Biotransformation of cortisone with Rhodococcus rhodnii: Synthesis of new steroids

Article abstract of DOI:10.3390/molecules26051352

Cortisone is a steroid widely used as an anti-inflammatory drug able to suppress the immune system, thus reducing inflammation and attendant pain and swelling at the site of an injury. Due to its numerous side effects, especially in prolonged and high-dose therapies, the development of the pharmaceutical industry is currently aimed at finding new compounds with similar activities but with minor or no side effects. Biotransformations are an important methodology towards more sustainable industrial processes, according to the principles of “green chemistry”. In this work, the biotransformation of cortisone with Rhodococcus rhodnii DSM 43960 to give two new steroids, i.e., 1,9β,17,21-tetrahydoxy-4-methyl-19-nor-9β-pregna-1,3,5(10)-trien-11,20-dione and 1,9β,17,20β,21- pentahydoxy-4-methyl-19-nor-9β-pregna-1,3,5(10)-trien-11-one, is reported. These new steroids have been fully characterized.

Full text of DOI:10.3390/molecules26051352

molecules
Article
Biotransformation of Cortisone with Rhodococcus rhodnii:
Synthesis of New Steroids
Federico Zappaterra 1 , Stefania Costa 1,
*
, Daniela Summa 1 , Valerio Bertolasi 2, Bruno Semeraro 3,
Paola Pedrini 1, Raissa Buzzi 1 and Silvia Vertuani 4
1
Department of Life Sciences and Biotechnology, University of Ferrara, Via L. Borsari 46, 44121 Ferrara, Italy;
federico.zappaterra@unife.it (F.Z.); daniela.summa@unife.it (D.S.); pdp@unife.it (P.P.);
raissa.buzzi@unife.it (R.B.)
Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Via L. Borsari 46,
44121 Ferrara, Italy; valerio.bertolasi@unife.it
GATE SRL, Via L. Borsari 46, 44121 Ferrara, Italy; bsemeraro@gategreen.it
Department of Life Sciences and Biotechnology, Master Course in Cosmetic Science and
Technology (COSMAST), University of Ferrara, Via L. Borsari 46, 44121 Ferrara, Italy; silvia.vertuani@unife.it
Correspondence: stefania.costa@unife.it; Tel.: +39-0532-455-708
2
3
4
*
Abstract: Cortisone is a steroid widely used as an anti-inflammatory drug able to suppress the
immune system, thus reducing inflammation and attendant pain and swelling at the site of an injury.
Due to its numerous side effects, especially in prolonged and high-dose therapies, the development
of the pharmaceutical industry is currently aimed at finding new compounds with similar activities
but with minor or no side effects. Biotransformations are an important methodology towards more
sustainable industrial processes, according to the principles of “green chemistry”. In this work, the
biotransformation of cortisone with Rhodococcus rhodnii DSM 43960 to give two new steroids, i.e.,
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Zappaterra, F.; Costa, S.;
Summa, D.; Bertolasi, V.; Semeraro, B.;
Pedrini, P.; Buzzi, R.; Vertuani, S.
Biotransformation of Cortisone with
Rhodococcus rhodnii: Synthesis of New
Steroids. Molecules 2021, 26, 1352.
https://doi.org/10.3390/
1,9
β,17,21-tetrahydoxy-4-methyl-19-nor-9
β-pregna-1,3,5(10)-trien-11,20-dione and 1,9β,17,20β,21-
pentahydoxy-4-methyl-19-nor-9
been fully characterized.
β-pregna-1,3,5(10)-trien-11-one, is reported. These new steroids have
Keywords: biotransformation; cortisone; Rhodococcus rhodnii; steroids
molecules26051352
Academic Editors: Ik-Soo Lee and
Hyun Jung Kim
1. Introduction
Steroid drugs are used for various therapeutic applications and represent the most
marketed drug category after antibiotics, with an annual production of over one million
tons. To date, there are about 300 steroid-based drugs on the market, and this is constantly
Received: 18 January 2021
Accepted: 25 February 2021
Published: 3 March 2021
growing [1]. In this field, corticosteroids are a group of hormones produced by the adrenal
gland cortex. This type of molecule is used for its anti-inflammatory, immunosuppressive
properties and for its effects on metabolism. In particular, steroids belonging to the glu-
cocorticoid class can control the metabolism of carbohydrates, lipids, and proteins, while
mineralocorticoids control the concentration of electrolytes and, consequently, the amount
of water present in the blood [2].
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Glucocorticoids play an important role in anti-inflammatory and immunosuppressive
therapy [3] and are widely used in the treatment of allergic reactions and inflammatory
and autoimmune diseases, as well as in the prevention of rejection in transplants and the
treatment of hematological neoplasia [4].
Unfortunately, the use of these drugs (also known as corticosteroids) has a long series
of side effects. On the other hand, it is known that even small structural differences can
significantly modify the power and duration of an action.
Copyright:
© 2021 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 (https://
creativecommons.org/licenses/by/
4.0/).
Besides, the glucocorticoids exhibit important interactions with drugs that affect the
levels of potassium in the blood (e.g., diuretics and laxatives) [
5] and with drugs that
decrease cortisone in the blood (e.g., rifampicin, carbamazepine, phenobarbital, phenytoin,
Molecules 2021, 26, 1352. https://doi.org/10.3390/molecules26051352
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1352
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primidone, and lithium) [
6
,7
]. The occurrence of such side effects is greater the higher the
dosage and the duration of the treatment. Additionally, the side effects are more common
in the case of systemic therapy (oral or injective) and rare for local therapies (with creams,
ointments, and eye drops), where they are generally used at low dosages. Moreover, the
glucocorticoids—in particular, cortisol and cortisone—are insoluble in water, and this limits
their therapeutic use. Therefore, the steroidal compounds with anti-inflammatory action,
which potentially do not present all the above-mentioned disadvantages, are investigated.
In this field, to produce drugs with greater therapeutic activity and fewer side effects,
the development of synthetic compounds deriving structurally from progesterone is of
interest (Figure 1).
Figure 1. Chemical structure of progesterone.
It is known that even minimal structural differences of these molecules can signifi-
cantly change the potency and duration of corticoid actions. In this field, several patents
describe steroid compounds, mainly characterized by the presence of a substituent group
in position C2, with cellular modulation activity and antiproliferative and antiangiogenic
effects [
context, to bypass the problems arising from chemical synthesis, the biotransformation
reactions of steroid molecules have been studied in depth over the past 70 years [ ]. This
8]. These compounds are obtained through traditional chemical processes. In this
9
approach offers several advantages over chemical synthesis, such as the region- and/or
stereospecificity of the rations and the eco-compatibility of the processes, as it works in
mild reaction conditions and aqueous solvents. Therefore, the production of steroids and
hormones is one of the best examples of the successful application of microbial technol-
ogy in large industrial processes [10]. Research activities in this field began in the 1950s,
following the identification of the pharmacological effects of cortisol and progesterone
and with the identification of the enzyme hydroxylation activity in position C11 of the
microbial genus Rhizopus [11]. Since then, multiple microbial bioconversion processes of
steroids and sterols have been reported in the literature; these biotransformations have
provided adequate tools for the large-scale production of analogs of natural or modified
steroids [12,13].
In this field, the ability of certain microorganisms to produce various metabolites is
well-known and not readily achievable by conventional methods, and this practice has
proven to be indispensable for the pharmaceutical industry [14].
To maximize the yield of the reaction, understood as the total conversion of the
substrate into the desired product, selected strains of a particular microorganism are
generally transformed to optimize the expression of the genes of interest and/or block
certain metabolic pathways to accumulate the pursued intermediates.
In this context, the ability of Actinobacteria to biotransform and, in some cases, to
partially or totally degrade steroid rings or side chains has turned out to be of great
interest [15,16].
Actinobacteria (also called Actinomycetes or Mycobacteria, as bacteria similar to filamen-
tous fungi) are a group of Gram-positive bacteria (with the sole exception of Actinoplanes)
that includes five subclasses and 35 families [17]. These strains have been widely studied
and their biotransformation capacities confirmed in various types of transformations of
steroid rings as the reduction of C-C and C-O double bonds, oxidation of alcoholic func-

Molecules 2021, 26, 1352
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tions, dehydrogenation, hydroxylation, and in many cases, also in lateral-chain degradation
processes or the total degradation of the steroid ring [18].
In particular, Rhodococcus, which belong to the order of Actinomycetales, are Gram-
positive, forced aerobic, nonmobile, nonsporulating bacteria and are usually found in
soil and aquatic environments and are very important for industrial use. Several studies
have shown that these strains can grow both in mesophilic [19] and in psychrophilic
conditions [20].
Rhodococci are also able to degrade various organic compounds, including ones highly
toxic and recalcitrant; moreover, they are equipped with enzymes that catalyze biologically
relevant reactions such as the biodesulfurization of fossil fuels [21], the degradation of
polychlorinated biphenyls (PCBs) [22], and the use of a large variety of other organic
compounds as sources of energy [19]. They are also able to degrade halogenated hydrocar-
bons with long or short chains [23] and to metabolize numerous halogenated differently
substituted aromatic and heteroaromatic compounds [24], bioactive steroids [25], and
acrylamide [26]. Rhodococci are also known for their ability to biotransform a wide range
of steroids via hydroxylation or Bayer-Villiger oxidation, while the Corynebacterium and
Nocardia genera are known to provide dehydrogenation products [18].
In previous works, the biotransformations of bile acids with Rhodococcus ruber to give
new 9,10-secosteroids [15], and of cortisone and cortisol with Rhodococcus coprophilus to
give
a preliminary screening of different strains of Rhodococcus, from which it emerged that
Rhodococcus rhodnii was able to biotransform cortisone ( ) by producing compounds dif-
∆
1-dehydrogenation [27] products, have been reported. These works derive from
1
ferent from the classic biotransformation products (i.e., prednisone or hydroxyl reduction
reactions in position C20) [27].
In this work, the biotransformation of cortisone (
new steroid compounds, i.e., 1,9 ,17,21-tetrahydroxy-4-methyl-19-nor-9
trien-11,20-dione ( ) and 1,9 ,17,20 ,21-pentahydroxy-4-methyl-19-nor-9β
1
) with Rhodococcus rhodnii to give
-pregna-1,3,5(10)-
-pregna-1,3,5(10)-
β
β
6
β
β
trien-11-one (7), is reported (Scheme 1).
2. Results
2.1. Biotransformation of Cortisone with Rhodococcus rhodnii (24 h)
Semipreparative cultures were necessary to isolate and identify the biotransformation
products. Although the biotransformation time (24 h) was rather short, the yields of
the product were almost quantitative; in fact, no residual cortisone was observed, and
1,9
0.07 g, 70%) and 1,9
11-one) (0.041 0.004 g, 20%) were obtained. In this case, the product recovery yield
β
,17,21-tetrahydoxy-4-methyl-19-nor-9
β
-pregna-1,3,5(10)-trien-11,20-dione
6
(0.146
±
β
,17,20 ,21-pentahydoxy-4-methyl-19-nor-9
β
β-pregna-1,3,5(10)-trien-
7
±
was not quantitative, as the downstream processes of extraction and purification of the
products on a silica gel column led to a loss of about 5-10%. An improvement in yield could
be obtained by trying to optimize the recovery process of the biotransformation products.
As regards the course of the reaction, samples were taken at 0, 2, 4, 6, 8, and 24 h
from the administration of the substrate (
matography) analysis, only the appearance of a band corresponding to prednisone (
shown, while the cortisone band was still very evident. At 4 h, a further band appeared in
correspondence with the 20 -hydroxy prednisone ( ) standard. After 6 h from the admin-
istration of the substrate, the bands of products and were clearly visible, even if a large
1
). After 2 h from the TLC (Thin Layer Chro-
2) was
β
3
2
3
part of the unreacted cortisone remained, a situation which remained almost unchanged
even at 8 h. After 24 h of biotransformation, the only visible bands were those relating to
products 1,9
) and 1,9 ,17,20
The structure of compound
β
,17,21-tetrahydroxy-4-methyl-19-nor-9
,21-pentahydroxy-4-methyl-19-nor-9
was determined by NMR analysis (Tables 1 and 2),
β
-pregna-1,3,5(10)-trien-11,20-dione
(6
β
β
β
-pregna-1,3,5(10)-trien-11-one ( ).
7
6
mass spectrometry, IR, and X-ray investigation (Table 3 and Figure 2). The only difference
between compounds 6 and 7 was the reduction of the C20-carbonyl group.

Molecules 2021, 26, 1352
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The stereochemistry of the C20-hydroxyl group was determined by the comparison of
the commercial standard spectrum of 17α,20β,21-trihydroxy-1,4-pregnadiene-3,11-dione
(20 -hydroxy-prednisone) that was isolated as an intermediate [28]. Based on the degra-
β
3
dation pathway of cortisone, we hypothesized the formation mechanism of products
6 and
7, as shown in Scheme 1.
Scheme 1. Biotransformation of cortisone (1) with Rhodococcus rhodnii.
Compounds
particular, the aromatization of the A ring and the displacement of the angular methyl group
from the C19 position ( 1.32 ppm) in cortisone to the C4 position in the new compounds
2.08 and 2.09 ppm for and , respectively). The aromatization of ring A was shown
by the presence of the signals C2-H ( 6.90 ppm) and C3-H ( 6.40 ppm) for product and
by the absence of signals C1-H and C9-H present in cortisone. Minimal differences were
present in compound (C2-H 6.45 ppm and C20-H 3.48 ppm). A further diagnostic
6 and 7 showed substantial structural variations concerning cortisone—in
δ
(
δ
6
7
δ
δ
6
7
δ
δ
signal of the change in the structure of ring A was represented by the signal shift relative
to the hydrogen atom in the C4 position of the cortisone, as this atom was replaced by the
methyl group (C19) following the rearrangement of the molecule after hydroxylation and
the opening of ring B.
As far as the hydroxyl groups visible from the NMR spectra in DMSO–d6 are con-
cerned, the appearance of the signal relating to the OH group in position C20 in product
7
at 4.20 ppm was noted, the signal absent in compounds and as they presented a group
1
6
in this position carbonyl. Further confirmation of this data was provided by the signal
at 3.48 ppm of the 1H-NMR spectrum of product
Supplementary Materials).
7, absent in the other two spectra (see
As prednisone has proven to be four to five times more active than cortisone and
hydrocortisone [29] due to the alteration of the conformation of ring A, compounds and
could also have an anti-inflammatory capacity comparable or superior to the starting
substrate; this would be a huge advantage in terms of the amount of drugs that should
be given. Furthermore, products and , inside the steroid structure, present ring A in a
phenolic form; this structure could also confer antioxidant properties to these molecules.
6
7
6
7
Based on the suggested biotransformation pathway of cortisone, products
2
and
3
are
potential precursors of products 6 and 7, as shown in Scheme 1.
1
In this mechanism, the
∆
1-dehydrogenation of cortisone (catalyzed by a
∆
-
hydroxysteroid dehydrogenase) and the reduction of the hydroxyl group in the C20 posi-
tion, catalyzed by a 20β-hydroxysteroid dehydrogenase, were the first reactions involved

Molecules 2021, 26, 1352
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to give prednisone
the C9 position by the 3-ketosteroid-9
that, by the spontaneous retro-aldol cleavage of ring B, gave the aromatic 9,10-secosteroids
5a . These intermediates were not isolated. In particular, we highlighted the nucleophilic
2
and 20β-hydroxy-prednisone
3. Subsequently, the hydroxylation of
α
-hydroxylase (KSH) afforded the intermediates 4a, b
,
b
attack of the benzenic ring on the carbonyl in the C9 position with the formation of the
ring B and the recovery of the hydroxyl function in C9, as shown by the signals at 4.74 and
4.68 ppm for products 6 and 7, respectively.
2.2. Biotransformation of Cortisone with Rhodococcus rhodnii (6 h)
The objective of carrying out biotransformations at short times was to confirm the
first biotransformation step of compounds
2
and
3
and the stereochemistry of the hydroxyl
group present in position C20 of the new product
after only 6 h.
7; for this reason, the reaction was stopped
Compound
3
is known in the literature and is commercially available. As was done in
corresponded to 20 -prednisone. The
, was confirmed by
a previous study, the purification of intermediate
3
β
stereochemistry of the C20 reduction and, consequently, of product
3
comparing the 1H-NMR data (i.e., 4.61 (C20-H) and 4.52 (C21-H) ppm in C5D5N) and 13C-
NMR data (i.e., 65.0 (C21) and 72.5 (C20) ppm) with the data recorded for the commercially
available compound.
This product could be an interesting derivative of the bioactive prednisone. The
yields obtained in these biotransformations, although of relative importance concerning
the purpose of this experiment, were as follows: prednisone 2 (0.06
±
0.003 g, 30%), 20
β-
hydroxy-prednisone 3 (0.02
60%).
±
0.002 g, 10%), and unreacted cortisone 1 (0.12 0.005 g,
±
As can be seen from the yields, given the short times of biotransformation, 60% of
cortisone was not yet biotransformed.
2.3. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assays
Product
6 and product 7 were tested for the determination of cell viability on the
human HaCaT keratinocyte lines. The test was conducted by exposing HaCaT cells to
different concentrations (from 5 to 500 µM) of the “product 6 and 7” test samples for 24 h
at 37 ◦C, and then, cell viability was measured by means of the MTT test. The negative
control (0.1% DMSO culture medium) was tested simultaneously with the substances. Both
substances examined were not cytotoxic at any of the tested concentrations, since they did
not decrease the vitality of the keratinocytes compared to the control culture.
Figure 2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test about the cell viability of the aneuploid
immortal keratinocyte c ell line after exposure to product ) and product ). The values were expressed as a percentage
with respect to the control (C = 100%) to the vehicle alone and reported as mean ± SEM.
6
(a
7 (b

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3. Discussion
To bypass all or part of the side effects deriving from the cortisone therapies, the
pharmaceutical industry is increasingly looking for new compounds with therapeutic
characteristics similar to those of cortisone but with fewer side effects.
Due to the numerous harmful and dangerous effects of the reagents used in the
chemical synthesis of new compounds, research is increasingly turning towards greener
approaches. To overcome all the problems deriving from chemical synthesis, the potential of
microbial steroid biotransformation has been known for several decades, as its application
offers several advantages over chemical synthesis.
In the present work, the bacterium R. rhodnii biotransformed cortisone
produce the new steroids 6 and 7.
1 in 24 h to
The proposed mechanism of formation of products 6 and 7 (Scheme 1) in the initial
steps was supported and followed partially the degradation pathway of cortisone by
Actinobacteria [18] (Scheme 2).
Scheme 2. Degradation pathway of cortisone by Actinobacteria [18].
Steroids with a 3-oxo-4-ene structure, such as cortisone, are subjected to the cleavage of
ring B. The ∆
1-dehydrogenation of cortisone to give prednisone is the initial stage that leads
to the complete elimination of the C17 chain with the 11-keto-ADD (androstadienedione)
formation. This is a classic step in the degradation of other steroids, such as sitosterol,
testosterone, and bile acids [30]. 11-Keto-ADD is subjected to hydroxylation at the C9
position catalyzed by 3-ketosteroid-9
α-hydroxylase (KSH), a two-component iron–sulfur-
containing monooxygenase [31 33]. This reaction leads to the spontaneous cleavage of the
–
ring B by a retro-aldol cleavage and the formation of the aromatic 9,10-secosteroids, which
undergo further degradation [34].
This degradation pathway sustained the proposed mechanism of C9-hydroxylation of
prednisone
formation of cortisone after 6 h. The C9-hydroxylated intermediates 4a
rearranged to secosteroids 5a b. The nucleophilic attack of the aromatic ring on the car-
bonyl in C9 ld to the subsequent B ring closure with the formation of new compounds
(70%) and (20%). The yields obtained did not correspond to 100%, as it was classic
2
and prednisolone
3
, whose presence was confirmed stopped by the biotrans-
,
b
spontaneously
,
6
7
that, in the extraction and purification of the products, there was a weight loss of about
5–10%. Furthermore, the path that led to the degradation of the compounds probably
began at this level; in fact, some studies have been conducted in which the duration of the
biotransformation was extended up to 72 h, with a decrease in the yield of the products
obtained, so we hypothesized that the degradation mechanism started after about 24 h [18].

Molecules 2021, 26, 1352
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The structures of compounds
6
and
7
allowed us to think that they could maintain an
anti-inflammatory capacity, as they had a carbonyl function in position C11. Furthermore,
products and , inside the steroid structure, presented ring A in a phenolic form; this
6
7
structure could also confer antioxidant properties to these molecules.
To date, the biotransformation approach is the only synthesis method of these molecules;
therefore, if in the future, it will be demonstrated that these new steroids have therapeutic
activity, it will be possible to produce them using a method that follows the guidelines of
“green chemistry”.
About the reduction product at the level of the carbonyl group in C20, the stereochem-
istry of the hydroxyl group was carried out by comparing the NMR spectra of its precursor
with a commercial standard.
4. Materials and Methods
4.1. Chemicals and Rhodococcus Strain
Cortisone was purchased from Sigma-Aldrich (St. Louis, MO, USA). Plate count broth
(PCB) is commercially available (Oxoid, (Basingstoke, UK)). 20β-hydroxy-prednisone was
purchased from Toronto Research Chemical Inc. (Toronto, ON, Canada).
Rhodococcus rhodnii (DSM 43960) was purchased from the DSMZ (Leibniz Institute
DSMZ—German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig
Germany) company. The master cell bank of every strain was maintained at
−
20 ◦C in
cryovials in plate count broth medium (1 mL) mixed with glycerol (0.5 mL) as a cryoprotec-
tant agent. The working cell bank was conserved at 4 ◦C in plate count agar (PCA) slants
for 6 months and used for seed cultures.
4.2. Analytical Methods
Melting points were uncorrected and were determined on a 510 Buchi melting point
instrument. The 1H, 13C, and DEPT NMR spectra were recorded with a Varian Gemini 300
operating at 300 MHz (1H) and 75 MHz (13C) or with a Varian Mercury Plus 400 operating
at 400 MHz (1H) and 100 MHz (13C), respectively. The chemical shifts were referenced
to the residual solvent signals (CDCl3: δH 7.26, δC 77.0; CD3OD: δH 3.34, δC 49.0; and
DMSO-d6: δH 2.49, δC 39.9). The 2D NMR experiments (COSY, HMQC, and HMBC) were
obtained with a Varian Mercury Plus 400, operating at 400 MHz (1H) and 100 MHz (13C),
and were processed using the manufacturer’s software. Infrared spectra were acquired
with a Perkin-Elmer Spectrum 100 FTIR. Mass spectra were recorded using an LCQ Duo
(TermoQuest, San Jose, CA, USA), equipped with an electrospray ionization (ESI) source.
◦
Optical rotations were measured at 20 C in the stated solvent using a Perkin-Elmer model
241 polarimeter. TLC were performed on precoated silica gel plates (thickness 0.25 mm,
Merck), and the phosphomolybdic acid solution was used as the spray reagent to visualize
the steroid spots. Silica gel (Fluka, Kiesegel 60, 70–230 mesh) was used for preparative
column chromatography.
4.3. Biotransformation of Cortisone 1 with Rhodococcus rhodnii (24 h)
A loopful of Rhodococcus rhodnii from a culture on plate count agar (PCA) containing
glucose (1 g/L), yeast extract (2.5 g/L), tryptone (5 g/L), and agar (15 g/L) was inoculated
in three 50-mL Erlenmeyer flasks, closed with cotton plugs, in sterile plate count broth
(PCB) (20 mL) contai◦ning glucose (1 g/L), yeast extract (2.5 g/L), and tryptone (5 g/L)
and incubated at 30 C and 110 rpm in an orbital shaker for 48 h. The whole cultures
were introduced into 500-mL Erlenmeyer flasks containing 200 mL of sterile PCB, and
after 48 h incubation at 30 ◦C and 110 rpm, cortisone (0.2 g) in DMSO (2 mL) was added,
and the culture was maintained in the same conditions for 24 h. All tests were carried
out in triplicate for statistical significance. A blank sample was also incubated containing
all the reagents but no bacteria. The reaction was monitored by withdrawing samples
(1 mL) every 6 h up to 24 h of reaction and followed by TLC analysis (ethyl acetate as the
eluent). After 24 h, the cells were removed by centrifugation (5242 RCF, 20 min), and the

Molecules 2021, 26, 1352
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supernatant was extracted with ethyl acetate (3
×
200 mL). The organic layer was dried
over anhydrous Na2SO4, the solvent evaporated, and the crude mixture purified on a
chromatographic column (silica gel; ethyl acetate as the eluent). The biotransformation
yields were obtained by working out the percentage of the ratio between the moles of the
product obtained compared to the moles of the administered substrate.
4.4. Biotransformation (6 h) of Cortisone 1 with Rhodococcus rhodnii
The biotransformations of cortisone
1 with Rhodococcus rhodnii at 6 h were carried out
following the same protocols used for those at 24 h with the differences that the TLC analy-
ses were performed using ethyl acetate/cyclohexane 50/50 as the eluent, and the crude
mixture was purified on a chromatographic column (silica gel; ethyl acetate/cyclohexane
50/50 as the eluent).
4.5. Crystal Structure Determination
The crystal data of compound
nius Kappa CCD diffractometer with graphite monochromated Mo-K
datasets were integrated with the Denzo-SMN package [35] and corrected for Lorentz
and polarization effects. The structure was solved by direct methods using the SIR97 [36
6
were collected at room temperature using a No-
α
radiation. The
]
system of the programs and refined using full-matrix least-squares with all nonhydrogen
atoms anisotropically and hydrogens included on calculated positions, riding on their
carrier atoms, except those linked to the oxygen atoms that were refined isotropically.
Compound 6 crystallized in the monoclinic non-centrosymmetric space group, P21, and
the absolute configuration was determined reliably from the crystallographic data because
of the calculated Flack parameter of 0.1 [37].
All calculations were performed using SHELXL-2018/3 [37] and PARST [38] imple-
mented in the WINGX system of the programs. The crystal data are given in Table 1. The
ORTEP (Oak Ridge Thermal-Ellipsoid Plot Program) [39] view of the structure is shown in
Figure 3.
Figure 3. An ORTEP view of compound
6 showing the thermal ellipsoids at a 30% probability level.
The crystallographic asymmetric unit is built up by a molecule of compound
solvent ethyl acetate.
6 and a molecule of

Molecules 2021, 26, 1352
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Table 1. Crystallographic data.
Crystallographic Data
Compound 6
C21H26O6, C4H8O2
462.52
Formula
M
Space group
Crystal system
a/Å
P21
Monoclinic
7.9730(2)
16.3719(3)
9.4380(3)
90.00
b/Å
c/Å
◦
α/
◦
β/
100.9145(8)
90.00
◦
γ/
U/Å3
Z
1209.69(5)
2
T/K
295
−3
1.270
Dc/g cm
F(000)
496
−1
0.094
µ(Mo-Kα)/mm
Unique Reflections
Rint
6576
0.0366
Obs. Refl.ns [I ≥ 2σ(I)]
5759
◦
θmin–θ
/
3.71−30.00
−11,11;−21,22;−13,13
0.0426
max
hkl ranges
R(F2) (Obs. Refl.ns)
wR(F2) (All Refl.ns)
No. Variables
0.1143
318
Goodness of fit
1.043
−3
0.232; −0.022
1909504
∆$max; ∆$min /e Å
CCDC Deposition N.
4.6. Compound Data
17
α
,21-dihydroxy-1,4-pregnadiene-3,11,20-trione (prednisone) 2 showed the following:
m.p. (melting point) 234 ◦C; 1H-NMR (CDCl3): see Table 2; and 13C-NMR (CDCl3): see
Table 3.
17α,20β,21-trihydroxy-1,4-pregnadiene-3,11-dione (20β-hydroxy-prednisone) 3 showed
◦
the following: m.p. 240–242 C; 1H-NMR (CDCl3): see Table 2; and 13C-NMR (CDCl3): see
Table 3.
1,9
showed the following: m.p. 91–92 ◦C; [
2939, 1708, 1590; ESI-MS (negative ion mode): calcd for C21 H26 O6: 374.17. Found, m/z (%
β,17,21-tetrahydroxy-4methyl-19-nor-9β-pregna-1,3,5(10)-trien-11,20-dione (6) (Scheme 1)
α
]D: + 64,6 (c 1.0, Methanol); IR max (KBr) 3338,
ν
relative to the base peak): 373.27 (100) [M
−
H+]; 1H-NMR (DMSO-d6): see Table 4; and
13C-NMR (DMSO-d6): see Table 5.
1,9
β
,17,20
β
,21-pentahydroxy-4-methyl-19-nor-9
β
-pregna-1,3,5(10)-trien-11-one (
7) (Scheme 1)
◦
showed the following: m.p. 141–142 C; [
α
]D: +23.3 (c 1.0, Methanol); IR max (KBr) 3358,
ν

Molecules 2021, 26, 1352
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2931, 1698, 1590; ESI-MS (negative ion mode): calcd for C21 H28 O6: 376.18. Found, m/z
(% relative to the base peak): 375.13 (100) [M–H+]; 1H-NMR (DMSO-d6): see Table 4; and
13C-NMR (DMSO-d6): see Table 5.
1
Table 2. H-NMR data of 2 and 3 at 400 MHz in CDCl3, δ in ppm, and J in Hz.
2
3
H-C(1)
H-C(2)
H-C(3)
H-C(4)
H-C(5)
H-C(6)
H-C(7)
H-C(8)
H-C(9)
H-C(10)
H-C(11)
H-C(12)
H-C(13)
H-C(14)
H-C(15)
H-C(16)
H-C(17)
H-C(18)
H-C(19)
H-C(20)
H-C(21)
7.63 (d, J = 10.2 Hz)
6.19 (dd, J = 10.2 and 1.8 Hz)
7.67 (d, 1H, J = 10.2 Hz)
6.18 (dd, J = 10.3 and 1.9 Hz)
–
–
6.07 (t, J = 1.8 Hz)
6.06 (t, J = 1.26 Hz)
–
2.01 (m)
1.73 (m)
1.98 (m)
1.99 (d, J = 3.1 Hz)
–
–
1.92 (m)
1.81 (m)
1.93 (m)
2.45 (m)
–
–
–
2.38 (m)
–
2.52 (m)
–
2.06 (m)
1.94 (m)
1.92 (m)
1.69 (m)
0.67 (s)
1.42 (s)
–
1.90 (m)
1.82 (m)
2.01 (m)
1.60 (m)
0.81 (s)
1.43 (s)
3.76 (br s) (4.61, m in C5D5N)
3.76 (br s) (4.52, m in C5D5N)
4.24 (d, J = 20 Hz)
Table 3. 13C-NMR data of compounds 2 and 3 at 400 MHz in CDCl3, δ in ppm.
2
3
H-C(1)
H-C(2)
H-C(3)
H-C(4)
H-C(5)
H-C(6)
H-C(7)
H-C(8)
H-C(9)
H-C(10)
H-C(11)
H-C(12)
H-C(13)
H-C(14)
H-C(15)
H-C(16)
H-C(17)
H-C(18)
H-C(19)
H-C(20)
H-C(21)
158.2
127.8
188.6
124.7
170.9
34.9
33.3
37.4
61.1
44.1
211.4
51.1
52.3
50.8
24.1
35.2
89.1
16.2
19.3
212.7
67.8
158.4
127.7
188.7
124.6
171.2
35.0
34.8
37.7
61.3
44.1
213.3
52.7
52.8
49.5
24.5
33.4
85.0
15.9
19.3
72.5
65.0

Molecules 2021, 26, 1352
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1
Table 4. H-NMR data of compounds 1, 6, and 7 at 400 MHz in DMSO-d6, δ in ppm, and J in Hz.
1
6
7
H-C(1)
H-C(2)
H-C(3)
H-C(4)
H-C(5)
H-C(6)
H-C(7)
H-C(8)
H-C(9)
H-C(10)
H-C(11)
H-C(12)
H-C(13)
H-C(14)
H-C(15)
H-C(16)
H-C(17)
H-C(18)
H-C(19)
H-C(20)
2.54 (m)/1.63 (m)
–
–
1.91
–
5.61 (s)
–
2.26 (m)
1.91 (m)
1.91 (m)
2.12 (m)
–
6.90 (d, J = 8.1)
6.40 (d, J = 8.1)
6.90 (d, J = 8.1)
6.45 (d, J = 8.1)
–
–
–
–
2.45 (m) c/2.52 (m) c
2.62 (m) e/2.51 (m) e
2.12 (m)/1.65 (m) d
2.15 (m)/1.60 (m) f
1.62 (m) d
1.58 (m) f
–
–
–
–
–
–
–
2.85 (d, J = 10.8) a/1.94 (d, J = 10.8) b 2.81 (d, J = 10.6) a/2.03 (d, J = 10.6) b 2.68 (d, J = 10.6) a/2.40 (d, J = 10.6) b
–
–
–
2.25 (m)
2.33 (m)
1.76 (m)/1.32 (m)
2.64 (m)/1.63 (m)
2.50 (m) c
1.69 (m) a,d/1.24 (m) b
1.60 (m)b,f/1.15 (m) a
1.75 (m)b/1.45 (m) a
–
2.58 (m) b,c/1.50 (m) a
–
–
0.45 (s)
1.32 (s)
0.44 (s)
2.08 (s)
0.65 (s)
2.09 (s)
3.48 (br s)
3.35 (m) g/3.30 (m) g
–
–
4.44 (dd, J = 19.3, 6.6)/4.08 (dd, J =
4.36 (dd, J = 19.2, 6.4)/3.98 (dd, J =
H-C(21)
19.3, 5.3)
19.2, 5.3)
9.22 (s)
4.74 (s)
H-O(3)
H-O(9)
–
–
9.15 (s)
4.68 (s)
H-O(17)
H-O(20)
H-O(21)
5.57 (s)
5.42 (s)
3.85 (s)
4.20 (br d, J = 6.5)
–
–
4.65 (dd, J = 6.3, 5.1)
4.66 (dd, J = 6.4, 5.3)
4.38 (br s)
a
α proton; b β proton; and c, d, e, f, and g overlapping multiplets.
Table 5. 13C-NMR data of compounds 1, 6, and 7 at 100 MHz in DMSO-d6, δ in ppm.
1
6
7
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)
31.3
33.8
198.7
124.1
169.6
32.4
32.0
36.4
61.49
38.2
210.7
50.5
50.8
49.5
23.2
34.0
88.1
15.9
17.3
211.9
66.6
129.4
112.2
152.9
123.5
135.8
22.1
19.6
41.9
75.6
126.0
212.0
44.3
50.9
42.2
22.1
33.5
87.1
14.4
129.7
112.7
153.6
124.3
136.4
22.6
20.3
42.9
76.0
126.3
214.2
46.4
52.3
41.2
23.1
34.5
83.4
14.7
19.2
75.4
63.8
18.7
211.0
65.6
4.7. Cytotoxic Activity
HaCaT cells were cultured using Ham’s F-12, fetal bovine serum, DMEM high glucose,
penicillin/streptomycin, and L-glutamine. Cells were incubated at 37 ◦C for 24 h in 95%

Molecules 2021, 26, 1352
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air/5% CO2 until 80% confluency. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) colorimetric assay was performed to estimate cell viability [40]. Cells were
seeded in 96-well plates at a density of 2
24 h. Different concentrations (from 5 to 500
treatments. After 72 h, the medium was removed and washed with PBS twice; 20
×
104 cells/well in 200-
µ
L complete medium for
and were used as cell
L of
µm) of products
6
7
µ
MTT (5 mg/mL) was then added in each well, and the plates were incubated for 4 h at
◦
37 C. The medium was removed and replaced with 100-
µL DMSO to dissolve the formazan
crystals. The extent of MTT reduction was measured spectrophotometrically at 570 nm
using a microplate reader (680 XR, Bio-Rad, Hercules, CA, USA).
5. Conclusions
This work focused on the synthesis of two new compounds resulting from the bio-
transformation of cortisone catalyzed by Rhodococcus rhodnii (DSMZ 43960): 1,9
tetrahydroxy-4-methyl-19-nor-9 -pregna-1,3,5(10)-trien-11,20-dione ( ) and 1,9 ,17,20
pentahydroxy-4-methyl-19-nor-9β-pregna-1,3,5(10)-trien-11-one (7).
β
,17,21-
β
6
β
β,21-
The time of biotransformation (24 h) was rather short, and the yield of the product
was almost quantitative, as no residual cortisone was observed.
To confirm the correct chemical structure of these new products, X-ray studies were
carried out on product
6, while for the determination of the stereochemistry of the hydroxyl
group in the C20 position of product
7
, a comparison of the NMR spectra of its precursor
with the reference commercial standard was carried out.
In this work, a green synthesis method of these new compounds was proposed, and
currently, it is the only strategy that can be used both from the biotransformation point of
view and from that of the chemical synthesis.
The chemical structures of these new products present peculiarities that suggest
potential biological and therapeutic activity. The preliminary cytotoxicity tests obtained
by MTT assay gave promising results for the use of these new derivatives, even at the
highest concentrations tested. Further studies will be focused on the evaluation of the
anti-inflammatory activity and the potential antioxidant activity.
6. Patents
WO2017037081A1 is the patent resulting from the work reported in this manuscript.
Supplementary Materials: The following are available online: Figure S1: 1H-NMR product 6.
Figure S2: 13C-NMR product 6. Figure S3: DEPT-NMR product 6. Figure S4: COSY-NMR product 6.
Figure S5: Focused COSY-NMR product 6. Figure S6: HMBC-NMR product 6. Figure S7: HMQC-
NMR product 6. Figure S8: MS spectroscopy of product 6. Figure S9: IR spectroscopy of product
1
6. Figure S10: H-NMR product 7. Figure S11: 13C-NMR product 7. Figure S12: Focused 13C-NMR
product 7. Figure S13: DEPT-NMR product 7. Figure S14: COSY-NMR product 7. Figure S15:
HMBC-NMR product 7. Figure S16: focused HMBC-NMR product 7. Figure S17: HMQC-NMR
product 7. Figure S18: MS spectroscopy of product 7. Figure S19: IR spectroscopy of product 7.
Author Contributions: Conceptualization, S.C. and P.P.; methodology, F.Z.; software, V.B.; investi-
gation, F.Z., R.B., D.S., and B.S.; resources, S.V.; writing—original draft preparation, S.C.; writing—
review and editing, S.C.; supervision, P.P.; project administration, S.V.; and funding acquisition, S.V.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by COSMAST (S.V. Grant number 01/2020), University of
Ferrara.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented are available in the manuscript Supplementary
Materials.

Molecules 2021, 26, 1352
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Acknowledgments: All the authors deeply thank Giancarlo Fantin, who unfortunately passed away,
for the precious contributions given to the realization of this article and Paolo Formaglio for support
during the NMR analyses.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of compounds 6 and 7 are available from the authors.
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