Novel Deazaflavin Analogues Potently Inhibited Tyrosyl DNA Phosphodiesterase 2 (TDP2) and Strongly Sensitized Cancer Cells toward Treatment with Topoisomerase II (TOP2) Poison Etoposide.

Topoisomerase II (TOP2) poisons as anticancer drugs work by trapping TOP2 cleavage complexes (TOP2cc) to generate DNA damage. Repair of such damage by tyrosyl DNA phosphodiesterase 2 (TDP2) could render cancer cells resistant to TOP2 poisons. Inhibiting TDP2, thus, represents an attractive mechanism-based chemosensitization approach. Currently known TDP2 inhibitors lack cellular potency and/or permeability. We report herein two novel subtypes of the deazaflavin TDP2 inhibitor core. By introducing an additional phenyl ring to the N-10 phenyl ring (subtype 11) or to the N-3 site of the deazaflavin scaffold (subtype 12), we have generated novel analogues with considerably improved biochemical potency and/or permeability. Importantly, many analogues of both subtypes, particularly compounds 11a, 11e, 12a, 12b, and 12h, exhibited much stronger cancer cell sensitizing effect than the best previous analogue 4a toward the treatment with etoposide, suggesting that these analogues could serve as effective cellular probes.

Triazolopyrimidine and triazolopyridine scaffolds as TDP2 inhibitors.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2) mediated DNA damages and causes cellular resistance to clinically used TOP2 poisons. Inhibiting TDP2 can potentially sensitize cancer cells toward TOP2 poisons. Commercial compound P10A10, to which the structure was assigned as 7-phenyl triazolopyrimidine analogue 6a, was previously identified as a TDP2 inhibitor hit in our virtual and fluorescence-based biochemical screening campaign. We report herein that the hit validation through resynthesis and structure elucidation revealed the correct structure of P10A10 (Chembridge ID 7236827) to be the 5-phenyl triazolopyrimidine regioisomer 7a. Subsequent structure-activity relationship (SAR) via the synthesis of a total of 47 analogues of both the 5-phenyl triazolopyrimidine scaffold (7) and its bioisosteric triazolopyridine scaffold (17) identified four derivatives (7a, 17a, 17e, and 17z) with significant TDP2 inhibition (IC50 < 50 µM), with 17z showing excellent cell permeability and no cytotoxicity.

New fluorescence-based high-throughput screening assay for small molecule inhibitors of tyrosyl-DNA phosphodiesterase 2 (TDP2).

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2) mediated DNA damages and causes resistance to TOP2-targeted cancer therapy. Inhibiting TDP2 could sensitize cancer cells toward TOP2 inhibitors. However, potent TDP2 inhibitors with favorable physicochemical properties are not yet reported. Therefore, there is a need to search for novel molecular scaffolds capable of inhibiting TDP2. We report herein a new simple, robust, homogenous mix-and-read fluorescence biochemical assay based using humanized zebrafish TDP2 (14M_zTDP2), which provides biochemical and molecular structure basis for TDP2 inhibitor discovery. The assay was validated by screening a preselected library of 1600 compounds (Z' ≥ 0.72) in a 384-well format, and by running in parallel gel-based assays with fluorescent DNA substrates. This library was curated via virtual high throughput screening (vHTS) of 460,000 compounds from Chembridge Library, using the crystal structure of the novel surrogate protein 14M_zTDP2. From this primary screening, we selected the best 32 compounds (2% of the library) to further assess their TDP2 inhibition potential, leading to the IC50 determination of 10 compounds. Based on the dose-response curve profile, pan-assay interference compounds (PAINS) structure identification, physicochemical properties and efficiency parameters, two hit compounds, 11a and 19a, were tested using a novel secondary fluorescence gel-based assay. Preliminary structure-activity relationship (SAR) studies identified guanidine derivative 12a as an improved hit with a 6.4-fold increase in potency over the original HTS hit 11a. This study highlights the importance of the development of combination approaches (biochemistry, crystallography and high throughput screening) for the discovery of TDP2 inhibitors.

Spirotriazoline oxindoles: A novel chemical scaffold with in vitro anticancer properties.

The design and synthesis of a library of twenty-six spirotriazoline oxindoles and their in vitro evaluation as potential anticancer agents is reported. The antiproliferative activity of the synthesized compounds was assessed against four different cancer cell lines (HCT-116 p53(+/+), HCT-116 p53(-/-), MCF-7, and MDA-MB-231). Four spirotriazoline oxindoles showed selectivity against the four cancer cell lines tested over the non-cancer derived HEK 293T cell line. To characterize the molecular mechanisms involved in compound antitumoral activity, two spirotriazoline oxindoles were selected for further studies. Both compounds were able to induce apoptosis and cell cycle arrest at G0/G1 phase and upregulated p53 steady-state levels, while decreasing its main inhibitor MDM2, in HCT-116 cells. Importantly, cytotoxic effects induced by spirotriazoline oxindoles occurred in cancer cells without eliciting cell death in non-malignant CCD-18Co human colon fibroblasts. In addition, four spirotriazoline oxindoles showed selectivity against the triple-negative breast cancer cell line MDA-MB-231 with IC50 values of 3.5-6.7 µM. These results highlight the anticancer potential of spirotriazoline oxindoles, especially when dealing with aggressive and challenging triple-negative breast cancer.

In vitro targeting of colon cancer cells using spiropyrazoline oxindoles.

We report on the synthesis and biological evaluation of a library of twenty-three spiropyrazoline oxindoles. The antiproliferative activity of the chemical library was evaluated in HCT-116 p53(+/+) human colon cancer cell line with eight derivatives displaying good activities (IC50<15 µM). To characterize the molecular mechanisms involved in compound antitumoral activity, two spiropyrazoline oxindoles were selected for further studies. Both compounds were able to induce apoptosis and cell cycle arrest at G0/G1 phase and upregulated p53 steady-state levels, while decreasing its main inhibitor MDM2. Importantly, cytotoxic effects induced by spiropyrazolines oxindoles occurred in cancer cells without eliciting cell death in non-malignant CCD-18Co human colon fibroblasts. Additionally, we demonstrated that the combination of spiropyrazoline oxindole 2e with sub-toxic concentrations of the chemotherapeutic agent 5-fluorouracil (5-FU) exerted a synergistic inhibitory effect on HCT-116 colon cancer cell proliferation. Collectively, our results show the potential of spiropyrazoline oxindoles for development of novel anticancer agents.

Chemical Variations on the p53 Reactivation Theme.

Among the tumor suppressor genes, p53 is one of the most studied. It is widely regarded as the "guardian of the genome", playing a major role in carcinogenesis. In fact, direct inactivation of the TP53 gene occurs in more than 50% of malignancies, and in tumors that retain wild-type p53 status, its function is usually inactivated by overexpression of negative regulators (e.g., MDM2 and MDMX). Hence, restoring p53 function in cancer cells represents a valuable anticancer approach. In this review, we will present an updated overview of the most relevant small molecules developed to restore p53 function in cancer cells through inhibition of the p53-MDMs interaction, or direct targeting of wild-type p53 or mutated p53. In addition, optimization approaches used for the development of small molecules that have entered clinical trials will be presented.

Novel squaramides with in vitro liver stage antiplasmodial activity.

A structure-activity relationship study was performed with ten 8-aminoquinoline-squaramides compounds active against liver stage malaria parasites, using human hepatoma cells (Huh7) infected by Plasmodium berghei parasites. In addition, their blood-schizontocidal activity was assessed against chloroquine-resistant W2 strain Plasmodium falciparum. Compound 3 was 7.3-fold more potent than the positive control primaquine against liver-stage parasites, illustrating the importance of the squarate moiety to activity.

Titanium(IV)-catalyzed stereoselective synthesis of spirooxindole-1-pyrrolines.

A stereoselective cyclization between alkylidene oxindoles and 5-methoxyoxazoles has been developed using catalytic titanium(IV) chloride (as low as 5 mol %) to afford spiro[3,3'-oxindole-1-pyrrolines] in excellent yield (up to 99%) and diastereoselectivity (up to 99:1). Using a chiral scandium(III)-indapybox/BArF complex affords enantioenriched spirooxindole-1-pyrrolines where a ligand-induced reversal of diastereoselectivity is observed. This methodology is further demonstrated for the synthesis of pyrrolines from malonate alkylidene and coumarin derivatives.

Synthesis and evaluation of spiroisoxazoline oxindoles as anticancer agents.

Restoring p53 levels through disruption of p53-MDM2 interaction has been proved to be a valuable approach in fighting cancer. We herein report the synthesis and evaluation of eighteen spiroisoxazoline oxindoles derivatives as p53-MDM2 interaction inhibitors. Seven compounds showed an antiproliferative profile superior to the p53-MDM2 interaction inhibitor nutlin-3, and induced cell death by apoptosis. Moreover, proof-of-concept was demonstrated by inhibition of the interaction between p53 and MDM2 in a live-cell bimolecular fluorescence complementation assay.

Squaric acid/4-aminoquinoline conjugates: novel potent antiplasmodial agents.

We report the synthesis and structure-activity relationship (SAR) analysis of a series of hybrid compounds containing a squaric moiety conjugated with heterocyclic moieties from well-known antimalarials. This novel series of compounds presents improved antiplasmodial activity compared with the squaric derivatives described in our previous work. Three compounds, 8b (IC50 = 99 nM), 8c (IC50 = 95 nM), and 8d (IC50 = 105 nM) had greater in vitro potency than chloroquine 1 (IC50 = 140 nM) against chloroquine resistant Plasmodium falciparum. In addition, they were noncytotoxic against NIH 3T3 and Hek 293T cells.

Cell death targets and potential modulators in Alzheimer's disease.

Apoptosis is now recognized as a normal feature in the development of the nervous system and may also play a role in neurodegenerative disorders, such as Alzheimer's disease. Cell surface receptors, caspases, mitochondrial factors or p53 participate in the modulation and execution of cell death. Therefore, the ability to understand and manipulate the cell death machinery is an obvious goal of medical research. Potential therapeutic approaches to modulate disease by regulating apoptosis are being tested, and include the traditional use of small molecules to target specific players in the apoptosis cascade. As our understanding of apoptosis increases, further opportunities will arise for more specific therapies that will result in improved efficacy. This review focuses on molecular mechanisms of apoptosis in Alzheimer's disease and highlights the potential use of small molecule modulators to treat neurodegenerative disorders.