Store-operated calcium entry (SOCE) is important in the maintenance of calcium homeostasis and alterations in this mechanism are responsible for several pathological conditions, including acute pancreatitis. Since the discovery of SOCE, many inhibitors have been identified and extensively used as chemical probes to better elucidate the role played by this cellular mechanism. Nevertheless, only a few have demonstrated drug-like properties so far. Here, we report a class of biphenyl triazoles among which stands out a lead compound, 34, that is endowed with an inhibitory activity at nanomolar concentrations, suitable pharmacokinetic properties, and in vivo efficacy in a mouse model of acute pancreatitis.
Store-operated calcium entry (SOCE) is important in the maintenance of calcium homeostasis and alterations in this mechanism are responsible for several pathological conditions, including acute pancreatitis. Since the discovery of SOCE, many inhibitors have been identified and extensively used as chemical probes to better elucidate the role played by this cellular mechanism. Nevertheless, only a few have demonstrated drug-like properties so far. Here, we report a class of biphenyl triazoles among which stands out a lead compound, 34, that is endowed with an inhibitory activity at nanomolar concentrations, suitable pharmacokinetic properties, and in vivo efficacy in a mouse model of acute pancreatitis.
Acute pancreatitis
(AP) is an inflammatory life-threatening disorder.
It is characterized by autodigestion of the pancreas, which causes
inflammation, edema, vacuolization, necrosis, and, in the worst scenario,
induces injury of remote extrapancreatic organs. AP represents an
urgent and unmet need as it affects about 35 individuals per 100,000
person-years worldwide,[1] with a mortality
rate between 1.5 and 4.2%, and no effective pharmacological treatment
is available.[1,2]Among the triggers of AP
is an intracellular Ca2+ overload
in pancreatic acinar cells (PACs) that induces the uncontrolled release
of intracellular digestive proenzymes. While there are numerous mechanisms
that control intracellular Ca2+ concentrations, store-operated
Ca2+ entry (SOCE) appears to have a pivotal role in the
induction of Ca2+ overload in PACs.[3]SOCE[4] is represented by the influx
of
Ca2+ activated in response to the depletion of the stores
from the endoplasmic reticulum (ER)[5] and
is associated with the electrophysiological current named ICRAC (CRAC, calcium release-activated channel).[6] The exact molecular mechanism behind this cellular
event was elucidated between 2005 and 2006, when the principal components
of SOCE machinery, STIM and Orai, were discovered.[7] At present, three Orai isoforms (Orai1–3) and two
STIM isoforms (STIM1-2) are known. STIM is a single-span protein located
on the ER membrane and behaves as a sensor: the depletion of ER Ca2+ stores induces a conformational change of STIM that, after
oligomerization, interacts with Orai. The latter is a plasma membrane
Ca2+ channel that allows for Ca2+ influx from
the extracellular environment, eventually refilling the intracellular
Ca2+ stores.Other crucial proteins known to participate
in SOCE are transient
receptor potential canonical (TRPC) channels,[8] which were previously believed to be the primary contributors of
Ca2+ rise in PACs and therefore mainly responsible for
AP.[5b,9] Yet, more recent studies have demonstrated
that the metabolic alcohol products that are among the mediators of
acinar cell damage induce the opening of IP3Rs, Ca2+ channels located in the ER, resulting in the depletion of
the ER stores and in the activation of STIM1.[10] This event leads to Ca2+ entry through the Orai1 opening,
sustaining toxic intracellular Ca2+ elevation and pointing
to Orai1 as a key culpable for AP damage.[10]Gerasimenko et al. demonstrated that a selective
CRAC channel blocker, GSK-7975A (1, Figure ), with no inhibitory activity
on TRP-channel currents, is able to decrease the overload of cytosolic
Ca2+ in a concentration-dependent manner and to prevent
the activation of the necrotic cell death pathway in both mouse and
human PACs,[11] confirming the involvement
of Orai in AP and its druggability. Furthermore, GSK-7975A, together
with another SOCE inhibitor, CM4620 (2, Figure ), were demonstrated to be
protective in three different murine models of chemically induced
AP.[12] Based on these preclinical evidences,
CM4620 has entered clinical trials,[13] with
a Phase II trial for AP already completed and an ongoing Phase I/II
trial for a rare condition in which AP is triggered by asparaginase
treatment (asparaginase-associated AP). This rare condition (incidence
between 7 and 18%) is a well-known complication of childhood acute
lymphoblastic leukemia (ALL) treatment that is often responsible for
the early discontinuation of drug treatments.[3b] As the increase in Ca2+ induced by asparaginase and the
related necrosis of PACs depend on CRAC channels, recent findings
have described the inhibition of CRAC channels as the most promising
therapeutic approach in this pathology.[3a,14]
Figure 1
Negative modulators
of SOCE reported in the literature.
Negative modulators
of SOCE reported in the literature.Among the several medicinal chemistry campaigns aimed at developing
SOCE inhibitors,[15] in 2018 our research
group described a class of SOCE modulators, named pyrtriazoles,[16] that were designed based on a known chemical
probe for SOCE, Pyr6.[17] Among the reported
compounds, a promising candidate (3, Figure ) able to significantly ameliorate
cerulein-induced AP in rodents without signs of toxicity was identified.
Nevertheless, the pharmacokinetic (PK) profile of 3,
with its relatively short half-life (mouse, i.p., 1.3 h) and high
volume of distribution (32 L/kg), prompted us to undertake a medicinal
chemistry campaign aimed at developing more drug-like SOCE modulators.Among the previously reported SOCE modulators, Synta66 (4, Figure ) is a CRAC channel blocker able to inhibit ICRAC with an IC50 of 1.4–3.0 μM.[18] Although its precise mechanism on SOCE remains unknown,
assays performed in siRNA knock-down of Orai1 mast cells have suggested
that Synta66 might be selective for the channel.[18a] Furthermore, experiments in vascular smooth
muscle cells have demonstrated that it does not interfere with STIM1
clustering.[19] Thanks to its inhibitory
activity toward Orai1, an increasing number of in vitro and in vivo studies have used Synta66 as a chemical probe to gain better insight into ICRAC biology. Moreover, the compound is selective over
a panel of other ion channels or receptors, including Ca2+ ATPase pump, voltage-gated Ca2+ and Na+ channels,
and TRPC1/5 channels,[18a,18b,19] indicating this molecule as a reliable starting point to develop
new SOCE modulators.
Figure 2
Modifications of Synta66 moieties to synthesize
biphenyl
triazoles.
Modifications of Synta66 moieties to synthesize
biphenyl
triazoles.In the present contribution, we
describe a family of biphenyl triazoles
that inhibit SOCE and are endowed with potency in the nanomolar range,
good PK profile, and efficacy in counteracting cerulein-induced AP.
While the compounds had been initially designed as mere isosteres
of Synta66,[20] replacement
of the arylamide moiety with the triazole ring (Figure ) gave unpredictable results in terms of
structure–activity relationships (SARs) and unmasked the fact
that this represents a completely new class of modulators.
Results
and Discussion
SAR Study around 2-Fluoro-4-pyridine Gives
Less-Active Compounds
Compared to Synta66 on SOCE
Starting from the
structure of Synta66, the amide moiety was replaced with
a 1,4-disubstituted1,2,3-triazole ring by a click chemistry approach.[21] To this aim, azide 8 and alkyne 17 were prepared according to Schemes and 2. 8 was synthesized starting from (2,5-dimethoxyphenyl)boronic acid
and 4-bromoaniline, which reacted in a Suzuki cross-coupling reaction
to give intermediate 7. Compound 7 underwent
a diazotization-azidation reaction to afford the desired azide 8 with a yield of 60%. Alkyne 17 was prepared
from (2,5-dimethoxyphenyl)boronic acid and 4-bromobenzaldehyde, which,
after a Suzuki cross-coupling reaction, gave intermediate 16 that reacted in the presence of Bestmann–Ohira reagent to
give 17.
Reagents and conditions:
(a)
K2CO3, Pd(OAc)2, EtOH, DMF, 50 °C,
3 h, 99%. (b) Bestmann–Ohira reagent, K2CO3, MeOH, rt, 18 h, 82%. (c) Sodium ascorbate, CuSO4·5H2O, t-BuOH, H2O, 50 °C, 16
h, 60–99%.With these two compounds
in hand, two click reactions were performed
and compounds 9 and 18 (Table ), displaying the same substructures
as the reference compound Synta66, were obtained with
a yield of 31 and 60%, respectively. 9 and 18 were tested for activity on SOCE in HEK cells, a human embryonic
kidney (HEK) cell line, by fluorescence microscopy, as described elsewhere.[16] After 600 s, Ca2+ was added and intracellular
levels were measured. Compared to Synta66, which exhibits
an inhibition of 90.8 ± 1.7%, compound 18 inhibited
SOCE to a smaller extent (26.2 ± 6.5%), whereas 9 showed a percentage of −4.9 ± 21.3, indicating that
the molecule slightly increased Ca2+ entry compared to
control (Table ).
Therefore, the isosterical replacement of the arylamide moiety with
a triazole ring led to active molecules, although the activity was
significantly reduced compared to the parent compound Synta66.
Table 1
First Series of Compounds and Their
Biological Activity in HEK Cells
Prompted by this observation, we decided to investigate
the SAR
around the 2-fluoro-4-pyridine ring. To this aim, 10 additional molecules
were designed and synthesized starting from azide 8 and
alkyne 17 that were clicked with five different alkynes
and azides, respectively, affording compounds 10–14 (series 1A, Figure ) and 19–23 (series 1B, Figure ). All the synthesized triazoles were tested
as described above. Five compounds (9, 10, 11, 19, 20) evoked a variable
Ca2+ entry, leading to a remarkable standard error and
suggesting that they were not able to reliably inhibit SOCE (Table ). Moreover, only
four molecules (13, 14, 21, 23) out of 12 inhibited SOCE by a considerable level (arbitrarily
chosen to be >70%).The most active compound, 23 (87.8 ± 2.9% of
inhibition) showed an inhibitory activity comparable to Synta66 (90.8 ± 1.7%; Figure A). The effects of both compounds on SOCE were characterized
analyzing the area under the curve (AUC), peak amplitude, and slope.
As shown in Figure B, both Synta66 and 23 significantly reduced
AUC and peak amplitude compared to control. Whereas only Synta66 showed a significant effect on the slope, it was apparent that also 23 had a similar effect. To determine the IC50 value,
we obtained the concentration–response curves for both compounds
(Figure C). 23 showed an IC50 of 1.79 ± 0.14 μM,
revealing approximately a 1 order of magnitude lower potency compared
to Synta66 (IC50 = 228 ± 33 nM). Moreover, 23 was slightly cytotoxic, with a residual cell viability
of 71.8% at 10 μM, a characteristic shared by Synta66 (75.8 ± 8.0%). To assess the cytotoxicity profile of the biphenyl
triazoles, viability assays were performed on other molecules of the
first series (10 μM) and all the compounds showed a cell viability
comparable to 23 (data not shown).
Figure 3
Effect of Synta66 (4) and 23 on SOCE in HEK cells. (A) Average
Ca2+-traces of SOCE
in the absence or presence of Synta66 or 23 (10 μM). Traces are the average of 200 cells. (B) Evaluation
of the AUC, peak amplitude, and slope of the Ca2+-rise
of the Ca2+-traces in the absence or presence of Synta66 or 23. The graph shows the median and
IQR of the AUC, peak amplitude, and slope of the Ca2+-rise.
Mann–Whitney U test of compounds vs control (**p < 0.0075 ***p =
0.0002 ****p < 0.0001). (C) Concentration–response
curves of Synta66 and 23.
Effect of Synta66 (4) and 23 on SOCE in HEK cells. (A) Average
Ca2+-traces of SOCE
in the absence or presence of Synta66 or 23 (10 μM). Traces are the average of 200 cells. (B) Evaluation
of the AUC, peak amplitude, and slope of the Ca2+-rise
of the Ca2+-traces in the absence or presence of Synta66 or 23. The graph shows the median and
IQR of the AUC, peak amplitude, and slope of the Ca2+-rise.
Mann–Whitney U test of compounds vs control (**p < 0.0075 ***p =
0.0002 ****p < 0.0001). (C) Concentration–response
curves of Synta66 and 23.
SAR Study around 2,5-Dimethoxyphenyl Gives Compounds as Active
as Synta66 on SOCE
The above data demonstrate
that all the synthesized biphenyl triazoles showed a reduced activity
on SOCE compared to Synta66. We therefore synthesized
a second series of compounds (series 2A and 2B, Figure ) where the 2,5-dimethoxyphenyl ring, the
only structural motif that had been kept fixed in our preliminary
SAR, was extensively modified (Table ). Given that the most potent compound in the first
series featured a 3-carboxyphenyl ring, we decided to select this
moiety as the one to keep fixed. The choice was also guided by the
fact that this substructure was the preferred substitution in our
previous paper reporting pyrtriazoles (CIC-37, Figure ) and by the perception
that the 3-carboxyphenyl substrate is a privileged scaffold in SOCE
modulation.[16]
Table 2
Second
Series of Compounds and Their
Biological Activity in HEK Cells
To obtain the second series of biphenyl triazoles,
a Suzuki cross-coupling
reaction was exploited, starting from two aryl bromides, 26 and 43, that were coupled with different boronic acids. 26 and 43 were synthesized as depicted in Schemes and 4. Click chemistry reaction between azide 24,
prepared from 4-bromoaniline by diazotization-azidation protocol,
and alkyne 25 afforded the aryl bromide 26. Similarly, 43 was obtained by clicking alkyne 41, synthesized by reacting 4-bromobenzaldehyde in the presence
of the Bestmann–Ohira reagent, with azide 42.
Reagents and conditions:
(a)
Bestmann–Ohira reagent, K2CO3, MeOH,
rt, 18 h, 54%. (b) Sodium ascorbate, CuSO4·5H2O, t-BuOH, H2O, 50 °C, 16
h, 65%. (c) K2CO3, Pd(OAc)2, EtOH,
DMF, 80 °C, 6 h, 22–99%.Starting
from these two intermediates, 28 Suzuki reactions were
performed and compounds 27–40 (series 2A, Figure ) and 44–56 (series 2B, Figure ) were synthesized. One reaction was instead not successful.As described above, all the compounds were initially tested at
10 μM in HEK cells. This second series was significantly more
potent compared to the first, and several molecules showed a noteworthy
inhibitory activity, with percentage above 80% (data not shown). Therefore,
in order to better discriminate between the different candidates,
we decided to evaluate the effect of the compounds at 3 μM on
SOCE. For those compounds that displayed SOCE inhibitory activity
≥70%, cell viability assays, this time at 10 μM, were
then performed. For those molecules showing an inhibitory activity
≥70% and a cell viability ≥85%, the IC50 values
were calculated (Table ).The biological results highlighted that removal of both
the methoxy
substituents from positions 2′ and 5′ (27, 51.0 ± 31.9%; 44, 1.8 ± 3.1%), or the presence
of the solely 2′-methoxy substituent (28, 45.9
± 14.5%; 45, 12.0 ± 18.9%), caused a significant
reduction of activity compared to Synta66 with a remarkable
variability. On the other hand, the additional methoxy group at position
4′ made the inhibition rise to 50% (29, 56.4 ±
21.0%; 46, 53.0 ± 3.8%). When the same insertion
was performed at position 6′, for one compound a drop in inhibitory
activity occurred (30, 25.8 ± 22.3%), whereas for
the other one (47) an increase in SOCE was surprisingly
observed (−22.8 ± 0.9%, Figure A). The compound, tested at a concentration
of 3 μM, significantly increased the AUC of calcium entry and
the peak amplitude (Figure B), that is, represents a positive modulator of SOCE.
Figure 4
Effect of 47 on SOCE. (A) Average Ca2+-traces
of SOCE in the absence or presence of 47 (3 μM
in HEK cells). Traces are the average of 200 cells. (B) Evaluation
of the AUC, peak amplitude, and slope of the Ca2+-rise
in the absence or presence of 47. The graph shows the
median and IQR of the AUC, peak amplitude, and slope of the Ca2+-rise. Mann–Whitney U test of compound vs control (*p = 0.0286).
Effect of 47 on SOCE. (A) Average Ca2+-traces
of SOCE in the absence or presence of 47 (3 μM
in HEK cells). Traces are the average of 200 cells. (B) Evaluation
of the AUC, peak amplitude, and slope of the Ca2+-rise
in the absence or presence of 47. The graph shows the
median and IQR of the AUC, peak amplitude, and slope of the Ca2+-rise. Mann–Whitney U test of compound vs control (*p = 0.0286).Given the absence of effect on slope, it is highly likely
that
it affects channel closure or desensitization. Given that the focus
of this study was to identify novel SOCE negative modulators for the
treatment of AP, the profile of compound 47 was not investigated
further, but its discovery supports the idea that minor structural
modifications of SOCE inhibitors can interfere with channel gating
and produce activators, as already observed for pyrtriazoles (AL-2T
(57), NM-3G (58); Figure )[16] and for another
recently described SOCE enhancer (IA65 (59), Figure ).[22]47 therefore represents an enhancer of SOCE
from a third distinct class of modulators and provides grounds to
develop models to understand the mechanism by which this occurs.
Figure 5
Structures
of compound 47 and other positive modulators
of SOCE reported in the literature.
Structures
of compound 47 and other positive modulators
of SOCE reported in the literature.Compound 31 in which the methoxy group is removed
from position 2′ while bearing a 3′-methoxy substituent
was more active (85.1 ± 9.0%; Figure A) compared to Synta66, whereas
the counterpart 48 was less active (29.5 ± 22.7%).
The substitution of the methoxy group with a hydroxyl (32, 0.0 ± 13.1%) or with a thioether (33, 23.6 ±
33.7%; 49, 0.0 ± 0.7%) was instead not tolerated.
Compounds 34 and 50 with a 2′,3′-dimethoxy
phenyl substituent also showed a good activity (96.5 ± 2.4 and
93.3 ± 5.1%, respectively; Figure A), whereas if the two methoxy groups were fused together
to form a 1,4-dioxanyl ring, the activity was lower (35, 70.9 ± 7.9% Figure A; 51, 44.1 ± 17.1%). The 3′,5′-dimethoxy
phenyl substituent provided a good inhibitory activity, as in the
case of 36, which induced an inhibition of 73.8 ±
11.3% (Figure A).
On the other hand, 52 had a good inhibitory activity
but due to its remarkable variability (75.4 ± 28.8%), the compound
was not selected for further studies. The reduction of SOCE inhibition
was also observed with the 3′,4′-dimethoxy phenyl substituent
but to a less extent (37, 54.3 ± 6.3%; 53, 39.6 ± 7.6%). When the substituents in 3′ and 4′
were fused together in a six-member 1,4-dioxanyl ring, the activity
rose (38, 77.5 ± 8.2%, Figure A; 54, 81.1 ± 7.0%), whereas
the 1,3-dioxolanyl was not tolerated in the case of 55 (0.0 ± 4.3%) and led to a less active compound with a high
standard error in the case of 39 (67.3 ± 44.6%).
Finally, a 2′-fluoro-5′-methoxy phenyl ring provided
two compounds with remarkable activity on SOCE, 40 (74.7
± 6.3%) and 56 (88.9 ± 7.5%), both reported
in Figure A.
Figure 6
Effect of Synta66 and selected biphenyl triazoles
on SOCE in HEK cells. (A) Average Ca2+-traces of SOCE in
the absence or presence of Synta66 or biphenyl triazoles
(3 μM). Traces are the average of 200 cells. (B) Concentration–response
curves.
Effect of Synta66 and selected biphenyl triazoles
on SOCE in HEK cells. (A) Average Ca2+-traces of SOCE in
the absence or presence of Synta66 or biphenyl triazoles
(3 μM). Traces are the average of 200 cells. (B) Concentration–response
curves.For all selected compounds (31, 34, 35, 36, 38, 40, 50, and 56),
the detailed analyses of the AUC,
peak amplitude, and slope demonstrated that, similarly to Synta66, all compounds induced a drop in the three parameters, with 34 significantly reducing the AUC when compared to the reference
compound. All these data are reported in the Supporting Information.In summary, in the second series we were
able to discover eight
molecules with IC50 values in the nanomolar range (Figure B, Table ).
Triazole is an Indispensable
Feature of the New Class of Modulators
and Reduces Off-Target Effects on DHODH
To better elucidate
the role of the triazole ring in the interaction with SOCE machinery,
we synthesized analogues of 38 displaying the direct
(64) and the inverse (69) amides, according
to Schemes and 6. Suzuki cross-coupling reaction between (2,3-dihydrobenzo[b][1,4]dioxin-6-yl)boronic acid and 4-bromoaniline afforded
amine 61 that, after coupling with 3-(methoxycarbonyl)benzoic
acid and hydrolysis of the methyl ester, yielded compound 64. (2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)boronic acid
and methyl 4-iodobenzoate underwent a Suzuki cross-coupling reaction
and, after deprotection of the carboxylic group, afforded intermediate 66. Then, 66 was coupled with methyl 3-aminobenzoate
and the methyl ester hydrolyzed to give compound 69.
Reagents
and conditions: (a)
K2CO3, Pd(OAc)2, EtOH, DMF, 80 °C,
6 h. (b) NaOH, H2O, THF, 4 h, 60 °C, 85%. (c) EDCI,
DMAP, DIPEA, dry CH2Cl2, rt, 18 h, 63%. (d)
NaOH, H2O, THF, 4 h, 60 °C, 61%.The triazole ring is reputed to be a nonclassical bioisostere of
amides,[20,21] although we have shown in a number of occasions
that this is not necessarily always the case.[23] To investigate the function of the triazole in this setting, we
evaluated the amides of 38 (64 and 69). Both molecules displayed a significantly reduced activity
compared to the parent compound (Table ). It should be noticed that such a difference was
also observed when comparing Synta66 with its triazole-substituted
close analogues (9 and 18; Table ).
Table 3
Amide Analogues
of 38
Surprisingly, 64, despite its low activity
on SOCE,
showed a significant cytotoxicity, with a residual viability after
24 h of 50% at 10 μM in HEK cells, in contrast to its inverse
amide 69 and 38, that did not affect cell
viability. When attempting to rationalize this cytotoxicity, we noticed
that 64 was structurally closely related to dihydroorotate
dehydrogenase (DHODH) inhibitors.[24] Indeed,
a hDHODH inhibitor usually includes a lipophilic
moiety that guarantees the interaction with subsite 1 of the enzyme,
together with a carboxylate moiety that interacts with the Arg136
residue located in subsite 2, two structural features that can be
found in compound 64.More surprisingly, a recent
screening performed on an FDA database
has highlighted that teriflunomide (70, Figure ), a DHODH inhibitor approved
for multiple sclerosis,[25] is endowed with
a considerable inhibitory activity on SOCE (IC50 = 4.3
± 1.0 μM in HEK cells).[26] This
led to asking whether 64 was a DHODH inhibitor and whether
triazole-bearing analogues shared this feature. To investigate the
involvement of DHODH, the cytotoxic activities of the two compounds
bearing an amide substructure, 64 and 69, and of the five selected biphenyl triazoles, 31, 34, 36, 38, 40, were
evaluated after 72 h at a high concentration (50 μM) in HEK
cells. Alongside, two well-characterized DHODH inhibitors, teriflunomide
itself (70, Figure ) and brequinar (71, Figure )[27] were used as reference compounds. Gratifyingly, the viability profile
revealed that the biphenyl triazoles did not impair cell viability
even at these high concentrations. For the arylamide-bearing molecules
displaying a significant cytotoxicity (64 and Synta66), the involvement of the de novo pyrimidine synthesis
pathway was evaluated by supplementing the medium with an excess of
uridine that should counterbalance the effect of DHODH inhibition
by triggering the de novo pathway.[28] As expected, brequinar and teriflunomide were cytotoxic
and their effect was reverted by uridine addition. The cytotoxic effect
of 64 was also fully reverted by uridine, supporting
our hypothesis that this is a DHODH inhibitor and that the substitution
with the triazole ring reduces the off-target effects (Figure ). While this observation deserves
additional investigations, it questions whether other previously reported
inhibitors bearing an arylamide moiety might have promiscuous effects
on this enzyme. Indeed, most SOCE inhibitors bear an amide-linkage
as part of the pharmacophore.[15e] We preliminarily
tested CM4620 and found that it was cytotoxic at 50 μM in HEK
cells but this cytotoxicity was not reverted by uridine, suggesting
that it is not a DHODH inhibitor (not shown). A similar lack of effect
was also observable for Pyr6, while no other arylamide SOCE inhibitor
was tested.
Figure 7
Effects of the selected
compounds on DHODH. HEK cells were treated
for 72 h at the concentration of 50 μM with or without uridine
(100 μM). The graph shows average ± SEM of cell viability,
peak amplitude, and slope of the Ca2+-rise. A Student t-test was performed on compounds vs control
(****p < 4.27 × 10–6);
(terif: teriflunomide, breq: brequinar).
Effects of the selected
compounds on DHODH. HEK cells were treated
for 72 h at the concentration of 50 μM with or without uridine
(100 μM). The graph shows average ± SEM of cell viability,
peak amplitude, and slope of the Ca2+-rise. A Student t-test was performed on compounds vs control
(****p < 4.27 × 10–6);
(terif: teriflunomide, breq: brequinar).Overall, these data corroborate previous evidence that the amide
to triazole substitution is not merely bioisosteric (REF), as the
presence of the triazole prevents off-target effects on DHODH.
Biphenyl
Triazoles as Sodium Salts Are More Soluble Than Synta66
According to both potency and cell viability
of the second series of modulators, the five most promising candidates
were selected (31, 34, 36, 38, and 40), excluding those molecules that differed
from these candidates only for the orientation of the triazole ring
(50 and 56). To assess the druggability
of the molecules, their thermodynamic aqueous solubility was evaluated.
Unfortunately, the biphenyl triazoles showed poor aqueous solubility
(about 0.20 μg/mL, data not shown) comparable to that of Synta66 (0.28 μg/mL, Table ). To overcome this limitation, the candidates
were salified as sodium salts and their aqueous solubility was reassessed
(Table ). Briefly,
except for 36, all the tested biphenyl triazoles salts
were soluble in water in the 0.67–1.53 mg/mL range. The presence
of one or two methoxy substituents on the phenyl ring considerably
increased the solubility compared to the 1,4-dioxanyl moiety of 38 as well as the addition of a fluorine atom that slightly
improved the solubility of 40 compared to 31. Interestingly, the enhanced solubility given by the methoxy substituents
is minimally driven by the decrease in hydrophobicity but rather by
the disruption of the molecular symmetry, as shown by the 80-fold
increase in aqueous solubility of 34 compared to 36. In addition, to assess the solubilization of the selected
candidates in the aqueous vehicle used for in vivo administration, compounds 34 and 40 were
dissolved at the nominal concentration of 6 mg/mL in saline solutions
containing cosolvents (see methods section). Only 34 gave
a limpid solution in saline containing 10% dimethyl sulfoxide (DMSO)
+ 20% PEG400, whose title was confirmed by LC–UV analysis,
pointing to this compound as the best candidate for further in vivo evaluation.
Table 4
Aqueous Solubility
and Metabolic Stability
of the Selected Biphenyl Triazoles
Soluble
at 6 mg/mL in saline containing
10% DMSO + 20% PEG400.
Residual
substrate after 1 h incubation
in MLMs.
Soluble
at 6 mg/mL in saline containing
10% DMSO + 20% PEG400.Residual
substrate after 1 h incubation
in MLMs.
Biphenyl Triazoles Are
More Metabolically Stable Than Synta66
Next,
the in vitro metabolic
stability of the five candidates (31, 34, 36, 38, and 40) was evaluated
in mouse liver microsomes (MLMs) activated by NADPH by measuring the
substrate residual after 1 h. For comparative purposes, Synta66 was incubated in the same conditions. All the salified biphenyl
triazoles resulted in a quite stable microsomal oxidation, with a
residual substrate in the range of 75–94% after incubation
(Table ). By contrast, Synta66 resulted in a considerably less stable microsomal
metabolism, with a substrate residual of only 15% after incubation,
amide hydrolysis and O-demethylation metabolites
showing the most extensive transformations (data not shown).Next, the structural characterization of the metabolites of 34 was performed by high-resolution mass spectrometry (HRMS),
processing the raw data with a workflow aimed at drug metabolite identification
provided by Compound Discoverer 3.1 software (Thermo Scientific).
Overall, data analysis highlighted the occurrence of three main transformations: O-demethylation (M1) followed by oxidation (M2) and hydroxylation
(M3). Furthermore, incubation of 34 with MLMs in the
presence of uridine diphosphate glucuronic acid (UDPGA) gave the corresponding
acyl glucuronide metabolite G1 (Figure ). Interestingly, data analysis did not highlight the
formation of glutathione (GSH) adducts, suggesting that metabolism
is not driven toward the formation of reactive species. Full data
of metabolite structures and mass spectral data, as well as the metabolic
pathways, are given in the Supporting Information.
Figure 8
Metabolic biotransformation of compound 34 in MLMs.
Metabolic biotransformation of compound 34 in MLMs.
34 Is Effective In Vivo in AP
To further characterize the compound, a PK analysis
was performed
in mice. Briefly, mice were injected with 34 (i.v., 7
mg/kg, once) and serial blood sampling was performed. 34 showed a half-life of 3.2 h, with a clearance of 0.5 L/h/kg, a volume
of distribution of 2.3 L/kg, and a Cmax of 16.8 mg/L (see the Supporting Information for the full set of PK parameters).The PK profile of our
candidate prompted us to investigate its efficacy in a cerulein-induced
murine model of AP.[29] The compound was
administered 30 and 150 min after the first cerulein injection at
a dose of 10 mg/kg i.p. The hematoxylin/eosin (H&E) staining of
the pancreatic tissues collected 5 h after the first cerulein injection
demonstrated that the compound was able to significantly ameliorate
the histological scores, with reduction of inflammation and edema
typical of this disease (Figure ), as expected from SOCE inhibitors with profiles compatible
with systemic administration.[16]
Figure 9
Evaluation
of compound 34 in AP. H&E sections
of pancreatic tissues. Analysis was performed in a blinded manner
and data represent the mean ± SEM of 10 mice for each group.
***p < 0.001 versus Sham; ###p < 0.001 vs cerulein.
Evaluation
of compound 34 in AP. H&E sections
of pancreatic tissues. Analysis was performed in a blinded manner
and data represent the mean ± SEM of 10 mice for each group.
***p < 0.001 versus Sham; ###p < 0.001 vs cerulein.
Conclusions
This work stems from our previous discovery
that the pyrtriazole
derivative 3, originally designed from known pyrazole
inhibitors, is an inhibitor of SOCE (IC50 = 4.4 μM
± 1.2).[16] The compound demonstrated
efficacy in the cerulein-induced model of AP despite its short half-life
(i.p., 1.3 h). With the aim of discovering drug-like SOCE inhibitors
endowed with a better PK profile, the replacement of the amide with
the triazole ring in Synta66, another well-known SOCE
inhibitor extensively employed as chemical probe, was attempted.The synthetic strategy relied on a two-step process based on a
click chemistry reaction, followed by a Suzuki coupling. The performed
SAR study highlighted that the pharmacophore of this novel class of
modulators includes the phenyl ring bearing a methyl or methylene
ether group in the meta position, the phenyl ring featuring a carboxylic
group in the meta position, and the triazole ring. The latter, when
switched into the direct or inverse amide, not only leads to a decrease
in SOCE inhibition (64 and 69) but also
to a significant cytotoxicity (64), which may in part
be reconducted to the fact that arylamide substructures may act on
DHODH. The summary of the SAR investigations is schematized in Figure .
Figure 10
Graphical representation
of SAR study around Synta66.
Graphical representation
of SAR study around Synta66.Our efforts resulted in compound 34 that compared
to Synta66 (i) displays a slightly decreased potency
on SOCE (IC50 = 851 ± 54 nM vs 228 ± 33 nM) but,
importantly, no detectable cytotoxicity in HEK cells up to 60 μM;
(ii) shows a significantly higher in vitro metabolic
stability in MLMs (75% vs 15% of residual substrate
after 1 h); and (iii) is endowed with a carboxylic group that confers
high aqueous solubility in the sodium salt form (1528 μg/mL vs 0.28 μg/mL). This yields a favorable PK profile
in mice (i.v., t1/2 of 3.2 h) and efficacy in a mouse model
of cerulein-induced AP.
Experimental Section
Chemistry
General
Experimental Methods
Reagents and solvents
were used without further purification, although, if required, they
were distilled and stored on molecular sieves. Column chromatography
was performed on silica gel. The following instrumentation was used:
Stuart scientific SMP3 apparatus (melting point), FT-IR Thermo-Nicolet
Avatar, FT-IR Bruker Alpha II, Jeol ECP 300 MHz (1H NMR),
Bruker AVANCE Neo 400 MHz or Jeol ECP 300 MHz (13C NMR),
Thermo Finningan LCQ-deca XP-plus equipped with an ESI source and
an ion trap detector or mass spectrometry (Thermo Scientific Q-Exactive
Plus) equipped with a heated electrospray ionization source. Chemical
shifts are reported in parts per million (ppm). All lead compounds
displayed a purity of 95% or higher, determined by HPLC (see the Supporting Information). Boronic acids, azides,
and alkynes are commercially available or were synthesized following
procedures reported in the literature, except for compounds 73, 75, 77, and 78 (intermediates
for the synthesis of 18, 21, 9, and 13, respectively) that were synthesized as reported
in the Supporting Information.
2′,5′-Dimethoxy-[1,1′-biphenyl]-4-amine,
(7)
4-Bromoaniline 6 (2 g, 11.63
mmol) was solubilized in DMF (23 mL) and ethanol (23 mL) under nitrogen
atmosphere. 2,5-(Dimethoxyphenyl)boronic acid 5 (3.17
g, 17.44 mmol), Pd(OAc)2 (26.1 mg, 0.116 mmol), and K2CO3 (3.2 g, 23.26 mmol) were added in order. The
mixture was stirred at 80 °C for 3 h and at room temperature
overnight. The reaction was then filtered under vacuo over a pad of
celite, rinsed with ethanol, and evaporated. The crude product was
purified by column chromatography using petroleum ether/ethyl acetate
7:3 v/v as eluent, affording compound 7 as a yellow solid
(2.61 g, 11.40 mmol, 98%); 1H NMR (300 MHz, CDCl3): δ 7.39 (d, J = 6.9 Hz, 2H), 6.97–6.88
(m, 2H), 6.84 (s, 1H), 6.70 (d, J = 6.9 Hz, 2H),
3.82 (s, 3H), 3.76 (s, 3H). MS (ESI) m/z: 230 [M + H]+.
4′-Azido-2,5-dimethoxy-1,1′-biphenyl,
(8)
To a solution of 2′,5′-dimethoxy-[1,1′-biphenyl]-4-amine
(2 g, 8.73 mmol) in water (40 mL), HCl 37% (3.5 mL) was added dropwise
and the resulting mixture was cooled down at 0 °C. Then, a solution
of NaNO2 (0.60 g, 8.73 mmol) in water (2 mL) was added
and, after 10 min, a solution of NaN3 (0.68 g, 10.48 mmol)
in water (2 mL) was added dropwise. The reaction was stirred at room
temperature for 5 h, diluted with ethyl acetate, and washed with water
(2×). The organic layer was dried over sodium sulfate and the
volatile was removed under vacuo. The crude material was purified
by column chromatography using petroleum ether/ethyl acetate 98:2
v/v as eluent, yielding compound 8 as an orange solid
(1.33 g, 5.24 mmol, 60%); 1H NMR (300 MHz, CDCl3): δ 8.31 (d, J = 7.1 Hz, 2H), 7.75 (d, J = 7.1 Hz, 2H), 6.92–6.83 (m, 3H), 3.85 (s, 3H),
3.79 (s, 3H).
General Procedure A
Compounds 9–14 were prepared from a suspension of 8 (74 mg, 0.29 mmol,
1 equiv) in water (320 μL) and t-BuOH (320
μL) and the relative alkyne (0.29 mmol, 1 equiv). Reactions
were carried out overnight under vigorous stirring in the presence
of sodium ascorbate 1 M (29 μL) and copper sulfate pentahydrate
(0.0029 mmol, 0.01 equiv). Evaporation of the volatile and purification
by silica gel column chromatography was performed.
Following general procedure A, the reaction of 8 and
methyl 4-ethynylpicolinate, after purification (petroleum ether/ethyl
acetate 4:6 v/v as eluent), yielded methyl 4-(1-(2′,5′-dimethoxy-[1,1′-biphenyl]-4-yl)-1H-1,2,3-triazol-4-yl)picolinate as a yellow solid (39 mg,
0.09 mmol, 32%). The compound (39 mg, 0.09 mmol) was solubilized in
acetone (390 μL) and water (390 μL). NaOH (7.2 mg, 0.18
mmol) was added and the mixture was stirred at room temperature for
1 h. The volatile was then removed and the crude material was purified
by column chromatography using ethyl acetate/methanol 7:3 v/v as eluent,
yielding compound 13 as a pale yellow solid (21 mg, 0.05
mmol, 58%); mp 162–163 °C. 1H NMR (300 MHz,
DMSO-d6): δ 9.72 (s, 1H), 8.70–8.56
(m, 3H), 8.01 (d, J = 8.0 Hz, 2H), 7.75 (d, J = 8.0 Hz, 2H), 7.75 (s, 1H), 6.96 (m, 2H), 3.76 (s, 3H),
3.73 (s, 3H). IR (KBr) ν̅: 3158, 2932, 2858, 1726, 1499,
1225, 1075, 812, 758, 716 cm–1. MS (ESI) m/z: 403 [M + H]+.
To a solution of 4-bromobenzaldehyde 15 (500 mg, 2.70 mmol) in DMF (8 mL) and water (2 mL) (2,5-dimethoxyphenyl)boronic
acid 5 (540 mg, 2.97 mmol), Pd(OAc)2 (11.2
mg, 0.05 mmol) and K2CO3 (933 mg, 6.75 mmol)
were added in order under nitrogen atmosphere and stirred at 50 °C
for 3 h. The reaction was filtered under vacuo over a pad of celite,
diluted with diethyl ether, and washed three times with water. The
organic phase was dried over sodium sulfate and evaporated. Purification
by column chromatography (petroleum ether/ethyl acetate 98:2 v/v)
yielded compound 16 as an orange solid (647 mg, 2.67
mmol, 99%). 1H NMR (300 MHz, CDCl3): δ
10.06 (s, 1H), 7.89 (d, J = 7.7 Hz, 2H), 7.69 (d, J = 7.7 Hz, 2H), 7.08–6.91 (m, 3H), 3.79 (s, 3H),
3.73 (s, 3H). MS (ESI) m/z: 243
[M + H]+.
4′-Ethynyl-2,5-dimethoxy-1,1′-biphenyl,
(17)
To a solution of intermediate 16 (636 mg, 2.63 mmol) in MeOH (6 mL), K2CO3 (727
mg, 5.26 mmol) and dimethyl (1-diazo-2-oxopropyl)phosphonate (759
mg, 3.95 mmol) were added in order under nitrogen atmosphere. The
mixture was stirred at room temperature overnight, then the solvent
was removed, water was added, and the aqueous layer was extracted
with CH2Cl2 (3×). The organic phases were
collected, dried over sodium sulfate, and evaporated. Purification
by column chromatography (petroleum ether/ethyl acetate 98:2 v/v as
eluent) yielded compound 17 as a white solid (514 mg,
2.16 mmol, 82%). 1H NMR (300 MHz, CDCl3): δ
7.59–7.49 (m, 4H), 6.93–6.85 (m, 3H), 3.86 (s, 3H),
3.76 (s, 3H), 3.10 (s, 1H). MS (ESI) m/z: 239 [M + H]+.
General Procedure B
Compounds 18–23 were prepared from a suspension
of 17 (0.29 mmol, 1
equiv) in water (320 μL) and t-BuOH (320 μL)
and the relative azide (0.29 mmol, 1 equiv). Reactions were carried
out overnight under vigorous stirring in the presence of sodium ascorbate
1 M (30 μL) and copper sulfate pentahydrate (0.0029 mmol, 0.01
equiv). Evaporation of the volatile and purification by silica gel
column chromatography was performed.
To a solution
of 4-bromoaniline (3 g, 17.44 mmol) in water (77 mL) HCl 37% (7 mL)
was added dropwise and the resulting mixture was cooled down at 0
°C. Then, a solution of NaNO2 (1.20 g, 17.44 mmol)
in water (3 mL) was added and, after 10 min, a solution of NaN3 (1.36 g, 20.92 mmol) in water (3 mL) was added dropwise.
The reaction was stirred at room temperature for 3 h, diluted with
ethyl acetate, and washed with water (2×). The organic layer
was dried over sodium sulfate and the volatile was removed under vacuo.
Purification by column chromatography (petroleum ether/ethyl acetate
98:2 v/v as eluent) yielded compound 24 as an orange
solid (4.86 g, 14.13 mmol, 81%); 1H NMR (300 MHz, CDCl3): δ 7.53–7.44 (m, 2H), 6.97–6.88 (m,
2H).
To a suspension of 1-azido-4-bromobenzene 24 (2.78 g, 14.04 mmol) in water (26 mL) and t-BuOH (26 mL) 3-ethynylbenzoic acid 25 (2.05 g, 14.04
mmol) was added. Then, 1.4 mL of an aqueous solution of sodium ascorbate
1 M and copper sulfate pentahydrate (34.9 mg, 0.14 mmol) were added
and the mixture was vigorously stirred for 48 h. Evaporation and purification
by column chromatography (petroleum ether/ethyl acetate 2:8 v/v and
ethyl acetate/methanol 8:2 v/v as eluents) yielded compound 26 as a yellow solid (4.22 g, 12.27 mmol, 87%); 1H NMR (300 MHz, DMSO-d6): δ 9.54
(s, 1H), 8.51 (s, 1H), 8.14 (d, J = 7.7 Hz, 1H),
7.98–7.94 (m, 3H), 7.86–7.83 (d, J =
8.8 Hz, 2H), 7.60 (t, J = 7.7 Hz, 1H). MS (ESI) m/z: 343 [M – H]−.
General Procedure C
Compounds 27–40 were prepared from a solution of 26 (0.29 mmol, 1 equiv)
in DMF (750 μL) and ethanol (750 μL) under nitrogen atmosphere
in the presence of the relative boronic acid (0.44 mmol, 1.5 equiv).
Reactions were carried out at 80 °C overnight in the presence
of Pd(OAc)2 (0.0029 mmol, 0.01 equiv) and K2CO3 (0.58 mmol, 2 equiv). After filtration of the reaction
mixture under vacuo over a pad of celite and evaporation of the volatile,
purification by silica gel column chromatography was performed.
To a solution
of 4-bromobenzaldehyde (2.15 g, 11.62 mmol) in MeOH (22 mL) K2CO3 (3.21 g, 23.24 mmol) and dimethyl (1-diazo-2-oxopropyl)phosphonate
(2.61 g, 17.43 mmol) were added in order under nitrogen atmosphere.
The mixture was stirred at room temperature overnight, then the solvent
was removed under vacuo, water was added, and the aqueous layer was
extracted with CH2Cl2 (5×). The organic
phases were collected, dried over sodium sulfate, and evaporated.
Purification by column chromatography (petroleum ether/ethyl acetate
9:1 and petroleum ether/ethyl acetate 8:2 v/v as eluents) yielded
compound 41 as an orange solid (1.12 g, 6.26 mmol, 54%); 1H NMR (300 MHz, CDCl3): δ 7.45 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H),
3.11 (s, 1H). MS (ESI) m/z: 180
[M + H]+.
To a suspension of 1-bromo-4-ethynylbenzene
(1 g, 5.52 mmol) in water (6 mL) and t-BuOH (6 mL)
3-azidobenzoic acid (0.89 g, 5.52 mmol) was added. Then, 55 μL
of an aqueous solution of sodium ascorbate 1M and copper sulfate pentahydrate
(13.7 mg, 0.055 mmol) were added and the mixture was vigorously stirred
overnight. Evaporation and purification by column chromatography (petroleum
ether/ethyl acetate 3:7 v/v and ethyl acetate as eluents) yielded
compound 43 as a pale yellow solid (1.23 g, 3.59 mmol,
65%); 1H NMR (300 MHz, DMSO-d6): δ 9.50 (s, 1H), 8.46 (s, 1H), 8.19 (d, J = 7.6 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.78–7.69 (m, 3H). MS (ESI) m/z: 343 [M – H]−.
General Procedure D
Compounds 44–56 were prepared from a solution of 43 (0.29 mmol, 1 equiv)
in DMF (750 μL) and ethanol (750 μL) under nitrogen atmosphere
in the presence of the relative boronic acid (0.44 mmol, 1.5 equiv).
Reactions were carried out at 80 °C overnight in the presence
of Pd(OAc)2 (0.0029 mmol, 0.01 equiv) and K2CO3 (0.58 mmol, 2 equiv). After filtration of the reaction
mixture under vacuo over a pad of celite and evaporation of the volatile,
purification by silica gel column chromatography was performed.
To a solution of 4-bromoaniline 6 (300 mg, 1.74 mmol) in ethanol (1.5 mL) and DMF (1.5 mL), (2,3-dihydrobenzo[b][1,4]dioxin-6-yl)boronic acid 60 (313 mg,
1.74 mmol), Pd(OAc)2 (11.7 mg, 0.017 mmol) and K2CO3 (481 mg, 3.48 mmol) were added in order. The mixture
was heated at 80 °C for 6 h and then was left at rt overnight.
The mixture was filtered over a pad of celite and rinsed with methanol
and then the volatile was removed. Purification by column chromatography
(petroleum ether/ethyl acetate 8:2 v/v as eluent) yielded compound 61 as a dark yellow oil (339 mg, 1.49 mmol, 86%); 1H NMR (300 MHz, CDCl3): δ = 7.38 (d, J = 8.2 Hz, 2H), 7.11 (s, 1H), 7.06 (d, J = 8.5 Hz,
1H), 6.94 (d, J = 8.5 Hz, 1H), 6.72 (d. J = 8.2 Hz, 2H), 4.28–4.27 (m, 4H). MS (ESI) m/z: 228 [M + H]+.
Methyl 4-iodobenzoate 65 (200 mg, 0.76 mmol) was solubilized in ethanol (1.7 mL) and DMF
(1.7 mL) under nitrogen atmosphere. (2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)boronic acid 60 (137 mg, 0.76 mmol),
Pd(OAc)2 (5.1 mg, 0.0076 mmol), and K2CO3 (211 mg, 1.53 mmol) were added in order. The mixture was
heated at 80 °C for 6 h and then was left at rt overnight. The
reaction was filtered over a pad of celite and rinsed with methanol
and then the volatile was removed, yielding a dark yellow solid. The
crude product was used in the next step without further purification.
The intermediate was solubilized in THF (2.4 mL) and a solution of
NaOH (31 mg, 0.76 mmol) in water (2.4 mL) was added. The mixture was
heated at 60 °C for 4 h, then HCl 3 N was added until pH 4, and
the aqueous layer was extracted with ethyl acetate (×2). The
organic layers were dried over sodium sulfate and evaporated, yielding
compound 66 as a white solid (166 mg, 0.65 mmol, 85%); 1H NMR (300 MHz, CD3OD): δ = 8.02 (d, J = 7.1 Hz, 2H), 7.56 (d, J = 7.1 Hz, 2H),
7.10–7.07 (m, 2H), 6.89 (d, J = 8.2 Hz, 1H),
4.30–4.31 (m, 4H). MS (ESI) m/z: 255 [M – H]−.
Compound 68 (157 mg,
0.40 mmol) was solubilized in THF (1.7 mL) and a solution of NaOH
(16.1 mg, 0.40 mmol) in water (1.7 mL) was added. The mixture was
heated at 60 °C for 4 h and then was left at rt overnight. HCl
3 N was added until pH 4 and the aqueous layer was extracted with
ethyl acetate (×2). The collected organic phases were dried over
sodium sulfate and evaporated. Purification by column chromatography
(ethyl acetate as eluent) yielded compound 69 as a white
solid (91.5 mg, 0.24 mmol, 61%); mp 234–235 °C dec. 1H NMR (300 MHz, DMSO-d6): δ
10.39 (br s, 1H), 8.44 (s, 1H), 8.06–8.00 (m, 3H), 7.77 (d, J = 8.2 Hz, 2H), 7.69 (d, J = 7.4 Hz, 1H),
7.48 (t, J = 7.4 Hz, 1H), 7.25 (d, J = 8.2 Hz, 2H), 6.98 (d, J = 8.2 Hz, 1H), 4.30–4.29
(m, 4H). 13C NMR (101 MHz, DMSO-d6): δ 167.8, 165.8, 144.3, 144.2, 143.1, 139.9, 133.3,
132.7, 131.9, 129.3, 128.8, 126.5, 124.9, 124.8, 121.6, 120.3, 118.1,
115.9, 64.7, 64.6. IR (neat) ν̅: 3310, 2924, 1693, 1650,
1485, 1302, 1069, 811, 752, 677 cm–1. MS (ESI) m/z: 376 [M + H]+. HRMS (ESI) m/z: (M + H)+ calcd for C22H18NO5, 376.1179; found, 376.1172.
In Vitro Metabolism Studies
Phase
I and II (glucuronidation) incubations were performed in MLMs (pooled
male mouseCD-1, protein concentration: 20 mg/mL, purchased from Corning
B.V. Life Sciences—Amsterdam, The Netherlands) using the procedure
previously described[16] with the following
modifications: 5 μM substrate concentration for the determination
of the residual percentage and 50 μM for 34 metabolite
characterization by HRMS; when metabolic activation was studied, 3
mM GSH trapping agent was added in the incubation mixture.
Aqueous
Solubility
Thermodynamic aqueous solubility
was determined as follows: about 3 mg of the tested compound was weighed
and dissolved in 3 mL of deionized water. After vigorous mixing by
vortex followed by sonication for 5 min, the resulting supersaturated
solution was shaken horizontally overnight at 25 °C. After filtration
over a syringe filter (pore 0.22 μm, regenerate cellulose membrane),
100 μL of DMSO was added to 1 mL of the filtered solution. The
resulting solution was further diluted in water (typically 1:10) before
LC–UV analysis. Aqueous solubility was calculated comparing
the filtrate peak area to those of the tested compound DMSO solutions.
Solubility in aqueous media was also checked in the following vehicles
at the target concentration of 6 mg/mL: saline + 10% DMSO, saline
+ 10% DMSO + 20% PEG400, saline + 5% ethanol.
Biology
Compounds
A 50 mM stock solution of Synta66, CM4620, teriflunomide,
brequinar, and all the biphenyl triazoles
synthetized was dissolved in 100% DMSO and stored at +4/–20
°C. For each experiment, working concentrations of these compounds
were freshly prepared by diluting DMSO to 0.1% in different physiologic
solutions according to the experimental procedures (i.e., Krebs–Ringer buffer, culture medium, Locke solution).
Cell Culture and Calcium Imaging Experiments
Screening,
dose–response experiments, and calcium imaging experiments
were performed in HEK cells (ATCC, Rock, ville, MD, USA), as already
reported elsewhere.[16]
Viability assays were performed in HEK cells that were
plated in 24-well plates at the density of 20,000 cells per well.
After 24 h, the cells were treated for other 24 h with the selected
compounds. At the end of the treatments, the medium was removed and
substituted with 300 μL of MTT reagent (Sigma-Aldrich Inc.,
Italy) at the final concentration of 0.25 mg/mL for 60 min at 37 °C.
Reactions were then stopped and the crystals were solubilized by adding
isopropyl alcohol/HCl (1 M) (Sigma-Aldrich Inc., Italy), before reading
the absorbance at 570 nm, using the multiplate reader Victor3 V (PerkinElmer,
Milan, Italy). To evaluate the effects on the DHODH enzyme, HEK cells
were treated with the selected compounds in the absence or presence
of 100 μM uridine for 72 h.
PK Analysis and Analysis
of Pancreatitis
All animal
experiments observe the regulations in Italy (D.M. 116192) as well
as the EU regulations (O.J. of E.C. L 358/1 12/18/1986). Compound 34 was injected i.v. at a dose of 7 mg/kg in C57BL/6 mice.
Blood was collected after 5, 15, 30, 60, 120, 240, 360 min and 24
h. Aliquots of plasma samples were analyzed as previously reported.[16] AP was induced in mice by i.p. injections of
cerulein, as already reported elsewhere.[16]
Statistical Analysis
In in vitro experiments,
data are presented as mean ± SEM or Median and interquartile
range (IQR). The normality of data distributions was assessed using
the Shapiro–Wilk test. Parametric (unpaired t-test and one-way analysis of variance (ANOVA) followed by Tukey’s
post-hoc) or nonparametric (Mann–Whitney U test and one-way Kruskal–Wallis H test followed
by Dunn’s post-hoc) statistical analysis was used for comparisons
of data. All statistical assessments were two-sided and a value of P < 0.05 was considered statistically significant. Statistical
analyses were performed using GraphPad Prism software (GraphPad Software,
Inc., USA).In in vivo experiments, results
were analyzed by one-way ANOVA followed by a Bonferroni post hoc test
for multiple comparisons.
Authors: Iman Azimi; Ralph J Stevenson; Xuexin Zhang; Aldo Meizoso-Huesca; Ping Xin; Martin Johnson; Jack U Flanagan; Silke B Chalmers; Ryan E Yoast; Jeevak S Kapure; Benjamin P Ross; Irina Vetter; Mark R Ashton; Bradley S Launikonis; William A Denny; Mohamed Trebak; Gregory R Monteith Journal: ACS Pharmacol Transl Sci Date: 2020-01-13
Authors: Li Wen; Svetlana Voronina; Muhammad A Javed; Muhammad Awais; Peter Szatmary; Diane Latawiec; Michael Chvanov; David Collier; Wei Huang; John Barrett; Malcolm Begg; Ken Stauderman; Jack Roos; Sergey Grigoryev; Stephanie Ramos; Evan Rogers; Jeff Whitten; Gonul Velicelebi; Michael Dunn; Alexei V Tepikin; David N Criddle; Robert Sutton Journal: Gastroenterology Date: 2015-04-25 Impact factor: 22.682
Figure 1
Figure 2
Scheme 1
Scheme 2
Figure 3
Scheme 3
Scheme 4
Figure 4
Figure 5
Figure 6
Scheme 5
Scheme 6
Figure 7
Figure 8
Figure 9
Figure 10