-IR) are nonadrenergic binding sites recognized by the radioligands [3
H]-idazoxan and [3
-aminoclonidine, as well as and with the lowest affinity, by [3
-IR are found in the central and peripheral nervous system and many organs and tissues [4
]. The alteration of I2
-IR is a hallmark in a plethora of neurodegenerative illnesses, such as brain disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease, depression, and glial tumours [6
]. Compounds with a high affinity for I2
-IR modulate many physiological and pathological processes such as analgesia and inflammation [14
]. Two I2
-IR ligands, CR4056 and [13
C]BU99008, are progressing in clinical trials, unveiling the relevance of these receptors in therapeutics. The analgesic CR4056, described as the first-in-class I2
-IR ligand, is in phase II for osteoarthritis and postoperative dental pain, and [13
C]BU99008 is in phase I for PET diagnosis in patients suffering from AD (Figure 1
Despite the potential of I2
-IR in therapeutics, their complete pharmacological characterization is still in its infancy due to their high heterogeneous nature and the absence of structural details. The disclosure of their pharmacological role relies on their modulation by I2
-IR ligands and the observation of the physiological responses. Known I2
-IR ligands are scarce and with a limited structural pattern based on the 4,5-nonsubstituted-2-heterocyclic-2-imidazoline nucleus (e.g., idazoxan, tracizoline, 2-BFI), with the only exceptions of CR4056 and LSL60101 endowing an imidazole motif (Figure 1
]. In this context, we have contributed by increasing the arsenal of ligands with structurally unprecedented new families that showed outstanding affinities to I2
-IR devoid of α2
-AR) in human brain tissues.
Therefore, in 2017, we reported the synthesis and pharmacological characterization of a family of (2-imidazolin-4-yl)phosphonates that showed excellent affinity to I2
]. Considering that I2
-IR are increased in the patients that suffered from AD [24
], we explored the implications of a representative member of this family, the [1-(3-chloro-4-fluorobenzyl)-5,5-dimethyl-4-phenyl-4,5-dihydro-1H
-imidazol-4-yl]phosphonate, named as MCR5
, in neurodegenerative diseases (ND). Our concern in this field is due to the huge worldwide impact of ND, mainly AD [25
] and PD [26
]. The WHO estimates that more than 50 million people are suffering from AD and about 7 million from PD. Unfortunately, these numbers are dramatically increasing every year. Aging of the population and the lack of effective treatments place AD as one of the most serious unmet medical needs that humankind must face in the upcoming years [27
]. Avoiding the collapse of health systems and ameliorating the suffering of patients and families guide the scientific community efforts. As no new cognitive enhancer drug has reached the market in nearly two decades, identifying new therapeutic targets and entities is a priority for medicinal chemistry programs [28
]. Accordingly, we treated with MCR5
a murine model of age-related cognitive decline, the senescence-accelerated mouse prone 8 (SAMP8), resulting in a cognitive improvement and amelioration of hallmarks. This study constituted the first experimental evidence that demonstrated the potential of the modulation of I2
-IR as a new therapy for AD [29
]. Additionally, improvement in the behavioural and psychological symptoms of dementia, including fear–anxiety, depressive-like behaviour, and memory decline, present in most ND, were observed when treating an orally SAMP8 murine model with MCR5
]. There are a few studies that discuss the presence of the I2
-IR in post-mortem tissue from PD patients, but none describe its role in the development of PD [7
Driven by the excellent precedented pharmacological results and the possibility of opening new structural opportunities, we reported a family of bicyclic α-iminophosphonates synthetised by a [3+2]cycloaddition reaction [31
]. We selected representative diethyl (1RS
]pyrrole-1-phosphonate, named as B06
, that shares an α-phenyl-α-iminophosphonate function and a 3-chloro-4-fluorophenyl group with MCR5
(highlighted in red in Figure 1
), and it is endowed with high affinity and selectivity to I2
-IR. Structurally, B06
does not fit into the restricted substitution pattern of I2
-IR ligands, with its bicyclic α-iminophosphonate structure being the first described non-imidazoline/non-imidazole-containing compound, showing outstanding affinity to I2
displays in the membranes of the post mortem human frontal cortex, a brain area that shows an important density of I2
-IR and α2
-AR, a pKi
= 8.56 ± 0.32, and a ratio of I2
= 195 in competitive binding studies against the selective I2
-IR radioligand [3
H]2-BFI and the selective α2
-AR radioligand [3
H]RX821002. Note that the standard idazoxan showed a lower pKi
with a value of 7.41 ± 0.63 and non I2
selectivity. Considering the localization of I2
-IR in the central nervous system (CNS), the good ability of B06
to cross the blood−brain barrier (BBB) was an essential requirement with a Pe value in a parallel artificial membrane permeability assay (PAMPA) well above the threshold established for high BBB permeation. B06
was devoid of cytotoxicity in a plethora of culture cells. In silico studies offered confidence for performing in vitro assays that showed non-warnings in the physicochemical properties (solubility and chemical stability), microsomal stability, cytochromes inhibition, hERG inhibition, and plasma protein binding. A receptor characterization panel de-risked our representative compound evaluated in vivo, exhibiting hypothermic properties and neuroprotection in the murine model of age-related cognitive decline, the SAMP8, as well as in a murine model of AD, the 5xFAD [32
]. Thus, B06
exerts a beneficial effect by improving cognition and ameliorating anxiety-like behaviour in the SAMP8 murine model. Modulation of I2
-IR by B06
reduces neuroinflammation, oxidative stress and calcineurin protein levels in SAMP8 [34
To progress the promising candidate B06
in preclinical studies, here, we report parameters to be assessed in the early discovery process, such as physicochemical characteristics (chemical stability at different pH) and complete ADME parameters, such as plasma stability. To shed light on the biotransformation of B06
, we undertook studies considering the stability in mouse and human liver microsomes, pharmacokinetics (PK), in vitro and in vivo metabolic profile, and the identification of metabolites of B06
. To better describe the pharmacological features of B06
and considering the paucity of information on the role of I2
-IR ligands in PD, we used a model of dopaminergic neurodegeneration to evaluate how B06
could protect neurons. Furthermore, since most ND such as AD and PD are characterized by the presence of an exacerbated and sustained neuroinflammatory process, which is partly responsible for the development of the disease, we have completed the study of the in vitro biological activity of B06
using a cellular model of neuroinflammation (Figure 2
Overall, this study boosts B06 as a promising candidate for further preclinical development in AD indication or other ND diseases. The in-depth characterization of B06 will contribute to build a robust comprehensive understanding of the pharmacological implications of I2-IR and their ligands in therapeutics.
2. Results and Discussion
2.1. Solubility and Chemical Stability of B06 at Different pH
The solubility of a compound is an essential physicochemical property that drives drug absorption and optimizes its therapeutic exposure in plasma and the therapeutic target. The capabilities of the compound to be solved in different media will avoid misunderstanding in the pharmacological results. B06
showed good solubility in several media, methanol, acetonitrile, and water, and an excellent solubility of 92 µM was found in 1% DMSO and 99% PBS buffer [33
]. Furthermore, the chemical stability of B06
in buffers at different pH was undertaken to measure the degradation by non-enzymatic processes (most common hydrolysis and oxidation). The pH value of the gastrointestinal tract varies from acidic in the stomach to basic in the small and large intestine, and alterations of representative compounds need to be assessed. To this end, a test solution of B06
(1 μM, 0.1% final DMSO concentration) was incubated with buffer pH 2, 5 and 7.4 at 37 °C. Serial samples were taken at 0, 5, 15, 30, 60 and 120 min. All samples were added immediately to three volumes of methanol containing internal standard in a microtiter plate cooled to halt chemical degradation. All the samples were analysed by LC-MS/MS. The percentage of parent compound remaining at each time point relative to the 0 min sample was calculated from peak area ratios. The chemical stability assay returned the per cent parent compound remaining at each time point for B06
The study of the graphics reveals that B06
was stable at pH 2, without significant changes in the structure up to 120 min (Figure 3
, Table 1
in a pH 5 media proved to be stable up to 1 h, and 25% of the compound suffered structural changes during the next hour (Figure 4
, Table 2
). In neutral media (pH = 7.4), B06
remained unchanged during the first minute, and at 15 min, 25% of the compound was transformed. At 30 min and pH = 7.4, 75% of B06
was detectable, with half the amount at 60 min, and only 19% at 2 h (Figure 5
, Table 3
). This study gave us an overview of the chemical behaviour of B06
, depending on the pH of the media versus time.
2.2. Plasma Stability of B06 at Mouse Plasma
Stability in liver microsomes does not imply stability in plasma, where many hydrolytic enzymes are circulating. In previous studies, the mice microsomal percentage remanent of B06
at 60 min was 65.08, with a t ½ of 91.72 min [33
]. The assessment of the plasma stability avoids misleading in vitro data and complex interpretation of the in vivo PK. To this end, 10 µM B06
in plasma mouse at 37 °C was incubated at different times (0, 60, 180 and 360 min), and acetonitrile was added for precipitating plasma protein. Then, centrifugation led to a supernatant that was analysed for sample quantification. The remaining percentage for B06
was 100% at 0 and 10 min, 69.7% at 20 min, 48.3% at 40 min and 21.6% at 60 min. Although B06
displays transformation with time, the percentage of non-modified compounds enables its in vivo activities.
2.3. Metabolic Stability of B06 in Liver Microsomes
In vitro liver microsome metabolite identification studies are essential models for assessing metabolic profiling in early drug discovery. As the compound B06 progresses through the discovery–development–clinical continuum, the goals of the metabolite analysis may alter, as will the detail with which the experiment is conducted. Early in drug discovery, the compounds being synthesized frequently have poor PK characteristics; thereby, in vitro studies are performed using liver microsomes or hepatocytes as a model to assess the metabolic profiling of the compound.
There are several reasons as to why understanding the metabolic profile of a compound is important. First, the FDA Guidance for Safety Testing of Drug Metabolites [35
] recommends in vitro evaluation of interspecies differences in drug metabolism between humans and the animals expected to be used in preclinical safety assessments. If a metabolite is formed only in humans and it is absent in the animal test species, or if the metabolite is present at disproportionately higher levels in humans than in the animal species, then it may be necessary to assess the preclinical safety of the metabolite in question. Second, understanding the metabolic liability of a compound is important in directing chemistry. If a compound is rapidly cleared, then identifying which functional groups are undergoing metabolism will be valuable in understanding how the chemistry may be altered to reduce compound degradation.
2.3.1. Metabolic Stability of B06 in Human Liver Microsomes
The metabolic stability using human liver microsomes (HLM) is directly related to the elimination of the drug from the body and significantly affects efficacy and safety. In the assay, the reaction was initiated by adding HLM (1 mg/mL) to an equal volume of buffer solution containing the test compound and cofactors at the proper concentrations. Reactions without NADPH were also incubated to rule out non-NADPH metabolism or chemical instability in the incubation buffer. Verapamil was included as a positive control to monitor incubation course. All reactions were terminated using 60 µL of ice-cold acetonitrile at 0, 5, 15, 30, 45, and 60 min. The plates were centrifugated at 3500 rpm for 15 min. All experiments were conducted in triplicate. Samples were monitored for B06
disappearance by LC-MS in MRM mode according to a quantitation method for B06
. The percentage of the remaining parent compound and the time course of metabolic stability for verapamil, positive control, and B06
at each incubation time are displayed in Table S2 and Figure S1
and Table 4
and Figure S2
The analysis of the percentage of B06
remaining in minus NADPH cofactor incubations did not show the metabolism of the parent from nondependent NADPH enzymes. The present study indicates that B06
displays a half-life value equal to 28.4 min (Table S3
). Consequently, the predicted intrinsic clearance calculated for B06
is 23.5 mL/min/mg protein (Table S3
). Attending to the classification bands typically used for categorizing compounds into low, medium or high clearance (Table S4
showed hepatic clearance classified as medium clearance.
2.3.2. Metabolic Stability of B06 in Mouse Liver Microsomes
Metabolic stability of B06 in mouse liver microsomes (MLM) was carried out as in the previous section, but instead of using HLM, the study required MLM.
The percentages of the remaining parent compound and the time course of metabolic stability for verapamil, positive control, and B06
at each incubation time are displayed in Table S6 and Figure S3
and Table 5
and Figure S4
, respectively. B06
showed a half-life value equal to 16.23 min (Table S7
). The intrinsic clearance calculated for B06
was 153.7 mL/min/mg protein, being classified as high clearance (Table S8
Note that the half-life value and the intrinsic clearance for B06 outcomes discussed above are in the same range as in liver microsomes of both species, mice and humans.
2.4. Metabolic Profiling of B06 in Liver Microsomes
The objective of the study is to evaluate the formation of the main metabolites from B06 in HLM incubations and MLM incubations preceding the in vivo metabolic study, using these same incubations to identify the possible metabolites. The assay was initiated by adding HLM solution (1 mg/mL) or MLM solution (1 mg/mL) to an equal volume of buffer solution containing B06 and cofactors. Reactions without NADPH were also incubated to rule out non-NADPH metabolism or chemical instability in the incubation buffer. Verapamil was included as positive control to monitor incubation course. All reactions were terminated using 60 µL of ice-cold acetonitrile at 0, 5, 15, 30, 45, and 60 min. The plates were centrifugated at 3500 rpm for 15 min. All experiments were conducted in triplicates. Samples were analysed by LC-HMRS in positive ionization mode. Compound incubations without NADPH and compound reference standard were used as interference control for each incubation sample.
2.4.1. Metabolic Profiling of B06 in Human Liver Microsomes
The potential metabolite 1
, ethyl hydrogen [(1R,3aS,6aS)-5-(3-chloro-4-fluorophenyl)-4,6-dioxo-1-phenyl-1,3a,4,5,6,6a-hexahydropyrrolo[3,4-c]pyrrol-1-yl]phosphonate, was tentatively identified in HLM incubations from 15 to 60 min. The assigned molecular formula was C20
FPCl, resulting in the loss of C2
). The transformation can be accounted for in the hydrolysis, in a Phase I metabolic conversion, of the ethoxy group of the phophonate function. The hydrolysis of the phosphonate ester group to the corresponding ethyl hydrogen phosphonate derivative is a plausible reaction in the physiological aqueous media (Scheme 1
). In the metabolite chromatogram at 15 min, the metabolite detected was 8.4%, at 30 min was 17.8%, at 45 min was 26.2% and at 60 min was 36% of the area (page S5, Table S9
From the TOF MS/MS spectra at 15 min, several peaks were selected for assignment. The signal with the mass of the product B06
without 25 units, 451.2389, could involve the loss of a C2
H moiety. Other peaks selected for the assignment were 341.0498 that could be the fragment A
without the diethylphosphonate ester of B06
, and 170.0606 that could correspond to the fragment B
with a 3,4-dihydro-2H-pyrrole nucleus (Figure 6
2.4.2. Metabolic Profiling of B06 in Mouse Liver Microsomes
The objective of the study is to evaluate the formation of the main metabolites from B06 in MLM incubations.
, identified in the metabolic profile of B06
in HLM, was tentatively detected in MLM incubations from 5 to 60 min. In the metabolite chromatogram at 5 min, compound 1
was detected in 10.7%, at 15 min its area was equal to 30.9%, at 30 min its presence increased to 60.9%, at 45 min it was found to be 81.6%, and at 60 min it represented 88.9% of the area (Page S5, Table S10
). Note that the half-life value and the intrinsic clearance for B06
outcomes discussed above are in the same range in HLM and MLM. These results could be a sign of a similar stay time of B06
in the organism in both animals species.
2.5. Synthesis of the Metabolite Identified in the Metabolic Profiling of B06 in Human Liver Microsomes
Metabolite 1 proposed from the biotransformation via Phase I of B06 by HLM prompted us to undertake its synthesis for unequivocal identification. To this end, the diethyl phosphonate ester group of B06 was hydrolysed by treatment with trimethylsilyl bromide in dichloromethane furnishing the desired compound 1.
The synthesized compound 1, was compared to the potential metabolite identified in both liver microsome incubations preceding the in vivo metabolic study, showing the same retention time and spectral information. Consequently, in the liver microsomes of both species, mice and humans, a metabolism of phase I occurs through the hydrolysis of the phosphonate ester furnishing 1.
2.6. In Vitro Effects of B06 in a Preclinical Model of Neurodegeneration
2.6.1. Effects of B06 on Cell Viability in a Dopaminergic Cell Line
The process of neurodegeneration results in progressive changes in the structure or function of neurons, leading to devastating neurological conditions characteristic of AD and PD [36
]. Due to the lack of information about the role of I2
-IR ligands in PD development, first, we investigated the neuroprotective effect of B06
on the human dopaminergic cell line SH-SY5Y. The human neuronal dopaminergic cell line SH-SY5Y has been described as an ideal system for modelling in vitro the characteristics of dopaminergic neurons because they have many features of substantia nigra neurons [37
]. We first tested whether the compound per se could have a cytotoxic effect on cell cultures. For this purpose, we treated the cells with different concentrations of the compounds from 200 nM to 20 μM (Page S18, Figure S18
). None of the concentrations used were cytotoxic to the cells. Then, we conducted a dose–response experiment in order to select the lowest concentration of B06
at which the most beneficial effect was obtained (Figure 7
a). From this point on, we always use that concentration (1 μM) in our analysis. Now, we tested whether B06
is able to protect the SH-SY5Y cell line from cell death induced by the toxin 6-hydroxydopamine (6-OHDA), a well-established cell model for dopaminergic neurodegeneration in which our group has extensive experience [38
]. For a more in-depth analysis, the level of active caspase 3 was used to determine apoptosis (Figure 7
c,d). Qualitatively, our results indicated an increase in the number of SH-SY5Y cells expressing active caspase 3 within 16 h after treatment with 6-OHDA. and this effect was reversed by the treatment with B06
. A Western blot analysis was used to confirm and quantify these results (Figure 7
d). For comparison, two well-known I2
-IR ligands, idazoxan and 2-BFI, were included as standards (Scheme 1
). The results summarized in Figure 7
clearly show that the treatment of SH-SY5Y cells with compound B06
significantly rescued human dopaminergic cells from 6-OHDA-induced apoptosis. These results are in agreement with previous observations, suggesting that I2
-IR are dysregulated in patients with ND [6
], providing new data on their role in PD in particular.
2.6.2. Anti-Inflammatory Effects In Vitro of B06
Along with the neuronal loss, neuroinflammation is a process related to the onset of the disease, and it is an important contributor to the pathogenesis and progression of these disorders [39
]. Since cytotoxicity induced by the neurotoxin 6-OHDA is frequently accompanied by pronounced inflammatory activity, we next evaluated the potential anti-inflammatory effect of B06
by evaluating the production of nitrites in SH-SY5Y cultures treated with the neurotoxin. In line with the results obtained with the cell viability experiments, the treatment with B06
significantly reversed the increase in nitrite production elicited by 6-OHDA treatment (Figure 8
), suggesting a convincing anti-inflammatory effect of B06
. Notably, B06
demonstrated a significantly better anti-inflammatory effect in comparison with other I2
In the CNS, microglia and astrocytes play a key role in regulating inflammatory responses. Moreover, I2
-IR are widely distributed in the brain and primarily in glial cells [40
], suggesting that this receptor plays an important role in neuroinflammation. Therefore, to further evaluate the role of B06
in inflammatory reactions, we performed experiments in different cell-based assays that mimic in part the neuroinflammatory process in PD [41
]. We used primary cultures of astrocytes and microglia treated with bacterial lipopolysaccharide (LPS), a potent inflammatory agent. Then, the potential anti-inflammatory activity of B06
was tested by evaluating the production of nitrites on primary cultured glial cells. Cultures were incubated with 1 μM of the compound for 1 h, and then cells were treated with LPS for a further 24 h. When primary astrocytes and microglial cells were stimulated with LPS (Figure 8
), we observed a significant induction of nitrite production (approximately seven-fold) in the culture medium, an indicator of inflammation. Those cultures previously treated with B06
showed a significant decrease in nitrite production, showing better results when compared with I2
-IR ligands idazoxan and 2-BFI, thus confirming the role of B06
as a potent anti-inflammatory agent. As neuroinflammation is a hallmark of many neurodegenerative diseases, including AD and PD [42
may have particular relevance for treating disorders that involve inflammation of the nervous system.
2.7. In Vivo Pharmacokinetics of B06
The physicochemical and biochemical properties of a compound determine its time course after its administration. Parameters such as concentration in bloodstream and tissues, distribution, metabolization, and elimination are fundamental for the description of the pharmacological behaviour and therapeutic characteristics of B06. To this end, after the absence of in vitro warnings in the ADME studies and in vivo advantages of B06, we proceeded with the PK and metabolic profile studies. Note, due to the therapeutic indication addressed by B06 in the CNS, we considered the quantity of compounds that rise in brain tissues.
Previously, we reported hypothermia observed in adult rats when treated intraperitoneally with 20 and 35 mg/kg measured 1 and 2 h postinjection and in mice after repeated administration of 20 mg/kg for 5 days of B06
]. It is well stablished that hypothermia causes significant neuroprotection, which also led us to have a wide range of dose confidence for the following studies. Taking into account the absence of toxicity at high doses of B06
, even at 5 days, and the dose of 5 mg/kg in drinking water during 4 weeks employed in the cognitive studies, we considered performing the PK assays at 10 mg/kg, intraperitoneally.
Thus, two-month-old mice were treated with a single dose of 10 mg/kg intraperitoneally in ten groups (n = 3), for consecutive times: 0, 5, 15, 30 min and 1, 2, 4, 6, 8, 24 h. After treatment, mice were euthanized, and the plasma and brain were collected. The experimental groups were composed of three mice, and there was not toxicity in any of the animals. Furthermore, de visu observation after euthanasia showed no toxicity in organs or tissues. These data were in accordance with the previous absence of toxicity at higher doses.
2.7.1. Pharmacokinetics of B06 in Plasma
The plasma profile and PK parameters of B06
are shown in Figure 9
The maximum concentration in plasma (Cmax) was 0.950 ± 0.624 μg/mL and was reached at 15 min (Tmax). Plasma levels, although low, remained fairly consistent for 8 h post-administration and at 24 h drug concentration, were almost undetectable. The half-life time (t1/2) was 312.8 min and the AUC0–1440/AUC0–inf_obs ratio (0.97) showed that 24 h is a good interval to define the kinetic behaviour of B06 in plasma after intraperitoneal administration at a dose of 10 mg/kg.
The maximum mean concentration found in plasma does not exceed 1% of the drug administered in a single dose intraperitoneally. Notwithstanding the low levels, the administration of B06 in drinking water at a dose of 5 mg/kg to the murine models continued at effective levels for 4 weeks. The sustained intake for the animals and the treatment time could account for the beneficial effects of B06 in the SAMP8 and 5xFAD murine models by improving cognition and ameliorating the neurodegenerative condition. In addition, a reduction in the AD hallmarks was in accordance with the new state of the animals, pointing out the variations in biochemical pathways due to the interaction of B06 with a therapeutic target.
2.7.2. Pharmacokinetics of B06 in Brain
The quantification in the brain of B06
and the calculated PK parameters are shown in Figure 10
The B06 concentrations found in the brain show that in this organ, the drug profile is identical to that found in plasma, presenting equal Tmax and similar t1/2, 312.81 min in plasma and 256.95 min in the brain. The ratio between brain/plasma concentrations remains constant (approximately 10%). These results indicate the consistent correlation of B06 levels between plasma and the brain when the drug is administered at a dose of 10 mg/kg.
These data are according to previous PAMPA-BBB results, confirming in vivo the high capacity of the drug to penetrate the BBB.
2.8. Metabolic Profiling In Vivo of B06
Metabolite identification studies are essential in the early stages of drug discovery and during drug and clinical development and are strongly recommended by the FDA Guidance for Industry on Safety Testing of Drug Metabolites [35
]. After performing the liver microsomes studies reported above, we undertook the metabolite determination by seizing the PK study. When entering the body, B06
will undergo biotransformation via Phase I and Phase II metabolic pathways. In this framework, a study to understand the biotransformation details of B06
by identifying the main in vivo metabolites from B06
at different times (30 to 480 min) was performed. The aim was to discard toxic issues and add information on the behaviour underlying beneficial cognitive impairment in the murine model of neurodegeneration and AD after treatment with B06
. To this end, an aliquot of 50.0 µL mouse plasma and 150.0 µL of cold acetonitrile was added. After vortex mixing for one minute, the samples were centrifuged for 15 min at 13,300 rpm. An aliquot of 110.0 µL of the supernatant was transferred to an auto sampler vial to analyse by LC-HRMS in positive ionization mode.
In this condition, two potential metabolites of B06
, 2-hydroxyethyl ((1R,3aS,6aS)-5-(3-chloro-4-fluorophenyl)-4,6-dioxo-1-phenyl-1,3a,4,5,6,6a-hexahydropyrrolo[3,4-c]pyrrol-1-yl)phosphonate, named as 2,
were tentatively identified in vivo from 30 to 480 min. The assigned molecular formula was C22
FPCl, and taking into account the fragmentation patterns in the mass spectra, it seems that one of the oxidations occurred in one of the ethyl groups of the phosphonate ester, and the other oxidation could be predicted to be in the other ethyl group. These oxidations of Phase I accounted for the prochiral character of the phosphorous that when substituted by two ethoxy groups is lacking chirality. It is not a stereocentre, but when one of the substituents is altered, the phosphorous becomes prochiral; therefore, both ethoxy groups are different from the stereochemical point of view (Scheme 2
The low concentration of metabolites made detection in plasma difficult due to the fact that concentrations of the parental B06 molecule were also low. The potential metabolite of B06 at 30 min was 2.5% of an oxidized B06 derivative (m/z 495.0877), at 60 min was slightly increased to 2.8%, and at 120 min was detected in 5.8%. When increasing the time to 240 min, two tentative oxidation metabolites were found with slightly different retention times, 7.82 and 7.98 min, in the chromatogram in the conditions used. The new product at 7.98 min represented 1.5%, and the former oxidized metabolite (7.82 min) was 6.3%. At 360 min, only one of the oxidized B06 proposed metabolite was detected at 6.7%, and at 480 min, the percentage was 4.5% in the order of the previous determinations.
From the TOF MS/MS spectra at 480 min, several peaks were selected for assignment. The signal with the mass of the product B06
with an additional 17 units, 495.2703, could represent the gain of an hydroxyl group. Other peaks selected for the assignment were 341.0498 that may belong to fragment A
without the diethylphosphonate ester moiety of B06
, 142.0646 that may correspond to fragment C
, 2-phenyl-3,4-dihydro-2H-pyrrole, and 449.0453 that can be the mass of fragment D
with loss of C2
H moiety (Figure 11
The discovery of B06, away from the structural limits of previous I2-IR ligands, has opened the door for the comprehensive exploration of these receptors in a plethora of indications. To start with, oral treatment with B06 in murine models of neurodegeneration and AD prevented behavioural abnormalities and memory loss, reducing AD hallmarks, oxidative stress, and apoptotic and neuroinflammation markers. Preliminary ADME-Tox parameters and a safety panel proved that B06 is a suitable compound to advance in preclinical phases.
In detail, in this work, we provided information on the metabolic stability of B06 in human and mouse liver microsomes showing a medium hepatic clearance. The metabolic profile in liver microsomes lead to the proposal and unequivocal identification, after its synthesis, of a metabolite resulting from the monohydrolysis of the phosphonate ester function of the molecule. The in vivo PK study led to the calculation of relevant parameters and revealed a good correlation between the levels of B06 found in plasma and in the brain, although the low amounts of the compound quantified indicated a good penetration in the CNS.
Notably, this paper is the first study aimed to unveil the role that I2-IR ligands could have in PD. After finding the optimal dose, the neuroprotective role of B06 on the human dopaminergic cell line SH-SY5Y was determined. Furthermore, in primary cultures of astrocytes and microglia treated with LPS, B06 was shown to be an anti-inflammatory agent even better than other known ligands.
Overall, our results confirm that B06 is a promising candidate for neurodegenerative management due to its immunomodulatory and neuroprotective properties. B06 is a robust compound to be seriously considered as a modulator of I2-IR for characterizing the pharmacological profile of these receptors and for further and more advanced preclinical development in brain disorders such as AD and PD.