Examination of aminophenol-containing compounds designed as antiproliferative agents and potential atypical retinoids.

Retinoic acid (RA, 1), an oxidized form of vitamin A, binds to retinoic acid receptors (RAR) and retinoid X receptors (RXR) to regulate gene expression and has important functions such as cell proliferation and differentiation. Synthetic ligands regarding RAR and RXR have been devised for the treatment of various diseases, particularly promyelocytic leukemia, but their side effects have led to the development of new, less toxic therapeutic agents. Fenretinide (4-HPR, 2), an aminophenol derivative of RA, exhibits potent antiproliferative activity without binding to RAR/RXR, but its clinical trial was discontinued due to side effects of impaired dark adaptation. Assuming that the cyclohexene ring of 4-HPR is the cause of the side effects, methylaminophenol was discovered through structure-activity relationship research, and p-dodecylaminophenol (p-DDAP, 3), which has no side effects or toxicity and is effective against a wide range of cancers, was developed. Therefore, we thought that introducing the motif carboxylic acid found in retinoids, could potentially enhance the anti-proliferative effects. Introducing chain terminal carboxylic functionality into potent p-alkylaminophenols significantly attenuated antiproliferative potencies, while a similar structural modification of weakly potent p-acylaminophenols enhanced growth inhibitory potencies. However, conversion of the carboxylic acid moieties to their methyl esters completely abolished the cell growth inhibitory effects of both series. Insertion of a carboxylic acid moiety, which is important for binding to RA receptors, abolishes the action of p-alkylaminophenols, but enhances the action of p-acylaminophenols. This suggests that the amido functionality may be important for the growth inhibitory effects of the carboxylic acids.

Design and synthesis of a new orthogonally protected glutamic acid analog and its use in the preparation of high affinity polo-like kinase 1 polo-box domain - binding peptide macrocycles.

Targeting protein - protein interactions (PPIs) has emerged as an important area of discovery for anticancer therapeutic development. In the case of phospho-dependent PPIs, such as the polo-like kinase 1 (Plk1) polo-box domain (PBD), a phosphorylated protein residue can provide high-affinity recognition and binding to target protein hot spots. Developing antagonists of the Plk1 PBD can be particularly challenging if one relies solely on interactions within and proximal to the phospho-binding pocket. Fortunately, the affinity of phospho-dependent PPI antagonists can be significantly enhanced by taking advantage of interactions in both the phospho-binding site and hidden "cryptic" pockets that may be revealed on ligand binding. In our current paper, we describe the design and synthesis of macrocyclic peptide mimetics directed against the Plk1 PBD, which are characterized by a new glutamic acid analog that simultaneously serves as a ring-closing junction that provides accesses to a cryptic binding pocket, while at the same time achieving proper orientation of a phosphothreonine (pT) residue for optimal interaction in the signature phospho-binding pocket. Macrocycles prepared with this new amino acid analog introduce additional hydrogen-bonding interactions not found in the open-chain linear parent peptide. It is noteworthy that this new glutamic acid-based amino acid analog represents the first example of extremely high affinity ligands where access to the cryptic pocket from the pT-2 position is made possible with a residue that is not based on histidine. The concepts employed in the design and synthesis of these new macrocyclic peptide mimetics should be useful for further studies directed against the Plk1 PBD and potentially for ligands directed against other PPI targets.

Solid-Phase Azopeptide Diels-Alder Chemistry for Aza-pipecolyl Residue Synthesis To Study Peptide Conformation.

Solid-phase chemistry for the synthesis and Diels-Alder reaction of Fmoc-protected azopeptides has been developed and used to construct aza-pipecolyl (azaPip) peptides. Considering their ability to induce electronic and structural constraints that favor cis-amide isomer geometry and type VI ß-turn conformation in model peptides, azaPip residues have now been introduced into biologically relevant targets by this enabling synthetic method. Turn conformers were shown to be important for receptor affinity, selectivity, and activity by employing azaPip residues to study the conformational requirements of opioid and cluster of differentiation 36 receptor peptide ligands.

Azapeptide Synthesis Methods for Expanding Side-Chain Diversity for Biomedical Applications.

Mimicry of bioactive conformations is critical for peptide-based medicinal chemistry because such peptidomimetics may augment stability, enhance affinity, and increase specificity. Azapeptides are peptidomimetics in which the α-carbon(s) of one or more amino acid residues are substituted by nitrogen. The resulting semicarbazide analogues have been shown to reinforce ß-turn conformation through the combination of lone pair-lone pair repulsion of the adjacent hydrazine nitrogen and urea planarity. Substitution of a semicarbazide for an amino amide residue in a peptide may retain biological activity and add benefits such as improved metabolic stability. The applications of azapeptides include receptor ligands, enzyme inhibitors, prodrugs, probes, and imaging agents. Moreover, azapeptides have proven therapeutic utility. For example, the aza-glycinamide analogue of the luteinizing hormone-releasing hormone analogue Zoladex is a potent long-acting agonist currently used in the clinic for the treatment of prostate and breast cancer. However, the use of azapeptides was hampered by tedious solution-phase synthetic routes for selective hydrazine functionalization. A remarkable stride to overcome this bottleneck was made in 2009 through the introduction of the submonomer procedure for azapeptide synthesis, which enabled addition of diverse side chains onto a common semicarbazone intermediate, providing a means to construct azapeptide libraries by solution- and solid-phase chemistry. In brief, aza residues are introduced into the peptide chain using the submonomer strategy by semicarbazone incorporation, deprotonation, N-alkylation, and orthogonal deprotection. Amino acylation of the resulting semicarbazide and elongation gives the desired azapeptide. Since the initial report, a number of chemical transformations have taken advantage of the orthogonal chemistry of semicarbazone residues (e.g., Michael additions and N-arylations). In addition, libraries have been synthesized from libraries by diversification of aza-propargylglycine (e.g., A3 coupling reactions, [1,3]-dipolar cycloadditions, and 5-exo-dig cyclizations) and aza-chloroalkylglycine residues. In addition, oxidation of aza-glycine residues has afforded azopeptides that react in pericyclic reactions (e.g., Diels-Alder and Alder-ene chemistry). The bulk of these transformations of aza-glycine residues have been developed by the Lubell laboratory, which has applied such chemistry in the synthesis of ligands with promising biological activity for treating diseases such as cancer and age-related macular degeneration. Azapeptide analogues of growth hormone-releasing peptide-6 (His-d-Trp-Ala-Trp-d-Phe-Lys-NH2, GHRP-6) have for example been pursued as ligands of the cluster of differentiation 36 receptor (CD36) and show promising activity for the development of treatments for angiogenesis-related diseases, such as age-related macular degeneration, as well as for atherosclerosis. Azapeptides have also been employed to make a series of conformationally constrained second mitochondria-derived activator of caspase (Smac) mimetics that exhibit promising apoptosis-inducing activity in cancer cells. The synthesis of cyclic azapeptide derivatives was used to make an aza scan to study the conformation-activity relationships of the anticancer agent cilengitide, cyclo(RGDf-N(Me)V), and its parent counterpart cyclo(RGDfV), which exhibit potency against human tumor metastasis and tumor-induced angiogenesis. Innovations in the synthesis and application of azapeptides will be presented in this Account, focusing on the creation and use of side-chain diversity in medicinal chemistry.

Application of constrained aza-valine analogs for Smac mimicry.

Constrained azapeptides were designed based on the Ala-Val-Pro-Ile sequence from the second mitochondria-derived activator of caspases (Smac) protein and tested for ability to induce apoptosis in cancer cells. Diels-Alder cyclizations and Alder-ene reactions on azopeptides enabled construction of a set of constrained aza-valine dipeptide building blocks, that were introduced into mimics using effective coupling conditions to acylate bulky semicarbazide residues. Evaluation of azapeptides 7-11 in MCF-7 breast cancer cells indicated aza-cyclohexanylglycyine analog 11 induced cell death more efficiently than the parent tetrapeptide likely by a caspase-9 mediated apoptotic pathway. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 235-244, 2016.

Enhancing precision in colorectal cancer surgery: development of an LGR5-targeting RSPO1 peptide mimetic as a contrast agent for intraoperative fluorescence molecular imaging.

Colorectal cancer (CRC) is the third most common cancer worldwide. In the United States alone, CRC was responsible for approximately 52,550 deaths in 2023, with an estimated 153,020 new cases. CRC presents with synchronous peritoneal spread in 5-10% of patients, and up to 20-50% of patients with recurrent disease will develop metachronous colorectal cancer peritoneal metastatic (CRC-PM) disease. Eradication of the tumor, tumor margins and microscopic residual disease is paramount, as microscopic residual disease is associated with local recurrences, with 5-year survival rates of less than 35%. The success of resection and reduction of residual disease depends on the accuracy with which cancer cells and normal tissue can be intra-operatively distinguished. Fluorescence Molecular Imaging (IFMI) and tumor-targeted contrast agents represent a promising approach for intraoperative detection and surgical intervention. Proper target selection, the development of scalable imaging agents and enhanced real-time tumor and tumor microenvironment imaging are critical to enabling enhanced surgical resection. LGR5 (leucine-rich repeat-containing G-protein-coupled receptor 5), a colonic crypt stem cell marker and the receptor for the R-spondins (RSPO) in the Wnt signaling pathway, is also expressed on colorectal cancer stem cells (CSC) and on CRC tumors and metastases, suggesting it could be a useful target for imaging of CRC. However, there are numerous diverging reports on the role of LGR5 in CRC therapy and outcomes. Herein, we report on the synthesis and validation of a 37 amino acid RSPO1-mimetic peptide, termed RC18, that was specifically designed to access the R-spondin binding site of LGR5 to potentially be used for interoperative imaging of CRC-PM. The receptor-binding capabilities of the RC18 indicate that direct interactions with LGR5 neither significantly increased LGR5 signaling nor blocked RSPO1 binding and signal transduction, suggesting that the RSPO1-mimetic is functionally inert, making it an attractive contrast agent for intraoperative CRC-PM imaging.

Diversity-Oriented A3-Macrocyclization for Studying Influences of Ring-Size and Shape of Cyclic Peptides: CD36 Receptor Modulators.

Cyclic peptide diversity has been broadened by elaborating the A3-macrocyclization to include various di-amino carboxylate components with different Nε-amine substituents. Triple-bond reduction provided new cyclic peptide macrocycles with Z-olefin and completely saturated structures. Moreover, cyclic azasulfurylpeptides were prepared by exchanging the propargylglycine (Pra) component for an amino sulfamide surrogate. Examination of such diversity-oriented methods on potent cyclic azapeptide modulators of the cluster of differentiation 36 receptor (CD36) identified the importance of the triple bond as well as the Nε-allyl lysine and azaPra residues for high CD36 binding affinity. Cyclic azapeptides which engaged CD36 effectively reduced pro-inflammatory nitric oxide and downstream cytokine and chemokine production in macrophages stimulated with a Toll-like receptor-2 agonist. Studying the triple bond and amine components in the multiple-component A3-macrocyclization has given a diverse array of macrocycles and pertinent information to guide the development of ideal CD36 modulators with biomedical potential for curbing macrophage-driven inflammation.

Cyst Reduction in a Polycystic Kidney Disease Drosophila Model Using Smac Mimics.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited malady affecting 12.5 million people worldwide. Therapeutic options to treat PKD are limited, due in part to lack of precise knowledge of underlying pathological mechanisms. Mimics of the second mitochondria-derived activator of caspases (Smac) have exhibited activity as antineoplastic agents and reported recently to ameliorate cysts in a murine ADPKD model, possibly by differentially targeting cystic cells and sparing the surrounding tissue. A first-in-kind Drosophila PKD model has now been employed to probe further the activity of novel Smac mimics. Substantial reduction of cystic defects was observed in the Malpighian (renal) tubules of treated flies, underscoring mechanistic conservation of the cystic pathways and potential for efficient testing of drug prototypes in this PKD model. Moreover, the observed differential rescue of the anterior and posterior tubules overall, and within their physiologically diverse intermediate and terminal regions implied a nuanced response in distinct tubular regions contingent upon the structure of the Smac mimic. Knowledge gained from studying Smac mimics reveals the capacity for the Drosophila model to precisely probe PKD pharmacology highlighting the value for such critical evaluation of factors implicated in renal function and pathology.

Azasulfurylpeptide Modulation of CD36-Mediated Inflammation Without Effect on Neovascularization.

Modulation of the cluster of differentiation-36 receptor (CD36) has proven promising for dampening pro-inflammatory macrophage signaling. For example, azapeptides (e.g., 1 and 2) bind CD36 selectively with high affinity, mitigate Toll-like receptor (TLR) agonist-induced overproduction of nitric oxide (NO), and reduce pro-inflammatory cytokine and chemokine production in macrophages. Moreover, semicarbazides 1 and 2 inhibit microvascular sprouting mediated through CD36 in the choroid explant. Seeking a selective CD36 modulator that mediated inflammation without influencing neovascularization, a set of azasulfurylpeptides (e.g., 3a⁻e) were synthesized in which the semicarbazide was replaced by an N-aminosulfamide residue using a novel solid-phase approach. Notably, azasulfurylpeptide 3c diminished selectively CD36-mediated TLR-2-triggered inflammatory response without affecting neovascularization. Subtle chemical modification at the peptide backbone from a carbonyl to a sulfuryl residue has had a selective effect on biological activity providing a valuable probe for studying CD36 chemical biology.

Azopeptides: Synthesis and Pericyclic Chemistry.

Azopeptides possess an imino urea as an amino amide surrogate in the sequence. Azopeptides were synthesized by oxidation of aza-glycine residues and employed in pericyclic chemistry. Diels-Alder cyclizations and Alder-ene reactions on azopeptides enabled construction of constrained aza-pipecolyl and reactive aza-allylglycyl residues. X-ray crystallographic analyses of azopeptide 16a and azapeptides 30a and 35a provided insight into imino urea configuration and conformational affects of cycloalkane side chains at the semicarbazide α- and ß-nitrogen, respectively.