Design and Synthesis of Cajanine Analogues against Hepatitis C Virus through Down-Regulating Host Chondroitin Sulfate N-Acetylgalactosaminyltransferase 1.

There still remains a need to develop new anti-HCV agents with distinct mechanism of action (MOA) due to the occurrence of resistance to direct-acting antiviral agents (DAAs). Cajanine, a stilbenic component isolated from Cajanus cajan L., was identified as a potent HCV inhibitor by phenotypic screening in this work (EC50 = 3.17 ± 0.75 µM). The intensive structure optimization provided significant insights into the structure-activity relationships. Furthermore, the MOA study revealed that cajanine inhibited HCV replications via down-regulating a cellular protein chondroitin sulfate N-acetylgalactosaminyltransferase 1. In consistency with this host-targeting mechanism, cajanine showed the similar magnitude of inhibitory activity against both drug-resistant and wild-type HCV and synergistically inhibited HCV replication with approved DAAs. Taken together, our study not only presented cajanine derivatives as a novel class of anti-HCV agents but also discovered a promising anti-HCV target to combat drug resistance.

Diethylstilbestrol-scaffold-based pregnane X receptor modulators.

Due to its function as a regulator of drug-metabolizing enzymes and transporters, pregnane X receptor (PXR) represents an important factor involved in drug metabolism. In this work, we describe the discovery of diethylstilbestrol-based PXR modulators, which were designed from marine sulfated steroids with PXR agonistic activity, solomonsterols A and B, and our recently reported bazedoxifene scaffold-derived PXR antagonists. The methylated diethylstilbestrol derivative 1 displayed potent PXR agonistic activity with an EC50 value of 10.5 µM, whereas compounds 3, 4 and 6 (IC50 for 6 = 27.4 µM) and diethylstilbestrol (2) itself (IC50 = 14.6 µM) exhibited PXR antagonistic effects in HepG2 cells. The PXR modulatory effects of the synthesized diethylstilbestrol derivatives were further confirmed by the induction of PXR-regulated CYP3A4 expression with compound 1, as well as by the inhibition of the rifaximin-promoted up-regulation of CYP3A4 expression with 2 and its derivative 6.

Demonstrating the importance of polymer-conjugate conformation in solution on its therapeutic output: Diethylstilbestrol (DES)-polyacetals as prostate cancer treatment.

The design of improved polymeric carriers to be used in the next generation of polymer therapeutics is an ongoing challenge. Biodegradable systems present potential advantages regarding safety benefit apart from the possibility to use higher molecular weight (Mw) carriers allowing PK optimization, by exploiting the enhanced permeability and retention (EPR)-mediated tumor targeting. Within this context, we previously designed pH-responsive polyacetalic systems, tert-polymers, where a drug with the adequate diol-functionality was incorporated within the polymer mainchain. The synthetic, non-steroidal estrogen, diethylstilboestrol (DES) clinically used for the treatment of advanced prostate cancer was chosen as drug. In order to improve the properties of this tert-polymer, novel polyacetalic systems as block-co-polymers, with more defined structure have been obtained. This second generation polyacetals allowed higher drug capacity than the tert-polymer, a biphasic DES release profile at acidic pH and due to its controlled amphiphilic character readily formed micelle-like structures in solution. These features result in an enhancement of conjugate therapeutic value in selected prostate cancer cell models. Exhaustive physico-chemical characterization focusing on nanoconjugate solution behavior and using advanced techniques, such as, pulsed-gradient spin-echo NMR (PGSE-NMR) and small-angle neutron scattering (SANS), has been carried out in order to demonstrate this hypothesis. Clear evidence of significantly different conformation in solution has been obtained for both polyacetals. These results demonstrate that an adequate control on molecular or supramolecular conformation in solution with polymer therapeutics is crucial in order to achieve the desired therapeutic output.

Synthesis of 3'-methoxy-E-diethylstilbestrol and its analogs as tumor angiogenesis inhibitors.

3'-Methoxy-E-diethylstilbestrol (2), with the structural and original similarities to 2-methoxyestradiol (2-ME2, 1), was synthesized and screened against HUVEC and a series of human cancer cell lines including RL95-2, SKOV-3, MCF-7 and T-47D in vitro. The configuration of the title compound was determined via the single crystal X-ray diffraction of its benzoyl-ester derivative (10). The fact that 3'-methoxy-E-diethylstilbestrol and its analogues (8 and 11) showed potential antiangiogenesis and anti-tumor activities at a close level, whereas its ester derivative (10) did not display any cytotoxic activities on all the screening cell lines indicated that the core scaffold of 3'-methoxy-3,4-diphenylhexane and the exposed hydroxyl-groups in the structures are essential pharmacophores for their anti-tumor activities.

Discovery of a small molecule PDI inhibitor that inhibits reduction of HIV-1 envelope glycoprotein gp120.

Protein disulfide isomerase (PDI) is a promiscuous protein with multifunctional properties. PDI mediates proper protein folding by oxidation or isomerization and disrupts disulfide bonds by reduction. The entry of HIV-1 into cells is facilitated by the PDI-catalyzed reductive cleavage of disulfide bonds in gp120. PDI is regarded as a potential drug target because of its reduction activity. We screened a chemical library of natural products for PDI-specific inhibitors in a high-throughput fashion and identified the natural compound juniferdin as the most potent inhibitor of PDI. Derivatives of juniferdin were synthesized, with compound 13 showing inhibitory activities comparable to those of juniferdin but reduced cytotoxicity. Both juniferdin and compound 13 inhibited PDI reductase activity in a dose-dependent manner, with IC(50) values of 156 and 167 nM, respectively. Our results also indicated that juniferdin and compound 13 exert their inhibitory activities specifically on PDI but do not significantly inhibit homologues of this protein family. Moreover, we found that both compounds can inhibit PDI-mediated reduction of HIV-1 envelope glycoprotein gp120.

Methoxylated isoflavones, cajanin and isoformononetin, have non-estrogenic bone forming effect via differential mitogen activated protein kinase (MAPK) signaling.

Following a lead obtained from stem-bark extract of Butea monosperma, two structurally related methoxyisoflavones; cajanin and isoformononetin were studied for their effects in osteoblasts. Cajanin had strong mitogenic as well as differentiation-promoting effects on osteoblasts that involved subsequent activation of MEK-Erk and Akt pathways. On the other hand, isoformononetin exhibited potent anti-apoptotic effect in addition to promoting osteoblast differentiation that involved parallel activation of MEK-Erk and Akt pathways. Unlike genistein or daidzein, none of these two compounds appear to act via estrogen receptors in osteoblast. Once daily oral (by gavage) treatment for 30 consecutive days was given to recently weaned female Sprague-Dawley rats with each of these compounds at 10.0 mg kg(-1) day(-1) dose. Cajanin increased bone mineral density (BMD) at all skeletal sites studied, bone biomechanical strength, mineral apposition rate (MAR) and bone formation rate (BFR), compared with control. BMD levels at various anatomic positions were also increased with isoformononetin compared with control however, its effect was less potent than cajanin. Isoformononetin had no effect on the parameters of bone biomechanical strength although it enhanced MAR and BFR compared with control. Isoformononetin had very mild uterotrophic effect, whereas cajanin was devoid of any such effect. Our data suggest that cajanin is more potent than isoformononetin in accelerating peak bone mass achievement. To the best of our knowledge, this work represents the first attempt to elucidate structure-activity relationship between the two methoxylated isoflavones regarding their effects in osteoblasts and bone formation.

Polyacetal-diethylstilboestrol: a polymeric drug designed for pH-triggered activation.

Those polymer anticancer-drug conjugates currently undergoing clinical evaluation have a tripartite structure; a water-soluble polymer, an anticancer agent and a pendant linker. To simplify the construct it would be attractive to develop anticancer polymer therapeutics that contain the bioactive agent as an integral part of the polymer backbone. The aim of this study was to utilise the reaction between a divinyl ethers and diols, to synthesise polyacetals incorporating a drug with bis-hydroxyl functionality into the polymer backbone. Degradation of the polymer backbone in the acidic environment of the lysosome or the extracellular fluid of some tumours would then trigger drug release eliminating the need for a biodegradable linker. A tert-polymerisation approach was used to incorporate non-steroidal oestrogen diethylstilboestrol (DES) into the mainchain of water-soluble polyacetals synthesised using as co-monomer PEG of Mw 2900 or 3400 g/mol. When PEG2900 was used the resultant polymer had a Mw of 18,900 g/mol, a Mw/Mn of 1.9 and a DES loading 4.3 wt.%. With PEG3400 the polymer Mw was 43,000 g/mol, Mw/Mn=1.8 and it had a DES loading 4.7 wt.%. 1H-NMR confirmed the presence of two distinct sets of acetal peaks, which correspond to the two possible mainchain acetals; from PEG at 1.25-1.3(d) and 4.7-4.8(q) ppm and from DES at 1.55-1.6(d) and 5.4-5.5(q) ppm. These were consistent with the acetal signals observed for the non-water-soluble co-polymer DES: tri(ethylene glycol) divinyl ether (TEGDVE) (1 : 2, Mw=6859 g/mol, Mw/Mn=1.3). When evaluated in vitro, the DES-polyacetal displayed greater cytotoxicity than DES against human and murine tumour cell lines (IC50=48 and 420 microg/ml against MCF-7 human breast cancer cells and IC50=97 and 560 microg/ml against B16F10 murine melanoma cells, respectively). These polymers showed no significant haemolysis at concentrations up to 20 mg/ml confirming suitability for further in vivo evaluation. An enhanced rate of hydrolytic degradation of the polymer backbone was seen at pH 5.5, (65% trans-DES released in 96 h), compared to pH 7.4 (4% trans-DES released in 96 h). These bioresponsive DES-polyacetals tert-polymers are the first water-soluble anticancer polymeric drugs designed for acidic pH-triggered release of a drug incorporated into the polymer mainchain. Their in vitro characteristics suggest further in vivo evaluation is warranted.

Large-scale synthesis of the catechol metabolites of diethylstilbestrol and hexestrol.

Diethylstilbestrol (DES) and hexestrol (HES) are carcinogenic synthetic estrogens. The major metabolites of these compounds are their catechol derivatives, 3'-OH-DES and 3'-OH-HES. Oxidation of these metabolites leads to the electrophilic quinones, which are presumably involved in the tumor-initiating process. A synthetic route based on the McMurry coupling reaction was developed for the synthesis of 3'-OH-DES. Using commercially inexpensive starting materials, this compound was synthesized in four steps, and the cis and trans isomers were separated and identified. Following the same synthetic route, 3'-OH-HES was synthesized in five steps.

Preparation of the N-(4'-hydroxy-[3'-125I]iodophenethyl)-6-(4-O-diethylstilbestryl)hexanam ide for diethylstilbestrol radioimmunoassays.

The synthesis of a radioiodinated diethylstilbestrol (DES) derivative is described. This derivative was prepared by coupling the previously synthesized active ester of 6-(4-O-diethylstilbestryl)hexanoic acid with mono-[125I]iodotyramine in dry tetrahydrofuran (20 to 22 C, 16 hours). The mono-[125I]iodotyramine was prepared using a chloramine-T method and purified by paper electrophoresis. The final product, N-(4'-hydroxy-[3'-125I]iodophenethyl)-6-(4-O- diethylstilbestryl)hexanamide, was separated by thin-layer chromatography (cyclohexane/ethanol/NH4OH 2.5 N/acetone; 40:50:5:20, v/v/v/v); it was stable for 2 months in ethanol at 4 C and had a specific activity higher than 540 Ci/mmol. The [125I]DES amide synthesized was found to retain the immunoreactivity of DES, since it competed with [3H]DES or DES in an in vitro radioimmunoassay system for the binding sites of a rabbit anti-DES antibody; thus, it seems to be capable of replacing the tritiated tracer used so far in DES radioimmunoassays.

Diethylstilbestrol-linked cytotoxic agents: synthesis and binding affinity for estrogen receptors.

The syntheses of diethylstilbestrol derivatives with a C4 side chain at the double bond bearing various functional and potentially alkylating groups (9-25, 38-40, 43, 44) as well as the coupling product with daunorubicin (41) are described. Derivatives with free phenolic groups show easy isomerization to (Z)-stilbenes and styrenes, which could be minimized with silyl protecting groups. Estrogen receptor binding is decreased by polar groups such as carboxylic acids (10) as well as sterically demanding substituents.

Peroxidase-mediated binding of diethylstilbestrol analogs to DNA in vitro: a possible role for a phenoxy radical.

In order to investigate the role of peroxidase-mediated metabolic activation in the mechanism of carcinogenicity of diethylstilbestrol (DES), a series of 14C-labelled analogs of DES was synthesized and their binding to DNA upon oxidation by peroxidases from horseradish or mouse uterus was studied in vitro. The compounds chosen for this study were the erythro and threo form of hexestrol (HES), the E,E- and Z,Z-isomer of dienestrol (DIES) and the mono- and dimethyl ether of DES. Non-extractable binding to DNA was observed for all compounds with at least one free hydroxyl group independent of the stilbene structure. The extent of binding was highest for the HES isomers and for E,E-DIES, whereas Z,Z-DIES and the monomethyl ether were bound to about the extent of DES. These findings imply that the formation of a phenoxy free radical is sufficient for non-extractable DNA binding and the stilbene structure is not required for peroxidase-mediated activation of DES.

Synthesis and evaluation of catechol analogs of diethylstilbestrol on a hormone-dependent human mammary carcinoma implanted in nude mice.

3,4-Bis(3',4'-diacetoxyphenyl)hex-3-ene (5a), 4(4'-acetoxyphenyl)-3(3',4'-diacetoxyphenyl)hex-3-ene (5b), and 4(3'-acetoxyphenyl)-3(3',4'-diacetoxyphenyl)hex-3-ene (5c) were synthesized according to the method of Dodds et al. (1939). All compounds inhibited the interaction of 3H-estradiol with its receptor. The relative binding affinity (RBA) values increased in the order: 5a (1.0) less than 5c (7.6) less than 5b (21.7). In the immature mouse uterine weight bioassay, the uterotrophic activity of 5a-c was only weak. 5a and 5c, but not 5b, exhibited significant antiuterotrophic properties. All compounds significantly inhibited the growth of a postmenopausal hormone-dependent human mammary carcinoma serially implanted in nude mice.

Synthesis of metabolic intermediates of diethylstilbestrol.

This report details the synthesis of 1) 3,4,4'-trihydroxy-alpha, alpha'-diethyl-trans-stilbene; 2) 3,4-bis-(p-hydroxyphenyl)-trans-3-hexenol; 3) 3,4-bis-(p-hydroxyphenyl)-2,4-cis,cis-hexadienol; 4) 3,4-bis-(3'-methoxy-4'-hydrophenyl)-trans-3-hexene; 5) 3,4-bis-(3',4'-dimethoxyphenyl)-trans-3-hexene. These compounds are suspected metabolites of diethylstilbestrol.

Synthesis of novel hexestrol and diethylstilbestrol derivatives as potential anticancer and estrogenic agents.

Two novel series of potential anticancer agents derived from hexestrol and diethylstilbestrol have been synthesized. The first includes several alkylating agents containing sulphonic esters and nitrogen mustard functions attached through various chains to only one phenolic group in hexestrol. The second contains the N1-acetyl-N4-substituted thiosemicarbazide moieties attached to one phenolic group hexestrol or to the two phenolic oxygens in diethylstilbestrol. The tests of some of the products for antileukemic activity in P 388 Lymphocytic Leukemia indicated no significant activity over the parent nuclei. The estrogenic activity of some representative examples of the thiosemicarbazides was found to be dependent on the nature of the N4-substituent in the thiosemicarbazide moiety.

A novel fluorinated derivative of diethylstilbestrol.

The synthesis of the diethylstilbestrol (DES) derivative with fluorine atoms present in the positions ortho to the hydroxyl in each ring is described. In vitro studies in a system containing horse radish peroxidase/H2O2 demonstrate extensive oxidation of tetrafluorodiethylstilbestrol to the corresponding dienestrol derivative. Tetrafluorodiethylstilbestrol and DES had comparable in vivo uterotropic activities at a dose of 100 microgram/kg. Competitive binding experiments demonstrated 20-25 fold reduced interaction with the mouse uterine estrogen receptor. This compound may be useful as an experimental estrogen in distinguishing between the biological and toxic effects of DES.

Carbon-14-labeled diethylstilbestrol synthesis by the McMurry method: concurrent formation of hexestrol.

Isotopically labeled diethylstilbestrol prepared from p-methoxypropiophenone by coupling with TiCl3-LiA1H4 contained approximately 50% hexestrol. Nonlabeled preparations and a subsequent labeled preparation had less than 2% hexestrol in them. Initial examinations for purity by gas-liquid chromatography, thin-layer chromatography, and isotope dilution did not show the presence of hexestrol. Specific ion monitoring of a gas chromatography-mass spectrometry determination revealed the presence of the deithylstilbestrol-hexestrol mixture.