Synthesis of New Steroidal Inhibitors of P-Glycoprotein-Mediated

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Synthesis of New Steroidal Inhibitors of P-glycoprotein-mediated Multidrug Resistance and Biological Evaluation on K562/R7 Erythroleukemia Cells Marc Rolland de Ravel, Ghina Alameh, Maxime Melikian, Zahia Mahiout, Agnès EmptozBonneton, Eva-Laure Matera, Thierry Lomberget, Roland Barret, Luc Rocheblave, Nadia Walchshofer, Sonia Beltran, Lucienne El Jawad, Elisabeth Mappus, Catherine Grenot, Michel Marie PUGEAT, Charles Dumontet, Marc LeBorgne, and Claude Yves Cuilleron J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501676v • Publication Date (Web): 29 Jan 2015 Downloaded from http://pubs.acs.org on February 1, 2015

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Journal of Medicinal Chemistry

LeBorgne, Marc; Université de Lyon - UCB Lyon 1 - ISPB, EA 4446 B2C : "Biomolecules, Cancer and Chemoresistances", Cuilleron, Claude; Université de Lyon, Université Lyon 1,Inserm U863, ISPB-Faculté de Pharmacie, Laboratoire de Chimie Organique

ACS Paragon Plus Environment


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Synthesis of New Steroidal Inhibitors of Pglycoprotein-mediated Multidrug Resistance and Biological Evaluation on K562/R7 Erythroleukemia Cells

Marc Rolland de Ravel,†§ Ghina Alameh,§ Maxime Melikian,§ Zahia Mahiout,§ Agnès EmptozBonneton,§ Eva-Laure Matera,† Thierry Lomberget,‡§ Roland Barret,‡§ Luc Rocheblave,‡§ Nadia Walchshofer,‡§ Sonia Beltran,§ Lucienne El Jawad,§ Elisabeth Mappus,§ Catherine Grenot,§ Michel Pugeat,‡ Charles Dumontet, † Marc Le Borgne,‡§ and Claude Yves Cuilleron*,§ †

Centre de Recherche en Cancérologie de Lyon - Université Claude Bernard Lyon 1, INSERM

U1052-CNRS UMR5286, Centre Léon Bérard-Cheney D, 28 rue Laënnec, 69373, Lyon Cedex 08, France §

INSERM-U863 Hormones stéroïdes et protéines de liaison, Université Claude Bernard Lyon 1,

8 Avenue Rockefeller, 69373, Lyon Cedex 08, France


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‡

Université de Lyon, Université Lyon 1, Faculté de Pharmacie, ISPB, EA 4446 Biomolécules

Cancer et Chimiorésistances, SFR Santé Lyon-Est CNRS UMS3453-INSERM US7, 8 Avenue Rockefeller, F-69373 Lyon Cedex 8, France

This paper is dedicated to the memories of Christiane Villard (1945-1993) and Daniel Royan (1942-1998).

ABSTRACT A simple route for improving the potency of progesterone as a modulator of P-gp-mediated multidrug resistance was established by esterification and/or etherification of hydroxylated 5α/βpregnane-3,20-dione or 5β-cholan-3-one precursors. X-ray crystallography of representative 7α-, 11α-

and

17α-(2’R/S)-O-tetrahydropyranyl

ether

diastereoisomers

revealed

different

combinations of axial-equatorial configurations of the anomeric oxygen. Substantial stimulation of accumulation and chemosensitization was observed on K562/R7 erythroleukemia cells resistant to doxorubicin, especially using 7α,11α-O-disubstituted derivatives of 5α/β-pregnane3,20-dione, among which the 5β-H-7α-benzoyloxy-11α-(2’R)-O-tetrahydropyranyl ether 22a revealed promising properties (accumulation index: 2.9, IC50: 0.5 µM versus 1.2 and 10.6 µM for progesterone), slightly overcoming those of verapamil and cyclosporin A. Several 7α,12α-Odisubstituted derivatives of 5β-cholan-3-one proved even more active especially the 7α-Omethoxymethyl-12α-benzoate 56 (accumulation index: 3.8, IC50: 0.2 µM). The panel of modulating effects from different O-substitutions at a same position suggests a structural


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influence of the substituent completing a simple protection against stimulating effects of hydroxyl groups on P-gp-mediated transport.

INTRODUCTION Multidrug resistance (MDR), either intrinsic or acquired, remains a major limit for cancer chemotherapy. The predominant mechanism is a reduced drug accumulation in cells consecutive to an active efflux by energy-dependent transporters belonging to the ATP-binding cassette (ABC) family.1 The main contribution comes from P-glycoprotein (P-gp/ABCB1) which can export structurally unrelated cytotoxic drugs as well as a wide variety of compounds including synthetic or natural hormonal steroids.2 P-gp is often overexpressed in resistant tumors but is also present in many normal cells. P-gp expression was found to be stimulated under the influence of the Pregnane Xenobiotic Receptor (PXR).3 Other MDR proteins such as the Multidrug Resistance Protein 1 (MRP1/ABCC1) and the Breast Cancer Resistance Protein (BCRP/ABCG2) can also contribute to drug efflux. Moreover, non-P-gp-mediated MDR mechanisms have been reported.4 The structure of human P-gp consists of a single-chain protein of 1280 amino acids organized in a spatial arrangement extrapolated from the X-ray structure of mouse P-gp.5-6 Considerable efforts have been devoted to the development of molecules able to inhibit the efflux of cytotoxic drugs by ABC transporters, focused especially on P-gp modulators for which structure-activity relationships have also been proposed7-11 but most often revealed side-effects not compatible for approval in therapy.12-13 Among the wide number of candidate structures for


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developing inhibitors of P-gp-mediated drug efflux, several natural and synthetic steroids have been investigated. The first report about the role of progesterone as a moderately active inhibitor of P-gp efflux14 stimulated numerous further studies. Investigations on the inhibiting effects of progesterone derivatives and metabolites revealed that the nontoxic 5β-dihydroprogesterone but not 5αdihydroprogesterone, was nearly as active as progesterone15-16 while 17α-hydroxy-5βdihydroprogesterone was reported to enhance P-gp inhibition without interacting with glucocorticoid receptors.17 Progesterone has been the target of numerous structural modifications to stimulate its P-gp inhibiting effects. Improvements were reported after introduction of optimized 7α-thiophenyl substituents also preventing interaction with progesterone receptors,18 or of 11α-benzoate and carbamate substituents.19-20 Synthetic drugs structurally related to progesterone and modified at the 17β-acetyl side-chain were also employed.21 A few isolated natural products with pregnane skeletons have been reported as P-gp inhibitors.22-25 P-gp inhibiting properties were also found for 21-dimethylaminobenzoate derivatives of two glucocorticoid receptor agonists26 as well as for several synthetic glucocorticoids.27-28 Many other compounds related to steroidal pharmaceuticals were also found able to overcome MDR such as the antiprogestin drug mifepristone (RU486)29 bearing a 11-dimethylaminophenyl substituent and their related estradiol analogs,30 as well as progestins31 and the steroidal antiestrogens ICI 182,78032 and 164,384.32 A few reports have mentioned MDR inhibition properties of bile acid or salt derivatives such as glycocholic acid,33 deoxycholate,34,35 taurolithocholate, taurochenodeoxycholate and glycochenodeoxycholate and ursodeoxycholate.36 Mechanisms associated with alterations in the fluidity of the plasma membrane have been postulated for some of the bile salts effects.37


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The importance of steroids either as inhibitors or transported substrates has justified structureactivity studies. Investigations on corticosteroid transport by murine mdr1-P-gp variants have identified mutations in the transmembrane domain 4-6 of the first half of the protein altering the recognition of 17α-hydroxyl and/or 20-ketone groups of steroid inhibitors.17,38 Comparative studies using different synthetic glucocorticoids revealed structural features which significantly influenced their transport by P-gp,39 completing those established for other interacting steroid structures.15,16,40 3D-QSAR studies41 and homology modeling/docking approaches42 have been developed. Fluorescence-quenching experiments have suggested interactions of steroid modulators in the proximity of the ATP-binding site40,43 potentially associated with ATPasedependent conformational changes controlling P-gp-mediated drug transport.44 Therefore attempts have been made to generate P-gp inhibitors with mixed functions that are able to interfere with both ATPase and modulator sites.45-48 The mechanisms underlying modulation of P-gp transport as well as ATPase activity appear complex owing to the involvement of several interacting binding sites according to ligand structures.49-51 The present study was undertaken with the view to explore whether very simple chemical modifications such as esterification and etherification of mono- or dihydroxylated steroidal precursors derived from progesterone, 5α- or 5β-dihydroprogesterone as well as from cholic acid series could provide structure-activity criteria which are useful for improving modulation of Pgp-mediated drug efflux. The use of hydroxylated precursors related either to known physiological steroids or their metabolites and the versatility of chemical modifications of hydroxyl groups also appeared particularly favorable for future structural optimizations aimed at maintaining a strong inhibiting potency in vivo without stimulating unwanted interactions or intrinsic toxicity beyond clinically acceptable limits.


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CHEMISTRY The three monosubstituted 11α-, 17α- and 21-O-tetrahydropyranyl (OTHP) ether derivatives of progesterone (Scheme 1) were prepared by tetrahydropyranylation of their commercially available hydroxyl precursors followed by preparative thin-layer chromatography (TLC) purification which was able to separate the 11α- and 17α- (2’R/S)OTHP diastereoisomers 1a,b and 2a,b but not those of the 21-(2’R+S)OTHP ether 3. The access to the monosubstituted 7α-, 11α-, 17α-OTHP ether and 7α-benzoate derivatives of 5β-dihydroprogesterone (Scheme 2) required a preliminary synthesis of the corresponding hydroxyl precursors. Catalytic hydrogenation of 6α,7α-epoxy-pregn-4-ene-3,20-dione 4 afforded 7α-hydroxy-5β-pregnane-3,20-dione 5 according to a reported procedure.52 Benzoylation of this precursor gave the 7α-benzoate 6 while tetrahydropyranylation led to the 7α-(R+S)OTHP ether 7 as a mixture of R/S isomers which could not be separated by TLC. Catalytic hydrogenation of 11α-hydroxyprogesterone led to 11α-hydroxy-5β-pregnane-3,20-dione53 8 which, after tetrahydropyranylation and TLC, provided the separated 11α-(R/S)OTHP isomers 9a,b. A similar protocol applied to 17α-hydroxyprogesterone led to 17α-hydroxy-5β-pregnane-3,20-dione38 10 and to the separated 17α-(R/S)OTHP isomers 11a,b. A first synthetic scheme leading to 7α,11α-O-disubstituted derivatives of both 5α/β-pregnane3,20-dione series relied upon a 11α-O-tert-butyldimethylsilyloxy (OTBDMS)-5-en-7-one intermediate (Scheme 3) prepared from 11α-hydroxyprogesterone. This enone was converted to a 5-ene-3,20-bis-ketal 12, then protected as the 11α-OTBDMS ether 13. Allylic oxidation with the CrO3-(pyridine)2 complex54 led to the 5-en-7-one 14. Catalytic hydrogenation55 of this enone


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gave similar proportions of 5α-pregnan-7-one 15a and 5β-pregnan-7-one 15b which could not be separated. Stereoselective reduction of these unseparated 7-ketones with lithium tri-secbutylborohydride (L-Selectride)56 afforded a mixture of 5β- and 5α-pregnan-7α-ol derivatives 16 and 17 readily separated by flash-chromatography. Benzoylation of the 7α-ol precursor 16 led to the 7α-benzoate 18 which, after cleavage of the 11-silyl group with tetra-n-butylammonium fluoride, gave the 11α-ol derivative 19. Acidolysis of the ketal groups provided 7α-benzoyloxy11α-hydroxy-3,20-dione 20 which was benzoylated to give the 7α,11α-dibenzoate 21. Tetrahydropyranylation of the 11α-ol precursor 20 followed by flash-chromatography led to the separated 11α-(R/S)OTHP isomers 22a,b. The monobenzoate 20 was also saponified to the 7α,11α-diol derivative 23 then converted to the 7α,11α-bis-OTHP ether which could be separated by TLC as the two 11α-(R/S) isomers 24a,b without separation of the 7α-(R/S) isomers. Benzoylation of the 7α-ol precursor 17 led to the 7α-benzoate 25 which, after desilylation, gave the 7α-benzoyloxy-11α-ol derivative 26. Acidolysis of the ketal group provided the 7α,11αdihydroxy-3,20-dione 27 then converted either to the 7α,11α-dibenzoate 28 or to the separated 7α-benzoyloxy-11α-(R/S)OTHP isomers 29a,b. A second synthetic scheme was designed to lead exclusively to 7α-OTHP-11α-benzoate derivatives of 5β-pregnan-3,20-dione (Scheme 4) according to the one employed for obtaining the 7α-OTHP ether 7. The 11α-OTBDMS ether 30 prepared from 11α-hydroxyprogesterone was dehydrogenated to the 4,6-dien-3-one 31 then converted to the 6α,7α-epoxide 32. Catalytic hydrogenation of epoxide 32 afforded the 5β-pregnan-7α-ol precursor 33. This compound was converted to the 7α-(R+S)OTHP ether 34 recovered as an unseparated mixture of R/S isomers. Desilylation gave the 7α-(R+S)OTHP-11α-ol derivative 35 which, after benzoylation and TLC


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separation, provided the two separated 11α-benzoyloxy-7α-(R/S)OTHP isomers 36a,b. Acidolysis of the unseparated mixture of isomers 36a,b led to the 7α-hydroxy-11α-benzoate 37. The synthesis of 11α,17α-O-disubstituted derivatives of 5β-pregnane-3,20-dione (Scheme 5) required an access to the 11α,17α-diol precursor 41. Mild acidolysis of 3,20-bisethylenedioxypregn-5-en-11α-ol derivative 12 led to the 3-ethylenedioxy-11α-hydroxypregn-5en-20-one 38. Oxygenation of this 20-ketone in the presence of NaH and triethylphosphite afforded the 17α-hydroxy-20-ketone 39. 57 Acidolysis of the ketal group led to the 4-en-3-one 40 which was reduced by catalytic hydrogenation exclusively to the 11α,17α-dihydroxy-5βpregnane-3,20-dione 41. This 11α,17α-diol precursor was converted by a first partial tetrahydropyranylation step to a 11α-mono-OTHP ether then separated by TLC as the 11α(R/S)OTHP isomers 42a,b. A second tetrahydropyranylation step of the remaining 17α–hydroxyl group afforded 11α,17α-bis-OTHP derivatives 43 showing a partial interconversion of the previously separated 11α-(R/S) isomers. Preparative TLC allowed the recovery of fractions enriched in three of the four possible isomers, characterized by their 1H/13C NMR spectra and liquid chromatography-mass spectrometry (LC-MS) profiles (cf Supporting Information). A first partially purified fraction, contained the 11α-(S),17α-(R) isomer 43a as the major product (c.a. 70%) mainly contaminated with the 11α-(S),17α-(S) isomer 43c. A second impure fraction consisted in a mixture of the 11α-(R),17α-(R) isomer 43b as the major product (c.a. 35%) contaminated with decreasing amounts of isomers 43a (c.a. 30%) and 43c (c.a. 25%). A third nearly pure fraction contained only the isomer 43c (c.a. 95%). The 11α-(R),17α-(S) isomer 43d could not be isolated as an independent fraction but was identified by NMR as the main byproduct in the two fractions enriched in isomers 43a or 43b. Owing to difficulties encountered for further purification (including attempts using reverse-phase HPLC hardly transposable at a


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preparative scale), preliminary biological assays were restricted to the three available major fractions respectively containing enriched isomers 43a, 43b and the pure isomer 43c. The access to 7α,12α-disubstituted derivatives of either methyl 3-oxo-5β-cholan-24-oate series or their 24-CH2OMe analogs (Scheme 6) relied on 7α,12α-dihydroxy-3-one precursors. Methyl cholate 44 was selectively oxidized with the Ag2CO3/Celite reagent58 to the 7α,12αdihydroxy-3-ketone 45. Benzoylation of this diol precursor gave the 7α,12α-dibenzoate 46 while tetrahydropyranylation afforded the 7α,12α-bis-OTHP derivative 47 as an unseparated mixture of R/S isomers. In order to avoid the formation of R/S diastereoisomers, an attempt was made to use the 5,6-dihydro-4-methoxy-2H-pyran etherification reagent.59 Etherification of the 7α,12αdiol derivative 45 with an excess of this reagent remained incomplete even after 48 h at room temperature and gave 7α,12α-bis-O-(5’,6’-dihydro-2’H-pyran-4’-yloxy)ether (ODHPE) 48 as the major product. This ODHPE derivative was identified as an unsaturated dihydropyranyl ether adduct homologous to the etherification reagent having lost the angular methoxy group of the initial tetrahydropyranyl ether adduct, as established by 1H and 13C NMR and confirmed by mass spectrometry. Selective mono-tetrahydropyranylation of the 7α,12α-diol precursor 45 with a limited amount of dihydropyran resulted in an incomplete reaction leading to a mixture of compounds containing the 7α-hydroxy-12α-mono-OTHP ether 49 as the major product, the 7αmono-OTHP-12α-ol regioisomer 50 as the minor product and trace amounts of 7α,12α-diol derivative 45 and 7α,12α-bis-OTHP ether 47. This mixture was benzoylated and the resulting benzoates were purified by preparative TLC to give the two 12α-benzoyloxy-7α-(R/S)OTHP isomers 51a,b and the two 7α-benzoyloxy-12α-(S/R)OTHP isomers 52a,b. An alternative access to the 12α-benzoyloxy-7α-(R/S)OTHP isomers 51a,b was obtained via selective benzoylation of the 12α-hydroxyl group followed by tetrahydropyranylation of the 7α-hydroxyl group (Rolland


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de Ravel, unpublished results). Selective mono-O-methoxymethylation of the 7α,12α-diol precursor 45 was undertaken using a stoichiometric amount of chloromethyl methyl ether which provided a mixture of compounds containing the 7α-hydroxy-12α-mono-O-methoxymethyl (OMOM) ether 53 as the major product and its 7α-mono-OMOM-12α-ol regioisomer 54 as the minor product. These 7α-and 12α-ol derivatives were separated by flash-chromatography and benzoylated to give the 7α-benzoyloxy-12α-OMOM ether 55 and the 7α-OMOM-12α-benzoate 56. The conversion of the 24-COOMe group of methyl cholate 44 to a 24-CH2OMe ether was made in three steps by partial silylation to the 3-OTBDMS ether 57, reduction with lithium aluminum hydride to the 24-ol intermediate 58 and by selective etherification of this alcohol with MeI-NaH to the 24-O-methyl ether 59. Desilylation afforded the 3α,7α,12α-triol 60 which was selectively oxidized at the 3-position with the Ag2CO3/Celite reagent58 to give the 7α,12αdihydroxy-3-ketone 61. Complete benzoylation of this substrate led to the 7α,12α-dibenzoate 62. Selective mono-tetrahydropyranylation of the same diol derivative 61 afforded a mixture containing the 7-mono-(R+S)OTHP-12α-ol derivative 63, the 7α-hydroxy-12-mono-(R+S)OTHP ether 64 (major product) and the 7α,12α-bis-OTHP ether 65 which could all be separated by preparative TLC. Benzoylation of the 12α-ol precursor 63 followed by chromatographic separation provided the 12α-benzoyloxy-7α-(R/S)OTHP isomers 66a,b. A similar treatment of the 7α-ol derivative 64 the 7α-benzoyloxy-12α-(S/R)OTHP isomers 67a,b.

STRUCTURAL ASSIGNMENTS


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Optical rotations. A preliminary attempt for the assignment of (R) and (S) configurations of the 11α-OTHP isomers of progesterone 1a and 1b was made by correlation of optical rotations with those of (R/S)OTHP isomers derived either from adenosine60 or from (S)-(+)-methyl mandelate61

for

which

absolute

stereochemistries

had

been

established

from

the

radiocristallographic structure of the less dextrorotatory (S) isomer.61 This suggested the same (S) stereochemistry for the less dextrorotatory 11α-OTHP isomer 1b and therefore a similar trend for the other pairs of OTHP isomers. X-ray crystallography studies. The structural assignments concerning both the tetrahydropyranyl ether R/S isomerism and the 5β-H stereochemistry of pregnane skeletons were unambiguously assessed from radiocristallographic data established for three characteristic representative pairs of 7α-, 11α- and 17α-(R/S)OTHP isomers, two of which in 5β-pregnane3,20-dione series and the third one in pregn-4-ene-3-one series (Figure 1). No adequate crystalline material could be obtained for 5β-cholane derivatives. The 7α-OTHP isomer 36a revealed a (R) configuration associated with an equatorial O-linkage to the THP ring. The diastereoisomer 36b revealed a (S) configuration associated with an axial O-linkage linkage to the THP ring as reported for the (2’S)OTHP derivative of adenosine.60 The 11α-OTHP diastereoisomers 22a and 22b were confirmed respectively as (R) and (S) isomers, both with a same equatorial O-linkage to the THP ring. Moreover, the 5β-H configuration was clearly observable for these two pairs of isomers. The 17α-OTHP-pregn-4-ene-3-one diastereoisomers 2a and 2b were confirmed respectively as (R) and (S) isomers, both with the same axial Olinkage to the THP ring, in keeping with previous conformational assignments on similar 17α(R/S)OTHP isomers.62 These structures revealed not only the absolute stereochemistries of 7α-, 11α- and 17α-(R/S)OTHP diastereoisomers but also showed three different combinations of


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orientations of ether linkages between the steroid hydroxyl group and the tetrahydropyranyl ether ring. These differences might result from steric constraints not predictable from NMR data on anomeric effects reported for 2-alkoxytetrahydropyrans.63 An additional hypothesis of conformational differences between crystalline and solubilized forms cannot be excluded. These differences of orientations of ether links with the THP rings may therefore not be safely extrapolated to the other homologous structural analogs reported in this study. Assignments for 1H and

13

C NMR shift. NMR assignments were based mainly on

correlations with NMR data reported in literature and on correlations between the effects of homologous structural modifications reported in this study. The mentioned structures take into account the configurations of the 7α-,11α- and 17α-(R/S)OTHP diastereoisomers deduced from radiocristallographic data. In the absence of crystal structures for 12α-OTHP ethers, putative (R) and (S) configurations opposite to those made for 11α-OTHP ethers were assigned on the assumption that the characteristic higher deshielding of the vicinal 12-methylene group by the 11α-(R) isomer might be exerted on the vicinal 11-methylene by the 12α-(S) isomer for 12αOTHP ethers by keeping with the change in the order of optical rotations. Assignments for the carbon atom resonances of the steroid skeleton were made using data reported for related hydroxysteroid analogs.64-70 The stereochemistries of 7α-O-substituted-5αdihydroprogesterone derivatives 28 and 29a,b were also confirmed by comparison with NMR data for 5α-cholestan-7α/β-ol/acetate isomers according to the original article66 erroneously transcribed for 7β-isomers in a reference review on 13C NMR of steroids.64 Assignments for the carbon atom resonances of 7α-, 11α-, 12α-, and 17α-OTHP ether rings were made by comparison with reported 1H/13C NMR data on steroidal OTHP ethers.71,72 A


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strong characteristic difference between the (R) and (S) isomers was observed for the dioxygenated C-2’ carbon atom resonance which was found either at ca. 100-103 ppm for 7α(R)-, 11α-(R)- and 12α-(S)OTHP isomers or at ca. 95-98 ppm for the opposite isomers. Such a marked difference did not appear for 17α-OTHP ethers that showed C-2’ at ca. 96-97 ppm for both R/S isomers with only a very slight downfield shift (< 1 ppm) for the (R) isomer. The 7α-, 11α-, 12α- and 17α-(R/S)OTHP isomers were found to influence the 13C resonances of carbon atoms of the steroid skeleton at short and long distance as well as of those of other substituents. In all cases the 7α-, 11α- and 17α-OTHP (R) isomers and the 12α-OTHP (S) isomer could be readily differentiated from the opposite isomers by a much stronger deshielding of the steroidal carbon atom bearing the OTHP substituent (ca. 7-8 ppm for 11α-OTHP, ca. 5 ppm for 7α- and 12α-OTHP ethers but only ca. 3 ppm for 17α-OTHP ether) as well as of the adjacent steroidal methylene carbon atom (ca. 3-4 ppm). 1

H NMR data revealed few additional characteristic effects of (R/S)OTHP isomers. For the

7α-, 11α-, 12α- and 17α-OTHP ethers, R/S isomerism induced slight shifts mainly on H-2’ of the OTHP ether group, on the steroidal H-7, H-11 and H-12 protons, and on the 18-, 19- and 21-Me groups. Combining these effects helped to characterize the isomers present in the bis-OTHP ether derivatives 43a-d, 24a,b and 47. For all 7α-O-substituted 3-oxo-5β-H-derivatives, the reported characteristic deshielding of the 1H NMR signal of the axial 4α-H52,53 was observed between 2.9 and 3.5 ppm.

BIOLOGICAL RESULTS AND DISCUSSION


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Protocols of biological evaluation. The evaluation of the relative potencies of the different substituted steroid derivatives to modulate P-gp-mediated multidrug resistance was made by using K562/R7 erythroleukemia cells which are highly resistant to doxorubicin. The identity of ABC transporters was analyzed by RT-qPCR which revealed that these cells contain a high level of ABCB1 mRNA (308192 copies normalized to G3PDH gene) indicating a predominant presence of P-gp, confirmed by Western blot (cf Supporting Information). Conversely, the amount of ABCC1 mRNA corresponding to MRP1 was very low (1664 copies) while the mRNAs for ABCC2/MRP2) or ABCG2/BCRP remained hardly detectable (< 70 copies). The screening protocol was based on four criteria: (i) short-term effects on intracellular accumulation of daunorubicin measured by flow cytometry after 1 h incubation (expressed as an accumulation index representing the ratio of daunorubicin fluorescence in the presence and absence of steroidal modulator), (ii) long-term effects on chemosensitization of doxorubicin cytotoxicity measured with the classical 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide reagent (MTT) for the assay of cell survival (expressed as IC50 value for doxorubicin), including comparisons with the reference modulators cyclosporin A and verapamil as well as the two established steroid modulators, progesterone and RU486,29,30 (iii) restoration of chemosensitivity to doxorubicin of resistant K562/R7 cells versus sensitive K562 parental cells, (iv) evaluation of intrinsic toxicity of modulators on parental K562 non-resistant cells. Accumulation assays were performed at high concentrations of both daunorubicin (10 µM) and tested modulator (10 µM) in order to include the detection of molecules which have low or very moderate activities. For the most efficient steroid modulators, the accumulation was also measured using lower 5 µM concentrations, at which the reference modulator, cyclosporin A, was found to restore a maximal accumulation of daunorubicin similar to the one observed in


14


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Page 16 of 56

sensitive cells. Chemosensitization assays were performed at a low 1 µM concentration of steroid modulators using a concentration range of doxorubicin comprised between 0.1 and 100 µM. Intrinsic cytotoxicities of modulators were evaluated in sensitive K562 parental cells using high concentrations (10 µM) of modulators (Tables 1 and 2).

Accumulation assays (daunorubicin) and chemosensitization assays (doxorubicin cytotoxicity). Pregn-4-ene-3,20-dione derivatives. A preliminary evaluation of the effects of progesterone on daunorubicin accumulation in K562/R7 cells revealed only a very moderate modulating activity on P-gp-mediated drug efflux (Table 1) confirmed by a weak chemosensitization of doxorubicin cytotoxicity whereas both 11α- and 17α-hydroxyprogesterone were inactive. Evaluations of the monosubstituted 11α-, 17α- and 21-OTHP ether derivatives of progesterone showed 11α-(S)OTHP isomer 1b and 17α-(R/S)OTHP isomers 2a,b increased accumulation (Table 1) similarly to the reference 11-p-dimethylaminophenyl steroid RU486.29,30 0n the other hand, the weak effects on both accumulation and chemosensitization of the 21(R+S)OTHP ether 3 contrasted with the efficiency of 21-p-dimethylaminobenzoate derivatives of dichlorisone or 17-deoxydexamethasone.26 5β-Pregnane-3,20-dione derivatives. With a view to facilitate chemical modifications such as the introduction of stable 7-O-alkoxy/acyloxy substituents as well as to limit potential interactions with hormone receptors,17,18 the progesterone skeleton was replaced by the saturated 5β-dihydroprogesterone skeleton.


15

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A preliminary comparison between unsubstituted 5β-dihydroprogesterone and progesterone revealed similar activities on daunorubicin accumulation whereas both 11α- and 17α-hydroxy5β-dihydroprogesterone 8 and 10 were inactive (Table 1). The monosubstituted 7α-(R+S)-, 11α-(R/S)- and 17α-(R/S)OTHP derivatives of 5βdihydroprogesterone 7, 9a,b and 11a,b, were all more efficient on accumulation than 5βdihydroprogesterone (Table 1). Only minor differences of both accumulation and chemosensitization were found according to 11α- or 17α-substituent positions while chemosensitization was improved for (S) isomers in both cases. The 7α-OTHP ether 7 proved to be more efficient than the 7α-benzoate 6 on both accumulation and chemosensitization. No attempt was made to prepare the 21-OTHP ether owing to the poor efficiency of the progesterone analog 3. These preliminary results on O-monosubstituted steroids stimulated a strategy focused towards the access to 7α,11α- or 11α,17α-O-disubstituted derivatives of 5β-dihydroprogesterone in order to evaluate possible beneficial additive/synergic effects, using simple benzoate ester and (R/S)OTHP ether substituents. The 7α,11α-dibenzoate 21 induced a moderate increase of accumulation but a nearly two-fold increase of chemosensitization as compared with the effects of the 7α-benzoate 6. The two 7α,11α-bis-[(R+S),(R/S)]OTHP

isomers

24a,b

induced

similar

moderate

effects

on

accumulation, comparable with those observed for the homologous 7α,11α-dibenzoate 21 as well as for the corresponding 7α-(R+S)- and 11α-(R/S)OTHP isomers 7 and 9a,b. On the other hand, both isomers 24a,b stimulated chemosensitization much more strongly than the dibenzoate 21 and the 11α-(R/S)OTHP isomers 9a,b devoid of a 7α-OTHP substituent.


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The 11α,17α-bis-OTHP ether containing the four isomers 43a-c showed a significant activity only for the TLC fraction enriched up to 70% of 11α-(S),17α-(R) isomer 43a. This fraction produced a moderate effect on accumulation associated with a nearly four-fold increase of chemosensitization as compared with the 11α-(S)- and 17α-(R)OTHP isomers 9b and 11a, despite similar accumulations. This improvement is in keeping with the strong potency of the 11,17α-bis-p-dimethylaminophenyl steroid RU4995330 as compared with the mono-11-pdimethylaminophenyl steroid RU486. A more refined strategy consisted to replace only one of the two ester groups of the 7α,11αdibenzoate 21 by a tetrahydropyranyl ether. A highly significant improvement of modulating properties was observed for the hetero-O-disubstituted 7α-benzoyloxy-11α-(R)OTHP ether 22a which induced a three-fold increase of both accumulation and chemosensitization (accumulation index: 2.9, IC50: 0.5 µM) as compared with the dibenzoate 21. The 11α-(S)OTHP diastereoisomer 22b proved to be much less active with an effect on accumulation close to that of dibenzoate 21 but associated with a much lower efficiency on chemosensitization (Table 1). On the other hand, changing the 7α-benzoate of compound 21 to obtain the 7α-(R)OTHP-11αbenzoate 36a brought an increase of accumulation nearly reaching the high level observed for its regioisomer 22a . A lower effect was found for the 7α-(S) diastereoisomer 36b. However, the 7α(R/S) isomers 36a,b showed only weak effects on chemosensitization. 5α-H-Dihydroprogesterone derivatives. The 7α,11α-dibenzoate 28 induced a moderate accumulation identical to that of its 5β-H analog 21 but was associated with a much more potent chemosensitizing effect (IC50: 0.4 µM). The replacement of the 11α-benzoate group by the 11α(R)OTHP ether in compound 29a decreased both accumulation and chemosensitization in


17

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contrast to the strong improvements found for the 5β-H analog 22a. The 11α-(S)OTHP isomer 29b induced the same accumulation as its 5β-H analog 22b but associated with an improvement of chemosensitization, although of moderate intensity. A comparison of the influence of the two 11α-(R/S)OTHP isomers between the 5α- and 5β-dihydroprogesterone series revealed a more favorable role of the 11α-(R)OTHP isomers for the isomeric pairs 29a,b and 22a,b, but not for the 5β-H-isomers 24a,b. 5β-cholan-3-one derivatives. Similar evaluations were extended to methyl 7α,12α-Odisubstituted 3-oxo-5β-cholan-24-oate derivatives (Table 2). The 7α,12α-dibenzoate 46 was found far less efficient in both accumulation and chemosensitization assays than its 5βdihydroprogesterone analog 21. In contrast, the 7α,12α-bis-OTHP ether 47, despite the absence of separation of R/S isomers, induced a marked increase of both accumulation and chemosensitization (accumulation index : 3.5, IC50 : 0.7 µM). These effects differ from the modest accumulation associated with a stronger chemosensitization observed for the 7α,11α-bisOTHP-5β-dihydroprogesterone 11α(R/S) analogs 24a,b. A parallel evaluation of the 7α,12α-bisODHPE analog 48 (accumulation index: 3.7, IC50: 0.6 µM) revealed a slightly stronger improvement of both accumulation and chemosensitization than observed with the 7α,12α-bisOTHP ether 47 although a possibility remains that increased effects could occur for compound 47 after separation of OTHP isomers. Replacing the 12α-benzoate group of the 7α,12α-dibenzoate 46 by a 12α-OTHP ether significantly increased both accumulation and chemosensitization for the 12-(R)OTHP ether 52b whereas a stronger increase of accumulation was associated to a weak influence on chemosensitization for the 12-(S) isomer 52a. Much more important effects resulted from


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replacing the 7α-benzoate of compound 46 by a 7α-OTHP ether, as shown by the strong improvements of accumulation and chemosensitization found for either the 7-(R) isomer 51a (accumulation index: 2.9, IC50: 0.4 µM) or the 7-(S) isomer 51b (accumulation index: 3.3, IC50: 0.4 µM) leading to effects challenging those of the above-mentioned active compound, 7αbenzoyloxy-11α-(R)OTHP-5β-dihydroprogesterone 22a. A further extension was made by replacing one of the two 7α,12α-dibenzoate groups of compound 46 by a OMOM ether having a more simple dioxygenated structure than an OTHP ether.

The

12α-OMOM

ether

55

showed

increased

levels

of

accumulation

and

chemosensitization slightly above those found for the corresponding 12α-(S/R)OTHP isomers 52a,b. The 7α-OMOM ether 56 produced the most significant effects observed in this study on both accumulation and chemosensitization (accumulation index: 3.8, IC50: 0.2 µM). A complementary study was undertaken using a more limited number of similarly Osubstituted 5β-cholan-3-one analogs in which the terminal 24-COOMe ester group of the side chain was converted into a 24-CH2OMe ether group, potentially more resistant to hydrolysis, thus providing a potential advantage for use in vivo. The 7α,12α-dibenzoate ether 62 proved to be a poorly effective modulator as found for its homologous dibenzoate 46 of the 24-COOMe series. The 7α-benzoyloxy-12α-(S/R)OTHP isomers 67a,b, induced significant increases of accumulation, as observed for 24-COOMe analogs. In contrast, the two-fold increase on chemosensitization previously found for 12-(R) isomer in the 24-COOMe series was observed in this case for the 12-(S) isomer 67a. The 7α-(R/S)OTHP-12α-benzoate isomers 66a,b both induced a marked improvement of accumulation as observed for their 24-COOMe analogs 51a,b but associated with an opposite


19

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order of effects of (R/S) isomerism and a much lower stimulation of chemosensitization which remained comparable with the levels found for the 7α-(R/S)OTHP-11α-benzoate isomers 36a,b. A comparison was established with the modulating properties of the hydroxyl steroidal precursors in the view to determine whether some positions were more favorable either to increase activity after esterification or etherification or to limit losses of activity after hydrolysis. Among the hydroxyl precursors evaluated in this study, a slight activity on accumulation was observed for the 7α,12α-diol derivative 45, the 7α-benzoyloxy-11α-ol derivative 20 and the 7αOMOM-12α-ol derivative 54. In contrast, the 7α-hydroxy-11α-benzoate 37 and 7α-hydroxy12α-OMOM ether 53 were able to stimulate a significant accumulation. Benzoylation of the 11α- and 12α-hydroxyl groups leading to compounds 21 and 56 produced a much stronger effect on accumulation than benzoylation of the 7α-hydroxyl group leading to compounds 21 and 55. Such a difference suggests a lesser sensitivity to the presence of the more hindered 7α-hydroxyl group. Blocking hydroxyl groups by ester or ether groups always increased accumulation except for the 7α,12α-dibenzoates 46 and 62 for which accumulation remained low. Restoration of chemosensitivity to doxorubicin of resistant R7 cells versus sensitive K562 parental cells. Four representative O-disubstituted steroidal modulators were tested, two of the 5β-dihydroprogesterone series: the 7α-benzoyloxy-11α-(R)OTHP ether 22a and 7α,11α-bis[(R+S), (S)]OTHP ether 24b and two of the methyl 3-oxo-5β-cholan-24-oate series: the 7α,12αbis-ODHPE derivative 48 and 7α-OMOM,12α-benzoate 56, all with a strong potency to increase both accumulation and chemosensitization. The levels of cytotoxicity of doxorubicin on resistant K562/R7 cells restored after chemosensitization with these four modulators were compared with the cytotoxicity of doxorubicin alone on the sensitive parental K562 cells. As shown by the dose-


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Page 22 of 56

response curves (Figure 2) a large proportion of sensitivity could be restored at a low 1 µM modulator concentration, according to the respective chemosensitization potencies but without reaching the level of sensitive parental K562 cells. Intrinsic toxicity. The intrinsic toxicity of steroidal modulators, expressed as percentage of surviving cells (Tables 1 and 2) was measured on the nonresistant parental K562 cell line after incubation for 72 h at a high 10 µM concentration, leading to values ranging from 43.5 % (most toxic compound 56) up to 100% (nontoxic compound 22b). The 5β-dihydroprogesterone skeleton appeared more favorable (values above 60% for all tested derivatives) than the 5βcholan-3-one skeleton. Among the most potent chemosensitizing 5β-dihydroprogesterone derivatives, the 7α,11α-bis-[(R+S),(R)]OTHP isomer 24a (86.7% cell survival) appeared as the less toxic one, followed by the 7α-benzoyloxy-11α-(R)OTHP ether 22a (80.8%). Among the Odisubstituted methyl 3-oxo-5β-cholan-24-oate derivatives, a cell survival above 60% was observed only for the two strongly efficient 7α,12α-bis-ODHPE derivative 48 (76.3%) and 7α(R+S),12α-(R+S)-bis-OTHP ether 47 (63.2%). A higher toxicity was observed for the two very active 7α-(R/S)OTHP-12α-benzoate isomers 51a,b (51.3 and 44.6%) and for 7α-OMOM-12αbenzoate 56 (43.5%). Among the O-disubstituted 24-methoxy-5β-cholane derivatives only the 7α-benzoyloxy-12α-(S)OTHP ether 67a showed a low intrinsic toxicity (75.8% cell survival) but had only a weak modulating potency. However, at the 1 µM concentration employed for the evaluation of chemosensitization potency on resistant K562/R7 cells, none of the compounds reported in this study showed any significant toxic effects in systematic controls made in the absence of the cytotoxic agent doxorubicin.


21

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CONCLUSION The introduction of O-tetrahydropyranylethers, O-(5’,6’-dihydro-2’H-pyran-4’-yloxy)ether and O-methoxymethyl ethers as well as of benzoate esters on appropriate steroidal hydroxyl precursors, was found able to provide a simple access to potent modulators of P-gp-mediated drug efflux, much more efficient than progesterone on K562/R7 MDR cells, especially using conveniently O-disubstituted derivatives. The use of OTHP ethers resulted in various efficiencies according to the position, the R/S isomerism and the possible occurrence of different axial/equatorial configurations of the anomeric oxygen as shown in this study by X-ray crystallography of monosubstituted steroid models. This kind of modifications offers an alternative or complementary approach to reported methods for obtaining steroid modulators based either on 7α-p-thiophenylamino groups introduced on progesterone18 or on the favorable p-dimethylamino phenyl group7 present in 7- or 11-monosubstituted derivatives of estradien-3one or of estradiol, on a 11,17-disubstituted estradiol derivative29,30 or on 21-monosubstituted corticoid derivatives.26 The classification of the efficiency of steroid derivatives was made according to three criteria of cytotoxic drug accumulation, chemosensitization and intrinsic toxicity (Tables 1 and 2). The O-disubstituted steroidal derivatives were found particularly apt to provide P-gp modulators more potent than the reference steroid RU486. The most efficient compound in 5βdihydroprogesterone series was the 7α-benzoyloxy-11α-(R)OTHP ether 22a. A stronger efficiency was found in the 5β-cholan-3-one series for the 7α-OMOM-12α-benzoate 56, followed by the 7α,12α-disubstituted derivatives 48, 47 and 51b, but was associated with an increased intrinsic toxicity.


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For several other compounds, accumulation did not appear to be well correlated with chemosensitization. Chemosensitization could be either relatively too strong, as found for the 5α-H-7α,11α-dibenzoate 28 and the 7α,11α-bis-[(R+S),(R/S)]OTHP isomers 24a,b or relatively too weak, as for the 7α-(R/S)OTHP-11α-benzoate isomers 36a,b, the 7α-benzoyloxy-12α(S)OTHP isomer 52a and the 7α-(R/S)OTHP-12α-benzoate isomers 66a,b. This lack of correlation, also reported for RU486,30 raises unresolved questions, especially concerning the evolution

of

the

intracellular

doxorubicin

concentration

along

the

time-scale

of

chemosensitization effects, well beyond that of the short-time fluorescence measurements of accumulation. The long-term accumulation effects and/or the activation of mechanisms contributing to chemosensitization might be influenced by the structural sensitivity of modulators to metabolic transformations and to chemical or biochemical hydrolysis of ether or ester groups according to their position and stereochemistry. Moreover, little is known on the mechanisms of P-gp inhibition which might differ according to the structures of modulators and to the possible existence of different sites of interaction yet to be characterized. For more detailed biological investigations in view of in vivo studies, the 5βdihydroprogesterone series were chosen as a first priority, particularly the 5β-H-7α-benzoyloxy11α-(R)OTHP ether 22a which is more efficient on both accumulation and chemosensitization than the three tested reference modulators RU486, verapamil and cyclosporin A. This lead compound has a strong effect on accumulation, potentially representative of a strong inhibition of P-gp drug-transport. Other advantages of compound 22a are its low intrinsic toxicity, its absence of binding to progesterone receptors and its moderate activation of PXR (Alameh, G. unpublished results). Other promising candidates from 5β-cholan-3-one series might benefit


23

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from further optimizations especially modifications of the C17-side-chain including conversion to a 17β-acetyl group in the view to decrease intrinsic toxicity. Most of these results indicate that inhibition of P-gp-mediated drug efflux is very sensitive to the structural features of steroidal modulators. Such a sensitivity suggests direct interactions with privileged parts of P-gp in keeping with numerous studies claiming either a competition at the drug efflux site(s)6 or an interaction at modulation site(s) inducing conformational transmission effects.72 However, the assessment and identification of binding sites for modulators as well as the validation of structure-activity relationships might be complicated by site heterogeneity according to the structures of the modulator and of the effluxed drug17,38,49 and by the lack of data concerning correlations between binding parameters and P-gp inhibition potency. The localization of interaction sites using affinity labeling probes derived from the most efficient modulators identified in this study may facilitate molecular modeling experiments using the recently reported 3D structure of mouse P-gp6 so as to improve the design of further generations of nontoxic adjuvants for cancer chemotherapies.

EXPERIMENTAL SECTION Chemistry. The synthetic protocols and physico-chemical data, including 1H/13C NMR and HRMS characterizations of all steroid compounds, are available free-of-charge as Supporting Information.


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Biology and biochemistry. Cell culture. The human erythroleukemic K562 cell line and the resistant K562/R7 MDR cell line, obtained by treatment of K562 cells with doxorubicine.74 Cells were cultured in RPMI 1640 supplemented with 10% foetal calf serum, L-glutamine (2 mM), glucose (0.3%), sodium pyruvate (1 mM), penicillin (200 U/mL), and streptomycin (100 µg/mL). Cells were maintained at 37 °C in a 5% CO2 atmosphere. Media and supplements were obtained from Invitrogen-Gibco (Paisley, UK), culture flasks from BD-Falcon (Meylan, France). Isolation of RNA, and RT-qPCR. Total RNA was extracted from K562/R7 cells (RNeasy Mini kit; Qiagen). First-strand cDNAs were first synthesized from 5 µg of total RNA in the presence of 50 units of the Superscript TM II reverse transcriptase using both random hexamers and oligo (dT) primers (Invitrogen Kit). qPCR was performed in a final volume of 20 µl containing 5 µl of a 60-fold dilution of the RT reaction medium, 15 µl of reaction buffer from the FastStart DNA Master Plus SYBR Green Kit (Roche Diagnostics), and 10 pmol of the specific forward and reverse primers75 (Operon Biotechnologies, Germany). Standard curves were prepared for each target and reference gene. Each assay was performed in duplicate, and validation of the qPCR runs was assessed by evaluation of the melting temperature of the products and by the slope and error obtained with the standard curve. The analyses were performed using Light-Cycler software (Roche Diagnostics). Results are expressed as relative levels after normalization by G3PDH mRNA.76 Intracellular accumulation of daunorubicin measured by flow cytometry. The accumulation of daunorubicin in the presence of steroid modulators in resistant K562/R7 cells was measured by flow cytometry using a reported procedure.77 Cells (1.10-6 cells/tube) were incubated for 1 h at 37 °C in 1 mL RPMI 1640 medium containing daunorubicin at 10 µM in the presence or absence of steroids at 10 µM. The cells were then washed twice with ice-cold PBS and kept on ice until


25

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analysis by flow cytometry on a FACS-II (Becton-Dickinson Corp., Mountain View, CA). Assays were performed in duplicate in at least three separate experiments. Cyclosporin A, an inhibitor of P-gp, was used as a positive control.78 Cytotoxicity assay using MTT reagent. Cell viability was determined on exponentially growing resistant K562/R7 cells using the MTT assay. This assay is based on the conversion by metabolically active cells of the MTT reagent into formazan crystals. K562/R7 cells (8 000/well) were seeded into 96-well plates and incubated with 100 µL of RPMI medium for 24 h. For each assay, steroid or control drug (1 µM) were added in the absence or presence of eight concentrations of doxorubicin (ranging from 10-7 to 10-4 M) in a final volume of 200 µL of medium. After incubation for 72 h, 20 µL of MTT reagent (5 mg/mL in PBS buffer) were added to each well and the plate was further incubated for 4 h at 37 °C, allowing viable cells to change the yellow MTT into dark-blue formazan crystals. Supernatants were carefully discarded and 100 µL of isopropanol containing 10% HCl 1 M were added to dissolve the formazan crystals. Absorbance in each well was determined at 570 nm using a microplate reader (MultisKan EX, Thermo Electron). Assays and controls, using support medium only, were performed in triplicates in at least three separate experiments. Results were expressed as the percentage of surviving cells in each well compared to untreated cells.78 IC50 values, defined as the concentration of doxorubicin inhibiting cell growth by 50%, were estimated using the Sigma Plot 11.0 software. Intrinsic Cytotoxicity of Steroid Inhibitors. K562 cells (8 000/well) were seeded into 96-well plates and incubated with 100 µL of RPMI medium for 24 h. Steroids (10 µM) were then added in a final volume of 200 µL of medium. After 72 h incubation, cells were treated with MTT as


26


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Page 28 of 56

described above. Each assay was performed in triplicate and results were expressed as the percentage of surviving cells in each well compared to untreated cells. Statistical Analysis. Statistical analysis was performed using three replicates in at least three separate experiments. The data, expressed as the mean ± SE, were analyzed by one-way ANOVA using the Sigma Plot 11.0 software. Differences for which p < 0.05 were considered significant.


27

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Figure 1. ORTEP views of diastereoisomeric 17α-(R/S)OTHP ethers 2a/b, 11α-(R/S)OTHP ethers 22a /b and 7α-(R/S)OTHP ethers 36a/b.


28


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2a

2b

22a

22b

36a

36b


29

Page 31 of 56

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Figure 2. Restoration of doxorubicin sensitivity of K562/R7 cells by steroid modulators. Cell viability of K562/R7 resistant cells treated with doxorubicin (0.1 to 100 µM) in absence () or presence of 1 µM of 48 (■), of 24b (), of 22a (▼), of 56 () and of K562 sensitive cells treated with doxorubicin alone (----) was measured by MTT assay after a 72 h incubation as described in the experimental section.


30


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Scheme 1. Synthesis of O-monosubstituted OTHP derivatives of 11α-OH-, 17α-OH- and 21OH-progesteronea

a

Reagents and conditions: (a) DHP, TsOH, THF, rt.


31

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Scheme 2. Synthesis of O-monosubstituted derivatives of 7α-OH-, 11α-OH- and 17α-OH-5βdihydroprogesteronea

a

Reagents and conditions: (a) m-CPBA, CHCl3; (b) H2-cat Pd/C; (c) BzCl, py; (d) DHP, TsOH,

THF.


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Page 34 of 56

Scheme 3. Synthesis of 7α,11α-O-disubstituted derivatives of 5α- and 5β-dihydroprogesterone via a 5-en-7-one intermediatea

a

Reagents and conditions: (a) ethyleneglycol, TsOH, toluene; (b) TBDMSCl, imidazole, DMF;

(c) CrO3-(py)2, DCM; (d) H2, cat Pd/C, dioxane-EtOH- py; (e) L-selectride, THF; (f) BzCl, py; (g) TBAF, THF; (h) TsOH, acetone; (i) DHP, TsOH, THF; (j) K2CO3 MeOH.


33

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Scheme 4. Synthesis of 7α,11α-O-disubstituted derivatives of 5β-dihydroprogesterone via a 4,6dien-3-one intermediatea

a

Reagents and conditions: (a) TBDMSCl, imidazole, DMF; (b) chloranil, t-BuOH (c) m-CPBA,

DCM; (d) H2, cat Pd/C, dioxane-EtOH-py; (e) DHP, TsOH, THF; (f) TBAF, THF; (g) BzCl, py; (h) TsOH, acetone.


34


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Page 36 of 56

Scheme 5. Synthesis of 11α,17α-O-disubstituted derivatives of 5β-dihydroprogesteronea

a

Reagents and conditions: (a) TsOH, acetone (2 h; rt); (b) O2, NaH, t-BuOH-DMF, P(OEt)3; (c)

TsOH, acetone (48 h, rt); (d) H2, cat Pd/C, dioxane-EtOH-py; (e) DHP, TsOH, THF.


35

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 6. Synthesis of 7α,12α-O-disubstituted derivatives of methyl 3-oxo-5β-cholan-24-oate and 24-methoxy 3-oxo-5β-cholanea OH cholic acid

a

c (46); d (47); e (48); d (49,50);

b

d, c (51,52); f (53,54); f,c (55,56) HO

OH H 44 (95%)

O

g (57); g, h (58);

O

H 45 (94%)

OH 44

OR1

COOMe

OH

R

H

CH2OMe

d, c (66,67)

H

57 (99%) 58 (92%) 59 (86%)

OH R = COOMe R = CH2OH R = CH2OMe

HO

O

OH

H 60 (71%)

H

O

O -ODHPE

O CH2OCH3 -OMOM

OR2

H

61 (77%)

O

a

Bz THP [R+S] DHPE H THP [R+S] THP [R/S] Bz H MOM Bz MOM

c (62); d (63-65);

g, h, i (59) TBDMSO

46 (46%) Bz THP [R+S] 47 (49%) 48 DHPE (62%) 49 THP [R+S] 50 H 51a,b (68%,62%) Bz 52a,b (27%,34%) THP [S/R] 53 (29%) MOM 54 H (17%) MOM 55 (13%) Bz 56 (60%)

OR1

CH2OMe b

j

OR2

R2

R1

COOMe

62 (79%) 63 (18%) 64 (30%) 65 (4%) 66a,b (35%,32%) 67a,b (21%,27%)

R1

R2

Bz H THP [R+S] THP [R+S] Bz THP [S/R]

Bz THP [R+S] H THP [R+S] THP [R/S] Bz

Reagents and conditions: (a) MeOH, HCl; (b) Ag2CO3/Celite, toluene; (c) BzCl, py; (d) DHP,

TsOH, THF; (e) (4-MeO)DHP, TsOH, THF; (f) MOMCl, (i-Pr)2NEt, DCM (g) TBDMSCl, imidazole, DMF; (h) LAH, diethylether; (i) CH3I, NaH-THF; (j) TBAF, THF.


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Table 1. Modulation of resistance of K562/R7 cells to doxorubicin by pregn-4-ene- and pregnane-3,20-dione derivatives and intrinsic toxicities of modulators on K562 sensitive cells Daunorubicin accumulationa Daunorubicin fluorescence ratio with/without modulator

Modulators

Compd Structure

IC50 Doxorubicinb (µM)

Intrinsic toxicity of modulators

MTT assay (1 µM modulator conc)

% of surviving K562 cells (10 µM modulator conc)

Rankc

Rankc

Rankc

pregn-4-ene-3,20-dione derivatives progesterone

1.2 ± 0.05d

22

10.6 ± 2.2d

25

81.6 ± 8.1d

9

11α-OH progesterone

1.0 ± 0.07

24

17α-OH progesterone

1.0 ± 0.09

24

1b

11α-(S)OTHP

2.4 ± 0.06

12

2.1 ± 0.6

15

2a

17α-(R)OTHP

2.4 ± 0.09

12

2b

17α-(S)OTHP

2.8 ± 0.1

8

3

21-(R+S)OTHP 5β β -pregnane-3,20-dione derivatives

1.6 ± 0.1

19

6.7 ± 2.2

23

5β-pregnane-3,20-dione

1.2 ± 0.1

22

6

7α-OBz

2.2 ± 0.1

14

3.1 ± 0.5

20

7

7α-(R+S)OTHP

2.4 ± 0.08

12

1.9 ± 0.6

13

8

11α-OH

1.0 ± 0.03

24

9a

11α-(R)OTHP

2.5 ± 0.1

11

3.4 ± 0.4

21

9b

11α-(S)OTHP

2.5 ± 0.1

11

2.9 ± 0.3

19

10

17αOH

1.1 ± 0 04

23

11a

17α-(R)OTHP

2.3 ± 0.05

13

2.8 ± 0.6

18

11b

17α-(S)OTHP

2.4 ± 0.1

12

2.0 ± 0.2

14

20

7α-OBz,11α-OH

1.4 ± 0.07

21

6.9 ± 0.7

24

92.9 ± 3.7

5

21

7α,11α-diOBz

2.5 ± 0.2

11

1.6 ± 0.3

11

66.4 ± 3.8

18

22a

7α-OBz,11α-(R)OTHP

2.9 ± 0.15

7

0.5 ± 0.05

3

80.8 ± 1.8

11

22b

7α-OBz,11α-(S)OTHP

2.3 ± 0.05

13

2.8 ± 0.3

18

100 ± 2.0

1

23

7α,11α-diOH

1.1 ± 0.02

23

24a

7α,11α- bis-[(R+S),(S)]OTHP

2.6 ± 0.1

10

0.4 ± 0.1

2

86.7 ± 0.6

6

24b

7α,11α- bis-[(R+S),(S)]OTHP

2.5 ± 0.05

11

0.4 ± 0.02

2

84.4 ± 1.0

8

36a

7α-(R)OTHP,11α-OBz

2.8 ± 0.2

8

1.6 ± 0.1

11

61.5 ± 0.8

20

36b

7α-(S)OTHP,11α-OBz

2.6 ± 0.2

10

1.3 ± 0.1

9

70.5 ± 2.6

16

37

7α-OH,11α-OBz

2.3 ± 0.05

13

0.8 ± 0.1

6

81.1 ± 3.9

10

41

11α,17α-diOH

1.0 ± 0.05

24


37

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


11α,17α-bis-[(S),(R)]OTHP 5α α-pregnane-3,20-dione derivatives

2.5 ± 0.2

11

5α-pregnane-3,20-dione

1.0 ± 0.05

24

28

7α,11α-diOBz

2.5 ± 0.1

29a


29b


43a

0.8 ± 0.1

6

67.9 ± 0.3

17

11

0.4 ± 0.1

2

71.1 ± 2.6

15

2.4 ± 0.1

12

0.9 ± 0.1

7

77.8 ± 0.5

12

2.3 ± 0.1

13

2.1 ± 0.6

15

Cyclosporin A

2.6 ± 0.05

10

0.7 ± 0.2

5

40.5 ± 0.4

32

RU486

2.4 ± 0.1

12

0.9 ± 0.1

7

95.6 ± 2.1

3

Verapamil

1.9 ± 0.04

16

0.6 ± 0.1

4

93.3 ± 1.8

4

reference modulators

a

Statistical significance: p < 0.05 for a difference ≥ 0.3 between fluorescence ratios. bStatistical

significance: p < 0.05 for a difference ≥ 0.3 between IC50 values. cGlobal rank order for both Tables 1 and 2 according to decreasing efficiencies. dValues expressed as the mean ± SE.


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Page 40 of 56

Table 2. Modulation of resistance of K562/R7 cells to doxorubicin by 5β-cholan-3-one derivatives and intrinsic toxicities of modulators on K562 sensitive cells

Modulators

Compd Structure

Daunorubicin accumulationa Daunorubicin fluorescence ratio with/without modulator

IC50 Doxorubicinb (µM) MTT assay (1 µM modulator conc)

Rankc

Intrinsic toxicity of modulators % of surviving K562 cells (10 µM modulator conc)

Rankc

Rankc

methyl 3-oxocholate derivatives 45

7α,12α-diOH

1.5 ± 0.07d

20

46

7α,12α-diOBz

1.5 ± 0.04

20

2.4 ± 0.4d

17

57.3 ± 0.7d

21

47

7α,12α-bis-(R+S)OTHP

3.5 ± 0.1

3

0.7 ± 0.1

5

63.2 ± 1.7

19

48

7α,12α-bis-ODHPE

3.7 ± 0.1

2

0.6 ± 0.1

4

76.3 ± 2.5

13

51a

7α-(R)OTHP-12α-OBz

2.9 ± 0.1

7

0.4 ± 0.05

2

51.3 ± 3.1

26

51b

7α-(S)OTHP-12α-OBz

3.3 ± 0.04

5

0.4 ± 0.1

2

44.6 ± 1.3

30

52a


2.7 ± 0.1

9

2.1 ± 0.4

15

53.5 ± 2.0

25

52b


2.1 ± 0.1

15

1.1 ± 0.3

8

56.4 ± 2.3

22

53

7α-OH,12α-OMOM

2.1 ± 0.07

15

1.6 ± 0.4

11

84.7 ± 4.6

7

54

7α-OMOM, 12α-OH

1.8 ± 0.08

17

2.9 ± 0.5

19

95.7 ± 5.9

2

55

7α-OBz,12α-OMOM

2.9 ± 0.1

7

0.9 ± 0.2

7

49.7 ± 4.2

29

56

3.8 ± 0.1 7α-OMOM, 12α-OBz 24-methoxy 3-oxocholane derivatives

1

0.2 ± 0.04

1

43.5 ± 0.8

31

62

7α,12α-diOBz

1.7 ± 0.05

18

5.5 ± 1.7

22

50.7 ± 1.5

28

66a

7α-(R)OTHP-12α-OBz

3.4 ± 0.1

4

1.8 ± 0.6

12

51 ± 2.5

27

66b

7α-(S)OTHP-12α-OBz

3.1 ± 0.1

6

1.5 ± 0.4

10

56.3 ± 2.0

23

67a


2.2 ± 0.1

14

2.2 ± 0.7

16

75.8 ± 4.0

14

67b


2.4 ± 0.03

12

1.1 ± 0.2

8

54.4 ± 1.3

24

Cyclosporin A

2.6 ± 0.05

10

0.7 ± 0.2

5

40.5 ± 0.4

32

RU486

2.4 ± 0.1

12

0.9 ± 0.1

7

95.6 ± 2.1

3

Verapamil

1.9 ± 0.04

16

0.6 ± 0.1

4

93.3 ± 1.8

4

reference modulators


39

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a

Statistical significance: p < 0.05 for a difference ≥ 0.3 between fluorescence ratios. bStatistical

significance: p < 0.05 for a difference ≥ 0.3 between IC50 values. cGlobal rank order for both Tables 1 and 2 according to decreasing efficiencies. dValues expressed as the mean ± SE.


40


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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ASSOCIATED CONTENT Supporting Information. Experimental section (chemical synthesis); 1H/13C NMR spectra of the most relevant active steroids (2a,b, 22a,b, 24a,b, 28, 29a, 36a,b, 43a, 47, 48, 51a,b, 55, 56; 2D-1H/13C HSQC-NMR spectrum of steroid 22a; X-ray diffraction, crystal data and refinement parameters for the three pairs of tetrahydropyranyl ether R/S isomers 2a,b, 22a,b and 36a,b; LC-MS profiles of TLC fractions enriched in isomers 43a-d; preparation of plasma membranes; Western blotting of membrane P-gp. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; Phone (+33) 47877 2859; Fax (+33) 47877 7158. Funding Sources This study was supported by research funds from INSERM and Université Claude Bernard Lyon 1. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT


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Page 44 of 56

The authors are very grateful to Drs G. Dayan and A. Di Pietro for fruitful discussions and their help in initial studies of steroid P-gp interactions which led to undertake this investigation. The authors are indebted to Dr B. Fenet for NMR measurements, Drs D. Bouchu and F. Albrieux for HRMS determinations, Ecole Normale Supérieure-Lyon for optical rotation determinations and Dr. E. Jeanneau for X-ray diffraction studies. The authors wish to acknowledge the helpful suggestions made by Mrs. A. Wackertapp and Dr. J. Wackertapp.

ABBREVIATIONS ABC, ATP-binding cassette; BCRP, Breast Cancer Resistance Protein (or ABCG2); DHP, 3,4dihydro-2H-dihydropyran,

ODHPE,

O-(5’,6’-dihydro-2’H-pyran-4’-yloxy)ether;

MRP1,

Multidrug Resistance Protein 1 (or ABCC1); MRP2, Multidrug Resistance Protein 2 (or ABCC2); MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; OMOM, 4-Omethoxymethyl; OTBDMS, O-tert-butyldimethylsilyl; OTHP, O-tetrahydropyranyl; QSAR, quantitative structure-activity relationship; qPCR, real-time PCR; RT-qPCR, reversetranscription qPCR; TBDMSCl, O-tert-butyldimethylsilyl chloride; TsOH, p-toluenesulfonic acid.


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254x190mm (96 x 96 DPI)


Synthesis of New Steroidal Inhibitors of P-Glycoprotein-Mediated

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