Novel peptidomimetics: synthesis, characterization, and molecular modeling studies

Since PD-1/PD-L1 plays a crucial role as immune checkpoint molecules in many cancers, developing immunotherapy with peptidomimetics that block these molecules is an important option in anticancer therapy. In this study, several peptidomimetic compounds were screened from the literature using computational methods, and it was determined that 2-amino-benzothiazole-based peptidomimetics show affinity for the active site of PD-L1. In the light of molecular modelling studies, six molecules with free binding energies ranging from -6.7 to -7.2 kcal/mol were chosen from the library of 2-amino-benzothiazole-based peptidomimetics for synthesis. These synthesized six compounds were characterized with 1 H-NMR, 13 C-NMR, FTIR and HRMS. Molecular modelling studies showed that these novel 2-amino-benzothiazole-based peptidomimetics may be potential checkpoint inhibitors for cancer immunotherapy.


Introduction
Immune checkpoint is a sort of signal to regulate the processes of T cells in immune responses.There are two types of signals based on antigen recognition by T cells; stimulatory signals to stimulate immune progression or inhibitory signals to suppress immune progression.
Programmed cell death 1 receptor (PD-1) and its ligand programmed cell death ligand 1 (PD-L1) are a type of inhibitory immune checkpoint molecules.PD-1 is found on the cell surface of T cells while PD-L1 is expressed in dendritic cells, T cells, B cells, macrophages, and cancer cells.When PD-1 receptor binds to its ligand PD-L1, the immune response of T-cells is supressed.Increased PD-L1 expression within the tumor microenvironment has been identified as a hindrance to the antitumoral T-cell response.Many experimental studies on PD-1 blockers confirmed that these molecules are clinically effective in various malignancies and proved that these inhibitors are promising anticancer agents. 1 Immunotherapy, a type of anticancer treatment, involves the utilization of inhibitors that specifically target the interaction of immune checkpoint molecules.Immune checkpoint inhibitors used in immunotherapy can be in the form of peptide-based, non-peptide small molecule, or monoclonal antibodies (mAbs)2.In the context of immunotherapy, five mAbs targeting PD-1 (Nivolumab, Pembrolizumab) and PD-L1 (Atezolizumab, Avelumab, Durvalumab) have been approved by the FDA for use in anticancer therapy in the clinic. 3,4Although immunotherapy based on mAbs targeting the PD-1/PD-L1 pathway offers unique clinical responses, these inhibitors have some limitations, such as high production costs, poor oral bioavailability, long half-life, poor tissue and tumor penetration. 5The area where antibodies typically engage with proteins consists of peptide chains.The PD-1/PD-L1 features a partially flat and hydrophobic interaction surface which significantly constrains the development of novel inhibitors. 6One of the approaches to overcome these challenges is the development of peptidomimetics as an alternative to mAbs.
Progress is currently being made in the development of small molecules, particularly peptidomimetics, that block PD-1/PD-L1. 7Peptidomimetics are derived from the modification of peptide sequences by replacing the peptide backbone with a molecular scaffold to improve biological properties.Such compounds are designed to be highly stable, selective, and metabolically bioavailable.So, peptidomimetics appear to be an important tool for targeting protein-protein interactions such as immune checkpoint molecules in drug discovery studies.Consequently, in immunotherapy, the exploration of peptidomimetic compounds as potential antagonists to inhibit the PD-1/PDL-1 interaction is contemplated as an alternative approach.
The present study aims to design and synthesize novel peptidomimetic compounds directed towards the PD-1/PDL-1 pathway.Numerous peptidomimetic compounds were screened from the literature using computational methods, and it was determined that 2-amino-benzothiazole-based peptidomimetics (Figure 1) display a pronounced affinity for the active site of PD-L1.In the light of docking studies, a library of 2-aminobenzothiazole-based peptidomimetics was constructed, which are specific to PD-L1 and six of these compounds (8a-b, 9a-b and 10a-b) were chosen for synthesis.The six synthetic compounds were characterized with 1 H-NMR, 13 C-NMR, FTIR and HRMS.In the second step, the synthesis of intermediates 5a-b were achieved with 75-82% yield as a result of the substitution reaction of methyl 4-hydroxybenzoate and substituted benzylic halides 4a-b in basic medium.Following this, substituted benzoic acid intermediates 6a-b were obtained in high yields, ranging from 95% to 97%, as a result of basic hydrolysis of the ester groups.Substituted benzoic acid intermediates in the presence of diethylaminosulfide trifluoride (DAST) formed substituted benzoyl fluorides 7a-b in good yields by nucleophilic fluoridation (Scheme 2).In this reaction, the disappearance of the OH band in the FTIR spectrum of compounds 7a-b was accompanied by a noticeable shift in the frequency of the C=O group.In the final step, the reaction of the intermediate 2-aminobenzothiazole derivatives with substituted benzoyl fluoride was carried out in the presence of diisopropylethylamine (DIPEA).The obtained compound 8a-b was subjected to alkylation in a basic medium, resulting in the formation of compound 9a-b with a yield of 68-70%.Two different methods were employed for the reduction of compound 9a-b molecules.One of these methods involves SnCl2-catalyzed reduction in the presence of acid, while the other involves Fecatalyzed reduction.Accordingly, peptidomimetic compounds 10a and 10b were obtained with yields of 50% and 70%, respectively (Scheme 3). 10,11 AUTHOR(S)

Molecular docking studies
In molecular docking studies, the best binding affinity and receptor-ligand interaction of all compounds were evaluated.Good interactions of the newly synthesized peptidomimetic compounds within the target PD-L1 receptor active site are given in Table 2. Compounds 10a and 9b showed good binding affinity towards PD-L1 with free binding energies ΔG:-7.2 kcal/mol and -7.1 kcal/mol, respectively.The interaction diagrams of compounds 10a and 9b were shown in Figures 2 and 3.The best docking interactions of compounds 10a and 9b with the PD-L1 are shown in Figures 4 and 5.
Compound 10a interacted with amino acids ILE A: 54 and VAL A: 68 via pi-sigma bonds with alkyl groups.On the other hand, - stacked interaction was observed with TYR A:56 amino acid via the benzothiazole ring.Moreover, compound 10a implicated van der Waals interactions with MET A: 115, VAL A: 76, ASN A: 63, HIS A: 78, LYS A:62, ASP A: 61, HIS A:69, GLU A: 72, GLU A:71, ASP A:73, GLN A:66 and SER A: 117.Compound 9b demonstrated a - interaction with MET A:115, while also engaging in a -alkyl interaction with the alkyl group of ALA A:12.In the 2D diagram of interactions between PD-L1 and 9b, Van der Waals interactions were observed with SER A:117, ILE A:54, VAL A:68, GLN A:66, LYS A:62, ASN A:63, ASP A:61, VAL A:76, ASP A:122, TYR A:123 and ILE A:116.In addition to all these interactions, it forms a - bond with TYR A:56 amino acid.The radius of gyration (Rg) measures the compactness of the protein in which also shows the stability of the inhibitors with its complexes.Rg profiles of the PD-L1 receptor alone and its complexes with the two hits (9b and 10a) are plotted as shown in the figures (Figure 6).All the systems show low Rg trends in between 1.24 Å and 1.32 Å over 200 ns so that the protein kept stable throughout the simulation.This means that 9b and 10a are perfect inhibitor candidates for PD-L1 receptor.

Conclusions
In this study, six new 2-amino-benzothiazole-based peptidomimetics (8a-b, 9a-b, 10a-b) that could bind with high affinity to PDL1, which is known to be overexpressed on various cancer such as breast cancer, lung cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, pancreatic cancer, ovarian cancer, and bladder cancer, were identified through docking studies.Then, molecular docking simulations were performed for these compounds.RMSD and RG results were obtained for PD-L1 receptor-ligand complexes.These six peptidomimetics were synthesized, purified in high yield and Page 10 of 14 © AUTHOR(S) characterized by 1 H-NMR, 13 C-NMR, HRMS and FTIR.This study showed that, from an in-silico perspective, these peptidomimetics may be suitable candidates as immune checkpoint inhibitors for the PD-1/PD-L1 pathway.We conclude that these new peptidomimetics will give promising results in cancer treatment in the light of preclinical studies planned to be conducted in the future.

Experimental Section
General.All utilized chemicals and solvents were of analytical grade, sourced from commercial vendors (Merck, Sigma-Aldrich, Acros Organics) and used without further purification.The solvents employed in chromatography were of technical grade and underwent distillation prior to utilization.The compounds 1-3 and 5b-7b, which were used as starting materials, were synthesized according to our previously published work. 12 1H NMR and 13 C NMR spectra were recorded at 500 MHz and 126 MHz, respectively.DMSO-d6 was used as a solvent, and Me4Si was used as the internal standard.FTIR spectra were recorded on a Perkin-Elmer FTIR System Spectrum BX (Massachusetts, USA) over the range 4000-500 cm −1 spectrophotometer.TLC spots were marked under UV light (254 nm, t = 25 °C).The synthesized compounds were characterized by an Agilent 6530 Q-TOF mass spectrometer equipped with an Electrospray Ionization (ESI) source.
Copies of NMR, MS and FTIR spectra of the synthesized compounds are given in the Supporting Information.

Preparation of 4-(3-nitrobenzyloxy)benzoic acid (6a)
. 9 4-(3-Nitro-benzyloxy)-benzoic acid methyl ester 5a (2.63 mmol) was dissolved in 1,4-dioxane (30 mL).NaOH solution (2.75 mL of 1.4 M) was added dropwise and stirred for 2 hours at 70 °C.At the end of the reaction, the dioxane was removed under reduced pressure and the residue was acidified to pH 2 with HCl solution (1 M).The precipitate was separated by vacuum filtration.After washing with water, dried under vacuum.Yield: 95%.Procedure for the synthesis of 10a. 11The corresponding intermediate 9a (1 equiv., 0.15 mmol) was dissolved in methanol (20 mL).SnCl2 (4 equiv., 0.6 mmol) was dissolved in HCl (37%, 5 mL) and added dropwise to the reaction medium.The reaction was stirred for 5 hours at 70 o C. At the end of the reaction, the methanol was evaporated under reduced pressure.The precipitate was dissolved in distilled water and the pH of the solution was brought to 11 with 1% NaOH solution.The resulting suspension mixture was precipitated by centrifugation, extracted with DCM (30 mL x 3) and distilled water (30 mL x 3).The organic phase was dried over Na2SO4 and the solvent was evaporated under reduced pressure.The product was washed with cold methanol and dried under vacuum.Yield: 50%.

4-(3-Aminobenzyloxy)-N-benzyl-N-[6-(4-t-butylphenoxy)benzothiazol-2-yl] benzamide (10a
mL) and acetic acid (10 mL).HCl (37%, 1 mL) and Fe powder (9 mmol) were added into the mixture.The reaction was then continued in an ultrasonic bath at rt.At the end of the reaction, the ethanol was evaporated under reduced pressure.The pH of the mixture was brought to 11 with 1% NaOH solution, extracted with DCM (30 mL x 3) and distilled water (30 mL x 3).The organic phase was dried over Na2SO4 and the solvent was evaporated under reduced pressure.The product was washed with cold methanol and dried under vacuum.Yield: 70%.

Molecular docking
PD-L1 is a crucial protein targeted in colorectal and breast cancer microenvironments in docking studies. 13tructural Bioinformatics Research Collaboration (RCSB) Protein Data Bank was used for the selection of the targeted receptor and the 5j80 coded receptor was preferred. 14In receptors crystal structure, PD-L1 and its inhibitor were observed as a complex.Docking processes were carried out between the relevant pdb-coded receptors and the candidate inhibitors synthesized, and the region where the inhibitor in the receptor complex binds was used as the binding site. 15Candidate molecules were drawn with the ChemDraw program

Figure 6 .
Figure 6.Root-mean-square deviation (RMSD) and radius of gyration (Rg) profiles displayed over 200 ns simulation of the PDl_1 receptor alone and its complexes with 9b and 10a.