Discovery of N-aryl-9-oxo-9H-fluorene-1-carboxamides as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 1. Structure–activity relationships of the carboxamide group

N-(2-Methylphenyl)-9-oxo-9H-fluorene-1-carboxamide (2a) was identified as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 2a was found to be active with sub-micromolar potencies for both caspase induction and growth inhibition in T47D human breast cancer, HCT116 human colon cancer, and SNU398 hepatocellular carcinoma cancer cells. It arrested HCT116 cells in G₂/M followed by apoptosis as assayed by the flow cytometry. Structure–activity relationship (SAR) studies of the carboxamide group identified the lead compound N-(2-(1H-pyrazol-1-yl)phenyl)-9-oxo-9H-fluorene-1-carboxamide (6s). Compound 6s, with increased aqueous solubility, was found to retain the broad activity in the caspase activation assay and in the cell growth inhibition assay with sub-micromolar EC₅₀ and GI₅₀ values in T47D, HCT116, and SNU398 cells, respectively.     

Discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-(E)-2,3,6,7-tetrahydro-1,4-thiazepines as a new series of apoptosis inducers using a cell- and caspase-based HTS assay

We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.     

Discovery of substituted 4-anilino-2-(2-pyridyl)pyrimidines as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay. Part 1: structure-activity relationships of the 4-anilino group

A series of 4-anilino-2-(2-pyridyl)pyrimidines has been discovered as a new class of potent inducers of apoptosis using a cell-based HTS assay. Compound 5a was found to arrest T47D cells in G2/M and induced apoptosis. SAR studies showed that a small and electron-donating group at the meta-position of the anilino ring is important for activity. A 20-fold increase in potency, from hit compound 4-(3-methoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5a) to lead compound 4-(2,5-dimethoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5l), was obtained through the SAR studies. Compound 5l is highly active with an EC50 value of 18 nM in the caspase activation assay in T47D breast cells. Interestingly, 5a and other meta-mono-substituted compounds were active against T47D cells but were not active against H1299 and HT29 cells, while 5l and other 2,5-disubstituted compounds were active against all the three cells. In a tubulin polymerization assay, compound 5l inhibited tubulin polymerization with an IC50 value of < 0.5 microM, while 5a was not active up to 50 microM.     

Discovery of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. Part 1: Structure-activity relationships of the 1- and 3-positions

1-Benzoyl-3-cyanopyrrolo[1,2-a]quinoline (2a) was identified as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 2a had good activity against several breast cancer cell lines but was much less active against several other cancer cell lines. SAR studies of 2a found that substitution at the 4-position of the 1-benzoyl group was important for activity. Replacing the 3-cyano group by an ester or ketone group led to inactive compounds. Interestingly, 4-substituted analogs such as 1-(4-(1H-imidazol-1-yl)benzoyl)-3-cyanopyrrolo[1,2-a]quinoline (2k) were found to be broadly and highly active in the caspase activation assay as well as in the cell growth inhibition assay with low nM EC(50) and GI(50) values in human breast cancer cells T47D, human colon cancer cells HCT116, and hepatocellular carcinoma cancer cells SNU398. Compound 2a was found not to inhibit tubulin polymerization up to 50 microM, while 2k was found to inhibit tubulin polymerization with an IC(50) value of 5 microM, indicating that certain substituents at the 4-position of the 1-benzoyl group can change the mechanism of action.     

Discovery of substituted 4-anilino-2-arylpyrimidines as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay. 2. Structure–activity relationships of the 2-aryl group

As a continuation of our efforts to discover and develop the apoptosis inducing 4-anilino-2-(2-pyridyl)pyrimidines as potential anticancer agents, we explored replacing the 2-pyridyl group by other aryl groups. SAR studies showed that the 2-pyridyl group can be replaced by a 3-pyridyl, 4-pyridyl and 2-pyrazinyl group, and that the SAR for the anilino group was similar to that of the 2-pyridyl series. However, replacement of the 2-pyridyl group by a phenyl group, a 3,5-dichloro-4-pyridyl group, or a saturated ring led to inactive compounds. Several potent compounds, including 2f, 3d, 3j and 4a, with EC₅₀ values of 0.048–0.024 μM in the apoptosis induction assay against T47D cells, were identified through the SAR studies. In a tubulin polymerization assay, compound 2f, which was active against all the three cell lines tested (T47D, HTC116 and SNU398), inhibited tubulin polymerization with an IC₅₀ value of 0.5 μM, while compound 2a, which was active against T47D cells but not active against HTC116 and SNU398 cells, was not active in the tubulin assay at up to 50 μM.     

Discovery and SAR of indole-2-carboxylic acid benzylidene-hydrazides as a new series of potent apoptosis inducers using a cell-based HTS assay

A series of indole-2-carboxylic acid benzylidene-hydrazides has been identified as a new class of potent apoptosis inducers through a novel cell-based caspase HTS assay. The screening hit, 5-chloro-3-methyl-indole-2-carboxylic acid (4-nitrobenzylidene)-hydrazide (3a), was found to arrest T47D cells in G(2)/M and to induce apoptosis as measured by the flow cytometric analysis assay. A SAR study was carried out by modification of the substitutions on the indole and benzene rings. Substitution at the 3-position of the indole ring was found to be important for apoptotic activity. A 20-fold increase of apoptotic activity was achieved from screening hit 3a to 5-methyl-3-phenyl-indole-2-carboxylic acid (4-methylbenzylidene)-hydrazide (9a) and 5-chloro-3-phenyl-indole-2-carboxylic acid (4-nitrobenzylidene)-hydrazide (9b), with EC(50) value of 0.1microM in the caspase activation assay in T47D breast cancer cells. Compound 9b also was found to be highly active in a standard growth inhibition assay with a GI(50) value of 0.9microM in T47D cells. Compound 3a and its analogs were found to inhibit tubulin polymerization, which is the most probable primary mechanism of action of these compounds.     

Discovery and structure-activity relationship of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as a new series of potent apoptosis inducers

We report the discovery and SAR study of a series of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as potent inducers of apoptosis. N-(3-Acetylphenyl)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-amine (2) was discovered through our cell- and caspase-based HTS assays as an inducer of apoptosis. Compound 2 is active against cancer cells derived from several human solid tumors, with EC(50) values ranging from 400 to 700 nM. SAR study of hit 2 led to the discovery of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as a novel series of potent apoptosis inducers, with 1,3-dimethyl-N-(4-propionylphenyl)-1H-pyrazolo[3,4-b]quinolin-amine (6b) having EC(50) values ranging from 30 to 70 nM in cancer cells. These compounds also demonstrated potent activity in the cell growth inhibition assay, with GI(50) values of 16-42 nM for compound 6b.     

Apoptosis induction in HCT116 cells by cytochalasins isolated from the fungus Daldinia vernicosa.

We examined the induction of apoptosis by cytochalasin (cc) derivatives (1-14) isolated from the Japanese fungus Daldinia vernicosa to HCT116 human colon cancer cell line based on their cytotoxicity, DNA ladder and DNA fragmentation ratio in agarose gel electrophoresis, and morphological changes. Most cc derivatives tested here induced apoptosis. Particularly cytochalasin 1 (cc1), monoacetate of 1 (cc1Ac), and cc14 were the most potent apoptosis inducers. These apoptotic activities were stronger than that of cytochalasin D as a known apoptosis inducer in HCT116 cell. However, cc4 and cc12 induced necrosis. The structure-activity relationship including their cytotoxicity will be discussed.     

Discovery of substituted N′-(2-oxoindolin-3-ylidene)benzohydrazides as new apoptosis inducers using a cell- and caspase-based HTS assay

We report the discovery of a series of substituted N′-(2-oxoindolin-3-ylidene)benzohydrazides as inducers of apoptosis using our proprietary cell- and caspase-based ASAP HTS assay. Through SAR studies, N′-(4-bromo-5-methyl-2-oxoindolin-3-ylidene)-3,4,5-trimethoxybenzohydrazide (3g) was identified as a potent apoptosis inducer with an EC₅₀ value of 0.24 μM in human colorectal carcinoma HCT116 cells, more than a 40-fold increase in potency from the initial screening hit N′-(5-bromo-2-oxoindolin-3-ylidene)-3,4,5-trimethoxybenzohydrazide (2a). Compound 3g also was found to be highly active in a growth inhibition assay with a GI₅₀ value of 0.056 μM in HCT116 cells. A group of potentially more aqueous soluble analogs were prepared and found to be highly active. Among them, compound 4e incorporating a methyl piperazine moiety was found to have EC₅₀ values of 0.17, 0.088 and 0.14 μM in human colorectal carcinoma cells HCT116, hepatocellular carcinoma cancer SNU398 cells and human colon cancer RKO cells, respectively. Compounds 3g and 4e were found to function as inhibitors of tubulin polymerization.     

Discovery of 4-anilino-N-methylthieno[3,2-d]pyrimidines and 4-anilino-N-methylthieno[2,3-d]pyrimidines as potent apoptosis inducers

We report the discovery of N-((benzo[d][1,3]dioxol-5-yl)methyl)-6-phenylthieno[3,2-d]pyrimidin-4-amine (2a) as an apoptosis inducer using our proprietary cell- and caspase-based ASAP HTS assay, and SAR study of HTS hit 2a which led to the discovery of 4-anilino-N-methylthieno[3,2-d]pyrimidines and 4-anilino-N-methylthieno[2,3-d]pyrimidines as potent apoptosis inducers. Compounds 5d and 5e were the most potent with EC₅₀ values of 0.008 and 0.004 μM in T47D human breast cancer cells, respectively. Compound 5d was found to be highly active in the MX-1 breast cancer model. Functionally, compounds 5d and 5e both induced apoptosis through inhibition of tubulin polymerization.     

Discovery of 4-aryl-2-oxo-2H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay

As a continuation of our efforts to discover and develop the apoptosis inducing 4-aryl-4H-chromenes as potential anticancer agents, we explored the removal of the chiral center at the 4-position and prepared a series of 4-aryl-2-oxo-2H-chromenes. It was found that, in general, removal of the chiral center and replacement of the 2-amino group with a 2-oxo group were tolerated and 4-aryl-2-oxo-2H-chromenes exhibited SAR similar to 4-aryl-2-amino-4H-chromenes. The 4-aryl-2-oxo-2H-chromenes with a N-methyl pyrrole fused at the 7,8-positions were highly active with compound 2a having an EC(50) value of 13 nM in T47D cells. It was found that an OMe group was preferred at the 7-position. 7-NMe(2), 7-NH(2), 7-Cl and 7,8 fused pyrido analogs all had low potency. These 4-aryl-2-oxo-2H-chromenes are a series of potent apoptosis inducers with potential advantage over the 4-aryl-2-amino-4H-chromenes series via elimination of the chiral center at the 4-position.     

Discovery of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 2: Structure–activity relationships of the 4-, 5-, 6-, 7- and 8-positions

As a continuation of our efforts to discover and develop the apoptosis inducing 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as potential anticancer agents, we explored substitutions at the 4-, 5-, 6-, 7- and 8-positions of pyrrolo[1,2-a]quinoline. SAR studies showed that substitution at the 6-position by a small group such as Cl resulted in potent compounds. Substitutions at the 5- and 8-positions were tolerated while substitutions at the 4- and 7-position led to inactive compounds. Several compounds, including 2c, 3a, 3b and 3f, were found to be highly active against human breast cancer cells T47D with EC₅₀ values of 0.053–0.080 μM, but much less active against human colon cancer cells HCT116 and hepatocellular carcinoma cancer cells SNU398 in the caspase activation assay. Compound 3f also was found to be highly active with a GI₅₀ value of 0.018 μM against T47D cells in a growth inhibition assay.     

Synthesis, biological evaluation and molecular docking studies of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide derivatives as inhibitors of HDAC activity

In present study, a series of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide derivatives (5a-8d) were designed, synthesized, and evaluated for HDAC inhibition and tumor cell antiproliferation. All of these compounds are reported for the first time, the chemical structures of these compounds were confirmed by means of (1)H NMR, ESI-MS and elemental analyzes. Among the compounds, compound 8c showed the most potent biological activity against HCT116 cancer cell line (IC(50) of 0.42 ± 0.02 μM for HDAC-1 and IC(50)=0.62 ± 0.02 for HCT116). Docking simulation was performed to position compound 8c into the HDAC active site to determine the probable binding model. The results of antiproliferative assay and western-blot demonstrated that compound 8c with potent inhibitory activity in tumor growth inhibition may be a potential anticancer agent against HCT116 cancer cell.     

Synthesis and monoamine transporter binding properties of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes

A series of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes were prepared and evaluated for affinities at dopamine, serotonin, and norepinephrine transporters using competitive radioligand binding assays. The 2beta-[3'-(substituted benzyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (3a-h) showed high binding affinities for the dopamine transporter (DAT). The IC(50) values ranged from 5.9 to 22nM. On the other hand, the 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (4a-h), with IC(50) values ranging from 65 to 173nM, were approximately 3- to 25-fold less potent than the corresponding 2beta-[3'-(substituted benzyl)isoxazol]tropanes. All tested compounds were selective for the DAT relative to the norepinephrine transporter (NET) and serotonin transporter (5-HTT). 3Beta-(4-Methylphenyl)-2beta-[3'-(4-fluorobenzyl)isoxazol-5-yl]tropane (3b) with IC(50) of 5.9nM at the DAT and K(i)s of 454 and 113nM at the NET and 5-HTT, respectively, was the most potent and DAT-selective analog. Molecular modeling studies suggested that the rigid conformation of the isoxazole side chain in 4a-h might play an important role on their low DAT binding affinities.     

New potent steroid sulphatase inhibitors based on 6-(1-phenyl-1H-1,2,3-triazol-4-yl)naphthalen-2-yl sulphamate derivatives

In the present work, we report a new class of potent steroid sulphatase (STS) inhibitors based on 6-(1-phenyl-1H-1,2,3-triazol-4-yl)naphthalen-2-yl sulphamate derivatives. Within the set of new STS inhibitors, 6-(1-(1,2,3-trifluorophenyl)-1H-1,2,3-triazol-4-yl)naphthalen-2-yl sulphamate 3L demonstrated the highest activity in the enzymatic assay inhibiting the STS activity to 7.98% at 0.5 µM concentration. Furthermore, to verify whether the obtained STS inhibitors are able to pass through the cellular membrane effectively, cell line experiments have been carried out. We found that the lowest STS activities were measured in the presence of compound 3L (remaining STS activity of 5.22%, 27.48% and 99.0% at 100, 10 and 1 nM concentrations, respectively). The measured STS activities for Irosustat (used as a reference) were 5.72%, 12.93% and 16.83% in the same concentration range. Moreover, a determined IC₅₀ value of 15.97 nM for 3L showed that this compound is a very promising candidate for further preclinical investigations.     

Novel retinoic acid 4-hydroxylase (CYP26) inhibitors based on a 3-(1H-imidazol- and triazol-1-yl)-2,2-dimethyl-3-(4-(phenylamino)phenyl)propyl scaffold

Retinoic acid (RA), the biologically active metabolite of vitamin A, is used medicinally for the treatment of hyperproliferative diseases including dermatological conditions and cancer. The antiproliferative effects of RA have been well documented as well as the limitations owing to toxicity and the development of resistance to RA therapy. RA metabolism inhibitors (RAMBAs or CYP26 inhibitors) are attracting increasing interest as an alternative method for enhancing endogenous levels of retinoic acid in the treatment of hyperproliferative disease. Here the synthesis and inhibitory activity of novel 3-(1H-imidazol- and triazol-1-yl)-2,2-dimethyl-3-(4-(phenylamino)phenyl)propyl derivatives in a MCF-7 CYP26A1 microsomal assay are described. The most promising inhibitor methyl 2,2-dimethyl-3-(4-(phenylamino)phenyl)-3-(1H-1,2,4-triazol-1-yl)propanoate (6) exhibited an IC(50) of 13 nM (compared with standards Liarozole IC(50) 540 nM and R116010 IC(50) 10 nM) and was further evaluated for CYP selectivity using a panel of CYP with >100-fold selectivity for CYP26 compared with CYP1A2, 2C9 and 2D6 observed and 15-fold selectivity compared with CYP3A4. The results demonstrate the potential for further development of these potent inhibitors.     

Novel azolyl-(phenylmethyl)]aryl/heteroarylamines: potent CYP26 inhibitors and enhancers of all-trans retinoic acid activity in neuroblastoma cells

The synthesis and potent inhibitory activity of novel 4-[(imidazol-1-yl and triazol-1-yl)(phenyl)methyl]aryl-and heteroaryl amines versus a MCF-7 CYP26A1 cell assay is described. Biaryl imidazole ([4-(imidazol-1-yl-phenyl-methyl)-phenyl]-naphthalen-2-yl-amine (8), IC(50)=0.5 microM; [4-(imidazol-1-yl-phenyl-methyl)-phenyl]-indan-5-yl-amine (9), IC(50)=1.0 microM) and heteroaryl imidazole derivatives ((1H-benzoimidazol-2-yl)-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (15), IC(50)=2.5 microM; benzooxazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (16), IC(50)=0.9 microM; benzothiazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (17), IC(50)=1.5 microM) were the most potent CYP26 inhibitors. Using a CYP26A1 homology model differences in activity were investigated. Incubation of SH-SY5Y human neuroblastoma cells with the imidazole aryl derivative 8, and the imidazole heteroaryl derivatives 16 and 17 potentiated the atRA-induced expression of CYP26B1. These data suggest that further structure-function studies leading to clinical development are warranted.     

Discovery and structure–activity relationships of (2-(arylthio)benzylideneamino)guanidines as a novel series of potent apoptosis inducers

1-(2-(2,5-Dimethoxyphenylthio)benzylidene)semicarbazide (2a) was discovered as a potent apoptosis inducer through our cell based HTS assay. SAR study led to the discovery of a more aqueous soluble analog (2-(2,5-dimethoxyphenylthio)-6-methoxybenzylideneamino)guanidine (5e) with EC₅₀ value of 60 nM in the caspase activation assay and GI₅₀ value of 62 nM in the growth inhibition assay in T47D cells. Compound 5e was found to be an inhibitor of tubulin polymerization and efficacious in a MX-1 breast tumor model.     

Cacospongionolide and scalaradial, two marine sesterterpenoids as potent apoptosis-inducing factors in human carcinoma cell lines

Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects of scalaradial and cacospongionolide, two sesterterpenoids from Cacospongia scalaris and Fasciospongia cavernosa marine sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot. The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116 (human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation. Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-κB subunits, nuclear level. In conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising compounds to inhibit cancer cell proliferation.