Discovery of an Aldo-Keto reductase 1C3 (AKR1C3) degrader.

Aldo-keto reductase 1C3 (AKR1C3) is a protein upregulated in prostate cancer, hematological malignancies, and other cancers where it contributes to proliferation and chemotherapeutic resistance. Androgen receptor splice variant 7 (ARv7) is the most common mutation of the AR receptor that confers resistance to clinical androgen receptor signalling inhibitors in castration-resistant prostate cancer. AKR1C3 interacts with ARv7 promoting stabilization. Herein we report the discovery of the first-in-class AKR1C3 Proteolysis-Targeting Chimera (PROTAC) degrader. This first-generation degrader potently reduced AKR1C3 expression in 22Rv1 prostate cancer cells with a half-maximal degradation concentration (DC50) of 52 nM. Gratifyingly, concomitant degradation of ARv7 was observed with a DC50 = 70 nM, along with degradation of the AKR1C3 isoforms AKR1C1 and AKR1C2 to a lesser extent. This compound represents a highly useful chemical tool and a promising strategy for prostate cancer intervention.

Aldo-Keto Reductase 1C3 Inhibitor Prodrug Improves Pharmacokinetic Profile and Demonstrates In Vivo Efficacy in a Prostate Cancer Xenograft Model.

Aldo-keto reductase 1C3 (AKR1C3) is overexpressed in castration-resistant prostate cancer where it acts to drive proliferation and aggressiveness by producing androgens. The reductive action of the enzyme leads to chemoresistance development against various clinical antineoplastics across a range of cancers. Herein, we report the continued optimization of selective AKR1C3 inhibitors and the identification of 5r, a potent AKR1C3 inhibitor (IC50 = 51 nM) with >1216-fold selectivity for AKR1C3 over closely related isoforms. Due to the cognizance of the poor pharmacokinetics associated with free carboxylic acids, a methyl ester prodrug strategy was pursued. The prodrug 4r was converted to free acid 5r in vitro in mouse plasma and in vivo. The in vivo pharmacokinetic evaluation revealed an increase in systemic exposure and increased the maximum 5r concentration compared to direct administration of the free acid. The prodrug 4r demonstrated a dose-dependent effect to reduce the tumor volume of 22Rv1 prostate cancer xenografts without observed toxicity.

Studies on diketopiperazine and dipeptide analogs as opioid receptor ligands.

Using the structure of gliotoxin as a starting point, we have prepared two different chemotypes with selective affinity to the kappa opioid receptor (KOR). Using medicinal chemistry approaches and structure-activity relationship (SAR) studies, structural features required for the observed affinity were identified, and advanced molecules with favorable Multiparameter Optimization (MPO) and Ligand Lipophilicity (LLE) profiles were prepared. Using the Thermal Place Preference Test (TPPT), we have shown that compound2 blocks the antinociceptive effect of U50488, a known KOR agonist. Multiple reports suggest that modulation of KOR signaling is a promising therapeutic strategy in treating neuropathic pain (NP). As a proof-of-concept study, we tested compound 2 in a rat model of NP and recorded its ability to modulate sensory and emotional pain-related behaviors. Observed in vitro and in vivo results suggest that these ligands can be used to develop compounds with potential application as pain therapeutics.

Selective carbonic anhydrase IX and XII inhibitors based around a functionalized coumarin scaffold.

Inhibition of specific carbonic anhydrase (CA) enzymes is a validated strategy for the development of agents to target cancer. The CA isoforms IX and XII are overexpressed in various human solid tumors wherein they play a critical role in regulating extracellular tumor acidification, proliferation, and progression. A series of novel sulfonamides based on the coumarin scaffold were designed, synthesized and characterized as potent and selective CA inhibitors. Selected compounds show significant activity and selectivity over CA I and CA II to target the tumor-associated CA IX and CA XII with high inhibition activity at the single digit nanomolar level. Twelve compounds were identified to be more potent compared with acetazolamide (AAZ) control to inhibit CA IX while one was also more potent than AAZ to inhibit CA XII. Compound 18f (Ki's = 955 nM, 515 nM, 21 nM and 5 nM for CA's I, II, IX, and XII, respectively) is highlighted as a novel CA IX and XII inhibitor for further development.

Access to Highly Strained Tricyclic Ketals Derived from Coumarins.

Synthesis of highly strained fused substituted dihydrobenzopyran cyclopropyl lactones derived from coumarin carboxylates are reported. The substrate scope tolerates a variety of 6- and 8-substituents on the coumarin ring. Substitution at the 5- or 7-position is resistant to tricyclic lactone formation except with 7-methyl substitution. Benzamide-containing coumarins afford the tricyclic ketal. A plausible mechanism is proposed for the formation of the fused lactone: intramolecular rearrangement of trans cyclopropyl methyl ketones with phenolic acetate via the formation of a hemiacetal.

Synthesis and biological evaluation of N, N-dialkylcarboxy coumarin-NO donor conjugates as potential anticancer agents.

A series of nitric oxide (NO) donor furoxan conjugates of N, N-dialkylcarboxy coumarins have been synthesized as potential anticancer agents. The synthesized compounds have been tested for their in vitro antiproliferative activities on various cancer and noncancerous cell lines. The candidate derivatives exhibit selectivity towards cancer cells with excellent activities in low nM to µM concentrations. In vitro mechanistic studies indicate that the candidate compounds generate substantial NO, inhibit colony formation, and cause apoptosis in cancer cells. A preliminary in vivo tolerance study of the lead candidate 10 in mice indicates that it is well-tolerated, evidenced by zero mortality and normal body weight gains in treated mice. Further translation of the lead derivative 10 using MDA-MB-231 based tumor xenograft model shows good tumor growth reduction.

Synthesis and biological evaluation of a novel anticancer agent CBISC that induces DNA damage response and diminishes levels of mutant-p53.

A novel nitrogen mustard CBISC has been synthesized and evaluated as an anticancer agent. CBISC has been shown to exhibit enhanced cell proliferation inhibition properties against mutant p53 cell lines colorectal cancer WiDr, pancreatic cancer (MIAPaCa-2 and PANC-1), and triple negative breast cancer (MDA-MB-231 and MDA-MB-468). In vitro mechanism of action studies revealed perturbations in the p53 pathway and increased cell death as evidenced by western blotting, immunofluorescent microscopy and MTT assay. Further, in vivo studies revealed that CBISC is well tolerated in healthy mice and exhibited significant in vivo tumor growth inhibition properties in WiDr and MIAPaCa-2 xenograft models. These studies illustrate the potential utility of CBISC as an anticancer agent.

Synthesis, in vitro, and in vivo evaluation of novel N-phenylindazolyl diarylureas as potential anti-cancer agents.

Novel N-phenylindazole based diarylureas have been designed, synthesized and evaluated as potential anticancer agents. In vitro cell viability studies of these derivatives illustrate good potency with IC50 values in the range of 0.4-50 µM in several cancer cell lines including murine metastatic breast cancer 4T1, murine glioblastoma GL261, human triple negative breast cancer MDA-MB-231, human pancreatic cancer MIAPaCa-2, and human colorectal cancer cell line WiDr. The ester group in the lead compound 8i was modified to incorporate amino-amides to increase solubility and stability while retaining biological activity. Further in vitro studies reveal that lead candidates inhibit tube length in HUVEC cells. In vivo systemic toxicity studies indicate that these candidate compounds are well tolerated in mice without any significant side effects. Anticancer efficacy studies in WiDr tumor xenograft and 4T1 tumor syngraft models demonstrate that the lead candidate 11 exhibits significant antitumor properties as a single agent in these tumor models.

Synthesis and biological evaluation of arylphosphonium-benzoxaborole conjugates as novel anticancer agents.

Arylphosphonium-benzoxaborole conjugates have been synthesized as potential mitochondria targeting anticancer agents. The synthesized compounds have been tested for their effects on cell viability in various solid tumor cell lines including breast cancer 4T1 and MCF-7, pancreatic cancer MIAPaCa-2 and colorectal adenocarcinoma WiDr. Compound 6c is designated as a lead compound for further studies due to its enhanced effects on cell viability in the above-mentioned cell lines. Seahorse Xfe96 based metabolic assays reveal that the lead candidate 6c inhibits mitochondrial respiration in 4T1 and WiDr cell lines as evidenced by the reduction of mitochondrial ATP production and increase in proton leak. Epiflourescent microscopy experiments also illustrate that 6c causes significant mitochondrial fragmentation in 4T1 and WiDr cells, morphologically consistent with programmed cell death. Our current studies illustrate that arylphosphonium-benzoxaborole conjugates have potential to be further developed as anticancer agents.

Development of Novel Analogs of the Monocarboxylate Transporter Ligand FACH and Biological Validation of One Potential Radiotracer for Positron Emission Tomography (PET) Imaging.

Monocarboxylate transporters 1-4 (MCT1-4) are involved in several metabolism-related diseases, especially cancer, providing the chance to be considered as relevant targets for diagnosis and therapy. [18F]FACH was recently developed and showed very promising preclinical results as a potential positron emission tomography (PET) radiotracer for imaging of MCTs. Given that [18F]FACH did not show high blood-brain barrier permeability, the current work is aimed to investigate whether more lipophilic analogs of FACH could improve brain uptake for imaging of gliomas, while retaining binding to MCTs. The 2-fluoropyridinyl-substituted analogs 1 and 2 were synthesized and their MCT1 inhibition was estimated by [14C]lactate uptake assay on rat brain endothelial-4 (RBE4) cells. While compounds 1 and 2 showed lower MCT1 inhibitory potencies than FACH (IC50 = 11 nM) by factors of 11 and 25, respectively, 1 (IC50 = 118 nM) could still be a suitable PET candidate. Therefore, 1 was selected for radiosynthesis of [18F]1 and subsequent biological evaluation for imaging of the MCT expression in mouse brain. Regarding lipophilicity, the experimental log D7.4 result for [18F]1 agrees pretty well with its predicted value. In vivo and in vitro studies revealed high uptake of the new radiotracer in kidney and other peripheral MCT-expressing organs together with significant reduction by using specific MCT1 inhibitor α-cyano-4-hydroxycinnamic acid. Despite a higher lipophilicity of [18F]1 compared to [18F]FACH, the in vivo brain uptake of [18F]1 was in a similar range, which is reflected by calculated BBB permeabilities as well through similar transport rates by MCTs on RBE4 cells. Further investigation is needed to clarify the MCT-mediated transport mechanism of these radiotracers in brain.

Development of Novel Silyl Cyanocinnamic Acid Derivatives as Metabolic Plasticity Inhibitors for Cancer Treatment.

Novel silyl cyanocinnamic acid derivatives have been synthesized and evaluated as potential anticancer agents. In vitro studies reveal that lead derivatives 2a and 2b have enhanced cancer cell proliferation inhibition properties when compared to the parent monocarboxylate transporter (MCT) inhibitor cyano-hydroxycinnamic acid (CHC). Further, candidate compounds exhibit several-fold more potent MCT1 inhibition properties as determined by lactate-uptake studies, and these studies are supported by MCT homology modeling and computational inhibitor-docking studies. In vitro effects on glycolysis and mitochondrial metabolism also illustrate that the lead derivatives 2a and 2b lead to significant effects on both metabolic pathways. In vivo systemic toxicity and efficacy studies in colorectal cancer cell WiDr tumor xenograft demonstrate that candidate compounds are well tolerated and exhibit good single agent anticancer efficacy properties.

Novel N,N-dialkyl cyanocinnamic acids as monocarboxylate transporter 1 and 4 inhibitors.

Potent and dual monocarboxylate transporter (MCT) 1 and 4 inhibitors have been developed for the first time as potential anticancer agents based on α-cyanocinnamic acid structural template. Candidate inhibitors 1-9 have been evaluated for in vitro cell proliferation against MCT1 and MCT4 expressing cancer cell lines. Potential MCT1 and MCT4 binding interactions of the lead compound 9 have been studied through homology modeling and molecular docking prediction. In vitro effects on extracellular flux via glycolysis and mitochondrial stress tests suggest that candidate compounds 3 and 9 disrupt glycolysis and OxPhos efficiently in MCT1 expressing colorectal adenocarcinoma WiDr and MCT4 expressing triple negative breast cancer MDA-MB-231 cells. Fluorescence microscopy analyses in these cells also indicate that compound 9 is internalized and concentrated near mitochondria. In vivo tumor growth inhibition studies in WiDr and MDA-MB-231 xenograft tumor models in mice indicate that the candidate compound 9 exhibits a significant single agent activity.

Development and radiosynthesis of the first 18 F-labeled inhibitor of monocarboxylate transporters (MCTs).

Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) are involved in tumor development and progression. Their expression levels are related to clinical disease prognosis. Accordingly, both MCTs are promising drug targets for treatment of a variety of human cancers. The noninvasive imaging of these MCTs in cancers is regarded to be advantageous for assessing MCT-mediated effects on chemotherapy and radiosensitization using specific MCT inhibitors. Herein, we describe a method for the radiosynthesis of [18 F]FACH ((E)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylic acid), as a novel radiolabeled MCT1/4 inhibitor for imaging with PET. A fluorinated analog of α-cyano-4-hydroxycinnamic acid (FACH) was synthesized, and the inhibition of MCT1 and MCT4 was measured via an L-[14 C]lactate uptake assay. Radiolabeling was performed by a two-step protocol comprising the radiosynthesis of the intermediate (E)/(Z)-[18 F]tert-Bu-FACH (tert-butyl (E)/(Z)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylate) followed by deprotection of the tert-butyl group. The radiofluorination was successfully implemented using either K[18 F]F-K2.2.2 -carbonate or [18 F]TBAF. The final deprotected product [18 F]FACH was only obtained when [18 F]tert-Bu-FACH was formed by the latter procedure. After optimization of the deprotection reaction, [18 F]FACH was obtained in high radiochemical yields (39.6 ± 8.3%, end of bombardment (EOB) and radiochemical purity (greater than 98%).

Synthesis and Evaluation of Baylis-Hillman Reaction Derived Imidazole and Triazole Cinnamates as Antifungal Agents.

Allylic acetates derived from Baylis-Hillman reaction undergo efficient nucleophilic isomerization with imidazoles and triazoles to provide imidazolylmethyl and triazolylmethyl cinnamates stereoselectively. Antifungal evaluation of these derivatives against Cryptococcus neoformans exhibits good minimum inhibitory concentration values. These compounds exhibit low toxicity in proliferating MCF-7 breast cancer cell line. Structure activity relationship studies indicate that halogenated aromatic derivatives provide better antifungal activity.

Synthesis and biological evaluation of 2-alkoxycarbonylallyl esters as potential anticancer agents.

The reaction of carboxylic acids with Baylis-Hillman reaction derived α-bromomethyl acrylic esters readily provide 2-(alkoxycarbonyl)allyl esters in good to excellent yields. These functionalized allyl esters have been evaluated for their cell proliferation inhibition properties against breast cancer (MDA-MB-231 and 4T1) and pancreatic cancer (MIAPaCa-2) cell lines to explore their potential as anticancer agents. Several of the synthesized derivatives exhibit good potency against all three cancer cell lines. Our structure activity relationship (SAR) studies on 2-carboxycarbonyl allyl esters indicate that substituted aromatic carboxylic acids provide enhanced activity compared to substituted aliphatic carboxylic acid analogs. Di- and tri-allyl esters derived from di-and tri-carboxylic acids exhibit higher inhibition of cell proliferation than mono esters. Further SAR studies indicate that the double bond in the 2-(alkoxycarbonyl)allyl ester is required for its activity, and there is no increase in activity with increased chain length of the alkoxy group. Two lead candidate compounds have been identified from the cell proliferation inhibition studies and their preliminary mechanism of action as DNA damaging agents has been evaluated using epifluorescence and western blot analysis. One of the lead compounds has been further evaluated for its systemic toxicity in healthy CD-1 mice followed by anticancer efficacy in a triple negative breast cancer MDA-MB-231 xenograft model in NOD-SCID mice. These two in vivo studies indicate that the lead compound is well tolerated in healthy CD-1 mice and exhibits good tumor growth inhibition compared to breast cancer drug doxorubicin.

Synthesis and evaluation of functionalized benzoboroxoles as potential anti-tuberculosis agents.

Several derivatives of aminobenzoboroxole have been prepared starting from 2-boronobenzaldehyde. All of these derivatives have been evaluated for their anti-mycobacterial activity on Mycobacterium smegmatis and cytotoxicity on breast cancer cell line MCF7. Based on these studies, all the tested molecules have been found to be generally non-toxic and benzoboroxoles with unsubstituted (primary) amines have been found to exhibit good anti-mycobacterial activity. Some of the key compounds have been evaluated for their anti-tubercular activity on Mycobacterium tuberculosis H37Rv using 7H9 and GAST media. 7-Bromo-6-aminobenzoboroxole 4 has been identified as the lead candidate compound for further development.

Coumarin carboxylic acids as monocarboxylate transporter 1 inhibitors: In vitro and in vivo studies as potential anticancer agents.

Novel N,N-dialkyl carboxy coumarins have been synthesized as potential anticancer agents via inhibition of monocarboxylate transporter 1 (MCT1). These coumarin carboxylic acids have been evaluated for their in vitro MCT1 inhibition, MTT cancer cell viability, bidirectional Caco-2 cell permeability, and stability in human and liver microsomes. These results indicate that one of the lead candidate compounds 4a has good absorption, metabolic stability, and a low drug efflux ratio. Systemic toxicity studies with lead compound 4a in healthy mice demonstrate that this inhibitor is well tolerated based on zero animal mortality and normal body weight gains compared to the control group. In vivo tumor growth inhibition studies in mice show that the candidate compound 4a exhibits significant single agent activity in MCT1 expressing GL261-luc2 syngraft model but doesn't show significant activity in MCT4 expressing MDA-MB-231 xenograft model, indicating the selectivity of 4a for MCT1 expressing tumors.

Synthesis, in vitro, and in vivo evaluation of novel functionalized quaternary ammonium curcuminoids as potential anti-cancer agents.

Novel functionalized quaternary ammonium curcuminoids have been synthesized from piperazinyl curcuminoids and Baylis-Hillman reaction derived allyl bromides. These molecules are found to be highly water soluble with increased cytotoxicity compared to native curcumin against three cancer cell lines MIAPaCa-2, MDA-MB-231, and 4T1. Preliminary in vivo toxicity evaluation of a representative curcuminoid 5a in healthy mice indicates that this molecule is well tolerated based on normal body weight gains compared to control group. Furthermore, the efficacy of 5a has been tested in a pancreatic cancer xenograft model of MIAPaCa-2 and has been found to exhibit good tumor growth inhibition as a single agent and also in combination with clinical pancreatic cancer drug gemcitabine.

Monocarboxylate transporter 1 inhibitors as potential anticancer agents.

Potent monocarboxylate transporter 1 inhibitors (MCT1) have been developed based on α-cyano-4-hydroxycinnamic acid template. Structure-activity relationship studies demonstrate that the introduction of p-N, N-dialkyl/diaryl, and o-methoxy groups into cyanocinnamic acid has maximal MCT1 inhibitory activity. Systemic toxicity studies in healthy ICR mice with few potent MCT1 inhibitors indicate normal body weight gains in treated animals. In vivo tumor growth inhibition studies in colorectal adenocarcinoma (WiDr cell line) in nude mice xenograft models establish that compound 27 exhibits single agent activity in inhibiting the tumor growth.