Lanifibranor

Design, Synthesis, and Evaluation of a Novel Series of Indole Sulfonamide Peroxisome Proliferator Activated Receptor (PPAR) α/γ/δ Triple Activators: Discovery of Lanifibranor, a New Antifibrotic Clinical Candidate

Abstract

The present research endeavors meticulously detail the comprehensive identification and subsequent innovative synthesis of a distinct class of chemical entities, specifically novel indole sulfonamide derivatives. These meticulously crafted compounds were rigorously evaluated for their unique capacity to effectively activate all three crucial peroxisome proliferator activated receptor (PPAR) isoforms. This investigation is particularly pertinent given the established and multifaceted roles that PPARs play in regulating various metabolic, inflammatory, and cellular differentiation processes, thereby positioning their modulation as a compelling and strategic approach for therapeutic intervention in numerous disease states.

The foundational genesis of this discovery initiative commenced with the identification of a pivotal lead compound, designated as compound 4. This initial compound was specifically recognized for its potent and selective activity as a PPAR alpha activator. Its discovery was the direct result of an extensive and high-throughput screening (HTS) campaign, a sophisticated and systematic methodology employed to rapidly assess the biological or biochemical activity of a vast array of compounds. This comprehensive screening was meticulously conducted against our institution’s proprietary screening library, an invaluable and diverse collection of chemical structures. The successful identification of compound 4 provided a crucial molecular anchor, serving as an indispensable starting point for the subsequent, more refined phases of chemical optimization.

Following this initial success, a rigorous and systematic optimization process was diligently initiated. This comprehensive medicinal chemistry endeavor encompassed a series of iterative structural modifications, synthetic pathways, and detailed biological evaluations. Each step was meticulously designed to progressively refine and enhance the pharmacological profile of the lead series. This diligent and scientifically driven progression ultimately culminated in the triumphant discovery of lanifibranor, also formally designated as IVA337, which is referred to as compound 5 in this context. Lanifibranor distinguishes itself remarkably as a moderately potent yet exceptionally well-balanced pan PPAR agonist. This attribute of balanced activation across the three distinct PPAR isoforms—alpha, delta, and gamma—is profoundly significant, as it strongly suggests a potentially broader and more holistic therapeutic impact compared to agents that exhibit selectivity for only a single isoform. Furthermore, an exhaustive assessment of this optimized compound unequivocally revealed an excellent safety profile, a paramount consideration for any prospective therapeutic agent, thereby indicating a highly favorable tolerability for clinical application.

The promising attributes and potential of compound 5 were subsequently subjected to rigorous preclinical validation through a series of comprehensive studies, meticulously designed and executed across both in vitro and in vivo experimental models. These extensive investigations conclusively demonstrated a robust and compelling biological activity for lanifibranor. Critically, the observed therapeutic effects were particularly pronounced and highly relevant within sophisticated experimental models specifically engineered to mimic the intricate and complex pathophysiology of nonalcoholic steatohepatitis (NASH). NASH represents a significant and growing global unmet medical need, characterized by progressive liver inflammation, damage, and fibrosis, making targeted and effective interventions absolutely essential. The consistent and strong performance of compound 5 in these highly pertinent preclinical models provides compelling and unequivocal evidence, strongly suggesting its substantial therapeutic potential for patients currently afflicted with nonalcoholic steatohepatitis, thereby offering a significant beacon of hope for managing this challenging and debilitating disease.

Introduction

Nonalcoholic Steatohepatitis, commonly referred to as NASH, represents a formidable and increasingly prevalent multifactorial liver disease that progresses through multiple distinct stages. Its clinical significance is underscored by its association with a heightened risk of cardiovascular mortality, a leading cause of death globally, and its well-established propensity to advance to severe and life-threatening liver pathologies, including cirrhosis and hepatocellular carcinoma. The hallmark of NASH lies in a constellation of characteristic histopathological alterations within the liver parenchyma. These include the accumulation of fat, a condition known as steatosis, coupled with the presence of inflammation, cellular ballooning degeneration, necroinflammation, and the insidious development of perisinusoidal fibrosis. Among these pathological features, the stage of fibrosis stands out as the most critical prognostic indicator, serving as the strongest independent predictor for both all-cause and disease-specific mortality in individuals diagnosed with NASH. While the precise and intricate mechanisms underpinning the pathogenesis of NASH are not yet fully elucidated, a broad scientific consensus firmly establishes that underlying metabolic disorders and the initial accumulation of hepatic steatosis are pivotal in the disease’s initiation and subsequent progression.

Peroxisome proliferator-activated receptors, or PPARs, constitute a vital subfamily within the broader nuclear receptor family. These sophisticated molecular switches exert profound influence over a diverse array of physiological processes. Their multifaceted roles extend beyond the critical regulation of lipid and glucose metabolism to encompass the intricate control of inflammatory responses and the complex processes of fibrogenesis. Given their central involvement in these key pathways, extensive and sustained scientific endeavors have been dedicated to identifying potent and selective agonists for PPARs, with the ambitious goal of developing pharmacologically useful agents for the management of various metabolic diseases. Historically, two prominent chemical families have emerged as well-described modulators in this context. The thiazolidinediones, exemplified by compounds such as rosiglitazone and pioglitazone, are particularly renowned for their capacity to regulate glucose metabolism. Concurrently, the fibrate class of compounds, including fenofibrate and bezafibrate, have been widely recognized for their efficacy in controlling lipid profiles.

All three principal PPAR subtypes—PPAR alpha (NR1C1), PPAR delta (NR1C2), and PPAR gamma (NR1C3)—function as ligand-activated nuclear receptors. These isoforms exhibit distinct patterns of tissue expression and cellular functions, reflecting their specialized roles within the body’s complex regulatory networks. The PPAR alpha isoform, for instance, is abundantly expressed in hepatocytes, the primary cells of the liver. Here, it plays a crucial role in facilitating fatty acid transport and beta-oxidation, processes essential for energy metabolism, and concurrently acts to dampen inflammatory responses, contributing to hepatic homeostasis. The PPAR delta isoform, in contrast, is more widely distributed, found in most liver cell types, including hepatocytes, hepatic stellate cells (HSC), and Kupffer cells. This isoform contributes significantly to the intricate regulation of both glucose and lipid metabolism while also exhibiting potent anti-inflammatory properties, particularly through its active promotion of M2-stage Kupffer cell polarization, a phenotype associated with resolving inflammation and tissue repair. The PPAR gamma isoform is predominantly expressed in adipose tissue, where it orchestrates adipocyte differentiation, enhances glucose uptake, promotes the efficient storage of triglycerides, leads to a reduction in circulating plasma free fatty acids, and crucially induces the secretion of adiponectin, an anti-inflammatory cytokine with systemic metabolic benefits. Moreover, the activation of PPAR gamma effectively increases the insulin sensitivity of several vital organs, including the liver, thereby contributing to improved glycemic control. Notably, both PPAR gamma and PPAR delta are expressed, albeit to varying extents, in hepatic stellate cells, which are recognized as primary cellular drivers of liver fibrosis. Within these cells, PPAR gamma is understood to play a critical role in maintaining HSCs in a quiescent, non-fibrogenic state, thereby mitigating the progression of liver scarring.

Based on the distinct yet complementary roles of each PPAR isoform in metabolic regulation, inflammation, and fibrogenesis, it can be logically posited that a therapeutic strategy combining the activation of PPAR alpha, PPAR delta, and PPAR gamma could offer an innovative and highly efficacious approach to prevent both the initiation and progression of NASH. Such a comprehensive approach would address a broad spectrum of the multifaceted parameters involved in the intricate pathology of the disease.

It is important to acknowledge that numerous PPAR agonists developed in the past were ultimately discontinued. Many of these compounds were characterized by exceptionally potent activation of PPARs, often exhibiting an unbalanced selectivity profile across the different targeted isoforms. This disproportionate and strong potency could potentially lead to a wide spectrum of undesirable side effects, as the over-regulation of gene expression and the activation of numerous pathways, some of which might be associated with toxicity, could disrupt physiological balance. Conversely, moderately potent PPAR agonists, such as fenofibrate, bezafibrate, and pioglitazone, have historically demonstrated good tolerability and safety when administered to a large population of patients. Informed by these valuable insights, our research strategy deliberately focused on identifying and developing moderately potent and exquisitely well-balanced modulators of all three PPAR isoforms. This judicious approach aimed to precisely target the NASH-related cells and pathways of interest while simultaneously optimizing the therapeutic margin, thereby maximizing efficacy and minimizing potential adverse effects.

Our initial discovery trace for this novel class of compounds originated from an indole hit, specifically compound 4. This key molecule was successfully identified through an extensive high-throughput screening campaign conducted using our proprietary compound collection. The screening employed human PPAR transactivation assays, a sophisticated cell-based system designed to measure the activation of these receptors. This particular indole derivative demonstrated robust agonist activity on PPAR alpha, exhibiting a half-maximal effective concentration (EC50) of 260 nM and a maximum efficacy (Emax) of 101%. However, its overall pan-PPAR profile was notably unbalanced, displaying significantly lower potency and efficacy on both PPAR delta (EC50 = 1912 nM, Emax = 56%) and PPAR gamma (EC50 = 1558 nM, Emax = 43%).

The molecular architecture of compound 4 presents notable similarities with an established pharmacophoric scaffold that is frequently described in the extensive literature on PPAR agonists. This archetypal structure typically comprises an acidic head group, critical for receptor binding, which is meticulously linked to a hydrophobic tail group via a linker segment of varying length and inherent flexibility. As previously articulated, there remains a clear and urgent unmet medical need in the challenging therapeutic area of fibrotic diseases, particularly NASH. The overarching objective of our rigorous optimization efforts was therefore the discovery of novel pan-PPAR agonist drugs that would offer a significantly improved safety margin when compared to existing PPAR gamma selective or dual PPAR alpha/gamma compounds. This comprehensive and strategic drug discovery program ultimately led to the successful identification of lanifibranor, also formally known as IVA337, designated as compound 5. Lanifibranor is characterized as a moderately potent and exceptionally well-balanced pan-PPAR agonist, distinguished by an excellent safety profile. Currently, this promising compound is undergoing phase 2 clinical trials for the treatment of NASH, representing a significant advancement in the potential therapeutic landscape for this debilitating condition.

Results and Discussion

Chemistry

The detailed synthetic pathways employed to generate the various indole compounds examined in this study are thoroughly delineated across several schemes. The initial stage of synthesis involved a palladium-catalyzed Sonogashira cross-coupling reaction. This highly efficient reaction was conducted between appropriately substituted iodo-nitrobenzenes and a diverse array of alkynes, resulting in the successful formation of the nitroalkyne intermediates, collectively referred to as 6. Subsequently, these nitroalkynes underwent a crucial reduction step in the presence of tin(II) chloride (SnCl2), a mild and selective reducing agent. This transformation yielded the corresponding aminoalkynes, designated as 7. Following this, the anilines were subjected to a sulfonylation reaction, which was effectively carried out in the presence of the desired sulfonyl chloride, utilizing pyridine as the solvent to facilitate the reaction. The final indole products, labeled as 9, were expertly obtained through a cyclization reaction of their alkyne precursors, compounds 8. This cyclization was catalyzed by copper acetate (Cu(OAc)2), a known reagent for such transformations, and performed either under reflux conditions in dichloroethane (CH2Cl2) or, for enhanced efficiency, under microwave irradiation at temperatures ranging from 130-150°C. The protective ester groups on these compounds were then removed through a deprotection reaction involving lithium hydroxide (LiOH) in a binary solvent system of tetrahydrofuran (THF) and water, ultimately yielding the target carboxylic acid compounds, 4 and 10.

In instances where the requisite nitro starting material was not commercially available, an alternative synthetic strategy was employed, initiating the synthesis from the corresponding commercially accessible iodoaniline precursors bearing the desired substitutions. The sulfonylation reaction in this pathway was executed with various aryl sulfonyl chlorides in pyridine. Frequently, an undesired over-reaction was observed, leading to the formation of a mixture comprising both disulfonylated and monosulfonylated compounds. This challenge was conveniently circumvented by the direct treatment of the crude reaction mixture with a 3 M potassium hydroxide (KOH) solution, which effectively converted the undesirable bis-capped product back to the desired iodosulfonamide, compound 11. Subsequent Sonogashira cross-coupling reactions were conducted in the presence of bis(triphenylphosphine)palladium(II) dichloride (Pd(PPh3)2Cl2), copper iodide (CuI), and diethylamine (Et2N) in dimethylformamide (DMF) under microwave heating, a process that directly afforded the indole derivatives 12. A final saponification step then provided the target derivatives, identified as 13.

A third synthetic sequence commenced with a Sonogashira reaction between readily available substituted iodoanilines and tert-butyl hex-5-ynoate, yielding compounds 14. These compounds were subsequently treated with Cu(OAc)2 under microwave conditions at 130°C for 40 minutes, which efficiently provided the indole structures 15. A deprotonation step utilizing sodium hydride (NaH), followed by the addition of various arylsulfonyl chlorides, generated the corresponding sulfonyle indoles. These intermediates were then subjected to a deprotection reaction with trifluoroacetic acid (TFA), readily affording the desired derivatives, designated as 16.

Compound 22 was specifically synthesized according to a distinct synthetic pathway. This route initiated with the sulfonylation of compound 7b, utilizing 4-fluoro-3-nitro-benzenesulfonyl chloride in a mixture of pyridine and 2-(dimethylamino)pyridine (DMAP), a reaction that produced compound 17. Compound 17 was subsequently cyclized in the presence of Cu(OAc)2 to form the indole 18. A nucleophilic substitution of the fluorine atom was then achieved using aqueous ammonia, leading to the formation of compound 19. The benzimidazole moiety 21 was constructed by first reducing the nitro group of an intermediate using iron powder in acetic acid, followed by a cyclization reaction in the presence of formic acid. Finally, a hydrolysis reaction with LiOH in a THF-water mixture yielded the target compound 22.

In vitro Structure Activity Relationship

The compounds synthesized were systematically evaluated for their human PPARs in vitro potency and efficacy. This assessment was conducted using a cell-based Gal-4 transactivation assay. Specifically, COS-7 cells, which are simian in origin and contain the SV40 gene, were transfected with expression plasmids encoding the ligand-binding domains (LBD) of either PPAR alpha, PPAR delta, or PPAR gamma. The maximum efficacy (Emax) of each test compound was carefully measured relative to established reference agonists: fenofibric acid for PPAR alpha, rosiglitazone (1a) for PPAR gamma, and GW501516 for PPAR delta. This comparative approach allowed for a standardized evaluation of their pharmacological profiles.

Investigating the Chain Linker

To establish fundamental structure-activity relationship (SAR) information, the initial phase of optimization focused on analogs possessing different types of linkers between the indole moiety and the acidic head group. This systematic variation revealed notable changes in the activity profiles across the various PPAR subtypes. As previously observed, compound 4, characterized by a two-carbon atom unit linker, exhibited potent PPAR alpha transactivation activity but displayed considerably weaker activity against both PPAR delta and PPAR gamma. Extending this linker to a three-carbon atom chain, as seen in compound 10b, resulted in a discernible decrease in PPAR alpha activity. Crucially, however, this modification simultaneously led to an increased activity for both PPAR delta and PPAR gamma, suggesting a shift in selectivity. Further lengthening the linker to a four-carbon atom unit chain, exemplified by compound 10c, or the strategic introduction of a heteroatom within the chain, as in compound 13a, regrettably led to a complete loss of activity across all PPAR isoforms. Consistent with established knowledge regarding PPAR alpha selective compounds, the incorporation of a substituent at the alpha position of the carbonyl group, specifically in compound 13b, indeed resulted in a selective PPAR alpha agonist profile. Ultimately, compound 10b, with its optimal three-carbon atom chain, demonstrated the most desirable balanced pan-PPAR profile in terms of potency, albeit with partial efficacy, typically in the range of 50%. This finding underscored the critical importance of linker length and composition in modulating the pan-PPAR activation profile.

Investigating the Substitution of the Indole

To further elucidate the activity and pan-PPAR profile within this chemical series, the subsequent phase of investigation concentrated on the strategic substitution patterns of the indole ring. Initially, the 5-position of the indole was thoroughly explored. It was observed that only hydrophobic substituents at this position, such as the 5-Chloro group in compound 10b, the 5-trifluoromethyl group in compound 16a, and the 5-methyl group in compound 13c, yielded active compounds. Specifically, the trifluoromethyl (CF3) substitution in compound 16a resulted in a dual PPAR alpha/gamma agonist profile, exhibiting potency and efficacy very similar to the parent compound 4. However, its pan-PPAR profile remained less balanced compared to compound 10b, which, at this stage, continued to demonstrate the most favorable balance across all three isoforms. In stark contrast, the introduction of a 5-methoxy group in compound 10d rendered the compound entirely inactive, highlighting the stringent requirements for hydrophobic character at this position. Additionally, a series of more sterically hindered alkoxy chains were synthesized (data not presented), but these modifications unfortunately proved to be completely detrimental to the desired pan-PPAR activity. The investigation also extended to other positions of the indole scaffold (data not presented). It was found that substitutions at the 6-position were generally unfavorable for PPAR activity, while modifications at the 4-position consistently led to a completely misbalanced pharmacological profile, further emphasizing the specific structural demands for optimal pan-PPAR agonism.

Investigating the Nature of the Aryl Sulfonamide

Our initial efforts in this phase were directed towards understanding the impact of substitutions on the phenyl sulfonamide moiety. It was consistently observed that substitutions at the ortho position, exemplified by compounds 16d and 16g, predominantly resulted in a drastic reduction of agonistic activity across all three PPAR subtypes. Conversely, exploration of the meta and para positions of the phenyl ring, using both electron-donating and electron-withdrawing groups, successfully yielded active compounds. Interestingly, these compounds consistently exhibited a tendency towards increased PPAR gamma activity. Furthermore, no significant difference in activity or profile was observed between the electron-donating group (as in 16b) and the electron-withdrawing group (as in 16e) when introduced at the meta position. A similar trend was noted for the para-substituted compounds, 16c, 16f, and 16i. Therefore, it was concluded that meta and para substitutions on the phenyl ring produced quite similar profiles, and no discernible electronic effect could be clearly attributed to this specific part of the molecule.

As detailed earlier, the three-carbon atom spacer chain consistently led to compounds demonstrating moderate potency, yet importantly, they exhibited the highly desired balanced pan-PPAR agonist activity. Similar favorable profiles were consistently obtained when the C5 indole position was substituted with either a trifluoromethyl or a chloro group. Ultimately, it became unequivocally clear that meta and para substitutions on the aryl sulfonamide ring were the most advantageous options for achieving the desired balanced pan-PPAR profile.

The role of the phenyl group, situated at what is referred to as the “south part” of the molecule, was further investigated by exploring various fused ring systems. As anticipated, a fused ring system that mimicked an ortho substitution pattern led to a drastic decrease in overall PPAR activity. For instance, the 1-naphthyl sulfonamide derivative, compound 10g, showed significantly reduced efficacy on PPAR gamma and completely lacked activity on PPAR delta. Consequently, efforts were then refocused on other fused rings designed to mimic meta or para substitutions, as these had previously shown greater promise for achieving a pan-PPAR balanced profile. Surprisingly, the 2-naphthyl analogue, compound 10f, displayed very low potency across all three PPARs. At this juncture, it was observed that non-conjugated fused rings, such as the benzo[1,3]dioxole in compound 10e and the N-methyl benzomorpholine in compound 13d, yielded highly active compounds but with an undesirable imbalance in potency, strongly favoring PPAR gamma. In stark contrast, the conjugated fused ring benzothiazole analogue, compound 5, demonstrated high efficacy across all PPAR subtypes and, critically, presented the most balanced profile in terms of potency, making it a standout candidate.

At this pivotal stage, the decision was made to undertake a more in-depth investigation into the potential of the benzothiazole moiety, which involved introducing a series of different heteroaromatic rings. It is important to note that the 5-sulfonylbenzothiazole isomer, specifically 4-[1-(1,3-benzothiazol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoic acid, could not be prepared due to inherent chemical stability issues associated with its corresponding sulfonyl chloride intermediate. To thoroughly explore the influence of substitution at position 2 of the benzothiazole ring, we strategically synthesized methylated derivatives of both benzothiazole isomers: the 1,3-benzothiazol-5-ylsulfonyl compound 10i and the 1,3-benzothiazol-6-ylsulfonyl compound 10h. These methylated derivatives, however, proved to be considerably less efficient than compound 5 on both PPAR alpha (with Emax values of less than 20% for 10h, 22% for 10i, compared to 106% for 5) and PPAR delta (Emax values of 27% for 10h, 20% for 10i, compared to 105% for 5). While they retained some efficacy against PPAR gamma (Emax values of 36% for 10h, 39% for 10i, compared to 79% for 5), their overall PPAR activity profile was found to be completely unbalanced. Ultimately, it became evident that utilizing the other isomer of the benzothiazole offered no advantage, as the resulting PPAR activity profile was consistently misbalanced. Replacing the methyl group at position 2 of the benzothiazole with an amino group, as in compound 10j, allowed for a partial regain of activity on PPAR alpha and PPAR delta. However, this modification also significantly increased the unbalance towards PPAR gamma, exhibiting an exceptionally low EC50 of 41 nM for that isoform. Shifting to the corresponding benzoxazole analogue, compound 10k, resulted in a compound that was well-balanced for PPAR delta and PPAR gamma, but displayed notably reduced potency on PPAR alpha. In conclusion for this particular segment of the investigation, all the tested substitutions at the 2-position (compounds 10h, 10i, 10j, and 10k) consistently led to unbalanced PPAR profiles. Consequently, the unsubstituted benzothiazole compound 5 remained the superior option with respect to the desired balanced pan-PPAR profile. Further, alternative 5,6-heteroaromatic ring-containing compounds, such as the benzimidazole 22 and benzothiadiazole 10l, either exhibited low potency or presented an unbalanced pharmacological profile. In summary, all the derivatives produced in this phase demonstrated inferior efficacy, potency, or an unbalanced profile when compared directly to compound 5. These compelling findings strongly prompted us to retain the benzothiazole substituent on the “south part” of the molecule as the most preferred option for achieving the desired pan-PPAR profile.

Reverting to the core indole ring investigation, the 5-CF3 substitution on the indole, which had previously shown favorable characteristics in the earlier phenyl series (compound 16a), surprisingly proved to be less efficient in this more advanced benzothiazole series. While compound 10m, with the 5-CF3 group, showed similar potency to compound 5, its overall efficacy was diminished (82% vs 106% for PPARα, 79% vs 105% for PPARδ, 70% vs 79% for PPARγ). Therefore, only the 5-Chloro analog, compound 5, consistently retained the desired balanced pan-PPAR profile, affirming its optimized structure.

Physicochemical Properties and Pharmacokinetic Profiling

The encouraging and highly promising in vitro results obtained from this novel series of indolesulfonamide derivatives provided a strong impetus for further investigation, specifically to ascertain their potential as viable compounds for preclinical development. A critical step in this progression involved a thorough examination of the drug-like properties of compound 5, identified as the most promising candidate, to determine its overall suitability for advanced studies.

Among the pivotal determinants of oral bioavailability, aqueous solubility and permeability stand out as two major contributing factors. Despite exhibiting relatively low aqueous solubility, a characteristic that might initially appear disadvantageous, compound 5, much like its progenitor, the original hit compound 4, displayed an exceptionally robust permeability profile. This superior permeability, when rigorously compared against a well-established reference compound like Warfarin, suggests an efficient transport across biological membranes, which can often compensate for limited aqueous dissolution, thereby facilitating systemic absorption. This advantageous combination of properties hints at the potential for effective oral administration, laying a solid foundation for its progression through preclinical stages.

Functional Activity: Beta-Oxidation in Human Hepatocytes and Mouse Myoblast Cell Line

To comprehensively assess the intricate functional activity of the peroxisome proliferator activated receptors across all three subtypes, a meticulously designed panel of in vitro assays was employed. Fatty acid beta-oxidation, a fundamental metabolic process, was strategically utilized as a sensitive and reliable cellular marker for evaluating the activation of both PPAR alpha and PPAR delta. These specific activities were, in turn, rigorously assessed within two distinct and relevant cellular models: the human hepatoma cell line (HuH7) for hepatic activity, and the mouse myoblast cell line (C2C12) for muscular metabolic responses. Concurrently, the functional activity pertaining to PPAR gamma was precisely quantified in human adipocytes, specifically through the measurement of the expression levels of key target genes, namely adiponectin and adipocyte protein 2 (aP2), with rosiglitazone at a concentration of 1 µM serving as the established reference standard.

The results of these functional assays unequivocally demonstrated that compound 5 induced a significant and dose-dependent activation of both PPAR alpha and PPAR delta. This was clearly evidenced by increasing levels of beta-oxidation observed in both the HuH7 human hepatoma cells and the C2C12 mouse myoblast cells, findings that were remarkably consistent with the earlier transactivation data. Specifically, compound 5 robustly induced a 45% increase in beta-oxidation at a concentration of 1 µM in HuH7 cells and elicited a profound and significant effect in C2C12 cells at 3 µM. In sharp contrast, the initial hit compound 4 only showed beta-oxidation activation in HuH7 cells at a much higher concentration of 10 µM and exerted no discernible effect on C2C12 cells, underscoring the substantial improvement achieved with compound 5. Indeed, the most favorable dose-response profile for beta-oxidation, across both human hepatocytes and mouse myoblasts, was definitively observed with compound 5 when directly compared to compound 4. A similar conclusive observation could be drawn from the induction of the PPAR gamma target genes, adiponectin and aP2. A clear and robust dose-response effect was consistently observed, although a minor decrease in expression was noted at the very highest concentration tested, 30 µM. This slight attenuation at maximum concentration could potentially be attributed to the partial precipitation of the compound in the assay medium, a common occurrence at supra-pharmacological concentrations, rather than a true loss of activity.

In vivo Pharmacokinetic Studies

Spurred by the compelling and highly encouraging in vitro results, a crucial next step involved determining the comprehensive pharmacokinetic parameters of the most promising candidate compound 5 in an established laboratory model, specifically the C57Bl6 mouse strain. In this study, a cohort of animals was administered compound 5 orally at a dose of 10 mg/kg, and plasma samples were meticulously collected over an extensive 24-hour period to capture its systemic exposure and disposition. The preliminary data derived from this investigation strongly suggested that a once-daily dosing regimen would be appropriate for this compound.

Notably, compound 5 was detected in plasma at concentrations well within the microgram range, achieving a maximum serum concentration (Cmax) of 10.7 µg/mL. This peak concentration significantly surpassed its in vitro half-maximal effective concentration (EC50) values, indicating a robust systemic exposure that is highly conducive to therapeutic activity. This excellent plasma exposure aligns remarkably well with the previously measured solubility, human Caco-2 permeability data, and the observed absorption characteristics, collectively affirming a highly satisfactory overall pharmacokinetic profile. These robust findings provided solid and compelling evidence that this new generation of pan-PPAR derivatives, particularly compound 5, was exceptionally well-suited for further rigorous evaluation in relevant animal disease models, thereby paving the way for preclinical efficacy studies.

In vivo Pharmacological Studies

db/db Mouse Model

Before embarking on detailed in vivo disease models, it was essential to confirm that compound 5 exhibited comparable potency and efficacy for both human and mouse PPARs. This critical cross-species validation was achieved through rigorous chimeric Gal-4 human or mouse PPAR-mediated reporter gene assays, which definitively established a consistent pharmacological profile, thereby ensuring the translational relevance of mouse models to human physiology.

The anti-diabetic effects of compound 5 were comprehensively evaluated in the db/db mouse model, a widely recognized and well-characterized obese rodent model of type 2 diabetes. This particular model reliably recapitulates several key pathological features of human type 2 diabetes, including severe insulin resistance, pronounced hypertriglyceridemia, and marked hyperglycemia, making it an ideal platform for assessing novel anti-diabetic agents.

In this study, compounds were administered orally once daily over a period of 5 days for compound 5 and 10 days for compound 4, to homozygous C57BL/Ks-db male mice. Blood samples were systematically collected from the retro-orbital sinus prior to the initiation of treatment and again 4 hours after the final gavage. Serum was then meticulously processed to measure circulating levels of triglycerides and glucose. The results were subsequently expressed as a percentage variation on the final day relative to the untreated control group, providing a clear indication of therapeutic impact.

As would be anticipated from a pan-PPAR agonist with its broad metabolic modulating capabilities, treatment of db/db mice with compound 5 induced a profound, dose-dependent, and statistically significant decrease in circulating glucose levels. Specifically, a remarkable 40% reduction was observed at a dose of 10 mg/kg, escalating to an even more significant 58% reduction at 30 mg/kg. In the very same study, the pathologically abnormal plasma triglycerides levels, a characteristic metabolic derangement observed in this disease model, were markedly corrected following treatment with compound 5. A substantial 33% reduction was achieved at 10 mg/kg, further improving to a 45% reduction at 30 mg/kg. In stark contrast, compound 4, the initial hit compound from which this series was developed, demonstrated no statistically significant effect on triglycerides levels. While it did induce a moderate decrease in circulating glucose at 10 mg/kg, this effect was only modestly improved at 30 mg/kg, clearly highlighting the superior efficacy and more comprehensive metabolic benefits conferred by compound 5.

Carbon Tetrachloride (CCl4)-Induced Liver Fibrosis in Mice

Liver fibrosis stands as a critical and progressively detrimental hallmark of NASH, and its presence is robustly associated with long-term overall mortality in affected patients. This pathological process is intricately characterized by an excessive and uncontrolled synthesis of extracellular matrix (ECM) components, primarily composed of collagen and fibronectin, which accumulate to form scar tissue within the liver. The CCl4-induced liver fibrosis model in mice is a well-established and extensively documented experimental system, widely employed to investigate the potential anti-fibrotic properties of various pharmacological agents. In this model, the evaluation of anti-fibrotic activity is primarily determined by the quantitative measurement of collagen deposition within the liver tissue, providing a direct assessment of scar reduction.

In this investigation, collagen deposition, meticulously quantified by picrosirius red (PSR) staining on liver histological sections, was significantly elevated in CCl4-vehicle treated mice when compared to mice that received saline injections, confirming the successful induction of fibrosis. Crucially, treatment with compound 5, administered orally once daily at doses of 10 and 30 mg/kg, produced a profound and dose-dependent reduction in collagen deposition, demonstrating its potent anti-fibrotic efficacy. In parallel, and entirely consistent with the expected systemic effects of pan-PPAR activation, compound 5 also elicited a significant reduction in plasma triglycerides levels, a key biochemical marker indicative of PPAR alpha and delta engagement. Furthermore, a significant elevation of plasma adiponectin levels was observed, serving as a robust marker of PPAR gamma engagement. These concerted effects on both fibrosis and metabolic markers underscore the broad therapeutic potential of compound 5 in addressing multiple facets of NASH pathophysiology.

Haemodilution, Plasma Volume Expansion, and Heart Weight Increase in Rats

A critical aspect of evaluating novel therapeutic agents involves a thorough assessment of their safety profile, particularly in comparison to existing drugs. In this regard, compound 5 distinguished itself remarkably when contrasted with first-generation PPAR gamma agonists, such as muraglitazar, tesaglitazar, or rosiglitazone. Unlike these earlier compounds, compound 5 demonstrated no discernible effect on hematocrit, plasma volume, or heart weight, even at elevated doses. This is a highly significant finding, as the three other PPAR gamma agonists included in this comparative study consistently induced a significant reduction in hematocrit levels and a substantial increase in plasma volume after 4 weeks of treatment at their highest respective doses. Furthermore, these comparator compounds, but unequivocally not compound 5, exhibited a marked increase in heart weight after 6 weeks of treatment, a concerning adverse effect often associated with potent PPAR gamma activation. The absence of these adverse cardiovascular and hematological effects with compound 5 highlights its superior safety profile, a critical advantage for long-term therapeutic application.

Xray PPARγ Structure with Compound 5 in Comparison to Rosiglitazone

A wealth of structural information regarding PPAR gamma has been meticulously documented within the protein data base. Classically, the ligand-binding domain (LBD) of PPARs is characterized by a sophisticated architecture comprising 12 alpha-helices and 4 beta-stranded sheets. The ligand binding pocket (LBP) of PPAR gamma is particularly notable for its large volume, approximately 1300 ų, often adopting distinct Y- or T-shapes, which allows for considerable flexibility in ligand accommodation. Diverse binding modes have been observed for various ligands within PPAR gamma’s LBP, with molecules occupying different sub-locations within the pocket, and in some intriguing cases, even accommodating two ligands concurrently.

Rosiglitazone, a potent PPAR gamma agonist, exemplifies a specific binding conformation. It typically binds in a U-shape, effectively wrapping itself around Helix 3 of the receptor. The thiazolidinedione head group of rosiglitazone, which functionally mimics the carboxylic acid moiety commonly found in PPAR’s natural ligands such as fatty acids, forms critical direct interactions with Helix 12 through multiple hydrogen bonds. Specifically, the nitrogen atom of the thiazolidinedione engages with Tyrosine 473, while its two carbonyl groups form hydrogen bonds with Histidine 323 and Histidine 449.

In contrast to rosiglitazone, compound 5 adopts a distinctly different binding posture within the PPAR gamma LBP. It does not directly interact with Helix 12 in the same manner as rosiglitazone. Nevertheless, it effectively stabilizes a conformationally active form of PPAR gamma. Compound 5 assumes a T-shaped conformation, which partially overlaps with the U-shaped binding site occupied by rosiglitazone. Critically, compound 5 also extends into and fills an additional sub-pocket, engaging in specific hydrogen bond interactions between its carboxylic acid group and both Glutamic acid 343 and Arginine 288. This unique T-shaped binding mode, distinct from the U-shape of potent full agonists, provides a structural basis for its differentiated pharmacological profile, potentially contributing to its balanced activity and improved safety.

Cofactors Recruitment by Compound 5 vs. Rosiglitazone

Accumulating evidence from both genetic and pharmacological investigations increasingly suggests that moderately activating compounds can lead to more favorable therapeutic outcomes compared to agents that induce full receptor activation. These important observations have translated into the conceptual framework of PPAR partial agonists and selective PPAR modulators (SPPARMs), which are distinguished by their ability to induce differential cofactor recruitment profiles, ultimately resulting in distinct patterns of gene expression. To meticulously compare the cofactor recruitment profiles induced by compound 5 and rosiglitazone, a time-resolved fluorescence energy transfer (TR-FRET) assay was employed, utilizing a comprehensive panel of co-activators and co-repressors, thereby allowing for the precise determination and comparative assessment of their respective potency and efficacy.

A comparative analysis unequivocally revealed significant differences in cofactor recruitment between compound 5 and rosiglitazone, affecting both potency and efficacy. For instance, the co-repressors nuclear receptor corepressor 1 (NCoR-ID1) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) were both effectively “derecruited” by both compound 5 and rosiglitazone. However, compound 5 exhibited a approximately 10-fold lower potency in this derecruitment process compared to rosiglitazone. Specifically, the half-maximal effective concentrations (EC50) for derecruitment were 4097 nM for NCoR-ID1 and 1140 nM for SMRT with compound 5, in contrast to the much lower EC50 values of 326 nM for NCoR-ID1 and 95 nM for SMRT with rosiglitazone.

Substantial differences in potency were also discerned for the recruitment of several co-activators and other regulatory proteins, including nuclear receptor coactivator 3 (NCoA3), steroid receptor coactivator 1 (SRC-1), PPAR gamma coactivator-1 alpha (PGC1 alpha), receptor interacting protein 140 (RIP140), and proline-rich nuclear receptor coactivator 1 (PNRC1). When considering efficacy, compound 5 consistently demonstrated a partial agonist activity for PGC1 alpha and ligand-dependent corepressor (LCOR), further highlighting its nuanced mechanism of action. Moreover, compound 5 exhibited a greater efficacy for PNRC1 recruitment in comparison to rosiglitazone. These findings, when considered collectively with the earlier transactivation data, definitively established that compound 5 can be characterized as a moderately potent and truly partial agonist for PPAR gamma, leading to distinct and beneficial differential gene expression patterns, as clearly demonstrated by its effects on aP2 and adiponectin.

In summation, the moderate potency of compound 5, combined with its unique and differentiated co-regulator recruitment profile, and its well-balanced activity across all three PPAR isoforms, collectively provide a comprehensive mechanistic explanation for its remarkably favorable safety profile when compared to other conventional PPAR gamma agonists. This distinct pharmacological signature positions compound 5 as a promising therapeutic agent with an improved risk-benefit profile.

Conclusion

In summary, this research endeavor has meticulously described the innovative synthesis and subsequent rigorous optimization of a novel series of indole compounds. These derivatives are uniquely characterized by their well-balanced activity across all three crucial PPAR subtypes. Notably, compound 5, identified as the lead candidate, demonstrated exceptional anti-hyperglycemic and hypolipidemic efficacy within the established db/db mouse model, a robust testament to its metabolic benefits. Furthermore, it exhibited significant anti-fibrotic activity in the mouse CCl4-induced liver fibrosis model, underscoring its potential in addressing a critical pathological component of liver disease. Importantly, and in stark contrast to several other PPAR gamma agonists that are often associated with dose-limiting side effects, compound 5 displayed no adverse effects on hematocrit, plasma volume, or heart weight in rat studies, indicating a superior safety margin. These highly encouraging and compelling results have provided strong justification and motivation for the continued development of lanifibranor, or compound 5, as a promising therapeutic agent for the comprehensive treatment of nonalcoholic steatohepatitis.

Experimental Section

Chemistry

General Methods: All chemical reagents and solvents utilized throughout these synthetic procedures were procured from commercial suppliers and were used directly without any further purification unless explicitly stated otherwise. Each final compound synthesized was rigorously analyzed and confirmed to possess a purity greater than 95%, as determined by liquid chromatography-mass spectrometry (LC/MS) analysis, ensuring the high quality of the materials for subsequent biological testing. Comprehensive characterization of all final compounds involved detailed analyses using 1H NMR and 13C NMR spectroscopy, employing Bruker Avance spectrometers operating at 300 MHz, 400 MHz, or 500 MHz. Chemical shifts are consistently reported in parts per million (ppm), denoted as δ, and were precisely calibrated using the resonance of the undeuterated solvent as an internal standard. Melting points were accurately determined on a hot stage apparatus and are presented uncorrected. The LC/MS analyses were performed on either a Waters ZQ mass spectrometer coupled to an Agilent 1100 series chromatography system, which included a photodiode array UV detector, or on a Waters UPLC Acquity system, which featured a photodiode array UV detector, an evaporative light scattering detector (ELSD), and a single quadrupole mass spectrometer (SQD). For the first chromatographic method, the gradient profile initiated at 5% mobile phase B, progressively increasing to 90% B over 9 minutes, maintaining 90% B for 2 minutes, then decreasing from 90% B to 10% B over 1 minute, and finally holding at 10% B for 3 minutes, all at a consistent flow rate of 0.6 mL/min. Mobile Phase A consisted of 0.05% trifluoroacetic acid in water, while Mobile Phase B comprised 0.05% trifluoroacetic acid in acetonitrile. Separations were achieved using an Uptispher OBD column with dimensions of 50 x 2 mm and a particle size of 3 µm, maintained at a temperature of 45 °C. UV detection was performed using a diode array detector (DAD) at wavelengths between 210-260 nm, with mass spectrometry detection employing electrospray ionization (ESI) in both positive and negative modes. A second chromatographic method employed a gradient starting at 5% B for 0.1 minutes, then increasing from 5% B to 95% B over 2.2 minutes, holding at 95% B for 0.2 minutes, and then rapidly returning to 5% B over 0.49 minutes, maintaining a flow rate of 0.8 mL/min. Mobile Phase A for this method was 0.1% acetic acid in water, and Mobile Phase B was 0.1% acetic acid in acetonitrile. An ACQUITY UPLC BEH C18 column, 50 x 2.1 mm with 1.7 µm particles, was used at 45 °C. UV detection was again performed with a DAD at 210-260 nm, and MS detection used ESI in both positive and negative modes.

3-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]propanoic acid (4)

The synthesis of 3-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]propanoic acid, designated as compound 4, commenced with 180 mg (0.46 mmol) of compound 9a. This starting material was carefully dissolved in a mixture of 16 mL of tetrahydrofuran and 4 mL of water. To this solution, 20 mg (0.48 mmol, 1.05 equivalents) of lithium hydroxide monohydrate (LiOH.l H2O) were added, initiating the saponification reaction. The reaction mixture was then stirred diligently for 3 hours at ambient room temperature to ensure complete hydrolysis of the ester. Following this reaction period, the mixture was concentrated under reduced pressure to remove volatile solvents. Water was subsequently added to the residue, and the aqueous solution was acidified with a 1 N hydrochloric acid solution, which facilitated the precipitation of the carboxylic acid product. The resultant white precipitate was then efficiently extracted into ethyl acetate. The organic phase, containing the desired product, was meticulously dried over magnesium sulfate to remove any residual water and then concentrated under reduced pressure. This careful workup yielded 160 mg of the expected product as a pale yellow solid, corresponding to an excellent yield of 93%. The compound exhibited a melting point in the range of 165-168°C. Its mass spectral analysis confirmed a molecular ion peak at m/z: 362 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) displayed characteristic signals at δ 2.72 (triplet, J=7.3 Hz, 2H), 3.24 (triplet, J=6.8 Hz, 2H), 6.59 (singlet, 1H), 7.31 (doublet of doublets, J=8.8, 2.2 Hz, 1H), 7.59 (multiplet, 3H), 7.70 (multiplet, 1H), 7.83 (multiplet, 2H), 8.02 (doublet, J=9.0 Hz, 1H), and a broad singlet at 12.35 (1H) corresponding to the acidic proton. The 13C NMR spectrum (126 MHz, DMSO-d6) further supported the structure with signals at δ 173.3, 142.4, 137.4, 134.8, 134.7, 130.8, 130.0, 128.3, 126.1, 124.0, 120.0, 115.6, 108.4, 32.4, and 24.0.

4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-chloro-indol-2-yl]butanoic acid (5)

This highly significant compound, identified as 4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-chloro-indol-2-yl]butanoic acid, designated as 5, was meticulously prepared following the same comprehensive synthetic methodology as previously established for compound 4. The process initiated with the precursor 12e, which underwent a crucial saponification reaction. This involved treating the ester with lithium hydroxide in a mixed solvent system comprising tetrahydrofuran and water. The reaction mixture was subjected to continuous agitation at room temperature for a defined period, allowing for the complete hydrolysis of the ester moiety. Subsequent purification and workup procedures yielded the desired carboxylic acid derivative with a commendable yield of 57%. The purified compound presented as a pristine white solid, exhibiting a sharp melting point in the range of 182-185°C. Its identity and purity were unequivocally confirmed through analytical techniques. Liquid chromatography-mass spectrometry, utilizing method a, indicated a retention time of 5.67 minutes and a deprotonated molecular ion peak at m/z 433 [M-H]-. Further structural elucidation was achieved through proton nuclear magnetic resonance (1H NMR) spectroscopy (500 MHz, DMSO-d6), which displayed characteristic chemical shifts at δ 1.95 (multiplet, 2H), 2.36 (triplet, J=7.3 Hz, 2H), 3.09 (triplet, J=7.5 Hz, 2H), 6.62 (singlet, 1H), 7.32 (doublet of doublets, J=8.9, 2.1 Hz, 1H), 7.57 (doublet, J=2.1 Hz, 1H), 7.85 (doublet of doublets, J=8.6, 1.85 Hz, 1H), 8.10 (doublet, J=8.9 Hz, 1H), 8.20 (doublet, J=8.6 Hz, 1H), 8.98 (doublet, J=1.9 Hz, 1H), 9.66 (singlet, 1H), and a broad singlet at 12.14 (1H). The carbon-13 nuclear magnetic resonance (13C NMR) spectrum (75 MHz, DMSO-d6) further corroborated the proposed structure with signals observed at δ 174.1, 162.5, 156.1, 143.0, 134.7, 134.6, 133.9, 130.9, 128.3, 124.2, 123.9, 123.4, 122.8, 119.9, 115.7, 108.7, 32.9, 27.7, and 23.6.

methyl 5-(2-amino-5-chloro-phenyl)pent-4-ynoate (7a)

The preparation of methyl 5-(2-amino-5-chloro-phenyl)pent-4-ynoate, compound 7a, involved a meticulous two-step synthetic sequence. The initial step entailed a palladium-catalyzed Sonogashira cross-coupling reaction. To a robust reaction vessel, 35.5 g (125 mmol) of 4-chloro-2-iodo-nitrobenzene, serving as the electrophilic coupling partner, was combined with a significant volume of 510 mL of triethylamine, which functioned as both solvent and base. Catalytic quantities of tetrakis(triphenylphosphine)palladium, specifically 2.88 g (2.5 mmol, 0.02 equivalents), and cuprous iodide, 0.72 g (3.75 mmol, 0.03 equivalents), were then introduced, followed by 50 mL of dimethylformamide to ensure homogeneity. Subsequently, 14 g (125 mmol) of methyl pent-4-ynoate, the alkyne coupling partner, was carefully added at room temperature. The reaction mixture was then allowed to stir continuously for an extended period of 24 hours at ambient temperature, facilitating the Sonogashira coupling. After the reaction, 100 mL of toluene was added, and the volatile solvents were removed under reduced pressure to concentrate the crude product. The residue was then partitioned between ethyl acetate and a 1 N hydrochloric acid solution. The organic layer, containing the desired methyl 5-(5-chloro-2-nitro-phenyl)pent-4-ynoate, compound 6a, was meticulously washed with water, dried over magnesium sulfate, and concentrated under reduced pressure. This intermediate, 6a, was deemed sufficiently pure for direct use in the subsequent reduction step without further purification.

The second crucial step involved the reduction of the nitro group to an amino group. In a clean round-bottomed flask, 90.6 g (400 mmol, 5 equivalents) of stannous chloride was combined with 70 mL of ethyl acetate and 22 mL of ethanol. This mixture was subjected to stirring for 15 minutes at room temperature to ensure proper mixing and dissolution. A solution of 21.5 g (80 mmol) of the crude nitroalkyne 6a, dissolved in 120 mL of ethyl acetate, was then introduced slowly and cautiously to the stannous chloride mixture. The resulting reaction mixture was then stirred continuously for a full 24 hours at room temperature, allowing the complete reduction of the nitro functionality. To quench the reaction and facilitate product isolation, the mixture was carefully poured into a vigorously stirred biphasic system consisting of 200 g of ice and 200 mL of a 1N sodium hydroxide solution. The aqueous layer was then extracted twice with fresh portions of ethyl acetate to maximize product recovery. The combined organic phases were subsequently washed with water to remove any residual impurities, dried over magnesium sulfate, and concentrated under reduced pressure to yield a crude oil. This oil was meticulously purified by column chromatography on silica gel, employing a solvent system of cyclohexane/ethyl acetate (80/20; v/v) as the eluent. This purification yielded 9.1 g of the desired compound 7a as a clear yellow solid, representing a 30% yield over the two steps. The solid exhibited a melting point of 67°C. Its identity was confirmed by LC/MS (method b), showing a retention time of 1.59 minutes and a protonated molecular ion at m/z 238 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 2.68 (multiplet, 4H), 3.63 (singlet, 3H), a broad singlet at 5.45 (2H) corresponding to the amino protons, 6.67 (doublet of doublets, J=8.3, 1.2 Hz, 1H), and a multiplet at 7.02 (2H).

methyl 6-(2-amino-5-chloro-phenyl)hex-5-ynoate (7b)

Compound 7b, methyl 6-(2-amino-5-chloro-phenyl)hex-5-ynoate, was synthesized following an analogous synthetic strategy to that described for compound 7a. The initial Sonogashira cross-coupling reaction was performed utilizing 4-chloro-2-iodo-nitrobenzene and methyl hex-5-ynoate as the starting materials, which were transformed into intermediate 6b. This intermediate was then subjected to the subsequent nitro group reduction to yield compound 7b. The overall synthetic sequence provided the desired product in a 41% yield. Analytical characterization by LC/MS (method b) confirmed its identity, showing a retention time of 1.67 minutes and a protonated molecular ion peak at m/z 252 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed distinct signals at δ 1.83 (multiplet, 2H), 2.48 (multiplet, 4H), 3.59 (singlet, 3H), a singlet at 5.40 (2H) for the amino protons, 6.67 (doublet, J=8.6 Hz, 1H), 7.03 (doublet of doublets, J=8.6, 2.4 Hz, 1H), and 7.08 (doublet, J=2.4 Hz, 1H).

methyl 7-(2-amino-5-chloro-phenyl)hept-6-ynoate (7c)

The synthesis of methyl 7-(2-amino-5-chloro-phenyl)hept-6-ynoate, compound 7c, was executed using a method similar to that employed for compound 7a. This involved the Sonogashira coupling of 4-chloro-2-iodo-nitrobenzene with methyl hept-6-ynoate to form intermediate 6c, followed by the efficient reduction of the nitro group. This two-step process successfully furnished compound 7c in a 68% yield. The purified product was obtained as a yellow solid, with a melting point of 66°C. LC/MS analysis (method b) revealed a retention time of 1.76 minutes and a deprotonated molecular ion at m/z 264 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) exhibited signals at δ 1.55 (multiplet, 2H), 1.68 (multiplet, 2H), 2.36 (triplet, J=7.4 Hz, 2H), 2.47 (triplet, J=7.0 Hz, 2H), 3.59 (singlet, 3H), a singlet at 5.40 (2H) for the amine protons, 6.67 (doublet, J=8.6 Hz, 1H), 7.03 (doublet of doublets, J=8.6, 2.6 Hz, 1H), and 7.07 (doublet, J=2.6 Hz, 1H).

methyl 6-(2-amino-5-methoxy-phenyl)hex-5-ynoate (7d)

Compound 7d, methyl 6-(2-amino-5-methoxy-phenyl)hex-5-ynoate, was synthesized analogously to compound 7a. The initial Sonogashira coupling involved 2-iodo-4-methoxy-1-nitro-benzene and methyl hex-5-ynoate, leading to intermediate 6d, which was subsequently reduced to afford compound 7d. The synthetic route provided the desired product in a 40% yield. This compound was isolated as a brown oil. The 1H NMR spectrum (300 MHz, DMSO-d6) showed signals at δ 1.81 (multiplet, 2H), 2.47 (triplet, J=7.5 Hz, 2H), 2.48 (multiplet, 2H), 3.60 (singlet, 3H), 3.62 (singlet, 3H), a singlet at 4.80 (2H) for the amino protons, 6.62 (multiplet, 1H), and 6.68 (multiplet, 2H).

methyl 6-[2-amino-5-(trifluoromethyl)phenyl]hex-5-ynoate (7e)

The preparation of methyl 6-[2-amino-5-(trifluoromethyl)phenyl]hex-5-ynoate, designated as 7e, followed the general two-step procedure outlined for compound 7a. The Sonogashira coupling reaction was performed using 2-iodo-1-nitro-4-(trifluoromethyl)benzene and methyl hex-5-ynoate to generate the corresponding nitroalkyne intermediate, 6e. This intermediate was then efficiently reduced to yield compound 7e, which was obtained in a 50% yield. The purified product presented as a brown oil. The 1H NMR spectrum (300 MHz, DMSO-d6) featured characteristic signals at δ 1.81 (multiplet, 2H), 2.47 (triplet, J=7.5 Hz, 2H), 2.48 (multiplet, 2H), 3.60 (singlet, 3H), a singlet at 5.98 (2H) for the amine protons, 6.78 (doublet, 1H), and 7.31 (multiplet, 2H).

methyl 5-[2-(benzenesulfonamido)-5-chloro-phenyl]pent-4-ynoate (8a)

The synthesis of methyl 5-[2-(benzenesulfonamido)-5-chloro-phenyl]pent-4-ynoate, compound 8a, involved a targeted sulfonylation reaction. A precisely prepared solution of 1.2 g (5 mmol) of the aminoalkyne 7a in 15 mL of pyridine was carefully treated with 0.77 mL (6 mmol, 1.2 equivalents) of benzenesulfonyl chloride, which served as the electrophilic sulfonating agent. The reaction mixture was subsequently stirred for 1 hour at room temperature, allowing the efficient formation of the sulfonamide bond. Upon completion, the mixture was concentrated under reduced pressure to remove the solvent. The residual oil obtained was then subjected to meticulous purification via column chromatography on silica gel. A solvent system consisting of cyclohexane/ethyl acetate (8/2; v/v) was employed as the eluent to isolate the desired product, yielding 1.8 g of compound 8a as a beige solid, representing an excellent 95% yield. LC/MS analysis (method b) confirmed its identity, showing a retention time of 1.82 minutes and a protonated molecular ion at m/z 378 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 2.56 (singlet, 4H), 3.65 (singlet, 3H), a complex multiplet between 7.28-7.36 (3H), a further multiplet between 7.54-7.72 (5H), and a singlet at 9.69 (1H) corresponding to the sulfonamide proton.

methyl 6-[2-(benzenesulfonamido)-5-chloro-phenyl]hex-5-ynoate (8b)

Compound 8b, methyl 6-[2-(benzenesulfonamido)-5-chloro-phenyl]hex-5-ynoate, was prepared following the same sulfonylation procedure as described for compound 8a. The synthesis began with the aminoalkyne 7b, which was reacted with benzenesulfonyl chloride. This reaction successfully yielded compound 8b in a 66% yield. The purified product was obtained as an orange solid, exhibiting a melting point of 90°C. LC/MS analysis (method b) confirmed its structure, showing a retention time of 1.87 minutes and a deprotonated molecular ion at m/z 390 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) presented signals at δ 1.72 (multiplet, 2H), 2.34 (triplet, J=7.1 Hz, 2H), 2.43 (triplet, J=7.4 Hz, 2H), 3.62 (singlet, 3H), 7.26 (doublet, J=8.4 Hz, 1H), 7.36 (multiplet, 2H), 7.54 (multiplet, 2H), 7.63 (multiplet, 1H), and 7.69 (multiplet, 2H).

methyl 7-[2-(benzenesulfonamido)-5-chloro-phenyl]hept-6-ynoate (8c)

The sulfonylation of aminoalkyne 7c with benzenesulfonyl chloride was carried out in a manner analogous to the preparation of compound 8a, yielding methyl 7-[2-(benzenesulfonamido)-5-chloro-phenyl]hept-6-ynoate, designated as 8c. The reaction provided compound 8c in a 54% yield. The product was isolated as a colorless oil. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed signals at δ 1.55 (multiplet, 4H), 2.33 (multiplet, 4H), 3.60 (singlet, 3H), 7.22 (multiplet, 3H), 7.61 (multiplet, 5H), and a singlet at 9.75 (1H) for the sulfonamide proton.

methyl 6-[2-(benzenesulfonamido)-5-methoxy-phenyl]hex-5-ynoate (8d)

Compound 8d, methyl 6-[2-(benzenesulfonamido)-5-methoxy-phenyl]hex-5-ynoate, was synthesized from the aminoalkyne 7d following the established sulfonylation procedure akin to that for compound 8a. This reaction efficiently provided the desired product in a 66% yield. Compound 8d was isolated as an orange solid. LC/MS analysis (method b) confirmed its identity with a retention time of 1.66 minutes and a protonated molecular ion at m/z 388 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) exhibited signals at δ 1.70 (multiplet, 2H), 2.29 (triplet, J=7.1 Hz, 2H), 2.43 (triplet, J=7.4 Hz, 2H), 3.62 (singlet, 3H), 3.70 (singlet, 3H), 6.78 (doublet, J=2.9 Hz, 1H), 6.86 (doublet of doublets, J=8.9, 2.9 Hz, 1H), 7.12 (doublet, J=8.9 Hz, 1H), 7.51 (multiplet, 2H), 7.61 (multiplet, 3H), and a singlet at 9.47 (1H) corresponding to the sulfonamide proton.

methyl 6-[2-(1,3-benzodioxol-5-ylsulfonylamino)-5-chloro-phenyl]hex-5-ynoate (8e)

The preparation of methyl 6-[2-(1,3-benzodioxol-5-ylsulfonylamino)-5-chloro-phenyl]hex-5-ynoate, compound 8e, was accomplished by reacting aminoalkyne 7b with 1,3-benzodioxole-5-sulfonyl chloride, closely mirroring the sulfonylation method used for compound 8a. This reaction proceeded with high efficiency, affording compound 8e in an excellent 89% yield. The product was obtained as a brown oil. LC/MS analysis (method a) indicated a retention time of 6.19 minutes and a protonated molecular ion at m/z 436 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) showed signals at δ 1.76 (multiplet, 2H), 2.40 (triplet, J=7.1 Hz, 2H), 2.44 (triplet, J=7.3 Hz, 2H), 3.61 (singlet, 3H), 6.15 (singlet, 2H), 7.01 (multiplet, 1H), 7.19 (multiplet, 2H), 7.26 (multiplet, 1H), 7.36 (multiplet, 2H), and a singlet at 9.66 (1H) for the sulfonamide proton.

methyl 6-[5-chloro-2-[(2-methyl-1,3-benzothiazol-6-yl)sulfonylamino]phenyl]hex-5-ynoate (8h)

Methyl 6-[5-chloro-2-[(2-methyl-1,3-benzothiazol-6-yl)sulfonylamino]phenyl]hex-5-ynoate, identified as 8h, was synthesized via a sulfonylation reaction starting from aminoalkyne 7b and 2-methyl-1,3-benzothiazole-6-sulfonyl chloride, following the general procedure established for compound 8a. The reaction successfully produced compound 8h in a 68% yield. The purified compound presented as a white solid, with a melting point ranging from 103-106°C. LC/MS analysis (method a) confirmed its molecular weight and purity, showing a retention time of 6.17 minutes and a protonated molecular ion at m/z 463 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed signals at δ 1.63 (multiplet, 2H), 2.26 (triplet, J=7.8 Hz, 2H), 2.37 (triplet, J=7.4 Hz, 2H), 2.83 (singlet, 3H), 3.61 (singlet, 3H), 7.29 (multiplet, 3H), 7.73 (doublet of doublets, J=8.6, 1.8 Hz, 1H), 8.01 (doublet, J=8.6 Hz, 1H), 8.42 (doublet, J=1.8 Hz, 1H), and a singlet at 9.89 (1H) for the sulfonamide proton.

methyl 6-[5-chloro-2-[(2-methyl-1,3-benzothiazol-5-yl)sulfonylamino]phenyl]hex-5-ynoate (8i)

The preparation of methyl 6-[5-chloro-2-[(2-methyl-1,3-benzothiazol-5-yl)sulfonylamino]phenyl]hex-5-ynoate, compound 8i, was achieved through the sulfonylation of aminoalkyne 7b with 2-methyl-1,3-benzothiazole-5-sulfonyl chloride. This reaction adhered to the general methodology previously described for compound 8a, resulting in compound 8i in a 42% yield. The purified product was obtained as a yellow solid, exhibiting a melting point between 68-72°C. LC/MS analysis (method a) confirmed its molecular structure, indicating a retention time of 6.25 minutes and a protonated molecular ion at m/z 463 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) showed characteristic signals at δ 1.60 (multiplet, 2H), 2.22 (triplet, J=7.1 Hz, 2H), 2.35 (triplet, J=7.4 Hz, 2H), 2.83 (singlet, 3H), 3.61 (singlet, 3H), 7.33 (multiplet, 3H), 7.63 (doublet of doublets, J=8.5, 1.8 Hz, 1H), 8.15 (doublet, J=1.6 Hz, 1H), 8.21 (doublet, J=8.5 Hz, 1H), and a singlet at 9.94 (1H) for the sulfonamide proton.

methyl 6-[2-[(2-amino-1,3-benzothiazol-6-yl)sulfonylamino]-5-chloro-phenyl]hex-5-ynoate (8j)

Compound 8j, methyl 6-[2-[(2-amino-1,3-benzothiazol-6-yl)sulfonylamino]-5-chloro-phenyl]hex-5-ynoate, was synthesized via the sulfonylation reaction of aminoalkyne 7b with 2-amino-1,3-benzothiazole-6-sulfonyl chloride. The procedure was consistent with the general methods applied for the synthesis of compound 8a, and this reaction proved highly efficient, affording compound 8j in a remarkable 96% yield. The purified product manifested as an orange solid, with a melting point observed in the range of 60-65°C. LC/MS analysis (method a) confirmed the compound’s identity, displaying a retention time of 5.37 minutes and a protonated molecular ion at m/z 464 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) featured signals at δ 1.70 (multiplet, 2H), 2.33 (triplet, J=7.1 Hz, 2H), 2.42 (triplet, J=7.4 Hz, 2H), 3.61 (multiplet, 3H), 7.33 (multiplet, 4H), 7.48 (doublet of doublets, J=8.8, 1.9 Hz, 1H), a broad singlet at 7.96 (2H), 8.04 (doublet, J=1.9 Hz, 1H), and a singlet at 9.60 (1H) for the sulfonamide proton.

methyl 6-[2-[(2-amino-1,3-benzoxazol-6-yl)sulfonylamino]-5-chloro-phenyl]hex-5-ynoate (8k)

The synthesis of methyl 6-[2-[(2-amino-1,3-benzoxazol-6-yl)sulfonylamino]-5-chloro-phenyl]hex-5-ynoate, compound 8k, involved the sulfonylation of aminoalkyne 7b with 2-amino-1,3-benzoxazole-6-sulfonyl chloride, following the general synthetic approach for compound 8a. This particular reaction provided compound 8k in a modest 9% yield. The purified product was obtained as a white solid, exhibiting a melting point of 135°C. LC/MS analysis (method a) confirmed its molecular structure with a retention time of 5.22 minutes and a protonated molecular ion at m/z 448 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) showed signals at δ 1.70 (multiplet, 2H), 2.33 (triplet, J=7.1 Hz, 2H), 2.42 (triplet, J=7.4 Hz, 2H), 3.61 (singlet, 3H), 7.29 (multiplet, 4H), 7.45 (doublet of doublets, J=8.2, 1.8 Hz, 1H), 7.59 (doublet, J=1.8 Hz, 1H), a broad singlet at 7.86 (2H), and a broad singlet at 9.57 (1H) for the sulfonamide proton.

methyl 6-[2-(2,1,3-benzothiadiazol-5-ylsulfonylamino)-5-chloro-phenyl]hex-5-ynoate (8l)

Compound 8l, methyl 6-[2-(2,1,3-benzothiadiazol-5-ylsulfonylamino)-5-chloro-phenyl]hex-5-ynoate, was synthesized by reacting aminoalkyne 7b with 2,1,3-benzothiadiazole-5-sulfonyl chloride, closely adhering to the sulfonylation protocol utilized for compound 8a. This reaction yielded compound 8l in a 24% yield. The purified product was isolated as a yellow oil. LC/MS analysis (method a) confirmed its identity, showing a retention time of 6.57 minutes and a protonated molecular ion at m/z 450 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed signals at δ 1.58 (multiplet, 2H), 2.20 (triplet, J=7.1 Hz, 2H), 2.33 (triplet, J=7.4 Hz, 2H), 3.60 (singlet, 3H), 7.35 (multiplet, 3H), 7.92 (doublet of doublets, J=9.1, 1.8 Hz, 1H), 8.31 (doublet, J=9.3 Hz, 1H), 8.37 (multiplet, 1H), and a singlet at 10.30 (1H) for the sulfonamide proton.

methyl 6-[2-(1,3-benzothiazol-6-ylsulfonylamino)-5-(trifluoromethyl)phenyl]hex-5-ynoate (8m)

The preparation of methyl 6-[2-(1,3-benzothiazol-6-ylsulfonylamino)-5-(trifluoromethyl)phenyl]hex-5-ynoate, compound 8m, was carried out by sulfonylation of aminoalkyne 7e with 1,3-benzothiazole-6-sulfonyl chloride, following the general synthetic methods outlined for compound 8a. This reaction provided compound 8m in a 59% yield. The purified product was obtained as an orange gum. LC/MS analysis (method a) confirmed its structure, showing a retention time of 6.15 minutes and a protonated molecular ion at m/z 483 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) exhibited signals at δ 1.68 (multiplet, 2H), 2.33 (triplet, J=7.2 Hz, 2H), 2.38 (doublet, J=7.4 Hz, 2H), 3.61 (singlet, 3H), 7.58 (multiplet, 3H), 7.89 (doublet of doublets, J=8.6, 1.9 Hz, 1H), 8.24 (doublet, J=8.6 Hz, 1H), 8.69 (doublet, J=1.9 Hz, 1H), a singlet at 9.62 (1H), and a broad singlet at 10.22 (1H) for the sulfonamide protons.

methyl 3-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]propanoate (9a)

The synthesis of methyl 3-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]propanoate, designated as 9a, involved a targeted cyclization reaction. A solution containing 300 mg (0.79 mmol) of the sulfonamidoalkyne 8a was meticulously prepared in 35 mL of 1,2-dichloroethane. To this solution, 15 mg (0.08 mmol, 0.1 equivalents) of copper acetate were introduced, serving as the catalyst for the intramolecular cyclization. The reaction mixture was then subjected to reflux conditions for an extended period of 24 hours, with continuous stirring to ensure efficient conversion. Upon completion of the reaction, the solvent was carefully removed under reduced pressure, yielding a residual viscous solid. This solid was subsequently purified by column chromatography on silica gel, employing a solvent system of toluene/ethyl acetate (9/1; v/v) as the eluent. This purification step successfully provided 230 mg of compound 9a as a clear yellow solid, representing a 77% yield. The compound exhibited a melting point in the range of 93-96°C. LC/MS analysis (method b) confirmed its identity with a retention time of 1.92 minutes, though no ionization was observed for the mass spectrometry. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 2.81 (triplet, J=7.4 Hz, 2H), 3.28 (triplet, J=7.1 Hz, 2H), 3.61 (singlet, 3H), 6.60 (singlet, 1H), 7.32 (doublet of doublets, J=8.9, 2.2 Hz, 1H), 7.58 (multiplet, 3H), 7.71 (multiplet, 1H), 7.82 (doublet, J=8.5 Hz, 2H), and 8.02 (doublet, J=8.9 Hz, 1H).

methyl 4-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]butanoate (9b)

Compound 9b, methyl 4-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]butanoate, was synthesized following a cyclization procedure analogous to that described for compound 9a. The starting material for this transformation was sulfonamidoalkyne 8b, which underwent intramolecular cyclization catalyzed by copper acetate. This reaction successfully yielded compound 9b in an 81% yield. The purified product was obtained as a white solid, exhibiting a melting point between 109-112°C. LC/MS analysis (method b) confirmed its molecular structure, displaying a retention time of 2.01 minutes and a deprotonated molecular ion at m/z 390 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) presented signals at δ 1.96 (multiplet, 2H), 2.43 (triplet, J=7.4 Hz, 2H), 3.02 (triplet, J=7.1 Hz, 2H), 3.59 (singlet, 3H), 6.62 (singlet, 1H), 7.32 (doublet of doublets, J=8.8, 2.2 Hz, 1H), 7.58 (multiplet, 3H), 7.70 (multiplet, 1H), 7.80 (multiplet, 2H), and 8.04 (doublet, J=9.0 Hz, 1H).

methyl 5-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]pentanoate (9c)

The synthesis of methyl 5-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]pentanoate, compound 9c, was carried out using a cyclization method similar to that employed for compound 9a. The starting material, sulfonamidoalkyne 8c, was subjected to intramolecular cyclization catalyzed by copper acetate, resulting in the formation of compound 9c in a 75% yield. The purified product was isolated as a beige solid, with a melting point ranging from 95-98°C. LC/MS analysis (method b) confirmed its identity, showing a retention time of 2.09 minutes and a protonated molecular ion at m/z 406 [M+H]+. The 1H NMR spectrum (400 MHz, DMSO-d6) displayed signals at δ 1.63 (multiplet, 2H), 1.70 (multiplet, 2H), 2.36 (triplet, J=7.3 Hz, 2H), 2.99 (triplet, J=6.9 Hz, 2H), 3.59 (singlet, 3H), 6.61 (singlet, 1H), 7.31 (doublet of doublets, J=9.0, 2.2 Hz, 1H), 7.57 (multiplet, 3H), 7.70 (multiplet, 1H), 7.81 (doublet, J=8.6 Hz, 2H), and 8.04 (doublet, J=9.0 Hz, 1H).

methyl 4-[1-(benzenesulfonyl)-5-methoxy-indol-2-yl]butanoate (9d)

The preparation of methyl 4-[1-(benzenesulfonyl)-5-methoxy-indol-2-yl]butanoate, compound 9d, involved an efficient copper acetate-catalyzed cyclization under microwave irradiation. A solution containing 1.14 g (2.95 mmol) of the sulfonamidoalkyne 8d was prepared in 10 mL of 1,2-dichloroethane. To this solution, 540 mg (2.98 mmol, 1.01 equivalents) of copper acetate were added. The mixture was then subjected to microwave heating at an elevated temperature of 150°C for a duration of 30 minutes, with continuous stirring to facilitate the reaction. Upon completion, the reaction mixture was meticulously filtered, and the solvent was subsequently evaporated to dryness, yielding 1.06 g of compound 9d as a brown solid, representing an excellent 93% yield. The purified compound exhibited a melting point between 80-82°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.15 minutes and a protonated molecular ion at m/z 388 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.96 (multiplet, 2H), 2.42 (triplet, J=7.4 Hz, 2H), 3.00 (triplet, J=7.5 Hz, 2H), 3.59 (singlet, 3H), 3.74 (singlet, 3H), 6.55 (singlet, 1H), 6.88 (doublet of doublets, J=9.1, 2.7 Hz, 1H), 7.00 (doublet, J=2.7 Hz, 1H), 7.54 (multiplet, 2H), 7.65 (multiplet, 1H), 7.74 (multiplet, 2H), and 7.91 (doublet, J=9.1 Hz, 1H).

methyl 4-[1-(1,3-benzodioxol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoate (9e)

The preparation of methyl 4-[1-(1,3-benzodioxol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoate, compound 9e, was accomplished via the cyclization of sulfonamidoalkyne 8e, following a synthetic methodology analogous to that described for compound 9d. This reaction afforded compound 9e with an outstanding 97% yield. The purified product was isolated as a white solid, displaying a melting point in the range of 97-103°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.71 minutes and a protonated molecular ion at m/z 436 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.96 (multiplet, 2H), 2.44 (triplet, J=7.4 Hz, 2H), 3.02 (triplet, J=7.3 Hz, 2H), 3.59 (singlet, 3H), 6.14 (singlet, 2H), 6.60 (singlet, 1H), 7.04 (doublet, J=8.3 Hz, 1H), 7.27 (doublet, J=2.1 Hz, 1H), 7.30 (doublet of doublets, J=9.0, 2.2 Hz, 1H), 7.42 (doublet of doublets, J=8.3, 2.1 Hz, 1H), 7.58 (doublet, J=2.2 Hz, 1H), and 8.03 (doublet, J=9.0 Hz, 1H).

methyl 4-[5-chloro-1-(2-naphthylsulfonyl)indol-2-yl]butanoate (9f)

The synthesis of methyl 4-[5-chloro-1-(2-naphthylsulfonyl)indol-2-yl]butanoate, compound 9f, was initiated by reacting naphthalene-2-sulfonyl chloride with aminoalkyne 7b, following the general sulfonylation procedure outlined for compound 8a. This initial step generated the sulfonamidoalkyne intermediate 8f, which was subsequently used without further purification in the cyclization step. The cyclization of 8f, carried out under conditions similar to those for compound 9d, successfully afforded compound 9f with a 71% yield. The purified product was obtained as a white solid, with a melting point of 116-118°C. LC/MS analysis (method b) confirmed its molecular structure, indicating a retention time of 2.19 minutes and a deprotonated molecular ion at m/z 440 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) featured characteristic signals at δ 1.98 (multiplet, 2H), 2.45 (triplet, J=7.4 Hz, 2H), 3.10 (triplet, J=7.4 Hz, 2H), 3.57 (singlet, 3H), 6.62 (multiplet, 1H), 7.31 (doublet of doublets, J=9.0, 2.2 Hz, 1H), 7.57 (doublet, J=2.0 Hz, 1H), 7.63 (doublet of doublets, J=8.8, 2.0 Hz, 1H), 7.72 (multiplet, 2H), 8.00 (doublet, J=9.0 Hz, 1H), 8.06 (doublet, J=9.0 Hz, 1H), 8.12 (doublet, J=9.0 Hz, 1H), 8.23 (doublet, J=9.0 Hz, 1H), and 8.72 (doublet, J=2.0 Hz, 1H).

methyl 4-[5-chloro-1-(1-naphthylsulfonyl)indol-2-yl]butanoate (9g)

The synthesis of methyl 4-[5-chloro-1-(1-naphthylsulfonyl)indol-2-yl]butanoate, designated as 9g, commenced with the reaction of naphthalene-1-sulfonyl chloride with aminoalkyne 7b, following the established sulfonylation procedure akin to that for compound 8a. This initial step yielded the sulfonamidoalkyne intermediate 8g, which was then directly utilized in the subsequent cyclization without further purification. The intramolecular cyclization of 8g, carried out under conditions similar to those described for compound 9d, successfully afforded compound 9g in a 44% yield. The purified product was obtained as a white solid, with a melting point in the range of 94-97°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 2.17 minutes and a deprotonated molecular ion at m/z 440 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) featured characteristic signals at δ 1.86 (multiplet, 2H), 2.35 (triplet, J=7.4 Hz, 2H), 2.89 (triplet, J=7.4 Hz, 2H), 3.53 (singlet, 3H), 6.70 (singlet, 1H), 7.30 (doublet of doublets, J=8.8, 2.2 Hz, 1H), a complex multiplet between 7.65 (5H), 7.94 (doublet, J=9.2 Hz, 1H), 8.13 (multiplet, 1H), and 8.34 (multiplet, 2H).

methyl 4-[5-chloro-1-[(2-methyl-1,3-benzothiazol-6-yl)sulfonyl]indol-2-yl]butanoate (9h)

The preparation of methyl 4-[5-chloro-1-[(2-methyl-1,3-benzothiazol-6-yl)sulfonyl]indol-2-yl]butanoate, designated as 9h, involved the cyclization of sulfonamidoalkyne 8h, following a synthetic methodology analogous to that described for compound 9d. This reaction efficiently provided compound 9h in a 74% yield. The purified product was obtained as a white solid, exhibiting a melting point between 151-153°C. LC/MS analysis (method a) confirmed its molecular structure, displaying a retention time of 6.95 minutes and a protonated molecular ion at m/z 463 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) featured characteristic signals at δ 1.96 (multiplet, 2H), 2.44 (triplet, J=7.4 Hz, 2H), 2.83 (singlet, 3H), 3.07 (triplet, J=7.3 Hz, 2H), 3.58 (singlet, 3H), 6.61 (singlet, 1H), 7.31 (doublet of doublets, J=9.0, 2.2 Hz, 1H), 7.57 (doublet, J=2.2 Hz, 1H), 7.78 (doublet of doublets, J=8.8, 2.0 Hz, 1H), 8.01 (doublet, J=8.8 Hz, 1H), 8.08 (doublet, J=8.8 Hz, 1H), and 8.81 (doublet, J=2.0 Hz, 1H).

methyl 4-[5-chloro-1-[(2-methyl-1,3-benzothiazol-5-yl)sulfonyl]indol-2-yl]butanoate (9i)

The synthesis of methyl 4-[5-chloro-1-[(2-methyl-1,3-benzothiazol-5-yl)sulfonyl]indol-2-yl]butanoate, compound 9i, was performed through the cyclization of sulfonamidoalkyne 8i, adopting a synthetic approach similar to that utilized for compound 9d. This reaction afforded compound 9i in an excellent 77% yield. The purified product was obtained as a beige solid, with a melting point in the range of 136-138°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.92 minutes and a protonated molecular ion at m/z 463 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.98 (multiplet, 2H), 2.45 (triplet, J=7.3 Hz, 2H), 2.81 (singlet, 3H), 3.08 (triplet, J=7.5 Hz, 2H), 3.59 (singlet, 3H), 6.62 (singlet, 1H), 7.32 (doublet of doublets, J=8.9, 2.2 Hz, 1H), 7.57 (doublet, J=1.9 Hz, 1H), 7.75 (doublet of doublets, J=8.5, 1.9 Hz, 1H), 8.11 (doublet, J=8.9 Hz, 1H), and a multiplet at 8.26 (2H).

methyl 4-[1-[(2-amino-1,3-benzothiazol-6-yl)sulfonyl]-5-chloro-indol-2-yl]butanoate (9j)

Compound 9j, methyl 4-[1-[(2-amino-1,3-benzothiazol-6-yl)sulfonyl]-5-chloro-indol-2-yl]butanoate, was synthesized through the cyclization of sulfonamidoalkyne 8j, following a synthetic method akin to that described for compound 9d. This reaction provided compound 9j in a 43% yield. The purified product was obtained as a yellow solid, with a melting point ranging from 235-239°C. LC/MS analysis (method b) confirmed its molecular structure, displaying a retention time of 1.80 minutes and a protonated molecular ion at m/z 464 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) featured signals at δ 1.96 (multiplet, 2H), 2.44 (triplet, J=7.4 Hz, 2H), 3.05 (triplet, J=7.5 Hz, 2H), 3.59 (singlet, 3H), 6.58 (singlet, 1H), 7.31 (multiplet, 2H), 7.60 (multiplet, 2H), 8.06 (multiplet, 3H), and 8.31 (multiplet, 1H).

methyl 4-[1-[(2-amino-1,3-benzoxazol-6-yl)sulfonyl]-5-chloro-indol-2-yl]butanoate (9k)

The preparation of methyl 4-[1-[(2-amino-1,3-benzoxazol-6-yl)sulfonyl]-5-chloro-indol-2-yl]butanoate, compound 9k, involved the cyclization of sulfonamidoalkyne 8k, following a synthetic procedure similar to that used for compound 9d. This reaction successfully yielded compound 9k in a 46% yield. The purified product was obtained as a yellow solid, exhibiting a melting point of 238°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.79 minutes and a protonated molecular ion at m/z 448 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.97 (multiplet, 2H), 2.44 (triplet, J=7.5 Hz, 2H), 3.05 (triplet, J=7.4 Hz, 2H), 3.59 (singlet, 3H), 6.58 (singlet, 1H), 7.27 (multiplet, 2H), 7.55 (multiplet, 2H), 7.83 (singlet, 1H), 8.03 (singlet, 2H), and 8.07 (doublet, J=9.0 Hz, 1H).

methyl 4-[1-(2,1,3-benzothiadiazol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoate (9l)

The synthesis of methyl 4-[1-(2,1,3-benzothiadiazol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoate, designated as 9l, was accomplished through the cyclization of sulfonamidoalkyne 8l, following a synthetic method analogous to that described for compound 9d. This reaction afforded compound 9l in an excellent 83% yield. The purified product was obtained as a yellow solid, with a melting point in the range of 103-106°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.97 minutes and a protonated molecular ion at m/z 450 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) featured characteristic signals at δ 1.98 (multiplet, 2H), 2.46 (triplet, J=7.4 Hz, 2H), 3.10 (triplet, J=7.3 Hz, 2H), 3.58 (singlet, 3H), 6.67 (singlet, 1H), 7.34 (doublet of doublets, J=8.9, 2.2 Hz, 1H), 7.59 (doublet, J=1.9 Hz, 1H), 7.81 (doublet of doublets, J=9.2, 1.9 Hz, 1H), 8.13 (doublet, J=8.9 Hz, 1H), a multiplet between 8.25 (1H), and 8.81 (multiplet, 1H).

methyl 4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-(trifluoromethyl)indol-2-yl]butanoate (9m)

The preparation of methyl 4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-(trifluoromethyl)indol-2-yl]butanoate, compound 9m, involved the cyclization of sulfonamidoalkyne 8m, following a synthetic procedure similar to that used for compound 9d. This reaction successfully yielded compound 9m in a 49% yield. The purified product was obtained as a white solid, exhibiting a melting point between 116-121°C. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.99 (multiplet, 2H), 2.47 (triplet, J=7.7 Hz, 2H), 3.11 (triplet, J=7.4 Hz, 2H), 3.58 (singlet, 3H), 6.78 (singlet, 1H), 7.63 (doublet, J=8.8 Hz, 1H), 7.89 (doublet of doublets, J=8.8, 2.1 Hz, 1H), 7.93 (singlet, 1H), 8.22 (doublet, J=8.8 Hz, 1H), 8.30 (doublet, J=8.8 Hz, 1H), 9.02 (doublet, J=2.1 Hz, 1H), and a singlet at 9.66 (1H).

4-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]butanoic acid (10b)

Compound 10b, 4-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]butanoic acid, was synthesized following the established saponification procedure as described for compound 4. Starting from the ester precursor 9b, this reaction efficiently yielded compound 10b in a 92% yield. The purified product was obtained as a pale pink solid, exhibiting a melting point in the range of 198-202°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.77 minutes and a deprotonated molecular ion at m/z 376 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) displayed characteristic signals at δ 1.93 (multiplet, 2H), 2.33 (triplet, J=7.2 Hz, 2H), 3.03 (triplet, J=7.5 Hz, 2H), 6.62 (singlet, 1H), 7.31 (doublet of doublets, J=9.0, 2.0 Hz, 1H), 7.57 (multiplet, 3H), 7.70 (multiplet, 1H), 7.81 (doublet, J=7.5 Hz, 2H), 8.04 (doublet, J=9.0 Hz, 1H), and a broad singlet at 12.10 (1H). The 13C NMR spectrum (126 MHz, DMSO-d6) further supported the structure with signals at δ 174.1, 143.1, 137.4, 134.8, 134.7, 131.0, 129.9, 128.3, 126.1, 123.9, 120.0, 115.7, 108.7, 33.0, 27.6, and 23.5.

5-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]pentanoic acid (10c)

The synthesis of 5-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]pentanoic acid, compound 10c, was carried out by saponification of the ester precursor 9c, following the same procedure as described for compound 4. This reaction afforded compound 10c in a 79% yield. The purified product was isolated as a white solid, with a melting point in the range of 144-148°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.80 minutes and a deprotonated molecular ion at m/z 390 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) displayed characteristic signals at δ 1.60 (multiplet, 2H), 1.70 (multiplet, 2H), 2.27 (triplet, J=7.3 Hz, 2H), 2.99 (triplet, J=7.1 Hz, 2H), 6.61 (singlet, 1H), 7.31 (doublet of doublets, J=8.9, 2.3 Hz, 1H), 7.58 (multiplet, 3H), 7.70 (multiplet, 1H), 7.81 (multiplet, 2H), 8.04 (doublet, J=9.0 Hz, 1H), and a broad singlet at 12.03 (1H). The 13C NMR spectrum (101 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.3, 143.4, 137.4, 134.8, 134.7, 131.0, 129.9, 128.2, 126.1, 123.9, 119.9, 115.7, 108.6, 33.3, 28.0, 27.8, and 24.1.

4-[1-(benzenesulfonyl)-5-methoxy-indol-2-yl]butanoic acid (10d)

Compound 10d, 4-[1-(benzenesulfonyl)-5-methoxy-indol-2-yl]butanoic acid, was synthesized through the saponification of the ester precursor 9d, following the general procedure established for compound 4. This reaction efficiently provided compound 10d in a 94% yield. The purified product was obtained as a white solid. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.26 minutes and a protonated molecular ion at m/z 374 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) exhibited signals at δ 1.94 (multiplet, 2H), 2.32 (triplet, J=7.1 Hz, 2H), 3.00 (triplet, J=7.2 Hz, 2H), 3.74 (singlet, 3H), 6.54 (singlet, 1H), 6.88 (doublet of doublets, J=9.1, 2.7 Hz, 1H), 7.00 (doublet, J=2.7 Hz, 1H), 7.54 (multiplet, 2H), 7.65 (multiplet, 1H), 7.75 (multiplet, 2H), 7.91 (doublet, J=9.1 Hz, 1H), and a broad singlet at 12.06 (1H). The 13C NMR spectrum (75 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 156.2, 141.9, 137.5, 134.4, 130.7, 130.6, 129.7, 126.0, 115.1, 112.5, 109.8, 103.2, 55.2, 32.9, 27.7, and 23.7.

4-[1-(1,3-benzodioxol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoic acid (10e)

The preparation of 4-[1-(1,3-benzodioxol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoic acid, compound 10e, was accomplished by saponification of the ester precursor 9e, following the established procedure for compound 4. This reaction afforded compound 10e in an excellent 96% yield. The purified product was obtained as a white solid, with a melting point in the range of 154-156°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.87 minutes and a deprotonated molecular ion at m/z 420 [M-H]-. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.93 (multiplet, 2H), 2.35 (triplet, J=6.6 Hz, 2H), 3.03 (triplet, J=7.6 Hz, 2H), 6.14 (singlet, 2H), 6.60 (singlet, 1H), 7.04 (doublet, J=8.2 Hz, 1H), 7.29 (multiplet, 2H), 7.43 (doublet of doublets, J=8.2, 2.1 Hz, 1H), 7.58 (doublet, J=1.9 Hz, 1H), 8.03 (doublet, J=8.9 Hz, 1H), and a broad singlet at 12.11 (1H). The 13C NMR spectrum (75 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 152.6, 148.2, 143.0, 134.6, 130.9, 130.2, 128.1, 123.8, 122.7, 119.8, 115.7, 108.7, 108.4, 105.8, 103.0, 32.9, 27.6, and 23.5.

4-[5-chloro-1-(2-naphthylsulfonyl)indol-2-yl]butanoic acid (10f)

Compound 10f, 4-[5-chloro-1-(2-naphthylsulfonyl)indol-2-yl]butanoic acid, was synthesized through the saponification of the ester precursor 9f, following the general procedure established for compound 4. This reaction afforded compound 10f in an excellent 90% yield. The purified product was obtained as a white solid, exhibiting a melting point in the range of 166-169°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.96 minutes and a deprotonated molecular ion at m/z 426 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) displayed characteristic signals at δ 1.95 (multiplet, 2H), 2.36 (triplet, J=7.4 Hz, 2H), 3.11 (triplet, J=7.5 Hz, 2H), 6.61 (singlet, 1H), 7.31 (doublet of doublets, J=9.0, 2.2 Hz, 1H), 7.56 (doublet, J=2.0 Hz, 1H), 7.72 (multiplet, 3H), 8.00 (doublet, J=7.9 Hz, 1H), 8.06 (doublet, J=9.02 Hz, 1H), 8.12 (doublet, J=9.02 Hz, 1H), 8.23 (doublet, J=7.9 Hz, 1H), 8.72 (doublet, J=1.8 Hz, 1H), and a singlet at 12.19 (1H). The 13C NMR spectrum (101 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.2, 143.1, 134.8, 134.7, 134.3, 131.4, 130.9, 130.2, 129.9, 129.7, 128.2, 128.2, 128.1, 127.9, 123.9, 120.6, 119.9, 115.7, 108.6, 33.0, 27.7, and 23.6.

4-[5-chloro-1-(1-naphthylsulfonyl)indol-2-yl]butanoic acid (10g)

Compound 10g, 4-[5-chloro-1-(1-naphthylsulfonyl)indol-2-yl]butanoic acid, was synthesized through the saponification of the ester precursor 9g, following the general procedure established for compound 4. This reaction afforded compound 10g in an excellent 89% yield. The purified product was obtained as a white solid, exhibiting a melting point in the range of 206-210°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.94 minutes and a deprotonated molecular ion at m/z 426 [M-H]-. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.84 (multiplet, 2H), 2.26 (triplet, J=7.3 Hz, 2H), 2.90 (triplet, J=7.5 Hz, 2H), 6.70 (singlet, 1H), 7.30 (doublet of doublets, J=8.9, 2.2 Hz, 1H), 7.61 (multiplet, 2H), 7.69 (multiplet, 3H), 7.93 (doublet, J=8.9 Hz, 1H), 8.13 (multiplet, 1H), 8.35 (multiplet, 2H), and a broad singlet at 12.09 (1H). The 13C NMR spectrum (101 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.0, 143.4, 135.7, 135.2, 134.0, 133.8, 130.3, 129.5, 129.0, 128.1, 127.8, 127.6, 127.0, 124.7, 123.9, 122.7, 120.2, 115.4, 107.8, 32.9, 27.3, and 23.2.

4-[5-chloro-1-[(2-methyl-1,3-benzothiazol-6-yl)sulfonyl]indol-2-yl]butanoic acid (10h)

The preparation of 4-[5-chloro-1-[(2-methyl-1,3-benzothiazol-6-yl)sulfonyl]indol-2-yl]butanoic acid, compound 10h, was accomplished through the saponification of the ester precursor 9h, following the general procedure established for compound 4. This reaction afforded compound 10h in an excellent 85% yield. The purified product was obtained as a white solid, with a melting point in the range of 163-165°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.78 minutes and a deprotonated molecular ion at m/z 447 [M-H]-. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.94 (multiplet, 2H), 2.35 (triplet, J=7.3 Hz, 2H), 2.82 (singlet, 3H), 3.08 (triplet, J=7.2 Hz, 2H), 6.61 (singlet, 1H), 7.31 (doublet of doublets, J=9.0, 2.2 Hz, 1H), 7.57 (doublet, J=2.2 Hz, 1H), 7.78 (doublet of doublets, J=8.7, 2.1 Hz, 1H), 8.01 (doublet, J=8.7 Hz, 1H), 8.08 (doublet, J=9.0 Hz, 1H), 8.82 (doublet, J=2.1 Hz, 1H), and a singlet at 12.11 (1H). The 13C NMR spectrum (101 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 173.9, 156.2, 143.0, 136.2, 134.7, 133.1, 130.9, 128.2, 123.9, 123.5, 122.9, 122.2, 119.9, 115.7, 108.6, 32.9, 27.7, 23.6, and 20.1.

4-[5-chloro-1-[(2-methyl-1,3-benzothiazol-5-yl)sulfonyl]indol-2-yl]butanoic acid (10i)

Compound 10i, 4-[5-chloro-1-[(2-methyl-1,3-benzothiazol-5-yl)sulfonyl]indol-2-yl]butanoic acid, was synthesized through the saponification of the ester precursor 9i, following the general procedure established for compound 4. This reaction afforded compound 10i in an excellent 98% yield. The purified product was obtained as a white solid, exhibiting a melting point of 164°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.01 minutes and a deprotonated molecular ion at m/z 447 [M-H]-. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.94 (multiplet, 2H), 2.35 (triplet, J=7.3 Hz, 2H), 2.81 (singlet, 3H), 3.08 (triplet, J=7.5 Hz, 2H), 6.62 (singlet, 1H), 7.32 (doublet of doublets, J=8.8, 2.0 Hz, 1H), 7.57 (doublet, J=2.0 Hz, 1H), 7.75 (doublet of doublets, J=8.8, 2.0 Hz, 1H), 8.10 (doublet, J=8.8 Hz, 1H), 8.25 (doublet, J=8.8 Hz, 1H), 8.28 (doublet, J=2.0 Hz, 1H), and a broad singlet at 12.14 (1H). The 13C NMR spectrum (101 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 171.6, 152.2, 143.1, 141.8, 135.1, 134.8, 131.0, 128.3, 124.0, 124.0, 121.2, 120.0, 119.5, 115.8, 108.9, 32.9, 27.7, 23.6, and 19.9.

4-[1-[(2-amino-1,3-benzothiazol-6-yl)sulfonyl]-5-chloro-indol-2-yl]butanoic acid (10j)

The preparation of 4-[1-[(2-amino-1,3-benzothiazol-6-yl)sulfonyl]-5-chloro-indol-2-yl]butanoic acid, compound 10j, was achieved through the saponification of the ester precursor 9j, following the general procedure established for compound 4. This reaction afforded compound 10j in a 49% yield. The purified product was obtained as a white solid, exhibiting a melting point in the range of 155-162°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.55 minutes and a deprotonated molecular ion at m/z 448 [M-H]-. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.93 (multiplet, 2H), 2.35 (triplet, J=6.7 Hz, 2H), 3.06 (triplet, J=7.4 Hz, 2H), a broad singlet at 4.28 (2H), 6.58 (singlet, 1H), 7.29 (doublet of doublets, J=8.9, 2.0 Hz, 1H), 7.36 (doublet, J=8.6 Hz, 1H), 7.56 (doublet, J=2.0 Hz, 1H), 7.62 (doublet of doublets, J=8.6, 2.0 Hz, 1H), 8.05 (doublet, J=8.9 Hz, 1H), a broad singlet at 8.27 (1H), and 8.33 (doublet, J=2.0 Hz, 1H). The 13C NMR spectrum (101 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.2, 171.1, a broad signal at 154.0, 143.1, 134.7, 131.0, a broad signal at 130.5, 129.4, 128.1, 124.7, 123.8, 121.0, 119.9, 116.7, 115.8, 108.3, 33.1, 27.7, and 23.6.

4-[1-[(2-amino-1,3-benzoxazol-6-yl)sulfonyl]-5-chloro-indol-2-yl]butanoic acid (10k)

Compound 10k, 4-[1-[(2-amino-1,3-benzoxazol-6-yl)sulfonyl]-5-chloro-indol-2-yl]butanoic acid, was synthesized through the saponification of the ester precursor 9k, following the general procedure established for compound 4. This reaction afforded compound 10k in a 76% yield. The purified product was obtained as a yellow solid, exhibiting a melting point of 220°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.03 minutes and a deprotonated molecular ion at m/z 432 [M-H]-. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.93 (multiplet, 2H), 2.35 (triplet, J=7.0 Hz, 2H), 3.05 (triplet, J=8.4 Hz, 2H), 6.57 (singlet, 1H), 7.26 (doublet, J=9.0 Hz, 1H), 7.30 (doublet of doublets, J=9.0, 2.2 Hz, 1H), 7.55 (multiplet, 2H), 7.83 (doublet, J=2.2 Hz, 1H), 8.03 (singlet, 2H), 8.07 (doublet, J=9.0 Hz, 1H), and a singlet at 12.09 (1H).

4-[1-(2,1,3-benzothiadiazol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoic acid (10l)

The preparation of 4-[1-(2,1,3-benzothiadiazol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoic acid, compound 10l, was achieved through the saponification of the ester precursor 9l, following the general procedure established for compound 4. This reaction afforded compound 10l in an excellent 92% yield. The purified product was obtained as a brown solid, with a melting point in the range of 172-175°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.03 minutes and a deprotonated molecular ion at m/z 434 [M-H]-. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.95 (multiplet, 2H), a triplet between 2.28-2.41 (2H, J=7.7 Hz), 3.10 (triplet, J=7.4 Hz, 2H), 6.67 (singlet, 1H), 7.33 (doublet of doublets, J=8.9, 2.2 Hz, 1H), 7.59 (doublet, J=2.2 Hz, 1H), 7.81 (doublet of doublets, J=9.3, 2.0 Hz, 1H), 8.13 (doublet, J=8.9 Hz, 1H), a multiplet between 8.20-8.29 (1H), 8.82 (doublet, J=2.0 Hz, 1H), and a broad singlet at 12.10 (1H). The 13C NMR spectrum (101 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.2, 155.4, 152.4, 143.1, 137.9, 134.7, 131.0, 128.5, 124.2, 124.1, 123.9, 121.8, 120.1, 115.8, 109.1, 32.9, 27.7, and 23.6.

4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-(trifluoromethyl)indol-2-yl]butanoic acid (10m)

Compound 10m, 4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-(trifluoromethyl)indol-2-yl]butanoic acid, was synthesized through the saponification of the ester precursor 9m, following the general procedure established for compound 4. This reaction afforded compound 10m in an excellent 97% yield. The purified product was obtained as a yellow solid, with a melting point in the range of 173-181°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.79 minutes and a deprotonated molecular ion at m/z 467 [M-H]-. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.96 (multiplet, 2H), 2.38 (triplet, J=7.3 Hz, 2H), 3.12 (triplet, J=7.4 Hz, 2H), 6.78 (singlet, 1H), 7.62 (doublet of doublets, J=8.8, 1.6 Hz, 1H), 7.91 (multiplet, 2H), 8.22 (doublet, J=8.8 Hz, 1H), 8.30 (doublet, J=8.8 Hz, 1H), 9.02 (doublet, J=1.6 Hz, 1H), 9.66 (singlet, 1H), and a broad singlet at 12.12 (1H). The 13C NMR spectrum (126 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 162.7, 156.2, 143.4, 137.9, 134.8, 133.9, 129.4, 124.6 (quartet, Jc,F=272.2 Hz), 124.4 (quartet, Jc,F=31.7 Hz), 124.3, 123.5, 123.1, 120.6 (quartet, Jc,F=2.7 Hz), 117.9 (quartet, Jc,F=2.7 Hz), 114.9, 109.0, 32.9, 27.6, and 23.5.

N-(4-chloro-2-iodo-phenyl)benzenesulfonamide (11a)

The synthesis of N-(4-chloro-2-iodo-phenyl)benzenesulfonamide, compound 11a, involved a two-stage process. Initially, a solution of 2 g (7.89 mmol) of 4-chloro-2-iodoaniline was prepared in 30 mL of pyridine. To this solution, 1.21 mL (9.5 mmol, 1.2 equivalents) of benzenesulfonyl chloride were added dropwise at 0°C, under continuous stirring to ensure controlled reaction. The reaction mixture was then allowed to warm to room temperature and stirred for an extended period of 16 hours, facilitating the formation of the desired sulfonamide. Following this, the mixture was concentrated under reduced pressure to remove pyridine. The residual oil was dissolved in 50 mL of ethyl acetate, and the resulting solution was washed with water, dried over magnesium sulfate, and concentrated under reduced pressure to yield the crude product. This crude material was then taken up in 60 mL of dioxane and subjected to further treatment with 19 mL of a 3 M potassium hydroxide solution under gentle reflux for 8 hours. This step was crucial for converting any undesired bis-sulfonylated byproducts back to the mono-sulfonamide. After this reflux period, the solvent was removed under reduced pressure, and the residue was dissolved in water. The solution was then carefully acidified to pH 2 using a dilute hydrochloric acid solution, which caused the precipitation of the purified sulfonamide. The precipitate was collected by filtration, thoroughly washed with water, and subsequently dried, affording 2.79 g of the expected product as a white solid, representing an excellent 90% yield. The compound exhibited a melting point of 126-128°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.75 minutes and a deprotonated molecular ion at m/z 392 [M-H]-. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 7.00 (doublet, J=8.6 Hz, 1H), 7.40 (doublet of doublets, J=8.6, 2.5 Hz, 1H), 7.59 (multiplet, 2H), 7.68 (multiplet, 3H), 7.89 (doublet, J=2.5 Hz, 1H), and a singlet at 9.88 (1H) for the sulfonamide proton.

N-(2-iodo-4-methyl-phenyl)benzenesulfonamide (11c)

Compound 11c, N-(2-iodo-4-methyl-phenyl)benzenesulfonamide, was synthesized following the two-stage procedure as detailed for compound 11a. The starting materials used were 2-bromo-4-methylaniline and benzenesulfonyl chloride, which were reacted to yield the desired product in a 95% yield. The purified compound was obtained as a beige solid. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.46 minutes and a protonated molecular ion at m/z 326 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 2.25 (singlet, 3H), 7.03 (doublet, J=8.2 Hz, 1H), 7.13 (multiplet, 1H), 7.41 (doublet, J=1.4 Hz, 1H), 7.58 (multiplet, 2H), 7.69 (multiplet, 3H), and a singlet at 9.77 (1H) for the sulfonamide proton.

N-(4-chloro-2-iodo-phenyl)-4-methyl-2,3-dihydro-1,4-benzoxazine-6-sulfonamide (11d)

The preparation of N-(4-chloro-2-iodo-phenyl)-4-methyl-2,3-dihydro-1,4-benzoxazine-6-sulfonamide, compound 11d, was accomplished using the sulfonylation procedure described for compound 8a. This reaction involved 4-chloro-2-iodoaniline and 4-methyl-2,3-dihydro-1,4-benzoxazine-6-sulfonyl chloride as the starting materials, yielding compound 11d in an 80% yield. The purified product was obtained as a beige solid, with a melting point of 159°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.81 minutes and a protonated molecular ion at m/z 465 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 2.80 (singlet, 3H), 3.28 (multiplet, 2H), 4.29 (multiplet, 2H), 6.79 (doublet, J=9.5 Hz, 1H), 6.92 (multiplet, 2H), 7.03 (doublet, J=8.8 Hz, 1H), 7.41 (doublet of doublets, J=8.8, 2.6 Hz, 1H), 7.89 (doublet, J=2.6 Hz, 1H), and a singlet at 9.50 (1H) for the sulfonamide proton.

N-(4-chloro-2-iodo-phenyl)-1,3-benzothiazole-6-sulfonamide (11e)

Compound 11e, N-(4-chloro-2-iodo-phenyl)-1,3-benzothiazole-6-sulfonamide, was synthesized following the sulfonylation procedure as described for compound 8a. The reaction involved 4-chloro-2-iodoaniline and 1,3-benzothiazole-6-sulfonyl chloride, yielding compound 11e in a 60% yield. The purified product was obtained as a yellow solid, with a melting point of 184°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.47 minutes and a protonated molecular ion at m/z 450 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 7.03 (doublet, J=8.4 Hz, 1H), 7.31 (doublet of doublets, J=8.8, 2.6 Hz, 1H), 7.82 (multiplet, 2H), 8.22 (doublet, J=8.8 Hz, 1H), 8.58 (doublet, J=1.8 Hz, 1H), a singlet at 9.59 (1H), and a broad singlet at 9.95 (1H).

methyl 2-[[1-(benzenesulfonyl)-5-chloro-indol-2-yl]methoxy]acetate (12a)

The synthesis of methyl 2-[[1-(benzenesulfonyl)-5-chloro-indol-2-yl]methoxy]acetate, compound 12a, was conducted in a microwave reaction tube to enhance efficiency. A mixture comprising 600 mg (1.52 mmol) of the sulfonamidoiodoarene 11a and 0.5 mL of dimethylformamide was carefully prepared. To this mixture, catalytic amounts of cuprous iodide, 14 mg (0.02 mmol, 0.013 equivalents), and bis(triphenylphosphine)dichloropalladium, 27 mg (0.04 mmol, 0.026 equivalents), were added, along with 293 mg (2.29 mmol, 1.5 equivalents) of the methyl ester of (2-propynyloxy)acetic acid, which served as the alkyne coupling partner. Finally, 0.5 mL of diethylamine was introduced. The reaction mixture was then subjected to microwave heating at an elevated temperature of 130°C for a controlled duration of 15 minutes. After the heating cycle, the mixture was cooled to room temperature and subsequently hydrolyzed with water. The desired product was then extracted three times with ethyl acetate, and the combined organic phases were meticulously washed with water, dried over magnesium sulfate, and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel, utilizing a solvent system of cyclohexane/ethyl acetate (90/10; v/v) as the eluent. This purification yielded 0.42 g of compound 12a as a yellow solid, representing a 71% yield. The compound exhibited a melting point of 98-100°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.36 minutes and a sodium adduct molecular ion at m/z 416 [MNa]+. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 3.68 (singlet, 3H), 4.25 (singlet, 2H), 4.97 (singlet, 2H), 6.88 (singlet, 1H), 7.37 (doublet of doublets, J=8.9, 2.2 Hz, 1H), 7.57 (multiplet, 2H), 7.70 (multiplet, 2H), and 8.00 (multiplet, 3H).

methyl 4-[1-(benzenesulfonyl)-5-methyl-indol-2-yl]butanoate (12c)

Compound 12c, methyl 4-[1-(benzenesulfonyl)-5-methyl-indol-2-yl]butanoate, was synthesized following a procedure analogous to that described for compound 12a. The reaction involved starting with sulfonamidoiodoarene 11c and methyl hex-5-ynoate. This transformation successfully yielded compound 12c in a 36% yield. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.64 minutes and a protonated molecular ion at m/z 372 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.98 (multiplet, 2H), 2.35 (singlet, 3H), 2.43 (multiplet, 2H), 3.02 (triplet, J=7.4 Hz, 2H), 3.61 (singlet, 3H), 6.55 (singlet, 1H), 7.11 (doublet of doublets, J=8.5, 1.4 Hz, 1H), 7.28 (singlet, 1H), 7.55 (multiplet, 2H), 7.66 (multiplet, 1H), 7.76 (multiplet, 2H), and 7.91 (doublet, J=8.5 Hz, 1H).

methyl 4-[5-chloro-1-[(4-methyl-2,3-dihydro-1,4-benzoxazin-6-yl)sulfonyl]indol-2-yl]butanoate (12d)

The preparation of methyl 4-[5-chloro-1-[(4-methyl-2,3-dihydro-1,4-benzoxazin-6-yl)sulfonyl]indol-2-yl]butanoate, compound 12d, was achieved by reacting sulfonamidoiodoarene 11d with methyl hex-5-ynoate, following a synthetic methodology similar to that described for compound 12a. This reaction proceeded with high efficiency, yielding compound 12d in an excellent 90% yield. The purified product was obtained as a white solid, with a melting point in the range of 139-140°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 2.08 minutes and a protonated molecular ion at m/z 463 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.96 (multiplet, 2H), 2.43 (triplet, J=7.3 Hz, 2H), 2.77 (singlet, 3H), 3.02 (triplet, J=7.5 Hz, 2H), 3.24 (multiplet, 2H), 3.59 (singlet, 3H), 4.23 (multiplet, 2H), 6.58 (singlet, 1H), 6.76 (doublet, J=8.4 Hz, 1H), 6.87 (doublet, J=2.2 Hz, 1H), 7.00 (doublet of doublets, J=8.4, 2.2 Hz, 1H), 7.30 (doublet of doublets, J=8.8, 2.2 Hz, 1H), 7.57 (doublet, J=2.2 Hz, 1H), and 8.06 (doublet, J=8.8 Hz, 1H).

methyl 4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-chloro-indol-2-yl]butanoate (12e)

The synthesis of methyl 4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-chloro-indol-2-yl]butanoate, compound 12e, was accomplished by reacting sulfonamidoiodoarene 11e with methyl hex-5-ynoate, following a synthetic procedure analogous to that described for compound 12a. This reaction yielded compound 12e in a quantitative yield, indicating excellent conversion. The purified product was obtained as a white solid. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.52 minutes and a protonated molecular ion at m/z 449 [M+H]+. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.97 (multiplet, 2H), 2.51 (triplet, J=7.3 Hz, 2H), 3.08 (triplet, J=7.4 Hz, 2H), 3.58 (singlet, 3H), 6.63 (singlet, 1H), 7.32 (doublet of doublets, J=8.9, 2.2 Hz, 1H), 7.58 (doublet, J=2.2 Hz, 1H), 7.84 (doublet of doublets, J=8.8, 2.0 Hz, 1H), 8.09 (doublet, J=8.9 Hz, 1H), 8.20 (doublet, J=8.8 Hz, 1H), 8.97 (doublet, J=2.0 Hz, 1H), and a singlet at 9.65 (1H).

2-[[1-(benzenesulfonyl)-5-chloro-indol-2-yl]methoxy]acetic acid (13a)

The preparation of 2-[[1-(benzenesulfonyl)-5-chloro-indol-2-yl]methoxy]acetic acid, compound 13a, was achieved through the saponification of the ester precursor 12a, following the general procedure established for compound 4. This reaction proceeded with high efficiency, affording compound 13a in an excellent 98% yield. The purified product was obtained as a white solid, with a melting point in the range of 140-142°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.61 minutes and a deprotonated molecular ion at m/z 378 [M-H]-. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 4.14 (singlet, 2H), 4.96 (singlet, 2H), 6.87 (singlet, 1H), 7.36 (doublet of doublets, J=9.0, 2.2 Hz, 1H), 7.57 (multiplet, 2H), 7.70 (multiplet, 2H), 8.01 (multiplet, 3H), and a broad singlet at 12.79 (1H). The 13C NMR spectrum (75 MHz, DMSO-d6) further corroborated the structure with signals at δ 171.2, 138.7, 137.1, 134.8, 134.5, 130.2, 129.7, 128.3, 126.7, 124.7, 120.7, 115.5, 110.8, 67.0, and 65.2.

4-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]-2,2-dimethyl-butanoic acid (13b)

Compound 13b, 4-[1-(benzenesulfonyl)-5-chloro-indol-2-yl]-2,2-dimethyl-butanoic acid, was synthesized following a two-step procedure analogous to that described for compound 12a. The reaction commenced with sulfonamidoiodoarene 11a and 2,2-dimethyl-5-hexynoic acid, leading to the formation of the indole derivative, which was subsequently saponified. This overall synthetic sequence yielded compound 13b in a 32% yield. The purified product was obtained as a brown solid, exhibiting a melting point of 242°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.52 minutes and a deprotonated molecular ion at m/z 404 [M-H]-. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.20 (singlet, 6H), 1.90 (multiplet, 2H), 2.95 (multiplet, 2H), 6.63 (singlet, 1H), 7.33 (doublet of doublets, J=8.8, 2.2 Hz, 1H), 7.59 (multiplet, 3H), 7.76 (multiplet, 3H), 8.06 (doublet, J=8.8 Hz, 1H), and a singlet at 12.26 (1H). The 13C NMR spectrum (101 MHz, DMSO-d6) further corroborated the structure with signals at δ 178.4, 143.5, 137.4, 134.8, 134.7, 131.0, 130.0, 128.2, 126.0, 123.9, 119.9, 115.7, 108.4, 41.2, 38.7, 24.9, and 24.2.

4-[1-(benzenesulfonyl)-5-methyl-indol-2-yl]butanoic acid (13c)

The preparation of 4-[1-(benzenesulfonyl)-5-methyl-indol-2-yl]butanoic acid, compound 13c, was accomplished through the saponification of the ester precursor 12c, following the general procedure established for compound 4. This reaction afforded compound 13c in a 7% yield. The purified product was obtained as a beige solid. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.67 minutes and a deprotonated molecular ion at m/z 356 [M-H]-. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 2.04 (multiplet, 2H), 2.34 (singlet, 3H), 2.51 (multiplet, 2H), 3.01 (multiplet, 2H), 6.52 (singlet, 1H), 7.10 (doublet, J=8.5 Hz, 1H), 7.27 (singlet, 1H), 7.56 (multiplet, 2H), 7.67 (singlet, 1H), 7.76 (multiplet, 2H), 7.90 (doublet, J=8.5 Hz, 1H), and a singlet at 12.12 (1H).

4-[5-chloro-1-[(4-methyl-2,3-dihydro-1,4-benzoxazin-6-yl)sulfonyl]indol-2-yl]butanoic acid (13d)

Compound 13d, 4-[5-chloro-1-[(4-methyl-2,3-dihydro-1,4-benzoxazin-6-yl)sulfonyl]indol-2-yl]butanoic acid, was synthesized through the saponification of the ester precursor 12d, following the general procedure established for compound 4. This reaction afforded compound 13d in an excellent 80% yield. The purified product was obtained as a white solid, exhibiting a melting point in the range of 164-166°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.84 minutes and a deprotonated molecular ion at m/z 447 [M-H]-. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.93 (multiplet, 2H), 2.33 (triplet, J=7.1 Hz, 2H), 2.78 (singlet, 3H), 3.02 (triplet, J=7.5 Hz, 2H), 3.24 (multiplet, 2H), 4.23 (multiplet, 2H), 6.57 (singlet, 1H), 6.76 (doublet, J=8.4 Hz, 1H), 6.88 (doublet, J=2.2 Hz, 1H), 7.01 (doublet of doublets, J=8.4, 2.2 Hz, 1H), 7.30 (doublet of doublets, J=8.8, 2.2 Hz, 1H), 7.57 (doublet, J=2.2 Hz, 1H), 8.06 (doublet, J=8.8 Hz, 1H), and a broad singlet at 12.41 (1H). The 13C NMR spectrum (101 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 148.5, 143.1, 137.1, 134.9, 130.9, 129.6, 127.9, 123.6, 119.8, 115.9, 115.8, 115.7, 108.3, 108.1, 64.7, 47.1, 37.8, 33.0, 27.6, and 23.5.

tert-butyl 6-[2-amino-5-(trifluoromethyl)phenyl]hex-5-ynoate (14a)

The preparation of tert-butyl 6-[2-amino-5-(trifluoromethyl)phenyl]hex-5-ynoate, compound 14a, was achieved through a Sonogashira cross-coupling reaction, following the procedure described for compound 12a. The starting materials for this reaction were 2-iodo-4-(trifluoromethyl)aniline and tert-butyl hex-5-ynoate. This transformation successfully yielded compound 14a in a 64% yield. The purified product was obtained as a brown solid. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.92 minutes and a protonated molecular ion at m/z 328 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.40 (singlet, 9H), 1.78 (triplet, J=7.3 Hz, 2H), 2.36 (triplet, J=7.3 Hz, 2H), 2.50 (multiplet, 2H), a singlet at 5.98 (2H) for the amino protons, 6.78 (doublet, J=8.5 Hz, 1H), 7.31 (doublet of doublets, J=8.7, 2.0 Hz, 1H), and 7.36 (doublet, J=2.0 Hz, 1H).

tert-butyl 6-(2-amino-5-chloro-phenyl)hex-5-ynoate (14b)

Compound 14b, tert-butyl 6-(2-amino-5-chloro-phenyl)hex-5-ynoate, was synthesized following a Sonogashira cross-coupling reaction procedure analogous to that described for compound 12a. The reaction involved 4-chloro-2-iodoaniline and tert-butyl hex-5-ynoate as the starting materials. This transformation proceeded with high efficiency, yielding compound 14b in an excellent 95% yield. The purified product was obtained as a brown oil. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.93 minutes and a protonated molecular ion at m/z 294 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.40 (singlet, 9H), 1.77 (multiplet, 2H), 2.35 (triplet, J=7.3 Hz, 2H), 2.49 (triplet, J=7.7 Hz, 2H), a singlet at 5.40 (2H) for the amino protons, 6.68 (doublet, J=8.6 Hz, 1H), 7.04 (doublet of doublets, J=8.6, 2.3 Hz, 1H), and 7.09 (doublet, J=2.3 Hz, 1H).

tert-butyl 4-[5-(trifluoromethyl)-1H-indol-2-yl]butanoate (15a)

The preparation of tert-butyl 4-[5-(trifluoromethyl)-1H-indol-2-yl]butanoate, compound 15a, was accomplished through an intramolecular cyclization reaction, following a synthetic methodology similar to that described for compound 9d. The starting material for this transformation was compound 14a, which underwent cyclization catalyzed by copper acetate under microwave irradiation. This reaction successfully yielded compound 15a in a 68% yield. The purified product was obtained as a brown solid. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 2.05 minutes and a deprotonated molecular ion at m/z 326 [M-H]-. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.39 (singlet, 9H), 1.91 (multiplet, 2H), 2.25 (triplet, J=7.5 Hz, 2H), 2.76 (triplet, J=7.5 Hz, 2H), 6.32 (singlet, 1H), 7.29 (doublet, J=8.6 Hz, 1H), 7.45 (doublet, J=8.6 Hz, 1H), 7.80 (singlet, 1H), and a singlet at 11.39 (1H) for the indole N-H proton.

tert-butyl 4-(5-chloro-1H-indol-2-yl)butanoate (15b)

Compound 15b, tert-butyl 4-(5-chloro-1H-indol-2-yl)butanoate, was synthesized through an intramolecular cyclization reaction, following a synthetic procedure analogous to that described for compound 9a. The starting material for this transformation was compound 14b, which underwent cyclization. This reaction successfully yielded compound 15b in a 64% yield. The purified product was obtained as a brown solid. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 2.02 minutes and a deprotonated molecular ion at m/z 292 [M-H]-. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.39 (singlet, 9H), 1.89 (multiplet, 2H), 2.24 (triplet, J=7.4 Hz, 2H), 2.72 (triplet, J=7.5 Hz, 2H), 6.13 (doublet, J=1.4 Hz, 1H), 6.98 (doublet of doublets, J=8.6, 2.1 Hz, 1H), 7.27 (doublet, J=8.6 Hz, 1H), 7.44 (doublet, J=2.1 Hz, 1H), and a broad singlet at 11.11 (1H) for the indole N-H proton.

4-[1-(benzenesulfonyl)-5-(trifluoromethyl)indol-2-yl]butanoic acid (16a)

The synthesis of 4-[1-(benzenesulfonyl)-5-(trifluoromethyl)indol-2-yl]butanoic acid, designated as 16a, involved a multi-step reaction sequence. To a solution of 115 mg (0.35 mmol) of the indole precursor 15a in 0.5 mL of dimethylformamide, 28 mg (0.7 mmol, 2 equivalents) of sodium hydride were added at 0°C, and the reaction mixture was stirred for 2 minutes at room temperature to deprotonate the indole nitrogen. Subsequently, 123 mg (0.7 mmol, 2 equivalents) of benzenesulfonyl chloride, dissolved in 0.7 mL of dimethylformamide, was added to the reaction mixture. The mixture was then stirred for 24 hours at room temperature to allow for the N-sulfonylation. After this, the reaction mixture was carefully poured into a biphasic system consisting of ice and a saturated ammonium chloride (NH4Cl) solution. The desired product was then extracted with ethyl acetate, and the combined organic phases were dried over magnesium sulfate and concentrated under reduced pressure. The crude residue, containing the tert-butyl ester, was then dissolved in 0.8 mL of dichloromethane, and 0.2 mL of trifluoroacetic acid was added to effect the deprotection of the tert-butyl ester. This mixture was stirred at room temperature for 5 hours. Following complete deprotection, the solvents were evaporated, and the resulting residue was meticulously purified by high-performance liquid chromatography (HPLC), utilizing an acetonitrile/water gradient mixture as the eluent. This purification yielded the expected compound 16a as a beige solid, with a 43% yield over the multiple steps. The compound exhibited a melting point of 209°C. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.81 minutes and a deprotonated molecular ion at m/z 410 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) displayed characteristic signals at δ 1.95 (multiplet, 2H), 2.35 (triplet, J=7.4 Hz, 2H), 3.06 (triplet, J=7.3 Hz, 2H), 6.77 (singlet, 1H), 7.60 (multiplet, 3H), 7.71 (multiplet, 1H), 7.86 (doublet, J=8.5 Hz, 2H), 7.94 (multiplet, 1H), 8.25 (doublet, J=8.8 Hz, 1H), and a broad singlet at 12.17 (1H). The 13C NMR spectrum (126 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 143.4, 138.0, 137.4, 134.9, 130.1, 129.4, 126.2, 124.5 (quartet, JCF=272.8Hz), 124.4 (quartet, JCF=31.6Hz), 120.6 (quartet, JCF=3.5Hz), 117.9 (quartet, JCF=4.0Hz), 114.9, 109.0, 32.9, 27.6, and 23.5.

4-[1-(3-methoxyphenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid (16b)

Compound 16b, 4-[1-(3-methoxyphenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid, was synthesized following the multi-step procedure described for compound 16a. The reaction commenced with indole precursor 15a and 3-methoxybenzenesulfonyl chloride. This transformation successfully yielded compound 16b in a 46% yield. The purified product was obtained as a white solid. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.85 minutes and a deprotonated molecular ion at m/z 440 [M-H]-. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.95 (multiplet, 2H), 2.36 (triplet, J=7.3 Hz, 2H), 3.06 (triplet, J=7.4 Hz, 2H), 3.77 (singlet, 3H), 6.78 (singlet, 1H), 7.28 (multiplet, 2H), 7.38 (multiplet, 1H), 7.50 (multiplet, 1H), 7.63 (doublet of doublets, J=8.9, 1.6 Hz, 1H), 7.94 (singlet, 1H), 8.25 (doublet, J=9.2 Hz, 1H), and a broad singlet at 12.15 (1H). The 13C NMR spectrum (126 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 159.6, 143.5, 138.5, 138.0, 131.4, 129.4, 124.6 (quartet, JCF=271.8Hz), 124.4 (quartet, JCF=31.8Hz), 120.6 (quartet, JCF=3.3Hz), 120.5, 118.1, 117.9 (quartet, JCF=4.0Hz), 114.9, 111.1, 109.1, 55.7, 32.9, 27.5, and 23.5.

4-[1-(4-methoxyphenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid (16c)

The preparation of 4-[1-(4-methoxyphenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid, compound 16c, was achieved following the multi-step procedure described for compound 16a. The reaction involved indole precursor 15a and 4-methoxybenzenesulfonyl chloride. This transformation successfully yielded compound 16c in a 48% yield. The purified product was obtained as a white solid. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.83 minutes and a deprotonated molecular ion at m/z 440 [M-H]-. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.94 (multiplet, 2H), 2.35 (triplet, J=7.3 Hz, 2H), 3.06 (triplet, J=7.4 Hz, 2H), 3.79 (singlet, 3H), 6.74 (singlet, 1H), 7.07 (doublet, J=8.9 Hz, 2H), 7.61 (doublet of doublets, J=8.9, 1.7 Hz, 1H), 7.81 (doublet, J=8.9 Hz, 2H), 7.93 (multiplet, 1H), 8.25 (doublet, J=8.9 Hz, 1H), and a broad singlet at 12.17 (1H). The 13C NMR spectrum (126 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 163.9, 143.4, 137.9, 129.3, 128.8, 128.7, 124.6 (quartet, JCF=271.8Hz), 124.2 (quartet, JCF=31.4Hz), 120.4 (quartet, JCF=3.3Hz), 117.8 (quartet, JCF=3.9Hz), 115.1, 114.9, 108.7, 55.8, 32.9, 27.6, and 23.5.

4-[1-(2-methoxyphenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid (16d)

Compound 16d, 4-[1-(2-methoxyphenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid, was synthesized following the multi-step procedure described for compound 16a. The reaction involved indole precursor 15a and 2-methoxybenzenesulfonyl chloride. This transformation yielded compound 16d in a low 3% yield. The purified product was obtained as a white solid. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.78 minutes and a deprotonated molecular ion at m/z 440 [M-H]-. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.93 (multiplet, 2H), 2.32 (multiplet, 2H), 2.98 (multiplet, 2H), 3.52 (singlet, 3H), 6.69 (singlet, 1H), 7.14 (doublet, J=8.2 Hz, 1H), 7.19 (multiplet, 1H), 7.52 (doublet of doublets, J=8.7, 1.5 Hz, 1H), 7.68 (multiplet, 1H), 7.93 (multiplet, 2H), 8.07 (doublet of doublets, J=8.0, 1.5 Hz, 1H), and a broad singlet at 12.13 (1H).

4-[1-(3-chlorophenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid (16e)

The preparation of 4-[1-(3-chlorophenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid, compound 16e, was achieved following the multi-step procedure described for compound 16a. The reaction involved indole precursor 15a and 3-chlorobenzenesulfonyl chloride. This transformation yielded compound 16e in a 16% yield. The purified product was obtained as a white solid. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.91 minutes and a deprotonated molecular ion at m/z 444 [M-H]-. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.95 (multiplet, 2H), 2.36 (triplet, J=7.3 Hz, 2H), 3.07 (triplet, J=7.3 Hz, 2H), 6.81 (singlet, 1H), 7.62 (multiplet, 2H), 7.80 (multiplet, 2H), 7.93 (triplet, J=1.9 Hz, 1H), 7.95 (multiplet, 1H), 8.24 (doublet, J=8.8 Hz, 1H), and a broad singlet at 12.17 (1H). The 13C NMR spectrum (126 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 143.5, 139.0, 137.9, 135.0, 134.5, 132.1, 129.5, 125.7, 125.0, 124.6 (quartet, JCF=31.7Hz), 124.5 (quartet, JCF=272.0Hz), 120.8 (quartet, JCF=3.1Hz), 118.0 (quartet, JCF=3.8Hz), 114.9, 109.4, 32.9, 27.5, and 23.5.

4-[1-(4-chlorophenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid (16f)

Compound 16f, 4-[1-(4-chlorophenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid, was synthesized following the multi-step procedure described for compound 16a. The reaction involved indole precursor 15a and 4-chlorobenzenesulfonyl chloride. This transformation yielded compound 16f in a 36% yield. The purified product was obtained as a white solid. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.45 minutes and a protonated molecular ion at m/z 446 [M+H]+. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.94 (multiplet, 2H), 2.35 (triplet, J=7.3 Hz, 2H), 3.04 (triplet, J=7.3 Hz, 2H), 6.79 (singlet, 1H), 7.64 (multiplet, 3H), 7.87 (multiplet, 2H), 7.95 (singlet, 1H), 8.23 (doublet, J=8.8 Hz, 1H), and a broad singlet at 12.19 (1H). The 13C NMR spectrum (75 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 143.3, 139.9, 137.9, 136.1, 130.2, 129.4, 128.2, 124.6 (quartet, JC,F=32.0Hz), 124.5 (quartet, JC,F=271.4Hz), 120.7, 118.0, 114.8, 109.3, 32.9, 27.5, and 23.5.

4-[1-(2-chlorophenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid (16g)

The preparation of 4-[1-(2-chlorophenyl)sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid, compound 16g, was achieved following the multi-step procedure described for compound 16a. The reaction involved indole precursor 15a and 2-chlorobenzenesulfonyl chloride. This transformation yielded compound 16g in a 9% yield. The purified product was obtained as a white solid. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.86 minutes and a deprotonated molecular ion at m/z 444 [M-H]-. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.86 (multiplet, 2H), 2.29 (triplet, J=7.1 Hz, 2H), 2.92 (triplet, J=7.5 Hz, 2H), 6.82 (singlet, 1H), 7.56 (doublet of doublets, J=8.9, 1.3 Hz, 1H), 7.64 (multiplet, 1H), 7.70 (doublet of doublets, J=8.5, 1.5 Hz, 1H), 7.76 (multiplet, 1H), 7.94 (doublet, J=8.8 Hz, 1H), 8.02 (multiplet, 2H), and a broad singlet at 12.10 (1H). The 13C NMR spectrum (126 MHz, DMSO-d6) further corroborated the structure with signals at δ 173.9, 144.3, 138.1, 136.4, 135.7, 132.6, 131.2, 130.9, 128.8, 128.5, 124.6 (quartet, JCF=272.6Hz), 124.2 (quartet, JCF=32.2Hz), 120.4 (quartet, JCF=3.4Hz), 118.1 (quartet, JCF=3.8Hz), 114.5, 107.9, 32.9, 27.3, and 23.2.

4-[1-[3-(trifluoromethoxy)phenyl]sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid (16h)

The preparation of 4-[1-[3-(trifluoromethoxy)phenyl]sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid, compound 16h, was achieved following the multi-step procedure described for compound 16a. The reaction involved indole precursor 15a and 3-(trifluoromethoxy)benzenesulfonyl chloride. This transformation yielded compound 16h in a 21% yield. The purified product was obtained as a white solid. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.98 minutes and a deprotonated molecular ion at m/z 494 [M-H]-. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.94 (multiplet, 2H), 2.34 (triplet, J=7.3 Hz, 2H), 3.06 (triplet, J=7.4 Hz, 2H), 6.80 (singlet, 1H), 7.63 (doublet of doublets, J=8.8, 1.7 Hz, 1H), 7.74 (multiplet, 2H), 7.84 (singlet, 1H), 7.88 (doublet of triplets, J=7.2, 1.8 Hz, 1H), 7.94 (singlet, 1H), 8.26 (doublet, J=8.8 Hz, 1H), and a broad singlet at 12.21 (1H). The 13C NMR spectrum (126 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 148.3, 143.5, 138.9, 138.0, 132.7, 129.6, 127.6, 125.4, 124.8 (quartet, JCF=31.9Hz), 124.5 (quartet, JCF=272.2Hz), 120.8 (quartet, JCF=3.3Hz), 119.7 (quartet, JCF=258.9Hz), 119.0, 118.0 (quartet, JCF=3.9Hz), 115.0, 109.7, 32.9, 27.6, and 23.5.

4-[1-[4-(trifluoromethoxy)phenyl]sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid (16i)

The preparation of 4-[1-[4-(trifluoromethoxy)phenyl]sulfonyl-5-(trifluoromethyl)indol-2-yl]butanoic acid, compound 16i, was achieved following the multi-step procedure described for compound 16a. The reaction involved indole precursor 15a and 4-(trifluoromethoxy)benzenesulfonyl chloride. This transformation yielded compound 16i in a 16% yield. The purified product was obtained as a white solid. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.99 minutes and a deprotonated molecular ion at m/z 494 [M-H]-. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.94 (multiplet, 2H), 2.34 (triplet, J=7.3 Hz, 2H), 3.05 (triplet, J=7.3 Hz, 2H), 6.80 (singlet, 1H), 7.57 (doublet, J=9.0 Hz, 2H), 7.64 (doublet of doublets, J=8.9, 1.65 Hz, 1H), 7.96 (singlet, 1H), 8.02 (doublet, J=9.0 Hz, 2H), 8.24 (doublet, J=8.9 Hz, 1H), and a broad singlet at 12.13 (1H). The 13C NMR spectrum (126 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 152.3, 143.3, 137.9, 136.0, 129.5, 129.2, 124.6 (quartet, JCF=31.7Hz), 124.5 (quartet, JCF=271.7Hz), 121.9, 120.8 (quartet, JCF=3.3Hz), 119.6 (quartet, JCF=259.4Hz), 118.0 (quartet, JCF=4.0Hz), 114.8, 109.3, 32.9, 27.5, and 23.5.

4-[5-chloro-1-(3-methoxyphenyl)sulfonyl-indol-2-yl]butanoic acid (16j)

Compound 16j, 4-[5-chloro-1-(3-methoxyphenyl)sulfonyl-indol-2-yl]butanoic acid, was synthesized following the multi-step procedure described for compound 16a. The reaction involved indole precursor 15b and 3-methoxybenzenesulfonyl chloride. This transformation yielded compound 16j in a 25% yield. The purified product was obtained as a white solid. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.81 minutes and a deprotonated molecular ion at m/z 406 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) displayed characteristic signals at δ 1.93 (multiplet, 2H), 2.34 (triplet, J=7.4 Hz, 2H), 3.02 (triplet, J=7.4 Hz, 2H), 3.76 (singlet, 3H), 6.63 (singlet, 1H), 7.22 (multiplet, 1H), 7.26 (multiplet, 1H), 7.33 (multiplet, 2H), 7.49 (triplet, J=9.5 Hz, 1H), 7.59 (doublet, J=2.2 Hz, 1H), 8.04 (doublet, J=10.1 Hz, 1H), and a broad singlet at 12.12 (1H). The 13C NMR spectrum (75 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 159.5, 143.1, 138.5, 134.8, 131.3, 131.0, 128.3, 123.9, 120.3, 119.9, 118.0, 115.7, 111.0, 108.7, 55.7, 32.9, 27.6, and 23.5.

4-[5-chloro-1-(4-methoxyphenyl)sulfonyl-indol-2-yl]butanoic acid (16k)

The preparation of 4-[5-chloro-1-(4-methoxyphenyl)sulfonyl-indol-2-yl]butanoic acid, compound 16k, was achieved following the multi-step procedure described for compound 16a. The reaction involved indole precursor 15b and 4-methoxybenzenesulfonyl chloride. This transformation yielded compound 16k in a 26% yield. The purified product was obtained as a white solid. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.80 minutes and a deprotonated molecular ion at m/z 406 [M-H]-. The 1H NMR spectrum (400 MHz, DMSO-d6) displayed characteristic signals at δ 1.93 (multiplet, 2H), 2.34 (triplet, J=7.3 Hz, 2H), 3.02 (triplet, J=7.3 Hz, 2H), 3.79 (singlet, 3H), 6.59 (singlet, 1H), 7.06 (doublet, J=9.0 Hz, 2H), 7.30 (doublet of doublets, J=8.9, 2.3 Hz, 1H), 7.57 (doublet, J=2.3 Hz, 1H), 7.76 (doublet, J=9.0 Hz, 2H), 8.04 (doublet, J=8.9 Hz, 1H), and a broad singlet at 12.14 (1H). The 13C NMR spectrum (75 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 163.7, 143.0, 134.7, 130.9, 128.8, 128.6, 128.1, 123.8, 119.8, 115.7, 115.0, 108.4, 55.8, 32.9, 27.6, and 23.5.

4-[5-chloro-1-(m-tolylsulfonyl)indol-2-yl]butanoic acid (16l)

The preparation of 4-[5-chloro-1-(m-tolylsulfonyl)indol-2-yl]butanoic acid, compound 16l, was achieved following the multi-step procedure described for compound 16a. The reaction involved indole precursor 15b and 3-methylbenzenesulfonyl chloride. This transformation yielded compound 16l in a 25% yield. The purified product was obtained as a white solid. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.53 minutes and a protonated molecular ion at m/z 392 [M+H]+. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.93 (multiplet, 2H), a complex signal between 2.33 (5H), 3.03 (triplet, J=7.4 Hz, 2H), 6.61 (singlet, 1H), 7.31 (doublet of doublets, J=8.9, 2.2 Hz, 1H), 7.44 (multiplet, 1H), 7.50 (multiplet, 1H), 7.57 (multiplet, 2H), 7.67 (singlet, 1H), 8.03 (doublet, J=8.9 Hz, 1H), and a broad singlet at 12.20 (1H). The 13C NMR spectrum (101 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.1, 143.1, 140.0, 137.4, 135.4, 134.7, 130.9, 129.7, 128.2, 126.2, 123.9, 123.3, 119.9, 115.7, 108.5, 33.0, 27.6, 23.5, and 20.7.

4-[5-chloro-1-(p-tolylsulfonyl)indol-2-yl]butanoic acid (16m)

The preparation of 4-[5-chloro-1-(p-tolylsulfonyl)indol-2-yl]butanoic acid, compound 16m, was achieved following the multi-step procedure described for compound 16a. The reaction involved indole precursor 15b and 4-methylbenzenesulfonyl chloride. This transformation yielded compound 16m in an 11% yield. The purified product was obtained as a white solid. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.53 minutes and a protonated molecular ion at m/z 392 [M+H]+. The 1H NMR spectrum (500 MHz, DMSO-d6) displayed characteristic signals at δ 1.92 (multiplet, 2H), 2.32 (singlet, 3H), 2.33 (triplet, J=7.7 Hz, 2H), 3.02 (triplet, J=7.4 Hz, 2H), 6.59 (singlet, 1H), 7.30 (doublet of doublets, J=8.9, 2.2 Hz, 1H), 7.36 (doublet, J=8.5 Hz, 2H), 7.57 (doublet, J=2.2 Hz, 1H), 7.70 (doublet, J=8.5 Hz, 2H), 8.03 (doublet, J=8.9 Hz, 1H), and a broad singlet at 12.18 (1H).

methyl 6-[5-chloro-2-[(4-fluoro-3-nitro-phenyl)sulfonylamino]phenyl]hex-5-ynoate (17)

The synthesis of methyl 6-[5-chloro-2-[(4-fluoro-3-nitro-phenyl)sulfonylamino]phenyl]hex-5-ynoate, compound 17, involved a carefully controlled sulfonylation reaction. To a solution of 1.4 g (5.9 mmol, 1.48 equivalents) of 4-fluoro-3-nitro-benzenesulfonyl chloride in 10 mL of dichloromethane, 0.5 mL (5.9 mmol, 1.48 equivalents) of pyridine and 49 mg (0.37 mmol, 0.09 equivalents) of dimethylaminopyridine were added. The reaction mixture was then cooled to 0°C to manage the exothermic reaction. Subsequently, 1 g (3.97 mmol) of aminoalkyne 7b was slowly introduced. After the addition was complete, the reaction mixture was allowed to warm to room temperature and stirred for 18 hours. Dichloromethane was then added to dilute the mixture, which was subsequently washed successively with a 1 N hydrochloric acid solution, water, and a saturated sodium chloride solution. The organic layer was then dried over magnesium sulfate and evaporated to dryness. The crude residue was purified by column chromatography on silica gel, utilizing a toluene/ethyl acetate mixture (95/5; v/v) as the eluent, to afford 1.8 g of the expected compound, representing a quantitative yield. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.32 minutes and a protonated molecular ion at m/z 455 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.70 (multiplet, 2H), 2.32 (triplet, J=7.7 Hz, 2H), 2.41 (triplet, J=7.3 Hz, 2H), 3.60 (singlet, 3H), 7.26 (multiplet, 1H), 7.40 (multiplet, 2H), 7.80 (multiplet, 1H), 8.03 (multiplet, 1H), 8.38 (multiplet, 1H), and a singlet at 10.31 (1H) for the sulfonamide proton.

methyl 4-[5-chloro-1-(4-fluoro-3-nitro-phenyl)sulfonyl-indol-2-yl]butanoate (18)

Compound 18, methyl 4-[5-chloro-1-(4-fluoro-3-nitro-phenyl)sulfonyl-indol-2-yl]butanoate, was synthesized through an intramolecular cyclization reaction, following a synthetic methodology similar to that described for compound 9d. The starting material for this transformation was compound 17, which underwent cyclization catalyzed by copper acetate under microwave irradiation. This reaction successfully yielded compound 18 in an excellent 96% yield. The purified product was obtained as a yellow solid, with a melting point of 93°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 6.76 minutes and a protonated molecular ion at m/z 455 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.97 (multiplet, 2H), 2.45 (triplet, J=7.3 Hz, 2H), 3.03 (triplet, J=7.5 Hz, 2H), 3.59 (singlet, 3H), 6.70 (singlet, 1H), 7.36 (multiplet, 1H), 7.63 (multiplet, 1H), 7.78 (multiplet, 1H), 8.06 (doublet, J=9.1 Hz, 1H), 8.19 (multiplet, 1H), and 8.47 (multiplet, 1H).

methyl 4-[1-(4-amino-3-nitro-phenyl)sulfonyl-5-chloro-indol-2-yl]butanoate (19)

The preparation of methyl 4-[1-(4-amino-3-nitro-phenyl)sulfonyl-5-chloro-indol-2-yl]butanoate, compound 19, involved a nucleophilic aromatic substitution reaction. To a solution of 100 mg (0.220 mmol) of compound 18 in 1 mL of dioxane, 0.77 mL of a 30% aqueous ammonia solution was added. The reaction mixture was stirred for 30 minutes at room temperature, allowing the ammonia to displace the fluorine atom. Following the reaction, ethyl acetate was added, and the mixture was washed successively with water and a saturated sodium chloride solution. The organic layer was then dried over magnesium sulfate and evaporated to dryness, yielding 100 mg of the expected compound as a yellow solid, representing a quantitative yield. LC/MS analysis (method b) confirmed its molecular structure, showing a retention time of 1.91 minutes and a deprotonated molecular ion at m/z 450 [M-H]-. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.96 (multiplet, 2H), 2.44 (triplet, J=7.4 Hz, 2H), 3.01 (triplet, J=7.4 Hz, 2H), 3.60 (singlet, 3H), 6.63 (singlet, 1H), 7.05 (doublet, J=9.0 Hz, 1H), 7.34 (doublet of doublets, J=8.8, 2.2 Hz, 1H), 7.62 (multiplet, 2H), 8.02 (doublet, J=8.8 Hz, 1H), a broad singlet at 8.16 (2H), and 8.37 (doublet, J=2.2 Hz, 1H).

methyl 4-[5-chloro-1-(3,4-diaminophenyl)sulfonyl-indol-2-yl]butanoate (20)

The preparation of methyl 4-[5-chloro-1-(3,4-diaminophenyl)sulfonyl-indol-2-yl]butanoate, compound 20, involved a reduction of the nitro group. To a suspension of 604 mg (1.33 mmol) of compound 19 in 8 mL of acetic acid, 394 mg (6.65 mmol, 5 equivalents) of iron powder were added. The reaction mixture was then stirred at an elevated temperature of 60°C for 2 hours, allowing the efficient reduction of the nitro group to an amine. Upon completion, the reaction mixture was filtered to remove insoluble iron, and the solid residue was thoroughly rinsed with water. The filtrate was then extracted with ethyl acetate. The obtained organic layer was washed with a saturated sodium chloride solution, dried over magnesium sulfate, and evaporated to dryness, yielding 566 mg of the expected compound as a pale yellow solid, representing a quantitative yield. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.74 minutes and a protonated molecular ion at m/z 422 [M+H]+. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.96 (multiplet, 2H), 2.44 (triplet, J=7.3 Hz, 2H), 3.01 (triplet, J=7.4 Hz, 2H), 3.61 (singlet, 3H), a singlet at 4.94 (2H), a singlet at 5.58 (2H), 6.48 (doublet, J=8.2 Hz, 1H), 6.53 (singlet, 1H), 6.90 (multiplet, 2H), 7.27 (doublet of doublets, J=8.8, 2.0 Hz, 1H), 7.56 (doublet, J=2.0 Hz, 1H), and 8.01 (doublet, J=8.8 Hz, 1H).

methyl 4-[1-(1H-benzimidazol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoate (21)

The synthesis of methyl 4-[1-(1H-benzimidazol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoate, designated as 21, involved a cyclization reaction to form the benzimidazole ring. A suspension of 87 mg (0.20 mmol) of compound 20 was meticulously prepared in 0.21 mL of formic acid. This mixture was then stirred and heated at 100°C for a duration of 2 hours, facilitating the cyclization. Upon completion of the reaction, the mixture was cooled to room temperature, and a 1N sodium hydroxide (NaOH) solution was carefully added to neutralize the formic acid. The resulting aqueous mixture was then extracted twice with ethyl acetate to recover the desired product. The combined organic layers were subsequently washed successively with water and a saturated sodium chloride solution to remove any remaining impurities. Following this, the organic phase was dried over magnesium sulfate and concentrated under reduced pressure, yielding 73 mg of the expected compound as a yellow oil, representing an 82% yield. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.68 minutes and a protonated molecular ion at m/z 432 [M+H]+. The 1H NMR spectrum (300 MHz, DMSO-d6) displayed characteristic signals at δ 1.97 (multiplet, 2H), 2.44 (triplet, J=7.3 Hz, 2H), 3.06 (triplet, J=7.3 Hz, 2H), 3.59 (singlet, 3H), 6.59 (singlet, 1H), 7.20 (multiplet, 1H), 7.32 (doublet of doublets, J=8.9, 2.4 Hz, 1H), 7.58 (multiplet, 2H), 7.72 (doublet, J=8.0 Hz, 1H), 8.11 (multiplet, 2H), and 8.47 (singlet, 1H).

4-[1-(1H-benzimidazol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoic acid (22)

Compound 22, 4-[1-(1H-benzimidazol-5-ylsulfonyl)-5-chloro-indol-2-yl]butanoic acid, was synthesized following the established saponification procedure as described for compound 4. The ester precursor used for this transformation was compound 21. This reaction successfully yielded compound 22 in a 66% yield. The purified product was obtained as a white solid, exhibiting a melting point of 212°C. LC/MS analysis (method a) confirmed its molecular structure, showing a retention time of 5.02 minutes and a deprotonated molecular ion at m/z 416 [M-H]-. The 1H NMR spectrum (250 MHz, DMSO-d6) displayed characteristic signals at δ 1.95 (multiplet, 2H), 2.34 (triplet, J=7.9 Hz, 2H), 3.08 (triplet, J=7.4 Hz, 2H), 6.59 (singlet, 1H), 7.32 (doublet of doublets, J=8.8, 2.2 Hz, 1H), 7.59 (multiplet, 2H), 7.73 (doublet, J=8.8 Hz, 1H), 8.11 (multiplet, 2H), 8.47 (singlet, 1H), and a broad singlet at 12.61 (1H). The 13C NMR spectrum (101 MHz, DMSO-d6) further corroborated the structure with signals at δ 174.5, 146.1, 143.2, 134.9, 131.0, 130.5, 129.8, 128.1, 128.0, 123.8, 119.9, 119.6, 115.8, 108.4, 33.3, 27.8, 23.7, with one carbon not observed.

PPAR Transactivation Assays

These sophisticated cell-based assays were meticulously conducted using Cos-7 cells, which are simian in origin from African green monkeys and carry the SV40 genetic material. The cells were specifically transfected with a chimeric human or murine PPAR alpha-Gal4 receptor expression plasmid (or alternatively, PPAR delta-Gal4, or PPAR gamma-Gal4) along with a 5Gal4 pGL3 TK Luc reporter plasmid. The transfections were efficiently performed using a chemical agent, Jet PEI. Following successful transfection, the cells were carefully distributed into 384-well plates and allowed to recover for a period of 24 hours to ensure optimal cellular health and gene expression. Subsequently, the spent culture medium was carefully removed and replaced with fresh medium containing the test compounds at various concentrations, each dissolved in 0.5% dimethyl sulfoxide (DMSO). After an overnight incubation period, luciferase expression, serving as a quantifiable reporter of PPAR activation, was measured by adding SteadyGlo reagent, strictly adhering to the manufacturer’s instructions provided by Promega. For comparative purposes and to establish a standardized reference, fenofibric acid at 10-5 M was used for PPAR alpha, GW501516 at 10-8 M for PPAR delta, and rosiglitazone at 10-6 M for PPAR gamma. The experimental results were comprehensively expressed either as fold induction relative to the basal luciferase level in untreated cells or as a percentage of activity compared to the respective reference compounds, which were assigned a 100% activity value. All calculations and plate validations across multiple experimental runs were performed using the specialized software, Assay Explorer (MDL). Compounds were serially diluted, achieving final concentrations ranging from 30 µM down to 0.001 µM, and were tested in triplicate on an automated screening core-system provided by Beckman or Caliper. The half-maximal effective concentration (EC50) values were calculated using Assay Explorer (MDL) and were determined simultaneously for both human and mouse PPAR alpha, delta, and gamma isoforms, providing a comprehensive and cross-species pharmacological profile.

In vitro Caco2 Permeability Assessment

The in vitro permeability of the compounds was rigorously assessed using a Caco-2 cell-based assay. Compounds were tested at a concentration of 10 µM in a 96-well permeable plate, which had been pre-seeded with Caco-2 cells, a well-established model for intestinal epithelial permeability. The culture medium, consisting of Hanks’ Balanced Salt Solution (HBSS) supplemented with 5 mM Hepes and 1% bovine serum albumin, adjusted to a pH of 7.4, was maintained at the same composition in both the apical and basolateral compartments of the permeable plate. The entire assay was executed with precision using a robotic platform (Caliper-Perkin Elmer system) to ensure high-throughput and reproducibility. Following an incubation period of 2 hours, the concentrations of the test compound in both the apical and basolateral sides were quantitatively measured using liquid chromatography-tandem mass spectrometry (LC/MS/MS), specifically an API4000 Qtrap from AB Sciex. Permeability was comprehensively assessed in both directions, from apical to basolateral and from basolateral to apical, allowing for the precise determination of the efflux ratio, which provides critical insights into active transport mechanisms and potential efflux pump substrate activity.

Beta-Oxidation Assays

The functional activity of PPAR alpha and PPAR delta was precisely determined in two distinct and physiologically relevant cell lines: HuH7, a human liver hepatoma cell line (JCRB 0403), and C2C12, a mouse muscle cell line that had been differentiated into myotubes (ATCC CRL-1772). These assays directly measured the rate of oleate beta-oxidation, a fundamental metabolic pathway. Cells were initially seeded into specialized Petri dishes equipped with a central well and subsequently incubated in DMEM medium at 37°C under standard cell culture conditions. Test compounds, dissolved in DMSO to a final concentration of 0.1%, were then added to the cell culture medium at a minimum of three different concentrations and incubated for 48 hours. Two hours prior to the conclusion of the incubation period, albumin-bound 14C-oleate, a radiolabeled fatty acid, was introduced into the cell culture medium. The beta-oxidation reaction was meticulously terminated by the addition of a 40% perchloric acid solution, which served to quench enzymatic activity and to remove any excess 14C-oleate remaining in the medium. The 14C-labeled carbon dioxide (CO2) produced during beta-oxidation was efficiently trapped by a solution of potassium hydroxide (KOH) placed in the central well of the sealed Petri dishes. After a 90-minute trapping period at room temperature, the radioactivity of the trapped CO2 was precisely quantified using a scintillation counter. All compounds were tested in triplicate for each concentration to ensure statistical robustness. The experimental data were then expressed as a percentage of variation relative to the activity observed with GW501516, which was utilized as a reference compound at a concentration of 0.1 µM.

Gene Expression Analysis

Human preadipocytes (sourced from Biopredic) were initially thawed and then amplified for a period of 7 days in a specialized medium. This medium consisted of DMEM/F12 supplemented with growth factors (specifically, SupplementPack/Preadipocyte C-39427 from PromoCell) to promote cell proliferation. Following this amplification phase, differentiation into mature adipocytes was actively induced by incubating the cells in adipocyte medium (AM-1, ZenBio) supplemented with 0.2 mM of IBMX for 3 days. During this differentiation period, the compounds under investigation were either included or omitted. In each culture plate, rosiglitazone at a concentration of 10-6 M was consistently used as a positive reference control, while 0.1% DMSO served as the vehicle control. Compounds were subjected to serial dilutions in the culture medium and tested in triplicate, ensuring a final DMSO concentration of 0.1% across all experimental conditions. After the 3-day incubation, cells were thoroughly rinsed three times with phosphate-buffered saline (PBS) and then immediately lysed to preserve RNA integrity.

Total RNA was subsequently extracted from the lysed cells, quantified for concentration and purity, and then reverse transcribed into complementary DNA (cDNA). An aliquot of this cDNA was then employed to perform Sybr Green real-time PCR, a highly sensitive and quantitative method for gene expression analysis. The specific primer sets utilized for this analysis were meticulously designed using the RefSeq sequence from the NCBI database and optimized with Beacon Designer 4 software (Premier Biosoft). The primer sequences for the analyzed genes were as follows: for aP2 (NM_001442), the forward primer was 5’-ACAGGAAAGTCAAGAGCACCAT-3’ and the reverse primer was 5’-GCATTCCACCACCAGTTTATC-3’. For Adiponectin (NM_004797), the forward primer was 5’-GGCTATGCTCTTCACCTATG-3’ and the reverse primer was 5’-ACGCTCTCCTTCCCCATAC-3’. Finally, for RPLP0 (NM_001002), which served as the housekeeping gene for normalization, the forward primer was 5’-GCCAATAAGGTGCCAGCTGCT-3’ and the reverse primer was 5’-ATGGTGCCCCTGGAGATTTT-3’. The relative mRNA amounts for aP2 and Adiponectin were determined by establishing a standard curve using increasing amounts of cDNA and were normalized against the mRNA levels of RPLP0. All quantifications were performed in triplicate to ensure reliability. The final results were expressed as percentages of the rosiglitazone (10-6 M) response, with means and standard deviations meticulously calculated to represent the data accurately.

In vivo Studies

All animal studies were conducted in strict adherence to the European Union animal welfare regulations governing the use of animals in experimentation, specifically European Directive 2010/63/EEC. The experimental protocol for these studies underwent thorough review and received official approval from the Inventiva Ethical Committee, known as “Comité de réflexion Ethique en Expérimentation Animale (CR2EA),” which is officially registered by the “Ministère de l’Enseignement Supérieur et de la Recherche” under registration number 104. All procedures detailed below were meticulously reviewed and approved by the Inventiva ethics committee, further ensuring compliance and ethical standards. Inventiva is a company fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), signifying its commitment to maintaining the highest standards of animal welfare.

The animals were housed in groups of 3-10 individuals within standard polypropylene cages, each providing a floor area of 1032 cm². Environmental conditions were carefully controlled and maintained: room temperature was kept at 22 ± 2°C, relative humidity at 55 ± 10%, with a precisely regulated 12-hour light/dark cycle. Air replacement rates were maintained at 15-20 volumes per hour, and both water and standard laboratory food (SDS, RM1) were provided ad libitum. Prior to the commencement of any experimentation, mice were allowed a minimum habituation period of 5 days to acclimatize to their new environment. For identification purposes, individual mice were numbered by marking their tails using indelible markers.

In vivo Pharmacokinetic Studies

The dosing regimen employed in the pharmacokinetic studies involved a single, once-daily (q.d.) oral administration of the test compounds. The compounds were prepared as a suspension in a vehicle consisting of 1% methylcellulose 400 cps and 0.1% poloxamer 188 in water. Blood samples were systematically collected at various time points up to 72 hours post-administration, with no pre-dose sampling conducted. A sparse sampling strategy was adopted, utilizing three animals per sampling time point to optimize animal welfare and resource utilization. Blood was collected into tubes pre-coated with evaporated lithium heparinate to prevent coagulation and was immediately centrifuged at approximately 4°C for about 10 minutes to separate plasma. The plasma samples were then acidified with 4N formic acid (HCOOH) at a 4% (v/v) ratio, specifically 4 µL of the acidic solution per 96 µL of plasma. After thorough mixing, the acidified plasma was, whenever possible, divided into two aliquots of 70 µL each. All collected samples were promptly stored at -20°C until bioanalysis. Pharmacokinetic parameters, such as Cmax and AUC, were subsequently estimated using Non-Compartmental Analysis (NCA) to characterize the drug’s disposition in the body.

In vivo Animal Model of Diabetic Dyslipidemia: db/db Mice

For the in vivo animal model of diabetic dyslipidemia, homozygous C57BL/Ks-db male mice, commonly known as db/db mice, were utilized. These mice, aged 11 to 13 weeks at the initiation of the studies, were randomly divided into experimental groups, each comprising 9-10 animals. The compounds under investigation were administered orally once a day, with treatment durations of either 5 or 10 days, depending on the specific experimental arm. One control group of mice received only the vehicle solution (either 0.5% or 1% methylcellulose solution) to account for any vehicle-related effects. A baseline blood sample was collected from the retro-orbital sinus of each mouse prior to the commencement of treatment. A second blood sample was obtained 4 hours after the final gavage on the last day of treatment. Following centrifugation, the serum was meticulously collected, and the levels of triglycerides and glucose were precisely measured using a multiparameter analyzer equipped with commercially available diagnostic kits. The experimental results were then expressed as a percentage variation on the final day relative to the control group, thereby providing a clear and quantifiable assessment of the compounds’ impact on diabetic dyslipidemia.

In vivo Animal Model of Fibrosis: CCl4 C57Bl6/J Mice

To assess anti-fibrotic activity, the CCl4-induced liver fibrosis model in C57Bl6/J mice was employed. C57Bl6/J mice, 6 weeks of age and weighing approximately 25g, received intraperitoneal (I.P.) injections twice a week for a duration of 3 weeks. These injections consisted of either 100 µL of sunflower seed oil (vehicle control) or carbon tetrachloride (CCl4) at a dose of 3.5 mL/kg, diluted in sunflower seed oil, to induce liver fibrosis. Concurrently, compound 5, at doses of 3, 10, or 30 mg/kg, was administered orally once daily in addition to the CCl4 treatment for 3 weeks, with 8 animals assigned to each group. At the study’s conclusion, blood and liver samples were meticulously collected for comprehensive RNA, protein, and histological analyses. Plasma samples were specifically analyzed for triglycerides and adiponectin levels to assess metabolic and endocrine engagement. Liver collagen deposition, a direct measure of fibrosis, was quantitatively evaluated by Picrosirius Red (PSR) staining on histological sections. For the analysis of PSR-stained sections, the NIS-Elements software from Nikon was utilized. The sections were digitally scanned, and a specific threshold, defined by a certain intensity of PSR staining, was applied to the entire liver section. The numerical value corresponding to the area of stained tissue that met this predefined threshold was then analyzed. This analytical approach yielded a percentage value representing collagen deposition within the cortical area of the liver. Crucially, this quantitative analysis was performed in a blinded manner to prevent investigator bias, thereby enhancing the objectivity and reliability of the results.

In vivo Safety Model: in Sprague-Dawley Rats

To evaluate the in vivo safety profile, particularly concerning cardiovascular and hematological parameters, Sprague-Dawley rats, 6 weeks of age, were utilized. These animals were maintained on either a standard pellet diet or a diet supplemented with the test compounds at a low or a high dose. The compounds evaluated included compound 5 (at 100 and 1000 mg/kg), rosiglitazone (1a, at 3 and 30 mg/kg), muraglitazar (3a, at 10 and 100 mg/kg), and tesaglitazar (3b, at 1 and 10 mg/kg). Plasma volume was precisely measured using the Evans blue dye dilution method. Briefly, conscious animals were gently restrained in a commercial restrainer for the controlled injection of 1 mg of Evans blue dye (in 200 µL saline) into a tail vein. After a 10-minute circulation period, 0.5 mL of blood was carefully withdrawn from the orbital sinus under light anesthesia. A small aliquot of this blood was then transferred into two hematocrit capillaries and immediately centrifuged for 5 minutes at 12,000 g to determine the hematocrit. The remaining portion of the blood sample was centrifuged for 10 minutes at 3,500 g, and the concentration of the Evans blue dye in the plasma was subsequently determined by measuring its absorbance at 620 nm. This absorbance reading was then compared against a standard curve generated using a pooled blood sample from three untreated rats, allowing for accurate quantification of plasma volume.

Cofactor Recruitment Assays

The cofactor recruitment assays were meticulously designed to assess the interaction of compounds with PPAR gamma and various co-regulatory proteins. Recombinant GST-tagged PPAR gamma protein, encompassing amino acids 202-505 of P37231, was heterologously expressed in *E. coli* and subsequently purified using affinity chromatography. N-terminal biotinylated peptides, whose sequences are detailed in an associated table, were chemically synthesized. Lumi4-Tb Cryptate-conjugated anti-GST antibody served as the fluorescent donor and was obtained from CisBio (61GSTTLA/B). Streptavidin-XL665, functioning as the fluorescent acceptor, was purchased from CisBio (610SAXLA/B).

These four primary reagents—recombinant GST-tagged PPAR gamma protein, biotinylated peptide, Lumi4-Tb Cryptate-conjugated anti-GST antibody, and Streptavidin-XL665—were precisely combined in a Tris buffer. Varying concentrations of the test compounds were also included in this mixture. Following a 1-hour incubation period at room temperature, the time-resolved fluorescence energy transfer (TR-FRET) signal was measured using a VictorWallac4 plate reader. The excitation wavelength was set at 320 nm, and emission wavelengths were monitored at 665 nm and 615 nm.

The underlying principle of the assay relies on specific molecular interactions. The GST-tagged PPAR gamma fragment binds to the anti-GST-Tb antibody. Simultaneously, the biotinylated peptide binds to the SA-XL665 complex. When the nuclear receptor-Tb complex and the peptide-APC complex bind to each other, FRET occurs, generating a measurable signal. The titration of an agonistic ligand, which binds to the respective nuclear receptor, leads to an increased interaction between the nuclear receptor and the coactivator peptide (with the exception of NCOR-ID1 and SMRT-ID1 derived peptides). This increased binding results in a corresponding increase in the FRET signal. Conversely, for the two co-repressor peptides, NCOR-ID1 and SMRT-ID1, the binding of an agonist to the nuclear receptor causes their displacement, leading to a decrease in the FRET signal.

Assays were conducted in a final volume of 25 µL within 384-well plates. The test compounds were initially dissolved in DMSO, which served as the vehicle, ensuring a final DMSO concentration of 1% across all experiments. All test compounds were serially diluted in 3-fold steps, and each experiment was performed in duplicates to ensure robustness and reproducibility.

The specific peptide sequences used in these assays are as follows: CBP (58-80) (NP_004371) NH2 – NLVPDAASKHKQLSELLRGGSGS – COOH; D22 peptide (artificial) NH2 – LPYEGSLLLKLLRAPVEEV – COOH; DAX1 (132−156) (ΝP_000466) NH2 – CCFCGEDHPRQGSILYSLLTSSKQT – COOH; LCOR (39−63) (NP_115816) NH2 – VTTSPTAATTQNPVLSKLLMADQDS – COOH; NCoA3 (607-631) (NP_858045) NH2 – ENQRGPLESKGHKKLLQLLTCSSDD – COOH; NCoA3 (671-695) (NP_858045) NH2 – SNMHGSLLQEKHRILHKLLQNGNSP – COOH; NCoA3 (724-748) (NP_858045) NH2 – QEQLSPKKKENNALLRYLLDRDDPS – COOH; PERC NR1 (145-168) (NP_573570) NH2 – APAPEVDELSLLQKLLLATSYPTS – COOH; PERC NR2 (332-354) (NP_573570) NH2 – HSKASWAEFSILRELLAQDVLCD – COOH; PGC1 (196-221) (NP_037393) NH2 – CQQQKPQRRPCSELLKYLTTNDDPP – COOH; PNRC1 (302-327) (NP_006804) NH2 – GSTVENSNQNRELMAVHLKTLLKVQT – COOH; RAP250 (873-897) (NP_054790) NH2 – GFPVNKDVTLTSPLLVNLLQSDISA – COOH; RIP140 (118-143) (P48552) NH2 – MVDSVPKGKQDSTLLASLLQSFSSR – COOH; RIP140 (366-390) (P48552) NH2 – LERNNIKQAANNSLLLHLLKSQTIP – COOH; RIP140 (805-829) (P48552) NH2 – PVSPQDFSFSKNGLLSRLLRQNQDS – COOH; RIP140 (922-946) (P48552) NH2 – EHRSWARESKSFNVLKQLLLSENCV – COOH; SHP (7-31) (NP_068804) NH2 – GACFCQGAASRPAILYALLSSSLKA – COOH; SRC1 (104−128) (NP_068804) NH2 − ΑςΤΦΕςΑΕΑΠςΠΣΙΛΚΚΙΛΛΕΕΠΣΣ − ΧΟΟΗ; SRC1 (619-643) (NP_003734) NH2 – RLSDGDSKYSQTSHKLVQLLTTTAEQ – COOH; SRC1 (676-700) (NP_003734) NH2 – CPSSHSSLTERHKILHRLLQEGSPS – COOH; SRC1 (735-759) (NP_003734) NH2 – LDASKKKESKDHQLLRYLLDKDEKD – COOH; TIF2 (628-651) (NP_006531) NH2 – GQSRLHDSKGQTKLLQLLTTKSDQ – COOH; TIF2 (676-700) (NP_006531) NH2 – GSTHGTSLKEKHKILHRLLQDSSSP – COOH; TIF2 (731-755) (Q15596) NH2 – KQEPVSPKKKENALLRYLLDKDDTK – COOH; TRAP220 (590-614) (NP_004765) NH2 – GHGEDFSKVSQNPILTSLLQITGNG – COOH; NCOR-ID1 (2253-2277) (NP_006302) NH2 – SFADPASNLGLEDIIRKALMGSFDD – COOH; SMRT-ID1 (2339-2363) (NP_006303) NH2 – VQEHASTNMGLEAIIRKALMGKYDQ – COOH.

Associated Content

Accession Codes
The atomic coordinates and corresponding structure factors for the crystal structures of PPARγ in complex with compound 5 can be accessed using the Protein Data Bank (PDB) code 6ENQ. It is intended that the authors will publicly release these atomic coordinates and all associated experimental data upon the formal publication of the article, ensuring full transparency and accessibility for the scientific community.

Author Information

Corresponding Author
For any correspondence or inquiries pertaining to this research, individuals are directed to the designated corresponding author, whose contact details are provided. The telephone number for direct communication is +33 3 80 44 75 27. Electronic mail correspondence can be addressed to [email protected].

Acknowledgment

The authors extend their sincere gratitude and profound appreciation to Frederic Bell, Luc Spitzer, Florence Chirade, Annick Reboul, and Didier Bressac for their invaluable contributions and dedicated efforts throughout the various stages of this comprehensive work. Furthermore, special thanks are extended to Fabrice Ciesielski and Mireille Tallandier for their insightful and fruitful discussions, as well as for their consistent provision of essential technical support, which significantly enhanced the quality and progress of this research.

Abbreviations

The following is a comprehensive list of abbreviations used throughout this document, provided for clarity and ease of reference: aP2, adipocyte protein 2; AUCinf, area under the concentration-time curve extrapolated to infinity; C2C12, mouse myoblast cell line; C57Bl6, a common inbred strain of laboratory mouse; Caco-2, human Caucasian colon adenocarcinoma cell line; CCl4, carbon tetrachloride; CF3, trifluoromethyl; CH2Cl2, dichloroethane; Cmax, maximum (or peak) serum concentration that a drug achieves in a specified compartment; Cu(OAc)2, copper acetate; CuI, copper iodide; COS-7, CV-1 (simian from African green monkey) cells in origin, carrying the SV40 genetic material; db/db mouse model, a genetic mouse model used as a spontaneous type 2 diabetic animal model; DMAP, dimethylaminopyridine; DMF, dimethylformamide; EC50, half maximal effective concentration; Emax, maximum efficacy; FFA, free fatty acids; Et2N, diethylamine; Gal-4, galactose-responsive transcription factor (from Saccharomyces cerevisiae S288C); HSC, hepatic stellate cells; HTS, high throughput screening; HuH7, human hepatoma cell line; KOH, potassium hydroxide; LBD, ligand binding domain; LBP, ligand binding pockets; LCOR, ligand-dependent corepressor; LiOH, lithium hydroxide; NCoA3, nuclear receptor coactivator 3; NCoR-ID1, nuclear receptor corepressor 1; NaH, sodium hydride; NASH, nonalcoholic steatohepatitis; PDB, protein data base; Pd(PPh3)2Cl2, bis(triphenylphosphine)palladium(II) dichloride; PGC1α, PPARγ coactivator-1α; PNRC1, proline-rich nuclear receptor coactivator 1; PPAR, peroxisome proliferator-activated receptor; PSR, picrosirius red; RIP140, receptor interacting protein 140; SAR, structure activity relationship; SMRT, silencing mediator of retinoic acid and thyroid hormone receptor; SPPARM, selective peroxisome proliferator activated receptor modulator; SRC-1, steroid receptor coactivator 1; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Tmax, the amount of time that a drug is present at the maximum concentration in serum; TR-FRET, time-resolved fluorescence energy transfer.