3-((R)-4-(((R)-6-(2-Bromo-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholin-2-yl)propanoic Acid (HEC72702), a Novel Hepatitis B Virus Capsid Inhibitor Based on Clinical Candidate GLS4

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

3‑((R)‑4-(((R)‑6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholin-2-

yl)propanoic Acid (HEC72702), a Novel Hepatitis B Virus Capsid

Inhibitor Based on Clinical Candidate GLS4

Qingyun Ren,*^,†,‡Xinchang Liu,^†,‡Guanghua Yan,^†,‡Biao Nie,^†Zhifu Zou,^†,‡Jing Li,^†Yunfu Chen,^†

Yu Wei,^†Jianzhou Huang,^†,‡Zhonghua Luo,^†Baohua Gu,^†Siegfried Goldmann,^†,‡Jiancun Zhang,^§

and Yingjun Zhang*^,†

^†The State Key Laboratory of Anti-Infection Drug Development, HEC Pharma Group, Dong Guan 523871, China

^‡Anti-infection Innovation Department, New Drug Research Institute, HEC Pharma Group, Dong Guan 523871, China

^§Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China

S

* Supporting Information

ABSTRACT: The inhibition of hepatitis B virus (HBV) capsid

assembly is a novel strategy for the development of chronic

hepatitis B (CHB) therapeutics. On the basis of the preclinical

properties and clinical results of GLS4, we carried out further

investigation to seek a better candidate compound with appropriate

anti-HBV potency, reduced hERG activity, decreased CYP enzyme

induction, and improved pharmacokinetic (PK) properties. To this

end, we have successfully found that morpholine carboxyl

analogues with comparable anti-HBV activities to that of GLS4

showed decreased hERG activities, but they displayed strong

CYP3A4 induction in a concentration-dependent manner, except

for morpholine propionic acid analogues. After several rounds of

modiﬁcation, compound 58 (HEC72702), which had an (R)-

morpholine-2-propionic acid at the C6 position of its dihydropyr-

imidine core ring, was found to display no induction of the CYP1A2, CYP3A4, or CYP2B6 enzyme at the high concentration of

10 μM. In particular, it demonstrated a good systemic exposure and high oral bioavailability and achieved a viral-load reduction

greater than 2 log in a hydrodynamic-injected (HDI) HBV mouse model and has now been selected for further development.

treatment of CHB and reduce drug resistance, the development

of novel agents with diﬀerent therapeutic targets besides viral

polymerase is highly necessary.⁶

INTRODUCTION

■

HBV infection is a serious global public-health problem, with

more than 350 million people, especially in Asia-Paciﬁc regions,

chronically infected by this small enveloped DNA virus. The

disease progression for many of the infected persons will ﬁnally

develop into more life-threatening diseases, such as liver failure,

cirrhosis, or hepatocellular carcinomas (HCC).^1,2It has been

estimated that HBV infections directly or indirectly account for

the deaths of 600 000 patients annually. Current treatment

regimens rely mainly on the use of interferon-α (IFN) and

nucleoside- or nucleotide-based reverse-transcriptase inhibitors.

However, the clinical use of IFN and peg-IFN is limited by

their low response rates (20−30%) among CHB patients and a

slew of side eﬀects, such as ﬂu-like symptoms, anemia,

The HBV capsid plays indispensable roles in viral DNA

synthesis from the pregenome and in intracellular traﬃcking.

HBV persistence and transmission require HBV replication,

which depends on the assembly of a nucleocapsid composed of

capsid proteins, reverse transcriptase, and pregenomic RNA.⁷

Furthermore, the encapsidation of HBV pregenomic RNA

(pgRNA) is an evolutionarily constrained process,⁸and this

genetic stability makes HBV-capsid formation a better drug

target against various genotypes of HBV polymerase-resistant

mutants.⁹

Some phenylpropenamide derivatives, such as 1 (AT-61), 2

(AT-130),^10,11the sulfamoylbenzamide 3 (DVR23),¹²and 4

(isothiaﬂudine),¹³shown in Figure 1, have been shown to

inhibit pgRNA packaging, which leads to the formation of

leucopenia, thrombocytopenia, anorexia, and depression.³

A

major drawback of the nucleotide analogues is the emergence

of drug resistance during long-term treatment, with approx-

imately 70% of patients becoming resistant to lamivudine;⁴

resistance to entecavir also occurs among lamivudine-pretreated

patients at a rate of 43% after 4 years.⁵In order to improve the

Received: December 28, 2017

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Figure 1. Chemical structures of the reported HBV-capsid inhibitors.

pgRNA-free empty capsids. These agents were found to be

potent against HBV replication in various human hepatoma cell

lines. Among them, NVR-3778 (unreported structure), a

sulfamoylbenzamide compound, has progressed into phase I,

and the clinical-phase Ib results show that at a dosage of 600

mg twice daily (b.i.d.), this compound could reduce serum

HBV-DNA levels to 1.72 log 10.¹⁴Jassen has also completed

the phase I clinical trials of JNJ-379 (unreported structure),

which is reported to possess the same mechanism as NVR-3778

but with improved eﬃcacy and a better safety proﬁle.¹⁵

risk, because of its moderate inhibition of the hERG channel

(hERG IC₅₀= 1.34 μM, Table 1). However, after careful

reasoning and assessment, we believe that GLS4 should be safe

enough for humans because of following results: (i) the safety

window of GLS4 is large enough (hERG IC₅₀/EC₅₀> 1000),

(ii) no cardiac toxicity is associated with hERG in 6 month

repeated toxicity tests with mice and 9 month repeated toxicity

tests with beagle dogs, and (iii) no obvious side eﬀects have

been observed so far from monitoring cardiac-toxicity-

associated indicators (QT durations) in clinical trials. In

conclusion, GLS4 was found to be safe enough in clinical trials,

and continuing clinical trials are currently underway. However,

having the potential safety risk of the use of GLS4 in the long-

term treatment of HBV patients in mind, we aimed to search

for a second-generation backup compound for GLS4. Thus, we

set out with the goal of ﬁnding a backup compound for GLS4

with the appropriate anti-HBV potency, signiﬁcantly reduced

hERG activity, decreased induction of the CYP enzyme, and

improved pharmacokinetic (PK) properties. Herein, we report

the discovery of compound 58, which appeared to be free of all

the drawbacks associated with GLS4 and has been selected for

further development as an oral anti-HBV-infection agent.

Bay 41-4109 (5, Figure 1) has been reported to disrupt or

misdirect the assembly of the HBV capsid, which leads to

capsid depletion and then termination of the viral replication

process.^16,17Bay 41-4109 was shown to prevent HBV infections

in a transgenic HBV model by reducing HBV viral DNA in the

liver and plasma, but it was found to be hepatotoxic at high

doses in rats; thus, the follow-up studies of this compound were

suspended.^18,19A currently reported compound in clinical stage

I with a similar mechanism as Bay 41-4109 was RG7689

(unreported structure), and preclinical experiments of this

compound showed that it could suppress viral replication

continually and prevent the formation of new cccDNA.^20,21

GLS4 (Figure 1) also possesses the same mechanism as

BAY41-4109, having been developed as a capsid inhibitor, and

is currently in the phase II clinical stage. The preclinical anti-

HBV eﬃcacy, mechanism of action, and pharmacological

properties of GLS4 have been recently published in several

articles.²²⁻²⁵It was reported that GLS4 demonstrated potent

inhibitory activities against wild and various drug-resistant HBV

strains in a HepG2-cell-transfection assay. However, it was

found that GLS4 alone could not achieve anticipated blood

concentrations to show the desired antiviral eﬀect in phase I

clinical trials, which likely resulted from its fast metabolism and

moderate induction of the CYP3A4 enzyme (see Table 3). To

address this problem, a combination of GLS4 and ritonavir

(RTV) was used in the ensuing clinical trials. This approach

proved to be successful, with signiﬁcant increases in the plasma

concentrations in the healthy volunteers and substantial

decreases in HBV-DNA levels in CHB patients.

RESULTS AND DISCUSSION

■

Structure and hERG Activity. To ameliorate the hERG

inhibition of GLS4, we started to search for a backup

compound for GLS4. We set to design and synthesize new

derivatives with reduced or diminished hERG activity.

Common approaches to decrease hERG activity include

structural modiﬁcations, formation of zwitterions, control of

lipophilicity, and attenuation of the pK_aof the molecule.²⁸To

this end, we ﬁrst separated the racemate of GLS4 and found

that the R and S enantiomers showed no diﬀerence in their

hERG activities (Table 1). Then, we replaced the dihydropyr-

imidine core with triazine and several other fused heterocycles

to give compounds 6−9. The dihydropyrimidine core was

oxidized to give pyrimidine compound 10, and N-alkylation

with a hydrophilic hydroxyethyl halide gave compound 11. As

shown in Figure 2, although these compounds displayed

Some drugs have been withdrawn from the market because

of prolonged QT intervals due to hERG inhibition.^26,27At ﬁrst

glance, GLS4 might cause some concerns of a QT-prolongation

decreased hERG inhibitions (for compound 10, hERG IC₅₀

10 μM), they also exhibited dramatic drops in their anti-HBV

activities (EC₅₀> 16.4 μM). Thus, we started to modify the

>

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Table 1. Anti-HBV Activities and hERG Activities of Molecules Generated via C2 and C4 Modiﬁcations

a

EC₅₀represents the mean values for the reduction of HBV-DNA levels by 50% in HepG.2.2.15 cells. Experiments were done in duplicate with

b

variations of <15%. hERG IC₅₀represents the mean values for the 50% inhibition of hERG in CHO cells (N = 3). Cisapride was used as a positive

control, and the maximum concentration used was 10 μM. IC₅₀> 10 μM is reported if the inhibition at 10 μM is lower than 50%.

peripheral substituents while keeping the dihydropyrimidine

core ring unchanged.

Thus, we made some small changes on the substituents at the

C4 phenyl and found that these changes had no signiﬁcant

eﬀect on anti-HBV activity (GLS4, 12, 13, and 14; Table 1).

These compounds, however, showed no obvious improvement

in their hERG activities. It was also observed that the hERG

activities of these compounds increased with increasing cLogP.

The order of the hERG activities of compounds GLS4 to 14

was as follows: 13 (hERG IC₅₀= 0.30 μM, cLogP = 5.17) >

GLS4 (hERG IC₅₀= 1.34 μM, cLogP = 4.75) > 12 (hERG IC₅₀

= 2.31 μM, cLogP = 4.60) > 14 (hERG IC₅₀= 4.04 μM, cLogP

= 4.45). Replacement of the 2-bromine atom on the 4-phenyl

group in GLS4 with NO₂or phenyl gave compounds 15 and

16, but these two compounds were less active against HBV

than GLS4, and their hERG activities showed no obvious

improvements.

Our previous studies indicated that the ester group at the C5

position of GLS4 (see structure of GLS4 for the numbering

convention) contacted a limited hydrophobic pocket so that

polar and large groups were generally not tolerated.²⁹After

investigating various ester substitutions at the C5 position, we

found that only the methyl ester and ethyl ester were well

tolerated. We therefore decided to focus on conducting further

modiﬁcations at the C2, C4, and C6 positions of the core

dihydropyrimidine structure to improve or retain anti-HBV

activity while decreasing hERG-inhibitory activity. The reported

cocrystal structure (PDB ID 2G34)³⁰indicated that the C4

position of the core dihydropyrimidine was located in a

conserved and large hydrophobic pocket, and we also found

that bulky groups, such as biphenyl groups, and hydrophilic

substituents, such as carboxyl groups, were not tolerated.²⁹

Next, we investigated the modiﬁcation at the C2 position.

Replacing the C2 thiazolyl with thiadiazolyl and thiophenyl

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Figure 2. Previous design structure of molecules from GLS4.

resulted in compounds 17 and 18, respectively. These two

compounds showed similar anti-HBV activities to each other

but decreased activities in comparison with GLS4. In the

hERG-activity studies, compound 17 showed decreased hERG

inhibition (IC₅₀= 3.49 μM), whereas compound 18 showed

increased hERG activity (IC₅₀= 0.9 μM). Apparently,

lipophilicity was a key determinant for the cLogP of the 2-

thiophenyl compound, 18, being much higher than that of

compound 17 (cLogP = 6.05 vs 3.66). Further replacement of

the thiazolyl group in GLS4 with pyridine, N-methyl-imidazole,

triazole, and substituted thiazole led to compounds 19, 20, 21,

23, and 24, but none of these compounds showed obvious

improvements in hERG inhibition. However, we found that

modiﬁcation with a tetrazole at the C2 position (compound

22) showed decreased hERG activity (IC₅₀> 10 μM) but at the

expense of decreased anti-HBV activity (EC₅₀> 16.4 μM). The

3-methyl acetate of thiazole 24 was hydrolyzed to carboxylic

acid to give compound 25, which showed decreased hERG

activity (IC₅₀> 10 μM), but this compound also lost the anti-

HBV activity. On the basis of the above results, we believed that

it would be diﬃcult to obtain compounds with optimal anti-

HBV activities and low hERG activities when the structure

modiﬁcations were made on the C2 and C4 positions of

dihydropyrimidine, and importantly, it seemed possible to

reduce hERG activity when a hydrophilic group, such as

carboxylic acid or tetrazole, was introduced.

On the basis of the preliminary SARs at the C2 and C4

positions, we decided to introduce less lipophilic or hydrophilic

groups to the C6 position by replacing the morpholine moiety

of GLS4 (Table 2). First, the substitution of the morpholine

group with thiomorpholine monoxide or dioxide led to

compounds 26 and 27 whose anti-HBV activities were about

10 times less than that of GLS4. The more hydrophilic

thiomorpholine monoxide, 26, showed less hERG inhibition

than thiomorpholine dioxide, 27. It was further conﬁrmed that

the hERG activity could be adjusted by lipophilicity, and thus

some hydrophilic groups, such as phenylalanine and proline,

were introduced. The resultant compounds, 28 and 29,

exhibited moderate anti-HBV activities and lower hERG

activities. The common “bioisosteres” of proline, piperdine

carboxylic acid and piperazine carboxylic acid, on the C6

position led to compounds 30 and 31, which showed decreased

hERG inhibitions and lower anti-HBV activities. It seemed

reasonable that compounds 30 and 31 showed decreased anti-

HBV activities because a polar carboxylic acid substituent was

not tolerated in the large hydrophobic binding pocket located

at the interface of the capsid dimers. However, to our delight,

the introduction of various morpholine carboxyl groups at the

C6 position, aﬀording compounds 32, 33, and 34, resulted in

comparable anti-HBV activities to that of GLS4 and lower

hERG inhibitions (hERG IC₅₀> 10 μM). Moreover, an

additional methyl group, as in compound 35 (EC₅₀= 0.0093

μM), resulted in a 5-fold higher potency than that of 32 (EC₅₀

= 0.043 μM). In contrast, the dimethyl morpholine carboxylic

acid analogue, 36, demonstrated reduced anti-HBV activity

(EC₅₀= 3.924 μM), suggesting that the substituent position of

the methyl was important.

Further derivatization of the carboxylic acid of 32 gave rise to

esters 37 and 38, carboxamides 39 and 40, sulfonamide 41, and

alcohols 42 and 43, and all of these compounds maintained low

hERG activities. Among them, 37, 38, 39, 42, and 43 showed

increased or comparable anti-HBV potencies to that of GLS4.

Large substitutions on the morpholine carboxyls of the ester

and carboxamide were did not result in good anti-HBV

activities, as in 40 and 41. Although the ester 37 and the

carboxamide 39 displayed favorable increases in anti-HBV

activities and signiﬁcantly decreased hERG activities as

compared with those of 32 and GLS4, further development

of these compounds (37−43) was not continued because of

their high liver-microsome clearances (data not shown).

Further studies revealed that both the substitution position

and chain length of the carboxyl group were crucial for anti-

HBV activity. The corresponding morpholine-2-carboxyl

analogue, 44, was much less active than 32, and the extended

acetic acid analogues, 45 and 46, also showed reduced anti-

HBV activities. In addition, to our surprise, the extended

propanoic acid analogues, 47 and 48, showed comparable anti-

HBV activities to that of 32 and decreased hERG activities

(hERG IC₅₀> 10 μM).

To further investigate the relationship between anti-HBV

activity and hERG activity, a series of compounds with good

anti-HBV activities and low hERG activities in Table 2 were

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Table 2. Anti-HBV Activities and hERG Activities of Molecules Generated via C6 Modiﬁcations

a,b

The deﬁnitions of EC₅₀and hERG IC₅₀are the same as those described in Table 1.

selected for chiral separation to obtain the chiral isomers to be

screened.

(R)-49, showed the most potent anti-HBV activity, which was

almost 400-fold more potent than that of the C4 S-isomer, (S)-

49 (0.0057 vs 2.432 μM). This was in accordance with the

result that (R)-GLS4 also showed 400-fold greater potency

than (S)-GLS4 (0.0098 vs 3.367 μM). In addition, we further

conﬁrmed that the (S)-morpholine-3-carboxylic acid analogue

demonstrated better anti-HBV activity than the corresponding

(R)-morpholine-3-carboxylic acid analogue. For example, the

(S)-morpholine-3-carboxylic acid analogue, (R)-49, showed

nearly 50-fold greater potency than the corresponding (R)-

morpholine-3-carboxylic acid analogue, (R)-50 (0.0057 vs

0.292 μM).

All compounds with carboxyl groups in Table 3 showed no

hERG inhibition at concentrations as high as 30 μM, except

compound 54, which bore an alcohol group and showed an

hERG IC₅₀of 24.2 μM. These data further reveal the essential

role of carboxyl groups in reducing the hERG activities of the

analogues of GLS4.

Chirality is important for anti-HBV activity. It was found that

the C4 R-conﬁguration analogues have better anti-HBV

activities than their corresponding C4 S-isomers. For example,

without changing the C6 substituent, (S)-morpholine-3-

carboxylic acid, the C4 R-conﬁguration of compound 49,

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Table 3. Anti-HBV Activities and hERG Activities of Selected Chiral Isomers

a

b

The deﬁnition of EC₅₀is the same as that described in Table 1. hERG IC₅₀represents the mean values for the 50% inhibition of hERG in HEK-

293 cells (N = 3). Quinidine is used as a positive control, and the maximum compound concentration used is 30 μM. IC₅₀> 30 μM is reported if the

inhibition at 30 μM is lower than 50%.

Substitutions on the C4 phenyl ring aﬀect activity, and the

following order of anti-HBV activity was found: 2-Cl-4-Cl (49,

EC₅₀= 0.0086 μM) > 2-Br-4-F (53, EC₅₀= 0.019 μM) > 2-Cl-

4-F (51, EC₅₀= 0.024 μM), which is in agreement with earlier

modiﬁcation trend (such as GLS4 and compounds 12 and 13

in Table 1). In addition, compounds with ethyl esters at the 5-

position of dihydropyrimidine demonstrated about 2-fold

higher potencies than compounds with methyl esters, as with

compounds 51 and 52 (0.024 vs 0.047 μM). After chiral

separation, the R-enantiomer of (R)-53 showed a nearly 2-fold

greater potency than racemic compound 53 (0.01 vs 0.019

μM), and the same situation occurred with (R)-49 than 49

(0.0057 vs 0.0086 μM). An additional methyl introduction near

the morpholine carboxyl group of (R)-53 (EC₅₀= 0.01 μM) led

to 55, which also showed a 2-fold improvement with an EC₅₀of

0.005 μM. With a gem-dimethyl substituent on the other side

of morpholine carboxyl, as in (R)-53, the anti-HBV potency

was dramatically decreased, as compound 56 had an EC₅₀of

0.914 μM. The 4,4-diﬂuoroprolino carboxyl analogue, 57, was

highly active, with an EC₅₀of 0.005 μM, a potency comparable

to those of (R)-52 and 55. The substitution position of the

propionic acid on morpholine had no consistent inﬂuence on

anti-HBV activity, as compound 58 showed reduced activity

compared with 60, whereas 59 showed increased activity

compared with 61. Additionally, the inﬂuence of the absolute

conﬁguration of a propionic acid group on anti-HBV activity

was studied in compounds 59 versus 58 and 61 versus 60

(EC₅₀values of 0.094 versus 0.039 μM and 0.154 versus 0.008

μM, respectively). Once again, these analogues in Table 3

revealed that the substitution position on the morpholine

moiety (such as morpholine-2-carboxylic acid vs morpholine-3-

carboxylic acid), the chain length of the carboxyl group (such as

formic acid vs propionic acid), and the absolute conﬁguration

of the carboxylic acid group (such as R-carboxylic acid vs S-

carboxylic acid) directly inﬂuenced anti-HBV activity but had

no obvious inﬂuence on hERG activity, as all of these

compounds showed no sign of hERG inhibition (IC₅₀> 30

μM). This is probably because of the introduction of the

carboxyl group, which led to reduced lipophilicity and basicity

or a certain amount of zwitterions.

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Table 4. Relative Induction Values of the CYP3A4 Enzyme by the Selected Compounds

a

Experiments were done in human primary hepatocytes (N = 3). The positive control had a minimum of a 2-fold increase in enzyme activity. The

cutoﬀ of a true inducer of P450 activity in vitro is generally considered to be 40% of the positive control.

Induction Eﬀect of CYP3A4. It was reported that the

CYP3A4 induction of 4-H heteroaryldihydropyrimidine (HAP)

analogues was likely related to the 4-H HAP core, as over 250

compounds of 4-H HAP were screened and most of them were

found to have CYP3A4-induction liabilities.²¹Our previous

studies also showed that most of the GLS4-like compounds

possessed potent inductions of CYP3A4 enzymes (>40%

increases compared with the positive control rifampicin at 10

μM). Although the series of compounds shown in Tables 2 and

3 showed favorable potencies and decreased hERG inhibitions,

we continued to investigate their CYP-enzyme-induction

properties. After excluding the nonactive compounds in Tables

2 and 3 and taking into consideration the eﬀects of diﬀerent

substituents on the C2, C4, and C6 positions and the eﬀects of

the chiral isomers, some representative compounds were

selected for further studies of their induction activities on the

CYP3A4 enzyme. As shown in Table 4, the more potent

enantiomer displayed higher induction activity than the less

potent enantiomer. For example, (R)-49 showed a 103.5%

increase of CYP3A4-enzyme activity, whereas (S)-49, which has

weaker anti-HBV activity, showed no induction (<40% increase

compared with rifampicin). A similar trend was observed with

the anti-HBV activities and induction abilities of the GLS4

enantiomers, R-GLS4 and S-GLS4. In addition, the compounds

induced the CYP3A4 enzyme in a concentration-dependent

manner. For example, compound 53 displayed no induction of

CYP3A4 at lower concentrations (0.05−0.5 μM), but it showed

serious induction eﬀects at the higher concentration of 1 μM.

At a concentration of 1 μM, the selected compounds with

potent anti-HBV activities, such as the morpholine-3-carboxylic

acid analogue, 53; derivatives of 53 such as 55 and the

carboxamide 40; and the 4,4-diﬂuoroproline carboxylic acid

analogue, 57, also showed strong CYP3A4 inductions.

Minor variations of the C4 phenyl showed no obvious

CYP3A4-induction changes, as compounds 49 (2-Cl-4-Cl), 53

(2-Br-4-F), and (R)-52 (2-Cl-4-F) displayed similar induction

eﬀects on CYP3A4 at the same concentration of 1 μM. To

investigate the eﬀect of the 2-heterocycle substituent (C2

position) on CYP3A4 induction, we replaced the thiazole group

with N-methyl imidazole to obtain compound 62. Although

this compound showed no signiﬁcant induction of CYP3A4 at

the concentration of 1 μM, it was found to be a strong inducer

conferring a 50.8% increase at a concentration of 10 μM. The

extended carboxyl analogues of 46, which possessed a

morpholine acetic acid at the C6 position, also showed dose-

dependent CYP3A4 induction. In addition, it is surprising to

ﬁnd that compound 58, which possessed an (R)-morpholine-2-

propionic acid at the C6 position displayed inconspicuous

induction of CYP3A4 at either concentration of 1 or 10 μM.

On the basis of the observation that the (R)-morpholine-2-

propionic acid analogue, 58, possessed good anti-HBV activity

and decreased induction of the CYP3A4 enzyme, we further

tested the anti-HBV activities of several compounds with

morpholine-propionic acid moieties and tested their induction

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Table 5. Anti-HBV Activities and Relative Induction Values of the CYP3A4 Enzyme by Selected Compounds

a

b

The deﬁnition of EC₅₀is the same as that described in Table 1. The deﬁnition of % of positive control is the same as that described in Table 4.

eﬀects on the CYP3A4 enzyme at concentrations of 1 μM. The

results revealed that both the substitution position and

stereochemistry of the propionic acid group were crucial for

the anti-HBV activity and ability to induce the CYP3A4

enzyme. As shown in Table 5, the (S)-morpholine-2-propionic

acid analogue, 59, showed about 3-fold-reduced anti-HBV

activity than 58 while displaying the same induction of the

CYP3A4 enzyme as 58. In addition, the anti-HBV activity of

(R)-morpholine-3-propionic acid analogue 61 was 20 times

lower than that of the S-analogue, compound 60, but 61

showed a little more CYP3A4-enzyme induction than 60

(>40% for 61 vs 17.9% for 60). It was also found that the

methyl ester analogues, 63, 64, and 65, which had diﬀerent

substituents on their C4 phenyls, showed decreased induction

eﬀects compared with that of 58 and lower anti-HBV activities

than that of 58. It was very fulﬁlling to ﬁnd that the (S)-

morpholine-3-propionic acid analogue, 60, not only exhibited

greater potency than the (R)-morpholine-2-propionic acid

analogue, 58, with an EC₅₀value of 0.008 μM, but it also

displayed a weak induction eﬀect. The (S)-diﬂuoropyrrolidine-

2-propionic acid analogue, 66, had a slightly decreased

induction ability and less anti-HBV activity compared with

those of 60 and 58. On the basis of the above results, we

concluded that the morpholine propionic acid played an

essential role in decreasing the CYP3A4-induction capability, as

unsubstituted morpholine, morpholine formic acid, and

morpholine acetic acid analogues showed strong induction

eﬀects on the CYP3A4 enzyme.

Molecular-Docking Studies. In our previously published

study, we demonstrated that GLS4 could prevent the normal

assembly of core proteins and lead to aberrant formation of the

capsid.²⁴Furthermore, in an AAV-HBV model (chronic

infection), GLS4 treatment signiﬁcantly reduced intrahepatic

HBc levels as determined by immunohistochemistry (IHC) of

liver sections (unpublished data). As derivatives of GLS4 with

similar structures and in the class of 4-H HAP compounds,

compound 58 and the other analogues were supposed to have

similar mechanisms of action as GLS4.

Klumpp²⁵has recently reported the cocrystal structure of the

Cp149 Y132A-mutant hexamer in complex with compound

NVR-010-001-E2 (an analogue of GLS4), which supports the

result that the introduction of a morpholine group on BAY41-

4109 could improve anti-HBV activity. In the article, Klumpp

proposed that optimizations focusing on the replacement of

this morpholine group would lead to improved protein

interactions. To explore the interaction mode of the GLS4

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Figure 3. Optimal-binding model for compounds 58 (left) and 60 (right) in the active site of the capsid docked by the SURFLEX module. The

ligand is shown in sticks, and some key residues are shown in lines. Hydrogen bonds are shown in dashed lines.

Table 6. Anti-HBV Activities, Microsome Stabilities, and Pharmacokinetics of Selected Compounds in Rats

a

b

The deﬁnition of EC₅₀is the same as that described in Table 1. Scaled intrinsic clearances in human liver microsomes (HLM), mouse liver

c

microsomes (MLM) and dog liver microsomes (DLM). Experiments were run in duplicate, with variations of <10%. Single-dose-pharmacokinetics

(SDPK) studies of selected compounds were carried out in rats according to standard procedures. The major parameters, including the area under

the curve (AUC), plasma clearance (CL), volume of distribution at steady state (V_ss), and oral bioavailability (F) are reported.

analogues with the active site of the HBV capsid protein, several

molecular-docking-simulation studies were carried out by using

the SURFLEX module of the SYBYL package. The crystal

coordinates of the capsid (PDB ID 5E0I) were downloaded

from protein data bank (www.rcsb.org), and compounds 58

and 60 were used as ligand examples.

As shown in Figure 3, both compounds are well docked in

the active site of the crystal coordinate of 5E0I. For both

compounds, the substituted phenyl group ﬁts into the well-

deﬁned, 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 substituted phenyl group,

large substituents are generally not well tolerated. The thiazole

moiety on each of these two compounds is coplanar with the

dihydropyrimidine core, as anticipated, and is buried in a

hydrophobic environment created by the aromatic residues

Phe23, Pro25, Tyr118, Trp102, and Thr128. Moreover, a

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a

Table 7. Pharmacokinetics of GLS4 and 58 in Beagle Dogs

compd

GLS4

dose (mg/kg)

AUC_0−24h(h·ng/mL)

C_max(ng/mL)

C₂₄(ng/mL)

t_1/2(h)

V_ss(L/kg)

CL (mL/min/kg)

16.6

F (%)

iv (2)

po (5)

iv (2)

po (5)

1472

1008

1423

391

15

9

17.9

11.5

5.71

5.1

15.83

23.8

58

16 540

47 900

2050

6330

47

232

0.76

1.93

114.1

a

Single-dose pharmacokinetics (SDPK) studies of selected compounds were carried out in beagle dogs according to standard procedures. The major

parameters, including the area under the curve (AUC), maximal concentration (C_max), concentration 24 h after the oral dose (C₂₄), half-life (t_1/2),

plasma clearance (CL), volume of distribution at steady state (V_ss), and oral bioavailability (F), are reported.

water-bridged HB network exists between the 2-thiazole and

Leu140, and a π−π interaction is found between the thiazole

residue and Phe23. The N3 atom of the dihydropyrimidine ring

forms a hydrogen bond with the main-chain nitrogen of

Trp102. The ester moiety on each of these two compounds

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. Compared with 58, 60 has achieved an

approximately 5-fold improvement in antiviral activity, which

can be clariﬁed from the diﬀerent binding eﬀects of these two

compounds with the core protein dimer. As shown in Figure 3,

the carboxylic acid moiety of 60 makes a water-bridged HB

interaction with Ser121, whereas the carboxylic acid moiety of

58 cannot make HB interactions with Ser121 in the binding

site. Interestingly, the oxygen atom of the morpholino group of

compound 58 could form a double water-bridged HB

interaction with Ser121, though it shows reduced activity

relative to that of 60. Apart from the docking simulation, which

showed that compound 58 could interact with the HBV-capsid-

protein dimer, the activity of compound 58 against HBV-capsid

assembly was also demonstrated in an HBV-capsid-assembly-

quenching assay, in which it had an IC₅₀value of 0.14 μM.

Microsome Stability and PK. On the basis of their anti-

HBV activities, hERG inhibitions, and CYP3A4-enzyme

inductions, a few analogues were selected for further in vitro

liver-microsome-stability tests and in vivo pharmacokinetic

studies in rats. Previous investigations indicated that the major

metabolic pathway of GLS4 was often associated with N−C

and O−C cleavages of the 6-morpholine moiety. It was

hypothesized that the introduction of substituents on the

morpholine would lead to decreased metabolism and improved

exposure. As shown in Table 6, (R)-53 showed improved

metabolic stability in three types of liver microsomes compared

with that of GLS4 and correspondingly increased systemic

exposure. It should be noted that the oral exposure increased

much more than the exposure by the intravenous route, which

suggested that the introduction of the polar carboxylic acid also

led to higher aqueous solubility. Thus, compound (R)-53

displayed better oral bioavailability. In addition, compound 55,

which had an additional methyl substitution on the side of the

carboxylic acid, showed more metabolic stability than (R)-53

and also displayed increased systemic exposure compared with

that of (R)-53 whether by intravenous (iv) or oral (po)

administration. These results indicated that when the metabolic

hot spots on the morpholine group of GLS4 were blocked, the

plasma concentration and systemic exposure of the parent drug

would increase.

dog-, and human-liver-microsome assays. In the experiment, all

four of these compounds were more stable in the human and

dog liver microsomes than in the mouse liver microsomes.

Compound 60 was highly potent in vitro and showed no

induction of the CYP3A4 enzyme, but its low systemic

exposure prevented its further development. In addition,

compounds 59 and 66 also showed higher clearances and

lower systemic exposures in rats. To our delight, compound 58

displayed greatly increased systemic exposures and low

clearances by both intravenous (iv) and oral (po) routes even

though its metabolic stability in mouse liver microsomes was

not satisfactory. We reasoned that 58 would show much higher

exposure when dosed in large animals, such as beagle dogs, as it

was stable in dog and human liver microsomes.

The PK proﬁle of 58 was further evaluated in beagle dogs. As

shown in Table 7, 58 exhibited longer half-lives of 5.7 h (iv)

and 5.1 h (po) as well as a lower plasma clearance of 1.93 mL/

min/kg, and it also showed a high systemic exposure with areas

under the curves (AUC) of 16 540 h·ng/mL (iv) and 47 900 h·

ng/mL (po) with an oral bioavailability (F) of 114.1%.

Although the half-life of compound 58 is shorter than that of

GLS4, the drug exposure and the concentration 24 h after the

oral dose (C₂₄, 232 ng/mL) for compound 58 were much

higher than those of GLS4 (AUC = 1008 h·ng/mL, C₂₄= 9 ng/

mL). Considering GLS4 is dosed b.i.d. in combination with

ritonavir to increase its drug exposure in the current clinical

study, both once daily (q.d.) and b.i.d. dosings of compound 58

will be evaluated in the further clinical experiments to

determine the best dosing regimen. The good systemic

exposure and oral bioavailability of 58 in beagle dogs suggested

that this compound could have desirable PK properties in

human, as the liver microsome stability of 58 displayed similar

tendencies in beagle dogs and human liver microsomes.

Since compound 58 demonstrated the optimal overall proﬁle

in its in vitro potency, in vitro metabolic stability, PK

properties, hERG inhibition, and CYP3A4 induction, it was

studied further for its induction eﬀects on other CYP enzymes.

As shown in Table 8, this compound was not a CYP1A2,

CYP3A4, or CYP2B6 inducer in primary human hepatocytes as

determined by the corresponding CYP1A2-, CYP3A4-, and

CYP2B6-enzyme levels (<40% increases compared with the

positive control) at either low concentrations of l μM or higher

concentrations of 10 μM. This unique property reduces the

potential risk of drug−drug interactions when HBV patients are

treated.

On the basis of these results, compound 58 was selected as

the candidate compound and was further assessed against HBV

infections in the hydrodynamic-injection (HDI) mouse

model.²⁸In this study, female BALB/c mice were hydro-

dynamically injected with replication-competent HBV-DNA

plasmids through the tail vein. Entecavir (ETV, 0.1 mg/kg) was

used as the positive control. Twenty hours after plasmid

In addition, both the substitution position and stereo-

chemistry of the propionic acid group were crucial for

metabolic stabilities and pharmacokinetic properties. The

metabolic stabilities of the analogues with propionic acid

groups (58, 59, 60, and 66) were assessed in vitro in mouse-,

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dependent reduction of HBV DNA in the plasma of infected

mice when the compound was orally administered b.i.d. at 50

mg/kg (mpk), and 100 mpk, respectively. In comparison to the

vehicle control group, the treatment with 50 and 100 mpk of 58

b.i.d. had achieved an over 2 log viral-load reduction in the

plasma of the HDI mice on day 4. The levels of HBV DNA in

the mouse livers were determined 8 days after the hydro-

dynamic injections of the HBV DNA plasmids. Consistent with

the results in the mouse plasma, with oral dosings of compound

58 at 50 or 100 mpk b.i.d., HBV-DNA copy numbers were

markedly reduced by over 100-fold in the liver, demonstrating a

strong anti-HBV eﬀect. The maximum eﬃcacy of 58 was

comparable to the maximum eﬃcacy of ETV in this model.

It was believed that compound 58 and the other analogues of

GLS4 would have activities against other HBV genetypes

because of the mechanisms of action of these compounds and

the conservation of the capsid protein sequence. The inhibition

experiments of compound 58 against other HBV genetypes is

still ongoing. Another compound, (R)-52, showed almost the

same activity against four genetypes (A, B, C, and D; shown in

the Supporting Information). In addition, other reported capsid

inhibitors, such as JNJ-379, were also shown to be active against

full-length genotype-A−H clinical isolates in vitro.³¹

Compound 58 exhibited low CYP inhibition, with IC₅₀> 10

μM, against the six major CYP enzymes (1A2, 2C9, 2D6, 3A4,

2C19, and 2B6) and was also not a time-dependent CYP

inhibitor. The in vitro toxicology proﬁles of compound 58 were

favorable, with no signs of hERG inhibition (IC₅₀> 30 μM),

and it was less cytotoxic to HepG2 cells, with a CC₅₀value of

96.76 μM determined by a CellTiter-Blue assay. It was also

negative in preliminary in vitro safety evaluations such as Ames

as well as micronucleus-test (MNT) assays. A 14-day in vivo

toxicology assessment in mice supported the further develop-

ment of compound 58, which showed a supportive safety

margin as deﬁned by the ratio of the plasma exposure (C_maxand

AUC) in mice at the NOAEL versus the exposure at the

projected human eﬃcacious dose.

Table 8. Relative Induction Values of the CYP1A2, CYP2B6,

and CYP3A4 Enzymes by 58

a

concentration

% of positive

control

enzyme subtype

(μM)

1A2 positive control (omeprazole, 50

1

10

1

4.19 (<40%)

2.96 (<40%)

−13.2 (<40%)

−10.6 (<40%)

11.8 (<40%)

10.0 (<40%)

μM)

3A4 positive control (rifampicin, 25

μM)

10

1

2B6 positive control (phenobarbital,

1000 μM)

10

a

Experiments were run in human primary hepatocytes (N = 3).

Positive controls induced a minimum of 2-fold increases in enzyme

activities. The cutoﬀ of a true positive sign of the induction of P450

activity in vitro is generally considered to be 40% of the positive

control.

injection, the mice were orally dosed with a blank vehicle or

diﬀerent doses of compound 58 for 8 days. Plasma and liver

samples were collected at the indicated time points for HBV-

DNA quantiﬁcation by real-time quantitative polymerase chain

reaction (qPCR).

A PK study was done, and the eﬀective dose of the

compound was estimated in the same BALB/c mice prior to

the anti-HBV-activity HDI experiment. Considering the short

half-life of compound 58, b.i.d. doses were chosen in the HDI

mouse-model experiment. The mice were given compound 58

in a dosage of 12.5 mg/kg, and the drug concentrations in the

plasma and liver were assayed at diﬀerent times. The plasma

C₂₄(the concentration 24 h after dosage) was 849 ng/mL and

the liver C₂₄was 361 ng/g, which were much higher than the

EC₅₀(0.039 μM) of compound 58. Considering the plasma

protein binding and linear correlation between the concen-

tration and dosage, the dosages for the HDI mouse-model

experiment were set to 50 and 100 mg/kg.

The hydrodynamic-injection mouse model was established

for HBV to transiently replicate in the mouse liver. Inhibition of

HBV replication can thus be studied during this transient time

of about 8 days. As shown in Figure 4, 58 demonstrated a dose-

Figure 4. Eﬀects of treatment with 58 (HEC72702) on the inhibition of HBV replication in the HDI mouse model. Mice (N = 6) were orally dosed

with the vehicle control, compound 58 (HEC72702, 50 or 100 mpk, b.i.d.), or ETV. (A) Levels of HBV DNA in plasma. At 2, 4, 6, and 8 days

postinfection, the serum HBV of all the treatment group (groups 2−4) were quantiﬁed and showed signiﬁcant reductions in HBV DNA compared

with that of the vehicle control. Error bar indicate the standard error of the mean (N = 6). *** indicates p < 0.001 for 50 mpk HEC72702 b.i.d. vs

the vehicle, ^###indicates p < 0.001 for 100 mpk HEC72702 b.i.d. vs the vehicle, and ^&&&indicates p < 0.001 for 0.1 mpk ETV q.d vs the vehicle. (B)

Quantiﬁed levels of HBV DNA in mouse livers. All the treatment groups (group 2−4) had signiﬁcant reductions in liver HBV DNA compared with

the vehicle control. *** indicates p < 0.0001.

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a

Scheme 1. Synthesis of Compound 58

a

Reagents and conditions: (a) 1.2 equiv NaOAc, EtOH, reﬂux, 16 h; (b) recrystallization with ethanol; (c) 2 equiv Li, EtOH, reﬂux, 2 h; (d) 1.1

equiv NBS, CCl₄, reﬂux, 1 h; (e) K₂CO₃, EtOH, 25 °C, 2 h.

The capsid inhibitor is diﬀerent from nucleoside or

nucleotide analogues such as tenofovir (TDF) and entecavir

(ETV) in its viral inhibition mechanism. It is believed that a

combination of these two kinds of drugs would beneﬁt HBV

treatment. As shown above, compound 58 displayed low CYP

inhibition (IC₅₀> 10 μM) against the six major enzymes, and it

was not a CYP (1A2, 2B6 and 3A4) inducer. These properties

would reduce the potential risk of drug−drug interactions of 58

and the nucleoside and nucleotide analogues. However,

whether there will be drug−drug interactions needs to be

further studied.

Chemistry. As exempliﬁed by the synthesis of compound

58, the synthetic approach to obtain targeted dihydropyr-

imidines was outlined in Scheme 1. First, a three-component

reaction of thiazole-2-carboxamidine hydrochloride, 2-bromo-4-

ﬂuorobenzaldehyde, and (S)-1-isopropoxy-1-oxopropan-2-yl 3-

oxobutanoate (58I-1) was conducted to give the racemic

dihydropyrimidine 58I-2. After the recrystallization of the crude

product of racemic dihydropyrimidine 58I-2, the enantiomeri-

cally pure (R)-58I-2 could be obtained, and the absolute

stereochemistry of (R)-58I-2 was unambiguously determined

by an X-ray-diﬀraction study. The enantiomerically pure (R)-

58I-2 was then reacted with lithium to aﬀord enantiomerically

pure (R)-58I-3 as the desired intermediate. Bromination of

(R)-58I-3 gave (R)-58I-4, which was ﬁnally reacted with (R)-3-

(morpholin-2-yl) propanoic acid to provide 58 after

puriﬁcation. This short and concise synthetic route was

successfully applied to the synthesis of the other dihydropyr-

imidine analogues described in this paper, which greatly

facilitated and expedited our SAR studies.

regard to CYP-enzyme induction, we found that unsubstituted

morpholine, morpholine carboxylic acid, and morpholine acetic

acid analogues displayed strong CYP3A4 inductions in

concentration-dependent manners, and we found that the

morpholine propionic acid analogues showed decreased

induction liabilities. Furthermore, the substitution position

and stereochemistry of the propionic acid groups were crucial

for the pharmacokinetic properties. In particular, 58, which

possessed an (R)-morpholine-2-propionic acid at the C6

position of the dihydropyrimidine core ring, displayed a very

weak induction of CYP3A4, even at the higher concentration of

10 μM, and it also displayed a greatly increased systemic

exposure and a low clearance both in rats and in beagle dogs.

Finally, compound 58 exhibited a desirable overall proﬁle as

a drug candidate in terms of its in vitro potency, in vitro

metabolic stability, PK properties, and hERG inhibition.

Further, it was not a CYP1A2, CYP3A4, or CYP2B6 inducer

in primary human hepatocytes, avoiding the potential risk of

drug−drug interactions in HBV patients. In the in vivo studies,

compound 58 dose-dependently reduced HBV DNA in both

the plasma and livers of HBV-infected HDI mice, achieving an

over 2 log viral-load reduction on day 4 with 50 mpk b.i.d.

dosings. Safety evaluations, including an Ames toxicity study

and oral toxicity study in mice, indicated that compound 58

was safe enough. On the basis of these results, compound 58

will be selected for further development as an oral anti-HBV-

infection agent.

EXPERIMENTAL SECTION

■

Synthetic-Chemistry General Comments. 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 a sodium wire and

CaH₂, respectively. ¹H NMR and ¹³C NMR spectra were recorded on

a Bruker-600, Bruker-500, or Bruker-400 spectrometer (Bruker,

Billerica, MA) and were referenced to the residual protio signals in

CDCl₃at 7.26 ppm or DMSO-d₆at 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, brs = broad singlet, d = doublet, t = triplet, q =

quartet, dd = doublet of doublets, m = multiplet), coupling constant

(Hz). HRMS analyses were performed on an Agilent 6530 Q-TOF

mass spectrometer, and ESIMS was carried out on an Agilent 6120

mass spectrometer (Agilent Technologies, Santa Clara, CA). TLC was

CONCLUSION

■

In summary, GLS4 in combination with RTV is currently in

clinical phase II for chronic HBV infections and has

demonstrated good eﬃcacy. On the basis of the preclinical

and clinical properties of GLS4, we obtained novel GLS4

derivatives with the appropriate anti-HBV potencies, signiﬁ-

cantly reduced hERG activities, decreased CYP enzyme

inductions, and improved pharmacokinetic (PK) properties.

After the extensive modiﬁcation of GLS4, we have found that

the morpholine carboxyl analogues showed decreased hERG

activities and comparable anti-HBV activities to GLS4. With

L

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performed on Huanghai HSGF 254 silica gel plates (China). Silica gel

60 H (200−300 mesh), manufactured by Qingdao Haiyang Chemical

Group Company (Qingdao, China), was used for general chromatog-

raphy. The purities of all the ﬁnal derivatives for biological testing were

conﬁrmed to be >95%, as determined using an Agilent 1200 series

HPLC instrument at 278 nm, using the following conditions: an

Alltima C18 column (4.6 × 250 mm, 5 μm; Hichrom, Reading, U.K.);

solvent A, 10 mM KH₂PO₄(pH 3.0); solvent B, acetonitrile; a

gradient from 20−80% B (0 min, 20% B; 15 min, 80% B; 25 min, 80%

B). Note that almost all the compounds possess similar dihydropyr-

imidine core structures, and representative compounds were selected

for testing the ¹³C NMR.

The synthesis and characterization of compounds 12−25 were

reported in published article²⁹and in the Supporting Information,

although their hERG activities were reported in this article. For

compounds 26−66, except for those described speciﬁcally, all the

analogues were prepared in analogy to 58 from commercially available

building blocks.

133.04, 126.58, 123.60, 123.50, 123.40, 121.63, 121.31, 121.20, 121.06,

120.95, 116.89, 116.78, 116.68, 116.55, 67.64, 66.75, 61.71, 58.36,

52.99, 33.09, 30.76, 23.75, 14.49.

1-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-(thia-

zol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-4-methylpipera-

zine-2-carboxylic Acid (31). HRMS: calcd (M) 565.0795, exptl

1

(MH⁺) 566.0866. H NMR (400 MHz, methanol-d₄) δ 7.95 (s, 1H),

7.72 (s, 1H), 7.51−7.38 (m, 2H), 7.11 (t, J = 8.2 Hz, 1H), 6.14 (d, J =

10.2 Hz, 1H), 4.28−4.13 (m, 1H), 4.09−3.88 (m, 3H), 3.72−3.54 (m,

1H), 3.20−3.05 (m, 2H), 2.97−2.78 (m, 2H), 2.70−2.52 (m, 3H),

2.45 (d, J = 10.6 Hz, 2H), 1.18−1.11 (m, 3H). ¹³C NMR (100 MHz,

methanol-d₄) δ 166.12, 162.73, 162.18, 160.25, 143.33, 143.20, 140.52,

140.04, 131.00, 123.31, 122.63, 119.41, 119.16, 114.93, 114.72, 62.17,

59.72, 59.62, 58.09, 55.16, 53.73, 53.53, 44.35, 13.18, 13.02.

4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-(thia-

zol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-3-car-

boxylic Acid (32). HRMS: calcd (M) 552.0478, exptl (MH⁺)

1

553.0567. H NMR (600 MHz, methanol-d₄) δ 7.97 (t, J = 3.0 Hz,

1H), 7.79 (d, J = 2.9 Hz, 1H), 7.52−7.45 (m, 1H), 7.42 (dd, J = 8.4,

2.5 Hz, 1H), 7.12 (td, J = 8.4, 2.6 Hz, 1H), 6.17 (s, 1H), 4.55−4.43

(m, 1H), 4.35−4.25 (m, 1H), 4.17−4.00 (m, 4H), 3.96−3.81 (m, 2H),

3.75−3.64 (m, 1H), 3.47−3.35 (m, 1H), 2.83 (s, 1H), 1.15 (td, J = 7.1,

2.7 Hz, 3H). ¹³C NMR (100 MHz, methanol-d₄) δ 165.76, 162.93,

161.75, 160.45, 147.24, 143.57, 139.65, 131.13, 131.5, 124.10, 122.56,

122.46, 119.62, 119.38, 115.02, 114.81, 68.03, 65.73, 63.33, 62.97,

59.89, 55.05, 49.94, 13.11.

E t h y l 4 - ( 2 - B r o m o - 4 - ﬂ u o r o p h e n y l ) - 6 - ( ( 1 -

oxidothiomorpholino)methyl)-2-(thiazol-2-yl)-1,4-dihydro-

pyrimidine-5-carboxylate (26). HRMS: calcd (MH⁺) 540.0301,

1

exptl (MH⁺) 541.360. H NMR (400 MHz, CDCl₃) δ 9.51 (s, 1H),

7.85 (s, 1H), 7.47 (s, 1H), 7.34 (d, J = 6.7 Hz, 2H), 6.97 (s, 1H), 6.21

(s, 1H), 4.22 (d, J = 17.0 Hz, 1H), 4.06 (s, 2H), 3.91 (d, J = 16.7 Hz,

1H), 3.45−3.26 (m, 2H), 3.04 (s, 4H), 2.86−2.71 (m, 2H), 1.15 (t, J =

13.2 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃) δ 166.02, 162.87, 162.23,

160.57, 145.13, 144.26, 143.14, 139.65, 130.51, 130.46, 129.92, 123.36,

123.69, 120.30, 120.27, 120.11, 114.98, 114.84, 98.70, 60.78, 60.05,

58.83, 56.34, 46.77, 14.14.

4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-(thia-

zol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-3-car-

boxylic Acid (33). HRMS: calcd (M) 508.0983, exptl (MH⁺)

1

509.1078. H NMR (600 MHz, methanol-d₄) δ 7.97 (t, J = 2.9 Hz,

E t h y l 4 - ( 2 - B r o m o - 4 - ﬂ u o r o p h e n y l ) - 6 - ( ( 1 , 1 -

dioxidothiomorpholino)methyl)-2-(thiazol-2-yl)-1,4-dihydro-

pyrimidine-5-carboxylate (27). HRMS: calcd (MH⁺) 556.0250,

1H), 7.80 (d, J = 2.9 Hz, 1H), 7.53−7.45 (m, 1H), 7.24 (dd, J = 8.7,

2.4 Hz, 1H), 7.08 (td, J = 8.4, 2.6 Hz, 1H), 6.19 (s, 1H), 4.55−4.40

(m, 1H), 4.35−4.25 (m, 1H), 4.13−4.05 (m, 4H), 3.94−3.83 (m, 2H),

3.75−3.65 (m, 1H), 3.48−3.37 (m, 1H), 2.84 (s, 1H), 1.15 (td, J = 7.1,

3.1 Hz, 4H). ¹³C NMR (100 MHz, methanol-d₄) δ 171.32, 165.73,

163.09, 161.68, 160.61, 147.22, 143.61, 137.91, 133.21, 133.10, 131.25,

131.16, 124.13, 116.42, 116.17, 114.44, 114.23, 67.95, 65.64, 63.29,

59.92, 55.04, 49.89, 13.03.

1

exptl (MH⁺) 557.0345. H NMR (400 MHz, CDCl₃) δ 9.38 (s, 1H),

7.88 (d, J = 3.0 Hz, 1H), 7.48 (d, J = 3.0 Hz, 1H), 7.35 (dd, J = 8.2, 2.1

Hz, 1H), 7.28 (t, J = 7.3 Hz, 1H), 6.97 (td, J = 8.3, 2.2 Hz, 1H), 6.22

(s, 1H), 4.34 (d, J = 17.1 Hz, 1H), 4.11−4.01 (m, 2H), 3.97 (d, J =

17.1 Hz, 1H), 3.33−3.09 (m, 8H), 1.15 (t, J = 7.1 Hz, 3H). ¹³C NMR

(150 MHz, CDCl₃) δ 165.95, 162.67, 162.27, 160.61, 144.43, 144.13,

143.26, 139.48, 139.46, 130.47, 130.42, 123.42, 123.39, 123.33, 115.01,

114.87, 99.22, 60.16, 58.80, 55.13, 51.57, 51.40, 14.12.

4-((6-(2,4-Dichlorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-

yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-3-carboxylic

1

Acid (34). HRMS: calcd (M) 524.0688, exptl (MH⁺) 525.0771. H

2-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-(thia-

NMR (600 MHz, methanol-d₄) δ 7.97−7.96 (m, 1H), 7.79 (s, 1H),

7.51−7.41 (m, 2H), 7.32 (d, J = 8.3 Hz, 1H), 6.18 (s, 1H), 4.53−4.42

(m, 1H), 4.33−4.22 (m, 1H), 4.18−3.99 (m, 4H), 3.94−3.83 (m, 2H),

3.69 (d, J = 19.7 Hz, 1H), 3.42 (s, 1H), 2.78 (d, J = 31.5 Hz, 1H), 1.15

(td, J = 7.0, 2.8 Hz, 3H). ¹³C NMR (150 MHz, methanol-d₄) δ 171.59,

167.93, 165.69, 165.66, 161.65, 161.63, 147.43, 143.63, 143.58, 140.44,

133.74, 133.25, 133.15, 132.20, 130.99, 130.97, 128.97, 128.91, 128.47,

127.58, 127.51, 124.19, 67.71, 63.26, 62.91, 59.95, 55.04, 54.88, 49.89,

49.19, 13.05.

(2R,3S)-4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-

2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-2-methyl-

morpholine-3-carboxylic Acid (35). The synthetic procedure was

described in the Supporting Information. HRMS: calcd (M) 566.0635,

exptl (MH⁺) 567.0731. ¹H NMR (600 MHz, methanol-d₄) δ 7.95 (t, J

= 3.4 Hz, 1H), 7.81−7.73 (m, 1H), 7.47−7.42 (m, 1H), 7.41−7.38 (m,

1H), 7.12−7.08 (m, 1H), 6.14 (d, J = 18.0 Hz, 1H), 4.43−4.32 (m,

1H), 4.06−3.97 (m, 3H), 3.94 (d, J = 11.3 Hz, 1H), 3.89−3.78 (m,

2H), 3.21 (s, 1H), 3.01−2.78 (m, 2H), 1.32 (d, J = 6.2 Hz, 3H), 1.12

(t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz, methanol-d₄) δ 173.39,

167.01, 164.31, 163.12, 163.03, 161.82, 161.79, 148.37, 148.09, 145.03,

144.98, 141.18, 141.01, 140.97, 132.56, 132.48, 132.42, 125.46, 125.36,

123.97, 123.89, 123.80, 121.06, 120.98, 120.82, 120.74, 116.48, 116.39,

116.27, 116.18, 75.46, 75.31, 73.57, 73.49, 66.69, 66.52, 61.30, 57.86,

56.95, 56.63, 52.67, 52.51, 18.77, 14.50.

zol-2-yl)-3,6-dihydropyrimidin-4-yl)amino)-3-phenylpropanoic

1

Acid (28). HRMS: calcd (MH⁺) 586.0686, exptl (MH⁺) 587.077. H

NMR (400 MHz, DMSO-d₆) δ 8.03 (s, 2H), 7.58 (d, J = 7.6 Hz, 1H),

7.50−7.40 (m, 1H), 7.36−7.17 (m, 6H), 5.97 (d, J = 3.5 Hz, 1H), 4.27

(s, 2H), 3.95 (q, J = 6.9 Hz, 2H), 3.30−2.99 (m, 3H) 0.05 (t, J = 7.0

Hz, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 165.33, 162.34, 162.07,

160.69, 144.54, 140.52, 131.97, 129.80, 129.77, 128.95, 127.36, 122.64,

120.16, 119.99, 116.11, 115.98, 60.22, 56.50, 19.02, 14.42

1-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-(thia-

zol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)pyrrolidine-2-car-

boxylic Acid (29). HRMS: calcd (M) 536.0529, exptl (MH⁺)

1

537.0569. H NMR (400 MHz, methanol-d₄) δ 7.97−7.88 (m, 2H),

7.56−7.52 (m, 1H), 7.47−7.44 (m, 1H), 7.27−7.14 (m, 1H), 6.19 (s,

1H), 4.78 (d, J = 14.6 Hz, 1H), 4.54−4.38 (m, 1H), 4.30−4.17 (m,

1H), 4.11 (q, J = 7.0 Hz, 2H), 4.05−3.87 (m, 1H), 3.31−3.23 (m,

1H), 2.61−2.45 (m, 1H), 2.40−2.26 (m, 1H), 2.26−2.13 (m, 1H),

2.12−1.98 (m, 1H), 1.15 (t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz,

methanol-d₄) δ 165.20, 163.33, 161.42, 161.38, 160.84, 144.22, 144.15,

139.04, 131.77, 131.62, 131.54, 125.30, 121.99, 121.89, 119.84, 119.80,

119.59, 119.55, 115.54, 115.48, 115.33, 115.27, 72.03, 69.22, 60.51,

55.59, 55.37, 29.01, 23.56, 13.02.

1-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-(thia-

zol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)piperidine-2-car-

boxylic Acid (30). HRMS: calcd (M) 550.0686, exptl (MH⁺)

1

551.0757. H NMR (400 MHz, methanol-d₄) δ 7.98 (d, J = 2.9 Hz,

4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-(thia-

zol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-6,6-dimethylmor-

pholine-3-carboxylic Acid (36). HRMS: calcd (M) 580.0791, exptl

(MH⁺) 581.0875. ¹H NMR (600 MHz, methanol-d₄) δ 7.96−7.89 (m,

1H), 7.75 (d, J = 2.8 Hz, 1H), 7.49−7.38 (m, 2H), 7.15−7.08 (m,

1H), 6.15 (d, J = 13.5 Hz, 1H), 4.18 (s, 1H), 4.13−3.97 (m, 5H), 3.43

1H), 7.88 (s, 1H), 7.62−7.50 (m, 1H), 7.50−7.39 (m, 1H), 7.20−7.16

(m, 1H), 6.19 (s, 1H), 4.75−4.58 (m, 2H), 4.09 (q, J = 6.9 Hz, 2H),

3.96−3.51 (m, 2H), 3.24−3.11 (m, 1H), 2.30−2.10 (m, 2H), 2.03−

1.72 (m, 4H), 1.14 (t, J = 7.0 Hz, 3H). ¹³C NMR (100 MHz,

methanol-d₄) δ 166.51, 164.73, 162.73, 162.23, 145.69, 140.43, 133.12,

M

DOI: 10.1021/acs.jmedchem.7b01914

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Journal of Medicinal Chemistry

Article

(d, J = 41.7 Hz, 1H), 3.04−2.93 (m, 1H), 2.48−72.42 (m, 1H), 1.42−

1.31 (m, 6H), 1.14 (t, J = 7.1 Hz, 3H). ¹³C NMR (150 MHz, MeOD)

δ 176.00 (s), 167.37, 163.78, 163.30, 163.21, 162.13, 149.00, 148.72,

144.65, 144.61, 141.58, 141.39, 132.37, 132.31, 129.86, 125.15, 125.12,

124.07, 124.01, 120.97, 120.94, 120.81, 120.77, 116.25, 116.11, 72.98,

71.30, 69.10, 65.48, 64.44, 61.14, 61.12, 60.90, 59.09, 56.40, 56.07,

26.28, 26.07, 24.39, 24.13, 14.53.

125.48, 123.91, 123.84, 121.03, 120.92, 120.87, 120.76, 116.49, 116.35,

116.23, 72.90, 69.49, 67.37, 66.65, 66.13, 65.63, 61.29, 55.93, 51.85,

51.31, 14.52.

Ethyl 4-(2,4-Dichlorophenyl)-6-((3-(hydroxymethyl)-

morpholino)methyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-

carboxylate (42). HRMS: calcd (M) 510.0895, exptl (MH⁺)

1

511.0973. H NMR (400 MHz, methanol-d₄) δ 7.96 (d, J = 2.1 Hz,

1H), 7.75 (d, J = 2.5 Hz, 1H), 7.51−7.36 (m, 2H), 7.30 (d, J = 7.8 Hz,

1H), 6.18 (s, 1H), 4.36−4.23 (m, 1H), 4.15−4.01 (m, 3H), 3.94 (d, J

= 8.6 Hz, 1H), 3.85−3.62 (m, 5H), 2.95−2.75 (m, 1H), 2.74−2.51 (m,

2H), 1.15 (t, J = 7.0 Hz, 3H). ¹³C NMR (100 MHz, methanol-d₄) δ

175.16, 167.03, 164.30, 163.13, 163.10, 161.82, 147.93, 144.92, 141.05,

141.02, 132.48, 132.44, 132.39, 132.35, 125.54, 125.51, 123.99, 123.97,

123.90, 123.87, 121.07, 120.82, 116.42, 116.40, 116.21, 116.19, 111.37,

75.59, 75.33, 66.26, 66.05, 61.51, 61.34, 57.97, 56.07, 55.82, 53.92,

53.72, 14.51.

Methyl 4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-

2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-

3-carboxylate (37). HRMS: calcd (M) 566.0635, exptl (MH⁺)

1

567.0729. H NMR (400 MHz, CDCl₃) δ 9.72 (d, J = 28.9 Hz, 1H),

7.87 (d, J = 1.9 Hz, 1H), 7.45 (d, J = 2.6 Hz, 1H), 7.39−7.29 (m, 2H),

6.99−6.94 (m, 1H), 6.20 (s, 1H), 4.28 (dd, J = 17.6, 8.5 Hz, 1H),

4.15−3.96 (m, 5H), 3.85 (s, 2H), 3.80 (d, J = 6.2 Hz, 3H), 3.57−3.46

(m, 1H), 3.25−3.11 (m, 1H), 2.60−2.50 (m, 1H), 1.15 (t, J = 6.8 Hz,

3H). ¹³C NMR (150 MHz, CDCl₃) δ 171.10, 170.99, 166.17, 166.13,

163.00, 162.97, 162.18, 160.53, 146.24, 146.08, 144.50, 144.47, 143.26,

143.23, 140.00, 139.98, 139.94, 139.92, 130.70, 130.64, 130.57, 130.51,

123.31, 123.25, 123.21, 123.15, 123.00, 122.97, 120.20, 120.10, 120.03,

119.94, 114.99, 114.91, 114.85, 114.77, 98.12, 97.93, 69.18, 67.29,

62.76, 62.66, 59.91, 59.89, 58.91, 58.75, 55.03, 54.84, 52.13, 49.82,

49.17, 14.16.

Isopropyl 4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbon-

yl)-2-(thiazol-2-yl)-3,6-dihydro-pyrimidin-4-yl)methyl)-

morpholine-3-carboxylate (38). HRMS: calcd (M) 594.0948, exptl

(MH⁺) 595.1021. ¹H NMR (400 MHz, methanol-d₄) δ 7.97 (d, J = 2.1

Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.11−7.07

(m, 1H), 6.16 (s, 1H), 5.15−5.06 (m, 1H), 4.32 (J = 14.9 Hz, 1H),

4.12−3.98 (m, 5H), 3.83 (s, 2H), 3.55 (d, J = 35.9 Hz, 1H), 3.24−3.11

(m, 1H), 2.61−2.50 (m, 1H), 1.27 (d, J = 6.1 Hz, 6H), 1.15 (t, J = 7.0

Hz, 3H). ¹³C NMR (150 MHz, methanol-d₄) δ 171.78, 171.71, 167.43,

163.73, 163.47, 163.42, 162.08, 148.29, 148.15, 146.49, 146.39, 144.62,

144.59, 141.42, 141.40, 132.19, 132.13, 125.03, 124.23, 124.16, 120.94,

120.78, 116.18, 116.04, 99.05, 98.96, 70.35, 70.29, 70.14, 70.04, 68.41,

64.16, 63.95, 61.06, 59.92, 59.87, 56.10, 55.84, 50.76, 50.20, 49.14,

49.00, 48.86, 22.10, 22.09, 22.07, 22.05, 14.57.

E t h y l 4 - ( 2 - B r o m o - 4 - ﬂ u o r o p h e n y l ) - 6 - ( ( 3 -

carbamoylmorpholino)methyl)-2-(thiazol-2-yl)-1,4-dihydro-

pyrimidine-5-carboxylate (39). HRMS: calcd (M) 551.0638, exptl

(MH⁺) 552.0726. ¹H NMR (400 MHz, methanol-d₄) δ 7.99−7.94 (m,

1H), 7.74 (s, 1H), 7.50−7.35 (m, 2H), 7.16−7.05 (m, 1H), 6.15 (d, J

= 10.3 Hz, 1H), 4.21 (q, 11.6 Hz, 1H), 4.11−3.97 (m, 3H), 3.93−3.69

(m, 4H), 3.09−2.87 (m, 1H), 2.65−2.47 (m, 1H), 1.47 (s, 1H), 1.15

(t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, methanol-d₄) δ 174.80,

167.47, 163.59, 161.74, 146.65, 144.63, 139.61, 132.30, 132.22, 131.25,

124.93, 123.51, 121.01, 120.94, 120.76, 120.70, 116.29, 116.28, 116.22,

116.17, 116.07, 116.01, 70.61, 68.03, 66.80, 61.11, 59.72, 55.13, 52.68,

52.56, 14.52.

Ethyl 4-(2-Bromo-4-ﬂuorophenyl)-6-((2-(hydroxymethyl)-

morpholino)methyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-

carboxylate (43). HRMS: calcd (M) 538.0686, exptl (MH⁺)

1

539.0753. H NMR (400 MHz, CDCl₃) δ 9.62 (d, J = 7.1 Hz, 1H),

7.86 (d, J = 2.9 Hz, 1H), 7.46 (d, J = 3.0 Hz, 1H), 7.37−7.29 (m, 2H),

6.97 (t, J = 8.1 Hz, 1H), 6.21 (s, 1H), 4.16−3.97 (m, 4H), 3.94−3.80

(m, 3H), 3.77−3.57 (m, 2H), 2.85−2.75 (m, 1H), 2.68 (t, J = 11.2 Hz,

1H), 2.50−2.30 (m, 1H), 2.06 (s, 1H), 1.15 (t, J = 7.1 Hz, 3H). ¹³C

NMR (150 MHz, CDCl₃) δ 166.15, 162.92, 162.21, 160.55, 145.86,

145.82, 144.37, 144.36, 143.20, 143.19, 139.89, 139.87, 139.84, 130.56,

130.51, 123.31, 123.25, 123.11, 123.09, 120.21, 120.05, 114.98, 114.84,

109.99, 98.33, 98.28, 76.11, 66.63, 66.55, 63.78, 59.93, 58.78, 58.77,

56.63, 54.85, 54.57, 53.34, 53.06, 14.15.

4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-(thia-

zol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-2-car-

boxylic Acid (44). HRMS: calcd (M) 552.0478, exptl (MH⁺)

1

553.0542. H NMR (400 MHz, methanol-d₄) δ 7.95 (s, 1H), 7.77 (s,

1H), 7.53−7.36 (m, 2H), 7.12 (t, J = 6.8 Hz, 1H), 6.17 (s, 1H), 4.41−

4.21 (m, 2H), 4.18−4.06 (m, 4H), 3.89 (t, J = 8.8 Hz, 1H), 3.49−3.24

(m, 1H), 3.01 (t, J = 14.0 Hz, 1H), 2.92−2.69 (m, 2H), 1.14 (t, J = 6.3

Hz, 3H). ¹³C NMR (100 MHz, methanol-d₄) δ 175.16, 167.03, 164.30,

163.13, 163.10, 161.82, 147.93, 144.92, 141.05, 141.02, 132.48, 132.44,

132.39, 132.35, 125.54, 125.51, 123.99, 123.97, 123.90, 123.87, 121.07,

120.82, 116.42, 116.40, 116.21, 116.19, 111.37, 75.59, 75.33, 66.26,

66.05, 61.51, 61.34, 57.97, 56.07, 55.82, 53.92, 53.72, 14.51.

2-(4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholin-3-

yl)acetic Acid (45). HRMS: calcd (M) 566.0636, exptl (MH⁺)

1

567.0713. H NMR (600 MHz, methanol-d₄) δ7.96 (s, 1H), 7.77 (s,

1H), 7.52−7.38 (m, 2H), 7.11 (t, J = 7.2 Hz, 1H), 6.17 (s, 1H), 4.40−

4.04 (m, 4H), 4.02−3.71 (m, 4H), 3.23 (s, 1H), 3.04 (s, 1H), 2.70−

2.65 (m, 3H), 1.22−1.08 (m, 3H). ¹³C NMR (150 MHz, methanol-d₄)

δ 174.25, 165.84, 162.46, 161.91, 160.78, 147.27, 143.40, 139.79,

131.00, 123.97, 122.61, 119.59, 119.55, 119.41, 119.38, 114.95, 114.81,

69.48, 59.83, 56.61, 53.20, 53.16, 52.84, 50.46, 49.84, 13.12.

Ethyl 4-(2-Bromo-4-ﬂuorophenyl)-6-((3-(methylcarbamoyl)-

morpholino)methyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-

carboxylate (40). HRMS: calcd (M) 565.0795, exptl (MH⁺)

1

566.0886. H NMR (400 MHz, methanol-d₄) δ 7.98 (d, J = 2.8 Hz,

2-(4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholin-2-

yl)acetic Acid (46). HRMS: calcd (M) 566.0635, exptl (MH⁺)

1H), 7.74 (s, 1H), 7.51−7.36 (m, 2H), 7.10 (t, J = 8.2 Hz, 1H), 6.15

(d, J = 10.5 Hz, 1H), 4.27−3.99 (m, 3H), 3.99−3.68 (m, 5H), 3.31−

3.23 (m, 1H), 3.07−2.85 (m, 1H), 2.78 (d, J = 20.5 Hz, 3H), 2.62−

2.49 (m, 1H), 1.14 (td, J = 6.8, 2.0 Hz, 3H). ¹³C NMR (100 MHz,

methanol-d₄) δ 171.29, 166.04, 162.15, 146.37, 146.29, 143.23, 130.94,

130.86, 123.55, 123.29, 122.68, 119.59, 119.29, 114.85, 114.64, 98.49,

69.09, 68.81, 66.58, 66.49, 65.82, 59.70, 58.32, 53.54, 51.34, 24.92,

13.12.

1

567.0729. H NMR (600 MHz, methanol-d₄) δ 7.96 (d, J = 3.1 Hz,

1H), 7.76 (d, J = 2.9 Hz, 1H), 7.46−7.40 (m, 2H), 7.16−7.10 (m,

1H), 6.17 (s, 1H), 4.14−4.04 (m, 4H), 3.98−3.91 (m, 2H), 3.86−3.81

(m, 1H), 3.09 (s, 1H), 2.77 (s, 1H), 2.61−2.43 (m, 3H), 2.32 (s, 1H),

1.15 (t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz, methanol-d₄) δ 174.62,

167.33, 164.22, 163.33, 161.74, 144.69, 141.24, 132.28, 132.19, 125.12,

124.12, 121.00, 120.75, 116.37, 116.16, 73.93, 67.52, 61.16, 59.04,

57.59, 54.01, 39.55, 14.52.

3-(4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholin-3-

yl)propanoic Acid (47). HRMS: calcd (M) 580.0791, exptl (MH⁺)

581.0861. ¹H NMR (600 MHz, acetone-d₆) δ 9.94 (s, 1H), 7.98 (d, J =

3.1 Hz, 1H), 7.80 (d, J = 3.1 Hz, 1H), 7.54−7.52 (m, 1H), 7.42 (dd, J

= 8.5, 2.6 Hz, 1H), 7.15 (td, J = 8.5, 2.6 Hz, 1H), 6.18 (s, 1H), 4.32 (d,

J = 18.1 Hz, 1H), 4.05−4.01 (m, 3H), 3.93 (dd, J = 11.4, 2.8 Hz, 1H),

3.83−3.80 (m, 1H), 3.74−3.70 (m, 1H), 3.50−3.47 (m, 1H), 2.92−

2.86 (m, 1H), 2.67−2.61 (m, 2H), 2.52−2.47 (m, 1H), 2.45−2.37 (m,

Ethyl 4-(2-Bromo-4-ﬂuorophenyl)-6-((3-((methylsulfonyl)-

carbamoyl)morpholino)methyl)-2-(thiazol-2-yl)-1,4-dihydro-

pyrimidine-5-carboxylate (41). The synthetic procedure was

described in the Supporting Information. HRMS: calcd (M)

1

629.0414, exptl (MH⁺) 630.0525. H NMR (600 MHz, methanol-

d₄) δ 7.97 (d, J = 3.0 Hz, 1H), 7.79 (d, J = 2.9 Hz, 1H), 7.53−7.45 (m,

1H), 7.45−7.38 (m, 1H), 7.13 (td, J = 8.4, 2.3 Hz, 1H), 6.17 (d, J = 3.9

Hz, 1H), 4.40 (d, J = 16.9 Hz, 1H), 4.17−4.03 (m, 4H), 3.93−3.81

(m, 3H), 3.58 (s, 1H), 3.31−3.16 (m, 4H), 2.76 (s, 1H), 1.15 (t, J =

7.1 Hz, 3H). ¹³C NMR (150 MHz, MeOD) δ 172.76, 167.16, 163.90,

163.15, 162.24, 148.49, 144.93, 140.98, 132.61, 132.56, 132.38, 129.88,

N

DOI: 10.1021/acs.jmedchem.7b01914

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

1H), 2.11−2.08 (m, 1H), 1.85−1.80 (m, 1H), 1.13 (t, J = 7.1 Hz, 3H).

¹³C NMR (150 MHz, acetone-d₆) δ 173.59, 165.45, 162.83, 162.11,

525.0722. H NMR (400 MHz, methanol-d₄) δ 7.94 (s, 1H), 7.73 (s,

1H), 7.43 (d, J = 9.5 Hz, 2H), 7.29 (d, J = 7.9 Hz, 1H), 6.16 (s, 1H),

4.17−3.95 (m, 5H), 3.93−3.77 (m, 2H), 3.74−3.68 (m, 1H), 3.51 (s,

1H), 3.30−3.16 (m, 2H), 1.14 (t, J = 6.8 Hz, 3H). ¹³C NMR (100

MHz, methanol-d₄) δ 172.96, 167.11, 163.09, 148.80, 144.92, 141.89,

135.09, 134.65, 132.32, 130.35, 128.87, 125.50, 69.35, 67.11, 64.33,

61.51, 61.33, 56.28, 50.61, 14.44.

160.47, 147.63, 144.17, 143.48, 140.71, 131.29, 131.23, 123.73, 122.84,

122.78, 119.54, 119.38, 115.34, 115.20, 96.43, 70.08, 66.78, 59.80,

59.67, 59.28, 58.69, 53.49, 53.45, 52.41, 13.66.

3-(4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholin-2-

yl)propanoic Acid (48). HRMS: calcd (M) 580.0791, exptl (MH⁺)

581.086.¹H NMR (600 MHz, acetone-d₆) δ 9.72 (s, 1H), 7.97 (d, J =

2.9 Hz, 1H), 7.79 (t, J = 3.2 Hz, 1H), 7.53−7.49 (m, 1H), 7.44 (dd, J =

8.5, 2.5 Hz, 1H), 7.15−7.11 (m, 1H), 6.19 (s, 1H), 4.10−3.99 (m,

3H), 3.97−3.88 (m, 2H), 3.75−3.71 (m, 1H), 3.69−3.65 (m, 1H),

2.99−2.73 (m, 2H), 2.51−2.33 (m, 3H), 2.23 (t, J = 10.5 Hz, 1H),

1.84−1.71 (m, 2H), 1.13 (t, J = 7.1 Hz, 3H). ¹³C NMR (150 MHz,

acetone-d₆) δ 173.63, 173.55, 165.38, 162.77, 162.73, 162.15, 160.50,

146.46, 146.40, 144.24, 144.22, 143.47, 143.45, 140.60, 131.14, 131.11,

131.09, 131.05, 123.75, 123.70, 122.94, 122.90, 122.88, 119.66, 119.49,

115.14, 115.10, 115.00, 114.96, 97.76, 97.62, 74.83, 74.80, 66.47,

66.40, 59.35, 59.34, 58.76, 58.73, 58.49, 58.30, 56.35, 56.28, 53.27,

53.03, 13.64.

(3S)-4-((6-(2-Chloro-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-3-

carboxylic Acid (51). HRMS: calcd (M) 508.0983, exptl (MH⁺)

1

509.1089. H NMR (600 MHz, methanol-d₄) δ 7.97 (t, J = 2.8 Hz,

1H), 7.80 (d, J = 2.7 Hz, 1H), 7.51−7.45 (m, 1H), 7.24 (dt, J = 8.7, 2.5

Hz, 1H), 7.10−7.05 (m, 1H), 6.19 (s, 1H), 4.54−4.44 (m, 1H), 4.32−

4.25 (m, 1H), 4.17−4.02 (m, 4H), 3.95−3.83 (m, 2H), 3.69 (d, J =

16.4 Hz, 1H), 3.44 (s, 1H), 2.83 (s, 1H), 1.15 (td, J = 7.1, 2.6 Hz, 3H).

¹³C NMR (100 MHz, methanol-d₄) δ 172.85, 167.09, 164.46, 163.04,

161.99, 148.67, 145.02, 144.97, 139.34, 134.66, 134.57, 134.47, 132.66,

132.57, 125.56, 117.87, 117.81, 117.62, 117.56, 115.86, 115.79, 115.65,

115.58, 69.33, 66.99, 64.83, 64.48, 56.45, 56.29, 51.32, 50.68, 14.44.

(3S)-4-((6-(2-Chloro-4-ﬂuorophenyl)-5-(methoxycarbonyl)-

2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-

3-carboxylic Acid (52). HRMS: calcd (M) 494.0827, exptl (MH⁺)

(3S)-4-((6-(2,4-Dichlorophenyl)-5-(ethoxycarbonyl)-2-(thia-

zol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-3-car-

boxylic Acid (49). HRMS: calcd (M) 524.0688, exptl (MH⁺)

1

495.0931. H NMR (600 MHz, methanol-d₄) δ 7.97 (t, J = 2.6 Hz,

1

525.0749. H NMR (600 MHz, methanol-d₄) δ 8.00−7.94 (m, 1H),

1H), 7.79 (d, J = 2.7 Hz, 1H), 7.50−7.44 (m, 1H), 7.24 (dt, J = 8.7, 2.4

Hz, 1H), 7.08−7.04 (m, 1H), 6.17 (s, 1H), 4.52−4.42 (m, 1H), 4.34−

4.22 (m, 1H), 4.16−4.00 (m, 2H), 3.95−3.83 (m, 2H), 3.70−3.67 (m,

1H), 3.62 (s, 3H), 3.42 (s, 1H), 2.82 (s, 1H). ¹³C NMR (100 MHz,

methanol-d₄) δ 172.92, 167.60, 164.47, 163.07, 162.00, 148.80, 144.99,

144.95, 139.06, 134.73, 134.65, 134.55, 132.50, 132.40, 125.50, 117.97,

117.90, 117.72, 117.65, 115.81, 115.75, 115.60, 115.54, 73.53, 69.46,

67.12, 64.79, 64.47, 62.27, 56.42, 56.32, 51.72, 51.36, 14.40.

7.79 (s, 1H), 7.48 (s, 1H), 7.45 (t, J = 9.3 Hz, 1H), 7.31 (d, J = 8.4 Hz,

1H), 6.18 (s, 1H), 4.53−4.42 (m, 1H), 4.35−4.23 (m, 1H), 4.17−4.09

(m, 1H), 4.08−4.04 (m, 3H), 3.93−3.84 (m, 2H), 3.69 (d, J = 19.4

Hz, 1H), 3.42 (s, 1H), 2.79 (d, J = 26.1 Hz, 1H), 1.15 (td, J = 7.1, 2.8

Hz, 3H). ¹³C NMR (100 MHz, methanol-d₄) δ 172.80, 167.07, 167.04,

163.04, 163.02, 148.81, 148.77, 144.99, 144.94, 141.87, 141.82, 135.09,

134.62, 134.53, 132.34, 130.34, 130.28, 128.95, 128.88, 125.55, 69.40,

69.28, 67.10, 64.67, 64.33, 61.34, 56.42, 56.27, 51.28, 50.62, 14.44.

(S)-4-(((R)-6-(2,4-Dichlorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-3-

carboxylic Acid ((R)-49). HRMS: calcd (M) 524.0688, exptl (MH⁺)

(S)-4-(((R)-6-(2-Chloro-4-ﬂuorophenyl)-5-(methoxycarbon-

yl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-

morpholine-3-carboxylic Acid ((R)-52). HRMS: calcd (M)

1

494.0827, exptl (MH⁺) 495.0910. H NMR (600 MHz, methanol-

1

525.0771. H NMR (400 MHz, methanol-d₄) δ 7.94 (d, J = 2.1 Hz,

d₄) δ 7.97 (d, J = 3.1 Hz, 1H), 7.79 (d, J = 3.0 Hz, 1H), 7.47−7.45 (m,

1H), 7.24 (dd, J = 8.7, 2.5 Hz, 1H), 7.06 (td, J = 8.4, 2.5 Hz, 1H), 6.17

(s, 1H), 4.52 (d, J = 16.8 Hz, 1H), 4.28 (d, J = 16.3 Hz, 1H), 4.16−

4.06 (m, 2H), 3.93−3.85 (m, 2H), 3.71 (s, 1H), 3.62 (s, 3H), 3.44 (s,

1H), 2.78 (s, 1H). ¹³C NMR (150 MHz, methanol-d₄) δ 171.58,

166.16, 162.68, 161.63, 161.03, 147.54, 143.61, 137.74, 133.29, 133.22,

131.10, 131.04, 124.22, 116.54, 116.38, 114.36, 114.22, 67.82, 65.48,

63.01, 60.14, 54.95, 50.36, 49.29.

1H), 7.76 (d, J = 1.6 Hz, 1H), 7.44 (d, J = 12.0 Hz, 2H), 7.28 (d, J =

8.1 Hz, 1H), 6.18 (s, 1H), 4.52 (d, J = 17.1 Hz, 1H), 4.27 (d, J = 17.1

Hz, 1H), 4.15−4.02 (m, 4H), 3.87 (s, 2H), 3.70 (s, 1H), 3.42 (s, 1H),

2.76 (d, J = 8.1 Hz, 1H), 1.14 (t, J = 6.9 Hz, 3H). ¹³C NMR (150

MHz, methanol-d₄) δ 172.93, 167.01, 163.00, 148.85, 144.95, 141.86,

135.08, 134.58, 132.36, 130.32, 128.89, 125.60, 69.20, 66.94, 64.31,

61.51, 61.34, 55.93, 50.63, 14.45

(S)-4-(((S)-6-(2,4-Dichlorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-3-

carboxylic Acid ((S)-49). HRMS: calcd (M) 524.0688, exptl (MH⁺)

525.0790. ¹H NMR (600 MHz, methanol-d₄) δ 8.22 (dd, J = 18.6, 3.0

Hz, 2H), 7.55 (dd, J = 5.2, 3.1 Hz, 2H), 7.42 (dd, J = 8.4, 2.0 Hz, 1H),

6.32 (s, 1H), 5.08 (d, J = 17.2 Hz, 1H), 4.42 (d, J = 11.4 Hz, 1H), 4.08

(q, J = 7.1 Hz, 2H), 3.95 (dd, J = 12.3, 3.5 Hz, 1H), 3.81−3.78 (m,

2H), 3.75−3.69 (m, 1H), 3.39−3.34 (m, 1H), 3.08 (s, 1H), 2.92−2.87

(m, 1H), 1.15−1.13 (m, 3H). ¹³C NMR (100 MHz, methanol-d₄) δ

175.16, 167.03, 164.30, 163.13, 163.10, 161.82, 147.93, 144.92, 141.05,

141.02, 132.48, 132.44, 132.39, 132.35, 125.54, 125.51, 123.99, 123.97,

123.90, 123.87, 121.07, 120.82, 116.42, 116.40, 116.21, 116.19, 111.37,

106.06, 75.59, 75.33, 66.26, 66.05, 61.51, 61.34, 57.97, 56.07, 55.82,

53.92, 53.72, 14.51.

(3S)-4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-3-

carboxylic Acid (53). HRMS: calcd (M) 552.0478, exptl (MH⁺)

1

553.0568. H NMR (600 MHz, methanol-d₄) δ 7.97 (t, J = 2.8 Hz,

1H), 7.79 (t, J = 2.7 Hz, 1H), 7.52−7.45 (m, 1H), 7.42 (dt, J = 8.4, 2.5

Hz, 1H), 7.14−7.10 (m, 1H), 6.17 (s, 1H), 4.55−4.44 (m, 1H), 4.38−

4.23 (m, 1H), 4.17−4.01 (m, 4H), 3.95−3.84 (m, 2H), 3.70 (d, J =

17.6 Hz, 1H), 3.53−3.36 (m, 1H), 2.84−2.73 (m, 1H), 1.15 (td, J =

7.1, 2.4 Hz, 3H). ¹³C NMR (100 MHz, methanol-d₄) δ 174.31, 167.27,

164.23, 163.29, 163.24, 161.75, 148.83, 147.90, 144.90, 144.88, 141.30,

141.17, 141.14, 132.52, 132.44, 132.35, 125.22, 123.94, 123.85, 121.00,

120.91, 120.75, 120.66, 116.39, 116.33, 116.18, 116.12, 100.84, 70.00,

69.72, 67.40, 67.24, 66.34, 65.49, 61.22, 61.19, 58.43, 56.19, 51.94,

51.18, 14.53.

(S)-4-(((R)-6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-

2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-

3-carboxylic Acid ((R)-53). HRMS: calcd (M) 552.0478, exptl

(MH⁺) 553.0585. ¹H NMR (600 MHz, methanol-d₄) δ 8.11 (d, J = 3.0

Hz, 1H), 8.06 (d, J = 3.0 Hz, 1H), 7.67 (dd, J = 8.7, 5.9 Hz, 1H), 7.51

(dd, J = 8.4, 2.5 Hz, 1H), 7.22 (td, J = 8.4, 2.5 Hz, 1H), 6.29 (s, 1H),

4.65 (d, J = 16.2 Hz, 1H), 4.38 (t, J = 4.1 Hz, 1H), 4.30−4.19 (m, 3H),

4.16−4.10 (m, 2H), 4.08−3.98 (m, 2H), 3.89−3.82 (m, 1H), 3.40−

3.34 (m, 1H), 1.16 (t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz,

methanol-d₄) δ 170.12, 168.39, 165.70, 164.97, 162.47, 158.91, 151.71,

146.23, 146.15, 139.16, 139.12, 133.62, 133.53, 128.19, 123.94, 123.84,

121.50, 121.25, 116.94, 116.72, 108.83, 66.99, 66.54, 64.96, 64.57,

63.25, 62.23, 58.32, 56.04, 55.95, 54.64, 51.76, 14.39.

(3R)-4-((6-(2,4-Dichlorophenyl)-5-(ethoxycarbonyl)-2-(thia-

zol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-3-car-

boxylic Acid ((R)-50). HRMS: calcd (M) 524.0688, exptl (MH⁺)

1

525.0761. H NMR (400 MHz, methanol-d₄) δ 8.02 (d, J = 3.0 Hz,

1H), 7.88 (d, J = 3.0 Hz, 1H), 7.51 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 8.4

Hz, 1H), 6.21 (s, 1H), 4.58−4.42 (m, 2H), 4.17 (dd, J = 11.9, 3.4 Hz,

1H), 4.13−4.04 (m, 3H), 4.00−3.86 (m, 3H), 3.53−3.46 (m, 1H),

3.04−2.93 (m, 1H), 1.16 (t, J = 7.1 Hz, 3H).¹³C NMR (150 MHz,

CDCl₃) δ 171.30, 165.05, 159.98, 147.72, 146.78, 143.97, 138.80,

134.62, 133.24, 130.81, 129.67, 127.98, 125.28, 68.00, 65.73, 63.48,

60.76, 55.35, 53.11, 50.28, 13.95.

(R)-4-(((S)-6-(2,4-Dichlorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-3-

carboxylic Acid ((S)-50). HRMS: calcd (M) 524.0688, exptl (MH⁺)

O

DOI: 10.1021/acs.jmedchem.7b01914

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

E t h y l 4 - ( 2 - B r o m o - 4 - ﬂ u o r o p h e n y l ) - 6 - ( ( ( R ) - 3 -

(hydroxymethyl)morpholino)methyl)-2-(thiazol-2-yl)-1,4-dihy-

dropyrimidine-5-carboxylate (54). HRMS: calcd (M) 538.0686,

exptl (MH⁺) 539.0734. ¹H NMR (600 MHz, methanol-d₄) δ 7.96 (t, J

= 3.3 Hz, 1H), 7.74 (d, J = 2.8 Hz, 1H), 7.49−7.37 (m, 2H), 7.09 (t, J

= 7.9 Hz, 1H), 6.16 (d, J = 2.9 Hz, 1H), 4.48−4.45 (m, 1H), 4.14−

3.98 (m, 3H), 3.96−3.91 (m, 1H), 3.88−3.69 (m, 4H), 3.68−3.62 (m,

1H), 2.93−2.77 (m, 1H), 2.73−2.65 (m, 1H), 2.64−2.53 (m, 1H),

1.15 (t, J = 7.1 Hz, 3H). ¹³C NMR (150 MHz, methanol-d₄) δ 166.10,

166.08, 162.32, 162.07, 160.67, 147.69, 147.37, 144.99, 143.22, 140.16,

140.13, 140.06, 140.04, 130.83, 130.77, 130.70, 123.60, 123.58, 119.54,

119.50, 119.37, 119.33, 114.80, 114.66, 97.48, 96.94, 68.78, 68.76,

66.74, 66.67, 61.74, 61.24, 59.63, 59.45, 59.06, 58.49, 58.43, 53.65,

52.91, 51.93, 51.80, 13.17.

product (compound (R)-58l-2) as a yellow solid (1.22 g, 24%). The

absolute stereochemistry of (R)-58l-2 was determined by an X-ray-

diﬀraction study (see the Supporting Information).

[α]²_D⁵= −39.26 (c = 0.2955 g/100 mL, MeOH)

MS (ESI, positive ion) m/z: 510.0 [M + H]⁺; ¹H NMR (600 MHz,

DMSO-d₆): δ 10.0 (s, 1H), 7.97 (d, 1H), 7.90 (d, 1H), 7.54 (dd, 1H),

7.37 (dd, 1H), 7.25 (td, 1H), 5.99 (s, 1H), 4.83−4.76 (m, 2H), 2.49

(s, 3H), 1.32 (d, 3H), 1.04−1.02 (m, 6H).

(R)-Ethyl 4-(2-Bromo-4-ﬂuorophenyl)-6-methyl-2-(thiazol-2-yl)-

1,4-dihydropyrimidine-5-carboxylate (58I-3). Anhydrous ethanol

(44.6 g) and lithium (0.243 g, 0.035 mol) were added to a ﬂask in

turn. The mixture was stirred at 43 °C until the lithium was consumed

entirely, and then a solution of (R)-58l-2 (5.1 g, 0.01 mol) in ethanol

(44.6 g) was added. The reaction mixture was allowed to warm to 78

°C and was stirred for 1.5 h under a nitrogen atmosphere. After the

reaction, the reaction mixture was cooled to 10 °C, kept at 10 °C, and

stirred for 8 h. The mixture was ﬁltered. The ﬁltrate was washed with

anhydrous ethanol (5 g) and water (50 g) in turn, and then dried in

vacuo at 60 °C for 8 h to obtain the product (compound 58l-3) as a

yellow solid (2.97 g, 70%).

(2R,3S)-4-(((R)-6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycar-

bonyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-2-

methylmorpholine-3-carboxylic Acid (55). HRMS: calcd (M)

1

566.0635, exptl (MH⁺) 567.0724. H NMR (600 MHz, methanol-d₄)

δ 7.97 (d, J = 3.0 Hz, 1H), 7.78 (d, J = 2.6 Hz, 1H), 7.47−7.42 (m,

2H), 7.13 (td, J = 8.4, 2.1 Hz, 1H), 6.15 (s, 1H), 4.36 (d, J = 16.6 Hz,

1H), 4.07−4.02 (m, 4H), 3.90−3.83 (m, 2H), 3.25 (s, 2H), 2.90 (s,

1H), 1.34 (d, J = 6.2 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H). ¹³C NMR (150

MHz, methanol-d₄) δ 171.58, 165.58, 162.52, 161.67, 160.86, 146.99,

143.65, 139.77, 131.15, 131.09, 124.05, 122.55, 122.49, 119.65, 119.49,

115.0, 114.85, 109.99, 73.90, 71.95, 65.07, 59.92, 55.70, 51.28, 17.29,

13.11.

(3S)-4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-6,6-dimethyl-

morpholine-3-carboxylic Acid (56). HRMS: calcd (M) 580.0791,

exptl (MH⁺) 581.0873. ¹H NMR (600 MHz, methanol-d₄) δ 7.93 (d, J

= 3.1 Hz, 1H), 7.76 (d, J = 3.0 Hz, 1H), 7.49−7.47 (m, 1H), 7.41 (dd,

J = 8.4, 2.6 Hz, 1H), 7.11 (td, J = 8.4, 2.5 Hz, 1H), 6.17 (s, 1H), 4.35

(d, J = 17.2 Hz, 1H), 4.14−3.99 (m, 5H), 3.49 (s, 1H), 3.00 (d, J =

11.5 Hz, 1H), 2.54−2.48 (m, 1H), 1.36 (s, 3H), 1.33 (s, 3H), 1.15 (t, J

= 7.1 Hz, 3H). ¹³C NMR (100 MHz, methanol-d₄) δ 175.16, 167.03,

164.30, 163.09, 161.82, 147.93, 144.92, 141.05, 141.02, 132.48, 132.44,

132.39, 132.35, 125.54, 125.51, 123.99, 123.97, 123.90, 123.87, 121.07,

120.82, 116.42, 116.40, 116.21, 116.19, 111.37, 75.59, 75.33, 66.26,

66.05, 61.51, 61.34, 57.97, 56.07, 55.82, 53.92, 53.72, 14.51.

(S)-1-(((R)-6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-

2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-4,4-diﬂuor-

opyrrolidine-2-carboxylic Acid (57). HRMS: calcd (M) 572.0341,

exptl (MH⁺) 573.0399. ¹H NMR (400 MHz, methanol-d₄) δ 7.92 (d, J

= 2.9 Hz, 1H), 7.73 (d, J = 2.9 Hz, 1H), 7.48−7.40 (m, 2H), 7.17−

7.07 (m, 1H), 6.15 (s, 1H), 4.45 (d, J = 16.3 Hz, 1H), 4.20 (d, J = 16.3

Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 3.96 (t, J = 7.9 Hz, 1H), 3.66 (q, J =

11.3 Hz, 1H), 3.24 (q, 11.3 Hz, 1H), 2.89−2.72 (m, 1H), 2.65−2.52

(m, 1H), 1.14 (t, J = 7.1 Hz, 3H). ¹³C NMR (150 MHz, methanol-d₄)

δ 174.51, 167.22, 163.81, 163.15, 162.16, 148.62, 147.38, 144.75,

141.33, 141.31, 132.34, 132.28, 130.57, 128.93, 127.29, 125.11, 124.06,

123.99, 120.98, 120.82, 116.32, 116.18, 64.99, 61.83, 61.63, 61.44,

61.24, 58.77, 53.86, 40.35, 40.18, 40.01, 14.51.

General Procedures for the Synthesis of 3-((R)-4-(((R)-6-(2-

Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)-

3,6-dihydropyrimidin-4-yl)methyl)morpholin-2-yl)propanoic

Acid (58). (R)-(S)-1-Isopropoxy-1-oxopropan-2-yl 4-(2-Bromo-4-

ﬂuorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-

carboxylate ((R)-58I-2). 2-Thiazolecarboxamidine hydrochloride

(1.64 g, 0.01 mol), 2-bromo-4-ﬂuorobenzaldehyde (2.03 g, 0.01

mol), (S)-1-isopropoxy-1-oxopropan-2-yl 3-oxobutanoate (2.16 g, 0.01

mol), anhydrous sodium acetate (0.82 g, 0.01 mol), and n-propanol

(10 mL) were added in turn to a ﬂask. The mixture was stirred at 80

°C for 16 h. After the reaction, the mixture was cooled to 30 °C, kept

at 30 °C, and stirred for 6 h. The resulting mixture was ﬁltered. The

ﬁlter cake was washed with water (330 mL) and dried in vacuo at 60

°C for 8 h to obtain the crude product (compound 58l-2). To the

crude product, n-propanol (19.2 g) was added. The mixture was

heated until the crude product dissolved completely, then it was

cooled to 30 °C, kept at 30 °C, and stirred and allowed to crystallize

for 3 h. The resulting mixture was ﬁltered. The ﬁlter cake was washed

with n-propanol (2 g) and dried in vacuo at 60 °C for 8 h to obtain the

[α]²_D⁵= −80.71 (c = 0.3023 g/100 mL, MeOH)

MS (ESI, positive ion) m/z: 424.0 [M + H]⁺; ¹H NMR (400 MHz,

DMSO-d₆): δ 9.88 (s, 1H), 7.97 (d, 1H), 7.89 (d, 1H), 7.54 (dd, 1H),

7.35 (dd, 1H), 7.23 (td, 1H), 5.96 (s, 1H), 3.93 (q, 2H), 2.46 (s, 3H),

1.03 (t, 3H).

(R)-Ethyl 4-(2-Bromo-4-ﬂuorophenyl)-6-(bromomethyl)-2-(thia-

zol-2-yl)-1,4-dihydropyrimidine-5-carboxylate (58I-4). (R)-Ethyl 4-

(2-bromo-4-ﬂuorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4-dihydropyri-

midine-5-carboxylate (compound 58l-3, 4.24 g, 0.01 mol) and CCl₄

(80 mL), followed by NBS (1.96 g, 0.011 mol), were added to a ﬂask

at 70 °C. The mixture was stirred for 30 min. After the reaction, the

mixture was cooled and ﬁltered. The ﬁltrate was concentrated to

obtain the product (compound 58l-4) as a yellow solid (3.42 g, 68%).

MS (ESI, positive ion) m/z: 503.9 [M + H]⁺; ¹H NMR (600 MHz,

DMSO-d₆): δ 10.23 (s, 1H), 8.01 (d, 1H), 7.98 (d, 1H), 7.62 (dd,

1H), 7.42 (dd, 1H), 7.29 (td, 1H), 6.01 (s, 1H), 4.79 (br, 2H), 4.01 (q,

2H), 1.08 (t, 3H).

3-((R)-4-(((R)-6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholin-2-yl)-

propanoic Acid (58). (R)-Ethyl 4-(2-bromo-4-ﬂuorophenyl)-6-(bro-

momethyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate

(compound 58l-4, 0.05 g, 0.001 mol), anhydrous ethanol (10 g), and

(R)-3-(morpholin-2-yl)propanoic acid (0.175 g, 0.0011 mol) were

added to a ﬂask. The mixture was stirred at 25 °C under a nitrogen

atmosphere for 6 h. After the reaction, the mixture was concentrated.

Ethyl acetate (10 g) was added to the residue, and the resulting

mixture was washed with water (15 mL × 2). The organic layer was

concentrated to give 3-((R)-4-(((R)-6-(2-bromo-4-ﬂuorophenyl)-5-

(ethoxycarbonyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-

morpholin-2-yl)propanoic acid (compound 58) as tawny oil (0.38 g,

1

65%). HRMS: calcd (M) 580.0791, exptl (MH⁺) 581.086. H NMR

(600 MHz, DMSO-d₆) δ 12.04 (s, 1H), 9.65 (s, 1H), 8.02 (d, J = 3.1

Hz, 1H), 7.94 (d, J = 3.1 Hz, 1H), 7.56 (dd, J = 8.5, 2.6 Hz, 1H),

7.40−7.38 (m, 1H), 7.22 (td, J = 8.5, 2.6 Hz, 1H), 6.03 (s, 1H), 3.99−

3.92 (m, 2H), 3.90−3.87 (m, 3H), 3.58 (td, J = 11.2, 1.9 Hz, 1H),

3.53−3.47 (m, 1H), 2.80−2.75 (m, 2H), 2.35 (td, J = 11.4, 3.0 Hz,

1H), 2.31−2.23 (m, 2H), 2.02 (t, J = 10.5 Hz, 1H), 1.69−1.56 (m,

2H), 1.06 (t, J = 7.1 Hz, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ

174.69, 165.64, 162.46, 162.07, 160.42, 146.84, 144.36, 144.09, 140.70,

140.68, 131.41, 131.35, 125.14, 123.08, 123.01, 120.14, 119.98, 116.03,

115.89, 97.59, 74.92, 66.54, 59.86, 58.66, 58.29, 56.26, 53.26, 29.98,

28.52, 14.46.

3-((S)-4-(((R)-6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbon-

yl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-

morpholin-2-yl)propanoic Acid (59). HRMS: calcd (M) 580.0791,

1

exptl (MH⁺) 581.0862. H NMR (600 MHz, acetone-d₆) δ 9.72 (s,

1H), 7.97 (d, J = 3.1 Hz, 1H), 7.80 (d, J = 3.0 Hz, 1H), 7.55−7.49 (m,

1H), 7.44 (dd, J = 8.5, 2.6 Hz, 1H), 7.13 (td, J = 8.4, 2.5 Hz, 1H), 6.19

P

DOI: 10.1021/acs.jmedchem.7b01914

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Journal of Medicinal Chemistry

Article

(s, 1H), 4.09 (d, J = 16.8 Hz, 1H), 4.03 (q, J = 7.1 Hz, 2H), 3.95 (d, J

= 16.6 Hz, 1H), 3.90 (d, J = 11.1 Hz, 1H), 3.74−3.68 (m, 2H), 2.98

(d, J = 8.6 Hz, 1H), 2.74 (d, J = 7.3 Hz, 1H), 2.54−2.41 (m, 3H), 2.37

(s, 1H), 2.29−2.20 (m, 1H), 1.83−1.78 (m, 2H), 1.13 (t, J = 7.1 Hz,

3H). ¹³C NMR (150 MHz, acetone-d₆) δ 173.66, 165.38, 162.77,

162.16, 160.51, 146.47, 144.28, 143.46, 140.59, 131.17, 131.12, 123.77,

122.92, 122.86, 119.65, 119.49, 115.15, 115.01, 97.80, 74.81, 66.37,

59.38, 58.72, 58.45, 56.30, 53.01, 13.64.

3-((3S)-4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-

2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholin-

3-yl)propanoic Acid (60). HRMS: calcd (M) 580.0791, exptl (MH⁺)

581.086. ¹H NMR (600 MHz, acetone-d₆) δ 9.93 (s, 1H), 7.98 (d, J =

3.1 Hz, 1H), 7.80 (d, J = 3.1 Hz, 1H), 7.54−7.51 (m, 1H), 7.42 (dd, J

= 8.5, 2.6 Hz, 1H), 7.16 (td, J = 8.5, 2.6 Hz, 1H), 6.18 (s, 1H), 4.33 (d,

J = 18.0 Hz, 1H), 4.04−4.01 (m, 3H), 3.93 (dd, J = 11.4, 2.7 Hz, 1H),

3.83−3.80 (m, 1H), 3.77−3.70 (m, 1H), 3.51−3.47 (m, 1H), 3.01 (s,

1H), 2.90 (d, J = 10.8 Hz, 1H), 2.70−2.60 (m, 2H), 2.51−2.46 (m,

1H), 2.44−2.37 (m, 1H), 1.86−1.78 (m, 1H), 1.13 (t, J = 7.1 Hz, 3H).

¹³C NMR (150 MHz, acetone-d₆) δ 173.65, 165.47, 162.80, 162.12,

3.90 (m, 3H), 3.79 (t, J = 10.5 Hz, 1H), 3.73−3.68 (m, 1H), 3.61 (s,

3H), 2.93−2.77 (m, 2H), 2.55 (t, J = 10.1 Hz, 1H), 2.47−2.33 (m,

2H), 2.17 (t, J = 9.5 Hz, 1H), 1.76 (q, J = 7.1 Hz, 2H). ¹³C NMR (101

MHz, methanol-d₄) δ 175.91, 166.37, 162.94, 161.82, 160.47, 146.79,

145.70, 143.37, 137.88, 137.85, 133.46, 133.36, 130.82, 130.73, 123.72,

116.55, 116.30, 114.27, 114.05, 98.21, 74.84, 66.20, 57.98, 56.39,

55.69, 53.02, 50.25, 29.54.

3-((R)-4-(((R)-6-(2,4-Dichlorophenyl)-5-(methoxycarbonyl)-2-

(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholin-2-

yl)propanoic Acid (65). HRMS: calcd (M) 538.0844, exptl (MH⁺)

539.0488. ¹H NMR (600 MHz, acetone-d₆) δ 9.78 (s, 1H), 7.97 (d, J =

3.1 Hz, 1H), 7.79 (d, J = 3.1 Hz, 1H), 7.49 (d, J = 2.1 Hz, 1H), 7.46

(d, J = 8.4 Hz, 1H), 7.31 (dd, J = 8.4, 2.0 Hz, 1H), 6.19 (s, 1H), 4.00

(q, J = 17.2 Hz, 2H), 3.95−3.92 (m, 1H), 3.73 (td, J = 11.1, 2.2 Hz,

1H), 3.75−3.71 (m, 1H), 3.58 (s, 3H), 2.86 (t, J = 12.6 Hz, 2H),

2.50−2.37 (m, 3H), 2.13 (t, J = 10.5 Hz, 1H), 1.77−1.74 (dd, J = 14.1,

7.4 Hz, 2H). ¹³C NMR (150 MHz, acetone-d₆) δ 173.54, 165.80,

162.59, 146.87, 144.63, 143.51, 141.15, 133.52, 133.00, 130.99, 129.07,

127.69, 123.79, 96.72, 74.80, 66.46, 58.28, 56.51, 56.42, 53.24, 50.38.

3-((S)-1-(((R)-6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbon-

yl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-4,4-di-

ﬂuoropyrrolidin-2-yl)propanoic Acid (66). The synthetic proce-

dure was described in the Supporting Information. HRMS: calcd (M)

160.48, 147.69, 144.26, 143.50, 140.66, 131.30, 131.24, 123.79, 122.83,

122.77, 119.55, 119.39, 115.36, 115.22, 96.47, 70.04, 66.75, 59.81,

59.33, 58.64, 53.46, 52.40, 13.66.

3-((3R)-4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-

2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholin-

3-yl)propanoic Acid (61). HRMS: calcd (M) 580.0791, exptl (MH⁺)

581.0862. ¹H NMR (600 MHz, acetone-d₆) δ 9.93 (s, 1H), 7.99 (d, J =

3.1 Hz, 1H), 7.79 (d, J = 3.1 Hz, 1H), 7.52−7.49 (m, 1H), 7.43 (dd, J

= 8.5, 2.6 Hz, 1H), 7.11 (td, J = 8.4, 2.6 Hz, 1H), 6.18 (s, 1H), 4.37 (d,

J = 17.9 Hz, 1H), 4.05−4.01 (m, 3H), 3.89 (dd, J = 11.4, 2.7 Hz, 1H),

3.81−3.80 (m, 1H), 3.76−3.70 (m, 1H), 3.58−3.55 (m, 1H), 2.99−

2.96 (m, 1H), 2.72−2.69 (m, 1H), 2.63−2.55 (m, 1H), 2.48−2.35 (m,

2H), 2.00−1.94 (m, 1H), 1.86−1.80 (m, 1H), 1.13 (t, J = 7.1 Hz, 3H).

¹³C NMR (150 MHz, acetone-d₆) δ 173.51, 165.44, 162.86, 162.12,

1

600.0654, exptl (MH⁺) 601.0723. H NMR (600 MHz, acetone) δ

7.95 (d, J = 3.1 Hz, 1H), 7.81 (d, J = 3.1 Hz, 1H), 7.59−7.56 (m, 1H),

7.44 (dd, J = 8.5, 2.6 Hz, 1H), 7.16 (td, J = 8.5, 2.6 Hz, 1H), 6.19 (s,

1H), 4.29 (q, J = 17.1 Hz, 2H), 4.03 (q, J = 7.1 Hz, 2H), 3.64−3.59

(m, 1H), 3.25−3.21 (m, 1H), 3.10−3.02 (m, 1H), 2.70−2.59 (m, 1H),

2.58−2.50 (m, 1H), 2.49−2.41 (m, 1H), 2.28−2.16 (m, 2H), 1.93−

1.86 (m, 1H), 1.12 (t, J = 7.1 Hz, 3H).¹³C NMR (150 MHz, acetone-

d₆) δ 173.78, 165.37, 162.73, 162.19, 160.54, 147.13, 144.38, 143.44,

140.64, 140.61, 131.40, 131.35, 129.81, 128.19, 126.54, 123.96, 122.77,

122.71, 119.57, 119.41, 115.34, 115.20, 97.99, 63.21, 63.18, 62.10,

61.91, 61.71, 59.46, 58.15, 51.84, 40.60, 40.44, 40.28, 29.27, 27.73,

13.60.

160.48, 147.66, 147.59, 144.25, 143.48, 140.67, 131.19, 131.13, 123.73,

122.94, 122.88, 119.63, 119.47, 115.11, 114.97, 96.52, 69.62, 66.66,

59.32, 59.29, 58.81, 53.35, 51.28, 13.66.

Molecular Docking. For docking purposes, the crystallographic

coordinates of the HBcAg (PDB ID 5E0I) were obtained from the

protein data bank (www.rcsb.org). Hydrogen atoms were added to the

structure to allow for appropriate ionization at physiological pH. The

protonated state of several important residues, such as His106, His142,

Tyr599, Glu751, and His640, were adjusted by using SYBYL7.3

(Tripos, St. Louis, MO) to favor the formation of reasonable hydrogen

bonds with the ligand. Molecular-docking analysis was carried out by

the SURFLEX module of the SYBYL package in order to explore the

interaction model for the active site of HBcAg with its ligand. All

atoms located within the range of 6.0 Å from any atom of the original

ligand were selected for the active-site analysis, and the corresponding

amino acid residue was, therefore, involved in the active-site analysis

even if only one of its atoms was selected. Other default parameters

were adopted in the SURFLEX-docking calculations. All calculations

were performed on a CCNU Grid based computational environment

(CCNU Grid website: http://www.202.114.32.71:8090/ccnu/chem/

platform.xml).

(3S)-4-((6-(2-Bromo-4-ﬂuorophenyl)-5-(ethoxycarbonyl)-2-

(1-methyl-1H-imidazol-2-yl)-3,6-dihydropyrimidin-4-yl)-

methyl)morpholine-3-carboxylic Acid (62). The synthetic proce-

dure was described in the Supporting Information. HRMS: calcd (M)

1

549.1023, exptl (MH⁺) 550.1090. H NMR (600 MHz, acetone-d₆) δ

7.46−7.43 (m, 1H), 7.42−7.39 (m, 1H), 7.21 (d, J = 4.5 Hz, 1H),

7.11−7.07 (m, 1H), 7.03 (s, 1H), 6.17 (d, J = 8.8 Hz, 1H), 4.34 (d, J =

17.7 Hz, 1H), 4.22 (d, J = 17.7 Hz, 1H), 4.18−4.11 (m, 1H), 4.05−

4.01 (m, 3H), 3.99 (d, J = 7.2 Hz, 3H), 3.88−3.76 (m, 2H), 3.67−3.54

(m, 1H), 3.26−3.21 (m, 1H), 2.62−2.44 (m, 1H), 1.13 (t, J = 7.1 Hz,

3H). ¹³C NMR (150 MHz, acetone-d₆) δ 171.71, 165.55, 165.54,

162.05, 160.41, 148.22, 143.00, 140.18, 140.16, 140.06, 140.04, 138.71,

138.69, 130.31, 130.25, 127.55, 127.54, 125.94, 125.92, 123.10, 123.05,

123.03, 119.86, 119.84, 119.69, 119.68, 114.76, 114.72, 114.63, 114.59,

95.59, 95.33, 69.22, 69.13, 67.04, 66.99, 62.12, 59.18, 58.16, 58.11,

54.79, 49.31, 48.37, 35.50, 13.68.

3-((R)-4-(((R)-6-(2-Bromo-4-ﬂuorophenyl)-5-(methoxycar-

bonyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-

morpholin-2-yl)propanoic Acid (63). HRMS: calcd (M) 566.0635,

In Vitro Biological Testing. General Considerations. All ﬁnal

compounds were examined for known classes of assay-interference

compounds (pan assay-interference compounds, PAINS),³²which

were classiﬁed as negative.

1

exptl (MH⁺) 567.0707. H NMR (600 MHz, acetone-d₆) δ 9.76 (s,

1H), 7.97 (d, J = 3.1 Hz, 1H), 7.79 (d, J = 3.1 Hz, 1H), 7.49−7.47 (m,

1H), 7.44 (dd, J = 8.5, 2.6 Hz, 1H), 7.11 (td, J = 8.4, 2.6 Hz, 1H), 6.17

(s, 1H), 4.00 (q, J = 17.2 Hz, 2H), 3.96−3.92 (m, 1H), 3.73 (td, J =

11.3, 2.3 Hz, 1H), 3.69−3.65 (m, 1H), 3.58 (s, 3H), 2.89−2.84 (m,

2H), 2.50−2.37 (m, 3H), 2.13 (t, J = 10.5 Hz, 1H), 1.77−1.74 (m,

2H). ¹³C NMR (150 MHz, acetone-d₆) δ 173.56, 165.88, 162.73,

162.67, 162.16, 160.51, 146.77, 146.69, 144.40, 144.36, 143.49, 140.31,

140.29, 130.99, 130.94, 123.74, 122.99, 122.92, 119.79, 119.63, 115.10,

114.96, 97.27, 74.8’1, 66.47, 58.66, 58.28, 56.44, 56.41, 53.24, 50.35.

3-((R)-4-(((R)-6-(2-Chloro-4-ﬂuorophenyl)-5-(methoxycar-

bonyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-

morpholin-2-yl)propanoic Acid (64). HRMS: calcd (M) 522.1140,

exptl (MH⁺) 523.1240. ¹H NMR (400 MHz, methanol-d₄) δ 7.96 (d, J

= 3.0 Hz, 1H), 7.75 (d, J = 3.0 Hz, 1H), 7.43−7.39 (m, 1H), 7.23 (dd,

J = 8.7, 2.3 Hz, 1H), 7.05 (td, J = 8.4, 2.3 Hz, 1H), 6.17 (s, 1H), 4.09−

In Vitro Anti-HBV Activity in HepG2.2.15. HBV replicon cells,

HepG2.2.15, were seeded into 96-well plates at 4 × 10⁵cells/well,

treated with cell-culture medium (RPMI 1640 containing 10% FBS, 2

mM ^L-glutamine, 100 units/mL penicillin, and 10 μg/mL

streptomycin), and incubated overnight at 37 °C. The control and

test compounds were 3-fold diluted with medium and added into the

plates with a 0.5% ﬁnal DMSO concentration. The plates were then

incubated at 37 °C in a 5% carbon dioxide atmosphere and refreshed

with compound-containing medium every 3 days. On day 7, the

HepG2.2.15 supernatant was collected and HBV DNA was extracted

using the QIAamp 96 DNA Blood Kit (QIAGEN 51161, Hilden,

Germany). The HBV DNA was quantiﬁed by real-time PCR. The

primers and probe used were as follows:

Q

DOI: 10.1021/acs.jmedchem.7b01914

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

HBV forward primer: 5′-GTGTCTGCGGGCGTTTTATCA-3′

HBV reverse primer: 5′-GACAAACGGGCAACATACCTT-3′

HBV probe: 5′FAM-CCTCTKCATCCTGCTGCTAT-

GCCTCATC-3′

an additional 24 h incubation. The total treatment period was 48 h.

After the treatment period of 48 h, the incubation solution was

aspirated. Hepatocytes were washed twice with HBSS, followed by the

addition of the enzyme-substrate working solution. The hepatocytes

were incubated for 30 min.

After the incubation, 75 μL of the samples were aspirated and

quenched with 150 μL of the stop solution. The resulting mixtures

were shaken for 10 min, which was followed by centrifugation at 4 °C

and 3220g for 20 min. For each, an aliquot of the supernatant were

mixed with 0.1% FA in water at a ratio of 1:4. The resulting samples

were shaken for 10 min prior to analysis by LC-MS/MS. The

concentrations of the three metabolites of the CYP-enzyme substrate,

1′-hydroxymidazolam, hydroxybupropion, and acetaminophen, in the

human hepatocytes were quantitatively determined by LC-MS/MS

after the proteins were precipitated.

A standard curve was generated by plotting Ct value versus the

amount of the HBV-plasmid standard, and the quantity of each sample

was estimated on the basis of the Ct-value projection on the standard

curve; the percent of HBV inhibition by compound was calculated

using the following equation:

inh% = (1 − sample HBV quantity

/DMSO‐control HBV quantity) × 100%

The EC₅₀were plotted with GraphPad Prism 4 using the equation

below.

In Vivo Eﬃcacy in the Hydrodynamic-Injection (HDI) Mouse

Model. This study was conducted by WuXi AppTec Company, Ltd.

(Shanghai, China). All the procedures in the study were in compliance

with local animal-welfare legislation, the Guide for the Care and Use of

Laboratory Animals, and protocols approved by the WuXi AppTec

Institutional Animal Care and Use Committee (IACUC).

Female BALB/c mice (6−7 weeks old, 16−18 g) that were speciﬁc-

pathogen free were supplied by Shanghai Lingchang Biotechnology

Company, Ltd. (Shanghai, China). The study was approved by the

WuXi IACUC (IACUC protocol R20160125-Mouse). On day 0, all

the mice were hydrodynamically injected with pAAV2-HBV1.3mer

plasmid DNA (genotype D) in a volume of normal saline equivalent to

8% of a mouse’s body weight within 5 s through a tail vein. From days

1 to 7, the mice in all the groups were orally dosed with the blank

vehicle, 0.1 mg/kg entecavir, 50 mg/kg HEC72702, or 100 mg/kg

HEC72702 at the indicated frequency. Plasma and liver samples were

collected at the indicated time points for HBV-DNA quantiﬁcation by

real-time PCR.

sigmoidal dose‐response (variable slope), Y

₅₀−X)×hill slope

= bottom + (top − bottom)/(1 + 10^{(log EC}

)

where X is the logarithm of the concentration, and Y is the response. Y

starts at the bottom and goes to the top with a sigmoid shape. This is

identical to the “four parameter logistic equation”.

hERG assay. In vitro electrophysiology manual-patch-clamp assays

of selected compounds on hERG channels were conducted in

PharmaCore Laboratories, Ltd. (Jiangsu, China) in compliance with

their Standard Operating Procedures. In detail, HEK293 cells were

perfused with an extracellular solution, and membrane currents were

recorded by a HEKA EPC-10 USB patch-clamp ampliﬁer and a

PATCHMASTER acquisition program (HEKA Instruments Inc.,

Lambrecht, Germany) at room temperature. The electrophysiolog-

ical-voltage protocol for the hERG-current recordings was the

following: Cells were voltage clamped at a holding potential of −80

mV. The hERG current was activated by depolarizing at +20 mV for 2

s, after which the current was taken back to −50 mV for 5 s to remove

the inactivation and observe the deactivating tail current. The

stimulation frequency was once every 15 s. The current amplitude

was the peak value of the tail current. The channel current was

recorded in the whole-cell recording mode. The extracellular ﬂuid was

perfused and recorded continuously until stabilization. Then, the

extracellular ﬂuid containing the tested compound was perfused and

recorded until the inhibition of the hERG current reached a steady

state. Data were analyzed using PATCHMASTER V2 × 60 (HEKA

Instruments Inc., Lambrecht, Germany) and Origin 8.5 (OriginLab

Corporation, Northampton, MA) software programs and Microsoft

Excel.

Microsomal-Stability Assay. Microsomes were preincubated with a

test compound for 10 min at 37 °C in 100 mM potassium phosphate

buﬀer (pH 7.4). The reactions were initiated by adding NADPH to

give a ﬁnal incubation volume of 45 μL. The incubations contained 1

μM test compound, 0.5 mg/mL liver microsomal protein, and 2 mM

NADPH in 100 mM potassium phosphate buﬀer (pH 7.4). After

incubation times of 0, 15, 30, and 60 min at 37 °C, stop solution was

added (150 μL/well) to terminate the reaction. Following

precipitation and centrifugation, the amounts of compound remaining

in the samples were determined by LC-MS/MS.

Cytochrome-P450 (Cyp450)-Induction-Screening Assay: Enzyme-

Activity Induction. Cryopreserved hepatocytes were thawed and

counted to determine their yield, and their viability was measured.

Hepatocytes at a concentration of 0.7 million cells/mL were

transferred to collagen-coated 48-well plates for attachment (0.2 mL

viable cells/well). After the hepatocytes attached to the collagen

matrix, the plating medium was replaced with an incubation medium

containing 2% (v/v) Matrigel (sandwich medium), and the

hepatocytes were incubated until their use. All incubations were

conducted at 37 °C, 5% CO₂, and 95% humidity.

After the cultures were established, the sandwich medium was

removed and the hepatocytes were treated for 24 h with incubation

solutions that contained a vehicle (0.1% DMSO) and a test compound

or were a negative control or a positive control. The incubation

solution was aspirated and replaced the appropriate fresh solution for

Pharmacokinetic (PK) Analysis. All pharmacokinetic studies in

male Sprague−Dawley (SD) rats and beagle dogs were conducted

according to protocols approved by the Animal Care and Use

Committee at the Laboratory Animal Center of HEC Pharma Group.

Brieﬂy, male SD rats were obtained from Hunan SJA Laboratory

Animal Company, Ltd. (China) and maintained on a 12 h light−dark

̈

cycle with ad libitum access to food and water. Non-naive beagle dogs

supplied by Beijing Marshall Biotechnology Company, Ltd. (Beijing,

China) were used in this study. The animals were conﬁrmed healthy

before being assigned to the study. Each animal was given a unique

identiﬁcation number marked on the ear and written on the cage card.

The compounds were evaluated in Sprague−Dawley rats (6−7

weeks old, 190−240 g), which had fasted overnight before their

dosings, and their food was returned 4 h after their dosings. There

were three rats in the iv group and three rats in the po group, all of

which had blood samples taken (iv group, 1 mg/kg; po group, 5 mg/

kg). The compounds were dissolved in 5% DMSO, 5% solutol, and

90% saline for iv and po doses. Blood samples were collected at each

time point, placed into tubes containing sodium heparin, and

centrifuged at 12 000 rpm for 2 min at 4 °C to separate the plasma

from the samples. Following centrifugation, the resulting plasma was

transferred to clean tubes and stored frozen at −80 °C pending

bioanalysis. Plasma concentrations were determined by LC-MS/MS,

and the data were analyzed by a noncompartmental module of a

WinNonlin 6.3. Any BLQs were omitted from the calculations.

All dogs (7−9 months old, 8−12 kg) fasted overnight before their

ﬁrst dosings; their food was returned immediately after the oral dosing

and at 4 h post the ﬁrst dosing. Reverse-osmosis (RO) water was

available to all the animals, ad libitum. There were three dogs in the iv

group and three dogs in the po group, all of which had blood samples

taken (iv group, 1 mg/kg; po group, 5 mg/kg). The compounds were

dissolved in 5% DMSO, 5% solutol, and 90% saline for the iv and po

doses. Blood samples were collected at each time point, placed into

tubes containing sodium heparin, and centrifuged at 12 000 rpm for 2

min at 4 °C to separate the plasma from the samples. Following

centrifugation, the resulting plasma was transferred to clean tubes and

stored frozen at −80 °C pending bioanalysis. Plasma concentrations

R

DOI: 10.1021/acs.jmedchem.7b01914

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

were determined by LC-MS/MS, and the data were analyzed by a

noncompartmental module of a WinNonlin 6.3. Any BLQs were

omitted from the calculations.

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ASSOCIATED CONTENT

■

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.7b01914.

Detailed experimental procedures for the synthesis of

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AUTHOR INFORMATION

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Corresponding Authors

*Tel.: +86 18676907182, E-mail: renqingyun@hec.cn (Q.R.).

*Tel.: +86 13925592278, E-mail: zhangyingjun@hec.cn (Y.Z.).

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ORCID

Qingyun Ren: 0000-0003-0000-0417

Siegfried Goldmann: 0000-0002-4776-0360

Notes

The authors declare no competing ﬁnancial interest.

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ACKNOWLEDGMENTS

■

This project was supported by the National Major Scientiﬁc

and Technological Special Project for “Signiﬁcant New Drugs

Development” during the Thirteenth Five-Year Plan Period of

China (nos. 2017ZX09201006004 and 2016ZX09101065). We

thank Dr. Wenchao Yang at the Central China Normal

University for the molecular-docking analysis. In addition, we

thank Wuxi AppTec for working together with us on the in

vitro and in vivo anti-HBV-activity experiments, Shanghai

Chem Explorer and Pharma Core Laboratories for the hERG-

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ABBREVIATIONS USED

■

HBV, hepatitis B virus; CHB, chronic hepatitis B; HAP,

heteroaryldihydropyrimidine; HBsAg, HBV surface antigen;

pgRNA, pregenomic RNA; cccDNA, covalently closed circular

̀

DNA; hERG, human ether-a-go-go-related gene; ADMET,

absorption, distribution, metabolism, excretion, and toxicity;

HDI, hydrodynamic injection; ETV, entecavir; CYP, cyto-

chrome P450; NBS, N-bromosuccinimide; PE, petroleum

ether; EA, ethyl acetate; HPLC, high-performance liquid

chromatography; IPA, 2-propanol; PD, pharmacodynamics;

PK, pharmacokinetic; qPCR, quantitative polymerase chain

reaction; q.d., once daily; b.i.d., twice daily; HLM, human liver

microsome; MLM, mouse liver microsome; DLM, dog liver

microsome.

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J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

3-((R)-4-(((R)-6-(2-Bromo-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholin-2-yl)propanoic Acid (HEC72702), a Novel Hepatitis B Virus Capsid Inhibitor Based on Clinical Candidate GLS4

DOI: 10.1021/acs.jmedchem.7b01914

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