An effective therapeutic approach based on the anti-inflammatory action of hydrogen sulfide (H2S) and inhibition of carbonic anhydrases (CAs) IX and XII is proposed here for the management of arthritis. H2S is a human gasotransmitter that modulates inflammatory response at low concentrations. Inhibition of CAs IX and XII can repristinate normal pH in the acidic inflamed synovial fluid, alleviating arthritis symptoms. We report here the design of H2S donor─CA inhibitor (CAI) hybrid derivatives. The latter were tested in vitro as inhibitors of human CAs I, II, IV, IX, and XII, showing a markedly increased inhibition potency/isoform selectivity compared to the CAI synthetic precursors. The best compounds demonstrated the ability to consistently release H2S and produce a potent pain-relieving effect in a rat model of arthritis. Compound 26 completely reverted the pain state 45 min after administration with enhanced antihyperalgesic effect in vivo compared to the single H2S donor, CAI fragment, or their co-administration.
An effective therapeutic approach based on the anti-inflammatory action of hydrogen sulfide (H2S) and inhibition of carbonic anhydrases (CAs) IX and XII is proposed here for the management of arthritis. H2S is a human gasotransmitter that modulates inflammatory response at low concentrations. Inhibition of CAs IX and XII can repristinate normal pH in the acidic inflamed synovial fluid, alleviating arthritis symptoms. We report here the design of H2S donor─CA inhibitor (CAI) hybrid derivatives. The latter were tested in vitro as inhibitors of human CAs I, II, IV, IX, and XII, showing a markedly increased inhibition potency/isoform selectivity compared to the CAI synthetic precursors. The best compounds demonstrated the ability to consistently release H2S and produce a potent pain-relieving effect in a rat model of arthritis. Compound 26 completely reverted the pain state 45 min after administration with enhanced antihyperalgesic effect in vivo compared to the single H2S donor, CAI fragment, or their co-administration.
Rheumatoid arthritis (RA) and juvenile
idiopathic arthritis (JIA)
are inflammatory diseases affecting people over sixty and under sixteen
years old, respectively, characterized by chronic inflammation of
the synovial membrane, that leads to articular cartilage and juxta-articular
bone destruction and, eventually, deformity. Both conditions are accompanied
by pain.[1−3] The pathophysiology of RA and JIA is unknown to date
but was shown to be related to the previous activation of endothelial
cells with the new blood vessel growth and a hyperplastic expansion
of the synovial membrane that invades the periarticular bone at the
cartilage–bone junction, leading to bone erosions and cartilage
degradation.[1−3] This process promotes the migration in loco of macrophages and leukocytes (T cells, B cells, and monocytes)
that release pro-inflammatory cytokines, such as the tumor necrosis
factor and interleukin-6 (IL-6), stimulating the production of several
secondary modulators: the nuclear factor κB ligand (RANKL),
which is responsible for the osteoclast differentiation and osteoblasts
apoptosis, prostaglandins, and matrix metalloproteinases, that mediate
the symptoms of the disease including pain, swelling, and degradation
of cartilage and bone.[1,2,4]An important piece of evidence is that the patients with RA showed
the concomitant presence of a lower pH (6.5) in the synovial fluid
compared to healthy patients or affected by osteoporosis, which is
linked to inflammatory reaction and direct damage to the cartilage
and tissue around the joint.[5−8] Furthermore, it was demonstrated that the synovial
fluid becomes acidic as the intensity of the inflammatory response
increases, and the more intense the inflammatory reaction was the
more acidic the synovial fluid pH became, provoking pain.[5]Several isoforms of the metalloenzymes
carbonic anhydrases (Cas
and EC 4.2.1.1), that catalyzed the reversible hydration of carbon
dioxide into bicarbonate and proton ions, have been associated with
articular inflammatory diseases.[9,10] In detail, CA I was
found to be overexpressed in the synovium of the patients with ankylosing
spondylitis, and transgenic mice that overexpressed CA I showed aggravated
joint inflammation and destruction.[11,12] Furthermore,
antibodies to CA III and IV have been identified in RA.[8] The latter study also showed that CA activity
in RA was significantly higher than that of control groups. Most importantly,
in 2016, Cimaz et al. demonstrated that isoforms CA IX and XII, which
are usually related to hypoxic tumors, are also overexpressed in the
inflamed synovium of patients affected by JIA.[13]Hydrogen sulfide is the third gasotransmitter of
the human body,
along with nitric oxide (NO) and carbon monoxide (CO), which have
been widely investigated for biomedical purposes over the last three
decades.[14] Its endogenous synthesis is
attributed to three enzymes with different subcellular and tissue
distribution called cystathionine-β-synthase, cystathionine-γ-lyase,
and 3-mercaptopyruvate sulfurtransferase, which use l-cysteine, l-homocysteine, and 3-mercaptopyruvate, respectively, as the
substrate.[14−17] Once synthesized by a cell, this ubiquitous small gaseous signaling
molecule performs a paracrine action that involves at least 200 neighboring
cells, thanks to its lipophilic nature which allows it to cross the
membranes.[18] Hydrogen sulfide has not a
specific receptor or signaling pathway, but it triggers many cellular
effectors in a cell/tissue/species-dependent way, being involved in
various and important physiological processes such as modulation of
vascular tone and blood pressure, neurotransmission and nociception,
angiogenesis, cardiac function, various leukocytic functions, penile
erectile function, scavenger action, acting as an adenosine 5′-triphosphate
(ATP) synthesis stimulator, and so forth.[14,18]Numerous studies demonstrated that the pathogenesis of many
diseases,
including several inflammatory disorders, is related to hydrogen sulfide
deficiency, and the treatment with H2S-donating molecules
improves symptoms.[14,19−28]Based on this evidence, hydrogen sulfide donor—CA inhibitor
hybrid derivatives are here proposed to combine the action of H2S and inhibitors of specific human (h) CA isoforms for the
treatment of inflammation, specifically RA. In fact, inhibition of
the hCA isoforms, whose activity is abnormal on the synovial membrane,
was shown to repristinate the normal pH of the synovial fluid, alleviating
the RA and JIA symptoms (pain, cartilages, and bony erosion).[29−33] On the other hand, the released H2S suppresses the inflammatory
process on multiple levels; improves symptoms such as redness, swelling,
heat, pain, and loss of function, blocking leucocyte adhesion, and
promoting neutrophils apoptosis, analgesia, and reparation of the
damage.[14,19] Moreover, H2S prevents the formation
of the complex RANKL-RANK (ligand–receptor activator of nuclear
factor kappa-Β), blocking the transcription of pro-inflammatory
cytokines, osteoclast differentiation, and osteoblast apoptosis, protecting
bone erosion.[4,22,34] A similar study was recently reported by some of us over the development
of effective CAIs incorporating scaffolds that release carbon monoxide
(based on the dicobalt hexacarbonyl complex), another gasotransmitter
showing pharmacological application for the management of inflammation.[30]
Results and Discussion
Drug Design and Chemistry
The design of H2S donor—CAI hybrids took into account that H2S
is also a rather toxic gas. In fact, if inhaled at concentrations
of >20 ppm, it enters the bloodstream and tissues determining the
blockage of the cell respiratory chain by binding the cytochrome C
oxidase iron, inducing oxidative stress, and blocking the synthesis
of ATP, which is fatal at a concentration of 1000 ppm.[15] For these reasons, it is important to control
the H2S release in order to achieve an optimal concentration
for the therapeutic effect and not poisoning. Among the numerous H2S-releasing scaffolds reported in the literature, which showed
different gasotransmitter releasing properties, the drug design strategy
here proposed was initiated with the 4-substituted 3H-1,2-dithiole-3-thione chemotype (DTT, Figure ),[35] chosen on the basis of the target therapeutic action and resulting
necessary H2S release rate. As for including CAI scaffolds
into the molecular hybrids, the choice fell on the benzenesulfonamide
chemotype which furnishes optimal hCA inhibition potency and was shown
to be suitable for drug design strategies aimed at enhancing isoform
selectivity.[36,37]
Figure 1
Rational design of H2S releaser—CAI
hybrid derivatives
for the management of inflammation.
Rational design of H2S releaser—CAI
hybrid derivatives
for the management of inflammation.The synthesis of the H2S releaser—CAI
hybrids
starts with the preparation of the 3H-1,2-dithiole-3-thione
core, which bears a p-COOH-phenyl moiety at position
4 for the subsequent hybridization with the CAI scaffold. Initially,
4-isopropylbenzoic acid was protected as ethyl ester 1, using SOCl2 in dry ethanol (EtOH) (Scheme ). The cyclization to 3H-1,2-dithiole-3-thione was obtained by treating derivative 1 in melted sulfur at 220 °C, to give intermediate 2, whose ester group was thus hydrolyzed with H2SO4 in CH3COOH at 100 °C, yielding the
carboxylic acid 3.
Scheme 1
Synthesis of the Intermediate 3H-1,2-Dithiole-3-thione 3
Reagents and conditions:
(a)
SOCl2, dry EtOH, 0 → 60 °C, 6 h, 92%; (b) S8, 135 → 220 °C, 6–8 h, 75%; (c) H2SO4 9 M, CH3COOH, 100 °C, 4 h, 81%.
Synthesis of the Intermediate 3H-1,2-Dithiole-3-thione 3
Reagents and conditions:
(a)
SOCl2, dry EtOH, 0 → 60 °C, 6 h, 92%; (b) S8, 135 → 220 °C, 6–8 h, 75%; (c) H2SO4 9 M, CH3COOH, 100 °C, 4 h, 81%.The coupling reaction of 3 with primary
amine derivatives
of benzenesulfonamides [e.g. sulfanilamide, 4-(aminomethyl)benzenesulfonamide,
4-(2-aminoethyl)benzenesulfonamide] with several coupling agents and
reaction conditions unexpectedly always led to the undesired attack
on the scaffold thione group with the formation of the imine-like
derivatives A–C even at very low temperatures
(Scheme ).
Scheme 2
Formation
of Side Products A–C Upon Amide Coupling
of Carboxylic Acid 3 with Primary Amine Benzenesulfonamide
Derivatives
As a result, it was supposed that the use of
secondary amines benzenesulfonamide
derivatives, such as 4–18, could prevent the formation
of these undesired side products in the coupling reaction conditions
used in our experiments (Scheme ). Hence, 4-(2-aminoethyl)benzenesulfonamide was treated
by reductive amination in presence of the proper aromatic aldehydes
and sodium borohydride in dry methanol (MeOH) or, alternatively, by
nucleophilic substitution with the appropriate halides in dry dimethylformamide
(DMF), to give secondary amines 4–18. The latter
was coupled with carboxylic acid 3 by using PyBOP as
the coupling agent and DIPEA as a base in dry DMF at room temperature
(r.t.).
Scheme 3
Synthesis of 3H-1,2-Dithiole-3-thione Benzenesulfonamide
Hybrid Derivatives 19–33
The synthesized compounds 19–33 were tested for their inhibitory action against hCA isoforms I,
II, IV, IX, and XII, by a stopped-flow kinetic assay using acetazolamide
(AAZ) as standard.[38] The cytosolic
hCA I and II are considered off-target for the CAI anti-inflammatory
therapeutic application because they are ubiquitous and responsible
for most CAI side effects. In contrast, hCAs IV, IX, and XII were
the target isoforms being overexpressed on the synovial membrane and
linked to inflamed conditions.[29−33,39]Table gathers the inhibition constants (KIs) of compounds 19–33 against
the panel of hCAs, whereas the selectivity index (SI) against hCA
IV, IX, and XII over the off-target isoforms hCA I and II are collected
in Table .
Table 1
Inhibition Data of Human CA Isoforms
hCA I, II, IV, IX, and XII with Sulfonamides 19–33 Reported Here and the Standard Sulfonamide Inhibitor AAZ by a Stopped-Flow CO2 Hydrase Assay[38]
Mean from three different assays,
by a stopped-flow technique (errors were in the range of ±5–10%
of the reported values).
Table 2
SI of Sulfonamides 19–33 and Standard AAZ Against the anti-inflammatory Human
CA Isoforms IV, IX, and XII versus the Ubiquitous Cytosolic Ones CA
I and II
SI
cmpd
CA I/IV
CA II/IV
CA I/IX
CA II/IX
CA I/XII
CA II/XII
19
0.18
0.11
19.2
11.1
79.4
49.5
20
<0.53
<0.06
115
13.0
81.5
9.2
21
12.8
6.9
229
124
561
303
22
7.4
3.5
403
192
597
284
23
>216.4
104
>248
120
>1136
548
24
1.2
0.3
188
43.9
448
105
25
<0.7
<0.09
204
25.1
226
27.9
26
1.6
0.7
1633
719
870
383
27
1.4
0.4
44.0
11.7
29.0
7.7
28
3.0
0.7
47.9
11.7
105
25.7
29
17.9
23.9
156
209
858
1148
30
>41.0
18.3
>641
286
>1428
637
31
>208
51.7
>4167
103
>1204
299
32
18.0
2.7
34.9
5.3
462
69.6
33
0.8
0.11
15.0
2.1
45.2
6.4
AAZ
3.4
0.17
10.0
0.5
43.9
2.2
Mean from three different assays,
by a stopped-flow technique (errors were in the range of ±5–10%
of the reported values).Most tested compounds displayed weak inhibition against
the off-target
hCA I, with KI values in a rather flat
micromolar range (KIs of 2215–10000
μM), except derivatives 19 and 28 that
inhibited this ubiquitous isoform in the high nanomolar range (KI of 770.7–890.3 nM). In contrast, all
tested compounds produced a greater hCA II inhibition when compared
to hCAI (KIs in the range 218.5–4826
nM), with the exception of compound 29. In fact, derivatives 19, 20, 27, 28, and 32 inhibit hCA II significantly below the micromolar range
(KIs of 218.5–660.9 nM). It should
be noted that the elongation of the N-substituent from a benzyl (19) to a phenethyl (32) decreases significantly
the inhibitory action against hCA I (KI from 770.7 to 4387 nM) and with minor extent against hCA II (KI from 479.9 to 660.9 nM). Interestingly, the
only tertiary amide derivative not bearing an aromatic scaffold on
the N-substituent, that is the nitrile 33 (R = CH2CN), shows the greatest efficacy against hCAs I
and II (KIs of 429.2 and 60.4 nM, respectively).
It can be speculated that the loss of steric encumbrance of 33 in comparison to other derivatives favors the ligand binding
in the narrow active sites of these ubiquitous isoforms. As a matter
of fact, all substituents included on the outer phenyl ring of compound 19 decrease the inhibitory action against the cytosolic isoforms,
with the only exception being the dimethoxy derivative 28 against hCA II (KI of 479.9 vs 218.5
nM).As for hCA IV, the p-CN-phenyl and benzo[b]thiophen-3-yl derivatives 23 and 31 only exhibit KI values below 100 nM
(KIs of 46.2 and 48.0 nM), resulting in the most potent
and also the first and the second most selective compounds against
hCA IV over hCA II (SI of 104 and 52, respectively).Compounds 21, 28–30, 32, and 33 produce instead hCA IV inhibition in the high
nanomolar range (KIs of 243.2–523.1
nM), while inhibitors 19, 22, 26, and 27 acted in the low micromolar span (KIs of 1042–4339 nM)and up to no inhibition detected
below 10 μM for 20 and 25.Notably,
hCA IX and XII were the isoforms most inhibited by all
3H-1,2-dithiole-3-thione derivatives, with most KIs collected in a narrow range for both isoforms
(KIs spanning between 2.4–50.3
nM and 5.7–32.9 nM for hCA IX and XII, respectively). It should
be stressed that the target isozymes IX and XII show in fact rather
roomier active sites when compared to other hCAs, which can favor
the accommodation and binding of the bulky tertiary amide derivatives 19–32 after the binding of the sulfonamide moiety to
the zinc ion. In particular, compounds 31 and 26 were the most potent and selective hCA IX inhibitors with a single
digit (KIs of 2.4 and 4.1 nM, respectively,
and hCA II/hCA IX SI of 1034 and 719, respectively). As with hCAs
I and II, the elongation of the N-substituent from 19 to 32 led the KI above
100 nM (KI = 125.7 nM). All other hybrid
compounds exhibited medium nanomolar range hCA IX inhibition with KI values between 15.6 and 50.3 nM.On
the other hand, hCA XII was inhibited with single-digit KIs by a greater subset of compounds (19, 21, 23, 24, 26, and 28–33) (KIs
in the range 7.0–9.8 nM). Among these, derivatives 30 (KI = 7.0 nM) and 26 (KI = 7.7 nM) were the most potent hCA XII inhibitors,
while 29 (KI = 8.4 nM) and 30 (KI = 7.0 nM) were the most
selective hCA XII inhibitors over the main off-target hCA II (SI of
1148 and 637, respectively). Compounds 20, 22, 25, and 27 inhibited instead hCA XII
in the medium nanomolar range with KI values
between 12.9 and 76.4 nM.Overall, the obligatory choice of
coupling bulky secondary instead
of primary amine benzenesulfonamide derivatives to the H2S donor nucleus, due to side products formation in the planned synthetic
pathway, resulted to be a good option for the achievement of potent
hCA IX and XII inhibitors also showing a high selectivity over off-target
hCAs. In fact, the incorporation of N-alkyl aromatic substituents
led the 3H-1,2-dithiole-3-thione derivatives to prefer
the binding to the roomier active site of hCA IX and XII with respect
to other hCA isoforms. In fact, it can be noted that the 2-ethylcyano
derivative 33 showing the least steric encumbrance within
the series exhibited a significant loss of target/off-target selectivity
of action in comparison to most other such hybrid compounds, with
the hCA II/IX SI being only 2.1.It should be also stressed
that the secondary amine benzenesulfonamide
derivatives 4–18 do not show notable CA inhibition
profiles both in terms of potency and isoform selectivity, as previously
reported,[32,33] and here further investigated. Table S1, Supporting Information collects the
inhibition profiles of 4–18 against hCA I, II,
IV, IX, and XII, while the related SI values were reported in Table S2, Supporting Information. Generally,
benzenesulfonamide derivatives 4–18 were medium
to high nanomolar inhibitors of hCA I (KI = 82.3–373.8 nM), II (KI = 47.3–201.6
nM), IX (45.3–119.9), and XII (KI = 46.2–113.2 nM), and weak inhibitors of hCA IV with inhibition
constant (KI) values in the low micromolar
range (0.9–7.2 μM). The 1-naphthyl derivative 15 stood out as the most promising one, according to inhibition potency
(hCA IX, KIs of 45.3; hCA XII, 54.7 nM)
and selectivity against hCAs IX and XII over off-target isoforms (CA
I/IX SI = 5.8; CA I/XII SI = 4.8; CA II/IX SI = 4.0; and CA II/XII
SI = 3.2). It is worth noting that, as previously shown,[35,36] most such single CAI derivatives did not show any relevant isoform
selectivity, but instead exhibit a rather flat inhibition profile
against target and off-target isozymes.
Evaluation of H2S Release
The H2S releasing properties of the 4-substituted-3H-1,2-dithiole-3-thione
precursor 3 and the hybrid derivatives showing the best
CA inhibition profiles (in terms of potency and selectivity against
the target isoforms), that are 26, 29, 30, and 31, were evaluated by a spectrophotometric
methylene blue (MB+) assay adapted from previously reported
methods.[40,41] The released H2S, trapped by
zinc acetate to form zinc sulfide, reacts with N,N-dimethyl-1,4-phenylenediamine in the presence of iron(III)
to yield methylene blue, which can be easily quantified by measuring
absorbance at 667 nm (MB+ form) or at 748 nm (MBH2+).[42,43] The compound H2S release in different
conditions was monitored over time and quantified according to a calibration
curve with Na2S (1–300 μM). Incubation of
compound 3 and the hybrid derivatives in phosphate buffer
at 37 °C either in the presence or absence of thiol compounds
(cysteine or glutathione) produced no or a barely detectable H2S release, respectively. Therefore, trigger mechanisms other
than hydrolysis and activation with thiols were investigated, such
as enzymatic/metabolic triggers in rat tissue homogenates as reported
in 2011 by Li et al. with similar such compounds.[41] Incubation of 3, 26, 29, 30, and 31 at 37 °C in rat liver homogenate
resulted in an almost immediate and sustained release of H2S over the full 90 min incubation period, implying that the most
released gas from the molecules occurred as a result of a metabolic
event. At present, the nature of this activation step is not known. Table S3, Supporting Information lists the parameters
of Cmax (the highest concentration of
H2S released) and t1/2 (the
time at which half the Cmax is reached)
from the tested compounds. The parent compound 3 showed
the highest H2S release in terms of the rate and Cmax. Its H2S release peaked at a
concentration of 12.1 μM after 20 min approximately and remained
as such with slight fluctuations for the residual analysis time (Figure ), with a t1/2 of 3.1 min.
Figure 2
H2S releasing profile of compounds 3, 26, 29, 30, and 31 (150
μM). Compounds were incubated at 37 °C in rat liver homogenate.
H2S concentration was determined at timed intervals spectrophotometrically.
Data are expressed as means ± SEM of three independent assays.
H2S releasing profile of compounds 3, 26, 29, 30, and 31 (150
μM). Compounds were incubated at 37 °C in rat liver homogenate.
H2S concentration was determined at timed intervals spectrophotometrically.
Data are expressed as means ± SEM of three independent assays.In comparison to precursor 3, the
tertiary amide derivatives 26, 29, 30, and 31 exhibited
a weaker H2S release (Figure ), with an overall superimposable trend for
all compounds, implying that the amide substituents barely affect
the compound’s ability to release H2S. The latter
seems instead in part affected by the carboxylic acid (3) to amide (26, 29, 30, and 31) conversion. The H2S Cmax values of the amide derivatives settle in the range of
7.9–8.4 μM, with t1/2 in
the range of 4.3–5.0 min (Table S3, Supporting Information). The gasotransmitter release of the tested
hybrid compounds overall reached a plateau after approximately 25
min. The H2S release profile of the DTT derivatives here
tested is consistent with that of previously described similar such
derivatives, although differences exist in the compound chemical structures
and in the adopted spectrophotometric protocol. As a matter of fact,
in 2007 Li et al. showed in vitro consistent H2S release
profiles of the 5-substituted DTT compound ADTOH and
its diclofenac derivative, when incubated in rat liver homogenate.[41] The same authors evaluated successively ADTOH
and the 4-substituted analogue ACS48 (compound 3 in the
present work) for their H2S donating properties in the
cell, also showing a sustained release.[44] In 2017, Li et al. also evaluated, in vitro, a similar H2S release for ADTOH and some such nonsteroidal anti-inflammatory
derivatives.[45] The authors deduced a hydrolytic
trigger for the compound gasotransmitter release, but the reducing
agent TECP, that is tris(2-carboxyethyl)phosphine, was actually used.
In 2018 Giustarini et al. also demonstrated the sustained H2S release of 3 (ACS48), in vivo, by monitoring the gasotransmitter
concentration in plasma of rats treated intravenously with such a
DTT and some of its derivatives.[35] As a
result, to the best of our knowledge, the 4-substituted DDT 3 and amide derivatives, such as 19–33, were here assessed for the first time for their H2S
release, in vitro.
In Vivo Studies
On the basis of the superimposable
H2S releasing profiles of all tested hybrid derivatives, 26, 29, 30, and 31 were
also evaluated for their anti-inflammatory effect in a rat model of
arthritis induced by the intra-articular injection of complete Freund’s
adjuvant (CFA) in the Paw pressure and Incapacitance tests.[33] Moreover, the synthetic precursors 3, as the single H2S releasing molecule, and 15, as the most potent single CAI warhead against the target hCAs IX
and XII, were tested to evaluate the hybrid efficacy with respect
to the single agents, and their coadministration. The CFA model induces
pain and articular damage characterized by diffuse lesions caused
by an immune reaction towards antigens of connective tissue or joint.[46] The primary target of this response is the collagen
that can be disrupted by T cells activation. The pathogenesis is characterized
by augmented levels of pro-inflammatory cytokines,[47] neutrophil infiltration and subsequent synovia hyperplasia
with a total ablation of the joint space that is replaced by hyperplastic
tissue.[48]The pain-relieving properties
of compounds were evaluated after a single per os administration in a dose ranging from 1 to 30 mg/kg when pain and
articular lesions were well established (day 14 from the induction
of the damage). The Paw pressure test was used to measure the hypersensitivity
in response to a mechanical noxious stimulus (Figure ). In the group treated with CFA (red line),
the weight tolerated on the ipsilateral paw decreased to 33 g in comparison
to the control value of 65 g (black line) in the untreated rats (vehicle
+ vehicle). Compound 26 evoked an antihypersensitivity
effect in a dose-dependent manner. In particular, the dose of 30 mg/kg
counteracted the mechanical hyperalgesia 45 min after injection, showing
a mild effect also at 60 min. The lower doses were still active and
even showed a lower efficacy (Figure A). Compounds 29, 30, and 31 were active in reducing the mechanical hyperalgesia evoked
by CFA intra-articular (i.a.) injection with no dose-response effect
between the dose of 10 and 30 mg/kg. Both dosages significantly increased
the weight tolerated by the animals on the ipsilateral paw in comparison
to CFA-treated animals starting from 15 min up to 45 min after treatment.
For all these three hybrids, the lower dose of 1 mg/kg was ineffective
(Figure B–D). Figure E reported the comparison
between the antihyperalgesic effect evoked by hybrid 30 30 mg/kg in comparison to the equimolar doses of its single portions,
that is the H2S donor 3 and the CAI 15, selected for being the most potent inhibitor of hCAs IX
and XII among the secondary amine derivatives 4–18. The effect of 30 was also compared to the co-administration
of 3 and 15. Despite 30 not
standing as the best hybrid compound in terms of in vivo efficacy
in the Paw pressure test, it clearly showed a greater antihyperalgesic
action with respect to 3, 15, and their
co-administration, highlighting the advantage of our strategy intended
to merge in a single-molecular entity the two therapeutically active
portions (Figure E).
Unlike the hybrid derivatives, the single H2S donor 3 evoked a rapid antihyperalgesic effect that peaked at 15
min and totally disappeared after 45 min. Such a trend might be partially
justified by the greater H2S release rate detected for
compound 3 (Figure ) in comparison to the other amide derivatives. Interestingly,
compound 15, which possesses CA inhibition properties
only, showed a very weak antihyperalgesic action at the dose here
evaluated (17.71 mg/kg) equimolar to the active dose of the hybrid
compounds (30 mg/kg). As a matter of fact, in the previous study regarding
CO releaser—CAI hybrid compounds, a benzenesulfonamide CAI
derivative inducing a potent inhibition of the target hCAs IX and
XII, referred to as 3a,[30] showed
a detectable pharmacological action in the Paw pressure test only
when administered at a 4-fold higher molar dose than 15. The different dosages administered for 15 (present
study) and 3a (past study)[30] depend on the 4-fold lower dose necessary for the best hybrid H2S releaser here reported (26) to achieve a pharmacological
action even higher than the best hybrid CAI—CO releaser (referred
as 5b)[30] in the Paw pressure
test. In contrast to the rapid action of the H2S donor 3, the CAI 3a exhibited a peak of antihyperalgesic
at 45 min.[30]
Figure 3
Acute pain-relieving
effect of hybrid 26, 29–31 (A–D)
H2S donor 3 and CAI 15 (E) in
an adjuvant-induced arthritis model. The compounds were administered
in a range of doses from 1 to 30 mg/kg, while 3 and 15 were administered
at the equimolar dose of 30 mg/kg of hybrid derivative 30 (13.23 and 17.71, respectively). Compounds were suspended in 1%
CMC and orally administered. The measurements of mechanical hyperalgesia
were accomplished on day 14 after CFA injection by the Paw pressure
test. The values reported are the mean of 8 rats performed in 2 different
experimental sets. ^̂P < 0.01 vs vehicle
+ vehicle-treated animals; *P < 0.05 and **P < 0.01 vs CFA + vehicle group.
Acute pain-relieving
effect of hybrid 26, 29–31 (A–D)
H2S donor 3 and CAI 15 (E) in
an adjuvant-induced arthritis model. The compounds were administered
in a range of doses from 1 to 30 mg/kg, while 3 and 15 were administered
at the equimolar dose of 30 mg/kg of hybrid derivative 30 (13.23 and 17.71, respectively). Compounds were suspended in 1%
CMC and orally administered. The measurements of mechanical hyperalgesia
were accomplished on day 14 after CFA injection by the Paw pressure
test. The values reported are the mean of 8 rats performed in 2 different
experimental sets. ^̂P < 0.01 vs vehicle
+ vehicle-treated animals; *P < 0.05 and **P < 0.01 vs CFA + vehicle group.To exclude possible toxicity induced by the acute
treatment with
the described hybrids, autonomic, behavioral, and neurological parameters
were assessed by the Irwin test. As reported in Table S4, Supporting Information the highest dose of all compounds
did not alter the endpoints analyzed.The hybrid derivatives 26, 29, 30, and 31 were
also tested against postural
unbalance, a typical feature of monolateral pain, using the Incapacitance
test as an evaluation of spontaneous pain since no direct noxious
or non-noxious stimuli are applied by the operator (Figure ). The difference between the
weight bore on the contralateral and the ipsilateral paw was significantly
increased up to 55 g (red line) in CFA-treated rats in comparison
to the 2 g of the control group (black line). As in the Paw pressure
test, similar results were obtained in this behavioral measurement.
Compounds 26, 29, and 31 reduced
spontaneous pain in a dose-dependent manner with a peak of efficacy
between 30 and 45 min after treatment (Figure A,B,D). Again, compound 26 demonstrated
the greatest antihyperalgesic action almost completely reverting spontaneous
pain after 45 min. Compound 30 oddly showed a slightly
greater efficacy at 10 mg/kg in comparison to the dose of 30 mg/kg,
while the lowest dose was ineffective (Figure C). In the Incapacitance test, compound 30 induced a greater antihyperalgesic action than the co-administration
of 3 and 15 (Figure E). Nonetheless, in this test the action
of compound 3 alone also exceeded that of the combination
therapy and was comparable to that of the hybrid analogue 30 at its optimal dose (Figure E). Again, the antihyperalgesic effect of the H2S releaser 3 rapidly peaked at 15 min, already disappearing
after 30 min. Again, in absence of effects detected in vivo for 15 at the used dose, the CAI 3a previously reported[30] can be used to prove the antihyperalgesic action
of a single CAI warhead in the Incapacitance test. In the latter,
compound 3a, tested at a 4-fold greater dose than 15, exhibited a weak action with a peak at 45 min.
Figure 4
Acute pain-relieving
effect of the hybrid compounds 26, 29–31 (A–D) H2S donor 3 and CAI 15 (E) in an adjuvant-induced arthritis
model. Hybrids were administered in a range of doses from 1 to 30
mg/kg, while 3 and 15 were administered
at the equimolar dose of 30 mg/kg of hybrid 30 (13.23
and 17.71, respectively). Compounds were suspended in 1% CMC and orally
administered. The measurements of spontaneous pain were accomplished
on day 14 after CFA injection by the Incapacitance test. The values
reported are the mean of 8 rats performed in 2 different experimental
sets. ^̂P < 0.01 vs vehicle + vehicle-treated
animals; *P < 0.05 and **P <
0.01 vs CFA + vehicle group.
Acute pain-relieving
effect of the hybrid compounds 26, 29–31 (A–D) H2S donor 3 and CAI 15 (E) in an adjuvant-induced arthritis
model. Hybrids were administered in a range of doses from 1 to 30
mg/kg, while 3 and 15 were administered
at the equimolar dose of 30 mg/kg of hybrid 30 (13.23
and 17.71, respectively). Compounds were suspended in 1% CMC and orally
administered. The measurements of spontaneous pain were accomplished
on day 14 after CFA injection by the Incapacitance test. The values
reported are the mean of 8 rats performed in 2 different experimental
sets. ^̂P < 0.01 vs vehicle + vehicle-treated
animals; *P < 0.05 and **P <
0.01 vs CFA + vehicle group.Relevantly, also in the Incapacitance test, the
antihyperalgesic
action of the best H2S releaser hybrid 26 significantly
exceeded, according to a 4-fold lower administered dose (30 vs 100
mg/kg, and greater molecular weight), that induced by the best hybrid
CAI—CO releaser (5b), which in turn was comparable
to the reference drug ibuprofen 100 mg/kg.[30]In both in vivo tests, the antihyperalgesic action of the
evaluated
hybrid derivatives reached a maximum effect after 30 min post-administration
with the exception of compound 26, which showed better
efficacy and prolonged action over analogues 29–31. The H2S release of the hybrid derivatives, detected
by a methylene blue assay in a sealed system, showed a plateau after
25 min approximately. In contrast, a more sustained H2S
release was notified in vivo (open system) for similar such derivatives,[41] which might account for an anti-inflammatory
action peak in the range 30–45 min. On the other hand, it can
be supposed that the trend of the hybrid in vivo action is consistent
with a biological response being the result of the merged H2S release (peak at 15 min) and target CA inhibition (peak at 45 min
as previously shown).[30]The detected
enzymatic/metabolic H2S release trigger
for the DTT scaffold, rather than hydrolysis and thiol activation,
might be an advantage for the setup of such an anti-inflammatory therapy.
In fact, it can be supposed that the DTT derivatives start the gasotransmitter
release only after absorption has occurred.On the basis of
the multitarget or hybridization approach here
proposed, the exceptionally improved antihyperalgesic profile of the
hybrid derivatives over the single agents and their co-administration
could be rationalized as upgraded pharmacokinetics driving both pharmacophores
(H2S donor and CAI), combined as a unique molecule, to
the target inflamed site, where a strong synergistic effect takes
place. Additionally, it can be speculated that the pharmacophore hybridization
enhances drug absorption, which may be instead diminished in case
of a coadministration.
Conclusions
H2S is a ubiquitous small gaseous
signaling molecule,
playing an important role in many physio-pathological processes. For
instance, H2S-donating molecules attracted enormous interest
as they were able to promote the resolution of inflammation at the
low endogenous gas concentration. On the other hand, some of us recently
showed that a panel of CA isoforms, with CAs IX and XII as the main
targets, are implicated in the pathogenesis of inflammation, and their
inhibitors can repristinate the normal pH in the acidic inflamed synovial
fluid, alleviating arthritis symptoms.Hence, we described herein
for the first time the synthesis and
biological evaluation of new hybrid derivatives possessing a CAI warhead
of the sulfonamide type conjugated to a DTT core as an H2S donating molecule. All hybrid compounds were tested in vitro as
inhibitors of human (h) CA I, II, IV, IX, and XII, showing a marked
increase in isoform-selectivity upon the proposed hybridization strategy
over the CAI synthetic precursors. Compounds 26 and 31 were the most potent and selective hCA IX inhibitors with
single-digit KIs of 4.1 and 2.4 nM, respectively,
and hCA II/hCA IX SI of 1034 and 719, respectively. Also, 26 and 31 were among the best hCA XII inhibitors with KIs of 7.7 and 8.0 nM, respectively, and hCA
II/hCA XII SI of 383 and 299, respectively. The best among such compounds, 26, 29, 30, and 31,
along with the precursor and previously reported DTT 3, were shown to consistently release H2S by a spectrophotometric
methylene blue method. Compounds 26, 29, 30, and 31 induced a pain-relieving effect in
a rat model adjuvant-induced arthritis with a single i.a. injection
of CFA. Compound 26 showed the greatest efficacy in vivo
completely reverting pain 45 min after treatment both in the Paw pressure
and Incapacitance tests. All tested compounds demonstrated a markedly
greater antihyperalgesic action compared to treatment with the single
H2S donor 3, the CAI fragment 15, and their co-administration. A comparison was thus made with previously
reported CAI—CO releaser hybrid derivatives.[30] Relevantly, the performance of the best H2S
donor hybrid 26 significantly exceeded, according to
a 4-fold lower administered dose (30 vs 100 mg/kg), that induced by
the best hybrid CAI—CO releaser (5b), which in
turn was comparable to the reference drug ibuprofen 100 mg/kg. Compound
toxicity induced by the acute treatment was excluded from evaluating
the animal autonomic, behavioral, and neurological parameters by the
Irwin test. In conclusion, the hybridization drug design approach
here proposed drastically improved the anti-inflammatory profile of
the hybrid derivatives over the single agents and their coadministration
and provided new outstanding biomedical tools for the development
of new potent anti-inflammatory treatment against arthritis based
on H2S release and CA inhibition.
Experimental Section
Chemistry
Anhydrous solvents and all reagents were
purchased from Sigma-Aldrich, Alfa Aesar, and TCI. All reactions involving
air- or moisture-sensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringes techniques to transfer
solutions. Nuclear magnetic resonance (1H NMR, 13C NMR, 19F NMR) spectra were recorded using a Bruker Advance
III 400 MHz spectrometer in dimethylsulfoxide (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; brs,
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 (EtOAc)/n-hexane or MeOH/dichloromethane
(DCM) were used as eluents. Melting points (mp) were measured in open
capillary tubes with a Gallenkamp MPD350.BM3.5 apparatus and are uncorrected.
All compounds were >95% pure by HPLC analysis, performed by using
a Waters 2690 separation module coupled with a photodiode array detector
(PDA Waters 996) and as column a Nova-Pak C18 4 μm 3.9 mm ×
150 mm (Waters) silica-based reverse phase column. The sample was
dissolved in acetonitrile 10%, and an injection volume of 45 μL
was used. The mobile phase, at a flow rate of 1 mL/min, was a gradient
of water + trifluoroacetic acid (TFA) 0.1% (A) and acetonitrile +
TFA 0.1% (B), with steps as follows: (A%:B%), 0–10 min 90:10,
10–25 min gradient to 60:40, 26–28 min isocratic 20:80,
and 29–35 min isocratic 90:10. TFA 0.1% in water as well in
acetonitrile was used as the counterion.
Synthesis of Ethyl 4-Isopropylbenzoate (1)
Thionyl chloride (1.8 equiv) was added dropwise to a solution of
4-isopropylbenzoic acid (12.7 mmol, 1.0 equiv) in EtOH (30 mL) cooled
at 0 °C, and the reaction mixture was stirred to 60 °C for
6 h. The solvent was evaporated under a vacuum, and the crude was
dissolved in EtOAc (40 mL) and washed with water and NaHCO3 sat. sol. (30 mL × 2). The organic layer was dried over Na2SO4, filtered, and evaporated under a vacuum. The
obtained colorless oil was purified with flash chromatography (EtOAc
20%/Hex) to give 1. Yield 92%; δH (400
MHz, DMSO-d6): 7.78 (d, J = 8.1 Hz, 2H, Ar–H), 7.29 (d, J = 8.2 Hz, 2H, Ar–H), 4.19 (q, J = 7.1 Hz, 2H, CH2), 2.86 (hept, J = 6.9 Hz, 1H, CH), 1.21 (t, J = 7.1 Hz, 3H, CH2CH3), 1.11
(d, J = 6.9 Hz, 6H, CH(CH3)2). m/z (ESI positive)
192.1 [M + H]+.
Synthesis of Ethyl 4-(3-Thioxo-3H-1,2-dithiol-4-yl)benzoate
(2)
Compound 1 (10.4 mmol, 1.0
equiv) was added dropwise to stirred melted sulfur (10 equiv) at 135
°C, and the reaction mixture was stirred at 220 °C for 6–8
h. The temperature is lowered to 135 °C, and after the addition
of 20 mL of a toluene/acetone mixture (3:7), the suspension was triturated
at r.t. The solvent was evaporated under vacuum, and the residue was
purified with flash chromatography (EtOAc 20%/Hex) to give 2 as a brown powder. Yield 75%; δH (400 MHz, DMSO-d6): 9.26 (s, 1H, =CH), 8.02 (d, J = 8.5 Hz, 2H, Ar–H), 7.73 (d, J = 8.5 Hz, 2H, Ar–H), 4.34 (q, J = 7.1 Hz, 2H, CH2), 1.33 (t, J = 7.1 Hz, 3H, CH3). m/z (ESI positive)
281.9 [M + H]+.
Synthesis of 4-(3-Thioxo-3H-1,2-dithiol-4-yl)benzoic
Acid (3)
H2SO4 9 M (16
mL) was added to a solution of 2 (7.09 mmol, 1.0 equiv)
in acetic acid (50 mL), and the reaction mixture was stirred at 100
°C for 4 h. After cooling at r.t. and adding slush, the mixture
was extracted with MeOH 10%/DCM (40 mL × 3) and washed with HCl
0.1 M (1 × 30 mL). The organic layer was dried over Na2SO4, filtered, and evaporated under a vacuum. The obtained
residue was triturated with diethyl ether, collected by filtration,
and purified with flash chromatography (EtOAc 50%/Hex) to give 3 as a brown-orange powder. Yield 81%; mp 235–236 °C;
δH (400 MHz, DMSO-d6):
12.94 (bs, 1H, exchange with D2O, COOH), 9.25 (s, 1H, =CH), 8.00 (d, J = 8.3 Hz, 2H, Ar–H), 7.71 (d, J = 8.2 Hz, 2H, Ar–H). m/z (ESI positive) 253.9 [M + H]+.
General Synthetic Procedures for Secondary Amines 4–18
Procedure 1
The appropriate aldehyde (1.1 equiv) was
added to a solution of 4-(2-aminoethyl)benzenesulfonamide (4,99 mmol,
1.0 equiv) in dry MeOH (20 mL), and the mixture was heated under stirring
to reflux temperature for 0.5–4 h. Sodium borohydride (1.6
equiv) was added portion-wise at 0 °C, and the reaction mixture
was heated under stirring to reflux temperature for 0.5–3 h.
The solvent was evaporated under a vacuum, water was added (25 mL)
to it, and it was neutralized with HCl 1 M. The suspension was filtered,
and the collected powder was occasionally purified with flash chromatography
(MeOH 5%/DCM) to give the compounds 4–16.
Procedure 2
Triethylamine (1.2 equiv) and the appropriate
halide (1.1 equiv) were added to a solution of 4-(2-aminoethyl)benzenesulfonamide
(4,99 mmol, 1.0 equiv) in dry DMF (3 mL) at r.t. and the mixture was
stirred at 60 °C for 8 h (17) or r.t. for 0.5 h
(18). The reaction mixture was quenched by the addition
of water (10 mL) and extracted with DCM (20 mL × 3). The organic
layer was collected, washed with brine (30 mL × 3), dried over
Na2SO4, filtered, and evaporated under vacuum
to give a yellow (17) or white (18) powder.
4-(2-(Benzylamino)ethyl)benzenesulfonamide (4)
Compound 4 was obtained as white powder according
to the general procedure 1 reported earlier using benzaldehyde (1.1
equiv). Yield 96%; mp 173–175 °C; silica gel TLC R 0.08 (TFA/MeOH/DCM 3/5/92%
v/v). δH (400 MHz, DMSO-d6): 7.76 (d, J = 8.1 Hz, 2H, Ar–H), 7.42 (m, 7H, Ar–H), 7.32 (s, 2H, exchange
with D2O, SO2NH2, overlap with signal at 7.42), 4.04 (s, 2H, CH2), 3.07 (m, 2H, CH2), 2.97 (m,
2H, CH2). δC (100 MHz,
DMSO-d6): 145.87, 142.74, 141.80, 129.99,
129.05, 128.86, 127.46, 126.55, 53.77, 50.91, 36.50. m/z (ESI positive) 291.1.
Compound 18 was obtained as a white powder
according to the general procedure 2 reported earlier using 3-chloropropionitrile
(1.1 equiv). Yield 85%; silica gel TLC R 0.15 (TFA/MeOH/DCM 3/5/92% v/v). δH (400 MHz, DMSO-d6): 7.72 (d, J = 8.0 Hz, 2H, Ar–H), 7.41 (d, J = 8.0 Hz, 2H, Ar–H), 7.27 (s,
2H, exchange with D2O, SO2NH2), 2.76 (m, 6H, 3 x CH2),
2.57 (t, J = 6.6 Hz, 2H, CH2). δC (100 MHz, DMSO-d6): 145.72, 142.88, 130.14, 126.68, 121.19, 50.83, 45.66, 36.59,
18.88. m/z (ESI positive) 254.0
[M + H]+.
General Synthetic Procedure for the H2S Releaser-CAI
Hybrids 19–33
The appropriate secondary
amine 4–18 (0.98 equiv), DIPEA (2.2 equiv), and
PyBOP (1.1 equiv) were added to a solution of 3 (0.98
mmol, 1.0 equiv) in DMF dry (1 mL), and the reaction mixture was stirred
o.n. at r.t. The reaction was quenched with water, and the formed
precipitate was collected by filtration and washed with NaHCO3 sat. sol. and water. The obtained residue was purified with
flash chromatography (MeOH 1%/DCM) to give the compounds 19–33.
Compound 33 was obtained as an orange powder according
to the general procedure reported earlier using 18 (1.1
equiv). Yield 41%; mp 154–155 °C. δH (400
MHz, DMSO-d6): 9.25 (s, 1H, =CH), 7.50 (m, 8H, Ar–H), 7.26 (s,
2H, exchange with D2O, SO2NH2, overlap with signal at 7.50), 3.79 (m, 4H, 2 x CH2), 2.96 (m, 4H, 2 × CH2). δC (100 MHz, DMSO-d6): 214.12, 213.99, 171.19, 170.88, 160.36, 160.09, 147.78,
147.51, 142.98, 142.79, 142.18, 141.73, 136.66, 136.52, 134.95, 134.84,
133.00, 129.77, 129.41, 128.96, 128.38, 127.66, 126.96, 126.57, 126.25,
119.55, 119.39, 50.59, 49.71, 45.17, 42.27, 34.41, 33.35, 16.11, 16.02.
ESI-HRMS (m/z): [M + H]+ calcd for C21H19N3O3S4, 490.0309; found, 490.0312.An Applied Photophysics stopped-flow
instrument has been used for assaying the CA-catalyzed CO2 hydration activity.[38] 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 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 15 min at r.t. before 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,[39] and represent the mean from at least three different
determinations. The enzyme concentrations were in the range of 3–11
nM. All hCA isoforms were recombinant ones obtained in-house, as reported
earlier.[32,33]
Spectrophotometric Evaluation of H2S Release
Rat liver was homogenized in ice-cold 50 mM potassium phosphate buffer
(pH 7.4).[41] Optimal w/v ratios of 1/20
were determined from preliminary experiments (data not shown). The
3H-1,2-dithiole-3-thione derivatives were stocked
as 30 mM solution in DMSO. The assay mixture (400 μL) contained
the DTT compound incubated at 150 μM in phosphate buffer either
in the presence or absence of a thiol derivative (such as l-cysteine and glutathione at 1.0 mM concentration) or in fresh rat
liver homogenate (upon a further 1 to 10 dilution with phosphate buffer)
in airtight vials at 37 °C for timed intervals (0–90 min).
The released H2S concentration was measured as described
previously.[45] Briefly, after incubation,
zinc acetate (1% w/v; 200 μL) was injected to trap the released
H2S, followed by trichloroacetic acid (10% w/v, 200 μL)
to precipitate the proteins present in the mixture and stop the H2S release reaction. Baseline H2S concentration
was determined in incubates upon addition of trichloroacetic acid
directly to the tissue homogenate in absence of the compound. Thus, N,N-dimethyl-p-phenylenediamine
sulfate in HCl 5 M (20 mM; 200 μL) and FeCl3 in HCl
1.2 M (30 mM; 200 μL) were added to the mixture, and the absorbance
of the formed methylene blue was measured after 30 min at 25 °C
at 667 nm using a Varian Cary 300 UV–Visible spectrophotometer.
The sample H2S concentration was calculated against a calibration
curve of sodium sulfide in the concentration range 1–250 μM,
and results, produced in triplicate, were plotted as released [H2S] versus time.
In Vivo Antinociceptive Study
Animals
Sprague Dawley rats (Envigo, Varese, Italy)
weighing 200–250 g at the beginning of the experimental procedure
were used. Animals were housed in the Centro Stabulazione Animali
da Laboratorio (CeSAL, University of Florence) and used at least 1
week after their arrival. Four rats were housed per cage (size 26
cm × 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 European Community guidelines for animal care [DL
116/92, application of the European Communities Council Directive
of November 24, 1986 (86/609/EEC)]. The ethical policy of the University
of Florence complies with the Guide for the Care and Use of Laboratory
Animals of the U.S. 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 Italian Ministry of Health (no. 517/2017-PR) and from the
Animal Subjects Review Board of the University of Florence. Experiments
involving animals have been reported according to ARRIVE guidelines.[49] All efforts were made to minimize animal suffering
and to reduce the number of animals used.
Complete Freund’s Adjuvant-Induced Arthritis
Articular damage was induced by injection of Complete Freund’s
Adjuvant (CFA; Sigma-Aldrich, St Louis, MO, USA), containing 1 mg/mL
of heat-killed and dried Mycobacterium tuberculosis in paraffin oil and mannide monooleate, into the tibiotarsal joint.[50] Briefly, the rats were lightly anesthetized
with 2% isoflurane, the left leg skin was sterilized with 75% ethyl
alcohol, and the lateral malleolus was located by palpation. A 28-gauge
needle was then inserted vertically to penetrate the skin and turned
distally for insertion into the articular cavity at the gap between
the tibiofibular and tarsal bone until a distinct loss of resistance
was felt. A volume of 50 μL of CFA was then injected (day 0).
Control rats received 50 μL of saline solution (day 0) in the
tibiotarsal joint.
Administration of Compounds
The hybrid derivatives 26, 29, 30, and 31 (1,
10, and 30 mg/kg) were suspended in a 1% solution of carboxymethylcellulose
(CMC) sodium salt and acutely per os administered
starting from day 14 after CFA i.a. injection, when arthritis was
well established. The H2S releaser 3 and CAI 15 were also administered alone or in coadministration at
the equimolar dose of compound 30 30 mg/kg (13.23 and
17.71 mg/kg, respectively). The antihypersensitivity effects were
evaluated over time by the Paw pressure and Incapacitance tests.
Paw Pressure Test
The nociceptive threshold of rats
was determined with an analgesimeter (Ugo Basile, Varese, Italy),
according to the method described by Leighton et al.[51] and Maresca et al.,[52] (doi:
10.1111/jphp.12828). Briefly, a constantly increasing pressure was
applied to a small area of the dorsal surface of the hind paw using
a blunt conical probe by a mechanical device. Mechanical pressure
was increased until vocalization or a withdrawal reflex occurred while
rats were lightly restrained. Vocalization or withdrawal reflex thresholds
were expressed in grams. Rats scoring below 40 g or over 75 g during
the test before drug administration were rejected (25%).
Incapacitance Test
Weight-bearing changes were measured
using an incapacitance apparatus (Linton Instrumentation, Norfolk,
U.K.) to detect changes in postural equilibrium after a hind limb
injury.[53,54] Rats were trained to stand on their hind
paws in a box with an inclined plane (65° from horizontal). This
box was placed above the incapacitance apparatus. This allowed us
to independently measure the weight that the animal applied on each
hind limb. The value reported for each animal is the mean of five
consecutive measurements. In the absence of hind limb injury, rats
applied an equal weight on both hind limbs, indicating postural equilibrium,
whereas an unequal distribution of weight on the hind limbs indicated
a monolateral decreased pain threshold. Data are expressed as the
difference between the weight applied to the limb contralateral to
the injury and the weight applied to the ipsilateral limb (Δ
weight).
Toxicity and the Irwin Test
Toxicity was evaluated
after a single acute administration of 26, 29, 30, and 31 at 30 mg/kg. Animals were
observed for 24 h. For the Irwin test, each rat was individually placed
in a transparent cage (26 × 41 cm), and 26 neurobehavioral or
physiological parameters were systematically assessed according to
Irwin (1968)[55] and already reported by
Berrino and colleagues in similar studies.[30] Behavioral, autonomic, and neurological manifestations produced
by compound administration in rats were evaluated: motor displacement,
motor reflexes, stereotypies, grooming, reaction to painful or environmental
stimuli (analgesia and irritability), startle response, secretions,
excretions, respiratory movements, skin color and temperature, piloerection,
exophthalmos, eyelid and corneal reflexes, muscle tone, ataxia, tremors,
head twitches, jumps, convulsions, Straub tail, and other signs or
symptoms. For postural reflexes (righting reflex) and other signs
such as piloerection, exophthalmia (exaggerated protrusion of the
eyeball), ataxia, tremors, and Straub tail, only presence or absence
was recorded. Skin color was evaluated qualitatively (pale, red, or
purple); other signs were evaluated semiquantitatively, according
to the observer’s personal scale (0 to +4, −4 to 0,
or −4 to +4). The terms sedation and excitation express the
final interpretation of a group of signs: reduced motor activity,
reduced startle response, eyelid ptosis, and reduced response to manual
manipulation, for the former; and increased motor activity, increased
startle response, increased response to manual manipulation, and exophthalmia,
for the latter. Hyperactivity includes running, jumping, and attempting
to escape from the container. Trained observers were not informed
about the specific treatment of each animal group carried out in this
test.
Liver Collection
At the end of the behavioral measurements,
control animals were sacrificed by decapitation. The liver was collected
and immediately frozen in liquid nitrogen to determine the release
of H2S by spectrophotometric analysis as described above.
Statistical Analysis
Behavioral measurements were performed
on eight rats for each treatment carried out in two different experimental
sets. Results were expressed as the mean (SEM) with a one-way analysis
of variance. A Bonferroni’s significant difference procedure
was used as a post hoc comparison. P-values of <0.05 or <0.01 were considered significant. Data
were analyzed using the Origin 9 software (OriginLab, Northampton,
MA, USA).
Authors: John L Wallace; Rory W Blackler; Melissa V Chan; Gabriela J Da Silva; Wagdi Elsheikh; Kyle L Flannigan; Iulia Gamaniek; Anna Manko; Lu Wang; Jean-Paul Motta; Andre G Buret Journal: Antioxid Redox Signal Date: 2014-04-15 Impact factor: 8.401
Authors: Ling Li; Giuseppe Rossoni; Anna Sparatore; Lin Chiou Lee; Piero Del Soldato; Philip Keith Moore Journal: Free Radic Biol Med Date: 2006-12-16 Impact factor: 7.376
Figure 1
Scheme 1
Scheme 2
Scheme 3
Figure 2
Figure 3
Figure 4