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 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.     

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 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 (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 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 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.     

Structure-activity relationships of 2-, 4-, or 6-substituted estrogens as aromatase inhibitors

Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic target for the selective lowering of estrogen levels in patients with estrogen-dependent breast cancer. To develop a novel class of aromatase inhibitors, we tested series of 2- and 4-substituted (halogeno, methyl, formyl, methoxy, nitro, and amino) estrones (7 and 9), as well as series of 6alpha- and 6beta-substituted (alkyl, phenalkyl, and alkoxy) estrones (13 and 14), and their estradiol analogs (8, 10, 11, and 12) as aromatase inhibitors. All of the inhibitors examined blocked the androstenedione aromatization in a competitive manner. Introduction of halogeno and methyl functions at C-2 of estrone as well as that of a phenalkyl or methyl function at the C-6alpha or C-6beta position markedly increased affinity to aromatase (apparent K(i) value=0.10-0.66 microM for the inhibitors versus 2.5 microM for estrone). 6alpha-Phenylestrone (13c) was the most powerful inhibitor among the estrogens studied, and its affinity was comparable to that of the androgen substrate androstenedione. Estradiol analogs were much weaker inhibitors than the corresponding estrone compounds in each series, indicating that the 17-carbonyl group plays a critical role in the formation of a thermodynamically stable enzyme-inhibitor complex.     

Discovery of potent, soluble and orally active TRPV1 antagonists. Structure-activity relationships of a series of isoxazoles

Systematic optimisation of a poorly soluble lead series of isoxazole-3-carboxamides was conducted. Substitution of the 4-position with specific polar functionality afforded the requisite balance of potency, solubility and physicochemical properties. Compound 21a was found to be efficacious in the rat Capsaicin Hargreaves assay following oral administration.     

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.     

Structure-activity relationships of novel anti-malarial agents. Part 7: N-(3-benzoyl-4-tolylacetylaminophenyl)-3-(5-aryl-2-furyl)acrylic acid amides with polar moieties

In a previous report, we have provided evidence that novel anti-malarial compounds based on 2,5-diaminobenzophenone farnesyltransferase inhibitors might benefit from the presence of a polar moiety at the para position of the terminal phenyl of the arylfurylacryloyl partial structure. Here, we demonstrate that different moieties with hydrogen bond acceptor properties lead to equipotent or even improved anti-malarial activity in comparison to the nitro group described before.     

Structure-activity relationships of novel anti-malarial agents. Part 4: N-(3-benzoyl-4-tolylacetylaminophenyl)-3-(5-aryl-2-furyl)acrylic acid amides

In a previous report, we have described novel anti-malarial compounds based on a 2,5-diaminobenzophenone scaffold. Here, we have invesigated acryloyl derivatives carrying a biaryl structure consisting of a terminal aryl residue and a central 2-furyl ring. Several compounds were obtained in the series of para-substituted phenylfurylacryloyl derivatives that displayed improved anti-malarial activity in comparison to earlier described derivatives. From the structure-activity relationships it can be deduced that there has to be a lipophilic moiety in the para-position of the terminal phenyl residue. Furthermore, there are indications that, alternatively, activity may benefit from the presence of a polar moiety with hydrogen bond acceptor properties.     

Synthesis and SAR of 5-, 6-, 7- and 8-aza analogues of 3-aryl-4-hydroxyquinolin-2(1H)-one as NMDA/glycine site antagonists

A series of 5-, 6-, 7- and 8-aza analogues of 3-aryl-4-hydroxyquinolin-2(1H)-one was synthesized and assayed as NMDA/glycine receptor antagonists. The in vitro potency of these antagonists was determined by displacement of the glycine site radioligand [(3)H]5,7-dicholorokynurenic acid ([(3)H]DCKA) in rat brain cortical membranes. Selected compounds were also tested for functional antagonism using electrophysiological assays in Xenopus oocytes expressing cloned NMDA receptor (NR) 1A/2C subunits. Among the 5-, 6-, 7-, and 8-aza-3-aryl-4-hydroxyquinoline-2(1H)-ones investigated, 5-aza-7-chloro-4-hydroxy-3-(3-phenoxyphenyl)quinolin-2-(1H)-one (13i) is the most potent antagonist, having an IC(50) value of 110 nM in [(3)H]DCKA binding and a K(b) of 11 nM in the electrophysiology assay. Compound 13i is also an active anticonvulsant when administered systemically in the mouse maximum electroshock-induced seizure test (ED(50)=2.3mg/kg, IP).     

Discovery of 5-(4-methylpiperazin-1-yl)-2-nitroaniline derivatives as a new class of SIRT6 inhibitors

SIRT6 is a deacetylase of histone H3 and inhibitors of SIRT6 have been thought as potential agents for treatment of diabetes. Herein we report the discovery of a series of new SIRT6 inhibitors containing the skeleton 1-phenylpiperazine. Among them, compound 5-(4-methylpiperazin-1-yl)-2-nitroaniline (6d) is the most potent one, which showed an IC₅₀ value of 4.93 μM against SIRT6 in the Fluor de Lys (FDL) assay. It displayed K_D values of 9.76 μM and 10 μM in surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) assays, respectively. In selectivity assay, 6d showed no activity against other members of the HDAC family (SIRT1-3 and HDAC1-11) at concentrations up to 200 µM. In a mouse model of type 2 diabetes, 6d could significantly increase the level of glucose transporter GLUT-1, thereby reducing blood glucose. Overall, this study provides a promising lead compound for subsequent drug discovery targeting SIRT6.     

Discovery of potent,selective, and orally bioavailable PDE5 inhibitor: Methyl-4-(3-chloro-4-methoxybenzylamino)-8-(2-hydroxyethyl)-7-methoxyquinazolin-6-ylmethylcarbamate (CKD 533)

In a continuing effort to discover novel PDE5 inhibitors, we have successfully found quinazolines with 4-benzylamino substitution as potent and selective PDE5 inhibitors. Initial lead compound (1) was found to be easily metabolized when incubated with human liver microsomes mainly through C6 amide hydrolysis. Blocking of this metabolic hot spot led to discovery of 10 (CKD533) which is highly potent, selective and orally efficacious in conscious rabbit model for erectile dysfunction and now is undergoing preclinical toxicology study.     

Structure-activity relationships of novel anti-malarial agents. Part 6: N-(4-arylpropionylamino-3-benzoylphenyl)-[5-(4-nitrophenyl)-2-furyl]acrylic acid amides

We have demonstrated that the p-trifluoromethylphenylpropionylamino residue at the 2-position of the core structure leads to an active benzophenone-type anti-malarial agent. The attempt to improve water solubility by introduction of an amino group into the alpha-position of the arylpropionyl residue resulted in decreased activity.     

Discovery of 3-aryl-3-methyl-1H-quinoline-2,4-diones as a new class of selective 5-HT6 receptor antagonists

A 5,7-dichloro-3-phenyl-3-methyl-quinoline-2,4-dione (11a) has been identified in a random screen as a lead for 5-HT(6) antagonist. During the lead optimization process, several analogs were synthesized and their biological activities were investigated. Within this series, several compounds display high binding affinity and selectivity for the 5-HT(6) receptor. In particular, 3-(4-hydroxyphenyl)-3-methyl-quinoline-2,4-dione (12f) exhibits high affinity (K(i)=12.3 nM) for 5-HT(6) receptor with good selectivity over other serotonin and dopamine (D(1)-D(4)) receptor subtypes. In a functional adenylyl cyclase stimulation assay, this compound exhibited considerable antagonistic activity (IC(50)=0.61 microM).     

Structure-activity relationships of 2-chloro-N6-substituted-4'-thioadenosine-5'-N,N-dialkyluronamides as human A3 adenosine receptor antagonists

On the basis of potent and selective A(3) adenosine receptor (AR) antagonist, 2-chloro-N(6)-(3-iodobenzyl)-4'-thioadenosine-5'-N,N-dimethyluronamide, structure-activity relationships were studied for a series of 5'-N,N-dialkyluronamide derivatives, synthesized from D-gulonic gamma-lactone. From this study, it was revealed that removal of the hydrogen bond-donating ability of the 5'-uronamide was essential for the pure A(3)AR antagonism. 5'-N,N-Dimethyluronamide derivatives exhibited higher binding affinity than larger 5'-N,N-dialkyl or 5'-N,N-cycloalkylamide derivatives, indicating that steric factors are crucial in binding to the human A(3)AR. A N(6)-(3-bromobenzyl) derivative 6c (K(i)=9.32 nM) exhibited the highest binding affinity at the human A(3)AR with very low binding affinities to other AR subtypes.