molecules

Article Synthesis and Physicochemical Characterization of the Process-Related Impurities of Eplerenone, an Antihypertensive Drug

Iwona Dams 1,*, Agata Biało ´nska 2, Piotr Cmoch 1, Małgorzata Krupa 1, Anita Pietraszek 1, Anna Ostaszewska 1 and Michał Chody ´nski 1

1 R&D Chemistry Department, Pharmaceutical Research Institute, Rydygiera 8, 01-793 Warsaw, Poland; [email protected] (P.C.); [email protected] (M.K.); [email protected] (A.P.); [email protected] (A.O.); [email protected] (M.C.) 2 Faculty of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383 Wrocław, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-22-456-3929; Fax: +48-22-456-3838

Received: 28 July 2017; Accepted: 11 August 2017; Published: 15 August 2017

Abstract: Two unknown impurities were observed during the process development for multigram-scale synthesis of eplerenone (Inspra®). The new process-related impurities were identified and fully characterized as the corresponding (7β,11α,17α)-11-hydroxy- and (7α,11β,17α) -9,11-dichloroeplerenone derivatives 12a and 13. Seven other known but poorly described in the literature eplerenone impurities, including four impurities A, B, C and E listed in the European Pharmacopoeia 8.4 were also detected, identified and fully characterized. All these contaminants result from side reactions taking place on the steroid ring C of the starting 11α-hydroxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (12) and the key intermediate (7α,17α)-9(11)-enester 7, including epimerization of the C-7 asymmetric center, oxidation, dehydration, chlorination and lactonization. The impurities were isolated and/or synthesized and fully characterized by infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry/electrospray ionization (HRMS/ESI). Their 1H- and 13C-NMR signals were fully assigned. The molecular structures of the eight impurities, including the new (7β,11α,17α)-11-hydroxy- and (7α,11β,17α)-9,11-dichloroeplerenone related substances 12a and 13, were solved and refined using single-crystal X-ray diffraction (SCXRD). The full identification and characterization of these impurities should be useful for the quality control and the validation of the analytical methods in the manufacture of eplerenone.

Keywords: selective aldosterone blocker; hypertension; steroids; eplerenone; impurities; spectroscopic methods; crystal structure

1. Introduction Eplerenone (2, Figure1) is a cardiovascular drug indicated for the treatment of essential hypertension and congestive heart failure that, in contrast to its predecessor spironolactone 1 (Figure1) demonstrates a high degree of selectivity for the aldosterone receptor and a low-binding affinity for progesterone and androgen receptors [1–8]. As a result of the presence of a 9,11-epoxide group in the eplerenone structure, its selectivity for the aldosterone receptor is enhanced and the drug minimizes the risk of adverse hormonal effects and provides important clinical benefits not previously available with spironolactone 1. Treatment with eplerenone is associated with reductions in blood pressure and improved survival (15% reduction in total mortality) for patients with heart failure who are in stable condition after a myocardial infarction. The product was originally developed by scientists at

Molecules 2017, 22, 1354; doi:10.3390/molecules22081354 www.mdpi.com/journal/molecules Molecules 2017, 22, 1354 2 of 26 Molecules 2017, 22, 1354 2 of 26 Molecules 2017, 22, 1354 2 of 26 originally developed by scientists at Ciba-Geigy AG (Basel, Switzerland) and launched in the US in ® originally2003 by Pharmacia developed (Sandwich, by scientists Kent, at Ciba-GeigyUK) under theAG trade (Basel, name Switzerland) Inspra . and launched in the US in Ciba-Geigy AG (Basel, Switzerland) and launched in the US in 2003 by Pharmacia (Sandwich, Kent, 2003 by Pharmacia (Sandwich, Kent, UK) under the trade name Inspra®. UK) under the trade name Inspra®.

Figure 1. Chemical structures of the aldosterone receptor antagonists spironolactone (Aldactone®, 1) Figureand eplerenone 1. Chemical (Inspra structures®, 2). of the aldosterone receptor antagonists spironolactone (Aldactone®, 1) Figure 1. Chemical structures of the aldosterone receptor antagonists spironolactone (Aldactone®, 1) and eplerenone (Inspra®, 2). and eplerenone (Inspra®, 2). Eplerenone contains sensitive 9,11α-epoxide, 17α-γ-lactone and 7α-carbomethoxy moieties that, Eplerenonedepending contains containson the sensitive sensitiveconditions, 9,11 9,11 αmay-epoxide,α-epoxide, hydrol 17 αyze17-γα-lactone -γor-lactone epimerize and and 7α-carbomethoxy 7toα -carbomethoxyafford degradation moieties moieties that, or dependingthat,epimerization depending on theproducts on conditions, the [9–13].conditions, may The hydrolyze SCXRDmay hydrol orstru epimerizectureyze orof toepimerizeeplerenone afford degradation toconfirms afford ordegradationthe epimerization relative cisor productsepimerizationconfiguration [9–13 ].productsof The the SCXRD 9[9–13].α,11 structureα-epoxide The SCXRD of ring eplerenone struandcture the confirms of7 αeplerenone-carbomethoxy the relative confirmscis substituentconfiguration the relative [14–17]. of thecis 9configurationManufactureα,11α-epoxide ofof ringeplerenone the and 9α the,11 is 7αα -epoxidealways-carbomethoxy accompaniedring and substituent theby side7α [14-carbomethoxy –reactions17]. Manufacture leading substituent to of the eplerenone unwanted [14–17]. is alwaysManufactureimpurities accompanied that of vary eplerenone bywith side the reactionsis different always leading startingaccompanied to ma theterials unwanted by andside impuritiesreactionreactions conditions thatleading vary toused. with thethe Mostunwanted different of the startingimpuritiespatents and materials that literature vary and with reactioninformation the different conditions dealing starting used. withMost thema terialspreparation of the and patents reaction of eplerenone and literatureconditions are information based used. on Most the dealing useof the of withpatentscanrenone the and preparation derivatives.literature ofinformation eplerenoneAs stereogenic dealing are basedcenters with onthe thein preparation eplerenone use of canrenone of eplerenoneprecursors derivatives. aregive based rise As on stereogenicto the various use of centerscanrenoneprocess-related in eplerenone derivatives. impurities, precursors As stereogenicincluding give rise centers todiastereo- various in process-relatedeplerenoneand regioisomers precursors impurities, of give includingstarting rise tomaterials, diastereo- various andprocess-relatedintermediates, regioisomers by-productsimpurities, of starting and materials,including the final intermediates, drugdiastereo- substance, by-productsand theregioisomers manu andfacture the finalof of eplerenonestarting drug substance, materials, with thethe manufactureintermediates,required stereochemistry of by-products eplerenone and withand pharmaceutical th thee requiredfinal drug stereochemistrygrade substance, purity the is a andmanu significant pharmaceuticalfacture challenge. of eplerenone grade In designing purity with isthe aa significantrequiredsynthesis stereochemistry of challenge. eplerenone In designingfrom and canrenonepharmaceutical a synthesis derivatives, of grade eplerenone puritythe principal fromis a significant canrenone challenges challenge. derivatives, are the Instereoselective the designing principal a challengessynthesisintroduction of are eplerenone of the the stereoselective carbomethoxy from canrenone introduction substituent derivatives, of at the the carbomethoxy theC-7 principalα position substituentchallenges of the steroid are at the the skeleton C-7 stereoselectiveα position and the of theintroductionregioselective steroid skeleton of dehydration the andcarbomethoxy the of regioselective the 11 substituentα-hydroxy dehydration groupat the [1,9,16–21].C-7 ofα the position 11α -hydroxy of the groupsteroid [1 skeleton,9,16–21]. and the regioselectiveInIn 1984,1984, GrobGrob dehydration etet al.al. [[17,18]17 of,18 the] fromfrom 11α Ciba-Geigy-hydroxyCiba-Geigy group AGAG accomplishedaccomplished[1,9,16–21]. thethe firstfirst synthesissynthesis ofof eplerenoneeplerenone 9(11) byby employingemployingIn 1984, Grob NagataNagata et al. hydrocyanationhydrocyanation [17,18] from Ciba-Geigy ofof ∆Δ9(11)-canrenone-canrenone AG accomplished ( (33)) as as the the the key first step synthesis (Scheme of 11), ),eplerenone butbut withwith moderatebymoderate employing stereoselectivitystereoselectivity Nagata hydrocyanation inin thethe 7 7αα-cyano-cyano of derivativeΔ derivative9(11)-canrenone4 4formation formation (3) as (7the (7α/7α key/7ββ≈ ≈step 4:1) (Scheme necessitating 1), but tedious with columnmoderatecolumn chromatographicchromatographic stereoselectivity in separations.separations. the 7α-cyano The The derivative original original 4EP EP formation 122232 122232 B1(7 B1α /7patent β ≈ 4:1) does necessitating not disclose tedious any informationcolumninformation chromatographic withwith respect respect to theseparations. to purity the of thepurityThe material original of obtained,the EP material122232 its purification B1 obtained, patent todoes pharmaceutical-gradeits notpurification disclose anyto orinformationpharmaceutical-grade the removal with of impurities. respect or the removalto the ofpurity impurities. of the material obtained, its purification to pharmaceutical-grade or the removal of impurities.

SchemeScheme 1.1.The The original original eplerenone eplerenone synthesis synthesis from from Ciba-Geigy Ciba-Geigy AG (Basel, AG Switzerland)(Basel, Switzerland) [17,18]. Reagents [17,18]. and Conditions: (1) Et AlCN, THF; (2) DIBAL-H, benzene; (3) CrO ,H SO , acetone; (4) CH N , CH Cl ; Reagents and Conditions2 : (1) Et2AlCN, THF; (2) DIBAL-H, benzene;3 2 (3)4 CrO3, H2SO4,2 acetone;2 2 (4)2 (5)Scheme m-CPBA, 1. The CH originalCl or H eplerenoneO , Cl CCN, sy KnthesisHPO from, CH ClCiba-Geigy. AG (Basel, Switzerland) [17,18]. CH2N2, CH2Cl2; (5)2 m-CPBA,2 2 2 CH23Cl2 or H2O2, Cl43CCN,2 K22HPO4, CH2Cl2. Reagents and Conditions: (1) Et2AlCN, THF; (2) DIBAL-H, benzene; (3) CrO3, H2SO4, acetone; (4)

CH2N2, CH2Cl2; (5) m-CPBA, CH2Cl2 or H2O2, Cl3CCN, K2HPO4, CH2Cl2.

Molecules 2017, 22, 1354 3 of 26 Molecules 2017, 22, 1354 3 of 26

InIn alternativealternative eplerenone eplerenone synthesis synthesis by Ngby etNg al. [et9], theal. 7[9],α-carbomethoxy the 7α-carbomethoxy group was group introduced was stereoselectivelyintroduced stereoselectively via 4,6-bishydrocyanation via 4,6-bishydrocyanation of 11α-hydroxycanrenone of 11α-hydroxycanrenone (9, Scheme2), but(9, Scheme regioselective 2), but dehydrationregioselective ofdehydration the intermediate of the intermediate (7α,11α,17α)-11-hydroxyester (7α,11α,17α)-11-hydroxyester12 was problematic 12 was problematic giving the regioisomericgiving the regioisomeric (7α,17α)-11(12)-enester (7α,17α)-11(12)-enester7b apart from 7b the apart main (7fromα,17 αthe)-9(11)-enester main (7α,177αproduct)-9(11)-enester (Table1 ).7 Theproduct 7α/7 (Tableβ diastereoisomeric 1). The 7α/7β puritydiastereoisomeric of (7α,11α,17 purityα)-12 obtainedof (7α,11α by,17 theα)- cleavage12 obtained of diketone by the cleavage11 with sodiumof diketone methoxide 11 with in refluxingsodium methanolmethoxide was in not refluxing reported; methanol however, was the literature not reported; sources however, on syntheses the ofliterature related 7sourcesα-carboalkoxy on syntheses stereoidal of spirolactonesrelated 7α-carboalkoxy provide information stereoidal on spirolactones their 7β-carboalkoxy provide diastereoisomersinformation on their resulting 7β-carboalkoxy from basic diastereoisomers epimerization of theresulting steroid from C-7 αbasicasymmetric epimerization center underof the thesteroid same C-7 conditionsα asymmetric as used center for diketoneunder the11 samecleavage conditions [22]. as used for diketone 11 cleavage [22].

Scheme 2. Synthesis of of EP EP from from G. G. D. D. Searle Searle & & Co. Co. (Chicago, (Chicago, IL, IL, USA) USA) [9]. [9 Reagents]. Reagents and and Conditions Conditions: (1):

(1)AspergillusAspergillus ochraceus ochraceus; (2); (2)LiCl, LiCl, DMF, DMF, Et3N, Et3 N,Me Me2CO,2CO, (CH (CH3)2C(OH)CN;3)2C(OH)CN; (3) HCl, (3) HCl, MeOH, MeOH, H2O; H 2(4)O; (4)MeONa, MeONa, MeOH; MeOH; (5) (5)SO SO2Cl2,Cl ImH,2, ImH, THF; THF; (6) H (6)2O H2,2 KO22HPO,K2HPO4, Cl34CCONH, Cl3CCONH2. 2.

When the authors authors repeated repeated the the preparation preparation taught taught inin WO WO 9825948 9825948 A2 A2 it was it was observed observed that that the thepurity purity of the of resulting the resulting eplerenone eplerenone was low was and low atte andmpts attempts to purify to purifythe resulting the resulting product product to a level to ameeting level meeting the requisite the requisite specifications specifications for use as for a pharmaceutical use as a pharmaceutical active were active unsuccessful. were unsuccessful. Although Althoughthe epimeric the 11-hydroxyester epimeric 11-hydroxyester (7β,11α,17 (7αβ)-,1112aα ,17wasα)- not12a wasreported not reportedby Ng et by al. Ng [9], et al.or [any9], or other any otherliterature literature sources, sources, it contaminated it contaminated the main the main (7α,17 (7α)-9(11)-enester,17α)-9(11)-enester 7 product.7 product. In particular, In particular, the theregioisomeric regioisomeric (7α (7,11αα,11,12αα,12,17αα,17)-11,12-epoxyesterα)-11,12-epoxyester 2b2b (Imp.(Imp. E), E), the the new new (7(7αα,11,11ββ,17α)-9,11-dichloro derivative 13 andand thethe 77αα,9:21,17-dicarbolactone,9:21,17-dicarbolactone 14 (Imp.(Imp. A) were difficultdifficult to removeremove fromfrom thethe finalfinal product.product. All these contaminants resu resultedlted from side reactions taking place on the steroid ring C of the (7(7α,11,11α,17α)-11-hydroxy derivative ( 12) and thethe keykey intermediateintermediate (7(7αα,17,17αα)-9(11)-enester)-9(11)-enester 7,, includingincluding epimerization of the C-7 C-7αα asymmetricasymmetric center, center, oxidation, oxidation, dehydrat dehydration,ion, chlorination chlorination and and lactonization. lactonization. Recently, wewe havehave developeddeveloped anan improved,improved, scalable,scalable, cost-effectivecost-effective andand environment-friendlyenvironment-friendly technologytechnology for thethe industrial-scaleindustrial-scale synthesis of eplerenoneeplerenone ((2)) fromfrom commerciallycommercially availableavailable 1111αα-hydroxy-7-hydroxy-7α-(methoxycarbonyl)-3-oxo-17αα-pregn-4-ene-21,17-carbolactone-pregn-4-ene-21,17-carbolactone ( (1212),), based based on thethe lastlast two steps steps of of the the general general route route described described by byNg Ng et al. et al.(Scheme (Scheme 2) [9].2)[ During9]. Duringthe process the processdevelopment, development, two new and two seven new known and seven process-rela knownted process-related impurities of eplerenone impurities were of eplerenone observed, wereand/or observed, synthesized and/or and fully synthesized characterized, and includ fullying characterized, four impurities including A, B, C fourand E impurities listed in the A, B,European C and EPharmacopoeia listed in the European8.4 [23]. Pharmacopoeia The new 8.4impurities [23]. The were new impuritiesidentified wereas identified11α-hydroxy-7 as 11αβ-(methoxycarbonyl)-3-oxo-17-hydroxy-7β-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactoneα-pregn-4-ene-21,17-carbolactone (12a) (12a) and 9,11β-dichloro-7α-(methoxycarbonyl)-3-oxo-17αα-pregn-4-ene-21,17-carbolactone-pregn-4-ene-21,17-carbolactone ( (1313).). To To the best ofof our knowledge,knowledge, thethe 11-hydroxyester 11-hydroxyester12a 12aand and the the 9,11-dichloro 9,11-dichloro derivative derivative13 are13 neware new compounds compounds that havethat have never never been been identified identified before. before. The synthesessyntheses of thethe startingstarting (7(7αα,11,11αα,17,17α)-11-hydroxyester)-11-hydroxyester 12 and thethe keykey intermediateintermediate (7(7α,17,17α)-9(11)-enester 7 are broadly described in the literatureliterature sources [[9,16–21,24];9,16–21,24]; however, their comprehensive structure elucidation and confirmation is still missing and SCXRD studies are reported here for the first time. As starting materials and intermediates in active pharmaceutical

Molecules 2017, 22, 1354 4 of 26 comprehensive structure elucidation and confirmation is still missing and SCXRD studies are reported here for the first time. As starting materials and intermediates in active pharmaceutical ingredient (API) synthesis often afford numerous impurities affecting the quality of the final drug product, their comprehensiveMolecules 2017 structural,, 22,, 13541354 elucidation and confirmation is essential for impurities identification4 of 26 and characterization. A complete physicochemical characterization, not only for an API, but also starting materialsingredient and key (API) synthetic synthesis intermediates, often afford numerous became impurities a requirement affecting of the both quality the of U.S. the final Food drug and Drug product, their comprehensive structural elucidation and confirmation is essential for impurities Administrationproduct, their (FDA) comprehensive and the European structural Medicine elucidation Agency and confirmation (EMA). The is essential five other for impuritiesimpurities listed identification and characterization. A complete physicochemical characterization, not only for an in Table1, i.e., the isomeric (7 β,17α)-9(11)-enester 7a and (7α,17α)-11(12)-enester 7b, the isomeric API, but also starting materials and key synthetic intermediates, became a requirement of both the (7β,11αU.S.,17α Food)-9,11-epoxyester and Drug Administrati2a (Imp.on E)(FDA) and and (7 αthe,11 Europeanα,12α,17 αMedicine)-11,12-epoxyester Agency (EMA).2b The(Imp. five B) and the 7α,9:21,17-dicarbolactoneother impurities listed 14in (Imp.Table A),1, werei.e., mentionedthe isomeric elsewhere (7β,17α)-9(11)-enester in the literature, 7a and mainly as part of(7 HPLCα,17α)-11(12)-enester studies; however, 7b, theirthe isomeric syntheses, (7β methods,11α,17α)-9,11-epoxyester of removalfrom 2a the(Imp. final E) productand and comprehensive(7α,11α,12 structuralα,17α)-11,12-epoxyester elucidation 2b and (Imp. confirmation B) and the 7 wereα,9:21,17-dicarbolactone not disclosed [9, 1214 ].(Imp. The A), determination were mentioned elsewhere in the literature, mainly as part of HPLC studies; however, their syntheses, of a drugmentioned substance elsewhere impurity in the profile, literature, including mainly as starting part of materials,HPLC studies; by-products, however, their intermediates syntheses, and methods of removal from the final product and comprehensive structural elucidation and potentialconfirmation degradation were products, not disclosed is critical [9,12]. for The the determ safetyination assessment of a drug of APIsubstance and manufacturingimpurity profile, process thereof.including In any API, starting it is necessarymaterials, by-products, to study the intermediates impurity profile, and potential and control degradation it during products, the preparation is of the pharmaceutical.critical for the safety Asindicated assessment in of the API ICH and guidelines, manufacturing any process impurity, thereof. formed In any at a API, level it of is≥ 0.10% with respectnecessary to the to study API, the should impurity be identified, profile, and synthesizedcontrol it during and the characterized preparation of thoroughlythe pharmaceutical. [25,26 ]. Only As indicated in the ICH guidelines, any impurity, formed at a level of ≥0.10% with respect to the two eplerenoneAs indicated impurities, in the ICH i.e., guidelines, 7α,9:21,17-dicarbolactone any impurity, formed14 at (Imp.a level A)of ≥ and0.10% 11,12-epoxy with respect ester to the2b (Imp. API, should be identified, synthesized and characterized thoroughly [25,26]. Only two eplerenone B), are accepted at a level greater than 0.10%, i.e., maximum 0.3%, in accordance with pharmacopoeial impurities, i.e., 7α,9:21,17-dicarbolactone 14 (Imp. A) and 11,12-epoxy ester 2b (Imp. B), are acceptanceaccepted criteria at a [ 22level]. greater than 0.10%, i.e., maximum 0.3%, in accordance with pharmacopoeial acceptance criteria [22]. Table 1. Structures of the process-related impurities of eplerenone (2). Table 1. StructuresStructures ofof thethe process-relatedprocess-related impuritiesimpurities ofof eplerenoneeplerenone ((2). Chemical Structure Name Chemical Structure Name

11α-hydroxy-711α-hydroxy-7α-(methoxycarbonyl)-3-oxo-17α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactoneα-pregn-4-ene-21,17-carbolactone

12

11α-hydroxy-711α-hydroxy-7β-(methoxycarbonyl)-3-oxo-17β-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactoneα-pregn-4-ene-21,17-carbolactone

12a

7α-(methoxycarbonyl)-3-oxo-177α-(methoxycarbonyl)-3-oxo-17α-pregna-4,9(11)-diene-21,17-carbolactoneα-pregna-4,9(11)-diene-21,17-carbolactone

1 7 (Imp. C) C) 1

7β-(methoxycarbonyl)-3-oxo-177β-(methoxycarbonyl)-3-oxo-17α-pregna-4,9(11)-diene-21,17-carbolactoneα-pregna-4,9(11)-diene-21,17-carbolactone

7a

7α-(methoxycarbonyl)-3-oxo-177α-(methoxycarbonyl)-3-oxo-17α-pregna-4,11(12)-diene-21,17-carbolactoneα-pregna-4,11(12)-diene-21,17-carbolactone

7b

Molecules 2017, 22, 1354 5 of 26

Molecules 2017,, 22,, 13541354 Table 1. Cont. 5 of 26

Chemical StructureTable 1. Cont. Name

β α α 9,11β9,11-dichloro-7-dichloro-7-dichloro-7α-(methoxycarbonyl)-3-oxo-17-(methoxycarbonyl)-3-oxo-17-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone-pregn-4-ene-21,17-carbolactone-pregn-4-ene-21,17-carbolactone

13 13

3-oxo-173-oxo-17α-pregn-4-ene-7-pregn-4-ene-7α-pregn-4-ene-7α,9:21,17-dicarbolactone,9:21,17-dicarbolactoneα,9:21,17-dicarbolactone

1 14 (Imp.(Imp. A) A) 11

9,11α9,11-epoxy-7-epoxy-7α-epoxy-7β-(methoxycarbonyl)-3-oxo-17-(methoxycarbonyl)-3-oxo-17β-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone-pregn-4-ene-21,17-carbolactoneα-pregn-4-ene-21,17-carbolactone

1 2a (Imp.(Imp. E) E) 11

11α,12,1211α-epoxy-7,12-epoxy-7α-epoxy-7α-(methoxycarbonyl)-3-oxo-17-(methoxycarbonyl)-3-oxo-17α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone-pregn-4-ene-21,17-carbolactoneα-pregn-4-ene-21,17-carbolactone

1 2b (Imp.(Imp. B) B) 11 1 Impurities listed in the European Pharmacopoeia 8.4 [23]. 1 Impurities11 ImpuritiesImpurities listed listedlisted in inin the thethe EuropeanEuEuropean Pharmacopoeia 8.4 8.4 [23]. [23]. The present paper deals with synthesis, physicochemicalicochemical characterizationcharacterization andand comprehensivecomprehensive Thestructural present elucidation paper deals and with confirmation synthesis, of physicochemicalthe process-related characterizationeplerenone impurities and found comprehensive in samples of the key intermediate (7α,17α)-9(11)-enester 7 and eplerenone bulk drug substance structuralsamples elucidation of the andkey confirmationintermediate (7 ofα,17,17 theα)-9(11)-enester process-related 7 eplerenoneandand eplerenoneeplerenone impurities bulkbulk drugdrug found substancesubstance in samples of manufactured according to the modified and optimized procedure described in Searle Co. patent [9] the key intermediatemanufactured (7accordingα,17α)-9(11)-enester to the modified7 andand optimized eplerenone procedure bulk drug described substance in Searle manufactured Co. patent according[9] (Scheme 2, 12 asas startingstarting material).material). ApartApart fromfrom thethe startingstarting materialmaterial 12,, thethe keykey intermediateintermediate enesterenester to the modified7 andand thethe andfinalfinal optimizedproductproduct 2,, sevenseven procedure process-relatedprocess-related described eplerenone in Searle impurities Co. patent 2a–b [9,, ]7a (Scheme–b,, 12a,, 132, andand12 as 14starting material).were Apart fully from characterized the starting by material IR, NMR12, theand key MS. intermediate The molecular enester structures7 and the of finalthe productepimeric2 , seven process-related11-hydroxyesters eplerenone (7α,11,11 impuritiesα,17,17α)-12 2aandand–b ,(7(77aβ,11,11–bα, ,17,1712aα,)-1312aand,, thethe14 isomericisomericwere fully (7(7α,17,17 characterizedα)-9(11)-enester by 7 IR,,, NMR and MS.(7 Theβ,17,17 molecularα)-9(11)-enester structures 7a andandof (7(7 theα,17,17 epimericα)-11(12)-enester 11-hydroxyesters 7b,, thethe isomericisomeric (7α,11 (7(7βα,11,11,17αα,17,17)-α12)-9,11-epoxyesterand (7β,11α,17 α)-12a, 2a and (7α,11α,12α,17α)-11,12-epoxyester 2b, and the new (7α,11β,17α)-9,11-dichloro derivative 13, the isomeric2a andand (7(7(7α,11,11,17αα,12,12)-9(11)-enesterα,17,17α)-11,12-epoxyester7, (7β,17 2bα,, )-9(11)-enesterandand thethe newnew (7(7α,11,117aβ,17,17andα)-9,11-dichloro (7α,17α)-11(12)-enester derivative 13,, 7b, the were solved and refined using SCXRD. The full identification and characterization of these isomeric (7β,11α,17α)-9,11-epoxyester 2a and (7α,11α,12α,17α)-11,12-epoxyester 2b, and the new compounds should be useful for the quality control and the validation of the analytical methods in (7α,11β,17the αmanufacture)-9,11-dichloro of eplerenone. derivative 13, were solved and refined using SCXRD. The full identification and characterization of these compounds should be useful for the quality control and the validation of the analytical2. Results methods and in Discussion the manufacture of eplerenone. Dehydration of 11-hydroxy steroids is commonly used for the introduction of the 9,11-double 2. Resultsbond and into Discussion the steroid skeleton. In a preferred technological embodiment, the (7α,17,17α)-9(11)-enester 7 is synthesized directly by in situ replacement of the 11α-hydroxy group of the ester (7α,11α,17α)-12 Dehydrationisis synthesizedsynthesized of directlydirectly 11-hydroxy byby inin situsitu steroids replacementreplacement is commonly ofof thethe 1111α-hydroxy used for group the introduction of the ester (7α of,11,11 theα,17,17α 9,11-double)-12 with halogen followed by thermal 9,11-dehydrohalogenation. The nucleophilic substitution of the α α bond into11α the-hydroxy steroid group skeleton. is effected In a preferredby reactiontechnological with sulfuryl halide embodiment, at about the−70 (7°C, ,17after)-9(11)-enester which a 7 is synthesizedhydrogen directly halide scavenger by in situ is replacementadded. In manufact of theuring 11α technology-hydroxy of group eplerenone of the developed ester (7α by,11 theα,17 α)-12 with halogenauthors, followed the 11α-hydroxyester by thermal 12 9,11-dehydrohalogenation. andand imidazoleimidazole werewere dissolveddissolved The simultaneouslysimultaneously nucleophilic inin substitution anhydrousanhydrous of the 11α-hydroxytetrahydrofuran group is effectedand cooled by to reaction −10 °C. Sulfuryl with sulfuryl chloride halide was adde at aboutd slowly− 70and◦ C,the after reaction which mixture a hydrogen was allowed to warm to room temperature, and then stirred for a time sufficient to complete the halide scavengerwas allowed is to added. warm to In room manufacturing temperature, and technology then stirredstirred of forfor eplerenone aa timetime sufficientsufficient developed toto completecomplete by the thethe authors, elimination reaction, typically about 1 h. The key intermediate 7 waswas isolatedisolated inin aa crudecrude formform byby the 11α-hydroxyester 12 and imidazole were dissolved simultaneously in anhydrous tetrahydrofuran dichloromethane extraction followed by evaporation of the solvent and recrystallized twice, from and cooled to −10 ◦C. Sulfuryl chloride was added slowly and the reaction mixture was allowed to warm to room temperature, and then stirred for a time sufficient to complete the elimination reaction, typically about 1 h. The key intermediate 7 was isolated in a crude form by dichloromethane extraction followed by evaporation of the solvent and recrystallized twice, from ethanol and a mixture Molecules 2017, 22, 1354 6 of 26 of dichloromethane/diethyl ether respectively, to give the pure (7α,17α)-9(11)-enester 7 (71% yield) as white crystals. The dehydration step produced the three main side products, i.e., the regioisomeric (7β,17α)- 11(12)-enester 7b, the new (7α,11β,17α)-9,11-dichloro derivative 13 and the 7α,9:21,17-dicarbolactone 14 (Imp. A), which were isolated chromatographically from the recrystallization mother liquors of 7 by elution with varying mixtures of ethyl acetate/dichloromethane. Further gradient elution with acetonitrile/toluene afforded the new 11-hydroxyester (7β,11α,17α)-12a, which was isolated in only a small yield, apart from the unreacted (7α,11α,17α)-12 isomer used as the starting material. The commercial 11-hydroxyester (7α,11α,17α)-12 was synthesized according to the procedure described by Ng et al. [9] and the chemical purity declared by the manufacturer was 98%. The novel 11-hydroxyester (7β,11α,17α)-12a was not previously detected by chromatographic or spectroscopic methods in its commercially available (7α,11α,17α)-12 isomer. However, the literature data on syntheses of related 7α-carboalkoxy steroidal spirolactones [22] allowed us to assume that the new 11-hydroxyester (7β,11α,17α)-12a originates from the fourth step of the Scheme2 synthesis in which sodium methoxide reacts with diketone 11 to afford the (7α,11α,17α)-12 isomer as the main product of the cleavage, which is contaminated with the (7β,11α,17α)-12a epimer resulting from the competitive basic epimerization of 12. The 11-hydroxyester (7β,11α,17α)-12a was synthesized independently by basic epimerization of the (7α,11α,17α)-12 isomer. As expected, sodium methoxide in methanol solution converted the C-7α ester 12 into an epimeric mixture of the C-7α 12 and C-7β 12a esters. The pure C-7β ester 12a was isolated chromatographically from the crude mixture of epimers 12 and 12a by 10–50% acetonitrile/toluene gradient elution to give (7β,11α,17α)-12a (49% yield) as a white solid. A sample of 12a was also isolated by column chromatography from recrystallization mother liquors of the commercial 11-hydroxyester (7α,11α,17α)-12. Even a small amount of the newly detected (7β,11α,17α)-11-hydroxyester 12a is hardly separable from its (7α,11α,17α)-12 isomer by large scale chromatography or recrystallization, therefore, the commercial 11-hydroxyester (7α,11α,17α)-12 was used further without purification. As the two 11-hydroxyesters of the C-7α and C-7β series, respectively 12 and 12a, are crucial for impurities formation during multi-gram scale synthesis of eplerenone, they were subjected to comprehensive structural elucidation and confirmation. The [M + Na]+ values, m/z 439.2110 and 439.2080, obtained for both 11-hydroxyesters 12 and 12a correspond to C24H32O6Na. The NMR data for epimeric hydroxyesters 12 and 12a are given in Table2. The 1H-/13C-NMR chemical shifts assignment was made after careful and precise 2D NMR spectra analysis and the data obtained for the known isomer 12 are in full compliance with those presented by Preisig et al. [24]. The 2D NOESY experiments allowed discrimination between epimeric structures 12 and 12a and fully confirmed the stereochemistry at the C-7 atom in both isomers. Blue arrows in Figure2 show the most important NOE effects involving H7 proton, simultaneously clearly indicating the 7α or 7β positioned carbomethoxy group. The strong H7-H8 NOE effect is observed for the compound 12 with β positioned H7 proton, whereas α situated H7 proton in 12a is involved in two significant H9-H7 and H7-H14 NOE effects. Additionally, the strong NOE effect between H9 and H14 protons for both 12 and 12a epimers is observed. Comparison of the NMR data for epimeric structures 12 and 12a revealed that some of the 1H/13C nuclei shieldings within the steroid rings B, C and D are related to the α or β configuration of the C-7 atom. The 1H shielding increase of 0.5 ppm for H7, 0.71 ppm for H9 and 0.28 ppm for H14 nuclei is observed when passing from the compound 12 with 7α positioned carbomethoxy group to its 7β epimer 12a. Additionally, the change of configuration at the C7 is accompanied by H8 (0.35 ppm) shielding decrease. Simultaneously, both diasterotopic H15 protons of the epimer 12a with 7β positioned carbomethoxy group became equal having the same proton chemical shift (1.49 ppm). In the case of 13C-NMR data the opposite effect is observed, the transition from 12 to 12a leads to the shielding decrease of 6.6 ppm for C7, 4.8 ppm for C9 and 3.4 ppm for C14 nuclei. Surprisingly, the change of configuration from 7α in 12 to 7β in 12a is related with no change of the shielding of the C8 nucleus and decreasing of shielding by 2.7 ppm for C23 carbon of the carbomethoxy substituent. Molecules 2017, 22, 1354 7 of 26 Molecules 2017, 22, 1354 7 of 26

(a) (b)

Figure 2. Crucial 1H- and 13C-NMR chemical shifts and NOE effects for 11-hydroxyesters: (a) Figure 2. Crucial 1H- and 13C-NMR chemical shifts and NOE effects for 11-hydroxyesters: (7α,11α,17α)-12; (b) (7β,11α,17α)-12a. (a) (7α,11α,17α)-12;(b) (7β,11α,17α)-12a.

1 13 Table 2. The 1H- and 13C-NMR spectral data in CDCl3 for 11-hydroxyesters (7α,11α,17α)-12 and Table 2. The H- and C-NMR spectral data in CDCl3 for 11-hydroxyesters (7α,11α,17α)-12 and (7β,11α,17α)-12a. (7β,11α,17α)-12a. 1H-NMR (1) 13C-NMR Proton 121H-NMR (1) 12a Carbon13C-NMR 12 12a ProtonH-1a 2.02 12 m 12a 2.07 m Carbon C-1 37.3 12 37.1 12a H-1aH-1b 2.802.02 mm 2.07 2.71 m m C-1 C-2 34.1 37.3 34.2 37.1 H-1bH-2a 2.342.80 mm 2.71 2.33 m m C-2 C-3 199.5 34.1 199.9 34.2 H-2aH-2b 2.462.34 mm 2.33 2.43 m m C-3 C-4 126.1 199.5 125.0 199.9 H-2bH-4 2.46 5.69 ms 2.43 5.75 m s C-4 C-5 167.7 126.1 167.2 125.0 H-4 5.69 s 5.75 s C-5 167.7 167.2 H-6aH-6a 2.52 mm 2.37 2.37 m m C-6 C-6 36.3 36.3 37.3 37.3 H-6bH-6b 2.64 2.64 ddd ddd (1.9, 5.8,5.8, 14.6)14.6) 2.62 2.62 m m C-7 C-7 42.4 42.4 49.0 49.0 H-7H-7 2.81 mm 2.31 2.31 m m C-8 C-8 37.0 37.0 37.0 37.0 H-8H-8 1.79 mm 2.14 2.14 m m C-9 C-9 52.6 52.6 57.4 57.4 H-9 1.93 m 1.22 t (10.4) C-10 39.4 39.6 H-11H-9 1.934.04 mm 1.22 4.10 mt (10.4) C-11 C-10 39.4 69.2 39.6 68.5 H-12aH-11 4.041.42 mm 1.42 4.10 m m C-12 C-11 69.2 43.4 68.5 43.5 H-12bH-12a 1.421.85 mm 1.83 dd 1.42(4.6, 11.6)m C-13 C-12 43.4 46.2 43.5 46.6 H-14H-12b 1.851.65 m 1.83 1.37 dd m(4.6, 11.6) C-14 C-13 46.2 45.3 46.6 48.7 H-15aH-14 1.651.41 mm 1.49 1.37 m m C-15 C-14 45.3 22.5 48.7 22.5 H-15b 1.73 m 1.49 m C-16 35.4 35.6 H-16aH-15a 1.411.88 mm 1.80 ddd (6.9, 1.49 8.8, m 14.3 ) C-17 C-15 22.5 95.0 22.5 94.5 H-16bH-15b 2.26 ddd 1.73 (3.6, m 12.1, 14.5) 2.21 ddd (4.4, 1.49 11.4, m 14.3) C-18 C-16 35.4 15.4 35.6 16.1 H-18H-16a 1.88 1.01 m s 1.80 ddd 1.03 (6.9, s 8.8, 14.3 ) C-19 C-17 95.0 18.4 94.5 18.4 H-19H-16b 2.26 ddd (3.6, 1.36 s12.1, 14.5) 2.21 ddd 1.40 (4.4, s 11.4, 14.3) C-20 C-18 15.4 31.2 16.1 31.4 H-20a 1.95 m 1.93 ddd (7.5, 9.4, 13.0) C-21 29.1 29.2 H-20bH-18 2.39 1.01 ms 2.37 1.03 m s C-22 C-19 18.4176.5 18.4 176.5 H-21aH-19 2.50 1.36 ms 2.49 ddd (11.6, 1.40 13.1, s 17.7) C-23 C-20 31.2172.7 31.4 175.4 H-21bH-20a 1.952.57 mm 2.56 1.93 ddd ddd (6.2, (7.5, 9.3, 9.4, 17.7) 13.0) C-24 C-21 29.1 51.6 29.2 52.1 H-24H-20b 2.39 3.65 m s 3.69 2.37 s m C-22 176.5 176.5 H-21a 2.501 Values m in parentheses 2.49 givedddδ (11.6,, multiplicity 13.1, 17.7) and J in Hz. C-23 172.7 175.4 H-21b 2.57 m 2.56 ddd (6.2, 9.3, 17.7) C-24 51.6 52.1 The NMRH-24 structure3.65 assignments s of the isomeric 3.69 s 11-hydroxyesters (7 α,11α ,17α)-12 and (7β,11α,17α)-12a were confirmed1 Values in byparentheses X-ray analysis give δ, multiplicity (Figure3 and and Table J in Hz.3). The SCXRD structures cis trans α α confirmedThe NMR the relativestructure -assignments and -configurations of the isomeric of the11-hydroxyesters 11 -hydroxy and(7α,11 7 α-carbomethoxy,17α)-12 and 12 12a α 12 (7substituentsβ,11α,17α)- in12a wereand confirmed, respectively. by X-ray The analysis carbomethoxy (Figure group 3 and is situatedTable 3). at The the SCXRD C-7 position structures in β 12a α 12 12a confirmedand the C-7 theposition relative in cis- ,and whereas trans the-configurations hydroxy group of adopts the 11 theα-hydroxy C-11 position and 7 inα-carbomethoxy both and α substituentsisomers. The in dehydration 12 and 12a, of respectively. 11 -hydroxy The steroids carbomethoxy leads predominantly group is situated to the formationat the C-7α of position the double in α α 7 12bond and between the C-7β C-9 position and C-11 in carbons12a, whereas of the the steroid hydroxy skeleton. group As adopts expected, the theC-11 (7α ,17position)-9(11)-enester in both 12 12 andwas 12a the mainisomers. product The dehydration of the 11-hydroxyester of 11α-hydroxydehydration steroids leads with predom sulfurylinantly chloride to inthe the formation presence of of the double bond between C-9 and C-11 carbons of the steroid skeleton. As expected, the (7α,17α)-9(11)-enester 7 was the main product of the 11-hydroxyester 12 dehydration with sulfuryl

MoleculesMolecules2017 2017, 22, 22, 1354, 1354 8 of8 26 of 26

chloride in the presence of imidazole; however, a considerable amount of the regioiosmeric imidazole;(7α,17α)-11(12)-enester however, a considerable 7b, as a result amount of competitive of the regioiosmeric 11,12-dehydration (7α,17α of)-11(12)-enester 12, was also isolated7b, as from a result ofthe competitive recrystallization 11,12-dehydration mother liquors of 12 of, 7 was. also isolated from the recrystallization mother liquors of 7.

(a) (b)

Figure 3. Molecular structure of the 11-hydroxyesters: (a) (7α,11α,17α)-12 and (b) (7β,11α,17α)-12a. Figure 3. Molecular structure of the 11-hydroxyesters: (a) (7α,11α,17α)-12 and (b) (7β,11α,17α)-12a.

Table 3. Summary of crystallographic data and structure refinement for the 11-hydroxyesters Table 3. Summary of crystallographic data and structure refinement for the 11-hydroxyesters (7α,11α,17α)-12 and (7β,11α,17α)-12a. (7α,11α,17α)-12 and (7β,11α,17α)-12a. Identification Code 12 12a IdentificationChemical formula Code C24H 1232O6 C24H 12a32O6 Molecular weight 416.50 g/mol 416.50 g/mol Chemical formula C24H32O6 C24H32O6 Temperature 100(2) K 100(2) K Molecular weight 416.50 g/mol 416.50 g/mol Wavelength 0.71073 Å 0.71073 Å Temperature 100(2) K 100(2) K Crystal system, space group Orthorhombic, P212121 Orthorhombic, P212121 Wavelength a = 6.7560(2)0.71073 Å Å a = 12.2862(3) 0.71073 Å Å

Crystal system, space group Orthorhombic,b = 16.1436(5) P2 Å1 2121 Orthorhombic, b = 12.4532(3) P2Å 12121 c = 19.0947(6) Å c = 13.3060(4) Å Unit cell dimensions a = 6.7560(2) Å a = 12.2862(3) Å α = 90° α = 90° b = 16.1436(5) Å b = 12.4532(3) Å β = 90 ° β = 90° Unit cell dimensions c =γ 19.0947(6) = 90° Å c =γ13.3060(4) = 90° Å Volume 2082.59(11)α = 90◦ Å3 2035.85(9)α = 90 Å◦ 3 Z, Calculated density 4, 1.328β = mg/m 90 ◦ 3 4, 1.359β =mg/m 90◦ 3 Absorption coefficient 0.094 0.096 γ = 90◦ γ = 90◦ F(000) 896 896 Volume 2082.59(11) Å3 2035.85(9) Å3 Crystal size 0.30 × 0.20 × 0.20 mm 0.30 × 0.20 × 0.10 mm 3 3 ThetaZ range, Calculated for data density collection 2.13°–25.68°4, 1.328 mg/m 2.26°–25.67°4, 1.359 mg/m Absorption coefficient−8 ≤ 0.094 h ≤ 8 −14 ≤ 0.096h ≤ 14 LimitingF(000) indices −19 ≤ 896k ≤ 19 −15 ≤ k 896 ≤ 15 Crystal size 0.30−×230.20 ≤ l ≤× 230.20 mm 0.30−×160.20 ≤ l ≤× 160.10 mm Reflections collected/unique 21,220/3973 11,318/3856 Theta range for data collection 2.13◦–25.68◦ 2.26◦–25.67◦ semi-empirical from semi-empirical from Absorption correction equivalents−8 ≤ h ≤ 8 equivalents−14 ≤ h ≤ 14 Limiting indices Max. and min. transmission 0.9814−19 and≤ k0.99723≤ 19 0.9904−15 and≤ k0.9716≤ 15 Refinement method full-matrix− least-squares23 ≤ l ≤ 23 on F2 full-matrix −least-squares16 ≤ l ≤ 16 on F2 ReflectionsData/restraints/parameters collected/unique 3973/0/275 21,220/3973 3856/1/275 11,318/3856 Goodness-of-fit on F2 1.156 1.041 Absorption correction semi-empirical from equivalents semi-empirical from equivalents Final R1/wR2 indices [I > 2sigma(I)] 0.0360/0.0921 0.0311/0.0721 Max.R1/wR2 and indices min. transmission (all data) 0.9814 0.0481/0.1132 and 0.99723 0.0361/0.0756 0.9904 and 0.9716 LargestRefinement diff. peak method and hole full-matrix 0.191 and least-squares −0.341 e/Å3 on F2 full-matrix0.172 and least-squares −0.212 e/Å3 on F2 Data/restraints/parameters 3973/0/275 3856/1/275 Additionally,Goodness-of-fit the on 9,11-dehydrationF2 of the1.156 residual 11-hydroxyester 1.041(7β,11α,17α)-12a contaminatingFinal R1/wR2 theindices starting [I > 2sigma( 12 isomerI)] afforded 0.0360/0.0921 the epimeric (7β,17α)-9(11)-enester 0.0311/0.0721 7a, which was also isolated chromatographically from recrystallization mother liquors of 7. It was synthesized R1/wR2 indices (all data) 0.0481/0.1132 0.0361/0.0756 independently by epimerization of the (7α,17α)-9(11)-enester 7 with sodium methoxide in methanol, Largest diff. peak and hole 0.191 and −0.341 e/Å3 0.172 and −0.212 e/Å3 or dehydration of the 11-hydroxyester (7β,11α,17α)-12a with sulfuryl chloride and imidazole, and

Molecules 2017, 22, 1354 9 of 26

Additionally, the 9,11-dehydration of the residual 11-hydroxyester (7β,11α,17α)-12a contaminatingMolecules 2017, 22, 1354 the starting 12 isomer afforded the epimeric (7β,17α)-9(11)-enester 7a, which9 wasof 26 also isolated chromatographically from recrystallization mother liquors of 7. It was synthesized independentlythen purified byby epimerizationcolumn chromatography of the (7α,17 αwith)-9(11)-enester 5–20% ethyl7 with acetate/dichloromethane sodium methoxide in methanol, gradient orelution dehydration to give the of pure the 11-hydroxyester (7β,17α)-9(11)-enester (7β,11 7aα,17, withα)- 12a37%with or 85% sulfuryl yield respectively, chloride and as imidazole, a white solid. and then purifiedThe [M by+ Na] column+ values, chromatography m/z 421.1986, with 421.2006, 5–20% ethyland 421.1988, acetate/dichloromethane obtained for the gradient free isomeric elution toenesters give the 7, pure7a and (7 β7b,17 correspondα)-9(11)-enester to C247aH,30 withO5Na. 37% The or full 85% NMR yield data respectively, confirming as athe white structure solid. of the knownThe enester [M + Na] 7 were+ values, reportedm/z 421.1986, by Grob 421.2006,et al. [17]; and however 421.1988, several obtained 1H and for the13C freesignals isomeric were enesters assigned7, 7areversibly.and 7b correspond The proper to 1H/ C2413HC30 chemicalO5Na. The shift full assignment NMR data confirmingfor isomeric the enesters structure 7, 7a of theand known 7b was enester based 7onwere the reportedcareful analysis by Grob of et 2D al. [NMR17]; however experiments several and1H is and given13C in signals Table were 4. Similarly assigned to reversibly. the epimeric The properhydroxyesters1H/13C 12 chemical and 12a shift, the assignment NOE effects for involving isomeric enestersH7 proton7, 7aallowedand 7b distinctionwas based between on the careful 7α (7 analysisand 7b) and of 2D 7β NMR (7a) diastereoisomers experiments and (Figure is given 4). in The Table strong4. Similarly H7-H8 NOE to the effect epimeric was hydroxyestersnoted for isomers12 and7 and12a 7b, thewith NOE β positioned effects involving H7 proton, H7 whereas proton allowed α situated distinction H7 in 7a between is involved 7α (in7 andH7-H147b) andNOE 7 βeffect(7a) diastereoisomersdisturbed by H7-H15 (Figure 4interaction.). The strong Additionally, H7-H8 NOE the effect significant was noted H9-H14 for isomers NOE7 andeffect7b forwith 7bβ positionedregioisomer H7 was proton, observed, whereas αwhilesituated H8 H7proton in 7a isof involved the epimers in H7-H14 7 and NOE 7b effect is involved disturbed byin H7-H15H8-H6 interaction.interaction. Additionally,Similarly to the the epimeric significant hydroxyesters H9-H14 NOE 12 effect and for 12a7b, someregioisomer of the 1 wasH/13C observed, nuclei shieldings while H8 protonwithin ofthe the steroid epimers rings7 and B,7b C isand involved D are inrelated H8-H6 to interaction. the α or β Similarly configuration to the epimericof the C-7 hydroxyesters atom. The 112H andshielding12a, some increase of the of1 H/0.713 ppmC nuclei for H7 shieldings and 0.22 within ppm the for steroid H8 nuclei rings was B, C observed and D are when related passing to the α fromor β configurationthe compound of 7 the with C-7 7 atom.α positioned The 1H shielding carbomethoxy increase group of 0.7 ppmto its for7β H7epimer and0.22 7a. ppmSimilarly for H8 to nuclei 12a, both was observeddiasterotopic when H15 passing protons from of the the compound epimer 7a7 with with 7 α7βpositioned positioned carbomethoxy carbometho groupxy group to its became 7β epimer equal7a. Similarlyhaving the to 12asame, both proton diasterotopic chemical H15 shift protons (1.54 ofppm). the epimer In the7a casewith of 7 β13positionedC-NMR the carbomethoxy opposite effect group is becameobserved, equal the havingtransition the from same 7 proton to 7a leads chemical to the shift shielding (1.54 ppm). decrease In the of case 5.9 ppm of 13C-NMR for C7 and the opposite2.4 ppm effectfor C14 is observed,nuclei. The the change transition of configuration from 7 to 7a leadsfrom 7 toα thein 7 shielding to 7β in 7a decrease is related of 5.9 with ppm no forchange C7 and of the 2.4 ppmshielding for C14 of the nuclei. C8 nucleus The change and decreasing of configuration of shielding from 7α byin 2.37 to ppm 7β in for7a C23is related carbon with of carbomethoxy no change of thesubstituent. shielding ofThe the dehydration C8 nucleus and of decreasingthe epimeric of shielding 11-hydroxyesters by 2.3 ppm for12/ C2312a carbonto the of corresponding carbomethoxy substituent.9(11)-enesters The 7/ dehydration7a caused the of thestrong epimeric 1H shielding 11-hydroxyesters decrease12 of/12a 89.7to ppm/84.7 the corresponding ppm for 9(11)-enesters C9 and 49.8 7ppm/51.9/7a caused ppm the strongfor C111H carbons, shielding respectively. decrease of 89.7The ppm/84.7 transition ppm from for 12 C9/12a and to 49.8 7/7a ppm/51.9 also resulted ppm forin C11medium carbons, shielding respectively. increase The of transition 10.5/10.8 from ppm12 for/12a C12to and7/7a shieldingalso resulted decrease in medium of 8.8/7.3 shielding ppm increasefor C19 ofand 10.5/10.8 3.4/3.4 ppm forfor C12C8, andwhereas shielding for C10 decrease (1.1/1.3 of 8.8/7.3 ppm), ppmC13 for(1.7/1.9 C19 andppm) 3.4/3.4 and ppmC14 (2.2/3.2 for C8, whereasppm) a forweak C10 increasing (1.1/1.3 ppm), effect C13 was (1.7/1.9 observed. ppm) andThe C14competitive (2.2/3.2 ppm) dehydration a weak increasing of 12 to effect the wasregioisomeric observed. The11,12-enester competitive 7b dehydration resulted in ofthe12 strongto the regioisomeric1H shielding 11,12-enesterdecrease of 57.37b resulted ppm for in theC11 strong and 90.41H shieldingppm for decreaseC12 nuclei. of 57.3Similarly ppm for to C11the hydroxyesters and 90.4 ppm for12 C12and nuclei.12a, the Similarly change toof theconfiguration hydroxyesters from12 7andα in12a 7 to, the 7β changein 7a is ofrelated configuration with no from change 7α in of7 theto 7 shieldingβ in 7a is related of the withC8 nucleus no change and of decreasing the shielding of ofshielding the C8 nucleus by 2.3 andppm decreasing for C23 carbon of shielding of the bycarbomethoxy 2.3 ppm for C23substituent. carbon of Additionally, the carbomethoxy the shielding substituent. increase Additionally, of 4.5 ppm the shieldingfor C8 carbon increase was of observed 4.5 ppm forwhen C8 carbonpassing was from observed epimeric when 9(11)-enesters passing from 7 epimericand 7a to 9(11)-enesters regioisomeric7 and11-enester7a to regioisomeric 7b. 11-enester 7b.

(a) (b) (c)

1 13 Figure 4.4. CrucialCrucial 1H- andand 13C-NMR chemicalchemical shiftsshifts andand NOE NOE effects effects for for esters: esters: (a ()a (7) (7α,17α,17α)-9(11)-eneα)-9(11)-ene7; (7b; )(b (7)β (7,17β,17α)-9(11)-eneα)-9(11)-ene7a ;(7ac;) ( (7c) α(7,17α,17α)-11-eneα)-11-ene7b 7b. .

Molecules 2017, 22, 1354 10 of 26 Molecules 2017, 22, 1354 10 of 26 Table 4. The 1H- and 13C-NMR spectral data for esters (7α,17α)-9(11)-ene 7, (7β,17α)-9(11)-ene 7a and (7α,17α)-11-ene 7b. 1 13 Table 4. The H- and C-NMR1 spectralH-NMR (1) data for esters (7α,17α)-9(11)-ene 7, (7β,1713C-NMRα)-9(11)-ene 7a and (7α,17Protonα)-11-ene 7b. 7 7a 7b Carbon 7 7a 7b H-1a 2.18 m 2.12 m 1.87 m C-1 33.7 33.9 34.9 H-1b 2.24 m 1H-NMR (1) 2.12 m 2.15 m C-2 34.213C-NMR 34.2 33.7 H-2a 2.53 m 2.50 m 2.45 m C-3 198.6 198.6 198.7 Proton 7 7a 7b Carbon 7 7a 7b H-2b 2.53 m 2.50 m 2.45 m C-4 125.7 124.6 126.8 H-1aH-4 5.71 2.18 d m(2.0) 5.75 2.12 d m (2.0) 1.87 5.81 m s C-1 C-5 166.6 33.7 165.5 33.9 166.3 34.9 H-1bH-6a 2.242.55 mm 2.43 2.12dd (3.8, m 14.6) 2.15 2.67 m m C-2 C-6 35.734.2 36.2 34.2 35.6 33.7 H-2a 2.53 m 2.50 m 2.45 m C-3 198.6 198.6 198.7 H-2bH-6b 2.82 ddd 2.53 (2.0, m 5.3, 15.0) 2.85 ddd 2.50 (2.0, m 13.2, 14.6) 2.45 2.67 m m C-4 C-7 125.7 43.8 49.7 124.6 42.1 126.8 H-4H-7 5.71 2.98 d (2.0)m 5.75 2.28 d (2.0) m 5.81 2.89 s m C-5 C-8 166.6 40.4 40.4 165.5 35.9 166.3 H-6aH-8 2.552.48 mm 2.43 dd 2.70 (3.8, m 14.6) 2.67 2.24 m m C-6 C-9 142.3 35.7 142.1 36.2 49.2 35.6 H-6bH-9 2.82 ddd (2.0, - 5.3, 15.0) 2.85 ddd (2.0, - 13.2, 14.6) 2.67 2.44 m m C-7 C-10 40.543.8 40.9 49.7 38.8 42.1 H-7H-11 2.985.65 mm 2.28 5.68 m m 5.59 2.89 dd m(2.1, 10.2) C-8 C-11 119.0 40.4 120.4 40.4 126.5 35.9 H-8 2.48 m 2.70 m 2.24 m C-9 142.3 142.1 49.2 H-12a 1.87 m 1.88 m 5.89 dd (2.9, 10.2) C-12 32.9 32.7 133.8 H-9 - - 2.44 m C-10 40.5 40.9 38.8 H-11H-12b 5.652.20 mm 5.68 2.17 m m 5.59 dd (2.1, - 10.2) C-11 C-13 119.0 44.5 44.7 120.4 49.0 126.5 H-12aH-14 1.871.49 mm 1.88 1.50 m m 5.89 dd (2.9,1.95 10.2)m C-12 C-14 43.132.9 45.5 32.7 44.1 133.8 H-12bH-15a 2.201.49 mm 2.17 1.54 m m 1.57- m C-13 C-15 23.344.5 23.8 44.7 21.8 49.0 H-14H-15b 1.491.89 mm 1.50 1.54 m m 1.95 1.87 m m C-14 C-16 35.443.1 35.4 45.5 35.7 44.1 H-15aH-16a 1.491.87 mm 1.54 1.81 m m 1.57 1.95 m m C-15 C-17 95.123.3 94.5 23.8 93.9 21.8 H-15b 1.89 m 1.54 m 1.87 m C-16 35.4 35.4 35.7 H-16b 2.31 m 2.26 m 2.20 m C-18 14.1 14.3 18.6 H-16a 1.87 m 1.81 m 1.95 m C-17 95.1 94.5 93.9 H-16bH-18 2.31 0.95 ms 2.26 0.96 m s 2.20 1.05 m s C-18 C-19 27.214.1 25.7 14.3 17.3 18.6 H-18H-19 0.951.39 ss 0.96 1.42 s s 1.05 1.16 s s C-19 C-20 31.427.2 31.2 25.7 32.3 17.3 H-19H-20a 1.92 1.39 m s 1.42 1.93 s m 1.16 2.00 s m C-20 C-21 29.231.4 29.1 31.2 28.7 32.3 H-20aH-20b 1.922.29 mm 1.93 2.30 m m 2.00 2.21 m m C-21 C-22 176.5 29.2 176.4 29.1 176.7 28.7 H-20bH-21a 2.292.50 mm 2.30 2.51 m m 2.21 2.61 m m C-22 C-23 172.6176.5 174.9 176.4 173.0 176.7 H-21a 2.50 m 2.51 m 2.61 m C-23 172.6 174.9 173.0 H-21bH-21b 2.59 2.59 ddd (6.0, 9.4,9.4, 17.8)17.8) 2.60 2.60 m m 2.53 2.53 m m C-24 C-24 51.4 52.1 52.1 51.6 51.6 H-24H-24 3.59 ss 3.72 3.72 s s 3.66 3.66 s s 1 1 ValuesValues in parentheses givegive δδ,, multiplicitymultiplicity and andJ Jin in Hz. Hz. The NMR structure assignments of the isomeric (7α,17α)-9(11)-enester 7, (7β,17α)-9(11)-enester 7a Theand (7 NMRα,17α structure)-11(12)-enester assignments 7b were of confirmed the isomeric by X-ray (7α,17 analysisα)-9(11)-enester (Figure 5 and7, (7 Tablesβ,17α 5)-9(11)-enester and 6). The 7a and (7SCXRDα,17α)-11(12)-enester structures confirmed7b were the confirmed presence of by the X-ray 9,11-double analysis bond (Figure in 57 andand Tables7a and5 the and 11,12-double6). The SCXRD structuresbond confirmedin 7b. The carbomethoxy the presence ofgroup the 9,11-doubleis situated at bond the C-7 inα7 andposition7a and in 7 the and 11,12-double 7b and at the bond C-7βin 7b. The carbomethoxyposition in 7a. group is situated at the C-7α position in 7 and 7b and at the C-7β position in 7a.

(a) (b)

Figure 5. Molecular structure of the isomeric 9(11)-enesters: (a) (7α,17α)-7 and (b) (7β,17α)-7a. Figure 5. Molecular structure of the isomeric 9(11)-enesters: (a) (7α,17α)-7 and (b) (7β,17α)-7a.

Table 5. Summary of crystallographic data and structure refinement for (7α,17α)-11(12)-enester 7b Tableand 5. Summary (7α,11β,17α of)-9,11-dichloro crystallographic derivative data and13. structure refinement for (7α,17α)-11(12)-enester 7b and (7α,11β,17α)-9,11-dichloro derivative 13. Identification Code 7b 13

Chemical formula C24H30O5 C24H31Cl2O5 Identification Code 7b 13 Molecular weight 398.48 g/mol 470.39 g/mol Chemical formula C24H30O5 C24H31Cl2O5 Temperature 100(2) K 100(2) K Molecular weight 398.48 g/mol 470.39 g/mol Wavelength 0.71073 Å 0.71073 Å Temperature 100(2) K 100(2) K Crystal system, space group Orthorhombic, P212121 Orthorhombic, P212121 Wavelength 0.71073 Å 0.71073 Å a = 6.2322(5) Å a = 9.2383(4) Å UnitCrystal cell dimensions system, space group Orthorhombic, P2 2 2 Orthorhombic, P2 2 2 b = 15.8874(13) Å1 1 1 b = 14.2496(6)1 1 1Å a = 6.2322(5) Å a = 9.2383(4) Å

b = 15.8874(13) Å b = 14.2496(6) Å Unit cell dimensions c = 20.5238(15) Å c = 16.3898(7) Å α = 90◦ α = 90◦ β = 90◦ β = 90◦ γ = 90◦ γ = 90◦ Molecules 2017, 22, 1354 11 of 26

Table 5. Cont.

Identification Code 7b 13 Volume 2032.1(3) Å3 2157.59(16) Å3 Z, Calculated density 4, 1.302 mg/m3 4, 1.448 mg/m3 Absorbtion coefficient 0.090 0.336 F(000) 856 996 Crystal size 0.30 × 0.15 × 0.15 mm 0.30 × 0.10 × 0.10 mm Theta range for data collection 2.36◦–26.37◦ 1.89◦–25.68◦ −7 ≤ h ≤ 7 −11 ≤ h ≤ 11 Limiting indices −19 ≤ k ≤ 19 −17 ≤ k ≤ 17 −25 ≤ l ≤ 25 −19 ≤ l ≤ 19 Reflections collected/unique 35,771/4149 21,917/4092 Absorption correction semi-empirical from equivalents semi-empirical from equivalents Max. and min. transmission 0.9866 and 0.9735 0.9671 and 0.9058 Refinement method full-matrix least-squares on F2 full-matrix least-squares on F2 Data/restraints/parameters 4149/0/265 4092/0/283 Goodness-of-fit on F2 1.017 1.024 Final R1/wR2 indices [I > 2sigma(I)] 0.0319/0.0883 0.0368/0.0698 R1/wR2 indices (all data) 0.0337/0.0903 0.0515/0.0749 Largest diff. peak and hole 0.295 and −0.135 e/Å3 0.231 and −0.244 e/Å3

Table 6. Summary of crystallographic data and structure refinement for the isomeric 9(11)-enesters (7α,17α)-7 and (7β,17α)-7a.

Identification Code 7 7a

Chemical formula C24H30O5 C24H30O5 Molecular weight 398.48 g/mol 398.48 g/mol Temperature 100(2) K 100(2) K Wavelength 0.71073 Å 0.71073 Å

Crystal system, space group Orthorhomblic, P212121 Orthorhomblic, P212121 a = 6.296(2) Å a = 12.099(3) Å b = 15.468(3) Å b = 12.310(3) Å Unit cell dimensions c = 21.088(3) Å c = 13.497(3) Å α = 90◦ α = 90◦ β = 90◦ β = 90◦ γ = 90◦ γ = 90◦ Volume 2053.7(8) Å3 2010.2(8) Å3 Z, Calculated density 4, 1.289 mg/m3 4, 1.317 mg/m3 Absorbtion coefficient 0.089 0.091 F(000) 856 856 Crystal size 0.41 × 0.32 × 0.29 mm 0.60 × 0.39 × 0.31 Theta range for data collection 3.183◦−28.719◦ 3.310◦−36.896◦ −8 ≤ h ≤ 8 −15 ≤ h ≤ 20 Limiting indices −19 ≤ k ≤ 16 −19 ≤ k ≤ 20 −27 ≤ l ≤ 28 −22 ≤ l ≤ 22 Reflections collected/unique 14,340/4925 36,105/9538 Absorption correction None None Max. and min. transmission 0.975 and 0.964 0.972 and 0.947 Refinement method full-matrix least-squares on F2 full-matrix least-squares on F2 Data/restraints/parameters 4925/0/262 9538/0/262 Goodness-of-fit on F2 0.929 0.975 Final R1/wR2 indices [I > 2sigma(I)] 0.0475/0.1011 0.0484/0.1205 R1/wR2 indices (all data) 0.0673/0.1071 0.0622/0.1259 Largest diff. peak and hole 0.189/−0.220 e/Å3 0.453/−0.303 e/Å3 Molecules 2017, 22, 1354 12 of 26

Sulfuryl chloride used for dehydration of 11α-hydroxy steroids is also known as a chlorinating agent and, under some conditions, applied for chlorination of steroid double bonds [27,28]. Indeed, the novel (7α,11β,17α)-9,11-dichloro derivative 13, formed as a result of competitive chlorine addition to the newly formed 9,11-double bond of the key intermediate (7α,17α)-9(11)-enester 7, was isolated chromatographically from the recrystallization mother liquors of 7. It was also synthesized by chlorination of the pure enester 7 with sulfuryl chloride in the presence of pyridine in chlorobenzene, and then purified by column chromatography with 5–30% EtOAc/CH2Cl2 gradient elution to give the pure (7α,11β,17α)-9,11-dichloro derivative 13 (41% yield) as a white solid. The novel 9,11-dichloro impurity 13 was formed by the nucleophilic attack of a chloride anion on the intermediate chloronium cation, a structural analogue of the eplerenone epoxide ring. No other isomers of 13 were obtained. The [M + Na]+ value, m/z 491.1386 obtained for the 9,11-dichloroderivative 13 corresponds to C24H30O5NaCl2. The stereochemistry at the C9 and C11 carbons of the dichloro derivative 13 was established on the basis of 1D/2D NOESY experiments, which indicated the relative trans configuration of the C-9α and C-11β positioned chlorine atoms. The addition of chlorine to the 9,11-double bond of enester 7 caused significant changes in the 1H/13C nuclei shieldings within the steroid rings A, B, C and D of the dichloro derivative 13 (Table7).

1 13 Table 7. The H- and C-NMR spectral data in CDCl3 for (7α,11β,17α)-9,11-dichloro derivative 13 and 7α,9:21,17-dicarbolactone 14.

1H-NMR (1) 13C-NMR Proton 13 14 Carbon 13 14 H-1a 2.27 m 1.88 m C-1 29.6 29.6 H-1b 2.77 m 2.27 m C-2 33.8 33.2 H-2a 2.50 m 2.49 m C-3 197.8 197.1 H-2b 2.50 m 2.49 m C-4 125.7 129.4 H-4 5.89 d (1.8) 5.86 d (2.0) C-5 163.7 161.5 H-6a 2.60 m 2.65 dd (4.0, 15.4) C-6 32.4 34.5 H-6b 2.93 m 2.80 d (15.4) C-7 40.9 45.4 H-7 2.83 m 2.68 m C-8 41.1 45.1 H-8 2.90 m 2.44 d (11.2) C-9 86.6 90.0 H-9 - - C-10 47.1 43.7 H-11a 4.68 dd (2.0, 4.8) 1.84 m C-11 59.5 22.4 H-11b - 2.05 ddd (2.0, 4.7, 15.2) C-12 37.0 26.9 H-12a 1.98 m 1.56 m C-13 45.0 44.0 H-12b 2.54 m 1.61 m C-14 41.3 42.7 H-14 2.46 m 1.33 m C-15 23.6 23.4 H-15a 1.57 m 1.49 m C-16 35.3 35.2 H-15b 2.32 m 1.84 m C-17 95.9 94.8 H-16a 1.97 m 1.92 m C-18 17.2 13.4 H-16b 2.33 m 2.31 m C-19 26.1 19.1 H-18 1.30 s 0.99 s C-20 31.5 31.0 H-19 1.86 s 1.40 s C-21 29.1 29.0 H-20a 2.06 m 1.91 m C-22 176.3 176.2 H-20b 2.39 m 2.35 ddd (3.6, 5.6, 13.0) C-23 172.3 176.5 H-21a 2.48 m 2.60 ddd (6.7, 9.2, 17.7) C-24 52.2 - H-21b 2.60 m 2.51 m H-24 3.61 s - 1 Values in parentheses give δ, multiplicity and J in Hz.

The strong shielding increase of 55.7 ppm for C9 and 59.5 ppm for C11 nuclei was observed when passing from 7 to 13. Minor shielding increase of 4.1 ppm for C1, 2.9 ppm for C5, 3.3 ppm for C6, 2.9 ppm for C7 and 1.8 ppm for C14 and shielding decrease of 6.6 ppm for C10 nuclei were also noted. The introduction of chlorine into C9 and C11 positions of 7 also resulted in shielding increase of 0.97 ppm for H11 and shielding decrease of 0.18 ppm for H4, 0.42 ppm for H8 and 0.97 for H14 Molecules 2017, 22, 1354 13 of 26 Molecules 2017, 22, 1354 13 of 26

Molecules 2017, 22, 1354 13 of 26 nucleinuclei in dichlorodichloro derivativederivative 13.. Noteworthy, thethe diasterotopicdiasterotopic effecteffect observed for H15 protons of 7 (0.4(0.4nuclei ppm)ppm) in increasedincreased dichloro derivative toto 0.750.75 ppmppm 13. forforNoteworthy, thethe samesame protonstheprotons diasterotopic inin 1313.. SimilarlySimilarly effect observed toto thethe otherother for H15 compoundscompounds protons of withwith 7 77α(0.4-carbomethoxy-carbomethoxy ppm) increased group, to 0.75 the ppm β positioned for the same H7 protonsproton inin dichlorodichloro13. Similarly derivativederivative to the other 13 isis compounds involvedinvolved inin with strongstrong H7-H8H7-H87α-carbomethoxy NOENOE effecteffect (Figure(Figure group,6 6).the). TheThe β positioned NMRNMR structurestructure H7 proton assignment assignment in dichloro of of the the derivative novel novel (7 (7 13αα,11,11 is βinvolvedβ,17α)-9,11-dichloro in strong derivativederivativeH7-H8 NOE 1313was effectwas confirmed confirmed(Figure 6). by The X-rayby NMRX-ray analysis structure analysis (Figure assignment (Fig7 andure Table7 of and the5). novelTable The SCXRD (7 5).α,11 Theβ structure,17 αSCXRD)-9,11-dichloro confirmed structure theconfirmedderivative presence the of13 presence thewas two confirmed of chlorine the two by atoms chlorine X-ray at theanalysis atoms C-9 α at(Figand theure C-9 C-11 7α β andandpositions C-11Tableβ 5).positions of theThe steroidSCXRD of the ring steroidstructure C in ring the relativeC inconfirmed thetrans relative theconfiguration presencetrans configuration of andthe two the chlorine C-7 andα thepositioned atoms C-7α positionedat carbomethoxythe C-9α carbomethoxyand C-11 substituent.β positions substituent. of the steroid ring C in the relative trans configuration and the C-7α positioned carbomethoxy substituent.

(a(a) ) (b(b) )

1 13 FigureFigureFigure 6.6. 6.Crucial Crucial 1H- 1H- and and 13 13C-NMRC-NMRC-NMR chemicalchemical shifts and NOE NOE effects effectseffects for: for:for: ( a((a)a )(7) (7(7αα,11α,11,11β,17ββ,17,17α)-9,11-dichloroαα)-9,11-dichloro)-9,11-dichloro derivativederivativederivative 13 13;(; (;b ()b 7) α7α,9:21,17-dicarbolactone,9:21,17-dicarbolactone 1414..

(a) (b) (a) (b) Figure 7. Molecular structure of (a) (7α,17α)-11-enester 7b and (b) (7α,11β,17α)-9,11-dichloro FigureFigure 7.7. MolecularMolecular structure structure of (a) (7(7α,17,17α)-11-enester 7b andand ((bb)) (7(7αα,11,11ββ,17,17αα)-9,11-dichloro)-9,11-dichloro derivative 13. derivativederivative 1313.. The acidic conditions applied for the dehydration of the 11α-hydroxy group in 12 resulted in competitiveTheThe acidic lactonizationconditions applied appliedbetween for for the the dehydration dehydrationhydroxyl and of of thecarbomethoxy the 11 11α-hydroxyα-hydroxy groups group group inand in12 12resultedleadresulted to in incompetitive7 competitiveα,9:21,17-dicarbolactone lactonization lactonization 14between (Imp. between A), whichthe the hydrox tends hydroxyl toyl be and formed and carbomethoxy carbomethoxy even in the presence groups groups of tracesand and of lead free toto 77αwater.,9:21,17-dicarbolactone,9:21,17-dicarbolactone The dicarbolactone 1414 14(Imp.(Imp. was A), A),isolated which which chromatographically tends tends to tobe be formed formed even from even in the the in recrystallization thepresence presence of traces of mother traces of free of freewater.liquors water. The of Thedicarbolactone 7 dicarbolactone. It was also 14 14wassynthesizedwas isolated isolated chromatographicallyindependently chromatographically from fromthe from mesylatethe the recrystallization recrystallization of the starting mother liquorsliquors11α-hydroxyester ofof 7. It7. was It also was12 by synthesized reactionalso synthesized with independently acetic acidindependently in from the pres theence mesylate from of sodium the of the mesylateacetate, starting and 11of αthen -hydroxyesterthe purified starting 1211αbyby-hydroxyester column reaction chromatography with 12 aceticby reaction acid with inwith 5–30% the acetic presence ethyl acid acetate/ in of the sodiumdichloromethane presence acetate, of sodium andgradient thenacetate, elution purified and to then afford by purified column the chromatographybypure column 7α,9:21,17-dicarbolactone chromatography with 5–30% with ethyl 14 5–30% (83% acetate/dichloromethane yield) ethyl asacetate/ a whitedichloromethane solid. gradient gradient elution elution to afford to afford the pure the 7pureα,9:21,17-dicarbolactone 7αThe,9:21,17-dicarbolactone [M + Na]+ value,14 (83% m14/z yield)(83% 407.1847, yield) as a white obtainedas a white solid. for solid. the dicarbolactone 14 corresponds to C23H28O5Na. The+ 1H-/+ 13C-NMR chemical shifts assignment for dilactone 14 is presented in Table 7. TheThe [M[M + Na]+ Na]value, value,m/z 407.1847,m/z 407.1847, obtained obtained for the dicarbolactonefor the dicarbolactone14 corresponds 14 corresponds to C23H28O5 Na.to 1 13 TheC23TheH128H-/ Odetailed5Na.13C-NMR The analysis 1H-/ chemical13C-NMR of 2D shifts NMRchemical assignment spectra, shifts especially forassignment dilactone H- for14C isdilactoneHMBC presented and 14 inisNOESY Tablepresented7 .experiments, The in detailed Table 7. analysisTheunambiguously detailed of 2D analysis NMR confirmed spectra, of 2D the especiallyNMR structure spectra, consisting1H-13 especiallyC HMBC of the andtwo1H-13 γ NOESYC-lactone HMBC experiments,rings. and TheNOESY second unambiguously experiments, γ-lactone unambiguouslymoiety results confirmedfrom internal the lactonizationstructure consisting between of 9 theα-hydroxy two γ-lactone and 7α rings.-carbomethoxy The second groups γ-lactone of moiety results from internal lactonization between 9α-hydroxy and 7α-carbomethoxy groups of

Molecules 2017, 22, 1354 14 of 26 confirmed the structure consisting of the two γ-lactone rings. The second γ-lactone moiety results from internal lactonization between 9α-hydroxy and 7α-carbomethoxy groups of intermediate ester, formed by water addition to the 9,11-double bond of enester 7 during the dehydration step of 12. This significant change in the steroid structure entails numerous changes in the 1H/13C nuclei shielding. The shielding increase of 46.8 ppm for C11 and 16.5 ppm for C12 and decrease of 37.4 ppm for C9 nuclei were observed when passing from hydroxyester 12 to the dicarbolactone 14. Minor shielding increase of 7.7 ppm for C1 and 6.2 ppm for C5 was also noted, whereas for C8 shielding decrease of 8.1 ppm was observed. Similarly to the other compounds of the series with 7α positioned carbomethoxy group, the H7 proton of the dicarbolactone 14 is involved in two strong H7-H8 and H6-H7 NOE effects (Figure6). The purification of the key intermediate (7α,17α)-9(11)-enester 7 by large-scale recrystallization only partially removes the impurities formed during the dehydration of the starting 11α-hydroxyester 12. Thus, in a preferred technological embodiment, the key intermediate enester 7 was isolated in a crude form by dichloromethane extraction followed by evaporation of the solvent, and used directly in the following step of the process, which was the epoxidation of the 9,11-double bond to produce the eplerenone. Thus, the crude enester 7 was dissolved in dichloromethane and epoxidized with hydrogen peroxide in the presence of disodium hydrogen phosphate and trichloroacetamide. The crude product was recrystallized from ethanol and 2-butanone to give the pharmaceutical grade eplerenone. Apart from the (7α,11β,17α)-9,11-dichloro derivative 13 and the 7α,9:21,17-dicarbolactone 14 (Imp. A), which were present in the crude enester 7, the epoxidation step produced the two further impurities, i.e., the isomeric (7β,11α,17α)-9,11-epoxyester 2a and (7α,11α,12α,17α)-11,12-epoxyester 2b, which were isolated chromatographically from the recrystallization mother liquors of 2. The trichloroacetamide is known to preferentially epoxidize the more highly substituted double bond, e.g., the 9,11-olefin. In the case of the crude 9,11-enester 7 contaminated with the 11,12-enester 7b, the 9,11-epoxide should be formed with the minimal competitive epoxidation of the 7b isomer yielding the 11,12-epoxide as the by-product. As expected, the chromatography of the recrystallization mother liquors of 2 with 1–10% acetone/dichloromethane gradient elution gave the (7α,11α,12α,17α)-11,12-epoxyester 2b, which was undoubtedly formed via epoxidation of the (7α,17α)-9(11)-enester 7b. It was also synthesized independently by epoxidation of the enester 7b with hydrogen peroxide in the presence of disodium hydrogen phosphate and trichloroacetamide, and purified by column chromatography to afford the pure 11,12-epoxyester 2b (60% yield) as a white solid. Further gradient elution of the recrystallization mother liquors of 2 with 1–10% acetone/dichloromethane gave the isomeric (7β,11α,17α)-9,11-epoxyester 2a, which was formed by epoxidation of the (7β,17α)-9(11)-enester 7a contaminating the crude intermediate enester 7. It was also synthesized independently by epoxidation of the enester 7a with hydrogen peroxide in the presence of disodium hydrogen phosphate and trichloroacetamide, or basic epimerization of eplerenone (2), and then purified by column chromatography to give the pure (7β,11α,17α)-9,11-epoxyester 2a, with 69% or 49% yield respectively, as a white solid. The recrystallization of the crude eplerenone (2) from ethanol removes the new 9,11-dichloro derivative 13, the isomeric (7β,11α,17α)-9,11-epoxyester 2a and the residuals of the unreacted isomeric (7β,17α)-9(11)-enester 7a and (7α,17α)-9(11)-enester 7b. The recrystallization from 2-butanone removes the 7α,9:21,17-dicarbolactone 14 (Imp. A) and affords the pharmaceutical grade eplerenone (2). The NMR data of eplerenone were presented in literature sources many times; however the full and correct 1H/13C chemical shifts assignment confirming its structure has never been done. Although the complete assignment of 1H-/13C-NMR signals was presented by Grob et al. [17], some of the signals were assigned reversibly. The careful analysis of 2D NMR experiments, including HSQC, HMBC and NOESY measurements, allowed to assign signals to the corresponding protons and carbons of the isomeric epoxy esters 2, 2a and 2b unambiguously, and thus confirm the structure of eplerenone and its isomers (Table8). Blue arrows in Figure8 show the most important NOE effects involving H7 proton, simultaneously indicating the 7α or 7β positioned carbomethoxy group. The strong H7-H8 NOE effect is observed for the compounds 2 and 2b with β positioned H7 proton, whereas α situated H7 proton in 2a is involved in the strong interaction with H14. The epoxidation of the 9,11-double bond caused several shielding Molecules 2017, 22, 1354 15 of 26 effects observed for 1H/13C nuclei within the steroid rings A, B and C. The strong shielding increase of 77.1/75 ppm for C9, 67.6/66.8 ppm for C11 carbons and 2.5/2.4 ppm for H11 protons was observed when passing from enesters 7/7a to epoxides 2/2a, respectively. Minor shielding increase of 7/6.5 ppm for C1, 5.9/6.1 ppm for C14, 2.6/3.4 for C7, 1.8/1.6 ppm for C8, 2/2 ppm for C12 and 0.7/0.4 for C13 carbons was also noted. The transition from enesters 7/7a to epoxides 2/2a caused shielding increase of 0.76/0.66 ppm for one of the H1 protons and 0.36/0.31 ppm for one of the H12 protons, whereas for H-14 protons shielding decrease of 0.4/0.14 ppm was noted. Similarly to the epimeric hydroxyesters 12/12a and enesters 7/7a, some of the 1H/13C nuclei shielding within the steroid rings B, C and D are related to the α or β configuration of the C7 atom. The 1H shielding increase of 0.22 ppm for H7 and 0.25 ppm for H14 nuclei is observed when passing from the compound 2 with 7α positioned carbomethoxy group to its 7β epimer 2a. Additionally, the change of configuration at the C7 is accompanied by weak shielding decrease of 0.1 ppm for H8. Simultaneously, both diasterotopic H15 protons of the epimer 2a with 7β positioned carbomethoxy group became equal having the same proton chemical shift (1.51 ppm). In the case of 13C-NMR data the opposite effect is observed, the transition from 2 to 2a leads to the shielding decrease of 5.1 ppm for C7, 1.9 ppm for C9 and 2.2 ppm for C14 nuclei. The change of configuration from 7α in 2 to 7β in 2a is also related with shielding decrease of 2.1 ppm for C23 carbon of the carbomethoxy substituent. The competitive epoxidation of the 11,12-enester 7b to the 11,12-epoxyester 2b resulted in the strong shielding increase of 76.1 ppm for C11 and 77.1 ppm for C12 nuclei, whereas for C9 (3.3 ppm), C18 (4.4 ppm) and C14 (6.7 ppm) medium effects were observed. The strong shielding increase of 2.57 and 2.84 ppm was noted for H11 and H12 protons, respectively. Minor changes in increasing of shielding for H7 (0.1 ppm), H8 (0.24 ppm) and H9 (0.4 ppm) protons were also observed. Similarly to the enesters 7, 7a and 7b, the shielding increase of 3.6 ppm and 3.8 ppm for C8 carbon was noted when passing from epimeric 9,11-epoxides 2 and 2a to regioisomeric 11,12-epoxyester 2b.

Table 8. 1H- and 13C-NMR spectral data for (7α,11α,17α)-9,11-epoxyester 2, (7β,11α,17α)-9,11-epoxyester 2a and (7α,11α,12α,17α)-11,12-epoxyester 2b.

1H-NMR 1 13C-NMR Proton 2 2a 2b Carbon 2 2a 2b H-1a 1.42 m 1.46 ddd (2.8, 5.2, 13.2) 2.02 m C-1 26.7 27.4 34.7 H-1b 2.19 m 2.07 m 2.30 ddd (2.6, 5.0, 13.4) C-2 33.0 33.2 33.4 H-2a 2.43 m 2.39 m 2.47 dddd (0.8, 2.6, 5.0, 17.5) C-3 197.9 197.9 198.2 H-2b 2.47 m 2.44 m 2.52 ddd (5.0, 14.3, 17.5) C-4 127.0 126.8 126.6 H-4 5.89 s 5.83 d (1.8) 5.81 s C-5 165.0 163.1 165.3 H-6a 2.72 m 2.90 m 2.64 m C-6 34.8 36.2 35.5 H-6b 2.72 m 2.89 ddd (2.2, 12.7, 15.3) 2.64 m C-7 41.2 46.3 41.7 H-7 2.89 m 2.67 ddd (3.9, 11.2, 12.6) 2.79 m C-8 38.6 38.8 35.0 H-8 2.49 dd (4.7, 11.2) 2.59 m 2.00 m C-9 65.2 67.1 45.9 H-9 - - 2.04 m C-10 39.7 40.0 38.2 H-11 3.12 d (5.4) 3.28 d (5.4) 3.02 d (4.0) C-11 51.4 53.6 50.4 H-12a 1.70 dd (5.4, 14.6) 1.72 dd (5.4, 14.6) 3.05 d (4.0) C-12 30.9 30.7 56.7 H-12b 1.84 d (14.6) 1.86 d (14.6) - C-13 43.8 44.3 47.2 H-14 1.89 m 1.64 m 1.92 m C-14 37.2 39.4 37.4 H-15a 1.51 m 1.51 m 1.51 m C-15 22.0 22.4 21.2 H-15b 2.03 m 1.51 m 1.90 m C-16 34.9 35.1 34.5 H-16a 1.88 m 1.76 m 1.90 m C-17 94.5 93.9 94.0 H-16b 2.21 m 2.15 m 2.18 m C-18 16.1 16.5 14.2 H-18 1.02 s 1.02 s 1.09 s C-19 22.2 21.0 17.9 H-19 1.50 s 1.54 s 1.30 s C-20 30.8 30.9 31.9 H-20a 1.94 ddd (7.0, 9.6, 13.2) 1.89 m 2.00 m C-21 28.8 29.0 28.8 H-20b 2.31 ddd (6.5, 9.5, 13.2) 2.28 ddd (6.7, 9.5, 13.2) 2.73 ddd (7.5, 8.9, 12.9) C-22 176.1 176.1 176.7 H-21a 2.50 ddd (6.9, 9.4, 17.9) 2.50 m 2.60 m C-23 172.5 174.6 172.5 H-21b 2.60 ddd (6.7, 9.6, 17.9) 2.60 m 2.60 m C-24 51.5 52.2 51.7 H-24 3.65 s 3.71 s 3.66 s 1 Values in parentheses give δ, multiplicity and J in Hz. Molecules 2017, 22, 1354 16 of 26 Molecules 2017, 22, 1354 16 of 26

Molecules 2017, 22, 1354 16 of 26

(a) (b) (c)

1 13 Figure 8. Crucial 1H- and 13C-NMR chemical shifts and NOE effects for: (a) (7α,11α,17α)-9,11-epoxyester Figure 8. Crucial (aH-) and C-NMR chemical shifts and(b) NOE effects for: (a) (7α,11α,17(αc))-9,11-epoxyester 2; (b) (7β,11α,17α)-9,11-epoxyester 2a and (c) (7α,11α,12α,17α)-11,12-epoxyester 2b. 2;(b) (7β,11α,17α)-9,11-epoxyester 2a and (c) (7α,11α,12α,17α)-11,12-epoxyester 2b. Figure 8. Crucial 1H- and 13C-NMR chemical shifts and NOE effects for: (a) (7α,11α,17α)-9,11-epoxyester The [M2; (b )+ (7 Na]β,11α+ ,17values,α)-9,11-epoxyester m/z 437.1941 2a and and (c) (7 α437.1942,,11α,12α,17 obtainedα)-11,12-epoxyester for the 2b two. isomeric epoxyesters 2 The [M + Na]+ values, m/z 437.1941 and 437.1942, obtained for the two isomeric epoxyesters and 2b correspond to C24H30O6Na. The [M + H]+ value, m/z 415.2113, obtained for the third isomeric 2 and 2bThecorrespond [M + Na]+ tovalues, C H m/zO 437.1941Na. The and [M 437.1942, + H]+ obtainedvalue, m for/z the415.2113, two isomeric obtained epoxyesters for the 2 third epoxyester 2a corresponds24 30to 6C24H31O6. The NMR structure assignments of the isomeric and 2b correspond to C24H30O6Na. The [M + H]+ value, m/z 415.2113, obtained for the third isomeric isomeric epoxyester 2a corresponds to C24H31O6. The NMR structure assignments of the isomeric (7β,11epoxyesterα,17α)-9,11-epoxyester 2a corresponds 2a andto C (724αH,1131Oα6. ,12Theα,17 NMRα)-11,12-epoxyester structure assignments 2b were ofconfirmed the isomeric by X-ray (7β,11α,17α)-9,11-epoxyester 2a and (7α,11α,12α,17α)-11,12-epoxyester 2b were confirmed by X-ray analysis(7β,11 (Figureα,17α)-9,11-epoxyester 9 and Table 9). The2a and SCXRD (7α,11 αst,12ructuresα,17α)-11,12-epoxyester confirmed the presence 2b were confirmed of the 9α ,11by αX-ray-epoxide analysis (Figure9 and Table9). The SCXRD structures confirmed the presence of the 9 α,11α-epoxide ring inanalysis 2a and (Figure 11α,12 9 αand-epoxide Table 9). ring The in SCXRD 2b. The structures carbomethoxy confirmed group the presence is situated of the at 9 αthe,11 αC-7-epoxideα position α α α inring 2b inring and2a in andat 2a the 11and C-7 ,1211βα position,12-epoxideα-epoxide in ring 2a ring. in in2b 2b. The. The carbomethoxy carbomethoxy groupgroup is is situated situated at at the the C-7 C-7α positionposition in 2b andin 2b at theand C-7at theβ positionC-7β position in 2a in. 2a.

(a) (a) (b)( b) Figure 9. Molecular structure of the isomeric epoxides: (a) (7β,11α,17α)-9,11-epoxy-2a and (b) Figure 9. Molecular structure of the isomeric epoxides: (a) (7β,11αα,17αα)-9,11-epoxy-2a and (b) Figure(7α 9.,11αMolecular,12α,17α)-11,12-epoxy- structure2b of. the isomeric epoxides: (a) (7 ,11 ,17 )-9,11-epoxy-2a and (b) (7α,11α,12α,17α)-11,12-epoxy-2b. Table 9. Summary of crystallographic data and structure refinement for the isomeric Table(7 9.β9.,11 SummaryαSummary,17α)-9,11-epoxyester ofof crystallographic crystallographic 2a and (7α,11 dataα ,12data andα,17 αand structure)-11,12-epoxyester structure refinement refinement2b. for the for isomeric the isomeric (7β,11α, (7βα,11α,17α)-9,11-epoxyester 2aα andα (7αα,11αα,12α,17α)-11,12-epoxyester 2b. 17 )-9,11-epoxyesterIdentification2a Codeand (7 ,11 ,12 ,17 )-11,12-epoxyester 2a2b. 2b IdentificationChemical formulaCode C24H 2a30O6 C24H30O 2b6 IdentificationMolecular weight Code 414.48 g/mol 2a 414.48 g/mol 2b Chemical formula C24H30O6 C24H30O6 MolecularChemicalTemperature weight formula 414.48 100(2) Cg/mol24 KH 30O 6 100(2) 414.48C K24 g/molH30O6 Wavelength 0.71073 Å 0.71073 Å TemperatureMolecular weight 100(2) 414.48 K g/mol 100(2)414.48 g/molK Crystal system, space group Monoclininc, P21 Orthorhomblic, P212121 WavelengthTemperature 0.71073 100(2) Å K 0.71073 100(2) Å K a = 6.0294(5) Å a = 6.377(2) Å Crystal system,Wavelength space group Monoclininc,b = 10.5733(8)0.71073 P2Å Å1 Orthorhomblic, b = 15.477(3) 0.71073 Å P Å212121 Crystal system, space groupca == 16.3548(12) 6.0294(5) Monoclininc, ÅÅ P2 c Orthorhomblic,= 20.886(4) a = 6.377(2) Å ÅP 2 2 2 Unit cell dimensions 1 1 1 1 b = 10.5733(8)α = 90° Å bα = = 15.477(3) 90° Å a = 6.0294(5) Å a = 6.377(2) Å c =β 16.3548(12) = 99.604(2)° Å cβ = = 20.886(4) 90° Å Unit cell dimensions b = 10.5733(8) Å b = 15.477(3) Å αγ == 90° γ = α90° = 90° Unit cell dimensions 3 3 Volume β1028.02(14) = c99.604(2)° = 16.3548(12) Å Å2061.4(9) cβ = = 20.886(4) Å90° Å 3◦ 3 ◦ Z, Calculated density 2, 1.339γ = 90°mg/mα = 90 4, 1.336 γmg/m =α 90°= 90 Absorbtion coefficient 0.095 0.095 Volume 1028.02(14)β = 99.604(2) Å3 ◦ 2061.4(9)β = 90 Å◦3 F(000) 444 888 Z, Calculated density 2, 1.339 mg/mγ ◦3 4, 1.336γ mg/m◦ 3 Crystal size 0.30 × 0.20 × 0.02= 90 mm 0.60 × 0.27 × 0.12= 90mm Absorbtion coefficient 0.095 0.095

F(000) 444 888 Crystal size 0.30 × 0.20 × 0.02 mm 0.60 × 0.27 × 0.12 mm

Molecules 2017, 22, 1354 17 of 26

Table 9. Cont.

Identification Code 2a 2b Volume 1028.02(14) Å3 2061.4(9) Å3 Z, Calculated density 2, 1.339 mg/m3 4, 1.336 mg/m3 Absorbtion coefficient 0.095 0.095 F(000) 444 888 Crystal size 0.30 × 0.20 × 0.02 mm 0.60 × 0.27 × 0.12 mm Theta range for data collection 2.30◦–27.48◦ 3.21◦–25.23◦ −7 ≤ h ≤ 7 −7 ≤ h ≤ 7 Limiting indices −13 ≤ k ≤ 13 −18 ≤ k ≤ 18 −21 ≤ l ≤ 21 −25 ≤ l ≤ 24 Reflections collected/unique 36785/4712 9774/3635 Absorption correction semi-empirical from equivalents None Max. and min. transmission 0.9981 and 0.9720 0.989 and 0.945 Refinement method full-matrix least-squares on F2 full-matrix least-squares on all F2 Data/restraints/parameters 4712/1/274 3635/0/271 Goodness-of-fit on F2 1.093 1.057 Final R1/wR2 indices [I > 2sigma(I)] 0.0391/0.1014 0.0371/0.0942 R1/wR2 indices (all data) 0.0408/0.1024 0.0427/0.0964 Largest diff. peak and hole 0.261 and −0.179 e/Å3 0.220 and −0.192 e/Å3

3. Experimental Section

3.1. General Information 11α-Hydroxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (12, 98%) was manufactured by Hangzhou Pharma Chemicals Ltd. (Hangzhou, China) according to synthetic procedure described by Ng et al. [9] (Scheme2). Sulfuryl chloride (97%), imidazole (99%), 2,2,2-trichloroacetamide (99%), disodium hydrogen phosphate (≥99%), hydrogen peroxide (35% in H2O) were purchased from Sigma-Aldrich (Munich, Germany) and CHEMPUR (Piekary Sl´ ˛askie, Poland) chemical companies. Deionized water was prepared using a MilliQ plus purification system (Millipore, Bradford, PA, USA). Potassium bromide (FT-IR grade) and deuterated chloroform were purchased from Merck KGaA (Darmstad, Germany). The course of all reactions and the purity of products were checked by thin-layer chromatography (TLC). Analytical TLC was performed on silica gel DC-Alufolien Kieselgel 60 F254 (Merck KGaA), with mixtures of toluene, ethyl acetate, acetone, dichloromethane and acetonitrile, in various ratios as developing systems. Compounds were detected by spraying the ◦ plates with 1% Ce(SO4)2/2%H3[P(Mo3O10)4] in 10% H2SO4 followed by heating to 120 C. Column chromatography was carried out on silica gel (Kieselgel 60, 40–63 µm, 230–400 mesh, Merck) with mixtures of ethyl acetate, toluene, acetone, acetonitrile and dichloromethane in varying ratios as eluents.

3.2. Optical Rotation Optical rotations were measured with a Perkin Elmer 341 automatic polarimeter (Perkin Elmer, Norwalk, CT, USA) in CH2Cl2 solutions as the solvents with percent concentrations.

3.3. Melting Point Melting points were determined with a MEL-TEMP II capillary melting point apparatus (Laboratory Devices, Holliston, MA, USA). Molecules 2017, 22, 1354 18 of 26

3.4. FT-IR Spectroscopy FT-IR spectra were taken for KBr pellets on a Nicolet Impact 410 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

3.5. NMR Spectroscopy

The NMR spectra of all the compounds were measured in CDCl3 solutions with a Varian VNMRS-600 (600 MHz for 1H-NMR and 150 MHz for 13C-NMR; Varian Inc., Palo Alto, CA, USA) at temperature 298 K using TMS as internal standard. All 1H-/13C-NMR resonance signals were assigned using results of 2D experiments [g-COSY (1H-1H), g-HSQC (1H-13C) and g-HMBC (1H-13C)] in gradient versions. The 1H-NMR chemical shifts were determined as centres of the correlation spots in the 1H domain of the 2D 1H-13C HSQC experiments. Relative configuration and stereochemistry at the C-7 and other atoms was established on the basis of 1D and 2D NOESY spectra. Concentration of all solutions used for measurements was about 20–30 mg of compounds in 0.6 mL of solvent.

3.6. Mass Spectrometry HRMS spectra were recorded on an AMD 604 Inectra GmbH (AMD Inectra GmbH, Harpstedt, Germany) and a Mariner PE Biosystem ESI-TOF (PerSeptive Biosystems/Applied Biosystems, Waltham, MA, USA) spectrometers.

3.7. X-ray Analysis The X-ray diffraction data for 2b, 7, 7b, 12, 12a and 13 were collected using a KM4CCD-axis diffractometer (Kuma Diffraction, Wrocław, Poland) with graphite-monochromated MoK radiation and equipped with an nitrogen gas-flow apparatus (Oxford Cryosystems, Oxford, UK). The data were corrected for Lorentz and polarization effects. The multi-scan absorption correction was applied for 12, 12a and 13. Data reduction and analysis were carried out with the Oxford Diffraction Ltd. (Wrocław, Poland) suit of programs [29,30]. The structures were solved by direct methods approach using the SHELXS97 [31] program and refined with the SHELXL97 [32]. The X-ray diffraction data for 2a and 7b were collected using the Kappa APEX II Ultra (Bruker, Billerica, MA, USA) controlled by APEX II software [33], equipped with MoKα rotating anode X-ray source (λ = 0.71073 Å, 50.0 kV, 22.0 mA) monochromatized by multi-layer optics and APEX-II CCD detector. The experiments were carried out at 100 K using the Oxford Cryostream cooling device. Indexing, integration and initial scaling were performed with SAINT [34] and SADABS [35] software. The structures were solved by direct methods approach using the SHELXS97 [31] program and refined with the SHELXL97 [32]. Multi-scan absorption correction has been applied in the scaling procedure. Crystal data and refinement details for 2b, 7, 7a–b, 12, 12a and 13 are listed in Tables3,5,6 and9. The deposition numbers CCDC 1502629 (2a), CCDC 1565292 (2b), CCDC 1565294 (7), CCDC 1565293 (7a), CCDC 1502628 (7b), CCDC 1502627 (12), CCDC 1502631 (12a) and CCDC 1502630 (13) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from via http://www.ccdc.cam.ac.uk/conts/ retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336-033; E-mail: [email protected]).

3.8. Syntheses

3.8.1. 11α-Hydroxy-7β-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (12a, Scheme3) 11-Hydroxyester (7α,11α,17α)-12 (5.0 g, 12.0 mmol) was added to a suspension of MeONa (1.30 g, 24.0 mmol) in anhydrous MeOH (25 mL) and then refluxed for 24 h. The mixture was cooled to room temperature and 3 M aqueous HCl solution (150 mL) was added. The product was extracted with CH2Cl2 (3 × 25 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a mixture of epimers 12 and 12a as a yellowish Molecules 2017, 22, 1354 18 of 26

in gradient versions. The 1H-NMR chemical shifts were determined as centres of the correlation spots in the 1H domain of the 2D 1H-13C HSQC experiments. Relative configuration and stereochemistry at the C-7 and other atoms was established on the basis of 1D and 2D NOESY spectra. Concentration of all solutions used for measurements was about 20–30 mg of compounds in 0.6 mL of solvent.

3.6. Mass Spectrometry HRMS spectra were recorded on an AMD 604 Inectra GmbH (AMD Inectra GmbH, Harpstedt, Germany) and a Mariner PE Biosystem ESI-TOF (PerSeptive Biosystems/Applied Biosystems, Waltham, MA, USA) spectrometers.

3.7. X-ray Analysis The X-ray diffraction data for 2b, 7, 7b, 12, 12a and 13 were collected using a KM4CCD-axis diffractometer (Kuma Diffraction, Wrocław, Poland) with graphite-monochromated MoK radiation and equipped with an nitrogen gas-flow apparatus (Oxford Cryosystems, Oxford, UK). The data were corrected for Lorentz and polarization effects. The multi-scan absorption correction was applied for 12, 12a and 13. Data reduction and analysis were carried out with the Oxford Diffraction Ltd. (Wrocław, Poland) suit of programs [29,30]. The structures were solved by direct methods approach using the SHELXS97 [31] program and refined with the SHELXL97 [32]. The X-ray diffraction data for 2a and 7b were collected using the Kappa APEX II Ultra (Bruker, Billerica, MA, USA) controlled by APEX II software [33], equipped with MoKα rotating anode X-ray source (λ = 0.71073 Å, 50.0 kV, 22.0 mA) monochromatized by multi-layer optics and APEX-II CCD detector. The experiments were carried out at 100 K using the Oxford Cryostream cooling device. Indexing, integration and initial scaling were performed with SAINT [34] and SADABS [35] software. The structures were solved by direct methods approach using the SHELXS97 [31] program and refined with the SHELXL97 [32]. Multi-scan absorption correction has been applied in the scaling procedure. Crystal data and refinement details for 2b, 7, 7a–b, 12, 12a and 13 are listed in Tables 3, 5, 6 and 9. The deposition numbers CCDC 1502629 (2a), CCDC 1565292 (2b), CCDC 1565294 (7), CCDC 1565293 (7a), CCDC 1502628 (7b), CCDC 1502627 (12), CCDC 1502631 (12a) and CCDC 1502630 (13) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336-033; E-mail: [email protected]).

3.8. Syntheses

3.8.1. 11α-Hydroxy-7β-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (12a, Scheme 3) 11-Hydroxyester (7α,11α,17α)-12 (5.0 g, 12.0 mmol) was added to a suspension of MeONa (1.30 g, 24.0 mmol) in anhydrous MeOH (25 mL) and then refluxed for 24 h. The mixture was cooled to room Moleculestemperature2017, 22 and, 1354 3 M aqueous HCl solution (150 mL) was added. The product was extracted19 with of 26 CH2Cl2 (3 × 25 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a mixture of epimers 12 and 12a as a yellowish white whitefoam. foam.The crude The crude product product was waspurified purified by bycolumn column chromatography chromatography over over silica silica gel gel with with 10–50% MeCN/PhCH33 gradient elutionelution to to give give (7 β(7,11β,11α,17α,17α)-α12a)-12a(2.45 (2.45 g, 49% g, 49% yield) yield) as a white as a solid,white a mixturesolid, a ofmixture12 and of12a 12 (0.56and 12a g) and (0.56 the g) (7andα,11 theα,17 (7α)-,1112α(1.27,17α)- g).12 (1.27 g).

Molecules 2017, 22, 1354 19 of 26 Scheme 3. Synthesis of the 11-hydroxyester (7(7ββ,11α,17α)-12a. (7β,11α,17α)-12a: Rf = 0.40 for 60% MeCN/PhCH3. M.p. 250–252 °C. α = +21.49 (c 1.0, − CHCl3). FT-IR (pellets, KBr) ν (cm 1): 3511, 2978, 2940, 2872, 2832, 1775,◦ 1736,20 1652, 1608, 1482, 1447, (7β,11α,17α)-12a: Rf = 0.40 for 60% MeCN/PhCH3. M.p. 250–252 C. [α]D = +21.49 (c 1.0, CHCl3). 1417,FT-IR 1361, (pellets, 1300, KBr) 1275,ν 1170,(cm− 11107,): 3511, 1047, 2978, 1003, 2940, 914, 2872,871, 789, 2832, 742, 1775, 672, 1736, 596, 509, 1652, 470. 1608, HRMS 1482, (ESI): 1447, calcd. 1417, for C24H32O6Na [M + Na]+ 439.20911, found 439.2110. 1H- and 13C-NMR spectra, see Table 2. 1361, 1300, 1275, 1170, 1107, 1047, 1003, 914, 871, 789, 742, 672, 596, 509, 470. HRMS (ESI): calcd. for (7α,11α,17α)-12: R+f = 0.33 for 60% MeCN/PhCH13. M.p. 228–22913 °C (lit. 229–230 °C [24]). α = C24H32O6Na [M + Na] 439.20911, found 439.2110. H- and C-NMR spectra, see Table2. −1 +40.68 (c 1.0, CHCl3) (lit. α = +18.9 (c 0.0027, CHCl3) [24]). FT-IR (pellets,◦ KBr) ν (cm◦ ): 3422, 3035,20 (7α,11α,17α)-12: Rf = 0.33 for 60% MeCN/PhCH3. M.p. 228–229 C (lit. 229–230 C[24]). [α]D = 2985, 2954, 2933, 2897, 2877,20 1759, 1719, 1662, 1617, 1469, 1443, 1376, 1269, 1234, 1211,−1 1181, 1144, +40.68 (c 1.0, CHCl3) (lit. [α] = +18.9 (c 0.0027, CHCl3)[24]). FT-IR (pellets, KBr) ν (cm ): 3422, 3035, D + 1059,2985, 1017, 2954, 999, 2933, 926, 2897, 866, 2877, 852, 1759,787, 677, 1719, 606, 1662, 533, 1617, 514, 1469,466. HRMS 1443, 1376, (ESI): 1269, calcd. 1234, for C 1211,24H32O 1181,6Na 1144,[M + Na] 1059, 439.20911, found 439.2080. 1H- and 13C-NMR spectra, see Table 2. + 1017, 999, 926, 866, 852, 787, 677, 606, 533, 514, 466. HRMS (ESI): calcd. for C24H32O6Na [M + Na] 439.20911, found 439.2080. 1H- and 13C-NMR spectra, see Table2. 3.8.2. Purification of the commercial (7α,11α,17α)-11-hydroxyester 12 and isolation of the (73.8.2.β,11α Purification,17α)-11-hydroxyester of the Commercial 12a impurity (7α,11 α,17α)-11-hydroxyester 12 and Isolation of the (7β,11α,17α)-11-hydroxyester 12a Impurity The commercial 11-hydroxyester (7α,11α,17α)-12 (32.5 g) was purified by recrystallization from a mixtureThe commercial of AcOEt/MeCN/CH 11-hydroxyester2Cl2 (8:4:1) (7α,11 αto,17 affordα)-12 (32.5the pure g) was 12 purified (13.98 g, by 43% recrystallization yield) as a fromwhite a crystals.mixture ofThe AcOEt/MeCN/CH recrystallization 2motherCl2 (8:4:1) liquors to afford of 12 the, containing pure 12 (13.98 a mixture g, 43% of yield) epimers as a white 12 and crystals. 12a, were The subjectedrecrystallization to column mother chromatography liquors of 12, containing over silica a mixture gel with of epimers10-50% 12MeCN/PhCHand 12a, were3 gradient subjected elution to column to givechromatography (7β,11α,17α)- over12a (0.91 silica g) gel as with a white 10–50% solid, MeCN/PhCH a mixture of3 12gradient and 12a elution (14.4 tog) giveand (7(7βα,11,11αα,17,17αα)-)-12a12 (2.67(0.91 g)g). asThe a white characterization solid, a mixture data of of12 12and and12a 12a(14.4 were g) and identical (7α,11 αin,17 allα )-aspects12 (2.67 with g). The those characterization obtained in experimentdata of 12 and 3.8.1.12a were identical in all aspects with those obtained in experiment 3.8.1.

3.8.3.3.8.3. 9,11 9,11ββ-dichloro-7-dichloro-7αα-(methoxycarbonyl)-3-oxo-17-(methoxycarbonyl)-3-oxo-17αα-pregn-4-ene-21,17-carbolactone-pregn-4-ene-21,17-carbolactone (13 (13, Scheme, Scheme 4)4 ) AA solutionsolution ofof (7 α(7,17α,17α)-9(11)-enesterα)-9(11)-enester7 (2.5 7 (2.5 g, 6.27 g, mmol)6.27 mmol) in anhydrous in anhydrous pyridine pyridine (5.1 mL, 62.7(5.1 mmol) mL, ◦ 62.7and mmol) chlorobenzene and chlorobenzene (25 mL) was (25 cooled mL) was to 0 cooledC. SO to2Cl 02 °C.(1.0 SO mL,2Cl2 12.54 (1.0 mL, mmol) 12.54 was mmol) added was dropwise added ◦ dropwiseand the mixture and the was mixture stirred was for stirred 30 min for at 030C. min The at cooling 0 °C. The bath cooling was removed bath was and removed the stirring and wasthe stirringcontinued was for continued 1 h at room for temperature. 1 h at room The temperature. reaction mixture The reaction was diluted mixture with was H2 Odiluted (100 mL) with and H the2O (100product mL) wasand extractedthe product with was CH extracted2Cl2 (3 × with50 mL). CH2Cl2 (3 × 50 mL).

SchemeScheme 4. 4. SynthesisSynthesis of of the the (7 (7αα,11,11ββ,17,17αα)-9,11-dichloro)-9,11-dichloro derivative derivative 1313. .

The combined organic extracts were washed with aqueous saturated NaHCO3 solution (150 The combined organic extracts were washed with aqueous saturated NaHCO3 solution (150 mL) mL) followed by H2O (150 mL), dried over anhydrous Na2SO4, filtered and concentrated under followed by H O (150 mL), dried over anhydrous Na SO , filtered and concentrated under reduced reduced pressure2 to give a brown foam. The crude product2 4 was purified by column chromatography pressure to give a brown foam. The crude product was purified by column chromatography over silica over silica gel with 5–30% AcOEt/CH2Cl2 gradient elution to afford 9,11-dichloro impurity 13 (1.21 g, 41% yield) as white crystals. Rf = 0.27 for 15% AcOEt/CH2Cl2. M.p. 230–231 °C. α = +123.30 (c 1.0, − CHCl3). FT-IR (pellets, KBr) ν (cm 1): 3020, 2987, 2949, 2901, 2880, 1765, 1717, 1657, 1616, 1424, 1361, 1343, 1243, 1213, 1187, 1159, 1103, 1036, 1014, 965, 916, 868, 777, 663, 641, 601, 511. HRMS (ESI): calcd. for C24H30O5NaCl2 [M + Na]+ 491.13625, found 491.1386. 1H- and 13C-NMR spectra, see Table 7.

3.8.4. 3-Oxo-17α-pregn-4-ene-7α,9:21,17-dicarbolactone (14, Imp. A, Scheme 5) A suspension of 11-hydroxyester (7α,11α,17α)-12 (5.0 g, 12.0 mmol) in anhydrous pyridine (25 mL) was cooled to 5 °C and MsCl (1.1 mL, 14.4 mmol) was added dropwise. A mixture was stirred at room temperature for 2 h followed by pyridine evaporation under reduced pressure. The residue was suspended in CH2Cl2 (25 mL), washed subsequently with aqueous 1 M HCl solution (3 × 25 mL), H2O (25 mL) and brine (25 mL). The organic phase was dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure.

Molecules 2017, 22, 1354 20 of 26

gel with 5–30% AcOEt/CH2Cl2 gradient elution to afford 9,11-dichloro impurity 13 (1.21 g, 41% yield) ◦ 20 as white crystals. Rf = 0.27 for 15% AcOEt/CH2Cl2. M.p. 230–231 C. [α]D = +123.30 (c 1.0, CHCl3). FT-IR (pellets, KBr) ν (cm−1): 3020, 2987, 2949, 2901, 2880, 1765, 1717, 1657, 1616, 1424, 1361, 1343, 1243, 1213, 1187, 1159, 1103, 1036, 1014, 965, 916, 868, 777, 663, 641, 601, 511. HRMS (ESI): calcd. for + 1 13 C24H30O5NaCl2 [M + Na] 491.13625, found 491.1386. H- and C-NMR spectra, see Table7.

3.8.4. 3-Oxo-17α-pregn-4-ene-7α,9:21,17-dicarbolactone (14, Imp. A, Scheme5) A suspension of 11-hydroxyester (7α,11α,17α)-12 (5.0 g, 12.0 mmol) in anhydrous pyridine (25 mL) was cooled to 5 ◦C and MsCl (1.1 mL, 14.4 mmol) was added dropwise. A mixture was stirred at room temperature for 2 h followed by pyridine evaporation under reduced pressure. The residue was suspended in CH2Cl2 (25 mL), washed subsequently with aqueous 1 M HCl solution (3 × 25 mL), H2O (25 mL) and brine (25 mL). The organic phase was dried over anhydrous Na2SO4, filtered and Moleculesevaporated 2017, under22, 1354 reduced pressure. 20 of 26

Scheme 5. Synthesis of the 7,9:21,17-dilactone 14 (Imp. A). Scheme 5. Synthesis of the 7,9:21,17-dilactone 14 (Imp. A).

The crude product was purified by column chromatography over silica gel with 0.5–2% The crude product was purified by column chromatography over silica gel with 0.5–2% MeOH/CH2Cl2 gradient elution to afford mesylate (5.04 g, 85% yield) as white crystals. A mixture of MeOH/CH2Cl2 gradient elution to afford mesylate (5.04 g, 85% yield) as white crystals. A mixture of mesylate (2.5 g, 5.05 mmol), AcONa (4.14 g, 50.5 mmol), 80% AcOH (15 mL) and H2O (5 mL) was mesylate (2.5 g, 5.05 mmol), AcONa (4.14 g, 50.5 mmol), 80% AcOH (15 mL) and H2O (5 mL) was heated heated◦ at 70 °C for 3 h followed by AcOH evaporation under reduced pressure. Saturated aqueous at 70 C for 3 h followed by AcOH evaporation under reduced pressure. Saturated aqueous Na2CO3 Na2CO3 solution (25 mL) was added to the residue and the product was extracted with CH2Cl2 (3 × × 25solution mL). (25The mL) combined was added organic to the phases residue were and thewashed product with was brine extracted (100 withmL), CH dried2Cl2 over(3 25anhydrous mL). The combined organic phases were washed with brine (100 mL), dried over anhydrous Na SO , filtered and Na2SO4, filtered and concentrated under reduced pressure. The crude product 2was4 purified by concentrated under reduced pressure. The crude product was purified by column chromatography over column chromatography over silica gel with 5–30% AcOEt/CH2Cl2 gradient elution to afford silica gel with 5–30% AcOEt/CH Cl gradient elution to afford 7α,9:21,17-dicarbolactone 14 (1.61 g, 7α,9:21,17-dicarbolactone 14 (1.612 g,2 83% yield) as a white solid. Rf = 0.20 for 15% AcOEt/CH2Cl2. ◦ 20 83% yield) as a white solid. Rf = 0.20 for 15% AcOEt/CH2Cl2. M.p. 244–245−1 C. [α] = −2.41 (c 1.0, M.p. 244–245 °C. α = −2.41 (c 1.0, CHCl3). FT-IR (pellets, KBr) ν (cm ): 2964, 2875,D 1770, 1674, ν −1 1627,CHCl 1452,3). FT-IR 1383, (pellets, 1344, 1268, KBr) 1196,(cm 1177,): 2964, 1157, 2875, 1110, 1770, 1047, 1674, 971, 1627,914, 874, 1452, 814, 1383, 722, 1344, 657, 1268,593, 509. 1196, HRMS 1177, 1157, 1110, 1047, 971, 914, 874, 814, 722, 657, 593, 509. HRMS (ESI): calcd. for C H O Na [M + Na]+ (ESI): calcd. for C23H28O5Na [M + Na]+ 407.18289, found 407.1847. 1H- and 13C-NMR23 spectra,28 5 see Table 7. 407.18289, found 407.1847. 1H- and 13C-NMR spectra, see Table7. 3.8.5. 7α-(Methoxycarbonyl)-3-oxo-17α-pregna-4,9(11)-diene-21,17-carbolactone (7, Imp. C, Scheme 6) 3.8.5. 7α-(Methoxycarbonyl)-3-oxo-17α-pregna-4,9(11)-diene-21,17-carbolactone (7, Imp. C, Scheme6) Imidazole (13.08 g, 192.08 mmol) was added to a solution of 11-hydroxyester (7α,11α,17α)-12 Imidazole (13.08 g, 192.08 mmol) was added to a solution of 11-hydroxyester (7α,11α,17α)-12 (20.0 g, 48.02 mmol) in THF (150 mL) and cooled to −10 °C. SO2Cl2 (8.2 mL, 100.84 mmol) was added (20.0 g, 48.02 mmol) in THF (150 mL) and cooled to −10 ◦C. SO Cl (8.2 mL, 100.84 mmol) was added dropwise and the mixture was stirred for 30 min. at −10 °C. The2 cooling2 bath was then removed and dropwise and the mixture was stirred for 30 min. at −10 ◦C. The cooling bath was then removed and the stirring was continued for 1 h at room temperature. The reaction mixture was diluted with H2O the stirring was continued for 1 h at room temperature. The reaction mixture was diluted with H2O (100 mL) and the product was extracted with CH2Cl2 (3 × 50 mL). The combined organic extracts (100 mL) and the product was extracted with CH2Cl2 (3 × 50 mL). The combined organic extracts were were washed with aqueous saturated NaHCO3 solution (100 mL), dried over anhydrous Na2SO4, washed with aqueous saturated NaHCO solution (100 mL), dried over anhydrous Na SO , filtered filtered and concentrated under reduced 3pressure to give a yellowish white foam. 2 4 and concentrated under reduced pressure to give a yellowish white foam. The crude product was recrystallized from ethanol and a mixture of dichloromethane/diethyl ether to give (7α,17α)-9(11)-enester 7 (13.58 g, 71% yield). Rf = 0.17 for 15% AcOEt/CH2Cl2. M.p. ◦ ◦ 20 −1 204–206 C (lit. 205–206 C[17]). [α]D = +2.75 (c 1.0, CHCl3). FTIR (KBr) ν (cm ): 3052, 2967, 2908, 2875, 1774, 1732, 1666, 1622, 1463, 1438, 1421, 1378, 1332, 1268, 1166, 1090, 1012, 966, 920, 868, 834, 770, + 1 665, 614, 531, 475. HRMS (ESI): calcd. for C24H30O5Na [M + Na] 421.19855, found 421.1986. H- and 13C-NMR spectra, see Table4.

Scheme 6. Synthesis of the (7α,17α)-9(11)-enester 7.

The crude product was recrystallized from ethanol and a mixture of dichloromethane/diethyl ether to give (7α,17α)-9(11)-enester 7 (13.58 g, 71% yield). Rf = 0.17 for 15% AcOEt/CH2Cl2. M.p. −1 204–206 °C (lit. 205–206 °C [17]). α = +2.75 (c 1.0, CHCl3). FTIR (KBr) ν (cm ): 3052, 2967, 2908, 2875, 1774, 1732, 1666, 1622, 1463, 1438, 1421, 1378, 1332, 1268, 1166, 1090, 1012, 966, 920, 868, 834, 770, 665, 614, 531, 475. HRMS (ESI): calcd. for C24H30O5Na [M + Na]+ 421.19855, found 421.1986. 1H- and 13C-NMR spectra, see Table 4.

3.8.6. 7β-(methoxycarbonyl)-3-oxo-17α-pregna-4,9(11)-diene-21,17-carbolactone (7a, Schemes 7 and 8)

Method 1 The (7α,17α)-9(11)-enester 7 (3.2 g, 8.03 mmol) was added to a suspension of MeONa (1.52 g, 28.11 mmol) in anhydrous MeOH (20 mL) and then refluxed for 24 h. The mixture was cooled to room temperature and aqueous 3 M HCl solution (100 mL) was added. The product was extracted

Molecules 2017, 22, 1354 20 of 26

Scheme 5. Synthesis of the 7,9:21,17-dilactone 14 (Imp. A).

The crude product was purified by column chromatography over silica gel with 0.5–2% MeOH/CH2Cl2 gradient elution to afford mesylate (5.04 g, 85% yield) as white crystals. A mixture of mesylate (2.5 g, 5.05 mmol), AcONa (4.14 g, 50.5 mmol), 80% AcOH (15 mL) and H2O (5 mL) was heated at 70 °C for 3 h followed by AcOH evaporation under reduced pressure. Saturated aqueous Na2CO3 solution (25 mL) was added to the residue and the product was extracted with CH2Cl2 (3 × 25 mL). The combined organic phases were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography over silica gel with 5–30% AcOEt/CH2Cl2 gradient elution to afford 7α,9:21,17-dicarbolactone 14 (1.61 g, 83% yield) as a white solid. Rf = 0.20 for 15% AcOEt/CH2Cl2. −1 M.p. 244–245 °C. α = −2.41 (c 1.0, CHCl3). FT-IR (pellets, KBr) ν (cm ): 2964, 2875, 1770, 1674, 1627, 1452, 1383, 1344, 1268, 1196, 1177, 1157, 1110, 1047, 971, 914, 874, 814, 722, 657, 593, 509. HRMS (ESI): calcd. for C23H28O5Na [M + Na]+ 407.18289, found 407.1847. 1H- and 13C-NMR spectra, see Table 7.

3.8.5. 7α-(Methoxycarbonyl)-3-oxo-17α-pregna-4,9(11)-diene-21,17-carbolactone (7, Imp. C, Scheme 6) Imidazole (13.08 g, 192.08 mmol) was added to a solution of 11-hydroxyester (7α,11α,17α)-12 (20.0 g, 48.02 mmol) in THF (150 mL) and cooled to −10 °C. SO2Cl2 (8.2 mL, 100.84 mmol) was added dropwise and the mixture was stirred for 30 min. at −10 °C. The cooling bath was then removed and the stirring was continued for 1 h at room temperature. The reaction mixture was diluted with H2O (100 mL) and the product was extracted with CH2Cl2 (3 × 50 mL). The combined organic extracts wereMolecules washed2017, 22 ,with 1354 aqueous saturated NaHCO3 solution (100 mL), dried over anhydrous Na212SO of 264, filtered and concentrated under reduced pressure to give a yellowish white foam.

SchemeScheme 6. 6. SynthesisSynthesis of of the the (7 (7αα,17,17αα)-9(11)-enester)-9(11)-enester 77..

3.8.6.The 7β-(methoxycarbonyl)-3-oxo-17 crude product was recrystallizedα-pregna-4,9(11)-diene-21,17-carbolactone from ethanol and a mixture of dichloromethane/diethyl (7a, Schemes7 and8) ether to give (7α,17α)-9(11)-enester 7 (13.58 g, 71% yield). Rf = 0.17 for 15% AcOEt/CH2Cl2. M.p. −1 204–206Method °C 1 (lit. 205–206 °C [17]). α = +2.75 (c 1.0, CHCl3). FTIR (KBr) ν (cm ): 3052, 2967, 2908, 2875, 1774, 1732, 1666, 1622, 1463, 1438, 1421, 1378, 1332, 1268, 1166, 1090, 1012, 966, 920, 868, 834, The (7α,17α)-9(11)-enester 7 (3.2 g, 8.03 mmol) was added to a suspension of MeONa (1.52 g, 770, 665, 614, 531, 475. HRMS (ESI): calcd. for C24H30O5Na [M + Na]+ 421.19855, found 421.1986. 1H- 28.11 mmol) in anhydrous MeOH (20 mL) and then refluxed for 24 h. The mixture was cooled to room andMolecules 13C-NMR 2017, 22 spectra,, 1354 see Table 4. 21 of 26 temperature and aqueous 3 M HCl solution (100 mL) was added. The product was extracted with CH Cl (3 × 25 mL). The combined organic phases were dried over anhydrous Na SO , filtered and 3.8.6.with2 7CHβ2-(methoxycarbonyl)-3-oxo-172Cl2 (3 × 25 mL). The combinedα-pregna-4,9(11)-diene-21,17-carbolactone organic phases were dried over anhydrous (7a, Schemes2 Na4 2SO 74, andfiltered 8) concentratedand concentrated under under reduced reduced pressure pressure to give to agive brownish a brownish foam. foam. Method 1 The (7α,17α)-9(11)-enester 7 (3.2 g, 8.03 mmol) was added to a suspension of MeONa (1.52 g, 28.11 mmol) in anhydrous MeOH (20 mL) and then refluxed for 24 h. The mixture was cooled to room temperature and aqueous 3 M HCl solution (100 mL) was added. The product was extracted

SchemeScheme 7.7. Synthesis of the (7β,17α)-9(11)-enester 7a..

The crude mixture of products was purified by column chromatography over silica gel with The crude mixture of products was purified by column chromatography over silica gel with 5–20% AcOEt/CH2Cl2 gradient elution to give (7β,17α)-9(11)-enester 7a (1.18 g, 37% yield) as a white 5–20% AcOEt/CH Cl gradient elution to give (7β,17α)-9(11)-enester 7a (1.18 g, 37% yield) as a white solid, a mixture of2 epimers2 7 and 7a (0.78 g) and (7α,17α)-9(11)-enester 7 (1.12 g). solid, a mixture of epimers 7 and 7a (0.78 g) and (7α,17α)-9(11)-enester 7 (1.12 g). (7β,17α)-9(11)-enester 7a: Rf = 0.34 for 15% AcOEt/CH2Cl2. M.p. 167–168 °C. α◦ = +0.5020 (c 1.0, (7β,17α)-9(11)-enester 7a: Rf −= 0.34 for 15% AcOEt/CH2Cl2. M.p. 167–168 C. [α]D = +0.50 CHCl3). FT-IR (pellets, KBr) ν (cm 1): 3053, 2970, 2883, 2847, 1773, 1731, 1671, 1618, 1435, 1369, 1299, (c 1.0, CHCl ). FT-IR (pellets, KBr) ν (cm−1): 3053, 2970, 2883, 2847, 1773, 1731, 1671, 1618, 1435, 1369, 1265, 1188, 31159, 1090, 1017, 990, 910, 868, 816, 792, 769, 665, 602, 521, 480. HRMS (ESI): calcd. for 1299, 1265, 1188, 1159, 1090, 1017, 990, 910, 868, 816, 792, 769, 665, 602, 521, 480. HRMS (ESI): calcd. C24H30O5Na [M + Na]+ 421.19855, found 421.2006. 1H- and 13C-NMR spectra, see Table 4. The for C H O Na [M + Na]+ 421.19855, found 421.2006. 1H- and 13C-NMR spectra, see Table4. The characterization24 30 5 data of 7 were identical in all aspects with those obtained in experiment 3.8.5. characterization data of 7 were identical in all aspects with those obtained in experiment 3.8.5.

Method 2

ImidazoleImidazole (1.31 (1.31 g, g, 19.20 19.20 mmol) mmol) was was added added to a to solution a solution of 11-hydroxyester of 11-hydroxyester (7β,11 α(7,17β,11α)-α12a,17α(2.0)-12a g, ◦ 4.80(2.0 mmol)g, 4.80 inmmol) THF (25in THF mL) (25 and mL) cooled and to cooled−20 C. to SO −202Cl °C.2 (0.8 SO mL,2Cl2 10.08(0.8 mL, mmol) 10.08 was mmol) added was dropwise added ◦ anddropwise the mixture and the was mixture stirred was for 30stirred min. for at − 3020 min.C. Theat −20 cooling °C. The bath cooling was then bath removed was then and removed the stirring and wasthe stirring continued was for continued 1 h at room for temperature.1 h at room temper The reactionature. The mixture reaction was mixture diluted withwas diluted H2O (10 with mL) H and2O the(10 productmL) and wasthe product extracted was with extracted CH2Cl2 with(3 × CH25 mL).2Cl2 (3 × 25 mL). The combined organic extracts were washed with aqueous saturated NaHCO3 solution (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a yellowish white foam. The crude product was purified by column chromatography over silica gel with 5–20% AcOEt/CH2Cl2 gradient elution to give (7β,17α)-9(11)-enester 7a (1.62 g, 85% yield). The characterization data of 7a were identical in all aspects with those obtained in Method 1.

Scheme 8. Synthesis of the (7β,17α)-9(11)-enester 7a.

The combined organic extracts were washed with aqueous saturated NaHCO3 solution (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a yellowish white foam. The crude product was purified by column chromatography over silica gel with 5–20% AcOEt/CH2Cl2 gradient elution to give (7β,17α)-9(11)-enester 7a (1.62 g, 85% yield). The characterization data of 7a were identical in all aspects with those obtained in Method 1.

3.8.7. 7α-(Methoxycarbonyl)-3-oxo-17α-pregna-4,11(12)-diene-21,17-carbolactone (7b) The recrystallization mother liquors of 7 (9.81 g) were chromatogaphed on silica gel using varying mixtures of ethyl acetate, dichloromethane, acetonitrile and toluene. Early cuts of the 5–30% AcOEt/CH2Cl2 gradient elution afforded (7β,17α)-9(11)-enester 7a (0.48 g) and (7α,17α)-11(12)-enester 7b (0.73 g) as white crystalline products. An analytical sample of 7b was prepared by a recrystallization from ethanol. Succeeding cuts of 5–30% AcOEt/CH2Cl2 gradient elution gave (7α,11β,17α)-9,11-dichloro impurity 14 (0.84 g), 7,9:21,17-dilactone 13 (1.16 g) and (7α,17α)-9(11)-enester 7 (2.75 g) as white crystalline products. Finally, crystalline 11-hydroxyesters (7β,11α,17α)-12a (0.17 g) and (7α,11α,17α)-12 (0.33 g) were obtained on further gradient elution with 10–50% MeCN/PhCH3. (7α,17α)-11(12)-enester 7b: Rf = 0.29 for 15% AcOEt/CH2Cl2. M.p. 232–234 °C.

Molecules 2017, 22, 1354 21 of 26 with CH2Cl2 (3 × 25 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a brownish foam.

Scheme 7. Synthesis of the (7β,17α)-9(11)-enester 7a.

The crude mixture of products was purified by column chromatography over silica gel with 5–20% AcOEt/CH2Cl2 gradient elution to give (7β,17α)-9(11)-enester 7a (1.18 g, 37% yield) as a white solid, a mixture of epimers 7 and 7a (0.78 g) and (7α,17α)-9(11)-enester 7 (1.12 g). (7β,17α)-9(11)-enester 7a: Rf = 0.34 for 15% AcOEt/CH2Cl2. M.p. 167–168 °C. α = +0.50 (c 1.0, − CHCl3). FT-IR (pellets, KBr) ν (cm 1): 3053, 2970, 2883, 2847, 1773, 1731, 1671, 1618, 1435, 1369, 1299, 1265, 1188, 1159, 1090, 1017, 990, 910, 868, 816, 792, 769, 665, 602, 521, 480. HRMS (ESI): calcd. for C24H30O5Na [M + Na]+ 421.19855, found 421.2006. 1H- and 13C-NMR spectra, see Table 4. The characterization data of 7 were identical in all aspects with those obtained in experiment 3.8.5.

Method 2

Imidazole (1.31 g, 19.20 mmol) was added to a solution of 11-hydroxyester (7β,11α,17α)-12a (2.0 g, 4.80 mmol) in THF (25 mL) and cooled to −20 °C. SO2Cl2 (0.8 mL, 10.08 mmol) was added dropwise and the mixture was stirred for 30 min. at −20 °C. The cooling bath was then removed and theMolecules stirring2017 ,was22, 1354 continued for 1 h at room temperature. The reaction mixture was diluted with22 H of2 26O (10 mL) and the product was extracted with CH2Cl2 (3 × 25 mL).

Scheme 8. Synthesis of the (7β,17,17α)-9(11)-enester 7a.

3.8.7.The 7α-(Methoxycarbonyl)-3-oxo-17 combined organic extracts wereα-pregna-4,11(12)-diene-21,17-carbolactone washed with aqueous saturated NaHCO (37b solution) (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a yellowish whiteThe foam. recrystallization The crude product mother was liquors purified of by7 (9.81column g) chromatography were chromatogaphed over silica on silicagel with gel 5–20% using varyingAcOEt/CH mixtures2Cl2 gradient of ethyl acetate,elution dichloromethane,to give (7β,17α acetonitrile)-9(11)-enester and toluene.7a (1.62 Earlyg, 85% cuts ofyield). the 5–30% The AcOEt/CHcharacterization2Cl2 gradient data of 7a elution were affordedidentical(7 inβ ,17all αaspects)-9(11)-enester with those7a (0.48obtained g) and in (7Methodα,17α)-11(12)-enester 1. 7b (0.73 g) as white crystalline products. An analytical sample of 7b was prepared by a 3.8.7.recrystallization 7α-(Methoxycarbonyl)-3-oxo-17 from ethanol. Succeedingα-pregna-4,11(12)-diene-21,17-carbolactone cuts of 5–30% AcOEt/CH2Cl2 gradient (7b) elution gave (7α,11β,17α)-9,11-dichloro impurity 14 (0.84 g), 7,9:21,17-dilactone 13 (1.16 g) and (7α,17α)-9(11)-enester The recrystallization mother liquors of 7 (9.81 g) were chromatogaphed on silica gel using Molecules7 (2.75 g) 2017 as, white22, 1354crystalline products. Finally, crystalline 11-hydroxyesters (7β,11α,17α)-12a (0.1722 of 26 g) varying mixtures of ethyl acetate, dichloromethane, acetonitrile and toluene. Early cuts of the 5–30% and (7α,11α,17α)-12 (0.33 g) were obtained on further gradient elution with 10–50% MeCN/PhCH3. AcOEt/CH 2Cl2 gradient elution afforded (7β,17−1 α)-9(11)-enester 7a (0.4820 g) and α = +33.42 (c 1.0, CHCl3). FT-IR (pellets, KBr) ν (cm ): 3028, 2971, 2949,◦ 2878, 1775, 1723, 1664, (7α,17α)-11(12)-enester 7b: Rf = 0.29 for 15% AcOEt/CH2Cl2. M.p. 232–234 C. [α]D = +33.42 (c 1.0, (7α,17α)-11(12)-enester 7b (0.73 g)− 1as white crystalline products. An analytical sample of 7b was 1616,CHCl 1465,3). FT-IR 1438, (pellets, 1380, 1334, KBr) 1267,ν (cm 1195,): 3028,1172, 2971,1048, 2949,1013, 2878,918, 868, 1775, 788, 1723, 716, 1664, 669, 1616,525, 454. 1465, HRMS 1438, (ESI): 1380, prepared by a recrystallization from ethanol. Succeeding cuts of 5–30% AcOEt/CH2Cl2 gradient calcd.1334, 1267,for C 1195,24H30O 1172,5Na [M 1048, + Na] 1013,+ 421.1991, 918, 868, found 788, 716, 421.1988. 669, 525, 1H- 454. and HRMS 13C-NMR (ESI): spectra, calcd. forsee CTableH 4.O Na elution gave (7α,11β,17α)-9,11-dichloro impurity 14 (0.84 g), 7,9:21,17-dilactone 13 (1.1624 30g) 5and [M +The Na] +characterization421.1991, found data 421.1988. of 7, 17aH-, 12 and, 12a13C-NMR, 13 and spectra, 14 were see identical Table4. in all aspects with those (7α,17α)-9(11)-enester 7 (2.75 g) as white crystalline products. Finally, crystalline 11-hydroxyesters obtainedThe in characterization experiments 3.8.1–3.8.6, data of 7 ,respectively.7a, 12, 12a, 13 and 14 were identical in all aspects with those (7β,11α,17α)-12a (0.17 g) and (7α,11α,17α)-12 (0.33 g) were obtained on further gradient elution with obtained in experiments 3.8.1–3.8.6, respectively. 3.8.8.10–50% 11 αMeCN/PhCH,12α-Epoxy-73.α (7-(methoxycarbonyl)-3-oxo-17α,17α)-11(12)-enester 7b: Rf α= -pregn-4-ene-21,17-carbolactone0.29 for 15% AcOEt/CH2Cl2. M.p. 232–234 °C. ( 3.8.8.2b, Imp. 11α B,,12 Schemeα-Epoxy-7 9) α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (2b, Imp. B, Scheme9) 2,2,2-Trichloroacetamide (0.52 g, 3.17 mmol) and Na2HPO4 (0.35 g, 2.48 mmol) were added to a stirred2,2,2-Trichloroacetamide solution of (7α,17α)-11(12)-enester (0.52 g, 3.17 7b mmol) (0.55 and g, 1.38 Na 2mmol)HPO4 in(0.35 CH g,2Cl 2.482 (15 mmol) mL). The were mixture added was to a cooledstirred solutionto 15 °C of (7andα,17 Hα2)-11(12)-enesterO2 (35%, 8.4 mL)7b (0.55was g,added 1.38 mmol) dropwise in CH. 2AfterCl2 (15 being mL). Thestirred mixture at room was ◦ temperaturecooled to 15 forC and 48 Hh,2 theO2 (35%,mixture 8.4 was mL) diluted was added with dropwise. H2O (15 AftermL) and being the stirred product at room was temperatureextracted with for CH48 h,2Cl the2 (3 mixture × 10 mL). was dilutedThe combined with H2 Oorganic (15 mL) extracts and the were product washed was extracted in succession with CH with2Cl 23%(3 ×aqueous10 mL). NaThe2SO combined3 solution organic (25 mL), extracts 1 M wereaqueous washed NaOH in succession solution (20 with mL), 3% 1 aqueous M aqueous Na2SO HCl3 solution solution (25 (20 mL), mL) 1 andM aqueous brine (25 NaOH mL). solution (20 mL), 1 M aqueous HCl solution (20 mL) and brine (25 mL).

Scheme 9. Synthesis of the (7 α,11,11α,12,12α,17,17α)-11,12-epoxyester)-11,12-epoxyester 2b (Imp.(Imp. B). B).

The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced The organic phase was dried over anhydrous Na SO , filtered and concentrated under reduced pressure to give a yellowish white foam. The crude2 411,12-epoxide was purified by column pressure to give a yellowish white foam. The crude 11,12-epoxide was purified by column chromatography over silica gel with 1–10% Me2CO/CH2Cl2 gradient elution to give 2b (0.34 g, 60% chromatography over silica gel with 1–10% Me2CO/CH2Cl2 gradient elution to give 2b (0.34 g, yield) as a white solid. Rf = 0.37 for 10% Me2CO/CH2Cl2. M.p. 209–210 °C. α = +24.63 (c 1.0, ◦ [α]20 60% yield) as a white solid. Rf = 0.37−1 for 10% Me2CO/CH2Cl2. M.p. 209–210 C. D = +24.63 (c 1.0, CH2Cl2). FT-IR (pellets, KBr) ν (cm ): 3531, 3430, 3024, 2952, 2880, 1780, 1725, 1664, 1614, 1463, 1437, CH Cl ). FT-IR (pellets, KBr) ν (cm−1): 3531, 3430, 3024, 2952, 2880, 1780, 1725, 1664, 1614, 1463, 1437, 1421,2 1404,2 1381, 1335, 1295, 1268, 1237, 1194, 1174, 1072, 1034, 1011, 977, 962, 920, 880, 836, 784, 664, 1421, 1404, 1381, 1335, 1295, 1268, 1237, 1194, 1174, 1072, 1034, 1011, 977, 962, 920, 880, 836, 784, 664, 629, 605, 524, 497, 454. HRMS (ESI): calcd. for C24H30O6Na [M + Na]+ 437.1940, found 437.1942. 1H- and 13C-NMR spectra, see Table 8.

3.8.9. 9,11α-Epoxy-7β-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (2a, Imp. E, Schemes 10 and 11).

Method 1 Eplerenone (3.1 g, 7.48 mmol) was added to a suspension of MeONa (1.41 g, 26.17 mmol) in anhydrous MeOH (25 mL) and then refluxed for 24 h. The mixture was cooled to room temperature and 3 M aqueous HCl solution (150 mL) was added. The product was extracted with CH2Cl2 (3 × 25 mL).

Scheme 10. Synthesis of the (7β,11α,17α)-9,11-epoxyester (2a, Imp. E).

The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a yellowish white solid. The crude mixture of epimers 2 and 2a was

Molecules 2017, 22, 1354 22 of 26

−1 α = +33.42 (c 1.0, CHCl3). FT-IR (pellets, KBr) ν (cm ): 3028, 2971, 2949, 2878, 1775, 1723, 1664, 1616, 1465, 1438, 1380, 1334, 1267, 1195, 1172, 1048, 1013, 918, 868, 788, 716, 669, 525, 454. HRMS (ESI): calcd. for C24H30O5Na [M + Na]+ 421.1991, found 421.1988. 1H- and 13C-NMR spectra, see Table 4. The characterization data of 7, 7a, 12, 12a, 13 and 14 were identical in all aspects with those obtained in experiments 3.8.1–3.8.6, respectively.

3.8.8. 11α,12α-Epoxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (2b, Imp. B, Scheme 9)

2,2,2-Trichloroacetamide (0.52 g, 3.17 mmol) and Na2HPO4 (0.35 g, 2.48 mmol) were added to a stirred solution of (7α,17α)-11(12)-enester 7b (0.55 g, 1.38 mmol) in CH2Cl2 (15 mL). The mixture was cooled to 15 °C and H2O2 (35%, 8.4 mL) was added dropwise. After being stirred at room temperature for 48 h, the mixture was diluted with H2O (15 mL) and the product was extracted with CH2Cl2 (3 × 10 mL). The combined organic extracts were washed in succession with 3% aqueous Na2SO3 solution (25 mL), 1 M aqueous NaOH solution (20 mL), 1 M aqueous HCl solution (20 mL) and brine (25 mL).

Scheme 9. Synthesis of the (7α,11α,12α,17α)-11,12-epoxyester 2b (Imp. B).

The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a yellowish white foam. The crude 11,12-epoxide was purified by column chromatography over silica gel with 1–10% Me2CO/CH2Cl2 gradient elution to give 2b (0.34 g, 60% yield) as a white solid. Rf = 0.37 for 10% Me2CO/CH2Cl2. M.p. 209–210 °C. α = +24.63 (c 1.0, − CHMolecules2Cl2).2017 FT-IR, 22, (pellets, 1354 KBr) ν (cm 1): 3531, 3430, 3024, 2952, 2880, 1780, 1725, 1664, 1614, 1463, 231437, of 26 1421, 1404, 1381, 1335, 1295, 1268, 1237, 1194, 1174, 1072, 1034, 1011, 977, 962, 920, 880, 836, 784, 664, 629, 605, 524, 497, 454. HRMS (ESI): calcd. for C24H30O6Na [M + Na]+ 437.1940, found 437.1942. 1H- 629, 605, 524, 497, 454. HRMS (ESI): calcd. for C H O Na [M + Na]+ 437.1940, found 437.1942. 1H- and 13C-NMR spectra, see Table 8. 24 30 6 and 13C-NMR spectra, see Table8.

3.8.9.3.8.9. 9,11 9,11αα-Epoxy-7-Epoxy-7ββ-(methoxycarbonyl)-3-oxo-17-(methoxycarbonyl)-3-oxo-17αα-pregn-4-ene-21,17-carbolactone-pregn-4-ene-21,17-carbolactone (2a, Imp. E, (Schemes2a, Imp. E,10 Schemesand 11). 10 and 11).

MethodMethod 1 1 EplerenoneEplerenone (3.1 (3.1 g, g, 7.48 7.48 mmol) mmol) was was added added to to a a suspension of of MeONa MeONa (1.41 (1.41 g, g, 26.17 26.17 mmol) mmol) in in anhydrousanhydrous MeOH MeOH (25 (25 mL) mL) and and then then refluxed refluxed for for 24 24 h. h. The The mixture mixture was was cooled cooled to to room room temperature temperature and and 33 M M aqueous aqueous HCl HCl solution solution (150 (150 mL) mL) was was ad added.ded. The The product product wa wass extracted extracted with with CH CH2Cl2 Cl(3 2× (325 ×mL).25 mL).

SchemeScheme 10. 10. SynthesisSynthesis of of the the (7 (7ββ,11,11αα,17,17αα)-9,11-epoxyester)-9,11-epoxyester ( (2a2a,, Imp. Imp. E). E). Molecules 2017, 22, 1354 23 of 26 The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated The combined organic phases were dried over anhydrous Na SO , filtered and concentrated under reduced pressure to give a yellowish white solid. The crude mixture2 4 of epimers 2 and 2a was underpurified reduced by column pressure chromatography to give a yellowish over silica white gel solid. with The 1–10% crude Me mixture2CO/CH of2Cl epimers2 gradient2 and elution2a was to purifiedgive (7β,11 byα column,17α)-9,11-epoxyester chromatography 2a over(1.52 silica g, 49% gel withyield) 1–10% as a white Me2CO/CH solid, 2aCl mixture2 gradient of elution2 and 2a to give(0.55 (7g)β and,11α (7,17αα,11)-9,11-epoxyesterα,17α)-9,11-epoxyester2a (1.52 2 g, (0.77 49% g). yield) as a white solid, a mixture of 2 and 2a (0.55 g) and (7(7αβ,11,11αα,17,17αα)-9,11-epoxyester)-9,11-epoxyester 2 2a(0.77: R g).f = 0.43 for 10% Me2CO/CH2Cl2. M.p. 249–251 °C − (lit. 254–258 °C [9]). α = +12.53 (c 1.0, CHCl3). FT-IR (pellets, KBr) ν (cm 1): 3030, 2972,◦ 2953, 2929, (7β,11α,17α)-9,11-epoxyester 2a: Rf = 0.43 for 10% Me2CO/CH2Cl2. M.p. 249–251 C (lit. 254–258 ◦2883, 1774,20 1734, 1674, 1619, 1436, 1384, 1296, 1269, 1195, 1165, 1080,−1 1013, 905, 866, 797, 659, 576, 521, C[9]). [α]D = +12.53 (c 1.0, CHCl3). FT-IR (pellets, KBr) ν (cm ): 3030, 2972, 2953, 2929, 2883, 1774, 1734,414. HRMS 1674, 1619, (ESI): 1436, calcd. 1384, for 1296,C24H31 1269,O6 [M 1195, + H] 1165,+ 415.21152, 1080, 1013, found 905, 415.2113. 866, 797, 1H- 659, and 576, 13C-NMR 521, 414. spectra, HRMS see Table 8. The characterization+ data of 2 were identical in1 all aspects13 with those obtained in (ESI): calcd. for C24H31O6 [M + H] 415.21152, found 415.2113. H- and C-NMR spectra, see Table8. Theexperiment characterization 3.8.10. data of 2 were identical in all aspects with those obtained in experiment 3.8.10.

Method 2

2,2,2-Trichloroacetamide2,2,2-Trichloroacetamide (0.98(0.98 g,g, 6.026.02 mmol)mmol) andand NaNa22HPOHPO44 (1.21 g, 8.53 mmol) were added to a stirred solution of (7(7ββ,17α)-11(12)-enester 7a (2.0 g, 5.025.02 mmol)mmol) inin CHCH22Cl2 (50(50 mL). The mixture was ◦ cooled toto 15 15C °C and and H2 OH2 2(35%,O2 (35%, 4.2 mL) 4.2 wasmL) added was dropwise.added dropwise After being. After stirred being at room stirred temperature at room fortemperature 48 h, the for mixture 48 h, the was mixture diluted was with diluted H2O (20with mL) H2O and (20 themL) product and the wasproduct extracted was extracted with CH with2Cl2 (CH3 ×2Cl252 mL(3 ×). 25 The mL). combined The combined organic organic extracts extracts were washed were washed in succession in succession with 3% with aqueous 3% aqueous Na2SO3 solutionNa2SO3 solution (50 mL), (50 1 M mL), aqueous 1 M aqueous NaOH solution NaOH (50solution mL), 1(50 M mL), aqueous 1 M HClaqueous solution HCl (50 solution mL) and (50 brine mL) (50and mL). brine (50 mL).

Scheme 11. Synthesis of the (7β,11α,17α)-9,11-epoxyester (2a, Imp. E).

The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a yellowish white foam. The crude product was purified by column pressure to give a yellowish white foam. The crude product was purified by column chromatography chromatography over silica gel with 1–10% Me2CO/CH2Cl2 gradient elution to give the (7β,11α,17α)-9,11-epoxyester 2a (1.44 g, 69% yield) as a white solid. The characterization data of 2a were identical in all aspects with those obtained in Method 1.

3.8.10. 9,11α-Epoxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (eplerenone, 2) (without purification of the enester 7, Scheme 12) Imidazole (6.53 g, 96.04 mmol) was added to a solution of 11-hydroxyester (7α,11α,17α)-12 (10.0 g, 24.01 mmol) in THF (100 mL) and cooled to −10 °C. SO2Cl2 (4.1 mL, 50.42 mmol) was added dropwise and the mixture was stirred for 30 min. at −10 °C. The cooling bath was then removed and the stirring was continued for 1 h at room temperature.

Scheme 12. Synthesis of the (7α,11α,12α,17α)-9,11-epoxyester 2.

The reaction mixture was diluted with H2O (100 mL) and the product was extracted with CH2Cl2 (3 × 50 mL). The combined organic extracts were washed with aqueous saturated NaHCO3

Molecules 2017, 22, 1354 23 of 26 purified by column chromatography over silica gel with 1–10% Me2CO/CH2Cl2 gradient elution to give (7β,11α,17α)-9,11-epoxyester 2a (1.52 g, 49% yield) as a white solid, a mixture of 2 and 2a (0.55 g) and (7α,11α,17α)-9,11-epoxyester 2 (0.77 g). (7β,11α,17α)-9,11-epoxyester 2a: Rf = 0.43 for 10% Me2CO/CH2Cl2. M.p. 249–251 °C −1 (lit. 254–258 °C [9]). α = +12.53 (c 1.0, CHCl3). FT-IR (pellets, KBr) ν (cm ): 3030, 2972, 2953, 2929, 2883, 1774, 1734, 1674, 1619, 1436, 1384, 1296, 1269, 1195, 1165, 1080, 1013, 905, 866, 797, 659, 576, 521, 414. HRMS (ESI): calcd. for C24H31O6 [M + H]+ 415.21152, found 415.2113. 1H- and 13C-NMR spectra, see Table 8. The characterization data of 2 were identical in all aspects with those obtained in experiment 3.8.10.

Method 2

2,2,2-Trichloroacetamide (0.98 g, 6.02 mmol) and Na2HPO4 (1.21 g, 8.53 mmol) were added to a stirred solution of (7β,17α)-11(12)-enester 7a (2.0 g, 5.02 mmol) in CH2Cl2 (50 mL). The mixture was cooled to 15 °C and H2O2 (35%, 4.2 mL) was added dropwise. After being stirred at room temperature for 48 h, the mixture was diluted with H2O (20 mL) and the product was extracted with CH2Cl2 (3 × 25 mL). The combined organic extracts were washed in succession with 3% aqueous Na2SO3 solution (50 mL), 1 M aqueous NaOH solution (50 mL), 1 M aqueous HCl solution (50 mL) and brine (50 mL).

Scheme 11. Synthesis of the (7β,11α,17α)-9,11-epoxyester (2a, Imp. E).

MoleculesThe2017 organic, 22, 1354 phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced24 of 26 pressure to give a yellowish white foam. The crude product was purified by column chromatography over silica gel with 1–10% Me2CO/CH2Cl2 gradient elution to give the over silica gel with 1–10% Me CO/CH Cl gradient elution to give the (7β,11α,17α)-9,11-epoxyester (7β,11α,17α)-9,11-epoxyester 22a (1.44 g,2 69%2 yield) as a white solid. The characterization data of 2a 2a (1.44 g, 69% yield) as a white solid. The characterization data of 2a were identical in all aspects with were identical in all aspects with those obtained in Method 1. those obtained in Method 1. 3.8.10. 9,11α-Epoxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (eplerenone, 2) 3.8.10. 9,11α-Epoxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (eplerenone, 2) (without purificationpurification ofof thethe enesterenester 77,, SchemeScheme 1212)) Imidazole (6.53 g, 96.04 mmol) was addedadded to aa solutionsolution ofof 11-hydroxyester11-hydroxyester (7α,11α,17α)-)-1212 (10.0(10.0 g, g, 24.01 mmol) in THF (100 mL) and cooled to◦ −10 °C. SO2Cl2 (4.1 mL, 50.42 mmol) was added 24.01 mmol) in THF (100 mL) and cooled to −10 C. SO2Cl2 (4.1 mL, 50.42 mmol) was added dropwise dropwiseand the mixture and the was mixture stirred was for 30stirred min. for at − 3010 min.◦C. Theat −10 cooling °C. The bath cooling was then bath removed was then and removed the stirring and wasthe stirring continued was for continued 1 h at room for 1 temperature. h at room temperature.

Scheme 12. Synthesis of the (7α,11α,12α,17α)-9,11-epoxyester 2.

The reactionreaction mixturemixture was was diluted diluted with with H2 OH (1002O (100 mL) mL) and theand product the product was extracted was extracted with CH with2Cl2 (3CH×2Cl502 mL).(3 × 50 The mL). combined The combined organic organic extracts extracts were washed were withwashed aqueous with aqueous saturated saturated NaHCO3 NaHCOsolution3 (100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a yellowish white foam. The crude (7α,17α)-9(11)-enester 7 was dissolved in CH2Cl2 (150 mL), followed by 2,2,2-trichloroacetamide (4.68 g, 28.81 mmol) and Na2HPO4 (5.79 g, 40.82 mmol) addition. The ◦ mixture was cooled to 15 C and H2O2 (35%, 40.0 mL) was added under vigorous stirring. The cooling bath was removed. After being stirred at room temperature for 18 h, H2O (50 mL) and crushed ice (50 g) were added to the reaction mixture and the layers were separated. The organic phase was washed with NaOH (0.5 M, 100 mL), HCl (0.5 N, 100 mL), brine (100 mL), dried over anhydrous Na2SO4 and filtered under reduced pressure to give a yellowish white foam. The crude product was recrystallized twice from ethanol and from 2-butanone to give the pharmaceutical grade (7α,11α,17α)-9,11-epoxyester ◦ 2 (5.20 g, 52% yield) as a white crystals. Rf = 0.27 for 10% Me2CO/CH2Cl2. M.p. 241–243 C ◦ 20 −1 (lit. 240–242 C[17]). [α]D = +1.55 (c 1.0, CHCl3). FTIR (pellets, KBr) ν (cm ): 2997, 2969, 2950, 2877, 1778, 1725, 1656, 1619, 1460, 1444, 1427, 1382, 1293, 1273, 1183, 1160, 1081, 1018, 985, 919, 848, + 797, 722, 694, 656, 543, 520, 460. HRMS (ESI): calcd. for C24H30O6Na [M + Na] 437.1940, found 437.1941. 1H- and 13C-NMR spectra, see Table8. The recrystallization mother liquors of 2 (4.1 g) were chromatographed on silica gel using varying mixtures of acetone and dichloromethane. Early cuts of the 1–10% Me2CO/CH2Cl2 gradient elution afforded the (7α,11β,17α)-9,11-dichloro impurity 13 (0.46 g) and the (7β,11α,17α)-9,11-epoxy ester 2a (0.19 g). Succeeding cuts of the 1–10% Me2CO/CH2Cl2 gradient elution gave the 7,9:21,17-dilactone 14 (0.85 g) and the (7α,11α,17α)-11,12-epoxy ester 2b (0.26 g). The characterization data of 2a, 2b, 13 and 14 were identical in all aspects with those obtained in experiments 3.8.3, 3.8.4, 3.8.8 and 3.8.9, respectively.

4. Conclusions Two new process-related impurities of the antihypertensive drug eplerenone (2) were synthesized and fully characterized. The impurities were identified as 11α-hydroxy-7β- (methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (12a) and 9,11β-dichloro-7α- (methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (13). Additionally, seven other eplerenone impurities poorly described in the literature, including four impurities A, B, C and E listed in the European Pharmacopoeia 8.4, were isolated and/or synthesized and fully characterized by Molecules 2017, 22, 1354 25 of 26

IR, NMR, HRMS/ESI and SCXRD. All the impurities resulted from side reactions taking place on the steroid rings B and C of the starting (7α,11α,17α)-11-hydroxyester 12 and the key intermediate (7α,17α)-9(11)-enester 7, including epimerization of the C-7 asymmetric center, oxidation, dehydration, chlorination and lactonization. The full identification and characterization of the impurities should be useful for the quality control and the validation of the analytical methods in the manufacture of eplerenone.

Acknowledgments: Financial support was provided by the European Union from the European Regional Development Fund (ERDF) under the Innovative Economy Operational Programme “Innovative technologies of cardiovascular medicines of special therapeutic and social importance” (POIG.01.03.01-14-062/09-00). The authors would like to kindly acknowledge Anna Witkowska and Krzysztof Kuziak from the R&D Analytical Chemistry Department, Pharmaceutical Research Institute, for recording of IR spectra and measuring optical rotations. The authors also wish to thank Maura Mali´nskaand Krzysztof Wo´zniakfrom Warsaw University for SCXRD analyses of the compounds 2a, 7a, 12, 12a and 13. Author Contributions: Iwona Dams designed and carried out the synthetic experiments, analyzed the data and wrote the paper. Michał Chody´nski, Małgorzata Krupa, Anita Pietraszek and Anna Ostaszewska carried out synthetic experiments. Agata Biało´nskaperformed X-ray analyses and resolved the molecular structure of the compounds 2b, 7 and 7b. Piotr Cmoch carried out NMR experiments and interpreted the spectra. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Rabasseda, X.; Silvestre, J.; Castañer, J. Eplerenone. Drugs Future 1999, 24, 488–501. [CrossRef] 2. De Gasparo, M.; Joss, U.; Ramjoué, H.P.; Whitebread, S.E.; Haenni, H.; Schenkel, L.; Kraehenbuehl, C.; Biollaz, M.; Grob, J.; Schmidlin, J.; et al. Three new epoxy-spirolactone derivatives: Characterization in vivo and in vitro. J. Pharmacol. Exp. Ther. 1987, 240, 650–656. 3. Delyani, J.A.; Rocha, R.; Cook, C.S.; Tolbert, D.S.; Levin, S.; Roniker, B.; Workman, D.L.; Sing, Y.L.; Whelihan, B. Eplerenone: A selective aldosterone receptor antagonist (SARA). Cardiovasc. Drug Rev. 2001, 19, 185–200. [CrossRef][PubMed] 4. Davis, K.L.; Nappi, J.M. The cardiovascular effects of eplerenone, a selective aldosterone-receptor antagonist. Clin. Ther. 2003, 25, 2647–2668. [CrossRef] 5. Burgess, E.D.; Lacourcière, Y.; Ruilope-Urioste, L.M.; Oparil, S.; Kleiman, J.H.; Krause, S.; Roniker, B.; Maurath, C. Long-term safety and efficacy of the selective aldosterone blocker eplerenone in patients with essential hypertension. Clin. Ther. 2003, 25, 2388–2404. [CrossRef] 6. Coleman, C.I.; Reddy, P.; Song, J.C.; White, C.M. Eplerenone. The first selective aldosterone receptor antagonist for the treatment of hypertension. Formulary 2002, 37, 514–524. 7. Muldowney, J.A.S.; Schoenhard, J.A.; Benge, C.D. The clinical pharmacology of eplerenone. Expert Opin. Drug Metab. Toxicol. 2009, 5, 425–432. [CrossRef][PubMed] 8. Dhillon, S. Eplerenone: A review of its use in patients with chronic systolic heart failure and mild symptons. Drugs 2013, 73, 1451–1462. [CrossRef][PubMed] 9. Ng, J.S.; Liu, C.; Anderson, D.K.; Lawson, J.P.; Wieczorek, J.; Kunda, S.A.; Letendre, L.J.; Pozzo, M.J.; Sing, Y.-L.L.; Wang, P.T.; et al. Process for the Preparation of 9,11-Epoxy Steroids and Intermediates Useful Therein. WO 9825948 A2, 18 June 1998. 10. Zhang, J.Y.; Fast, D.M.; Breau, A.P. A validated SPE-LC-MS/MS assay for eplerenone and its hydrolyzed metabolite in human urine. J. Pharm. Biomed. Anal. 2003, 31, 103–115. [CrossRef] 11. Babu, K.S.; Madireddy, V.; Indukuri, V.S. Degradation pathway for eplerenone by validated stability indicating UP-LC method. ISRN Pharm. 2012, 2012, 251248. [CrossRef] 12. Du, M.; Pan, C.; Chen, J.; Song, M.; Zhu, T.; Hang, T. A validated stability-indicating ultra performance liquid chromatography method for the determination of potential process-related impurities in eplerenone. J. Sep. Sci. 2016, 39, 2907–2918. [CrossRef][PubMed] 13. Zhang, J.Y.; Fast, D.M.; Breau, A.P. Development and validation of a liquid chromatography-tandem mass spectrometric assay for eplerenone and its hydrolyzed metabolite in human plasma. J. Chromatoqr. B 2003, 787, 333–344. [CrossRef] 14. Yang, Q.; Ye, W.-D.; Yuan, J.Y.; Nie, J.-J.; Xu, D.-J. Eplerenone ethanol solvate. Acta Cryst. 2008, E64, o829. [CrossRef] Molecules 2017, 22, 1354 26 of 26

15. Yang, Q.; Zhang, B.-Y.; Ye, W.-D.; Yuan, J.-Y.; Xu, D.-J.; Nie, J.-J. Crystal structure of 9α,11-epoxy-7α- (methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone. J. Chem. Crystallogr. 2008, 38, 659–661. [CrossRef] 16. Zhang, B.; Chen, H.; Tang, H.; Feng, H.; Li, Y. A diastereoselective synthesis of 7α-nitromethyl steroid derivative and its use for an efficient synthesis of eplerenone. Steroids 2012, 77, 1086–1091. [CrossRef] [PubMed] 17. Grob, J.; Boillaz, M.; Schmidlin, J.; Wehrli, H.; Wieland, P.; Fuhrer, H.; Rihs, G.; Joss, U.; de Gasparo, M.; Haenni, H.; et al. Stereoidal, aldosterone antagonists: Increased selectivity of 9α,11-epoxy derivatives. Helv. Chim. Acta 1997, 80, 566–585. [CrossRef] 18. Grob, J.; Kalvoda, J. 20-Spiroxanes and Analogues Having an Open Ring E, Process for Their Manufacture and Pharmaceutical Preparations Thereof. EP 122232 B1, 10 April 1984. 19. Pearlman, B.A.; Padilla, A.G.; Hach, J.T.; Havens, J.L.; Pillali, M.D. A new approach to the furan degradation problem involving ozonolysis of the trans-enedione and its use in a cost-effective synthesis of eplerenone. Org. Lett. 2006, 8, 2111–2113. [CrossRef][PubMed] 20. Wuts, G.M.; Anderson, A.M.; Ashford, S.W.; Goble, M.P.; White, M.J.; Beck, D.; Gilbert, I.; Hrab, R.E. A chemobiological synthesis of eplerenone. Synlett 2008, 3, 418–422. [CrossRef] 21. Zhang, B.; Chen, H.; Feng, H.; Li, Y. Stereoselective construction of a steroid 5α,7α-oxymethylene derivative and its use in the synthesis of eplerenone. Steroids 2011, 76, 56–59. [CrossRef][PubMed] 22. Weier, R.M.; Hofmann, L.M. 7α-Carboalkoxy steroidal spirolactones as aldosterone antagonists. J. Med. Chem. 1975, 18, 817–821. [CrossRef][PubMed] 23. Eplerenone. In European Pharmacopoeia 8.4; European Directorate for the Quality of Medicines: Strasburg, France, 2015; Volume 2765, pp. 4749–4750. 24. Preisig, C.L.; Laakso, J.A.; Mocek, U.M.; Wang, P.T.; Baez, J.; Byng, G. Biotransformations of the cardiovascular drugs mexrenone and canrenone. J. Nat. Prod. 2003, 66, 350–356. [CrossRef][PubMed] 25. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline: Impurities in New Drug Substances Q3A (R2). Currsent Step 4 Version. 24 October 2006. Available online: http://www.ich.org/fileadmin/Public_Web_Site/ICH_ Products/Guidelines/Quality/Q3A_R2/Step4/Q3A_R2__Guideline.pdf (accessed on 16 May 2016). 26. Substances for Pharmaceutical Use. In The European Pharmacopoeia 7.0; European Directorate for the Quality of Medicines: Strasbourg, France, 2010; p. 2034. 27. Kelly, R.W.; Sykes, P.J. Synthetic steroids. Part III. The preparation of 3β,15β,17β-trihydroxyandrost-5-ene and the attempted preparation of 3β,15α,17β-trihydroxyandrost-5-ene. J. Chem. Soc. C 1968, 416–421. [CrossRef] 28. Rachwal, S.; Druzgala, P.; Liu, Z.-Z.; Vlasak, J.; Brewster, M.E.; Pop, E. Chemistry of loteprednol etabonate and related steroids. II. Reactions at ring C and NMR structural studies of the resulting compounds. Steroids 1998, 63, 193–201. [PubMed] 29. CrysAlisPro, version 1.171.35.15; Agilent Technologies: Wrocław, Poland, 2011. 30. Oxford Diffraction. CrysAllis CCD and CrysAllis RED, versions 1.171.32.29; Oxford Diffraction Ltd.: Wrocław, Poland, 2008. 31. Sheldrick, G.M. Phase annealing in SHELX-90: Direct methods for larger structures. Acta Crystallogr. A 1990, 46, 467–473. [CrossRef] 32. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. 2008, A64, 112–122. [CrossRef][PubMed] 33. APEXII-2008, version 1.0; Bruker Nonius: Madison, WI, USA, 2007. 34. SAINT, version 7.34A; Bruker Nonius: Madison, WI, USA, 2007. 35. SADABS, Bruker AXS Area Detector Scaling and Absorption Correction, version 2008/1; Bruker AXS: Madison, WI, USA, 2008.

Sample Availability: Samples of the compounds 2, 2a–b, 7, 7a–b, 12, 12a, 13 and 14 are available from the authors.

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).