Synthesis and evaluation of a new series of tri-, di-, and mono-N-alkylcarbamylphloroglucinols as conformationally constrained inhibitors of cholesterol esterase

Abstract

1,3,5-Tri-N-alkylcarbamylphloroglucinols (1-4) are synthesized as conformationally constrained analogs of triacylglycerols (TGs) to probe Jenck's proximity effect in the cholesterol esterase inhibition. For the cholesterol esterase inhibition, inhibitors 1-4 are 220-760-fold more potent than 1,2,3-tri-N-alkylcarbamylglycerols (13-15) that are substrate analogs of TG. Comparison of tridentate inhibitors 1-4, bidentate inhibitors 3,5-di-N-n-alkylcarbamyloxyphenols (5-8) and monodentate inhibitors 5-N-n-alkylcarbamyloxyresorcinols (9-12) indicates that inhibitory potencies are as followed: tridentate inhibitor > bidentate inhibitor > monodentate inhibitor. The log k(i) and pK(i) values of tridentate inhibitors, bidentate inhibitors, and monodentate inhibitors are linearly correlated with the alkyl chain length indicating a common mechanism in each inhibition. Also, positive slopes of these correlations indicate that the longer chain inhibitors bind more tightly to the enzyme than the shorter ones. Molecular dockings of tridentate 1, bidentate 5, and monodentate 9 into the X-ray crystal structure of cholesterol esterase suggest that one carbamyl group in the cis form of the inhibitor binds to the acyl chain-binding site of the enzyme. The second carbamyl groups in the trans forms of inhibitors 1 and 5 bind to the second acyl chain-binding site of the enzyme. The third carbamyl group in the trans form of inhibitor 1 binds to the third acyl chain-binding site of the enzyme. Moreover, the configuration of the inhibitor in the enzyme-inhibitor complex is the (1,3,5)-(cis, trans, trans)-tricarbamate form that mimics the (+gauche, -gauche)-conformation of TG.

Figure 1

Kinetic scheme for the pseudo-substrate inhibition of inhibitors in the presence of substrate. E, enzyme (CEase); S, substrate (PNPB); ES, acylenzyme intermediate; I, inhibitors 1–17; EI, enzyme-inhibitor tetrahedral intermediate; EI′, carbamyl enzyme intermediate; P, the product from substrate reaction (4-nitrophenol); P′, the product from pseudo substrate reaction (alcohols); Q, the second product (carbamic acids).

Figure 2

Superimposition of tridentate inhibitor 1,3,5-tri-N-n-octylcarbamyl-phloroglucinol (1), cholesterol ester, and TG into the active sites of CEase that contains the catalytic triad, an oxyanion hole, ACS, SACS, TACS, and LGS based on the X-ray crystal structures., One of octylcarbamate moiety of inhibitor 1, the acyl chain of cholesterol ester, and the sn-1 or sn-3 acyl chain of TG are orientated to fit into ACS of the enzyme. The carbonyl oxygen atoms of these ACS-bound acyl or carbamyl groups of substrates or inhibitor are orientated to fit into the oxyanion hole of the enzyme, and the carbonyl carbon atoms of those are in the correct position for the attack by the Ser 194 of the enzyme. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

Five stable conformations for the glycerol backbone of TG.

Figure 4

Five stable conformations for the glycerol backbone of 1,2,3-tridecanoylglycerol and structure of tridentate inhibitor 1. The structure of tridentate inhibitor 1 mimics (+g, −g)-form. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 5

Chemical structures of inhibitors 1–17.

Figure 6

Nonlinear least-squares curve fittings of k_app versus inhibitor concentration ([I]) plots against Eq. (2) for the pseudo-substrate inhibition– of CEase by (A) 1,3,5-tri-N-n-octylcarbamylphloroglucinol (1), (B) 3,5-di-N-n-hexylcarbamyl- oxyphenol (7), and (C) 5-N-n-heptylcarbamyloxyresorcinol (10). For (A), k₂ = 0.0027 ± 0.0001 s⁻¹ and K_i = 15 ± 3 nM (R²= 0.965). For (B), k₂ = 0.0016 ± 0.0002 s⁻¹ and K_i = 110 ± 40 nM (R² = 0.913). For C, k₂ = 0.0018 ± 0.0002 s⁻¹ and K_i = 60 ± 20 nM (R² = 0.926). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 7

Comparisons of the k_i values of pseudo-substrate inhibitions of CEase by tridentate inhibitors 1–4, bidentate inhibitors 5–8, and monodentate inhibitors 9–12. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 8

The (A) pK_i and (B) log k_i values versus carbamyl carbon numbers (n) of inhibitors 1–12 for the pseudo-substrate inhibition of CEase. In (A), linear correlations between the pK_i and n values are observed (pK_i = 7.24 ± 0.09 + (0.10 ± 0.01)n and R = 0.979 for tridentate inhibitors 1–4; pK_i = 7.2 ± 0.2 + (0.10 ± 0.03)n and R = 0.933 for bidentate inhibitors 5–8; pK_i = 7.2 ± 0.2 + (0.09 ± 0.32)n and R = 0.916 for monodentate inhibitors 9–12). In (B), linear correlations between the log k_i and n values are observed (log k_i = 4.91 ± 0.07 + (0.05 ± 0.01)n and R = 0.957 for tridentate inhibitors 1–4; log k_i = 4.52 ± 0.06 + (0.077 ± 0.009)n and R = 0.986 for bidentate inhibitors 5–8; log k_i = 4.1 ± 0.2 + (0.11 ± 0.03)n and R = 0.934 for monodentate inhibitors 9–12). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 9

Linear correlations of the (A) pK_i, (B) log k₂, and (C) log k_i values with the substituent constant (σ*) of 1,2,3-tri-N-substituted carbamylglycerols (13–17) for the pseudo-substrate inhibition of CEase. In (A), pK_i = 6.1 ± 0.1 – (2.4 ± 0.5)σ* and R = 0.944. In (B), log k₂ = 3.75 ± 0.03 + (0.57 ± 0.09)σ* and R = 0.974. In (C), log k_i = 2.39 ± 0.04 – (1.8 ± 0.1)σ* and R = 0.929. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 10

Internal changes between (1,3,5)-(trans, trans, trans)- and (1,3,5)-(cis, trans, trans)-tricarbamate rotamers of inhibitor 1.

Figure 11

Molecular docking of tridentate inhibitor 1, with the mode of free rotation around the carbamyl CN partial double bond, into the active sites of X-ray crystal structure of CEase: (A) the active site view and (B) the view from the entrance (mouth) of the enzyme. The configuration of the inhibitor after docked is the (1,3,5)-(cis, trans, trans)-tricarbamate form. The carbamyl carbonyl carbon atom of the inhibitor is close to S194 of the catalytic triad, and the carbamyl ester oxygen atom of the inhibitor is closed to H435 of the catalytic triad. The cis octylcarbamyl moiety of the inhibitor binds to ACS of the enzyme. The other two octylcarbamyl groups of the inhibitor, in the trans forms, bind to SACS and TACS of the enzyme. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 12

Superimpositions of structures of tridentate 1 (yellow), bidentate 5 (turquoise), and monodentate 9 (mangenta) that have been automatically docked into the X-ray crystal of CEase 1AQL by AutoDock program., – View from the active site (A) and from the entrance (mouth) (B) of the enzyme. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]