Structure-Optimization and Structure-Activity Relationship of Diphenylpyrazinyl Aminoalkoxyacetic Acid Derivatives: Piperidine Substitution and Hydrophobic Modification as Key Strategies for IP Receptor Agonists

Structure-Optimization and Structure-Activity Relationship of Diphenylpyrazinyl Aminoalkoxyacetic Acid Derivatives: Piperidine Substitution and Hydrophobic Modification as Key Strategies for IP Receptor Agonists | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Structure-Optimization and Structure-Activity Relationship of Diphenylpyrazinyl Aminoalkoxyacetic Acid Derivatives: Piperidine Substitution and Hydrophobic Modification as Key Strategies for IP Receptor Agonists Xianrong Cai, Juping Cheng, Yuhe Wang, Guiying Liu, Xiaoyan Ma, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8764373/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract To develop novel, highly potent, and safe IP receptor agonists, two series of diphenylpyrazinyl amino alkoxy acetic acid derivatives were designed and synthesized. Combined with antiplatelet aggregation assays, molecular docking, and molecular dynamics simulations, a systematic investigation was conducted on their structure-activity relationships (SAR). The results revealed that replacing the N -isopropyl group and N -butyl side chain of MRE-269 with a piperidine ring represented the optimal modification strategy, as it enhanced the hydrophobic interactions between the derivatives and the IP receptor while reduce the flexibility of their alkylene chains. The additional introduction of small hydrophobic groups (e.g., methyl and dimethyl groups) onto the alkylene chain could significantly improve the derivatives’ antiaggregatory activity, which represents a key pharmacodynamic feature of this class of compounds. In conclusion, this study clarifies the SAR of these diphenylpyrazinyl amino alkoxy acetic acid derivatives and provides critical insights for the rational design of IP receptor agonists. IP receptor agonists Diphenylpyrazinyl aminoalkoxyacetic acids Structure-Activity Relationship Antiaggregatory Activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Prostacyclin (prostaglandin I 2 , PGI 2 ) is an endogenous bioactive substance produced by endothelial and vascular smooth muscle cells[ 1 , 2 ]. PGI2 has a range of physiological effects, including vasodilation, inhibition of platelet aggregation, promotion of vascular smooth muscle cell differentiation and inhibition of their proliferation[ 3 – 5 ]. Meanwhile, PGI 2 has been shown to reduce pulmonary blood pressure and bronchial hyper-responsiveness, improving pulmonary function[ 6 ]. These biological effects are mainly mediated by binding to the prostacyclin receptor (IP receptor) on the surface of smooth muscle cells and activating downstream signaling pathways. Hence, the IP receptor has emerged as a key therapeutic target for vascular diseases such as pulmonary arterial hypertension (PAH). Prostacyclin, prostacyclin analogs, and IP receptor agonists (including selexipag, epoprostenol, beraprost, iloprost, and treprostinil) have been clinically utilized for the treatment of PAH[ 7 , 8 ]. Selexipag (1, Fig. 1 ) is a first-in-class, orally active, non-prostanoid IP receptor agonist[ 9 ]. It was approved by the U.S. Food and Drug Administration (FDA) in 2015 for the treatment of PAH, with the aim of delaying disease progression and reducing the risk of hospitalization for PAH[ 10 , 11 ]. Following oral administration, selexipag is rapidly metabolized to form its active metabolite MRE-269[ 12 ], which has been confirmed as the major contributor to the drug's pharmacological effects[ 13 ], with mean elimination half-life of 9.4–14.2 h in the human body[ 14 ]. Pharmacological studies have demonstrated that MRE-269 exhibits high selectivity for the IP receptor, with a binding affinity (K i = 20 nM) approximately 130-fold higher than that for other prostaglandin receptors (prostaglandin E receptors, EP 1-4 ; prostaglandin D receptor, DP 1 ; prostaglandin F 2 receptor, FP; and thromboxane A2 receptor, TP)[ 15 , 16 ]. Structural biology studies have verified that this high selectivity is attributed to the π-π stacking interaction formed between the biphenylpyrazine moiety of MRE-269 and the Tyr281 residue of the IP receptor[ 17 ]. The most common adverse reactions of selexipag are similar to those of prostacyclin analogs, mainly including headache, diarrhea, nausea, and jaw pain[ 18 ]. To develop IP receptor agonists with high potency, high selectivity, and long-lasting pharmacological effects, structural modification and optimization were conducted on MRE-269 as the lead compound in our previous research. Specifically, we designed and synthesized a series of 2-cyclic amino-5,6-diphenylpyrazine derivatives by substituting the N -isopropyl group of MRE-269 with cyclic amines of varying sizes, while preserving the key pharmacophoric scaffolds (the oxyacetic acid group and diphenylpyrazine scaffolds intact), which are structural elements critical to the antiaggregatory effects and specificity of MRE-269[ 19 ]. Among these newly synthesized derivatives, active compounds featuring piperidine ring substitution were successfully identified[ 20 ]. In the present work, we continue the aforementioned structural optimization strategy and further expand the modification dimensions. Still taking MRE-269 as the lead compound, we designed two structural modification strategies: (1) side-chain engineering, involving elongation, shortening, or the insertion of oxygen atoms into the N -butyl side chain; and (2) cyclic amine incorporation, modifying the N -isopropyl group and N -butyl side chain with various cyclic amines. Based on these strategies, two series of derivatives were synthesized, diphenylpyrazinyl isopropylamine alkoxyacetic acid (series 3 ) and diphenylpyrazinyl cycloaminoalkoxyacetic acid (series 4 ) (Fig. 2 ). To systematically elucidate the effect of structural modifications on activity, we comprehensively investigated the structure-activity relationships (SAR) of these derivatives by integrating platelet aggregation assays in vitro, molecular docking and conventional molecular dynamics (cMD) simulation techniques. The purpose of this study is to clarify the effects and molecular mechanisms of side chain engineering and cyclic amine substitution on IP receptor agonistic activity through systematic SAR analysis, thereby providing a solid scientific basis and clear design ideas for the directed structural optimization of IP receptor agonists in future research. Results and Discussion Synthesis of the Designed Library The synthesis of all 29 target molecules followed the outlined pathways in Scheme 1 (series 3 ) and Scheme 2 (series 4 ). Key synthetic steps included phase-transfer alkylation of diphenylpyrazin-2-yl isopropylamino alcohols with tert-butyl bromoacetate, followed by reduction of tert-butyl esters to the corresponding alcohols using LiAlH₄. The final hydrolysis step converted tert-butyl esters into the free acids. For series 3 , diphenylpyrazinyl isopropylamino alkoxyacetic acids 3 were synthesized via basic hydrolysis of tert-butyl esters 8 , 10 and 12 Esters 8 were prepared by alkylating diphenylpyrazin-2-yl isopropylamino alcohols 7 with tert-butyl bromoacetate (or its derivatives) under phase-transfer conditions. The alcohols 7 were synthesized by coupling 2-chloro-5,6-dimethylpyrazine 6 with the corresponding isopropylamine derivatives 5 . Reduction of esters 8 with lithium aluminum hydride (LiAlH 4 ) afforded alcohols 9 , which were then alkylated with tert-butyl bromoacetate to give esters 10 . Subsequent reduction of 10 provided alcohol 11 , and re-alkylation with tert-butyl bromoacetate afforded ester 12 (Scheme 1). In parallel, for series 4 , we synthesized the modified diphenylpyrazinyl carboxylic acids ( 4 ) through a similar alkylation strategy. The synthesis involved alkylating diphenylpyrazin-2-yl cyclic amino alcohols 14 with tert-butyl bromoacetate under analogous phase-transfer conditions. The alcohols 14 were prepared by coupling 2-chloro-5,6-diphenylpyrazine 6 with various cyclic amino alcohols 13 . The resulting esters 15 were reduced with LiAlH₄ to furnish alcohols 16 , which were subsequently alkylated with tert-butyl bromoacetate to give esters 17 . Hydrolysis of 15 or 17 provided the corresponding acids 4 (Scheme 2). Inhibitory A ctivity A gainst P latelet A ggregation Following the synthesis, the compounds were tested for inhibition of ADP‑induced platelet aggregation in rabbit platelet‑rich plasma, and IC₅₀ values were obtained from dose‑response curves (Figure 3). MRE-269, used as a positive control to evaluate the inhibitory effect on platelet aggregation, exhibits an IC₅₀ value of 6.2 µM. Among the 29 structurally modified derivatives, the diphenylpyrazinyl cyclic amino alkoxy acetic acids derivatives 4i-R and 4o show antiaggregatory activity comparable to that of MRE-269, with corresponding IC₅₀ values of 9.2 and 6.5 µM respectively. Notably, the diphenylpyrazinyl isopropylamino alkoxy acetic acids derivatives 3g and 3i, as well as the diphenylpyrazinyl cyclic amino alkoxy acetic acids derivatives 4q-R and 4q-S, demonstrate significantly superior antiaggregatory activity relative to MRE-269, with IC₅₀ values of 2.8, 2.0, 1.25 and 0.15 µM respectively. It is also noteworthy that the antiaggregatory activity of derivative 4q-S is far stronger than MRE-269, with a 40-fold increase in activity. As can be inferred from the aforementioned activity data, the insertion of an oxygen atom into the N -butyl side chain of MRE-269 exerts an adverse effect on the antiaggregatory activity, with derivative 3c exhibiting an IC₅₀ value of 20 μM. Then both elongation and shortening of the side chain of MRE-269 or 3c exert an adverse effect on the antiaggregatory activity, as exemplified by derivatives 3a, 3b, 3d and 3e. However, the introduction of hydrophobic groups (i.e., methyl or ethyl) onto the alkoxyl side chain of 3c can compensate for the loss of activity caused by such side chain elongation. As exemplified by derivatives 3g, 3h, and 3i, their antiaggregatory activities are increased by multiple folds compared with that of 3c. Therefore, the incorporation of hydrophobic groups into the side chain is likely to be crucial for enhancing the antiaggregatory activity of derivatives. Additionally, derivatives of MRE-269 that contain a four-membered cyclic amine structure substituting of the N -isopropyl and N -butyl side chains completely lose their antiaggregatory activity (4a, 4b, 4c and 4d). In contrast, derivatives with a five-membered (4f-R and 4i-R) or six-membered cyclic amine structure (4o and 4q) retain potent antiplatelet aggregatory activity. This activity difference is likely attributable to the ability of five- and six-membered cyclic amine moieties to mimic the functional moiety of the N -isopropyl group in MRE-269. Notably, six-membered cyclic amine derivatives show stronger antiaggregatory activity than five-membered ones. This enhanced activity may be attributed to the more extensive hydrophobic interactions between the six-membered cyclic amine structure and the IP receptor. Molecular Docking Studies and Molecular Dynamics Simulations Based on the antiaggregatory activities of the synthesized derivatives, we employed molecular docking and cMD simulations to establish the structure-activity relationships (SARs) of the synthesized diphenylpyrazinyl isopropylamine alkoxyacetic acid and diphenylpyrazinyl cycloaminoalkoxyacetic acid derivatives as IP receptor agonists. To establish the SARs, we first employed molecular docking to delineate the binding modes of these derivatives. First, we redocked MRE-269 into the binding site of the IP receptor to confirm the reliability of the docking protocol implemented in MOE's dock application. The top-ranked redocking binding pose of MRE-269 aligned well with the native pose of its cryo‑EM structure (Figure 4A), with a root-mean-square deviation (RMSD) value of 0.93 Å (RMSD < 2 Å)[21] and a corresponding docking score of −10.9 kcal/mol (Table S1). Subsequently, we docked both active and inactive structural derivatives into the binding site of the IP receptor in accordance with the same docking protocol. Subsequently, conducting cMD simulations to further validate and complement the structural insights. As shown in Figure 4, the binding modes of all active derivatives are similar to those of MRE-269, with favorable docking scores (Table S1). Both key structural groups of the active derivatives, namely the diphenylpyrazinyl group and the carboxyl end, overlap well with the corresponding structure of MRE-269 (Figures 4B, 4C and 4D). The electrostatic interaction between the carboxyl end of IP receptor agonists and the positively charged binding site is considered the major driving force for agonist-receptor binding[17]. As depicted in the interaction diagram, the carboxyl ends of the active derivatives form hydrogen bonds and salt bridge with the conserved structural motif Y75-S168-R279. The π-π stacking between the phenyl group of the diphenylpyrazine moiety and Tyr281 is recognized as the key structural feature determining the selectivity of MRE-269 and its structural derivatives[17]. Among all active structural derivatives, the benzene ring maintained π-π stacking with Try281. Docking simulations were also conducted for the inactive derivatives. When the alkoxy side chain attached to the amine moiety is short, the carboxyl end fails to form key electrostatic interactions with the conserved structural motif Y75-S168-R279, resulting in poor docking scores (Table S1). For instance, derivatives 3a, 3b (Figure 5A), 4a, 4c, 4g, and 4m (Figure 5B) feature shortened alkoxy side chains relative to MRE-269. Consequently, docking results indicate that these compounds fail to establish electrostatic interactions with the motif Y75-S168-R279 and correspondingly lose their antiaggregatory activity. On the other hand, when the alkoxy side chain on the amine is extended, derivatives 3d, 3e and 3f, the antiaggregatory activity of the derivatives is diminished. As demonstrated by the docking results, although the diphenylpyrazine moieties and carboxyl ends aligned well with those of MRE-269, the intermediate alkoxy side chain adopts an unnatural curled conformation (Figure 5C), which may result in high intramolecular conformational energy. Therefore, based on the structural characteristics of the series of synthesized derivatives, the optimal distance of the linear alkoxy side chain between the amino and carboxyl terminals should be 6-7 atoms. In an alternative structural modification strategy, the N -isopropyl group and N -butyl side chain of MRE-269 were substituted with azetidine, pyrrolidine, and piperidine, respectively. Among the resulting derivatives, the analog modified with the piperidine showed remarkably enhanced antiaggregatory activity. This phenomenon may be associated with the steric volume of the hydrophobic group on the amine. The N -isopropyl group of MRE-269 engages with the hydrophobic pocket formed by Ser20, Met23 and Val71 (Figure 6). Mutations of Met23 and Val71 to alanine both resulted in reduced inhibitory activity of MRE-269 in cAMP accumulation assay[17], which confirms the indispensable role of hydrophobic interactions participated by these two residues in modulating the activity of IP receptor agonists. The N -isopropyl group and N -butyl side chain were substituted with azetidine, as exemplified by compounds 4b and 4d. Molecular docking results showed that the core structural motifs of 4b and 4d aligned well with those of the reference compound MRE-269, and the side chain architectures of these derivatives were structurally comparable to that of MRE-269 (Figure 6A). Despite this high degree of structural overlap, the reduced steric volume of azetidine compared with the N -isopropyl group was presumably responsible for the attenuated hydrophobic contacts formed with the surrounding hydrophobic pocket of the target protein. Energy decomposition calculations based on cMD simulations further indicated that Val71 exerted a negligible favorable energetic contribution to the binding of derivative 4b (Figure 7). In addition, cMD simulations result demonstrated that although the diphenylpyrazine moiety of 4b retained interactions with the adjacent amino acid residue, the energetic contributions of the surrounding residues Phe278 and Tyr281 to its binding were markedly diminished. Most importantly, the key amino acid Arg279 exerted an unfavorable energetic contribution to the binding of 4b, implying that the carboxyl end of 4b failed to form electrostatic interactions with Arg279. Moreover, the prominent RMSD fluctuations of 4b throughout the simulation period indicated its inability to maintain binding at the active site (Figure S1B). Following the replacement of the N -isopropyl group and N -butyl side chain with pyrrolidine and piperidine rings, whose steric volumes are comparable to that of the N -isopropyl group. As exemplified by derivatives 4f-R, 4o and 4q, these derivatives retained their hydrophobic interactions with the surrounding hydrophobic pocket (Figures 6B, 6C and 6D). The energy decomposition analysis further confirmed that Met23 and Val71 contributed favorable binding energies to 4f-R, 4o and 4q-R that were comparable to those contributed to MRE-269 (Figure 7). However, we noted that the electrostatic interaction between the pyrrolidine modified derivative 4f-R and the key amino acid Arg279 was significantly attenuated (Figure 7). In contrast, the piperidine modification did not elicit any adverse effect on the energetic contributions of other key amino acids. This finding underscores that the piperidine modification strategy achieves cyclic structural optimization, while preserving the key interactions between the derivatives and the receptor’s binding site. In addition, piperidine modification concurrently facilitated the replacement of the N -butyl side chain of MRE-269. While preserving the length of the side chain, this cyclic modification effectively reduced the conformational flexibility of the side chain. The 300 ns cMD simulations demonstrated that the RMSD fluctuation amplitudes of 4o and 4q-R were smaller than those of 4f-R and 3i featuring with flexible linear side chains, confirming that bound conformations of 4o and 4q-R exhibited enhanced dynamic stability (Figure S1B). Collectively, these results indicate that replacing the N -isopropyl group and N -butyl side chain with piperidine not only retained the hydrophobic pharmacophore but also constrained the conformational flexibility of the alkylene chain. Common Feature-Based Pharmacophore Models Pharmacophore model was generated based on the common structural features of MRE-269 and its active derivatives, using the Pharmacophore Query Editor of MOE. The Pharmacophore model demonstrated that the essential pharmacophoric features for the activity of MRE-269 derivatives include, in addition to the previously validated aromatic ring regions of the diphenylpyrazine scaffold, a hydrophobic substituent at the nitrogen atom adjacent to the pyrazine ring, and a terminal electrostatic moiety[19, 22]. Notably, the active derivatives uncovered an additional hydrophobic group on the alkylene linker (Figure S2). And this hydrophobic group serves as a critical pharmacophoric feature of the active derivative. Derivative 3c, obtained by inserting an oxygen atom into the alkylene linker of MRE-269, showed an almost complete loss of activity. In contrast, derivatives 3g, 3h, and 3i, prepared by introducing methyl, ethyl, and dimethyl hydrophobic groups onto the alkoxyl side chain of 3c respectively, exhibited a marked increase in antiaggregatory activity, with IC₅₀ reaching 2.8, 11.7, and 2.0 μM. Among them, the antiaggregatory activity of 3i was nearly 10-fold higher than that of 3c (Figure 3). Similarly, derivative 4q (IC₅₀ = 0.75 μM), derived from the introduction of dimethyl group onto the piperidine ring of 4o, also displayed a 11-fold enhancement in antiaggregatory activity. Molecular docking studies showed that 4q shared a similar binding mode via the dimethyl group on the alkoxyl side chains with 3g, 3h and 3i, and these hydrophobic moieties could directly interact with Trp169 (Figures 4B, 4D, S2B and S2C). However, 300 ns cMD simulation results revealed distinctly different binding modes of the dimethyl groups between 3i and 4q-R (Figure S3). Specifically, the dimethyl group on the alkoxyl side chain of 3i oriented toward transmembrane domain 7 (TM7) and formed stable hydrophobic interactions with hydrophobic amino acid residues including Met23, Leu275 and Phe278 (Figure S3A). Further binding free energy decomposition analysis confirmed that, compared with MRE-269, the favorable energy contribution of Met23 to 3i binding was significantly enhanced (-0.97 kcal/mol for MRE-269, -2.40 kcal/mol for 3i). In contrast, the dimethyl group on the piperazine ring of 4q-R was oriented toward transmembrane domain 2 (TM2). Meanwhile, the introduction of the piperazine ring induced the carboxyl end to adopt an upward conformation. This conformation could not only form electrostatic interactions with Arg279 but also establish hydrogen bonds with Trp169 and Ser168 located on extracellular loop 2 (ECL2) (Figure S3D). Binding free energy decomposition results showed that the favorable energy contribution of Trp169 to 4q-R binding exhibited a particularly remarkable increase (-1.65 kcal/mol for MRE-269, -3.28 kcal/mol for 4q-R). Furthermore, small-sized hydrophobic group on the alkylene chains were well tolerated. For instance, derivative 3h, obtained by introducing an ethyl group, exhibited a 5 fold decrease in antiaggregatory activity compared with 3i. This phenomenon is attributed to the steric restriction of the hydrophobic pocket, which can only accommodate small hydrophobic group (Figure 6). Conclusions In this study, MRE-269, the active metabolite of selexipag, was used as the lead compound to design and synthesize two series of derivatives, diphenylpyrazinyl isopropylamine alkoxyacetic acid (Series 3 ) and diphenylpyrazinyl cycloaminoalkoxyacetic acid (Series 4 ). The SAR of these derivatives was systematically investigated via platelet aggregation inhibition assays combined with molecular docking and cMD simulations. Specifically, replacing the N -isopropyl group and N- butyl side chain of MRE-269 with pyrrolidine or piperidine rings (yielding derivatives 4f-R, 4i-R, 4o, and 4q) retained hydrophobic interactions with IP receptor residues Met23 and Val71 due to matching steric volume. Among these modifications, piperidine ring substitution emerged as the optimal strategy. It enhanced the binding affinity with Met23 and Val71 by expanding the hydrophobic interaction surface. Meanwhile, replacing partial alkylene chain with piperidine ring rigidified the alkylene chain linker, thereby reducing its conformational flexibility. Furthermore, this study confirmed that small hydrophobic substituents (e.g., methyl and dimethyl groups) on the alkylene chain serve as key pharmacophores for MRE-269 derivatives. For instance, the hydrophobic groups on the alkoxyl side chains of active derivatives such as 3g and 3i can achieve spatial matching with the hydrophobic pocket of the IP receptor, thereby enhancing local hydrophobic interactions. Derivative 4q-S (the S-isomer of 4q), generated by integrating the two aforementioned modification strategies, exhibited not only potent antiaggregatory activity but also excellent safety profiles and superior pharmacokinetic stability[ 20 ]. In rat models, 4q-S demonstrated favorable oral bioavailability and a relatively prolonged plasma residence time. Moreover, it was non-toxic in cardiotoxicity assessments (hERG channel inhibition assay) (Table S4) and genotoxicity assays (Mini-Ames Test) (Table S3). Collectively, these pharmacodynamic, pharmacokinetic, and safety properties indicate that 4q-S holds promising druggability. These findings delineate clear SAR guidance for the rational design of IP receptor agonists. Specifically, this study can provide critical structural insights to support the development of novel, highly potent, and low-toxicity IP receptor agonists for the treatment of PAH. Declarations Acknowledgements The authors are grateful to Cloud Scientific for access to the MOE software. Competing Interests All other authors declare no potential conflicts of interest. Funding This work was supported by the National Science Foundation of China (Grant No. 82373761), the Chongqing Natural Science Foundation (Grant No. CSTB2025NSCO-GPX0347) and the Sichuan Natural Science Foundation (NSFSC) (Grant No. 2024NSFSC0634). Author information Authors and Affiliations School of Chemistry Engineering, Sichuan University of Science & Engineering, No.180 Xueyuan Street, Huixing Road, Zigong 643000, China Xianrong Cai, Juping Cheng, Yuhe Wang, Guiying Liu, Xiaoyan Ma, Qingquan Fu & Xiaoli An Chongqing Key Laboratory of High Active Traditional Chinese Medicine Delivery System, Chongqing Medical and Pharmaceutical College, No. 82, University Town Middle Road, Shapingba District, Chongqing 410331, China Zhenqiang Mu Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Xianrong Cai, Xiaoli An, Juping Cheng and Yuhe Wang . The first draft of the manuscript was written by Xianrong Cai and Xiaoli An, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Corresponding authors Correspondence to Zhenqiang Mu, Xiaoli An Supplementary data Supplementary data to this article can be found online at https://doi.org/. References Alfranca A, Iñiguez MA, Fresno M, et al. (2006) Prostanoid signal transduction and gene expression in the endothelium: role in cardiovascular diseases. Cardiovascular research 70: 446–456 Pluchart H, Khouri C, Blaise S, et al. (2017) Targeting the Prostacyclin Pathway: Beyond Pulmonary Arterial Hypertension. Trends in pharmacological sciences 38: 512–523 Narumiya S (2007) Physiology and pathophysiology of prostanoid receptors. Proc Jpn Acad Ser B Phys Biol Sci. 83: 296–319 Vane J, Corin RE (2003) Prostacyclin: A Vascular Mediator. European Journal of Vascular and Endovascular Surgery 26: 571-578 Kothapalli D, Stewart SA, Smyth EM, et al. (2003) Prostacylin Receptor Activation Inhibits Proliferation of Aortic Smooth Muscle Cells by Regulating cAMP Response Element-Binding Protein- and Pocket Protein-Dependent Cyclin A Gene Expression. Molecular Pharmacology 64: 249-258 Idzko M, Hammad H, Van Nimwegen M, et al. (2007) Inhaled iloprost suppresses the cardinal features of asthma via inhibition of airway dendritic cell function. Journal of Clinical Investigation 117: 464-472 Zeng C, Liu J, Zheng X, et al. (2023) Prostaglandin and prostaglandin receptors: present and future promising therapeutic targets for pulmonary arterial hypertension. Respiratory Research 24. Sharma M, Paudyal V, Syed SK, et al. (2025) Management of Pulmonary Arterial Hypertension: Current Strategies and Future Prospects. Life 15. Duggan ST, Keam SJ, Burness CB (2016) Selexipag: A Review in Pulmonary Arterial Hypertension. American Journal of Cardiovascular Drugs 17: 73-80 Scott LJ (2016) Selexipag: First Global Approval. 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(2008) A Long-Acting and Highly Selective Prostacyclin Receptor Agonist Prodrug, 2-{4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide (NS-304), Ameliorates Rat Pulmonary Hypertension with Unique Relaxant Responses of Its Active Form, {4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic Acid (MRE-269), on Rat Pulmonary Artery. The Journal of Pharmacology and Experimental Therapeutics 326: 691-699 Asaki T, Kuwano K, Morrison K, et al. (2015) Selexipag: An Oral and Selective IP Prostacyclin Receptor Agonist for the Treatment of Pulmonary Arterial Hypertension. Journal of Medicinal Chemistry 58: 7128-7137 Wang JJ, Jin S, Zhang H, et al. (2024) Molecular recognition and activation of the prostacyclin receptor by anti-pulmonary arterial hypertension drugs. Sci. Adv. 10: eadk5184 Baldoni D, Bruderer S, Muhsen N, et al. (2015) Bioequivalence of different dose-strength tablets of selexipag, a selective prostacyclin receptor agonist, in a multiple-dose up-titration study. Int. Journal of Clinical Pharmacology and Therapeutics 53: 788 - 798 Stoll F, Liesener S, Hohlfeld T, et al. (2002) Pharmacophore Definition and Three-Dimensional Quantitative Structure-Activity Relationship Study on Structurally Diverse Prostacyclin Receptor Agonists. Mol Pharmacol. 62: 1103-1111 Cai X, Lin G, Tang J, et al. (2025) Synthesis and Evaluation of Diphenylpyrazine Cyclic Amine Derivatives as IP Receptor Agonists. ACS Medicinal Chemistry Letters 16: 1772-1779 Allen WJ, Rizzo RC (2014) Implementation of the Hungarian Algorithm to Account for Ligand Symmetry and Similarity in Structure-Based Design. Journal of Chemical Information and Modeling 54: 518-529 Asaki T, Hamamoto T, Sugiyama Y, et al. (2007) Structure–activity studies on diphenylpyrazine derivatives: A novel class of prostacyclin receptor agonists. Bioorganic & Medicinal Chemistry 15: 6692-6704 Scheme Scheme 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Supportinginformations.docx scheme1.png Scheme 1. Reagents: (a) 5-chloro-2,3-diphenylpyrazine 6, NaOH, MeOH, 90 ℃; (b) t -butyl 2-bromoacetate derivatives, n -Bu 4 NHSO 4 , aq KOH, toluene; (c) LiOH, MeOH; (d) LiAlH 4 , THF, 0~20 ℃; (e) t -butyl 2-bromoacetate, n -Bu 4 NHSO 4 , aq KOH, toluene. scheme2.png Scheme 2. Reagents: (a) 5-chloro-2,3-diphenylpyrazine 6, NaOH, MeOH, 90 o C; (b) t -butyl 2-bromoacetate derivatives, n -Bu 4 NHSO 4 , aq KOH, toluene; (c) LiOH, MeOH; (d) LiAlH 4 , THF, 0~20 o C; (e) t -butyl 2-bromoacetate, n -Bu 4 NHSO 4 , aq KOH, toluene. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 18 May, 2026 Reviews received at journal 15 May, 2026 Reviewers agreed at journal 13 May, 2026 Reviewers agreed at journal 10 May, 2026 Reviewers invited by journal 01 Mar, 2026 Editor assigned by journal 01 Mar, 2026 Submission checks completed at journal 25 Feb, 2026 First submitted to journal 02 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8764373","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":600418831,"identity":"34eea972-1a87-4b25-b4a0-bf3b5f489a5c","order_by":0,"name":"Xianrong Cai","email":"","orcid":"","institution":"Sichuan University of Science \u0026 Engineering","correspondingAuthor":false,"prefix":"","firstName":"Xianrong","middleName":"","lastName":"Cai","suffix":""},{"id":600418832,"identity":"c2e72feb-98f9-44e4-bf24-06c27cdf6b91","order_by":1,"name":"Juping Cheng","email":"","orcid":"","institution":"Sichuan University of Science \u0026 Engineering","correspondingAuthor":false,"prefix":"","firstName":"Juping","middleName":"","lastName":"Cheng","suffix":""},{"id":600418833,"identity":"4d7b669e-530a-487e-bb3e-6f9158c3910a","order_by":2,"name":"Yuhe Wang","email":"","orcid":"","institution":"Sichuan University of Science \u0026 Engineering","correspondingAuthor":false,"prefix":"","firstName":"Yuhe","middleName":"","lastName":"Wang","suffix":""},{"id":600418834,"identity":"ae1b8aa6-c536-4284-b2ec-4ad2cad5969c","order_by":3,"name":"Guiying Liu","email":"","orcid":"","institution":"Sichuan University of Science \u0026 Engineering","correspondingAuthor":false,"prefix":"","firstName":"Guiying","middleName":"","lastName":"Liu","suffix":""},{"id":600418835,"identity":"9369a5ca-94e0-4e5e-80c3-7da0e7f37b1f","order_by":4,"name":"Xiaoyan Ma","email":"","orcid":"","institution":"Sichuan University of Science \u0026 Engineering","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Ma","suffix":""},{"id":600418836,"identity":"0de2b6f8-aa1e-427b-a9c8-80b81bab3da5","order_by":5,"name":"Qingquan Fu","email":"","orcid":"","institution":"Sichuan University of Science \u0026 Engineering","correspondingAuthor":false,"prefix":"","firstName":"Qingquan","middleName":"","lastName":"Fu","suffix":""},{"id":600418837,"identity":"6a32b595-1992-4bb2-93b6-6011104b26fa","order_by":6,"name":"Xiaoli An","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIie3OMQrCMBSA4RcKnaJd6+IZ4iYo9iophbh07lwXpx6gxUv0CK8EHJs10A6KF+jYQcHiIg6adnPIB28IvJ8XAMv6Qy4Awa7fvp9G3jBVkYkJySIFkNSVExLWZAwpVQFTEqFLJHin1JC0Z47+uglLLTjJawl+i4ZER4iMNpxpypzZUQLz+e8k0GGK3K2Hj6nOeYxJXlfQRVJiDA4ZlwheHbIoLLRgVVbvqa+NSby63ftdMFfyeumTzdLLDckHHIZO2Lcsy7K+eQJzN0uzsM7lXgAAAABJRU5ErkJggg==","orcid":"","institution":"Sichuan University of Science \u0026 Engineering","correspondingAuthor":true,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"An","suffix":""},{"id":600418838,"identity":"92ba2a51-b369-410d-96cd-c4dd7f281fe7","order_by":7,"name":"Zhenqiang Mu","email":"","orcid":"","institution":"Chongqing Medical and Pharmaceutical College","correspondingAuthor":false,"prefix":"","firstName":"Zhenqiang","middleName":"","lastName":"Mu","suffix":""}],"badges":[],"createdAt":"2026-02-02 11:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8764373/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8764373/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104069574,"identity":"a747eee4-f0a4-4602-97d4-1e554b3abbbf","added_by":"auto","created_at":"2026-03-06 11:33:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15823,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures of Selexipag and MRE-269.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8764373/v1/c3e8e7c1705f242fc12b39d3.png"},{"id":104069573,"identity":"f68bb120-d46d-4229-88c7-cf7eccd8c0cc","added_by":"auto","created_at":"2026-03-06 11:33:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38278,"visible":true,"origin":"","legend":"\u003cp\u003eDesign of diphenylpyrazinyl amino alkoxy acetic acids derivatives.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8764373/v1/fa85ec7257821961fd88b74d.png"},{"id":104403436,"identity":"5d72fdb5-ff6a-4a1f-9924-68deb9e16ab1","added_by":"auto","created_at":"2026-03-11 12:18:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27750,"visible":true,"origin":"","legend":"\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e values of the designed compounds on ADP-induced platelet aggregation.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8764373/v1/2671e95841b3a8ec61b3e900.png"},{"id":104403437,"identity":"1b2eab74-ff45-4b2f-ad49-8897c7383fca","added_by":"auto","created_at":"2026-03-11 12:18:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":355899,"visible":true,"origin":"","legend":"\u003cp\u003eSuperposition of the docking binding modes of MRE-269 and its active derivatives with the native pose (gray) of its cryo‑EM structure (PDB ID: 8X79). Ligands are shown as sticks, and residues as lines. A: Re-docking binding mode of MRE-269 (green); B: Docking binding modes of 3g (yellow), 3h (orange), and 3i (blue); C: Docking binding modes of 4f-R (pink) and 4i-R (darkgreen); D: Docking binding modes of 4o (aquamarine), 4q-R (mediumpurple), and 4q-S (plum red).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8764373/v1/3d5956485287b651948a7c4c.png"},{"id":104069582,"identity":"280a6d6e-902d-41e7-9cfc-5641bb58a5a8","added_by":"auto","created_at":"2026-03-06 11:33:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":356877,"visible":true,"origin":"","legend":"\u003cp\u003eSuperposition of the docking binding modes of inactive structural derivatives with the native pose (gray) of its cryo‑EM structure. A: Docking binding mode of 3a (magenta) and 3b (gold); B: Docking binding modes of 4a (seagreen), 4c (olivedrab), 4g (chartreuse) and 4m (paleturquoise); C: Docking binding modes of 3d (forestgreen) , 3e (darkturquoise) and 3f (khaki); D: Docking binding modes of 4h (purple), 4n (darkorchid), and 4p (plum).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8764373/v1/2ecc068a9c87adff303d4228.png"},{"id":104403456,"identity":"9a8d5c8d-5fc9-40d9-8eab-299c0dce78e5","added_by":"auto","created_at":"2026-03-11 12:18:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":678641,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction between the hydrophobic binding pocket (cyan surface) of the IP receptor and N-isopropyl group of MRE-269 (gray). A, Docking binding mode of 4b (darkcyan) and 4d (moccasin). B, Docking binding mode of 4f-R (pink). C, Docking binding mode of 4o (aquamarine). D, Docking binding mode of4q-R (mediumpurple) and 4q-S (plum red).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8764373/v1/cc25d3435c5a1773b4cc007b.png"},{"id":104069581,"identity":"d422c3b9-722f-4735-a34f-01da04db5b67","added_by":"auto","created_at":"2026-03-06 11:33:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":179811,"visible":true,"origin":"","legend":"\u003cp\u003ePer-residue energetic contribution difference for MRE-269, derivatives 4q-R, 4o, 4f-R, 3i and 4b.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8764373/v1/d2cdb920b59868e21ab74926.png"},{"id":104409266,"identity":"954274c9-d344-4325-9fab-49f76bd08b12","added_by":"auto","created_at":"2026-03-11 12:44:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2341258,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8764373/v1/220241f6-0e72-4a8d-858b-0422cdfc9085.pdf"},{"id":104069583,"identity":"eec271b4-ad5b-494e-b661-f108c0aea437","added_by":"auto","created_at":"2026-03-06 11:33:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":56012641,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformations.docx","url":"https://assets-eu.researchsquare.com/files/rs-8764373/v1/43de856e979b43d43d3c173f.docx"},{"id":104069577,"identity":"af2acd41-d24d-4422-998b-b7a18049af5f","added_by":"auto","created_at":"2026-03-06 11:33:27","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":74180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e Reagents: (a) 5-chloro-2,3-diphenylpyrazine \u003cstrong\u003e6\u003c/strong\u003e, NaOH, MeOH, 90 ℃; (b) \u003cem\u003et\u003c/em\u003e-butyl 2-bromoacetate derivatives, \u003cem\u003en\u003c/em\u003e-Bu\u003csub\u003e4\u003c/sub\u003eNHSO\u003csub\u003e4\u003c/sub\u003e, aq KOH, toluene; (c) LiOH, MeOH; (d) LiAlH\u003csub\u003e4\u003c/sub\u003e, THF, 0~20 ℃; (e) \u003cem\u003et\u003c/em\u003e-butyl 2-bromoacetate, \u003cem\u003en\u003c/em\u003e-Bu\u003csub\u003e4\u003c/sub\u003eNHSO\u003csub\u003e4\u003c/sub\u003e, aq KOH, toluene.\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-8764373/v1/4afc66fe4fffa036242cb9f7.png"},{"id":104069580,"identity":"67c8a1a7-c959-4087-874a-332bc19bf572","added_by":"auto","created_at":"2026-03-06 11:33:27","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":107480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 2.\u003c/strong\u003e Reagents: (a) 5-chloro-2,3-diphenylpyrazine \u003cstrong\u003e6\u003c/strong\u003e, NaOH, MeOH, 90 \u003csup\u003eo\u003c/sup\u003eC; (b) \u003cem\u003et\u003c/em\u003e-butyl 2-bromoacetate derivatives, \u003cem\u003en\u003c/em\u003e-Bu\u003csub\u003e4\u003c/sub\u003eNHSO\u003csub\u003e4\u003c/sub\u003e, aq KOH, toluene; (c) LiOH, MeOH; (d) LiAlH\u003csub\u003e4\u003c/sub\u003e, THF, 0~20 \u003csup\u003eo\u003c/sup\u003eC; (e) \u003cem\u003et\u003c/em\u003e-butyl 2-bromoacetate, \u003cem\u003en\u003c/em\u003e-Bu\u003csub\u003e4\u003c/sub\u003eNHSO\u003csub\u003e4\u003c/sub\u003e, aq KOH, toluene.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"scheme2.png","url":"https://assets-eu.researchsquare.com/files/rs-8764373/v1/a7662ef083b5ac543009cc2f.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structure-Optimization and Structure-Activity Relationship of Diphenylpyrazinyl Aminoalkoxyacetic Acid Derivatives: Piperidine Substitution and Hydrophobic Modification as Key Strategies for IP Receptor Agonists","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProstacyclin (prostaglandin I\u003csub\u003e2\u003c/sub\u003e, PGI\u003csub\u003e2\u003c/sub\u003e) is an endogenous bioactive substance produced by endothelial and vascular smooth muscle cells[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. PGI2 has a range of physiological effects, including vasodilation, inhibition of platelet aggregation, promotion of vascular smooth muscle cell differentiation and inhibition of their proliferation[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Meanwhile, PGI\u003csub\u003e2\u003c/sub\u003e has been shown to reduce pulmonary blood pressure and bronchial hyper-responsiveness, improving pulmonary function[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These biological effects are mainly mediated by binding to the prostacyclin receptor (IP receptor) on the surface of smooth muscle cells and activating downstream signaling pathways. Hence, the IP receptor has emerged as a key therapeutic target for vascular diseases such as pulmonary arterial hypertension (PAH). Prostacyclin, prostacyclin analogs, and IP receptor agonists (including selexipag, epoprostenol, beraprost, iloprost, and treprostinil) have been clinically utilized for the treatment of PAH[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSelexipag (1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) is a first-in-class, orally active, non-prostanoid IP receptor agonist[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It was approved by the U.S. Food and Drug Administration (FDA) in 2015 for the treatment of PAH, with the aim of delaying disease progression and reducing the risk of hospitalization for PAH[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Following oral administration, selexipag is rapidly metabolized to form its active metabolite MRE-269[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], which has been confirmed as the major contributor to the drug's pharmacological effects[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], with mean elimination half-life of 9.4\u0026ndash;14.2 h in the human body[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Pharmacological studies have demonstrated that MRE-269 exhibits high selectivity for the IP receptor, with a binding affinity (K\u003csub\u003ei\u003c/sub\u003e = 20 nM) approximately 130-fold higher than that for other prostaglandin receptors (prostaglandin E receptors, EP\u003csub\u003e1-4\u003c/sub\u003e; prostaglandin D receptor, DP\u003csub\u003e1\u003c/sub\u003e; prostaglandin F\u003csub\u003e2\u003c/sub\u003e receptor, FP; and thromboxane A2 receptor, TP)[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Structural biology studies have verified that this high selectivity is attributed to the π-π stacking interaction formed between the biphenylpyrazine moiety of MRE-269 and the Tyr281 residue of the IP receptor[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The most common adverse reactions of selexipag are similar to those of prostacyclin analogs, mainly including headache, diarrhea, nausea, and jaw pain[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo develop IP receptor agonists with high potency, high selectivity, and long-lasting pharmacological effects, structural modification and optimization were conducted on MRE-269 as the lead compound in our previous research. Specifically, we designed and synthesized a series of 2-cyclic amino-5,6-diphenylpyrazine derivatives by substituting the \u003cem\u003eN\u003c/em\u003e-isopropyl group of MRE-269 with cyclic amines of varying sizes, while preserving the key pharmacophoric scaffolds (the oxyacetic acid group and diphenylpyrazine scaffolds intact), which are structural elements critical to the antiaggregatory effects and specificity of MRE-269[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Among these newly synthesized derivatives, active compounds featuring piperidine ring substitution were successfully identified[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present work, we continue the aforementioned structural optimization strategy and further expand the modification dimensions. Still taking MRE-269 as the lead compound, we designed two structural modification strategies: (1) side-chain engineering, involving elongation, shortening, or the insertion of oxygen atoms into the \u003cem\u003eN\u003c/em\u003e-butyl side chain; and (2) cyclic amine incorporation, modifying the \u003cem\u003eN\u003c/em\u003e-isopropyl group and \u003cem\u003eN\u003c/em\u003e-butyl side chain with various cyclic amines. Based on these strategies, two series of derivatives were synthesized, diphenylpyrazinyl isopropylamine alkoxyacetic acid (series \u003cb\u003e3\u003c/b\u003e) and diphenylpyrazinyl cycloaminoalkoxyacetic acid (series \u003cb\u003e4\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To systematically elucidate the effect of structural modifications on activity, we comprehensively investigated the structure-activity relationships (SAR) of these derivatives by integrating platelet aggregation assays in vitro, molecular docking and conventional molecular dynamics (cMD) simulation techniques. The purpose of this study is to clarify the effects and molecular mechanisms of side chain engineering and cyclic amine substitution on IP receptor agonistic activity through systematic SAR analysis, thereby providing a solid scientific basis and clear design ideas for the directed structural optimization of IP receptor agonists in future research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003ch4\u003e\u003cstrong\u003eSynthesis of the Designed Library\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThe synthesis of all 29 target molecules followed the outlined pathways in Scheme 1 (series \u003cstrong\u003e3\u003c/strong\u003e) and Scheme 2 (series \u003cstrong\u003e4\u003c/strong\u003e). Key synthetic steps included phase-transfer alkylation of diphenylpyrazin-2-yl isopropylamino alcohols with tert-butyl bromoacetate, followed by reduction of tert-butyl esters to the corresponding alcohols using LiAlH₄. The final hydrolysis step converted tert-butyl esters into the free acids.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor series \u003cstrong\u003e3\u003c/strong\u003e, diphenylpyrazinyl isopropylamino alkoxyacetic acids \u003cstrong\u003e3\u003c/strong\u003e were synthesized via basic hydrolysis of tert-butyl esters \u003cstrong\u003e8\u003c/strong\u003e, \u003cstrong\u003e10\u003c/strong\u003e and \u003cstrong\u003e12\u003c/strong\u003e Esters \u003cstrong\u003e8\u003c/strong\u003e were prepared by alkylating diphenylpyrazin-2-yl isopropylamino alcohols \u003cstrong\u003e7\u0026nbsp;\u003c/strong\u003ewith tert-butyl bromoacetate (or its derivatives) under phase-transfer conditions. The alcohols \u003cstrong\u003e7\u003c/strong\u003e were synthesized by coupling 2-chloro-5,6-dimethylpyrazine \u003cstrong\u003e6\u003c/strong\u003e with the corresponding isopropylamine derivatives \u003cstrong\u003e5\u003c/strong\u003e. Reduction of esters \u003cstrong\u003e8\u003c/strong\u003e with lithium aluminum hydride (LiAlH\u003csub\u003e4\u003c/sub\u003e) afforded alcohols \u003cstrong\u003e9\u003c/strong\u003e, which were then alkylated with tert-butyl bromoacetate to give esters \u003cstrong\u003e10\u003c/strong\u003e. Subsequent reduction of \u003cstrong\u003e10\u003c/strong\u003e provided alcohol \u003cstrong\u003e11\u003c/strong\u003e, and re-alkylation with tert-butyl bromoacetate afforded ester \u003cstrong\u003e12\u003c/strong\u003e (Scheme 1).\u003c/p\u003e\n\u003cp\u003eIn parallel, for series \u003cstrong\u003e4\u003c/strong\u003e, we synthesized the modified diphenylpyrazinyl carboxylic acids (\u003cstrong\u003e4\u003c/strong\u003e) through a similar alkylation strategy. The synthesis involved alkylating diphenylpyrazin-2-yl cyclic amino alcohols \u003cstrong\u003e14\u003c/strong\u003e with tert-butyl bromoacetate under analogous phase-transfer conditions. The alcohols \u003cstrong\u003e14\u0026nbsp;\u003c/strong\u003ewere prepared by coupling 2-chloro-5,6-diphenylpyrazine \u003cstrong\u003e6\u003c/strong\u003e with various cyclic amino alcohols \u003cstrong\u003e13\u003c/strong\u003e. The resulting esters \u003cstrong\u003e15\u003c/strong\u003e were reduced with LiAlH₄ to furnish alcohols \u003cstrong\u003e16\u003c/strong\u003e, which were subsequently alkylated with tert-butyl bromoacetate to give esters \u003cstrong\u003e17\u003c/strong\u003e. Hydrolysis of \u003cstrong\u003e15\u003c/strong\u003e or \u003cstrong\u003e17\u003c/strong\u003e provided the corresponding acids \u003cstrong\u003e4\u0026nbsp;\u003c/strong\u003e(Scheme 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibitory\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003ectivity\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003egainst\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003elatelet\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003eggregation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the synthesis,\u0026nbsp;the compounds were tested for inhibition of ADP‑induced platelet aggregation in rabbit platelet‑rich plasma, and IC₅₀ values were obtained from dose‑response curves (Figure 3). MRE-269, used as a positive control to evaluate the inhibitory effect on platelet aggregation, exhibits an IC₅₀ value of 6.2 \u0026micro;M. Among the 29 structurally modified derivatives, the diphenylpyrazinyl cyclic amino alkoxy acetic acids derivatives 4i-R and 4o show antiaggregatory activity comparable to that of MRE-269, with corresponding IC₅₀\u003csub\u003e\u0026nbsp;\u003c/sub\u003evalues of 9.2 and 6.5 \u0026micro;M respectively. Notably, the diphenylpyrazinyl isopropylamino alkoxy acetic acids derivatives 3g and 3i, as well as the diphenylpyrazinyl cyclic amino alkoxy acetic acids derivatives 4q-R and 4q-S, demonstrate significantly superior antiaggregatory activity relative to MRE-269, with IC₅₀ values of 2.8, 2.0, 1.25 and 0.15 \u0026micro;M respectively. It is also noteworthy that the antiaggregatory activity of derivative 4q-S is far stronger than MRE-269, with a 40-fold increase in activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs can be inferred from the aforementioned activity data, the insertion of an oxygen atom into the \u003cem\u003eN\u003c/em\u003e-butyl side chain of MRE-269 exerts an adverse effect on the antiaggregatory activity, with derivative 3c exhibiting an IC₅₀ value of 20 \u0026mu;M. Then both elongation and shortening of the side chain of MRE-269 or 3c exert an adverse effect on the antiaggregatory activity, as exemplified by derivatives 3a, 3b, 3d and 3e.\u0026nbsp;However, the introduction of hydrophobic groups (i.e., methyl or ethyl) onto the alkoxyl side chain of 3c can compensate for the loss of activity caused by such side chain elongation. As exemplified by derivatives 3g, 3h, and 3i, their antiaggregatory activities are increased by multiple folds compared with that of 3c. Therefore, the incorporation of hydrophobic groups into the side chain is likely to be crucial for enhancing the antiaggregatory activity of derivatives.\u003c/p\u003e\n\u003cp\u003eAdditionally, derivatives of MRE-269 that contain a four-membered cyclic amine structure substituting of the \u003cem\u003eN\u003c/em\u003e-isopropyl and \u003cem\u003eN\u003c/em\u003e-butyl side chains completely lose their antiaggregatory activity (4a, 4b, 4c and 4d). In contrast, derivatives with a five-membered (4f-R and 4i-R) or six-membered cyclic amine structure (4o and 4q) retain potent antiplatelet aggregatory activity. This activity difference is likely attributable to the ability of five- and six-membered cyclic amine moieties to mimic the functional moiety of the \u003cem\u003eN\u003c/em\u003e-isopropyl group in MRE-269. Notably, six-membered cyclic amine derivatives show stronger\u0026nbsp;antiaggregatory activity\u0026nbsp;than five-membered ones. This enhanced activity\u0026nbsp;may be attributed to\u0026nbsp;the more extensive hydrophobic interactions between the six-membered cyclic amine structure and the IP receptor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Docking Studies and Molecular Dynamics Simulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the antiaggregatory activities of the synthesized derivatives, we employed molecular docking and cMD simulations to establish the structure-activity relationships (SARs) of the synthesized diphenylpyrazinyl isopropylamine alkoxyacetic acid and diphenylpyrazinyl cycloaminoalkoxyacetic acid derivatives as IP receptor agonists.\u003c/p\u003e\n\u003cp\u003eTo establish the SARs, we first employed molecular docking to delineate the binding modes of these derivatives. First, we redocked MRE-269 into the binding site of the IP receptor to confirm the reliability of the docking protocol implemented in MOE\u0026apos;s dock application. The top-ranked redocking binding pose of MRE-269 aligned well with the native pose of its\u0026nbsp;cryo‑EM\u0026nbsp;structure (Figure 4A), with a root-mean-square deviation (RMSD) value of 0.93 \u0026Aring; (RMSD \u0026lt; 2 \u0026Aring;)[21]\u0026nbsp;and a corresponding docking score of \u0026minus;10.9 kcal/mol (Table S1). Subsequently, we docked both active and inactive structural derivatives into the binding site of the IP receptor in accordance with the same docking protocol. Subsequently, conducting cMD simulations to further validate and complement the structural insights.\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 4, the binding modes of all active derivatives are similar to those of MRE-269, with favorable docking scores (Table S1). Both key structural groups of the active derivatives, namely the diphenylpyrazinyl group and the carboxyl end, overlap well with the corresponding structure of MRE-269 (Figures 4B, 4C and 4D). The electrostatic interaction between the carboxyl end of IP receptor agonists and the positively charged binding site is considered the major driving force for agonist-receptor binding[17]. As depicted in the interaction diagram, the carboxyl ends of the active derivatives form hydrogen bonds and salt bridge with the conserved structural motif Y75-S168-R279. The \u0026pi;-\u0026pi; stacking between the phenyl group of the diphenylpyrazine moiety and Tyr281 is recognized as the key structural feature determining the selectivity of MRE-269 and its structural derivatives[17]. Among all active structural derivatives, the benzene ring maintained \u0026pi;-\u0026pi; stacking with Try281.\u003c/p\u003e\n\u003cp\u003eDocking simulations were also conducted for the inactive derivatives. When the alkoxy side chain attached to the amine moiety is short, the carboxyl end fails to form key electrostatic interactions with the conserved structural motif Y75-S168-R279, resulting in poor docking scores (Table S1). For instance, derivatives 3a, 3b (Figure 5A), 4a, 4c, 4g, and 4m (Figure 5B) feature shortened alkoxy side chains relative to MRE-269. Consequently, docking results indicate that these compounds fail to establish electrostatic interactions with the motif Y75-S168-R279 and correspondingly lose their antiaggregatory activity. On the other hand, when the alkoxy side chain on the amine is extended, derivatives 3d, 3e and 3f, the antiaggregatory activity of the derivatives is diminished. As demonstrated by the docking results, although the diphenylpyrazine moieties and carboxyl ends aligned well with those of MRE-269, the intermediate alkoxy side chain adopts an unnatural curled conformation (Figure 5C), which may result in high intramolecular conformational energy. Therefore, based on the structural characteristics of the series of synthesized derivatives, the optimal distance of the linear alkoxy side chain between the amino and carboxyl terminals should be 6-7 atoms.\u003c/p\u003e\n\u003cp\u003eIn an alternative structural modification strategy, the \u003cem\u003eN\u003c/em\u003e-isopropyl group and \u003cem\u003eN\u003c/em\u003e-butyl side chain of MRE-269 were substituted with azetidine, pyrrolidine, and piperidine, respectively. Among the resulting derivatives, the analog modified with the piperidine showed remarkably enhanced antiaggregatory activity. This phenomenon may be associated with the steric volume of the hydrophobic group on the amine. The \u003cem\u003eN\u003c/em\u003e-isopropyl group of MRE-269 engages with the hydrophobic pocket formed by Ser20, Met23 and Val71 (Figure 6). Mutations of Met23 and Val71 to alanine both resulted in reduced inhibitory activity of MRE-269 in cAMP accumulation assay[17], which confirms the indispensable role of hydrophobic interactions participated by these two residues in modulating the activity of IP receptor agonists.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eN\u003c/em\u003e-isopropyl group and \u003cem\u003eN\u003c/em\u003e-butyl side chain were substituted with azetidine, as exemplified by compounds 4b and 4d. Molecular docking results showed that the core structural motifs of 4b and 4d aligned well with those of the reference compound MRE-269, and the side chain architectures of these derivatives were structurally comparable to that of MRE-269 (Figure 6A). Despite this high degree of structural overlap, the reduced steric volume of azetidine compared with the \u003cem\u003eN\u003c/em\u003e-isopropyl group was presumably responsible for the attenuated hydrophobic contacts formed with the surrounding hydrophobic pocket of the target protein. Energy decomposition calculations based on cMD simulations further indicated that Val71 exerted a negligible favorable energetic contribution to the binding of derivative 4b (Figure 7). In addition, cMD simulations result demonstrated that although the diphenylpyrazine moiety of 4b retained interactions with the adjacent amino acid residue, the energetic contributions of the surrounding residues Phe278 and Tyr281 to its binding were markedly diminished. Most importantly, the key amino acid Arg279 exerted an unfavorable energetic contribution to the binding of 4b, implying that the carboxyl end of 4b failed to form electrostatic interactions with Arg279. Moreover, the prominent RMSD fluctuations of 4b throughout the simulation period indicated its inability to maintain binding at the active site (Figure S1B).\u003c/p\u003e\n\u003cp\u003eFollowing the replacement of the \u003cem\u003eN\u003c/em\u003e-isopropyl group and \u003cem\u003eN\u003c/em\u003e-butyl side chain with pyrrolidine and piperidine rings, whose steric volumes are comparable to that of the \u003cem\u003eN\u003c/em\u003e-isopropyl group. As exemplified by derivatives 4f-R, 4o and 4q, these derivatives retained their hydrophobic interactions with the surrounding hydrophobic pocket (Figures 6B, 6C and 6D). The energy decomposition analysis further confirmed that Met23 and Val71 contributed favorable binding energies to 4f-R, 4o and 4q-R that were comparable to those contributed to MRE-269 (Figure 7). However, we noted that the electrostatic interaction between the pyrrolidine modified derivative 4f-R and the key amino acid Arg279 was significantly attenuated (Figure 7). In contrast, the piperidine modification did not elicit any adverse effect on the energetic contributions of other key amino acids. This finding underscores that the piperidine modification strategy achieves cyclic structural optimization, while preserving the key interactions between the derivatives and the receptor\u0026rsquo;s binding site.\u003c/p\u003e\n\u003cp\u003eIn addition, piperidine modification concurrently facilitated the replacement of the \u003cem\u003eN\u003c/em\u003e-butyl side chain of MRE-269. While preserving the length of the side chain, this cyclic modification effectively reduced the conformational flexibility of the side chain. The 300 ns cMD simulations demonstrated that the RMSD fluctuation amplitudes of 4o and 4q-R were smaller than those of 4f-R and 3i featuring with flexible linear side chains, confirming that bound conformations of 4o and 4q-R exhibited enhanced dynamic stability (Figure S1B). Collectively, these results indicate that replacing the\u003cem\u003e\u0026nbsp;N\u003c/em\u003e-isopropyl group and \u003cem\u003eN\u003c/em\u003e-butyl side chain with piperidine not only retained the hydrophobic pharmacophore but also constrained the conformational flexibility of the alkylene chain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCommon Feature-Based Pharmacophore Models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePharmacophore model was generated based on the common structural features of MRE-269 and its active derivatives, using the Pharmacophore Query Editor of MOE. The Pharmacophore model demonstrated that the essential pharmacophoric features for the activity of MRE-269 derivatives include, in addition to the previously validated aromatic ring regions of the diphenylpyrazine scaffold, a hydrophobic substituent at the nitrogen atom adjacent to the pyrazine ring, and a terminal electrostatic moiety[19, 22]. Notably, the active derivatives uncovered an additional hydrophobic group on the alkylene linker\u0026nbsp;(Figure\u0026nbsp;S2). And this hydrophobic group serves as a critical pharmacophoric feature of the active derivative. Derivative 3c, obtained by inserting an oxygen atom into the\u0026nbsp;alkylene\u0026nbsp;linker of MRE-269, showed an almost complete loss of activity. In contrast, derivatives 3g, 3h, and 3i, prepared by introducing methyl, ethyl, and dimethyl hydrophobic groups onto the\u0026nbsp;alkoxyl side chain\u0026nbsp;of 3c respectively, exhibited a marked increase in antiaggregatory activity, with IC₅₀ reaching 2.8, 11.7, and 2.0\u0026nbsp;\u0026mu;M. Among them, the antiaggregatory activity of 3i was nearly 10-fold higher than that of 3c (Figure 3). Similarly, derivative 4q (IC₅₀\u003csub\u003e\u0026nbsp;\u003c/sub\u003e= 0.75 \u0026mu;M), derived from the introduction of dimethyl group onto the piperidine ring of 4o, also displayed a 11-fold enhancement in antiaggregatory activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMolecular docking studies showed that 4q shared a similar binding mode via the dimethyl group on the alkoxyl side chains with 3g, 3h and 3i, and these hydrophobic moieties could directly interact with Trp169 (Figures 4B, 4D, S2B and S2C). However, 300 ns cMD simulation results revealed distinctly different binding modes of the dimethyl groups between 3i and 4q-R (Figure S3). Specifically, the dimethyl group on the alkoxyl side chain of 3i oriented toward transmembrane domain 7 (TM7) and formed stable hydrophobic interactions with hydrophobic amino acid residues including Met23, Leu275 and Phe278 (Figure S3A). Further binding free energy decomposition analysis confirmed that, compared with MRE-269, the favorable energy contribution of Met23 to 3i binding was significantly enhanced (-0.97 kcal/mol for MRE-269, -2.40 kcal/mol for 3i).\u003c/p\u003e\n\u003cp\u003eIn contrast, the dimethyl group on the piperazine ring of 4q-R was oriented toward transmembrane domain 2 (TM2). Meanwhile, the introduction of the piperazine ring induced the carboxyl end to adopt an upward conformation. This conformation could not only form electrostatic interactions with Arg279 but also establish hydrogen bonds with Trp169 and Ser168 located on extracellular loop 2 (ECL2) (Figure S3D). Binding free energy decomposition results showed that the favorable energy contribution of Trp169 to 4q-R binding exhibited a particularly remarkable increase (-1.65 kcal/mol for MRE-269, -3.28 kcal/mol for 4q-R).\u003c/p\u003e\n\u003cp\u003eFurthermore, small-sized hydrophobic group on the alkylene chains were well tolerated. For instance, derivative 3h, obtained by introducing an ethyl group, exhibited a 5 fold decrease in antiaggregatory activity compared with 3i. This phenomenon is attributed to the steric restriction of the hydrophobic pocket, which can only accommodate small hydrophobic group (Figure 6).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, MRE-269, the active metabolite of selexipag, was used as the lead compound to design and synthesize two series of derivatives, diphenylpyrazinyl isopropylamine alkoxyacetic acid (Series \u003cb\u003e3\u003c/b\u003e) and diphenylpyrazinyl cycloaminoalkoxyacetic acid (Series \u003cb\u003e4\u003c/b\u003e). The SAR of these derivatives was systematically investigated via platelet aggregation inhibition assays combined with molecular docking and cMD simulations.\u003c/p\u003e \u003cp\u003eSpecifically, replacing the \u003cem\u003eN\u003c/em\u003e-isopropyl group and \u003cem\u003eN-\u003c/em\u003ebutyl side chain of MRE-269 with pyrrolidine or piperidine rings (yielding derivatives 4f-R, 4i-R, 4o, and 4q) retained hydrophobic interactions with IP receptor residues Met23 and Val71 due to matching steric volume. Among these modifications, piperidine ring substitution emerged as the optimal strategy. It enhanced the binding affinity with Met23 and Val71 by expanding the hydrophobic interaction surface. Meanwhile, replacing partial alkylene chain with piperidine ring rigidified the alkylene chain linker, thereby reducing its conformational flexibility. Furthermore, this study confirmed that small hydrophobic substituents (e.g., methyl and dimethyl groups) on the alkylene chain serve as key pharmacophores for MRE-269 derivatives. For instance, the hydrophobic groups on the alkoxyl side chains of active derivatives such as 3g and 3i can achieve spatial matching with the hydrophobic pocket of the IP receptor, thereby enhancing local hydrophobic interactions.\u003c/p\u003e \u003cp\u003eDerivative 4q-S (the S-isomer of 4q), generated by integrating the two aforementioned modification strategies, exhibited not only potent antiaggregatory activity but also excellent safety profiles and superior pharmacokinetic stability[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In rat models, 4q-S demonstrated favorable oral bioavailability and a relatively prolonged plasma residence time. Moreover, it was non-toxic in cardiotoxicity assessments (hERG channel inhibition assay) (Table S4) and genotoxicity assays (Mini-Ames Test) (Table S3). Collectively, these pharmacodynamic, pharmacokinetic, and safety properties indicate that 4q-S holds promising druggability.\u003c/p\u003e \u003cp\u003eThese findings delineate clear SAR guidance for the rational design of IP receptor agonists. Specifically, this study can provide critical structural insights to support the development of novel, highly potent, and low-toxicity IP receptor agonists for the treatment of PAH.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to Cloud Scientific for access to the MOE software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll other authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Science Foundation of China (Grant No. 82373761), the Chongqing Natural Science Foundation (Grant No. CSTB2025NSCO-GPX0347) and the Sichuan Natural Science Foundation (NSFSC) (Grant No. 2024NSFSC0634).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchool of Chemistry Engineering, Sichuan University of Science \u0026amp; Engineering, No.180 Xueyuan Street, Huixing Road, Zigong 643000, China\u003c/p\u003e\n\u003cp\u003eXianrong Cai, Juping Cheng, Yuhe Wang, Guiying Liu, Xiaoyan Ma, Qingquan Fu \u0026amp; Xiaoli An\u003c/p\u003e\n\u003cp\u003eChongqing Key Laboratory of High Active Traditional Chinese Medicine Delivery System, Chongqing Medical and Pharmaceutical College, No. 82, University Town Middle Road, Shapingba District, Chongqing 410331, China\u003c/p\u003e\n\u003cp\u003eZhenqiang Mu\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by\u003cstrong\u003e\u0026nbsp;Xianrong Cai, Xiaoli\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;An,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eJuping Cheng\u003c/strong\u003e and \u003cstrong\u003eYuhe Wang\u003c/strong\u003e. The first draft of the manuscript was written by\u003cstrong\u003e\u0026nbsp;Xianrong Cai\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;Xiaoli\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;An,\u003c/strong\u003e and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Zhenqiang Mu, Xiaoli An\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data to this article can be found online at https://doi.org/.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlfranca A, I\u0026ntilde;iguez MA, Fresno M, et al. (2006) Prostanoid signal transduction and gene expression in the endothelium: role in cardiovascular diseases. Cardiovascular research 70: 446\u0026ndash;456\u003c/li\u003e\n \u003cli\u003ePluchart H, Khouri C, Blaise S, et al. (2017) Targeting the Prostacyclin Pathway: Beyond Pulmonary Arterial Hypertension. Trends in pharmacological sciences 38: 512\u0026ndash;523\u003c/li\u003e\n \u003cli\u003eNarumiya S (2007) Physiology and pathophysiology of prostanoid receptors. Proc Jpn Acad Ser B Phys Biol Sci. 83: 296\u0026ndash;319\u003c/li\u003e\n \u003cli\u003eVane J, Corin RE (2003) Prostacyclin: A Vascular Mediator. European Journal of Vascular and Endovascular Surgery 26: 571-578\u003c/li\u003e\n \u003cli\u003eKothapalli D, Stewart SA, Smyth EM, et al. (2003) Prostacylin Receptor Activation Inhibits Proliferation of Aortic Smooth Muscle Cells by Regulating cAMP Response Element-Binding Protein- and Pocket Protein-Dependent Cyclin A Gene Expression. Molecular Pharmacology 64: 249-258\u003c/li\u003e\n \u003cli\u003eIdzko M, Hammad H, Van Nimwegen M, et al. (2007) Inhaled iloprost suppresses the cardinal features of asthma via inhibition of airway dendritic cell function. Journal of Clinical Investigation 117: 464-472\u003c/li\u003e\n \u003cli\u003eZeng C, Liu J, Zheng X, et al. (2023) Prostaglandin and prostaglandin receptors: present and future promising therapeutic targets for pulmonary arterial hypertension. Respiratory Research 24.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSharma M, Paudyal V, Syed SK, et al. (2025) Management of Pulmonary Arterial Hypertension: Current Strategies and Future Prospects. Life 15.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDuggan ST, Keam SJ, Burness CB (2016) Selexipag: A Review in Pulmonary Arterial Hypertension. American Journal of Cardiovascular Drugs 17: 73-80\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Scott LJ (2016) Selexipag: First Global Approval. Drugs 76: 413-418\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Sitbon O, Channick R, Chin KM, et al. (2015) Selexipag for the Treatment of Pulmonary Arterial Hypertension. New England Journal of Medicine 373: 2522-2533\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Mubarak KK (2010) A review of prostaglandin analogs in the management of patients with pulmonary arterial hypertension. Respiratory Medicine 104: 9-21\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Bruderer S, Hurst N, Remenova T, et al. (2017) Clinical pharmacology, efficacy, and safety of selexipag for the treatment of pulmonary arterial hypertension. Expert Opinion on Drug Safety 16: 743-751\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Kaufmann P, Okubo K, Bruderer S, et al. (2015) Pharmacokinetics and Tolerability of the Novel Oral Prostacyclin IP Receptor Agonist Selexipag. American Journal of Cardiovascular Drugs 15: 195-203\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Kuwano K, Hashino A, Noda K, et al. (2008) A Long-Acting and Highly Selective Prostacyclin Receptor Agonist Prodrug, 2-{4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide (NS-304), Ameliorates Rat Pulmonary Hypertension with Unique Relaxant Responses of Its Active Form, {4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic Acid (MRE-269), on Rat Pulmonary Artery. The Journal of Pharmacology and Experimental Therapeutics 326: 691-699\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Asaki T, Kuwano K, Morrison K, et al. (2015) Selexipag: An Oral and Selective IP Prostacyclin Receptor Agonist for the Treatment of Pulmonary Arterial Hypertension. Journal of Medicinal Chemistry 58: 7128-7137\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Wang JJ, Jin S, Zhang H, et al. (2024) Molecular recognition and activation of the prostacyclin receptor by anti-pulmonary arterial hypertension drugs. Sci. Adv. 10: eadk5184\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Baldoni D, Bruderer S, Muhsen N, et al. (2015) Bioequivalence of different dose-strength tablets of selexipag, a selective prostacyclin receptor agonist, in a multiple-dose up-titration study. Int. Journal of Clinical Pharmacology and Therapeutics 53: 788 - 798\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Stoll F, Liesener S, Hohlfeld T, et al. (2002) Pharmacophore Definition and Three-Dimensional Quantitative Structure-Activity Relationship Study on Structurally Diverse Prostacyclin Receptor Agonists. Mol Pharmacol. 62: 1103-1111\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Cai X, Lin G, Tang J, et al. (2025) Synthesis and Evaluation of Diphenylpyrazine Cyclic Amine Derivatives as IP Receptor Agonists. ACS Medicinal Chemistry Letters 16: 1772-1779\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Allen WJ, Rizzo RC (2014) Implementation of the Hungarian Algorithm to Account for Ligand Symmetry and Similarity in Structure-Based Design. Journal of Chemical Information and Modeling 54: 518-529\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Asaki T, Hamamoto T, Sugiyama Y, et al. (2007) Structure\u0026ndash;activity studies on diphenylpyrazine derivatives: A novel class of prostacyclin receptor agonists. Bioorganic \u0026amp; Medicinal Chemistry 15: 6692-6704\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-computer-aided-molecular-design","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcam","sideBox":"Learn more about [Journal of Computer-Aided Molecular Design](http://link.springer.com/journal/10822)","snPcode":"10822","submissionUrl":"https://submission.nature.com/new-submission/10822/3","title":"Journal of Computer-Aided Molecular Design","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"IP receptor agonists, Diphenylpyrazinyl aminoalkoxyacetic acids, Structure-Activity Relationship, Antiaggregatory Activity","lastPublishedDoi":"10.21203/rs.3.rs-8764373/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8764373/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo develop novel, highly potent, and safe IP receptor agonists, two series of diphenylpyrazinyl amino alkoxy acetic acid derivatives were designed and synthesized. Combined with antiplatelet aggregation assays, molecular docking, and molecular dynamics simulations, a systematic investigation was conducted on their structure-activity relationships (SAR). The results revealed that replacing the \u003cem\u003eN\u003c/em\u003e-isopropyl group and \u003cem\u003eN\u003c/em\u003e-butyl side chain of MRE-269 with a piperidine ring represented the optimal modification strategy, as it enhanced the hydrophobic interactions between the derivatives and the IP receptor while reduce the flexibility of their alkylene chains. The additional introduction of small hydrophobic groups (e.g., methyl and dimethyl groups) onto the alkylene chain could significantly improve the derivatives\u0026rsquo; antiaggregatory activity, which represents a key pharmacodynamic feature of this class of compounds. In conclusion, this study clarifies the SAR of these diphenylpyrazinyl amino alkoxy acetic acid derivatives and provides critical insights for the rational design of IP receptor agonists.\u003c/p\u003e","manuscriptTitle":"Structure-Optimization and Structure-Activity Relationship of Diphenylpyrazinyl Aminoalkoxyacetic Acid Derivatives: Piperidine Substitution and Hydrophobic Modification as Key Strategies for IP Receptor Agonists","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-06 11:33:19","doi":"10.21203/rs.3.rs-8764373/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-18T15:35:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T12:25:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62636015779855183087964804324791151056","date":"2026-05-13T08:14:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204782712218008398716347210881858223003","date":"2026-05-10T20:35:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-01T21:52:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-01T21:51:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-26T04:41:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Computer-Aided Molecular Design","date":"2026-02-02T11:07:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-computer-aided-molecular-design","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcam","sideBox":"Learn more about [Journal of Computer-Aided Molecular Design](http://link.springer.com/journal/10822)","snPcode":"10822","submissionUrl":"https://submission.nature.com/new-submission/10822/3","title":"Journal of Computer-Aided Molecular Design","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2050825b-8528-423f-875d-8bdcf1126d34","owner":[],"postedDate":"March 6th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-18T15:35:29+00:00","index":68,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T12:25:16+00:00","index":64,"fulltext":""},{"type":"reviewerAgreed","content":"62636015779855183087964804324791151056","date":"2026-05-13T08:14:40+00:00","index":63,"fulltext":""},{"type":"reviewerAgreed","content":"204782712218008398716347210881858223003","date":"2026-05-10T20:35:53+00:00","index":58,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-06T11:33:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-06 11:33:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8764373","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8764373","identity":"rs-8764373","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}