Isolation and characterization of a trypsin-like protease from Trichoderma viride.

A serine endopeptidase with a molecular mass of 25 kDa has been purified from the culture filtrate of Trichoderma viride to electrophoretic homogeneity. The isoelectric point was determined at 7.3. Two carboxyl sites at Arg22 and Lys29 of the oxidized insulin B-chain were cleaved, and peptidyl-p-nitroanilide substrates with Lys or Arg at the P1 position were also hydrolyzed by the enzyme. These results suggest that the specificity of T. viride protease is similar to that of trypsin. However, the hydrolytic activity toward casein of T. viride protease was less than that of porcine trypsin. The amino-terminal sequence of the enzyme protein is similar to that of bovine trypsin. It seems that the trypsin of T. viride is a protease which is promising for the substitution of animal trypsin in the food industry and in medicine at this stage.

Practical asymmetric synthesis of a selective endothelin A receptor (ETA) antagonist.

[structure: see text]. A practical, chromotography-free asymmetric synthesis was developed for the large scale preparation of an endothelin receptor antagonist 2. This synthesis includes a new efficient process for the preparation of 6-bromo-2,3-dihydrobenzofuran, a stereoselective conjugate addition of an aryllithium followed by stereospecific addition of the Grignard reagent of the top aryl bromide, and an aminophosphate-mediated sterospecific intramolecular enolate alkylation, which led to the formation of the five-membered ring bearing three contiguous asymmetric centers.

Synthesis of a muscarinic receptor antagonist via a diastereoselective Michael reaction, selective deoxyfluorination and aromatic metal-halogen exchange reaction.

An efficient synthesis of a structurally unique, novel M(3) antagonist 1 is described. Compound 1 is conveniently disconnected retrosynthetically at the amide bond to reveal the acid portion 2 and the amine fragment 3. The synthesis of key intermediate 2 is highlighted by a ZnCl(2)-MAEP complex 19 catalyzed diastereoselective Michael reaction of dioxolane 7 with 2-cyclopenten-1-one (5) to establish the contiguous quaternary-tertiary chiral centers and a subsequent geminal difluorination of ketone 17 using Deoxofluor in the presence of catalytic BF(3).OEt(2). The synthesis of the amine moiety 3 is highlighted by the discovery of a novel n-Bu(3)MgLi magnesium-halogen exchange reaction for selective functionalization of 2,6-dibromopyridine. This new and practical metalation protocol obviated cryogenic conditions and upon quenching with DMF gave 6-bromo-2-formylpyridine (26) in excellent yield. Further transformations afforded the amine fragment 3 via reductive amination with 35, Pd-catalyzed aromatic amination, and deprotection. Finally, the highly convergent synthesis of 1 was accomplished by coupling of the two fragments. This synthesis has been used to prepare multi-kilogram quantities of the bulk drug.

Seaweed prevents breast cancer?

To investigate the chemopreventive effects of seaweed on breast cancer, we have been studying the relationship between iodine and breast cancer. We found earlier that the seaweed, wakame, showed a suppressive effect on the proliferation of DMBA (dimethylbenz(a)anthracene)-induced rat mammary tumors, possibly via apoptosis induction. In the present study, powdered mekabu was placed in distilled water, and left to stand for 24 h at 4 degrees C. The filtered supernatant was used as mekabu solution. It showed an extremely strong suppressive effect on rat mammary carcinogenesis when used in daily drinking water, without toxicity. In vitro, mekabu solution strongly induced apoptosis in 3 kinds of human breast cancer cells. These effects were stronger than those of a chemotherapeutic agent widely used to treat human breast cancer. Furthermore, no apoptosis induction was observed in normal human mammary cells. In Japan, mekabu is widely consumed as a safe, inexpensive food. Our results suggest that mekabu has potential for chemoprevention of human breast cancer.

Pharmacological properties of (2R)-N-[1-(6-aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide: a novel mucarinic antagonist with M(2)-sparing antagonistic activity.

We evaluated the pharmacological profiles of (2R)-N-[1-(6- aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide(compound A), which is a novel muscarinic receptor antagonist with M(2)-sparing antagonistic activity. Compound A inhibited [(3)H]NMS binding to cloned human muscarinic m1, m2, m3, m4, and m5 receptors expressed in Chinese hamster ovary cells with K(i) values (nM) of 1.5, 540, 2.8, 15, and 7.7, respectively. In isolated rat tissues, compound A inhibited carbachol-induced responses with 540-fold selectivity for trachea (K(B) = 1.2 nM) over atria (K(B) = 650 nM). In in vivo rat assays, compound A inhibited acetylcholine-induced bronchoconstriction and bradycardia with intravenous ED(50) values of 0.022 mg/kg and >/=10 mg/kg, respectively. Furthermore, in dogs, compound A (0.1-1 mg/kg p.o.) dose dependently shifted the methacholine concentration-respiratory resistance curves. In mice, compound A (10 mg/kg i.v.) did not inhibit oxotremorine-induced tremor. The brain/plasma ratio (K(p)) of compound A (3 mg/kg i.v.) was 0.13 in rats; this K(p) was less than that of scopolamine (1.7) and darifenacin (0.24). The inhibition of compound A (3 mg/kg i.v.) on ex vivo binding in rat cerebral cortex was almost similar to that of NMS. These findings demonstrate that compound A has high selectivity for M(3) receptors over M(2) receptors, displays a potent, oral M(3) antagonistic activity without inhibition of central muscarinic receptors because of low brain penetration. It is well known that central muscarinic antagonists may have diverse CNS effects, and M(2) receptors regulate cardiac pacing and act as autoreceptors in the lung and bladder. Thus, compound A may have fewer cardiac or CNS side effects than nonselective compounds.

A novel class of inhibitors for human and rat steroid 5alpha-reductases: synthesis and biological evaluation of indoline and aniline derivatives. III.

While searching for novel nonsteroidal inhibitors of human and rat prostatic 5alpha-reductases, we found a new series of indoline and aniline derivatives that showed potent inhibitory activities for both enzymes. Among them, 3-chloro-4-¿[1-(4-phenoxybenzyl)indolin-5-yl]oxylbenzoic acid (2e, YM-36117) showed a more potent inhibitory activity for the human enzyme than ONO-3805 with an IC50 value of 5.3 nM and a reduced rat prostatic dihydrotestosterone (DHT) concentration by oral administration. The synthesis and the structure-activity relationships of these indoline and aniline derivatives are presented.

Bone metabolic analysis in patients with primary hyperparathyroidism.

Surgical treatment for primary hyperparathyroidism (PHPT) improves not only the calcium and phosphate metabolism but also the bone metabolism. This study was conducted to analyze the bone metabolism after PHPT operations. Bone mineral density (BMD) was measured by dual-photon absorptiometry in 50 patients before and after operation. Osteocalcin (OC) and alkaline-phosphatase activity (Alp) in serum were measured before and after surgery as markers of bone formation, and urinary deoxypiridinorine (DPD) as an index of osteoclast activity. The 50 patients under study were 40 women (80%) and ten men (20%). Increases in BMD at the lumbar spine were remarkable at three months following operation. Slow but steady progress was made until six months, reaching a plateau thereafter. The increase in BMD of lumbar spine was approximately 10%. Urinary DPD was the most sensitive among the three bone metabolic markers. Although serum Alp and OC remained high after operation, urinary DPD was normalized earlier. The discrepancy of bone formation and resorption was shown after operation and this contributed to the increases in BMD in the first six months.

[Surgical treatment for infective endocarditis with multiple embolic complications].

A 59-year-old man was transferred to our hospital because of severe right leg pain, dyspnea and anuria. Due to severe cyanosis of the legs below the knees and severe hyperkalemia, he had undergone embolectomy of the right femoral artery and hemodialysis. Medical treatment for infective endocarditis was started after the first operation, because transesophageal echo cardiography revealed severe aortic regurgitation and massive vegetation of the aortic valve. Amputation of the right leg below the knee and of the left leg below the Lisfranc joint was performed after 19 days and aortic valve replacement with patch closure of a perivalvular abscess was performed one month after the first operation. The post-operative course was uneventful. He was weaned from hemodialysis and the follow-up echocardiographic study revealed no vegetation.

Discovery of a muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors among 2-[(1S,3S)-3-sulfonylaminocyclopentyl]phenylacetamide derivatives.

In the course of developing a metabolically stable M3 receptor antagonist from the prototype antagonist, J-104129 (1), introduction of certain substituents into the cyclopentane ring of 1 was found to be effective not only in improving metabolic stability but also in greatly enhancing the subtype selectivity. Among the cyclopentane analogues, sulfonamide derivatives (10f) and (10g) displayed 160- and 310-fold selectivity for M3 over M2 receptors, and both were significantly more selective than the prototype antagonist (120-fold). Subsequent derivatization of the sulfonamide series led to the highly selective M3 receptor antagonists (10h, 10i and 10j) with >490-fold selectivity for M3 over M2 receptors. Among them, p-nitrophenylsulfonamide (J-107320, 10h) exhibited 1100-fold selectivity for M3 receptors (Ki = 2.5 nM) over M2 receptors (Ki = 2800 nM) in the human muscarinic receptor binding assay using [3H]-NMS as a radio ligand.