Synthesis of linear and cyclic guazatine derivatives endowed with antibacterial activity

Article abstract of DOI:10.1016/j.bmcl.2014.09.081

Antibiotic resistance has reached alarming levels in many clinically-relevant human pathogens, and there is an increasing clinical need for new antibiotics active on drug-resistant Gram-negative pathogens who rapidly evolve towards pandrug resistance phenotypes. Here, we report on two related classes of guanidinic compounds endowed with antibacterial activity. The two best compounds (9a and 13d) exhibited the most potent antibacterial activity with MIC values ranging 0.12-8 μg/ml with most tested pathogens, including both Gram-positive and Gram-negative bacteria. Interestingly, MIC values were not affected (1-8 μg/ml) when measured using recent clinical isolates with various antibiotic resistance determinants. The results reported herein identify guazatine derivatives as an interesting starting point for the optimization of a potentially novel class of antibacterial agents.

Full text of DOI:10.1016/j.bmcl.2014.09.081

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5525–5529
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of linear and cyclic guazatine derivatives endowed with
antibacterial activity
Giorgio Maccari a, Stefania Sanfilippo a, Filomena De Luca b, Davide Deodato a, Alexandru Casian a,
Maria Chiara Dasso Lang a, Claudio Zamperini a, Elena Dreassi a, Gian Maria Rossolini b,
Jean-Denis Docquier b, Maurizio Botta a,c,d,
⇑
a Department of Biotechnology, Chemistry and Pharmacy, University of Siena, I-53100 Siena, Italy
b Department of Medical Biotechnology, University of Siena, I-53100 Siena, Italy
c Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Building, Suite 333,
1900 N 12th Street, Philadelphia, PA 19122, USA
d Lead Discovery Siena s.r.l, Via Vittorio Alfieri 31, I-53019 Castelnuovo Berardenga, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Antibiotic resistance has reached alarming levels in many clinically-relevant human pathogens, and there
is an increasing clinical need for new antibiotics active on drug-resistant Gram-negative pathogens who
rapidly evolve towards pandrug resistance phenotypes. Here, we report on two related classes of guan-
idinic compounds endowed with antibacterial activity. The two best compounds (9a and 13d) exhibited
Received 19 August 2014
Revised 25 September 2014
Accepted 26 September 2014
Available online 14 October 2014
the most potent antibacterial activity with MIC values ranging 0.12–8
including both Gram-positive and Gram-negative bacteria. Interestingly, MIC values were not affected
(1–8 g/ml) when measured using recent clinical isolates with various antibiotic resistance determi-
lg/ml with most tested pathogens,
Keywords:
Guanidinic compounds
Antibacterial
Drug resistant bacteria
Macrocycles
l
nants. The results reported herein identify guazatine derivatives as an interesting starting point for the
optimization of a potentially novel class of antibacterial agents.
Ó 2014 Elsevier Ltd. All rights reserved.
The treatment of bacterial infections caused by multi-drug,
extensively-drug or even pan-drug resistant strains is becoming
challenging for health care practitioners. The prevalence of drug-
resistant isolates has reached alarming levels in many significant
human pathogens, among both Gram-negative and Gram-positive
pathogens. These are often referred to as ‘ESKAPE’ organisms,
which include Enterococcus faecium, Staphylococcus aureus, Klebsi-
ella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa
and Enterobacter spp.1–5 This term emphasizes that these organ-
isms have the potential to escape most of the available antibacte-
rial therapies. It has been reported that, in U.S.A. hospitals, more
people currently die of methicillin-resistant S. aureus than of
HIV/AIDS and tuberculosis combined.6–9 Moreover, pandrug-resis-
tant strains are frequently reported emerging in clinically-relevant
Gram-negative pathogens, such as P. aeruginosa, A. baumannii and
K. pneumoniae, and may represent a dramatic pace forward the
return to the pre-antibiotic era.5,10–12
drugs, such as colistin, often burdened by a significant toxicity
and severe side effects. As illustrated by the growing concern
expressed by many public health agencies and authorities, the
need for new therapies and drugs active against resistant strains
should be urgently addressed to eventually overcome severe con-
sequences for global Human Health.
Our research group has been involved for years into in-depth
studies on linear and cyclic derivatives of guazatine, some of which
showed broad-spectrum antifungal properties.13–18 The potential
antibacterial properties of a series of such compounds was evalu-
ated with a panel of different bacteria, including both type strains
and clinical isolates (showing various antibiotic susceptibility pro-
file), allowing the identification of some active molecules. A quite
large number of compounds bearing guanidine moieties have been
reported in the literature as broadly active agents against microbial
pathogens, in particular against parasitic fungi.14,15,19–24 The anti-
bacterial activity of such compounds was not, at our best knowl-
edge, previously evaluated. In this work, compounds from two
series were tested on 13 different bacterial species, including some
ESKAPE organisms.
Due to both the lack of investment in antibiotic R&D and the
increased spread of resistant strains, the therapeutic options are
diminishing and might be limited, in some cases, to suboptimal
Described compounds (Table 1) can be divided in two classes of
guazatine derivatives, linear (Scaffold A) and cyclic (Scaffold B).
Although the syntheses of these compounds involved some
⇑
Corresponding author. Tel./fax: +39 0577 234306.
E-mail address: botta.maurizio@gmail.com (M. Botta).
http://dx.doi.org/10.1016/j.bmcl.2014.09.081
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

5526
G. Maccari et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5525–5529
common steps, they differs remarkably due to the different reactiv-
ity of the intermediates.
NBoc
NHBoc
NBoc
N
i
R
N
N
1
N
N
The guanylating agents used for each synthesis have been
obtained through Mitsunobu reaction between the desired alcohol
and the di-Boc-pyrazole-1-carboximidamide (Scheme 1).25 Com-
pounds 9a–d and 12a–e have been described elsewhere.14,22,24
Linear compounds 5a–c have been obtained following the
straightforward protocol described in Scheme 2. Starting material
bis(hexamethylene)triamine was subsequently guanylated with
the appropriate guanylating agent furnishing, after Boc cleavage,
the desired derivatives 5a–c.
ROH
Boc
2
Scheme 1. Synthesis of guanylating compounds 2. Reagent and conditions: DIAD,
PPh3, THF dry, 0 °C to reflux, 12 h.
The synthesis of compounds 9a–h, bearing longer alkyl chains,
is reported in Scheme 3 starting from the common intermediates
6a–c already described in a previous work.14,21,24
NBoc
+
N
H2N
N
H
NH2
4
4
BocN R1
N
2
After Cbz deprotection the primary amines 7a–c were guanylat-
ed and Boc-deprotected leading to the desired diguanidines 9a–h.
On the other hand, cyclization was obtained as reported in
Scheme 4, by refluxing the common intermediate 6 in THF leading
i
NBoc
N
R1
H2N
R2
N
H
N
H
4
4
Boc
Table 1
3a-b
Structures of the synthesized compounds
NBoc
N
H
+
N
NH2
O
N
R3
N
N
+
i
H2
H
N
H
N
H
N
Boc
HN
N
• 2 CF3COO-
R1
R2
R2
n
n
H
N
H
N
• 3 CF3COO-
+
+
2
N
NH2
NH2
n
+
Scaffold A
NH2
NBoc
N
NBoc
N
Scaffold B
R2
R1
N
N
H
N
H
4
4
R1
R2
H
R3
H
Boc
H
Boc
Compd
Scaffold
A
n
4a-c
5a
4
ii
5b
A
A
A
4
4
6
H
H
H
NH2
NH2
R2
R1 3CF3COO
5c
H
H
N
H
N
H
N
N
H
N
H
4
4
H2
9a14,22
5a-c
9b22
9c22
A
A
6
6
H
H
H
Scheme 2. Synthesis of compounds 5a–c. Reagents and conditions: (i) DIPEA,
CH3CN/MeOH, 50 °C, 12 h, (ii) TFA, DCM, rt, 8 h.
H
H
9d22
9e
A
A
6
6
H
H
R3
H
R1
R3
N
H
N
H
N
Boc
N
a=
b=
H
H
R1
CH3
Cbz
6
6
9f
A
A
6
6
H
H
CH3
CH3
NBoc
c=
H
6a-c
9g
i
R3
H
Boc
N
9h
A
6
H
CH3
H2N
N
N
R1
6
6
NBoc
13a24
13b14
13c
B
B
B
6
6
6
H
H
H
H
H
H
7a-c
NBoc
N
R2
N
ii
Boc
2
N
13d14
B
6
H
H
R3
N
Boc
H
N
H
N
Boc
N
13e24
13f
B
B
6
4
H
H
H
H
N
R2
R1
6
+
6
8a-h
NBoc
NBoc
iii
13g
B
4
H
H
R3
N
H2
N
H
N
H
N
13h
13i
B
B
4
4
H
H
H
H
NHR1
R2
6
6
• 3 CF3COO-
+
+
NH2
NH2
9a-h
13j
B
4
H
H
Scheme 3. Synthesis of linear compounds 9a–h. Reagent and conditions: (i) H2 Pd/
C, 2-propanol, HCl, rt, 5 h; (ii) DIPEA, CH3CN, 50 °C, 12 h, (iii) TFA, DCM, rt, 8 h.

G. Maccari et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5525–5529
5527
to the formation of the desired macrocycles with good yield. The
Cbz protecting group was then cleaved by hydrogenation affording
the corresponding primary amine 11a–b. This latter compound
was then guanylated with the appropriate N-substituted pyra-
zole-1-carboximidamide leading, after Boc cleavage, to the desired
derivatives 13a–j. The purity of the compounds was assessed by
means of HPLC as described in Supplementary information.
Aminooctanoic acid 14 was selected as building block for the
synthesis of compounds 9f–h (Scheme 5). The starting material
14 was reacted with acyl chloride in dry MeOH affording 15 which
free amino group was protected using benzyl chloroformate. The
ester moiety of 16 was then reduced with DIBAL-H in DCM to give
the intermediate 17. The latter was reacted with p-toluenesulfonyl
chloride, DMAP and Et3N in DCM to give 18. The methyl amino
moiety was introduced as described by Sharpless and Gao by react-
ing compound 18, previously dissolved in THF, with 40% aqueous
methylamine.26
Compound 20 was obtained in quantitative yield by reacting 19
with 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine in DCM and
Et3N.26 The deprotection of the primary amine of 20 was done by
hydrogenolysis using 10% Pd/C and H2 atmosphere. Since the inter-
mediate 21 is unstable as free amine, during the hydrogenolysis we
used 36% HCl in order to block the amino group in a hydrochloride
salt form. To obtain the linear intermediate 6b, the aldehyde 22
was reacted with 21 in a reductive amination reaction using
triacetoxyborohydride as mild reductive agent in dry DCE.27 The
subsequent functionalization of compound 6b was accomplished
as described in Scheme 3.
All compounds were obtained as trifluoroacetic salts and have
been tested in this form. Totally, we synthesized twenty-one guaz-
atine derivatives as reported in Table 1, eleven belonging to the lin-
ear family and ten to the macrocyclic one. Among the latters, five
have an eight carbons side chain (13a–e) and five a six carbons side
chain (13f–j). Various groups have been attached to the guanidine
moiety looking for a structure activity relationship (see Table 2).
Bacterial strains, including representatives of both Gram-posi-
tive and Gram-negative bacteria, as well as clinical isolates show-
ing various level of antibiotic resistance, were obtained from the
ATCC or CCUG culture collections or present in the authors’ collec-
tion of clinical isolates.28–31 Compounds were resuspended in
dimethyl sulfoxide (DMSO) at a final concentration of 100 mg/ml
and subsequently diluted in the culture medium. The minimum
inhibitory concentrations (MICs) of the compounds were deter-
mined using the micro-dilution broth method using Mueller–Hin-
ton broth as recommended by the Clinical Laboratory Standards
Institute (CLSI).32 Bacterial inoculum was 5 Â 104 CFU/well. MICs
were recorded after 16–18 h incubation at 35–37 °C.
Compounds 9a and 13d showed potent and broad-spectrum
antibacterial activity, being active against representatives of both
Gram-positive and Gram-negative bacteria. Furthermore, the mac-
rocyclic compound bearing the cyclopropylmethyl group, com-
pound 13a, is only moderately active against Gram-positive
bacteria and almost inactive against Gram-negatives. Similarly,
compound 9b, which can be considered the linear analogue of
compound 13d, is also completely inactive against both Gram-neg-
ative and Gram-positive organisms.1–5
H
N
H
N
H
N
NHBoc
NBoc
Cbz
n
6
6a, 6d
Linear compound 9e bearing symmetric substitutions on the
two guanidinic moieties also was also found to be inactive. Similar
results were obtained for linear compound 5a, bearing shorter
chains linking the central nitrogen and the guanidinic moieties.
Overall, these data suggest that the nature of the substituents
and the distance between the guanidinic moieties control the
activity of the linear compounds. This hypothesis is further sup-
ported by analyzing compounds 9f–h, which showed lower anti-
bacterial activity, as compared to their demethylated counterparts.
Macrocyclic compounds 13f–j characterized by a shorter side
chain were found completely inactive on Gram-negative bacteria,
and only moderately active on Gram-positives. This finding sug-
gests that, in these macrocyclic compounds, the distance between
the two guanidinic moieties might be determinant for their activ-
ity, and this information might be important to allow the design of
optimized compounds.
Interestingly, the antibacterial potency of compounds 9a and
13d was poorly affected when MIC values were determined with
recent multi-drug resistant clinical isolates, such as carbapenem-
ase-producing Gram-negative pathogens and vancomycin-resis-
tant staphylococci (see Table 3). The apparent absence of cross-
resistance with existing mechanisms is interesting and further
could support the potential of such compounds to address antibi-
otic resistance in relevant pathogens, although their mechanism
of action has not been elucidated yet.
H
N
i
NBoc
O
HN
H
N
N
Cbz
n
10a-b
H
N
NBoc
O
ii
HN
N
NH2
n
11a-b
NBoc
iii
R1
N
N
N
H
N
Boc
NBoc
O
2
HN
H
N
Boc
N
N
R1
n
12a-j
NBoc
iv
H
N
+
In vitro ADME properties (apparent permeability in gastrointes-
tinal model, water solubility, microsomal stability) for the most
active compound 9a were evaluated (see Table 4).33,34 The parallel
artificial membrane permeability assays (PAMPA) reveal a low
value of apparent permeability (Papp) at physiological pH, while,
in duodenal alkaline condition (pH 10) an higher Papp was found,
as expected for a positively ionizable compound.35 Noteworthy,
compound 9a shows a solubility of 0.966 0.022 g/L in pure water
and metabolic stability, measured by means of human liver micro-
somal proteins, higher than 99%. Moreover, compound 9a binds
NH2
HN
O
H
N
H
N
• 2 CF3COO-
n = 4, 6
N
R1
n
+
13a-j
NH2
Scheme 4. Synthesis of cyclic compounds 13a–j. Reagents and conditions: (i) THF,
reflux, 12 h; (ii) H2 Pd/C, 2-propanol, HCl, rt, 5 h; (iii) DIPEA, CH3CN, 50 °C, 12 h, (vi)
TFA, DCM, rt 8 h.

5528
G. Maccari et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5525–5529
O
6
O
O
ii
i
Cbz
Cl-
+H3N
O
N
H
O
H2N
OH
6
6
16
17
15
14
iii
iv
v
N
H
CbzHN
CbzHN
OTs
CbzHN
OH
6
6
6
18
19
vi
viii
O
NBoc
NHBoc
NBoc
NHBoc
vii
+
Cl- +
N
H
CbzHN
ix
H3N
N
CbzHN
6
6
6
22
21
20
NBoc
N
H
6b
N
NHBoc
CbzHN
6
6
Scheme 5. Synthesis of intermediate 6b. Reagent and conditions: (i) acetyl chloride, dry MeOH, reflux, 12 h; (ii) CbzCl, NaHCO3, H2O/THF 1:1, À7 °C–rt, 12 h; (iii) 1 M DIBAL-
H, DCM, 0 °C–rt, 12 h; (iv) 4-toluenesulfonyl chloride, TEA, DMAP, DCM, 0 °C–rt, 12 h; (v) 40% CH3NH2, THF, 65 °C 5 h; (vi) 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine,
TEA, DCM, 12 h; (vii) H2 1 atm, Pd/C 10%, HCl 36%, 2-propanol, 4 h; (viii) DMP, NaHCO3, DCM, rt, 5 h (ix) NaBH(OAc)3, dry DCE, 16 h.
Table 2
Antibacterial activity of tested compounds against representative strains of Gram-positive and Gram-negative bacteria
Bacterial strain
MIC (
lg/ml)
9a
9b
>256 64 128 >256 >256 64
>256 64 256 >256 >256 n.d. 64 64
9c 9d
9e
5a
13a 13b 13c 13d 13e 13f
13g 13h 13i
256 256 >256 >256 256 256 256 128
n.d. >256 >256 >256 >256 >256 >256 >256 256
64 64
13j
9f
9g
9h
Acinetobacter baumannii ATCC 17978
Aeromonas hydrophila ATCC 7966
Elizabethkingia meningoseptica CCUG
4310
4
8
32
32 32
8
4
64
>256 n.d. >256 >256 >256 n.d. 128 256 32 n.d. 256 >256 >256 >256 >256 32
Escherichia coli CCUGT
0.5
1
8
0.5
1
4
128
256 16 32
>256 64 256 >256 >256 >256 256 >256
64
8
16
>256 >256 n.d. 64 64
>256 256 128 32 32
0.5 n.d. 256 256 >256 >256 256 >256 >256 >256
Klebsiella pneumoniae ATCC 13833
Pseudomonas aeruginosa ATCC 27853
Bacillus subtilis ATCC 6633
Enterococcus faecalis ATCC 19433
Staphylococcus aureus ATCC 25923
Staphylococcus epidermidis ATCC
14990
1
8
128 >256 >256 >256 >256 >256
>256 >256 >256 >256 >256 >256
8
8
16
32
32
32
4
4
>256 >256
>256 16
4
32
4
8
4
8
0.5
1
1
16
16
64
64
64
64
256 256 >256
16
256 16 16
128 32
128 128 16
4
8
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
64
0.5
4
4
>256 256
8
4
4
0.5 16
16
16
32
32
16
8
16
32
Streptococcus pyogenes ATCC 12344
<0.125 16
1
2
>256 32
32
2
2
0.5
4
32
8
16
16
8
8
16
16
n.d.: Not determined.
Table 3
Antibacterial activity of compounds 9a and 13d on a panel of Gram-negative and Gram-positive clinical isolates, showing various antimicrobial susceptibility profiles
Bacterial strains
Resistance profile
MIC (lg/ml)
9a
13d
Acinetobacter baumannii AC-54/97
Achromobacter xylosoxidans AX 22
Alcaligenes faecalis 424/98
Enterobacter cloacae VA-417/02
Klebsiella pneumoniae 7023
Pseudomonas aeruginosa 101/1477
Pseudomonas aeruginosa VR-143/97
Stenotrophomonas maltophilia 634/08
Staphylococcus aureus ATCC 43300 (MRSA)
Staphylococcus aureus ATCC 700699 (VanA)
Staphylococcus haemolyticus SI-6/2011
Staphylococcus warneri SI-5/2011
Staphylococcus hominis SI-7/2011
PEN, ES-CEPH, CARB, AZT, AG, FQ, FOS, SXT
PEN, ES-CEPH, CARB, AG
ES-CEPH, CARB, MON
PEN, CEPH, CARB, AG, FQ
PEN, ES-CEPH, CARB, AG, FQ, SXT
PEN, ES-CEPH, CARB, AG
PEN, ES-CEPH, CARB, MON, AG, FQ
PEN, ES-CEPH, CARB, MON, AG, SXT, FOS
PEN
GLY
AG
PEN, AG
LNZ
4
4
4
2
2
8
8
4
4
4
2
2
2
4
8
4
1
1
8
8
4
n.d.
n.d.
n.d.
n.d.
n.d.
The class of drugs for which the isolate was resistant to is indicated in column 2 (PEN, penicillins; ES-CEPH, expanded-spectrum cephalosporins; CARB, carbapenems; AZT;
aztreonam [a monobactam]; AG, aminoglycosides; FQ, fluoroquinolones; SXT, trimethoprim/sulfamethoxazole; FOS, fosfomycin; GLY, glycopeptides; LNZ, linezolid). Strains
were from either ATCC, our strain collection or described in Pereira et al., 2007 and references therein, Rossolini et al., 2000 and Cagnacci et al., 2008.29–31
n.d.: Not determined.

G. Maccari et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5525–5529
5529
Table 4
PAMPA assay results for compound 9a rifaximin and chloramphenicol
References and notes
1. Frazee, B. W.; Lynn, J.; Charlebois, E. D.; Lambert, L.; Lowery, D.; Perdreau-
Remington, F. Ann. Emerg. Med. 2005, 45, 311.
2. Giske, C. G.; Monnet, D. L.; Cars, O.; Carmeli, Y. Antimicrob. Agents Chemother.
2008, 52, 813.
Papp (nm/s)
% membrane retention
pH 7.4
pH 10.0
pH 7.4
pH 10.0
9a
9.5 6.0
0.06 0.01
0.30 0.5
166.5 7.0
0.12 0.09
0.57 0.10
8.8 0.5
0.0
60.6 0.4
0.0
3. Rice, L. B. J. Infect. Dis. 2008, 197, 1079.
Rifamixin
Chloramphenicol
4. Spellberg, B.; Guidos, R.; Gilbert, D.; Bradley, J.; Boucher, H. W.; Scheld, W. M.;
Bartlett, J. G.; Edwards, J., Jr. Clin. Infect. Dis. 2008, 46, 155.
5. Theuretzbacher, U. Int. J. Antimicrob. Agents 2012, 39, 295.
6. Boucher, H. W.; Corey, G. R. Clin. Infect. Dis. 2008, 46, S344.
7. Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L. B.;
Scheld, M.; Spellberg, B.; Bartlett, J. Clin. Infect. Dis. 2009, 48, 1.
8. Klevens, R. M.; Edwards, J. R.; Tenover, F. C.; McDonald, L. C.; Horan, T.; Gaynes,
R. Clin. Infect. Dis. 2006, 42, 389.
0.0
0.0
strongly to human serum albumins with a Kd of 26.52 7.2 lM, the
value is comparable to that of known bioactive compounds such as
Diazepam.36
To better assess the potential of compound 9a, which shows the
most potent antibacterial activity, its cytotoxicity was investigated.
The CC50 values against HeLa cells after 24 h and 48 h of incubation
9. Klevens, R. M.; Morrison, M. A.; Fridkin, S. K.; Reingold, A.; Petit, S.; Gershman,
K.; Ray, S.; Harrison, L. H.; Lynfield, R.; Dumyati, G.; Townes, J. M.; Craig, A. S.;
Fosheim, G.; McDougal, L. K.; Tenover, F. C. Emerg. Infect. Dis. 2006, 12,
1991.
10. Livermore, D. M. J. Antimicrob. Chemother. 2009, 64, i29.
11. Nordmann, P.; Poirel, L.; Toleman, M. A.; Walsh, T. R. J. Antimicrob. Chemother.
2011, 66, 689.
12. Martinez, J. L.; Baquero, F. Ups. J. Med. Sci. 2014, 119, 68.
13. Dreassi, E.; Zizzari, A. T.; D’Arezzo, S.; Visca, P.; Botta, M. J. Pharm. Biomed. Anal.
2007, 43, 1499.
14. Manetti, F.; Castagnolo, D.; Raffi, F.; Zizzari, A. T.; Rajamaki, S.; D’Arezzo, S.;
Visca, P.; Cona, A.; Fracasso, M. E.; Doria, D.; Posteraro, B.; Sanguinetti, M.;
Fadda, G.; Botta, M. J. Med. Chem. 2009, 52, 7376.
15. Castagnolo, D.; Schenone, S.; Botta, M. Chem. Rev. 2011, 111, 5247.
16. Berlinck, R. G.; Burtoloso, A. C.; Kossuga, M. H. Nat. Prod. Rep. 2008, 25,
919.
17. Berlinck, R. G.; Burtoloso, A. C.; Trindade-Silva, A. E.; Romminger, S.; Morais, R.
P.; Bandeira, K.; Mizuno, C. M. Nat. Prod. Rep. 2010, 27, 1871.
18. Berlinck, R. G.; Trindade-Silva, A. E.; Santos, M. F. Nat. Prod. Rep. 2012, 29, 1382.
19. Matthijs, S.; Adriaens, P. A. J. Clin. Periodontol. 2002, 29, 1.
20. Cona, A.; Manetti, F.; Leone, R.; Corelli, F.; Tavladoraki, P.; Polticelli, F.; Botta, M.
Biochemistry 2004, 43, 3426.
21. Botta, M.; Raffi, F.; Visca, P. WO 200,911,303,3A2; 2009, p 27.
22. Manetti, F.; Cona, A.; Angeli, L.; Mugnaini, C.; Raffi, F.; Capone, C.; Dreassi, E.;
Zizzari, A. T.; Tisi, A.; Federico, R.; Botta, M. J. Med. Chem. 2009, 52,
4774.
23. Feng, E.; Ye, D.; Li, J.; Zhang, D.; Wang, J.; Zhao, F.; Hilgenfeld, R.; Zheng, M.;
Jiang, H.; Liu, H. ChemMedChem 2012, 7, 1527.
24. Sanguinetti, M.; Sanfilippo, S.; Castagnolo, D.; Sanglard, D.; Posteraro, B.;
Donzellini, G.; Botta, M. ACS Med. Chem. Lett. 2013, 4, 852.
25. Mitsunobu, O.; Yamada, M. Bull. Chem. Soc. Jpn. 1967, 40, 2380.
26. Katritzky, A. R.; Rogovoy, B. V. ARKIVOC 2005, 49.
27. Abdel-Magid, A. F.; Mehrman, S. J. Org. Process Res. Dev. 2006, 10, 971.
28. Riccio, M. L.; Pallecchi, L.; Fontana, R.; Rossolini, G. M. Antimicrob. Agents
Chemother. 2001, 45, 1249.
29. Pereira, M.; Perilli, M.; Mantengoli, E.; Luzzaro, F.; Toniolo, A.; Rossolini, G. M.;
Amicosante, G. Microbiol. Drug Resist. 2000, 6, 85.
30. Rossolini, G. M.; Docquier, J. D. In Enzyme-Mediated Resistance to Antibiotics:
Mechanisms, Dissemination, and Prospects for Inhibition; Bonomo, R. A.,
Tolmasky, M., Eds.; ASM Press, 2007; p 115.
were 92.6 0.7 lM and 62.0 0.3 lM, respectively. Although the
cytotoxicity would ideally be improved with the future series, it
is encouraging that the observed CC50 values are P70-fold higher
than the MIC value observed for E. coli and K. pneumoniae.
Presently, it was neither possible to assess whether cyclic and
linear compounds had a similar mechanism of action, nor to iden-
tify the target at the molecular level. Nevertheless, these com-
pounds do not show a lytic effect at concentrations up to 10Â
MIC on E. coli, suggesting that they would not act through an aspe-
cific membrane permeabilization mechanism. It is reasonable to
think that the mode of action of our compounds can be specific,
as suggested by the loss of activity due to small changes in the sub-
stituent attached to the guanidinic moiety.
The broad spectrum activity of 9a and 13d, which also covers
Gram-negative bacteria (including MDR clinical isolates of patho-
gens currently evolving towards XDR and PDR resistance pheno-
types) make them promising candidates for future studies.
Accordingly, more chemical modifications of the linker, such as
introduction of less flexible groups and new functionalizations of
the guanidinic group will be explored to further optimize these
starting hits. Moreover, a larger effort will be made to elucidate
the mode of action of both linear and cyclic guazatine derivatives
reported herein. In conclusion, we have identified original guanid-
inic compounds showing a promising potent and broad-spectrum
in vitro antibacterial activity, which might represent a valuable
starting point to obtain optimized analogues with enhanced
drug-like properties.
31. Cagnacci, S.; Gualco, L.; Roveta, S.; Mannelli, S.; Borgianni, L.; Docquier, J. D.;
Dodi, F.; Centanaro, M.; Debbia, E.; Marchese, A.; Rossolini, G. M. J. Antimicrob.
Chemother. 2008, 61, 296.
Acknowledgments
32. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow
Aerobically: Approved Standard; Wayne, PA 19087 USA: Clinical and Laboratory
Standards Institute, 2012.
We thank the University of Siena and Lead Discovery Siena S.r.l.
for financial support.
33. Sugano, K.; Hamada, H.; Machida, M.; Ushio, H. J. Biomol. Screen. 2001, 6, 189.
34. Wohnsland, F.; Faller, B. J. Med. Chem. 2001, 44, 923.
35. Permeability classification: high >200 nm/s, medium 100–200 nm/s, low
<100 nm/s.
36. Parikh, H. H.; McElwain, K.; Balasubramanian, V.; Leung, W.; Wong, D.; Morris,
M. E.; Ramanathan, M. Pharm. Res. 2000, 17, 632.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09.
081.