Synthetic analogs of green tea polyphenols as proteasome inhibitors

BACKGROUND: Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study. MATERIALS AND METHODS: GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay. RESULTS: (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells. CONCLUSIONS: Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.     

Methylation suppresses the proteasome-inhibitory function of green tea polyphenols

Under physiological conditions, biotransformation reactions, such as methylation, can modify green tea polyphenols (GTPs) and therefore limit their in vivo cancer-preventive activity. Although a recent study suggested that methylated polyphenols are less cancer-protective, the molecular basis is unknown. We previously reported that ester bond-containing GTPs, for example (-)-epigallocatechin-3-gallate [(-)-EGCG] or (-)-epicatechin-3-gallate [(-)-ECG], potently and specifically inhibit the proteasomal chymotrypsin-like activity. In this study, we hypothesize that methylated GTPs have decreased proteasome-inhibitory abilities. To test this hypothesis, methylated (-)-EGCG and (-)-ECG analogs that can be found in vivo were synthesized and studied for their structure-activity relationships (SARs) using a purified 20S proteasome. The addition of a single methyl group on (-)-EGCG or (-)-ECG led to decreased proteasome inhibition and, as the number of methyl groups increased, the inhibitory potencies further decreased. These SARs were supported by our findings from in silico docking analysis published recently. Previously, we synthesized a peracetate-protected (-)-EGCG molecule, Pro-EGCG (1), to enhance its cellular permeability and stability, and current HPLC analysis confirms conversion of Pro-EGCG (1) to (-)-EGCG in cultured human leukemic Jurkat T cells. Furthermore, in this study, peracetate-protected forms of methylated GTPs were added in intact Jurkat T cells to observe the intracellular effects of methylation. Peracetate-protected, monomethylated (-)-EGCG induced greater cellular proteasome inhibition and apoptosis than did peracetate-protected, trimethylated (-)-EGCG, consistent with the potencies of the parent methylated analogs against a purified 20S proteasome. Therefore, methylation on GTPs, under physiological conditions, could decrease their proteasome-inhibitory activity, contributing to decreased cancer-preventive effects of tea consumption.     

A potential prodrug for a green tea polyphenol proteasome inhibitor: evaluation of the peracetate ester of (-)-epigallocatechin gallate [(-)-EGCG

Green tea has been shown to have many biological effects, including effects on metabolism, angiogenesis, oxidation, and cell proliferation. Unfortunately, the most abundant green tea polyphenol (-)-epigallocatechin gallate or (-)-EGCG is very unstable in neutral or alkaline medium. This instability leads to a low bioavailability. In an attempt to enhance the stability of (-)-EGCG, we introduced peracetate protection groups on the reactive hydroxyls of (-)-EGCG (noted in text as 1). HPLC analysis shows that the protected (-)-EGCG analog is six times more stable than natural (-)-EGCG under slightly alkaline conditions. A series of bioassays show that 1 has no inhibitory activity against a purified 20S proteasome in vitro, but exhibits increased proteasome-inhibitory activity in intact leukemic cells over natural (-)-EGCG, indicating an intercellular conversion. Inhibition of cellular proteasome activity by 1 is associated with induction of cell death. Therefore, our results indicate that the protected analog 1 may function as a prodrug of the green tea polyphenol proteasome inhibitor (-)-EGCG.     

Docking studies and model development of tea polyphenol proteasome inhibitors: applications to rational drug design

Previously, we demonstrated that natural and synthetic ester bond-containing green tea polyphenols were potent and specific non-peptide proteasome inhibitors. However, the molecular mechanism of inhibition is currently unknown. Here, we report that inhibition of the chymotrypsin activity of the 20S proteasome by (-)-epigallocatechin-3-gallate (EGCG) is time-dependent and irreversible, implicating acylation of the beta5-subunit's catalytic N-terminal threonine (Thr 1). This knowledge is used, along with in silico docking experiments, to aid in the understanding of binding and inhibition. On the basis of these docking experiments, we propose that (-)-EGCG binds the chymotrypsin site in an orientation and conformation that is suitable for a nucleophilic attack by Thr 1. Consistently, the distance from the electrophilic carbonyl carbon of (-)-EGCG to the hydroxyl group of Thr 1 was measured as 3.18 A. Furthermore, the A ring of (-)-EGCG acts as a tyrosine mimic, binding to the hydrophobic S1 pocket of the beta5-subunit. In the process, the (-)-EGCG scissile bond may become strained, which could lower the activation energy for attack by the hydroxyl group of Thr 1. This model is validated by comparison of predicted and actual activities of several EGCG analogs, either naturally occurring, previously synthesized, or rationally synthesized.     

Transetherification-mediated E-ring opening and stereoselective "Red-Ox" modification of furostan

We have developed a novel E-ring opening method for furostan, and applied it to prepare D-ring modified steroids, which can be used to synthesize cephalostatin analogs.     

Synthesis and SAR of novel conformationally restricted oxazolidinones possessing gram-positive and fastidious gram-negative antibacterial activity. Part 2: Amino substitutions on heterocyclic D-ring system

A novel series of conformationally restricted oxazolidinones was synthesized, in which the heterocyclic D ring was substituted with various amino groups. Several analogs exhibited potent activity against both gram-positive and fastidious gram-negative organisms. Certain amino-substituted analogs also exhibited improved aqueous solubility compared to the corresponding un-substituted heterocyclic D-ring analogs.     

Relationship between the methylation status of dietary flavonoids and their growth-inhibitory and apoptosis-inducing activities in human cancer cells

Flavonoids are polyphenolic compounds widely distributed in the plant kingdom. Compelling research indicates that flavonoids have important roles in cancer chemoprevention and chemotherapy possibly due to biological activities that include action through anti-inflammation, free radical scavenging, modulation of survival/proliferation pathways, and inhibition of the ubiquitin-proteasome pathway. Plant polyphenols including the green tea polyphenol (-)-epigallocatechin gallate or (-)-EGCG, and the flavonoids apigenin, luteolin, quercetin, and chrysin have been shown to inhibit proteasome activity and induce apoptosis in human leukemia cells. However, biotransformation reactions to the reactive hydroxyl groups on polyphenols could reduce their biological activities. Although methylated polyphenols have been suggested to be metabolically more stable than unmethylated polyphenols, the practical use of methylated polyphenols as cancer preventative agents warrants further investigation. In the current study, methylated and unmethylated flavonoids were studied for their proteasome-inhibitory and apoptosis-inducing abilities in human leukemia HL60 cells. Methylated flavonoids displayed sustained bioavailability and inhibited cellular proliferation by arresting cells in the G(1) phase. However, they did not act as proteasome inhibitors in either an in vitro system or an in silico model and only weakly induced apoptosis. In contrast, unmethylated flavonoids exhibited inhibition of the proteasomal activity in intact HL60 cells, accumulating proteasome target proteins and inducing caspase activation and poly(ADP-ribose) polymerase cleavage. We conclude that methylated flavonoids lack potent cytotoxicity against human leukemia cells and most likely have limited ability as chemopreventive agents.     

Antiproliferative, low-calcemic, fluorinated sulfone analogs of 1alpha,25-dihydroxyvitamin D3: chemical synthesis and biological evaluation

Novel fluorinated sulfone analogs of the hormone 1alpha,25-dihydroxyvitamin D(3) have been designed and synthesized in order to study the biological effects of fluorine incorporation at the terminus of the C,D-ring side chain. Although biologically active 26,27-hexafluorinated 1alpha,25-dihydroxyvitamin D(3) analogs have been synthesized previously, this investigation reports the first successful fluorinated series in which trifluoromethyl sulfone analogs present a favorable biological profile. This study shows that two new analogs featuring incorporation of a synthetically simple single trifluoromethyl sulfone group have significantly increased antiproliferative activity while causing desirably low in vivo calciuria relative to that of calcitriol. Incorporation of additional fluorines, as in a perfluorobutyl analog, results in a loss of antiproliferative activity.     

Design, synthesis, and activity of achiral analogs of 2-quinolones and indoles as non-thiol farnesyltransferase inhibitors

Beginning with the structure of tipifarnib (1), a series of inhibitors of FTase have been synthesized by transposition of the D-ring to the imidazole and subsequent modification of the 2-quinolone motif. The compounds in the new series may be achiral and have structural features that allow for analogs that are difficult or impossible to make in the tertiary carbon-based tipifarnib series. The most potent compound (4d) is 4 times more active in vitro against FTase than tipifarnib.     

Strong inhibition of estrone-3-sulfatase activity by pregnenolone 16alpha-carbonitrile but not by several analogs lacking a 16alpha-nitrile group

In recent years, development of potent inhibitors for estrogen sulfatases has become an actively pursued strategy for chemoprevention and/or chemotherapy of estrogen-dependent human breast cancers. We report here our findings that pregnenolone 16alpha-carbonitrile (PCN) is a potent inhibitor of estrone-3-sulfatase activity of rats and also humans. PCN inhibited in a concentration-dependent manner the desulfation of estrone-3-sulfate catalyzed by liver microsomal and nuclear fractions of female Sprague-Dawley rats. The inhibition of estrone-3-sulfatase activity in these two subcellular fractions showed a biphasic pattern, with a highly sensitive phase seen at 78 nM to 1.25 microm of PCN followed by a markedly less-sensitive phase at > 2.5 microm of PCN. Interestingly, several of PCN's structural analogs without a 16alpha-nitrile group showed little or no inhibitory effect on rat liver microsomal E(1)-3-sulfatase activity. Double-reciprocal analysis showed that the inhibition of rat liver microsomal E(1)-3-sulfatase activity by PCN was essentially competitive in nature. When microsomes from six human term placentas were tested for their E(1)-3-sulfatase activity, PCN showed a similar biphasic inhibition of placental E(1)-3-sulfatase. Likewise, several of its structural analogs showed little or no inhibitory effect on placental E(1)-3-sulfatase activity. Computational analysis of the D-ring structure of PCN and other structurally similar analogs used in the study suggests that the potent sulfatase-inhibiting activity of PCN may be partly due to its unique steric orientation and size of the 16alpha-nitrile group. This knowledge may be useful for the rational design of more potent steroidal inhibitors of E(1)-3-sulfatase by introducing an additional nitrile group to their C16alpha-position.     

Synthesis and evaluation of novel podophyllotoxin analogs

Because prior studies have shown inconsistency between structure-activity relationships for podophyllotoxin derivatives as topoisomerase II inhibitors and cytotoxic agents, eight novel podophyllotoxin analogs were synthesized to further explore the effects of structural variations on both A and D rings on activity. The new compounds contain a 4,5-dimethoxy substituted A ring and opened D-ring variants and were prepared by appropriate functional and stereochemical operations at the methylenedioxy group, C7, C8, and C8'. Four compounds (15, 18, 21 and 22) demonstrated noticeable inhibitory activity against A549, DU145, KB and KBvin tumor cells, and the most active compound 18 showed IC(50) values less than 10 μg/mL.     

过氧化心肌线粒体产生的脂类自由基及（－）－EGCG的抑制作用

本文利用ＥＳＲ技术研究了心肌线粒体酶修饰下的脂质过氧化和脂类自由基,以及（－）－ＥＧＣＧ的抑制作用。结果表明：４－ＰＯＢＮ能捕集ｌｉｐｏｘｙｇｅｎｇｓｅ诱发心肌线粒体产生的自由基,得到６条线谱的脂类自由基和４条线谱捕集物,（－）－ＥＧＣＧ对该体系中使用的１ｉｐｏｘｙｅｎａｓｅ活性无影响,对所产生的自由基有明显的清除作用,并呈量效关系。对脂质过氧化的抑制作用,在本试验浓度范围内随浓度增加变化不大,最大抑制率约２０％。     

Synthesis and antimitotic activity of novel 2-methoxyestradiol analogs--Part II

The syntheses and antimitotic activity of several novel 18a-homo-analogs of 2-methoxyestradiol are described. Structural modifications of the parent 2-methoxy-18a-homoestradiol include introduction of unsaturation in the D-ring and methylation of the 17-OH. Of seven analogs synthesized, one has demonstrated superior biological activities compared to 2-methoxyestradiol. The relationship between biological activity and the conformational preference of the 13-ethyl group as determined by computational analysis is discussed.     

Inhibitory effects of green tea catechins on the activity of human matrix metalloproteinase 7 (matrilysin)

Inhibitory effects of green tea catechins and their derivatives on the matrilysin-catalyzed hydrolysis of a synthetic substrate, (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR], were examined. The 10 catechins examined were classified into three groups according to their inhibition potency. Catechins with a galloyl group at the 3 position, including a major component of green tea catechin, (-)-epigallo-3-catechin gallate [(-)-EGCG], were the most potent inhibitors and inhibited matrilysin in a non-competitive manner with K(i) values of 0.47-1.65 micro M. The inhibitory potency of (-)-EGCG was not influenced by the presence of an inhibitor, ZnCl(2), suggesting that the inhibitions of matrilysin by (-)-EGCG and by ZnCl(2) might be independent of each other. The inhibitory effects of green tea catechins suggest that a high intake of green tea might be effective for the prevention of tumor metastasis and invasion in which matrilysin is concerned.     

Synthetic and biosynthetic studies of porphyrins: III. Structures of the intermediates between uroporphyrinogen-III and coproporphyrinogen-III: Synthesis of fourteen heptacarboxylic,hexacarboxylic, and pentacarboxylic porphyrins related to uroporphyrin-III

Four heptacarboxylic, six hexacarboxylic, and four pentacarboxylic porphyrins related to uroporphyrin-III by decarboxylation of one, two, or three of the acetic acid side chains have been synthesised as their methyl esters by application of the MacDonald or b-oxobilane methods, as appropriate. Comparison (mixed mp, “mixed” nmr spectra, and hplc) of the synthetic materials with the methyl esters of hepta-, hexa-, and pentacarboxylic porphyrins isolated from natural sources showed that the structures of the latter corresponded to the D-ring methyl, the DA-dimethyl, and the DAB-trimethyl analogs of uroporphyrin-III. Because the naturally occurring porphyrins arise by oxidation of intermediate porphyrinogens, we conclude that the enzymic decarboxylation of uroporphyrinogen-III to coproporphyrinogen-III takes place in a preferred sequential clockwise fashion (both in normal and abnormal metabolism) starting with the acetic acid moiety on the D-ring and followed by those on the A, B, and C rings.     

D-ring modified estrone derivatives as novel potent inhibitors of steroid sulfatase

A series of novel D-ring modified derivatives of estrone was synthesized and tested as inhibitors of steroid sulfatase (STS). The steroidal D-ring was cleaved via an iodoform reaction and thermal condensation of the resulting marrianolic acid derivative gave 16,17-seco-estra-1,3,5(10)-triene-16,17-imide derivatives, where a piperidinedione moiety is in place of the D-ring. This synthetic approach was found to give a higher overall yield than the literature method of Beckmann rearrangement. A range of alkyl side chains have been introduced on the nitrogen atom of the imido-ring and the corresponding 3-O-sulfamates synthesized. The new D-ring modified estrone derivatives bearing a propyl (39) and a 1-pyridin-3-ylmethyl (46) moiety had IC(50) values of 1 nM when tested in placental microsomes for the inhibition of STS. These compounds are therefore up to 18-fold more potent than EMATE, the very first highly potent irreversible steroidal STS inhibitor.     

Strigolactone analogs derived from ketones using a working model for germination stimulants as a blueprint

Strigolactones are important signaling compounds in the plant kingdom. Here we focus on their germination stimulatory effect on seeds of the parasitic weeds Striga and Orobanche spp. and more particularly on the design and synthesis of new active strigolactone analogs derived from simple cyclic ketones. New analogs derived from 1-indanone, 1-tetralone, cyclopentanone, cyclohexanone and a series of substituted cyclohexanones (including carvone and pulegone) are prepared by formylation of the ketones with ethyl formate followed by coupling with a halo butenolide. Both enantiomers of the analog derived from 1-tetralone have been prepared by employing a homochiral synthon for the coupling reaction. For three other strigolactone analogs the antipodes have been obtained by chromatography on a chiral column. All analogs have an appreciable germinating activity towards seeds of Striga hermomonthica and Orobanche crenata and O. cernua. Stereoisomers having the same configuration at the D-ring as in naturally occurring strigol have a higher stimulatory effect than the corresponding antipodes. The analogs obtained from 1-indanone and 1-tetralone have an activity comparable with that of the well known stimulant GR 24. Analogs derived from 2-phenyl-cylohexanone, carvone and pulegone also have a good germinating response. The results show that the working model for designing new bioactive strigolactones is applicable.     

Design and synthesis of novel D-ring fused steroidal heterocycles

Using dehydroepiandrosterone as the starting material, we have synthesized a series of steroid analogs possessing a D-ring fused with heterocycles which are pyridine, imidazo [2,1-b]thiazoles or substituted thiazole imines. All the final structures are first reported and identified by NMR and MS spectroscopys, the yields of these products are moderate to good and the reaction conditions are mild. The cytotoxicity of the synthesized compounds against EC-109(human esophageal carcinoma), EC-9706(human esophageal carcinoma), MGC-803(human gastric carcinoma) were investigated.     

Site-directed mutations of human growth hormone that selectively modify its lactogenic activity and binding properties

Two novel analogs of human (h) GH, 1) Des-7-hGH (Arg8Met, Asp11Ala) in which the Arg8 was substituted by Met and Asp11 by Ala, and 2) bovine (b) GH/hGH hybrid II (MetAla 1-13/14-191, Ala11Asp) composed of 13 N-terminal amino acid of bGH and elongated by two amino acids (Met-Ala-1-13) and 14-191 amino acids of hGH, were constructed and expressed in Escherichia coli. CD spectra indicated that the alpha-helix content of the purified proteins was similar to that of the native hormone. Both analogs retained their full ability to stimulate the proliferation of Nb2 lymphoma cells, and their binding to the lactogen receptors in homogenate of Nb2 cells and in microsomal fraction from bovine lactating mammary gland was only slightly reduced. However, their ability to bind to the somatogen receptors in intact IM-9 lymphocytes and bovine liver was reduced by 7- to 11-fold (bGH/hGH hybrid II) and 20- to 30-fold (Des-7-hGH). Both analogs were able to down-regulate the respective lactogen and somatogen receptors in intact Nb2 and IM-9 cells. The galactopoietic activity of both analogs in the lactating bovine mammary explants bioassay was almost completely abolished, and the bGH/hGH hybrid II exhibited a remarkable antagonistic activity. These results further indicate that the lactogen receptors in different species or organs are not identical. We have shown that the new recombinant analogs of hGH that recognize both somatogen and lactogen receptors but have modified postreceptor effects are helpful in elucidating these differences.