SummaryHepsin, a liver-enriched novel serine protease, has been implicated in participating with normal cell growth, embryogenesis, and blood coagulation pathway. To study its function in vivo, we have disrupted the mouse hepsin gene by homologous recombination. Targeted disruption of the hepsin gene and ablation of hepsin message were demonstrated by Southern blotting, Northern blotting and RT-PCR analysis. Homozygous hepsin −/− mice were viable, fertile, and exhibited no gross abnormalities, as judged by the size, weight and blood coagulation (PT) assays. However, the serum concentration of the bone form of alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase of the hepsin −/− mice was mildly elevated, in spite of no obvious pathological change of hepatocytes. To examine whether hepsin is involved in controlling cell growth in adult tissues, 70% hepatectomy was applied to the hepsin −/− mice. Liver regeneration proceeded normally in the hepsin −/− mice as judged by the liver mass restoration rate. These results suggest that loss of hepsin function causes no effect in cell growth and embryogenesis in vivo, which is in contradiction to the studies using in vitro cell culturing system. Moreover, gross mass regeneration of liver after damage proceeds normally in the absence of functional hepsin.

A series of 4-aryl-N-arylcarbonyl-2-aminothiazoles of scaffold 4 was designed and synthesized as Hec1/Nek2 inhibitors. Structural optimization of 4 led to compound 32 bearing C-4' 4-methoxyphenoxy and 4-(o-fluoropyridyl)carbonyl groups that showed low nanomolar in vitro antiproliferative activity (IC50: 16.3-42.7 nM), high intravenous AUC (64.9 μM·h, 2.0 mg/kg) in SD rats, and significant in vivo antitumor activity (T/C = 32%, 20 mg/kg, IV) in mice bearing human MDA-MB-231 xenografts. Cell responses resulting from Hec1/Nek2 inhibition were observed in cells treated with 32, including a reduced level of Hec1 coimmunoprecipitated with Nek2, degradation of Nek2, mitotic abnormalities, and apoptosis. Compound 32 showed selectivity toward cancer cells over normal phenotype cells and was inactive in a [(3)H]astemizole competitive binding assay for hERG liability screening. Therefore, 32 is as a good lead toward the discovery of a preclinical candidate targeting Hec1/Nek2 interaction.

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Through the syntheses of its C-1 desvinyl, C-7 methylene, C-7 exocyclic ethylidene, and various C-3 phenylmethyl analogues, the structure−activity relationship of antimitotic ottelione A (4) against tubulin and various cancer cells was established. The results indicated that compound 4 was a colchicine-competitive inhibitor and that the C-1 vinyl group is unnecessary for its potency, whereas the C-7 exocyclic double bond is essential, possibly because of its irreversible interaction with tubulin. Further optimization of the substituents on the phenylmethyl group at the C-3 position generated compound 10g with a 3′-fluoro-4′-methoxyphenylmethyl substituent, which was 6−38-fold more active against MCF-7, NCI-H460, and COLO205 cancer cells relative to 4. Results from in vitro tubulin polymerization assay confirmed the potency of compounds 4, 10g, and 11a. KEYWORDS: ottelione A, antimitotic, microtubule, tubulin, anticancer A fter the success of antimitotic taxanes and vinca alkaloids as anticancer drugs, 1,2 antimitotic agents that target the colchicine-binding site to inhibit tubulin polymerization have attracted much attention. Many structurally diverse compounds binding to the colchicine-binding site have been discovered from natural or synthetic sources. 3,4 Despite no compound of this class having been approved for clinical use, the prototype colchicine (1, Figure 1) has been used to treat gout for many years.5 Combretastatin A-4 (2) 6−9 and ABT-751 (3) 10,11 in Figure 1 are representative examples of colchicine-competitive inhibitors currently in clinical trials to treat various cancers.Ottelione A (4, Figure 1), isolated from the fresh water plant Ottelia alismoides collected in the Nile Delta, 12 is among the most potent natural products that possess in vitro antiproliferative activity. Its IC 50 values lie in the pM−nM range against a panel of 60 human cancer cell lines through acting as a colchicine-competitive inhibitor. 12,13 Because of its promising antitumor activity and intriguing core structure with four stereogenic centers, several synthetic groups have investigated the total synthesis of 4, 14−21 but no analogue has been reported and assayed for anticancer activity, perhaps because of the many steps in its preparation.We already achieved an enantioselective total synthesis of 4 using our α-carbonyl radical cyclization method. 21 The bicyclo[4.3.0]nonane core structure was constructed via radical cyclization. The C-3 3′-hydroxy-4′-methoxyphenylmethyl group, C-1 vinyl group, and C-7 exocyclic double bond were then sequentially introduced. We thus envisaged that our scheme could be adaptable for the preparation of various analogues of 4, which would be useful to establish its structure−activity relationship.As shown in Scheme 1, we first prepared compounds 10a−g and 11a−c according to our scheme for the total synthesis of 4. For economy, these compounds were all synthesized in their racemic forms. Compound 5, prepared via α-carbonyl radical cyclization, 21 served as the starting material. H...

In this paper, we present a copper­(I)-catalyzed nitrile-addition/N-arylation ring-closure cascade for the synthesis of 5,11-dihydro-6H-indolo­[3,2-c]­quinolin-6-ones from 2-(2-bromophenyl)-N-(2-cyanophenyl)­acetamides. Using CuBr and t-BuONa in dimethylformamide (DMF) as the optimal reaction conditions, the cascade reaction gave the target products, in high yields, with a good substrate scope. Application of the cascade reaction was demonstrated on the concise total syntheses of alkaloid isocryptolepine. Further optimization of the products from the cascade reaction led to 3-chloro-5,12-bis­[2-(dimethylamino)­ethyl]-5,12-dihydro-6H-[1,3]­dioxolo­[4′,5′:5,6]­indolo­[3,2-c]­quinolin-6-one (2k), which exhibited the characteristic DNA topoisomerase-I inhibitory mechanism of action with potent in vitro anticancer activity. Compound 2k actively inhibited ARC-111- and SN-38-resistant HCT-116 cells and showed in vivo activity in mice bearing human HCT-116 and SJCRH30 xenografts. The interaction of 2k with the Top–DNA cleavable complex was revealed by docking simulations to guide the future optimization of 5,11-dihydro-6H-indolo­[3,2-c]­quinolin-6-ones as topoisomerase-I inhibitors.