24 hours on MBEC pegs before exposure to the compounds for a
further 24 hours. After exposure the biofilms were dispersed by sonication and the CFU.mLꢀ1
determined via spot plating. Again all compounds were serially diluted in 100% DMSO before
addition to treatment wells (as outlined in MIC assays).
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Toxicity assays
Haemolysis assay
Sheep erythrocytes were exposed to various concentrations of 1, 27 and 69 as described
previously.30 Briefly, erythrocytes were washed three times in saline, reꢀsuspended to a final
concentration of ~1010 cells.mLꢀ1 and 5 ꢀL of compound dissolved in DMSO was added.
Erythrocytes were incubated at 37 °C for 30 minutes with gentle shaking before being put on ice for
5 minutes and centrifuged to pellet cells. The positive control was erythrocytes exposed to water
resulting in 100% lysis. After centrifugation the absorbance of the supernatant was recorded at 550
nm.
Tissue culture cytotoxicity
HEL 299 (ATCC CCLꢀ137) and Hep G2 (ATCC HBꢀ8065) human cell lines were maintained in
Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco Cat No: 12430) supplemented with 10%
(vol/vol) foetal bovine serum and 1% PenStrep (100 U/mL Penicillin and 100 ꢀg/mL Streptomycin)
and passaged ~ every 3 days. Assays were performed in duplicate in 96 well plates seeded with ~50
000 cells per well. Twenty four hours after seeding the plates, media was removed, washed once
with medium without antibiotics and fresh media was added. Two hours later, 1, 27 and 69 were
diluted in DMSO and added to each well at a concentration of 1% in doubling dilutions starting at
the same concentrations used for MIC testing. After 24 hours exposure WSTꢀ1 reagent was added
to each well at a final concentration of 10%. Absorbance at 450 nm was recorded after 1 hour of
incubation with WSTꢀ1 reagent. The GI50 values were determined via nonꢀlinear regression using
GraphPad Prism v6 software.
Synthetic Chemistry14
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Compounds 3, 4, 8, 9ꢀ11, 15ꢀ17, 21ꢀ23, 69, 71, S1, S3, and S5ꢀS7, S36, S37 were synthesized by
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an external contractor, EpiChem Pty Ltd (Melbourne, Australia).
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General Method A
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A suspension of the appropriate aldehyde (2.2 eq.) and N,Nꢀdiaminoguanidine hydrochloride
(1.0 eq.) in EtOH was heated at reflux for 16 h. The mixture was cooled and diluted with Et2O to
effect crystallization. The resulting precipitate was collected and washed with Et2O to afford the
carbonimidic dihydrazide.
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General Method B
A suspension of aldehyde (2.2 eq.) and N,Nꢀdiaminoguanidine hydrochloride (1.0 eq.) in EtOH
(5 mL) was subjected to microwave irradiation (150 W) at 100 °C for 10 minutes. 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.
2,2'-Bis[(4-chlorophenyl)methylene]carbonimidic dihydrazide hydrochloride (1) NCL812
Synthesized by according to General Method A from 4ꢀchlorobenzaldehyde to afford the title
compound as a white solid. Supplied by Neoculi Pty Ltd.
1H NMR (DMSOꢀd6) δ 12.04 (br. s, 2H), 8.48 (br. s, 1H), 8.37 (br. s, 2H), 7.97 (d, J = 8.6 Hz,
4H), 7.57 (d, J = 8.6 Hz, 4H).
2,2'-Bis(phenylmethylene)carbonimidic dihydrazide hydrochloride (2)
Synthesized according to General Method B from benzaldehyde to afford the title compound as a
white solid (88%).
M.P. 234–237°C. 1H NMR (DMSOꢀd6) δ 12.39 (br. s, 2H), 8.55 (s, 2H), 8.46 (s, 2H), 8.01 –
7.88 (m, 4H), 7.55 – 7.41 (m, 6H). 13C NMR (DMSOꢀd6) δ 152.9, 148.9, 133.3, 130.8, 128.8,
127.9. HRMS: m/z (calcd for C15H15N5, 265.1327; found 265.1246).
2,2'-Bis[(3-chlorophenyl)methylene]carbonimidic dihydrazide hydrochloride (3)
Synthesized according to General Method A from 3ꢀchlorobenzaldehyde to afford the title
compound as a white solid.
1H NMR (DMSOꢀd6) δ 12.17 (br. s, 2H), 8.61 (br. s, 2H), 8.38 (br. s, 2H), 8.16 (s, 2H), 7.83 (d, J
= 7.0 Hz, 2H), 7.56 – 7.50 (m, 4H).
2,2'-Bis[(2-chlorophenyl)methylene]carbonimidic dihydrazide hydrochloride (4)
Synthesized according to General Method A from 2ꢀchlorobenzaldehyde to afford the title
compound as a white solid.
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1H NMR (DMSOꢀd6) δ 12.37 (br. s, 2H), 8.83 (br. s, 2H), 8.63 (br. s, 2H), 8.44 – 8.39 (m, 2H),
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7.60 – 7.55 (m, 2H), 7.55 – 7.44 (m, 4H).
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2,2'-Bis[(4-bromophenyl)methylene]carbonimidic dihydrazide hydrochloride (5)
Synthesized according to General Method B from 4ꢀbromobenzaldehyde to afford the title
compound as a white solid (89%).
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M.P. 224–226°C. 1H NMR (DMSOꢀd6) δ 12.42 (br. s, 2H), 8.60 (s, 2H), 8.42 (s, 2H), 7.91 (d, J
= 8.5 Hz, 4H), 7.69 (d, J = 8.5 Hz, 4H). 13C NMR (DMSOꢀd6) δ 152.9, 147.7, 132.6, 131.7, 129.7,
124.2. HRMS: m/z (calcd for C15H13Br2N5, 422.9517; found 422.9320).
2,2'-Bis[(3-bromophenyl)methylene]carbonimidic dihydrazide hydrochloride (6)
Synthesized according to General Method B from 3ꢀbromobenzaldehyde to afford the title
compound as a white solid (44%).
M.P. 248–251°C. 1H NMR (DMSOꢀd6) δ 12.45 (br. s, 2H), 8.68 (s, 2H), 8.41 (s, 2H), 8.29 (s,
2H), 7.87 (d, J = 7.8 Hz, 2H), 7.70 – 7.63 (m, 2H), 7.44 (t, J = 7.9 Hz, 2H). 13C NMR (DMSOꢀd6) δ
153.0, 147.4, 135.7, 133.3, 130.9, 129.5, 127.6, 122.3. HRMS: m/z (calcd for C15H13Br2N5,
422.9517; found 422.9413).
2,2'-Bis[(2-bromophenyl)methylene]carbonimidic dihydrazide hydrochloride (7)
Synthesized according to General Method B from 2ꢀbromobenzaldehyde to afford the title
compound as a white solid (72%).
M.P. 267–270°C. 1H NMR (DMSOꢀd6) δ 8.84 (s, 2H), 8.69 (s, 2H), 8.41 (dd, J = 7.8, 1.7 Hz,
2H), 7.70 (dd, J = 8.0, 1.0 Hz, 2H), 7.48 (t, J = 7.3 Hz, 2H), 7.44 – 7.37 (m, 2H). 13C NMR
(DMSOꢀd6) δ 152.8, 147.3, 133.1, 132.4, 132.1, 128.5, 127.9, 123.9. HRMS: m/z (calcd for
C15H13Br2N5, 422.9517; found 422.9355).
2,2'-Bis[(4-fluorophenyl)methylene]carbonimidic dihydrazide hydrochloride (8)
Synthesized by according to General Method A from 4ꢀfluorobenzaldehyde to afford the title
compound as a white solid.
1H NMR (DMSOꢀd6) δ 12.11 (br. s, 1H), 8.52 (br. s, 2H), 8.40 (br. s, 2H), 8.05 – 7.96 (m, 4H),
7.39 – 7.30 (m, 4H).
2,2'-Bis[(3-fluorophenyl)methylene]carbonimidic dihydrazide hydrochloride (9)
Synthesized according to General Method A from 3ꢀfluorobenzaldehyde to afford the title
compound as a white solid.
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1H NMR (DMSOꢀd6) δ 12.08 (br. s, 2H), 8.38 (br. s, 2H), 7.97 – 7.94 (m, 2H), 7.69 – 7.67 (m,
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2H), 7.56 – 7.53 (m, 2H), 7.35 – 7.31 (m, 2H).
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2,2'-Bis[(2-fluorophenyl)methylene]carbonimidic dihydrazide hydrochloride (10)
Synthesized according to General Method A from 2ꢀfluorobenzaldehyde to afford the title
compound as a white solid.
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1H NMR (DMSOꢀd6) δ 12.18 (br. s, 2H), 8.65 (br. s, 2H), 8.58 (br. s, 2H), 8.34 (t, J = 7.6 Hz,
2H), 7.58 – 7.53 (m, 2H), 7.34 (t, J = 8.2 Hz 4H).
2,2'-Bis{[4-(trifluoromethyl)phenyl]methylene}carbonimidic dihydrazide hydrochloride (11)
Synthesized according to General Method A from 4ꢀtrifluoromethylbenzaldehyde to afford the
title compound as a white solid.
1H NMR (DMSOꢀd6) δ 12.32 (br. s, 2H), 8.69 (br. s, 2H), 8.49 (br. s, 2H), 8.18 (d, J = 7.8 Hz,
4H), 7.86 (d, J = 8.2 Hz, 4H).
2,2'-Bis[(4-hydroxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (12)
Synthesized according to General Method B from 4ꢀhydroxybenzaldehyde to afford the title
compound as a yellow solid (18%).
M.P. 246–248°C. 1H NMR (DMSOꢀd6) δ 11.92 (br. s, 2H), 10.13 (br. s, 2H), 8.28 (s, 4H), 7.75
(d, J = 8.5 Hz, 4H), 6.86 (d, J = 8.5 Hz, 4H). 13C NMR (DMSOꢀd6) δ 160.1, 152.4, 148.8, 129.6,
124.3, 115.6. LRMS: m/z (calcd for C15H15N5O2, 297.12; found 297.90).
2,2'-Bis[(3-hydroxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (13)
Synthesized according to General Method B from 3ꢀhydroxybenzaldehyde to afford the title
compound as a pale yellow solid (61%).
M.P. 121–123°C. 1H NMR (DMSOꢀd6) δ 8.00 (s, 2H), 7.26 – 7.08 (m, 6H), 6.98 – 6.43 (m, 4H).
13C NMR (DMSOꢀd6, DEPT) δ 157.6, 143.8, 136.8, 129.5, 118.0, 116.1, 113.0. HRMS: m/z (calcd
for C15H15N5O2, 297.1226; found 297.1114).
2,2'-Bis[(2-hydroxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (14)
Synthesized according to General Method B from 2ꢀhydroxybenzaldehyde as a white solid
(95%).
M.P. 263–266°C. 1H NMR (DMSOꢀd6) δ 12.19 (s, 2H), 10.25 (s, 2H), 8.70 (s, 2H), 8.34 (s, 2H),
8.06 (d, J = 7.8 Hz, 2H), 7.35 – 7.23 (m, 2H), 7.00 (d, J = 8.2 Hz, 2H), 6.87 (t, J = 7.5 Hz, 2H). 13C
NMR (DMSOꢀd6) δ 156.9, 152.5, 145.4, 132.0, 126.9, 119.5, 119.1, 116.3. HRMS: m/z (calcd for
C15H15N5O2, 297.1226; found 297.1125).
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2,2'-Bis[(4-methoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (15)
Synthesized according to General Method B from 4ꢀmethoxybenzaldehyde to afford the title
compound.
1H NMR (DMSOꢀd6) δ 11.78 (br. s, 2H), 8.31 (br. s, 3H), 7.87 (d, J = 8.6 Hz, 4H), 7.04 (d, J =
8.6 Hz, 4H), 3.83 (s, 6H).
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2,2'-Bis[(3-methoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (16)
Synthesized according to General Method A from 3ꢀmethoxybenzaldehyde to afford the title
compound as a white solid.
1H NMR (DMSOꢀd6) δ 11.99 (br. s, 2H), 8.48 (br. s, 2H), 8.36 (br. s, 2H), 7.56 (s, 2H), 7.46 –
7.38 (m, 4H), 7.08 – 7.05 (m, 2H), 3.84 (s, 6H).
2,2'-Bis[(2-methoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (17)
Synthesized according to General Method A from 2ꢀmethoxybenzaldehyde to afford the title
compound as a white solid.
1H NMR (400 MHz, DMSOꢀd6) δ 12.00 (br. s, 2H), 8.74 (br. s, 2H), 8.38 (br. s, 2H), 8.22 (d, J =
6.7 Hz, 2H), 7.50 – 7.45 (m, 2H), 7.14 (d, J = 8.2 Hz, 2H), 7.05 (t, J = 7.6 Hz, 2H), 3.88 (s, 6H).
2,2'-Bis{[4-(trifluoromethoxy)phenyl]methylene}carbonimidic dihydrazide hydrochloride (18)
Synthesized according to General Method B from 4ꢀtrifluoromethoxybenzaldehyde to afford the
title compound as a white solid (69%).
M.P. 258–261°C. 1H NMR (DMSOꢀd6) δ 12.49 (br. s, 2H), 8.65 (s, 2H), 8.49 (s, 2H), 8.10 (d, J
= 8.6 Hz, 4H), 7.47 (d, J = 8.3 Hz, 4H). 13C NMR (DMSOꢀd6) δ 153.0, 149.8 (d, J = 1.6 Hz), 147.4,
132.6, 129.9, 121.2, 120.0 (q, J = 256.9 Hz). LRMS: m/z (calcd for C17H13F6N5O2, 433.10; found
433.80).
2,2'-Bis{[4-(methylsulfanyl)phenyl]methylene}carbonimidic dihydrazide hydrochloride (19)
Synthesized according to General Method B from 4ꢀthiomethylbenzaldehyde to afford the title
compound as an offꢀwhite solid (73%).
M.P. 242–245°C. 1H NMR (DMSOꢀd6) δ 12.17 (br. s, 2H), 8.46 (s, 2H), 8.37 (s, 2H), 7.86 (d, J
= 8.2 Hz, 4H), 7.34 (d, J = 8.2 Hz, 4H), 2.53 (s, 6H). 13C NMR (DMSOꢀd6, DEPT) δ 152.7, 148.4,
142.0, 129.7, 128.2, 125.4, 14.2. LRMS: m/z (calcd for C17H19N5S2, 357.11; found 357.95).
2,2'-Bis{[4-(trifluoromethylsulfanyl)phenyl]methylene}carbonimidic dihydrazide hydrochloride
(20)
Synthesized according to General Method B from 4ꢀthiotrifluoromethylbenzaldehyde to afford
the title compound as a white solid (48%).
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M.P. 195°C (Decomp.). 1H NMR (DMSOꢀd6) δ 12.68 (s, 1H), 8.73 (s, J = 48.7 Hz, 1H), 8.53 (s,
1H), 8.11 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.1 Hz, 2H). 13C NMR (DMSOꢀd6) δ 153.06, 147.43,
136.20, 136.16, 129.52 (q, J = 308.0 Hz), 129.05, 125.20 – 125.01 (Unresolved quartet). LRMS:
m/z (calcd for C17H13F6N5S2, 465.05; found 465.60).
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2,2'-Bis[(4-methylphenyl)methylene]carbonimidic dihydrazide hydrochloride (21)
Synthesized according to General Method A from 4ꢀmethylbenzaldehyde to afford the title
compound as a white solid.
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1H NMR (DMSOꢀd6) δ 11.92 (br. s, 2H), 8.41 (br. s, 2H), 8.36 (br. s, 2H), 7.82 (d, J = 8.2 Hz,
4H), 7.30 (d, J = 7.8 Hz, 4H), 2.37 (s, 6H).
2,2'-Bis[(3-methylphenyl)methylene]carbonimidic dihydrazide hydrochloride (22)
Synthesized according to General Method A from 3ꢀmethylbenzaldehyde to afford the title
compound as a white solid.
1H NMR (DMSOꢀd6) δ 11.96 (br. s, 2H), 8.44 (br. s, 2H), 8.37 (br. s, 2H), 7.76 (s, 2H), 7.71 (d,
J = 7.8 Hz, 2H), 7.38 (t, J = 7.8 Hz, 2H), 7.31 (d, J = 7.8 Hz, 2H), 2.38 (s, 6H).
2,2'-bis[(2-methylphenyl)methylene]carbonimidic dihydrazide hydrochloride (23)
Synthesized according to General Method B from 2ꢀmethylbenzaldehyde to afford the title
compound as a white solid.
1H NMR (DMSOꢀd6) δ 11.99 (br. s, 2H), 8.73 (br. s, 2H), 8.41 (br. s, 2H), 8.19 (d, J = 7.8 Hz,
2H), 7.39 (t, J = 8.0 Hz, 2H), 7.30 (t, J = 7.8 Hz, 4H), 2.46 (s, 6H).
2,2'-Bis[(4-propylphenyl)methylene]carbonimidic dihydrazide hydrochloride (24)
Synthesized according to General Method B from 4ꢀpropylbenzaldehyde to afford the title
compound as a white solid (77%).
M.P. 218–222°C. 1H NMR (DMSOꢀd6) δ 8.60 – 8.30 (m, 4H), 7.84 (d, J = 8.1 Hz, 4H), 7.28 (d,
J = 8.1 Hz, 4H), 2.59 (t, J = 7.5 Hz, 4H), 1.64 – 1.54 (m, 4H), 0.88 (t, J = 7.3 Hz, 6H). 13C NMR
(DMSOꢀd6) δ 152.8, 148.8, 145.3, 130.9, 128.7, 127.9, 37.2, 23.9, 13.6. HRMS: m/z (calcd for
C21H27N5, 349.2266; found 349.2112).
2,2'-Bis[(4-butylphenyl)methylene]carbonimidic dihydrazide hydrochloride (25)
Synthesized according to General Method B from 4ꢀbutylbenzaldehyde to afford the title
compound as a pale yellow solid (80%).
M.P. 216–220°C. 1H NMR (DMSOꢀd6) δ 12.21 (br. s, 2H), 8.44 (s, 2H), 8.39 (s, 2H), 7.83 (d, J
= 8.2 Hz, 4H), 7.30 (d, J = 8.2 Hz, 4H), 2.63 (t, J = 7.7 Hz, 4H), 1.61 – 1.52 (m, 4H), 1.36 – 1.26
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(m, 4H), 0.90 (t, J = 7.3 Hz, 6H). 13C NMR (DMSOꢀd6) δ 152.7, 148.8, 145.5, 130.9, 128.7, 127.9,
34.8, 32.9, 21.7, 13.8. HRMS: m/z (calcd for C23H31N5, 377.2579; found 377.2336).
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2,2'-Bis{[4-(1-methylethyl)phenyl]methylene}carbonimidic dihydrazide hydrochloride (26)
Synthesized according to General Method B from 4ꢀisopropylbenzaldehyde to afford the title
compound as a white solid (28%).
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M.P. 193–196°C. 1H NMR (DMSOꢀd6) δ 8.62 – 8.29 (m, 4H), 7.85 (d, J = 8.2 Hz, 4H), 7.33 (d,
J = 8.2 Hz, 4H), 2.98 – 2.87 (m, 2H), 1.21 (d, J = 6.9 Hz, 12H). 13C NMR (DMSOꢀd6) δ 152.7,
151.4, 148.8, 131.0, 128.0, 126.7, 33.4, 23.6. HRMS: m/z (calcd for C21H27N5, 349.2266; found
349.2112).
2,2'-Bis{[4-(1,1-dimethylethyl)phenyl]methylene}carbonimidic dihydrazide hydrochloride (27)
Synthesized according to General Method B from 4ꢀtertꢀbutylbenzaldehyde to afford the title
compound as a white solid (50%).
M.P. 150–155°C. 1H NMR (DMSOꢀd6) δ 8.56 – 8.32 (m, 4H), 7.85 (d, J = 8.3 Hz, 4H), 7.49 (d,
J = 8.3 Hz, 4H), 1.31 (s, 18H). 13C NMR (DMSOꢀd6) δ 153.7, 152.7, 148.8, 130.7, 127.8, 125.6,
34.7, 31.0. HRMS: m/z (calcd for C23H31N5, 377.2579; found 377.2454).
2,2'-Bis{[4-(dimethylamino)phenyl]methylene}carbonimidic dihydrazide hydrochloride (28)
Synthesized according to General Method B from 4ꢀ(dimethylamino)benzaldehyde to afford the
title compound as an orange solid (80%).
M.P. 161–164°C. 1H NMR (DMSOꢀd6) δ 11.92 (br. s, 2H), 8.24 (s, 2H), 8.16 (s, 2H), 7.71 (d, J
= 8.9 Hz, 4H), 6.74 (d, J = 8.9 Hz, 4H), 2.98 (s, 12H). 13C NMR (DMSOꢀd6) δ 152.1, 151.8, 149.0,
129.2, 120.5, 111.5, 39.7. HRMS: m/z (calcd for C19H25N7, 351.2171; found 351.1966).
2,2'-Bis([1,1'-biphenyl]-4-ylmethylene)carbonimidic dihydrazide hydrochloride (29)
Synthesized according to General Method B from 4ꢀphenylbenzaldehyde to afford the title
compound as a yellow solid (81%).
M.P. 264–266°C. 1H NMR (DMSOꢀd6) δ 12.48 (br. s, 2H), 8.62 (s, 2H), 8.51 (s, 2H), 8.04 (d, J
= 7.5 Hz, 4H), 7.85 – 7.69 (m, 8H), 7.54 – 7.36 (m, 6H). 13C NMR (DMSOꢀd6) δ 152.8, 148.4,
142.2, 139.3, 132.5, 129.1, 128.5, 128.0, 127.0, 126.8. HRMS: m/z (calcd for C27H23N5, 417.1953;
found 417.1831).
2,2'-Bis([1,1'-biphenyl]-2-ylmethylene)carbonimidic dihydrazide hydrochloride (30)
Synthesized according to General Method B from biphenylꢀ2ꢀcarboxaldehyde to afford the title
compound as a yellow solid (89%).
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M.P. 129–133°C. 1H NMR (DMSOꢀd6) δ 12.27 (br. s, 2H), 8.49 (s, 2H), 8.44 – 8.27 (m, 4H),
7.57 – 7.44 (m, 10H), 7.41 – 7.33 (m, 6H). 13C NMR (DMSOꢀd6) δ 152.6, 147.2, 142.4, 138.8,
130.6, 130.4, 129.7, 128.7, 127.8, 127.6, 126.8. HRMS: m/z (calcd for C27H23N5, 417.1953; found
417.1709).
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2,2'-Bis[(3-ethynylphenyl)methylene]carbonimidic dihydrazide hydrochloride (31)
Synthesized according to General Method B from 4ꢀethynylbenzaldehyde to afford the title
compound as a yellow solid (93%).
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M.P. 220°C (Decomp.). H NMR (DMSOꢀd6) δ 12.27 (br. s, 2H), 8.58 (s, 2H), 8.42 (s, 2H),
7.96 (d, J = 8.3 Hz, 4H), 7.58 (d, J = 8.2 Hz, 4H), 4.39 (s, 2H). 13C NMR (DMSOꢀd6) δ 152.9,
148.0, 133.7, 132.0, 128.0, 123.7, 83.2, 83.0. LRMS: m/z (calcd for C19H15N5, 313.13; found
313.80).
2,2'-Bis[(2,5-difluorophenyl)methylene]carbonimidic dihydrazide hydrochloride (32)
Synthesized according to General Method B from 2,5ꢀdifluorophenylbenzaldehyde to afford the
title compound as a white solid (34%).
1H NMR (DMSOꢀd6) δ 12.83 (br s, 2H), 8.76 (s, 2H), 8.69 (s, 2H), 8.31 – 8.21 (m, 2H), 7.45 –
7.31 (m, 4H). 13C NMR (DMSOꢀd6) δ 158.5 (dd, J = 240.2, 1.2 Hz), 157.2 (dd, J = 247.2, 1.4 Hz),
152.8, 140.7, 122.5 (dd, J = 12.2, 8.9 Hz), 119.4 (dd, J = 25.1, 9.0 Hz), 117.8 (dd, J = 24.0, 8.8 Hz),
113.0 (dd, J = 25.9, 2.3 Hz). HRMS: m/z (calcd for C15H11F4N5, 337.0951; found 338.0877).
N',2-Bis((E)-4-chloro-2-fluorobenzylidene)hydrazine-1-carboximidhydrazide hydrochloride (33)
Synthesized according to General Method A from 4ꢀchloroꢀ2ꢀfluorobenzaldehyde to afford the
title compound as a white solid (39%).
1H NMR (DMSOꢀd6) δ 12.43 (s, 1H), 8.38 (t, J = 8.3 Hz, 1H), 7.61 (dd, J = 10.5, 1.9 Hz, 1H),
7.45 (dd, J = 8.6, 1.6 Hz, 1H). 13C NMR (DMSOꢀd6) δ 160.7 (d, J = 254.5 Hz), 152.8*, 140.8*,
136.3 (d, J = 10.8 Hz), 128.5, 125.3, 120.2 (d, J = 10.0 Hz), 116.7 (d, J = 24.7 Hz). *Poor resolution
failed to resolve doublets.
2,2'-Bis[(3,4-difluorophenyl)methylene]carbonimidic dihydrazide hydrochloride (34)
Synthesized according to General Method B from 3,4ꢀdifluorobenzaldehyde to afford the title
compound as a white solid (76%).
1H NMR (DMSOꢀd6) δ 8.68 (s, 2H), 8.43 (s, 2H), 8.28 – 8.16 (m, 2H), 7.77 – 7.64 (m, 2H), 7.58
– 7.46 (m, 2H). 13C NMR (DMSOꢀd6) δ 153.0, 150.8 (dd, J = 250.6, 13.0 Hz), 149.9 (dd, J = 245.9,
13.2 Hz), 146.6, 131.2 (dd, J = 6.4, 3.4 Hz), 126.0 (dd, J = 6.4, 2.8 Hz), 117.8 (d, J = 17.7 Hz),
115.7 (d, J = 18.5 Hz). HRMS: m/z (calcd for C15H11F4N5, 337.0951; found 338.0943).
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2,2'-Bis[(2,4-dichlorophenyl)methylene]carbonimidic dihydrazide hydrochloride (35)
Synthesized according to General Method B from 2,4ꢀdichlorobenzaldehyde to afford the title
compound as a white solid (47%).
1H NMR (DMSOꢀd6) δ 12.84 (br s, 2H), 8.84 (s, 2H), 8.74 (s, 2H), 8.46 (d, J = 8.6 Hz, 2H), 7.71
(d, J = 1.6 Hz, 2H), 7.54 (dd, J = 8.6, 1.2 Hz, 2H). 13C NMR (DMSOꢀd6) δ 152.7, 144.0, 135.9,
134.3, 129.7, 129.3, 129.3, 127.8. HRMS: m/z (calcd for C15H11Cl4N5, 402.9739; found 403.9786).
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2,2'-Bis[(2,6-dichlorophenyl)methylene]carbonimidic dihydrazide hydrochloride (36)
Synthesized according to General Method B from 2,6ꢀdichlorobenzaldehyde to afford the title
compound as a white solid (63%).
1H NMR (DMSOꢀd6) δ 12.94 (br s, 2H), 8.68 (s, 2H), 8.33 (s, 2H), 7.60 (d, J = 7.9 Hz, 4H)*,
7.49 (dd, J = 8.7, 7.4 Hz, 2H)*. 13C NMR (DMSOꢀd6) δ 153.0, 145.1, 134.1, 131.9, 129.9, 129.0.
HRMS: m/z (calcd for C15H11Cl4N5, 402.9739; found 403.9765). *Poorly resolved 1HꢀNMR
spectrum.
2,2'-Bis[(3,5-dichlorophenyl)methylene]carbonimidic dihydrazide hydrochloride (37)
Synthesized according to General Method B from 3,5ꢀdichlorobenzaldehyde to afford the title
compound as a white solid (84%).
1H NMR (DMSOꢀd6) δ 12.67 (br s, 2H), 8.81 (s, 2H), 8.40 (s, 2H), 8.06 (d, J = 1.8 Hz, 4H), 7.68
(t, J = 1.8 Hz, 2H). 13C NMR (DMSOꢀd6) δ 153.1, 146.1, 136.9, 134.6, 129.7, 126.2.
2,2'-Bis[(2-amino-4-chlorophenyl)methylene]carbonimidic dihydrazide hydrochloride (38)
Synthesized according to General Method B from 2ꢀaminoꢀ4ꢀchlorobenzaldehyde to afford the
title compound as a pale yellow solid (13%).
1H NMR (DMSOꢀd6) δ 11.71 (br s, 2H), 8.40 (s, 2H), 8.37 (s, 2H), 7.29 (d, J = 8.4 Hz, 2H), 6.87
(d, J = 2.0 Hz, 2H), 6.73 (br s, 4H), 6.59 (dd, J = 8.3, 2.0 Hz, 2H). 13C NMR (DMSOꢀd6) δ 152.1,
151.5, 148.9, 136.0, 134.7, 115.1, 114.5, 112.8.
2,2'-Bis{[2-(1-hydroxyethylamino)-4-chlorophenyl]methylene}carbonimidic dihydrazide
hydrochloride (39)
Synthesized according to General Method A from 4ꢀchloroꢀ2ꢀ[(1ꢀ
hydroxyethyl)amino]benzaldehyde to afford the title compound as a yellow solid (17%).
1H NMR (DMSOꢀd6) δ 11.86 (s, 1H), 8.41 (s, 2H), 7.36 (d, J = 6.8 Hz, 2H), 6.75 (s, 1H), 6.66
(d, J = 7.9 Hz, 1H), 3.44 (d, J = 6.9 Hz, 1H)*, 1.24 (t, J = 5.3 Hz, 3H), 1.07 (dt, J = 13.8, 6.8 Hz,
1H). *Signal eclipsed by water. 13C NMR (DMSOꢀd6) δ 152.0, 148.1, 136.9, 135.1, 114.7, 113.5,
109.7, 64.9, 56.0, 13.9.
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2,2'-bis[(2-acetamido-4-chlorophenyl)methylene]carbonimidic dihydrazide (40)
Synthesized according to General Method A from Nꢀ(5ꢀchloroꢀ2ꢀformylphenyl)acetamide to
afford the title compound as an offꢀwhite crystalline solid (18%).
M.P. 259°C (Decomp.). 1H NMR (DMSOꢀd6) δ 12.29 (s, 1H), 10.29 (s, 1H), 8.55 (s, 1H), 8.45
(s, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.71 (s, 1H), 7.35 (dd, J = 8.5, 1.8 Hz, 1H), 2.12 (s, 3H). 13C
NMR (DMSOꢀd6) δ 169.2, 152.8, 146.4, 138.4, 135.0, 129.7, 125.0, 124.7, 124.3, 23.6. LRMS: m/z
(calcd for C19H19Cl2N7O2, 447.10; found 447.80).
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2,2'-Bis[(2-hydroxy-3-methylphenyl)methylene]carbonimidic dihydrazide hydrochloride (41)
Synthesized according to General Method B from 2ꢀhydroxyꢀ3ꢀmethylbenzaldehyde to afford
the title compound as a pale yellow solid (93%).
M.P. 260°C (Decomp.). 1H NMR (DMSOꢀd6) δ 12.08 (br s, 2H), 9.35 (s, 2H), 8.66 (s, 2H), 8.48
(s, 2H), 7.67 (d, J = 7.2 Hz, 2H), 7.25 (d, J = 7.2 Hz, 2H), 6.88 (t, J = 7.6 Hz, 2H), 2.23 (s, 6H). 13C
NMR (DMSOꢀd6) δ 154.6, 152.1, 149.0, 133.4, 127.0, 125.7, 119.7, 119.1, 16.1. LRMS: m/z (calcd
for C17H19N5O2, 325.15; found 325.85).
2,2'-Bis(4-chloro-2-hydroxyphenylmethylene)carbonimidic dihydrazide hydrochloride (42)
Synthesized according to General Method A from 4ꢀchlorosalicylicaldehyde to afford the title
compound as a grey solid (22%).
1H NMR (DMSOꢀd6) δ 12.02 (s, 1H), 10.81 (s, 1H), 8.63 (s, 1H), 8.38 (s, 1H), 8.12 (d, J = 8.3
Hz, 1H), 7.13 – 6.84 (m, 2H). 13C NMR (DMSO) δ 157.5, 128.3, 119.5, 119.0, 115.9.
2,2'-Bis{[4-(dimethylamino)-2-hydroxyphenyl]methylene}carbonimidic dihydrazide (43)
Synthesized according to General Method A from 4ꢀ(dimethylamino)salicylic acid to afford the
title compound as a brown solid (62%).
M.P. 254°C (Decomp.). 1H NMR (DMSOꢀd6) δ 11.63 (s, 1H), 9.84 (s, 1H), 8.46 (s, 1H), 8.02 (s,
1H), 7.74 (d, J = 8.8 Hz, 1H), 6.30 (d, J = 7.4 Hz*, 1H), 6.17 (s, 1H), 2.94 (s, 6H). 13C NMR
(DMSOꢀd6) δ 158.0, 153.1, 151.4, 146.4, 128.4, 107.5, 104.4, 97.7, 39.7*. *Signal eclipsed by the
DMSO. LRMS: m/z (calcd for C19H25N7O2, 383.21; found 383.70). *Poorly resolved doublet gives
reduced coupling constant.
2,2'-Bis[(2,3-dihydroxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (44)
Synthesized according to General Method B from 2,3ꢀdihydroxybenzaldehyde to afford the title
compound as a yellow solid (75%).
M.P. 217°C (Decomp.). 1H NMR (DMSOꢀd6) δ 12.06 (br s, 2H), 9.71 (br s, 2H), 9.21 (br s, 2H),
8.70 (s, 2H), 8.30 (s, 2H), 7.50 (d, J = 7.9 Hz, 2H), 6.90 (d, J = 7.7 Hz, 2H), 6.70 (t, J = 7.7 Hz,
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2H). 13C NMR (DMSOꢀd6) δ 152.4, 146.0, 145.8, 145.7, 120.3, 119.2, 117.5, 117.2. HRMS: m/z
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(calcd for C15H15N5O4, 329.1124; found 330.1100).
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2,2'-Bis[(2,4-dihydroxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (45)
Synthesized according to General Method B from 2,4ꢀdihydroxybenzaldehyde to afford the title
compound as a pale yellow solid (73%).
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M.P. 234°C (Decomp.). H NMR (DMSOꢀd6) δ 11.81 (br s, 2H), 10.32 – 9.85 (m, 4H), 8.52 (s,
2H), 8.12 (s, 2H), 7.85 (d, J = 8.4 Hz, 2H), 6.43 (s, 2H), 6.33 (d, J = 8.5 Hz, 2H). 13C NMR
(DMSOꢀd6) δ 161.4, 158.5, 152.0, 145.7, 128.4, 111.2, 108.0, 102.4. HRMS: m/z (calcd for
C15H15N5O4, 329.1124; found 330.1116).
2,2'-Bis[(3,4-dihydroxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (46)
Synthesized according to General Method A from 3,4ꢀdihydroxybenzaldehyde to afford the title
compound as a pale yellow solid (21%).
M.P. 250°C (Decomp.). 1H NMR (DMSOꢀd6) δ 11.80 (br s, 2H), 10.30 – 9.80 (m, 4H), 8.52 (s,
2H), 8.12 (s, 2H), 7.84 (d, J = 8.6 Hz, 2H), 6.42 (d, J = 1.8 Hz, 2H), 6.33 (d, J = 8.6 Hz, 2H). 13C
NMR (DMSOꢀd6) δ 161.4, 158.5, 152.0, 145.7, 128.4, 111.2, 108.0, 102.4. HRMS: m/z (calcd for
C15H15N5O4, 329.1124; found 330.1133).
2,2'-Bis[(3-hydroxy-4-methoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (47)
Synthesized according to General Method B from 3ꢀhydroxyꢀ4ꢀmethoxybenzaldehyde to afford
the title compound as a yellow solid (61%).
M.P. 158ꢀ161°C. 1H NMR (DMSOꢀd6) δ 9.21 (br s, 2H), 8.42 – 8.17 (m, 4H), 7.43 (d, J = 1.9
Hz, 2H), 7.25 (dd, J = 8.4, 1.9 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 3.83 (s, 6H). 13C NMR (DMSOꢀ
d6) δ 152.5, 150.3, 148.9, 146.7, 126.2, 120.9, 113.8, 111.7, 55.7. HRMS: m/z (calcd for
C17H19N5O4, 357.1437; found 358.1371).
2,2'-Bis[(4-hydroxy-3-methoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (48)
Synthesized according to General Method B from 4ꢀhydroxyꢀ3ꢀmethoxybenzaldehyde to afford
the title compound as a yellow solid (69%).
M.P. 200ꢀ203°C. 1H NMR (DMSOꢀd6) δ 9.77 (br s, 2H), 8.36 (s, 2H), 8.29 (s, 2H), 7.58 (d, J =
1.5 Hz, 2H), 7.23 (dd, J = 8.1, 1.2 Hz, 2H), 6.87 (d, J = 8.1 Hz, 2H), 3.86 (s, 6H). 13C NMR
(DMSOꢀd6) δ 152.4, 149.7, 149.0, 148.1, 124.7, 122.9, 115.4, 110.4, 55.9. HRMS: m/z (calcd for
C17H19N5O4, 357.1437; found 358.1447).
2,2'-Bis[(3,4-dimethoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (49)
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Synthesized according to General Method B from 3,4ꢀdimethoxybenzaldehyde to afford the title
compound as a pale yellow solid (100%).
M.P. 218ꢀ220°C. 1H NMR (DMSOꢀd6) δ 12.09 (br s, 2H), 8.44 (s, 2H), 8.34 (s, 2H), 7.63 (d, J =
1.4 Hz, 2H), 7.33 (dd, J = 8.3, 1.6 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 3.86 (s, 6H), 3.81 (s, 6H). 13C
NMR (DMSOꢀd6) δ 152.5, 151.4, 149.2, 148.9, 126.0, 122.9, 111.4, 109.5, 55.9, 55.7. HRMS: m/z
(calcd for C19H23N5O4, 385.1750; found 386.1739).
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2,2'-Bis[(2,3,4-trihydroxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (50)
Synthesized according to General Method B from 2,3,4ꢀtrihydroxybenzaldehyde to afford the title
compound as a white solid (84%).
M.P. 225°C (Decomp.). 1H NMR (DMSOꢀd6) δ 11.75 (br s, 2H), 9.71 (br s, 2H), 9.15 (br s, 2H),
8.86 – 8.40 (m, 4H), 8.13 (s, 2H), 7.33 (d, J = 8.6 Hz, 2H), 6.42 (d, J = 8.6 Hz, 2H). 13C NMR
(DMSOꢀd6) δ 151.9, 149.1, 147.0, 146.8, 132.9, 118.1, 112.1, 108.1. HRMS: m/z (calcd for
C15H15N5O6, 361.1022; found 362.0995).
2,2'-Bis[(2,4,5-trihydroxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (51)
Synthesized according to General Method B from 2,3,5ꢀtrihydroxybenzaldehyde to afford the
title compound as an orange solid (82%).
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M.P. 260°C (Decomp.). H NMR (DMSOꢀd6) δ 11.72 (br s, 2H), 9.78 (br s, 2H), 9.45 (s, 2H),
8.48 (s, 2H), 8.34 (br s, 2H), 8.04 (s, 2H), 7.33 (s, 2H), 6.44 (s, 2H). 13C NMR (DMSOꢀd6) δ 152.0,
151.4, 150.2, 146.1, 138.9, 112.9, 110.2, 103.4. HRMS: m/z (calcd for C15H15N5O6, 361.1022;
found 362.1043).
2,2'-Bis[(3,4,5-trihydroxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (52)
Synthesized according to General Method B from 3,4,5ꢀtrihydroxybenzaldehyde to afford the
title compound as a pale brown solid (77%).
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M.P. 226°C (Decomp.). H NMR (DMSOꢀd6) δ 9.06 (br s, 6H), 8.25 – 8.01 (m, 4H), 6.83 (s,
4H). 13C NMR (DMSOꢀd6) δ 152.2, 149.7, 146.2, 136.5, 123.7, 107.4. HRMS: m/z (calcd for
C15H15N5O6, 361.1022; found 362.1044).
2,2'-Bis[(4,5-dihydroxy-3-methoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (53)
Synthesized according to General Method B from 4,5ꢀdihydroxyꢀ3ꢀmethoxybenzaldehyde to
afford the title compound as a pale brown solid (88%).
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M.P. 144°C (Decomp.). H NMR (DMSOꢀd6) δ 9.12 (br s, 4H), 8.29 (s, 2H), 8.20 (s, 2H), 7.10
(s, 2H), 6.93 (s, 2H), 3.84 (s, 6H). 13C NMR (DMSOꢀd6) δ 152.4, 149.5, 148.8, 145.8, 137.5, 123.7,
110.1, 103.1, 56.3. HRMS: m/z (calcd for C17H19N5O6, 389.1335; found 390.1275).
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2,2'-Bis[(4-hydroxy-3-nitrophenyl)methylene]carbonimidic dihydrazide hydrochloride (54)
Synthesized according to General Method B from 4ꢀhydroxyꢀ3ꢀnitrobenzaldehyde to afford the
title compound as a white solid (80%).
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M.P. 269°C (Decomp.). H NMR (DMSOꢀd6) δ 8.56 (s, 2H), 8.42 (d, J = 2.0 Hz, 2H), 8.38 (s,
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2H), 8.09 (dd, J = 8.7, 2.0 Hz, 2H), 7.29 (d, J = 8.7 Hz, 2H). C NMR (DMSOꢀd6) δ 153.6, 152.8,
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146.9, 137.6, 133.7, 124.7, 124.4, 119.3. HRMS: m/z (calcd for C15H13N7O6, 388.0917; found
387.0927).
2,2'-Bis[(2,3,4,5,6-pentafluorophenyl)methylene]carbonimidic dihydrazide hydrochloride (55)
Synthesized according to General Method B from 2,3,4,5,6ꢀpentafluorobenzaldehyde to afford
the title compound as a white solid (60%).
M.P. 234ꢀ236°C. 1H NMR (DMSOꢀd6) δ 8.64 (s, 2H), 8.36 (s, 2H). 13C NMR (DMSOꢀd6) δ
152.8, 146.0 (m), 143.4 (m), 142.7 (m), 140.1 (m), 138.6 (m), 138.1, 136.1 (m), 108.9 (td, J = 12.6,
3.8 Hz. HRMS: m/z (calcd for C15H5F10N5, 445.0358; found 446.0306).
2,2'-Bis[(2-bromo-4,5-dimethoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (56)
Synthesized according to General Method B from 2ꢀbromoꢀ4,5ꢀdimethoxybenzaldehyde to
afford the title compound as a white solid (80%).
M.P. 233ꢀ235°C. 1H NMR (DMSOꢀd6) δ 12.24 (br s, 2H), 8.66 (s, 2H), 8.49 (br s, 2H), 7.79 (s,
2H), 7.24 (s, 2H), 3.88 (s, 6H), 3.85 (s, 6H). 13C NMR (DMSOꢀd6) δ 152.4, 152.0, 148.7, 147.7,
124.0, 116.1, 115.5, 110.1, 56.3, 56.2. HRMS: m/z (calcd for C19H21Br2N5O4, 542.9940; found
543.9882).
2,2'-Bis[(3-bromo-4,5-dimethoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (57)
Synthesized according to General Method B from 3ꢀbromoꢀ4,5ꢀdimethoxybenzaldehyde to
afford the title compound as a white solid (45%).
M.P. 223ꢀ224°C. 1H NMR (DMSOꢀd6) δ 8.66 (s, 2H), 8.37 (s, 2H), 7.79 (d, J = 1.6 Hz, 2H),
7.68 – 7.62 (m, 2H), 3.92 (s, 6H), 3.78 (s, 6H). 13C NMR (DMSOꢀd6) δ 153.6, 152.8, 147.5, 147.2,
130.7, 123.8, 117.1, 111.8, 60.3, 56.5. HRMS: m/z (calcd for C19H21Br2N5O4, 542.9940; found
543.9850).
2,2'-Bis(cyclohexylmethylene)carbonimidic dihydrazide (58)
Synthesized according to General Method B from cyclohexane carboxaldehyde to afford the title
compound as an orange oil (36%).
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1H NMR (CDCl3) δ 7.16 (d, J = 4.7 Hz, 2H), 6.10 (br. s, 3H), 2.27 – 2.14 (m, 2H), 1.84 – 1.61
(m, 10H), 1.37 – 1.13 (m, 10H). 13C NMR (CDCl3) δ 157.2, 152.7 (br. s), 40.9, 30.6, 26.1, 25.7.
HRMS: m/z (calcd for C15H27N5, 277.2266; found 277.2169).
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2,2'-Bis(4-pyridinylmethylene)carbonimidic dihydrazide hydrochloride (59)
Synthesized according to General Method B from 4ꢀpyridinecarboxaldehdye to afford the title
compound as a yellow solid (74%).
M.P. 231–234°C. 1H NMR (DMSOꢀd6) δ 8.75 (d, J = 6.1 Hz, 4H), 8.68 (s, 2H), 8.47 (s, 2H),
8.07 (d, J = 6.1 Hz, 4H). 13C NMR (DMSOꢀd6) δ 154.4, 148.0, 145.3, 143.0, 122.1. LRMS: m/z
(calcd for C13H13N7, 267.12; found 267.90).
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2,2'-Bis(4-chloropyridin-3-ylmethylene)carbonimidic dihydrazide hydrochloride (60)
Synthesized according to General Method B from 4ꢀchloroꢀ3ꢀpyridine carboxaldehyde to afford
the title compound as a yellow solid (84%).
M.P. 269–272°C. 1H NMR (DMSOꢀd6) δ 12.63 (br. s, 2H), 8.90 (d, J = 2.1 Hz, 2H), 8.74 (s,
2H), 8.56 – 8.42 (m, 4H), 7.66 (d, J = 8.4 Hz, 2H). 13C NMR (DMSOꢀd6) δ 153.0, 151.7, 149.6,
145.0, 137.8, 128.9, 124.5. LRMS: m/z (calcd for C13H11Cl2N7, 335.05; found 335.60).
2,2'-Bis(2-aminopyridin-3-ylmethylene)carbonimidic dihydrazide hydrochloride (61)
Synthesized according to General Method B from 2ꢀaminoꢀ3ꢀpyridine carboxaldehyde to afford
the title compound as a yellow solid (49%).
M.P. 227–229°C. 1H NMR (DMSOꢀd6) δ11.94 (br. s, 2H), 8.44 (s, 2H), 8.36 (s, 2H), 8.07 (dd, J
= 4.8, 1.7 Hz, 2H), 7.72 (dd, J = 7.6, 1.4 Hz, 2H), 7.19 (s, 4H), 6.67 (dd, J = 7.5, 4.9 Hz, 2H). 13C
NMR (DMSOꢀd6) δ 156.1, 152.2, 150.4, 149.5, 141.2, 112.0, 110.1. LRMS: m/z (calcd for
C13H15N9, 297.15; found 297.70).
2,2'-Bis(3-phenyl-2-propenylidene)carbonimidic dihydrazide hydrochloride (62)
Synthesized according to General Method B from transꢀcinnamaldehyde to afford the title
compound as a yellow solid (63%).
M.P. 176ꢀ178°C. 1H NMR (DMSO) δ 12.38 (s, 1H), 8.25 (d, J = 10.2 Hz, 2H), 7.59 (d, J = 7.3
Hz, 2H), 7.39 (m, 3H), 7.20 (d, J = 16.1 Hz, 1H), 6.94 (dd, J = 16.1, 9.3 Hz, 1H). 13C NMR
(DMSOꢀd6) δ 152.3, 150.8, 141.0, 135.5, 129.4, 129.0, 127.2, 124.1. HRMS: m/z (calcd for
C19H19N5, 317.1640; found 318.1629).
2,2'-Bis[3-(4-methoxylphenyl)-2-propenylidene]carbonimidic dihydrazide hydrochloride (63)
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Synthesized according to General Method B from 4ꢀmethoxycinnamaldehyde to afford the title
compound as a yellow solid (51%).
M.P. 204ꢀ206°C. 1H NMR (DMSOꢀd6) δ 12.10 (br s, 2H), 8.23 – 8.07 (m, 4H), 7.55 (d, J = 8.7
Hz, 4H), 7.13 (d, J = 16.0 Hz, 2H), 6.99 (d, J = 8.7 Hz, 4H), 6.81 (dd, J = 16.0, 9.4 Hz, 2H), 3.79 (s,
6H). 13C NMR (DMSOꢀd6) δ 160.3, 152.1, 151.2, 140.8, 128.8, 128.2, 121.7, 114.5, 55.3. LRMS:
m/z (calcd for C21H23N5O2, 377.19; found 377.80).
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2,2'-Bis(2,3-diphenyl-2-propenylidene)carbonimidic dihydrazide hydrochloride (64)
Synthesized according to General Method A from 2,3ꢀdiphenylacrolein to afford the title
compound as a yellow solid (46%).
M.P. 137ꢀ140°C. 1H NMR (DMSOꢀd6) δ 12.12 (br s, 2H), 8.21 (s, 2H), 7.97 (s, 2H), 7.54 – 7.47
(m, 6H), 7.42 – 7.36 (m, 6H), 7.31 – 7.21 (m, 8H), 6.84 (d, J = 9.8 Hz, 2H). 13C NMR (DMSOꢀd6) δ
152.0, 150.4, 148.5*, 140.2, 137.6, 130.0, 129.0, 128.7, 128.7, 128.5, 127.4, 123.0. *Confirmed via
HSQC analysis. LRMS: m/z (calcd for C31H27N5, 469.23; found 469.85).
2,2'-Bis(2-naphthalenylmethylene)carbonimidic dihydrazide hydrochloride (65)
Synthesized according to General Method A from 2ꢀnaphthaldehyde to afford the title compound
as a white solid (79%).
M.P. 183ꢀ185°C. 1H NMR (DMSOꢀd6) δ 8.64 (s, 4H), 8.35 – 8.24 (m, 4H), 8.06 – 7.93 (m, 6H),
7.64 – 7.54 (m, 4H). 13C NMR (DMSOꢀd6) δ 152.9, 148.9, 134.1, 132.8, 131.2, 129.9, 128.5, 128.4,
127.9, 127.6, 126.9, 123.3. HRMS: m/z (calcd for C23H19N5, 365.1640; found 366.1740).
2,2'-Bis(1H-indol-5-ylmethylene)carbonimidic dihydrazide hydrochloride (66)
Synthesized according to General Method B from indoleꢀ5ꢀcarboxaldehyde to afford the title
compound as a pinkꢀbrown solid (64%).
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M.P. 269°C (Decomp.). H NMR (DMSOꢀd6) δ 11.97 (br s, 2H), 11.45 (s, 2H), 8.47 (s, 2H),
8.30 (s, 2H), 8.02 (s, 2H), 7.80 (dd, J = 8.6, 0.9 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 7.45 – 7.40 (m,
2H), 6.53 (s, 2H). 13C NMR (DMSOꢀd6) δ 152.4, 150.6, 137.4, 127.6, 126.7, 124.5, 121.8, 120.3,
111.9, 102.0. LRMS: m/z (calcd for C19H17N7, 343.15; found 343.80).
2,2'-Bis[(5-chlorobenzo[b]thien-3-yl)methylene]carbonimidic dihydrazide hydrochloride (67)
Synthesized according to General Method B from 5ꢀchloroꢀ1ꢀbenzothiopheneꢀ3ꢀcarbaldehyde to
afford the title compound as a pale yellow solid (90%).
M.P. 285ꢀ287°C. 1H NMR (DMSOꢀd6) δ 8.66 (s, 2H), 8.60 (d, J = 1.9 Hz, 2H), 8.52 (br s, 2H),
8.49 (s, 2H), 8.09 (d, J = 8.6 Hz, 2H), 7.50 (dd, J = 8.6, 2.0 Hz, 2H). 13C NMR (DMSOꢀd6) δ 152.9,
145.4, 138.9, 136.9, 136.5, 131.1, 129.5, 126.0, 125.0, 124.2. LRMS: m/z (calcd for C19H13Cl2N5S2,
445.00; found 445.75).
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2,2'-Bis[1-phenylethylidene]carbonimidic dihydrazide hydrochloride (68)
Synthesized according to General Method B from acetophenone to afford the title compound as a
white solid (60%).
M.P. 253ꢀ254°C. 1H NMR (DMSOꢀd6) δ 11.91 (br s, 2H), 8.66 (br s, 2H), 8.10 – 8.00 (m, 4H),
7.51 – 7.41 (m, 6H), 2.45 (s, 6H). 13C NMR (DMSOꢀd6) δ 154.2, 153.3, 136.7, 129.9, 128.3, 127.0,
15.0. LRMS: m/z (calcd for C17H19N5, 293.16; found 294.00).
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2,2'-Bis[1-(4-chlorophenyl)ethylidene]carbonimidic dihydrazide hydrochloride (69)
Synthesized according to General Method A from 4ꢀchlorobenzaldehyde to afford the title
compound.
1H NMR (DMSOꢀd6) δ 11.68 (br. s, 2H), 8.78 (br. s, 1H), 8.10 (d, J = 8.6 Hz, 4H), 7.52 (d, J =
8.6 Hz, 4H), 2.43 (s, 6H).
2,2'-Bis[1-(4-bromophenyl)ethylidene]carbonimidic dihydrazide hydrochloride (70)
Synthesized according to General Method B from 4ꢀbromobenzaldehyde to afford the title
compound as a white solid (94%).
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M.P. 299°C (Decomp.). H NMR (DMSOꢀd6) δ 11.63 (s, 2H), 8.76 (s, 2H), 8.01 (d, J = 8.6 Hz,
4H), 7.65 (d, J = 8.6 Hz, 4H), 2.41 (s, 6H). 13C NMR (DMSOꢀd6) δ 154.1, 152.4, 135.9, 131.3,
129.1, 123.6, 14.7. LRMS: m/z (calcd for C17H17Br2N5, 450.98; found 449.70).
2,2'-Bis{1-[4-(trifluoromethyl)phenyl]ethylidene}carbonimidic dihydrazide hydrochloride (71)
Synthesized according to General Method A from 4ꢀtrifluoromethylbenzaldehyde to afford the
title compound.
1H NMR (DMSOꢀd6) δ 11.71 (br. s, 2H), 8.91 (br. s, 1H), 8.28 (d, J = 7.8 Hz, 4H), 7.82 (d, J =
8.2 Hz, 4H), 2.49 (br. s, 6H).
2,2'-Bis[1-(4-chlorophenyl)propylidene]carbonimidic dihydrazide hydrochloride (72)
Synthesized according to General Method B from 4ꢀchloropropiophenone to afford the title
compound as a white solid (47%).
M.P. 246ꢀ247°C. 1H NMR (DMSOꢀd6) δ 11.68 (s, 2H), 8.78 (s, 2H), 8.09 (d, J = 8.4 Hz, 4H),
7.52 (d, J = 8.6 Hz, 4H), 2.92 (q, J = 7.5 Hz, 4H), 1.12 (t, J = 7.4 Hz, 6H). 13C NMR (DMSOꢀd6) δ
156.6, 154.2, 134.7, 134.2, 128.9, 128.5, 20.4, 10.9. LRMS: m/z (calcd for C19H21Cl2N5, 389.12;
found 389.90).
2,2'-Bis[1-(4-chlorophenyl)butylidene]carbonimidic dihydrazide hydrochloride (73)
Synthesized according to General Method B from 4ꢀchlorobutyrophenone to afford the title
compound as a white solid (26%).
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M.P. 246ꢀ247°C. 1H NMR (DMSOꢀd6) δ 12.18 (s, 2H), 8.71 (s, 2H), 8.08 (d, J = 8.0 Hz, 4H),
7.50 (d, J = 8.3 Hz, 4H), 3.01 – 2.87 (m, 4H), 1.58 – 1.46 (m, 4H), 1.01 (t, J = 7.1 Hz, 6H). 13C
NMR (DMSOꢀd6) δ 155.5, 154.2, 134.6, 128.9, 128.4, 28.7, 19.5, 13.6. LRMS: m/z (calcd for
C21H25Cl2N5, 417.15; found 417.80).
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2,2'-Bis[1-(4-chlorophenyl)pentylidene]carbonimidic dihydrazide hydrochloride (74)
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Synthesized according to General Method B from 1ꢀ(4ꢀchlorophenyl)pentanꢀ1ꢀone to afford the
title compound as a pale yellow solid (53%).
M.P. 140ꢀ142°C. 1H NMR (DMSOꢀd6) δ 12.09 (s, 2H), 8.69 (s, 2H), 8.07 (d, J = 8.6 Hz, 4H),
7.50 (d, J = 8.6 Hz, 4H), 3.01 – 2.88 (m, 4H), 1.49 – 1.39 (m, 8H), 0.88 (t, J = 6.6 Hz, 6H). 13C
NMR (DMSOꢀd6) δ 155.8, 154.4, 134.7, 129.0, 128.5, 28.3, 27.0, 22.0, 13.9. LRMS: m/z (calcd for
C23H29Cl2N5, 445.18; found 445.80).
2,2'-Bis[1-(4-chlorophenyl)-2-hydroxyethylidene]carbonimidic dihydrazide (75)
Synthesized according to General Method A from 1ꢀ(4ꢀchlorophenyl)ꢀ2ꢀhydroxyethanone to
afford the title compound as a yellow solid (6%).
1H NMR (DMSO) δ 8.67 (s, 2H), 7.97 (s, 4H), 7.50 (d, J = 8.6 Hz, 4H), 4.81 (s, 4H). LRMS: m/z
(calcd for C17H17Cl2N5O2, 393.08; found 393.60).
2,2'-Bis(1-phenyl-2-aminoethylidene)carbonimidic dihydrazide trihydrochloride (76)
Synthesized according to General Method B from 2ꢀaminoacetophenone hydrochloride to afford
the title compound as a white solid (8%).
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M.P. 250°C (Decomp.). H NMR (DMSOꢀd6) δ 10.87 (br s, 1H), 9.52 (br s, 1H), 8.76 (br s, 3H),
7.61ꢀ7.44 (m, 8H), 7.39 (br s, 3H), 7.26 (br s, 3H), 4.06 (br s, 2H). 13C NMR (DMSOꢀd6) δ 154.4,
150.8, 130.9, 130.2, 129.6, 127.6, 43.0. LRMS: m/z (calcd for C17H23N7, 324.25; found 323.75).
2,2'-Bis(phenylcarboxymethylene)carbonimidic dihydrazide hydrochloride (77)
Synthesized according to General Method B from phenylglycoxylic acid to afford the title
compound as an offꢀwhite crystalline solid (13%).
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M.P. 306°C (Decomp.). H NMR (DMSOꢀd6) δ 14.09 (s, 1H), 8.12 (d, J = 6.9 Hz, 2H), 7.97
(dd, J = 6.4, 3.0 Hz, 2H), 7.56 (ddd, J = 8.2, 6.7, 2.9 Hz, 7H). 13C NMR (DMSOꢀd6) δ 159.4, 151.3,
149.5, 143.0, 132.0, 131.9, 131.2, 130.2, 129.4, 128.4, 128.3, 128.2. LRMS: m/z (calcd for
C17H15N5O4, 353.11; found 351.75).
N',2-Bis((E)-1-(p-tolyl)ethylidene)hydrazine-1-carboximidhydrazide hydrochloride (78)
Synthesized according to General Method A from 1ꢀ(pꢀtolyl)ethanꢀ1ꢀone to afford the title
compound as an offꢀwhite crystalline solid (71%).
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M.P. 288°C (Decomp.). H NMR (DMSOꢀd6) δ 11.66 (s, 1H), 8.61 (s, 1H), 7.94 (d, J = 8.2 Hz,
2H), 7.25 (d, J = 8.1 Hz, 2H), 2.41 (s, 3H), 2.35 (s, 3H). 13C NMR (DMSOꢀd6) δ 154.0, 153.3,
139.7, 133.9, 129.0, 127.0, 20.9, 14.9.
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2,2'-Bis{1-[4-(t-butyl)phenyl]ethylidene}carbonimidic dihydrazide hydrochloride (79)
Synthesized according to General Method A from 4′ꢀ(tertꢀbutyl)acetophenone to afford the title
compound as a yellow crystalline solid.
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M.P. 310°C (Decomp.). H NMR (DMSOꢀd6) δ 11.74 (s, 1H), 8.60 (s, 1H), 7.95 (d, J = 8.6 Hz,
2H), 7.45 (d, J = 8.6 Hz, 2H), 2.42 (s, 3H), 1.31 (s, 9H). 13C NMR (DMSOꢀd6) δ 154.1, 153.3,
152.6, 134.0, 126.8, 125.0, 34.5, 30.9, 14.9.
2,2'-Bis[1-(4-piperazinylphenyl)ethylidene]carbonimidic dihydrazide hydrochloride (80)
Synthesized according to General Method A from 1ꢀ(4ꢀ(piperazinꢀ1ꢀyl)phenyl)ethanꢀ1ꢀone to
afford the title compound as a mustardꢀyellow solid (1%).
1H NMR (400 MHz, DMSOꢀd6) δ 7.73 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.9 Hz, 2H), 3.22 – 3.13
(m, 4H), 2.99 – 2.86 (m, 4H), 2.24 (s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ 156.9, 151.1, 147.7,
129.5, 126.8, 114.7, 47.9, 44.8, 13.3.
AUTHOR INFORMATION
Corresponding Authors:
* Professor Adam McCluskey, Email: Adam.McCluskey@newcastle.edu.au, Phone: (02) 4921
6486, Fax: (02) 4921 5472
* Associate Professor Darren Trott, Email: Darren.Trott@adelaide.edu.au, Phone: (08) 8521
2989, Fax: (08) 8313 7956.
Author Contributions:
¶ Rebecca J Abraham and Andrew J Stevens contributed equally
Acknowledgements
This research was supported by a Linkage Project grant from the Australian Research Council in
collaboration with Neoculi Pty. Ltd (LP110200770). We gratefully acknowledge the technical
assistance of Dr Jane Owens, Dr Steven Polyak, Geraldine LavenꢀLaw and Steven Rutherford in the
initial phases of this study.
Associated Content
Supporting Information Available: Experimental procedures, 1H and 13CNMR spectra for
compounds 1ꢀ80 and supplementary compounds S1ꢀS25. Details of the MRSA clinical isolates:
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Journal of Medicinal Chemistry
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clone/isolate name, type, source, multiꢀlocus sequence type (MLST), staphylococcal cassette
chromosome (SCCmec) type, clonal complex, PantonꢀValentine leukocidin status (PVL), and spa
type for isolates used in the present study. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Abbreviations
MIC: Minimum inhibitory concentration; MIC50: the drug concentration resulting in the growth
inhibition of the upper 50 percentile of a bacteria population; MIC90: the drug concentration
resulting in the growth inhibition of the upper 90 percentile of a bacteria population; MBC:
minimum bactericidal concentration; PMBN: polymyxin B nonapeptide; MRSA: methicillinꢀ
resistant Staphylococcus aureus; MSSA: methicillinꢀsensitive Staphylococcus aureus; VRE:
vancomycinꢀresistant Enterococcus; CLint: Intrinsic clearance rate; EH: microsomal predicted
hepatic extraction; ESKAPE: Enterococcus faecium, Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species;
ATCC: American Type Culture Collection
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