A new synthesis of the ORL-1 antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) and activity in a calcium mobilization assay

A new chiral synthesis of the ORL-1 antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (2, J-113397) was developed. J-113397 has a K(e)=0.85nM in an ORL-1 calcium mobilization assay and is 89-, 887-, and 227-fold selective for the ORL-1 receptor relative to the mu, delta, and kappa opioid receptors.     

Further studies on the pharmacological features of the nociceptin/orphanin FQ receptor ligand ZP120

ZP120 is a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In previous studies, the effects of ZP120 were found to be sensitive to J-113397 in mouse tissues while resistant to UFP-101 in rat tissues. The aim of this study was to further investigate the ZP120 pharmacological profile using mouse and rat preparations, J-113397 and UFP-101, as well as NOP receptor knockout (NOP(-/-)) mice. Electrically stimulated mouse and rat vas deferens were used to characterize the pharmacology of ZP120 in vitro. For in vivo studies the tail-withdrawal assay was performed in wild type (NOP(+/+)) and NOP knockout (NOP(-/-)) mice. In the mouse and rat vas deferens ZP120 mimicked the effects of N/OFQ showing higher potency but lower maximal effects. In both preparations, J-113397 antagonized N/OFQ and ZP120 effects showing similar pK(B) values ( approximately 7.8). UFP-101 antagonized the actions of N/OFQ (pK(B) values approximately 7.3) but did not modify the effects of ZP120. The inhibitory effects of N/OFQ and ZP120 were no longer evident in vas deferens tissues taken from NOP(-/-) mice. In NOP(+/+) mice subjected to the tail-withdrawal assay, ZP120 (1 nmol) mimicked the pronociceptive action of N/OFQ (10 nmol), producing longer lasting effects. The effects of both peptides were absent in NOP(-/-) animals. The NOP receptor ligand ZP120 is a high potency NOP selective partial agonist able to evoke long-lasting effects; its diverse antagonist sensitivity in comparison with N/OFQ may derive from different modality of binding to the NOP receptor.     

Further evidence for an involvement of nociceptin/orphanin FQ in the pathophysiology of Parkinson's disease: a behavioral and neurochemical study in reserpinized mice

The contribution of nociceptin/orphanin FQ (N/OFQ) to reserpine-induced Parkinsonism was evaluated in mice. A battery of motor tests revealed that reserpine caused dose-dependent and long-lasting motor impairment. Endogenous N/OFQ sustained this response because N/OFQ peptide (NOP) receptor knockout (NOP(-/-) ) mice were less susceptible to the hypokinetic action of reserpine than wild-type (NOP(+/+) ) animals. Microdialysis revealed that reserpine elevated glutamate and reduced GABA levels in substantia nigra reticulata, and that resistance to reserpine in NOP(-/-) mice was accompanied by a milder increase in glutamate and lack of inhibition of GABA levels. To substantiate this genetic evidence, the NOP receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H benzimidazol-2-one (J-113397) simultaneously reduced akinesia and nigral glutamate levels in reserpinized NOP(+/+) mice, being ineffective in NOP(-/-) mice. Moreover, repeated J-113397 administration in reserpinized mice resulted in faster recovery of baseline motor performance which was, however, accompanied by a loss of acute antiakinetic response. The short-term beneficial effect of J-113397 was paralleled by normalization of nigral glutamate levels, whereas loss of acute response was paralleled by loss of the ability of J-113397 to inhibit glutamate levels. We conclude that endogenous N/OFQ contributes to reserpine-induced Parkinsonism, and that sustained NOP receptor blockade produces short-term motor improvement accompanied by normalization of nigral glutamate release.     

A new synthetic approach to 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-benzimidazol-2-one(J-113397), the first non-peptide ORL-1 receptor antagonist..

An efficient approach to 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-benzimidazol-2-one (J-113397) 1, the first non-peptide ORL-1 receptor antagonist described in literature, is outlined. After construction of the piperidine framework through Dieckmann cyclization of the Michael adduct 8 of cyclooctylmethylamine to methyl acrylate, condensation with o-phenylendiamine produced the beta-enamino ester 2, which has been conveniently used to construct the benzimidazolone substituent at C-4. Catalytic hydrogenation of intermediate 11 followed by base-promoted cis--trans isomerization of the key compound 12 led to the formation of ester 13, which was converted to the racemic title compound by LiAlH(4) reduction. The pure enantiomers were obtained by chiral preparative HPLC separation using a derivatized cellulose-based stationary phase.     

Synthesis and biological activities of NB-506 analogues modified at the glucose group

A new indolocarbazole compound, NB-506 (1), modified at the glucose group yielded a beta-D-glucopyranoside, J-107,088 (2), which showed potent anticancer activity. A beta-D-ribofuranoside, J-109,534 (3), was found to be 6 times more potent than J-107,088 at inhibiting topoisomerase I.     

Discovery of triazolo [1,5-a] pyridine derivatives as novel JAK1/2 inhibitors

Small molecule JAK inhibitors have been demonstrated efficacy in rheumatoid arthritis, inflammatory bowel disease, and psoriasis with the approval of several drugs. Aiming to develop potent JAK1/2 inhibitors, two series of triazolo [1,5-a] pyridine derivatives were designed and synthesized by various strategies. The pharmacological results identified the optimized compounds J-4 and J-6, which exerted high potency against JAK1/2, and selectivity over JAK3 in enzyme assays. Furthermore, J-4 and J-6 effectively suppressed proliferation of JAK1/2 high-expression BaF3 cells accompanied with acceptable metabolic stability in liver microsomes. Therefore, J-4 and J-6 might serve as promising JAK1/2 inhibitors for further investigation.     

高产脂肪酸菌株的诱变筛选

本文研究了无花果丝孢酵母（Ｔｒｉｃｈｏｓｐｆｉｇｕｅｒｉａｅ）Ｊ—０４等６株微生物的脂肪酸在细胞内、细胞外及细胞表面的分布。用紫外线和硫酸二酯对Ｊ－０４进行复合诱变。用制霉菌素和琥珀酸钠筛选具有高渗透性和抗阻遏的突变菌株。诱变筛选的ＪＵ－０９菌株,其胞外酶活性提高了７６％。     

Inhibitory activity of nociceptin/orphanin FQ on capsaicin-induced bronchoconstriction in the guinea-pig

In vivo studies were conducted in the guinea-pig to investigate the activity of the selective ORL1 receptor agonist nociceptin/orphanin FQ against capsaicin-induced bronchoconstriction, a response mediated by the release of tachykinins from pulmonary sensory nerves. Anesthetized guinea-pigs were ventilated with a rodent ventilator and placed in a whole-body plethysmograph, and pulmonary resistance (R(L)) and dynamic lung compliance (C(Dyn)) were monitored. Intravenous administration of nociceptin/orphanin FQ (0.3 mg/kg) inhibited the capsaicin-induced bronchoconstriction. The new nonpeptide ORL1 receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) administered intravenously (1 mg/kg) produced a significant blockade of the inhibitory effect of nociceptin/orphanin FQ (0.3 mg/kg) on capsaicin-induced bronchoconstriction, whereas the nonselective opioid receptor antagonist naloxone (1 mg/kg) had no effect. Nociceptin/orphanin FQ (0.3 mg/kg) did not affect the bronchoconstriction induced exogenously by the tachykinin NK2 receptor agonist [beta-ala8]-neurokinin A (4-10). We conclude that nociceptin inhibits in vivo capsaicin-evoked tachykinin release from sensory nerve terminals in the guinea-pig by a prejunctional mechanism. This inhibitory action does not involve activation of opioid receptors.     

The small molecule Retro-1 enhances the pharmacological actions of antisense and splice switching oligonucleotides

The attainment of strong pharmacological effects with oligonucleotides is hampered by inefficient access of these molecules to their sites of action in the cytosol or nucleus. Attempts to address this problem with lipid or polymeric delivery systems have been only partially successful. Here, we describe a novel alternative approach involving the use of a non-toxic small molecule to enhance the pharmacological effects of oligonucleotides. The compound Retro-1 was discovered in a screen for small molecules that reduce the actions of bacterial toxins and has been shown to block the retrograde trafficking pathway. We demonstrate that Retro-1 can also substantially enhance the effectiveness of antisense and splice switching oligonucleotides in cell culture. This effect occurs at the level of intracellular trafficking or processing and is correlated with increased oligonucleotide accumulation in the nucleus but does not involve the perturbation of lysosomal compartments. We also show that Retro-1 can alter the effectiveness of splice switching oligonucleotides in the in vivo setting. These observations indicate that it is possible to enhance the pharmacological actions of oligonucleotides using non-toxic and non-lysosomotropic small molecule adjuncts.     

The synthesis of compound bands in Drosophila melanogaster salivary gland chromosomes

Small chromosome aberrations were utilized to construct compound bands in the 3C region of Drosophila melanogaster salivary gland chromosomes. The results imply that one should expect to find multiple functions associated with single bands, especially heavy bands. It is suggested that the natural occurrence of compound bands needs to be recognized and that exceptions to the one gene: one band correspondence are expected to occur.Journal Paper No. J-9459 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 1985     

Characterisation of the non-peptide nociceptin receptor agonist,Ro64-6198 in Chinese hamster ovary cells expressing recombinant human nociceptin receptors

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the opioid receptor-like receptor or nociceptin receptor (NOP). We have compared a novel non-peptide NOP agonist Ro64-6198 with N/OFQ in a series of GTPgamma35S binding and inhibition of forskolin stimulated cAMP formation assays. GTPgamma35S binding assays were performed in membranes prepared from Chinese hamster ovary cells expressing the recombinant human NOP (CHOhNOP). cAMP inhibition studies were performed in whole CHOhNOP cells. Both Ro64-6198 and N/OFQ stimulated GTPgamma35S binding with pEC50 values(95%CL) of 7.61(0.18) and 8.58(0.21) respectively. Both Ro64-6198 and N/OFQ inhibited cAMP formation with pEC50 values of 8.45(0.9) and 9.28(028) respectively. In each assay Ro64-6198 and N/OFQ were full agonists. Ro64-6198 stimulation of GTPgamma35S binding and inhibition of cAMP formation was competitively antagonised by the NOP antagonists [Nphe1]NC(1 - 13)NH2 (10microM), J-113397 (100nM) and III-BTD (1microM) with pKB values of 7.04(0.34) and 6.29(0.10), 8.65(0.34) and 7.90(0.30) and 7.59(0.22) and 7.60(0.22) respectively. Despite the slightly reduced potency of Ro64-6198 compared with N/OFQ, by virtue of high selectivity and relative metabolic stability this molecule will be of considerable use in studies of the actions of the NOP.     

Formation of heterosymplasts in human reticular cell cultures]

The cell fusion has been studied in human reticular cell cultures J-96 and J-41 treated with the Sendai virus or with polyethylene glycol 1000 and 6000. The J-96 cells have a high alkaline phosphatase activity, in J-41 cells the enzyme is not detectable. No heterogenous alkaline phosphatase activity was seen in the protoplasm of symplasts 18 hours after virus cell fusion. It has been shown with polyethylene glycol treatment that during the fusion of cells J-96 and J-41 the enzyme activity was spreading over the symplast protoplasm.     

Structure-activity relationships of trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenems. Part 2: J-111,225, J-114,870, J-114,871 and related compounds

Through further derivatization of J-111,347 (1a), a trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenem, undesired epileptogenicity in a rat intracerebroventricular assay (200 microg/rat) could be eliminated to afford J-111,225 (2a), J-114,870 (3a) and J-114,871 (3b) which preserved comparable broad antimicrobial activity.     

Discovery of novel trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenems

Novel trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenems were designed and synthesized to provide J-111,347 (1a) as the first example of an exceptionally broad-spectrum antibiotic, showing activity against methicillin-resistant Staphyloccocus aureus (MRSA) as well as Pseudomonas aeruginosa. Further derivation of 1a afforded J-111,225 (2a), J-114,870 (3a), and J-114,871 (3b). which showed improved safety profiles and retained broad-spectrum antibacterial activities.     

Lack of Cytosolic Carboxypeptidase 1 Leads to Subfertility due to the Reduced Number of Antral Follicles in pcd3J-/- Females

Females homozygous for the Purkinje cell degeneration mutation (pcd) are fertile, although the success rate is much lower than in the wild type. We performed detailed analysis of reproductive abnormalities of pcd females. The number of oocytes produced following exogenous gonadotropin treatment was much lower in pcd ^3J-/- females than in pcd ^3J+/+ females. Furthermore, the estrous cyclicity of pcd ^3J-/- females according to the appearance of the vagina was almost undetectable comparing to that of the wild type. Histological analyses and follicle counting of 4- and 8-week-old pcd ^3J-/- ovaries showed an increase in the number of secondary follicles and a decrease in the number of antral follicles, indicating that AGTPBP1/ CCP1 plays an important role in the development of secondary follicles into antral follicles. Consistent with a previous analysis of the pcd cerebellum, pcd ^3J-/- ovaries also showed a clear increase in the level of polyglutamylation. Gene expression analysis showed that both oocytes and cumulus cells express CCP1. However, Ccp4 and CCP6, which can compensate the function of CCP1, were not expressed in mouse ovaries. Failure of microtubule deglutamylation did not affect the structure and function of the meiotic spindle in properly aligning chromosomes in the center of the nucleus during meiosis in pcd ^3J-/- females. We also showed that the pituitary-derived growth and reproduction-related endocrine system functions normally in pcd ^3J-/- mice. The results of this study provide insight into additional functions of CCP1, which cannot be fully explained by the side chain deglutamylation of microtubules alone.     

Disruptive developmental effects of benzyl-1,3-benzodioxole derivatives on unparasitized and parasitized Manduca sexta larvae

Treatment of tobacco hornworm larvae with the benzyl-1,3-benzodioxole derivative J-2710 immediately after ecdysis to the fourth instar disrupted development either during the moult to the fifth instar or shortly thereafter. Larvae given topical applications of 100 μg J-2710 in 1 μl acetone suffered 100% mortality, often after secreting moulting fluid in large pockets between the epidermis and the cuticle later in the fourth instar. Larvae that successfully ecdysed had abnormalities of the mouthparts and cervix that interfered with normal feeding, inhibiting growth in the fifth instar. Larvae of the gregarious endoparasitic wasp Cotesia congregata (=Apanteles congregatus) frequently failed to emerge from host Manduca sexta larvae treated with high doses of J-2710, particularly when the host failed to feed normally. Less potent disruptive effects on Manduca and Cotesia were seen after treatment of larvae with the derivatives J-3370 and J-2581.No anti-juvenile hormone action of J-2710 was observed. J-2710-treated M. sexta larvae showed no precocious metamorphosis and the developmental effects of J-2710 were not prevented by co-application of the juvenile hormone analogue methoprene in doses ranging from 1 to 100 μg/larva. Moreover, J-2710 had no effect on the action of methoprene in the black larval assay for juvenile hormone-like activity, unlike results reported to occur using the Galleria wax wound assay.     

Mitochondrial DNA polymorphism in three Antillean island populations of the fruit bat, Artibeus jamaicensis

The Neotropical fruit bat, Artibeus jamaicensis, occurs throughout Latin America and on many islands in the Caribbean. Populations from Jamaica (in the Greater Antilles) to Barbados (in the Lesser Antilles) have been classified as a subspecies (A.j. jamaicensis) separate from that on the Lesser Antillean island of St. Vincent (A.j. schwartzi). Mitochondrial DNA (mtDNA) was isolated from 54 individuals collected on these islands, analyzed by digestion with restriction endonucleases, and the restriction sites were mapped. Three different mtDNA genotypes (16,000 +/- 200 bp) were identified: J-1 (16 animals from Jamaica, one from St. Vincent, 15 from Barbados), J-2 (two animals from Jamaica), and SV-1 (18 animals from St. Vincent, two from Barbados). The J-1 and J-2 genotypes were estimated to differ from each other by only 0.4%, but the SV-1 genotype differed from J-1 and J-2 by 8.1%-10.5%. The estimated sequence divergence between SV-1 and J-1 is unusually large for mammals that are regarded as conspecific. Restriction mapping showed that the differences among the genotypes (presence or absence of particular restriction sites) were located throughout the genome. The presence of the J-1 mtDNA genotype on Jamaica and on St. Vincent and Barbados (1,400 km away) demonstrates that maternal lineages in these bats are not necessarily confined to single islands or limited geographic regions. The presence of the J-1 mtDNA genotype within the A. j. schwartzi population on St. Vincent and the presence of the SV-1 genotype in two specimens of A. j. jamaicensis from Barbados document genetic exchange between subspecific populations on these islands, which are separated by 180 km of open water.     

Evaluation of the Antitumor Effects of BPR1J-340, a Potent and Selective FLT3 Inhibitor,Alone or in Combination with an HDAC Inhibitor,Vorinostat, in AML Cancer

Overexpression or/and activating mutation of FLT3 kinase play a major driving role in the pathogenesis of acute myeloid leukemia (AML). Hence, pharmacologic inhibitors of FLT3 are of therapeutic potential for AML treatment. In this study, BPR1J-340 was identified as a novel potent FLT3 inhibitor by biochemical kinase activity (IC₅₀ approximately 25 nM) and cellular proliferation (GC₅₀ approximately 5 nM) assays. BPR1J-340 inhibited the phosphorylation of FLT3 and STAT5 and triggered apoptosis in FLT3-ITD⁺ AML cells. The pharmacokinetic parameters of BPR1J-340 in rats were determined. BPR1J-340 also demonstrated pronounced tumor growth inhibition and regression in FLT3-ITD⁺ AML murine xenograft models. The combination treatment of the HDAC inhibitor vorinostat (SAHA) with BPR1J-340 synergistically induced apoptosis via Mcl-1 down-regulation in MOLM-13 AML cells, indicating that the combination of selective FLT3 kinase inhibitors and HDAC inhibitors could exhibit clinical benefit in AML therapy. Our results suggest that BPR1J-340 may be further developed in the preclinical and clinical studies as therapeutics in AML treatments.     

Synthesis and biological activities of NB-506 analogues: Effects of the positions of two hydroxyl groups at the indole rings

In the course of a study of 6-N-amino-substituted analogues of NB-506 (1), a more potent anticancer drug, J-109,404 (2), in which the formyl group of NB-506 was replaced with a 1,3-dihydroxypropane group, was reported. A study of further modification in the positions of two hydroxyl groups at the indole rings of 2 resulted in the discovery of a 2,10-dihydroxy analogue, J-107,088 (3), which is a promising anticancer agent with a broader therapeutic window than J-109,404.