Discovery of RG7834: The First-in-Class Selective and Orally Available Small Molecule Hepatitis B Virus Expression Inhibitor with Novel Mechanism of Action

Article abstract of DOI:10.1021/acs.jmedchem.8b01245

Chronic hepatitis B virus (HBV) infection is a serious public health burden, and current therapies cannot achieve satisfactory cure rate. There are high unmet medical needs of novel therapeutic agents with differentiated mechanism of action (MOA) from the

Full text of DOI:10.1021/acs.jmedchem.8b01245

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Article
Discovery of RG7834: The First-in-Class Selective
and Orally Available Small Molecule Hepatitis B Virus
Expression Inhibitor with Novel Mechanism of Action
Xingchun Han, Chengang Zhou, Min Jiang, Yongguang Wang, Jianhua Wang,
Zhanling Cheng, Min Wang, Yongqiang Liu, Chungen Liang, Jianping Wang, Zhanguo
Wang, Robert Weikert, Wenzhe Lv, Jianxun Xie, Xin Yu, Xue Zhou, Souphalone
Luangsay, Hong C Shen, Alexander V Mayweg, Hassan Javanbakht, and Song Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01245 • Publication Date (Web): 04 Oct 2018
Downloaded from http://pubs.acs.org on October 4, 2018
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Discovery of RG7834: The FirstꢀinꢀClass
Selective and Orally Available Small Molecule
Hepatitis B Virus Expression Inhibitor with Novel
Mechanism of Action
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Xingchun Han, † Chengang Zhou, † Min Jiang, † Yongguang Wang, † Jianhua Wang, † Zhanling
Cheng,† Min Wang, † Yongqiang Liu, † Chungen Liang, † Jianping Wang, † Zhanguo Wang, † Robert
Weikert, ‡ Wenzhe Lv, & Jianxun Xie, & Xin Yu, ǂ Xue Zhou, ǂ Souphalone Luangsay, § Hong C. Shen, †
Alexander V. Mayweg, † Hassan Javanbakht, § Song Yang*, †
ǂ
Department of †Medicinal Chemistry, &Pharmaceutical Sciences, Discovery Virology, Roche
Innovation Center Shanghai, Roche Pharma Research and Early Development, Bld 5, 720 Cailun
Road, Shanghai, 201203 China,
‡
§
Department of Medicinal Chemistry, Discovery Virology, Roche Innovation Center Basel, Roche
Pharma Research and Early Development, Grenzacherstrasse 124, CHꢀ4070 Basel, Switzerland
Abstract: Chronic hepatitis B virus (HBV) infection is a serious public health burden and current
therapies cannot achieve satisfactory cure rate. There are high unmet medical needs of novel
therapeutic agents with differentiated mechanism of action (MOA) from the current standard of care.
RG7834, a compound from the dihydroquinolizinone (DHQ) chemical series, is a firstꢀinꢀclass highly
selective and orally bioavailable HBV inhibitor which can reduce both viral antigens and viral DNA
with a novel mechanism of action. Here we report the discovery of RG7834 from a phenotypic
screening and the structureꢀactivity relationship (SAR) of the DHQ chemical series. RG7834 can
selectively inhibit HBV but not other DNA or RNA viruses in a virus panel screening. Both in vitro
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and in vivo profiles of RG7834 are described herein, and the data support further development of this
compound as a chronic HBV therapy.
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Keywords: RG7834, chronic HBV, inhibition of HBsAg expression, inhibition of HBV DNA
production, dihydroquinolizinone (DHQ), viral gene expression, novel mechanism of action,
humanized mice, SAR
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1. Introduction
Hepatitis B virus (HBV) infection is a serious public health burden worldwide with over 250 million
people chronically infected, defined as hepatitis B surface antigen (HBsAg) positive. About one
quarter of the patients are likely to develop serious liver diseases such as cirrhosis and hepatocellular
carcinoma (HCC). It was reported that hepatitis B resulted in 887 000 deaths in 2015, mostly from
complications (including cirrhosis and HCC). 1
In clinics, a high level of HBsAg is considered as one of the hallmarks of chronic HBV infection.
HBsAg may contribute to exhaustion of antiviral CD8+ Tꢀcell2ꢀ5 and it negatively regulates HBVꢀ
specific immune responses.6ꢀ8 Persistence of HBsAg is the principal marker of risk for developing
chronic liver diseases and HCC later in life.1 HBsAg loss, which is defined as HBV “functional cure”
and subsequently seroꢀconversion with detectable antiꢀHBs antibody levels are considered an ideal
end point for HBV therapy.9, 10 As such, agents that reduce HBsAg levels will provide potential
therapies for chronic HBV infection and reduce the risk of developing severe liver diseases.
The current therapies for HBV infection include nucleos(t)ide analogues (lamivudine, tenofovir or
entecavir (ETV) etc.) and interferons (IFNs). Although these two types of therapies can reduce viral
load (HBV DNA), neither can reduce HBsAg levels effectively.11 Moreover, the nucleos(t)ides
therapies require a lifelong treatment in most patients and the interferon based therapy is poorly
tolerated. Thus, there are huge unmet medical needs to develop novel medicines with differentiated
mechanism of action (MOA) to improve the HBV cure rate.
In a previous report,12 we described the biological profile of RG7834, a novel, orally bioavailable
small molecule from dihydroquinolizinone (DHQ) chemical series which selectively reduced HBV
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antigens (both HBsAg and HBeAg) as well as HBV DNA both in vitro and in vivo. To the best of our
knowledge, this is the first small molecule which displayed significant HBsAg reduction in the HBVꢀ
infected human liver chimeric uPAꢀSCID mice model. RG7834 is highly selective for HBV, which
was demonstrated in an antiꢀviral assay against a panel of 15 DNA and RNA viruses including human
immunodeficiency virus(HIV) and hepatitis C virus (HCV). The mechanism is clearly different from
the current therapies like nucleos(t)ides, which have no significant effect on HBV antigens reduction.
Moreover, the MOA of RG7834 is different from the reported HBsAg inhibitors in recent years. For
example, HBꢀ0259 could inhibit HBsAg secretion but have no effect on HBV DNA or HBeAg
secretion.13, 14 A benzimidazole derivative, BM601, was another reported HBsAg inhibitor which
inhibited HBsAg secretion but not production. In addition, BM601 had no effect on HBV DNA
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16ꢀ18
replication and HBeAg secretion.15 A series of fluorine substituent nucleoside analogues (FNCs)
were reported as inhibitors of HBsAg, HBV DNA and HBeAg, but they were also active against HIV
and HCV, and not specific HBV inhibitors as RG7834.
O
N
OH
F
Cl
O
N
O
O
N
N
N
H
O
Cl
HBF-0259
RG7834
HO
N
OH
H
Cl
Cl
N
N
OH
N
N
O
N
O
N
N
N
3
HO
F
FNC analogue
BM601
Figure 1. Structures of RG7834 and reported HBsAg inhibitors
In this article, we report the discovery of RG7834 from a phenotypic screening and the preclinical
data supporting its progression for further development.
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2. Results and Discussion
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2.1. Primary SAR of Dihydroquinolizinones (DHQs)
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The research for novel HBsAg inhibitors started by performing a phenotypic highꢀthroughput
screening (HTS) of a library containing approximately one million compounds in a cellꢀbased assay
detecting the extracellular HBsAg level. From this screening, compound 1 with dihydroquinolizinone
(DHQ) scaffold was identified as a singleton hit. It reduced the level of secreted HBsAg and HBV
DNA with IC50 values of 0.5 ꢀM and 0.2 ꢀM, respectively, in HBVꢀexpressing HepG2.215 cells. To
confirm the antiviral activity, compound 1 was then evaluated in differentiated HepaRG (dHepaRG)
cells and showed inhibition of HBsAg expression with IC50 value of 0.88 ꢀM. Hence compound 1
was considered as a valid starting point.
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O
O
1
OH
3
11
8
O
10
N
O
9
6
1
We subsequently explore the structureꢀactivity relationship (SAR) with hit expansion utilizing the
analogues of compound 1 in the Roche collection. This effort led to the identification of compound
2−4 (Table 1), which showed that the substituents at C6, C9 and C10 had a significant impact on the
potency. Without the C6 methyl, compound 2 showed a ~5ꢀfold potency drop. Removal of the C10
methoxy resulted in a ~10ꢀfold potency reduction (3 vs 1), and further removal of the C9 methoxy (4)
resulted in a complete loss of antiꢀHBV activity.
With these observations we first synthesized several analogues with structure variations in the left part
(C8−C10 positions) and evaluated their antiꢀHBV activities. The results are summarized in Table 1. It
turned out that the activity was sensitive to the substituents at C9 and C10 positions. Small groups like
methyl and chloro at C10 position gave improved potency (5, 6 compared to 1), but with elongation or
size growth of the substituents, compounds 7-10 showed gradually reduced potency. At the C9
position, demethylation (11) or removal of the methoxy (12) led to a dramatic potency loss, while
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larger groups ethoxy and benzyloxy brought a 5ꢀfold enhancement of potency (13, 14 vs. 1). These
observations indicated that some large groups could be accommodated at the C9 position or may even
help to improve antiꢀHBV activity. In addition, a bromo at the C9 position was well tolerated (15 vs
3). Further derivatization of the bromo of 15 gave compounds 16, 17 and 18, in which the pyrrolidinyl
analogue 17 showed much improved potency with an IC50 of 0.08 ꢀM. Methylamino analogue 18
containing a ꢀNꢀH at the C9 position showed lower potency compared to that of 3, suggesting that a
hydrogen bond donor is not favored. Compared to 13, analogue 19 with a hydroxy at the C10 positon
showed much reduced antiꢀHBV activity. Combined with the C10 SAR findings mentioned above, the
observations suggested that a small hydrophobic group was preferred at the C10 position. We also
explored the C8 and C11 positions, and it seemed that these two positions were less sensitive to
substituents compared to C9 and C10, as the C8 chloro analogue 20 and C11 methoxy analogue 21
afforded similar potency as that of 3.
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Table 1. Effects on HBsAg Reduction of Analogs 1-24
IC50
(ꢀM)a
0.87
2.64
4.97
52
CC50
(ꢀM)b
>50
>50
>50
>50
>50
>50
>50
R1
R2
R3
R4
R5
ID
1
2
3
4
5
6
7
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀOMe
ꢀOMe
ꢀH
ꢀOMe
ꢀOMe
ꢀOMe
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀMe
ꢀH
ꢀMe
ꢀMe
ꢀMe
ꢀMe
ꢀMe
ꢀH
ꢀMe
ꢀCl
ꢀOMe
ꢀOMe
ꢀOMe
0.24
0.29
1.2
ꢀOEt
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ꢀH
ꢀH
ꢀH
ꢀOMe
ꢀOMe
ꢀOMe
ꢀH
ꢀH
ꢀH
ꢀMe
ꢀMe
ꢀMe
1.98
29.
>50
>50
>50
8
ꢀOBn
9
>50
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ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀOMe
ꢀOMe
ꢀOMe
ꢀOMe
ꢀH
ꢀOH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀMe
ꢀMe
ꢀMe
ꢀMe
ꢀMe
7.24
7.67
0.097
0.10
4.75
>50
>50
>50
>50
>50
11
12
13
14
15
ꢀOEt
ꢀOBn
ꢀBr
ꢀH
ꢀH
ꢀH
ꢀMe
0.58
>50
16
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀMe
ꢀMe
0.081
8.99
>50
>50
17
18
ꢀH
ꢀOH
ꢀH
ꢀOEt
ꢀH
ꢀCl
ꢀH
ꢀMe
ꢀMe
ꢀMe
1.86
5.03
4.56
>50
>50
>50
19
20
21
ꢀH
ꢀOMe
ꢀOMe
ꢀOMe
ꢀH
ꢀH
ꢀH
ꢀH
ꢀOMe
ꢀOMe
ꢀOMe
ꢀOMe
ꢀOMe
ꢀOMe
ꢀH
ꢀH
ꢀH
0.11
0.049
10.9
>50
>50
>50
22
(R)-22
(S)-22
ꢀH
ꢀH
ꢀOMe
ꢀOMe
ꢀOMe
ꢀOMe
ꢀH
ꢀH
0.068
0.052
>50
>50
23
24
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a IC50 is the geometric mean value for reduction of HBsAg by 50% in HepG.2.2.15 cells. bCC50 is the geometric
mean value for cytotoxicity measured by CCKꢀ8 in HepG.2.2.15 cells.
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In parallel to the SAR exploration in the left part, we also synthesized compounds 22-24 (Table 1) to
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explore the tolerability of larger alkyl groups at the C6 position. It was encouraging to see that
compound 22 showed a ~5ꢀfold increase in potency compared to 1. Then, in order to further
understand the antiꢀHBV activity of this new scaffold, we conducted chiral separation of 22 by
supercritical fluid chromatography (SFC), and the two enantiomers showed marked difference in antiꢀ
HBV activities. The absolute configuration of (S)ꢀ22 was confirmed by single crystal Xꢀray studies
(see the Supporting Information), and optical rotation was measured. In the crystal structure, an
intramolecular hydrogen bond is formed between the carboxyl group with the carbonyl at the C2
position. The enantiomer (R)ꢀ(+)ꢀ22 was active in HepG2.2.15 with an IC50 value of 0.049 ꢀM, while
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the enantiomer (S)ꢀ(ꢀ)ꢀ22 was much less active with an IC50 value of 10.9 ꢀM. Propyl and isobutyl
substituents (23, 24) showed slightly better potency than 22.
2.2. Further SAR exploration at the C9 and C10 positions
Having identified the favorable substituents in both the left part and the C6 position, and to quickly
improve the antiꢀHBV activity in this series, we decided to prepare compounds with combination of
these substituents. The results are shown in Table 2. To our delight, the combination the C9
benzyloxy and C6 ethyl (25) led to an IC50 value of 0.039 ꢀM, ~2 fold more potent than that of 14 and
22. Then, consistent with the SAR finding, compound 26 also showed a ~2ꢀfold increase in potency
compared to that of 13 and 22.
Table 2. Further SAR at the C9 and C10 Positions: Effects on HBsAg Reduction and
Physicochemical Properties of 25-52
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ID
R2
R3
IC50
CC50
LYSA
MLM
HLM
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(ꢀM)a
0.039
0.043
(ꢀM)a
>50
(ꢀg/mL)b
<1.0
(ꢀL/min/mg)c
(ꢀL/min/mg)d
ꢀOMe
ꢀOMe
ꢀOBn
ꢀOEt
3.6
5.3
1.4
1.3
25
26
>50
25.0
8
9
ꢀOMe
ꢀOMe
0.020
0.018
>50
>50
36.0
15.0
2.3
5.7
0.33
0
27
28
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29
30
ꢀOMe
ꢀOMe
0.013
0.015
>50
>50
3.0
3.0
2.5
1.6
1.3
4.8
ꢀOMe
ꢀOMe
0.043
0.059
20.7
>50
<1.0
67.9
10.7
31
32
12
36
3.8
0.23
1.7
33
34
ꢀOMe
ꢀOMe
0.090
0.37
>50
>50
0.63
11.0
17.0
16.7
1.9
4.2
6.7
ꢀOMe
ꢀCN
0.33
1.94
0.67
0.024
>50
>50
>50
>50
35
36
37
38
ꢀ
ꢀ
ꢀ
ꢀ
ꢀEt
ꢀOMe
10.0
9.0
22.2
23.6
2.5
1.5
ꢀOMe
0.013
>50
39
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ꢀOMe
ꢀOMe
26.1
3.9
>50
>50
ꢀ
ꢀ
ꢀ
40
41
>470
1.9
0
10
11
12
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ꢀOMe
ꢀOMe
5
>50
>50
36
3.4
2.3
0
42
43
0.18
>470
3.3
ꢀOMe
ꢀOMe
3.1
>50
>50
>510
>535
6.5
3.1
3.2
3.0
44
45
0.16
ꢀOMe
ꢀOMe
ꢀOMe
ꢀOMe
0.56
0.2
>50
>50
>50
>50
172
101
225
63.5
3.5
9.5
0
8.7
1.8
2.4
2.5
46
47
48
49
0.064
0.019
4.0
50
ꢀOMe
0.22
>50
208
4.6
0.096
ꢀOMe
ꢀOMe
0.028
0.056
>50
>50
71
97
2.0
0
51
52
0.97
0.72
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aDefinitions are the same as those described in Table 1. bLyophilization solubility assay (LYSA) (ꢁg/mL).
cMouse liver microsome (MLM) (mL/min/kg). dHuman liver microsome (HLM).
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As C9 and C10 substituents showed marked impact on antiꢀHBV activity, especially C9 substituent
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was indicated as an important moiety which potentially could help to improve the activity, we then
extensively explored the SAR at these two positions, with fixed ethyl group at C6. Based on 26,
elongation of the ethoxy to propoxy resulted in 2ꢀfold increase in potency (27 vs 26), but further
elongation to butoxy did not give appreciable improvement (28 vs 27). Interestingly, still with four
carbon at C9, isobutoxy (29) and cyclopropylmethoxy (30) afforded improved potency with IC50
values of 0.013 ꢀM and 0.015 ꢀM. Larger substituent (31) or difluoroethoxy (32) were also well
tolerated, but isopropoxy group resulted in 2ꢀfold potency drop (33 vs 26), suggesting that steric bulk
close to C9 oxygen was not favored. Replacement of the C9 ethoxy with propyl or cyano group, or
cyclization of C9 and C10 substituents led to significant potency drop (34-37 vs 26). At C10 position,
considering the SAR observations mentioned above, we replaced the methoxy with ethyl (38) and
cyclopropyl (39). The two compounds were very active against HBsAg in HepG2.2.15 cells with
IC50 values of 0.024 ꢀM and 0.013 ꢀM, respectively.
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
The aboveꢀdiscussed SAR study indicated that appropriate hydrophobic groups at C6, C9 and C10
positions helped to dramatically improve the antiꢀHBV activity in HepG2.2.15 cells. However,
combination of these hydrophobic groups also led to unfavorable physiochemical properties. For
example, although analogue 29 and 30 demonstrated >2ꢀfold increase in potency compared to that of
26, they also showed significantly reduced aqueous solubility in lyophilization solubility assay
(LYSA). Moreover, the potent analogue 39 showed not only reduced aqueous solubility, but also
reduced microsomal stability compared to that of 26. Therefore, in an attempt to improve the
physiochemical properties, we explored the tolerability of solubilizing groups at C9 position. The
results showed that amide (40), reversed amide (41) and methanesulfonamide (42) at terminal of the
C9 side chain were not tolerated, but without –NꢀH, lactam analogue gave better activity (43 vs 41).
Pyrroline (44) brought very high aqueous solubility (LYSA > 510 ꢀg/mL), but also led to dramatic
potency drop. Compared to 44, morpholine analogue 45 with lower basicity showed higher antiꢀHBV
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5
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7
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9
activity while keeping high solubility. With a terminal hydroxy group, analogue 46 and 47 showed
good solubility but moderate antiꢀHBV activity. Notably, ether side chain analogues 48–52 appeared
to give balanced properties. Among these modifications, methoxypropoxy analogue 49 demonstrated
better antiꢀHBV activity and higher aqueous solubility than that of 26, which increased the potential
for further optimization.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.3. SAR study at the C3 position
Table 3. SAR study at C3 position: Effects on HBsAg Reduction of Compounds 53-63,
compared with Compound 25
IC50
CC50
(ꢀM)a
>50
ID
R
(ꢀM)a
0.039
>50
ꢀOH
25
53
ꢀNH2
>50
19
>50
>50
54
55
>50
>50
>50
56
6.4
43
>50
>50
57
58
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5
6
7
8
13
>50
>50
59
60
6.6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.52
19
>50
48.7
>50
61
62
63
0.98
aDefinitions are the same as those described in Table 1.
As part of our SAR study, we also synthesized a number of compounds to replace the C3 carboxylic
acid. The results in Table 3 showed that replacement of the carboxylic acid with primary amide (53)
or tertiary amides (55, 56) led to a complete loss of activity. Secondary amides 57–59 afforded weak
activity in HepG2.2.15 cells. A methyl group adjacent to the amide nitrogen was beneficial to
potency (60 vs 58, 61 vs 59). Meanwhile, bioisostere replacement of the C3 carboxylic acid was
explored as well. Hydroxamic acid analogue 62 was only weakly active with an IC50 of 19 ꢀM, while
the acylsulfonamide analogue 63 maintained a moderate IC50 of 0.98 ꢀM.
2.4. Further SAR at the C6 position
Table 4. Further SAR at the C6 position: Effects on HBsAg Reduction and Physicochemical
Properties of Compounds 64-77, Compared with Compound 49
MLM
HLM
ID
R5
IC50
CC50
LYSA
(mL/min/kg) a (mL/min/kg) a
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(ꢀM)a
(ꢀM)a
(ꢀg/mL) a
4
5
6
7
8
9
9.4
5.0
13
9.3
0
1.9
49
64
0.019
0.009
0.003
3.15
>50
63.5
71
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
>50
>50
>50
>50
>50
1.1
(S)-64
82
3.7
(RG7834)
(R)-64
65
85
0.77
0.63
0.03
0.001
0.002
5.0
10
14.
66
0.039
0.074
>50
>50
119
192
0.92
8.4
0.76
4.6
67
68
0.005
0.13
>50
>50
137
195
6.2
6.3
0
69
70
0.91
OH
OH
0.006
0.002
>50
>50
51
8.6
0
0.75
0
71
>515
(S)-71
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6
7
8
OH
(R)-71
0.088
0.010
>50
>50
>550
ꢀ
0
0
ꢀ
ꢀ
72
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.064
0.80
>50
>50
>50
7.0
5.5
0
73
74
75
6.0
ꢀ
ꢀ
0.092
<1.0
20
6.2
0.010
0.003
>50
>50
1.0
5.0
10
10
0
76
77
1.1
aDefinitions are the same as those described in Table 2.
Finally, we carried out further SAR exploration at the C6 position with fixed methoxypropoxy at C9
as shown in Table 4. Growth of the C6 substituent into isopropyl led to > 2ꢀfold increase in potency
(64 vs 49) with an IC50 value of 0.009 ꢀM. It was also notable that compound 64 showed comparable
solubility with that of 49. Racemic compound 64 was then separated by SFC, and the absolute
configuration was confirmed by the Xꢀray study. Consistent with the enantioselective antiꢀHBV
activity observed on the two enantiomers of 22, the enantiomer (S)ꢀ(+)ꢀ64 (RG7834, Figure 2) had
higher potency with an IC50 value of 0.003 ꢀM. Bulkier substituent analogues 65 and 66 were the
most potent ones with an IC50 values of 0.001 ꢀM and 0.002 ꢀM, respectively, without chiral
separation. However, unfortunately, the increased hydrophobicity also led to solubility reduction.
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5
6
7
8
9
Trifluoroethyl (67) and alkoxy (68) were tolerated, and with gemꢀdimethyl adjacent to the C6
position, alkoxy compound 69 showed much improved potency and solubility compared to compound
49. Addition of a hydroxyl group on the C6 substituent of 49 resulted in potency drop, but extended
hydroxyl analogue 71 had similar potency as compound 49. Moreover, the enantiomer (S)ꢀ(+)ꢀ71
showed very high aqueous solubility (LYSA > 515 ꢀg/mL). C6 aromatic substituents (71ꢀ76) were
also explored, and thienyl 70 and 71 showed a high potency. Not surprisingly, the solubility of the C6
aromatic analogues was generally low.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Xꢀray structure of RG7834
2.5. In vitro anti-HBV profile and selectivity of RG7834 and molecular targets for DHQ series
Besides the potent activity in inhibition of HBsAg expression, RG7834 also displayed a very potent
activity in inhibition of HBV DNA production in HepaG2.2.15 cells with an IC50 < 0.13 nM while the
IC50 of the nucleoside agent lamivudine (3ꢀTC) was around 25 nM.
In HBVꢀinfected dHepaRG cells, RG7834 inhibited HBsAg, HBeAg and HBV DNA at a single
digital nM concentration, while (R)ꢀ64, the enantiomer of RG7834, did not show significant inhibition
against all the three viral markers at concentration up to 1 ꢀM (Table 5). The nucleoside drug ETV
showed very potent activity in inhibition of HBV DNA production with an IC50=0.06 nM but no
activity against HBsAg and HBeAg.12 The different antiviral profile between RG7834 and ETV
indicated a differentiated MOA of RG7834 compare with nucleoside drugs.
Table 5. Antiviral activity of RG7834, (R)-64 and ETV in HBV-infected dHepaRG cells
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IC50 (nM)
4
Compound
5
6
7
8
HBsAg
2.8
HBeAg
2.6
HBV DNA
RG7834
(R)ꢀ64
ETV
3.2
>1000
0.06
9
>1000
>1
>1000
>1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To evaluate the antiviral selectivity of RG7834, the compound was tested in a panel of 15 different
DNA and RNA viruses. The IC50 of RG7834 against all these viruses was higher than 10 ꢀM which
demonstrated the high specificity of RG7834 against HBV.12
To get better understanding of the MOA, we tried different approaches to identify the molecular
target(s) for DHQ chemical series. Finally, the nonꢀcanonical poly(A) RNA polymerases, PAPꢀ
associated domainꢀcontaining protein 5 and 7 (PAPD5 and PAPD7) were identified as protein targets
for DHQ compounds. These two proteins had not been reported previously as targets for HBV
therapy. The detailed information about the targets will be reported separately.
2.6. DMPK, Early Safety and in vivo Efficacy Assessment of RG7834
RG7834 was selected for further physicochemical and ADME characterization based on its favorable
antiꢀHBV activity, cytotoxicity, solubility and liver microsome stability. With favorable measured
logD value of 1.28 and pKa value of 5.79 (acidic), RG7834 showed very good permeability with a
P
app(AꢀB) of 12.8 × 10−6 cm s−1 and Papp ratio of 1.3 in Cacoꢀ2 assay. The unbound fractions of
RG7834 in human and mouse plasma were determined to be 32.8% and 35.2%, respectively. The
mouse single dose pharmacokinetics (SDPK) profile of RG7834 was evaluated in male BALB/C mice
following intravenous (i.v.) and oral (p.o.) administration. The results are summarized in Table 6.
RG7834 had moderate plasma clearance (Cl) (41.9 mL min−1 kg−1) and good oral bioavailability (F)
(62%) in mice. RG7834 also demonstrated satisfactory oral exposure, and particularly, good liver
exposure which was 4ꢀfold higher than that in plasma. Although we did not have free liver drug
concentration, the reasonably high total concentration of RG7834 in liver was considered as a
desirable attribute for antiꢀHBV drugs because liver is the target organ in chronic HBV infections.
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Table 6. Single-Dose Pharmacokinetics of RG7834 in micea
4
5
6
7
8
9
dose CL
Vss
t1/2
Plasma
Cmax
Plasma
AUC(0ꢀ∞)
(h⋅ng/mL)
398
F
Plasma
Liver
Liver
AUC(0ꢀt) ratio
(mg/ (mL/m (L/k (h)
(%)
AUC(0ꢀt)
(h⋅ng/mL)
Cmax
AUC(0ꢀt)
(h⋅ng/mL)
(Liver/Plasma)
kg)
in/kg)
41.9
g)
(ng/mL)
(ng/mL)
i.v.
p.o.
po
1
2.07 0.71 466
1.48 322
2037
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
490
62
14.5
4366
10987
17265
4.0
a The singleꢀdose pharmacokinetics (SDPK) study of RG7834 was carried out in male BALB/C mice according
to standard procedures. Major parameters, including plasma clearance (CL), volume of distribution at steady
state (Vss), t1/2, maximal concentration (Cmax), area under the curve (AUC), and oral bioavailability (F) are
reported.
The cynomolgus monkey SDPK study of RG7834 was conducted subsequently, and it showed low
plasma clearance (4.6 mL min−1 kg−1) following intravenous administration. RG7834 also showed
good oral bioavailability (57%) with a halfꢀlife of 4.9 hours following oral administration.
Simultaneously we also assessed the in vitro safety profile of RG7834. In the in vitro drugꢀdrug
interaction (DDI) assessment, RG7834 showed IC50 values of >50 ꢀM against CYP3A4, CYP2D6 and
CYP2C9. It had no time dependent inhibition (TDI) issue and no CYP induction issue. Moreover,
RG7834 demonstrated an excellent in vitro toxicology profile, showing no inhibition of hERG
channel, no flag for glutathione adduction (GSH), and clean in Ames assay (mutagenicity) and
micronucleus test (MNT, clastogenicity).
Figure 3. Effect of RG7834 and ETV on HBV viral markers in HBVꢀinfected Human liver chimeric uPAꢀSCID
mice. HBV DNA, HBsAg and HBeAg were determined in serum from HBVꢀinfected uPA/SCID mice during
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the 21 days’ treatment and followꢀup period up to 35 days. (figure reprinted from Journal of Hepatology12 with
permission)
6
7
The in vivo antiꢀHBV efficacy was evaluated in HBVꢀinfected human liver chimeric uPAꢀSCID mice
8
9
with ETV as a control. After 21 days of treatment with 4 mg/kg twice daily dosing, RG7834 reduced
both HBsAg and HBeAg significantly while ETV did not show clear efficacy on the reduction of the
two antigens. RG7834 also reduced serum HBV DNA by 0.6 log10 with 4 mg/kg treatment and ETV
showed more pronounced HBVꢀDNA reduction (Figure 3). The in vivo results are consistent with the
in vitro observation, and the detailed biological results were reported recently.12
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3. Chemistry.
The synthesis of these molecules was carried out as shown in the various schemes. A general
synthetic route for the C3 carboxylic acid DHQs is shown in Scheme 1. The Henry reaction of
aldehyde G1 and nitroalkane G2 afforded nitroalkene G3. Reduction of G3 by LiAlH4 gave a key
intermediate amine G4. This intermediate could also be prepared by another route, namely, the
coupling reaction of bromide G5 with ketone G6 to afford G7, which then underwent reductive
amination to afford amine G4. Subsequently, the treatment of G4 with formic acid generated
formamide G8, which then underwent BischlerꢀNapieralski cyclization to produce 3,4ꢀ
dihydroisoquinoline G9. The further cyclization employing ethyl 2ꢀ(ethoxymethylene)ꢀ3ꢀoxoꢀ
butanoate G10 yielded diastereoisomers G11. Ester G12 was obtained by oxidation of G11 mixture
with pꢀchloranil. Finally, basic hydrolysis afforded desired acid G13. Although this synthetic route
was employed for the synthesis of most of the analogues, other routes for specific analogues will be
given in the Support Information.
Scheme 1
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G2
R5CH2NO2
5
6
LiAlH4, THF
AcONH
7
G1
G3
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pd2(dba)3, Xantphos
base, solvent
G4
NH4OAc , NaBH3CN
HCOOH, solvent
G5
G7
G6
POCl3, solvent
G10
G8
G9
G11
p-chloranil
solvent
LiOH, H2O, solvent
G12
G13
During exploration of the C9 position, another synthetic route was used to quickly introduce various
substituents (Scheme 2). Deꢀprotection of 25 afforded phenol G14, which underwent substitution
reaction to give ester G16. Lithium hydroxide hydrolysis produced desired acid G17.
Scheme 2
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3
4
5
6
7
Pd/C, H2, solvent
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25
G14
R6-Q
G15
K2CO3
Q is Cl, Br, I, O-mesyl or O-tosyl
LiOH, H2O
G17
G16
In addition, analogues 53ꢀ63 were prepared using analogue 25 as starting material as shown in
Scheme 3. Coupling reaction of 25 with amine G18 in the presence of HATU afforded desired amide
G19.
Scheme 3
G18
HATU, Et3N, DMF
25
G19
4. Conclusion
In summary, we have discovered DHQ chemical series from a phenotypic screening as a novel class
of HBV inhibitors with differentiated MOA from current therapies. PAPD5 and PAPD7 were
identified as molecular targets for this chemical series. The SAR and structureꢀproperty relationship
(SPR) of DHQ series were explored systematically. Of the two enantiomers, one is much more potent
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than the other and the absolute configuration of two compounds (RG7834 and (S)ꢀ22) was established
by Xꢀray crystal structures.
6
7
8
9
The representative lead compound RG7834 displayed potent (single digital nM IC50) inhibitory
activity against HBsAg, HBeAg and HBV DNA in HBVꢀinfected hepatocytes while no cytotoxicity
was observed up to 50 ꢀM. Moreover, RG7834 is highly specific for HBV showing no inhibitory
activity against other 15 viruses in a panel screening. In addition to favourable physicoꢀchemical and
in vitro/in vivo DMPK properties, RG7834 also displayed a favourable in vitro safety profile. This
compound was well tolerated in preclinical in vivo toxicology studies including a 4ꢀday in vivo
toxicology study in rats, and 4ꢀweek GLP studies in rat and monkey (data not shown). Furthermore,
RG7834 demonstrated unprecedented PD efficacy in the human liver chimeric uPAꢀSCID mouse
model. All the data together supported moving RG7834 forward into clinical investigation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
Synthetic Chemistry General Comments. All of the intermediates were purified by silica gel
chromatography using either a Biotage or an ISCO CombiFlash chromatography instrument. All of
the final compounds were purified by preparative HPLC (prepꢀHPLC) on a reversedꢀphase column
using a Waters XBridge OBD Phenyl (30 mm × 100 mm, 5 ꢁm) or OBD RP18 (30 mm × 100 mm, 5
ꢁm) column under acidic conditions (A, 0.1% formic acid in H2O; B, 0.1% formic acid in acetonitrile)
or basic conditions (A, 0.1% ammonia in H2O; B, acetonitrile). For SFC chiral separation, the
intermediates were separated using a chiral column (Daicel Chiralpak IC, 30 mm × 250 mm, 5 ꢁm) on
a Mettler Teledo SFCꢀMultigram system (solvent system of 95% CO2 and 5% IPA (0.5% TEA in
IPA), backpressure of 100 bar, UV detection at 254 nm). NMR spectra were obtained using Bruker
AVANCE 400 MHz spectrometer, operating at 400.13 MHz (1H) and 100.62 MHz (13C). LC−MS
spectra were obtained using a MicroMass Platform LC (Waters Alliance 2795ꢀZQ2000). Optical
rotation was measured using a Rudolph Autopol V automatic polarimeter at a wavelength of 589 nm.
Highꢀresolution mass spectra were obtained on Xevo G2ꢀXSꢀQTOF mass spectrometer (Waters,
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5
6
7
8
9
Manchester, U.K.) equipped with an electrospray ionization source. All of the starting materials were
obtained commercially. 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.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General Procedure for the Preparation of G4. Method A. A mixture of aldehyde G1(12.4 mmol)
and ammonium acetate (12.4 mmol) in toluene (40 mL) was refluxed with a DeanꢀStark trap for 2
hours. Then G2 (62 mmol) was added and the resultant mixture was refluxed for additional 36 hours.
The mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate
(100 mL). The resultant solution was washed with water (60 mL), and then dried over anhydrous
Na2SO4 and then concentrated. The residue was purified by column chromatography to give G3.
To a mixture of LiAlH4 (30 mmol) in THF (15 mL) was added a solution of G3 (10 mmol) in
THF (20 mL) dropwise in an iceꢀwater bath. The mixture was refluxed for 6 hours and then stirred at
o
room temperature for additional 16 hours. Then water (1.1 g) was added dropwise at 0 C, and then
followed by addition of 15% NaOH aqueous solution (1.1 mL) and water (3.3 mL). The resultant
mixture was filtered, and the filtrate was concentrated to afford crude G4 which was used in the next
step without further purification.
Method B. To a mixture of G5 (80 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.8
mmol), 9,9ꢀdimethylꢀ4,5ꢀbis(diphenylphosphino)xanthene (1.6 mmol) and tꢀBuONa (264 mmol) in
o
THF (200 mL) was added G6 (240 mmol). The resultant mixture was heated to 50 C for 3 hours
under argon atmosphere, and then cooled to room temperature and filtered. The filtrate was
concentrated under reduced pressure. The residue was purified by column to afford G7 as brown oil
which was directly used for the next step without further purification.
To a mixture of G7 (57.4 mmol) and ammonium acetate (861 mmol) in CH3OH (160 mL) was
added NaBH3CN (7.23 g, 114.8 mmol) in portions at 0 oC. The resultant mixture was stirred from 0 oC
to room temperature overnight and then basified by 2 M NaOH aqueous solution to pH 12ꢀ14. The
mixture was extracted with CH2Cl2 (300 mL x 3), and the combined organic layers were washed with
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brine, and then dried over anhydrous Na2SO4 and then concentrated to give G4 as yellow oil which
was directly used for the next step without purification.
6
7
General Procedure for the Preparation of G8. A solution of G4 (54.4 mmol) and formic acid (1
8
9
o
mL) in ethyl formate (100 mL) was heated at 90 C overnight, and then concentrated to give G8 as
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yellow oil which was directly used for the next step without purification.
General Procedure for the Preparation of G9. To a solution of G8 (52 mmol) in acetonitrile (150
o
mL) was added POCl3 (62.4 mmol). The resultant mixture was heated at 90 C for 2 hours and then
concentrated. The residue was dissolved in acetonitrile (50 mL), and then cooled to 0oC. Ammonium
hydroxide was added dropwise at 0 oC to basify the mixture. The mixture was extracted with CH2Cl2
(200 mL x 5), and the combined organic layers were washed with brine, and then dried over
anhydrous Na2SO4 and then concentrated to give crude G9 as darkꢀgreen oil which was directly used
for the next step without purification.
General Procedure for the Preparation of G11. A mixture of G9 (46 mmol) and ethyl 2ꢀ
o
(ethoxymethylene)ꢀ3ꢀoxoꢀbutanoate G10 (138 mmol) in EtOH (150 mL) was heated to 100 C
overnight. The mixture was concentrated, and the residue was purified by flash column
chromatography to afford G11 as red oil which was directly used in the next step without further
purification.
General Procedure for the Preparation of G12. A mixture of G11 (25 mmol) and pꢀchloranil (25
mmol) in DME (100 mL) was heated to 70 oC for 3 hours under argon atmosphere. Removed the
solvent and the residue was dissolved in CH2Cl2 (150 mL) and then washed with saturated NaHCO3
aqueous solution (100 mL x 5). The separated organic layer was washed with water and brine, and
then dried over anhydrous Na2SO4, and then concentrated. The residue was purified by flash column
chromatography to afford G12 as brown oil.
General Procedure for the Preparation of G13. To a mixture of G12 (4.8 mmol) in CH3OH (32
mL) and H2O (8 mL) was added lithium hydroxide monohydrate (19.2 mmol). The resultant reaction
mixture was stirred at room temperature for 3 hours. The mixture was diluted with CH2Cl2, and then
acidified by 1 M hydrochloric acid to pH 2ꢀ3, and then extracted with CH2Cl2 (100 mL x 3). The
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combined organic layers were washed with brine, and then dried over anhydrous Na2SO4 and then
concentrated. The residue was washed with EtOH/Et2O to afford G13 as a white or yellow solid.
General Procedure for the Preparation of G14. A mixture of ethyl 9ꢀbenzyloxyꢀ6ꢀethylꢀ10ꢀ
methoxyꢀ2ꢀoxoꢀ6,7ꢀdihydrobenzo[a]quinolizineꢀ3ꢀcarboxylate (25, 5.2 g) and 10% palladium on
carbon (300 mg) in THF/MeOH (1/1, 40 mL) was stirred under hydrogen atmosphere for 12 hours.
The mixture was filtered through celite pad, and filtrate was concentrated under reduced pressure to
afford G14 as yellow solid (4.2 g).
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General Procedure for the Preparation of G16. To a solution of G14 (0.29 mmol) in DMF was
o
added G15 (0.87 mmol) and K2CO3 (0.58 mmol). The mixture was stirred for 2 hours at 80 C, and
then cooled to room temperature and filtered. The filtrate was concentrated in vacuo to give crude
G16 which was used for the next step without further purification.
General Procedure for the Preparation of G17. To a solution of crude G16 (0.25 mmol) in MeOH
(9 mL) and water (3 mL) was added LiOH.H2O (36.7 mg). The mixture was stirred for 2 hours and
then concentrated under reduced pressure. The residue was dissolved in water (5 mL), and then
acidified with 6 M hydrochloric acid and filtered. The filter cake was dried in vacuo to give G17.
General Procedure for the Preparation of G19. To a solution of 9ꢀbenzyloxyꢀ6ꢀethylꢀ10ꢀmethoxyꢀ
2ꢀoxoꢀ6,7ꢀdihydrobenzo[a]quinolizineꢀ3ꢀcarboxylic acid (25 0.2 mmol) in DMF (10 mL) was added
HATU (0.3 mmol), triethylamine (100 µL) and amine G18 (2.2 mmol). The resultant solution was
stirred at room temperature for 3h and then purified by preparative HPLC to afford G19.
For the Following Compounds, Except Described Specifically, All the Analogues Are Prepared
in Analogy to General Procedure from the Commercially Available Building Blocks.
1
9,10-Dimethoxy-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (1). H NMR
(400 MHz, DMSOꢀd6): δ 8.83 (s, 1H), 7.54 (s, 1H), 7.47 (s, 1H), 7.02 (s, 1H), 4.95 (m, 1H), 3.89 (s,
3H), 3.86 (s, 3H), 3.40ꢀ3.28 (m, 1H), 2.91(dd, 1H), 1.20 (d, 3H). MS obsd. (ESI+) [(M+H)+]: 316.
1
9-Methoxy-6,10-dimethyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (5). H NMR
(400 MHz, DMSOꢀd6): δ 8.83 (s, 1H), 7.91 (s, 1H), 7.30 (s, 1H), 6.99 (s, 1H), 4.96 (m, 1H), 3.89 (s,
3H), 3.39 (dd, 1H), 2.95 (d, 1H), 2.21 (s, 3H), 1.20 (d, 3H). MS obsd. (ESI+) [(M+H)+]: 300.
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10-Chloro-9-methoxy-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (6). H
NMR (400MHz, CDCl3): δ 8.29 (br. s, 1H), 7.51 (s, 1H), 6.75 (s, 1H), 6.61 (br. s, 1H), 4.25 ꢀ 4.18 (m,
1H), 3.55 (s, 3H), 3.07 ꢀ 2.97 (m, 1H), 2.62 ꢀ 2.50 (m, 1H), 0.90 (br. s, 3H). MS obsd. (ESI+)
[(M+H)+]: 320.
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10-Ethoxy-9-methoxy-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (7). H
NMR (400 MHz, CD3OD): δ 8.80 (s, 1H), 7.45 (s, 1H), 7.29 (s, 1H), 7.02 (s, 1H), 4.18 (m, 2H), 3.95
(s, 3H), 3.43 (m, 2H), 2.90 (m, 1H), 1.47 (t, 3H), 1.35 (d, 3H). MS obsd. (ESI+) [(M+H)+]: 330.
9-Methoxy-6-methyl-2-oxo-10-propoxy-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (8). 1H
NMR (400 MHz, CDCl3): δ 8.56 (s, 1H), 7.22 (s,1H), 7.10 (s, 1H), 6.77 (s, 1H), 4.56 (m, 1H), 4.05 (t,
2H), 3.97 (s, 3H), 3.43 (m ,1H), 2.85 (m, 1H), 1.94 (m, 2H), 1.40 (d, 3H), 1.11 (t, 3H). MS obsd.
(ESI+) [(M+H)+]: 344.
10-Benzyloxy-9-methoxy-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (9).
1H NMR (400 MHz, CDCl3): δ 8.56 (s, 1H), 7.44 (m, 5H), 7.25 (s, 1H), 6.95 (s, 1H), 6.79 (s, 1H),
5.22 (s, 2H), 4.55 (m, 1H), 3.99 (s, 3H), 3.43 (dd ,1H), 2.85(d, 1H), 1.38 (d, 3H). MS obsd. (ESI+)
[(M+H)+]: 392.
9-Methoxy-10-(2-methoxyethoxy)-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic
acid (10). 1H NMR (400 MHz, CDCl3): δ 8.80 (s, 1H), 7.52 (m, 1H), 7.25 (s, 1H), 6.95 (s, 1H), 4.71
(q, 1H), 4.24 ꢀ 4.12 (m, 2H), 3.89 (s, 3H), 3.70 (t, 2H), 3.4 (s, 3H), 3.33 (m, 1H), 2.99 (d, 1H), 1.38 (t,
3H). MS obsd. (ESI+) [(M+H)+]: 360.
9-Hydroxy-10-methoxy-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (11).
1H NMR (400 MHz, CD3OD): δ 8.80 (s, 1H), 7.47 (s, 1H), 7.33 (s, 1H), 6.83 (s,1H), 4.91ꢀ4.83 (m,
1H), 3.99 (s, 3H), 3.45ꢀ3.39 (m, 1H), 2.91ꢀ2.87 (m, 1H), 1.35 (d, 3H). MS obsd. (ESI+) [(M+H)+]:
302.
10-Methoxy-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (12). 1H NMR
(400 MHz, CDCl3): δ 8.60 (s, 1H), 7.26 (d, 2H), 7.21 (s, 1H), 7.09 (dd, 1H), 4.58 (m, 1H), 3.90 (s,
3H), 3.43 (dd, 1H), 2.90 (d, 1H), 1.38 (d, 3H). MS obsd. (ESI+) [(M+H)+]: 286.
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9-Ethoxy-10-methoxy-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (13). H
NMR (400 MHz, DMSOꢀd6): δ 8.82 (s, 1H), 7.52 (s, 1H), 7.46 (s, 1H), 7.00 (s, 1H), 4.97ꢀ4.91 (m,
1H), 4.11 (q, 2H), 3.88 (s, 3H), 3.34ꢀ3.30 (m, 1H), 2.91ꢀ2.87 (m, 1H), 1.37 (t, 3H), 1.19 (d, 3H). MS
obsd. (ESI+) [(M+H)+]: 330.
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9-Benzyloxy-10-methoxy-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (14).
1H NMR (400 MHz, DMSOꢀd6): δ 8.84 (s, 1H), 7.56 (s, 1H), 7.48ꢀ7.35 (m, 6H), 7.15 (s, 1H), 5.18 (s,
2H), 5.01ꢀ4.91 (m, 1H), 3.89 (s, 3H), 3.41ꢀ3.37 (m, 1H), 2.91ꢀ2.87 (m, 1H), 1.20 (d, 3H). MS obsd.
(ESI+) [(M+H)+]: 392.
9-Bromo-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (15). 1H NMR
(400MHz, CDCl3): δ 8.59 (s, 1H), 7.71 ꢀ 7.58 (m, 2H), 7.54 (s, 1H), 7.19 (s, 1H), 4.65 ꢀ 4.54 (m, 1H),
3.55 ꢀ 3.43 (m, 1H), 2.95 (dd, 1H), 1.39 (d, 3H). MS obsd. (ESI+) [(M+H)+]: 334.
1
9-Ethoxy-10-hydroxy-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (19). H
NMR (400MHz, DMSOꢀd6): δ 9.26 (br. s, 1H), 8.81 (s, 1H), 7.36 (s, 1H), 7.04 (s, 1H), 6.96 (s, 1H),
4.92 (t, 1H), 4.13 (q, 2H), 3.32 (dd, 1H), 2.89 ꢀ 2.81 (m, 1H), 1.38 (t, 3H), 1.19 (d, 3H). MS obsd.
(ESI+) [(M+H)+]: 316.
1
8-Chloro-9-methoxy-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (20). H
NMR (400 MHz, CDCl3): δ 8.60 (s, 1H), 7.74 (d, 1H), 7.07 (s, 1H), 7.04 (d, 1H), 4.61 (m, 1H), 4.06
(s, 3H), 3.46 (m, 1H), 3.27 (m, 1H), 1.38 (d, 3H). MS obsd. (ESI+) [(M+H)+]: 320.
9,11-Dimethoxy-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (21). 1H NMR
(400 MHz, DMSOꢀd6): δ 8.83 (s, 1H), 7.54 (s, 1H), 6.69 (d, 1H), 6.64 (d, 1H), 4.91ꢀ4.88 (m, 1H),
3.95 (s, 3H), 3.88(s, 3H), 3.34ꢀ3.30 (m, 1H), 2.93ꢀ2.89 (m, 1H), 1.17 (d, 3H). MS obsd. (ESI+)
[(M+H)+]: 316.
1
6-Ethyl-9,10-dimethoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (22). H NMR
(400 MHz, DMSOꢀd6): δ 8.81 (s, 1H), 7.53 (s, 1H), 7.47 (s, 1H), 7.04 (s, 1H), 4.72 (m, 1H), 3.88 (s,
3H), 3.85 (s, 3H), 3.36 (dd, 1H), 3.02 (d, 1H), 1.52ꢀ1.44 (m, 2H), 0.81 (t, 3H). MS obsd. (ESI+)
[(M+H)+]: 330. HRMS: calcd (MH+) 330.1342, exp (MH+) 330.1341.
(6R)-(+)-6-Ethyl-9,10-dimethoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid ((R)-
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22). 1H NMR (400 MHz, DMSOꢀd6): δ 8.81 (s, 1H), 7.53 (s, 1H), 7.47 (s, 1H), 7.04 (s, 1H), 4.72 (m,
4
5
1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.36 (dd, 1H), 3.02 (d, 1H), 1.52ꢀ1.44 (m, 2H), 0.81 (t, 3H). MS obsd.
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7
8
20
(ESI+) [(M+H)+]: 330. [
α]
= +121.21 (0.165%, CH3CN), the absolute stereochemistry was
D
9
determined by the Xꢀray diffraction study of its (6S)ꢀenantiomer (S)ꢀ22.
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(6S)-(-)-6-Ethyl-9,10-dimethoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid ((S)-
22). 1H NMR (400 MHz, DMSOꢀd6): δ 8.81 (s, 1H), 7.53 (s, 1H), 7.47 (s, 1H), 7.04 (s, 1H), 4.72 (m,
1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.36 (dd, 1H), 3.02 (d, 1H), 1.52ꢀ1.44 (m, 2H), 0.81 (t, 3H). MS obsd.
(ESI+) [(M+H)+]: 330, the absolute stereochemistry was determined by Xꢀray diffraction study.
1
,10-Dimethoxy-2-oxo-6-propyl-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (23). H NMR
(400MHz, CDCl3): δ 8.51 (s, 1H), 7.21 (s, 1H), 7.11 (s, 1H), 6.77 (s, 1H), 4.40 ꢀ 4.26 (m, 1H), 3.99 (s,
3H), 3.98 (s, 3H), 3.44 (dd, 1H), 2.94 (d, 1H), 1.70 ꢀ 1.51 (m, 2H), 1.40 ꢀ 1.19 (m, 2H), 0.90 (t, 3H).
MS obsd. (ESI+) [(M+H)+]: 344.
6-Isobutyl-9,10-dimethoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (24). 1H
NMR (400MHz, DMSOꢀd6): δ 8.76 (s, 1H), 7.53 (s, 1H), 7.48 (s, 1H), 7.05 (s, 1H), 4.88 (m, 1H),
3.88 (s, 3H), 3.85 (s, 3H), 3.31 (d, 1H), 2.99 (d, 1H), 1.46 (qd, 1H), 1.34 (t, 2H), 0.88 (d, 3H), 0.83 (d,
3H). MS obsd. (ESI+) [(M+H)+]: 358.
9-Benzyloxy-6-ethyl-10-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (25).
1H NMR (400 MHz, CDCl3): δ 8.50 (s, 1H), 7.46ꢀ7.30 (m, 5H), 7.21 (s, 1H), 7.10 (s, 1H), 6.78 (s,
1H), 5.22 (d, 2H), 4.24ꢀ4.19 (m, 1H), 3.96 (s, 3H), 3.37 (d, 1H), 2.88 (d, 1H), 1.86ꢀ1.59 (m, 2H), 0.90
(t, 3H). MS obsd. (ESI+) [(M+H)+]: 406. HRMS: calcd (MH+) 406.1655, exp (MH+) 406.1652.
9-Ethoxy-6-ethyl-10-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (26). 1H
NMR (400MHz, CDCl3): δ 8.52 (s, 1H), 7.20 (s, 1H), 7.10 (s, 1H), 6.76 (s, 1H), 4.31 ꢀ 4.15 (m, 3H),
3.96 (s, 3H), 3.42 (dd, 1H), 2.94 (d, 1H), 1.66ꢀ1.62 (m, 2H), 1.54 (t, 3H), 0.94 (t, 3H). MS obsd.
(ESI+) [(M+H)+]: 344. HRMS: calcd (MH+) 344.1498, exp (MH+) 344.1499.
1
6-Ethyl-10-methoxy-2-oxo-9-propoxy-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (27). H
NMR (400 MHz, DMSOꢀd6): δ 8.81 (s, 1H), 7.52 (s, 1H), 7.46 (s, 1H), 7.04 (s, 1H), 4.70 (m, 1H),
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4.04ꢀ3.98 (m, 2H), 3.88(s, 3H), 3.33 (d, 1H), 3.00 (d, 1H), 1.77 (m, 2H), 1.55ꢀ1.35 (m, 2H), 0.99 (t,
3H), 0.87 (m, 3H). MS obsd. (ESI+) [(M+H)+]: 358.
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9
9-Butoxy-6-ethyl-10-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (28). 1H
NMR (400 MHz, CDCl3): δ 8.51 (s, 1H), 7.18 (s, 1H), 7.09 (s, 1H), 6.75 (s, 1H), 4.25ꢀ4.21 (m, 1H),
4.10 (dt, 2H), 3.94 (s, 3H), 3.41 (d, 1H), 2.93 (d, 1H), 1.92ꢀ1.85 (m, 2H), 1.70ꢀ1.58 (m, 2H), 1.58ꢀ
1.49 (m, 2H), 1.01 (t, 3H), 0.93 (t, 3H). MS obsd. (ESI+) [(M+H)+]: 372.
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6-Ethyl-9-isobutoxy-10-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (29).
1H NMR (400MHz, CDCl3): δ 8.50 (s, 1H), 7.20 (s, 1H), 7.09 (s, 1H), 6.75 (s, 1H), 4.27 ꢀ 4.17 (m,
1H), 3.95 (s, 3H), 3.86 (d, 2H), 3.46 ꢀ 3.36 (m, 1H), 2.97 ꢀ 2.90 (m, 1H), 2.28 ꢀ 2.17 (m, 1H), 1.74 ꢀ
1.62 (m, 2H), 1.10 (d, 6H), 0.94 (t, 3H). MS obsd. (ESI+) [(M+H)+]: 372.
9-(Cyclopropylmethoxy)-6-ethyl-10-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic
acid (30). 1H NMR (400MHz, CDCl3): δ 8.51 (s, 1H), 7.20 (s, 1H), 7.10 (s, 1H), 6.74 (s, 1H), 4.23 (q,
1H), 4.00 ꢀ 3.90 (m, 5H), 3.40 (s, 1H), 2.93 (d, 1H), 1.74 ꢀ 1.63 (m, 2H), 1.44 ꢀ 1.34 (m, 1H), 0.94 (t,
3H), 0.77 ꢀ 0.69 (m, 2H), 0.46 ꢀ 0.38 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 370.
9-(2-Cyclohexylethoxy)-6-ethyl-10-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic
acid (31). 1H NMR (400 MHz, CDCl3): δ 8.50 (s, 1H), 7.18 (s, 1H), 7.08 (s, 1H), 6.74 (s, 1H), 4.25ꢀ
4.20 (m, 1H), 4.11 (dt, 2H), 3.93 (s, 3H), 3.41 (dd, 1H), 2.92 (d, 1H), 1.82ꢀ1.52 (m, 9H), 1.34ꢀ1.17
(m, 4H), 1.05ꢀ0.97 (m, 2H), 0.93 (t, 3H). MS obsd. (ESI+) [(M+H)+]: 426.
9-(2,2-Difluoroethoxy)-6-ethyl-10-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic
acid (32). 1H NMR (400MHz, CDCl3): δ 8.53 (s, 1H), 7.25 (s, 1H), 7.12 (s, 1H), 6.83 (s, 1H), 6.32 (t,
0.3H), 6.19 (t, 0.53H), 6.05 (t, 0.32H), 4.41 ꢀ 4.17 (m, 3H), 3.96 (s, 3H), 3.43 (d, 1H), 2.96 (d, 1H),
1.84 ꢀ 1.65 (m, 2H), 0.94 (t, 3H). MS obsd. (ESI+) [(M+H)+]: 380.
6-Ethyl-9-isopropoxy-10-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (33).
1H NMR (400 MHz, CDCl3): δ 8.51 (s, 1H), 7.19 (s, 1H), 7.09 (s, 1H), 6.76 (s, 1H), 4.68ꢀ4.65 (m,
1H), 4.25ꢀ4.21 (m, 1H), 3.93 (s, 3H), 3.40 (d, 1H), 2.92 (d, 1H), 1.69ꢀ1.60 (m, 2H), 1.45 (d, 3H), 1.43
(d, 3H ), 0.93 (t, 3H). MS obsd. (ESI+) [(M+H)+]: 358.
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6
7
8
9
6-Ethyl-10-methoxy-2-oxo-9-propyl-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (34). 1H
NMR(400 MHz, DMSOꢀd6): δ 8.84 (s, 1H), 7.57 (s, 1H), 7.51 (s, 1H), 7.19 (s, 1H), 4.73 (m, 2H),
3.91 (s, 3H), 2.99 (d, 1H), 1.62ꢀ1.53 (m, 2H), 1.51ꢀ1.39 (m, 5H), 0.94ꢀ0.88 (m, 2H), 0.79 (m, 3H). MS
obsd. (ESI+) [(M+H)+]: 342.
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6-Ethyl-2-oxo-6,7,10,11-tetrahydro-12H-[1,5]benzodioxepine[7,6-a]quinolizine-3-carboxylic acid
1
(36). H NMR (400 MHz, METHANOLꢀd4) δ ppm 8.75 (s, 1H), 7.59 (s, 1H), 7.24 (s, 1H), 6.99 (s,
1H), 4.59 (br d, 1H), 4.20 ꢀ 4.38 (m, 4H), 3.36 ꢀ 3.44 (m, 1H), 3.05 (dd, 1H), 2.20 ꢀ 2.36 (m, 2H), 1.50
ꢀ 1.70 (m, 2H), 0.92 (s, 3H). MS obsd. (ESI+) [(M+H)+]: 342.
6-Ethyl-2-oxo-6,7,10,11-tetrahydro-[1,4]benzodioxino[7,6-a]quinolizine-3-carboxylic acid (37).
1H NMR (400 MHz, CHLOROFORMꢀd) δ ppm 8.49 (s, 1 H), 7.29 (s, 1 H), 7.04 (s, 1 H), 6.81 (s, 1
H), 4.31 ꢀ 4.42 (m, 3 H), 4.17 ꢀ 4.27 (m, 1 H), 3.31 ꢀ 3.40 (m, 1 H), 2.92 (dd, 1 H), 1.55 ꢀ 1.74 (m, 3
H), 0.93 (t, 3 H). MS obsd. (ESI+) [(M+H)+]: 328. HRMS: calcd (MH+) 328.1185, exp (MH+)
328.1180.
1
6,10-Diethyl-9-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (38). H NMR
(400 MHz, CD3OD): δ 8.74 (s, 1H), 7.74 (s, 1H), 7.28 (s, 1H), 6.99 (s, 1H), 4.59 (m, 1H), 3.97 (s,
3H), 3.43 (m ,1H), 3.13 (d, 1H), 2.73 (m, 2H), 1.64 (m, 2H), 1.24 (t, 3H), 0.93 (t, 3H). MS obsd.
(ESI+) [(M+H)+]: 328. HRMS: calcd (MH+) 328.1549, exp (MH+) 328.1546.
10-Cyclopropyl-6-ethyl-9-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (39).
1H NMR (400 MHz, CDCl3): δ 8.49 (s, 1H), 7.23 (s, 1H), 7.08 (s, 1H), 6.73 (s, 1H), 4.23 (m, 1H),
3.97 (s, 3H), 3.42 (m, 1H), 2.98 (m, 1H), 2.17 (m, 1H), 1.64 (m, 2H), 1.03 (m, 2H), 0.95 (t, 3H), 0.70
(m, 2H). MS obsd. (ESI+) [(M+H)+]: 340.
9-(2-Amino-2-oxo-ethoxy)-6-ethyl-10-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-
carboxylic acid (40). 1H NMR (400 MHz, METHANOLꢀd4) δ 8.07 (s, 1H), 7.39 (s, 1H), 7.01 (s, 1H),
6.97 (s, 1H), 4.37ꢀ4.44 (m, 1H), 4.17 (m, 2H), 3.95 (s, 3H), 3.54ꢀ3.66 (m, 2H), 3.36ꢀ3.46 (m, 3H),
3.04 (m, 1H), 1.54ꢀ1.71 (m, 2H), 0.93 (t, 3H). MS obsd. (ESI+) [(M+H)+]: 373.
9-(2-Acetamidoethoxy)-6-ethyl-10-methoxy-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic
1
acid (41). H NMR (400MHz, CDCl3): δ 8.51 (s, 1H), 7.21 (s, 1H), 7.10 (s, 1H), 6.81 (s, 1H), 6.24
29
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