Coumarin-Based Aldo-Keto Reductase Family 1C (AKR1C) 2 and 3 Inhibitors

Abstract

A series of 7-substituted coumarin derivatives have been characterized as pan-aldo-keto reductase family 1C (AKR1C) inhibitors. The AKR1C family of enzymes are overexpressed in numerous cancers where they are involved in drug resistance development. 7-hydroxy coumarin ethyl esters and their corresponding amides have high potency for AKR1C3 and AKR1C2 inhibition. Coumarin amide 3 a possessed IC₅₀ values of 50 nM and 90 nM for AKR1C3 and AKR1C2, respectively, and exhibits 'drug-like' metabolic stability and half-life in human and mouse liver microsomes and plasma. Compound 3 a was employed as a chemical tool to determine pan-AKR1C2/3 inhibition effects both as a radiation sensitizer and as a potentiator of chemotherapy cytotoxicity. In contrast to previously reported pan-AKR1C inhibitors, 3 a demonstrated no radiation sensitization effect in a radiation-resistant prostate cancer cell line model. Pan-AKR1C inhibition also did not potentiate the in vitro cytotoxicity of ABT-737, daunorubicin or dexamethasone, in two patient-derived T-cell ALL and pre-B-cell ALL cell lines. In contrast, a highly selective AKR1C3 inhibitor, compound K90, enhanced the cytotoxicity of both ABT-737 and daunorubicin in the T-cell ALL cell line model. Thus, the inhibitory profile required to enhance chemotherapeutic cytotoxicity in leukemia may be AKR1C isoform and drug specific.

Keywords: AKR1C2 Inhibitor; AKR1C3 Inhibitor; Drug Resistance; Leukemia; Prostate Cancer.

Figure 1:

Structures of known coumarin-based AKR inducers/inhibitors.

Figure 2.

Docking pose and predicted hydrogen bond interactions (green dotted lines) of compounds 3a (Gold) and chromene-based AKR1C3 inhibitor 2j (magenta). A) 3a docked into the crystal structure of AKR1C3. B) 3a docked into the crystal structure of AKR1C2.C) 2j docked into the crystal structure of AKR1C3. D) Overlay of 3a and 2j.

Figure 3.

A) In-vitro metabolism of 3a in mouse (MLM) and human liver microsomes (HLM), values represent the mean ±SEM of n = 3 experiments. NC = negative control (without NADPH). B) In-vitro mouse plasma stability of 3a, values represent the mean ±SD of n = 3 experiments.

Figure 4:

Effect of compounds 3a and 3c on cell proliferation. A) AKR1C3 expressing DuCap Wt cells in the presence of 1 nM Δ4An, 30 μM 3c or indomethacin (Indo). B) AKR1C3 null LNCaP cells in the presence of 3a at indicated concentrations. C) AKR1C3 null LNCaP cells in the presence of 3c at indicated concentrations. D) WPMY-1 non-malignant prostate stromal cells in the presence of 3a at indicated concentrations. E) WPMY-1 non-malignant prostate stromal cells in the presence of 3c at indicated concentrations. Values represent the mean ±SD of n = 3 experiments. *, p <0.05; ****, p <0.0001 by two-way ANOVA.

Figure 5:

Clonogenic survial in 22Rv1 cells treated with A) 10 μM 3a and B) K90 and then irradiated with 2Gy X-rays. Values represent the mean ±SD of n = 3 experiments.

Figure 6.

Selective AKR1C3 inhibitor, K90, but not pan-AKR1C inhibitor, 3a, potentiated the cytotoxicity of ABT-737 and daunorubicin in patient-derived ALL leukemia cell lines, in vitro. A, B) Structure, and cytotoxic dose-response, of 3a, alone and in combination with ABT-737, daunorubicin, or dexamethasone in the COG-LL-317h T-cell ALL and TX-LL-057h pre-B-cell ALL cell lines. C) Structure, and cytotoxic dose-response of K90, alone and in combination with ABT-737, daunorubicin, or dexamethasone in COG-LL-317h T-cell ALL cells. Cytotoxicity assayed using DIMSCAN assay. Values represent the mean ±SEM of n = 2 experiments performed in triplicate.

Scheme 1:

Synthesis of substituted coumarin carboxylic acid derivatives 1a-j.

Scheme 2:

Synthesis of 7-hydroxy coumarin benzyl amides 2a-2f.

Scheme 3:

Synthesis of 7-hydroxy coumarin aliphatic amides 3a-3e.

Scheme 4:

Synthesis of prenyl coumarin (4a-b) and cinnamic acid coumarin (4c) derivatives.

Scheme 5:

Synthesis of phenyl sulfonyl coumarin derivative.