'9“- nun~ "3“»qu “nu-q- m- «nu-nu .4 .-n -\ IVSIER SITY UBRAR'ES m 1111111111111111111111111111 111111 21111 A 193101 1 This is to certify that the dissertation entitled Synthesis and in Vitro Metabolism of Soybean Isoflavones presented by Yu—Chen Chang has been accepted towards fulfillment of the requirements for Ph.D. degree in Horticulture Major professor Date June 22, 1995 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE N RETURN BOX to remove this Mum your record. To AVOID F|I1IEB Mom on or baton dds duo. DATE DUE‘ DATE DUE DATE DUE ' . ‘1’ .r N! Ann“ ‘ “- }( .1- ‘V‘ ‘ 1 “11" - __ _ ’— 1 * 41/1/12 MSU IsAn Affirmative Action/Emil Opportunity 1m W3 SYNTHESIS AND IN VITRO METABOLISM OF SOYBEAN ISOFLAVONES By Yu—Chen Chang A DISSERTATION Submitted to Michigan State University in partial fiilfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1995 ABSTRACT SYNTHESIS AND IN VITRO METABOLISM OF SOYBEAN ISOFLAVONES By Yu-Chen Chang Soybean dietsareknowntodecreasethemnnberoftumors in rats induced by chemical carcinogens or by irradiation. Also, epidemiological studies indicated a negative correlation between the soybean consumption and breast cancer incidence. Published data showed that soybean isoflavones inhibited the growth of human breast cancer cells in vitro. However, in vivo studies of pure compounds were not available due to the unavailability of these compounds in substantial quantities. Several metabolites of genistein and daidzein were detected in lmman mine, nevertheless, their metabolic pathways remained unknown. It is our hypothesis that the isoflavone metabolites detected in the human urine are produced fi’om the isoflavones in food by intestinal bacteria. Also, these metabolites may function as better anticancer agents than their parent isoflavones present in soy products. A rapid two-step syntheses of isoflavones daidzein, genistein, formononetin and biochanin A, were accomplished. The intermediate ketones were first synthesized from commercially available and low cost starting materials. The ketones were then cyclized in a conventional microwave oven within 2 min to yield the respective isoflavones. The structures of the ketones and isoflavones were confirmed by their 11-1- and ‘3 C-NMR spectra This method provided a convenient and faster way to yield larger quantities of genistein and daidzein for biological studies. Synthetic daidzein and genistein were incubated with human fecal bacteria under anaerobic conditions. Dihydrodaidzein, benzopyran-4,7-diol, 3-(4-hydroxyphenyl) and equol were isolated fi'om the fermentation broth of daidzein Only one metabolite, dihydrogenistein, was isolated and characterized from the fermentation broth of genistein. The metabolites isolated from this fermentation study were identical to the metabolites detected in human urine. The metabolites isolated and characterized from the fermentation studies were synthesized in appreciable quantities for their eficacy studies. Equol, 5,7,4'- nihydroxyisoflavan, 4,7,4'-t1ihydroxyisoflavan, dihydrodaidzein, and dihydrogenistein were synthesized either from daidzein or genistein by hydrogenation. Antifimgal, antibacterial, mosquitocidaL nematicidal and anticancer activities of these compounds were evaluated. Equol was the most active compound among all the metabolites assayed. Equol was also found to be anticarcinogenic in assays with mutant Saccharonwces cerevr'siae stains. 7a my W. pew-at iv ACKNOWLEDGMENT I would like to thank my major advisor, Dr. Mmaleedharan Nair, for his encouragement and help to firlfill my dream. He brought me into the research field of bioactive natural products and Opened a magnificent view in float of me. I would also like to express my appreciation to my guidance committee members, Dr. Wayne Loescher, Jack Kelly, J. Ian Gray and William Helferich, for their advises and kindness. Iwould also like to thank Dr. Amitabh Chandra who has been extremely helpfirl since the first day when I joined Dr. Nair’s research group. Without his help, all the research work would have taken much longer than they had. I would like to thank Dr. Long Le in NMR Facility of Michigan State University for his assistance and Beverly Chamberlin in Mass Spectrometry Facility of Michigan State University for collecting mass spectra for me. Marshall Elson is the first friend I made in the building. I learned English, American culture and food fi'om him, which could never be done without a kind fiiend. I am also glad to knowotherfiiendsinthebuilding, MarkKelm, DiZhang, Jennifeeryer, LavettaNewell, Alex and Catherine Fernandez, Mario Mandujano, Joseph Masabni and Bebecca Baughan. My husband, Pao-Chi, deserves my greatest thank. We have known each other for morethantenyears. Heseernsto know memorethanldo. He always knowswhatisgood for me and put that on top of his priority. Without his encouragement and understanding, I would never have the opportunity to write this dissertation Thank you, thank everybody. TABLE OF CONTENTS LIST OF FIGURES ............................................ x LIST op TABLES ............................................. xiii LIST OF ABBREVIATIONS ..................................... xiii LIST OF APPENDICES ........................................ xiv CHAPTERI — Literature Review Dietary Fat and Breast Cancer ............................. 1 Soybeans and Breast Cancer ............................... 2 Isoflavone Contents of Soybeans and Soy Products ------------- 4 Biological Activities of Isoflavones ......................... 10 Metabolism Studies of Soybeans and Isoflavones --------------- 12 Synthesis of Isoflavones .................................. 19 CHAPTER II _ Introduction .................................. 25 vii CHAPTER III — Microwave—Mediated Synthesis of Anticarcinogenic Isoflavones from Soybeans Abstract .............................................. 29 Introduction ............................................ 30 15mm ........................................... 3 2 Remus and Won .................................... 36 CHAPTER IV -— Metabolism of Daidzein and Genistein Anticarcinogens by Intestinal Bacteria Abstract .............................................. 40 Introduction ........................................... 41 Experimental .......................................... 42 Results and Drscussron .................................... 48 CHAPTER V -— Reduction Products of Anticancer Daidzein and Genistein And Their Biological Activity Abstract ............................................. 52 Introduction ............................................ 53 him-mental ........................................... 54 Results and Discussion .................................... 51 CHAPTER VI _ Summary and Conclusions ........................ 7o viii ix fish! I Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 1.6 Figure 1.7 Figure 1.8 Figure 1.9 /Figure 3.1 Figure 3.2 Figure 4.1 LIST OF FIGURES SOYbeaD ISOflaVOIIOIdS ........................... 5 Human metabolism of endogenous and exogenous mmmms 00000000000000000000000000 13 Types of conjugates of metabolites excreted to the urine ° ° ° 15 Animal metabolites of daidzein and genistein detected intheufinefi-ommtsandmman ooooooooooooooooooo l7 ThesynthesisofgenisteinasreportedbyR C. Shrinerand C. J. Hull (1945).]. Org. Chem. 10, 288-291. ---------- 20 The synthesis of isoflavones as reported by L. Yoder, E. Cheng and W. Burroughs (1954) Iowa Acad SCI. 61’ 271-277 ....................... 21 The synthesis of biochanin A as reported by J. Bass (1976) J. C. S. Chem. Comm, 78-79. --------- 22 The synthesis of daidzein and formononetin as reported by A Petler and S. Foot (1976) Synthesis, 326. ----------- 24 The synthesis of daidzein and formononetin as reported by H. Seikizake et al. (1993) Biol. Pharm. Bull. 16(7), 698-701: 25 Soybean isoflavones ooooooooooooooooooooooooooooo 3 1 Synthesis of isoflavones ooooooooooooooooooooooooooo 37 Metabolites isolated fi'om fermentation broth of daidzein and genistein incubated with human fecal bacteria ----------- 44 Figure 4.2 Figm'e 5.1 Figure 5.2 Figure 5.3 Production of metabolites of daidzein and genistein by ercal bacteriaat 72h ........................ 51 Reduction products of daidzein and genistein ----------- 63 Efl‘eet of camptothecin and equol at 10 and 100 ppm, respectively, on the cell growth of yeast strains JN394, JN394 t1 and JN394 t2” ................................... 68 Efi‘ect of equol at various concentration on the cell growth of yeast strains JN394, JN394 t1 and JN394 t2_5 ----------- 69 LIST OF TABLES Table 1.1 Isoflavones in soybeans and soy flour ----------------- Table 1.2 Distribution of isoflavones in two varieties of soybeans 000000000000000000000000000000000000000 Table 1.3 Isoflavoncs in soy products .......................... ACN BF3-Et20 BI-II CFU DMBA DMF DMSO EIMS EGF HPLC MeSOzCl MNU NMR PDA PDA PDGF Pd/C PTK SI-IBG THF TLC VAM VLC lH-NMR l3C-NMR 6 dd J rnp. m/z rel. int. LIST OF ABBREVIATIONS Aeetonitrile Boron trifluoride etherate Brain heart infirsion media Colony forming unit 7,12-dimethylbenz[a]anthrancene Dirnethyl forrnamide Dirnethyl sulfoxide Electron impact ionization mass spectrometer Epidermal growth factor High performance liquid chromatography Methanesulfonyl chloride N—methyl-N-nitrosourea Nuclear magnetic resonance Photodiode array detector Potato dextrose agar Platelet-derived growth factor Palladium on active carbon Protein tyrosine kinase Sex hormone binding globulin Tetrahydrofuran Thin layer chromatography Vesicular-arbuscular micorrhiza Vacuum liquid chromatography Proton nuclear magnetic resonance "Carbon nuclear magnetic resonance Chemical shifts doublet of doublet Coupling constant Melting point Mass-to-charge ratio Relative intensity APPENDIX I APPENDIX 11 APPENDIX III APPENDD( IV APPENDIX V APPENDIX VI APPENDIX VII LIST OF APPENDICES Isolation and purification of daidzein and its metabolites fi’om the fermentation broth ........................... 84 Isolation and purification of genistein and its metabolites fi'0m thC fermentation broth ........................... 85 HPLC of daidzein and genistein and their metabolites -------- 86 HPLC of the fermentation broth of daidzein --------------- 87 HPLC of the fermentation broth of genistein -------------- 88 Hydrogenation scheme for daidzein ..................... 39 Hydrogenation scheme for genistein ..................... 90 xiv CHAPTERI Literature Review Breastcancerisoneofthe mostprominent cancersamongwomen inthe United States. Approximately 1 in 8 women is diagnosed with breast cancer each year. Several epidemiological studies revealed that the incidence of breast cancer among women is higher in Western countries and lower in Asia (Haenszel and Kuribara, 1968; Armstrong and dell, 1975; Lee et al., 1991; Aldercreutz, 1990). When women with low risk of breast cancer immigratedto countrieswithahighmcidenceofbreast cancer,theirbreast cancerriskbecame similar to the risk of host-country women (Haenszel and Kurihara, 1968; Staszewski et al., 1971; Aldercreutz, 1990; Locke and King, 1980; King and Locke, 1980; King et al., 1985; Dunn, 1977; Buell, 1973, Tominage, 1985). These observations suggest that breast cancer occurrence in these women is associated with environmental difl‘erences as well as with genetic difi‘erences (Barnes et al., 1990). Dietary Fat and Breast Cancer High fat and high calorie diets are correlated positively with breast cancer occurrence (Drasarandlrving 1973; ArmstrongandDoll, 1975). Apositivecorrelation offat intake and incidence of breast cancer was observed by several researchers (Talamini et al., 1984; Lubin 1 2 et al, 1986; Hislop et al., 1986). Also, several animal feeding studies confirmed the efl‘ects of dietary fat on the occurrence ofbreast cancer. In one study, rats were fed with diets containing high levels (20 %) of polyunsaturated fatty acids. The results showed a significant increase in the incidence and number of tumors induced by carcinogens and a decrease in tumor latency of these rats compared with rats fed with 5 °/o fat (Carrol et al., 1986; 1p and Sinha, 1981; Ip et al., 1985;). Similarly, a positive correlationowas observed between the total fat intake and plasma estrone and estradiol levels among American and Oriental women (Goldin et al., 1986). It was found that the gut microbial B—glucuronidase activity was significantly increased with high fat diets and resulted in enhanced enterohepatic circulation of estrogens along with high plasma levels of androgens and estrogens (Goldin et al., 1982). However, the epidenriological studies of the relationship between dietary fat intake and breast cancer risk are controversial (Graham et al., 1982). Willett et a1. (1987) reported that there is no positive correlation between dietary fat intake and the risk of breast cancer among U. S. women 34 to 59 years of age and having no history of cancers. Similarly, association between fat consumption and the occurrence of breast cancer was not observed among Japanese women (Hirohata et al., 1987). Soybeans and Breast Cancer One of the dietary factors which contribute to a lower breast cancer risk among Asian and the US women is the inclusion of soy products in their diet. It was reported that there is an inverse correlation between soy intake and the risk of breast cancer among 142,857 women in Japan observed for 17 years (Messina et al., 1994). Another study of the breast 3 cancer in Singapore among Chinese women with and without breast cancer revealed a negative correlation between high soy diet and the risk of breast cancer (Lee et al., 1991). Aldercreutz ct al.(l986"’, 1987, 1988) reported a strong positive correlation of the plasma sex-hormone binding globulin (SHBG) concentration, urinary phytoestrogen and lignanexcretionwithfiberintakeinvegetariansand omnivores, and anegative correlation for theseobservationsandbreast cancer. The SHBGisanimportanttransporter ofestrogens and androgens. A higher SHBG concentration in plasma results in lower bioavailability of sex hormones in the circulatory system The plasma levels of estrogens was 3-fold higher among American women than among Oriental women studied for breast cancer occurrence (Golding et al., 1986). Itwasshownthatchimpanzeesareresistanttobreast cancer and contain large amounts of phytoestrogens and their metabolites in the urine (Aldercreutz et al. 1986'). Similar excretion of phytoestrogens was observed fi'om human vegetarians. Only small amounts of lignans and phytoestrogens were found in the urine fiom breast cancer patients. Therefore, it was suggested that the phytoestrogens present in vegetables and soybeans may play an important role in the prevention of breast cancer in chimpanzees and human vegetarians (Aldercreutz et al., 1986”). Inrats, soybeandietsreducedmamrnarynnnoroccrmenceinducedbyirradiation (Troll et a1, 1980) and the carcinogen, N-methyl-N-nitrosourea (MNU) (Barnes et al., 1988). Troll et al. ( 1980) suggested that the protease-inhibitory activity of soy-rich diets may contribute to the reduction of cancer. However, Barnes et al. (1990) deactivated the protease inhibitors 4 present in soybean by autoclaving the soybean chips for 40 min When rats were fed these autoclaved soybean chips, the appearance of mammary tumors induced by the carcinogens, MNU or 7,12-dimethylbenz[a]anthrancene (DMBA), were not observed. There was no difi‘erence in the degree of inhibition between non-autoclaved and autoclaved soybean chips. Therefore, the protease inhibitors were not responsible for the tumor-reducing efi‘ect of soybean. Isoflavone Contents of Soybeans and Soy Products Soybeans contain high levels of isoflavonoids (Figure 1.1), various quantitative analysis of isoflavone contents in soybeans and soy foods have been reported. 1Naim et al. (1974) analyzed soy syrup obtained as a residue fi'om the extraction of commercially available defatted soybean flakes. The results showed that all the isoflavones in soyflakes were present as glycosides with less than 1 °/o as of their aglycones. I The percentage of isoflavones in soybean was about 0.25 %, of which 64 % was genistin, 23 % daidzin and 13 % glycitein 7- o-p-glyooside (Table 1.1).] Eldridge and Kwolek (1933) also analyzed the isoflavonoid contents in many soybean varieties grown in difi‘erent locations in Illinois, USA during 1980. Significant interactions among variety and location were observed for the total and individual isoflavonoid content. Anatomical distribution of soybean isoflavones in two variety of soybeans, Amsoy and Tiger, also was described by Eldridge and Kwolek (1983) (Table 1.2). It was found that hypocotyl and the hull contained 90 and 1 % of the total isoflavones, respectively. Therefore, dehulled soybean have little efi‘ect on the total isoflavonoid content. It was reported that the isoflavones predominantly exist as the 7-O-glucoside 6"-malonyl R. R. R. R. OH H H OH O-Glu H H OH OH H H OMe OH H OH OH O-Glu H OH OH OH H OH OMe OH OMe H OH O-Glu OMe H OH O-6"-0MalGlu H H OH O—6"-0MalGlu H OH OH O-6'-OMalGlu OMe H OH O-6"-OAcGlu H H OH O-6"-OAcGlu H OH OH O-6"-OAcGlu OMe H OH CI-LOH CILOCOCI-LCOOH O on o __ HO OH O-Glu O-6"-OMalGlu Figure. 1.1 Biochanin A Glycitein Glycitin 6'-O—Malonyldaidzin 6'-O-Malonylgenistin 6'-O-Malmylglycitin 6"-O-Acetyldaidzin o'onoaylgonisdn 6"-O-Acetylglycitin .0 O Ou6'-OAcGlu Soybean isoflavonoids so: >8 .o 8.5!. no ... eased o5 .3. 2.: .nmoovxeio .8..on8 .nmSoxacoo 83850 63.2....» .nB 0296...... .m» .8532 .88 was 322 8830 u .s 853 5.05:: .50: >3 ” .> Seam o5 as... 23. .580. ”s .82.. ofifinx .58.. .38: auto .8885... i685: 2.. e350 05 atom .85.. .858: ” ... 382. 05 >85. n .. coogoo .0 .933 c3055 .2 u _ gun 32 85m 82 was .8. m8; 8.: amotom «8. .392... as. 532 82:8 son .8 .rna 2.88 52.0206 2: 3.82. 82. 538020.... «mu m. .82. 82. 53.8208 m... 9.8.3 35.3. 5.9.6...sz o. 5 3735 92-0.8 £250...sz 8 «Snow 33.2 canaoazoe «we no code So... 52.0 678 one 33.“. 0.88.8 5580 $3 ode o. P 2..» 23.: 53.3 98.5 «~88 53.. £620 01 .. E... are. casino 9.... 82. 99.8 «.35 o: no cannon. .s s a 2 ... ._ _ £89.38. 600 :95 50: >8 ecu 20230» .0 «29:00 20:05:02 E 0.8... .838 .3 .530 nook 95‘ 223: a .3 8.95. .5. .0 a. «more... «.29 mom. «.8: men: o.o« 0.2 .83 a.» o.« «...« ha a. no 52350 9.0 o. p p m; 52.0sz 3 .1 3; ca. 3 a... cannon o.8« as: 3 no as o.« source 3 5 38 2.8 8383.93.5320 «.8. QB «.8» 38. o... no :38 In”: >022 Mg. ~02? com... >025. 3:03:00. 5.8330 582:... .5: A .0 8 :95 g8 .o 8.6.3.. o2 :. 8:888. .o 8.52.35 «.. some 8 coniugates in soybean tissue (Graham, 1990; Kudou et al., 1991). Wang and Murphy (1994) analyzed the isoflavonoid content of several American and Japanese soybean varieties and confirmed that 6”-malonylgenistin, 6"-malonyldaidzin genistin and daidzin represented 83 to 93 % of the total isoflavone content. Among them, 6"-malonylgenistin was the major isoflavone constituent and represented 25 - 42 % of the total isoflavones. There was a significant difi‘erence in the total and individual isoflavone concentrations among difi‘erent cropyears. Also,theisoflavonewmanswuemtgreaflyinflumcedbythegrowthlocafions. The isoflavone contents of soy flour samples were reported by Eldridge (1983) and Barnes et al. (1994). Barnes ct al. (1994) found that most of the isoflavones present in soy productwereextractedwith80%aqueousACN orMeOHatroomternperatureforZh At higher temperatures, the amounts of B-glycoside conjugates and aglycones were increased at the expense of 6"-malonylisoflavones. The conversion of isoflavone 6”-Malonylglycosides to B-glycosides and aglycones was greatly increased when temperature was elevated to 80°C. Eldridge (1983) analyzed the isoflavones in 10 commercial samples of soy flour afier extracting them with 80 % MeOH, (Table 1.1). The conversion of isoflavone 6"- malonylglycosides to B-glycosides and aglycones upon heating explained the large variation shown in the results by Barnes et al (1994) and Eldridge (1983) (T able 1.1). The isoflavone contents of various soy products were published by other researchers as well (Table 1.3). Murphy (1982) compared her results with the published data (Table 1.1) and concluded that there was a decrease in genistin content in processed soybean food products. Similar conclusions were described by Wang and Murphy (1994”). Generally, the 8.5 .o as... 58:5 3.. p.382 3 one... .5055 n =3 22 .o .22.. 565.5 . ..., as .o 653 :38; u 3 88.588 223 c .... 85.6 2. ...: .8882... £88. 2.3 58.5 u s 2033.. 300.. .0 30:3 565:: .2 . ....E >8 .o 355 58E: . ... 8.28. 522.. >8 .o .88.. 505.5 n .. oz. 29.6 68 2&6 66 296 .< 2“. .3 .z 2a .3 ..oo 2:2. Siam 52. 83.8. 522.. ..om . . .8383 32932. 329...; «8.3.5:. «BU-Es salons; «8.868 .BPSEum «8.3.94.5 823838 B E o.« «.3.» «.2 2 52.0208 ... B 3 s.«.«.« at .8 3 cn¢.cooo .... s > z ... .. . 8.82.88. Boo :95 90.605 >8 5 3:05:02 0.? 23.. 10 total isoflavone contents in soy food products are lower than soybean itself. 7-0-glucoside 6"-0-acetates were produced during heat treatment of soybeans (Farmakalidis and Murphy, 1985'). Therefore, the higher content of 6"-0-acetylisoflavones should be expected in extensively processed soy food. Biological Activities of Isoflavones The isoflavonoids daidzein, genistein, formononetin and biochanin A are estrogenic in sheep (Shutt et al., 1968), rats and guinea pigs (Farmakalidis and Murphy, 1984; Farmakalidis et al., 1985”). In sheep, the estrogenic activity of genistein was about 10" times than that of diethylstilbesn'ol administered inn'anmscularly (Shutt et al., 1968; Braden et al., 1967). Both genistein and biochanin A inhibited human stomach cancer cell proliferation in vitro (Yanagihara et al., 1993). It was suggested that the anticancer activity of genistein was estrogen-receptor independent The growth of the human breast carcinoma cell lines, MDA- 468 (estrogen-receptor negative), and MCF-7 and MCF—7-D-40 (estrogen-receptor positive) were inhibited in vitro by genistein (Peterson and Barnes, 1991). Biochanin A and daidzein are less inhibitory to the cancer cell growth when compared to genistein. Also, the isoflavones, genistin and daidzin, were not efi‘ective as anticarcinogens. It was shown that singlet oxygen played an important role in mutagenesis and carcinogenesis, particularly in tumor promotion Genistein strongly inhibited H202 formation both in vivo and in vitro (Wei, et al., 1993).. Genistein was reported as an inhibitor for protein tyrosine kinase (PTK) and DNA synthesis (Akiyama et al, 1987; Dean et a1, 1989; Sit et al., 1991). Cell growth is controlled l 1 bymanygrowthfactors, suchasepidermal growth factor (EGF) and platelet-derived growth fictor (PDGF). Non-proliferating cells treated with EGF will start proliferating again The receptor protein for EGF was found to be associated with specific PTK activity. The phosphorylafimoftymdmisamevanmnormflcdls.Phosphotymsineacmmfor only one in 2,000 of the phosphate molecules linked to proteins. However, in cancer cells, the amount of phosphotyrosine increased dramatically (Bishop, 1986). Also, several oncogene productswerefoundtobethe mutant forms ofEGF receptor. Hence, the inhibition ofPTK activitybecameanimportant property ofcompomdstobeusedasamicarcinogens. Recently, Uclnm et al. (1995) published on the biotherapy of B-cell precursor leukemia by a complex of genistein with a monoclonal antibody of the B-cell-specific receptor, a member of the protein tyrosine kinases. This genistein-antibody immunoconjugate was over 1500 times more efl‘ective than unconjugated genistein at inhibiting the PTK activity of B-cell specific receptor and causing decreased tyrosine phosphorylation in leukemia cells. It was proposed that more genistein molecules were delivered into leukemia cells by the genistein-antibody immunoconjugate. Also, the immunoconjugates brought genistein molecule closer to the protein tyrosine kinase which enhanced the PTK inhibition of genistein. Genistein also was reported as DNA topoisomerase II inhibitor (Markovits et al., 1989; Corbett et al., 1993). DNA topoisomerases catalyze the interconversion of topoisomers, the wound or unwound DNA strains. DNA strains are tightly wound to form chromosomes. The binding of topoisomerase on DNA untangles the DNA strains. This allows the DNA replication and transcription which are essential for cell growth. Drugs, acting as DNA topoisomerase poisons, can produce the cleavable DNA-topoisomerasedrug complex and 12 curse site-specific breakage of the chromosomal DNA. This event inhibits DNA replication, RNA synthesis and cell division, and eventually leads to cell death (Liu,1990). Cancerous cells contain larger amount of topoisomerases which makes them good targets for topoisomerase inhibitors. Therefore, tapoisomerase I (cleaves one strain of DNA) and II (deavestwosnamsofDNAMtnaaedagrendealofhnaeumcancerreswchmthe1990s. ThereisahomologyinthesequenceofPTK and humanDNAtopoisomerase 11. Both PTK and DNA topoisomerase II are involved in the formation of phosphate ester between a phosphate group of a nucleotide and the hydroxyl group of tyrosine. This might be the reason that both enzymes, responding to the same inhibitor (Markovits et al., 1989). The isoflavonoids, daidzein, formononetin, genistein and biochanin A, exhibit other important biological activities. Formononetin and biochanin A were reported as the signal molecules for vesicular-arbosmlar mycorrhiza (VAM)-host plant symbiosis (Nair et al., 1991; Siqueira et al., 1991'; Safir et al., 1992) and as herbicide safeners (Siqueira et al., 1991 ). Also, genistein and daidzein, isolated from the soybean (Glycine max L.) root extract, were responsible for the induction of Brandyrhizobim japom'cum nod genes (Kosslak et al., 1987). Antifimgal and antioxidant activities also were reported for soybean isoflavonoids (Ikehata et al., 1968; Naim et al., 1974; Murakami et al., 1984; Kramer et al., 1984). Metabolism Studies of Soybeans and Isofavones The lower gut is one of the major metabolic sites of the human body (Figure 1.2). In vitro metabolism studies revealed that the metabolisms accomplished by gut flora are mainly 13 ave—59:00 maoeowoxm v.8 maceowovcm— .«0 833802 5:52 NA 2:!"— 8...8%5 «:28 .23 05.5 _ ~33 - >260— _ , W , > EEBSoE 0:55.... 8»: 0:8: .850 w - a \k 0.5 _ _ 82m anon. 4T 82m befits. A > 4/ 8:205 muaaaaoo maoaomovfi be... as 338:. EEBSeE omega—m 8:82? 35.8“? eczema—.8 28 2:95. 2:». 8289:»- Engage—oceans 23835.8 9:5 x 88". AI_ 8:32.033 4... 2.53... ..26 AT ..oaasm _ 4 wagon—Eco goeomoxm l4 hydrolysis and reductions. Most oxidation and conjugation reactions are carried out by the liver (Gorrod, 1978). Endogenous or exogenous chemicals entering the human body are metabohzedfirstbygmbaaaiamdthenabmrbedmdnansponedtothefivathrough enterohepatic circulation for further transformation The major role of liver enzymes is to transform these chemicals into more polar metabolites in order to excrete them into urine. The most common conjugates present in human and other mammalian species are glucuronides (Figure 1.3). There are several reasons for the formation of these conjugates in biological systems. They are: (r) the easy supply of carbohydrate as a conjugating agent; (ii) the high polarity of the polyhydroxy and carboxylic acid groups of the glucuronic acid; (iii) the lower toxicity and higher sohrbility of the glucuronide compormds; and (iv) the wide range of compounds that can be the substrates of UDP-glucuronyltransferase. Other conjugates include glycosides, sulfate esters, methylates, thiocyanate acetylates, amino acids and mercaptun'c acid conjugates (Figure 1.3). These polar metabolites are either excreted into the urine or into the gut with bile salts, where these compounds are metabolized further by intestinal microorganisms. The intestinal microorganisms contain important enzymes such as B-glucuronidase, B-galactosidase and B-glucosidase to metabolize chemicals or their conjugates. The chemical conjugates present in food must be hydrolyzed before they can be absorbed into the circulation system. Therefore, these glycosidases play an important role in the release and bioavailability of biologically active aglycones. The incidence of colon cancer was significantly lower in germofi'ee rats than in conventional rats when both were treated with DMBA carcinogen. Dietary fat had no efl‘ect on tumor incidence in germ-free rats. However, conventional rats fed with high-fat diets were 15 OH 0 —@ PM I reactions Phase 11 reactions CH,OH COOH o OH 0 OH OH OH HO HO OH OH -D-Glucose -D-Glucuronic acid -30; -CH, Sulfate Methyl -SCN . Thiocyanate ('30 ——glycrne -S-CH.,-CIIH NH —_glutamate -COCH3 AMY] Mereapturic acid Figure 1.3 Types of conjugates of metabolites excreted to the urine 16 much more susceptible to the carcinogenic efi‘ect of DMBA (Gorbach and Goldin, 1990). These observations further emphasized the role of intestinal bacterial metabolism \Aldercreutzetal. (l984)reportedthatlmmansubjectstreatedwithantimicrobial drugs exhibit an increase in the excretion of conjugated steroid hormones in feces, and a decrease in the excretion of unconjugated steroid hormones in the urine. These results indicate that the intestinal bacteria play an important role in the metabolism of steroidal hormones. A number ofreductivereactionsofandrogensandestrogemwereobserved when incubated with human fecal bacteria under anaerobic conditions (Lombardi et al., 1978; Jarvenpaa, et al., 1980). Metabolic studies of soybean isoflavones showed that p-ethyl phenol was isolated as theonlymetaboliteofgenisteinandbiochaninAinthemine ofsheep (Batterham et al.,1965; Braden et al., 1967; Shutt et al., 1970; Batterham et al., 1971) (Figure 1.4). Equol and 0- desmethyl angolensin were identified as the metabolites in the urine when the sheep were fed with daidzein and formononetin (Batterham et al., 1965; Batterham et al., 1971; Shutt et al. 1971; Shutt and Braden, 1968). The runrinal fluid was capable of metabolizing biochanin A to genistein, and formononetin to daidzein and equol during fermentation (Dickinson et al., 1988) Similar results were obtained with the urine and plasma of guinea-pigs and Sprague- Dawley rats fed with soy flour (Shutt and Braden, 1968; Axelson et al., 1984). In the urine of rats fed with soy flour, both equol and daidzein were characterized as monoglucuronide conjugates (Axelson et al., 1982'). Also, daidzin, the glucoside of daidzein, present in soy flour was confirmed as a precursor of equol (Axelson et al., 1984). Yasuda, et al. (1994) 17 IO gig-3. a-.. $0 a O O: 58:: can 38 Eon.“ 2E: 05 E 3.08% 52m_:ow v5 532% we 8509302 v; oSwE Ag: assuring = .... 18 reportedthatthemetabolitespruaninthemineandbilefluid oftheratsfedwith daidzin and daidzein were glucuronide and monosulfate and disulfate conjugates of daidzein. The excretion of equol was undectable in the urine of germ-flee rats on soy diets, whereas the urine of conventional rats contained equol. This confirmed the bacterial transformation of daidzein to equol in rats (Axelson and Setchell, 1981). Daidzein, equol and O—desmethyl angolensin were identified as the monoglucuronide conjugates in human urine (Axelson et al., 1982”; Bannwart et al., 1984‘"; Axelson et al., 1934). The majority of equol excreted in human urine was monoglucuronide conjugate and a small amount occurred as monosulfate or disulfate conjugates (Axelson ’et al, 1982”). Similar reslults were observed for daidzein excreted in human urine (Axelson et al, 1984). Kelly et al (1993) confirmed the presence of equol, O-desmethyl angolensin, dihydrodaidzein, 6'-hydnoxy-0-desmethyl angolensin, dehydro-O-desmethyl angolensin, benaopyran-4,7-diol, 3-(4-hydroxyphenyl) and tetrahydrodaidzein as the metabolites of daidzein (Figure 1.4). Dihydrogenisteinwasthe only metabolite ofgenistein from human urine. p-Ethyl phenol was detected as the metabolite of genistein in the urine of rumens (Batterham et al., 1965; Batterham et al., 1971; Shutt et al. 1971; Shutt and Braden, 1968). However, it was not detectedinhumanmine. Themetabolism studiesofdaidzeinand genistein inhumans showed a large variation in the production of metabolites. Also, quantitative analysis of the metabolites revealed that an inverse relationship was observed between the production of equol and O-desmethyl angolensin. 19 Synthesis of Isoflavones GENISTEIN.— Genistein was synthesized from phloroglucinol and p-methoxyphenyl acetonitrileasfollowingthemethod ofShrinerandI-hrll (1945) (Figure 1.5). A stream ofdry HCl gas was passed through an ethereal solution of phloroglucinol and p-methoxyphenyl acetonitrile until saturation for two days. The ether layer then was decanted and the resulting precipitate was refluxed with 2 % HCl for 4 h The resulting ketone was crystallized upon cooling. The ketone was condensed with ethyl formate and metallic sodium followed by acidification. Biochanin A then was hydrolyzed with HI to yield genistein. Amodified procedurewas reported laterbyYoderet aL (1954) (Figure 1.6). The same step was employed for the production of the ketone as above, using phloroglucinol and p- hydroxypherryl acetonitrile. The ketone then was treated with ethyl oxalyl chloride in pyridine followed by alkalic hydrolysis. The 2-carboxylic acid of genistein thus afi‘ordcd was decarboxylated to produce genistein was achieved by pyrolysis. BIOCHANIN A— The total synthesis of biochanin A was reported by Shriner and Hull (1945) (Figure 1.5). The method reported by Yoder et al. (1954) for the synthesis of genistein was used also for the synthesis of biochanin A by substituting p-methoxyphenyl acetonitrile. Thecyclization ofketonealsowasaccomplishedbyheating it with dimethyl forrnamide (DMF), boron trifluoride etherate (BF3-Et20) and methanesulfonyl chloride (MeSO,Cl) (Bass, 1976) (Figure 1.7). This method provided a convenient cyclization of the ketone with .833 .2 $25 .80 .s Ga: :3. a .0 one 555 .o .m 3 3:32 mm 3835» no ammo—aim 2F m4 253 25288.» 38:§xo5».2-q £8er vhzox H U - .m - A83 6: flWoszéz _ me o: .50 «2 £000: 35033802.“ m0 :0 O: n . .RSR .3 new new 9:3 $3: Eweefim .3 new ago .m an?» .4 3 vow—098 ma mo=o>aa8_ me 3258? 2:. w._ oSwE 1 #8886 2 86 n J. m u .m nineseee not... at: _§$e§2a niota .m each: mote £3228. .espsam :u so .. .m so .. .z Eugenie mo u J 383.5%. mo u i o .m J. .‘39 u Q..- + 9 mo 0: .x no on 55: ..aseu anew .m. U .563: .mmam H .m 3 vow—once 8 < 8:283 mo $8583 2:. Rm— 833 0 his T l + $8 $5 Gnome: cam—mam =0 OE 23 difi'erent substituents to produce corresponding isoflavones. DAIDZEIN.— Daidzein was synthesized fi'om resorcinol by replacing phloroglucinol in the synthesis ofgenistein (Yoder et al. 1954) (Figure 1.6). An alternative cyclization ofthe ketone was achieved by the cyclization of the intermediate ketone with dimethoxy- dimethylaminomethane in DMF for 3 h (Pelter and Foot, 1976) (Figure 1.8). Modified Vilsmeier-Haack reaction using POCl3 and DMF as reagent also was used to cyclize the corresponding ketones to form daidzein and formononetin (Kagal et al., 1962). Seldzaki et al. (1993) reported a new procedure for synthesis of isoflavones using the condensation of corresponding acetophenone and aldehyde to produce the key intermediate tetrahydropyranyl-chalcone (Figure 1.9). These chalcones were oxidized and cyclized with thallium (III) nitrate trihydrate in methanol. Using this method, 19 isoflavones were synthesized, including daidzein and formononetin, but not genistein and biochanin A FORMONONETIN.— The methods used for the synthesis of daidzein described earlier were utilized also for the synthesis of formononetin (Kagal et al., 1962; Pelter and Foot, 1976; Seldzaki et al. 1993) (Figure 1.6, 1.8, and 1.9). 6mm 2&5“wa GS: 8cm .m Ea cog—om .< .3 BEES ma goeoaonuom 2a 832% .«o $3553 2? m _ Bum—m % 56.8555 020 u rm 020 u r: 553 so use. no I J £00 5 z 98 +££UMV|©I nzoo 25 .83$ .63 .35 Sui .33 $8: .a .e nausea d E 3.82 a neeeeeeéee as 53%.. .6 385% 2:. 3 use: gcoeogom 020 n J— Eng no u «a .06: .: 32.5.5 ease a: 53:5: 220 u «a no u rm use u rm mo"! w o = u 0 6/ 39m 0:0 \ HU/ 01 + 20v— :O 0: J— \©\ IO 0: CHAPTERI] Introduction Approximately 1 in 8 women is diagnosed with breast cancer in the United States during their life time. Epidemiological studies found that when women living in countries with low breast cancer risk immigrate to the countries where women have higher breast cancerincidence, thebreast cancerincidenceofthe immigrantsbecame similarto the women in the host countries. This suggests that breast cancer is associated with environmental as well as genetic factors. In many instances, diets are correlated with the incidence of breast cancer. Among all the dietary factors, most researchers believed that fat is the most important factor. Also, many animal feeding experiments confirmed the detrimental effect of dietary fat. However, some epidemiological studies did not observe correlation between the fat intake and breast cancer incidence. Another dietary factor that may be related to the breast cancer incidence is soybean intake. Epidemiological studies found that Japanese women have much lower breast cancer rate than the women in the United States. Other studies in Asian countries such as Japan, 26 27 Taiwan, Chinaand Singaporeindicatedanegativeconelationbetweenthesoybeanintakeand breastcancerocarrrence. InTaiwmnwasesfimatedthatthedailyconsumptionofsoybean was 35 g/person and provided 35 mg or more of soybean isoflavones, such as genistein and daidzein However, intheUnited States, the soybean intakeislessthanZydayperperson Inanimalexperiments, ratswerefedwithsoybeandietandcarcinogens. Itwas obsavedmmlessnmomwaehflucedbychamwwdnogmsorhradiafionmwybeanfed rats. Thisstudysuggestedthatthelowerbreastcancerriskmaybeassociatedwiththe higher soybean consumption by both humans and laboratory animals. It was reported that the isoflavones present in soybean such as genistein and daidzein, theaglyconesofgenistinanddaidzin, inhibitedthegrowthofbreast cancercellbothinvitro and in vivo. However, most of the soybean isoflavones are very expensive or not commercially available. This seriously limited the evaluation of these isoflavones as anticarcinogens. Therefore, the first objective of our research was focussed on the synthesis of soybean isoflavones in large quantities. The availability of these isoflavones will allow firrther eficacy tests including animal feeding studies. Many bioactive metabolites are produced in the large intestine by bacterial metabolism. Various metabolites of genistein and daidzein have been detected in the urine fi’orn sheeps, rats and humans. However, the metabolic pathways of these compounds have not been evaluated. Since most of the metabolites of soybean isoflavones detected in biological systems are their reduction products of isoflavones, it is possible that these isoflavones are metabolized initially by the intestinal bacteria. Therefore, the second part of this dissertation 28 contained the study of the metabolic pathways of genistein and daidzein Daidzein and genistein were incubated with human fecal bacteria under anaerobic conditions. The metabolites produced in the fermentation broth were isolated and characterized by spectral methods. nnposnblemnthemeubohteepromoedinvivommvinoreedingecpenmenuwnh isoflavones are more biologically active than their parent compormds. These metabolites were detected in small quantities in the biological systems and their biological activities were not reported so far. To determine the biological activities of these metabolites of daidzein and genistein, we have synthesized the metabolites of daidzein and genistein detected in the biological systems by the hydrogenation ofeither genistein or daidzein The chapter v ofthis dissertation describes the antibacterial, antifungal, mosquitocidal, nematicidal and anticancer activities of the hydrogenation products of daidzein and genistein. Q CHAPTER III ' Microwave-Mediated Synthesis of Anticarcinogenic Isoflavones from Soybeans Abstract Soybean isoflavonoids, 7,4'-dihydroxyisoflavone (daidzein), 7-hydroxy-4'- methoxyisoflavone (formononetin), 5,7,4'-trihydroxyisoflavone (genistein) and 5,7-dihydroxy- 4'-methoxyisoflavone (biochanin A), were synthesized with high yields by cyclization of their corresponding ketones in a conventional microwave oven. ' Published: Chang, Y.-C., Nair, M G., Santell, R C. And Helferich, W. (1994) Microwave-Mediated Synthesis of Anticarcinogenic Isofiavones from Soybeans. J. Agric. Food Chem. 42, 1869-1871. 29 30 Introduction Soybean isoflavonoids, daidzein, formononetin, genistein and biochanin A (Figure 3.1), are reported to have important biological activities, such as being the signal molecules for vesicular-arbuscular mycorrhiza infection on host plants (Nair et al., 1991; Siqueira et al., 1991'; Safir et al., 1992); herbicide safening efi‘ect (Siqueira et al., 1991 ); estrogenic and anticarcinogenic activities in sheep and rats (Braden et al., 1967; Troll et al., 1980) and they have been implicated in the inhibition ofgrowth ofhuman breast cancer cells (Peterson and Barnes, 1991). Epidemiological studies showed that the incidence of breast cancer is high in North America and North West Europe (Drasar and Irving, 1973). Diets normally are consideredanimportantfactor, breast cancerishighlycorrelatedwithahighfatandanimal protein diets (Drasar and Irving, 1973; Lee et al., 1991). It was reported that women consuming high levels of isoflavonoid-containing diets have lower breast cancer incidence than the women with low intake of such diets (Aldercreutz et al., 1988; Aldercreutz, 1990; Lee et al., 1991; Messina and Messina, 1991; Messina and Barnes, 1991; Setchell et al., 1984). However, pro-clinical studies of these important compounds have not been evaluated adequately due to the limited quantities of isoflavonoids available. Most of the soybean isoflavonoids are very expensive which seriously limits their evaluation as potential anticarcinogens. Therefore, the source of these isoflavones became our first priority when investigating them as anticarcinogens. The reported syntheses of many soybean isoflavonoids including genistein, biochanin A, daidzein and formononetin are very time-consuming (Bass, 1976; Baker et al., 1953; 31 RI= OH, Rf H, 1g= OH Daidzein R,= O—Glu, &= H, lg= OMe Daidein Rl= OH, R,= H, Rf OMe, Formononetin R,=OH, Ig=OH, RJ=OH Genistein R,= O-Glu, R,= OH, R,=OH Genistin R,= OH, Rf OH, Rf Ome, Biochanin A Figure 3.1. Soybean isoflavones 32 Farkas et al, 1971; Pelter and Foot, 1976; Shriner and Hull, 1945; Yoder et al., 1954). The use of the microwave energy in organic syntheses are now popular (Abramovitch, 1991). It was shown that commercial microwave-oven treatment dramatically reduced the reaction times of many organic reactions such as the Diels-Alder and Claisen reactions (Giguere et al., 1986)anda-vinle-lactamsymhesis(Baniketal., 1992). Inthispaper, wedeseribeefi‘ective and rapid synthses of daidzein, formononetin, genistein and biochanin A by cyclizing their corresponding ketones using an unmodified microwave oven. Experimental INSTRUMENTS—1H and 13C NMR spectra were recorded on Varian VXR 300 and 500 MHz spectrometers, respectively, in CD3OD or erMSO solution at ambient temperature. The noncorrected melting points were recorded on a Thomas Model 40 micro hot-stage apparatus. CHEMICALs.—Resorcinol, 4-hydroxyphenylacetic acid, BF3 etherate, N,N-dimethyl- formamide dimethyl acetal, 4-methoxyphenylacetic acid, phloroglucinol, 4-hydroxyphenyl acetonitrile, and methanesulfonyl chloride were purchased fiom Aldrich Chemical Company (Milwaukee, WI, USA); 4-HYDROXYBENZYL 2,4-DIHYDROXYPHENYL KETONB, l .—Resorcinol (2.9 g) was added to a mixture containing 4-hydroxyphenylacetic acid (2 g) and BF3 etherate (4.5 ml). The reaction mixture was refluxed for 10 min, cooled and treated with saturated aqueous NaOAc (30 ml) and NaI-ICO3 (15 ml), respectively. The precipitate formed was filtered ofi: 33 washed with water, dried, and then washed with war, to give yellow needle-like crystals (2.8 g, 88%); mp. 188-190°C; ‘H—NMR ((219,013) 6 7.82 (lI-L'd, J= 8.7 Hz, H—6), 7.08 (2H, dd, J= 6.6, 2.1 Hz, H-2', 6'), 6.72 (1H, dd, J= 6.6, 2.1 Hz, H-3', 5'), 6.35 (1H, dd, J= 8.7, 2.4 Hz, H-5), 6.24 (1H, d, J=2.1 Hz, H-3), 4.09 (2H, s, -CH,-); ”C-NMR (€19,019) 6 202.66 (CO), 164.80 (0.2), 164.52 (0.4), 155.26 (04), 132.65 (C-l'), 129.51 (c-2', C-6'), 125.38 (C-6), 114.58 (03', 05), 111.72 (C-l), 107.43 (C-S), 101.87 (C-3), 42.83 (-CH,-). DADZEm.——-N,N-dimethylformamide dimethyl acetal (0.5 ml) and THF (0.5 ml) was added to a pressure-resistant vial containing compound 1 (40.9 mg). The reaction mixture was heated in a microwave for 2 min at medium energy, yielding a red solution Methanol (2 ml) was added to the reaction product and evaporated to dryness in vacuo. The crude producttlmsobtaindwaspurifiedbypreparative'ILC (CHClJMeOH 9:1) and recrystallized from aqueous methanol to give daidzein (27.8 mg, 71 %); mp. 290°C (decomposed); 1H- NMR (dg-DMSO) 6 8.27 (1H, s, H-2), 7.95 (1H, d, l= 9.0 Hz, H-5), 7.36 (2H, d, J= 6.6, 1.8 Hz, H-2', 6'), 6.92 (1H, dd, J= 8.7, 2.4 Hz, H—6), 6.84 (1H, d, J= 2.1 Hz, H—8), 6.79 (1H, d, J= 6.6, 1.8 Hz, H-3', 5'); l3C-NMR (dc-DMSO) 6 174.64 (C-4), 162.44 (C-4'), 157.38 (C-8a), 157.11 (C-7), 130.00 (C-2', C-6'), 127.22 (C-5), 123.46 (C-3), 122.52 (C-l'), 116.62 (C-4a), 115.05 (C-6), 114.90 (03', 05'), 102.04 (08). 4-ME‘IHOXYBENZYL 2,4-DIHYDROXYPHENYL KETONE, 2.—The same synthetic procedure was employed as in compound 1 by substituting 4—methoxyphenylacetic acid (2.2 g) instead of 4-hydroxyphenylacetic acid. The corresponding ketone was yellow needle-like crystals (2.47 g, 51%); mp. 159-163°C; ‘H-NMR (CD,OD) o 7.94 (1H, d, J= 9 Hz, H-6), 34 7.20 (2H, d, J= 8.4 Hz, H-2', 6'), 6.87 (2H, d, J= 8.7 Hz, H-3', 5'), 6.33 (2H, dd, J= 8.8, 2.7 Hz), 6.25 (1H, d, J= 2.1 Hz, H-3), 4.20 (2H, s,-CH,-), 3.71 (3H, s, ~0Me); ”C-NMR (03,019) 6 202.40 (CO), 164.89 (0.2), 164.64 (0.4), 157.99 (04), 133.49 (C-l), 130.43 (C-2', C-6'), 126.93 (C-6), 113.79 ((2.3; (2.5), 112.40 (C-l), 108.20 (05), 102.44 (C-3), 54.94 (-OMe), 43.16 (-CH,-). FW.—Usingthesameprocedmeasfordaidzein, compound 2 (40 mg) was converted to formononetin The crude precipitate was recrystallized fiom aqueous methanol to giveformononetin (32.3 mg, 91%); ‘H-NMR (do-DMSO) o 8.30 (1H, s, H-2), 7.95 (1H, d, J= 9 Hz, H-5), 7.49 (2H, d, J= 8.4 Hz, H-2', H-6'), 6.97 (2H, d, J= 8.7 Hz, H-3', H-5'), 6.92 (1H, dd, J= 8.7, 2.1 Hz, H-6), 6.84 (1H, d, J= 1.8 Hz, H-8), 3.76 (3H, s, -OMe); ”C- NMR (dg-DMSO) 6 174.50 (04), 163.67 (04'), 158.89 (C-8a), 157.57 (0.7), 152.87 (C-2), 130.00 (C-2', C-6'), 127.09 (C-S), 124.34 (03), 123.04 (C-l'), 116.02 (C-4a), 115.56 (C-6), 113.54 (C-3', C-5'), 102.00 (C-8), 55.10 (-OMe). 4-HYDROXYBENZYL 2,4,6—‘1'RII-1YDROXYPHENYL KETONE, 3.—Phloroglucinol (1.0 g) and 4-hydroxyphenyl acetonitrile (1.1 g) in ether (10 ml) was cooled in an ice bath and saturated with a stream of HCl gas (HCl was produced by reacting NaCl and cone. 11,804). Thereactionmbcturewasrefiigeratedfor 12h, saturated againwithHClgasand refi'igerated foranother 12 h. Aafier decanting the ether, the precipitatewaswashed furtherwith ether. The white precipitate thus obtained was refluxed with 2% aqueous HCl (20 ml) for 3 h and cooled. The solution was extracted twice with ether (50 ml each), and the organic layer was neutralized with saturated NaHCO, solution The ether was removed in vacuo to give yellow 35 needle-like crystals (1.0 g, 46.5%); mp. 258-262°C; lH-NMR(CD,OD)15 7.05 (2H, d, J= 8.1 Hz, H-3', H-S'), 6.69 (2H, d, J=8.1, H-2', H-6'), 5.80 (2H, s, H-3, H-S), 4.26 (2H, s, - c112); 1’0.th (CD,OD) 6 205.08 (CO), 166.24 (C-2, C—6), 164.80 (0.4), 156.90 (C-4'), 131.66 (02; C-6'), 128.20 (C-l'), 115.98 (C-3'. C-5'), 95.83 (C-3, C-5), 49.54 (-CH,-). GEMS’I‘EIN.—-BF3 etherate (1 ml) was added to a solution containing DMF (2 ml) and compound3(50mg)inabeaker. Thereactionmixturewas heatedinamicrowavefor 15 sec using low energy, followed by the addition of methanesulfonyl chloride (CH,SOzCl, 1 ml). The resulting product was heated in a microwave again for 1 min at low energy. A ight yellowish precipitate, obtained by the addition of water (100 ml) into the reaction mixture, wascentrifilgcd, washedwithwater(10mlx3) and recrystallizedfiom aqueous methanolto give genistein (43 mg, 80 %); mp. 291-296°C; lH-NMR (dG-DMSO) 6 8.30 (1H, s, H-2), 7.35 (2H, dd, J= 6.6, 1.8 Hz H-2', H-6'), 6.80 (2H, dd, J= 6.6, 1.8 Hz, H-8), 6.21 (ll-I, d, J= 1.8 Hz, H-6); l3C-NMR (dg-DMSO) 6 180.68 (C-4), 164.77 (C-4'), 162.47 (C-5), 158.06 (C- 88), 157.86 (C-7), 154.44 (C-2), 130.64 (C-2', C-6'), 122.75 (C-3), 121.69 (C-l'), 115.51 (C- 3', 5'), 104.93 (C-4a), 99.43 (C-8), 94.13 (C-6). 4-MEIHOXYBEN2YL 2,4,6—1‘RlHYDROXYPI-IENYL KE'I‘ONE, 4.—Using 4-hydroxypherlyl acetonitrile (1 g) and phloroglucinol (1 g), a synthetic procedure similar to that used with compound 3 was employed to synthesize Compound 4, yellow plate-like crystals (0.88 g, 47 %); mp. 195-197°C; lH-NMR (CD,OD) 6 7.15 (2H, d, J= 8.7 Hz, H-2',H-6'), 6.83 (2H, d, J= 8.4 Hz, H-3', H-S'), 5.80 (2H, s, H—3, H-S), 4.89 (2H, s, -CHz-), 3.76 (3H, s, -OMe); 1’C- NMR (CD30D) 6 205.0 (CO), 165.80 (C-2, C-6), 165.75 (C-4), 159.77 (C-4'), 131.67 (C-2', 36 C-6'), 130.22 (C-l'), 114.71 (C-3; C-5'), 105.29 (C-l), 95.87 (C-3, C-5), 49.84 (-CH,-). BIOCHANIN A—Biochanin A (45.2 mg, 86%) was synthesized fi'om compound 4 (50.5 mg) using the same procedure as for genistein. mp. 180—184°C; lH-NMR (dg-DMSO, 300MHz) o 8.36 (1H, s, H-2), 7.48 (2H, d, J= 8.4 Hz, H-2', H-6'), 6.99 (2H. d. J= 8.7 Hz, H-3', H-S'), 6.38 (1H, d, J=2.4 Hz, H-8), 6.22 (1H, d, J= 2.1 Hz, H-6), 3.77 (3H, -OMe); ”C- NMR (erMso, 500MHz) 6 180.07 (C-4), 164.29 (C-4'),l61.97 (06), 159.15 (C-8a), 157.56 (07), 154.17 (C-2), 130.15 (C-2', 06'), 122.90 (03), 121.95 (C-l'), 113.69 (C-3', C-5'), 104.45 (C-4a), 98.99 (C-8), 93.67 (C—6), 55.135 (-OMe). Results and Discussion 4-Hydroxybenzyl 2,4—dihydroxyphenyl ketone (1), as needle-like crystals (88%), was synthesized by refluxing resorcinol and 4-hydroxyphenylacetic acid with boron trifluoride etherate (BF3-Et20) (Figure 3.2). The ABX pattern in A ring of l and a singlet of 2 protons ofthe benzylic methylene group at 4.09 ppm were confirmed by the 1H-NMR spectrum. The synthesis ofthis ketone was reported by Shriner and Hull (1945) and Yoder et al. (1954) by saturating a solution Of resorcinol and 4-hydroxyphenyl acetonitrile in ether with dry HCl over a period of three days. Pelter and Foot (1976) repOrted that the cyclization of l to daidzeincanbeachievedbyrefluxing l withN,N-dimethylforrnamide dimethyl acetal inDMF for 3 h, yielding 76 % daidzein. Using N,N-dimethylformamide dimethyl acetal and THF as the solvent, daidzeinwasobtained(71%)fiom l undermcdium microwave energyfor2 min, which gave an overall yield of 57 %. To prevent the evaporation ofN,N-dimethylformamide dimethylacetalduringthecyclizationofl,thereactionwascarriedoutinasealedvial. The 37 < £5385 £8350 ago—88.8..— $83.5 . 8:26—33 mags—H62 .«O 285:.3 «.«. oSwE u—stV 3:6qu 0: Odes— xQ ., rm £8 20 mo 2O :6 :08 mo :80 rm am :0 =0 :0 38 resrrltingproductafierrecrystallintion, didnot showthe—CH,- signalappearedat4.09ppm inketonel. TheH-23ignalwasat68.2,asasinglet,inits‘H-NhtIRspectrum Both lH-and L"C-»NMR spectra of the product were identical to an authentic sample of daidzein Similarly, 4-methoxybenzyl 2,4-dihydroxyphenyl ketone (2) was synthesized (51 %), asinthecaseofcompound l, byreplacingthe starting material ,4-hydroxyphenylacetic acid, with 4-methoxyphenylacetic acid. Using N,N-dimethylformamide dimethyl acetal and THF asthesolvent, formonomtinwasproduccd (91%, overall 45 %) bythe cyclization of2 under medium microwave energy for 1 min, as in for daidzein. The 1H» and 13C-NMR spectra of 2 were similar to l and contained the signal for a methoxy group. The 1H-- and l3C—NMR spectra of formononetin were identical to the published data (Nair et al., 1991). Syntheses of ketones 3 and 4, for genistein and biochanin A synthesis, respectively, were conducted by modification of the procedure reported by Yoder et al. (1954). The 4- hydroxybenzyl 2,4,6—trihydroxyphenyl ketone (3) was produced by bubbling dry HCl into a solution of phloroglucinol and p-hydroxyphenyl acetonitrile in dry ether, with 46 % yield. Compound 3 gave distinct singlets of two protons each at 6 6.69 and 6 4.26 for H-2' and H- 6' and the methylene protons, respectively, in the 1H-NMR spectrum. The cyclization of 3 with BF, etherate in DMF and metlulnesulfonyl chloride (Bass, 1976) also was accomplished in a microwave oven for 2 min at low energy and afl‘orded genistein in high purity (80 %, overall 36 %). The structure of genistein was confirmed by 1H- and l3C-NMR spectra Using identical procedures used for the synthesis of 3, 4-methoxybenzyl 2,4,6- trihydroxyphenyl ketone (4), for biochanin A synthesis, was prepared (47 %) fiom p- 39 methoxyphenyl acetonitrile and phloroglucinol as the starting materials. Similarly, the cyclization of 4 to biochanin A (86 %, overall 40 %) was carried out in a microwave-oven usingthesameconditionsasusedinthegenisteinsynthesis. Thestructuresof4and biochanin A were confirmed by their ‘H- and l3C-NMR spectra Microwave conversion of ketones 2, 3, 4, to formononetin, genistein and biochanin A afl‘orded superior yields than the reported values of 85 % ( Pelter and Foot, 1976), 74 % (Y Oder et al., 1954) and 65 % (Bass, 1976), respectively. The microwave conversion of the ketoneltodaidzeinwith71 %yieldiscomparabletotheyield reportedbyPelterandFoot (1976). The overall yields for the isoflavones are not available fiom published reports. However, the overall yield of isoflavones daidzein, formononetin, genistein and biochanin A under microwave conditions were 57, 40, 36 and 40 %, respectively. Our synthesis of these isoflavones has the advantages of reduced cost and time consumption. For example, the 4- hydroxyphenylacetic acid used for the synthesis of 1 is considerably cheaper than its corresponding nitrile. The syntheses of these soybean isoflavonoids, especially genistein and daidzein, in substantial quantities using less expensive reagents in a very short time facilitate their in vivo evaluation as anticarcinogens for human medicine. CHAPTERIV' Metabolism of Daidzein And Genistein Anticarcinogens by Intestinal Bacteria Abstract Isoflavones, daidzein {1} andgenistein {2}, were fermented with human fecal bacteria under anaerobic conditions. Dihydrodaidzein {3}, benzopyran-4,7-diol, 3-(4-hydroxyphenyl) {4} and equol {5} were isolated fi'om the fermentation broth of daidzein Only one metabolite, dihydrogenistein {6}, was isolated and characterized fi'om the fermentation broth of genistein. The metabolites 3 - 6 were identified by spectral methods. ' Accepted for publication by Journal of Natural Products 40 41 Introduction Endogenous and exogenous chemicals such as estrogens (Jarvenpaa et al., 1980), androgens (Lambardi et al., 1978), and safllower yellow B (Meselhy et al., 1993) are metabolizedbyintestinalbacteia. Thebiologicalactivitiesofthese compounds canbealtered dramaticallybythemetabolismbyintestinalbacteria. Ofienthesemetabolitesaremutagenic (Shu et al., 1991; Gorbach and Goldin, 1990). Soybean products are an integral part of human diet in Asian countries. It was implicated that soybean, especially the isoflavones daidzeinandgelisteinpresertinit, mayprovideprotectionagainstbreast cancerand filnction as anticarcinogens (Aldercreutz, 1988). The animal metabolism studies of daidzein and genistein present in soybean diets were canied out in sheep, rats and humans. However, the metabolism of pure daidzein and genistein in humans is not reported yet. Several isoflavone metabolites have been detected in urine collected fi'om human subjects on soy diets. They were identified as dihydrodaidzein 0-desmethyl angolensin, glycitein, 6'-hydroxy-0-desmethyl angolensin, equol dihydrogenistein, and dehydro-O- desmethyl angolensin ((Braden et al., 1967; Axelson and Setchell, 1981; Axelson et al., 1982‘”; Bannwart er al., 1984; Bannwart et al., 1988 ; Kelly et al., 1993; Yasuda er al., 1994) as glucuronide or sulfate conjugates (Axelson et al., 1982"; Yasuda er al., 1994). Equol was the major metabolite identified in the urine of sheep, guinea-pigs (Shutt and Branden, 1968) and humans (Axelson et al., 1982'). Equol also was detected in the fermentation broth of soy protein incubated with human fecal bacteria (Setchell et al., 1984). In another study, the germ-free rats did not excrete equol upon feeding soy diets, whee as 42 conventional rats’ urine contained equo1(Axelson and Setchell, 1981). This suggested that intestinal bacteria metabolized the isoflavone daidzein in soy diets to equal. Flavonoidsque'cetin, kaempfe'olandnaringeninlmde'wert C-ring cleavage at C-3 and C-4 upon the incubation with Clostrr'dr'ran strains isolated from human intestinal bacteria (Winter et al., 1991). A similar fragmentation of isoflavonoids by intestinal bacteria is not known. p-Ethyl phenol was the major metabolite of genistein in the urine of ruminants (Batte'harn er al., 1965), but not in human urine (Kelly et al., 1993). Using gc-ms, Kelly et al. (1993) confirmed the presence ofdihydrodaidzein and tetrahydrodaidzein in human urine. Dihydrodaidzein was detected in the urine fi'om all human subjects studied, whereas tetrahydrodaidzeinwasfoundonlyintheurinefiomasinglenlbject. Also, only one intwelve subjects excreted dihydrogenistein in a trace amounts. These data suggested that the metabolism of daidzein and genistein vary in humans. It is important to note that there are no published data on the intestinal metabolism of isoflavones, an important class of antioxidants present in many foods. Therefore, we have conducted an in vitro metabolism study of daidzein and genistein using human fecal bacteria Experimental GENERAL—1H and ‘3 C-NMR spectra were recorded on a Varian VXR 500 MHz spectrometers in CD,OD solution at ambient temperature. Mass spectra were acquired on a JEOL HX-l 10 double-focusing mass spectrometer (JEOL,Tokyo, Japan). Sep-Pak cartridges (C-18) were purchased fi'om Waters (Milford, Massachusetts, USA). HPLC 43 analyses were performed with an HPLC system equipped with automatic gradient controller, autosampler and photodiode array detector (PDA) (Waters, Milford, Massachusetts, USA). Recycling Preparative HPLC (LC-20) and C-18 reverse phase column (Jaigel, S-343-15; 15 pm, 250 x 20 mm) were purchased fiom Japan Analytical Industrial Ltd., (Tokyo, Japan). 018 reverse phase capcell pak columns (AG-120 S-S um, 30 % carbon loading 5 11m, 250 x10 mm and AG-120 S-5 11m, 30 % carbon loading, 5 11m, 250 x 4.6 mm) were purchased from Shiseido Co. Ltd. (Tokyo, Japan). CHEMICALS AND WAHON MEDIA—Daidzein and genistein were synthesized in our laboratory (Chang et al., 1994). Compounds 3, 5 and 6 were isolated firm the fermentationofboth l and2 andusedas standardsforthequantification ofthese compounds in the fermentation media. BHI dehydrated media was purchased fiom Difco Lab (Detroit, MI, USA) and vitamin K heme, cystine chloride and resazurine were purchased fi'om Aldrich Chemical Company (Milwaukee, WI, USA); IN mROANAEROBIC FERMENTA‘I'ION.—Commercially available BHI media (3.7 g / 25 ml) was supplemented with vitamin K (20 ul/ 100 ml) and heme solution (1 ml / 100 ml media from 50 mg / 100 ml stock solution) and mixed with 5 mg of the isoflavone, daidzein orgenistein. Cystine chloride (50 mg/ 100 ml) and resazurine (0.4 ml/ 100 ml mediafiom 25 mg / 100 ml stock solution) were used as reducing agent and O2 indicator, respectively (Holdmen et al., 1977). The pH of the media was adjusted to 7 with 1N NaOH solution and autoclaved for 15 min under anaerobic conditions. Fresh human feces (1 g) was suspended 4 R=H » 5 R=H Figlue 4.1 Metabolites isolated fiom fermentation broth of daidzein and genistein incubated with human fecal bacteria 45 in pro-reduced BHI media (10 ml). Pro-reduced supplemented BHI media (25 ml) was inoculated with the fecal suspension (0.5 ml) and incubated for 3 days at 37’C under anaerobicconditions. Afier3 daysofincubation, 1 mlofthefementationbrothwassamplcd and analyzed by HPLC. The remaining fermentation broth was lyophilized for the isolation and purification of metabolites. PURlFICATIONOF FERMENTA‘I‘ICN PRODUCTS.—_-Fementation broth of daidzein (125 ml) was lyophilized, and the resulting solid was extracted with hexane/CHCl, (1:1, 25 ml x 2). The hexane/CHCl, extract was discarded. The residue then was extracted with MeOH (25 ml x 2). The MeOH extract was evaporated to dryness under vacuum and the residue was purified on a recycling preparative HPLC using C-18 reverse phase column The solvent system, MeOH/H,O, 60:40, was used as the mobile phase under isocratic condition at a flow rateof3 ml/min ThemetabolitesweredetectedrmderuvatZlOnm Thefractions containing isoflavoneanditsmetabolitesweepmifiedfintherbyI-IPLC onaC-18 reversephasecapcell pak column using MeOH/Hzo, 40:60, as the mobile phase at a flow rate of 1 ml/min The compounds were monitored by a photodiode array detector (PDA) at 210 nm. Lyophilized fermentation broth of genistein (100 ml) was purified by vacuum liquid chromatography with CHCl,,MeOH, 4: 1, as the solvent system. Fractions containing isoflavones and their metabolites wee combined and purified by recycling preparative HPLC on a C-18 reverse phase colunm using MeOH/Hzo, 60:40, as the mobile phase at a flow rate of 3 ml/min The metabolites were monitored under uv at 210 nm. HPLC ANALYSIS OF ISOFLAVONES AND mam summaries—The fermentation broth 45 (1 ml) was passedthrough a C—18 Sep-Pak cartridge which was pro-conditioned with MeOH (5 ml) and H20 (10 ml). The cartridge then was washed successively with water (5 ml), ACN/[1,0, 30:70, (1 ml), and finally with ACN/Hp, 90:10, (2 ml). The ACN/11,0, 90:10, eluatewasanalyzedforisoflavonesandtheirmetabolitesonaC-IS reversephasecapcellpak column The mobile phase was ACN and H20 under a linear gradient ofACN/H,O 30:70 to 100%ACN(final)in 15 minataflowrate of0.5 ml/min. Thecolumnwaselutedwith 100%ACNforanadditional 10min 'I‘hecompormdsweremonitoredusingaPDAdetector and the data were collected at 200-360 nm and processed to obtain the results at 210 nm. The isoflavones have comparable absorption maxima at 210 and 262 nm. The HPLC analysis ofthe isoflavones and their metabolites were carried out at 210 run, since uv spectra ofthe metabolites showed the absorption maxima at 210 nm. The metabolites formed during the fementation of compound 1 and 2 were monitored by withdrawing samples (1 ml) from the fermentation broth at 24, 48 and 72 h, respectively. Cahbrationcurvesforcompound l -3,5and6weecreatedbyanalyzingtherespective solution by HPLC as mentioned above. The solutions were prepared by the serial dilution of respective stock solutions to afford 0.39, 0.78, 1.56, 3.12, 6.25, 12.5 and 25.0 ug/ml concentration, respectively. Calibration curves were generated by Millenium 2010 chromatograph manager by the following equation: y = A + Bx, where y = response calculated for the standard peak at 210 nm; A = intercept ofcalibration curve; B -= slope of calibration curve; x = the amount of standard, respectively. DIHYDRODAIDZE1N,3.-— ’H-NMR: o 7.74 (1H, d, J=8.5 Hz, H-S), 7.08 (2H, d, J=8.5 47 Hz, H-3', 5'), 6.74 (2H, d, J=8.5 Hz, H-2', 6'), 6.48 (1H, dd, J=8.75, 2.0 Hz, H—6), 6.31 (1H, d, J=2.5 Hz, H—8), 4.57 (1H, dd, J=11.5, 5.5 Hz, H-2a), 4.53 (1H, dd, J=11.25, 8.0 Hz, H- 2b), 3.83 (1H, dd, J=8.o, 5.5 Hz, H-3); “C-NMR' 6 194.07 (C-4), 166.89(C-4'), 165.54 (C- sa), 157.96 (C-7), 130.64 (C-2', 6'), 130.30 (C-S), 128.10 (C-l'), 116.56 (C-4a), 115.11 (C- _ 6), 103.66 (0.3; 5'), 103.48 (C-8), 73.18 (C-2), 52.56 (C-3). BENZDPYRAN—4,7—DIOL, 3-(4-HYDROXYPHENYL), 4.— ‘H-Nm 6 7.82 (1H, d, J=8.5 Hz, H-5), 7.09 (2H, d, J=8.5 Hz H-2', 6'), 6.72 (2H, J=8.5 Hz, H-3', 5'), 6.33 (1H, dd, J=9.0, 2.5 Hz, H-6), 6.22 (1H, d, J=2.0 Hz, H-8), 4.10 (2H, s, H-2). EQUOL, 5.—EIMS m/z (rel. int): 242 (82), 120 (100); ‘H-NMR: o 7.18 (2H, d, J=9 Hz, H-2',6'), 6.88 (1H, d, J=8.0 Hz, H-S), 6.76 (2H, d, J=8.5 Hz, H-3', 5'), 6.33 (1H, dd, J=8.5, 2.5 Hz H-6), 6.24 (1H, d, J=2.5 Hz H-8), 4.19 (1H, ddd, J=10.5, 3.5, 2.0 Hz, H.2a), 3.91 (1H, dd, J=10.5, 10.5 Hz, H-2b), 3.05 (1H, mm, H-3), 2.87 (II-1, dd, J=15.8, 10.0 Hz, H-4a), 2.82 (1H, ddd, J=16.0, 6.0, 1.5 Hz, H-4b); “C-NMK 5 155.70 (C-4' or G7), 155.42 (C-4' or C-7), 154.39 (C-8a), 131.97 (C-l'), 129.25 (C-S), 127.41 (C-2', 6'), 114.52 (C-3', 5'), 112.69 (c-4a), 107.20 (C-6), 101.90 (C-8), 70.29 (C-2), 37.53 (C-3), 31.12 (C-4). DIHYDROGENIS‘I‘EIN, 6.—EIMS m/z (rel. int): 272 (25), 153 (100); lH-NMR: 6 7.10 (2H, d, J=9.0 Hz, H-2", 6"), 6.75 (2H, d, J=9.0 Hz, H—3", 5"), 5.83 (2H, s, H-3', 5'), 4.51 (1H, dd, J=11.5, 5.0 Hz, H-2a), 4.44 (1H, dd, J=11.5, 7.5 Hz, H-2b), 3.83 (1H, dd, J=7.5, 4.5 Hz, H-3); 13C-NMR- 6 196.50 (C-4), 166.71 (C-4'), 164.00 (C-5), 162.92 (C-8a), 156.16 (C-7), 128.84 (C-2', 6'), 125.88 (C-l'), 114.65 (C-3', 5'), 101.48 (C-4a), 95.33 (C-8), 94.10 (C—6), 70.77 (C-2), 49.74 (C-3). 48 Results and Discussion Fermentation studies of endogenous and erogenous chemicals with fecal bacteria have provided valuable information to elucidate their metabolic pathways. The incubation of daidzein with human feces afforded compounds 3 - 5 (Figure 4.1). After 72 h of incubation, the fermentation broth of compound 1 was purified. The band with a higher R, value than daidzein was collected and further purified by HPLC to afi‘ord 3. 1H-NMR spectra of this compound showed the presence of two aromatic rings with an ABX substitution pattern in one ring and para-substitution in the other. The dd signals appearing at 4.57 and 4.53 ppm in3wereassignedtotheH—2 protons. Aoneproton, dd, at 3.83 ppm confirmed the presence ofH-3 in compound 3. The absence of an olefinic proton in 3 , appeared at 8.02 ppm in 1, indicating that the double bond between C-2 and G3 in compound 1 was reduced. Therefore, the lH--NMR data of compound 3 indicated that it is a metabolite of daidzein. The lH-NMRspectraofcompound4 showedthattheprotonsofringAandB in4 gave similar multiplicity and chemical shifts to the ring A and B protons in daidzein. The H-2 singlet appearing at 8.02 ppm in daidzein was absent in 4. Therefore, the olefinic bond between C-2 and C-3 in daidzein was reduced to yield 4. A two-proton singlet at 4.10 ppm in compound 4 was assigned to H-2 protons. This confirmed the presence of a double bond between C-3 and C-4 in 4 and the structure of 4 as benzopyran-4,7-diol, 3-(4—hydroxyphenyl). Athird metabolite, isolated fiomthefermentation broth ofdaidzein and purified by recycling preparative HPLC, was compound 5. The lH-NMR spectral data of compound 5 was identical to the published values for equol (Aldercreutz, et al., 1986‘). 49 The only metabolite isolated fiom the fermentation broth of genistein was compound 6. lH-NMRsignalsof6inthearomaticregionwassimilartothatofgenistein Theabsence ofasingletat8.30 ppmin6indicatedthattheolefinicbondbetwcenC-2andC—3ingenistein wasreduced Threeddsignalsappearedat4.51,4.44and3.83ppmin6weeassignedtoH- 2a, H-2b and H-3 protons, respectively. The production of metabolites during the fermentation of isoflavones l and 2 with humanfecalbacteriawasmonitoredbyHPLC(Appe1dicesIII, IVand V). Compound3 was the major metabolite during the 72-h fermentation of compound 1 (Figure 4.2). Compound Swasthemajormetabolitereportedfordaidzeininlmmanurine fi'om those whose consumed the soy diet. In our studies, the fecal bacterial metabolism of daidzein afi‘ordcd compound 3 in higher yield than 5. This indicated that compound 3 was metabolized further to compound 5 prior to excretion, as evidenced by previous in vitro metabolism studies (Setchell et al., 1984). Since compound 4 was isolated in a very small quantity, the quantification of this compound was not carried out. The estrogenic activity of genistein in ruminants was considerably lower when given intranlminally than intramuscularly (Braden et al., 1967). This implied that the metabolism of genistein by rumen fluid may be responsible for the lack of estrogenic activity. p-Ethyl phenol, the reported end-product ofgenistein metabolism, was detected in the urine of sheep fed with soy diets. This compound was not detected in our fermentation studies when gelisteinwasincubatedwithhuman feces. TheHPLC analysis ofthe fermentationproducts ofgenistein with fecal bacteria showed that the amount of genistein declined rapidly during 50 24 it However, a corresponding increase in the amount of compound 6 was not observed (Figure 4.2). This indicated that genistein was metabolized to several other compounds that were not detected in our study. We did not isolate any other metabolite from the fermentation broth of genistein. 250 '- 200 Concanhflon (up/ml) cub uh or o or o o o l l l ——-.-— 1 + 5 Figure 4.2 51 * ~4- ? l l 24 48 72 Incubation Time (h) —+—-2—+—3 +6 Production of metabolites of daidzein and genistein by human fecal bacteria at 72 h. 1: Daidzein; 2: Genistein; 3: Dihydrodaidzein; 5: Equol; 6: Dihydrogenistein CHAPTER V' Metabolites of Anticancer Daidzein and Genistein And Their Biological Activities Abstract Daidzein and genistein, the isoflavones present in soybean, were synthesized earlier to study their metabolism in humans. The metabolites detected were mostly reduction products of daidzein or genistein We have synthesized these metabolites to evaluate their eficacy. Equol {3}, 5,7,4'-trihydroxyisoflavan {5}, 4,7,4'-trihydroxyisoflavan {6}, dihydrodaidzein {8}, and dihydrogenistein {9}, wee synthesized either fiom daidzein {l} or genistein {2} by hydrogenation and characterized by spectral methods. During acetylation and NMR experiments, compormd 9 was converted to an intermediate enol form, a novel compound 10. Antifungal, antibacterial, mosquitocidal, nematicidal and inhibition of topoisomerase activities of these compounds were evaluated. Equol {3} was the most active compound among the five metabolites assayed. ° Accepted for publication by Journal of Natural Products 52 53 Introduction Several epidemiological studies has shown that soybean products reduced the incidence of breast cancer in women (Messina et al., 1994; Lee et al, 1991). Animal studies also revealed that soybean isoflavonoids daidzein {l} and genistein {2} are responsible for this protective efl‘ect Troll et al, 1980; Barnes et al., 1988; Messina et al., 1994). Singlet oxygen species play an important role in mutagenesis and carcinogenesis, particularly in tumor promotion (Wei et al., 1993). Tyrosine-kinase activity is associated with growth factors which are involved in the uncontrolled growth of cancer cells (Chang and Geahlen, 1992). Genistein has been implicated by inhibiting H202 formation both in vivo and in vitro studies (Wei etal., 1993). Also, it acts as an inhibitor for tyrosine kinase and DNA synthesis (Dean et al., 1989; Sit et al., 1991). Soybean diets were able to lower mammary tumor in rats induced by radiation (Troll et al., 1980) or by the carcinogen, N-methyl-N-nitrosourea (MNU) (Barnes et al., 1988). Isoflavonoids present in soybean are metabolized in the digestive track (Setchell et al., 1984) and are considered to reduce the incidence of breast cancer (Messina et al., 1994). Several metabolites are detected in the urine fi'om human subjects on soybean diet (Kelly et al., 1993). The major metabolites of daidzein {1} and genistein {2} are their reduction products, equol {3}, 7,4'-dihydroxyisoflavanone {8}, tetrahydrodaidzein {6} and 0-desmethyl angolensin and 5,7,4'-t1ihydroxyisoflavanone {9}. The biological activities of these metabolites, isolated from physiological samples in trace quantities, have not been evaluated. We have now synthesized compounds 3, 6, 8 and 9 to evaluate their biological 54 activities. Thesynflredsofisoflwmonesandisoflwmsfiomdaidzeinandgemstdnweereponed earlier (Inoue, 1964; Szabo, 1973; Lamberton et al., 1978). Hydrogenation reactions of isoflavones wee non-selective and often resulted in complex mixtures (Inoue, 1964; Szabo, 1973; Lamberton et al., 1978). A selective catalytic hydrogenation of several isoflavones to their corresponding isoflavanones was published (Krishnamurty and Sathanarayana, 1986). Daidzein {l} and genistein {2} were not used to produce isoflavanones by previous researches (Krishnamurty and Sathanarayana, 1986). In this paper, we report the synthesis of several hydrogenation products of daidzein and genistein and their biological activities. Experimental GENERAL—‘H- and 13C-NMR spectra were recorded on a Varian VXR 300 and 500 MI-Iz spectrometers (V arian, California, USA), respectively, at ambient temperature. The melting points were recorded on a Thomas model 40 micro hot-stage apparatus and were not corrected. Mass spectra were acquired on a JEOL FIX-110 double focusing mass spectrometer (JEOL, Tokyo, Japan). Preparative silica gel TLC plates were purchased fi'om Analtech Inc. (Newark, Delaware, USA). Recycling preparative HPLC LC-20 and C-18 reverse phase column (Jaigel, S-343-15; 15 11m, 250 x 20 mm) were purchased fiem Dychrom (Santa Clara, California, USA) CHEMICALS AND CELL CULTURE mama—Daidzein {l} and genistein {2} were synthesized in our laboratory (Chang et al., 1994). Pd/C and Pd/BaSO, (5%) were purchased 55 fiemAldrichCherficalCompanylemrkceWrsconsin, USA). YMG (yeast extract4 g/L, maltose 10 g/L, glucose 4 g/L and agar 12 gm, PDA (potato dextrose agar) and Emmons (neopeptone 10¢,glucoseZOglLandagar15 g/L)mediaweepreparcdaspublished (Nair et al, 1989) and the ingredients were purchased fiom Difco Lab (Detroit, Michigan, USA). NG medium (NaC13.0 yL, bacto peptone 2.5 g/L, cholesterol 1 ml/L fiom 5 mg/ml stock solution, CaCl2 1 ml/L fi'om 1 M stock solution, MgSO, 1 ml fiom 1 M stock solution, and potassiumphosphatebufi‘eZS ml/L ofstock solution containing KH2PO, 11.97 g/lOOml and KJIPO4 2.09 g/100 ml). YPDAmedium (yeast extract 20 g/L, peptone 10 glL and dextrose 20 g/L, and adenine sulfate 2 ml/L fiom 0.5% stock sohrtion) were purchased from Difco Lab (Detroit, Michigan, USA). Adenine sulfate and camptothecin were purchased fi’om Aldrich Chemical Company (Milwaukee, Wisconsin, USA). HYDROGENATION OF ISOFLAVONES.—A solution of daidzein {1} or genistein {2} in EtOH orglacial acetic acid was flushed with H2 for 15 min and then added to a pro-reduced ethanolic orglacial acetic acid sohrtion containing 5% Pd/C. The reaction mixture was stirred at room temperature under H2 atmosphere until the sioflavone was not detectable on silica gel TLC. The reaction mixture was filtered through a celite bed and the resulting solution was dried under vacuum. EQUOL {3}—The product fi'omthe hydrogenation ofdaidzein {1} in glacial acetic acid was recrystallized fiorn MeOI-I/H,O to yield 3 (46.7 %); mp. 150-152°; EIMS m/z (rel. int): 242 (82), 120 (100); lH-NMR (CD,OD) 6 7.18 (2H, d, J=9 Hz, H-2‘,6'), 6.88 (ll-I, d, J=8.0 Hz, H-S), 6.76 (2H, d, J=8.5 Hz), 6.33 (1H, dd, J=8.5, 2.5 Hz, H-6), 6.24 (1H, d, J=2.5Hz, 56 H-8), 4.19 (1H, ddd, J=10.5, 3.5, 2.0 Hz, H-2a), 3.91 (1H, dd, J=10.5, 10.5 Hz, H-2b), 3.05 (1H, m, H-3), 2.87 (1H, dd, J=15.8, 10.0 Hz, H-4a), 2.82 (1H, ddd, J=16.0, 6.0, 1.5 Hz, H- 4b); "(l-NMR (CD,OD) 6 155.70 (04' or C7), 155.42 (04' or C7), 154.39 (C-8a), 131.97 (01'), 129.25 (C5), 127.41 (02', 6‘), 114.52 (C-3', 5'), 112.69 (C-4a), 107.20 (C-6), 101.90 (C8), 70.29 (C2), 37.53 (C3), 31.12 (C4). 7,4'-DIME'IHOXYBQUOL {4}—A sohrtion of compound 3 (20.4 mg) in acetone (15 ml) was stirred with K,CO, (5 g) for 15 min and refluxed with dimethyl sulfate (50 111) for 8 h. Thereactionmixturewascooledtoroomtemperamre, theresultingsolutionthenfiltered and the resulting solution was dried under vacuum. The product was dissolved in CHCl, (50 ml) and washed with H20 (20 ml x 2) followed by washing with saturated NaHCO, solution (25 ml x 1) andH,O (25 ml x2). The resulting CHCl, sohrtionwas evaporated under vacuum and the white precipitate was recrystallized from MeOH to give needle-like crystals (16 mg); mp. 112-113"; lH-NMR(CDC1,) 6 7.17 (2H, d, J=8.4 Hz, H-2', 6'), 6.99(1H, d, J=7.8 Hz, H- 5), 6.90 (2H, d, J=8.7 Hz H-3', 5'), 6.49 (1H, dd, J=8.4, 2.7 Hz, H-6), 6.43 (II-I, d, J=2.7 Hz, H-8), 4.31 (II-I, ddd, J=10.5, 3.0, 1.5 Hz, H-Za), 3.98 (1H, dd, J=10.2, 10.2 Hz, H-2b), 3.81 (3H, s, OMe), 3.78 (3H, s, OMe), 3.17 (1H, m, H-3), 2.95 (2H, d, J=8.4 Hz, H-4). 4, 7,4'-TRIHYDROXYISOFLAVAN {6} .—Amorphous powder, produced fi'om the hydrogenation ofdaidzein {1} using ethanol as the solvent (60 %); mp. 204-208°; EIMS m/z (rel. int): 240 (100); ‘H—NMR (CD,OD) o 7.21 (1H, d, J=8.5 Hz, H-S), 7.07 (2H, d, J=8.5 Hz, H-2',6'), 6.72 (2H, d, J=8.5 Hz, H-3', 5'), 6.40 (1H, dd, J=8.5, 2.0 Hz, H-6), 6.22 (1H, d, J=2.0 Hz, H-8), 4.74 (1H, d, J=7.5 Hz, H-4), 4.25 (ll-I, dd, J=11, 3.5 Hz, H—2a), 4.16 (1H, 57 dd, J=10.75, 8.0 Hz, H-2b), 2.99 (1H, ddd, J=7.5, 7.5, 3.5 Hz, H-3); l"’C-N1\dl?t(CD,OD)6 157.42 (C-Sa), 155.62, 155.20 (C-4' and C7), 130.39 (01'), 129.59 (C5), 128.56 (C-2', 6'), 114.86 (C-3',5'), 114.72 (C4a), 108.19 (C6), 101.88 (C-8), 67.70 (C4), 68.23 (C2), 45.96 (C3). 4,7,4'-TR1ACEI'YLISOH.AVAN {7}.—To a solution of compound 6 (9.8 mg) in pyridine (1 ml), acetic anhydride (200 11]) was added and left in the dark at room temperature for 24 h. The reaction mixture was dried under vacuum and purified by preparative TLC using CHCl,/McOH, 12:1, as the mobile phase to give amorphous white solid (14.6 mg) ; mp. 115- 117°; ‘H-NMR(CDC1,) 6 7.34 (2H, dd, J=8.5, 2.5, H-2',6'), 7.22 (1H, dd, J=8.5, 2.5 Hz, H- 5), 7.04 (2H, dd, J=8.75, 2.5 Hz, H-3',5'), 6.68 (1H, ddd, J=8.5, 2.5, 2.0 Hz, H-6), 6.65 (1H, d, J=2.5 Hz, H-8), 6.13 (1H, dd, J=5.25, 2.5 Hz H-4), 4.45 (2H, m, H-2), 3.39 (1H, m, H-3), 2.26 (6H, s, CH,- of C-7, 4'), 2.05 (3H, s, CH,- of C-4) DIHYDRODAIDZEIN {8}.—The reaction mixture from the hydrogenation of daidzein {1} inethanol ande/BaSO,waspurified onarecycling preparative HPLC (C-18 column, mobile phase H,O:MeOH 30:70 at a flow rate of 2 ml/min, detected at 210 nm) and yielding compound 8 (41.4 %) as the major product; mp. 198-200°; ‘H-NMR (CD,OD) 6 7.74 (1H, d, J=9.0 Hz, H—S), 7.07 (21-1, d, J=8.5 Hz, H-3', 5'), 6.74 (2H, d, J=9.0 Hz, H-2', 6'), 6.50 (ll-I, dd, J=8.75, 2.0 Hz, H-6), 6.33 (1H, d, J=2.5 Hz H-8), 4.56 (1H, dd, J=11.5, 5.5 Hz, H-2a), 4.52 (1H, dd, J=11.25, 8.0 Hz, H-2b), 3.82 (1H, dd, J=8.0, 5.5 Hz, H-3); 13C-NMR (CD,OD) 6 194.07 (C-4), 166.89(C-4'), 165.54 (C-8a), 157.96 (C7), 130.90 C-3', 5'), 130.64 (C-2', 6'), 130.30 (C5), 128.10(C-1'), 116.56 (C-4a), 115.11 (C-6), 103.66 (C8), 58 73.18 (C-2), 52.56 (C3). DII-IYDROGENISTEIN {9}.—The reaction product fi'om the hydrogenation of genistein in {2} ethanol and Pd/C was purified by preparative TLC using solvent system (10:1) CHCleeOH The major component was recrystallized fi'om MeOH/H,O and gave colorless needle-like crystals, compound 9 (67 %); mp. 196-198"; EIMS m/z (rel. int.): 272 (25), 153 (100); lH--NMR (CD,OD) 6 7.09 (2H, d, J=8.5 Hz, H-2", 6"), 6.75 (2H, d, J=8.5 Hz H-3", 5"), 5.88 (2H, 8, H3, 5'), 4.51 (1H, dd, J=ll.5, 5.0 Hz, H-2a), 4.47 (1H, dd, J=11.5, 7.75 Hz H-2b), 3.86 (1H, dd, J=7.75, 5.5 Hz, H—3); 13C-NMR(CD,OD) 6 196.50 (C-4), 166.71 (C-4'), 164.00 (C5), 162.92 (C-8a), 156.16 (C7), 128.84 (C-2', 6'), 125.88(C-1'), 114.65 (C-3', 5'), 101.48 (C4a), 95.33 (C-8), 94.10 (C6), 70.77 (C2), 49.74 (C-3). 5,7,4'-TR1HYDROXYISOFLAVAN {S} .—The hydrogenation of genistein {2} with Pd/C in glacial acetic acid yielded a mixture of two compounds. The reaction mixture was purified by preparative TLC developed with (10:1) CHCl,/MeOH. The compound with the similar Rfvalue to genistein was identified as compound 9 (17.8 %). The second compound with a low Rfvalue was reaystallized from MeOH/H,O and gave needle-like crystals, compound 5 (27.0 %); mp. 209-211°; EIMS m/z (rel. int): 258 (58), 139 (100); 1H-NMR(CD,OD) 6 7.12 (2H, d, J=8.5 Hz, H-2', 6'), 6.79 (2H, d, J=9.0 Hz H-3', 5'), 5.97 (1H, d, J=2.5 Hz, H-6), 5.86 (1H, d, J=2.5 Hz H-8), 4.18 (II-I, ddd, J=10.5, 3.5, 2.0 Hz, H-Za), 3.89 (1H, dd, J=10.0, 10.0 Hz H-2b), 3.02 (1H, m, H-3), 2.88 (ll-I, ddd, J=16.0, 5.5, 2.0 Hz H-4a), 2.61 (1H, dd, J=16.0, 10.75 Hz H-4b); l3C-NMR (CD,OD) 6 155.43 (C-4'), 155.36 (C-S), 155.13 (C-8a), 155.10 (C7), 132.45 (C-l'), 127.49 (C-2', 6'), 114.59 (C-3', 5'), 101.09 (C- 59 4a), 94.37 (C-8), 93.95 (C-6), 70.08 (C-2), 37.10 (C-3), 25.82 (C-4). 4,5,7,4'-TE'1‘RAHYDROXYISOFLAVANONE {10} .—Compound 9 (12 mg) in d,-pyridine (0.75 ml)wastreatedwith d4-acetic acid (100121) in anNMRtube and its ‘H—NMR spectrum was recorded; ‘H—NMR (d,.pytidine and 100 pl of d,-acetic acid) 6 7.20 (2H, d, J=8.7 Hz, H-2', 6'), 7.05 (2H, d, J=8.7 Hz, H-3', 5'), 6.36 (1H, d, J=1.8 Hz, H-6, 8), 4.48 (1H, d, J=12.0 Hz H-2a), 4.43 (1H, d, J=12.0 Hz, H-2b). 4,5,7,4'-TEI'RAACETA'I'EISOFLAVANONE {11 } .—Acetic anhydride (200 111) was added to a solution of compound 9 (20 mg) in pyridine (1 ml) and stored in the dark at room temperature for 24 h. Crushed ice was added into“ the reaction mixture and the white precipitate formed was isolated by centrifugation. This precipitate was then recrystallized from MeOH to give colorless needle-er crystals, compound 11 (22 mg); mp. 191-192°; ‘H-NMR(CDC1,)6 7.36 (2H, d, J=8.7 Hz H-2', 6'), 7.11 (2H, d, J=8.7 Hz H-3', 5'), 6.66 (1H, d, J=2.1 Hz H-6), 6.47 (1H, d, J=2.4 Hz H-8), 5.03 (2H, s, H-2), 2.31 (3H, s, CH,), 2.29 (3H, s, CH,), 2.27 (3H, s, CH,), 2.10 (3H, s, CH,); l3C-N1vfl{(CDCl,) 6 168.77 (C=O), 167.97 (C=O), 167.92 (C=O), 167.29 (C=O), 156.13 (C-8a), 150.72 (C-4'), 149.83 (C-S), 145.38 (C-7), 136.14 (C-4), 130.74 (C-l'), 128.58 (C-2', 6'), 121.31 (C-3', 5'), 111.29 (C-3), 109.88 (C8), 107.54 (C-6), 68.55 (C2), 20.62 (2 x CH3), 20.49 (CH3). 20.08 (CH,). Antimicrobial assay—Antifimgal and antibacterial assays of compounds 1, 2, 3, 5, 6, 8, 9 and 11 were carried out according to the procedure reported earlier (Nair et al., 1989). Cultures of F usan’wn oaysponan (MSU-SM-1322), Fusarr'ran monilrfonne (MSU-SM- 1323 ), Gleosporum spp. and Rhizoctom’a spp. were grown on potato dextrose agar (PDA) 60 medium Cultures of Candida albicans and Aspergillusflam (MSU strains) wee grown on YMG medium, and cultures of Swivlocxars spider-mitts (ATCC 25923), Steptococcus aureus(MSU strain), andEschericln’acoIi(ATCC 25922) were gown on Emmons medium. Cell or spore suspension of the test organisms were made by adding 10 ml of sterile safimsohrfionmafilflygowneunnemaPeuidishandsfiningwithaglassrodgenfly. The cell concentration was adjusted to 10‘ colony forming units per milliliter (CFU/ml). Bioassay platesweemadebyspreadingevenlyofthecellsuspension(100 ul)inPet1idishescontaining appropriate medium (20 ml). The test compound (250 ugZS 111 of DMSO) was spotted carefully in the center of the bioassay plates and the plates were incubated at 27°C for 72 h mmofmhibidonchamaeizedbytheabsmceofnnemrgammngowth,msmeawred. MOSQUTI‘OCIDAL AND NEMATICIDAL ASSAYS.—The mosquito larvae, Aedes aegwtii, (Michigan State University, courtesy of Dr. Raikhel) were used to test the insecticidal property of compounds 1, 2, 3, 5, 6, 8, 9 and 11 using the procedure reported earlier (Nair et al., 1989). About 200 mosquito eggs, (Aedes aegptr’i), were placed in 500 ml distilled and sonicated water and hatched. Fifteen mosquito larvae (4 days old, 4th instar) were placed in 980 u] of water in a test tube. DMSO (20 111) solutions containing various concentration of test compounds were added into each test tube. Control received pure DMSO (20 111). The test tubes were covered and left at room temperature. The number of dead larvae were recorded at 2-, 4-, 6-, 24-, and 48-h intervals. Each treatment was repeated in triplicate. Nematicidal activity was carried out on Panagrellus redivr‘vus and Caenorhabditrs elegans using the procedure reported earlier (Nair et al., 1993). The nematode suspension 61 (NO medium) (48 111) containing 20-30 nematodesatvarious developmental stageswere transfercd into each well of a 96-well tissue culture plate. Test compounds 1, 2, 3, 5, 6, 8, 9andllinDMSO(2ul)wereaddedtoeachwellandmixedgently. Eachtreatrnentwas repeatedmuiphcate.1hemouflmedplatesweehddmahunudchmbemdmonafitywas recorded at 2-, 4-, 6-, 24-, and 48-h intervals. TOPOISOMERASE ASSAYS.-—Saccharamyces cerevr‘siae mutant cell cultures, JN394, JN394 t1 and JN394 t“, were supplied by Dr. John Nitiss of St. Jude Children’s Research Hospital (Jannatipour et al., 1993; Nitiss et al., 1993). JN394 is hypersensitive to topoisomerase I poisons due to the mutations that destroyed the RAD52 repair pathway. JN3 94 t1 is isogenic to JN394 except that top] gene is deleted. The deletion of top! gene results inthelack ofthe response to topoisomeaselpoisons. JN394 t,,, the cell culture that carries the tap2-5 gene, is resistant to the topoisomerase II poisons but responds to the topoisomerase I poisons. The organisms were cultured in Petri dishes containing YPDA medium (20 ml). The cells fi'om a fully gown plate wee suspended in saline solution (10 ml). The cell suspension was diluted to obtain 5 x 10‘ CFU/ml. YPDA liquid media (1.95 ml) wee inoculated with 25 111 of the cell suspension (5 x 106 CFU/ml) from IN394, JN394 t1 and JN394 ,t, , respectively. Test compounds, daidzein {l}, genistein {2}, dihydrodaidzein {8} and dihydrogenistein {9}, were dissolved in DMSO and were added to the test tubes (25 1.11) to give the final concentration at 250 ppm. Each treatment was repeated in triplicates (data not shown). The positive control, a top-I poison, camptothecin, was tested at 10 ppm Since 62 equol {3} showed an excellent activity in the preliminary plate assay, it was tested at concentrations of 100, 50, 25, and 10 ppm. The test tubes containing cell cultures and compoundswereincubatedat27°for24h Attheendofincubafionpeiodaserialdilution ofeachcellsuspensionwasprepared. Analiquot(100 ul)fi'omeachdilutionwasspread evenlyonaPetridishcontainingYPDAmediaandincubatedat27°for72h Thcnumber ofcoloniesweecountedattheendofthe incubationperiod and evaluated fortheactivityof test compounds (Figure 5.1). Results and Discussion Equol {3} (Figure 5.1) was produced by the hydrogenation ofdaidzein {l} in glacial acetic acid with Pd/C as the catalyst (Appendix VI). The disappearance of C-ring olefinic proton chemical shift at 6 8.27 indicated that the olefinic bond between C-2 and C-3 of 1 (Chang et al., 1994) was reduced. Also, the hydrogenation product gave l3C-NMR chemical shifts at 6 70.27, 37.53 and 31.12 which were assigned to C-2, C-3 and C-4, respectively. The absence ofC=O signal in the 13C-NMR spectrum of 3 confirmed it as equol. Methylation of 3 afi‘orded a dimethoxy product, 4. Compound 3 was previously synthesized by the reduction of 0,0-diacetyl-daidzein followed by the hydrolysis of the resulting product in ethanolic NaOH (Lamberton et al., 1978). Aldeereutz et al. (1986”) reported the synthesis of equol {3} by the hydrogenation of daidzein {1} in EtOH. In our laboratory, this procedure yielded only 4,7,4'-trihydroxy- isoflavan {6} (Appendix VI). TheH-Z proton of6 in its 1H-NMR spectrum appeared as two dd at 4.25 and 4.16 ppm, respectively. The ddd at 2.99 ppm in 6 was assigned to the H-3 63 R, R. R. R. R. OH H OH 3 OH H OH H OH OH OH 4 OMe H OMe H s OH OH OH H 6 OH H OH OH 7 OAc H OAc OAe R, , O R2 R4 R.’ a R. R. R. R. R. R. OH H OH 10 OH OH OH OH OH OH OH 11 OM OM OM OM Figure 5.1 Reduction products Of daidzein and genistein 54 proton. 'I’he"C-NMRspectrumof6gaveasignalat667.70andwasassignedtoC-4which confirmedthatthe C=Ogoupindaidzein {l} waspartiallyreduced. Acetylation of6 gave a triacetate, 7, and provided an additional evidence for the existence of C-4 -OH goup in 6. The hydrogenation of daidzein {1} over Pd/BaSO4 in EtOH yielded 8 as the major product (Appendix VI). l3C-NMR of compound 8 showed a signal at 6 194.07 which indicate that the C=0 group at C-4 was not reduced. Also, lH-NMR of8 did not give the olefinic proton signal at 6 8.02. The H-2 protons appeared as dd at 4.56 and 4.52 ppm, respectively, and H-3 proton as dd at 3.82 ppm Therefore, the NMR data confirmed the edsterceofaC=OgoupatC-4andthereduction ofthe olefinic bond between C-2 and C-3 in compound 8. Compound9wasproducedbythehydrogenationofge1istein {2} inethanol overPd/C (AppendixVII). 'I'hiscompoundgaveRwahlesimilarto genistein {2} on silica gel TLC and its structure was confirmed by 1H and13 C-NMR spectra However, hydrogenation Of genistein {2} inglacialaceticacidoverPd/C yieldedamixtureofcompoundsSand9dueto partial hydrogenation (Appendix VII). Compound 5 had a lower R{ on TLC than 9. The structure of compound 5 was identified as 5,7,4'-t1ihydroxyisoflavan. Acetylation of compound 9 in pyridine and acetic anhydride gave interesting results. Purification and characterimtion ofthe acetylated product 11 confirmed the presence offour hydroxylgoupsincompormd ll eventhough compound 9 had onlythree-OH goups. The H-2 proton of 11 in its 1H-NMR spectrum appeared as a 2H singlet at 6 5.03. The signals at 136.14 and 111.29 ppm were assigned to the olefinic carbons C-3 and C-4 formed by the as enolization of the C-4 carbonyl goup. Thetetlaacetate ll wasyieldedfi'omtheenol 10. Thisindicatedthat during acetylation condition the entire keto form was converted to the enol, compound 10. The enolization of 9 was also observed during NMR experiment with d,-py1idine as the solvent. The addition of 100 111 of d4-acetic acid to a d,-pyridine solution of 9 caused an instant enolization of 9 to 10. The H-2a and H-2b signals of 10 appeared as doublets at 4.48 and 4.43 ppm, respectively. However, the enol form was unstable and converted completely to the keto form during isolation and purification. The same was true when the tetraacetate, 11, was hydrolyzed to produce compound 10. The enolization of compound 9 was absent in CD,OD/d,-acetic acid and observed only in d,-pyridine as the solvent. The corresponding isoflavanone 8 did not enolize in d,-pyridine and d,-acetic acid. The enolization of 9 in pyridine/acetic acid may be induced by the 5-OH goup in the A-ring complexing with the pyridine solvent. It is possible that 10 can exist in biological systems as a metabolite of genistein {2}. Compound 3 showed gowth inhibition for F usarr'um oxym, F usariran monilg‘forme, Gleoqrorum app, Rhizoctonia app. and Aspergr'llusflavrar (all fungi), Candida albicans (yeast) and bacteria Staphylococcus epidermidis, Streptococcus arrests, and Escherichia coli at 250 rig in plate assays. Mosquitocidal (Aedes aegwtr’r’ larve) and nematicidal (Panagrellus redivr'vus and Caemrhabditzls elegans) assays with 3 showed 100% mortality at 250 ppm within 24 h. Compound 9 inhibited the gowth of all microorganisms tested at 250 11g concentration, excluding A. flaws and E coli. Mosquitocidal or nematicidal 66 activitieswere not observed for 9. Compounds 1, 2, 5, 6, 8, and 11 were not active against bacteria, firngi, yeast, mosquito larvae or nematodes tested. Genistein {2} is reported to have topoisomease II inhibitory activity (Markovitis et al., 1989; Corbett et al., 1993). Therefore we have evaluated both topoisomerase I and II activities for compound 1 - 9 using mutant yeast strains JN394, JN394 t1 and JN394 5,, (Jannatipour et al., 1993; Nitiss et al., 1993). In our experiments, the inhibitory concentration (1C,o ) for genestein {2} was 250 ppm for JN394. Also, it inhibited the gowth OfJN3 94tl and IN 3 949,, by 30%. Compounds 4 - 7 did not show topoisomerase inhibition in preliminary plate assay. Camptothecin, a topo-I poison, was used as the positive control against JN394 and JN394 t,_, at 10 ppm. At this concentration it showed 97% inhibition for the gowth of JN394 and JN394t,,,. Carnptothecin did not inhibit the gowth of JN394tl (Figure 5.2). Compounds 8 and 9 inhibited the gowth OfJN394 t,,,, but had no efl‘ect on JN394. Compound 3 inhibited the growth of these yeast strains with IC,(, at 50ppm (Figure 5.3). Various compounds are identified as topoisomerase inhibitors (Constantinou et al., 1995; Kawada et al., 1995). These include camptothecin and etOpocide, the topoisomerase I and II inhibitors, respectively. Drugs, acting as DNA topoisomerase inhibitors, will produce cleavable DNA-topoisomerase-drug complex and cause site-specific cleavage of chromosomal DNA (Liu, 1990). This event will inhibit DNA replication, RNA synthesis and cell division which eventually leadsto cell death (Liu,1990). Cancerous cells contain larger quantities of topoisomerases, therefore, the inhibitory activities of certain chemicals on topoisomerases 67 have attr’actedageatdealofinterestincancerresearchrecently(Lh1,I990). Ourresults revealed that equol {3} inhibited the gowth of JN394 and JN394 t,,, at 50 ppm concentration and indicated that it is similar to other topoisomerase I drugs (Jannatipour et al., 1993; Nitiss et al., 1993). 68 130 104 ' Control 78 ' a - Camptothecin O 52 " ;/ ’///////. Equal Z? /// , 9' 0 an V/lfi a”; . JN394 JN394 t1 JN394 12-5 100ppm of camptothecin and equal Figure 5.2 Effect ofcamptothecin and equol at 10 and 100 ppm, respectively, on the cell gowth of yeast strains JN394, JN394 t, and JN394 t,, * Cell numberx 100 69 150 120 . é — JN394 g 90- 0 ---e 5 .lN394t1 6 a 60 ’ —-— JN394 12.5 a: 30 ~ 0 0 20 40 60 80 100 120 Concentration of equol in ppm Figure 5.3 Efi‘ect of equol at various concentration on the cell gowth of yeast strains JN394, JN394 t1 and JN394 t,,, ----------- CHAPTERVI Summary and Conclusions The third chapter of this dissertation describes the synthesis of anticarcinogenic isoflavones, genistein, daidzein and their 4-methoxy derivatives, biochanin A and formononetin These compormds wee previously synthesized by other researchers, however, the synthetic procedures were time consuming and costly. We have developed a two-step synthetic route for the production of daidzein and genistein which reduced both time and expenses dramatically. In the first step, the corresponding ketones for daidzein and genistein were produced by Friedal-Crafi acylation reactions using commercially available starting materials, resorcinol, phloroglucinol, 4-hydroxyphenyl acetic acid and 4-hydroxyphenyl acetonitrile, respectively. The second step involved the cyclization of the ketone by heating it with N, N-dimethylforrnamide dimethyl acetal and THF in a conventional microwave oven for 2 min to yield daidzein at 71 % yield. Similarly, genistein was synthesized by the cyclization of its corresponding ketone by heating with DMF, methanesulfonyl chloride and BF, etherate in a conventional microwave oven for 2 min. The yield of genistein was 80 %. Several isoflavone metabolites such as equol, O-desrnethyl angolensin, dihydrodaidzein, 6'-hydroxy-0-desmethyl angolensin, dehydro-O-desmethyl angolensin, benzopyran-4,7-diol- 3-(4-hydroxyphenyl) and tetrahydrodaidzein and dihydrogenistein were detected in the urine fiom human subjects on soy diet. The pathways for the formation of isoflavone metabolites have not been studied. In order to confirm the hypothesis that the isoflavone metabolites detected in human urine were the results of intestinal bacterial metabolism, genistein and daidzein were incubated, respectively, with human feces under anaerobic conditions for 72 70 71 h. The metabolites produced were isolated and characterized by 1H-NMR spectra Three metabolites, dihydrodaidzein, benz0pyran-4,7-diol-3-(4-hydroxyphenyl) and equol, were isolatedfi'omthefemertationbrothofdaidzein These metabolites were previously detected in human urine by other researchers. Dihydrogenistein was the only metabolite of genistein reported in the urine from human subjects on soy diets. We have isolated dihydrogenistein fi'omthefermentationbrothofgenisteinwith human feces. Another metabolite ofgenistein, p—ethylphenol, found in the urine of sheeps was not detected in the urine fi'om humans. Similarly, we did not isolate this compound fiom the fermentation broth of genistein with human feces. Thisindicatedthatmetabolismofgenisteinweenotsimilarinhumansand sheeps. The metabolites formed during the fermentation of daidzein and genistein with human feces during 72 h period were monitored. The results indicated that the amount of daidzein decreased considerably in 24 h Dihydrodaidzein, the major metabolite of daidzein, was detected within 24 h Equol was detected only in smaller quantities at 72 it Therefore, it is possible that equol was derived from dihydrodaidzein. However, additional experiments are required to confirm this hypothesis. Even though the level of genistein declined rapidly in 24 h, a corresponding increase in dihydrogenistein was not observed. This suggests that genistein was metabolized to other compound(s) which were not identified in our studies. The metabolites isolated from fermentation broth of daidzein or genistein with human feces were in small quantities and were not suficient to carry out biological studies. Therefore, they were synthesized from either genistein or daidzein by hydrogenation using Pd/C as the catalyst in glacial acetic acid or ethanol as the solvent. The 1H-NMR spectra of these synthetic products were identical to the compounds isolated fiom fermentation broth of daidzein and genistein. Also, antibacterial, antifungal, mosquitocidal, nematicidal and anticancer activities of these synthetic metabolites were determined. Antibacterial activity of isoflavone metabolites were carried out on Staplpvlococcus epidermidis, Streptococcus aureus and Escherichia coli. Equol inhibited the gowth of all the bacteria tested at 250 11g Dihydrogenistein inhibited the gowth ofStqzln'Iococcus and 72 Streptococcus but not on E. coli at 250 pg. Other metabolites did not show antibacterial Test fungi, Fumriwn W0", Fusrm'wn moniliforme, Gleam-um 3p. Rhizoctonia spp. and Asperigillus flavus were used to determine antifirngal activity of the isoflavone metabolites. Equol inhibited the gowth of all the fungi tested at 250 11g. Except on A. flaws, dilrydrogenisteininhibitedthegowthoffimgitested at 250 pg. Othermetabolites did not show antibacterial activity. Equol was also active in Candida albicans at 250 pg Equol gave 100 % mortality when tested on mosquito larvae, Aedes aegwtii, and nematodes, PanagreIIu rediviws and Caenorhabditr's elegans within 24 h at 250 ppm. Other isoflavone metabolites did not show mosquitocidal or nematicidal activities. MutantSaccharono’cescereviHae strainsweeusedto determinetheanticanceractivity of the isoflavone metabolites. Topoisomerases are the enzymes that catalyze the interconversion oftopoisomers. This interconversion is a crucial step in living organisms for DNA replication, RNA and protein synthesis. The interference of topoisomerase activity will eventually lead to cell death. Cancerous cells proliferate continuously and hence, contain more topoisomerases. Therefore, topoisomerase poisons are potential anticancer drugs. In the anticancer activity assay, three mutant S. cerevisiae strains, JN394, JN394tl and JN394t,_ ,, that carry difi'erent genotype and respond to top-I or top-II poisons were used. All the DNA repair genes have been deleted from the mutant S. cerevisiae strains which makes them sensitive to DNA damage caused by UV light or chemicals. JN394tl was derived fi'om JN394 and with the tap-I gene deleted. Therefore, the gowth of IN 3 94tl is not inhibited by top-I drugs. JN394t,,, was similar to JN394 and carrying the mutant top-II gene. Therefore, JN394t,_, is resistant to top-II drugs. The known top-I poison camptothecin was used as the positive control Equol showed a similar inhibition pattern as camptothecin in the assays. All other metabolites showed weak or no activity in this assay and hence additional eficacy studies were not conducted for these compounds. The synthesis ofgenistein, daidzein, biochanin A and formononetin in large quantities- allowed us to conduct further research on these isoflavones as anticarcinogens and 73 antioxidants. Compoundspresentiningested foodscanbemetabolizedatvariousmetabolic sites such as liver and intestine. Our fermentation studies of genistein and daidzein with human feces confirmed the hypothesis that the isoflavone metabolites of daidzein and gemstempresentmsoybeandeteaedmhummufineweredaivedbymtesfinalbaaeda Various biological activities of the metabolite equol have been reported by other researchers. 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Lyophilized 2.Extracted with Hexane/CHCI, 1:1 (25 m1 x 2) l ' . Hexane/CHCl, Extract; Residue . Dis 1 l Extracted with MeOH (25 ml x 2) Residue; MeOH Extract Discarded I Purified by recycling preparative hplc (LC-20) on a reverse phase C-18 column with MeOH/H,O, 60:40, 3 ml/min under isocratic condition Fr. 1 Fr. 2 Fr. 3 Fr. 4 Fr. 5 Fr. 6 Fr. 7 Purified by Purifiedby . Semi-preparative hplc 533““me hplc on a reverse phase C-l8 column “81118 the same condition with ACN/11,0,40:60,1m1/min as formpomds 1 m3 under isocratiI condition I 1 3 4 85 APPENDIX II Isolation and purification of genistein and its metabolites fi'om the fermentation broth Fermentation Broth (50.5 mg in 100 ml media) 1. Lyophilized 2. Extracted with MeOH (150 ml x 2) MeOH extract Residue; Discarded 1. Dired under vacuum 2. Suspended in CHCl/MeOH 4:1 3. Silica gel VLC (50 g) CHCl/MeOH CHClp’LlcOH CHCl/McOH CHCl/MeOH 4:1, 20 ml; 4:1, 50 m1 4:1, 100 ml 1:01, 300 m1; Discarded L I Discarded 1. Dried under vacuum 2. Purified by Recycling Preparative hplc reverse phase C-18 column with MeOH/Hp 70:30, 3 mein under isocratic condition I 7 2 6 AU APPENDIX III HPLC ofdaidzeinandgenisteinandtheirmetabolites 1.00: 1.60- 5 1.40- 1.20- 6 1 3 1.00- . 0.80- 2 0.50- 0.40- - ‘ 0.20 moo-R i I ' a.'oo' ' ioloo ‘ izloo ' 14:00 ' isloo ' isloc ' 201065 22:06 ' asloo Minutes HPLC condition: Column: C-18 reverse phase column (4.6 x 250 mm) Mobile phase: ACN and 11,0 under linear gradient of ACN/1110 30:70 to ACN 100%(final)in 15 min. ThecolumnwaselutedwithACN 100% for an addition 10 min. ‘ Flow rate: 0.5 ml/min Detection: Monitored by a PDA detector and the data was collected at 200 - 360nmandprocessedtoobtaintheresultsat210mn 1: Daidzein; 2: Genistein; 3: Dihydrodaidzein; s: Equol; s: Dihydrogenistein 87 APPENDIX IV HPLC of the fermentation broth of daidzein 0.50- A0 0.20- 0.10- 0.00- 1o.oo' ' '12100' ' '1sloo' ' '16100' ' '1sloo' ' '2oloo' ' '22100' . Hirsute: HPLC condition: Column: C-l8 reverse phase column (4.6 x 250 mm) Mobile phase: ACN and 1120 under linear gradient of ACN/[LO 30:70 to ACN 100 °/o (final) in 15 min. The column was eluted with ACN 100 % for an addition 10 min. Flow rate: 0.5 ml/min Detection: Monitored by a PDA detector and the data was collected at 200 - 360mnandprocessedtoobtaintherenrltsat210nm. 1: Daidzein; 3: Dihydrodaidzein; 4: Benzopyran-4,7-diol, 3-(4-hydroxyphenyl); 5: Equal 88 APPENDIX V HPLC of the fermentation broth of genistein 1. 00- 0.30- 0 . 60- 0. 40- 2 o. 20- 5 [L 0'00: ‘ 'A/\0 ‘1 OJ ‘Ll LK-L 10.00' .' '12100' '14200' ' '1sloo' ' '1sloo' ' '2oloo' ' '22100' Minutes HPLC condition: Column: C-18 reverse phase column (4.6 x 250 mm) Mobile phase: ACN and H20 under linear gradient of ACN/11,0 30:70 to ACN 100 % (final) in 15 min. The column was eluted with ACN 100 °/o for an addition 10 min. Flow rate: 0.5 ml/min Detection: Monitored by a PDA detector and the data was collected at 200 - 360 nm and processed to obtain the results at 210 nm. 2: Genistein; 6: Dihydrogenistein 89 APPENDIX VI Hydrogenation scheme for daidzein Pd/C, H, GlacialAceticAcid RT. 24 h H0 0 4,7,4'-Trihydroxyisoflavan Pd/BsSO EtOH RT., 21 h H0 0 .. 3. ...S. mom i .23 gag—FEE.§KW Engage—vain ._ 3.. ..S. :8. ..z .02 8 aged 2% 38... 386 _ $.93 Sofie—ow com 080:8 massage—i: => EQZmnE/w IV. LIBRRR ES MICHIGAN STATE UN \llllWIm“INI‘IWWWW 31293014 1111111 mi 172427