Dipeptide analogues of fluorinated aminophosphonic acid sodium salts as moderate competitive inhibitors of cathepsin C

Dipeptide analogues of α- and β-fluorinated aminophosphonates 5 and 7 were obtained from the corresponding ʟ-amino acids [23,24] 1. In the key step of the synthesis fluorine was introduced to the corresponding α- and β-fluorinated aminophosphonates 4, 6 (Scheme 1) by regioselective deoxyfluorination reactions of α-hydroxy-β-aminophosphonates 2 [24-26].

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Scheme 1: Synthetic strategy towards 5 and 7.

Next, the conditions for the solvolysis were carefully assembled (Scheme 2). The optimized reaction conditions included 8 equiv of trimethylsilyl bromide (TMSBr) and freshly distilled methylene chloride as a solvent. In each case the reactions were carried out at room temperature overnight under an argon atmosphere. The next day, the solvent, volatile byproducts, and TMSBr residues were thoroughly evaporated. Time of solvolysis reactions varies in the literature, ranging from 10 minutes to several hours [27-29]. In our case the alcoholysis was carried out for 30 minutes. During this process, disappearance of the brownish or yellowish color of the compounds was observed. According to the literature, addition of diethyl ether in the next step should make precipitation more efficient [10]. This was done for compound 8a, but no improvement was observed. Much better results were obtained with additional double wash of the precipitate with methanol combined with evaporation of the solvent under reduced pressure. As a result of the reactions carried out, the dipeptide analogues of α- and β-fluorinated aminophosphonic acids 8 and 10 were obtained. All the samples were solids, with very poor solubility in water and organic solvents such as DMSO and MeOH.

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Scheme 2: Synthesis of 9 and 11. (a) R = -CH3; (b) R = -CH(CH3)2; (c) R = -CH2CH(CH3)2; (d) R = -CH(CH3)CH2CH3, (e) R = -CH2Ph; i) (a) 5 or 7 (1 equiv), TMSBr (8 equiv), CH2Cl2, rt, 20 h, (b) MeOH, 30 min.; ii) 8 or 10 (1 equiv), 1 M NaOH (2 equiv), H2O, 15 min.

The final step of the synthesis was the reaction of the resulting phosphonic acids 8 and 10 with a 1 M aqueous NaOH solution. Based on the literature data, alternatively to this method [30-32], phosphonic acids can be passed through an ion exchange column [33]. The reactions of compounds 8 and 10 with 1 M NaOH were carried out at room temperature (Scheme 2). When a clear solutions were obtained, the reaction was carried out for another 15 minutes. The solutions were then concentrated under reduced pressure. The precipitated salts were washed with methanol [31] and the solvent evaporation procedure was repeated. Sublimation drying (lyophilization) was carried out, obtaining white powders with a yield of 99% in each case. The resulting sodium salts of phosphonic acids 9 and 11 were subjected to 1H, 13C, 19F, and 31P NMR spectroscopic analysis as well as mass spectrometry (MS) confirming their purity. The spectroscopic data of 9 and 11 are in agreement with the literature data of the starting esters 5 and 7 literature data [23,24]. A very good correlation of chemical shifts was also observed in the 13C NMR spectra for the key signals from the C1 and C2 atoms (Table 1). Each sample was pure; no byproducts were present.

Table 1: The 13C NMR chemical shifts of C1 and C2 carbon atoms.

  [Graphic 1] R = 5 C1 [ppm] 9 C1 [ppm] 5 C2 [ppm] 9 C2 [ppm] (a) -CH3 89.56 94.72 45.66 47.77 (b) -CH(CH3)2 88.97 93.30 54.77 57.29 (c) -CH2CH(CH3)2 91.33 95.36 48.62 50.22 (d) -CH(CH3)CH2CH3 88.89 92.17 54.45 56.33 (e) -CH2Ph 88.05 94.79 51.39 52.93   [Graphic 2] R = 7 C1 [ppm] 11 C1 [ppm] 7 C2 [ppm] 11 C2 [ppm] (a) -CH3 49.51 54.13 89.00 92.58 (b) -CH(CH3)2 47.55 50.45 98.03 99.23 (c) -CH2CH(CH3)2 50.00 54.09 92.21 94.76 (d) -CH(CH3)CH2CH3 47.16 49.91 95.16 96.08 (e) -CH2Ph 48.69 53.55 93.24 96.21
Kinetic studies

Due to the homology and similar structural requirements, bovine cathepsin C is often used in research as a model for human cathepsin C as it was well documented by Poręba et al. [34] in the study of the substrate specificity of these two mammalian cathepsins. They showed the best fit of amino acids with larger side to the S1 pocket of the enzyme. In contrast, the S2 pocket preferably accommodates amino acids having short aliphatic side-chains, but also recognizes aromatic amino acids, preferably phenylalanine. To study the structural requirements of the S1 binding site of the enzyme, we synthesized a series of ten dipeptide analogues of fluorinated aminophosphonic acid sodium salts 9, 11 with phenylalanine at the N-terminus and evaluated their inhibitory activity against bovine cathepsin C. Inhibition kinetics were carried out at 37 °C for 10 minutes in acetate buffer at pH 5. Changes in product concentration versus time were monitored spectrophotometrically at λ = 405 nm. Seven of the tested compounds were moderate competitive inhibitors with millimolar inhibitory activity (Table 2). Three of them at higher concentrations precipitated from 0.1 M acetate buffer at pH 5.0. Dipeptide analogues of α-fluorinated aminophosphonic acid sodium salts 9 were more active against cathepsin C than β-fluorinated analogues 11.

Table 2: Inhibitory constants of the studied of α- and β-fluorinated aminophosphonic acid sodium salts towards bovine cathepsin C.

Dipeptide [Graphic 3]
9
KI ± SD [mM] [Graphic 4]
11
KI ± SD [mM] Phe-Ala
(a) -CH3 0.603 ± 0.1 0.733 ± 0.087 Phe-Val
(b) -CH(CH3)2 0.0951 ± 0.05 1.869 ± 0.171 Phe-Leu
(c) -CH2CH(CH3)2 0.309 ± 0.066 0.847 ± 0.38 Phe-Ile
(d) -CH(CH3)CH2CH3 0.273 ± 0.15 up to a concentration of 0.37 mM, it does not inhibit activity;
at a higher concentration, it precipitates Phe-Phe
(e) -CH2Ph precipitates in a buffer precipitates in buffer

The dipeptide analogue of α-fluorinated aminophosphonic acid sodium salt bearing the valine residue as a second amino acid in the chain (9b) showed the greatest inhibitory power (Figure 1). The type of inhibition and the inhibition constant were determined from the Dixon-type linearization of Equation 1 [35]. For each of the simple data, Equation 1 determines the slope factor a, whereby a weighted fit was used. The statistical weight for each point in the above-mentioned transformation 1/V0,i = f(I) is equal to [Graphic 5].

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Figure 1: Dixon plot for the hydrolysis of Gly-Phe-pNA substrate catalyzed by bovine cathepsin C in the presence of 9b (T = 37 °C, pH 5.0).

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where:

Vmax = maximum reaction velocity, KM = Michaelis constant, Kic = competitive inhibitory constant, Kiu = uncompetitive inhibitory constant, [S] = concentration of the substrate, [I] = concentration of the inhibitor.

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