Novel Pyrrolidine Derivatives of Budesonide as Long Acting Inhaled Corticosteroids for the Treatment of Pulmonary Inflammatory Diseases

Article abstract of DOI:10.1021/acs.jmedchem.7b01873

Inhaled corticosteroids (ICSs) represent the first line therapy for the treatment of asthma and are also extensively utilized in chronic obstructive pulmonary disease. Our goal was to develop a new ICS with a basic group, which can allow solid state feature modulation, achieving at the same time high local anti-inflammatory effect and low systemic exposure. Through a rational drug design approach, a new series of pyrrolidine derivatives of budesonide was identified. Within the series, several compounds showed nanomolar binding affinity (K_i) with GR that mostly correlated with the effect in inducing GR nuclear translocation in CHO cells and anti-inflammatory effects in macrophagic cell lines. Binding and functional cell-based assays allowed identifying compound 17 as a potent ICS agonist with a PK profile showing an adequate lung retention and low systemic exposure in vivo. Finally, compound 17 proved to be more potent than budesonide in a rat model of acute pulmonary inflammation.

Full text of DOI:10.1021/acs.jmedchem.7b01873

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Article
Novel pyrrolidine derivatives of budesonide as long acting inhaled
corticosteroids for the treatment of pulmonary inflammatory diseases
Eleonora Ghidini, Gessica Marchini, Anna Maria Capelli, Chiara Carnini,
Valentina Cenacchi, Alessandro Fioni, Fabrizio Facchinetti, and Fabio Rancati
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01873 • Publication Date (Web): 09 May 2018
Downloaded from http://pubs.acs.org on May 10, 2018
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Novel pyrrolidine derivatives of budesonide
as long acting inhaled corticosteroids for the
treatment of pulmonary inflammatory diseases
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Eleonora Ghidini,*,§ Gessica Marchini,§ Anna Maria Capelli,‡ Chiara Carnini,§
Valentina Cenacchi,# Alessandro Fioni,# Fabrizio Facchinetti,§ and Fabio Rancati*,§
§Chemistry Research and Drug Design, §Pharmacology and Toxicology, #Pharmacokinetics
Biochemistry and Metabolism, ‡Computational chemistry
Chiesi Farmaceutici S.p.A, Nuovo Centro Ricerche, Largo Belloli 11/a, 43122 Parma, Italy
Eleonora Ghidini, Corporate Preclinical R&D, Chemistry Research & Drug Design, Chiesi
Farmaceutici, Largo Belloli 11/a, 43122 Parma, Italy, phone +39 0521 279913, fax +39 0521
279880, e-mail: e.ghidini@chiesi.com
Fabio Rancati, Corporate Preclinical R&D, Chemistry Research & Drug Design, Chiesi Farmaceutici,
Largo Belloli 11/a, 43122 Parma, Italy, phone +39 0521 279784, fax +39 0279880, e-mail:
f.rancati@chiesi.com
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ABSTRACT:Inhaled corticosteroids (ICS) represent the first line therapy for the treatment of asthma
and are also extensively utilized in chronic obstructive pulmonary disease. Our goal was to develop
a new ICS with a basic group which can allow solid state feature modulation, achieving at the same
time high local antiinflammatory effect and low systemic exposure. Through a rational drug design
approach, a new series of a pyrrolidine derivatives of budesonide was identified. Within the series,
several compounds showed nanomolar binding affinity (Ki) with GR that mostly correlated with the
effect in inducing GR nuclear translocation in CHO cells and anti-inflammatory effects in
macrophagic cell lines. Binding and functional cell-based assays allowed identifying compound 17
as a potent ICS agonist with a PK profile showing an adequate lung retention and low systemic
exposure in vivo. Finally, compound 17 proved to be more potent than budesonide in a rat model of
acute pulmonary inflammation.
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INTRODUCTION
Inhaled corticosteroids (ICS) continue to be the pharmacotherapeutic cornerstone for the treatment
of asthma in both adults and children.1 In addition, ICS are also utilized in chronic obstructive
pulmonary disease (COPD) in combinations with bronchodilators.2 ICS exert their effects by binding
and activating the glucocorticoid receptor (GR) that translocates to the nucleus and modulates
inflammatory gene expression. Increasing asthma disease severity, treatment intensity is stepped up,
initially by raising the ICS dose, in combination with long acting β2-adrenoceptor agonists, and
eventually by adopting oral corticosteroids (CS).3 Systemic exposure to CS is typically associated
with adverse effects, including diabetes, hypertension, myopathies, and osteoporosis.4 Inhaled
corticosteroids exert their anti-inflammatory activity locally in the airways, and hence this can be
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dissociated from their potential to cause systemic adverse effects. However, a degree of
bioavailability to systemic circulation, whether it be through lung absorption or via oral absorption
from a swallowed fraction of the inhaled dose of ICS, inevitably occurs.5 This may raise safety
concerns, especially in elderly and pediatric patients as well as in patients requiring high dose
regimen.6 Hence, there is the need for a better safety/tolerability profile of ICS through maximization
of local anti-inflammatory potency, while minimizing systemic exposure in order to achieve a higher
therapeutic index. To date, only few novel steroidal molecules showing significant improved
properties with respect to current drugs have been reported. 7,8,9,10 An example is fluticasone furoate
1, the last ICS that entered the market, structurally very similar to fluticasone propionate 2, but
showing an enhanced pharmacological profile e.g. duration of action.11,12
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Figure 1. Reference inhaled corticosteroids.
Consequently, the medicinal chemistry strategy, applied to the development of new ICS, aimed to
obtain compounds characterized by: 1) potent anti-inflammatory effect upon topical administration;
2) long persistence in the lung with no significant accumulation; 3) minimal systemic exposure; 4)
high clearance and formation of inactive metabolites; 5) compatibility with inhaler dosing devices
and with common excipients such as lactose to make the compound suitable for a dry powder
formulation largely used by COPD patients. In this scenario, the physical form (i.e. crystallinity) of
the compound needs to be investigated and considered for compound selection at an early stage of
the project in order to limit the risk of failure in next stage of development.13 This ‘inhalation by
design’ approach underlines the importance of tuning of the physico-chemical properties with the
aim of delivering compounds with long persistence in the lung to increase the duration of action and
limit systemic side-effects.14 Among ICS currently utilized in clinical practice, budesonide (3, Figure
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1) is effective and extensively used in the management of adult and childhood asthma.15 Starting from
the budesonide core structure we investigated whether structural modifications either in the
polycyclic scaffold or in the ketonic chain could lead to compounds with an improved potency, an
increased lung retention and eventually a longer duration of action. We further expanded the
exploration by introducing different decorations, following a rational drug design approach which
exploited the X-ray structure of the human GR-binding domain in complex with fluticasone furoate.16
The general formula of the compounds synthesized is shown in Figure 2. The pyrrolidine ring is
attractive being versatile for the modulation of physicochemical properties that could influence the
drug release rate in the lungs. Indeed basicity and lipophilicity of the pyrrolidine ring can be
modulated by the introduction of suitable substituents (R, R1) as well as salification in order to obtain
compounds suitable for lung delivery.17 On the other hand, the synthesis s of compounds bearing a
substituted, condensed pyrrolidine ring (Figure 2) required an intense investigation for the
identification of synthetic pathways suitable for structure-activity relationships (SAR) exploration
typical of a medicinal chemistry program.
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Considering biological data of our previously developed ICS isoxazoline series10 and the examples
reported in the literature,18,19 it was evident that prednisolone derivatives carrying fluorine atoms at
positions 4b and 12 show higher receptor-binding affinity at the GR compared to unsubstituted
analogue compound (see Figure 2 for compound numbering). For this reason the medicinal chemistry
investigation described here was focused on 4b,12-difluoro substituted scaffold. Here we report the
stereoselective synthesis and the pharmacological profile of a new series of ICS featuring a
pyrrolidine ring as replacement of the 1,3-dioxolane ring of budesonide.
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Figure 2. General formula of newly synthesized compounds.
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A novel series of pyrrolidine derivatives acting as potent GR agonists was identified and a compound
selected for its high anti-inflammatory potency in vitro, displayed prolonged lung retention and
efficacy in counteracting lipopolysaccharide-induced pulmonary inflammation upon intratracheal
administration in rats.
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SYNTHESIS
From the synthetic point of view, the introduction of a pyrrolidine ring required to develop a highly
stereo and regioselective 1,3-dipolar cycloaddition20 reacting α,ß-unsaturated intermediates 4 or 5
with a 1,3-dipole or a suitable precursor, as depicted in Scheme 1 and 2. Intermediates 4 and 5 were
prepared starting from commercially available difluprednate following a method established in our
laboratory.21 Intermediate 5 was reacted with a suitable azomethine ylide precursor 6,22 that under
acid catalysis led to the final products 7 and 8, as described in Scheme 1.
Scheme 1. Preparation of Compounds 7-8 a
aReagents and conditions: (a) TFA 0.01%, Xylene, 140 °C, 1 h; (b) Candida Antarctica Lipase,
ethanol, 37 °C, overnight.
To overcome the restriction due to the limited number of azomethine ylide precursors commercially
available, a new and more versatile synthetic approach was developed and reported in Scheme 2.
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Scheme 2. Preparation of Compounds 11 and 12a
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aReagents and conditions: (a) Dioxane, 0.01% TFA, 110 °C, 1 h; (b) vinyl chloroformate, ACN,
NaHCO3, 50 °C, 2 h; (c) 2 M HCl Dioxane, rt, 2 h, followed by treatment with MeOH, 40 °C, 1 h;
(d) HCl, water, dioxane, 50 °C, 7.5 h.
The N-benzyl pyrrolidine intermediate 9, obtained reacting 5 and N-(methoxymethyl)-N-
(trimethylsilylmethyl) benzylamine (6a) at high temperature in presence of TFA, was debenzylated
upon reaction with vinyl carbamoyl chloride followed by deprotection of the carbamate 10 with
hydrochloric acid. The 3 steps synthesis gave the unsubstituted pyrrolidine 11, in a 10% overall yield
and finally, after acetyl deprotection in acidic conditions, 12. Compound 11 can be easily converted
into different families of final products: their synthesis involves first the NH functionalization with a
suitable reagent followed by hydrolysis of the acetate ester that can be accomplished under acidic or
basic conditions depending on the substrate as further reported.
In order to investigate the influence of the R group (Figure 2) on biological activity, analogues 13-26
were synthesized (Scheme 3). Alkylation with alkyl bromides or chlorides gave compounds 13-19.
Compound 20-23 were prepared using the Eschweiler-Clarke reaction.23 The direct N-arylation was
more challenging: a method described in the literature24,25 encompasses the reaction with a benzyne
precursor. The reaction of 11 with 2-trimethylsilyl-phenyl trifluoromethane sulfonates in the presence
of cesium fluoride led to compound 24. The Chan-Lam reaction26 was also exploited reacting 11 with
the suitable aryl boronic acids leading to 25 and 26 as single regioisomer.
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Scheme 3. Preparation of Compounds 13-26a
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aReagents and conditions: (a) Alk-Br or Alk-Cl, Base, DCM; (b) HCOOH, MW; (c) CsF, ACN, rt,
overnight; (d) Cu(OAc)2, TEA, DCM, rt, 15 days; (e) K2CO3, MeOH, 0°C, 1 h; (f) HCl 3N Dioxane,
60 °C, 2 h.
Compounds 27-29 were prepared reacting intermediate 11 with the suitable acyl or sulfonyl chloride
(Scheme 4).
Scheme 4. Preparation of Compounds 27-29a
aReagents and conditions: (a) RCOCl, TEA, DCM, rt, 1 h or NaHCO3, ACN, 50 °C, 2h; (b) K2CO3,
MeOH, 0 °C, 1 h; (c) HCl 3N Dioxane, 60 °C, 2 h.
Exploration of the 6b-side chain proved to be more challenging. The first modification considered
was the conversion of the hydroxyl group of the 2-hydroxyacetyl substituent of budesonide into
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different functional groups such as fluoromethylketone, ester, amide and thioester, the last one present
in fluticasone furoate. As described in Scheme 5, the key step to obtain the fluoromethyl ketone
side-chain is the conversion of the mesylate into fluoro27 to give intermediates 30, then transformed
into 31 that was converted into secondary amine 32, following the same approach as described for
the synthesis of 11. This intermediate was converted into 33 using the same reaction conditions
previously described for 24.
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Scheme 5. Preparation of Compounds 31 and 33a
aReagents and conditions: (a) MsCl, DIPEA, ACN, rt, 30 min.; (b) TBAF, KF, 60 °C, 4 h; (c) 0.01%
TFA Dioxane reflux 1 h; (d)Vinyl chloroformate, ACN, NaHCO3, 1 h, 50 °C; (e) HCl dioxane, DCM,
rt, disappearance starting intermediate; (f) CsF, ACN, rt, 24 h.
The introduction of the fluticasone-like side chain required the synthesis of suitable acid precursor.
With the aim of evaluating the effect of this particular side chain modification, three different
functionalizations of the N atom of the pyrrolidine ring were considered: the corresponding acids
were obtained treating intermediates 9, 34 and 35 under basic conditions21 as described in Scheme 6.
In such a way, intermediate carboxylic acids 36-38 were obtained in a good yield.
Scheme 6. Preparation of Compounds 36-38a
aReagents and conditions: (a) NaOH, THF, rt, 24 h.
Having the desired carboxylic acid in hand, the scope of this investigation was not limited to the
preparation of the fluoromethyl thio ester, but it was also extended to the preparation of several
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derivatives showing different physico-chemical properties, as described in Scheme 7. The methyl
ester 39 was obtained by treatment of the corresponding acid 36 with dimethyl carbonate in the
presence of DBU, while amides 40-41 were easily obtained reacting the suitable amine in the presence
of HATU as condensing agent. The fluoro-methyl ester 42 was obtained by alkylation with
fluorobromomethane. The synthesis of the fluoromethyl thioesters foresaw reaction of the carboxylic
acid with HATU, followed by reaction with sodium hydrogen sulfide, NaHS, to give thioesters
employed to synthesize the carbothioic acid derivative, followed by alkylation with
bromochloromethane and substitution of chlorine first with iodine and then with fluorine to give 43
or by bromofluoromethane in dimethylformamide to give 44.28
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Scheme 7. Preparation of Compounds 39-44a
aReagents and conditions: (a) (CH3O)2CO, DBU, 90 °C, 3 days; (b) (CH3)2NH or benzylamine,
HATU, NMM, DMF, at 55 °C, 1 h; (c) BrCH2F, Na2CO3, DMF, -20 °C, 1 h; (d) HATU, NMM,
NaHS, 70 °C, 3h; (e) BrCH2Cl, rt, 1 h 30 min.; NaI, acetone; AgF, ACN, 3 steps; (f) BrCH2F,
DMF, rt, 1.5 h.
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The synthesis of the methyl ketone 48 was the most complicated and was carried out as described in
Scheme 8. Intermediate 10 was hydrolyzed under basic conditions, to preserve the vinyl carbamate,
then converted into 21-methanesulfonate, intermediate 45, followed by treatment with NaI to give
the methyl ketone 46.29 Hydrolysis of the carbamate followed by alkylation of 47 gave the desired
product 48.
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Scheme 8. Preparation of Compounds 54a
aReagents and conditions: (a) K2CO3, MeOH. (b) MsCl, DIPEA, DCM. (c) NaI, acetone. (d)
HCl/Dioxane (e) MeOH. (f) TEA, DCM.
RESULTS and DISCUSSION
In order to figure out the role played by the substituent on the nitrogen atom in modulating the binding
affinity at GR, compounds 7, 8, 13-26 were designed and synthesized by keeping fixed R1 group to
hydroxyacetyl (Table 1). The binding affinities (Ki) to GR have been commonly used to identify
compounds that bind to the GR receptor, although a linear relationship between binding and evoked
functional response at the cellular level has not always been verified.10 For this reason, in our SAR
analysis, we routinely utilized nuclear translocation as a key parameter for conpound progression,
being translocation of GR to the nucleus a necessary step for triggering the anti-inflammatory
response.
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The unsubstituted pyrrolidine 12 that served as key intermediate to expand the SAR at nitrogen was
also tested. It showed poor affinity to GR suggesting that its higher basicity together with its low
lipophilicity is not tolerated and confirming the importance of a lipophylic substituent to properly fit
the lipophilic pocket in the binding site. In the light of this result, aromatic and planar rings as well
as alkyl decorations were installed on the nitrogen in order to modulate its basicity, together with the
size, shape and lipophilic character of this part of the molecule.
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As shown in Table 1, GR binding affinity (Ki) of budesonide-like compounds lie in the range 0.39-
1.90 nM showing high affinity for GR receptor, similar to that shown by budesonide 3. However, Ki
values cover a very narrow range, making not possible a discrimination among compounds. On the
contrary, the GR NT assay data were more spread in a wide range allowing SAR analysis.
Table 1. GR Binding Affinity (Ki), Nuclear Translocation (EC50) Data, Calculated cLogD and
pKa of Budesonide-like Compounds 7, 8, 12-29.
NT EC50c (nM)
(95% CI)
Compound
R
Kia, b[nM]
cLogD7.4 pKa
1
-
0.51 (0.44-0.57) 0.4 (0.3-0.6)
-
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3
12
-
H
1.30 (0.62-1.8)
40%d
0.66
3.5 (2.0-6.0)
n.d.e
1.7 (1.4-2.1)
-
-
0.60
2.63
8.82
6.96
7
8
(0.45-0.95)
n.d.e
1.3 (0.5-3.2)
1.8 (1.1-2.9)
3.28
3.30
6.72
6.64
0.76
(0.28-1.95)
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NT EC50c (nM)
(95% CI)
Compound
R
Kia, b[nM]
cLogD7.4 pKa
0.66
(0.17-2.42)
5.3 (3.7-7.5)
3.30
2.96
6.43
7.27
13
0.52
(0.18-1.43)
11.3 (8.3-15.4)
14
0.52
(0.17-1.62)
8.4 (4.0-17.5)
63.9 (51.0-80.2)
54.9 (42.2-71.5)
15.2 (15.3-18.7)
3.4 (2.9-4.0)
3.03
1.26
0.61
1.57
2.48
7.11
4.11
7.91
7.83
7.98
15
16
20
19
n.d.e
n.d.e
n.d.e
1.90
(0.52-6.76)
18
0.57
(0.46-0.71)
4.6 (2.9-7.1)
4.90
6.40
22
23
0.71
(0.38-1.33)
31.2 (24.3-40.0)
2.1 (1.3-3.5)
2.59
3.31
3.42
6.95
0.62
(0.25-1.52)
21
0.39
(0.12-1.28)
1.3 (0.64-2.9)
5.8 (3.4-9.9)
1.13
3.57
8.82
4.06
24
25
0.52
(0.03-6.10)
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21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NT EC50c (nM)
(95% CI)
Compound
R
Kia, b[nM]
cLogD7.4 pKa
0.67
(0.52-0.76)
8.5 (4.8-8.6)
3.34
4.42
26
0.66
(0.41-1.00)
44.1 (29.6-68.0)
2.65
27
n.d.e
248 (80.7-767.3) 1.42
28
29
0.66
(0.48-0.86)
7.4 (5.7-9.7)
2.18
3
b
aConcentration displacing 50% of GR bound [ H]Dexamethasone. Data in parenthesis express
confidence intervals. cData in parenthesis express confidence intervals. d% Inhibition at the highest
concentration used in this assay (1000 nM). en.d.: not determined.
The insertion of an unsubstituted benzyl group as in compound 7 allowed both high binding and
efficacy which was either comparable or superior to that of budesonide 3 in the same tests.
The introduction of substituents at position 4- or 3- of the phenyl ring is generally well tolerated,
although small group like chlorine (compound 8, EC50=1.3 nM; compound 17, EC50=1.8 nM) made
compounds slightly more potent than trifluoromethyl (compound 13, EC50=5.3 nM) at para-position
on the phenyl ring. Introduction of electron donating groups causes a decrease in potency when
compared with electron withdrawing substituents, as observed for compounds 14 and 15.
Replacement of the phenyl ring with a pyridine fragment is not tolerated. The presence of the electron
lone-pair of pyridine's nitrogen likely disrupts the interaction of compound 16 with the GR,
demonstrating again how the presence of polar groups in this region is detrimental to the translocation
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efficacy. Derivatives carrying small alkyl group decoration, such as methyl (compound 20, EC50=54.9
nM) or cyclopropylmethyl (compound 19, EC50=15 nM) fragment, exhibit lower potency than
analogues carrying a benzyl group. This is likely due to the lower lypophilicity of compound 20
(cLogD = 0.611) with respect to compound 7 (cLogD = 2.62). The incorporation of a larger alkyl
substituent as in compound 18, which carries an isopentyl group, suggests that longer alkyl groups
are nicely accommodated in the lipophilic receptor binding pocket. The increased size of aromatic
portion led to a slight reduction in potency in the GR NT assay, as observed for compounds 22 and
23. Additional substituents characterized by different length were introduced at position 4 of the
benzyl group to gather information about shape and size requirements of the binding site. As a result,
compound 21, showing a good binding affinity and potency in NT assay, was identified. Further
exploration aimed to a selection of an N-aryl pyrrolidine sub-series, as reported in Table 1. Compound
24 exhibited good affinity and potency at GR whereas the introduction of a chlorine in m- or p-
position, as in compounds 25 and 26, caused a slight drop in NT potency. Conversion of the
pyrrolidine basic nitrogen into an amide or carbamate derivative, compounds 27 and 28, did not seem
to be tolerated; in particular, despite the comparable binding affinities, these derivatives showed very
low potency in the NT assay. Only sulfonamide derivative 29 retained a good potency in the NT
assay.
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13
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16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
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40
41
42
43
44
45
46
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48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. GR Binding affinity (Ki) and Nuclear Translocation (NT EC50), Calculated cLogD and
pKa of Compounds with Side Chain Modification at R1.
NT EC50 (nM)c
(95% CI)
Compound
R
R1
Ki [nM]a,b
cLogD7.4
pKa
0.52
(0.12-2.38)
3.4 (2.5-4.5)
2.99
6.87
31
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22
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25
26
0.52
(0.23-1.14)
0.71
(0.28-1.85)
0.57
(0.22-1.28)
3.2 (2.4-4.2)
1.59
3.10
3.70
8.74
7.12
6.90
33
39
5.7 (3.9-8.6)
7.7 (5.2-11.3)
48
42
0.57
(0.21-1.47)
20.8 (15.5-27.9)
3.90
6.58
1.1
(0.62-1.8)
9.8
(3.8-24.8)
n.d.d
40.3 (26.0-62.0)
50.0 (29.4-85.0)
45.0 (29.5-69.6)
2.26
3.80
n.d.d
7.30
7.05
n.d.d
40
41
43
44
4.8
(3.1-7.6)
57.0 (18.0-178.0) 4.46
n.d.d
3
aConcentration displacing 50% of GR bound [ H]Dexamethasone. Data in parenthesis refer to
b
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confidence intervals. cData in parenthesis express confidence intervals. dn.d.: not determined.
Exploration of side chain requirements at position 6b (R1, Table 2) was performed to improve the
potency in the NT assay and tune compounds’ properties. The replacement of the OH group of the
hydroxyacetyl substituent, typical of budesonide, with F, exemplified by compounds 31 and 33
resulted in a limited drop of potency in comparison with compounds 7 and 24, respectively. The lack
of a hydrogen bond interaction with the receptor for the methyl ester derivative 39 and the methyl
ketone analogue 48 caused a potency reduction in the NT assay.
Some other ester (OCH2F) and amide derivatives were also synthesized in order to re-establish the
hydrogen bond interaction with the GR receptor. While ester 42 and amide 40 appear tolerated in the
binding assay, amide 41 is much less potent, confirming the observation that the receptor does not
accept bulky groups in this position. Unfortunately, all the three compounds proved to be inactive in
NT functional assay. Finally, introduction of the fluoromethylthio ester in lateral chain (R1 = SCH2F)
did not result in an increase in compound affinity or potency in the NT assay, as observed for
compounds 43 and 44. To gain additional structural information on ligand-receptor complexes it was
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decided to perform a docking study to rationalize the similar binding affinity observed for two
compounds with a markedly different dimension of the substituent on the pyrrolidine nitrogen. The
crystal structure of human GR in complex with fluticasone furoate 1 was used as docking site and
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compound 17, carrying
a
m-chlorobenzyl, and compound 21, carrying
a
4-((4-
hydroxyphenylthio)methyl)benzyl residue, structurally different from the other compounds, were
chosen. Both these derivatives cannot be docked within the GR cavity even when the VDW radii of
the binding site amino acids were artificially reduced to simulate their flexibility. Visual inspection
of the binding site residues suggested that the rotamer of Met560 likely prevents compound 17 from
binding. Consequently, the amino acids lying within 5 Å from each atom of co-crystallized
fluticasone furoate 1 were allowed to alter their side chain conformations during the induced fit
docking simulations. As a result, 17 was nicely accommodated in the GR binding site when
Met5603 was moved from -177 degrees as in the X-ray structure (Figure 3, right) to +68 degrees of
the IFD pose (Figure 3, left) whereas the conformations of Tyr735 and Gln642 side chains as well as
those of the other key binding site amino acids (Asn564, Arg611, Gln570) were totally conserved.
Figure 3. Left: Induced fit docking (IFD) pose of compound 17 bound to the GR crystal structure
(PDB code: 3CLD). Right: crystal structure of compound 1 bound to GR.
Binding of the bulkier derivative 21 also required the alteration of Tyr735 1, moving from -166
degrees in the X-ray structure (Figure 4, residue colored by atom type) to -78 degrees in the IFD pose
(Figure 4, residue in yellow). Hence, Tyr735 appears to behave as the gate of the GR binding site.
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Finally, whilst the chloro-phenyl group of compound 17 can be buried inside GR hydrophobic binding
site (Figure 3), the phenol fragment of compound 21 is projected towards the solvent (Figure 4).
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7
8
9
To fully rationalize the experimental results with the computational studies, docking study of
budesonide was carried out and results are included in the Supporting Information.
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Figure 4. IFD pose of compound 21 bound to the GR crystal structure (3CLD PDB code). M560,
Y735 and Q642 rotamers identified with induced fit docking simulation are shown in yellow, overlaid
to the X-ray ones colored by atom type.
For a more detailed examination of this new series of corticosteroid pyrrolidine derivatives, their
selectivity against mineralocorticoid receptor (MR) was evaluated for few representative compounds
and data are shown in Table 3.
Table 3. Nuclear Translocation (EC50) for the GR and the MR of selected compounds.
Compound MR NT
GR NT
Ratio
MR NT/GR NT
EC50 (nM) a EC50 (nM) a
1
>3000
4.8 (4.3-5.4)
2.5 (1.5-3.5)
3.2 (1.9-5.8)
1.6 (0.8-2.7)
>3000
0.4 (0.3-0.6)
3.5 (2.0-6.0)
>3000
1.3
3
17
13
21
40
43
44
1.8 (1.1-2.9)
1.3
5.3 (3.7-7.5)
0.6
2.1 (1.3-3.5)
0.8
40.3 (26.0-62.0)
45.0 (29.5-69.6)
57.0 (18.0-178.0)
>50
>50
>50
>3000
>3000
a Data in parenthesis refer to confidence intervals.
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Compounds 13, 17 and 21 featuring the same side chain of budesonide 3 show the same low selctivity,
while compounds 40, 43 and 44 are more selective. This data confirmed that the removal of CH2OH
in side chain R1 is important to get selctivity versus MR.30
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To correlate potency in evoking nuclear translocation with the anti-inflammatory properties of each
compound, the inhibitory effects on nitric oxide release was evaluated in RAW 264.7 macrophagic
cells. Compounds selected either for their potency in the NT assay or for their particular structural
characteristics were tested and their IC50 values (concentration that inhibits 50% of NO production
compared to control) are reported in Table 4.
Table 4. Inhibition of LPS-induced NO Release in RAW264.7 Macrophagic cellsa
Cpd.
IC50 (nM)b
Cpd.
IC50 (nM) b
3
0.82 (0.52-1.3)
0.15 (0.08-0.31)
0.25 (0.12-0.51)
0.30 (0.21-0.42)
0.30 (0.29-0.31)
0.16 (0.01-2.97)
0.08 (0.05-0.11)
0.22 (0.18-0.26)
0.44 (0.32-0.60)
1
0.1 (0.01-0.86)
0.33 (0.11-0.98)
11.90 (4.17-34.3)
0.34 (0.10-1.20)
0.40 (0.12-1.30)
0.66 (0.13-3.26)
3.68 (1.34-10.15)
1.38 (0.20-9.36)
1.40 (1.30-1.60)
7
22
23
24
25
26
29
33
48
8
13
14
15
17
18
21
aValues are the average of experiments performed in triplicate. bConcentration that inhibits 50% of
NO production compared to control. Data in parenthesis express confidence intervals.
All compounds tested exhibited nanomolar/subnanomolar potency in RAW264.7 macrophages,
confirming that the NT assay well translates into a functional inhibition of an inflammatory response
in cells playing a major role in orchestrating immune responses. Among them, compound 17 showed
a potency similar to fluticasone furoate 1, one of the most potent compound in this assay model. As
highlighted in the introduction,13 the availability of a suitable crystalline form at an early stage of the
project is a very important feature for a compound designed for inhalation delivery, as it could result
in a lower risk of failure during the development phase. Besides its promising biological profile,
compound 17 exhibited a marked propensity to crystallize as a free base, which supported the
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progression of this compound to in vivo studies. One important aspect to consider for optimal inhaled
therapy is the prolonged duration of action as consequence of a high lung retention of the compound
that could be affected by its physical form. The presence of a basic center in these compounds could
have a high impact on their pharmacokinetic profile especially when converted into salts, normally
characterised by higher solubility. To evaluate this aspect, both compound 17 and its hydrochloride
salt were administered as liquid formulation to rats at the dose of 1 mol/kg by intratracheal route,
the mean lung and plasma pharmacokinetic parameters are reported in Table 5 and PK profiles
represented in Figure 5.
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Table 5. Main plasma and lung pharmacokinetic parameters in rat after intratracheal
administration of 17 as base, 17 as hydrochloride salt, budesonide 3 and fluticasone furoate 1
at 1 µmol/kg.
a
b
Compound
Matrix
Cmax
AUClast
MRTlast
(nmol/mL or (h*nmol/mLor (h)
nmol/g) ±SD
h*nmol/g)
1
plasma
lung
0.045 ± 0.01
0.050
1.4
9.5
77.10 ± 18.50 1049
0.127 ± 0.065 11.60
18.67 ± 12.47 110.03
3
plasma
lung
1.20
0.71
1.4
16.7
1.1
17 free base
plasma
lung
0.11 ± 0.02
64.26 ±15.60
0.090 ± 0.03
9.03 ± 1.58
0.118
871.20
0.120
9.0
17 hydrochloride salt plasma
lung
6.4
aAUClast: area under the curve, indicating the exposure up to the last quantified sampling time.
bMRTlast: mean residence time, from 0 up to the last quantified sampling time.
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17 base, plasma
17 base, lung
17 hydrochloride, plasma
17 hydrochloride, lung
3, plasma
100
10
3, lung
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1
0.1
0.01
0.001
0
8
16
24
Time (h)
32
40
48
Figure 5. Rat plasma and lung levels after intratracheal administration of compound 17 as base or as
hydrochloride salt and of reference compound 3 at 1 µmol/kg dose.
After intratracheal administration, the lung mean residence time (MRTlast) and lung exposure
(AUClast) were higher for compound 17 free base compared to 17 hydrochloride salt andbudesonide
3 ( Figure 5 and Table 5). Compound 17 systemic exposure resulted to be comparable for the free
base and hydrochloride salt and 100-fold lower than budesonide (in terms of AUC values); compound
17 is similar to 1 in terms of peak and exposure, but it shows a longer MRT (16.7 h vs 9.5 h).
The remarkably higher level and persistence of the compound 17 in the lung when administered as
free base suggested that this form is more suitable for once daily administration than the
hydrochloride salt and therefore it was progressed to in-vivo study in a model of neutrophilic lung
inflammation in the rat. This model is commonly used to investigate the anti-inflammatory activity
of compounds developed for the treatment of inflammatory airway diseases, such as corticosteroids
and phosphodiesterase 4 (PDE4) inhibitors.31,32 Compound 17, when administered to rats as
suspension via the intratracheal route 1 hour before LPS intratracheal challenge, proved to be more
potent than budesonide 3 in counteracting neutrophilic infiltration in the bronchoalveolar lavage
(Figure 6, ID50=0.097 µmol/kg vs ID50=0.34 µmol/kg). Moreover, compound 17 proved to be
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effective even when dosed 24 h before LPS challenge (˃50 % inhibition at ID80), showing that the
duration of action is in line with the remarkable lung retention shown by the PK profile. In the same
in vivo assay compound 3 did not show any significant residual activity at 24 hours post-dose while
compound 1 showed a sustained duration of action (>80% inhibition at ID80, data not shown).
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Figure 6. In vivo anti-inflammatory effect of compound 17 (Left) and compound 3 (budesonide,
Right) in a model of LPS-induced lung inflammation in the rat. Data are shown as mean ±standard
error of the mean. **p˂0.01 vs vehicle-treated, LPS-challenged group. Kruskal-Wallis test followed
by Dunn’s multiple comparison test.
Conclusions
Through a rational drug design approach a new series of a glucocorticoid pyrrolidine derivatives of
budesonide 3 with high affinity with GR was identified. Within the series, several compounds showed
a potent effect in inducing GR nuclear translocation and anti-inflammatory properties in macrophagic
cell cultures. In particular, compound 17 exhibited a pharmacokinetic profile suitable for topical
pulmonary administration, and higher potency than budesonide in an in vivo rat model of acute
pulmonary inflammation. Therefore, compound 17 could be a potential candidate for the development
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as an inhaled anti-inflammatory agent for treating pulmonary inflammatory diseases such as asthma
5
6
and COPD.
7
8
EXPERIMENTAL SECTION
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CHEMISTRY
All commercially available chemicals and solvents were used without further purification. Reactions
1
were performed under an atmosphere of nitrogen. All new compounds gave satisfactory H NMR,
1
LC/MS and mass spectrometry results. H NMR spectra were recorded on a Bruker ARX 300 (300
MHz) NMR spectrometer or on a Varian MR-400 (400 MHz) spectrometer, using residual signal of
deuterated NMR solvent as internal reference.
LC-MS (ESI+ ionization) analysis were performed on a Waters Micromass ZQ2000 instrument:
column, Acquity UPLC BEH C18 5Ox2.1mmx1.7μm ESI pos, 3.2KV, 25V; gradient elution 0_100%
B/A over 4 min with 1 min hold (solvent A, 95% water/5% MeCN/0.05% HCOOH; solvent B, 95%
MeCN/5% water/0.05% HCOOH; flow rate, 0.6 mL/min; bpi detection wavelength).
High resolution mass spectrometry (HRMS) was performed on a Thermo scientific QExactive as an
additional criterion of compounds identity (in ESI MS+).
The purity of tested compounds, determined by analytical UPLC, was >95%.
Column chromatography was performed on silica gel (200-300 mesh).
Preparative HPLC purifications were performed using the following method:
Waters Corporation purification system equipped with a XTerra Prep MS C18 Column (5 µm, 19 x
150 mm, Waters), 11 minutes gradient of 0-100% solvent B, where solvent A is
water:MeCN:HCOOH 95:5:0.05 and solvent B is water:MeCN:HCOOH 5:95:0.05.
The octanol/water partition coefficient cLogD and Ka were calculated using ACD Lab Percepta
software 2016.
The synthesis of starting compounds 4, 5 and of all the intermediates and/or acetyl precursors are
described in the Supporting Information.
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(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-Benzyl-4b,12-difluoro-5-hydroxy-6b-(2-hydroxy-
acetyl)-4a,6a-dimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8-aza-
pentaleno[2,1-a]phenanthren-2-one (7).
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A mixture of intermediate 4 (95 mg, 0.185 mmol), N-benzyl-1-methoxy-N-((trimethylsilyl)
methyl)methanamine (500 mg) and then xylene (5 ml) containing 0.01% of TFA was placed in a
closed vessel and heated at 140 °C for 1 h. The solvent was removed under vacuum and the residue
was purified by silica gel chromatography (DCM to DCM/AcOEt=6:4 V/V) leading to 7 (52 mg,
0.102 mmol, 54.9% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.08-7.44 (m, 7H),
6.29 (dd, J=1.53, 10.08 Hz, 1H), 6.12 (s, 1H), 5.51-5.76 (m, 1H), 5.41 (br s, 1H), 4.85 (br t, J=5.59
Hz, 1H), 4.05-4.28 (m, 3H), 3.39-3.47 (m, 2H), 3.26-3.29 (m, 1H), 3.14 (br d, J=5.70 Hz, 1H),
2.80-2.93 (m, 1H), 2.18-2.41 (m, 2H), 1.93-2.07 (m, 2H), 1.77-1.86 (m, 1H), 1.50-1.75 (m, 3H),
1.44-1.50 (m, 3H), 1.28-1.40 (m, 1H), 0.82-0.88 (m, 2H). LC-MS (ESI POS): 512.14 (MH+)ESI-
HRMS m/z: 512.2614 [M + H]+ (calcd for C30H35F2NO4, 512.2612).D20 = +50.8 c= 0.3,
MeOH).
(4aS,4bR,5S,6aS,6bS,9aR,8R,10aS,10bS,12S)-8-(4-Chloro-benzyl)-4b,12-difluoro-5-hydroxy-
6b-(2-hydroxy-acetyl)-4a,6a-dimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-
4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (8).
Following synthetic protocol and chromatographic conditions for 7 and starting from 4 and N-p-
chloro-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine, Cpd 8 was obtained (69 mg,
0.126 mmol, 39.4 % yield) as off white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.10-7.53 (m, 5H),
6.29 (br d, J=10.08 Hz, 1H), 6.12 (s, 1H), 5.50-5.79 (m, 1H), 5.40 (br d, J=2.19 Hz, 1H), 4.85 (br t,
J=5.70 Hz, 1H), 4.18-4.30 (m, 1H), 4.05-4.18 (m, 2H), 3.42 (s, 2H), 3.28 (br s, 1H), 3.09-3.20 (m,
1H), 2.87 (br t, J=8.22 Hz, 1H), 2.53-2.58 (m, 1H), 2.21-2.38 (m, 2H), 1.93-2.05 (m, 2H), 1.78-1.88
(m, 1H), 1.50-1.73 (m, 3H), 1.48 (s, 3H), 1.29-1.40 (m, 1H), 0.81-0.89 (m, 3H).
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LC-MS (ESI POS): 546.04 (MH+). ESI-HRMS m/z: 546.2228 [M + H]+ (calcd for
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C30H34ClF2NO4, 546.2223)D20= +83.5 (CHCl3, c 0.23).
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(4aR,4bS,5S,6aS,6bS,9aR,10aS,10bS)-5-Hydroxy-6b-(2-hydroxy-acetyl)-4a,6a-dimethyl-
4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8-aza-pentaleno[2,1-
a]phenanthren-2-one hydrochloride (12).
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A mixture of 11 (130 mg, 0.260 mmol), conc. HCl (1 mL, 32.9 mmol) in Dioxane (4ml) and Water
(4 ml) was stirred at 50 °C for 7.5 h. The solvent was evaporated and the crude was triturated with
EtOH to afford Cpd 12 (30 mg, 0.066 mmol, 25.2% yield) as a off white solid.
1H NMR (300 MHz, DMSO-d6)  ppm 9.08 (br. s., 2 H), 7.25 – 7.30 (m, 1 H), 6.27 – 6.33 (m, 1 H),
6.12 (s, 1 H), 5.54 – 5.60 (m, 1 H), 5.47 - 5.82 (m, 1 H), 5.11 (br. s., 1 H), 4.33 - 4.52 (m, 1 H), 4.07
- 4.31 (m, 2 H), 3.33 - 3.51 (m, 3 H), 2.92 - 3.16 (m, 2 H), 2.57 - 2.67 (m, 1 H), 2.11 - 2.30 (m, 1 H),
1.57 - 1.95 (m, 5 H), 1.49 (s, 3 H), 1.42 - 1.54 (m, 1 H), 0.94 (s, 3 H). LC-MS (ESI POS): 421.97
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(MH+). []D = +64.8 (c 0.2, H2O).
General procedure for the hydrolysis of acyl derivatives. Method A.
(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-
4a,6a-dimethyl-8-(4-trifluoromethyl-benzyl)-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-
tetradecahydro-4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (13).
Acetic acid 2-[(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-difluoro-5-hydroxy-4a,6adimethyl-
2-oxo-8-(4-trifluoromethyl-benzyl)-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8-
aza-pentaleno[2,1-a]phenanthren-6b-yl]-2-oxo-ethyl ester (114 mg, 0.183 mmol) was dissolved in
Dioxane (2 ml) and aqueous HCl 3 N (1 ml) and the reaction mixture was warmed at 60 °C for 2 h
under stirring. After evaporation of the solvent, the reaction mixture was poured in NaHCO3 and
extracted with AcOEt. The organic phase was washed with brine, dried over Na2SO4 and
evaporated; the residue was purified by silica gel chromatography (DCM to DCM/AcOEt=6:4
V/V), Cpd 13 was obtained (44 mg, 0.076 mmol, 41.4 % yield) as an off-white solid. 1H NMR (400
MHz, DMSO-d6) δ 7.64 (d, J=8.11 Hz, 2H), 7.43 (d, J=8.11 Hz, 2H), 7.25 (d, J=10.52 Hz, 1H),
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6.29 (dd, J=1.75, 10.08 Hz, 1H), 6.12 (s, 1H), 5.50-5.78 (m, 1H), 5.34-5.44 (m, 1H), 4.86 (t, J=5.81
Hz, 1H), 4.09-4.30 (m, 3H), 3.46-3.60 (m, 2H), 3.05-3.22 (m, 1H), 2.89 (t, J=8.33 Hz, 1H), 2.54-
2.61 (m, 1H), 2.37-2.43 (m, 1H), 2.21-2.31 (m, 1H), 1.95-2.07 (m, 2H), 1.80-1.87 (m, 1H), 1.51-
1.74 (m, 3H), 1.46-1.50 (m, 3H), 1.30-1.40 (m, 1H), 0.86 (s, 3H). LC-MS (ESI POS): 580.36
(MH+). ESI-HRMS m/z: 580.2485 [M + H]+ (calcd for C31H34F5NO4, 580.2481). []D20 = + 72.5
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(c = 0.45, MeOH).
General procedure for the hydrolysis of acyl derivatives. Method B.
(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-
4a,6a-dimethyl-8-(3-methyl-benzyl)-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-
4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (14).
A solution of acetic acid 2-[(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-difluoro-5-hydroxy-
4a,6a-dimethyl-8-(3-methyl-benzyl)-2-oxo-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-
4aH-8-aza-pentaleno[2,1-a]phenanthren-6b-yl]-2-oxo-ethyl ester (130 mg, 0.229 mmol) in MeOH (8
ml) was degassed bubbling nitrogen for 15 minutes and cooled to 0 °C. Then K2CO3 (15.83 mg, 0.115
mmol) was added and the mixture was stirred at 0 °C with continuous degassing for 1 h. The reaction
mixture was partitioned between a NaHCO3 solution and AcOEt. The combined organic phases were
concentrated and purified by silica gel chromatography (DCM/MeOH from 99:1 to 98:2) to yield 14
(91 mg, 0.173 mmol, 76 % yield) as an off-white foam. 1H NMR (400 MHz, DMSO-d6) δ 7.25 (d,
J=10.08 Hz, 1H), 7.12-7.19 (m, 1H), 6.96-7.06 (m, 3H), 6.23-6.42 (m, 1H), 6.07-6.18 (m, 1H), 5.52-
5.76 (m, 1H), 5.33-5.46 (m, 1H), 4.80-4.90 (m, 1H), 4.02-4.30 (m, 3H), 3.40 (q, J=13.37 Hz, 2H),
3.26-3.30 (m, 1H), 3.08-3.19 (m, 1H), 2.84 (t, J=8.22 Hz, 1H), 2.47 (br s, 1H), 2.36-2.42 (m, 1H),
2.26-2.35 (m, 1H), 2.18-2.25 (m, 3H), 1.98-2.12 (m, 2H), 1.78-1.87 (m, 1H), 1.51-1.74 (m, 3H), 1.49
(s, 3H), 1.31-1.40 (m, 1H), 0.86 (s, 3H). LC-MS (ESI POS): 526.30 MH+. ESI-HRMS m/z: 526.2769
[M + H]+ (calcd for C31H37F2NO4, 526.2763). []D25 + 81.0 (c=0.33, MeOH).
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(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-Difluoro-5-hydroxy-6b-(2-hydroxy-
acetyl)-4a,6a-dimethyl-8-(4-methyl-benzyl)-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-
tetradecahydro-4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (15).
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Following Method B, Cpd 15 was obtained (61 mg, 0.116 mmol, 47.4 % yield) as an off white
foam. 1H NMR (DMSO-d6, 400 MHz) δ 7.25 (d, J=10.1 Hz, 1H), 7.08 (s, 4H), 6.19-6.42 (m, 1H),
6.12 (s, 1H), 5.47-5.80 (m, 1H), 5.40 (br d, 1H, J=2.4 Hz), 4.62-4.93 (m, 1H), 3.90-4.39 (m, 3H),
3.38 (s, 2H), 3.09-3.19 (m, 1H), 2.85 (t, J=8.3 Hz, 1H), 2.50-2.71 (m, 2H), 2.35-2.41 (m, 1H), 2.25
(s, 3H), 1.90-2.08 (m, 2H), 1.85-1.91 (m, 1H), 1.50-1.73 (m, 3H), 1.48 (s, 3H), 1.34 (br dd, J=6.4,
12.1 Hz, 1H), 0.85 (s, 3H). LC-MS (ESI POS): 526.32 MH+. ESI-HRMS m/z: 526.2767 [M + H]+
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(calcd for C31H37F2NO4, 526.2763). []D + 89.3 (c=0.33, MeOH).
(4aS,4bR,5S,6aS,6bS,9aR,10aS,12S)-4b,12-Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-
4a,6adimethyl-8-pyridin-3-ylmethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-
4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (16) .
Following Method A, Cpd 16 was obtained (40 mg, 0.078 mmol, 30.9 % yield) as a white solid after
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purification by silica gel chromatography (AcOEt/MeOH=97:3 V/V). H NMR (DMSO-d6, 400
MHz) δ 8.41 (br s, 2H), 7.59 (br d, J=7.5 Hz, 1H), 7.03-7.44 (m, 2H), 6.29 (dd, J=1.5, 10.3 Hz, 1H),
6.12 (s, 1H), 5.48-5.79 (m, 1H), 5.41 (br d, J=2.2 Hz, 1H), 4.86 (br t, J=5.5 Hz, 1H), 4.01-4.32 (m,
3H), 3.48 (br s, 2H), 3.15 (br q, J=5.3 Hz, 1H,), 2.86 (br t, J=8.1 Hz, 1H), 2.54 (br s, 1H), 2.21-2.42
(m, 2H), 1.90-2.13 (m, 2H), 1.26-1.89 (m, 9H), 0.84-0.91 (m, 3H). LC-MS (ESI POS): 513.2 (MH+).
ESI-HRMS m/z: 513.2561 [M + H]+ (calcd for C29H34F2N2O4, 513.2565). []D25 =+64.40 (c=0.3,
MeOH).
(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-(3-Chloro-benzyl)-4b,12-difluoro-5-hydroxy-6b-
(2-hydroxy-acetyl)-4a,6a-dimethy-l4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-
4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (17).
Following Method B, Cpd 17 was obtained (376 mg, 0.689 mmol, 71 % yield) as a crystalline
white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.17-7.44 (m, 5H), 6.21-6.32 (m, 1H), 6.12 (s, 1H),
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5.50-5.73 (m, 1H), 5.37 (br s, 1H), 4.87 (br s, 1H), 3.90-4.39 (m, 3H), 3.40-3.52 (m, 2H), 3.08-3.20
(m, 1H), 2.85 (br t, J=8.2 Hz, 1H), 2.53-2.64 (m, 1H), 2.41 (br d, J=10.1 Hz, 1H), 2.21-2.32 (m, 1H),
1.99-2.08 (m, 2H), 1.81-1.92 (m, 1H), 1.5-1.7 (m, 3H), 1.48 (s, 3H), 1.34 (br dd, 1H, J=6.4, 12.1 Hz),
0.86 (s, 3H). LC-MS (ESI POS): 546.31 (MH+). ESI-HRMS m/z: 546.2236 [M + H]+ (calcd for
C30H34ClF2NO4, 546.2231). []D20 = +68.8 (c=0.5 CHCl3).
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(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-(3-Chloro-benzyl)-4b,12-difluoro-5-hydroxy-6b-
(2-hydroxy-acetyl)-4a,6a-dimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-
4aH-8-aza-pentaleno [2,1a]phenanthren-2-one hydrochloride (17.HCl).
Hydrochloric acid 4M in dioxane (156 µl, 0.623 mmol) was added to a clear solution of compound
17 as a base (68 mg, 0.125 mmol) in dioxane (8 ml). A solid immediately precipitated. The mixture
was stirred for 15 minutes, then the solid was filtered. The amount of recovered product was very
low, so the mother liquor was evaporated and the crude was triturated with AcOEt to give 17 as
hydrochloride salt (53 mg, 0.091 mmol, 73% yield) as an off white solid. 1H NMR (300 MHz,
DMSO-d6)  ppm 10.23 (br. s., 1 H), 7.05 - 7.83 (m, 5 H), 6.30 -6.34 (m, 1 H), 6.14 (s, 1 H), 5.58 -
5.65 (m, 1 H), 5.42 - 5.83 (m, 1 H), 4.11 - 4.57 (m, 5 H), 3.75 - 3.90 (m, 1 H), 3.42 - 3.69 (m, 2 H),
2.99 - 3.17 (m, 1 H), 2.78 - 2.92 (m, 1 H), 1.92 - 2.42 (m, 4 H), 1.56 - 1.86 (m, 3 H), 1.50 (s, 3 H),
1.38 - 1.48 (m, 1 H), 0.88 (s, 3 H). LC-MS (ESI POS): 546.23 MH+. []D25 + 22.63 (c 0.35 MeOH).
(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-
4a,6a-dimethyl-8-(3-methyl-butyl)-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-
8-aza-pentaleno[2,1-a]phenanthren-2-one (18).
Following Method A, Cpd 18 was obtained (48 mg, 0.098 mmol, 65.1 % yield) as a whitish solid.
1H NMR (300 MHz, DMSO-d6)  ppm 7.26 (d, J=10.1 Hz, 1H), 6.29 (dd, J=1.7, 10.1 Hz, 1H), 6.11
(s, 1 H), 5.47 - 5.83 (m, 1 H), 5.34 - 5.45 (m, 1 H), 4.68 - 4.91 (m, 1 H), 4.03 - 4.43 (m, 3 H), 3.06 -
3.18 (m, 1 H), 2.77 - 2.89 (m, 1 H), 2.12 - 2.45 (m, 8 H), 1.71 - 2.05 (m, 3 H), 1.58 (s, 2 H), 1.49 (s,
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3 H), 1.30 – 1.35 (m, 3 H), 0.86 (s, 3 H), 0.75 – 0.95 (m, 6 H). LC-MS (ESI POS): 491.28 (MH+). ESI-
HRMS m/z: 492.2922 [M + H]+ (calcd for C28H39F2NO4, 492.2920). []D20 =+50.48 (c=0.5, MeOH).
(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-Cyclopropylmethyl-4b,12-difluoro-5-hydroxy-6b-
(2-hydroxy-acetyl)-4a,6a-dimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-
4aH-8-azapentaleno[2,1-a]phenanthren-2-one (19).
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Following Method B, Cpd 19 was obtained (75 mg, 0.158 mmol, 53.0 % yield) as an off white
solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.25 (d, J=10.7 Hz, 1H), 6.30 (d, J=1.8 Hz, 1H), 6.10 (s,
1H), 5.48-5.72 (m, 1H), 5.32-5.40 (m, 1H), 4.83 (t, J=5.5 Hz, 1H), 4.29-4.40 (m, 1H), 4.10-4.18 (m,
2H), 3.11-3.22 (m, 1H), 2.99 (t, J=8.4 Hz, 1H), 2.69 (br d, J=9.9 Hz, 1H), 2.30 (br d, J=10.1 Hz,
1H), 2.20-2.31 (m, 1H), 2.12-2.21 (m, 1H), 2.00-2.12 (m, 1H), 1.82-2.00 (m, 3H), 1.7-1.8 (m, 1H),
1.52-1.60 (m, 1H), 1.48 (s, 3H), 1.38-1.42 (m, 1H), 0.86 (s, 3H), 0.71-0.82 (m, 1H), 0.34-0.42 (m,
2H), 0.04-0.02 (m, 2H). LC-MS (ESI POS): 476.25 MH+. ESI-HRMS m/z: 476.2608 [M + H]+
(calcd for C27H35F2NO4, 476.2612). []D25 =+ 72.4 (c 0.5 MeOH).
(4aS,4bR,5S,6aS,6bS,9aR,10aS,12S)-4b,12-Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-4a,6a,8-
trimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8-aza-pentaleno[2,1-
a]phenanthren-2-one (20).
Following Method A, Cpd 20 was obtained (35 mg, 0.080 mmol, 45.2 % yield) as an off-white solid
after trituration in AcOEt/Et2O. 1H NMR (400 MHz, DMSO-d6) δ 7.29 (d, J=10.08 Hz, 1H), 6.30 (dd,
J=1.64, 10.19 Hz, 1H), 6.11 (s, 1H), 5.40-5.84 (m, 2H), 4.99-5.35 (m, 1H), 4.01-4.59 (m, 3H), 3.53-
3.91 (m, 2H), 2.86-3.18 (m, 2H), 2.58-2.86 (m, 3H), 2.48-2.49 (m, 1H), 1.59-2.29 (m, 6H), 1.46-1.53
(m, 3H), 1.18-1.31 (m, 2H), 0.80-0.93 (m, 2H). LC-MS (ESI POS): 436.2 (MH+). ESI-HRMS m/z:
436.2294 [M + H]+ (calcd for C24H31F2NO4, 436.2299 ). ]D20 = +51.73 (c=0.3 MeOH).
(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS, 12S)-4b,12-Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-
8-[4-(4-hydroxy-phenylsulfanylmethyl)-benzyl]-4a,6a-dimethyl4b,5,6,6a,6b,7,8,9,9a,10,
10a,10b, 11,12-tetradecahydro-4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (21).
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Following Method B, Cpd 21 was obtained (52 mg, 0.080 mmol, 27.0 % yield), as an off-white
solid. 1H NMR (400 MHz, DMSO-d6)  ppm 9.51 (s, 1 H), 7.57 – 6.81 (m, 7 H), 6.67 (d, J=8.3 Hz,
2 H), 6.29 (dd, J=10.1, 1.8 Hz, 1 H), 6.12 (s, 1 H), 5.53 - 5.78 (m, 1 H), 5.41 (br. s., 1 H), 4.73 - 4.94
(m, 1 H), 4.05 – 4.30 (m, 3 H), 3.99 (s, 2 H), 3.39 (br. s., 2 H), 3.03 - 3.23 (m, 1 H), 2.70 - 2.93 (m, 1
H), 2.42 – 2.60 (m, 2 H), 2.15 - 2.40 (m, 2 H), 1.91 - 2.10 (m, 2 H), 1.40 – 1.80 (m, 7 H), 1.32 – 1.38
(m, 1 H), 0.86 (s, 3 H). LC-MS (ESI POS): 650.41 MH+. ESI-HRMS m/z: 650.2750 [M + H]+ (calcd
for C37H41F2NO5S, 650.2746). []D25 + 62.13 (C 0.16 MeOH).
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(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-Benzo[b]thiophen-2-ylmethyl-4b,12-difluoro-5-
hydroxy-6b-(2-hydroxy-acetyl)-4a,6a-dimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-
tetradecahydro-4aH-8-aza-pentaleno[2,1-a ]phenanthren-2-one (22).
Following Method B, Cpd 22 was obtained (75 mg, 0.132 mmol, 63.9 % yield) as an off-white solid.
1H NMR (DMSO-d6, 400 MHz) δ 7.85 (d, J=7.9 Hz, 1H), 7.72 (d, J=7.0 Hz, 1H), 7.23-7.35 (m, 3H),
7.22 (s, 1H), 6.29 (dd, J=1.9, 10.2 Hz, 1H), 6.13 (s, 1H), 5.53-5.75 (m, 1H), 5.38-5.43 (m, 1H), 4.86
(t, J=5.8 Hz, 1H), 4.21-4.43 (m, 2H), 4.01-4.27 (m, 2H), 3.74-3.92 (m, 2H), 3.18 (br d, J=5.7 Hz,
1H), 2.95 (t, J=8.3 Hz, 1H), 2.65 - 2.69 (m, 1 H) 2.61 (br d, J=10.1 Hz, 1H), 2.23-2.34 (m, 1H), 2.15
(dd, J=5.4, 8.9 Hz, 1H), 2.04 (br dd, J=5.5, 11.8 Hz, 1H), 1.79-1.87 (m, 1H), 1.51-1.79 (m, 3H), 1.49
(s, 3H), 1.33-1.42 (m, 1H), 0.87 (s, 3H). LC-MS (ESI POS): 568.28 MH+. ESI-HRMS m/z: 568.2333
[M + H]+ (calcd for C32H35F2NO4S, 568.2328). []D25 + 94.9 (c=0.35, MeOH).
4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-
4a,6a-dimethyl-8-quinolin-2-ylmethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-
4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (23).
Following Method B, Cpd 23 was obtained (68 mg, 0.121 mmol, 56.2 % yield) as a light orange
solid. 1H NMR (400 MHz, DMSO-d6) δ 8.29 (d, J=8.55 Hz, 1H), 7.93 (dd, J=1.53, 8.55 Hz, 2H), 7.72
(t, J=7.13 Hz, 1H), 7.52-7.60 (m, 1H), 7.46 (d, J=8.33 Hz, 1H), 7.25 (d, J=10.08 Hz, 1H), 6.29 (dd,
J=1.64, 10.19 Hz, 1H), 6.13 (s, 1H), 5.55-5.75 (m, 1H), 5.40 (br d, J=2.19 Hz, 1H), 4.87 (t, J=5.81
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