Non-steroidal estrogen-receptor antagonists

Abstract

This invention relates generally to non-steroidal estrogen-receptor (ER) antagonists that are useful as therapeutics in the treatment of advanced stage breast cancer. More particularly, this invention relates to the cloning and heterologous expression of genes encoding ER antagonists. This invention also relates to the use of such expression to overproduce the ER antagonist and analogs thereof.

Description

FIELD OF THE INVENTION

This invention relates generally to non-steroidal estrogen-receptor (ER) antagonists that are useful as therapeutics in the treatment of advanced stage breast cancer. More particularly, this invention relates to the cloning and heterologous expression of genes encoding ER antagonists. This invention also relates to the use of such expression to overproduce the ER antagonist and analogs thereof.

BACKGROUND OF THE INVENTION

Non-steroidal estrogen-receptor (ER) antagonists are useful in the treatment of advanced stage breast cancer. One class of ER antagonists that has been shown to be particularly useful are the R1128 substances (Y. Hori et al., The Journal of Antibiotics, 46(7): 1055-1062.) These substances are anthraquinone natural products with in vitro and in vivo potency approaching that of Tamoxifen. However, many of these types of ER antagonists exhibit variable effects in different target tissue. For example, tamoxifen acts as an ER antagonist in breast tissue, but an ER agonist in bone (Love, R. R. et al., New England Journal of Medicine 26: 852-856 (1992)) and uterine tissue (Kedar, R. P. et al., Lancet 28: 1318-1321 (1992)). Likewise, Raloxifene is an ER antagonist in breast tissue and an ER agonist in bone but not in uterine tissue (Black, L., et al., J. Clin. Invest. 93: 63-69 (1994)).

Estrogen receptors are transcription factors that bind to estrogen response elements in the promoter region of certain estrogen-related genes. Thus, estrogen-like ligands are useful to modulate transcription of these genes (Katzenellenbogen, J. A. and Katzenellenbogen B. S. (1996) Chem. Biol. 3: 529-536.) Recent studies have suggested that the tissue-dependent variability exhibited by these ligands may be due to differential ligand binding and/or activation of the two ER subtypes, ER-α and ER-β, which each have different tissue expression patterns and ligand binding characteristics (Mosselman, S. et al. (1996) FEBS Letters 392: 49-53.) It has also been reported that the ligand-bound ERs may modulate transcription activation by binding to different types of DNA enhancer elements, such as the AP1 transcription factors (Paech, K. et al. (1997) Science 277: 1508-1510).

Accordingly, there is thus a significant need for new ER antagonists which can inhibit ER function in a predictable way at both ER-dependent and AP 1-dependent DNA binding sites.

SUMMARY OF THE INVENTION

The present invention includes the estrogen receptor antagonist shown in FIG. 3 . Other aspects of the invention are found throughout the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of the R1128 compounds A (FIG. 1 A), B (FIG. 1 B), C ( FIG. 1C ) and D (FIG. 1 D).

FIG. 2 depicts the biosynthetic pathway leading to the R1128 compounds A-D.

FIG. 3 depicts the product, HU235.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the cloning and expression of the R1128 gene cluster in a manner that favors production of alternatively cyclized derivatives of the R1128 anthraquinones.

The isolation, fermentation, structures and properties of R1128 A-D (shown in FIGS. 1A to D) were described in 1993. See Hori, Y. et al. (1993) J. Antibiot. 46: 1055-1062 (Part I); 1063-1068 (Part II); and 1069-1075 (Part III). In Part II, the authors compared their pharmacological properties to tamoxifen, the non-steroidal estrogen-receptor antagonist that is a leading choice for treatment of advanced breast cancer. These molecules, which were identified as potent ER antagonists, had IC 50 values in the range of 0.1-0.3 mM. Although these activities were approximately 7-17 fold less than that of tamoxifen, their selectivity (50-fold better binding to ER as compared to the androgen receptor) and low toxicity (significant inhibition of tumor volume in a subrenal capsule assay at 100 mg/kg; no acute toxicity in mice or rats at 500 mg/kg) profiles were promising. (Yori, H. et al., supra, Part III.) Accordingly, it appears that manipulating the structures of natural tamoxifen-like products offers an opportunity to optimize their pharmacological properties.

In order to further study the pharmacological properties of R1128 as a first step towards engineering the structure to enhance its pharmacological properties, the present invention provides for a cloning procedure that allows the capture of the entire R1128 biosynthetic gene cluster and its expression in a model heterologous host, Streptomyces lividans K4-114 (Ziermann, R., et al. (1999) BioTechniquies 20: 106-110. Although described herein as an exemplary cloning procedure, it should be understood by one of skill in the art that other known cloning procedures and hosts can also be utilized to clone the compounds describe herein using routine cloning and expression optimization methodologies.

Recent studies on bacterial aromatic polyketide biosynthesis have demonstrated considerable promise with respect to altering four aspects of product structure: primer unit incorporation, chain length, degree and regiochemistry of reduction, and regiochemistry of cyclization. (R. McDaniel, et al., Nature 375: 549-554 (1995); and L. Han, et al., J. Bacteriol. 180:4481-4486 (1988).) Most of these features are potentially attractive targets for manipulation in the R1128 family of ER antagonists. For example, the incorporation of an additional ketide unit in the polyketide backbone could lead to the introduction of an aliphatic ketone functionality in place of the variable alkyl group. Likewise, elimination of the C-9 hydroxyl has been accomplished in similar systems by co-expression of a suitable ketoreductase with regiospecificfty towards the C-9 carbonyl of the nascent polyketide backbone. Finally, of particular interest is the variable functional group in the R1128 anthraquinone series, which is presumably derived from alternative primer unit incorporation in the polyketide backbone. The recent identification of a single protein as the principal determinant of primer unit specificity in bacterial aromatic polyketide biosynthesis suggests that it should also be possible to predictively alter this functionality in the natural product.

A minimal prerequisite for biosynthetic engineering of natural products is the availability of cloned genes, which are typically clustered and coordinately regulated in microorganisms. An ideal strategy for cloning a desired gene cluster would involve its direct identification through heterologous expression in a biologically friendly host. Building on the pioneering studies of Hopwood and coworkers (Malpartida, F. et al. (1984) Nature 309: 462-464), recent studies have successfully demonstrated the ability of an organism, such as S. lividans, to produce heterologous aromatic polyketides (Hong, S. T., et al. (1997) J. Bacteriol. 179: 470-476; and Gould, S. J., et al. (1998) 51: 52-57.) However, due to the limitations of the integrative vectors used in such efforts, genomic libraries had to be pre-screened for candidate cosmids carrying putative polyketide genes prior to their transfer into S. lividans.

Accordingly, other vector systems capable of exhibiting a larger DNA carrying capacity for DNA are more preferred for use in the present invention. The pHU204 vector, which is a 12.16 kb derivative of pRM5, has such properties (McDaniel, R., et al. (1993) Science 262: 1546-1550.) Among other features, it has the well-characterized SCP2* replicon. While its carrying capacity as a cosmid cloning vector in E. coli is limited to 40 kb inserts, SCP2* vectors have been used to clone significantly larger fragments in S. lividans.

The organization of the R1128 gene cluster is shown below in Table I and the sequence is given below in Seq. ID No. 1

TABLE I
Organization of the R1128 Gene Cluster
Gene				Length	Length	Start	Stop
No.	Gene Function	Start	Stop	(bp)	AA	Codon	Codon
				0
1	Chain Length factor	1623	376	1248	416	ATG	TAG
2	ketosynthase	2873	1620	1254	418	ATG	TGA
3	Acyltransferase	3067	4113	1047	349	ATG	TGA
4	Acetyl-CoA carboxylase subunit 3	6207	4822	1386	462	ATG	TGA
	(Biotin carboxylase)
5	Acetyl-CoA carboxylase subunit 2	6651	6214	438	146	ATG	TAG
	(Biotin carrier protein)
6	Acetyl-CoA carboxylase subunit 1	8901	7165	1737	579	ATG	TGA
7	Acyl Carrier Protein 1	9182	8922	261	87	ATG	TGA
8	Priming ketosynthase	9448	10467	1020	340	ATG	TGA
9	Aromatase/Cyclase	10903	11412	510	170	ATG	TGA
10	Cyclase 2	11409	12179	771	257	GTG	TAG
11	Oxygenase 1	12183	13757	1575	525	ATG	?
12	Amidase	13754	15277	1524	508	ATG	TGA
13	Oxygenase 2	15270	16823	1554	518	GTG	TGA
14	Acyl Carrier Protein 2	16861	17109	249	83	ATG	TGA
15	Transcriptional regulator (has a 1 bp	17249	18060	812	271	GTG	TGA
	frameshift)
16	Transporter (has a 1 bp frameshift)	18154	18923	770	257	ATG	TGA
17	Oxygenase/Transporter	18920	20122	1203	401	GTG	TAG
18	Activator	21003	20185	819	273	ATG	TGA
19	Repressor	22124	21357	768	256	ATG	TGA
20	Activator	22255	22992	738	246	ATG	TGA

When this gene cluster was isolated and expressed as described in Example I, an alternatively cyclized, optically active product designated HU 235 ( FIG. 2 ) was also produced. Because a mutation in the cloned gene structure abolishes production of HU 235, but still allows for production of R1128B and C, this suggests that the bioactive natural products might actually be side products, rather than the principal products, of the biosynthetic pathway shown in FIG. 3 .

EXAMPLES

Example 1

Cloning of the R1128 Gene Cluster

Anticipating that the R1128 gene cluster was under 40 kb in size, a cosmid library was constructed in pHU204 (McDaniel, R., et al. (1993) Science 262: 1546-1550) using genomic DNA derived from Streptomyces sp. R1128, the R1128 producer. The entire library was transformed without prior screening into S. lividans K4-114. Several pigmented colonies were identified; one of them, which was designated K4-114/pHU235, was cultivated on a larger scale (500 mL on semi-solid R5 agar medium). After neutral extraction and pre-purification using column chromatography, two major UV-active peaks were collected from reversed-phase HPLC, dried, and analyzed. Mass-, 1H and 13C NMR spectra revealed that one of the compounds was identical to R1128C (FIG. 1 C). The purified yield of the compound was 0.5 mg/L. The second compound, designated HU235 (FIG. 2 ), was produced in considerably greater quantifies (16 mg/L), and was structurally characterized by 1H, 13C, 1H, 1H-COSY, HMBC, HMQC, ROESY-NMR, and IR spectroscopy. Mass spectrometry was performed with the acetylated derivatives 6 and 7. The polyketide origin of HU235 was established by feeding of 1,2-13C sodium acetate, which gave the expected incorporation pattern as observed in the 13C NMR spectrum. It was initially suspected that HU235 was a shunt product resulting from low activity of a late-stage enzyme in the R1128 pathway. However, optical rotation analysis revealed that the compound HU235 was optically active ([a]D=33.0°), suggesting that it arose from stereocontrolled cyclization of a common biosynthetic intermediate ( FIG. 1 a ). To test this hypothesis, we subjected cosmid pHU235 to in vitro transposon mutagenesis (R. McDaniel, et al., Nature 375: 549-554 (1995). Transformation of the resulting pool of mutagenized cosmids into S. lividans led to the identification of one clone, Tn164, that produced both R1128B and R1128C in 1.4 mg/L and 1.6 mg/L, respectively, while completely abolishing HU235 production. The result confirms that one or more enzymes encoded within the biosynthetic gene cluster are involved in the final stages of HU235 biosynthesis. If so, then the biologically active R1128 metabolites are probably side products of a biosynthetic pathway whose principal product is HU2353

Example 2

Isolation and Characterization of Products

To further study the products obtained in Example 1, the cultures were extracted with EtOAc. The extract was dried with MgSO4, evaporated, and subjected to column chromatography (SiO2, Hexane/EtOAc 1:1). The yellow fractions were dried and further separated on a preparative reverse phase HPLC column (25×150 mm, C18-IP column, Beckman, isocratic run with H2O (1% HOAc)/MeCN 55:45, flow 20 ml/min, UV-detection at I=250 and 350 nm. Three major UV-active peaks (tR=10.2, 22.5, and 28.3 min) were collected, dried, and analyzed. Spectral data of HU235 (5): [a]D (in CH3OH, c=0.63)+33.0°; 1H NMR (500 MHz, (CD 3 )2SO): d=16.10 (s, 1 H-O-C(13)), 10.23 (s, 1 H-O-C(9)), 9.81 (s, 1 H-O-C(11)), 4.84 (s, 1 H-O-C(3)), 2.95 (2 d, J=16.3, 2 H-C(4)), 2.58 (dd, J=8.1, 3.0, 1 H-C(16)), 1.75 (m, 1 H-C(2′)), 1.64 (m, 1 H-C(1′)), 1.57 (m, 1 H-C(1′)), 1.25 (s, 3 H-C(2)), 0.95 (d, J=6.4, 3 H-C(3′)), 0.91 (d, J=6.6, 3 H-C(3′)). 13C NMR (125 MHz, (CD3)2SO, (coupling constants of 13C-enriched material, 100 MHz, (CD3)2SO)): d=206.4 (C(15), J (C,C)=39.9), 165.3 (C(13), J (C,C)=63.0), 161.3 (C(9), J (C,C)=65.0), 159.1 (C(11) J (C,C)=62.5), 140.8 (C(7), J (C,C)=58.2), 136.2 (C(5), J (C,C)=64.9), 115.9 (C(6), J (C,C)=64.9), 106.9 (C(14), J (C,C)=63.0), 106.4 (C(12)), J (C,C)=62.5), 101.7 (C(8), J (C,C)=58.2), 101.1 (C(10), J (C,C)=65.0), 71.2 (C(3), J (C,C)=36.8), 55.1 (C(16), J (C,C)=39.9), 41.2 (C(4), J (C,C)=36.8), 34.4 (C(1′)), 28.0 (C(2)), 26.5 (C(2′)), 22.5 (C(3′)), 21.8 (C(3′)); IR (KBr):˜=3448m, 2956m, 2553w, 1638s, 1597s, 1458w, 1420w, 1384w, 1163m, 1095w, 917m, 848m. Acetylation of HU235: HU235 (5 mg) was stirred in 1 ml pyridine/Ac2O (3:2) for 2 h, the mixture was poured into 2 N HCl and extracted with ether. Two products were isolated after chromatographic separation (hexane-EtOAc 3:2). HU235Ac2 (6): 1H NMR (400 MHz, CDC13): d=7.33 (d, J=2.1, 1 H), 7.02 (s, 1 H), 6.87 (d, J=2.1, 1 H 3.21 (d, J=16.5, 1 H), 3.00 (d, J=16.5, 1 H), 2.60 (dd, J=8.3, 4.0, 1 H), 2.38 (s, 3 H), 2.33 (s, 3 H), 1.78 (m, 1 H), 1.66-1.53 (2 m, 2 H), 1.37 (s, 3 H), 0.99 (d, J=6.4, 3 H), 0.93 (d, J=6.5, 3 H); MS (FAB+): m/z (%): 459 (15) [M-H+2 Na+], 437 (100) [M+Na+]; 415 (20) [MH+]; HRMS (FAB+) calcd. for C23H26O7 Na+437.1576, found 437.1589. HU235Ac3 (7): 1H-NMR (400 MHz, CDC13): d=7.57 and 7.55 (2 s, 1 H), 7.48 and 7.45 (2 d, J=2.1, 1 H), 6.95 and 6.93 (2 d, J=2.1, 1 H), 3.31 (m, 1 H), 3.15 (m, 1 H), 2.58 (m, 1 H), 2.47 and 2.45 (2 s, 3 H), 2.40 and 2.38 (2 s, 3 H), 2.33 and 2.31 (2 d, 3 H), 1.65-1.51, 1.78 (m, 1 H), 1.66-1.53, 1.42-1.34 (2 m, 3 H), 1.36 and 1.34 (2 s, 3 H), 0.96-0.84 (m, 6 H); MS (FAB+): m/z (%): 479 (100) [M+Na+]; HRMS (FAB+): calcd. for C25H28O8 Na+479.1682, found 479.1695.

Numerous modifications may be made to the foregoing systems without departing from the basic teachings thereof. Although the present invention has been described in substantial detail with reference to one or more specific embodiments, those of skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the invention as set forth in the claims which follow. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference.

1 1 23673 DNA Human misc_feature (1)...(23623) n = A,T,C or G 1ccaccggggc tcgtccccgt gccgcaccgg gtcggttgtt cgtgtcgcgt cgccctgcga 60cgacgcggca gtcactccgt gcggcggcaa accgggtttc acacgtcggt ggttcgcacc 120gacgggcgtg ctcccggtcg tgggtcgagc ccggccgggc gtcgggcggc ggcggttcgc 180gccgtcgtcc gtcgctccgg ccacgccggc ctcccccgtt gccgcgcgga tgcgcgacga 240cgggaggccg gccagggtcc cggcggcggt ggttcgcacc gtcgcccggc cctccgagat 300cgaggtcccg gtggtcgtgg gccgccgacg tgcgccgcgc ggtcgcgtcg ggtcgcatgt 360cggtcggctc ggcttctaca gttcggggtt catcgcgcgg accacgacgg ccgcgttgaa 420cccgccgtgg ccgcgggcca gcaccagcac cgtccgcacg cgcgcgttgc gcggttcgga 480cagcacgagg tcgagcccgt agtcggccgg cacgtcggtg gtgttgggcg tcggcgggac 540gacgtcgtcg cggatcgaca acagcgcggc caccacgtcg agcggcggcc cgcccgagtt 600gaggcgtccc accaggctct tgggcgccgt caccggcacc gcgccggccc cgaacacctc 660gttgatcgcc tcggcttcgg cgcggtccag gtccggtacg ccggccgcgt ccgcgaacac 720cgcgtccacg tcctcgggcc gcacgcccgc gtcgtcgagc gcgaggcggg ccgcgcgggc 780cagccccggc ggccgttcgg agccgggcgg cgggtcgaag gtggccgcgt agcctgcgat 840ctcgccgtag agcgtggccg cgccccgggc ccgggcggcg cgttcgtcct ccaggacgag 900gatggccccg ccctcccccg gcacgtagcc gtcggcggcc gcgtcgaacg gccggtaggc 960cagctcgggt tcggtcgcgc gggagatccg tccggtggtc aggtggctga cgtatcccca 1020cgggtcgagc gccgagtcgc atccgccggt gaccacgagg tcgatgccgc gccgcaggtt 1080gcggcgcgcg tggccgatgg cgtcgaggcc gccggcttgt tcgccgacca ggacgccgct 1140gggcccgcgc atgccgtgcc ggatggacaa ctggccggtg ttgacggcgt agaaccacgc 1200gaacgactcg tagacgctga cgtgttgcgg tcccttggtc cagagcttgt gtacctcgcc 1260gtgggtgaac tcgaagccgc cgatggcgct ggaggtgacg acgcccatcg agtagtcgac 1320gagtgcctgc gggtcgacgc cggcgtcgtc gagcgcgctc tggccggcga cgagggcgag 1380tcgcgtgacg cggtcggtct gcggcaacag ccttcccgga atgtgctgtt cggcctcgaa 1440gccgagtatc tggccggcga gtcgggacgg gaatccggac gcgtcgtagc ggtgcagcgg 1500gtggatgccg gagcgtccgc gcagcgtggc cttccagtac tcctcggtgc cgaagccgtt 1560gggggcaacg gcgccgaggc cggtgacgac ggcgcgccgc gtcgtggggg ctgcctgggt 1620catcgcgcgt cctcctgaag gcggcgcagc accatcgcgc tctggaagcc accgaagccg 1680ctgccgaccg acagcacggt gtcggtgcgg tgttcgcggg cggtcaccgg cacgtagtcc 1740aggtcgcatt cggggtcgga ctgggtcagg ttcgcggtcg gcggcacgac gttgtgctgg 1800atcgcgagga cgctggcggc gatctcgatg gagccgatcg cgccgagcga gtgcccgatc 1860atcgacttga tggagctgac cgggacgtcg taggcgtggt cgccgagggc gaccttgaac 1920gcgttggtct cgtgccggtc gttctgcttg gtcccggacc cgtgggcgtt gatgtagtcc 1980accgccgtcg ggtcgatgcg cgattcgttc agcgcgtagg tgatggcgtc ggccatctcc 2040cgtccgtcgg ccttgagtcc ggtcatgtgg tacgcgttgc agcgggtcgc gtagccggtg 2100acctcggcgt agatgtgcgc gccgcgcgcc ttggcgtgct cgtactcctc gaggacgaac 2160atcgccgccc cctcgccgat cacgaatccg ctgcgggtca ggtcgaacgg cctcgacgcg 2220gactcgggtt cgtcgttgcg cggggtggtg gccttgatgg cgtcgaagca cgcgacggcg 2280atcggcgaga tcggcgcgtc ggtggcgccg gtgaccatga tgtcggcggt gccctcgacg 2340atgagctggt aggcgtagcc gaccgagtcc aggcccgagg tgcagccgga ggacacgatg 2400gtcgcggggc cctccgcgcc gacgctccag gccacttcgg cggcgatcgc gccgggcgtg 2460aagtagtcgt acatgtgcgg cgtggcgtag tcggcgtcga ccacccagtc gcgtccggag 2520tcggagagca cgaggtattc gcgttcgagg ctcgtggtgg cgccgacggc gctgccgacg 2580ctgactccga tccgggtcgg gtcgaggtcg gcgaacgcca gtccgctgtc ggcggcgcag 2640tcccgcgcgc tggcgacggc gaacagcgac gcgcggtcca ggcggcggat ctcgcggtgc 2700gtgaagccgg ccaggaccgg gtcgaagtcg cattccgcgg ccacctggga ccggaacggc 2760gacgggtcga agtgggagat gcgccgggtg gcggtgcggc ctgaggtgag caggtcccag 2820aaccccttgg ttccggtgcc gcccggggcc acgaccccca tcccggtgat caccgctcgt 2880cgttccaaga caagcctcca gcggtggtgt cggttgttgc tgtgctcttc gagcgtgcgt 2940ggcgcgactc gaaccgtgct caaggacgga tcgcgcggag tcgcgggccg ttgtcgagtg 3000ggggtcgagt ggcgtgcgcg agcctcgtgg acccctgtag atgtactcgg gtcgaggaga 3060cctccgatga cgatcagact ggttccgccg cgccgccgtg tcgccctgct gctgccgggg 3120cagggctcgc aacaccccgg tatggggctg gagttgtacg gctacgacgg tgtgttcacc 3180gagacgatgg acgagatgtt cgagctgctc gggccggtgg acggcgcgtc gctgcgcgcc 3240gactggctgg ccggcgacgc gctcgcgccg gagctgctga acgacgcgag ccgcgcgcag 3300ccgctgttgt tcgcggtcga gtacgcgctg ggccgcagcc tgttgtcgca gggcgtcgac 3360gtggacgtgc tgctcgggca cagcgtcggc gagttgtcgg cggcggcgtt ggccggcgtc 3420gtcgacctgc cgggcgcggc gcgcgtgatg gcggcccgga ccgcggcgtt ggccgcgctt 3480cccccgggcg ggatgctcgc ggtcgcggcg accgccgagg agttggcgcc gttcgtggcc 3540gccgagcacg tcgagatcgg cgtggtggtg ggcgcgttga acgcgccgcg gcagaccgta 3600ctggcggggc ccgagccgcg gttgaccgag gtggagggcg aactgcgggc ggccggtgtg 3660atgttccgtc gggtgccggc gcggcagccg ttccactccc ccgcgtcggc gccggccgcc 3720gaggtggtgg agaaggtgtt gtccggggtg acgttgcgcg agccgacgat cccgatcatg 3780tcgacgcact cgggcgccta cgtgacccgc gagcaggccc tcgacccggc cttctgggcc 3840tggcacttcg tgcgcgtggt gttgttctgg ccggcgttgg acgcgctgct ctccgaggcc 3900gaccacgcgc tcgtcgagct gggcccgggc caggagctga cggtgctggc gcgccggcac 3960aaggcggtcc ggtcgggcgc cagcacggtg cgcgcggtgt tgccgcgcgg ggcgcgcggt 4020acgcgggagg cgtgggacga cgcgctggcc gaactggccg agtacaccgg gccggccggg 4080ggtgtgccgc tcgcctgcgc ggcggaacgg tgaacgtggc actccgccgc gggcggcgcc 4140gccaacgccc ttgagaagcc cggaccgtgg cggtggtccc gtgtcctcgt caccttccgg 4200tgggcgacgt cgcctcctac attggcgggt aaggcggcga ggacatggag gctcggatga 4260gcggtgcctg ggtgcgcagt gccgacggac tctcgttgat ccgcgcggtg gagatcgtct 4320ccctgagggt caacggcacc gccctcgaat gcgactcgcg cctggcccgc gccttcgtcc 4380tggccaccgg cgccgaggag gatctggtcc ggggcgcacg ggagttgatc gagacgctgg 4440cccgactgcc gcacgacgcc tacatcgtct cggccgatgt gaccgacggg cgtgtcgtgt 4500gggcgctctc cgagatcggc ccggtcgagg acgacccgcc gtacgccgcg ggcgggcagg 4560cttacggggc gaccggccag gtgtacggcg cgggcatgcc cggtaggccg atgccgggga 4620tgccgatggg gggcgcgtgg ccgccctggg gcaacccgcc gccgcacagg tgagcctcgg 4680gtccggcaca tcccgttccg ggcccggccc ccgcctcgcg gtacgcgaac acccccgccc 4740ggcccgcggc gtcgtcgttc gcgggccggg cggaggcggg ggtcggcgtg cctgggagca 4800ccgccgggca cgccgaccgc ttcacgcgga caggggcact cccgtccgcg cgttccgggc 4860ctcggcggcg agcatctcgt cgacgaggga ggtgctgtgg cgtccggcgg cgaacttgtc 4920gttgtcgaac acctcgcgca ggaacccgat ggtggtgcgc acccgttggc cctcgacctc 4980gaactggtcg agcgcgcggc gcatgcgggc gagggcctgt tcgcggtccg gtgcccacac 5040cacgagcttg gccagcagcg agtcgtacga cgcgggcacg gtgtacccgg cgtgcacgtg 5100tgtgtcgacg cgcacgaacg ggccgccggc gggcgtgaac gtgtcgatca gccctggcgt 5160cggcacgaat ccgcgcgtcg ggtcctcggc gttgatgcgg cactccatgg acacgccgtt 5220gggtcggatg tcttcctggc gcaccgtcag cggacggccg gcggcgacca ggatctgctc 5280gcgcaccagg tcgatgccgg tgaccatctc tgtgaccggg tgctcgacct ggatacggca 5340gttgacctcc atgaaggaga acgttccgtc gggcgcgagc aggaactcga acgtgccggc 5400gccgacgtag ccgaccgcgc gcgccccggc gacggccgcc tcgcccatcc gggcggcgag 5460gccgtccgga agccggggcc cgggcgtctc ctcgacgagc ttctggtgcc ggcgctgcac 5520ggagcagtcg cgttcgccca ggtggacgac gttgccgtag cggtcggcga gcacctggat 5580ctcgacgtgc cgggcctcgg gcaggtagcg ctcgacgtac acccgcccgt cgccgaacac 5640cgccaacgcg gtggcctggg tgcggcggaa cgtgtcgggg aagtcgtcgc tggagtggac 5700caccgacatg ccgcgcccgc cgccgccggc cgccgccttg acgatgacgg ggtagccgat 5760cgtgtcggcg agcgcgtgcg cctcgcggtc ggagagcggg tcgaggctgc cgggcagcag 5820cggaaggccg gcctgcgaca tgatcgaccg ggcggacgcc ttgttcccga gccgcgacat 5880cacctcggcg ggcgggccga tgagggtgat gccctcgtcc tcgcacgcct cggcgaagtc 5940cgggtcctcg gacaggaatc cgtagccggg gtggaccgcc tcgacgccgg tctgcacggc 6000ggcctggagc acggcggcgg cgttgaggta gctggcgcgc ggcggggcgg ggccgacgca 6060cacggcctcg tccgcgaacc gcacggcggc ggagtggcgg tcggcatcgg agtgcacgac 6120gacggtccgg atgccgagct cccgacaggt cgcggcgaca cgcagcgcga tctcgccgcg 6180gttggcgatg agtaccttct cgaacatgtc gggctacgcg ccttccacgg ggaccagcgc 6240gacgagcggc tcgccgtact cgacctggtc gccgttggcg cgcaggatcg cggacacccg 6300gccgggacgg tcggcctcga cggggagcat gagcttcatc gcctcgacga tgccgatctg 6360ttgaccgggg cggaccacgt cgccgacccg cacgaacggc gcggcgccgg gctcggtggc 6420gtggtagaac acgccgacgc tgggcgcgca cacctggtgc gcggtcggct ccgccggggg 6480cgcgggcacg ggcggcggcg cggcgacggc gggcacggtg gcgacgggaa cggcggacac 6540gacgggctgg ggcaccgccg gggcctgcgg cgcctcccag gacacctcga tcccgacgcc 6600gtccgcgcgc acgttgagcc gcgtgggccg ctcgggcaac gcttcgagca tctcgatgac 6660ggagtgccgc agggtgttca acaaccgcgc ggcgtcctcg tcggcgccct cgccgccgcg 6720taccgcgggg tcggcgatcg gggtgatggt cacggtgtct ccttctcctt gattcctgta 6780cgtctacgcg gccgcgccac ggacggcccc tcgggacaca ccacctgcgc ggccgccccg 6840accccgtgcg cgagcgccac ggcgagcccg cccgagagca cgagccgaaa cccgaccccg 6900accacacccg cgacggtcga cggggagtcc tcccccggcc gcacgtggtg ggcgcgaagc 6960aacgcccgcg aaacgggctg aacggcttcg acgaggggct gaggcgtcat gccgtcacca 7020cacacgcgcg ggtcgcgcgc accaggccga agcagcgagg cggcggggcc gcggatgcga 7080ggacgccgct gggcgacgcg gcgactcgag gcaccgacac cccgccgtca cccccacaac 7140cgccgcgcgc agcgccggtg ggtctcacgt cgggtcgcct ttcatggcgg tgatctcggc 7200gtcggtggtg cgttccgcgc catagcggcg gaagcgggcc cagcggtcgg cgaccaactg 7260gtggccgtcg aggggggaca actccgcgag gccggccgcg atcgcgtcgc ggagggcgtc 7320ggcggcgcgc agcgggtcgg ctccggtgcc gccttcgggt tccgggagga cgccgtcgac 7380gatgcccagg ccgaggagtt cgcgggcggt gaccctcagc gcgcgggcgg cacgcccggc 7440ggcggcgggg tcctgccaca ggatggccgc gcagccctcg gggctgatga ccgagtagat 7500gccgttctcg aacatcagga cacggttggc gacggcgagc gcgagcgccc cgccgctgcc 7560gccctcgccg gtgacgacgg tgacgaccgg cacgcgcagg cccgccatca ggcgcaggtt 7620ctcggcgatg gcgacggcct ggccctgctc ctcggcgacc gcgccggggt aagcgccggg 7680ggtgtcgacg aacgtgacga ccgggagacc gagtttgtcg gcgagccgca tgatccgggc 7740ggccttgcgg tagcccgacg ggatcggcat gccgaagttg cggcggcgca gctcctgcgc 7800gtcgtggccc ttctggtggc cgatgacggc gacgggtcgg ccgccgaagc gggccaggcc 7860ggcgacgatg gcggggcagt cgcccgagat gcggtcgccg cgcagttcga cgaagtcctc 7920gcacagcagc gcgatgtagt cgagcgtggt gggccggtcg atggcccgcg cggcgcgcac 7980ctggcggctg gggtcgtgcg cggcgaggtc ggcggggtcg acgacgtacg gcgatgcccc 8040cgcgtccagg ccgccgcgtc gggccggttc cccgtccccg cccttgggcc gcttcgcgcc 8100ggcccgcagc aggcgggcca gttcgtcgcg cagcgcgcgg cgcggcacga tcaggtcgat 8160caggccgtgt tcgaggaggt attcggcggt ctggaactcc ggcggcagtt cctcgcggat 8220ggtctgcgcg atgacccgcc ggccggcgaa gccgagacgc gcgccgggtt cggcgacgat 8280gacgtcgcac agggtcgcga aggacgccgc gacgccgccg taggtggggt cggtgaccag 8340cgagatggtc aacacgccgg cctcgtccag ttgcccgagg gcggcgctgg tcttggccat 8400ctgcatgagc gcgacggcgc cttcctgcat gcgcgcgccg ccggacgcgc tgacgatcag 8460cagcggcagg tgttcggcga gcgcggtctc ggcggcgagg gtgatcagtt cgccgaccgc 8520gccgcccagg ctgccgccga ggaacgcgaa gtccatcgcg gcgacgacga gcggctggcc 8580gtggatccgg ccggtcacga tgacgacggc ctcgtcgagc ccggtggcgg cacgcgcggc 8640ggcgagccgc gcggggtagg gcttggtgtc ggtgaagccg agcacgtcga aggtgcgcac 8700gtcgttgcgg atcggctcgg cggagccggg gtcgaggagc tggtcgaggc gttcgcgggc 8760gccgaccggg tggtgtttgg cgcattcggg gcagacccgc aggccgcggg cccagcgctt 8820gccgtacacg gcggcgcggc agttgccgca ctgcacccag tcggcgacgc cggtcgtggg 8880ccggatggcg gtttcggtca tgtcgttgtc tccctcggtt ctcagcccgc ggtctcgacg 8940agcaggccgt tgacgaacgc gaccatgtcg cgcggtgtct ccaggccgtc gatgtcgtcg 9000tcggagatgg cgacgccgcg ctcgtcctgg atccgggtga cgacctcgta gacggcgagc 9060gagtcgaggc cgaggtccag gaacggggtg gccgcgccgg tctcgtcgag ctggaccgcg 9120tcgtcggtgc cgacgcacgc gtcgatcagc cgcttgaggt cgtcgagcgt gaaggggtcc 9180atgggttcgt cctcccggtg gtctggcttc ctgccgaacg cagacagcca ctgtcggccg 9240gccggatgga ggtctggtcg agagccgctg gatgtgcgcc ggagggcgcg gcgtcggctt 9300cgagtcggct tccggcgggc ttcgaggggg cgccgcgcat gctcggtgcg cgcccctctc 9360ccgccggacg ccgccggacg cccggcgggt cccagacggg gttcaggcgg gtgtcaggcg 9420agaccagaac cgcgaaaggt ggtcgcgatg cccggacttc gggttcccga gcggcgtttc 9480agccgtgttc tcggggtcgg ttcgtaccgg ccccgccgtg aggtgagcaa caaggaggtg 9540tgcacctgga tcgactcgac ggaggagtgg atcgagacgc gcaccgggat caggtcgcgg 9600cggatcgccg agccggacga gacgatccag gtgatggggg tggccgcgag ccgccgcgcg 9660ctggagcacg ccggcgtgga tccggccgag atcgacctgg tcgtggtctc cacgatgacc 9720aacttcgtgc acacgccgcc gctttcggtc gcgatcgcgc acgagctggg tgccgacaac 9780gccggcgggt tcgacctgtc ggcggcgtgc gcggggttct gccacgcgtt gtcgatcgcg 9840gcggacgcgg tggagtccgg cggttcgcgg cacgtgctcg tggtggcgac ggagcggatg 9900accgacgtga tcgacctggc ggaccggagc ctgtcgttcc tgttcggcga cggcgccggc 9960gcggccgtgg tcggcccgtc cgacgtgccc ggcatcggtc cggtggtgcg cggcatcgac 10020ggcaccggcc tgggctcgct gcacatgagc agttcgtggg accagtacgt cgaggacccc 10080tcggtgggcc ggccggcgct ggtgatggac ggcaagcggg tgttccgctg ggcggtcgcg 10140gacgtcgtcc cggcggcccg cgaggcgctc gaggtggccg gcctgaccgt cggggacctg 10200gtggcgttcg tcccgcacca ggccaacctg cggatcatcg acgtgctggt ggaccggctc 10260ggcgtgccgg agcacgtggt cgtctcgcgc gacgcggagg acaccggcaa cacctcgtcg 10320gcgtcggtgg ccctggccct ggaccggctg gtgcgctccg gcgcggtgcc cggcggcggg 10380cccgcgctga tgatcggttt cggcgccggg ctgagctacg cgggccaggc tctactgctt 10440cccgacccgc cgtccactcc ggcttgagcg gcgttctagc cgccgcccgc aagctcggca 10500tcaaccccag tcggccgcat cacccgtcaa ggagatccac cgtgatcgtg cacaccctga 10560tctaccgttt cccggcctcc gtcccgcagg aggacctgga cgcgttcttc gacgcggcgc 10620gcgagctggt gctgagcacc ggcctgatga acgcgttcga cgtcaagccg cacctgtggc 10680tgcccgccga cgaccgcgcc cgcggcatga cggccggcta catcgtgcag ttcgtgtgcg 10740acgacctgga cgcgctcgcg aagttctccg agctgcccac cgtctacgac ttcatcacgg 10800actggaaggc ccgcctgcag ttcgaggcgg cgtacgccaa ccacgaggcg ctcgacctca 10860cccccaccgc ctgagcccgc ccaaccaccg aggagcaccc tcatgcgtca cgtcgaacac 10920accgtcaccg tagcggcccc ggccgacctg gtctgggagg tgctggccga tgtgctcggc 10980tacgccgaca tcttcccgcc gaccgagaag gtcgagatcc tcgaggaggg tcagggctac 11040caggtcgtcc gcctgcacgt ggacgtcgcc ggcgagatca acacctggac ctcgcgccgc 11100gacctggacc cggcccgccg cgtcatcgcc taccggcagt tggagacggc cccgatcgtc 11160gggcacatga gcggcgagtg gcgcgccttc acgctggacg ccgagcgcac ccaactggtg 11220ctcacccacg acttcgtgac gcgcgcggcc ggcgacgacg gcctggtggc cggcaagctc 11280acccccgacg aggcgcgcga gatgctggag gcggtcgtcg agcgcaacag cgtcgccgac 11340ctgaacgcgg tgctcggcga ggccgagcgt cgggtgcgcg cggccggcgg cgtcgggacg 11400gtgaccgcgt gagcggccgc aagacgttcc tcgacctgag cttcgcgacc cgggacaccc 11460ccagcgaggc caccccggtc gtcgtcgacc tgctcgacca cgtgaccggc gcgacggtgc 11520tcgggctgag ccccgaggac ttccccgacg gcatggccat ctcgaacgag acggtcaccc 11580tcaccacgca caccggcacg cacatggacg cgccgctgca ctacggcccg ctcagcggcg 11640gcgtgccggc caagtcgatc gaccaggtcc cgctggagtg gtgctacggc ccgggcgtgc 11700gcctggacgt gcggcacgtg ccggccggcg acggcatcac cgtcgaccac ctcaacgccg 11760cgctcgacgc cgccgagcac gacctggcgc ccggcgacat cgtcatgttg tggaccggcg 11820cggacgccct gtggggcacc cgcgagtacc tgtccacctt ccccggactg accggcaagg 11880gaacgcagtt cctggtcgag gcgggcgtga aggtgatcgg catcgacgcg tggggcctgg 11940accggccgat ggccgcgatg atcgaggagt accggcgtac cggcgacaag ggcgccttgt 12000ggccggcgca cgtctacggg cgcacccgcg aatacctcca gttggagaag ctcaacaacc 12060tgggcgcgct gcccggcgcc accggatacg acatcagttg cttcccggtc gccgtcgcgg 12120gcaccggtgc gggctggacc cgggtggtcg ccgtgttcga gcaggaagag gaggactagt 12180ccatggacgc cgacgtcatc atcgccggtg ccggccccac cggactcatg ctggccgccg 12240agctgcggct ggccggcgcc gacgtgctga tcctggagct gctgccggag cccaccggcc 12300agtcgcgcgc gctcggcatc aacccgcgcg ccgtcgaact gctggacgcg cgcggcctga 12360tgggccggtt cgccgaccag cacaccatca ccaactcgca ctacggcgcg ctgcaccgcc 12420cgctggactt cagccggctc gacaccgcgc acggcgtcat gatggtgccg cagcgccgca 12480ccgagaagga actgggcgag tgggcccggg ggttgggcgc acgaatactg cacgggcaca 12540ccgtggtcgg actgacccag tacgccgacc gggtcgaggt cgaggccgac acgtccgagg 12600gcacacgcac cttcaccgcg cggtacgtgg tcggcgccga cggcagccgc agcagtgtgc 12660ggcagttgtc cggcatcggc tacccgggcg tgccgtcgac cgtcgacgcg ctcatgtgcg 12720acgtgtcggg cctggacctg gccttcaagt tcttcgagcg caacgaccgt ggcttgtggg 12780cggtgttccc ggtcggcgac ggcgtcttcc gggtcgtggt ctacgcgttc gaccgcccgc 12840cggtgcgcgg cgcggacgcg ccctccttcg aggaggtcaa cgacgcggcc cgcaccattg 12900cggggatcga cctgaccggc ggcacgccgc actggctgag ccggcacggc aacaccaccc 12960ggatcgccga ccggttccgc gccgagcggg tgttcctggc cggcgacgcg gcgcacgtgg 13020tcccgccggc gggcggccag ttgctgacca ccgggctgca cgacgcgacc aacctgggct 13080ggaagctcgg cgcggcggtc gccggctggg ggtccgacga cctgctcgac agctaccagg 13140acgagcggta cccggtcgcc gagcggatca tcctcaacgc gcgcgtgcag aacctgctca 13200tgtcgggcgg cctggacatc gccgcgctgc gcgaactgtt caccgagctg gtcgagttcg 13260acgaggtgaa cttctacctg tcgggtcagc tcagcgcggt gtcggtgcgc tacgacgtcg 13320gcggcgacca cccgatgctg ggcctggggc tgccgcaccg cacgctggag accgacgcgg 13380gcgaggtgac caccaccgag ctgctgcggt cggcgcgcgg gctgcttctg gaggtcgggg 13440acgcgcctgg cttcgcggac ctggtcgcgc cgtacgcgga ccgggtggac gtccgggtcg 13500cgcgcgtccc ggccggctcc cccgggttcg acggggtcac cgccgtgctg gtccgcccgg 13560acacgcacgt cgcgtgggtc gccgagaccc ccgcgggcgc cgacgcggcg gacgcggacc 13620gcgacgcgct cggcaccgcg ctgcgccgct ggttcggcgc gccggccgac acgaactccg 13680gcgcggaatc cggtacggag tccggcacgc agaccgccgc gcccgtcggc gccgggaccg 13740ccgggggcac gccatgaccc tcgacgacgg cgcggtcctc gcgcccgccg ccgtcctgac 13800ggcggcgctg cgccgccggg agatctccag ccgcgaactg ctcgacctgt acctggcccg 13860cgtcgaggcg gtgaacccgg cgctgaacgc cgtggtcacc ctcgacgtgg aacgggcccg 13920ccgcgaggcg gccgaggcgg accgcgcgac ggcggccggc gcccgtaccg ggccgctgca 13980cgggctgccg atgacggtca aggacaccct ggagaccgag ggtctgcgca ccacggccgg 14040cgccgcggaa ctggccgagc acgtgccggc ccgcgacgcg gactcggtgg cgctgctgcg 14100ccgcgcgggc gcggtggtgt tcggcaagac caacaccgcc acctacgcct ccgacgcgca 14160gacctacaac ccggtgttcg gcaccaccaa caacccgtgg gacacctccc gcgcgccggg 14220cgggtcctcg ggcggcgcgg cggccgcgct cgccgccggg ctgacgtcgc tggaactggg 14280cggcgagctg tccggttcgg cgcgctaccc ggcgcactgc tgcggcgtgt tcgcgctgcg 14340gccgtccttc gggatcgtgc cgatgcgcgg ccacatcccg cgccaaccgg ggtcgctgaa 14400gaccaacgac atggtgacgc tggcgccgct cgcccgcagc gcggccgacc tcgacctgct 14460gctcgacgtg ctggcccggc cggccgccga caaggcgccc gcgtggcgcc tggacctgcc 14520cgcgccgcgc gccgaatcgc tgggcgggct gcgcgtgggg ctgtggctgg acgacccgct 14580gtgcccggtc gacgccgagg tcggcgacgt gctggcggcg gcggtcgacg ccctcaaggg 14640caccggcgtc gcacggctgg cggacgtcac gccggtcgac cgcgcgacgc acgaccggct 14700ctacgagcgg ctgttgcagg gcgcggtcag tctcggactg cccgccccgg tgtacgcggc 14760caaccaggcc gcggccgccg cgctcgcccc cgacgacgac tcgccgcgcg cgtcgttcct 14820gcgcgccgcg acgctgtcgc accgcgactg gctggcggcc gacgaggagc gcgagcacca 14880gcgggcccgt tgggccgagg tgttcgacga gttcgacgtc gtcctgtcgc ccgtcgcgcc 14940ggtcgtcgcg acgccgcacg accagcgtcc cgacctgtcc gcgcggcgga tcaccgtcaa 15000cggcgcccaa cggccctact gggacatcat ccgctggacc agcccggcca ccgcggccgg 15060cctgccggcc gcgtccgtcc cggtgggggt ggcccgttcg ggcctgccgg tcgggctcca 15120ggtggtcggc ccgcacctgc acgaccgcac cgtcacgtgg ttcgcccggc gtgtctcgga 15180gctgctcggc ggcttccagg ccccgcccct gcgcgccgcc gggaacgacg gggcgtcccc 15240cggatcatca cgaacggaag gagcggaccg tgcctgagtc cgacggcatc gacggcacct 15300accccgtcgt catcgcgggc ggcggcccgg tcggcaccat gctggcgtgc gggctgcggc 15360aggccggcgt cgacgttctg gtgctcgaac gccgcaccga gcccgacctg ttgccgcgcg 15420ccggctcgat gggcccgctg gcgttcgagg cgctggaacg gctcggcctg cacgacgcgc 15480tgctggccgc cgagcaggac acgctcgccg agtacggcaa gatgttcgcc gactgggcgg 15540ccaagaaggg catcggccag ccggccaccc gctccgcgcc caaggagcac ttcgcgggcc 15600tggagaagat cgacccggcc cgccgcaccg accccgagcg ccgccgggtg cgcgtcgagc 15660agccggtgct cgaggagatc ctgcaccggc acgcggtgtc gctcggcgcg gtgatcctgc 15720gcggccacga gatcgtcggc gtgttccagg acgaggagcg cgtcatcgtc gacgtgcgca 15780ccgtcgacgg gaccgtcgcc caggtgacca cgcagtacct ggtcggcgcc gacggcgcgg 15840acagcagcgt gcgcggcttc gtcggcttcg acttccccgg caccgacgcg accgtcaccg 15900gccgcatggc ggtcgtggag ttggccgacg aggagaaact ctccccgggc ttccactaca 15960cgccggtcgg cctgtacgtg cacggcctgg gcgtcaaccg gctgtcgacg gtcgagttcg 16020acggtccgcc gaccgacgac gaggacatga ccgccgagga gctgcagggc agcatccgac 16080gggtcagcgg cacggacgtg acgatcagcg agatgtcctc gggcacccgc tggatcgaca 16140cggcgcggca ggcgtccgcg taccggctcg gccgggtgct gctggccggc gacgcggcgc 16200acgtgtacgc gccggtcggc ggccagggcc tcaacgtcgg cctggtcgac gcggccaacc 16260tcgtgtggaa gctggccgcg caggtacagc gctgggcgcc gcactatctc ctcgacacct 16320acggttcgga gcgctacccg gtcgccgcgc ggctgttgca gaacacccgc gcgcagatcg 16380cgctcatgca ccccgacccg cagaccaccg cgctgcgcga gatgttcgag gaactgctcg 16440acatcgacga ggtgcaccgc acgatcgccg acatgatggc cggaatggac gtgcggtacg 16500ccgtcgaggg cgagcatccg ctcctcggca cgctggtccc ggacgccaag ctcaccggcg 16560gcaaggccgc cgacctgtgg gcggacgggc gcggcgtgct cgtcgacttc gccgaccgcg 16620cggacgtgcg cgacgcggtc ggcgcgtggt cggcgcgggt caacgtcgtc acgttgcccg 16680gcgcgcgcga ggacgtcgac gcgctgctgg tccggcccga cggctgcgtg gtgtgggcgc 16740tgcccaccgg cgcggcgctc gccgacgcgc ccccgaccga ggccctgacc acgtggttcg 16800gacgcgcccc gcgcgtgtcc tgagcaatcc caccccatcg cgaaggagat tccgaacacc 16860atgacgatcg acgacctgcg caggatcctg accgagtgcg ccggcgagga cgagtccgtc 16920gacctgggcg gcgacatcct ggacacgccg ttcaccgagc tgggctacga ctcgctcgcg 16980ctgatggaga ccgccgcccg catcgagcag gagttcggtg tcgcgatacc ggacgacgag 17040ttcgccgaac tggcgacgcc gcgtgcggtg ttggccgcgg tctccacggc ggtctcggcc 17100gcggcctgag ccgcggcccg gcccccgggt tccgaggcgc ggtgcttgcg gtgcccgcgc 17160tgccgcggtg ccgcagcccg gttccgcggc cggaatccgg ccaccgtctc ccgtgtcccg 17220cccccatcgg ctcaggggcg ggacacgggt gtgttccgcg ccgcgactcg cccccgagcg 17280tgccggtgtg aggtaatcgt cccgggaaag ggaaggcgtc gccatgtcgc tcggtcgggt 17340gctcgtcgtc gacgacgaac ccaagatccg catgatcgtc cggggttacc tcgaggccga 17400cggctacaag gtcgtcgagg cgtgccacgg tcgcnaccgg cccgacttgt tccggacggc 17460ctgcgcgcca tcgtgcgcga ccggcccgac ctggtcgtcc tggacgtgat gttgcccggc 17520ctggacggct tcgaggtgct gcgccggttg cgcgccaccg tcgaccggac cccggtcatc 17580ctgctgaccg cgcgcgccga ggaggtggac cgggtcatcg ggttcaccgc cggcggcgac 17640gactacgtca ccaagccctt cagcgcccgc gaactcgcct tgcgggtccg cgcggtgctg 17700cgccgctgcc agccggacga gcgggccgcc gagcccgacg acacgctgcg cttcgccgac 17760atcgagatcg acccgaccgc gcgggtcgtc accgtcgacg gcgcccccgt cgggccgctg 17820accgcactgg acttcgacct gctgctcgca ctcgcccgcg cgcccggacg cgtcttcacc 17880cgccaccggc tcctcgaaca cgtctggggc cgcgacgcgt tcggcgacga ccgggtggtg 17940gacgtgcacg tgcgcacact gcgccgcgcg ctcggggacg acgccgccgc gccgcgattc 18000gtcgggacgg tgcgcgcggt cggctaccgg ttcctggccg gcgccgccgc ccacgcctga 18060gccggcgagt gtgagactcg ggcgagcccc gcttcacaca agcttcacgg agcgcaccgc 18120cgtcgtcaca cgaactcccg gtgtgctgga ggcatgacaa ccaccgaagc ggcacctccc 18180cccgcagcgt ccacgcgcgt ggccgcctcc gtcgtcgccg gcgtcaagtc ctacggttcg 18240ggcgacgacg agatccgcgc gctcgacggc gtgaccatag agttcgcctc cggcgagttc 18300accgccgtca tgggcccctc cggctcgggc aagtcgaccc tgatgcactg cgcggcgggc 18360ctggaccggc tcacctcggg ccacgcgtcg cgtacatcgg cgacaccgac ctcggcaccc 18420tgccggaccg cgagttgacg ttgctgcgcc gcagccgggt cggcttcgtc ttccaggcgt 18480acaacctcat ctccacgctc aacgcgcggg agaacatcct gctgccgcag aacctcgccg 18540gcgtgcgccc ggacctggag ttcctgggcg aggtggtgga ccggctcggg ctcggcgcgc 18600gtctgcggca ccgccccggc gagttgtccg gcgggcagca gcagcgggtc gcggtggcgc 18660gcgccctggt cagcaggccg gacatcgtct tcgccgacga gccgaccggc aacctggaca 18720gcgccgcgag caccgaggtg ctggagttcc tgcgcggcac cgcgaacgag ctgtgccaga 18780ccatcgtgat ggtcacccac gacccgcaca ccgccgccta cgccgaccgc gcggtgttcc 18840tcgccgacgg ccgggtcgtc gacagcatcg ccgcccccac cgccgaagcg gtcctggagc 18900gtatgaaggg cttccgaagg tgaacggttt ggtgcgcgcc gcgctcgccg gtgtgtgggt 18960gcgccgcgct cggtatgcga tgaccgtcgc ggcggtcgtc ctgtcggtca tgttcatgac 19020cgccaccctc atgctcacca gcggtctggg cgaccgcgag aaggaccaga tcacccgggc 19080cggcgccggc gtcgacgcgg tggtgcgcgg cccggtggtc gccgacccgg agggcgggcc 19140cggcgaggtg cagacgcccg cgcgggtgcc ggtcgacgac gcggtgctcg cggcggtacg 19200ggcgaccccc ggcgtgagcg cggcggccgg cagcgtcaac ggctatgcgc gcctggcccg 19260ttccgggtcg cggctgctcg gtttcgagga cgccacccgc gacgtgggct cggcgtggat 19320cgacgacgac gcgctgaatc cgtatcggct cgccgagggc cacgcgccgc acggcgcggg 19380cgaggtcgtc gtggacgcgg ccgccgcgcg ccgcgaccac atctccgtcg gcgaacggtt 19440gtcgctgctg accccgagcg ggcgcggcga cgtgacggtc gtgggcgtgg cccgctacgt 19500cggcgacgat gggccggcgg acaagagcgc tgtgctgctc gacgcggccg tcgcgcccgg 19560cctgttcggg cacgcgggcg gctacgaggc catcctggtg cgcggcgccg gcgggcacga 19620cgcggcggca cgcgaggcat tggcgacctc ggtggggcgg acggtcgcgg ccgtgccgcg 19680cgccgggccg gtgccggcac cgggcgcggt cgccgggccg gtgtggacgg gcgagctgcg 19740cgacgcggtc ggcaccgcgt acgacaccct cacgatcatc ctggacggcg cggcgttcat 19800cgcgctgctg gtcggcgccg cgctggtgca caacacgttc accgccgtca ccgcgcaacg 19860gctgcgcgaa ctggcgctga tgcgggcact gggcgccacc cggcggcagg tcaccgcgtc 19920ggtgctggcc gaggcggcgg tggtcggcgc ggtggccggc gtgctcggga tagcgctggg 19980cgcgctcgcc gcgcggatcc ggcgcatgcg cttggcgatc tgctcgactt cggtgacctg 20040ggcccgttcg aagatcaggt tgagcagccg gtctttcatc ggttgcctcc tcgtgacgaa 20100gggaaacggg agccccgggt agggccgggg cttccgtgct caccggacgg gcggtccgcc 20160aaccgcgccg tccgtcggcc ggagtcaccc gaccagttgg tactcgcggc cttccagcgc 20220gggatcggcg tcgaggatcg cgcgctgcaa ctggtgcagc cacggcgacg gttcgaggcc 20280gagctcgtcg agcatcgtca ggcgcagccg ctggaacacg tccagcgcgt gggtgcgcct 20340gccgcaccgg tacagggcca gcatgagctg gccgtgcagg ccctcgtgcg tccggtcgag 20400cgcggcgagc ccggacagct cgtccacgat cgtgtagtgc cggccgagcg cgaggtcgat 20460ctggatgcga cggtcgacca ccgcgcgacg ccggccttcg aggcggagga tctccgcttc 20520gaggaccggg ccgacgtgca cgtcgaccaa ggcggggccg cgccacagcg cgacggcctg 20580ccgtaagagt tccgaggagg tgcggtagtc gccctcctcg cacttgcgca tgcccaggtt 20640cgcgaggcgg tcgaactcga ccacgtccga catgccgtcc gggctggcca acaggtagcc 20700ggtcccacac gtcatgatga tttccttggc gcgcgccttg gagttctccg ccgacgttcc 20760cgcgaccagt cgcttcgcga tacggttccg gatatgcaga atataagtct gcaacgtcgt 20820cgaagcgctc ttgggaaggt ccacgggcca cagctcgtct cggattctcc gagtcgtgac 20880cacgttgttc gaattgactg ccagcagcgt cagaaggcta cgcggcttgg gggcgctcgg 20940gacgacggtc aagccgtcca gatcgacctg cagcggtccc agtatgccga tatccaccat 21000catcccccga tgtgaataca tccttggagc ggctgtcatt gggtttttga cctgcgattt 21060tgtggacggt aattcccctc ttcccctccc ttgcggagca ctttccggta ccgtcgcacg 21120cgggtcgcca tcccgatctc ggacactatc acccggtgcg gaactgccgt gttgccggaa 21180cgtaaaaatg gctgccaata ggacggttgg tgcactgccg ggctcattcg cgacggtccg 21240acgaccggaa atggcggtta tcagggcagc agaaacgacc cgccagtgtg cgggtctgga 21300atattctgtt ctggagggtc ctgcgggaac tccgtcgaaa atcggaggga aaaccctcag 21360gaatcgacgg gggtcgcggt cagaccgttg atcaggacgt tgaccgcgaa atcgaagcgt 21420tgttccgcgg tgccgtaggt caggtgttcc gagagtgccg ccagggtcgg atagcggccg 21480gactccaggg cgtcgaggac gtcgacgatg cccgaggcgc gccggcgctc ggactgcgcc 21540tcgtccaact ggtggtcggt cccgcccggg cttccgttcg aacccgccgc gccggccggg 21600gcgccggtcg tcgggtcggc gggtcgggcg acgccttcca ggctggcctc gtacgcgacc 21660gccgaggtgt agaggccgag caggtcgcac gcccacgccg cggtctgcgc cgagacgccg 21720ccggccagca gcagcgccgt ggtgcgttcc gcgatcgcga tggacgccgg gccggtgggg 21780atgcggccca tcgagacctg ggcgatgccc gggtgcgcgt tcatggcagc caccgtcaag 21840cgcagcagtt cgaccaactg gctgcgccag cgcgccggtt cgatcgggtc gaggggaacc 21900gtcgacaaca ccgcgtcgag cgcctggcgc agcaggtcgt cgcggttgtc gacgtagacg 21960tacaacgacg ccggtccggt gtcgagctcg ttggcgaccc ggcgcatggt gaccgacgag 22020agtccttcgc tctgcaggat gcgcagagct gtctcgacga ccacctcctg gctgaggggc 22080gccttggcgg ggcgggctct ggtactgcgc gaagtgacgg ccatgcggcg agtgtagcag 22140ttgacacgaa tgttgttcgt gacagacgat gttcgtaacg aacaacgttc gttacgaaca 22200gcgtttgccg ctctcgggcc cctgtccggc ccactcttcc gccaaggaaa cgccatggat 22260tccaacccga tcacccttcc cgcgaacgga ccggtggccg aaccattggg cgagtcgccg 22320gccgagccac ccggcaccgg accggcgccg tacgcggctc gctggatggt cctcgccgtc 22380gtcttggtcg ccgaggtcat ggacctgctc gacgcgacca tcaccagcgt cgccgcaccg 22440tcgatgtcgg cgccgcgtac accctgccgt tcgccgtctt cctcatcacc gccggccgac 22500tcggcgacat ctggggccgg cgccggctct tcgtcatcgg cgcgggcggc ttcacgatcg 22560cgtccgcgtt gtgcgcgctg gcgaactcgc cggaaatgct gatcaccacc cgcgcgctgc 22620agggcgcgct cggcgcgctg ctcatcccgc agggcttcgg gctgatcaag gaggtcttcc 22680cggacaagga actgggcaag gcgttcgcgc tgttcggccc ggtcatgggc ctgtcggcgg 22740tgctcggccc gatcatcggc ggcgcgctgg tggacggcga cctgttcggc accggctggc 22800ggatgatctt cctgatcaac ctgccgctcg gtgtcctcgc cgtactcggc gcgttccgct 22860acatgccgac cggcgcgcgg atccggtgtg ctcaacgccg tccagcagtt cgccaacgcg 22920atcggcgtcg cggtcgtcgc gacgatcttc ttcgcctacc tcgagacggc cacacctcct 22980ccgaggcgat gacccgcacc gccgtcgcca cctgcgggct gctcgcgctg gcgttcctcg 23040ccggcttcgc gctgccgcgc acggcacggg aagaaggcca ctagccggcc gccccggccg 23100cgtcgtcgcg gccacacggt ggccgcaccc tggccacacc cctggaaaag ccggtcggac 23160cgggcacgca ccggcggcac tcgcggtgcg tcacgtcccc ggtccggccg gcccggccga 23220ccgccctcgg ccctcaagcg cggtcccctc cgtgaccgtg accggtgacg cgcctcgcgt 23280cccacctccg ccgccactcc ggcccgtccg ccccgggcat caccgcaccg acgaacgcgc 23340caggagcaca tcacgtgtcc acgcaccgca cccccgagcc cgaacgcgcg gccgagcacc 23400gcgaccgcga acgccttcgg ggccacgatc gagaccgaga ccgcgaccga gaccggatgc 23460cacgccggat cggcgcgggt ttcgcgcgtg gtccccacgt ccacaccacg cacgtcctga 23520agaccgagca gatcctcgac gtcgccgccg cgcacttcgg tgtcgagccg cccgagtgcc 23580ggctcgtcga cgaggccgtc gaacacaccg ggaaacgcct cgcgttgaag gaggtgtacg 23640cgatctggga ctagggtccg gattttttct aga 23673

Claims

1. An estrogen receptor antagonist given by the formula: