pubs.acs.org/jmc Article Novel Antagonist of the Type 2 Lysophosphatidic Acid Receptor (LPA2), UCM-14216, Ameliorates Spinal Cord Injury in Mice Nora Khiar-Fernández,† Debora Zian,† Henar Vázquez-Villa, R. Fernando Martínez, Andrea Escobar-Peña, Román Foronda-Sainz, Manisha Ray, Maria Puigdomenech-Poch, Giovanni Cincilla, Melchor Sánchez-Martínez, Yasuyuki Kihara, Jerold Chun, Rubeǹ López-Vales, María L. López-Rodríguez,* and Silvia Ortega-Gutiérrez* Cite This: J. Med. Chem. 2022, 65, 10956−10974 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Spinal cord injuries (SCIs) irreversibly disrupt spinal connectivity, leading to permanent neurological disabilities. Current medical treatments for reducing the secondary damage that follows the initial injury are limited to surgical decompression and anti-inflammatory drugs, so there is a pressing need for new therapeutic strategies. Inhibition of the type 2 lysophosphatidic acid receptor (LPA2) has recently emerged as a new potential pharmacological approach to decrease SCI- associated damage. Toward validating this receptor as a target in SCI, we have developed a new series of LPA2 antagonists, among which compound 54 (UCM- 14216) stands out as a potent and selective LPA2 receptor antagonist (Emax = 90%, IC50 = 1.9 μM, KD = 1.3 nM; inactive at LPA1,3−6 receptors). This compound shows efficacy in an in vivo mouse model of SCI in an LPA2-dependent manner, confirming the potential of LPA2 inhibition for providing a new alternative for treating SCI. ■ INTRODUCTION A spinal cord injury (SCI) is defined as damage to the spinal However, the current medical reality is that there is no treatment for acute SCI because methylprednisolone, which cord that provokes a temporary or permanent impairment of was the standard treatment for acute SCI, is no longer used for its function. It has negative consequences for the physical and the management of spinal cord trauma in many countries social well-being of patients and imposes an important based on several reports demonstrating its lack of therapeutic economic burden to the individual and the health care system. efficacy and its undesirable side effects related to immunosup- SCI can have traumatic or nontraumatic origins. The former pression and gastrointestinal bleeding.4 Hence, it is evident happens when an external physical impact acutely harms the there is a crucial need to develop new treatments for SCI. In spinal cord, whereas the latter is associated with disease this regard, there is a consensus in that primary injury cannot development, such as a tumor, an infection, or a neuro- be therapeutically addressed, but secondary cell damage events degenerative process. Regardless of the etiology, the primary that occur after SCI could be susceptible to therapeutic injury damages cells and initiates a complex secondary cascade intervention. Hence, much research effort has focused on delineation of the receptor pathways responsible for the of secondary degeneration characterized by ischemia, ex- irreversible cellular damage that occurs after SCI, because they citotoxicity, and inflammatory processes that lead to the death could represent new therapeutic targets for novel drug of neurons and glial cells. This process is followed by a treatments. In this context, bioactive lipids have recently reorganization of the structural architecture of the spinal cord emerged as major players in the initiation and maintenance of and by the formation of glial scars that, together with the poor the pro-inflammatory environment that prevent tissue repair capacity of the central nervous system (CNS) to promote and recovery of homeostasis.5 Among them, lysophosphatidic remyelination and axonal growth, causes irreversible neuro- acid (LPA, 1-acyl-sn-glycerol-3-phosphate) has received an logical deficits. Considering the negative impact of SCI, it is clear that prevention of the primary injury is desirable, as would an efficacious treatment to minimize secondary injury Received: January 11, 2022 events to prevent functional impairments. The last several Published: August 10, 2022 years have witnessed an important advancement of the field, with the development of different experimental neuro- protective and neuroregenerative therapies that have been translated from preclinical studies into clinical trials.1−3 © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.jmedchem.2c00046 10956 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 1. Structure of the LPA2 antagonists C35, H2L5186303, and Ki16425. Figure 2. Design of new LPA2 antagonists 2, 3, and 10−13. increasing attention.6,7 Although LPA can refer to multiple and might represent a new therapeutic avenue. Here we report different species of lysophospholipids with saturated (16:0, the development of the most potent and selective LPA2 18:0) and unsaturated (16:1, 18:1, 18:2, 20:4) acyl chains, in antagonist described so far, compound UCM-14216 (54), the context of SCI, LPA 18:1 (1-oleoyl-sn-glycerol-3- which has an IC50 value of 1.9 μM as an LPA2 antagonist, a KD phosphate) appears to be the most important form.8 The value of 1.3 nM, and a selectivity over other LPA receptor increase in LPA levels in the CNS after traumatic injury has subtypes (LPA1 and LPA3−6, with 10-fold selectivity in terms of detrimental effects, as it has been confirmed by experiments IC50 value with respect to LPA1 and LPA3 and 10-fold showing that intraspinal injection of LPA leads to inflamma- selectivity versus LPA6 and >50-fold selectivity versus LPA4 tion and demyelination.8 However, taking into account that and LPA5 in terms of KD). In addition, this compound LPA can activate at least six different receptors (LPA1−6) that significantly improves motor recovery in an in vivo model of belong to the G protein-coupled receptor (GPCR) super- SCI, supporting the importance of LPA2 for the treatment of family,9−11 the next important step is to determine which SCI. specific receptor subtype(s) is responsible for the deleterious effect of pathological LPA exposure. In this regard, the importance of LPA1 as a target for the treatment of SCI has been well established,8,12 but this receptor does not account for ■ RESULTS AND DISCUSSION Within a broad project focused on the discovery of new ligands all the effects observed with LPA. Very recently, LPA2 has been for LPA receptors,18 we started our search of potent and postulated as a key receptor in mediating the effects of LPA in selective LPA2 antagonists through an in-house screen using a SCI.13 However, its validation has been hampered by the lack functional assay to detect calcium mobilization in cells stably of selective antagonists. Currently, only two compounds (C35 transfected with the LPA2 receptor in which the compounds and H2L5186303, Figure 1) have been characterized as potent under study were added at a fixed dose of 10 μM and the cells (IC50 values at LPA2 of 0.017 and 0.0089 μM, respectively) were subsequently stimulated with LPA at the same and selective LPA2 antagonists (IC50 values >50 μM at LPA1 concentration. We considered active those compounds able and LPA3 for C35 and 1.23 and 27.3 μM at LPA1 and LPA3 for to reduce the LPA-mediated calcium response by at least 30%. H2L5186303).14,15 However, their selectivity profile versus the Among all tested molecules, compound 1 (Figure 2) showed a other LPA receptors (LPA4−6), pharmacokinetic properties, consistent antagonist signal at LPA2 receptor, absence of and in vivo efficacy have not been studied. Another tool significant agonist activity at this receptor (Figure S1), and compound widely used to study the effect of blocking LPA selectivity versus LPA1 and LPA3 receptors, so it was selected receptor signaling is Ki16425 (Figure 1), but although it has as our initial hit. However, its moderate antagonism at LPA2 at good in vitro potency, this derivative is a nonselective 10 μM (Emax = 48 ± 9%) led us to carry out a systematic antagonist with submicromolar activity at LPA1 and LPA3 structural exploration of this compound with the aim of and lower affinity at LPA2 (IC50 values of 0.34, 0.93, and 6.5 improving its biological activity. μM, respectively)16 and with limited in vivo activity that may Structure−Activity Relationship (SAR) Study of Hit 1. reflect its short half-life.17 First, we tried to establish the relative importance of the New potent and selective LPA2 antagonists could enable the different parts of the molecule for the LPA2 antagonist activity. validation of this receptor as a target for the treatment of SCI We started by studying the influence of the chlorophenoxy 10957 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 1. Synthesis of Compounds 2, 3, and 10−13a a Reagents and conditions: (a) CH(OEt)3, 70% HClO4, H2O, rt, 13 h, 47%; (b) methyl bromoacetate, K2CO3, acetone, reflux, 3 h, 24−98%; (c) (i) 65% N2H4·H2O, EtOH, reflux, 30 min, 77−99%; (ii) 2 M NaOH, EtOH, reflux, 12 h, 67−99%; (d) (i) SOCl2, toluene, 110 °C, 16 h, 99%; (ii) resorcinol, BF3·Et2O, DCM, reflux, 4−5 h, 19−25%; (e) acetic anhydride, Et3N, NaOAc, 140 °C, 2−3 h, 83−99%; (f) conc. HCl, EtOH, reflux, 2 h, 75−99%. group by removing the whole moiety or just the halogen atom similar to the one previously followed for compound 3 starting with the synthesis of compounds 2 and 3 (Figure 2). from the corresponding chlorophenoxyacetic acid and Compound 2 was prepared from commercially available 1- resorcinol (Scheme 1). (2,4-dihydroxyphenyl)ethanone by treatment with triethyl Determination of the LPA2 antagonist character of orthoformate and perchloric acid. Then, resulting hydrox- compounds 10 and 11 revealed that whereas the former did ychromone 4 was alkylated with methyl bromoacetate and not improve the antagonist activity of the initial hit 1 [Emax (1) treated with an excess of hydrazine to obtain desired derivative = 48%; Emax (10) = 45%], the latter increased the LPA2 2 (Scheme 1), through opening of the pyrone ring and antagonist activity at the maximum concentration [Emax (11) = subsequent formation of pyrazole ring. The reaction with 60%] (Table 1). These results suggested that the chlorine hydrazine promoted the simultaneous transformation of the atom was tolerated at the three positions, with the best result ester group to the corresponding hydrazide, which was obtained for the para derivative, so it may be possible that the hydrolyzed to obtain the target carboxylic acid. With respect introduction of a second chlorine atom allowed further to compound 3, its synthesis started with a Friedel−Crafts improvement of activity. Accordingly, compounds 12 and 13 acylation between 2-phenoxyacetyl chloride and resorcinol. were synthesized (Scheme 1) and tested for LPA2 activity Next, the Kostanecki−Robinson reaction between the resulting (Table 1). Determination of their antagonist character revealed ketone 6 and acetic anhydride afforded chromone 7 in a good that introduction of the 2,4-dichlorophenoxy moiety yielded an yield, which was, after hydrolysis of the acetyl group in acid excellent LPA2 antagonist [Emax (13) = 84%; IC50 (13) = 5.5 media, alkylated with methyl bromoacetate to obtain μM; Figure S2], with similar LPA2 antagonist activity to intermediate 9. Finally, treatment with hydrazine gave target Ki16425, used as the reference ligand (Table 1). Also, to rule compound 3 (Scheme 1). Antagonist activity assays revealed out the existence of partial agonism, we measured the agonist that compound 2 was inactive at LPA2 (Emax = 7 ± 3%) activity of compounds 3 and 10−13 at LPA2 receptors, and whereas derivative 3 showed a low activity at LPA2 (Emax = 20 none of them was able to induce any significant activation of ± 7%, Table 1), highlighting the need not only of the chlorine the receptor at 10 μM concentration (see Figure S1 for the atom but also of the whole phenoxy system for the LPA2 result obtained for compound 13, which is representative of antagonist activity. Hence, we studied the influence of the the rest of the compounds). position of the chloro substituent with the synthesis of At this point, we considered that a detailed study of the compounds 10 and 11, where the chlorine atom was located in molecular interactions involved in the affinity of compound 13 a meta or para position, respectively (Figure 2). These for LPA2 could help us to rationalize the activity results and syntheses were accomplished following a synthetic route also shed some light on the binding site of the compound. 10958 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 1. Antagonist Activities of Compounds 1, 3, 10−13, and Ki16425 at LPA1‑3 a Emax = maximum blockade effect of the activation induced by 10 μM of LPA (18:1, 1-oleoyl-sn-glycerol-3-phosphate) at a concentration of the compound under study of 10 μM. bFor Emax > 70%, IC50 values are expressed as mean ± s.e.m, from a minimum of two independent experiments, performed in triplicate. cN.E., no effect was observed at the highest concentration of compound tested (10 μM). Hence, we built a homology model of LPA2 using the disclosed pocket since it cannot simultaneously reach glycine 257, crystal structure of the LPA1 as a template.19 The best docking alanine 284, and leucine 111 as compound 13 does through its pose of compound 13 in the LPA2 receptor model (Figure 3A) two chlorine atoms (Figure 3B). suggests that the phenolic hydroxy group interacted with two The importance of the phenolic hydroxy group was hydrogen bonds with arginine 107 and glutamine 108 and the confirmed through the synthesis of compound 30 (Scheme carboxylic acid group is engaged in two salt bridges with lysines 2), which was obtained starting with a Williamson reaction 22 and 278 (Figure 3A). The dichlorophenoxy moiety lies in a between 2-bromo-1-(4-methoxyphenyl)ethanone and 2,4- hydrophobic pocket surrounded by leucine 261, leucine 111, dichlorophenol under microwave (MW) irradiation, using glutamine 108, glycine 257, tryptophan 254, alanine 284, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base. Then, tyrosine 85 and phenylalanine 280. The chlorine atom in the 2- treatment of the intermediate 31 with 1,1-dimethoxy-N,N- position points to residues leucine 111 and glutamine 108, dimethylethanamine yielded enaminone 32, which was reacted while the one in the 4-position points to residues glycine 257 with hydrazine to obtain pyrazole 33. Finally, removal of the and alanine 284 (Figure 3A). Also, the oxygen atom of the methoxy group followed by O-alkylation with methyl phenoxy moiety forms a hydrogen bond with glutamine 108. bromoacetate and hydrolysis of the ester gave the target Compound 3 adopts a similar pose to compound 13, but its pyrazole 30 (Scheme 2), which was basically inactive at the phenoxy moiety does not completely fill the hydrophobic LPA2 antagonist assay, with an Emax value of only 11%. 10959 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article and lysine 22 can take place in compound 13 but not in derivative 38 due to the binding conformation induced by the four-unit spacer. In addition, a key hydrogen bond established between the phenolic hydroxy group of compound 13 and arginine 107 is missing in the binding of compound 38 to LPA2 (Figure 4). Further confirmation of the importance of the carboxylic acid interactions was obtained with the synthesis of compounds 42−45, where the carboxylic acid moiety was replaced by hydroxy, methoxy, methyl ester or carboxamide groups, respectively. The synthesis of these compounds started from chromone 25, which by reaction with hydrazine yielded pyrazole 42 that was further methylated to give 43. Alternatively, O-alkylation of chromone 25 with methyl bromoacetate or bromoacetamide followed by pyrazole ring formation yielded compounds 44 and 45 (Scheme 4). Biological evaluation of all these compounds (Table 3) revealed that only the methyl ester derivative 44 showed a good activity value (Emax = 74 ± 7%; IC50 = 11.6 ± 0.4 μM). To further discard the existence of partial agonism, derivatives 42−45 were tested for their capacity to activate the LPA2 receptor and none of them induced any appreciable effect at a concentration of 10 μM. We next studied the effect of changes in the pyrazole ring. Specifically, we introduced an N-methyl group (compound 47), removed the methyl group located at position 5 of the heterocycle (compound 48), and replaced the pyrazole by an isoxazole ring (derivative 49). Direct methylation reaction of 13 provided N-methylated analogue 47 (Scheme 5), whereas the preparation of compound 48 started with the reaction of intermediate 17 with methanesulfonyl chloride, in the presence of the boron trifluoride diethyl etherate complex to obtain the corresponding hydroxychromone 50. Alkylation of this intermediate with ethyl bromoacetate and reaction with hydrazine, afforded desired pyrazole 48 (Scheme 5). Finally, isoxazole analogue 49 was obtained from chromone 29 using Figure 3. (A) LPA1 (PDB ID 4Z35)-derived homology model of hydroxylamine (Scheme 5). Biological evaluation of the LPA2R in complex with compounds 3 (in purple) and 13 (in white). compounds (Table 4) revealed the importance of the methyl The phenolic hydroxy group of both compounds is engaged in two group in position 5 of the pyrazole ring for the antagonist hydrogen bonds with arginine 107 and glutamine 108 and the activity, since derivative 48 exhibited a moderate Emax value of carboxylic acid group is engaged in two salt bridges with lysines 22 35%. With respect to derivatives 47 and 49, they showed good and 278. Also, the oxygen atom of the phenoxy moiety forms a hydrogen bond with glutamine 108. (B) Phenoxy moiety of the two activity at LPA2 but also a decrease in selectivity, since they compounds lies in the same hydrophobic pocket but compound 3, display some antagonist character at LPA3 (Table 4). None of represented here with a C-purple surface representation, cannot reach them showed any activity as LPA2 agonists at a concentration simultaneously residues glycine 257, alanine 284, and leucine 111 as of 10 μM. compound 13 does, represented here as C-white mesh representation. In sum, these results indicated that derivative 13 was the best compound identified so far. Hence, we studied its Next, we focused our attention on the influence of the pharmacokinetic profile. First, we estimated the membrane distance between the oxygen atom and the carboxylic acid permeability using the parallel artificial membrane permeability group in compound 13. To determine the optimum length of assay (PAMPA) and its metabolic stability in mouse and the methylenic chain that separates these two moieties, we human liver microsomes (MLMs and HLMs, respectively). In synthesized compounds 36−38, which have 2−4 methylenes these assays, compound 13 showed a moderate permeability in the linker (Scheme 3). value (P) of 0.11 × 10−6 cm/s, considering as reference values None of the synthesized compounds showed any activity as P < 1 × 10−7 cm/s for low permeable compounds and P > 1 × LPA2 agonists at 10 μM concentration and, in all cases, 10−5 cm/s for highly permeable molecules. The metabolic increasing the distance between the carboxylic acid group and stability was also moderate, with a half-life (t1/2) of about 60 the rest of the molecule resulted in decreased LPA2 antagonism min in HLMs and 16 min in MLMs. Hence, it would be activity (Table 2). The worst result was obtained for desirable to improve these parameters to obtain an optimized compound 38, bearing the longest chain (n = 4) with an compound suitable for in vivo efficacy experiments. Emax value of 21% compared to the 84% of derivative 13. This Optimization of Compound 13. We initially addressed decrease in activity can be rationalized by the docking model the optimization of derivative 13 with the replacement of between compound 38 and LPA2 (Figure 4), which shows that chlorine atoms by fluorine in compound 53 (Scheme 6), as a key salt bridge interaction between the carboxylic acid group this change usually involves an improvement of the 10960 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 2. Synthesis of Compound 30a a Reagents and conditions: (a) 2,4-dichlorophenol, DBU, DMF, MW, 140 °C, 30 min, 80%; (b) 1,1-dimethoxy-N,N-dimethylethanamine, 90 °C, 4 h, 52%; (c) 65% N2H4·H2O, EtOH, reflux, 1 h, 44%; (d) BBr3, DCM, −78 °C to rt, 21 h, 70%; (e) methyl bromoacetate, K2CO3, DMF, −20 °C to rt, 16 h, 32%; (f) 1 M NaOH, 1,4-dioxane, 60 °C, 2 h, 99%. Scheme 3. Synthesis of Compounds 36−38a a Reagents and conditions: (a) methyl acrylate, DMAP, MW, 150 °C, 2.5 h, 10%; (b) (i) 65% N2H4·H2O, EtOH, reflux, 30 min, 24−86%; (ii) 2 M NaOH, EtOH, reflux, 12 h, 86−99%; (c) methyl 4-bromobutanoate or methyl 5-bromopentanoate, K2CO3, acetone, reflux, 3−5 h, 80−82%. pharmacokinetic parameters.20,21 Also, considering that the LPA2 antagonist, with an Emax of 90% and an IC50 value of 1.9 free carboxylic acid could be responsible for the moderate μM (Figure S2), values that are superior to the ones showed by permeability, it was replaced by the (bio)isostere tetrazole its analogue 13 (Table 5). None of these compounds showed (compounds 54 and 55, Scheme 6). Tetrazole is among the any agonist activity at LPA2 (see Figure S1 for the result most commonly employed carboxylic acid isosteres22 because obtained for compound 54, representative of the rest of the its planarity and acidity closely resemble those of carboxylic compounds). acids (pKa = 4.5−4.9). In addition, tetrazolate anions are more Docking studies of these compounds showed how the lipophilic than the corresponding carboxylates and they exhibit docking pose of compound 54 is very similar to that of slightly different electrostatic potential and charge distribution compound 13 by replacing the carboxylate moiety with its due to the delocalization of the negative charge over the five- tetrazole ring (Figure 5A). In fact, the tetrazole moiety of membered ring system. Then, synthesis of difluorinated compound 54 perfectly reproduces the interactions of the derivative 53 was carried out following a similar route to the carboxylic acid of compound 13, substituting salt bridges for one described for compound 13 but starting with 2,4- hydrogen bonds with lysines 22 and 278. With respect to the difluorophenoxyacetic acid (Scheme 6). With respect to the replacement of chlorine by fluorine in compounds 53 and 55, tetrazole derivatives 54 and 55, they were prepared by the dichloro and difluorophenoxy moieties lie in the same alkylation of the intermediate chromones 25 and 58, hydrophobic pocket (Figure 5B). However, the substitution of respectively, with 2-bromoacetonitrile followed by sequential chlorine by fluorine atoms provokes a change in the treatment with hydrazine and sodium azide to build the orientation of the aromatic ring and hinders the difluor- corresponding pyrazole and tetrazole rings, respectively ophenoxy moiety from simultaneously reaching residues (Scheme 6). leucine 111 and alanine 284 as observed for compounds 13 Biological evaluation of compounds 53−55 indicated that and 54 through their two chlorine atoms (Figures 3 and 5B). derivative 54 showed the best results, being the most potent To experimentally validate the proposed docking model, we 10961 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 2. Antagonist activities of compounds 13, 36−38, and cytometry using a primary antibody against HA and the Ki16425 at LPA1‑3 appropriate fluorescent secondary antibody (Figure 6A) and the antagonist capacity of compound 54 was determined in cells transfected with each mutant. The obtained results indicate that replacement of any of the four amino acids (lysines 22 and 278, arginine 107 and glutamine 108) by alanine involved the lost of the antagonist activity of compound 54 (Figure 6B), thus confirming the importance of the proposed interactions. The data indicate that Emax (%)a [IC50 (μM)]b substitution of lysine 22, arginine 107, and glutamine 108 by compd n LPA1 LPA2 LPA3 alanine basically abolished the capacity of compound 54 to 13 1 N.E.c 84 ± 3 N.E. bind LPA2 receptor (since no significant agonist nor antagonist [5.5 ± 0.7] activity was observed in the mutant receptors). However, the 36 2 N.E. 67 ± 7 N.E. exchange of lysine 278 by alanine completely switched the 37 3 N.E. 43 ± 12 N.E. functional activity of the receptor since compound 54 behaved 38 4 N.E. 21 ± 8 N.E. as an agonist in this mutant receptor. Overall, these data Ki16425 97 ± 4 92 ± 3 99 ± 3 suggest that amino acids 22, 107, and 108 are important for [0.8 ± 0.2] [1.2 ± 0.6] [1.6 ± 0.5] binding, whereas lysine 278 seems to be involved in the a Emax = maximum blockade effect of the activation induced by 10 μM functional activity of the receptor. of LPA (18:1, 1-oleoyl-sn-glycerol-3-phosphate) at a concentration of Noteworthy, compound 54 kept the receptor selectivity the compound under study of 10 μM. bFor Emax > 70%, IC50 values versus LPA1 and LPA3 (Table 5), so it emerged as an excellent are expressed as mean ± s.e.m, from a minimum of two independent candidate for in-depth pharmacological characterization. experiments, performed in triplicate. cN.E., no effect was observed at In-Depth Characterization of Compound 54. First, we the highest concentration of compound tested (10 μM). determined the membrane permeability using the PAMPA assay and the in vitro metabolic stability of the compound. The obtained results showed a good permeability value (P = 6.1 × 10−6 cm/s) and also increased stability in comparison with analog 13, with t1/2 values of 50 ± 6 and 97 ± 15 min for MLMs and HLMs, respectively (Table 6). These in vitro values correlated with the results obtained in the in vivo pharmacokinetic (PK) study, which was carried out to determine the suitability of the compound to reach the CNS in therapeutically relevant doses. For this aim, compound 54 was administered intraperitoneally (i.p.) at a dose of 25 mg/kg. Then, at different postinjection times (between 0.5 and 4 h), plasma, brain, and spinal cord samples were taken and the levels of compound 54 were measured using high-performance liquid chromatography coupled to mass spectrometry (HPLC- MS). These experiments confirmed the presence of compound 54 at significant levels in spinal cord and brain, with the maximum levels reached at one hour postadministration. These data (Table 7) indicate that antagonist 54 can readily cross the blood brain barrier and it is therefore an excellent candidate to validate the role of LPA2 antagonism, at least as a proof of principle, in an in vivo model of SCI. Figure 4. LPA1 (PDB ID 4Z35)-derived homology model of LPA2 in In addition, the binding affinity of the compound for LPA2 complex with compound 13 (in white) and compound 38 (in purple). was evaluated by means of a free solution assay-compensated Docking of compound 13 in the model suggests that the carboxylic interferometric reader (FSA-CIR) technique,23−26 showing a acid of the compound is involved in two salt bridge interactions with binding equilibrium constant (KD) value of 1.3 nM. As a lysine 22 and lysine 278; however, derivative 38 cannot form the salt positive control, LPA showed a KD value of 6.7 nM to LPA2. bridge with lysine 22. Docking results also suggest that the phenolic The analogous assay carried out for LPA4−6 provided 10-fold hydroxy group of compound 13 establishes a hydrogen bond with arginine 107 and another one with glutamine 108, whereas derivative selectivity versus LPA6 and >50-fold selectivity versus LPA4 38 can only establish hydrogen bonds with glutamine 108. For and LPA5 (Figure S3), making compound 54 (UCM-14216) compound 13, salt bridges are colored in yellow and hydrogen bonds the most potent and selective LPA2 antagonist described so far. in blue, whereas for derivative 38, they are colored in orange and pink, In Vivo Efficacy Study of Compound 54 in an SCI respectively. Mouse Model. Since LPA2 activation plays harmful actions after SCI, we finally assessed whether compound 54 protects carried out point mutation experiments to confirm the against locomotor deficits in a spinal cord contusion injury importance of the most relevant residues (lysines 22 and model. It has been established that the LPA2 receptor is 278, arginine 107, and glutamine 108). Thus, we transfected constitutively expressed at very low levels in spinal cord and its McA-RH7777 cells with plasmids containing the correspond- transcripts are up-regulated during the first days after injury, ing N terminus HA-tagged LPA2 mutant (K22A, R107A, returning to basal levels by day 7.13 This suggests that LPA- Q108A, or K278A). Transfection efficacy was assessed by flow LPA2 signaling in the injured spinal cord mainly occurs during 10962 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 4. Synthesis of Compounds 42−45a a Reagents and conditions: (a) 65% N2H4·H2O, EtOH, reflux, 30 min, 65−80%; (b) CH3I, K2CO3, acetone, 65 °C, 6 h, 20%; (c) methyl bromoacetate or bromoacetamide, K2CO3, acetone, reflux, 3 h, 51−67%; (d) 2 M NaOH, EtOH, reflux, 12 h, 90%; (e) CH3OH, cat. H2SO4, reflux, 16 h, 85%. Table 3. Antagonist Activities of Compounds 13, 42−45, and Ki16425 at LPA1‑3 a Emax = maximum blockade effect of the activation induced by 10 μM of LPA (18:1, 1-oleoyl-sn-glycerol-3-phosphate) at a concentration of the compound under study of 10 μM. bFor Emax > 70%, IC50 values are expressed as mean ± s.e.m, from a minimum of two independent experiments, performed in triplicate. cN.E., no effect was observed at the highest concentration of compound tested (10 μM). the first week postinjury. Hence, we hypothesized that in the study). It is conceivable that this concentration is even administration of compound 54 for 10 days could block higher in the injured mice, as SCI results in increases LPA-LPA 2 signaling in the injured spinal cord, and permeability of the blood-spinal cord barrier.27 Then, this consequently, improve the outcome of SCI, as observed after dose was selected as the minimal capable of potentially eliciting genetic deletion of Lpa2.13 The in vivo PK study suggested that the sought biological effects and simultaneously avoiding side an i.p. dose of 25 mg/kg was enough to reach significant levels effects related with the use of higher concentrations. of the compound one hour after administration (3.3 ng/mg Accordingly, mice were treated daily with compound 54 (25 tissue are equivalent approximately to a concentration of 2 μM mg/kg, i.p.) starting at 1 h following lesion and subsequently in the spinal cord considering the volume of the sections used for 10 consecutive days, and locomotor performance was 10963 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 5. Synthesis of Compounds 47-49a a Reagents and conditions: (a) CH3I, NaH, THF, rt, 16 h, 31%; (b) CH3SO2Cl, BF3·Et2O, DMF, 100 °C, 1.5 h, 94%; (c) ethyl bromoacetate, K2CO3, acetone, reflux, 3 h, 91%; (d) (i) 65% N2H4·H2O, EtOH, reflux, 30 min, 99%; (ii) 2 M NaOH, EtOH, reflux, 12 h, 58%; (e) (i) NH2OH· HCl, pyridine, EtOH, 85 °C, 12 h; (ii) p-toluenesulfonic acid, EtOH, 78 °C, 5 h, 40%; (f) 1 M NaOH, 1,4-dioxane, 60 °C, 16 h, 91%. Table 4. Antagonist Activities of Compounds 13, 47−49, and Ki16425 at LPA1‑3 Emax (%)a [IC50 (μM)]b compd R R′ X LPA1 LPA2 LPA3 c 13 Me H N N.E. 84 ± 3 [5.5 ± 0.7] N.E. 47 Me Me N N.E. 73 ± 10 47 ± 7 48 H H N N.E. 35 ± 8 N.E. 49 Me H O N.E. 58 ± 3 71 ± 4 Ki16425 97 ± 4 [0.8 ± 0.2] 92 ± 3 [1.2 ± 0.6] 99 ± 3 [1.6 ± 0.5] a Emax = maximum blockade effect of the activation induced by 10 μM LPA (18:1, 1-oleoyl-sn-glycerol-3-phosphate) at a concentration of the compound under study of 10 μM. bFor Emax > 70% and selectivity at LPA2 receptor, IC50 values are expressed as mean ± s.e.m, from a minimum of two independent experiments, performed in triplicate. cN.E., no effect was observed at the highest concentration of compound tested (10 μM). assessed by using the Basso Mouse Scale (BMS). BMS is the with more frequent administration (i.e., twice a day), longer gold standard test used for locomotor scoring after SCI in duration or greater dose of the compound. which two blinder observers score the mouse motor perform- Importantly, the observed locomotor improvement is largely ance based on a nine-point scale.28 As shown in Figure 7A, mediated by the action of the compound at the LPA2 receptor, mice treated with compound 54 displayed significant improve- because administration of the same dose of compound to LPA2 ment in locomotor recovery after SCI. Bonferroni’s post hoc null mice undergoing SCI did not induce any significant effect analysis revealed that motor skills were significantly enhanced (Figure 7B). These results clearly validate the LPA2 receptor as in the injured mice that had been treated with compound 54 a valuable therapeutic target for the treatment of SCI. for 10 days at 25 mg/kg, from day 35 postinjury onward. At the end of the follow up (day 50 postinjury) mice treated with vehicle showed plantar placement of the hind paw but no ■ CONCLUSIONS In this work, we report the synthesis of the most potent and weight-bearing stepping (BMS score 3.0 ± 0.2). In contrast, selective LPA2 antagonist identified to date, compound 54 mice treated with compound 54 displayed occasional or (UCM-14216), with functional Emax and IC50 values of 90% frequent stepping (BMS of 4.1 ± 0.3). We do not discard that and 1.9 μM, respectively, and a KD value of 1.3 nM at LPA2 the therapeutic actions of the compound 54 could be enhanced and functional selectivity against other LPA receptors 10964 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 6. Synthesis of Compounds 53−55a a Reagents and conditions: (a) (i) SOCl2, toluene, 110 °C, 16 h, 99%; (ii) resorcinol, BF3·Et2O, DCM, reflux, 4 h, 10%; (b) acetic anhydride, Et3N, NaOAc, 140 °C, 2.5 h, 71%; (c) conc. HCl, EtOH, reflux, 2 h, 99%; (d) methyl bromoacetate, K2CO3, acetone, reflux, 3 h, 99%; (e) 65% N2H4· H2O, EtOH, reflux, 30 min, 21−99%; (f) 2 M NaOH, EtOH, reflux, 12 h, 61%; (g) bromoacetonitrile, K2CO3, acetone, reflux, 3−5 h, 77−93%; (h) NaN3, NH4Cl, DMF, reflux, 16 h, 52−72%. Table 5. Antagonist Activities of Compounds 13, 53−55, and Ki16425 at LPA1‑3 a Emax = maximum blockade effect of the activation induced by 10 μM of LPA (18:1, 1-oleoyl-sn-glycerol-3-phosphate) at a concentration of the compound under study of 10 μM. bFor Emax > 60%, IC50 values are expressed as mean ± s.e.m, from a minimum of two independent experiments, performed in triplicate. cN.E., no effect was observed at the highest concentration of compound tested (10 μM). 10965 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 5. (A) LPA1 (PDB ID 4Z35)-derived homology model of LPA2 in complex with compounds 13 (in white) and 54 (in turquoise). The tetrazole moiety of compound 54 interacts in a similar manner to the carboxylic acid group of compound 13. Thus, the carboxylic acid forms salt bridges with lysines 22 and 278 (in yellow) and the tetrazole ring establishes hydrogen bonds with these two residues (in blue). The rest key interactions are maintained in both compounds. (B) LPA1-derived homology model of LPA2 in complex with compounds 13 (in white), 53 (in orange), 54 (in turquoise), and 55 (in yellow). The dichlorophenoxy and difluorophenoxy moieties of compounds 13 and 53−55 lie in the same hydrophobic pocket. However, the substitution of chlorine for fluorine provokes a change in the orientation of the aromatic ring and prevents compounds 53 and 55 from reaching residues leucine 111 and alanine 284. Figure 6. (A) Cell surface expression of each mutant LPA2 receptor was assessed by flow cytometry using an anti-HA antibody raised in mice followed by an antimouse antibody conjugated to Alexa 488 in McA-RH7777 cells transiently transfected with mock plasmid (CTL, control) or with plasmids containing the indicated LPA2 mutant receptor. (B) Capacity of compound 54 (10 μM) to block the activation induced by 10 μM of LPA (18:1, 1-oleoyl-sn-glycerol-3-phosphate) in the indicated mutant LPA2 receptor. Values are expressed as mean ± s.e.m, from two independent experiments performed in triplicate. Values obtained for the four point mutations have differences statistically significant (p < 0.01) with respect to the value obtained for the wild type (WT) receptor. #Compound 54 behaved as an agonist of the LPA2 K278A mutant (able to induce 75 ± 5% activation at 10 μM, being the stimulation produced by 10 μM LPA normalized to 100%). (LPA 1,3−6 ). In addition, compound 54 has a good SCI. SCI mainly affects to young and otherwise healthy adults, pharmacokinetic profile both in vitro and in vivo, reaching who suffer from a lack of efficacious treatments. Current pharmacologically relevant levels in the CNS, where the site of treatments are generally palliative, limited to analgesic and action is located. Furthermore, it shows efficacy in an acute in anti-inflammatory drugs, underscoring high medical need for vivo mouse model of SCI being inactive in LPA2 knockout new pharmacological strategies that might be accessed by LPA2 mice undergoing the same model, thus supporting the antagonists to ameliorate SCI physiopathology and improve involvement of LPA2 in the secondary damage that follows neurological outcomes. Further study of UCM-14216 and 10966 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 6. In Vitro Pharmacokinetic Profile of Compounds 13 Analytical thin-layer chromatography (TLC) was run on Supelco and 54 silica gel plates (silica gel 60 F254) with detection by UV light (254 nm) and 5% ninhydrin solution in ethanol or 10% phosphomolybdic stability (t1/2, min)a acid solution in ethanol. Products were purified by flash compd MLMs HLMs P (cm/s)b clogPc chromatography on glass columns using silica gel (60 Å pore size, −6 230−400 mesh particle size from Supelco) or using a Varian 971-FP 13 16 ± 8 61 ± 10 0.1 × 10 4.14 system with cartridges of silica gel (Varian, 50 μm size particle). 54 50 ± 6 97 ± 15 6.1 × 10−6 3.29 All compounds were obtained as oils, except for those whose a Data for stability in mouse and human liver microsomes (MLMs and melting points (mp) are indicated, which were solids. Mp values were HLMs, respectively) are expressed as the mean ± s.e.m. from five determined on a Stuart Scientific electrothermal apparatus. Infrared independent experiments performed in duplicate. bP = permeability (IR) spectra were measured on a Bruker Tensor 27 instrument value; reference values consider P < 10−7 cm/s for low permeability equipped with a Specac ATR accessory of 5200−650 cm −1 compounds and P > 10−5 cm/s for highly permeable molecules. transmission range; frequencies (ν) are expressed in cm−1. c Values obtained with the ACDLabs Percepta software (version 6.0). Nuclear magnetic resonance (NMR) spectra were recorded at rt on a Bruker Avance III 700 MHz (1H, 700 MHz; 13C, 175 MHz), Bruker Table 7. In Vivo Levels of Compound 54 at Different Post- Avance 500 MHz (1H, 500 MHz; 13C, 125 MHz) or Bruker DPX 300 injection Times MHz (1H, 300 MHz; 13C, 75 MHz) instrument at the Universidad Complutense de Madrid (UCM) NMR core facility. 19F-NMR spectra concentration of compound 54 (ng/mg tissue) after the were recorded on a Bruker DPX 300 MHz. Chemical shifts (δ) are indicated postinjection time (h)a expressed in parts per million relative to the residual solvent peak for 1 sample 0.5 1 2 4 H and 13C nucleus (CDCl3: δH = 7.26, δC = 77.2; MeOH-d4: δH = 3.31, δC = 49.0; DMSO-d6: δH = 2.50, δC = 39.5), and coupling plasma 130 ± 50 41 ± 5 38 ± 1 ND constants (J) are in hertz (Hz). The following abbreviations are used spinal cord 0.40 ± 0.06 3.3 ± 0.3 NDb ND to describe peak patterns when appropriate: s (singlet), d (doublet), t brain 0.10 ± 0.02 28 ± 4 0.08 ± 0.01 ND (triplet), q (quadruplet), m (multiplet), br (broad), and app a Mice received a single injection of compound 54 (25 mg/kg, i.p.). (apparent). 2D NMR experiments (COSY, HMQC, and HMBC) Samples were taken at the indicated postinjection times and of representative compounds were carried out to assign protons and immediately frozen, and the levels of compound were then analyzed carbons of new structures; for those carbons displaying very broad by HPLC-MS. Data are the means ± s.e.m. from three independent signals in 13C NMR spectra, the corresponding chemical shifts were samples. bND, not detected. established by their correlation peaks in HSQC and HMBC spectra (Figure S4 shows the numbered structures used in the structural characterization by NMR of all final compounds). High-resolution other related compounds could provide novel approaches to mass spectrometry (HRMS) was carried out on a FTMS Bruker treat SCI and possibly other traumatic CNS injuries. APEX Q IV spectrometer in electrospray ionization (ESI) or matrix- ■ EXPERIMENTAL SECTION Synthesis. Unless stated otherwise, starting materials, reagents, assisted laser desorption ionization (MALDI) mode at UCM’s mass spectrometry facilities. For all final compounds, purity was determined by HPLC-MS and and solvents were purchased as high-grade commercial products from satisfactory chromatograms confirmed a purity of at least 95%. HPLC- Sigma-Aldrich, Alfa Aesar, Acros, Fluka, Panreac or Scharlab, and MS analysis was performed using an Agilent 1200LC-MSD VL were used without further purification. Dichloromethane (DCM) and instrument. LC separation was achieved with an Eclipse XDB-C18 (5 tetrahydrofurane (THF) were dried using a Pure Solv Micro 100 Liter μm, 4.6 mm × 150 mm) or a Zorbax SB-C3 column (5 μm, 2.1 mm × solvent purification system. Triethylamine and pyridine were dried 50.0 mm) together with a guard column (5 μm, 4.6 mm × 12.5 mm). over KOH and distilled prior to its use. All nonaqueous reactions were Mobile phase consisted of A (95:5 water/acetonitrile) and B (5:95 performed under an argon atmosphere in oven-dried glassware unless water/acetonitrile) with 0.1% formic acid as solvent modifier. otherwise stated. MW irradiation reactions were carried out on a Gradients are indicated in Table S1. MS analysis was performed Biotage Initiatior 2.5 reactor, using Biotage vials sealed with an with an ESI source. The capillary voltage was set to 3.0 kV and the aluminum/Teflon crimp top, which can be exposed to a maximum of fragmentor voltage was set at 72 eV. The drying gas temperature was 250 °C and 20 bar internal pressure. 350 °C, the drying gas flow was 10 L/min, and the nebulizer pressure Figure 7. Compound 54 significantly improves locomotor recovery after SCI in LPA2 wild type mice but not in LPA2 deficient mice. Effect of i.p. injection of 54 (25 mg/kg) or vehicle on locomotor recovery in (A) C57bl/6 mice and (B) LPA2 deficient mice quantified using the 0 (absence of movement) to 9 (completely normal locomotor behavior) Basso Mouse Scale (BMS). Data are expressed as mean ± s.e.m. and correspond to seven animals per group in A and five animals per group in B. * p < 0.05 compared with vehicle-treated group (two-way repeated measures ANOVA with Bonferroni’s post hoc test for multiple comparisons). 10967 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article was 20 psi. Spectra were acquired in positive or negative ionization hydrazide derivative was taken to next step (General Procedure 6) mode from 100 to 1000 m/z and in UV-mode at four different without further purification. wavelengths (210, 230, 254, and 280 nm). General Procedure 6: Hydrolysis of Hydrazide Derivatives. To a General Procedure 1: Friedel−Crafts Acylation. (a) Preparation of solution of the corresponding hydrazide obtained according to the aryloxyacetyl chloride: to a solution of the corresponding General Procedure 5 (1 equiv.) in the minimum amount of 96% aryloxyacetic acid (1 equiv.) in anhydrous toluene (5.5 mL/mmol) ethanol was added 2 M NaOH (0.8 mL/mmol), and the reaction was was added thionyl chloride (2.8 mL/mmol) and the reaction mixture refluxed for 12 h. After cooling to rt, the mixture was diluted with was refluxed for 16 h. After this time, the excess of thionyl chloride ethyl acetate and acidified with 1 M HCl until pH 6. The aqueous and toluene were evaporated under reduced pressure, affording the phase was extracted with ethyl acetate (×2) and the combined organic corresponding aryloxyacetyl chloride in quantitative yield. (b) layers were washed with brine, dried over Na2SO4, filtered, and Friedel−Crafts acylation: to a cooled (0 °C) stirred solution of the concentrated under reduced pressure. The residue was purified by corresponding freshly prepared aryloxyacetyl chloride (1 equiv.) and flash chromatography to yield target carboxylic acid derivatives 2, 3, resorcinol (1.1 equiv.) in anhydrous DCM (1.5 mL/mmol), boron 10−13, 36−38, 48, and 53. trifluoride diethyl etherate (1.3 mL/mmol) was added. The reaction General Procedure 7: Synthesis of Tetrazole Derivatives. To a was stirred at 0 °C for 10 min and then at 90 °C until starting material solution of the corresponding nitrile (1 equiv.) in anhydrous DMF was consumed (TLC, 4−5 h). The reaction vessel was then cooled in (15 mL/mmol), NH4Cl (1.5 equiv.), and NaN3 (1.5 equiv.) were an ice bath and the mixture poured into an excess of ice water. The added, and the reaction was refluxed overnight. Then, the mixture was aqueous phase was extracted with DCM (×2), and the combined filtered to remove salts, and the resulting solution was acidified until organic layers were washed with brine, dried over Na2SO4, filtered and pH 3 with 1 M HCl and extracted with ethyl acetate (×2). The concentrated under reduced pressure. The residue was purified by combined organic phases were washed with a 1:1 mixture of water/ flash chromatography to yield the corresponding 2,4-dihydroxyphe- brine, dried with Na2SO4, filtered, and concentrated under reduced nylethanones 6, 14−17, and 56. pressure. The residue was purified by flash chromatography to afford General Procedure 2: Synthesis of Chromones by Kostanecki− the corresponding tetrazoles 54 and 55. Robinson Acylation. A mixture of the corresponding 2,4-dihydrox- [3-Hydroxy-4-(1H-pyrazol-3-yl)phenoxy]acetic Acid (2). Follow- yphenylethanone (1 equiv.), freshly distilled acetic anhydride (0.6 ing general procedures 5 and 6, pyrazole 2 was obtained from mL/mmol), triethylamine (0.8 mL/mmol), and anhydrous sodium chromone 5 (29 mg, 0.12 mmol) in 98% yield. Chromatography: acetate (2.4 equiv.) was stirred at 140 °C until the reaction was DCM to DCM/methanol, 8:2. Mp: 92−94 °C. Rf: 0.79 (DCM/ completed (TLC, 2−3 h). Afterward, cold water was added and the methanol, 7:3). IR (ATR): ν 3404 (OH, NH); 1623 (C�O). 1H mixture was extracted with DCM (×2). The combined organic phases NMR (DMSO-d6, 700 MHz): δ 4.35 (s, 2H, CH2); 6.41−6.43 (m, were washed with brine, dried over Na2SO4, filtered, and concentrated 2H, H2, H6); 6.71 (br s, 1H, H4′); 7.56 (d, J = 8.7, 1H, H5); 7.80 (br under reduced pressure, affording the corresponding acetoxychro- s, 1H, H5′); 11.05 (br s, 1H, OH); 13.13 (br s, 1H, NH). 13C NMR mones 7, 18−21, and 57, which were used without further (DMSO-d6, 175 MHz): δ 66.6 (CH2); 101.3 (C4′); 102.0 (C2); 106.3 purification. (C6); 110.0 (C4); 127.3 (C5); 130.0 (C5′); 150.3 (C3′); 156.3 (C3); General Procedure 3: Hydrolysis of Acetoxychromone Deriva- 159.1 (C1); 171.5 (C�O). HRMS (ESI, m/z): calcd for C11H9N2O4 tives. To a solution of the appropriate acetoxychromone (1 equiv.) in [M-H]−: 233.0568; found: 233.0559. HPLC (method B, tR, min): the minimum amount of absolute ethanol was added conc. HCl (0.6 10.97. mL/mmol), and the reaction was refluxed for 2 h. After cooling to rt, [3-Hydroxy-4-(5-methyl-4-phenoxy-1H-pyrazol-3-yl)phenoxy]- acetic Acid (3). Following general procedures 5 and 6, pyrazole 3 was the mixture was diluted with ethyl acetate and washed with a obtained from chromone 9 (10 mg, 0.03 mmol) in 99% yield. saturated aqueous solution of NaHCO3 and brine. The organic phase Chromatography: DCM to DCM/methanol, 7:3. Mp: >180 °C was dried over Na2SO4, filtered, and concentrated under reduced (decomp.). Rf: 0.92 (DCM/methanol, 7:3). IR (ATR): ν 3440 (OH, pressure, affording the corresponding hydroxychromones 8, 22−25, NH); 1636 (C�O). 1H NMR (methanol-d4, 700 MHz): δ 2.11 (s, and 58, which were used without further purification. 3H, CH3); 4.38 (s, 2H, CH2); 6.32 (dd, J = 8.7, 2.2, 1H, H6); 6.46 (d, General Procedure 4: Alkylation of Hydroxychromone Deriva- J = 2.2, 1H, H2); 6.88−6.91 (m, 2H, H2″, H6″); 6.99 (t, J = 7.4, 1H, tives. To a solution of the corresponding hydroxychromone (1 H4″); 7.26−7.29 (m, 2H, H3″, H5″); 7.56 (d, J = 8.8, 1H, H5). 13C equiv.) in anhydrous acetone (15 mL/mmol) was added K2CO3 (2 NMR (methanol-d4, 175 MHz): δ 8.8 (CH3); 67.9 (CH2); 103.3 equiv), and the reaction mixture was refluxed for 30 min. Then, a (C2); 107.0 (C6); 110.6 (C4); 115.9 (C2″, C6″); 123.2 (C4″); 128.7 solution of the appropriate bromoderivative (1.1−4.3 equiv.) in (C5); 130.8 (C3″, C5″); 133.6 (C4′, C5′); 159.7 (C3); 160.6 (C1, C1″); anhydrous acetone (1 mL/mmol) was added and the mixture was 177.2 (C�O); C3′ not observed. HRMS (ESI, m/z): calcd for refluxed until consumption of starting material (TLC, 3−5 h). Next, C18H15N2O5 [M-H]−: 339.0986; found: 339.0975. HPLC (method B, cold water was added and acetone was removed under reduced tR, min): 21.34. pressure. The aqueous residue was extracted with DCM (×2), and the {4-[4-(3-Chlorophenoxy)-5-methyl-1H-pyrazol-3-yl]-3- combined organic phases were washed with brine, dried with Na2SO4, hydroxyphenoxy}acetic Acid (10). Following the general procedures filtered, and concentrated under reduced pressure. The residue was 5 and 6, pyrazole 10 was obtained from chromone 26 (49 mg, 0.64 purified by flash chromatography to afford the corresponding mmol) in 67% yield. Chromatography: ethyl acetate to ethyl acetate/ alkylated chromones 5, 9, 26−29, 40, 41, 46, 51, 59, 60, and 61. methanol, 8:2. Mp: >240 °C (decomp.). Rf: 0.18 (ethyl acetate/ General Procedure 5: Synthesis of Pyrazole Derivatives by methanol, 8:2). IR (ATR): ν 3346 (OH, NH); 1585 (C�O). 1H Reaction with Hydrazine. A solution of the corresponding chromone NMR (methanol-d4, 700 MHz): δ 2.12 (s, 3H, CH3); 4.42 (s, 2H, or enaminone (1 equiv.) in absolute ethanol (5 mL/mmol) at 40 °C CH2); 6.35 (dd, J = 8.8, 2.5, 1H, H6); 6.47 (d, J = 2.5, 1H, H2); 6.83 was treated with a solution of hydrazine monohydrate (65%, 0.18 (ddd, J = 8.4, 2.3, 0.6, 1H, H6″); 6.90 (t, J = 2.2, 1H, H2″); 7.00 (dd, J mL/mmol) in absolute ethanol (1.3 mL/mmol), and the mixture was = 7.9, 1.5, 1H, H4″); 7.25 (t, J = 8.2, 1H, H5″); 7.50 (d, J = 8.7, 1H, refluxed until the reaction was completed (TLC, 0.5−2 h). After H5). 13C NMR (methanol-d4, 175 MHz): δ 8.7 (CH3); 67.5 (CH2); cooling to rt, the mixture was concentrated under reduced pressure 103.5 (C2); 107.1 (C6); 110.5 (C4); 114.5 (C6″); 116.4 (C2″); 123.4 and the residue was dissolved with ethyl acetate and acidified with 1 (C4″); 128.7 (C5); 132.0 (C5″); 133.2 (C4′/C5′), 135.3 (C4′/C5′); M HCl until pH 6. The aqueous phase was extracted with ethyl 136.3 (C3″); 140.7 (C3′); 158.1 (C3); 160.5, 160.6 (C1, C1″); 176.6 acetate (×2), and the combined organic layers were washed with (C�O). HRMS (ESI, m/z): calcd for C18H14ClN2O5 [M-H]−: brine, dried over Na2SO4, filtered, and concentrated under reduced 373.0597; found: 373.0577. HPLC (method B, tR, min): 14.21. pressure. The residue was purified by flash chromatography to yield {4-[4-(4-Chlorophenoxy)-5-methyl-1H-pyrazol-3-yl]-3- the corresponding pure pyrazoles 33, 42, 45, 62, and 63. For those hydroxyphenoxy}acetic Acid (11). Following general procedures 5 chromones or enaminones bearing an ester group, the resulting and 6, pyrazole 11 was obtained from chromone 27 (14 mg, 0.04 10968 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article mmol) in 99% yield. Chromatography: DCM to DCM/methanol, 8:2. = 6.5, 2H, OCH2); 6.36 (dd, J = 8.8, 2.6, 1H, H6); 6.53 (d, J = 2.6, Mp: 121−123 °C. Rf: 0.85 (DCM/methanol, 7:3). IR (ATR): ν 1627 1H, H2); 6.59 (d, J = 8.9, 1H, H6″); 7.03 (dd, J = 8.9, 2.6, 1H, H5″); (C�O). 1H NMR (methanol-d4, 700 MHz): δ 2.22 (s, 3H, CH3); 7.45 (d, J = 2.5, 1H, H3″); 7.56 (d, J = 8.7, 1H, H5). 13C NMR 4.69 (s, 2H, CH2); 6.42 (dd, J = 8.9, 2.5, 1H, H6); 6.51 (d, J = 2.5, (methanol-d4, 175 MHz): δ 8.6 (CH3); 34.5 (CH2CO); 63.4 1H, H2); 6.96 (d, J = 9.0, 2H, H2″, H6″); 7.30 (d, J = 9.0, 2H, H3″, (OCH2); 102.6 (C2); 106.8 (C6); 109.0 (C4); 115.7 (C6″); 123.6 H5″); 7.62 (d, J = 8.8, 1H, H5). 13C NMR (methanol-d4, 175 MHz): δ (C2″); 127.6 (C4″); 127.7 (C5); 128.0 (C5″); 130.5 (C3″); 133.0, 8.5 (CH3); 65.9 (CH2); 103.3 (C2); 107.3 (C6); 108.3 (C4); 117.7 135.0 (C4′, C5′); 140.0 (C3′); 152.4 (C1″); 158.0 (C3); 160.0 (C1); (C2″, C6″); 128.9 (C4″); 130.3 (C5); 130.9 (C3″, C5″); 134.1, 137.3 174.0 (C�O). HRMS (ESI, m/z): calcd for C19H15Cl2N2O5 [M- (C4′, C5′); 138.3 (C3′); 157.6 (C1″); 158.1 (C3); 161.6 (C1); 171.0 H]−: 421.0364; found: 421.0370. HPLC (method B, tR, min): 23.38. (C�O). HRMS (ESI, m/z): calcd for C18H14ClN2O5 [M-H]−: 4-{4-[4-(2,4-Dichlorophenoxy)-5-methyl-1H-pyrazol-3-yl]-3- 373.0591; found: 373.0577. HPLC (method B, tR, min): 23.78. hydroxyphenoxy}butanoic Acid (37). Following general procedures {4-[4-(3,4-Dichlorophenoxy)-5-methyl-1H-pyrazol-3-yl]-3- 5 and 6, pyrazole 37 was obtained from chromone 40 (12 mg, 0.03 hydroxyphenoxy}acetic Acid (12). Following general procedures 5 mmol) in 99% yield. Chromatography: ethyl acetate to ethyl acetate/ and 6, pyrazole 12 was obtained from chromone 28 (38 mg, 0.09 methanol, 8:2. Rf: 0.56 (ethyl acetate/methanol, 1:1). IR (ATR): ν mmol) in 92% yield. Chromatography: ethyl acetate to ethyl acetate/ 3431 (NH/OH); 1669 (C�O); 1263 (C−O−C). 1H NMR methanol, 8:2. Mp: >240 °C (decomp.). Rf: 0.10 (ethyl acetate/ (methanol-d4, 700 MHz): δ 2.00−2.04 (m, 2H, CH2); 2.13 (s, 3H, methanol, 9:1). IR (ATR): ν 3406 (OH, NH); 1586 (C�O); 1258, CH3); 2.43 (t, J = 7.4, 2H, CH2CO); 3.96 (t, J = 6.3, 2H, OCH2); 1174 (C−O−C). 1H NMR (methanol-d4, 700 MHz): δ 2.13 (s, 3H, 6.35 (dd, J = 8.7, 2.6, 1H, H6); 6.45 (d, J = 2.5, 1H, H2); 6.69 (d, J = CH3); 4.41 (s, 2H, CH2); 6.37 (dd, J = 8.7, 2.5, 1H, H6); 6.47 (d, J = 8.9, 1H, H6″); 7.14 (dd, J = 8.9, 2.5, 1H, H5″); 7.48 (d, J = 8.8, 1H, 2.5, 1H, H2); 6.86 (dd, J = 9.0, 3.0, 1H, H6″); 7.06 (d, J = 3.0, 1H, H5); 7.52 (d, J = 2.5, 1H, H3″). 13C NMR (methanol-d4, 175 MHz): δ H2″); 7.41 (d, J = 9.0, 1H, H5″); 7.46 (d, J = 8.7, 1H, H5). 13C NMR 7.9 (CH3); 26.1 (CH2); 31.7 (CH2CO); 68.1 (OCH2); 103.2 (C2); (methanol-d4, 175 MHz): δ 8.6 (CH3); 67.6 (CH2); 103.5 (C2); 107.1 (C6); 109.1 (C4); 116.8 (C6″); 124.1 (C2″); 128.3 (C5, C4″); 107.2 (C6); 110.3 (C4); 116.2 (C6″); 118.1 (C2″); 126.4 (C4″); 128.7 129.1 (C5″); 131.1 (C3″); 133.1, 134.2 (C4′, C5′); 154.0 (C1″); 158.2 (C5); 132.4 (C5″); 133.1 (C4′/C5′); 134.1 (C4′/C5′, C3″); 158.9 (C3, (C3); 161.3 (C1); 178.1 (C�O); C3′ not observed. MS (ESI, m/z): C1″); 160.8 (C1); 176.9 (C�O); C3′not observed. HRMS (ESI, m/ 435.0, 437.1, 439.0 [M-H]−. HPLC (method B, tR, min): 23.33. z): calcd for C18H13Cl2N2O5 [M-H]−: 407.0207; found: 407.0198. 5-{4-[4-(2,4-Dichlorophenoxy)-5-methyl-1H-pyrazol-3-yl]-3- HPLC (method B, tR, min): 15.86. hydroxyphenoxy}pentanoic Acid (38). Following general procedures {4-[4-(2,4-Dichlorophenoxy)-5-methyl-1H-pyrazol-3-yl]-3- 5 and 6, pyrazole 38 was obtained from chromone 41 (140 mg, 0.30 hydroxyphenoxy}acetic Acid (13). Following general procedures 5 mmol) in 86% yield. Chromatography: hexane/ethyl acetate 9:1 to and 6, pyrazole 13 was obtained from chromone 29 (45 mg, 0.11 hexane/ethyl acetate 1:1. Rf: 0.64 (ethyl acetate/methanol, 8:2). IR mmol) in 99% yield. Chromatography: DCM to DCM/methanol, 8:2. (ATR): ν 2923, 2852 (NH, OH); 1703 (C�O); 1242, 1191 (C−O− Mp: >196 °C (decomp.). Rf: 0.93 (DCM/methanol/acetic acid, C). 1H NMR (methanol-d4, 700 MHz): δ 1.74−1.78 (m, 4H, 2CH2); 7:3:0.01). IR (ATR): ν 3413 (OH, NH); 1619 (C�O); 1182 (C− 2.13 (s, 3H, CH3); 2.35 (t, J = 6.7, 2H, COCH2); 3.94 (t, J = 5.5, 2H, O−C). 1H NMR (methanol-d4, 300 MHz): δ 2.14 (s, 3H, CH3); 4.60 OCH2); 6.34 (dd, J = 8.7, 2.6, 1H, H6); 6.44 (d, J = 2.5, 1H, H2); 6.68 (s, 2H, CH2); 6.36 (dd, J = 8.7, 2.5, 1H, H6); 6.47 (d, J = 2.5, 1H, (d, J = 8.9, 1H, H6″); 7.14 (dd, J = 8.9, 2.5, 1H, H5″); 7.48 (d, J = 8.8, H2); 6.68 (d, J = 8.9, 1H, H6″); 7.14 (dd, J = 8.9, 2.5, 1H, H5″); 7.49− 1H, H5); 7.52 (d, J = 2.5, 1H, H3″). 13C NMR (methanol-d4, 175 7.53 (m, 2H, H5, H3″). 13C NMR (methanol-d4, 125 MHz): δ 8.7 MHz): δ 8.3 (CH3); 22.8, 29.8 (2CH2); 34.7 (COCH2); 68.5 (CH3); 66.5 (CH2); 103.5 (C2); 107.1 (C6); 110.6 (C4); 116.8 (C6″); (OCH2); 103.2 (C2); 107.1 (C6); 109.8 (C4); 116.8 (C6″); 124.1 124.1 (C2″); 128.3 (C4″); 128.6 (C5); 129.1 (C5″); 131.1 (C3″); (C2″); 128.3 (C5); 128.6 (C4″); 129.1 (C5″); 131.1 (C3″); 133.2 (C5′); 133.2, 134.3 (C4′, C5′); 139.7 (C3′); 153.9 (C1″); 158.1 (C3); 160.4 135.5 (C4′); 140.4 (C3′); 154.0 (C1″); 156.0 (C3); 161.4 (C1); 177.5 (C1); 173.8 (C�O). HRMS (ESI, m/z): calcd for C18H13Cl2N2O5 (C�O). HRMS (ESI, m/z): calcd for C21H19Cl2N2O5 [M-H]−: [M-H]−: 407.0207; found: 407.0192. HPLC (method B, tR, min): 449.0749; found: 449.0741. HPLC (method B, tR, min): 18.34. 14.90. 4-[4-(2,4-Dichlorophenoxy)-5-methyl-1H-pyrazol-3-yl]benzene- 4-(2,4-Dichlorophenoxy)-3-(4-carboxymethoxyphenyl)-5-meth- 1,3-diol (42). Following general procedure 5, pyrazole 42 was yl-1H-pyrazole (30). A mixture of pyrazole 35 (10 mg, 0.02 mmol, 1 obtained from chromone 25 (82 mg, 0.24 mmol) in 65% yield. Mp: equiv.) and 1 M NaOH (30 μL, 0.03 mmol, 1.5 equiv.) in 1,4-dioxane 206−208 °C. Rf: 0.23 (ethyl acetate/methanol, 6:4). IR (ATR): ν (1 mL) was stirred at 60° for 2 h. After cooling to rt, the mixture was 3293 (OH, NH); 1250 (C−O−C). 1H NMR (CDCl3, 700 MHz): δ treated with 1 M HCl until acidic pH and extracted with ethyl acetate 2.12 (s, 3H, CH3); 6.21 (dd, J = 8.5, 2.1, 1H, H6); 6.34 (d, J = 2.0, (2×). The combined organic phases were washed with brine, dried 1H, H2); 6.66 (d, J = 8.9, 1H, H6″); 7.11 (dd, J = 8.9, 2.4, 1H, H5″); over Na2SO4, filtered, and concentrated under reduced pressure. The 7.40 (d, J = 8.6, 1H, H5); 7.50 (d, J = 2.4, 1H, H3″). 13C NMR crude was purified by flash chromatography (ethyl acetate/methanol, (CDCl3, 175 MHz): δ 8.7 (CH3); 103.9 (C2); 108.0 (C6); 108.9 8:2 to ethyl acetate/methanol, 7:3) to yield pure compound 30 in (C4); 116.8 (C6″); 124.1 (C2″); 128.3 (C4″); 128.6 (C5); 129.1 (C5″); 99% yield. Mp: 158−160 °C. Rf: 0.13 (ethyl acetate/methanol, 7:3). 130.9 (C4′/C5′); 131.1 (C3″); 133.0 (C4′/C5′); 153.9 (C3′); 158.1 IR (ATR): ν 3321 (NH/OH); 1649 (C�O); 1020 (C−O−C). 1H (C1″); 159.6 (C1, C3). HRMS (ESI, m/z): calcd for C16H13Cl2N2O3 NMR (methanol-d4, 700 MHz): δ 2.11 (s, 3H, CH3); 4.63 (s, 2H, [M + H]+: 351.0298; found: 351.0303. HPLC (method B, tR, min): CH2); 6.66 (d, J = 8.9, 1H, H6″); 6.92 (d, J = 9.0, 2H, H2, H6); 7.12 21.80. (dd, J = 8.9, 2.5, 1H, H5″); 7.50 (d, J = 2.5, 1H, H3″); 7.62 (d, J = 9.0, 2-[4-(2,4-Dichlorophenoxy)-5-methyl-1H-pyrazol-3-yl]-5-me- 2H, H3, H5). 13C NMR (methanol-d4, 175 MHz): δ 9.2 (CH3); 66.1 thoxyphenol (43). A mixture of pyrazole 42 (25 mg, 0.07 mmol, 1 (CH2); 115.9 (C2, C6); 116.7 (C6″); 124.1 (C2″); 124.4 (C4); 128.2 equiv.), potassium carbonate (21 mg, 0.15 mmol, 2.1 equiv.) and (C3, C5); 128.3 (C4″); 129.1 (C5″); 131.1 (C3″); 133.7, 137.5 (C4′, iodomethane (10 μL, 0.13 mmol, 1.8 equiv.) in acetone (0.84 mL) C5′); 139.0 (C3′); 154.1 (C1″); 159.5 (C1); 173.0 (C�O). HRMS was stirred at 65 °C for 6 h. After cooling to rt, the solvent was (ESI, m/z): calcd for C18H13Cl2N2O4 [M-H]−: 391.0258; found: evaporated under reduced pressure and the residue was dissolved in 391.0247. HPLC (method A, tR, min): 17.53. ethyl acetate and washed with water and brine. The organic phase was 3-{4-[4-(2,4-Dichlorophenoxy)-5-methyl-1H-pyrazol-3-yl]-3- dried over Na2SO4, filtered, and concentrated under reduced pressure. hydroxyphenoxy}propanoic Acid (36). Following general procedures The crude was purified by flash chromatography (hexane to hexane/ 5 and 6, pyrazole 36 was obtained from chromone 39 (13 mg, 0.03 ethyl acetate, 7:3) to yield pure compound 43 in 20% yield. Mp: mmol) in 97% yield. Chromatography: ethyl acetate/methanol, 8:2. 220−222 °C. Rf: 0.50 (ethyl acetate/methanol, 6:4). IR (ATR): ν Rf: 0.83 (ethyl acetate/methanol, 8:2). IR (ATR): ν 2955, 2941 (NH, 3352 (OH, NH); 1252 (C−O−C). 1H NMR (methanol-d4, 700 OH); 1653 (C�O); 1223 (C−O−C). 1H NMR (methanol-d4, 700 MHz): δ 2.15 (s, 3H, C5′CH3); 3.85 (s, 3H, OCH3); 6.18 (dd, J = 8.6, MHz): δ 2.20 (s, 3H, CH3); 2.78 (t, J = 6.5, 2H, CH2CO); 4.22 (t, J 2.5, 1H, H4); 6.31 (d, J = 2.4, 1H, H6); 6.68 (d, = 8.9, 1H, H6″); 7.14 10969 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article (dd, J = 8.9, 2.5, 1H, H5″); 7.37 (d, J = 8.6, 1H, H3); 7.53 (d, J = 2.5, (ATR): ν 3187 (OH, NH); 1627 (C�O); 1226 (C−O−C). 1H 1H, H3″). 13C NMR (methanol-d4, 175 MHz): δ 8.1 (C5′CH3); 52.1 NMR (methanol-d4,, 500 MHz): δ 4.42 (s, 2H, CH2); 6.39 (dd, J = (OCH3); 104.0 (C6); 107.9 (C4); 109.1 (C2); 116.8 (C6″); 124.1 8.8, 2.5, 1H, H6); 6.49 (d, J = 2.5, 1H, H2); 6.84 (d, J = 8.9, 1H, H6″); (C2″); 128.35 (C3); 128.4 (C4″); 129.1 (C5″); 131.1 (C3″); 132.8, 7.16 (dd, J = 8.9, 2.5, 1H, H5″); 7.50 (d, J = 2.5, 1H, H3″); 7.57 (d, J = 133.3 (C4′, C5′); 141.6 (C3′); 153.9 (C1″); 158.4, 159.5 (C1, C5). 8.7, 1H, H5); 7.61 (s, 1H, H5′). 13C NMR (methanol-d4, 125 MHz): δ HRMS (ESI, m/z): calcd for C17H15Cl2N2O3 [M + H]+: 365.0454; 67.8 (CH2); 103.5 (C2); 107.2 (C6); 110.1 (C4); 117.8 (C6″); 124.7 found: 365.0458. HPLC (method B, tR, min): 21.90. (C2″); 128.7 (C4″); 128.9 (C5, C5′); 129.1 (C5″); 131.1 (C3″); 136.6 Methyl {4-[4-(2,4-dichlorophenoxy)-5-methyl-1H-pyrazol-3-yl]- (C4′); 154.4 (C1″); 157.9 (C3); 160.8 (C1); 176.5 (C�O); C3′ not 3-hydroxyphenoxy} Acetate (44). To a solution of pyrazole 13 observed. HRMS (ESI, m/z): calcd for C17H11Cl2N2O5 [M-H]−: (293 mg, 0.94 mmol) in methanol (3.70 mL) was added conc. H2SO4 393.0050; found: 393.0017. HPLC (method B, tR, min): 21.11. (31 μL), and the reaction was refluxed overnight. The mixture was {4-[4-(2,4-Dichlorophenoxy)-3-methyl-1,2-oxazol-3-yl]-3- diluted with ethyl acetate, washed with water and brine, dried over hydroxyphenoxy}acetic Acid (49). To a solution of isoxazole 52 (7 Na2SO4, filtered, and concentrated under reduced pressure. The crude mg, 0.02 mmol, 1 equiv.) in the minimum amount of 1,4-dioxane was was purified by flash chromatography (hexane/ethyl acetate, 9:1 to added 1 M NaOH (0.20 mL, 0.20 mmol, 10 equiv.), and the reaction hexane/ethyl acetate, 7:3) to yield pure compound 44 in 85% yield. was heated at 60 °C overnight. After cooling to rt, the solvent was Mp: >210 °C (decomp.). Rf: 0.98 (DCM/methanol/acetic acid, removed under reduced pressure. The residue was dissolved with 8:2:0.1). IR (ATR): ν 3297 (OH, NH); 1634 (C�O); 1016 (C−O− ethyl acetate and washed with a 1 M HCl solution and brine, dried C). 1H NMR (methanol-d4, 500 MHz): δ 2.21 (s, 3H, C5′CH3); 3.77 over Na2SO4, filtered, and concentrated under reduced pressure, (s, 3H, OCH3); 4.69 (s, 2H, CH2); 6.41 (dd, J = 8.7, 2.6, 1H, H6); affording the corresponding isoxazole 49 in 91% yield. Mp: >210 °C 6.49 (d, J = 2.5, 1H, H2); 6.75 (d, J = 8.9, 1H, H6″); 7.17 (dd, J = 8.9, (decomp.). Rf: 0.15 (ethyl acetate/methanol/acetic acid, 7:3:0.01). IR 2.5, 1H, H5″); 7.55 (d, J = 8.8, 1H, H5); 7.57 (d, J = 2.5, 1H, H3″). 13C (ATR): ν 2919 (OH); 1729 (C�O); 1175 (C−O−C). 1H NMR NMR (methanol-d4, 125 MHz): δ 8.5 (C5′CH3); 52.6 (OCH3); 65.9 (methanol-d4, 500 MHz): δ 2.13 (s, 3H, CH3); 4.63 (s, 2H, OCH2); (CH2); 103.4 (C2); 107.2 (C6); 108.8 (C4); 116.9 (C6″); 124.3 (C2″); 6.41 (d, J = 2.4, 1H, H2); 6.51 (dd, J = 8.7, 2.5, 1H, H6); 6.87 (d, J = 129.0 (C4″); 129.3 (C5); 129.7 (C5″); 131.3 (C3″); 133.6, 137.5 (C4′, 8.9, 1H, H6″); 7.17 (dd, J = 8.9, 2.5, 1H, H5″); 7.38 (d, J = 8.7, 1H, C5′); 142.2 (C3′); 153.3 (C1″); 158.2 (C3); 161.3 (C1); 171.0 (C� H5); 7.47 (dd, J = 2.5, 1H, H3″). 13C NMR (methanol-d4, 125 MHz): O). HRMS (ESI, m/z): calcd for C19H15Cl2N2O5 [M-H]−: 421.0363; δ 9.2 (CH3); 65.9 (OCH2); 103.2 (C2); 107.3 (C6); 108.5 (C4); found: 421.0343. HPLC (method B, tR, min): 26.27. 117.6 (C6″); 124.4 (C2″); 128.7 (C4″); 128.8 (C5″); 131.0 (C5); 131.1 2-{4-[4-(2,4-Dichlorophenoxy)-5-methyl-1H-pyrazol-3-yl]-3- (C3″); 132.2 (C4′); 153.4 (C1″); 156.7 (C5′); 158.1, 158.9 (C3, C3′); hydroxyphenoxy}acetamide (45). Following the general procedure 162.5 (C1); 172.4 (C�O). HRMS (MALDI, m/z): calcd for 5, pyrazole 45 was obtained from chromone 46 (24 mg, 0.06 mmol) C18H13Cl2NO6 [M]+: 409.0120; found: 409.0127. HPLC (method B, in 80% yield. Rf: 0.50 (ethyl acetate/methanol, 9:1). IR (ATR): ν tR, min): 11.48. 3413 (OH, NH); 1619 (C�O); 1182 (C−O−C). 1H NMR {4-[4-(2,4-Difluorophenoxy)-5-methyl-1H-pyrazol-3-yl]-3- (methanol-d4, 700 MHz): δ 2.14 (s, 3H, CH3); 4.44 (s, 2H, CH2); hydroxyphenoxy}acetic Acid (53). Following general procedures 5 6.42 (d, J = 8.6, 1H, H6); 6.53 (d, J = 2.5, 1H, H2); 6.68 (d, J = 8.9, and 6, compound 53 was obtained from chromone 59 (65 mg, 0.17 1H, H6″); 7.14 (dd, J = 8.9, 2.4, 1H, H5″); 7.52 (d, J = 8.7, 1H, H5); mmol) in 61% yield. Chromatography: ethyl acetate to ethyl acetate/ 7.52 (d, J = 2.4, 1H, H3″). 13C NMR (methanol-d4, 175 MHz): δ 7.9 methanol 8:2. Mp: >210 °C (decomp.). Rf: 0.15 (ethyl acetate/ (CH3); 67.8 (CH2); 103.6 (C2); 107.1 (C6); 110.9 (C4); 116.7 (C6″); methanol 8:2). IR (ATR): ν 3400 (OH); 1620 (C�O); 1246, 1190 124.1 (C2″); 128.4 (C4″, C5); 129.1 (C5″); 131.1 (C3″); 133.2, 136.3 (C−O−C). 1H NMR (methanol-d4, 700 MHz): δ 2.14 (s, 3H, CH3); (C5′, C4′); 142.5 (C3′); 153.9 (C1″); 158.3 (C3); 159.8 (C1); 173.9 4.43 (s, 2H, CH2); 6.37 (dd, J = 9.0, 2.5, 1H, H6); 6.47 (d, J = 2.5, (C�O). HRMS (ESI, m/z): calcd for C18H14Cl2N3O4 [M-H]−: 1H, H2); 6.71 (td, J = 9.2, 5.3, 1H, H6″); 6.74−6.77 (m, 1H, H5″); 406.0440; found: 406.0356. HPLC (method B, tR, min): 15.39. 7.09 (ddd, J = 11.5, 9.0, 3.0, 1H, H3″); 7.53 (d, J = 8.5, 1H, H5). 13C {4-[4-(2,4-Dichlorophenoxy)-1,5-dimethyl-1H-pyrazol-3-yl]-3- NMR (methanol-d4, 175 MHz): δ 8.6 (CH3); 67.5 (CH2); 103.4 hydroxyphenoxy}acetic Acid (47). To a solution of pyrazole 13 (66 (C2); 106.0 (dd, J = 27.5, 22.0, C3″); 107.1 (C6); 110.5 (C4); 111.8 mg, 0.16 mmol, 1 equiv.) in anhydrous THF (0.70 mL) was added (dd, J = 23.0, 3.5, C5″); 117.2 (d, J = 9.5, C6″); 128.6 (C5); 133.5, sodium hydride (60% dispersion in mineral oil, 8 mg, 0.32 mmol, 2 1135.7 (C4′, C5′); 140.5 (C3′); 143.9 (dd, J = 10.5, 3.0, C1″); 153.0 equiv.) at 0 °C and the mixture was stirred for 3 h, allowing it to reach (dd, J = 248.5, 12.0, C2″); 158.1 (C3); 158.5 (dd, J = 241.5, 10.0, rt during this time. Next, iodomethane (20 μL, 0.16 mmol, 1 equiv.) C4″); 160.7 (C1); 176.3 (C�O). HRMS (ESI, m/z): calcd for was added and the reaction was stirred at rt for 16 h. After this time, 1 C18H13F2N2O5 [M-H]−: 375.0798; found: 375.0792. HPLC (method M HCl was added until neutral pH and the organic solvent was B, tR, min): 13.33. evaporated. The residue was extracted with ethyl acetate (3×). The 2-[4-(2,4-Dichlorophenoxy)-5-methyl-1H-pyrazol-3-yl]-5-[1H-tet- combined organic phases were washed with brine, dried over Na2SO4, razol-5-yl)methoxy] Phenol (54). Following general procedure 7, filtered, and concentrated under reduced pressure to obtain pure compound 54 was obtained from pyrazole 62 (262 mg, 0.70 mmol) in compound 47 in 31% yield. Chromatography: hexane/ethyl acetate, 72% yield. Chromatography: ethyl acetate/methanol, 9:1 to ethyl 4:6 to ethyl acetate. Rf: 0.16 (ethyl acetate/methanol/acetic acid, acetate/methanol, 6:4. Mp: 180−182 °C. Rf: 0.19 (ethyl acetate/ 9:1:0.01). IR (ATR): ν 2929 (OH); 1629 (C�O); 1261 (C−O−C). methanol, 8:2). IR (ATR): ν 2921 (NH, OH); 1238 (C−O−C). 1H 1 H NMR (methanol-d4, 700 MHz): δ 2.16 (s, 3H, C5′CH3); 3.86 (s, NMR (methanol-d4, 300 MHz): δ 2.14 (s, 3H, CH3); 5.40 (s, 2H, 3H, NCH3); 4.49 (s, 2H, CH2); 6.34 (dd, J = 8.8, 2.5, 1H, H6); 6.46 CH2); 6.49 (dd, J = 8.7, 2.6, 1H, H4); 6.59 (d, J = 2.6, 1H, H6); 6.68 (d, J = 2.5, 1H, H2); 6.67 (d, J = 8.9, 1H, H6″); 7.13 (dd, J = 8.9, 2.5, (d, J = 8.9, 1H, H6″); 7.14 (dd, J = 8.9, 2.5, 1H, H5″); 7.52−7.56 (m, 1H, H5″); 7.46 (d, J = 2.5, 1H, H5); 7.53 (d, J = 8.7, 1H, H3″). 13C 2H, H3, H3″). 13C NMR (methanol-d4, 75 MHz): δ 8.5 (CH3); 60.8 NMR (methanol-d4, 175 MHz): δ 8.2 (C5′CH3); 37.2 (NCH3); 67.2 (CH2); 103.7 (C6); 107.0 (C4); 111.4 (C2); 116.8 (C6″); 124.1 (C2″); (CH2); 103.5 (C2); 107.2 (C6); 110.5 (C4); 116.7 (C6″); 124.0 (C2″); 128.4 (C4″); 128.8 (C5″); 129.1 (C3); 131.1 (C3″); 133.3, 134.9 (C4′, 128.2 (C4″); 128.5 (C5″); 129.2 (C5); 131.2 (C3″); 133.0, 133.4 (C5′, C5′); 140.2 (C3′); 153.9 (C1″); 156.9 (Ctetrazole); 158.3 (C1); 159.8 C4′); 141.2 (C3′); 153.8 (C1″); 158.3 (C3); 160.6 (C1); 172.9 (C� (C5). HRMS (MALDI, m/z): calcd for C18H15Cl2N6O3 [M + H]+: O). HRMS (MALDI, m/z): calcd for C19H16Cl2N2O5 [M]+: 433.0504; found: 433.0506. HPLC (method C, tR, min): 10.38. 422.0436; found: 422.0433. HPLC (method B, tR, min): 16.60. 2-[4-(2,4-Difluorophenoxy)-5-methyl-1H-pyrazol-3-yl]-5-[1H-tet- {4-[4-(2,4-Dichlorophenoxy)-1H-pyrazol-3-yl]-3- razol-5-yl)methoxy] Phenol (55). Following general procedure 7, hydroxyphenoxy}acetic Acid (48). Following general procedures 5 compound 55 was obtained from pyrazole 63 (344 mg, 0.96 mmol) in and 6, pyrazole 48 was obtained from chromone 51 (27 mg, 0.07 52% yield. Chromatography: ethyl acetate/methanol, 9:1 to ethyl mmol) in 58% yield. Chromatography: ethyl acetate/methanol, 9:1 to acetate/methanol, 6:4. Mp: >210 °C (decomp.). Rf: 0.47 (hexane/ ethyl acetate/methanol, 8:2. Rf: 0.10 (ethyl acetate/methanol, 7:3). IR ethyl acetate, 4:6). IR (ATR): ν 2931 (OH, NH); 1252, 1190 (C− 10970 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article O−C). 1H NMR (methanol-d4, 300 MHz): δ 2.15 (s, 3H, CH3); 5.40 concentration was maintained at 25 μg/mL with 0, 0.08, 0.4, 2, 10, 50, (s, 2H, OCH2); 6.51 (dd, J = 8.7, 2.6, 1H, H4); 6.59 (d, J = 2.6, 1H, and 250 nM concentrations of the compound in a final buffer H6); 6.72−6.77 (m, 2H, H5″, H6″); 7.10 (td, J = 9.5, 8.3, 2.5, 1H, composition of 0.25% DMSO/PBS. Receptor-compound mixture was H3″); 7.60 (d, J = 8.6, 1H, H3). 13C NMR (methanol-d4, 75 MHz): δ incubated at rt for about an hour on a shaker and then filled in a 8.5 (CH3); 60.8 (OCH2); 103.7 (C6); 106.0 (dd, J = 27.5, 22.0, C3″); dropix sample well tray in the format of reference then sample and 107.0 (C4); 110.4 (C2); 111.7 (dd, J = 26.4, 3.3, C5″); 117.2 (dd, J = finally introduced to CIR using an automated Mitos Dropix 9.5, 1.9, C6″); 128.8 (C3); 133.7, 134.6 (C4′, C5′); 141.2 (C3′); 143.8 (Dolomite Microfluidics, UK) sample introducer. The detailed (dd, J = 11.1, 3.2, C1″); 153.1 (dd, J = 248.7, 12.2, C2″); 155.3 description of the CIR was mentioned elsewhere.31,32 It is a benchtop (Ctetrazole); 158.3 (C1); 158.6 (dd, J = 241.8, 10.2, C4″); 159.7 (C5). RI reader that combined a compensated interferometer with a Mitos 19 F-NMR (methanol-d4, 282 MHz): −132.5, −120.5. HRMS (ESI, Dropix (an automated droplet generator) and a syringe pump. The m/z): calcd for C18H15F2N6O3 [M + H]+: 401.1095; found: 401.1105. compensated interferometer, which consisted of a diode laser, one or HPLC (method C, tR, min): 9.20. two mirrors, one glass capillary, and a CCD camera, measures the RI Evaluation of Receptor Activation by Ca2+ Mobilization change from a solution undergoing conformational and/or hydration Assay. Cells stably expressing the corresponding LPA1−3 receptor alteration compared to a reference (with no such binding events). were grown as described previously.18 Changes in intracellular ΔRI is measured by capturing the translational shifts in backscattered calcium levels were measured by using the fluorescent calcium light interference fringes produced from the interaction between an sensitive dye Fluo-4 NW (Invitrogen). RH7777 cells or B103 cells expanded beam profile of the laser and a capillary filled with droplets were plated on poly-D-lysine or collagen coated, respectively, black- of sample-reference solutions. The positional shift of the back- wall clear-bottom 96-well plates (Corning) at a density of 50 000 scattered fringes, which is equivalent to molecular interaction, was cells/well and cultured overnight. The culture medium was then quantified using fast Fourier transform of selected bright fringes replaced with 100 μL of Fluo-4 NW dye loading solution containing captured in a CCD camera. The data acquisition and analysis were 2.5 μM of probenecid and incubated for 30 min at 37 °C followed by performed using a LabVIEW interface designed at the laboratory. an additional 30 min at rt. Then, 20 μL of the test compound from a In Silico Experiments. Docking calculations were performed 6× stock solution in assay buffer were added and fluorescence was using Autodock433 [using: ga_num_evals (depending on the number measured during 120 s after which 10 μM of LPA (18:1, 1-oleoyl-sn- of rotatable bonds) = 6 310 000 (for compounds 3, 13, 53, 54, 55) glycerol-3-phosphate) was added and wells were monitored for and 25 000 000 (for compound 38), ga_run = 100 and all the other additional 120 s. Fluorescence changes were registered in a FluoStar parameters set to their default values]. The LPA2 receptor model was Optima instrument (BMG Labtech) at 525 nm using an excitation generated using SwissModel34 and the crystal structure with PDB ID wavelength of 494 nm. Ca2+ transient increase was quantified by 4Z3519 as a template. The generated model was prepared for docking calculating the difference between maximum (stimulation with LPA using pdb 2pqr35,36 with the propka37,38 protonation option at a pH of 10 μM) and baseline values for each well, and antagonist activity was 7.4 and the peoepb force field.39 All the analyzed compounds were quantified by determining the percentage of the signal suppression modeled using RDKIT (Open-source cheminformatics) and its caused by the compound under study with respect to the Ca2+ protonation state adjusted at pH 7.4 by the ChemAxon cxcalc increase induced by LPA (which was considered 100%). As positive module (command line version of ChemAxon’s Calculator Plugins, controls, 10 μM LPA and 10 μM ionomycin were included in every v16.10.24.0, 2016). Binding mode pictures were created using PyMOL experiment. At this concentration, LPA induced a response about 30− v2.5.0. 33% of the one shown by ionomycin, which is in agreement with Mutagenesis Experiments. Amino terminal hemagglutinin previously described results.29 The data presented are from two to (HA)-tagged LPA2 point mutants K22A, R107A, Q108A, and four independent experiments carried out in triplicate or quad- K278A containing pcDNA3.1 plasmids were provided by GenScript. ruplicate. Dose−response curves were generated and IC50 values For expression of the different constructs, McA-RH7777 (CRL-1601, calculated by nonlinear regression analysis using Prism software ATCC) cells were selected, as they have been previously used for version 5 (GraphPad Software Inc., San Diego, CA, USA). point mutation experiments of LPA receptors.40 Cells were grown in The agonist activity at LPA2 receptors was determined at 10 μM Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) concentration for all final compounds as previously described.18 fetal bovine serum and 1% (v/v) penicillin-streptomycin and kept at Binding Affinity at LPA4−6. A free solution assay,23,24 where the 37 °C and 5% CO2. Cells were transiently transfected with the lysophosphatidic acid receptor (LPA2, 4−6) containing nanovesicles different plasmids using lipofectamine and following the manufac- (of 110−130 nm size as measured by dynamic light scattering) and turer’s procedure. Successful transfection was confirmed by flow compound under study are freely moving into solution was prepared cytometry analysis. For these experiments, 1 × 105 cells were to determine the equilibrium binding constants (KD) in a native resuspended in 50 μL of PBS with 2 mM EDTA and 0.5% BSA. Anti- environment of the binding partners (ligand/compound−receptor). HA antibody (Santa Cruz, sc-7392; 1 μg per 1 × 106 cells) was added The assay was analyzed using a benchtop Compensated Interfero- and cells were incubated for 30 min at rt with shaking. Cells were metric Reader (CIR) that measured the light refractive index (ΔRI) centrifuged, washed with buffer, and incubated with antimouse Alexa change from binding-induced conformational and/or hydration Fluor 488 (Invitrogen, 1:5000) for 30 min at rt with shaking and changes produced by real time binding events in a sample (receptor protected from light. Cells were centrifuged, washed with buffer, containing nanovesicles plus compound) and compared to a resuspended in 0.3 mL of buffer and analyzed by flow cytometry in a nonbinding reference (RI matched buffer plus compound).30 Finally, FACScalibur instrument (Becton Dickinson) at the UCM’s the interferometric signal from vector nanovesicles binding (non- microscopy and flow cytometry unit. After confirming the transfection specific) to compound was subtracted from the LPA2,4−6 containing by flow cytometry, calcium mobilization experiments were carried out nanovesicles binding (total) to compound, to determine the specific as previously described. binding interactions of the compounds to LPA2,4−6. The concen- Permeability and Microsomal Stability. These studies were tration-dependent change in RI (ΔRI) signal from the compounds to carried out as previously described with minor modifications.41,42 The LPA2,4−6 or vector was fitted using the single site total vs nonspecific assessment of the membrane permeability of synthesized compounds binding isotherm using GraphPad Prism. Specific KD values were and propranolol and metoprolol as reference compounds was determined by fitting the total minus nonspecific signal to a single site performed in a commercially available 96-well Corning Gentest binding isotherm. precoated PAMPA plate system (Cultek S.L.U., Spain). Prior to use, The detailed free solution assay method was described the precoated PAMPA plate system was warmed to rt for 30 min and previously.25,26 All the compounds were dissolved in 100% DMSO, 300 μL of 200 μM solution of tested compound in 2% DMSO in PBS aliquoted, and frozen at −80 °C for 1 week. The compound dilution (pH 7.4) were added into wells in the receiver (donor) plate. Then, series was freshly prepared in 0.5% DMSO/PBS (pH 7.4) to keep the 200 μL of PBS were added into wells in the filter (acceptor) plate. maximum compound in solution. In the final assay, total protein The filter plate was placed on the receiver plate by slowly lowering the 10971 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article precoated PAMPA plate until it sits on the receiver plate. The were anesthetized by intramuscular injection with a mixture of assembly was incubated at rt for 5 h, and then buffer samples were ketamine and xylazine (90:10 mg/kg). A laminectomy was performed collected carefully from each plate. The final concentrations of at the 11th thoracic vertebrae and the exposed spinal cord was compound in both donor and acceptor wells were analyzed by HPLC- contused using the Infinite Horizon Impactor device (Precision MS and quantification was estimated by using the peak area Scientific Instrumentation) using a force of 60 kdynes. Only mice integration normalized with an internal standard. Permeability value showing a spinal cord tissue displacement ranging between 450 and of the compounds was calculated using the following formula: P (cm/ 550 μm were selected. One hour after injury, compound 54 was s) = {−ln[1 − CA(t)/Ceq]}/[A(1/VD + 1/VA)t], where A = filter area injected intraperitoneally (25 mg/kg) which was then repeated daily (0.3 cm2), VD = donor well volume (0.3 mL), VA = acceptor well for 10 consecutive days. volume (0.2 mL), t = incubation time (s), CA(t) = compound concentration (μM) in the acceptor well at time t, CD(t) = compound concentration (μM) in donor well at time t, and Ceq = [CD(t)VD + CA(t)VA]/(VD + VA). Assays were performed in duplicate, and the ■ ASSOCIATED CONTENT * Supporting Information sı compound was tested in two different plates on different days. The Supporting Information is available free of charge at For measuring the stability in mouse and human liver microsomes, https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c00046. compounds were incubated at 37 °C at a final concentration of 1 or 5 Representative agonist curves for compounds 1, 13, and μM in PBS, respectively, together with a solution of nicotinamide 54 (Figure S1); dose−activity curves for compounds 13, adenine dinucleotide phosphate (NADPH) in PBS (final concen- 44, and 53−55 (Figure S2); representative plots of FSA- tration of 2 mM) and a solution of MgCl2 in PBS (final concentration of 5 mM). Reactions were initiated by the addition of a suspension of CIR signal vs ligand concentration for the determination mouse liver microsomes (MLMs) (male CD-1 mice pooled, Sigma- of affinity constants of compound 54 (Figure S3); Aldrich) or human liver microsomes (HLMs) (male human pooled, numbered structures used in the structural character- Sigma-Aldrich), respectively, at a final protein concentration of 1 mg/ ization by NMR of all final compounds (Figure S4); mL. The solutions were vortexed and incubated at 37 °C. Aliquots of HPLC gradients employed in HPLC-MS analysis of all 100 μL were quenched at time zero and at seven points ranging to 2 h tested compounds (Table S1); detailed synthetic (MLM) or 4 h (HLM) by pouring into 100 μL of ice-cold procedures and characterization data of all intermedi- acetonitrile. Quenched samples were centrifuged at 10 000g for 10 ates; NMR spectra and HPLC trace analysis of final min, and the supernatants were filtered through a polytetrafluoro- compounds (PDF) ethylene (PTFE) membrane syringe filter (pore size of 0.2 μm, 13 mm in diameter, GE Healthcare Life Sciences). The relative PDB files of the structure of the LPA2 receptor and disappearance of the compound under study over the course of the select compounds from this study (ZIP) incubation was monitored by HPLC-MS using SIM mode. Molecular formula strings (CSV) Concentrations were quantified by measuring the area under the peak ([M + H]+) normalized with an internal standard and converted to the percentage of compound remaining, using the time zero peak area value as 100%. The natural logarithm of the remaining ■ AUTHOR INFORMATION Corresponding Authors percentage versus time data for each compound was fit to a linear Silvia Ortega-Gutiérrez − Departamento de Química regression, and the slope was used to calculate the degradation half- Orgánica I, Facultad de Ciencias Químicas, Universidad life (t1/2). Complutense de Madrid, Madrid E-28040, Spain; Determination of the In Vivo Levels of Compound. orcid.org/0000-0002-0257-6754; Email: siortega@ Compound 54 was administered intraperitoneally (25 mg/kg) in adult female 12−16 weeks old C57Bl/6J mice. At 1, 2, and 4 h after ucm.es drug administration (n = 3 for each time and sample), mice were María L. López-Rodríguez − Departamento de Química sacrificed and their brains, spinal cords, and blood were obtained. The Orgánica I, Facultad de Ciencias Químicas, Universidad brain and spinal cord were immediately frozen and kept at −80 °C Complutense de Madrid, Madrid E-28040, Spain; until analysis. Blood was allowed to clot at rt for 30 min and orcid.org/0000-0001-8607-1085; Email: mluzlr@ucm.es centrifuged at 4 °C for 10 min at 16 000g. Serum was transferred to a clean polypropylene tube and stored at −80 °C until analysis. For Authors analysis, a volume of cold acetonitrile was added to the serum. The Nora Khiar-Fernández − Departamento de Química Orgánica sample was incubated in an ice bath for 10 min and centrifuged at 4 I, Facultad de Ciencias Químicas, Universidad Complutense °C for 10 min at 16 000g. The resulting organic layer was filtered de Madrid, Madrid E-28040, Spain through a PTFE filter (0.2 μm, 13 mm diameter, Fisher Scientific) Debora Zian − Departamento de Química Orgánica I, and 20 μL of the sample analyzed by LC-MS/MS at the UCM’s Mass Facultad de Ciencias Químicas, Universidad Complutense de Spectrometry CAI. Separation was performed using a Phenomenex Gemini 5 μm C18 110A 150 × 2 mm column (run time 8 min; flow Madrid, Madrid E-28040, Spain 0.5 mL/min; gradient: 0.5 min 10% Phase B, 2 min 60% Phase B, Henar Vázquez-Villa − Departamento de Química Orgánica I, 4.5−6 min 100% Phase B, 7−8 min 10% Phase B; Phase A: water with Facultad de Ciencias Químicas, Universidad Complutense de formic acid 0.1%; Phase B: acetonitrile). The entire LC eluent was Madrid, Madrid E-28040, Spain; orcid.org/0000-0001- directly introduced to an electrospray ionization (ESI) source 7911-3160 operating in the positive ion mode for LC MS/MS analysis on a R. Fernando Martínez − Departamento de Química Orgánica Shimadzu LCMS8030 triple quadrupole mass spectrometer coupled I, Facultad de Ciencias Químicas, Universidad Complutense to UHPLC with an oven temperature of 31.5 °C. The mass de Madrid, Madrid E-28040, Spain; orcid.org/0000- spectrometer ion optics were set in the multiple reaction monitoring 0002-3278-6074 mode and the transition selected for quantification was 432.90 > Andrea Escobar-Peña − Departamento de Química Orgánica 215.10 (CE: −30 V). Spinal Cord Injury In Vivo Model. All Surgical Procedures Were I, Facultad de Ciencias Químicas, Universidad Complutense Approved by the Universitat Autònoma De Barcelona Animal Care de Madrid, Madrid E-28040, Spain Committee (CEEAH 4273) and followed the guidelines of the Román Foronda-Sainz − Departamento de Química Orgánica European Commission on Animal Care (EU Directive 2010/63/EU). I, Facultad de Ciencias Químicas, Universidad Complutense Adult female C57Bl/6J mice (10−12 weeks old) and LPA2 null mice de Madrid, Madrid E-28040, Spain 10972 https://doi.org/10.1021/acs.jmedchem.2c00046 J. Med. Chem. 2022, 65, 10956−10974 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Manisha Ray − Translational Neuroscience Initiative, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, ■ ABBREVIATIONS USED BMS, Basso Mouse Scale; CNS, central nervous system; Emax, California 92037, United States maximal receptor activation; FSA-CIR, free solution assay- Maria Puigdomenech-Poch − Departament de Biologia Cel· compensated interferometric reader; GPCR, G protein- lular, Fisiologia i Immunologia, Institut de Neurociències, coupled receptor; HLM, human liver microsome; IC50, Centro de Investigación Biomédica en Red sobre concentration of compound that inhibit the 50% of the Enfermedades Neurodegenerativas (CIBERNED), activation induced by LPA; i.p., intraperitoneally; LPA, Universitat Autònoma de Barcelona, Barcelona E-08193, lysophosphatidic acid; MLM, mouse liver microsome; MW, Spain microwave; N.E., no effect; P, permeability; PK, pharmacoki- Giovanni Cincilla − Molomics, Barcelona E-08028, Spain; netic; SCI, spinal cord injury; s.e.m., standard error of the orcid.org/0000-0002-5242-0707 mean Melchor Sánchez-Martínez − Molomics, Barcelona E-08028, Spain; Burua Scientific, Sant Pere de Ribes E-08810, Spain; orcid.org/0000-0002-3674-8577 Yasuyuki Kihara − Translational Neuroscience Initiative, ■ REFERENCES (1) Ahuja, C. 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M.P.-P. and R.L.-V. performed the in vivo experiments and (9) Choi, J. W.; Herr, D. R.; Noguchi, K.; Yung, Y. C.; Lee, C. W.; analyzed data. All authors have given approval to the final Mutoh, T.; Lin, M. E.; Teo, S. T.; Park, K. E.; Mosley, A. N.; Chun, J. version of the manuscript. LPA receptors: subtypes and biological actions. Annu. Rev. Pharmacol. Toxicol. 2010, 50, 157−186. Notes (10) Kihara, Y.; Maceyka, M.; Spiegel, S.; Chun, J. Lysophospholipid The authors declare no competing financial interest. receptor nomenclature review: IUPHAR Review 8. Br. J. Pharmacol. 2014, 171, 3575−3594. ■ ACKNOWLEDGMENTS This work has been supported by the Spanish Ministerio de (11) González-Gil, I.; Zian, D.; Vázquez-Villa, H.; Ortega-Gutiérrez, S.; López-Rodríguez, M. L. The status of the lysophosphatidic acid receptor type 1 (LPA1R). MedChemComm. 2015, 6, 13−26. 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