Combination of Betulinic Acid Fragments and Carbonic Anhydrase Inhibitors-A New Drug Targeting Approach

Abstract

Human carbonic anhydrase IX (hCA IX) is a zinc(II)-dependent metalloenzyme that plays a critical role in the conversion of carbon dioxide and water to protons and bicarbonate. It is a membrane-bound protein with an extracellular catalytic center that is predominantly overexpressed in solid hypoxic tumors. Sulfamates and sulfonamides, for example acetazolamide (AZA), have been used to inhibit hCA IX in order to improve the response to solid hypoxic tumors. In the present study, we propose a new drug targeting approach by attaching the natural cytotoxic substances betulin and betulinic acid (BA) via a linker to sulfonamides. The conjugate was designed with different spacer lengths to accumulate at the target site of hCA IX. Computational and cell biological studies suggest that the length of the linker may influence hCA IX inhibition. Cytotoxicity tests of the newly synthesized bifunctional conjugates 3, 5, and 9 show effective cytotoxicity in the range of 6.4 and 30.1 µM in 2D and 3D tumor models. The hCA IX inhibition constants of this conjugates, measured using an in vitro enzyme assay with p-nitrophenyl acetate, were determined in a low µM-range, and all compounds reveal a significant inhibition of hypoxia-induced CA activity in a cell-based assay using the Wilbur-Anderson method. In addition, the cells respond with G1 increase and apoptosis induction. Overall, the dual strategy to produce cytotoxic tumor therapeutics that inhibit tumor-associated hCA IX was successfully implemented.

Keywords: Carbonic anhydrase IX inhibition; betulin and betulinic acid; dual tumor targeting agents.

Figure 1

(a) Binding pocket of hCA IX (PDB code: 3iai) with the essential amino acids of the hydrophobic and hydrophilic site (hCA II numbering). The coloring of the surface is based on the hydrophobicity (brown: hydrophobic, grey: neutral, blue: hydrophilic). The zinc(II) ion is shown as a green sphere in the center of the binding pocket. (b) Sulfonamide binding mechanism of hCA IX.

Figure 2

Schematic illustration of the concept of a bifunctional conjugate of betulin/BA, a linker, and CA IX inhibitor fragments.

Scheme 1

Synthesis of the sulfonamides of betulin (5) and BA (3) and the triterpenoid-acetazolamide conjugates (9 and 12). (a) Dry DCM, acetic anhydride, triethylamine (3 equiv.), DMAP (catal.), 20 °C for 24 h; (b) dry DCM, oxalyl chloride, taurine amide, triethylamine, overnight, 20 °C; (c) pyridine, DMAP, succinic anhydride, 5 h reflux; (d) DMF, HOBT, DCC, taurine amide, 15 h, 20 °C; (e) (i) DCM, oxalyl chloride, 2 h, (ii) DMF, DMAP (cat.) 6; (f) (i) DCM, oxalyl chloride, 20 min (ii) 0 °C, diglycol amine; (g) pyridine, DMAP, succinic anhydride, 5 h reflux; (h) (i) DCM, oxalyl chloride, 2 h, (ii) DMF, DMAP (cat.) 6.

Scheme 2

AZA and inhibitory fragments 6–8.

Figure 3

Graphical representation of Annexin V analysis of the breast cancer cell line MCF-7 after (a) 24 h, (b) 48 h, and (c) 72 h of treatment with compounds 3, 5, and 9 and an untreated control. (d) Summary of early and late apoptotic cells of all three time points. Double the IC₅₀ concentration was used for treatment.

Figure 4

Graphical representation of the cell cycle analysis of the human breast cancer cell line MCF-7 after a treatment period of (a) 24 h, (b) 48 h, and (c) 72 h with compounds 3, 5, and 9 and an untreated control. (d) Comparison of another cell line (human melanoma; A375) with compound 3 with a treatment period of 24, 48, and 72 h. Double the IC₅₀ concentration was used for the treatment.

Figure 5

Activity of hypoxia induced hCA IX in a cell-based system measured using the Wilbur–Anderson method. Mean relative Wilbur–Anderson units/mg protein (relative WAU/mg protein) from at least three independent biological replicates are shown. p values indicate significant differences from untreated hypoxic cells (** p ≤ 0.01; *** p ≤ 0.001).

Figure 6

Docking poses of the betulin derivatives 5 (a) and 12 (b) in the binding pocket of hCA IX superimposed on the crystal structure of AZA (PDB code: 3iai). The surface of the binding pocket was colored by hydrophobicity (blue: hydrophilic, grey: neutral and brown: lipophilic). The zinc(II) ion is represented as a green sphere in the center of the binding pocket.

Figure 7

Histograms of distances between the C17 of betulin scaffold and the N atom of the sulfonamide inhibitor for compounds 9 and 12.