Robenidine Analogues as Gram-Positive Antibacterial Agents

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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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Journal of Medicinal Chemistry

Robenidine analogues as Gram-positive antibacterial agents 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

1 ACS Paragon Plus Environment


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Abstract 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 4Cl isosteres with 4-F (8), 3-F (9), 3-CH3 (22), 4-C(CH3)3 (27) (23.7-71 µM) and 3-Cl (3), 4-CH3 (21), 4-CH(CH3)2 (26) (8.1-13.0 µM). Imine carbon alkylation identified a methyl/ethyl binding pocket which also accommodated a CH2OH moiety (75; 2,2'-bis[1-(4-chlorophenyl)-2hydroxyethylidene]carbonimidic dihydrazide). Analogues 1, 27 (2,2'-bis{[4-(1,1dimethylethyl)phenyl]methylene}carbonimidic dihydrazide) and 69 (2,2'-bis[1-(4chlorophenyl)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 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 T1/2 > 247 min, CLint 247min; EH < 0.22; CLint < 7µL/min/mg protein

MRSA



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Figure 1. Chemical structure of Robenidine (1, NCL812).

Figure 2: Time-kill assay of (A) 1 (Robenidine), (B) 27 and (C) 69; against methicillin-sensitive (left) and methicillin-resistant (right) strains of S. aureus. Cultures were inoculated with 106 CFU/mL bacteria and exposed to increasing concentrations of compounds for 8 hours. Samples were taken at 4 and 8 hours to determine viable cell numbers. Key: closed circles (•) – control, open circles (○) - MIC, closed diamonds (♦) – 2× MIC, open diamonds (◊) – 4× MIC, closed triangles () - 8× MIC and grey broken line – detection limit. Error ± SD.


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Scheme 1. Reagents and conditions: (i) 1,3-diaminoguanidine hydrochloride, EtOH, reflux, 16 h.

Scheme 2. Reagents and conditions: (i) N-substituted isocyanate or O-substituted chloroformate, iPrNEt2, CH3CN, reflux, 2.5 h (for full detail of ‘R’ see supplementary data).

Scheme 3. Reagents and conditions: (i) 1,3-diaminoguanidine hydrochloride, EtOH, reflux, 16 h.



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Table 1. The inhibition of MRSA, VRE, E. coli and P. aeruginosa growth by 1,3-aminoguanidine Schiff base analogues possessing mono-substituted aromatic rings (1 – 31).

MIC50 (µM)a Compound

R

MRSA

VRE

E. coli

Ampicillin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

4-Cl H 3-Cl 2-Cl 4-Br 3-Br 2-Br 4-F 3-F 2-F 4-CF3 4-OH 3-OH 2-OH 4-OCH3 3-OCH3 2-OCH3 4-OCF3 4-SCH3 4-SCF3 4-CH3 3-CH3 2-CH3 4-(CH2)2CH3 4-(CH2)3CH3 4-CH(CH3)2 4-C(CH3)3 4-N(CH3)2 4-Ph 2-Ph 4-CCH

183 8.1 8.1 47 47 36.5 192 192 36.0 88 187 61 96 9.1 24.4 121 83 232 9.1 24.2 30.9 282 -

11.5 4.7 10.8 15.4 23.7 71 11.4 288 288 240 88 358 85 12.7 135 12.1 24.4 61 73 193 13.0 29.0 30.9 106 17.2

45.8 -b -

P. aeruginosa 366 -

-

-

a

MIC50 = drug concentration resulting in growth inhibition of the upper 50 percentile of a bacteria population; b ‘-’ = Inactive, IC50 > 400 µM.


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Table 2. The inhibition of MRSA, VRE, E. coli and P. aeruginosa growth by 1,3-diaminoguanidine Schiff base analogues possessing di-, tri- and poly-substituted aromatic rings (32 - 57).

Compound

R

32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

2,5-F 2-F, 4-Cl 3,4-F 2,4-Cl 2,6-Cl 3,5-Cl 2-NH2, 4-Cl 2-NHCH(OH)CH3, 4-Cl 2-NHCOCH3, 4-Cl 2-OH, 3-CH3 2-OH, 4-Cl 2-OH, 4-N(CH3)2 2,3-OH 2,4-OH 3,4-OH 3-OH, 4-OCH3 3-OCH3, 4-OH 3,4-OCH3 2,3,4-OH 2,4,5-OH 3,4,5-OH 3-OCH3, 4,5-OH 3-NO2, 4-OH 2,3,4,5,6-F 2-Br, 4,5-OCH3 3-Br, 4,5-OCH3

MRSA -b 6.1 40.1 5.0 66 80 229 88 244 161b 161 161 -

VRE 7.4 10.7 6.3 98 45 76b 262 163 161b 322 -

MIC50 (µM)a E. coli -

P. aeruginosa 322 -

a

MIC50 = drug concentration resulting in growth inhibition of the upper 50 percentile of a bacteria population; b ‘-’ = Inactive, IC50 > 400 µM; b active against only one of the two strains examined within either MRSA or VRE.



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Table 3. Inhibition of MRSA and VRE by 1,3-diaminoguanidine Schiff bases bearing extended linkers, non-aromatic and isosteric phenyl ring replacements (58 – 67). MIC50 (µM)a R

MRSA

VRE

58

408

306

59

-b

-

60

172

86c

61

-

-

62

-

-

63

309

-

64

61c

-

65

239

159c

66

31.6

42

67

-

33.1c

a

MIC50 = drug concentration resulting in growth inhibition of the upper 50 percentile of a bacteria population; b ‘-’ = Inactive, IC50 > 400 µM; c active against only one of the two strains examined within either MRSA or VRE.


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Table 4. Inhibition of MRSA and VRE by 1,3-diaminoguanidine Schiff base analogues (68 – 80) bearing imine substitution.

MIC50 (µM)a MRSA VRE CH3 H 18.2 49 68 CH Cl 7.5 3.8 69 3 Br 16.4 24.6 CH3 70 CH3 CF3 -b 17.2 71 CH2CH3 Cl 56 18.7 72 Cl (CH2)2CH3 73 (CH2)3CH3 Cl 74 CH2OH Cl 20.3 15.2 75 CH2NH2 H 76 COOH H 77 CH3 CH3 11.2 8.4 78 C(CH3)3 4.5 36.2 CH3 79 CH3 piperazin-1-yl 243 278 80 a MIC50 = drug concentration resulting in growth inhibition of the upper 50 percentile of a bacteria population; b ‘-’ = Inactive, IC50 > 400 µM. Compound

R

R′



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Table 5. MIC90 and MBC concentrations for 1, 27, 69 and ampicillin against 20 clinically isolated MRSA and 4 ATCC MSSA bacterial strains. MIC90 range a 1 27 69 Ampicillin

5.4 4.8 – 38.6 1.3 – 5.0 0.3 – 143

S. aureus (n = 24) 2% Serum b 50% Serum

43.1 38.6 10.0 -c

µM >250 >250 >250 -

MBC

MIC:MBC

5.4 – 10.8 4.8 – 38.6 2.5 – 5.0 2.0 – 143

1 (96%) 1 (87.5%) 1 (87.5%) -

Range a

a MIC90 = drug concentration resulting in growth inhibition of the upper 90 percentile of a bacteria population; b MIC90 in serum determined using S. aureus ATCC 29213 only; c ‘-’ = not determined

Table 6. MIC90 for 1, 27 69 and clofazimine against E. coli ATCC 25922 and P. aeruginosa ATCC 27853. E. coli 25922 MIC90 a 1 27 69 Clofazimine

>250 >250 >250 >250

+PMBNb µM 21.6 >250 10.0 4.2-8.4

P. aeruginosa 27853 MIC90 a +PMBN >250 >250 >250 >250

21.6 >250 5.0 8.4

a

MIC90 = drug concentration resulting in growth inhibition of the upper 90 percentile of a bacteria population; b PMBN – polymyxin B nonapeptide


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Table 7. Physicochemical and metabolism results for NCL812 (1), NCL099 (27) and NCL177 (75). Physicochemical Parameters

Compound MW

PSA (Å2)

FRB

Metabolism Parameters

HBA

HBD

1

334.20

72.6

4

3

3

27

337.54

72.6

6

3

3

75

394.26

113.1

6

5

5

pKa

5.0 – imine 1.2 – amine 5.1 - imine 2.4 – amine 5.1 – imine

LogD pH 3.0

Solubility (µg/mL) pH 2.0 pH 6.5

pH 7.4

Species

3.7

4.5

6.3 - 12.5

5.3

247a 131

CLint (µL/min/mg protein)

Robenidine Analogues as Gram-Positive Antibacterial Agents

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