Discovery and Evaluation of Active Site-Directed, Potent, and Selective Sulfophenyl Acetic Amide-Based Inhibitors for the Laforin Phosphatase

Abstract

Lafora disease is a rare and fatal progressive myoclonus epilepsy characterized by the accumulation of insoluble glycogen deposits in the brain and peripheral tissues. Mutations in the gene encoding the glycogen phosphatase laforin result in Lafora disease. Currently, there are no laforin-specific chemical probes, limiting our understanding of the roles of laforin in glycogen metabolism and other cellular processes. Here, we identified sulfophenyl acetic amide (SPAA), as a novel nonhydrolyzable phosphotyrosine mimetic for laforin inhibition. Using fragment-based and scaffold-hopping strategies, we discovered several highly potent and selective active site-directed laforin inhibitors. Among them, compound 9c displayed a K_i value of 1.9 ± 0.2 nM and more than 8,300-fold preference for laforin. Moreover, these inhibitors efficiently block laforin-mediated glucan dephosphorylation inside the cell and possess favorable pharmacokinetic properties in mice. These chemical probes will enable further investigation of the roles of laforin in normal physiological processes and in diseases.

Graphical Abstract

INTRODUCTION

Lafora progressive myoclonus epilepsy, also known as Lafora disease, is an autosomal recessive, severe neurodegenerative disorder that is classified as a neurological glycogen storage disease¹. It is characterized by the accumulation of abnormal, water-insoluble, glycogen-like inclusions called Lafora bodies in nearly all tissues of affected individuals^{2, 3}. The onset of Lafora disease typically occurs in childhood or adolescence with refractory epilepsy and myoclonus, and the condition rapidly deteriorates with the patient experiencing childhood dementia, ultimately leading to the demise of the patient within a decade of the initial symptoms^2–4. Unfortunately, Lafora disease can only be managed for a limited period with palliative care. Indeed, there is currently no effective treatment available that can cure or treat the disease^{1, 5, 6}. The underlying cause of the disease is associated with the activity of two proteins, the glycogen phosphatase laforin and the E3-ubiquitin ligase malin, which form a functional complex^7–10. Mutations in the gene encoding either protein result in poorly branched, hyperphosphorylated glycogen, which subsequently precipitates, aggregates, and accumulates into Lafora bodies^11–15. These Lafora bodies are observed in cells from nearly all tissues with neurons and astrocytes being especially sensitive to these aggregates and the Lafora bodies are the pathological agent driving the neurodegeneration and eventual fatality observed in individuals with Lafora disease. While considerable progress has been made in recent years, the exact physiological roles of both laforin and malin proteins, as well as the underlying mechanisms by which their absence leads to the formation of Lafora bodies and the development of Lafora disease, are not yet fully understood^{4, 5}. Consequently, understanding the precise functions of these proteins and unraveling the reasons behind the disease manifestation in the absence of laforin or malin has been the subject of intense investigation.

As a member of the protein tyrosine phosphatase (PTP) superfamily¹⁶, laforin consists of a catalytic dual-specificity phosphatase domain located in the C-terminus and a carbohydrate-binding domain in the N-terminus^{7, 17–19}. Similar to other PTPs, laforin utilizes a conserved CX₅R motif (CNAGVGR, residues 265–271) at its active site to catalyze the hydrolysis of phosphomonoester bonds in the substrates²⁰. Laforin is the only known human glycogen phosphatase, playing an important role in the cellular process of glycogen metabolism. Its absence results in progressive hyperphosphorylated and aberrant branched glycogen, which accumulates into neurotoxic Lafora bodies^{3, 21}. While laforin’s primary role as a glycogen phosphatase is well investigated, recent studies have unveiled its extensive influence beyond glycogen metabolism. Laforin can also serve as an adaptor protein in multiple cellular pathways, playing a significant role in modulating diverse processes, including signal transduction, cellular differentiation, and stress responses^21–28. Furthermore, evidence suggests that laforin is linked to pulmonary fibrosis and it may also possess tumor suppressor properties, with its protein phosphatase activity being intricately linked to cancer biology^29–33. These studies have implicated laforin in the regulation of crucial signaling pathways associated with tumor development and progression. We surmised that potent, selective, and bioavailable laforin inhibitors would serve as invaluable chemical probes to shed new light on the roles of laforin in glycogen metabolism, cellular signaling pathways, and diseases. Unfortunately, no laforin inhibitor exists to enable pharmacological investigation of laforin in different biological processes and testing of various therapeutic hypotheses.

The goal of this study was to develop potent and selective laforin inhibitors that could be deployed as chemical probes to interrogate the functions of laforin in health and diseases. To that end, we employed fragment-based and scaffold-hopping strategies that ultimately led to the identification and development of a series of highly potent and selective sulfophenyl acetic amide (SPAA)-based inhibitors targeting the active site of laforin. The most potent inhibitor, 9c, displayed outstanding potency with an IC₅₀ value of 4.0 ± 0.3 nM for laforin. Importantly, compound 9c exhibited remarkable selectivity, exhibiting an over 8,300-fold preference towards laforin over a panel of 15 mammalian PTPs. Through careful kinetic analysis, it was determined that compound 9c acted as a reversible and competitive active site-directed inhibitor of laforin, with a K_i value of 1.9 ± 0.2 nM. This inhibitory activity was further confirmed through binding assays and molecular modeling analyses, credentialing these compounds as robust laforin inhibitors. One of the significant aspects of the work is that these inhibitors possessed favorable pharmacokinetic properties and effectively blocked laforin-mediated glucan phosphatase activity inside the cell. These findings highlight the potential utility of these laforin inhibitors in determining the fundamental roles of laforin in cell biology and Lafora disease. Moreover, our work further demonstrates the feasibility of obtaining highly potent and selective PTP active site-directed inhibitors that are also capable of penetrating cell membranes.

RESULTS AND DISCUSSION

Development of Potent and Selective Laforin Active Site-Directed Inhibitors

The development of active site-directed small molecule probes for members of the PTP family has posed significant challenges for the field. Two key obstacles have been encountered in this pursuit³⁴. Firstly, the high degree of amino acid sequence similarity within the catalytic domain, particularly the pTyr-binding pocket, among the PTPs has made it difficult to obtain compounds that can selectively inhibit the desired target without affecting closely related family members. Secondly, the positively charged environment of the PTP catalytic site presents a unique dilemma in drug discovery. Compounds with high affinity for PTP active site typically require ionizable functional groups, which often results in low cell permeability and limited bioavailability. Addressing these issues effectively is crucial for the successful development of PTP-targeted therapeutics. To tackle the selectivity problem, we have pioneered a novel approach that involves the design and synthesis of molecules that interact not only with the active site but also with nearby peripheral pockets unique to the desired PTP target. This strategy allows for a more precise and selective inhibition of the intended PTP target, while minimizing off-target inhibition of related family members^35–37. To overcome the insufficient cell permeability and bioavailability hurdle, we have focused on identifying nonhydrolyzable phosphotyrosine (pTyr) mimetics that possess the necessary polarity to bind effectively to the PTP catalytic site, while also harboring the potential propensity to penetrate cell membranes³⁴. Application of these strategies has led to the development of numerous potent and selective active site-directed PTP inhibitors with excellent efficacy in cellular systems, and some of them even in animal models, demonstrating their potential as valuable chemical probes and therapeutic agents^{34, 38–48}.

We recently identified α-sulfophenyl acetic amide (SPAA), a fragment derived from the FDA-approved drug Cefsulodin, as a novel nonhydrolyzable pTyr mimetic⁴⁹. While the included sulfonic acid has high water solubility, it can present challenges in terms of cellular membrane permeability. By modifying sulfonic acid derivatives, particularly by incorporating lipophilic groups, their drug-like properties, such as cellular membrane permeability can be improved⁵⁰. Notably, several marketed drugs containing sulfonic acid demonstrate favorable druglikeness, including Cefsulodin, Penicillins, and Tauroursodeoxycholic acid. Indeed, by incorporating lipophilic groups, sulfonic acid has been successfully utilized in the development of PTP inhibitors^{44, 51, 52}. We have also demonstrated that PTP inhibitors carrying the SPAA scaffold exhibit excellent cellular and in vivo efficacies^{42, 49, 53–55}. Here, we aimed to leverage the SPAA scaffold as a starting point for the development of inhibitors targeting laforin. We first assessed the ability of SPAA to inhibit the laforin catalyzed reaction. We observed that (S)-SPAA (i.e. compound 3 in Table 1), exhibited inhibitory activity against laforin with an IC₅₀ of 51.7 ± 3.3 μM. Further kinetic studies showed that compound 3 is competitive with p-nitrophenyl phosphate (pNPP), a substrate of laforin, and the K_i value for compound 3 was determined to be 24.7 ± 1.3 μM (Supporting Information, Figure S1). These results indicate that the SPAA fragment likely binds to the active site of laforin, consistent with previous observations that the SPAA motif targets the PTP active site^{49, 54}. To enhance the potency and selectivity of SPAA-based inhibitors for laforin, we embarked on a strategy to capture additional interactions beyond the catalytic pocket. To achieve this goal, we systematically synthesized three series of compounds based on the SPAA scaffold (Figure 1). These compounds were created by reacting (S)-sulfophenyl acetic acid with a diverse range of amines varying in size, charge, and lipophilicity. This approach enabled us to incorporate appropriate linkers and structural diversity, thereby enhancing the potency and selectivity of the SPAA-based inhibitors for laforin, ultimately optimizing the compounds for their intended target.

Table 1.

IC₅₀ value (μM) of SPAA-based laforin inhibitors 3, 3a-3d, 5a, 5b, 8a-8l^a

ID	R	laforin IC50/μM	ID	R	laforin IC50/μM
3	H	51.7 ± 3.3	8d		1.84±0.49
3a		4.05±0.65	8e		0.99±0.28
3b		4.72±0.81	8f		3.14±0.57
3c		2.65±0.41	8g		0.95±0.37
3d		0.65±0.15	8h		0.20±0.04
5a		10.9±3.2	8i		9.33±1.02
5b		0.31±0.08	8j		0.15±0.02
8a		2.07±0.52	8k		0.11±0.01
8b		0.15±0.03	8l		0.16±0.02
8c		0.80±0.15

^aIC₅₀ values were determined from three independent measurements.

Figure 1.

Design and optimization of SPAA-based laforin inhibitors. 3 different linkers were applied to create 3 series of derivatives, incorporating a total of 20 distinct tail fragments.

We then began our effort on optimizing the activity of SPAA-based laforin inhibitors by introducing an array of aniline derivatives with varying substitutions (Series 1, Figure 1). Compounds 3a-3d were synthesized through the reaction between phenyl sulfoacetyl chloride 2 and the corresponding anilines in the presence of DIEA. The resulting compounds were purified using HPLC (Series 1, Scheme 1). Their inhibitory activity toward laforin was quantified by their IC₅₀ values against the laforin-catalyzed hydrolysis of pNPP (Table 1). Compounds 3a-3c demonstrated laforin inhibitory activity in the low micromolar range, showing more than a 10-fold improvement in IC₅₀ values compared to their parent compound 3. These results indicate that the aromatic substitutions on the amide position of SPAA likely make additional interactions with laforin beyond the active site. Interestingly, compound 3d, which features an N-(2-aminophenyl)-[1,1’-biphenyl]-4-carboxamide functionalized aniline motif, displayed an even higher binding affinity for laforin, with an IC₅₀ of 0.65 ± 0.15 μM. This was at least 4-fold more potent than compounds 3a-3c. Taking inspiration from the performance of compound 3d, we proceeded to assess other substituted 2-aminophenyl benzamide motifs (Series 2, Figure 1). Through the condensation of compound 4 with the corresponding benzoic acids in the presence of HOBt, HBTU, and DIEA (Series 2, Scheme 1), we obtained derivatives 5a and 5b. Notably, compound 5b showed a 2-fold improvement in potency compared to compound 3d, with an IC₅₀ value against laforin of 0.31 ± 0.08 μM (Table 1). On the other hand, compound 5a, which lacks a substituent on the distal phenyl ring, displayed approximately 16-fold lower potency than compound 3d.

Scheme 1.

Synthesis of SPAA-based laforin inhibitors 3a-3d, 5a, 5b, and 8a-8n^a

^aReaction conditions: (a) SOCl₂, reflux, overnight, 100%; (b) corresponding aniline, DIEA, DCM, r.t., 12h, 87–90%; (c) o-phenylenediamine, DIEA, DCM, r.t., 12h, 62%; (d) corresponding benzoic acid, HOBt, HBTU, DIEA, DMF, r.t., overnight, 65–90%; (e) DIEA, DCM, r.t., 1h, 72%; (f) corresponding aniline, HOBt, HBTU, DIEA, DMF, r.t. overnight, 52–75%.

In order to explore the potential for a more efficient linker, we proceeded to replace the amide functional group with oxamide, resulting in the synthesis of derivatives 8a-8c (Series 3, Figure 1). To prepare these compounds, compound 2 reacts with 2-((2-aminophenyl)amino)-2-oxoacetic acid (6) in the presence of DIEA, leading to the formation of compound 7. Finally, derivatives 8a-8c were obtained through the condensation of compound 7 with the corresponding anilines in the presence of HOBt, HBTU, and DIEA (Series 3, Scheme 1). Their IC₅₀ values against laforin were then determined and included in Table 1. Compound 8c exhibited a slight decrease in IC₅₀ value compared to 3d. However, the other two derivatives showed improvements in potency. Compound 8a demonstrated approximately 5-fold greater potency than 5a, while compound 8b achieved 2-fold improvement compared to 5b, with an IC₅₀ value of 0.15 ± 0.03 μM. These findings suggest that the oxamide linker may be preferred for binding with the enzyme, showcasing enhanced potency. To further investigate the structure-activity relationship (SAR) of substituted anilines attached to the oxamide linker, we synthesized eight additional derivatives, namely 8d-8k, using the same methodology. These derivatives featured various substituents on the aniline phenyl ring, ranging from halogen, alkyl, and alkynyl to aryl functional groups. The key findings of the SAR analysis are summarized in Table 1. The introduction of hydrophilic groups to the aniline ring led to decreased potency in the derivatives, as observed in compounds 8f and 8i. Conversely, hydrophobic substitutions were found to be favorable, as exemplified by compounds 8e, 8g, and 8h, which exhibited IC₅₀ values against laforin in the sub-micromolar range. Notably, derivatives with an additional hydrophobic phenyl ring as a substituent attached to the aniline demonstrated even higher potency, as evidenced by compound 8j and 8l, which displayed an IC₅₀ value of 150 nM and 160 nM, respectively. Furthermore, the addition of a chloro group at the meta-position of the aniline in compound 8j led to compound 8k, which showed an improved inhibitory activity against laforin with an IC₅₀ of 110 nM. Taken together, this SAR study revealed that hydrophobic substituents on the aniline ring, particularly when accompanied by an additional phenyl ring, contribute to enhanced potency. The incorporation of a chloro group at the meta-position of the aniline further augmented the inhibitory activity, making compound 8k the most potent laforin inhibitor in this series.

Steady state kinetic analyses were conducted to characterize compound 8k with pNPP as a substrate. The resulting Lineweaver-Burk plots revealed 8k as a competitive inhibitor of laforin as expected (Figure 2A). The K_i value for compound 8k was 63.0 ± 6.1 nM, signifying its strong binding affinity for the enzyme. Incubation of compound 8k with laforin for 30 min prior to the addition of substrate afforded the same inhibition constant as when pNPP and inhibitor were mixed simultaneously, indicating that compound 8k is a reversible inhibitor of laforin. Next, we evaluated the ability of compound 8k to inhibit the glucan phosphatase activity of laforin using the phosphorylated carbohydrate amylopectin as the substrate. To accomplish this, we incubated laforin with amylopectin in the presence and absence of the inhibitor, and the release of inorganic phosphate was detected using malachite green⁵⁶. Notably, compound 8k inhibited laforin’s glucan phosphatase activity with an IC₅₀ value of 131 nM (Figure 2B), similar to that measured with pNPP as a substrate (IC₅₀ = 110 nM).

Figure 2:

A) Compound 8k is a reversible and competitive inhibitor of laforin with pNPP as a substrate. Lineweaver-Burk plot for compound 8k mediated laforin inhibition. Compound 8k concentrations were 0 nM (▲), 50 nM (■), and 100 nM (●). The K_i value of 63.0 ± 6.1 nM was determined from three independent measurements. B) Inhibition of the glucan phosphatase activity of laforin by compound 8k. A malachite green assay was performed using amylopectin as substrate, robust dose-dependent inhibition was observed with an IC₅₀ = 131 nM. Values were measured in triplicate and are reported as the average +/− standard deviation.

To exclude the possibility that compound 8k inhibits laforin through non-specific aggregation, we conducted additional experiments to measure its potency in the presence of detergent Triton X-100, which is known to diminish promiscuous inhibitory effect by nonspecific aggregators^{57, 58}. Satisfactorily, we found that the presence of Triton X-100 had no impact on compound 8k mediated laforin inhibition (Supporting Information, Figure S2). The IC₅₀ value for 8k measured in the presence of TritonX-100 was 100 ± 0.10 nM, which is identical to the IC₅₀ of 110 nM determined in the absence of the detergent, indicating that compound 8k does not inhibit laforin activity through nonspecific aggregation.

Next, we assessed the specificity of compound 8k by measuring its inhibitory activity against a panel of 15 mammalian PTPs. This panel included various cytosolic PTPs such as SHP2, SHP1, PTP1B, LYP, HePTP, STEP, and TC-PTP, as well as receptor-like PTPs like PTPα, PTPβ, PTPδ, LAR, and CD45, along with the dual specificity phosphatases VHR and YopH, and the low molecular weight PTP. The results, presented in Table S1 (Supporting Information), show the IC₅₀ values of compound 8k for all tested PTPs are greater than 50 μM, with > 450-fold selectivity for laforin. This finding highlights the specific and targeted inhibitory action of compound 8k towards laforin, reinforcing its potential as a promising candidate for selective laforin inhibition.

Cellular Inhibition of Laforin-Mediated Glucan De-phosphorylation by Compound 8k.

To determine the cellular efficacy of the laforin inhibitor compound 8k, we assessed glycogen architecture, including total abundance, chain length distribution, and phosphate content, in A549 lung adenocarcinoma cells following treatment with compound 8k. After a 24-hour incubation with 8k, cells were fixed and treated with isoamylase to cleave glycogen α−1,6-glycosidic bonds and release linear glucose polymers from glycogen. These polymers were then assessed for chain length distribution and phosphate content using MALDI-MSI. Consistent with laforin phosphatase inhibition, 8k treatment significantly increased the phosphate content among all glucose chain lengths (Figure 3A). The data confirm both cell permeability and target engagement by 8k in a cellular context. Furthermore, 8k increased the abundance of longer-chain glucose polymers (Figure 3B). These results are consistent with the role of laforin as a major regulator of glycogen turnover in these cells and demonstrate the utility of using 8k to explore laforin biology in other systems.

Figure 3.

Compound 8k blocks laforin-mediated glucan de-phosphorylation. A549 lung adenocarcinoma cells were treated with 1 μM 8k for 24h followed by fixation, treatment with isoamylase to cleave glucose polymers, and MALDI-MSI to assess glycogen abundance, phosphate content, and architecture. A) 8k increased the phosphate content among all glucose chain lengths; B) 8k increased the abundance of longer chain glucose polymer.

Pharmacokinetic Properties of SPAA-based Laforin Inhibitors

To enable applications of these laforin inhibitors for in vivo studies, we initiated an evaluation of their pharmacokinetic properties in mice. Intraperitoneal administration of compound 8k at a dosage of 10 mg/kg resulted in a C_max (maximum concentration) value of 2.25 μM, which was more than 20-fold higher than its IC₅₀ value (Table 2). However, we observed rapid clearance of the compound in vivo, leading to a short half-life (t_1/2) of approximately 0.4 hours. In an attempt to identify the specific motif or functional group responsible for this rapid clearance, we conducted the same pharmacokinetic assay using compound 8l and compound 4 as controls. The results revealed a t_1/2 of 1.2 hours for compound 8l and 0.9 hours for compound 4, respectively. Therefore, it can be speculated that the key motif present in compound 4 was primarily responsible for its rapid clearance. Moreover, it is known that the diaminobenzene motif in compound 4 can be readily oxidized by P450 enzymes⁵⁹. Given the need to improve the pharmacokinetic profiles while maintaining the interactions with laforin, we decided to explore a feasible approach for optimization. Scaffold hopping⁶⁰, a strategy known to be effective in such situations, was considered. Our initial plan involved utilizing a saturated cis-1,2-diaminocyclohexane scaffold to replace the 1,2-diaminobenzene motif in our most potent inhibitor, compound 8k. This substitution resulted in compound 9a, which exists as a mixture of two cis- diastereomers: (2S,5R,6S)-9a and (2S,5S,6R)-9a (Table 2). This strategy aimed to preserve the desired interactions with laforin while maintaining a higher blood drug concentration for an extended period. Strikingly, compound 9a displayed approximately 2-fold higher potency compared to its parental compound 8k, exhibiting an IC₅₀ value of 67 ± 9 nM against laforin (Table 3). Subsequent pharmacokinetic analyses indicated that compound 9a possessed a half-life of approximately 1.0 hour, which was about 2.5-fold longer than that of compound 8k. Furthermore, the maximum concentration of compound 9a in serum reached 7.45 μM, a significant increase of approximately 3.3-fold compared to that of compound 8k when administered via the intraperitoneal (IP) route. Additionally, when comparing compound 8k and 8l, we observed that replacing the ether group with a methylene group also resulted in an approximately 3-fold extension of the half-life. Encouraged by these findings, we applied the same approach to compound 9a, leading to the synthesis of compound 9b. As expected, compound 9b showed an extended half-life of 2.5 hours (Table 2). Remarkably, compound 9b also exhibited a higher affinity for laforin, with an IC₅₀ value of 20 ± 2 nM (Table 3). The improved properties displayed by compound 9b suggest that this strategy holds promise for the development of laforin inhibitors with enhanced pharmacokinetic properties.

Table 2.

Pharmacokinetic properties of some typical SPAA-based laforin inhibitors

ID	Structure	parameter	Cmax (μM)	tmax (hour)	t1/2 (hour)
8k		# 10 mg/kg	2.25	0.5	0.4
8l		# 10 mg/kg	1.68	1	1.2
4		# 10 mg/kg	8.75	1	0.9
9a		# 10 mg/kg	7.45	1	1.0
9b		# 10 mg/kg	2.01	1	2.5
9c		# 10 mg/kg	7.68	1	1.2

Table 3.

IC₅₀ value (μM) of SPAA-based laforin inhibitors 9a-h^a

ID	R	laforin IC50/μM	ID	R	laforin IC50/μM
9a		0.067±0.009	9e		0.099±0.028
9b		0.020±0.002	9f		0.261±0.083
9c		0.004±0.0003	9g		0.289±0.149
9d		0.078±0.007	9h		> 0.5

^aIC₅₀ values were determined from three independent measurements.

Compound 9c is the most potent and selective laforin inhibitor

Based on the promising improvements in potency and pharmacokinetic properties observed by utilizing cis-1,2-diaminocyclohexane as a linker, we were motivated to further explore the potential of developing even more potent laforin inhibitors. This led us to evaluate additional tail anilines that shared structural similarities with compounds 9a and 9b. As a result, we successfully synthesized compounds 9c-9h using the same synthetic approach as described in Scheme 2. As shown in Table 3, compounds 9c-9g, which featured a flexible distal phenyl ring, exhibited significant inhibitory activity against laforin. However, compound 9h, which possessed fused phenyl rings as the tail fragment, displayed poor potency, with an IC₅₀ value exceeding 0.5 μM. Among these compounds, compound 9c emerged as the most potent laforin inhibitor, showcasing an impressive IC₅₀ value of 4.0 ± 0.3 nM. To exclude the possibility of non-specific laforin aggregation induced by compound 9c, we also conducted an experiment to measure its potency in the presence of the detergent Triton X-100. Similar to the findings for compound 8k, it was observed that the presence of Triton X-100 had no effect on the inhibitory activity of compound 9c against laforin (Supporting Information, Figure S3). The IC₅₀ value of compound 9c remained unchanged at 3.9 ± 0.4 nM in the presence of Triton X-100, indicating that compound 9c does not inhibit laforin activity through nonspecific aggregation.

Scheme 2.

Synthesis of SPAA-based laforin inhibitors 9a-9i^a

^aReaction conditions: (a) DIEA, DCM, r.t., overnight, 73%; (b) corresponding aniline, HOBt, HBTU, DIEA, DMF, r.t. overnight, 62–81%.

We then determined the mode of compound 9c mediated laforin inhibition by steady state kinetic analyses, varying the substrate pNPP and inhibitor concentrations. The Lineweaver Burk plots revealed 9c as a classic competitive inhibitor, affecting the apparent K_m value, while V_max was unchanged (Figure 4A). The calculated K_i value for compound 9c was 1.9 ± 0.2 nM, further confirming its potent inhibitory activity against laforin. This finding is again consistent with the expectation that SPAA-based inhibitors target the PTP active site, due to the known pTyr mimetic properties of the sulfophenyl acetic amide moiety^{42, 49, 54}.

Figure 4:

A) Lineweaver-Burk plot for compound 9c mediated laforin inhibition at three different concentrations (0 nM (●), 1 nM (▲), and 4 nM (■)). pNPP was used as the substrate. The data show that compound 9c inhibits the laforin catalyzed pNPP hydrolysis in a competitive manner (K_i = 1.9 ± 0.2 nM). B) The MST measurement shows the binding of compound 9c to laforin labeled with fluorescent dye (RED-tris-NTA dye). A K_d value of 2.1 ± 0.6 nM for laforin and compound 9c was determined. Data are shown as (mean ± SD) of three independent trials.

To gain a deeper understanding of the binding properties of compound 9c with laforin, we employed MicroScale Thermophoresis (MST), an immobilization-free method used for characterizing biomolecular interactions by measuring the motion of fluorescent molecules along a microscopic temperature gradient^{61, 62}. In this assay, the laforin protein labeled with RED-tris-NTA dye was incubated with varying concentrations of compound 9c (ranging from 0.001 to 100 nM). The binding of a small molecule such as compound 9c to the labeled protein (laforin) alters the chemical microenvironment, resulting in changes in the fluorescence intensity of the probe. Using this approach, we determined a K_d (dissociation constant) value of 2.1 ± 0.6 nM for laforin and compound 9c, based on three independent experiments (Figure 4B). This K_d value determined by MST is similar to the K_i obtained by steady state kinetics (Figure 4A) and provides further confirmation of the highly potent binding affinity of compound 9c for laforin.

To assess the specificity of compound 9c for laforin, we determined its ability to inhibit a panel of 15 mammalian PTPs that cover all major subgroups in the PTP superfamily. This panel included cytosolic PTPs such as SHP2, SHP1, PTP1B, LYP, HePTP, STEP, and TC-PTP, receptor-like PTPs including PTPα, PTPβ, PTPδ, LAR, and CD45, dual specificity phosphatases VHR and YopH, and the low molecular weight PTP. The results, summarized in Table 4, revealed that compound 9c displayed exceptional selectivity for laforin, exhibiting a greater than 8,300-fold selectivity over all other tested PTPs. This remarkable selectivity underscores the specific interaction of compound 9c with laforin and distinguishes it as a highly specific laforin inhibitor.

Table 4.

Selectivity of 9c against a panel of PTPs^a

Enzyme	IC50/μM	Enzyme Class	Enzyme	IC50/μM	Enzyme Class
laforin	0.004±0.0003	Dual Specificity PTPs	SHP2	33.2±5.4	Non-Receptor PTPs
VHR	> 50		SHP1	> 50
YopH	> 50		PTP1B	> 50
PTPα	> 50	Receptor PTPs	LYP	> 50
PTPβ	> 50		HePTP	> 50
PTPδ	> 50		STEP	> 50
LAR	> 50		TC-PTP	> 50
CD45	> 50		LMW-PTP	> 50	LMW PTP

^aIC₅₀ values were determined from three independent measurements.

Since compound 9c is a mixture of two cis-diastereomers ((2S,5R,6S)-9c and (2S,5S,6R)-9c, d.r. = 100:77), we made efforts to separate them and further evaluate their IC₅₀ values against laforin individually. Through normal-phase chromatography, we successfully obtained the pure diastereomers, 9c-diaster1 and 9c-diaster2 (¹H NMR and ¹³C NMR Spectra see pages S26-S27, SI), which exhibited IC₅₀ values of 2.5 ± 0.2 nM and 7.7 ± 0.6 nM, respectively. Both diastereomers demonstrated high potency against laforin within the same nM range, with 9c-diaster1 being approximately three times more potent than 9c-diaster2. These findings are in line with the observed IC₅₀ value (4.0 ± 0.3 nM) for 9c. To the best of our knowledge, compound 9c-diaster1 represents the most potent laforin inhibitor reported to date, further highlighting its significant potential as a chemical probe for laforin.

Molecular modeling of the interactions of compounds 8k and 9c with laforin

To understand the potential interactions between these inhibitors and laforin, molecular modeling was performed using Glide⁶³. For this work, the crystal structure of laforin (PDB ID 4RKK) was selected, as previous studies suggested the requirement of dimerization to maintain laforin activity^{64, 65}. Laforin inhibitors were docked into the active site using Glide in standard precision mode, with up to 10 poses saved per molecule. The resulting poses for each analog could be separated into two distinct groups (Group 1 and 2) (Figure 5A), both of which were taken forward. All docked poses were minimized using the Prime MM-GBSA function in Maestro⁶⁶, and the binding energetics were computed using the OPLS4 force field to determine the calculated binding affinity⁶⁷. The lowest energy pose for each analog was determined for both Group 1 and 2, and these values were plotted against the compound’s pIC₅₀, resulting in a correlation coefficient of 0.663 and 0.258 for Group 1 and 2, respectively (Figure 5B & 5C), giving us greater confidence in the accuracy of the poses observed in Group 1.

Figure 5.

A) Representative predicted docking poses. Most of the compounds presented in this study could be separated into two different groups of docking poses. Representative docking poses for 8k with laforin (grey cartoon, PDB ID: 4RKK) could be separated into Group 1 (green sticks) and Group 2 (pink sticks). B & C) Predicted Δg_bind vs. pIC₅₀ for presented inhibitors vs laforin. Poses were separated into representative Groups 1 and 2.

Figure 6 shows representative poses for two key analogs investigated in this study, namely compounds 8k and 9c ((2S,5R,6S)-diastereomer). These analogs are predicted to bind to the maltohexaose binding site of laforin, as depicted in Figure 6A, which aligns with the experimentally determined competitive mode of inhibition. In the case of compound 9c, extensive hydrogen bonding is predicted between its sulfonic acid moiety and the backbone amide hydrogens of laforin active site residues Ala268, Val270, Gly271, and Arg272 (Figure 5B). Additionally, interaction is anticipated to occur between the sulfonic acid and the guanidine sidechain of Arg272. These hydrogen bonds constitute the majority of specific interactions between compound 9c and laforin. Furthermore, a hydrogen bond is predicted to form between one of the cyclohexyl amide protons and the carboxylate oxygen of Asp235 away from the active site. Moreover, a 𝜋-𝜋 stacking interaction is predicted between the distal phenyl ring of compound 9c and the sidechain of Trp196 in the vicinity of the active site. Finally, a halogen bond is projected to occur between the chlorine atom of 9c and the backbone amide hydrogen of Asp235, as illustrated in Figure 6B. Compound 8k, as depicted in Figure 6C, exhibits many of the same interactions as compound 9c. The sulfonic acid moiety of 8k is predicted to engage in an extensive hydrogen bond network with Ala268, Val270, Gly271, and Arg272, with an additional hydrogen bond forming between the sulfonic acid and the guanidine sidechain of Arg272. Similar to compound 9c, the amide nitrogen attached to the phenyl core of 8k is predicted to establish a hydrogen bond with the sidechain carboxylate of Asp235. However, there are some differences between compound 8k and 9c. The oxamide linker of compound 8k is also projected to form a productive interaction with laforin, with a hydrogen bond predicted to occur with the sidechain carbonyl oxygen of Asp197 adjacent to the active site. Additionally, the terminal phenyl ring of compound 8k is predicted to engage in an edge-face 𝜋-𝜋 interaction with Trp196. These interactions further contribute to the binding of compound 8k to laforin. Interestingly, the predicted pose reveals that the chlorine atom of 8k does not form any specific interactions. This observation aligns with the observed biochemical data, where compound 8k demonstrates much lower potency compared to 9c. Moreover, the phenyl core of 8k and the cyclohexyl core of 9c are not predicted to establish significant interactions with laforin. This finding aligns with the biochemical data, as compound 8k and 9a, which possess a phenyl and cyclohexyl core, respectively, demonstrate a minimal difference in IC₅₀ values, with less than a 2-fold variation. These results indicate that, in most cases, the substitution of a saturated ring with an unsaturated ring does not eliminate any specific protein-ligand interactions. These predicted poses provide a valuable rationale to support the feasibility and effectiveness of the scaffold hopping approach utilized in this study.

Figure 6.

A) Compound 9c ((2S,5R,6S)-diastereomer, green sticks) is predicted to bind to the maltohexaose (cyan sticks) binding groove of laforin (grey cartoon and surface, PDB ID: 4RKK). B & C) 3D interaction diagram of compound 9c ((2S,5R,6S)-diastereomer)/ 8k with laforin. An extensive hydrogen bond network (yellow dashed line) is predicted to occur between the active site of laforin and the sulfonic acid moiety of 9c (green sticks) or 8k (magenta sticks). Additional hydrogen bonds are predicted to form between the oxamide linker of 8k and the side chains of Asn267 and Asp197. A halogen bond interaction (cyan dashed line) is proposed to occur between the chlorine atom of 9c and the backbone amide proton of Asp235. A 𝜋-𝜋 stacking interaction (red dashed line) is proposed to occur between the distal phenyl ring of 9c (green sticks) or 8k (magenta sticks) and the sidechain of Trp196.

To further confirm the accuracy of our structural modeling, we synthesized two additional analogs, 9i and 9j (for the synthesis, see Supplementary Information, Scheme S1), to investigate whether the predicted key interactions truly influence the binding affinity of compound 9c with laforin. The IC₅₀ values of these compounds against laforin are presented in Table 5. Compound 9i, lacking both the distal phenyl ring and chlorine atom substitutions on the aniline core, exhibited an IC₅₀ value of 9.87 ± 1.62 μM. The absence of these moieties led to a 2,450-fold reduction in efficacy, indicating that these interactions play a crucial role in forming key protein-ligand interactions, as supported by the proposed docking model. In contrast, compound 9j, lacking the α-sulfonic acid group that is intended to mimic pTyr, displayed no inhibition against laforin even at 50 μM. These findings strongly support the validity of our docking-based structural modeling.

Table 5.

IC₅₀ value (μM) of laforin inhibitors 9c, 9i, and 9j^a

ID	Structure	Laforin IC50/μM
9c		0.004 ± 0.0003
9i		9.87 ± 1.62
9j		> 50

^aIC₅₀ values were determined from three independent measurements.

To further rationalize the selectivity profile of compound 9c based on our structural modeling, an abbreviated sequence alignment was performed for human laforin and all other PTPs from the selectivity panel. The results, depicted in Figure 7, revealed an interesting finding. The residue Trp196, which participates in the 𝜋-𝜋 stacking interaction with the distal phenyl ring of compound 9c, is unique to laforin and is absent in all other tested PTP enzymes. This indicates that this particular residue likely plays a crucial role in conferring selectivity for our inhibitor. It is worth noting that the other residues involved in hydrogen bonding with the inhibitor scaffold are conserved, particularly the P-loop residues that establish an extensive hydrogen bond network with the sulfonic acid moiety.

Figure 7.

Abbreviated sequence alignment for human laforin and all PTPs from the selectivity panel showing key selectivity residue Trp196 marked with a black arrow. Uniprot accession codes for laforin: O95278; SHP1: P29350; SHP2: Q06124; PTP1B: P18031; LYP: A0A0B4J1S7; HePTP: P35236; STEP: P54829; TCPTP: P17706; PTPα: P18433; PTPβ: P23467; PTPδ: B2GV87; LAR: P10586; CD45: P08575; VHR: P51452; YopH: P08538; LMWPTP: P24666. Key: white block with black text = different residues; red text = similar residues; blue frame = similarity across group of residues. Figure generated using ESPript 3.0.

CONCLUSIONS

Members of the PTP family perform vital tasks in cellular processes such as growth, differentiation, metabolism, migration, and survival¹⁶. Malfunction of PTPs is connected to the occurrence and advancement of a range of human diseases, including cancer, diabetes, autoimmune disorders, and neurodegenerative disorders^68–71. Laforin plays a crucial role in the regulation of glycogen metabolism, its absence results in progressive glycogen hyper-phosphorylation^{22, 24}. Apart from its glycogen phosphatase activity, laforin interacts with malin to form a functional complex where laforin recruits and recognizes substrates that are then ubiquitinated by malin⁴. The absence of either laforin or malin leads to glycogen that is poorly branched and excessively phosphorylated. As a consequence, this abnormal form of glycogen tends to precipitate, aggregate, and accumulate within cells, forming structures known as Lafora bodies which is a hallmark of Lafora disease. Although laforin’s role in removing phosphate from glycogen is well-established, the specific effects of phosphate on normal glycogen homeostasis and how hyper-phosphorylation disrupts glycogen regulation leading to the formation of Lafora bodies are subjects of ongoing investigation. Additionally, the broader role of laforin in other physiological pathways as well as its potential connections to cancer biology and lung fibrosis are evolving. Due to the lack of potent and selective laforin inhibitors, the majority of findings have been based on comparisons between wild-type and laforin-deficient mouse models. As a result, numerous intricate details and underlying mechanisms regarding how laforin functions in normal physiology and in diseases remain elusive. Furthermore, the extent to which the glycogen phosphatase activity of laforin contributes to these processes remains uncertain.

In order to gain deeper insights into its biological function, it is crucial to develop highly potent and selective inhibitors specifically targeting laforin. Although targeting allosteric sites presents a promising alternative for developing selective PTP inhibitors with better pharmacological properties⁷⁰, the discovery of allosteric inhibitors has been serendipitous, and as of now, there is no established method for systematically identifying them at scale. Consequently, there is a compelling need for a more systematic and scalable approach to discovering PTP inhibitors. We have advanced a novel paradigm for the acquisition of potent and selective PTP inhibitors by targeting both the PTP active site and unique pockets in the vicinity of the active site^35–37. These and other efforts have produced many of the most potent and selective PTP inhibitors and degraders available today, some of them even with excellent in vivo efficacy in animal models of oncology and other diseases^{34, 38–48}.

Here we report the development of a novel class of sulfophenyl acetic amide (SPAA)-based inhibitors for laforin. Our approach involved targeting both the laforin active site and peripheral sites near the catalytic pocket using fragment-based and scaffold-hopping strategies. This allowed us to enhance the binding affinity and selectivity of the inhibitors towards laforin. Using this approach, we identified a highly potent and selective laforin inhibitor, 9c, with an IC₅₀ value of 4.0 ± 0.3 nM and over 8,300-fold selectivity over a panel of 15 mammalian PTPs. Kinetic analysis revealed that compound 9c acts as a reversible and competitive inhibitor of laforin, with a K_i value of 1.9 ± 0.2 nM. Modeling compounds into a published structure of laforin offered a solid correlation between the biochemical efficacy and predicted binding affinity, providing insights into the binding mode, potency, and selectivity of laforin inhibition by 8k and 9c. Notably, compound 8k effectively blocked laforin-mediated glucan phosphorylation inside the cell. These findings establish the SPAA-based inhibitors as the first class of truly specific and highly efficacious laforin inhibitors. Moreover, compounds 9a, 9b, and 9c exhibit favorable pharmacokinetic properties in mice. Collectively, these molecular attributes qualify these compounds as chemical probes for laforin because they meet the specific criteria suggested for a small molecule probe^{72, 73}. Given its extremely high potency, selectivity, and favorable pharmacokinetic properties, these well-characterized chemical probes will allow more precise analysis than ever before of the biological roles of laforin in glycogen metabolism, Lafora disease, and other cellular processes. The work also further solidifies SPAA as a privileged non-hydrolyzable phosphotyrosine mimetic for the development of active site-directed, highly potent, and selective inhibitors for the PTPs.

EXPERIMENTAL SECTION

General Synthetic Procedures and Reagents.

Unless otherwise specified, all reagents were purchased from commercial suppliers and used directly without further purification. Analytical thin-layer chromatography (TLC) was performed on 0.25 mm silica gel 60-F₂₅₄. Column chromatography was performed using KP-SIL silica gel (Biotage, USA), and flash column chromatography was performed on Biotage prepacked columns using the automated flash chromatography system Biotage Isolera One. The ¹H and ¹³C NMR spectra were recorded on a Bruker AVANCE 500 MHz instrument. Chemical shifts for Proton magnetic resonance spectra (¹H NMR) were quoted in parts per million (ppm) referenced to the appropriate solvent peak or 0.0 ppm for tetramethylsilane (TMS). The following abbreviations were used to describe peak splitting patterns when appropriate: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multriplet, dd = doublet of doublet. Coupling constants, J, were reported in hertz unit (Hz). Chemical shifts for 13C NMR were reported in ppm referenced to the center line at 39.52 of DMSO-d. HPLC purification was carried out on a Waters Delta 600 equipped with a Sunfire Prep C18 OBD column (30 mm/150 mm, 5 μm) with methanol-water (both containing 0.1% TFA) as mobile phase (gradient: 50–100% methanol, flow 10 mL/min). Low-resolution mass spectra and purity data were obtained using an Agilent Technologies 6470 series, triple quadrupole LC/MS. The purity of all final tested compounds was determined to be >95% (UV, λ = 254 nm). High-resolution mass spectra (HRMS) were recorded on an Agilent Mass spectrometer using ESI-TOF (electrospray ionization-time of flight).

Synthesis of (S)-Sulfophenylacetyl Chloride (2).

To a round-bottom flask was added (S)-2-phenyl-2-sulfoacetic acid (3.0 g, 13.88 mmol) and thionyl chloride (16.51 g, 138.75 mmol), the resulting mixture was stirred at 80 °C overnight. Then, the removal of extra thionyl chloride by rotary evaporator gave the corresponding product (S)-sulfophenylacetyl chloride as a brown oil which was directly used for the next step without purification.

General Procedure for the Synthesis of Compounds 3a-3d.

To the corresponding amine (1.1 mmol) and DIEA (0.54 mL, 3.0 mmol) in DCM (15 mL) was slowly added (S)-sulfophenylacetyl chloride (0.235 g, 1.0 mmol) and DCM (5 ml) solution. The resulting mixture was stirred at r.t. for 12 hours and was monitored by LC/MS. After completion of the reaction, the solvent DCM was completely removed by rotary evaporator. Then the residue was subjected to column chromatography/HPLC for purification, and the corresponding product was obtained as a white or off-white powder (>95% purity).

(S)-2-((3,5-dibromo-4-methylphenyl)amino)-2-oxo-1-phenylethanesulfonic acid (3a).

Off-white powder; (402 mg, 87% yield). ¹H NMR (500 MHz, DMSO) δ 10.44 (s, 1H), 7.89 (s, 2H), 7.59 – 7.58 (m, 2H), 7.28 – 7.26 (m, 3H), 4.78 (s, 1H), 2.43 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 166.5, 139.0, 134.8, 130.6, 130.0, 127.4, 127.0, 124.2, 121.8, 71.6, 22.6. Mass spectra (ESI) m/z: 460.0 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₁₅H₁₂Br₂NO₄S: 459.8854, found: 459.8845.

(S)-2-([1,1’-biphenyl]-4-ylamino)-2-oxo-1-phenylethanesulfonic acid (3b).

White powder; (331 mg, 90% yield). ¹H NMR (500 MHz, CDCl₃) δ 10.4 (s, 1H), 7.69 – 7.58 (m, 8H), 7.43 (t, J = 7.6 Hz, 2H), 7.33 – 7.24 (m, 4H), 4.80 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 166.0, 139.7, 138.6, 135.2, 134.8, 129.9, 128.9, 127.4, 127.0, 127.0, 126.6, 126.2, 119.3, 71.8. Mass spectra (ESI) m/z: 366.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₀H₁₆NO₄S: 366.0800, found: 366.0809.

(S)-2-((4-(1H-imidazol-1-yl)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (3c).

Off-white powder; (311 mg, 87% yield). ¹H NMR (500 MHz, DMSO) δ 10.60 (s, 1H), 9.65 (t, J = 1.3Hz, 1H), 8.26 (t, J = 1.7 Hz, 1H), 7.90 (t, J = 1.6 Hz, 1H), 7.84 – 7.83 (m, 2H), 7.76 – 7.73 (m, 2H), 7.59 – 7.58 (m, 2H), 7.30 – 7.24 (m, 3H), 4.80 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 166.5, 140.2, 135.1, 134.3, 130.0, 129.6, 127.3, 127.0, 122.6, 120.8, 120.7, 119.7, 71.8. Mass spectra (ESI) m/z: 356.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₁₇H₁₄N₃O₄S: 356.0705, found: 356.0715.

(S)-2-((2-([1,1’-biphenyl]-4-ylcarboxamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (3d).

Off-white powder; (428 mg, 88% yield). ¹H NMR (500 MHz, DMSO) δ 10.42 (s, 1H), 9.88 (s, 1H), 8.10 (d, J = 8.5 Hz, 2H), 7.82 (dd, J = 7.9, 1.7 Hz, 1H), 7.79 – 7.74 (m, 4H), 7.63 (dd, J = 7.7, 1.7 Hz, 1H), 7.56 (dd, J = 6.8, 2.8 Hz, 2H), 7.53 – 7.48 (m, 2H), 7.43 (dt, J = 3.7, 1.5 Hz, 1H), 7.25 – 7.18 (m, 5H), 4.72 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 166.94, 165.68, 143.14, 139.39, 135.16, 133.34, 132.69, 130.00, 129.22, 128.78, 128.22, 127.60, 127.08, 127.04, 126.57, 126.35, 126.26, 125.94, 125.83, 124.62, 123.10, 72.22. Mass spectra (ESI) m/z: 485.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₇H₂₁N₂O₅S: 485.1171, found: 485.1171.

Synthesis of (S)-2-((2-aminophenyl)amino)-2-oxo-1-phenylethanesulfonic acid (4).

To o-phenylenediamine (1.50 g, 13.88 mmol) and DIEA (7.41 mL, 41.65 mmol) in DCM (40 mL) was slowly added (S)-phenyl sulfoacetyl chloride (2.98 g, 13.88 mmol) and DCM (20 ml) solution. The resulting mixture was stirred at r.t. for 12 hours and was monitored by LC/MS. After completion of the reaction, the solvent DCM was completely removed by rotary evaporator. Then the residue was subjected to column chromatography for purification, and product (S)-2-((2-aminophenyl)amino)-2-oxo-1-phenylethanesulfonic acid was obtained as off-white solid (2.65 g, 62% yield, >95% purity). ¹H NMR (500 MHz, DMSO) δ 10.22 (s, 1H), 7.60 (d, J = 6.5 Hz, 2H), 7.32 – 7.24 (m, 5H), 7.24 – 7.17 (m, 2H), 4.90 (s, 1H). Mass spectra (ESI) m/z: 307.2 (M + H)⁺.

General Procedure for the Synthesis of Compounds 5a and 5b.

To (S)-2-((2-aminophenyl)amino)-2-oxo-1-phenylethanesulfonic acid (100 mg, 0.33 mmol) and DIEA (0.175 mL, 0.98 mmol) in DMF (10 mL) was added corresponding benzoic acid (0.36 mmol), HOBt (53 mg, 0.39 mmol) and HBTU (149 mg, 0.39 mmol). The resulting mixture was stirred at r.t. overnight and was monitored by LC/MS. After completion of the reaction, the solvent DMF was completely removed by rotary evaporator. Then the residue was subjected to column chromatography/HPLC for purification, and the corresponding product was obtained as an off-white powder (>95% purity).

(S)-2-((2-benzamidophenyl)amino)-2-oxo-1-phenylethanesulfonic acid (5a).

Off-white powder; (88 mg, 90% yield). ¹H NMR (500 MHz, DMSO) δ 10.38 (s, 1H), 9.82 (s, 1H), 8.00 (dd, J = 5.1, 3.3 Hz, 2H), 7.79 (dd, J = 7.9, 1.7 Hz, 1H), 7.61 (dd, J = 7.7, 1.6 Hz, 1H), 7.59 – 7.52 (m, 3H), 7.48 (t, J = 7.6 Hz, 2H), 7.25 – 7.15 (m, 5H), 4.71 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 166.94, 165.99, 135.13, 134.49, 132.57, 131.62, 129.96, 129.93, 128.37, 128.03, 127.58, 127.08, 126.31, 125.88, 124.64, 123.19, 72.18. Mass spectra (ESI) m/z: 408.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₁H₁₇N₂O₅S: 409.0858, found: 409.0867.

(S)-2-oxo-2-((2-(4-phenoxybenzamido)phenyl)amino)-1-phenylethanesulfonic acid (5b).

Off-white powder; (78 mg, 65% yield). ¹H NMR (500 MHz, DMSO) δ 10.39 (s, 1H), 9.72 (s, 1H), 8.02 (d, J = 8.8 Hz, 2H), 7.74 (dd, J = 7.6, 2.0 Hz, 1H), 7.65 (dd, J = 7.5, 2.0 Hz, 1H), 7.54 (dd, J = 6.5, 3.1 Hz, 2H), 7.50 – 7.44 (m, 2H), 7.27 – 7.17 (m, 6H), 7.15 – 7.11 (m, 2H), 7.03 – 6.97 (m, 2H), 4.72 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 166.93, 165.02, 159.94, 155.55, 135.09, 132.23, 130.38, 130.24, 130.15, 129.94, 129.01, 127.47, 126.97, 125.97, 125.62, 124.64, 124.53, 123.27, 119.86, 117.12, 72.04. Mass spectra (ESI) m/z: 501.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₇H₂₁N₂O₆S: 501.1120, found: 501.1110.

Synthesis of 2-((2-aminophenyl)amino)-2-oxoacetic acid (6).

To benzene-1,2-diamine (1.0 g, 9.25 mmol) and DIEA (4.8 mL, 27.74 mmol) in DCM (200 mL) was slowly added methyl chlorooxoacetate (1.13 g, 1.80 mmol). The resulting mixture was stirred at r.t. for 1 hour and then was concentrated by rotary evaporator. The mixture was purified by column chromatography eluting with dichloromethane/methanol 10:1 v/v to give methyl 2-((2-aminophenyl)amino)-2-oxoacetate (1.15 g, 64% yield). To a solution of methyl 2-((2-aminophenyl)amino)-2-oxoacetate (500 mg, 2.57 mmol) in methanol (40 ml) and H₂O (40 ml), KOH (1.16 g, 20.6 mmol) was added. The obtained mixture was stirred at r.t. for 1 hour. The mixture was brought to 0°C, and carefully acidified with 1N HCl until pH ~5 to furnish product 2-((2-aminophenyl)amino)-2-oxoacetic acid. Subsequently, it was purified by column chromatography, and the corresponding product was obtained as a white solid (351 mg, 75% yield). Mass spectra (ESI) m/z: 179.1 (M - H)⁻.

Synthesis of (S)-2-oxo-2-((2-(2-phenyl-2-sulfoacetamido)phenyl)amino)acetic acid (7).

To compound 6 (384 mg, 2.13 mmol) and DIEA (1.10 mL, 6.39 mmol) in DCM (30 mL) was slowly added (S)-sulfophenylacetyl chloride (0.5 g, 2.13 mmol) and DCM (10 ml) solution. The resulting mixture was stirred at r.t. for 12 hours and was monitored by LC/MS. After completion of the reaction, the solvent DCM was completely removed by rotary evaporator. Then the residue was subjected to the column for purification, and the corresponding product was obtained as a colorless oil (581 mg, 72% yield, >95% purity). Mass spectra (ESI) m/z: 377.1 (M - H)⁻.

General Procedure for the Synthesis of Compounds 8a-8n.

(S)-2-oxo-2-((2-(2-phenyl-2-sulfoacetamido)phenyl)amino)acetic acid (7) (100 mg, 0.26 mmol), HOBt (43 mg, 0.31 mmol) and HBTU (120 mg, 0.31 mmol) were dissolved in dry DMF (10 ml). The mixture was stirred at room temperature for 15 minutes. Then the corresponding aniline (0.29 mmol) and DIEA (0.14 ml, 0.79 mmol) were added, and the resulting mixture was stirred at room temperature overnight and monitored by LC/MS. After completion of the reaction, DMF was removed by a rotary evaporator, the residue was subjected to column chromatography/HPLC for purification, and the corresponding product was obtained as a white or off-white powder (>95% purity).

(S)-2-oxo-2-((2-(2-oxo-2-(o-tolylamino)acetamido)phenyl)amino)-1-phenylethanesulfonic acid (8a).

Off-white powder; (89 mg, 72% yield). ¹H NMR (500 MHz, DMSO) δ 10.49 (s, 1H), 10.25 (s, 1H), 10.04 (s, 1H), 7.82 (dd, J = 8.1, 1.3 Hz, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.55 (dd, J = 8.0, 1.5 Hz, 2H), 7.47 (dd, J = 7.9, 1.4 Hz, 1H), 7.28 – 7.22 (m, 6H), 7.21 – 7.13 (m, 2H), 4.67 (s, 1H), 2.25 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 166.90, 159.58, 158.36, 141.90, 137.42, 135.28, 134.56, 132.06, 131.84, 130.55, 129.91, 127.63, 126.33, 125.98, 124.67, 124.26, 123.13, 118.24, 72.20, 17.70. Mass spectra (ESI) m/z: 466.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₃H₂₀N₃O₆S: 466.1073, found: 466.1081.

(S)-2-oxo-2-((2-(2-oxo-2-((4-phenoxyphenyl)amino)acetamido)phenyl)amino)-1-phenylethanesulfonic acid (8b).

Off-white powder; (81 mg, 56% yield). ¹H NMR (500 MHz, DMSO) δ 10.78 (s, 1H), 10.44 (s, 1H), 10.26 (s, 1H), 7.90 (d, J = 9.0 Hz, 2H), 7.80 (dd, J = 8.1, 1.2 Hz, 1H), 7.56 (d, J = 7.0 Hz, 2H), 7.51 (dd, J = 7.9, 1.2 Hz, 1H), 7.39 (dd, J = 8.5, 7.5 Hz, 2H), 7.31 – 7.20 (m, 5H), 7.16 – 7.10 (m, 1H), 7.06 (d, J = 9.0 Hz, 2H), 7.04 – 6.99 (m, 2H), 4.73 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 166.91, 159.35, 158.17, 157.20, 152.86, 135.15, 133.64, 132.89, 130.09, 129.90, 128.42, 127.56, 126.96, 126.71, 126.20, 124.72, 123.23, 122.18, 119.32, 118.17, 71.98. Mass spectra (ESI) m/z: 544.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₈H₂₂N₃O₇S: 544.1178, found: 544.1185.

(S)-2-((2-(2-([1,1’-biphenyl]-4-ylamino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (8c).

White powder; (105 mg, 75% yield). ¹H NMR (500 MHz, DMSO) δ 10.80 (s, 1H), 10.45 (s, 1H), 10.23 (s, 1H), 7.96 (d, J = 8.7 Hz, 2H), 7.79 (dd, J = 8.1, 1.1 Hz, 1H), 7.73 – 7.65 (m, 4H), 7.56 (d, J = 7.1 Hz, 2H), 7.50 (d, J = 7.7 Hz, 1H), 7.46 (t, J = 7.7 Hz, 2H), 7.35 (t, J = 7.4 Hz, 1H), 7.30 – 7.18 (m, 5H), 4.72 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 166.94, 159.39, 158.47, 139.69, 137.29, 136.25, 135.19, 132.94, 129.94, 129.05, 128.50, 127.61, 127.35, 127.03, 126.78, 126.53, 126.27, 124.81, 123.42, 120.87, 72.05. Mass spectra (ESI) m/z: 528.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₈H₂₂N₃O₆S: 528.1229, found: 528.1236.

(S)-2-((2-(2-((4-chlorophenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (8d).

White powder; (85 mg, 66% yield). ¹H NMR (500 MHz, DMSO) δ 10.85 (s, 1H), 10.42 (s, 1H), 10.21 (s, 1H), 7.89 (d, J = 8.9 Hz, 2H), 7.79 (dd, J = 8.1, 1.2 Hz, 1H), 7.54 (d, J = 6.9 Hz, 2H), 7.47 (dd, J = 7.9, 1.3 Hz, 1H), 7.44 (d, J = 8.9 Hz, 2H), 7.30 – 7.18 (m, 5H), 4.70 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 166.86, 159.23, 158.54, 136.86, 135.18, 133.05, 129.93, 129.43, 128.77, 128.36, 127.60, 127.02, 124.74, 123.29, 122.08, 121.43, 72.06. Mass spectra (ESI) m/z: 486.1 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₂H₁₇ClN₃O₆S: 486.0527, found: 486.0533.

(S)-2-((2-(2-((3-chloro-4-fluorophenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (8e).

Off-white powder; (92 mg, 69% yield). ¹H NMR (500 MHz, DMSO) δ 10.48 (s, 1H), 10.25 (s, 1H), 10.03 (s, 1H), 7.82 (dd, J = 8.1, 1.3 Hz, 1H), 7.59 (d, J = 7.3 Hz, 1H), 7.54 (dd, J = 8.0, 1.4 Hz, 2H), 7.47 (dd, J = 7.9, 1.4 Hz, 1H), 7.28 – 7.23 (m, 5H), 7.21 – 7.13 (m, 2H), 4.66 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 166.88, 159.56, 158.34, 135.27, 133.09, 131.98, 130.53, 129.89, 127.71, 127.61, 127.01, 126.30, 125.96, 124.65, 124.22, 123.11, 120.14, 118.22, 72.20. Mass spectra (ESI) m/z: 504.1 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₂H₁₆ClFN₃O₆S: 504.0432, found: 504.0441.

(S)-2-((2-(2-((4-fluoro-3-(methoxycarbonyl)phenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (8f).

White powder; (85 mg, 61% yield). ¹H NMR (500 MHz, DMSO) δ 11.01 (s, 1H), 10.41 (s, 1H), 10.25 (s, 1H), 8.53 (dd, J = 6.5, 2.8 Hz, 1H), 8.09 – 8.02 (m, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 7.2 Hz, 2H), 7.49 (d, J = 7.9 Hz, 1H), 7.38 (dd, J = 10.5, 9.1 Hz, 1H), 7.29 – 7.18 (m, 5H), 4.70 (s, 1H), 3.88 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 166.95, 159.02, 158.56, 156.56, 135.17, 134.29, 132.98, 129.93, 128.38, 127.60, 127.12, 127.02, 126.87, 126.33, 124.80, 123.29, 123.22, 118.22, 117.66, 72.01, 52.61. Mass spectra (ESI) m/z: 528.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₄H₁₉FN₃O₈S: 528.0877, found: 528.0869.

(S)-2-((2-(2-((3-ethynylphenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (8g).

White powder; (79 mg, 63% yield). ¹H NMR (500 MHz, DMSO) δ 10.82 (s, 1H), 10.42 (s, 1H), 10.23 (s, 1H), 8.00 (t, J = 1.7 Hz, 1H), 7.88 (ddd, J = 8.3, 2.1, 0.9 Hz, 1H), 7.78 (dd, J = 8.1, 1.3 Hz, 1H), 7.57 – 7.52 (m, 2H), 7.48 (dd, J = 7.9, 1.4 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.31 – 7.17 (m, 6H), 4.71 (s, 1H), 4.20 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 167.28, 167.26, 159.52, 159.01, 138.44, 135.51, 133.30, 130.27, 129.67, 128.75, 128.19, 127.94, 127.88, 127.36, 127.19, 126.65, 125.15, 123.68, 123.63, 122.50, 121.50, 83.73, 81.28, 72.34. Mass spectra (ESI) m/z: 476.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₄H₁₈N₃O₆S: 476.0916, found: 476.0925.

(S)-2-oxo-2-((2-(2-oxo-2-((4-propylphenyl)amino)acetamido)phenyl)amino)-1-phenylethanesulfonic acid (8h).

White powder; (68 mg, 52% yield). ¹H NMR (500 MHz, DMSO) δ 10.55 (s, 1H), 10.35 (s, 1H), 10.16 (s, 1H), 7.74 – 7.67 (m, 3H), 7.50 (d, J = 7.0 Hz, 2H), 7.45 (dd, J = 7.9, 1.3 Hz, 1H), 7.26 – 7.12 (m, 7H), 4.67 (s, 1H), 2.45 (dt, J = 3.5, 1.7 Hz, 2H), 1.58 – 1.47 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 166.90, 159.50, 158.22, 142.10, 138.49, 135.16, 132.83, 129.73, 128.64, 127.61, 127.03, 126.18, 124.84, 123.49, 122.79, 120.43, 72.02, 36.69, 24.12, 13.63. Mass spectra (ESI) m/z: 494.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₅H₂₄N₃O₆S: 494.1386, found: 494.1380.

(S)-2-((2-(2-((4-(1H-imidazol-1-yl)phenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (8i).

Off-white powder; (82 mg, 60% yield). ¹H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 10.47 (s, 1H), 10.28 (s, 1H), 9.77 (s, 1H), 8.31 – 8.27 (m, 1H), 8.09 (d, J = 9.0 Hz, 2H), 7.93 – 7.91 (m, 1H), 7.85 (d, J = 9.0 Hz, 2H), 7.56 (d, J = 7.0 Hz, 2H), 7.49 (d, J = 6.9 Hz, 1H), 7.29 – 7.17 (m, 6H), 4.77 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 166.94, 159.09, 158.79, 139.35, 138.87, 135.08, 132.91, 130.99, 130.55, 129.93, 128.40, 127.63, 127.08, 124.89, 123.68, 122.69, 121.45, 120.99, 118.24, 71.87. Mass spectra (ESI) m/z: 518.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₅H₂₀N₅O₆S: 518.1134, found: 518.1125.

(S)-2-((2-(2-((4-(benzyloxy)phenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (8j).

White powder; (101 mg, 68% yield). ¹H NMR (500 MHz, DMSO) δ 10.62 (s, 1H), 10.38 (s, 1H), 10.23 (s, 1H), 7.77 (d, J = 9.1 Hz, 2H), 7.59 – 7.53 (m, 2H), 7.51 (d, J = 7.2 Hz, 1H), 7.45 (t, J = 7.5 Hz, 3H), 7.41 – 7.37 (m, 2H), 7.34 – 7.19 (m, 6H), 7.03 (d, J = 9.1 Hz, 2H), 5.10 (s, 2H), 4.74 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 167.06, 159.44, 157.95, 155.28, 137.21, 135.09, 132.62, 131.10, 129.91, 128.07, 127.96, 127.87, 127.63, 127.07, 126.07, 124.37, 123.63, 121.94, 114.93, 71.87, 69.51. Mass spectra (ESI) m/z: 558.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₉H₂₄N₃O₇S: 558.1335, found: 558.1342.

(S)-2-((2-(2-((4-(benzyloxy)-3-chlorophenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (8k).

White powder; (96 mg, 61% yield). ¹H NMR (500 MHz, DMSO) δ 10.79 (s, 1H), 10.39 (s, 1H), 10.24 (s, 1H), 8.03 (d, J = 2.5 Hz, 1H), 7.77 (dd, J = 9.0, 2.3 Hz, 2H), 7.55 (d, J = 7.0 Hz, 2H), 7.50 – 7.46 (m, 3H), 7.41 (t, J = 6.6 Hz, 2H), 7.35 (d, J = 7.3 Hz, 1H), 7.28 – 7.19 (m, 6H), 5.21 (s, 2H), 4.72 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 166.97, 159.16, 158.19, 150.45, 136.70, 135.16, 132.85, 131.78, 129.92, 128.60, 128.48, 128.06, 127.66, 127.57, 126.99, 126.76, 126.22, 124.80, 123.37, 122.01, 121.30, 120.30, 114.53, 71.94, 70.32. Mass spectra (ESI) m/z: 592.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₉H₂₃ClN₃O₇S: 592.0945, found: 592.0951.

(S)-2-((2-(2-((3-benzylphenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (8l).

White powder; (78 mg, 54% yield). ¹H NMR (500 MHz, DMSO) δ 10.63 (s, 1H), 10.39 (s, 1H), 10.22 (s, 1H), 7.80 (s, 1H), 7.76 (dd, J = 8.1, 1.3 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.59 – 7.53 (m, 2H), 7.49 (dd, J = 7.9, 1.2 Hz, 1H), 7.34 – 7.22 (m, 9H), 7.21 – 7.17 (m, 2H), 7.03 (d, J = 7.6 Hz, 1H), 4.72 (s, 1H), 3.95 (s, 2H). ¹³C NMR (126 MHz, DMSO) δ 167.35, 159.66, 158.68, 142.29, 141.49, 138.23, 135.51, 133.13, 130.26, 129.18, 128.91, 127.93, 127.33, 127.04, 126.48, 125.41, 125.16, 123.83, 121.18, 118.63, 72.30, 41.72. Mass spectra (ESI) m/z: 542.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₉H₂₄N₃O₆S: 542.1368, found: 542.1359.

(S)-2-((2-(2-((3-chloro-4-phenoxyphenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (8m).

White powder; (110 mg, 72% yield). ¹H NMR (500 MHz, DMSO) δ 10.97 (s, 1H), 10.45 (s, 1H), 10.28 (s, 1H), 8.18 (d, J = 2.5 Hz, 1H), 7.88 (dd, J = 8.9, 2.5 Hz, 1H), 7.81 (dd, J = 8.1, 1.2 Hz, 1H), 7.58 – 7.53 (m, 2H), 7.49 (d, J = 7.9 Hz, 1H), 7.37 (dd, J = 8.7, 7.4 Hz, 2H), 7.29 – 7.20 (m, 5H), 7.20 – 7.16 (m, 1H), 7.14 – 7.08 (m, 1H), 6.97 – 6.92 (m, 2H), 4.72 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 166.93, 159.02, 158.48, 157.04, 147.51, 135.23, 135.15, 133.02, 130.13, 129.90, 128.25, 127.56, 126.97, 126.84, 126.34, 124.97, 124.69, 123.17, 122.13, 122.10, 120.83, 116.87, 71.96. Mass spectra (ESI) m/z: 578.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₈H₂₁ClN₃O₇S: 578.0789, found: 578.0781.

(S)-2-((2-(2-((3-chloro-4-(3,5-difluorophenoxy)phenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonic acid (8n).

White powder; (112 mg, 69% yield). ¹H NMR (500 MHz, DMSO) δ 11.05 (s, 1H), 10.46 (s, 1H), 10.29 (s, 1H), 8.23 (d, J = 2.5 Hz, 1H), 7.96 (dd, J = 8.8, 2.1 Hz, 1H), 7.85 – 7.80 (m, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.9 Hz, 1H), 7.27 – 7.18 (m, 5H), 7.00 – 6.96 (m, 1H), 6.74 – 6.70 (m, 2H), 4.73 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 167.33, 164.61, 162.65, 159.62, 158.95, 146.37, 136.71, 135.50, 133.38, 130.27, 128.62, 127.95, 127.36, 126.73, 125.71, 125.10, 123.39, 122.68, 120.71, 102.01, 101.13, 99.10, 72.28. Mass spectra (ESI) m/z: 614.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₈H₁₉ClF₂N₃O₇S: 614.0600, found: 614.0608.

Synthesis of 2-((2-aminocyclohexyl)amino)-2-oxoacetic acid (6a).

To cis-cyclohexane-1,2-diamine (1.0 g, 8.76 mmol) and DIEA (4.54 mL, 26.27 mmol) in DCM (200 mL) were slowly added methyl chlorooxoacetate (1.07 g, 8.76 mmol). The resulting mixture was stirred at r.t. for 1 hour and then was concentrated by rotary evaporator. The mixture was purified by column chromatography eluting with dichloromethane/methanol 10:1 v/v to give methyl 2-((2-aminocyclohexyl)amino)-2-oxoacetate (1.03 g, 59% yield). To a solution methyl 2-((2-aminocyclohexyl)amino)-2-oxoacetate (500 mg, 2.5 mmol) in methanol (40 ml) and H₂O (40 ml), KOH (1.12 g, 19.98 mmol) was added. The obtained mixture was stirred at r.t. for 1 hour. The mixture was brought to 0°C, and carefully acidified with 1N HCl until pH ~5 to furnish product 2-((2-aminocyclohexyl)amino)-2-oxoacetic acid. Subsequently, it was purified by column chromatography, and the corresponding product was obtained as white solid (325 mg, 70% yield). Mass spectra (ESI) m/z: 185.1 (M - H)⁻.

Synthesis of (S)-2-oxo-2-((2-(2-phenyl-2-sulfoacetamido)cyclohexyl)amino)acetic acid (7a).

To compound 6a (397 mg, 2.13 mmol) and DIEA (1.10 mL, 6.39 mmol) in DCM (30 mL) was slowly added (S)-sulfophenylacetyl chloride (0.5 g, 2.13 mmol) and DCM (10 ml) solution. The resulting mixture was stirred at r.t. for 12 hours and was monitored by LC/MS. After completion of the reaction, the solvent DCM was completely removed by rotary evaporator. Then the residue was subjected to the column for purification, and the corresponding product was obtained as a colorless oil (596 mg, 73% yield, >95% purity). Mass spectra (ESI) m/z: 383.2 (M - H)⁻.

General Procedure for the Synthesis of Compounds 9a-9i.

(S)-2-oxo-2-((2-(2-phenyl-2-sulfoacetamido)cyclohexyl)amino)acetic acid (7a) (100 mg, 0.26 mmol), HOBt (43 mg, 0.31 mmol) and HBTU (120 mg, 0.31 mmol) were dissolved in dry DMF (10 ml). The mixture was stirred at room temperature for 15 minutes. Then the corresponding aniline (0.29 mmol) and DIEA (0.14 ml, 0.79 mmol) were added, and the resulting mixture was stirred at room temperature overnight and was monitored by LC/MS. After completion of the reaction, DMF was removed by a rotary evaporator, the residue was subjected to column chromatography/HPLC for purification, and the corresponding product was obtained as a white or off-white powder (>95% purity).

(S)-2-((2-(2-((4-(benzyloxy)-3-chlorophenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid (9a), a mixture of two cis-diastereomers:

(S)-2-(((1R,2S)-2-(2-((4-(benzyloxy)-3-chlorophenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5R,6S)-9a) and (S)-2-(((1S,2R)-2-(2-((4-(benzyloxy)-3-chlorophenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5S,6R)-9a). White powder; (111 mg, 71% yield); dr value (100: 12). ¹H NMR (500 MHz, DMSO) δ 10.66 (s, 1H), 8.71 (d, J = 8.5 Hz, 1H), 8.37 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 2.6 Hz, 1H), 7.75 (dd, J = 9.0, 2.6 Hz, 1H), 7.48 (d, J = 7.1 Hz, 2H), 7.44 – 7.39 (m, 4H), 7.37 – 7.32 (m, 1H), 7.25 (d, J = 9.1 Hz, 1H), 7.20 – 7.12 (m, 3H), 5.20 (s, 2H), 4.50 (s, 1H), 4.20 – 4.13 (m, 1H), 3.95 – 3.87 (m, 1H), 1.79 – 1.61 (m, 4H), 1.60 – 1.33 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 168.06, 159.47, 158.30, 150.36, 136.69, 135.53, 131.73, 129.92, 128.56, 128.02, 127.63, 127.39, 126.67, 121.99, 121.22, 120.23, 114.45, 71.66, 70.26, 50.42, 47.05, 28.71, 26.79, 23.32, 20.32. ¹H NMR (500 MHz, DMSO) δ 10.64 (s, 1H), 8.71 (d, J = 8.5 Hz, 1H), 8.32 (d, J = 8.6 Hz, 1H), 7.98 (d, J = 2.6 Hz, 1H), 7.72 (dd, J = 9.0, 2.6 Hz, 1H), 7.48 (d, J = 7.1 Hz, 2H), 7.44 – 7.39 (m, 4H), 7.37 – 7.32 (m, 1H), 7.25 (d, J = 9.1 Hz, 1H), 7.20 – 7.12 (m, 3H), 5.19 (s, 2H), 4.48 (s, 1H), 4.20 – 4.13 (m, 1H), 3.95 – 3.87 (m, 1H), 1.79 – 1.61 (m, 4H), 1.60 – 1.33 (m, 4H). Mass spectra (ESI) m/z: 598.3 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₉H₂₉ClN₃O₇S: 598.1415, found: 598.1408.

(S)-2-((2-(2-((3-benzylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid (9b), a mixture of two cis-diastereomers:

(S)-2-(((1R,2S)-2-(2-((3-benzylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5R,6S)-9b) and (S)-2-(((1S,2R)-2-(2-((3-benzylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5S,6R)-9b). Off-white powder; (103 mg, 72% yield); dr value (100: 76). ¹H NMR (500 MHz, DMSO) δ 10.49 (s, 1H), 8.70 (s, 1H), 8.35 (d, J = 7.9 Hz, 1H), 7.78 (s, 1H), 7.59 (t, J = 9.1 Hz, 1H), 7.44 – 7.39 (m, 2H), 7.32 – 7.06 (m, 9H), 7.02 (d, J = 7.6 Hz, 1H), 4.45 (s, 1H), 4.18 – 4.11 (m, 1H), 3.93 (s, 3H), 1.79 – 1.60 (m, 4H), 1.59 – 1.34 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 167.98, 159.57, 158.38, 141.86, 141.13, 137.84, 135.36, 129.84, 129.63, 128.76, 128.52, 127.43, 126.66, 126.09, 124.97, 120.75, 118.17, 71.81, 53.69, 47.11, 41.36, 28.67, 26.51, 23.16, 20.38. ¹H NMR (500 MHz, DMSO) δ 10.45 (s, 1H), 8.69 (s, 1H), 8.31 (d, J = 8.5 Hz, 1H), 7.76 (s, 1H), 7.59 (t, J = 9.1 Hz, 1H), 7.44 – 7.39 (m, 2H), 7.32 – 7.06 (m, 9H), 6.99 (d, J = 7.6 Hz, 1H), 4.48 (s, 1H), 4.18 – 4.11 (m, 1H), 3.91 (s, 3H), 1.79 – 1.60 (m, 4H), 1.59 – 1.34 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 167.79, 159.51, 158.27, 141.86, 141.13, 137.84, 135.53, 129.95, 129.68, 128.76, 128.52, 127.57, 126.86, 126.09, 124.97, 120.75, 118.25, 71.53, 50.27, 45.92, 41.94, 28.46, 26.82, 23.20, 20.69. Mass spectra (ESI) m/z: 548.3 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₉H₃₀N₃O₆S: 548.1855, found: 548.1865.

(S)-2-((2-(2-((3-chloro-4-phenoxyphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid (9c), a mixture of two cis-diastereomers:

(S)-2-(((1R,2S)-2-(2-((3-chloro-4-phenoxyphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethane-1-sulfonic acid ((2S,5R,6S)-9c) and (S)-2-(((1S,2R)-2-(2-((3-chloro-4-phenoxyphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethane-1-sulfonic acid ((2S,5S,6R)-9c). Off-white powder; (109 mg, 72% yield); dr value (100: 77). ¹H NMR (500 MHz, DMSO) δ 10.83 (s, 1H), 8.71 (d, J = 8.6 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 2.5 Hz, 1H), 7.83 (dd, J = 9.0, 2.6 Hz, 1H), 7.45 – 7.40 (m, 2H), 7.39 – 7.34 (m, 2H), 7.28 – 7.10 (m, 5H), 6.95 – 6.90 (m, 2H), 4.50 (s, 1H), 4.21 – 4.14 (m, 1H), 3.94 – 3.88 (m, 1H), 1.76 – 1.64 (m, 4H), 1.61 – 1.33 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 168.06, 159.44, 158.68, 157.05, 147.52, 135.55, 135.23, 130.17, 129.99, 127.44, 126.96, 124.93, 123.26, 122.16, 122.04, 120.84, 116.93, 71.72, 50.51, 47.05, 29.25, 26.80, 23.35, 20.35. ¹H NMR (500 MHz, DMSO) δ 10.81 (s, 1H), 8.71 (d, J = 8.6 Hz, 1H), 8.36 (d, J = 8.5 Hz, 1H), 8.12 (d, J = 2.5 Hz, 1H), 7.80 (dd, J = 9.3, 2.9 Hz, 1H), 7.45 – 7.40 (m, 2H), 7.39 – 7.34 (m, 2H), 7.28 – 7.10 (m, 5H), 6.95 – 6.90 (m, 2H), 4.48 (s, 1H), 4.21 – 4.14 (m, 1H), 3.94 – 3.88 (m, 1H), 1.76 – 1.64 (m, 4H), 1.61 – 1.33 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 167.96, 159.30, 158.56, 157.05, 147.47, 135.65, 135.30, 130.17, 129.64, 127.66, 126.73, 124.93, 123.26, 122.16, 122.09, 120.80, 116.93, 71.60, 50.46, 47.05, 28.76, 26.67, 23.31, 20.12. Mass spectra (ESI) m/z: 584.2 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₈H₂₇ClN₃O₇S: 584.1258, found: 584.1266.

9c-diaster1:

¹H NMR (500 MHz, DMSO) δ 10.83 (s, 1H), 8.72 (d, J = 9.0 Hz, 1H), 8.36 (d, J = 8.6 Hz, 1H), 8.13 (d, J = 2.5 Hz, 1H), 7.81 (dd, J = 9.0, 2.5 Hz, 1H), 7.43 (d, J = 6.9 Hz, 2H), 7.40 – 7.34 (m, 2H), 7.29 – 7.24 (m, 2H), 7.22 – 7.15 (m, 1H), 7.15 – 7.08 (m, 2H), 6.92 (d, J = 7.8 Hz, 2H), 4.48 (s, 1H), 4.19 – 4.15 (m, 1H), 3.94 – 3.89 (m, 1H), 1.76 – 1.70 (m, 2H), 1.68 – 1.63 (m, 2H), 1.56 – 1.37 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 167.93, 159.25, 158.51, 157.01, 147.41, 135.31, 135.21, 130.11, 129.60, 127.59, 126.88, 124.87, 123.18, 122.03, 121.97, 120.74, 116.87, 71.56, 50.81, 46.99, 29.21, 26.43, 23.74, 20.44.

9c-diaster2:

¹H NMR (500 MHz, DMSO) δ 10.84 (s, 1H), 8.71 (d, J = 8.5 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 2.5 Hz, 1H), 7.83 (dd, J = 8.9, 2.5 Hz, 1H), 7.44 (dd, J = 7.8, 1.5 Hz, 2H), 7.40 – 7.33 (m, 2H), 7.20 – 7.15 (m, 2H), 7.13 – 7.06 (m, 1H), 6.93 (dd, J = 8.6, 0.9 Hz, 2H), 4.51 (s, 1H), 4.22 – 4.16 (m, 1H), 3.96 – 3.89 (m, 1H), 1.75 – 1.66 (m, 2H), 1.63 – 1.55 (m, 2H), 1.51 – 1.35 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 168.06, 159.40, 158.65, 157.16, 147.47, 135.55, 135.21, 130.13, 129.96, 127.39, 126.68, 124.88, 123.21, 122.12, 122.01, 120.79, 116.89, 71.65, 50.49, 47.03, 28.72, 26.75, 23.31, 20.15.

(S)-2-oxo-2-((2-(2-oxo-2-((4-phenoxyphenyl)amino)acetamido)cyclohexyl)amino)-1-phenylethanesulfonic acid (9d), a mixture of two cis-diastereomers:

(S)-2-(((1R,2S)-2-oxo-2-((2-(2-oxo-2-((4-phenoxyphenyl)amino)acetamido)cyclohexyl)amino)-1-phenylethanesulfonic acid ((2S,5R,6S)-9d) and (S)-2-(((1S,2R)-2-oxo-2-((2-(2-oxo-2-((4-phenoxyphenyl)amino)acetamido)cyclohexyl)amino)-1-phenylethanesulfonic acid ((2S,5S,6R)-9d). Brown powder; (89 mg, 62% yield); dr value (100: 96). ¹H NMR (500 MHz, DMSO) δ 10.62 (s, 1H), 8.70 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 9.1 Hz, 2H), 7.45 – 7.41 (m, 2H), 7.39 – 7.35 (m, 2H), 7.27 – 7.08 (m, 4H), 7.03 – 6.96 (m, 4H), 4.48 (s, 1H), 4.20 – 4.11 (m, 1H), 3.93 – 4.11 (m, 1H), 1.78 – 1.60 (m, 4H), 1.59 – 1.34 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 168.05, 159.69, 158.32, 157.20, 152.92, 135.53, 133.61, 130.14, 129.90, 127.63, 126.94, 123.33, 122.23, 119.25, 118.28, 71.80, 50.73, 47.17, 29.26, 26.92, 23.76, 20.49. ¹H NMR (500 MHz, DMSO) δ 10.59 (s, 1H), 8.69 (s, 1H), 8.33 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 9.1 Hz, 2H), 7.45 – 7.41 (m, 2H), 7.39 – 7.35 (m, 2H), 7.27 – 7.08 (m, 4H), 7.03 – 6.96 (m, 4H), 4.47 (s, 1H), 4.20 – 4.11 (m, 1H), 3.93 – 4.11 (m, 1H), 1.78 – 1.60 (m, 4H), 1.59 – 1.34 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 167.93, 159.62, 158.23, 157.20, 152.85, 135.36, 133.61, 130.14, 129.68, 127.51, 126.77, 123.33, 122.17, 119.25, 118.24, 71.55, 50.36, 47.11, 28.74, 26.52, 23.31, 20.15. Mass spectra (ESI) m/z: 550.3 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₈H₂₈N₃O₇S: 550.1648, found: 550.1641.

(S)-2-((2-(2-((4-benzylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid (9e), a mixture of two cis-diastereomers:

(S)-2-(((1R,2S)-2-((2-(2-((4-benzylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5R,6S)-9e) and (S)-2-(((1S,2R)-2-((2-(2-((4-benzylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5S,6R)-9e). Off-white powder; (96 mg, 67% yield); dr value (100: 71). ¹H NMR (500 MHz, DMSO) δ 10.52 (s, 1H), 8.66 (d, J = 5.3 Hz, 1H), 8.37 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.43 (t, J = 6.4 Hz, 2H), 7.31 – 7.13 (m, 10H), 4.53 (s, 1H), 4.17 – 4.11 (m, 1H), 3.91 (s, 3H), 1.76 – 1.57 (m, 4H), 1.56 – 1.31 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 168.25, 159.66, 158.35, 141.44, 137.61, 135.71, 135.41, 129.85, 129.00, 128.76, 128.55, 127.63, 126.95, 126.07, 120.68, 71.55, 50.66, 47.29, 40.68, 29.20, 26.90, 23.49, 20.34. ¹H NMR (500 MHz, DMSO) δ 10.47 (s, 1H), 8.64 (d, J = 4.7 Hz, 1H), 8.30 (d, J = 8.6 Hz, 1H), 7.69 (d, J = 8.5 Hz, 2H), 7.43 (t, J = 6.4 Hz, 2H), 7.31 – 7.13 (m, 10H), 4.50 (s, 1H), 4.17 – 4.11 (m, 1H), 3.90 (s, 3H), 1.76 – 1.57 (m, 4H), 1.56 – 1.31 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 167.99, 159.58, 158.20, 141.44, 137.52, 135.75, 135.31, 129.64, 129.00, 128.76, 128.55, 127.52, 126.95, 126.07, 120.58, 71.44, 50.44, 47.18, 40.68, 28.69, 26.07, 23.25, 20.15. Mass spectra (ESI) m/z: 548.3 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₉H₃₀N₃O₆S: 548.1855, found: 548.1863.

(S)-2-((2-(2-((3-benzoylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid (9f), a mixture of two cis-diastereomers:

(S)-2-(((1R,2S)-2-((2-(2-((3-benzoylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5R,6S)-9f) and (S)-2-(((1S,2R)-2-((2-(2-((3-benzoylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5S,6R)-9f). Off-white powder; (107 mg, 73% yield); dr value (100: 70). ¹H NMR (500 MHz, DMSO) δ 10.86 (s, 1H), 8.70 (d, J = 8.4 Hz, 1H), 8.39 (d, J = 7.9 Hz, 1H), 8.32 (t, J = 1.7 Hz, 1H), 8.11 – 8.04 (m, 1H), 7.79 – 7.73 (m, 2H), 7.72 – 7.66 (m, 1H), 7.61 – 7.47 (m, 4H), 7.44 – 7.38 (m, 2H), 7.25 – 7.20 (m, 1H), 7.15 – 7.06 (m, 2H), 4.45 (s, 1H), 4.20 – 4.10 (m, 1H), 3.97 – 3.87 (m, 1H), 1.80 – 1.60 (m, 4H), 1.59 – 1.33 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 194.17, 168.01, 159.48, 154.61, 141.68, 137.93, 137.17, 136.38, 135.54, 132.89, 129.89, 129.80, 128.73, 127.62, 126.96, 126.81, 124.64, 121.68, 71.82, 50.35, 47.16, 29.21, 27.28, 23.54, 20.10. ¹H NMR (500 MHz, DMSO) δ 10.83 (s, 1H), 8.67 (d, J = 9.0 Hz, 1H), 8.35 (d, J = 8.6 Hz, 1H), 8.27 (t, J = 1.8 Hz, 1H), 8.11 – 8.04 (m, 1H), 7.79 – 7.73 (m, 2H), 7.72 – 7.66 (m, 1H), 7.61 – 7.47 (m, 4H), 7.44 – 7.38 (m, 2H), 7.25 – 7.20 (m, 1H), 7.15 – 7.06 (m, 2H), 4.48 (s, 1H), 4.20 – 4.10 (m, 1H), 3.97 – 3.87 (m, 1H), 1.80 – 1.60 (m, 4H), 1.59 – 1.33 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 194.17, 167.70, 159.38, 154.44, 141.68, 137.59, 137.06, 136.38, 135.35, 132.89, 129.89, 129.80, 128.73, 127.47, 126.92, 126.72, 124.57, 121.68, 71.51, 50.29, 47.11, 28.67, 26.58, 23.27, 20.03. Mass spectra (ESI) m/z: 562.3 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₉H₂₈N₃O₇S: 562.1648, found: 562.1656.

(S)-2-((2-(2-((4-benzoylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid (9g), a mixture of two cis-diastereomers:

(S)-2-(((1R,2S)-2-((2-(2-((4-benzoylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5R,6S)-9g) and (S)-2-(((1S,2R)-2-((2-(2-((4-benzoylphenyl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5S,6R)-9g). Off-white powder; (119 mg, 81% yield); dr value (100: 66). ¹H NMR (500 MHz, DMSO) δ 10.93 (s, 1H), 8.71 (s, 1H), 8.46 (d, J = 7.9 Hz, 1H), 8.04 (d, J = 8.8 Hz, 2H), 7.81 – 7.70 (m, 4H), 7.69 – 7.65 (m, 1H), 7.60 – 7.53 (m, 2H), 7.43 (t, J = 6.4 Hz, 2H), 7.29 – 7.11 (m, 3H), 4.48 (s, 1H), 4.23 – 4.14 (m, 1H), 4.00 – 3.87 (m, 1H), 1.82 – 1.62 (m, 4H), 1.60 – 1.34 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 194.78, 169.10, 159.73, 155.96, 141.88, 137.44, 132.54, 130.89, 129.91, 129.55, 128.61, 127.60, 127.41, 126.97, 126.70, 119.85, 71.76, 50.82, 47.34, 28.70, 25.30, 23.42, 22.22. ¹H NMR (500 MHz, DMSO) δ 10.91 (s, 1H), 8.72 (s, 1H), 8.41 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 8.8 Hz, 2H), 7.81 – 7.70 (m, 4H), 7.69 – 7.65 (m, 1H), 7.60 – 7.53 (m, 2H), 7.43 (t, J = 6.4 Hz, 2H), 7.29 – 7.11 (m, 3H), 4.49 (s, 1H), 4.23 – 4.14 (m, 1H), 4.00 – 3.87 (m, 1H), 1.82 – 1.62 (m, 4H), 1.60 – 1.34 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 194.78, 168.65, 159.41, 155.56, 141.77, 137.44, 132.47, 130.89, 129.91, 129.55, 128.53, 127.60, 127.41, 126.86, 126.70, 119.80, 71.73, 50.48, 47.06, 27.56, 25.06, 22.85, 22.15. Mass spectra (ESI) m/z: 562.3 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₉H₂₈N₃O₇S: 562.1648, found: 562.1658.

(S)-2-((2-(2-((9H-fluoren-2-yl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid (9h), a mixture of two cis-diastereomers:

(S)-2-(((1R,2S)-2-((2-(2-((9H-fluoren-2-yl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5R,6S)-9h) and (S)-2-(((1S,2R)-2-((2-(2-((9H-fluoren-2-yl)amino)-2-oxoacetamido)cyclohexyl)amino)-2-oxo-1-phenylethanesulfonic acid ((2S,5S,6R)-9h). Off-white powder; (109 mg, 76% yield); dr value (100: 73). ¹H NMR (500 MHz, DMSO) δ 10.65 (s, 1H), 8.72 (t, J = 8.3 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.12 (s, 1H), 7.88 – 7.79 (m, 3H), 7.57 (dd, J = 7.4, 3.4 Hz, 1H), 7.49 – 7.42 (m, 2H), 7.39 – 7.34 (m, 1H), 7.30 – 7.15 (m, 4H), 4.53 (s, 1H), 4.21 – 4.14 (m, 1H), 3.98 – 3.91 (m, 3H), 1.79 – 1.63 (m, 4H), 1.62 – 1.34 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 168.15, 159.73, 158.42, 143.65, 143.08, 140.93, 137.60, 136.78, 135.51, 129.94, 129.63, 127.64, 127.47, 126.87, 126.49, 125.17, 120.18, 119.34, 117.21, 71.64, 50.47, 47.21, 36.65, 28.75, 26.89, 23.76, 20.36. ¹H NMR (500 MHz, DMSO) δ 10.62 (s, 1H), 8.72 (t, J = 8.3 Hz, 1H), 8.36 (d, J = 8.6 Hz, 1H), 8.12 (s, 1H), 7.88 – 7.79 (m, 3H), 7.57 (dd, J = 7.4, 3.4 Hz, 1H), 7.49 – 7.42 (m, 2H), 7.39 – 7.34 (m, 1H), 7.30 – 7.15 (m, 4H), 4.50 (s, 1H), 4.21 – 4.14 (m, 1H), 3.98 – 3.91 (m, 3H), 1.79 – 1.63 (m, 4H), 1.62 – 1.34 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 167.97, 159.62, 158.28, 143.65, 143.08, 140.93, 137.50, 136.78, 135.33, 129.94, 129.63, 127.64, 127.47, 126.94, 126.77, 125.17, 120.18, 119.25, 117.09, 71.56, 50.73, 47.09, 36.65, 29.27, 26.55, 23.31, 20.13. Mass spectra (ESI) m/z: 546.3 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₉H₂₈N₃O₆S: 546.1699, found: 546.1690.

(S)-2-oxo-2-((2-(2-oxo-2-(phenylamino)acetamido)cyclohexyl)amino)-1-phenylethanesulfonic acid (9i), a mixture of two cis-diastereomers:

(S)-2-(((1R,2S)-2-oxo-2-((2-(2-oxo-2-(phenylamino)acetamido)cyclohexyl)amino)-1-phenylethanesulfonic acid ((2S,5R,6S)-9i) and (S)-2-(((1S,2R)-2-oxo-2-((2-(2-oxo-2-(phenylamino)acetamido)cyclohexyl)amino)-1-phenylethanesulfonic acid ((2S,5S,6R)-9i). Off-white powder; (93 mg, 78% yield); dr value (100: 67). ¹H NMR (500 MHz, DMSO) δ 10.55 (s, 1H), 8.75 – 8.68 (m, 1H), 8.38 (d, J = 7.9 Hz, 1H), 7.82 (d, J = 7.7 Hz, 2H), 7.45 – 7.40 (m, 2H), 7.39 – 7.31 (m, 2H), 7.30 – 7.10 (m, 4H), 4.45 (s, 1H), 4.16 (s, 1H), 3.99 – 3.85 (m, 1H), 1.83 – 1.28 (m, 8H). ¹³C NMR (126 MHz, DMSO) δ 168.25, 159.73, 158.62, 140.33, 137.80, 137.05, 129.89, 128.77, 127.59, 124.51, 120.42, 71.84, 49.15, 47.09, 27.77, 26.90, 23.25, 20.43. ¹H NMR (500 MHz, DMSO) δ 10.52 (s, 1H), 8.75 – 8.68 (m, 1H), 8.34 (d, J = 8.6 Hz, 1H), 7.80 (d, J = 7.7 Hz, 2H), 7.45 – 7.40 (m, 2H), 7.39 – 7.31 (m, 2H), 7.30 – 7.10 (m, 4H), 4.47 (s, 1H), 4.16 (s, 1H), 3.99 – 3.85 (m, 1H), 1.83 – 1.28 (m, 8H). ¹³C NMR (126 MHz, DMSO) δ 167.97, 159.67, 158.54, 140.33, 137.80, 136.95, 129.89, 128.77, 127.44, 124.51, 120.42, 71.57, 48.98, 47.05, 27.52, 26.50, 23.29, 20.46. Mass spectra (ESI) m/z: 458.3 (M - H)⁻. HRMS (ESI-TOF): m/z [M - H]⁻ calcd. for C₂₂H₂₄N₃O₆S: 458.1386, found: 458.1378.

Synthesis of N-(2-aminocyclohexyl)-2-phenylacetamide (4b).

2-phenylacetic acid (2b) (200 mg, 3.67 mmol), HOBt (238 mg, 1.76 mmol), and HBTU (668 mg, 1.76 mmol) were dissolved in dry DMF (20 ml). The mixture was stirred at room temperature for 15 minutes. Then cis-1,2-cyclohexanediamine (184 mg, 1.62 mmol) and DIEA (0.785 ml, 4.41 mmol) were added, and the resulting mixture was stirred at room temperature overnight and was monitored by LC/MS. After completion of the reaction, DMF was removed by a rotary evaporator, the residue was subjected to column chromatography for purification, and the corresponding product was obtained as a white powder (260 mg, 76% yield, >95% purity). Mass spectra (ESI) m/z: 233.3 (M + H)⁺.

Synthesis of methyl 2-oxo-2-((2-(2-phenylacetamido)cyclohexyl)amino)acetate (6b).

To N-(2-aminocyclohexyl)-2-phenylacetamide (200 mg, 0.86 mmol) and DIEA (0.46 mL, 2.58 mmol) in DCM (20 mL) was slowly added methyl chlorooxoacetate (106 mg, 0.86 mmol). The resulting mixture was stirred at r.t. for 1 hour and then was concentrated by rotary evaporator. The mixture was purified by column chromatography eluting with dichloromethane/methanol 30:1 v/v to give methyl 2-oxo-2-((2-(2-phenylacetamido)cyclohexyl)amino)acetate as a white powder (241 mg, 88% yield, >95% purity). Mass spectra (ESI) m/z: 319.3 (M + H)⁺.

Synthesis of 2-oxo-2-((2-(2-phenylacetamido)cyclohexyl)amino)acetic acid (7b).

To a solution of compound 6b (150 mg, 0.47 mmol) in methanol (40 ml) and H₂O (40 ml), KOH (1.06 g, 18.85 mmol) was added. The obtained mixture was stirred at r.t. for 1 hour. The mixture was brought to 0°C, and carefully acidified with 1N HCl until pH = 1 to furnish product 2-oxo-2-((2-(2-phenylacetamido)cyclohexyl)amino)acetic acid. Subsequently, it was subjected to HPLC purification, and the corresponding product was obtained as a colorless oil (102 mg, 71% yield, >95% purity). Mass spectra (ESI) m/z: 303.2 (M - H)⁻.

Synthesis of N1-(3-chloro-4-phenoxyphenyl)-N2-(2-(2-phenylacetamido)cyclohexyl)oxalamide (9j).

2-oxo-2-((2-(2-phenylacetamido)cyclohexyl)amino)acetic acid (7b) (100 mg, 0.33 mmol), HOBt (53 mg, 0.39 mmol) and HBTU (150 mg, 0.39 mmol) were dissolved in dry DMF (10 ml). The mixture was stirred at room temperature for 15 minutes. Then 3-chloro-4-phenoxyaniline (80 mg, 0.36 mmol) and DIEA (0.234 ml, 1.31 mmol) were added, the resulting mixture was stirred at room temperature over night and was monitored by LC/MS. After completion of the reaction, DMF was removed by rotary evaporator, the residue was subjected to column chromatography/HPLC for purification, and the corresponding product was obtained as White powder (122 mg, 73% yield, >95% purity). ¹H NMR (500 MHz, DMSO) δ 10.93 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 2.5 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.84 (dd, J = 8.9, 2.5 Hz, 1H), 7.41 – 7.33 (m, 2H), 7.24 (d, J = 4.4 Hz, 4H), 7.19 – 7.08 (m, 3H), 6.96 – 6.90 (m, 2H), 4.06 – 4.00 (m, 1H), 3.98 – 3.91 (m, 1H), 3.47 (s, 2H), 1.81 – 1.58 (m, 4H), 1.53 – 1.32 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 170.45, 159.01, 158.65, 156.94, 147.61, 136.64, 135.06, 130.13, 128.97, 128.21, 126.28, 124.86, 123.27, 122.21, 121.96, 120.85, 116.94, 49.51, 47.74, 42.40, 28.40, 27.04, 22.70, 20.85. Mass spectra (ESI) m/z: 506.3 (M + H)⁺. HRMS (ESI-TOF): m/z [M + H]⁺ calcd. for C₂₈H₂₉ClN₃O₄: 506.1847, found: 506.1854.

Molecular Modeling Studies.

The molecular modeling studies were performed using the previously published crystal structure of human laforin (PDB ID 4RKK)⁶⁴. The Maestro suite version 2021–3 (Schrödinger, LLC, New York, NY) was used for the modeling studies. The protein was prepared using the protein preparation workflow. First, S266 was mutated to cysteine to match the construct used in the biochemical activity assays. The maltohexaose chains were removed from the structure, and the protein was ionized at a pH of 7.0 ± 1.0 to match the conditions used in the biochemical assay. Hydrogen bond assignments were then optimized according using PROPKA, and the cysteine thiol was manually ionized. Finally, the protein was minimized by converging heavy atoms to an RMSD of 0.3 Å using the OPLS4 force field, and all waters further than 5 Å from heteroatoms were removed.

The receptor grid was generated using the receptor grid generation module. The center of the grid was placed within chain A defined by the X, Y, and Z coordinates 52.47, 71.22, and 122.78 respectively and the grid box was 14 by 14 by 14 Å. A positional constraint was defined, requiring the placement of a charged acceptor within a 3 Å radius of the co-crystallized phosphate.

Ligands were prepared in the LigPrep module using the OPLS4 force field. Ionization states were generated at pH 7.0 ± 1.0 using Epik to match biochemical assay conditions. All combinations of stereoisomers were generated. After ligand preparation, the output was manually filtered to retain only the cis-isomers for the cyclohexyl core series.

Docking was performed using SP-Glide. During the docking experiment, a constraint was included requiring the presence of a charged hydrogen-bond acceptor, such as a sulfonic acid, within a 3 Å sphere defined by the co-crystallized phosphate ion. The inclusion of this constraint was supported by the following: compound 9j does not contain a sulfonic acid and is inactive up to a concentration of 50 μM, and during compound characterization, 9c was determined to act as a competitive inhibitor. Taken together, these data support the inclusion of the constraint and increase the reliability of the resulting binding poses predicted by docking. Since compound 9c is a mixture of two cis-isomers/diastereomers, we performed docking studies for both diastereomers using the same approach. 10 poses were generated for each ligand. Among these, only (2S,5R,6S)-9c exhibited a binding mode similar to that of 8k, aligning well with the existing SAR. In contrast, the binding mode of (2S,5S,6R)-9c did not well correspond with the existing SAR, therefore we did not include this docking study in the manuscript. Consequently, we used (2S,5R,6S)-9c for the modeling studies. The generated docking poses were then post-processed using the Prime MM-GBSA module according to the default settings.

Protein expression.

Expression and purification of His-tagged Laforin was performed as previously described⁵⁴. Laforin was expressed in BL21 E coli cells. Protein expression was induced by 0.5 mM IPTG and harvested after overnight incubation at 18 °C. The cells were pelleted by centrifugation at 6,000 rpm at 4 °C for 15 minutes and resuspended in 40 mL of 20 mM Tris buffer, pH 8.0, with 500 mM NaCl and 5 mM imidazole. The suspension was lysed by sonication under 0 °C for 30 min with 1 mM PMSF. The bacteria lysate was centrifuged at 15,000 rpm at 4 °C for 30 minutes. The supernatant was collected and incubated with 1 mL of Ni-NTA beads washed with the same 20 mM Tris buffer by end-over-end rotation for 1.5 h at 4 °C. The mixture was flowed through a gravity flow column, and the beads were washed with 20 mM Tris buffer, pH 8.0, 500 mM NaCl, and 20 mM imidazole. Laforin was eluted with 20 mM Tris buffer, pH 8.0, with 500 mM NaCl and 300 mM imidazole. Laforin was concentrated by centrifugation and further purified by going through a Sephadex G75 column. Purified laforin was stored in 20 mM Tris buffer, pH 8.0 with 150 mM NaCl and 1 mM DTT. Other PTPs for selectivity include SHP1 PTP domain, SHP2 PTP domain, PTP1B, LYP, HePTP, STEP, TCPTP, PTPα-D1D2, PTPβ-D1, PTPδ, LAR, CD45-D1D2, VHR, YopH, and LWMPTP were expressed and purified using the same method.

IC₅₀ Determination and K_i Determination.

PTP activity was measured using p-nitrophenyl phosphate (pNPP) in DMG buffer (50 mM 3,3-dimethylglutarate, 1 mM EDTA, NaCl to adjust the ionic strength to 0.15 M) at pH 7.0, 25 °C with a working volume of 200 μL. The reaction was initiated by adding laforin (final concentration 2.0 nM) to the solution containing pNPP (final concentration of 4.0 mM, K_m value) and 1.5-diluted compounds. The reaction was incubated for 80 minutes and quenched with 5 N NaOH The amount of product p-nitrophenol (pNPP) was determined from the absorbance at 405 nm detected by a Clariostar microplate spectrophotometer using a molar extinction coefficient of 18,000 M ⁻¹cm⁻¹. IC₅₀ was calculated by fitting the data using GraphPad 9.2.0.

To evaluate the selectivity, the IC₅₀ of compounds against SHP1 PTP domain, SHP2 PTP domain, PTP1B, LYP, HePTP, STEP, TCPTP, PTPα-D1D2, PTPβ-D1, PTPδ, LAR, CD45-D1D2, VHR, YopH, and LWMPTP were tested under the same condition as used for laforin except that the final enzyme concentration was 20 nM and pNPP was at K_m concentrations.

All assays were performed with and without 0.01% Triton to eliminate aggregation effects; each assay was independently repeated 3 times and the results were averaged.

The inhibition constants (K_i) for the inhibitors against laforin were determined at pH 7.0 and 25 °C. In the presence of 0, 1, or 4 nM of compound and 1 nM of laforin, the initial rate for 1.7–20 mM pNPP was measured by following the production of p-Nitrophenol (UV absorbance at 405 nm) as described above; the data were fitted using GraphPad 9.2.0. Kinetics to obtain the inhibition constant and to assess the mode of inhibition.

Pharmacokinetics Study.

Animal dosing and sample collection for pharmacokinetic studies are presented below. The compounds were first dissolved in DMSO to make a 20 mg/ml solution. Then the solution was further diluted to a 2 mg/ml solution for which the formulation is 10% DMSO - 90% PBS. Each mouse was administered a single IP dose of 10 mg/kg. The volume of each injection was about 100 μL according to the weight of mouse. At different time points (0.5 h, 1 h, 2 h, 3 h, 6, 24 h), blood samples (50 μL) were collected and centrifuged to get the serum. The serum (10 μL) was then mixed with acetonitrile (20 μL) and centrifuged. The supernatant was collected and subjected to Liquid Chromatography/Mass Spectrometry analysis. The Liquid Chromatography/Mass Spectrometry (LC/MS) analysis was carried out on an Agilent 1260 analytic HPLC system and an Agilent 6470 Triple Quadrupole MS detector, equipped with a Kinetex 2.6um C18 column (3 mm X 50 mm), eluted with 0–100% MeOH-H₂O with 0.1% (w/v) formic acid at 0.7 mL/min flow-rate (gradient method: 1.2 min 0–10% MeOH linear-gradient, 1.5 min 10–90% MeOH linear-gradient, followed by 1.3 min 90–100% MeOH, followed by 2.5 min 100% MeOH), MS detector were set at single ion mode (SIM), monitoring the negative charge according to the compound molecular weight. The pharmacokinetic parameters were calculated in GraphPad Prism 6.

All animal experiments were approved by the Purdue University Institutional Animal Care and Use Committee. For the pharmacokinetic investigation, the compound’s concentration in the blood over time was determined by collecting blood samples using the tail-clip method at 0.5, 1, 2, 3, 6, and 24-hour post-compound injection. The study for each compound was conducted on three inbred C57BL/6J mice (8–10 weeks old; ~25 g each), including two males and one female (total 18 mice).

MST Assay.

MST experiments were performed on a NanoTemper Monolith NT.115 with blue/red filters using Monolith NT His-Tag Labeling Kit. Samples were prepared in PBS-T buffers. The laforin protein (1 nM) was incubated with RED-tris-NTA dye (0.5 nM) at room temperature for 30 min followed by centrifuge at 4 °C and 15,000 g for 10 min. Compound 9c of 100 nM to 0.001 nM were dispensed in PCR tubes (20 μL in each tube). Labeled protein of equal volume was added and mixed with compounds in each tube, and the solution was loaded into standard capillaries. Measurements were performed at room temperature using 40% MST power with laser off/on times of 5 s and 20 s, respectively. Data analysis was performed using the GraphPad Prism 9.2.0 software.

Malachite Green Phosphatase Assay.

Release of phosphate from potato amylopectin was determined as done previously^{22, 56, 74, 75} with the following modifications. Briefly, phosphate release was monitored using the PiColorLock^™ Phosphate Detection Reagent (Novus Biologicals), a malachite green-based assay. For time course assays, 5 nM recombinant Hs-laforin was incubated with 90 μg solubilized potato amylopectin (Sigma-Aldrich), supplied as a powder; solubilized at a stock concentration of 5 mg/mL using alcohol/alkaline method⁵⁶ in 1X phosphatase buffer in a final volume of 80 μL at pH 6.5. For Hs-laforin kinetic characterization, 5 nM Hs-laforin was incubated with varying amylopectin concentrations for 10 mins. All reactions were terminated by the addition of 20 μL (0.25 initial reaction volume) of the PiColorLock Gold solution with Accelerator in a 100:1 ratio of Gold solution to the accelerator. After 5 minutes, 8 μL stabilizer solution (0.1 initial reaction volume) was added and the reaction was allowed to develop for 30 mins at r.t. before the absorbance of each reaction was measured at 635 nm using a Synergy HTX Multi-Mode Reader (BioTek). Absorbance was converted to pmol P_i release using a P_i absorbance standard curve. Data points are presented as the mean of three independent replicates, each consisting of three technical replicates.

Cellular Assay.

A549 cells were seeded at 60% confluency in 8-well glass chamber slides (Nunc; Lab-Tek II) and allowed to adhere overnight in DMEM + 10% FBS + 1% Penicillin/Streptomycin. The next day, cells were treated with media containing 1 μM 8k or an equal volume of DMSO, and cells were returned to the incubator for 24h. For harvest, cells were washed twice with 0.1x PBS and placed on dry ice for 5 minutes. Slides were then transferred to a vacuum desiccator for 2 hours before fixing with 10% neutral buffered formalin for 20 min. Cells were then washed twice with 70% ethanol and returned to the vacuum desiccator to dry for another 2 hours. Slides were stored at −80 until further processed.

Glycogen chain length and phosphate quantification.

Glycogen analyses were done using MALDI mass spectrometry using the method previously described^{32, 33, 76}. Briefly, the monolayer cell line in chamber wells was sprayed using an HTX spray station, which was used to coat the slide with 0.2 mL of an aqueous solution containing isoamylase (3 units per slide). The spray nozzle was set to 45°C with a spray velocity of 900 m/min. After enzyme application, the slides were incubated at 37°C for 2 hours in a humidified chamber and subsequently dried using a vacuum desiccator before matrix application. The matrix solution [α-cyano-4-hydroxycinnamic acid (0.021 g CHCA in 3 mL 50% acetonitrile/50% water with 12 μL of 25% TFA)] was applied using the HTX spray. A Waters Synapt G2-Si high-definition mass spectrometer equipped with traveling wave ion mobility was used to detect and separate glycogen and N-glycans. The laser was operated at 1000 Hz with an energy of 200 AU and a spot size of 75 μm, and the mass range was set to 500–3000 m/z. Data were acquired using Masslynx v4.2 software. MALDI images were generated with HDI software v1.5 (Waters Corp), using built-in peak integration to account for mass drift during the MALDI run. Relative abundance corresponding to glycogen peaks was extracted from HDI software and plotted using GraphPad Prism.

Supplementary Material

¹H and ¹³C NMR spectra for the final compounds (3d, 5a, 5b, 8a-8n, and 9a-9j) tested for biological activity. This material is available free of charge via the Internet at http://pubs.acs.org.

Molecular Formula Strings (CSV)

ABBREVIATIONS

DCM

dichloromethane

DIEA

N,N-Diisopropylethylamine

DMF

N,N-dimethylformamide

DMSO

dimethyl sulfoxide

dr

diastereomeric ratio

HBTU

Hexafluorophosphate Benzotriazole Tetramethyl Uronium

HOBt

Hydroxybenzotriazole

pNPP

p-nitrophenyl phosphate

PTP

protein tyrosine phosphatase

SPAA

sulfophenyl acetic amide