Design, Synthesis, and Evaluation of Trivalent PROTACs Having a Functionalization Site with Controlled Orientation.

Trivalent PROTACs having a functionalization site with controlled orientation were designed, synthesized, and evaluated. Based on the X-ray structure of BRD protein degrader MZ1 (1) in complex with human VHL and BRD4BD2, we expected that the 1,2-disubstituted ethyl group near the JQ-1 moiety in MZ1 (1) could be replaced by a planar benzene tether as a platform for further functionalization. To test this hypothesis, we first designed six divalent MZ1 derivatives, 2a-c and 3a-c, by combining three variations of substitution patterns on the benzene ring (1,2-, 1,3-, and 1,4-substitution) and two variations in the number of ethylene glycol units (2 or 1). We then tested the synthesized compounds for the BRD4 degradation activity of each. As expected, we found that 1,2D-EG2-MZ1 (2a), an MZ1 derivative with 1,2-disubstituted benzene possessing two ethylene glycol units, had an activity profile similar to that of MZ1 (1). Based on the structure of 2a, we then synthesized and evaluated four isomeric trivalent MZ1 derivatives, 15a-15d, having a tert-butyl ester unit on the benzene ring as a handle for further functionalization. Among the four isomers, 1,2,5T-EG2-MZ1 (15c) retained a level of BRD4 depletion activity similar to that of 2a without inducing a measurable Hook effect, and its BRD4 depletion kinetics was the same as that of MZ1 (1). Other isomers were also shown to retain BRD4 depletion activity. Thus, the trivalent PROTACs we synthesized here may serve as efficient platforms for further applications.

Calofolic Acid-A from Calophyllum scriblitifolium Bark Has Vasorelaxant Activity via Indirect PKA Activation Caused by PI-3 Kinase Inhibition in Rat Vascular Smooth Muscle Cells.

Previously, we isolated 2R,3S,15R-calofolic acids (CAs) from Calophyllum scriblitifolium bark, which showed vasorelaxant activity on phenylephrine (PE)-precontracted rat aortic rings. Although the effect was suggested to be induced via an extracellular Ca2+-independent manner and mainly acts on vascular smooth muscle, the exact mechanism of action of CAs remained unclear. Thus, this study investigated the detailed mechanism of calofolic acid-A (CA-A) induced vasorelaxation in an aortic ring specimen using rat vascular smooth muscle cells (VSMCs). The levels of PE-induced phosphorylation on MLC Ser19 decreased in VSMCs pretreated with CA-A. CA-A also decreased the phosphorylation of MYPT1 Thr696 and MYPT1 Thr853. On the other hand, CA-A increased the PE-induced phosphorylation of MYPT1 Ser695 and MYPT1 Ser668, which are reported to be phosphorylated by a cAMP-dependent protein kinase (PKA). CA-A slightly increased PKA substrate phosphorylation in a concentration-dependent manner. Furthermore, CA-A enhanced isoproterenol (ISO)-induced cAMP accumulation and PKA substrate phosphorylation. Treatment with PI-3 kinase (PI3K) inhibitor, LY294002, enhanced ISO-induced cAMP accumulation and PKA substrate phosphorylation in the same manner as CA-A treatment. Furthermore, CA-A was found to directly inhibit PI3K enzyme activity in a dose-dependent manner. Taken together, the present study indicated that CA-A induces vasorelaxation through an indirectly activated PKA-MYPT1 pathway caused by inhibition of PI3K activity.

Bisleuconothines B-D, Modified Eburnane-Aspidosperma Bisindole Alkaloids from Leuconotis griffithii.

Three new bisindole alkaloids, bisleuconothines B-D (1-3), were isolated from the bark of Leuconotis griffithii. Their structures were elucidated by 1D and 2D NMR spectroscopy and DFT calculations. Bisleuconothine B (1) is the first monoterpene indole alkaloid dimer featuring bridges between both C-16-C-10' and C-2-O-C-9'. All compounds were deemed noncytotoxic (IC50 > 10 µM) when tested against A549 human lung adenocarcinoma cells.

Syntheses and anti-inflammatory activity of azamollugin derivatives.

Oxomollugin (2) is a degradation product of mollugin (1) and a potent inhibitor of NO-production including nuclear factor kappa B signals. In our endeavor to develop a potent anti-inflammatory compound, we synthesized several aza-derivatives of oxomollugin (2) and evaluated their NO-production inhibitory activity. Azamollugin (3) showed a potent inhibitory activity, and its activity (IC50 0.34µM) was proved to be more potent than that of oxomollugin (2, IC50 1.3µM).

Bisleuconothine A Induces Autophagosome Formation by Interfering with AKT-mTOR Signaling Pathway.

We have previously reported that bisleuconothine A (Bis-A), a novel bisindole alkaloid isolated from Leuconotis griffithii, showed cytostatic activity in several cell lines. In this report, the mechanism of Bis-A-induced cytostatic activity was investigated in detail using A549 cells. Bis-A did not cause apoptosis, as indicated by analysis of annexin V and propidium iodide staining. Expression of all tested apoptosis-related proteins was also unaffected by Bis-A treatment. Bis-A was found to increase LC3 lipidation in MCF7 cells as well as A549 cells, suggesting that Bis-A cytostatic activity may be due to induction of autophagy. Subsequent investigation via Western blotting and immunofluorescence staining indicated that Bis-A induced formation but prevented degradation of autophagosomes. Mechanistic studies showed that Bis-A down-regulated phosphorylation of protein kinase B (AKT) and its downstream kinase, PRAS40, which is an mTOR repressor. Moreover, phosphorylation of p70S6K, an mTOR-dependent kinase, was also down-regulated. Down-regulation of these kinases suggests that the increase in LC3 lipidation may be due to mTOR deactivation. Thus, the cytostatic activity shown by Bis-A may be attributed to its induction of autophagosome formation. The Bis-A-induced autophagosome formation was suggested to be caused by its interference with the AKT-mTOR signaling pathway.

Chrotacumines G-J, chromone alkaloids from Dysoxylum acutangulum with osteoclast differentiation inhibitory activity.

Four new chromone alkaloids, chrotacumines G-J (1-4), have been isolated from the barks of Dysoxylum acutangulum. Their structures and absolute configurations were elucidated on the basis of NMR and CD data. Chrotacumines G and J (1 and 4) showed osteoclast differentiation inhibitory activity in a dose dependent manner.

Epicutaneous administration of papain induces IgE and IgG responses in a cysteine protease activity-dependent manner.

BACKGROUND: Epicutaneous sensitization to allergens is important in the pathogenesis of not only skin inflammation such as atopic dermatitis but also "atopic march" in allergic diseases such as asthma and food allergies. We here examined antibody production and skin barrier dysfunction in mice epicutaneously administered papain, a plant-derived occupational allergen belonging to the same family of cysteine proteases as mite major group 1 allergens. METHODS: Papain and Staphylococcus aureus V8 protease were patched on the backs of hairless mice. Transepidermal water loss was measured to evaluate the skin barrier dysfunction caused by the proteases. Papain or that treated with an irreversible inhibitor specific to cysteine proteases, E64, was painted onto the ear lobes of mice of an inbred strain C57BL/6. Serum total IgE levels and papain-specific IgE and IgG antibodies were measured by ELISA. RESULTS: Papain and V8 protease patched on the backs of hairless mice caused skin barrier dysfunction and increased serum total IgE levels, and papain induced the production of papain-specific IgG1, IgG2a, and IgG2b. Papain painted onto the ear lobes of C57BL/6 mice induced papain-specific IgE, IgG1, IgG2c, and IgG2b, whereas papain treated with E64 did not. IgG1 was the most significantly induced papain-specific IgG subclass among those measured. CONCLUSIONS: We demonstrated that the epicutaneous administration of protease not only disrupted skin barrier function, but also induced IgE and IgG responses in a manner dependent on its protease activity. These results suggest that protease activity contained in environmental sources contributes to sensitization through an epicutaneous route.

Isovincathicine from Catharanthus roseus induces apoptosis in A549 cells.

A dimeric indole alkaloid, isovincathicine consisting of an aspidosperma type and modified iboga with C-7-C-20 connection type skeletons was first isolated from Catharanthus roseus, and the structure including stereochemistry was elucidated on the basis of spectroscopic data as well as DP4 statistical analysis. Isovincathicine inhibited cell proliferation in A549 cells. We investigated the detailed mode of action of isovincathicine-induced inhibitory effects on cell proliferation in A549 cells. Flow cytometric analysis showed that isovincathicine-treated cells accumulated in the G2 phase after 24 h, and the percentage of cells showing cell death increased after 48 h. Western blotting also showed increased expression of BimEL, an apoptosis-related protein, and decreased expression of Mcl-1 and Bcl-xL. Isovincathicine was suggested to induce apoptosis in A549 cells by a mechanism is similar to that of vinblastine.

Vincarostine A, a novel anti-malarial trimeric monoterpenoid indole alkaloid from Catharanthus roseus.

A novel trimeric monoterpenoid indole alkaloid, vincarostine A (1) consisting of an aspidosperma-iboga-aspidosperma type skeleton, was isolated from the whole plant of Catharanthus roseus. The structure including absolute stereochemistry was elucidated on the basis of 2D NMR data and CD spectrum. Vincarostine A (1) showed anti-malarial activity.

Vincazalidine A, a unique bisindole alkaloid from Catharanthus roseus.

A new dimeric indole alkaloid, vincazalidine A consisting of an aspidosperma type and a modified iboga type with 1-azatricyclo ring system consisting of one azepane and two piperidine rings coupled with an oxazolidine ring was isolated from Catharanthus roseus, and the structure including absolute stereochemistry was elucidated on the basis of spectroscopic data as well as DP4 statistical analysis. Vincazalidine A induced G2 arrest and subsequent apoptosis in human lung carcinoma cell line, A549 cells.

Oxomollugin, an oxidized substance in mollugin, inhibited LPS-induced NF-κB activation via the suppressive effects on essential activation factors of TLR4 signaling.

Oxomollugin is a degraded product of mollugin and was found to be an active compound that inhibits LPS-induced NF-κB activation. In this study, we investigated the inhibitory activity of oxomollugin, focusing on TLR4 signaling pathway, resulting in NF-κB activation. Oxomollugin inhibited the LPS-induced association of essential factors for initial activation of TLR4 signaling, MyD88, IRAK4 and TRAF6. Furthermore, oxomollugin showed suppressive effects on LPS-induced modification of IRAK1, IRAK2 and TRAF6, LPS-induced association of TRAF6-TAK1/TAB2, and followed by IKKα/ß phosphorylation, which critical in signal transduction leading to LPS-induced NF-κB activation. The consistent results suggested that oxomollugin inhibits LPS-induced NF-κB activation via the suppression against signal transduction in TLR4 signaling pathway.The activities of oxomollugin reported in this study provides a deeper understanding on biological activity of mollugin derivatives as anti-inflammatory compounds.

Ceramicines U-Z from Chisocheton ceramicus and structure-antimalarial activity relationship study.

Ceramicines are a series of limonoids which were isolated from the barks of Malaysian Chisocheton ceramicus (Meliaceae), and were known to show various biological activity. Six new limonoids, ceramicines U-Z (1-6), with a cyclopentanone[α]phenanthrene ring system with a ß-furyl ring at C-17 were isolated from the barks of C. ceramicus. Their structures were determined on the basis of the 1D and 2D NMR analyses, and their absolute configurations were investigated by CD spectroscopy. Ceramicine W (3) exhibited potent antimalarial activity against Plasmodium falciparum 3D7 strain with IC50 value of 1.2 µM. In addition, the structure-antimalarial activity relationship (SAR) of the ceramicines was investigated to identify substituent patterns that may enhance activity. It appears that ring B and the functional groups in the vicinity of rings B and C are critical for the antimalarial activity of the ceramicines. In particular, bulky ester substituents with equatorial orientation at C-7 and C-12 greatly increase the antimalarial activity.

Cryptadine C, a new C27N3-type Lycopodium alkaloid from Lycopodium cryptomerinum.

A new C27N3-type Lycopodium alkaloid consisting of two decahydroquinolines and a piperidine, cryptadine C (1) was isolated from Lycopodium cryptomerinum. The structure and relative configuration of 1, which is related to those of cryptadines A and B, lycoperine A, and hupercumine A, was elucidated on the basis of spectroscopic data. Cryptadine C showed moderate inhibitory activity against acetylcholinesterase.

Antifungal Susceptibility Testing for Microsporum canis from Cats in Japan.

Control of infection caused by Microsporum canis in pet animals are important for prevention of zoonosis. Treatments for animal dermatophytosis have generally consisted of itraconazole (ITZ) and terbinafine (TRF); however, a TRF-resistant M. canis strain from a case of feline dermatophytosis has been reported. In the present study, we examined the in vitro susceptibility of clinical isolates of M. canis to new antifungal drugs, such as ravuconazole (RVZ) and luliconazole (LCZ). The results indicated that RVZ and LCZ are more effective than ITZ and TRF. Therefore, oral administration of RVZ or topical application of LCZ may serve as new treatment options.

Antimalarial ceramicines Q-T from Chisocheton ceramicus.

Ceramicines are a series of limonoids that were isolated from the bark of Malaysian Chisocheton ceramicus (Meliaceae) and were known to show various biological activity. Four new limonoids, ceramicines Q-T (1-4) were isolated from the barks of C. ceramicus, and their structures were determined on the basis of the 1D and 2D NMR analyses in combination with calculated 13C chemical shift data. Ceramicines Q-T (1-4) were established to be new limonoids with a cyclopentanone[α]phenanthren ring system with a ß-furyl ring at C-17, and without a tetrahydrofuran ring like ceramicine B, which is characteristic of known ceramicines. Ceramicine R (2) exhibited potent antimalarial activity against Plasmodium falciparum 3D7 strain with IC50 value of 2.8 µM.

Quassinoids from Eurycoma longifolia Jack roots and their potential inhibitory activity against human benign prostatic hyperplasia cells (BPH-1) and testosterone-induced BPH rat model.

Benign prostate hyperplasia (BPH) is an enlargement of the prostate gland, because of hormonal changes in aging males which contribute significantly to excessive proliferation over apoptosis of prostatic cells. The anti-proliferative and induced apoptotic activities of Eurycoma longifolia quassinoids on cancer cell lines could be promising therapeutic targets on BPH. Hitherto, no report of the quassinoids against BPH problem was available. In this study, a systematic phytochemical fractionation of the root extract, TAF2 was performed, which led to the discovery of nine previously described C20 quassinoids (1-9). Two undescribed C20 (10 and 12) and one undescribed (11) C19 quassinoids were identified by detailed NMR and HR-ESI-MS data analysis. Their absolute configurations were assigned by ECD spectral analysis. The quassinoids (1-12) were tested for inhibitory activity against the proliferation of human BPH-1 and human skin Hs27 fibroblast cells cultured in vitro. 1, 2 and 3 at 10 µM significantly reduced BPH-1 cell viability and were cytotoxic to Hs27 fibroblast cells. 2 was selected for further study of anti-BPH activity against testosterone induced BPH rats. At 5 mg/kg, 2 reduced the rat prostatic weight and prostatic index, consistent with the decrease in papillary acini number and epithelial thickness of the prostate tissues. These quassinoids may be potential anti-BPH compounds that require further studies.

Alstiphyllanines I-O, ajmaline type alkaloids from Alstonia macrophylla showing vasorelaxant activity.

Seven new ajmaline type alkaloids, alstiphyllanines I-O (1-7) were isolated from the leaves of Alstonia macrophylla together with six related alkaloids (8-13). Structures and stereochemistry of 1-7 were fully elucidated and characterized by 2D NMR analysis. A series of alstiphyllanines I-O (1-7) as well as the known ajmaline type alkaloids (8-13) showed that they relaxed phenylephrine (PE)-induced contractions against rat aortic ring. Among them, vincamedine (10) showed potent vasorelaxant activity, which may be mediated through inhibition of Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCs) and/or receptor-operated Ca(2+) channels (ROCs) as well as partially mediated the NO release from endothelial cells. The presence of substituents at both N-1 and C-17 may be important to show vasorelaxation activity.

New tricyclic alkaloids, cassiarins G, H, J, and K from leaves of Cassia siamea.

Four new alkaloids, cassiarins G, H, J, and K (1-4) which showed moderate antiplasmodial activity against Plasmodium falciparum 3D7, were isolated from the leaves of Cassia siamea (Leguminosae) and the structures of 1-4 were elucidated by 1D- and 2D-NMR analysis and X-ray crystallographic analysis.

Cuspidans A and B, two new stilbenoids from the bark of Gnetum cuspidatum.

Cuspidan A (1), a new stilbene sestermer consisting of a resveratrol, an oxyresveratrol, and a 3,5-dihydroxyphenylmethanol constituent units and cuspidan B (2), a new tri-cyclic stilbene monomer were isolated from the bark of Gnetum cuspidatum. The structures and configurations of 1 and 2 were elucidated on the basis of 2D-NMR correlations.