3-([4-(Acetylamino)phenyl]methoxy-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic Acid

Abstract

Overexpression of protein phosphatase 5 (PP5) is linked to tumor cell growth, making it a candidate for small-molecule drug therapy. Since the PP2A domain has been selectively inhibited using functionalized scaffolds that maximize contacts, a similar approach is proposed to work for PP5. As cantharidin’s demethylated cousin, norcantharidin, is a potent but unselective phosphatase inhibitor that can be prepared in just two synthetic steps, the bicyclic scaffold holds promise as an attractive target upon functionalization. Our hypothesis targets PP5 selectivity through derivatives of norcantharidin with functionalized attachments for optimal active-site binding. The methodology offers a promising platform for developing PP5-selective anticancer therapeutics. The approach reported herein exploits anhydride reactivity to yield a carboxylic acid derivative as our next-generation inhibitor of PP5. The methodology offers groundwork for future optimization of norcantharidin-based drug candidates with improved tumor selectivity, potency, and synthetic feasibility.

1. Introduction

Over the past two decades, cancer research has focused on protein kinases and phosphatases, which regulate phosphorylation states crucial for cell cycle control. Deregulation of these pathways can lead to tumorigenesis. While kinases have been primary targets, serine/threonine phosphatases, particularly phosphoprotein phosphatase 5 (PP5), are emerging as promising therapeutic targets [1]. PP5, encoded on human chromosome 19 and conserved across eukaryotes, uses its tetratricopeptide (TPR) domain for protein–protein interactions and influences key signaling pathways [2].

PP5 binds the glucocorticoid receptor (GR), inhibiting transcription of antiproliferative factors like p21. A truncated PP5 containing only the TPR domain still inhibited GR function, suggesting non-catalytic roles. PP5 also dephosphorylates p53, reducing its stability and transcriptional activity, while p53 negatively regulates PP5 expression [3]. Suppression of PP5 enhances GR-mediated transcription and reduces cancer cell growth, underscoring its role in downregulating antiproliferative pathways. Additionally, PP5 disrupts DNA repair by dephosphorylating DNA-PK, promotes survival under hypoxic conditions via ASK-1 inhibition, and correlates with breast cancer progression in tissue analyses [4].

Selective phosphatase inhibitors have proven valuable potent PP2A inhibitors, as demonstrated by fostriecin, microcystin, and okadaic acid (Figure 1) [5]. However, each system, while highly selective, suffers from poor synthetic accessibility, highlighting the need for viable selective PP5 inhibitors.

Cantharidin, a natural toxin from blister beetles, is a semi-selective phosphatase inhibitor with notable PP5 activity due to specific active site interactions [6]. Cantharidin’s demethylated cousin, norcantharidin, offers more opportunities as the assembly of this platform can be done in two synthetic steps (Scheme 1) at a fraction of the cost and at nearly quantitative yield when comparing the use of maleic anhydride (R = H, assembly of norcantharidin) and dimethyl maleic anhydride (R = CH₃, assembly of cantharidin).

As we have documented, modifications at positions 5 and 6 of the bicyclic scaffold of norcantharidin improve PP5 selectivity [7,8,9]. The synthetic overhead, however, when comparing a strategy involving the ring-opening of the anhydride moiety to derivatization of the diene prior to cycloaddition is unmatched when considering the former. As such, modification of the anhydride to furnish a carboxylic acid derivative was conducted, making this platform an even stronger candidate for further development, whereby adjusting, if needed, the linker and adding site-specific functionality can improve cytotoxicity against cancer cells, guiding rational design of next-generation PP5 inhibitors.

2. Results and Discussion

Aryl scaffolds, specifically those derived from benzene, are fundamental in drug discovery due to their unique combination of structural simplicity and chemical stability. The planar, six-membered ring offers a robust and synthetically viable framework that resists metabolic degradation, ensuring molecular integrity within biological systems [10]. This stability, combined with the scaffold’s planarity, facilitates strong interactions such as π-π stacking, hydrophobic, and van der Waals forces with enzyme-active sites or receptor pockets, enhancing binding affinity and selectivity [11]. Furthermore, what is perhaps more significant is benzene’s scaffold, which provides multiple sites for functionalization, enabling fine-tuning of pharmacokinetic and pharmacodynamic properties to optimize drug efficacy and safety [12].

Coupled to its favorable chemical and biological properties, access to functionalized derivatives, such as acetamido derivatives, makes this an attractive entry point. Acetamido derivatives are widely available and allow for the efficient generation of compound libraries for high-throughput screening [13]. The prevalence of systems bearing this unique platform underscores their compatibility with biological targets and has proved successful in drug development [14], having this scaffold as an indispensable and versatile foundation for designing next-generation inhibitors with optimized therapeutic profiles.

Scheme 2 offers our approach toward the assembly of next-generation inhibitors bearing carboxylic acid derivatives upon ring opening of the anhydride moiety. Treatment of norcantharidin with a solution of N-[4-(hydroxymethyl)phenyl]acetamide in acetonitrile in the presence of the general base pyridine afforded the desired product 1 in 64% isolated yield. Access to all the spectral data can be found within the Supplementary Materials.

Unique to the assembly of this system was how compound 1 was isolated. As all the materials used are soluble in warm ethyl acetate, simple trituration of the crude reaction mixture afforded analytically pure material upon filtration and evaporation of the solvent. The ease at which functionalized appendages can be added to the bicyclic scaffold via a ring-opening of the anhydride motif only highlights norcantharidin’s synthetic versatility and offers the opportunity of modular scaffold modifications to improve pharmacological properties in one step.

3. Materials and Methods

All spectra were obtained as solutions in (CD₃)₂SO having the following field strength: ¹H NMR (500 MHz) and ¹³C NMR (125 MHz). The NMR that generated the spectra was a JEOL ECA-500 spectrometer (JEOL Ltd., Tokyo, Japan) using JEOL Delta^TM Version 6.1.0 (MAC) software (JEOL Ltd., Tokyo, Japan). Chemical shifts are reported in parts per million (ppm). Chemical shifts are referenced to δ 0.00 ppm (TMS) in ¹H NMR and ¹³C NMR. Infrared spectra are reported in wavenumbers (cm⁻¹) and recorded using a JASCO FT/IR-4100 equipped with an Attenuated Total Reflectance (ATR) accessory (JASCO, Tokyo, Japan). For the synthetic procedures performed, all hazardous materials were handled while wearing protective gloves, protective clothing, and eye protection, and conducted in the hood. Additional considerations consisted of the following: TLC analyses were performed on flexible aluminum-backed TLC plates with silica gel and a fluorescent indicator. Detection was conducted by UV absorption (254 nm). Solutions were concentrated in vacuo using a rotary evaporator. The HRMS data were generated on a Waters SYNAPT (Waters Corporation, Milford, MA, USA) at the Mass Spectrometry Laboratory, which is part of the School of Chemical Sciences at the University of Illinois Urbana-Champaign. The chemicals used for this synthetic procedure were the following: acetonitrile, ethyl acetate, N-[4-(hydroxymethyl)phenyl]acetamide, 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (norcantharidin), and pyridine and were reagent-grade or better.

4. Experimental

3-([4-(Acetylamino)phenyl]methoxy-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic Acid (1)

N-[4-(hydroxymethyl)phenyl]acetamide (490 mg, 3.0 mmol, 1.0 equiv.) was added to a 15 mL round-bottomed flask (RBF) equipped with a magnetic stir bar. Acetonitrile (3.0 mL) was then added to the RBF at room temperature. After adding pyridine (0.5 mL, 2.1 equiv.), the reaction mixture was externally warmed until solution was observed. Norcantharidin (489 mg, 2.9 mmol, 1.0 equiv.) was added to the RBF and the resulting solution was left to stir overnight. Within 5 min, product formation, in the form of a white precipitate, began to appear. After 20 h, the reaction mixture was transferred to a 35 mL Erlenmeyer flask and triturated with approximately 50 mL hot ethyl acetate. Once the slurry was allowed to cool to room temperature, the flask was placed in an ice bath for 10 min. The mixture was filtered and rinsed with cold ethyl acetate (5 × 2 mL). The collected white crystalline solid (mp 165–174 °C) weighed 620 mg, corresponding to a 64% isolated yield. ¹H NMR (500 MHz, (CD₃)₂SO); 12.30 (br s, 1H, -COOH), 9.99 (s, 1H, -NH-), 7.56 (d, J = 8.0 Hz, 2H, =CH-), 7.27 (d, J = 8.5 Hz, 2H, =CH-), 4.96 (d, J = 12.0 Hz, 1H, -CHO-), 4.85 (d, J = 12.0 Hz, 1H, -CHO-), 4.70 (m, 2H, -CH₂O-), 3.03 (dd, J = 10.0 Hz, J = 3.0 Hz, 2H, -CHCH-), 2.04 (s, 3H, -CH₃), 1.53 (m, 4H, -CH₂CH₂-). ¹³C NMR (125 MHz, (CD₃)₂SO); 172.4, 171.1, 168.4, 139.1, 130.4, 128.8, 118.8, 77.9, 77.6, 65.5, 52.0, 50.9, 28.5 (both CH₂′s), 24.0. IR (Attenuated Total Reflectance); 3303, 3007, 2992, 2984, 2880, 1732, 1689, 1654, 1519, 1257, 1229, 1198, 1189, 1007, 966, 818, 562 cm⁻¹. HRMS (LC-MS) m/z: [M + H]⁺ calcd for C₁₇H₁₉NO₆ 334.1291; found 334.1281.