Design and Synthesis of Arf1-Targeting γ-Dipeptides as Potential Agents against Head and Neck Squamous Cell Carcinoma

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer-related deaths and calls for new druggable targets. We have previously highlighted the critical role of ADP-ribosylation factor-1 (Arf1) activation in HNSCC. In the present study, we address the question whether targeting Arf1 could be proposed as a valuable strategy against HNSCC. Methods: We rationally designed and synthesized constrained ATC-based (4-amino-(methyl)-1,3-thiazole-5-carboxylic acid) γ-dipeptides to block Arf1 activation. We evaluated the effects of these γ-dipeptides in HNSCC cells: The cell viability was determined in 2D and 3D cell cultures after 72 h treatment and Arf1 protein levels and activity were assessed by GGA3 pull-down and Western blotting assays. Results: Targeting Arf1 offers a valuable strategy to counter HNSCC. Our new Arf1-targeting compounds revealed a strong in vitro cytotoxicity against HNSCC cells, through inhibiting Arf1 activation and its downstream pathways. Conclusions: Arf1-targeting γ-dipeptides developed in this study may represent a promising targeted therapeutic to improve managing the HNSCC disease.


I. General conditions
Commercially available reagents and solvents were used without any further purification. Reactions were monitored by HPLC with an analytical Chromolith Speed Rod RP-C18 185 Pm column (50 X 4.6 mm, 5 μm) using a flow rate of 5.0 ml/min, and gradients from 100/0 to 0/100 eluents A/B over 3 (condition A) or 5 min (condition B), in which eluents A = H2O / TFA 0.1% and B = CH3CN / TFA 0.1%. Detection was performed at λ = 214 and 254 nm with a photodiode array detector. The retention times are reported as follows: LC: tR = [min]. Analytical thin-layer chromatography (TLC) was performed with aluminium-backed silica gel plates coated with a 0.2 mm thickness of silica gel or with aluminium oxide 60 F254, neutral. Column chromatography was performed using 60 Å 40-63 mesh silica gel. The 1 H and 13 C NMR spectra were recorded at room temperature (RT) in deuterated solvents, using a Bruker AC-300 spectrometer, or a Bruker Avance 600 AVANCE III spectrometer equipped with a 5 mm quadruple-resonance probe ( 1 H, 13 C, 15 N, 31 P). The chemical shifts (δ) are given in parts per million relative to tetramethylsilane (TMS) or by using CHCl3, CD3OD and DMSO as references (respectively 7.26, 3.31 and 2.5 ppm for 1 H spectrum and 77.16, 49.0 and 39.52 ppm for 13 C spectrum). Coupling constants (J) are reported in hertz (Hz). Standard abbreviations indicating multiplicity were used as follows: s (singlet), br (broad), d (doublet), dd (doublet doublet), t (triplet), q (quartet), m (multiplet). Infrared (IR) spectra have been recorded on a Perkin Elmer Spectrum One spectrometer. LC-MS spectra (ESI) were recorded with a Quattro micro ESI triple quadrupole mass spectrometer (Micromass, Manchester, UK). The HPLC separations were done using an analytical Chromolith Speed Rod RP-C18 185 Pm column (50 X 4.6 mm, 5 μm) and an Alliance HPLC System (Waters, Milford, USA) at a flow rate of 3.0 ml/min, and a gradient of 100/0 to 0/100 eluents A/B over 2.5 min (solvent A = H2O / HCOOH 0.1% and solvent B = CH3CN /HCOOH 0.1%). High-resolution mass spectrometric analyses (HRMS) were performed with a time-of-flight (TOF) mass spectrometer fitted with an electrospray ionization source (ESI). All measurements were performed in the positive-ion mode. Melting points were recorded with a capillary melting point apparatus. All reactions were carried out under an atmosphere of nitrogen unless otherwise indicated.

II. Synthetic procedures and characterizations
ATC 4a,d was prepared according to the procedure previously reported by our group. [7,8] Intermediates 1a,b and N-Fmoc-ATC-OBn 5a,b and N-Fmoc-ATC-O-dimethylallyl 4c,d, were previously described.
Imidazolide Formation: In a 250 ml two-neck flask under a nitrogen atmosphere was dissolved the Fmoc-AA-OH (28.59 mmol, 1.0 equiv.) in 100 ml dry THF. Then, CDI (5.10 g, 31.45 mmol, 1.1 equiv.) was added in three portions. A catalytic amount of DMAP (105 mg, 0.857 mmol, 0.03 equiv.) was added 3 min later, and the solution was stirred for 1 h at room temperature (RT).

Condensation:
The imidazolide solution was added dropwise to the enolate solution at -78 °C over 5 min. After stirring for 15 min (HPLC/TLC monitoring), the mixture was removed from the cold bath and poured onto a solution of 10 % aqueous citric acid (200 ml) until pH 7. The crude was extracted twice with 100 ml EtOAc. The combined organic layers were washed with water then with 100 ml saturated NaHCO3 solution and brine (3 x 150 ml), dried on MgSO4, filtered, and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel, with a gradient of cyclohexane/ EtOAc from 95/5 vv to 50/50 vv over 10 column volumes.

Synthesis of bromo-β-keto esters:
To a solution of the β-keto ester 1a,b (2.87 mmol, 1.0 equiv.) in CH3CN (20 ml) was added magnesium perchlorate (210 mg, 0.95 mmol, 0.3 equiv.). The solution was stirred at -45 °C for 10 min. A solution of NBS (537 mg, 3.01 mmol, 1.1 equiv.) in CH3CN (15 ml) was then added dropwise over 5 min. The reaction was complete after few minutes stirring at -45 °C (HPLC monitoring). The mixture was diluted with Et2O (40 ml) and washed twice with 40 ml water and with brine (3 x 20 ml). The organic layer was dried with MgSO4 and filtered. The solvent was removed under reduced pressure to give the crude product, which was used in the next step without further purification.

General Procedures C of deprotections
C1: General procedure for dimethyl allyl removal [7,8] Pd(PPh3)4 (0.0245 g, 0.0212 mmol, 0.03 equiv.) was dispersed into a solution of O-dimethyl allyl ester (0.706 mmol, 1.0 equiv.) and PhSiH3 (0.261 ml, 2.12 mmol, 3 equiv.) in 30 ml of anhydrous THF under nitrogen atmosphere. After stirring for 3 hours (HPLC monitoring), the reaction was stopped by adding a mix of diethyl ether (10 ml) and ethyl acetate (2ml). A white solid formed in the medium was collected by filtration and was used without any further purification.
C2: General procedure for Fmoc removal Fmoc derivative (1.23 mmol, 1.0 equiv.) was solubilized in 25 ml of diethylamine / DMF (1:9 vv) and stirred for 30 min at RT. The solvent was evaporated and the crude was used without any further purification.
3b,c were deprotected following the general procedure C1 to afford the free acid 4b,c which was used without any further purification. The acidic intermediate 4b,c (1.703 g, 3.772 mmol, 1 equiv.) was converted to its cesium salt by dissolving in MeOH (50 ml) and addition of a solution of 20 % mv cesium carbonate in water (3.0 ml, 0.615 g, 1.886 mmol, 0.5 equiv.). The solvent was removed under reduced pressure then the product was dissolved in DMF (20 ml). Benzyl bromide (0.496 ml, 4.149 mmol, 1.1 equiv.) was added to the mixture. The solution was stirred 1 hour at RT and the DMF was evaporated under reduced pressure. The solid residue was partitioned between AcOEt (120 ml) and water (80 ml). The organic layer was washed with water (1 x 80 ml) and brine (1 x 80 ml) then dried with MgSO4 and filtered. The solvent was evaporated under reduced pressure to yield the crude product. Purification by chromatography on a silica gel column with a gradient of cyclohexane/ EtOAc from 95/5 to 50/50 afforded 6a',b'.  5, 21.9, 23.4, 25.1, 45.2, 47.4, 48.9, 66.8, 67.3, 120.

Synthesis of N-benzyl amide 5a'
Scheme S5 : Synthesis of ATC 5a' . reagents and conditions: a) Pd(PPh3)4, PhSiH3, THF, RT, 3h; c) IBCF, DIEA, benzylamine, THF, 0°C to RT,2h. 3b was deprotected following the general procedure C1 to afford the free acid 4b which was used without any further purification. The acidic intermediate 4b (0.556 g, 1.23 mmol, 1 equiv.) was dissolved in anhydrous THF (30 ml). The solution was cooled to 0 o C and isobutylchloroformate (IBCF, 0.177 ml, 1.36 mmol, 1.2 equiv.) and DIEA (0.234 ml, 1.36 mmol, 1.2 equiv.) were added in one portion. The reaction was stirred for 10 minutes at 0 o C then 30 minutes at RT then benzylamine (0.270 ml, 2.46 mmol, 2 equiv.) was added and the reaction was stirred at RT for 1 hour. THF was evaporated under reduced pressure and the crude was dissolved in DCM (60 ml) and washed with a saturated solution of NaHCO3 (2 x 30 ml) and brine (2 x 30 ml). The organic layer was dried with MgSO4 and filtered, and the solvents were evaporated under reduced pressure. The crude was purified by chromatography on a silica gel column with a gradient of cyclohexane/ EtOAc from 95/5 to 70/30 afforded 5a' with a yield of 95% (0.64 g). 5b' was obtained starting from 4c and benzylamine by a slight modification of the previous procedure. Indeed the use of IBCF did not lead to the targeted compound 5b'. We opted then for the use of EDC coupling agent.