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bkirogcntc Activity ot Wi Analogs ana rolycmorinatea bipnenyis Joel Bitman* and Helene C. Cecil Because of the geometric similarity of DDT to the synthetic estrogen, uilbcstrol, DDr and 52 related compounds were tested in a sensitive estrogen assay In rnls. Estrogenic activity wax evaluated using the |g-hr glycogen response or the immature rat uterus. Diphcnylmcthime, diph nylcthanc. and triphcnyl. methane compounds wet : active when a p- of/>'-posilion was unoccupied or occupied by an hydroxy or methoxy group. Halide or Alkyl groups in the ?,/>' positions rendered the compounds inactive. Poly chlorinated biphenyls and polychlorinated tri phenyls, compounds which ore environmental pol lutants of industrial origin, were cstrogcnitalfy active. Phcnolphthalol and phenofphthalcin, com pounds which arc used as laxatives in drug prepara tions, were also cstrogenically active since they con tain the appropriate />,/>'-dihydro*y structures. Stereo models indicated that p.p'-dihydroxy com pounds of the active nuclei would have internudear distances or the hydroxyl groups which would ap proximate those of the natural steroidal estrogens and the synthetic stilbene estrogens. n 1945 Solmiscn published an excellent and comprehen (p-chloropheny!)cthylene; p,p'-DDMU, l-ch(oro-2,2-bis- I sive 117-page review of the synthetic estrogens and the (p-chlorophcnyOcthyicne: and p,p'-DDA, 2,2-bis(/vchlororelation between their structure and activity. In the phenyOaccticacid. 25 years since Solmsscn's review, DDT, a chlorinated hydro Table II: e.p'-DDT, l,l,l-trichloro-2-(/>-chlofOpheny|).2- carbon with a geometric similarity to the synthetic estrogens, (o-chlorophenyl)elh*ne; o.p'-DDE, 1,1 -dichloro-I^/xl 'oro- has been widely used throughout the world for pest control. phenyl),2<(o-chlorophcnylX'thyiene; n.p'-DDMU, I-ch 'oro- The recent demonstration by Welch ei at. (1969) of the estro 2-0>-chlorophenyl), 2-(r>-chlorophenyl)eihylcne; o,/'-DDD, genic activity of o,p'-DDT and our own investigations (Bit- I, I -dichloro -2 -(p-chlorophcnyl), 2 (o - chlorophcnyl)cthane; man ft at., 1968) have prompted us to investigate a scries of m/.DDD, 1,1 -dichloro-2-(p-chlorophenyl),2-(m-chloro- DDT analogs, homologs, and structurally related compounds phenylXIhore and p.p'-Mcthoxychlor, l,l,Mrichloro-2,2-bis in an attempt to determine relatirnships of structure to estro (p-mcthoxyphenylXthanc. genic activity. Table III: Compound 20,1,1-Diphcnylmcthanc; 21,1,1* METHODS Dichloro-1,1 -diphcnylmcthanc; 22, 1,1 -bis(p-bromophcnylX methane; 25, Kphcnyl)-l-(/>-hydroxyphcnyl)rnrthane; 24, We used the sensitive 18-hr glycogen response of the rat 1,1 -bis(p-hydroxyphcnyl)mclhanc; 25, l-(p-hydroxyphcnyl)- uterus at n measure of estrogenic activity (Bitman ft at,, 1-O-methoxyphcnyDmclhane; 26, MphcnyO-Mp-mcihoxy- 1965). The potency of active compounds is reported in o-hydroxyphcnyl)mcihane; 27, l,l*bis(o-hydroxypbcnyl)- terms of the minimal subcutaneous dose which will increase methane; 28, l,1-bis(n*hydroxy*m-chIofophenyl)mcthnnc; glycogen to a level significantly different from'control. The 29, KphcnylXl-(p<hlorophenylH-methanoi; 50, l.l-bis(p- 18-hr glycogenic response is illustrated in Figure 1, in which chk>rophcnyl)-l-mcthanol; 51, 2,2'-dihydroxybemophcnooc; the dose-response curve for o,p'-DDT is represented. The 32, 2,4-dihydroxvbcnzophcnone; 33, 4,4'-dihydroxybcnzo- steeper response line for glycogen, as compared to uterine phenone; and 34, 2t2'-dihydroxy-4,4'-dimc(hoxybenzophe- weight, Is readily apparent. none. Test substances were dissolved in olive oil or an aqueous Sources of the compounds used in this study were: Nos. ethanol solution and injected subcutaneously at a screening 4, 5, 16--Rohm and Haas, Philadelphia; 9, 13--Dr. G. F. dose rate of 8 mg per rot. Immature female Wistar rats Fries, U.S. Dept. Agr., BcItsviHc; 17--E. K. Du Pont de (21-25 days old; 56-48 g)were kilted 18 hr after the injection; Nemours & Co., Inc., Wilmington; 18--Sigma Chemical Co.. uteri were quickly excised, weighed, and analyzed for glycogen St. Louis; 24, 27, 35, 42--K A K Laboratories, Inc., Plain- by the enthrone procedure (Setflcr ei at., 1950). Substances view, N.Y.; 38--Eastman Kodak Co., Rochester; 45-33-- showing activity were tested further at dosage levels to 0.05 Monsanto Co., St. Louis. All other compounds were pur mg. Statistical comparisons were made using Student's t test chased from the Aldrich Chemical Co., Inc., Milwaukee. with correction for unequal group size. Purity, as given by the manufacturers, was better than 99%. ABBREVIATIONS ` Table I: p,p'-DDT, l,l,t-(richloro-2,2-bis(p-chlorophcnylXihane; Tctrachloro-DDT, I,1,1,2-tclrachloro-2,2-bisOnrhlorophenyitethanc; p.p'-DFDT, l,l,l-trichloro-2,2-bis(p-fliorophenyl)clhanc; p.p'-Pcrthane, 1,1,1 -trichloro-2.2btrip'dhylphenylXlhnnc; p,p'-Kdthanc, M,l-lrichlofo-2,2bh(/KhlorophcnylX`thane: p,/>'-DDTF, l,l,I-trifluoro-2,2Msfp-chlorophcnylX'fhanc; p,p'-DDD, !,!-dichloro-2,2-bis(p-chlorophcnylX'thanc; p.p'-DDE, l,l-dkhk>ro-2,2-bii* Animal Husbandry Research Division, Bdtsville. Md. 20705 * To whom correspondence should be addressed. RESULTS AND DISCUSSION The natural estrogens are steroids which contain a phenolic ring A and an oxygen function at the C position, while the synthetic estrogens, which are stilbene derivatives, contain two phenolic rings (Figure 2). It is apparent that active eslrogcnicity is dependent upon the presence of al least one phenolic hydroxy ring structure. In most estrogen texts thine compounds arc active in the microgram or stibmicrogrnm range. , In contrast to this, the chlorinated hydrocarbon pesticides related to DDT are only active as estrogens in milligram amounts, a 1000-fold difference. The DDT analogs arc not phenolic, but they may give rise to aromatic phenolic sub stitution during metabolic conversions in the animal. The IIP9 l AQR. POOD CHEM., VOL. It. NO. , 1970 HONS 087005 FlBr* I. DoM-rttpwiM retationshlpr uterine weight, (Jycofen, and o.p'-DDT DDT analogs ore compounds or the diphcnylcihanc typo (Figure 2). Other analogs tested were compounds of the diphcnylmcthnnc or triphcnylmethnnc series. We have also examined polychlorinated biphenyls and polychlorinated tri phenyls, compounds which have become increasingly im plicated as environmental pollutants of industrial origin. W have not included in this study any steroids, synthetic estrogens, or anti-estrogens of the Miibenc structure, and have excluded almost all compounds of the coumarin. isoflavone, anthracene, and phenanthrene type. DtPHENYLETHANB COMPOUNDS ^'Psdtlsm Occupied by Halide or Alkyl. The com pounds evaluated in Table I arc diphenyiethane derivatives in TeMe I, Diphenyiethane Compounds with p,p'*Poettlom Occupied by Halide or Alkyl Groups R Ns, X R R' Name 1a 2a aH --cct, -ecu p,p'-DDT Tctrachloro-DDT 1p H -CCI, />,/>'-DFDT a4 CHiCHt H -CCI, p.p'-Pcrthane $ OH --CCI, P,p'-Kd(hane 6a 7a H -CF, p,p'-DDTF H --CHCt, p.p'-DDO a 9 Cal 10 a --ca, /,*'DDE ~CHC! p.p'-DDMU h' -COOH p,p'-DDA . * M.B.D. minimum effective dose. * t - inactive. Activity MJE.D.* mg 4 4 F 1* I* F 1* P P P which the p.p'-positions arc occupied by halide or alkyl groups. Almost all were dpvoid of estrogenic activity; p,p'DDT (empd I) and tctrachloro-DDT (empd 2) exhibited a slight glycogenic response. It appears that halide or alkyl substitutions in the /,p'-posiiion* were stable, and during me tabolism in the animai body, little if any p,p'-phenolic hydroxy compounds are produced. p- rp,p' Position Occupied by --H or--OCH. When one of the para positions of the aromatic ring is substituted by a hy drogen or methoxy group, the compound exhibits estrogenic - activity (Table II). Potency is of a low order of magnitude, being approximately 1000 times less active than compounds of the stilbene series, but is similar in potency to coumarin and isoflavone estrogens (BickolT et a/., I960). The minimum effective dose (MED) of dlcthylsiilbestrol which elicited a glycogen response was 0.1 pg, as compared to the most active compound of Table II, o,p'-DDT, empd It, whose MED was 0.23 mg. The phenolic character of the natural and synthetic estro gen* has demonstrated the dependency of estrogcnicity upon the presence of a phenolic structure. The aromatic rings of the active compounds of Tabic It are open, i.e., they have a p- or p'-position occupied by -- H and may give rise to phenolic substitution during metabolism. There also appears to be a requirement for the cthano chain to be inert, f.e, either the Irichlorocthanc (-CH-CCU) or the vinyl halide group (> CCU) must also be present (empds II, 12,13). Thus, empds 14,13, and 19, containing more reactive 2-carbon chain con figurations, are inactive, even though one of the aromatic rings could be hydroxylated to the phenolic structure. We have concluded that rapid in vivo metabolism of these compounds ia responsible for their lack of activity. In the stiibestrol series (Solmssen, 1945) and in the coumarin aeries (Bickoff ei a/., I960) of estrogens, p,p'-dimethoxy com pounds are less active than comparable p,p'-dihydroxy com pounds. In the chlorinated diphenyiethane series (Table II), mw p,p`inethoxy compound, methoxychlor (empd 17) was HONS 0 8 7 0 0 6 TfeMtSL Dlphaiylitbat Compounds wttfc or /tVPtiHlw OwiyM by --H or --QCH. p'<0>-c-^>p R' Cmm Nat 9 p* . o ~R------------ R7 11 H i> H 13 H 14 H 15 H ~cai a a a aa H -ecu -CCI, a --CHC1 a H --CHC1, m*Cl H -CHC1, mH HH 17 OCH. OCH. H H --ecu H -ecu 18 19 OCH, H OCHi H *H'-a H H -ecu --CHO o-Cl M.E.D. miitimurn effective dose 11 inactive. Name a,p'-DOT .p'*DDE cp'-DDMU o.p'-DDD m.p'-DDD l.1,I*Trchloro-2,2-bis(phenyl)ethene p.p'-Methoxychlor Tech. Methoxychlor (/./'- + o,p'-) 2,2-Bii(oehlorophenyl)acetaldehyde Activity M.E.D.* mg 0.25 4 8 1* P 1 4 1 1* approximately it active at other compounds which might give rile lo phenolic hydroxy substitution on metabolism. Tech- leal methoxychlor (empd 18), which may contain an <?,/'mathoxychlor, was four times mire active than pure p,p'- MPHENYLMBTHANE COMPOUNDS; MNBOPHBNONE COMPOUNDS A series of diphcnylmcthane compounds was examined to determine structural correlates of estrogenic activity (Table III). Active compounds contained either one or two p-hydroxy or p-methoxy groups (empds 23, 24, 25, 32, 33, 34). -- ------ -- - - - * Table 111. Dlphcnybnethanc Compounds; Banaophenona Compounds o p<0^-c-<^p Na. 9 Gramm 9' k Activity fc1 M.K.D.* mg . Diphenytmethane derive!ivas JO H 21 H 22 Br 23 H M OH 25 OCH, 25 OCH* 27* H 28< H 29 H 90 a 31 H H H Br OH OH OH H H H a a H HH a ci HH HH H H NH H 'H HH HH H OH H OH 1* P I* 2 1 4 P P 1* P P P Banzophenone derivatives 32 H OH 33 OH OH 34 OCHi OCH, 2 2 4 * M.B.D. minimum effective dot*. * 1 - inactive. *bU(n-hy> draayphtnyi)mstlMM. 4 biitwhydroxy, mcMorophenyl}msthane. The most active compound was p,p'-dihydroxydiph< nylmethane which elicited a glycogenic response at the I mg dose level. Soimssen (1945) reported activity for this compour d at the 100 mg level, but the differences in bioassay proced ires could explain part of this difference in result. In the diphenylethanc scries, compounds with e /?-hydrrgcn and a stable ethane chain were metabolized to active estrogens, probably containing a p-hydroxy structure, in contrast, di phcnylmcthane compounds with a p-hydropen were not active, probably being metabolized rapidly at the methane linkage and excreted from the body. Denzophcnonc derivatives, which contain the more stable ketone structure at the methane carbon, were active if a p-hydroxy was present (Tab)c HI). DIPHENYL PROPANE COMPOUNDS; TRIPHENYL METHANe COMPOUNDS Two diphenylpropane compounds were active (empds 35 and 36), the p,p'-hydroxy compound exhibiting much greater activity than a p,p'-dimcthoxy compound. Dihydroxy di phenyl propane (empd 35)wasasactivea&o,p'-DDT. Soims sen (1945) found that this compound was active at a 100 mg dose level. Since the p,p'-dihydroxy structure appeared to be the struc ture conferring activity, phcnoiphlhaloi, a phenyl substituted diphenylmethane compound containing p,p'-dihydroxy groups was tested. Phenoiphthalol was as potent as any compound of the types studied. Ring closure, as in phenoiphthalcin, resulted in a 20-fold loss in potency. These compounds are not known to have estrogenic activity and are extensively used as laxatives in a number of drug preparations. In Table IV two miscellaneous derivatives which bear some relation to closed ring diphenylmethane structures are in cluded: ftuorene and 9,10-dimethylamhrnccne. Both of these compounds were inactive when tested at dose levels up to 8 mg per rat. BIPHENYL AND TRIPHENYL COMPOUNDS v Two hydroxy biphenyl compounds were active but only at the 4 and 8 mg dose levels (Table V). In a series of poly chlorinated biphenyls, the compounds containing up to 48% chlorine were active. A; judged from glc chromatograms, It 10 AOft. FOOD CHEhi.. VOL. NO. v, M M0NS 08700? 0-0: H-H: IITIANC Mit tut DlfMlNYUTHAHI 0-0: H-H: mX io.X SfHKINC t*.X imX -c-P' DieMINYlMITMANC Ml DirHINTtnOTAHI -jf* TRIfHINYIMITHANV A< ItFHCNYl 0-0: Atoft t.fX TRimiNYl Aioft 14.2X ftlYHCNYI A to ft r.z X H* K: nuX IAOX r.iX 0-0: AioC 4iX H-H: r.oX AioC toiX uiX Figure 1. Structural formulae of eatraginte compound* these products are crude mixtures containing a number of compounds. A polychlorinated triphcnyl containing 42% chlorine was found to be more active, at a I mg dose level. CORRELATIONS BETWEEN CHEMICAL STRUCTURE AND BIOLOGICAL ACTIVITY Schuelcr (1946) and his coworkers (Fisher el a/., 1952; Keniling and Schueler, 1950) have theorized tfat a rather large, rigid, lipoid soluble molecular structure with two active hydrogen-bond forming groups located at an optimum dis Atance of 14.S units from each other would be estrogenic. They further stated that potency is decreased as the distance between group* it decreased or increased. While DDT possesses a relatively large, rigid, lipoid soluble molecular constitution, it docs not present active hydrogen A,atoms at the hypothesized optimum distance of 14.5 how ever. The presence of the electronegative chlorine atoms in the /v'-orienwtions would prohibit the existence of active h)drogun, If these p.p' chlorine atoms were metabolized to groups possessing active hydrogen, the possibility of estrogen action would exist. The general lack of estrogenic activity of /,/>'-DDT analogs suggest* that such metabolism docs not occur readily in the biological situations studied thus far. Conversely, the activity of n./'-DDT raises interesting theoretical relationships between chemical constitution and estrogenic activity. The ./>' chlorine atoms are not at the hypothesized optimum distance. The exact nature of (he active estrogen structure arising from o.p'*DDT, if it is not Vi'.DDT itself, might provide important information relating to the spatial configuration of an active estrogen. Drdding Stereomodcls were constructed of many of the active structures to determine whether consistent stereochemi cal factors were present. Internuclear distances were mea sured and were given in Figure 2 for both O to O atoms of assumed dihydroxy compounds, and for the H to H distance nf the hydroxyl groups. Interatomic distances for the Dreiding models of e&iranc and stiibene were found to be much Table JV* , WphWjbropana and TnpnenylmcthaM CwnpoMM Activity No. Formula CM, 35 2.2-BWp-hy- 0.25 droxyphentH, yi)propMa 36 1.3-Dkhloro- 4 U-bistpmethoxy, m-methylpKmyUpropane 37 Phenol- 0.2 phthalol . jsr 31 Phenol- 4 phthaldn 39 Fluorene P 40 MI.E.D. minimum effective dote. 9,10-Dimeth* ylanthraccno P Inactive. HONS 08700* l J. AGR. FOOD CMEM., VOL. IS. NO. 4. WO till T*M V. Biphenyl end Triphenyl Compound! H2> Nfc Name 41 o,ff'-Oiphcnol 2,2'-Dihydroxytfiphenyl 47 p,p'-R(phcnui Poiyehlormnied 4ADihydroxydiphenyl lliphenyl (I'd!) 43 |C1I Aroclor 1221 21% Chlorine 44 PCII Aroctar 12)2 32 % a 45 PCD Atoclnr 1242 42% Cl 46 PCn Aroelor 1248 48% Cl PCU Aroclor 1234 $4% a 48 PCD Aroclor 1260 60% Cl 49 PCD Aroclor 1262 62% U 1 30 PCR Aroclor 1268 68% a 31 PCB Aroclor 4463 60% PCB, 40% polychlori- nated triphenyl (PCI), 63% Cl 32 PCT Aroclor 5442 42% a 33 PCT Aroclor 3460 60*/. CI M.8.0. minimum effective Jose. * 1 inactive. Activity M.E.D.* mg 4 8 8 8 8 8 1* P P 1* I* 1 P Amailer than the 14.5 quoted by Keasling and Schueler A(1950). The 10.9 we found agree closely with the X-ray crystallographic data of Norton rt at. (1965,1964), who found AI0.9S for 173-cstradiol. This discrepancy in interatomic dif ferences may he related to the improved accuracy of the cur rent atomic models, when eompnreri to those used in I9S0. The dlphcnyleihanc, dtphenylmiihanc. diphcnylpropane, IriphenylnH'thftne, biphenyl, and Iriphcnvl compounds all Ahave interatomic distances of 9.4 to 10.3 for the most likely Olo Oaubsiilutions. The II to H internuclvar distances of the Ahydroxyl groups range from 9.1 to 11.1 in these compounds. Both the 0--0 and H--H internuclear distances, therefore, rc only slightly smaller than corresponding bond distance* In natural and synlheliceslrogena. The structural observations regarding estrogenic tc.ivity In the compounds studied indicated that activity ia conferred when a p- or p'-posilion is unoccupied (--H), or it tubatituted by --OH or --OCH *. Halide, or alkyl groups, occupying the p.p'-positions render the compounds estrogenicaJly inactive. A stable ethane chain was found to be necessary for activity, e.g., (he trirhloroClhanc or the inert vinyl halide group; if either C of the ethane chain beara an oxygen function (alco hol, aldcnydu, or acid), the compound it metabolized ami no estrogenic activity is observed. Some polychlorinated bi phenyl and Itiphcnyt compounds exhibited estrogenic activity. Measurements of intcrnuclcar distances of Drcidin s stcric Amodels indicated that active sites would be 9-11 apart, a range similar to those found in natural and synthetic estro gens. Quantitatively similar estrogenic activity was obtained with a series of diphenylmethane or triphenylmethane deriva tives which contained p-OH functions. Correlations of struc ture will activity suggest that the active estrogens derived from o^'-analogs of DDT are p-phenolic metabolites. LITERA/URE CITED Biekoff, b. M,, Livingston, A. L., Book, A, N., Arch. Biothem. H, 262 (I960), Bitman, i, Cecil, H. C, Harris, $. Fries, Q. F., Science 162,371 (1968). Bitman, Cecil. H. C., Mench, M. L., Wrenn,T. R., tndocrtm atagy 71. 63 (1965). Fisher, A. L.. Keasling. H. H.. Schueler. F. W., Free. 5or. i tp. Bin. AM. *.4)9(19321. Keasling. H. ((..Schueler. F. W.,/ Amer. Pharm.Au.i9,17(1950). Norton. D. A.. Karlha, (. , I.u, C. T,, Acta Crytt. 16,89 (1963). Norton. D. A., knrihn, G.. I.u, C. T.. Ana Crytt. IT. 77 (1964). Schueler, F. W,, Science 103, 221 (1946). Scificr. S.. Dayton, S., Novic, U., Muntwylcr, E., Arch. Bhchem. 25, 191 (1930). Sobmsen. U. V.. Chem. Rev. 37,481 (1943). Welch. K. M.. Levin. W., Cooney, A. H., Toxicol. Appt. Pharmacol. 14, 358(1969). Receivedfor review Jam 25, 1970. Accepted Augmtt IS, 1970. \ \ (\ J.L. 1111 I. AO*. FOOD CHEM. VOL. I*. NO. , I,TO MQNS 087009