Novel class of benzoic acid ester derivatives as potent PDE4 inhibitors for inhaled administration in the treatment of respiratory diseases

Article

pubs.acs.org/jmc

Novel Class of Benzoic Acid Ester Derivatives as Potent PDE4

Inhibitors for Inhaled Administration in the Treatment of Respiratory

Diseases

Elisabetta Armani,*^,†Gabriele Amari,^†Andrea Rizzi,^†Renato De Fanti,^†Eleonora Ghidini,^†

Carmelida Capaldi,^†Laura Carzaniga,^†Paola Caruso,^†Matilde Guala,^‡,§Ilaria Peretto,^‡,∥

Elena La Porta,^‡,⊥Pier T. Bolzoni,^†Fabrizio Facchinetti,^†Chiara Carnini,^†Nadia Moretto,^†

Riccardo Patacchini,^†Franco Bassani,^†Valentina Cenacchi,^†Roberta Volta,^†Francesco Amadei,^†

Silvia Capacchi,^†Maurizio Delcanale,^†Paola Puccini,^†Silvia Catinella,^†Maurizio Civelli,^†

and Gino Villetti^†

^†Chiesi Farmaceutici S.p.A., Nuovo Centro Ricerche, Largo Belloli 11/a, 43122 Parma, Italy

^‡NiKem Research Srl, Via Zambeletti, 25, 20021 Baranzate, Milano, Italy

S

* Supporting Information

ABSTRACT: The ﬁrst steps in the selection process of a new

anti-inﬂammatory drug for the inhaled treatment of asthma

and chronic obstructive pulmonary disease are herein

described. A series of novel ester derivatives of 1-(3-

(cyclopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)-2-(3,5-di-

chloropyridin-4-yl) ethanol have been synthesized and

evaluated for inhibitory activity toward cAMP-speciﬁc

phosphodiesterase-4 (PDE4). In particular, esters of variously

substituted benzoic acids were extensively explored, and

structural modiﬁcation of the alcoholic and benzoic moieties

were performed to maximize the inhibitory potency. Several

compounds with high activity in cell-free and cell-based assays

were obtained. Through the evaluation of opportune in vitro ADME properties, a potential candidate suitable for inhaled

administration in respiratory diseases was identiﬁed and tested in an in vivo model of pulmonary inﬂammation, proving its

eﬃcacy.

The development of ﬁrst-generation PDE4 inhibitors, such as

rolipram (1), has been hampered by the occurrence of

mechanism-associated side eﬀects, nausea and emesis, at eﬀective

anti-inﬂammatory doses.⁵Second-generation inhibitors, roﬂu-

milast⁶(2) and cilomilast⁷(3), were generally better-tolerated,

but this was likely after administration of doses that did not

achieve sustained PDE4 inhibition^8,9(Figure 1). The recent

registration of roﬂumilast in Europe and the U.S. has conﬁrmed

INTRODUCTION

■

The phosphodiesterase 4 (PDE4) enzyme is highly expressed in

inﬂammatory and immune cell types relevant for the patho-

genesis of airway inﬂammatory disorders.¹Indeed, PDE4

inhibition, resulting in an increase of intracellular cAMP

concentration, exerts a broad range of anti-inﬂammatory eﬀects,

including the inhibition of cellular traﬃcking and microvascular

leakage, cytokine and chemokine release, reactive oxygen species

production, and cell adhesion-molecule expression.²PDE4 is

also expressed in lung structural cells: ﬁbroblasts, airway

epithelial and endothelial cells, and airway and vascular smooth

muscle cells. Therefore, PDE4 inhibitors have the potential to

alleviate not only pulmonary inﬂammation but also ﬁbrotic

airway and vascular remodeling and mucociliary dysfunction

(mucus hypersecretion and decreased mucus transport), which

are pathophysiological features common to various pulmonary

diseases.³These beneﬁcial eﬀects of PDE4 inhibitors have been

demonstrated in a broad array of in vitro and in vivo experimental

models of airway inﬂammation.⁴Consequently, there has been

great interest in developing PDE4 inhibitors for the treatment of

respiratory diseases.

Figure 1. Structures of rolipram (1) (ﬁrst-generation PDE4 inhibitor)

and roﬂumilast (2) and cilomilast (3) (second-generation PDE4

inhibitors for oral administration).

Received: October 4, 2013

Published: January 8, 2014

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Figure 2. Structures of the most signiﬁcant PDE4 inhibitors for inhaled administration.

Figure 3. AMP bound to PDE4B (orange, hydrophobic region; gray, metal region; and turquoise, solvent exposed region) (PDB ID: 1TB5).

the therapeutic utility of targeting PDE4 in severe chronic

obstructive pulmonary disease (COPD) patients and has

renewed interest in this target, prompting the search for third-

generation inhibitors endowed with a greater therapeutic

window.¹⁰

the circulation,¹⁵and PDE4 inhibitors may interfere with local

regulation of acid secretion in the gastrointestinal tract.¹⁶

A diﬀerent strategy to achieve a larger therapeutic window

between eﬃcacy and side eﬀects involves drug administration via

inhalation directly to lung tissue. Topically administered

compounds require much lower doses to block local PDE4

activity than oral drugs and should lack the adverse eﬀects

associated with systemic exposure. This idea prompted the

clinical development of a number of inhaled inhibitors such as 4

(AWD-12-281),¹⁷5 (CP-325366),¹⁸6 (UK-500,001),¹⁹and 7

(GSK-256066)²⁰(Figure 2).

Unfortunately, a lack of eﬃcacy in phase II trials in asthma and

COPD led to the discontinuation of the development of 4

(AWD-12-281), 5 (CP-325366), and 6 (UK-500,001).²¹Given

the suboptimal anti-inﬂammatory activity displayed by these

compounds in cell-based assays, it can be hypothesized that their

clinical eﬃcacy has been compromised by the administration of

doses lower than the ones required to achieve adequate and

sustained inhibition of PDE4 in the lung tissue. 7 (GSK-256066),

A number of strategies are being followed to achieve this

objective. The synthesis of compounds selective for the PDE4B

isoform^11,12or that are devoid of aﬃnity for the high-aﬃnity

rolipram binding site¹³has been pursued. However, because of

the lack of very selective compounds, it remains elusive whether

these approaches may be a viable option for improving the

therapeutic window of PDE4 inhibitors. It has been hypothesized

that compounds characterized by a limited blood-brain barrier

penetration could be endowed with a reduced emetic proﬁle.

Nevertheless, this approach has also been questioned because

compounds that preferentially distribute to the brain are

relatively free from emesis.¹⁴Moreover, the chemoreceptor

trigger zone in the brain stem is accessible to the free drug within

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which is at least 3 orders of magnitude more potent than the

other inhaled compounds tested in the clinic, showed promising

eﬃcacy in an allergen-challenge study in mild asthmatics and in a

4 week study in COPD patients at a dose of 87.5 μg/day. In the

latter study, a signiﬁcant inhibition of markers of PDE4 activity

was demonstrated in sputum cells, suggesting that the

administration of extremely potent compounds may be necessary

to aﬀect the activity of PDE4 in the lung tissue signiﬁcantly.²²

We report here the discovery of a novel series of potent PDE4

inhibitors, which are characterized by an in vitro proﬁle that is

promising for inhaled administration. Such PDE4 inhibitors

speciﬁcally suitable for inhaled administration at a low dose could

oﬀer a solution to the issue of emesis and nausea for long-term

treatment of chronic airway diseases and could also improve the

clinical eﬃcacy. Our medicinal chemistry strategy was initially

based on the analysis of the PDE4 catalytic binding pocket. This

includes three subdomains: a hydrophobic region, which

contains residues Gln443 and Asn395 that are important for

nucleotide recognition, a metal region where two cations (Zn²⁺

and Mg²⁺) are linked to polar residues and to six coordinated

water molecules, and a solvent-exposed region, which is generally

ﬁlled by water molecules. In Figure 3, the crystal structure of

PDE4B in complex with the hydrolytic product AMP (PDB ID:

1TB5) reveals that the main interactions of the endogenous

ligand are formed exclusively in the hydrophobic pocket and in

the metal region of the catalytic binding pocket.²³The adenine

fragment of AMP forms four hydrogen bonds with residues

Gln443 and Asn395 in the hydrophobic region, the ribose ring is

shared by the hydrophobic and metal regions, and the phosphate

group interacts near the metals. The left part of Figure 3 shows

the solvent-exposed region in turquoise; additional binding

energy could be achieved through interacting with the residues of

the solvent-exposed region.

interaction with all three regions of the catalytic binding pocket

(Figure 3), resulting in potentially improved inhibitory potency.

Starting from the structure of roﬂumilast, we replaced the amide

linker connecting the two aromatic systems with a hydroxy-

ethylene one, which oﬀered an anchoring point for the

introduction of an additional portion potentially oriented to

the solvent-exposed region. Ester derivatives of 1-(3-(cyclo-

propylmethoxy)-4-(diﬂuoromethoxy)phenyl)-2-(3,5-dichloro-

pyridin-4-yl) ethanol were synthesized, and the benzoic acid

ester class was extensively explored. In such compounds, the

dialkyloxyphenyl and the dichloropyridinyl groups could ensure

the interaction with the hydrophobic region and the metal region

of the PDE4 protein, similar to the binding mode of roﬂumilast,²³

whereas the ester portion could potentially interact with the

solvent-exposed region. The importance of the stereochemistry

of the alcoholic carbon and the eﬀect of N-oxidation of the

pyridine ring were investigated, and several patterns of

substitution were considered on the benzoic acid portion.

Calculated LogD_7.4and selected in vitro ADME properties were

investigated for a subset of potent compounds to identify a

candidate suitable for inhaled administration.

Chemistry. The synthetic pathway employed for the

synthesis of ketone and enolic ester derivatives (14 and 15) is

shown in Scheme 1. The preparation of acyl chloride 11 was

accomplished starting from commercially available 3,4-dihydrox-

ybenzaldehyde 8. Selective alkylation of the 4-hydroxy group

using sodium chlorodiﬂuoroacetate and NaOH led to the

formation of 4-diﬂuoromethoxy intermediate 8a, alternatively

purchased from a commercial supplier. Further alkylation at

position 3 with (bromomethyl)cyclopropane gave aldehyde 9,

which was oxidized to acid 10 by sodium chlorite and sulfamic

acid in acetic acid. Intermediate 10 was ﬁnally converted into

corresponding acyl chloride 11 by treatment with thionyl

chloride. 3,5-Dichloro-4-methylpyridine 13 was obtained by

methylation of commercially available 3,5-dichloropyridine 12

with methyl iodide in the presence of freshly prepared LDA. The

lithium salt of 13 (obtained by means of LDA in THF) was

coupled with 0.3 equiv of acyl chloride 11 at −70 °C to aﬀord

ketone 14. When an excess of 11 was employed, ester 15 was

obtained.

Because an inhaled administration could reduce the side eﬀects

of PDE4 inhibitors, particular attention should be given to the

physicochemical properties that can aﬀect the level of drug in

plasma. High lipophilicity is an important physicochemical

property for high plasma protein binding,²⁴which can contribute

to the reduction of unwanted systemic side eﬀects. A comparison

of the predicted LogD_7.4values²⁵for the PDE4 inhibitors,

included in Figures 1 and 2 and Table 1, conﬁrmed that most

Esters of ( )-16 were prepared following the synthetic

protocol in Scheme 2. Alcohol ( )-16 was obtained from

aldehyde 9 and intermediate 13 in the presence of potassium tert-

butoxide at −30 °C. Esters ( )-18 and ( )-19a−e were

obtained by coupling intermediate ( )-16 with suitable

carboxylic acids using EDC and DMAP.

Enantiomeric separation of ( )-18 was obtained by semi-

preparative HPLC (Chiralcel OD column, hexane/iPrOH 95:5)

to aﬀord single enantiomers (+)-18 (ﬁrst elution) and (−)-18

(second elution) (Scheme 3). Oxidation of racemic ( )-18 with

mCPBA gave the corresponding N-oxide as a racemic mixture,

which was resolved by semipreparative chiral HPLC (Chiralcel

OD, hexane/iPrOH 50:50) to give single enantiomers (+)-20

(ﬁrst eluting compound) and (−)-20 (second eluting com-

pound) (Scheme 3).

Table 1. Calculated logD_7.4Values for Oral and Inhaled PDE4

Drug Candidates

a

compd

administration

calculated LogD_7.4

rolipram

oral

2.00

2.30

roﬂumilast

cilomilast

oral

−0.78

4.92

AWD-12-281

CP-325366

UK-500,001

GSK-256066

inhalation

4.22

5.05

2.18

a

ACD/Labs logD v.11.

The absolute conﬁguration of these compounds was then

assigned. Racemic alcohol ( )-21, obtained by oxidation of

( )-16 with mCPBA, was condensed with (R)-naproxen to

aﬀord a mixture of two diastereoisomers that were separated by

preparative HPLC (Scheme 4). By means of single-crystal X-ray

diﬀraction (Supporting Information) it was possible to

determine the structure of compound (R*,S**)-22 (Figure 4),

revealing that the absolute conﬁguration at the alcoholic

inhaled clinical candidates are more lipophilic than the oral ones.

Even if, in principle, a high lipophilicity could reduce the free

fraction available for activity in the lung, there are also cases that

suggest that a high lipophilicity is compatible with a good eﬃcacy

in the lung (i.e., the clinical eﬃcacy of Fluticasone Furoate²⁶with

a predicted LogD_7.44.10). Therefore, the aim of our project was

the identiﬁcation of a novel class of PDE4 inhibitors endowed

with high plasma protein binding and able to undertake extended

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a

Scheme 1. Preparation of Derivatives 14 and 15

a

Reagents and conditions: (a) CF₂ClCOO⁻Na⁺, NaOH, DMF, H₂O, 120 °C, 2 h, 44%; (b) (bromomethyl)cyclopropane, K₂CO₃, THF, reﬂux, 14 h,

97%; (c) H₂NSO₃H, NaClO₂, CH₃COOH, H₂O, rt, 1 h, 97%; (d) SOCl₂, toluene, reﬂux, 2 h, 100%; (e) iPr₂NH, BuLi, −10 °C, 30 min, 12, −20 °C,

30 min, then MeI, −70 °C to rt, 68%; (f) 13, LDA (1.8 M in THF), −70 °C, 30 min; 11 (0.3 equiv), −70 °C, 15 min, 20%; (g) 13, LDA (1.8 M in

THF), −70 °C, 30 min, then 11 (2 equiv), −70 °C to rt, 30 min, 30%.

a

Scheme 2. Preparation of Alcohol ( )-16 and Esters ( )-18 and ( )-19a−e

a

Reagents and conditions: (a) tBuOK, THF, −30 °C, 1 h, 75%; (b) EDC, DMAP, DCM or DMF, rt.

stereogenic center was (S). The chemical correlations

summarized in Scheme 5, by means of reactions not involving

the chiral center (i.e., reduction of N-oxide, cleavage of naproxen

ester, esteriﬁcation with acid 10, and oxidation with mCPBA),

allowed the assignment of absolute conﬁguration to compounds

(R)-(+)-18, (S)-(−)-18, (R)-(+)-20, and (S)-(−)-20 as well as

to the related alcohols. Retention of conﬁguration was further

conﬁrmed by esteriﬁcation of (S)-(+)-16 with (R)-naproxen,

giving (R**,S*)-23 (Scheme 5, step c).

A synthetic pathway for the large-scale preparation of

enantiomerically pure (S)-21 was optimized as shown in Scheme

6. Racemic alcohol ( )-21 was coupled with (S)-(+)-acetyl-

mandelic acid to aﬀord a couple of diastereoisomers, which were

separated by fractional crystallization from Et₂O. The less soluble

(S*,S**)-24 isomer was obtained in a diastereomeric ratio >95%

after several crystallizations, whereas its epimer, (S*,R**)-24,

was recovered from mother liquors and crystallized from iPrOH.

Solvolysis in a mixture of methanol and aqueous NaHCO₃

aﬀorded enantiomerically pure (S)-21 and (R)-21.

The preparation of esters (S)-26a−s was achieved by coupling

alcohol (S)-21 with suitable carboxylic acids in the presence of

EDC and DMAP as condensing agents (Scheme 7). Benzoic

acids 25a−s were either commercially available or were prepared

as detailed in the Supporting Information. Aniline derivative (S)-

26t was obtained by reduction of nitro analogue (S)-26q with

SnCl₂hydrate, whereas hydroxyl derivative (S)-26u was

prepared by catalytic hydrogenation of benzyl derivative (S)-

26r with palladium on barium sulfate as catalyst. Reduction of

aldehyde (S)-26s with sodium borohydride in THF gave

hydroxymethyl derivative (S)-26v, whereas oxidation of (S)-

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Scheme 3. Preparation of Enantiopure Compounds (+)-18,

a

(−)-18 and (+)-20, (−)-20

a

Figure 4. X-ray structure of (R*,S**)-22.

Reagents and conditions: (a) chiral HPLC, column: Chiralcel OD,

eluant: hexane/iPrOH 95:5, ﬂow: 18 mL/min; (b) mCPBA, DCM, rt,

18 h, 45%; (c) chiral HPLC, column: Chiralcel OD, eluant: hexane/

iPrOH 1:1, ﬂow: 18 mL/min.

subsequent coupling with (S)-21 followed by cleavage with HCl

aﬀorded sulfonamides (S)-36a−c.

26s with sodium chlorite and sulfamic acid in acetic acid gave

carboxylic acid (S)-26w.

Sulfonamides (S)-38a−e were obtained by sulfonylation of

corresponding anilines (S)-37a−e according to Scheme 10.

Anilines (S)-37a−e could be prepared either by reduction of the

nitro analogues or by deprotection of the N-Boc-protected

derivatives obtained by coupling alcohol (S)-21 with suitable

intermediates. For the detailed synthesis of intermediates (S)-

37a−e, see the Supporting Information.

Sulfonamides (S)-32a−l were prepared as described in

Scheme 8. Intermediates 27a−l were either commercially

available or were prepared as detailed in the Supporting

Information. Sulfonilation of anilines 27a−l with sulfonyl

chlorides in pyridine gave sulfonamides 28a−j. N-Boc protection

followed by hydrolysis of methyl esters 29a−j under basic

conditions gave acids 30a−j, which were coupled with (S)-21 to

obtain intermediates (S)-31a−j. The removal of Boc protection

under acidic conditions led to ﬁnal compounds (S)-32a−j.

Compounds (S)-32k−l were alternatively prepared by direct

hydrolysis of intermediates 28k−l followed by coupling with

alcohol (S)-21, which avoided the protecting and deprotecting

steps.

Phenol (S)-44 was obtained as summarized in Scheme 11.

Aldehyde 40, obtained by condensation of isovanillin (39) with

N-Boc-(S)-proline, was converted into acid 41 by oxidation with

sodium chlorite and sulfamic acid in acetic acid. After coupling

with (S)-21 followed by acidic removal of the Boc protecting

group, aﬀording diester (S)-43, a mild basic hydrolysis of the

proline ester gave phenolic derivative (S)-44.

Biology and ADME. The U937 monocytic cell line was used

as source of PDE4 enzyme for the in vitro evaluation of

compound inhibitory activity in a cell-free assay. Inhibitory

activity was determined by assaying cAMP disappearance from

the incubation mixture, and it was expressed as an IC₅₀value.

A partially modiﬁed synthetic route was followed for

compounds (S)-36a−c (Scheme 9): acids 34a−c were prepared

by hydrogenolysis of corresponding benzylester intermediates

33a−c (synthesis described in the Supporting Information). The

a

Scheme 4. Synthesis of (R)-Naproxen Derivatives (R*,R**)-22 and (R*,S*)-22

a

Reagents and conditions: (a) mCPBA, DCM, rt, 2 h, 76%; (b) EDC, DMAP, DMF, rt, o/n, 88%; (c) preparative HPLC.

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Scheme 5. Chemical Correlations for the Assignment of Absolute Conﬁguration to Alcohols and to the Corresponding Final

a

Esters

a

Reagents and conditions: (a) Zn, NH₄Cl, THF, H₂O, rt, 5 min, quantitative; (b) tBuOK, MeOH, THF, rt, 20 h, 76%; (c) (R)-naproxen, EDC,

DMAP, DMF, rt, 20 h, 78%; (d) 10, EDC, DMAP, DMF or DCM, rt; (e) mCPBA, DCM, rt, 20 h, 68%; (f) mCPBA, DCM, rt 68%.

a

Scheme 6. Large-Scale Preparation of Enantiomerically Pure Alcohol (S)-21

a

Reagents and conditions: (a) EDC, DMAP, DCM, rt; (b) trituration with Et₂O followed by crystallization from iPrOH, 34% yield; (c) mother

liquor evaporation followed by crystallization from iPrOH, 23% yield; (d) aqueous sat. NaHCO₃, MeOH, rt, 24 h, 86%.

a

Scheme 7. Preparation of Compounds (S)-26a−w

a

Reagents and conditions: (a) EDC, DMAP, DCM or DMF, rt or heating; (b) SnCl₂·2H₂O, THF, 40 °C, 7 h, 53%; (c) H₂, 5% Pd on BaSO₄,

EtOAc, 30 psi, 1 h, rt, 78%; (d) NaBH₄, THF, rt, 24 h, 28%; (e) NH₂SO₃H, NaClO₂, AcOH, H₂O, 3 h, rt, 50%.

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a

Scheme 8. Synthesis of Sulfonamides (S)-32a−l

a

Reagents and conditions: (a) RSO₂Cl, pyridine or DCM/pyridine, rt; (b) Boc₂O, DMAP, DCM, rt; (c) 1 N NaOH, MeOH, rt to 50 °C; (d) 1 N

LiOH, THF, rt; (e) (S)-21, EDC, DMAP, DCM or DMF rt; (f) 4 M HCl in dioxane and DCM or HCl 4 M in EtOAc, rt; (g) conditions for

hydrolysis of compound 28l: KOH, abs. EtOH, reﬂux, 16 h.

a

Scheme 9. Preparation of Sulfonamides (S)-36a−c

a

Reagents and conditions: (a) Boc₂O, DMAP, DCM, rt; (b) H₂, 10% Pd/C, MeOH, 30 psi, rt; (c) (S)-21, EDC, DMAP, DCM or DMF, rt; (d) 4 N

HCl in dioxane, DCM or 4 M HCl in EtOAc, rt.

a

Scheme 10. Preparation of Sulfonamides (S)-38a−e

a

Reagents and conditions: (a) MeSO₂Cl, pyridine, DCM or CHCl₃, rt.

a

Scheme 11. Preparation of Compound (S)-44

a

Reagents and conditions: (a) Boc-L-proline, EDC, DMAP, DMF, rt, 95%; (b) NaClO₂, H₂NSO₃H, AcOH, H₂O, rt, 76%; (c) (S)-21, EDC, DMAP,

DMF, rt, 1 h, 95%; (d) HCl 4 M in EtOAc, rt, 6 h, 75%; (e) aqueous sat. NaHCO₃, MeOH, rt, 3 h, 95%.

The assay for the in vitro evaluation of compound activity in

peripheral blood mononuclear cells (PBMCs) was based on the

known inhibitory activity exerted by PDE4 inhibitors on

lipopolyshaccarides (LPS)-induced tumor necrosis factor-alpha

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Figure 5. Identiﬁcation of hit compound ( )-18.

(TNF-α) release. The inhibitory activity was determined by

measuring LPS-induced TNF-α production after incubation of

human PBMCs with diﬀerent concentrations of the test

compounds, and it was expressed as IC₅₀values.

Table 2. Inhibitory Activity of Compounds 15 and ( )-18 in

PDE4 Cell Free Assay

In vitro ADME developability parameters of selected

compounds were explored by evaluating the stability in S9 rat

and human lung, human and rat plasma protein binding (PPB),

and permeability in Caco-2 cells.

Selectivity versus other PDE isoforms, binding kinetics to the

PDE4 isoform, stability in human and rat lung slices and in

human and rat plasma, Calu-3 permeability, and eﬃcacy in OVA-

induced lung eosinophilia in guinea pigs were evaluated for one

selected compound.

All methods are described in the Experimental Section.

RESULTS AND DISCUSSION

■

a

Data are the mean SD of three to four experiments performed in

SAR Analysis. According to the earlier-described medicinal

chemistry strategy, we started evaluating unsaturated structures

that maintain the same geometry as roﬂumilast and contain a

third aryl substituent, potentially able to establish additional

interactions, such as enolethers, enolesters, enolcarbamates, α,β-

unsaturated ketones, and α,β-unsaturated esters (Figure 5). It

was found that benzoic enolester derivatives were the only

compounds displaying appreciable activity in a cell-free assay

(data not shown).

duplicate.

assay. As reported in Table 3, all of the derivatives with longer

aliphatic chains and/or featuring aromatic residues (19b−e)

Table 3. Structure and Inhibitory Activity of Compounds

( )-19a−e in PDE4 Cell Free Assay

A more detailed investigation of the enolester class²⁷allowed

the identiﬁcation of symmetrically substituted compound 15 as

the most interesting compound (cell-free IC₅₀= 1.18 nM).

Despite this encouraging result, the enolester class presented

some issues because of the diﬃculty in controlling E/Z

isomerism during the synthesis and in avoiding partial isomer-

ization during storage. For these reasons and to investigate if the

double bond was required for PDE4 inhibition, the replacement

of the enolester scaﬀold with simple ester derivatives was

evaluated.

The substitution pattern shown by enolester 15 was applied to

ester ( )-18, causing a slight decrease in cell-free assay activity

(( )-18 IC₅₀= 3.45 nM, Table 2). Selectivity versus other PDE

isoforms was evaluated by testing ( )-18 at the concentration of

10 μM against the enzymatic activity of human PDE5, PDE3, and

PDE2, with resulting inhibitions of 58, 18, and 0%, respectively.

Given its potency and isoform selectivity, compound ( )-18 was

considered a good starting point for further structure−activity

relationship (SAR) investigations.

a

compd

R

cell-free IC₅₀(nM)

( )-19a

( )-19b

( )-19c

( )-19d

( )-19e

methyl

4.09 0.83

1.74 0.55

1.34 0.49

1.06 0.28

1.14 0.06

propyl

phenylpropyl

benzyl

phenyl

a

Data are the mean SD of three to four experiments performed in

duplicate.

proved to be more active than acetic ester 19a. Esteriﬁcation with

benzoic acid derivatives was then adopted for the investigation

reported in this article because of its high synthetic versatility to

allow an extensive exploration of the acidic portion.

To conﬁrm our computational hypothesis regarding the

relevance of additional interactions with the solvent-exposed

region in the enzyme catalytic site, diﬀerent esters from

commercially available carboxylic acids featuring aliphatic chains

and/or aromatic groups were prepared and tested in the cell-free

To verify the importance of stereochemistry for this class of

compounds, our exploration then focused on chiral resolution of

( )-18. The two enantiomers were separated, and it was found

that the (−) enantiomer, having an (S) conﬁguration, was

signiﬁcantly more active than the corresponding (+)-(R)

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enantiomer (Table 4) (for the assignment of absolute

conﬁguration, see the previous section).

its alcoholic fragment on the roﬂumilast structure in 1XMU,

whereas the (R) enantiomer was not able to obtain a good

overlay (Figure 6).

It is known that in humans roﬂumilast is metabolized to the

pharmacologically active compound roﬂumilast N-oxide,²⁸

which is almost as potent as roﬂumilast itself.²⁹Compounds

(R)-20 and (S)-20, N-oxide analogues of derivatives (R)-18 and

(S)-18, were therefore prepared. As reported in Table 4, the

introduction of an N-oxide group in the dichloropyridine moiety

unexpectedly and strongly enhanced the inhibitory activity in

comparison with roﬂumilast. Moreover, it was observed that the

stereochemistry−activity relationship observed for nonoxidized

compounds was maintained in N-oxide analogues, with the (S)

enantiomer showing the best activity result. To determine the

inhibitory activity exerted by the synthesized compounds in a

relevant cellular system that takes into account their cellular

penetration capacity, the inhibition of LPS-induced TNF-α

production from PBMCs was evaluated. Compound (S)-20

(PBMC IC₅₀= 1.10 nM, Table 4) was about 5-fold less active in

the cell-based assay than in the cell-free assay, indicating a

moderate diﬃculty in penetrating the cell. It is important to note

that N-oxide alcohol (S)-21 was about 50-fold less active in the

cell-free assay and about 450-fold less active in the cell-based

assay than corresponding ester (S)-20 (Table 4), proving that

ester 20 is stable under the assay conditions, as was also

conﬁrmed by HPLC−MS analysis of the tested samples (data

not shown). Hence forth, the functional eﬀects of compounds as

PDE4 inhibitors in the in vitro model of PBMCs was evaluated to

select the best compounds; however, the cell-free assays were

also performed as a preliminary evaluation of the enzymatic

activity.

Table 4. Inhibitory Activity in Cell-Free and Cell-Based

Assays of Compounds (R)-18, (S)-18, (R)-20, (S)-20, and (S)-

21

a

Data are the mean SD of three to four experiments performed in

Insertion of substituents on the benzoic acid portion was

examined, and mono substitution at the para position was

considered (Table 5). The best para-substituted compounds thus

obtained, in terms of PBMCs activity, were p-methoxyderivative

(S)-26f (PBMC IC₅₀= 0.47 nM) and p-sulfonamido derivative

(S)-36a (PBMC IC₅₀= 0.13 nM). The disubstituted benzoic acid

derivatives of (S)-26f and (S)-36a were investigated, as shown in

Tables 6 and 7, respectively. In Table 6, the introduction of

diﬀerent substituents at position 3 of the benzoic acid portion

already bearing a para-methoxy group was examined. Dialkoxy-

b

duplicate. Data are presented as IC₅₀values followed by their 95%

conﬁdence intervals obtained by nonlinear regression.

The two enantiomers were docked in the catalytic site of the

PDE4B structure (PDB ID: 1XMU) in an attempt to explain the

diﬀerence in their potency. The 1XMU crystal structure was

selected because its cocrystallized ligand, roﬂumilast, shares the

same disubstituted catechol scaﬀold as our benzoic acid ester

derivatives. The (S) enantiomer showed a good superposition of

Figure 6. Binding poses of compounds (R)-18 (left) and (S)-18 (right) in the PDE4B catalytic site. The docked ligands (green) are overlaid on the

crystallographic binding conformation of roﬂumilast (light blue).

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Table 5. Structure and Inhibition Activity in Cell-Free and

Cell-Based Assays of Para-Monosubstituted Benzoic Acid

Esters of (S)-21

Table 7. Structure and Inhibitory Activity in Cell-Free and

Cell-Based Assays of Disubstituted Benzoic Acid Esters of (S)-

21 Featuring a Sulfonamide Substituent at Position 4

a

b

compd

R

cell-free IC₅₀(nM)

PBMCs IC₅₀(nM)

(S)-26a

(S)-26b

(S)-26c

(S)-26d

(S)-26e

(S)-26f

(S)-36a

H

0.072 0.03

0.16 0.06

0.10 0.03

0.20 0.06

0.41 0.28

0.14 0.07

0.05 0.03

1.71 (0.85−3.30)

1.85 (1.32−2.61)

0.66 (0.33−1.31)

21.00 (5.12−90.07)

2.10 (0.67−6.60)

0.47 (0.16−1.36)

0.13 (0.05−0.37)

cell-free

a

b

compd

R

IC₅₀(nM)

PBMCs IC₅₀(nM)

Cl

(S)-36a

(S)-36b

(S)-32a

(S)-32b

(S)-32c

(S)-36c

(S)-32d

(S)-32k

3-H

0.05 0.03

0.06 0.02

0.04 0.01

0.56 0.33

0.05 0.02

0.03 0.01

0.06 0.02

0.26 0.03

0.13 (0.05−0.37)

0.38 (0.11−1.5)

0.06 (0.01−0.38)

0.99 (0.52−1.88)

0.41 (0.35−0.48)

1.05 (0.75−1.50)

0.43 (0.15−1.24)

Me

CF₃

CN

3-OMe

3-OCH₂cyclopropyl

3-OBn

OMe

3-Me

NHSO₂Me

a

3-CH₂OH

Data are the mean SD of three to four experiments performed in

duplicate. Data are presented as IC₅₀values followed by their 95%

conﬁdence intervals obtained by nonlinear regression.

b

2-OMe

2-OCH₂cyclopropyl

1.96 (0.16−24.01)

a

Data are the mean SD of three to four experiments performed in

duplicate. Data are presented as IC₅₀values followed by their 95%

conﬁdence intervals obtained by nonlinear regression.

b

Table 6. Structure and Inhibitory Activity in Cell-Free and

Cell-Based Assays of Disubstituted Benzoic Acid Esters of (S)-

21 Featuring a Methoxy Substituent at Position 4

tolerated, whereas N-dimethylation in (S)-26m did not

signiﬁcantly aﬀect the enzymatic activity in the cell-free assay

but reduced the cell-based assay activity, probably because of

limited cell penetration.

The introduction of a second substituent on 4-sulfonamido

analogue (S)-36a at position 2 or 3 was considered, as shown in

Table 7. Small substituents were tolerated or well accepted at

both positions 3 and 2, whereas the bulky benzyloxy group at

position 3 in (S)-32b caused a considerable reduction of potency.

The cyclopropylmethoxy group, with an intermediate size

between that of the methoxy and benzyloxy substituents, was

well-accepted at position 3 in (S)-32a, whereas at position 2 in

(S)-32k, it caused a signiﬁcant loss of activity in both the cell-free

and cell-based assays. The hydroxymethyl group at the 3 position

in (S)-36c caused the highest drop in activity between the cell-

free and cell-based assays, whereas the 3-cyclopropylmethoxy

group resulted in compound (S)-32a having the same activity in

the cell-free and cell-based assays and showing the best PBMC

activity among the 4-sulfonamido derivatives. The cyclo-

propylmethoxy group at position 3 of the benzoic portion

increased the potency of both para-methoxy (26h) and para-

sulfonamido (32a) derivatives. Therefore, an in-depth examina-

tion of its role was carried out, and the activity data of the

compounds bearing a cyclopropylmethoxy group at position 3

and variable substituents at position 4 are summarized in Table 8.

All of the compounds were endowed with high potency in both

the cell-free and cell-based assays. Again, (S)-32a and (S)-26h

emerged as the most potent compounds. From the comparison

of (S)-26h with analogue (S)-26f, devoid of the cyclo-

propylmethoxy group at position 3, the contribution of such a

group to the potency was clearly evident. Indeed, (S)-26h was 3-

fold more potent than (S)-26f in the cell-free assay and 11-fold

more potent in the cell-based assay (Table 6).

a

b

compd

R

cell-free IC₅₀(nM) PBMCs IC₅₀(nM)

(S)-26f

(S)-26g

(S)-26h

(S)-26i

(S)-44

H

0.14 0.07

0.07 0.03

0.05 0.02

0.21 0.08

0.08 0.04

0.05 0.02

0.08 0.02

0.04 0.01

0.03 0.01

0.47 (0.16−1.36)

0.12 (0.08−0.17)

0.04 (0.02−0.09)

1.36 (0.63−2.90)

0.75 (0.48−1.20)

0.96 (0.30−2.14)

0.67 (0.22−2.21)

0.06 (0.03−0.16)

0.25 (0.12−0.53)

1.85 (0.72−4.73)

OMe

OCH₂cyclopropyl

OPh

OH

(S)-26j

(S)-26k

(S)-32f

(S)-26l

(S)-26m

OCOMe

NHCOMe

NHSO₂Me

SO₂NHMe

SO₂NMe₂

a

Data are the mean SD of three to four experiments performed in

duplicate. Data are presented as IC₅₀values followed by their 95%

conﬁdence intervals obtained by nonlinear regression.

b

substituted derivatives (S)-26g and (S)-26h proved to be very

active; in particular, compound (S)-26h, with a cyclopropylme-

thoxy substituent in the meta position, displayed an excellent

inhibitory activity in both cell-free and cell-based assays. The

replacement of 3-alkoxy groups by a hydroxy or an acetyloxy

group, as in (S)-44 and (S)-26j, respectively, reduced the

PBMCs activity, as did the insertion of an acetamido portion in

(S)-26k; the introduction of the more rigid and lipophilic

phenoxy substituent in (S)-26i resulted in a more pronounced

activity decrease in both assays. The combination of a 4-methoxy

group with a 3-sulfonamide group strongly enhanced the activity

of (S)-32f (IC₅₀= 0.06 nM) in PBMCs, aﬀording a very potent

compound. Reverted N-methylsulfonamide (S)-26l was well-

To investigate the class of sulfonamide derivatives further,

diﬀerent compounds featuring this moiety in the meta or ortho

position on the benzoic acid portion were then examined (Tables

9 and 10). Among 4-substituted 3-sulfonamides (Table 9),

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Table 8. Structure and Inhibitory Activity in Cell-Free and

Cell-Based Assays of Disubstituted Benzoic Acid Esters of (S)-

21 Featuring a Cyclopropylmethoxy Substituent at Position 3

Table 10. Structure and Inhibitory Activity in Cell-Free and

Cell-Based Assays of Disubstituted Benzoic Acid Esters of (S)-

21 Featuring a Sulfonamide Substituent at Position 2

a

b

compd

R

cell-free IC₅₀(nM)

PBMCs IC₅₀(nM)

cell-free

a

b

(S)-38d

(S)-38e

(S)-32l

4-OMe

5-OMe

6-OMe

0.73 0.18

0.40 0.02

6.83 1.82

>60

compd

R

IC₅₀(nM)

PBMCs IC₅₀(nM)

(S)-20

OCHF₂

OMe

NO₂

0.22 0.15

0.05 0.02

0.21 0.12

0.09 0.04

0.31 0.22

0.24 0.12

0.04 0.01

0.15 0.07

0.04 0.01

0.08 0.01

0.05 0.01

1.10 (0.67−1.90)

0.04 (0.02−0.09)

0.69 (0.45−1.04)

0.11 (0.04−0.30)

0.63 (0.18−2.20)

0.21 (0.11−0.43)

0.06 (0.01−0.38)

0.34 (0.19−0.57)

0.85 (0.40−1.79)

0.69 (0.10−4.70)

0.30 (0.11−0.84)

(S)-26h

(S)-26q

(S)-26t

(S)-26n

(S)-26o

(S)-32a

(S)-32e

(S)-26u

(S)-26v

(S)-26w

a

Data are the mean SD of three to four experiments performed in

duplicate. Data are presented as IC₅₀values obtained by nonlinear

regression.

b

NH₂

NMe₂

NHCOMe

NHSO₂Me

NHSO₂cyclopropyl

OH

3-sulfonamido series, the best results were therefore obtained

with the additional substituent in position 4. The limited

exploration of 2-sulfonamido derivatives (Table 10) evidently

highlighted that such a substituent is not tolerated in position 2,

expecially with regard to cell-based activity.

Among the tested derivatives, a number of compounds

showing excellent PDE4 potency were identiﬁed, and a subset

was selected for further characterization (Table 11). In the cell-

based assay, all of the selected compounds, having PBMC IC₅₀<

0.2 nM, performed much better than roﬂumilast in the same

assay (PBMC IC₅₀= 3.8 nM; conﬁdence interval: 2.9−5.1).

In Vitro ADME Characterization. Some in vitro ADME

properties and experimental LogD_7.4of the selected compounds

were evaluated to identify PDE4 inhibitors suitable for inhaled

administration.

CH₂OH

COOH

a

Data are the mean SD of three to four experiments performed in

duplicate. Data are presented as IC₅₀values followed by their 95%

conﬁdence intervals obtained by nonlinear regression.

b

Table 9. Structure and Inhibitory Activity in Cell-Free and

Cell-Based Assays of Disubstituted Benzoic Acid Esters of (S)-

21 Featuring a Sulfonamide Substituent at Position 3

Experimental LogD_7.4, which is an estimation of lipophilicity

and can give an indication on the solubility of the compounds,

and half-life in human and rat lung S9, which is a model of the

metabolic stability in lung, were chosen as in vitro properties

predictive of long retention in the lung in vivo. Plasma protein

binding (PPB), which is a measure of the absorbed compound

free fraction in the systemic circulation, and Caco-2 permeability,

which predicts the oral bioavailability of the swallowed fraction,

were taken as properties that can have a role, even if not

exhaustive, in limiting unwanted side eﬀects.

As summarized in Table 11, the measured LogD_7.4values of

the selected compounds were comparable to measured LogD_7.4

values of inhaled PDE4 inhibitor clinical candidates 4 (AWD-12-

281, LogD_7.4= 3.97), 5 (CP-325366, LogD_7.4= 3.50), and 6

(UK-500,001, LogD_7.4= 4.27) as well as to the inhaled

corticosteroid ﬂuticasone furoate (LogD_7.4= 4.43). As

anticipated by the LogD values, the PPB was in general very

high in both human (>99%) and rat (>98%) plasma for all of

these compounds. The tested compounds showed good stability

in rat and especially in human lung S9 fraction (the remaining

percent at 1 h incubation in human S9 was about 100%).

Concerning the Caco-2 data, it was observed that the presence of

the cyclopropylmethoxy substituent proved to be crucial to

confer low permeability to compounds (S)-32a and (S)-38a.

Compound (S)-32a proved to be the most interesting

derivative, displaying the best combination of high potency,

high lung stability, low permeability, and very high PPB, thus

satisfying our medicinal chemistry strategy for a drug suitable for

a

b

compd

R

cell-free IC₅₀(nM) PBMCs IC₅₀(nM)

(S)-32f

(S)-38a

(S)-38b

(S)-32g

(S)-38c

(S)-32h

(S)-32i

(S)-26p

(S)-32j

4-OMe

0.04 0.01

0.13 0.01

0.32 0.01

0.03 0.01

0.17 0.03

0.06 0.01

0.04 0.01

0.40 0.03

0.18 0.04

0.06 (0.03−0.16)

0.12 (0.04−0.41)

0.53 (0.22−1.25)

0.96 (0.54−1.70)

0.13 (0.04−0.42)

0.92 (0.40−2.13)

0.89 (0.52−1.50)

4.90 (2.76−8.50)

1.31 (0.38−4.50)

4-OCH₂cyclopropyl

4-OBn

4-Me

5-OMe

5-OCH₂cyclopropyl

6-OMe

6-OCH₂cyclopropyl

2-OCH₂cyclopropyl

a

Data are the mean SD of three to four experiments performed in

duplicate. Data are presented as IC₅₀values followed by their 95%

conﬁdence intervals obtained by nonlinear regression.

b

methoxy derivative (S)-32f was the most potent in both assays.

Replacement of the cyclopropylmethoxy group with a benzyloxy

one in (S)-38b negatively aﬀected the inhibitory potency, as

observed in the 4-sulfonamido series ((S)-32b, Table 7).

Interestingly, compound (S)-38c was comparably potent in the

cell-free and cell-based assays, whereas compound (S)-32h

displayed a 3-fold improved cell-free activity but an 8-fold loss in

PBMC potency. A remarkable loss of potency was also found for

6-cyclopropylmethoxy analogue (S)-26p, whereas the same

group was slightly better-tolerated in position 2 ((S)-32j). In the

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Table 11. ADME Proﬁle of Selected Compounds

rat ADME data

lung S9 stability

1 h

human ADME data

lung S9 stability

1 h

activity

PPB

bound remain.

(%)

PPB

Caco-2

perm.

level

cell-free

PBMC

bound remain.

a

compd

R₁

R₂

IC₅₀(nM) IC₅₀(nM) LogD_7.4

(%)

t_1/2(min)

(%)

t_1/2(min)

(S)-

32a

4-NHSO₂CH₃

3-OCH₂cyclopropyl

0.04

0.13

0.04

0.17

0.07

0.06

0.12

0.06

0.13

0.12

4.48

4.23

3.60

n.t.

99.20

88

189

99.98

100

>280

low

(S)-

38a

4-OCH₂cyclopropyl

4-OCH₃

3-NHSO₂CH₃

3-OCH₃

99.28

99.41

98.38

99.85

76

85

n.t.

78

105

277

99.87

99.75

99.81

99.85

100

n.t.

>280

low

(S)-

32f

high

(S)-

38c

5-NHSO₂CH₃

4-OCH₃

medium

high

(S)-

3-OCH₃

4.29

112

100

>280

26g

a

General absorption classiﬁcation for permeability values: <10 nm/s, low level; 10−50 nm/s, medium level; and >50 nm/s, high level.

Figure 7. Binding pose of compound (S)-32a in the PDE4B catalytic site (orange, hydrophobic region; gray, metal region; and turquoise, solvent-

exposed region) (PDB ID: 1XMU).

inhaled administration. Therefore, it was selected for further

characterization.

Given these premises, an extended in vitro characterization of

compound 32a was performed. When tested against the

enzymatic activity of bovine PDE1 and PDE6 and human

PDE2, PDE3, and PDE5, compound 32a showed IC₅₀values >1

μM. With an IC₅₀value of 0.04 nM for 32a in the PDE4

enzymatic assay, our data reveal that 32a is >20 000-fold selective

versus PDE4 in comparison with other PDE isoforms. Binding

kinetics experiments were performed using human recombinant

catalytic domains of PDE4B and PDE4D isoenzymes and labeled

[¹⁴C]32a as ligand. Binding of [¹⁴C]32a (100 nM) to both

PDE4BCat and PDE4DCat is rapid, reaching saturation in

approximately 10 min, with K_onvalues of 1.44 × 10⁶M⁻¹min⁻¹

and 1.69 × 10⁶M⁻¹min⁻¹for PDE4BCat and PDE4DCat,

respectively. We were not able to compete [¹⁴C]32a oﬀ of these

enzymes, even when using an excess of unlabeled 32a (50 μM)

with incubation times up to 20 h at room temperature. Although

In Silico, in Vitro, and in Vivo Characterization of

Compound 32a. To evaluate the interactions in the binding site

of the PDE4 protein, compound (S)-32a was docked in the

PDE4B catalytic site. Compound (S)-32a interacts with all three

regions of the catalytic binding site (Figure7). The catechol

scaﬀold ﬁnds good contacts in the hydrophobic region (orange),

and its oxygen atoms form two hydrogen bonds with residue

Gln443, consistent with what was observed for roﬂumilast.²³The

3,5-dichloro-pyridine 1-oxide ring is placed near the metal region

and forms hydrogen bonds with the backbone of the Met347

residue and with a coordinated water molecule. The 3-

(cyclopropylmethoxy)-4-(methylsulfonamido)benzoic acid por-

tion is placed in the solvent-exposed region, and a hydrogen

bond is formed with the backbone of the Met431 residue.

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we could not calculate an exact K_offvalue for 32a, the dissociation

t_1/2can be estimated as >20 h, suggesting a prolonged activity of

this compound upon engagement with the PDE4 enzyme.

Pulmonary stability of 32a was conﬁrmed in rat and human lung

slices, where the compound proved to be highly stable (t_1/2= 172

and 237 min in rat and human, respectively). Interestingly, 32a

proved to be extremely stable when incubated in plasma (100%

remaining after 1 h incubation both in rat and human plasma).

These in vitro data suggest that this compound is also potentially

highly stable under conditions associated with increased

hydrolytic and proteolytic activities in the lung parenchyma

(i.e., COPD patients). The permeability of 32a across airway

epithelium was studied in Calu-3 cells; negligible transport was

measured in this in vitro model of pulmonary absorption,

indicating a low potential of the compound for systemic

availability.

CONCLUSIONS

■

A new class of benzoic acid ester derivatives of (S)-3,5-dichloro-

4-(2-(3-(cyclopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)-2-

hydroxyethyl)pyridine 1-oxide ((S)-21) was discovered. To

optimize the inhibitory activity toward cAMP-speciﬁc phospho-

diesterases-4 (PDE4), starting from initial hit ( )-18 the most

relevant SAR results were achieved by the oxidation of the

dichloropyridine ring to the N-oxide derivative and by chiral

resolution, with the (S) enantiomer displaying higher potency.

Subsequently, substitutions at the benzoic acid portion were

considered, and the introduction of alkoxy and sulfonamide

groups further improved the activity. Among the most potent

derivatives, investigation of in vitro ADME properties allowed

the selection of a compound, namely, 32a, with a proﬁle suitable

for inhaled administration (slow K_off, high PPB, low Calu-3

permeability, and high stability in lung tissue). Anti-inﬂammatory

eﬃcacy upon topical administration of compound 32a was

demonstrated in an in vivo model of allergen-induced pulmonary

inﬂammation.

The in vitro results for compound 32a are supportive of a

compound that is stable, long lasting in the lung, and

characterized by minimized systemic exposure.

The in vivo eﬃcacy of 32a was assessed in a well-known

asthma model:³⁰ovalbumin (Ova)-induced lung eosinophilia in

guinea pigs (GP), an animal species where the in vitro ADME

parameters of 32a are comparable to the ones in human and rat.

(The percent remaining at 1 h in both GP plasma and GP lung S9

was 100%, and GP PPB was 99.1%.) When administered

intratracheally as dry powder formulation 2 h before Ova

challenge, compound 32a dose-dependently inhibited lung

eosinophilia with an eﬃcacious dose (ED₅₀) of 0.016 μmol/kg

(Figure 8).

EXPERIMENTAL SECTION

■

Chemistry. Reagents and starting materials were commercially

available. All solvents and chemicals were used as purchased without

further puriﬁcation. ¹H NMR spectra were recorded on a Bruker AC 200

(200 MHz) spectrometer, a Bruker ARX 300 (300 MHz) spectrometer,

or a Varian MR-400 (400 MHz) spectrometer equipped with a self-

shielded z-gradient coil, 5 mm ¹H/ⁿX broad-band probehead for reverse

detection, deuterium digital lock channel unit, and quadrature digital

detection unit with transmitter oﬀset frequency shift. Chemical shifts are

reported as δ downﬁeld in parts per million (ppm) and are referenced to

tetramethylsilane (TMS) as the internal standard in the ¹H NMR

measurements. Coupling constants (J values) are given in hertz (Hz),

and multiplicities are reported using the following abbreviation (s,

singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; and nd,

not determined). The pulse programs were taken from the Varian and

Bruker software libraries.

Chiral analyses were performed on a Chiralcel OD-H analytical

column or on a Chiralcel OD analytical column.

Preparative HPLC puriﬁcations were performed using the following

methods:

(1) Waters Corporation UV puriﬁcation system equipped with a

Waters Symmetry Preparative C18, 17 μm, 19 × 300 mm, 30 min

gradient of 5−100% solvent B, where solvent A is 90% H₂O, 10%

acetonitrile, and 0.05% TFA and solvent B is 10% H₂O, 90%

acetonitrile, and 0.05% TFA;

(2) Waters Corporation puriﬁcation system equipped with a Water

X-Terra Preparative, 5.0 μm, 19 × 150 mm, 20 min gradient of

5−100% solvent B, where solvent A is 90% H₂O, 10%

acetonitrile, and 0.05% TFA and solvent B is 10% H₂O, 90%

acetonitrile, and 0.05% TFA;

Figure 8. Dose-dependent inhibition of OVA-induced eosinophilia with

a 2 h predose with compound 32a.

(3) Varian Corporation UV puriﬁcation system equipped with a X-

Terra Preparative, 5.0 μm, 19 × 150 mm, Waters column,

isocratic elution with H₂O/MeOH = 25:75, ﬂow = 9.0 mL/min.

The eﬃcacy achieved at the 2 h predosing time point is similar

to the one observed after treatment with the steroid ﬂuticasone

furoate at a dose of 0.3 μmol/kg (90%). The protective eﬀect was

long lasting and was also observed when the compound was

administered 24 h before allergen challenge (data not shown).

The in vivo anti-inﬂammatory activity of compound 32a

indicates that a successful medicinal chemistry strategy can

combine a robust anti-inﬂammatory eﬀect upon topical

administration with properties such as an high plasma protein

binding and low Calu-3 permeability, with the aim of reducing

systemic exposure and providing potential advantages for the

safety proﬁle.

Chiral separations were performed by chiral HPLC using a

semipreparative Chiralcel OD column, 10 μm, 250 × 4.6 mm.

UV purity and m/z of compounds were assessed by ESI+ LC−MS

analysis performed on an Acquity UPLC BEH C18, 1.7 μm, 50 × 2.1

mm column with a Micromass ZQ2000 Waters instrument equipped

with an ACQ-PDA Waters detector. UV purity was also assessed on an

XTerra MS C18, 2.5 μm, 1.6 × 2.5 mm column with an Alliance 2695

Waters instrument equipped with a 2996 PAD Waters detector. All of

the compounds tested in biological assays had a purity >95%.

Speciﬁc rotation of compounds was measured with a polarimeter

(PerkinElmer, model 241) at the sodium D line (589 nm), 20 °C, 1 dm

path length. Reactions were monitored by TLC using 0.25 mm Merck

silica gel plates (60 F254) or LC−MS analysis; column chromatography

805

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was performed on Merck silica gel 60 (0.063−0.2 mm). Anhydrous

solvents were purchased from Aldrich and used as received. Brine refers

to a saturated aqueous solution of NaCl. Unless otherwise speciﬁed,

solutions of common inorganic salts used in workups are aqueous

solutions.

Synthesis of 4-Diﬂuoromethoxy-3-hydroxybenzaldehyde

(8a). To a solution of 3,4-dihydroxybenzaldehyde (16.6 g, 120 mmol)

and sodium chlorodiﬂuoroacetate (18.3 g, 120 mmol) in DMF (150

mL) and water (3 mL) was added sodium hydroxide (4.8 g, 120 mmol).

The mixture was heated at 120 °C and stirred at this temperature for 2 h.

The solvent was removed by vacuum distillation, and to the residue was

added aqueous HCl (20 mL). The mixture was extracted with Et₂O and

washed with brine. The solvent was removed under reduced pressure,

and the crude product was puriﬁed by chromatography on silica gel

(hexane/EtOAc = 80:20) to aﬀord 8 as a white solid (10 g, 52.8 mmol,

44%). LC−MS (ESI+) m/z 189.2 (MH⁺).

Synthesis of 3-Cyclopropylmethoxy-4-diﬂuoromethoxyben-

zaldehyde (9). To a solution of compound 8 (10 g, 53 mmol) in THF

(100 mL) was added potassium carbonate (44 g, 105 mmol), and the

mixture was cooled to 0 °C. A solution of bromomethylcyclopropane

(11 mL, 116.6 mmol) in THF (50 mL) was added, and the reaction was

heated to reﬂux under stirring for 7 h. Fresh bromomethylcyclopropane

(5.5 mL, 58.3 mmol) was added, and the heating was continued for a

further 7 h. The reaction mixture was cooled to rt, and 2 N sodium

hydroxide (100 mL) was added. The aqueous layer was extracted with

DCM and dried over Na₂SO₄. The solvent was removed under reduced

pressure to aﬀord crude compound 9 (12 g, 50 mmol, 97%), which was

used without further puriﬁcation. LC−MS (ESI+) m/z 243.3 (MH⁺).

Synthesis of 3-Cyclopropylmethoxy-4-diﬂuoromethoxyben-

zoic Acid (10). Compound 9 (12 g, 50 mmol) and sulfamic acid (7.3 g,

75 mmol) were dissolved in glacial acetic acid (50 mL), and a solution of

sodium chlorite (8.2 g, 75 mmol) in water (15 mL) was added. The

reaction mixture was stirred at rt for 1 h, and then water (300 mL) was

added. The precipitated solid was ﬁltered and dried at 40 °C under

vacuum, aﬀording compound 10 (12 g, 48 mmol, 97% yield). LC−MS

(ESI+) m/z 256.9 (MH⁺).

Synthesis of 3-Cyclopropylmethoxy-4-diﬂuoromethoxyben-

zoyl Chloride (11). To a solution of compound 10 (12 g, 48 mmol) in

toluene (100 mL) was added thionyl chloride (25 mL, 344 mmol)

dropwise, and the reaction mixture was heated to reﬂux for 2 h. The

mixture was evaporated to dryness, and crude 11 was used without

further puriﬁcation (13.2 g, 48 mmol, quantitative yield).

Synthesis of 3,5-Dichloro-4-methylpyridine (13). A solution of

diisopropylamine (70 mL, 500 mmol) in dry THF (500 mL) was cooled

to −10 °C, and buthyl lithium (2.5 N in hexane, 210 mL, 525 mmol) was

added dropwise under stirring. After 30 min, the solution was cooled to

−20 °C, and a solution of 3,5-dichloropyridine 12 (66.6 g, 450 mmol) in

THF (200 mL) was added dropwise. The resulting solution was stirred

at −10 °C for 30 min, cooled to −70 °C, and added dropwise to a

solution of iodomethane (50 mL, 800 mmol) in THF (100 mL). The

reaction mixture was allowed to warm to rt, quenched with water (100

mL), and extracted with diethyl ether (3 × 100 mL). The combined

organic layers were dried over Na₂SO₄and evaporated to dryness. The

crude product was crystallized from aqueous EtOH than from hexane to

aﬀord 13 (49.9 g, 306 mmol, 68%) as a white solid. LC−MS (ESI+) m/z

162.0 (MH⁺).

Synthesis of 1-(3-Cyclopropylmethoxy-4-diﬂuoromethoxy-

phenyl)-2-(3,5-dichloro-pyridin-4-yl)-ethanone (14). A solution

of 13 (1.0 g, 6.2 mmol) in dry THF (10 mL) was cooled to −78 °C, and

a 1.8 M solution of lithium diisopropylamide in tetrahydrofuran (3.5

mL, 6.3 mmol) was then added dropwise under stirring, keeping the

temperature below −70 °C. The resulting solution was stirred for 30

min, and a solution of 11 (0.43 g, 1.6 mmol) in dry THF (10 mL) was

then added dropwise, maintaining the temperature below −70 °C. After

stirring for 15 min, crushed ice (20 g) was added followed by 200 mL of

water. The mixture was extracted with ethyl acetate, and the solvent was

dried over Na₂SO₄and evaporated. The crude product was crystallized

from petroleum ether/EtOAc 6:4 to yield 14 as a white solid (130 mg,

0.32 mmol, 20%). LC−MS (ESI+) m/z 402.20 (MH⁺). ¹H NMR (200

MHz, CDCl₃) δ 8.53 (s, 2H), 7.47−7.81 (m, 2H), 7.33 (m, 1H), 6.77 (t,

J = 75.00 Hz, 1H), 4.65 (s, 2H), 3.97 (d, J = 6.65 Hz, 2H), 1.17−1.44 (m,

1H), 0.60−0.75 (m, 2H), 0.24−0.51 (m, 2H).

Synthesis of 3-Cyclopropylmethoxy-4-diﬂuoromethoxy-

benzoic Acid (Z)-1-(3-Cyclopropylmethoxy-4-diﬂuorome-

thoxy-phenyl)-2-(3,5-dichloro-pyridin-4-yl)-vinyl Ester (15). A

solution of 13 (2.06 g, 12.7 mmol) in dry THF (30 mL) was cooled to

−78 °C, and a 1.8 M solution of lithium diisopropylamide in THF (14.8

mL, 26.7 mmol) was added dropwise, keeping the temperature below

−70 °C. The resulting solution was stirred for 30 min, a solution of 11

(7.02 g, 25.4 mmol) in dry THF (20 mL) was then added, and the

mixture was allowed to warm to rt. After stirring for 30 min, the reaction

was quenched with water and extracted with EtOAc. The organic layer

was dried over Na₂SO₄and evaporated, and the resulting oil was puriﬁed

by ﬂash chromatography on silica gel (petroleum ether/EtOAc from 9:1

to 7:3), yielding 15 as a white solid (2.4 g, 3.8 mmol, 30%). LC−MS

(ESI+) m/z 642.2 (MH⁺). ¹H NMR (400 MHz, acetone) δ 8.52 (s, 2H),

7.58−7.71 (m, 2H), 7.53 (d, J = 1.32 Hz, 1H), 7.20−7.34 (m, 3H), 7.07

(t, J = 75.00, 75.00 Hz, 1H), 7.04 (s, 1H), 6.98 (t, J = 75.00 Hz, 1H), 4.00

(dd, J = 7.03, 4.83 Hz, 4H), 1.12−1.53 (m, 2H), 0.50−0.70 (m, 4H),

0.24−0.47 (m, 4H).

Synthesis of Racemic 1-(3-Cyclopropylmethoxy-4-diﬂuoro-

methoxy-phenyl)-2-(3,5-dichloro-pyridin-4-yl)-ethanol

(( )-16). A solution of 9 (5.00 g, 20.64 mmol) and 13 (2.57 g, 15.86

mmol) in 50 mL of dry THF was cooled to −30 °C. Potassium tert-

butilate (1.96 g, 17.47 mmol) was added portionwise, maintaining the

temperature between −30 and −20 °C, and the dark red solution was

stirred at −30 °C for 1 h. A saturated aqueous solution of NH₄Cl (50

mL) was then added, maintaining the temperature between −5 and −10

°C (the color turned to yellow). The mixture was extracted with EtOAc,

and the organic layer was dried over Na₂SO₄. The solvent was

evaporated to dryness, the residue was triturated with petroleum ether/

EtOAc 8:2, and the solid was ﬁltered and dried, obtaining ( )-16 as a

white solid (4.83 g, 11.95 mmol, 75%). LC−MS (ESI+) m/z 404.16

(MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 8.56 (s, 2H), 7.11 (d, J = 7.9

Hz, 1H), 7.03 (d, J = 1.8 Hz, 1H), 6.88 (dd, J = 8.2, 1.8 Hz, 1H), 7.01 (t, J

= 74.8 Hz, 1H), 5.59 (d, J = 4.7 Hz, 1H), 4.91 (dt, J = 8.1, 5.1 Hz, 1H),

3.88 (dd, J = 10.3, 6.7 Hz, 1H), 3.82 (dd, J = 10.3, 7.0 Hz, 1H), 3.28 (dd, J

= 12.9, 8.5 Hz, 1H), 3.11 (dd, J = 12.9, 5.6 Hz, 1H), 1.04−1.34 (m, 1H),

0.47−0.68 (m, 2H), 0.20−0.47 (m, 2H).

Synthesis of Racemic 1-(3-(Cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3,5-dichloropyridin-4-yl)ethyl 3-

(Cyclopropylmethoxy)-4-(diﬂuoromethoxy)benzoate (( )-18).

A mixture of ( )-16 (250 mg, 0.62 mmol), acid 10 (160 mg, 0.62

mmol), EDC (356 mg, 1.86 mmol), and DMAP (0.038 mg, 0.31 mmol)

in dry DCM (8 mL) was stirred at rt for 3 days. The reaction mixture was

treated with a saturated solution of NH₄Cl and extracted with DCM.

The organic phase was dried over Na₂SO₄, and the solvent was removed.

The residue was puriﬁed by ﬂash chromatography on silica gel (hexane/

EtOAc from 90:10 to 80:20) to aﬀord ( )-18 as an oﬀ-white solid (307

mg, 0.476 mmol, 77%). LC−MS (ESI+) m/z 644.1 (MH⁺). ¹H NMR

(300 MHz, CDCL₃) δ 8.48 (s, 2H), 7.66 (dd, J = 8.4, 1.9 Hz, 1H), 7.58

(d, J = 2.1 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H),

7.08 (dd, J = 8.2, 1.8 Hz, 1H), 7.04 (d, J = 2.1 Hz, 1H), 6.72 (t, J = 74.8

Hz, 1H), 6.63 (t, J = 74.8 Hz, 1H), 6.30 (dd, J = 10.3, 4.1 Hz, 1H), 3.86−

4.00 (m, 4H), 3.79 (dd, J = 13.5, 10.3 Hz, 1H), 3.38 (dd, J = 13.6, 4.3 Hz,

1H), 1.14−1.42 (m, 2H), 0.54−0.75 (m, 4H), 0.28−0.47 (m, 4H).

Resolution of Compound ( )-18: (S)-(−)-1-(3-(Cyclopropyl-

methoxy)-4-(diﬂuoromethoxy)phenyl)-2-(3,5-dichloropyridin-

4-yl)ethyl 3-(cyclopropylmethoxy)-4-(diﬂuoromethoxy)-

benzoate ((S)-18) and (R)-(+)-1-(3-(Cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3,5-dichloropyridin-4-yl)ethyl 3-

(cyclopropylmethoxy)-4-(diﬂuoromethoxy)benzoate ((R)-18).

Four-hundred milligrams of ( )-18 was separated by chiral HPLC on

a semipreparative Chiralcel OD column (hexane/iPrOH 95:5, ﬂow = 18

mL/min) to aﬀord the two enantiomers.

First-eluting enantiomer: t_R8.2 min, (R)-(+)-18 (white solid, 128 mg,

20

32%). [α]_D= +29.31 (c = 0.5, MeOH). LC−MS (ESI+) m/z 644.1

(MH⁺). NMR equal to ( )-18.

Second-eluting enantiomer: t_R9.4 min, (S)-(−)-18 (white solid, 126

20

mg, 32%). [α]_D= −27.96 (c = 0.5, MeOH). LC−MS (ESI+) m/z

644.1 (MH⁺). NMR equal to ( )-18.

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General Procedure for the Synthesis of Compounds 19a−e.

Synthesis of Racemic 1-(3-(Cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3,5-dichloropyridin-4-yl)ethyl Acetate

(( )-19a). To a mixture of ( )-16(0.100 g, 0.247 mmol), EDC

(0.142 g, 0.742 mmol), and DMAP (0.030 g, 0.247 mmol) in DCM (10

mL) was added 17a (0.018 mL, 0.322 mmol), and the reaction was

stirred at rt for 4 h. The mixture was diluted with DCM and washed with

1 N HCl, 1 N NaHCO₃, and brine, the organic phase was dried over

Na₂SO₄, and the solvent was evaporated. The crude product was

puriﬁed by ﬂash chromatography on silica gel cartridge (petroleum

ether/EtOAc from 90:10 to 80:20), aﬀording ( )-19a as a white solid

(0.092 g, 0.206 mmol, 83%). LC−MS (ESI+) m/z 446.04 (MH⁺). ¹H

NMR (300 MHz, DMSO-d₆) δ 8.60 (s, 2H), 7.18 (d, J = 8.2 Hz, 1H),

7.06 (d, J = 1.8 Hz, 1H), 6.97 (dd, J = 8.2, 1.8 Hz, 1H), 7.06 (t, J = 74.8

Hz, 1H), 6.03 (dd, J = 9.1, 4.7 Hz, 1H), 3.90 (d, J = 6.7 Hz, 2H), 3.54

(dd, J = 13.6, 9.2 Hz, 1H), 3.31 (dd, J = 13.5, 4.7 Hz, 1H), 1.97 (s, 3H),

1.08−1.34 (m, 1H), 0.48−0.70 (m, 2H), 0.27−0.47 (m, 2H).

crude product was puriﬁed by preparative HPLC (method 1) to yield

racemic compound ( )-20 (69 mg, 45%). LC−MS (ESI+) m/z 660.1

(MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 8.55 (s, 2H), 7.62 (dd, J =

8.5, 2.1 Hz, 1H), 7.59 (d, J = 1.8 Hz, 1H), 7.04−7.37 (m, 4H), 7.23 (t, J =

74.4 Hz, 1H), 7.06 (t, J = 74.8 Hz, 1H), 6.19 (dd, J = 9.5, 4.3 Hz, 1H),

3.85−4.04 (m, 4H), 3.62 (dd, J = 14.2, 9.5 Hz, 1H), 3.34 (dd, J = 14.2,

4.0 Hz, 1H), 1.12−1.37 (m, 2H), 0.47−0.70 (m, 4H), 0.22−0.47 (m,

4H).

Four-hundred milligrams of ( )-20 was subjected to chiral HPLC on

a semipreparative Chiralcel OD column (hexane/iPrOH 50:50). The

ﬁrst-eluting enantiomer, t_R9.5 min, was collected, evaporated, and

triturated with a mixture of hexane/iPrOH 50:50 to aﬀord (R)-(+)-20 as

a white solid (45 mg, 11%). LC−MS (ESI+) m/z 660.1 (MH⁺). NMR

equal to ( )-20. [α]_D²⁰= +30.86 (c = 0.5, MeOH). The second-eluting

enantiomer, t_R15 min, was collected, evaporated, and triturated with a

mixture of hexane/iPrOH 50:50 to aﬀord (S)-(−)-20 as a white solid

(50 mg, 13%). LC−MS (ESI+) m/z 660.1 (MH⁺). NMR equal to

Compounds 19b−e were synthesized following the same procedure

starting from corresponding carboxylic acids 17b−e using DCM or

DMF as solvent.

20

( )-20. [α]_D= −33.88 (c = 0.5, MeOH). NMR in agreement with

( )-20.

Synthesis of 1-(3-Cyclopropylmethoxy-4-diﬂuoromethoxy-

phenyl)-2-(3,5-dichloro-1-oxy-pyridin-4-yl)-ethanol (( )-21).

Compound ( )-16 (13.0 g, 32.18 mmol) was dissolved in DCM (250

mL), m-chloroperbenzoic acid (77% w/w, 16.5 g, 74 mmol) was added,

and the resulting solution was stirred at rt for 2 h. Na₂S₂O₃(25.4 g, 160

mmol) was added, and the mixture was vigorously stirred for 1 h. The

solid residue was removed by ﬁltration, the ﬁltrate was washed with 1 N

NaOH, the organic phase was dried over Na₂SO₄, and the solvent was

evaporated to aﬀord ( )-21 as a white solid, which was used in the next

steps without further puriﬁcation (10.3 g, 24.5 mmol, 76%). LC−MS

(ESI+) m/z 420.1 (MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 8.51 (s,

2H), 7.11 (d, J = 8.2 Hz, 1H), 7.05 (d, J = 1.8 Hz, 1H), 6.88 (dd, J = 8.2,

1.8 Hz, 1H), 7.01 (t, J = 74.8 Hz, 1H), 5.59 (d, J = 3.5 Hz, 1H), 4.84 (dd,

J = 8.2, 5.6 Hz, 1H), 3.89 (dd, J = 10.3, 7.0 Hz, 1H), 3.84 (dd, J = 10.3, 6.7

Hz, 1H), 3.18 (dd, J = 13.2, 8.2 Hz, 1H), 3.02 (dd, J = 13.5, 5.6 Hz, 1H),

1.03−1.35 (m, 1H), 0.46−0.67 (m, 2H), 0.24−0.46 (m, 2H).

Synthesis of 3,5-Dichloro-4-((S)-2-(3-(cyclopropylmethoxy)-

4-(diﬂuoromethoxy)phenyl)-2-((R)-2-(6-methoxynaphthalen-

2-yl)propanoyloxy)ethyl)pyridine 1-Oxide ((R*,S**)-22) and

3,5-Dichloro-4-((R)-2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-((R)-2-(6-methoxynaphthalen-2-

yl)propanoyloxy)ethyl)pyridine 1-Oxide ((R*,R**)-22). Com-

pound ( )-21 (420 mg, 1.0 mmol) was dissolved in dry DMF (6

mL), and (R)-naproxene (231 mg, 1.0 mmol), EDC (290 mg, 1.50

mmol), and DMAP (122 mg, 1.0 mmol) were added. The orange

solution was stirred at rt overnight, water (100 mL) was then added, and

the precipitate was collected by ﬁltration. The wet solid was dissolved in

EtOAc (100 mL), and the solution was dried over Na₂SO₄and

evaporated to dryness. The residue was puriﬁed by ﬂash chromatog-

raphy on silica gel (petroleum ether/EtOAc 45:55) to aﬀord 560 mg

(0.88 mmol, 88%) of a diastereomeric mixture as a white solid. One-

hundred milligrams of this mixture was dissolved in 3 mL of methanol

and separated by preparative HPLC (method 3).

Racemic 1-(3-(Cyclopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)-

2-(3,5-dichloropyridin-4-yl)ethyl Butyrate (( )-19b). Acid 17b (1

equiv), EDC (1 equiv), DMAP (1 equiv), and DMF, rt, 3 h; puriﬁcation

by trituration with heptane, white solid, 62%. LC−MS (ESI+) m/z 474.2

1

(MH⁺). H NMR (200 MHz, CDCl₃) δ 8.46 (s, 2H), 7.07−7.24 (m,

1H), 6.80−7.04 (m, 2H), 6.62 (t, J = 75.00 Hz, 1H), 6.11 (dd, J = 9.54,

4.45 Hz, 1H), 3.88 (d, J = 6.99 Hz, 2H), 3.49−3.73 (m, 1H), 3.27 (dd, J

= 13.99, 4.45 Hz, 1H), 2.11−2.43 (m, 2H), 1.44−1.68 (m, 2H), 1.12−

1.38 (m, 1H), 0.82 (t, J = 7.31 Hz, 3H), 0.60−0.70 (m, 2H), 0.22−0.50

(m, 2H).

Racemic 1-(3-(Cyclopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)-

2-(3,5-dichloropyridin-4-yl)ethyl 4-Phenylbutanoate (( )-19c). Acid

17c (1 equiv), EDC (1 equiv), DMAP (1 equiv), and DMF, rt, 3 h;

puriﬁcation by trituration with heptane, white solid, 67%. LC−MS (ESI

1

+) m/z 550.3 (MH⁺). H NMR (200 MHz, CDCl₃) δ 8.17−8.69 (m,

2H), 7.05−7.25 (m, 6H), 6.91−7.02 (m, 2H), 6.62 (t, J = 75.00 Hz, 1H),

6.11 (dd, J = 9.54, 4.45 Hz, 1H), 3.86 (d, J = 6.99 Hz, 2H), 3.46−3.70

(m, 1H), 3.05−3.35 (m, 1H), 2.39−2.67 (m, 2H), 2.13−2.37 (m, 2H),

1.68−2.00 (m, 2H), 1.13−1.34 (m, 1H), 0.57−0.76 (m, 2H), 0.14−0.50

(m, 2H).

Racemic 1-(3-(Cyclopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)-

2-(3,5-dichloropyridin-4-yl)ethyl 2-Phenylacetate (( )-19d). Acid

17d (1 equiv), EDC (1 equiv), DMAP (1 equiv), and DMF, rt, 3 h;

puriﬁcation by trituration with heptane, white solid, 83%. LC−MS (ESI

1

+) m/z 522.2 (MH⁺). H NMR (200 MHz, CDCl₃) δ 8.42 (s, 2H),

7.04−7.23 (m, 6H), 6.74−6.94 (m, 2H), 6.60 (t, J = 75.00 Hz, 1H), 6.10

(dd, J = 9.54, 4.45 Hz, 1H), 3.65−3.89 (m, 2H), 3.43−3.62 (m, 3H),

3.05−3.34 (m, 1H), 1.09−1.40 (m, 1H), 0.57−0.77 (m, 2H), 0.11−0.42

(m, 2H).

Racemic 1-(3-(Cyclopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)-

2-(3,5-dichloropyridin-4-yl)ethyl Benzoate (( )-19e). Acid 17e (1.3

equiv), EDC (3 equiv), DMAP (3 equiv), and DCM, rt, 16 h;

puriﬁcation by ﬂash chromatography on silica gel (petroleum ether/

EtOAc from 90:10 to 80:20), white solid, 86%. LC−MS (ESI+) m/z

508.1 (MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 8.59 (s, 2H), 7.94−

8.01 (m, 2H), 7.62−7.71 (m, 1H), 7.48−7.57 (m, 2H), 7.21 (d, J = 1.8

Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 7.10 (dd, J = 8.2, 1.8 Hz, 1H), 7.06 (t, J

= 74.8 Hz, 1H), 6.30 (dd, J = 9.7, 4.4 Hz, 1H), 3.93 (d, J = 6.7 Hz, 2H),

3.73 (dd, J = 13.8, 9.7 Hz, 1H), 3.45 (dd, J = 13.8, 4.4 Hz, 1H), 1.11−

1.30 (m, 1H), 0.51−0.62 (m, 2H), 0.29−0.40 (m, 2H)

Synthesis of (R)-3,5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-

4-(diﬂuoromethoxy)benzoyloxy)-2-(3-(cyclopropylmethoxy)-

4-(diﬂuoromethoxy)phenyl)ethyl)pyridine 1-Oxide ((R)-(+)-20)

and (S)-3,5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)benzoyloxy)-2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)ethyl)pyridine 1-Oxide ((S)-(−)-20).

To a solution of compound ( )-18 (150 mg, 0.233 mmol) in DCM

(8 mL) was added m-chloroperbenzoic acid (77% w/w, 250 mg, 1.12

mmol), and the resulting mixture was stirred at rt for 2 h. The mixture

was then diluted with DCM and washed with 1 N NaOH. The organic

phase was dried over Na₂SO₄, and the solvent was evaporated. The

First-eluting diastereoisomer: t_R21 min, (R*,S**)-22, white solid (35

1

mg, 35%). LC−MS (ESI+) m/z 632.1. (MH⁺). H NMR (200 MHz,

CDCl₃) δ 8.08 (2H, s, H-pyr), 7.63 (2H, d, J = 8.30 Hz, H-ortho

naphthyl), 7.51 (1H, d, J = 1.46 Hz, H-meta naphthyl), 7.22−7.13 (3H,

m, 2 H-ortho meta naphthyl + 1H-meta naphthyl), 6.95 (1H, d, J = 8.31

Hz, H-ortho phenyl), 6.90−6.52−6.14 (1H, t, J = 75.20 Hz, CHF₂), 6.64

(1H, dd, J = 8.30, 1.95 Hz, H-ortho meta phenyl), 6.54−6.52 (1H, m, H-

meta phenyl), 5.97 (1H, dd, J = 9.76, 4.39 Hz, *CH-CH₂Ar), 3.93 (3H, s,

OCH₃), 3.80 (1H, q, J = 14.16, 7.33 Hz, *CH-CH₃), 3.48−3.24 (3H, m,

OCH₂+ H-α of CH₂-Ar), 3.48−3.24 (1H, dd, J = 13.67, 4.39 Hz, H-β of

CH₂-Ar), 1.48 (3H, d, J = 7.32 Hz, CH₃-CH), 1.10−0.94 (1H, m,

cyclopropyl CH), 0.58−0.49 (2H, m, cyclopropyl CH₂), 0.21−0.13

(2H, m, cyclopropyl CH₂) (the assignment of absolute conﬁguration by

XRD is reported in the Supporting Information).

Second-eluting diastereoisomer: t_R30 min, (R*,R**)-22, white solid

(45 mg, 45%). (ESI+) m/z 632.1 (MH⁺). ¹H NMR (200 MHz, CDCl₃)

δ 7.70−7.55 (5H, m, 2H + 3H, 2H-pyr + 2H-ortho naphthyl + 1H-meta

naphthyl), 7.24−7.08 (4H, m, 2H-ortho meta naphthyl + 1H-meta

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naphthyl + 1H-ortho phenyl), 6.99−6.61−6.24 (1H, t, J = 75.20 Hz,

CHF₂), 6.95 (1H, dd, J = 8.30, 1.95 Hz, H-ortho meta phenyl), 6.90 (1H,

d, J = 1.95 Hz, H-meta phenyl), 6.06 (1H, dd, J = 10.74, 4.40 Hz, *CH-

CH₂Ar), 3.96 (3H, s, OCH₃), 3.79−3.71 (3H, m, 2H + 1H, OCH₂+

*CH-CH₃), 3.39 (1H, dd, J = 14.16, 10.74 Hz, H-α of CH₂-Ar), 3.00

(1H, dd, J = 14.16, 4.39 Hz, H-β of CH₂-Ar), 1.45 (3H, d, J = 7.32 Hz,

CH₃-CH), 1.32−1.17 (1H, m, cyclopropyl CH), 0.70−0.61 (2H, m,

cyclopropyl CH₂), 0.39−0.31 (2H, m, cyclopropyl CH₂). m/z = 632.1

Synthesis of 4-((S)-2-((S)-2-Acetoxy-2-phenylacetoxy)-2-(3-

(cyclopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)-3,5-

dichloropyridine 1-Oxide ((S*,S*)-24 and 4-((R)-2-((S)-2-Ace-

toxy-2-phenylacetoxy)-2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)ethyl)-3,5-dichloropyridine 1-Oxide

((S*R**)-24). A mixture of ( )-21 (19.95 g, 46.43 mmol), (S)-

acetylmandelic acid (9.22 g, 47.52 mmol), EDC (18 g, 94.24 mmol), and

DMAP (2.89 g, 23.69 mmol) in dry DCM (300 mL) was stirred at rt

overnight. A 5% aqueous solution of NaHCO₃(200 mL) was added, and

the aqueous phase was extracted with DCM. The organic layer was dried

over Na₂SO₄, and the solvent was evaporated under reduced pressure to

give 32 g of a mixture of diastereoisomers that was repeatedly triturated

and sonicated with Et₂O to obtain a solid enriched with diastereoisomer

(S*,S**)-24. This solid was crystallized from iPrOH and ﬁltered to give

9.65 g of pure (S*,S**)-24 (yield 69.4%). The diastereomeric purity was

determined by analytical chiral HPLC performed on a Chiracel OD

column (hexane/isopropanol 40:60, ﬂow 0.45 mL/min, t_R27.2 min).

LC−MS (ESI+) m/z 596.2 (MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ

8.57 (s, 2H), 7.18−7.51 (m, 5H), 7.07 (d, J = 8.8 Hz, 1H), 7.03 (t, J =

74.5 Hz, 1H), 6.74 (d, J = 1.8 Hz, 1H), 6.74 (dd, J = 8.8, 2.1 Hz, 1H),

5.95 (dd, J = 9.7, 4.7 Hz, 1H), 5.86 (s, 1H), 3.72 (dd, J = 10.3, 7.0 Hz,

1H), 3.60 (dd, J = 10.0, 6.7 Hz, 1H), 3.41 (dd, J = 14.1, 10.0 Hz, 1H),

3.24 (dd, J = 14.4, 5.0 Hz, 1H), 2.13 (s, 3H), 1.06−1.28 (m, 1H), 0.46−

0.70 (m, 2H), 0.27−0.40 (m, 2H). [α]_D²⁰= +14 (c = 0.5, MeOH).

Mother liquors from triturations were collected and evaporated to

give a solid mixture enriched in diastereoismer (S*,R**)-24, which was

crystallized from iPrOH to give 6.4 g of pure (S*,R**)-24 (yield 46%) as

a white solid. The diastereomeric purity was determined by analytical

chiral HPLC performed on a Chiracel OD column (hexane/isopropanol

40:60, ﬂow = 0.45 mL/min, t_R21.6 min). LC−MS (ESI+) m/z 596.2

(MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 8.27 (s, 2H), 7.27−7.45 (m,

5H), 7.20 (d, J = 8.2 Hz, 1H), 7.08 (d, J = 2.0 Hz, 1H), 7.00 (dd, J = 8.2,

1.8 Hz, 1H), 7.08 (t, J = 74.2 Hz, 1H), 5.97 (dd, J = 10.4, 4.3 Hz, 1H),

5.85 (s, 1H), 3.93 (dd, J = 10.3, 7.0 Hz, 1H), 3.89 (dd, J = 10.3, 7.9 Hz,

1H), 3.33 (dd, J = 14.4, 10.9 Hz, 1H), 3.17 (dd, J = 14.1, 4.1 Hz, 1H),

2.07 (s, 3H), 1.14−1.38 (m, 1H), 0.50−0.71 (m, 2H), 0.21−0.47 (m,

2H). [α]_D²⁰= +26 (c = 0.5, MeOH).

Oxide ((S)-26a). A mixture of (S)-21 (100 mg, 0.238 mmol), acid 25a

(58 mg, 0.476 mmol), EDC (136 mg, 0.714 mmol), and DMAP (32 mg,

0.262 mmol) in DMF (10 mL) was stirred at rt overnight. The mixture

was diluted with water and extracted with EtOAc. The organic phase was

washed twice with 1 N HCl and dried over Na₂SO₄. The solvent was

removed, and the crude product was puriﬁed by crystallization from

EtOH to aﬀord (S)-26a as a white solid (100 mg, 0.190 mmol, 80%).

1

LC−MS (ESI+) m/z 524.3 (MH⁺). H NMR (400 MHz, acetone) δ

8.25 (s, 2H), 8.08 (d, J = 7.50 Hz, 2H), 7.60−7.71 (m, 1H), 7.47−7.56

(m, 2H), 7.31 (d, J = 1.76 Hz, 1H), 7.15−7.27 (m, 2H), 6.68−6.91−7.12

(t, 1 H, CHF₂), 6.36 (dd, J = 9.48, 4.63 Hz, 1H), 3.98 (d, J = 6.62 Hz,

2H), 3.75 (dd, J = 14.11, 9.70 Hz, 1H), 3.45 (dd, J = 14.11, 4.85 Hz, 1H),

1.27 (m, 1H), 0.52−0.66 (m, 2H), 0.32−0.44 (m, 2H).

Compounds 26b−s were synthesized following the same procedure

starting from corresponding carboxylic acid 25b−s using DCM or DMF

as solvent. The preparation of noncommercially available acids is

detailed in the Supporting Information.

(S)-3,5-Dichloro-4-(2-(4-chlorobenzoyloxy)-2-(3-(cyclopropylme-

thoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)pyridine 1-Oxide ((S)-26b).

Acid 25b (1.3 equiv), EDC (3 equiv), DMAP (1 equiv), and DCM, rt,

overnight; puriﬁcation by ﬂash chromatography on silica gel (petroleum

ether/EtOAC from 60:40 to 40:60) followed by trituration with EtOH,

1

white solid, 45% yield. LC−MS (ESI+) m/z 558.1 (MH⁺). H NMR

(300 MHz, DMSO-d₆) δ 8.54 (s, 2H), 7.87−8.12 (m, 2H), 7.49−7.72

(m, 2H), 7.22 (d, J = 2.0 Hz, 1H), 7.21 (d, J = 7.9 Hz, 1H), 7.09 (dd, J =

8.2, 2.1 Hz, 1H), 7.06 (t, J = 74.8 Hz, 1H), 6.21 (dd, J = 9.1, 4.4 Hz, 1H),

3.93 (d, J = 6.7 Hz, 2H), 3.62 (dd, J = 14.1, 9.4 Hz, 1H), 3.35 (dd, J =

14.1, 4.4 Hz, 1H), 1.06−1.35 (m, 1H), 0.47−0.70 (m, 2H), 0.16−0.45

(m, 2H). [α]_D²⁰= −54.09 (c = 0.5, DCM).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(4-methylbenzoyloxy)ethyl)pyridine 1-

Oxide ((S)-26c). Acid 25c (1.3 equiv), EDC (3 equiv), DMAP (1

equiv), and DCM, rt, overnight; puriﬁcation by trituration with EtOH,

1

white solid, 59% yield. LC−MS (ESI+) m/z 538.3 (MH⁺). H NMR

(300 MHz, DMSO-d₆) δ 8.54 (s, 2H), 7.79−7.98 (m, 2H), 7.31−7.43

(m, 2H), 7.21 (d, J = 2.1 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 7.09 (dd, J =

8.4, 1.9 Hz, 1H), 7.06 (t, J = 74.8 Hz, 1H), 6.21 (dd, J = 9.4, 4.4 Hz, 1H),

3.93 (d, J = 7.0 Hz, 2H), 3.61 (dd, J = 14.2, 9.5 Hz, 1H), 3.34 (dd, J =

14.2, 4.5 Hz, 1H), 2.38 (s, 3H), 1.04−1.38 (m, 1H), 0.45−0.66 (m, 2H),

0.24−0.45 (m, 2H). [α]_D²⁰= −39.92 (c = 0.5, DCM).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(4-(triﬂuoromethyl)benzoyloxy)ethyl)-

pyridine 1-Oxide ((S)-26d). Acid 25d (1 equiv), EDC (3 equiv), DMAP

(0.5 equiv), and DCM, rt, 24 h; puriﬁcation by chromatography on silica

gel (DCM/EtOAc 80:20) followed by trituration with iPrOH, white

1

solid, 51% yield. LC−MS (ESI+) m/z 591.9 (MH⁺). H NMR (300

Synthesis of (S)-1-(3-Cyclopropylmethoxy-4-diﬂuorome-

thoxy-phenyl)-2-(3,5-dichloro-1-oxy-pyridin-4-yl)-ethanol ((S)-

(+)-21. Compound (S*,S**)-24 (6.42 g, 10.77 mmol) was suspended

in methanol (350 mL), and a saturated solution of NaHCO₃(175 mL)

was added. The mixture was vigorously stirred at rt overnight, diluted

with DCM (700 mL), and washed with a 5% aqueous solution of

NaHCO₃(300 mL). The organic layer was dried over Na₂SO₄and

evaporated under vacuum. The residue was triturated with Et₂O and

ﬁltered to aﬀord (S)-(+)-21 (3.88 g, 9.24 mmol, 86%) as a white solid

with an enantiomeric purity >99%. The enantiomeric purity was

determined by analytical chiral HPLC performed on a Chiracel OD

column (hexane/isopropanol 30:70, ﬂow = 0.35 mL/min, t_R22.3 min).

LC−MS (ESI+) m/z 420.1 (MH⁺). ¹H NMR equal to ( )-21.

MHz, DMSO-d₆) δ 8.54 (s, 2H), 8.19 (m, 2H), 7.91 (m, 2H), 7.24 (d, J

= 2.1 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 7.11 (dd, J = 8.2, 1.8 Hz, 1H),

7.07 (t, J = 74.8 Hz, 1H), 6.24 (dd, J = 9.4, 4.4 Hz, 1H), 3.94 (d, J = 7.0

Hz, 2H), 3.65 (dd, J = 14.1, 9.4 Hz, 1H), 3.37 (dd, J = 14.2, 4.5 Hz, 1H),

20

1.11−1.32 (m, 1H), 0.49−0.65 (m, 2H), 0.26−0.45 (m, 2H). [α]_D

−30.4 (c = 0.7, MeOH).

=

(S)-3,5-Dichloro-4-(2-(4-cyanobenzoyloxy)-2-(3-(cyclopropylme-

thoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)pyridine 1-Oxide ((S)-26e).

Acid 25e (1.1 equiv), EDC (1.5 equiv), DMAP (1.1 equiv), and DMF,

rt, 24 h, then 70 °C, 2 h; puriﬁcation by preparative HPLC (method 2),

oil, 61% yield. LC−MS (ESI+) m/z 549.3 (MH⁺). ¹H NMR (200 MHz,

CDCl₃) δ 8.02−8.22 (m, 4H), 7.67−7.84 (m, 2H), 7.16−7.25 (m, 1H),

6.93−7.15 (m, 2H), 6.63 (t, J = 75.00 Hz, 1H), 6.16−6.44 (m, 1H),

3.85−4.06 (m, 2H), 3.72 (dd, J = 14.16, 10.25 Hz, 1H), 3.35 (dd, J =

14.16, 4.39 Hz, 1H), 1.06−1.39 (m, 1H), 0.56−0.76 (m, 2H), 0.00−0.52

(m, 2H).

Synthesis of (R)-1-(3-Cyclopropylmethoxy-4-diﬂuorome-

thoxy-phenyl)-2-(3,5-dichloro-1-oxy-pyridin-4-yl)-ethanol ((R)-

(−)-21). This compound was prepared by hydrolysis of corresponding

ester (S*,R**)-24 following the procedure described earlier. The

product was puriﬁed by two triturations with Et₂O followed by a further

trituration with DCM to aﬀord (R)-(−)-21 as a white solid in 88% yield

with an enantiomeric purity >99%. The enantiomeric purity was

determined by analytical chiral HPLC performed on a Chiracel OD

column (hexane/isopropanol 30:70, ﬂow = 0.35 mL/min, t_R24.0 min).

LC−MS (ESI+) m/z 420.1 (MH⁺). ¹H NMR equal to ( )-21.

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(4-methoxybenzoyloxy)ethyl)pyridine

1-Oxide ((S)-26f). Acid 25f (1.8 equiv), EDC (3 equiv), DMAP (1

equiv), and DCM, rt, 48 h; puriﬁcation by trituration with MeOH, white

1

solid, 53% yield. LC−MS (ESI+) m/z 554.1 (MH⁺). H NMR (300

MHz, DMSO-d₆) δ 8.54 (s, 2H), 7.87−8.05 (m, 2H), 7.20 (d, J = 2.1 Hz,

1H), 7.20 (d, J = 8.5 Hz, 1H), 7.08 (dd, J = 8.5, 2.1 Hz, 1H), 7.01−7.08

(m, 2H), 7.06 (t, J = 74.8 Hz, 1H), 6.20 (dd, J = 9.5, 4.3 Hz, 1H), 3.93 (d,

J = 7.0 Hz, 2H), 3.84 (s, 3H), 3.60 (dd, J = 13.9, 9.5 Hz, 1H), 3.33 (dd, J

General Procedure for the Preparation of Compounds (S)-

26a−s. Synthesis of (S)-4-(2-(Benzoyloxy)-2-(3-(cyclopropylme-

thoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)-3,5-dichloropyridine 1-

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= 14.1, 4.4 Hz, 1H), 1.04−1.32 (m, 1H), 0.45−0.66 (m, 2H), 0.27−0.45

(m, 2H). [α]_D²⁰= −45.51 (c = 0.5, DCM).

DMSO-d₆) δ 8.53 (s, 2H), 8.33 (d, J = 2.1 Hz, 1H), 8.19 (dd, J = 8.7, 2.2

Hz, 1H), 7.36 (d, J = 8.8 Hz, 1H), 7.17−7.25 (m, 3H), 7.07 (m, J = 8.5,

2.1 Hz, 1H), 7.07 (t, J = 74.8 Hz, 1H), 6.20 (dd, J = 9.5, 4.3 Hz, 1H), 3.99

(s, 3H), 3.96 (dd, J = 10.3, 7.0 Hz, 1H), 3.91 (dd, J = 10.3, 6.7 Hz, 1H),

3.63 (dd, J = 14.4, 9.7 Hz, 1H), 3.35 (dd, J = 14.1, 4.1 Hz, 1H), 2.42 (d, J

= 4.7 Hz, 3H), 1.15−1.31 (m, 1H), 0.48−0.66 (m, 2H), 0.28−0.44 (m,

2H). [α]_D²⁰= −45.93 (c = 0.5, MeOH).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3-(N,N-dimethylsulfamoyl)-4-

methoxybenzoyloxy)ethyl)pyridine 1-Oxide ((S)-26m). Acid 25m (1.2

equiv), EDC (2 equiv), DMAP (1.1 equiv), and DMF, rt, overnight;

puriﬁcation by crystallization from EtOH/hexane, white solid, 80%

yield. LC−MS (ESI+) m/z 683.2 (MH⁺). ¹H NMR (400 MHz, CDCl₃)

δ 8.59 (d, J = 2.20 Hz, 1H), 8.07−8.26 (m, 3H), 7.19 (d, J = 8.07 Hz,

1H), 6.94−7.11 (m, 3H), 6.63 (t, J = 75.00 Hz, 1H), 6.23 (dd, J = 10.18,

4.13 Hz, 1H), 4.02 (s, 3H), 3.84−3.96 (m, 2H), 3.63−3.80 (m, 4H),

3.14−3.38 (m, 4H), 1.24−1.40 (m, 1H), 0.58−0.75 (m, 2H), 0.39 (q, J =

5.01 Hz, 2H).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3,4-dimethoxybenzoyloxy)ethyl)-

pyridine 1-Oxide ((S)-26g). Acid 25g (4 equiv added portionwise over 6

h), EDC (4 equiv), DMAP (1 equiv), and DCM, rt, 24 h; puriﬁcation by

ﬂash chromatography on silica gel (DCM/EtOAc from 80:20 to 70:30)

followed by dissolution in EtOAc, washing with aqueous NaHCO₃,

evaporation, and treatment with MeOH, white solid, 84% yield. LC−MS

(ESI+) m/z 584.1 (MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 8.56 (s,

2H), 7.63 (dd, J = 8.5, 2.1 Hz, 1H), 7.45 (d, J = 2.1 Hz, 1H), 7.14−7.27

(m, 2H), 7.07 (d, J = 7.9 Hz, 2H), 6.81 (t, J = 74.8 Hz, 1H), 6.19 (dd, J =

9.8, 4.3 Hz, 1H), 3.88−3.99 (m, 2H), 3.81 (s, 3H), 3.84 (s, 3H), 3.61

(dd, J = 14.1, 10.0 Hz, 1H), 3.34 (dd, J = 14.4, 4.4 Hz, 1H), 1.14−1.28

(m, 1H), 0.51−0.63 (m, 2H), 0.29−0.41 (m, 2H). [α]_D²⁰= −37.71 ( c =

0.5, DCM).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-

methoxybenzoyloxy)ethyl)pyridine 1-Oxide ((S)-26h. Acid 25h (1.7

equiv), EDC (3 equiv), DMAP (1 equiv), and DCM, rt, overnight;

puriﬁcation by trituration with MeOH, white solid, 74% yield. LC−MS

(ESI+) m/z 624.1 (MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 8.55 (s,

2H), 7.61 (dd, J = 8.4, 1.9 Hz, 1H), 7.41 (d, J = 2.1 Hz, 1H), 7.16−7.23

(m, 2H), 7.02−7.11 (m, 2H), 7.06 (t, 1H), 6.18 (dd, J = 9.7, 4.1 Hz, 1H),

3.93 (d, J = 6.7 Hz, 2H), 3.85 (s, 3H), 3.84 (d, J = 5.9 Hz, 2H), 3.60 (dd, J

= 14.1, 9.7 Hz, 1H), 3.33 (dd, J = 14.4, 4.4 Hz, 1H), 1.05−1.31 (m, 2H),

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-

(dimethylamino)benzoyloxy)ethyl)pyridine 1-Oxide ((S)-26n). Acid

25n (2 equiv), EDC (2 equiv), DMAP (1.2 equiv), and DMF, rt,

overnight; puriﬁcation by preparative HPLC (method 2), white solid,

1

15% yield. LC−MS (ESI+) m/z 637.1 (MH⁺). H NMR (200 MHz,

CDCl₃) δ 8.14 (s, 2H), 7.53−7.71 (m, 1H), 7.40 (m, 1H), 7.09−7.22

(m, 1H), 6.92−7.08 (m, 2H), 6.72−6.89 (m, 1H), 6.61 (t, J = 75.00 Hz,

1H), 6.09−6.33 (m, 1H), 3.79−4.03 (m, 4H), 3.55−3.78 (m, 1H),

3.20−3.42 (m, 1H), 2.95 (s, 6H), 1.00−1.46 (m, 2H), 0.66 (d, J = 6.84

Hz, 4H), 0.11−0.44 (m, 4H). [α]_D²⁰= −63.33 (c = 1, CHCl₃).

(S)-4-(2-(4-Acetamido-3-(cyclopropylmethoxy)benzoyloxy)-2-(3-

(cyclopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)-3,5-di-

chloropyridine 1-Oxide ((S)-26o). Acid 25o (1 equiv), EDC (3 equiv),

DAMP (0.5 equiv), and DCM, rt, 3 days; puriﬁcation by ﬂash

chromatography on silica gel (DCM/EtOAc from 80:20 to 100%

EtOAc), white solid, 26% yield. LC−MS (ESI+) m/z 651.1 (MH⁺). ¹H

NMR (300 MHz, DMSO-d₆) δ 9.16 (s, 1H), 8.55 (s, 2H), 8.20 (d, J =

8.5 Hz, 1H), 7.59 (dd, J = 8.4, 1.9 Hz, 1H), 7.51 (d, J = 1.8 Hz, 1H),

7.15−7.24 (m, 2H), 7.07 (dd, J = 8.2, 1.8 Hz, 1H), 7.06 (t, J = 74.8 Hz,

1H), 6.18 (dd, J = 9.5, 4.3 Hz, 1H), 3.79−4.09 (m, 4H), 3.60 (dd, J =

14.1, 9.7 Hz, 1H), 3.34 (dd, J = 14.1, 4.4 Hz, 1H), 2.16 (s, 3H), 1.14−

1.37 (m, 2H), 0.49−0.73 (m, 4H), 0.24−0.48 (m, 4H). [α]_D²⁰= −26.22

(c = 0.5, MeOH).

20

0.49−0.69 (m, 4H), 0.23−0.45 (m, 4H). [α]_D= −37.56 (c = 0.6,

DCM).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(4-methoxy-3-phenoxybenzoyloxy)-

ethyl)pyridine 1-Oxide ((S)-26i). Acid 25i (1.2 equiv), EDC (2 equiv),

DMAP (0.5 equiv), and DCM, rt, 48 h; puriﬁcation by chromatography

on silica gel (DCM/EtOAc 80:20), white solid, 43% yield. LC−MS (ESI

+) m/z 645.9 (MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 8.50 (s, 2H),

7.84 (dd, J = 8.5, 2.1 Hz, 1H), 7.52 (d, J = 2.3 Hz, 1H), 7.33−7.42 (m,

2H), 7.29 (d, J = 8.8 Hz, 1H), 7.16−7.21 (m, 2H), 7.12 (m, J = 7.3, 7.3

Hz, 1H), 7.03 (dd, J = 8.2, 2.1 Hz, 1H), 6.87−6.96 (m, 2H), 7.05 (t, J =

74.8 Hz, 1H), 6.15 (dd, J = 9.7, 4.4 Hz, 1H), 3.90 (d, J = 7.0 Hz, 2H),

3.85 (s, 3H), 3.55 (dd, J = 14.2, 9.5 Hz, 1H), 3.30 (dd, J = 12.9, 4.4 Hz,

1H), 1.09−1.34 (m, 1H), 0.47−0.65 (m, 2H), 0.26−0.43 (m, 2H).

[α]_D²⁰= −36.4 (c = 0.5, DCM).

(S)-4-(2-(3-Acetoxy-4-methoxybenzoyloxy)-2-(3-(cyclopropylme-

thoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)-3,5-dichloropyridine 1-

Oxide ((S)-26j). Acid 25j (1.2 equiv), EDC (2 equiv), DMAP (1.1

equiv), and DCM, rt, overnight; puriﬁcation by crystallization from

MeOH, white solid, 33,7% yield. LC−MS (ESI+) m/z 612.4 (MH⁺). ¹H

NMR (400 MHz, CDCl₃) δ 8.14 (s, 2H), 7.90 (d, J = 1.94 Hz, 1H), 7.71

(d, J = 1.94 Hz, 1H), 7.18 (d, J = 8.26 Hz, 1H), 6.96−7.08 (m, 3H),

6.43−6.62−6.81 (t, 1H, CHF₂), 6.20−6.30 (m, 1H), 3.84−3.96 (m,

5H), 3.60−3.73 (m, 1H), 3.27−3.38 (m, 1H), 2.34 (s, 3H), 1.19−1.36

(m, 1H), 0.66 (d, J = 6.80 Hz, 2H), 0.38 (d, J = 5.83 Hz, 2H).

(S)-4-(2-(3-Acetamido-4-methoxybenzoyloxy)-2-(3-(cyclopropyl-

methoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)-3,5-dichloropyridine 1-

Oxide ((S)-26k). Acid 25k (1.5 equiv), EDC (1.5 equiv), DMAP (1.5

equiv), and DMF, rt, 1 h; puriﬁcation by trituration with petroleum

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(2-(cyclopropylmethoxy)-5-

(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-Oxide ((S)-26p).

Acid 25p (1 equiv), EDC (1 equiv), DMAP (1 equiv), and DCM, rt,

overnight; puriﬁcation by preparative HPLC (method 1), white solid,

1

10% yield. LC−MS (ESI+) m/z 687.1 (MH⁺). H NMR (300 MHz,

DMSO-d₆) δ 9.54 (s, 1H), 8.53 (s, 2H), 7.55 (d, J = 2.6 Hz, 1H), 7.36

(dd, J = 9.1, 2.6 Hz, 1H), 7.20 (d, J = 8.2 Hz, 1H), 7.08−7.17 (m, 3H),

7.06 (t, J = 74.8 Hz, 1H), 6.18 (dd, J = 8.2, 5.3 Hz, 1H), 3.79−3.98 (m,

4H), 3.53 (dd, J = 14.2, 8.7 Hz, 1H), 3.39 (dd, J = 17.6, 7.6 Hz, 1H), 2.92

(s, 3H), 0.99−1.32 (m, 2H), 0.42−0.67 (m, 4H), 0.04−0.42 (m, 4H).

[α]_D²⁰= −9.90 (c = 0.4, MeOH).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-

nitrobenzoyloxy)ethyl)pyridine 1-Oxide ((S)-26q). Acid 25q (1 equiv),

EDC (3 equiv), DMAP (0.5 equiv), and DCM, rt, overnight;

puriﬁcation by ﬂash chromatography on silica gel (DCM/EtOAc

90:10), light yellow solid, 66% yield. LC−MS (ESI+) m/z 639.1 (MH⁺).

¹H NMR (300 MHz, DMSO-d₆) δ 8.55 (s, 2H), 7.97 (d, J = 8.2 Hz, 1H),

7.75 (d, J = 1.5 Hz, 1H), 7.69 (dd, J = 8.2, 1.5 Hz, 1H), 7.25 (d, J = 1.8

Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 7.10 (dd, J = 8.5, 2.1 Hz, 1H), 7.07 (t, J

= 74.8 Hz, 1H), 6.21 (dd, J = 9.4, 4.4 Hz, 1H), 4.06−4.18 (m, 2H),

3.84−4.03 (m, 2H), 3.64 (dd, J = 14.1, 9.7 Hz, 1H), 3.36 (dd, J = 14.1,

4.4 Hz, 1H), 1.08−1.38 (m, 2H), 0.46−0.72 (m, 4H), 0.25−0.46 (m,

4H). [α]_D²⁰= −32.94 (c = 0.49, MeOH).

1

ether, white solid, 92% yield. LC−MS (ESI+) m/z 611.0 (MH⁺). H

NMR (400 MHz, acetone) δ 9.11 (s, 1H), 8.64 (br. s., 1H), 8.24 (s, 2H),

7.77 (dd, J = 8.38, 2.21 Hz, 1H), 7.33 (d, J = 1.76 Hz, 1H), 7.15−7.23

(m, 1H), 7.05−7.14 (m, 2H), 6.89 (t, J = 75.00 Hz, 1H), 6.33 (dd, J =

9.48, 4.63 Hz, 1H), 3.97−4.10 (m, 2H), 3.95 (s, 3H), 3.70 (dd, J = 14.11,

9.70 Hz, 1H), 3.42 (dd, J = 14.11, 4.41 Hz, 1H), 2.13−2.23 (s, 3H),

1.23−1.37 (m, 1H), 0.60 (dd, J = 7.94, 1.76 Hz, 2H), 0.33−0.47 (m,

2H).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(4-methoxy-3-(N-methylsulfamoyl)-

benzoyloxy)ethyl)pyridine 1-Oxide ((S)-26l). Acid 25l (1.2), EDC

(1.3), DMAP (1.4 equiv), TEA (2.5 equiv), and DCM, rt, 48 h;

puriﬁcation by ﬂash chromatography on silica gel (DCM/MeOH 98:2)

followed by preparative HPLC (method 1), evaporation, dissolution in

EtOAc, washing with aqueous NaHCO₃, and evaporation, white solid,

(S)-4-(2-(4-(Benzyloxy)-3-(cyclopropylmethoxy)benzoyloxy)-2-(3-

(cyclopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)-3,5-di-

chloropyridine 1-Oxide ((S)-26r). Acid 25r (1.3 equiv), EDC (1.5

equiv), DMAP (1.2 equiv), and DMF, rt, 2 h, 80% yield, used without

1

52% yield. LC−MS (ESI+) m/z 647.2 (MH⁺). H NMR (300 MHz,

809

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Journal of Medicinal Chemistry

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puriﬁcation. LC−MS (ESI+) m/z 700.5 (MH⁺). ¹H NMR (400 MHz,

acetone) δ 8.26 (s, 2H), 7.63−7.71 (m, 1H), 7.58 (d, J = 2.21 Hz, 1H),

7.52 (d, J = 7.06 Hz, 2H), 7.28−7.46 (m, 4H), 7.11−7.24 (m, 3H),

6.70−6.90−7.10 (t, 1H, CHF₂), 6.26−6.35 (m, 1H), 5.25 (s, 2H), 3.89−

4.06 (m, 4H), 3.66−3.78 (m, 1H), 3.33−3.49 (m, 1H), 1.28 (d, J = 6.62

Hz, 2H), 0.52−0.67 (m, 4H), 0.39 (t, J = 5.07 Hz, 4H).

Synthesis of (S)-4-(2-(4-Carboxy-3-(cyclopropylmethoxy)-

benzoyloxy)-2-(3-(cyclopropylmethoxy)-4-(diﬂuoromethoxy)-

phenyl)ethyl)-3,5-dichloropyridine 1-Oxide ((S)-26w). To a

solution of (S)-26s (40 mg, 0.064 mmol) in acetic acid (2 mL) was

added a solution of NaClO₂(10 mg, 0.088 mmol) in H₂O (0.5 mL)

dropwise, and the resulting mixture was stirred at rt for 3 h and then

partitioned between EtOAc and water. The organic layer was dried over

Na₂SO₄, and the solvent was removed. The crude product was puriﬁed

by crystallization from a mixture of EtOH/petroleum ether to aﬀord

(S)-26w as a white solid (20 mg, 0.031 mmol, yield 50%). LC−MS (ESI

+) m/z 639.4 (MH⁺). ¹H NMR (400 MHz, CDCl₃) δ 8.29 (d, J = 8.22

Hz, 1H), 8.15 (s, 2H), 7.78 (dd, J = 8.07, 1.32 Hz, 1H), 7.60 (d, J = 1.17

Hz, 1H), 7.21 (d, J = 8.22 Hz, 1H), 7.00−7.11 (m, 2H), 6.64 (t, J = 75.00

Hz, 1H), 6.29 (dd, J = 9.98, 4.11 Hz, 1H), 4.13 (d, J = 7.34 Hz, 2H),

3.84−4.01 (m, 2H), 3.63−3.79 (m, 1H), 3.34 (dd, J = 14.38, 4.11 Hz,

1H), 1.33−1.45 (m, 1H), 1.20−1.33 (m, 1H), 0.61−0.84 (m, 4H),

0.31−0.52 (m, 4H).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-

formylbenzoyloxy)ethyl)pyridine 1-Oxide ((S)-26s). Acid 25s (1.2

equiv), EDC (2 equiv), DMAP (1.1 equiv), and DMF, rt, 3 h,, light

yellow solid, 92% yield, used without puriﬁcation. LC−MS (ESI+) m/z

1

622.4 (MH⁺). H NMR (400 MHz, CDCl₃) δ 10.59 (s, 1H), 8.15 (s,

2H), 7.84−7.96 (m, 1H), 7.65−7.73 (m, 1H), 7.55−7.59 (m, 1H),

7.15−7.23 (m, 1H), 6.96−7.12 (m, 2H), 6.41−6.63−6.85 (t, 1H,

CHF₂), 6.20−6.34 (m, 1H), 3.85−4.12 (m, 4H), 3.59−3.76 (m, 1H),

3.26−3.40 (m, 1H), 1.21−1.41 (m, 2H), 0.52−0.77 (m, 4H), 0.29−0.46

(m, 4H).

Synthesis of (S)-4-(2-(4-Amino-3-(cyclopropylmethoxy)-

benzoyloxy)-2-(3-(cyclopropylmethoxy)-4-(diﬂuoromethoxy)-

phenyl)ethyl)-3,5-dichloropyridine 1-Oxide ((S)-26t). A mixture

of (S)-26q (595 mg, 0.931 mmol) and SnCl₂dihydrate (840 mg, 3.72

mmol) in THF (30 mL) was heated at 40 °C for 1 h. Additional SnCl₂

dihydrate (840 mg, 3.72 mmol) was added, and the mixture was heated

for a further 6 h. The solvent was removed under reduced pressure, and

EtOAc and aqueous sat. NaHCO₃were added to the residue. The

inorganic precipitate was removed by ﬁltration through a Celite pad, and

the biphasic ﬁltrate was separated. The organic layer was washed with

brine and dried over Na₂SO₄, the solvent was evaporated under vacuum,

and the crude product was ﬁrst puriﬁed by ﬂash chromatography on

silica gel (DCM/EtOAc 80:20) and then by trituration with EtOH to

aﬀord 200 mg of (S)-26t as a light yellow solid (36% yield). LC−MS

(ESI+) m/z 608.9 (MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 8.54 (s,

2H), 7.40 (dd, J = 8.4, 1.9 Hz, 1H), 7.29 (d, J = 6.2 Hz, 1H), 7.14−7.23

(m, 2H), 7.04 (dd, J = 8.2, 1.8 Hz, 1H), 7.05 (t, J = 74.8 Hz, 1H), 6.65 (d,

J = 8.2 Hz, 1H), 6.14 (dd, J = 9.8, 4.3 Hz, 1H), 5.61 (s, 2H), 3.74−4.04

General Procedure for the Preparation of Intermediates

28a−l. Synthesis of 3-Cyclopropylmethoxy-4-methanesulfonylami-

no-benzoic Acid Methyl Ester (28a). To a solution of 27a (8.86 g, 40.04

mmol) in dry pyridine (80 mL) under a N₂atmosphere was added

methansulfonyl chloride (4.04 mL, 52.19 mmol), and the mixture was

stirred at rt for 18 h. The reaction mixture was evaporated to dryness,

and the crude product was partitioned between 1 N HCl (500 mL) and

DCM (200 mL). The organic layer was dried over Na₂SO₄and

evaporated to yield 28a (11.7 g, 39.08 mmol, yield 98%). No puriﬁcation

was requried. LC−MS (ESI+) m/z 300.0 (MH⁺).

Compounds 28b−l were synthesized following the same procedure

starting from corresponding aniline derivatives 27b−l and suitable

sulfonyl chlorides. In some cases, DCM or chloroform were used as

solvent in the mixture with pyridine.

General Procedure for the Preparation of Intermediates

29a−j. Synthesis of 3-Cyclopropylmethoxy-4-(N-tert-butoxycarbon-

yl-N-methanesulfonyl)-amino-benzoic Acid Methyl Ester (29a). To a

solution of intermediate 28a (3.0 g, 10.0 mmol) in DCM (150 mL) were

added DMAP (1.22 g, 10 mmol) and Boc₂O (2.18 g, 10 mmol), and the

mixture was stirred at rt for 1 h. The reaction mixture was washed with

5% aqueous HCl (2 × 50 mL), the organic layer was dried over Na₂SO₄,

and the solvent was removed under reduced pressure. The residue was

triturated with Et₂O and ﬁltered to aﬀord 29a, which was used in the

next steps without further puriﬁcation (4.0 g, 10.0 mmol, quantitative

yield). LC−MS (ESI+) m/z 400.2 (MH⁺).

(m, 4H), 3.55 (dd, J = 13.9, 9.5 Hz, 1H), 3.30 (dd, J = 13.9, 4.4 Hz, 1H),

20

1.13−1.32 (m, 2H), 0.47−0.69 (m, 4H), 0.15−0.46 (m, 4H). [α]_D

−42.29 (c = 0.5, MeOH).

=

Synthesis of (S)-3,5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-

4-(diﬂuoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-

hydroxybenzoyloxy)ethyl)pyridine 1-Oxide ((S)-26u). To a

solution of (S)-26r (1.093 g, 1.56 mmol) in EtOAc (20 mL) was

added a catalytic amount of 5% Pd on BaSO₄, and the mixture was

hydrogenated in a Parr apparatus at 35 psi for 1 h at rt. The catalyst was

ﬁltered oﬀ, and the ﬁltrate was evaporated to dryness. The crude product

was purifed by crystallization from a mixture of EtOH/petroleum ether

to aﬀord (S)-26u as a white solid (742 mg, 1.22 mmol, 78%). LC−MS

(ESI+) m/z 610.0 (MH⁺). ¹H NMR (400 MHz, acetone) δ 8.40−8.56

(m, 1H), 8.26 (s, 2H), 7.62 (dd, J = 8.32, 1.91 Hz, 1H), 7.49−7.57 (m,

1H), 7.29 (d, J = 1.83 Hz, 1H), 7.13−7.22 (m, 2H), 6.90 (m, 2H), 6.30

(dd, J = 9.77, 4.58 Hz, 1H), 3.90−3.99 (m, 4H), 3.71 (dd, J = 14.19, 9.77

Hz, 1H), 3.41 (dd, J = 14.19, 4.58 Hz, 1H), 1.22−1.35 (m, 2H), 0.47−

0.66 (m, 4H), 0.29−0.46 (m, 4H).

Compounds 29b−j were synthesized following the same procedure

starting from corresponding aniline derivatives 28b−j.

Hydrolysis Method A: General Procedure for the Preparation

of Intermediates 30a, 30c, 30e, and 30g. Synthesis of 3-

Cyclopropylmethoxy-4-(N-tert-butoxycarbonyl-N-methanesulfon-

yl)-amino-benzoic Acid (30a). Compound 29a (4.0 g, 10.0 mmol) was

dissolved in MeOH (100 mL), 1 N NaOH (15 mL, 15 mmol) was

added, and the mixture was stirred at rt for 1 h and heated at 50 °C for a

further 2 h. The reaction mixture was diluted with EtOAc and washed

with 1 N HCl. The organic layer was dried over Na₂SO₄, and the solvent

was removed under reduced pressure to aﬀord 30a (3.5 g, 7.78 mmol,

yield 78%). LC−MS (ESI+) m/z 408.0 (MNa⁺).

Synthesis of (S)-3,5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-

4-(diﬂuoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-

(hydroxymethyl)benzoyloxy)ethyl)pyridine 1-Oxide ((S)-26v).

To a solution of (S)-26s (380 mg, 0.610 mmol) in THF (20 mL) was

added NaBH₄(6.93 mg, 0.183 mmol), and the mixture was stirred at rt

for 24 h. The reaction mixture was diluted with EtOAc and washed twice

with brine; the organic layer was dried over Na₂SO₄and evaporated to

dryness. The crude product was puriﬁed by chromatography on silica gel

(DCM/EtOAc from 8:2 to 1:1) to yield (S)-26v as a white solid (105

mg, 0.168 mmol, 28%). LC−MS (ESI+) m/z 623.9 (MH⁺). ¹H NMR

(300 MHz, DMSO-d₆) δ 8.55 (s, 2H), 7.61 (dd, J = 7.9, 1.5 Hz, 1H),

7.53 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 1.5 Hz, 1H), 7.17−7.25 (m, 2H),

7.08 (dd, J = 8.5, 2.1 Hz, 1H), 7.06 (t, J = 74.8 Hz, 1H), 6.20 (dd, J = 9.5,

4.5 Hz, 1H), 5.18 (t, J = 5.6 Hz, 1H), 4.57 (d, J = 5.9 Hz, 2H), 3.83−4.01

Intermediates 30c, 30e, and 30g were synthesized following the same

procedure starting from corresponding esters 29c, 29e, and 29g.

Hydrolysis Method B: General Procedure for the Preparation

of Intermediates 30b, 30d, 30f, and 30h−j. Synthesis of 3-(N-(tert-

Butoxycarbonyl)methylsulfonamido)-4-methoxybenzoic Acid (30f).

To a solution of intermediate 29f (0.882 g, 2.454 mmol) in THF (15

mL) was added aqueous 1 N LiOH (2.94 mL, 2.94 mmol),and the

reaction was stirred at rt for 24 h. The mixture was acidiﬁed with 1 N

HCl and extracted with EtOAc. The organic phase was washed with

brine and dried over Na₂SO₄, the solvent was removed under reduced

pressure, and the residue was triturated with Et₂O to aﬀord 30f as a white

solid (0.487 g, 1.410 mmol, yield 57.5%). LC−MS (ESI+) m/z 367.9

(MNa⁺).

(m, 4H), 3.61 (dd, J = 14.1, 9.7 Hz, 1H), 3.34 (dd, J = 13.8, 4.4 Hz, 1H),

Intermediates 30b, 30d, and 30h−j were synthesized following the

same procedure starting from corresponding esters 29b, 29d, and 29h−

j.

20

1.05−1.36 (m, 2H), 0.43−0.65 (m, 4H), 0.12−0.46 (m, 4H). [α]_D

−32.4 (c = 0.7, DCM).

=

810

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General Procedure for the Preparation of Intermediates (S)-

31a−j. Synthesis of (S)-4-(2-(4-(N-(tert-Butoxycarbonyl)-

methylsulfonamido)-3-(cyclopropylmethoxy)benzoyloxy)-2-(3-(cy-

clopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)-3,5-dichloro-

pyridine 1-Oxide ((S)-31a). A mixture of alcohol (S)-21 (5.85 g, 13.93

mmol), acid 30a (5.36 g, 13.93 mmol), EDC (8.00 g, 41.78 mmol), and

DMAP (0.850 mg, 6.97 mmol) in DCM (250 mL) under a N₂

atmosphere was stirred at rt for 5 h. The reaction mixture was diluted

with DCM and washed with 1 N HCl and then with 5% NaHCO₃. The

organic layer was dried over Na₂SO₄, and the solvent was removed

under vacuum. The crude product was puriﬁed by ﬂash chromatography

on silica gel (from 100% DCM to DCM/EtOAc 75:25), aﬀording (S)-

31a as a white solid (6.69 g, 8.50 mmol, yield 61%). LC−MS (ESI+) m/

z 787.3 (MH⁺).

7.25 (m, 2H), 6.92−7.13 (m, 2H), 6.63 (t, J = 75.00 Hz, 1H), 6.12−6.39

(m, 1H), 3.91 (d, J = 6.84 Hz, 4H), 3.70 (dd, J = 13.67, 10.25 Hz, 1H),

3.31 (dd, J = 13.92, 4.15 Hz, 1H), 2.55 (m, 1H), 1.12−1.39 (m, 4H),

1.00 (dd, J = 8.06, 2.20 Hz, 2H), 0.56−0.84 (m, 4H), 0.18−0.52 (m,

4H).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(4-methoxy-3-(methylsulfonamido)-

benzoyloxy)ethyl)pyridine 1-Oxide ((S)-32f). From (S)-31f, 4 M HCl

in EtOAc, rt, 2 h; puriﬁcation by crystallization from EtOH, white solid,

72%. LC−MS (ESI+) m/z 647.5 (MH⁺). ¹H NMR (400 MHz, CDCl₃)

δ 8.24 (d, J = 2.05 Hz, 1H), 8.15 (s, 2H), 7.83 (dd, J = 8.66, 1.91 Hz, 1H),

7.17 (d, J = 8.22 Hz, 1H), 7.09 (m, 1H), 7.02 (dd, J = 8.22, 1.76 Hz, 1H),

6.96 (d, J = 8.51 Hz, 1H), 6.83 (m, 1H), 6.44−6.63−6.82 (t, 1 H, CHF₂),

6.27 (dd, J = 10.12, 3.96 Hz, 1H), 3.87−3.99 (m, 5H), 3.69 (dd, J =

14.09, 10.27 Hz, 1H), 3.30 (dd, J = 13.79, 4.11 Hz, 1H), 2.98 (s, 3H),

1.22−1.34 (m, 1H), 0.60−0.70 (m, 2H), 0.39 (q, J = 4.89 Hz, 2H).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(4-methyl-3-(methylsulfonamido)-

benzoyloxy)ethyl)pyridine 1-Oxide ((S)-32g). From (S)-31g, 4 M HCl

in EtOAc, rt, 1 h; puriﬁcation by crystallization from EtOH, white solid,

72%. LC−MS (ESI+) m/z 631.4 (MH⁺). ¹H NMR (200 MHz, DMSO-

d₆) δ 9.25 (s, 1H), 8.53 (s, 2H), 7.91 (d, J = 1.46 Hz, 1H), 7.76 (dd, J =

7.81, 1.46 Hz, 1H), 7.32−7.45 (m, 1H), 7.14−7.26 (m, 2H), 6.91−7.12

(m, 2H), 6.21 (dd, J = 9.52, 4.15 Hz, 1H), 3.92 (d, J = 6.84 Hz, 2H),

Compounds (S)-31b−j were synthesized following the same

procedure starting from corresponding acid derivatives 30b−j and

using DCM or DMF as solvent.

General Procedure for the Preparation of Compounds (S)-

32a−j. Synthesis of (S)-3,5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-

4-(diﬂuoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-

(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-Oxide ((S)-32a).

Compound (S)-31a (4.6 g, 5.85 mmol) was dissolved in DCM (150

mL), and a solution of 4 M HCl in dioxane (40 mL, 160 mmol) was

added dropwise. The reaction was stirred at rt overnight and then

evaporated to dryness. The residue was puriﬁed by crystallization from

EtOH to aﬀord (S)-32a as a white solid (3 g, 4.36 mmol, 75%). LC−MS

(ESI+) m/z 687.3 (MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 9.16 (br s,

1H), 8.55 (s, 2H), 7.58 (dd, J = 8.2, 1.8 Hz, 1H), 7.49 (d, J = 1.8 Hz, 1H),

7.40 (d, J = 8.2 Hz, 1H), 7.16−7.25 (m, 2H), 7.07 (dd, J = 7.9, 1.8 Hz,

1H), 7.06 (t, J = 74.8 Hz, 1H), 6.18 (dd, J = 9.7, 4.4 Hz, 1H), 3.80−4.12

(m, 4H), 3.61 (dd, J = 14.2, 9.8 Hz, 1H), 3.33 (dd, J = 14.1, 3.5 Hz, 1H),

3.11 (s, 3H), 1.25−1.42 (m, 1H), 1.12−1.25 (m, 1H), 0.48−0.73 (m,

4H), 0.21−0.48 (m, 4H). [α]_D²⁰= −47 (c = 0.4, MeOH).

3.47−3.73 (m, 1H), 3.37 (d, J = 4.39 Hz, 1H), 3.00 (s, 3H), 2.38 (s, 3H),

20

1.11−1.28 (m, 1H), 0.48−0.65 (m, 2H), 0.25−0.42 (m, 2H). [α]_D

−38.67 (c = 1, CHCl₃).

=

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-5-

(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-Oxide ((S)-32h).

From (S)-31h, 4 M HCl in dioxane, DCM, rt, 2 h; puriﬁcation by

preparative HPLC (method 2), white solid, 45%. LC−MS (ESI+) m/z

1

687.5 (MH⁺). H NMR (400 MHz, acetone) δ 8.64−8.92 (br s, 1H),

Compounds (S)-32b−j were similarly prepared starting from

corresponding acids (S)-31b−j.

8.27 (s, 2H), 7.59−7.72 (m, 1H), 7.28−7.38 (m, 2H), 7.13−7.25 (m,

3H), 6.92 (t, J = 75.00 Hz, 1H), 6.33 (dd, J = 9.70, 4.41 Hz, 1H), 3.99

(dd, J = 6.84, 1.98 Hz, 2H), 3.91 (d, J = 7.06 Hz, 2H), 3.74 (dd, J = 14.55,

9.70 Hz, 1H), 3.43 (dd, J = 14.11, 4.41 Hz, 1H), 3.05 (s, 3H), 1.16−1.36

(m, 2H), 0.52−0.71 (m, 4H), 0.29−0.47 (m, 4H).

(S)-4-(2-(3-(Benzyloxy)-4-(methylsulfonamido)benzoyloxy)-2-(3-

(cyclopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)-3,5-di-

chloropyridine 1-Oxide ((S)-32b). From (S)-31b, 4 M HCl in EtOAc,

rt, 3 h, not puriﬁed, yellow solid, 71%. LC−MS (ESI+) m/z 723.6

(MH⁺). ¹H NMR (400 MHz, acetone) δ 8.27 (2s, 3H), 7.67−7.78 (m,

2H), 7.51−7.66 (m, 3H), 7.34−7.47 (m, 3H), 7.25−7.32 (m, 1H),

7.12−7.24 (m, 2H), 6.90 (t, J = 75.00 Hz, 1H), 6.24−6.43 (m, 1H),

5.22−5.39 (s, 2H), 3.90−4.07 (m, 2H), 3.65−3.80 (m, 1H), 3.37−3.48

(m, 1H), 3.07 (s, 3H), 1.17−1.35 (m, 1H), 0.51−0.67 (m, 2H), 0.30−

0.44 (m, 2H).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(2-methoxy-5-(methylsulfonamido)-

benzoyloxy)ethyl)pyridine 1-Oxide ((S)-32i). From (S)-31i, 4 M HCl

in dioxane, DCM, rt, 5 days; puriﬁcation by ﬂash chromatography on

silica gel (from 100% EtOAc to EtOAc/MeOH 95:5) followed by

preparative HPLC (method 1), white solid, 27%. LC−MS (ESI+) m/z

647.3 (MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 9.54 (s, 1H), 8.54 (s,

2H), 7.56 (d, J = 2.6 Hz, 1H), 7.40 (dd, J = 8.9, 2.8 Hz, 1H), 7.21 (d, J =

8.2 Hz, 1H), 7.17 (d, J = 2.3 Hz, 1H), 7.15 (d, J = 8.9 Hz, 1H), 7.05 (dd, J

= 8.4, 1.9 Hz, 1H), 7.07 (t, J = 74.8 Hz, 1H), 6.20 (dd, J = 8.9, 4.8 Hz,

1H), 3.94 (dd, J = 10.3, 7.0 Hz, 1H), 3.89 (dd, J = 10.3, 7.0 Hz, 1H), 3.78

(s, 3H), 3.51 (dd, J = 14.1, 9.1 Hz, 1H), 3.31 (dd, J = 14.1, 4.7 Hz, 1H),

2.92 (s, 3H), 1.13−1.35 (m, 1H), 0.49−0.66 (m, 2H), 0.26−0.43 (m,

2H). [α]_D²⁰= −9.90 (c = 0.4, MeOH).

(S)-3, 5-Dichloro-4-(2-(2-(cyclopropylmethoxy)-3-

(methylsulfonamido)benzoyloxy)-2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)ethyl)pyridine 1-Oxide ((S)-32j). From (S)-

31j, 4 M HCl in dioxane, DCM, rt, overnight; puriﬁcation by preparative

HPLC (method 1), white solid, 31%. LC−MS (ESI+) m/z 687.2

(MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ 8.99 (s, 1H), 8.56 (s, 2H),

7.57 (ddd, J = 7.9, 1.5, 0.6 Hz, 1H), 7.50 (dd, J = 7.9, 1.5 Hz, 1H), 7.16−

7.25 (m, 3H), 7.05−7.11 (m, 1H), 7.07 (t, J = 74.8 Hz, 1H), 6.19 (dd, J =

9.1, 4.7 Hz, 1H), 3.93 (d, J = 6.7 Hz, 2H), 3.59 (dd, J = 14.1, 9.7 Hz, 1H),

3.49−3.59 (m, 2H), 3.33 (dd, J = 14.1, 4.7 Hz, 1H), 3.10 (s, 3H), 1.14−

1.27 (m, 1H), 0.95−1.12 (m, 1H), 0.49−0.67 (m, 2H), 0.24−0.46 (m,

4H), 0.01−0.16 (m, 2H). [α]_D²⁰= −39.46 (c = 0.5, MeOH).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3-methyl-4-(methylsulfonamido)-

benzoyloxy)ethyl)pyridine 1-Oxide ((S)-32c). From (S)-31c, 4 M HCl

in EtOAc, rt, 1 h; puriﬁcation by crystallization from chloroform/iPr₂O,

1

white solid, 72%. LC−MS (ESI+) m/z 631.4 (MH⁺). H NMR (200

MHz, DMSO-d₆) δ 8.55 (s, 2H), 7.93 (m, 2H), 7.48 (d, J = 7.81 Hz,

1H), 7.00−7.28 (m, 4H), 6.08−6.27 (m, 1H), 3.93 (d, J = 6.84 Hz, 2H),

3.60 (m, 4H), 3.31−3.41 (m, 1H), 2.28 (s, 3H), 1.21 (m, 1H), 0.43−

0.65 (m, 2H), 0.20−0.39 (m, 2H). [α]_D²⁰= −58 (c = 1, CHCl₃).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(2-methoxy-4-(methylsulfonamido)-

benzoyloxy)ethyl)pyridine 1-Oxide ((S)-32d). From (S)-31d, 4 M HCl

in EtOAc, rt, 2 h; puriﬁcation by preparative HPLC (method 2), white

1

solid, 35% yield. LC−MS (ESI+) m/z 647.5 (MH⁺). H NMR (400

MHz, acetone) δ 8.76−9.30 (m, 1H), 8.25 (s, 2H), 7.83 (d, J = 8.38 Hz,

1H), 7.28 (d, J = 1.76 Hz, 1H), 7.12−7.23 (m, 2H), 7.03−7.10 (m, 1H),

6.94 (dd, J = 8.38, 2.21 Hz, 1H), 6.90 (t, J = 75.00 Hz, 1H), 6.32 (dd, J =

9.04, 5.07 Hz, 1H), 3.93−4.05 (m, 2H), 3.87 (s, 3H), 3.64 (dd, J = 14.11,

9.26 Hz, 1H), 3.35−3.45 (m, 1H), 3.11 (s, 3H), 1.19−1.34 (m, 1H),

0.52−0.67 (m, 2H), 0.32−0.47 (m, 2H).

Synthesis of (S)-3,5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-

4-(diﬂuoromethoxy)phenyl)-2-(2-(cyclopropylmethoxy)-4-

(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-Oxide ((S)-

32k). To a solution of 28k (150 mg, 0.5 mmol) in MeOH (10 mL)

was added 6 N NaOH (0.167 mL, 1.0 mmol),and the reaction was

heated at 80 °C for 16 h. After addition of 6 N NaOH (0.167 mL, 1.0

mmol), the mixture was heated for a further 16 h. MeOH was removed

(S)-3,5-Dichloro-4-(2-(4-(cyclopropanesulfonamido)-3-

(cyclopropylmethoxy)benzoyloxy)-2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)ethyl)pyridine 1-Oxide ((S)-32e). From (S)-

31e, 4 M HCl in EtOAc, rt, 1.5 h; puriﬁcation by crystallization from

EtOH, white solid, 45%. LC−MS (ESI+) m/z 713.6 (MH⁺). ¹H NMR

(200 MHz, CDCl₃) δ 8.15 (s, 2H), 7.66 (m, 2H), 7.45 (m, 1H), 7.14−

811

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Journal of Medicinal Chemistry

Article

under reduced pressure, and the remaining aqueous solution was

acidiﬁed with 2 N HCl and extracted with EtOAc. The organic phase was

dried over Na₂SO₄and evaporated under vacuum. The crude residue

(130 mg) was dissolved in DCM (10 mL), (S)-21 (0.230 g, 0.547

mmol) was added followed by EDC (0.087 g, 0.456 mmol) and DMAP

(0.056 g, 0.456 mmol), and the reaction was stirred at rt overnight. 1 N

HCl was added, and the organic phase was separated, dried over

Na₂SO₄, and evaporated to dryness. The crude product was puriﬁed by

preparative HPLC (method 1), aﬀording (S)-32k as a white solid (0.095

g, 0.138 mmol, 30.3%). LC−MS (ESI+) m/z 687.2 (MH⁺). ¹H NMR

(300 MHz, DMSO-d₆) δ 10.18 (s, 1H), 8.54 (s, 2H), 7.74 (d, J = 8.5 Hz,

1H), 7.19 (d, J = 8.2 Hz, 1H), 7.15 (d, J = 1.5 Hz, 1H), 7.12 (dd, J = 8.2,

1.8 Hz, 1H), 6.87−6.93 (m, 1H), 6.76−6.86 (m, 2H), 7.05 (t, J = 74.8

Hz, 1H), 6.16 (dd, J = 8.5, 5.3 Hz, 1H), 3.74−4.01 (m, 4H), 3.53 (dd, J =

13.8, 8.8 Hz, 1H), 3.10 (s, 3H), 1.11−1.37 (m, 2H), 0.44−0.66 (m, 4H),

0.24−0.44 (m, 4H). [α]_D²⁰= −7.24 (c = 0.5, MeOH).

Synthesis of (S)-3,5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-

4 - ( d i ﬂ u o r o m e t h o x y ) p h e n y l ) - 2 - ( 2 - m e t h o x y - 6 -

(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-Oxide ((S)-

32l). To a solution of KOH (764 mg, 11.57 mmol) in EtOH (5 mL)

was added compound 28l (300 mg, 1.157 mmol), and the reaction was

heated to reﬂux for 8 h. Additional KOH (764 mg, 11.57 mmol) was

added, and the heating was continued for 8 h. The solvent was

evaporated under vacuum, and the residue was dissolved in water and

acidiﬁed with concentrated HCl to pH 2. The precipitate was collected

by ﬁltration, washed with water, and dried to aﬀord 250 mg of acid

intermediate. To a solution of this acid (100 mg, 0.408 mmol), EDC

(196 mg, 1.021 mmol), and DMAP (41.6 mg, 0.340 mmol) in dry DCM

(10 mL) was added (S)-21 (143 mg, 0.340 mmol), and the resulting

solution was stirred at rt for 3 h. The solvent was evaporated, and the

residue was partioned between EtOAc and sat. NaHCO₃. The organic

phase was washed with 1 N HCl and brine, dried over Na₂SO₄, and

evaporated. The residue was puriﬁed by ﬂash chromatography on silica

gel (DCM/MeOH/aq. 33% NH₄OH 95:5:0.5) followed by trituration

with iPr₂O to aﬀord (S)-32l as a white solid (85 mg, 0.131 mmol, 34%).

LC−MS (ESI+) m/z 647.14 (MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ

9.17 (br s, 1H), 8.52 (s, 2H), 7.44 (t, J = 8.2 Hz, 1H), 7.17 (d, J = 8.2 Hz,

1H), 7.13 (d, J = 2.1 Hz, 1H), 7.00−7.07 (m, 2H), 7.00 (d, J = 8.5 Hz,

1H), 7.05 (t, J = 74.8 Hz, 1H), 6.09 (dd, J = 8.2, 6.2 Hz, 1H), 3.92 (dd, J =

10.3, 7.0 Hz, 1H), 3.87 (dd, J = 10.3, 7.0 Hz, 1H), 3.74 (s, 3H), 3.52 (dd,

J = 13.8, 8.5 Hz, 1H), 3.31−3.37 (m, 1H), 2.83 (s, 3H), 1.08−1.38 (m,

1H), 0.49−0.70 (m, 2H), 0.19−0.43 (m, 2H). [α]_D²⁰= −22.96 (c = 0.5,

DCM).

General Procedure for the Preparation of Intermediates

34a−c. Synthesis of 4-(N-(tert-Butoxycarbonyl)-

methylsulfonamido)benzoic Acid (34a). A mixture of compound

33a (445 mg, 1.46 mmol), Boc₂O (350 mg, 1.60 mmol), and DMAP

(196 mg, 1.60 mmol) in DCM (30 mL) was stirred at rt for 2 h. The

reaction mixture was diluted with DCM and washed with 1 N HCl; the

organic phase was dried over Na₂SO₄, and the solvent was removed

under vacuum. The residue was dissolved in MeOH (60 mL), and 10%

Pd/C (60 mg) was added. The mixture was hydrogenated in a Parr

apparatus at 20 psi for 1 h. The catalyst was ﬁltered oﬀ, and the ﬁltrate

was evaporated to dryness, aﬀording 34a (450 mg, 98%), which was

used without puriﬁcation. LC−MS (ESI+) m/z 316.2 (MH⁺).

Compounds 34b−c were synthesized following the same protocol

and starting from corresponding intermediates 33b−c.

Preparation of Intermediates 35a−c. Compounds (S)-35a−c

were synthesized following the protocol described for the preparation of

compound (S)-31a starting from corresponding acids 34a−c.

Preparation of Compounds 36a−c. Compounds (S)-36a−c were

synthesized following the protocol described for the preparation of

compound (S)-32a starting from corresponding Boc-protected

intermediates (S)-35a−c.

1.8 Hz, 1H), 7.20 (d, J = 8.2 Hz, 1H), 7.07 (dd, J = 8.2, 1.8 Hz, 1H), 7.06

(t, J = 74.8 Hz, 1H), 6.19 (dd, J = 9.4, 4.4 Hz, 1H), 3.93 (d, J = 7.0 Hz,

2H), 3.60 (dd, J = 14.2, 9.5 Hz, 1H), 3.33 (dd, J = 14.4, 4.7 Hz, 1H), 3.09

(s, 3H), 1.09−1.38 (m, 1H), 0.45−0.71 (m, 2H), 0.21−0.45 (m, 2H).

[α]_D²⁰= −45.06 (c = 0.5, MeOH).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3-methoxy-4-(methylsulfonamido)-

benzoyloxy)ethyl)pyridine 1-Oxide ((S)-36b). From (S)-35b, 4 M HCl

in EtOAc, rt, 3 h; puriﬁcation by crystallization from EtOAc, white solid,

57%. LC−MS (ESI+) m/z 647.5 (MH⁺). ¹H NMR (400 MHz, CDCl₃)

δ 7.83 (s, 2H), 7.33−7.41 (m, 1H), 7.21−7.26 (m, 1H), 7.17 (m, 1H),

6.81−6.89 (m, 1H), 6.76−6.80 (m, 1H), 6.70 (m, 2H), 6.29 (t, J = 75.00

Hz, 1H), 5.87−5.99 (m, 1H), 3.48−3.71 (m, 5H), 3.24−3.43 (m, 1H),

2.90−3.02 (m, 1H), 0.81−1.03 (m, 1H), 0.23−0.44 (m, 2H), −0.16−

0.18 (m, 2H).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂ uoromethoxy)phenyl)-2-(3-(hydroxymethyl)-4-

(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-Oxide ((S)-36c).

From (S)-35c, 4 M HCl in dioxane, DCM, rt, overnight; puriﬁcation

by ﬂash chromatography on silica gel (DCM/MeOH 98:2), white solid,

78%. LC−MS (ESI+) m/z 646.9 (MH⁺). ¹H NMR (300 MHz, DMSO-

d₆) δ 9.25 (s, 1H), 8.54 (s, 2H), 8.04 (d, J = 2.1 Hz, 1H), 7.89 (dd, J = 8.5,

2.1 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.17−7.23 (m, 2H), 7.03−7.10 (m,

1H), 7.06 (t, J = 74.8 Hz, 1H), 6.21 (dd, J = 9.2, 4.3 Hz, 1H), 5.52 (s,

1H), 4.63 (s, 2H), 3.93 (d, J = 7.0 Hz, 2H), 3.61 (dd, J = 14.1, 9.4 Hz,

1H), 3.35 (dd, J = 14.4, 4.7 Hz, 1H), 3.10 (s, 3H), 1.13−1.37 (m, 1H),

20

0.48−0.72 (m, 2H), 0.24−0.43 (m, 2H). [α]_D= −40.42 (c = 0.5,

MeOH).

General Protocol for the Preparation of Compounds (S)-38a−

e by Sulfonylation of the Corresponding Aniline Intermediates.

Synthesis of (S)-3,5-Dichloro-4-(2-(4-(cyclopropylmethoxy)-3-

(methylsulfonamido)benzoyloxy)-2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)ethyl) Pyridine 1-Oxide ((S)-38a). Com-

pound (S)-37a (40 mg, 0.06 mmmol) was dissolved in CHCl₃(2 mL),

and the solution was cooled to 0 °C. Methanesulfonyl chloride (8 mg,

0.07 mmol) and dry pyridine (0.5 mL) were added, and the reaction was

stirred at 0−10 °C for 4 h. Aqueous 1 M HCl (2 mL) was added, and the

mixture was extracted with CHCl₃. The organic layer was dried over

Na₂SO₄and evaporated under vacuum. The residue was puriﬁed by

crystallization from EtOH to aﬀord (S)-38a as a white solid (31 mg,

0.045 mmol, 68%). LC−MS (ESI+) m/z 687.2 (MH⁺). ¹H NMR (400

MHz, acetone) δ 8.12−8.37 (m, 3H), 7.95 (s, 1H), 7.87 (dd, J = 8.60,

1.98 Hz, 1H), 7.31 (d, J = 1.76 Hz, 1H), 6.68−7.25 (m, 4H), 6.34−6.30

(m, 1H), 3.92−4.16 (m, 4H), 3.68−3.78 (m, 1H), 3.44 (d, J = 4.41 Hz,

1H), 3.00−3.05 (s, 3H), 1.17−1.46 (m, 2H), 0.61 (dd, J = 12.57, 7.72

Hz, 4H), 0.40 (t, J = 4.63 Hz, 4H).

Compounds (S)-38b−e were synthesized in a similar way starting

from corresponding aniline intermediates (S)-37b−e.

(S)-4-(2-(4-(Benzyloxy)-3-(methylsulfonamido)benzoyloxy)-2-(3-

(cyclopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)-3,5-di-

chloropyridine 1-Oxide ((S)-38b). From (S)-37b, CHCl₃, rt, 2 h;

puriﬁcation by preparative HPLC (method 2), white solid, 89%. LC−

MS (ESI+) m/z 723.6 (MH⁺). ¹H NMR (400 MHz, acetone) δ 8.19−

8.31 (m, 3H), 8.08 (s, 1H), 7.87 (dd, J = 8.60, 1.98 Hz, 1H), 7.55 (d, J =

7.06 Hz, 2H), 7.12−7.47 (m, 7H), 6.67−6.91−7.11 (t, 1 H, CHF₂), 6.33

(dd, J = 9.26, 4.41 Hz, 1H), 5.23−5.39 (m, 2H), 3.99 (dd, J = 7.06, 3.97

Hz, 2H), 3.72 (dd, J = 14.11, 9.70 Hz, 1H), 3.42 (dd, J = 14.11, 4.41 Hz,

1H), 2.99 (s, 3H), 1.27 (br s, 1H), 0.60 (dd, J = 7.94, 1.32 Hz, 2H),

0.32−0.45 (m, 2H).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(3-methoxy-5-(methylsulfonamido)-

benzoyloxy)ethyl)pyridine 1-Oxide ((S)-38c). From (S)-37c, CHCl₃, rt,

2 h; puriﬁcation by preparative HPLC (method 2), white solid, 66%.

1

LC−MS (ESI+) m/z 646.9 (MH⁺). H NMR (400 MHz, acetone) δ

8.69−8.86 (m, 1H), 8.28 (s, 2H), 7.68 (m, 1H), 7.29−7.40 (m, 2H),

7.18−7.25 (m, 1H), 7.16 (dd, J = 5.29, 2.21 Hz, 2H), 6.92 (t, J = 75.00

Hz, 1H), 6.29−6.36 (m, 1H), 3.99 (dd, J = 7.06, 2.21 Hz, 2H), 3.87 (s,

3H), 3.69−3.79 (m, 1H), 3.39−3.47 (m, 1H), 3.05 (s, 3H), 1.21−1.34

(m, 1H), 0.60 (dd, J = 8.16, 1.54 Hz, 2H), 0.38 (d, J = 5.73 Hz, 2H).

[α]_D²⁰= −59.33 (c = 1, CHCl₃).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(4-(methylsulfonamido)benzoyloxy)-

ethyl)pyridine 1-Oxide ((S)-36a). From (S)-35a, 4 M HCl in dioxane,

DCM, rt, 3 days; puriﬁcation by trituration with EtOH, white solid, 70%.

LC−MS (ESI+) m/z 617.0 (MH⁺). ¹H NMR (300 MHz, DMSO-d₆) δ

10.35 (br s, 1H), 8.55 (s, 2H), 7.94 (m, 2H), 7.27 (m, 2H), 7.20 (d, J =

812

dx.doi.org/10.1021/jm401549m | J. Med. Chem. 2014, 57, 793−816

Journal of Medicinal Chemistry

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(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(4-methoxy-2-(methylsulfonamido)-

benzoyloxy)ethyl)pyridine 1-Oxide ((S)-38d). From (S)-37d, DCM, rt,

2 h; puriﬁcation by preparative HPLC (method 2), beige solid, 35%.

Hz, 1H), 3.42 (dd, J = 14.11, 4.41 Hz, 1H), 1.27 (br s, 1H), 0.53−0.67

(m, 2H), 0.34−0.46 (m, 2H). [α]_D²⁰= −41.04 (c = 1, CHCl₃).

Biology and ADME. In Vitro Determination of PDE4 Inhibitory

Activity in the Cell-Free Assay. PDE4 activity was determined in U937

human monocytic supernatants cells lysate (ECACC, UK). Cells were

cultured and harvested, and the supernatant fraction was prepared

essentially as described by Torphy.³¹All of the materials used for cell

culture were from Gibco (Monza, Italy). U937 cells were grown at 37 °C

and 5% CO₂in RPMI-1640 with GlutaMAX-I medium supplemented

with 10% fetal bovine serum and 100 μg/mL of pen/strep. Cells were

harvested and washed twice by centrifugation (150g, 8 min) in cold PBS.

Washed cells were resuspended in cold Krebs−Ringer−Henseleit buﬀer

at a ﬁnal concentration 20 × 10⁶cells/mL and sonicated. After

centrifugation at 15000g for 20 min, the supernatants were pooled,

divided into aliquots, and stored at −80 °C. PDE4 activity was

determined in cells supernatants by assaying cAMP disappearance from

the incubation mixtures. The concentration of the test compounds

ranged between 10⁻¹²and 10⁻⁶M. Reactions were stopped by enzyme

heat inactivation (2.5 min at 100 °C), and residual cAMP content was

determined using the LANCE cAMP Detection kit from PerkinElmer

(Milan, Italy) following the manufacturer’s instructions. IC₅₀values

were determined from concentration−response curves by nonlinear

regression analysis using the Solver tool in the Excel program

(Microsoft, Milan, Italy) and a four-parameters logistic equation.

In Vitro Determination of PDE4 Inhibitory Activity in the PBMCs

Assay. The assay was based on the known inhibitory activity exerted by

PDE4 inhibitors on LPS-induced TNF-α release.³²PDE4 inhibitors

were solubilized in DMSO at 5 mM (by sonication for 30 min and

incubation for 30 min at 37 °C), diluted in RPMI culture medium, and

added to wells at a range of concentrations (ﬁnal DMSO concentration

= 0.2%). Human peripheral blood mononuclear cells (PBMCs) were

purchased from Lonza (Basel, CH). PBMCs were washed, resuspended

in RPMI 1640 medium (w/o phenol red) supplemented with 10% FBS,

2 mM glutamine, 100 U penicillin, and 100 μg/mL streptomycin

(Invitrogen), plated in 96-well tissue culture plates at a density of 10⁵

cells/well, and grown in an atmosphere of 95% air and 5% CO₂at 37 °C.

Cells were treated with diﬀerent concentrations of PDE4 inhibitors

(10⁻¹⁵to 10⁻⁶M, ﬁnal DMSO concentration = 0.2%), stimulated with

lipolysaccharide (LPS) from Escherichia coli at a ﬁnal concentration of 3

ng/mL, and incubated for 18 h in RPMI (w/o phenol red)

supplemented with 10% FBS. Human TNFα in the supernatant was

assayed using a paired antibody quantitative ELISA kit (Bender

MedSystems GmbH, Vienna, Austria). IC₅₀values were determined

from concentration−response curves by nonlinear regression analysis

using GraphPad Prism v.6 (GraphPad Software, La Jolla, CA, USA) and

a four-parameter logistic equation.

Rat and Human Lung S9 Stability. Test compounds were incubated

in duplicate at a concentration of 1 μM with lung S9 fraction (0.5 mg

protein/mL) in Dulbecco’s buﬀer (pH 7.4) at 37 °C in the presence of 1

mM NADPH. At diﬀerent time points (0, 15, 30, and 60 min), 50 μL

aliquots were taken, added to 80 μL of ice-cold acetonitrile and 20 μL of

1 μM warfarin in acetonitrile (injection check) to stop the reaction, and

centrifuged. The supernatants were analyzed by LC−MS/MS for

unchanged compound.

Test compounds were incubated with lung S9 fraction in Dulbecco’s

buﬀer in the absence of NADPH and in Dulbecco’s buﬀer alone for 0

and 60 min as controls. 7-Ethoxyresoruﬁn at a concentration of 1 μM

was incubated with the S9 fraction as a positive control for phase I

activity. Control samples were processed the same as the test-compound

samples.

Plasma Protein Binding. Five micromolar solutions of standards and

compounds were prepared in plasma from a 500 μM stock solution.

Plasma solutions (200 μL) were equilibrated with 200 μL of phosphate

buﬀer (pH 7.4) for 5 h at 37 °C under slow rotation in a Dianorm

equilibrium dialysis apparatus. After the incubation period, the solutions

were taken from the cells and extracted with ACN/MeOH (with

Verapamil 100 nM as IS). Forty microliters of dialyzed plasma was

added to 40 μL of blank PBS and extracted with 320 μL of the solvent

solution. Solutions were then vortexed and centrifuged at 3000 rpm for

25 min, and the supernatants were analyzed by LC−MS/MS.

1

LC−MS (ESI+) m/z 647.5 (MH⁺). H NMR (400 MHz, acetone) δ

10.23−10.41 (m, 1H), 8.18−8.37 (m, 3H), 7.11−7.39 (m, 4H), 6.92 (t, J

= 75.00 Hz, 1H), 6.67−6.84 (m, 1H), 6.19−6.42 (m, 1H), 3.85−4.09

(m, 5H), 3.68−3.83 (m, 1H), 3.37−3.53 (m, 1H), 3.12 (s, 3H), 1.20−

1.37 (m, 1H), 0.60 (d, J = 7.94 Hz, 2H), 0.38 (d, J = 4.41 Hz, 2H).

(S)-3, 5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-4-

(diﬂuoromethoxy)phenyl)-2-(5-methoxy-2-(methylsulfonamido)-

benzoyloxy)ethyl)pyridine 1-Oxide ((S)-38e). From (S)-37e, DCM, rt,

2 h; puriﬁcation by crystallization from EtOH, beige solid, 70% yield.

1

LC−MS (ESI+) m/z 647.2 (MH⁺). H NMR (400 MHz, acetone) δ

9.62 (br s, 1H), 8.29 (s, 2H), 7.77 (d, J = 3.09 Hz, 1H), 7.60 (d, J = 8.82

Hz, 1H), 7.36 (d, J = 1.32 Hz, 1H), 7.16−7.31 (m, 3H), 6.92 (t, J = 75.00

Hz, 1H), 6.36 (dd, J = 9.70, 4.85 Hz, 1H), 4.00 (dd, J = 6.84, 1.10 Hz,

2H), 3.91 (s, 3H), 3.81 (dd, J = 14.33, 9.92 Hz, 2H), 3.47 (dd, J = 14.11,

4.41 Hz, 2H), 2.94 (s, 3H), 1.21−1.39 (m, 1H), 0.52−0.66 (m, 2H),

0.52−0.66 (m, 2H).

Synthesis of (S)-1-tert-Butyl 2-(5-formyl-2-methoxyphenyl)-

pyrrolidine-1,2-dicarboxylate (40). To a solution of 39 (2.1 g, 14

mmol) in DMF (20 mL) were added N-Boc-^L-proline (3 g, 14 mmol),

EDC (3 g, 15 mmol), and DMAP (2 g, 16 mmol). The mixture was

stirred at rt for 24 h was then partitioned between an aqueous saturated

solution of K₂CO₃and iPr₂O. The organic layer was dried over Na₂SO₄,

and the solvent was removed under vacuum to give 40 as an oil (4.8 g,

98%), which was used without puriﬁcation. LC−MS (ESI+) m/z 350.2

(MH⁺).

Synthesis of (S)-3-(1-(tert-Butoxycarbonyl)pyrrolidine-2-car-

bonyloxy)-4-methoxybenzoic Acid (41). Compound 40 (4.8 g,

13.7 mmol) was dissolved in acetic acid (50 mL), and sulfamic acid (1.7

g, 17 mmol) was added. A solution of sodium chlorite (3 g, 33 mmol) in

water (20 mL) was added dropwise, and the reaction was stirred at rt for

30 min. The mixture was partitioned between water and EtOAc. The

organic layer was dried over Na₂SO₄and evaporated under vacuum. The

residue was dissolved in CHCl₃and precipitated with hexane. The solid

was ﬁltered and triturated in iPr₂O/hexane 1:1 to aﬀord 41 as a white

solid (3.8 g, 10.4 mmol, 76%). LC−MS (ESI+) m/z 366.2 (MH⁺).

Synthesis of 4-((S)-2-(3-((S)-1-(tert-Butoxycarbonyl)-

pyrrolidine-2-carbonyloxy)-4-methoxybenzoyloxy)-2-(3-(cy-

clopropylmethoxy)-4-(diﬂuoromethoxy)phenyl)ethyl)-3,5-di-

chloropyridine 1-Oxide ((S)-42). A mixture of alcohol (S)-21 (0.3 g,

0.71 mmol), acid 41 (0.3 g, 0.82 mmol), DMAP (0.15 g, 1.2 mmmol),

and EDC (0.3 g, 1.5 mmol) in DMF (2.5 mL) was stirred at rt overnight.

1 M HCl was added, and the mixture was extracted with EtOAc. The

organic layer was washed with 0.5 M K₂CO₃, dried over Na₂SO₄, and

evaporated to dryness to obtain crude (S)-42, which was used in the next

step without puriﬁcation (520 mg, 0.677 mmol, 95%). LC−MS (ESI+)

m/z 767.4 (MH⁺).

Synthesis of 3,5-Dichloro-4-((S)-2-(3-(cyclopropylmethoxy)-

4-(diﬂuoromethoxy)phenyl)-2-(4-methoxy-3-((S)-pyrrolidine-

2-carbonyloxy)benzoyloxy)ethyl)pyridine 1-Oxide Hydrochlor-

ide ((S)-43). Crude (S)-42 (0.5 g, 0.6 mmol) was suspended in 4 M HCl

in EtOAc (2 mL) and stirred at rt for 2 h. The solvent was removed

under reduced pressure, and the residue was puriﬁed by crystallization

from hexane/EtOAc 2:1 to yield (S)-43 as a white solid (380 mg, 0.57

mmol, 95%). LC−MS (ESI+) m/z 703.4 (MH⁺).

Synthesis of (S)-3,5-Dichloro-4-(2-(3-(cyclopropylmethoxy)-

4 - ( d i ﬂ u o r o m e t h o x y ) p h e n y l ) - 2 - ( 3 - h y d r o x y - 4 -

methoxybenzoyloxy)ethyl)pyridine 1-Oxide ((S)-44). To a

solution of (S)-43 (0.12 g, 0.18 mmol) in MeOH was added a saturated

solution of aqueous NaHCO₃, and the reaction mixture was stirred at rt

for 2 h. 1 M HCl was added, and the mixture was extracted with EtOAc.

The organic layer was dried over Na₂SO₄, and the solvent was

evaporated under vacuum. The crude was puriﬁed by crystallization

from EtOH/hexane 1:1 to yield (S)-44 as a white solid (90 mg, 0.16

mmol, 88%). LC−MS (ESI+) m/z 570.1 (MH⁺). ¹H NMR (400 MHz,

acetone) δ 8.25 (s, 2H), 7.94 (s, 1H), 7.47−7.66 (m, 2H), 7.12−7.36

(m, 3H), 7.09−6.90−6.71 (t, 1H, CHF₂), 7.03 (d, J = 8.38 Hz, 1H), 6.31

(dd, J = 9.48, 4.63 Hz, 1H), 3.89−4.01 (m, 5H), 3.71 (dd, J = 13.89, 9.48

813

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Caco-2 Membrane Permeability Assay. Caco-2 cells (ECACC)

were cultured in DMEM, 10% FCS, 1% NEAA, 10 mM Hepes buﬀer, 50

U/mL penicillin, and 50 μg/mL streptomycin. For transport studies,

200 000 cells/well were seeded on Millicell 24-well cell culture plates.

After 24 h of incubation at 37 °C and 5% CO₂, the medium was changed

with Enterocyte Diﬀerentiation Medium (Becton Dickinson), which

allows Caco-2 cells to establish a diﬀerentiated enterocyte monolayer

within 3 days. The transport across the Caco-2 monolayer was

determined by adding a 10 μM solution of compound in DMEM (1%

ﬁnal concentration of DMSO) to the apical side; after 2 h of incubation

at 37 °C, the basolateral side, the apical side, and the starting solutions

were analyzed and quantiﬁed by LC−MS/MS. The experiment was

performed using buﬀers at diﬀerent pH (6.5 apical vs 7.4 basolateral) to

mimic physiological conditions better. The P_app, expressed in nanome-

ters per second, was calculated as follows: P_app= J/C_o, where J = ﬂux

(dX/dt per A) and C_o= donor concentration (μM) at t = 0; dX/dt =

change in mass (X, nmol) per time (t, sec) and A = ﬁlter surface area

(cm²). The rank order of apparent permeability of the test compound

was compared with that of known reference compounds tested in the

same experiment, including caﬀeine (as a probe for transcellular

transport) and cimetidine (as a P-gp substrate). The general absorption

classiﬁcation for P_appvalues in Caco-2 assay were <10 nm/s, low level;

10−50 nm/s, medium level; and >50 nm/s, high level.

bicarbonate. Testosterone was incubated under the same conditions to

assess the metabolic viability of the fresh slices (n = 2). The

concentration of each test compound was assessed by LC−MS/MS

after an incubation period of 0, 20, 40, 60, 90, and 120 min.

Calu-3 Membrane Permeability. Calu-3 cells were maintained at 37

°C, 5% CO₂, and 90−98% relative humidity in 75 cm²culture ﬂasks with

MEM Medium. The cells were passaged once a week using a trypsin/

EDTA solution. About 2.5 × 10⁶cells were seeded per ﬂask. The cells at

a grown density of 1 × 10⁵Calu-3 cells per 1 cm²on transwell ﬁlters

(polyester, with a permeation area of 1.12 cm²and pore diameter of 0.4

μm²).

For the transport experiments, Calu-3 cells were seeded on Transwell

ﬁlter inserts, which were placed into 12-well ﬂat-bottom culture plates.

The inserts (apical compartments) were supplied with 0.5 mL and the

outer wells (basal compartments) with 1.5 mL of MEM culture medium.

The cells were cultured at 37 °C, 5% CO₂, and 90−98% relative

humidity in MEM culture medium for 3 days. The cells were cultured for

a further 11 to 27 days until they formed conﬂuent monolayers. The

transport across the monolayer was determined by adding three

diﬀerent concentrations (1.5, 3.5, and 7 μM) to the apical side. During

incubation, samples was taken from the receiver compartment at ﬁve

time points, 0, 30, 60, 90, and 120 min, and the test compounds in the

samples were quantiﬁed by LC−MS/MS.

Plasma Stability. Test compounds were dissolved in DMSO at a

concentration of 5 mM and then added to plasma to reach a ﬁnal

concentration of 5 μM. Test compounds were incubated for 120 min at

37 °C. At t = 0 and after 15, 30, 60, 90, and 120 min incubation, sample

aliquots were taken, treated with a 2-fold volume of chilled acetonitrile,

and centrifuged. The supernatants were analyzed by LC−MS/MS.

In Vivo Pharmacology. ED₅₀Determination of Inhibition of

Ovalbumin-Induced Lung Eosinophilia in Guinea Pigs. Male

Dunkin−Hartley guinea pigs were actively sensitized with ovalbumin

(OVA, grade III; Sigma). On days 1 and 11, 20 μg of Ova in 5%

aluminum hydroxide adjuvant suspended in 0.5 mL of saline was

injected intraperitoneally. On day 20 after the ﬁrst sensitization, animals

were challenged with a 1 h aerosol of OVA (10 μg mL⁻¹) dissolved in

saline to provoke an inﬂux of eosinophils into the airways. The sham

control group was exposed to an aerosol of saline. The dose−response

curve and the duration of action of compound 32a were investigated by

administering the drug 2 and 24 h before antigen challenge, respectively.

Sham animals and control Ova group received lactose as vehicle. For

each time point, eight drug- and vehicle-treated animals were used.

Intratracheal administration of compound 32a was performed by

placing the micronized drug blended with lactose into a dry powder

insuﬄator (Penn Century, Philadelphia, PA, USA). The tip of the

insuﬄator was then inserted into the lower part of the trachea of animals

under isoﬂurane anesthesia (4% in O₂), and the powder was blown into

the airways during the inspiration phase with 4 mL of air. The control

groups received a corresponding amount of lactose (10 mg/kg body wt).

Bronchoalveolar lavage (BAL) collection was carried out by instilling 5

mL of saline into the lungs via a tracheal cannula in terminally

anaesthetised animals. Total cell counts of the BAL ﬂuid samples were

performed using a Neubaur hemocytometer. Cytospin smears of BAL

samples were prepared by centrifugation at 200 rpm for 5 min at room

temperate and stained using a DiﬀQuik stain system (Dade Behring).

Diﬀerential cell count was performed using oil-immersion microscopy.

A dose-response curve was constructed for the inhibition of Ova-

induced lung eosinophilia at 2 h predosing, and half-maximal eﬀect

doses (ED₅₀) were estimated from the ﬁtted curve.

PDEs Enzyme Assays. PDE1 was puriﬁed from bovine brain.³³PDE2,

PDE3, and PDE5 were puriﬁed from human platelets.³⁴PDE6 was

puriﬁed from bovine retina.³⁵The radiometric assay method is a

modiﬁcation of the two-step method of Thompson and Appleman.³⁶

Compound 32a was tested atﬁve concentrations (1% ﬁnal DMSO

concentration) in duplicate with a starting concentration of 30 μM and a

1:10 serial dilution against human PDEs.

LogD_7.4Measurement. LogD_7.4values were measured in the n-

octanol/buﬀer partition system at room temperature (25 3 °C) via the

shake-ﬂask method.³⁷Test compounds, after equilibrating overnight

between presaturated partition phases, were analyzed in each phase via

LC−UV after dilution with CH₃OH. The LogD_7.4values are the means

of at least three independent partition experiments employing diﬀerent

n-octanol/buﬀer volume ratios. The chosen buﬀer was 50 mM MOPS

(3-morpholinopropanesulfonic acid), pH 7.4, adjusted to 0.15 M ionic

strength with KCl. LC−UV elution conditions were optimized for each

analyte. The column used was a Phenomenex Kinetex C18 100 Å 50 ×

2.1 mm, 5 μm particle size. Solvent A was water (0.1% formic acid).

Solvent B was acetonitrile (0.1% formic acid). Flow rate was 0.8 mL/

min. The standard deviations of the LogD_7.4values were all ≤ 0.04.

Binding Kinetics. Recombinant human PDE4BCat and PDE4DCat

enzymes were prepared from baculovirus-infected Sf9 cells. The assay

buﬀer for K_onand K_offexperiments was 30 mM Tris, pH 7.4, 50 mM

NaCl, 5 mM MgCl2, 0.01% Tween 20, and 0.01% BSA. K_onexperiments

were conducted by mixing PDEs or uninfected protein with 100 nM

[¹⁴C]32a for 0, 2.5, 5, 10, 20, 30, 45, 60, and 90 min at room

temperature. After the incubation, the reactions were placed into a

centrifugal concentrator (Vivaspin 500, 10 kDa cut oﬀ), centrifuged for

30 min at 15 000g, and subsequently washed three times with 500 μL of

assay buﬀer. The retained proteins were mixed with scintillation ﬂuid

and counted. K_offexperiments were conducted by mixing PDE proteins

with 1 μM [¹⁴C]32a for 30 min at room temperature. The reactions

were then centrifuged in Vivaspins for 30 min at 15 000g and washed ﬁve

times with assay buﬀer. Aliquots of the retained protein and ﬁltrates were

mixed with scintillation ﬂuid and counted. The [¹⁴C]32a -bound

proteins were divided into appropriate aliquots and mixed with 32a (50

μM) for 0, 5, 10, 15, 20, 30, 45, 60, and 90 min and 20 h at room

temperature. After incubation, the reactions were centrifuged in

Vivaspins, washed, and counted as described for the K_onexperiments.

Rat and Human Lung Slices Stability. Fresh lung slices from both

species were preincubated for 1 h in preincubation medium (DMEM/F-

12, 10% fetal calf serum, penicillin/streptomycin, ITS, and glutamine) at

37 °C in a O₂/CO₂(95%/5%) humidiﬁed atmosphere; at the end of the

1 h preincubation period, the test compounds were incubated for 2 h

with liver slices and lung slices of both species (three replicates with

human slices and four replicates with rat slices) at 1 μM in MEM-

EARLE medium without phenol red and ^L-glutamine but with sodium

Molecular Modeling. PDB crystal structures were prepared with

the Protein Preparation Wizard in Maestro.³⁸The binding poses of

compounds (R)-18, (S)-18, and (S)-32a in the PDE4B catalytic site

(PDB ID: 1XMU) were obtained by applying the following docking

procedure. For each ligand, ﬁve preliminary poses were generated in

Glide³⁹with the standard precision (SP) mode using the formation of

hydrogen bonds with key residue Gln443 in the hydrophobic region as a

constraint, the poses were reﬁned with the accurate extra-precision (XP)

method, and the docking pose with the best XP GScore value was

selected for each ligand.

814

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Article

ASSOCIATED CONTENT

REFERENCES

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S

* Supporting Information

XRD single-crystal analysis of compound (R*,S**)-22; chemical

correlation details depicted in Scheme 5; synthesis and

characterization of intermediates 25i, 25o−s, 27h, 27j, 27k,

33a−c, and (S)-37a−e; and characterization of intermediates

28b−l, 29b−j, 30b−e, 30g−j, 31b−l, 34b−c, and 35a−c. This

material is available free of charge via the Internet at http://pubs.

acs.org.

AUTHOR INFORMATION

■

Corresponding Author

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*E-mail: e.armani@chiesi.com; Tel.: +39 0521 279477; Fax: +39

0521 279880.

Present Addresses

^§Aptuit, via A. Fleming, 4, 37135 Verona, Italy.

^∥Sicor Srl, via Terrazzano 77, 20017 Rho, Milan, Italy.

^⊥Sintetica S.A, via Penate, 5, 6850 Mendrisio, Switzerland.

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Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

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We thank Professors Marco Mor and Silvia Rivara for their

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ABBREVIATIONS USED

■

ACN, acetonitrile; AcOH, acetic acid; ADME, absorption,

distribution, metabolism, and excretion; AMP, adenosine

monophosphate; BAL, bronchoalveolar lavage; BOC, tert-

butoxycarbonyl; Boc₂O, di-tert-butyl dicarbonate; BSA, bovine

serum albumin; BuLi, butyl lithium; cAMP, cyclic adenosine

monophosphate; cat, catalytic domain; compd, compound;

COPD, chronic obstructive pulmonary disease; DCM, dichloro-

methane; DIAD, diisopropyl azodicarboxylate; DMAP, 4-

dimethylaminopyridine; DMEM, Dulbecco’s modiﬁed Eagle’s

medium; DMF, N,N-dimethylformamide; DMSO, dimethyl

sulfoxide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-

carbodiimide); EDTA, ethylendiaminetetraacetic acid; ESI,

electrospray ionization; Et₂O, diethyl ether; EtOAc, ethyl

acetate; EtOH, ethyl alcohol; FBS, fetal bovine serum; FCS,

fetal calf serum; GP, guinea pig; HPLC, high-performance liquid

chromatography; iPr₂NH, diisopropylamine; iPrO₂, diisopropyl

ether; iPrOH, isopropyl alcohol; ITS, insulin, transferrin,

selenium; LARBS, low-aﬃnity rolipram binding site; LC−MS,

liquid chromatography−mass spectrometry; LDA, lithium

diisopropylamide; LPS, lipopolysaccharides; mCPBA, meta-

chloroperoxybenzoic acid; MeI, iodomethane; MEM, minimum

essential medium; MEM-EARLE, minimum essential medium

with Earle’s salts; MeOH, methyl alcohol; MeSO₂Cl, meth-

ansulphonyl chloride; NADPH, nicotinamide adenine dinucleo-

tide phosphate reduced form; NEAA, nonesssential amino acids;

NMR, nuclear magnetic resonance; OVA, ovalbumin; PBMC,

peripheral blood mononuclear cell; PBS, phosphate buﬀered

saline; PDE, phosphodiesterase; PDE4, phosphodiesterase-4;

Pgp, P-glycoprotein; PPB, plasma protein binding; PPh₃,

triphenylphosphine; RPMI, Roswell Park Memorial Institute;

t_R, retention time; S₉, lung S₉fraction; tBuOK, potassium tert-

butoxide; TEA, triethylamine; THF, tetrahydrofuran; TLC, thin-

layer chromatography; TNF-α, tumor necrosis factor alpha;

XRD, X-ray diﬀraction

815

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dx.doi.org/10.1021/jm401549m | J. Med. Chem. 2014, 57, 793−816

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Novel class of benzoic acid ester derivatives as potent PDE4 inhibitors for inhaled administration in the treatment of respiratory diseases

DOI: 10.1021/jm401549m

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Full text of DOI:10.1021/jm401549m

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