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Oxidative Coupling of 17 -Estradiol: Inventory of Oligomer
Products and Configuration Assignment of Atropoisomeric
C4-Linked Biphenyl-Type Dimers and Trimers
Alessandro Pezzella,* Liliana Lista, Alessandra Napolitano, and Marco d’Ischia Department of Organic Chemistry and Biochemistry, University of Naples “Federico II” Complesso Universitario Monte S. Angelo, Via Cinthia 4, I-80126 Naples, Italy The oxidation chemistry of 17 -estradiol (1) is of central relevance to the nongenomic effects of
estrogens and offers valuable prospects in the search for novel steroidal scaffolds of academic and
industrial interest. Herein, we report the results of a detailed investigation into the nature of the
oligomer products formed by phenolic oxidation of 1. Of the oxidants tested, the peroxidase/H2O2
system proved to be the most effective in inducing conversion of 1 to a complex mixture of oligomer
species. Repeated chromatographic fractionation followed by extensive 2D NMR and mass
spectrometric analysis allowed identification of a series of phenolic coupling products comprising,
besides the C2-symmetric dimers 2 and 3, a 2,4′ dimer (4), two O-linked dimers (5, 6), and the
novel trimers 7-9. All 4-linked biphenyl-type oligomers, i.e., 3 and 7-9, occurred as couples of
atropoisomers, reflecting steric hindrance at biphenyl linkages. For all atropoisomers, absolute
configuration was established by the exciton chirality method and the interconversion energy was
determined by dynamic NMR. These results provide the first systematic inventory of oxidative
coupling products of 1 and lay the foundation for future studies aimed to develop novel estrogen
derivatives based on oligomeric scaffolds.
Introduction
by, their potent antioxidant5 and free radical scavengingcapacity.6 In view of that, a detailed elucidation of the 17 -Estradiol (1) and structurally related estrogens
structural modifications suffered by 1 in oxidative set-
possess both carcinogenic1 and neuroprotective2 proper- tings is central for the understanding of the nongenomic ties that have been attributed to the inherent susceptibil- effects of estrogens. In addition, beyond the specific ity of the phenolic A-ring to enzymatic or chemical relevance to the steroid sector, the oxidation of estrogen oxidation.3 In particular, the involvement of 1 in breast
compounds represents an attractive research issue be- and other human cancers would be the result of metabolic cause of its potential as convenient entry to complex conversion to the 2- and 4-hydroxy derivatives, termed functionalized scaffolds of academic and industrial inter- the catechol estrogens, and the corresponding o-quinones, est, e.g. in asymmetric synthesis7 and supramolecular which can induce the critical initiation step of tumori- chemistry,8 in the quest for innovative lead compounds genesis via adduct formation with DNA and depurination in anticancer therapy,9 or for liquid crystal prepara- processes.4 On the other hand, the neuroprotective action tions,10 where 1 and related compounds are commonly
of estrogens would be related to, or at least complemented (5) Behl, C.; Skutella, T.; Lezoualc’h, F.; Post, A.; Widmann, M.; * To whom correspondence should be addressed. Fax: +39-081- Newton, C. J.; Holsboer F. Mol. Pharmacol. 1997, 51, 535-541.
(6) (a) Dhandapani, K. M.; Brann, D. W. Biol. Reprod. 2002, 67,
(1) Yager, J. D. J. Natl. Cancer Inst. Monogr. 2000, 27, 67-73.
1379-1385. (b) Prokai, L.; Prokai-Tatrai, K.; Perjesi, P.; Zharikova, Yager, J. D.; Liehr, J. G. Annu. Rev. Pharmacol. Toxicol. 1996, 36,
A. D.; Perez, E. J.; Liu, R.; Simpkins, J. W. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 11741-11746.
(2) (a) Bisagno, V.; Bowman, R. E.; Luine, V. E. Endocrine 2003,
(7) (a) Kaufmann, M. D.; Grieco, P. A.; Bougie, D. W. J. Am. Chem.
21, 33-41. (b) Kajta, M.; Beyer, C. Endocrine 2003, 21, 3-9. (c) Picazo,
Soc. 1993, 115, 11648-11649. (b) Enev, V. S.; Ewers, Ch. L. J.; Harre,
O.; Azcoitia, I.; Garcia-Segura, L. M. Brain Res. 2003, 990, 20-27. (d)
M.; Nickisch, K.; Mohr, J. T. J. Org. Chem. 1997, 60, 364-369. (c)
Czlonkowska, A.; Ciesielska, A.; Joniec, I. Med. Sci. Monit. 2003, 9,
Strickler, R. R.; Schwan, A. L. Tetrahedron: Asymmetry 1999, 10,
247-256. (e) Moosmann, B.; Behl, C. Proc. Natl. Acad. Sci. U.S.A. 1999,
4065-4069. (d) Soulivong, D.; Matt, D.; Ziessel, R. J. Chem. Soc., Chem.
Commun. 1999, 393-394. (e) Kostova, K.; Genov, M.; Philipova, V.
(3) (a) Bolton, J. L.; Yu, L.; Thatcher, G. R. Methods Enzymol. 2004,
Tetrahedron: Asymmetry 2000, 11, 3253-3256.
378, 110-123. (b) Green, P. S.; Yang, S. H.; Simpkins, J. W. Novartis (8) (a) Davis, A. P.; Joos, J.-B. Coord. Chem. Rev. 2003, 240, 143-
Found. Symp. 2000, 230, 202-220.
156. (b) de Jong, M. R.; Knegtel, R. M. A.; Grootenhuis, P. D. J.; (4) (a) Cavalieri, E. L.; Rogan, E. G.; Chakravarti, D. Cell Mol. Life Huskens, J.; Reinhoudt, D. N. Angew. Chem., Int. Ed. Engl. 2002, 41,
Sci. 2002, 59, 665-681. (b) Convert, O.; Van Aerden, C.; Debrauwer,
1004-1008. (c) Smith, D. K.; Diederich, F.; Zingg, A. Math. Phys. Sci. L.; Rathahao, E.; Molines, H.; Fournier, F.; Tabet, J.-C.; Paris, A. Chem. 1999, 527, 261-272.
Res. Toxicol. 2002, 15, 754-764. (d) Markushin, Y.; Zhong, W.;
(9) (a) Lakhani, N. J.; Sarkar, M. A.; Venitz, J.; Figg, W. D.
Cavalieri, E. L.; Rogan, E. G.; Small, G. J.; Yeung, E. S.; Jankowiak Pharmacotherapy. 2003, 23, 165-172. (b) Schumacher, G.; Neuhaus,
R. Chem. Res. Toxicol. 2003, 16, 1107-1117.
P. J. Cancer Res. Clin. Oncol. 2001, 7, 405-410
10.1021/jo0492665 CCC: $27.50 2004 American Chemical Society J. Org. Chem. 2004, 69, 5652-5659
Oxidative Coupling of 17 -Estradiol employed. The close bearing on the field of phenolic By contrast, a substantial substrate consumption was coupling, which is central to several areas of organic observed with the peroxidase/H2O2 system, with forma- chemistry, further warrants exploration of the oxidation tion of a number of products whose chromatographic and spectral properties were suggestive of oligomer species.
Yet, despite the many prospects offered by oxidative The mechanistic background provided by the extensive manipulation of estrogens, current knowledge in the field literature on peroxidase-catalyzed oxidation of phenols16 is surprisingly limited. The only known oxidation prod- and the occurrence of peroxidase in mammalian tissues ucts include, besides the catechol estrogens, a 10 - responsive to estrogen activity, such as uterus,17 war- hydroxyestra-1,4-dien-3-one derivative arising by peracid- ranted investigation of such reaction as a paradigm to induced photooxygenation or oxidation by Fenton detail the behavior of this estrogen on oxidation. Accord- reagent,6b a series of benzylic oxidation species of estrone ingly, we decided to embark on the isolation and detailed methyl ether,11 and two dimers obtained by chemical and characterization of the products formed by peroxidase/ enzymatic oxidation of 1, namely, the symmetric 2,2′ and
H2O2 oxidation of 1. As the medium, phosphate buffer at
4,4′ dimers.12 Other studies have appeared reporting pH 7.4 with little methanol to favor estrogen solubiliza- formation of oligomer species by oxidation of 1, but their
tion was preferably used. Two-phase systems, e.g. water- characterization relied only on evaluation of chemical ethyl acetate,12 proved of limited utility and were not physical properties.13 More recently, a convenient syn- thetic access to O-linked dimers of 1 was reported14 in
In a typical preparative scale reaction, 1 at 0.3 mM
the frame of a study of the NADPH-dependent metabo- concentration was allowed to react with peroxidase (1 lism of 1 by human liver microsomes and cytochrome
U/mL) and hydrogen peroxide (2 mol equiv). After 60 min, P450 enzymes. These latter studies and the vast body of with >98% substrate consumption, the mixture was literature on the oxidative coupling of phenols15 suggest extracted with ethyl acetate following careful acidification that oxidative conversion of 1 and related estrogens in
to pH 5.0. PLC fractionation afforded seven main chro- vivo can lead to an array of oligomeric products, yet their nature and biological properties have remained so far 0.22, and 0.10 (eluant A) designated A-G, in that order.
poorly elucidated. This study was therefore concerned Of these, only fraction D consisted of a single species pure with an investigation of the reaction behavior of 1 with
enough for spectroscopic analysis, whereas fractions A, various oxidizing systems. Specific goals were to gain a B, and D-F required further fractionation. The most systematic insight into the modes of oxidative coupling polar band G was made of chromatographically ill-defined of 1 and to provide a detailed structural characterization
products, and their identity was not investigated.
of the oligomer products for future in vitro and in vivo Spectral data (1H and 13C NMR) of the product from band D were in agreement with a C2-symmetric dimer
(molecular ion peak at m/z 542). Homo- and hetero-
nuclear correlation experiments allowed straightforward
formulation of the product as 2.
Results and Discussion
In preliminary experiments, the ability of various chemical and enzymatic oxidants to induce conversion
of 1 to oligomer products was briefly investigated under
different reaction conditions. With the chemical oxidants
tested, i.e., persulfate, ferricyanide, and ceric ammonium
nitrate, little or no substrate conversion was observed
(HPLC and TLC evidence) in aqueous buffers or biphasic
media in a broad range of pH values. With ferricyanide,
slow substrate consumption was obtained only in 0.1 M
NaOH, as previously reported.12
(10) (a) Brandenburger, F.; Matthes, B.; Seifert, K.; Strohriegl, P.
Liq. Cryst. 2001, 28, 1035-1039. (b) McHugh, C.; Tomlin, D. W.;
Bunning, T. J. Macromol. Chem. Phys. 1997, 198, 2387-2395.
(11) Modica, E.; Bombieri, G.; Colombo, D.; Marchini, N.; Ronchetti, F.; Scala, A.; Toma, L. Eur. J. Org. Chem. 2003, 2964-2971.
Chromatographic band A consisted of an intimate (12) Ius, A.; Meroni, G.; Ferrara, L. J. Steroid Biochem. 1977, 8,
mixture of two closely related species which could be (13) (a) Norymberski, J. K. FEBS Lett. 1977, 76, 231-234. (b)
Norymberski, J. K. FEBS Lett. 1978, 86, 163-166. (c) Norymberski,
(16) (a) Uyama, H.; Kobayashi, S. Curr. Org. Chem. 2003, 7, 1387-
J. K. J. Steroid Biochem. Mol. Biol. 1991, 39, 73-81.
1397. (b) Uyama, H.; Kobayashi, S. J. Mol. Catal. B: Enzymol. 2002,
(14) Lee, A. J.; Sowell, J. W.; Cotham, W. E.; Zhu, B. T. Steroids 19-20, 117-127. (c) Adam, W.; Lazarus, M.; Saha-Moller, C. R.; 2004, 69, 61-65.
Weichold, O.; Hoch, U.; Haring, D.; Schreier, P. Adv. Biochem. Eng./ (15) (a) Kobayashi, S.; Higashimura, H. Prog. Polym. Sci. 2003, 28,
Biotechnol. 1999, 63 (Biotransformations), 73-108.
1015-1048. (b) Schmid, A.; Hollmann, F.; Buehler, B. Enzyme Cataly- (17) (a) Jellinck, P. H.; McNabb, T.; Cleveland, S.; Lyttle, C. R. Adv. sis in Organic Synthesis, 2nd ed.; Drauz, K., Waldmann, H., Eds.; Enzyme Regul. 1976, 14, 447-465. (b) O’Brien, P. J. Chem. Biol.
Interact. 2000, 129, 113-139.
J. Org. Chem, Vol. 69, No. 17, 2004 5653
separated by preparative HPLC. The products showed TABLE 1. Selected NMR Data for Compounds 5 and 6
nearly identical 1H NMR spectra featuring in the aro- matic region two doublets (J ) 8.8 Hz) at around δ 7.3 2-symmetric 4,4′ dimer 3. On
this basis, the two products were regarded as atropoiso- mers arising by a restricted rotation around the sterically crowded biphenyl 4,4′ linkage. No appreciable intercon- version of the rotational isomers was observed by heating to 110 °C at which temperature the products began to decompose significantly. This implies that the activation energy barrier is greater than 22.5 kcal mol-1.
The constituents of chromatographic band C as purified by HPTLC displayed very similar 1H NMR spectra showing in the aromatic region two singlets at about δ 7.0 and 6.8 and two doublets (J ) 8.4 Hz) at about δ 7.3and 6.9, suggesting two atropoisomers of a 2,4′-linked showed very close proton spectra and were regarded as dimer (4). This view was confirmed by dynamic 1H NMR
atropoisomers. Both displayed in the aromatic region an experiments. Line shape analysis at a temperature ABX spin system and three singlets around δ 6.7, 6.9, around coalescence allowed calculation of the mean and 7.0, consistent with a trimer in which a central lifetime of the atropoisomers, and a free energy of estradiol unit is linked to the 2-position of an outer activation of 21.5 ( 0.5 kcal mol-1 was determined by moiety and to the oxygen of the other unit. On the basis of the HMBC correlation data, it was possible to assign
the signals at δ 7.04 and 6.75, for the faster HPLC eluted
compound, to the H-1 and H-4 protons of the same
estradiol unit. The shielding effect caused by the oxygen
bridge, observed also in dimers 5 and 6, and the presence
of a weak but well discernible cross-peak between the
proton resonance at δ 7.04 and a substituted C-4 carbon
resonance at δ 124.3 (see Table 2) allowed straightfor-
ward assignment of the former signal to the H-1 proton
of the central unit leading eventually to assign the
The two components of chromatographic band B were trimers structure 7. In these, the atropoisomerism is
separated by preparative HPLC. The mass and NMR apparently the result of the restricted rotation around data led to straightforward formulation of the compounds the 2,4′ linkage. NMR line shape analysis around the as the O-linked dimers 5 and 6.14 Extensive 2D proton-
coalescence temperature allowed calculation of a free proton and proton-carbon correlation experiments al- energy of activation of 20.9 ( 0.4 kcal mol-1.
lowed complete assignment of the aromatic resonances(see Table 1).
Of the four main HPLC-separable products in band F, those eluting at 24 and 80 min (eluant II) interchangedon heating, suggesting again an atropoisomer relation-ship, whereas those eluting at 34 and 37 min (eluant II)were not affected by heating to 100 °C.
The aromatic region of the 1H NMR spectrum of the products eluted at 24 and 80 min displayed five singlets,a feature which was compatible with the trimeric struc- All products from chromatographic bands E and F ture 8. Complete assignment of the proton and carbon
exhibited molecular ion peaks at m/z 812 in the EI-MS signals in the aromatic region was achieved on the basis spectrum indicating trimeric structures. Products from of the data of the correlation experiments. In particular, band E as obtained in pure form by HPLC separation in the case of the slower eluting isomer (8b), the singlets
5654 J. Org. Chem., Vol. 69, No. 17, 2004
Oxidative Coupling of 17 -Estradiol TABLE 2. Selected 1H-13C NMR Correlation Data for
TABLE 3. Selected 1H-13C NMR Correlation Data for
Compound 7a18
Compound 8b18
a Numbering as shown in formula 7.
TABLE 4. Selected 1H-13C NMR Correlation Data for
Compound 9b18
at δ 7.21 and δ 6.76 were assigned to H-1 and H-4 protons of the same unit, respectively, on the basis of the 3J and 2J long-range contacts exhibited with C-3 and C-2 at δ 151.1 and 123.0, respectively. A 3J contact between the latter carbon and the H-1′ proton at δ 7.32 of another unit provided support to the 2,2′ linkage between two of the trimer units (see Table 3). The free energy activation for the interconversion was calculated as 21.3 ( 0.5 kcal was compatible with either of the two trimeric structures
in which the estradiol units were linked through the 2,4′:
2′,4′′ or the 2,2′:4′,4′′ positions. The lack of appreciable
interconversion on heating, observed also for the atro-
poisomers of the 4,4′ dimer 3, strongly argued in favor
The 1H and 13C spectra of the other two constituents of structure 9. This assignment was confirmed by analy-
of band F were likewise very similar. The aromatic sis of the proton-carbon correlation spectra showing regions of the proton spectra diplayed three singlets and contacts matching those of the 2,2′ subunit of trimer 8
two doublets (J ) 8.4 Hz), a pattern of resonance that Interestingly, when the oxidation of 1 was carried out
with the substrate at 0.3 µM concentration, that is, at a
concentration close to physiological values, substrate
consumption was >95% at 1 h and the main reaction
products were the dimers 2 and 4, whereas the O-linked
dimers 5 and 6 were formed only in very small amounts
and dimer 3 and trimers 7-9 were below detection limits.
For comparative purposes, the oxidation of 1 with
manganese dioxide in chloroform was briefly investi-
gated. Under these conditions, a smooth oxidation of 1
(>99% consumption after 20 h) was observed with
formation of dimers 3 and 4 as main species (about 30%
overall formation yields) but with no detectable 2, 5, and
6. The different product patterns obtained at lower
J. Org. Chem, Vol. 69, No. 17, 2004 5655
FIGURE 1. CD spectra of compounds 3a (A), 4a (B), and 7a (C).
substrate concentration, or using manganese dioxide in
chloroform, suggest that the generation and mode of
coupling of phenoxyl radicals is under the influence of
several factors. For example, tenuous steric factors may
become significant under high dilution conditions, thus
accounting for the lack of formation of the relatively
hindered dimer 3, whereas solvent effects may explain
the prevalence of C-coupling products, i.e. dimers 3 and
4 in chloroform, also furnishing a suggestion for prepara-
tive purposes requiring regiochemically more restricted
products patterns.
The sterically hindered biphenyl linkage in 3, 4, and
7-9 represents a stereogenic element which adds to those
already present in 1. For all isolated products featuring
such structural system, configuration at the biphenyl
linkage (and thus absolute stereochemistry) was estab-
lished by the exciton chirality method on the basis of the
Cotton effect associated with the phenolic transition 1La,
whose vector nearly overlaps that joining the C10-C3-O
centers.19 This transition is observed at around 220 nm
in 1 in EtOH, but the formation of biphenyl linkages and
the presence of other substituents, such as the O-linked
unit in 7a,b, cause shift to longer wavelengths. The
FIGURE 2. CD spectra of compounds 8a (A) and 9a (B).
relative directions of the 1La transition dipoles and of thebiphenyl bonds allowed assignment of positive screwness all isolated products, whereas the Cotton effect at ca. 280 configuration (P) to those isomers exhibiting positive nm (transition 1Lb) was less defined for nearly all Cotton effect independently from the regiochemistry of products, with the exception of those featuring 4,4′- the biphenyl linkage. Indeed, geometry optimization of the oligomer structures (MM+) showed that the dihedral On this basis, the negative Cotton effect of the first angle between the planes of the aromatic rings of the HPLC eluted atropoisomer of 4 and 7 (i.e. 4a and 7a)
biphenyl system has an absolute value ranging from 43° indicates a negative helical orientation of phenol transi- 22′ to 45° 15′ for 2,4′ linkages and from 90° 02′ to 94° 92′ tion moments that means an M molecular chirality, while for 4,4′ linkages. On the basis of the angles between 1La the first eluted isomer of 3 has the P configuration
transition vectors and the dihedral intersection line, trigonometric calculations gave the range +37 to +77° In the case of 8 (i.e. 2,2′:4′,2′′-triestradiol) and 9 (i.e.
for the angle between the dipole transition moments in 2,2′:4′,4′′-triestradiol) the first eluted isomers share the the case of a positive dihedral angle. These values are M configuration at the 4′,2′′ biphenyl linkage and at the significantly smaller than 110°, which is the theoretical 4′,4′′ linkage, respectively (Figure 2 A,B).
zero point at which for polyphenyl systems featuring Mechanistically, formation of oligomer products 3-9
right-handed screwness19c the sign of the exciton split of by peroxidase/H2O2 promoted oxidation of 1 can be
CD Cotton effect changes from positive to negative, interpreted as involving generation and coupling of allowing straightforward molecular configuration assign- phenoxyl radicals from 1. In the presence of H2O2, ferric
peroxidase (ground state) generates the ferryl π cation The choice of 1La transition arises also from the clearly (compound I) via two electron oxidation. Compound I can defined monosignated Cotton effect at around 230 nm in then be reduced to compound II, the ferryl form of theenzyme, which has higher oxidative equivalents than the (18) “a” and “b” lettering denotes the faster and the slower HPLC resting ferric form.17b Both compounds I and II can oxidize the phenolic moiety of 1 to give the phenoxyl
(19) (a) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy- Exciton Coupling in Organic Stereochemistry; University Science Books: Mill Valley, CA, 1983. (b) Dong, J. G.; Akritopoulou-Zanze, I.; From inspection of the SOMO and Mulliken spin Guo, J.; Berova, N.; Nakanishi, K.; Harada, N. Enantiomer 1997, 2,
densities of the phenoxyl radical of 1 reported in a
397-409. (c) Hanazaki, I.; Akimoto, H. J. Am. Chem. Soc. 1972, 94,
4102-4106.
previous study,20 no appreciable difference was antici- 5656 J. Org. Chem., Vol. 69, No. 17, 2004
Oxidative Coupling of 17 -Estradiol pated in the reactivity of 1 through the 2 and 4 positions,
absorbance values in the range 0.1-0.2 at 220 nm. 1H (13C) in accord with experimental evidence. Coupling through NMR spectra were recorded at 400.1 (100.6) MHz. 1H-1H the oxygen center is clearly a reflection of the high spin COSY, 1H-13C HMQC, and 1H-13C HMBC experiments were density at this site, in conformity with the known run at 400.1 MHz using standard pulse programs from theBruker library. For electron impact (EI-MS) and high resolu- patterns of oxidative coupling of phenols.
tion (HR-MS) mass spectra samples were ionized with a 70eV beam and the source was taken at 180-280 °C.
Conclusions
Analytical and preparative TLC analyses were performed on F254 0.25 and 0.5 mm silica gel plates or high performance The analytical and structural undertaking described TLC (HPTLC) using 40:60 cyclohexanes-ethyl acetate (eluant herein fills an important gap in the current knowledge A) or 98:2 chloroform-methyl alcohol (eluant B).
of the oxidation chemistry of estrogens and, more in Analytical and preparative HPLC was performed with an general, of natural phenolic compounds. Highlights of instrument equipped with a UV detector set at 280 nm.
this study include (a) the first isolation and complete Octadecylsilane-coated columns, 4.6 × 250 mm or 22 × 250 characterization of trimeric steroids linked through C-C mm, 5 µm particle size, were used for analytical or preparative and C-O-C bonds, (b) the first example, to the best of runs, respectively. Flow rates of 1 or 15 mL/min were used.
Different isocratic and gradient elution conditions were used our knowledge, of atropoisomerism in steroidal systems, generated by steric hindrance to free rotation at 2,4′- and acetonitrile (eluant II); 90:10 H2O-acetonitrile (solvent A), 4,4′-biphenyl linkages, and (c) the exploitation of peroxi- acetonitrile (solvent B), 0-5 min 30% B, 5-30 min 30-55% dase/H2O2 as an efficient and clean oxidizing system in Oxidation of 1 by the Peroxidase/H2O2 System: Gen-
From the biomedical point of view, the present results eral Procedure. To a solution of 1 (5 mg, 1.9 × 10-5 mol) in
offer an improved background to elucidate the chemical methanol (5 mL) were added 0.1 M phosphate buffer, pH 7.4 nature and fate of the products derived from the anti- (60 mL), and peroxidase (1 U/mL) sequentially. The mixture oxidant and radical scavenging reactions or from oxida- was then treated with hydrogen peroxide in aliquots (8 × 2.5 × 10-6 mol) every 10 min while being kept under stirring at tive changes of the estrogens at sites of inflammation and room temperature. At different time intervals the reaction was active metabolic transformation. In the light of the carefully acidified at pH 5.0 and extracted three times with suggested role of 1 as OH radical scavenger, generation
ethyl acetate (3 × 60 mL). The combined organic layers were of these oligomers may represent an alternative outcome dried over sodium sulfate and analyzed by HPLC (eluant III) of the radical scavenging action in addition to quinol and TLC (eluant A). In other experiments the reaction was formation.6b Oligomers 5 and 6 resemble the photodeg-
carried out as above with the substrate at 3 × 10-7 M radation products of ethinyl estradiol,21 and their forma- concentration using peroxidase (0.02 U/mg) and hydrogenperoxide (1 mol equiv) tion by autoxidation and photodegradation of estradiol- Oxidation of 1 by MnO
containing drugs can be predicted. C 2. A solution of 1 (10 mg, 3.7 ×
10-5 mol) in chloroform (10 mL) was treated with MnO2 (64 bear considerable similarity to stereochemically related mg, 8 × 10-4 mol) and kept overnight at room temperature.
products22 currently under scrutiny because of their The solid was removed by centrifugation, and the mixture was antiestrogenic activity and may represent attractive taken to dryness, taken up in methanol, and analyzed by prototypes/leads for the rational design of new bioactive HPLC (eluant III) and TLC (eluant A).
Isolation of Compounds 2-9. For preparative purposes,
Finally, atropoisomeric estradiol oligomers are analo- reaction of 1 with peroxidase/H2O2 was run as described above
gous to para-polyaryls, which exhibit attractive structural using 500 mg (1.84 × 10-3 mol) of the starting material at 3.0 ×10-4 M concentration. After workup of the reaction mixture, features, such as helicity, and other connected unusual the residue obtained (480 mg) was fractionated by PLC (eluant chemical-physical properties underlying a number of A) to give seven fractions. Fraction A (15 mg, R ) A) was further purified by preparative HPLC (eluant II) to The extension of the scope and utility of the oxidation give pure 3a (5 mg, t )
8 min, eluant II, 1% yield) and 3b (5
chemistry of estrogens is currently a matter of concern 17 min, eluant II, 1% yield). Fraction B (25 mg, Rf 0.55 eluant A) was fractionated by PLC (eluant I) to give pure
5 (8 mg, t )
27 min, eluant II, 1.6% yield) and 6 (8 mg, tr
29 min, eluant II, 1.6% yield). Fraction C (15 mg, R ) 0.45 Experimental Section
eluant A) was purified by HPTLC (eluant B) to afford 4a (5
General Methods. 17 -Estradiol (1), manganese(IV) diox-
9 min, eluant II, 1% yield) and 4b (5 mg, tr
ide activated 5 µm (85%), and hydrogen peroxide (30% w/w eluant II, 1% yield). Fraction D (20 mg, Rf solution in water) were used as obtained. Horseradish peroxi- consisted of pure 2 (tr
0.33, eluant A) was purified by preparative 2O2 oxidoreductase; EC 1.11.1.7) type II and mushroom tyrosinase (EC 1.14.18.1) were used.
HPLC (eluant II) to afford 7a (3 mg, tr
UV spectra were performed using a diode array spectro- 0.6% yield) and 7b (3 mg, tr
photometer. CD spectra were taken on spectropolarimeter at 25 °C using solutions of the products in ethanol exhibiting preparative HPLC (eluant II) to give four bands corresponding
to pure 8a (3 mg, t )
24 min, eluant II,, 0.6% yield), 8b
80 min, eluant II, 0.6% yield), 9a (3 mg, tr
(20) Pezzella, A.; Manini, P.; Di Donato, P.; Boni, R.; Napolitano, min, eluant II, 0.6% yield), and 9b (3 mg, t )
A.; Palumbo, A.; d’Ischia, M. Bioorg. Med. Chem. 2004, 12, 2927-2936.
II, 0.6% yield). Fraction G (35 mg, R ) (21) Segmuller, B. E.; Armstrong, B. L.; Dunphy, R.; Oyler, A. R. J. Pharm. Biomed. Anal. 2000, 23, 927-37.
found to consist of a complex pattern of species and was not (22) (a) Rabouin, D.; Perron, V.; N’Zemba, B.; C-Gaudreault, R.; Berube, G. Bioorg. Med. Chem. Lett. 2003, 13, 557-560 (b) Portoghese,
2,2′-Bis[estra-1,3,5(10)-trien-3,17 -diol] (2). UV [λmax (CH3-
P. S. J. Med. Chem. 1992, 35, 1927-1937.
OH)]: 288 nm. 1H NMR (CD3OD), δ (ppm): 0.75 (s, 3H × 2), (23) (a) Berresheim, A. J.; Mueller, M.; Muellen, K. Chem. Rev. 1999,
99, 1747-1785. (b) Martin, R. E.; Diederich, F. Angew. Chem., Int. 1.0-1.8 (m, 8H × 2), 1.9-2.1 (m, 4H × 2), 2.15 (m, 1H × 2), Ed. Engl. 1999, 38, 1351-1377.
2.25 (m, 1H × 2), 2.85 (m, 1H × 2), 3.64 (m, 1H × 2), 6.31 (s, J. Org. Chem, Vol. 69, No. 17, 2004 5657
1H × 2), 7.14 (s, 1H × 2). 13C NMR (CD3OD), δ (ppm): 12.6 (2 139.7 (C), 147.4 (C), 155.7 (C). EI/MS (m/z): 542, [M]+.
× CH3), 24.1 (2 × CH2), 29.3 (2 × CH2), 31.5 (2 × CH2), 31.8 HREIMS (m/z): calcd mass for C36H46O4, 542.3396; found, (4 × CH2), 38.8 (2 × CH2), 41.1 (2 × CH), 45.1 (2 × C), 46.1 (2 × CH), 52.1 (2 × CH), 83.3 (2 × CH), 118.1 (2 × CH), 127.0 (2 2-[[(17 )-17-Hydroxy-19-norpregna-1,3,5(10)-trien-3-yl]-
× C), 130.3 (2 × CH), 134.7 (2 × C), 139.1 (2 × C), 153.0 (2 × oxy]-4,2′-bis[estra-1,3,5(10)-trien-3,17 -diol] (7a). UV [λmax
C). EI/MS (m/z): 542, [M]+. HREIMS (m/z): calcd mass for (CH3OH)]: 288 nm. 1H NMR (CDCl3), δ (ppm): 0.78 (s, 3H), C36H46O4, 542.3396; found, 542.3401.
0.79 (s, 3H) 0.80 (s, 3H), 1.1-1.7 (m, 21H), 1.70-1.85 (m, 3H), 4,4′-Bis[estra-1,3,5(10)-trien-3,17 -diol] (3a). UV [λ
1.85-2.00 (m, 5H), 2.1-2.2 (m, 5H), 2.2-2.3 (m, 3H), 2.35 (m, 2H), 2.45 (m, 1H), 2.55 (m, 1H), 2.85 (m, 4H), 3.74 (m, 3H), 3OH)]: 288 nm. 1H NMR (CDCl3), δ (ppm): 0.80 (s, 3H × 2), 1.1-1.7 (m, 8H × 2), 1.78 (m, 1H × 2), 1.95 (m, 1H × 2), 6.75 (s, 1H), 6.76 (d, J ) 2.4 Hz, 1H), 6.80 (dd, J ) 8.4, 2.4 2.11 (m, 1H × 2), 2.15-2.30 (m, 2H × 2), 2.30-2.40 (m, 2H × Hz, 1H), 6.98 (s, 1H), 7.04 (s, 1H), 7.23 (d, J ) 8.4, 1H). 13C 2), 3.73 (t, J ) 8.2 Hz, 1H × 2), 6. 87 (d, J ) 8.8 Hz, 1H × 2), NMR (CDCl3), δ (ppm): 11.9 (CH3), 23.9 (CH2), 27.1 (CH2), 7.30 (d, J ) 8.8 Hz, 1H × 2). 13C NMR (CDCl 2), 27.9 (CH2), 30.5 (CH2), 31.4 (CH2), 31.7 (CH2), 37.5 (CH), 38.9 (C), 39.5 (C), 44.0 (CH), 44.8 (CH), 45.1 (CH), 50.9 3), 23.9 (2 × CH2), 27.1 (2 × CH2), 27.8 (2 × CH2), (CH), 82.7 (CH), 115.4 (CH), 116.4 (CH), 117.8 (CH), 118.4 2), 31.4 (2 × CH2), 37.5 (2 × CH2), 39.1 (2 × CH), 44.0 (2 × C), 45.0 (2 × CH), 50.9 (2 × CH), 82.6 (2 × CH), (CH), 119.8 (C), 124.3 (C), 127.4 (CH), 128.3 (CH), 132.3 (C), 113.6 (2 × CH), 119.9 (2 × C), 128.0 (2 × CH), 134.4 (2 × C), 133.6 (C), 133.9 (C), 134.3 (C), 136.0 (C), 139.3 (C), 142.2 (C), 137.8 (2 × C), 152.0 (2 × C). EI/MS (m/z): 542, [M]+. HREIMS 144.6 (C), 151.6 (C), 155.9 (C). EI/MS (m/z): 812, [M]+.
HREIMS (m/z): calcd mass for C54H68O6, 812.5016; found, 3b. UV [λmax (CH3OH)]: 288 nm. 1H NMR (CDCl3), δ (ppm):
7b. UV [λ
0.81 (s, 3H × 2), 1.1-1.7 (m, 8H × 2), 1.79 (m, 1H × 2), 1.99 (ppm): 0.77 (s, 3H), 0.78 (s, 3H) 0.79 (s, 3H), 1.1-1.7 (m, 24 (s, 1H × 2), 2.12 (m, 1H × 2), 2.15-2.30 (m, 2H × 2), 2.30- H), 1.7-2.0 (m, 5H), 2.0-2.3 (m, 8H), 2.40 (m, 2H), 2.50 (m, 2.40 (m, 2H × 2), 3.74 (t, J ) 8.2 Hz, 1H × 2), 6. 86 (d, J ) 8.8 2H), 2.87 (m, 4H), 3.72 (m, 3H), 6.75 (d, J ) 2.4 Hz, 1H), 6.77 Hz, 1H × 2), 7.32 (d, J ) 8.8 Hz, 1H × 2). EI/MS (s, 1H), 6.81 (dd, J ) 8.4, 2.4 Hz, 1H), 6.98 (s, 1H), 7.03 (s, (m/z): 542, [M]+. HREIMS (m/z): calcd mass for C36H46O4, 1H), 7.24 (d, J ) 8.4, 1H). EI/MS (m/z): 812, [M]+. HREIMS (m/z): calcd mass for C54H68O6, 812.5016; found, 812.5026.
2,4′-Bis[estra-1,3,5(10)-trien-3,17 -diol] (4a). UV [λmax
2,2′:4′,2”-Tris[estra-1,3,5(10)-trien-3,17 -diol] (8a). UV
(CH3OH)]: 288 nm. 1H NMR (CDCl3), δ (ppm): 0.79 (s, 6H), [λmax (CH3OH)]: 288 nm. 1H NMR (CDCl3), δ (ppm): 0.78 (s, 1.1-1.8 (m, 16H), 1.9-2.0 (m, 4H), 2.10 (m, 2H), 2.25 (m, 2H), 3H), 0.80 (s, 6H), 1.1-1.9 (m, 24H), 1.9-2.0 (m, 6H), 2.1-2.4 2.32 (m, 2H), 2.50 (m, 2H), 2.85 (m, 2H), 3.73 (m, 2H), 6.77 (s, (m, 10H), 2.42 (m, 2H), 2.90 (m, 3H), 3.73 (m, 3H), 6.76 (s, 1H), 6.85 (d, J ) 8.4 Hz, 1H), 7.00 (s, 1H), 7.29 (d, J ) 8.4 Hz, 1H), 6.79 (s, 1H), 7.04 (s, 1H), 7.20 (s, 1H), 7.32 (s, 1H).
1H). 13C NMR (CDCl3), δ (ppm): 11.9 (CH3), 23.9 (CH2), 27.3 EI/MS (m/z): 812, [M]+. HREIMS (m/z): calcd mass for (CH2), 27.8 (CH2), 28.0 (CH2), 28.3 (CH2), 30.4 (CH2), 31.4 C54H68O6, 812.5016; found, 812.5005.
(CH2), 31.7 (CH2), 37.50 (CH2), 37.52 (CH2), 39.0 (CH), 39.5 8b. UV [λmax (CH3OH)]: 288 nm. 1H NMR (CDCl3), δ
(CH), 44.0 (C), 44.8 (CH), 45.0 (CH), 50.8 (CH), 83.3 (CH), (ppm): 0.77 (s, 3H), 0.78 (s, 6H), 1.1-1.9 (m, 24H), 1.9-2.0 113.6 (CH), 116.7 (CH), 117.4 (C), 120.5 (C), 127.9 (CH), 128.3 (m, 5H), 2.0-2.2 (m, 5H), 2.2-2.5 (m, 8H), 2.90 (m, 3H), 3.73 (CH), 133.3 (C), 134.5 (C), 136.9 (C), 140.1 (C), 152.3 (C). EI/ (m, 3H), 6.76 (s, 1H), 6.78 (s, 1H), 7.04 (s, 1H), 7.21 (s, 1H), MS (m/z): 542, [M]+. HREIMS (m/z): calcd mass for C36H46O4, 7.32 (s, 1H). 13C NMR (CDCl3), δ (ppm): 11.8 (CH3), 23.9 (CH2), 24.0 (CH2), 24.5 (CH2), 27.2 (CH2), 27.9 (CH2), 29.3 (CH2), 30.1 4b. UV [λmax (CH3OH)]: 288 nm. 1H NMR (CDCl3), δ
(CH2), 30.4 (CH2), 30.5 (CH2), 31.4 (CH2), 37.5 (CH2), 39.1 (CH), (ppm): 0.78 (s, 6H), 1.1-1.8 (m, 16H), 1.9-2.1 (m, 4H), 2.1- 39.4 (CH), 39.6 (CH), 44.0 (C), 44.8 (CH), 45.1 (CH), 50.9 (CH), 2.3 (m, 4H), 2.3-2.5 (m, 4H), 2.92 (m, 2H), 3.72 (m, 2H), 6.78 82.7 (CH), 116.9 (CH), 118.2 (CH), 121.9 (C), 122.5 (C), 123.0 (s, 1H), 6.87 (d, J ) 8.4 Hz, 1H), 6.99 (s, 1H), 7.29 (d, J ) 8.4 (C), 128.6 (CH), 128.9 (CH), 130.0 (CH), 133.3 (C), 133.9 (C), Hz, 1H). EI/MS (m/z): 542, [M]+. HREIMS: calcd mass for 137.4 (C), 138.3 (C), 140.0 (C), 150.9 (C), 151.1 (C). EI/MS C36H46O4, 542.3396; found, 542.3401.
(m/z): [M]+. HREIMS (m/z): calcd mass for C54H68O6, 812.5016; 2-[[(17 )-17-Hydroxy-19-norpregna-1,3,5(10)-trien-3-yl]-
oxy]estra-1,3,5(10)-trien-3,17 -diol (5). UV [λ
2,2′:4′,4”-Tris[estra-1,3,5(10)-trien-3,17 -diol] (9a). UV
max (CH3OH)]: 288 nm. 1H NMR (CDCl3), δ (ppm): 0.79 (s, 1.6 (m, 16H), 1.6-1.8 (m, 2H), 1.90 (m, 2H), 1.95 (m, 1H), 2.0- 3H), 0.80 (s, 3H), 0.81 (s, 3H), 1.2-1.8 (m, 24H), 1.8-2.0 (m, 2.2 (m, 5H), 2.32 (m, 1H), 2.83 (m, 3H), 3.71 (m, 2H), 6.70 (d, 6H), 2.0-2.2 (m, 4H), 2.2-2.4 (m, 8H), 2.89 (m, 3H), 3.74 (m, J ) 2.4 Hz, 1H), 6.74 (s, 1H), 6.76 (dd, J ) 8.4, 2.4 Hz, 1H), 3H), 6.76 (s, 1H), 6.88 (d, J ) 8.4 Hz, 1H), 7.22 (s, 1H), 7.32 6.87 (s,1H), 7.22 (d, J ) 8.4 Hz, 1H). 13C NMR (CDCl (d, J ) 8.4, 1H), 7.34 (s, 1H). EI/MS (m/z): [M]+. HREIMS 3), 23.9 (CH2), 27.0 (CH2), 27.1 (CH2), 27.9 2), 28.0 (CH2), 29.9 (CH2), 30.4 (CH2), 31.4 (CH2), 37.4 λmax (CH3OH)]: 288 nm. 1H NMR (CDCl3), δ (ppm): 0.79 (s, 3H), 0.80 (s, 6H), 1.1-1.8 (m, 24H), 1.8-2.0 2), 37.5 (CH2), 39.40 (CH), 39.45 (CH), 44.0 (C), 44.8 (CH), 50.8 (CH), 82.6 (CH), 114.8 (CH), 116.7 (CH), 117.6 (CH), 117.7 (m, 5H), 2.1-2.2 (m, 5H), 2.2-2.5 (m, 8H), 2.8-2.9 (m, 3H), (CH), 127.4 (CH), 133.7 (C), 134.2 (C) 135.8 (C), 139.3 (C), 141.5 3.73 (m, 3H), 6.76 (s, 1H), 6.89 (d, J ) 8.4 Hz, 1H), 7.22 (s, (C), 146.2 (C), 156.0 (C). EI/MS (m/z): 542, [M]+. HREIMS 1H), 7.33 (d, J ) 8.4, 1H), 7.36 (s, 1H). 13C NMR (CDCl3), δ 3), 11.9 (CH3), 23.8 (CH2), 24.0 (CH2), 24.5 4-[[(17 )-17-Hydroxy-19-norpregna-1,3,5(10)-trien-3-yl]-
(CH2), 26.5 (CH2), 28.1 (CH2), 29.7 (CH2), 30.5 (CH2), 31.1 oxy]estra-1,3,5(10)-trien-3,17 -diol (6). UV [λ
(CH2), 31.4 (CH2), 37.5 (CH2), 38.6 (CH), 39.5 (CH), 42.1 (C), 44.0 (CH), 44.9 (CH), 48.8 (CH), 50.8 (CH), 82.7 (CH), 118.3 3), δ (ppm): 0.78 (s, 6H), 1.1-1.8 (m, 16H), 1.8-1.9 (m, 2H), 1.9-2.0 (m, 2H), 2.0-2.2 (m, 4H), 2.33 (CH), 118.6 (CH), 118.9 (C), 122.6 (C), 122.7 (C), 128.2 (CH), (m, 2H), 2.40 (m, 1H), 2.73 (m, 1H), 2.81 (m, 2H), 3.73 (m, 130.1 (CH), 130.2 (CH), 132.6 (C), 134.3 (C), 136.3 (C), 136.5 2H), 6.60 (d, J ) 2.4 Hz, 1H), 6.62 (dd, J ) 8.4, 2.4 Hz, 1H), (C), 138.2 (C), 148.0 (C), 151.5 (C), 152.1 (C). EI/MS (m/z): 812, 6.86 (d, J ) 8.4, 1H), 7.10 (d, J ) 8.4 Hz, 1H), 7.17 (d, J ) 8.4 [M]+. HREIMS (m/z): calcd mass for C54H68O6, 812.5016; found, 24.6 (CH2), 27.0 (CH2), 28.0 (CH2), 28.2 (CH2), 30.5 (CH2), 31.4(CH Acknowledgment. This study was carried out in
2), 37.4 (CH2), 39.0 (CH), 39.5 (CH), 44.0 (C), 44.8 (CH), 50.8 (CH), 82.7 (CH), 112.8 (CH), 113.8 (CH), 115.6 (CH), 123.6 the frame of MIUR (“Neurosteroidi e loro modificazioni (CH), 127.5 (CH), 131.5 (C), 134.4 (C), 135.5 (C), 139.4 (C), ossidative e nitrosative nel sistema nervoso dei cefa- 5658 J. Org. Chem., Vol. 69, No. 17, 2004
Oxidative Coupling of 17 -Estradiol lopodi”, PRIN 2002, “Sostanze naturali ed analoghi Supporting Information Available: 1H NMR spectra
sintetici ad attivita` antitumorale”, PRIN 2003) and or selected regions of compounds 2-9 and 1H-13C HMBC
Regione Campania projects (L.R. 5, a. 2002). We thank spectra of compounds 3a, 4a, 5, 7a, 8a, and 9a. This
the “Centro Interdipartimentale di Metodologie Chimico- material is available free of charge via the Internet at Fisiche” (CIMCF, University of Naples Federico II) for NMR and mass facilities. We thank Mrs. SilvanaCorsani for technical assistance.
J. Org. Chem, Vol. 69, No. 17, 2004 5659

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