Alkyl-guanidine Compounds as Potent Broad-Spectrum Antibacterial Agents: Chemical Library Extension and Biological Characterization

Article abstract of DOI:10.1021/acs.jmedchem.8b00619

Nowadays, the increasing of multidrug-resistant pathogenic bacteria represents a serious threat to public health, and the lack of new antibiotics is becoming a global emergency. Therefore, research in antibacterial fields is urgently needed to expand the currently available arsenal of drugs. We have recently reported an alkyl-guanidine derivative (2), characterized by a symmetrical dimeric structure, as a good candidate for further developments, with a high antibacterial activity against both Gram-positive and Gram-negative strains. In this study, starting from its chemical scaffold, we synthesized a small library of analogues. Moreover, biological and in vitro pharmacokinetic characterizations were conducted on some selected derivatives, revealing notable properties: broad-spectrum profile, activity against resistant clinical isolates, and appreciable aqueous solubility. Interestingly, 2 seems neither to select for resistant strains nor to macroscopically alter the membranes, but further studies are required to determine the mode of action.

Full text of DOI:10.1021/acs.jmedchem.8b00619

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Article
Alkyl-guanidine compounds as potent broad-spectrum antibacterial
agents: chemical library extension and biological characterization.
CAROLINA PASERO, ILARIA D'AGOSTINO, FILOMENA DE LUCA, Claudio Zamperini, davide deodato,
GIUSEPPINA IVANA TRUGLIO, FILOMENA SANNIO, ROSITA DEL PRETE, TERESA FERRARO, Daniela
Visaggio, Arianna Mancini, Mario B. Guglielmi, Paolo Visca, Jean-Denis Docquier, and Maurizio Botta
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00619 • Publication Date (Web): 28 Sep 2018
Downloaded from http://pubs.acs.org on September 30, 2018
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Alkyl-guanidine compounds as potent broad-spectrum
antibacterial agents: chemical library extension and
biological characterization.
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#,1
#,1
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Carolina Pasero; Ilaria D’Agostino; Filomena De Luca; Claudio Zamperini; Davide
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Deodato; Giuseppina I. Truglio; Filomena Sannio; Rosita Del Prete; Teresa Ferraro; Daniela
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Visaggio; Arianna Mancini; Mario B. Guglielmi; Paolo Visca; Jean-Denis Docquier;
Maurizio Botta.*1,3,6
1 Department of Biotechnology, Chemistry, and Pharmacy, University of Siena, I-53100 Siena,
Italy
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Department of Medical Biotechnology, University of Siena, I-53100 Siena, Italy
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Lead Discovery Siena s.r.l., Via Vittorio Alfieri 31, I-53019 Castelnuovo Berardenga, Italy
4 Department of Sciences, Roma Tre University, Rome 00146, Italy
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Biogem, Research Institute, Ariano Irpino, AV, Italy
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Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, BioLife
Science Building, Suite 333, 1900 North 12th Street, Philadelphia, Pennsylvania 19122, United
States of America
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#
The authors equally contributed to the work
*
Corresponding author
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KEYWORDS
Antibacterial; broad-spectrum; dimer; guanidine; polycationic; bactericidal; drug-resistant bacteria;
haemolytic assay.
ABSTRACT
Nowadays, the increasing of multi-drug-resistant pathogenic bacteria represents a serious threat to
public health and the lack of new antibiotics is becoming a global emergency. Therefore, the research
in the antibacterial field is urgently needed to expand the currently available arsenal of drugs. We
have recently reported an alkyl-guanidine derivative (2), characterized by a symmetrical dimeric
structure, as a good candidate for further developments, with a high antibacterial activity against both
Gram-positive and Gram-negative strains. In this study, starting from its chemical scaffold, we
synthesized a small library of analogues. Moreover, biological and in vitro pharmacokinetic
characterizations were conducted on some selected derivatives, revealing notable properties: broad-
spectrum profile, activity against resistant clinical isolates and appreciable aqueous solubility.
Interestingly, 2 seems neither to select for resistant strains nor to macroscopically alter the
membranes, but further studies are required to determine the mode of action.
INTRODUCTION
The availability of effective antimicrobial chemotherapies is critical in many areas of medicine,
being fundamental to treat or prevent nosocomial infections, especially during invasive surgery, in
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cancer chemotherapy and in elderly or immune-compromised patient treatments. However, the rise
of antibiotic resistance, in part due to overuse or misuse of antibiotics, is now compromising the
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effectiveness of such common and relatively cheap drugs. Today antibiotic-resistant bacteria
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accounts for 700,000 infections worldwide, and are responsible for over 25,000 deaths per year in the
European Union only, which World Health Organization (WHO) identified as a major public health
issue of the 21st century.2,3
Antibiotic resistance has been developing worldwide in major clinical bacterial pathogens causing
common hospital-acquired infections, such as pneumonia, or urinary tract and bloodstream infections.
Critical issues are represented by methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-
resistant enterococci (VRE), or multidrug-resistant Gram-negative bacteria, including Klebsiella
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pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa or Acinetobacter baumannii. The
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WHO Global Report on Surveillance of Antimicrobial Resistance 2014 reported that Gram-negative
pathogens such as Escherichia coli and Klebsiella pneumoniae have developed resistance to more
than 50% of commonly used antibacterial drugs.6–10 Concomitantly, the investment in the discovery
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and development of novel antibacterials were largely considered unattractive for big pharma, since
unlike chronic pathologies, drugs against infectious diseases were used for therapeutic treatments that
take a limited period. This lack of interest concerns not only the developing countries but also the
12
developed ones. This limited the introduction of new antibiotics in the clinical practice especially
those active against Gram-negative bacteria. Carbapenems and, subsequently, colistin became
essential last resort drug for the treatment of infections caused by multi-drug resistant bacteria but are
gradually losing their efficacy, as we are now facing the emergence of extensively- or pan-drug
resistant isolates. Only very recently several new antibacterial agents (mainly new β-lactam-β-
lactamase inhibitor combinations such as ceftazidime-avibactam or meropenem-vaborbactam) were
introduced in the clinical practice. However, not only resistance to these new generation drugs have
already been detected, but they are not active against all antibiotic-resistant bacteria (such as for
instance, New Delhi Metallo-β-lactamase-1 (NDM-1)-producing K. pneumoniae).13–15
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The majority of current drugs have the same well-understood target. For this reason, there is an
urgent need to expand our currently available arsenal of antibiotics, possibly with some new classes
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of compounds showing a novel mechanism of action. Only a few drugs showing the latter property
and active on Gram-negative pathogens are actually in the stage of clinical development and include
murepavadin and ACHN-745, which both target the bacterial lipopolysaccharide
biosynthesis/transport machinery.
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In the literature polyguanidines have attracted considerable attention for their antibacterial
activity.16,17 The supposed mechanism of action of guanidine compounds is based on the strong
electrostatic interaction between their positive charges and the electronegative envelope of bacteria.
This immediate binding to the components of the cytoplasmic membrane or the cell wall leads to the
loss of bio-functions of phospholipids or to the disruption of the membrane and eventually the cell
death.18–21
Our research group has investigated a series of alkyl-guanidines as potent antibacterial agents
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against both Gram-positive and Gram-negative clinically relevant bacterial pathogens. In our recent
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study on dimeric and trimeric isomers of a linear alkyl-guanidine derivative (compound 1, Figure
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) we identified compound 2 as the most potent derivative, with Minimum Inhibitory Concentration
(MIC) values ranging from 1 to 8 µg/mL on representative Gram-positive and Gram-negative
bacterial strains.
Figure 1. Structures of monomer (1) and its dimeric derivative (2).
Encouraged by these good results obtained we decided to better investigate the chemical class of
dimers, structurally analogous to compound 2 (Figure 1) in order to obtain a small chemical library
that would allow us to perform some preliminary structure-activity relationship (SAR) considerations.
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With this purpose, we designed some possible modifications by changing the length of the alkyl
chains and the substituent on the guanidine moieties.
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RESULTS
Chemistry.
The design of the new class of dimeric antibacterial agents consisted in the connection between two
(bis)guanidinic building blocks: a monomer and an appropriate carbamoyl derivative, as reported in
our previous work.23 Our first synthetic strategy started from a commercial product, namely
Bis(octamethylene)triamine, that was subsequently bis-guanylated with the appropriate guanylating
2
2
agents, providing the desired monomers. Unfortunately this reagent was no longer commercially
available, so an alternative synthetic approach that bypassed its use was developed.23
Aimed to the generation of a new series of dimers, a more efficient and more convenient synthetic
pathway was set up, in order to obtain an important intermediate, the triamine derivative (Scheme 1),
with few and high yielded steps. However, its preparation represented a great challenge and it was
not trivial because, despite its simple chemical structure, this compound is highly polar for the
presence of a secondary and two primary amino groups that make the purification step very difficult.
Two previously described strategies for the synthesis of bis(8-aminooctyl)amine (6a) start both from
1
,8-diaminooctane and use Raney Nickel23 or nitric acid at high temperature,24 respectively, to
perform the dimerization of the starting material. Unfortunately, they are hardly practicable in an
academic laboratory, also for economic and sustainability reasons. Taking into account all the
disadvantages of these pathways, a new cheap and convenient method was set up in our research
group. In this way, the whole synthetic process became more accessible and versatile. It is based on
a straightforward four-steps route and represents a modification of a recently published procedure.25
We preferred azido groups, in place of the cyano moiety used by Zhang et al., in order to insert the
terminal amino functions. We thus obtained the final product under milder conditions, higher yields
and avoiding the use of a high amount of dangerous sodium cyanide and expensive Raney Nickel,
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used as a catalyst for cyano groups reduction. The development of this synthetic approach is fairly
significant considering its versatility. On confirmation of that, it was easily readapted for the 10 C
atoms derivative. A bis-alkylation of benzylamine with bromoazide 3a-b (as already described in our
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previous work) was performed to obtain diazide derivatives 4a-b. Reduction of the azide moieties
was accomplished by Staudinger reaction, giving compounds 5a-b. This very mild protocol involves
the formation of a triphenyl-phosphazide intermediate, by treatment with triphenylphosphine,
followed by loss of nitrogen gas with the formation of an iminophosphorane. Aqueous work up leads
to the amine 5a-b and the very stable phosphine oxide. 26 Then, a debenzylation of the central amine
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through an acid-catalyzed hydrogenation over Palladium followed, affording the triamines 6a-b, as
shown in Scheme 1.
Scheme 1. Synthesis of triamines 6a-b.a
aReagents and conditions: (i) NaN
, DMF, 50 °C, 16 h; (ii) Benzylamine, KI, K
CO , 1-BuOH, 115
3
2
3
°
C, 20 h; (iii) PPh , H O, THF, r. t., 16 h; (iv) H
3
2
2
, Pd/C, AcOH, i-PrOH, r. t., 16 h. Where a indicates
compounds with n = 6 and b indicates compounds with n = 8.
The obtained triamines 6a-b or the commercial bis(hexamethylene)triamine were thus guanylated
as shown in Scheme 2: the appropriate guanylating agents (21a-d or 24) were used in the right
stoichiometric ratio, in order to obtain monomers with the alkyl substitution on one (i, ii), two (iii) or
no (iv) guanidine functions or an N-methyl amidinourea moiety (v in Scheme 2). The synthesis and
characterization of N,N’-Di-Boc-N-alkyl-pyrazole-1-carboxamidine 21a-d have been already
described,22,27 but reported again, together with the procedure for 24, in Supporting Information
Scheme S 1 and S 2 respectively.
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Scheme 2. Synthesis of monomers.a
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aReagents and conditions: (i) 1,3-Bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea,
THF/MeOH, 50 °C, 16 h; (ii) appropriate guanylating agent 21a-d), DIPEA, THF, r.t., 16 h; (iii)
N,N’-Di-Boc-N-alkyl-pyrazole-1-carboxamidine, THF/MeOH, r.t., 16 h; (iv) 1,3-Bis(tert-
butoxycarbonyl)-2-methyl-2-thiopseudourea, THF/MeOH, r. t., 16 h; (v) 23 , DIPEA, THF/MeOH,
r. t., 16 h.
A coding system to identify monomers, carbamoyl derivatives and dimers is adopted, each
monomer being named with an alphanumeric code consisting in three parts. The first number (6, 8 or
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0) indicates the number of C atoms in the alkyl chains, while the following two letters identify the
substituents (legend shown in Table 1) present on the two guanidine moieties of the monomer. The
carbamoyl derivatives are identified through the code of the corresponding monomer with an asterisk;
the dimers with the respective codes of their constituting building blocks separated by a slash and the
deprotected final compounds followed by the letter “s” (for “salt”) after the dimeric code. In
Supporting Information, Table S 1, the correspondence between each compound and its code is
shown, e.g., compound 2 is identified with the code: 8CH*/8CH s.
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Table 1. Legend of the symbol identifying each substituent.
Symbol C
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B
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U
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The resulting monomers were converted into the corresponding carbamoyl derivatives, the
dimerization occurred and the dimers were deprotected under acidic conditions, as previously
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described in Zamperini et al and reported in Scheme 3.
Scheme 3. Synthesis of dimers.a
aReagents and conditions: (i) Triphosgene, DIPEA, dry THF, 0 °C to r.t., 1 h; (ii) appropriate
monomer 8-20, DIPEA, NaI, dry DCM, 40 °C, 48 h; (iii) TFA, dry DCM, r. t.; 7 h.
Compound 66 with N-methyl amidinourea moieties on the two terminals was synthesized as
reported in Scheme 4. The reaction involves the carbonyl group of the Boc on the two non-substituted
guanidines and the methylamine as the nucleophilic reagent, starting from the intermediate 64 (Boc-
protected compound 2), already described by Zamperini et al.23
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Scheme 4. Synthesis of amidinourea compound 66. a
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aReagents and conditions: (i) CH
r.t., 7 h.
NH aq., DIPEA, THF, 80 °C, 24 h; (ii) TFA 10%, dry DCM,
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Evaluation of antibacterial activity of new substituted alkyl-guanidines.
The antibacterial activity of the newly synthesized compounds 50-63, 66 and the intermediate 45
was evaluated on a panel of 8 representative organisms, including four Gram-positive and four Gram-
negative type strains (as reported in Table 2). Although the compounds synthesized in this series
were not enough to conduct an exhaustive SAR analysis, data reported in Table 2 allowed us to do
some preliminary but interesting observations, in terms of the length of the alkyl chains, the type and
the number of the substituents on the guanidine functions and other significant modifications.
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Table 2. MICs of the dimer chemical library and control antibiotics (colistin, vancomycin and
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5
5
5
6
daptomycin) on representative Gram-positive and Gram-negative bacteria.a
n, n : 4, 6, 8
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
X, Y, Z, W:
C: cyclopropylmethyl;
H: hydrogen; E: ethyl;
M: methyl; B: benzyl;
U: CONHCH
3
*
: carbamoyl intermediate
s: trifluoroacetate salt
B.
E.
S.
A.
P.
aeruginosa
ATCC
27853
S. aureus
ATCC
K.
subtilis faecalis
ATCC ATCC
pyogenes baumannii E. coli
ATCC
12344
Cpd Cpd Code
pneumoniae
ATCC 13833
ATCC
17978
CCUGT
2
5923 SEP
6
633
19433
1
2
5
5
8CHs
64
>64
2
>64
2
>64
1
>64
8
64
2
>64
2
>64
8
8CH*/8CHs
0.5
0
1
10CH*/10CHs 2
4
8
4
64
128
16
8
16
16
2
8
64
256
16
8
6CH*/6CHs
8EH*/8EHs
4
1
32
1
4
2
32
2
52
1
0.5
0.5
2
5
5
5
5
5
3
4
5
6
7
8MH*/8MHs 0.5
1
0.5
2
1
4
8BH*/8BHs
8CC*/8CCs
8EE*/8EEs
8BB*/8BBs
8HH*/8HHs
8CC*/8HHs
6CC*/6CHs
6EE*/6EHs
8CC*/6CCs
8CC*/8UUs
8CU/8CUs
2
2
8
4
2
16
64
32
8
8
4
16
0.5
8
2
8
16
2
4
0.5
4
1
0.25
1
16
16
16
16
64
256
64
8
4
4
4
4
58
32
2
2
2
1
4
32
4
32
16
128
>256
128
16
32
0.5
-
5
6
6
6
6
9
0
1
2
3
8
8
4
4
4
16
64
8
4
2
8
16
128
4
16
4
32
2
4
64
4
1
2
4
2
2
4
4
66
8
16
-
8
16
-
16
1
8
8
Colistin
-
-
0.5
-
0.5
-
Vancomycin
0.5
1
1
0.5
-
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Daptomycin
1
1
0.5
0.12
-
-
-
-
a
- : not determined. MICs (μg/mL) are expressed as median values calculated from experiments
performed at least in triplicate. Data for compounds 1 and 2 are from Zamperini et al.23
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
With regard to the alkyl chains, we increased the length of the alkyl chains to 10 C atoms or
decreased it to 6 C atoms, synthesizing homologous of 2 (50 and 51, respectively). Observing the
MIC values of compounds 2, 50 and 51, we could identify 8 as the optimal number of carbons in the
alkyl chains. In fact, whereas the increasing of two carbon atoms (50) determined a modest reduction
of the activity, the decreasing to 6 carbon atoms (51) resulted in a complete loss of antibacterial
potency, in particular against Gram-negative strains, where high MIC values were often recorded.
Then, the cyclopropylmethyl substituents of 2 were replaced with ethyl, methyl or benzyl groups
(compounds 52, 53 and 54 respectively): ethyl and methyl groups were chosen as substituents because
of their smaller alkyl size whereas benzyl moiety was inserted to hinder and make more lipophilic the
N-termination of the guanidine moiety. These compounds retain the activity trend of 2. Thus, the
nature of the alkyl substituent did not appear to affect considerably the antibacterial profile. In
particular, by comparing the MIC values of these compounds, we observed that in general the
activities of these compounds were a bit worse than the one of 2, but the difference was never higher
than one or two dilutions. The activity of 52 and 53 against the Gram-positive Staphylococcus aureus
resulted to be higher than the one of 2, as also seen for the activity of 54 against the Gram-negative
Burkholderia cepacea. Again, we detected a significant improvement of activity for 53 and 54 against
the Gram-negative Acinetobacter faecalis with a MIC value lower than 0.125 µg/mL.
Moreover, in order to understand the influence of the number of guanidine substitutions on the
antibacterial properties and starting from 2, we synthesized compounds characterized by four alkyl
substituents, such as cyclopropylmethyl (55), ethyl (56) or benzyl (57) groups, or no one (58) and an
analogous compound of 2 with a symmetry axis instead of a plane, yielding a dimer composed by
two different monomers (59). Comparing their MIC values, we observed some interesting SARs: both
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5 and 58 (with the cyclopropylmethyl substituent on all or no guanidine functions, respectively)
showed a moderate activity. Compound 59 was synthesized through the dimerization of two
symmetric monomers, one completely substituted, while the other no, to evaluate if this structural
inversion would affect the activity. Despite our expectations, the comparison between the
antimicrobial profiles of 2 and 59 revealed that the inversion compromised the activity of the hit
compound, although their superficial charge was the same. Moreover, it was evident that 57 (with
four benzyl substituents) was only a little more active than 55 (with four cyclopropylmethyl
substituents) and 56 (with four ethyl substituents), while 58 was less active against almost all the
tested bacterial strains. The obtained data highlighted that the increasing of the number or lipophilicity
of substituents did not lead to any improvement of antibacterial activity, thus leading to the conclusion
that the optimal number of substitutions was two.
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compounds with three, instead of two, substituted guanidines (60 and 61) were also evaluated and
despite maintaining a significant antibacterial activity on Gram-positive pathogens (MIC values
ranging from 2 to 64 µg/mL), compound 61 (three ethyl substituents) showed a very poor activity on
Gram-negatives.
The hybrid dimer 62, characterized by monomers with different length of carbon chains, exhibited,
when compared to compound 2 a narrower spectrum of activity on Gram-negatives, with a higher
activity (MIC range, 8-16 µg/mL) on Enterobacteriaceae.
Furthermore, we assumed that a decreasing in pKa value could improve the selectivity towards
bacterial cells, so we designed two compounds with a lower predicted pKa characterized by the
transformation of the guanidine functions into N-methyl amidinoureas (63 and 66). This required the
development of a new synthetic strategy. These two dimers showed to be active against almost all
tested strains, even if a decrease of the antibacterial potency was found in particular against the Gram-
negative Acinetobacter baumannii and Pseudomonas aeruginosa.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Then, in order to understand if a Boc-cleavage occurred in bacterial cells, the intermediate 45 was
also tested. However, it was found to be inactive with MIC values ≥ 256 µg/mL for all the tested
bacterial strains.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Little chemical modifications were performed furnishing a library of compounds active against
almost all the tested bacterial strains, showing in some cases a broad-spectrum activity. Eventually,
we did not observe a significant change of the antibacterial profile and the hit compound 2 still
remains the most potent compound, except for S. aureus ATCC 25923 and A. faecalis 424/98, on
which 53 shows better MIC values.
Further biological characterization of selected compounds.
The antibacterial properties of selected analogues of compound 2 were further investigated on
recent antibiotic-resistant clinical isolates, including pandrug-resistant Klebsiella pneumoniae (Table
3
). Most interestingly, all compounds retained much of their activity on specific pathogens regardless
of the resistance phenotype, and particularly colistin-resistant K. pneumoniae. This indicates that,
despite the polycationic nature of both colistin (a polymyxin antibacterial peptide) and our alkyl-
guanidines, the latter likely show a different mechanism of action, and are not susceptible to
alterations of the Lipopolysaccharide (LPS) layer determining a decrease of the negative net charge
on the bacterial cell surface.28
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Table 3. MICs of selected compounds on Gram-negative antibiotic-resistant clinical isolates.a
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cpd
2
52
53
54
55
56
Cpd Code
8CH*/8CHs 8EH*/8EHs 8MH*/8MHs 8BH*/8BHs 8CC*/8CCs 8EE*/8EEs
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A.
baumannii
AC-54/97
2
-
4
-
8
8
B. cepacia
SI-R2
1
1
6
32
4
32
4
16
4
32
4
32
2
E. cloacae
VA-417/02
K.
pneumoniae 2
SI-081*
4
8
8
8
4
8
8
S.
maltophilia 16
16
16
32
634/08
a
-: not determined; * clinical isolate with pandrug-resistant phenotype. MICs (μg/mL) are
expressed as median values calculated from experiments performed at least in triplicate.
The Minimal Bactericidal Concentration (MBC) values of most active compounds, although
measured only for E. coli and A. baumannii, resulted identical to that of the MIC, indicating a strong
bactericidal activity of these compounds (data not shown). This was further confirmed by a time-kill
curve analysis, in which the bactericidal activity of dimer 2 (4 µg /mL, 2 x MIC) was shown to be ≥4
log10 in one hour and thus superior to that of colistin (Supporting Information, Figure S 1).
Furthermore, in order to estimate the possible effect of plasma protein binding (PPB) on
antibacterial activity of alkyl-guanidine derivatives, compound 2 was tested on E. coli MG1655 in
Mueller Hinton Broth (MHB) supplemented with 10% complement-free human serum (HS). E. coli
MG1655 was completely inhibited by 2 μg/mL of compound 2 (MIC; Table S 2 in Supporting
Information) while 1 μg/mL did not affect the growth profile (1/2 MIC; Figure S 2 in Supporting
Information). The addition of 10% HS to MHB moderately increased its MIC value (2-fold), although
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
growth E. coli MG1655 was clearly retarded in MHB-HS supplemented with 2 μg/mL of compound
(MIC; Figure S 2A in Supporting Information). This result indicates that HS component(s) can
reduce, but not abrogate, the efficacy of compound 2. An obvious candidate is human serum albumin
2
2
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
HSA), the most abundant plasma protein (concentration 35 – 50 g/L, which acts as a trap for a
30
variety of ligands. Since bovine serum albumin (BSA) shares 76% sequence identity with HSA, the
susceptibility of E. coli MG1655 to compound 2 was tested in MHB supplemented with increasing
concentrations of BSA. Remarkably, the addition of BSA (5 to 40 mg/mL) to MHB did not alter the
MIC value of compound 2 (Figure S 2B in Supporting Information), suggesting that other serum
proteins (e.g. alpha-1-acid glycoprotein, AGP) could eventually affect the activity of compound 2 in
HS. In fact, the binding assays performed on HSA and AGP (reported in ADME paragraph)
confirmed a high affinity to this latter, without compromising the antibacterial activity. These results
are compatible with a future development of the alkylguanidine derivatives as antibacterial agents. In
fact, also if incremented, the observed MIC value for compound 2 on the tested E. coli strain in
presence of plasma proteins can be considered a reliable prediction of a maintained antibacterial
activity in vivo studies.
Considering the polycationic nature of our compounds, a potential mechanism of action could be
represented by the impairment of the structural integrity of the bacterial cytoplasmic (inner)
membrane or the outer membrane of Gram-negative organisms. Thus, the first hypothesis was tested
using a simple enzyme-based whole assay, in which the activity of a cytoplasmic enzyme on a poorly
permeable chromogenic substrate was evaluated. Indeed, the chromogenic substrate o-nitrophenyl-β-
D-galactoside (ONPG), colourless, releases the yellow o-nitrophenol (ONP) upon enzymatic
hydrolysis but only reaches the cytoplasm following permeability defects, such as those acting on
membrane integrity, such as colistin and other antimicrobial peptides (AMP).31,32 The assay was
conducted for some rationally selected compounds (1, 2, 50, 55 and 58) in three bacterial strains
characterized by physiological or induced high-level of intracellular production of β-galactosidase.
Interestingly, as shown in Figure 2, none of the tested compounds, besides sodium dodecyl sulfate
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(SDS) which was used as a positive control, apparently induced significant levels of OPNG
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
conversion, indicating that they did not cause a macroscopic alteration of the bacterial membrane.
However, this assay does not allow to exclude the possibility that the tested compounds could
generate very small, microstructural, damages in the inner membrane.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Effect of selected compounds on bacterial permeability. Time-depending production of o-
nitrophenol from ONPG by A) Escherichia coli MG1655; B) Acinetobacter baumannii
ATCC19606T harboring plasmid pMP220::PrrnB; C) Pseudomonas aeruginosa ATCC15692
harboring plasmid pMP220::PrrnB, untreated (negative control) and upon exposure to 256 µg/mL of
each compound or 1% of SDS (positive control), as indicated.
The possibility to affect the outer membrane integrity of Gram-negative species was investigated
by an additional specific fluorescence assay. Three Gram-negative bacteria were exposed to
increasing concentrations of the selected compounds, colistin or dimethylsulfoxide (DMSO), as
positive and negative controls, respectively. Then, the addition of 1-N-phenylnaphthylamine (NPN)
followed. This lipophilic dye weakly fluoresces in aqueous environments but it emits strong
fluorescence in the hydrophobic ones, thus enabling to detect a potential outer membrane damage. In
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
fact, the LPS external layer of the outer membrane of Gram-negative bacteria acts as a permeability
barrier to lipophilic substances like NPN. Disruption of outer membrane integrity makes
phospholipids of the internal outer membrane layer accessible to NPN, allowing fluorescence
3
3
0
1
2
3
4
5
6
7
8
9
0
1
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3
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8
9
0
1
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
emission by the probe. Notably, all the tested compounds caused a dose-dependent increase in the
NPN fluorescence emission for all Gram-negative species tested (Figure 3). Interestingly,
compounds 2, 50 and 55 caused higher fluorescence emission than colistin, whereas any fluorescence
was detected in the presence of DMSO. In contrast, compound 1 showed the lowest activity on the
outer membrane. These results indicate that all compounds perturb the Gram-negative outer
membrane, although to a different extent.
Figure 3. Effect of selected compounds on Gram-negative outer membrane integrity. The uptake of
NPN (measured as relative fluorescence unit, RFU) by A) E. coli MG1655, B) A. baumannii
T
ATCC19606 and C) P. aeruginosa ATCC15692 in the presence of increasing concentrations of each
selected compound. Colistin or DMSO were used as positive and negative controls, respectively. Data
are the mean ± standard deviation of triplicate experiments.
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As another potential mechanism of action, the depolarization of the cytoplasmic membrane (thus
affecting the proton motive force) was also investigated. Thus, compound 2 was tested for its ability
to depolarize E. coli membranes using the fluorescent dye 3,3’-dipropylthiacarbocyanine (DiSC -5),
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
essentially as described by Breeuwer and Wand.18,34 In this assay, compound 2 was unable to restore
any fluorescence after incubation of bacterial cells at a concentration 2 x MIC (4 μg/mL), thus
suggesting that membrane depolarization was likely not the prevalent mechanism accounting for its
fast bactericidal activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Biofilm is a very complex microbial architecture that allows bacteria to attach to surfaces and to
form aggregates, encasing them into a matrix composed of self-produced extracellular polymers.
Biofilm makes microorganisms more resistant to antibacterial treatments, harsh environments and
human immunity. One of the current strategies to prevent the formation of biofilms is the treatment
with AMPs like colistin, polymixins and gramicidin. 35,36 These molecules are able to bind
electrostatically the negative charged LPS through their cationic amino acids (lysine and arginine)
and to stabilize this binding through hydrophobic interactions between the hydrophobic amino acids
37
of the peptide and fatty acyl chains of LPS. Our compounds, being polycationic and amphiphilic,
bear both the polar and nonpolar functions to let us hypothesizing a similar trend. For these reasons,
in-depth analyses were performed to evaluate their behaviour toward the biofilm, revealing that they
are able neither to inhibit the formation of biofilm (assessed through the crystal violet assay) nor to
alter the three-dimensional structure of biofilm (Table S 2 and Figures S 3 and S 4 in Supporting
Information).
Furthermore, considering that our monomer derivatives are endowed with the inhibition of Maize
Poly-Amino Oxidase (mPAO) with a micromolar activity, as already published,38 we strongly
hypothesize a possible involvement of the dimeric derivatives in the bacterial polyamine pathways.
Indeed, we supposed that our dimers could be a possible substrate of some poly-amino transporters,
present in all kind of cells, also in bacteria ones,39,40 since they show an amphiphilic structure like the
polyamines. Thus, compound 2 was already tested on E. coli mutants in which the genes encoding
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
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5
5
5
5
5
5
6
-
-
-
-
the main components of polyamine transport were inactivated (mutants potA , potB , potC , potD ,
-
-
-
-
-
41
potE , potF , potG , potH and potI mutants from the Keio library). Comparing to the activity on E.
T
coli CCUG , the MIC values on the mutant strains were unaffected. This does not completely
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
invalidate our hypothesis because the assay results could be explained in two ways, being the
polyamine transport systems different for spermidine (potABCD) and putrescine (potGHIF): dimer 2
could interact with neither potABCD nor potGHIF or could interact with both the systems so when
one is abolished by a single mutation, it can bind the other one. Unfortunately, a limited literature in
the field of bacterial polyamines is available so far, but ours is just a preliminary assumption and
further studies are certainly needed to better investigate this issue.
In order to evaluate the propensity of dimer 2 to select for resistant mutants, and potentially
T
6
7
investigate the resistance mechanism(s), E. coli CCUG (starting inoculum, 10 – 10 CFU/mL) was
subcultured sequentially in MHB containing increasing concentrations of compound 2 (4-32 μg/mL
corresponding to 2-16 x MIC). No growth could be observed at concentrations higher than 32 μg/mL,
after up to 48 hours. Cells recovered from the various cultures were plated on agar plates containing
the same concentration of compound 2. Surprisingly, the phenotypic analysis of these isolates did not
show the acquisition of a stable resistant phenotype, as the 2 MIC values measured for 25 randomly-
selected isolates was essentially identical to that of the parental strain. This would indicate that 2
could induce the formation of persisters in the bacterial population, rather than selecting stable
mutants with an acquired mutation, but that this tolerance phenomenon could only promote survival
at concentrations equal or below to 32 µg/mL.
Haemolytic activity and cytotoxicity of representative compounds.
The same rationally selected compounds (50, 55 and 58), in addition to 1 and 2, have been
investigated about their behaviour towards human membranes. Haemolysis of red blood cells is often
42
used as a tool to study the effect of compounds on mammalian cell integrity. Each compound was
tested at 64 µg/mL on human erythrocytes recovered from healthy donors with different blood groups;
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.2% Triton X-100 and 1.6 % DMSO were used as positive and negative controls, respectively. As
shown by data reported in Supporting Information, Table S 3, the assay revealed that there was no
evidence of haemolysis, with the exception of compound 50, that showed a significant percentage of
haemolysis ranging from 41 to 54%, depending on the blood group. This is interesting considering
that the only compound with haemolytic activity showed a low antibacterial activity, indicating that
the length of the alkyl chain could be very important for selectivity. For the other compounds, the
obtained values are in a range of 0-7%, in agreement as preliminary described for 2 in a previous
6
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1
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23
assay. Only minor blood group-dependent haemolytic differences were observed for all compounds.
The potential cytotoxicity of compound 2 was further evaluated on the commercially available
HeLa cells. When tested at 16 μg/mL, it showed only a minimal cytotoxicity after 24 hours of
incubation. Cytotoxicity was more evident at 256 μg/mL, although it did not exceed 60% after 24
hours (Figure 4), indicating that the selectivity towards bacterial cells should be improved.
Figure 4. Cytotoxicity of compound 2 on HeLa cells. The figure shows the cytotoxicity (%) after 5
and 24 hours of incubation when 2 was tested at 16 μg/mL (black) or 256 μg/mL (grey). The
cytotoxicity control is shown in white.
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Preliminary ADME characterization of hit compound 2.
In the end, in vitro preliminary absorption, distribution, metabolism, excretion (ADME) properties
for the most active compound 2 was evaluated (Table 4). It was found to be metabolically stable, as
measured by means of human liver microsomal proteins, and very soluble in pure water, probably
thanks to its positive charges at physiological pH (7.4), allowing a future intravenous administration
development. Moreover, the distribution constant logD measurement at pH 7.4 revealed a good value,
comparable to the ones of daptomycin and vancomycin and is in agreement with the experimental
solubility found. The indirect fluorescence method was used for PPB assays. This protocol was
validated by using known drugs (see Experimental session) characterized by variable interaction with
HSA and AGP. The main goal of drug binding assays is the determination of plasma-bounded drug
fraction at therapeutic concentrations. During the discovery stage of a research project, this
information could help to classify drugs as high, intermediate or poorly bound to plasmatic proteins.
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The drugs that are highly bound, such as warfarin and diazepam, show dissociation rate constant (K
d
)
of less than 100 μM, while the poorly bound drugs, as paracetamol, are characterized by a K value
d
higher than 1 mM. Compound 2 shows a good affinity to the AGP, as expected for molecules that
bear positive charges at physiological pH but it has a low percentage of bond (Bmax) values, as
reported in Table 4.
Thus, a low maximal percentage of bond and a slow dissociation rate of compound 2 are considered
as permissive conditions to the predicted in vivo ADME.43–45 Moreover, as above-reported, kill curve
experiments in presence of albumin or serum, showed that the PPB does not impair restrictively the
antibacterial activity.
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Table 4. Preliminary ADME assays result for compound 2
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1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
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5
5
6
Metabolic
stability
HSA
AGP
Solubility
g/L]b
logD (pH 7.4)c
K [μM]d
Bmax (%)d K [μM]d
Bmax (%) d
[
d
d
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%)a
(
>
99
0.292 ± 0.022 -0.79
23.27 ± 14.6 14.0
0.11 ± 0.06 30.2
aThe human liver microsome stability is expressed as a percentage of the unmodified parent drug.
b
c
Aqueous solubility was determined by means of the LC-MS method. Distribution coefficient 1-
d
octanol/ TRIS buffer pH 7.4 . K
d
and Bmax values are measured by means of the indirect fluorescence
method.
The parallel artificial membrane permeability assay (PAMPA), validated with known drugs
2
2
(rifamixin and chloramphenicol) revealed a low apparent permeability (Papp) at physiological pH,
as expected since ionic compounds cannot pass across the membrane (Table 5): dimer 2 is, in fact, a
trifluoroacetic salt with four net positive charges on the guanidine moieties and no pH adjustments
46
have been performed during the assay. Since this experiment, from Kansy et al, replaces cellular
membranes (phospholipids, enzymes, proteins, sterols) with a barrier made of only phospholipids
(phosphatidylcholine in our case), it correlates only with passive diffusion and can give false results
for compounds that are substrate of membrane enzymes or transporters (active permeability). In fact,
it is known from the literature that organic cations, such as endogenous substances bearing guanidine
and amine functions, can cross biological membranes through apical transport mechanisms, like H+
antiports and P-glycoprotein G (P-gp), and the less studied basolateral ones. In general, a family of
organic cation transporters (OCT) has been cloned and studied to give more information about
absorption and secretion of cationic compounds.47–49 To evaluate if compound 2 is a substrate of this
kind of transporters, in-depth permeability experiments were conducted. In this frame, we opted for
the Caco-2 permeability assays that are based on the use of a polarized cell monolayer. It is the gold
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standard in vitro test system, since it is quicker to use, convenient, and it produces more reproducible
data than animal studies, giving an effective assessment of the permeability (Papp) of new compounds.
Caco-2 is the best known immortal human colon carcinoma cell line as a model of the epithelial cell
layer permeability barrier that compounds encounter in the small intestine. This cell line is
characterized by a particular morphology that allows multiple permeability mechanisms and it
provides the opportunity to investigate various permeability pathways. This model resembles the
morphology of gastrointestinal epithelial cells for the presence of microvilli on the apical surface and
for the expression of certain enzymes and membrane transporters, such as P-gp, breast cancer
resistance protein (BCRP), and multidrug resistance protein 2 (MRP2). These characteristics make
Caco-2 cells the most suitable and frequent used line to conduct permeability experiments and to rank
compounds, according to the Biopharmaceutics Classification System (BCS) by Food and Drug
Administration (FDA), as low, medium or high permeability compounds.50–53 We calculated the Papp
apical-to-basolateral and basolateral-to-apical by means of two different experiments, placing the test
compound in the apical or in the basolateral side buffers. The first value provides information about
the permeability in the absorptive direction, furnishing a quite reliable model of the gastrointestinal
absorption. Compound 2 showed a very low permeability, in accordance with PAMPA results. In
fact, the low Papp(A>B) found (Table 5) indicates that it is not absorbed through passive diffusion (as
already showed by PAMPA experiment), but also paracellular permeation and active transport are
not involved in its absorption. This corresponds to a low oral bioavailability, that can be overcame
by intravenous administration, as it happens for the majority part of natural drugs, as example those
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4
antibiotics active against chemo-resistant organisms, such as colistimethate (prodrug of colistin,
indicated for treatment of infection by multi-drug resistant Gram-negative strains, often used as a last
resort), daptomycin (for treatment of nosocomial Gram-positive infections including MRSA and VRE
strains) and vancomycin (for treatment of Gram-positive bacterial infections). The opposite
experiment was performed to study the permeability of compound 2 by cell membrane transporters
in the efflux direction. Also in this case, we obtained a very low Papp. In Table 5, we reported the
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ratio between Papp in the B>A and the A>B directions: this value, known as efflux ratio, is 0.65,
because the permeability values are approximatively the same (in the same range), highlighting that
compound 2 could permeate only by passive or paracellular diffusions, not being substrates of active
transporters for the cellular uptake or efflux. In fact, in the first case it should have been A>B/B>A)
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≥
2 and in the second one B>A/A>B) ≥2.45,55,56 Considering that the efflux is a serious issue for an
antibiotic, not allowing it to reach the appropriate concentration in the cell (MIC) to have antibacterial
efficacy and provoking the rise of resistant strains, a low efflux is a relevant finding.
Table 5. Apparent Permeability Coefficients obtained by PAMPA and Caco-2 cell line experiments
and efflux ratio for dimer 2, atenolol (characterized by a low permeability) and propranolol (classified
as a highly permeable compound), as reference compounds.
Apparent
Caco-2
Permeability
Efflux ratiob
Permeabilitya
Cpd
(PAMPA)
Papp(A>B) / Papp(B>A)
-6
Papp(A>B) [10 cm/s]
-6
P
app [10 cm/s]
2
1.60 ± 0.43
0.19 ± 0.09
0.65
Rifamixin
Chloramphenicol
Atenolol
0.06 ± 0.01
-
0.30 ± 0.50
-
-
-
0.13 ± 0.04
22.4 ± 5.4
0.37
0.40
Propranolol
aA, apical; B, basolateral; Papp(A>B) is the Papp in the apical-to-basolateral direction.
bB>A/A>B is the ratio of the basolateral-to-apical and the apical-to-basolateral permeation rate.
Results are obtained from the average values of Papp (A>B) and (B>A).
CONCLUSION
Recent findings23 highlighted a notable antibacterial activity of alkyl-guanidine oligomers,
serendipitously originated from a monomeric compound. In particular, the symmetric dimer 2 was
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identified as a potent broad-spectrum bactericidal. In this work, we present a chemical library of
fifteen derivatives (50-63 and 66) which have been tested allowing us to perform some preliminary
SAR considerations. Indeed, it clearly appears that the optimal length of the aliphatic linker should
include 8 carbon atoms, whereas the nature and the number of the substituents on the guanidine
moieties do not considerably affect the antibacterial properties of these compounds. Thus, the
biological data indicate that almost all the synthesized derivatives show a strong bactericidal activity
against a panel of Gram-positive and in some cases against Gram-negative pathogens. Despite the
structural changes, dimer 2 still remains the best compound in antibacterial activity terms. Moreover,
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2
and some rationally selected derivatives turn out to be active also against drug-resistant clinical
isolates, acting through a bactericidal behavior similar to that of colistin, i.e. by not causing a
macroscopic permeabilization of bacterial membranes or cell lysis. They were also investigated about
their behavior towards human cell membranes, through haemolytic assays, revealing that there is no
evidence of haemolysis, with the exception of 50. Furthermore, extended biological investigations of
dimer 2 were performed, such as the evaluation of a possible interaction with polyamine transport
systems and the propensity to select for resistant mutants, revealing the induction of persisters
generation, without selecting stable mutants. The nature of these molecules strongly suggests that
they could act on the membranes by electrostatic interactions, even if macroscopic alterations of the
bacterial membrane were not observed, we cannot exclude the generation of microstructural damages.
In fact, they can perturb the Gram-negative outer membrane and affect neither the proton motive
force nor the ability of bacteria to form the biofilm. Finally, the in vitro ADME properties of dimer 2
was investigated: it was found to be metabolically stable, very soluble in pure water and it showed a
low binding to plasma proteins that does not impair the activity and low passive and active
permeability.
In summary, we synthesized a small library of alkyl-guanidine symmetric dimers as bactericidal
agents, in some cases characterized by a broad-spectrum activity, including on drug-resistant clinical
isolates. These compounds are generally not haemolytic, moderately toxic and do not apparently
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select for stable resistant mutants, as found for the hit compound 2. Notably, ADMET properties of
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2
make it suitable to proceed with further optimization, including the improvement of selectivity.
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EXPERIMENTAL SECTION
1
1
. CHEMISTRY
.1 General Chemistry.
All commercially available chemicals and solvents were used as purchased. DCM was dried over
calcium hydride and THF was dried over sodium and benzophenone prior to use. Anhydrous reactions
were run under a positive pressure of dry nitrogen. Chromatographic separations were performed on
1
13
columns packed with silica gel (230-400 mesh, for flash technique). H NMR and C NMR were
recorded at 400 and 100 MHz respectively on a Bruker AC200F spectrometer and are reported in
parts per million (δ scale) and internally referenced to the CDCl
3
or CD
3
OD signal, respectively at δ
7
.24 ppm and 3.31 ppm. Chemical shifts for carbon are reported in parts per million (δ scale) and
referenced to the carbon resonances of the solvent (CDCl
3
at δ 77.00 and CD OD at δ 49.00 ppm).
3
Data are shown as following: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m =
multiplet and/or multiplet resonances, br = broad), coupling constant (J) in Hertz (Hz) and integration.
Mass spectra (LC-MS) were acquired using an Agilent 1100 LC-MSD VL system (G1946C) by direct
injection with a 0.4 mL/min flow rate using a binary solvent system of 95/5 MeOH/H O. UV
2
detection was monitored at 254 nm. Mass spectra were acquired in positive mode scanning over the
mass range 105-1500 m/z, using a variable fragmentor voltage of 10-70 mV.
1
.2 Determination of the purity.
The purity of final products (50-63 and 66) was 95% or higher and it was assessed by HPLC-UV-
MS, using an Equivalence 3 C18 column (ACE EQV-8977: 150 x 4.6 mm, 5 μm particle size) at a
flow rate of 0.6 mL/min with a linear gradient elution from 100/0 to 50/50 v/v CH
3
CN (formic acid
0
.1% v/v)/H O (formic acid 0.1% v/v). UV detection was monitored at 210 nm. Mass spectra were
2
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acquired in positive mode scanning over the mass range 105-1500 m/z, using a fragmentor voltage of
0 mV.
7
1
1
.3 Synthetic procedures for compounds 3-49 are reported in Supporting Information.
.4 General procedure for synthesis of dimer salts 50-63.
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Protected dimeric compound 36-49 (0.046 mmol) was dissolved in dry DCM (3.6 mL) and TFA
10%, 0.4 mL) was added. The reaction mixture was stirred at room temperature for 7.5h. Then the
(
solvent was evaporated and the crude was dissolved and evaporated several times first with MeOH
to remove TFA residue. No further purification followed, the product was obtained a colourless oil.
1
.4.1
1,3‐bis(10‐carbamimidamidodecyl)‐1,3‐bis({10‐[N'‐
(cyclopropylmethyl)carbamimidamido]decyl})urea trifluoroacetate salt (10CH*/10CHs, 50).
1H NMR (CD
3
OD) δ (ppm): 0.26 (m, 4H); 0.57 (m, 4H); 1.05 (m, 2H); 1.34 (m, 48H); 1.50 (m, 8H);
1
3
1
.56 (m, 8H); 2.20 (bs, 1H); 3.05 (d, 4H, J = 6.8 Hz); 3.15 (m, 12H); 3.30 (m, 4H). C NMR
CD OD) δ (ppm): 2.41; 9.53; 26.23; 26.60; 27.51; 28.40; 28.48; 28.86; 29.05; 29.10; 29.20; 40.99;
1.10; 45.77; 46.87; 47.08; 47.30; 47.51; 47.73; 47.94; 48.15; 156.01; 166.00. LCMS m/z (ES+) =
(
3
4
4
2+
3+
4+
79.1 [M + 2H] ; 319.8 [M + 3H] ; 240.1 [M + 4H] YIELD: quantitative
1
.4.2
1,3‐bis(6‐carbamimidamidohexyl)‐1,3‐bis({6‐[N'‐
1
(cyclopropylmethyl)carbamimidamido]hexyl})urea trifluoroacetate salt (6CH*/6CHs, 51). H
NMR (CD
6H); 1.55 (m, 16H); 3.05 (d, 4H, J = 7.2 Hz); 3.16 (m, 16H). C NMR (CD
6.09; 9.53; 22.71; 26.05; 26.28; 27.46; 28.38; 29.26; 41.02; 42.16; 45.80; 155.60; 165.40. LCMS m/z
3
OD) δ (ppm): 0.27 (q, 4H, J = 4.8 Hz); 0.58 (q, 4H, J = 5.6 Hz); 1.06 (m, 2H); 1.34 (m,
1
3
1
3
OD) δ (ppm): 4.36;
2
+
3+
(
ES+) = 367.1 [M + 2H] ; 245.1 [M + 3H] YIELD: quantitative
1
.4.3
1,3‐bis(8‐carbamimidamidooctyl)‐1,3‐bis[8‐(N'‐ethylcarbamimidamido)octyl]urea
1
trifluoroacetate salt (8EH*/8EHs, 52). H NMR (CD
3
OD) δ (ppm): 1.20 (t, 6H, J = 7.2 Hz); 1.33
1
3
(
m, 32H); 1.50 (m, 8H); 1.55 (m, 8H); 3.14 (m, 12H); 3.21 (q, 4H, J = 7.2 Hz); 3.30 (m, 4H). C
NMR (CD
3
OD) δ (ppm): 12.96; 22.71; 26.16; 26.52; 27.49; 28.35; 28.43; 28.82; 28.92; 29.18; 29.24;
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9.40; 35.91; 40.97; 41.02; 46.87; 59.65; 62.04; 62.89; 124.64; 164.88; 165.23. LCMS m/z (ES+) =
5
7
6
7
8
9
+
2+
3+
4+
93.0 [M + H] ; 397.1 [M + 2H] ; 265.1 [M + 3H] ; 199.2 [M + 4H] YIELD: quantitative
.4.4 1,3‐bis(8‐carbamimidamidooctyl)‐1,3‐bis[8‐(N'‐methylcarbamimidamido)octyl]urea
trifluoroacetate salt (8MH /8MHs, 53). H NMR (CD
1
*
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
OD) δ (ppm): 1.34 (m, 32H); 1.51 (m, 8H);
13
1
2
.56 (m, 8H); 2.83 (s, 6H); 3.14 (q, 12H, J = 7.2 Hz); 3.30 (m, 4H). C NMR (CD
3
OD) δ (ppm):
6.17; 26.53; 26.74; 27.50; 28.38; 28.92; 29.95; 40.97; 128.09; 156.74; 157.17; 161.28; 165.83.
2+
3+
4+
LCMS m/z (ES+) = 383.0 [M + 2H] ; 255.8 [M + 3H] ; 192.1 [M + 4H] YIELD: quantitative
.4.5 1,3‐bis[8‐(N'‐benzylcarbamimidamido)octyl]‐1,3‐bis(8‐carbamimidamidooctyl)urea
trifluoroacetate salt (8BH /8BHs, 54). H NMR (CD
3.13 (q, 8H, J = 7.2 Hz); 3.19 (t, 8H, J = 6.8 Hz); 4.41 (s, 4H); 7.30 (m, 6H); 7.36 (m, 4H). C NMR
CD OD) δ (ppm): 22.30; 26.25; 26.59; 26.93; 27.61; 28.46; 28.87; 28.99; 29.30; 29.50; 41.16; 44.44;
26.60; 127.42; 127.86; 128.38; 136.60; 139.10; 155.90; 157.15; 162.00. LCMS m/z (ES+) = 459.0
1
*
1
3
OD) δ (ppm): 1.33 (m, 32H); 1.53 (m, 16H);
1
3
(
3
1
2
+
3+
4+
[
M + 2H] ; 306.4 [M + 3H] ; 230.1 [M + 4H] YIELD: quantitative
1
.4.6
1,1,3,3‐tetrakis({8‐[N'‐cyclopropylmethyl)carbamimidamido]octyl})urea
1
trifluoroacetate salt (8CC*/8CCs, 55). H NMR (CD
3
OD) δ (ppm): 0.27 (q, 8H, J = 4.8 Hz); 0.57
(
q, 8H, J = 8.0 Hz); 1.05 (m, 4H); 1.34 (m, 32H); 1.51 (m, 8H); 1.58 (m, 8H); 3.05 (d, 8H, J = 7.2
13
Hz); 3.06 (t, 8H, J = 7.2 Hz); 3.17 (t, 8H, J = 7.2 Hz). C NMR (CD
3
OD) δ (ppm): 2.41; 9.53; 26.19;
2
1
6.55; 27.52; 28.46; 28.86; 28.95; 29.24; 29.39; 41.09; 45.78; 124.09; 155.78; 163.66; 163.75;
2
+
3+
4+
67.03. LCMS m/z (ES+) 477.5 [M + 2H] ; 318.7 [M + 3H] ; 239.3 [M + 4H] YIELD:
quantitative
.4.7
1
1,1,3,3‐tetrakis[8‐(N'‐ethylcarbamimidamido)octyl]urea
trifluoroacetate
salt
1
(8EE*/8EEs, 56). H NMR (CD
3
OD) δ (ppm): 1.20 (t, 12H, J = 7.2 Hz); 1.34 (m, 32H); 1.50 (m,
1
3
8
H); 1.55 (m, 8H); 3.13 (t, 8H, J = 7.2 Hz); 3.16 (t, 8H, J = 7.2 Hz); 3.22 (q, 8H, J = 7.2 Hz). C
NMR (CD OD) δ (ppm): 12.97; 26.18; 26.55; 27.51; 28.44; 28.84; 28.94; 29.24; 29.39; 35.91; 41.03;
3
2+
3+
1
24.63; 155.88; 164.88; 165.23. LCMS m/z (ES+) = 425.1 [M + 2H] ; 284.0 [M + 3H] ; 213.4 [M
4
+
+
4H] YIELD: quantitative
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