Design, Synthesis, and Evaluation of Tetrahydropyrrolo[1,2-c]pyrimidines as Capsid Assembly Inhibitors for HBV Treatment

Article abstract of DOI:10.1021/acsmedchemlett.7b00288

The discovery of novel tetrahydropyrrolo[1,2-c]pyrimidines derivatives from Bay41-4109 as hepatitis B virus (HBV) inhibitors is herein reported. The structure-activity relationship optimization led to one highly efficacious compound 28a (IC₅₀ = 10 nM) with good PK profiles and the favorite L/P ratio. The hydrodynamic injection model in mice clearly demonstrated the efficacy of 28a against HBV replication.

Full text of DOI:10.1021/acsmedchemlett.7b00288

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Letter
Design, Synthesis and Evaluation of Tetrahydropyrrolo[1,2-
c]pyrimidines as Capsid Assembly Inhibitors for HBV treatment
Xiaolin Li, Kai Zhou, Haiying He, Qiong Zhou, Ya Sun, Lijuan Hou, Liang Shen, Xiaofei Wang,
Yuedong Zhou, Zhen Gong, Shibo He, Huangtao Jin, Zhengxian Gu, Shuyong Zhao, Long Zhang,
chunyan Sun, Shansong Zheng, Zhe Cheng, Yidong Zhu, Minghui Zhang, Jian Li, and Shuhui Chen
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00288 • Publication Date (Web): 24 Aug 2017
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Design, Synthesis and Evaluation of Tetrahydropyrrolo[1,2-
c]pyrimidines as Capsid Assembly Inhibitors for HBV treatment
Xiaolin Li1, Kai Zhou1, Haiying He1*, Qiong Zhou1, Ya Sun 1, Lijuan Hou1, Liang Shen1, Xiaofei
Wang1, Yuedong Zhou1, Zhen Gong1, Shibo He1, Huangtao Jin1, Zhengxian Gu1, Shuyong Zhao2, Long
Zhang2, Chunyan Sun2, Shansong Zheng2, Zhe Cheng2 , Yidong Zhu2, Minghui Zhang2,
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Jian Li1, Shuhui Chen1
1WuXi AppTec (Shanghai) Co., Ltd, 288 FuTe Zhong Road, Shanghai, 200131, P. R. China
2Shandong Provincial Key Laboratory of small molecular targeted drugs, Qilu Pharmaceutical Co., Ltd. No. 243 Gong Ye
Bei Road, Jinan, Shandong Province, 250100, P. R. China
KEYWORDS Hepatitis B virus (HBV), Tetrahydropyrrolo[1,2-c]pyrimidines, Capsid assembly inhibitor
ABSTRACT: The discovery of novel tetrahydropyrrolo[1,2ꢀc]pyrimidines derivatives from Bay41_4109 as hepatitis B virus inhibꢀ
itors is herein reported. The SAR optimization led to one highly efficacious compound 28a (IC50 = 10 nM) with good PK profiles
and favorite L/P ratio. The Hydrodynamic Injection (HDI) model in mice clearly demonstrated the efficacy of 28a against HBV
replication.
Background and introduction
Therefore, it is crucial to develop safer and more effective
antiꢀHBV agents with novel mechanisms 6ꢀ8
.
Hepatitis B is a potentially lifeꢀthreatening liver disease
caused by the hepatitis B virus (HBV) infection. Over 350
million people have chronical HBV infection . Chronic HBV
Heteroaryldihydropyrimidines (HAPs), discovered by a cell
cultureꢀbased screening, were identified as a novel class of
HBV inhibitors targeting capsid assembly and RNA packaging
9. It was reported that HAPs could enhance the rate of assemꢀ
bly and cause the formation of aberrant capsid, thus lead to
decreased production of virions 10. However, HAPs were hyꢀ
drophobic due to their special chemical structures 11,12. The
poor water solubility significantly affected the drug candiꢀ
date’s oral PK properties and eventually limited the chance of
being developed into a pharmaceutical product.
1
infection can lead to liver cirrhosis and hepatocellular carciꢀ
noma (HCC)2. Currently, there are two types of antiꢀHBV
agents
in
the
market:
immune modulators (IFNꢀα) and nucleosi(t)des as reverse
transcriptase inhibitors (Lamivudine, Entecavir, Telbivudine,
3
Adefovir and Tenofovir ) . However, the useage of IFNꢀα is
limited due to its low efficacy, high cost and serious side efꢀ
fects; while nucleos(t)ides often result in drugꢀresistance after
longꢀterm treatment4,5. Moreover, both IFNꢀα and nucleꢀ
os(t)ide analogs cannot eradicate chronic HBV infection.
GLS4 (Scheme 1), derived from BAY41-4109 (Scheme 1),
is currently in phase II clinical trial as an antiꢀHBV agent,
which also targets the HBV capsid formation [13ꢀ16]. GLS4 was
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ACS Medicinal Chemistry Letters
as potent as the prototypeꢀBAY41-4109. Moreover, it can
inhibit both wildꢀtype and Adefovir resistant strains of HBV in
cell culture. However, the poor oral bioavailability of GLS4
might become a hurdle for further development.
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Scheme 1 Chemical structures of BAY41-4109, GLS4
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We herein report the approaches for the design, synthesis,
biological evaluation of
a
novel series of 3,5,6,7ꢀ
tetrahydropyrrolo[1,2ꢀc]pyrimidine derivatives as HBV capsid
assembly inhibitors.
The derivatives (Scheme 2) were designed based on HAPs.
Although it was reported that blocking the hydrogen on pyrriꢀ
midine with alkyl group resulted in loss of antiviral activity17,18
we discovered that the fused ring analogs could maintain
moderate to excellent potency.
,
Scheme 2 Chemical structures of the derivatives
The analogues in table 1 were evaluated by the cellꢀbased
antiꢀHBV assay. The HepG2.2.15 cells were seeded at 4 × 104
cells/well in a 96ꢀwell plate (100 ꢁl/well) in seeding medium
and incubated overnight at 37 °C in a 5% CO2 humid atmosꢀ
phere. Next day, compounds were added to the cell culture.
Three days later, cells were replenished with fresh medium.
After another 3ꢀday incubation, cell culture supernatants were
collected and stored at −80 °C until HBV DNA was used.
HBV DNA was extracted from supernatants with QIAamp 96
DNA Blood Kit and quantified by qPCR.
* Mean value of three experiments
Scheme 3: Synthetic route of 2
The IC50 data in table 1 showed the fused ring analogs can
keep moderate activity. Among them, compound 2 possessed
best potency against HBV.
A general synthetic route for compound 2 is illustrated in
Scheme 3. The THP protected ethyl 3ꢀoxoheptanoate was reꢀ
acted with amidine 10 and the substituted benzaldehyde to
produce the cyclized compound 12 under the Biginelli condiꢀ
tion 19. Compound 12 was deprotected with pꢀTsOH to afford
compound 13, followed by cyclization in the presence of
MsCl/Et3N to generate the racemic compound 2.
Utilizing the most potent compound 2 as a starting point,
we firstly investigated the effects of different R1 groups as
shown in Table 2. The SAR analysis indicated that the thiazole
group is the best moiety in this region.
Table 1 Anti-HBV activity of the fused ring analogs in
HepG 2.2.15 cells
Table 2 Anti-HBV activity in HepG2.2.15 cells via R1 modi-
fication
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We explored the further SARꢀbased optimization of comꢀ
pound 2 at R2 position (Table 3). 2ꢀBromoꢀ4ꢀfluorophen(15a)
provided a comparable activity (IC50 = 0.21 ꢁM). Replaceꢀ
ment of the phenyl at 2, 3 or 4 position can retain the potency,
such as 2ꢀchloroꢀ3ꢀfluorophenyl (15b, IC50 = 0.31 ꢁM). Howꢀ
ever, it led to less potency if substituted at 2,5 or 2, 6 positions,
for example, 2ꢀchloroꢀ5ꢀfluorophenyl (15c) and 2ꢀchloroꢀ6ꢀ
fluorophenyl (15d). 2,4ꢀDifluorophenyl (15e, IC50 = 0.51 ꢁM)
and 3,4ꢀdifluorophenyl (15f, IC50 = 0.43 ꢁM) analogs reꢀ
mained similar potency. No significant potency improvement
was observed by introducing methyl group at the 2ꢀposition of
phenyl (15g, IC50 = 0.45 ꢁM). The replacement of 2ꢀbromoꢀ4ꢀ
fluorophen with 1ꢀmethylꢀ1Hꢀpyrazolor thiazol, resulted in the
loss of potency, such as compound 15m or 15k. Besides，
introduction of pyridine analogues (15i and 15j) at R2 position
also led to the loss of potency.
Based on the above result, further SAR was investigated at
R3 region (Table 4). We found it led to loss of potency by reꢀ
placing ethyl with 2ꢀmethoxyethyl carboxylate (16c) and 2ꢀ
(dimethylamino)ethyl carboxylate (16d), but no difference if
replaced with methyl group (16a). The decline in potency was
also observed when the ethyl group of 16a was replaced with
the similar methoxymethyl group (16b) or 2,2ꢀdifluoroethyl
group (16e). Furthermore, replacement of the ethyl carboxꢀ
ylate with Nꢀmethoxy carboxylate (16f, IC50 = 5.0 ꢁM) did not
maintain the potency.
* Mean value of three experiments
Table 4: Anti-HBV activity in HepG2.2.15 cells via R3
modification
Table 3: Anti-HBV activity in HepG2.2.15 cells via R2
modification
* Mean value of three experiments
Interestingly, when we investigated the substituents at the
five membered ring, we found the compound 26 and 27 with
Boc group also showed good potency with IC50 = 50 nM and
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30 nM, respectively (Table 5). So we did further exploration at percritical CO2/15% EtOH at 100 mL/min rate. The absolute
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this region and identified the methylsulfonyl analogs such as
28a, 29a and 30 possessed excellent activities. The absolute
stereochemistry of 30 was determined by the single crystal Xꢀ
ray study (see supporting information). Compared to (3R,6R)
isomers such as 28b (IC50 = 60 nM) and 29b (IC50 = 70 nM),
the (3R,6S) isomers (28a and 29a) were 6~7ꢀfold more potent.
Based on the efficacy of 28a and 29a, there was no difference
if methyl ester replaced by ethyl ester. We kept the ethyl ester
moiety and conducted the optimization on the pyrrolo ring, but
hydroxyl (31 ), N,NꢀDimethyl (32) and the methyl (33) anaꢀ
logs did not keep potency.
stereochemistry of (−)ꢀenantiomer 20 was determined by its
rotation value as reported21. Bromination of compound 20
generated 21, which was reacted with 22 via three steps to
give compound 23. The subsequent reduction of 23 with
NaBH4 afforded compound 24. Compound 25 was generated
by cyclization of 24 in the presence of MsCl, Et3N. The comꢀ
pounds 26 and 27 were seperated by the of SFC separation
with higher polarity of 27. Compound 27 reacted with
HCl/EtOAc to give the amine intermediate, then coupled with
MsCl to generate the compound 28a.
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The computer modeling on interaction of compound 28a
with HBV capsid protein.
Table 5: Anti-HBV activity in HepG2.2.15 cells
The dihydropyrimidine compounds can potentially bind to
the interface of HBV core proteins, disrupting the formation of
HBV capsid, therefore, blocking the lifecycle of HBV 22–24. To
better understand the SAR, some of the analogues in this work
were docked into the crystal structure of HBV capsid (PDB
code 5GMZ)
The binding pose of sulphonamide compound 28a in core
protein is shown in Figure 1. The F, Clꢀ phenyl occupies a
compact and hydrophobic pocket which consists of Thr33,
Ile105, Leu30, Pro25, Trp102 in one of the core proteins
(chain B), and Val124, Arg127 and Thr128 in the other core
protein (chain C). This pocket is very tight and hydrophobic,
where the fluorine, chlorine, bromine analogues are potent, but
polar and bulky groups are generally not well tolerated (Table
3). The dihydropyrimidine can catch a key hydrogen bond to
Trp102, as well as have hydrophobic interactions with Leu140
and Val124. On the other hand, the dihydropyrimidine core
has a unique conformation, which facilitates the F, Clꢀphenyl
binding to the tight pocket with an optimal dihedral angle. The
thiazole can form hydrophobic interaction with Pro25, Thr128,
* Mean value of three experiments
as well as π−π stacking with Phe23 and Tyr118. Some heteroꢀ
cycles with high polarity, such as pyrrozole and pyridine, can
reduce activity. Moreover, saturated rings may even signifiꢀ
cantly drop activity (Table 2). Most of these interactions lie in
the deep pocket, stabilizing the conformation of 28a in the
interface of HBV core proteins.
To gain deeper insights into the mechanisms of our comꢀ
pounds, we did the evaluation activity of 28a, Bay41_4109
and GLS4 against HBV capsid assembly in the capsid quenchꢀ
ing assay. 28a, Bay41_4109 and GLS4 had IC50 values of 0.8,
0.43 and 1.2 µM, respectively, which proved the similar casꢀ
pid assembly inhibition mechanism.
The fused pyrrolidine can form hydrophobic interactions
with Leu140, Phe110 and Val124, moreover, it acts as a rigid
linker, which can deliver a hydrophilic tail to a region near
solvent. Given the high lipophilicity of lead compound 2, the
free space around this region make it possible to introduce
hydrophilic groups to improve the solubility and pharmaꢀ
cokineitcs properties. The vicinity of this hydrophilic region
has Leu140 and Ser141, where two amides on main chain and
a hydroxyl on side chain point to the inner pocket, forming a
hydrogen bond donor rich region. The sulphonamide potentialꢀ
ly can catch a hydrogen bond to Ser141, stabilizing the conꢀ
formation of compound 28a in the binding pocket (Figure 1).
For the potent analogue 26, its carbamate tail can also catch a
hydrogen bond to Ser141, and its tꢀBu can have additional
hydrophobic interactions with Pro134 and Ile139. However,
those compounds with less hydrogen bond acceptors on the
tail (31, see table 5) and even basic compounds (32) may lose
the hydrogen bond at this region, thus become less potent. For
the flexibility of pyrrolidine, both R and S enantiomers are
tolerated in the pocket, but generally the compounds in S conꢀ
figuration can catch hydrogen bonds and fit binding pocket
better. Substitutions at position 7 of tetrahydropyrrolopyrimiꢀ
dine are very close to the thiazole ring, which can change its
Scheme 4: Synthetic route of 28a
A general synthetic route for compound 28a is illustrated in
Scheme 4. The compound 10 reacted with 2ꢀClꢀ4ꢀFꢀ
benzaldehyde and ethyl 3ꢀoxobutanoate (17) to generate the
cyclized compound 18 under the Biginelli condition. The enꢀ
antiopure 20 was obtained through SFC chiral separation of
the stereo mixtures eluting with a mixed solvent of 85% suꢀ
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conformation and affect protein binding. For example, the
addition of methyl in 33 significantly drops activity.
(1 mpk)
Vdss (L/kg)
4.1±0.6
56±7
2.0±0.2
21±3
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8
Cl
(mL/min/kg)
AUC0ꢀlast (µMꢂh)
0.6±0.1
0.7±0.1
1.7±0.2
4.7±0.3
Cmax (µM)
AUC0ꢀlast (µMꢂh)
F%
PO a *
(10 mpk)
0.8±0.1
9.1±1.5
14%
54%
a. The single-dose pharmacokinetics of GLS4 and compound 28a was carried out in
female Balb/c mice(n=3) according to standard procedures; *vehicle: 1.00 mg/mL in
0.5%HEC.
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Figure 1. The putative binding pose of compound 28a in HBV
capsid. The core proteins, chain B and C, are shown in magenꢀ
ta and cyan cartoon. The ligand is shown as ballꢀand stick,
where carbon atoms are in green. (a) A view from the left
hand side of the entrance to binding site; (b) A view from the
right hand side; (c) The binding pocket of the interface of core
proteins; (d) 2D plot of compound 28a in HBV capsid. The
figures were prepared by pymol 25 and maestro 26 packages.
Figure 2. The effect of treatment of GLS4 and 28a on inhibiꢀ
tion of HBV replication in HDI mice. Mice (n=6 per group)
were orally dosed with vehicle (0.5% HEC), Compound 28a
(50 mpk, bid) or GLS4 (100 mpk, bid). The levels of HBV
DNA in plasma (A) and liver (B) were determined by qPCR.
Error bars represent standard error. Statistical analysis was
performed by Student's t test.
Physical properties, ADME/PK profiles of GLS4 and 28a:
Compound 28a showed desirable in vitro potency and physꢀ
icochemical properties (Table 6). In the in vitro DDI assessꢀ
ment, compound 28a displayed limited risk on CYP inhibition
in a panel of five enzymes (CYP1A2, 2C9, 2C19, 2D6 and
3A4). 28a exhibited moderate plasma clearance (Cl) (21 mL
min−1 kg−1) with good oral bioavailability of 54% in mice,
while the oral bioavailability of GLS4 was only 14%.
The in vivo antiviral activity of 28a was evaluated in a hyꢀ
drodynamic injection (HDI) HBV mouse model27 . In considꢀ
eration of higher oral exposure of 28a than GLS4, 28a was
dosed at 50 mpk, BID versus GLS4 at 100 mpk, BID. As
shown in Figure 2, 28a demonstrated statistically significant
reduction (p < 0.01) of HBV DNA in both mouse plasma and
liver. In comparison with the vehicle group, 50 mpk (BID) of
compound 28a treatment achieved 2.05 and 2.57 log viral
DNA reduction in mouse plasma on days 3 and 5, respectively
(Figure 2A), while 100 mpk (BID) of reference compound
GLS4 treatment achieved 0.35 and 1.56 log viral DNA reducꢀ
tion in mouse plasma on days 3 and 5, respectively. Treatment
of the mice with 28a and GLS4 reduced 0.68 and 0.91 log
viral load of DNA in mouse liver on day 7, respectively (Figꢀ
ure 2B).
** p < 0.01.
CONCLUSIONS:
In summary, we have described the successful structural opꢀ
timization of HAPs to 3,5,6,7ꢀtetrahydropyrrolo[1,2ꢀ
c]pyrimidines as a novel class of HBV capsid assembly inhibiꢀ
tors. Some of these newly developed derivatives demonstrated
highly potent in vitro activities against HBV replication.
Structural optimization resulted in the identification of one
lead compound 28a with IC50 value of 5 nM in HepG2.2.15
cells. Compound 28a also showed good oral bioavailability
with favorite L/P ratio (10:1) in mice. The HBV HDI mouse
model demonstrated that 28a significantly reduced HBV DNA
in both mouse plasma and liver. These results demonstrate that
28a is a promising lead compound for further optimization.
ASSOCIATED CONTENT
Supporting Information
Synthetic procedures, analytical data, assay protocol, inhibitor
mechanism of action are available free of charge on the ACS
Publications website.
Table 6: Physical properties, ADME/PK profiles
AUTHOR INFORMATION
Compound ID
MW/tPSA
GLS4
508/76
4.7/4.4
14
28a
484/100
2.7/ 2.8
22
Corresponding Author
CLogP / LogD
KS (µM)
* Eꢀmail: he_haiying@wuxiapptec.com
Liver/Plasma ratio of
AUC0ꢀ4h(µMꢂh)
ABBREVIATIONS
9
10
KS, kinetic solubility; PK, pharmacokinetics; L/P ratio, Livꢀ
er/Plasma ratio of AUC; SAR, structureꢀactivity relationship; CYP,
CYP (µM)
1A2/2C9/2C19/2D6/3A4
>50/16/4/52/2
>50/ 28/ 39/ >50/11
IV
T1/2 (h)
1.5±0.1
1.1±0.1
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ture−Activity Relationships and Optimization of Heterocyclic
Substituents. J. Med. Chem. 2008, 51, 1719.
19. Kappe, C. O. 100 years of the biginelli dihydropyrimidine synꢀ
thesis. Tetrahedron. 1993, 49, 6937.
20. Liu, N ; Zhao, F ;Zhan, P ; Liu, X . Review of small synthetic
Cytochrome P450; hERG, human etherꢀaꢀgoꢀgo related gene; IV,
intravenous; PO, per oral; AUC, area under curve; mpk, mg/kg
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