Development of Novel Alkoxyisoxazoles as Sigma-1 Receptor Antagonists with Antinociceptive Efficacy

Article abstract of DOI:10.1021/acs.jmedchem.6b00571

A novel series of sigma (σ) receptor ligands based on an alkoxyisoxazole scaffold has been designed and synthesized. Preliminary receptor binding assays identified highly potent (K_i < 1 nM) and selective σ1 ligands devoid of binding interactions with the monoamine transporters DAT, NET, and SERT. In particular, compound 53 was shown to possess significant antinociceptive activity in the mouse formalin-induced inflammation pain model when administered intraperitoneally at 40 and 80 mg/kg. Initial pharmacokinetics evaluation indicated an excellent brain exposure following oral dosing in mice, suggesting that further investigation into the use of alkoxyisoxazoles as σ1 ligands for antinociception is warranted. This study supports the notion that selective σ1 antagonism could be a useful strategy in the development of novel antipain therapy.

Full text of DOI:10.1021/acs.jmedchem.6b00571

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Article
Development of Novel Alkoxyisoxazoles as Sigma-1
Receptor Antagonists with Anti-Nociceptive Efficacy
Hao Sun, Min Shi, Wei Zhang, Yue-Ming Zheng, Yazhou Xu, Jun-Jie Shi, Ting Liu,
Hendra Gunosewoyo, Tao Pang, Zhao-Bing Gao, Fan Yang, Jie Tang, and Li-Fang Yu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00571 • Publication Date (Web): 16 Jun 2016
Downloaded from http://pubs.acs.org on June 19, 2016
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Development of Novel Alkoxyisoxazoles as Sigma-1
Receptor Antagonists with Anti-Nociceptive Efficacy
Hao Sun,† Min Shi, † Wei Zhang, † YueꢀMing Zheng, § YaꢀZhou Xu, ‡ JunꢀJie Shi, † Ting Liu, † Hendra
Gunosewoyo, ┴ Tao Pang, ‡ ZhaoꢀBing Gao, § Fan Yang, † Jie Tang, *, †, LiꢀFang Yu *, †
†Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development,
School of Chemistry and Molecular Engineering, East China Normal University, 3663 North
Zhongshan Road, Shanghai 200062, China
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§CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 501 Hai Ke Road, Shanghai 201203, China.
‡Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, Nanjing 210009, PR China
┴School of Pharmacy, Faculty of Health Sciences, Curtin University, Bentley, Perth, WA 6102, Australia
Shanghai Key Laboratory of Green Chemistry and Chemical Process, School of Chemistry and
Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062,
China
ABSTRACT
A novel series of sigma (σ) receptor ligands based on an alkoxyisoxazole scaffold has been designed
and synthesized. Preliminary receptor binding assays identified highly potent (Ki < 1 nM) and selective σ1
ligands devoid of binding interactions with monoamine transporters DAT, NET and SERT. In particular,
compound 53 was shown to possess significant antiꢀnociceptive activity in the mouse formalinꢀinduced
inflammation pain model when administered intraperitoneally at 40 and 80 mg/kg. Initial
pharmacokinetics evaluation indicated an excellent brain exposure following oral dosing in mice,
suggesting that further investigation into the use of alkoxyisoxazoles as σ1 ligands for antiꢀnociception is
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warranted. This study supports the notion that selective σ1 antagonism could be a useful strategy in the
development of novel antiꢀpain therapy.
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INTRODUCTION
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Sigma (σ) receptors belong to a unique class of receptors that is distinct from the opioid and NꢀmethylꢀDꢀ
aspartate/phencyclidine (NMDA/PCP) receptors. Initially classified as an opioid receptor subtype by
Martin in 19761 and subsequent confusion with the PCP binding site of NMDA receptor, σ receptors are
nowadays referred to as central nervous system (CNS) proteins with no homology to known Gꢀprotein
coupled receptors but are capable of influencing opioid actions. Two types of σ receptors have been
identified, namely σ1 (25–29 kDa) and σ2 (18–22 kDa), both of which have been implicated in the
pathophysiology of various neurological disorders2 and cancer3, respectively.
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The cloned σ1 receptor4 encodes for a protein of 223 amino acid with two transmembrane domains
located at the mitochondrialꢀassociated endoplasmic reticulum membrane (MAM)5 and regulates
endoplasmic reticulum (ER)–mitochondrion calcium signaling and cell survival.6 Sigma 1 receptor
ligands trigger the translocation of σ1 receptor from MAM to other parts of the cell thereby modulating
the activities of various ion channels, receptors, or kinases, thus affecting each stage of neuronal
transmission. Su et al. reported that σ1 receptors modulate inositol 1,4,5ꢀtrisphosphate (IP3) function by
forming a trimeric complex with IP3 receptor and its inhibitory protein ankyrin B.7 Acute binding of σ1
receptor agonist was found to remove ankyrin B from IP3 receptor thereby enhancing the calcium release
from ER into the cytosol in NGꢀ108 cells. Moreover, σ1 receptor agonists are likely to exert their effects
by dissociating σ1 receptor chaperones from bindingꢀimmunoglobulin protein (BiP).8 This is in contrast to
the σ1 receptor antagonists which do not affect the σ1 receptorꢀBiP association and rather stabilize this
complex. The σ2 receptor on the other hand has not yet been cloned but is known to be overexpressed in
aggressive tumor cells. Unlike the σ1 receptor that readily translocates upon activation, σ2 receptor is
localized within the progesterone receptor membrane component 1 (PGRMC1) protein known to be
associated with cell proliferation.3 The σ2 receptor is regarded as a reliable biomarker in oncology as
proliferating cancer cells are found to have a 10ꢀfold higher density of σ2 receptor compared to the
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dormant cancer cells.3 The progress towards understanding of the precise function of σ receptors however,
has been hampered partly due to the lack of conclusive evidence on certain endogenous ligand(s) that
selectively bind to these receptors. N,NꢀDimethyltryptamine (DMT) was suggested to be a low potency
endogenous ligand for the σ1 and σ2 receptors.9 Other endogenous σ1 ligands such as Dꢀerythroꢀ
sphingosine and sphinganine have also been studied. (+)ꢀPentazocine (1) (Ki, σ1 = 3.1 nM, Ki, σ2 = 1542
nM) and DTG (2, 1,3ꢀdiꢀoꢀtolylguanidine, Ki, σ1 = 15 nM, Ki, σ2 = 28.4 nM) are now commonly used as the
radioligand of choice to study the σ1 and σ2 receptor binding.
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Recent clinical and preclinical findings suggested the implication of σ1 receptor in various CNS
disorders. This is best demonstrated by reports on the use of various σ1 receptor antagonists in drug
addiction,10 including alcohol, cocaine, and methamphetamine, as well as HIVꢀassociated neurocognitive
disorder and pain.11, 12 Interestingly, σ1 receptor agonists are also reported to possess beneficial effects in
neurodegenerative disorders,13 such as stroke,14 Alzheimer’s disease,15 pseudobulbar affect,16 depression
and anxiety disorders17. Of particular interest, a considerable body of evidence lends support to the
hypothesis that antagonism of σ1 receptor results in antinociceptive effect. The σ1 receptor knockꢀout
mouse showed decreased pain responses in the formalin test and no mechanical hypersensitivity following
capsaicin sensitization or sciatic nerve injury.18, 19 Pasternak group demonstrated that σ1 receptor agonist
(+)ꢀpentazocine reduced the analgesic effect of ꢀ opiate morphine but not the inhibition of gastrointestinal
transit.20 The administration of classical σ1 receptor antagonist haloperidol (3) and its metabolites resulted
in antinociceptive effects in the mouse formalin test.21 The most clinically advanced selective σ1 receptor
antagonist based on a morpholinyl pyrazole scaffold S1RA (Eꢀ52862, 4) is currently in the phase II trials
for treatments of diverse pain states.22 Although inactive when administered alone, this compound
enhanced the antinociceptive effect of morphine in mouse tail flick test and thus proposed as opioid
adjuvants, offering novel approach in pain management.23 Other recently reported selective σ1 receptor
antagonists showing antinociceptive effects in animal models of pain include the 4ꢀaminotriazole 5，
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hexahydroꢀ2Hꢀpyrano[3,2ꢀc]quinoline 6, and 5ꢀchloroꢀ2ꢀ(4ꢀchlorophenyl)ꢀ4ꢀmethylꢀ6ꢀ(3ꢀ(piperidinꢀ1ꢀ
yl)propoxy)pyrimidine 7.24ꢀ26
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During our previous campaign on potent and selective α4β2 partial agonists, compounds 8 and 9 were
identified as novel selective σ1 ligands resulting from a comparative analysis of the pharmacophoric
elements required for σ1 and nicotinic ligands.27 Compounds 8 and 9 displayed high affinity for the σ1
receptor (8: Ki, σ1 = 33 nM, Ki, σ2 = 1472 nM; 9: Ki, σ1 = 4.1 nM, Ki, σ2 = 1312 nM) in the National
Institute of Mental HealthꢀPsychoactive Drug Screening Program (NIMHꢀPDSP) broad screening panel of
42 common CNS neurotransmitter transporters and receptors, while possessing good to moderate affinity
for DAT (8: Ki, DAT = 24 nM; 9: Ki, DAT = 373 nM) and NET (8: Ki, NET = 191 nM; 9: Ki, NET = 203
nM). Considering the good preliminary ADMEꢀTox profile and brain uptake for this series of compounds,
current efforts are undertaken to ultimately identify selective σ1 antagonists and assess their analgesic
properties alone or in combination with clinicallyꢀused opioids. Herein, we report the rational design and
synthesis of isoxazoleꢀbased compounds as σ1 ligands. Highly potent and selective analogues devoid of
binding interactions with DAT or NET are identified, with the pharmacokinetic properties and in vivo
antiꢀnociceptive efficacy assessed for the best compound.
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Figure 1. Selected σ receptor ligands.
RESULTS AND DISCUSSION
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Chemistry
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Analogues of alkoxyisoxazoles 8 and 9 were designed based on the following principles: 1) adherence
with the σ1 receptor pharmacophore requirements; 2) improve selectivity over nicotinic receptors and
monoamine transporters; 3) compounds can be accessed from readily available starting materials. We
previously reported that 3ꢀalkoxyisoxazoles, originally developed for α4β2 nicotinic acetylcholine
receptors, could be readily switched upon slight chemical modifications to a scaffold that is selective for
σ1 receptor.27, 28 In this study, three regions of the isoxazoles based on the previously identified selective
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σ1 ligand compound 9 were selected for SAR investigation: hydrophobic region, linker L, and the Nꢀ
region (Figure 2), keeping the core isoxazole intact.
The 3ꢀalkoxyisoxazoles 15–33 were synthesized starting from commercially available dimethyl 2ꢀ
butynedioate (10) in 5–8 steps utilizing the synthetic routes shown in Scheme 1. Alkyne 10 and Nꢀ
hydroxyurea underwent a [2+3] intermolecular cycloaddition to form isoxazole 11, which was
subsequently reacted with Bocꢀprotected 2(S)ꢀpyrrolidinylmethanol via Mitsunobu reaction to form ester
12. Basic hydrolysis or lithium borohydride reduction of ester 12 furnished the acid 13 and primary
alcohol 14 respectively. Final compounds 15–17 were synthesized through condensation of acid 13 with
phenol or indoline followed by deprotection of Boc group. Alcohol 14 was initially converted to the
iodide and subsequently reacted with benzenethiol or substituted phenols to form the thioether 18 and
aromatic ethers 20–33. Carbamate 19 was prepared by treatment of intermediate alcohol 14 with
phenylisocyanate. After acidic deprotection final compounds 18–33 were obtained as trifluoroacetates or
hydrochlorides.
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Scheme 1a
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a
Reagents and conditions: (a) Nꢀhydroxyurea, 1,5ꢀdiazabicyclo[5.4.0]undecꢀ5ꢀene, MeOH, 0 °C, then
HCl; (b) 1ꢀ(tertꢀbutoxycarbonyl)ꢀ2(S)ꢀpyrrolidinylmethanol, diisopropyl azodicarboxylate, PPh3, THF,
0 °C to rt; (c) NaOH, THF, rt; (d) DCC, DMAP, phenol, CH2Cl2, rt; (e) TFA/CH2Cl2, rt, or HCl/EtOAc, rt;
(f) EDC, DMAP, aniline or indoline, rt; (g) LiBH4, THF, 0 °C to rt; (h) I2, PPh3, imidazole, CH2Cl2, 0 °C
to rt; (i) phenol, or naphthalenꢀ2ꢀol, or substituted phenol, K2CO3, DMF, rt; (j) PhSH, K2CO3, DMF, rt; (k)
PhNCO, DMAP, CH2Cl2, rt.
Compounds 34–39 were synthesized in a similar manner to parent compound 927, 28 from methyl 3ꢀ
hydroxyisoxazoleꢀ5ꢀcarboxylate 11 with corresponding alcohols 40–43 (Scheme 2). The Nꢀalkylated
analogues 44–59 were synthesized via reductive amination with the corresponding aldehydes or ketones.
All final compounds were purified by HPLC or recrystallization as trifluoroacetates or hydrochlorides
respectively.
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Scheme 2a
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9
R
R
a
O
O
OH
OH
O
O
N
N
N
H
N
O
N
O
H
40
44: R = H
45: R = 3-F
9: R = H
21
: R = 3-F
24: R = 3-Cl
46: R = 3-Cl
a
a
O
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O
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H
N
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N
O
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O
H
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N
HN
O
OH
HN
O
O
O
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N
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O
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O
OH
b or
c
R1
O
O
R1
R1
N
R2
N
O
N
N
N
O
H
H
43
36-39
49-59
a Reagents and conditions: (a) HCHO, CF3CH2OH, NaBH4, 40 °C; (b) aldehyde or ketone, Na(CN)BH3,
CH3CO2H, CH3OH, rt; (c) (ClCH2CH2)2O, NaI, K2CO3, DMF.
Scheme 3a
a Reagents and conditions: (a) KHCO3, DMF, ꢀ10 °C to rt then 1N HCl; (b) i. NꢀBoc piperazine, tBuOH,
Na2CO3, reflux; ii. imidazole, I2, PhMe, 110 °C; (c) LiBH4, THF; (d) PPh3, imidazole, I2, rt; (e) phenol,
K2CO3, DMF, rt; (f) HCl, EtOAc, rt.
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Compounds 65–69 were synthesized by employing similar strategy as described in Scheme 3. 1,1ꢀ
1
2
Dibromoformaldoxime 60 and tertꢀbutyl acrylate 61 underwent intermolecular cycloaddition under basic
condition to form intermediate dihydroisoxazole 62. Additionꢀelimination with appropriate amine in basic
condition, followed by oxidation with iodine afforded isoxazole 63. This intermediate was reduced with
lithium borohydride and reacted with iodine/triphenylphosphine to give halide 64. Subsequent reaction
with phenol in the presence of potassium carbonate and Boc deprotection gave the desired final
compounds 65–69.
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In Vitro Characterization—Radioligand Binding Studies29
In vitro binding affinities of compounds 15–33 at σ1 and σ2 receptors were determined by the standard
[3H]pentazocine and [3H]DTG binding assay with rat brain homogenate and P12 cells respectively (Table
1). Keeping the pyrrolidine group intact on 3ꢀisoxazole position, we first looked at the effects of changing
linker L from a methylene ether to ester (compound 15), amide (compounds 16 and 17), thioether 18 and
carbamate 19. Ester 15 turned out to be completely inactive both at σ1 and σ2 receptors while the amide
16 had approximately 30ꢀfold reduction on the affinity to σ1 receptor. Substituting the benzene ring for a
bulkier indoline resulted in a 9ꢀfold improvement in σ1 binding affinity (16 vs 17), but still 3ꢀfold less
potent compared to the original methylene ether 9. Thioether or carbamate linkers (18 and 19) also
resulted in a decrease in σ1 affinity (~16ꢀ to 74ꢀfold vs compound 9), therefore prompted us to retain
linker L as a methylene ether and investigate the effects of phenyl substitution (R1).
The hydrophobic region R1 typically occupied by a benzene ring is generally deemed an important
pharmacophoric element as seen in most of σ1 ligands.30 In our previous work, we demonstrated that
replacing a benzene group at R1 with small aliphatic groups, e.g. methyl and cyclopropylmethyl, was
deleterious for σ1 affinity.27 A 5ꢀchloropyridyl substitution also resulted in a less potent σ1 ligand (Ki σ1
103 nM, σ2 >10,000 nM). Next iteration of SAR studies was thus focused on assessing the effects of
electron withdrawing group (EWG; F, Cl, Br, CF3) and electron donating group (EDG; OMe, Me)
substituents on the benzene ring. A fluoro or chloro monosubstitution (compounds 20–25) generally
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retained affinity at σ1 receptor, with no higher than a 3ꢀfold decrease or increase relative to parent
compound 9. Unfortunately, this was accompanied with an unwanted improvement at σ2 receptor binding,
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with the single exception of 3ꢀchloroꢀsubstituted compound 24 (Ki σ1 2.5 nM, σ2 >10,000 nM).
Monobromo or monotrifluoromethyl substitution (compounds 26–29) resulted in a very similar trend to
those observed with monofluoro compounds. Introduction of a methoxy group at 3ꢀ or 4ꢀposition resulted
in 8ꢀfold and 51ꢀfold reduction respectively in σ1 affinity (compounds 30 and 31). A 3ꢀmethyl
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susbstitution gave an equipotent compound to parent compound 9 in terms of σ1 affinity, but a more
potent binder at σ2 receptor (Ki σ2 337 nM). Substitution of the benzene ring with a sterically bulkier
naphthalene yielded a more potent σ2 receptor binder and less potent affinity at σ1 (Ki σ1 36 nM, σ2 151
nM). In almost all of the tested analogues 20–33, affinity at σ2 receptor was improved upon
monosubstitution with an EWG or EDG, with the exception of 3ꢀchloroꢀsubstituted compound 24 (Ki σ1
2.5 nM, σ2 >10,000 nM) and 3ꢀmethoxyꢀsubstituted compound 30 (Ki σ1 31 nM, σ2 >10,000 nM).
Table 1. Binding affinities of 3ꢀalkoxyisoxazole ligands at σ1 and σ2 receptors. a
Ki，σ1 (nM) b
7.0 ± 1.1
Ki，σ2 (nM)
19.6 ± 2.2
1312e
Compound
L1
R1
ꢀ
LogBBd
0.124
ꢀ
3
9
C6H5
4.1 ± 0.7
ꢀ0.255
O
C6H5
C6H5
NAc
NA
NA
ꢀ0.390
ꢀ0.618
15
16
O
130 ± 18
13.9 ± 1.6
NA
ꢀ0.542
ꢀ0.052
17
18
C6H5
65.4 ± 10.5
1033 ± 142
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1
2
C6H5
299 ± 48
NA
ꢀ0.785
19
3
4
5
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7
8
9
2ꢀFꢀC6H4
3ꢀFꢀC6H4
6.0 ± 0.8
1.4 ± 0.2
4.4 ± 0.6
2.7 ± 0.4
2.6 ± 0.3
13.0 ± 1.5
3.2 ± 0.4
18.3 ± 1.7
6.6 ± 0.6
13.3 ± 1.2
31.1 ± 3.6
210 ± 19
4.0 ± 0.5
396 ± 72
277 ± 32
471 ± 54
165 ± 30
NA
ꢀ0.242
ꢀ0.212
ꢀ0.212
ꢀ0.072
ꢀ0.072
ꢀ0.072
ꢀ0.050
ꢀ0.050
ꢀ0.031
ꢀ0.031
ꢀ0.325
ꢀ0.325
ꢀ0.179
20
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32
4ꢀFꢀC6H4
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2ꢀClꢀC6H4
3ꢀClꢀC6H4
4ꢀClꢀC6H4
3ꢀBrꢀC6H4
4ꢀBrꢀC6H4
3ꢀCF3ꢀC6H4
4ꢀCF3ꢀC6H4
3ꢀOCH3ꢀC6H4
4ꢀOCH3ꢀC6H4
3ꢀCH3ꢀC6H4
139 ± 16
85.9 ± 11.8
137 ± 25
88 ± 12
91.7 ± 10.3
NA
675 ± 138
341 ± 39
36.6 ± 4.1
153 ± 17
ꢀ0.077
33
aSee Experimental Section. Radioligands: σ1: [3H]ꢀ(+)ꢀpentazocine; σ2: [3H]DTG (ditolylguanidine). bThe
Ki values for compound 9 are cited from the literature.27 c NA: not active, defined as < 50% binding in the
d
primary assay at 10 ꢀM. LogBB values were calculated from the Clark’s equation: LogBB = ꢀ
0.0148×PSA + 0.152×CLogP + 0.139.31 e Data shown as average of Ki values from two measurements:
665 ± 29 nM and 1958 ± 392 nM.
Subsequent SAR studies were focused on assessing replacement of the nitrogenꢀcontaining group R2,
keeping the linker L and hydrophobic group R1 as methylene ether and benzene or 3ꢀsubstituted benzene
respectively. Compound 34, which is an Rꢀenantiomer of the parent compound 9, was found to be ~10ꢀ
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fold less potent at the σ1 receptor and 3ꢀfold more potent at the σ2 receptor. Replacement with 4ꢀ
1
2
methylpiperidine or ringꢀopened aminopropyl group resulted in deleterious effects on the σ1 affinity
(compounds 35 and 37), which could be explained by the nonꢀideal positioning of the nitrogen according
to the proposed pharmacophore model shown in Figure 2, which consists of: 1) a substituted basic
nitrogen; 2) a primary and secondary hydrophobic regions at a distance of approximately 6 to 10 Å and
2.5 to 3.9 Å from the nitrogen atom.32 A shorter aminoethyl or Nꢀmethylaminoethyl substitution
(compounds 36 and 38) partially restored binding affinity at σ1 receptor, with σ1 Ki values of 63 nM and
80 nM respectively, as the nitrogen fits better with the proposed pharmacophore. NꢀEthylaminoethyl
substitution (compound 39) resulted in a significant increase of σ1 binding affinity (Ki = 3.9 nM), while
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
weakly binding to the σ2 receptor with a Ki value of 975 nM.
We next examined the effect of Nꢀalkylation to the binding affinity profiles (44–59). NꢀMethyl
pyrrolidine/piperidine analogues generally slightly improved the binding affinity at σ1 receptor relative to
the corresponding unsubstituted pyrrolidines/piperidine (44 vs 9, 45 vs 21, 46 vs 24, 47 vs 34, 48 vs 35).
Compound 47 was a less potent binder at σ1 compared to its enantiomer compound 44, suggesting Sꢀ
configuration of pyrrolidine moiety is favored for σ1 binding. In contrast, further Nꢀmethylation of
compounds 38 and 39 resulted in a significant decrease of binding affinity (49 vs 38 and 50 vs 39).
Gratifyingly, bulkier cycloaliphatic substituents (compounds 51–54) resulted in compounds with low
singleꢀdigit nanomolar to subnanomolar Ki values at the σ1 receptor. The cyclopropyl analogue 53 was
found to possess the most superior σ2/σ1 ratio, whereas the cyclohexyl analogue 54 was the most potent
binder at σ1 receptor with a Ki value of 0.3 nM. 3ꢀFluoro or 3ꢀchloro substitutions at the hydrophobic
region R1 for these two compounds (55–58) yielded compounds with very similar binding profiles.
However, binding affinity to the σ2 receptor also increased with Ki values of less than 100 nM for the
cyclopentyl and cyclohexyl compounds (compounds 52 and 54). This observation is consistent with SAR
findings in the 1ꢀarylpyrazoles series of σ ligands in which only small cyclic amines analogues possessed
sufficient selectivity for σ1 vs σ2 receptor.33 As the ethoxymorpholine group is favored in 1ꢀarylpyrazoles
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series, we attempted to attach the same appendage to our isoxazoleꢀcontaining ligands resulted in
compound 59. Unfortunately the potency was found to dramatically drop by 100ꢀfold (59 vs 9).
Table 2. Binding affinities of 22 isoxazole ligands at σ1 and σ2 receptors. a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Ki，σ1 (nM)b
Ki，σ2 (nM)b
Compound
3
R2
R3
LogBBd
ꢀ
ꢀ
7.0 ± 1.1
19.7 ± 2.2
0.124
H
H
33.0 ± 5.2
4.1 ± 0.7
1472e
1312f
ꢀ0.255
ꢀ0.255
8
9
H
H
37.4 ± 4.3
356 ± 33
508 ± 94
284 ± 39
ꢀ0.202
ꢀ0.253
34
35
HN
H
H
63.9 ± 10.2
2312 ± 318
NAc
NA
ꢀ0.617
ꢀ0.556
36
37
H
H
80.8 ± 14.7
3.7 ± 0.7
NA
ꢀ0.364
ꢀ0.283
38
39
987 ± 135
H
3ꢀF
3ꢀCl
H
3.4 ± 0.4
0.7 ± 0.1
1.4 ± 0.1
23.1 ± 2.6
665 ± 76
687 ± 125
371 ± 76
411 ± 56
ꢀ0.042
0.001
0.087
ꢀ0.042
44
45
46
47
H
H
96.4 ± 13.2
NA
216 ± 35
NA
ꢀ0.056
ꢀ0.143
48
49
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H
H
18.4 ± 2.9
1.8 ± 0.2
820 ± 113
307 ± 56
ꢀ0.063
ꢀ0.005
50
51
1
2
3
4
5
6
7
8
9
H
H
H
0.5 ± 0.06
0.8 ± 0.1
0.3 ± 0.05
73.7 ± 13.4
225 ± 25
0.080
0.004
0.165
52
53
54
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18.7 ± 2.1
3ꢀF
0.9 ± 0.1
1.1 ± 0.1
314 ± 57
123 ± 25
0.048
0.134
55
56
3ꢀCl
3ꢀF
3ꢀCl
H
0.5 ± 0.05
0.5 ± 0.04
403 ± 63
24.5 ± 5.0
15.1 ± 3.1
NA
0.208
0.295
ꢀ0.289
57
58
59
aSee Experimental Section. Radioligands: σ1: [3H]ꢀ(+)ꢀpentazocine; σ2: [3H]DTG (ditolylguanidine).
b
The Ki values for 8 and 9 are cited from the literature.27 c not active, defined as < 50% binding in the
d
primary assay at 10ꢀM. LogBB values were calculated from the Clark’s equation: LogBB = ꢀ
0.0148×PSA + 0.152×CLogP + 0.139.31 e Data shown as average of Ki values from two measurements:
f
1288 ± 128 nM and 1657 ± 329 nM. Data shown as average of Ki values from two measurements: 665 ±
29 nM and 1958 ± 392 nM.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
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17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. σ1 receptor pharmacophore model and proposed match of substructures with pharmacophore
elements for compounds 8, 9, 53, and 54.
Table 3. Binding affinities of 5 isoxazole ligands at σ1 and σ2 receptors. a
Compound
X
ꢀ
R
ꢀ
Ki, σ1 (nM)
7.0 ± 1.1
5.3 ± 1.0
36.5 ± 3.3
63.9 ± 10.2
NAb
Ki, σ2 (nM)
19.6 ± 2.2
221 ± 30
687 ± 125
2202 ± 297
NA
LogBBc
0.124
3
NH
NH
NCH3
O
H
Cl
H
H
H
ꢀ0.272
ꢀ0.142
ꢀ0.054
ꢀ0.228
0.118
65
66
67
68
69
CH2
NA
NA
b
aSee Experimental Section. Radioligands: σ1: [3H]ꢀ(+)ꢀpentazocine; σ2: [3H]DTG (ditolylguanidine).
NA: not active, defined as < 50% binding in the primary assay at 10 ꢀM. c LogBB values were calculated
from the Clark’s equation: LogBB = ꢀ0.0148×PSA + 0.152×CLogP + 0.139.31
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We also examined briefly the effects of replacing the 3ꢀalkyoxy groups with cyclic amines as shown in
compounds 65–69. Piperazineꢀsubstituted analogue 65 had a very similar Ki value to the parent compound
9 at σ1 receptor but improved binding affinity to the σ2 receptor. 3ꢀChloro substitution at the hydrophobic
1
2
3
4
5
6
7
8
9
region R1 (compound 66) resulted in a 7ꢀfold decrease in σ1 affinity. NꢀMethylpiperazine substitution
(compound 67) gave a much less potent σ1 binder (Ki, σ1 = 64 nM, Ki, σ2 = 2,147 nM). Morpholine, or
piperidine substitution was found to be deleterious for affinity at both σ receptors (68 and 69).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Selectivity Studies at Selected Neurotransmitter Transporters
A broadꢀrange screening study was carried out previously for compounds 8 and 9 to determine their
global selectivity profile among 42 other CNS neurotransmitter receptors and transporters, including
serotonin receptors, dopamine receptors, GABA receptors, biogenic amine transporters, adrenergic
receptors, muscarinic receptors, opioid receptors, and histamine receptors.27 We previously found that
compounds 8 and 9 not only bind to σ1 receptor (Ki = 33 and 4.1 nM) but also showed moderate binding
affinities at two of the biogenic amine transporters DAT (Ki = 24 and 373 nM) and NET (Ki = 191 and
203 nM), respectively. Gratifyingly, no appreciable binding was observed for DAT, NET and SERT using
10 ꢁM concentration of compounds 53 and 54, indicating that these two compounds have excellent
selectivity for σ1 receptor over DAT, NAT and SERT. (Table 4.) The other three tested compounds 20, 23
and 66 were found to retain some binding affinities to at least one of the three transporters tested.
Table 4. Binding affinities of selected ligands at NET, DAT, and SERT.a
Compound
GBR12909
Desipramine
Amitryptiline
8
Ki, DAT (nM)b
Ki, NET (nM)
Ki, SERT (nM)
Ki, σ1 (nM)
8.8 ± 3.8
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2.5 ± 0.4
ꢀ
ꢀ
ꢀ
5.4 ± 0.9
NAc
NA
ꢀ
24 ± 0.95
373 ± 24
191 ± 10
203 ± 12
33 ± 5.2
4.1 ± 0.7
9
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NA
NA
NA
NA
1082 ± 148
2109 ± 289
NA
NA
2946
NA
6.1 ± 0.8
2.7 ± 0.4
0.8 ± 0.1
0.3 ± 0.05
5.3 ± 0.9
20
23
53
54
65
1
2
3
4
5
6
7
8
NA
NA
497 ± 297
163 ± 37
NA
9
10
11
aSee Experimental Section. DAT: Dopamine Transporter; NET: Norepinephrine Transporter; SERT:
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Serotonin Transporter. Radioligands: σ1: [3H]ꢀ(+)ꢀpentazocine; σ2: [3H]DTG (ditolylguanidine); DAT:
b
[3H]WIN35428; NET: [3H]nisoxetine; SERT: [3H]citalopram. The Ki values for 8–9 are cited from the
literature.27 c not active, defined as < 50% binding in the primary assay at 10 ꢀM. Ki values were
determined for those targets where the binding efficacy at 10 ꢁM was greater than 50%.
Functional Profile
To date, there is still an unmet need of high throughput functional assays that readily identify σ1 ligands
as an agonist, partial agonist or antagonist. Competition binding studies of the examined ligands with
phenytoin, an allosteric modulator of σ1 receptor, has recently emerged as an accepted method26, 33 to
differentiate σ1 agonists and antagonists.34 In this assay, phenytoin is believed to induce a conformational
change in the receptor to shift the equilibrium towards the active state, thereby increasing the affinity of
putative agonists but not of the antagonists.34 Six selected compounds (4, 9, 21, 24, 51, 53) were subjected
to this assay and found to bind less potently to the σ1 receptor in the presence of phenytoin, indicating
that they behave as antagonists (See Supporting Information Table 1).
In Vivo Analgesic Effects—Mouse Formalin Test
Compound 53 was further advanced to the in vivo analgesic studies given its in vitro potency, selectivity,
functional profiles, as well as LogBB value. Antiꢀnociceptive efficacy was assessed using the mouse
formalin test, a classic model in which formalin solution is injected to the hind paw of the mouse and a
biphasic pain response characterized by analysing the behavior of licking and flinching of the affected
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hind paw.35, 36 The first phase (0–10 min) reflects mostly nociceptive pain whereas the second phase (11–
45 min) represents the inflammatory responses. Traditional analgesics as well as σ1 receptor antagonists
have been reported to attenuate the licking and flinching behavior. In this study, mice were administered
compounds 53 (40 and 80 mg/kg i.p.) or selected σ1 antagonist 4 as a positive control (80 mg/kg i.p.) and
the time spent licking the paw after injection was recorded as shown in Figure 3. Drug administration
produced a reduction of paw licking behavior in mouse, suggestive of an antiꢀnociceptive effect.
Compound 53 (80 mg/kg) or 4 (80 mg/kg) significantly attenuated paw licking behavior in both phases in
mice, while 40 mg/kg of compound 53 markedly reduced paw licking in phase II but not in phase I
compared to the vehicle group.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Analgesic effects of compound 53 and 4 in phase I (0–10 min) and phase II (11−45) of the
mouse formalin test at the dose of 80 mg/kg. All drugs were administered intraperitoneally; each column
and vertical line represents mean ± SEM of the values obtained in at least 6 animals. Statistically
significant differences:* p < 0.05, ** p < 0.01, *** p < 0.001; vs vehicle using unpaired, twoꢀtailed tꢀtest).
Rotarod Test.
The rotarod test is widely used to evaluate the motor coordination of rodents. In this study, we utilized this
assay to rule out the possibility that the observed efficacy of compound 53 in the formalin test was due to
the interference with motor coordination. As shown in Figure 4, pretreatment with pregabalin (40 mg/kg)
significantly decreased the rotarod latency of mice at 60, 90, and 120 min compared to the vehicle group.
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Compound 53 did not significantly alter the time mice remained on the rotarod at both 40 and 80 mg/kg
1
2
3
suggesting no interference with motor coordination.
4
5
6
7
300
8
9
10
11
12
13
14
15
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40
41
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
**
200
100
0
***
***
vehicle (saline, i.p.)
cmpd 53 (40 mg/kg, i.p.)
cmpd 53 (80 mg/kg, i.p.)
pregabalin (40 mg/kg, i.p.)
Figure 4. Dose response effect of 53 and pregabalin on rotarod test. Data obtained from 8–10 mice per
group and expressed as mean ± SEM latency (s) to fall down from rod. Statistically significant
differences: * p < 0.05, ** p < 0.01, *** p < 0.001 vs vehicle (TwoꢀWay ANOVA followed by Bonferroni
test).
In Vitro PAMPA Data
The oral absorption of seven representative compounds was initially evaluated using parallel artificial
membrane permeability assay (PAMPA), which is an in vitro model of passive, transcellular permeation
widely used in the pharmaceutical industry. The in vitro permeability (Pe) of compounds 4, 9, 21, 24, 44,
53, 65 and the control drugs verapamil and atenolol through a lipid extract of porcine brain was
determined using PBS as the solvent. Compounds with a Pe above 4.0×10ꢀ6 cm·sꢀ1 are considered possibly
penetrating into the CNS by passive diffusion (CNS+), whereas compounds with Pe below 2.0×10ꢀ6 cm·sꢀ
are considered to be impenetrant (CNSꢀ).37 Those with Pe values ranging from 2.0×10ꢀ6 cm·sꢀ1 to
1
4.0×10ꢀ6 cm·sꢀ1 are categorized as uncertain (CNS±). Compound 65 was found to possess the greatest
permeability with a Pe value of 7.36×10ꢀ6 cm·sꢀ1(SI Table 2.), while all the other tested compounds have
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Pe values between 2×10ꢀ6 cm·sꢀ1 and 4×10ꢀ6 cm·sꢀ1, including compound 4, which is currently in phase II
1
2
3
clinical trial.
4
5
In Vivo Pharmacokinetic Studies of Compound 53
6
7
8
9
To determine the ability of these alkoxyisoxazole compounds to penetrate the BBB, compound 53 was
selected for in vivo pharmacokinetic studies in mouse. Compound 53 (40 mg/kg) was orally administered
to KM mice before collection of plasma and brain samples at the following time points: 0.167, 0.5, 1, 2, 4,
6, and 8 h (n = 3/group). The concentrations of compound 53 in the brain and plasma samples were
determined by a developed LC−MS/MS method. The plasma and brain oral halfꢀlives of compound 53
were found to be 2.64 and 2.52 h respectively (Table 5). The AUC0−∞ values were 113 h·ng·mLꢀ1 and 403
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
h·ng·gꢀ1 in the plasma and brain respectively, with the brain/plasma ratio of 3.6. These data suggest that
compound 53 is absorbed rapidly following oral administration and readily crosses the BBB.
Table 5. Pharmacokinetics parameters of 53 in KM mice (40 mg/kg, po)
Plasma PK Parameters
Kel, hꢀ1
Plasma
0.263
2.64
0.167
193
Brain PK Parameters
Kel, hꢀ1
Brain
0.276
2.52
0.167
395
Ratio (Brain/Plasma)
ꢀ
t1/2, h
t1/2, h
ꢀ
tmax, h
tmax, h
ꢀ
Cmax, ng·mLꢀ1
Cmax, ng·gꢀ1
2.0
3.6
3.6
6.3
6.0
ꢀ
AUC0ꢀ8, h·ng·mLꢀ1
AUC0ꢀinf, h·ng·mLꢀ1
AUMC0ꢀ8, h·h·ng·mLꢀ1
AUMC0ꢀinf, h·h·ng·mLꢀ1
MRTPO, h
108
AUC0ꢀ8, h·ng·gꢀ1
AUC0ꢀinf, h·ng·gꢀ1
AUMC0ꢀ8, h·h·ng·gꢀ1
AUMC0ꢀinf, h·h·ng·gꢀ1
MRTPO, h
373
113
403
91.5
157
578
935
1.39
2.32
Conclusions
A total of 46 compounds were designed and synthesized based on the previously identified
alkoxyisoxazole scaffold as potent and selective σ1 receptor ligands. Binding assays at both σ1 and σ2
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receptors revealed analogues with subnanomolar potencies at the σ1 receptor. Selected compounds were
tested for their binding profiles at DAT, NET and SERT transporters. Compound 53 was found to be the
most promising compound overall given its in vitro binding profile, LogBB value and permeability in
PAMPA assay. Administration of σ1 antagonist 53 at 80 mg/kg (i.p.) was found to demonstrate
statistically significant analgesic effects in mouse formalin test. Combined with the acceptable
pharmacokinetic parameters and its excellent brain/plasma ratio, this study overall supports the notion of
the use of σ1 antagonists for novel treatment in pain.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
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48
49
50
51
52
53
54
55
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57
58
59
60
Experimental Section
General Methods: Starting materials, reagents, and solvents were purchased from commercial suppliers
and used without further purification, unless otherwise stated. Anhydrous THF and CH2Cl2 were obtained
by distillation over sodium wire or CaH2, respectively. All nonꢀaqueous reactions were run under a
nitrogen atmosphere with exclusion of moisture from reagents, and all reaction vessels were ovenꢀdried.
The progress of reactions was monitored by TLC on SiO2. Spots were visualized by their quenching of the
fluorescence of an indicator admixed to the SiO2 layer, or by dipping into KMnO4 solution followed by
heating. SiO2 for column chromatography (CC) was of 200−300 mesh particle size, and an EtOAc/hexane
1
mixture or gradient was used unless stated otherwise. H NMR spectra were recorded at a spectrometer
frequency of 400 MHz, 13C NMR spectra at 101 MHz. Chemical shifts are reported in δ (ppm) using the δ
0 signal of tetramethylsilane (TMS) as internal standards. High resolution mass spectra were performed
using a Bruker ESIꢀTOF highꢀresolution mass spectrometer. Purities of final compounds (> 98%) were
established by analytical HPLC, which was carried out on an Agilent 1200 HPLC system with a
ZORBAX Eclipse XDBꢀC18 column, with detection at 220 or 254 nm on a variable wavelength detector
G1365D; flow rate = 1.4 mL/min; gradient of 0 to 100% methanol in water (both containing 0.05 vol % of
TFA) in 25 min. Final products were purified by preparative HPLC (Beckman GOLD126P) under the
following conditions: column, Atlantis T3 5 ꢁm, 250 x 4.6 mm; flow, 3 mL/min; all solvents containing
0.05 vol% TFA; UV detection at 254 nm and 220 nm; Gradient I: 8–100% MeOH in water in 30 min,
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100% for 4 min, return to 8% in 20 min. The optical rotation experiments were performed using an
Autopol VI polarimeter.
1
2
3
4
5
6
7
8
General Procedure for the Reduction of Isoxazolecarboxylic Acid Esters to Alcohols (Method A). To
a solution of isoxazolecarboxylic acid esters (0.8 mmol) in anhydrous THF (20 mL) was added LiBH4 (4
mmol) with ice cooling under Ar. After stirring overnight at rt, saturated aqueous NH4Cl solution was
added with ice cooling. Extraction with EtOAc (2×30 mL), drying over Na2SO4, followed by evaporation
under vacuum afforded the residue which was further purified by flash chromatography to give the
alcohol.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
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General Procedure for the Esterification of Isoxazolecarboxylic Acid with Phenol or Alcohol
(Method B). To a stirred solution of isoxazoleꢀ5ꢀcarboxylic acid (0.3 mmol) and phenol (or alcohol) (0.3
mmol) in 10 mL CH2Cl2 was added DCC (98 mg, 0.47 mmol) and DMAP (19.2 mg, 0.16 mmol) under
N2. After stirring overnight at rt, the reaction mixture was evaporated under vacuum and the residue was
purified by flash chromatography to give the ester product.
General Procedure for the Deprotection of NꢀBocꢀAmines to Afford HCl Salts (Method C). To a
solution of the NꢀBoc protected precursor (1 mmol) in CH2Cl2 (5 mL) was added HCl/EtOAc (4 mol/L, 2
mL) under N2 with ice cooling. The mixture was stirred overnight at rt, after which the solvent was
evaporated and the residue was triturated with diethyl ether (20 mL). The resultant solid was filtered off to
give the HCl salt.
General Procedure for the Deprotection of NꢀBocꢀAmines to Afford TFA Salts (Method D). To a
solution of the NꢀBoc protected precursor (1 mmol) in CH2Cl2 (5 mL) was added TFA (0.5 mL) under N2
with ice cooling. The mixture was stirred overnight at rt. After the solvent was evaporated, the residue
was purified by preparative HPLC. Following evaporation under vacuum, the residue was dissolved in
distilled water (about 2−3 mL) and the solution was lyophilized to obtain the TFA salt.
General Procedure for the Amidation of Isoxazoleꢀ5ꢀCarboxylic Acid (Method E). To a stirred
solution of isoxazoleꢀ5ꢀcarboxylic acid (1.6 mmol) and amine (1.6 mmol) in 20 mL CH2Cl2 was added
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EDC·HCl (2.08 mmol) and 3ꢀmethylpyridine (2.24 mmol) under N2. After stirring overnight at rt, the
reaction mixture was evaporated and the residue was purified by flash chromatography to give the amide
product.
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General Procedure for the Preparation of Phenyl Ethers or Thioether from Iodides (Method F). To
a stirred solution of an iodide (1 mmol) and phenol or thiophenol (2 mmol) in anhydrous DMF (4 mL)
was added K2CO3 (6 mmol) under N2. After stirring overnight at rt, saturated aqueous NH4Cl solution was
added. The mixture was extracted with EtOAc (2×30 mL), and the combined organic phases were washed
with water (3×20 mL), dried over Na2SO4, and evaporated. The residue was purified by flash
chromatography to give the phenyl ether or thioether product.
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General Procedure for the Mitsunobu Reaction of Alcohol with Hydroxyisoxazole (11) to Afford
Alkoxyisoxazoles (Method G). To a stirred solution of alcohol (1.2 mmol), 11 (2.0 mmol), and PPh3 (2.0
mmol) in anhydrous THF (20 mL) was added diisopropyl azodicarboxylate (1.5 mmol) dropwise with ice
cooling under N2. After stirring overnight at rt, the solvent was evaporated, and the residue was dissolved
in EtOAc (30 mL). The solution was washed with water (20 mL) and brine (15 mL), dried over Na2SO4,
filtered, and concentrated under vacuum. The residue was purified by flash chromatography to give the
alkoxyisoxazole product.
General Procedure for the Preparation of Iodides from Alcohols (Method H). To a stirred solution of
crude alcohol (0.7 mmol), imidazole (1.05 mmol), and PPh3 (1.05 mmol) in anhydrous CH2Cl2 (10 mL)
was added I2 (1.05 mmol) with ice cooling under N2. After stirring overnight at rt, the solvent was
evaporated and the residue was purified by flash chromatography to give the iodide.
General Procedure for the Preparation of NꢀMethyl Analogues from Primary or Secondary Amines
(Method I). To a solution of primary or secondary amine (0.5 mmol) and HCHO (0.5 mL) in CF3CH2OH
(5 mL) was added NaBH4 (0.6 mmol) at 40 °C under N2. After stirring for 2 h at 40 °C, the solvent was
evaporated and extracted with EtOAc (3×10 mL). The combined organic layers were washed with water
(20 mL) and brine (15 mL), dried over Na2SO4, filtered, and concentrated under vacuum. The residue was
purified by flash chromatography. To a solution of the Nꢀmethyl compound in CH2Cl2 (3 mL) was added
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HCl/EtOAc (4 mol/L, 1 mL) under N2 with ice cooling. The mixture was stirred overnight at rt and the
solvent evaporated to give the HCl salt.
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General Procedure for the Preparation of NꢀAlkyl Analogues from Secondary Amines (Method J).
To a stirred solution of secondary amine (0.43 mmol), aldehyde or ketone (0.5 mmol) and acetic acid
(0.05 mL) in CH3OH (10 mL) was added Na(CN)BH3 (0.6 mmol) at rt under N2. After stirring overnight
at rt, the solvent was evaporated and the residue was extracted with EtOAc (3×10 mL). The combined
organic layers were washed with water (20 mL) and brine (15 mL), dried over Na2SO4, filtered, and
concentrated under vacuum. The residue was then purified by flash chromatography. To a solution of the
Nꢀalkyl compound in CH2Cl2 (3 mL) was added HCl/EtOAc (4 mol/L, 1 mL) under N2 with ice cooling.
The mixture was stirred overnight at rt and the solvent evaporated to give the HCl salt.
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Methyl (S)ꢀ3ꢀ((1ꢀ(tertꢀButoxycarbonyl)pyrrolidinꢀ2ꢀyl)methoxy)isoxazoleꢀ5ꢀcarboxylate (12). This
compound was obtained from 1ꢀ(tertꢀbutoxycarbonyl)ꢀ2(S)ꢀazetidinylmethanol and 1128 employing
1
Method G. White solid; yield 41%. H NMR (400 MHz, DMSOꢀd6) δ 7.10 (s, 1H), 4.31–4.29 (m, 1H),
4.24–4.20 (m, 1H), 4.09–3.98 (m, 1H), 3.88 (s, 3H), 3.23–3.21 (m, 2H), 2.13–1.94 (m, 1H), 1.95–1.83 (m,
13
2H), 1.81–1.71 (m, 1H), 1.39 (s, 9H). C NMR (101 MHz, CDCl3) δ 176.9, 171.8, 160.5, 157.1, 100.7,
80.1, 70.6, 56.1, 52.8, 46.4, 28.5, 23.6, 22.6.
(S)ꢀ3ꢀ((1ꢀ(tertꢀButoxycarbonyl)pyrrolidinꢀ2ꢀyl)methoxy)isoxazoleꢀ5ꢀcarboxylic Acid (13). To a stirred
solution of 12 (326 mg, 1.0 mmol) in 10 mL THF was added NaOH (1 M, 4 mmol). After stirring for 4h
at rt, the pH of the solvent was adjusted to 1 with hydrochloric acid (1 M). The reaction mixture was
extracted with EtOAc (2×30 mL) and the combined organic layers were dried over Na2SO4, filtered, and
concentrated under vacuum. The residue was purified by flash chromatography to give the title compound
1
as white solid in 87% yield. H NMR (400 MHz, CDCl3) δ 8.95 (s, 1H), 6.54 (s, 1H), 4.33 (m, 1H), 4.16
(m, 2H), 3.46 (m, 2H), 1.98 (m, 2H), 1.91 (m, 2H), 1.48 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 171.5,
160.7, 158.2, 155.2, 100.7, 80.8, 60.6, 55.9, 41.9, 30.3, 28.5, 27.0.
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tertꢀButyl (S)ꢀ2ꢀ(((5ꢀ(hydroxymethyl)isoxazolꢀ3ꢀyl)oxy)methyl)pyrrolidineꢀ1ꢀcarboxylate (14). This
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3
1
compound was obtained from 12 employing Method A. Colorless oil; yield 91%. H NMR (400 MHz,
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7
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CDCl3) δ 5.90 (s, 1H), 4.74 (m, 1H), 4.60 (s, 2H), 4.29 (m, 1H), 4.12 (m, 2H), 3.36 (m, 2H), 1.94 (m, 3H),
1.86 (m, 1H), 1.45 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 173.3, 171.7, 154.6, 92.8, 79.8, 69.8, 60.4, 56.8,
56.4, 46.6, 28.4, 22.9.
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Phenyl (S)ꢀ3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazoleꢀ5ꢀcarboxylate Hydrochloride (15). This compound
was obtained from 13 and phenol in two steps employing Method B and C. White solid; yield 41%;
purity 99.2%. [α]D20 +18.23 (c = 0.13, MeOH); 1H NMR (400 MHz, D2O) δ 7.47 (t, J = 8.0 Hz, 2H), 7.35
(t, J = 8.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 2H), 7.00 (s, 1H), 4.61 (dd, J = 12.0, 4.0 Hz, 1H), 4.53–4.31 (m,
1H), 4.07 (m, 1H), 3.34 (t, J = 8.0 Hz, 2H), 2.36–2.14 (m, 1H), 2.16–1.94 (m, 2H), 1.93–1.70 (m, 1H); 13C
NMR (101 MHz, DMSOꢀd6) δ 171.1, 159.8, 154.6, 149.4, 129.8, 126.7, 121.5, 101.7, 69.3, 57.5, 45.4,
26.1, 23.3. HRMS (ESI): Calcd for C15H16N2O4 [M+H]+, 289.1183; Found 289.1207.
(S)ꢀNꢀPhenylꢀ3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazoleꢀ5ꢀcarboxamide
Hydrochloride
(16).
This
compound was obtained from 13 and aniline in two steps employing Method E and C. White solid; 52%
yield; purity 98.6%. [α]D20 +10.21 (c = 0.10, MeOH); 1H NMR (400 MHz, DMSOꢀd6) δ 10.74 (br s, 1H),
9.26 (br s, 1H), 8.90 (s, 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.39 (t, J = 8.0 Hz, 2H), 7.17 (t, J = 6.0 Hz, 1H),
7.09 (s, 1H), 4.54 (dd, J = 12.0, 4.0 Hz, 1H), 4.47–4.32 (m, 1H), 4.14–3.83 (m, 1H), 3.24 (t, J = 8.0 Hz,
2H), 2.22–2.05 (m, 1H), 2.05–1.85 (m, 2H), 1.83–1.65 (m, 1H); 13C NMR (101 MHz, DMSOꢀd6) δ 171.0,
164.1, 153.8, 137.5, 128.8, 124.8, 120.7, 98.0, 69.0, 57.6, 45.4, 26.0, 23.3; HRMS (ESI): Calcd for
C15H18N3O3 [M+H]+, 288.1343; Found 288.1353.
(S)ꢀIndolinꢀ1ꢀyl(3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazolꢀ5ꢀyl)methanone Hydrochloride (17). This
compound was obtained from 13 and indoline in two steps employing Method E and C. White solid; yield
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78%; purity 99.0%. [α]D +11.43 (c = 0.14 , MeOH); H NMR(400 MHz, DMSOꢀd6) δ 9.49 (br s, 2H),
8.12 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 7.13 (t, J = 8.0 Hz, 1H), 7.01
(s, 1H), 4.57–4.43 (m, 2H), 4.36 (t, J = 8.0 Hz, 2H), 4.01–3.91 (m, 1H), 3.22 (m, 4H), 2.21–2.07 (m, 1H),
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13
2.04–1.84 (m, 2H), 1.81–1.67 (m, 1H); C NMR (101 MHz, DMSOꢀd6) δ 170.7, 164.1, 154.6, 142.0,
132.8, 127.2, 125.1, 125.0, 116.9, 99.0, 68.9, 57.1, 49.1, 45.0, 27.9, 26.4, 23.2; HRMS (ESI): Calcd for
C17H20N3O3 [M+H]+, 314.1499; Found 314.1522.
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(S)ꢀ5ꢀ((Phenylthio)methyl)ꢀ3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazole
Trifluoroacetate
(18).
This
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compound was obtained from 14 and benzenethiol in two steps employing Method F, D, and Gradient I.
White solid; yield 24%; purity 98.4%. [α]D20 +13.49 (c = 0.16, MeOH); 1H NMR (400 MHz, DMSOꢀd6) δ
9.25 (br s, 1H), 8.77 (s, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.34 (t, J = 8.0 Hz, 2H), 7.24 (t, J = 8.0 Hz, 1H),
6.11 (s, 1H), 4.40 (m, 1H), 4.36 (s, 2H), 4.30–4.19 (m, 1H), 3.88 (m, 1H), 3.19 (m, 2H), 2.16–2.01 (m,
1H), 2.02–1.78 (m, 2H), 1.78–1.57 (m, 1H); 13C NMR (101 MHz, DMSOꢀd6) δ170.9, 170.6, 158.2 (TFA),
134.3, 129.1, 128.7, 126.6, 116.1 (TFA), 93.9, 68.3, 57.6, 45.3, 27.6, 26.0, 23.3; HRMS (ESI): Calcd for
C15H19N2O2S[M+H]+, 291.1162; Found, 291.1170.
(S)ꢀ(3ꢀ(Pyrrolidinꢀ2ꢀylmethoxy)isoxazolꢀ5ꢀyl)methyl phenylcarbamate Trifluoroacetate (19). To a
stirred solution of alcohol 14 (0.8 mmol) and DMAP (1.5 mmol) in 20 mL CH2Cl2 was added
isocyanatobenzene (1.0 mmol) under N2. After stirring for 5 h at rt, the reaction mixture was evaporated
and the residue was purified by flash chromatography to give Bocꢀprotected carbamate processor. The
title compound was obtained by employing Method D and Gradient I. White solid; yield 40% in two
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steps; purity 99.0%. [α]D +11.67 (c = 0.16, MeOH); H NMR (400 MHz, DMSOꢀd6) δ 9.92 (br s, 1H),
9.48 (br s, 1H), 8.98 (s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.38–7.21 (m, 2H), 7.09–6.97 (m, 1H), 6.40 (s, 1H),
5.20 (s, 2H), 4.45 (dd, J = 12.0, 4.0 Hz, 1H), 4.33 (dd, J = 12.0, 8.0 Hz, 1H), 3.94 (m, 1H), 3.22 (s, 2H),
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2.25–2.03 (m, 1H), 2.04–1.81 (m, 2H), 1.82–1.63 (m, 1H). C NMR (101 MHz, DMSOꢀd6) δ 170.8,
168.8, 158.3 (TFA), 152.5, 138.6, 128.8, 122.8,118.3, 115.2 (TFA), 95.2, 68.5, 57.5, 56.5, 45.2, 26.0,
23.3; HRMS (ESI): Calcd for C16H20N3O4 [M+H]+, 318.1448; Found, 318.1444.
(S)ꢀ5ꢀ((2ꢀFluorophenoxy)methyl)ꢀ3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazole Hydrochloride (20). This
compound was obtained from 14 and 2ꢀfluorophenol in two steps employing Method F and C. White solid;
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yield 37%; purity 98.0%. [α]D +13.32 (c = 0.16, MeOH); H NMR (400 MHz, DMSOꢀd6) δ 9.90 (br s,
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Journal of Medicinal Chemistry
1H), 9.39 (br s, 1H), 7.37–7.19 (m, 2H), 7.14 (m, 1H), 7.00 (m, 1H), 6.47 (s, 1H), 5.27 (s, 2H), 4.42 (m,
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2H), 3.89 (m, 1H), 3.17 (m, 2H), 2.22–1.99 (m, 1H), 1.97–1.73 (m, 2H), 1.78–1.54 (m, 1H); C NMR
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(101 MHz, DMSOꢀd6) δ 170.8, 168.5, 151.7 (d, JCꢀF = 248.0 Hz), 145.2 (d, JCꢀF =10.0 Hz) 124.8 (d, JCꢀF
=
7
8
4.0 Hz), 122.2 (d, JCꢀF = 7.0 Hz), 116.2 (d, JCꢀF = 19.2 Hz), 115.6, 95.8, 68.4, 61.3, 57.3, 44.9, 26.1, 23.2;
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HRMS (ESI): Calcd for C15H17FN2O3 [M+H]+, 293.1309; Found, 293.1311.
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(S)ꢀ5ꢀ((3ꢀFluorophenoxy)methyl)ꢀ3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazole Hydrochloride (21). This
compound was obtained from 14 and 3ꢀfluorophenol in two steps employing Method F and C. White solid;
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yield 36%; purity 97.8%. [α]D +9.33 (c = 0.10, MeOH); H NMR (400 MHz, CDCl3) δ 7.45–7.05 (m,
1H), 6.78–6.58 (m, 3H), 5.99 (s, 1H), 5.00 (s, 2H), 4.39–4.24 (m, 1H), 4.22–4.15 (m, 1H), 3.71–3.56 (m,
1H), 3.16–2.95 (m, 2H), 2.02–1.94 (m, 1H), 1.93–1.76 (m, 2H), 1.70–1.52 (m, 1H); 13C NMR (101 MHz,
CDCl3) δ 171.8, 168.4, 163.0 (d, JCꢀF = 136.2 Hz), 159.1 (d, JCꢀF = 10.8 Hz), 130.6 (d, JCꢀF = 9.9 Hz),
108.7(d, JCꢀF = 11.1 Hz), 102.9 (d, JCꢀF = 25.3 Hz), 95.3, 71.8, 61.8, 60.5, 57.2, 46.2, 27.6, 24.9.
HRMS(ESI): Calcd for C15H17FN2O3 [M+H]+, 293.1309; Found, 293.1311.
(S)ꢀ5ꢀ((4ꢀFluorophenoxy)methyl)ꢀ3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazole Hydrochloride (22). This
compound was obtained from 14 and 4ꢀfluorophenol in two steps employing Method F and C. White solid;
yield 36%; purity 98.1%. [α]D20 +10.67 (c = 0.10 , MeOH); 1H NMR (400 MHz, DMSOꢀd6) δ 9.77 (br s,
1H), 9.26 (br s, 1H), 7.17 (m, 2H), 7.10–7.01 (m, 2H), 6.44 (s, 1H), 5.18 (s, 2H), 4.48–4.31 (m, 2H), 3.89
(m, 1H), 3.19 (m, 2H), 2.09 (m, 1H), 2.00–1.79 (m, 2H), 1.71 (m, 1H); 13C NMR (101 MHz, DMSOꢀd6) δ
170.8, 169.1, 157.0 (d, JCꢀF = 236.9 Hz), 153.7 (d, JCꢀF = 2.0 Hz), 116.3 (d, JCꢀF = 8.1 Hz), 115.9 (d, JCꢀF
=
23.2 Hz), 95.6, 68.5, 61.1, 57.4, 45.1, 26.2, 23.3. HRMS (ESI): Calcd for C15H17FN2O3 [M+H]+,
293.1309; Found, 293.1311.
(S)ꢀ5ꢀ((2ꢀChlorophenoxy)methyl)ꢀ3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazole Hydrochloride (23). This
compound was obtained from 14 and 2ꢀchlorophenol in two steps employing Method F and C. White solid;
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1
yield 71%; purity 98.4%. [α]D +14.71 (c = 0.14, MeOH); H NMR (400 MHz, DMSOꢀd6) δ 9.57 (br s,
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2H), 7.46 (d, J = 8.0 Hz, 1H), 7.33 (m, 2H), 7.02 (m, 1H), 6.48 (s, 1H), 5.31 (s, 2H), 4.60–4.36 (m, 2H),
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4.15–3.77 (m, 1H), 3.19 (m, 2H), 2.20–2.04 (m, 1H), 2.01–1.81 (m, 2H), 1.79–1.60 (m, 1H). C NMR
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(101 MHz, DMSOꢀd6) δ 170.8, 168.6, 152.7, 130.2, 128.3, 122.5, 121.5, 114.4, 95.8, 68.4, 61.3, 57.3,
45.0, 26.1, 23.2; HRMS (ESI): Calcd for C15H18ClN2O3 [M+H]+, 309.1000; Found, 309.1014.
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(S)ꢀ5ꢀ((3ꢀChlorophenoxy)methyl)ꢀ3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazole Trifluoroacetate (24). This
compound was obtained from 14 and 3ꢀchlorophenol in two steps employing Method F, D, and Gradient I.
White solid; yield 51%; purity 98.1%. [α]D20 +7.44 (c = 0.14, MeOH); 1H NMR (400 MHz, DMSOꢀd6) δ
9.49 (br s, 1H), 8.98 (s, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.14 (m, 1H), 7.09–6.97 (m, 2H), 6.44 (s, 1H), 5.24
(s, 2H), 4.44 (dd, J = 12.0, 4.0 Hz, 1H), 4.37–4.28 (m, 1H), 3.93 (m, 1H), 3.21 (m, 2H), 2.21–2.04 (m,
1H), 2.03–1.82 (m, 2H), 1.80–1.60 (m, 1H); 13C NMR (101 MHz, DMSOꢀd6) δ 170.8, 168.7, 160.9
(TFA), 158.3, 133.8, 131.0, 121.5, 115.4 (TFA), 114.9, 113.9, 95.6, 68.5, 60.8, 57.5, 45.2, 26.0, 23.3.;
HRMS (ESI): Calcd for C15H18ClN2O3 [M+H]+, 309.1000; Found, 309.1014.
(S)ꢀ5ꢀ((4ꢀChlorophenoxy)methyl)ꢀ3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazole Hydrochloride (25). This
compound was obtained from 14 and 4ꢀchlorophenol in two steps employing Method F and C. White solid
yield 55%; purity 97.1%. [α]D20 +5.95 (c = 0.14, MeOH); 1H NMR (400 MHz, DMSOꢀd6) δ 9.44 (s, 1H),
8.93 (s, 1H), 7.36 (d, J = 12.0 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 6.44 (s, 1H), 5.21 (s, 2H), 4.44 (dd, J =
12.0, 4.0 Hz, 1H), 4.33 (dd, J = 12.0, 8.0 Hz, 1H), 3.94 (m, 1H), 2.17–2.06 (m, 1H), 2.02–1.84 (m, 2H),
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1.77–1.64 (m, 1H); C NMR (101 MHz, DMSOꢀd6) δ 170.8, 168.9, 156.2, 129.3, 125.2, 116.6, 95.5,
68.5, 60.8, 57.6, 45.3, 26.0, 23.3; HRMS (ESI): Calcd for C15H18ClN2O3 [M+H]+, 309.1000; Found,
309.0996.
(S)ꢀ5ꢀ((3ꢀBromophenoxy)methyl)ꢀ3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazole Hydrochloride (26). This
compound was obtained from 14 and 3ꢀbromophenol in two steps employing Method F and C. White
solid; yield 40%; purity 98.0%. [α]D20 +9.79 (c = 0.16, MeOH); 1H NMR (400 MHz, DMSOꢀd6) δ 9.60 (s,
1H), 9.11 (s, 1H), 7.35–7.24 (m, 2H), 7.19 (d, J = 8.0 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.47 (s, 1H), 5.25
(s, 2H), 4.44 (dd, J = 12.0, 4.0 Hz, 1H), 4.37 (dd, J = 12.0, 8.0 Hz 1H), 3.90 (m, 1H), 3.19 (m, 2H), 2.19–
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Journal of Medicinal Chemistry
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2.02 (m, 1H), 2.02–1.83 (m, 2H), 1.81–1.59 (m, 1H); C NMR (101 MHz, DMSOꢀd6) δ 170.7, 168.7,
158.2, 131.4, 124.4, 122.1, 117.5, 114.2, 95.6, 68.3, 60.6, 57.6, 45.2, 25.8, 23.2; HRMS (ESI): Calcd for
C15H18BrN2O3 [M+H]+, 353.0495; Found, 353.0516.
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(S)ꢀ5ꢀ((4ꢀBromophenoxy)methyl)ꢀ3ꢀ(pyrrolidinꢀ2ꢀylmethoxy)isoxazole Hydrochloride (27). This
compound was obtained from 14 and 4ꢀbromophenol in two steps employing Method F and C. White
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solid; yield 34%; purity 98.2%. [α]D +12.41 (c = 0.19, MeOH); H NMR (400 MHz, DMSOꢀd6) δ 8.96
(br s, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 6.45 (s, 1H), 5.22 (s, 2H), 4.45 (dd, J = 12.0,
4.0 Hz, 1H), 4.32 (dd, J = 12.0, 8.0 Hz, 1H), 3.93 (m, 1H), 3.21 (t, J = 6.0 Hz, 1H), 2.18–2.05 (m, 1H),
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2.02–1.83 (m, 2H), 1.82–1.61 (m, 1H); C NMR (101 MHz, DMSOꢀd6) δ 170.8, 168.8, 158.3 (TFA),
156.6, 132.2, 117.1, 115.6 (TFA), 113.0, 95.5, 68.5, 60.7, 57.6, 45.3, 26.0, 23.3; HRMS (ESI): Calcd for
C15H18BrN2O3 [M+H]+, 353.0495; Found, 353.0523.
(S)ꢀ3ꢀ(Pyrrolidinꢀ2ꢀylmethoxy)ꢀ5ꢀ((3ꢀ(trifluoromethyl)phenoxy)methyl)isoxazole
Trifluoroacetate
(28). This compound was obtained from 14 and 3ꢀ(trifluoromethyl)phenol in two steps employing Method
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F, D, and Gradient I. Pale yellow liquid; yield 30%; purity 98.5%. [α]D +13.96 (c = 0.16, MeOH); H
NMR (400 MHz, DMSOꢀd6) δ 9.59 (br s, 1H), 9.06 (br s, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.37 (m, 2H), 6.47
(s, 1H), 5.32 (s, 2H), 4.45 (dd, J = 8.0, 4.0 Hz, 1H), 4.34 (dd, J = 10.0, 4.0 Hz, 1H), 3.96 (m, 1H), 3.23 (t,
J = 6.0 Hz, 2H), 2.18–2.06 (m, 1H), 2.04–1.81 (m, 2H), 1.81–1.61 (m, 1H); 13C NMR (101 MHz, DMSOꢀ
d6) δ 170.8, 168.6, 158.6 (TFA) , 157.7, 130.8, 130.2 (q, JCꢀF = 31.6 Hz), 123.8 (q, JCꢀF = 271.0 Hz),
119.0, 118.0 (q, J CꢀF = 4.0 Hz), 115.6 (TFA), 111.4 (q, JCꢀF = 4.0 Hz), 95.6, 68.4, 60.8, 57.5, 45.2, 26.0,
23.3; HRMS (ESI): Calcd for C16H18F3N2O3 [M+H]+, 343.1264; Found, 343.1281.
(S)ꢀ3ꢀ(Pyrrolidinꢀ2ꢀylmethoxy)ꢀ5ꢀ((4ꢀ(trifluoromethyl)phenoxy)methyl)isoxazole
Trifluoroacetate
(29). This compound was obtained from 14 and 4ꢀ(trifluoromethyl)phenol in two steps as white solid in
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30% yield employing Method F, D, and Gradient I. Purity 95.6%. [α]D +9.24 (c = 0.18, MeOH); H
NMR (400 MHz, DMSOꢀd6) δ 9.40 (br s, 1H), 8.96 (br s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 12.0
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