Synthesis and physicochemical characterization of the process-related impurities of eplerenone, an antihypertensive drug

Article abstract of DOI:10.3390/molecules22081354

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.

Full text of DOI:10.3390/molecules22081354

molecules
Article
Synthesis and Physicochemical Characterization of
the Process-Related Impurities of Eplerenone, an
Antihypertensive Drug
Iwona Dams 1,*, Agata Białon´ska 2, Piotr Cmoch 1, Małgorzata Krupa 1, Anita Pietraszek 1,
Anna Ostaszewska 1 and Michał Chodyn´ski 1
1
R&D Chemistry Department, Pharmaceutical Research Institute, Rydygiera 8, 01-793 Warsaw, Poland;
p.cmoch@ifarm.eu (P.C.); m.krupa@ifarm.eu (M.K.); a.pietraszek@ifarm.eu (A.P.);
a.ostaszewska@ifarm.eu (A.O.); m.chodynski@ifarm.eu (M.C.)
Faculty of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383 Wrocław, Poland;
2
agata.bialonska@chem.uni.wroc.pl
*
Correspondence: i.dams@ifarm.eu; 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
11
α
-hydroxy-7
α
-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (12) and the key
intermediate (7
α
,17 )-9(11)-enester , including epimerization of the C-7 asymmetric center,
α
7
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 (
hypertension and congestive heart failure that, in contrast to its predecessor spironolactone
demonstrates a high degree of selectivity for the aldosterone receptor and a low-binding affinity for
progesterone and androgen receptors [ ]. As a result of the presence of a 9,11-epoxide group in the
2
, Figure 1) is a cardiovascular drug indicated for the treatment of essential
1
(Figure 1)
1–8
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
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Ciba-Geigy AG (Basel, Switzerland) and launched in the US in 2003 by Pharmacia (Sandwich, Kent,
UK) under the trade name Inspra®.
Figure 1. Chemical structures of the aldosterone receptor antagonists spironolactone (Aldactone®,
and eplerenone (Inspra®, 2).
1)
Eplerenone contains sensitive 9,11
depending on the conditions, may hydrolyze or epimerize to afford degradation or epimerization
products [ 13]. The SCXRD structure of eplerenone confirms the relative cis configuration of the
,11 -epoxide ring and the 7 -carbomethoxy substituent [14 17]. Manufacture of eplerenone is
α-epoxide, 17α-γ-lactone and 7α-carbomethoxy moieties that,
9
–
9
α
α
α
–
always accompanied by side reactions leading to the unwanted impurities that vary with the different
starting materials and reaction conditions used. Most of the patents and literature information dealing
with the preparation of eplerenone are based on the use of canrenone derivatives. As stereogenic
centers in eplerenone precursors give rise to various process-related impurities, including diastereo-
and regioisomers of starting materials, intermediates, by-products and the final drug substance, the
manufacture of eplerenone with the required stereochemistry and pharmaceutical grade purity is a
significant challenge. In designing a synthesis of eplerenone from canrenone derivatives, the principal
challenges are the stereoselective introduction of the carbomethoxy substituent at the C-7
the steroid skeleton and the regioselective dehydration of the 11α-hydroxy group [1,9,16–21].
In 1984, Grob et al. [17 18] from Ciba-Geigy AG accomplished the first synthesis of eplerenone
by employing Nagata hydrocyanation of ) as the key step (Scheme 1), but with
9(11)-canrenone (
moderate stereoselectivity in the 7 -cyano derivative formation (7
/7β ≈ 4:1) necessitating tedious
α position of
,
∆
3
α
4
α
column chromatographic separations. The original EP 122232 B1 patent does not disclose any
information with respect to the purity of the material obtained, its purification to pharmaceutical-grade
or the removal of impurities.
Scheme 1. The original eplerenone synthesis from Ciba-Geigy AG (Basel, Switzerland) [17,18]. 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
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In alternative eplerenone synthesis by Ng et al. [
stereoselectively via 4,6-bishydrocyanation of 11
dehydration of the intermediate (7 ,11 ,17
regioisomeric (7 ,17 )-11(12)-enester 7b apart from the main (7
The 7 /7 diastereoisomeric purity of (7 ,11 ,17 )-12 obtained by the cleavage of diketone 11 with
sodium methoxide in refluxing methanol was not reported; however, the literature sources on syntheses
of related 7 -carboalkoxy stereoidal spirolactones provide information on their 7 -carboalkoxy
asymmetric center under
9
], the 7
α
-carbomethoxy group was introduced
α
-hydroxycanrenone (
)-11-hydroxyester 12 was problematic giving the
,17 )-9(11)-enester product (Table 1).
9, Scheme 2), but regioselective
α
α
α
α
α
α
α
7
α
β
α
α
α
α
β
diastereoisomers resulting from basic epimerization of the steroid C-7
α
the same conditions as used for diketone 11 cleavage [22].
Scheme 2. Synthesis of EP from G. D. Searle & Co. (Chicago, IL, USA) [9]. Reagents and Conditions:
(1) Aspergillus ochraceus; (2) LiCl, DMF, Et3N, Me2CO, (CH3)2C(OH)CN; (3) HCl, MeOH, H2O;
(4) MeONa, MeOH; (5) SO2Cl2, ImH, THF; (6) H2O2, K2HPO4, Cl3CCONH2.
When the authors repeated the preparation taught in WO 9825948 A2 it was observed that
the purity of the resulting eplerenone was low and attempts to purify the resulting product to
a level meeting the requisite specifications for use as a pharmaceutical active were unsuccessful.
Although the epimeric 11-hydroxyester (7
other literature sources, it contaminated the main (7
the regioisomeric (7 ,11 ,12 ,17 )-11,12-epoxyester 2b (Imp. E), the new (7
derivative 13 and the 7 ,9:21,17-dicarbolactone 14 (Imp. A) were difficult to remove from the final
product. All these contaminants resulted from side reactions taking place on the steroid ring C of the
(7 ,11 ,17 )-11-hydroxy derivative (12) and the key intermediate (7 ,17 )-9(11)-enester , including
β,11α
,17
α
)-12a was not reported by Ng et al. [
,17 )-9(11)-enester product. In particular,
,11 ,17 )-9,11-dichloro
9], or any
α
α
7
α
α
α
α
α
β
α
α
α
α
α
α
α
7
epimerization of the C-7α asymmetric center, oxidation, dehydration, chlorination and lactonization.
Recently, we have developed an improved, scalable, cost-effective and environment-friendly
technology for the industrial-scale synthesis of eplerenone (
11 -hydroxy-7 -(methoxycarbonyl)-3-oxo-17 -pregn-4-ene-21,17-carbolactone (12), based on the
last two steps of the general route described by Ng et al. (Scheme 2) [ ]. During the
2) from commercially available
α
α
α
9
process development, two new and seven known process-related impurities of eplerenone
were observed, and/or synthesized and fully characterized, including four impurities A,
B, C and E listed in the European Pharmacopoeia 8.4 [23].
identified as 11 -hydroxy-7 -(methoxycarbonyl)-3-oxo-17 -pregn-4-ene-21,17-carbolactone (12a) and
9,11 -dichloro-7 -(methoxycarbonyl)-3-oxo-17 -pregn-4-ene-21,17-carbolactone (13). To the best of
The new impurities were
α
β
α
β
α
α
our knowledge, the 11-hydroxyester 12a and the 9,11-dichloro derivative 13 are new compounds that
have never been identified before.
The syntheses of the starting (7
α
,11
α
,17
α
)-11-hydroxyester 12 and the key intermediate
16 21 24]; however, their
(7α
,17 )-9(11)-enester are broadly described in the literature sources [9, – ,
α
7

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
comprehensive structural elucidation and confirmation is essential for impurities identification and
characterization. A complete physicochemical characterization, not only for an API, but also starting
materials and key synthetic intermediates, became a requirement of both the U.S. Food and Drug
Administration (FDA) and the European Medicine Agency (EMA). The five other impurities listed
in Table 1, i.e., the isomeric (7
(7 ,11 ,17 )-9,11-epoxyester 2a (Imp. E) and (7
the 7 ,9:21,17-dicarbolactone 14 (Imp. A), were mentioned elsewhere in the literature, mainly as
part of HPLC studies; however, their syntheses, methods of removal from the final product and
comprehensive structural elucidation and confirmation were not disclosed [ 12]. The determination
β,17
α
)-9(11)-enester 7a and (7
α
,17α)-11(12)-enester 7b, the isomeric
β
α
α
α
,11 ,12 ,17
α
α
α)-11,12-epoxyester 2b (Imp. B) and
α
9,
of a drug substance impurity profile, including starting materials, by-products, intermediates and
potential degradation products, is critical for the safety assessment of API and manufacturing process
thereof. In any API, it is necessary to study the impurity profile, and control it during the preparation
of the pharmaceutical. As indicated in the ICH guidelines, any impurity, formed at a level of ≥0.10%
with respect to the API, should be identified, synthesized and characterized thoroughly [25,26]. Only
two eplerenone impurities, i.e., 7α,9:21,17-dicarbolactone 14 (Imp. A) and 11,12-epoxy ester 2b (Imp.
B), are accepted at a level 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).
Chemical Structure
Name
11α-hydroxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone
12
12a
11α-hydroxy-7β-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone
7α-(methoxycarbonyl)-3-oxo-17α-pregna-4,9(11)-diene-21,17-carbolactone
7β-(methoxycarbonyl)-3-oxo-17α-pregna-4,9(11)-diene-21,17-carbolactone
7α-(methoxycarbonyl)-3-oxo-17α-pregna-4,11(12)-diene-21,17-carbolactone
7 (Imp. C) 1
7a
7b

Molecules 2017, 22, 1354
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Table 1. Cont.
Chemical Structure
Name
9,11β-dichloro-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone
13
3-oxo-17α-pregn-4-ene-7α,9:21,17-dicarbolactone
14 (Imp. A) 1
2a (Imp. E) 1
2b (Imp. B) 1
9,11α-epoxy-7β-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone
11α,12α-epoxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone
1
Impurities listed in the European Pharmacopoeia 8.4 [23].
The present paper deals with synthesis, physicochemical characterization and comprehensive
structural elucidation and confirmation of the process-related eplerenone impurities found in samples of
the key intermediate (7
to the modified and optimized procedure described in Searle Co. patent [
material). Apart from the starting material 12, the key intermediate enester and the final product
process-related eplerenone impurities 2a 7a 12a 13 and 14 were fully characterized by IR, NMR
and MS. The molecular structures of the epimeric 11-hydroxyesters (7 ,11 ,17 )-12 and (7 ,11 ,17 )-12a
the isomeric (7 ,17 )-9(11)-enester , (7 ,17 )-9(11)-enester 7a and (7 ,17 )-11(12)-enester 7b, the
isomeric (7 ,11 ,17 )-9,11-epoxyester 2a and (7 ,11 ,12 ,17 )-11,12-epoxyester 2b, and the new
(7 ,11 ,17 )-9,11-dichloro derivative 13, were solved and refined using SCXRD. The full identification
α,17α)-9(11)-enester
7
and eplerenone bulk drug substance manufactured according
] (Scheme 2, 12 as starting
, seven
9
7
2
–b,
–b,
,
α
α
α
β
α
α
,
α
α
7
β
α
α
α
β
α
α
α
α
α
α
α
β
α
and characterization of these compounds should be useful for the quality control and the validation of the
analytical methods in the manufacture of eplerenone.
2. Results and Discussion
Dehydration of 11-hydroxy steroids is commonly used for the introduction of the 9,11-double
bond into the steroid skeleton. In a preferred technological embodiment, the (7α,17α)-9(11)-enester
7
is synthesized directly by in situ replacement of the 11 -hydroxy group of the ester (7
α
α
,11 ,17 )-12
α
α
with halogen followed by thermal 9,11-dehydrohalogenation. The nucleophilic substitution of the
◦
11
α
-hydroxy group is effected by reaction with sulfuryl halide at about
−
70 C, after which a hydrogen
halide scavenger is added. In manufacturing technology of eplerenone developed by the authors,
the 11
and cooled to
α
-hydroxyester 12 and imidazole were dissolved simultaneously in anhydrous tetrahydrofuran
◦
−
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 was isolated in a crude form by dichloromethane
extraction followed by evaporation of the solvent and recrystallized twice, from ethanol and a mixture
7

Molecules 2017, 22, 1354
6 of 26
of dichloromethane/diethyl ether respectively, to give the pure (7
white crystals.
α
,17
α
)-9(11)-enester
7
(71% yield) as
The dehydration step produced the three main side products, i.e., the regioisomeric (7
11(12)-enester 7b, the new (7 ,11 ,17 )-9,11-dichloro derivative 13 and the 7 ,9:21,17-dicarbolactone
14 (Imp. A), which were isolated chromatographically from the recrystallization mother liquors of
by elution with varying mixtures of ethyl acetate/dichloromethane. Further gradient elution with
acetonitrile/toluene afforded the new 11-hydroxyester (7 ,11 ,17 )-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 (7 ,11 ,17 )-12 was synthesized according to the procedure
described by Ng et al. [ ] and the chemical purity declared by the manufacturer was 98%. The novel
11-hydroxyester (7 ,11 ,17 )-12a was not previously detected by chromatographic or spectroscopic
methods in its commercially available (7 ,11 ,17 )-12 isomer. However, the literature data on
syntheses of related 7 -carboalkoxy steroidal spirolactones [22] allowed us to assume that the new
11-hydroxyester (7 ,11 ,17 )-12a originates from the fourth step of the Scheme 2 synthesis in which
sodium methoxide reacts with diketone 11 to afford the (7 ,11 ,17 )-12 isomer as the main product of
the cleavage, which is contaminated with the (7 ,11 ,17 )-12a epimer resulting from the competitive
basic epimerization of 12. The 11-hydroxyester (7 ,11 ,17 )-12a was synthesized independently
by basic epimerization of the (7 ,11 ,17 )-12 isomer. As expected, sodium methoxide in methanol
solution converted the C-7 ester 12 into an epimeric mixture of the C-7α 12 and C-7β 12a esters.
The pure C-7 ester 12a was isolated chromatographically from the crude mixture of epimers 12
and 12a by 10–50% acetonitrile/toluene gradient elution to give (7 ,11 ,17 )-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 (7 ,11 ,17 )-12. Even a small amount of the newly
detected (7 ,11 ,17 )-11-hydroxyester 12a is hardly separable from its (7 ,11 ,17 )-12 isomer by large
scale chromatography or recrystallization, therefore, the commercial 11-hydroxyester (7 ,11 ,17 )-12
was used further without purification. As the two 11-hydroxyesters of the C-7 and C-7 series,
β,17α)-
α
β
α
α
7
β
α
α
α
α
α
9
β
α
α
α
α
α
α
β
α
α
α
α
α
β
α
α
β
α
α
α
α
α
α
β
β
α
α
α
α
α
β
α
α
α
α
α
α
α
α
α
β
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
Table 2. 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 Figure 2 show the most important NOE effects involving H7 proton, simultaneously clearly
indicating the 7
the compound 12 with
α
or 7
β
positioned carbomethoxy group. The strong H7-H8 NOE effect is observed for
positioned H7 proton, whereas situated H7 proton in 12a is involved in two
β
α
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
0.71 ppm for H9 and 0.28 ppm for H14 nuclei is observed when passing from the compound 12 with
positioned carbomethoxy group to its 7 epimer 12a. Additionally, the change of configuration
at the C7 is accompanied by H8 (0.35 ppm) shielding decrease. Simultaneously, both diasterotopic
α or β
configuration of the C-7 atom. The 1H shielding increase of 0.5 ppm for H7,
7
α
β
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
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(a)
(b)
Figure 2. Crucial 1H- and 13C-NMR chemical shifts and NOE effects for 11-hydroxyesters:
(a) (7α,11α,17α)-12; (b) (7β,11α,17α)-12a.
1
Table 2. The H- and 13C-NMR spectral data in CDCl3 for 11-hydroxyesters (7
α
,11α
,17α
)-12 and
(7β,11α,17α)-12a.
13C-NMR
12
1H-NMR (1)
12
Proton
12a
Carbon
12a
H-1a
H-1b
H-2a
H-2b
H-4
H-6a
H-6b
H-7
2.02 m
2.80 m
2.34 m
2.46 m
5.69 s
2.07 m
2.71 m
2.33 m
2.43 m
5.75 s
2.37 m
2.62 m
2.31 m
2.14 m
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
37.3
34.1
199.5
126.1
167.7
36.3
42.4
37.0
52.6
39.4
69.2
43.4
46.2
45.3
22.5
35.4
95.0
15.4
18.4
31.2
29.1
176.5
172.7
51.6
37.1
34.2
199.9
125.0
167.2
37.3
49.0
37.0
57.4
39.6
68.5
43.5
46.6
48.7
22.5
35.6
94.5
16.1
18.4
31.4
29.2
176.5
175.4
52.1
2.52 m
2.64 ddd (1.9, 5.8, 14.6)
2.81 m
H-8
H-9
1.79 m
1.93 m
4.04 m
1.42 m
1.85 m
1.65 m
1.41 m
1.73 m
1.22 t (10.4)
4.10 m
1.42 m
H-11
H-12a
H-12b
H-14
H-15a
H-15b
H-16a
H-16b
H-18
H-19
H-20a
H-20b
H-21a
H-21b
H-24
1.83 dd (4.6, 11.6)
1.37 m
1.49 m
1.49 m
1.80 ddd (6.9, 8.8, 14.3 )
2.21 ddd (4.4, 11.4, 14.3)
1.03 s
1.88 m
2.26 ddd (3.6, 12.1, 14.5)
1.01 s
1.36 s
1.95 m
2.39 m
2.50 m
2.57 m
3.65 s
1.40 s
1.93 ddd (7.5, 9.4, 13.0)
2.37 m
2.49 ddd (11.6, 13.1, 17.7)
2.56 ddd (6.2, 9.3, 17.7)
3.69 s
1
Values in parentheses give δ, multiplicity and J in Hz.
The NMR structure assignments of the isomeric 11-hydroxyesters (7
,11 ,17 )-12a were confirmed by X-ray analysis (Figure 3 and Table 3). The SCXRD structures
confirmed the relative cis- and trans-configurations of the 11 -hydroxy and 7 -carbomethoxy
substituents in 12 and 12a, respectively. The carbomethoxy group is situated at the C-7 position in 12
and the C-7 position in 12a, whereas the hydroxy group adopts the C-11 position in both 12 and 12a
isomers. The dehydration of 11 -hydroxy steroids leads predominantly to the formation of the double
bond between C-9 and C-11 carbons of the steroid skeleton. As expected, the (7 ,17 )-9(11)-enester
was the main product of the 11-hydroxyester 12 dehydration with sulfuryl chloride in the presence of
α,11α,17α)-12 and
(7β
α
α
α
α
α
β
α
α
α
α
7

Molecules 2017, 22, 1354
8 of 26
imidazole; however, a considerable amount of the regioiosmeric (7
α
,17
α
)-11(12)-enester 7b, as a result
of competitive 11,12-dehydration of 12, 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.
Table 3. Summary of crystallographic data and structure refinement for the 11-hydroxyesters
(7α,11α,17α)-12 and (7β,11α,17α)-12a.
Identification Code
Chemical formula
Molecular weight
Temperature
12
C24H32O6
12a
C24H32O6
416.50 g/mol
100(2) K
416.50 g/mol
100(2) K
Wavelength
0.71073 Å
0.71073 Å
Crystal system, space group
Orthorhombic, P212121
a = 6.7560(2) Å
b = 16.1436(5) Å
c = 19.0947(6) Å
Orthorhombic, P212121
a = 12.2862(3) Å
b = 12.4532(3) Å
c = 13.3060(4) Å
Unit cell dimensions
◦
◦
α = 90
α = 90
◦
◦
β = 90
β = 90
◦
◦
γ = 90
γ = 90
2082.59(11) Å3
4, 1.328 mg/m3
0.094
2035.85(9) Å3
4, 1.359 mg/m3
0.096
Volume
Z, Calculated density
Absorption coefficient
F(000)
896
896
Crystal size
0.30 × 0.20 × 0.20 mm
0.30 × 0.20 × 0.10 mm
◦
◦
◦
◦
Theta range for data collection
2.13 –25.68
2.26 –25.67
−8 ≤ h ≤ 8
−19 ≤ k ≤ 19
−23 ≤ l ≤ 23
−14 ≤ h ≤ 14
−15 ≤ k ≤ 15
−16 ≤ l ≤ 16
Limiting indices
Reflections collected/unique
Absorption correction
21,220/3973
11,318/3856
semi-empirical from equivalents
0.9814 and 0.99723
full-matrix least-squares on F2
3973/0/275
semi-empirical from equivalents
0.9904 and 0.9716
full-matrix least-squares on F2
3856/1/275
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
1.156
1.041
Final R1/wR2 indices [I > 2sigma(I)]
R1/wR2 indices (all data)
Largest diff. peak and hole
0.0360/0.0921
0.0311/0.0721
0.0481/0.1132
0.191 and −0.341 e/Å3
0.0361/0.0756
0.172 and −0.212 e/Å3

Molecules 2017, 22, 1354
9 of 26
Additionally, the 9,11-dehydration of the residual 11-hydroxyester (7
contaminating the starting 12 isomer afforded the epimeric (7 ,17 )-9(11)-enester 7a, which was
also isolated chromatographically from recrystallization mother liquors of . It was synthesized
independently by epimerization of the (7 ,17 )-9(11)-enester with sodium methoxide in methanol,
or dehydration of the 11-hydroxyester (7 ,11 ,17 )-12a with sulfuryl chloride and imidazole, and
β,11α,17α)-12a
β
α
7
α
α
7
β
α
α
then purified by column chromatography with 5–20% ethyl acetate/dichloromethane gradient elution
to give the pure (7β,17α)-9(11)-enester 7a, with 37% or 85% yield respectively, as a white solid.
The [M + Na]+ values, m/z 421.1986, 421.2006, and 421.1988, obtained for the free isomeric enesters
7,
7a and 7b correspond to C24H30O5Na. The full NMR data confirming the structure of the known enester
were reported by Grob et al. [17]; however several 1H and 13C signals were assigned reversibly. The
proper 1H/13C chemical shift assignment for isomeric enesters
7a and 7b was based on the careful
analysis of 2D NMR experiments and is given in Table 4. Similarly to the epimeric hydroxyesters 12
and 12a, the NOE effects involving H7 proton allowed distinction between 7 and 7b) and 7 7a
diastereoisomers (Figure 4). The strong H7-H8 NOE effect was noted for isomers and 7b with
positioned H7 proton, whereas situated H7 in 7a is involved in H7-H14 NOE effect disturbed by H7-H15
interaction. Additionally, the significant H9-H14 NOE effect for 7b regioisomer was observed, while H8
proton of the epimers and 7b is involved in H8-H6 interaction. Similarly to the epimeric hydroxyesters 12
and 12a, some of the 1H/13C nuclei shieldings within the steroid rings B, C and D are related to the
or
configuration of the C-7 atom. The 1H shielding increase of 0.7 ppm for H7 and 0.22 ppm for H8 nuclei was
observed when passing from the compound with 7 positioned carbomethoxy group to its 7 epimer 7a
Similarly to 12a, both diasterotopic H15 protons of the epimer 7a with 7 positioned carbomethoxy group
became equal having the same proton chemical shift (1.54 ppm). In the case of 13C-NMR the opposite
effect is observed, the transition from to 7a leads to the shielding decrease of 5.9 ppm for C7 and 2.4
ppm for C14 nuclei. The change of configuration from 7 in to 7 in 7a is related with no change of
the shielding of the C8 nucleus and decreasing of shielding by 2.3 ppm for C23 carbon of carbomethoxy
substituent. The dehydration of the epimeric 11-hydroxyesters 12 12a to the corresponding 9(11)-enesters
7a caused the strong 1H shielding decrease of 89.7 ppm/84.7 ppm for C9 and 49.8 ppm/51.9 ppm for
7
7,
α
(7
β ( )
7
β
α
7
α
β
7
α
β
.
β
7
α
7
β
/
7/
C11 carbons, respectively. The transition from 12/12a to 7/7a also resulted in medium shielding increase
of 10.5/10.8 ppm for C12 and shielding decrease of 8.8/7.3 ppm for C19 and 3.4/3.4 ppm for C8, whereas
for C10 (1.1/1.3 ppm), C13 (1.7/1.9 ppm) and C14 (2.2/3.2 ppm) a weak increasing effect was observed.
The competitive dehydration of 12 to the regioisomeric 11,12-enester 7b resulted in the strong 1H shielding
decrease of 57.3 ppm for C11 and 90.4 ppm for C12 nuclei. Similarly to the hydroxyesters 12 and 12a, the
change of configuration from 7α in 7 to 7β in 7a is related with no change of the shielding of the C8 nucleus
and decreasing of shielding by 2.3 ppm for C23 carbon of the carbomethoxy substituent. Additionally, the
shielding increase of 4.5 ppm for C8 carbon was observed when passing from epimeric 9(11)-enesters
7
and 7a to regioisomeric 11-enester 7b.
(a)
(b)
(c)
)-9(11)-ene 7;
Figure 4. Crucial 1H- and 13C-NMR chemical shifts and NOE effects for esters: (
(b) (7β,17α)-9(11)-ene 7a; (c) (7α,17α)-11-ene 7b.
a) (7α
,17
α

Molecules 2017, 22, 1354
10 of 26
Table 4. The 1H- and 13C-NMR spectral data for esters (7
α
,17α)-9(11)-ene 7, (7β,17α)-9(11)-ene 7a and
(7α,17α)-11-ene 7b.
1H-NMR (1)
13C-NMR
7
Proton
7
7a
7b
Carbon
7a
7b
H-1a
H-1b
H-2a
H-2b
H-4
H-6a
H-6b
H-7
2.18 m
2.24 m
2.53 m
2.53 m
5.71 d (2.0)
2.55 m
2.12 m
2.12 m
2.50 m
2.50 m
1.87 m
2.15 m
2.45 m
2.45 m
5.81 s
2.67 m
2.67 m
2.89 m
2.24 m
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
33.7
34.2
33.9
34.2
34.9
33.7
198.7
126.8
166.3
35.6
42.1
35.9
49.2
38.8
126.5
133.8
49.0
44.1
21.8
35.7
93.9
18.6
17.3
32.3
28.7
176.7
173.0
51.6
198.6
125.7
166.6
35.7
43.8
40.4
142.3
40.5
119.0
32.9
44.5
43.1
23.3
35.4
95.1
14.1
27.2
31.4
29.2
176.5
172.6
51.4
198.6
124.6
165.5
36.2
49.7
40.4
142.1
40.9
120.4
32.7
44.7
45.5
23.8
35.4
94.5
14.3
25.7
31.2
29.1
176.4
174.9
52.1
5.75 d (2.0)
2.43 dd (3.8, 14.6)
2.85 ddd (2.0, 13.2, 14.6)
2.28 m
2.82 ddd (2.0, 5.3, 15.0)
2.98 m
H-8
H-9
2.48 m
-
5.65 m
1.87 m
2.20 m
1.49 m
1.49 m
1.89 m
1.87 m
2.31 m
0.95 s
1.39 s
1.92 m
2.29 m
2.50 m
2.70 m
-
5.68 m
1.88 m
2.17 m
1.50 m
1.54 m
1.54 m
1.81 m
2.26 m
0.96 s
1.42 s
1.93 m
2.30 m
2.51 m
2.60 m
2.44 m
H-11
H-12a
H-12b
H-14
H-15a
H-15b
H-16a
H-16b
H-18
H-19
H-20a
H-20b
H-21a
H-21b
H-24
5.59 dd (2.1, 10.2)
5.89 dd (2.9, 10.2)
-
1.95 m
1.57 m
1.87 m
1.95 m
2.20 m
1.05 s
1.16 s
2.00 m
2.21 m
2.61 m
2.53 m
3.66 s
2.59 ddd (6.0, 9.4, 17.8)
3.59 s
3.72 s
1
Values in parentheses give δ, multiplicity and J in Hz.
The NMR structure assignments of the isomeric (7
and (7 ,17 )-11(12)-enester 7b were confirmed by X-ray analysis (Figure 5 and Tables 5 and 6). The SCXRD
structures confirmed the presence of the 9,11-double bond in and 7a and the 11,12-double bond in 7b
The carbomethoxy group is situated at the C-7α position in 7 and 7b and at the C-7β position in 7a.
α,17α)-9(11)-enester 7, (7β,17α)-9(11)-enester 7a
α
α
7
.
(a)
(b)
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 (7
(7α,11β,17α)-9,11-dichloro derivative 13.
α
,17
α
)-11(12)-enester 7b and
Identification Code
Chemical formula
Molecular weight
Temperature
7b
C24H30O5
13
C24H31Cl2O5
470.39 g/mol
100(2) K
398.48 g/mol
100(2) K
Wavelength
0.71073 Å
0.71073 Å
Crystal system, space group
Orthorhombic, P212121
a = 6.2322(5) Å
b = 15.8874(13) Å
c = 20.5238(15) Å
Orthorhombic, P212121
a = 9.2383(4) Å
b = 14.2496(6) Å
c = 16.3898(7) Å
Unit cell dimensions
◦
◦
α = 90
α = 90
◦
◦
β = 90
β = 90
◦
◦
γ = 90
γ = 90

Molecules 2017, 22, 1354
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Table 5. Cont.
Identification Code
Volume
7b
2032.1(3) Å3
4, 1.302 mg/m3
0.090
13
2157.59(16) Å3
4, 1.448 mg/m3
0.336
Z, Calculated density
Absorbtion coefficient
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
−19 ≤ k ≤ 19
−25 ≤ l ≤ 25
−11 ≤ h ≤ 11
−17 ≤ k ≤ 17
−19 ≤ l ≤ 19
Limiting indices
Reflections collected/unique
Absorption correction
35,771/4149
21,917/4092
semi-empirical from equivalents
0.9866 and 0.9735
full-matrix least-squares on F2
4149/0/265
semi-empirical from equivalents
0.9671 and 0.9058
full-matrix least-squares on F2
4092/0/283
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
1.017
1.024
Final R1/wR2 indices [I > 2sigma(I)]
R1/wR2 indices (all data)
Largest diff. peak and hole
0.0319/0.0883
0.0368/0.0698
0.0337/0.0903
0.295 and −0.135 e/Å3
0.0515/0.0749
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
Chemical formula
Molecular weight
Temperature
7
C24H30O5
7a
C24H30O5
398.48 g/mol
100(2) K
398.48 g/mol
100(2) K
Wavelength
0.71073 Å
0.71073 Å
Crystal system, space group
Orthorhomblic, P212121
a = 6.296(2) Å
b = 15.468(3) Å
c = 21.088(3) Å
Orthorhomblic, P212121
a = 12.099(3) Å
b = 12.310(3) Å
c = 13.497(3) Å
Unit cell dimensions
◦
◦
α = 90
α = 90
◦
◦
β = 90
β = 90
◦
◦
γ = 90
γ = 90
2053.7(8) Å3
4, 1.289 mg/m3
0.089
2010.2(8) Å3
4, 1.317 mg/m3
0.091
Volume
Z, Calculated density
Absorbtion coefficient
F(000)
856
856
Crystal size
0.41 × 0.32 × 0.29 mm
0.60 × 0.39 × 0.31
3.310 −36.896
◦
◦
◦
◦
Theta range for data collection
3.183 −28.719
−8 ≤ h ≤ 8
−19 ≤ k ≤ 16
−27 ≤ l ≤ 28
−15 ≤ h ≤ 20
−19 ≤ k ≤ 20
−22 ≤ l ≤ 22
Limiting indices
Reflections collected/unique
Absorption correction
14,340/4925
36,105/9538
None
None
Max. and min. transmission
Refinement method
0.975 and 0.964
full-matrix least-squares on F2
4925/0/262
0.972 and 0.947
full-matrix least-squares on F2
9538/0/262
Data/restraints/parameters
Goodness-of-fit on F2
0.929
0.975
Final R1/wR2 indices [I > 2sigma(I)]
R1/wR2 indices (all data)
Largest diff. peak and hole
0.0475/0.1011
0.0673/0.1071
0.189/−0.220 e/Å3
0.0484/0.1205
0.0622/0.1259
0.453/−0.303 e/Å3

Molecules 2017, 22, 1354
12 of 26
Sulfuryl chloride used for dehydration of 11
α
-hydroxy steroids is also known as a chlorinating
agent and, under some conditions, applied for chlorination of steroid double bonds [27
the novel (7 ,11 ,17 )-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 (7 ,17 )-9(11)-enester , was isolated
chromatographically from the recrystallization mother liquors of . It was also synthesized by
chlorination of the pure enester 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 (7 ,11 ,17 )-9,11-dichloro derivative 13 (41% yield) as a white solid. The novel 9,11-dichloro
,28]. Indeed,
α
β
α
α
α
7
7
7
α
β
α
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-9
α 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 (Table 7).
Table 7. The 1H- and 13C-NMR spectral data in CDCl3 for (7
and 7α,9:21,17-dicarbolactone 14.
α
,11β,17α
)-9,11-dichloro derivative 13
13C-NMR
1H-NMR (1)
Proton
13
14
Carbon
13
14
H-1a
H-1b
H-2a
H-2b
H-4
H-6a
H-6b
H-7
2.27 m
2.77 m
2.50 m
2.50 m
5.89 d (1.8)
2.60 m
2.93 m
2.83 m
2.90 m
-
1.88 m
2.27 m
2.49 m
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
29.6
33.8
197.8
125.7
163.7
32.4
40.9
41.1
86.6
47.1
59.5
37.0
45.0
41.3
23.6
35.3
95.9
17.2
26.1
31.5
29.1
176.3
172.3
52.2
29.6
33.2
197.1
129.4
161.5
34.5
45.4
45.1
90.0
43.7
22.4
26.9
44.0
42.7
23.4
35.2
94.8
13.4
19.1
31.0
29.0
176.2
176.5
-
2.49 m
5.86 d (2.0)
2.65 dd (4.0, 15.4)
2.80 d (15.4)
2.68 m
H-8
H-9
2.44 d (11.2)
-
1.84 m
2.05 ddd (2.0, 4.7, 15.2)
H-11a
H-11b
H-12a
H-12b
H-14
H-15a
H-15b
H-16a
H-16b
H-18
H-19
H-20a
H-20b
H-21a
H-21b
H-24
4.68 dd (2.0, 4.8)
-
1.98 m
2.54 m
2.46 m
1.57 m
2.32 m
1.97 m
2.33 m
1.30 s
1.56 m
1.61 m
1.33 m
1.49 m
1.84 m
1.92 m
2.31 m
0.99 s
1.86 s
1.40 s
2.06 m
2.39 m
2.48 m
2.60 m
3.61 s
1.91 m
2.35 ddd (3.6, 5.6, 13.0)
2.60 ddd (6.7, 9.2, 17.7)
2.51 m
-
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 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 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
7
7

Molecules 2017, 22, 1354
13 of 26
nuclei in dichloro derivative 13. Noteworthy, the diasterotopic effect observed for H15 protons of
(0.4 ppm) increased to 0.75 ppm for the same protons in 13. Similarly to the other compounds with
-carbomethoxy group, the positioned H7 proton in dichloro derivative 13 is involved in strong
H7-H8 NOE effect (Figure 6). The NMR structure assignment of the novel (7 ,11 ,17 )-9,11-dichloro
derivative 13 was confirmed by X-ray analysis (Figure 7 and Table 5). The SCXRD structure confirmed
the presence of the two chlorine atoms at the C-9 and C-11 positions of the steroid ring C in the
relative trans configuration and the C-7α positioned carbomethoxy substituent.
7
7
α
β
α
β
α
α
β
(a)
(b)
) (7
Figure 6. Crucial 1H- and 13C-NMR chemical shifts and NOE effects for: (
derivative 13; (b) 7α,9:21,17-dicarbolactone 14.
a
α,11β,17α)-9,11-dichloro
(a)
(b)
) (7 ,11β,17α)-9,11-dichloro
Figure 7. Molecular structure of (
derivative 13.
a) (7
α
,17α
)-11-enester 7b and (
b
α
The acidic conditions applied for the dehydration of the 11
in competitive lactonization between the hydroxyl and carbomethoxy groups and lead to
,9:21,17-dicarbolactone 14 (Imp. A), which tends to be formed even in the presence of traces of
free water. The dicarbolactone 14 was isolated chromatographically from the recrystallization mother
liquors of 7. It was also synthesized independently from the mesylate of the starting 11 -hydroxyester
α-hydroxy group in 12 resulted
7
α
α
12 by reaction with acetic acid in the presence of sodium acetate, and then purified by column
chromatography with 5–30% ethyl acetate/dichloromethane gradient elution to afford the pure
7α,9:21,17-dicarbolactone 14 (83% yield) as a white solid.
The [M + Na]+ value, m/z 407.1847, obtained for the dicarbolactone 14 corresponds to C23H28O5Na.
1
The H-/13C-NMR chemical shifts assignment for dilactone 14 is presented in Table 7. The detailed
1
analysis of 2D NMR spectra, especially H-13C HMBC and NOESY experiments, unambiguously

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 7
proton of the dicarbolactone 14 is involved in two strong H7-H8 and H6-H7 NOE effects (Figure 6).
The purification of the key intermediate (7 ,17 )-9(11)-enester by large-scale recrystallization
only partially removes the impurities formed during the dehydration of the starting 11 -hydroxyester
12. Thus, in a preferred technological embodiment, the key intermediate enester was isolated in a
α positioned carbomethoxy group, the H7
α
α
7
α
7
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 (7
were present in the crude enester
the isomeric (7 ,11 ,17 )-9,11-epoxyester 2a and (7
isolated chromatographically from the recrystallization mother liquors of
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 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 with
1–10% acetone/dichloromethane gradient elution gave the (7 ,11 ,12 ,17 )-11,12-epoxyester 2b, which
was undoubtedly formed via epoxidation of the (7 ,17 )-9(11)-enester 7b. It was also synthesized
α
,11
β
,17
α
)-9,11-dichloro derivative 13 and the 7
, the epoxidation step produced the two further impurities, i.e.,
,11 ,12 ,17 )-11,12-epoxyester 2b, which were
. The trichloroacetamide
α,9:21,17-dicarbolactone 14 (Imp. A), which
7
β
α
α
α
α
α
α
2
7
2
α
α
α
α
α
α
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
2a, which was formed by epoxidation of the (7
intermediate enester . 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 ( ), and then purified by column chromatography to give the pure
(7 ,11 ,17 )-9,11-epoxyester 2a, with 69% or 49% yield respectively, as a white solid. The recrystallization
of the crude eplerenone ( ) from ethanol removes the new 9,11-dichloro derivative 13, the isomeric
)-9,11-epoxyester 2a and the residuals of the unreacted isomeric (7 ,17 )-9(11)-enester 7a and
)-9(11)-enester 7b. The recrystallization from 2-butanone removes the 7 ,9:21,17-dicarbolactone
2
with 1–10% acetone/dichloromethane gave the isomeric (7β,11α,17α)-9,11-epoxyester
β
,17 )-9(11)-enester 7a contaminating the crude
α
7
2
β
α
α
2
(7β,11α,17α
β
α
(7α,17α
α
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 (Table 8).
Blue arrows in Figure 8 show the most important NOE effects involving H7 proton, simultaneously
indicating the 7
the compounds
α
or 7
and 2b with
β
positioned carbomethoxy group. The strong H7-H8 NOE effect is observed for
positioned H7 proton, whereas situated H7 proton in 2a is involved
2
β
α
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
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 7a to epoxides 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
7a, some of the 1H/13C nuclei shielding within the steroid rings B, C and D are related to
the or
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 with 7 positioned carbomethoxy group to
its 7 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 7
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
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
in to 7 in 2a is also related with shielding decrease of 2.1 ppm for C23 carbon of the carbomethoxy
7/7a to epoxides 2/2a, respectively. Minor shielding increase of 7/6.5 ppm
7/
2/
/
7/
α
β
2
α
β
β
2
7α
2
β
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 (7
α,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
H-1b
H-2a
H-2b
H-4
H-6a
H-6b
H-7
1.42 m
2.19 m
2.43 m
2.47 m
5.89 s
2.72 m
2.72 m
2.89 m
1.46 ddd (2.8, 5.2, 13.2)
2.02 m
2.30 ddd (2.6, 5.0, 13.4)
2.47 dddd (0.8, 2.6, 5.0, 17.5)
2.52 ddd (5.0, 14.3, 17.5)
5.81 s
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
26.7
33.0
197.9
127.0
165.0
34.8
41.2
38.6
65.2
39.7
51.4
30.9
43.8
37.2
22.0
34.9
94.5
16.1
22.2
30.8
28.8
176.1
172.5
51.5
27.4
33.2
197.9
126.8
163.1
36.2
46.3
38.8
67.1
40.0
53.6
30.7
44.3
39.4
22.4
35.1
93.9
16.5
21.0
30.9
29.0
176.1
174.6
52.2
34.7
33.4
198.2
126.6
165.3
35.5
41.7
35.0
45.9
38.2
50.4
56.7
47.2
37.4
21.2
34.5
94.0
14.2
17.9
31.9
28.8
176.7
172.5
51.7
2.07 m
2.39 m
2.44 m
5.83 d (1.8)
2.90 m
2.89 ddd (2.2, 12.7, 15.3)
2.67 ddd (3.9, 11.2, 12.6)
2.59 m
2.64 m
2.64 m
2.79 m
2.00 m
H-8
H-9
2.49 dd (4.7, 11.2)
-
3.12 d (5.4)
1.70 dd (5.4, 14.6)
1.84 d (14.6)
1.89 m
-
2.04 m
H-11
H-12a
H-12b
H-14
H-15a
H-15b
H-16a
H-16b
H-18
H-19
H-20a
H-20b
H-21a
H-21b
H-24
3.28 d (5.4)
1.72 dd (5.4, 14.6)
1.86 d (14.6)
1.64 m
3.02 d (4.0)
3.05 d (4.0)
-
1.92 m
1.51 m
1.90 m
1.90 m
2.18 m
1.09 s
1.51 m
2.03 m
1.88 m
2.21 m
1.02 s
1.50 s
1.51 m
1.51 m
1.76 m
2.15 m
1.02 s
1.54 s
1.30 s
2.00 m
2.73 ddd (7.5, 8.9, 12.9)
2.60 m
1.94 ddd (7.0, 9.6, 13.2)
2.31 ddd (6.5, 9.5, 13.2)
2.50 ddd (6.9, 9.4, 17.9)
2.60 ddd (6.7, 9.6, 17.9)
3.65 s
1.89 m
2.28 ddd (6.7, 9.5, 13.2)
2.50 m
2.60 m
3.71 s
2.60 m
3.66 s
1
Values in parentheses give δ, multiplicity and J in Hz.

Molecules 2017, 22, 1354
16 of 26
(a)
(b)
(c)
α,11α,17α)-9,11-epoxyester
Figure 8. Crucial 1H- and 13C-NMR chemical shifts and NOE effects for: (
a) (7
2; (b) (7β,11α,17α)-9,11-epoxyester 2a and (c) (7α,11α,12α,17α)-11,12-epoxyester 2b.
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
2
isomeric epoxyester 2a corresponds to C24H31O6. The NMR structure assignments of the isomeric
(7 ,11 ,17 )-9,11-epoxyester 2a and (7 ,11 ,12 ,17 )-11,12-epoxyester 2b were confirmed by X-ray
analysis (Figure 9 and Table 9). The SCXRD structures confirmed the presence of the 9 ,11 -epoxide
ring in 2a and 11 ,12 -epoxide ring in 2b. The carbomethoxy group is situated at the C-7 position in
β
α
α
α
α
α
α
α
α
α
α
α
2b and at the C-7β position in 2a.
(a)
(b)
Figure 9. Molecular structure of the isomeric epoxides: (
(7α,11α,12α,17α)-11,12-epoxy-2b.
a) (7β,11α,17α)-9,11-epoxy-2a and (b)
Table 9. Summary of crystallographic data and structure refinement for the isomeric (7
17α)-9,11-epoxyester 2a and (7α,11α,12α,17α)-11,12-epoxyester 2b.
β,11α,
Identification Code
Chemical formula
Molecular weight
Temperature
2a
2b
C24H30O6
C24H30O6
414.48 g/mol
100(2) K
414.48 g/mol
100(2) K
Wavelength
0.71073 Å
0.71073 Å
Crystal system, space group
Monoclininc, P21
a = 6.0294(5) Å
b = 10.5733(8) Å
c = 16.3548(12) Å
Orthorhomblic, P212121
a = 6.377(2) Å
b = 15.477(3) Å
Unit cell dimensions
c = 20.886(4) Å
◦
◦
α = 90
α = 90
◦
◦
β = 99.604(2)
β = 90
◦
◦
γ = 90
γ = 90

Molecules 2017, 22, 1354
17 of 26
Table 9. Cont.
Identification Code
2a
2b
2061.4(9) Å3
4, 1.336 mg/m3
0.095
1028.02(14) Å3
2, 1.339 mg/m3
Volume
Z, Calculated density
Absorbtion coefficient
F(000)
0.095
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
−13 ≤ k ≤ 13
−21 ≤ l ≤ 21
−7 ≤ h ≤ 7
−18 ≤ k ≤ 18
−25 ≤ l ≤ 24
9774/3635
Limiting indices
Reflections collected/unique
Absorption correction
36785/4712
semi-empirical from equivalents
0.9981 and 0.9720
full-matrix least-squares on F2
4712/1/274
None
Max. and min. transmission
Refinement method
0.989 and 0.945
full-matrix least-squares on all F2
Data/restraints/parameters
Goodness-of-fit on F2
3635/0/271
1.093
1.057
Final R1/wR2 indices [I > 2sigma(I)]
R1/wR2 indices (all data)
Largest diff. peak and hole
0.0391/0.1014
0.0371/0.0942
0.0408/0.1024
0.261 and −0.179 e/Å3
0.0427/0.0964
0.220 and −0.192 e/Å3
3. Experimental Section
3.1. General Information
11
manufactured by Hangzhou Pharma Chemicals Ltd. (Hangzhou, China) according to synthetic
procedure described by Ng et al. [ ] (Scheme 2). Sulfuryl chloride (97%), imidazole (99%),
2,2,2-trichloroacetamide (99%), disodium hydrogen phosphate ( 99%), hydrogen peroxide (35% in H2O)
α-Hydroxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (12, 98%) was
9
≥
´
were purchased from Sigma-Aldrich (Munich, Germany) and CHEMPUR (Piekary Sla˛skie, 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)
1
at temperature 298 K using TMS as internal standard. All H-/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
2a), CCDC 1565292 (2b), CCDC 1565294 (
12), CCDC 1502631 (12a) and CCDC 1502630 (13) contain the supplementary crystallographic data for
–
b
,
12
,
12a and 13 are listed in Tables 3, 5, 6 and 9. The deposition numbers CCDC 1502629
7), CCDC 1565293 (7a), CCDC 1502628 (7b), CCDC 1502627
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: deposit@ccdc.cam.ac.uk).
3.8. Syntheses
3.8.1. 11
α
-Hydroxy-7β-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (12a, Scheme 3)
11-Hydroxyester (7
α
,11 ,17 )-12 (5.0 g, 12.0 mmol) was added to a suspension of MeONa (1.30 g,
α
α
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
19 of 26
white foam. The crude product was purified by column chromatography over silica gel with 10–50%
MeCN/PhCH3 gradient elution to give (7 ,11 ,17 )-12a (2.45 g, 49% yield) as a white solid, a mixture
of 12 and 12a (0.56 g) and the (7α,11α,17α)-12 (1.27 g).
β
α
α
Scheme 3. Synthesis of the 11-hydroxyester (7β,11α,17α)-12a.
)-12a: Rf = 0.40 for 60% MeCN/PhCH3. M.p. 250–252 C. [α]2D0 = +21.49 (c 1.0, CHCl3).
(cm−1): 3511, 2978, 2940, 2872, 2832, 1775, 1736, 1652, 1608, 1482, 1447, 1417,
◦
(7
β,11α,17α
FT-IR (pellets, KBr)
ν
1361, 1300, 1275, 1170, 1107, 1047, 1003, 914, 871, 789, 742, 672, 596, 509, 470. HRMS (ESI): calcd. for
1
C24H32O6Na [M + Na]+ 439.20911, found 439.2110. H- and 13C-NMR spectra, see Table 2.
(7
α,11α,17α =
)-12: Rf = 0.33 for 60% MeCN/PhCH3. M.p. 228–229 ◦C (lit. 229–230 ◦C [24]). [α]2D0
+40.68 (c 1.0, CHCl3) (lit. [α]2D0 = +18.9 (c 0.0027, CHCl3) [24]). FT-IR (pellets, KBr)
ν
(cm−1): 3422, 3035,
2985, 2954, 2933, 2897, 2877, 1759, 1719, 1662, 1617, 1469, 1443, 1376, 1269, 1234, 1211, 1181, 1144, 1059,
1017, 999, 926, 866, 852, 787, 677, 606, 533, 514, 466. HRMS (ESI): calcd. for C24H32O6Na [M + Na]+
1
439.20911, found 439.2080. H- and 13C-NMR spectra, see Table 2.
3.8.2. Purification of the Commercial (7α,11α,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
mixture of AcOEt/MeCN/CH2Cl2 (8:4:1) to afford the pure 12 (13.98 g, 43% yield) as a white crystals. The
recrystallization mother liquors of 12, containing a mixture of epimers 12 and 12a, were subjected to column
chromatography over silica gel with 10–50% MeCN/PhCH3 gradient elution to give (7
(0.91 g) as a white solid, a mixture of 12 and 12a (14.4 g) and (7 ,11 ,17 )-12 (2.67 g). The characterization
data of 12 and 12a were identical in all aspects with those obtained in experiment 3.8.1.
β,11α,17α)-12a
α
α
α
3.8.3. 9,11β-dichloro-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (13, Scheme 4)
A solution of (7
α
,17α
)-9(11)-enester (2.5 g, 6.27 mmol) in anhydrous pyridine (5.1 mL, 62.7 mmol)
7
and chlorobenzene (25 mL) was cooled to 0 ◦C. SO2Cl2 (1.0 mL, 12.54 mmol) was added dropwise
and the mixture was stirred for 30 min at 0 ◦C. The cooling bath was 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).
Scheme 4. Synthesis of the (7α,11β,17α)-9,11-dichloro derivative 13.
The combined organic extracts were washed with aqueous saturated NaHCO3 solution (150 mL)
followed by H2O (150 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced
pressure to give a brown foam. The crude product was purified by column chromatography over silica

Molecules 2017, 22, 1354
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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]D20 = +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
C24H30O5NaCl2 [M + Na]+ 491.13625, found 491.1386. H- 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 (7
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
α
,11
α
,17
α
)-12 (5.0 g, 12.0 mmol) in anhydrous pyridine (25 mL)
◦
suspended in CH2Cl2 (25 mL), washed subsequently with aqueous 1 M HCl solution (3
H2O (25 mL) and brine (25 mL). The organic phase was dried over anhydrous Na2SO4, filtered and
evaporated under reduced pressure.
×
25 mL),
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
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
×
25 mL). The
silica gel with 5–30% AcOEt/CH2Cl2 gradient elution to afford 7
α
,9:21,17-dicarbolactone 14 (1.61 g,
83% yield) as a white solid. Rf = 0.20 for 15% AcOEt/CH2Cl2. M.p. 244–245 ◦C. [α]D20
=
−
2.41 (c 1.0,
CHCl3). FT-IR (pellets, KBr)
ν
(cm−1): 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]+
1
407.18289, found 407.1847. H- 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 (7
α
,11
α
,17
α
)-12
◦
(20.0 g, 48.02 mmol) in THF (150 mL) and cooled to
dropwise and the mixture was stirred for 30 min. at
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 were
−
10 C. SO2Cl2 (8.2 mL, 100.84 mmol) was added
−
10 ◦C. The cooling bath was then removed and
×
washed with aqueous saturated NaHCO3 solution (100 mL), dried over anhydrous Na2SO4, filtered
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 (7
α
,17
α
)-9(11)-enester 7 (13.58 g, 71% yield). Rf = 0.17 for 15% AcOEt/CH2Cl2. M.p.
(cm−1): 3052, 2967, 2908,
204–206 ◦C (lit. 205–206 ◦C [17]). [α]D20 = +2.75 (c 1.0, CHCl3). FTIR (KBr)
ν
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 Table 4.

Molecules 2017, 22, 1354
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Scheme 6. Synthesis of the (7α,17α)-9(11)-enester 7.
-(methoxycarbonyl)-3-oxo-17 -pregna-4,9(11)-diene-21,17-carbolactone (7a, Schemes 7 and 8)
3.8.6. 7
β
α
Method 1
The (7
α
,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 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 (7
β,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).
)-9(11)-enester 7a: Rf = 0.34 for 15% AcOEt/CH2Cl2. M.p. 167–168 C. [α]2D0 = +0.50
◦
(7β,17α
(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.
1
for C24H30O5Na [M + Na]+ 421.19855, found 421.2006. H- 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 the stirring
was continued for 1 h at room temperature. The reaction mixture was diluted with H2O (10 mL) and
the product was extracted with CH2Cl2 (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 (7
β,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.

Molecules 2017, 22, 1354
22 of 26
Scheme 8. Synthesis of the (7β,17α)-9(11)-enester 7a.
3.8.7. 7α-(Methoxycarbonyl)-3-oxo-17α-pregna-4,11(12)-diene-21,17-carbolactone (7b)
The recrystallization mother liquors of (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 (7 ,17 )-9(11)-enester 7a (0.48 g) and (7 ,17 )-11(12)-enester
7
β
α
α
α
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
(7
7 (2.75 g) as white crystalline products. Finally, crystalline 11-hydroxyesters (7
and (7 ,11 ,17 )-12 (0.33 g) were obtained on further gradient elution with 10–50% MeCN/PhCH3.
(7 ,17
)-11(12)-enester 7b: Rf = 0.29 for 15% AcOEt/CH2Cl2. M.p. 232–234 ◦C [α]D20 = +33.42 (c 1.0,
CHCl3). FT-IR (pellets, KBr)
(cm−1): 3028, 2971, 2949, 2878, 1775, 1723, 1664, 1616, 1465, 1438, 1380,
α,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
β,11α,17α)-12a (0.17 g)
α
α
α
α
α
.
ν
1334, 1267, 1195, 1172, 1048, 1013, 918, 868, 788, 716, 669, 525, 454. HRMS (ESI): calcd. for C24H30O5Na
1
[M + Na]+ 421.1991, found 421.1988. H- 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 solut◦ion of (7
α,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]20 = +24.63 (c 1.0,
D
CH2Cl2). FT-IR (pellets, KBr)
ν
(cm−1): 3531, 3430, 3024, 2952, 2880, 1780, 1725, 1664, 1614, 1463, 1437,
1421, 1404, 1381, 1335, 1295, 1268, 1237, 1194, 1174, 1072, 1034, 1011, 977, 962, 920, 880, 836, 784, 664,

Molecules 2017, 22, 1354
23 of 26
1
629, 605, 524, 497, 454. HRMS (ESI): calcd. for C24H30O6Na [M + Na]+ 437.1940, found 437.1942. H-
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
purified by column chromatography over silica gel with 1–10% Me2CO/CH2Cl2 gradient elution to
give (7 ,11 ,17 )-9,11-epoxyester 2a (1.52 g, 49% yield) as a white solid, a mixture of and 2a (0.55 g)
and (7α,11α,17α)-9,11-epoxyester 2 (0.77 g).
(7 ,11 ,17 )-9,11-epoxyester 2a: Rf = 0.43 for 10% Me2CO/CH2Cl2. M.p. 249–251 C (lit. 254–258
]). [α]2D0 = +12.53 (c 1.0, CHCl3). FT-IR (pellets, KBr) (cm−1): 3030, 2972, 2953, 2929, 2883, 1774,
2 and 2a was
β
α
α
2
◦
β
α
α
◦C [
9
ν
1734, 1674, 1619, 1436, 1384, 1296, 1269, 1195, 1165, 1080, 1013, 905, 866, 797, 659, 576, 521, 414. HRMS
1
(ESI): calcd. for C24H31O6 [M + H]+ 415.21152, found 415.2113. H- 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 (7
β
,17
α
)-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).
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 chromatography

Molecules 2017, 22, 1354
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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. 9,11
α-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 (7
α
,11
α
,17
α
)-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
10 C. The cooling bath was then removed and the stirring
◦
and the mixture was stirred for 30 min. at
−
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
50 mL). The combined organic extracts were washed with aqueous saturated NaHCO3 solution
(3
×
(100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a
yellowish white foam. The crude (7 ,17 )-9(11)-enester was dissolved in CH2Cl2 (150 mL), followed
α
α
7
by 2,2,2-trichloroacetami◦de (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 (7
α
,11
α
,17
α
)-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
(lit. 240–242 C [17]). [α]2D0 = +1.55 (c 1.0, CHCl3). FTIR (pellets, KBr)
ν
(cm−1): 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
1
437.1941. H- and 13C-NMR spectra, see Table 8. 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 (7 ,11 ,17 )-9,11-dichloro impurity 13
(0.46 g) and the (7 ,11 ,17 )-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 (7 ,11 ,17 )-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
α
β
α
β
α
α
α
α
α
,
,
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 11
-pregn-4-ene-21,17-carbolactone 12a and 9,11
-pregn-4-ene-21,17-carbolactone (13).
α
-hydroxy-7
β
-
-
(methoxycarbonyl)-3-oxo-17
α
(
)
β-dichloro-7
Additionally, seven other
α
(methoxycarbonyl)-3-oxo-17
α
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 (7 ,11 ,17 )-11-hydroxyester 12 and the key intermediate
(7 ,17 )-9(11)-enester , including epimerization of the C-7 asymmetric center, oxidation, dehydration,
α
α
α
α
α
7
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 Malin´ska and Krzysztof Woz´niak from 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 Chodyn´ski, Małgorzata Krupa, Anita Pietraszek and Anna Ostaszewska carried out
synthetic experiments. Agata Białon´ska performed 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.
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Sample Availability: Samples of the compounds
2, 2a–b, 7, 7a–b, 12, 12a, 13 and 14 are available from the authors.
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