Human carbonic anhydrase (CA; EC 4.2.1.1) isoforms II and VII are implicated in neuronal excitation, seizures, and neuropathic pain (NP). Their selective inhibition over off-target CAs is expected to produce an anti-NP action devoid of side effects due to promiscuous CA modulation. Here, a drug design strategy based on the observation of (dis)similarities between the target CA active sites was planned with benzenesulfonamide derivatives and, for the first time, a phosphorus-based linker. Potent and selective CA II/VII inhibitors were identified among the synthesized phenyl(thio)phosphon(amid)ates 3-22. X-ray crystallography depicted the binding mode of phosphonic acid 3 to both CAs II and VII. The most promising derivatives, after evaluation of their stability in acidic media, were tested in a mouse model of oxaliplatin-induced neuropathy. The most potent compound racemic mixture was subjected to HPLC enantioseparation, and the identification of the eutomer, the (S)-enantiomer, allowed to halve the dose totally relieving allodynia in mice.
Human carbonic anhydrase (CA; EC 4.2.1.1) isoforms II and VII are implicated in neuronal excitation, seizures, and neuropathic pain (NP). Their selective inhibition over off-target CAs is expected to produce an anti-NP action devoid of side effects due to promiscuous CA modulation. Here, a drug design strategy based on the observation of (dis)similarities between the target CA active sites was planned with benzenesulfonamide derivatives and, for the first time, a phosphorus-based linker. Potent and selective CA II/VII inhibitors were identified among the synthesized phenyl(thio)phosphon(amid)ates 3-22. X-ray crystallography depicted the binding mode of phosphonic acid 3 to both CAs II and VII. The most promising derivatives, after evaluation of their stability in acidic media, were tested in a mouse model of oxaliplatin-induced neuropathy. The most potent compound racemic mixture was subjected to HPLC enantioseparation, and the identification of the eutomer, the (S)-enantiomer, allowed to halve the dose totally relieving allodynia in mice.
Neuropathic pain (NP)
is pain initiated by a damage or ailment
of the peripheral or central somatosensory system.[1] The prevalence of NP in the population is estimated to
be 6.9–10% and amounts to 20–25% of all chronic pain
cases.[2,3] Numbness, needle sensation and tingling,
paresthesia, and neurological sensory deficits are NP main symptoms,
which can occur both at the central and peripheral nervous systems.
Peripheral neuropathies can derive from viral infections and traumatic,
post-surgical, or diabetic neuropathies, while central pain syndromes
are usual in patients who suffered from multiple sclerosis, spinal
cord injury, or stroke.[4] The syndromes
can manifest in the form of spontaneous, continuous (or paroxysmal),
or evoked pain. The latter can be in turn distinguished in hyperalgesia,
in case it is initiated by painful stimuli and allodynia, if triggered
by non-noxious stimuli. Neuropathic pain is challenging to treat because
of the heterogeneity and complexity of signals and symptoms. Indeed,
the use of traditional analgesics (e.g., nonsteroidal anti-inflammatory
drugs) or weak opioids is associated to weak outcomes in NP patients
as these drugs do not target the types of symptoms correlated with
the disease.[4] The lack of specific drugs
for treating NP is partially overcome with serendipitously discovered
agents of the anticonvulsant and antidepressant classes, although
actually only 40–60% of patients attain satisfactory pain alleviation
upon treatment.[3] The first-line treatment
comprises some types of antidepressants such as tricyclic antidepressants,
dual norepinephrine/serotonin reuptake inhibitors, topical analgesics
(lidocaine), and calcium channel α2-δ ligands as anticonvulsants
(pregabalin and gabapentin).[1,5−7] Second-line treatments are represented by effective opioids and
tramadol, while other antidepressants, antiepileptics (among which
topiramate, zonisamide, and lacosamide), N-methyl-d-aspartate receptor antagonists, topical capsaicin, and mexiletine
can be used as third-line treatment.[8−10] Alternative pharmacological
strategies are being actually explored to overcome the difficulties
met in the treatment of most NP patients. Among the numerous promising
new targets/drug classes identified are histamine H4 receptor (H4R)
agonists, agonists of type 1 lysophosphatidic acid receptor, glial
cell activity modulators, μ-opioid receptor (MOP) agonists,
and carbonic anhydrase inhibitors (CAIs).[11−14]Carbonic anhydrases (CAs;
EC 4.2.1.1) are widely expressed in the
central nervous system (CNS) and choroid plexus.[15,16] Of the 15 human (h) CAs, CA II is the most physiologically relevant
isoform and is abundantly expressed in oligodendrocytes, myelin sheaths,
astrocytes, choroid plexus, and myelinated tracts of brain.[15,17] The membrane-bound CA IV is located in layers III and VI in thalamus,
hippocampus, and cortex as well as on the luminal surface of cerebral
capillaries present in the blood–brain barrier.[18] Likewise, CA VII is highly expressed in thalamus,
hippocampus, and cortex. CA VII is deemed a brain-associated CA isozyme
as it is predominantly expressed in the CNS while missing in most
other systems.[15,18]The link between CAs and
NP was recently established by the groups
of Kaila and Price.[19,20] The CA-catalyzed production of
HCO3– significantly affects the biochemistry
and physiology of neurotransmitters, such as γ-aminobutyric
acid (GABA). Injuries in peripheral nerves negatively impact spinal
GABAergic networks by reducing the neuron-specific potassium chloride
(K+–Cl–) cotransporter (KCC2).
A potential strategy to mitigate this process was validated, which
implicates CA inhibition as it can decrease bicarbonate-related depolarization
through GABAA receptors in case the KCC2 function is compromised.
Moreover, Ruusuvuori et al. have provided insights into the role of
CAs in the modulation of the neuronal signaling and showed that isoforms
II and VII are the only cytosolic isozymes present in both somata
and dendrites of mature CA1 pyramidal neurons.[21,22] As CAs are implicated in maintaining the bicarbonate gradient that
results in the efflux of HCO3– ions through
GABAA receptors, this study corroborated the involvement
of CA VII as well as CA II in neuronal excitation and seizures.[21]Hence, a structure-based drug design strategy
based on benzenesulfonamide
derivatives including, for the first time, a phosphorus-based linker
is here reported to provide potent and possibly selective CA II/VII
inhibitors in search of new tools for the management of neuropathicpain.
Results and Discussion
Drug Design and Chemistry
The spreading
interest of
novel therapeutic agents based on CA inhibition or continued validation
of specific hCAs as drug targets requires that selective inhibitors
are developed.[23] However, all clinically
used CAIs, among which primary sulfonamides and sulfamates investigated
in the therapy of neuropathic pain such as acetazolamide, methazolamide,
topiramate, zonisamide, celecoxib, and furosemide, are predominantly
nonselective with respect to all human isoforms.[17] Most recent drug design studies on CAIs involved the tumor-associated
CA IX and CA XII and culminated with the entrance of the phenylureidobenzenesulfonamideSLC-0111 in phase Ib/II clinical trials for the treatment
of advanced, metastatic solid tumors as a monotherapy or in combination
with other agents such as gemcitabine.[23,24] In contrast,
few compounds with pronounced inhibitory selectivity and/or potency
toward hCA II and hCA VII have been reported to date.[23,25]In this study, by observing the significant similarity in
amino acid composition and active site architecture existing between
hCA II and hCA VII (Figure ), a new series of CAIs were designed, which can concomitantly
target both isozymes with the aim of achieving a neuropathic pain-relieving
action.[15,26]
Figure 1
Comparison between the active site of (A) hCA
II (PDB ID: 3HS4) and (B) hCA VII
(PDB ID: 3ML5) in complex with acetazolamide (AAZ). Residues different
between the two enzymes are boxed in blue for hCA II and orange for
hCA VII. The target amino acid Phe131 from both active sites is underlined.
Comparison between the active site of (A) hCA
II (PDB ID: 3HS4) and (B) hCA VII
(PDB ID: 3ML5) in complex with acetazolamide (AAZ). Residues different
between the two enzymes are boxed in blue for hCA II and orange for
hCA VII. The target amino acid Phe131 from both active sites is underlined.In detail, CA II and CA VII share a Phe residue
in position 131
(Figure ). This residue
is known to play an important role in the binding of many CAIs[26,27] and thus was taken as the reference point to explore the chemical
space in the middle portion of the CA active site where differences
exist between CAs II and VII (Figure ) as well as with other CA isoforms. Benzenesulfonamide
derivatives were thus designed according to the tail approach that,
in recent years, has been mainly applied by using urea-based linkers.[23,26] Such an approach consists in appending a variety of chemical frameworks
to the main CA inhibitory scaffold with the aim of diversely targeting
the medium/outer regions of the CA active sites where most amino acid
variability exists.[23] Nonetheless, the
arylureidobenzenesulfonamide (Figure A) was not deemed to possess an easily accessible chemical
versatility to include a variety of substituents for studying the
binding to the active site middle region, that is, nearby Phe131.
In fact, such a scaffold has been chiefly adopted for exploring the
chemical space in the outer region of the binding cavity.[28,29] Hence, in this context a phosphorus-based linker (Figure B) was adopted for the following
reasons:[30] (i) because of the electronic
properties of the phosphorus atom, an additional substituent can be
included on the spacer while holding the benzenesulfonamide and the
aromatic ring interacting with Phe131 (i.e., the outer ring of the
benzenesulfonamide derivative in Figure B); (ii) the extra substituent is appended
in the middle of the derivative structure, a suitable position for
interaction with the medium portion of the binding cavity (Figure B); (iii) by using
phenylphosphonic dichlorides as reagents, a wide variety of substituents
can be easily appended to the spacer in the form of (thio)phosphon(amid)ates;
and (iv) finally, the presence of a chiral center on the phosphorus
atom could induce an enantiomeric binding mode.
Figure 2
(A) Schematic representation
of phenylureidobenzenesulfonamides
bound to the CA II active site. (B) Drug design of benzene (thio)phosphon(amid)ates
as CA II and VII inhibitors.
(A) Schematic representation
of phenylureidobenzenesulfonamides
bound to the CA II active site. (B) Drug design of benzene (thio)phosphon(amid)ates
as CA II and VII inhibitors.On the basis of the drug design, a series of (thio)phosphon(amid)ates
were designed and synthesized using 4-hydroxybenzenesulfonamide 1 and phenylphosphonic dichloride 2 as starting
materials (Scheme ). The choice of 1 in place of sulfanilamide, which
is commonly derivatized to yield benzenesulfonamide series, was related
to its major reactivity with phosphonic chlorides than the aromatic
amine, thus producing higher yields as well as ease of purification.[31] The snap addition of phenylphosphonic dichloride 2 to a solution of 4-hydroxybenzenesulfonamide 1 and triethylamine in anhydrous THF at −5 °C yielded
intermediate A. In contrast, a greater rate of double
attack of 1 into phenylphosphonic dichloride was observed
raising the temperature over r.t., and thus, these conditions were
used to achieve compound 10. Otherwise, the addition
of various nucleophiles, among which (thio)alcohols, (thio)phenols,
and aliphatic and aromatic amines, to the reaction mixture containing A and stirring thereof at r.t. yielded (thio)phosphonates 3–9 and 11–14 and phosphonamidates 15–22 (Table ). The adopted synthetic
pathway provided the compounds as racemic mixtures, with the exception
of bis-sulfonamide 10 and, when deprotonated, phenylphosphonic
acid 3. All derivatives were purified by column chromatography
using silica gel and MeOH/DCM gradients and fully characterized by 1H NMR, 13C NMR, 31P NMR, and HRMS (Supporting Information). According to literature
data, peak picking of 31P signals in NMR spectra reported
the various ranges of signals given by phosphorus atoms of different
types. 31P signals of alkyl phosphonates 3–8 spanned from 14.31 to 16.73 ppm, while those
of phosphonic acid 3 and aryl phosphonates 9 and 10 lied in the range 12.01–12.49 ppm. In
contrast, 31P signals of alkyl thiophosphonates 11–13 were found between 42.64 and 43.65 ppm, whereas
the thiophenol derivative 14 showed a signal at 39.97
ppm. Primary and secondary phosphonamidates 15–17 and 21 reported 31P peaks in the
range 21.20–22.23 ppm, the tertiary derivatives 18–20 between 18.85 and 19.65 ppm, and the aromatic
amine derivative at 15.37 ppm.
Scheme 1
Synthesis of (Thio)phosphonates 3–14 and Phosphonamidates 15–22
Table 1
Inhibition Data of Human CA Isoforms
I, II, IV, VA, VB, VII, IX, and XII with (Thio)phosphon(amid)ates 3–22 and the Standard Acetazolamide (AAZ) by a Stopped-Flow CO2 Hydrase Assay[32]
KIa (nM)
cmp
CA I
CA II
CA IV
CA VA
CA VB
CA VII
CA IX
CA XII
3
196.9 ± 11.2
37.9 ±
2.1
220.1 ± 16.2
76.3 ± 5.2
52.8 ± 3.4
16.1 ± 1.1
26.1 ± 1.4
30.8 ± 2.3
4
494.9 ± 35.4
11.4
± 0.8
259.2 ± 14.5
51.7 ±
4.0
24.3 ± 1.5
36.0 ± 1.9
16.2 ± 0.9
47.2 ± 3.1
5
397.5 ± 26.7
36.6 ± 2.4
424.6 ± 35.6
142.6
± 8.3
40.1 ± 2.6
38.2 ±
2.5
18.7 ± 1.1
22.4 ± 1.5
6
307.8 ± 18.1
12.8 ± 0.7
1196 ± 102
100.8 ± 5.9
19.8 ± 1.1
6.2
± 0.4
17.5 ± 1.2
42.0 ±
3.2
7
415.3 ±
29.0
3.6 ± 0.2
1322 ± 124
82.1 ± 5.7
38.4 ± 2.5
12.2 ± 0.8
22.6 ± 1.6
26.7
± 1.8
8
779.7
± 44.8
28.5 ± 1.9
1644 ±
113
59.3 ± 4.1
67.2 ± 3.8
25.0 ± 1.2
12.1 ± 0.7
35.7 ± 2.8
9
971.1 ± 62.7
15.2 ± 1.0
2619
± 153
470.4 ± 33.6
22.7 ±
1.4
31.4 ± 1.8
13.2 ± 0.9
24.6 ± 1.9
10
670.7 ± 40.1
3.2 ± 0.2
360.3 ± 29.5
284.3 ± 19.3
15.0
± 0.9
7.7 ± 0.5
19.7 ±
1.4
16.9 ± 0.9
11
726.7 ± 49.3
30.2 ± 2.1
1389 ± 89
94.3 ± 5.8
96.7 ± 5.7
41.5 ± 2.8
27.0
± 2.0
34.1 ± 2.5
12
8345 ± 552
234.1
± 15.3
4164 ± 342
641.9 ±
45.7
224.6 ± 13.4
100.8 ± 6.3
51.2 ± 3.6
47.8 ± 3.4
13
3264 ± 258
64.8 ± 3.7
4456 ± 298
150.7
± 10.4
58.4 ± 4.1
75.5 ±
5.7
25.0 ± 1.6
21.3 ± 1.6
14
1635 ± 101
34.9 ± 2.3
2633 ± 186
382.0 ± 26.5
28.7 ± 1.6
15.7
± 1.2
6.6 ± 0.4
39.2 ±
2.5
15
195.1 ±
15.7
17.9 ± 1.0
292.4 ± 22.1
41.3 ± 3.2
12.6 ± 0.8
18.9 ± 1.0
20.9 ± 1.2
15.4
± 0.8
16
759.4 ± 57.2
26.2 ± 1.6
1516
± 99
85.2 ± 6.2
33.7 ±
2.4
20.8 ± 1.4
3.2 ± 0.2
17.3 ± 0.9
17
521.3 ± 37.5
17.5 ± 1.1
371.1 ± 26.4
106.8 ± 5.9
49.1
± 2.7
13.5 ± 0.8
19.5 ±
1.4
46.8 ± 3.1
18
242.1 ± 18.6
1.7 ± 0.1
307.6 ± 19.2
78.3 ± 4.7
25.6 ± 1.3
9.3 ± 0.6
28.9 ±
2.1
58.1 ± 4.5
19
317.9 ± 17.0
7.9 ± 0.5
120.1 ± 8.3
43.9 ± 3.1
27.4 ± 1.6
30.5 ± 2.4
11.1
± 0.6
27.6 ± 1.3
20
390.8 ± 29.6
9.1
± 0.6
76.9 ± 5.2
164.1 ±
12.5
47.3 ± 3.4
17.0 ± 1.3
61.9 ± 5.1
25.3 ± 1.4
21
1705.9 ± 117
52.9 ± 2.9
643.9 ± 46.2
59.1
± 4.0
68.7 ± 4.8
15.4 ±
1.0
2.6 ± 0.2
19.7 ± 1.2
22
817.9 ± 62.4
2.4 ± 0.1
859.9 ± 58.3
184.6 ± 14.1
24.0 ± 1.5
5.5
± 0.3
19.6 ± 1.1
20.9 ±
1.3
AAZ
250 ±
15
12 ± 1
74 ± 6
63 ± 4
54 ± 3
2.5 ± 0.2
25 ± 2
5.7 ± 0.5
Inhibition data are expressed as
means ± SEM of three different assays.
Inhibition data are expressed as
means ± SEM of three different assays.
Carbonic Anhydrase Inhibition
(Thio)phosphon(amid)ates 3–22 were evaluated for their inhibition
against the cytosolic CAs I, II, and VII, the mitochondrial CAs VA
and VB, and the membrane-associated CAs IV, IX, and XII by a stopped-flow
method that measures CO2 hydrase activity.[32]AAZ was used as the reference drug. The following
SAR can be worked out from the data reported in Table .Overall, the adopted structure-based
drug design strategy produced a satisfactory result in terms of inhibition
potency against the target CAs II and VII as well as selectivity over
the other ubiquitous and off-target isoform CA I. The latter isozyme
was in fact feebly inhibited by (thio)phosphon(amid)ates 3–22 with KI values
(inhibition constants) spanning in the high nanomolar up to low micromolar
range (195.1–8345 nM). Phosphonic acid 3 and primary
phosphonamidate 15 stood out as the most potent CA I
inhibitors presumably because of the low steric hindrance produced
within one of the narrowest active sites among hCAs. As a matter of
fact, compounds bearing phenyl (9, 14, and 22) and benzyl (13 and 21) R groups
as well as the lauryl derivative 12 acted as the weakest
CA I inhibitors (KI values of 817.9–8345
nM).The target CA II was effectively inhibited by compounds 3–22 with KI values
below 100 nM (KI values of 1.7–64.8
nM), except for derivative 12, which showed again the
lowest CA inhibition (a KI of 234.1 nM). KI values even below 10 nM were exhibited by
propargyl and bis-sulfamoyl phosphonates 7 and 10 and phosphonamidates bearing a pyrrolidine (18), morpholine (19), N-methylpiperazine
(20), and aniline (22). It should be noted
that all other compounds inhibit CA II with KI values below 50 nM with the exceptions of the benzyl derivatives 13 and 21 (KI values
of 64.8 and 52.9 nM).SAR of compounds 3–22 against
CA IV is rather flat. This isozyme was the least inhibited by the
new reported derivatives, most of which did not inhibit CA IV up to
500 nM. KI values lying in the range 120–450
nM were however estimated for phosphonates 3–5 and 10 and phosphonamidates 15 and 17–19, whereas N-methylpiperazine 20 uniquely achieved a KI of 76.9 nM.The mitochondrial CAs VA and VB show
two distinct levels of inhibition
with compounds 3–22. While CA VB
was inhibited in a comparable range with CA II (KI values between 12.6 and 224.6 nM), a drop of inhibition
was measured for CA VA (KI values of 41.3–641.9
nM) but without approaching the high nanomolar to low micromolar KI values detected against CAs I and IV. Methyl
phosphonate 4 (a KI of 51.7
nM), simple phosphonamidate 15 (a KI of 41.3 nM), and morpholine derivative 19 (a KI of 43.9 nM) stood out as the best CA VA inhibitors
herein, whereas a drop of efficacy was consistently observed with
thiophosphonates 12–14 (KI values of 150.7–641.9 nM). Phosphonamidate 15 was also detected as the most effective inhibitor against
CA VB (a KI of 12.6 nM) in a comparable
manner with bis-benzenesulfonamide 10 (a KI of 15.0 nM). The aliphatic thiophosphonates 11 and 12 showed to act as the worst CA VB inhibitors
with KI values of 96.7 and 224.6 nM. The
remaining compounds showed KI values spanning
in a rather narrow range between 20 and 60 nM.As CA II, the
other target isozyme CA VII was efficiently inhibited
by compounds 3–22 as KI values did not raise above 50 nM (spanning from 5.5
to 41.5 nM), with the exception of thiophosphonates 12 and 13 (KI values of 100.8
and 75.5 nM). The isopropyl (6) and bis-sulfamoyl (10) compounds among phosphonates and pyrrolidine (18) and aniline (22) derivatives among phosphonamidates
acted as the most potent CA VII inhibitors with KI values below 10 nM.Although not presenting a
Phe residue in position 131, but instead
a Val or Ala, respectively, the tumor-associated CAs IX and XII were
potently inhibited by compounds 3–22. In fact, these isozymes display two of the roomiest active sites
among hCAs, which enable the accommodation and adjustment of a plethora
of chemotypes and moieties from CAIs. Interactions with Phe131 might
be indeed substituted by hydrophobic contacts with Val and Ala residues
that summed up with other ligand/target hydrophobic interactions within
the binding pocket account for low nanomolar inhibitory effectiveness.
It should be stressed that, for instance, as the lauryl derivative 12, the weak inhibitor against other screened isoforms showed
a 50 nM efficacy against CAs IX and XII, owing to a likely favored
adaptation within their binding sites, which show rather extended
lipophilic pockets. CA IX and CA XII showed rather flat inhibition
profiles with KI values spanning in the
ranges 2.6–61.9 and 15.4–58.1 nM.Interestingly,
a subset of derivatives can be selected, which showed
a preferred inhibition against the target CAs II and VII over all
other isoforms, particularly CAs I, IV, and VA: isopropyl (6), propargyl (7), and bis-sulfamoyl (10) phosphonates and phosphonamidates bearing pyrrolidine (18), N-methylpiperazine (20), and aniline
(22). It is fair to stress that these compounds showed
selectivity for CAs II and VII above one order of magnitude solely
over CAs I, IV, and VA. In contrast, the selectivity index was lower
for CAs II and VII over CAs VB, IX, and XII, which are, however, minor
off-target isoforms with respect to the ubiquitous CA I.
X-ray Crystallography
To understand the molecular basis
responsible for the inhibition efficiency of phosphonate/phosphonamidate
benzenesulfonamides against CA II and CA VII, the crystal structure
of these two isoforms was solved in adduct with phenylphosphonic acid 3. This compound based on the ChemAxon software[36] has a pKa value
of 1.62 and, therefore, is assumed to bind the enzyme at physiological
pH and in crystallization conditions in a deprotonated, nonchiral
form. Crystals of the CA adducts with 3 were obtained
by soaking experiments as previously reported for other sulfonamide
CA inhibitors.[33−35] Data were collected to resolution values of 1.26
and 1.94 Å for CA II/3 and CA VII/3 complexes, respectively. The structures were analyzed by difference
Fourier techniques and refined to Rwork and Rfree values of 14.6 and 17.4% for
the CA II/3 complex and 18.8 and 22.6% for the CA VII/3 one (Table S1). Analysis of the
electron density maps at various stages of the crystallographic refinement
showed in both complexes the binding of one molecule of the inhibitor
within the enzyme active site.The electron density maps were
very well defined for both proteins and for the entire inhibitor in
the case of CA VII/3. On the contrary, in the case of
the CA II/3 complex, a less defined electron density
was associated to the inhibitor phenyl tail, indicating a higher mobility
of this part of the molecule within the CA II active site (Figure ).
Figure 3
σA-weighted |2Fo
– Fc| map (contoured at 1.0 σ)
relative to the inhibitor molecule in (A) CA II/3 and
(B) CA VII/3 adducts. The zinc ion coordination and residues
with a distance of less than 4.0 Å from the inhibitor are also
reported. Continuous lines show zinc ion coordination, whereas dashed
lines indicate potential hydrogen bonds.
σA-weighted |2Fo
– Fc| map (contoured at 1.0 σ)
relative to the inhibitor molecule in (A) CA II/3 and
(B) CA VII/3 adducts. The zinc ion coordination and residues
with a distance of less than 4.0 Å from the inhibitor are also
reported. Continuous lines show zinc ion coordination, whereas dashed
lines indicate potential hydrogen bonds.As shown in the structural superposition reported in Figure A, the inhibitor adopts the
same orientation within the two active sites, with only a small shift
of the phenyl tail. In detail, in both structures, the benzenesulfonamide
moiety binds the active site in the same conformation adopted in other
CA/benzenesulfonamide complexes, coordinating the catalytic zinc ion,
forming two hydrogen bonds with residue Thr199, and establishing many
hydrophobic interactions with residues located on the bottom of the
cavity (Figure and Table S2). The phosphonate group points toward
the hydrophilic region of the active site[13] and forms in both structures several water-mediated hydrogen bonds
with enzyme residues (Figure B,C). Finally, the phenyl tail is oriented toward the hydrophobic
side of the active site[13] establishing
many hydrophobic interactions with residues delimiting this region
(Figures and 4A and Table S2) and interacting
with residue Phe131 as hypothesized in the design. The comparable
number and nature of the interactions established by the inhibitor
in the two active sites might explain the similarity of the inhibition
constants against the two isoforms (Table S2). On the basis of the orientations of the phosphonateoxygens depicted
in Figures and 4, it can be supposed that the substituents included
in the P-based linker, both in the (S)- and (R)-enantiomers, are directed toward and binded the medium
and target portion of the CA active site (Figure B).
Figure 4
(A) Structural superposition of compound 3 when bound
to CA II (green) with the same compound when bound to CA VII (magenta).
The surface representation of CA II is also shown with the hydrophobic
region of the active site in red and the hydrophilic one in blue.
(B, C) Details of the polar interactions established by inhibitor 3 within the active site of (B) CA II and (C) CA VII. Water
molecules are represented as red spheres, continuous lines indicate
zinc ion coordination, whereas dashed lines indicate hydrogen bond
distances.
(A) Structural superposition of compound 3 when bound
to CA II (green) with the same compound when bound to CA VII (magenta).
The surface representation of CA II is also shown with the hydrophobic
region of the active site in red and the hydrophilic one in blue.
(B, C) Details of the polar interactions established by inhibitor 3 within the active site of (B) CA II and (C) CA VII. Water
molecules are represented as red spheres, continuous lines indicate
zinc ion coordination, whereas dashed lines indicate hydrogen bond
distances.
Drug Stability Study
The selected subset of compounds
(6, 7, 10, 18, 20, and 22) was promoted to in vivo evaluation of their neuropathic pain-relieving action. As the drug
administration would have been performed per os, a drug stability
study was carried out to verify the endurance of the compounds to
different media such as phosphate buffer (PBS), a pH 2 aqueous solution
(i.e., HCl(aq) 10 mM) to mimic the stomach environment
of the animal, and human plasma (H-Pl) to check the efficiency of
hydrolytic enzymes against such phosphonic ester/amide derivatives.
The study was performed by coupling liquid chromatography to a mass
spectrometer working in the tandem mass spectrometry mode (LC–MS/MS).The collected data indicate that the tested derivatives exhibited
stability to all the tested matrices/solutions (Figures S1–S6, Supporting Information), with the exception
of derivative 18 that showed a quick degradation (t1/2 of approximately 2 min) in the pH 2 solution
(Figure S4, Supporting Information).Absorption, distribution, metabolism, excretion, and toxicity (ADMET)
properties for 6, 7, 10, 20, and 22 were also predicted by the online
server PreADMET[37] and are reported in Table S4, Supporting Information.
In
Vivo Neuropathic Pain-Relieving Action
The selected
compounds except 18, shown to be unstable
in acidic media, were evaluated in vivo for their
properties in relieving neuropathic pain induced by the anticancer
drug oxaliplatin that is characterized by a relevant neurotoxicity
in a high percentage of patients.[38] This
platinum derivative has become a valid option as adjuvant therapy
in several types of cancer, but as a common side effect, it provokes
a painful neuropathy associated with characteristic nervous system
alterations that reduce the patients’ quality of life.[39] The pain-relieving effect obtained with the
CAIs was compared to that achieved with the reference drugs AAZ and duloxetine, and the results are shown in Figure . Ten oxaliplatin
injections decreased the licking latency to 9.0 ± 0.5 s in comparison
to 18.1 ± 0.3 s of the control group (vehicle + vehicle-treated
animals). Compound 6, acutely per os administered in
a range doses from 10 to 100 mg kg–1, evoked a dose-dependent
antineuropathic effect that lasted up to 45 min after treatment with
a peak of efficacy at the same time point (Figure A). A better antineuropathic profile was
obtained with compound 7 (Figure B), which also showed a dose-dependent antinociceptive
effect. The higher dose (100 mg kg–1) was able to
totally counteract oxaliplatinhypersensitivity 30 min after treatment
with a pain-relieving effect that continued up to 45 min. The 30 mg
kg–1 dose also statistically amplified the pain
threshold at 15 and 30 min. Likewise, notable results were attained
by administration of derivatives 20 and 22, as depicted in Figure D,E. Compound 10 evoked a maximum effect 30 min
after administration and uniquely at the dose of 100 mg kg–1 (Figure C). The
reference CAIAAZ was also moderately active at the dose
of 100 mg kg–1 but solely at 15 min after injection
(a licking latency of 15.8 ± 0.7 s) (Figure F).
Figure 5
(A–F) Effect of acute administration
of carbonic anhydrase
inhibitors 6, 7, 10, 20, 22, AAZ, and duloxetine on oxaliplatin-induced
neuropathic pain in the mouse cold plate test. Oxaliplatin (2.4 mg
kg–1) was i.p. administered for 5 consecutive days
every week for 2 weeks. Compounds were dissolved in 1% CMC and per
os administered acutely when neuropathy was well established (day
15). Each value represents the means ± SEM of 12 mice collected
in two different experimental sets. Statistical analysis is one-way
ANOVA followed by Bonferroni’s post-hoc comparison. **P < 0.s01 vs vehicle + vehicle-treated animal; ^P < 0.05 and ^^P <
0.01 vs oxaliplatin + vehicle-treated animals.
(A–F) Effect of acute administration
of carbonic anhydrase
inhibitors 6, 7, 10, 20, 22, AAZ, and duloxetine on oxaliplatin-induced
neuropathic pain in the mousecold plate test. Oxaliplatin (2.4 mg
kg–1) was i.p. administered for 5 consecutive days
every week for 2 weeks. Compounds were dissolved in 1% CMC and per
os administered acutely when neuropathy was well established (day
15). Each value represents the means ± SEM of 12 mice collected
in two different experimental sets. Statistical analysis is one-way
ANOVA followed by Bonferroni’s post-hoc comparison. **P < 0.s01 vs vehicle + vehicle-treated animal; ^P < 0.05 and ^^P <
0.01 vs oxaliplatin + vehicle-treated animals.The weak activity of compound 10, although a strong
dual CA II and VII inhibition, can be related to its low water solubility
with respect to the other derivatives due to the presence of a double
benzenesulfonamide scaffold. Barring 10, derivatives 7 and 22, which totally reverted allodynia in vivo after 30 min post-administration, are the most potent
CA II inhibitors among the four remaining compounds. The slightly
less effective pain-relieving action of 22 with respect
to 7, although 2-fold more potent as the CA VII inhibitor,
can be related to its minor water solubility (as predicted in silico, Table S4). In contrast,
compound 6, 4-fold less potent as the CA II inhibitor
than 7 and 22, but as active as 22 as the CA VII inhibitor, showed a significantly inferior in vivo efficacy. As a result, excluding other participating
factors, a greater implication of CA II than CA VII could be speculated.
Surprising is the case of methylpiperazine derivative 20, that is, it is the worst CA II and VII inhibitors among the five
compounds but exhibited the quickest in vivo action,
reverting the pain sensation after only 15 min. This is likely due
to its greater water solubility, which favors its dissolution after
per os administration.The pain-relieving efficacy of the most
active derivatives 7, 20, and 22 was comparable (at
30 mg kg–1) or even greater (at 100 mg kg–1) than that induced by duloxetine (when dosed at 15 mg kg–1, a neuropathic pain-relieving dose in animals[40] (Figure F), a standard drug clinically used for the management of chemotherapy-induced
neuropathy.[41] It should be thus stressed
that further comparison between the doses of these phosphon(amid)ates
and duloxetine cannot be done as such compounds did not share the
same mechanism of action.
Chiral Resolution and Assignment of Absolute
Configuration of
Compound 7
The racemic mixture of the most in vivo effective compound, namely, compound 7, was subjected to enantioseparation, accomplished by HPLC on the
immobilized-type CHIRALPAK IA chiral stationary phase under the normal-phase
mode (Figure ). Both
enantiomers could be collected in multimilligram quantities, by performing
repetitive injections of 1 mg of racemic sample on a 250 mm ×
10 mm I.D. CHIRALPAK IA column. As shown in Figure A, the semipreparative HPLC resolution was
achieved in nonoverlapping band conditions. The analytical control
of the collected fractions gave an enantiomeric excess of both enantiomers
higher than 98%.
Figure 6
Chiral resolution of compound 7 by chiral
HPLC.
Figure 7
(A) Typical chromatogram illustrating the enantioseparation
of
1 mg of 7 on the 250 mm × 10 mm I.D. CHIRALPAK IA
column. Mobile phase: n-hexane/EtOH/MeOH/TFA 60:35:5:0.1
(v/v/v/v); flow rate: 4.5 mL min–1; temperature:
15 °C; detector: UV at 260 nm. (B) ORD and (C) ECD spectra of
the enantiomers of 7 recorded in ethanol at 25 °C
(solid lines) and calculated (dashed lines). Green and red lines refer
to the first and second eluted enantiomers, respectively.
Chiral resolution of compound 7 by chiral
HPLC.(A) Typical chromatogram illustrating the enantioseparation
of
1 mg of 7 on the 250 mm × 10 mm I.D. CHIRALPAK IA
column. Mobile phase: n-hexane/EtOH/MeOH/TFA 60:35:5:0.1
(v/v/v/v); flow rate: 4.5 mL min–1; temperature:
15 °C; detector: UV at 260 nm. (B) ORD and (C) ECD spectra of
the enantiomers of 7 recorded in ethanol at 25 °C
(solid lines) and calculated (dashed lines). Green and red lines refer
to the first and second eluted enantiomers, respectively.To determine the absolute configuration and then the enantiomer
elution order on the chiral chromatographic support, the chiroptical
properties (i.e., CD and ORD spectra) of the enantiomers isolated
by HPLC were measured and compared with the calculated ones (Figure B,C).A detailed
description of the adopted procedure is provided in
the Experimental Section.
Carbonic Anhydrase
Inhibition of Enantiomers of Compound 7
(R)- and (S)-enantiomers of compound 7 were evaluated for their inhibition
against the cytosolic CAs I,
II, and VII, the mitochondrial CAs VA and VB, and the membrane-related
CAs IV, IX, and XII. The following considerations can be made from
the data reported in Table .
Table 2
Inhibition Data of Human CA Isoforms
I, II, IV, VA, VB, VII, IX, and XII with Compound 7 as
the Racemic Mixture and its (R)- and (S)-Enantiomers Using AAZ as the Standarda
KI (nM)
cmp
CA I
CA II
CA IV
CA VA
CA VB
CA VII
CA IX
CA XII
7
415.3
± 29.0
3.6 ± 0.2
1322 ±
124
82.1 ± 5.7
38.4 ± 2.5
12.2 ± 0.8
22.6 ± 1.6
26.7 ± 1.8
(R)-7
365.4 ± 23.4
56.4 ± 3.8
1984 ± 153
66.5 ± 4.6
66.1 ± 3.5
71.3
± 4.5
47.2 ± 2.8
49.2 ±
3.2
(S)-7
564.1 ± 42.7
0.78 ± 0.06
746.5 ± 38.4
108.3 ± 8.6
19.2 ± 1.5
1.1
± 0.1
10.5 ± 1.4
17.3 ±
1.3
Inhibition data are expressed as
means ± SEM of three different assays.
Inhibition data are expressed as
means ± SEM of three different assays.Enantiomer ( resulted
to be the
eutomer within the racemic mixture against most screened CA isoforms,
which are CAs II, IV, VB, VII, IX, and XII. Unique exceptions are
CAs I and VA, against which ( showed KI values of 365.4 and 66.5 nM, respectively,
arising as the eutomer. Astonishingly, the CA inhibition profile of ( was even improved when compared to 7 in terms of potency and selectivity for the target CAs over
off-target ones. Indeed, CA II was inhibited at a subnanomolar value
of 0.78 nM by (, whereas the KI against CA VII reached the single-digit value
of 1.1 nM. Moreover, the KI of ( halved against CA VB and CA IX with respect
to 7, while an inferior difference was measured between KI values of ( and 7 as the racemic mixture against CA XII (17.3 and
26.7 nM, respectively).
In Vivo Neuropathic Pain-Relieving
Action with ( and (
The in vivo action
of the two enantiomers
of compound 7 was thus assessed in the same mouse model
of neuropathic pain when hyperalgesia was well established (Figure ). Searching for
a dose reverting the allodynia and expecting one of them having a
greater efficacy than 7 as the racemate, a dose of 50
mg kg–1 was used. According to in vitro outcomes, the study identified (, the eutomer, since it counteracted oxaliplatin-induced hyperalgesia
comparably to 7 but notably by the half dose. Surprisingly,
50 mg kg–1( was
totally ineffective. This might indicate that a CA II and/or VII inhibition
of a certain intensity (KI values of ( against CAs II and VII dropped by 15
and 6 times, respectively, with respect to 7) is necessary
to produce an action, which relieves neuropathic pain.
Figure 8
Effect of acute administration
of eutomers ( and ()-7, in comparison
with 7 on oxaliplatin-induced
neuropathic pain in the mouse cold plate test. Oxaliplatin (2.4 mg
kg–1) was i.p. administered for 5 consecutive days
every week for 2 weeks. Compounds were dissolved in 1% CMC and per
os administered acutely when neuropathy was well established (day
15). Each value represents the means ± SEM of 12 mice collected
in two different experimental sets. Statistical analysis is one-way
ANOVA followed by Bonferroni’s post-hoc comparison. **P < 0.01 vs vehicle + vehicle-treated animals; ^P < 0.05 and ^^P <
0.01 vs oxaliplatin + vehicle-treated animals.
Effect of acute administration
of eutomers ( and ()-7, in comparison
with 7 on oxaliplatin-induced
neuropathic pain in the mousecold plate test. Oxaliplatin (2.4 mg
kg–1) was i.p. administered for 5 consecutive days
every week for 2 weeks. Compounds were dissolved in 1% CMC and per
os administered acutely when neuropathy was well established (day
15). Each value represents the means ± SEM of 12 mice collected
in two different experimental sets. Statistical analysis is one-way
ANOVA followed by Bonferroni’s post-hoc comparison. **P < 0.01 vs vehicle + vehicle-treated animals; ^P < 0.05 and ^^P <
0.01 vs oxaliplatin + vehicle-treated animals.
Conclusions
NP is induced by ailments or lesion existing
in the CNS, which
produces the sensation of pain in the brain. New promising drug classes
are necessary because of difficulties met in treating NP in a vast
majority of patients. The human CA isoforms II and VII are implicated
in neuronal excitation, seizures, and NP. Therefore, their selective
inhibition over off-target CAs is expected to produce an anti-NP action
devoid of side effects, which arise by promiscuous CA inhibition.
In this study, a drug design strategy was planned to produce selective
CA II and VII inhibitors. Benzenesulfonamide derivatives with a phosphorus-based
linker were adopted for the first time with the aim of pursuing the
tail approach for the exploration of the middle portion of the CA
active site cavity. A subset of potent and selective CA II/VII inhibitors
was identified among the synthesized phenyl(thio)phosphon(amid)ates 3–22. It is fair to stress that these
compounds showed selectivity for CAs II and VII above one order of
magnitude solely over CAs I, IV, and VA. In contrast, the selectivity
index was lower for CAs II and VII over CAs VB, IX, and XII, which
are, however, minor off-target isoforms with respect to the ubiquitous
CA I.The efficacy of the adopted drug design strategy was confirmed
at the molecular level by X-ray crystallographic studies depicting
the binding mode of phosphonic acid 3 to both CAs II
and VII. The most promising derivatives for potent and preferred inhibition
against the target CAs II and VII over all other isoforms, after evaluation
for their stability in acidic media, were tested in a mouse model
of oxaliplatin-induced neuropathy. The most potent compound (7) racemic mixture was also subjected to semipreparative HPLC
resolution, and the identification of the eutomer for CA II and VII
inhibition ( even allowed to halve
the dose totally reverting allodynia in mice from 100 to 50 mg kg–1.These results testify the efficacy of the
adopted drug design strategy
toward CA II/VII inhibitors for the treatment of NP and overall support
the potentiality of the (thio)phosphon(amid)ate linker for yielding
potent and selective CA inhibitors for therapeutic applications.
Experimental Section
Chemistry
Anhydrous
solvents and all reagents were
purchased from Sigma-Aldrich, Fluorochem, and TCI. All reactions involving
air- or moisture-sensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringe techniques to transfer
solutions. Nuclear magnetic resonance (1H NMR, 13C NMR, and 31P NMR) spectra were recorded using a Bruker
Avance III 400 MHz spectrometer in DMSO-d6. Chemical shifts are reported in parts per million (ppm), and the
coupling constants (J) are expressed in hertz (Hz).
Splitting patterns are designated as follows: s, singlet; d, doublet;
sept, septet; t, triplet; q, quadruplet; m, multiplet; bs, broad singlet;
and dd, double of doublets. The assignment of exchangeable protons
(OH and NH) was confirmed by the
addition of D2O. Analytical thin-layer chromatography (TLC)
was carried out on Sigma-Aldrich silica gel F-254 plates. Flash chromatography
purifications were performed on Sigma-Aldrich silica gel 60 (230–400
mesh ASTM) as the stationary phase, and ethyl acetate/n-hexane or MeOH/DCM was used as eluents. Melting points (mp) were
measured in open capillary tubes with a Gallenkamp MPD350.BM3.5 apparatus
and are uncorrected.Compounds 3–22 were ≥95% pure. The purity of the final compounds was determined
by an Agilent 1200 liquid chromatography system composed by an autosampler,
binary pumps, a column oven, and a diode array detector (LC-DAD) operating
in the UV range (210–400 nm). The operating conditions are
reported in the Supporting Information.
The following chromatographic parameters were employed to check the
purity of the compounds: (i) column, Luna PFP length = 50 mm, internal
diameter = 2 mm; particle size = 3 μm purchased from Phenomenex
(Bologna, Italy); (ii) the eluents used were 10 mM formic acid and
5 mM ammonium formate in water solution (solvent A) and 10 mM ammonium
formate and 5 mM formic acid in methanol (solvent B); (iii) the flow
rate and injection volume were 0.25 mL min–1 and
5 μL, respectively. The elution gradient is shown in Table S3, Supporting Information. The HPLC chromatograms
are depicted in Figures S7–S27,
Supporting Information.The high-resolution mass spectrometry
(HRMS) analysis was performed
with a Thermo Finnigan LTQ Orbitrap mass spectrometer equipped with
an electrospray ionization source (ESI). The analysis was carried
out by introducing, via a syringe pump at 10 μL min–1, the sample solution (1.0 μg mL–1 in mQ
water/acetonitrile 50:50) and acquired the signal of the positive
ions. These experimental conditions allow the monitoring of protonated
molecules of the studied compounds ([M + H]+ species) that
were measured with a proper dwell time to achieve 60,000 units of
resolution at full width at half-maximum (FWHM). Elemental compositions
of compounds were calculated on the basis of their measured accurate
masses, accepting only results with an attribution error of less than
2.5 ppm and a noninteger RDB (double bond/ring equivalents) value,
to consider only the protonated species.[42]
General Synthetic Procedure of Compounds (3–22)
Phenylphosphonic dichloride 2 (1.2
equiv) was quickly added to a solution of 4-hydroxybenzenesulfonamide 1 (0.3 g, 1.0 equiv) and trimethylamine (2.2 equiv) in dry
THF (10 mL) at −5 °C under a nitrogen atmosphere, and
the resulting suspension was stirred for 0.5 h at 0 °C. The proper
reactant was added to the mixture and that was stirred at r.t. for
1 h. The reaction mixture was concentrated in vacuo, and the obtained residue was purified by silica gel column chromatography
eluting with MeOH/DCM to afford the title compounds 3–23 as powders.
4-Sulfamoylphenyl Hydrogen
Phenylphosphonate (3)
Compound 3 was obtained according to the
general procedure earlier reported using H2O (5.0 equiv),
phenylphosphonic dichloride 2 (1.2 equiv), 4-hydroxybenzenesulfonamide 1 (0.3 g, 1.0 equiv), and trimethylamine (2.2 equiv) in dry
THF (10 mL). The reaction mixture was concentrated in vacuo, and the obtained residue was purified by silica gel column chromatography
eluting with 4% MeOH in DCM to afford the title compound as a white
solid. 55% yield; mp 183–184 °C; silica gel TLC Rf, 0.11 (MeOH/CH2Cl2 15%
v/v); δH (400 MHz, DMSO-d6): 7.32 (m, 4H, partial exchange with D2O, Ar-H + SO2NH2), 7.55
(m, 2H, Ar-H), 7.63 (m, 1H, Ar-H), 7.87 (m, 4H, Ar-H); δC (100
MHz, DMSO-d6): 121.56 (d, J3CP = 4.9), 128.50, 129.48 (d, J2CP = 14.7), 131.05 (d, J1CP = 185.8), 132.21 (d, J3CP = 9.9), 133.16 (d, J4CP = 3.2), 140.77, 154.27 (d, J2CP = 6.6); δP (162 MHz, DMSO-d6): 12.01; ESI-HRMS (m/z):
[M + H]+ calcd for C12H13NO5PS, 314.0252; found, 314.0247.
Methyl (4-Sulfamoylphenyl)
Phenylphosphonate (4)
Compound 4 was obtained according to the
general procedure earlier reported using MeOH (5.0 equiv), phenylphosphonic
dichloride 2 (1.2 equiv), 4-hydroxybenzenesulfonamide 1 (0.3 g, 1.0 equiv), and trimethylamine (2.2 equiv) in dry
THF (10 mL). The reaction mixture was concentrated in vacuo, and the obtained residue was purified by silica gel column chromatography
eluting with 4% MeOH in DCM to afford the title compound as a white
waxy solid. 28% yield; mp 92–93 °C; silica gel TLC Rf, 0.31 (MeOH/CH2Cl2 5%
v/v); δH (400 MHz, DMSO-d6): 3.87 (d, J = 11.4, 3H, OCH3), 7.38 (s, 2H, exchange with D2O, SO2NH2), 7.41 (m, 2H, Ar-H), 7.64 (m, 2H, Ar-H), 7.75 (m, 1H, Ar-H), 7.81 (m, 4H, Ar-H); δC (100
MHz, DMSO-d6): 54.29 (d, J2CP = 6.0), 121.64 (d, J3CP = 4.4), 126.95 (d, J1CP = 187.8), 128.74, 129.95 (d, J2CP = 15.2), 132.63 (d, J3CP = 10.4), 134.39 (d, J4CP = 3.1), 141.61, 153.29 (d, J2CP = 6.6); δP (162 MHz, DMSO-d6): 16.73; ESI-HRMS (m/z):
[M + H]+ calcd for C13H15NO5PS, 328.0408; found, 328.0402.
Ethyl (4-Sulfamoylphenyl)
Phenylphosphonate (5)
Compound 5 was obtained according to the general procedure
earlier reported using EtOH (5.0 equiv), phenylphosphonic dichloride 2 (1.2 equiv), 4-hydroxybenzenesulfonamide 1 (0.3
g, 1.0 equiv), and trimethylamine (2.2 equiv) in dry THF (10 mL).
The reaction mixture was concentrated in vacuo, and
the obtained residue was purified by silica gel column chromatography
eluting with 4% MeOH in DCM to afford the title compound as a white
waxy solid. 33% yield; mp 98–99 °C; silica gel TLC Rf, 0.52 (MeOH/CH2Cl2 10%
v/v); δH (400 MHz, DMSO-d6): 1.32 (t, J = 7.0, 3H, CH2CH3), 4.25 (m, 2H, OCH2), 7.38 (s, 2H, exchange with D2O, SO2NH2), 7.40 (m, 2H, Ar-H), 7.62
(m, 2H, Ar-H), 7.73 (m, 1H, Ar-H), 7.86 (m, 4H, Ar-H); δC (100
MHz, DMSO-d6): 17.05 (d, J3CP = 6.1), 64.01 (d, J2CP = 5.9), 121.71 (d, J3CP = 4.8), 127.65 (d, J1CP = 188.6), 128.77, 129.97 (d, J2CP = 15.3), 132.59 (d, J3CP = 10.3), 134.33 (d, J4CP = 3.2), 141.55, 153.40 (d, J2CP = 6.7); δP (162 MHz, DMSO-d6): 15.36; ESI-HRMS (m/z): [M + H]+ calcd for C14H17NO5PS, 342.0565;
found, 342.0560.
Compound 22 was obtained according
to the general procedure earlier reported using aniline (3.0 equiv),
phenylphosphonic dichloride 2 (1.2 equiv), 4-hydroxybenzenesulfonamide 1 (0.3 g, 1.0 equiv), and trimethylamine (2.2 equiv) in dry
THF (10 mL). The reaction mixture was concentrated in vacuo, and the obtained residue was purified by silica gel column chromatography
eluting with 4% MeOH in DCM to afford the title compound as a white
solid. 32% yield; mp 187–188 °C; silica gel TLC Rf, 0.38 (MeOH/CH2Cl2 10%
v/v); δH (400 MHz, DMSO-d6): 6.91 (m, 1H, Ar-H), 7.13 (m, 2H, Ar-H), 7.21 (m, 2H, Ar-H), 7.36 (s, 2H, exchange with
D2O, SO2NH2), 7.46
(m, 2H, Ar-H), 7.60 (m, 2H, Ar-H), 7.67 (m, 1H, Ar-H), 7.85 (m, 2H, Ar-H), 7.93 (m, 2H, Ar-H); δC (100
MHz, DMSO-d6): 118.86 (d, J3CP = 6.9), 121.75 (d, J3CP = 4.5), 122.21, 128.61, 129.73 (d, J2CP = 14.5), 129.96, 130.62 (d, J1CP = 172.9), 132.32 (d, J2CP = 10.4), 133.64 (d, J4CP = 2.5), 141.29, 141.39 (d, J4CP = 1.2), 153.61 (d, J2CP = 7.6); δP (162 MHz, DMSO-d6): 15.37; ESI-HRMS (m/z):
[M + H]+ calcd for C18H18N2O4PS ,389.0724; found, 389.0716.
Carbonic Anhydrase
Inhibition
An Applied Photophysics
stopped-flow instrument has been used for assaying the CA-catalyzed
CO2 hydration activity.[32] Phenol
red (at a concentration of 0.2 mM) has been used as an indicator,
working at the absorbance maximum of 557 nm, with 20 mM HEPES (pH
7.5) as a buffer and 20 mM Na2SO4 (for maintaining
constant the ionic strength), following the initial rates of the CA-catalyzed
CO2 hydration reaction for a period of 10–100 s.
The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters and inhibition constants.
For each inhibitor, at least six traces of the initial 5–10%
of the reaction have been used for determining the initial velocity.
The uncatalyzed rates were determined in the same manner and subtracted
from the total observed rates. Stock solutions of the inhibitor (0.1
mM) were prepared in distilled-deionized water, and dilutions up to
0.01 nM were done thereafter with the assay buffer. Inhibitor and
enzyme solutions were preincubated together for 1 h at room temperature
prior to the assay, to allow for the formation of the E–I complex.
The inhibition constants were obtained by nonlinear least-squares
methods using PRISM 3 and the Cheng–Prusoff equation, as reported
earlier,[43,44] and represent the mean from at least three
different determinations. All CA isoforms were recombinant ones obtained
in-house as reported earlier.[30]
Drug Stability
Study
Chemicals
Acetonitrile (Chromasolv), methanol (Chromasolv),
formic acid (MS grade), ammonium formate, HCl, NaCl, KCl, Na2HPO4·2H2O, KH2PO4 (Reagent grade), and verapamil hydrochloride (analytical standard,
used as the internal standard) were purchased from Sigma-Aldrich (Milan,
Italy). MilliQ water 18 MΩ was obtained from the Millipore’s
Simplicity system (Milan, Italy). Phosphate buffer solution (PBS)
was prepared by adding 8.01 g L–1 NaCl, 0.2 g L–1 KCl, 1.78 g L–1 Na2HPO4·2H2O, and 0.27 g L–1 KH2PO4. Human plasma was collected from a healthy
male volunteer; each plasma batch was kept at −80 °C until
use.
Instrumental
The LC–MS/MS analysis was carried
out using a Varian 1200L triple quadrupole system (Palo Alto, CA,
USA) equipped with two ProStar 210 pumps, a ProStar 410 autosampler,
and an electrospray source (ESI) operating in positive ions. Raw data
were collected and processed by Varian Workstation version 6.8 software.
A G-Therm 015 thermostatic oven was used to maintain the samples at
37 °C during the test of degradation. An ALC microcentrifuge
4214 was employed to centrifuge the samples.
LC–MS/MS Methods
The chromatographic parameters
employed to analyze the samples were tuned to minimize the run time.
The column having a PFP 30 mm length, 2 mm internal diameter, and
3 μm particle size was used, at a constant flow of 0.25 mL min–1, employing a binary mobile phase elution gradient.
The eluents used were 10 mM formic acid and 5 mM ammonium formate
in water solution (solvent A) and 10 mM ammonium formate and 5 mM
formic acid in methanol (solvent B) according to the elution gradient
as follows: initial at 90% solvent A, which was then decreased to
10% in 4.0 min, kept for 3.0 min, returned to initial conditions in
0.1 min, and maintained for 3.0 min for reconditioning, to a total
run time of 10.0 min.The column temperature was maintained
at 30 °C, and the injection volume was 5 μL. The ESI source
was operated in the positive ion mode, using the following setting:
5 kV needle, 42 psi nebulizing gas, 600 V shield, and 20 psi drying
gas at 280 °C. The analyses were acquired in multiple reaction
monitoring (MRM), and the ion transitions are reported in Table .
Table 3
MRM Parameters
compound
precursor ion (m/z)
quantifier ion (m/z) [CE (V)]
qualifier ion (m/z) [CE (V)]
6
356
297 [20]
314 [10]
7
352
335 [15]
131 [25]
10
469
452 [15]
280 [35]
18
367
194 [30]
350 [20]
20
396
223 [25]
296 [40]
22
389
308 [20]
296 [30]
ISTD
455
165 [25]
303 [25]
Standard Solutions
Stock solutions
of analytes and
verapamil hydrochloride (internal standard or ISTD) were prepared
in acetonitrile at 1.0 mg mL–1 and stored at 4 °C.
The ISTD working solution was prepared in acetonitrile at 15 μg
mL–1 (ISTD solution). The spiked solutions of each
analyte were prepared separately, by diluting the respective stock
solutions in mQ water/acetonitrile 80:20 (v/v) solution, to obtain
a final concentration of 10 μM.
Sample Preparation
The sample was prepared by adding
10 μL of spiked solution to 100 μL of tested matrix (PBS
or human plasma or 10 mM HCl) in microcentrifuge tubes. The obtained
solutions correspond to 1 μM analyte. Each set of samples was
incubated in triplicate at four different times: 0, 30, 60, and 120
min at 37 °C. Therefore, the matrix stability profile of each
analyte was represented by a batch of 12 samples (4 incubation times
× 3 replicates). After the incubation, the samples were added
with 300 μL of ISTD solution and centrifuged (room temperature
for 5 min at 800g). The supernatants were transferred
in autosampler vials and added with 0.6 mL of mQ water. The final
solutions were analyzed by the LC–MS/MS methods described above.Native hCA II was produced and
purified as previously described.[45] For
hCA VII, a mutated form where the cysteine residues in positions 183
and 217 were mutated to serines was used for crystallographic studies
since we previously reported that this mutant was more suitable for
crystallization experiments.[46] For both
enzymes, crystals were obtained by the hanging drop vapor diffusion
technique, using a precipitant solution containing 1.3 M sodium citrate
and 60 mM Tris-HCl, pH 8.0 for CA II and 25% (w/v) polyethylene glycol
3350, 0.2 M ammonium acetate, and 0.1 M Tris-HCl, pH 8.5 for CA VII.
Crystals of enzyme–inhibitor complexes were prepared by soaking
enzyme crystals in precipitant solutions containing 10 mM inhibitor
and using 1/18 h as the soaking time for CA II/VII crystals, respectively.X-ray diffraction data for both CA II/3 and CA VII/3 adducts were collected at 100 K, using a Rigaku MicroMax-007
HF generator producing Cu Kα radiation and equipped with a Saturn
944 CCD detector. Prior to cryogenic freezing, crystals were transferred
to the respective precipitant solution with the addition of 10/25%
(v/v) glycerol for hCA II/VII. Data were integrated, merged, and scaled
using HKL2000.[47] Crystal parameters and
data collection statistics for both adducts are reported in Table S1.Phasing and refinement of the
complexes were carried out with REFMAC5[48] using as starting models the previously solved
hCA II (PDB ID: 6H29)[33] and hCA VII (PDB ID: 6G4T)[46] structures with water molecules and ligands removed. In
both structures, the inhibitor molecule was clearly visible in the
difference Fo – Fc map from the early stages of refinement.
Restraints on inhibitor bond angles and distances were taken from
the Cambridge Structural Database,[49] and
topology file was generated using the PRODRG2 server.[50] Standard restraints were used on protein bond angles and
distances throughout refinement. Several rounds of manual rebuilding
were performed using the program O[51] with
careful inspection of the 2Fo – Fc and Fo – Fc electron
density maps. The geometric restraints of the final models were analyzed
using the programs PROCHECK[52] and WHATCHECK.[53] The refinement statistics of the final models
are summarized in Table S1. The atomic
coordinates of the complexes have been deposited in the Protein Data
Bank with accession codes 6SDS and 6SDT.
Animals
Male
CD-1 albino mice (Envigo, Varese, Italy) weighing approximately 22–25
g at the beginning of the experimental procedure were used. Animals
were housed in Ce.S.A.L (Centro Stabulazione Animali da Laboratorio,
University of Florence) and used at least 1 week after their arrival.
Ten mice were housed per cage (size 26 × 41 cm); animals were
fed a standard laboratory diet and tap water ad libitum and kept at 23 ± 1 °C with a 12 h light/dark cycle, light
at 7 a.m. All animal manipulations were carried out according to the
Directive 2010/63/EU of the European Parliament and of the European
Union Council (22 September 2010) on the protection of animals used
for scientific purposes. The ethical policy of the University of Florence
complies with the Guide for the Care and Use of Laboratory Animals
of the US National Institutes of Health (NIH publication no. 85-23,
revised 1996; University of Florence assurance number: A5278-01).
Formal approval to conduct the experiments described was obtained
from the Animal Subjects Review Board of the University of Florence.
Experiments involving animals have been reported according to ARRIVE
guidelines.[54] All efforts were made to
minimize animal suffering and to reduce the number of animals used.
Oxaliplatin-Induced
Neuropathic Pain Model and Pharmacological
Treatments
Mice treated with oxaliplatin (2.4 mg kg–1) were administered intraperitoneally (i.p.) on days 1 and2, 5–9,
and 12–14 (10 i.p. injections).[55] Oxaliplatin was dissolved in 5% glucose solution. Control animals
received an equivalent volume of vehicle. Behavioral tests were performed
starting from day 15. Acetazolamide (AAZ; 100 mg kg–1), duloxetine (15 mg kg–1), compounds 6, 7, 10, 20, and 22 (10–100 mg kg–1), and enantiomers
()-7 and ()-7, both 50 mg kg–1, were suspended in 1% carboxymethylcellulose sodium salt (CMC, Sigma-Aldrich,
Milan, Italy) and per os (p.o.) acutely administered. Behavioral tests
were carried out before and after (15, 30, 45, and 60 min) compound’s
injection.
Cold Plate
Thermal allodynia was
assessed using the
cold plate test. With minimal animal–handler interaction, mice
were taken from home cages and placed onto the surface of the cold
plate (Ugo Basile, Varese, Italy) maintained at a constant temperature
of 4 °C ± 1 °C. Ambulation was restricted by a cylindrical
Plexiglas chamber (diameter: 10 cm; height: 15 cm), with an open top.
A timer controlled by foot pedal began timing response latency from
the moment the mouse was placed onto the cold plate. Pain-related
behavior (licking of the hind paw) was observed, and the time (seconds)
of the first sign was recorded. The cutoff time of the latency of
paw lifting or licking was set at 30 s.[56]
Statistical Analysis
Behavioral measurements were performed
on 12 mice for each treatment carried out in two different experimental
sets. Results were expressed as means ± SEM. The analysis of
variance of behavioral data was performed by one-way ANOVA, and a
Bonferroni’s significant difference procedure was used for
post-hoc comparison. P values of less than 0.05 or
0.01 were considered significant. Investigators were blind to all
experimental procedures. Data were analyzed using the “Origin
9” software (OriginLab, Northampton, USA).
Enantioseparation
and Assignment of Absolute Configuration
Semipreparative
HPLC separations of the enantiomers of 7 was carried
out on the commercially available 250 mm × 10 mm
I.D. CHIRALPAK IA (Chiral Technologies Europe, Illkirch, France) column
using the mixture n-hexane/EtOH/MeOH/TFA 60:35:5:0.1
(v/v/v/v) as a mobile phase. The temperature was set at 15 °C.
The flow rate was 4.5 mL min–1. HPLC apparatus consisted
of a PerkinElmer (Norwalk, CT, USA) 200 LC pump equipped with a Rheodyne
(Cotati, CA, USA) injector, a 5 mL sample loop, an HPLC PerkinElmer
oven, and a PerkinElmer detector. The signal was acquired and processed
by Clarity software (DataApex, Prague, Czech Republic). The amount
of racemic samples resolved for single chromatographic run was 1 mg.The CD spectra of enantiomers of 7, dissolved in ethanol
(concentration about 0.40 mg mL–1) in a 0.1 cm path
length quartz cell at 25 °C, were measured by using a Jasco model
J-700 spectropolarimeter. The spectra are average computed over three
instrumental scans, and the intensities are presented in terms of
ellipticity values (mdeg).Specific rotations were measured
at 589, 578, 546, 436, 365, and
302 nm by a PerkinElmer polarimeter model 241 equipped with Na/Hg
lamps. The volume of the cell was 1 mL, and the optical path was 10
cm. The system was set at a temperature of 20 °C.The in silico procedure adopted to achieve the
assignment of absolute configuration can be summarized in the following
five steps: (i) conduction of a conformational search based on molecular
mechanic calculations (MM) performed on the structure of 7 characterized by (R)-configuration followed by
the selection of the more representative conformations inside an energetic
window of 4 kcal mol–1; (ii) further optimization
at the AM1 semiempirical level of theory of the conformations selected
from the MM search again followed by the selection of the structures
found inside an energetic window of 1.2 kcal mol–1(four conformations), which corresponds to an overall Boltzmann population
of 93.6%; (iii) energy minimization performed in a vacuum at the B3LYP/6-31G*
level of theory of the four AM1 structures coming from the previous
step, which collapsed to just one final conformation (maximum difference
in energy stability amounting to 0.06 kcal mol–1 and root-mean-square deviation (RMSD) of atomic positions, with
exclusion of hydrogens, lesser than 0.3 Å); (iv) conclusive optimization
at the M06/6-31G* level of theory of the most stable conformation
found in the previous B3LYP minimization step; (v) quantum mechanical
simulation of both electronic circular dichroism (ECD) and optical
rotatory dispersion (ORD) assessed for the conformation of 7 of (R)-configuration achieved in the previous fourth
step.The conformational search quoted in the first step of
the procedure
was carried out through MM calculations (force field: MMFF94), according
to the systematic algorithm implemented in the computer program Spartan
10 v.1.1.0 (Wavefunction Inc., 18401 Von Karman Avenue, Suite 370,
Irvine, CA 92612, USA). The conditions adopted in this analysis were
as follows: (a) all the rotatable bonds varied; (b) 40 kJ mol–1 as the maximum energy gap from the lowest energy
geometry imposed for kept conformations; (c) R2 ≥ 0.9, the criterion adopted to define conformers
as duplicates in the analysis of similarity between conformations.
Similarly, the structure optimizations mentioned in steps from 2 to
4 were again carried out by means of the program Spartan 10 v.1.1.0.Instead, simulation of both ECD and ORD spectra has been performed
through the algorithms implemented in the Amsterdam Density Functional
(ADF) package v. 2007.01. The options set for such calculations were
as follows: single point at the BLYP level of theory, employing the
QZ4P large core basis set; ethanol as the solvent; 30 singlet and
triplet excitations; the diagonalization method: Davidson; velocity
representation; scaling factor, 0.90; and peak width, 8.0. Optical
rotation values, [α], have been
assessed at six different wavelengths (n), in the
range 400–589 nm. The found values, scaled of a factor 10,
were as follows: [α]400 = 45.5°; [α]438 = 33.6°; [α]476 = 26.1°; [α]513 = 21.1°; [α]551 = 17.6°; and
[α]589 = 14.8°.The final simulated ECD
and ORD spectra of ()-7, superimposed on the experimental ones corresponding
to the isolated enantiomers of 7, have been reported
in Figure B,C. By
inspection of the resulting plots, it can be reasonably inferred that
to the first and second eluted enantiomers of 7 (green
and red lines in Figure B,C) have to be assigned the configurations (R)
and (S), respectively.
Authors: Fabrizio Carta; Lorenzo Di Cesare Mannelli; Melissa Pinard; Carla Ghelardini; Andrea Scozzafava; Robert McKenna; Claudiu T Supuran Journal: Bioorg Med Chem Date: 2015-02-21 Impact factor: 3.641
Authors: Anna Di Fiore; Emanuela Truppo; Claudiu T Supuran; Vincenzo Alterio; Nina Dathan; Fatemeh Bootorabi; Seppo Parkkila; Simona Maria Monti; Giuseppina De Simone Journal: Bioorg Med Chem Lett Date: 2010-07-16 Impact factor: 2.823
Authors: Laura Micheli; Lara Testai; Andrea Angeli; Donatello Carrino; Alessandra Pacini; Francesco Margiotta; Lorenzo Flori; Claudiu T Supuran; Vincenzo Calderone; Carla Ghelardini; Lorenzo Di Cesare Mannelli Journal: Int J Mol Sci Date: 2022-06-02 Impact factor: 6.208
Authors: Alma Fuentes-Aguilar; Penélope Merino-Montiel; Sara Montiel-Smith; Socorro Meza-Reyes; José Luis Vega-Báez; Adrián Puerta; Miguel X Fernandes; José M Padrón; Andrea Petreni; Alessio Nocentini; Claudiu T Supuran; Óscar López; José G Fernández-Bolaños Journal: J Enzyme Inhib Med Chem Date: 2022-12 Impact factor: 5.051
Figure 1
Figure 2
Scheme 1
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8