New Cathepsin D Inhibitorswith Hydroxyethylamine Isosteres: Preparation and Characterization

Abstract

The lysosomal aspartyl protease, cathepsin D, has been suggested to play a role in the metastatic potential of several types of cancer. Cathepsin D is secreted by malignant cells, and is believed to be involved in the breakdown of the extracellular matrix. High levels of active cathepsin D have been found in colon cancer, prostate cancer, uterine cancer and ovarian cancer. Also cathepsin D has recently been associated with the development of Alzheimer's disease. Hydroxyethyl isosteres with cyclic tertiary amine have proven to be clinically useful as inhibitors of aspartyl proteases similar to cathepsin D in activity, such as the HIV-1 aspartyl protease. In the present study twenty-eight compounds containing (hydroxyethyl)amine isosteres with cyclic tertiary amines have been synthesized. These compounds show significant activity as cathepsin D inhibitors, many with IC(50) values in the nanomolar range. For example, the compounds that contain hydroxyethylamines where the amine is formed from N-piperazine-2-carboxylic acid methyl ester, 4y-bb, show IC(50) values ranging from 2.5 to 15 nM.

New Cathepsin D Inhibitor Library Utilizing Hydroxyethyl Isosteres with Cyclic Tertiary Amines

“…The novel chiral aminoalkyl epoxides (Precursor C ) were first reported by Evans and coworkers [40] and have been subsequently used in the preparation of several HIV-1 aspartyl protease inhibitors with hydroxyethyl amine isosteres. In the earlier phase of this project [35, 36, 41] we successfully prepared BOC-protected chiral amino epoxides starting from commercially available corresponding BOC-amino acids. In this paper, we utilized a similar approach for the preparation of the aminoalkyl epoxides with only slight modifications.…”

“…The synthetic inhibitors were screened for their inhibition of cathepsin D (Table 1 ) by fluorometric methods [36, 39] using a fluorometric assay of human liver cathepsin D with picomolar accuracy. The commercially available peptide substrate Ac-Glu-Glu(Edans)-Lys-Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly-Lys(Methyl Red)-Glu-NH 2 was used in the fluorometric assays of cathepsin D at an excitation wavelength of 340 nm with a 430 nm cutoff filter for emission.…”

“…Elemental analyses demonstrating compound purities of more than 95% were performed by Galbraith Laboratories, Knoxville, TN. Butoxy-carbonyl (BOC)-protected chiral amino epoxides (Precursor C) were prepared as described in our earlier work [35, 36, 41] using a slight modification of a procedure reported by Evans and coworkers [40]. …”

“…excitation λ 340 nm , emission λ 430 nm Substrate: Ac-Glu-Glu(Edans)-Lys-Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly-Lys(Methyl Red)-Glu-NH 2 * Compound also screened using hemoglobin as substrate and reported in our earlier work [35, 36]. …”

“…Our earlier work [35, 36] involved the development of compounds similar in structure to those created in the combinatorial libraries of Kick’s and Ellman’s [29-31] but which contain cyclized tertiary amines, like those in palinavir. Model studies [37] utilizing AMBER 8 [38] demonstrated that N -piperazine ( S) -hydroxyethyl amine with a 2-carboxylic amide in the axial position (Figure 3 ) would hold the compound into a preferred conformation for cathepsin D inhibition.…”