Hidenori Yoshizawa

A novel acetylenic tricyclic bis-(cyano enone) potently induces phase 2 cytoprotective pathways and blocks liver carcinogenesis induced by aflatoxin

A novel acetylenic tricyclic bis-(cyano enone), TBE-31, is a lead compound in a series of tricyclic compounds with enone functionalities in rings A and C. Nanomolar concentrations of this potent multifunctional molecule suppress the induction of the inflammatory protein, inducible nitric oxide synthase, activate phase 2 cytoprotective enzymes in vitro and in vivo, block cell proliferation, and induce differentiation and apoptosis of leukemia cells. Oral administration of TBE-31 also significantly reduces formation of aflatoxin-DNA adducts and decreases size and number of aflatoxin-induced preneoplastic hepatic lesions in rats by >90%. Because of the two cyano enones in rings A and C, TBE-31 may directly interact with DTT and protein targets such as Keap1 that contain reactive cysteine residues. The above findings suggest that TBE-31 should also be tested for chemoprevention and chemotherapy in relevant models of cancer and against other chronic, degenerative diseases in which inflammation and oxidative stress contribute to disease pathogenesis.

Tricyclic compounds containing nonenolizable cyano enones. A novel class of highly potent anti-inflammatory and cytoprotective agents.

Forty-four novel tricycles containing nonenolizable cyano enones (TCEs) were designed and synthesized on the basis of a semisynthetic pentacyclic triterpenoid, bardoxolone methyl, which is currently being developed in phase II clinical trials for the treatment of severe chronic kidney disease in diabetic patients. Most of the TCEs having two different kinds of nonenolizable cyano enones in rings A and C are highly potent suppressors of induction of inducible nitric oxide synthase stimulated with interferon-γ and are highly potent inducers of the cytoprotective enzymes heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Among these compounds, (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile ((±)-31) is the most potent in these bioassays in our pool of drug candidates including semisynthetic triterpenoids and synthetic tricycles. These facts strongly suggest that an essential factor for potency is not a triterpenoid skeleton but the cyano enone functionality. Notably, TCE 31 reduces hepatic tumorigenesis induced with aflatoxin in rats. Further preclinical studies and detailed mechanism studies on 31 are in progress.

Synthesis of a novel dicyano abietane analogue: a potential antiinflammatory agent.

From a structure-activity relationship perspective, the new abietane 5 having cyano groups at C-2 and C-13 and a phenolic ring C has been synthesized and evaluated biologically because the related compound 4 has high potency in inflammation models in vitro and in vivo. Compound 5 was synthesized from 8, which was obtained in five steps from the known compound 9, via an unexpected aromatization caused by the addition of PhSeCl and subsequent oxidation/elimination of the selenated intermediate 14 with H2O2.

Indazole-based potent and cell-active Mps1 kinase inhibitors: rational design from pan-kinase inhibitor anthrapyrazolone (SP600125)

Monopolar spindle 1 (Mps1) is essential for centrosome duplication, the spindle assembly check point, and the maintenance of chromosomal instability. Mps1 is highly expressed in cancer cells, and its expression levels correlate with the histological grades of cancers. Thus, selective Mps1 inhibitors offer an attractive opportunity for the development of novel cancer therapies. To design novel Mps1 inhibitors, we utilized the pan-kinase inhibitor anthrapyrazolone (4, SP600125) and its crystal structure bound to JNK1. Our design efforts led to the identification of indazole-based lead 6 with an Mps1 IC50 value of 498 nM. Optimization of the 3- and 6-positions on the indazole core of 6 resulted in 23c with improved Mps1 activity (IC50 = 3.06 nM). Finally, application of structure-based design using the X-ray structure of 23d bound to Mps1 culminated in the discovery of 32a and 32b with improved potency for cellular Mps1 and A549 lung cancer cells. Moreover, 32a and 32b exhibited reasonable selectivities over 120 and 166 kinases, respectively.

A unique hinge binder of extremely selective aminopyridine-based Mps1 (TTK) kinase inhibitors with cellular activity

Mps1, also known as TTK, is a dual-specificity kinase that regulates the spindle assembly check point. Increased expression levels of Mps1 are observed in cancer cells, and the expression levels correlate well with tumor grade. Such evidence points to selective inhibition of Mps1 as an attractive strategy for cancer therapeutics. Starting from an aminopyridine-based lead 3a that binds to a flipped-peptide conformation at the hinge region in Mps1, elaboration of the aminopyridine scaffold at the 2- and 6-positions led to the discovery of 19c that exhibited no significant inhibition for 287 kinases as well as improved cellular Mps1 and antiproliferative activities in A549 lung carcinoma cells (cellular Mps1 IC₅₀=5.3 nM, A549 IC₅₀=26 nM). A clear correlation between cellular Mps1 and antiproliferative IC₅₀ values indicated that the antiproliferative activity observed in A549 cells would be responsible for the cellular inhibition of Mps1. The X-ray structure of 19c in complex with Mps1 revealed that this compound retains the ability to bind to the peptide flip conformation. Finally, comparative analysis of the X-ray structures of 19c, a deamino analogue 33, and a known Mps1 inhibitor bound to Mps1 provided insights into the unique binding mode at the hinge region.

AN EFFICIENT SYNTHESIS OF TRICYCLIC COMPOUNDS, (+/-)-(4abeta8abeta10aalpha)-1,2,3,4,4a,6,7,8,8a,9,10,10a-DODECAHYDRO-1,1,4a-TRIMETHYL-2-OXOPHENANTHRENE-8a-CARBOXYLIC ACID, ITS METHYL ESTER, AND (+/-)-(4abeta,8abeta10aalpha)-3,4,4a,6,7,8,8a,9,10,10a-DECAHYDRO-8a-HYDROXYMETHYL-1,1,4a-TRIMETHYLPHENANTHREN-2(1H)-ONE.

Our ongoing efforts for the improvement of anti-inflammatory and antiproliferative activity of oleanolic acid analogues led us to discover 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO, 1) and related compounds.1 In connection with these investigations, we have found that tricyclic compounds with similar enone functionalities in rings A and C are also a novel class of highly active inhibitors of nitric oxide (NO) production in mouse macrophages.2 In particular, bis-cyano enone (±)-2 is orally active in a preliminary in vivo inflammation model.2 In addition, we have found that (+)-2 having the opposite configuration to that of CDDO shows 10 times higher inhibitory activity than (-)-2 on NO production in mouse macrophages.3 These results encouraged us to design and synthesize analogues of 2. Thus, we focused our attention on the modifications of the C-8a position, because some biologically active natural products have functionalities at the same position (e.g., anti-tumor quassinoids4). For our projected synthesis of C-8a functionalized TBE compounds, the simple tricycles 3-5 are potentially very desirable intermediates. We envisioned preparing 3-5 from the known acid 65,6 by standard reductive methylation.7 However, attempts to reductively methylate acid 6 with 5-7 equivalents of lithium in liquid ammonia containing no proton donor, followed by esterification with diazomethane gave 4 in 30% yield (average of 7 experiments, the yield fluctuates) along with many by-products. These by-products caused serious difficulty for the purification of 4. An attempt with one equivalent of tert-butanol gave similar results as without a proton donor. Attempts to reductively methylate methyl ester 7,6 which is prepared from 6 with diazomethane, using 10 equivalents of lithium in liquid ammonia containing no proton donor gave the desired compounds 3-5 in low yield along with several by-products including enones 6 and 8. After much experimentation, we have found that the addition of one equivalent of water dramatically improves this reductive methylation reaction. Thus, the reductive methylation of 7 using 7.2 equivalents of lithium and one equivalent of water followed by quenching the excess lithium with isoprene, and then methyl iodide at -78 °C cleanly produced 3-5 in 38%, 15%, and 36% yields (total 89%), respectively. The yields are reproducible and we have prepared 3-5 several times by this procedure. These compounds can be easily separated by extracting the acid with aqueous base, followed by column chromatography (see Experimental Section). Also, they were easily converted to a single compound. For example, oxidation (e.g., Jones reagent and RuO2-NaIO4 etc.) of alcohol 5 gave acid 3, and both acid 3 and methyl ester 4 were converted to alcohol 5 in three steps (ketalization, reduction with LiAlH4, and deketalization). Acid 3 may be an important intermediate for the synthesis of abietane and totarane diterpenoids.

A new method for regioselective synthesis of a broad-spectrum parenteral S-3578-related cephalosporin bearing an imidazo[4,5-b]pyridinium derivative at C-3'

A broad-spectrum S-3578-related cephalosporin, 7β-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-fluoromethoxyiminoacetamido]-3-[1-(3-methylaminopropyl)-1H-imidazo[4,5-b]pyridinium-4-yl]methyl-3-cephem-4-carboxylate sulfate was regioselectively synthesized in a good yield using diaminopyridine derivative bearing a dimethylformamidine group.