Design and synthesis of orally bioavailable 4-methyl heteroaryldihydropyrimidine based hepatitis B virus (HBV) capsid inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00879

Targeting the capsid protein of hepatitis B virus (HBV) and thus interrupting normal capsid formation have been an attractive approach to block the replication of HBV viruses. We carried out multidimensional structural optimizations based on the heteroaryldihydropyrimidine (HAP) analogue Bay41-4109 (1) and identified a novel series of HBV capsid inhibitors that demonstrated promising cellular selectivity indexes, metabolic stabilities, and in vitro safety profiles. Herein we disclose the design, synthesis, structureactivity relationship (SAR), cocrystal structure in complex with HBV capsid proteins and in vivo pharmacological study of the 4-methyl HAP analogues. In particular, the (2S,4S)-4,4-difluoroproline substituted analogue 34a demonstrated high oral bioavailability and liver exposure and achieved over 2 log viral load reduction in a hydrodynamic injected (HDI) HBV mouse model.

Full text of DOI:10.1021/acs.jmedchem.6b00879

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Article
Design and Synthesis of Orally Bioavailable 4-Methyl
Heteroaryldihydropyrimidine Based Hepatitis B Virus (HBV) Capsid Inhibitors
Zongxing Qiu, Xianfeng Lin, Mingwei Zhou, Yongfu Liu, Wei Zhu, Wenming Chen, Weixing Zhang, Lei Guo,
Haixia Liu, Guolong Wu, Mengwei Huang, Min Jiang, Zhiheng Xu, Zheng (Joe) Zhou, Ning Qin, Shuang
Ren, Hongxia Qiu, Sheng Zhong, Yuxia Zhang, Yi Zhang, Xiaoyue Wu, Liping Shi, Fang Shen, Yi Mao, Xue
Zhou, Wengang Yang, Jim Zhen Wu, Guang Yang, Alexander V Mayweg, Hong C Shen, and Guozhi Tang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00879 • Publication Date (Web): 26 Jul 2016
Downloaded from http://pubs.acs.org on July 28, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Shen, Hong; Roche R&D Center China, Medicinal chemistry
Tang, Guozhi; Roche Pharma Research and Early Development,
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Design and Synthesis of Orally Bioavailable 4-Methyl
Heteroaryldihydropyrimidine Based Hepatitis B Virus
(HBV) Capsid Inhibitors
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Zongxing Qiu, Xianfeng Lin, Mingwei Zhou, Yongfu Liu, Wei Zhu, Wenming Chen,
ǂ,‡
ǂ,‡
ǂ,‡
ǂ,‡
ǂ,‡
ǂ,‡
Weixing Zhang, Lei Guo, Haixia Liu, Guolong Wu, Mengwei Huang, Min Jiang, Zhiheng
ǂ,†
ǂ,†
ǂ,†
ǂ,ǁ
ǂ,ǁ
ǂ,ǁ
ǂ,ǁ
Xu, Zheng Zhou, Ning Qin, Shuang Ren, Hongxia Qiu, Sheng Zhong, Yuxia Zhang, Yi
ǂ,ǁ
ǂ,ǁ
ǂ,§
ǂ,§
ǂ,§
ǂ,§
ǂ,§
Zhang, Xiaoyue Wu, Liping Shi, Fang Shen, Yi Mao, Xue Zhou, Wengang Yang, Jim Z.
ǂ,§
ǂ,§
ǂ,‡
ǂ,‡
ǂ,‡,*
Wu, Guang Yang, Alexander V. Mayweg, Hong C. Shen, and Guozhi Tang
ǂ
‡
Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Medicinal
†
ǁ
§
Chemistry, Chemical Biology, Pharmaceutical Sciences, Discovery Virology, 720 Cailun Road,
Shanghai, 201203 China
ABSTRACT—Targeting the capsid protein of hepatitis B virus (HBV) and thus interrupting normal
capsid formation has been an attractive approach to block the replication of HBV viruses. We carried
out multidimensional structural optimizations based on the heteroaryldihydropyrimidine (HAP)
analogue Bay41-4109 (1) and identified a novel series of HBV capsid inhibitors that demonstrated
promising cellular selectivity indexes, metabolic stabilities, and in vitro safety profiles. Herein we
disclose the design, synthesis, structure−activity relationship (SAR), co-crystal structure in complex
with HBV capsid proteins, and in vivo pharmacological study of the 4-methyl HAP analogues. In
*
Tel.: +86 21 28946723. Fax: +86 21 50790292. E-mail: gordon.tang@roche.com
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particular, the (2S, 4S)-4,4-difluoroproline substituted analogue 34a demonstrated high oral
bioavailability and liver exposure, and achieved over 2-log viral load reduction in a hydrodynamic
injected (HDI) HBV mouse model.
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Keywords— hepatitis B virus (HBV), capsid inhibitor, heteroaryldihydropyrimidine, capsid assembly
INTRODUCTION
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Chronic hepatitis B virus (HBV) infection remains a major threat to public health despite the
fact that HBV vaccination programs have been extensively implemented in the past. There are about
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50 million chronic HBV carriers in the world and disease progression among many of them often
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leads to cirrhosis and hepatocellular carcinoma. It has been estimated that HBV infection directly or
indirectly accounts for an annual death of 600,000 patients. Nevertheless, current treatment options are
limited to interferons (IFNs) and nucleos(t)ides-based reverse transcriptase inhibitors that do not offer a
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satisfactory clinical cure rate for chronic HBV carriers. For example, the response rate in HBeAg-
positive patients following 4-5 years of treatment with nucleos(t)ides is about 0-10%, and 8-15% with
pegylated IFN-α or pegylated IFN-α plus lamivudine, whereas the response rate in HBeAg-negative
patients is even lower. Of note, nucleos(t)ide therapies typically require lifetime treatment to prevent
viral rebound while IFNs require parenteral administration and are associated with adverse effects such
as ‘flu-like’ symptoms. To achieve a higher HBV cure rate than current standard of care, a
3, 4
multipronged approach with new molecular entities is very likely required.
As such, there is a
tremendous unmet medical need to identify and develop novel, efficacious and safe anti-HBV agents
with diverse mechanisms of action.
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(Bay41-4109)
2 (GLS4): R = Et
(NVR-010001-E2): R = Me
4 (AT-61)
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(DVR-23)
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Figure 1. Chemical structures of reported HBV capsid inhibitors.
As a structural component of the viral nucleocapsid, the HBV capsid protein plays multiple
roles in the HBV lifecycle including reverse transcription of pre-genomic RNA (pgRNA), formation
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and intracellular trafficking of relaxed circular-DNA, and subviral particle formation.
To date a
number of reported capsid inhibitors (or effectors) are able to interrupt normal capsid assembly and
thus inhibit HBV DNA synthesis (Figure 1). Among them, Bay 41-4109 (1),
a
heteroaryldihydropyrimidine (HAP) compound, is a seminal HBV capsid inhibitor that has been well
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studied in the past. Mechanistic and biostructural studies indicate that 1 can fit into a hydrophobic
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pocket located at the interface of capsid dimers and trigger aberrant capsid assembly and aggregation.
Treatment of virus-producing HepG2.2.15 cells with 1 leads to reduction of HBV DNA as well as
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dose-dependent depletion of core proteins and nucleocapsids. However, further development of 1 has
been hindered by several factors including in vitro and in vivo toxicity and limited efficacy in animal
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models.
substituents and 2-thioazolyl groups attached to the core scaffold.
potent and less cytotoxic in in vitro tests, 2 demonstrated similar in vivo effects as 1 in a mouse
GLS-4 (2) and 3 are a new generation of HAP analogues featuring 6-morpholine
1
1−13
Although reported to be more
1
1
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model. Acrylamide 4 (previously reported as AT-61 but with the wrong E-configuration),
1
6
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sulfamoylbenzamide DVR23 (5), and isothiafludine (6) are the other class of capsid inhibitors that
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have distinct effects on the HBV capsid assembly process from that of HAPs. Mechanistically,
compounds 4−6 also bind to capsid proteins and accelerate the capsid assembly process, but they
usually result in normal-sized and pgRNA-free empty capsids. Sometimes referred to as pgRNA
encapsidation blockers, 4−6 inhibit HBV DNA synthesis without the capsid depletion effects of
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HAPs.
Whether the in-cell capsid depletion induced by HAPs provides an extra benefit to treat
HBV infection or not remains to be investigated in relevant animal models and eventually in a clinical
setting.
To develop novel, orally bioavailable and in vivo efficacious anti-HBV agents, we attempted to
explore the chemistry space of HAPs from benchmark analogue 1 with goals to enhance anti-HBV
activity, reduce cytotoxicity, and improve metabolic stability and in vitro safety profiles.
RESULTS AND DISCUSSION
Section 1. Design and Synthesis of 4-Methyl HAPs. Because of the lack of high resolution
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co-crystal structures of capsid protein and HAPs at the time, an information-driven approach was
carried out to optimize the HAP series represented by 1. Firstly, metabolite identification studies of 1
upon incubation with human and mouse liver microsomes (HLM and MLM) indicated that
aromatization of the dihydropyrimidine core (M-2H) is a major metabolic pathway. When evaluated in
1
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HepDE19 cells that support high levels of HBV DNA replication, 1 demonstrates anti-HBV activity
with an EC of 0.14 µM. The corresponding CC value of 1 is 13.4 µM in the same cell line. Further
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structure-activity relationship (SAR) study of 4-H HAPs revealed that 2-Cl on the 4-phenyl head group
is indispensable to their anti-HBV activities and that the 3,5-difluoropyridinyl moiety is chemically
susceptible to nucleophilic S Ar displacements (unpublished results). Taken together, it was
N
anticipated that 4-methyl-substituted HAPs would not be susceptible to aromatization, and that the
methyl group could also take a position close to the 2-Cl substituent of 1 and recapitulate its role in
capsid binding (Figure 2). Indeed, predicted lowest energy conformations of 4-methyl HAP 7a (4-S
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enantiomer as shown) and 4-H HAPs like 1 overlay well with one another and the 4-methyl group of
a points in the same direction as the 2-Cl substituent of 1. Further conformation analysis also
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suggested that the 4-phenyl group of 7a remains orthogonal to the HAP core and that the 2-thiazolyl
moiety is essentially co-planar with the HAP core due to the formation of an intramolecular hydrogen
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bond and 1,4-N−S interaction. Thus, a series of 4-methyl-substituted HAP analogues including 7a−c
were synthesized and evaluated in HepDE19 cells.
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Figure 2. Predicted lowest energy conformations of 1 (A) and (S)-enantiomer of 7a (B) in vacuum. Force field MMFF94x
was applied in Molecular Operating Environment (MOE), CCG. An intramolecular hydrogen-bonding (HB) interaction
between 1-NH and 2-heteroaryl group is indicated. Molecules are presented as ball and stick with electrostatic surface
included. Carbon, oxygen, nitrogen, fluorine and chlorine atoms are shown in gray, red, blue, green and dark green colors,
respectively.
Generally, 4-methyl HAP analogues can be prepared from β-ketoester 8 and a variety of
benzaldehydes 9 according to the procedures shown in Scheme 1. Briefly, an aldol condensation of 8
and 9 was carried out and the resulting products were treated with CH CuLi to give 1,4-Michael
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addition products 10. Ketoesters 10 were then treated by a two-step phenylselenation and oxidation to
give α, β-unsaturated esters 11, which were separated and used in the subsequent step as a mixture of
E/Z-isomers. The condensation and ring cyclization reactions of 11 and amidines 12 turned out quite
sluggish and complicated, in part because of high steric hindrance and decomposition of 11 at high
temperature. To minimize the decomposition issue, olefins 11 were dissolved in NMP and added
dropwise into a stirring mixture of 12 and NaHCO in NMP that was pre-warmed to 120 °C . By this
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method, the methyl-substituted HAP scaffold with a quaternary carbon was successfully built up from
1 and 12 in moderate to high yields.
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Scheme 1. Synthesis of 4-Alkyl-substituted HAP Analogues 7a−j
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11 (E/Z)
7ai
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7j
a
Reagents and conditions: (a) piperidine, HOAc, EtOH; (b) CH CuLi, THF, −78 °C; (c) PhSeCl, NaH, THF; (d) H O /H O,
3
2
2
2
DCM; (e) NaHCO , NMP, 120 °C, 2 h, 30−70%; (f) NaBH , EtOH; (g) SOCl , DCM, 90% for 2 steps; (h) 8, NaH, THF,
3
4
2
6
0%; (i) thiazole-2-carboxamidine, AcOK, i-PrOH, 120 °C, 15%.
Section 2. SAR at the 4-Phenyl Head Group of 4-Methyl HAPs. Analogues 7a and 7b
moderately reduced HBV DNA in cellular activity tests with EC50 values of 11.6 µM and 7.1 µM,
respectively (Table 1). On the other hand, they were also much less cytotoxic to HepDE19 cells with
CC values above 100 µM and had good aqueous solubility in lyophilized solubility assays (LYSA).
50
To confirm the anti-HBV activity of this new HAP scaffold, we conducted chiral separation of 7a by
supercritical fluid chromatography (SFC) and determined that enantiomer (S)-7a is active in HepDE19
cells with an EC of 6.8 µM. The absolute configuration of (S)-7a was confirmed by single crystal X-
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ray studies, and it is clear that the core structures and 4-phenyl head groups of (S)-7a and 1 are well
aligned to each other (see the Supporting Information). Notably, analogue 7c with the same 2-Cl, 4-F
phenyl group of 1 was less active than 7a and 7b and was more cytotoxic to HepDE19 cells with a
CC of 82.5 µM. The 2-Cl substituent of 7c may occupy a position opposite to the 4-methyl group, an
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orientation that may not favor target binding. Furthermore, 4-methyl HAPs may generally have
different SAR and chemistry space relative to 1, especially at the 4-phenyl head group. Thus, additional
structural explorations of the 4-methyl HAPs were carried out in an attempt to rescue their in vitro anti-
HBV activities.
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Table 1. The Effects on HBV DNA Reduction and Physicochemical Properties of 7a−j
7
aj
(S)-7a
(R)-7a
1
2
3
a
b
c
d
e
ID
R
R
R
EC50
CC50
SI
logP
4.6
LYSA
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1
H
2-Cl, 4-F
0.14
13.4
96
7
a
Me
Me
Me
4-F
4-F
4-F
11.6
6.8
>100
>100
>100
>9
4.2
4.2
4.4
22
(S)-7a
7b
>15
>14
ND
178
7.1
7c
Me
Me
Me
Me
Me
Me
Me
Et
2-Cl, 4-F
H
28.1
35.9
26.8
>100
>100
>100
4.1
82.5
>100
43.6
3
>3
2
4.8
4.0
4.6
2.5
4.0
3.5
4.4
4.6
18
ND
14
7
d
7
e
4-Cl
7
f
4-SO
2
Me
>100
>100
>100
85.9
-
365
106
87
7
7
g
4-OMe
4-CN
-
h
-
7
i
3-F, 4-F
4-F
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-
50
7j
>100
96.0
ND
a
EC50 values [µM] for the reduction of HBV DNA in HepDE19 cells. The levels of HBV DNA in supernatant were
determined by dot-blot experiments in duplicate runs, with variation < 15%.
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CC values [µM] measured by CCK-8 in HepDE19 cells. Experiments were in duplicate runs with variation < 15%.
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Selectivity index (SI) of CC /EC in HepDE19 cells.
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Predicted partition coefficient between octanol phase and water phase (K logP).
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Lyophilized solubility assay (LYSA) from duplicate runs, mean values reported in µg/mL with variation < 10%).
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A number of analogues with structure variations at the 4-phenyl head group (substitution at the
C4 of the HAP core) were prepared and evaluated for their anti-HBV activities (Table 1). SAR analysis
indicated that the p-F substituent on the 4-phenyl group is critical for anti-HBV activity and that
replacements like −H, −SO Me, −OMe and −CN reduce potency in HepDE19 cells (analogues
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including 7d and 7f−h). Just like 7c, analogue 7e had inferior cytotoxicity and metabolic stability
−
1
−1
profiles with MLM of 85 mL min kg , presumably due to their high lipophilicity (K logP ≥ 4.6).
ow
On the other hand, polar and electron-withdrawing substituents such as −SO Me, −CN, −CO H and
2
2
amide groups improved the metabolic stability and solubility profiles of 4-methyl HAPs. For example,
−
1
−1
analogues 7f and 7h showed lower intrinsic clearance with MLM of 57 and 75 mL min kg ,
respectively. Introducing a second fluorine substitution at the m-position improved the potency of
analogue 7i relative to 7a, with an EC of 4.1 µM. However, 7i was also highly lipophilic (KowlogP =
5
0
−
1
−1
4.4) with MLM of 83 mL min kg . We speculated that the head groups sit in a hydrophobic pocket
making close molecular contacts with HBV capsid proteins such that minor structural modifications
can have profound effects on anti-HBV activity.
Considering the SAR findings discussed above, we explored the tolerability of larger alkyl
groups including ethyl at the 4-position of HAPs. To synthesize the 4-ethyl-substituted HAP analogue
7j, ketone 13 was reduced with NaBH and the resulting alcohol was converted to substituted benzyl
4
chloride 14 (Scheme 1). Chloride 14 was then treated with 8 and NaH to afford β-ketoester 15. By
using the same methods as described previously, tetra-substituted olefin 16 was obtained and it was
added into a mixture of thiazole-2-carboxamidine and KOAc in isopropanol at 120 °C to give analogue
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
j. Unforutnately, 7j was inactive in HepDE19 cells (Table 1). Thus the 4-methyl HAP core was fixed
in subsequent explorations of structure-property relationships focused on the 2-heteroaryl and 5-, 6-
positions of the HAP core.
Section 3. SAR of 2-Heteroaryls in 6-Morpholine-substituted 4-Methyl HAPs. As in the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1, 22
case of 4-H HAPs like 2 and 3,
a solubilizing 6-morpholine substituent may help improve in vitro
anti-HBV potency yet its impact on the 4-methyl HAPs remained to be investigated. Accordingly, 6-
morpholine substituted analogues 18a−k were prepared from racemic or optically pure intermediates 7
(Scheme 2). As an efficient method, 4-methyl HAPs were treated with (Boc) O anhydride and N, N'-
2
dimethylaminopyridine (DMAP) and the N-Boc protected products were treated with NBS and
azobisisobutyronitrile (AIBN) at 50 °C to give bromides 17. Without purification, bromides 17 were
substituted with morpholine and the products were treated with trifluoroacetic acid (TFA) to give
analogues 18a−k in over 40% yields.
Generally, the 6-morpholine substituted 4-methyl HAPs demonstrated better cellular activities
than their parent compounds in HepDE19 cells. For example, enantiomer (S)-18a was 40 times more
active than (S)-7a with an EC50 of 0.17 µM (Table 2). 3,4-Difluoro-substituted analogue (S)-18b
significantly reduced HBV DNA with an EC50 of 70 nM in HepDE19 cells. However, 3-Cl, 4-F-
substituted analogue 18c was over 100 times less active than (S)-18b with an EC of 8.1 µM and a
5
0
CC of 67.7 µM. In agreement with previous SAR observations, enantiomers (R)-18a and (R)-18b had
50
moderate to weak anti-HBV activities with EC values of 26.8 µM and 65.8 µM, respectively.
50
a
Scheme 2. Synthesis of 4-Methyl HAP Analogues 18a−k
a, b
c, d
7
17
18ak
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a
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4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Reagents and conditions: (a) (Boc) O, DMAP, DCM, rt, overnight; (b) NBS, AIBN, CCl , 50 °C, 2 h; (c) morpholine,
2 4
DCM, 50 °C, 1 h; (d) TFA, DCM, rt.
(S)-18a and (S)-18b are highly potent anti-HBV inhibitors with selectivity margins in HepDE19
cells that meet our primary goals for in vitro potency. However, they have poor metabolic stability and
limited oral availability. For example, the mouse liver microsome (MLM) clearance of (S)-18a and (S)-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
−
1
−1
1
8b was 81 and 89 mL min kg , respectively. The oral bioavailability (F) of (S)-18a was about 1.5%
in a mouse single dose pharmacokinetics (SDPK) study. We therefore set out to improve the metabolic
stability of HAPs and to identify orally bioavailable analogues with sufficient target organ exposure for
in vivo efficacy studies.
In an attempt to improve microsomal stability and physiochemical properties, the 2-thiazolyl
group was replaced with polar heteroaryls to reduce lipophilicity, and analogues such as 18d−j were
prepared accordingly. A number of these analogues had increased microsomal stability relative to (S)-
1
8a and also were expected to have good aqueous solubility based on LYSA results of > 300 µg/mL
(
(
Table 2). Both N-methyl imidazolyl and 5-F pyridinyl groups were tolerated with EC values < 1 µM
5
0
analogues 18d and 18f), whereas oxazolyl (18e), pyrimidinyl (18i) and pyridazinyl (18j) resulted in
lower potencies than (S)-18a. Our results also suggested that polar substituents cannot be tolerated on
the heteroaryls, for example, 5-methoxypyridinyl analogue 8g was 25 times less active than 18f. In
addition, aliphatic groups were not tolerated at the 2-position of HAPs, leading to a total loss of activity
(analogue 18k).
Table 2. The Effects on HBV DNA Reduction and Physicochemical Properties of 18a−k
1
8ak
(S)-18a (X = H)
S)-18b (X= F)
(R)-18a (X = H)
(R)-18b (X = F)
(
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8
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1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
a
a
EC50
µM]
CC50
LYSA
2
3
ID
R
R
SI
logP
3.0
[
[µM]
[µg/mL]
(
S)-18a
4-F
0.17
>100
>588
14
(
R)-18a
S)-18b
R)-18b
8c
4-F
26.8
0.07
65.8
8.1
>100
>100
>100
67.7
>4
3.0
3.2
3.2
3.6
16
7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
3-F, 4-F
3-F, 4-F
3-Cl, 4-F
>1429
(
-
ND
3
1
8
1
8d
4-F
0.65
>100
>154
2.6
435
1
8e
4-F
4-F
10.1
0.61
>100
>100
>10
2.7
3.0
336
2
1
8f
>164
18g
4-F
3.65
> 100
> 27
3.3
22
1
8h
8i
18j
8k
4-F
4-F
15.6
2.95
>100
>100
>6
2.9
1.9
77
1
>34
424
4-F
4-F
3.65
>100
>100
>27
-
2.5
3.0
341
ND
1
>100
a
Definitions are the same as those described in Table 1.
Section 4. SAR at 5-position of 4-Methyl HAPs. The 5-ester group appeared to have unclear
roles in target binding. In our SAR studies of the 5-ester group, we found that direct chemistry
conversions of the ester moiety were problematic. Neither hydrolysis nor reduction of analogues like 7
gave desired products even under harsh conditions, probably due to conjugation effects with the HAP
core and high steric hindrance of the ester moiety. To address these issues, t-butyl β-ketoester 19 was
used as the starting material and t-butyl ester 20 was prepared by means of the same method as shown
in Scheme 1. Treatment of 20 with TFA smoothly afforded acid 21 and further functionalization was in
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turn carried out (Scheme 3). In one case, 21 was treated with 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU) and ammonia to give primary amide 22, which was
then converted to 5-CN analogue 23 upon treatment with trifluoroacetic acid anhydride (TFAA). On
the other hand, amide analogues such as 24 and 25 were prepared by treating 21 with N, N'-
carbonyldiimidazole (CDI) and then specified amines at elevated temperature. Amide 25 was treated
with polyphosphoric acid (PPA) at 120 °C to give 5-oxazole-substituted analogue 26. Finally,
analogues 27a−e were prepared by converting their respective precursors to bromide intermediates and
then subsequently treating with morpholine. Besides exploring the polarity and heteroaryl vectors like
oxazole, we also looked into steric factors via isopropyl ester 27f, which was synthesized with
isopropyl β-ketoester as the initial starting material.
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 3. Synthesis of HAP Analogue 27a−e
a
b
c
4
1
9
20
21 (R = H)
22
23
h
h
h
d, f
4
27b
2
7a (R = morpholinyl)
27c
d, e
g
2
2
4
25
26
h
h
7d
27e
a
Reagents and conditions: (a) TFA, DCM, 3 h, 100%; (b) HATU, NH , NEt , DCM, 40%; (c) TFAA, DCM, 3 h, 87%; (d)
3
3
CDI, THF; (e) CH NH ∙HCl, NEt , CH CN, 90 °C, 80%; (f) 2,2-dimethoxyethanamine, CH CN, 0.5 h, 120 °C, MW, 90%;
3
2
3
3
3
(
g) PPA, 2 h, 120 °C, 30%; (h) NBS, AIBN, CCl , 50 °C, 2 h then morpholine, DCM, 50 °C, 1 h, >20% over 2 steps.
4
The cellular activities, physicochemical properties and microsome stabilities of analogues
2
7a−f are shown in Table 3. Polar groups such as −CO H and amides improved microsome stability as
2
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
well as solubility but were not tolerated with EC values > 100 µM in HepDE19 cells (analogues
50
2
7a/b/d). Heteroaryls and bulky groups like isopropyl ester were also detrimental to both cellular
potency and metabolic stability. For example, analogues 27e and 27f showed moderate anti-HBV
activities and narrow selectivity margins, with reduced metabolic stabilities in liver microsomes. In
contrast, the less lipophilic 5-CN analogue 27c had a better selectivity margin and microsome stability
than 27e and 27f, with an EC of 2.7 µM in HepDE19 cells.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
Table 3. The Effects on HBV DNA Reduction and Physicochemical Properties of 27a−f
27a-f
a
a
a
EC50
µM]
CC50
LYSA
5
a
a
b
ID
7a
7b
27c
7d
R
SI
logP
MLM
[
[µM]
[µg/mL]
2
−CO
H
2
>100
61.8
2.7
>100
-
-
1.0
1.9
2.4
>504
6
2
>100
>100
515
6
59
79
−CN
>37
-
2
>100
>100
1.9
573
77
2
7e
2.0
6.1
42.3
74.7
21
3.4
3.9
263
1
89
88
27f
12.2
a
b
Definitions are the same as those described in Table 1.
−1
−1
Scaled intrinsic clearance [mL min kg ] in mouse liver microsome (MLM). Experiments were run in duplicate, with
variation < 10%.
Section 5. SAR at the 6-position of 4-Methyl HAPs. As part of our effort to optimize the 4-
methyl HAP series, we sought to address the potential metabolic liability of the 6-morpholine moiety
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that is often associated with N−C and O−C cleavage. Thus, we developed a highly efficient and easily
scalable synthesis of 7a. First, tetrasubstituted olefin 29 was obtained by one-step indium (III)-
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
3
catalyzed condensation of β-ketoester 8 and alkyne 28 in 40% yield (Scheme 4). Second, the
condensation of 29 and thiazole-2-carboxamidine was carried out as described before and the product
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
a was converted to bromide intermediate 30 for chemical diversification. Finally, in the case of N-
linked analogues, 30 was treated with selected amines or (sulfon)amides (32) and complementary bases
such as K CO and t-BuOK in DMF. The resulting products were then treated with TFA to give 31a−r
2
3
(
Table 4). For the synthesis of 6-amino acid substituted analogues 31s−t, amino esters were alkylated
with bromide 30, followed by LiOH-mediated hydrolysis and finally removal of the N-Boc protecting
group. These analogues were then tested in HepDE19 cells.
a
Scheme 4. Synthesis of N-linked and O-linked Analogues 31a−w
a
b
8
28
29 (E/Z)
7a
e, f, g
c, d
7a
3
1a-t
32
N-linker
30
h, f, g
33
31u-w
O-linker
a
Reagents and conditions: (a) In(OTf) , o-xylene, 120 °C, 1 h, 40%; (b) thiazole-2-carboxamidine, NaHCO , NMP, 120 °C,
3
3
2
h, 60%; (c) (Boc) O, DMAP, DCM, rt, overnight; (d) NBS, CCl , 50 °C, 2 h, 82% for 2 steps; (e) amine 32, K CO or t-
2 4 2 3
BuOK [for (sulfon)amides], DMF, 40 °C, 3 h; (f) LiOH, MeOH/H O, rt, 2h for hydrolysis of the amino ester group; (g)
2
TFA, DCM, rt, 2 h; (h) alcohol 33, NaH, THF, rt, overnight.
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9
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Substitution of the morpholine group with piperidine and piperazine, common “bioisosteres” of
morpholine, led to a complete loss of activity (analogues 31a−b). Gem-dimethyl morpholine was well
tolerated (31c), suggesting that some steric bulk could be accommodated. However, a
conformationally-constrained bridged morpholine led to a significant potency drop (31d). Finally,
lactams were clearly detrimental and both 31e and 31f had weak cellular activities.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Without knowing the molecular interactions that the morpholine group plays in capsid binding,
we attempted to identify fragments that may resemble the role of the morpholine oxygen atom. The 4-
hydroxyl piperidine analogue 31g was weakly active with an EC of 65.2 µM, while analogues with
50
thiomorpholine dioxide and 4,4-difluoro piperidine substitutions (31h and 31i) were about 10 times
more potent than 31g. By shifting the gem-difluoro substituents to the 3-position of the piperidine ring,
the anti-HBV potency was further improved as analogue 31j had an EC50 of 2.1 µM. Next, 3,3-
difluoropyrrolidine-substituted analogue 31m was prepared and showed a 15-fold improvement over
3
1j with an EC50 value of 0.14 µM. In contrast, 3-CF -substituted pyrrolidine analogue 31n had
3
moderate potency with an EC of 8.1 µM and 3-hydroxyl pyrrolidine analogue 31k was only weakly
5
0
active. Notably, sulfonamide analogue 31p had better activities than lactams 31e and 31f, and groups
like imidazole and azetidine were not tolerated (analogues 31q−r).
Table 4. The Effects on HBV DNA Reduction and Physicochemical Properties of 31a−w
3
1a-w
a
a
a
a
EC50
CC50
LYSA
MLM
4
ID
R
SI
logP
−1
−1
[µM]
µM]
[µg/mL]
[mL min kg ]
31a
>100
>100
-
-
4.7
2.7
282
88
3
1b
>100
>100
250
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3
4
5
6
7
8
9
3
1c
1d
1e
0.19
17.3
66.6
350
>6
3.4
3.2
13
88
84
3
>100
564
3
65.3
70.2
>100
>100
-
-
2.4
2.1
440
34
80
81
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
1f
31g
31h
65.2
6.4
>100
>100
>100
-
3.2
1.8
4.5
593
15
81
66
88
>16
>14
3
1i
1j
7.1
< 1
3
2.1
>100
>100
92
>48
-
4.5
2.7
4.0
ND
503
5
89
83
89
3
1k
64.7
0.14
31m
31n
31p
657
8.1
46
6
5.1
3.3
13
88
79
16.7
>100
>6
125
3
1q
>100
>100
>100
>100
>100
>100
-
-
-
3.9
2.9
0.6
338
ND
ND
69
88
31r
3
1s
1t
ND
3
21.3
8.1
>100
>100
98
>5
>12
15
1.3
3.4
4.3
>510
30
42
79
81
3
1u
1v
3
6.4
19
3
1w
24.4
>100
>4
3.5
>649
24
a
Definitions are the same as those described in Table 1.
In addition, structural explorations with noncyclic amines were carried out. Among those
modifications, the α-aminocyclopropanecarboxylic acid substituted analogue 31t demonstrated
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
moderate cellular potency with an EC50 of 21.3 µM. In particular, 31t had significantly improved
solubility and metabolic stability compared to other analogues with LYSA > 510 µg/mL and MLM of
−
1
−1
42 mL min kg , respectively. It seems that the c-Pr moiety is important for cellular activity as
sarcosine substituted analogue 31s was inactive. Subsequently, we applied the SAR learning from the
N-linked series and prepared O-linked analogues by treating bromide 30 with alcohols 33 and NaH,
followed by the removal of the N-Boc protecting group by TFA (Scheme 4). 31u−w had moderate anti-
HBV activities, and carboxylic acid analogue 31w had highly attractive physicochemical properties
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
−
1
−1
with LYSA > 649 µg/mL and MLM of 24 mL min kg (Table 4).
a
Scheme 5. Design and Synthesis of Amino Acid Substituted HAPs 34a−f
cyclized AA
*S
*S
*S
*R
34a (W = -CF -)
3
1m (W = -CF -)
31t
2
34d (W = -CF -)

2
2
3
3
4b (W = -OCH -)
(S)-18a (W = -OCH -)
2
34e (W = -OCH -)
2
2
4c (W = -CH -)
d, e
2
a, b, c
*
e
*S
*S
*S
W = -CF2-
*
*S
3
5
(S)-7a
36
34f
a
Reagents and conditions: (a) (Boc) O, DMAP, DCM, rt, overnight; (b) NBS, CCl , 50 °C, 2 h, 82% for 2 steps; (c) amino
2
4
ester, K CO , DMF, 40 °C, 3 h; (d) LiOH, MeOH, rt, 2h; (e) TFA, DCM, rt, 2 h.
2
3
Section 6. Design of 6-Cyclic Amino Acid Substituted 4-Methyl HAPs. Analogues 31c and
1m were highly potent in vitro but lacked sufficient systemic exposure for in vivo efficacy studies,
3
because of their lipophilic characteristics. A logical next step was to combine their 6-substituents with
that of carboxylic acid-based analogues 31t and 31w, which had moderate activities but nevertheless
promising physicochemical properties. Thus, the 6-cyclic amino acid substituted analogues 34a−e were
synthesized from optically pure intermediate (S)-7a according to the procedures shown in Scheme 5.
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Esters 36 were obtained in medium to high yields after N-Boc protection of (S)-7a, bromination and
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
subsequent S 2 reaction with chiral amino esters 35. Esters 36 were then hydrolyzed with LiOH and
N
the resulting acid intermediates were treated with TFA to give desired products.
Among them, the (2S, 4S)-4,4-difluoroproline substituted analogue 34a demonstrated
significantly improved potency and solubility profiles with an EC50 of 0.084 µM and LYSA > 645
µg/mL (Table 5). Importantly, 34a had a selectivity index (SI) of 1905-fold in HepDE19 cells, a
significant improvement over 1. The (2R, 4S)-4,4-difluoroproline substituted analogue 34c was
significantly less active than its diastereoisomer 34a with an EC50 of 6.6 µM, as was the (2R, 4S)-
morpholine acid analogue 34d in comparison to diastereoisomer 34b. Interestingly, the (2R, 4S)-
isomers had better stability profiles than the corresponding (2S, 4S)-isomers in both human and mouse
liver microsome stability tests (analogues 34a−d). The gem-difluoro substituents and the carboxylic
acid moiety appear to be important for anti-HBV activity since the (2S, 4S)-proline analogue 34e lost
activity and the ester analogue 34f was a few times less active than 34a.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. The Effects on HBV DNA Reduction and Physicochemical Properties of 6-Amino Acid Derivatives 34a−f
3
4a-f
a
a
a
a
a
EC50
µM]
CC50
LYSA
MLM
HLM
4
a
a
ID
R
SI
logP
1.2
−1
−1
−1
−1
[
[µM]
[µg/mL]
[mL min kg ] [mL min kg ]
3
4a
0.084
0.77
6.6
160
1905
>357
12
>645
84
80
60
12
4.1
0
34b
34c
>100
82.3
0.14
1.2
>633
>659
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
34d
34e
34f
3.0
>100
>100
>100
>15
-
0.14
1.4
>633
>589
ND
61
14
90
0
>100
0.66
2.3
23
>151
3.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Definitions are the same as those described before. HLM, human liver microsomal test.
Section 7. Co-crystal Structure of 34a in Complex with HBV Capsid Protein. To gain
additional SAR understanding and elucidate the molecular interaction network of 4-methyl HAPs with
the HBV capsid protein, we carried out crystallization studies with those amino acid-based analogues.
A high resolution co-crystal structure of 34a in complex with Y132A mutant capsid protein was
obtained with diffraction up to 1.7Å (PDB 5GMZ). The 4-methyl HAP scaffold of 34a fits into the
hydrophobic pocket of the capsid protein dimer-dimer interface consistent with the 5Å-complex
8
, 22
structure of HAP1, a close analogue of 1.
It is clear that the 2-thiazolyl group of 34a is co-planar
with the dihydropyrimidine core as anticipated and is buried in a well-defined hydrophobic cavity
formed by residues Phe23, Pro25, Tyr118, Trp102, Thr128’ and Ala132’ (Figure 3). In addition, 4-
methyl substitution on the core and the p-fluorophenyl group of 34a occupy an adjacent and larger
hydrophobic pocket and make van der Waals (VDW) contacts with residues including Pro25, Leu30,
Thr33, Trp102, Ile105, Ser106, Val124’, Arg127’, and Thr128’. Given the limited space around the p-
fluorophenyl group, large and polar substituents are generally not well tolerated and they typically
result in loss of activity for analogues such as 7f−h. While an o-substituent on the 4-phenyl group is
expected to take an opposite position to the 4-methyl group on the core, it will elicit steric clash with
the side chain of Thr128 and makes analogues such as 7c much less active. Importantly, the methyl
group of the 5-ester contacts the outer rim of a pocket formed by Leu37 and Thr109 so that polar and
large groups may not be favored at this position (examples 27a−f).
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4
The 4,4-difluoroproline group is partially solvent-exposed, however, the gem-fluoro
substituents interact with Trp125’, Thr128’, Arg133’ and Pro134’ and the carboxylic acid moiety make
bidentate HB interactions with Ser141. Those interactions could contribute to the high binding affinity
of 34a to capsid proteins and its increased anti-HBV activity. In contrast, amino ester analogue 34f and
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
R-diastereoisomer 34c cannot make bidentate HB interactions with Ser141 in the binding site and
both of them had reduced activities relative to 34a. Moreover, a water-bridged HB network exists
between the 2-thiazole and carboxyl groups of 34a (Figure 3). This well-defined water molecule, as
well as the HB formed between the dihydropyrimidine core and Trp102, are also noted in the recent co-
crystal structure of the N-terminal assembly domain of the HBV capsid protein in complex with 4-H
12
HAP analogue 3 (PDB 5E0I). Interestingly, besides the molecule of 34a binding to the dimer-dimer
interface, an additional molecule was identified at the A-B spike probably due to the excess of ligand
during the crystallization setup (Figure. S3 in Supporting Information).
Figure 3. Binding site of 34a with HBV capsid protein Y132A. The compound binding pocket at B-C interface is shown as
an exemplary site. Chain B and Chain C of Y132A-34a structure are colored in cyan and magenta respectively. Compound
34a is highlighted in green stick. Residues within 4 Å radius are shown as lines. Red spheres represent water molecules in
the pocket. Black dash lines indicate hydrogen bonds between 34a and core protein.
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8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Section 8. DMPK and In Vivo Efficacy of 34a. Amino acid analogue 34a had desirable in
2
vitro potency and physicochemical properties with a polar surface area (PSA) of 85 Å and a measured
logD of 0.25. Since the “zwitterionic” structural features of 34a are often associated with permeability
issues, we carried out mechanistic and SDPK studies of 34a. Compound 34a had a Papp (A→B) of 1.9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
−
6
−1
×
10 cm s and P ratio of 3.6 in the Caco-2 assay. The unbound fraction of 34a was determined to
app
be 1.9% and 8.1% in human and mouse plasma, respectively. The SDPK profile of 34a was evaluated
in CD-1 mice following intravenous (i.v.) and oral (p.o.) administration. 34a had moderate plasma
−
1
−1
clearance (Cl) (55.3 mL min kg ) with an oral bioavailability (F) of 36% in mice (Table 6). In
particular, 34a had a high exposure in the liver with an area under the curve (AUC(0−t)) of 107000 μg
−1
−1
L
hr and very limited tissue distribution to lung, heart and brain. The liver exposure of 34a was
about 20 times higher than that in plasma by oral administration. In contrast, 1 is not stable in liver
homogenate so its liver exposure could not be determined (data not included). The preferential liver
distribution of 34a could be a useful characteristic in treating chronic HBV infections, where the
disease organ is the liver.
In addition, 34a exhibits low CYP inhibition with IC s > 30 μM against five major CYP
5
0
enzymes (3A4, 2D6, 2C9, 2C19, 1A2) and was also not a time dependent CYP inhibitor. Moreover,
34a did not significantly inhibit the hERG ion channel with IC20 > 10 µM and was negative in
preliminary in vitro safety evaluations such as glutathione (GSH) adduct, Ames, as well as
micronucleus test (MNT) assays. In contrast, compound 1 and non-carboxylic acid 4-methyl HAP
analogues (S)-18a and 31m were tested positive in GSH adduct assays. Our mechanistic studies
indicated that 34a induces capsid assembly (or aggregation) of the N-terminal capsid assembly domain
(
Cp149) in a concentration dependent manner, in agreement with previous reports on the 4-H HAP-
7
, 13
based capsid inhibitors (see the Supporting Information).
a
Table 6. ADME Parameters of 4-Methyl HAP 34a in CD-1 Mice
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Plasma_C
F
Dose
CL
Vss
Plasma_AUC(0-t) Liver_Cmax
Liver_AUC(0-t)
t1/2 (h)
−1
−1
−1
−1
−1
−1
(mg/kg)
(mL min kg )
(L/kg)
max (μg/L)
(µg L hr )
(μg/L)
(µg L hr )
(%)
8
9
i.v.
5
55.3
0.22
0.18
0.8
12400
2470
1500
5420
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
p.o.
50
41200
107000
36
a
The single-dose pharmacokinetics (SDPK) study of 34a was carried out in CD-1 mice according to standard procedures.
Major parameters, including plasma clearance (Cl), volume of distribution at steady state (Vss), t1/2 (i.v.), maximal
concentration (Cmax), area under the curve (AUC), and oral bioavailability (F) are reported.
Finally, the in vivo efficacy of 34a was evaluated in BALB/c mice that underwent
24
hydrodynamic injection (HDI) with replication-competent HBV DNA plasmid. Entecavir (ETV, 0.1
mg/kg) was used as the positive control in the HDI experiment. HBV replication was quickly built up
in the liver of HDI mice after injection of the HBV plasmid and the levels of HBV DNA were
determined by real-time PCR. As shown in Figure 4A, 34a demonstrated dose-dependent reduction of
HBV DNA in the plasma of infected mice when the compound was orally administered twice a day
(b.i.d.) at 12.5 mg/kg (mpk), 25 mpk and 50 mpk, respectively. In comparison to the vehicle control
group, treatment with 50 mpk of 34a b.i.d. achieved over 2-log viral load reduction in the plasma of
HDI mice on day 5.
The levels of HBV DNA in mouse liver were determined 5 days post hydrodynamic injection
of the HBV DNA plasmid (Figure 4B). The liver DNA was isolated and subjected to real-time PCR for
quantification of HBV DNA. Consistent with the results in mouse plasma, 34a dose-dependently
reduced HBV DNA levels in the mouse liver, achieving levels comparable to the positive control on
day 5 with oral dosing at 50 mpk b.i.d.
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9
1
1
1
1
1
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3
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3
3
3
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0
1
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4
5
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7
8
9
0
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3
4
5
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7
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9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. The levels of HBV DNA in the plasma (A) and liver (B) of infected mice. Treatments with the vehicle control,
4a and ETV are included. The HBV DNA was quantified by real-time PCR. Error bars represent standard error. Statistical
3
analysis was done by Student's t test. * P< 0.001.
CONCLUSIONS
In summary, we have identified a novel class of 4-methyl HAP-based HBV capsid inhibitors by
an information-driven strategy. A highly efficient synthetic method was developed for SAR and
structure−property relationship (SPR) studies with the aim to improve the in vitro anti-HBV activity
and cytotoxicity profile, and to optimize physicochemical properties. Structural optimization resulted
in the identification of (2S, 4S)-4,4-difluoroproline substituted analogue 34a, which exhibited an EC50
value of 84 nM and an over 1900-fold selectivity margin in HepDE19 cells. In particular, this kind of
zwitterionic compound had good plasma stability and oral bioavailability with high liver exposure in
mice. In in vivo studies, 34a dose-dependently reduced HBV DNA in both the plasma and liver of
HBV-infected HDI mice, achieving over 2-log viral load reduction on day 5 with 50 mpk b.i.d. dosing.
Future optimizations of 34a include core modifications and peripheral substituent changes with the
specific aim to reduce efficacious doses.
In the course of SAR exploration, it was noted that minor changes in HAP structure can have
profound effects on the capsid assembly or aggregation process and thus inhibition of HBV DNA
synthesis. A co-crystal structure of 34a and Y132A mutant capsid protein was obtained with 1.7Å
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resolution, clearly elucidating the binding mode of 34a and importantly, the role that the (2S)-4,4-
difluoroproline group plays in protein-ligand interactions. Specifically, the fluorine atoms make
important VDW contacts with Trp125’, Thr128’, Arg133’ and Pro134’ of the capsid protein and the
carboxylic acid moiety forms bidentate HB interactions with Ser141. The SAR, biostructure, SPR,
DMPK and in vivo efficacy studies of 4-methyl HAP analogues like 34a provide valuable information
for further structural optimizations of the HAP scaffold in our endeavor to identify and develop highly
efficacious and safe HBV capsid inhibitors, the results of which will be published in due course.
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
Synthetic Chemistry General Comments. All of the intermediates were purified by silica gel
chromatography using either a Biotage SP1 system or an ISCO CombiFlash chromatography
instrument. All of the final compounds were purified by preparative HPLC (prep-HPLC) on a reversed-
phase column using a Waters XBridge OBD Phenyl (30 mm × 100 mm, 5 μm) or OBD RP18 (30 mm
× 100 mm, 5 μm) column under acidic conditions (A, 0.1% formic acid in H O; B, 0.1% formic acid in
2
acetonitrile) or basic conditions (A, 0.01% ammonia in H O; B, acetonitrile). For SFC chiral separation,
2
the intermediates were separated using a chiral column (Daicel Chiralpak IC, 30 mm × 250 mm, 5 μm)
on a Mettler Teledo SFC-Multigram system (solvent system of 95% CO and 5% IPA (0.5% TEA in
2
IPA), backpressure of 100 bar, UV detection at 254 nm). Optical rotation was measured using a
Rudolph Autopol V automatic polarimeter at a wavelength of 589 nm. LC−MS spectra were obtained
using a MicroMass Platform LC (Waters Alliance 2795-ZQ2000). NMR spectra were obtained using a
Bruker Avance 400 MHz NMR spectrometer. All of the starting materials were obtained commercially,
and amidines 12 were prepared in house according to literature reported methods (see the Supporting
1
Information). All of the final compounds have purities greater than 95% based upon LC−MS and H
NMR analyses. All of the reported yields are for isolated products and are not optimized.
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1
1
1
1
1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
General Synthetic Procedures for 4-Methyl HAP Analogues 7a−i: A mixture of methyl 3-
oxobutanoate (compound 8, 10 mmol) and a benzaldehyde derivative 9 (10 mmol), piperidine (0.5
mmol) and AcOH (0.5 mmol) in anhydrous EtOH (100 mL) was stirred at rt for 12 h. After removal of
the solvent, the residue was purified by flash chromatography to afford the aldol condensation product,
which was then dissolved in 50 mL of anhydrous THF and added dropwise to a fresh prepared THF
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
solution of CH CuLi (2 eq.) at −78 °C. The reaction mixture was stirred for 1 h and quenched with
3
saturated NH Cl solution. The aqueous solution was extracted with EtOAc and the combined organic
4
phase was concentrated. The residue was purified by flash column chromatography to give β-ketoester
10.
To a solution of NaH (60%, 15 mmol) and 10 (10 mmol) in anhydrous THF (100 mL) was added
a solution of phenylselenyl chloride (15 mmol) in THF (20 mL), and the reaction mixture was stirred at
rt for 1 h. A mixture of pentene/ether (v/v =1/1, 60 mL) and 30 mL of saturated NaHCO solution were
3
added to the flask. The organic layer was separated and then treated with H O (30%, 2 mL) in 30 mL
2
2
of DCM. The mixture was stirred at rt for 1 h and diluted with DCM before workup. Tetra-substituted
olefin 11 was obtained as yellowish oil after purification by flash chromatography.
A mixture of amidine 12 (10 mmol) and NaHCO (20 mmol) in 15 mL of NMP was preheated to
3
120 °C , and to this stirring mixture was added dropwise a solution of 11 (10 mmol) in NMP (5 mL).
The reaction mixture was stirred at 120 °C for 2 h before it was cooled to rt and diluted with water. The
aqueous phase was extraced with EtOAc and the combined organic phase was washed with brine, dried
over anhydrous Na SO and concentrated. The residue was purified by flash column chromatography
2
4
to afford 7a−i as powder.
Methyl (4S)-(4-fluorophenyl)-4,6-dimethyl-2-(2-methylthiazol-4-yl)-1H-pyrimidine-5-carboxylate [(S)-
2
0
7
a]. (S)-7a was obtained by chiral SFC separation. Optical rotation: [α]D = 75.6 (1.0 mg/mL, MeOH).
+
+
+
+
1
MS: calcd (MH ) 346.1, exp (MH ) 346.1. HRMS: calcd (MH ) 346.1020, exp (MH ) 346.1020. H
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NMR (Methanol-d ) δ 7.93 (d, J = 3.3 Hz, 1H), 7.72 (d, J = 3.3 Hz, 1H), 7.52−7.46 (m, 2H), 7.07−7.00
4
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
m, 2H), 3.45 (s, 3H), 2.28 (s, 3H), 1.90 (s, 3H).
Methyl 2-(3,5-difluoro-2-pyridyl)-4-(4-fluorophenyl)-4,6-dimethyl-1H-pyrimidine-5-carboxylate (7b).
+
+
+
+
1
MS: calcd (MH ) 376.1, exp (MH ) 376.1. HRMS: calcd (MH ) 376.1267, exp (MH ) 376.1269. H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NMR (Methanol-d ) δ 8.46 (d, J = 2.5 Hz, 1 H), 7.71 (ddd, J = 9.8, 8.6, 2.3 Hz, 1 H), 7.53 (dd, J = 9.1,
4
5.3 Hz, 2 H), 7.04 (t, J = 8.8 Hz, 2 H), 3.45 (s, 3 H), 2.24 (s, 3 H), 1.92 (s, 3 H).
Methyl 4-(2-chloro-4-fluoro-phenyl)-4,6-dimethyl-2-thiazol-2-yl-1H-pyrimidine-5-carboxylate (7c).
+
+
1
MS: calcd (MH ) 380.1, exp (MH ) 380.1. H NMR (Methanol-d ) δ 7.95 (d, J = 3.2 Hz, 1H), 7.72 (d,
4
J = 3.2 Hz, 1H), 7.70−7.67 (m, 1H), 7.15−7.08 (m, 2H), 3.51 (s, 3H), 2.3 (s, 3H), 1.95 (s, 3H).
+
Methyl 4,6-dimethyl-4-phenyl-2-thiazol-2-yl-1H-pyrimidine-5-carboxylate (7d). MS: calcd (MH )
+
1
3
7
1
28.1, exp (MH ) 328.1. H NMR (Methanol-d ) δ 7.93 (d, J = 3.3 Hz, 1H), 7.73 (d, J = 3.3 Hz, 1H),
4
.49 (dd, J = 8.5, 1.1 Hz, 2H), 7.32 (t, J = 7.7 Hz, 2H), 7.24−7.17 (m, 1H), 3.42 (s, 3H), 2.28 (s, 3H),
.92 (s, 3H).
Methyl 4-(4-chlorophenyl)-4,6-dimethyl-2-thiazol-2-yl-1H-pyrimidine-5-carboxylate (7e). MS: calcd
+
+
1
(MH ) 362.1, exp (MH ) 362.1. H NMR (Methanol-d ) δ 7.93 (d, J = 3.0 Hz, 1H), 7.71 (d, J = 3.0 Hz,
4
1
H), 7.44 (d, J = 8.6 Hz, 2H), 7.31 (d, J = 8.6 Hz, 2H), 3.45 (s, 3H), 2.29 (s, 3 H), 1.88 (s, 3H).
Methyl 4,6-dimethyl-4-(4-methylsulfonylphenyl)-2-thiazol-2-yl-1H-pyrimidine-5-carboxylate (7f). MS:
+
+
1
calc’d (MH ) 436.1, exp (MH ) 436.1. H NMR (Methanol-d ) δ 7.94 (d, J = 3.3 Hz, 1H), 7.91 (d, J =
4
8
1
.3 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 7.71 (d, J = 3.3 Hz, 1H), 3.45 (s, 3H), 3.12 (s, 3H), 2.34 (s, 3H),
.93 (s, 3H).
Methyl 4-(4-methoxyphenyl)-4,6-dimethyl-2-thiazol-2-yl-1H-pyrimidine-5-carboxylate (7g). MS: calcd
+
+
1
(MH ) 358.1, exp (MH ) 358.1. H NMR (Methanol-d ) δ 7.93 (d, J = 3.0 Hz, 1H), 7.72 (m, 1H), 7.39
4
(m, 2H), 6.88 (d, J = 8.6 Hz, 2H), 3.79 (s, 3H), 3.44 (s, 3H), 2.26 (s, 3H), 1.89 (s., 3H).
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9
1
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1
1
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2
2
2
2
2
2
2
2
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3
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Methyl 4-(4-cyanophenyl)-4,6-dimethyl-2-thiazol-2-yl-1H-pyrimidine-5-carboxylate (7h). MS: calcd
+
+
+
+
1
(
MH ) 353.1, exp (MH ) 353.1. HRMS: calcd (MH ) 353.1067, exp (MH ) 353.1068. H NMR
(Methanol-d ) δ 7.93 (d, J = 3.3 Hz, 1H), 7.73−7.62 (m, 5H), 3.44 (s, 3H), 2.33 (s, 3H), 1.90 (s, 3H).
4
Methyl 4-(3,4-difluorophenyl)-4,6-dimethyl-2-thiazol-2-yl-1H-pyrimidine-5-carboxylate (7i). MS:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
+
+
1
calcd (MH ) 364.1, exp (MH ) 364.1. HRMS: calcd (MH ) 364.0926, exp (MH ) 364.0925. H NMR
(Methanol-d ) δ 7.94 (d, J = 3.0 Hz, 1H), 7.72 (d, J = 3.3 Hz, 1H), 7.36−7.14 (m, 3H), 3.48 (s, 3H),
4
2
.30 (s, 3H), 1.86 (s, 3H).
Synthesis of 4-Ethyl HAP Analogue 7j. To a mixture of 1-(4-fluorophenyl)propan-1-one
(compound 13, 0.1 mol) in 100mL of EtOH was added NaBH (0.2 mol) and the mixture was stirred at
4
rt for 2 h. After removal of the solvent, the residue was treated with saturated Na CO solution and
2
3
extracted with EtOAc. The organic phase was washed with brine, dried over anhydrous Na SO and
2
4
concentrated. The crude product was dissolved in 200 mL of DCM and to this solution was added
thionyl chloride (0.2 mol) at 0 °C . After 3 h of stirring at rt, the mixture was concentrated and benzyl
chloride 14 was obtained as yellowish oil.
To a mixture of 8 (2.9 mmol) in THF (20 mL) was added NaH (60%, 3.5 mmol) and the
reaction mixture was stirred at 0 °C for 20 min. Chloride 14 (2.9 mmol) was added into the flask and
the resulted mixture was stirred at rt for 12 h before it was quenched with 5 mL of brine. The organic
layer was separated and concentrated. The residue was purified by flash chromatography to afford
+
methyl 2-acetyl-3-(4-fluorophenyl)pentanoate (15) in 60% yield. MS: exp (MH ) 253.3.
Methyl 2-acetyl-3-(4-fluorophenyl)pent-2-enoate (16) was obtained as a mixture of E/Z isomers from
1
5 by the two-step phenylselenation and oxidation reactions as described previously. To a mixture of
thiazole-2-carboxamidine hydrochloride (0.4 mmol) and AcOK (0.4 mmol) in 5 mL of isopropanol was
added a solution of 16 (0.4 mmol) in 2 mL of isopropanol at 120 °C . The reaction mixture was stirred
at 120 °C for 3 h before concentrated in vacuum. The residue was purified by prep-HPLC to afford 7j
as yellowish solid in 15% yield.
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Methyl 4-ethyl-4-(4-fluorophenyl)-6-methyl-2-thiazol-2-yl-1H-pyrimidine-5-carboxylate (7j). MS:
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
+
+
1
calcd (MH ) 360.1, exp (MH ) 360.1. HRMS: calcd (MH ) 360.1182, exp (MH ) 360.1191. H NMR
(Methanol-d ) δ 7.97 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.49−7.45 (m, 2H), 7.04−7.00 (m,
4
2
H), 3.51 (s, 3H), 2.65 (m, 1H), 2.30(s, 3H), 2.04 (m, 1H), 1.00 (m, 3H)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
1
2
3
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7
8
9
0
1
2
3
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5
6
7
8
9
0
General Procedures for the Synthesis of 6-Morpholine Substituted Analogues 18a−k. To a
solution of intermediates 7 (3 mmol) and DMAP (1.5 mmol) in 20 mL of DCM was added Boc O (4.5
2
mmol), and the mixture was stirred at rt overnight. The organic phase was washed with water and brine,
dried over anhydrous Na SO and concentrated. The residue was purified by flash chromatography to
2
4
afford the N-Boc protected intermediate in quantitative yield. The product was dissolved in 30 mL of
CCl . To this solution was added NBS (3 mmol) and the mixture was brought to 50 °C . AIBN (30 mg)
4
was added to the flask and the mixture was stirred at 50 °C for 2 h. The mixture was filtered through
silic gel and washed with EtOAc. The filtrate was concentrated to afford 17 as yellowish powder and it
was used for the next reaction directly.
To a solution of 17 (1 mmol) in 20 mL of DCM was added morpholine (0.5 mL) and the
mixture was stirred at 50 °C for 1 h. After concentration, the residue was dissolved in EtOAc and
washed with 1N HCl and brine. The organic phase was concentrated and the residue was dissolved in
20 mL of DCM. To this solution was added TFA (1 mL) and the mixture was stirred at rt for 2 h. After
removal of the solvent, the residue was purified by prep-HPLC to afford 18a−k.
(
S)-4-(4-Fluoro-phenyl)-4-methyl-6-morpholin-4-ylmethyl-2-thiazol-2-yl-1,4-dihydro-pyrimidine-5-
carboxylic acid methyl ester [(S)-18a]. (S)-18a was obtained in together with (R)-18a by the SFC
20
+
chiral separation of racemic 18a. Optical rotation: [α]D = 70.5 (1.0 mg/mL, MeOH). MS: calcd (MH )
+
+
+
1
4
31.1, exp (MH ) 431.1. HRMS: calcd (MH ) 431.1548, exp (MH ) 431.1550. H NMR (Methanol-d )
4
δ 7.96 (d, J = 3.0 Hz, 1H), 7.74 (d, J = 3.3 Hz, 1H), 7.52−7.43 (m, 2H), 7.03 (t, J = 8.8 Hz, 2H), 3.78
t, J = 4.4 Hz, 4H), 3.64 (d, J = 17.1 Hz, 1H), 3.60 (d, J = 16.2 Hz, 1H), 3.46 (s, 3H), 2.55−2.60 (m,
4H), 1.90 (s, 3H).
(
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