Synthesis of 1-O-acyl- and 1-oxo-kamebanin analogues and their cytotoxic activity.

A series of 1-O-acyl- and 1-oxo-kamebanin analogues were prepared from kamebanin, isolated from Rabdosia excisa and their cytotoxicity was assayed on HL60 promyelocytic leukemia cells and HCT116 human colon cancer cells. The structure-activity relationship study showed that the presence of 1-O-acyl groups of a C3-C5 carbon chain increased the cytotoxic activity.

Semisynthesis of Antitrypanosomal p-Quinone Analog Possesing the Komaroviquinone Pharmacophore.

A p-quinone analog having the komaroviquinone pharmacophore fused with a more conformationally flexible cycloheptane ring, was semisynthesized from natural demethlsalvicanol isolated from Perovskia abrotanoides via four steps in 26% overall yield. The IC50 for the antitrypanosomal activity of the analog was 0.55 µM.

Garcinielliptone G from Garcinia subelliptica Induces Apoptosis in Acute Leukemia Cells.

Cytotoxicity and apoptosis-inducing properties of compounds isolated from Garcinia subelliptica leaves were investigated. The hexane-soluble portion of MeOH extracts of G. subelliptica leaves that showed cytotoxic activity was separated to yield seven compounds 1-7. Chemical structure analysis using NMR spectroscopy and mass spectrometry confirmed that compound 1 was canophyllol, and compounds 2-7 were garcinielliptones N, O, J, G, F, and garcinielliptin oxide, respectively. Among them, garcinielliptone G (5) showed growth inhibition by causing apoptosis in THP-1 and Jurkat cells derived from human acute monocytic leukemia and T lymphocyte cells, respectively. Apoptosis induced by garcinielliptone G (5) was demonstrated by the detection of early apoptotic cells with fluorescein-labeled Annexin V and increases in cleaved caspase-3 and cleaved PARP protein levels. However, the addition of caspase inhibitor Z-VAD-FMK did not affect growth arrest or apoptosis induction. These results suggest that garcinielliptone G (5) can induce both caspase-3 activation and caspase-independent apoptosis. Therefore, garcinielliptone G (5) may be a potential candidate for acute leukemia treatment.

RA-XXV and RA-XXVI, Bicyclic Hexapeptides from Rubia cordifolia L.: Structure, Synthesis, and Conformation.

Two RA-series bicyclic hexapeptides, RA-XXV (4) and RA-XXVI (5), which have no N-methyl group at Tyr-5, were isolated from the roots of Rubia cordifolia L. Their amino acid compositions and sequences were determined by interpretation of MS, and 1D and 2D NMR data and their relative structures were elucidated by XRD analysis of 4 and RA-XXVI acetate (6). The absolute stereochemistry of 4 was established by the total synthesis of 4, and that of 5, by the chemical correlation with 4. Peptides 4 and 5 exhibited cytotoxicity toward human promyelocytic leukemia HL-60 (IC50 =0.062 and 0.066 µm, respectively) and human colonic carcinoma HCT-116 (IC50 =0.028 and 0.051 µm, respectively) cell lines. Analysis of the conformational structures of 4 and 6 in the crystalline state and those of 4 and 5 in solution revealed that the N-methyl group at Tyr-5 functions to make this series of peptides preferentially adopt the active conformation.

1O, 20O-diacetyl kamebakaurin protects against acetaminophen-induced hepatotoxicity in mice.

The present study aimed to investigate the protective effects of kamebakaurin (KA) and 1O, 20O-diacetyl kamebakaurin (Ac2KA) on acetaminophen (APAP)-induced hepatotoxicity and compare the hepatoprotective mechanisms of the two chemicals. Seven-week-old male C57BL/6J mice were orally administered KA, Ac2KA, or an ethanol/olive oil emulsion once per day for 7-days. Twenty-four hours after the final administration, the mice were fasted and then intraperitoneally injected with 450 mg/kg APAP or saline. At 16 h after injection, the mice were euthanized and blood samples were collected for plasma analysis. Pretreatment with KA and Ac2KA significantly attenuated APAP-induced hepatic injury. The protective effect of Ac2KA was stronger than that of KA. These two chemicals attenuated oxidative stress, inflammatory cytokine production, c-jun N-terminal kinase activation, and receptor-interacting protein (RIP)-3 activation. Ac2KA also decreased APAP-induced RIP-1 activation and nuclear factor kappa B (NF-κB) p65 translocation. Moreover, Ac2KA repressed mRNA expression of Cyp1a2/2e1 in the liver. Our results showed that KA and Ac2KA exerted protective effects against APAP-induced hepatotoxicity. The responsible mechanisms may be related to the chemicals' antioxidant activity and the inhibition of c-jun N-terminal kinase activation and RIP-3 activation. The effects of Ac2KA included those of KA, as well as RIP-1 inactivation, NF-κB inhibition, and Cyp inhibition.

Methyl dehydroabietate counters high fat diet-induced insulin resistance and hepatic steatosis by modulating peroxisome proliferator-activated receptor signaling in mice.

The aim of this study was to investigate the therapeutic effects of methyl dehydroabietate (mDA) on adipocyte differentiation in 3T3-L1 preadipocytes and obesity characteristics induced by high-fat diet (HFD) in mice. Adipocyte differentiation in 3T3-L1 cells was evaluated after 14 days of incubation with mDA. mDA enhanced adipocyte differentiation in 3T3-L1 cells. For the in vivo evaluation, five-week-old male C57BL/6J mice were fed HFD or normal CE-2 diet (control) for eight weeks. During the experimental period, mice were administered mDA (50 mg/kg, p.o.) as an olive oil emulsion (containing 10% ethanol), and body weights were measured weekly. At the end of the experiment, the mice were euthanized after 16 h fasting period, and plasma samples were collected. The liver, kidney, and epididymal adipose tissues were collected and weighed. It significantly decreased body weight, adipose tissue weight, and plasma levels of glucose, insulin, leptin, and pro-inflammatory cytokines compared with that in the HFD group, and markedly reduced the impairment in glucose tolerance in obese mice. Furthermore, mDA reduced HFD-induced adipocyte hypertrophy and the formation of hepatic lipid droplets. Moreover, it induced the expression of proliferator-activated receptor alpha (PPARα) in the liver and PPARγ in the adipose tissues. Our findings demonstrate that mDA reduces obesity-induced glucose and insulin tolerance by inducing PPAR expression.

Suppressive effect of kamebakaurin on acetaminophen-induced hepatotoxicity by inhibiting lipid peroxidation and inflammatory response in mice.

BACKGROUND: Kamebakaurin (KA) is an ent-kaurane diterpenoid known to have anti-inflammatory potential. In the current study, we investigated whether pretreatment with KA could ameliorate acetaminophen (APAP)-induced hepatotoxicity by inhibiting the anti-inflammatory response in mice. METHODS: Seven-week-old C57BL/6J mice were orally administered KA or olive oil emulsion for seven days. Twenty-four hours after the last KA or olive oil administration, the mice were intraperitoneally injected with 400mg/kg APAP or saline under feed deprived condition. The mice from each group were euthanized and bled for plasma analysis 24h after the injection. RESULT: APAP increased plasma levels of hepatic injury markers (i.e., alanine aminotransferase and aspartate aminotransferase), lipid peroxidation, and pro-inflammatory cytokines. Pretreatment with KA reduced the magnitude of APAP-induced increases in plasma levels of hepatic injury markers, lipid peroxidation, and inflammatory response. In addition, KA exhibited antioxidant capacity in a dose-dependent manner, with slight reactive oxygen species scavenging activity. CONCLUSION: Our results indicate that KA has the ability to protect the liver from APAP-induced hepatotoxicity, presumably by both inhibiting the inflammatory response and oxidative stress.

RA-dimer†B, a New Dimeric RA-series Cyclopeptide Incorporating Two Different Types of Cycloisodityrosine Units, from Rubia cordifolia†L.

RA-dimer B, a new cytotoxic RA-series peptide, was isolated from the roots of Rubia cordifolia L. Its structure was elucidated on the basis of spectroscopic analysis to be a dimeric cyclopeptide composed of deoxybouvardin and allo-RA-V. Those two cyclopeptide units are connected by an ether linkage between the phenolic oxygen atom of deoxybouvardin and the ϵa carbon atom of Tyr-6 of allo-RA-V. RA-dimer B was synthesized by the coupling reaction of deoxybouvardin with the boronic acid derivative of allo-RA-V, and subsequent deprotection, confirming the relative stereochemistry and establishing the absolute configuration of this peptide. RA-dimer B showed cytotoxic activity against human promyelocytic leukaemia HL-60, human colonic carcinoma HCT-116, and human renal cell carcinoma ACHN cells with IC50 values of 0.59, 0.54, and 0.74 µm, respectively.

Semisynthesis of salviandulin E analogues and their antitrypanosomal activity.

A series of analogues of salviandulin E, a rearranged neoclerodane diterpene originally isolated from Salvia leucantha (Lamiaceae), were prepared and their in vitro activity against Trypanosoma brucei brucei was evaluated with currently used therapeutic drugs as positive controls. One of the 19 compounds prepared and assayed in the present study, butanoyl 3,4-dihydrosalviandulin E analogue was found to be a possible candidate for an antitrypanosomal drug with fairly strong antitrypanosomal activity and lower cytotoxicity.

The current status of tailor-made medicine with molecular biomarkers for patients with clear cell renal cell carcinoma.

Appropriate use of multiple reliable molecular biomarkers in the right context will play a role in tailormade medicine of clear cell renal cell carcinoma (RCC) patients in the future. A total of 11,056 patients from 53 studies were included in this review. The article numbers of the each evidence levels, using the grading system defined by the Oxford Centre for Evidence-based Medicine, in 1b, 2a, 2b, and 3b were 5 (9%), 18 (34%), 29 (55%), and 1 (2%), respectively. The main goal of using biomarkers is to refine predictions of tumor progression, pharmacotherapy responsiveness, and cancer-specific and/or overall survival. Currently, carbonic anhydrase (CA9) and vascular endothelial growth factor (VEGF) in peripheral blood and p53 in tumor tissues are measured to predict metastasis, while VEGF-related proteins in peripheral blood are used to assess pharmacotherapy responsiveness with sunitinib. Furthermore, interleukin 8, osteopontin, hepatocyte growth factor, and tissue inhibitors of metalloproteinases-1 in peripheral blood enable assessment of responsiveness to pazopanib treatment. Other reliable molecular biomarkers include von Hippel­Lindau gene alteration, hypoxia-inducible factor-1a, CA9, and survivin in tumor tissues and VEGF in peripheral blood for predicting cancer-specific survival. In the future, studies should undergo external validation for developing tailored management of clear cell RCC with molecular biomarkers, since individual institutional studies lack the generalization and consistency required to maintain accuracy among different patient series.

Aza-cycloisodityrosine analogue of RA-VII, an antitumor bicyclic hexapeptide.

An aza-cycloisodityrosine analogue of RA-VII, 3, was designed and synthesized. The key aza-cycloisodityrosine unit was prepared by copper(II)-acetate-mediated intramolecular phenylamine/arylboronic acid coupling of dipeptide followed by connection with the tetrapeptide segment to afford a hexapeptide. Subsequent macrocyclization of the hexapeptide with EDC · HCl and HOOBt under dilute conditions gave 3. Analogue 3 showed significant cytotoxic activity against human promyelocytic leukemia HL-60 cells and human colon carcinoma HCT-116 cells, but its activity was weaker than that of parent peptide RA-VII (1).

Absolute structures of stemona-lactam S and tuberostemospiroline, alkaloids from stemona tuberosa.

A new alkaloid, stemona-lactam S, and a known alkaloid, tuberostemospiroline, were isolated from the roots of Stemona tuberosa LOUR. (Stemonaceae). Their structures and absolute stereochemistry were established by X-ray crystallography and vibrational circular dichroism.

Conformational folding of mycobacterial methoxy- and ketomycolic acids facilitated by α-methyl trans-cyclopropane groups rather than cis-cyclopropane units.

The oxygenated long-chain mycolic acids from many mycobacteria are characterized by the presence of mid-chain cyclopropane groups, which can have either cis-configuration or trans-configuration with an adjacent methyl branch. To determine the effect of these functional groups on mycolic acid conformation, surface pressure (π) versus mean molecular area isotherms of methoxy- (MeO-) mycolic acids (MAs) from Mycobacterium kansasii, Mycobacterium tuberculosis (Mtb) Canetti and Mtb H37Ra, and of keto-MAs from Mycobacterium avium-intracellulare complex (MAC) and Mtb H37Ra were recorded and analysed. The MeO- and keto-MAs from Mtb H37Ra, containing scarcely any trans-cyclopropyl groups, apparently took no fully folded 'W-form' conformations. Keto-MA from MAC, whose trans-cyclopropyl group content is nearly 90 %, showed a very solid W-form conformation. MeO-MAs from M. kansasii and Mtb Canetti gave stable W-form conformations at lower temperatures and surface pressures and extended conformations at higher temperatures and surface pressures; their W-form conformation was not as stable as expected from their cis-cyclopropyl group content, probably because they had a wide range of constituent homologues. Energy level calculations of cis- or α-methyl trans-cyclopropane-containing model molecules and computer simulation studies confirmed the superior folding properties of the latter functional unit. The present results were compared with those of MeO- and keto-MAs from Mtb and from M. bovis Bacillus Calmette-Guérin (BCG) reported previously. Among the oxygenated MAs, those having higher trans-cyclopropane content tended to take W-form conformations more firmly, implying that the meromycolate proximal intra-chain α-methyl trans-cyclopropane groups facilitated MA folding more than cis-cyclopropane groups.

Abietane diterpenoids and a sesquiterpene pyridine alkaloid from Euonymus lutchuensis.

Four new abietane diterpenoids (1-4), a new 9(10→20)-abeo-abietane diterpenoid (5), and a new sesquiterpene pyridine alkaloid (6) were isolated from the roots of Euonymus lutchuensis along with 19 known compounds. The structures of the new compounds were elucidated by interpretation of the spectroscopic data.

Goniolandrene A and B from Goniothalamus macrophyllus.

Goniothalamus macrophyllus (Blume) Hook. f. & Thoms. is a plant widely distributed in Malaysia. The aim of this study is to identify compounds from the roots of G. macrophyllus. The ground roots were extracted with aqueous methanol and partitioned sequentially with n-hexane, chloroform and butanol. Purification from this extracts afforded six compounds with two new compounds, namely goniolandrene-A (1), -B (2). The absolute configuration of goniolandrene B (2) was established by circular dichrosim. The compounds were cytotoxic against the P388 cells with IC50 values ranging from 0.42 to 160 µM. Goniothalamin (3) exhibited the highest inhibition of 0.42 µM.

Value of metastin receptor immunohistochemistry in predicting metastasis after radical nephrectomy for pT1 clear cell renal cell carcinoma.

KISS-1 is a metastasis-suppressor gene of human melanoma, and encodes metastin, which was identified as the ligand of a G-protein-coupled receptor (metastin receptor). The precursor protein is cleaved to 54 amino acids, which may be further truncated into carboxy-terminal fragments. Previous studies showed that lack of metastin receptor in clear cell renal cell carcinoma (RCC) is associated with tumor progression, but the prediction of metastasis in patients with pT1 clear cell RCC after radical nephrectomy is difficult. The objective of this study was to evaluate the usefulness of metastin receptor immunohistochemistry in predicting metastasis after nephrectomy for pT1 clear cell RCC. After verification of the correlation between immunostaining and mRNA expression, we evaluated the clinical value of metastin receptor immunohistochemistry. Fifty-four patients were enrolled in this study; following radical nephrectomy, seven patients were found to have lung metastasis. The sensitivity, specificity, positive predictive value, and negative predictive value with negative immunostaining of metastin receptor were 85.7, 97.6, 46.2, and 97.6 %, respectively. Metastasis-free survival rates were significantly higher in patients with positive staining (97.6 %) than in patients with negative staining (53.8 %) (P < 0.001). In univariate analysis for metastasis-free survival, negative immunostaining of metastin receptor was a significant risk factor for metastasis (P = 0.001). Furthermore, negative immunostaining of metastin receptor was an independent predictor for metastasis in multivariate analysis (hazard ratio, 3.735; 95 % CI 0.629-22.174; P = 0.002). In conclusion, our study suggests that negative expression of metastin receptor in clear cell RCC is significantly related to metastasis.

Synthesis of [Tyr-5-Ψ(CH2NMe)-Tyr-6]RA-VII, a reduced peptide bond analogue of RA-VII, an antitumor bicyclic hexapeptide.

A reduced peptide bond analogue of RA-VII, [Tyr-5-Ψ(CH(2)NMe)-Tyr-6]RA-VII (3), was designed and synthesized. The key reduced cycloisodityrosine unit was prepared by reduction of the cycloisodityrosine derived from natural RA-VII, followed by connection with the tetrapeptide segment to afford a hexapeptide. Subsequent macrocyclization of the hexapeptide with FDPP under dilute conditions gave 3. Analogue 3 showed cytotoxic activity against P-388 cells, but its activity was much weaker than that of parent peptide RA-VII.

Isolation, structure determination, and synthesis of allo-RA-V and neo-RA-V, RA-series bicyclic peptides from Rubia cordifolia L.

Two bicyclic hexapeptides, allo-RA-V (4) and neo-RA-V (5), and one cyclic hexapeptide, O-seco-RA-V (6), were isolated from the roots of Rubia cordifolia L. Their gross structures were elucidated on the basis of spectroscopic analysis and X-ray crystallography of compound 5. The absolute stereochemistry of compounds 4 and 5 were established by their total syntheses, and the absolute stereochemistry of compound 6 by chemical correlation with deoxybouvardin (3). Comparison of the 3D structures of highly active RA-VII (1) with less-active compounds 4 and 5 suggests that the orientation of the Tyr-5 and/or Tyr-6 phenyl rings plays a significant role in their biological activity. The isolation of peptides 4-6, along with compound 3, and the comparison of their structures seem to indicate that peptide 6 may be the common precursor to bicyclic peptides 3-5 in the plant.

Monoclonal antibodies from Rubia cordifolia against antitumor cyclohexapeptide deoxybouvardin and their use in immunoassay.

An immunoassay system was established for the estimation of the quantity of an antitumor cyclohexapeptide, deoxybouvardin (RA-V) from Bouvardia ternifolia (Cav.) Schlecht, Rubia cordifolia L., and R. akane Nakai (Rubiaceae). First, RA-V was converted into a protein conjugate to make it an effective antigen. In the conjugate the molecular ratio between RA-V and the carrier protein was 5.9:1. The splenocytes from the mouse immunized with the conjugate were then fused with mouse myeloma cells to produce hybridoma, secreting monoclonal antibodies (MAbs) against RA-V. Two clones were isolated, one producing MAb IgG(1) and the other MAb IgG(2b), both having a κ light chain. The resultant MAbs were evaluated for their sensitivity and cross-reactivity.

Cyclization vs. cyclization/dimerization in o-amidostilbene radical cation cascade reactions: the amide question.

The n-butyramido, isobutyramido, benzamido, and furancarboxamido functions profoundly modulate the electronics of the stilbene olefinic and NH groups and the corresponding radical cations in ways that influence the efficiency of the cyclization due presumably to conformational and stereoelectronic factors. For example, isobutyramido- stilbene undergoes FeCl(3) promoted cyclization to produce only indoline, while n-butyramidostilbene, under the same conditions, produces both indoline and bisindoline.