Development of an Aryloxazole Class of Hepatitis C Virus Inhibitors Targeting the Entry Stage of the Viral Replication Cycle

Article abstract of DOI:10.1021/acs.jmedchem.7b00561

Reliance on hepatitis C virus (HCV) replicon systems and protein-based screening assays has led to treatments that target HCV viral replication proteins. The model does not encompass other viral replication cycle steps such as entry, processing, assembly and secretion, or viral host factors. We previously applied a phenotypic high-throughput screening platform based on an infectious HCV system and discovered an aryloxazole-based anti-HCV hit. Structure-activity relationship studies revealed several compounds exhibiting EC₅₀ values below 100 nM. Lead compounds showed inhibition of the HCV pseudoparticle entry, suggesting a different mode of action from existing HCV drugs. Hit 7a and lead 7ii both showed synergistic effects in combination with existing HCV drugs. In vivo pharmacokinetics studies of 7ii showed high liver distribution and long half-life without obvious hepatotoxicity. The lead compounds are promising as preclinical candidates for the treatment of HCV infection and as molecular probes to study HCV pathogenesis.

Full text of DOI:10.1021/acs.jmedchem.7b00561

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Article
The Development of an Aryloxazole Class of Hepatitis C Virus
Inhibitors Targeting the Entry Stage of the Viral Replication Cycle
Shanshan He, Kelin Li, Billy Lin, Zonyi Hu, Jingbo Xiao, Xin Hu, Amy Q.
Wang, Xin Xu, Marc Ferrer, Noel Southall, Wei Zheng, Jeffrey Aubé, Frank
J. Schoenen, Juan J Marugan, T Jake Liang, and Kevin J. Frankowski
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 21 Jun 2017
Downloaded from http://pubs.acs.org on June 21, 2017
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The Development of an Aryloxazole Class of Hepatitis C Virus Inhibitors
Targeting the Entry Stage of the Viral Replication Cycle
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a
b, c
a
a
d
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Shanshan He, Kelin Li, Billy Lin, Zongyi Hu, Jingbo Xiao, Xin Hu, Amy Q. Wang, Xin
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d
d
d
b, c
b
Xu, Marc Ferrer, Noel Southall, Wei Zheng, Jeffrey Aubé, Frank J. Schoenen, Juan J.
d
a*
b, c *
Marugan, T. Jake Liang and Kevin J. Frankowski
a
Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases,
b
National Institutes of Health, 10 Center Drive, Bethesda, Maryland 20892 ꢀ1800; University of
c
Kansas Specialized Chemistry Center, University of Kansas, Lawrence, KS 66047; Current
address: Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School
of Pharmacy, University of North Carolina at Chapel Hill, 125 Mason Farm Road, Marsico Hall,
d
Chapel Hill, NC 27599ꢀ7363; Division of Preclinical Innovation, National Center for
Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive,
Rockville, MD, 20850; * Coꢀcorresponding authors.
KEYWORDS
Hepatitis C, antivirals, oxazole, phenotypic screening
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ABSTRACT
Reliance on hepatitis C virus (HCV) replicon systems and proteinꢀbased screening assays has led
to treatments that target HCV viral replication proteins. The model does not encompass other
viral replication cycle steps, such as entry, processing, assembly and secretion, or viral host
factors. We previously applied a phenotypic highꢀthroughput screening platform based on an
infectious HCV system and discovered an aryloxazoleꢀbased antiꢀHCV hit. Structureꢀactivity
relationship studies revealed several compounds exhibiting EC values below 100 nM. Lead
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compounds showed inhibition of the HCV pseudoparticle entry, suggesting a different mode of
action from existing HCV drugs. Hit 7a and lead 7ii both showed synergistic effects in
combination with existing HCV drugs. In vivo pharmacokinetics studies of 7ii showed high liver
distribution and long halfꢀlife without obvious hepatotoxicity. The lead compounds are
promising as preclinical candidates for the treatment of HCV infection and as molecular probes
to study HCV pathogenesis.
INTRODUCTION
Hepatitis C virus (HCV) leads to chronic infection in 80% of patients, and disease progression
1
can eventually cause liver cancer or cirrhosis following decades of asymptomatic infection.
HCV is the major underlying cause for liver transplants and is responsible for significant
2
healthcare costs. The prevalence of HCV has been estimated at around 200 million people
3
4
worldwide, and, to date, no effective vaccine has been developed. The historical treatment
regimen for HCV involved combination therapy of ribavirin with pegylated interferon, a poorly
tolerated course of treatment with only moderate success in achieving a sixꢀmonth postꢀtreatment
sustained virological response (SVR), an undetectable viral RNA level six months after
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treatment cessation. The recent approval of several smallꢀmolecule directꢀacting antivirals
5
(
DAAs) has dramatically improved the standard of care for HCV. These drugs target the viral
proteins (NS3/4A protease, NS5B polymerase and NS5A) involved in the replication stage of
HCV infection. Although these treatments offer renewed hope toward curing HCV infection, the
price of the medicines is prohibitively expensive for many highꢀrisk populations, such as
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intravenous drug users, prisoners and those in the developing world. Furthermore, these agents
can lead to rapid development of viral resistance if the virus is not fully eradicated during the
treatment regimen.
The hegemony of replication inhibitors as treatments for HCV infection is a direct consequence
of the available methods to screen for inhibitors of the virus. Current HCV inhibitors have
mostly been discovered through either the replicon assay, which measures the replication of
isolated viral RNA, or proteinꢀbased assays using HCV proteins involved in viral replication
(
e.g. NS3, NS5A or NS5B). On the other hand, a cellꢀbased infectious HCV assay platform can
cover the complete spectrum of potentially druggable targets in all stages of the HCV replication
cycle, and allow for the development of inhibitors acting on different phases of the viral life
cycle less prone to mutation. More importantly, targeting several key processes in the viral
replication cycle may not only increase antiviral efficacy, but also reduce the capacity of the
virus to develop resistance to the compound. We have developed such an assay platform based
7
on a HCV infectious cell culture system. The assay was adapted for the highꢀthroughput
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screening of a 350,000ꢀmember compound collection, affording 149 validated hit compounds.
Herein we describe the optimization, preliminary mode of action (MOA) studies, and detailed
biopharmaceutical and pharmacokinetic (PK) properties for an aryloxazole class of compounds
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discovered during the screening campaign, and demonstrate that the class is promising for further
pharmaceutical development.
RESULTS AND DISCUSSION
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Chemistry. The resynthesized aryloxazole HTS hit compound and most SAR analogues were
constructed according to the general route in Scheme 1. The aryloxazole cores 4 were assembled
using the method of Goto et al. from commercial benzaldehydes 1 and butaneꢀ2,3ꢀdione monoꢀ
9
oxime 2 via the Nꢀoxide intermediates 3. Derivatization of 4 with isonipecotic acid provided the
carboxylic acids 5, which were coupled with the diamine fragments 6 using
diisopropylcarbodiimide and 1ꢀhydroxybenzotriazole (HOBt) hydrate to afford the final
analogues 7, 13 or 18. The coupling reactions were conducted either at room temperature in
DCM or at 100 °C using microwave irradiation in MeCN, depending on when during the SAR
campaign the analogues were synthesized. Detailed synthetic protocols for individual final
analogues are provided in the Experimental Section. The final compounds were purified by
either flash chromatography or reverseꢀphase, preparativeꢀscale, massꢀdirected high performance
liquid chromatography (HPLC) and their purity was assessed by analyticalꢀscale HPLC under
analogous conditions. The thiazole core 10 was synthesized via condensation of 3ꢀchlorobutaꢀ2ꢀ
one and thioamide 8 (Scheme 2). Chlorination with Nꢀchlorosuccinimide followed by
derivatization with isonipecotic acid and diamine fragment 6a, as described above afforded the
final analogue 13. The desmethyl oxazole core was constructed via condensation and
monodehalogenation of 1,3ꢀdichloroacetone with benzamide 14 to directly afford the
choromethyloxazole scaffold 16 (Scheme 3). Derivatization with isonipecotic acid and diamine
fragments 6, as described above afforded the final analogues 18. The 3ꢀpiperidinylpropylamines
6
that were not commercially available were readily synthesized by a short twoꢀstep sequence
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(
Scheme 4). Briefly, addition of the appropriately substituted piperidine to acrylonitrile followed
by Raney nickelꢀmediated nitrile reduction under a hydrogen atmosphere afforded the requisite
diamine fragments 6. Full synthetic protocols for individual diamine fragments are provided in
the Supporting Information.
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a
Scheme 1. General synthetic route to aryloxazole analogues 7.
a
Reagents and conditions: (a) HCl (4M in dioxane), AcOH, 0 °C to rt; (b) POCl , DCE, reflux;
(g)
3
(
c) KOH, EtOH, isonipecotic acid, rt; (d) HOBt hydrate, DMAP, diisopropylcarbodiimide,
DCM, rt; or HOBt hydrate, diisopropylcarbodiimide, MeCN, µW irradiation, 100 °C, 10 min.
a
Scheme 2. Synthetic route to thiazole analogue 13.
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Reagents and conditions: (a) iPrOH, µW irradiation, 120 °C, 1 h, 65% yield; (b) NCS, MeCN,
60 °C, 2 h, 82% yield; (c) KOH, EtOH, isonipecotic acid, rt, 78% yield; (d) HOBt hydrate,
diisopropylcarbodiimide, MeCN, µW irradiation, 100 °C, 10 min, 45% yield.
a
Scheme 3. Representative synthetic route to desmethyloxazole analogues; synthesis of 18a.
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F
F
Me
Me
F
Me
Cl
O
OH
a
b
+
N
N
O
Cl
NH2
O
O
Cl
N
O
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H
H
1
4
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Me
c
H N
N
2
Me
6
a
Me
Me
N
Me
HN
N
O
O
N
H
18a
a
Reagents and conditions: (a) toluene, 140 °C, 5 h, 57% yield; (b) KOH, EtOH, isonipecotic
acid, rt, 93% yield; (d) HOBt hydrate, DMAP, diisopropylcarbodiimide, DCM, rt, 60% yield.
a
Scheme 4. General synthetic route to diamine fragments 6.
a
Reagents and conditions: (a) acrylonitrile, formamide, water; (b) Raney nickel, H2 (g), 200 psi,
MeOH, NH3 (MeOH)
.
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Structure-activity relationship studies. Shown in Figure 1, the structural elements that were
investigated during the structureꢀactivity relationship (SAR) study are highlighted in the HTS hit
molecule (PubChem CID3244725). The majority of the structural analogues focused on varying
either arylꢀring substitution or substitution on, or replacement of, the piperidine moiety, the
molecular fragments on the left and right ends of the compound hit, respectively. All analogues
were screened for inhibition of HCV infection in the cellꢀbased HCVꢀLuc assay and
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counterscreened for cytotoxicity in Huh 7.5.1 cells (ATPlite assay). In addition, most analogues
were profiled in three assays to assess in vitro biopharmaceutical properties: rat microsomal
stability, cell permeability and aqueous kinetic solubility. Tables 1 to 3 summarize the results of
these SAR investigations. The stereochemistry on the dimethylꢀsubstituted piperidine ring of the
HTS hit (PubChem CID3244725) was undefined in the registered structure. Thus, we began our
SAR investigation by synthesizing both the cis and trans stereoisomers of the hit compound, 7a
and 7b, respectively. The trans stereoisomer 7b was synthesized and screened as a racemic
mixture. Both isomers were equipotent in the HCVꢀLuc assay and showed favorable aqueous
solubility, however, 7a possessed slightly lower cytotoxicity and appeared to have better stability
in the rat liver microsomes. Replacing the 3,5ꢀdimethyl substitution with bulkier 3,5ꢀdiethyl (7c)
or 3,3,5,5ꢀtetramethyl substitution (7d) also afforded potent analogues, though with slightly
greater cytotoxicity. Moreover, analogues exploring substitution on the piperidine moiety
revealed potency to be dependent on both the size and location of substituents on the heterocycle
ring. Sufficiently bulky substitution at the 4ꢀposition afforded potent analogues (7e and 7f),
while a single methylꢀgroup substituent at this position (7g), afforded less potent analogues.
Consistent with this trend, the unsubstituted piperidine 7h was even less potent. While the cisꢀ
2,6ꢀdimethyl substituted analogue 7i did not improve the potency, the 3ꢀsubstituted analogues
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(
racemic compounds 7j to 7l) possessed slightly improved potency compared to the 3,5ꢀ
disubstituted compound 7a. The cisꢀdisubstituted compounds (e.g. 7a) however do not possess
any asymmetric centers (meso compounds), and were therefore screened as single stereoisomers
rather than enantiomeric mixtures. The constrained analogue 7m did not provide any
improvement in potency or cytotoxicity and added unnecessary structural complexity. In all the
above cases, increased steric bulk on the piperidine also correlated with decreased microsomal
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stability. Decreasing or increasing the tether length by one –CH group in the diamine fragment
2
(7n and 7o, respectively), did not improve the potency and we retained the threeꢀcarbon linker
for the remaining SAR investigation. Ringꢀopening of the piperidine to the dialkyl amine 7p
afforded a slightly less potent analogue, possibly suggesting that the piperidine constraint might
not be required. However, limited exploration of more structurally drastic piperidine
replacements was detrimental to potency (7q to 7s).
Figure 1. Summary of structural modifications explored in optimizing the aryloxazole hit and
structure of the selected lead analogue 18a.
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Table 1. Effect of varying the diamine fragment on potency, cytotoxicity and in vitro
biopharmaceutical properties.
structure
cellꢀbased assay activity
in vitro pharmacokinetic assays
rat liver
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entry/
cmpd
selectivity
index
CC50/EC50
PAMPA
aqueous
potency, EC50 cytotoxicity, CC50
microsome
stability
a
a
permeability solubility
(1×10 cm/s) (µg/ml)
(µM)
(µM)
ꢀ6
(
)
t
1/2 (min)
2
R
0
0
.138 ± 0.134
(n=8)
7
7
a
13.4 ± 2.258
9.390 ± 0.348
97
>30.0
489.1
553.7
53.9
.050 ± 0.025
188
14
>69.0
b
7
c
0.181 ± 0.104
0.083 ± 0.046
0.056 ± 0.015
0.066 ± 0.029
0.225 ± 0.160
0.473 ± 0.300
0.120 ± 0.023
0.037 ± 0.006
0.054 ± 0.013
3.467 ± 0.266
4.377 ± 0.260
4.283 ± 0.135
10.153 ± 0.393
12.267 ± 0.586
18.1 ± 0.800
19
53
5.5
1267
>1397
395.6
241.4
424.7
34.5
ND
7d
5.7
ND
7
e
76
8.8
>71.0
>69.0
>67.0
>65.0
>69.0
>67.0
>71
7
f
154
55
>30.0
>30.0
>30.0
>30.0
>30.0
19.4
7g
7
h
38
7
i
H
N
21.300 ± 1.277
15.000 ± 1.572
5.823 ± 1.541
178
405
108
55.4
N
7
j
ND
7
k
ND
H
N
N
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l
0.094 ± 0.018
4.640 ± 0.243
49
2.3
3
54.9
7
m
0.106 ± 0.034
0.156 ± 0.035
0.113 ± 0.065
10.867 ± 0.252
24.437 ± 4.502
23.133 ± 4.155
103
157
205
7.4
>30.0
12
330.9
712.4
287.8
>71
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7n
>67.0
>71.0
7
o
H
N
N
7
7
p
q
0.341 ± 0.142 30.467 ± 4.225
89
61
>30.0
>30.0
913.1
432.4
>65.0
>65.0
1
.063 ± 0.727
65.300 ± 3.387
19.933 ± 2.159
27.233 ± 2.060
7
r
1.621 ± 1.426
2.789 ± 2.800
12
10
>30.0
>30.0
<3.2
14
>71.0
>67.0
7s
a
Average of three separate assays (unless otherwise noted) ± standard deviation
ND, not determined
The relationship between activity and aryl ring substitution is summarized in Table 2. Changing
the position of the 2ꢀmethyl substituent on the aryl ring to either the 3ꢀ or 4ꢀposition (7t and 7u,
respectively) had no significant effect on potency, however the 4ꢀmethyl analogue 7u had
increased cytotoxicity. 2ꢀEthyl substitution (7v) or various arrangements of dimethyl substituted
aryl analogues (7w to 7aa) all possessed a satisfactory potency of less than 100 nM. Both 7v and
the 3,5ꢀdimethylꢀsubstituted analogue 7w possessed reduced microsomal stability compared to
other analogues. Further increasing the substitution to triꢀ and pentaꢀmethyl substituted aryl
analogues (7bb and 7cc, respectively) afforded less potent compounds than the monoꢀ or diꢀ
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1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
methyl substituted analogues. The activities for the monoꢀhalogensubstituted analogues (7dd to
7gg) were on par with the activities for the most potent 2ꢀmethyl aryl analogues. The diꢀhalo
analogues (7hh to 7mm) were generally less potent; the notable exception being the 3,4ꢀdichloro
analogue 7ii, which possessed a potency of 13 nM, ultimately the most potent analogue
synthesized in this study. Other than 7ii and the 3,5ꢀdifluoroꢀsubstituted analogue 7jj, dihaloꢀ
substitution reduced the microsomal stability. While either monoꢀhalogenꢀ or methylꢀsubstitution
had been found to afford analogues with attractive potency (< 100 nM), the combination of both
mono halogenꢀ and methylꢀsubstitution in a single analogue (7nn to 7ss) afforded analogues of
only modest potency (124–370 nM) and lower microsomal stability than either substitution in
isolation. Methoxy substitution (7tt to 7zz) was uniformly detrimental to the potency. The
naphthyl analogues (7aaa and 7bbb) both possess potency below 100 nM, however the
anthracene analogue (7ccc) was significantly less potent (357 nM).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Effect of varying the aryl substitution on potency, cytotoxicity and in vitro
biopharmaceutical properties.
structure
cellꢀbased activity and cytotoxicity
in vitro biopharmaceutical property
entry/
cmpd
selectivity
index
rat liver
microsomes t1/2
(min)
7
{x}
potency, EC50 cytotoxicity, CC50
permeability solubility
a
a
ꢀ6
(
µM)
(µM)
(1×10 cm/s)
(µg/ml)
(CC50/EC50)
1
2
R
R
7
t
0.096 ± 0.027
0.070 ± 0.038
0.111 ± 0.035
16.483 ± 3.963
4.333 ± 2.012
9.323 ± 0.784
172
62
>30.0
>30.0
17
279.1
274.3
106.1
>69.0
7
u
v
>69.0
55.9
7
84
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Journal of Medicinal Chemistry
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9
7
w
0.091 ± 0.037
0.093 ± 0.022
0.076 ± 0.016
0.037 ± 0.031
0.098 ± 0.29
0.190 ± 0.094
0.149 ± 0.074
0.057 ± 0.025
0.094 ± 0.028
0.041 ± 0.025
0.053 ± 0.029
0.196 ± 0.024
0.013 ± 0.008
0.421 ± 0.229
0.539 ± 0.343
21.033 ± 4.910
6.910 ± 0.834
4.113 ± 0.122
4.05 ± 0.108
231
74
3.1
>30.0
>30.0
<30.0
30
253.9
219.4
353.1
864
55.9
>71.0
>71.0
>71.0
56.1
7
7
7
x
y
z
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
54
109
42
7aa
4.070 ± 0.130
6.573 ± 1.253
8.183 ± 0.671
16.500 ± 1.735
4.210 ± 0.201
11.517 ± 6.370
12.800 ± 2.307
13.233 ± 0.252
3.17 ± 0.635
ND
7
bb
35
>30
ND
ND
ND
7
cc
55
455.2
352.4
615.1
>635.0
ND
ND
7dd
289
45
15
>72.0
>72.0
>72.0
ND
7
ee
>30.0
>30.0
28.1
2.9
7
ff
281
242
68
7
gg
7
hh
ND
>77.0
ND
7ii
244
78
>30
21
522
7
jj
32.667 ± 1.159
12.633 ± 2.307
82.4
>72.0
ND
7
kk
23
3.9
129.9
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7
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
ll
0.220 ± 0.144
0.193 ± 0.086
6.497 ± 0.166
30
2.1
<1.0
62.4
7mm
12.200 ± 0.173
12.750 ± 1.569
16.411 ± 0.501
11.700 ± 0.361
4.907 ± 0.364
63
103
44
1.4
2.9
5.4
4.4
2
ND
196.3
650.6
ND
>83.0
>74.0
52.6
>74
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.124 ± 0.087
(n=6)
7
nn
0
.370 ± 0.179
n=2)
7
oo
(
7
7
pp
qq
0.220 ± 0.060
0.124 ± 0.059
53
40
ND
>76
7rr
0.326 ± 0.083
0.226 ± 0.088
0.351 ± 0.167
0.243 ± 0.195
0.577 ± 0.238
0.428 ± 0.057
12.033 ± 0.723
13.000 ± 0.458
14.900 ± 0.520
15.833 ± 0.513
37.000 ± 2.946
16.700 ± 0.265
37
58
42
65
64
39
25
1.8
4.4
3.5
3.3
8.6
4.6
12
ND
233.8
245.3
51.0
ND
65.1
7
ss
>72.0
>83.0
>76.0
>74.0
>73.0
>76.0
7tt
7
uu
7vv
7ww
197.7
ND
7xx
7yy
3.110 ± 0.199 77.133 ± 12.596
0.440 ± 0.264
57.233 ± 3.213
>100
130
>3
>30.0
>30.0
81.9
10.4
>76.0
>72.0
7
zz
33.983 ± 24.162
7
aaa
0.052 ± 0.021
4.307 ± 0.479
83
>30.0
ND
>74.0
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7
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9
7
bbb
0.097 ± 0.024
0.357 ± 0.147
4.057 ± 0.177
4.277 ± 0.110
42
12
4.9
22
547.1
ND
>76.0
62.1
7
ccc
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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28
29
30
31
32
33
34
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49
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a
Average of three separate assays (unless otherwise noted) ± standard deviation
ND, not determined
We also explored limited structural alterations to the scaffold core, as summarized in Table 3.
The thiazole analogue 13 displayed similar potency to the hit, and, with no clear advantage,
further thiazole analogues were not explored. The desmethyl oxazole analogues 18a and 18b
were found to be among the most potent analogues, suggesting that the methyl group on the
oxazole may not be essential for activity. The cisꢀdimethyl piperidine analogue 18a also
possessed a higher selectivity index (SI = CC / EC ) than that of either 7a or 7ii (SI = 97, 244
5
0
50
and 341 for 7a, 7ii and 18a, respectively).
Table 3. Effect of core scaffold modifications on potency, cytotoxicity and in vitro
biopharmaceutical properties.
cellꢀbased activity and cytotoxicity
in vitro biopharmaceutical property
entry/
cmpd
structure
selectivity
index
CC50/EC50
rat liver
microsomes
t1/2 (min)
potency, EC50 cytotoxicity, CC50
permeability solubility
(1×10 cm/s) (µg/ml)
a
a
ꢀ6
(µM)
(µM)
(
)
1
3
0.081 ± 0.041 7.053 ± 0.788
87
7.4
ND
>71
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7
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
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4
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
18a
0.038 ± 0.037 12.967 ± 0.851
341
14.1
215.4
>70
N
O
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
O
Me
NH
18b
0.055 ± 0.020 6.195 ± 0.587
113
2.5
276.4
>72
N
a
Average of three separate assays (unless otherwise noted) ± standard deviation
ND, not determined
During this SAR study, we established a clear relationship between substitution on the terminal
piperidine moiety and onꢀtarget activity; highly potent analogues all contained apparent steric
bulk at the 3ꢀ and/or 4ꢀposition of the piperidine. While a single methyl group at the 3ꢀposition
gave highly potent analogues, the 4ꢀposition required more steric bulk, gem dimethyl or
isopropyl substitution to provide equipotent analogues. The aryl moiety distal to the piperidine of
the chemotype also allowed some flexibility in substitution. A single substituent (e.g. methyl or
chloro) at the ortho position provided highly potent analogues. Disubstituted analogues, either at
the 3,5ꢀ or 3,4ꢀpositions also afforded highly potent compounds, with these analogues also
possessing improved stability in the microsomal stability assay. The limited exploration of
changes to the oxazole core indicates that modest changes in this portion of the molecule are
tolerated, however more extensive investigation would be needed to establish a more detailed
analysis. In summary, we have prepared a number of highly potent lead compounds with low
cytotoxicity and promising in vitro biopharmaceutical properties.
In vitro profiling of anti-HCV activity and selectivity. During the SAR studies, a number of
promising compounds were identified and three analogues were chosen for further biological
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evaluation and characterization. Figure 2 shows the representative titration curves for the antiꢀ
HCV activity and cytotoxicity of selected leads. Selectivity indices between 103 and 341 were
achieved for these compounds, showing a significant therapeutic index. The analogue used for a
particular profiling study switched over the course of the project from 7nn to 7ii once the latter
compound emerged as the more potent and selective analogue. The intent of the profiling results
is to illustrate the potential of this compound class for further development or mechanistic
investigation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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43
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
ii
7nn
18a
EC50 0.0197 µM
CC50 5.03 µM
EC50 0.110 µM
CC50 14.1 µM
EC50 0.0188 µM
CC50 13.0 µM
1
1
20
00
120
100
80
60
40
20
0
120
100
80
8
6
4
0
0
0
60
40
20
0
20
0
-
20
-20
-20
-3
-2
-1
0
1
2
-3
-2
-1
0
1
2
-3
-2
-1
0
1
2
Log [Concentration] (µM)
Log [Concentration] (µM)
Log [Concentration] (µM)
Figure 2. Titration curves for antiꢀHCV activity (EC , triangle in blue) and cytotoxicity (CC ,
50
50
circle in red) of selected leads. Cellꢀculture adapted HCV (HCVcc) harboring a luciferase
reporter gene (HCVꢀLuc, genotype 2a) was used to infect Huh7.5.1 cells in the presence of
increasing concentrations of test compound. Viral infection and replication were measured by
luciferase signal 48 h after treatment. Cytotoxicity was evaluated in parallel with the ATPꢀbased
cell viability assay (ATPlite). The results are mean from three replicates ± SEM. The EC and
5
0
CC values were calculated with GraphPad Prism 5.0 software using nonlinear regression.
50
Curves and values for each compound were from single, representative experiments.
To determine whether this class of compounds possessed a distinct antiviral mode of action from
those of the known HCV inhibitors, the activity of the hit compound 7a and the selected lead
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Journal of Medicinal Chemistry
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compounds were measured at 10 µM in a series of assays focused on discrete phases of the viral
replication cycle (Table 4). The HCVꢀLuc antiviral activity and cytotoxicity were confirmed at
the same concentration for comparison. In the HCV singleꢀcycle infection assay, singleꢀround
infectious HCV defective particles (HCVsc, genotype 2a) can infect and replicate but do not
assemble new virions, thus this assay detects compounds with inhibitory activity to HCV
replication cycle events prior to assembly. The activities of 7a and the selected lead compounds
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7ii, 7nn and 18a in the HCVsc infection assay were equivalent to the values obtained in the
phenotypic assay, suggesting an earlyꢀstage target of the viral replication cycle such as HCV
entry and/or RNA replication. Therefore, HCV subgenomic replicon assays evaluate whether
compounds target viral RNA replication. All compounds showed less than 40% inhibitory effect
on HCV replication in GT 1b replicon cell line, except for 7ii possibly due to cytotoxicity as
suggested in the ATPlite assay result. This would indicate that replicationꢀstage targets are not
responsible for the HCV inhibition. HCV pseuodoparticles (HCVpp, GT 1a and 1b) utilize
defective retroviral particles that display HCV envelope glycoproteins. HCVpp are a wellꢀ
10
established surrogate system that mimics the entry stage of cellꢀculture adapted HCV (HCVcc).
We thus utilized the HCVpp assay to assess the effect of compound treatment on viral entry.
Viral pseudoparticles from vesicular stomatitis virus (VSVpp) were used to test the effect of
compounds on an unrelated virus. The hit 7a showed potent inhibitory activity (< 30% RLU of
control) in HCVpp GT 1a assay and moderate inhibition (~50% RLU) in VSVpp assay. The lead
compounds also exhibited potent to moderate inhibition in HCVpp GT 1a and 1b assays. The
combined profile for 7a and the lead compounds suggests that the chemotype is targeting an
early stage of the viral replication cycle, likely by inhibiting HCV entry, based on the HCVpp
activity.
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Journal of Medicinal Chemistry
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7
1
Table 4. Activity of selected leads in a series of HCV lifecycle assays.
a
%
RLU
d
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Entry
HCV
HCVpp
b
e
f
HCVꢀLuc
ATPlite
HCVsc
replicon
GT 1b
MLVpp
VSVpp
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
GT 1a GT 1b
7
a
2.16 ± 0.508 90.0 ± 3.0 1.63 ± 1.57 75.2 ± 4.9 21.9 ± 14.7 16.8 ± 6.5 48.7 ± 3.85 61.5 ± 5.2
ii 0.27 ± 0.07 4.88 ± 0.19 3.92 ± 1.51 9.1 ± 3.0 14.9 ± 11.6 25.3 ± 6.4 57.7 ± 2.4 28.0 ± 3.1
nn 0.72 ± 0.32 90.4 ± 1.5 3.29 ± 2.06 83.3 ± 7.0 76.8 ± 7.7 16.6 ± 10.5 102 ± 5 77.7 ±12.0
8a 0.59 ± 0.39 74.1 ± 3.0 4.14 ± 1.94 65.2 ± 3.2 35.1 ± 5.8 31.7 ± 8.5 111 ± 6 78.0 ± 4.9
7
7
1
1
Activity of compound (10 µM) in a series of assays to assess efficacy in different stages of the viral life
cycle. Results are presented as percentage response compared to the untreated control. Relative
luminescence units; HCV singleꢀcycle infection assay; HCV subgenomic replicon assay; HCV
pseudoparticle assay; murine leukemia virus pseudoparticle assay; vesicular stomatitis virus
pseudoparticle assay.
a
b
c
d
e
f
In vitro toxicity profiling against a panel of fifty CNS-relevant targets. Compound 7nn was
evaluated in a panel of fifty CNSꢀrelevant receptors and neurotransmitter transporters by the
Psychoactive Drug Screening Program (PDSP) at the University of North Carolina, Chapel Hill
(
Figure 3). The results show that 7nn only has sub micromolar affinity for four targets across the
fiftyꢀcomponent panel tested. The highest affinity was for the serotoninergic receptor 5ꢀHT2B
11, 12
(
415 nM), which has been implicated in the development of myofibroblast proliferation.
While any cardiac issue is of concern, the selectivity for 7nn against the other targets in the
PDSP panel and the modest affinity for the 5ꢀHT2B receptor bode well that this offꢀtarget
activity could be minimized with further compound development. The receptor profiling of other
compounds will be conducted following additional characterization of select lead compounds for
further development.
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2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
-HT1A 5-HT1B 5-HT1D 5-ht1e 5-HT2A 5-HT2B 5-HT2C 5-HT3
5-ht5a
5-HT6 5-HT7 Alpha1A
415
Alpha1B Alpha1D Alpha2A Alpha2B Alpha2C Beta1 Beta2 Beta3 BZP Rat Brain Site
D1
D2
D3
M2
1,416
575
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
D4
D5
DAT
DOR
GABAA
H1
H2
H3
H4
KOR
M1
M3
V2
443
1,208
M4
M5
mGlur5 MOR
NET oxytocin PBR
SERT
Sigma 1
Sigma 2 V1A
V1B
1,749
1,289
2,495
1,806
984
Key:
Ki > 10 μM or primary screen missed
Figure 3. Profile of representative analogue 7nn against a panel of fifty CNSꢀrelevant targets.
Values are Ki determinations (nM) of radioligand binding in a displacement assay. Primary
screen assays are singleꢀpoint experiments to determine percent radioligand displacement at 10
µM of test compound. The threshold for hit or miss in the primary screen was 50% displacement.
In vitro combination profiling with different classes of anti-HCV drugs. We investigated the
combination of 7ii with different classes of antiꢀHCV drugs. The HCVꢀLuc and the ATPlite
assays were performed in the presence of various concentrations of 7ii in combination with
various concentrations of each drug (Table 5). A synergistic effect was indicated if the
combination led to a greater HCV inhibitory effect than either of them alone in a concentrationꢀ
dependent manner without a toxic effect on cell viability. Log volumes of synergy or antagonism
were generated according to the Bliss independence model by using the MacSynergy II
1
3
14
program. The results were also analyzed with the CalcuSyn program, in which the
combination indices were calculated from combination of 7ii and the tested drug at or near their
EC values when tested alone. The hit compound 7a was tested following the same protocol.
50
We found that 7ii was synergistic with ribavirin, sofosbuvir, telaprevir, daclatasvir, cyclosporin
A, and boceprevir without significant cytotoxicity, supporting its potential for use in combination
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therapy with these drugs. Similar synergistic effects were observed with compound 7a, except
for nearly additive effect in combination with sofosbuvir. The observed synergistic effects
suggest that the aryloxazole analogues likely operate via a distinct mode of action from that of
any one of these drugs. The mechanism of action of ribavirin is mediated through host antiviral
response. Telaprevir and boceprevir are NS3/4A protease inhibitors and daclatasvir inhibits HCV
NS5A. Cyclosporin A targets virus RNA replication. Together, this collection of HCV drugs
cover the known modes of action for currently available therapeutics, thus this chemotype is
attractive for further development.
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Table 5. Synergistic activity of 7a and 7ii with selected HCV therapeutics.
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Entry
Program
Parameter
Ribavirin
Sofosbuvir
Telaprevir
Daclatasvir
Cyclosporin A
0.625 ± 0.136
Boceprevir
N.D.
a
CI value 0.770 ± 0.189 1.13 ± 0.12 0.775 ± 0.108 0.510 ± 0.082
CalcuSyn
Synergy
volume
b
++
+/ꢀ
+
++
+++
+
+++
N.D.
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a
Synergy
volume
MacSynergy
CalcuSyn
c
+++
+++
++
0.867 ± 0.115
+
N.D.
0.782 ± 0.104
++
a
CI value 0.745 ± 0.084 0.759 ± 0.076 0.740 ± 0.102 0.831 ± 0.133
Synergy
b
++
++
++
++
+
7
ii
volume
Synergyc
MacSynergy
+++
+++
+++
++
++
volume
a
Values are mean ± SEM of combination indices (CI) obtained from combinations of the tested drug with 7a or 7ii at or near their
EC values when tested alone (n ≥ 6).
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b
The level of synergy is defined as the following: “+/ꢀ” means nearly additive (0.9 ≤ CI < 1.1), “+” means minor synergy (0.8 ≤ CI <
0.9), “++” means moderate synergy (0.7 ≤ CI < 0.8) and “+++” means strong synergy (CI < 0.7).
c
The levels of synergy are defined as the following: “+/ꢀ” means nearly additive (0 ≤ log volume < 2) “+” means minor synergy (2 ≤
log volume < 5), “++” means moderate synergy (5 ≤ log volume <9) and “+++” means strong synergy (log volume > 9).
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In vivo pharmacokinetic studies in mice at multiple doses. We evaluated the in vivo
pharmacokinetics and tissue distribution of 7ii in a mouse model after a single dose of 10 mg/kg
and 1 mg/kg through the intraperitoneal (i.p.) route of administration (Figure 4). Excellent liver
distribution was observed at both doses, as shown in the liver/plasma AUC0ꢀ24h ratio of 41 and
224, respectively. Slow clearance in plasma was observed at both doses, leading to halfꢀlives
longer than 24 h. When dosed at 1 mg/kg, the liver concentration of 7ii throughout 24 h post
administration (3.33 ~ 9.82 ꢁM) was more than 100ꢀfold of its in vitro EC values (0.013 ꢁM).
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0
In light of the above results, we further evaluated the pharmacokinetics properties of 7ii at 0.1
mg/kg and elongated the study time to 168 h (7 days). 7ii retained excellent liver distribution at
this dose during the first 24 h post administration and during the total 168 h study (liver/plasma
AUC0ꢀ24h and AUC0ꢀ168h = 399 and 448, respectively). A reasonably long halfꢀlife was observed
in liver (t1/2 = 77 h) as well as in plasma (t1/2 = 26 h). At a dose as low as 0.1 mg/kg, the
concentration of 7ii in liver reached at 0.39 ꢁM in the first 5 min, and remained at least 7ꢀfold
above its EC values (0.013 ꢁM) throughout the 168 h. Alanine aminotransaminase (ALT) level
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in the mouse serum was monitored to detect any potential hepatotoxicity effect (Figure 4C).
Regardless of the dose, the ALT levels were around or below 80 U/L at most time points with a
few exceptions of higher ALT at random time points from individual mice. These elevations are
unlikely due to the effect of the compound because similar random elevations were noted in mice
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treated with vehicle only (Figure 4C). Moreover, there was not an obvious correlation between
the ALT levels and the liver concentration of compound 7ii. Overall, the representative analog
7ii exhibited high liver distribution and long halfꢀlife without obvious hepatotoxicity indicated
by ALT level in the mouse model.
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Figure 4. Pharmacokinetics studies of compound 7ii in mouse model. (A) Mean liver (green
triangle) and plasma (red dot) concentrationꢀtime profiles of compound 7ii after administration
of a single i.p. dose at 10 mg/kg, 1 mg/kg and 0.1 mg/kg at indicated time points. Compound
concentration of 7ii was measured by UPLCꢀMS/MS methods and is shown in means ± SEM (n
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=
3 per time point). (B) Pharmacokinetic parameters of compound 7ii. AUClast = AUC0ꢀ24h or
AUC0ꢀ168h depending on the sample collection interval. N.D., not determined. (C) Alanine
aminotransaminase (ALT) levels of the mouse serum samples collected during the
pharmacokinetics study. Result for each mouse was shown with scatter plots and error bars show
means ± SEM.
In vitro antiviral specificity profiling against a panel of thirteen viruses. To access whether
this chemotype exhibits nonꢀspecific antiviral effects against viruses other than HCV, we carried
out an antiviral screen with representative lead compound 7nn against 13 viruses utilizing the
nonꢀclinical and preꢀclinical services program offered by the National Institute of Allergy and
Infectious Diseases
(www.niaid.nih.gov/labsandresources/resources/dmid/invitro/Pages/invitro.aspx). The 13 types
of viruses are hepatitis B virus, HCV replicon, herpes simplex virusꢀ1, human cytomegalovirus,
vaccinia virus, dengue virus, influenza A (H1N1) virus, respiratory syncytial virus, SARS
coronavirus, poliovirus 3, Rift Valley fever virus, Tacaribe virus, and Venezuelan equine
encephalitis virus. As shown in the supplementary materials, compound 7nn had little or no
activity (selective index ≤10 and/or EC > 2 ꢁM) against all the above viruses. These results
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suggest that this series of compounds is selectively active against HCV infection.
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CONCLUSIONS
We have presented the development of a new class of HCV inhibitors. The SAR study generated
a number of potent analogues with low cytotoxicity and a promising preliminary PK profile in
mice, the species potentially used for the efficacy evaluation. The compound class appears to act
via a mode of action distinct from that of HCV inhibitors currently approved for HCV therapy.
Such HCV inhibitors with a novel mechanism targeting entry could offer a lower probability of
developing resistant virus strains during treatment as well as provide an additional weapon
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against nonresponsive cases. The attractiveness of HCV entry as an antiꢀHCV target is
18ꢀ20
evidenced by multiple recent efforts in developing HCV entry inhibitors.
oral, pangenotypic HCV treatment with the shortest possible course of treatment, it would be of
benefit to target multiple viral or host targets simultaneously. Studies toward determining the
molecular target and validating the efficacy in vivo are underway and will be reported in due
course.
To attain an all
EXPERIMENTAL SECTION
General synthesis and analysis experimental details. All reagents were used as received from
the following suppliers: Alfa Aesar, Ark Pharm, Aldrich, and Fisher Scientific. Acetonitrile and
THF were purified using the Innovative Technology PureSolv solvent purification system. The
1
13
H and C spectra were recorded on a Bruker Avance 400 MHz or 500 MHz spectrometer.
Chemical shifts are reported in parts per million and were referenced to residual proton solvent
13
signals. C multiplicities were determined with the aid of an APT pulse sequence, differentiating
the signals for methyl (CH ) and methyne (CH) carbons as “d” from methylene (CH ) and
3
2
quaternary (C) carbons as “u”. The infrared (IR) spectra were acquired as thin films using a
universal ATR sampling accessory on a Thermo Scientific Nicolet iS5 FTꢀIR spectrometer and
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ꢀ1
the absorption frequencies are reported in cm . Microwave syntheses were conducted in a
Biotage Initiator constant temperature microwave synthesizer. Flash column chromatography
separations were performed using the Teledyne Isco CombiFlash R using RediSep R silica gel
F
F
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columns. TLC was performed on Analtech UNIPLATE silica gel GHLF plates (gypsum
inorganic hard layer with fluorescence). TLC plates were developed using iodine vapor.
Automated preparative RP HPLC purification was performed using an Agilent 1200 Massꢀ
Directed Fractionation system (Prep Pump G1361 with gradient extension, makeꢀup pump
G1311A, pH modification pump G1311A, HTS PAL autosampler, UVꢀDAD detection G1315D,
fraction collector G1364B, and Agilent 6120 quadrapole spectrometer G6120A). The preparative
chromatography conditions included a Waters XꢀBridge C column (19 × 150 mm, 5 µm, with
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19 × 10ꢀmm guard column), elution with a water and acetonitrile gradient, which increases 20%
in acetonitrile content over 4 min at a flow rate of 20 mL/min (modified to pH 9.8 through
addition of NH OH by auxiliary pump), and sample dilution in DMSO. The preparative gradient,
4
triggering thresholds, and UV wavelength were selected according to the analytical RP HPLC
analysis of each crude sample. The analytical method used an Agilent 1200 RRLC system with
UV detection (Agilent 1200 DAD SL) and mass detection (Agilent 6224 TOF). The analytical
method conditions included a Waters Aquity BEH C column (2.1 × 50 mm, 1.7 µm) and
18
elution with a linear gradient of 5% acetonitrile in pH 9.8 buffered aqueous ammonium formate
to 100% acetonitrile at 0.4 mL/min flow rate. Compound purity was measured on the basis of
peak integration (area under the curve) from UV/Vis absorbance (at 214 nm), and compound
identity was determined on the basis of mass analysis. Compounds used for assays or biological
studies have HPLC purity >95%, with the exception of 7yy (purity = 94.2%). The analytical
HPLC system used is a dedicated instrument for assessing compound purity and routinely detects
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impurities as low as 0.1% that elute within the detection window. Any compounds with a
measured purity of 100% were thus conservatively assigned a purity of >99.8%. Any compounds
purified by reverseꢀphase, preparative HPLC utilized the same solvent gradient and column
material as the analytical conditions to minimize the possibility of impurities that were not
detected in the analytical method. All final compounds were inspected for functional groups
known to contribute PAINS liabilities and none were found.
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General procedure A-1: coupling of carboxylic acid fragment 5 or 12 and amine fragment
6. To a mixture of oxazolecarboxylic acid 5 or thiazolecarboxylic acid 12, amine 6 (1.0–2.0
equiv.) and HOBt (1.0 equiv.) in MeCN (1.5 to 4 mL, ca. 0.1 M) was added diisopropyl
o
carbodiimide (2.0 equiv.). The microwave vial was capped and irradiated at 100 C for 10 min.
After cooling to rt, the solvent was removed and the residue purified by silica gel
chromatography (eluents 0 to 20% MeOH + 0 to 2% NH OH
in CH Cl ) to afford the
2 2
4
(aq)
coupled product.
General procedure A-2: coupling of oxazolecarboxylic acid 5 and amine fragment 6. To a
mixture of oxazolecarboxylic acid 5, amine 6 (1.0–2.0 equiv.) and HOBt (1.0 equiv.) in MeCN
(1.5 to 4 mL, ca. 0.1 M) was added diisopropyl carbodiimide (2.0 equiv.). The microwave vial
o
was capped and irradiated at 100 C for 10 min. After cooling to rt, the solvent was removed and
the residue purified by Cꢀ18 functionalized silica chromatography (eluents 5 to 100% MeCN in
deionized water with 0.5 % NH OH (aq)) to afford the coupled product 7.
4
General procedure A-3: coupling of oxazolecarboxylic acid 5 or 17 and amine fragment 6.
To a mixture of oxazolecarboxylic acid 5 or 17, amine 6 (1.0–2.0 equiv.), DMAP (0.1 equiv.)
and HOBt (1.0 equiv.) in CH Cl (1.5 to 4 mL, ca. 0.05 M) was added diisopropyl carbodiimide
2
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(
5.0 equiv.). The reaction was stirred at rt for 16 h, the solvent was removed and the residue
purified by silica gel chromatography (eluents 0 to 20% MeOH + 0 to 2% NH OH (aq) in CH Cl )
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2
to afford the coupled product 7.
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General procedure A-4: coupling of oxazolecarboxylic acid 5 and amine fragment 6. To a
mixture of oxazolecarboxylic acid 5, amine 6 (1.0–2.0 equiv.), DMAP (0.1 equiv.) and HOBt
(
1.0 equiv.) in CH Cl (1.5 to 4 mL, ca. 0.05 M) was added diisopropyl carbodiimide (5.0
2 2
equiv.). The reaction was stirred at rt for 16 h, the solvent was removed and the residue purified
by automated preparative RP HPLC purification as described in the general experimental section
to afford the coupled product 7.
Synthesis of aryloxazole final analogues.
N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((5-methyl-2-(o-tolyl)oxazol-4-
yl)methyl)piperidine-4-carboxamide 7a. 1ꢀ((5ꢀMethylꢀ2ꢀ(oꢀtolyl)oxazolꢀ4ꢀ
yl)methyl)piperidineꢀ4ꢀcarboxylic acid (41 mg, 0.13 mmol) and 3ꢀ(cisꢀ3,5ꢀdimethylpiperidinꢀ1ꢀ
yl)propanꢀ1ꢀamine (22 mg, 0.13 mmol) were reacted according to general procedure Aꢀ1 to
1
afford the product as a white solid (46 mg, 0.098 mmol, 75% yield). H NMR (400 MHz, CDCl )
3
δ 0.53 (q, J = 12.3 Hz, 1H), 0.86 (d, J = 6.6 Hz, 6H), 1.39 (t, J = 11.0 Hz, 2H), 1.58–1.85
(
complex, 9H), 1.97–2.14 (m, 3H), 2.39 (s, 3H), 2.42 (t, J = 6.2 Hz, 2H), 2.65 (s, 3H), 2.85–2.89
m, 2H), 3.01–3.05 (m, 2H), 3.33 (q, J = 6.1 Hz, 2H), 3.48 (s, 2H), 7.22–7.32 (m, 3H), 7.91–7.94
(
1
3
(m, 1H); C NMR (101 MHz, CDCl , APT pulse sequence) δ d (CH, CH ): 10.4, 19.6, 21.8,
3
3
31.4, 43.5, 125.8, 128.7, 129.4, 131.4; u (C, CH ): 24.9, 29.1, 40.0, 42.1, 53.1, 53.9, 58.3, 61.8,
2
ꢀ1
1
26.9, 132.4, 137.0, 145.5, 159.8, 174.8; IR 1541, 1646, 2950 cm ; HRMS calcd for
+
C H N O [M + H] 467.3381; found 467.3373; HPLC purity: >99.8%.
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