Discovery of Benzoazepinequinoline (BAQ) Derivatives as Novel, Potent, Orally Bioavailable Respiratory Syncytial Virus Fusion Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b01394

A novel benzoazepinequnoline (BAQ) series was discovered as RSV fusion inhibitors. BAQ series originated from compound 2, a hit from similarity-based virtual screening. In SAR exploration, benzoazepine allowed modifications in the head moiety. Benzylic sulfonyl on benzoazepine and 6-Me on quinoline were crucial for good anti-RSV activity. Although the basic amine in the head portion was crucial for anti-RSV activity, the attenuated basicity was required to reduce V_ss. Introducing oxetane to the head portion led to discovery of compound 1, which demonstrated single-digit nM anti-RSV activity against different RSV strains, reasonable oral exposure in plasma, and 78-fold higher exposure in lung. Compound 1 also displayed 1 log viral reduction in a female BALB/c mice RSV model by b.i.d. oral dosing at 12.5 mg/kg. A single resistant mutant at L138F in fusion protein proved compound 1 to be a RSV fusion inhibitor.

Full text of DOI:10.1021/acs.jmedchem.8b01394

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Article
Discovery of Benzoazepinequinoline (BAQ) Derivatives as Novel,
Potent, Orally Bioavailable Respiratory Syncytial Virus Fusion Inhibitors
Xiufang Zheng, Chungen Liang, Lisha Wang, Baoxia Wang, Yongfu Liu, Song Feng, Jim
Zhen Wu, Lu Gao, Lichun Feng, Li Chen, Tao Guo, Hong C Shen, and Hongying Yun
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01394 • Publication Date (Web): 19 Oct 2018
Downloaded from http://pubs.acs.org on October 19, 2018
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Discovery of Benzoazepinequinoline (BAQ)
Derivatives as Novel, Potent, Orally Bioavailable
Respiratory Syncytial Virus Fusion Inhibitors
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Xiufang Zheng†, Chungen Liang†, Lisha Wang†, Baoxia Wang†, Yongfu Liu†, Song Feng†, Jim Zhen Wu†,
Lu Gao†, Lichun Feng†, Li Chen†, Tao Guo‡, Hong C. Shen† and Hongying Yun†, *
†Roche Pharma Research and Early Development，Roche Innovation Center Shanghai,Bldg 5, 720 Cailun
Road, 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, Benzoazepine, Quinoline,
Oxetane
ABSTRACT: A novel benzoazepinequnoline (BAQ) series was discovered as RSV fusion inhibitors. BAQ
series was originated from compound 2, a hit from similarity-based virtual screening. In SAR exploration,
benzoazepine allowed modifications in the head moiety. Benzylic sulfonyl on benzoazepine and 6-Me on
quinolone were crucial for good anti-RSV activity. Although the basic amine in the head portion was crucial
for anti-RSV activity, the attenuated basicity was required to reduce Vss. Introducing oxetane to the head
portion led to discovery of compound 1, which demonstrated single-digit nM anti-RSV activity against
different RSV strains, reasonable oral exposure in plasma and 78-fold higher exposure in lung. Compound
1 also displayed > 2 log viral reduction in a female BALB/c mice RSV model by BID oral dosing at 12.5
mg/kg. A single resistant mutant at L138F in fusion protein proved compound 1 to be a RSV fusion inhibitor.
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Introduction
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As a negative-sense single-stranded RNA virus, human respiratory syncytial virus (RSV) belongs
to the family of Paramyxoviridae.1 The most common cause of human RSV infection is acute upper and
lower respiratory tract infection in infants and young children. Almost all children by age of three can be
infected by RSV at least once.2 RSV infection is normally mild and associated with upper respiratory tract
symptoms in normal adults and elder children. However, populations associated with high-risk factors, such
as premature birth, congenital heart disease, chronic pulmonary diseases, and immunocompromised
conditions, are more prone to mortality and morbidity due to lower respiratory tract infections. Severe RSV
infection often leads to bronchiolitis and pneumonia with an increased chance of morbidity or mortality in
young children and immunocompromised adults.2,3 Furthermore, the sequela of severe RSV infection at
young age is recurrent wheezing or asthma,4 and RSV-related mortality rate becomes higher in high-risk
populations.5,6
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Although numerous attempts in inactivated subunits and live-attenuated vaccination are under
development, there is no available vaccine for RSV infection. Palivizumab, a humanized monoclonal
antibody against RSV fusion protein, was approved for prophylaxis in high-risk infants in 1998. However,
palivizumab showed no efficacy in the treatment of established RSV infection.7 Ribavirin, an aerosol
formulation comprising ribavirin, is the only approved antiviral therapy for RSV infection. Due to limited
efficacy and side effects, it is rarely used in clinic.8 As such, safe and effective therapy for RSV infection
is a high unmet medical need.
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OH
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5
6
7
HO
HO
N
N
N
N
O
O
N
N
H2N
N
N
N
N
N
N
N
H
N
H
N
H
8
N
OH
9
JNJ-2408068
TMC-353121
BMS-433771
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N N
N N
N
N
O
N
N
N
N
O
S
N
N
HN
NH2
OH
N
N
N
N
N
O
O
H
F
F
F
N
N
Cl
OH
OH
NH2
N
O
Cl
N
S
O
N
OH
MDT-637
JNJ-53718678
7
GS-5806
Figure 1. Selected RSV fusion inhibitors under development.
RSV Fusion (F) protein is a surface glycoprotein on the viral envelope. The F protein plays a major
role for the virus entry into host cells together with the G surface glycoprotein.9 Depletion of the RSV G
protein can only reduce the efficiency of entry process but cannot terminate the entry process. Moreover,
the RSV F protein also promotes syncytia formation between infected cell and adjacent cells. Inhibition of
viral entry and spread by targeting RSV F protein has emerged as a promising treatment for RSV infected
patients. Small molecule RSV F protein inhibitors have great potential to decrease the duration and severity
of respiratory symptoms, and the subsequent risk of prolonged hospitalization and complications.
In the past, several structurally different and potent RSV F protein inhibitors were reported.10-13
A
number of RSV F inhibitors had successfully progressed to preclinical stage, but only a few had entered
clinical development.14 JNJ-2408068 was a picomolar RSV F inhibitor (EC50: 0.16 nM) with low
cytotoxicity against several lab strains. However its long tissue retention time in the tested species hindered
its further development.11 Another picomolar RSV F inhibitor, TMC-353121, exhibited anti-RSV efficacy
in inhibiting RSV replication prophylactically and therapeutically.12 BMS-433771 was the first RSV F
inhibitor to demonstrate oral bioavailability,13 but was terminated for further development likely due to
business reason. VP-14637, a nanomolar RSV F inhibitor, was formulated and used as an inhaled dry
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powder product called MDT-637 in Phase I trials. This molecule demonstrated good tolerance in healthy
human subjects.15 Most recently, GS-5806 achieved proof-of-concept in human RSV challenge studies and
showed a significant viral load reduction (4.2 log10) and a significant disease severity reduction.16 In a
Phase IIa trial with the RSV-A Memphis 37b virus inoculated healthy adults, daily oral dosing JNJ-
53718678, a picomolar RSV F inhibitor, reduced viral load and clinical severity, which established clinical
proof of concept of RSV treatment by using RSV F inhibitor. 17
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In our endeavors to develop highly potent RSV F inhibitors, we used a similarity-based virtual
screening approach and generated several hits as chemistry starting points. In our similarity-based virtual
screening, MOS (2D similarity) and ROCS (3D similarity) were used in parallel to screen the Roche Smart
library. JNJ-2408068, TMC-353121and BMS-433771 were chosen as reference compounds for the virtual
screening. In our previous publication, we reported compound 7 as a validated hit as RSV F inhibitor by
this approach.18
In this article, we describe the discovery of benzoazepinequnoline (BAQ) series derived from
compound 2, another hit from the similarity-based virtual screening approach (Figure 2).
Rh
HN
HN
NH2
OH
N
Ring expansion
R2
R1
N
N
N
X
2
BAQ
Figure 2. BAQ series originated from ring expansion of compound 2.
Results and Discussion
As aforementioned, BAQ series was originated from compound 2. We were delighted to see that
the reasonable anti-RSV activity of compound 2 in CPE assay (EC50=0.22 M). Further exploration
disclosed that ring size reduction to 5-membered ring 3 resulted in a total loss of anti-RSV activity and
strong cytotoxicity. The anti-RSV activity remained the same with ring expansion to a 7-membered ring
(4, EC50=0.22 M).18
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Table 1. SAR results of R2 of the scaffold derived from compound 2a
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6
7
8
9
HN
N
NH2
OH
R2
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Compd
2
R2
EC50 (µM)a
CC50 (µM)
TIb
N
N
0.22
21
95
3
4
>100
0.22
2.9
21
0
N
95
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.
To explain the correlation of ring size to anti-RSV activity, we did the conformation analysis
(Figure 3). Compound 3 (a 5-membered ring analogue) adopted almost a planar conformation. Compound
2 (a 6-membered ring analogue) adopted a conformation with nearly 40-degree dihedral angle between
quinoline and tetrahydroisoquinoline. Finally, compound 4 (a 7-membered ring analogue) adopted a
conformation with almost 90-degree dihedral angle between quinoline and benzoazepine. These
observations gave us a hint that the active RSV inhibitors may prefer a non-planar scaffold with a dihedral
angle between two ring systems up to 90 degrees.
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Figure 3. Conformation of 2, 3 and 4 at the lowest energy.
Compound 2 in orange, compound 3 in pink and compound 4 in green.
An exploration on the head portion (Rh) of compound 2 revealed a flat SAR (Table 2). All the
modifications on Rh resulted in a significant loss of antiviral activity. Furthermore, derivatives with basic
terminals (2b, 2d and 2f) demonstrated better antiviral activity than the non-basic terminal derivatives (2a,
2c and 2g). The chain length between the two amino groups of Rh also impacted on activity. For example,
two-carbon linker derivatives (2d and 2f) provided better anti-RSV activity than a three- and a four-carbon
linker derivatives (2b and 2e).
Table 2. SAR exploration of the head portion (Rh) of the scaffold derived from compound 2a
Rh
HN
N
N
Compd
2
Rh
EC50 (µM)a
CC50 (µM)
TIb
NH2
0.22
21
95
OH
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OH
2a
2b
2c
22
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8.4
89
3
1
0
OH
NH2
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> 100
O H
NH2
2d
2e
2.3
15
7
0
NH2
> 100
7.4
H
N
NH2
NH
2f
2.3
15
> 100
80
43
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H
N
NH2
2g
O
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.
Further profiling of compound 2 revealed a very low permeability (0  10-6 cm/s) in the PAMPA
assay, which indicated a very low cellular permeability potential. Thus compound 2 was not suitable for
oral dosing.
Considering the flat SAR of the derivatives of compound 2 on Rh and poor permeability of
compound 2, we turned our attention to compound 4 (BAQ). Compound 4 showed identical activity and TI
to compound 2. However, different from compound 2, compound 4 allowed further modification on Rh
without sacrificing potency. For example, compound 5, a close analogue to 2d, showed the identical good
activity to compound 4. Moreover, the PAMPAof compound 5 was 0.99, which was categorized as medium
to high cell permeability.
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Table 3. Head portion (Rh) exploration on the scaffold derived from compound 4a
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Rh
HN
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N
N
PAMPA Peff
(10-6 cm/s)
Compd
Rh
EC50 (µM)a CC50 (µM)
TIb
NH2
NH2
4
5
0.22
0.20
21
23
95
0
OH
115
0.99
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.
Subsequently, the rat single-dose pharmacokinetics (SDPK) of compound 5 showed reasonable
oral exposure with moderate oral bioavailability (Table 4). Both in vitro rat liver microsomal clearance and
in vivo clearance were medium to high, which needed to be further optimized. The high volume of
distribution (Vss) was most likely due to the lipophilic basic nature of compound 5.
Table 4. The DMPK profile of compound 5a
RLMb
AUC0-INF
AUC0-INF
F (%)
in vivo CL
Compd
5
(mL/min/kg)
(iv 2 mg/kg) (po 10 mg/kg)
Vss (L/kg)
(mL/min/kg)
(h*ng/mL)
(h*ng/mL)
23 (medium)
324
510
89 (high)
106
32
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a The SDPK study in male Wistar rats was 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), rat liver microsomal clearance (RLM).
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In the resistant mutation selection study, a single mutant at D331N in RSV F protein was identified
in compound 5 resistant virus at passage 7. The anti-RSV activity was dropped from 0.20 M to 2.3 M in
resistant virus. It proved that compound 5 was targeting on RSV fusion protein.
Encouraged by the good potency, reasonable oral absorption, and modeling understanding, we
selected compound 5 as a new chemistry starting point for further optimization. Chemistry modification
was initiated at R1 on quinoline. Selected data are summarized in Table 5. A Cl-walk on quinolone revealed
that 6-position was critical for antiviral potency. SAR exploration at the 6-position disclosed that the 6-Me
analogue (5f) demonstrated the best potency and the largest TI although it still showed strong cytotoxicity
like the other R1 derivatives. The cytotoxicity cannot be addressed by optimizing R1 only, but was
eventually addressed by the combined optimization on both R1 and X, which was described in Table 6.
Table 5. SAR exploration of R1 of the quinolone scaffold derived from compound 5a
NH2
HN
6
R1
N
N
Compd
R1
EC50 (µM)a
CC50 (µM)
TIb
5
H
0.20
6.6
23
23
115
3
5a
5b
5-Cl
6-Cl
0.12
>3.2
>27
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5c
5d
5e
5f
7-Cl
8-Cl
>3.2
1.1
7.5
25
2
23
15
129
5
6-OMe
6-Me
0.32
0.073
1.5
4.8
9.4
8.0
6.7
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5g
5h
5i
6-F
6-CF3
6-SO2Me
1.1
6
5.6
2
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.
As described in Table 4, compound 5 showed medium to high in vitro and in vivo clearance. Based
on the metabolic identification (Met ID) study in mouse and rat liver microsomes (Figure 4), the
benzoazepine moiety was identified as a metabolic “hot spot” generating 90% of metabolites in rat liver
microsomes. In particular, the benzylic position (X in Figure 2) and benzene ring of benzoazepine were
highly likely to be accountable for metabolism. In the head moiety, amide and acid were generated by
oxidation of the carbon adjacent to the terminal amine. All these Met ID results cast lights on how to further
optimize compound 5.
Figure 4. Met ID of compound 5 in rat and mouse liver microsomes.
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Our optimization strategy focused on reducing the electronic density of benzene (Table 6). Two
approaches were explored. The first was to introduce fluorine to benzene to block a metabolic “hot spot”,
which unfortunately failed (6f and 6g). The other approach was to replace the benzyl carbon (X in Figure
2) with heteroatoms, which was proven to be very successful. In particular, introducing the electron
withdrawing group SO2 led to discovery of compounds 6a and 6h (R1 = H or Me and X = SO2), which led
to significantly improved metabolic stability. The liver microsomal clearance in tested species were low.
In addition, compounds 6a and 6h also demonstrated superior anti-RSV activity and good TI compared to
other modifications. Compound 6h demonstrated the best anti-RSV activity (0.004 M) and TI (16,500),
as shown in Table 6.
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Table 6. X optimization impacts on both potency and XLM
NH2
HN
R1
R2
3'
N
N
X
HLM/RLM/MLM
(mL/min/kg)c
10/23/19
0/3/0
Compd
R1
R2
X
EC50 (µM)a CC50 (µM)
TIb
5
H
H
H
H
H
H
H
CH2
SO2
O
0.20
0.011
0.23
23
94
115
8,545
>43
106
52
6a
6b
6c
6d
H
>10
6.6
12
14/20/25
NA
Me
Me
CF2
NH
0.062
0.23
5/43/19
HN
6e
Me
H
0.31
55
177
0/21/11
N
H
O
6f
Me
Me
3’-F
2’-F
CH2
CH2
0.46
0.10
>3.2
>3.2
>7
8/33/31
6g
>32
6/160/24
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6h
Me
H
SO2
0.004
66
16,500
5/8/1
5
6
7
8
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. cScaled intrinsic clearance of compounds in human, rat and mouse
liver microsomes (HLM, RLM or MLM).
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In the next round of optimization, the head portion (Rh) was re-explored with the optimal 6-Me and
X as SO2 (Table 7). We observed a much broader tolerance of substitution on Rh. The length of alkyl chain
between two amino groups was identified to be crucial for antiviral activity. Compound 6h and compound
7a were optimal for excellent anti-RSV activity (EC50=0.004 M and 0.001 M, respectively). Methyl
substitution on either terminal amine (7d and 7e) or alkyl chain (7f to 7i) reduced the ant-RSV activity.
Replacing the terminal amine by heterocycles (from 7j to 7m) significantly reduced the anti-RSV activity.
Oxetanylation of terminal amine (7s) led to much reduced anti-RSV activity. Acetylation of the terminal
amine (7p) totally removed the anti-RSV activity. Clearly introducing electron withdrawing group at the
terminal amine would most likely reduce anti-RSV activity. In addition, replacing terminal amine with
hydroxyl group (7q) resulted in a 10-fold decrease in anti-RSV activity compared to compound 6h.
It was noteworthy that oxetane analogues (7r and 1) both demonstrated excellent anti-RSV
activity at single-digit nM level (EC50=0.005 M and 0.002 M, respectively). Compound 1 also
demonstrated the highest therapeutic index (TI >50,000) throughout the whole chemical series in the CPE
assay.
a
Table 7. SAR exploration at the Rh position with fixed 6-Me and X = SO2
Rh
HN
N
N
S O
O
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Compd
6h
Rh
EC50 (µM)a
0.004
CC50 (µM)
TIb
NH2
66
21
92
21
21,500
21,000
13,142
778
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7a
0.001
NH2
NH2
7b
0.007
7c
0.027
NH2
H
N
7d
7e
0.014
0.037
29
89
2,071
2,405
N
7f
NH2
NH2
0.010
25
2,500
7g
7h
7i
0.014
0.025
0.012
86
25
23
6,143
1,000
1,917
NH2
NH2
NH
7j
0.15
0.54
40
267
N
O
7k
>100
>185
N
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7l
0.28
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N
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N
7m
0.071
324
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O
7n
7o
0.039
0.026
>100
22
>2,564
846
NH2
NH2
O
H
N
7p
7q
7r
3.1
66
>100
86
21
O
O H
0.041
0.005
>2,439
17,200
O
NH2
H
N
7s
1
0.031
0.002
>100
>100
3,226
O
NH2
>50,000
O
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.
Since compound 1 was identified with the best potency and therapeutic index, it was further
profiled against different RSV strains in CPE assay. To our delight, compound 1 demonstrated consistent
and high anti-RSV activity against different RSV strains in CPE and plaque reduction assays. Regarding
selectivity, compound 1 showed no activity to influenza H1N1 in CPE assay (Table 8), which indicated
that it did not target on a broad-antiviral host target.
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Table 8. Anti-RSV activity and selectivity of compound 1a
5
6
7
8
9
RSV Long
EC50 (µM)
0.002
RSV A2
EC50 (µM)
0.004
RSV B
EC50 (µM)
0.002
Plaque reduction Influenza H1N1
Compd
1
EC90 (µM)
EC50 (µM)
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0.003
100
a EC50: the concentration of compound that reduced 50% cytopathic effect of RSV Long, A2 and B strains, plaque
reduction in long strain and influenza H1N1 strain infected HEp-2 cells.
ADME and in vivo efficacy assessment of selected BAQs.
The physicochemical properties and ADME of selected BAQs were listed in Table 9. The mlLogD
had correlation with pKb of the head amine except for compound 7q. Compound 7q, the one without
ionizable head amine showed high lipophilicity (mlLogD), good permeability but low aqueous solubility.
On the contrary, all the other terminal amino analogues demonstrated good solubility. In term of pKb, it
was known that oxetane as a carbonyl surrogate can reduce pKb of adjacent amine.20 Introducing oxetane
next to the terminal amine in compound 1 led to the reduction of the pKb more than 2 log units compared
to any other analogues containing a terminal amino group. The impact of oxetane on the pKb was quickly
diminished with the elongated distance to terminal amine such as compound 7r. Compounds 1 and 7r
demonstrated high clearance in liver microsomal clearance assay. Except for compound 6h, all the tested
analogues showed good permeability in PAMPA assay. In addition, the plasma protein binding of tested
compounds were medium to high.
Table 9: Physicochemical and ADME properties of compounds 1, 5, 6h, 7q and 7r.
pKbb
Lysac
HLM/RLM/MLM
PAMPAe
PPBf
Compd mlLogDa
head amine (µg/mL) d (mL min−1 kg−1)
(10−6 cm/s) (human/mouse)
1
5
2.20
0.73
8.0
482
17/95/204
10/23/19
0.73
0.99
2.4/11
NA
10.4
>460
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6h
7q
7r
0.88
2.57
1.12
10.4
none
9.9
>466
9
5/8/1
0.12
1.53
0.59
7.8/8
NA
36/81/120
12/37/23
>480
3/10
9
a
b
d
Machine learning LogD. Predicted by MoKa v2.0. c Lyophilisation solubility assay (LYSA) (µg/mL). Scaled
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e
intrinsic clearance of compounds in human, rat and mouse liver microsomes (HLM, RLM or MLM). Parallel
Artificial Membrane Permeability Assay (10-6 cm/s). e Plasma protein binding assay (% unbound fraction).
In the SDPK study in male Wistar rats (Table 10), compounds 1, 5, 7q and 7r all demonstrated
reasonable oral exposure. Compound 6h did not show any oral exposure, which could be due to its low
cellular permeability. The most important finding was that the Vss of compound 1 was as low as that of
compound 7q, which did not contain a basic head group. It is well known that basic lipophilic molecules
typically lead to a high volume of distribution, which may cause unexpected accumulation of molecule in
undesired tissues. Hence the project team made the decision to choose compound 1 for further
characterization.
Table 10. Rat SDPK profiles of selected BAQ compoundsa
Compd
t1/2 (iv, h)
1
5
6h
4.5
17
7q
1.0
2.5
7r
4.1
20
1.7
4.1
12
73
Vss (iv, L/kg)
CL (iv)
51
106
324
510
97
346
0
33
53
(mL min−1 kg−1)
AUC(0-∞)
Plasma
686
558
717
466
(iv, µg/L*hr)
AUC(0-∞)
1640
1267
(po, µg/L*hr)
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F (%)
16
32
0
59
29
5
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7
8
a The SDPK study in male Wistar rats was carried out according to the standard procedures. (iv: 2 mg/kg; po: 10
mg/kg). Major parameters, including plasma clearance (CL), volume of distribution at steady state (Vss), T1/2 (i.v.),
area under the cure (AUC), and oral bioavailability (F) are reported.
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Since the in vivo anti-RSV model was run in mice, the additional SDPK in IRC mice was run with
compound 1 at different doses (Table 11). The Vss in mouse SDPK was very close to the one measured in
rat SDPK. The oral exposure was over dose proportional, which could be due to the saturation of metabolic
enzymes at high doses. In the medium dose (200 mg/kg), the lung exposure was also analyzed. It was very
interesting to see the exposure of lung was 78-fold higher than plasma. It is known that basic drugs are
bound to certain binding sites containing phosphatidylserine.21 Phosphatidylserine is highly expressed in
the surface of epithelium cells in lung. Compound 1 could accumulate in lung as a counter ion to
phosphatidylserine, which is quite acidic. This is perhaps the most convincing explanation of high exposure
of compound 1 in lung. To our RSV fusion inhibitor project, high exposure in lung was an advantage. As
we previously mentioned, an overly high Vss might lead to unexpected accumulation and potential toxicity
risk for basic molecule. We utilized oxetane to control pKb of head amine and minimized the potential risk
of toxicity.
Table 11. Mouse SDPK profiles of compound 1a
Dose (mg/kg)
t1/2 (po, h)
50
200
2.3
450
1.3
2.9
Vss (iv, L/kg)
CL (iv) (mL min−1 kg−1)
AUC(0-∞) (iv, µg/L*hr)
AUC(0-∞) (po, µg/L*hr)
F (%)
4.7
222
150
9,884
66
Plasma
1,352
36
44,532
132
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Lung
AUC(0-∞) (po, µg/L*hr)
NA
NA
769,641
NA
NA
AUC(0-∞), lung/AUC(0-∞), plasma (po)
78
7
8
9
a The SDPK study in male ICR mice was carried out according to the standard procedures. (iv: 2 mg/kg; po: 50, 150,
450 mg/kg). Major parameters, including plasma clearance (CL), volume of distribution at steady state (Vss), T1/2 (i.v.),
area under the cure (AUC), and oral bioavailability (F) are reported.
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Finally, compound 1 was tested in a mouse RSV model (Figure 5). This compound demonstrated
1 log unit of viral titer reduction at 12.5 mg/kg by oral dosing twice a day (BID). The anti-RSV efficacy
was dose dependent. The RSV titer was below detectable limit at 50 mg/kg and 150 mg/kg, po, BID.
Typically, an antiviral drug needs to have enough exposure to maintain antiviral activity, and one criterion
is that the plasma Cmin is higher than IC90. In our case, the oral T1/2 of 50 mg/kg was only 1.25 h, the total
plasma drug concentration was below IC90 after 8 hours. It could be assumed that at 12.5 mg/kg, po, BID
dosing, the in vivo efficacy was not likely to be observed. However, considering the high lung exposure of
compound 1, we speculated that the observed good anti-RSV efficacy at such a low dose was a result of the
high lung exposure and longer lung T1/2 of compound 1. Unfortunately, we were unable to test the drug
concentration in lung in RSV mouse model due to the limited amount of samples. We plan to provide more
evidences on the relationship of pKb and lung distribution in our succeeding manuscript.
4
3.5
3
2.5
2
1.5
1
Baseline
0.5
0
Vehicle
150 mg/kg
50 mg/kg
25 mg/kg
12.5 mg/kg
Figure 5. In vivo Reduction of RSV Titer in Mouse Lung with compound 1 (po, BID)a
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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 compound 1 for 4 days, the mice were euthanized with CO2, and lungs were harvested
and analyzed for viral titer.
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There were more examples carrying oxetane with diverse modifications on 6-position and SO2
replacement in our previous publication.19 Among those modification, compound 1 remained the top
molecule considering anti-RSV activity, TI, DMPK and toxicity profiles.
In the resistant mutation selection study, a single mutant at L138F in RSV F protein was identified
in compound 1 resistant virus at the passage 7. The anti-RSV activity of compound 1 was dropped from 2
nM to 2.56 M, which proved that the target of compound 1 was indeed RSV fusion protein.
The single resistant mutation study provided us the opportunity to seek the potential binding site of
compound 1 in RSV F protein. Actually, L138 located quite close to the binding pocket of RSV pre-fusion
protein with JNJ-2408068 reported by McLellan, et al.22 We docked compound 1 into the same threefold-
symmetric cavity in profusion RSV F-protein. We observed that the 6-Me entered into a small hydrophobic
pocket and picked more hydrophobic interaction with protein, such as Met 396. The oxygen atoms of
sulfone formed strong hydrophobic interaction with Phe 140. The model suggested that the oxetane moiety
did not form any interaction with the protein, and it mainly controlled the conformation. All these 3 factors
possibly accounted for 100-fold potency increase compared to original hit, compound 2. The docking model
also demonstrated that the Leu 138 was very close to the binding pocket, and L138F mutation may change
the conformation of the binding pocket. This appeared to elucidate the huge drop of the antiviral activity of
compound 1 to this resistant virus.
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a)
b)
Figure 6. Docking model of compound 1 binding to a three-fold symmetric cavity in prefusion RSV F
glycoprotein (the docking procedure was described in the supporting information).
a) Side view of the compound 1 in the binding pocket of prefusion FSV F glycoprotein (shown in white). Leu138 was
shown as a stick with color in orange. Compound 1 was shown as ball-and-stick, 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 1 with RSV F main-chain and side-chain atoms were shown as blue and green dotted lines,
respectively. And the arrowhead pointed to the acceptor.
Chemistry
As exemplified by the synthesis of compound 1, BAQ deriatives described herein were mostly prepared
according to the procedure shown in Scheme 1. Starting with 2-sulfanylbenzoic acid 8, esterification with
methanol gives methyl 2-sulfanylbenzoate 9, which was followed by annulation with 2-chloro ethylamine
to afford 3,4-dihydro-1,4-benzothiazepin-5(2H)-one 10. Reduction of compound 10 with LiAlH4 provided
2,3,4,5-tetrahydro-1,4-benzothiazepine 11, which was converted to 13 via acetylation and oxidation. The
subsequent deacylation afforded the desired building block 14, which underwent a coupling reaction with
2,4-dichloro-6-methyl-quinoline to generate the key intermediate 15. Another coupling with 3-
(aminomethyl)-N,N-dibenzyloxetan-3-amine yielded compound 16, which was finally de-benzylated to
afford 1 after HPLC purification. The structure of compound 1 could be successfully assigned by
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spectroscopic analysis on the basis of 1 H, 13C, H-H COSY, H-C HSQC, and H-C HMBC NMR experiments.
This efficient synthetic route was successfully applied to the synthesis of the BAQ analogues described in
this paper.
9
10
11
12
13
14
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21
22
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Scheme 1a
O
H
O
O
H
N
N
a
b
c
HO
HS
O
S
HS
S
8
9
10
11
O
H
O
N
d
e
f
g
N
N
S
O
S
O
S
O
O
12
13
14
Bn
NH2
N Bn
Cl
HN
HN
O
O
N
h
i
N
N
N
N
N
S
S
O
S
O
O
O
O
O
15
16
1
aReagents and conditions: (a) H2SO4, MeOH, reflux, 18 h, 91% (crude); (b) 2-chloroethanamine hydrochloride,
NaH, DMF, rt, overnight, 47%; (c) LiAlH4, THF, reflux, 18 h, 90%; (d) Ac2O, Et3N, DCM, 0 oC to rt, 1 h; (e)
mCPBA, DCM, 0 oC to rt, 1 h; (f) NaOH, EtOH/H2O, reflux, overnight, 77%; (g) 2,4-dichloro-6-methyl-quinoline,
1-butanol, MW., 160 oC, 2 h; (h) 3-(aminomethyl)-N,N-dibenzyloxetan-3-amine, PdCl2(dppf), dppf, t-BuONa,
dioxane, MW., 120 oC, 1 h, 65%; (i) Pd(OH)2, H2, MeOH, rt, 16 h, 17%.
Conclusions
In summary, we have discovered a novel series of BAQ derivatives as RSV fusion inhibitors
derived from compound 2, a similarity-based virtual screening hit. In the SAR exploration of the BAQ
series, the combination of sulfonyl group at the benzylic position of benzoazepine and 6-Me on quinolone
led to singledigit nM anti-RSV compounds (1, 6h, 7a, 7b and 7r). Sulfonyl group replacement at the
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benzylic position of benzoazepine also significantly improved the metabolic stability (compound 6a vs.
compound 5). Although the basic terminal amine on the head region was crucial for anti-RSV activity, it
was discovered that reduced basicity was important for reducing Vss. Oxetane was introduced adjacent to
the terminal NH2, whichled to discovery of compound 1 with much reduced Vss and intact anti-RSV activity.
Compound 1 was the most balanced molecule in the BAQ series. It demonstrated good anti-RSV activity
against different RSV strains in CPE assays (IC50: 2 nM in long strain, 4 nM in A2 strain, and 2 nM in B
strain), and reasonable oral exposure in plasma and 78-fold higher exposure in lung. It also demonstrated
good anti-RSV activity (> 2 log units) in a female BALB/c mice model by BID oral dosing at 12.5 mg/kg.
In the resistant mutation selection study, a single mutant at L138F in RSV F protein was identified in
compound 1 resistant virus, which confirmed compound 1 as a RSV fusion inhibitor. Based on its favorable
in vitro and in vivo properties, compound 1 was considered as a promising clinical candidate for RSV
treatment.
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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) KP-
SIL 60 Å, particle size: 40-60 µM; ii) CAS registry NO: Silica Gel: 63231-67-4, particle size: 47-60 micron
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 X BridgeTM Perp C18
(5 µm, OBDTM 30 × 100 mm) column or SunFireTM Perp C18 (5 µm, OBDTM 30 × 100 mm) column. LC/MS
spectra were obtained using a MicroMass Plateform LC (WatersTM alliance 2795-ZQ2000). Standard
LC/MS conditions were as follows (running time 6 minutes): 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)+.
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The microwave assisted reactions were carried out in a Biotage Initiator Sixty. NMR Spectra were obtained
using Bruker Avance 400MHz. 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 HPLC,
LC/MS, and 1H NMR analyses. All of the reported yields are for isolated products and are not optimized.
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General Synthetic Procedures for the Synthesis of BAQ Series with N-[(3-Aminooxetan-3-yl)methyl]-
2-(1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-6-methylquinolin-4-amine (1) Exemplified.
o
To a cooled solution of concentrated sulfuric acid (72 g) in methanol (1.5 L) at 0 C, was added 2-
sulfanylbenzoic acid (8, 300 g, 1.95 mol) in portions under argon atmosphere. After being refluxed with
stirring for 18 hours, the reaction mixture was concentrated in vacuo. The residue was diluted with water
(800 mL), basified with a saturated aqueous solution of sodium bicarbonate to about pH 7, and extracted
with dichloromethane (600 mL  3). The combined organic layers were washed with brine (800 mL), dried
over sodium sulfate, filtered and concentrated in vacuo to afford methyl 2-sulfanylbenzoate (9, 300 g, yield
91%) as a light yellow oil, which was used for the next step without further purification.
To a cooled solution of methyl 2-sulfanylbenzoate (9, 200 g, 1.19 mol) in tetrahydrofuran and N,N-
dimethylformamide (2 L, V/V = 1/1) was added 2-chloroethanamine hydrochloride (138 g, 1.19 mol) at 0
oC followed by sodium hydride (143 g, 3.57 mol, 60% in mineral oil) in portions. After being stirred at
room temperature overnight, the reaction mixture was poured into ice-water and extracted with ethyl acetate
(900 mL  4). The organic layers were combined, washed with brine (900 mL  3), dried over sodium
sulfate and concentrated in vacuo. The residue was stirred in a mixture of ethyl acetate and petroleum ether
(300 mL, V/V = 1/1) for 1 hour. The solid was collected by filtration and dried in vacuo to afford 3,4-
dihydro-1,4-benzothiazepin-5(2H)-one (10, 100 g, yield 47%).
To a bottle containing a cooled suspension of lithium aluminum hydride (44 g, 1.17 mol) in dry
tetrahydrofuran (1.5 L) was added 3,4-dihydro-1,4-benzothiazepin-5(2H)-one (10, 150 g, 0.84 mol) in
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portions at 0 oC. After being refluxed for 18 hours, the reaction mixture was cooled to 0 oC, followed by
addition of water (25 mL) dropwise. The reaction mixture was then filtered through a pad of celite and
washed with dichloromethane. The filtrate was dried over sodium sulfate and evaporated in vacuo to afford
2,3,4,5-tetrahydro-1,4-benzothiazepine (11, 125 g, yield 90%), which was used for the next step without
further purification.
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To a solution of 2,3,4,5-tetrahydro-1,4-benzothiazepine (11, 5 g, 30.3 mmol) in dry dichloromethane (100
mL) was added triethylamine (5.06 mL, 36.3 mmol) at room temperature, followed by the dropwise addition
of acetic anhydride (3.43 mL, 36.3 mmol) at 0 oC under nitrogen. The resulting solution was stirred for 1
hour whilst allowing the temperature to rise slowly to room temperature. The mixture was washed with
brine (50 mL × 2), dried over sodium sulfate, filtered and concentrated in vacuo to afford 1-(2,3-dihydro-
1,4-benzothiazepin-4(5H)-yl)ethanone (12, 6.28 g, quantitative yield) as a yellow oil, which was used for
next step without further purification.
To a cooled solution of 1-(2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)ethanone (12, 6.27 g, 30.2 mmol) in
dichloromethane (100 mL) was added a suspension of 3-chloroperoxybenzoic acid (20.9 g, 90.8 mmol,
75% purity) in dichloromethane (50 mL) at 10 oC. After the addition, the resulting mixture was stirred for
1 hour whilst allowing the temperature to rise slowly to room temperature. The mixture was washed with a
saturated aqueous solution of sodium carbonate (100 mL  2), a saturated aqueous solution of sodium sulfite
(100 mL  2) and brine (100 mL) in sequence. The organic layer was dried over sodium sulfate, filtered
and concentrated in vacuo. The residue was stirred in diethyl ether (50 mL) and the solid was collected by
filtration and dried in vacuo to afford 1-(1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)ethanone
(13, 6 g, yield 83%) as a white powder.
To a solution of 1-(1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)ethanone (13, 6 g, 25 mmol) in
ethanol (25 mL) was added sodium hydroxide (5 g, 125 mmol) and water (28 mL). The mixture was
refluxed overnight and then concentrated in vacuo. The residue was extracted by ethyl acetate (60 mL  4).
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The combined organic layers were extracted by hydrochloric acid (80 mL, 3 N). The acidic aqueous layer
was washed with ethyl acetate (60 mL  2), then basified with a saturated aqueous solution of sodium
bicarbonate to pH > 7, and extracted with ethyl acetate (60 mL  4). The combined organic layers were
dried over sodium sulfate, filtered and concentrated in vacuo to afford 2,3,4,5-tetrahydro-1,4-
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benzothiazepine1,1-dioxide (14, 3.8 g, yield 77%), MS obsd. (ESI+) [(M+H)+] 198. H NMR (400 MHz,
DMSO-d6) δ ppm 7.89 (dd, J = 1.2, 7.6 Hz, 1 H), 7.56 (t, J = 7.6 Hz, 1 H), 7.47 (t, J = 7.6 Hz, 1 H), 7.42
(d, J = 7.6 Hz, 1 H), 4.04 (s, 2 H), 3.32 - 3.30 (m, 2 H), 3.30 - 3.25 (m, 2 H), 2.64 (s, 1 H).
To a solution of 2,4-dichloro-6-methyl-quinoline (2.12 g, 10 mmol) in 20 mL of 1-butanol was added
2,3,4,5-tetrahydro-1,4-benzothiazepine1,1-dioxide (14, 2.16 g, 11 mmol). The mixture was stirred at 160
ºC under microwave irradiation for 2 hours. After being cooled to room temperature, the mixture was
diluted with dichloromethane, washed with brine, dried over anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure, and the residue was purified by chromatography to give 8-(4-chloro-6-
methyl-quinolin-2-yl)-6,7,8,9-tetrahydro-5-thia-8-aza-benzocycloheptene 5,5-dioxide (15, 3.7 g, yield
99%), which was used for next step without further purification. MS obsd. (ESI+) [(M+H)+] 373.
A mixture of 4-(4-chloro-6-methylquinolin-2-yl)-8-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine 1,1-
dioxide (15, 100 mg, 0.27 mmol), sodium tert-butoxide (52 mg, 0.54 mmol), 1,1'-
bis(diphenylphosphino)ferrocene-palladium(II)dichloride
(22
mg,
0.027
mmol),
1,1'-
bis(diphenylphosphino)ferrocene (15 mg, 0.027 mmol) and 3-(aminomethyl)-N,N-dibenzyloxetan-3-amine
(76 mg, 0.27 mmol) in 1,4-dioxane (4 mL) was stirred at 120 ºC under microwave irradiation for 1 hour.
The resulting mixture was concentrated in vacuo. The residue was purified by preparative HPLC to afford
N-[[3-(dibenzylamino)oxetan-3-yl]methyl]-2-(1,1-dioxo-3,5-dihydro-2H-1lambda6,4-benzothiazepin-4-
yl)-6-methyl-quinolin-4-amine (16, 108 mg, yield 65%) as a white powder. MS obsd. (ESI+) [(M+H)+] 619.
A
mixture of N-[[3-(dibenzylamino)oxetan-3-yl]methyl]-2-(1,1-dioxo-3,5-dihydro-2H-1lambda6,4-
benzothiazepin-4-yl)-6-methyl-quinolin-4-amine (108 mg, 0.17 mmol), 10% palladium hydroxide on active
carbon (14 mg) in methanol (6 mL) was stirred for 16 hours at room temperature under hydrogen
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atmosphere (1 bar). The resulting mixture was concentrated in vacuo. The residue was purified by
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preparative
HPLC
to
afford
N-[(3-aminooxetan-3-yl)methyl]-2-(1,1-dioxido-2,3-dihydro-1,4-
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benzothiazepin-4(5H)-yl)-6-methylquinolin-4-amine (1, 13 mg, yield 17%). m.p. 229.3  229.7 C. MS
obsd. (ESI+) [(M+H)+] 439. HRMS calcd [(M+H)+] 439.17984, measured [(M+H)+] 439.17988. 1H NMR
(400 MHz, DMSO-d6) δ ppm 7.96 (d, J = 7.07 Hz, 1 H), 7.88 (dd, J = 1.26, 7.83 Hz, 1 H), 7.69 (s, 1H),
7.62 (dt, J = 1.14, 7.52 Hz, 1 H), 7.47 (t, J = 7.67 Hz, 1 H), 7.34 (d, J = 8.34 Hz, 1 H), 7.25 (dd, J = 1.52,
8.59 Hz, 1 H), 6.45 (br t, J = 5.56 Hz, 1 H), 6.20 (s, 1 H), 5.09 (br s, 2 H), 4.45 (d, J = 6.06 Hz, 2 H), 4.32
- 4.42 (m, 2 H), 3.63 (br t, J = 4.55 Hz, 2 H), 3.55 (d, J = 5.31 Hz, 2 H), 2.53 - 2.81 (m, 2 H), 2.36 (s, 3 H).
13C NMR (101 MHz, DMSO-d6) δ 155.6, 151.6, 146.5, 141.1, 137.7, 133.5, 133.3, 131.2, 129.9, 128.2,
127.5, 127.0, 120.5, 115.5, 85.5, 82.2, 57.0, 55.3, 51.9, 49.7, 45.0, 41.0, 40.9, 40.8, 21.4.
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For the following BAQ series, except described specifically, all the analogues are prepared in analogy
to 1 from the commercially available building blocks.
1-Amino-3-[[2-(3,4-dihydro-1H-isoquinolin-2-yl)-4-quinolyl]amino]propan-2-ol (2). MS obsd. (ESI+)
[(M+H)+] 349. 1H NMR (400 MHz, CD3OD) δ ppm 8.21 (d, J = 8.08 Hz, 1 H), 7.88 (d, J = 8.34 Hz, 1 H),
7.78 (t, J = 7.71 Hz, 1 H), 7.50 (t, J = 7.71 Hz, 1 H), 7.34 - 7.41 (m, 1 H), 7.28 - 7.34 (m, 3 H), 6.23 (s, 1
H), 4.88 - 4.93 (m, 2 H), 4.29 (br d, J = 6.06 Hz, 1 H), 3.96 (t, J = 5.81 Hz, 2 H), 3.60 - 3.78 (m, 2 H), 3.23
- 3.31 (m, 1 H), 3.16 (t, J = 5.81 Hz, 2H), 3.04 (br dd, J = 9.98, 12.25 Hz, 1 H).
3-[[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]amino]propane-1,2-diol (2a). MS obsd. (ESI+)
[(M+H)+] 350. 1H NMR (400 MHz, CDCl3) δ ppm 7.75 (br d, J = 8.31 Hz, 1 H), 7.60 (br d, J = 8.19 Hz, 1
H), 7.37 (br t, J = 7.70 Hz, 1 H), 7.27 - 7.31 (m, 1 H), 7.18 - 7.25 (m, 3 H), 7.11 (t, J = 7.57 Hz, 1 H), 5.69
(br s, 1 H), 4.77 (s, 2 H), 4.09 - 4.18 (m, 1 H), 3.84 - 3.95 (m, 3 H), 3.78 (dd, J = 5.26, 11.37 Hz, 1 H), 3.24
- 3.51 (m, 2 H), 3.01 (t, J = 5.75 Hz, 2 H).
N'-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]propane-1,3-diamine (2b). MS obsd. (ESI+)
[(M+H)+] 333. 1H NMR (400 MHz, CD3OD) δ ppm 8.22 (d, J = 8.38 Hz, 1 H), 7.88 (d, J = 7.82 Hz, 1 H),
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7.77 (t, J = 7.76 Hz, 1 H), 7.49 (t, J = 7.44 Hz, 1 H), 7.29 - 7.40 (m, 4 H), 6.11 (s, 1 H), 4.90 - 4.94 (m, 2
H), 3.96 (t, J = 5.93 Hz, 2 H), 3.71 (t, J = 7.03 Hz, 2 H), 3.10 - 3.31 (m, 4 H), 2.22 (quin, J = 7.37 Hz, 2 H).
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1-[[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]amino]propan-2-ol (2c). MS obsd. (ESI+)
[(M+H)+] 334. 1H NMR (400 MHz, CD3OD) δ ppm 7.97 - 8.13 (m, 1 H), 7.60 - 7.85 (m, 2 H), 7.45 (ddd,
J = 1.10, 7.11, 8.32 Hz, 1 H), 7.18 - 7.33 (m, 4 H), 6.12 (s, 1 H), 4.80 - 4.86 (m, 2 H), 4.15 (dt, J = 4.74,
6.67 Hz, 1 H), 3.89 (t, J = 5.95 Hz, 2 H), 3.41 - 3.55 (m, 2 H), 3.10 (t, J = 5.95 Hz, 2 H), 1.30 (d, J = 6.39
Hz, 3 H).
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N'-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]ethane-1,2-diamine (2d). MS obsd. (ESI+)
[(M+H)+] 319. 1H NMR (400 MHz, CD3OD) δ ppm 8.04 - 8.28 (m, 1 H), 7.84 - 7.99 (m, 1 H), 7.79 (dt, J
= 1.16, 7.73 Hz, 1 H), 7.51 (ddd, J = 1.16, 7.09, 8.25 Hz, 1 H), 7.29 - 7.37 (m, 4 H), 6.16 (s, 1 H), 4.90 -
4.93 (m, 2 H), 3.84 - 4.03 (m, 4 H), 3.35 - 3.43 (m, 2 H), 3.04 - 3.31 (m, 2 H).
N'-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]butane-1,4-diamine (2e). MS obsd. (ESI+)
[(M+H)+] 347. 1H NMR (400 MHz, CD3OD) δ ppm 8.22 (d, J = 7.91 Hz, 1 H), 7.87 (d, J = 8.19 Hz, 1 H),
7.77 (t, J = 7.78 Hz, 1 H), 7.42 - 7.56 (m, 1 H), 7.29 - 7.39 (m, 4 H), 6.07 (s, 1 H), 4.90 - 4.92 (m, 2 H),
3.95 (t, J = 5.93 Hz, 2 H), 3.63 (t, J = 6.60 Hz, 2 H), 3.12 - 3.31 (m, 2 H), 3.06 (br t, J = 7.27 Hz, 2 H), 1.83
- 1.99 (m, 4 H).
1-[2-[[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]amino]ethyl]guanidine (2f). The synthetic
1
procedure was described in the Supporting Information. MS obsd. (ESI+) [(M+H)+] 361. H NMR (400
MHz, CD3OD) δ ppm 8.12 (d, J = 8.31 Hz, 1 H), 7.82 (d, J = 8.31 Hz, 1 H), 7.72 (t, J = 7.64 Hz, 1 H), 7.44
(t, J = 7.58 Hz, 1 H), 7.25 - 7.34 (m, 4 H), 6.09 (s, 1 H), 4.86 (s, 2 H), 3.92 (t, J = 5.81 Hz, 2 H), 3.75 (br t,
J = 5.69 Hz, 2 H), 3.62 (br t, J = 5.62 Hz, 2 H), 3.11 (t, J = 5.75 Hz, 2 H).
2-[[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]amino]ethylurea (2g). The synthetic procedure was
described in the Supporting Information. MS obsd. (ESI+) [(M+H)+] 362. 1H NMR (400 MHz, CD3OD) δ
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ppm 8.00 (d, J = 8.19 Hz, 1 H), 7.76 (d, J = 8.31 Hz, 1 H), 7.67 (t, J = 7.70 Hz, 1 H), 7.19 - 7.41 (m, 5 H),
6.16 (s, 1 H), 4.86 (s, 2 H), 3.92 (t, J = 5.87 Hz, 2 H), 3.35 - 3.58 (m, 4 H), 3.09 (t, J = 5.81 Hz, 2 H).
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1-Amino-3-[(2-isoindolin-2-yl-4-quinolyl)amino]propan-2-ol (3). MS obsd. (ESI+) [(M+H)+] 335. H
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NMR (400 MHz, CD3OD) δ ppm 7.86 - 8.06 (m, 1 H), 7.68 - 7.77 (m, 1 H), 7.58 (s, 1 H), 7.38 - 7.46 (m,
2 H), 7.31 - 7.38 (m, 2 H), 7.23 - 7.29 (m, 1 H), 5.90 (s, 1 H), 4.89 - 4.97 (m, 4 H), 3.98 - 4.18 (m, 1 H),
3.43 - 3.58 (m, 2 H), 3.01 (dd, J = 3.73, 13.02 Hz, 1 H), 2.85 (dd, J = 7.95, 12.96 Hz, 1 H).
1-Amino-3-[2-(1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-quinolin-4-ylamino]-propan-2-ol (4). MS
obsd. (ESI+) [(M+H)+] 363. 1H NMR (400 MHz, CD3OD) δ ppm 8.05 - 8.03 (d, J = 7.6 Hz, 1 H), 7.78 -
7.76 (d, J = 7.6 Hz, 1 H), 7.72 - 7.68 (m, 1 H), 7.52 - 7.50 (m, 1 H), 7.44 - 7.40 (m, 1 H), 7.23 - 7.19 (m, 3
H), 6.12 (s, 1 H), 4.97 (s, 2 H), 4.16 - 4.11 (m, 3 H), 3.56 - 3.55 (d, J = 6 Hz, 2 H), 3.20 - 3.16 (dd, J = 2.8,
12.8 Hz,1 H), 3.11 - 3.08 (m, 2 H), 2.99 - 2.94 (m, 1 H), 1.99 - 1.98 (m, 2 H).
N-[2-(1,3,4,5-Tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine (5). MS obsd.
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(ESI+) [(M+H)+] 333. HRMS calcd [(M+H)+] 333.20737, measured [(M+H)+] 333.20677. H NMR (400
MHz, CD3OD) δ ppm 7.91 - 7.89 (m, 1 H), 7.62 - 7.60 (m, 1 H), 7.52 - 7.45 (m, 2 H), 7.21 - 7.12 (m, 3 H),
5.96 (s, 1 H), 4.10 (s, 2 H), 3.51 - 3.47 (t, J = 6.0 Hz, 1 H), 3.04 - 2.98 (m, 5 H), 2.84 (s, 2 H), 1.94 - 1.91
(m, 2 H).
N-[5-Chloro-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine (5a). MS
obsd. (ESI+) [(M+H)+] 367. 1H NMR (400 MHz, CD3OD) δ ppm 7.46 - 7.42 (m, 2 H), 7.27 - 7.23 (m, 1 H),
7.16 - 7.11 (m, 3 H), 7.02 (66, J = 7.6, 1.2 Hz, 1 H), 5.97 (s, 1 H), 4.79 (s, 2 H), 4.13 (br s, 2 H), 3.38 - 3.21
(m, 2 H), 3.04 - 3.02 (m, 2 H), 2.96 (t, J = 6.0 Hz, 2 H), 1.91 - 1.86 (m, 2 H).
N-[6-Chloro-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine (5b). MS
obsd. (ESI+) [(M+H)+] 367. 1H NMR (400 MHz, CD3OD) δ ppm 7.87 (d, J = 2.4 Hz, 1 H), 7.47 - 7.42 (m,
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2 H), 7.34 (dd, J = 9.0, 2.2 Hz, 1 H), 7.15 - 7.09 (m, 3 H), 5.99 (s, 1 H), 4.76 (s, 2 H), 4.11 (br s, 2 H), 3.38
- 3.32 (m, 2 H), 3.00 - 2.93 (m, 4 H), 1.87 (t, J = 5.2 Hz, 2 H).
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N-[7-Chloro-2-(1,3,4,5-tetrahydro-2-benzazepin-2-yl)-4-quinolyl]ethane-1,2-diamine (5c). MS obsd.
(ESI+) [(M+H)+] 367. 1H NMR (400 MHz, CD3OD) δ ppm 7.71 (d, J = 8.84 Hz, 1 H), 7.48 (d, J = 2.02 Hz,
1 H), 7.38 (d, J = 7.07 Hz, 1 H), 7.09 - 7.13 (m, 1 H), 7.02 - 7.07 (m, 2 H), 6.97 (dd, J = 2.02, 8.84 Hz, 1
H), 5.88 (s, 1 H), 4.66 (s, 2 H), 4.04 (br s, 2 H), 3.37 (s, 1 H), 3.29 - 3.31 (m, 1 H), 2.85 - 2.94 (m, 4 H),
1.80 (br s, 2 H).
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N-[8-Chloro-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine (5d). MS
obsd. (ESI+) [(M+H)+] 367. 1H NMR (400 MHz, CD3OD) δ ppm 7.24 (d, J = 8.0 Hz, 1 H), 7.60 (d, J = 6.8
Hz, 1 H), 7.55 (d, J = 7.6 Hz, 1 H), 7.13 - 7.09 (m, 3 H), 6.98 (t, J = 8.0 Hz, 1 H), 6.03 (s, 1 H), 4.89 (s, 2
H), 4.20 (br s, 2 H), 3.46 (t, J = 6.0 Hz, 2 H), 3.07 - 3.02 (m, 4 H), 1.92 (t, J = 5.2 Hz, 2 H).
N-[6-Methoxy-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine
(5e).
MS obsd. (ESI+) [(M+H)+] 363. 1H NMR (400 MHz, CD3OD) δ ppm 7.60 (d, J = 9.2 Hz, 1 H), 7.50 (d, J =
6.4 Hz, 1 H), 7.42 (d, J = 2.4 Hz, 1 H), 7.25 - 7.18 (m, 4 H), 6.01 (s, 1 H), 4.93 (s, 2 H), 4.13 (br s, 2 H),
3.89 (s, 3 H), 3.53 (t, J = 6.4 Hz, 2 H), 3.10 - 3.01 (m, 4 H), 1.96 (s, 2 H).
N-[6-Methyl-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2 diamine (5f). MS
obsd. (ESI+) [(M+H)+] 347. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.68 (s, 1 H), 7.55 (d, J = 6.8 Hz, 1 H),
7.29 (d, J = 8.4 Hz, 1 H), 7.21 (d, J = 6.8 Hz, 1 H), 7.13 - 7.05 (m, 3 H), 6.67 (br s, 1 H), 6.00 (s, 1 H), 4.79
(s, 2 H), 4.11 (br s, 2 H), 3.52 (s, 2 H), 3.05 - 2.99 (m, 4 H), 2.35 (s, 3 H), 1.77 (s, 2 H).
N-[6-Fluoro-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine (5g). MS
obsd. (ESI+) [(M+H)+] 351. 1H NMR (400 MHz, CD3OD) δ ppm 7.60 - 7.52 (m, 2 H), 7.47 (d, J = 6.8 Hz,
1 H), 7.26 - 7.21 (m, 1 H), 7.17 - 7.12 (m, 3 H), 6.04 (s, 1 H), 4.82 (s, 2 H), 4.15 (br s, 2 H), 3.45 (t, J = 6.4
Hz, 2 H), 3.06 - 2.99 (m, 4 H), 1.91 (t, J = 5.4 Hz, 2 H).
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