Discovery of (3-Phenylcarbamoyl-3,4-dihydro‑2H‑pyrrol-2- yl)phosphonates as Imidazoline I2 Receptor Ligands with Anti- Alzheimer and Analgesic Properties Andrea Bagán, Alba López-Ruiz, Soǹia Abás, M. Carmen Ruiz-Cantero, Foteini Vasilopoulou, Teresa Taboada-Jara, Christian Griñán-Ferré, Merce ̀ Pallas̀, Carolina Muguruza, Rebeca Diez-Alarcia, Luis F. Callado, José M. Entrena, Enrique J. Cobos, Belén Pérez, José A. Morales-García, Elies Molins, Steven De Jonghe, Dirk Daelemans, José Brea, Cristina Val, M. Isabel Loza, Elena Hernández-Hernández, Jesuś A. García-Sevilla, M. Julia García-Fuster, Caridad Díaz, Rosario Fernández-Godino, Olga Genilloud, Milan Beljkas,̌ Slavica Oljacǐc,́ Katarina Nikolic, and Carmen Escolano* Cite This: J. Med. Chem. 2025, 68, 2551−2573 Read Online ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: Imidazoline I2 receptors (I2-IRs) are altered in Alzheimer’s disease (AD) patients and are associated with analgesia. I2-IRs are not structurally described, and their pharmacological characterization relies on their modulation by highly affine ligands. Herein, we describe the synthesis of (3- phenylcarbamoyl-3,4-dihydro-2H-pyrrol-2-yl)phosphonates en- dowed with relevant affinities for I2-IRs in human brain tissues. The optimal ADME and pharmacokinetic profile of a selected compound, 12d, secured its in vivo exploration in a senescence accelerated prone 8 mice revealing improvement in the cognitive impairment and unveiling the mechanism of action by analyzing specific AD biomarkers. The treatment of a capsaicin-induced mechanical hypersensitivity murine model with 12d revealed analgesic properties devoid of motor coordination issues. The target engagement of 12d was demonstrated by suppression of the analgesic effect by pretreatment with idazoxan. Overall, 12d is a putative candidate for advancing preclinical phases and supports the modulation of I2-IRs as an innovative approach for therapeutics. ■ INTRODUCTION At the end of the 20th century, scientists identified imidazoline I2 receptors (I2−IRs), nonadrenergic binding sites for imidazo- lines, as relevant biological targets. I2−IRs are widely distributed in the brain and are found in organs such as the heart and the liver.1−3 The dysregulation of the levels of I2−IRs is a hallmark in human brain disorders encompassing glial tumors,4,5 depres- sion,6,7 Alzheimer’s disease (AD),8 Huntington’s disease,9 and Parkinson’s disease (PD).10,11 More recently, I2−IRs have been validated as promising new targets in analgesia12 and AD diagnostics, after the progression in clinical trials of ligands CR4056 (1, Figure 1) and [11C]BU99008 (2, Figure 1). CR4056 (1),13 the first-in-class I2−IRs ligand, is in a phase II multisite randomized placebo-controlled clinical trial for knee osteoarthritis patients, and has also been reported to have a promising role in AD therapeutics.14 We have described sex differences in the antidepressant-like response and molecular events induced by CR4056 (1) in rats.15 [11C]BU99008 (2) is a clinical candidate in phase I for Positron Emission Tomography diagnostics in patients suffering from PD and AD.11,16 From the molecular point of view, I2−IRs are nondescribed heterogeneous proteins that are identified by their high affinity for the radiolabeled ligand [3H]idazoxan (4), and with less affinity for [3H]clonidine and [3H]p-aminoclonidine.3 Due to the absence of structural data on I2−IRs, their pharmacological characterization relies on the discovery of high affine and selective I2−IRs ligands. Unfortunately, the known I2−IRs ligands are scarce,17 and their structures are largely restricted to imidazole heterocycles, such as CR4056 (1) and LSL60101 (3, Figure 1), 2-heterocyclic-2-imidazolines, such as [11C]BU99008 (2), idazoxan (4, Figure 1), tracizoline (5, Figure 1), BU224 (6, Figure 1) and 2-BFI (7, Figure 1) and Received: July 16, 2024 Revised: November 19, 2024 Accepted: December 18, 2024 Published: January 17, 2025 Articlepubs.acs.org/jmc © 2025 The Authors. Published by American Chemical Society 2551 https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 This article is licensed under CC-BY 4.0 D ow nl oa de d vi a 88 .2 6. 77 .8 3 on M ar ch 1 7, 2 02 5 at 2 0: 41 :0 8 (U T C ). Se e ht tp s: //p ub s. ac s. or g/ sh ar in gg ui de lin es f or o pt io ns o n ho w to le gi tim at el y sh ar e pu bl is he d ar tic le s. https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Andrea+Baga%CC%81n"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Alba+Lo%CC%81pez-Ruiz"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="So%CC%80nia+Aba%CC%81s"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="M.+Carmen+Ruiz-Cantero"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Foteini+Vasilopoulou"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf 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https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Luis+F.+Callado"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Luis+F.+Callado"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Jose%CC%81+M.+Entrena"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Enrique+J.+Cobos"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Bele%CC%81n+Pe%CC%81rez"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Jose%CC%81+A.+Morales-Garci%CC%81a"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Elies+Molins"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Steven+De+Jonghe"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Steven+De+Jonghe"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Dirk+Daelemans"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Jose%CC%81+Brea"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Cristina+Val"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf 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https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Rosario+Ferna%CC%81ndez-Godino"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Olga+Genilloud"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Milan+Beljkas%CC%8C"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Milan+Beljkas%CC%8C"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Slavica+Oljac%CC%8Cic%CC%81"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Katarina+Nikolic"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf 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https://pubs.acs.org/jmc?ref=pdf https://pubs.acs.org/jmc?ref=pdf https://acsopenscience.org/researchers/open-access/ https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ other imidazoline derivatives such as phenyzoline and diphenyzoline.18 Being aware of the importance of discovering I2−IRs ligands to build a comprehensive understanding of the pharmacological implications of I2−IRs, we took the challenge of developing structurally original ligands. Thus, we published that (2- imidazolin-4-yl)phosphonates derivatives were endowed with outstanding binding affinity and selectivity for I2−IRs.19 The improvement in the condition and amelioration of AD hallmarks in a murine model of age-related cognitive decline model, the senescence-accelerate mouse prone 8 (SAMP8) treated with a representative compound, the diethyl [(1-(3-chloro-4-fluoro- benzyl)-5,5-dimethyl-4-phenyl-4,5-dihydro-1H-imidazol-4-yl]- phosphonate named MCR5 (8, Figure 2), was the first in vivo experimental evidence that brought forward I2−IRs as a new therapeutic target for neurodegeneration,20 depression,21 and vascular disease.22 To support the potential of I2−IRs ligands in AD, we reported a disease-modifying single therapy of a murine familial AD model (5xFAD) treated with LSL60101 (3).23 We also synthesized and pharmacologically characterized, hetero- cyclic N1 or N2-linked imidazole skeleton compounds as new I2−IRs ligands, and reported the amelioration of the cognitive impairment and synaptic plasticity after the treatment of 5xFAD model with 2-(benzo[b]thiophene-2-yl)-1H-imidazole hydro- chloride.24,25 More recently, we took on the challenge of moving beyond the imidazoline/imidazole ring to bicyclic iminophosphonates as a source of new high affine I2−IRs ligands. A selected compound, (1RS,3aSR,6aSR)-5-(3-chloro-4-fluorophenyl)-4,6- dioxo-1-phenyl-1,3a,4,5,6,6a-hexahydropyrrolo[3,4-c]pyrrole- 1-phosphonate, named B06 (9, Figure 2), ameliorated the cognitive decline and improved the behavior of two murine models, SAMP8 and 5xFAD.26,27 Furthermore, B06 (9) showed promising in vitro ADME-Tox properties, anti-inflammatory properties, and neuroprotective properties in an in vitromodel of PD.28 These bicyclic iminophosphonates are highly function- alized and we approximate their structural modification by adding nucleophiles to the imine functional group. Selected compounds with an additional indole substituent of the newly accessed bicyclic phosphoprolines family, displayed an interest- ing affinity for I2−IRs and rescued the neurodegenerative condition of a transgenic AD Caenorhabditis elegans model (BIN05, 10, Figure 2).29 This success prompted us to continue exploring the synthetic transformation opportunities of bicyclic iminophosphonates and the pharmacological characteristics of the new compounds. In particular, the opening of the ring embodying the imide functional group attracted our attention and disclosed a new transformation leading to unprecedented (3-phenylcarbamoyl- 3,4-dihydro-2H-pyrrol-2-yl)phosphonates. The new com- pounds were completely characterized from the stereochemical point of view after performing X-ray crystallography of selected compounds. We evaluated the pharmacological profile of new compounds in human tissues by competitive studies using the selective radioligand [3H]2-BFI ([3H]7) and, its selectivity versus the related α2-adrenergic receptor (α2-ARs) through competition studies with the selective α2-ARs radioligand [3H]RX821002 (2-methoxyidazoxan). Analogously, the selec- tivity versus the related target, imidazoline I1 receptors (I1−IRs), was assessed for the most promising compounds through competition studies using [3H]clonidine. After a screening cascade, the representative compound, diethyl [(2RS,3RS)-3- ((3-chloro-4-fluorophenyl)carbamoyl)-2-phenyl-3,4-dihydro- 2H-pyrrol-2-yl)phosphonate (12d, Figure 2), was selected for further in vitro and in vivo studies. ■ RESULTS AND DISCUSSION Chemistry. Synthesis and Structural Characterization. The starting bicyclic iminophosphonates (Scheme 1, compound 9 and compounds with the general structure 11) were prepared by diastereoselective [3 + 2]cycloaddition reaction of a diethyl isocyanomethylphosphonate derivative and an N-substituted maleimide following procedures described by us.26 The opening of the imide was performed by treatment of the bicyclic derivatives 9 and 11 with a solution of NaOH 0.05 M in a mixture 2:1 THF/H2O (Scheme 1). To define the scope of the reaction, the absence, or the presence of a substituent in the α- phosphonate position (R1 = aryl, substituted aryl, alkyl), and different substituents in the nitrogen atom of the imide (R2= aryl, substituted aryl or cycloalkyl) were considered. To this end, Figure 1. Structure of the I2−IRs ligands: CR4056 (1), [11C]BU99008 (2), LSL60101 (3), idazoxan (4), tracizoline (5), BU224 (6), and 2- BFI (7). Figure 2. (A)Outline of the previous work and structure of compounds MCR5 (8), B06 (9), and BIN05 (10), and milestones achieved. (B) Outline of the present work. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2552 https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig2&ref=pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as the starting bicyclic derivatives with the α-phosphonate position (R1) occupied with a hydrogen atom, 11a and 11b (Table 1, entries 1 and 2, respectively), with a phenyl group 11c, 9, 11e- 11h (Table 1, entries 3 to 8, respectively), with a 4-substituted phenyl group 11i, 11j and 11k, 11l (p-fluoro, Table 1, entries 9 and 10, and p-methoxy, Table 1, entries 11 and 12, respectively), with a methyl group 11m (Table 1, entry 13) and with a benzyl group, 11n and 11o (Table 1, entries 14 and 15), were subjected to the general reaction conditions to yield the 3-substitutedcar- bamoyl-3,4-dihydro-2H-pyrrol-2-yl)phosphonates (12a-12o), in 19−75% yields (Table 1). To confirm that the relative configuration of the stereocenters in the bicyclic iminophosphonates was retained, the X-ray crystallography of monocrystals of compounds 12b, 12d and 12h was performed. The cis relationship between the hydrogen atom in position 2 in 12b, and the phenyl group in 12d and 12h, and the phenylcarbamoyl substituent in position 3 was unambiguously confirmed (Figure 3). The stereochemistry of the other compounds (12, Scheme 1) was assigned by comparison of their 1H and 13C NMR spectra (pages S3−S17, Tables S1 and S2, discussion on the spectra data S20). The reactionmechanism occurred irrespective of the presence or absence of a substituent in the α-phosphonate position or the type of substituent (phenyl, alkyl, or benzyl). To understand the molecular transformations to yield final compounds with the general structure 12, a putative mechanism is depicted in Scheme 2. The regioselective nucleophilic attack of the anion hydroxide to the carbonyl (4-CO) produced the opening of the imide, leading to an intermediate having a carboxylic acid at the position 3 of an imine functional group that, in the reaction conditions, underwent decarboxylation leading to the mono- cyclic final structures. These final products (general structure 12 in Scheme 1) are unprecedented and offer a wide range of synthetic transformations due to the high degree of function- alization that will be explored in the future. I2−IR Binding Activity and Structure−Activity Rela- tionships. The pharmacological activity of the compounds 12a-12o depicted in Scheme 1 was evaluated through competition binding studies against the selective I2−IRs radioligand [3H]-2-BFI, ([3H]7,) and the selective α2-ARs radioligand [3H]RX821002. The studies were performed in membranes from the post-mortem human frontal cortex, a brain area that shows an important density of I2−IRs and α2-ARs. 30 The inhibition constant (Ki) for each compound was obtained and it is expressed as the corresponding pKi (Table 2). The selectivity for these two receptors was determined by the I2/α2 index, calculated as the antilogarithm of the ratio between pKi values for I2−IRs and pKi values for α2-ARs (Table 2). Competition experiments against [3H]2-BFI, ([3H]7,) were monophasic for most of the compounds (for exceptions 12a, 12b, 12f, 12h, 12l and 12n, Table 2). As reference compounds, we used idazoxan (4), a non- selective compound, with well-established affinity for I2−IRs Scheme 1. General Structures of the Starting Materials (11) and Final Products (12)a aReagents and conditions: (i) NaOH 0.05M, 2:1 THF/H2O, r. t., 2.5 h. Table 1. Description of the Different Substituents, R1 and R2, Number of the Starting Materials and Final Products, and Yield of the Reaction.a Entry SM R1 R2 Comp Yield (%) 1 11a H Ph 12a 51 2 11b H 3-Cl,4-FPh 12b 44 3 11c Ph Ph 12c 70 4 9 Ph 3-Cl,4-FPh 12d 76 5 11e Ph 4-ClPh 12e 50 6 11f Ph 4-PhPh 12f 50 7 11g Ph 4-CF3Ph 12g 50 8 11h Ph cyclohexyl 12h 73 9 11i 4-FPh Ph 12i 50 10 11j 4-FPh 3-Cl,4-FPh 12j 50 11 11k 4-MeOPh Ph 12k 50 12 11l 4-MeOPh 3-Cl,4-FPh 12l 50 13 11m Me Ph 12m 31 14 11n Bn Ph 12n 19 15 11o Bn cyclohexyl 12o 57 aSM = starting material. Figure 3. X-ray structures of 12b, 12d and 12h. Scheme 2. Putative Mechanism for the Formation of Compounds with the General Structure 12 from 11.a aReagents and conditions: (i) NaOH 0.05 M, 2:1 THF/H2O, r. t., 2.5 h Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2553 https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=sch1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=sch1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig3&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig3&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig3&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig3&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=sch2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=sch2&ref=pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as (monophasic curve, pKi = 7.41 ± 0.63) and α2-ARs (pKi = 8.35 ± 0.16) (Table 2, entry 1), as well as the clinical candidates CR4056 (1)31 and BU99008 (2)32 that were synthesized following described procedures. We reported the affinity of both clinical candidates for human I2−IRs (Table 2, entries 2 and 3, respectively).26 Additionally, as the structurally closest com- pound, we selected our representative compound B06 (9) (Table 2, entry 4). The affinity of these compounds for I2−IRs is better described with a biphasic curve, CR4056 (1), pKiH I2 = 7.72 ± 0.31 (29% occupancy) and pKiL I2 = 5.45 ± 0.15 (Table 2, entry 2), BU99008 (2), pKiH I2 = 6.89 ± 0.21 (51% occupancy) and pKiL I2 = 3.82 ± 0.30 (Table 2, entry 3), and B06 (9), pKiH I2 = 8.61 ± 0.28 (37% occupancy) and pKiL I2 = 4.29 ± 0.20 (Table 2, entry 4). The compounds with the general structure 12 where R1 is a hydrogen atom, and R2 is an aryl derivative, 12a and 12b, were not selective (I2/α2) and fitted best into a two-site model of binding with pKiH I2 = 8.70 ± 1.29 (Ki = 1.99 nM, 25% Table 2. I2−IRs and α2-ARs Binding Affinities (pKi) of Four Previously Reported Compounds 25 and New Compounds. aSelectivity I2−IRs/α2-ARs expressed as the antilog (pKi I2−IRs- pKi α2-ARs). bThe best fit of the data for CR4056 1, BU99008 2, B06 9, 12a, 12b, 12f, 12h and 12l was to a two-site binding model of binding with high pKi (pKiH) and low pKi (pKiL) affinities for both binding sites respectively. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2554 https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=tbl2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=tbl2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=tbl2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=tbl2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=tbl2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=tbl2&ref=pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as occupancy) and pKiL I2 = 3.89 ± 0.62 and pKiH I2 = 8.66 ± 0.43 (Ki = 2.18 nM, 28% occupancy) and pKiL I2 = 5.12 ± 0.24, respectively. The two compounds depicted better affinity for the I2−IRs than the three standard compounds (Table 2, entries 1− 3), but no I2/α2 selectivity. As a first structural approximation, we turned our attention to compounds bearing a quaternary center in the α-position by including a phenyl group. This modification was highly deleterious for the affinity in compound 12c (R1, R2 = Ph, pKi 3.52± 0.64, Table 2, entry 7). Tomaintain the homology with 12b (R1= H, R2= 3-Cl,4-FPh) compound 12d (R1= Ph, R2= 3-Cl,4-FPh) was evaluated. A remarkable benefit was observed by an increase in the affinity to a pKi = 9.98 ± 0.74 (Ki = 1.05 nM) with no I2/α2 selectivity (Table 2, entry 8). The introduction of halogen atoms in the R2 = Ph, as in 12e (R2 = 4-ClPh), was highly deleterious for the affinity (Table 2, entry 9). Compound 12f (R2 = 4-PhPh) showed an affinity property that fits best with a two-site model of binding, pKiH I2 = 7.88 ± 0.38 (35% occupancy) and pKiL I2 = 5.26 ± 0.27 (Table 2, entry 10), without overpassing the standard I2−IRs ligands (Table 2, entries 1−3). The presence of a 4-CF3 substituent in the phenyl R2 substituent in the compound 12g led to an affinity (pKi 7.04 ± 0.28) in the range of the idazoxan (4) but with an improved selectivity ratio of 51 (I2/α2). After exploring compounds 12c, 12d, 12e, 12f, and 12g with N-aryl substituents, we synthesized compound 12h with a N- cyclohexyl substituent showing a biphasic curve with a pKiH I2 = 8.93 ± 0.38 but with only a 15% occupancy and pKiL I2 = 3.18 ± 0.29 (Table 2, entry 12). Next, we considered modifications in the α-phenyl substituent. For maintaining the homology with the first considered compounds (12a and 12c, R2 = Ph, and 12b and 12d, R2 = 3-Cl,4-FPh), we synthesized compounds 12i and 12j (R2 = Ph, and 3-Cl,4-FPh, respectively) with a 4-fluorine atom in the α-phenyl substituent and compounds 12k and 12l (R2 = Ph, and 3-Cl,4-FPh, respectively) with an electron donating group, 4-methoxy, in the α-phenyl substituent. The presence of an additional fluorine atom did not restore the affinity with a pKi I2 = 5.03± 0.41 for 12i (Table 2, entry 13) and pKi I2 = 3.71 ± 0.27 for 2j (Table 2, entry 14). The 4- methoxyphenyl group was not the right choice in the case of compound 12k (pKi I2 = 3.07 ± 0.47, Table 2, entry 15) but showed better activity in compound 12l, considering the biphasic fitted curve pKiH I2 = 10.20 ± 0.31 (23% occupancy) and pKiH I2 = 3.75 ± 0.13. Finally, the substitution of an α-phenyl group for an alkyl substituent, R1 =Me in compound 12m displays negligible pKi I2 (Table 2, entry 17). Compound 12n where the alkyl substituent is a benzyl group and R2 is phenyl exhibit a biphasic fitted curve, pKiH I2 = 8.99 ± 0.37, with 33% occupancy and pKiL I2 = 3.04 ± 0.33 and a remarkable selectivity I2/α2 6530 (Table 2, entry 18). The affinity drops in compound 12o bearing the α-benzyl subtituent and with an R2 cyclohexyl substituent, pKi I2 = 5.22 ± 0.14 (Table 2, entry 19). Selectivity for I2−IR Versus I1−IR. To further evaluate the selectivity of new compounds, apart from considering α2-ARs (Table 2), we assessed the affinity of selected compounds for the related I1−IRs. The competition binding site assays for I1−IRs, using [3H]clonidine as radioligand, were conducted in membranes obtained from the rat kidney using as reference moxonidine, a known I1−IRs selective compound. Compounds 12a, 12b, 12d, 12h, 12l and 12n, with an affinity value (pKi or pKiH) for the I2−IRs above 8 were selected (Table 2). The results are summarized in Table S18, and only deserved a mention, the values of pKi 7.84 ± 0.24 and 7.58 ± 0.47 for compounds 12a and 12c, respectively. Therefore, there was not significant interaction with I1−IRs highlighting the I2−IRs selective behavior of the herein reported family. This fact allows us to exclude that the new compounds may have secondary effects on blood pressure through the activation of I1−IRs.33 Three-Dimensional Quantitative Structure−Activity Relationship Study. A comprehensive three-dimensional quantitative structure−activity relationship (3D-QSAR) study was performed to gain insight into the structural features that are critical for the activity and selectivity for I2−IRs of new compounds with the general structure 12 (Scheme 1). Also, a comparison of these results was carried out with already established 3D-QSAR models of starting materials (bicyclic iminophosphonates, Scheme 1).26 The results of the external validation confirmed that both 3D- QSAR models reliably predict I2−IRs and α2-ARs activity (Tables S19 and S20). The key variables that correlate with I2− IRs activity and selectivity are further discussed and evaluated. Var200 (TIP-TIP: 17.6 Å − 18.0 Å) is observed between the steric hot spots around the para-substituents of the phenyl ring (such as fluorine, trifluoromethyl and phenyl) and the steric hot spots around the ethoxy group. This variable appears in almost all the most active compounds (12d, B06, etc) (Figure 4A), suggesting that the introduction of substituents at the para- position of the phenyl ring can positively correlate with the overall I2−IRs activity. Furthermore, this variable is absent in the least active compound, 12k, which has no substitutions on the phenyl ring, supporting this conclusion. Likewise, the contribution of para-substitution to I2−IRs activity can be explained by var507 (N1-TIP:15.6 Å − 16.0 Å), which emphasizes the importance of the optimal distance between the steric hot spots around the para-substituents of the phenyl ring and the hydrogen-accepting group (the nitrogen atom of 1- pyrroline or carbonyl group) in the most active compound 12d. Notably, this variable is also not present in the least active compound, 12k, nor is var200. The significance of the phenyl ring attached to the amide group for I2−IRs activity is evidenced by var350 (DRY-TIP: 15.2 Å − 15.6 Å), which reflects the optimal distance between the steric hot spots around the phenyl ring and the hydrophobic regions around the ethoxy group. In contrast, this variable is absent in compounds that have cyclohexane substituents instead of the phenyl ring, such as 12h and 12n, which exhibit low I2− IRs activity (pKi I2 = 3.57 and pKi I2 = 5.22, respectively). In addition, the negative impact of cyclohexane on I2−IRs activity is underlined by unfavorable var291 (DRY-N1:12.4 Å −12.8 Å) that is observed in 12h and 12n (Figure 4B) between the hydrogen accepting group (nitrogen atom of 1-pyrroline) and the hydrophobic regions around the cyclohexane ring. This led us to conclude that the phenyl ring is important for I2−IRs activity. Finally, the introduction of para-substituents on the phenyl ring (C-5 of 1-pyrroline) correlates negatively with I2−IRs activity, as shown by the unfavorable var264 (DRY-N1:1.6 Å − 2.0 Å) and the unfavorable var182 (TIP-TIP: 10.4 Å−10.8 Å) in the less active compounds of the data set (12j and 12k, Figure 4C). Var264 indicates the negative influence of the distance between the hydrogen-accepting group of 1-pyrroline (the nitrogen atom) and the hydrophobic regions around the para- substituents of the phenyl ring, such as fluorine and methoxy group, on the I2−IRs activity. In parallel, var182 points out the negative effects of the distance between steric hot spots of these substituents and ethoxy groups on the overall I2−IRs activity. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2555 https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as The 3D-QSAR (α2-ARs) model identifies var476 (N1-TIP: 5.6 Å −6.0 Å) as one of the key factors contributing to α2-ARs activity (Figure 4D). This variable represents the optimal distance between a hydrogen-accepting group, such as a carbonyl group, and a hot spot region around the ethoxy group. In contrast, the 3D-QSAR (I2−IRs) model does not highlight the carbonyl group as critical for I2−IRs activity. This suggests that changes to this part of the molecule could potentially decrease α2-ARs activity while increasing selectivity for I2−IRs. Two variables, var430 (O-TIP:12.0 Å − 12.4 Å) and var416 (O-TIP:16.4 Å −16.8 Å), indicate the positive correlation between the presence of a hydrogen-donating group −the nitrogen atom of the amide - and α2-ARs activity, suggesting that the substitution of this atom is likely to decrease α2-ARs activity while being important for selectivity toward I2−IRs, bearing in mind that the 3D-QSAR (I2−IRs) model does not imply the importance of this structural feature for I2−IRs activity (Figure 4D). In summary, we find that the presence of phenyl rings (positions C-4 and C-5 of 1-pyrroline) and ethoxy groups positively correlates with I2−IRs activity, as shown by var351 and var291. The introduction of para-substituents into the phenyl ring at position C-4 can have a favorable effect on I2−IRs activity, as indicated by the favorable var200 and var507, whereas substitution of the phenyl ring at C-5 leads to a decrease in I2−IRs activity, as shown by the unfavorable var264 and var182. Finally, the modification of the amide group could be important to increase I2−IRs activity and selectivity, which is consistent with the influence of var476, var430 and var416 on α2-ARs activity. Similar to the previously described 3D-QSAR model,26 the phenyl ring was identified as a critical structural feature for I2− IRs activity, especially with respect to the para-substituents on the phenyl ring (C-4 position of the 1-pyrroline moiety). In contrast to the previous 3D-QSAR model, which suggested that meta- and para-substituents on the phenyl ring enhance both I2− IRs activity and selectivity, the current model shows that these substituents are indeed essential for I2−IRs activity but have no effect on selectivity. This result is consistent with experimental data, as the most active molecule for α2-ARs, 12d, has these substituents. Furthermore, the new model shows that para- substitution of the phenyl ring at the C-5 position can have negative effects on the overall I2−IRs activity and should not be considered in the further development of inhibitors. In conclusion, the 3D-QSAR models show that strategic modification of the phenyl rings and selective substitution patterns as well as modification of the amide group can increase I2−IRs activity while improving selectivity toward α2-ARs, providing valuable guidance for the development of more selective I2−IR ligands. In Silico ADMET Analysis of Physicochemical and Pharmacokinetic Parameters. In silico prediction of absorption, distribution, metabolism, excretion and toxicity (ADMET) aims to evaluate the individual pharmacokinetic properties of the investigated drugs, which is an important step in lead optimization. The pharmacokinetic profiles of the compounds were determined using the ADMET Predictor software,34 while the physicochemical parameters were determined using the online program SwissADME.35 The predicted parameters are listed in Tables S21 and S22. From the results of the calculations performed, we can conclude that all the compounds studied have good water solubility and lipophilicity. The compounds presented here fulfill the Lipinski’s Rule of 5, which speaks for their drug-like properties and potential chance of oral bioavailability. In addition, the polarity of the compounds was evaluated by TPSA (topological polar surface area) and the results showed that the studied compounds had higher polarity than CR4056 (1), BU99008 (2), and idazoxan (4). In terms of pharmacokinetic properties, all molecules were found to exhibit high permeation into the BBB. Compared to standard molecules such as BU99008 (2) and idazoxan (4), all studied compounds showed lower levels of unbound drug in plasma. In addition, this family showed a lower metabolic CYP risk than idazoxan (4) and a lower TOX risk compared to CR4056 (1), BU99008 (2), and idazoxan (4). P-gp Figure 4. (A1). Favorable var200: TIP-TP and favorable var507: N1-TIP of compound 12d (3D-QSAR I2−IRs model); (A2) Favorable var200: TIP- TIP of compound B06 9; the steric hot spots (TIP) are presented in green andH-bond acceptor regions in blue. (B1) Unfavorable var291: DRY-N1 of compound 12h (3D-QSAR I2−IRs model); (B2) Unfavorable var291: DRY-N1 of compound 12n (3D-QSAR I2−IRs model); the hydrophobic regions (DRY) are labeled in yellow and H-bond acceptor regions in blue; (C1) Favorable var182: TIP-TIP and favorable var264: DRY-N1 of compound 12k (3D-QSAR I2−IRs model); (C2) Favorable var182: TIP-TIP of compound 12j (3D-QSAR I2−IRs model); the hydrophobic regions (DRY) are labeled in yellow, the steric hot spots (TIP) are presented in green and H-bond acceptor regions in blue. (D) Favorable var416: O-TIP, favorable var430: O-TIP and favorable var476: N1-TIP of compound 12d (3D-QSAR α2-ARs model); the steric hot spots (TIP) are presented in green, H-bond acceptor regions in blue and H-bond donor regions in red. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2556 https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig4&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig4&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig4&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig4&ref=pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as is thought to play an important role in drug distribution and resistance to CNS drugs. The investigated compounds were not identified as potential substrates for P-gp transporters. Finally, the compounds studied did not show a significantly different probability of inhibiting hERG channels compared to idazoxan (4). As there were no caveats in this theoretical study, we are confident to perform further in vitro and eventually in vivo experiments to evaluate the new compounds as neuroprotective agents. PAMPA-BBB Permeation Assay. Considering the location of I2−IRs in the CNS, a good ability to cross the BBB is an essential requirement for developing effective I2−IRs ligands with potential therapeutic applications in the neuroprotective field. For this reason, the in vitro permeability (Pe) of all the novel compounds was determined by using the PAMPA-BBB permeability assay (Table S24). Most of the new compounds prepared were well above the threshold established by Di et at.36 for high BBB permeation (Pe > 5.198× 10−6 cm s−1), while 12b, 12k and 12m had values considered of uncertain BBB permeation (CNS ± : 5.198 > Pe (10−6 cm s−1) > 2.054). Cytotoxicity. Since compounds 12a and 12d displayed high binding affinity values for I2−IRs (Table 2, entries 5 and 8), they were selected for a broad cytotoxicity screening against different mammalian cell lines, such as HeLa (human cervix carcinoma), Vero (African green monkey kidney), MDCK (Mandin-Darby canine kidney), and MT4 (human T-lymphocyte). None of the two compounds showed any cytotoxicity at 100 μM, the highest concentration tested. In addition, compounds 12a, 12c, 12d, 12f, 12g, 12h, 12i, 12j, 12k and 12l lacked cytotoxicity (CC50 > 100 μM) when evaluated in a panel of cancer cell lines including LN-229, Caspan-1, Hap-1, HCT-116, NCI-H460, DND-41, HL-60, K-562 and Z-138 cell lines. Moreover, compound 12d was also tested in MRC-5 (human embryonic lung fibroblast) cells and no cytotoxicity was observed (CC50 > 100). Considering its affinity for I2−IRs, noncytotoxicity and predicted BBB-penetrance, we selected compound 12d for further characterization as I2−IRs ligand. ADME-DMPK Profiling of 12d. The determination of the following parameters is recommended for predicting drug absorption and avoiding misleading in vivo results. The solubility of 12dwas excellent (>100 μM) in amixture of 1%DMSO and a 99% PBS buffer. The chemical stability of 12d in buffers at different pHs was undertaken to assess its degradation by no- enzymatic processes (e.g., 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 the representative compound 12d need to be assessed. To this end, a test solution of 12d (1 μM, 0.1% final DMSO concentration) was incubated with a buffer at pH 2, 5, and 7.4 at 37 °C. The analysis of the samples taken at 0, 5, 30, 60, and 120 min by LC-MS/MS reveals that 12d was stable at the pHs and times studied (Tables S25−S27). The permeability across a human colon carcinoma cell line Caco-2monolayer indicated an optimal prediction for the intestinal absorption of 12d. The parameters of microsomal stability, plasma stability, and protein binding were evaluated in different species (human, mouse, and rat), ensuring confidence in 12d to progress. The concentration for the inhibition of cytochromes [CYP1A2, CYP2C9, CYP2C19, CYP2A4 (BFC and DBF), and CYP2D6] was 10 μM, far away from the nanomolar Ki of the affinity of 12d for the I2−IRs. The inhibition of the hERG potassium channel was negligible considering the nanomolar range of actuation of 12d. The results of this study warrant the confidence in the progression of 12d in further studies (see the parameters and conditions of the assays in the Supporting Information). In Vitro Effects of 12b in a Preclinical Model of Neurodegeneration. Neurodegenerative diseases are primar- ily characterized by neuronal death accompanied by a strong neuroinflammatory process that contributes to disease pro- gression. Therefore, we utilized two well-known in vitro models of neurodegeneration and neuroinflammation. One model is based on the use of the neurotoxin 6-hydroxydopamine (6- OHDA), which induces cytotoxic stress in SH-SY5Y neuronal cultures. The other model uses lipopolysaccharide (LPS) as an inducer of glial hyperactivation in primary astrocyte and microglial cultures, mimicking the inflammatory process characteristic of neurodegenerative disorders. Initially, we assessed the potential cytotoxic effect of the compound 12d itself. For this, we treated neuronal and glial cultures with various concentrations of the compound. The results of cell survival assays and nitrite measurement tests, as an indicator of inflammation, demonstrate that the compound is nontoxic at the assessed concentrations (Figure S6). Subsequently, we evaluated the neuroprotective capacity of the compound 12d in a neuronal damage model. The results shown in Figure 5 demonstrate that compound exerts a significant neuroprotective Figure 5.Neuroprotective effect of compound 12d. SH-SY5Y cultures were exposed to 6-OHDA (35 μM) for at least 18 h. Previously, some cultures were pretreated 1h before with different concentrations of the compound 12d. CR4056 (1) (1 μM)was used as a reference. Basal cultures were treated with vehicle. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay, and nitrite production in the cell supernatant was quantified using the Griess reaction. The reported values represent the mean ± SD obtained from triplicate determinations repeated at least three times. Statistical analysis was performed: *p ≤ 0.05, **p ≤ 0.01 (versus 6-OHDA- treated cultures); ### p ≤ 0.001 (compared to basal cultures). Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2557 https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig5&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig5&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig5&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig5&ref=pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as effect against 6-OHDA-induced damage at all tested concen- trations. This analysis also included the clinical candidate CR4056 (1) at 1 μM concentration as a reference. When we assessed the neuroprotective effect (Figure 5), we observed that compound 12d also exhibited anti-inflammatory properties in this neuronal damage model, significantly reducing nitrite levels produced after injury. In neurodegenerative pathologies, glial cells are the main inducers of inflammatory processes; upon activation, they produce pro-inflammatory factors that contribute to neuronal death and disease progression. In a subsequent analysis, we used primary cultures of astrocytes (Figure 6A) and microglia (Figure 6B) isolated from the murine cerebral cortex. The results presented in Figure 6 demonstrate that compound 12d, at a concentration of 5 μM, exerts a considerable anti-inflammatory effect. This is evidenced by the reduced nitrite levels in culture and the marked decrease in the production of pro-inflammatory agents such as cyclo- oxygenase-2 (COX-2) and tumor necrosis factor-α (TNFα) compared to LPS-stimulated glial cultures. Pharmacokinetics of 12d. The resulting plasma PK parameters of 12d are shown in Table 3. The maximum mean concentration found in plasma (2.61 ± 0.64 μg/mL) was reached at 15 min after the drug administration in a single oral dose of 30 mg/kg. Compound 12d is found in plasma after 5 min of administration and rapidly disappears from the plasma, due to its high distribution throughout the organism. The AUC levels between the last sampling and infinite were very similar to each other. Notwithstanding plasma levels do not remain very high during the sampling period, it should be noted that the administration of 12d in drinking water at a dose of 5 mg/kg to the murine model (SAMP8) continued at effective levels for 4 weeks. These efficacy results could also be explained by the elimination half- life (176 ± 36.2 min) which will allow reaching effective levels in the brain during treatment. In the PK study, the quantitation in mouse brain samples was assessed, and after a single oral administration of 30 mg/kg, a maximum concentration of 3.19 ± 1.01 μg/mL was detected at 15 min (Cmax). From this moment on, concentrations decreased significantly but remained detectable throughout the sampling interval. These values support the presumed high distribution of 12d throughout the body and its rapid elimination at a single dose as well as the levels sustained during administration through water. Figure 6. Role of compound 12d in an in vitro neuroinflammation model. Glial cells, astrocytes (A) and microglial cells (B) were isolated frommurine cortex and stimulated with bacterial LPS (10 μg/mL) in the presence of the compound 12d (at different doses). CR4056 1 (1 μM) was used as a reference and basal cultures were treated with vehicle. After 24h in culture, nitrite production was determined in the supernatant of glial cells using the Griess reaction. Values presented represent the mean ± SD from triplicate determinations, repeated at least three times. Statistical analysis revealed significant differences: *p ≤ 0.05, **p ≤ 0.01 compared to LPS-treated cultures; ###p ≤ 0.001 compared to basal cultures. Immunocytochemical analysis of the expression of pro-inflammatory factors in primary glial cultures is shown, cyclooxygenase 2 (COX-2, green) and tumor necrosis factor alpha (TNFα, red). Cell nuclei were stained with DAPI. Scale bar, 20 μM. Table 3. Basic PK Parameters were Calculated.a Pharmacokinetic parameters of 12d in plasma t1/2β (min) 176 ± 36.2 Tmax (min) 15 ± 0 Cmax (μg/mL) 2.61 ± 0.64 AUC0‑ 1440 (μg/mL*min) 197.43 ± 16.58 AUC0‑ ∞ (μg/mL*min) 199.23 ± 16.25 Cl (mg/kg)/(μg/mL)/min 0.15 ± 0.01 Vd (mg/kg)/(μg/mL) 38.24 ± 8.25 Pharmacokinetic parameters of 12d in brain t1/2β (min) 252 ± 74.7 Tmax (min) 15 ± 0 Cmax (μg/mL) 3.19 ± 1.01 AUC0‑ 1440 (μg/mL*min) 117.62 ± 34.51 AUC0‑ ∞ (μg/mL*min) 118.83 ± 35.02 aCmax: maximum observed concentration, Tmax: time of maximum observed concentration, t1/2β: terminal elimination half-life, AUC0‑ 1440: area under the curve from zero to the last sampling time, AUC0‑ ∞: area under the curve from zero extrapolated to infinity, CI: plasma clearance, Vd: volume of distribution. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2558 https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig6&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig6&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig6&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig6&ref=pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as For details on the method validation for quantification of 12d in mouse plasma and in mouse brain (Supporting Information pages S87−S93). Selectivity. In an Alzheimer’s MoA panel (Eurofins)37 with 41 assays, including neurotransmitters, glutamate excitotoxicity, bioprocess of β-amyloid, tau hyperphosphorylation, and neuro- inflammation, compound 12d did not give significant results on the interaction with the targets included (Table S40). Hypothermic Effects of 12d in Mice. It is well precedented that several I2−IRs ligands induce hypothermia in rodents.3,19,24,26,38 Similar to what was recently reported for B06 (9),26 acute 12d (20mg/kg) induced hypothermia in adult CD1 mice (ranging from −1.1 to −3.8 °C in temperature drop) as measured 1 h postinjection (Figure 7A). Repeated admin- istration of 12d (20 mg/kg, 5 days) showed persistent hypothermic effects in mice, observed consistently from days 1 to 5 of treatment (Figure 7B), and contrarily to what was previously observed for other I2−IRs ligands, that developed tolerance to the acute hypothermic effects.19,20,26 As mentioned in our prior publications,19,20,24,26,39 the hypothermic effects induced by 12d might be mediated and/or related to the neuroprotective potential of I2−IRs ligands. In this context, prior data suggested a putative role of hypothermia in mediating neuroprotection, such as in cerebral ischemia,40 or following stroke or brain injury, proposing a way to improve the neurological outcome under various pathological conditions in the clinic,41,42 and presenting these novel I2−IRs ligands as good therapeutic candidates. Neurochemical Effects of 12d in Mice Hippocampus. We have recently shown that the acute administration of some I2−IRs ligands to rodents, besides inducing hypothermia, decreased the brain content of FADD multifunctional protein.19,20,26 As previously described, FADD is an adaptor of cell death receptors with a dual role in the brain, since it can mediate both pro- and antiapoptotic/neuroprotective actions in rodents.43 The present results failed to show an acute regulation of FADD by treatment with compound 12d, but revealed a 25% decrease in hippocampal FADD content following the repeated drug paradigm (Figure 8). This significant decrease, together with the still present hypothermic effect observed at this time point (Figure 7B), suggests that a more prolonged treatment might be needed for this compound to mediate some of its neuroplastic or neuroprotective actions through the regulation of this key brain marker, in contrast to the acute effects observed for other I2−IRs compounds.19,20,26 In this line of thought, we evaluated other putative neurochemical markers that could parallel the effects observed for 12d and that have been previously shown to be regulated in conjunction with FADD, such as Cdk5 and pERK/ERK.19,44 However, the results, as reported in Figure 11, showed no changes in the protein content of Cdk5 or pERK/ERK by 12d, reinforcing the particular role of FADD in the effects mediated by I2−IRs ligands and in parallel to their induction of hypothermia. Cognitive Effects in the In Vivo Senescence Accel- erated Prone 8 (SAMP8) Mice Treated with 12d. The SAMP8 mouse model is a well-characterized inbred selected strain to showADpathological characteristics fully present at 5− 6 months of age, including cognitive and behavioral deficits such as memory impairment.45−47 Thus, at the selected age of 10 months, SAMP8 mice provide a severe AD pathological landscape suitable for evaluating the drug effects. The treatment with compound 12d (5 mg/kg for 4 weeks, p. o.) improved cognitive parameters measured through Novel Object Recog- nition and Novel Object Location Tests (NORT and OLT, respectively). NORT short- and long-term memory showed a significant Discrimination Index (DI) increase indicating an amelioration of working memory. Moreover, spatial memory determined by OLT was also significantly improved in SAMP8 (Figure 9). These results agreed with those previously published with other I2−IRs ligands in SAMP8 and in 5XFAD, a mice model of familial AD.20,21,23,27 MolecularMechanisms Driving 12d Treatment Neuro- protection in SAMP8Mice. Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) maintains the homeostasis of 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) in the brain, two key second messengers that regulate a broad range of intracellular processes and neurocognitive functions, specifically memory and cognition, associated with AD.48 Ca2+/calm- odulin-dependent protein kinase II (CaM kinase II or CaMKII) is also regulated by the Ca2+/calmodulin complex, also involved in many signaling cascades, and is thought to be an important mediator of learning and memory.49 12d was able to reduce the protein content in the hippocampus of SAMP8, pointing out to be responsible for cognitive improvement shown after 12d treatment (Figure 10 A, D, E). In fact, PDE1 and CaMKII inhibitors gained interest in the treatment of neurodegenerative diseases.50 In addition, 12d treatment increased the protein levels of hippocalcin (HPCA), which is involved in long-term depression in hippocampal neurons and required for normal spatial learning (Figure 10 B, E).51 Of note, HPCA prevented Amyloid-beta toxicity.52 12d also increases protein levels of synaptotagmin 7 (SYT7) (Figure 10 C, E) calcium sensor involved in neuronal membrane trafficking, a transcendental process in neurodegenerative diseases with high synaptic vulnerability, accordingly we found a sparse increase in postsynaptic density 95 (PSD95) and Synaptophysin albeit did not reach significance (data not shown). In this string of evidence, neurotrophic response elements were also modulated by 12d treatment in SAMP8. Gene expression for Tropomyosin receptor kinase A (TrkA) and Figure 7. Acute and repeated effects of compound 12d at core body temperature in mice. (A) The hypothermic effect was induced by acute treatment with 12d (20mg/kg, i.p., 1 h). Columns are means± SEM of the difference in body temperature (°C) (Δ, 1 h minus basal value) for 12d-treated mice (n = 12) and vehicle-treated control mice (n = 13). Symbols represent individual values for each mouse. ***p < 0.001 vs control group (Student’s t test, t = 5.588, df = 23). (B) Hypothermic effects observed during the repeated treatment with 12d (20 mg/kg, i.p., 5 days). Columns are means ± SEM of the daily difference in body temperature (°C) (Δ, 1 h minus basal value) for 12d-treated mice (n = 6) and vehicle-treated control mice (n = 6). **p < 0.01 or ***p < 0.001 vs control group (two-way repeated measures ANOVA followed by Sidak’s multiple comparisons test). Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2559 https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig7&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig7&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig7&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig7&ref=pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as B (TrkB) was significantly increased, and, in parallel, a reduction in Neural growth factor (Ngf) and Brain-derived neurotrophic factor (Bdnf), its respective ligands, gene expression after 4 weeks of treatment in SAMP8 hippocampus (Figure 10F) demonstrating the neuroprotective effect of 12d in a mice model of cognitive decline and indicating a putative role as a new therapeutic approach for neurodegenerative conditions. Antiallodynic Effects of 12d in Capsaicin-Induced Mechanical Hypersensitivity Model and the Rotarod Test. Besides to its role in neurodegenerative diseases, I2−IRs are well-known by their analgesic properties. Indeed, CR4056 (1) is an effective analgesic against knee osteoarthritis.13 Therefore, in addition to the above-mentioned studies related with neuroprotection/neurodegeneration, we next explored the ability of 12d for reducing pain in a murine model. The enhanced pain sensitivity in the area surrounding capsaicin injection is known to result from central sensitization, which is a process that plays a pivotal role in chronic pain development.53 In fact, capsaicin-induced mechanical hyper- sensitivity has been used to study drug effects in central sensitization not only in rodents,54,55 but also in humans.56 We first tested the effect of CR4056 (1), a prototypical I2−IRs agonist, in capsaicin-induced mechanical hypersensitivity (allodynia) in mice. Nonsensitized mice showed a response latency to the mechanical stimulation of approximately 45 s. This response latency markedly decreased in mice intraplantarly treated with capsaicin, denoting the development of tactile allodynia (Figure S13A). CR4056 (1) (1.25−10 mg/kg, p.o.) induced a dose-dependent and nearly full reversal of capsaicin- induced allodynia (Figure S13A). To our knowledge, this is the first report on the antiallodynic effects of an I2−IRs agonist on this behavioral model of central sensitization. Then, we tested the effects of the association of CR4056 (1) with idazoxan (4) (3 mg/kg, s.c.), a well-known (albeit nonselective) I2−IRs antagonist.57 The administration of this latter compound fully suppressed the antiallodynic effect of CR4056 (1) (Figure Figure 8. Acute and repeated effects of compound 12d on the content of hippocampal neurochemical markers (FADD, Cdk5, pERK/ERK) in mice. (A) Acute (20 mg/kg, i.p., 1 h, n = 6) and repeated (20 mg/kg, i.p., 5 days, n = 6) effects induced by 12d. Columns are means ± SEM of the target protein (%Control (C), n = 7−6). Symbols represent individual values for eachmouse. *p = 0.0025 vs respective C group (Student’s t-test, t = 2.644, df = 10). (B) Representative immunoblots depicting the labeling of FADD, Cdk5 and pERK/ERK for each treatment group. Figure 9. Results obtained from NORT, both short- (A) and long-term memory (B), and OLT (C) evaluations in SAMP8. Control mice group and SAMP8 treated with the compound 12d mice group represented as the summary of DI. Values represented are mean ± Standard error of the mean (SEM); (n = 8 per group); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2560 https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig8&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig8&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig8&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig8&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig9&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig9&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig9&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig9&ref=pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as S13B). Therefore, these results suggest that the antiallodynic effect of CR4056 (1) is mediated through I2−IRs agonism. We then tested the effect of 12d (5−20mg/kg, s.c.), clonidine (0.05 mg/kg, s.c.) and gabapentin (10−40 mg/kg, s.c.). Both clonidine and gabapentin have known analgesic effects in human patients,58,59 and have been previously reported to decrease capsaicin-induced mechanical hypersensitivity in mice.55,58 However, mechanisms of their analgesic effects differ, since whereas clonidine is a prototypical α2-ARs agonist, with low affinity for imidazoline receptors,60 gabapentin acts through the modulation of α2δ auxiliary subunits of calcium channels61 (i.e., not related to actions on adrenergic or imidazoline receptors). We show here that not only clonidine and gabapentin, but also 12d, induced dose-dependent and full antiallodynic effects (Figure 11A). We also evaluated the in vivo effects of the association of 12d and clonidine with idazoxan (4) and its methoxy analog RX 821002 (both at 3 mg/kg, s.c.). Whereas idazoxan (4) is known to be an antagonist at both I2−IRs and α2- AR, RX 821002 is a selective α2-AR antagonist without affinity for imidazoline receptors.62 We selected doses of 12d and clonidine that induced the maximum antiallodynic effect (i.e., 12d 20 mg/kg and clonidine 0.05 mg/kg). The administration of idazoxan (4) fully reversed the antiallodynic effect of 12d, without modifying the effect induced by clonidine, suggesting that idazoxan (4) (in our experimental conditions) is acting primarily through I2−IRs but not α2-ARs. Conversely, RX 821002 completely reversed the effect of clonidine (as expected), but without significantly modifying the effect of 12d (Figure 11B). Altogether, these results suggest that 12d induces its antiallodynic effect through I2−IRs agonism without the participation of α2-AR. Finally, we also found that idazoxan (4) was unable to modify the antiallodynic effect of gabapentin (40 mg/kg) (Figure 14B). These latest results further validate the specificity of idazoxan in reversing only the effect of I2-ARs agonism. The rotarod test is a standard test to assess motor coordination in rodents. Drugs that affect rotarod latency are thought to impair motor coordination.63 We tested animals treated with 12d and gabapentin in rotarod performance, at doses able to reverse approximately the same degree of mechanical hypersensitivity (20 and 40 mg/kg, respectively). Administration of the vehicle of the drugs did not alter rotarod latency at any time point tested during the 4 h evaluation period, in comparison to the baseline value (time 0 in Figure 12). Figure 10. Representative Western blots and quantifications for PDE1B, HPCA, SYT7, and CAMKII (A − E). Values in bar graphs are adjusted to 100% for protein levels of the SAMP8 Control group. Representative gene expression forTrkA, Ngf, TrkB, andBdnf (F). Gene expression levels were determined by real-time PCR. Values represented are mean ± Standard error of the mean (SEM); (n = 3− 5 per group); *p < 0.05; **p < 0.01. Figure 11. Reduction of capsaicin-induced mechanical hypersensitivity by the administration of 12d, clonidine and gabapentin in mice. (A) Dose dependency of the antinociceptive effects induced by the subcutaneous (s.c.) administration of 12d, clonidine and gabapentin. (B) Effects of 12d, clonidine or gabapentin alone and associated with the administration of the I2−IRs antagonist idazoxan (4) or its methoxy analog RX 821002 (both at 3 mg/kg, s.c.). Values are the mean ± SEM obtained from 7−9 animals per group: **p < 0.01 vs nonsensitized animals treated with the vehicle of the drugs tested; ## p < 0.01 vs capsaicin-injectedmice treated with the vehicle of the drugs tested; †† p < 0.01 12d-treated animals associated with idazoxan (4) or its solvent, and clonidine-treated animals associated with RX 821002 or its solvent (one-way ANOVA followed by Bonferroni test). Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2561 https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig10&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig10&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig10&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig10&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig11&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig11&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig11&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig11&ref=pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as Similarly, animals administered with compound 12d showed no change in the latency to fall in comparison to the baseline value or to vehicle-treated mice, at any time point tested (Figure 12). Assessment of drug-induced motor functioning is relevant for the interpretation of the results from tests of nociception, as nonanalgesic treatments affecting motor coordination might attenuate nociceptive responses (which are motor responses) inducing false analgesic-like effects.63 Therefore, our results on the effects of 12d on capsaicin-induced mechanical hyper- sensitivity cannot be attributed to motor impairment. In contrast to the effects of 12d, gabapentin induced a marked decrease in the rotarod latency which peaked 45 min after its administration but that was still observable even 2 h after its administration (Figure 12). These data agree with previous rodent studies showing that gabapentin and other gabapenti- noids have an impact on rotarod performance,64,65 but also with the clinical effects of this type of drugs on human patients, since it is well-known that the most frequent side effects of gabapentinoids include fatigue, dizziness, sedation, somnolence, and ataxia. These effects occur in the range of the analgesic effects and have a clinical relevance limiting the use of the gabapentinoids.58 ■ CONCLUSIONS Following with our interest in the description of new I2−IRs ligands, we have accessed to a family of (3-phenylcarbamoyl-3,4- dihydro-2H-pyrrol-2-yl)phosphonates from previously reported bicyclic iminophosphonates. The evaluation of the affinity for I2−IRs in human tissues by a competition binding assay using radiolabeled compounds, led to the selection of representative 12d to continue with its pharmacological characterization. Compound 12d was endowed with optimal ADME parameters and showed a neuroprotective role on the human cell line SH- SY5Y and an anti-inflammatory activity in primary cultures of astrocytes and microglia treated with LPS. After the determination of its pharmacokinetic profile, the treatment of mice with 12d confirmed a hypothermic effect and a decrease in the content of FADD protein, a key signaling mediator of neuroprotective actions. The I2−IRs also fostered an improve- ment in the condition, spatial memory and working memory, of the murine SAMP8 model by a modification in the Ca2+/ calmodulin-dependent protein kinase II. Finally, compound 12d induced antiallodynic effects attributable to I2−IRs agonism in a behavioral model of central sensitization, and at doses not affecting motor coordination, unlike gabapentin, which induced a prominent motor impairment at therapeutic doses. To sum up, we have delivered a new pharmacological tool for characterizing the physiopatological implications of I2−IRs. This new I2−IRs ligand, with an unprecedent structure and endowed with optimal in vivo properties may be progressed toward early preclinical phases, either in neurodegenerative diseases or as analgesic. ■ EXPERIMENTAL SECTION Chemistry. General Information. Reagents, solvents and starting products were acquired from commercial sources. The term “concentration” refers to the vacuum evaporation using a Büchi rotavapor. When indicated, the reaction products were purified by “flash” chromatography on silica gel (35−70 μm) with the indicated solvent system. The melting points were measured in a MFB 59510 M Gallenkamp instruments. IR spectra were performed in a spectropho- tometer Nicolet Avantar 320 FTR-IR or in a Spectrum Two FT-IR Spectrometer, and only noteworthy IR absorptions (cm−1) are listed. NMR spectra were recorded in CDCl3 at 400 MHz (1H) and 100.6 MHz (13C), and chemical shifts are reported in δ values downfield from TMS or relative to residual chloroform (7.26 ppm, 77.0 ppm) as an internal standard. Data are reported in the following manner: chemical shift, multiplicity, coupling constant (J) in hertz (Hz), integrated intensity and assignment (when possible). The coupling constants (J) described in the 13C spectra referred to the carbon-phosphor couplings (JCP). Multiplicities are reported using the following abbreviations: s, singlet; d, doublet; dd, doublet of doublets; ddd, double double of doublets; dq, doble quadruplet; t, triplet; quin, quintet; m, multiplet; br s, broad signal, app, apparent. Assignments and stereochemical determinations are given only when they are derived from definitive two-dimensional NMR experiments (g-HSQC−COSY). Mass Spec- trometry was performed using LC/MSD TOF Agilent Technologies G1969A spectrometer by the Mass Spectrometry for Molecular Characterization Unit (Biomolecular Analysis Unit), from Scientific and Technological Centers (CCiTUB), Universitat de Barcelona. The elemental analyses were carried out in a Flash 1112 series Thermofinnigan elemental microanalyzator (A5) to determine C, H, and N. HPLC-MS (Agilent 1260 Infinity II) analysis was conducted on an Poroshell 120 EC-C15 (4.6 mm x50 mm, 2.7 μm) at 40 °C. Mobile phase (A: H20 + 0.05% formic acid and B: ACN + 0.05% formic acid) using a gradient elution. Flow rate 0.6 mL/min. The DAD detector was set at 254 nm and the injection volume was 5 μL and oven temperature 40 °C. All compounds are >95% pure by HPLC. General Procedure for the Opening of the Imide Ring in Basic Media. A solution of bicyclic α-iminophosphonates (11a−11n) in 0.05 M NaOH in THF/H2O 2:1 was stirred at room temperature for 2.5 h. The reaction mixture was concentrated in vacuo, water (2 mL) was added, and the pH of the mixture was acidified (pH = 3) with a 1 M solution of HCl. The reaction mixture was extracted with EtOAc, the organic phases were combined, dried over Na2SO4, filtered, and concentrated to give a residue that was purified by column chromatography to afford pure product 12a−12o. Diethyl [(2RS,3RS)-3-(Phenylcarbamoyl)-3,4-dihydro-2H-pyrrol- 2-yl]phosphonate (12a). Following the general procedure, 11a (100 mg, 0.28 mmol) and a solution of 0.05 M NaOH in THF/H2O 2:1 (7 mL) gave 12a (47 mg, 51%) as a white solid, after column chromatography (CH2Cl2/MeOH 97.5:2.5). An analytical sample was recrystallized in EtOAc. Mp 123−125 °C (EtOAc). IR (NaCl) 3265, 2983, 1686, 1601, 1444, 1232, 1028, 755, 693 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.41 (dt, J = 15.4, 7.0 Hz, 6H, OCH2CH3), 2.90 (ddd, J = 18.4, 10.4, 3.5 Hz, 1H, H-4), 3.31−3.39 (m, 1H, H-4), 3.41− Figure 12. Effect of 12d and gabapentin on motor coordination. The latency to fall-down from the rotarod was recorded in each mouse immediately before (time 0) and at several times after the subcutaneous (s.c.) administration of 12d (20 mg/kg), gabapentin (40 mg/kg) or their vehicle. Values are the mean ± SEM from 8 animals: ∗p < 0.05, ∗∗p < 0.01 between the values at time 0 and after drug administration; ##p < 0.01 between gabapentin- and vehicle-treated groups on the same time after treatment (2-way repeated measures ANOVA followed by Bonferroni test). Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2562 https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig12&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig12&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig12&ref=pdf https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?fig=fig12&ref=pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as 3.54 (ddt, J = 20.9, 10.4, 8.0 Hz, 1H, H-3), 4.18−4.34 (m, 4H, OCH2CH3), 4.45−4.53 (ddt, J = 17.4, 8.0, 2.7 Hz, 1H, H-2), 7.06−7.10 (tt, J = 7.3, 1.2 Hz, 1H, ArH), 7.27−7.33 (tt, 2H, J = 7.4 Hz, 2.0 Hz, ArH), 7.60 (d, J = 7.1 Hz, 2H, ArH), 7.65 (t, J = 2.6 Hz, 1H, H-5), 9.53 (br s, 1H, NH). 13C NMR (101 MHz, CDCl3) δ 16.4 (t, J = 4.9 Hz, OCH2CH3), 40.1 (C-4), 43.3 (C-3), 63.4 (dd, J = 7.4, 3.5 Hz, OCH2CH3), 72.3 (d, J = 162.0 Hz, C-2), 119.5 (2CHAr), 123.9 (CHAr), 128.8 (2CHAr), 138.5 (C-ipso), 169.0 (d, J = 16.5 Hz, C-5), 169.1 (CO). HRMS C15H22N2O4P [M + H]+ 325.1308; found, 325.1312. Anal. Cald. for C15H21N2O4P·1/4H2O: C, 54.79; H, 6.59; N, 8.52; found, C,54.60; H, 6.54; N, 8.36%. Diethyl (2RS,3RS)- 3-[(3-Chloro-4-fluorophenylcarbamoyl)-3,4- dihydro-2H-pyrrol-2-yl]phosphonate (12b). Following the general procedure, 11b (210 mg, 0.52 mmol) and 0.05 MNaOH in THF/H2O 2:1 (13 mL) gave 12b (86.8 mg, 44%) as a white solid after column chromatography (CH2Cl2/MeOH, 97.5:2.5). An analytical sample was recrystallized in EtOAc. Mp 120−123 °C (EtOAc). IR (NaCl) 3268, 2986, 1689, 1501, 1220, 1054, 1028, 973 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.42 (dt, J = 23.9, 7.0 Hz, 6H, OCH2CH3), 2.93 (dd, J = 18.0, 9.5 Hz, 1H, H-4), 3.32−3.39 (m, 2H, H-4), 3.40−3.48 (ddt, J = 20.9, 10.4, 8.2 Hz, 1H, H-3), 4.13−4.35 (m, 4H, OCH2CH3), 4.46−4.35 (m, 1H, H-2), 7.07 (t, J = 8.8 Hz, 1H, ArH), 7.42 (ddd, J = 9.0, 4.3, 2.5 Hz, 1H, ArH), 7.66 (t, J = 2.9 Hz, 1H, H-5), 7.77 (dd, J = 6.6, 2.6 Hz, 1H, ArH), 9.77 (br s, 1H, NH). 13C NMR (101 MHz, CDCl3) δ 16.4 (t, J = 4.6 Hz, OCH2CH3), 39.6 (d, J = 5.9 Hz, C-4), 43.3 (C-3), 63.4 (dd, J = 13.4, 6.6 Hz, OCH2CH3), 72.3 (d, J = 162.6 Hz, C-2), 116.4 (d, J = 22.0 Hz, CHAr), 119.0 (d, J = 7.0 Hz, CHAr), 121.0 (d, J = 18.0 Hz, C-ipso), 121.5 (CHAr), 135.2 (d, J = 3.0 Hz, C-ipso), 154.5 (d, J = 245.0 Hz, C- ipso), 168.0 (CO), 169.0 (d, J = 12.5 Hz, C-5). HRMS C15H20ClFN2O4P [M + H]+ 377.0836; found, 377.0828. Purity 100% (tR = 4.153 min). Diethyl [(2RS,3RS)-2-Phenyl-3-(phenylcarbamoyl)-3,4-dihydro- 2H-pyrrol-2-yl]phosphonate (12c). Following the general procedure, 11c (144 mg, 0.34 mmol) and 0.05MNaOH in THF/H2O 2:1 (8 mL) gave 12c (94 mg, 70%) as a white solid after column chromatography (EtOAc/MeOH, 95:5). An analytical sample was recrystallized in EtOAc. Mp 117−122 °C (EtOAc). IR (NaCl) 3267, 2981, 1692, 1600, 1548, 1443, 1225, 1028, 968, 755 cm−1. 1H NMR (400MHz, CDCl3) δ 1.08 (t, J = 7.1 Hz, 3H, CH2CH3), 1.36 (t, J = 6.9 Hz, 3H, CH2CH3), 2.87 (ddt, J = 18.4, 9.5, 1.2 Hz, 1H, H-4), 3.32 (dddd, J = 18.4, 8.2, 6.3, 1.0 Hz, 1H, H-4), 3.79−3.94 (complex signal, 2H, H-3 and CH2CH3), 4.06−4.15 (m, 1H, CH2CH3), 4.16−4.30 (m, 2H, CH2CH3), 7.04 (tt, J = 7.4, 1.3 Hz, 1H, ArH), 7.17−7.24 (m, 5H, ArH), 7.28−7.31 (m, 2H, ArH), 7.51−7.54 (m, 2H, ArH), 8.03 (dt, J = 3.8, 1.2 Hz, H-5), 9.24(br s, 1H, NH). 13C NMR (101 MHz, CDCl3) δ 16.0 (d, J = 5.8 Hz, CH2CH3), 16.4 (d, J = 5.8 Hz, CH2CH3), 39.5 (d, J = 5.0 Hz, C-4), 50.0 (d, J = 3.0 Hz, C-3), 63.5 (d, J = 8.0 Hz, CH2CH3), 64.8 (d, J = 7.5 Hz, CH2CH3), 83.9 (d, J = 162.2 Hz, C-2), 119.8 (2CHAr), 123.9 (CHAr), 127.2 (d, J = 7.2 Hz, 2 CHAr), 128.0 (d, J = 0.8 Hz, 2CHAr), 128.1 (d, J = 1.6 Hz, CHAr) 128.7 (2CHAr), 133.6 (C-ipso), 138.0 (C-ipso), 166.8 (d, J = 4.1 Hz, CO), 169.2 (d, J = 15.0 Hz, C-5). HRMS C21H26N2O4P [M +H]+ 401.1626; found, 401.1625. Anal. Cald. for C21H25N2O4P: C, 62.99; H, 6.29; N, 7.00; found, C, 63.76; H, 6.31; N, 6.55%. Purity 98.2% (tR = 4.176 min). Diethyl [(2RS,3RS)-3-((3-Chloro-4-fluorophenyl)carbamoyl)-2- phenyl-3,4-dihydro-2H-pyrrol-2-yl]phosphonate (12d). Following the general procedure, 9 (99.3 mg, 0.21 mmol) and 0.05 M NaOH in THF/H2O 2:1 (5 mL) gave 12d (71 mg, 76%) as a white solid after column chromatography (EtOAc/MeOH, 95:5). An analytical sample was recrystallized in EtOAc. Mp 136−138 °C (EtOAc). IR (NaCl) 3266, 2923, 2853, 1694, 1500, 1220, 1056, 1029, 971 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.07 (td, J = 7.1, 0.8 Hz, 3H, CH2CH3), 1.36 (t, J = 7.0 Hz, 3H, CH2CH3), 2.84−2.92 (ddt, J = 18.5, 9.4, 1.6 Hz, 1H, H- 4), 3.30 (dddd, J = 18.5, 7.5, 6.5, 1.0 Hz, 1H, H-4), 3.81−3.93 (complex signal, 2H, H-3 and CH2CH3), 4.03−4.11 (m, 1H, CH2CH3), 4.13− 4.28 (m, 2H, CH2CH3), 6.97 (t, J = 8.8 Hz, 1H, ArH), 7.13 (ddd, J = 8.5, 4.0, 2.5 Hz, 1H, ArH), 7.18−7.21 (m, 3H, ArH), 7.40 (dd, J = 6.6, 2.6 Hz, 1H ArH), 7.51−7.54 (m, 2H, ArH), 8.03 (dt, J = 4.0, 1.2 Hz, 1H, H-5), 9.57 (br s, 1H, NH). 13CNMR (101MHz, CDCl3) δ 16.0 (d, J = 5.8 Hz, CH2CH3), 16.4 (d, J = 5.7 Hz, CH2CH3), 39.9 (d, J = 4.1 Hz, C-4), 49.7 (d, J = 3.0 Hz, C-3), 63.6 (d, J = 8.1 Hz, CH2CH3), 65.0 (d, J = 7.6 Hz, CH2CH3), 84.3 (d, J = 160.9 Hz, C-2), 116.3 (d, J = 22.1 Hz, CHAr), 119.4 (d, J = 6.8 Hz, CHAr), 120.7 (d, J = 18.5 Hz, C-ipso), 121.8 (CHAr), 127.2 (d, J = 7.0 Hz, 2CHAr), 128.2 (2CHAr), 128.3 (CHAr), 133.6 (C-ipso), 134.7 (d, J = 3.2 Hz, C-ipso), 155.7(d, J = 246.3 Hz, C-ipso), 167.4 (d, J = 5.1 Hz, CO), 169.3 (d, J = 14.7 Hz, C- 5). HRMS C21H24ClFN2O4P [M + H]+ 453.1143; found, 453.1141. Anal. Cald. for C21H23ClN2O4P: C, 55.70; H, 5.12; N, 6.19; found, C, 55.82; H, 5.09; N, 5.93%. Purity 99.3% (tR = 4.496 min). Diethyl [(2RS,3RS)-3-((4-Chlorophenyl)carbamoyl)-2-phenyl-3,4- dihydro-2H-pyrrol-2-yl]phosphonate (12e). Following the general procedure, 11e (100mg, 0.22mmol) and a solution of 0.05MNaOH in THF/H2O 2:1 (7 mL) gave 12e (47 mg, 50%) as a white solid, after column chromatography (CH2Cl2/MeOH, 97.5:2.5). An analytical sample was recrystallized in EtOAc. Mp 130−132 °C (EtOAc). IR (ATR) 3300, 3263, 2984, 1662, 1540, 1491, 1251, 1036, 977, 752, 704 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.09 (td, J = 7.1, 0.7 Hz, 3H, OCH2CH3), 1.38 (td, J = 7.1, 0.6 Hz, 3H, OCH2CH3), 2.82−2.90 (dddd, J = 18.5, 9.6, 1.4, 0.7 Hz 1H, H-4), 3,27−3.36 (dddd, J = 18.4, 8.7, 6.2 0.9 Hz, 1H, H-4), 3.75−3.84 (quin, J = 9.0 Hz, 1H, H-3), 3.88− 3.98 (complex signal, 1H, OCH2CH3), 4.08−4.16 (m, 1H, OCH2CH3), 4.18−4.29 (m, 2H, OCH2CH3), 7.17−7.20 (m, 5H, ArH), 7.26−7.29 (m, 2H, ArH), 7.45−7.48 (m, 2H, ArH), 8.04 (d, J = 3.7 Hz, 1H, H-5), 9.37 (s, 1H, NH). 13C NMR (101 MHz, CDCl3) δ 16.2 (d, J = 5.8 Hz, OCH2CH3), 16.6 (d, J = 5.7 Hz, OCH2CH3), 39.3 (d, J = 5.5 Hz, C-4), 50.4 (d, J = 2.6 Hz, C-3), 63.8 (d, J = 8.0 Hz, OCH2CH3), 65.1 (d, J = 7.7 Hz, OCH2CH3), 83.9 (d, J = 163.2 Hz, C- 2), 121.0 (2CHAr), 127.2 (d, J = 7.2 Hz, 2CHAr), 128.3 (d, J = 0.6 Hz, 2CHAr), 128.4 (d, J = 1.5 Hz, CHAr), 128.8 (2CHAr), 128.9 (C-ipso), 133.4 (C-ipso), 136,8 (C-ipso), 166.9 (d, J = 3.2 Hz, CO), 169.3 (d, J = 15.3 Hz, C-5). HRMS C21H25ClN2O4P [M + H]+ 435.1235; found, 435.1237. Anal. Cald. for C21H24ClN2O4P: C, 58.00; H, 5.56; N, 6.44; found, C, 58.45; H, 5.34; N, 6.35%. Purity 98.1% (tR = 4.455 min). Diethyl [(2RS,3RS)-3-((1,1′-Biphenyl)-4-ylcarbamoyl)-2-phenyl- 3,4-dihydro-2H-pyrrol-2-yl]phosphonate (12f). Following the general procedure, 11f (100mg, 0.20mmol) and a solution of 0.05MNaOH in THF/H2O 2:1 (7 mL) gave 12f (47 mg, 50%) as a white solid, after column chromatography (EtOAc/hexane, 70:30). An analytical sample was recrystallized in EtOAc. Mp 194−195 °C (EtOAc). IR (ATR) 3248, 3058, 2923, 1686, 1537, 1485, 1222, 1027, 968, 764, 697 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.09 (td, J = 7.1, 0.7 Hz, 3H, OCH2CH3), 1.39 (td, J = 7.0, 0.6 Hz, 3H, OCH2CH3), 2.85−2.92 (ddd, J = 18.5, 9.6, 0.8Hz, 1H, H-4), 3.31−3.39 (dddd, J = 18.4, 8.8, 6.1, 0.8 Hz, 1H, H-4), 3.79−3.88 (quin, J = 9.3 Hz, 1H, H-3), 3.89−3.99 (m, 1H, OCH2CH3), 4.10−4.18 (m, 1H, OCH2CH3), 4.19−4.33 (m, 2H, OCH2CH3), 7.19−7.22 (m, 3H, ArH), 7.31 (tt, J = 7.4, 1.2 Hz, 1H, ArH), 7.38−7.43 (m, 4H, ArH), 7.48 (t, J = 2.1 Hz, 1H, ArH), 7.49− 7.53 (m, 3H, ArH), 7.54−7.57 (m, 2H, ArH), 8.06 (dt, J = 3.6, 1.3 Hz, 1H, H-5), 9.30 (s, 1H, NH). 13C NMR (101 MHz, CDCl3) δ 16.2 (d, J = 5.7 Hz, OCH2CH3), 16.6 (d, J = 5.7 Hz, OCH2CH3), 39.3 (d, J = 5.5 Hz, C-4), 50.5 (d, J = 2.7 Hz, C-3), 63.8 (d, J = 7.9 Hz, OCH2CH3), 65.0 (d, J = 7.6 Hz, OCH2CH3), 83.8 (d, J = 163.3 Hz, C-2), 120.2 (2CHAr), 126.9 (2CHAr), 127.1 (CHAr), 127.3 (d, J = 7.1Hz, CHAr), 127.5 (2CHAr), 128.3 (2CHAr), 128.4 (2CHAr), 128.8 (2CHAr), 133.5 (C-ipso), 136.8 (C-ipso), 137.5 (C-ipso), 140.8 (C-ipso), 166.8 (d, J = 3.0 Hz, CO), 169.4 (d, J = 15.4 Hz, C-5). HRMS C27H30N2O4P [M + H]+ 477.1938; found, 477.1940. Anal. Cald. for C27H29N2O4P: C, 68.06; H, 6.13; N, 5.88; found: C, 67.94; H, 6.28; N, 5.73%. Purity 97.2% (tR = 4.680 min). Diethyl [(2RS,3RS)-2-Phenyl-3-((4-(trifluoromethyl)phenyl)- carbamoyl)-3,4-dihydro-2H-pyrrol-2-yl]phosphonate (12g). Follow- ing the general procedure, 11g (100 mg, 0.20 mmol) and a solution of 0.05 M NaOH in THF/H2O 2:1 (7 mL) gave 12g (47 mg, 50%) as a white solid, after column chromatography (EtOAc/hexane, 70:30). An analytical sample was recrystallized in EtOAc. Mp 137−139 °C (EtOAc). IR (ATR) 3269, 3087, 2990, 1698, 1560, 1493, 1335, 1222, 1163, 1023, 978, 898, 796 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.09 (td, J = 7.0, 0.8 Hz, 3H, OCH2CH3), 1.37 (td, J = 7.1, 0.6 Hz, 3H, OCH2CH3), 2.84−2.92 (ddt, J = 18.4, 9.6, 1.3 Hz, 1H, H-4), 3.28− 3.37(dddd, J = 18.4, 8.6, 6.2, 1.0 Hz, 1H, H-4), 3.90 (quin, J = 9.2, 1H, Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2563 pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as H-3), 3.92−3.99 (m, 1H, OCH2CH3), 4.11−4.17 (m, 1H, OCH2CH3), 4.19−4.29 (m, 2H, OCH2CH3), 7.17−7.20 (m, 3H, ArH), 7.27−7.35 (dt, J = 15.8, 7.8 Hz, 2H, ArH), 7.48−7.51 (m, 3H, ArH), 7.60 (s, 1H, ArH), 8.06 (dt, J = 3.7, 1.2 Hz, 1H, H-5), 9.60 (s, 1H, NH). 13C NMR (101 MHz, CDCl3) δ 16.2 (d, J = 5.8 Hz, OCH2CH3), 16.6 (d, J = 5.7 Hz, OCH2CH3), 39.3 (d, J = 5.3 Hz, C-4), 50.4 (d, J = 2.9 Hz, C-3), 63.8 (d, J = 7.9 Hz, OCH2CH3), 65.1 (d, J = 7.6 Hz, OCH2CH3), 83.9 (d, J = 162.9Hz, C-2), 116.7 (q, J = 4.0Hz, CHAr), 120.6 (q, J = 3.8 Hz, CHAr), 123.0 (d, J = 1.2 Hz, CHAr), 125.9 (d, J = 273.5 Hz, CF3), 127.2 (d, J = 7.1 Hz, 2CHAr), 128.3 (d, J = 0.6 Hz, 2CHAr), 128.5 (d, J = 1.5 Hz, CHAr), 129.3 (CHAr), 131.1 (q, J = 32.0 Hz, CCF3), 133.4 (C-ipso), 138.6 (C-ipso), 167.3 (d, J = 3.5 Hz, CO), 169.3 (d, J = 15.3 Hz, C-5). HRMS C22H25F3N2O4P [M + H]+ 469.1499; found, 469.1502. Anal. Cald. for C22H25ClFN2O5P: C, 56.41; H, 5.16; N, 5.98; found, C, 56.65; H, 5.28; N, 5.82%. Diethyl [(2RS,3RS)-3-(Cyclohexylcarbamoyl)-2-phenyl-3,4-dihy- dro-2H-pyrrol-2-yl]phosphonate (12h). Following the general proce- dure, 11h (116mg, 0.27mmol) and 0.05MNaOH in THF/H2O 2:1 (6 mL) gave 12h (80 mg, 73%) as a white solid after column chromatography (EtOAc/MeOH, 95:5). An analytical sample was recrystallized in EtOAc. Mp 143−148 °C (EtOAc). IR (NaCl) 3269, 2934, 2854, 1640, 1560, 1240 cm−1. 1H NMR (400 MHz, CDCl3) δ 0.90−1.25 (m, 5H, CH2cycl), 1.08 (td, J = 7.2 0.6 Hz, 3H, OCH2CH3), 1.31 (td, J = 7.1, 0.5 Hz, 3H, OCH2CH3), 1.50−1.73 (m, 5H, CH2cycl), 2.83−2.91 (ddq, J = 18.4, 9.6, 1.0 Hz, 1H, H-4), 3.28 (dtd, J = 18.4, 7.0, 1.0 Hz, 1H, H-4), 3.34−3.42 (m, 1H, CHcycl), 3.56−3.65 (ddd, J = 18.6, 9.6, 7.1 Hz, 1H, H-3), 3.82−3.91 (m, 1H, OCH2CH3), 4.07 (m, 1H, OCH2CH3), 4.10−4.24 (m, 2H, OCH2CH3), 6.54 (d, J = 7.6 Hz, 1H,NH), 7.23−7.31 (m, 3H, ArH), 7.54−7−57 (m, 2H, ArH), 7.98 (d, J = 4.2 Hz, H-5). 13C NMR (101 MHz, CDCl3) δ 16.0 (d, J = 5.6 Hz, OCH2CH3), 16.3 (d, J = 5.8 Hz, OCH2CH3), 24.7 (2CH2cycl), 25.5 (CH2cycl), 32.3 (CH2cycl), 32.9 (CH2cycl), 40.8 (d, J = 3.7 Hz, C-4), 48.2 (CHcycl), 48.7 (d, J = 2.9 Hz, C-3), 63.2 (d, J = 7.9 Hz, OCH2CH3), 64.4 (d, J = 7.5 Hz, OCH2CH3), 84.4 (d, J = 159.8 Hz, C- 2), 127.6 (d, J = 7.1 Hz, 2CHAr), 127.8 (d, J = 1.0 Hz, 2CHAr), 127.9 (d, J = 1.8Hz, CHAr), 134.4 (d, J = 1.0Hz, C-ipso), 167.8 (d, J = 6.3Hz, CO), 169.2 (d, J = 14.2 Hz, C-5). HRMS C21H32N2O4P [M + H]+ 407.2096; found, 407.2094. Purity 99.7% (tR = 4.487 min). Diethyl [(2RS,3RS)-2-(4-Fluorophenyl)-3-(phenylcarbamoyl)-3,4- dihydro-2H-pyrrol-2-yl]phosphonate (12i). Following the general procedure, 11i (100mg, 0.23mmol) and a solution of 0.05MNaOH in THF/H2O 2:1 (7 mL) gave 12i (47 mg, 50%) as a white solid, after column chromatography (EtOAc/hexane, 70:30). An analytical sample was recrystallized in EtOAc. Mp 188−190 °C (EtOAc). IR (ATR) 3262, 3145, 2988, 1659, 1550, 1443, 1227, 1049, 971, 753, 691 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.10 (t, J = 7.0 Hz, 3H, OCH2CH3), 1.35 (t, J = 7.1 Hz, 3H, OCH2CH3), 2.84−2.92 (ddt, J = 18.5, 9.6, 1.2 Hz, 1H, H-4), 3.32 (dt, J = 18.4, 6.7 Hz, 1H, H-4), 3.75−3.84 (quin, J = 8.4 Hz, 1H, H-3), 3.87−3.96 (m, 1H, OCH2CH3), 4.07−4.16 (m, 1H, OCH2CH3), 4.17−4.30 (m, 2H, OCH2CH3), 6.89 (t, J = 8.6 Hz, 2H, ArH), 7.07 (tt, J = 7.3, 1.3 Hz, 1H, ArH), 7.24 (d, J = 8.3 Hz, 2H, ArH), 7.32 (d, J = 7.6 Hz, 2H, ArH), 7.48−7.52 (ddd, J = 8.9, 5.2, 1.7 Hz, 2H, ArH), 8.03 (d, J = 3.8 Hz, 1H, H-5), 9.12 (s, 1H, NH). 13C NMR (101 MHz, CDCl3) δ 16.3 (d, J = 5.7 Hz, OCH2CH3), 16.6 (d, J = 5.6 Hz, OCH2CH3), 39.7 (d, J = 5.0 Hz, C-4), 50.2 (d, J = 3.3 Hz, C-3), 63.8 (d, J = 8.0 Hz, OCH2CH3), 65.0 (d, J = 7.6 Hz, OCH2CH3), 83.5 (d, J = 163.0 Hz, C-2), 115.3 (d, J = 21.5 Hz, 2 CHAr), 119.8 (2CHAr), 124.2 (CHAr), 129.0 (2CHAr), 129.2 (d, J = 7.0 Hz, CHAr), 129.3 (d, J = 7.1 Hz, CHAr), 129.6 (d, J = 2.8 Hz, C-ipso), 137.9 (C-ipso), 163.8 (d, J = 248.9 Hz, C-ipso), 166.8 (d, J = 3.8 Hz, CO), 169.6 (d, J = 15.1 Hz, C- 5). HRMS C21H25FN2O4P [M +H]+ 419.1530; found, 419.1526. Anal. Cald. for C21H24FN2O4P: C, 60.28; H, 5.78; N, 6.70; found, C, 60.29; H, 6.00; N, 6.26%. Purity 97.0% (tR = 4.234 min). Diethyl [(2RS,3RS)-3-((3-Chloro-4-fluorophenyl)carbamoyl)-2-(4- fluorophenyl)-3,4-dihydro-2H-pyrrol-2-yl]phosphonate (12j). Fol- lowing the general procedure, 11j (100 mg, 0.20 mmol) and a solution of 0.05 M NaOH in THF/H2O 2:1 (7 mL) gave 12j (47 mg, 50%) as a white solid, after column chromatography (EtOAc/hexane, 70:30). An analytical sample was recrystallized in EtOAc. Mp 176−178 °C (EtOAc). IR (ATR) 3264, 3069, 2986, 1684, 1499, 1397, 1226, 1187, 1024, 972, 805 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.12 (td, J = 7.0, 0.7 Hz, 3H, OCH2CH3), 1.38 (td, J = 7.0, 0.6 Hz, 3H, OCH2CH3), 2.83−2.91 (ddt, J = 18.2, 9.6, 1.2 Hz, 1H, H-4), 3.25−3.33 (dddd, J = 18.5, 8.5, 6.2, 1.0 Hz, 1H, H-4), 3.83 (quin, J = 9.4 Hz, 1H, H-3), 3.90− 3.99 (m, 1H, OCH2CH3), 4.08−4.16 (m, 1H, OCH2CH3), 4.18−4.29 (m, 2H, OCH2CH3), 6.90 (t, J = 8.4 Hz, 2H, ArH), 7.00 (t, J = 8.8 Hz, 1H, ArH), 7.17−7.21 (ddd, J = 8.9, 4.2, 2.6 Hz, 1H, ArH), 7.45−7.49 (m, 3H, ArH), 8.03 (dt, J = 3.7, 1.2 Hz, 1H, H-5), 9.45 (s, 1H, NH). 13C NMR (101MHz, CDCl3) δ 16.1 (d, J = 5.8Hz, OCH2CH3), 16.4 (d, J = 5.7 Hz, OCH2CH3), 39.2 (d, J = 5.2 Hz, C-4), 50.0 (d, J = 3.2 Hz, C-3), 63.8 (d, J = 8.1 Hz, OCH2CH3), 65.0 (d, J = 7.8 Hz, OCH2CH3), 83.2 (d, J = 163.3 Hz, C-2), 115.2 (d, J = 21.4 Hz, 2CHAr), 116.5 (d, J = 22.0 Hz, CHAr), 119.1 (d, J = 6.9 Hz, CHAr), 121.0 (d, J = 18.7 Hz, C-ipso), 121.6 (CHAr), 128.9 (d, J = 7.3 Hz, CHAr), 129.0 (d, J = 7.3 Hz, CHAr), 129.2 (d, J = 3.2Hz, C-ipso), 134.6 (d, J = 3.4Hz, C-ipso), 155.7 (d, J = 246.8 Hz, C-ipso), 162.7 (d, J = 249.7 Hz, C-ipso), 166.7 (d, J = 3.5Hz, CO), 169.4 (d, J = 15.3Hz, C-5). HRMSC21H23ClF2N2O4P [M + H]+ 471.1047; found, 471.1059. Anal. Cald. for C21H22ClF2N2O4P: C, 53.57; H, 4.71; N, 5.95; found, C, 54.06; H, 4.95; N, 5.81%. Diethyl [(2RS,3RS)-2-(4-Methoxyphenyl)-3-(phenylcarbamoyl)- 3,4-dihydro-2H-pyrrol-2-yl]phosphonate (12k). Following the gen- eral procedure, 11k (100 mg, 0.22 mmol) and a solution of 0.05 M NaOH in THF/H2O 2:1 (7 mL) gave 12k (47 mg, 50%) as a white solid, after column chromatography (EtOAc/hexane, 70:30). An analytical sample was recrystallized in EtOAc. Mp 105−107 °C (EtOAc). IR (ATR) 3299, 3144, 2987, 1659, 1550, 1443, 1240, 1032, 970, 832, 764 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.10 (t, J = 7.0 Hz, 3H, OCH2CH3), 1.37 (t, J = 7.0Hz, 3H,OCH2CH3), 2.87 (dd, J = 18.3, 9.6 Hz, 1H, H-4), 3.35 (ddd, J = 18.0, 8.2, 6.4 Hz, 1H, H-4), 3.68 (s, 3H, OCH3), 3.73−382 (quin, J = 9.2 Hz, 1H, H-3), 3.86−3.96 (m, 1H, OCH2CH3), 4.07−4.15 (m, 1H, OCH2CH3), 4.17−4.28 (m, 2H, OCH2CH3), 6.71 (d, J = 8.8 Hz, 2H, ArH), 7.05 (t, J = 7.4 Hz, 1H, ArH), 7.25 (t, J = 7.6 Hz, 2H, ArH), 7.36 (d, J = 7.6 Hz, 2H, ArH), 7.42 (dd, J = 8.8, 1.6 Hz, 2H, ArH), 8.01 (d, J = 3.6 Hz, 1H, H-5), 9.26 (s, 1H, NH). 13C NMR (101 MHz, CDCl3) δ 16.3 (d, J = 5.8 Hz, OCH2CH3), 16.6 (d, J = 5.7 Hz, OCH2CH3), 39.2 (d, J = 5.7 Hz, C-4), 50.3 (d, J = 3.3Hz, C-3), 55.2 (OCH3), 63.6 (d, J = 8.0Hz, OCH2CH3), 64.9 (d, J = 7.6 Hz, OCH2CH3), 83.3 (d, J = 163.2 Hz, C-2), 113.7 (2CHAr), 119.9 (2CHAr), 124.0 (CHAr), 125.4 (C-ipso), 128.6 (d, J = 7.1 Hz, 2CHAr), 128.9 (2CHAr), 138.2 (C-ipso), 159.4 (d, J = 1.4 Hz, C-ipso), 166.9 (d, J = 3.0 Hz, CO), 169.1 (d, J = 15.6 Hz, C-5). HRMS C22H28N2O5P [M + H]+ 431.1730; found, 431.1727. Anal. Cald. for C22H27N2O5P: C, 61.39; H, 6.32; N, 6.51; found, C, 61.14; H, 6.41; N, 6.34%. Purity 98.0% (tR = 4.156 min). Diethyl [(2RS,3RS)-3-((3-Chloro-4-fluorophenyl)carbamoyl)-2-(4- methoxyphenyl)-3,4-dihydro-2H-pyrrol-2-yl]phosphonate (12l). Following the general procedure, 11l (100 mg, 0.20 mmol) and a solution of 0.05 M NaOH in THF/H2O 2:1 (7 mL) gave 2l (47 mg, 50%) as a white solid, after column chromatography (EtOAc/hexane, 70:30). An analytical sample was recrystallized in EtOAc. Mp 121−123 °C (EtOAc). IR (ATR) 3257, 3073, 2981, 1689, 1499, 1218, 1027, 975, 828, 789 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.12 (t, J = 7.2, 0.9 Hz, 3H, OCH2CH3), 1.37 (td, J = 7.0, 0.6 Hz 3H, OCH2CH3), 2.79−2.87 (ddd, J = 18.4, 9.5, 1.4Hz, 1H, H-4), 3.25−3.33 (dddd, J = 18.4, 8.9, 6.2, 1.0 Hz, 1H, H-4), 3.71 (s, 3H, OCH3), 3.81 (quin, J = 9.0 Hz, 1H, H-3), 3.89−3.99 (m, 1H, OCH2CH3), 4.08−4.16 (m, 1H, OCH2CH3), 4.18−4.28 (m, 2H, OCH2CH3), 6.73 (d, J = 8.2Hz, 2H, ArH), 7.02 (t, J = 8.7 Hz, 1H, ArH), 7.21 (ddd, J = 8.9, 4.2 2.6 Hz, 1H, ArH), 7.38 (dd, J = 9.1, 1.7 Hz, 2H, ArH), 7.49 (dd, J = 6.6, 2.6 Hz, 1H, ArH), 8.01 (dt, J = 3.6, 1.1 Hz, 1H, H-5), 9.52 (s, 1H, NH). 13C NMR (101 MHz, CDCl3) δ 16.3 (d, J = 5.8 Hz, OCH2CH3), 16.6 (d, J = 5.6 Hz, OCH2CH3), 38.9 (d, J = 5.7 Hz, C-4), 50.3 (d, J = 3.4 Hz, C-3), 55.3 (OCH3), 63.8 (d, J = 7.9 Hz, OCH2CH3), 65.1 (d, J = 7.6 Hz, OCH2CH3), 83.2 (d, J = 163.5 Hz, C-2), 113.7 (2CHAr), 116.4 (d, J = 22.0 Hz, CHAr), 119.4 (d, J = 6.8 Hz, CHAr), 121.0 (d, J = 18.7 Hz, C- ipso), 122.0 (CHAr), 125.1 (C-ipso), 128.5 (d, J = 7.2 Hz, 2CHAr), 134.9 (d, J = 3.2 Hz, C-ipso), 155.8 (d, J = 246.3 Hz, C-ipso), 159.5 (d, J = 1.4Hz, C-ipso), 167.1 (d, J = 2.9Hz, CO), 169.1 (d, J = 15.6 Hz, C-5). HRMS C22H26ClFN2O5P [M + H]+ 483.1246; found, 483.1249. Anal. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2564 pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as Cald. for C22H25ClFN2O5P: C, 54.72; H, 5.22; N, 5.80; found: C, 54.91; H, 5.50; N, 5.65%. Diethyl [(2RS,3RS)-2-Methyl-3-(phenylcarbamoyl)-3,4-dihydro- 2H-pyrrol-2-yl]phosphonate (12m). Following the general procedure, 11m (62 mg, 0.17 mmol) and 0.05 MNaOH in THF/H2O 2:1 (4 mL) gave 12m (18 mg, 31%) as a white solid after column chromatography (AcOEt:MeOH, 95:5). An analytical sample was recrystallized in EtOAc. Mp 129−132 °C (EtOAc). IR (ATR) 3339, 2982, 2911, 1665, 1599, 1543, 1443, 1228, 1047, 1016, 955, 755, 691 cm−1. 1HNMR (400 MHz, CDCl3) δ 1.36 (t, J = 9.2 Hz, 3H, OCH2CH3), 1.38 (d, J = 14.9 Hz, 3H, CH3), 1.44 (t, J = 7.0 Hz, 3H, OCH2CH3), 2.75−2.85 (m, 1H, H-4), 3.43−3.57 (complex signal, 2H, H-3 and H-4), 4.16−4.26 (m, 2H, OCH2CH3), 4.29−4.39 (m, 2H, OCH2CH3), 7.06−7.10 (tt, J = 7.4, 1.2 Hz, 1H, ArH), 7.29−7.33 (t, J = 7.4 Hz, 2H, ArH), 7.58 (complex signal, 1H, ArH), 7.61 (m, 2H, H-5 and ArH) 9.65 (br s, 1H, NH).13C NMR (101 MHz, CDCl3) δ 16.4 (d, J = 5.7 Hz, OCH2CH3), 16.5 (d, J = 5.9 Hz, OCH2CH3), 17.3 (CH3), 38.2 (d, J = 6.7 Hz, C-4), 47.3 (d, J = 3.8 Hz, C-3), 63.5 (d, J = 7.7 Hz, OCH2CH3), 64.2 (d, J = 7.4 Hz, OCH2CH3), 76.7 (d, J = 168.6 Hz, C-2), 119.6 (2CHAr), 123.9 (CHAr), 128.9 (2CHAr), 138.4 (C-ipso), 166.7 (d, J = 16.6 Hz, C-5), 167.3 (d, J = 1.6 Hz, CO). HRMS C16H24N2O4P [M + H]+ 339.1475; found, 339.1468. Diethyl [(2RS,3RS)-2-Benzyl-3-(phenylcarbamoyl)-3,4-dihydro- 2H-pyrrol-2-yl]phosphonate (12n). Following the general procedure, 11n (45 mg, 0.10 mmol) and a solution of 0.05MNaOH in THF/H2O 2:1 (4 mL) gave 12n (8 mg, 19%) as a yellowish oil after column chromatography (EtOAc/hexane, 60:40). IR (ATR) 3263, 3196, 3032, 2925, 2853, 1688, 1621, 1601, 1555, 1496, 1443, 1301, 1253, 1218, 1050, 1023, 971, 792, 755, 729, 695 cm−1. 1HNMR (400MHz, CDCl3) δ 1.32−1.38 (dtd, J = 10.7, 7.1, 0.6 Hz, 6H, OCH2CH3), 2.50−2.57 (dddd, J = 18.6, 10.3, 5.7, 1.0 Hz, 1H, H-4), 2.60 (dd, J = 9.9, 1.4 Hz, 1H, H-4), 3.22−3.34 (m, 2H, CH2-benzyl), 3.53−3.63 (quin, J = 9.8 Hz, 1H, H-3), 4.16−4.31 (m, 4H, OCH2CH3), 7.10 (m, 3H, ArH), 7.18 (m, 3H, ArH), 7.36 (m, 2H, ArH), 7.70 (complex signal, 2H, ArH), 7.72 (complex signal, 1H, H-5), 10.28 (br s, 1H, NH). 13C NMR (101 MHz, CDCl3) δ 16.4 (d, J = 1.6 Hz, CH2CH3), 16.5 (d, J = 1.7 Hz, CH2CH3), 36.6 (CH2-benzyl), 39.3 (d, J = 7.6 Hz, C-4), 47.8 (d, J = 3.1 Hz, C-3), 63.6 (d, J = 7.8 Hz, CH2CH3), 64.4 (d, J = 7.8 Hz, CH2CH3), 79.7 (d, J = 164.5 Hz, C-2), 119.4 (2CHAr), 123.8 (CHAr), 127.0 (CHAr), 127.6 (2CHAr), 129.0 (2CHAr), 131.4 (2CHAr), 134.0 (d, J = 13.5 Hz, C-ipso), 138.8 (C-ipso), 166.7 (CO), 168.9 (d, J = 17.1 Hz, C-5). HRMS C22H28N2O4P [M + H]+ 415.1781; found, 415.1763. Purity: 95.2% (tR = 4.703 min). Diethyl [(2RS,3RS)-2-Benzyl-3-(cyclohexylcarbamoyl)-3,4-dihy- dro-2H-pyrrol-2-yl]phosphonate (12o). Following the general proce- dure, 11o (76mg, 0.17mmol) and a solution of 0.05MNaOH in THF/ H2O 2:1 (4 mL) gave 12o (41 mg, 57%) as a white solid, after column chromatography (EtOAc/hexane, 70:30). An analytical sample was recrystallized in EtOAc. Mp 119−121 °C (EtOAc). IR (ATR) 3522, 3294, 2928, 2856, 1638, 1541, 1238, 1216, 1025, 974, 959, 758, 697 cm−1. 1H NMR (400MHz, CDCl3) δ 1.28 (tdd, J = 7.1, 1.6 0.6 Hz, 6H, OCH2CH3), 1.33−1.48 (m, 5H, CH2cycl), 1.58−1.64 (m, 1H, CH2cycl), 1.71−1.81 (m, 2H, CH2cycl), 1.89−1.95 (m, 1H, CH2cycl), 2.00−2.08 (m, 1H, CH2cycl), 2.53−2.62 (ddd, J = 18.6, 10.2, 1.5 Hz, 1H, H-4), 2.66−2.77 (dddd, J = 16.1, 9.9, 6.2, 1.0 Hz, 1H, H-4), 3.09− 3.26 (complex signal, 2H, CH2-benzyl), 3.44 (dt, J = 19.3, 10.1 Hz, 1H, H-3), 3.81−3.90 (m, 1H, CHcycl), 3.98- 4.17 (m, 4H, OCH2CH3), 7.15−7.23 (m, 5H, ArH), 7.63 (br d, J = 3.2 Hz, 1H, NH), 7.82 (d, J = 7.8 Hz, 1H, H-5). 13C NMR (101 MHz, CDCl3) δ 16.4 (d, J = 5.7, 2OCH2CH3), 24.8 (d, J = 3.0 Hz, 2CH2cycl), 25.6 (CH2cycl), 32.9 (2CH2cycl), 36.8 (CH2-benzyl), 39.6 (d, J = 7.1 Hz, C-4), 47.1 (d, J = 3.0 Hz, C-3), 48.5 (CHcycl), 63.4 (d, J = 7.7 Hz, OCH2CH3), 63.8 (d, J = 7.7Hz, OCH2CH3), 79.9 (d, J = 164.9Hz, C-2), 126.7 (CHAr), 127.5 (2CHAr), 131.5 (2CHAr), 135.0 (d, J = 10.9 Hz, C-ipso), 167.9 (d, J = 1.9 Hz, CO), 168.9 (d, J = 16.8 Hz, C-5). HRMS C22H34N2O4P [M + H]+ 421.2251; found, 421.2266. Purity 97.0% (tR = 4.639 min). X-Ray Crystallographic Analysis of 12b, 12d, and 12h. Crystals of 12b, 12d, and 12h were obtained from slow evaporation of methanol solutions. Single crystal X-ray diffraction data sets for 12d and 12h were collected at 295 K up to a max 2ϑ of ca. 57° on a Bruker Smart APEX II diffractometer, usingmonochromaticMoKα radiation λ = 0.71073 Å and 0.3° separation between frames. Data integration was performed using SAINT V6.45A and SORTAV66 in the diffractometer package.67 The single crystal experiment for 12b was made on an Enraf Nonius CAD4 diffractometer using also MoKα radiation. The crystal and collection data and structural refinement parameters are given in the Tables S3−S17 in the Supporting Information. The structures were solved by direct methods using SHELXT-2014 and Fourier’s difference methods and refined by least-squares on F2 using SHELXL-2014/7 inside the WinGX program environment.68,69 Anisotropic displace- ment parameters were used for non-H atoms and the H atoms were positioned in calculated positions and refined riding on their parent atoms. Atom coordinates are given and bond distances and hydrogen bonds. All this information is detailed in the Tables S3−S17 in the Supporting Information. 12b crystallized in the triclinic P-1 space group (Ortep in Figure 3). The 1-pyrroline ring presented envelope conformation on C3 (E- form). The phenyl ring appears as flat. The stereocenters C2 and C3 exhibit identical quiralities, either R, R or S, S. Molecules are packed in dimers through the mutual N7−H7···O1 hydrogen bond. 12d crystallized in the triclinic P-1 space group (Ortep in Figure 3). The 1-pyrroline ring presented envelope conformation on C3. The phenyl rings appear as flat. The stereocenters C2 and C3 exhibit opposed quiralities either R, S or S, R, as the space group is centrosymmetric. Molecules are packed in dimers through the mutual N7−H7···O1 hydrogen bond. 12h crystallized in the monoclinic P21/a space group space group with four molecules per asymmetric unit (Ortep in Figure 3). All trifluoromethyl groups have been modeled as having two possible rotational disordered orientations (for two molecules, the disorder was about 50:50, and for the other two, 80:20 and 64:36, approximately). Also, one terminal ethyl group exhibited two different orientations (67:33). The five-membered rings present envelope conformations on C3, C23, C43 and C63. The six-membered rings appeared all as planar. The stereocenters C2 and C3 showed opposed quirality, as well as C22 and C23, C42 and C43 and C62 and C63 (S and R, respectively, for the asymmetric unit provided). Molecules are packed as two dimers through the N7−H7···O10 and N67−H67···O1 hydrogen bonds, for one of them, and through the N27−H27···O7 and N47−H47···O5 hydrogen bonds, for the other. Dimers are further linked by other hydrogen bonds of different types (C−H···F, C−H···O, C−H···N), amounting for a total of 23 identified formal HBs (see Table S17 for 12h) Crystallographic data for the reported structures has been deposited in the Cambridge Crystallographic Data Centre as supplementary publication, CCDC No. 2369628, 2369629, and 2369630. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK. Fax: +44 1223 336 033. Email: data_request@ccdc.cam.ac.uk. Web page: http:// www.ccdc. cam.ac.uk. Binding Studies. Preparation of Cellular Membranes. Human brain samples were obtained at autopsy in the Basque Institute of Legal Medicine, Bilbao, Spain. Samples from the prefrontal cortex (Brodmann’s area 9) were dissected at the time of autopsy and immediately stored at −70 °C until assay. The study was developed in compliance with policies of research and ethical review boards for post- mortem brain studies. For I1-IR experiments kidneys were obtained from male Sprague−Dawley rats (250−280 g). To obtain cellular membranes (P2 fraction) the different samples were homogenized using an ultraturrax in 10 volumes of homoge- nization buffer (0.25 M sucrose, 5 mM Tris−HCl, pH 7.4). The crude homogenate was centrifuged for 5 min at 1000 g (4 °C) and the supernatant was centrifuged again for 10 min at 40,000 g (4 °C). The resultant pellet was washed twice in 5 volumes of homogenization buffer and recentrifuged in similar conditions. Protein content was measured according to the method of Bradford using BSA as standard. Competition Binding Assays. The pharmacological activity of the compounds was evaluated through competition binding studies against the I2−IRs selective radioligand [3H]2-BFI, the I1−IRs selective radioligand [3H]clonidine, or the α2-adrenergic receptor selective Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2565 https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as radioligand [3H]RX821002. Specific binding was measured in 0.25 mL aliquots (50 mM Tris-HCl, pH 7.5) containing 100 μg of membranes, which were incubated in 96-well plates either with [3H]2-BFI (2 nM) for 45 min at 25 °C, [3H]clonidine (5 nM) for 45 min at 22 °C or [3H]RX821002 (1 nM) for 30 min at 25 °C, in the absence or presence of the competing compounds (10−12 to 10−3 M, 10 concentrations). [3H]clonidine experiments were performed in the presence of 10 μM adrenaline to preclude binding to α2-adrenoceptors. Incubations were terminated by separating free ligand from bound ligand by rapid filtration under vacuum (1450 Filter Mate Harvester, PerkinElmer) through GF/C glass fiber filters. The filters were then rinsed three times with 300 μL of binding buffer, air-dried (120 min), and counted for radioactivity by liquid scintillation spectrometry using a MicroBeta TriLux counter (PerkinElmer). Specific binding was determined and plotted as a function of the compound concentration. Nonspecific binding was determined in the presence of idazoxan (10−5 M), a compound with well stablished affinity for I2−IRs and α2-ARs, in [3H]2-BFI and [3H]RX821002 assays. To obtain the inhibition constant (Ki) analyses of competition experiments were performed by nonlinear regression using the GraphPad Prism program. Ki values were normalized to pKi values. I2−IRs/α2 selectivity index was calculated as the antilogarithm of the difference between pKi values for I2−IRs and pKi values for α2-AR. For [3H]clonidine experiments logIC50 values were calculated (the concentration of tested ligand that displaces 50% of specifically bound [3H]clonidine). 3D-QSAR study. Data Set Preparation. The three-dimensional Quantitative Structure Activity Relationship (3D-QSAR) study was performed on a data set consisting of 17 I2−IRs and 16 α2-ARs ligands (4 previously reported compounds: Idazoxan, CR40506, BU99008.25 and B06,26 and 15 new compounds, Table 1 and 2). The compounds reported here (Table 2) with pKi values between 3.07 and 9.98 for I2−IRs and 2.65−9.43 for α2-ARs were used for 3D- QSAR modeling. For all compounds studied, the dominant forms were determined at a phisiological pH of 7.4 using the programMarvin Sketch 5.5.1.0.70 The semiempirical/PM3 method (Parameterized Model revision 3)71,72 was applied in the first step of geometry optimization on selected dominant forms, followed by the ab initio Hartree−Fock/3-21G method,73 using the Gaussian 09 software,74 which is part of the ChemBio3D Ultra 13 program.75 The Pentacle program76 was used for the calculation of alignment- independent three-dimensional molecular descriptors (GRIND) and 3D-QSAR models building. The computation of GRIND descriptors is based on Molecular Interaction Fields (MIF) by using four different probes: O probe (hydrogen bond acceptor groups), N1 probe (hydrogen bond donor groups), TIP probe (the shape of molecule), and DRY probe (hydrophobic interactions). In the next step the ALMONDalgorithmwas applied for the extraction of themost relevant regions representing favorable interaction positions between ligand and probe. Finaly, the CLACC (Consistently Large Auto and Cross Correlation) algorithm with a smoothing window of 0.8 Å was applied to display node−node energies, between the same or a different probe, into auto- and cross-correlograms.77 The analyzed data set was divided into a training set for the creation of the 3D-QSAR model and a test set for the external validation of the model. The 3D-QSAR (I2−IRs) model was built with 17 compounds, 12 ligands in the training set and 5 ligands in the test set, while the 3D- QSAR (α2-ARs) model was built with 11 compounds in the training set and 5 compounds in the test set. Ligands with pKi values less than 3 were not used for 3D-QSAR analysis. The PCA (Principal Component Analysis) plot was used to select a test set of compounds, taking into account that the pKi values were homogeneously distributed over the entire range of activities studied. The most significant variables were selected using a Fractional Factorial Design (FFD), and the final 3D- QSAR models were created by Partial Least Square (PLS) regression. In Vitro Effects of 12b in a Preclinical Model of Neuro- degeneration. SH-SY5Y Cell Culture. SH-SY5Y human neuro- blastoma-derived cells (ATCC, Rockville, MD, USA, ref.CRL-2266) were cultured and maintained in DMEM medium (Sigma) supplemented with 2 mM glutamine (Sigma) and 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin under standard conditions. For cell viability assays, cells were seeded onto 96-well plates at a density of 3 × 10^4 cells per well. Upon reaching semiconfluence, cells were treated with 35 μM6-OHDA (Sigma) for 18 h. Primary Glial Cultures. Primary glial cultures were isolated from the cerebral cortex of 3-day-old mice as described previously.78 In brief, the cerebral cortex was dissected, dissociated, and incubated with trypsin/ EDTA (0.25%) at 37 °C for 1 h. Following centrifugation, the pellet was washed with HBSS (Gibco) and the cells were plated in poly-D-lysine (20 μg/mL) pretreated flasks (75 cm2). After 7−10 days, the flasks were agitated in an orbital shaker at 230 rpm for 4 h at 37 °C to isolate nonadherent microglial cells, which were then plated onto 24-well plates at a density of 3 × 10^5 cells per well. The flasks were subsequently replenished with DMEM and agitated in a horizontal shaker at 260 rpm at 37 °C. After overnight agitation, the supernatant, containing oligodendrocytes and some residual microglial cells, was removed. Astrocytes, which remained adherent, were collected and plated onto 96-well plates at a density of 3 × 10^5 cells per well for the neuronal survival studies, and onto 24-well plates (10 × 10^5 cells/ well) for the analysis of inflammation by immunofluorescence. The purity of the cultures exceeded 95%, as determined by immuno- fluorescence analysis using an antiglial fibrillary acidic protein (GFAP; clone G-A-5; Sigma-Aldrich) antibody to identify astrocytes, an anti- Iba1 (Wako) antibody to identify microglial cells, and an anti-O4 (Millipore) antibody as an oligodendrocyte marker. Treatments. SH-SY5Y, astrocytes and microglial cells seeded on 96- well plates were treated with compound 12d at different doses (0.5 μM to 20 μM). Some SH-SY5Y cultures were treated with the neurotoxic agent 6-hydroxydopamine (6-OHDA, 35 μM, Merck-Sigma) as a control for cellular damage, and glial cells with bacterial lip- opolysaccharide (LPS, 10 μg/mL, Merck-Sigma) as an inducer of inflammation. Then cellular viability and nitrite production (indicator of inflammation) was assessed using the MTT and the Griess assays, respectively. None of the concentrations of 12d tested were neurotoxic or pro-inflammatory at those doses. Then, cultures were treated for 1h with compound 12d at 1, 5, and 10 μM. Some cultures were treated with the well-known anti-inflammatory compound CR4056 (1 μM). Doses were chosen based on previous studies.27,28 Subsequently, SH- SY5Y cultures were exposed to 6-OHDA (35 μM) for 18 h. To determine the inflammatory state, glial cells subcultured in 96-well plates were exposed to LPS (10 μg/mL). Finally, the cellular viability and nitrite production of the cultures were assessed. Cell Viability Assay. Cell viability was determined using the 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophen- yl)-2H-tetrazolium (MTT) assay kit (Roche Diagnostic, GmbH) on SH-SY5Y cultures, based on the ability of viable cells to reduce yellow MTT to blue formazan. The extent of reduction of MTTwas quantified by absorbance measurement at 595 nm according to the manufacturer’s protocol. Each data point represents the mean ± SD of 6 replications in 3 different experiments and the results were expressed as a percentage of the control. Nitrite Measurement. After treatments, 100 μL of the cell culture supernatant (SH-SY5Y and glial cultures) was mixed with an equal volume of Griess reagent (consisting of 1% sulfanylamide and 0.1% naphthyl ethylene diamine in 5% phosphoric acid, sourced from Sigma- Aldrich, Madrid, Spain) at room temperature for 15 min. To determine the nitrite concentrations, a standard solution of sodium nitrite was employed. The absorbance of themixture wasmeasured at 492/540 nm using a microplate reader from Thermofisher (Madrid, Spain). The experiments were repeated a minimum of three times. Immunocytochemical Analysi. For immunofluorescence analysis, primary glial cultures, grown on glass coverslips in 24-well cell culture plates, were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde (Sigma), blocked in PBS containing 0.1% Triton X- 100 for 30 min at 37 °C, and incubated overnight at 4 °C with primary antibodies. The following antibodies were used: rabbit polyclonal anti- COX2 (Cayman) and mouse monoclonal anti-TNFα (Abcam). Cells were then incubated for 45 min at 37 °C with appropriate Alexa Fluor 488- and 546-conjugated secondary antibodies (Jackson Immuno Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2566 pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as Research). Nuclei were stained with DAPI. Coverslips were mounted with Vectashield Mounting Medium (Vector Laboratories). Images were captured using a Zeiss LSM710 laser scanning spectral confocal microscope equipped with Plan-Apochromat 63X/1.4. Confocal microscope settings were adjusted to produce the optimum signal-to- noise ratio. To compare fluorescence signals from different preparations, settings were fixed for all samples within the same analysis. Representative images of at least three independent experi- ments are shown. Statistical Analysis. In vitro models of neurodegeneration and neuroinflammation, Figures 4 and 5, are expressed as the mean ± standard deviation (SD) of six replicates determinations. The experiments were repeated at least three times, and consistent outcomes were obtained. Data analysis was initially performed using one-way analysis of variance (ANOVA). Subsequently, post hoc statistical analyses were conducted using the Tukey test, with a significance level set at p < 0.05. The SPSS statistical software package, version 20.0, for Windows (Chicago, IL, USA), was utilized for these analyses. In Vivo Pharmacokinetics. The pharmacokinetic study was carried on in male CD1 mice (Envigo Laboratories) with a body weight between 40 to 50 g (n = 3−4 per group). Animals were randomized to be included in the treated or control groups. A single intraperitoneal dose of 12d (30 mg/kg, 10 mL/kg) was administered early in the morning (between 8 and 11 a.m.) without anesthesia. The compound was dissolved in 10% of 2-hydroxypropyl-β-cyclodextrin in physiological saline. Mice were monitored for signs of pain or distress during the time between injection and euthanasia. Mice were sacrificed by cervical dislocation and blood (0.6 mL) was collected at different time points (0 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 2 h, 3 h, 4 h, 6 h, 8 and 24 h after injection) in tubes with serum gel and clotting activator (Sarstedt Micro tube 1.1 mL Z-Gel). Samples were centrifugated at 10.000 rpm for 10 min to obtain plasma and stored at −80 °C up to analysis of compound concentration by UPLC-MS/MS. Experimental procedures were in line with the Directive 2010/63/EU and approved by the Institutional Animal Care and Generalitat de Catalunya (#10291, 1/28/2018). Hypothermic Effects Evaluation: Changes in Core Body Temperature. Twenty-five adult CD-1 mice (30−40 g), bred in the animal facility of the University of the Balearic Islands, were used in this study. All experimental procedures were performed following the ARRIVE (McGrath and Lilley, 2015) and standard ethical guidelines (European Communities Council Directive 86/609/EEC; Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research, National Research Council 2003), and were approved by the Local Bioethics Committee (UIB CAIB). All efforts were made to minimize the number of mice used and their suffering. Mice had continuous access to a regular diet and tap water while being housed in standard cages under specific conditions (22 °C, 70% humidity, and 12 h light/dark cycle, lights on at 8:00 h). Prior to drug treatment, mice were habituated to the experimenter by being handled and weighed for 2 days. For the acute treatment, mice received (i.p.) either a single dose of 12b (20mg/kg, n = 6) or vehicle (1mL/kg of DMSO, n = 7). For the repeated treatment, mice received a daily injection (i.p.) of 12b (20 mg/kg, n = 6) or vehicle (n = 6) for 5 consecutive days. Changes on rectal temperature were calculated by subtracting the values obtained with a rectal probe connected to a digital thermometer (Compact LCD display thermometer, SA880−1M, RS, Corby, UK) 1 h post-treatment minus before drug treatment (baseline values). Mice were sacrificed right after the last temperature recording, and the hippocampus was freshly dissected and kept at −80 °C until further neurochemical analyses were performed. In Vivo Treatment in SAMP8Mice.Male and female SAMP8 (n = 16, 10-month-old) were used to perform behavioral and molecular analyses. Animals were randomly divided into control (n = 8), and 12d (5 mg·kg−1·day−1) (n = 8). The animals had free access to food and water and were kept under standard temperature conditions (22 ± 2 °C) and 12-h light/dark cycles (300/0 lx). Compounds were dissolved in 1.8% (2-hydroxypropyl)-β-cyclodextrin and administered through drinking water. Control groups received water plus 1.8% (2- hydroxypropyl)-β-cyclodextrin during the treatment period. For the drug administration, dosages were calculated based on average daily water consumption recorded in each cage, and they were confirmed by recalculations once a week. Each animal’s weight was also recorded once a week during the treatment period and the drug dosages were recalculated, when necessary, based on the results obtained. After 4 weeks of treatment, Novel Object Recognition and Novel Object Location tests were performed to study the effects of treatment on working and spatial memory (Figure 9). Mice were also treated during this period and up to their euthanasia. All studies and procedures for the mouse behavior test of brain dissection and extractions followed the standard ethical guidelines (European Communities Council Directive 2010/63/EU and Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research, National Research Council 2003). Animal studies are reported in compliance with the ARRIVE guidelines,79 and were approved by Bioethical Committees from the University of Barcelona and the Government of Catalonia (#222/18). Novel Object Recognition Test. A modification of the novel object recognition test protocol was performed (Ennaceur &Delacour, 1988). In brief, mice were placed in a 90° two-arm (25 × 20 × 5 cm) black maze, with removable walls for easy cleaning and light intensity in midfield was 30 lx. Before the memory, trial mice were habituated to the apparatus for 10 min for 3 days. On day 4, the animals were submitted to a 10min acquisition trial, in which they were allowed to freely explore two identical objects located at the end of each arm (first trial� familiarization). After 2 h (for short-termmemory evaluation) and 24 h (for long-term memory evaluation) from the first trial, the mice were submitted to a 10 min retention trial, in which one of the two identical objects had been replaced by a novel one. The behavior was recorded, and the time that the mice spent exploring the new object (TN) and the old one (TO) was measured manually. Exploration was defined as sniffing or touching objects with the nose and/or forepaws. The discrimination index (DI) was calculated as (TN − TO)/(TN + TO). To avoid object preference biases, objects were alternated; 70% EtOH was used to clean the arms and objects after each trial to eliminate olfactory cues. Object Location Test. The test was performed in a cage (50 × 50 × 25 cm), in which three walls were white except one that was black and lasted 3 days. On day 1, mice were familiarized to the arena for 10 min. On day 2, two identical objects (A+A) were in front of the black wall, and the mice were freely allowed to explore both objects for 10 min (Trial 1−training phase). On day 3, after a retention period of 24 hmice were returned to the testing arena for another 10 min (Trial 2−testing phase) with one object moved to a different position (opposite direction toward the black wall) and were allowed to explore (Figure 6). The trials were recorded, and the object exploration time was measured manually. The time sniffing the object in the old position (PO) and the time exploring the object in the new position (TN) were evaluated. The DI defined as (PN-TO)/(PN+PO) was determined as an indicator of cognitive performance. For the elimination of olfactory cues, 70% EtOH was used to clean the testing arena after each trial. Western Blot Analysis for Neurochemical Markers. Following standardized Western blot procedures,19 total homogenates from each hippocampal sample were prepared and loaded (40 μg) into 10−12% SDS-PAGE polyacrilamide gels (Bio-Rad), and transferred onto nitrocellulose membranes to later label the content of key target proteins using primary antibodies. In particular, membranes were incubated overnight with anti-FADD (H-181, sc-5559, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cdk5 (Ab-2, DC17, Lab Vision, NeoMarkers, USA), anti-pERK1/2 (#9101L, Cell Signaling, MA, USA), anti-ERK (CEMI0112011, Clone 631122, R&D Systems, Biotechne, USA) anti-PDE1B (Santa Cruz/sc-393112); SYT7 (Santa Cruz/sc-293343); HPCA (Santa Cruz/sc393125); CAMKII (Abcam/ ab52476). Membranes were stripped and reprobed with β-actin antibody (clone AC-15, no. A1978, Sigma) or GAPDH (Millipore/ MAB374), since its content was used as a loading control (no differences between treatment groups, data not shown). Then, with the appropriate secondary antibody incubation (Goat-antimouse HRP conjugated, Biorad/170−5047, or Goat-antirabbit HRP conjugated, Biorad/170−6515), and posterior ECL detection system (Amersham, Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2567 pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as Buckinghamshire, UK), target proteins were visualized on autoradio- graphic films (Amersham ECL Hyperfilm), and immunoreactive bands were quantified (integrated optical density, IOD) by densitometric scanning (GS-800 Imaging Densitometer, Bio-Rad). The amount of target protein (e.g., FADD, Cdk5, pERK/ERK, CaMKII, SYT7) from each mouse treated with 12b was compared in the same gel with that of control mice (% change vs acute or repeated controls). Each brain sample (and target protein) was quantified at least in 2−4 gels, and the mean value was used as a final estimate. RNA Extraction and Gene Expression Determination. Total RNA isolation from hippocampal samples was performed using the TRIzol reagent according to the manufacturer’s instructions (Bioline Reagents, London, UK). The yield, purity, and quality of RNA were determined spectrophotometrically with a NanoDrop ND-1000 apparatus (Thermo Fisher, Waltham, MA, USA) and an Agilent 2100B Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA samples with 260/280 ratios and RINs higher than 7.5, respectively, were selected. RT-PCR was performed. Briefly, 2 μg of mRNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). SYBR Green real-time PCR was performed using a Step One Plus Detection System (Applied Biosystems) with SYBR Green PCRMaster Mix (Applied Biosystems). Each reaction mixture contained 6.75 μL of cDNA (with a concentration of 2 μg), 0.75 μL of each primer (with a concentration of 100 nM), and 6.75 μL of SYBR Green PCR Master Mix (2 × ) (Applied Biosystems). Data were analyzed utilizing the comparative cycle threshold (Ct) method (ΔΔCt), where the housekeeping gene level was used to normalize differences in sample loading and preparation. Normalization of expression levels was performed with β-actin for SYBR Green-based real-time PCR results. Each sample was analyzed in triplicate, and the results represented the n-fold difference of the transcript levels among different groups. The data were analyzed utilizing the comparative cycle threshold (Ct) (ΔΔCt) method, in which the levels of a housekeeping gene are used to normalize differences in sample loading and preparation. The normalization of expression levels was performed with β-actin. The primer sequences and TaqMan probes used in this study are presented in Tables S40− S41. Each sample was analyzed in duplicate and the results represent the n-fold difference in the transcript levels among different groups. Data Analysis.Data analysis, Figures 9 and 10, was performed using GraphPad Prism ver.9.2 software (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Data were expressed as the mean ± standard error of the mean (SEM) from at minimum eight samples per group for behavioral tests and four samples for molecular techniques. Means were compared with one-way analysis of variance followed by Tukey’s post hoc analysis. Comparison between groups was also performed by two-tail Student’s t test for independent samples. Statistical significance was considered when P-values were <0.05. In Vivo Analgesia Studies. Experimental Animals. Female CD-1 mice (Charles River, Barcelona, Spain) weighing 25−30 g (8−11 weeks old) were used to perform the pain studies. The mice were housed in colony cages (10 animals per cage) in a temperature-controlled room (22 ± 2 °C) with an automatic 12-h light/dark cycle (08:00−20:00). The mice were fed a standard laboratory diet and had free access to tap water until the beginning of the experiments. Envionmental enrichment (igloo and plastic tunnel) were placed in each cage to improve animal welfare. The behavioral tests were performed during the light phase (9:00−15:00). The mice were randomized to treatment groups, testing a balanced number of animals from several groups each day. Random testing was also conducted throughout the estrous cycle. Themice were handled in accordance with international standards (European Communities Council directive 2010/63), and the experimental protocols were approved by regional (Junta de Andaluciá) and institutional (Research Ethics Committee of the University of Granada) authorities. Drugs and Drugs Administration.The effects of the drugs tested on nociception were studied in a model of mechanical hypersensitiviy induced by capsaicin. The algogenic agent, capsaicin (Sigma-Aldrich Quiḿica S.A.), was dissolved in 1% DMSO in physiological sterile saline to a concentration of 0.05 μg/μL (i.e., 1 μg per mouse). Capsaicin solution was injected intraplantarly (i.pl.) into the right hind paw proximate to the heel, in a volume of 20 μL using a 1710 TLLHamilton microsyringe (Teknokroma, Barcelona, Spain) with a 301/2-gauge needle. Control animals were injected with the same volume of the vehicle of capsaicin. Drug solutions were prepared immediately before the start of the experiments. In addition to our compound 12d, we used CR4056 (1), synthesized following described procedures,31 as a control prototypical I2−IRs agonist.13 Clonidine (Sigma-Aldrich Quiḿica S.A.) was used as a prototypical α2 agonist, with low affinity for imidazoline receptors.60 Gabapentin (Abcam, UK), was used as a control drug active on central sensitization with known antinociceptive effects.55 In some experi- ments, the I2/α2 antagonist idazoxan (4)57 (Sigma-Aldrich Quiḿica S.A.) or ist methoxy analog, the α2‑AR agonist RX 821002 (Biogen Cientif́ica S.L., Madrid, Spain) were associated with other treatments to determine the mechanism involved in the antiallodynic effects.80 12d, CR4056 (1), and gabapentin were dissolved in DMSO 10% and cyclodextrin 40% in saline. Clonidine, idazoxan (4) and RX 821002 were dissolved in physiological sterile saline. CR4056 (1) was orally (p.o.) administrated in a volumen of 10 mL/kg by oral gavage. The other drugs were administered subcutaneously (s.c.) in a volume of 5 mL/kg into the interscapular area. When the effect of the association of several drugs was assessed, each s.c. administration was performed in different areas of the interscapular zone to avoid the mixture of the drug solutions and any physicochemical interaction between them. The researchers who performed the experiments were blinded to the treatment received by each animal. Evaluation of Capsaicin-Induced Secondary Mechanical Hyper- sensitivity.Animals were placed into individual test compartments for 2 h before the behavioral evaluation to habituate them to the test conditions. The compounds tested, or their vehicles, were administered as described above, and 30 min after drug administration, capsaicin or DMSO 1%, was intraplantarly (i.pl.) administered. Punctate mechan- ical stimulation was applied with a dynamic plantar aesthesiometer (Ugo Basile, Varese, Italy) at 15 min after the administration of capsaicin or its solvent (i.e., drug effects were evaluated 45 min after drug administration). Briefly, a nonflexible filament (0.5 mm diameter) was electronically driven into the ventral side of the paw previously injected with capsaicin or solvent (i.e., the right hind paw), at least 5 mm away from the site of the injection toward the fingers. The intensity of the stimulation was fixed at 0.5 g force, as described previously.54 When a paw withdrawal response occurred, the stimulus was automatically terminated, and the latency of the behavioral response was automatically recorded. The filament was applied three times, separated by intervals of 0.5 min, and the mean value of the three trials was considered the withdrawal latency of the animal. A cutoff time of 50 s was used. Rotarod Test.Motor coordination was assessed with an accelerating rotarod (Cibertec, Madrid, Spain), as previously described.81 Briefly, mice were required to walk against the motion of an elevated rotating drum at increasing speed (4−80 rpm over 5 min), and the latency to fall down was recorded using a cutoff time of 300 s. Mice were given three training sessions 24 h before drug testing. On the day of the test, rotarod latencies were measured immediately before administration of the drug or their solvent (time 0) and then several times (45, 90, 120, and 240 min) after the s.c. injection. As a comparison drug we used gabapentin (40 mg/kg, s.c.). ■ ASSOCIATED CONTENT *sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644. 1H and 13C NMR spectra, tables of representative data and discussion on the assignation of the signals; X-ray crystallography data of compounds 12b, 12d, and 12h; 3D-QSAR study and theoretical ADMET parameters; PAMPA-BBB studies; experimental details on physico- chemical properties, solubility, chemical stability at Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. Chem. 2025, 68, 2551−2573 2568 https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.4c01644/suppl_file/jm4c01644_si_001.pdf http://www.graphpad.com https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01644?goto=supporting-info pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as different pHs of 12d; complete details of in vitro biological methods of 12d: cytotoxicity assays, Caco-2 permeability, microsomal stability, plasma stability, protein binding, cytochrome inhibition, and hERG ion channel inhibition; complete details of experimental procedures/tables for in vivo experiments: pharmacokinetics, neurodegenerative preclinical model, SAMP8 studies, and capsaicin-induced mechanical hypersensitivity; molecular formula string and data (PDF) ■ AUTHOR INFORMATION Corresponding Author Carmen Escolano − Laboratory of Medicinal Chemistry (Associated Unit to CSIC), Department of Pharmacology, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona 08028, Spain; Institute of Biomedicine of the University of Barcelona (IBUB), University of Barcelona, Barcelona 08028, Spain; orcid.org/0000-0002-9117-8239; Phone: +34 934024542; Email: cescolano@ub.edu Authors Andrea Bagán − Laboratory of Medicinal Chemistry (Associated Unit to CSIC), Department of Pharmacology, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona 08028, Spain; Institute of Biomedicine of the University of Barcelona (IBUB), University of Barcelona, Barcelona 08028, Spain Alba López-Ruiz − Laboratory of Medicinal Chemistry (Associated Unit to CSIC), Department of Pharmacology, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona 08028, Spain; Institute of Biomedicine of the University of Barcelona (IBUB), University of Barcelona, Barcelona 08028, Spain Sònia Abás − Laboratory of Medicinal Chemistry (Associated Unit to CSIC), Department of Pharmacology, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona 08028, Spain M. Carmen Ruiz-Cantero − Laboratory of Medicinal Chemistry (Associated Unit to CSIC), Department of Pharmacology, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona 08028, Spain Foteini Vasilopoulou − Pharmacology Section, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, and Institut de Neurocieǹcies, University of Barcelona, Barcelona 08028, Spain Teresa Taboada-Jara − Pharmacology Section, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, and Institut de Neurocieǹcies, University of Barcelona, Barcelona 08028, Spain Christian Griñán-Ferré − Pharmacology Section, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, and Institut de Neurocieǹcies, University of Barcelona, Barcelona 08028, Spain; Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CiberNed), National Institute of Health Carlos III, Madrid 28029, Spain; orcid.org/0000-0002-5424-9130 Merce ̀ Pallàs − Pharmacology Section, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, and Institut de Neurocieǹcies, University of Barcelona, Barcelona 08028, Spain; Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CiberNed), National Institute of Health Carlos III, Madrid 28029, Spain; orcid.org/0000-0003-3095-4254 Carolina Muguruza−Department of Pharmacology, University of the Basque Country, UPV/EHU, 48940 Leioa, Bizkaia; Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM, Spain Rebeca Diez-Alarcia − Department of Pharmacology, University of the Basque Country, UPV/EHU, 48940 Leioa, Bizkaia; Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM, Spain; BioBizkaia Health Research Institute, Barakaldo, Bizkaia 48903, Spain Luis F. Callado − Department of Pharmacology, University of the Basque Country, UPV/EHU, 48940 Leioa, Bizkaia; Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM, Spain; BioBizkaia Health Research Institute, Barakaldo, Bizkaia 48903, Spain José M. Entrena − Animal Behavior Research Unit, Scientific Instrumentation Center, Parque Tecnológico de la Salud, University of Granada, Armilla, Granada 18100, Spain Enrique J. Cobos − Department of Pharmacology, Faculty of Medicine and Biomedical Research Center (Neurosciences Institute), Biosanitary Research Institute ibs.GRANADA, University of Granada, Granada 18016, Spain Belén Pérez − Department of Pharmacology, Therapeutic and Toxicology. Autonomous, University of Barcelona, Cerdanyola 08193, Spain José A. Morales-García − Department of Cell Biology. Faculty of Medicine, Computense University of Madrid. (UCM), Madrid 28040, Spain Elies Molins − Institut de Ciencia de Materials de Barcelona (CSIC), Campus UAB, Cerdanyola 08193, Spain; orcid.org/0000-0003-1012-0551 Steven De Jonghe − Molecular, Structural and Translational Virology Research Group, Rega Institute for Medical Research, Department of Microbiology, Immunology and Transplantation, Katholieke Universiteit Leuven, Leuven 3000, Belgium; orcid.org/0000-0002-3872-6558 Dirk Daelemans − Molecular Genetics and Therapeutics in Virology and Oncology Research Group, Rega Institute for Medical Research, Department of Microbiology, Immunology and Transplantation, Katholieke Universiteit Leuven, Leuven 3000, Belgium José Brea − Drug Screening Platform/Biofarma Research Group, CIMUS Research Center, University of Santiago de Compostela (USC), Santiago de Compostela 15782, Spain; orcid.org/0000-0002-5523-1979 Cristina Val − Drug Screening Platform/Biofarma Research Group, CIMUS Research Center, University of Santiago de Compostela (USC), Santiago de Compostela 15782, Spain M. Isabel Loza − Drug Screening Platform/Biofarma Research Group, CIMUS Research Center, University of Santiago de Compostela (USC), Santiago de Compostela 15782, Spain; orcid.org/0000-0003-4730-0863 Elena Hernández-Hernández − IUNICS, University of the Balearic Islands (UIB) and IdISBa, Palma de Mallorca 07122, Spain; Present Address: Department of Pharmacology, University of the Basque Country, UPV/ EHU, E-48940 Leioa, Bizkaia Jesús A. García-Sevilla − IUNICS, University of the Balearic Islands (UIB) and IdISBa, Palma de Mallorca 07122, Spain M. Julia García-Fuster − IUNICS, University of the Balearic Islands (UIB) and IdISBa, Palma de Mallorca 07122, Spain Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.4c01644 J. Med. 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https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Caridad+Di%CC%81az"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf pubs.acs.org/jmc?ref=pdf https://doi.org/10.1021/acs.jmedchem.4c01644?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as Caridad Díaz − Fundación MEDINA Centro de Excelencia en Investigación de Medicamentos Innovadores de Andalucía, Ganada 10016, Spain Rosario Fernández-Godino − Fundación MEDINA Centro de Excelencia en Investigación de Medicamentos Innovadores de Andalucía, Ganada 10016, Spain Olga Genilloud − Fundación MEDINA Centro de Excelencia en Investigación de Medicamentos Innovadores de Andalucía, Ganada 10016, Spain; orcid.org/0000-0002-4202-1219 Milan Beljkas ̌ − Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Belgrade 11000, Serbia Slavica Oljacǐc ́ − Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Belgrade 11000, Serbia Katarina Nikolic − Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Belgrade 11000, Serbia Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.4c01644 Author Contributions C.E. conceived the idea, project administration, supervision, analysis of the data. A.B., A.L.-R., and S.A. synthesized and chemically characterized the compounds. E.M. performed the X- ray crystallographic analysis. F.V., T.T.-J., C.G.-F., and M.P. designed and performed the in vivo experiments in SAMP8. C.M. and L.F.C. performed the in vitro I2-IRs binding studies. R.D.-A. and L.F.C. performed the in vitro I1-IRs binding studies. B.P. conducted the PAMPA-BBB studies. C.V., M.I.L., and J.M.B. carried out DMPK studies. S.J. and D.D. performed cytotoxicity studies. J.A.M.-G. designed the cell culture and performed the in vitro studies on neuroprotective and anti- inflammatory activity. E.H.-H., J.A.G.-S., andM.J.G.-F. designed and performed the in vivo hypothermia and Western blot experiments. C.D., R.G.-G. and O.G. carried out the pharmacokinetic study. M.C., R.-C., J.M.E., and E.J.C. designed and performed the in vivo analgesia experiments. A.L.-R. managed the experimental data and prepared the experimental and Supporting Information. C.G.-F., M.P., L.F.C., C.D., E.J.C., and M.J.G.-F. designed the in vivo experiments and funding acquisition. C. G.-F., M.P., J.A.M.-G., E.J.C., E. M., M.J.G.-F., C.D.S.O., M.B., and K. N. performed the 3DQSAR studies and calculated the ADME theoretical parameters. C.E. and A.L.-R. wrote the original draft. C.E. and A.L.-R. wrote, edited, and reviewed the manuscript with feedback from all the authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS This work was supported byMinisterio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación (Spain, PID2022-1380790B-I00 to C.E., and PID2021-138079OB-I00 to M.P.; MICIU/AEI/10.13039/501100011033 and FEDER, UE; and PDC2022-133441-I00 to C.E.; MICIU/AEI/ 10.13039/501100011033 Europea Next GenerationEU/ PRTR), Basque Government (IT-1512-22) and Generalitat de Catalunya (2021 SGR 00357). Financial support was provided for A.L.-R. (PRE2022-105091 ministerio de Ciencia e Innovación). E.H.-H. was first funded by the Margarita Salas Program (Ministerio de Universidades; Plan de Recuperación, Transformación y Resilencia; NextGenerationEU) with the participation of the University of the Balearic Islands, and currently holds grant FJC2022-048338-I, funded by MCIN/ AEI/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR. The authors acknowledge grant support from the Ministry of Science and Innovation, Spain, with funds from the European Union NextGenerationEU (PRTRC17.I1) and the Autonomous Community of Galicia within the framework of the 2023 Biotechnology Plan Applied to Health, Xunta de Galicia (ED431C 2022/20) and the European Regional Development Fund (ERDF). This study was partially supported by the Andalusian Regional Government (grant CTS- 109). Authors also thank the support of the Unit of Excellence “UNETE” from the University of Granada (reference UCE- PP2017-05). M.B., S.O., and K.N. acknowledge the Ministry of Science, Technological Development and Innovation, Republic of Serbia through two Grant Agreements with University of Belgrade-Faculty of Pharmacy No. 451-03-65/2024-03/200161 and No. 451-03-66/2024-03/200161. A.L. thanks to Grant PRE2022-105091 funded by MICIU/AEI/10.13039/ 501100011033. ■ ABBREVIATIONS I2−IRs, imidazoline I2 receptors; 5xFAD, murine familial AD model; α2-AR, α2-adrenergic receptor; FADD, Fas-associated protein with death domain; NORT, Novel Object Recognition; OLT, Novel Object Location Test; SAMP8, mouse model of accelerated aging and AD; Ki, inhibition constant; MoA, mechanism of action ■ REFERENCES (1) Bousquet, P.; Feldman, J.; Schwartz, J. Central Cardiovascular Effects of Alpha Adrenergic Drugs: Differences between Catechol- amines and Imidazolines. J. Pharmacol. Exp. 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