Discovery of (R)-2-(6-Methoxynaphthalen-2-yl)butanoic Acid as a Potent and Selective Aldo-keto Reductase 1C3 Inhibitor

Abstract

Type 5 17β-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ(4)-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively, in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogues and find that (R)-2-(6-methoxynaphthalen-2-yl)butanoic acid (in which the methyl group of R-naproxen was replaced by an ethyl group) acts as a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. R-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids and in the context of advanced prostate cancer R-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease.

Figure 1

Structure of naproxen analogues.

Figure 2

Inhibitory effect of compound 14a on AKR1C1–4.

Figure 3

Effect of compounds 14a (green) and 14b (blue) on AKR1C2 catalyzed reduction of 5α-DHT.

Figure 4

(a) Competitive Inhibition of AKR1C3 catalyzed oxidation of S-tetralol by 14a. (b) Competitive Inhibition of AKR1C3 catalyzed Reduction of Δ⁴-AD (Δ⁴-androstene-3,17-dione) by 14a.

Figure 5

Inhibition of COX-1 by naproxen analogues.

Figure 6

Effect of compound 14a on DHT induced AR gene expression. DHT alone (purple) and DHT plus 10 µM compound 14a.

Figure 7

Inhibition of testosterone formation in LNCaP-1C3 cells with compound 14a. (A) Conversion of 100 nM Δ⁴-AD to testosterone in LNCaP-AKR1C3 cells following digestion with β-glucurondiase. (B) Same experiment performed in the presence of 30 µM compound 14a. (C) Statistical analysis of n = 3 versus indomethacin as a positive control *p value <0.001; p value = 0.001.

Figure 8

Inhibition of Δ⁴-AD induced PSA expression in LNCaP-AKR1C3 cells by compound 14a. (A) Immunoblot. (B) Densitometric traces of immunoblots with normalization of PSA to β-tubulin for biological replicates (n = 3).

Figure 9

Alignment of 14b and 14a in the AKR1C3 active site. AKR1C3 residues (green), 14b (yellow), 14a (purple), Dotted line: possible hydrogen bond. OX: oxyanion site (residues highlighted in pink). Ligand alignments were performed using LigAlign plugin in Pymol. The template crystal structures of the AKR1C3· NADP⁺ complexes were chosen from the RCSB Protein Data Bank (PDB: 3UFY and 3R58) (also see Supporting Information).

Figure 10

Comparison of 14b and 14a binding to AKR1C2 and AKR1C3. 14b (yellow) and 14a (purple) binding modes in AKR1C2 (cyan) and AKR1C3 (green). Template crystal structure of the AKR1C2·NADP⁺ complex was taken from the RCSB Protein Data Bank (PDB: 4JQ1) (also see Supporting Information).

Figure 11

Kinetic mechanism of the reactions catalyzed by AKR1C3. (A) Oxidation reaction where the substrate S is S-tetralol and the product P is tetralone, and (B) reduction reaction where the substrate is Δ⁴-androstene-3,17-dione and the product is testosterone. S = substrate, P = product, and E = enzyme.

Scheme 1. Central Role of AKR1C3 in Androgen Biosynthesis in Prostate Cancera

^aDHEA = dehydroepiandrosterone, Δ⁴-AD = Δ⁴-androstene-3,17-dione, Adione = 5α-androstane-3,17-dione, Δ⁵-Adiol = Δ⁵-androstene-3β,17β-diol, 5α-DHT = 5α-dihydrotestosterone. Enzymes are referred to by their gene names and are italicized: HSD3B1 = 3β-hydroxysteroid dehydrogenase type 1, SRD5A 5α-reductase type 1 or type 2, AKR1C2 = type 3 3α-hydroxysteroid dehydrogenase, and HSD17B6 = 17β-hydroxysteroid dehydrogenase type 6.

Scheme 2. Synthesis of Racemic Naproxen Analogues 2–6a

^aReagents and conditions: (i) 48% HBr, AcOH, reflux, 3 h; (ii) TMSCl, CH₃OH, 25 °C, 2 h; (iii) (CF₃SO₂)₂O, Et₃N, DCM, 25 °C, 1 h; (iv) CH₂ = CHBF₃K, Cs₂CO₃, Pd(PPh₃)₄, Et₃N, EtOH, 50 °C, 16 h; (v) (OAc)₂Pd, t-But₃P, HCO₂H, 25 °C, 12 h; (vi) 3 M KOH/CH₃OH, reflux, 3 h; (vii) KOH/C₂H₅I, 25 °C, 30 min; (viii) 3 M KOH/CH₃OH, reflux, 2 h.

Scheme 3. Synthesis of Racemic Naproxen Analogues 7–11 and 13–15a

^aReagents and conditions: (i) Pd(PPh₃)₄, [(CH₃)₂CH]₃SiSH, C₆H₆, reflux, 4 h; (ii) TBAF, CH₃I, 25 °C, 2 h; (iii) 3 M KOH/CH₃OH, reflux, 2 h; (iv) mCPBA, DCM 0 °C, 1 h; (v) KHSO₅·0.5KHSO₄· 0.5K₂SO₄, (CH₃)₂C = O/H₂O 25 °C, 2 h; (vi) Mg, I₂, THF, reflux, 1 h; (vii) CH₃CH₂CHBrCO₂CH₃, THF, reflux, 2 h; (viii) 3 M KOH/CH₃OH, reflux, 2 h, (ix) CDI, CH₃SO₂NH₂, DBU, DCM, 25 °C, 4 h.