Bioassay-guided fractionation of a methanolic extract of a Thai crude drug, derived from heartwood of Anaxagorea luzonensis A. Gray (Annonaceae), resulted in the isolation of 8-isopentenylnaringenin (1) as an estrogen agonist with a activity of about an order of magnitude greater than genistein. Various flavonoids possessing isopentenyl side chains in the A-ring have been prepared and evaluated for their ability to bind estrogen receptor. In addition, enantiomers of 1 were separated and the respective enantiomers were assayed. These studies have demonstrated that the presence of an 8-isopentenyl group is an important factor for binding. Flavones, flavanones and flavonols having an isopentenyl substituent at C-8 exhibited an appreciable affinity for estrogen receptor. Conversely, isoflavones possessing an 8-isopentenyl substituent at C-8 did not show this activity. Movement of the isopentenyl group from position 8 to 6 resulted in loss of the activity. No significant difference was observed between 2(S)- and 2(R)-enantiomers of 1 in their binding affinity. Prenylflavonoids are reported to possess a wide range of biological activities; however, estrogenic activity has not been described.

To identify regions that are involved in the formation of the dihydropyridine receptor site of skeletal muscle L-type Ca2+ channels, the al subunit of the channel complex was specifically labeled with the 1,4-dihydropyridinereceptor-selective photoaffinity probe [3Hjdiazipine. Photoaffinity-labeled regions were identified by probing labeled proteolytic fragments with several anti-peptide antibodies recognizing different segments of the al sequence. Forty to 50% of the al-associated [3Hldiazipine label was contained in the tryptic fragment between Arg-988 and Ala-1023 derived from the loop between segments S5 and S6 in domain HI. This region corresponds to a portion of the channel that is believed to contribute to formation of the transmembrane pore. Twenty to 30% of the labeling occurred in a V8 protease fragment between Glu-1349 and Trp-1391. This fragment contains transmembrane segment S6 of domain IV and has previously been shown to form part of the drug receptor for phenylalkylamine Ca2+ antagonists. Our data suggest that the dihydropyridine receptor is formed by close apposition of two discontinuous regions of the al subunit sequence in domains Im and IV. In light of previous work localizing this receptor site to the extracellular surface of the lipid bilayer, it is proposed that amino acid residues at the extracellular surface in the loop connecting segments mSs and IIIS6 and at the extracellular end of segment IVS6 contribute to formation of the dihydropyridine receptor site. These drugs specifically bind with high affinity to distinct allosterically coupled receptor sites on the al subunit of the calcium channel complex as revealed by photoaffinity labeling (4-7). Neutron-diffraction and kinetic studies (8) suggest that DHPs approach their receptor site by lateral diffusion after partition into the lipid bilayer of the membrane. This binding domain is thought to be localized close to the outer surface of the al subunit as DHPs in a membraneimpermeable charged form only have access to their receptor site if applied from the extracellular side (9, 10). This is in contrast to phenylalkylamine Ca2+ antagonists, such as verapamil, that interact with an intracellular receptor site formed in part by the intracellular end of transmembrane helix IVS6 and/or the adjacent intracellular amino acid residues (11). Knowledge of the regions of the al subunit involved in the formation of the DHP receptor site would help to elucidate the molecular basis of channel modulation by DHPs and allosteric interaction between the DHP and phenylalkylamine binding domains. In this report, we define the location of the DHP binding domain within the al subunit of skeletal muscle Ca2" channels by photoaffinity labeling with the DHP-receptor-selective ligand trifluoroethyl diazirine [3H]diazipine (12, 13) and mapping of labeled proteolytic fragments using sequence-directed antibodies.

Biological evaluation of the extract prepared from the stem wood of Dracaena loureiri, a Thai folkloric medicine called "Chan-daeng", revealed a significant capacity to inhibit [3H]-estradiol binding to the estrogen receptor. During the course of separation, two novel (1 and 2) and two known retrodihydrochalcones (3 and 4), in addition to three known homoisoflavones (5, 6, and 7), were isolated. The structures of compounds 1 and 2 were established by NMR and MS studies by correlating their spectroscopic properties with those of 3 and 4. Each isolate was assessed for its estrogenic activity. Compounds 1 and 6 exhibited activity comparable to that of genistein and daidzein.

Naturally occurring phenylpropanoids, hinokiresinol (trans-hinokiresinol) and nyasol (cis-hinokiresinol) were found to possess appreciable estrogen receptor binding activity. Strong differences in activity were observed between the geometrical isomers and enantiomers. Among these, (3S)-cis-hinokiresinol displayed the highest activity, one order of magnitude greater than the activity of genistein. Furthermore, cis- and trans-hinokiresinol stimulated the proliferation of estrogen-dependent T47D breast cancer cells, and their stimulatory effects were blocked by an estrogen antagonist, indicating that the compounds are estrogen agonists. In addition, the absolute configuration of C-3 in (+)-cis-hinokiresinol has been assigned as S by comparison with the circular dichroism spectra of the hydrogenated products prepared from cis and trans ((3S)-trans-hinokiresinol: previously assigned) isomers. These results incidentally provide us with an unambiguous answer to contradictory reports regarding the assignment of the full stereochemisry of cis- and trans-hinokiresinol that have existed in the literature for more than two decades.

Activity-guided fractionation of twigs of Pistacia chinensis resulted in the isolation and characterization of two novel ingredients as potent estrogen agonists. On the basis of spectral analysis and comparison with a related compound their structures were elucidated as 3,3''-dimers of 4-aryldihydrocoumarins (3,4-dihydro-4-(4'-hydroxyphenyl)-7-hydroxycoumarin) differing only in the stereochemical disposition of the linkage between the two 4-arylcoumarin moieties. These compounds are the first examples of bis-flavonoids which have been proven to possess estrogen-like activity.