Discovery of Ziresovir as a Potent, Selective, and Orally Bioavailable Respiratory Syncytial Virus Fusion Protein Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.9b00654

Ziresovir (RO-0529, AK0529) is reported here for the first time as a promising respiratory syncytial virus (RSV) fusion (F) protein inhibitor that currently is in phase 2 clinical trials. This article describes the process of RO-0529 as a potent, selective, and orally bioavailable RSV F protein inhibitor and highlights the in vitro and in vivo anti-RSV activities and pharmacokinetics in animal species. RO-0529 demonstrates single-digit nM EC₅₀ potency against laboratory strains, as well as clinical isolates of RSV in cellular assays, and more than one log viral load reduction in BALB/c mouse model of RSV viral infection. RO-0529 was proven to be a specific RSV F protein inhibitor by identification of drug resistant mutations of D486N, D489V, and D489Y in RSV F protein and the inhibition of RSV F protein-induced cell-cell fusion in cellular assays.

Full text of DOI:10.1021/acs.jmedchem.9b00654

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Drug Annotation
Discovery of Ziresovir as a Potent, Selective and Orally
Bioavailable Respiratory Syncytial Virus Fusion Protein Inhibitor
Xiufang Zheng, Lu Gao, Lisha Wang, Chungen Liang, Baoxia Wang, Yongfu
Liu, Song Feng, Bo Zhang, Mingwei Zhou, Xin Yu, Kunlun Xiang, Li Chen,
Tao Guo, Hong C Shen, Gang Zou, Jim Zhen Wu, and Hongying Yun
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00654 • Publication Date (Web): 13 Jun 2019
Downloaded from http://pubs.acs.org on June 14, 2019
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Discovery of Ziresovir as a Potent, Selective and Orally
Bioavailable Respiratory Syncytial Virus Fusion Protein
Inhibitor
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Xiufang Zheng†, Lu Gao†, Lisha Wang†, Chungen Liang†, Baoxia Wang†, Yongfu Liu†, Song Feng†, Bo
Zhang†, Mingwei Zhou†, Xin Yu†, Kunlun Xiang†, Li Chen†, Tao Guo‡, Hong C. Shen†, Gang Zou⸙, Jim
Zhen Wu⸙, and Hongying Yun† *
†Roche Pharma Research and Early Development，Roche Innovation Center Shanghai, Building 5, 720
Cailun Road, Shanghai, 201203 China
⸙Ark Biosciences Inc. 780 Cailun Road, Suite 701, ZhangJiang Hitech Park, Pudong, Shanghai, 201203
China
‡International Discovery Service Unit, Research Service Division, WuXi AppTec (Shanghai) Co., Ltd.
Lane 31, Yiwei Road, Waigaoqiao, Shanghai, 200131 China
KEYWORDS: Respiratory Syncytial Virus (RSV), Antiviral, Fusion Inhibitors, Ziresovir, RO-0529,
AK0529
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ABSTRACT: Ziresovir (RO-0529, AK0529) is reported here for the first time as a promising respiratory
syncytial virus (RSV) fusion (F) protein inhibitor that currently is in phase 2 clinical trials. This article
describes the process of RO-0529 as a potent, selective and orally bioavailable RSV F protein inhibitor,
and highlights the in vitro and in vivo anti-RSV activities and pharmacokinetics in animal species. RO-
0529 demonstrates single-digit nM EC50 potency against laboratory strains as well as clinical isolates of
RSV in cellular assays, and more than one log viral load reduction in BALB/c mouse model of RSV viral
infection. RO-0529 was proven to be a specific RSV F protein inhibitor by identification of drug resistant
mutations of D486N, D489V, and D489Y in RSV F protein and the inhibition of RSV F protein-induced
cell-cell fusion in cellular assays.
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Introduction
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Human respiratory syncytial virus is a negative-sense single-stranded RNA virus and a member of
the family of Pneumoviridae.1 Human RSV infection causes acute upper and lower respiratory tract
infection in infants, children, elderly, and immunocompromised adults. Particularly, all children by age of
two will be infected by RSV and they can be re-infected in the succeeding RSV season.2 RSV infection
could be mild in healthy children and adults with symptoms of upper respiratory tract infection. However,
in the high-risk populations, such as premature infants, immunocompromised adults, chronic obstructive
pulmonary disease (COPD) and congestive heart failure (CHF) patients, upper respiratory tract infection
can develop into lower respiratory tract infection, and associated mortality and morbidity would occur. The
increased chance of morbidity or mortality is observed from severe RSV infection, causing bronchiolitis in
infants and pneumonia in adult patients.2,3 The sequela of severe RSV infection in the children population
could be recurrent wheezing or asthma.4 It should be noted that severe RSV infection causes around 3.4
million hospitalizations and around 160,000 deaths in children under 5 years old worldwide per year. 5,6
Ribavirin in an aerosol formulation is the only approved anti-RSV therapy. However, the limited
efficacy and genotoxicity of the drug have hindered its application in clinic.7 There is no available vaccine
for prophylaxis of RSV infection. Palivizumab was approved for passive prevention of RSV infection in
high-risk infants, which reduces patient’s hospitalization rate by only 50%. As a humanized monoclonal
antibody against RSV fusion protein, Palivizumab exhibits no efficacy in the treatment of RSV infection.8
A safe and effective therapy is urgently needed for treatment of RSV infection.
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RSV F protein is a glycoprotein expressed on the surface of viral envelope. It not only plays a
crucial role for the virus entry but also promotes syncytia formation between the infected cells and the
adjacent healthy cells.9 Inhibition of RSV F protein provided an opportunity to treat RSV infected patients.
Small molecule F protein inhibitors are capable of decreasing the severity and duration of respiratory
symptoms and cure the viral infection without the risk of prolonged hospitalization and complications.
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F
O
F
O
S
F
N
N
N
N
6
7
8
9
N
N
N
O
N
O
H
N
H2N
O
N
F
NH2
F
F
N
Cl
Cl
O
N
S
O
F
N
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GS-5806
RV521
JNJ-53718678
N N N N
N
N
NH2
N
N
N
N
HN
O
N
N
N
S
O
OH
OH
O
RO-0529 AK 29)
05
(
MDT-637
OH
Figure 1. RSV Fusion Inhibitors in Clinical Development.
In the last two decades, several structurally different RSV F protein inhibitors have been discovered
and reported,10-13 among of which several inhibitors had successfully progressed to clinical studies (Figure
1).14 JNJ-53718678 with a picomolar potency reduced viral load and clinical severity in a phase IIa human
viral challenge study in healthy adults. Drug resistant mutation studies mapped its target on F protein.15 GS-
5806, another picomolar F protein inhibitor, demonstrated efficacy in human RSV viral challenge studies
by significantly reduction of viral load (4.2 log10) and disease symptom score.16 Drug resistant mutation
studies proved its antiviral target as F protein.17 RV521, a single digital nanomolar F protein inhibitor,
demonstrated good potency on the reduction of viral load and mucous production in a phase IIa human viral
challenge study.18 VP-14637, an earlier generation of nanomolar F protein inhibitor, was formulated as an
inhaled dry powder called MDT-637, and was tested in phase I clinical trial.19
We conducted a high throughput screen, identified a promising benzoazepinequinoline (BAQ) hit
compound, and then underwent extensive lead identification to discover compound 1 as a promising lead
compound (Figure 2).20 In this article, we describe the discovery of Ziresovir (RO-0529, AK0529) as a
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potent, selective and orally bioavailable RSV F protein inhibitor, which is currently in phase II clinical
studies. 21
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Rh
Y
NH2
NH2
HN
N
HN
N
HN
N
O
N
O
N
N
R2
R1
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N
S O
O
S O
O
X
BAQ
1
RO-0529
(AK0529)
Figure 2. RO-0529 is originated from BAQ chemical series and optimized from Compound 1.
Results and Discussion
As aforementioned, compound 1 was identified as a lead compound from the BAQ chemical series
with a good potential to be a clinical development candidate. This compound showed good anti-RSV
activity with EC50 of 2 nM in CPE assay, and a much higher exposure (78-fold) in lung than plasma and
good in vivo efficacy in an BALB/c mouse RSV infection model.20 SAR exploration was conducted on the
basis of compound 1. We firstly modified the substitutions on quinoline (R1) and benzoazepine (R2) (Table
1). As described in our previous work, 6-Me was very important for the anti-RSV activity (1). Replacement
of 6-Me with 6-Cl significantly reduced anti-RSV activity (1a, EC50=34 nM). Replacement of 6-Me with
6-CH2OH (1b) maintained the activity but this compound did not possess cellular permeability. Introducing
mono-F or di-F to quinoline led to a loss of anti-RSV activity (1d-1f). More potent compounds were
discovered by substitution on R2, such as 1j and 1o (EC50=1 nM and 0.8 nM, respectively). Unfortunately,
further study revealed a very higher human liver microsomal clearance for these two compounds.
Table 1. SAR of compound 1 analogues
NH2
HN
5
R2
3'
4'
6
5'
R1
O
N
7
N
8
2'
S O
O
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Compd
R1
R2
EC50 (µM)a
CC50 (µM)
TIb
5
6
7
8
9
1
1a
1b
1c
1d
1e
1f
6-Me
6-Cl
H
H
0.002
0.034
0.002
>100
0.021
0.023
0.51
>100
87
>50,000
2,560
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6-CH2OH
6-C(Me)2OH
6-Me-7-F
5-F-6-Me
6-Me-7,8-di-F
6-Me
H
>100
>100
64
>50,000
<1
H
H
3,050
H
93
4,040
H
53
104
1g
1h
1i
2’-F
2’-OMe
3’-F
3’-OMe
3’-Me
3’-Cl
4’-F
4’-OMe
4’-OH
3’-OMe
3’-Me
0.003
0.35
35
11,700
>286
6-Me
>100
79
6-Me
0.01
7,900
1j
6-Me
0.001
0.005
0.027
0.072
0.003
0.0008
0.008
0.074
92
92,000
18,200
560
1k
1l
6-Me
91
6-Me
15
1m
1n
1o
1p
1q
6-Me
93
1,290
6-Me
90
30,000
>125,000
>12,500
1,280
6-Me
>100
>100
95
H
H
a EC50: the concentration of compound that reduced 50% cytopathic effect of RSV Long strain infected HEp-2 cells.
b
CC50: the concentration of compound that manifests cytotoxicity towards 50% of uninfected HEp-2 cells.
Therapeutic index (TI) is the ratio of CC50/EC50.
The next round of optimization was focused on the nitrogen walk of quinoline including removal
or introduction of an additional nitrogen to quinoline (Table 2). Moving nitrogen from 1-position (1) to 3-
position (2a) or adding one more nitrogen at 6-position (2b) of quinoline resulted in much decreased anti-
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RSV activity. Converting quinoline (1) to naphthalene (2c) totally deprived the analog of anti-RSV activity.
Finally, converting quinoline to quinazoline led to the discovery of RO-0529, which demonstrated similar
anti-RSV activity (EC50=3 nM) as compound 1.
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Table 2. Nitrogen Walk around Quinoline of Compound 1a
Compd
Rh
EC50 (µM)a CC50 (µM)
TIb
NH2
HN
O
N
1
0.002
0.22
>100
>50,000
N
S O
O
NH2
HN
O
N
N
2a
2b
81
370
8,250
<2
S O
O
NH2
HN
O
N
0.012
>31.6
0.003
99
71
N
N
S O
O
NH2
HN
O
2c
N
S O
O
NH2
HN
N
O
N
N
RO-0529
>100
>33,300
S O
O
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a EC50: the concentration of compound that reduced 50% cytopathic effect of RSV Long strain infected HEp-2 cells.
b
CC50: the concentration of compound that manifests cytotoxicity towards 50% of uninfected HEp-2 cells.
6
7
Therapeutic index (TI) is the ratio of CC50/EC50.
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An SAR exploration on the quinazoline scaffold of RO-0529 was listed in Table 3. It was found
that the SAR of quinazoline was very similar to quinoline (compound 1). In the head portion (Rh), an
oxetane substitution offered better anti-RSV activity than gem-di-Me (3a) and cyclobutyl (3c). Compared
to cycloproyl analog (3b), RO-0529 showed much larger TI (> 33,000 vs. 1,250). 6-Me demonstrated its
importance for good anti-RSV activity compared to 6-H (3d, EC50=20 nM). Although replacement of SO2
with SO (3e) maintained the anti-RSV activity (EC50=4 nM) and excellent TI, it was not further developed
considering the oxidative conversion potential of SO (3e) to SO2 in both in vitro and in vivo evaluation.
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Table 3. SAR Exploration on the Scaffold Derived from RO-0529a
Rh
HN
R1
N
N
N
X
Compd
Rh
R1
X
EC50 (µM)a CC50 (µM)
TIb
NH2
RO-0529
Me
SO2
0.003
>100
>33,000
O
NH2
NH2
3a
3b
Me
Me
SO2
SO2
0.016
0.004
20
13
1,250
3,250
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NH2
NH2
NH2
3c
3d
3e
Me
H
SO2
SO2
SO
0.10
0.02
21
210
>100
>100
>5,000
>25,000
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O
O
H
0.004
a EC50: the concentration of compound that reduced 50% cytopathic effect of RSV Long strain infected HEp-2 cells.
CC50: the concentration of compound that manifests cytotoxicity towards 50% of uninfected HEp-2 cells. b Therapeutic
index (TI) is the ratio of CC50/EC50.
Selection of RO-0529 as a clinical candidate:
Compound 1 and RO-0529 were both chosen as potential clinical candidates. In order to select a
superior compound as the clinical candidate, we profiled both molecules in in vitro and in vivo anti-RSV
activities, TI, DMPK and early safety profile. Between them, RO-0529 stood out.
Table 4 listed single dose pharmacokinetics (SDPK) results of compound 1 and RO-0529 in
Sprague-Dawley rats. RO-0529 exhibited better oral exposure and bioavailability than compound 1. Take
all the results into consideration, RO-0529 was selected as the clinical candidate.
Table 4. Rat SDPK Profile of Compound 1 and RO5500529a
1
RO-0529
Compound
Rat SDPK iv @ 2 mg/kg & po @10 mg/kg
AUC0-24h
442
906
(po) (h*ng/mL)
in vivo CL
51
58
(mL/min/kg)
1.7
1.2
T1/2, iv (h)
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4.1
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3.9
32
Vss (L/kg)
F (%)
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a The SDPK study in male Wistar-Han rats were carried out according to the standard procedures described in the
supporting information. Plasma clearance (CL), volume of distribution at steady state (Vss), area under the curve
(AUC), oral bioavailability (F).
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In vitro and in vivo anti-RSV activities of RO-0529
RO-0529 was tested against RSV strains of both Long, A2 and B18537 subtypes in the CPE assay.
It demonstrated high potency with EC50 values in the single-digit nanomolar level (Table 5). In order to
study the efficacy against clinical strains of RSV, clinical isolates of RSV A subtype (1177) and B subtype
(821) were collected from Chongqing Children’s Hospital in the RSV season from 2011 to 2012. In the
cytopathic effect assays, both the drug showed similar activities in the clinical strains compared with
laboratory strains of RSV (Long and B18537), which suggests that RO-0529 might be broadly active with
a similar activity against both RSV A and B strains as well as clinical isolates (Table 5). RO-0529 showed
no activity against influenza H1N1, human parainfluenza virus and rhinovirus in the CPE assay (data not
shown), which indicates that its antiviral activity is RSV specific.
Table 5. Anti-RSV Activity of RO-0529a
Clinical
isolates
Clinical
isolates
RSV
Long
RSV
B18537
CPE
Influenza
RSV Long
CPE
RSV A2
CPE
H1N1
CPE
EC50
(µM)
Antivir
Chongqing Chongqing
Plaque
reduction
EC90
al assay EC50/EC90 EC50/EC90
(µM) (µM)
1177 RSV
A subtype
CPE
821 RSV
B subtype
CPE
EC50/EC90
(µM)
(µM)
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EC50/EC90
(µM)
EC50/EC90
(µM)
7
8
9
RO-
0529
0.003/0.005 0.002/0.003 0.002/0.003 0.004/0.007 0.003/0.008
0.010
> 100
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a EC50: the concentration of compound that reduced 50% cytopathic effect of RSV Long and B strains in the presence
of 10% FBS, plaque reduction in long strain and influenza H1N1 strain infected HEp-2 cells.
Furthermore, the effect of fetal bovine serum (FBS) on drug potency in the CPE assay using RSV
Long strain was examined. The results indicated that there was no significant decrease in anti-RSV potency
in the presence of 20% and 40% FBS (CPE EC50/EC90 = 0.003/0.008 M with 40% FBS), comparing with
potency under standard cell culture conditional in the presence of 10% FBS (Table 5).
In the CD-1 mice SDPK study, RO-0529 demonstrated a high tissue distribution to lung than
plasma (Table 6). The lung exposure (AUC(0-t)) is nearly 8-fold higher than that of plasma exposure. The
T1/2 of lung is 3-time longer of that of plasma, suggesting a favorable lung targeted drug profile.
Table 6. CD-1 Mice SDPK Profile of RO-0529
Tissue/plasma
AUC(0-24)
AUC(0-24) Ratio
T1/2
(h)
Tmax
(h)
Cmax
PO (150 mg/kg/day)
Plasma
(μg/L*h)
(μg/L*h)
(μg/L)
8,380
1
1.02
3.31
0.25
1
5,090
Lung
72,400
8.6
22,700
The in vivo efficacy against RSV was examined in female BALB/c mice. RO-0529 was orally
dosed in 12 mice at 12.5 mg/kg and 50 mg/kg, twice a day (BID at 0 h and 8 h each day) for 4 days. It
revealed that > 1 log unit of viral titer reduction in the lung of infected mice was achieved at the dose level
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as low as 12.5 mg/kg. Further increase dose to 50 mg/kg can reduce viral titer to 1.9 log units compared to
vehicle (Figure 4).
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Figure 4. In vivo Reduction of RSV Titer in Mouse Lung with RO-0529a
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a Female BALB/c mice were used for in vivo efficacy assay. Animals were anesthetized and to the
animals were intranasally administrated RSV long strain (5 105 plaque-forming units [PFU]). After oral
administration of RO-0529 BID for 4 days, the mice were euthanized with CO2, and lungs were harvested
and analyzed for viral titer.
The pharmacokinetics of RO-0529 was studied in mouse, rat, dog and monkey (Table 7). The in
vitro liver microsomal clearance (Mic CL) and hepatocyte clearance (Hep CL) showed a good correlation
using microsome preparations of mouse, rat and dog, respectively. In monkey and human, in vitro liver Mic
CL was higher than Hep CL. The in vivo plasma clearance was much higher than liver blood flow in mouse，
suggesting the possibility of high blood to plasma ratio, or extrahepatic metabolic clearance, such as urinary
or biliary excretion. In general, the in vitro Hep CL and in vivo CL correlated well with each other in the
rest of species, suggesting that the metabolic clearance is the major clearance pathway in the rat, dog, and
monkey.
Table 7. Pharmacokinetics of RO-0529 in Nonclinical Species
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Liver Mic CLa
Hep CLa
in vivo CL
AUC0-24h, po
Vss
T1/2, iv
Species
F%
(L/kg)
6.3
(h)
0.4
1.2
2.8
1.0
NA
(mL/min/kg) (mL/min/kg) (mL/min/kg)
(ng*h/mL)
1,970
906
CD-1 mouseb
SD ratb
74
40
12
40
13
60
42
10
23
4.3
277
58
66
32
3.9
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Beagle dogc
Monkeyc
Human
36
6.7
1,170
1,440
NA
26
26
1.9
22
NAd
NA
NA
a The in vitro microsomal CL and hepatocytes CL based on incubation with corresponding species microsomes and
b
hepatocytes. Mouse (5 mg/kg, iv; 50 mg/kg, po) and rat (2 mg/kg, iv; 10 mg/kg, po) SDPK with solution in 5%
c
DMSO, 40% PEG400 and 55% 0.9% sodium chloride. Beagle dog (1 mg/kg, iv; 10 mg/kg, po) and cynomolgus
monkey (2 mg/kg, iv; 10 mg/kg, po) SDPK with solution in 12.5% captisol in water. Major parameters, including
plasma clearance (CL), volume of distribution at steady state (Vss), T1/2 (iv), area under the cure (AUC), and oral
bioavailability (F) are reported. d NA: not available.
To identify the molecular target of the inhibitor, drug resistant mapping studies were performed,
and drug resistant mutants were selected in the presence of RO-0529. Resistant viral isolates were obtained
after several passages with increasing concentration of the compound. The RSV genome from the resistant
isolates were amplified by reverse-transcription-PCR and sequenced in order to determine the viral protein
that was targeted by the inhibitor. Each of the resistant isolates harbored a different single nucleotide
substitution in the F gene, resulting in D486N, D489A, D489V, D489Y amino acid changes in passage 3,
respectively. All the mutations rendered drug resistance to RO-0529 were located in the HRC region of the
C-terminal of RSV-F protein. The data indicated that the bona fide target of RO-0529 is RSV F protein.
The antiviral activity of RO-0529 was tested in the CPE assays using plaque-purified virus with D486N or
D489A mutants. The results were shown in Table 8.
Table 8. Effect of RO-0529 on the Wild Type and Mutant Strainsa
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CPE assay
RO-0529
EC50/EC90 (M) (WT)
0.003/0.005
EC50/EC90 (M) (D486N)
EC50/EC90 (M) (D489A)
>10/>10
2.1/10.0
7
8
9
a For the selected mutant strains of RSV, CPE assay was conducted as following. HEp-2 cells were seeded at 6000/well
in 96-well plates. The viral inoculation volume was 50 L and MOI was 0.04. The compound volume and the plates
were read at absorbance of reagent CCK-8 at the absorbance of 450 nm/650 nm.
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The mode of action of RO-0529 was further confirmed in an assay of inhibition of RSV F protein-
induced cell-cell fusion process as shown in Figure 5. Without RO-0529, RSV F protein induced cell fusion
process and generated syncytia formation shown in the red dot cycles (left figure). In the presence of 0.1
M of RO-0529, the syncytia formation induced by the RSV F protein was completely inhibited (right
figure). These results further confirmed that RO-0529 was a RSV F inhibitor, as there was no any other
RSV viral protein present except F protein.
Figure 5. RO-0529 Inhibited RSV F Protein-induced Cell-Cell Fusion Processa
a RSV-F expression was induced by 1μg/ml tetracycline in the RSV F protein stable cell line. Compound was serial
diluted and added into the RSV-F expressing cells. The cell-cell fusion was observed by microscopy at three days
after compound addition.
Battles et al 22 reported bona fide co-crystal structures for a number of RSV F inhibitors. All the
RSV F inhibitors tested by Battles were found to target the same RSV F microdomain and the escape
mutations observed in our study are known hot-spots of RSV pan-resistance to RSV F inhibitors. When
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RO-0529 was docked into the same RSV F microdomain, it took the same binding mode as the Compound
1 in the previous article20, which picks the similar interactions with the protein, such as the H-bond
interaction with Asp486 and the hydrophobic interaction with Phe488. Since two N atoms of the oxetane
side chain form the strong H-bond interaction with Asp486 (shown in orange in Figure 6a), the mutation of
this residue – D486N, will significantly reduce the potency. In addition, the side chain of one Asp489
(shown in yellow in Figure 6a) forms the hydrophobic interaction with the 6-methyl of the quinazoline ring.
Thus, mutations D489N & D489Y will also influence the potency of RO-0529. From these results, we
conclude that the modeling and experimental results are highly corroborative.
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a)
b)
Figure 6. Docking Model of Compound RO-0529 Binding to a Three-fold Symmetric Cavity in
Prefusion RSV F Protein.
a) Side view of the RO-0529 in the binding pocket of prefusion FSV F glycoprotein (shown in white). Asp486 is
shown as sticks with color in orange. Asp489 is shown as sticks with color in yellow. RO-0529 is shown in ball-and-
stick mode, with carbon atoms colored in green, nitrogen atoms in blue, and oxygen atoms in red; b) 2D ligand-
interaction diagram was generated in MOE. The interactions of compound RO-0529 with RSV F main-chain and side-
chain atoms are shown as blue and green dotted lines, respectively. The arrowheads are pointed to the acceptor.
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Clinical Development
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In 2014, Ark Biosciences licensed in RO-0529 from Roche for clinical development and renamed
the drug as AK0529. Several clinical studies with the drug have been completed, including two Phase 1
clinical studies in healthy adult volunteers in Australia23 and China24, respectively, and a Phase 1 human
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mass balance study in the United Kingdom.25 A global multi-center randomised double-blind placebo-
controlled study of orally administered AK0529 is performed. This study evaluates the drug’s safety,
tolerability, pharmacokinetics, pharmacodynamics, and antiviral effect in infant patients hospitalized with
RSV infection. The study is coded as Victor study, taking the term of “Viral Inhibition in Children for
Treatment of RSV”. WHO recently granted the common name of AK0529 as ziresovir. The final results
of the VICTOR study will be reported in due course.
Chemistry
Scheme 1a
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5a R1 = 6-Cl
Cl
5
5b R1 = 6-Me, 7-F
5c R1 = 5-F, 6-Me
5d R1 = 6-Me, 7,8-2F
5e R1 = 6-CO2CH3
6
H
N
R1
a
7
N
N
8
S
b
c
5f R1 = 6-CH2OH
5g R1 = 6-(CH3)2COH
S
4a
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d
Cl
N
6a R1 = 6-Cl
5
8
6b R1 = 6-Me, 7-F
6c R1 = 5-F, 6-Me
6d R1 = 6-Me, 7,8-2F
6e R1 = 6-CH2OH
6
7
R1
e
1a - 1f
N
6f R1 = 6-(CH3)2COH
S
O
O
aReagents and conditions: (a) substituted 2,4-dichloroquinoline, 1-butanol, 160 oC, microwave; (b) LiAlH4, THF, 0
oC to rt; (c) MeMgI, THF, 0 oC to rt; (d) mCPBA, DCM, 0 oC to rt; (e) 3-(aminomethyl)oxetan-3-amine,
PdCl2(dppf), dppf, t-BuONa, 1,4-dioxane, 120 oC, microwave.
Scheme 2a
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6g R1 = Me, R2 = 2'-F,
6h R1 = Me, R2 = 3'-F
6i R1 = Me, R2 = 3'-Cl
6j R1 = Me, R2 = 3'-Me
6k R1 = Me, R2 = 3'-OMe
6l R1 = Me, R2 = 4'-F
6m R1 = Me, R2 = 4'-OMe
6n R1 = H, R2 = 3'-OMe
6o R1 = H, R2 = 3'-Me
Cl
5'
2'
H
N
R1
4'
R2
a, b
5'
4'
2'
R2
3'
N
N
S
3'
S
4b R2 = 2'-F,
4c R2 = 3'-F
O
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O
4d R2 = 3'-Cl
4e R2 = 3'-Me
4f R2 = 3'-OMe
4g R2 = 4'-F
c
4h R2 = 4'-OMe
Bn
7g R1 = Me, R2 = 2'-F,
7h R1 = Me, R2 = 3'-F
7i R1 = Me, R2 = 3'-Cl
7j R1 = Me, R2 = 3'-Me
7k R1 = Me, R2 = 3'-OMe
7l R1 = Me, R2 = 4'-F
7m R1 = Me, R2 = 4'-OMe
7n R1 = H, R2 = 3'-OMe
7o R1 = H, R2 = 3'-Me
N Bn
HN
N
R1
d
O
5'
4'
2'
R2
1g, 1i - 1n, 1p, 1q
N
3'
e
1g
1h
1o
S
O
f
1m
O
aReagents and conditions: (a) substituted 2,4-dichloroquinoline, 1-butanol, 160 oC, microwave; (b) mCPBA, DCM,
0 oC to rt; (c) 3-(aminomethyl)-N,N-dibenzyloxetan-3-amine, Pd(OAc)2, Cs2CO3, toluene, 120 oC; (d) Pd(OH)2, H2,
TFA, MeOH, rt; (e) NaOMe, MeOH, 100 oC, microwave; (f) KOH, DMSO, 100 oC, microwave.
Compound 1, and intermediates 4a and 8 were prepared as described previously.20 Compounds 1a–1f
were prepared as shown in Scheme 1, and compounds 1g–1q were prepared as shown in Scheme 2.
Intermediates 5a–5g were prepared starting from the 1,4-benzothiazepine 4a. SNAr reactions with the
substituted 2,4-dichloroquinolines led to 5a–5e. Compound 5e was derivatized to alcohols 5f and 5g by
reduction and dimethylation, respectively. These intermediates were then oxidated to the corresponding
sulfones 6a–6f with mCPBA, followed by Buchwald-Hartwig coupling with 3-(aminomethyl)oxetan-3-
amine to afford compounds 1a–1f. Coupling of 2,4-dichloroquinolines with substituted 1,4-
benzothiazepines 4b–4h (see Supporting Information for their synthesis), followed by oxidation to afford
intermediates 6g–6o. The subsequent coupling reactions with 3-(aminomethyl)-N,N-dibenzyloxetan-3-
amine generated 7g–7o, and finally de-benzylation afforded the final compounds 1g, 1i–1o, 1p and 1q.
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Compounds 1h and 1o were obtained by further displacement of the corresponding F substituted 1g and
1m, respectively.
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Scheme 3a
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OTs
OH
N
HN
f
a
N
N
S
O
S
S
O
O
O
O
O
9a
8
11
b
d
g
Bn
Cl
Cl
N
Bn
HN
N
N
O
N
N
N
N
S
S
O
O
S
O
O
O
O
9b
12
10
c
e
h
2a
2c
2b
aReagents and conditions: (a) 2-(cyanomethyl)-5-methyl-benzoic acid, DMA, 170 oC; (b) POCl3, reflux; (c) 3-
(aminomethyl)oxetan-3-amine, PdCl2(dppf), dppf, t-BuONa, 1,4-dioxane, 150 oC, microwave; (d) 2,4-dichloro-1,6-
naphthyridine, 1-butanol, 160 oC, microwave; (e) 3-(aminomethyl)oxetan-3-amine, PdCl2(dppf), dppf, t-BuONa,
1,4-dioxane, 120 oC, microwave; (f) [3-(trifluoromethylsulfonyloxy)-1-naphthyl] 4-methylbenzenesulfonate,
Pd2(dba)3, dppf, t-BuONa, toluene, 110 oC, microwave; (g) 3-(aminomethyl)-N,N-dibenzyloxetan-3-amine, Pd(t-
Bu3P)2, Josiphos SL-J003-1, t-BuONa, toluene, 120 oC, microwave; (h) Pd(OH)2, H2, TFA, MeOH, rt.
Compounds 2a–2c (Table 2) were made as shown in Scheme 3. Reaction of intermediate 8 with 2-
(cyanomethyl)-5-methyl-benzoic acid led to 9a, which was then converted to chloride 9b. The coupling
with
2,4-dichloro-1,6-naphthyridine
or
[3-(trifluoromethylsulfonyloxy)-1-naphthyl]
4-
methylbenzenesulfonate gave intermediate 10 or 11, respectively. Buchwald-Hartwig coupling with 3-
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(aminomethyl)oxetan-3-amine led directly to compounds 2a and 2b, and with 3-(aminomethyl)-N,N-
dibenzyloxetan-3-amine followed by de-benzylated led to compound 2c.
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Scheme 4a
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NH2
14a R1 = Me, Rh =
N
H
Rh
Cl
NHBoc
R1
R1
14b R1 = Me, Rh =
N
H
a
N
N
NHBn
NH2
N
H
14c R1 = Me, Rh =
14d R1 = H, Rh =
N
Cl
N
Cl
13a R1 = H
13b R1 = Me
N
H
O
O
NH2
b
N
H
R1 = Me, Rh =
14e
N(Bn)2
N
H
14f R1 = Me, Rh =
O
Rh
N
R1
3a, 3d, 3e
N
c
NHBoc
NHBn
X= SO2, R1 = Me, Rh =
15b X= SO2, R1 = Me, Rh =
X= SO2, R1 = Me, Rh =
RO-0529, 3b, 3c
15a
N
H
N
N
H
X
N(Bn)2
N
H
15c
O
aReagents and conditions: (a) amine, Et3N, MeOH, rt; (b) Compounds 3a and 15b: (i) 4a, 1-butanol, 160 oC,
microwave; (ii) mCPBA, DCM, 0 oC to rt; Compounds 3d and 15c: 8, Et3N, DMF, 130 oC, microwave; Compound
3e: 2,3,4,5-tetrahydro-1,4-benzothiazepine 1-oxide, 1-butanol, 160 oC, microwave; Compound 15a: 8, NH4Cl,
EtOH, 70 oC; (c) Compounds RO-0529 and 3c: Pd(OH)2, H2, TFA, MeOH, rt; Compound 3b: TFA, DCM, rt.
In Scheme 4, 2,4-dichloroquinazoline 13 was used as the starting material for the synthesis of 3a–3e and
RO-0529 (Table 3). Displacement of the 4-Cl in 13 with different amines generated intermediates 14a–14f
in high yield which were then coupled with the corresponding intermediates 8 and 2,3,4,5-tetrahydro-1,4-
benzothiazepine 1-oxide to afford 3d, 3e, 15a and 15c directly, and with 4a followed by oxidation with
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mCPBA to generate 3a and 15b. Finally, deprotection of 15a–15c resulted in the final compounds 3b, 3c
5
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and RO-0529.
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Conclusions
In summary, ziresovir (RO-0529, AK0529) is evolved from compound 1, which was a lead
compound from the benzoazepine quinoline (BAQ) series discovered from a high throughput screen hit.
Although the difference of ziresovir and compound 1 is quite minor with only replacement of quinoline
with quinazoline moiety, the DMPKT properties are quite different between these two compounds.
Ziresovir demonstrates a larger safety window with the preferred pharmacokinetics compared to compound
1. As we had interpreted in the previous publication20, the introduction of oxetane to control basicity of the
terminal amine in the head portion is the highlight of the discovery efforts, which is important to reduce the
Vss with intact anti-RSV activity. Ziresovir demonstrated good in vitro antiviral activity to both RSV A and
B strains. In addition, it also demonstrated comparable antiviral activity to clinical isolated RSV strains
tested thus far. Due to the higher lung exposure, it demonstrated a good in vivo efficacy in the mice RSV
viral challenge model. In the drug resistant mutation selection study, D486N, D489V and D489Y in RSV
F protein were identified as the resistant viruses, which confirmed drug’s mechanism of action of targeting
RSV fusion protein. With the good and balanced preclinical profile of antiviral, DMPK and toxicology
properties, ziresovir has since moved to clinical development. The clinical results will be reported in due
course.
EXPERIMENTAL SECTION
Synthetic Chemistry General Comments. Intermediates and final compounds were purified by flash
chromatography using one of the following instruments: (i) Biotage SP1 system and the Quad 12/25
cartridge module; (ii) ISCO Combi-flash chromatography instrument. Silica gel brand and pore size: (i)
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KP-SIL 60 Å, particle size 40–60 µm; (ii) CAS registry no., silica gel, 63231-67-4, particle size 47–60 µm
silica gel; (iii) ZCX from Qingdao Haiyang Chemical Co., Ltd., pore 200–300 or 300–400. Intermediates
and final compounds were purified by preparative HPLC on reversed phase column using XBridge Perp
C18 (5 µm, OBD 30 mm × 100 mm) column or SunFire Perp C18 (5 µm, OBD 30 mm × 100 mm) column.
LC/MS spectra were obtained using a MicroMass Plateform LC (Waters Alliance 2795-ZQ2000).
Standard LC/MS conditions were as follows (running time 6 min). Acidic condition: A, 0.1% formic acid
in H2O; B, 0.1% formic acid in acetonitrile. Basic condition: A, 0.01% NH3·H2O in H2O; B, acetonitrile.
Neutral condition: A, H2O; B, acetonitrile. Mass spectra (MS): Generally only ions which indicate the
parent mass are reported, and unless otherwise stated the mass ion quoted is the positive mass ion (M+H)+.
The microwave assisted reactions were carried out in a Biotage Initiator Sixty. NMR spectra were
obtained using Bruker Avance 400 MHz. All reactions involving air-sensitive reagents were performed
under an argon atmosphere. Reagents were used as received from commercial suppliers without further
purification unless otherwise noted. All of the final compounds had purities greater than 95% based upon
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1
HPLC, LC/MS, and H NMR analyses. All of the reported yields are for isolated products and are not
optimized.
N-[(3-Aminooxetan-3-yl)methyl]-6-chloro-2-(1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-
yl)quinolin-4-amine (1a). To a solution of 2,4,6-trichloroquinoline (2 g, 8.6 mmol) in 20 mL of 1-butanol
was added 6,7,8,9-tetrahydro-5-thia-8-aza-benzocycloheptene (4a, 1.42 g, 8.6 mmol). The mixture was
stirred at 160 ºC under microwave irradiation for 2 h. After being cooled to room temperature, the mixture
was diluted with saturated NaHCO3 and extracted with DCM (50 mL). The organic layer was washed with
brine, dried over Na2SO4. The solvent was evaporated under reduced pressure, and the residue was purified
by flash column, eluting with a gradient of 0–10% MeOH in DCM to give compound 5a as a light yellow
solid (2.2 g, yield 71%), which was used for the next step directly.
To a solution of compound 5a (3 g, 8.31 mmol) in DCM (50 mL) was added mCPBA (3.6 g, 20.78 mmol)
at 0 oC. After the addition, the resulting mixture was stirred for 1 h while allowing the temperature to rise
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slowly to room temperature. The reaction mixture was quenched with saturated Na2SO3 and then washed
sequentially with saturated Na2CO3 (30 mL) and saturated brine (30 mL). The organic layer was dried over
Na2SO4, filtered, and then concentrated in vacuo. The residue was purified by flash column, eluting with a
gradient of 0–10% MeOH in DCM to give compound 6a as a white solid (2.7 g, yield 83%), which was
used for the next step directly.
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A mixture of compound 6a (539 mg, 1.37 mmol), PdCl2(dppf) (100 mg, 0.14 mmol), dppf (76 mg, 0.14
mmol), t-BuONa (263 mg, 2.74 mmol) and 3-(aminomethyl)oxetan-3-amine (140 mg, 1.37 mmol) in 1,4-
dioxane (10 mL) was stirred at 120 ºC under microwave irradiation for 1.5 h. The resulting mixture was
concentrated in vacuo and purified by preparative HPLC to afford compound 1a (70 mg, yield 11%). MS
obsd (ESI+) [(M+H)+] 459. 1H NMR (400 MHz, CD3OD) δ ppm 8.34 (s, 1H), 8.00–8.02 (t, J = 4.0, 2.4 Hz,
2H), 7.90–7.92 (d, J = 7.2 Hz, 1H), 7.62–7.65 (m, 1H), 7.54–7.56 (d, J = 9.2 Hz, 1H), 7.44–7.49 (m, 2H),
6.27 (s, 1H), 5.22 (s, 2H), 4.63–4.67 (m, 4H), 4.56 (br s, 2H), 3.78 (s, 2H), 3.60–3.63 (t, J = 4.8 Hz, 2H).
Compounds 1b–1f were prepared by a similar procedure to that described for compound 1a (Scheme 1).
[4-{[(3-Aminooxetan-3-yl)methyl]amino}-2-(1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-
yl)quinolin-6-yl]methanol (1b). MS obsd (ESI+) [(M+H)+] 455. HRMS calcd [(M+H)+] 455.1753,
1
measured [(M+H)+] 455.1754. H NMR (400 MHz, CD3OD) δ ppm 8.33 (br s, 1H), 8.02–8.11 (m, 2H),
7.93 (d, J = 7.33 Hz, 1H), 7.65–7.76 (m, 3H), 7.55 (t, J = 7.71 Hz, 1H), 6.26 (s, 1H), 5.32 (s, 2H), 4.72 (s,
2H), 4.63 (s, 4H), 4.57 (br s, 2H), 3.88 (s, 2H), 3.72 (br s, 2H).
2-[4-[(3-Aminooxetan-3-yl)methylamino]-2-(1,1-dioxo-3,5-dihydro-2H-1lambda6,4-benzothiazepin-
4-yl)-6-quinolyl]propan-2-ol (1c). MS obsd (ESI+) [(M+H)+] 483. HRMS calcd [(M+H)+] 483.2066,
measured [(M+H)+] 483.2066. 1H NMR (400 MHz, CDCl3) δ ppm 8.05 (dd, J = 1.26, 7.83 Hz, 1H), 7.69–
7.73 (m, 2H), 7.58–7.62 (m, 1H), 7.50–7.57 (m, 2H), 7.39 (dt, J = 1.26, 7.71 Hz, 1H), 6.04 (s, 1H), 5.51 (br
s, 1H), 5.16 (s, 2H), 4.56–4.63 (m, 4H), 4.45–4.71 (br s, 2H), 3.60 (d, J = 4.80 Hz, 4H), 1.62 (s, 6H).
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N-[(3-Aminooxetan-3-yl)methyl]-2-(1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-7-fluoro-
6-methylquinolin-4-amine (1d). MS obsd (ESI+) [(M+H)+] 457. HRMS calcd [(M+H)+] 457.1710,
measured [(M+H)+] 457.1709. 1H NMR (400 MHz, CD3OD) δ ppm 8.03–8.05 (d, J = 8.0 Hz, 1H), 7.88–
7.92 (t, J = 8.0, 8.8 Hz, 2H), 7.65–7.68 (m, 1H), 7.49–7.53 ( t, J = 7.6 Hz, 1H), 7.25–7.28 (d, J = 11.6 Hz,
1H), 6.20 (s, 1 H), 5.24 (s, 2 H), 4.48–4.66 (m, 6 H), 3.82 (s, 2 H), 3.64–3.66 (m, 2 H), 2.68 (s, 3 H).
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N-[(3-Aminooxetan-3-yl)methyl]-2-(1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-5-fluoro-
6-methylquinolin-4-amine (1e). MS obsd (ESI+) [(M+H)+] 457. HRMS calcd [(M+H)+] 457.1710,
measured [(M+H)+] 457.1716. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.97–7.99 (d, J = 7.2 Hz, 1H), 7.88–
7.90 (m, 1H), 7.62–7.66 (m, 1H), 7.47–7.51 ( t, J = 7.6 Hz, 1H), 7.24–7.29 (t, J = 8.4, 8.8 Hz, 1H), 7.18–
7.20 (d, J = 8.4 Hz, 1H), 6.57–6.61 (d, J = 16.0 Hz, 1H), 6.12 (s, 1H), 5.11 (s, 2H), 4.43–4.44 (d, J = 6.0
Hz, 2H), 4.37–4.38 (d, J = 6.0 Hz, 2H), 3.61–3.63 (t, J = 4.4, 4.8 Hz, 2H), 3.53 (s, 2H), 2.67–2.68 (t, J =
2.0, 1.6 Hz, 1H), 2.33–2.34 (t, J = 2.0, 1.6, 1H), 1.92 (s, 3H).
N-[(3-Aminooxetan-3-yl)methyl]-2-(1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-7,8-
difluoro-6-methylquinolin-4-amine (1f). MS obsd (ESI+) [(M+H)+] 475. HRMS calcd [(M+H)+]
475.1615, measured [(M+H)+] 475.1622. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.04 (br s, 1H), 7.88 (d, J
= 7.83 Hz, 2H), 7.62 (t, J = 6.82 Hz, 1H), 7.47 (t, J = 7.58 Hz, 1H), 7.18 (br s, 1H), 6.31 (br s, 1H), 5.16
(br s, 2H), 4.60 (d, J = 8.34 Hz, 2H), 3.97–4.05 (m, 2H), 3.82 (br s, 2H), 3.62 (br s, 2H), 3.17 (m, 2H), 2.33
(s, 3H).
N-[(3-Aminooxetan-3-yl)methyl]-2-(9-fluoro-1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-
6-methylquinolin-4-amine (1g). A mixture of 9-fluoro-2,3,4,5-tetrahydro-1,4-benzothiazepine (4b, 0.59
g, 3.2 mmol), 2,4-dichloro-6-methyl-quinoline (0.68 g, 3.2 mmol) and 1-butanol (4 mL) was stirred at 160
ºC under microwave irradiation for 2.5 h. The solvent was removed, and the residue was dissolved in a
mixture solvent of EtOH and DCM. After the DCM in the resulting mixture was removed under reduced
pressure, the resulting precipitate was collected by filtration, which was washed with diethyl ether and
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petroleum ether, dried in vacuo to afford 4-(4-chloro-6-methyl-2-quinolyl)-9-fluoro-3,5-dihydro-2H-1,4-
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benzothiazepine as a pale white solid (0.57 g, yield 50%). MS obsd (ESI+) [(M+H)+] 359.
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A mixture of 4-(4-chloro-6-methyl-2-quinolyl)-9-fluoro-3,5-dihydro-2H-1,4-benzothiazepine (0.39 g, 1.1
mmol) and NaIO4 (0.71 g, 3.3 mmol) in MeOH (15 mL) and water (6 mL) was stirred at room temperature
for 12 h. After removal of the solvent, the residue was dissolved into MeOH (15 mL), then cooled to 0 oC.
A solution of KMnO4 (0.17 g, 1.1 mmol) in water (6 mL) was added dropwise. After being stirred at 0 oC
for 2 h, the mixture was extracted with EtOAc. The organic layer was filtered through a pad of silica gel,
concentrated in vacuo to give compound 6g (0.40 g, yield 93%), which was used for the next step directly.
MS obsd (ESI+) [(M+H)+] 391.
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A mixture of compound 6g (250 mg, 0.64 mmol), Cs2CO3 (420 mg, 1.28 mmol), Pd(OAc)2 (14.4 mg, 0.064
mmol), and 3-(aminomethyl)-N,N-dibenzyloxetan-3-amine (270 mg, 0.96 mmol) in toluene (10 mL) was
stirred at 120 oC for 4 h. After being concentrated under reduced pressure, the residue was purified by flash
column, eluting with 5% MeOH and 0.5% EEt3N in DCM to afford compound 7g (200 mg, yield 50%).
MS obsd (ESI+) [(M+H)+] 637.4.
A mixture of compound 7g (200 mg, 0.31 mmol), palladium hydroxide (10% on carbon, 100 mg) and TFA
(0.2 mL) in MeOH (20 mL) was stirred under a hydrogen atmosphere for 12 h. After being basified with
saturated NaHCO3, the mixture was extracted with DCM. The organic layer was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The residue was purified by HPLC to afford compound
1g as a white solid (20 mg, yield 14%). MS obsd (ESI+) [(M+H)+] 457. HRMS calcd [(M+H)+] 457.1710,
measured [(M+H)+] 457.1719. 1H NMR (400 MHz, CD3OD) δ ppm 7.65–7.75 (m, 2H), 7.60 (td, J = 4.80,
7.96 Hz, 1H), 7.46 (d, J = 8.34 Hz, 1H), 7.32 (dd, J = 1.77, 8.59 Hz, 1H), 7.17 (dd, J = 8.34, 10.36 Hz, 1H),
6.15 (s, 1H), 5.20 (s, 2H), 4.53–4.86 (m, 4H), 4.45 (br s, 2H), 3.73–3.86 (m, 2H), 3.65 (s, 2H), 2.43 (s, 3H).
Compounds 1h–1q were prepared by a similar procedure to that described for compound 1g (Scheme 2).
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N-[(3-Aminooxetan-3-yl)methyl]-2-(9-methoxy-1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-
6-methylquinolin-4-amine (1h). A mixture of compound 1g (20 mg, 0.044 mmol), NaOMe (24 mg, 0.44
mmol) in MeOH (2 mL) was stirred at 100 oC under microwave irradiation for 20 min. After removal of
the solvent, the residue was purified by HPLC to afford compound 1h as a white solid (6 mg, 29%). MS
obsd (ESI+) [(M+H)+] 469. HRMS calcd [(M+H)+] 469.1910, measured [(M+H)+] 469.1909. 1H NMR (400
MHz, CD3OD) δ ppm 7.67 (s, 1H), 7.52–7.62 (m, 1H), 7.47 (d, J = 8.59 Hz, 1H), 7.41 (d, J = 7.07 Hz, 1H),
7.31 (dd, J = 1.89, 8.46 Hz, 1H), 7.16 (d, J = 7.83 Hz, 1H), 5.99 (s, 1H), 5.08 (s, 2H), 4.49–4.63 (m, 4H),
4.31 (t, J = 5.56 Hz, 2H), 3.81–3.98 (m, 5H), 3.54 (s, 2H), 2.43 (s, 3H).
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N-[(3-Aminooxetan-3-yl)methyl]-2-(8-fluoro-1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-
6-methylquinolin-4-amine (1i). MS obsd (ESI+) [(M+H)+] 457. HRMS calcd [(M+H)+] 457.1710,
measured [(M+H)+] 457.1700. 1H NMR (400 MHz, CD3OD) δ ppm 7.95 (dd, J = 5.18, 8.46 Hz, 1H), 7.64–
7.77 (m, 2H), 7.47 (d, J = 8.34 Hz, 1H), 7.25–7.43 (m, 2H), 6.20 (s, 1H), 5.19 (br s, 2H), 4.57–4.68 (m,
6H), 3.70 (s, 2H), 3.56–3.67 (m, 2H), 2.44 (s, 3H).
N-[(3-Aminooxetan-3-yl)methyl]-2-(8-methoxy-1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-
yl)-6-methylquinolin-4-amine (1j). MS obsd (ESI+) [(M+H)+] 469. HRMS calcd [(M+H)+] 469.1910,
measured [(M+H)+] 469.1913. 1H NMR (400 MHz, CD3OD) δ ppm 7.82 (d, J = 8.34 Hz, 2H), 7.56 (br s,
2H), 7.44 (br s, 1H), 7.18 (d, J = 8.59 Hz, 1H), 6.22 (s, 1H), 5.13–5.27 (m, 2H), 4.55–4.74 (m, 4H), 4.50
(br s, 2H), 3.86 (s, 3H), 3.72–3.81 (m, 2H), 3.60–3.71 (m, 2H), 2.46 (s, 3H).
N-[(3-Aminooxetan-3-yl)methyl]-6-methyl-2-(8-methyl-1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-
4(5H)-yl)quinolin-4-amine (1k). MS obsd (ESI+) [(M+H)+] 453. HRMS calcd [(M+H)+] 453.1960,
measured [(M+H)+] 453.1965. 1H NMR (400 MHz, CD3OD) δ ppm 7.82 (d, J = 7.83 Hz, 1H), 7.68 (d, J =
3.03 Hz, 2H), 7.41 (d, J = 6.57 Hz, 1H), 7.33 (d, J = 8.59 Hz, 1H), 7.25 (dd, J = 1.52, 8.59 Hz, 1H), 6.42
(t, J = 5.56 Hz, 1H), 6.19 (s, 1H), 5.05 (br s, 2H), 4.45 (d, J = 5.81 Hz, 3H), 4.39 (d, J = 6.06 Hz, 3H), 3.60
(t, J = 4.55 Hz, 2H), 3.55 (d, J = 5.31 Hz, 2H), 2.37 (s, 3H), 2.31 (s, 3H).
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N-[(3-Aminooxetan-3-yl)methyl]-2-(8-chloro-1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-
6-methylquinolin-4-amine (1l). MS obsd (ESI+) [(M+H)+] 473. HRMS calcd [(M+H)+] 473.1414,
measured [(M+H)+] 473.1426. 1H NMR (400 MHz, CD3OD) δ ppm 7.88–7.99 (m, 2H), 7.77 (s, 1H), 7.64
(dd, J = 2.27, 8.08 Hz, 1H), 7.53 (d, J = 8.59 Hz, 1H), 7.40 (d, J = 8.59 Hz, 1H), 6.19 (s, 1H), 5.21 (br s,
2H), 4.56–4.84 (m, 4H), 4.53 (br s, 2H), 3.75 (s, 2H), 3.68 (br s, 2H), 2.45 (s, 3H).
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N-[(3-Aminooxetan-3-yl)methyl]-2-(7-fluoro-1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-
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6-methylquinolin-4-amine (1m). MS obsd (ESI+) [(M+H)+] 457. H NMR (400 MHz, CD3OD) δ ppm
8.02 (dd, J = 5.43, 8.72 Hz, 1H), 7.65–7.79 (m, 2H), 7.46 (d, J = 8.34 Hz, 1H), 7.33 (d, J = 8.84 Hz, 1H),
7.17 (td, J = 2.65, 8.40 Hz, 1H), 6.17 (s, 1H), 5.17 (br s, 2H), 4.41–4.68 (m, 6H), 3.64–3.72 (m, 2H), 3.53–
3.64 (m, 2H), 2.44 (s, 3H).
N-[(3-Aminooxetan-3-yl)methyl]-2-(7-methoxy-1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-
yl)-6-methylquinolin-4-amine (1n). MS obsd (ESI+) [(M+H)+] 469. HRMS calcd [(M+H)+] 469.1910,
measured [(M+H)+] 469.1900. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.79 (d, J = 8.84 Hz, 1H), 7.68 (s,
1H), 7.57 (d, J = 2.53 Hz, 1H), 7.34 (d, J = 8.59 Hz, 1H), 7.21–7.30 (m, 1H), 6.96 (dd, J = 2.65, 8.72 Hz,
1H), 6.35 (t, J = 5.31 Hz, 1H), 6.19 (s, 1H), 5.04 (br s, 2H), 4.39–4.45 (m, 6H), 3.75–3.92 (m, 3 H), 3.45–
3.62 (m, 4H), 2.37 (s, 3H).
4-[4-[(3-Aminooxetan-3-yl)methylamino]-6-methyl-2-quinolyl]-1,1-dioxo-3,5-dihydro-2H-
1lambda6,4-benzothiazepin-7-ol (1o). A mixture of compound 1m (20 mg, 0.044 mmol), KOH (24 mg,
0.44 mmol) in DMSO (2 mL) was stirred at 100 oC under microwave irradiation for 20 min. After removal
of solvent, the residue was purified by HPLC to afford compound 1o as a white solid (6 mg, yield 30%).
MS obsd (ESI+) [(M+H)+] 455. HRMS calcd [(M+H)+] 455.1753, measured [(M+H)+] 455.1745. 1H NMR
(400 MHz, CD3OD) δ ppm 7.77 (d, J = 8.59 Hz, 1H), 7.67 (s, 1H), 7.45 (d, J = 8.59 Hz, 1H), 7.31 (dd, J =
1.64, 8.46 Hz, 1H), 7.18 (d, J = 2.53 Hz, 1H), 6.72 (dd, J = 2.27, 8.59 Hz, 1H), 6.19 (s, 1H), 5.03 (s, 2H),
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4.68 (d, J = 6.57 Hz, 2H), 4.61 (d, J = 6.57 Hz, 2H), 3.66 (s, 2H), 3.50 (br s, 2H), 3.30 (m, 2H), 2.43 (s,
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3H).
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N-[(3-Aminooxetan-3-yl)methyl]-2-(8-methoxy-1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-
yl)quinolin-4-amine (1p). MS obsd (ESI+) [(M+H)+] 455. HRMS calcd [(M+H)+] 455.1753, measured
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[(M+H)+] 455.1762. H NMR (400 MHz, CD3OD) δ ppm 7.74–7.91 (m, 2H), 7.38–7.58 (m, 3H), 7.03–
7.22 (m, 2H), 6.23 (s, 1H), 5.12 (br s, 2H), 4.46–4.70 (m, 6H), 3.81 (s, 3H), 3.70 (s, 2H), 3.52–3.65 (m,
2H).
N-[(3-Aminooxetan-3-yl)methyl]-2-(8-methyl-1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-
quinolin-4-amine (1q). MS obsd (ESI+) [(M+H)+] 439. HRMS calcd [(M+H)+] 439.1804, measured
[(M+H)+] 439.1795. 1H NMR (400 MHz, CD3OD) δ ppm 7.91 (d, J = 8.34 Hz, 1H), 7.84 (d, J = 7.58 Hz,
1H), 7.60–7.72 (m, 1H), 7.35–7.49 (m, 3H), 7.09 (ddd, J = 3.03, 5.18, 8.21 Hz, 1H), 6.52 (t, J = 5.43 Hz,
1H), 6.22 (s, 1H), 5.06 (br s, 2H), 4.45 (d, J = 6.06 Hz, 3H), 4.38 (d, J = 6.06 Hz, 3H), 3.61 (t, J = 4.80 Hz,
2H), 3.56 (d, J = 5.31 Hz, 2H), 2.31 (s, 3H).
N-[(3-Aminooxetan-3-yl)methyl]-3-(1,1-dioxo-3,5-dihydro-2H-1lambda6,4-benzothiazepin-4-yl)-7-
methyl-isoquinolin-1-amine (2a). A mixture of 2-(cyanomethyl)-5-methyl-benzoic acid (2 g, 11.4 mmol)
and 2,3,4,5-tetrahydro-1lambda6,4-benzothiazepine 1,1-dioxide (8, 2.3 g, 11.4 mmol) in DMA (10 mL)
was heated with stirring at 170 oC for 2 h. After being cooled to room temperature, the resulting mixture
was poured into water. The solid was collected by filtration and washed with ether, dried in vacuo to give
comppound 9a as a brown solid (1.24 g, yield 31%), which was used for the next step without further
purification. MS obsd (ESI+) [(M+H)+] 355
The compound 9a (1.24 g, 3.5 mmol) was dissolved in POCl3 (20 mL). The reaction mixture was stirred
under reflux for 5 h. The resulting mixture was poured into ice-water and extracted with EtOAc. The organic
layre was dried over Na2SO4 and concentred in vacuo. The residue was purified by flash column, eluting
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with 10% EtOAc in petroleum ether to give compound 9b as a brown solid (800 mg, yield 61%). MS obsd
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(ESI+) [(M+H)+] 373.
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A mixture of compound 9b (400 mg, 1.07 mmol), PdCl2(dppf) (79 mg, 0.107 mmol), dppf (60 mg, 0.107
mmol), t-BuONa (206 mg, 2.15 mmol) and 3-(aminomethyl)oxetan-3-amine (110 mg, 1.07 mmol) in 1,4-
dioxane (10 mL) was stirred at 150 ºC under microwave irradiation for 0.5 h. The resulting mixture was
poured into water and extracted with DCM. The organic layer was dried over Na2SO4 and concentrated in
vacuo. The residue was purified by flash column, eluting with a gradient of 0–10% MeOH in DCM to give
compound 2a as a white solid (150 mg, yield 32%). MS obsd (ESI+) [(M+H)+] 439. HRMS calcd [(M+H)+]
439.1804, measured [(M+H)+] 439.1805. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.88 (t, J = 7.9 Hz, 2H),
7.77 (s, 1H), 7.61 (dt, J = 1.2, 7.5 Hz, 1H), 7.4–7.5 (m, 1H), 7.31 (d, J = 8.3 Hz, 1H,), 7.22 (d, J = 8.4 Hz,
1H), 7.12 (t, J = 5.8 Hz, 1H), 6.13 (s, 1H), 4.99 (br s, 2H), 4.47 (d, J = 5.7 Hz, 2H), 4.32 (d, J = 5.7 Hz,
4H), 3.83 (d, J = 5.6 Hz, 2H), 3.59 (t, J = 4.9 Hz, 2H), 2.33 (s, 3H).
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N-[(3-Aminooxetan-3-yl)methyl]-2-(1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-1,6-
naphthyridin-4-amine (2b). Compound 2b was prepared in analogue to compound 1a (Scheme 3). MS
obsd (ESI+) [(M+H)+] 426. HRMS calcd [(M+H)+] 426.1600, measured [(M+H)+] 426.1592. 1H NMR (400
MHz, CD3OD) δ ppm 9.15 (s, 1H), 8.28–8.80 (d, J = 6.4 Hz, 1H), 7.95–8.00 (d, J = 1.2 Hz, 1H), 7.90–7.95
(d, J = 7.2 Hz, 1H), 7.58–7.62 (t, J = 0.8 Hz, 1H), 7.40–7.50 (m, 2H), 6.31 (s, 1H), 5.35 (s, 2H), 4.58–4.65
(m, 6H), 3.80 (s, 2H), 3.50–3.60 (t, J = 2.8 Hz, 2H).
3-[[[3-(1,1-Dioxo-3,5-dihydro-2H-1lambda6,4-benzothiazepin-4-yl)-1-
naphthyl]amino]methyl]oxetan-3-amine (2c). A mixture of [3-(trifluoromethylsulfonyloxy)-1-naphthyl]
4-methylbenzenesulfonate (5 g, 11.21 mmol), Pd2(dba)3 (1 g, 1.12 mmol), dppf (314 mg, 0.56 mmol), t-
BuONa (1.6 g, 16.82 mmol) and 2,3,4,5-tetrahydro-1lambda6,4-benzothiazepine 1,1-dioxide (8, 2.2 g,
11.21 mmol) in toluene (20 mL) was stirred at 110 ºC under microwave irradiation for 0.5 h. The resulting
mixture was diluted with water and extracted with EtOAc (50 mL × 3). The organic layer was washed with
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