Novel guanidine compound against multidrug-resistant cystic fibrosis-associated bacterial species

Article abstract of DOI:10.3390/molecules23051158

Chronic pulmonary infection is a hallmark of lung disease in cystic fibrosis (CF). Infections dominated by non-fermentative Gram-negative bacilli are particularly difficult to treat and highlight an urgent need for the development of new class of agents to combat these infections. In this work, a small library comprising thiourea and guanidine derivatives with low molecular weight was designed; these derivatives were studied as antimicrobial agents against Gram-positive, Gram-negative, and a panel of drug-resistant clinical isolates recovered from patients with CF. One novel compound, a guanidine derivative bearing adamantane-1-carbonyl and 2-bromo-4,6-difluouro-phenyl substituents (H-BDF), showed potent bactericidal activity against the strains tested, at levels generally higher than those exhibited by tobramycin, ceftazimide and meropenem. The role that different substituents exert in the antimicrobial activity has been determined, highlighting the importance of the halo-phenyl group in the guanidine moiety. The new compound displays low levels of cytotoxicity against THP-1 and A549 cells with a selective index (SI) > 8 (patent application PCT/IB2017/054870, August 2017). Taken together, our results indicate that H-BDF can be considered as a promising antimicrobial agent.

Full text of DOI:10.3390/molecules23051158

molecules
Article
Novel Guanidine Compound against
Multidrug-Resistant Cystic Fibrosis-Associated
Bacterial Species
Aamer Saeed 1,
*
ID , Alejandra Bosch 2, Marisa Bettiol 3, Diana L. Nossa González 4,
Mauricio Federico Erben 4,
*
and Yanina Lamberti 2,
*
ID
1
Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
CINDEFI (UNLP, CONICET-CCT La Plata), Departamento de Química, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, La Plata 1900, Argentina; bosch@quimica.unlp.edu.ar
Sala de Microbiología, Hospital de Niños Sor María Ludovica, La Plata 1900, Argentina;
microbiologialudovica@gmail.com
CEQUINOR (UNLP, CONICET-CCT La Plata), Departamento de Química, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, La Plata 1900, Argentina; diana.nossa@uptc.edu.co
Correspondence: asaeed@qau.edu.pk (A.S.); erben@quimica.unlp.edu.ar (M.F.E.);
ylamberti@quimica.unlp.edu.ar (Y.L.)
2
3
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 21 April 2018; Accepted: 8 May 2018; Published: 11 May 2018
Abstract: Chronic pulmonary infection is a hallmark of lung disease in cystic fibrosis (CF).
Infections dominated by non-fermentative Gram-negative bacilli are particularly difficult to
treat and highlight an urgent need for the development of new class of agents to combat
these infections. In this work, a small library comprising thiourea and guanidine derivatives
with low molecular weight was designed; these derivatives were studied as antimicrobial agents
against Gram-positive, Gram-negative, and a panel of drug-resistant clinical isolates recovered from
patients with CF. One novel compound, a guanidine derivative bearing adamantane-1-carbonyl and
2-bromo-4,6-difluouro-phenyl substituents (H-BDF), showed potent bactericidal activity against
the strains tested, at levels generally higher than those exhibited by tobramycin, ceftazimide
and meropenem. The role that different substituents exert in the antimicrobial activity has been
determined, highlighting the importance of the halo-phenyl group in the guanidine moiety. The new
compound displays low levels of cytotoxicity against THP-1 and A549 cells with a selective index (SI)
> 8 (patent application PCT/IB2017/054870, August 2017). Taken together, our results indicate that
H-BDF can be considered as a promising antimicrobial agent.
Keywords: antimicrobials; thioureas; guanidines; drug-resistant; cystic fibrosis
1. Introduction
In recent years, increasing infections due to antibiotic-resistant pathogens have made the formerly
routine therapy of many infectious diseases challenging, and in many instances, extremely difficult or
impossible to be eradicated [
1
–3]. Multidrug resistance is specially associated with respiratory tract
infection in cystic fibrosis (CF) [
4
] where opportunistic pathogens such as Pseudomonas aeruginosa,
Staphylococcus aureus, Stenotrophomonas maltophilia and species of the Burkholderia cepacia complex (Bcc)
infect patient’s lung and airways. Although for some patients the infection may occur only transiently,
their acquisition most typically results in a chronic infection with acute debilitating exacerbations,
causing a severe decline in respiratory function which contributes to disease progression and premature
mortality [
5,6]. In addition, they are important nosocomial pathogens affecting both immunocompetent
and immunocompromised patients, and are responsible for a considerable proportion of infections in
Molecules 2018, 23, 1158; doi:10.3390/molecules23051158
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1158
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patients in Intensive Care Units (ICUs) worldwide [
7]. Despite the emergence and dissemination of
resistant bacteria and the need of more effective therapies, the development of new antimicrobial agents
against these life-threatening infections is declining [ ]. The impermeable nature of Gram-negative
8
bacteria envelope, and the presence of multiple efflux pumps, in combination with other resistance
mechanisms, has made the discovery of new effective antibacterial drugs very difficult [9].
Thioureas as well as guanidines represent two important groups of compounds due to their wide
range of application as pharmaceutical agents. They possess a broad biological activity range including
anti-inflammatory, anticancer, antiviral, antiparasitic, antifungal and antimicrobial properties [10,11].
Such a diverse range of biochemical behavior can be attributed to their flexible structure and the
presence of nitrogen atoms in these molecules that make it possible to bear various substituents.
For instance, it is well known that the 1-aroyl-3-(substituted-2-benzothiazolyl) thioureas exhibit
potent antibacterial activity [12]. In addition, 1-(benzoyl)-3-(substituted) thioureas are antimicrobial
agents [13] and the fluorinated analogues exhibit good antifungal activity [14]. Furthermore, due to
efficient resonance stabilization of the charged protonated state, the guanidine groups have a relatively
high acid dissociation constant which makes them stronger bases better suited for stable electrostatic
interaction with the negative charged membranes of bacteria. This property improves the penetration
of guanidine-bearing compounds through membranes and thus their biological activity [15,16]. On the
other hand, the introduction of fluorine or appropriate fluorinated groups into organic compounds
has advanced over recent decades in medicinal chemistry. The incorporation of fluorine atoms may
contribute to increase metabolic stability, binding affinity and lipid solubility, thereby enhancing
rates of absorption and transport of drugs in vivo [17,18]. Several studies further indicated that the
incorporation of fluor and/or different electron withdrawing groups, such as bromo, chloro, acetyl,
and nitro groups, on aromatic rings results in an improvement in antibacterial activity [16,19–21].
Taking into account the aforesaid biological and synthetic significance of thioureas and guanidines
on one hand, and the multifunctional value of the electron withdrawing groups in drug design on
the other, the endeavor of the current work was to investigate the activity of newly synthesized
halophenyl substituted thioureas and guanidines against drug-resistant clinical isolates recovered
from patients with CF.
2. Materials and Methods
2.1. Reagents and Equipment
1-adamantane carboxylic acid, thionyl chloride, triethylamine, potassium thiocyanate, mercury(II)
chloride and substituted anilines were commercial products (Sigma-Aldrich, St. Louis, MO, USA)
and were used as received. Analytical grade (Merck, Kenilworth, NJ, USA) acetone and dimethyl
formamide, DMF, were dried and freshly distilled prior to use.
Melting points were recorded using a digital Gallenkamp (SANYO, Moriguchi, Japan) model
MPD.BM 3.5 apparatus and are uncorrected. 1H and 13C NMR spectra were determined in CDCl3 at 300
MHz and 75.4 MHz, respectively, using a Bruker spectrophotometer (Billerica, Middlesex, MA, USA).
FTIR spectra were acquired by a FTS 3000 MX spectrometer. Elemental analyses were conducted using
a LECO-183 CHNS analyzer (LECO Corporation, MI, USA). Thin layer chromatography (TLC) was
carried out on 0.25 mm silica gel plates (60 F254, Merck, Darmstadt, Germany). Visualization was
achieved by ultraviolet light.
2.2. Synthesis of Compounds
Thirteen compounds were synthesized and their structures were confirmed by a
combination of elemental analysis, infrared and nuclear magnetic resonance spectroscopy.
1-(Adamantane-1-carbonyl)-3-substituted thiourea compounds were prepared by the addition reaction
between adamantyl isothiocyanate with a variety of suitably substituted anilines [22
–25]. The starting
material 1-adamantane carbonyl chloride was obtained via the reaction of 1-adamantane carboxylic

Molecules 2018, 23, 1158
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acid with thionyl chloride. A solution of adamantane-1-carbonyl chloride in dry acetone
was treated with an equimolar quantity of potassium thiocyanate in dry acetone to yield the
adamantane-1-carbonyl-isothiocyanate as intermediate (Figure 1). A treatment of the latter with
an equimolar quantity of cyclohexylamine (for compound
1, Table 1) and a variety of substituted
anilines (compounds , Table 1) in acetone produced the thiourea derivatives. In a typical procedure,
2–7
a freshly distilled solution of adamantane-1-carbonyl chloride (10 mmol) in dry acetone (50 mL) was
added dropwise to a suspension of potassium thiocyanate (10 mmol) in acetone (30 mL) and the
reaction mixture was refluxed for 30 min under nitrogen. After cooling to room temperature, a solution
of the substituted aniline (10 mmol) in acetone (10 mL) was added and the resulting mixture refluxed
for 2–4 h. The reaction mixture was poured into cold water and the precipitated thioureas were
recrystallized from suitable solvents.
Figure 1. Synthesis of acyl thiourea and guanidine derivatives. Reagents and conditions: (i) Acyl
chlorides and KSCN in dry acetone, 2 h, reflux. (ii) Primary amines in dry acetone. (iii) HgCl2,
substituted aniline and Et3N in dry DMF.
Three 1-acyl-3-(2-bromo-4,6-difluoro-phenyl)thioureas (compounds
8–10, Table 1) were
synthesized in a similar way by treating the corresponding acyl chloride derivatives (1-naphthoyl
chloride, 2,4-dichloro-benzoyl chloride and 4-methyl-benzoyl chloride, respectively) with potassium
thiocyanate in dry acetone followed by the addition of 2-bromo-4,6-difluoro-aniline.
For the synthesis of guanidine derivatives (compounds 11–13, Figure 2), the general method
proposed by Vencato and coworkers [26] was applied (Figure 1). In a typical procedure triethylamine
(2.8 mL, 20 mmol) and selected anilines (10 mmol) were added successively to a stirred solution of
the corresponding 1-(adamantane-1-carbonyl)thiourea (10 mmol) in DMF (20 mL) at 10 ◦C followed
by the addition of mercury(II) chloride (2.72 g, 10 mmol). The reaction mixture was stirred at room
temperature for 12 h and then filtered to remove the HgS. The filtrate was extracted with EtOAc/H2O
(1:1) (3
×
5 mL), the organic phase dried over anhydrous Na2SO4, and concentrated in vacuum to
leave an oily residue which recrystallized on standing.
1-(Adamantane-1-carbonyl)-3-cyclohexylthiourea ). Yield 68%, semisolid; FT-IR (KBr,
3336 (NH), 3034 (Ar-CH), 2926 (CH2), 2909, 2849 (CH2, CH), 1675 (C=O), 1575, 1457, 1370 (C=S).
1H NMR (300 MHz, CDCl3):
13.08 (br s, 1H, NH, D2O exchangeable); 6.25 (1H, s, broad, NH);
4.09 (br s, 1H, NH, D2O exchangeable); 3.94 (1H, m, CH), 2.1 (br s, 3H, adamantane-CH), 1.95 (s, 6H,
adamantane-CH2), 1.94–2.02 (2H, dd, CH2), 1.60–1.76 (4H, m, CH2 2), 1.79 (m, 6H, adamantane-CH2)
(1
ν
, cm−1):
δ
×
1.18–1.45 (4H, m, CH2 × 2); 13C NMR (75 MHz, CDCl3): 179.1 (C=S); 178.46 (C=O), 54.37 (CH), 41.98,
41.90, 39.2, 38.5, 36.4, 36.0, 33.03 (CH2-4), 32.81 (CH2-2), 31.6, 28.0, 24.75 (CH2-3), 27.7, (adamantane-C)
25.41 (CH2-3), 24.75 (CH2-3); Anal. Calcd for C18H28N2OS (320.19): C, 67.46; H, 8.81; N, 8.74; S, 10.00%;
Found: C, 67.46; H, 8.81; N, 8.74; S, 10.00%.
1-(Adamantane-1-carbonyl)-3-phenylthiourea
3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1679 (C=O), 1575, 1457, 1375 (C=S); 1H NMR (300 MHz,
CDCl3): 12.71 (br s, 1H, NH, D2O exchangeable); 7.63 (br s, 1H, NH, D2O exchangeable); 7.23–7.33
(2 ν
). Yield 72%, mp 108–110 ◦C. FT-IR (KBr, , cm−1):
δ
(m, 2H, Ar); 7.38–7.43 (m, 2H, Ar), 8.40–8.48 (m, 1H, Ar); 2.08 (s, 3H, adamantane-CH), 1.69 (s, 6H,
adamantane-CH2), 1.58 (q, 6H, adamantane-CH2, J = 8.6 Hz); 13C NMR (75 MHz, CDCl3): 179.6 (C=S);
170.12 (C=O); 143.05 (C-9); 41.51, 39.25, 38.69, 38.49, 36.44, 36.14, 28.05, 27.86, 27.78, (adamantane-C);
Anal. Calcd for C18H22N2OS (314.45): C, 68.75; H, 7.05; N, 8.91; S, 10.20%; Found: C, 68.83; H, 7.10;
N, 8.98; S, 10.14%.

Molecules 2018, 23, 1158
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◦
1-(Adamantane-1-carbonyl)-3-(4-methyl-3-fluorophenyl)thiourea
(3
). Yield 69%, mp 174–176 C.
FT-IR (ν δ 12.47
, cm−1): 3436, 3034, 2909, 1675, 1585, 1457, 1368. 1H NMR (300 MHz, CDCl3):
(br s, 1H, NH, D2O exchangeable); 8.53 (br s, 1H, NH, D2O exchangeable); 7.19 (s, 1H, Ar), 7.59 (s,
1H, Ar), 7.81(d, 2H, J = 8.6 Hz, Ar), 2.37 (s, 3H, Ar-CH3) 2.14 (brs, 3H, adamantane-CH), 1.95 (s, 6H,
adamantane-CH2), 1.79 (q, 6H, adamantane-CH2, J = 8.6 Hz); 13C NMR (75 MHz, CDCl3): 178.9 (C=S),
177.1 (C=O), 161.7 (Ar), 136.7 (Ar), 135.1 (Ar), 136.7 (Ar), 129.7, 141.4, 124.2 (ArCs), 21.2 (Ar-CH3) 38.44,
36.14, 27.86, 21.78, (adamantane-C); Anal. Calcd for C19H23FN2OS (346.15): C, 65.87; H, 6.69; N, 8.09;
S, 9.25%; Found: C, 65.739; H, 6.72; N, 7.97; S, 9.23%.
◦
1-(Adamantane-1-carbonyl)-3-(2-nitrophenyl)thiourea
ν
(
4
). Yield 73%, mp 160–162 C. FT-IR (KBr,
, cm−1): 3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1682 (C=O), 1586, 1543 (NO2 asymmetric)
1457, 1368 (C=S), 1340 cm-1 (NO2 symmetric); 1H NMR (300 MHz, CDCl3):
12.71 (br s, 1H, NH, D2O
δ
exchangeable); 7.63 (br s, 1H, NH, D2O exchangeable); 7.23–7.33 (m, 2H, Ar); 7.38–7.43 (m, 2H, Ar);
2.08 (s, 3H, adamantane-CH), 1.69 (s, 6H, adamantane-CH2), 1.58 (q, 6H, adamantane-CH2, J = 8.6 Hz);
13C NMR (75 MHz, CDCl3): 179.6 (C=S); 170.12 (C=O); 143.05 (C-9); 41.51, 39.25, 38.69, 38.49, 36.44,
36.14, 28.05, 27.86, 27.78, (adamantane-C); Anal. Calcd for C18H21N3O3S (359.44): C, 60.15; H, 5.89;
N, 11.69; O, 13.35; S, 8.92%; Found: C, 60.21; H, 5.93; N, 11.71; S, 8.89%.
1-(Adamantane-1-carbonyl)-3-(4-acetyl-phenyl)thiourea
3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1679 (C=O), 1575, 1457, 1375 (C=S). 1H NMR (300 MHz,
CDCl3): 12.74 (br s, 1H, NH, D2O exchangeable), 9.83 (br s, 1H, NH, D2O exchangeable), 7.91 (d, 2H,
J= 8.6 Hz, Ar); 7.73 (d, 2H, J = 8.6 Hz, Ar), 2.3 (s, 3H, CH3CO), 2.08 (s, 3H, adamantane-CH), 1.69 (s, 6H,
adamantane-CH2), 1.58 (q, 6H, adamantane-CH2, J = 8.6 Hz); 13C NMR (75 MHz, CDCl3):
193.6 (CO),
(5 ν
). Yield 160–161 ◦C. FT-IR (KBr, , cm−1):
δ
δ
179.6 (C=S), 174.5 (C=O), 143.0, 138.0, 132.6, 127.8, 28.1 (CH3), 41.51, 39.25, 38.69, 38.49, 36.44, 36.14,
28.05, 27.86, 27.78, (adamantane-C); Anal. Calcd for C20H24N2O2S (356.47): C, 67.39; H, 6.79; N, 7.86;
S, 8.99%; Found: C, 67.42; H, 6.83; N, 7.81; S, 8.91%.
◦
1-(Adamantane-1-carbonyl)-3-(2,3-dichlorophenyl)thiourea
(KBr,
1H NMR (300 MHz, CDCl3):
(
6
). Yield 79%, mp 196–198 C. FT-IR
ν
, cm−1): 3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1675 (C=O), 1575, 1457, 1370 (C=S).
δ
12.74 (br s, 1H, NH, D2O exchangeable); 8.70 (br s, 1H, NH, D2O
exchangeable); 8.03 (d, 1H, J = 8.6 Hz Ar), 7.96 (d, 1H, J = 8.6 Hz Ar), 7.90 (d, 1H, J = 8.6 Hz Ar), 7.83 (d,
1H, J = 8.6 Hz Ar), 7.57 (m, 3H, Ar), 2.1 (br s, 3H, adamantane-CH), 2.03 (s, 6H, adamantane-CH2),
1.81 (q, 6H, adamantane-CH2, J = 8.6 Hz); 13C NMR (75 MHz, CDCl3): 178.9 (C=S); 134.10 (Ar), 128.6,
126.9 125.3,123.64, 121.67 (ArCs), 41.94, 41.90, 39.2, 38.6, 36.1, 36.0, 31.6, 28.0, 27.8, (adamantane-C);
Anal. Calcd for C18H2o Cl2N2OS (383.34): C, 56.40; H, 5.26; N, 7.31; S, 8.36%; Found: C, 56.40; H, 5.26;
N, 7.31; S, 8.36%.
1-(Adamantane-1-carbonyl)-3-(2-bromo-4,6-difluorophenyl)thiourea
FT-IR (KBr,
, cm−1): 3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1675 (C=O), 1575, 1457, 1370
(C=S). 1H NMR (300 MHz, CDCl3):
11.93 (br s, 1H, NH, D2O exchangeable), 9.61 (br s, 1H, NH, D2O
(7
). Yield 70%, mp 194–196 ◦C.
ν
δ
exchangeable), 7.48–7.44 (m, 1H, Ar), 7.29-7.22 (m, 1H, Ar), 2.08 (t, 10H, adamantane-H, J = 6.0 Hz),
1.80 (t, 6H, adamantane-H, J = 4.8 Hz); 13C NMR (75 MHz, CDCl3): 182.3 (C=S), 179.2 (C=O), 163.3,
160.6, 159.8, 157.2, 123.9, 115.8, 104.5, 103.8 (ArCs), 41.9, 37.6, 35.8, (adamantane-C); Anal. Calcd for
C18H19F2BrN2OS (429.32): C, 50.36; H, 4.46; N, 6.53; S, 7.47; Found: C, 50.24; H, 4.51; N, 6.57; S, 7.36%.
◦
1-(1-naphtyl)-3-(2-bromo-4,6-difluoro-phenyl)thiourea
ν
(
8
). Yield 81%, mp 174–176 C. FT-IR (KBr,
, cm−1): 3336 (NH), 3034 (Ar-CH), 1671 (C=O), 1585, 1451, 1372 (C=S). 1H NMR (300 MHz, CDCl3):
δ
11.98 (br s, 1H, NH, D2O exchangeable); 11.29 (br s, 1H, NH, D2O exchangeable); 8.89–6.71 (m, 9H,
Ar); 13C NMR (75 MHz, CDCl3): 168.9, 164.3, 145.1, 132.0, 134, 120.4, 125.1, 129.6, 116, 103.1 (ArCs);
Anal. Calcd for C18H11 F2BrN2OS (421.97): C, 51.32; H, 2.63; N, 6.65 S, 7.61%; Found: C, 51.24; H, 2.60;
N, S, 6.61, 7.57%.
◦
1-(2,4-dichloro-phenyl)-3-(2-bromo-4,6-difluoro-phenyl)thiourea
FT-IR (KBr,
(
9
). Yield 81%, mp 174–176 C.
ν
, cm−1): 3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1675 (C=O), 1575, 1457,

Molecules 2018, 23, 1158
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1370 (C=S). 1H NMR (300 MHz, CDCl3):
δ
12.07 (br s, 1H, NH, D2O exchangeable); 11.35 (br s, 1H,
NH, D2O exchangeable); 7.61 (s, 1H, Ar), 7.58 (d, 1H, J = 8.3 Hz, Ar), 7.58 (d, 1H, J = 8.3 Hz, Ar), 7.49 (s,
1H, Ar), 7.19 (s, 1H, Ar); 13C NMR (75 MHz, CDCl3): 181.9 (C=S); 170.3 (C=O), 168.2, 159.5, 134.10
(Ar), 141.4, 134.1, 130.1, 129.4, 127.3, 128.6, 126.9, 124.2, 119.7, 114.9 (ArCs); Anal. Calcd for C14H17 Cl2
F2BrN2OS (439.88): C, 38.21; H, 1.60; N, 6.37; S, 7.28%; Found: C, 37.28; H, 1.62; N, 6.33; S, 8.30%.
1-(4-methylphenyl)-3-(2-bromo-4,6-difluoro-phenyl)thiourea
(KBr,
, cm−1): 3436 (NH), 3034 (Ar-CH), 2909, 1675 (C=O), 1585, 1457, 1368 (C=S). 1H NMR (300 MHz,
CDCl3): 12.74 (br s, 1H, NH, D2O exchangeable); 11.31 (br s, 1H, NH, D2O exchangeable); 7.19 (s,
(
10). Yield 69%, mp 174–176 ◦C. FT-IR
ν
δ
1H, Ar), 7.59 (s, 1H, Ar), 7.81 (d, 2H, J = 8.6 Hz, Ar), 2.51 (s, 3H, Ar-CH3); 13C NMR (75 MHz, CDCl3):
178.9 (C=S), 173.1 (C=O), 134.10 (Ar), 181.7, 141.4, 130.1, 128.6, 126.9, 124.2, 119.7, 114.9 (ArCs), 19.4
(Ar-CH3); Anal. Calcd for C15H11 F2BrN2OS (385.97): C, 46.77; H, 2.88; N, 7.27; S, 8.32%; Found: C,
46.81; H, 2.92; N, 7.23; S, 8.28%.
1-(Adam◦antane-1-carbonyl)-2,3-bis(2-bromo-4,6-difluoro-phenyl)guanidine
(11). Yield 70%, mp
148–149 C. FT-IR (KBr,
ν
, cm−1): 3336, 3413, 3245, 3128, 3043, 3034, 2909, 2849, 1675, 1575, 1457,
1
1370. H NMR (300 MHz, CDCl3):
δ 9.79 (br s, 1H, NH, D2O exchangeable); 8.04 (br s, 1H, NH, D2O
exchangeable); 7.17–7.13 (m, 2H, Ar), 7.06–6.98 (m, 2H, Ar), 2.0 (br s, 3H, adamantane-H), 1.94–1.89
(br m, 3H, adamantane-H), 1.78–1.60 (br m, 10H, adamantane-H); 13C NMR (75 MHz, CDCl3): 178.2
(C=O), 174.2 (C=N), 154.9, 151.8, 148.6, 131.9, 114.6, 107.8, 103.2 (ArCs), 40.9, 37.9, 35.8, (adamantane-C);
Anal. Calcd for C24H21F4Br2N3O (603.2): C, 47.78; H, 3.51; N, 6.97%; Found: C, 48.1; H, 3.49; N, 7.01%.
1-(Adamantane-1-carbonyl)-2-(2-bromo-4,6-difluoro-phenyl)-3(2,6-di-bromo-4-fluoro-phenyl)
guanidine
(
12). Yield 70%, mp 144–145 ◦C. FT-IR (KBr,
ν
, cm−1): 3413, 3245, 3128, 3043, 3034, 2909,
δ 11.94 (br s, 1H, NH, D2O exchangeable);
1
2849, 1675, 1575, 1457, 1370. H NMR (300 MHz, CDCl3):
9.66 (br s, 1H, NH, D2O exchangeable); 7.48 (m, 1H, Ar), 7.23 (m, 2H, Ar), 7.01 (m, 1H, Ar), 1.99–1.84
(m, 10H, adamantane-H), 1.79–1.59 (m, 6H, adamantane-H); 13C NMR (75 MHz, CDCl3): 179.2 (C=O),
174.2 (C=N), 160.4, 159.8, 157.2, 151.9, 147.2, 140.2, 123.9, 115.8, 114.3, 104.5 (ArCs), 41.9, 37.6, 35.8
(adamantane-C); Anal. Calcd for C24H21F3Br3N3O (664.2): C, 43.40; H, 3.19; N, 6.33%; Found: C,
43.21.1; H, 3.52; N, 6.97%.
◦
1-(Adamantane-1-carbonyl)-2,3-bis-(2-nitro-phenyl)guanidine
(KBr,
CDCl3):
(
13). Yield 70%, mp 156 C. FT-IR
1
ν
, cm−1): 3336, 3413, 3245, 3128, 3043, 3034, 2909, 2849, 1675, 1575, 1457, 1370. H NMR (300 MHz,
δ
11.94 (br s, 1H, NH, D2O exchangeable); 9.66 (br s, 1H, NH, D2O exchangeable); 7.48 (m,
1H, Ar), 7.23 (m, 2H, Ar), 7.01 (m, 1H, Ar), 1.99–1.84 (m, 10H, adamantane-H), 1.79–1.59 (m, 6H,
adamantane-H); 13C NMR (75 MHz, CDCl3): 179.2 (C=O), 174.2 (C=N), 160.4, 159.8, 157.2, 151.9, 147.2,
140.2, 123.9, 115.8, 114.3, 104.5 (ArCs), 41.9, 37.6, 35.8 (adamantane-C); Anal. Calcd for C24H25N5O5
(463.5): C, 62.19; H, 5.44; N, 15.11%; Found: C, 61.97.1; H, 5.42; N, 6.93%.
Figure 2. Chemical structure of guanidine derivative compounds 11–13. Molecular weights (g/mol)
are shown in parentheses.

Molecules 2018, 23, 1158
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Table 1. Activities of newly obtained compounds and common antibiotics used in clinical treatments against Pseudomonas aeruginosa PAO1 and Burkholderia
cenocepacia J2315.
Molecular Weight
(g/mol)
P. aeruginosa PAO1
MIC (µg/mL) MBC (µg/mL)
B. cenocepacia J2315
MIC (µg/mL) MBC (µg/mL)
Chemical
Structure
Entry
R1
R2
R3
a
a
a
a
1
2
3
4
C10H15
C6H11
320.19
314.45
385.97
359.44
>128
nd
>128
nd
C10H15
C10H15
C10H15
C6H5
-
-
-
>128
>128
>128
nd
nd
nd
>128
>128
>128
nd
nd
nd
3-F-4-CH3-C6H3
2-NO2-C6H4
a
5
C10H15
4-CH3CO-C6H4
-
356.47
>128
nd
>128
nd
a
a
6
7
C10H15
2,3-di-Cl-C6H3
-
-
383.34
428.32
>128
>128
nd
nd
>128
>128
nd
nd
C10H15
2-Br-4,6-di-F-C6H2
b
8
9
2-Br-4,6-di-F-C6H2
-
-
421.97
439.88
>128
>128
nd
nd
>128
>128
nd
nd
C10H7
2,4-di-Cl-C6H3 2-Br-4,6-di-F-C6H2

Molecules 2018, 23, 1158
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Table 1. Cont.
Molecular Weight
P. aeruginosa PAO1
MIC (µg/mL) MBC (µg/mL)
B. cenocepacia J2315
MIC (µg/mL) MBC (µg/mL)
Chemical
Structure
Entry
R1
R2
R3
(g/mol)
10
4-CH3-C6H4
2-Br-4,6-di-F-C6H2
-
385.97
>128
nd
>128
nd
a
11
C10H15
2-Br-4,6-di-F-C6H2 2-Br-4,6-di-F-C6H2
603.2
0.5
4
2
8
Tobramycin
Meropenem
Ceftazimide
467.51
383.46
546.57
2
1
2
2
4
2
>128
8
16
>128
64
128
a
1-adamantyl, b-naphthyl

Molecules 2018, 23, 1158
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2.3. Bacterial Strains
The antibacterial activity of the compounds was tested against the reference strains
Escherichia coli ATCC25922, Bordetella bronchiseptica 9.73H+ [27], Pseudomonas aeruginosa ATCC15692,
Burkholderia cenocepacia J2315, Pandorea apista DSM16535, Staphyloccocus aureus ATCC6538, Bacillus cereus
ATCC10876. A total of forty non-fermenting Gram-negative bacilli and two Methicillin-Resistant
Staphylococcus aureus (MRSA) clinical isolates collected from sputum samples of patients with
CF attended at different hospitals and CF Centers in the period 2004 to 2017 were used in
this study. They were selected from the collection of microorganisms CAMPA (Colección Argentina de
Microorganismos Patógenos y Ambientales) of CINDEFI, at the Faculty of Exact Sciences in La Plata
University [28]. All Bcc isolates were identified by PCR-recA technology (amplification, PCR-recA
RFLP HaeIII, and sequencing). Additionally hisA, gyrB, or other gene from the current multilocus
sequence typing (MLST) scheme were sequenced when the identification remained ambiguous [29
,
30].
The isolates were maintained both as lyophilized and frozen at
−
80 ◦C in Trypticase-soy broth with
10% (v/v) glycerol until further analysis.
2.4. Antimicrobial Activity Assays
The in vitro susceptibility tests (Minimal inhibitory concentration (MIC) and minimal bactericidal
concentration (MBC) tests) were determined using the micro-dilution method according to guidelines
of the Clinical and Laboratory Standards Institute (CLSI) [31]. Briefly, serial two-fold dilutions of
each compound were prepared (final volume of 50
(Sarstedt, Nümbrecht, Germany) with Mueller Hinton (MH) broth. Each dilution series included
control wells without any compound and control wells without bacteria. Then, a total of 50 L of the
adjusted inoculum (approximately 5
105 cells/mL) in MH broth was added to each well. The MIC
µL) in 96-well polypropylene microtiter plates
µ
×
was taken as the lowest concentration of antimicrobial compound resulting in the complete inhibition
of visible growth after 18 h of incubation at 37 ◦C. Minimal bactericidal concentration (MBC) assay
was performed following MIC assay. After reporting the MIC assay value, 10 µL aliquots of the
medium were taken from wells with no visible bacterial growth. These were plated on LB agar and
incubated for 24 h to allow colony growth. The lowest concentration of the compound at which
no growth occurred on LB plates was denoted as the MBC. Results are mean values of at least two
independent determinations.
2.5. Checkerboard Assay
The activity of compound 11 in combination with meropenem, tobramycin and ciprofloxcin
was analyzed using the checkerboard broth dilution method [32] to determine the fractional
inhibitory concentration indices (FICIs), calculated as: FICI = (MICH-BDFcomb/MICH-BDFalone) +
(MICantibioticcomb/MICantibioticalone) (comb, combination). The calculated FICI was interpreted as
synergistic (FICI
according to the previously published methods [33].
≤
0.5), additive (0.5 < FICI < 1), indifferent (1
≤
FICI < 4.0), or antagonistic (FICI
≥
4.0),
2.6. Cytotoxicity Assays
A trypan blue exclusion assay [34] was performed to check the cytotoxicity of compound 11
against THP-1 human monocytic leukemia cells (ATCC, TIB-202, Manassas, VA, USA) and A549
alveolar epithelial cells (ATCC, CCL185, Rockville, MD, USA). Cells were routinely maintained
in Complete Medium RPMI-1640 and Dulbecco’s Modified Eagle’s medium (DMEM), respectively,
supplemented with 10% heat-inactivated fetal bovine serum (FBS). For the cytotoxicity assay, cells were
seeded at a density of 5
×
104 per well in a 9◦6 well plate and were incubated with serial dilutions
of compound 11 to a total of 200 µL, at 37 C in a humidified atmosphere of 5% CO2 for 24 h.
Two negative controls were included: cells in drug-free culture media and cells treated for 24 h with
the maximum concentration of the drug solvent used in the experiment (4% dimethyl sulfoxide).

Molecules 2018, 23, 1158
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Cells were subsequently stained with 0.2% trypan blue and incubated for 3 min at room temperature.
The number of dye-excluding cells was counted by microscopy. A minimum of 200 cells were counted
and the percent viability was calculated in comparison to the control. The IC50 value was defined as
the highest drug concentration at which 50% of the cells are viable relative to the control. Results are
mean values of at least five independent determinations. The selectivity index (SI) was calculated as
the ratio of IC50 and the MIC [35].
3. Results and Discussion
3.1. Chemistry
A series of 11 novel closely related compounds belonging to the thiourea family (compounds 1–10)
and a guanidine derivative (11) was prepared (Figure 1 and Table 1). Primary amines substituted with
different electron withdrawing groups were subjected to the addition reaction with isothiocyanates
in order to be transformed into the corresponding thioureas by using the general method originally
proposed by Douglas and Dains [22] (Figure 1). The substitution on both nitrogen positions
(1 and 3) of the thiourea group was varied in order to better understand the role of different
substituents in the biological activity. To rationalize this aspect, a series of closely related
1-(adamantane-1-carbonyl)-3-mono substituted thioureas was firstly prepared by taking into account
the well-known capacity of the adamantyl group to enhance antibacterial activity [36–40]. Thus,
several thioureas were prepared bearing the adamantyl group in R1 (compounds 1–7, Table 1).
Moreover, taking into account the improvement in antibacterial activity exerted by the presence
of phenyl groups substituted with electron withdrawing groups [16
thioureas (compounds 10, Table 1) was substituted in R2 with the 2-bromo-4,6-difluoro-phenyl
group. Finally, the effect of replacing the thiocarbonyl (C=S) with aryl-guanidino functionality
(Ar-N=C) was evaluated in compound , in which N-3 of the guanidine was substituted with the
,19–21], a second group of
7–
7
2-bromo-4,6-difluoro-phenyl group. To this end, the procedure proposed by Vencato et al. [26] was
applied and the acyl thiourea derivatives were treated with mercury(II) chloride under basic conditions
in the presence of 2-bromo-4,6-difluoroaniline to produce the corresponding guanidine derivative
(compound 11, Figure 1 and Table 1) [26,41].
Obtained compounds were purified by flash chromatography. FTIR, 1H-NMR and 13C-NMR
spectra and elemental analysis confirmed the identity of the products (see Materials and Methods).
1
In the H-NMR of most of the compounds, the characteristic signals of adamantyl moiety: a 6H
quartet at
singlet around 2.08 ppm (adamantane-CH), besides N-H amide and thioamide singlets at
and 12.7–13.0 ppm were clearly observed. In the 13C-NMR, characteristic signals for adamantyl
moiety at = 27.7, 36.1–36.4, 38.6–38.5 and 41.5 ppm, as well those at = 170–179 for carbonyl and
= 178–182 ppm for thiocarbonyl carbons, were observed. The acyl thioureas were also characterized
δ
= 1.75–1.79 ppm (adamantane-CH2), a 6H, singlet at 1.95–1.98 (adamantane-CH2) and a 3H,
δ
= 8.5–8.7
δ
δ
δ
by their IR spectra, with intense absorptions around 3300–3400 (νNH), 1670 (νC=O), 1580 (δNH),
and 1380 (νC=S) cm−1 [24,42,43].
The guanidine derivative 11 was characterized by two typical NH absorptions at ca. 3400 and
3240 cm−1, the C=O stretching at around 1670 cm−1 and the absence of thiocarbonyl stretching when
the FTIR spectra are compared with the corresponding thiourea reagent. The characteristic C=N
stretching modes of the guanidine group are observed as an intense absorption at ca. 1575 cm−1
In 1H-NMR, two broad NH singlets appeared besides the aromatic protons. The carbonyl carbons are
observed at 178–179 ppm in the 13C-NMR spectrum, while the (C=N-Ar) appeared upfield at 174 ppm
compared to the thiocarbonyl carbon.
.

Molecules 2018, 23, 1158
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3.2. Biological Activity
3.2.1. Antimicrobial Evaluation of Newly Synthesized Compounds
All obtained compounds were tested in vitro for their MIC and MBC against two
reference Gram-negative non-fermentative bacilli strains, Pseudomonas aeruginosa PAO1 and
Burkholderia cenocepacia J2315. These species play a critical role in morbidity and mortality associated
with CF and they were selected on the basis of their high level of resistance to a variety of antimicrobial
substances [44–47]. The results of antimicrobial activity are summarized in Table 1. The MIC and
MBC values of meropenem, tobramycin and ceftazidime, three commonly used antibiotics for the
treatment of chronic pulmonary bacterial infections [48], were analyzed in parallel. It is apparent
from the results that only the guanidine derivative 11, namely H-BDF, showed a MIC value less than
2 µg/mL, and comparable or superior activity than standard drugs. Interestingly, this compound has
the lowest MIC and MBC against B. cenocepacia J2315, a strain particularly resistant to meropenem [44].
A first look into structural activity relationship (SAR) indicates that, independent of the halogens
introduced in the phenyl group, thiourea derivatives have poor or no antimicrobial activity. However,
the replacement of thiourea in compound
7 for the guanidine group (compound 11) greatly improves
antimicrobial activity. We next evaluated the impact of introducing changes in the phenyl ring of
compound 11 in the biological activity. To this end, the guanidine derivatives 12 and 13 (Figure 2)
were synthesized and characterized. Compound 13 was designed to evaluate the effect of changing
the substitution of the halophenyl groups by the incorporation of another electron withdrawing group
(nitro) in N-2 and N-3, whereas compound 12 evaluates the effect of introducing a small change in N-3
by the substitution of bromine by fluorine in position 6.
The antimicrobial activity of the new compounds was tested against P. aeruginosa PAO1 and B.
cenocepacia J2315 as well as other Gram-negative and Gram-positive reference strains. As shown in
Table 2, when the phenyl group substituent of compound 11 was altered by the introduction of a nitro
group at the meta position (compound 13), the guanidine derivate completely lost its inhibition potency,
suggesting that not only the guanidine group but also the identity and/or position of the phenyl
substitutions are decisive for the antibacterial activity. Moreover, whereas compound 11 exhibited
very good inhibitory and bactericidal activity against all tested strains, compound 12, in which
the 2-bromo-4,6-difluoro-phenyl group in N-2 was substituted by 2,6-dibromo-4-fluoro-phenyl ring,
showed only moderate microbicidal activity, suggesting that the presence of fluorine atom in position
6 of the phenyl group in N-2 is critical to ensure high inhibition and bactericidal potency.
Table 2. Anitmicrobial activities of new compounds 11, 12 and 13 against Gram-negative and
Gram-positive bacteria—minimal inhibitory concentrations (MIC, µg/mL) and minimal bactericidal
concentration (MBC, µg/mL).
Compound
Organism
11 (H-BDF)
12
13
Tobramycin
Meropenem Ceftazimide
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
Gram-negative bacteria
Bordetella bronchiseptica 9.73H+
Escherichia coli ATCC25922
Pseudomonas aeruginosa PAO1
Burkholderia cenocepacia J2315
Pandorea apista DSM16535
0.5/2
1/2
0.5/4
2/8
16/64
64/64
32/>128
64/128
64/128
>128/>128
64/64
16/16
2/2
>128/>128
32/128
0.125/0.25
0.03125/0.0625
1/4
8/64
>128/nd
8/64
1/1
2/2
16/128
128/nd
>128/>128
>128/>128
>128/>128
>128/>128
1/2
Gram-positive bacteria
Staphyloccocus aureus ATCC6538
Bacillus cereus ATCC10876
0.25/1
2/2
8/64
64/64
>128/>128
>128/>128
2/2
8/32
<0.125/<0.25
<0.125/<0.25
8/8
1/1
nd: no data.

Molecules 2018, 23, 1158
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3.2.2. Cytotoxic Evaluation of H-BDF
As limited human cellular toxicity is an important feature for an antibiotic compound, the toxicity
of H-BDF was evaluated using the human monocytic leukemia cell line THP-1 and the human lung
epithelial cell line A549, commonly employed in toxicity evaluation of new compounds for pulmonary
application [49
,
50]. The IC50 for compound 11 was 38.4
±
5.4
µ
g/mL for A549 and 15.5
±
3.1 µg/mL
for THP-1 cells. On the basis of the MIC and IC50 values, the selectivity indices were calculated for
standard strains (Table 3). It is generally considered that the ratio for a good therapeutic index for
a drug should be >10, which is a cut-off point ensuring that overdose does not put the life of the
patient in danger [35]. Good SI values were obtained with compound 11 suggesting that H-BDF can
be considered as a promising antibacterial agent.
Table 3. Selective Indices (SI) of compound 11 against different cell lines.
Cells
Organisms
A549
THP-1
Gram-negative bacteria
Bordetella bronchiseptica 9.73H+
Escherichia coli ATCC25922
Pseudomonas aeruginosa PAO1
Burkholderia cenocepacia J2315
Pandorea apista DSM16535
76.8
38.4
76.8
19.2
38.4
30.9
15.45
30.9
7.7
15.45
Gram-positive bacteria
Staphyloccocus aureus ATCC6538
Bacillus cereus ATCC10876
153.6
19.2
61.8
7.7
3.2.3. Synergistic Effects between H-BDF and Conventional Antibiotics
Developments of alternate antibacterial strategies to potentiate the antimicrobial activity of
conventional antibiotics have become increasingly important due to the emerging threat of multi-drug
resistant infection [51]. As many clinical isolates exhibit resistance to meropenem, ciprofloxacin
and tobramycin, three of the different classes of antibiotics commonly used to treat CF pulmonary
exacerbations [52], we next studied the ability of H-BDF to potentiate the antimicrobial activity of
these antibiotics toward the multidrug-resistant strain B. cenocepacia J2315. To this end, the relationship
between H-BDF and meropenem, tobramycin, and ciprofloxacin was assessed via a standard
checkerboard assay [29]. Treatment with H-BDF reduced the minimum inhibitory concentration of
ciprofloxacin and meropenem below their clinical sensitivity breakpoints (
≤
4
µ
g/mL and
≤
1 µg/mL,
respectively). Fractional inhibitory concentration calculations revealed that H-BDF exhibited a
synergistic interaction with meropenem and ciprofloxacin with FICIs values of 0.3 and 0.4, respectively,
and an additive interaction with tobramycin with a FICI value of 0.75. This preliminary study suggests
that in addition to being used as antimicrobial agent alone, H-BDF has the potential to be used in
combination with other antibiotics.
3.2.4. Activity of Compound H-BDF against Multidrug-Resistant Clinical Isolates Recovered from
Respiratory Samples of CF Patients
Respiratory infections with opportunistic pathogens with intrinsic antibiotic resistance to most
clinically available antimicrobials are life-threatening in patients with CF [53–55]. Although P. aeruginosa
and S. aureus remain the most common pathogens in CF lung infections, other bacteria such as
species within the Bcc, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans, have emerged
as significant opportunistic human pathogens in the last decades [56–59]. To investigate whether
the guanidine derivative H-BDF would have clinical utility against current multidrug resistant
bacteria, we determined the MIC and MBC of compound 11 against thirty eight Bcc clinical isolates,

Molecules 2018, 23, 1158
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one Achromobacter xylosoxidans, one Stenotrophomonas maltophilia and two MRSA recovered from
sputum samples of CF patients and selected on the basis of their high level of resistance to a variety of
antimicrobial substances [55] (Table 4). MIC values of compound H-BDF were generally lower than
those of meropenem, ceftazimide and tobramycin. In total, 69% of Bcc clinical isolates had H-BDF
MIC values less than or equal to 4
to meropenem (MIC values g/mL), 49% were classified as susceptible to ceftazimide (MIC values
g/mL), and 2.6% of isolates were classified as susceptible to tobramycin (MIC values g/mL).
The activity of compound H-BDF against B. cenocepacia strains was impressive, with 92% susceptible
at 4 g/mL compared with only 31% susceptible to meropenem at 4 g/mL, and 69% susceptible
to ceftazimide at 8 g/mL (Table 4). Interestingly, some clinical isolates were resistant to more than
16 antibiotics, such as B. seminalis CBC040 [55] had H-BDF MIC values g/mL. Indeed, H-BDF
was active against two methicillin-resistant S. aureus clinical isolates with MIC values varying from
1 to 2 g/mL. In conclusion, compound H-BDF was active in vitro against a significant number of
multi-resistant clinical isolates recovered from CF patients.
µg/mL whereas only 41% of isolates were classified as susceptible
≤
4 µ
≤
8
µ
≤
4 µ
µ
µ
µ
≤
4 µ
µ

Molecules 2018, 23, 1158
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Table 4. Microbial susceptibility of multi-resistant isolates recovered from patients with cystic fibrosis.
H-BDF
Tobramycin
Meropenem
Ceftazidime
Clinical Isolates a
MIC (µg/mL)
MBC (µg/mL)
MIC (µg/mL)
MBC (µg/mL)
MIC (µg/mL)
MBC (µg/mL)
MIC (µg/mL)
MBC (µg/mL)
Achromobacter xylosoxidans
A. xylosoxidans HNA 001
0.125
0.25
R
nd
S
8
S
nd
Burkholderia cenocepacia
B. cenocepacia CAMPA 669
B. cenocepacia CAMPA 1533
B. cenocepacia CAMPA 1194
B. cenocepacia CAMPA 544
B. cenocepacia CAMPA 1771
B. cenocepacia CAMPA 817
B. cenocepacia CAMPA 548
B. cenocepacia CAMPA 825 (CBC 033) b
B. cenocepacia CAMPA538 (CBC 035) b
B. cenocepacia CAMPA 817
B.cenocepacia CAMPA 531
B.cenocepacia CAMPA 993 (CBC 024) b
B.cenocepacia HE001
0.25
4
2
2
8
2
2
4
2
2
1
1
4
2
16
4
8
16
8
4
16
4
8
S
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
S
R
R
R
I
R
R
I
nd
64
nd
nd
32
nd
nd
nd
16
nd
nd
nd
nd
R
S
R
S
R
S
S
S
S
S
S
S
R
nd
16
nd
8
nd
8
R
R
R
R
R
R
R
R
R
R
R
R
8
32
16
16
nd
nd
nd
I
R
S
S
R
4
4
64
Burkholderia cepacia
B. cepacia CAMPA 545
4
2
16
4
R
R
R
R
R
R
R
R
R
R
R
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
R
S
R
R
R
R
R
I
nd
8
S
S
R
R
R
S
R
I
R
R
R
16
16
nd
64
128
32
nd
64
64
nd
nd
B. cepacia CAMPA 233 (CBC 012) b
B. cepacia CAMPA 260
32
32
32
32
64
32
64
4
nd
nd
nd
nd
nd
nd
nd
8
32
32
32
64
32
64
64
4
B. cepacia CAMPA 914
B. cepacia CAMPA 886
B. cepacia CAMPA 998
B. cepacia CAMPA 1039
B. cepacia CAMPA 853 (CBC 001) b
B. cepacia CAMPA 860 (CBC 007) b
B. cepacia CAMPA 660
I
S
S
B. cepacia CAMPA 721 (CBC 011) b
2
32
64
Burkholderia contaminans
B. contaminans HNBC001
Burkholderia multivorans
0.25
1
R
nd
R
nd
S
nd
B. multivorans CAMPA 661(CBC 015) b
B. multivorans CAMPA 1530
2
2
4
2
2
4
2
4
4
8
4
8
8
16
4
8
R
R
R
R
R
R
R
R
nd
nd
nd
nd
nd
nd
nd
nd
S
R
S
S
S
S
S
S
4
nd
4
4
8
32
8
8
S
S
S
8
4
8
B. multivorans CAMPA 647 (CBC 017) b
B. multivorans CAMPA 653 (CBC 018) b
B. multivorans CAMPA 623(CBC 019) b
B. multivorans CAMPA 832 (CBC 020) b
B. multivorans CAMPA 987 (CBC 021) b
B. multivorans CAMPA 997 (CBC 022) b
S
8
R
R
R
R
nd
nd
nd
nd

Molecules 2018, 23, 1158
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Table 4. Cont.
H-BDF
Tobramycin
Meropenem
Ceftazidime
Clinical Isolates a
Burkholderia seminalis
MIC (µg/mL)
MBC (µg/mL)
MIC (µg/mL)
MBC (µg/mL)
MIC (µg/mL)
MBC (µg/mL)
MIC (µg/mL)
MBC (µg/mL)
B. seminalis CAMPA 231
32
32
4
nd
nd
8
R
R
R
R
nd
nd
nd
nd
I
S
I
nd
16
nd
nd
R
S
R
R
32
16
nd
nd
B. seminalis CAMPA 261 (CBC 039) b
B. seminalis CAMPA 475 (CBC 040) b
B. seminalis CAMPA 227
1
8
R
Burkholderia vietnamiensis
B. vietnamiensis CAMPA 992 (CBC 038) b
Staphylococcus aureus
32
nd
R
nd
S
8
S
16
S. aureus CAMPA 1909
S. aureus CAMPA 1908
2
1
16
4
128
32
nd
>128
>128
>128
nd
nd
>128
>128
nd
nd
Stenotrophomonas maltophilia
S. maltophilia CAMPA 1911
2
16
>128
nd
>128
nd
>128
nd
nd = non-determined. R= resistant, I = intermediate, S = sensible (according to the criteria set up by the CLSI). Meropenem (
g/mL S, 16 g/mL I, 32 g/mL R). Tobramycin ( g/mL S, 8 g/mL I, 16
g/mL R). a Isolates recovered from patients with chronic infections in the period 2004–2017.
Isolates recovered from patients with cystic fibrosis whose complete antibiotic susceptibilities to 17 antimicrobial agents were previously reported (reference [55]).
≤
4
µg/mL S, 8
µg/mL I,
≥
16 µg/mL R). Ceftazidime
(
≤
8
µ
µ
≥
µ
≤
4
µ
µ
≥
µ
b

Molecules 2018, 23, 1158
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4. Conclusions
We have reported the synthesis and preliminary evaluation of the antimicrobial activity of
13 novel thiourea and guanidine derivatives. The results evidenced that H-BDF, a guanidine
derivative bearing adamantane-1-carbonyl and two 2-bromo-4,6-di-fluoro-phenyl groups, can be
considered as a promising antimicrobial agent, since it exhibited higher in vitro antibacterial potency
against Gram-positive and Gram-negative reference strains than previously reported guanidine
compounds [10,11,15]. Moreover, the novel compound was active in vitro against a panel of
multidrug-resistant clinical isolates recovered from sputum samples of patients with CF. Preliminary
studies further suggest that H-BDF was able to significantly potentiate antibacterial synergy with
meropenem and ciprofloxacin. From the structure activity relationship, it can be concluded that the
antimicrobial activity depends mainly on the presence of a guanidine group. It has been proposed
that most of the biological properties of guanidine derivatives are related to their strong basicity
due to efficient resonance stabilization of the charged protonated state. The pKa of H-BDF was
not determined; however, it is expected that under physiological conditions, the guanidine group
exists mainly in its protonated form [60]. We can hypothesize that under this state, the guanidine
moiety may alter bacterial outer membrane permeability by binding to a negatively charged site in
the lipopolysaccharide layer, causing cell death. This mechanism of action have been proposed for
several guanidine derivatives with antibacterial activity [61]. Alternatively, the protonated forms
may interact with the active site of proteins and enzymes altering its function [11]. By analyzing the
role that different substituents exert in the antimicrobial activity, the importance of the halo-phenyl
group in the guanidine moiety was also demonstrated. The substituted fluorine in position 6 of the
phenyl group in N-2 may contribute to increase binding affinity and/or lipid solubility [18]. Also, the
electron-withdrawing group may activate the guanidine binding moiety to enhance its interaction
with amine groups present in the cell membrane. Future studies will be directed towards elucidating
the targets of H-BDF and the mechanisms of action.
Importantly, this compound displays low levels of cytotoxicity against THP-1 and A549 cell lines.
Future research will be performed to evaluate its efficacy and safety in animal models of infection in
order to validate its development as a novel antimicrobial.
5. Patents
“Antimicrobials compounds”. Patent application PCT/IB2017/054870, August 2017.
Author Contributions: Aamer Saeed, Alejandra Bosch , Yanina Lamberti and Mauricio Federico Erben conceived
and designed the experiments; Aamer Saeed, Diana L. Nossa González, Marisa Bettiol , and Yanina Lamberti
performed the experiments; Aamer Saeed, Alejandra Bosch , Diana L. Nossa González, Yanina Lamberti and
Mauricio Federico Erben analyzed the data; Alejandra Bosch and Marisa Bettiol contributed reagents and clinical
isolates; Yanina Lamberti, Aamer Saeed, Alejandra Bosch, and Mauricio Federico Erben wrote the paper.
Acknowledgments: This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica,
ANPCyT (PICT-2013-2130) and Universidad Nacional de La Plata (11/X794), Argentina. Y.L. and M.F.E. are
members of the Scientific Career of CONICET. D.L.N.G. is a doctoral fellow of CONICET. A.B. is member of
CIC PBA.
Conflicts of Interest: The authors declare no conflict of interest.
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