4,10-dihydro-5H-thieno[3,2-c][1]benzazepine derivatives and 9,10-dihydro-4H-thieno[2,3-c][1]benzazepine derivatives as orally active arginine vasopressin receptor antagonists.

Synthesis and structure-activity relationships (SAR) of arginine vasopressin receptor (AVP) antagonists are described. Potent and orally active compounds are prepared when tricyclic 10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine moiety in VPA-985 1 is replaced with a compound 7 or 12.     

Pyrrolo[3,2-c]pyridine derivatives as inhibitors of platelet aggregation

A series of pyrrolo[3,2-c]pyridines, isosteres of the antithrombotic drug ticlopidine, has been synthesized and evaluated in vitro for the ability to inhibit aggregation of human platelet-rich plasma induced by adenosin 5'-diphosphate (ADP). Structure-activity relationships showed their antiplatelet effects to be related to the lipophilicity.     

Pulvinones as bacterial cell wall biosynthesis inhibitors

Pulvinones were synthesized (>180) in arrays and evaluated as inhibitors of early stage cell wall biosynthesis enzymes MurA-MurD. Several pulvinones inhibited Mur enzymes with IC(50)'s in the 1-10 microg/mL range and demonstrated antibacterial activity against Gram-positive bacteria including methicillin-resistant Staphyloccus aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.     

Discovery of pyrrolo[3,2-c]quinoline-4-one derivatives as novel hedgehog signaling inhibitors

The Hedgehog (Hh) signaling pathway plays a significant role in the regulation of cell growth and differentiation during embryonic development. Since activation of the Hh signaling pathway is implicated in several types of human cancers, inhibitors of this pathway could be promising anticancer agents. Using high throughput screening, thieno[3,2-c]quinoline-4-one derivative 9a was identified as a compound of interest with potent in vitro activity but poor metabolic stability. Our efforts focused on enhancement of in vitro inhibitory activity and metabolic stability, including core ring conversion and side chain optimization. This led to the discovery of pyrrolo[3,2-c]quinoline-4-one derivative 12b, which has a structure distinct from previously reported Hh signaling inhibitors. Compound 12b suppressed stromal Gli1 mRNA expression in a murine model and demonstrated antitumor activity in a murine medulloblastoma allograft model.     

Synthesis and biological evaluation of thieno[3,2-c]pyrazol-3-amine derivatives as potent glycogen synthase kinase 3β inhibitors for Alzheimer’s disease

Glycogen synthase kinase 3β (GSK-3β) catalyses the hyperphosphorylation of tau protein in the Alzheimer’s disease (AD) pathology. A series of novel thieno[3,2-c]pyrazol-3-amine derivatives were designed and synthesised and evaluated as potential GSK-3β inhibitors by structure-guided drug rational design approach. The thieno[3,2-c]pyrazol-3-amine derivative 16b was identified as a potent GSK-3β inhibitor with an IC₅₀ of 3.1 nM in vitro and showed accepted kinase selectivity. In cell levels, 16b showed no toxicity on the viability of SH-SY5Y cells at the concentration up to 50 μM and targeted GSK-3β with the increased phosphorylated GSK-3β at Ser9. Western blot analysis indicated that 16b decreased the phosphorylated tau at Ser396 in a dose-dependent way. Moreover, 16b effectively increased expressions of β-catenin as well as the GAP43, N-myc, and MAP-2, and promoted the differentiated neuronal neurite outgrowth. Therefore, the thieno[3,2-c]pyrazol-3-amine derivative 16b could serve as a promising GSK-3β inhibitor for the treatment of AD.     

2-Phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole derivatives: new potent inhibitors of fMLP-induced neutrophil chemotaxis

It is well known that both acute and chronic autoimmune inflammatory disorders arise following a breakdown in control of neutrophil activation and recruitment. In the search for new anti-inflammatory agents, we synthesized some new 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole derivatives and tested them in vitro in order to evaluate their ability to interfere with human neutrophil functions. All tested compounds showed strong inhibition of fMLP-OMe-induced chemotaxis, although they appeared unable to block degranulation and the fMLP-OMe-induced respiratory burst, and were inactive in binding experiments.     

6-Phenyl-1H-imidazo[4,5-c]pyridine-4-carbonitrile as cathepsin S inhibitors

6-Phenyl-1H-imidazo[4,5-c]pyridine-4-carbonitrile analogues were identified as potent and selective cathepsin S inhibitor against both purified enzyme and in human JY cell based cellular assays. This core has a very stable thio-trapping nitrile war-head in comparison with the well reported pyrimidine-2-carbonitrile cysteine cathepsin inhibitors. Compound 47 is also very potent in in vivo mouse spleenic Lip10 accumulation assays.     

Synthesis and biological evaluation of 4'-(6,7-disubstituted-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol as potent Chk1 inhibitors

A new series of potent tricyclic pyrazole-based Chk1 inhibitors are described. Analogues disubstituted on the 6- and 7-positions show improved Chk1 inhibition potency compared with analogues with a single substituent on either the 6- or 7-position. Based on the lead compound 4'-(6,7-dimethoxy-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol (2), detailed SAR studies on the 6- and 7-positions were performed. 3'-morpholin-4'-yl-propoxy, pyridin-4'-ylmethoxy, pyridin-3'-ylmethoxy, 2'-(5'-ethyl-pyridin-2'-yl)-ethoxy, pyridin-2'-ylethoxy, (6'-methyl-pyridin-2'-yl)-propoxyethoxy, 2',3'-dihydroxyl-1'-yl-propoxy, and tetrahydro-furan-3'-yloxy have been identified as the best groups on the 6-position when the 7-position is substituted with methoxyl group. Pyridin-2'-ylmethoxy and pyridin-3'-ylmethoxy have been identified as the best substituents at the 7-position while the 6-position bearing methoxyl group. These compounds significantly potentiate the cytotoxicity of DNA-damaging antitumor agents in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and the camptothecin-induced S checkpoints, suggesting that their potent biological activities are mechanism-based through Chk1 inhibition.     

Design,synthesis and biological evaluation of fused naphthofuro[3,2-c] quinoline-6,7,12-triones and pyrano[3,2-c]quinoline-6,7,8,13-tetraones derivatives as ERK inhibitors with efficacy in BRAF-mutant melanoma

Approximately 60% of human cancers exhibit enhanced activity of ERK1 and ERK2, reflecting their multiple roles in tumor initiation and progression. Acquired drug resistance, especially mechanisms associated with the reactivation of the MAPK (RAF/MEK/ERK) pathway represent a major challenge to current treatments of melanoma and several other cancers. Recently, targeting ERK has evolved as a potentially attractive strategy to overcome this resistance. Herein, we report the design and synthesis of novel series of fused naphthofuro[3,2-c]quinoline-6,7,12-triones 3a-f and pyrano[3,2-c]quinoline-6,7,8,13-tetraones 5a,b and 6, as potential ERK inhibitors. New inhibitors were synthesized and identified by different spectroscopic techniques and X-ray crystallography. They were evaluated for their ability to inhibit ERK1/2 in an in vitro radioactive kinase assay. 3b and 6 inhibited ERK1 with IC50s of 0.5 and 0.19 µM, and inhibited ERK2 with IC50s of 0.6 and 0.16 µM respectively. Kinetic mechanism studies revealed that the inhibitors are ATP-competitive inhibitors where 6 inhibited ERK2 with a K_i of 0.09 µM. Six of the new inhibitors were tested for their in vitro anticancer activity against the NCI-60 panel of tumor cell lines. Compound 3b and 6 were the most potent against most of the human tumor cell lines tested. Moreover, 3b and 6 inhibited the proliferation of the BRAF mutant A375 melanoma cells with IC50s of 3.7 and 0.13 µM, respectively. In addition, they suppressed anchorage-dependent colony formation. Treatment of the A375 cell line with 3b and 6 inhibited the phosphorylation of ERK substrates p-90RSK and ELK-1 and induced apoptosis in a dose dependent manner. Finally, a molecular docking study showed the potential binding mode of 3b and 6 within the ATP catalytic binding site of ERK2.     

Synthesis of 5,6-dihydro-11H-benzo[5,6]-cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidine-N-cyanoguanidine derivatives as inhibitors of ras farnesyl protein transferase

A series of novel N-cyanoguanidine tricyclic farnesyl protein transferase (FPT) inhibitors was prepared. Replacement of a piperidine amide-group with a N-cyanoguanidine functionality increased FPT activity. X-ray crystal structure determination of 42 complexed with FPT revealed differences in the interactions of the amide and N-cyanoguanidine groups with the protein.     

1,3-Dioxo-4-methyl-2,3-dihydro-1H-pyrrolo[3,4-c]quinolines as potent caspase-3 inhibitors

Synthesis, biological evaluation and structure-activity relationships for a series of novel nonpeptide small molecule inhibitors of caspase-3 are described. Among the studied compounds, 8-sulfamide derivatives of 1,3-dioxo-4-methyl-2,3-dihydro-1H-pyrrolo[3,4-c]quinolines have been identified as potent inhibitors of caspases-3. The most active compound within this series (8f) inhibited caspase-3 with IC(50)=4 nM.     

[(4-Hydroxyl-benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)-amino]-acetic acid derivatives; HIF prolyl 4-hydroxylase inhibitors as oral erythropoietin secretagogues

A new series of PHD (HIF prolyl 4-hydroxylase) inhibitors was designed based on the X-ray co-crystal structure of FG-2216. Using a lead generation process, a series of [(4-Hydroxyl-benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)-amino]-acetic acid derivatives was developed as potent PHD2 inhibitors. This class of compounds also showed the ability to stabilize HIF-α, to stimulate EPO secretion in in vitro studies, and to increase hematocrit, red blood cell count, and hemoglobin levels in an animal efficacy study.     

Discovery of 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles and 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-pyridine-2'-carbonitriles as potent checkpoint kinase 1 (Chk1) inhibitors

An extensive structure-activity relationship study of the 3-position of a series of tricyclic pyrazole-based Chk1 inhibitors is described. As a result, 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles (4) and 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-pyridine-2'-carbonitriles (29) emerged as new lead series. Compared with the original lead compound 2, these new leads fully retain the biological activity in both enzymatic inhibition and cell-based assays. More importantly, the new leads 4 and 29 exhibit favorable physicochemical properties such as lower molecular weight, lower Clog P, and the absence of a hydroxyl group. Furthermore, structure-activity relationship studies were performed at the 6- and 7-positions of 4, which led to the identification of ideal Chk1 inhibitors 49, 50, 51, and 55. These compounds not only potently inhibit Chk1 in an enzymatic assay but also significantly potentiate the cytotoxicity of DNA-damaging agents in cell-based assays while they show little single agent activity. A cell cycle analysis by FACS confirmed that these Chk1 inhibitors efficiently abrogate the G2/M and S checkpoints induced by DNA-damaging agent. The current work paved the way to the identification of several potent Chk1 inhibitors with good pharmacokinetics that are suitable for in vivo study with oral dosing.     

Tetrahydropyrrolo[3,2-c]azepin-4-ones as a new class of cytotoxic compounds

Pyrroloazepinones 8a-j and 9a-j were designed by structural modification of lead compound 3. These compounds were tested on five tumor cell lines to determine the role of the azeto ring and the 2-methyl substituent in the cytotoxicity of compound 3. Our results show that compounds 8a-j (R1=CH3) have dramatically reduced cytotoxicity, resulting from the loss of the azeto moiety of lead compound 3. By contrast, azepinones 9a-j (R1=4-nitrophenyl) inhibited the proliferation of almost all cancer cell lines tested even though they lack the azeto ring. Preliminary SAR studies with these compounds revealed the importance of halogens at the para- or meta-position of the 1-phenyl moiety. Additionally, derivatives 9a (R2=H), 9e (R2=4-F), and 9g (R2=4-OMe) were selectively cytotoxic to U-251 cells. However, none of the pyrroloazepinones inhibited the enzymatic activity of CDK1/cyclin B, CDK5/p25, and GSK-3.     

Synthesis and antiproliferative evaluation of certain indolo[3,2-c]quinoline derivatives

The present report describes the synthesis and antiproliferative evaluation of certain indolo[3,2-c]quinoline derivatives. For the C₆ anilino-substituted derivatives, (11H-indolo[3,2-c]quinolin-6-yl)phenylamine (6a) was inactive. Structural optimization of 6a by the introduction of a hydroxyl group at the anilino-moiety resulted in the enhancement of antiproliferative activity in which the activity decreased in an order of para-OH, 7a > meta-OH, 8a > ortho-OH, 9a. For the C₆ alkylamino-substituted derivatives, 11a, 12a, 13a, 14a, and 15a exhibited comparable antiproliferative activities against all cancer cells tested and the skin Detroit 551 normal fibroblast cells. Three cancer cells, HeLa, A549, and SKHep, are very susceptible with IC₅₀ of less than 2.17 μM while PC-3 is relatively resistant to this group of indolo[3,2-c]quinolines. For the 2-phenylethylamino derivatives, compound 20a is active against the growth of HeLa with an IC₅₀ of 0.52 μM, but is less effective against the growth of Detroit 551 with an IC₅₀ of 19.32 μM. For the bis-indolo[3,2-c]quinolines, N,N-bis-[3-(11H-indolo[3,2-c]quinolin-6-yl)aminopropyl]amine hydrochloride (25) is more active than its N-methyl derivative 26 and the positive Doxorubicin. Mechanism studies indicated 25 can induce caspase-3 activation, γ-H2AX phosphorylation, cleavage of poly(ADP-ribose)polymerase and DNA fragmentation. These results provide evidence that DNA, topo I, and topo II are the primary targets of indolo[3,2-c]quinoline derivatives and that consequently inhibits proliferation and causes apoptosis in cancer cells.     

Design, synthesis, and antiproliferative activity of new 1H-pyrrolo[3,2-c]pyridine derivatives against melanoma cell lines. Part 2

A new series of diarylureas and diarylamides possessing 1H-pyrrolo[3,2-c]pyridine scaffold was designed and synthesized. Their in vitro antiproliferative activities against A375P human melanoma cell line and NCI-9 human melanoma cell line panel were tested. All the target compounds, except three amino derivatives 8g, h and 9h, demonstrated superior potencies against A375P to Sorafenib. In addition, compounds 8a and 9b-f demonstrated higher potencies than Vemurafenib against A375P. Compounds 8c and 9b were 7.50 and 454.90 times, respectively, more selective towards A375P melanoma cells over NIH3T3 fibroblasts. Furthermore, compounds 8d, e and 9a-d, f demonstrated very high potencies against the nine tested melanoma cell lines at the NCI. The bisamide derivatives 9a-c, f showed 2-digit nanomolar IC(50) values over different cell lines of the NCI-9 melanoma cell lines.     

Expeditious synthesis and cytotoxic activity of new cyanoindolo[3,2-c]quinolines and benzimidazo[1,2-c]quinazolines

Novel 6-cyanoindolo[3,2-c]quinoline and 6-cyanobenzimidazo[1,2-c]quinazoline derivatives have been synthesised by treatment of the appropriate aromatic amines with 4.5-dichloro-1,2,3-dithiazolium chloride 1 (Appel salt). The cytotoxicity and the effect of these compounds on cellular growth were measured.     

Identification of the 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives as highly selective PDE4B inhibitors

A PDE4B subtype selective inhibitor is expected to have a wider therapeutic window than non-selective PDE4 inhibitors. In this Letter, two series of 7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives and 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives were evaluated for their PDE4B subtype selectivity using human PDE4B2 and PDE4D2 full length enzymes. To improve their PDE4B selectivity over PDE4D, we optimized the substituents on the pyrimidine ring and the side chain phenyl ring, resulting in several derivatives with more than 100-fold selectivity for PDE4B. Consequently, we identified 2-(3-chloro-4-methoxy-phenyl)-5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative 54 as a highly selective PDE4B inhibitor, which had potent hPDE4B inhibitory activity with an IC₅₀ value of 3.0 nM and 433-fold PDE4B selectivity over PDE4D.     

Discovery of 1,8-disubstituted-[1,2,3]triazolo[4,5-c]quinoline derivatives as a new class of Hippo signaling pathway inhibitors

Inhibitors of the Hippo signaling pathway have been demonstrated to have a potential clinical application in cases such as tissue repair and organ regeneration. However, there is a lack of potent Hippo pathway inhibitors at present. Herein we report the discovery of a series of 1,8-disubstituted-[1,2,3]triazolo[4,5-c]quinoline derivatives as a new class of Hippo pathway inhibitors by utilizing a cell line-based screening model (A549-CTGF). Structure-activity relationship (SAR) of these compounds was also discussed. The most potent compound in the A549-CTGF cell assay, 11g, was then evaluated by real-time PCR and immunofluorescence assays. Overall, this study provides a starting point for later drug discovery targeting the Hippo signaling pathway.