Discovery of hepatitis B virus capsid assembly inhibitors leading to a heteroaryldihydropyrimidine based clinical candidate (GLS4)

Article abstract of DOI:10.1016/j.bmc.2016.12.017

Inhibition of hepatitis B virus (HBV) capsid assembly is a novel strategy for the development of chronic hepatitis B (CHB) therapeutics. Herein we described our lead optimization studies including the synthesis, molecular docking studies and structure-activity relationship (SAR) studies of a series of novel heteroaryldihydropyrimidine (HAP) inhibitors of HBV capsid assembly inhibitors, and the discovery of a potent inhibitor of HBV capsid assembly of GLS4 (ethyl 4-[2-bromo-4-fluorophenyl]-6-[morpholino-methyl]-2-[2-thiazolyl]-1,4-dihydro-pyrimidine-5-carboxylate) which is now in clinical phase 2. GLS4 demonstrated potent inhibitory activities in HBV HepG2.2.15 cell assay with an EC₅₀value of 1 nM, and it also exhibited high potency against various drug-resistant HBV viral strains with EC₅₀values in the range of 10–20 nM, more potent than the typical HBV polymerase inhibitors such as lamivudine, telbivudine, and entecavir. Pharmacokinetic profiles of GLS4 were favorable and safety evaluation including acute toxicity and repeated toxicity study indicated that GLS4 was safe enough to support clinical experiments in human.

Full text of DOI:10.1016/j.bmc.2016.12.017

Accepted Manuscript
Discovery of Hepatitis B Virus Capsid Assembly Inhibitors Leading to a Het-
eroaryldihydropyrimidine Based Clinical Candidate (GLS4)
Qingyun Ren, Xinchang Liu, Zhonghua Luo, Jing Li, Chaolei Wang, Siegfried
Goldmann, Jiancun Zhang, Yingjun Zhang
PII:
S0968-0896(16)31201-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.12.017
BMC 13444
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
16 November 2016
10 December 2016
Please cite this article as: Ren, Q., Liu, X., Luo, Z., Li, J., Wang, C., Goldmann, S., Zhang, J., Zhang, Y., Discovery
of Hepatitis B Virus Capsid Assembly Inhibitors Leading to a Heteroaryldihydropyrimidine Based Clinical
Candidate (GLS4), Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.12.017
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Discovery of Hepatitis B Virus Capsid Assembly Inhibitors Leading to a
Heteroaryldihydropyrimidine Based Clinical Candidate (GLS4)
Qingyun Ren a, b, Xinchang Liu a, b, Zhonghua Luo a, Jing Li a, Chaolei Wang a, b, Siegfried
Goldmann a,*, Jiancun Zhang c, Yingjun Zhang a,*
a Sunshine Lake Pharma Co., Ltd, Dong Guan, 523808, China
bAnti-infection Innovation Department, New Drug Research Institute, HEC Pharma Group, Dong Guan,
523871, China
cGuangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
Keywords: HBV, capsid assembly inhibitor, SAR, GLS4, pharmacokinetics, safety evaluation
ABSTRACT: Inhibition of Hepatitis B Virus (HBV) capsid assembly is a novel strategy for the development
of chronic hepatitis B (CHB) therapeutics. Herein we described our lead optimization studies including the
synthesis, molecular docking studies and structure-activity relationship (SAR) studies of a series of novel
Heteroaryldihydropyrimidine (HAP) inhibitors of HBV capsid assembly inhibitors, and the discovery of a
potent inhibitor of HBV capsid assembly of GLS4 (ethyl 4-[2-bromo-4-fluorophenyl]-6-[morpholino-methyl]-
2-[2-thiazolyl]-1,4-dihydro-pyrimidine-5-carboxylate) which is now in clinical phase 2. GLS4 demonstrated
potent inhibitory activities in HBV HepG2.2.15 cell assay with an EC50 value of 1 nM, and it also exhibited
high potency against various drug-resistant HBV viral strains with EC50 values in the range of 10-20 nM, more
potent than the typical HBV polymerase inhibitors such as lamivudine, telbivudine, and entecavir.
Pharmacokinetic profiles of GLS4 were favorable and safety evaluation including acute toxicity and repeated
toxicity study indicated that GLS4 was safe enough to support clinical experiments in human.
__________
1

*
Corresponding author. E-mail address: zhangyingjun@hecpharm.com (Yingjun, Zhang).
sigi@hecpharm.com (Siegfried Goldmann).
1. INTRODUCTION
HBV infection is a serious global public health problem. Currently, it was estimated that more than 350
million people worldwide are chronically infected with HBV virus, and each year about one million chronic
hepatitis patients develop into more life-threatening diseases such as liver failure, cirrhosis or hepatocellular
carcinoma (HCC).1, 2
Current treatment regimens for the patients with chronic HBV infection rely mainly on the use of interferon-
α(IFN) and nucleotide reverse transcriptase and polymerase inhibitors. However, clinical use of IFN and peg-
IFN was limited by its low response rates (20-30%) among CHB patients and a slew of side effects such as flu-
like symptoms, anemia, leucopenia, thrombocytopenia, anorexia and depression.3 A major drawback of the
nucleotide analogs is the emergence of drug resistance during long term treatment with approximately 70% of
4
patients becoming resistant to lamivudine and the resistance to entecavir also occurs at 43% after 4 years
among lamivudine-pretreated patients.5 In order to improve the treatment of CHB and reduce drug resistance,
development of novel agents with different therapeutic targets besides viral polymerase is still highly
necessary. 6
HBV capsid as a new target has recently been investigated, 7-8 the HBV capsid plays indispensable roles in
viral DNA synthesis from the pregenome and intracellular trafficking. Furthermore, as the encapsidation of
9-10
HBV pregenomic RNA (pgRNA) is an evolutionary constraint process,
the genetic stability makes HBV
capsid formation a better drug target against various genotypes of HBV polymerase resistant mutants.11
Some phenylpropenamide derivatives such as AT-61, sulfamoylbenzamide DVR23, and isothiafludine have
been shown to inhibit pgRNA packaging and led to the formation of pgRNA-free empty capsids, and were
found to be potent against HBV replication in various human hepatoma cell lines.12-13 Bay 41-4109 and Bay 39-
5493 have been reported to disrupt or misdirect the assembly of HBV capsid, which led to capsid depletion
and then terminated viral’s replication process. 14-19
2

Figure 1. Chemical structures of reported HBV capsid inhibitors
Bay 41-4109 was shown to prevent HBV infection in a transgenic HBV model by reducing HBV viral DNA
in the liver and the plasma 20-21 but was found hepato-toxic at high doses in rats, then the follow-up studies of
this compound were suspended.22 As part of our efforts of developing novel HBV capsid assembly inhibitor, an
optimization study based on Bayer's reported compounds was conducted. Herein, we described our lead
optimization studies including the synthesis, molecular docking studies and biological evaluation of a series of
novel HAP inhibitors of HBV capsid assembly. This led to the identification of a novel candidate 8n (GLS4)
with an EC50 of 1 nM against HBV ( gene type D) that is more than 50-fold more potent in the cellular
replication assay than the lead compound of Bay 41-4109 or Bay 39-5493. In addition, the compound 8n also
demonstrated good activity against various HBV mutants, including lamivudine, telbivudine, and entecavir
resistant mutants, DMPK evaluations and toxicology profiles were also reported in this article.
2. Result and discussion
Klumpp had reported the cocrystal structures of capsid protein and HAPs at 2015, but at the time we start
this project, there was lack of high resolution cocrystal structures of capsid protein and HAPs, 23-24 so an
3

information-driven approach was carried out to optimize the HAP series represented by Bay 41-4109 and Bay
39-5493. Previous studies indicated that the 3,5-difluoropyridinyl moiety of Bay 41-4109 is chemically
susceptible to nucleophilic SNAr displacements (unpublished results) while thiazole group at the 2-position of
Bay 39-5493 was more stable, then extensive studies were mainly performed on the modifications of R1, R2,
R3, X, and Y of dihydropyrimidine 8 as shown in Scheme 1.
2.1 Chemistry
There is a chiral center present in the dihydropyrimidine molecule, and it was reported that both Bay 41-
4109 and Bay 39-5493 are the (R)-enantiomer. The activity of the (S)-enantiomer was not reported. We first
synthesized the racemate of new derivatives and then studied their in vitro activities.
The general synthetic approach to obtain targeted dihydropyrimidines 6-8 was outlined in Scheme 1. The
dihydropyrimidine structures of 6a-d were obtained under classical Biginelli condition in moderate yields by
reacting thiazole-2-carboxamidine hydrochloride, substituted benzaldehyde and β-dicarbonyl compound.
However, direct hydrolysis of analogues like 6a-c can’t gave desired products even under harsh basic
conditions, probably due to conjugation effects with the core and steric hindrance of the eater moiety. To
obtain the carboxylic acid 6e which will be used as intermediate for further functionalization, tertbutyl ester 6d
4

was prepared and then converted to its corresponding carboxylic acid 6e under acidic condition with TFA. The
acid 6e was reacted with ammonia or methylamine in the presence of EDCI and HOAT to give compound 6f
and 6g. Compound 6e was coupled with BocNHNH2 under the same coupling conditions with HOAt and EDCI,
followed by deprotection with HCl to give the compound 6h. Treatment of compound 6 with NBS yielded the
bromomethyl compound 7, and further reaction of 7 with various alcohols or amines gave lead compounds 8a-
w.
For compounds 8ai-ak linked with 2-triazole or 2-tetrazole at 2-position, a classical Biginelli reaction of 2-
bromo-4-fluorobenzaldehyde, ethyl acetoacetate and urea was used to form dihydropyrimidone compound 9
(Scheme 2). Further reaction with NBS gave the corresponding 6-bromomethyl dihydropyrimidone 10.
Treatment with morpholine gave the 6-morpholinyl substituted dihydropyrimidones 11 followed by POCl3
treatment to provide the chlorinated compound 12, which can be reacted easily with triazole or tetrazole
analogues to furnish compounds 8ai-ak. All of the starting material were either commercially available or can
be readily prepared by the procedures described in the literature.25
5

2.2 Anti-HBV activities and structure-activity relationship studies
Starting from the racemate of Bay 39-5493, we retained the thiazole ring at the 2-position and first explored
the structure−activity relationship (SAR) of the 5-position (Table 1).
Table 1.In-Vitro Anti-HBV Activity of Dihydropyrimidine Bearing Different R1 at 5-Position
Compd
6a
R1
EC50 (μM)
0.25
0.15
6b
6c
>16.4
>16.4
6d
OH
>16.4
>16.4
>16.4
>16.4
0.10
6e
NH2
6f
6g
CH3NH
NH2NH
6h
Lamivudine
Compounds 6e and 6f-h with a carboxylic acid, carboxamide and hydrazide group at the 5-position of the
dihydropyrimidine respectively, showed no inhibition of HBV replication at the highest tested concentration of
16.4 μM, which suggested that a hydrophilic substitution at this position is unfavorable. In contrast, compound
with a methyl ester (6a) or ethyl ester (6b) at 5-position of the dihydropyrimidine demonstrated much
6

improved increase in potency, with EC50 of 0.25 and 0.15 μM respectively. We also found that more bulky
substituents like isopropyl ester (6c) or tert-butyl ester (6d) reduced activity.
On the basis of above results, while surveying ethoxycarbonyl group at the C5 position of the HAP core, a
number of analogues with structure variations at the C4 and C6 position were prepared and evaluated for their
anti-HBV activities (Table 2).
First, the methyl at C6 position of compound 6b was replaced by small groups such as amino, hydroxyl and
alkoxy-groups and it was found that compounds with hydrophilic substitution at the 6-position such as a
hydroxyl group (8a) and amino group (8f) demonstrated reduced activities. There was no obvious pattern
observed for alkoxy groups, for example, the activity of 6-methoxy analogue (8b) was lost while that of
compound 8c (6-ethoxy-derivative) and 8d (6-propoxy-derivative) remained approximately the same within a
narrow range of 0.8-1.2 μM. Compound 8e which incorporated a 2-methoxyethoxy showed improved
inhibitory activity with an EC50 of 0.2 μM. At the same time, compound 8g with a bulky and polar substituent
of 2-hydroxyethyl amino group also showed inhibitory activity with an EC50 of 0.9 μM. This suggested that the
sub-pocket of the protein for the 6-position site of the dihydropyrimidine ring is large enough to accommodate
bulky residue.
Based on these observation, we decided to place a more big morpholine residue which can be seen as
incorporated two 2-hydroxyethyl amino together at the 6-position. To our surprise, the inhibitory activity of
compound 8h with 6- morpholine moiety was 180 times more potent than compound 8g with an EC50 value of
0.005 μM. The thiomorpholine analogue 8k was less active with an EC50 of 0.25 μM, while analogues with
thiomorpholine oxide and thiomorpholine dioxide substitutions (8l and 8m ) were about 3 times more potent
than 8k. However, substitution of the morpholine group with piperdine and piperazine, commun “bioisosteres”
of morpholine, led to decreased activity of analogue 8i and complete loss of activity of analogue 8j. Compared
with the activity of compound 8h and it’s analogues 8i-m, it was proposed that the free electron pairs of the
oxygen in morpholinyl moiety (8h) seem to be more important for increasing activity possibly due to
additional hydrogen bonding.
7

Table 2.In-Vitro Anti-HBV Activity of Dihydropyrimidine Bearing Different R2, X and Y
Compd
8a
R2
Compd
8h
X
F
Y
Cl
Br
F
EC50 (μM)
>16.4
>16.4
0.8
EC50 (μM)
0.005
OH
8b
8n
F
0.001
8c
8o
F
0.026
8d
1.2
8p
H
Cl
0.016
0.2
5.1
0.9
H
H
Br
F
0.01
0.074
0.003
8e
8f
8q
8r
8s
NH2
8g
Cl
Cl
8h
8i
0.005
3.5
8t
8u
H
F
F
F
F
F
NO2
0.1
>16.4
>16.4
>16.4
0.5
8j
>16.4
0.25
0.1
8v
8k
8l
8w
Br
Br
(R)-8n
(S)-8n
8m
0.08
3.1
8

Replacing the chlorine atom in 8h with bromine and fluorine gave compounds 8n and 8o. Among them,
compound 8n exhibited a 5-fold improvement over 8h with an EC50 value of 0.001 μM while compound 8o
was over 5 times less active than compound 8n with an EC50 value of 0.026 μM. The effect of 4’-fluorine on
the phenyl ring was also investigated. Compounds possessing a second fluorine atom at 4’ position on the
phenyl such as 8h, 8n, and 8o, displayed more than 3 folds increase in potency compared with corresponding
4’-H phenyl analogues of 8p, 8q, and 8r.
Based on the result that compound 8n bearing 2-bromine substituent on the 4-phenyl group demonstrated
best activity. We envisaged that the pocket surface near this position was large enough to accommodate other
big residues. Thus, phenyl and substituted phenyls were introduced in 2’ position on the 4-phenyl group,
however, these compounds (8u-w) lost activity completely. At the same time, replacing the 2-bromine
substituent on the 4-phenyl group in 8q with bulky polar NO2 also gave compound 8t with 10 times less active.
The above facts indicated that the 4-phenyl group making close molecular contacts with HBV capsid proteins
such that minor structural modifications can have profound effects on activity.
Modification of the substituent at 2-position of dihydropyrimidine ring were further undertaken while the 2-
bromo-4-fluorophenyl moiety at the 4-position, the ethoxycarbonyl at the 5-position and the morpholine at the
6-position of the core ring (Table 3). It was surprising to find that an additional methoxyl group on thiazole
ring reduced activity greatly (8aa). Thiadiazolyl and thiophenyl group were tolerated with EC50 value of 0.035
μM ( 8ab) and 0.069 μM ( 8ad) while an additional methyl on thiadiazolyl group also led to complete loss of
activity of analogue 8ac. This means that additional substituent on thiazole and thiadiazole ring was
detrimental to their anti-HBV activities.
Replacement of the thiazolyl group in 8n by six-member ring such as pyridine and 3, 5-difluoropyridine
resulted in compounds 8ae and 8af. Compound 8ae showed moderate activity, while the difluoropyridine
compound of 8af showed excellent activity against HBV replication with EC50 value of 0.006μM. This is in
accordance with the early findings of Bay 41-4109.
Introduction of N-methyl-imidazole group at the 2-position of core pyrimidine ring led to compound 8ag
and this group can be tolerated with EC50 of 0.02 μM. As the imidazole moiety of 8ag is more polar and basic
than the thiazole group of 8n, this prompted us to introduce more hydrophilic moiety at 2-position to obtain
9

compound with increased solubility. However, the activity of compound 8ah with a triazole group was
dramatically decreased and the tetrazole rings were also not tolerated at the 2-position of core ring, leading to
total loss of activities (analogue 8aj and 8ak).
Table 3. In-Vitro Anti-HBV Activity of Dihydropyrimidine Bearing Different R3
Compd
R3
EC50 (μM) Compd
R3
EC50 (μM)
0.001
>16.4
0.035
0.006
8n
8af
8ag
8ah
8aa
8ab
0.02
4.3
8ac
8ad
8ae
>16.4
0.069
0.042
8ai
8aj
8ak
>16.4
>16.4
>16.4
Enantiomers of compound 8n were separated by chrial HPLC, and anti-HBV activities of two enantiomers
were tested in the same assay, it was found that (R)-enantiomer possesses higher activity against HBV with
EC50 of 0.5 nM while the EC50 of (S)-isomer was 3.1uM, there was almost 6000 folds gap between (R)- and
10

(S)- enantiomer (Table 2). A PK bridging study between racemate and enantiomers of compound 8n had been
evaluated in Mice and Beagle dogs by oral administration of racemate, (R)- and (S)- enantiomer, respectively.
These studies suggested that there is no biotransformation existed in-vivo between (R)- and (S)- enantiomer
(unpublished results), suggested that the racemate of 8n can be used as candidate compound for further
development considering the difficult in separating the single (R)-enantiomer at that time.
2.3 Influence of the NH Proton of the 1,4-Dihydropyrimidine Ring
The acidic NH proton of the 1, 4-dihydropyrimidine ring seems to be essential for anti HBV activity.
Replacement of the hydrogen with a methyl group led to completely loss of activity (Scheme 3, 8h to 13).
Oxidation of the 1, 4-dihydropyrimidine ring of compound 8n also led to inactive pyrimidine 14. Analysis of
the X-ray structure of the compound 8n (CCDC 1012347), intramolecular hydrogen bond from the N1-H to
the morpholine nitrogen and the thiazole nitrogen was detected (Figure 2). It was supposed that these
intramolecular hydrogen bonds stabilized the conformation of compound 8n. If the NH was replaced by N-Me
or the dihydropyrimidine core was oxidized, the intramolecular hydrogen bonds also were disappeared, then
the conformation or the dihedral angle between the dihydropyrimidine core and thiazole ring were changed,
which led to the completely lost of activity of compound 13 and 14.
Scheme 3.The essential role of NH on activity
11

Figure 2.X-ray crystal structure of compound 8n
2.4 Assessment of Activity of Compound 8n against Commercial nucleotide inhibitors.
As compound 8n showed the best activity among all the tested compounds, we compared the activity of
compound 8n with the lead compounds Bay41-4109 and Bay39-5493 and some nucleotides such as
lamivudine, adefovir, entecavir, telbivudine, and tenofovir, and the results were shown in Table 4. Compound
8n showed 48, 100, and 100 folds higher than Bay41-4109, Bay39-5493, and lamivudine in activity
respectively, also better than adefovir, entecavir, telbivudine, and tenofovir in the same HepG2.2.15 cell. The
CC50 of Compound 8n was >45μM, and the therapeutic index (CC50/EC50) for compound 8n was larger than
45000 in the HepG2.2.15 cell assay.
Table 4. Inhibitory Activity of Compound 8n (GLS4) Compared with Commercial Nucleotide Inhibitors.
Compounds
EC50 (nM)c
EC90 (nM)
CC50 (μM)
8na
1
6
>45
19.3
>45
1020
200
Bay41-4109a
Bay39-5493a
48
102
100
300
lamivudinea
100
2000
Adefovirb
entecavirb
telbivudineb
Tenofovirb
82.7
0.24
819.4
241.5
-
-
-
-
Nucleotide
30
>200
399
12

a
b
Tested in University of Heidelberg, Germany. The EC50 of adefovir, entecavir, telbivudine and tenofovir in
hepG 2.2.15 are tested in WuxiApptec, and the CC50 data were from FDA website.c the levels of HBV DNA in
cell supernatants were quantified by real-time PCR.
2.5 Assessment of Activity of Compound 8n against Polymerase Drug-Resistant HBV Strains.
As one of the limitations associated with the current nucleotide analogs approved for HBV treatment is the
emergence of drug resistant strains, it is important to evaluate the activity of new compound against drug
resistant HBV strains. Compound 8n, which possesses the most potent inhibitory activity, was tested against
the major classes of drug resistance HBV strains, including lamivudine, telbivudine, and entecavir resistant
mutants. It was surprised that compound 8n demonstrated potent inhibitory activity against both wild type and
polymerase drug-resistant mutants including lamivudine-resistant HBV strains (Table 5), telbivudine-resistant
HBV strains (Table 6) and entecavir-resistant HBV strains (Table 7). On the contrary, all the commercial
nucleotide analogs showed greatly reduced activity against their corresponding resistant mutants. The almost
equally effective activity of compound 8n against either wild type or drug-resistant mutants probably
confirmed the unique advantages of capsid inhibitors and also suggested that the assembly of capsid is
independent of the reverse transcriptase. Therefore, future studies of combination protocols using nucleotide
analogs together with capsid inhibitor such as compound 8n could produce a better strategy for the treatment
of patients who have developed polymerase drug resistant strains.
Table 5. Inhibitory Activity of Compound 8n against Lamivudine-Resistant HBV Strains
EC50 (uM)
HBV type
Lamivudine
8n
Intracellular DNA
Secreted DNA
Wild type
0.237
rtM204I
>20
rtM204I+V173L Wild type
rtM204I
>20
rtM204I+V173L
>20
0.055
0.02
>20
0.034
0.092
0.019
0.011
0.015
Table 6. Inhibitory Activity of Compound 8n against Telbivudine -Resistant HBV Strains
EC50 (uM)
HBV type
Telbivudine
Intracellular DNA
Secreted DNA
Wild type
N/A
rtM204I
>20
rtL180M+M204V Wild type
rtM204I
>20
rtL180M+M204V
>20
0.569
0.020
>20
0.012
0.092
0.033
0.010
0.019
8n
13

Table 7. Inhibitory Activity of Compound 8n against Entecavir -Resistant HBV Strains
EC50 (uM)
HBV type
Intracellular DNA
rtM204I+S202G
Secreted DNA
rtM204I+S202G
+M250V
>20
Wild
type
Wild
type
rtM204I+S202G
rtM204I+S202G
+M250V
>20
Entecavir
0.013
0.039
>20
0.014
0.034
>20
8n
0.087
0.046
0.012
0.018
1:The highest concentration in this assay was 20 μM. If the EC50 was greater than 20 μM or the inhibition at 20
μM is lower than 50%, the EC50 was not determined. 2: The levels of HBV DNA in cell supernatants (Secreted
DNA) or HBV DNA in cells (Intracellular DNA) were quantified by real-time PCR.
2.6 Molecular docking studies
Our mechanistic studies indicated that 8n induces capsid assembly (or aggregation) of the N-terminal capsid
assembly domain (Cp149) in a concentration dependent manner, which had published elsewhere 26. To explore
the interaction mode of compound 8n which was found to be best activity with the active site of HBV capsid
protein, several molecular docking simulation studies were carried out by using the SURFLEX module of
SYBYL package. Crystal co-ordinates of capsid (PDB ID 5E0I) was downloaded from protein data bank
(www.rcsb.org) and compound 8n as ligand example.
Figure 3. Optimal binding model for compound (R)-8n (left) and (S)-8n (right) into active site of capsid
docked by SURFLEX module, ligand and some key residues are shown in stick, hydrogen bonds are shown in
dashed lines.
As shown in Figure 3, compound (R)-8n was well docked in the active site of the crystal coordinate of 5E0I.
It was shown that the bromofluoro-substituted phenyl group of compound (R)-8n fits into a well-defined,
14

largely hydrophobic pocket created by Pro25, Asp29, Leu30, Thr33, Trp102, and Ile105, with the bromine
pointing toward the protein and deep into the hydrophobic pocket. Given the limited space around the
bromofluorophenyl group, large substituents are generally not well tolerated and they typically result in loss of
activity for analogues such as 8u−w. The thiazole moiety of (R)-8n is coplanar with the dihydropyrimidine
core as anticipated and is buried in a hydrophobic environment created by aromatic residues Phe23, Pro25,
Tyr118, Trp102, Thr128’, and Ala132’. The N3 atom of the dihydropyrimidine ring forms a hydrogen bond
with the main chain nitrogen of Trp102. The ester moiety of (R)-8n 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 6c−h).
Compared with BAY 41–4109, (R)-8n has achieved an ~100-fold improvement in antiviral activity, this is
mostly by the presence of an additional morpholino group, which adds significant interactions of the
compound with the “lid” structure of another core protein dimer and contacts with residues Val124’, Trp125’,
Thr128’, and Arg133’. In addition, there are significant differences with the interaction between compound
(S)-8n and protein, the core dihydropyrimidine can’t form hydrogen bond with Trp102, and other stable effects
such as the π-π interaction of thiazole residue with Phe23 also disappeared. These docking results provided
us a reasonable explanation for why compound (S)-8n decreased activity greatly compared with (R)-8n.
2.7 Pharmacokinetic profiles of compound 8n.
The pharmacokinetics of 8n was evaluated in Beagle dog following intravenous (iv) and oral (po)
administration. With the dosage of 5 mg/kg, 8n had moderate plasma clearance (CL) (17.9 mL min-1 kg-1) with
an oral bioavailability (F) of 15.6% in dog. After oral administration for once at different dose levels from 5.0
to 45mg/kg, the area under serum drug concentration (AUC0-24h) and the average maximum plasma
concentration (Cmax) of parent drug were all approximately dose dependent (Table 8). After administration for
7 days at 15mg/kg/d, the plasma concentration in beagle dogs reach steady state on day 7, the AUC0-24h and Cmax
were 0.87 and 0.72 times of the parameters in the beagle dogs treated for once. There was no evidence
indicated that the 8n accumulated in beagle dogs after multiple dosing.
Table 8. Pharmacokinetic Characteristics of Compound 8n
15

Dose
Cmax
ng/mg
—
AUC0-24h
ng.h/ml
4353
t1/2
h
CL
Vss
F
mg/kg
5（IV）
mL min-1 kg-1
L/kg
6.86
%
9.85
17.9
136
698
1181
524
695
6.91
7.32
6.29
9.47
—
—
—
—
—
—
—
—
15.6
—
5（PO）
2761
8466
2829
15（PO）
45（PO）
15（PO）*7d
—
—
In particular, 8n had a high exposure in the liver and very limited tissue distribution to lung, heart, and brain.
The liver exposure of 8n was about 10 times higher than that in plasma by oral administration. As the disease
organ of chronic HBV infections is the liver, the preferential liver distribution of 8n could be a useful
characteristic. Serum proteins can bind to and sequester many products and interfere with a product’s antiviral
activity. The unbound fraction of 8n was determined to be 0.1% and 1.8% in human and Beagle dog plasma,
respectively. In the presence of 0% human serum (HS), 5% HS, 10% HS, 20% HS, 40% HS, 50% HS, EC50s of
the test compound 8n are 3.497 nM, 14.49 nM, 17.1 nM, 38.36 nM, 30.98 nM and 34.46 nM respectively,
which means the activity of compound 8n decreased along with human serum concentration and decreased by
2.4 folds to 34.46 nM in 50% HS compared with 5% HS.
2.8 Safety Assessment of compound 8n.
Compound 8n exhibits low CYP inhibition with IC50 > 20μM against five major CYP enzymes (1A2, 2C9,
2C19, 2D6, 2E1 and 3A4) and was also not a time dependent CYP inhibitor. Moreover, 8n was negative in
preliminary in vitro safety evaluations such as Ames, as well as micronucleus test (MNT) assays. In the
concentration of 1 μM, 8n showed no significant effect (inhibition or agonism <50%) on the 36 selected targets
(Table 9). It suggested that 8n as an anti-HBV drug with new mechanism, had a strong target selectivity, and
may not make a significant effect on the other proteins in the body.
16

Table 9. Activity of 8n (GLS4) for different targets
Target
category
Target name
MMP-2
inh%
-3
Target category
Target name
SRPK1
inh%
-21
Serine/arginine
kinase
Proteasome
Trypsin
2
4
2
AKT1
CAMK2D
DAPK1
ERK1
-3
11
3
Protease
TACE
Serine/threonine
kinase
7
Phosphatase
ACP1
-4
1
PKCα
Agonism
-2
4
FXR
Antagonism
Agonism
ABL1
2
0
Nuclear
receptor
Epidermal Growth Factor
Receptor
5
LXR
β
Antagonism
-7
-13
4
FLT3
JAK2
4
25
-3
9
Tyrosine kinase
Acetylcholinesterase
Na/K ATP-enzyme
COX-1
SRC
-10
7
ZA70
SIRT1
Adenosine A1
1
Chemokine receptor
CXCR1/2
Other
Lipase
4
8
GPCR
MAO-A
iNOS
3
7
Dopamine D1
Platelet-activating factor
5 - serotonin
7
10
-1
-6
Neural phospholipase
UGT1A1
9
Ion channels
30
Calcium channel
We further conducted a short-term acute toxicity study on 8n. ICR mice were administered a single oral
dose of each formulated compound at 2000 mg/kg and 5000 mg/kg of 8n, or vehicle alone. Over 14 days, no
observable morbidity or mortality was observed, and the obvious adverse effect is weight loss which can be
recovered. The reasons for these were likely to the gastrointestinal tract irritation of drug (the solution was
17

highly acidic). In addition, gastric contents in mice induced reduced activity. Blood cell counts were not
affected in a statistically significant manner, although administration of 5000 mg/kg of 8n did show a trend of
reduction of platelets and neutrophils and along with an increase in lymphocytes. Serum chemistry was
unaffected.
A 4-week oral (gavage) toxicity study with a two-week recovery period was subsequently carried out with
Beagle dogs. In the study, Beagle dogs were administered 8n at oral gavage doses of 10, 50, 250 mg/kg/day
with control group for 4 weeks with a two-week recovery period. Drug-related effects included vomiting,
diarrhea, salivation, thin and reduced activity, these symptoms can be recovered. The main toxicity related to
8n was the reduction in body weight gain and decreases in mean food consumption, ALB and A/G, increase in
DBIL at mid and high dose. The target organs identified in this study were thymus thyroid and gastrointestinal
tract, although all these pathology changes can be recovered, the NOAEL was 10 mg/kg.
3. Conclusion
We investigated the SAR of a series of active compounds based on the HAP core structure, leading to the
identification of the novel and potent candidate 8n (GLS4). Molecular docking simulation studies were done to
give some reasonable explanations about the SAR studies. This compound of 8n demonstrated high potency
against wild and major drug-resistant HBV viral strains with EC50 values at nano molar level and was more
potent than the classical HBV polymerase inhibitor such as lamivudine, telbivudine, entecavir in the cellular
assays. The favorable pharmacokinetic profiles of 8n suggested that an effective dosing regimen can be
developed. Safety evaluation including acute toxicity study in mice and and 4-week oral toxicity study in
Beagle dogs indicated that 8n was safe enough to support clinical experiments in human. On the basis of these
results, further investigations of compound 8n for clinical study are ongoing and the results will be reported in
due course.
4. Experiment section
4.1 Chemistry
18

All commercially available starting materials and solvents were reagent grade and used without further
purification unless otherwise noted. Anhydrous THF and CH3CN were obtained by distillation over sodium
1
13
wire or CaH2, respectively. H NMR and C NMR spectra were recorded on Bruker-600, Bruker-500 or
Bruker-400 spectrometers, and were referenced to the residual protio signals in CDCl3 at 7.26 ppm or DMSO-
d6 at 2.50 ppm (1H NMR) and CDCl3 at 77.23 ppm or DMSO-d6 at 39.52 ppm (13C NMR). Data for NMR
spectra are reported as follows: chemical shift (δ ppm), multiplicity integration (s = singlet, br s = broad
singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, m = multiplet), coupling constant (Hz).
HRMS were performed on Agilent 6530 Q-TOF mass spectrometer, and ESIMS was carried out on a Agilent
6120 mass spectrometer. TLC was performed on Huanghai HSGF 254 silica gel plates (China). Silica gel 60 H
(200-300 mesh), manufactured by Qingdao Haiyang Chemical Group Co. (China), was used for general
chromatography. The purity of all final derivatives for biological testing was confirmed to be >95% as
determined using an Agilent 1200 series HPLC instrument at 278nm, using the following condition: Alltima
C18 column (4.6*250mm，5μm); solvent A, 10 mM KH2PO4 ( pH3.0 ); solvent B, acetonitrile; gradient, 0
min, 20% B; 15 min, 80% B; 25 min, 80% B. (Note: Almost all the compounds possess similar
dihydropyrimidine core structures, representative compounds were selected for testing 13C NMR).
4.2 Preparation of Compounds 6a-h
4.2.1. Tert-butyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-
carboxylate (6d)
To a suspension of thiazole-2-carboxamidine hydrochloride (5.4 g, 33 mmol), 2-chloro-4-
fluorobenzaldehyde (4.74 g, 30 mmol), NaOAc (2.71 g, 33 mmol) in anhydrous EtOH (60 mL) was added tert-
butyl 3-oxobutanoate (4.74 g, 33 mmol). The reaction mixture was refluxed at 80 ℃ under N2 atmosphere
overnight. Then the mixture was concentrated in vacuo and diluted with EtOAc, washed with water, brine, and
the combined organic phase was dried over Na2SO4,concentrated in vacuo to give a crude product, which was
purified by silica gel chromatography (petrol ether/ethyl acetate (V/V) = 5/1) to give a yellow solid (6.1 g,
50%).
19

1H NMR (600 MHz, CDCl3): δ7.82 (d, 1H, J = 4.2 Hz), 7.49 (br s, 1H), 7.33 (dd, 1H, J = 13.2 Hz, 9.6 Hz),
7.14 (dd, 1H, J = 12.6 Hz, 3.6 Hz), 6.95 (td, 1H, J = 12.6 Hz, 3.6 Hz), 6.12 (s, 1H), 2.54 (s, 3H), 1.30 (s, 9H);
MS-ESI: (ESI, pos.ion) m/z: 408.1 [M+1]+; HRMS (ESI) calcd for C19H19ClFN3O2S [M + H]+ 408.0871; found
408.0949; HPLC analysis: retention time = 23.19 min; peak area, 97.67%.
6a-c was prepared following a similar procedure to that described for 6d.
4.2.2.
Methyl
4-(2-chloro-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,
4-dihydropyrimidine-5-
carboxylate (6a)
1H NMR (600 MHz, DMSO-d6): δ9.97 (s, 1H), 7.96 (d, 1H, J = 3.0 Hz), 7.88 (d, 1H, J = 3.0 Hz), 7.39 (dd,
1H, J = 9.0 Hz, 2.4 Hz), 7.35 (dd, 1H, J = 9.0 Hz, 6.6 Hz), 7.17 (td, 1H, J = 8.4 Hz, 2.4 Hz), 5.98 (s, 1H), 3.49
(s, 3H), 2.47 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ166.2, 162.7, 160.8 (d, J= 246.0 Hz), 147.8, 144.6,
143.3, 138.7 (d, J= 3.0 Hz), 132.5 (d, J= 10.5 Hz), 130.6 (d, J= 9.0 Hz), 124.4, 116.6 (d, J= 25.5 Hz), 114.8
(d, J= 19.5 Hz), 96.2, 55.2, 50.9, 17.5; MS-ESI: (ESI, pos.ion) m/z: 366.0 [M + 1]+; HRMS (ESI) calcd for
C16H13ClFN3O2S [M + 1]+ 366.0401; found 366.0473; HPLC analysis: retention time = 18.17 min; peak area,
98.29%.
4.2.3.
Ethyl
4-(2-chloro-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,
4-dihydropyrimidine-5-
carboxylate (6b)
1H NMR (600 MHz, CDCl3): δ7.80 (d, 1H, J = 3.0 Hz), 7.46 (br s, 1H), 7.34 (dd, 1H, J = 8.4 Hz, 6.0 Hz),
7.11 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 6.91 (td, 1H, J = 8.4 Hz, 2.4 Hz), 6.17 (s, 1H), 4.05 (q, 2H, J = 6.0 Hz), 2.53
(s, 3H), 1.12 (t, 3H, J = 6.0 Hz); 13C NMR (150 MHz, DMSO-d6): δ165.6, 162.7, 160.8 (d, J= 246.0 Hz),
147.5, 144.4, 143.2, 139.1 (d, J= 2.8 Hz), 132.4 (d, J= 9.0 Hz), 130.8 (d, J= 9.0 Hz), 124.3, 116.4 (d, J= 24.0
Hz), 114.8 (d, J= 21.0 Hz), 96.6, 59.1 55.3, 17.4, 13.9; MS-ESI: (ESI, pos.ion) m/z: 380.1 [M+1]+; HRMS
(ESI) calcd for C17H15ClFN3O2S [M + 1]+ 380.0558; found 380.0631; HPLC analysis: retention time = 19.60
min; peak area, 98.85%.
20

4.2.4. Isopropyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-
carboxylate (6c)
1H NMR (600 MHz, CDCl3): δ7.76 (d, 1H, J = 3.0 Hz), 7.42 (br s, 1H), 7.33 (dd, 1H, J = 9.0 Hz, 6.0 Hz),
7.09 (dd, 1H, J = 8.4 Hz, 2.4 Hz), 6.90 (td, 1H, J = 8.4 Hz, 2.4 Hz), 6.15 (s, 1H), 4.90 (m, 1H), 2.49 (s, 3H),
1.18 (d, 3H, J = 6.0 Hz), 0.93 (d, 3H, J = 6.0 Hz); MS-ESI: (ESI, pos.ion) m/z: 393.9 [M+1]+; HRMS (ESI)
calcd for C18H17ClFN3O2S [M + 1]+ 394.0714; found 394.0781; HPLC analysis: retention time = 21.17 min;
peak area, 99.98%.
4.2.5. 4-(2-Chloro-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-carboxylic acid
(6e)
To a solution of 6d (5.18 g, 12.7 mmol) in DCM (60 mL) was added TFA (20 mL), the reaction mixture
was stirred at room temperature overnight. Then the reaction mixture was concentrated in vacuo, diluted with
water. Na2CO3 was added to form a salt. The aqueous phase was washed with EtOAc twice and then was
acidified by 2N HCl. The precipitate was collected by filtration and dried in vacuo to give a yellow power
(3.75 g, 84%).
1H NMR (600 MHz, DMSO-d6): δ11.90 (s, 1H), 9.83 (s, 1H), 7.95 (d, 1H, J = 3.0 Hz), 7.87 (br s, 1H), 7.38
(dd, 1H, J = 9.0 Hz, 2.4 Hz), 7.33 (dd, 1H, J = 8.4 Hz, 6.6 Hz), 7.17 (td, 1H, J = 8.4 Hz, 2.4Hz), 5.95 (s, 1H),
2.47 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ167.44, 162.79, 160.81 (d, J= 246.0 Hz), 147.09, 144.70,
143.23, 138.66 (d, J = 3.0 Hz), 132.74 (d, J= 10.5 Hz), 130.58 (d, J= 9.0 Hz), 124.34, 116.67 (d, J= 24.0 Hz ),
114.73 (d, J= 19.5 Hz), 96.85, 55.51, 17.48; MS-ESI: (ESI, pos.ion) m/z: 352.0 [M+1]+; HRMS (ESI) calcd
for C15H11ClFN3O2S [M + 1]+ 352.0245; found 352.0324; HPLC analysis: retention time = 13.36 min; peak
area, 99.56%.
4.2.6. 4-(2-Chloro-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-carbohydrazide
hydrochloride (6h).
To a solution of 6e (0.5 g, 1.42 mmol) in DCM (20 mL) was added Boc-hydrazine (0.21 g, 1.56 mmol),
EDCI (0.33 g, 1.7 mmol), HOAt (0.23 g, 1.7 mmol), DIPEA (0.37 g, 2.84 mmol). The mixture was stirred at
21

25℃ overnight. Then the mixture was diluted with EtOAc and was washed with water, 1N HCl, saturated
NaHCO3 solution, brine and dried over Na2SO4. After evaporation of the solvent, the residue was purified by a
silica gel column chromatography (petrol ether/ethyl acetate (V/V) = 5/1) to give a yellow solid (0.53 g, 80%).
The solid was diluted with EtOAc (5 mL), HCl in EtOAc (5 mL, 5M) was added. After 10 min, the product
precipitated from the solvent and then was collected by filtration, dried in vacuo to give a yellow power (0.39
g, 95%).
1H NMR (600 MHz, D2O): δ8.19 (s, 2H), 7.58 (dd, 1H, J = 8.4 Hz, 6.0 Hz), 7.36 (dd, 1H, J = 8.4 Hz, 2.4
Hz), 7.20 (td, 1H, J = 8.4 Hz, 2.4 Hz), 6.25 (s, 1H), 2.26 (s, 3H); 13C NMR (150 MHz, D2O): δ164.8, 162.8 (d,
J= 249.6 Hz), 151.1, 149.7, 145.7, 135.4, 133.6 (d, J= 11.0 Hz), 132.2 (d, J= 9.6 Hz), 131.7 (d, J= 3.0 Hz),
128.9, 117.8 (d, J= 25.2 Hz), 115.4 (d, J= 21.6 Hz), 107.5, 52.8, 15.8; MS-ESI: (ESI, pos.ion) m/z: 365.9
[M+1]+; HRMS (ESI) calcd for C15H13ClFN5OS [M + 1]+ 366.0513; found 366.0592; HPLC analysis: retention
time = 7.69 min; peak area, 96.56%.
6f-g was prepared following a similar procedure to that described for 6h.
4.2.7. 4-(2-Chloro-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-carboxamide
(6f).
1H NMR (600 MHz, DMSO-d6): δ9.37 (s, 1H), 7.94 (d, 1H, J = 3.0 Hz), 7.84 (d, 1H, J = 3.0 Hz), 7.42 (dd,
1H, J = 8.4 Hz, 6.6 Hz), 7.38 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 7.18 (td, 1H, J = 8.4 Hz, 2.4 Hz), 7.13 (br s, 1H),
6.90 (br s, 1H), 5.95 (s, 1H), 2.21 (s, 3H); MS-ESI: (ESI, pos.ion) m/z: 350.9 [M+1]+; HRMS (ESI) calcd for
C15H12ClFN4OS [M + 1]+ 351.0404; found 351.0480; HPLC analysis: retention time = 9.14 min; peak area,
99.21%.
4.2.8.
4-(2-Chloro-4-fluorophenyl)-N-6-dimethyl-2-(thiazol-2-yl)-1,
4-dihydropyrimidine-5-
carboxamide (6g).
1H NMR (600 MHz, CDCl3): δ7.84 (d, 1H, J = 3.6 Hz), 7.52 (d, 1H, J = 3.0 Hz), 7.47 (dd, 1H, J = 8.4 Hz,
6.0 Hz), 7.14 (dd, 1H, J = 8.4 Hz, 2.4 Hz), 6.97 (td, 1H, J = 8.4 Hz, 2.4 Hz), 6.05 (s, 1H), 5.79 (br s, 1H), 2.76
22

(d, 3H, J = 4.8 Hz), 2.43 (s, 3H); MS-ESI: (ESI, pos.ion) m/z: 365.0 [M+1]+; HRMS (ESI) calcd for
C16H14ClFN4OS [M + 1]+ 365.0561; found 365.0632; HPLC analysis: retention time = 9.37 min; peak area,
99.36%.
4.3 Preparation of Compounds 8a-r
4.3.1.
Ethyl
4-(2-chloro-4-fluorophenyl)-2-(thiazol-2-yl)-6-(thiomorpholinomethyl)-1,
4-
dihydropyrimidine-5-carboxylate (8k).
To a solution of 6b (1.90 g, 5 mmol) in CCl4 (60 mL) was added NBS (0.98 g, 5.5 mmol) in portions during
1 h at 60℃ under nitrogen atmosphere. Upon complete conversion, the reaction mixture was filtrated to
remove the byproduct, and the CCl4 solvent was concentrated in vacuo. The residue was dissolved in
anhydrous EtOH (10 mL), then thiomorpholine (0.31 g, 3 mmol) was added. The reaction mixture was stirred
at 25 ℃ for 2 h. Then the mixture was concentrated in vacuo, and the residue was purified by a silica gel
column chromatography (petrol ether /ethyl acetate (V/V) = 10/1) to give a yellow solid (0.78 g, 32%, two
steps).
1H NMR (600 MHz, CDCl3): δ9.62 (s, 1H), 7.84 (d, 1H, J = 3.0 Hz), 7.42 (br s, 1H), 7.27 (t, 1H, J = 7.2
Hz), 7.11 (dd, 1H, J = 9.0 Hz, 2.4 Hz ), 6.89 (t, 1H, J = 7.2 Hz), 6.19 (s, 1H), 4.03 (m, 3H), 3.81 (d, 1H, J =
17.4 Hz), 2.84 (s, 4H), 2.80 (s, 4H), 1.11 (t, 3H, J = 7.2 Hz); 13C NMR (150 MHz, CDCl3): δ166.2, 162.9,
161.5 (d, J= 247.5 Hz), 146.2, 144.6, 143.3, 138.3, 133.7 (d, J = 9.0 Hz), 130.8 (d, J= 9.0 Hz), 123.0, 116.9 (d,
J= 24.0 Hz), 114.3 (d, J= 24.0 Hz ), 97.7, 59.9, 57.3, 56.5, 55.3, 28.2, 14.1; MS-ESI: (ESI, pos.ion) m/z:
481.0 [M+1]+; HRMS (ESI) calcd for C21H22ClFN4O2S2 [M + 1]+ 481.0857; found 481.0920; HPLC analysis:
retention time = 13.31 min; peak area, 97.28%.
8a-w was prepared following a similar procedure to that described for 8k.
4.3.2. Ethyl 4-(2-chloro-4-fluorophenyl)-6-(hydroxymethyl)-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-
carboxylate (8a).
23

1H NMR (600 MHz, CDCl3): δ7.85 (d, 1H, J = 3.0 Hz), 7.52 (br s, 1H), 7.36 (dd, 1H, J = 8.4 Hz, 6.0 Hz),
7.13 (dd, 1H, J = 8.4 Hz, 2.4 Hz), 6.96 (td, 1H, J = 8.4 Hz, 1.8 Hz), 6.17 (s, 1H), 4.91 (m, 2H), 4.04 (q, 2H, J =
7.2 Hz), 1.13 (t, 3H, J = 7.2 Hz); MS-ESI: (ESI, pos.ion) m/z: 396.0 [M+1]+; HRMS (ESI) calcd for
C17H15ClFN3O3S [M + 1]+ 396.0507; found 396.0640; HPLC analysis: retention time = 17.93 min; peak area,
97.14%.
4.3.3. Ethyl 4-(2-chloro-4-fluorophenyl)-6-(methoxymethyl)-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-
carboxylate (8b).
1H NMR (600 MHz, CDCl3): δ9.08 (br s, 1H), 7.87 (s, 1H), 7.48 (s, 1H), 7.37 (t, 1H, J = 7.2 Hz), 7.12 (m,
1H), 6.92 (m, 1H), 6.22 (s, 1H), 4.81 (m, 2H), 4.02 (m, 2H), 3.55 (s, 3H), 1.13 (t, 3H, J = 7.2 Hz); MS-ESI:
(ESI, pos.ion) m/z: 410.0 [M+1]+; HRMS (ESI) calcd for C18H17ClFN3O3S [M + 1]+ 410.0663; found 410.0730;
HPLC analysis: retention time = 22.19 min; peak area, 99.97%.
4.3.4. Ethyl 4-(2-chloro-4-fluorophenyl)-6-(ethoxymethyl)-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-
carboxylate (8c).
1H NMR (600 MHz, DMSO-d6): δ9.10 (s, 1H), 8.00 (m, 1H), 7.94 (m, 1H), 7.42 (m, 2H), 7.18 (m, 1H),
6.05 (s, 1H), 4.80 (q, 2H, J = 15.6 Hz), 3.95 (m, 2H), 3.64 (m, 2H), 1.24 (t, 3H, J = 7.2 Hz), 1.04 (m, 3H); 13
C
NMR (150 MHz, DMSO-d6): δ165.0, 161.8, 160.9 (d, J= 246.0 Hz), 146.8, 144.1, 144.0, 138.5, 132.5 (d, J=
10.5 Hz), 131.1 (d, J= 9.0 Hz), 124.9, 116.5 (d, J= 24.0 Hz), 115.0 (d, J= 21.0 Hz), 95.5, 66.3, 66.0, 59.5,
55.4, 15.0, 13.9; MS-ESI: (ESI, pos.ion) m/z: 424.0 [M+1]+; HRMS (ESI) calcd for C19H19ClFN3O3S [M + 1]+
424.0820; found 424.0886; HPLC analysis: retention time = 24.53 min; peak area, 99.81%.
4.3.5. Ethyl 4-(2-chloro-4-fluorophenyl)-6-(propoxymethyl)-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-
carboxylate (8d).
1H NMR (600 MHz, DMSO-d6): δ9.11 (s, 1H), 7.99 (d, 1H, J = 3.0 Hz), 7.94 (d, 1H, J = 3.0 Hz), 7.43 (m,
2H), 7.19 (m, 1H), 6.04 (s, 1H), 4.81 (q, 2H, J = 15.6 Hz), 3.95 (m, 2H), 3.55 (m, 2H), 1.63 (q, 2H, J = 7.2
Hz), 1.04 (t, 3H, J = 7.2 Hz), 0.96 (t, 3H, J = 7.2 Hz); MS-ESI: (ESI, pos.ion) m/z: 437.9 [M+1]+; HRMS
24

(ESI) calcd for C20H21ClFN3O3S [M + 1]+ 438.0976; found 438.1042; HPLC analysis: retention time = 19.31
min; peak area, 99.88%.
4.3.6.
Ethyl
4-(2-chloro-4-fluorophenyl)-6-((2-methoxyethoxy)methyl)-2-(thiazol-2-yl)-1,4-
dihydropyrimidine-5-carboxylate (8e).
1H NMR (600 MHz, DMSO-d6): δ9.20 (s, 1H), 7.99 (d, 1H, J = 3.0 Hz), 7.94 (d, 1H, J = 3.0 Hz), 7.43 (m,
2H), 7.19 (td, 1H, J = 8.4 Hz, 2.4 Hz), 6.04 (s, 1H), 4.85 (q, 2H, J = 16.2 Hz), 3.95 (m, 2H), 3.73 (m, 2H), 3.55
(m, 2H), 3.28 (s, 3H), 1.04 (t, 3H, J = 7.2 Hz); 13C NMR (150 MHz, DMSO-d6): δ165.0, 161.8, 160.9 (d, J=
244.0 Hz), 147.0, 143.7, 143.5, 138.5 (d, J= 4.5 Hz), 132.5 (d, J= 10.5 Hz), 131.1 (d, J= 9.0 Hz), 124.8, 116.5
(d, J= 25.5 Hz), 115.0 (d, J= 19.5 Hz), 95.5, 71.0, 70.3, 66.6, 59.5, 58.2, 55.4, 13.9; MS-ESI: (ESI, pos.ion)
m/z: 453.9 [M+1]+; HRMS (ESI) calcd for C20H21ClFN3O4S [M + 1]+ 454.0925; found 454.0995; HPLC
analysis: retention time = 21.22 min; peak area, 98.95%.
4.3.7. Ethyl 6-(aminomethyl)-4-(2-chloro-4-fluorophenyl)-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-
carboxylate (8f).
1H NMR (600 MHz, DMSO-d6): δ8.28-8.04 (m, 5H), 7.54 (m, 1H), 7.44 (m, 1H), 7.21 (m, 1H), 6.00 (s,
1H), 4.21 (m, 2H), 3.99 (q, 2H, J = 7.2 Hz), 1.07 (t, 3H, J = 7.2 Hz); MS-ESI: (ESI, pos.ion) m/z: 395.1
[M+1]+; HRMS (ESI) calcd for C17H16ClFN4O2S [M + 1]+ 395.0667; found 395.0740; HPLC analysis:
retention time = 11.01 min; peak area, 99.64%.
4.3.8.
Ethyl
4-(2-chloro-4-fluorophenyl)-6-(((2-hydroxyethyl)amino)methyl)-2-(thiazol-2-yl)-1,4-
dihydropyrimidine-5-carboxylate (8g).
1H NMR (600 MHz, DMSO-d6): δ7.99 (d, 1H, J = 3.0 Hz), 7.92 (br s, 1H), 7.41 (m, 2H), 7.17 (td, 1H, J =
8.4 Hz, 2.4 Hz ), 6.03 (s, 1H), 4.62 (br s, 1H), 4.06 (d, 1H, J = 17.4 Hz), 4.01 (d, 1H, J = 17.4 Hz), 3.95 (m,
2H), 3.52 (s, 2H), 2.65 (m, 2H), 1.04 (t, 3H, J = 7.2 Hz); MS-ESI: (ESI, pos.ion) m/z: 439.0 [M+1]+; HRMS
(ESI) calcd for C19H20ClFN4O3S [M + 1]+ 439.0929; found 439.0855; HPLC analysis: retention time = 10.72
min; peak area, 98.90%.
25

4.3.9.
Ethyl
4-(2-chloro-4-fluorophenyl)-6-(morpholinomethyl)-2-(thiazol-2-yl)-1,
4-
dihydropyrimidine-5-carboxylate (8h).
1H NMR (600 MHz, DMSO-d6): δ9.67 (s, 1H), 8.03 (d, 1H, J = 3.0 Hz), 7.94 (d, 1H, J = 3.0 Hz), 7.41 (m,
2H), 7.17 (td, 1H, J = 8.4 Hz, 2.4 Hz), 6.05 (s, 1H), 3.97-3.85 (m, 4H), 3.67 (br s, 4H), 2.54 (m, 4H), 1.04 (t,
3H, J = 7.2 Hz); MS-ESI: (ESI, pos.ion) m/z: 465.1 [M+1]+; HRMS (ESI) calcd for C21H22ClFN4O3S [M + 1]+
465.1085; found 465.1149; HPLC analysis: retention time = 12.83 min; peak area, 97.98%.
4.3.10.
Ethyl
4-(2-chloro-4-fluorophenyl)-6-(piperidin-1-ylmethyl)-2-(thiazol-2-yl)-1,
4-
dihydropyrimidine-5-carboxylate (8i).
1H NMR (600 MHz, DMSO-d6): δ9.75 (s, 1H), 8.02 (d, 1H, J = 3.0 Hz), 7.93 (d, 1H, J = 3.0 Hz), 7.40 (m,
2H), 7.17 (td, 1H, J = 8.4 Hz, 2.4 Hz), 6.05 (s, 1H), 3.96 (m, 2H), 3.87 (d, 1H, J = 17.4 Hz), 3.80 (d, 1H, J =
17.4 Hz), 2.48 (br s, 4H), 1.59 (m, 4H), 1.45 (br s, 2H), 1.04 (t, 3H, J = 7.2 Hz); MS-ESI: (ESI, pos.ion) m/z:
463.1 [M+1]+; HRMS (ESI) calcd for C22H24ClFN4O2S [M + 1]+ 463.1293; found 463.1363; HPLC analysis:
retention time = 13.31 min; peak area, 97.28%.
4.3.11.
Ethyl
4-(2-chloro-4-fluorophenyl)-6-(piperazin-1-ylmethyl)-2-(thiazol-2-yl)-1,
4-
dihydropyrimidine-5-carboxylate (8j).
1H NMR (600 MHz, DMSO-d6): δ9.52 (s, 1H), 8.52 (d, 1H, J = 3.0 Hz), 7.95 (d, 1H, J = 3.0 Hz), 7.41 (m,
2H), 7.19 (td, 1H, J = 8.4 Hz, 2.4 Hz), 6.04 (s, 1H), 3.94 (m, 4H), 3.03 (br s, 4H), 2.73 (br s, 4H), 1.04 (t, 3H, J
= 7.2 Hz); MS-ESI: (ESI, pos.ion) m/z: 464.1 [M+1]+; HRMS (ESI) calcd for C21H23ClFN5O2S [M + 1]+
464.1245; found 464.1316; HPLC analysis: retention time = 12.03 min; peak area, 98.44%.
4.3.12. Ethyl 4-(2-chloro-4-fluorophenyl)-6-((1-oxidothiomorpholino)methyl)-2-(thiazol-2-yl)-1, 4-
dihydropyrimidine-5-carboxylate (8l).
1H NMR (600 MHz, DMSO-d6): δ9.58 (s, 1H), 8.01 (d, 1H, J = 3.0 Hz), 7.95 (d, 1H, J = 3.0 Hz), 7.42 (m,
2H), 7.18 (td, 1H, J = 8.4 Hz, J = 2.4 Hz), 6.06 (s, 1H), 4.04-3.92 (m, 4H), 3.07 (m, 2H), 2.95 (m, 2H), 2.83
(m, 3H), 2.72 (m, 1H), 1.05 (t, 3H, J = 7.2 Hz); MS-ESI: (ESI, pos.ion) m/z: 497.1.1 [M+1]+; HRMS (ESI)
26

calcd for C21H22ClFN4O3S2 [M + 1]+ 497.0806; found 497.0887; HPLC analysis: retention time = 14.47 min;
peak area, 99.06%.
4.3.13. Ethyl 4-(2-chloro-4-fluorophenyl)-6-((1,1-dioxidothiomorpholino)methyl)-2-(thiazol-2-yl)-1,4-
dihydropyrimidine-5-carboxylate (8m).
1H NMR (600 MHz, DMSO-d6): δ9.56 (s, 1H), 8.03 (d, 1H, J = 3.0 Hz), 7.94 (d, 1H, J = 3.0 Hz), 7.42 (m,
2H), 7.18 (td, 1H, J = 8.4 Hz, 2.4 Hz), 6.05 (s, 1H), 4.13 (d, 1H, J = 16.2 Hz), 4.02 (d, 1H, J = 16.2 Hz), 3.96
(m, 2H), 3.19 (m, 4H), 3.07 (br s, 4H), 1.04 (t, 3H, J = 7.2 Hz); 13C NMR (150 MHz, DMSO-d6): δ165.6,
162.5, 161.4 (d, J = 246.0 Hz), 146.4, 144.6, 144.1, 138.9, 133.0 (d, J = 10.5 Hz), 131.5 (d, J = 9.0 Hz), 125.2,
117.0 (d, J = 24.0 Hz), 115.4 (d, J = 21.0 Hz), 97.9, 60.0, 56.2, 53.9, 51.2, 51.1, 14.4; MS-ESI: (ESI, pos.ion)
m/z: 513.0 [M+1]+; HRMS (ESI) calcd for C21H22ClFN4O4S2 [M + 1]+ 513.0755; found 513.0818; HPLC
analysis: retention time = 18.02 min; peak area, 99.23%.
4.3.14.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-(morpholinomethyl)-2-(thiazol-2-yl)-1,
4-
dihydropyrimidine-5-carboxylate (8n).
1, To a suspension of 2-bromo-4-fluorobenzaldehyde (6.09 g, 30 mmol), thiazole-2-carboxamidine
hydrochloride (5.4 g, 33 mmol), AcONa (2.71 g, 33 mmol) in anhydrous EtOH (60 mL) was added ethyl 3-
oxobutanoate (4.29 g, 33 mmol). The reaction mixture was refluxed at 80 ℃ under N2 atmosphere overnight.
Then the mixture was concentrated in vacuo and diluted with EtOAc, washed with water, brine, and the
combined organic phase was dried over Na2SO4,concentrated in vacuo to give a crude product, which was
purified by silica gel chromatography (petrol ether/ethyl acetate (V/V) = 8/1) to give 7n as a yellow solid
(7.00 g, 55%).
1H NMR (500 MHz, DMSO-d6): δ9.92 (s, 1H), 7.97 (d, 1H, J = 3.0 Hz), 7.90 (d, 1H, J = 2.5 Hz), 7.55 (dd,
1H, J = 8.5 Hz, 3.0 Hz), 7.36 (dd, 1H, J = 8.5 Hz, 6.5 Hz), 7.23 (td, 1H, J = 8.5 Hz, 2.5 Hz), 5.97 (s, 1H), 3.94
(q, 2H, J = 7.0 Hz), 2.47 (s, 3H), 1.03 (t, 3H, J = 7.0 Hz); MS-ESI: (ESI, pos.ion) m/z: 426.0 [M+1]+.
27

2, To a solution of compound 7n (0.85 g, 2 mmol) in CCl4 (60 mL) was added NBS (0.41 g, 2.2 mmol) in
portions during 1 h at 60 ℃ under nitrogen atmosphere. Upon complete conversion, the reaction mixture was
filtrated to remove the byproduct, and the CCl4 solvent was concentrated in vacuo. The residue was dissolved
in anhydrous EtOH (10 mL), then morpholine (0.10 g, 1.2 mmol) was added. The reaction mixture was stirred
at 25 ℃ for 2 h. Then the mixture was concentrated in vacuo, and the residue was purified by a silica gel
column chromatography (petrol ether /ethyl acetate (V/V) = 10/1) to give a yellow solid (0.55 g, 54%, two
steps).
1H NMR (600 MHz, DMSO-d6): δ9.67 (s, 1H), 8.03 (d, 1H, J = 3.0 Hz), 7.94 (d, 1H, J = 3.0 Hz), 7.56 (dd,
1H, J = 2.4 Hz, 3.0 Hz), 7.40 (dd, 1H, J = 6.0 Hz, 6.0 Hz), 7.22 (td, 1H, J = 3.0 Hz, 6.0 Hz), 6.04 (s, 1H), 3.98-
3.87 (m, 4H), 3.67 (t, 4H, J = 4.2 Hz), 2.53-2.56 (m, 4H), 1.06 (t, 3H, J = 7.2 Hz); 13C NMR (125 MHz,
DMSO-d6): δ165.64, 162.51, 162.08, 160.43, 146.79, 144.37, 144.07, 140.68, 131.38(d, J = 8 Hz), 125.71,
123.05(d, J = 8 Hz), 120.05(d, J = 8 Hz), 115.94(d, J = 18.5 Hz), 115.87, 97.67, 66.89, 59.85, 58.67, 56.38,
53.67, 14.45; MS-ESI: (ESI, pos.ion) m/z: 511.0 [M+1]+; HRMS (ESI) calcd forC21H22BrFN4O3S [M + 1]+
511.0575 ; found 511.0619; HPLC analysis: retention time = 13.39 min; peak area, 99.95%.
4.3.15. Ethyl 4-(2,4-difluorophenyl)-6-(morpholinomethyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-
carboxylate (8o).
1H NMR (600 MHz, DMSO-d6): δ9.68 (s, 1H), 8.03 (d, 1H, J = 3.0 Hz), 7.95 (d, 1H, J = 3.0 Hz), 7.36 (q,
1H, J = 8.4 Hz), 7.19 (td, 1H, J = 10.8 Hz, 2.4 Hz), 7.02 (td, 1H, J = 8.4 Hz, 1.8 Hz), 5.90 (s, 1H), 3.98 (m,
2H), 3.89 (d, 1H, J = 16.8 Hz ), 3.82 (d, 1H, J = 16.8 Hz ), 3.66 (br s, 4H), 2.52 (m, 4H), 1.08 (t, 3H, J = 7.2
Hz);13C NMR (150 MHz, DMSO-d6): δ165.2, 162.0, 161.5 (dd, J= 244.5 Hz, 12.0 Hz), 159.5 (dd, J= 247.5
Hz, 12.0 Hz), 146.0, 144.4, 143.6, 130.8 (dd, J= 9.0 Hz, 6.0 Hz), 128.2 (dd, J= 15.0 Hz, 3.0 Hz), 124.7, 111.7
(dd, J= 21.0 Hz, 3.0 Hz), 103.9 (t, J= 27.0 Hz ), 96.5, 66.4, 59.4, 55.9, 53.1, 53.0 (d, J= 1.5 Hz), 13.9; MS-
ESI: (ESI, pos.ion) m/z: 449.1 [M+1]+; HRMS (ESI) calcd for C21H22F2N4O3S [M + 1]+ 449.1381 ; found
449.1461; HPLC analysis: retention time = 11.67 min; peak area, 99.86%.
28

4.3.16. Ethyl 4-(2-chlorophenyl)-6-(morpholinomethyl)-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-
carboxylate (8p).
1H NMR (600 MHz, DMSO-d6): δ9.65 (s, 1H), 8.02 (d, 1H, J = 3.0 Hz), 7.93 (d, 1H, J = 3.0 Hz), 7.44 (dd,
1H, J = 7.8 Hz, 1.2 Hz), 7.38 (dd, 1H, J = 7.8 Hz, 1.2 Hz), 7.31-7.25 (m, 2H), 6.09 (s, 1H), 3.97-3.86 (m, 4H),
3.67 (br s, 4H), 2.54 (m, 4H), 1.03 (t, 3H, J = 7.2 Hz);13C NMR (150 MHz, DMSO-d6): δ165.2, 162.1, 146.1,
144.0, 143.6, 141.9, 131.8, 129.7, 129.4, 129.0, 127.8, 124.7, 97.1, 66.4, 59.4, 56.3, 55.9, 53.2, 13.9. MS-ESI:
(ESI, pos.ion) m/z: 447.1 [M+1]+; HRMS (ESI) calcd forC21H23ClN4O3S [M + 1]+ 447.1179 ; found 447.1235;
HPLC analysis: retention time = 12.00 min; peak area, 98.25%.
4.3.17. Ethyl 4-(2-bromophenyl)-6-(morpholinomethyl)-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-
carboxylate (8q).
1H NMR (600 MHz, DMSO-d6): δ9.65 (s, 1H), 8.02 (d, 1H, J = 3.0 Hz), 7.93 (d, 1H, J = 3.0 Hz), 7.61 (d,
1H, J = 7.8 Hz), 7.35 (m, 2H), 7.18 (t, 1H, J = 7.8 Hz), 6.05 (s, 1H), 3.96-3.89 (m, 4H), 3.67 (br s, 4H), 2.54
(m, 4H), 1.04 (t, 3H, J = 7.2 Hz);13C NMR (150 MHz, DMSO-d6): δ165.2, 162.1, 146.2, 143.8, 143.6, 143.6,
132.7, 129.7, 129.2, 128.5, 124.6, 122.5, 97.3, 66.4, 59.3, 58.8, 55.9, 53.2, 13.9; MS-ESI: (ESI, pos.ion) m/z:
493.1 [M+1]+; HRMS (ESI) calcd for C21H23BrN4O3S [M + 1]+ 493.0654; found 493.0732; HPLC analysis:
retention time = 12.60 min; peak area, 98.01%.
4.3.18. Ethyl 4-(2-fluorophenyl)-6-(morpholinomethyl)-2-(thiazol-2-yl)-1, 4-dihydropyrimidine-5-
carboxylate (8r).
1H NMR (600 MHz, DMSO-d6): δ9.66 (s, 1H), 8.03 (d, 1H, J = 3.0 Hz), 7.94 (d, 1H, J = 3.0 Hz), 7.34-7.27
(m, 2H), 7.14 (m, 2H), 5.93 (s, 1H), 3.97 (m, 2H), 3.85 (dd, 2H, J = 16.8 Hz ), 3.66 (br s, 4H), 2.52 (m, 4H),
1.07 (t, 3H, J = 7.2 Hz);13C NMR (150 MHz, DMSO-d6): δ 165.3, 162.1, 159.5 (d, J= 244.5 Hz), 145.8,
144.4, 143.6, 131.6 (d, J = 15.0 Hz), 129.6 (d, J= 3.0 Hz), 129.3 (d, J= 7.5 Hz), 124.7, 124.7, 115.4 (d, J=
21.0 Hz ), 96.8, 66.4, 59.4, 55.9, 53.3 (d, J= 3.0 Hz), 53.1, 13.9; MS-ESI: (ESI, pos.ion) m/z: 431.1 [M+1]+;
HRMS (ESI) calcd forC21H23FN4O3S [M + 1]+ 431.1475 ; found 431.1557; HPLC analysis: retention time =
11.01 min; peak area, 99.45%.
29