Doi:10.1016/j.bmcl.2003.09.01

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3927-3931 Synthesis of 17-Estradiol Platinum(II) Complexes: Biological Caroline Descoˆteaux,a Jose'e Provencher-Mandeville,a Isabelle Mathieu,a Vale'rie Perron,a Sanat K. Mandal,b E'ric Asselina and Gervais Be'rube'a,* aDe'partement de Chimie-Biologie, Universite' du Que'bec a` Trois-Rivie`res, C.P. 500, Trois-Rivie`res, Que'bec, Canada G9A 5H7 bDivision of Science & Technology, College of the North Atlantic, Clarenville Campus, Clarenville, Newfoundland, Canada A5A 1V9 Received 19 June 2003; accepted 3 September 2003 Abstract-The synthesis of a novel series of 17b-estradiol-linked platinum(II) complexes is described. The new molecules are linkedwith an alkyl chain at position 16a of the steroid nucleus and bear a 16b-hydroxymethyl side chain. They are made from estrone infive chemical steps with an overall yield exceeding 28%. The biological activity of these compounds was evaluated in vitro onestrogen dependent and independent (ER+ and ERÀ) human breast cancers. The derivatives incorporating a 2-(20-amino-ethyl)pyridine ligand displayed good activity against the cell lines particularly when the connecting arm is 10 carbon atoms long.
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Several platinum coordination complexes such as cis- larly kidney toxicity and neurotoxicity, also limit the clinical utility of the drug.3 It is noteworthy that carbo- amine[1,1-cyclobutanedicarboxylato]-O,O0-platinum(II) platin is less toxic than cisplatin and can be given at a (carboplatin) are currently used in chemotherapy of much higher dose (up to 2000 mg/dose for carboplatin as neoplastic diseases (see Scheme 1).1 These complexes of compare to a typical dose of 100 mg/day for cisplatin).1 a non-essential heavy metal, exhibit a remarkable anti- However carboplatin is less effective than cisplatin.1,2 tumor effectiveness and a broad spectrum of activity.
Recent literature reviews present a broad overview of More recently, two other platinum(II) derivatives were the actual knowledge of platinum-based antitumor approved for use in some countries (see Scheme 1).
agents as well as their mechanisms of action.1,2 It is (trans-l-diaminocyclohexane)oxalatoplatinum(II) (oxali- generally accepted that the antitumor activity of plati- platin) has been approved for the secondary treatment num drugs is a consequence of their interaction with of metastatic colorectal cancer in France and other DNA. Cisplatin binds readily to guanine residues of DNA molecules thereby blocking replication and/or platinum(II) (nedaplatin) has received approval for use transcription.2,3 Cisplatin has proved very successful inthe treatment of a variety of human solid tumors suchas genitourinary and gynecologic tumors as well ashead, neck and lung tumors. Unfortunately, the devel-opment of cellular resistance to cisplatin in mammaliancells is common and is believed to occur via three mainmechanisms: (a) increased efficiency of repair of plati-num-DNA lesions, (b) increased detoxification by thiolcontaining scavenger molecules such as glutathione(GSH) and metallothionein (MT), and (c) decreasedcellular uptake of the drug.2 Its toxic effects, particu- *Corresponding author. Fax: +1-819-376-5084; e-mail: gervais_berube@uqtr.ca Scheme 1. Structure of the known platinum(II) complexes.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
C. Descoˆteaux et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3927-3931 in Japan.1 Unfortunately, oxaplatin and nedaplatinhave not shown any distinct advantages over cisplatinand carboplatin. Thus, the design of novel platinum(II)complexes with a broader spectrum of activity, lesstoxicity and improved selectivity towards cancerouscells is still of great importance.
This paper describes the straightforward synthesis of anew family of 17b-estradiol-linked Pt(II) complexes (seegeneral structure 1). The new molecules bear a 16b-hydroxymethyl side chain and a platinum(II) complexlocked in position 16a of the steroid nucleus. It alsoreports the in vitro cytotoxic activity of these com-pounds on estrogen dependent and independent (ER+and ERÀ) human breast cancer cell lines.
A retrosynthetic analysis of the target molecules is pre-sented in Scheme 2. As one can observe, the Pt(II) b-estradiol-linked platinum(II) complexes.
1 are derived from the 17b-estradiol amino- Reagents: (a) DHP, PPTs, CH2Cl2, 22 C, 24 h, 99%; (b) KH, dime- pyridine derivative 7 upon a complexation reaction with thyl carbonate, THF, reflux, 3 h, 98%; (c) Br(CH2)nBr, Et3N+BnClÀ, NaOH 10% p/v, CH2Cl2, reflux, 20 h, 80%; (d) (1) LiBH4, Et2O, 0 C, obtained by the stepwise combination of three key 3 h and 22 C, 24 h; (2) PPTs, EtOH, 22 C, 17 h, 60% (e) 2-amino- components. Hence, the aminopyridine 7 can easily be methyl pyridine (m=1) or 2-(20-aminoethyl)pyridine (m=2), CH3OH,reflux, 3 days, 95% crude; (f) K prepared from estrone, a a,o-dibromoalkane and a As shown in Scheme 3, the synthesis involves only five yield. Treatment of derivative 4 with a suitable a,o- chemical steps starting from estrone (2) as the steroid dibromoalkane under phase transfer catalyst (PTC) template. The 17b-estradiol Pt(II) complexes 1 were reaction conditions gave compound 5 in 80% yield. The obtained efficiently in high yield (28% overall) using a bromoalkane side chain was added to the less hindered a face of the molecule as shown by the presence of asingle peak for the 18-CH3 at d 0.93 in the 1H NMR spectrum and at d 14.9 ppm in the 13C NMR spectrum.
hydropyranyl ether (R=THP) under standard reaction Reduction of the b-ketoester moiety with lithium bor- conditions. Accordingly, estrone was treated with dihy- ohydride in dry ether at 0 C followed by the cleavage of dropyran in dichloromethane in the presence of pyr- the tetrahydropyranyl ether of derivative 5 gave the triol idinium p-toluenesulfonate.4 The yield of the protection 6.4 It was obtained in 60% overall yield as a single 17b- reaction is 99%. The derivative 3 was transformed into hydroxy isomer as shown by a sole signal for the 18- the b-cetoester 4 upon treatment with dimethyl carbon- CH3 at d 0.89 in the 1H NMR spectrum and at d 12.5 ate in the presence of a mixture of NaH/KH in dry tet- ppm in the 13C NMR spectrum. The stereochemistry of rahydrofuran.5,6 Derivative 4 was obtained with 98% the 17b-hydroxy function was confirmed by comparisonwith 13C NMR spectral data of known 17b- and 17a-estradiol derivatives.7 The final 17b-estradiol-linked Pt(II) complexes 1 wereobtained in a two-step chemical sequence.8 Firstly, thetriol 6 was treated with an excess 2-aminoalkylpyridineto give derivative 7 for a yield of 80-100%. Secondly,the triol-aminopyridine intermediates were treated withpotassium tetrachloroplatinate in a mixture of dime-thylformamide and water to give the corresponding17b-estradiol-linked Pt(II) complexes 1 with m=1 or 2and n=2, 4, 6, 8. Consequently, the new cytotoxicmolecules possess an alkyl side chain varying from 4 to10 carbon atoms long. Complexation of the amino-methylpyridine intermediates was readily confirmed by Scheme 2. Retrosynthetic analysis for the estradiol-linked Pt(II)complexes.
the presence of two signals at d 4.28 ppm and d 4.95 C. Descoˆteaux et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3927-3931 As shown by the MTT assays on the human breastcancer cell lines, the new Pt(II) complexes do not pre-sent any apparent specific toxicity towards ER+ breastcancer cells (Tables 1 and 2). The reference derivatives 8and 9 did not show any toxicity at the maximum (40mM) concentration tested (data not shown in the tables).
Hence, the linkage of this kind of cytotoxic moiety to a Scheme 4. Complexation of the aminomethylpyridine.
steroid nucleus improves the biological activity. One canspeculate that a large organic portion enhance the cel-lular penetration of the membranes to the nucleus. ThePt(II) complexes 1, m=1 are, in general, less toxic thanthose where m=2. This was previously observed with aseries of triphenylethylene Pt(II) complexes.8a Thelength of the side chain seems to be optimal at n=6 or 8for both types of aminopyridine analogues (m=1 or 2).
The derivatives with a short side chain (n=2) are Scheme 5. Structures of reference derivatives 8 and 9.
essentially inactive when compared with cisplatin. ThePt(II) complex with m=2 and n=8 is the most inter- ppm representing the methylene group locked in an esting derivative of the series. It presents an activity heteronuclear ring system of the final products instead three to four times greater than cisplatin on all types of of the initial singlet at d 3.93 ppm in the 1H NMR breast cancer cells (ER+ and ERÀ). These data confirm spectrum of the starting material (Scheme 4).
noethyl)pyridine ligand presents higher activity than Scheme 5 shows the structure of two known Pt(II) those bearing the 2-aminomethylpyridine ligand.
complexes (8 and 9) that were made as reference pro-ducts for the biological evaluation study.9 All new Molecular mechanics (MM2) and semi-empirical quan- compounds synthesized were characterized by IR and tum mechanical calculations (AM1)14 were used to study derivatives 1 (m=1, 2), 8 and 9. The conforma-tion studies were calculated in vacuo. It is observed that The toxicity of the 17b-estradiol-linked Pt(II) complexes the amino group of the reference compounds 8 and 9 was evaluated on four human breast tumor cell lines possess an electron density of À0.418 and À0.404, using the MTT colorimetric assay.11,12 The cytotoxicity respectively. For the corresponding E2-Pt(II) com- of the compounds was tested along with controls (cis- plexes, the amino group have an electron density of platin, 8 and 9) on both estrogen-receptor positive À0.357 and À0.348. This is in agreement with the theory (ER+, MCF-7 and ZR-75-1) and estrogen-receptor as a secondary amino group is more basic than a pri- negative (ERÀ, MDA-MB-231 and HS578T) human mary amino group. Hence, there is a better coordina- mammary carcinomas.13 The MTT assay was per- tion of the secondary amino groups with the platinum formed over an incubation period of 72 h.
atom. However, the predicted bond length, resulting Inhibitory concentrationa of 1 (m=1) and of cisplatin on both ER+ and ERÀ breast cancer cell lines aInhibitory concentration (IC50, mM) as obtained by the MTT assay. Experiments were performed in duplicates and the results represent themean ÆSEM of three independent experiments. NR=IC50>40 mM.
Inhibitory concentrationa of 1 (m=2) and of cisplatin on both ER+ and ER- breast cancer cell lines aInhibitory concentration (IC50, mM) as obtained by the MTT assay. Experiments were performed in duplicates and the results represent themean ÆSEM of three independent experiments. NR=IC50>40 mM.
C. Descoˆteaux et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3927-3931 Bond angles of selected platinum(II) complexes and of cis- showed that the combination of a side chain and the six- member ring [N(CH2)2N Pt] to derivatives 9 induce amuch greater structural change of the PtN2Cl2 core as compared to derivative 8, same chain length but form a five-member ring [N(CH2)N Pt]. This could account forthe discrepancies in cytotoxic activities observed for derivatives 1, m=1 and 1, m=2. This kind of estrogen- linked Pt(II) complexes could present several advan- tages over the known cisplatin analogues. Theoretically,the estrogenic portion of the molecule may direct thecytotoxic Pt(II) moiety towards the target cells in vivo,increasing specificity and reducing systemic toxicity.
Further research will be necessary to evaluate the com-plete biological potential of this novel class of 17b-estradiol-linked Pt(II) complexes.
This work was supported by the Universite' du Que'bec a`Trois-Rivie`res, NSERC summer grants to C.D. andJ.P.-M., and FRSQ to E'.A. We are grateful to Dr. G.
Sauve' and Mr. N. Le Berre, Pharmacor Inc. for the 1Hand 13C NMR spectra. We also thank Mrs. S. Parent,Mrs. V. Gagnon, and Mrs. M.-E`. St-Germain for bio-logical evaluation studies. Special thanks to Mr. D.
Figure 1. Superimposition of the most stable structures of E Rabouin for his involvement in the project.
m=1, n=8 (pink) and E2-Pt(II) m=2, n=8 (blue) showing structuralerror (RMS=0.3954).
from the combination of both factors basicity and steric 1. Wong, E.; Giandomenico, C. M. Chem. Rev. 1999, 99, hindrance, are similar on all the models studied. The difference in activity might be simply due to the con- 2. (a) Jamieson, E. R.; Lippard, S. J. Chem. Rev. 1999, 99, 2467. (b) Scozzafava, A.; Casini, A.; Supuran, C. T. Curr.
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In summary, this manuscript presents a facile synthesis of cytotoxic 17b-estradiol Pt(II) complexes. They are made form estrone in only five chemical transforma- hexyl)-1,3,5(10)-estratrien-3,17b-diol (6, n=4): IR (NaCl, tions with an overall yield exceeding 28%. Using this nmax, cmÀ1) 3355 (OH). 1H NMR (200 MHz, acetone-d6) d7.98 (1H, br s, OH), 7.08 (1H, d, J=8.6 Hz, 1-CH), 6.58 (1H, strategy a large variety of Pt(II) complexes could easily dd, J=2.7 Hz and J=8.6 Hz, 2-CH), 6.51 (1H, d, J=2.7 Hz, be synthesized either with an alkyl side chain or a poly- 4-CH), 4.33 (1H, br d, J=2.3 Hz, CHOH), 3.80-3.30 (4H, m, ethylene glycol side chain. Furthermore, other diamine OH, CH2OH), 3.50 (2H, t, J=7.0 Hz, CH2Br), 2.76 (2H, m, 6- ligands could, without difficulty, be coupled to the bro- CH2), 2.40-1.10 (21H, m, 3ÂCH, 9ÂCH2), 0.89 (3H, s, 18- mide intermediate 6. Molecular mechanics (MM2) and CH3). 13C NMR (200 MHz, acetone-d6): d 155.9, 138.4, 132.1, semi-empirical quantum mechanical calculations (AM1) 126.9, 116.0, 113.6, 90.6, 67.1, 48.5, 47.6, 45.8, 44.8, 40.2, 39.2, C. Descoˆteaux et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3927-3931 38.9, 34.8, 34.6, 33.7, 30.5, 28.9, 28.3, 27.2, 25.1, 12.5 (18-C).
47.5, 46.6, 45.8, 44.8, 40.5, 40.2, 39.2, 38.9, 34.7, 30.9, 30.9, Spectral data for 16b-hydroxymethyl-16a-[6-(2-pyridin-2-yl- ethylamino)-hexyl]-1,3,5(10)-estratrien-3,17b-diol platinum (II) (1, n=4, m=2): IR (NaCl, nmax, cmÀ1): 3600-3050 (O-H and N-H), 1609 (C¼C), 1241 and 1062 (C-O). 1HNMR (500 MHz, acetone-d6) d 9.13 (1H, d, J=5.5 Hz, a0-CH), 8.03 (1H, t, J=7.5 Hz, c0-CH), 7.98 (1H, s, OH), 7.53(1H, d, J=7.5 Hz, d0-CH), 7.43 (1H, t, J=6.5 Hz, b0-CH),7.08 (1H, d, J=8.4 Hz, 1-CH), 6.59 (1H, dd, J=1.3 Hz andJ=8.3 Hz, 2-CH), 6.53 (1H, s, 4-CH), 6.08 (1H, br s, NH),4.31 (1H, t, J=3.3 Hz, CH2OH), 3.72, 3.61, 3.45, 3.20 and 11. Carmichael, J.; DeGrapp, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer Res. 1987, 47, 943.
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d6) d 160.5 (pyridyl-C), 155.9 (a0-C), 154.4 (3-C), 140.1 (c0-C), 13. Horwitz, K. B.; Zava, D. T.; Thilagar, A. K.; Jensen, 138.4 (5-C), 132.1 (10-C), 127.0 (1-C), 125.6 (d0-C), 124.6 (b0- E. M.; McGuire, W. L. Cancer Res. 1978, 38, 2434.
C), 116.0 (4-C), 113.6 (2-C), 90.5 (CHOH), 67.2, 57.2, 48.4, 14. BioMedCAChe 6.0; Fujitsu Limited: USA, 2003.

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