Stereoselective Synthesis of Natural and Non-natural Thomsen-nouveau Antigens and Hydrazide Derivatives.

A selective glycosylation strategy enabling access to all stereochemical combinations of tumor associated Thomsen-nouveau (Tn) antigen, D-GalNAc-O-Ser/Thr, has been developed. The key component for selectivity is the phthalimide-protected D- or L-amino acid acceptors which allow access to α- or ß-anomers in excellent yields (72-96%) and selectivity (∼100%) when appropriate C-2 substitution is installed. The glycoamino acid intermediates were divergently converted to Tn-based carboxylates or to hydrazides by tandem Pd-C debenzylation followed by treatment with hydrazine hydrate or hydrazine hydrate treatment alone.

Dihydromethysticin from kava blocks tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis and differentially reduces DNA damage in A/J mice.

We have previously shown that kava and its flavokavain-free Fraction B completely blocked 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice with a preferential reduction in NNK-induced O (6)-methylguanine (O (6)-mG). In this study, we first identified natural (+)-dihydromethysticin (DHM) as a lead compound through evaluating the in vivo efficacy of five major compounds in Fraction B on reducing O (6)-mG in lung tissues. (+)-DHM demonstrated outstanding chemopreventive activity against NNK-induced lung tumorigenesis in A/J mice with 97% reduction of adenoma multiplicity at a dose of 0.05mg/g of diet (50 ppm). Synthetic (±)-DHM was equally effective as the natural (+)-DHM in these bioassays while a structurally similar analog, (+)-dihydrokavain (DHK), was completely inactive, revealing a sharp in vivo structure-activity relationship. Analyses of an expanded panel of NNK-induced DNA adducts revealed that DHM reduced a subset of DNA adducts in lung tissues derived from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the active metabolite of NNK). Preliminary 17-week safety studies of DHM in A/J mice at a dose of 0.5mg/g of diet (at least 10× its minimum effective dose) revealed no adverse effects, suggesting that DHM is likely free of kava's hepatotoxic risk. These results demonstrate the outstanding efficacy and promising safety margin of DHM in preventing NNK-induced lung tumorigenesis in A/J mice, with a unique mechanism of action and high target specificity.

Kava blocks 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in association with reducing O6-methylguanine DNA adduct in A/J mice.

We previously reported the chemopreventive potential of kava against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- and benzo(a)pyrene (BaP)-induced lung tumorigenesis in A/J mice during the initiation and postinitiation stages. In this study, we investigated the tumorigenesis-stage specificity of kava, the potential active compounds, and the underlying mechanisms in NNK-induced lung tumorigenesis in A/J mice. In the first experiment, NNK-treated mice were given diets containing kava at a dose of 5 mg/g of diet during different periods. Kava treatments covering the initiation stage reduced the multiplicity of lung adenomas by approximately 99%. A minimum effective dose is yet to be defined because kava at two lower dosages (2.5 and 1.25 mg/g of diet) were equally effective as 5 mg/g of diet in completely inhibiting lung adenoma formation. Daily gavage of kava (one before, during, and after NNK treatment) completely blocked lung adenoma formation as well. Kavalactone-enriched fraction B fully recapitulated kava's chemopreventive efficacy, whereas kavalactone-free fractions A and C were much less effective. Mechanistically, kava and fraction B reduced NNK-induced DNA damage in lung tissues with a unique and preferential reduction in O(6)-methylguanine (O(6)-mG), the highly tumorigenic DNA damage by NNK, correlating and predictive of efficacy on blocking lung adenoma formation. Taken together, these results demonstrate the outstanding efficacy of kava in preventing NNK-induced lung tumorigenesis in A/J mice with high selectivity for the initiation stage in association with the reduction of O(6)-mG adduct in DNA. They also establish the knowledge basis for the identification of the active compound(s) in kava.

Reduction in colon cancer risk by consumption of kava or kava fractions in carcinogen-treated rats.

Epidemiological studies suggest that kava reduces colon cancer risk. However, no experimental studies of the chemopreventive properties of kava toward colon cancer have been reported. Further, there are concerns regarding hepatotoxicity of kava. The goal of this study was to determine whether kava consumption reduces markers of colon cancer in an animal model and to study the safety of kava. An ethanolic extract and polar and nonpolar fractions of the kava extract were fed to rats for 12 days prior to, during, and after administration of dimethylhydrazine, a colon-specific carcinogen. After 14 wk, rats fed the nonpolar extract had a significant reduction in precancerous lesions [aberrant crypt (AC) foci (ACF)] as well as large (≥ 4 AC/ACF) sialomucin-only expressing foci, an indicator of greater tumorigenic potential, compared to the control group. Groups fed the ethanolic extract and polar kava fraction trended toward reductions in ACF and large sialomucin-only expressing foci. The combined kava groups had significantly fewer total AC, ACF, large ACF, and large sialomucin-only expressing foci compared to the control group. Histological examination found no hepatic lesions in animals consuming the kava diets, suggesting that kava is safe to consume. Our results support that kava may reduce colon cancer risk.

Quantitative determination of decursin, decursinol angelate, and decursinol in mouse plasma and tumor tissue using liquid-liquid extraction and HPLC.

The pyranocoumarin compound decursin and its isomer decursinol angelate (DA) are the major hydrophobic phytochemicals in the root of Angelica gigas Nakai (AGN, Korean Angelica), a major traditional medicinal herb. The ethanol extract of AGN and especially the purified decursin and DA have been shown to exhibit antitumor activities by our collaborative team and others. Although decursinol has been identified as a major hydrolysis metabolite of decursin and DA in vivo in previous pharmacokinetic studies with mouse and rat, other recently published results sharply disputed this conclusion. In this study, we set up a practical method for the concurrent analysis of decursin, DA, and decursinol in mouse plasma and tumor tissues by liquid-liquid extraction and HPLC-UV and applied the method to several animal experiments. Plasma or tumor homogenate was extracted directly with ethyl acetate. The extraction efficiency for decursin/DA (quantitated together) and decursinol was between 82-95 % in both mouse plasma and tumor homogenate. The lower limit of quantitation (LLOQ) was approximately 0.25 µg/mL for decursin/DA and 0.2 µg/mL for decursinol in mouse plasma. In a pilot pharmacokinetic study, male C57BL/6 mice were given a single dose of 4.8 mg decursin/DA mixture (~240 mg/kg) per mouse either by oral gavage or intraperitoneal injection. Maximum plasma concentrations for decursin/DA and decursinol were 11.2 and 79.7 µg/mL, respectively, when decursin/DA was administered via intraperitoneal injection, and 0.54 and 14.9 µg/mL via oral gavage. Decursin/DA and decursinol contents in the tumor tissues from nude mouse xenografts correlated very well with those in plasma. Overall, our results confirm the conclusion that the majority of decursin/DA hydrolyze to decursinol in rodent models with a tiny fraction remaining as the intact compounds administered.

A synthetic decursin analog with increased in vivo stability suppresses androgen receptor signaling in vitro and in vivo.

Targeting androgen receptor (AR) signaling with agents distinct from current antagonist drugs remains a rational approach to the prevention and treatment of prostate cancer (PCa). Our previous studies have shown that decursin and isomer decursinol angelate (DA), isolated from the Korean medicinal herb Angelica gigas Nakai, interrupt AR signaling and possess anti-PCa activities in vitro. In the LNCaP PCa cell model, these pyranoccoumarin compounds exhibit properties distinct from currently used antagonists (e.g., Casodex). However, both are rapidly de-esterified to decursinol, a partial AR agonist. We report here that a synthetic decursin analog, decursinol phenylthiocarbamate (DPTC), has greater in vivo stability than the parent compounds. DPTC-decursinol conversion was undetectable in mice. Furthermore, in LNCaP cells, DPTC decreased prostate specific antigen (PSA) expression, down-regulated AR abundance and mRNA and inhibited AR nuclear translocation. The effect of DPTC on AR and PSA mRNA and protein abundance was also observed in VCaP cells expressing wild type AR. DPTC inhibited growth of both PCa cell lines through G(1) cell cycle arrest and apoptosis, as did decursin and DA. Furthermore, i.p. administration of DPTC for 3 weeks suppressed the expression of AR target genes probasin and Nkx3.1 in mouse prostate glands. Overall, our data suggest that DPTC represents a prototype lead compound for development of in vivo stable and active novel decursin analogs for the prevention or therapy of PCa.

Preparation of penta-O-galloyl-ß-D-glucose from tannic acid and plasma pharmacokinetic analyses by liquid-liquid extraction and reverse-phase HPLC.

The gallotannin penta-O-galloyl-beta-D-glucose (PGG) has many biological activities including in vivo anti-cancer efficacy. We present in this paper a scaled-up protocol for its preparation in high purity from tannic acid by acidic methanolysis with typical yield of 15%. We also describe a method for the analysis of PGG in mouse plasma by HPLC and its application in preliminary pharmacokinetic studies. A liquid-liquid extraction (LLE) protocol was optimized for the extraction of PGG from mouse plasma. The extraction efficiency for PGG at 1 µg/mL in mouse plasma was 70.0±1.3% (n=5). The limit of detection (LOD) for PGG was approximately 0.2 µg/mL. Preliminary pharmacokinetic parameters of PGG following a single i.p. injection with 5% ethanol/saline vehicle in mice were established. The peak plasma PGG concentrations (C(max)) were approximately 3-4 µM at a dose of 0.5 mg per mouse (∼20 mg/kg) at 2 h post-injection (T(max)).

Penta-O-galloyl-beta-D-glucose induces G1 arrest and DNA replicative S-phase arrest independently of cyclin-dependent kinase inhibitor 1A, cyclin-dependent kinase inhibitor 1B and P53 in human breast cancer cells and is orally active against triple negative xenograft growth.

INTRODUCTION: Natural herbal compounds with novel actions different from existing breast cancer (BCa) treatment modalities are attractive for improving therapeutic efficacy and safety. We have recently shown that penta-1,2,3,4,6-O-galloyl-ß-D-glucose (PGG) induced S-phase arrest in prostate cancer (PCa) cells through inhibiting DNA replicative synthesis and G(1) arrest, in addition to inducing cell death at higher levels of exposure. We and others have shown that PGG through intraperitoneal (i.p.) injection exerts a strong in vivo growth suppression of human PCa xenograft models in athymic nude mice. This study aims to test the hypothesis that the novel targeting actions of PGG are applicable to BCa cells, especially those lacking proven druggable targets. METHODS: Mono-layer cell culture models of p53-wild type estrogen receptor (ER)-dependent MCF-7 BCa cells and p53-mutant ER-/progesterone receptor (PR)- and Her2-regular (triple-negative) MDA-MB-231 BCa were exposed to PGG for a comprehensive investigation of cellular consequences and molecular targets/mediators. To test the in vivo efficacy, female athymic mice inoculated with MDA-MB-231 xenograft were treated with 20mg PGG/kg body weight by daily gavage starting 4 days after cancer cell inoculation. RESULTS: Exposure to PGG induced S-phase arrest in both cell lines as indicated by the lack of 5-bromo2'-deoxy-uridine (BrdU) incorporation into S-phase cells as well as G(1) arrest. Higher levels of PGG induced more caspase-mediated apoptosis in MCF-7, in strong association with induction of P53 Ser(15) phosphorylation, than in MDA-MB-231 cells. The cell cycle arrests were achieved without an induction of cyclin dependent kinase (CDK) inhibitory proteins P21(Cip1) and P27(Kip1). PGG treatment led to decreased cyclin D1 in both cell lines and over-expressing cyclin D1 attenuated G(1) arrest and hastened S arrest. In serum-starvation synchronized MCF-7 cells, down-regulation of cyclin D1 was associated with de-phosphorylation of retinoblastoma (Rb) protein by PGG shortly before G(1)-S transition. In vivo, oral administration of PGG led to a greater than 60% inhibition of MDA-MB231 xenograft growth without adverse effect on host body weight. CONCLUSIONS: Our in vitro and in vivo data support PGG as a potential drug candidate for breast cancer with novel targeting actions, especially for a triple negative BCa xenograft model.

Identification of methysticin as a potent and non-toxic NF-kappaB inhibitor from kava, potentially responsible for kava's chemopreventive activity.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays an essential role in cancer development. The results of our recent chemopreventive study demonstrate that kava, a beverage in the South Pacific Islands, suppresses NF-kappaB activation in lung adenoma tissues, potentially a mechanism responsible for kava's chemopreventive activity. Methysticin is identified as a potent NF-kappaB inhibitor in kava with minimum toxicity. Other kava constituents, including four kavalactones of similar structures to methysticin, demonstrate minimum activities in inhibiting NF-kappaB.

Efforts towards the development of new antitubercular agents: potential for thiolactomycin based compounds.

Development of new chemotherapeutic drugs is the need of the hour to improve tuberculosis control, particularly in the developing world. In the last fourty years no new compound has been brought to the market for the treatment of tuberculosis. However, in recent years there is an enhanced activity in the research and development of new drugs for TB. Some compounds are presently in clinical development, while others are being investigated pre-clinically in an attempt to explore new molecules for the target based treatment of TB. Simultaneously some new targets are being identified and validated for their practical usefulness. Structures based on thiolactomycin could have considerable potential in the development of target based anti-TB agents. The present review provides an overview of the drugs that are being clinically used and the compounds that are in advanced stages of clinical as well as preclinical studies. We have also attempted to highlight the efforts that are being made in the development of new molecules based on thiolactomycin as lead compound, including studies from this laboratory.

Antitubercular agents. Part 2: new thiolactomycin analogues active against Mycobacterium tuberculosis.

Structurally modified analogues of naturally occurring antibiotic thiolactomycin, substituted at 4-position of the thiolactone ring have been prepared and evaluated for their antitubercular activity. Some of the compounds have exhibited potential activity against Mycobacterium tuberculosis.