Novel Pyrrolidine Derivatives of Budesonide as Long Acting Inhaled

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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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Journal of Medicinal Chemistry

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*,§ §

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: [email protected] 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: [email protected]

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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.

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 2 ACS Paragon Plus Environment

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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

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 3 ACS Paragon Plus Environment


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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. 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. 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. 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

a

Reagents 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

a

Reagents 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. 6 ACS Paragon Plus Environment

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Scheme 3. Preparation of Compounds 13-26a

a

Reagents 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

a

Reagents 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 7 ACS Paragon Plus Environment


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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. Scheme 5. Preparation of Compounds 31 and 33a

a

Reagents 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

a

Reagents 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 8 ACS Paragon Plus Environment

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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 Scheme 7. Preparation of Compounds 39-44a

a

Reagents 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. 9 ACS Paragon Plus Environment


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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. Scheme 8. Preparation of Compounds 54a

a

Reagents 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. As shown in Table 1, GR binding affinity (Ki) of budesonide-like compounds lie in the range 0.391.90 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)

cLogD7.4

pKa

0.51 (0.44-0.57) 1.30 (0.62-1.8) 40%d 0.66 (0.45-0.95)

0.4 (0.3-0.6) 3.5 (2.0-6.0) n.d.e 1.7 (1.4-2.1)

0.60 2.63

8.82 6.96

8

n.d.e

1.3 (0.5-3.2)

3.28

6.72

0.76 (0.28-1.95)

1.8 (1.1-2.9)

3.30

6.64

17

Compound

R

Kia, b[nM]

1 3 12

H

7



Compound

R

Kia, b[nM]

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cLogD7.4

pKa

13

0.66 (0.17-2.42)

5.3 (3.7-7.5)

3.30

6.43

14

0.52 (0.18-1.43)

11.3 (8.3-15.4)

2.96

7.27

15

0.52 (0.17-1.62)

8.4 (4.0-17.5)

3.03

7.11

16

n.d.e

63.9 (51.0-80.2)

1.26

4.11

20

n.d.e

54.9 (42.2-71.5)

0.61

7.91

19

n.d.e

15.2 (15.3-18.7)

1.57

7.83

18

1.90 (0.52-6.76)

3.4 (2.9-4.0)

2.48

7.98

22

0.57 (0.46-0.71)

4.6 (2.9-7.1)

4.90

6.40

23

0.71 (0.38-1.33)

31.2 (24.3-40.0)

2.59

3.42

0.62 (0.25-1.52)

2.1 (1.3-3.5)

3.31

6.95

24

0.39 (0.12-1.28)

1.3 (0.64-2.9)

1.13

8.82

25

0.52 (0.03-6.10)

5.8 (3.4-9.9)

3.57

4.06

21


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Compound

R

Kia, b[nM]


cLogD7.4

pKa 4.42

26

0.67 (0.52-0.76)

8.5 (4.8-8.6)

3.34

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

0.66 (0.48-0.86)

7.4 (5.7-9.7)

2.18

28

29 a

Concentration displacing 50% of GR bound [3H]Dexamethasone. bData 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 13 ACS Paragon Plus Environment


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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 pposition, 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. Table 2. GR Binding affinity (Ki) and Nuclear Translocation (NT EC50), Calculated cLogD and pKa of Compounds with Side Chain Modification at R1.

Compound 31

R

R1

Ki [nM]a,b 0.52 (0.12-2.38)

NT EC50 (nM)c (95% CI)

cLogD7.4

pKa

3.4 (2.5-4.5)

2.99

6.87


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3.2 (2.4-4.2)

1.59

8.74

5.7 (3.9-8.6)

3.10

7.12

7.7 (5.2-11.3)

3.70

6.90

48

0.52 (0.23-1.14) 0.71 (0.28-1.85) 0.57 (0.22-1.28) 0.57 (0.21-1.47)

20.8 (15.5-27.9)

3.90

6.58

42

1.1 (0.62-1.8) 9.8 (3.8-24.8) n.d.d

40.3 (26.0-62.0)

2.26

7.30

50.0 (29.4-85.0)

3.80

7.05

45.0 (29.5-69.6)

n.d.d

n.d.d

33 39

40 41 43

4.8 57.0 (18.0-178.0) 4.46 n.d.d (3.1-7.6) a Concentration displacing 50% of GR bound [3H]Dexamethasone. bData in parenthesis refer to 44

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 15 ACS Paragon Plus Environment


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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 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. 16 ACS Paragon Plus Environment

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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). 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.

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 a EC50 (nM) EC50 (nM) a 1 >3000 0.4 (0.3-0.6) 3 4.8 (4.3-5.4) 3.5 (2.0-6.0) 17 2.5 (1.5-3.5) 1.8 (1.1-2.9) 13 3.2 (1.9-5.8) 5.3 (3.7-7.5) 21 1.6 (0.8-2.7) 2.1 (1.3-3.5) 40 >3000 40.3 (26.0-62.0) 43 >3000 45.0 (29.5-69.6) 44 >3000 57.0 (18.0-178.0) a Data in parenthesis refer to confidence intervals. 17 ACS Paragon Plus Environment

Ratio MR NT/GR NT >3000 1.3 1.3 0.6 0.8 >50 >50 >50


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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 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) 1 0.1 (0.01-0.86) 7 0.15 (0.08-0.31) 22 0.33 (0.11-0.98) 8 0.25 (0.12-0.51) 23 11.90 (4.17-34.3) 13 0.30 (0.21-0.42) 24 0.34 (0.10-1.20) 14 0.30 (0.29-0.31) 25 0.40 (0.12-1.30) 15 0.16 (0.01-2.97) 26 0.66 (0.13-3.26) 17 0.08 (0.05-0.11) 29 3.68 (1.34-10.15) 18 0.22 (0.18-0.26) 33 1.38 (0.20-9.36) 21 0.44 (0.32-0.60) 48 1.40 (1.30-1.60) a Values 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 18 ACS Paragon Plus Environment

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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. 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. Cmax AUClasta MRTlastb (nmol/mL or (h*nmol/mLor (h) nmol/g) ±SD h*nmol/g) 1 plasma 0.045 ± 0.01 0.050 1.4 lung 77.10 ± 18.50 1049 9.5 3 plasma 0.127 ± 0.065 11.60 1.20 lung 18.67 ± 12.47 110.03 0.71 17 free base plasma 0.11 ± 0.02 0.118 1.4 lung 64.26 ±15.60 871.20 16.7 17 hydrochloride salt plasma 0.090 ± 0.03 0.120 1.1 lung 9.03 ± 1.58 9.0 6.4 a AUClast: area under the curve, indicating the exposure up to the last quantified sampling time. Compound

b

Matrix

MRTlast: mean residence time, from 0 up to the last quantified sampling time.



17 base, plasma 17 base, lung 17 hydrochloride, plasma 17 hydrochloride, lung 3, plasma

100

3, lung

Concentration (nmol/mL or nmol/g)

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

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10

1

0.1

0.01

0.001 0

8

16

24

32

40

48

Time (h)

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 20 ACS Paragon Plus Environment

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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).

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 21 ACS Paragon Plus Environment


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as an inhaled anti-inflammatory agent for treating pulmonary inflammatory diseases such as asthma and COPD. EXPERIMENTAL SECTION CHEMISTRY All commercially available chemicals and solvents were used without further purification. Reactions were performed under an atmosphere of nitrogen. All new compounds gave satisfactory 1H NMR, LC/MS and mass spectrometry results. 1H 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.


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


(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-Benzyl-4b,12-difluoro-5-hydroxy-6b-(2-hydroxyacetyl)-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 (7). 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+)ESIHRMS 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-hydroxy6b-(2-hydroxy-acetyl)-4a,6a-dimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (8). Following synthetic protocol and chromatographic conditions for 7 and starting from 4 and N-pchloro-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 C30H34ClF2NO4, 546.2223)D20= +83.5 (CHCl3, c 0.23). (4aR,4bS,5S,6aS,6bS,9aR,10aS,10bS)-5-Hydroxy-6b-(2-hydroxy-acetyl)-4a,6a-dimethyl4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8-aza-pentaleno[2,1a]phenanthren-2-one hydrochloride (12).

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. 1

H 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 (MH+). []D20 = +64.8 (c 0.2, H 2O). 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,11,12tetradecahydro-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,6adimethyl2-oxo-8-(4-trifluoromethyl-benzyl)-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8aza-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), 24 ACS Paragon Plus Environment

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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), 2.542.61 (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), 1.511.74 (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 (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-tetradecahydro4aH-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-hydroxy4a,6a-dimethyl-8-(3-methyl-benzyl)-2-oxo-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,11,12-tetradecahydro4aH-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), 5.525.76 (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-hydroxyacetyl)-4a,6a-dimethyl-8-(4-methyl-benzyl)-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12tetradecahydro-4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (15). 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]+ (calcd for C31H37F2NO4, 526.2763). []D25 + 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-tetradecahydro4aH-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 purification by silica gel chromatography (AcOEt/MeOH=97:3 V/V). 1H 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-tetradecahydro4aH-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), 26 ACS Paragon Plus Environment

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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). (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-tetradecahydro4aH-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-4aH8-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. 1

H 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+). ESIHRMS 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-tetradecahydro4aH-8-azapentaleno[2,1-a]phenanthren-2-one (19). 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,8trimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8-aza-pentaleno[2,1a]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), 3.533.91 (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). 28 ACS Paragon Plus Environment

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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). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-Benzo[b]thiophen-2-ylmethyl-4b,12-difluoro-5hydroxy-6b-(2-hydroxy-acetyl)-4a,6a-dimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12tetradecahydro-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. 1

H 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-tetradecahydro4aH-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 29 ACS Paragon Plus Environment


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Hz, 1H), 4.22-4.32 (m, 1H), 4.07-4.19 (m, 2H), 3.75 (d, J=1.97 Hz, 2H), 3.28 (br s, 1H), 3.14-3.23 (m, 1H), 2.94 (t, J=8.33 Hz, 1H), 2.55-2.68 (m, 2H), 2.24-2.35 (m, 1H), 2.13-2.20 (m, 1H), 2.03-2.12 (m, 1H), 1.78-1.86 (m, 1H), 1.51-1.76 (m, 3H), 1.49 (s, 3H), 1.34-1.43 (m, 1H), 0.87 (s, 3H). LC-MS (ESI POS): 563.35 MH+. ESI-HRMS m/z: 563.2720 [M + H]+ (calcd for C33H36F2N2O4, 563.2719). []D25 + 76.1 (c=0.32, MeOH). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)4a,6a-dimethyl-8-phenyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8azapentaleno[2,1-a]phenanthren-2-one (24). Following Method B, Cpd 24 was obtained (40 mg, 0.080 mmol, 82 % yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.26 (d, J=10.3 Hz, 1H), 7.14 (t, J=7.9 Hz, 2H), 6.58-6.69 (m, 3H), 6.29 (dd, J=1.9, 10.2 Hz, 1H), 6.08 (s, 1H), 5.51-5.73 (m, 1H), 5.41-5.50 (m, 1H), 4.92 (t, J=5.9 Hz, 1H), 4.43 (d, J=5.9 Hz, 1H), 4.17-4.24 (m, 1H), 4.13-4.21 (m, 1H), 3.43-3.52 (m, 1H), 3.34-3.41 (m, 1H), 3.25 (s, 2H), 3.05 (dd, J=2.3, 9.5 Hz, 1H), 2.18-2.28 (m, 1H), 1.92-2.01 (m, 2H), 1.78-1.92 (m, 2H), 1.60-1.69 (m, 1H), 1.48-1.52 (m, 4H), 0.99 (s, 3H). LC-MS (ESI POS): 498.13 MH+. ESIHRMS m/z: 498.2457 [M + H]+ (calcd for C29H33F2NO4, 498.2450). []D25 +29.6 (c= 0.2, MeOH). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-(3-Chloro-phenyl)-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-tetradecahydro4aH-8-aza-pentaleno[2,1-a]phenanthr en-2-one (25). Following Method B, Cpd 25 was obtained (45 mg, 0.085 mmol, 63.1 % yield) as an off-white solid. 1

H NMR (400 MHz, DMSO-d6) δ 7.26 (d, J=10.74 Hz, 1H), 7.14 (t, J=8.11 Hz, 1H), 6.63-6.69 (m,

2H), 6.54-6.59 (m, 1H), 6.29 (dd, J=1.75, 10.08 Hz, 1H), 6.05-6.11 (m, 1H), 5.51-5.72 (m, 1H), 5.45 (br d, J=1.97 Hz, 1H), 4.93 (t, J=5.92 Hz, 1H), 4.46 (dd, J=5.92, 18.42 Hz, 1H), 4.05-4.23 (m, 2H), 3.42-3.51 (m, 1H), 3.34-3.41 (m, 1H), 3.26-3.29 (m, 2H), 3.04-3.14 (m, 1H), 2.18-2.28 (m, 1H), 1.751.96 (m, 4H), 1.43-1.53 (m, 5H), 1.20-1.26 (m, 1H), 0.99 (s, 3H). LC-MS (ESI POS): 532.33 MH+.


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ESI-HRMS m/z: 532.2067 [M + H]+ (calcd for C29H32ClF2NO4, 532.2072). []D25 +18.1 (c= 0.4, MeOH). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-(4-Chloro-phenyl)-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-tetradecahydro4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (26). Following Method B, Cpd 26 was obtained (24 mg, 0.045 mmol, 78 % yield) as an off-white foam. 1

H NMR (300 MHz, DMSO-d6) δ ppm 7.26 (d, J=10.1 Hz, 1H), 7.10 - 7.22 (m, 2 H), 6.52 - 6.72 (m,

2 H), 6.29 (dd, J=1.7, 10.1 Hz, 1H), 6.09 (s, 1 H), 5.43 - 5.72 (m, 2 H), 4.93 (t, J=5.92 Hz, 1H), 4.45 (dd, J=5.9, 18.4 Hz, 1H), 4.10 - 4.27 (m, 2 H), 3.43 - 3.55 (m, 1 H), 3.34 - 3.43 (m, 3 H), 3.06 (br d, J=5.7 Hz, 1H), 2.59 - 2.67 (m, 1 H), 2.18 - 2.34 (m, 1 H), 1.71 - 2.00 (m, 4 H), 1.52-1.59 (m, 1 H), 1.50 (s, 3 H), 1.37 - 1.48 (m, 1 H), 0.99 (s, 3 H). LC-MS (ESI POS): 532.28 MH+. ESI-HRMS m/z: 532.2064 [M + H]+ (calcd for C29H32ClF2NO4, 532.2061). [D25 +19.3 (c= 0.26, MeOH). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)4a,6a-dimethyl-2-oxo-2,4b,5,6,6a,6b,7,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8azapentaleno[2,1-a]phenanthrene-8-carboxylic acid benzyl ester (27). Following Method A, Cpd 27 was obtained (28 mg, 0.050 mmol, 41.8 % yield) as a white solid after purification by silica gel chromatography (AcOEt/petroleum ether=70:30 V/V). 1H NMR (400 MHz, DMSO-d6) δ 7.09-7.47 (m, 6H), 6.29 (dd, J=1.75, 10.30 Hz, 1H), 6.12 (br s, 1H), 5.51-5.74 (m, 1H), 5.46 (br d, J=2.41 Hz, 1H), 4.88-5.11 (m, 3H), 4.33-4.46 (m, 1H), 4.02-4.22 (m, 2H), 3.343.60 (m, 3H), 3.20-3.29 (m, 3H), 2.15-2.27 (m, 1H), 1.70-1.92 (m, 4H), 1.48 (s, 3H), 1.34-1.43 (m, 1H), 0.95 (s, 3H). LC-MS (ESI POS): 556.38 (MH+). ESI-HRMS m/z: 556.2510 [M + H]+ (calcd for C31H35F2NO6, 556.2511). [] +D20 = +17.15 (c 0.33, MeOH). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-Benzoyl-4b,12-difluoro-5-hydroxy-6b-(2-hydroxyacetyl)-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 (28). 31 ACS Paragon Plus Environment


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Following Method B, Cpd 28 was obtained (49 mg, 0.093 mmol, 53% yield) as a white glassy solid. 1

H NMR (400 MHz, DMSO-d6) δ 7.12-7.53 (m, 6H), 6.22-6.37 (m, 1H), 6.12 (s, 1H), 5.53-5.73 (m,

1H), 5.48 (br s, 1H), 4.93-5.06 (m, 1H), 4.30-4.53 (m, 1H), 3.99-4.26 (m, 2H), 3.53-3.76 (m, 2H), 3.36-3.52 (m, 1H), 3.20-3.29 (m, 2H), 2.24 (br s, 1H), 1.66-1.97 (m, 4H), 1.51-1.59 (m, 1H), 1.48 (s, 3H), 1.22-1.37 (m, 1H), 0.94 (s, 3H). LC-MS (ESI POS): 526.39 (MH+). ESI-HRMS m/z: 526.2402 [M + H]+ (calcd for C30H33F2NO5, 526.2405). []D20 = + 6.3 (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-(thiophene-2-sulfonyl)-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12tetradecahydro-4aH-8-aza-pentaleno[2,1-a]phenanthren-2-one (29). Following Method A, Cpd 29 was obtained (37 mg, 0.065 mmol, 47 % yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.03 (dd, J=1.21, 4.93 Hz, 1H), 7.65 (dd, J=1.32, 3.73 Hz, 1H), 7.057.38 (m, 2H), 6.29 (dd, J=1.75, 10.08 Hz, 1H), 6.10 (s, 1H), 5.49-5.72 (m, 1H), 5.43-5.48 (m, 1H), 5.07 (t, J=5.81 Hz, 1H), 4.29-4.39 (m, 1H), 4.01-4.18 (m, 2H), 3.28 (br d, J=2.63 Hz, 2H), 3.17 (d, J=11.18 Hz, 1H), 3.01 (d, J=11.40 Hz, 1H), 2.80-2.89 (m, 1H), 2.11-2.22 (m, 1H), 1.85 (br d, J=15.57 Hz, 1H), 1.50-1.74 (m, 3H), 1.46 (s, 3H), 1.20-1.35 (m, 3H), 0.88 (s, 3H). LC-MS (ESI POS): 568.43 (MH+). ESI-HRMS m/z: 568.1639 [M + H]+ (calcd for C27H31F2NO6S2, 568.1634). []D20 = +28.09 (c 0.22, MeOH). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-Benzyl-4b,12-difluoro-6b-(2-fluoro-acetyl)-5hydroxy-4a,6a-dimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8azapentaleno[2,1-a]phenanthren-2-one (31). In a closed vessel to a mixture of compound 30 (125 mg, 0.329 mmol) and N-benzyl-1-methoxy-N((trimethylsilyl)methyl)methanamine (0.318 ml, 1.314 mmol) in 1,4-Dioxane (5 ml) , 1 drop of TFA (cat) was added and the mixture was stirred at 100 °C for 1 h. The reaction mixure was concentrated and purified by silica gel chromatography (DCM/AcOEt=7:3 V/V) and then by preparative HPLC to afford 31 (45 mg, 0.088 mmol, 26.7 % yield) as a white glassy solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.18-7.31 (m, 6H), 6.29 (dd, J=1.8, 10.1 Hz, 1H), 5.53-5.74 (m, 1H), 5.38-5.49 (m, 1H), 5.01-5.35 32 ACS Paragon Plus Environment

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(m, 2H), 4.08-4.20 (m, 1H), 3.47-3.61 (m, 2H), 3.05-3.14 (m, 1H), 2.92 (t, J=8.3 Hz, 1H), 2.52-2.63 (m, 2H), 2.37 (br d, J=10.1 Hz, 1H), 2.22-2.31 (m, 1H), 1.93-2.11 (m, 2H), 1.83 (br d, J=13.6 Hz, 1H), 1.52-1.84 (m, 3H), 1.48 (s, 3H), 1.32-1.40 (m, 1H), 0.90 (s, 3H). LC-MS (ESI POS): 514.39 MH+. ESI-HRMS m/z: 514.2568 [M + H]+ (calcd for C30H34F3NO3, 514.2569). []D25 + 84.2 (c 0.36 CHCl3). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-Difluoro-6b-(2-fluoro-acetyl)-5-hydroxy4a,6adimethyl-8-phenyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8azapentaleno[2,1-a]phenanthren-2-one (33). Cpd 32 as a hydrochloride salt (307 mg, 0.667 mmol) was suspended in Acetonitrile (12 ml), cesium fluoride (406 mg, 2.67 mmol), 2-(trimethylsilyl)phenyl-trifluoromethanesulfonate (0.194ml, 0.801 mmol) and water (1 drop) were added and the mixture was stirred at room temperature for 24 h. The reaction mixture was partitioned between water and AcOEt, the organic layer was separated, dried and concentrated. The crude was purified by silica gel chromatography (DCM/MeOH = 99:1 V/V) to yield 33 (94 mg, 0.188 mmol, 28.2 % yield) as an off-white foam. 1H NMR (DMSO-d6, 400 MHz) δ 7.26 (d, J=10.1 Hz, 1H), 7.15 (t, J=7.8 Hz, 2H), 6.6-6.7 (m, 3H), 6.28 (dd, J=1.8, 10.1 Hz, 1H), 6.08 (s, 1H), 5.43-5.74 (m, 3H), 5.0-5.2 (m, 1H), 4.18-4.23 (m, 1H), 3.49-3.54 (m, 2H), 3.32-3.40 (m, 1H), 3.22-3.35 (m, 1H), 3.03-3.11 (m, 1H), 2.54-2.65 (m, 1H), 2.20-2.31 (m, 1H), 1.82-2.01 (m, 4H), 1.40-1.52 (m, 5H), 1.03 (s, 3H). LC-MS (ESI POS): 500.31 MH+. ESI-HRMS m/z: 500.2411 [M + H]+ (calcd for C29H32F3NO3, 500.2407). []D25 + 24,1 (c=0.48, MeOH). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-Benzyl-4b,12-difluoro-5-hydroxy-4a,6a-dimethyl2-oxo-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8-aza-pentaleno[2,1-a] phenanthrene-6b-carboxylic acid methyl ester (39). Compound 36 (150 mg, 0.301 mmol) was dissolved in dimethyl carbonate (4 mL, 47.5 mmol), 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (DBU, 0.045 mL, 0.301 mmol) was added and the mixture was heated at 90 °C for 3 days. The reaction mixture was concentrated to dryness and purified by preparative HPLC to yield 45 mg of compound as TFA salt, which was further purified on a silica 33 ACS Paragon Plus Environment


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gel chromatography (DCM/MeOH=98:2 V/V) affording the titled compound 39, as TFA salt, (39 mg, 0.062 mmol, 20.7% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.07-7.63 (m, 6H), 6.32 (br d, J=9.87 Hz, 1H), 6.00-6.22 (m, 1H), 5.52-5.76 (m, 2H), 4.27-4.50 (m, 2H), 4.17 (br s, 1H), 3.593.76 (m, 4H), 3.21-3.29 (m, 1H), 3.07-3.18 (m, 1H), 2.80-2.93 (m, 1H), 2.59-2.70 (m, 1H), 2.24-2.36 (m, 1H), 2.01-2.15 (m, 1H), 1.54-1.79 (m, 4H), 1.50 (s, 3H), 1.40-1.48 (m, 1H), 0.86-1.00 (m, 3H). LC-MS (ESI POS): 512.18 MH+. ESI-HRMS m/z: 512.2612 [M + H]+ (calcd for C30H35F2NO4, 512.2607). []D25 + 32.08 (c 0.49, MeOH). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-Benzyl-4b,12-difluoro-5-hydroxy-4a,6a-dimethyl2-oxo-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8-aza-pentaleno[2,1a]phenanthrene-6b-carboxylic acid dimethylamide (40). A mixture of 36 (85 mg, 0.171 mmol), HATU (65.0 mg, 0.171 mmol) and N-Methylmorpholine (0.021 ml, 0.188 mmol) in dry DMF (3 ml) was stirred at 55 °C under nitrogen for 1h. Then 2M solution in THF dimethylamine (0.854 ml, 1.708mmol) was added and the mixture was stirred at room temperature overnight. The mixture was partitioned between AcOEt and water, then the organic layer was dried over Na2SO4 was concentrated. The crude was purified by silica gel chromatography (DCM/AcOEt=7: 3 to then AcOEt/MeOH=9:1 V/V) to give 40 (31 mg, 0.059 mmol, 34.6 % yield) as a white solid. 1H NMR (300 MHz, DMSO-d6)  ppm 6.91 - 7.42 (m, 6 H), 6.30 (dd, J=1.7, 10.1 Hz, 1H), 6.14 (s, 1 H), 5.48 - 5.79 (m, 1 H), 5.32 - 5.48 (m, 1 H), 4.08 - 4.31 (m, 1 H), 3.38 - 3.63 (m, 3 H), 2.98 - 3.15 (m, 1 H), 2.82 (s, 6 H), 2.05 - 2.47 (m, 5 H), 1.83 - 1.98 (m, 1 H), 1.54 - 1.83 (m, 4 H), 1.49 (s, 3 H), 1.22 - 1.39 (m, 1 H), 0.93 (s, 3 H). LC-MS (ESI POS): 525.41 MH+. ESI-HRMS m/z: 525.2929 [M + H]+ (calcd for C31H38F2N2O3, 525.2923). []D25 + 13.1 (c 0.27, DMSO). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-Benzyl-4b,12-difluoro-5-hydroxy-4a,6a-dimethyl2-oxo-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8-aza-pentaleno[2,1a]phenanthrene-6b-carboxylic acid benzylamide (41).


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Following the protocol described for 40 and starting from 36 and benzylamine, Cpd 41 was obtained (56 mg, 0.095 mmol, 30.4 % yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.91 (br t, J=5.92 Hz, 1H), 6.96-7.39 (m, 11H), 6.29 (dd, J=1.64, 10.19 Hz, 1H), 6.12 (s, 1H), 5.48-5.82 (m, 1H), 5.34-5.45 (m, 1H), 4.68-4.69 (m, 1H), 4.16-4.43 (m, 2H), 4.04-4.15 (m, 1H), 3.38-3.53 (m, 2H), 3.12-3.24 (m, 1H), 2.91 (t, J=8.22 Hz, 1H), 2.62-2.78 (m, 1H), 2.32-2.32 (m, 1H), 2.22-2.39 (m, 2H), 1.94-2.09 (m, 2H), 1.85 (br d, J=13.81 Hz, 1H), 1.52-1.69 (m, 2H), 1.43-1.51 (m, 3H), 1.26-1.38 (m, 1H), 0.90 (s, 3H). LC-MS (ESI POS): 587.26 MH+. ESI-HRMS m/z: 587.3093 [M + H]+ (calcd for C36H40F2N2O3, 587.3085). []D25 + 91.47 (c 0.34, MeOH). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-(3-Chloro-benzyl)-4b,12-difluoro-5-hydroxy4a,6a-dimethyl-2-oxo-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8azapentaleno[2,1-a]phenanthrene-6b-carboxylic acid fluoromethyl ester (42). Na2CO3 (189 mg, 1.786 mmol) was added to a stirred solution of compound 37 (190 mg, 0.357 mmol) in anhydrous N,N-dimethylformamide (3 ml) and, after stirring at room temperature for 15 minutes, the mixture was cooled to -20 °C under nitrogen. Bromofluoromethane (0.446 ml, 0.893 mmol) was added and the reaction stirred at -20 °C for 1 h, then the reaction mixture was allowed to warm to room temperature overnight. The reaction was partitioned between water and ethyl acetate. The organic layer was dried over anhydrous Na2SO4, filtered and evaporated in vacuo. The crude product was purified on a silica gel chromatography (Hexane/AcOEt=7:3 to 1:1 V/V), then it was triturated in H2O, recovered by filtration and dried to give 42 (94 mg, 0.167 mmol, 46.7% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.23-7.33 (m, 4H), 7.17 (d, J=7.2 Hz, 1H), 6.29 (dd, J=1.8, 10.1 Hz, 1H), 6.12 (s, 1H), 5.85 (dd, J=2.0, 16.4 Hz, 1H), 5.72 (br dd, J=2.2, 16.4 Hz, 1H), 5.54-5.61 (m, 1H), 5.51 (d, J=3.5 Hz, 1H), 4.11-4.2 (m, 1H), 3.42-3.55 (m, 2H), 3.09-3.21 (m, 1H), 2.93 (br t, J=8.3 Hz, 1H), 2.70 (br d, J=9.9 Hz, 1H), 2.53-2.62 (m, 1H), 2.42 (br d, J=10.1 Hz, 1H), 2.24-2.32 (m, 1H), 1.91-2.23 (m, 2H), 1.82-1.83 (m, 1H), 1.52-1.71 (m, 3H), 1.49 (s, 3H), 1.37 (br dd, J=6.5, 12.2 Hz, 1H), 0.96 (s, 3H). LC-MS (ESI POS): 564.29 MH+. ESI-HRMS m/z: 564.2132 [M + H]+ (calcd for C30H33ClF3NO4, 564.2123). []D25 + 78.6 (c 0.4, MeOH). 35 ACS Paragon Plus Environment


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(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-(3-Chloro-benzyl)-4b,12-difluoro-5-hydroxy-4a, 6a-dimethyl-2-oxo-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8-aza-pentaleno [2,1-a]phenanthrene-6b-carbothioic acid S-fluoromethyl ester Trifluoroacetate (43). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-(3-Chloro-benzyl)-4b,12-difluoro-5-hydroxy4a,6a-dimethyl-2-oxo-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8azapentaleno[2,1-a]phenanthrene-6b-carbothioic acid S-iodomethyl ester (50, 240 mg, 0.349 mmol) and silver(I)fluoride (89 mg, 0.698 mmol) in dry acetonitrile (15 ml) was stirred at room temperature in the dark under nitrogen for 1h 30 min. The mixture was decanted and the insoluble Ag salts were washed several times with dioxane. The combined organic layers were evaporated and the resulting crude was purified by preparative HPLC to give 43 (20 mg, 0.029 mmol, 8.26 % yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.08-7.75 (m, 5H), 6.32 (br d, J=8.33 Hz, 1H), 6.08-6.20 (m, 1H), 5.97-6.08 (m, 1H), 5.91 (br s, 1H), 5.49-5.76 (m, 2H), 4.05-4.66 (m, 3H), 3.76-3.98 (m, 1H), 3.18-3.42 (m, 3H), 2.63-3.05 (m, 2H), 2.19-2.35 (m, 1H), 1.98-2.18 (m, 1H), 1.92 (br d, J=13.37 Hz, 1H), 1.70 (br d, J=10.96 Hz, 2H), 1.49 (br s, 3H), 1.14-1.35 (m, 1H), 0.87-1.06 (m, 3H). LC-MS (ESI POS): 580.27 MH+. (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-Difluoro-5-hydroxy-4a,6a-dimethyl-8-(3methyl-butyl)-2-oxo-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8azapentaleno[2,1-a]phenanthrene-6b-carbothioic acid S-fluoromethyl ester (44). The intermediate 38 (183 mg, 0.383 mmol), HATU (160 mg, 0.421mmol) and N-methyl-morpholine (42.1 μl, 0.383 mmol) in DMF dry (3 ml) was stirred at 65 °C under nitrogen for 30 minutes. The solution was cooled down to room temperature, then anhydrous sodium hydrogensulfide (53.7 mg, 0.958 mmol) was added and the solution turned green and was stirred at room temperature for 1h. Bromofluoromethane 2M in DMF (575 μl, 1.150 mmol) 0.558 ml, 1.117 mmol), was added and the mixture turned immediately into dark yellow and was stirred at room temperature for 1 h. The reaction mixture was partitioned between AcOEt and brine, then the aqueous phase was extracted with AcOEt and the combined organic layers were dried over Na2SO4 and filtered. After evaporation of the 36 ACS Paragon Plus Environment

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solvent, the crude was purified by silica gel chromatography (AcOEt/petroleum ether=25:75 V/V) to afford 44 (60 mg, 0.114 mmol, 29.8 % yield) as a white solid. 1H NMR (300 MHz, DMSO-d6)  ppm 7.25 (d, J=10.08 Hz, 1H), 6.30 (br d, J=8.33 Hz, 1H), 6.11 (s, 1 H), 5.91 – 5.97 (m, 1 H), 5.86 – 5.92 (m, 1 H), 5.53 - 5.77 (m, 1 H), 5.50 – 5.57 (m, 1 H), 4.10 - 4.28 (m, 1 H), 3.13 - 3.25 (m, 1 H), 2.92 – 2.98 (m, 1 H), 2.79 – 2.85 (m, 1 H), 2.56 - 2.67 (m, 1 H), 2.35 – 2.40 (m, 1 H), 2.17 - 2.31 (m, 3 H), 1.88 - 2.09 (m, 3 H), 1.52 - 1.84 (m, 5 H), 1.49 (s, 3 H), 1.33 -1.38 (m, 1 H), 1.18 - 1.29 (m, 1 H), 0.95 (s, 3 H), 0.80 – 0.85 (m, 6 H). LC-MS (ESI POS): 526.39 MH+. ESI-HRMS m/z: 478.2765 [M + H]+ (calcd for C28H38F3NO3S, 478.2755). []D25 + 59.17 (c 0.29, CHCl3). (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-6b-Acetyl-8-(3-chloro-benzyl)-4b,12-difluoro-5hydroxy-4a,6a-dimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,11,12-tetradecahydro-4aH-8azapentaleno[2,1-a]phenanthren-2-one (48). A mixture of intermediate 47 (143 mg, 0.324 mmol), 1-(bromomethyl)-3-chlorobenzene (55.1 μl, 0.421 mmol) and Triethylamine (113 μl, 0.809 mmol) in dry DCM (10 ml) was stirred under nitrogen at room temperature overnight. The crude was purified by silica gel chromatography (AcOEt/petroleum ether=50:50 V/V) and by trituration with diisopropylether to afford 48 (79 mg, 0.149 mmol, 46.1 % yield) as a pale yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.23-7.35 (m, 4H), 7.17 (d, J=7.23 Hz, 1H), 6.29 (dd, J=1.64, 10.19 Hz, 1H), 6.12 (s, 1H), 5.53-5.73 (m, 1H), 5.385.45 (m, 1H), 4.16 (br dd, J=2.85, 6.14 Hz, 1H), 3.46 (s, 2H), 3.09-3.18 (m, 1H), 2.77 (t, J=8.11 Hz, 1H), 2.51-2.57 (m, 1H), 2.21-2.31 (m, 1H), 2.02-2.12 (m, 4H), 1.85-1.86 (m, 1H), 1.73-1.88 (m, 2H), 1.60-2.02 (m, 2H), 1.51-1.62 (m, 2H), 1.49 (s, 3H), 1.29-1.38 (m, 1H), 0.81-0.91 (m, 3H). LC-MS (ESI POS): 530.25 MH+. ESI-HRMS m/z: 530.2275 [M + H]+ (calcd for C30H34ClF2NO3, 530.2268). []D25 + 62.69 (c 0.26, MeOH). In vitro & In Vivo Pharmacology.



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The in vitro assays used to characterize biological activities of the GR ligands, including nuclear receptor binding following radioligand protocol, GR Nuclear Translocation and Raw RAW NO release assays, have been previously reported.10 Competitive binding assay. Human recombinant glucocorticoid receptor expressed in insect cells are used in modified potassium phosphate buffer pH 7.4. A 10 ug aliquot is incubated with 5 nM [3H]Dexamethasone and 2 mg/ml SPA beads for 24 h at 4 °C. Non-specific binding is estimated in the presence of 10uM Dexamethasone.

Receptors are counted to determine [3H]Dexamethasone specifically bound.

Compounds are screened at various concentrations. Ki values were calculated using the equation of Cheng and Prusoff using the observed IC50 of the tested compound, the concentration of radioligand employed in the assay, and the historical values for the KD of the ligand. Glucocorticoid Receptor (GR) translocation assay protocol. The cell-based GR-translocation assay in Enzyme Fragment Complementation format developed by DiscoveRx (Fremont, CA) was employed to quantitatively measure GR nuclear translocation. PathHunter CHO-K1 GR and PathHunter CHO-K1 MR cells were seeded in a 96-well plate at 15,000 cells/well in 100 L medium without antibiotics and 24 hours later the compounds were added (concentration ranging from 10-12M to 10-6M) for 3 h at 37 °C. Luminescence, estimated as relative light units (RLU), was detected by using a CENTRO LB 960 microplate reader (Berthold Technologies). Statistical analysis and determinations of EC50s were performed by using Prismversion 3.0 Graphpad Software (San Diego, CA). Cell culture. Murine macrophagic cell line (RAW264.7) was purchased from ATTC (Manassas, United States) and cultured in RPMI 1640 medium (w/o Phenol Red) supplemented with 10% FBS, 2 mM glutamine, 100 U penicillin and 100 g/ml streptomycin (Invitrogen), in an atmosphere of 5% CO2 at 37°C. PathHunter™ CHO-K1 GR and MR Cell Line stably expressing EA-NLS-NRS and the ProLabel-tagged glucocorticoid and mineralcorticoid receptor respectively were purchased from 38 ACS Paragon Plus Environment

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DiscoverX (CA, United States). Cells were cultured in F-12 Nutrient Mixture (HAM) supplemented with 10% Fetal Bovine Serum (Invitrogen) plus 2 mM L-glutamine and antibiotics (100 U/ml Penicillin, 100 g/ml Streptomycin, 300 g/ml Hygromycin B, and 500 g/ml G418/Geneticin) in an atmosphere of 5% CO2 at 37 °C. Primary human airway smooth muscle cells (ASMCs) were purchased from LONZA (Basel, Switzerland) and cultured in DMEM medium supplemented with 10% Fetal Bovine Serum, 2 mM glutamine, 100 U penicillin and 100 g/ml streptomycin (Invitrogen), in an atmosphere of 5% CO2 at 37 °C. Nitric measurement assay protocol. RAW264.7 cells were seeded in 0.3 ml RPMI (w/o Phenol Red) containing 10% FBS in 48-well tissue culture plates at the density of 7.5X104 cells/well and grown for 24 h at 37 °C with 5% CO2. Then cells were treated with different concentration of corticosteroids (10-11M-10-6 M, final DMSO concentration 0.1%) for 15 min. before stimulation with lipopolysaccharide from Escherichia coli (100 ng/ml as final concentration) and incubated for 18 hours in RPMI (w/o Phenol Red) supplemented with 10% FBS. Accumulation of nitrite in the medium was measured by a colorimetric assay method based on the Griess reaction. Briefly, samples were reacted with 1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride, and 2.5% phosphoric acid at room temperature for 10 min, and nitrite concentration was determined by absorbance at 540 nm in comparison with sodium nitrite as a standard. Compound potencies were expresses as concentration able to inhibit the half maximal (50%) NO release [IC50] in the dose-response curve obtained after stimulation with LPS (Lipopolysaccharide from Escherichia Coli, serotype 0111:B4 L3012, 046K4098, Sigma Aldrich). Lipopolysaccharide (LPS)-induced lung neutrophilia in rats. All experimental procedures involving animal research performed in this work were approved by Italian Ministry of Health (Prot.n° 186/2013 for LPS-induced lung neutrophilia assay and 201/2015 for PK studies) and complied with the European and Italian regulations for animal care and use of laboratory animals. Intratracheal administration of LPS induces intense lung inflammation, with 39 ACS Paragon Plus Environment


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macrophage activation, and recruitment of neutrophils to the interstitium, alveoli and the airways of rats. This response is driven by the local production of cytokines and chemokines and by the upregulation of adhesion molecules on circulating leukocytes and the pulmonary vascular endothelium and has been demonstrated to be highly sensitive to ICS inhibitory effect. Anaesthetised male Sprague-Dawley rats were challenged intratracheally with lipopolysaccharide (Escherichia Coli 0111:B4, Sigma Aldrich) and bronchhoalveolar lavage fluid (BALF) was collected 4 hours later as previously described. Control animals received an intratracheal challenge with saline only. Total cell counts and cell type differentiation in BALF were obtained using an automatic cell analyzing system (Sysmex, DASIT, Italy). For the evaluation of the anti-inflammatory activity compound 17 (0.01-1 μmol/Kg) or reference 3 (0.1-3 μmol/Kg) were administered intratracheally as suspension (0.2%Tween 80 in saline) 1 hour before LPS challenge. A dose-response curve was constructed for the inhibition of LPS-induced lung neutrophilia and half-maximal effect dose (ID50 dose) was extrapolated from the fitted log-linear regression analysis based on the individual inhibition data. Intratracheal administration and sample analysis. Compounds 17 as base and a hydrochloride salt were administered as suspension (0.2% Tween 80 in saline as vehicle) to rats at the dose of 1 mol/kg, with a volume of administration of 0.5 mL/kg. 48 male Sprague Dawley rats (Charles River, Calco, Italy), with a body weight of 270-310 g at the day of treatment, were anesthetized with sevoflurane (Sevorane; Abbott S.p.A., Latina, Italy), put in supine position on an adjustable table, and intratracheally dosed using a homemade air-jet device. After the administration, at fixed time points (0.083, 0.5, 1, 2, 4, 6, 24 and 48 hours after administration), three animals for each time point were anesthetized with sevoflurane and sacrificed by bleeding from abdominal aorta. Blood samples were put in heparinised plastic tubes and then centrifuged within 0.5 hour from the withdrawal to obtain plasma samples. After the sacrifice, lung tissues were immediately excised, washed with cold saline, accurately weighed and transferred into plastic tubes. Plasma and lung samples were stored at -80 ºC until the analysis. For the analysis, 40 ACS Paragon Plus Environment

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aliquots of 50 L plasma samples were added with 200 L of acetonitrile containing the internal standard (budesonide 500 ng/mL) and vortexed for 30 seconds. The obtained mixture was centrifuged at 12000 rpm for 5 min at 4C, then 5 L of supernatant were injected in the HPLC-MS/MS system for the analysis. Before the analytical determination, lung tissues were added with ammonium formiate 20 mM solution with a ratio of 10 mL buffer/g tissue and homogenized using Velp OV5 homogenizer (VELP Scientifica, Usmate, Italy). Then, 50 L of homogenate were added with 200 L of acetonitrile containing the internal standard (budesonide 500 ng/mL) and vortexed for 30 seconds. The obtained mixture was centrifuged at 12000 rpm for 5 min at 4C and 5 L of supernatant were injected into the HPLC-MS/MS system. For the analyte quantification, an API3200 mass spectrometer (SCIEX, Framingham, MA, United States) with ESI source and coupled to Prominence Shimadzu UFLC (Shimadzu Corp., Kyoto, Japan) was used. The chromatographic separation was performed in gradient mode with formic acid 0.1% in water (Solvent A) and formic acid 0.1% in acetonitrile (Solvent B), using a Gemini C18 column (5 m, 110A, 50x2.0 mm) equipped with a Gemini C18 precolumn (Phenomenex, Torrance, CA, United States). The spectrometer was used in ESI positive ion mode, monitoring the following transitions: 546→ 125 for compound 17 and 431→323 for compound 3, as IS. The calibration curves in plasma and lung were linear in the range 0.0018-3.66 M (with R2 higher than 0.9986) . The lung samples with an analyte concentration exceeding the ULOQ (3.66 M) were diluted with blank matrix in order to reach a concentration level within the linearity range. Modelling studies (Materials and Methods) Protein and ligands preparation The crystal structure of fluticasone furoate (FF) bound to the human GR was retrieved from the Brookhaven Protein Databank (PDB code: 3CLD)15 and was processed with the Protein Preparation Wizard in the Schrodinger suite 2016-1 (Schrödinger, LLC NY 2016) Hydrogen atoms were added followed by adjustment of bond orders for the ligand. The protonation and tautomeric states of Asp, 41 ACS Paragon Plus Environment


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Glu, Arg, Lys and His were adjusted to match a pH of 7.4. Only the hydrogens were minimized during “Protein minimization” step with Impref. Finally, all water molecules were deleted except for that bridging R611 and N564. The structures of compounds 17 and 21 were manually built within Maestro starting from the conformation of FF steroid core and subsequently energy minimized within LigPrep. Rigid docking Rigid docking studies were carried out with Glide SP. The binding site was defined as the centroid of the amino acids lying within 5 Å from each atom of co-crystallized FF. Softening the potential of the nonpolar parts of the receptor was done by scaling the van der Waals radii by a factor of 0.8. In addition, hydrogen bond interactions between R611 and N564 side chains and the carbonyl group at C3 of ring A were imposed as restraints throughout the simulations. Default values of all the other parameters were used. Induced Fit Docking Schrödinger induced fit docking (IFD) protocol was used to take into account the conformational changes induced by the ligands in the GR binding site. The receptor grid centre was set as the centroid of FF bound to GR (inner box size = 10 Å; the outer box was automatically set by the protocol). During Glide SP docking the scaling factors to soften the potentials of the receptors and ligands were set to 0.5 in both cases. A maximum of 20 poses was saved for each ligand. Then, all residues within 5.0 Å of ligand poses were refined using the Prime molecular dynamics module to allow for binding domain flexibility. After that, Glide SP was used for the re-docking step into the top 20 receptor structures generated within 30 kcal/mol of the best structure by the Prime refinement. IFD poses were ranked with default scoring function consisting of the GlideScore for the Glide Redocking plus 5% of the Prime Energy from the Prime Refinement. Finally, poses were visually inspected within Maestro. Pictures


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Pictures of docked ligands were generated with the PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC NY. Supporting Information: The Supporting Information is available free of charge on the ACS Publications website: Preparation of compounds 4, 5, 9, 10, 11, acetyl (R1) intermediates of compounds 13-16, 1929, compounds 30, 32, 34, 35, 36, 37, 38, 45, 46, 47, 49 and 50. Table of purity of final substituted pyrrolidines and 12. Overlay of budesonide 3 bound to human GR in the X-ray structure on the IFD top-ranked solution. 1

H NMR spectra of final substituted pyrrolidines.

Molecular formula strings and biological data. AUTHOR INFORMATION Corresponding Authors *For E.G.: Phone +39 0521 279913. Fax +39 0521 279880. E-mail: [email protected] *For F.R.: Phone +39 0521 279784, Fax +39 0521 279880, E-mail: [email protected] Notes The authors declare no competing financial interest. Abbreviations Used ACN, acetonitrile; Cpd, compound; COPD, chronic obstructive pulmonary disease; DBU, 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine;

DCM,

dichloromethane;

DMF,

N,N-

dimethylformamide; DMSO, dimethylsulfoxide; ESI, electrospray ionization; Et2O, diethylether; EtOAc, ethyl acetate; EtOH, ethyl alcohol; HATU, 1-(bis(dimethylamino)methylene)-1H[1,2,3]triazolo[4,5-b]pyridine-1-ium

3-oxide

hexafluorophosphate(V);

LC-MS,

liquid

chromatography–mass spectrometry; HPLC-MS, high performance liquid chromatography–mass spectrometry; LPS, lipopolysaccharides; MeOH, methyl alcohol; MRT, mean residence time; NMR,



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nuclear magnetic resonance; SPA, Scintillation proximity assays; TEA, triethylamine; THF, tetrahydrofuran; ULOQ, upper limit of quantitation. References 1. Aalbers, R.; Vogelmeier, C.; Kuna, P. Achieving asthma control with ICS/LABA: a review of strategies for asthma management and prevention. Respir. Med. 2016, 111, 1-7. 2. Barnes, P. J. Inflammatory mechanisms in patients with chronic obstructive pulmonary disease. J. Allergy Clin. Immunol. 2016, 138(1), 16-27. 3. Covar, R. A. Pivotal efficacy trials of inhaled corticosteroids in asthma. Ann. Allergy Asthma Immunol. 2016, 117(6), 582-588. 4. Ernst, P.; Suissa, S. Systemic effects of inhaled corticosteroids. Curr. Opin. Pulm. Med. 2012, 18, 85-89. 5. Barnes, P. J. Inhaled corticosteroids. Pharmaceuticals (Basel) 2010, 3(3), 514-540. 6. Pruteanu, A. I.; Chauhan, Bhupendrasinh F.; Zhang, L.; Prietsch, S. O. M.; Ducharme, F. M. Inhaled corticosteroids in children with persistent asthma: dose-response effects on growth. Cochrane Database Syst. Rev. 2014, 7, CD009878. 7. Kwon, T.; Heiman, A. S.; Oriaku, E. T.; Yoon, K.; Lee, H. J. New steroidal anti-inflammatory antedrugs: steroidal [16,17-d]-3-carbethoxyisoxazolines. J. Med. Chem. 1995, 38, 1048– 1051. 8. Park, K.-K.; Ko, D.-H.; You, Z.; Khan, M. O. F.; Lee, H. J. In vitro anti-inflammatory activities of new steroidal antedrugs: [16α,17α-d] isoxazoline and [16α,17α-d]-3-hydroxyiminoformyl isoxazoline derivatives of prednisolone and 9α-fluoroprednisolone. Steroids 2006, 71, 183–188. 9. Millan, D. S.; Ballard, S. A.; Chunn, S.; Dybowski, J. A.; Fulton, C. K.; Glossop, P. A.; Guillabert, E.; Hewson, C. A.; Jones, R. M.; Lamb, D. J.; Napier, C. M.; Payne-Cook, T. A.; Renery, E. R.; Selby, M. D.; Tutt, M. F.; Yeadon, M. Design and synthesis of long acting


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17. Glossop, P. A.; Lane, C. A. L.; Price, D. A.; Bunnage, M. E.; Lewthwaite, R. A.; James, K.; Brown, A. D.; Yeadon, M.; Perros-Huguet, C.; Trevethick, M. A.; Clarke, N. P.; Webster, R.; Jones, R. M.; Burrows, J. L.; Feeder, N.; Taylor, S. C. J.; Spence, F. J. Inhalation by design: novel ultra-long-acting β2-adrenoreceptor agonists for inhaled once-daily treatment of asthma and chronic obstructive pulmonary disease that utilize a sulfonamide agonist headgroup. J. Med. Chem. 2010, 53, 6640–6652. 18. Bowers, A.; Ringold, H. J. Steroids. XCII. Synthesis of halogenated steroid hormones. 2. 6αand 6β-Fluorotestosterone and 6α- and 6β-fluoroprogesterone. Tetrahedron 1958, 3, 14-27. 19. Toscano, L.; Grisanti, G.; Fioriello, G.; Barlotti, L. Synthesis and topical antiinflammatory properties of 17,21-bis(acetyloxy)-6β,9-difluoro-11β-hydroxypregna-1,4-diene-3,20-dione and related 2-halogenated compounds. J. Med. Chem. 1977, 20, 213-220. 20. Coldham, I.; Hufton, R. Intramolecular dipolar cycloaddition reactions of azomethine ylides. Chem. Rev. 2005, 105, 2765-2809. 21. Armani, E.; Ghidini, E.; Peretto, I.; Virdis, A. Isoxazolidine Derivatives. WO 2011029547, 2011. 22. Gerlach, K.; Hoffmann, H. M. R.; Wartchow, R. Non-stabilized azomethine ylides in [3 1 2] cycloadditions. Pyrrolidinylfuranones from (5S)-5-menthyloxy-4-vinylfuran-2(5H)-one. J. Chem. Soc. Perkin Trans. 1 1998, 3867-3872. 23. Li, J. J.; Corey, E. J. Eschweiler-Clarke Reductive Alkylation of Amines. Name Reactions for Functional Group Transformations; John Wiley & Sons: Hoboken, NJ, 2007; pp 86-111. 24. Fischer, C.; Koenig, B. Palladium- and copper-mediated N-aryl bond formation reactions for the synthesis of biological active compounds. Beilstein J. of Organic Chem. 2011, 7, 59-74. 25. Liu, Z.; Larock, R. C. Facile N-arylation of amines and sulfonamides and O-arylation of phenols and arenecarboxylic Acids. J. Org. Chem. 2006, 71(8), 3198-3209.


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Novel Pyrrolidine Derivatives of Budesonide as Long Acting Inhaled

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