Discovery of Novel 1-Cyclopentenyl-3-phenylureas as Selective, Brain Penetrant, and Orally Bioavailable CXCR2 Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.7b01854

CXCR2 has emerged as a therapeutic target for not only peripheral inflammatory diseases but also neurological abnormalities in the central nervous system (CNS). Herein, we describe the discovery of a novel 1-cyclopentenyl-3-phenylurea series as potent and CNS penetrant CXCR2 antagonists. Extensive SAR studies, wherein molecules' property forecast index (PFI) was carefully optimized for overall balanced developability profiles, led to the discovery of the advanced lead compound 68 with a desirable PFI. Compound 68 demonstrated good in vitro pharmacology with excellent selectivity over CXCR1 and other chemokine receptors. Rat and dog pharmacokinetics (PK) revealed good oral bioavailability, high oral exposure, and desirable elimination half-life of the compound in both species. In addition, the compound demonstrated dose-dependent efficacy in the in vivo pharmacology neutrophil infiltration "air pouch" model in rodents after oral administration. Further, compound 68 is a CNS penetrant molecule with high unbound fraction in brain tissue.

Full text of DOI:10.1021/acs.jmedchem.7b01854

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Article
Discovery of Novel 1-Cyclopentenyl-3-phenylureas as Selective,
Brain Penetrant and Orally Bioavailable CXCR2 Antagonists
Hongfu Lu, Ting Yang, Zhongmiao Xu, Xichen Lin, Qian Ding, Yueting
Zhang, Xin Cai, Kelly Dong, Sophie Gong, Wei Zhang, Metul Patel, Royston
C. B. Copley, Jianing Xiang, Xiaoming Guan, Paul Wren, and Feng Ren
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01854 • Publication Date (Web): 06 Feb 2018
Downloaded from http://pubs.acs.org on February 6, 2018
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Discovery of Novel 1ꢀCyclopentenylꢀ3ꢀphenylureas
as Selective, Brain Penetrant and Orally
Bioavailable CXCR2 Antagonists
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Hongfu Lu, Ting Yang, Zhongmiao Xu, Xichen Lin, Qian Ding, Yueting Zhang, Xin Cai,
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#
ǂ
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Kelly Dong, Sophie Gong, Wei Zhang, Metul Patel, Royston C. B. Copley, Jianing Xiang,
†
ǁ
,†
Xiaoming Guan, Paul Wren, Feng Ren*
†
Neurosciences Therapeutic Area Unit, GSK Pharmaceuticals R&D, 898 Halei Road, Zhangjiang
HiꢀTech Park, Pudong, Shanghai 201203, PR China.
§
R&D Projects Clinical Platforms and Sciences, GSK Pharmaceuticals R&D, 898 Halei Road,
Zhangjiang HiꢀTech Park, Pudong, Shanghai 201203, PR China.
‡
Platform Technology Sciences, GSK Pharmaceuticals R&D, 898 Halei Road, Zhangjiang Hiꢀ
Tech Park, Pudong, Shanghai 201203, PR China.
#
Platform Technology Sciences, GSK Pharmaceuticals R&D, Stevenage, Herts, SG1 2NY, UK.
ǂ
Platform Technology & Science, GSK Medicines Research Centre, Gunnels Wood Road,
Stevenage, Hertfordshire, SG1 2NY, UK.
ǁ
Neurosciences Therapeutic Area Unit, GSK Pharmaceuticals R&D, 1250 S. Collegeville Road,
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Collegeville, PA 19426, US.
ABSTRACT. CXCR2 has emerged as a therapeutic target for not only peripheral inflammatory
diseases but also neurological abnormalities in the central nervous system (CNS). Herein, we
describe the discovery of a novel 1ꢀcyclopentenylꢀ3ꢀphenylurea series as potent and CNS
penetrant CXCR2 antagonists. Extensive SAR studies, wherein molecules’ property forecast
index (PFI) was carefully optimized for overall balanced developability profiles, led to the
discovery of the advanced lead compound 68 with a desirable PFI. Compound 68 demonstrated
good in vitro pharmacology with excellent selectivity over CXCR1 and other chemokine
receptors. Rat and dog pharmacokinetics (PK) revealed good oral bioavailability, high oral
exposure, and desirable elimination halfꢀlife of the compound in both species. In addition, the
compound demonstrated doseꢀdependent efficacy in the in vivo pharmacology neutrophil
infiltration “air pouch” model in rodents after oral administration. Further, compound 68 is a
CNS penetrant molecule with high unbound fraction in brain tissue.
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KEYWORDS. CXCR2 antagonist, 1ꢀcyclopentenylꢀ3ꢀphenylurea, Pꢀgp, BCRP, CNS
penetration, PFI, air pouch
INTRODUCTION
CXCR2, a CXC chemokine receptor of the Gꢀproteinꢀcoupled receptor (GPCR) family, is a
sevenꢀtransmembrane protein with a 67‒70 kD molecular weight expressed on neutrophils,
granulocytes, NK cells, T lymphocytes, mast cells, monocytes, endothelial cells, megakarocytes,
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neurons, and oligodendrocytes. CXCR2 and its main cognate ligands CXCL1 (Groꢀα) and
CXCL8 (ILꢀ8) have been extensively reported as critical proꢀinflammatory components of
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neutrophil activation and chemotaxis, megakaryocytic proliferation, and angiogenesis.
Increasing evidence has emerged to implicate the important role of CXCR2 in numerous
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inflammatory disorders including chronic obstructive pulmonary disease (COPD) , rheumatoid
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arthritis (RA) , and atherosclerosis . Recently, CXCR2 inhibitor was reported to reduce
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metastases and augment antiꢀPD1 immunotherapy in KPC mice to result in prolonged survival.
Likewise, CXCR2 antagonist significantly enhanced the efficacy of immune checkpoint
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blockage (ICB) treatment in a mouse prostate cancer model. These preclinical data have raised
significant interest of CXCR2 as a therapeutic target for cancer immunotherapy. The correlation
between CXCR2 expression and human glioma was reported, and CXCR2 inhibition was found
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to mitigate glioma cell migration. In addition to peripheral immune system, CXCR2 also plays
a role in regulating neuronal activity and brain development even though its expression level is
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low in both human and rodent brains. Increasingly, CXCR2 has been recognized as a potential
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target for the treatment of CNS diseases such as multiple sclerosis and Alzheimer’s disease
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AD) , and others. Thus, discovery of small molecule CXCR2 antagonists with brain
penetration properties may open new opportunities to explore and tackle abnormalities in the
CXCL1 (Groꢀα)/CXCL8 (ILꢀ8) signaling pathway in CNS diseases.
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The first small molecule CXCR2 antagonists were reported in 1998, and since then a number
of chemical series of small molecule CXCR2 antagonists with diverse pharmacophores have
been developed (Figure 1). Among them, quite a few compounds have been progressed into
clinical trials for the treatment of neutrophilꢀinduced pulmonary diseases. Reparixin (1), an
allosteric inhibitor of CXCR1 with several hundredꢀfold selectivity over CXCR2, was reported
to block CXCL8ꢀ and CXCLꢀ1 mediated neutrophil (PMN) migration and was advanced into
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clinical trials via intravenous administration for delayed graft function and transplant rejection.
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Later the compound was reꢀpositioned for a clinical trial for breast cancer. A pyrimidineꢀbased
compound, AZD5069 (2), with reasonable oral bioavailability was progressed to clinical stage
for COPD and asthma and afterwards, a clinic study for cancer treatment was initiated.
Compound 2 proved to be a potent CXCR2 antagonist with around 100ꢀfold selectivity over
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CXCR1. Later, a bicyclic pyrimidine series was reported as dual CXCR2/CCR2 antagonists
and a representative, AZ10397767 (3), demonstrated nanomolar antagonism activity for both
17,18
CXCR2 and CCR2.
Navarixin (4, also known as SCH527123 or MKꢀ7123), bearing a novel
squaramide core structure, was described as a potent and selective antagonist of CXCR2 with 80ꢀ
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fold selectivity over CXCR1. A phase 2 clinical trial of compound 4 in COPD patients
demonstrated clinical benefit of the antiꢀinflammatory mechanism resulting from CXCR2
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antagonism.
Several other novel chemical series were also disclosed as CXCR2 antagonists
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including the 5ꢀcarbonitrile pyrimidines, 2ꢀaminoꢀquinoxalines, and boronic acid derivatives
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containing aminopyridines/aminopyrimidines. However, none of these structures was able to be
progressed to clinic testing. Among all the small molecule CXCR2 antagonists reported to date,
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the diarylurea series was the first disclosed and the successful chemical series from which a
couple of compounds had been advanced to the clinic. SBꢀ332235 (5), even though not
progressed to the clinic, was an important diarylurea early tool compound for mechanistic
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explorations of CXCR2 antagonism for COPD and AD. The first diarylureaꢀbased CXCR2
antagonist advanced to clinical trial is SBꢀ656933 (6), which has been tested in COPD and cystic
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fibrosis (CF) patients. Further development of compound 6 was discontinued due to lack of
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significant lung function improvement in clinics during the dosing regimen and in favor of a
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more attractive diarylurea analogue, Danirixin (7, also known as GSK1325756). Currently
compound 7 is in phase II clinical trial for COPD. Extensive literature reports have been focused
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on the small molecule CXCR2 antagonists for the treatment of peripheral indications. On the
other hand, however, little has been disclosed on brain penetration characteristics of compounds.
Most recently, a CNS penetrant CXCR2 antagonist based on the diarylurea skeleton (8) was
disclosed, and the compound promoted remyelination in mouse brain in the cuprizoneꢀinduced
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demyelination model. The series including compound 8 was reported to have low unbound
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fractions in plasma which might be attributed to its high lipophilicity (cLogP) and high
property forecast index (PFI). PFI, the sum of hydrophobicity value and number of aromatic
rings, has evolved as one of the key “drugꢀlikeness” measurements as supported by its
correlations with compound developability properties including solubility, membrane
permeability, plasma protein binding, cytochrome P450 inhibition, hERG binding, and
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promiscuity, etc.
Herein, we report the discovery of a novel 1ꢀcyclopentenylꢀ3ꢀphenylurea series as brain
penetrant and selective CXCR2 antagonists for the potential treatment of CNS diseases. In our
work, replacing one aromatic ring of the previously reported diarylureas with the aliphatic
cyclopentenyl group provides marked reduction of PFI thus significant improvement of the
developability profile (plasma protein binding, solubility, etc.) of the series.
RESULTS AND DISCUSSION
StructureꢀActivity Relationships and CNS Penetration. We started our exploration from the
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known CNS penetrant diarylurea series where compound 8 was identified as the lead.
However, compound 8 might suffer from an active demethylation metabolite issue wherein the
methyl group of the Nꢀmethyl piperidine was observed to be metabolized to a significant extend
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in vitro in human liver microsomes. Therefore, among the diarylurea analogues without the Nꢀ
methyl piperidine substitution, taken into consideration of compounds’ potency and “drugꢀ
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likeness” properties such as molecular weight (MW) and PFI, compound 9 (Table 1) was
selected as the chemistry starting point. Our initial work was focused on replacing the 2ꢀchloroꢀ
3ꢀfluorophenyl group (R, Table 1) of 9 with aliphatic rings to reduce PFI. The cyclohexyl
analogue (10) demonstrated much reduced PFI (6.7) compared to compound 9 (8.2), but
decreased CXCR2 antagonism activity (pIC = 7.3) by ~100 fold in the CXCR2 Tango assay,
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which determined compounds’ functional potency at recombinant CXCR2 receptors.
Introduction of methyl (11, pIC50 = 6.5) or methoxyl (12, pIC50 = 5.8) substitutions to the
cyclohexyl ring resulted in even lower potencies. 6ꢀMembered saturated heterocyclic rings
including tetrahydropyran (13, 14) and piperidine (15, pIC < 5.0) were not tolerated, indicating
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a hydrophobic pocket occupied by the R group. We then explored the ring size and found that the
ꢀmembered cyclopentane ring (16) demonstrated higher potency (pIC = 8.0) and lower PFI
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(6.0) than its 6ꢀmembered analogue (10). Efforts to further improve CXCR2 antagonist activity
by introducing hydrophobic and/or hydrophilic substitutions (17‒21) failed to provide any
compound with higher potency. The orthoꢀsubstitution (17, pIC50 = 8.0) demonstrated better
potency than the metaꢀsubstitution (18, pIC = 6.7). Increasing the size of the orthoꢀsubstitution
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resulted in decreased potency (19, pIC = 7.2), suggesting that small substitutions be preferred.
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The hydrophilic substitution was poorly tolerated (20, pIC50 = 6.6), further supporting the
existence of a hydrophobic binding pocket. The diꢀsubstitution at the orthoꢀposition of the
cyclopentanyl group resulted in low activity (21, pIC = 6.8), likely due to the unfavorable steric
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effect. Sterically less hindered spiro and fused analogues afforded better potencies (22, 23). As
expected, the hydrophilic pyrrolidine group led to an inactive compound (24, pIC < 5.0). We
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then started to explore the unsaturated rings to increase planarity which might better mimic
aromatic rings. Intriguingly, the cyclopentenyl ring afforded slightly improved potency (25,
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pIC = 8.2) compared with the cyclopentanyl analogue (16). A breakthrough was achieved when
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a methyl substitution was introduced to the αꢀposition of the cyclopentenyl group (26, pIC =
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9.4). The Cl substitution provided comparable potency (27, pIC = 9.0) whereas sterically more
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hindered and less hindered substitutions were less potent (28, 29). Diꢀsubstituted cyclopentenyl
groups (30, 31) were observed to result in decreased potency. Again, ring size was explored and,
similarly, 6ꢀmembered unsaturated rings (32‒34) were less potent than their 5ꢀmembered
counterparts. Thus, the 2ꢀmethylcyclopentenyl (26) was identified as the best rightꢀhandꢀside
(RHS) R group for future SAR studies.
Compounds in Table 1 were synthesized and tested as racemates. In order to further
understand the effect of cyclopentenyl stereochemistry on the CXCR2 antagonism activity,
several pairs of the single known (R)ꢀ and (S)ꢀenantiomers were designed and synthesized. It was
worth mentioning that the 2ꢀchlorocyclopentꢀ2ꢀenamine was synthetically more accessible in
both (R)ꢀ and (S)ꢀconfigurations comparing to the 2ꢀmethylcyclopentꢀ2ꢀenamine, thus the 2ꢀClꢀ
cyclopentenyl analogues were used for stereochemistry SAR exploration (Table 2). Generally,
the (R)ꢀisomers (35‒37) were observed to be ~100 fold more potent than the (S)ꢀisomers (35’‒
3
7’) as evaluated in the CXCR2 Tango assay. When R was oxetane, the potency difference
between the (R)ꢀisomer (38, pIC50 = 6.5) and the (S)ꢀisomer (38’, pIC50 = 5.6) was less
significant although still ~10 fold. Having established the SAR that the 2ꢀmethylcyclopentene
was the best RHS group and the (R)ꢀconfiguration was more favored than the (S)ꢀconfiguration,
future SAR studies were focused on analogues containing the (R)ꢀ2ꢀmethylcyclopentenyl ring.
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SAR studies were then focused on the leftꢀhandꢀside (LHS) especially on R and R
substitutions (Table 3). The aim was to balance the overall profile of the series including
potency, solubility, permeability, and Pꢀglycoprotein (Pꢀgp)/breast cancer resistance protein
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(
BCRP) efflux which are in vitro measurements for CNS penetration property . Exploration of
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the R substitution started from the tertꢀbutyl group (39), which demonstrated high CXCR2
antagonist potency (pIC = 9.0) in the Tango assay. It was further evaluated in the human whole
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blood (HWB) Groꢀα induced CD11b expression assay, which measured the functional potency at
the native CXCR2 receptor, and was found quite active (pIC = 6.5). In addition, compound 39
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showed high cell permeability (364 nm/s) and proved not to be a Pꢀgp/BCRP efflux substrate,
resulting in good CNS penetration. However, the compound demonstrated very low solubility
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1.2 µg/mL) in fasted state simulated intestinal fluids (FaSSIF), likely due to its high PFI (7.4).
Incorporating electron withdrawing group (EWG) such as F or CF to the tertꢀbutyl group
3
provided analogues (40, 41) with slightly reduced PFI (6.6 and 6.7, respectively) however, not
surprisingly, only marginal improvements in FaSSIF solubility were observed. Further reducing
PFI by replacing two methyl groups with two F led to compound 42 (PFI = 4.3) with much
improved solubility (50.8 µg/mL). Introduction of a dimethylamino group resulted in a
significant drop in CXCR2 antagonist potency (43, pIC = 6.9) even though its solubility was
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high (395.4 µg/mL). Another dimethylamino derivative (44) demonstrated improved potency
pIC = 8.7), however it proved to be a strong Pꢀgp/BCRP substrate (ER = 12.6) thus carried
(
50
high risk for adequate CNS penetration. Intrigued by the high solubility of 43 and 44, a series of
nitrogenꢀcontaining derivatives were synthesized and evaluated (45‒51). Among them,
compounds 50 and 51 demonstrated balanced overall profiles for further progression and the rest
of compounds were less attractive due to Pꢀgp/BCRP liabilities (45, 46) or low potency (47, 49).
2
Substitutions at the R position were also investigated. The acidity of the phenol group was
believed to be critical for the potency of the series, we thus limited our exploration to the EWG
only, in order to maintain or increase its acidity. Replacing the Cl of 39 with a stronger electronꢀ
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withdrawing CN group provided compound 52 with improved potency (pIC = 9.7), presumably
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due to the increased acidity of the phenol. In addition, 52 demonstrated much lower PFI and as a
result high solubility (685.6 ꢁg/mL). On the other hand, as the compound got more polar its
passive permeability decreased (90 nm/s). A similar profile was also observed with another CN
derivative (53) with even lower permeability (30 nm/s).
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Based on the overall properties especially potencies and Pꢀgp/BCRP profiles, several
representative analogues (40‒42, 50‒52) were selected for evaluation for in vivo CNS
penetration using a rat short oral absorption (SOA) model (Table 4). The brainꢀtoꢀblood ratio
(
K ) of the area under curve (AUC) of compound 40 was measured to be 0.16 after oral gavage
p
to rats. Intriguingly, its free unbound fraction in both brain (Fu,br = 3.4%) and blood (Fu,bl
=
1
.1%) were measured to be significantly higher than the diarylurea exemplars (e.g. compound
2
9
9
) which was likely due to its lower PFI. Increasingly, the brainꢀtoꢀblood ratio of free unbound
3
5
drug concentration (Kp,uu) has been described as a critical parameter for CNS penetration. K
p,uu
for compound 40 was determined to be 0.48. The trifluoromethyl analogue 41 also demonstrated
good CNS penetration in terms of both total drug exposure (K = 0.20) and free unbound drug
p
exposure (Kp,uu = 0.59). With a slightly higher PFI, its free unbound fractions in both brain (Fu,br
=
2.1%) and blood (Fu,bl = 0.70%) were slightly lower than compound 40. The nitrogenꢀ
containing analogues (50 and 51) demonstrated high peripheral drug exposure with dose
normalized area under curve (DNAUC) of 2580 and 11721 (ng·h/mL)/(mg/kg), respectively.
However, their K values were measured very low (0.06 and 0.03, respectively) and as a result
p
their Kp,uu could not be determined with meaningful values. Both 50 and 51 were thus considered
peripherally restricted compounds. Compound 52 with the CN substitution also demonstrated
low CNS penetration as evidenced by its low brainꢀtoꢀblood ratio of AUC (K = 0.03). It was
p
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noteworthy that all the compounds evaluated with low brain penetration showed PFI values < 5
(4.3, 3.7, 3.7 and 4.1 for compound 42, 50, 51 and 52, respectively), whereas compounds with
high brain penetration showed PFI values > 5 (6.6 and 6.7 for compound 40 and 41,
respectively). SAR suggested that, for this 1ꢀcyclopentenylꢀ3ꢀphenylurea series, compounds with
PFI > 5 might have better potentials to be brain penetrant. On the other hand, high PFI has been
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shown to be associated with low solubility, high protein binding, and other developability risks.
We thus focused our efforts on the compounds with PFIs between 5 and 7, for the purpose of
achieving balanced profiles of CNS penetration and developability (solubility, free unbound
fraction, etc.).
Due to low solubility for alkyl/haloalkyl groups and high efflux ratios for Nꢀcontaining groups
1
as R substitutions, Oꢀcontaining substituents were then investigated to balance solubility and
efflux ratio (Table 5). Compound 54 with a methoxyl substitution demonstrated balanced profile
of FaSSIF solubility (72.0 µg/mL) and Pꢀgp/BCRP efflux ratio (3.2), however its potency,
especially when measured in the human whole blood CD11b assay, was not optimal (pIC =
5
0
6
.4). Addition of a 4ꢀmembered oxetane ring (55) provided much improved CXCR2 antagonist
potency in the Tango assay whereas the solubility decreased dramatically (7.0 µg/mL).
Increasing the ring size (56‒58) failed to improve the potency and/or resulted in low solubility
(58). The HWB potency was slightly improved (pIC50 = 6.7) when the tetrahydropyran was
directly connected to the sulfonyl group (59) but low solubility was observed. Replacing the
methyl group of 59 with an ethyl group (60) led to higher solubility but lower HWB potency.
Introduction of F atom provided compounds (61‒63) with increased Pꢀgp/BCRP efflux liability.
Changing the position of oxygen atom in tetrahydropyran (64, 65) failed to provide compounds
with more balanced profile of solubility and Pꢀgp/BCRP efflux. We then switched to a 5ꢀ
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membered tetrahydrofuran ring with 5 < PFI < 6 (66‒68) and, to our delight, compound 68
demonstrated a balanced profile of solubility (21.0 µg/mL) and Pꢀgp/BCRP efflux ratio (2.0). In
addition, the HWB potency of 68 (pIC50 = 7.4) proved to be the highest amongst all the
compounds explored. Introduction of a F atom (69) resulted in an increased efflux ratio, similar
to the 6ꢀmembered tetrahydropyran analogues. The hydroxyl (70) and spiro (71) substitutions led
to high efflux ratios (26.8 and 25.5, respectively), likely due to their low PFIs (< 5).
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Based on their overall profiles including potency, solubility, and Pꢀgp/BCRP efflux,
compounds 56, 67, and 68 were selected for rat SOA studies for in vivo CNS penetration
evaluation (Table 6). Among them, compound 68 demonstrated the highest Kp,uu (0.63),
indicating a good in vivo brain penetration property. It showed a higher brain penetration
29
property comparing to the previously reported CNS penetrant compound (8) , the Kp,uu of which
was determined to be 0.18. In addition, the unbound fractions of compound 68 were measured to
be reasonable in both rat blood (Fu,bl = 2.7%) and human plasma (Fu,pl = 1.9%), which were more
than 10ꢀfold higher than the unbound fraction of compound 8 in human plasma (Fu,pl = 0.13%)
29
described previously. The balanced profile of CNS penetration and free unbound fraction of
compound 68 was likely due to its desirable PFI (5.8), supporting the belief that ideal values of
PFIs of the series are between 5 and 7 as discussed previously. It was noteworthy that the free
unbound fraction of 68 in brain (Fu,br = 12.3%) was observed to be much higher than in blood.
This phenomenon was consistent throughout the series and likely due to the acidic character of
the sulfone substituted phenol group unique within this 1ꢀcyclopentenylꢀ3ꢀphenylurea series. The
diastereoisomer of 68, compound 67, had the same PFI (5.8) thus its free unbound fractions in
both blood (Fu,bl = 3.6%) and brain (Fu,br = 10.6%) were similar to 68. However, the CNS
penetration properties of 67 measured as both K (0.08) and K (0.23) were less attractive.
p
p,uu
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Compound 56 demonstrated a good CNS penetration profile whereas its low exposure in blood
[
63.8 (ng·h/mL)/(mg/kg)] was a concern and prohibited further progression.
DMPK. Lead compounds 40 and 68 were further progressed in an in vivo PK study in rats
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following 1 mg/kg intravenous (IV) and 2 mg/kg oral administration (Table 7). Both compounds
showed low clearance and high exposure in rat, consistent with their profile from the in vivo rat
SOA study. The halfꢀlife (T ) of compound 68 (4.3 h) was longer than that of compound 40
1/2
(
2.5 h), likely due to its lower clearance (9.1 mL/min/kg) compared to 40 (15.7 mL/min/kg). The
DNAUC values for compounds 40 and 68 were determined to be 1071 and 1829
ng·h/mL)/(mg/kg), respectively. The oral exposure of both compounds was measured high with
(
DNAUC values of 961 (ng·h/mL)/(mg/kg) for 40 and 860 (ng·h/mL)/(mg/kg) for 68,
corresponding to 88% and 47% oral bioavailability, respectively. Compound 68 was selected as
the advanced lead compound due to its longer T , better solubility, and higher free unbound
1
/2
fraction compared to compound 40. An in vivo dog PK study was conducted for compound 68
and low clearance (5.6 mL/min/kg), reasonable T1/2 (3.6 h), high oral exposure [2298
(ng·h/mL)/(mg/kg)], and good oral bioavailability (76%) were observed, which were consistent
with the rat profile. In addition, the in vitro metabolic stability of compound 68 in human, rat,
and dog hepatocytes was determined (Table 8). The result revealed that the compound was most
stable in human hepatocytes [Cl = 0.79 (mL/min/g)] and least stable in rat hepatocytes [Cl =
int
int
7
.7 (mL/min/g)].
In Vitro Pharmacology in Mouse and Human Whole Blood. The ability of compound 68 to
inhibit CXCR2 receptor activation was assessed in whole blood Groꢀα induced CD11b
expression assays and quantified by flow cytometry. In the mouse whole blood assay (Figure 2a),
compound 68 inhibited Groꢀα induced CD11b expression with a IC value of 5.9 nM (N = 3),
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comparable to the control compound Navarixin
(IC = 8.1 nM). It was worth mentioning
50
that the maximal response of compound 68 was less than that of Navarixin, around two thirds of
CD11b inhibition driven by Navarixin at 10 µM, suggesting a partial inhibitory effect of
compound 68 in mouse whole blood. In contrast, in human whole blood CD11b assay (Figure 2b)
compound 68 (similar to Navarixin) fully inhibited Groꢀα induced CD11b expression with an
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IC value of 0.04 µM (N = 5) which was 17ꢀfold more potent than that of Navarixin (IC = 0.68
50
50
µM). In addition, compound 68 was 12ꢀfold more potent than the reported CNS penetrant
29
compound 8 (IC = 0.50 µM) in this HWB assay.
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In Vitro Chemokine Selectivity. Compound 68 demonstrated excellent selectivity for
CXCR2 antagonist activity over other CXC and CC chemokine receptors (Table 9). A total of 18
CXC and CC chemokine receptors including CXCR2 were evaluated in both agonism and
antagonism modes in βꢀarrestin assays and, among them, compound 68 was measured to be
inactive (IC s > 10 µM) in all assays except for CXCR2 and CXCR1. IC s of the compound on
50
50
CXCR2 and CXCR1 were 5.2 nM and 3.8 µM, respectively. Thus, compound 68 was determined
to be a highly selective CXCR2 antagonist with ~730ꢀfold selectivity over CXCR1 and >1900ꢀ
fold selectivity over all other chemokine receptors evaluated.
In Vivo Pharmacology in Mouse and Rat. The effects of compound 68 on peripheral
inflammatory cell infiltration induced by 2% carrageenan was tested using mouse and rat air
36
pouch models. As shown in Figure 3a, compound 68 significantly inhibited neutrophil
infiltration into mouse air pouch, as compared to vehicle (One way ANOVA), 4 h after
carrageenan induction at 1, 3, 10 mg/kg oral dosing twice daily (BID). Similar in vivo
pharmacology was observed in the rat air pouch model (Figure 3b). In this species, compound 68
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demonstrated a marked doseꢀdependent inhibition of neutrophil migration from 1 mg/kg to 3
mg/kg to 10 mg/kg BID oral dosing.
Synthetic Chemistry. Convergent synthetic routes for the syntheses of 1ꢀcyclopentenylꢀ3ꢀ
phenylureas were developed as illustrated in the syntheses of key compounds 40 and 68. Both
compounds were assembled from the common RHS chiral amine intermediate (75) and the
corresponding LHS aminophenol intermediates (82 for compound 40 and 85 for compound 68),
as shown in Scheme 1. Synthesis of chiral amine 75 was initiated from the commercial starting
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material, ketone 72. Enantioselective reduction of the ketone group of 72 catalyzed by (R)ꢀCBS
provided alcohol 73 in quantitative yield, and the enantiomeric excess (ee) was determined in the
next step. Stereospecific reversion of the chiral alcohol with phthalimide by Mitsunobu reaction
afforded chiral phthalimide intermediate 74 with 74% ee favoring the (R)ꢀisomer. Chiral
purification of the mixture by supercritical fluid chromatography (SFC) afforded
enantiomerically pure intermediate 74 as the (R)ꢀisomer with >98% ee. Treatment of 74 with
hydrazine at elevated temperature provided the common RHS intermediate, the chiral amine 75.
Synthesis of the lead compound 40 was initiated from the reported starting material, thiol 76
3
2
(
Scheme 2). 76 was treated with ethyl 2ꢀbromoꢀ2ꢀmethylpropanoate to afford the thiol ether 77,
which was then oxidized by mꢀCPBA to afford sulfone 78 in 64% yield over two steps.
Reduction of the ethyl ester of 78 with DIBALꢀH provided the alcohol 79. The hydroxyl group
of 79 was converted to trifluoromethanesulfonate (80) and, after treatment with TBAF, the Fꢀ
containing intermediate 81 was obtained in good yield (46% over three steps). Hydrolysis of the
benzoxazole moiety in strong acidic condition at elevated temperature gave the LHS
intermediate, aminophenol 82. Coupling of LHS 82 with the transient isocyanate intermediate
derived from RHS 75 provided the final compound 40 in good isolated yield (58%).
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For the synthesis of lead compound 68 (Scheme 3), the starting material 83 was prepared in
2
8
large quantities following a published procedure. Compound 83 was treated with methyl iodide
in the presence of LHMDS to afford the intermediate 84 as an enantiomeric mixture at the
tetrahydrofuran moiety. The benzoxazole group of 84 was hydrolyzed under acidic condition to
afford the LHS aminophenol 85 in good isolated yield over two steps (66%). Similarly, the LHS
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5 was coupled with the isocyanate derived from RHS 75 to produce the diastereomeric mixture
6 in high yield (96%). The two diastereomers of 66 were separated by SFC to afford the desired
single diastereomer 68 in 22% isolated yield (>93% de). The absolute stereochemistry of the 3ꢀ
methyltetrahydrofuranꢀ3ꢀyl group of compound 68 was established unambiguously as the (S)ꢀ
isomer from crystal structure.
Xꢀray Crystallography. The absolute configuration of compound 68 was determined using
single crystal Xꢀray diffraction techniques. The compound crystallizes with two independent
molecules in the asymmetric unit. One of these is shown in Figure 4. For both molecules, the 3ꢀ
methyltetrahydrofuranꢀ3ꢀyl groups are found to be disordered over two positions but the (S)ꢀ
configuration for this moiety is maintained within the model throughout (see Supporting
Information for further information). The chiral center of the 2ꢀmethylcyclopentꢀ2ꢀenamine
moiety was confirmed to be (R), consistent with the reported stereochemistry assignment
generated from (R)ꢀCBS reduction. Intermolecular hydrogen bonds between urea groups
alternately link the two independent molecules into chains within the structure. An
intramolecular interaction exists in both molecules between the hydroxyl group and a sulfonyl
oxygen atom.
CONCLUSIONS
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In summary, a series of novel 1ꢀcyclopentenylꢀ3ꢀphenylureas were discovered as potent, brain
penetrant, and orally available CXCR2 antagonists. Extensive SAR studies to improve solubility,
protein binding, and in vitro and in vivo CNS penetration properties by carefully optimizing
molecules’ PFI led to the discovery of the advanced lead compound 68. Compound 68
demonstrated desirable PFI (5.8), good solubility (21.0 µg/mL), and high free unbound fraction
in both blood (Fu,bl = 2.7%) and brain (Fu,br = 12.3%). Rat and dog PK studies revealed moderate
to good oral bioavailability (47% and 76%, respectively) with high oral exposure [DNAUC: 860
and 2298 (ng·h/mL)/(mg/kg), respectively] and desirable T (4.3 and 3.6 h, respectively) in both
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species. Compound 68 inhibited human whole blood Groꢀα induced CD11b expression with a
IC value of 0.04 µM, 17ꢀfold more potent than a reported clinical compound Navarixin (IC =
5
0
50
0
.68 µM). Screening of 68 on a panel of 18 chemokine receptors highlighted potent inhibition of
CXCR2 (IC = 5.2 nM) with ~730ꢀfold selectivity over CXCR1 and >1900ꢀfold selectivity over
5
0
all other chemokine receptors tested (IC s >10 µM). Compound 68 doseꢀdependently reduced
5
0
neutrophil infiltration in vivo in rat and mouse “air pouch” models upon oral administration (1, 3,
and 10 mg/kg BID). In addition, good CNS penetration profile of compound 68 was observed in
rat in vivo SOA study (Kp,uu = 0.63). Compared to the reported CNS penetrant compound 8,
compound 68 demonstrated much better potency (12 fold) in human whole blood assay,
significantly higher unbound fraction in human plasma (>10 fold), and improved brain
penetration as measured by Kp,uu (>3 fold). The compound might represent a promising candidate
for not only peripheral inflammatory diseases but also a range of CNS indications.
EXPERIMENTAL SECTION
Chemistry. The syntheses of the compounds 40, 66 and 68 are described below. Detailed
experimental procedures for the syntheses of all other compounds, their intermediates, and
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characterization data are provided in the Supporting Information. Liquid chromatography mass
spectrometry (LCMS) was used to determine the purity of compounds, and all tested compounds
were determined with ≥ 95% purity.
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Materials and Methods. Reagents were purchased from commercial suppliers and used
1
13
directly without further purification. H NMR and C NMR spectra were measured on Bruker
00/600 NMR spectrometer wherein CDCl (deuterochloroform), CD OD (deuterated methanol),
4
3
3
or DMSOꢀd6 (hexadeuterioꢀdimethyl sulfoxide) were used as the solvents and TMS
tetramethylsilane) was used as the internal standard. Chemical shifts ( ) were given in parts per
(
δ
million (ppm) downfield from the internal standard TMS signal. In NMR spectra, the following
abbreviations were used for multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, dd = doublet of doublets, dt = doublet of triplets, ddd = doublet of doublet of doublets,
br = broad. J was used to indicate the NMR coupling constant measured in Hertz. High
resolution mass (HRMS) measurement was achieved using an orthogonal acceleration timeꢀofꢀ
TM
flight (oaꢀTOF) SYNAPT G2 HDMS equipped with a positive mode of electrospray ionization
(ESI) (Waters, Manchester, UK). LCMS analysis was conducted on Agilent 1200SLꢀ6110 under
the acidic condition, water containing 0.05 % TFA/acetonitrile as the mobile phase on Agilent
SBꢀC18 column (1.8 µm, 4.6 x 30 mm), with MS and photodiode array detector (PDA). The
following conditions were used for LCMS: a gradient from 5 to 95% in 5 min (or 6 min) and
held at 95% for 1 min; UV detection at 214 and 254 nm; a flow rate of 1.5 ml/min; full scan;
mass range from 100 to 1000 amu. Column chromatography purifications were performed on
Isco or Biotage with a detector with UV wavelength at 254 nm and 280 nm, using a preꢀpacked
silica gel column.
The preparation of the target compounds 40, 66 and 68.
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Synthesis
of
(R)ꢀ1ꢀ(4ꢀchloroꢀ3ꢀ((1ꢀfluoroꢀ2ꢀmethylpropanꢀ2ꢀyl)sulfonyl)ꢀ2ꢀ
hydroxyphenyl)ꢀ3ꢀ(2ꢀmethylcyclopentꢀ2ꢀenꢀ1ꢀyl)urea (40). To a solution of (R)ꢀ2ꢀ
methylcyclopentꢀ2ꢀenamine, hydrochloride salt (75, 560 mg) in toluene (36 mL) was added
triphosgene (746 mg). The reaction mixture was refluxed for 6 hours, and then cooled to room
temperature to give the fresh prepared isocyanate solution. To a solution of 6ꢀaminoꢀ3ꢀchloroꢀ2ꢀ
0
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(
(1ꢀfluoroꢀ2ꢀmethylpropanꢀ2ꢀyl)sulfonyl)phenol (82, 100 mg) in pyridine (5 mL) was added the
above isocyanate solution in toluene (4 mL). The reaction mixture was stirred at room
temperature for overnight. The reaction mixture was concentrated and the resulting residue was
purified by prepꢀHPLC to give (R)ꢀ1ꢀ(4ꢀchloroꢀ3ꢀ((1ꢀfluoroꢀ2ꢀmethylpropanꢀ2ꢀyl)sulfonyl)ꢀ2ꢀ
hydroxyphenyl)ꢀ3ꢀ(2ꢀmethylcyclopentꢀ2ꢀenꢀ1ꢀyl)urea (40, 70 mg, 58% yield) as a white solid.
1
H NMR (400 MHz, DMSOꢀd ) δ 10.31 (s, 1H), 8.38 (d, J = 8.8 Hz, 1H), 8.16 (s, 1H), 7.12 (d, J
6
=
8.8 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H), 5.51 (br. s., 1H), 4.73 (s, 1H), 4.62 (s, 1H), 4.45‒4.57 (m,
1
3
1H), 2.10‒2.37 (m, 3H), 1.66 (s, 3H), 1.45‒1.57 (m, 1H), 1.35‒1.42 (m, 6H); C NMR (151
MHz, DMSOꢀd ) δ 155.1, 148.1, 140.6, 130.8, 127.4, 124.1, 124.0, 123.9, 116.6, 85.2, 84.0,
6
+
+
6
7.8, 58.0, 32.6, 30.0, 17.5, 14.3. LCMS(ESI) m/z 405 [M+H] . HRMS (EI) m/z: [M+H] calcd
for C H ClFN O S 405.1051, found 405.1045.
17 23
2
4
Synthesis of 1ꢀ(4ꢀchloroꢀ2ꢀhydroxyꢀ3ꢀ((3ꢀmethyltetrahydrofuranꢀ3ꢀyl)sulfonyl)phenyl)ꢀ3ꢀ
(
(R)ꢀ2ꢀmethylcyclopentꢀ2ꢀenꢀ1ꢀyl)urea (66). To a solution of 6ꢀaminoꢀ3ꢀchloroꢀ2ꢀ((3ꢀ
methyltetrahydrofuranꢀ3ꢀyl)sulfonyl)phenol (85, 3.0 g) in pyridine (20 mL) was added the fresh
prepared isocyanate solution (as described in the synthesis of 40). The mixture was stirred at
4
0 °C for 12 h. Iceꢀwater (30 mL) was added to the above solution and the mixture was extracted
with DCM (2×100 mL). The combined organic layers were dried over sodium sulphate, filtered
and concentrated in vacuo. The resulting residue was purified by silica gel column
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chromatography eluting with 0‒80% ethyl acetate in petroleum ether to give 1ꢀ(4ꢀchloroꢀ2ꢀ
hydroxyꢀ3ꢀ((3ꢀmethyltetrahydrofuranꢀ3ꢀyl)sulfonyl)phenyl)ꢀ3ꢀ((R)ꢀ2ꢀmethylcyclopentꢀ2ꢀenꢀ1ꢀ
1
yl)urea (66, 4.1 g, 96% yield) as a white solid. H NMR (400 MHz, DMSOꢀd ) δ 10.51 (s, 1H),
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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2
2
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8.37 (d, J = 9.0 Hz, 1H), 8.20 (s, 1H), 7.15 (d, J = 8.8 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 5.51 (s,
1H), 4.47‒4.58 (m, 1H), 4.33 (d, J = 10.0 Hz, 1H), 3.75‒3.89 (m, 2H), 3.60 (d, J = 10.0 Hz, 1H),
2.67 (dt, J = 13.3, 7.9 Hz, 1H), 2.09‒2.37 (m, 3H), 1.90‒2.02 (m, 1H), 1.66 (s, 3H), 1.44‒1.60
13
(
m, 4H); C NMR (101 MHz, DMSOꢀd ) δ 155.1, 148.0, 140.6, 131.0, 127.4, 124.1, 123.5,
6
+
1
17.1, 73.7, 73.3, 67.8, 58.0, 34.3, 32.6, 30.0, 20.3, 14.3. LCMS(ESI) m/z 415 [M+H] . HRMS
+
(
EI) m/z: [M+H] calcd for C H ClN O S 415.1094, found 415.1085.
1
8
24
2
5
Synthesis
of
1ꢀ(4ꢀchloroꢀ2ꢀhydroxyꢀ3ꢀ(((S)ꢀ3ꢀmethyltetrahydrofuranꢀ3ꢀ
yl)sulfonyl)phenyl)ꢀ3ꢀ((R)ꢀ2ꢀmethylcyclopentꢀ2ꢀenꢀ1ꢀyl)urea (68). The compound 1ꢀ(4ꢀ
chloroꢀ2ꢀhydroxyꢀ3ꢀ((3ꢀmethyltetrahydrofuranꢀ3ꢀyl)sulfonyl)phenyl)ꢀ3ꢀ((R)ꢀ2ꢀmethylcyclopentꢀ
2ꢀenꢀ1ꢀyl)urea (66, 3.0 g) was separated by supercritical fluid chromatograpy (SFC) and purified
by mass detected auto purification system (MDAP) under acidic condition to afford 1ꢀ(4ꢀchloroꢀ
2
ꢀhydroxyꢀ3ꢀ(((S)ꢀ3ꢀmethyltetrahydrofuranꢀ3ꢀyl)sulfonyl)phenyl)ꢀ3ꢀ((R)ꢀ2ꢀmethylcyclopentꢀ2ꢀ
enꢀ1ꢀyl)urea (68, 666 mg, 22% yield) as a white solid. HPLC: Chiralpak IC column (4.6*250
mm, 5 uM), 1:1 ACN/IPA (containing 0.5% DEA), CO flow rate: 2.55 mL/min; coꢀsolvent flow
2
1
rate: 0.45 mL/min; back pressure: 120 bar); t = 16.9 min; >93% de; H NMR (400 MHz,
r
DMSOꢀd ) δ 10.53 (s, 1H), 8.36 (d, J = 8.8 Hz, 1H), 8.17 (s, 1H), 7.14 (d, J = 8.8 Hz, 1H), 7.05
6
(d, J = 8.3 Hz, 1H), 5.51 (s, 1H), 4.49‒4.63 (m, 1H), 4.33 (d, J = 10.3 Hz, 1H), 3.76‒3.89 (m,
2
H), 3.61 (d, J = 10.0 Hz, 1H), 2.68 (dt, J = 13.3, 7.9 Hz, 1H), 2.10‒2.35 (m, 3H), 1.91‒2.02 (m,
13
1
H), 1.67 (s, 3H), 1.44‒1.58 (m, 4H); C NMR (151 MHz, DMSOꢀd ) δ 155.1, 148.0, 140.6,
6
131.0, 127.4, 124.1, 124.1, 123.5, 117.1, 73.7, 73.3, 67.8, 58.1, 34.4, 32.6, 30.0, 20.3, 14.3.
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+
+
LCMS(ESI) m/z 415 [M+H] ; HRMS (EI) m/z: [M+H] calcd for C H ClN O S 415.1094,
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found 415.1097.
The preparation of the intermediates 73–75, 77‒82, 84‒85.
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Synthesis of (S)ꢀ2ꢀmethylcyclopentꢀ2ꢀenol (73). To a solution of (R)ꢀCBS catalyst (1 M in
o
toluene, 31.8 mL) in anhydrous THF (20 mL) at ‒60 C was added 2ꢀmethylcyclopentꢀ2ꢀenone
(
72, 15.3 g) and BH ·THF (1 M in THF, 111 mL). The mixture was stirred at this temperature
3
for 1 h. Methanol (150 ml) was added to the reaction and the resulting mixture was diluted with
brine (150 mL). The aqueous layer was extracted with DCM (2×200 mL). The combined organic
layers were dried over Na SO , filtered and concentrated in vacuo to give (S)ꢀ2ꢀmethylcyclopentꢀ
2
4
1
2ꢀenol (73, 16.0 g) as a yellow oil. H NMR (400 MHz, CDCl ) δ 5.45 (s, 1H), 4.51 (s, 1H),
3
2
.42‒1.98 (m, 4H), 1.70 (s, 3H).
Synthesis of (R)ꢀ2ꢀ(2ꢀmethylcyclopentꢀ2ꢀenꢀ1ꢀyl)isoindolineꢀ1,3ꢀdione (74). To a solution of
(S)ꢀ2ꢀmethylcyclopentꢀ2ꢀenol (73, 16.0 g) and phthalimide (36.0 g) in THF (240 mL) was added
PPh (77.0 g) under nitrogen, followed by the addition of DIAD (63.4 mL) at 0 °C. The reaction
3
mixture was stirred at 0 °C for overnight. The solvent was removed in vacuo and the resulting
residue was directly purified by column chromatography eluting with petroleum ether/ethyl
acetate (10:1) to give a crude product (74% ee), which was further purified by supercritical fluid
chromatography (SFC) to afford (R)ꢀ2ꢀ(2ꢀmethylcyclopentꢀ2ꢀenꢀ1ꢀyl)isoindolineꢀ1,3ꢀdione (74,
1
0.6 g, 28% yield over two steps, >98% ee) as a white solid. Chiral HPLC (Column: ADꢀH,
4.6*250 mm, 5 ꢁm, MeOH/CO = 10%, column temperature: 40 °C, CO flow rate: 2.7
2
2
1
mL/min): t = 2.17 min, >98% ee; H NMR (400 MHz, CDCl ) δ 7.83 (dd, J = 5.4, 3.1 Hz, 2H),
R
3
7
.76‒7.61 (m, 2H), 5.68 (s, 1H), 5.21 (d, J = 7.4 Hz, 1H), 2.69 (dd, J = 5.7, 3.5 Hz, 1H), 2.42‒
+
2.30 (m, 2H), 2.22‒2.12 (m, 1H), 1.61 (s, 3H); LCMS(ESI) m/z 228 [M+H] .
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Synthesis of (R)ꢀ2ꢀmethylcyclopentꢀ2ꢀenamine (75). To a solution of (R)ꢀ2ꢀ(2ꢀ
methylcyclopentꢀ2ꢀenꢀ1ꢀyl)isoindolineꢀ1,3ꢀdione (74, 10.7 g) in ethanol (150 mL) was added
hydrazine (85% in water, 3.0 mL). After refluxing for 3 hours, the reaction mixture was cooled
to room temperature. The precipitate was filtered and the filter cake was rinsed with EtOH (10
mL). To the filtrate was added HCl in dioxane (4 M, 5 mL) and the mixture was concentrated.
The resulting residue was dissolved in water, and then freeze dried to afford (R)ꢀ2ꢀ
1
1
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methylcyclopentꢀ2ꢀenamine hydrochloride salt (75, 6.3 g, 100% yield) as a brown solid, which
1
was used in the next step without purification. H NMR (400 MHz, DMSOꢀd ) δ 8.27 (3 H, br.
6
s.), 5.68 (1 H, s), 3.87‒4.02 (1 H, m), 2.32‒2.48 (1 H, m), 2.15‒2.30 (2 H, m), 1.71‒1.85 (1 H,
m), 1.78 (3 H, s).
Synthesis
of
ethyl
2ꢀ((2ꢀ(tertꢀbutyl)ꢀ6ꢀchlorobenzo[d]oxazolꢀ7ꢀyl)thio)ꢀ2ꢀ
methylpropanoate (77). To a solution of 2ꢀ(tertꢀbutyl)ꢀ6ꢀchlorobenzo[d]oxazoleꢀ7ꢀthiol (76,
11.0 g) in DMF (80 mL) at room temperature was added ethyl 2ꢀbromoꢀ2ꢀmethylpropanoate (9.0
g) and K CO (2.9 g). After stirring at 60 °C for 3 h, the reaction mixture was diluted with ethyl
2
3
acetate (200 mL) and washed by brine (3×150 mL). The organic layer was dried over sodium
sulfate, filtered and concentrated in vacuo to afford ethyl 2ꢀ((2ꢀ(tertꢀbutyl)ꢀ6ꢀ
chlorobenzo[d]oxazolꢀ7ꢀyl)thio)ꢀ2ꢀmethylpropanoate (77, 14.8 g) as a white solid. LCMS(ESI)
+
m/z 356 [M+H] .
Synthesis
of
ethyl
2ꢀ((2ꢀ(tertꢀbutyl)ꢀ6ꢀchlorobenzo[d]oxazolꢀ7ꢀyl)sulfonyl)ꢀ2ꢀ
methylpropanoate (78). To a solution of ethyl 2ꢀ((2ꢀ(tertꢀbutyl)ꢀ6ꢀchlorobenzo[d]oxazolꢀ7ꢀ
yl)thio)ꢀ2ꢀmethylpropanoate (77, 14.8 g) in dichloromethane (200 mL) at room temperature was
added mꢀCPBA (20.6 g). The mixture was stirred at room temperature overnight. The reaction
mixture was added saturated aqueous Na SO solution and was washed with saturated sodium
2
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carbonate solution (100 mL) and brine (100 mL). The organic phase was dried over sodium
sulfate, filtered and concentrated in vacuo. The resulting residue was purified by column
chromatography eluting with 0‒40% ethyl acetate in petroleum ether to give ethyl 2ꢀ((2ꢀ(tertꢀ
butyl)ꢀ6ꢀchlorobenzo[d]oxazolꢀ7ꢀyl)sulfonyl)ꢀ2ꢀmethylpropanoate (78, 10.3 g, 64% yield over
0
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2
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4
5
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8
9
0
1
2
3
4
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7
8
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+
two steps) as a white solid. LCMS(ESI) m/z 388 [M+H] .
Synthesis of 2ꢀ((2ꢀ(tertꢀbutyl)ꢀ6ꢀchlorobenzo[d]oxazolꢀ7ꢀyl)sulfonyl)ꢀ2ꢀmethylpropanꢀ1ꢀol
(79). To a solution of ethyl 2ꢀ((2ꢀ(tertꢀbutyl)ꢀ6ꢀchlorobenzo[d]oxazolꢀ7ꢀyl)sulfonyl)ꢀ2ꢀ
methylpropanoate (78, 5.0 g) in THF (130 mL) under nitrogen at ‒50 °C was added DIBALꢀH (1
M in hexane, 64.5 mL). The reaction mixture was allowed to warm to room temperature and
stirred at room temperature for overnight. The reaction mixture was quenched with saturated
ammonium chloride (20 mL), and then partitioned between ethyl acetate (150 mL) and sodium
hydroxide solution (2 M, 150 mL). The organic phase was washed with saturated brine (150 mL),
dried over sodium sulphate and evaporated in vacuo to give 2ꢀ((2ꢀ(tertꢀbutyl)ꢀ6ꢀ
chlorobenzo[d]oxazolꢀ7ꢀyl)sulfonyl)ꢀ2ꢀmethylpropanꢀ1ꢀol (79, 4.5 g) as a white solid.
+
LCMS(ESI) m/z 346 [M+H] .
Synthesis of 2ꢀ((2ꢀ(tertꢀbutyl)ꢀ6ꢀchlorobenzo[d]oxazolꢀ7ꢀyl)sulfonyl)ꢀ2ꢀmethylpropyl
trifluoromethanesulfonate (80). To a solution of 2ꢀ((2ꢀ(tertꢀbutyl)ꢀ6ꢀchlorobenzo[d]oxazolꢀ7ꢀ
yl)sulfonyl)ꢀ2ꢀmethylpropanꢀ1ꢀol (79, 5.2 g) in dichloromethane (100 mL) under nitrogen at
room temperature was added pyridine (2.1 mL) and triflic anhydride (1 M in dichloromethane,
19.4 mL). The mixture was stirred at room temperature for 1 h, and then partitioned between
dichloromethane (100 mL) and brine (100 mL). The organic phase was dried over anhydrous
sodium sulphate and evaporated in vacuo to give 2ꢀ((2ꢀ(tertꢀbutyl)ꢀ6ꢀchlorobenzo[d]oxazolꢀ7ꢀ
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yl)sulfonyl)ꢀ2ꢀmethylpropyl trifluoromethanesulfonate (80, 6.5 g) as a white solid. LCMS(ESI)
+
m/z 478 [M+H] .
Synthesis
of
2ꢀ(tertꢀbutyl)ꢀ6ꢀchloroꢀ7ꢀ((1ꢀfluoroꢀ2ꢀmethylpropanꢀ2ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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5
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1
2
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1
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6
7
8
9
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1
2
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7
8
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yl)sulfonyl)benzo[d]oxazole (81). To a solution of 2ꢀ((2ꢀ(tertꢀbutyl)ꢀ6ꢀchlorobenzo[d]oxazolꢀ7ꢀ
yl)sulfonyl)ꢀ2ꢀmethylpropyl trifluoromethanesulfonate (80, 6.5 g) in THF (100 mL) at ‒15 °C
was added TBAF (1 M in THF, 27.2 mL) dropwise. The reaction mixture was allowed to warm
to 30 °C gradually and stirred for 2 h. The solvent was evaporated and the residue was
partitioned between ethyl acetate (100 mL) and saturated brine (100 mL). The organic phase was
dried over anhydrous sodium sulphate and evaporated in vacuo. The resulting residue was
purified by column chromatography by eluting with 0‒50% ethyl acetate in petroleum ether to
afford 2ꢀ(tertꢀbutyl)ꢀ6ꢀchloroꢀ7ꢀ((1ꢀfluoroꢀ2ꢀmethylpropanꢀ2ꢀyl)sulfonyl)benzo[d]oxazole (81,
+
2
.4 g, 46% yield over three steps) as a white solid. LCMS(ESI) m/z 348 [M+H] .
Synthesis of 6ꢀaminoꢀ3ꢀchloroꢀ2ꢀ((1ꢀfluoroꢀ2ꢀmethylpropanꢀ2ꢀyl)sulfonyl)phenol (82). To
a solution of 2ꢀ(tertꢀbutyl)ꢀ6ꢀchloroꢀ7ꢀ((1ꢀfluoroꢀ2ꢀmethylpropanꢀ2ꢀyl)sulfonyl)benzo[d]oxazole
81, 2.4 g) in 1,4ꢀdioxane (20 mL) was added 37% HCl solution (20 mL). The reaction mixture
was refluxed for 4 h, and then concentrated in vacuo. The residue was dissolved in ethyl acetate
20 mL) and the pH of the solution was adjusted to 8 with triethylamine. The organic phase was
(
(
washed with brine (20 mL), dried over sodium sulphate, and concentrated in vacuo. The
resulting residue was purified by column chromatography eluting with 0‒80% ethyl acetate in
petroleum ether to afford 6ꢀaminoꢀ3ꢀchloroꢀ2ꢀ((1ꢀfluoroꢀ2ꢀmethylpropanꢀ2ꢀyl)sulfonyl)phenol
1
(
82, 1.9 g, 98% yield) as a white solid. H NMR (400 MHz, CDCl )
δ
10.18 (1 H, s), 6.89 (1 H,
3
d, J = 8.8 Hz), 6.82 (1 H, d, J = 8.8 Hz), 4.62 (2 H, d, J = 41.6 Hz), 4.02 (2 H, br.s), 1.49 (6 H,
+
s). LCMS(ESI) m/z 282 [M+H] .
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Synthesis
of
2ꢀ(tertꢀbutyl)ꢀ6ꢀchloroꢀ7ꢀ((3ꢀmethyltetrahydrofuranꢀ3ꢀ
yl)sulfonyl)benzo[d]oxazole (84). To a solution of 2ꢀ(tertꢀbutyl)ꢀ6ꢀchloroꢀ7ꢀ((tetrahydrofuranꢀ3ꢀ
o
yl)sulfonyl)benzo[d]oxazole (83, 4.3 g) and methyl iodide (2.0 mL) in THF (100 mL) at ‒70 C
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was added LHMDS (1 M in THF, 31.3 mL). The reaction mixture was gradually warmed to
room temperature and stirred at room temperature for 30 min. The reaction mixture was diluted
with saturated NH Cl solution (100 mL) and extracted with ethyl acetate (2×150 mL). The
4
combined organic layers were dried over sodium sulphate, filtered and concentrated in vacuo to
afford 2ꢀ(tertꢀbutyl)ꢀ6ꢀchloroꢀ7ꢀ((3ꢀmethyltetrahydrofuranꢀ3ꢀyl)sulfonyl)benzo[d]oxazole (84,
+
4
.4 g). LCMS(ESI) m/z 358 [M+H] .
Synthesis of 6ꢀaminoꢀ3ꢀchloroꢀ2ꢀ((3ꢀmethyltetrahydrofuranꢀ3ꢀyl)sulfonyl)phenol (85). To
a solution of 2ꢀ(tertꢀbutyl)ꢀ6ꢀchloroꢀ7ꢀ((3ꢀmethyltetrahydrofuranꢀ3ꢀyl)sulfonyl)benzo[d]oxazole
84, 4.4 g) in 1,4ꢀdioxane (150 mL) was added 37% HCl solution (30.3 mL). The mixture was
refluxed overnight, and then concentrated in vacuo. The residue was dissolved in ethyl acetate
100 mL). After pH of the solution was adjusted to 8 with saturated NaHCO solution, the
(
(
3
organic layer was washed with saturated brine, dried over sodium sulphate, filtered and
concentrated in vacuo. The resulting residue was purified by column chromatography eluting
with 0‒80% ethyl acetate in petroleum ether to afford 6ꢀaminoꢀ3ꢀchloroꢀ2ꢀ((3ꢀ
1
methyltetrahydrofuranꢀ3ꢀyl)sulfonyl)phenol (85, 2.4 g, 66% yield over two steps). H NMR (400
MHz, DMSOꢀd ) δ 6.89‒6.96 (2 H, m), 4.32 (1 H, d, J = 10.0 Hz), 3.76‒3.89 (2 H, m), 3.60 (1
6
H, d, J = 10.0 Hz), 2.67 (1 H, dt, J = 13.3, 7.8 Hz), 1.93 (1 H, ddd, J = 13.1, 7.3, 5.3 Hz), 1.48 (3
+
H, s). LCMS(ESI) m/z 292 [M+H] .
Biology and DMPK. All studies were conducted in accordance with the GSK Policy on the
Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal
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Care and Use Committee either at GSK or by the ethical review process at the institution where
the work was performed. The human biological samples were sourced ethically and their
research use was in accord with the terms of the informed consents.
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CXCR2 Tango Assay. The assay measures ligandꢀinduced activation of the receptor CXCR2
in a stable cell line containing the recombinant human CXCR2 linked to a TEV protease site and
a Gal4ꢀVP16 transcription factor (Invitrogen). Ligand binding to the receptor results in the
recruitment of arrestin proteins (tagged with protease) to the receptor and triggers the release of a
tethered transcription factor. The transcription factor enters the nucleus and activates the
transcription of the reporter gene. The ability of a compound to inhibit CXCR2 activation is
indirectly assessed by measuring the reporter gene activity.
Cells were starved for 24 h at 37 ºC, 5% CO , harvested and reꢀsuspended in assay medium at
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a density ~200,000 cells/mL. 50 nL/well of testing compounds in 100% DMSO solution were
dispensed into the assay plate (Greiner 781090) using Echo 555 (Labcyte) with concentrationꢀ
response program. The cellꢀfree and unꢀstimulated control wells were loaded with 50 nL/well
pure DMSO to ensure that the DMSO concentration was constant across the plate for all assays.
5
0 ꢁL of assay medium, cell suspension without hCXCL1 (a CXCR2 ligand) and cell suspension
with 80 nM hCXCL1 were added to cellꢀfree control wells, unꢀstimulated control wells and the
rest of wells, respectively. The cells were incubated overnight at 37 ºC/5% CO and then 10 µL
2
of 6× substrate mixture [LiveBLAzer™ꢀFRET B/G substrate (CCF4ꢀAM) Cat # K1096 from
Invitrogen, Inc.] was dispensed into each well using Bravo and the plates were incubated at room
temperature for another 2 h in the dark before reading on EnVision using one excitation channel
(409 nm) and two emission channels (460 nm and 530 nm). The blue/green emission ratio (460
nm/530 nm) was calculated for each well, by dividing the cellꢀfree backgroundꢀsubtracted Blue
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emission values by the backgroundꢀsubtracted Green emission values. The concentration
response curves were fitted based on sigmoidal concentrationꢀresponse model to return IC and
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intrinsic activity (IA, percentage of maximum inhibition).
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Pꢀgp/BCRP Efflux Ratio (ER) and Cell Permeability Assay. Polarized MadinꢀDarby canine
kidney (MDCKII) cells stably transfected with human Pꢀgp (MDCKIIꢀMDR1 cell line) were
obtained from The Netherlands Cancer Institute (Amsterdam, The Netherlands). MDCKIIꢀ
MDR1 cells transduced with BacMam2ꢀBCRP according to experimental protocols reported
33b
previously were used for the in vitro transport studies in the Transwell system. The procedures
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of cell culture and transport studies were described previously. The transport of test compounds
(3 µM) was measured in duplicates in two directions [apical to basolateral (A → B) and
basolateral to apical (B→A)] or one direction [apical to basolateral (A→B)] after 90 min
incubation in the absence and presence of GF120918 (5 µM, a dual Pꢀgp and BCRP inhibitor).
The passive permeability (Pexact, nm/s) for test compounds across MDCKII monolayers were
3
3b
calculated using the equation reported previously. Efflux ratio (ER) is defined as the ratio of
permeability of basolateralꢀtoꢀapical to apicalꢀtoꢀbasolateral direction in the absence of
GF120918. The permeability ratio is defined as the ratio of permeability of apicalꢀtoꢀbasolateral
direction in the presence to absence of GF120918. The functionality of human Pꢀgp and BCRP
transporters in this in vitro system was qualified by typical ERs of amprenavir (a Pꢀgp specific
substrate) and dantrolene (a BCRP specific substrate) greater than 12 and 5, respectively.
Human Whole Blood CD11b Assay. The whole blood assay tested the compounds’ ability to
inhibit the upregulation of CD11b in GROꢀα stimulated neutrophils in human whole blood.
Blood was withdrawn by venipuncture from consented adults and poured into a Sterilin tube
containing an antiꢀcoagulant Heparin (10 uL/mL of blood). All the test compounds were
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screened at a top 10 µM final assay concentration, (1 in 3 serial dilution to provide 10ꢀpoint dose
response curves) with a final DMSO concentration to 0.25% after addition of blood.
10 uL of blood was transferred to the compound plate using a multiꢀchannel pipette, gently
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tapped and incubated for 15 min, at 37 C. After 15 min, the stimulant GROꢀα was diluted to 100
nM in 0.1% BSA (Albumin Bovine Serum)ꢀPBS and 5 uL is dispensed across the whole plate for
a final concentration of 33 nM. The plate was gently tapped and incubated further for 15 min at
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7 C. The plate was placed on ice for 1 min before addition of 10 uL of an antibody cocktail
consisting of CD11bꢀFITC (40 µg/mL) purchased from BioLegend (address: Cambridge
Bioscience, Munro House, Trafalgar Way, Bar Hill, Cambridge, UK) and CD16ꢀPE purchased
from BD Pharmingen (address: The Danby Building, Edmung Halley Road, Oxford Science
Park, Oxford, UK) (stock concentration 100 tests, 2 mL stock volume is diluted 1 in 5). The plate
was placed on ice for 1 h in the dark. The cells were then fixed using 200 µL/well of 1x FACS
(Flow Activated Cell Sorting) Lyse solution (Becton and DickinsonꢀBD) and on ice for 20 min
in the dark. The plate was centrifuged at 1600 rpm for 5 min and reꢀsuspended with 200 µL of
ice cold PBS. This step was repeated twice and on the final step the plate was reꢀsuspended with
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0 µL of ice cold PBS for flow cytometric analysis.
Samples were run on a Becton and Dickinson (BD)ꢀAcurri C6 Flow Cytometer using a
HyperCyt sampling apparatus (IntelliCyt) with a flow rate 2 µL/sec. CD11b upregulation is
monitored in neutrophils and identified using a combination of both side scatter and CD16
expression.
Mouse Whole Blood CD11b Assay. 50 µL freshly isolated whole blood with antiꢀcoagulant
heparin (10 uL/mL of blood) from C57 mice was pretreated with CXCR2 compounds at
concentrations from 1.5 nM to 10 µM in 96ꢀUꢀwellꢀplate (Corning, NY, US, Cat. #3799).
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Samples were gently mixed with multiꢀchannel pipette and incubated for 2 h in 37 °C incubator.
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5 µL bloodꢀcompound mixture in each well was transferred into 96ꢀVꢀwellꢀplate (Corning, NY,
US, Cat. #3897) for the secondary treatment with 100 nM murine CXCL1 (Pepro Tech, NJ, US,
Cat. #250ꢀ11) in DPBS (ThermoFisher, PA, USA, Cat. #14190250)/0.1% BSA (Sigma, MO, US,
Cat. #85040C). Samples were incubated for 10 min in 37 °C water bath with gentle agitation at 5
min. Then samples were placed on ice and fixed with 150 µL of ice cold CellFix solution (1%
formaldehyde/0.1% azide/DPBS further 1:4 diluted in Robosep buffer from Stemcell
Technologies, Vancouver, Canada, Cat. #20104) for 1 min. 50 µL of each sample was
transferred to 96 Vꢀwellꢀplate preloaded with 5 µL antiꢀCD11bꢀFITC (ThermoFisher, PA, US,
Cat. #11ꢀ0112ꢀ82) or isotype control of FITCꢀIgG2a (BD Biosciences, NJ, US, Cat. #553456).
Samples were gently mixed and incubated for 20 min in the dark on ice. 200 µL freshly prepared
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×Red blood lysis buffer (BD Biosciences, NJ, US, Cat. #555899) was added into antibodyꢀ
blood mixture for red blood cells lysis. This usually took 3‒5 min. Samples were centrifuged at
000 rpm for 5 min, and supernatant was discarded with replacement of 250 µL ice cold and
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sterile 1% FBS (ThermoFisher, PA, USA, Cat. #10099141)/DPBS.
Samples were gently mixed for FACS reading within 1hr with BD FACS Canto II flow
cytometer and FACsDiva Software. CD11b population was analyzed by firstly gating on the
granulocyte population on the FSC vs. SSC plot, and then gating on the CD16+ cells of
neutrophil within the granulocyte population. As CD16+ cells usually take up 95% of
granulocytes, this step is usually omitted. Therefore, mean FITC signal of granulocyte
population subtracted by mean fluorescence of isotype control can reflect specific CD11b
content.
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Rat SOA for CNS Penetration. The blood brain barriers (BBB) separate the brain from the
peripheral circulation. These barriers have tight junctions and also multiple efflux transporters to
prevent CNS exposure of compounds to reach their CNS molecular targets. CNS penetration
properties of CXCR2 compounds were evaluated in the male HanꢀWistar rats after oral gavage at
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mg/kg. Test compound was formulated as homogenous suspension in a dosing vehicle
containing DMSO: 1% methylcellulose (w/v) = 1:99. At each time point post dose (0.25 h, 0.5 h,
h, 2 h, 4 h, 7 h), blood samples (> 60 ꢁL/time point/each site) were collected via cardiac
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puncture, into separate EDTAꢀK (0.5 M) containers following anaesthesia with isofluorane.
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Exact 50 uL of blood samples were diluted with 150 ꢁL of diH O (1:3), snap frozen on dry ice
2
and further stored at ‒80 °C. Immediately after blood collection, the brains from all the animals
were excised, transferred suitable containers and immediately stored frozen. Brain were
homogenized with 3ꢀfold PBS (w/v) and further stored at ‒80 °C (dilution factor = 3.9).
Two separate calibration curves were prepared in blank blood/water mixture and brain
homogenate respectively. Study samples and their corresponding calibration curves were
extracted by protein precipitation. Exact 50 µL sample was aliquot into a clean extract plate and
then extracted with 150 µL of acetonitrile containing analytical internal standard. After mixing
and centrifugation, approximately 100 µL of supernatant was removed into an injection plate and
sample was further diluted with same volume of deionized water. Quantitation of test compound
was carried out by UPLC/MS/MS using an optimized methodology.
Systemic and brain exposure were estimated by AUC up to the last time point at 7 h. CNS
penetration potential (K ) of test compounds was evaluated from the ratio of AUCbrain/AUCblood
.
p
Kp,uu value was calculated using the measured K , free unbound compound fractions in blood and
p
brain.
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