4-(1,3-Dioxoisoindolin-2-yl)butyl(2R,4aS,6aS,12bR,14aS,14bR)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydropicene-2-carboxylate

Abstract

In this report, we describe the synthesis of a compound derived from the natural compound celastrol, which is connected to a phthalimide moiety via an ester linkage. The compound was fully characterized by proton (¹H), carbon-13 (¹³C), heteronuclear single-quantum coherence (HSQC), and distortionless enhancement by polarization transfer (DEPT) NMR. Ultraviolet–visible spectroscopy (UV-Vis), Fourier-transform infrared (FTIR), and elementary analysis were also performed.

1. Introduction

In recent years, celastrol (1) has emerged as a compound of considerable interest in the search for new therapeutic agents, owing to its broad pharmacological potential. Isolated from the roots of Tripterygium wilfordii, a key species in traditional Chinese medicine (TCM) with a long-standing history, celastrol has transitioned from a folk remedy to a subject of rigorous biomedical research. Although its medicinal use dates back centuries, the scientific community has only recently begun to elucidate the full extent of its biological activity, primarily through mechanistic studies conducted over the past decade.

Celastrol exhibits a broad spectrum of pharmacological effects, including anti-inflammatory [1], antioxidant [2], cardioprotective [3], and anti-rheumatic properties [4]. Moreover, it has demonstrated anticancer activity [5], neuroprotective potential in neurodegenerative conditions [6], and anti-obesity effects [7]. A particularly compelling aspect of celastrol’s bioactivity lies in its modulation of critical cellular signaling pathways involved in disease development. It has been shown to suppress the activation of NF-κB [8], disrupt the Hsp90–Cdc37 complex [9], and inhibit the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) [10], all of which are linked to inflammatory and proliferative diseases.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by persistent inflammation and progressive joint deterioration. With a global prevalence estimated between 0.25% and 1% [11], RA remains a significant public health burden. Despite considerable efforts to manage its symptoms, a definitive cure has yet to be found. The pathophysiology of RA is multifactorial, involving a complex interplay among immune cells (e.g., T cells, macrophages, fibroblasts) and molecular mediators [12]. Among these, calcium ions (Ca²⁺) play a central role in driving autoimmune responses and maintaining chronic inflammation [13]. SERCA, a key regulator of calcium homeostasis, has been studied for its inhibitory abilities not only in cancer research, where it leads to increased intracellular calcium, ATP depletion, and cell death [14], but also as a therapeutic target in inflammatory diseases such as RA [10,15]. Celastrol’s ability to target SERCA has attracted considerable attention due to its observed effects in reducing bone erosion and suppressing inflammatory arthritis in preclinical studies [10].

Although celastrol holds considerable therapeutic promise, its clinical translation is hindered by several challenges, such as its notably high toxicity, poor aqueous solubility, and chemical instability. These limitations have prompted extensive efforts in medicinal chemistry to develop structurally modified derivatives with improved drug-like properties (Figure 1, panel ii). Strategies such as esterification and amidation of the C29-carboxylic acid group (Figure 1, panel i) have been applied to enhance water solubility, reduce toxicity, and explore novel pharmacological activities [16,17,18].

Phthalimide-based scaffolds have garnered increasing interest in recent years among medicinal chemists due to their remarkable versatility and broad-spectrum biological activities [19]. These nitrogen-containing heterocyclic compounds, characterized by a phthalimide core, have served as privileged scaffolds in the development of new pharmacophores with diverse therapeutic applications. The structural rigidity and electron-withdrawing characteristics of the phthalimide ring system contribute to its favorable pharmacokinetic and pharmacodynamic properties, making it a suitable candidate for drug design [20]. Numerous studies have reported the antitumor activity of phthalimide derivatives, particularly through mechanisms involving the induction of apoptosis, inhibition of angiogenesis, and disruption of cell cycle progression [21]. For instance, thalidomide and its analogues, lenalidomide and pomalidomide, are clinically approved immunomodulatory agents that have revolutionized treatment paradigms for multiple myeloma and other hematologic malignancies [22].

Beyond oncology, phthalimide compounds have demonstrated promising antitubercular and antimalarial effects, often attributed to their ability to disrupt vital enzymatic pathways in Mycobacterium tuberculosis [23] and Plasmodium species [24], respectively. Their structural framework has also been utilized in the synthesis of potent antioxidants, which scavenge free radicals and mitigate the oxidative stress implicated in various chronic diseases. Furthermore, antimicrobial activity against both Gram-positive and Gram-negative bacterial strains has been documented, with several derivatives showing potential as alternatives to conventional antibiotics [25].

Additionally, the anti-inflammatory properties of phthalimide derivatives have been extensively investigated [26], with many compounds being observed to be capable of modulating key mediators such as prostaglandins, cytokines, and nitric oxide. These agents are being explored for the treatment of inflammatory and autoimmune disorders, including RA and inflammatory bowel disease. Taken together, the multifaceted biological profile of phthalimide-based moieties, combined with their synthetic accessibility and amenability to structural modification, positions them as a highly valuable chemotype in the ongoing search for novel therapeutic agents. Recently, a derivative of celastrol containing phthalimide moiety was synthesized via esterification and analyzed for its in vitro anticancer activity [27].

This study describes the synthesis of a new celastrol derivative whose scaffold has been linked to phthalimide derivatives (Scheme 1), which was designed and fully characterized by NMR, IR, UV-Vis, and elementary analyses.

Figure 1. i. Functional groups of celastrol (1) that are important for its biological activity; ii. Example of celastrol derivatives (a) [28], (b) [17], and (c–e) [16] reported in the literature; iii. Chemical structures of 2-(4-bromobutyl)isoindoline-1,3-dione (3) and compound 4 are reported in the inset of the figure.

Figure 1. i. Functional groups of celastrol (1) that are important for its biological activity; ii. Example of celastrol derivatives (a) [28], (b) [17], and (c–e) [16] reported in the literature; iii. Chemical structures of 2-(4-bromobutyl)isoindoline-1,3-dione (3) and compound 4 are reported in the inset of the figure.

2. Results and Discussion

The synthetic pathway toward 4-(1,3-dioxoisoindolin-2-yl)butyl(2R,4aS,6aS,12bR,14aS,14bR)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydropicene-2-carboxylate was initiated by the preparation of the precursor (3), as outlined in Scheme 1a.

Isoindoline-1,3-dione, also known as phthalimide, is widely recognized in synthetic organic chemistry due to its multifunctional utility in fields of pharmaceuticals, agrochemicals, and materials science. This compound acts as an amine synthon (^-NH₂ equivalent), enabling efficient access to primary amines. Following a reported procedure [29], 2-(4-bromobutyl)-isoidoline-1,3-dione 3 was prepared by the reaction of isoindoline-1,3-dione potassium salt 2 with 1,4-dibromobutane in DMF. The identity of 3 was confirmed by ¹H-, ¹³C-NMR, and DEPT-135 spectra (Supplementary Materials, Figures S1–S3).

Compound 3 was subsequently used in a coupling reaction to obtain the final ester product. The transformation was carried out under carbonate-mediated activation conditions, employing a modified protocol described by Jiang et al. [30]. Specifically, celastrol was dissolved in DMF and treated with potassium carbonate and a six-equivalents compound 3 (Scheme 1, step b), resulting in the formation of compound 4 with high yield (66.6%).

The structure of compound 4 was elucidated by NMR, IR, UV-Vis and elementary analyses. Concerning NMR analysis, aromatics proton signals corresponding to position 3′′–6′′ appeared in the δH 7.7–7.8 ppm region (Figures S4 and S5), while the methylene group gave rise to peaks between δH 3.5 and 4 ppm in the ¹H-NMR spectrum, consistent with the proposed molecular framework. Regarding the ¹³C-NMR spectrum, the disappearance of the characteristic carboxylic acid carbon peak at 182 ppm (present in compound 1), replaced by a new signal at 178.4 ppm attributable to the amide carbon, confirmed the successful transformation (Figure S6).

Further structural assignments were supported by 2D NMR experiments, including Heteronuclear single-quantum coherence spectroscopy (HSQC) and distortionless enhancement by polarization transfer (DEPT-135, DEPT-90 and DEPT-45), which enabled the classification of carbon environments in compound 4 (Table S1; Supplementary Figures S7–S10). A total of 41 carbon signals were observed, corresponding to six methyl groups, eleven methylene carbons, eight methines, and sixteen quaternary carbons.

The IR spectrum of compound 4 showed prominent bands at 3427 cm⁻¹ (O-H stretch), 2954 and 2926 cm⁻¹ (C-H stretch), and 1714 cm⁻¹ (amide C=O stretch of phthalimide moiety), along with a peak at 1595 cm⁻¹ corresponding to N-H bending (Figures S11 and S12). The UV-Vis spectral analysis further confirmed the compound’s identity, displaying a strong absorption band in the range of 228–242 nm and a weaker band at 426 nm, corresponding to an n→π^* transition) (Figure S13).

3. Materials and Methods

3.1. Chemistry

Silica gel (FCP 230–400 mesh) was used for column chromatography. Thin-layer chromatography (TLC) was carried out on Merck precoated silica gel 60 F254 plates (Merck, Darmstadt, Germany) and visualized with phosphomolybdic acid, iodine, or a UV lamp. All reagents were purchased from Bide Pharmatech., Ltd. (Shanghai, China).

¹H-NMR and ¹³C-NMR spectra were recorded in CDCl₃ at 25 °C on a Bruker Ascend^®-600 (Magnet System 600′54 Ascend LH, San Jose, CA, USA) NMR spectrometer (600 MHz for ¹H and 150 MHz for ¹³C). All chemical shifts (δ) are reported in the standard δ notation of parts per million (ppm) using the peak of the residual proton signals of CDCl₃ as an internal reference (CDCl₃, δH 7.26 ppm, δC 77.2 ppm).

UV-Vis analysis was performed by a Shimadzu UV–2600 (Osaka, Japan) with a 1 cm quartz cell and a slit width of 2.0 nm. The analysis was carried out using wavelengths in the range of 200–700 nm.

IR spectra were obtained using a Shimadzu, IRAffinity-1S Fourier transform infrared spectrometer (Osaka, Japan).

3.2. 2-(4-Bromobutyl) isoindoline-1, 3-dione (3)

A mixture of phthalimide potassium (2.0 g, 10.7 mmol) and 1, 4-dibromobutane (2.8 g, 12.9 mmol) in dry DMF (25 mL) was stirred at room temperature for 26 h. After that, the reaction mixture was extracted with CH₂Cl₂ and washed with water. The combined organic layer was dried over anhydrous MgSO₄, filtered, and evaporated in vacuo. The resulting residue was recrystallized in distilled water to afford 3 as a white solid with 90% yield. m.p.: 75–77 °C. ¹H-NMR (600 MHz, CD₃Cl): δ ppm: 7.5–7.83 (m, 2H), 7.71–7.72 (m, 2H), 3.72 (t, J = 6.7 Hz, 2H), 3.44 (t, J = 6.6 Hz, 2H), 1.90 (m, 2H), 1.83 (m, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ ppm: 168.51, 134.13, 132.18, 123.40, 37.09, 32.92, 29.97, 27.37. ESI-MS: ([M + H]⁺): 283.4.

The spectral characteristics are consistent with those of 3 in the literature [29].

3.3. 4-(1,3-Dioxoisoindolin-2-yl)butyl (2R,4aS,6aS,12bR,14aS,14bR)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydropicene-2-carboxylate (4)

Potassium carbonate (six equivalents) was added to a solution of celastrol (45 mg, 0.10 mmol) in dichloromethane (6 mL), and the mixture was stirred at room temperature for 12 h. The mixture was washed three times with water, and the organic phase was collected, dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo to yield the crude product. Purification was achieved via chromatography on a silica gel column (eluents: hexane/ethyl acetate 2:1). Yield 66.6%, ¹H NMR (600 MHz, CDCl₃) δ ppm:7.84 (dd, J = 5.4, 3.0 Hz, 2H), 7.71 (dd, J = 5.4, 3.0 Hz, 2H), 6.99 (d, J = 1.4 Hz, 1H), 6.50 (d, J = 1.6 Hz, 1H), 6.33 (d, J = 7.2 Hz, 1H), 4.04–3.82 (m, 2H), 3.70 (t, J = 7.1 Hz, 2H), 2.38 (d, J = 15.9 Hz, 1H), 2.19 (s, 3H), 2.12–1.83 (m, 4H), 1.80–1.72 (m, 5H), 1.69–1.46 (m, 7H), 1.42 (s, 3H), 1.38–1.32 (m, 1H), 1.24 (s, 3H), 1.15 (s, 3H), 1.07 (s, 3H), 0.97–0.92 (m, 1H), 0.52 (s, 3H); ¹³C-NMR (150 MHz, CDCl₃) δ ppm: 10.36 (CH₃), 18.59 (CH₃), 21.75 (CH₃), 25.48, 26.02, 27.35, 28.74, 29.76, 29.80, 29.89, 29.94, 30.64, 31.03, 31.70, 32.89, 33.58, 34.90, 36.47, 37.68, 38.32, 39.55, 40.51, 43.04, 44.36, 45.16, 63.85, 117.20, 118.25, 119.67, 123.41, 127.51, 132.17, 134.09, 134.19, 146.12, 164.80, 168.45, 170.07, 178.33, 178.46; UV-Vis (CH₂Cl₂) peaks 228 and 424 nm; IR (FTIR) 3332, 2931, 2870, 2360, 2106, 1643, 1589, 1442, 1288 and 1103 cm⁻¹. Calc. for C₄₁H₄₉NO₆: C, 75.2; H, 7.4; N, 2.1; O, 14.3

4. Conclusions

In this study, a celastrol derivative was synthesized and evaluated for its potential applications in cancer therapy and anti-inflammatory treatment. The compound was thoroughly characterized using NMR, UV-Vis, IR, and elementary analysis.