Design, Synthesis, and Biological Evaluation of Substituted 4,6-Dihydrospiro[[1,2,3]triazolo[4,5- b]pyridine-7,3′-indoline]-2′,5(3 H)-dione Analogues as Potent NS4B Inhibitors for the Treatment of Dengue Virus Infection

Article abstract of DOI:10.1021/acs.jmedchem.9b00698

A series of substituted 4,6-dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3′-indoline]-2′,5(3H)-dione analogues were synthesized and evaluated as potent dengue virus inhibitors. Throughout a structure-activity relationship exploration on the amide of the

Full text of DOI:10.1021/acs.jmedchem.9b00698

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Article
Design, Synthesis, and Biological Evaluation of Substituted
,6-Dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-
4
indoline]-2',5(3H)-dione Analogues as Potent NS4B
Inhibitors for the Treatment of Dengue Virus Infection
Jimin Xu, Xuping Xie, Na Ye, Jing Zou, Haiying Chen, Mark Andrew White, Pei-Yong Shi, and Jia Zhou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00698 • Publication Date (Web): 12 Aug 2019
Downloaded from pubs.acs.org on August 12, 2019
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Design, Synthesis, and Biological Evaluation of Substituted 4,6-
Dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione Analogues as
Potent NS4B Inhibitors for the Treatment of Dengue Virus Infection
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Jimin Xu, Xuping Xie, Na Ye, Jing Zou, Haiying Chen, Mark A. White, Pei-Yong
‡,§,
†,§,
Shi, * and Jia Zhou *
†
‡
Chemical Biology Program, Department of Pharmacology and Toxicology, Department of
§
Biochemistry and Molecular Biology, and Sealy Center for Structural Biology and Molecular
Biophysics, University of Texas Medical Branch, Galveston, Texas 77555, United States
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ABSTRACT:
A series of substituted 4,6-dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-
dione analogues were synthesized and evaluated as potent dengue virus inhibitors. Throughout a
structure-activity relationship exploration on the amide of the indolone moiety, a wide range of
substitutions were found to be well tolerated for chemical optimization at this position. Among
these compounds, 15 (JMX0254) displayed the most potent and broad inhibitory activities,
effective against DENV-1 to -3 with EC50 values of 0.78 μM, 0.16 μM and 0.035 μM,
respectively, while compounds 16, 21, 27-29, 47 and 70 exhibited relatively moderate to high
activities with low micromolar to nanomolar potency against all four serotypes. The biotinylated
compound 73 enriched NS4B protein from cell lysates in pull-down studies and the findings
together with the mutation investigations further validated dengue NS4B protein as the target of
this class of compounds. More importantly, compound 15 exhibited good in vivo
pharmacokinetic properties and efficacy in the A129 mouse model, indicating its therapeutic
potential against the dengue virus infection as a drug candidate for further preclinical
development.
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INTRODUCTION
Dengue is a viral disease vectored mainly by female Aedes mosquitoes that causes flu-like
symptoms with high fever, headache, vomiting, rash and joint/bone/muscle pain, and sometimes
it can develop into a potentially life-threatening complication called severe dengue, dengue
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hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Before 1970, only nine countries
had experienced severe dengue epidemics. Today, the disease is widespread globally in more
than 100 tropical and subtropical countries, putting almost half of the world’s population at risk,
and the Americas, South-East Asia and Western Pacific regions are the most seriously affected.2-
4
The incidence of dengue has grown dramatically with a 30-fold increase around the world over
5
the past 50 years. As a vast majority of cases are asymptomatic, the actual numbers of dengue
cases are underreported and many cases are misclassified. It is estimated that 390 million dengue
infections occur annually, of which 96 million manifest clinically (with any severity of disease),
including approximately 500,000 cases of severe dengue and 22,000 deaths worldwide.6, 7 The
burden of dengue is considerable, causing human suffering, strained health services and massive
economic losses. However, there remains no effective antiviral therapy for dengue fever, and
vector control is the main method to prevent dengue outbreaks.8-17 Recently, the first dengue
vaccine CYD-TDV, developed by Sanofi Pasteur, has been licensed in a number of countries but
has limited efficacy and safety issues.18, 19 Because of this, it is limited to individuals ranging
from 9-45 years of age and given as 3-dose series with 6 months between each dose. Moreover,
in September 2018, World Health Organization (WHO) updated its recommendations regarding
the use of CYD-TDV based on the evidence that seronegative vaccine recipients have an excess
risk of hospitalized and severe dengue compared to seronegative non-vaccinated individuals.20
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Therefore, there is an urgent need to develop safe and effective therapeutics for the treatment of
dengue virus infection.
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Figure 1. Previous work (SAR summary on DENV-2) and drug design for the current work.
Dengue viruses (DENV) belong to the genus Flavivirus within the Flaviviridae family,
and there are 4 distinct, but closely related, serotypes of the virus (DENV-1, DENV-2, DENV-3,
and DENV-4).21, 22 A geographic region may be affected by one or more DENV serotypes
simultaneously.23-25 Recovery from infection by one serotype provides lifelong immunity against
that particular serotype, while cross-immunity against the other serotypes is only partial and
temporary.26, 27 A second infection with a different dengue serotype is more likely to develop
severe dengue.28-32 Hence an ideal dengue antiviral should be effective against all four
3
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serotypes. The dengue viral genome is a positive single-strand RNA of approximately 11 kb in
length. This RNA encodes three structural proteins (capsid, envelope, and membrane) that form
the virus particle and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and
3
4
NS5) that are essential for replication of the virus. Dengue NS4B is a small integral membrane,
consisting of 248 amino acid residues. It is moderately conserved, with about 83-89% amino acid
similarity among the four dengue serotypes. Due to its high hydrophobicity, neither the crystal
nor NMR structure of flavivirus NS4B is currently available.35, 36 In the recent years, some
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progresses have been made to figure out the biological functions of NS4B, including its
engagement in host innate immunity and interactions with other viral proteins.37-45 However, the
exact mechanisms of flavivirus NS4B involved in the viral replication cycle remain elusive.
Despite many gaps in our knowledge of its structure and function, NS4B protein has become an
attractive therapeutic target and a relatively large number of NS4B inhibitors were discovered by
phenotypic screening.46-51 Our team discovered a series of spiropyrazolopyridone analogues as
potent inhibitors against DENV-2 and DENV-3, while these compounds have a lack of potency
against DENV-1 and DENV-4.51-53 Based on the previous structure-activity relationship (SAR)
studies as depicted in Figure 1, a narrow range of substitutions are tolerated on the core spiro
scaffold and only methylation on the amide of the indolone moiety improved the potency against
DENV-2. Herein, we reported our continued SAR optimization efforts mainly focused on two
amide moieties, aiming to elucidate the chemical space for the potential to acquire enhanced
potency of all four serotypes.
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RESULTS AND DISCUSSION
Chemistry. According to the previous work, the three component condensation of substituted
aminotriazoles, isatin derivatives, and Meldrum’s acid can provide the access to a series of
spirotrizolopyridone analogues for investigating the substitution on the two amide groups.51, 52
However, this condensation yields the final product as two enantiomers or diastereoisomers with
great discrepancy in potency, and it will bring a large amount of separation work to get each pure
and potent stereoisomer. Surprisingly, when 1.2 equivalent of methyl iodide was added, direct
methylation of pure diastereoisomer 1a afforded mono-methylated product 7 with a high yield of
7
6% (Scheme 1). The structure of methylated derivative 7 was subsequently determined by X-
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ray crystallography to confirm the methylation position and the absolute configurations of the
chiral centers. These results demonstrated that the amide of the indolone moiety has a higher
nucleophilic reactivity in comparison with the one of the pyridone moiety, and it provides a good
chance for a quick SAR investigation on this part using pure stereoisomer 1a as a starting
material. The synthesis of diastereoisomer 1a commenced with commercially available 1-(4-
chlorophenyl)ethanol (2), which was brominated followed by azidation to generate azide 3.52
This intermediate was treated with ethyl 2-cyanoacetate and sodium in ethanol to provide
aminocarboxylate 4, which was then converted to (S)-aminotriazole 6 via hydrolysis,
decarboxylation, and then chiral HPLC separation. Aminotriazole 6 was condensed with 5-
chloroisatin and Meldrum's acid in acetic acid to give the final products as two diastereoisomers
with a ratio of approximately 1:1, which were successfully separated by column chromatography
to afford pure stereoisomer 1a and 1b.
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Scheme 1. Synthesis of Compounds 1a and 7a
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aReagents and conditions: (a) i. PBr
-cyanoacetate, Na, EtOH, 85 °C, 5 h. (c) NaOH, EtOH/H
min; ii. chiral HPLC separation. (e) 5-chloroisatin, Meldrum's acid, AcOH, 100 °C, 4 h. (f) CH
NaH, DMF, 0 °C to r.t, 2 h.
, CH
Cl
, 0 °C to r.t, 4 h; ii. NaN
O, 85 °C, 4 h. (d) i. DMF, 200 °C, 20
, DMSO, r.t, 5 h. (b) ethyl
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Scheme 2. Synthesis of Substituted 4,6-Dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-
indoline]-2',5(3H)-dione Analogues 8-50 and 55-70a
a
Reagents and conditions: (a) RI or RBr, NaH, DMF, 0 °C to r.t or 50 °C. (b) TsOH, MeOH, r.t.
3
4
(c) PPh
3
, CBr
4
, DCM, 0 °C to r.t. (d) R R NH, K
2
CO
3
, CH CN, 60 °C ~ 80 °C.
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With pure stereoisomer 1a in hand, the effect of the substitution on the two amide
moieties was successfully explored. As outlined in Scheme 2, direct substitution of 1a with
corresponding
dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione analogues 8-41, 43-44,
6-51 and 53-54. Acid analogue 45 was accessed from ester 43 via hydrolysis. Depyranylation of
iodide
or
bromide
afforded
a
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substituted
4,6-
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THP ether 51 gave alcohol 42, which was then brominated to give bromide 52. Substitution of
bromide or chloride 48-50 and 52-54 with different amines afforded the corresponding amine
analogues 55-70. To assist the target validation and mechanistic studies of this series of
molecules, two biotinylated compounds 73 and 75 were designed and synthesized as the
chemical probe tools (Scheme 3). Condensation of D-(+)-Biotin (71) with methyl 8-
aminooctanoate was followed by hydrolysis to provide acid 72, which was then condensed with
alcohol 42 to give the biotinylated analogue 73. The biotinylated analogue 75 was prepared using
the same route as that for its diastereoisomer 73 starting from compound 1b for comparison.
Scheme 3. Synthesis of Biotinylated Analogues 73 and 75a
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aReagents and conditions: (a) i. methyl 8-aminooctanoate hydrochloride, EDCI, HOBt, DIEA,
DMF, r.t; ii. NaOH, MeOH/H O, r.t. (b) 42, EDCI, DMAP, DMF, r.t. (c) i. 2-(2-
2
bromoethoxy)tetrahydro-2H-pyran, NaH, DMF, 0 °C to r.t.; ii. TsOH, MeOH, r.t. (d) 72, EDCI,
DMAP, DMF, r.t.
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In Vitro Evaluation of DENV Inhibition. All new compounds were first screened against
DENV-2, and the active compounds screened out (DENV-2 EC50 < 1 μM) were further
evaluated against other three DENV serotypes. Considering the high hydrophobicity of NS4B
protein, we first introduced different alkyl groups on the amide of indolone or pyridone moiety
(Table 1). As expected, compound 7 with methyl on the amide of indolone showed increased
inhibitory activities against DENV-2 and DENV-3 (EC50 = 0.019 μM and 0.004 μM,
respectively) compared to the parent compound 1a. However, neither of these two compounds
showed potency against DENV-1 or DENV-4 (EC50 > 5 μM). Compound 8 with ethyl began to
show inhibitory activity against DENV-1 with an EC50 of 2.5 μM, while it maintained the same
level of potency against DENV-2 and DENV-3 (EC50 = 0.024 μM and 0.006 μM, respectively).
When we increased the length of alkyl, the corresponding compounds (9, 10 and 11) showed
improved potency against DENV-1 with EC50 values of 1.9 μM, 1.3 μM and 0.83 μM,
respectively. Nevertheless, n-hexyl group (11) resulted in a significant loss of potency against
DENV-2 and DENV-3 (EC50 = 0.81 μM and 0.14 μM, respectively); n-decyl group (12) led to a
complete loss of potency against DENV-2 (EC50 > 10 μM), while it showed potency against
DENV-1, -3 and -4 with EC50 values of 3.4 μM, 1.2 μM and 7.3 μM, respectively. These results
indicated that the long length of alkyl was unfavorable for DENV-2 potency. Compounds with
the branched alkyl groups (13-15) showed potency against DENV-1 to -3 with the same trend.
Among these compounds, 15 with isopentyl showed the most potent inhibitory activity against
DENV-1 with an EC50 of 0.78 μM, meanwhile maintaining a similar level of potency against
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DENV-2 and DENV-3 (EC50 = 0.16 μM and 0.035 μM, respectively). Unfortunately, all these
compounds showed no obvious potency against DENV-4 (EC50 > 5 μM). Interestingly,
compound 16 with 3,3-dimethylbutyl maintained potency against DENV-1 to -3 (EC50 = 1.1 μM,
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.87 μM and 0.082 μM, respectively), while also showing inhibitory activity against DENV-4
with EC50 of 3.9 μM. Compounds 17 with cyclopropylmethyl and 18-20 with unsaturated alkyls
all exhibited good potency against DENV-2 and DENV-3 and weak inhibitory activities against
DENV-1 and DENV-4. Compound 21 with isopropyl on the amide of pyridone also showed
broad inhibitory activities against DENV-1 to -4 with EC50 of 2.1 μM, 0.86 μM, 0.31 μM and 4.4
μM, respectively. However, considering the significant loss of potency against DENV-2 when
introducing the alkyl group on the amide of pyridone (21), the subsequent investigation was
mainly focused on the indolone moiety. Two isopropyl substitutions (22) further decreased the
antiviral activity against DENV-2 (EC50 > 10 μM) and DENV-3 (EC50 = 1.5 μM), while it
maintained potency against DENV-1 and DENV-4 (EC50 = 3.8 μM and 3.0 μM, respectively).
Compound 23 with four methyl groups (α-carbonyl carbon was di-methylated) resulted in
significant loss of potency against all four DENV serotypes (EC50 > 5 μM).
Table 1. Antiviral Activity and Cytotoxicity of Compounds 1a and 7-23
EC50 (μM) a
CC50
Compd
R1
H
R2
H
b
(μM)
DENV-1 DENV-2 DENV-3 DENV-4
>5.0 0.039 0.010 >5.0
1a
>10
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9
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>5.0
2.5
0.019
0.024
0.071
0.075
0.81
0.004
0.006
0.010
0.018
0.14
>5.0
>5.0
>5.0
>5.0
>5.0
7.3
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.9
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
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
1
0
1
1.3
1
0.83
3.4
12
>10
1.2
1
1
1
1
3
4
5
6
5.7
0.023
0.15
0.009
0.020
0.035
0.082
0.017
0.005
0.003
0.020
0.31
>5.0
>5.0
>5.0
3.9
1.3
0.78
1.1
0.16
0.87
17
3.1
0.11
>5.0
>5.0
>5.0
>5.0
4.4
1
1
2
2
8
9
0
1
2.1
0.038
0.069
0.063
0.86
>5.0
>5.0
2.1
H
22
3.8
>10
1.5
3.0
2
3
>5.0
>5.0
>5.0
>5.0
>10
aEC50 values were determined on Huh7 cells stably expressing DENV-1 to -4 replicon. CC50
values were measured using Huh7 cells stably expressing DENV-2 replicon.
b
To explore the effect of introducing aryl groups on the amide of indolone moiety,
compounds 24-37 were prepared and evaluated as shown in Table 2. Compounds 24 with benzyl
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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
and 25-26 with fluorobenzyl showed moderate potency against DENV-2 (EC50 = 0.14 ~ 0.29
μM) and DENV-3 (EC50 = 0.020 ~ 0.051 μM), weak potency against DENV-1 (EC50 = 2.0 μM,
5.0 μM and 10.0 μM, respectively), and no obvious potency against DENV-4. Compounds 27
with 4-chlorobenzyl and 28 with 4-chloro-3-fluorobenzyl resulted in a significant loss of potency
against DENV-2 (EC50 = 3.3 μM and 4.0 μM, respectively) and DENV-3 (EC50 = 0.20 μM and
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
.29 μM, respectively). However, these two compounds showed potency against both DENV-1
(EC50 = 2.2 μM and 1.5 μM, respectively) and DENV-4 (EC50 = 6.5 μM and 5.1 μM,
respectively). 3-Methoxybenzyl substitution (29) showed moderate potency against DENV-2 and
DENV-3 (EC50 = 0.83 μM and 0.087 μM, respectively) and weak potency against DENV-1 and
DENV-4 (EC50 = 2.6 μM and 6.2 μM, respectively). 3,5-Dimethoxybenzyl substitution (30)
displayed decreased potency against DENV-2 and DENV-3 (EC50 = 3.1 μM and 0.79 μM,
respectively) and maintained the same level of potency against DENV-1 and DENV-4 (EC50
=
3.4 μM and 7.4 μM, respectively). Interestingly, 3,5-di-tert-butylbenzyl substitution (31) showed
no obvious potency against DENV-2 (EC50 > 10 μM), while it maintained potency against
DENV-1 and DENV-4 (EC50 = 4.6 μM and 4.9 μM, respectively). Compound 32 with
homobenzyl exhibited improved potency against DENV-1 (EC50 = 1.1 μM) and decreased
potency against DENV-2 and DENV-3 (EC50 = 0.60 μM and 0.19 μM, respectively) in
comparison with 24. Excitingly, compounds 33-37 with pyridine moieties displayed highly
potent inhibitory activities against DENV-2 (EC50 = 0.020 ~ 0.15 μM) and DENV-3 (EC50
=
0.003 ~ 0.025 μM). However, no significant improvement of potency against DENV-4 was
observed for these compounds.
Table 2. Antiviral Activity and Cytotoxicity of Compounds 24-37
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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
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
EC50 (μM) a
DENV-1 DENV-2 DENV-3 DENV-4
CC50
Compd
R
1
R
2
b
(μM)
2
4
H
H
H
H
H
H
2.0
5.0
10.0
2.2
1.5
2.6
0.15
0.16
0.29
3.3
0.020
0.048
0.051
0.20
>5.0
10
>10
25
>10
>10
>10
>10
>10
26
>5.0
6.5
27
2
2
8
9
4.0
0.29
5.1
0.83
0.087
6.2
3
0
H
H
3.4
4.6
3.1
0.79
5.9
7.4
4.9
>10
>10
3
3
1
2
>10
H
H
H
H
H
H
1.1
>5.0
3.2
0.60
0.020
0.029
0.072
0.078
0.15
0.19
>5.0
>5.0
>5.0
>5.0
>5.0
>5.0
>10
>10
>10
>10
>10
33
0.003
0.021
0.025
0.006
0.011
3
3
4
5
>5.0
>5.0
>5.0
36
37
>10
aEC50 values were determined on Huh7 cells stably expressing DENV-1 to -4 replicon. CC50
b
values were measured using Huh7 cells stably expressing DENV-2 replicon.
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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
Next, a wide range of functional groups was investigated. As listed in Table 3, ether (38
and 39), acetal (41), alcohol (42), ester (43), 2-fluoroethyl (46) and 4-chlorobutyl (48) groups
were all well tolerated on the amide of indolone moiety, presenting potent inhibitory activities
against DENV-2 (EC50 = 0.037 ~ 0.29 μM) and DENV-3 (EC50 = 0.009 ~ 0.076 μM). As
expected, ether (40) and ester (44) on the amide of pyridone moiety resulted in a significant loss
of potency against DENV-2 (EC50 = 8.7 μM and > 10 μM, respectively), compared to the
corresponding substitutions on the amide of indolone moiety (39 and 43). Neither of these two
compounds showed potency against DENV-1 and DENV-4 (EC50 > 10 μM). The carboxyl group
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
(45) diminished the potency against DENV-2 (EC50 = 2.4 μM) in comparison of the
corresponding ester (43). Surprisingly, compound 47 with 3-bromopropyl showed potency
against all four serotypes DENV-1 to -4 (EC50 = 2.4 μM, 0.25 μM, 0.076 μM and 3.2 μM,
respectively). Substitutions with long carbon chain (49-50) showed decreased potency against
DENV-2, consistent with the trend observed before.
Table 3. Antiviral Activity and Cytotoxicity of Compounds 38-50
EC50 (μM) a
CC50
Compd
R
1
R
2
b
(μM)
>10
>10
>10
DENV-1 DENV-2 DENV-3 DENV-4
3
3
8
9
H
H
>5.0
>5.0
>10
0.092
0.040
8.7
0.019
0.009
0.91
>5.0
>5.0
>10
40
H
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41
H
H
H
>5.0
>5.0
>5.0
>10
NDc
>5.0
2.4
0.29
0.037
0.077
>10
0.076
0.010
0.011
2.7
>5.0
>5.0
>5.0
>10
ND
>10
>10
>10
>10
>10
>10
>10
>10
>10
4
4
4
4
2
3
4
5
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
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
H
H
H
H
H
H
H
2.4
ND
46
0.038
0.25
0.073
3.0
0.009
0.076
0.019
ND
>5.0
3.2
4
4
4
5
7
8
9
0
>5.0
ND
>5.0
ND
ND
>5.0
ND
ND
>10
aEC50 values were determined on Huh7 cells stably expressing DENV-1 to -4 replicon. CC50
values were measured using Huh7 cells stably expressing DENV-2 replicon. ND: not detected.
b
c
To improve the aqueous solubility, we attempted to introduce various alkylamino groups
and compounds 55-70 were designed, synthesized and evaluated (Table 4). Intriguingly, amino
analogs 55-62 with two carbon chain and 63-65 with three carbon chain all showed great potency
against DENV-2 (EC50 = 0.025 ~ 0.13 μM) and DENV-3 (EC50 = 0.001 ~ 0.030 μM). When the
length of carbon chain was increased from 4 to 10, the potency of the compounds (66-70) against
DENV-2 exhibited a declining trend. Among these compounds, 68 with 6-(dimethylamino)hexyl
group was effective against three serotypes DENV-1 to -3 (EC50 = 1.5 μM, 0.90 μM and 0.066
μM, respectively); 70 with 10-(dimethylamino)decyl group showed moderate to high potency
against all four serotypes DENV-1 to -4 (EC50 = 2.7 μM, 3.5 μM, 0.098 μM and 3.5 μM,
respectively). However, all these amino compounds except 70 lack potency against DENV-4.
Taken together, the substitution on the amide of indolone moiety was widely tolerated against
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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
DENV-2 and DENV-3, and various functional groups such as amino, hydroxyl, and ester with
proper carbon chain maintained the same level of potency against these two serotypes. The alkyl
groups with proper length are favorable for DENV-1 potency, while the long and bulky alkyl
groups are beneficial for DENV-4 potency. The substitutions on the amide of pyridone moiety or
on both amide moieties diminished the potency against DENV-2 and DENV-3. However,
compounds with the proper substitutions at these positions maintained the micromolar level of
potency against both DENV-1 and DENV-4. Unfortunately, during this SAR investigation, no
highly potent compounds were discovered against all four serotypes DENV-1 to -4 except a few
compounds such as 16, 21, 27-29, 47 and 70 with a relatively moderate potency.
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 4. Antiviral Activity and Cytotoxicity of Compounds 55-70
EC50 (μM) a
CC50
Compd
R
1
R
2
b
(
μM)
>10
>10
>10
>10
>10
>10
>10
DENV-1 DENV-2 DENV-3 DENV-4
5
5
H
H
H
H
H
H
H
>5.0
>5.0
>5.0
>5.0
>5.0
>5.0
>5.0
0.075
0.074
0.025
0.038
0.30
0.017
0.005
0.002
0.006
0.030
0.018
0.007
>5.0
>5.0
>5.0
>5.0
>5.0
>5.0
>5.0
56
5
7
58
5
9
60
0.077
0.067
6
1
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62
H
H
H
>5.0
>5.0
>5.0
0.12
0.025
0.13
0.022
0.001
0.012
>5.0
>5.0
>5.0
>10
>10
>10
6
6
3
4
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
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
6
6
6
5
6
7
H
H
H
>5.0
>5.0
>5.0
0.076
0.19
0.35
0.005
0.038
0.078
>5.0
>5.0
>5.0
>10
>10
>10
68
69
H
H
1.5
0.90
0.49
0.066
0.042
>5.0
>5.0
>10
>10
>5.0
7
0
H
2.7
3.5
0.098
3.5
>10
aEC50 values were determined on Huh7 cells stably expressing DENV-1 to -4 replicon. CC50
values were measured using Huh7 cells stably expressing DENV-2 replicon.
b
Biotinylated Compound 73 Can Pull Down NS4B Protein from Cell Lysates. Previous
studies revealed that the spiropyrazolopyridone derivatives inhibited DENV replication by
targeting NS4B protein.53 To facilitate the validation of NS4B as the target of this class of
compounds, a biotin group with a proper linker was added to compound 1a and its
diastereoisomer 1b, resulting in compounds 73 and 75 (Figure 2A). Consistent with previous
SAR results, the (R,S)-diastereoisomer 73 but not the (S,S)-diastereoisomer 75 remains active
against DENV-2 and DENV-3 with EC50 values of 0.041 μM and 0.017 μM, respectively
(
Figure 2B).51, 52 In addition, compound 73 has no significant activity against DENV-1 and
DENV-4 at the concentration up to 10 μM (data not shown). To further prove the association of
compound 73 with viral NS4B protein, a pull-down assay was performed by mixing compounds
with the lysates of DENV-2 infected cells. As shown in Figure 2C, only compound 73 but not
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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
7
5 can pull down viral NS4B protein, suggesting that this class of compounds likely target viral
NS4B protein.
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
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8
9
0
1
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3
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Antiviral activity, cytotoxicity and the association with NS4B of the biotinylated
compounds. (A) Chemical structures of biotinylated compound (R,S)-73 and its (S,S)-
diastereoisomer 75. (B) Antiviral activity and cytotoxicity of compound 73 and 75 in cell culture.
(C) Compound 73 is associated with DENV-2 viral NS4B protein. NS4B in the cell lysates and
eluates were detected by Western blot using a mouse monoclonal antibody against DENV-2
NS4B protein.
Compound 63 Resistance Maps to the Viral NS4B Protein. To further validate whether the
series of compounds generated in this study still target DENV NS4B protein, we raised resistant
virus by consecutively culturing DENV-2 with increasing concentrations of compound 63
(Figure 3A), a compound with excellent potency against DENV-2 and DENV-3. After twelve
rounds of selection, all four independent selections in the presence of compound 63 (selections
III to VI) showed increased EC50 values by >100 folds (Figure 3B). Sequencing of the entire
genome of the resistant viruses revealed that the amino acid change at position 63 (V63M or
V63L) in the NS4B protein occurred in those compound 63-treated isolates but not those DMSO-
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treated isolates (Figures 3B and 3C). The same mutation has been previously determined
responsible for the viral resistance to the spiropyrazolopyridone inhibitors.53 The data
demonstrated that DENV NS4B protein is still the target of this series of compounds. Moreover,
we evaluated the sensitivity of the P12 viruses (using P12-VI as an example) to compound 15, a
compound with excellent efficacy against three serotypes DENV-1 to -3 (Figure 3D). The
mutation NS4B V63L increased the viral resistance to compound 15 by 16 folds. Notably,
compound 15 remained effective against resistant viruses P12-VI with an EC50 of 2.0 μM,
suggesting that other sequence variations beyond V63 in NS4B protein may influence its
sensitivity to compound treatment.
1
1
1
1
1
1
1
1
1
1
2
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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
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
Figure 3. Mutations in NS4B confer to viral resistance to the inhibition by this series of
compounds. (A) Scheme of selection and validation of resistant viruses. See details in the
Method. DENV-2 strain NGC was used for the selection on Huh7 cells. (B) Resistant profile of
P12 mutant viruses. Mutations at position V63 of DENV-2 NS4B were consistently recovered
from four independent selections. DMSO (0.45%) was used as a negative control during
resistance selection. The resistance fold was calculated by comparing the sensitivities (indicated
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by EC50) of the resistant virus to the that of the P0 DENV-2. (C) NS4B protein sequence
alignment among four DENV serotypes. The sequences between positions 60 to 83 are shown.
V63 residue is highlighted. (D) Sensitivity of P12-VI to the treatment of compound 15. EC50
values were determined by viral reduction assay on Huh7 cells.
0
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In Vivo PK and Efficacy of Compound 15. Considering its excellent inhibitory activities
against three serotypes DENV-1 to -3, Compound 15 (JMX0254) was selected for further in vivo
PK and efficacy studies. First, plasma pharmacokinetics after 10 mg/kg intravenous and 20
mg/kg oral administration of compound 15 to male SD rats were characterized. As listed in
Table 5, compound 15 showed good oral exposure (6,240 ng∙h/mL) and oral bioavailability
(30%). Following an oral dose of 20 mg/kg, the rat plasma concentration of 15 reached a
maximum (1,517 ng/mL) at 1.0 h postdose administration.
Table 5. Pharmacokinetic Parameters of Compound 15 Following 20 mg/kg Oral and 10
mg/kg Intravenous Dosing in Ratsa
AUC0-∞
(ng∙h/mL)
T
(h)
max
C
max
CL
(L/h/kg)
V
ss
F
(%)
MRT (h)
t
1/2 (h)
(ng/mL)
1,517
(L/kg)
11.54
2.67
po
iv
6,240
3.46
2.28
2.24
2.29
1.00
3.57
0.82
30
10,334
10,334
aCmax, maximum concentration of drug in plasma; Tmax, time to maximum concentration of drug
in plasma; AUC, area under the curve (t = 0 to 24 h); MRT, mean residence time; Vss, volume of
distribution at steady state; CL, plasma clearance; t1/2, terminal half-life; F, absolute oral
bioavailability. Formulation for po and iv: DMSO: 20% HP-β-CD in saline = 1:9.
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Figure 4. In vitro activity and in vivo efficacy profile of compound 15 (JMX0254). (A) Antiviral
activity of compound 15 against DENV-2 strains NGC and D2Y98P. (B) Schematic diagram of
evaluating the efficacy of compound 15 against DENV-2 strain D2Y98P in vivo. 3-week old
6
A129 mice were subcutaneously (S.C.) inoculated with 10 PFU D2Y98P virus. Immediately,
the mice were administrated orally at 100 mg/kg twice daily (BID) with compound 15 or vehicle
for 3 days. On day 3 post-infection (p.i.), mice were retro-orbitally bled. Viremia were
determined by plaque assay. (C) Viremia on day 3 p.i. (D) Mouse body weight after infection.
The statistical significances between the group “vehicle + D2Y98P” and the group “compound
1
5 + D2Y98P” are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
We first confirmed the in vitro activity of 15 on DENV-2 strain D2Y98P, using a viral
titer reduction assay. As shown in Figure 4A, consistent with the screening results using the
replicon cell line, compound 15 inhibited both DENV-2 strains NGC (for in vitro study) and
D2Y98P (for in vivo study) in a dose-dependent manner on Huh7 cells, with EC50 values of
about 100 nM. The in vivo efficacy of compound 15 was then tested in a DENV-2 (strain
D2Y98P) viremia A129 mouse model (Figure 4B). Compared to vehicle control, the compound
15 treatment (when administrated orally at 100 mg/kg twice daily for 3 days) significantly
reduced viremia (2.4 log, p = 0.0051) (Figure 4C). In addition, this compound eliminated the
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morbidity caused by DENV-2 infection (Figure 4D). In summary, compound 15 clearly
demonstrated a promising efficacy profile in both cell culture and mouse model.
0
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CONCLUSION
In conclusion, we optimized a series of substituted 4,6-dihydrospiro[[1,2,3]triazolo[4,5-
b]pyridine-7,3'-indoline]-2',5(3H)-dione analogues as potent dengue NS4B inhibitors.
Throughout a SAR exploration on the amide of the indolone moiety, a wide range of
substitutions were found to be well tolerated at this position. Among these compounds, 15
(
JMX0254) showed the most potent and broad inhibitory activities, effective against DENV-1 to
3 with nanomolar to submicromolar EC50 values. Compounds 16, 21, 27-29, 47 and 70
-
displayed relatively moderate to high activities with low micromolar to nanomolar potency
against all four serotypes. The biotinylated compound 73 can enrich NS4B protein from cell
lysates in pull-down studies and the findings together with the mutation investigations further
validated dengue NS4B protein as the target of this class of compounds. More importantly,
compound 15 exhibited good in vivo pharmacokinetic properties and promising efficacy in the
A129 mouse model, indicating its therapeutic potential against the dengue virus infection as a
drug candidate for further preclinical development. The SAR studies also revealed that a new
chemical space where biotin was conjugated (e.g. 73 with maintained excellent potency) could
be utilized for further design and synthesis of novel protein degraders. Proteolysis-targeting
chimaera (PROTAC) technology utilizes bifunctional molecules, whereby one end binds to the
protein of interest while the other end hijacks cellular E3 ligase-mediated ubiquitination to
induce the degradation of the target protein.54, 55 Since only a transient binding event is sufficient
for protein degradation activity, the strong binding to an active site of NS4B protein based on the
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traditionally occupancy-driven pharmacology is likely not required. Such PROTAC strategy may
open the new avenue to tackle the challenges with respect to the enhanced activities towards all
four serotypes, and our effort along this line is currently ongoing.
1
1
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EXPERIMENTAL SECTION
General Chemistry Information. All commercially available starting materials and
solvents were reagent grade and used without further purification. Reactions were performed
under a nitrogen atmosphere in dry glassware with magnetic stirring. Preparative column
chromatography was performed using silica gel 60, particle size 0.063-0.200 mm (70-230 mesh,
flash). Analytical TLC was carried out employing silica gel 60 F254 plates (Merck, Darmstadt).
Visualization of the developed chromatograms was performed with detection by UV (254 nm).
1
13
NMR spectra were recorded on a Brucker-600 and Brucker-300 ( H, 600 & 300 MHz; C, 150
1
13
&
75 MHz) spectrometer. H and C NMR spectra were recorded with TMS as an internal
reference. Chemical shifts were expressed in ppm, and J values were given in Hz. High-
resolution mass spectra (HRMS) were obtained from Thermo Fisher LTQ Orbitrap Elite mass
spectrometer. Parameters include the following: Nano ESI spray voltage was 1.8 kV; Capillary
temperature was 275 °C and the resolution was 60,000; Ionization was achieved by positive
mode. Melting points were measured on a Thermo Scientific Electrothermal Digital Melting
Point Apparatus and uncorrected. Purities of final compounds were established by analytical
HPLC, which was carried out on a Shimadzu HPLC system (model: CBM-20A LC-20AD SPD-
20A UV/VIS). HPLC analysis conditions: Waters Bondapak C18 (300 × 3.9 mm); flow rate 0.5
mL/min; UV detection at 270 and 254 nm; linear gradient from 10% acetonitrile in water to
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1
00% acetonitrile in water in 20 min followed by 30 min of the last-named solvent (0.1% TFA
was added into both acetonitrile and water). All biologically evaluated compounds are > 95%
pure.
0
1
2
3
4
5
6
7
8
9
0
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2
3
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(R)-5'-Chloro-3-((S)-1-(4-chlorophenyl)ethyl)-4,6-dihydrospiro[[1,2,3]triazolo[4,5-
b]pyridine-7,3'-indoline]-2',5(3H)-dione (1a). Compound 1a was prepared by a reported route
decribed in Scheme 1. The title compound was obtained as an off-white solid. HPLC purity 95.9%
1
(t
R
= 17.21 min). H NMR (300 MHz, DMSO-d
6
) δ 11.22 (s, 1H), 10.72 (s, 1H), 7.47 (d, J = 8.4
Hz, 2H), 7.41 – 7.29 (m, 4H), 6.93 (d, J = 8.4 Hz, 1H), 5.85 (q, J = 6.9 Hz, 1H), 3.34 (d, J = 16.2
1
3
Hz, 1H), 2.61 (d, J = 16.2 Hz, 1H), 1.88 (d, J = 6.9 Hz, 3H). C NMR (75 MHz, DMSO-d
6
) δ
1
1
4
77.5, 168.5, 141.3, 139.3, 136.4, 132.7, 132.1, 128.8, 128.8 (2C), 128.5 (2C), 126.8, 126.1,
+
24.7, 111.4, 55.6, 46.7, 21.2. HRMS (ESI) calcd for C20
28.0679.
H16Cl
2
N
5
O 428.0681 (M + H) , found
2
(S)-5'-Chloro-3-((S)-1-(4-chlorophenyl)ethyl)-4,6-dihydrospiro[[1,2,3]triazolo[4,5-
b]pyridine-7,3'-indoline]-2',5(3H)-dione (1b). Compound 1b was prepared by a reported route
decribed in Scheme 1. The title compound was obtained as an off-white solid. HPLC purity 99.8%
1
(t
R
= 16.92 min). H NMR (300 MHz, DMSO-d
6
) δ 11.23 (s, 1H), 10.72 (s, 1H), 7.47 (d, J = 8.7
Hz, 2H), 7.41 – 7.30 (m, 4H), 6.93 (d, J = 8.4 Hz, 1H), 5.85 (q, J = 6.9 Hz, 1H), 3.34 (d, J = 16.2
1
3
Hz, 1H), 2.61 (d, J = 16.2 Hz, 1H), 1.87 (d, J = 7.2 Hz, 3H). C NMR (75 MHz, DMSO-d
6
) δ
1
1
77.5, 168.5, 141.3, 139.3, 136.4, 132.7, 132.1, 128.8, 128.8 (2C), 128.5 (2C), 126.8, 126.1,
+
24.7, 111.4, 55.6, 46.7, 21.2. HRMS (ESI) calcd for C20
H16Cl
2
N
5
O 428.0681 (M + H) , found
2
428.0677.
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(
R)-5'-Chloro-3-((S)-1-(4-chlorophenyl)ethyl)-1'-methyl-4,6-
dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione (7). To a solution of
(R)-5'-chloro-3-((S)-1-(4-chlorophenyl)ethyl)-4,6-dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-
1
1
1
1
1
1
1
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1
1
2
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2
2
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3
4
4
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5
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0
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7
,3'-indoline]-2',5(3H)-dione (1) (33 mg, 0.077 mmol) in anhydrous DMF (3 mL) was added
NaH (6 mg, 0.15 mmol, 60% dispersion in mineral oil) at 0 °C. The resulting mixture was stirred
at r.t. for 30 min. Then CH I (6 µL, 0.093 mmol) was added and the resulting mixture was stirred
at r.t. for 2 h till the reaction was completed monitored by TLC. The reaction mixture was poured
into NH Cl (aq., 15 mL) and extracted with EtOAc/MeOH (3:1, 2×45 mL). The organic phase
was washed with water (2 × 15 mL) and brine (15 mL), dried over Na SO and concentrated.
3
4
2
4
The residue was purified by preparative TLC developed by 60% EtOAc in hexane to afford
1
compound 7 as a white solid (26 mg, 76%). HPLC purity 98.5% (t
MHz, CDCl
2.1 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 5.54 (q, J = 6.9 Hz, 1H), 3.26 (s, 3H), 3.00 (s, 2H), 1.94 (d,
R
= 18.50 min). H NMR (300
3
) δ 10.45 (s, 1H), 7.36 (dd, J = 8.4, 2.1 Hz, 1H), 7.33 – 7.27 (m, 4H), 7.13 (d, J =
1
3
J = 6.9 Hz, 3H). C NMR (75 MHz, CDCl
3
) δ 175.9, 169.9, 142.2, 138.2, 135.5, 134.6, 130.9,
1
29.8, 129.3 (2C), 129.2, 128.2 (2C), 127.1, 124.4, 110.2, 57.7, 46.8, 40.8, 27.0, 21.8. HRMS
+
(
ESI) calcd for C21
H18Cl N
2 5
O 442.0838 (M + H) , found 442.0831.
2
(R)-5'-Chloro-3-((S)-1-(4-chlorophenyl)ethyl)-1'-ethyl-4,6-
dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione (8). Compound 8
(
20 mg, 93%) was prepared by a procedure similar to that used to prepare compound 7. The title
1
compound was obtained as a white solid. HPLC purity 99.4% (t
MHz, CDCl
R
= 18.58 min). H NMR (300
3
) δ 10.31 (s, 1H), 7.38 – 7.27 (m, 5H), 7.14 (d, J = 1.8 Hz, 1H), 6.90 (d, J = 8.4 Hz,
H), 5.56 (q, J = 6.9 Hz, 1H), 3.80 (q, J = 7.2 Hz, 2H), 3.00 (s, 2H), 1.96 (d, J = 6.9 Hz, 3H),
1
1
1
3
.31 (t, J = 7.2 Hz, 3H). C NMR (75 MHz, CDCl ) δ 175.5, 170.0, 141.4, 138.2, 135.4, 134.7,
3
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2
2
2
2
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31.1, 129.7, 129.4 (2C), 129.0, 128.2 (2C), 127.3, 124.7, 110.3, 57.8, 46.7, 40.9, 35.6, 21.8,
+
2.7. HRMS (ESI) calcd for C22
H20Cl
2
N
5
O
2
456.0994 (M + H) , found 456.0986.
(R)-5'-Chloro-3-((S)-1-(4-chlorophenyl)ethyl)-1'-propyl-4,6-
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
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9
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9
0
dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione (9). Compound 9
(20 mg, 90%) was prepared by a procedure similar to that used to prepare compound 7. The title
1
compound was obtained as a white solid. HPLC purity 99.6% (t
MHz, CDCl
H), 5.56 (q, J = 6.9 Hz, 1H), 3.84 – 3.53 (m, 2H), 3.04 (d, J = 16.5 Hz, 1H), 2.97 (d, J = 16.8
R
= 19.10 min). H NMR (300
3
) δ 10.36 (s, 1H), 7.37 – 7.27 (m, 5H), 7.13 (d, J = 2.1 Hz, 1H), 6.89 (d, J = 8.4 Hz,
1
1
3
Hz, 1H), 1.96 (d, J = 6.9 Hz, 3H), 1.83 – 1.71 (m, 2H), 0.98 (t, J = 7.5 Hz, 3H). C NMR (75
MHz, CDCl ) δ 175.9, 170.1, 141.7, 138.2, 135.4, 134.7, 131.2, 129.7, 129.4 (2C), 128.9, 128.2
2C), 127.4, 124.6, 110.5, 57.74, 46.7, 42.3, 40.9, 21.8, 20.8, 11.4. HRMS (ESI) calcd for
3
(
+
C
23
H22Cl
2
N
5
O 470.1151 (M + H) , found 470.1146.
2
(R)-1'-Butyl-5'-chloro-3-((S)-1-(4-chlorophenyl)ethyl)-4,6-
dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione (10). Compound 10
(20 mg, 88%) was prepared by a procedure similar to that used to prepare compound 7. The title
1
compound was obtained as a white solid. HPLC purity 97.7% (t
MHz, CDCl
R
= 19.65 min). H NMR (300
3
) δ 10.32 (s, 1H), 7.36 – 7.27 (m, 5H), 7.13 (d, J = 2.1 Hz, 1H), 6.89 (d, J = 8.4 Hz,
1H), 5.56 (q, J = 6.9 Hz, 1H), 3.85 – 3.64 (m, 2H), 3.03 (d, J = 16.5 Hz, 1H), 2.97 (d, J = 16.5
Hz, 1H), 1.96 (d, J = 7.2 Hz, 3H), 1.76 – 1.61 (m, 2H), 1.48 – 1.33 (m, 2H), 0.95 (t, J = 7.2 Hz,
1
3
3
H). C NMR (75 MHz, CDCl ) δ 175.8, 170.1, 141.7, 138.2, 135.4, 134.7, 131.2, 129.7, 129.4
3
(2C), 128.9, 128.2 (2C), 127.4, 124.6, 110.4, 57.8, 46.7, 40.9, 40.6, 29.5, 21.8, 20.2, 13.8.
+
HRMS (ESI) calcd for C24
H24Cl
2
N
5
O
2
484.1307 (M + H) , found 484.1303.
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(
R)-5'-Chloro-3-((S)-1-(4-chlorophenyl)ethyl)-1'-hexyl-4,6-
dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione (11). Compound 11
(21 mg, 87%) was prepared by a procedure similar to that used to prepare compound 7. The title
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
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4
4
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5
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compound was obtained as an off-white solid. HPLC purity 99.5 % (t
300 MHz, CDCl
Hz, 1H), 5.56 (q, J = 6.9 Hz, 1H), 3.81 – 3.65 (m, 2H), 3.00 (s, 2H), 1.96 (d, J = 7.2 Hz, 3H),
R
= 20.81 min). H NMR
(
3
) δ 9.98 (s, 1H), 7.38 – 7.27 (m, 5H), 7.13 (d, J = 2.1 Hz, 1H), 6.89 (d, J = 8.4
13
1
1
1
.76 – 1.63 (m, 2H), 1.42 – 1.25 (m, 6H), 0.87 (t, J = 6.9 Hz, 3H). C NMR (75 MHz, CDCl ) δ
3
75.8, 170.1, 141.7, 138.2, 135.4, 134.7, 131.1, 129.7, 129.4 (2C), 129.0, 128.2 (2C), 127.4,
24.6, 110.4, 57.8, 46.7, 40.9, 40.8, 31.5, 27.4, 26.6, 22.6, 21.8, 14.1. HRMS (ESI) calcd for
+
C
26
H
28Cl
2
N
5
O
2
512.1620 (M + H) , found 512.1614.
(R)-5'-Chloro-3-((S)-1-(4-chlorophenyl)ethyl)-1'-decyl-4,6-
dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione (12). Compound 12
(
22 mg, 83%) was prepared by a procedure similar to that used to prepare compound 7. The title
1
compound was obtained as a white solid. HPLC purity 99.5% (t
MHz, CDCl
H), 5.56 (q, J = 6.9 Hz, 1H), 3.81 – 3.63 (m, 2H), 3.04 – 2.93 (m, 2H), 1.96 (d, J = 6.9 Hz, 3H),
R
= 23.61 min). H NMR (300
3
) δ 10.39 (s, 1H), 7.37 – 7.27 (m, 5H), 7.13 (d, J = 2.1 Hz, 1H), 6.89 (d, J = 8.4 Hz,
1
1
3
1.76 – 1.63 (m, 2H), 1.42 – 1.20 (m, 14H), 0.87 (t, J = 6.6 Hz, 3H). C NMR (75 MHz, CDCl
3
)
δ 175.8, 170.1, 141.7, 138.2, 135.4, 134.7, 131.1, 129.7, 129.4 (2C), 128.9, 128.2 (2C), 127.3,
1
24.6, 110.4, 57.7, 46.7, 40.9, 40.9, 32.0, 29.6, 29.6, 29.4, 29.4, 27.4, 27.0, 22.8, 21.8, 14.2.
+
HRMS (ESI) calcd for C30
H36Cl
2
N
5
O
2
568.2246 (M + H) , found 568.2241.
(R)-5'-Chloro-3-((S)-1-(4-chlorophenyl)ethyl)-1'-isopropyl-4,6-
dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione (13). To a solution
of (R)-5'-chloro-3-((S)-1-(4-chlorophenyl)ethyl)-4,6-dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-
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Journal of Medicinal Chemistry
Page 28 of 70
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7
,3'-indoline]-2',5(3H)-dione (7) (42 mg, 0.099 mmol) in anhydrous DMF (3 mL) was added
NaH (16 mg, 0.40 mmol, 60% dispersion in mineral oil) at 0 °C. The resulting mixture was
stirred at r.t. for 30 min. Then 2-bromopropane (32 mg, 0.24 mmol) was added and the resulting
mixture was stirred at 50 °C for 24 h. The reaction mixture was cooled to r.t. and poured into
0
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8
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0
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9
0
NH
washed with water (2 × 15 mL) and brine (15 mL), dried over Na
vacuo. The residue was purified by preparative TLC developed by 40% EtOAc in hexane to
4
Cl (aq., 15 mL) and extracted with EtOAc/MeOH (3:1, 2 × 45 mL). The organic phase was
2
SO and concentrated in
4
afford compound 13 as an off-white solid (12 mg, 26%). HPLC purity 95.3% (t
1H NMR (300 MHz, CDCl
R
= 19.08 min).
3
) δ 10.24 (s, 1H), 7.35 – 7.28 (m, 5H), 7.14 (d, J = 2.1 Hz, 1H), 7.03
(d, J = 8.7 Hz, 1H), 5.56 (q, J = 6.9 Hz, 1H), 4.65 – 4.50 (m, 1H), 3.03 – 2.91 (m, 2H), 1.97 (d, J
13
=
7.2 Hz, 3H), 1.52 (d, J = 7.2 Hz, 6H). C NMR (75 MHz, CDCl
3
) δ 175.6, 170.1, 141.1,
1
38.1, 135.4, 134.7, 131.3, 129.5, 129.4 (2C), 128.7, 128.2 (2C), 127.6, 124.7, 111.7, 57.8, 46.7,
+
45.0, 41.0, 21.8, 19.5, 19.5. HRMS (ESI) calcd for C23
70.1146.
H22Cl
2
N
5
O
2
470.1151 (M + H) , found
4
(R)-5'-Chloro-3-((S)-1-(4-chlorophenyl)ethyl)-1'-isobutyl-4,6-
dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione (14). Compound 14
(13 mg, 57%) was prepared by a procedure similar to that used to prepare compound 7. The title
1
compound was obtained as a light yellow solid. HPLC purity 99.4% (t
300 MHz, CDCl
Hz, 1H), 5.56 (q, J = 6.9 Hz, 1H), 3.61 (dd, J = 13.8, 7.8 Hz, 1H), 3.50 (dd, J = 13.8, 7.2 Hz,
R
= 19.57 min). H NMR
(
3
) δ 10.36 (s, 1H), 7.36 – 7.27 (m, 5H), 7.12 (d, J = 1.8 Hz, 1H), 6.88 (d, J = 8.4
1
H), 3.06 (d, J = 16.5 Hz, 1H), 2.98 (d, J = 16.5 Hz, 1H), 2.25 – 2.10 (m, 1H), 1.97 (d, J = 6.9
13
Hz, 3H), 1.03 – 0.94 (m, 6H). C NMR (75 MHz, CDCl
3
) δ 176.1, 170.2, 142.0, 138.2, 135.3,
1
34.7, 131.2, 129.6, 129.4 (2C), 128.9, 128.2 (2C), 127.5, 124.5, 110.7, 57.8, 48.1, 46.7, 40.9,
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Journal of Medicinal Chemistry
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+
2
7.1, 21.8, 20.3, 20.2. HRMS (ESI) calcd for C24
84.1299.
H
24Cl
2
N
5
O 484.1307 (M + H) , found
2
4
(R)-5'-Chloro-3-((S)-1-(4-chlorophenyl)ethyl)-1'-isopentyl-4,6-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
4
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4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
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9
0
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7
8
9
0
dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione (15). Compound 15
(
18 mg, 77%) was prepared by a procedure similar to that used to prepare compound 7. The title
1
compound was obtained as an off-white solid. H NMR (300 MHz, CDCl
3
) δ 10.42 (s, 1H), 7.38
– 7.26 (m, 5H), 7.13 (d, J = 2.1 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 5.56 (q, J = 6.9 Hz, 1H), 3.87
–
3.60 (m, 2H), 2.99 (s, 2H), 1.97 (d, J = 6.9 Hz, 3H), 1.73 – 1.54 (m, 3H), 0.98 (d, J = 6.3 Hz,
1
3
6
H). C NMR (75 MHz, CDCl ) δ 175.7, 170.2, 141.6, 138.2, 135.4, 134.7, 131.1, 129.7, 129.4
3
(
2C), 128.9, 128.2 (2C), 127.3, 124.6, 110.4, 57.7, 46.7, 40.9, 39.2, 36.0, 26.1, 22.6, 22.5, 21.8.
+
HRMS (ESI) calcd for C25
H26Cl
2
N
5
O
2
498.1464 (M + H) , found 498.1457.
(R)-5'-Chloro-3-((S)-1-(4-chlorophenyl)ethyl)-1'-(3,3-dimethylbutyl)-4,6-
dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione (16). Compound 16
(29 mg, 65%) was prepared by a procedure similar to that used to prepare compound 7. The title
1
compound was obtained as a yellow solid. HPLC purity 99.6% (t
MHz, CDCl
H), 5.56 (d, J = 6.9 Hz, 1H), 3.84 – 3.64 (m, 2H), 2.99 (s, 2H), 1.95 (d, J = 6.9 Hz, 3H), 1.67 –
R
= 19.71 min). H NMR (300
3
) δ 10.50 (s, 1H), 7.39 – 7.26 (m, 5H), 7.13 (d, J = 2.1 Hz, 1H), 6.85 (d, J = 8.4 Hz,
1
1
3
1.50 (m, 2H), 1.02 (s, 9H). C NMR (75 MHz, CDCl
3
) δ 175.6, 170.1, 141.6, 138.3, 135.4,
1
3
34.6, 131.2, 129.7, 129.3 (2C), 128.9, 128.2 (2C), 127.2, 124.6, 110.2, 57.7, 46.7, 40.8, 40.4,
+
7.4, 30.1, 29.3 (3C), 21.8. HRMS (ESI) calcd for C26
H28Cl
2
N
5
O 512.1620 (M + H) , found
2
512.1617.
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