Amino acids, reagents and solvents were purchased from Bachem or Aldrich. Cyclic peptides were synthesized with the first Fmoc amino acid linked to a chloro-trityl polystyrene resin (TentaGel S Trt Cl from RAPP Polymere GmbH) or alternatively, 2-Chlorotrityl chloride resin (100–200 mesh, 1% DVB, 1.0–1.6 mmol Cl/g, from ABCR). Syntheses were carried out manually or alternatively automatic syntheses were carried out using an Initiator + Alstra microwave peptide synthesizer from Biotage or a Symphony synthesizer from PTI.
Peptides were prepared utilizing a synthetic strategy involving attachment of the Fmoc protected C-terminal amino acid e.g. Fmoc-d-Pro residue via the C-terminal carboxy group to the 2-Cl-Trt-resin and subsequent assembly of the linear peptide on the resin. Peptides were prepared by automated peptide synthesis, cleaved from the resin, subsequently cyclized in solution and purified by preparative HPLC. Typically, peptides were synthesized as follows: Fmoc removal was performed using 4-methylpiperidine/DMF 1:4 (2 × 5 min, 5 mL). The resin was washed with DMF (× 3) and DCM (× 4) for 10 min each. Coupling of AAs was performed using 2 eq. of Fmoc-AA-OH, 4.0 eq. of HATU and 8.0 eq. of DIPEA at room temperature. Subsequently, the resin was washed with DMF (× 3) and DCM (× 4). The linear peptide resin was cleaved from the resin by shaking at room temperature for 3 × 10 min then 1 × 5 min with HFIP/DCM (25:75) (6 mL each time). The resin was then washed with DCM (× 2) and the cleavage and washing solutions were filtered off, then concentrated to dryness in-vacuo. Subsequent to cleavage, cyclization was carried out in solution. Typically a 500 mL round-bottomed flask was equipped with a magnetic stirrer and stirring was carried out at 45 °C for several hours. Preparative reversed phase purification was carried out as follows: Column: Waters XBridge Prep C18 5 μm OBD, 30 × 250 mm; Waters Part No 186004025; Eluent A: 0.1% TFA in water; Eluent B:AcN; Flow: 30 mL/min; Gradient: 30% B linearly increasing in 30 min to 100% B, fractions collected with a Waters 2767 Sample Manager and UV detection with a Waters 2996 Photo Diode Array Detector linked to a Waters 2525 Binary Gradient Module Pump. Analytical UPLC-MS was carried out as follows: Peptide standard method: Waters Synapt G2; column: Acquity UPLC CSH C18, 1.7 μm, 2.1x100 mm at 80 °C, Eluent A: H2O + 0.05% TFA, B:AcN + 0.05% TFA, Gradient: hold 5% B for 0.2 min; from 5 to 98% B in 9.2 min, flow: 0.5 mL/min. Peptide non-polar method: Waters Synapt G2; column: Acquity UPLC BEH C4, 1.7 μm, 2.1 × 100 mm at 80 °C, Eluent A: H2O + 0.05% TFA, B:AcN + 0.05% TFA, Gradient: hold 5% B for 0.2 min; from 5 to 98% B in 4.8 min, flow: 0.5 mL/min.
In case of further MPLC purification, the peptide was typically dissolved in 6 mL water and purified on a medium pressure MPLC-C18 Column [Merck Lichroprep (15–25 µm) 26 × 100 mm] with 20 mL/min flow-rate, eluent A:Water, eluent B:AcN, gradient 100% A 2 min isocratic, 100% A 4 min increasing linearly to 70% A:30% B, 4 min isocratic 70% A:30% B, UV Detection: 225 nm providing the purified peptide as a white lyophilisate after lyophilization. For desalting, preparative HPLC fractions containing the peptide as a TFA salt are typically mixed with 0.6 g NaHCO3, concentrated to 50 mL and pumped over the water equilibrated MPLC-C18 column (Merck Lichroprep (15–25 µm) 26 × 100 mm) with 50 mL/min flow-rate, eluent A: Water, eluent B:AcN, gradient 100%A—2 min—> 100%A—30 s—10%A—3 min—> 0%A—4 min—> 0%A, UV Detection: 225 nm providing the de-salted peptide as a white lyophilisate after lyophilization.
Synthesis of Compound (1): [Phe-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro]: Cyclization Procedure
Crude H2N-Phe-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH (216 mg, 0.291 mmol) was dissolved in DCM (290 mL). HOAt (59.4 mg, 0.436 mmol), HATU (442 mg, 1.163 mmol) and 2,6-lutidine (1.016 mL, 8.72 mmol) were subsequently added and the reaction mixture was vigorously stirred. HPLC and UPLC-MS analysis after 1 h showed complete conversion. Solvent was removed in-vacuo and the crude product was dissolved in 2 mL NMP and 2 mL H2O. The crude product was purified by preparative HPLC providing pure cyclic peptide (110 mg, 152 mmol, 52% yield, > 99% % purity, (M + H)+ = 725.5, tR = 3.67 min) as a white lyophilisate.
Synthesis of Precursor of Compound (2): H2N-2-L-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH
Coupling was carried out as follows: TPTU (202 mg, 0.680 mmol) dissolved in 6 mL of NMP/DCE (1/1) to provide solution 1. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(pyridin-2-yl)propanoic acid, Fmoc-2-Pal-OH (264 mg, 0.680 mmol) was dissolved in 8 mL of NMP/DCE (1/1) and 479 μL of collidine was added. The solution was added to the resin and heated to 45 °C while stirring to provide solution 2. Then, solution 1 was added slowly (60 mL/h) in 10 min to the solubilized resin in solution 2. After the additon, the black reaction mixture was stirred for 5 min at RT. The cleavage of the resin was carried out for 2x15 min with a solution of 40% HFIP in DCM. All the fractions were collected together and volatiles were fully evaporated with high vacuum (DCM and HFIP). The crude linear peptide was then purified prior to cyclization by preparative HPLC (45% B for 7 min, 60% B for 7 min, 70% B for 2 min). The linear peptide H2N-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH (270 mg, 363 mmol, 61.3% yield, 100% purity, (M + H)+ = 743.5, tR = 1.579) was isolated as a white lyophilisate and the diastereomeric linear peptide H2N-D-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH (87 mg, 117 mmol, 19.7% yield, 100% purity, (M + H)+ = 743.5, tR = 1.604) was isolated additionally as a white lyophilisate.
Synthesis of Compound (2): [2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro]: Cyclization Procedure
The linear peptide H2N-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH was dissolved in 25 mL AcN, with DIPEA (2 eq) to provide solution 1. In 25 mL AcN, HATU (3 eq) and DIPEA (4 eq) were dissolved (this addition has to be done at the last minute) and this solution was heated to 45 °C while stirring to provide solution 2. Solution 1 was then added slowly (100 mL/h) in 15 min to the solution 2. After the additon, the orange reaction mixture was stirred for 5 min at RT. The crude cyclized peptide was purified by preparative HPLC to provide the cyclic peptide (132.5 mg, 183 mmol, 48.5% yield, 97% purity UPLC-MS-Non-Polar method, (M + H)+ = 726.5, tR = 3.92 min, 96% purity, UPLC-MS-Standard method, tR = 4.91 min) as a white lyophilisate. Chiral integrity of compound (2) was investigated by C.A.T. Tuebingen, verifying 98.82% 2-Pal and 1.18% undesired D-Pal enantiomer.
Synthesis of Building Block for Compound (3): Boc-NMe-2-Pal-OH
Boc-2-Pal-OH (500 mg, 1.784 mmol) was dissolved in 10 mL dry THF. The solution was cooled to 0 °C under vigorous stirring, then 4 eq. MeI (0.451 mL, 7.13 mmol) was added. Subsequently 5 eq of NaH (~ 356 mg, dispersion in oil) was added and after 30 min, a further 5 eq of NaH (~ 356 mg, dispersion in oil) was added to the solution at 0 °C. The solution was then warmed up to 45 °C and after 60 min, full conversion was observed. The solution was then cooled to 0 °C and 12 mL of AcOH (dilute) were added, then the solution was evaporated to dryness. The residue was then dissolved in 6 mL water and purified on a medium pressure MPLC-C18 Column (Merck Lichroprep (15–25 µm) 26 × 100 mm) with 20 mL/min flow-rate, eluent A:Water, eluent B:AcN, 100% A 2 min isocratic, 100% A 4 min increasing linearly to 70% A:30% B, 4 min isocratic 70% A:30% B. UV Detection: 225 nm providing the Boc-2-NMe-Pal)-OH as a white lyophilisate (320 mg, 1.139 mmol, 63.4% yield, 99% purity, (M + H)+ = 281.1, tR = 2.80 min), 1H NMR (400 MHz, DMSO-d6) δ 12.84 (s br, 0H), 8.53–8.44 (m, 1H), 7.73–7.69 (m, 1H), 7.29–7.16 (m, 2H), 4.97, 4.83 (1H) (dd, J = 10.4, 5.2 Hz) (dd, J = 10.7, 4.6 Hz), 3.30–3.13 (m, 2H), 2.69, 2.64 (s, 3H), 1.29, 1.24 (s, 9H).
Synthesis of Precursor of Compound (3): H2N-NMe-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH
The peptide-resin Fmoc-HN-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-Cl-Trt-Resin was swollen in DCM (+ 0.3% of DIPEA) for 15 min. Subsequently Fmoc was removed using 4-methylpiperidine/DMF 1:4 (2 × 5 min, 5 mL) and then the resin was washed. Then Boc-2-NMe-Pal-OH dissolved in 3 mL NMP/DCM (1/1), and 240 μL of collidine was added, and then this solution was combined with the suspended peptide resin. Subsequently, TPTU (1.5 eq) was dissolved in 5 mL NMP/DCM (1:1) and this solution was added slowly over 15 min to the peptide resin. After the addition, the reaction mixture was black colored. A test cleavage was carried out with a 40% solution of HFIP in DCM. UPLC-MS of the peptide identified the desired Boc protected linear peptide as a diastereomeric mixture, tR = 1.35 min, purity 30%, (M + H)+ = 858.7; tR = 1.38 min, purity 49%, (M + H)+ = 858.7. Then, the peptide resin was cleaved with a 40% solution of HFIP in DCM with shaking for 2 × 15 min. The volatiles were fully evaporated with high vacuum (DCM and HFIP) providing 395 mg of crude Boc protected linear peptide. The Boc protecting group was removed using 4 mL of a solution of TFA:H2O:AcN (50:25:25) which was added to the peptide and the mixture was heated at 50 °C. The reaction mixture was then stirred for 30 min and UPLC-MS showed completion of the reaction. Two peaks could be identified in UPLC, H2N-2-L-NMe-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH (tR = 1.673 min, 60.4% purity by UV) and H2N-2-D-NMe-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH (tR = 1.729 min, 28.4% UV). The diastereomeric linear peptide was purified by preparative HPLC providing the desired product H2N-2-L-NMe-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH, (70 mg, 92 mmol, 40.7% yield, > 99% purity, (M + H)+ = 758.1, tR = 1.69 min) and the diastereomer H2N-2-D-NMe-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH, (30 mg, 39.6 mmol, 17.4% yield, > 99% purity, (M + H)+ = 758.6, tR = 1.75 min) as white lyophilisates.
Synthesis of Compound (3): [NMe-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro]: Cyclization Procedure
The linear peptide (H2N-NMe-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH) was dissolved in 10 mL AcN, with DIPEA (2 eq) to provide solution 1. In 10 mL AcN, HATU (3 eq) and DIPEA (4 eq) were freshly dissolved and the solution was heated to 45 °C while stirring to provide solution 2. Then, solution 1 was added slowly (60 mL/h) during 10 min to solution 2. After the addition, the orange reaction mixture was stirred for 15 min at RT. The reaction was monitored by UPLC-MS after 15 min, tR = 2.00 min, purity 82.8% UV, UPLC-MS (Standard Peptide method): tR = 1.41 min, (M + H)+ = 740.5. The crude cyclized peptide was purified by preparative HPLC. UPLC analysis was carried out (tR = 1.99 min, purity > 99% UV) on the purified cyclic peptide obtained as a white lyophilisate (20.5 mg, 28 mmol, 29.8% yield, > 99% purity UPLC-MS (Non polar method), (M + H)+ = 740.5, tR = 4.05 min, 99% purity; UPLC-MS (standard method), tR = 5.03 min). Chiral integrity was successfully determined by analytical UPLC using a Luna C8 column, eluent A: water + 5 mM HCl, eluent B:AcN; gradient 22%B linearly increasing to 80% B in 2.5 min at 75 °C indicating 96.4% of the target cyclic peptide [2-NMe-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro] and 3.6% of the diastereomeric cyclic peptide [2-D-NMe-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro]. Interestingly, for compound (3) the GC method of determining chiral integrity was not successful.
Synthesis of Precursor of Compound (4): H2N-D-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH
The peptide-resin was first swollen in NMP (10 mL). The Fmoc-D-2-Pal-OH (2 eq) was dissolved in 10 mL NMP with HOBT (3 eq). DIC was added in 2 portions, at 0 min and after 30 min. The resin was washed and stored in the fridge overnight. A double coupling was made, with 1 eq Fmoc-D-2-Pal-OH and 1.5 eq DIC/HOBT. After 7 h, the resin was washed and a test cleavage was carried out. UPLC-MS analysis of the test cleavage showed two diastereomeric peaks at tR = 2.93 min and 2.95 min, 43% purity and 23% purity, (M + H)+ = 966. The Fmoc deprotection was performed using 10 mL 20% 4-methylpiperidine in NMP for 3 × 15 min, and the resin was washed with 2x NMP and 1x iPrOH. The cleavage of the peptide-resin was made 3 × 15 min with a 1% TFA solution in DCM (10 mL), to provide crude linear peptide (121 mg, 160 mmol, 46% yield, 27% purity, (M + H)+ = 744.5, tR = 2.44 min).
Synthesis of Compound (4): [D-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro]: Cyclization Procedure
The crude linear peptide H2N-D-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH was dissolved in 25 mL DCM with DIPEA (2 eq) to provide solution 1. In 80 mL MeCN, HATU (3 eq) and DIPEA (4 eq) were dissolved and the solution was warmed up to 45 °C while stirring to provide solution 2. Then, solution 1 was added slowly (60 mL/h) in 17 min to the warm solution 2. After the addition, the orange reaction mixture was stirred for 30 min at 45 °C. Subsequently, the volatiles were evaporated after 30 min to provide the crude cyclic peptide, UPLC-MS tR = 2.57 min, 27% purity UV, (M + H)+ = 726. The crude cyclic peptide was purified by preparative HPLC. Fractions were compared to the 2-Pal diastereomer previously synthesized by UPLC. The results confirmed that peptide in fractions 19 and 28 were the D-2-Pal diastereomer and peptide in fraction 21 is the 2-Pal diastereomer. The fractions were directly frozen and lyophilized, yielding [D-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro] as a white lyophilisate (15.2 mg, 21 mmol, 10.8% yield, > 99% purity, (M + H)+ = 726.5, UPLC tR = 1.455 min, UPLC-MS (standard method) 98% purity, tR = 4.85 min) and the diastereomeric product [2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro] as a white lyophilisate (3.3 mg, 4 mmol, 2.3% yield, > 99% purity, (M + H)+ = 726.5, tR = 1.460 min). Chiral integrity of compound (4) obtained by this route was investigated by C.A.T. Tuebingen GmbH, verifying 95.96% D-2-Pal and chiral integrity of compound (2) obtained by this route as a byproduct was additionally investigated verifying 89.85% 2-Pal enantiomer.
Synthesis of Compound (5): [3-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro]: Cyclization Procedure
A solution of H2N-3-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH (149 mg, 0.200 mmol), HOAt (40.8 mg, 0.300 mmol) and HATU (304 mg, 0.800 mmol) in DCM (200 mL) and 2,6-lutidine (0.699 mL, 6.00 mmol) was stirred at RT for 16 h then concentrated to dryness in-vacuo. The product was isolated by preparative HPLC. The pure fractions were combined and lyophilised, providing the cyclic peptide as a white lyophilisate (46.7 mg, 56 mmol, 27.8% yield, 98% purity UPLC-MS Standard method, (M + H)+ = 726.5, tR = 2.89 min, > 99% purity HPLC, tR = 6.17 min).
Synthesis of Compound (6): [4-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro]: Cyclization Procedure
The linear peptide H2N-4-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH was dissolved in AcN (20 mL) with DIPEA (2 eq) to provide solution 1. HATU (3 eq) and DIPEA (4 eq) were dissolved in 120 mL AcN and the fresh solution was warmed up to 45 °C while stirring to provide solution 2. Then solution 1 was added slowly (60 mL/h) in 15 min to the warm solution 2. After the additon, the yellow reaction mixture was cooled to RT and stirred for 18 h. The crude cyclic peptide was concentrated in-vacuo and UPLC-MS of the crude cyclized peptide was carried out (tR = 2.44 min, 50% purity UV, (M + H)+ = 726). The crude cyclic peptide was purified and UPLC of combined fractions showed a purity of 98.3% (tR = 1.66 min). The volatiles were evaporated in-vacuo and the mixture was frozen and lyophilized providing the cyclic peptide as a white lyophilisate (145.1 mg, 199 mmol, 74.7% yield, > 99% purity UPLC-MS Standard method, (M + H)+ = 726.5, tR = 2.55 min).
Analysis for Optical Purity
The degree of chiral integrity was determined by two different methods. As a first test analytical GC after deuteration of the hydrolyzed peptide product, as carried out at C.A.T. GmbH (Tuebingen, Germany), was employed. This method was successful in determining the % presence of the epimeric D-2-Pal-OH in the [2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro] cyclic peptide, and similarly, the % of the epimeric 2-Pal-OH in the [D-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro] sequence.
Concerning the synthesis of cyclic peptide (3), preparative HPLC separation of the protected linear precursor H-NMe-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH from the undesired H–D-2-NMe-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-OH was identified as being the superior approach. Quantification of the chiral integrity of this cyclic peptide was successfully achieved by an alternative method using an optimized UPLC analysis, where the two diastereomeric peptides [NMe-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro] and [NMe-D-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro] could be separated. In order to validate and optimize the UPLC method, a 1:1 mixture of [NMe-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro] and [NMe-D-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro] was prepared and analyzed using different conditions. An UPLC method using a Luna C8 column, combined with eluent A:water + 5 mM HCl, eluent B:AcN; gradient 22%B linearly increasing to 80%B in 2.5 min at 75 °C resulted as the optimal method illustrating 96.4% purity of the target cyclic peptide (3) [NMe-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro] and only 3.6% of the [NMe-D-2-Pal-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro] diastereoisomer.
Assays for Permeability and Solubility
Protocols for high-throughput equilibrium solubility, PAMPA and cellular permeability by the MDCK-LE and MDCK-MDR1 assays were followed as reported earlier (Lewis et al. 2015; Tatrai et al. 2019). The values relate to single experiments run in triplicates. For the MDCK assay, the LLOQ (lower limit of quantification) was 5 nM, and for PAMPA 2 nM, respectively. For both PAMPA and MDCK, the standard deviation in log units for those assays is 0.2 (1.6 fold on a linear scale). This has been estimated by looking at independent replicates across the assay dynamic range. For the determination of the apparent passive permeability, Papp, apical to basolateral was determined using MDCK-Low efflux cells (Sigma #8412903) and for the determination of efflux ratios Papp, apical to basolateral, basolateral to apical was determined using MDCK-MDR1 cells (Netherland Cancer Institute).
Molecular dynamics simulations were performed over 20 ns for every peptide using an implicit solvent model of water (Schaefer and Karplus 1996) generating a set of 20000 conformations for each as previously reported (Lewis et al. 2015). The initial 3-dimensional structures were generated using the program Corina (Sadowski et al. 1994). Before starting data collection, the peptides were minimized for 400 steps and heated to 300 K by performing 10 ps Langevin dynamics. A water model was applied for the dynamics, rather than a model representing the membrane interior (e.g., chloroform), since this leads to faster transitions between conformations in the simulations and better sampling of conformation space (Chen et al. 2008). For every structure thus obtained, we computed the 3-dimensional solvent-accessible polar surface area (SAPSA). By averaging over conformations observed between 10 and 20 ns, we determined the time-averaged SAPSA for the various peptides. Conformational changes and the dynamic formation of hydrogen bonds were facilitated by a high dielectric constant of 80 representing water as the solvent, and a low friction constant of 1/ps for performing constant temperature Langevin dynamics (Brooks et al. 1983). The SAPSA for each frame is determined as the area covered by the center of a solvent probe sphere with a radius of 1.4 Å in contact with oxygen, nitrogen, and their bonded hydrogens (Richmond 1984). For the solute atoms, we employ the same Bondi radii as are used to compute the conventional polar surface area (PSA) (Ertl et al. 2000).
Conditions for analytical supercritical fluid chromatography (SFC) were taken from previous publications (Goetz et al. 2014a, b). The Pirkle chiral stationary phase Chirex 3014, a silica bonded (S)-valine and (R)-1-(α-naphthyl)ethylamine with a urea linkage, was selected. A mobile phase of 15% methanol in supercritical CO2 enabled adequate separation in combination with a low-slope gradient. High retention times were mitigated by increasing the percentage of methanol in the eluent and the addition of the relatively weak salt pair ammonium formate. In the resulting chromatographic system, differences in retention depend upon hydrophobic interactions, the ability for hydrogen bonding, the presence of dipoles, and van der Waals interactions of the solutes with the stationary phase (Goetz et al. 2014a, b).
Potentiometric pKa Determination
Potentiometric ionization constants were determined on the commercial SiriusT3 instruments (Pion-inc.com) as described by Völgyi et al. (2007). Briefly, 0.3–1 mM of test solutions were titrated from pH 2 to 12 for bases or 12 to 2 for acids. Titrations were conducted at 25 °C and in 0.15 M ionic strength. Aqueous titrations were performed in triplicate in 0.15 M KCl, while sparingly soluble test compounds were titrated in 10–60 %wt methanol, 1,4-dioxane, or dimethyl sulfoxide co-solvent. A minimum of three titrations in varying amounts of co-solvent were performed for extrapolation to the aqueous pKa. For each titration, initial estimates of pKa values were obtained from Bjerrum difference plots (number of bound protons versus pH) and then were refined by a weighted non-linear least-squares procedure (Avdeef 1992, 1993) available in the instrument software. Experimental variability was determined from 389 duplicate measurements from different days and experimentalists, with a standard deviation of 0.28.
Solid peptide samples (2–3 mg) were dissolved in 600 μL CDCl3 and treated with dried aluminum oxide. All NMR data were collected on a Bruker Avance 600 MHz NMR instrument with a 5 mm TCI cryoprobe at 298 K. Titration experiments were carried out by serial addition of 10 to 50 μL of DMSO-d6 to 500 μL of CDCl3 samples with 10 μL increments. Chemical shift changes of amide protons were analyzed against the volume of DMSO-d6 added.
For rat PK, blood concentration versus time profiles were obtained from 3 male Sprague–Dawley rats. For intravenous PK, the compound was administered intravenously by bolus injection (0.5 mL/kg) at a dose of 1 mg/kg, solubilized in NMP (30%) and PEG200 (70%). For oral PK, after a washout period of 48 h, an oral gavage was administered to the same animals at a dose of 3 mg/kg at a dosing volume of 2.5 mL/kg. The oral dose was dispersed in water (99%), Tween80 (0.5%) and methylcellulose (0.5%), forming a homogenous suspension. For both rodents, blood samples (10–50 µL) were collected at 0.08 (i.v. only), 0.25, 0.5, 1, 2, 4, 7, and 24 h after dosing. Analyses of parent compound concentrations were carried out in blood using LC–MS/MS. An aliquot of 30 μL was taken and added to 200 μL AcN (including Glyburide (c = 50 ng/mL) as internal standard) for protein precipitation. Sample analysis was performed on a LC–MS/MS system consisting of an AB SCIEX API 5500 QTrap mass spectrometer equipped with a TurboIon SprayTM interface (Framingham, USA). The MS system was connected to a HTS CTC PAL auto-sampler (Zwingen, Switzerland) and to a Flux Rheos 2200 pump system (Reinach, Switzerland). The supernatants (2 µl) were injected directly onto the LC–MS/MS system for analysis. The test articles and its internal standard were separated with a Phenomenex Kinetex C18 (50 x 2 mm ID, 2.7 µm pore size). A binary gradient with a mobile phase consisting of water (A) and AcN (B) was used for the LC-separation. Both mobile phases (A) and (B) were acidified with 0.1% formic acid. The elution gradient program was as follows: [time (min), (% mobile phase B): (0, 95) (5.8, 85) (5.81, 99) (6.5, 99) (6.51, 95) (8, 95]. The column temperature was maintained at 50 °C using a column heater. Under these experimental conditions, the LLOQ was about 0.7 ng/mL.
All animals were maintained under standard housing conditions with access to standard pelleted food and water ad libitum, including throughout experiments. Animal experiments were conducted in accordance with Swiss national animal welfare regulations, under the ethically approved animal experimentation licenses authorized by the Cantonal Veterinary Authority of Basel city and the Federal Veterinary Office of Switzerland.