Synthesis of new linear guanidines and macrocyclic amidinourea derivatives endowed with high antifungal activity against Candida spp. and Aspergillus spp.

Article abstract of DOI:10.1021/jm900760k

New linear and cyclic guanidines were synthesized and tested in vitro for their antifungal activity toward clinically relevant strains of Candida species, in comparison to fluconazole. Macrocyclic compounds showed a minimum inhibitory concentration in the micromolar range and a biological activity profile in some cases better than that of fluconazole. One macrocyclic derivative was also tested against Aspergillus species and showed high antifungal activity comparable to that of amphotericin B and itraconazole.

Full text of DOI:10.1021/jm900760k

7376 J. Med. Chem. 2009, 52, 7376–7379
DOI: 10.1021/jm900760k
by variable oral absorption and low bioavailability. For these
reasons, a drug active against Candida and Aspergillus patho-
gens and endowed with high bioavailability would be highly
desirable. Accordingly, in recent years, new structural classes
of antifungals have been reported,4 among which guanidine
derivatives proved to have a very interesting inhibitory activ-
ity.5 As an example, guazatine (a mixture of guanidines and
polyamines used in agriculture as fungicide) was classified as a
moderately hazardous antifungal agent, while results from in
vivo animal studies demonstrated a high potential for guaza-
tine and related compounds as antifungal agents.6 In this
context, we have recently reported that single components of
guazatine (compounds 1-6, Figure 1) are able to act toward
albicans and non-albicans Candida strains,7 laying the founda-
tion for designing new agents endowed with antifungal activ-
ity. On the basis of such results, we focused our efforts on the
synthesis of guanidine and polyamine derivatives related to
guazatine with the aim of identifying new potential antifungal
agents. Herein, we describe the synthesis of novel linear
guanidine and macrocyclic amidinourea derivatives and their
biological evaluation against clinical isolates of Candida spp.
Moreover, one of the amidinourea derivatives (13d), with the
best antifungal activity toward the entire panel of Candida
isolates, was also tested in vitro for its activity toward fungal
isolates representative of clinically relevant Aspergillus spp.
The natural components 3 and 5 of guazatine,7 showing
good antifungal activity toward Candida strains, were chosen
as models for the synthesis of the new derivatives. The 1,17-
diamino-9-azaheptadecane 7 was selected as a building block
for the synthesis of guanidine derivatives and was reacted with
N,N0-di-Boc-S-methylisothiourea, leading to the monogua-
nylated intermediate 8 (Scheme 1).8 The aminoguanidine 8
was then reacted with different N,N0-di-Boc-N-alkyl-S-methyl-
isothioureas 9a-f (previously synthesized via Mitsunobu reac-
tion or via phase-transfer catalyzed N-alkylation reaction,
Scheme 2),9 leading to desired linear guanidines 10 and/or to
the macrocyclic amidinoureas 11, in agreement with our recent
studies on the synthesis of macrocyclic amidinoureas10 showing
that linear aminoguanidines bearing a di-Boc-guanidine and a
primary or secondary amine moiety, such as 8or 10,aregenerally
converted into macrocyclic amidinoureas when heated in THF.
In some cases, because of the different rates of the guanyla-
tion/cyclization steps of 8, the linear guanidines 10 were the
only product obtained. On the other hand, since the cycliza-
tion of 10 into 11 started as soon as the guanylation step was
completed, amidinoureas 11 were obtained in most cases as
the only/major product, together with only a small amount of
10.11 The reaction of 7 with an excess of the isothiourea 9f led
to 14. Compounds 10, 11, and 14 were finally reacted with
10% TFA to afford deprotected linear guanidines 12 and 15
and cyclic amidinourea 13 in quantitative yield. The reaction
mixtures of 12 and 13 were then purified by semipreparative
HPLC, and the structureofthe macrocyclicamidinoureas was
confirmed by exact mass analysis (MALDI-TOF).
Synthesis of New Linear Guanidines and
Macrocyclic Amidinourea Derivatives Endowed
with High Antifungal Activity against Candida
spp. and Aspergillus spp.†
Fabrizio Manetti,‡ Daniele Castagnolo,‡ Francesco Raffi,‡
Alessandra T. Zizzari,‡ Suvi Rajamaki,‡ Silvia D’Arezzo,§
Paolo Visca,§, Alessandra Cona, Maria Enrica Fracasso,^
Denise Doria,^ Brunella Posteraro,# Maurizio Sanguinetti,#
Giovanni Fadda,# and Maurizio Botta*,‡
‡
ꢀ
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi
di Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italy, §National
Institute for Infectious Diseases “Lazzaro Spallanzani” I.R.C.C.S.,
Rome, Italy, Department of Biology, University “Roma Tre”, Viale
Guglielmo Marconi 446, I-00146 Rome, Italy, ^Department of
Medicine and Public Health Section of Pharmacology, University of
ꢀ
Sacro Cuore, Largo F. Vito, 1 I-00168 Rome, Italy
#
Verona, Italy, and Istituto di Microbiologia, Universita Cattolica del
Received May 30, 2009
Abstract: New linear and cyclic guanidines were synthesized and
tested in vitro for their antifungal activity toward clinically relevant
strains of Candida species, in comparison to fluconazole. Macro-
cyclic compounds showed a minimum inhibitory concentration in
the micromolar range and a biological activity profile in some cases
better than that of fluconazole. One macrocyclic derivative was also
tested against Aspergillus species and showed high antifungal
activity comparable to that of amphotericin B and itraconazole.
The opportunistic human pathogen Candida albicans and
other non-albicans species have acquired considerable clinical
significance as infectious agents in immunocompromised
patients, being important causes of morbidity and mortality.1
The pathogenic species of Candida derive their importance
from the severity of their infections and from their ability to
develop resistance against a variety of antifungal agents.2 In
addition to candidosis, other invasive filamentous fungal
infections, such as aspergillosis, are a major problem for
certain groups of patients. Clinically, candidosis and asper-
gillosis account for 80-90% of systemic fungal infections in
immunocompromised patients. However, whereas many
drugs have proven to be effective against Candida infections,
aspergillosis (including infections caused by the mostly en-
countered A. fumigatus, A. flavus, and A. terreus) remains very
hard to overcome. The antifungal agents currently used in
therapy are azoles (e.g., fluconazole and itraconazole), poly-
enes (e.g., amphotericin B), echinocandins, and allylamines.3
Among them, azoles are fungistatic and orally active agents
against most yeasts and filamentous fungi. Moreover, fluco-
nazole shows good antifungal activity with relatively low
toxicity and is preferred as firstline antifungal therapy. How-
ever, it has suffered severe drug resistance and is not effective
against invasive aspergillosis,3b unlike amphotericin B and
itraconazole, although wide use of itraconazole is hampered
A total of eight clinical isolates of five different Candida
species (obtained from respiratory specimens, with each strain
representing a single isolate from a patient) were tested.
Regarding chemical components found in the guazatine
mixture, molecular doubling of the guanidinooctyl moiety
†For the centennial anniversary of the the Division of Medicinal
Chemistry of the American Chemical Society (MEDI).
*To whom correspondence should be addressed. Phone: þ39 0577
234306. Fax: þ39 0577 234333. E-mail: botta@unisi.it.
r
pubs.acs.org/jmc
Published on Web 08/03/2009
2009 American Chemical Society

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7377
Scheme 2a
a Reagents and conditions: (i) ROH, DIAD, PPh3, THF; (ii) RBr,
KOH, Bu4NBr, DCM, H2O.
Figure 1. Structures of major components isolated from guazatine
mixture.
C. krusei reference strain, C. parapsilosis 64E, C. glabrata, and
C. tropicalis. Activity of 12c was comparable to that of 12f
against C. albicans 15T, C. krusei 193T, and both the reference
and 81E strains of C. parapsilosis. Among cyclic derivatives,
13f, bearing the bulkiest acyclic side chain (a prenyl group),
showed interesting activity toward C. albicans, C. krusei, and
C. tropicalis (10-40 μM) and lower activity against C. para-
psilosis and C. glabrata (40-80 μM). By reduction of the size
of the unsaturated moiety to the corresponding unbranched
chain (the butenyl group of 13d), the activity underwent a
significant increase toward C. albicans (1.25-2.5 μM) and
C. tropicalis (1.25 μM) and toward C. krusei and C. parapsilosis
(5 μM). The lowest activity of 13d was toward C. glabrata
(20 μM). Shortening and branching the butenyl chain into an
isobutenyl moiety (13e) caused a dramatic loss of activity
against all fungal strains. The best activity for this compound
was found toward the reference and 15T strains of C. albicans
(20 μM). Transforming the isobutenyl side chain to a linear
propargyl moiety as in 13b restored good activity toward
C. albicans (with the exception of C. albicans 4T that was
resistant to such a compound) and C. tropicalis (5 μM).
Finally, aromatization of the side chain to a benzyl group
(13a) led to activity data comparable in some cases
(C. albicans and C. tropicalis) to those found for the butenyl
derivative 13d. In summary, both the butenyl and benzyl
derivatives showed the best antifungal activity, followed by
the propargyl analogue that retained interesting activity
toward a wide number of fungal strains. Moreover, it seems
that linear side chains of cyclic derivatives are better for
activity in comparison to branched moiety (compare the
prenyl compound 13f with the butenyl analogue 13d, and
the isobutenyl 13e with the propargyl derivative 13b). Fluco-
nazole, used as the reference compound, showed a 0.8 μM
activity toward the reference strain of C. albicans.12 Interest-
ing activity was also retained against C. parapsilosis and, in
part, toward C. tropicalis, while activity against the remaining
C. albicans strains and against C. krusei and C. glabrata was
unimportant. Biological results showed that 13a, 13d, and the
linear derivative 12c were characterized by very good activity
values toward all the strains tested (with the sole exception of
the standard C. parapsilosis). They also showed a biological
activity profile better than that of fluconazole against
C. albicans (except the reference strain), C. krusei, C. glabrata,
and C. tropicalis. In contrast, activity of 13a and 12c toward
the reference and the 81E strains of C. parapsilosis was in
general lower, while a significantly better activity was found
toward the 64E strain, in comparison to fluconazole.
Scheme 1a
a Reagents and conditions: (i) N,N0-di-Boc-S-methylisothiourea, THF/
MeOH (5:3), 50 ꢀC; (ii) N,N0-di-Boc-N-alkyl-S-methylisothioureas 9a-f,
THF, 70 ꢀC; (iii) 10% TFA, dry CH2Cl2, 24 h, room temp; (iv) THF/
MeOH (5:3), 9f, 50 ꢀC; (v) 10% TFA, dry CH2Cl2, 24 h, room temp.
Compounds: 9a-13a, R = benzyl; 9b-13b, R = propargyl; 9c-13c,
R=cyclopropylmethyl; 9-13d, but-2-enyl; 9e-13e, R=isobutenyl; 9f-
13f, R=prenyl.
of 1 by an amino linkage led to iminoctadine (3) that showed
measurable antifungal activity toward many of the fungal
strains (Table 1), with a very good inhibition of C. tropicalis
(1.25 μM).7 Moreover, introduction of an additional guani-
dino moiety instead of the central amino group led to 5, found
to be much more efficient than 3 against C. albicans, while
comparable or lower activity was measured toward the re-
maining fungal strains. Prenylation of one of the terminal
guanidino groups of 3 led to 12f, characterized by a general
decrease in activity, with the sole slight improvement of
activity toward C. albicans 15T. The corresponding double
prenylated 15 showed an additional decrease in antifungal
activity with respect to 3 and 12f, suggesting that prenylation
of terminal guanidino groups of linear compounds was detri-
mental for antifungal activity toward clinical or reference
strains tested. On the other hand, transforming the prenyl
group of 12f into a cyclopropylmethyl moiety (12c) allowed a
significant enhancement of activity toward C. albicans, the

7378 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Manetti et al.
Table 1. Anticandidal Activity of Guazatine Components and Linear and Cyclic Guanidino Derivatives
antifungal activity (MIC, μM)a
Candida species
Gb
3
5
12cc
12fc
13a
13b
13d
13e
20
13f
40
15
Fd
0.8
C. albicans ATCC 60193
C. albicans 4T
C. albicans 53T
40
20
40
80
20
20
10
20
20
40
10
80
80
5
5
20
10
10
20
5
80
80
2.5
2.5
2.5
5
2.5
80
5
2.5
1.25
2.5
1.25
5
>80
80
40
40
20
20
209
418
209
209
418
40
80
20
10
80
40
80
C. albicans 15T
C. krusei ATCC 14243
C. krusei 193T
2.5
80
40
40
40
80
80
5
20
40
20
10
>80
40
20
20
10
40
40
20
20
10
80
20
20
40
2.5
10
80
5
5
80
20
80
C. parapsilosis ATCC 34136
C. parapsilosis 64E
C. parapsilosis 81E
C. glabrata 70E
>80
40
>80
>80
>80
>80
>80
>80
40
5
>80
>80
>80
80
>80
>80
40
>80
>80
>80
>80
>80
6.5
5
32
13
20
40
20
20
5
5
80
20
20
1.25
80
20
209
52
C. tropicalis 86E
1.25
1.25
40
a MIC values were determined at 24 h both visually and spectrophotometrically. b G is the guazatine mixture. c Compounds 12d and 12e were isolated
in amounts not enough for assays. d F is fluconazole.
Table 2. Activity of 13d toward clinical isolates of Aspergillus spp.
antifungal activity (μM)a
compd
A. nigerd
A. nigere
A. fumigatusd
A. fumigatusg
A. versicolord
A. versicolorf
A. terreusg
A. flavusd
13d
AMBb
ITRAc
18
18
73
36
18
4
73
1
36
0.27
0.35
0.135
0.35
0.135
0.35
0.27
0.04
0.54
0.02
0.135
0.35
2.1
0.04
0.35
a MIC values were determined visually at 48 h. b AMB is amphotericin B. c ITRA is itraconazole. d Isolated from sputum. e Isolated from nasal swab.
f Isolated from skin. g Isolated from bronchoalveolar lavage fluid.
Morevoer, 13d proved to be active toward all the C. para-
psilosisstrains withMICa values better thanorcomparable to
that of fluconazole. Among the guanidine derivatives, the
most active compound 13d was selected and tested in vitro
against clinical isolates belonging to five different Aspergillus
species that were obtained from respiratory and nonrespira-
tory specimens. In all cases, 13d showed a very good activity
against Aspergillus species, even if lower than that of ampho-
tericin B and itraconazole (Table 2). In particular, the best
activity was found toward A. niger and A. versicolor, with
MIC ranging from 4 to 18 μM. In vitro cytotoxicity of 13d was
checked in human lymphocytes at 25 and 37 ꢀC with com-
pound concentrations between 0 and 50 μM. Preliminary
results suggest that 13d does not induce cellular damage at
doses up to 30 μM (100% viability), while it interacts with the
cellular membrane at the highest concentrations (40 and 50
M), thus reducing the lymphocyte viability to 78% and 43%,
respectively. As expected, temperature increases the toxic
effects.13 Under these experimental conditions, the therapeu-
tic indices (TI50) related to the various Candida species were
evaluated on the basis of the 50% viability and MIC50.
As a result, TI50 was between 38 and 2.4 μM at 25 ꢀC and
between 30 and 1.9 μM at 37 ꢀC. Hence, the broad spectrum
antifungal activity and high solubility in aqueous medium and
low cytotoxicity make 13d and its derivatives a very attractive
class of compounds in the treatment of topical and systemic
fungal infections.
In conclusion, this study demonstrates that analogues of
guazatine components have a high potential as antifungal
agents, being characterized by a low toxicity in preliminary
cell-based assays and by a good inhibitory profile against
common fungal pathogens, thus showing an activity profile
in many cases better than fluconazole. Fungi often infect
the skin surface and subsequently invade the stratum
corneum to avoid being shed from the skin surface by
desquamation. Pharmacologic agents applied to the surface
of the skin in the form of creams, lotions, or sprays readily
penetrate the stratum corneum to kill the fungi (fungicidal
agents) or at least render them unable to grow or divide
(fungistatic agents).14 In this context, biological activity
data reported here suggest a potential role of the new
guanidines for the topical treatment of Candida infections
and contribute to the active and challenging research on
development of novel antifungal agents. However, the
development of new antifungal compounds and the use of
alternative approaches in immunocompromised patients
should be pursued through well-designed laboratory and
clinical studies. For this purpose, additional studies are
required to clearly understand the mechanism of action of
guazatine derivatives and to define precisely the role of
a Abbreviations: G, guazatine mixture; F, fluconazole, MIC, mini-
mum inhibitory concentration; AMB, amphotericin B; ITRA, itraco-
nazole, LD50, lethal dose 50%; NOAEL, no observed adverse effect
level.

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7379
these new agents alone or in combination with other anti-
fungal agents. Further functional characterization of the hit
compounds 12c, 13a, and 13d is planned, and their results
will be reported in due time.
NOAEL of guazatine was 200 ppm, equivalent to 10 mg/kg bw per
day. In a 1-year study in dogs, the NOAEL was 25 ppm, equal to
0.8 mg/kg bw per day. Several studies in rats concluded that
guazatine is not genotoxic and carcinogenic, while in a study of
developmental toxicity in rabbits there were no signs of fetotoxicity
or teratogenicity. FAO Corporate Document Repository.
(7) Dreassi, E.; Zizzari, A. T.; D’Arezzo, S.; Visca, P.; Botta, M.
Analysis of guazatine mixture by LC and LC-MS and antimycotic
activity determination of principal components. J. Pharm. Biomed.
Anal. 2007, 46, 1499–1506.
Acknowledgment. We are grateful to the Italian Ministero
ꢀ
dell’Istruzione, dell’Universita e della Ricerca (Grant PRIN
2006030948).
(8) Delle Monache, G.; Botta, B.; Delle Monache, F.; Espinal, R.; De
Bonnevaux, S. C.; De Luca, C.; Botta, M.; Corelli, F.; Carmignani,
M. Novel hypotensive agents from Verbesina caracasana. 2.
Synthesis and pharmacology of caracasanamide. J. Med. Chem.
1993, 36, 2956–2963.
Supporting Information Available: General procedures and
experimental data for compounds 9-15. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
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protected guanidines using the Mitsunobu protocol. Tetrahedron
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