ACS Medicinal Chemistry Letters
Letter
The metalloenzyme carbonic anhydrases (CAs, EC 4.2.1.1)
recently identified in these protozoans are novel promising
targets for chemotherapeutic interventions.6,8,9 CAs catalyze the
reversible hydration of CO2 to bicarbonate and proton, a pivotal
reaction for all cells and complex organisms, which is also basic
in the growth and virulence of pathogenic microorganisms.9 CAs
from Trypanosoma cruzi (TcCA) and Leishmania donovani
chagasi (LdcCA) were cloned and characterized in 2013,10−12
resulting in the design of novel antiprotozoal agents that act by a
totally new mechanism of action and lack cross-resistance to
existing drugs. The α-CA TcCA is endowed with a very high
catalytic activity for the CO2 hydration reaction and was shown
to be inhibited in the nanomolar range by many types of CA
inhibitors (CAIs) such as aromatic/heterocyclic sulfona-
mides,10,13,14 sulfamates,10 thiols,10 anions,15 dithiocarba-
mates,15 hydroxamates,16 and benzoxaboroles.17 Thiols and
hydroxamates exhibited in vitro antitrypanosomal activity,
inhibiting the three phases of the pathogen’s life cycle.10,16
The β-CA LdcCA also features an effective catalytic activity and
was shown to be efficiently inhibited by sulfonamides and
heterocyclic thiols with nanomolar inhibition constants.12,18
Some such thiol derivatives displayed in vitro antileishmania
activity in preliminary assays being able to reduce parasites’
growth and causing their death.12 Identification of new
protozoans CAIs with effective antitrypasonomal or antileish-
mania activities is more than ever worth the endeavor due to
such targets’ remarkable druggability.
protozoa entities to be synergistically exploited to overcome
resistance issues displayed by single-targeted therapy.
The general synthetic strategy proposed by Minksztym24 for
the chemoselective mononitration of aminosulfonamides was
applied to a set of ten starting compounds being commercially
available (1−3, 7, 8) or yielded by methylation (4−6) or
deacetylation (17, 19) reactions (Schemes 1 and 2).
Scheme 1. Synthesis of Aromatic N-Nitrosulfonamides: (a)
H2O; (b) NH4OH(aq)
Scheme 2. Synthesis of Heteroaromatic N-Nitrosulfonamides
Nifurtimox and benznidazole have been the first effective
drugs for treating acute-phase human Chagas infection, with the
first being no longer available on the market because of
undesirable side effects.19 They feature heteroaromatic nitro
moieties that are pivotal for the antiprotozoa mechanism of
action. Parasite resistance arisen with benznidazole drove the
development of alternative therapies. Indeed, combined treat-
ment of benznidazole with drugs with different mechanisms of
action such as azoles, nitric oxide, or clomipramine could be a
strategy to improve the pharmacotherapy efficacy.6
Noteworthy, a new chemotype able to afford α- and β-CAs
inhibition was reported by us in 2016, namely, N-nitro-
sulfonamides.20 Interestingly, these latter were shown to inhibit
ubiquitous, off-targets isoforms, such as CA II, feebler than lead
sulfonamides though holding remarkable submicromolar
inhibition of the human (h) tumor-associated CA IX (α-CA)
and the β-CA from the pathogen fungus Malassezia Globosa.
Considering the above, we have herein extended the set of N-
nitrosulfonamides and screened them on a wider pattern of
human and pathogen (from protozoa and fungi) CAs, among
which are the targets α-TcCA and β-LdcCA.
Quenching and workup of the NH4NO3/H2SO4 based
nitration reaction was switched for the most unstable derivatives
16, 18, and 20 from water to NH4OH(aq) to generate the stable
ammonium salts instead of the zwitterion forms of N-
nitrosulfonamides (Schemes 1 and 2).25,26
The production of the silver salts of the derivatives was
achieved by different methods depending on the nature of the
compound or the form it was produced as in the previous step
(Scheme 3). Silver carbonate was used as the base (to remove
Scheme 3. Synthesis of Silver Salts of N-Nitrosulfonamides:
(c) Ag2CO3, H2O; (d) NaOH, AgNO3, H2O
Furthermore, we produced silver salts of all such derivatives
based on their marked effects against viruses, bacteria, fungi, and
protozoa.20 The antimicrobial behaviors of silver, silver ions, and
silver-containing compounds have long been investigated with
various antimicrobial mechanisms of action having been
proposed to date.21−24 The biologically active silver ion (Ag+)
irreversibly damages key enzyme systems in the cell membranes
of pathogens. Conversely, silver exhibits low toxicity in the
human body and minimal risk is expected due to clinical
exposure.21 Recently, silver nanoparticles (Ag-NPs) were
demonstrated to produce reactive oxygen species to which
Leishmania parasites are very sensitive.24 Moreover, the
commercially available antibiotic silver sulfadiazine shares a
wealth of features with silver N-nitrosulfonamides. These latter
derivatives are thus endowed with multiple potential anti-
the proton) and the source of silver ion in an aqueous phase in
case of zwitterion of amino aromatic compounds. NaOH/
AgNO3 was used for the zwitterion of the amino aliphatic
compound 15 and for ammonium salts 16, 18, and 20. All
compounds silver salts precipitated in aqueous phases and were
therefore recrystallized by the same solvent. All the obtained
derivatives were properly characterized by means of 1H NMR,
The inhibition profiles of the N-nitrosulfonamide derivatives
were evaluated against six α-CAs and three β-CA isoforms in
addition to acetazolamide (AAZ) as standard inhibitor, by a
stopped flow CO2 hydrase assay.27
Five human CA isoforms, among which the ubiquitous CA I
and II (involved in many physiopathological processes), the
B
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX