Discovery of N-methyl-4-(4-methoxyanilino)quinazolines as potent apoptosis inducers. Structure-activity relationship of the quinazoline ring.

As a continuation of our efforts to discover and develop apoptosis inducing N-methyl-4-(4-methoxyanilino)quinazolines as novel anticancer agents, we explored substitution at the 5-, 6-, 7-positions of the quinazoline and replacement of the quinazoline by other nitrogen-containing heterocycles. A small group at the 5-position was found to be well tolerated. At the 6-position a small group like an amino was preferred. Substitution at the 7-position was tolerated much less than at the 6-position. Replacing the carbon at the 8-position or both the 5- and 8-positions with nitrogen led to about 10-fold reductions in potency. Replacement of the quinazoline ring with a quinoline, a benzo[d][1,2,3]triazine, or an isoquinoline ring showed that the nitrogen at the 1-position is important for activity, while the carbon at the 2-position can be replaced by a nitrogen and the nitrogen at the 3-position can be replaced by a carbon. Through the SAR study, several 5- or 6-substituted analogs, such as 2a and 2c, were found to have potencies approaching that of lead compound N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine (1g, EP128495, MPC-6827, Azixa).

Cell-based apoptosis assays in oncology drug discovery.

IMPORTANCE OF THE FIELD: Screening compounds with a cell-based phenotypic approach complements target-based discovery programs because of the opportunity to investigate targets in the context of the cellular milieu and to discover novel targets. AREAS COVERED IN THIS REVIEW: Utilizing a cell-based apoptotic phenotype screen for discovery and optimization of apoptosis inducers and affirming activity as potential anticancer agents in vivo with xenograft models. Subsequently, chemical genetic tools are utilized to identify and validate previously unrecognized cancer targets. Case studies showing the various multidisciplinary approaches utilized for several years are reviewed. WHAT THE READER WILL GAIN: The interactive nature of the drug and target discovery processes, and insights that come from integration of cellular biology, medicinal chemistry and animal research. TAKE HOME MESSAGE: Phenotype proapoptotic screen followed by chemical genetics is useful for anticancer drug research, for the discovery of potential drugs and identification of druggable targets.

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. 2. Structure-activity relationships of the 9-oxo-9H-fluorene ring.

As a continuation of our studies of apoptosis inducing 9-oxo-9H-fluorene-1-carboxamides as potential anticancer agents, we explored modification of the 9-oxo-9H-fluorene ring. SAR studies showed that most changes to the 9-oxo-9H-fluorene ring were not well tolerated, except the 9H-fluorene (2b) and dibenzothiophene (2d) analogs, which were about twofold less active than the 9-oxo-9H-fluorene analog 2a. Significantly, introduction of substitutions at the 7-position of the 9-oxo-9H-fluorene ring led to compounds 5a-5c with improved activity. Compound 5a was found to have EC(50) values of 0.15-0.29 microM against T47D, HCT116, and SNU398 cells, about fivefold more potent than the original lead 2a. As opposed to the original lead compound 2a, compounds 5a-5b were active in a tubulin inhibition assay, indicating a change of mechanism of action. The potent azido analog 5c could be utilized for target identification.

Discovery of 3-aryl-5-aryl-1,2,4-oxadiazoles as a new series of apoptosis inducers. 2. Identification of more aqueous soluble analogs as potential anticancer agents.

As a continuation of our efforts to discover and develop the 3-aryl-5-aryl-1,2,4-oxadiazole series of apoptosis inducers as potential anticancer agents, we explored substitutions at the 2- and 3-positions of the 3-aryl group to improve the aqueous solubility properties and identify development candidates. A small substitution such as methyl or hydroxymethyl at the 2-position was well tolerated. This modification, in combination with a 3-substituted furan ring as the 5-aryl group, resulted in 4g and 4h, which have improved solubility properties. Compound 4g was found to have good in vivo efficacy in animal studies via intravenous administration.

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 EC(50) values of 0.053-0.080microM, 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 GI(50) value of 0.018microM against T47D cells in a growth inhibition assay.

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 EC(50) values of 0.008 and 0.004microM 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-4H-chromenes as potent apoptosis inducers using a cell- and caspase-based Anti-cancer Screening Apoptosis Program (ASAP): SAR studies and the identification of novel vascular disrupting agents.

Many cancer cells are known to have defects in the apoptosis machinery. Therefore identification of compounds that can activate or promote apoptosis in cancer cells is an attractive approach for targeted therapies. By applying a novel cell- and caspase-based Anti-cancer Screening Apoptosis Program (ASAP) HTS assay, 4-aryl-4H-chromenes were identified as potent apoptosis inducers. 4-Aryl-4H-chromenes were found to induce nuclear fragmentation and PARP cleavage, as well as to arrest cells at the G(2)/M stage followed by apoptosis as determined by the flow cytometry analysis assay in multiple human cell lines (e.g. Jurkat, T47D). These compounds were found to be highly active in the growth inhibition MTT assay, including for paclitaxel resistant, p-glycoprotein overexpressed, MES-SA/DX5 tumor cells. Functionally, they were found to be potent inhibitors of tubulin polymerization and to effectively inhibit the binding of colchicine to tubulin. In addition, several 4-aryl-4H-chromenes were also found to be effective vascular disrupting agents (VDA). One of the lead compounds, EPC2407, is currently in clinical trials as a novel tumor vascular disrupting agent.

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 G(2)/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 EC(50) and GI(50) values in T47D, HCT116, and SNU398 cells, respectively.

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 EC(50) value of 0.24microM 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 GI(50) value of 0.056microM 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 EC(50) values of 0.17, 0.088 and 0.14microM 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 N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine, a potent apoptosis inducer and efficacious anticancer agent with high blood brain barrier penetration.

As a continuation of our structure-activity relationship (SAR) studies on 4-anilinoquinazolines as potent apoptosis inducers and to identify anticancer development candidates, we explored the replacement of the 2-Cl group in our lead compound 2-chloro-N-(4-methoxyphenyl)-N-methylquinazolin-4-amine (6b, EP128265, MPI-0441138) by other functional groups. This SAR study and lead optimization resulted in the identification of N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine (6h, EP128495, MPC-6827) as an anticancer clinical candidate. Compound 6h was found to be a potent apoptosis inducer with EC(50) of 2 nM in our cell-based apoptosis induction assay. It also has excellent blood brain barrier penetration, and is highly efficacious in human MX-1 breast and other mouse xenograft cancer models.

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 EC(50) values of 0.048-0.024 microM 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 IC(50) value of 0.5 microM, 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 microM.

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 EC(50) value of 60 nM in the caspase activation assay and GI(50) 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.

Selective activation of apoptosis by a novel set of 4-aryl-3-(3-aryl-1-oxo-2-propenyl)-2(1H)-quinolinones through a Myc-dependent pathway.

Oncogene addiction due to Myc deregulation has been identified in a variety of tumor types. In order to identify pharmacological agents that cause selective apoptosis in tumors with deregulated Myc expression, we designed a cell-based screening assay based on our Anti-cancer Screening Apoptosis Program (ASAP) technology targeting increased activity in a "Myc-addicted" cancer cell panel. We have identified a novel set of substituted 4-aryl-3-(3-aryl-1-oxo-2-propenyl)-2(1H)-quinolinones that activates apoptosis in cancer cell lines with deregulated Myc, but show low activity in cell lines where Myc is not deregulated. Apoptosis induced by these compounds is rapid, and is associated with a significant downregulation of Myc protein. Selective knockdown of Myc levels in these cells by RNA interference increased sensitivity to apoptosis with compound treatment. By targeting the Myc pathway in Myc-addicted cancer cells, we have identified a novel class of apoptotic inducers that selectively and efficiently target cancer cells with deregulated Myc.

Discovery of (naphthalen-4-yl)(phenyl)methanones and N-methyl-N-phenylnaphthalen-1-amines as new apoptosis inducers using a cell- and caspase-based HTS assay.

We report the discovery of a series of (naphthalen-4-yl)(phenyl)methanones as potent inducers of apoptosis using our proprietary cell- and caspase-based ASAP HTS assay. Through SAR studies, a group of N-methyl-N-phenylnaphthalen-1-amines also were identified as potent inducers of apoptosis. (1-(Dimethylamino)naphthalen-4-yl)(4-(dimethylamino)phenyl)methanone (2a), one of the most potent analogs, had EC(50) values of 37, 49 and 44nM in T47D, HCT116 and SNU398 cells, respectively. Compound 2a also was highly active in a growth inhibition assay with an GI(50) value of 34nM in T47D cells. Functionally, compound 2a arrested HCT116 cells in G(2)/M followed by induction of apoptosis and inhibited tubulin polymerization.

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 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 2-chloro-N-(4-methoxyphenyl)-N-methylquinazolin-4-amine (EP128265, MPI-0441138) as a potent inducer of apoptosis with high in vivo activity.

Using a live cell, high-throughput caspase-3 activator assay, we have identified a novel series of 4-anilinoquinazolines as inducers of apoptosis. In this report, we discuss the discovery of 2-chloro-N-(4-methoxyphenyl)-N-methylquinazolin-4-amine, compound 2b (EP128265, MPI-0441138) as a highly active inducer of apoptosis (EC50 for caspase activation of 2 nM) and as a potent inhibitor of cell proliferation (GI50 of 2 nM) in T47D cells. Compound 2b inhibited tubulin polymerization, was effective in cells overexpressing ABC transporter Pgp-1, and was efficacious in the MX-1 human breast and PC-3 prostate cancer mouse models. In contrast to the SAR of 4-anilinoquinazolines as EGFR kinase inhibitors, the methyl group on the nitrogen linker was essential for the apoptosis-inducing activity of 4-anilinoquinazolines and substitution in the 6- and 7-positions of the quinazoline core structure decreased potency.

Synthesis of caged 2,3,3a,7a-tetrahydro-3,6-methanobenzofuran-7(6H)-ones: Evaluating the minimum structure for apoptosis induction by gambogic acid.

We have reported the discovery of gambogic acid (GA) as a potent apoptosis inducer and the identification of transferrin receptor as its molecular target. In order to understand the basic pharmacophore of GA for inducing apoptosis and to discover novel and simplified derivatives as potential anti-cancer agents, we explored the synthesis of caged 2,3,3a,7a-tetrahydro-3,6-methanobenzofuran-7(6H)-ones (4-oxatricyclo[4.3.1.0]decan-2-ones). Three types of 2,3,3a,7a-tetrahydro-3,6-methanobenzofuran-7(6H)-ones based on xanthone, 2-phenylchromene-4-one and benzophenone, were synthesized using a Claisen/Diels-Alder reaction cascade. All the reactions produced the targeted caged compound as well as its neo-isomer. The caged compounds based on xanthone and 2-phenylchromene-4-one were found to maintain the apoptosis inducing and cell growth inhibiting activity of GA, although with less potency. The caged compounds based on benzophenone were found to be inactive. Our study determined the minimum structure of GA for its apoptosis inducing activity, which could lead to the development of simple derivatives as potential anti-cancer drugs.

Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay. 4. Structure-activity relationships of N-alkyl substituted pyrrole fused at the 7,8-positions.

In our continuing effort to discover and develop apoptosis inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored the structure-activity relationship (SAR) of alkyl substituted pyrrole fused at the 7,8-positions. A methyl group substituted at the nitrogen in the 7-position of the pyrrole ring led to a series of potent apoptosis inducers with potency in the low nanomolar range. These compounds were also found to be low nanomolar or subnanomolar inhibitors of cell growth, and they inhibited tubulin polymerization, indicating that methylation of the 7-position nitrogen does not change the mechanism of action of these chromenes. Compound 2d was identified as a highly potent apoptosis inducer with an EC50 value of 2 nM and a highly potent inhibitor of cell growth with a GI50 value of 0.3 nM in T47D cells.

Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based HTS assay. Part 5: modifications of the 2- and 3-positions.

As a continuation of our efforts to discover and develop apoptosis inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored modifications at the 2- and 3-positions. It was found that replacement of the 3-cyano group by an ester, including methyl and ethyl ester, resulted in >200-fold reduction of activity. Conversion of the 2-amino group into an amide or urea resulted in 4- to 10-fold drop of activity. Similarly, converting the 2-amino group into a hydrogen resulted in 4- to 10-fold reduction of activity. Compound 3d was highly active with an EC(50) value of 29 nM and a GI(50) value of 6 nM in T47D cells. Importantly, the 2-H analog 3d was found to be much more stable under acidic conditions compared to the 2-NH(2) analog 3b, suggesting that 2-H analogs might have better bioavailability than the 2-NH(2) analogs.