Research paperExploration of novel macrocyclic dipeptide N-benzyl amides as proteasome inhibitors
Graphical abstract
Introduction
The eukaryotic 26S proteasome is a large (1.6–2.4 MDa) ATP-dependent proteolytic complex, composed of a cylindrical 20S core particle (CP) capped by two 19S regulatory complexes [1]. The 20S proteasome is the proteolytically active key element of the ubiquitin proteasome system (UPS) that directs the majority of intracellular protein degradation in eukaryotic cells. It is composed by four heptameric rings stacked in a α7β7β7α7 arrangement and contains three proteolytic subunits, β1, β2, and β5 [2], respectively. There are two main proteasome subtypes: the constitutive proteasome (cCP) which contains three catalytic subunits denoted as β1c, β2c, and β5c and the immunoproteasome (iCP) which contains three catalytic subunits denoted as β1i, β2i, and β5i [3]. Inhibition of the 20S proteasome leads to the accumulation of substrate proteins involved in signal transduction, antigen presentation, cell-cycle progression and apoptosis, and is preferentially cytotoxic to cancer cells [[4], [5], [6], [7], [8]]. Indeed, proteasome inhibitors are recognized as clinically effective anti-cancer agents, primarily for hematological malignancies [9]. To date, three proteasome inhibitors Bortezomib, Carfilzomib, and Ixazomib (Fig. 1) are FDA approved for the treatment of multiple myeloma (MM) and mantle cell lymphoma [[10], [11], [12]]. All three are covalent inhibitors with an electrophilic warhead at the C-terminal end of a peptidyl backbone for covalent attachment to the catalytic Thr1 residues of the proteasome [13]. However, the electrophilic warhead is often related to excessive reactivity, lack of specificity and instability, which is believed to be the major cause of side effects during therapy [12,14]. These inhibitors have also been unsuccessful in the treatment of solid cancers [15,16], with the lack of efficacy most likely due to their covalent binding to the proteasome, limiting their widespread tissue distribution. In contrast, non-covalent proteasome inhibitors do not possess an electrophilic warhead with less reactivity. Non-covalent and reversible binding mode ensure them with rapid binding and dissociation kinetics. These features may allow non-covalent inhibitors to overcome drawbacks arising in therapeutics of covalent ones.
Representative peptidyl non-covalent proteasome inhibitors have been reported. TMC-95A (4, Fig. 2) is a macrocyclic natural product isolated from Apiospora montagnei Sacc TC 1093, which potently inhibits all the three proteasome catalytic activities with preference for β5c activity (IC50 = 5.4 nM) [17,18]. However, the structural complexity of TMC-95A maybe one of important obstacle to its further development. Additionally, a series of 5-methoxy-1-indanone di-peptide benzyl amides have been reported, including CVT-659 (5, Fig. 2) which selectively inhibits β5c site with submicromolar potency (IC50 = 0.14 μM), but this compound displayed poor cellular activity (IC50 = 8 μM) [19]. Researchers from Millennium Pharmaceuticals, Inc. identified a series of di- and tripeptides (e. g. 6 and 7, Fig. 2) with potency and selectivity for both constitutive proteasome and immunoproteasome β5 sites [20]. Moreover, the X-ray structures of the inhibitors in complex with proteasome suggest that the occupancy of S1 and S3 pockets was crucial to inhibitor potency. Of note, the size of the hydrophobic benzyl group was well suited to S1 pocket, which may be a reason for the wide use of various substituted benzyl groups in peptidyl non-covalent proteasome inhibitors. Meanwhile, the 2-(neopentylamino)-2-oxoethyl group (as present in compound 6) has been reported to provide a near-optimal fit for the S3 binding pocket [[20], [21], [22]]. Other reported peptidyl non-covalent proteasome inhibitors include 2-aminobenzylstatine derivatives [23,24] and linear TMC-95A analogues [25,26]. Overall, linear peptide-based proteasome inhibitors remain to be the mainstream of this field [13,21,[27], [28], [29], [30]].
However, linear peptides are always thought to be unstable with unsatisfied pharmacokinetic profiles. In contrast, macrocyclic peptides often show distinct and (in comparison to linear peptides) superior pharmacokinetic and pharmacological properties [31,32]. Desirable properties introduced by the macrocycle include increased metabolic stability, cellular penetration and selectivity [[33], [34], [35]]. We have identified a series of oral and potent macrocyclic dipeptide epoxyketones as covalent proteasome inhibitors in our previous work [36]. Due to the disadvantages of the electrophilic warhead discussed, in this manuscript, we explored novel proteasome inhibitors based on the peptidyl macrocyclic skeleton of macrocyclic dipeptide epoxyketones, replacing the electrophilic warhead with substituted benzyl groups (Fig. 3). During further optimization, a series of macrocyclic dipeptide N-benzyl amides were designed and synthesized. Moreover, the anti-proliferative activity against various cancer cells of selected compounds were evaluated. In addition, we explored the differences in metabolic stability between our macrocyclic dipeptides and the corresponding linear analogues.
Section snippets
Chemistry
All the target compounds were synthesized by following routes as described in Scheme 1, Scheme 2, Scheme 3, Scheme 4, Scheme 5. Scheme 1, Scheme 2 show the synthetic routes of the intermediates which would be used in Scheme 3, Scheme 4, Scheme 5. Scheme 3, Scheme 4 show the synthetic routs of macrocyclic dipeptides 23a-23j and 31. Finally, Scheme 5 shows the synthetic rout of linear analogue 33.
The synthetic routes of terminal alkene-containing carboxylic intermediates 10a - 10e are displayed
Conclusions
In this study, a series of novel macrocyclic dipeptide N-benzyl amides were rationally designed, synthesized, and biologically evaluated. Most of them exhibited potent proteasomal inhibition, as well as excellent anti-proliferative activity against RPMI 8226, MM1S, and MV-4-11 cell lines. Compound 23h and 23i displayed potent and selective inhibition against β5c and β5i. Further studies found that compound 23h was more potent and more stable than its corresponding linear analogues, proving that
Experimental section
1H and 13C NMR spectra were recorded on Brüker 500 MHz spectrometer (Brüker Bioscience, Billerica, MA, USA) with CDCl3 or DMSO‑d6 as solvent. Chemical shifts (d) were reported in parts per million (ppm) relative to internal TMS, and coupling constants (J) were reported in Hertz (Hz). Splitting patterns were designated as singlet (s), broad singlet (brs), doublet (d), double doublet (dd), triplet (t), quartet (q) and multiplet (m). Melting points were determined using a Buchi B-540 capillary
Acknowledgments
We thank Jianyang Pan (Research and Service Center, College of Pharmaceutical Sciences, Zhejiang University) for performing NMR spectrometry for structure elucidation. This work was supported by grants from the key project of Zhejiang Provincial Natural Science Foundation of China (LZ15H300001), and the Science and Technology Commission of Shanghai Municipality (17431903000).
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