Novel Insights on Human Carbonic Anhydrase Inhibitors Based on Coumalic Acid: Design, Synthesis, Molecular Modeling Investigation, and Biological Studies

Abstract

Human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms IX and XII are overexpressed in solid hypoxic tumors, and they are considered as prognostic tools and therapeutic targets for cancer. Based on a molecular simplification of the well-known coumarin scaffold, we developed a new series of derivatives of the pyran-2-one core. The new compounds are endowed with potent and selective inhibitory activity against the tumor-related hCA isoforms IX and XII, in the low nanomolar range, whereas they are inactive against the two cytosolic off-targets hCA I and II. The compounds exhibiting the best hCA inhibition were further investigated against the breast adenocarcinoma cell line (MCF7) in hypoxic conditions, evaluating their ability to eventually synergize with doxorubicin. The compounds’ biocompatibility on healthy cells was also tested and confirmed on Human Gingival Fibroblasts (HGFs). Furthermore, the possible binding mode of all compounds to the active site of the tumor-associated human CA IX was investigated by computational techniques which predicted the binding conformations and the persistency of binding poses within the active site of the enzyme, furnishing relevant data for the design of tight binding inhibitors.

Synthetic Approaches to Novel Human Carbonic Anhydrase Isoform Inhibitors Based on Pyrrol-2-one Moiety

“…Moreover, it is widely recognized that hCA IX and XII isoforms, responsible for severe events connected with tumor progression and bad prognosis, appear overexpressed in the presence of a hypoxic environment. As a consequence, in order to overexpress hCA IX and XII isoforms in MCF7 cells, before cotreaments of doxo 2.5 μM and compounds 9 , 14 , 16 , and 20 , a hypoxic condition was reproduced by administering cobalt­(II) chloride hexahydrate (CoCl 2 ) at 100 μM for 48 h, chemically inducing HIF-1α, as already reported elsewhere . Cotreatments of doxo 2.5 μM + compounds 9 , 14 , 16 , and 20 were kept up to 72 h; next, the metabolic activity was measured by the MTT test.…”

“…As a consequence, in order to overexpress hCA IX and XII isoforms in MCF7 cells, before cotreaments of doxo 2.5 μM and compounds 9 , 14 , 16 , and 20 , a hypoxic condition was reproduced by administering cobalt(II) chloride hexahydrate (CoCl 2 ) at 100 μM for 48 h, chemically inducing HIF-1α, as already reported elsewhere. 56 Cotreatments of doxo 2.5 μM + compounds 9 , 14 , 16 , and 20 were kept up to 72 h; next, the metabolic activity was measured by the MTT test. The latter reveals that for both samples exposed to doxo 2.5 μM alone and to cotreatments of doxo 2.5 μM with compounds 9 , 14 , 16 , and 20 at 50 μM, a statistically significant reduction in cell metabolic activity is recorded with respect to cells exposed to DMSO+CoCl 2 , assumed as control ( Figure 8 A).…”

“…MCF7 were seeded in a tissue culture-treated 96-well plate at a density of 10,000/well and let adhere for 24 h. After 24 h, a fresh medium containing CoCl 2 100 μM was added to induce hypoxia, as reported elsewhere. 56 CoCl 2 was maintained within the culture medium for 48 h, renewing the medium every 24 h. After that, the treatment with doxo alone at 2.5 μM and in combination with newly synthesized compounds, namely, 9 , 14 , 16 , and 20 , at 50 μM, previously dissolved in DMSO, was started and kept up to 72 h. The final concentration of DMSO was established at 0.1% in all experimental points. Inner controls, represented by cells exposed to CoCl 2 alone, DMSO alone, and DMSO+CoCl 2 were also added to the experimental panel.…”

Novel Anthraquinone-Based Benzenesulfonamide Derivatives and Their Analogues as Potent Human Carbonic Anhydrase Inhibitors with Antitumor Activity: Synthesis, Biological Evaluation, and In Silico Analysis

“…Modulation of CA activity via CA inhibitors (CAIs) has been a validated clinical strategy for decades. For example, inhibitors targeting hCA I are utilized for retinal and cerebral edema, while inhibitors targeting hCA II are used as diuretics and anti-glaucoma agents [ 12 ], and hCA II is widely associated with various types of cancers, with a recent study finding that it is expressed in the endothelium of neovessels in melanoma, as well as esophageal, renal, and lung cancers [ 13 ]. In addition, hCA IX is related to cancer development; hCA IX is not highly expressed in most normal tissues but is abundant in gastric and gallbladder epithelial cells [ 14 ], and it is highly expressed in various solid tumors [ 15 , 16 ].…”

6‐Aminocoumarin oxime‐ether/sulfonamides as selective hCA IX and XII inhibitors: Synthesis, evaluation, and molecular dynamics studies

“…[11] There are four types of carbonic anhydrase inhibitors reported to date such as zinc binders (sulfonamides), Anchoring to zinc-coordinated water/hydroxide ion (phenols and polyamines), blocking the entrance of the active site (coumarins and lacosamide) and allosteric inhibitors. [12,13] The α-CAs are known to hydrolyze the lactone ring of coumarin into hydroxycinnamic acids, [14] which bind at the entrance of the binding site, thereby occluding the active site entrance and inhibiting the enzyme. [11,15] Natural and synthetic derivatives of the neo-flavonoids like coumarin, found in plants as secondary metabolites, are known to exhibit various therapeutic effects.…”