Discovery and Pre-Clinical Characterization of Third-Generation 4-H

Article pubs.acs.org/jmc

Discovery and Pre-Clinical Characterization of Third-Generation 4‑H Heteroaryldihydropyrimidine (HAP) Analogues as Hepatitis B Virus (HBV) Capsid Inhibitors Zongxing Qiu,†,§ Xianfeng Lin,†,§ Weixing Zhang,†,§ Mingwei Zhou,†,§ Lei Guo,†,§ Buelent Kocer,†,§ Guolong Wu,†,§ Zhisen Zhang,†,§ Haixia Liu,†,§ Houguang Shi,†,§ Buyu Kou,†,§ Taishan Hu,†,§ Yimin Hu,†,§ Mengwei Huang,†,§ S. Frank Yan,†,∥ Zhiheng Xu,†,∥ Zheng Zhou,†,∥ Ning Qin,†,∥ Yue Fen Wang,†,⊥ Shuang Ren,†,⊥ Hongxia Qiu,†,⊥ Yuxia Zhang,†,⊥ Yi Zhang,†,⊥ Xiaoyue Wu,†,⊥ Kai Sun,†,⊥ Sheng Zhong,†,⊥ Jianxun Xie,†,⊥ Giorgio Ottaviani,†,⊥ Yuan Zhou,†,# Lina Zhu,†,# Xiaojun Tian,†,# Liping Shi, †, # Fang Shen,†,# Yi Mao,†,# Xue Zhou,†,# Lu Gao,†,# John A. T. Young,‡,# Jim Zhen Wu,†,# Guang Yang,†,# Alexander V. Mayweg,†,§ Hong C. Shen,*,†,§ Guozhi Tang,*,†,§ and Wei Zhu*,†,§ †

Roche Innovation Center Shanghai, ‡Roche Innovation Center Basel, §Medicinal Chemistry, ∥Chemical Biology, ⊥Pharmaceutical Sciences, #Discovery Virology, Roche Pharma Research and Early Development, Bldg 5, 720 Cailun Road, Shanghai 201203, China S Supporting Information *

ABSTRACT: Described herein are the discovery and structure−activity relationship (SAR) studies of the third-generation 4-H heteroaryldihydropyrimidines (4-H HAPs) featuring the introduction of a C6 carboxyl group as novel HBV capsid inhibitors. This new series of 4-H HAPs showed improved anti-HBV activity and better drug-like properties compared to the first- and second-generation 4-H HAPs. X-ray crystallographic study of analogue 12 (HAP_R01) with Cp149 Y132A mutant hexamer clearly elucidated the role of C6 carboxyl group played for the increased binding affinity, which formed strong hydrogen bonding interactions with capsid protein and coordinated waters. The representative analogue 10 (HAP_R10) was extensively characterized in vitro (ADMET) and in vivo (mouse PK and PD) and subsequently selected for further development as oral anti-HBV infection agent.

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INTRODUCTION Hepatitis B virus (HBV) infection is a major public health problem. Currently, it is estimated that 240 million people worldwide are chronically infected with HBV. Approximately one-third of these individuals will die from the end stage of liver diseases caused by HBV infection such as cirrhosis, hepatocellular carcinoma, and liver failure, if left untreated.1,2 Current therapies include nucleos(t)ide analogues (lamivudine, adefovir, tenofovir, telbivudine, and entecavir) and interferon alpha (INF-α, including nonpegylated and pegylated). Although both therapies can reduce HBV DNA and normalize liver enzymes, neither treatment offers a high rate of clinical cure as defined by the loss of HBV surface antigen (HBsAg) (with or without seroconversion).2,3 In addition, IFN-based therapies are poorly © 2017 American Chemical Society

tolerated; while nucleos(t)ide analogues frequently require prolonged or possibly life-long treatment, and some are associated with a high risk of viral resistance. As such, there remains a tremendous unmet medical need to discover and develop efficacious and safe anti-HBV agents with novel mechanisms of action to improve disease cure rate.4,5 HBV capsid is formed by the assembly of HBV core proteins, and it protects the enclosed viral genome and creates the environment for reverse transcription of pregenomic RNA (pgRNA) to DNA. The assembly and disassembly of HBV capsids are essential steps in the HBV life cycle and interruption of the Received: January 18, 2017 Published: March 24, 2017 3352

DOI: 10.1021/acs.jmedchem.7b00083 J. Med. Chem. 2017, 60, 3352−3371

Journal of Medicinal Chemistry

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Figure 1. Chemical structures of representative HBV capsid assembly effectors.

cells with EC50 values in the range of 0.05−0.08 μM (internal and literature data6). Compound 3 also demonstrated in vivo anti-HBV efficacy in HBV transgenic mice16 and Alb-uPA/ SCID mice.17 Further development of 3 was not pursued with undisclosed reason. However, it was reported that 3 could induce hepatotoxicity in rats at high doses.18 We identified that 3 has an unfavorable cytotoxicity profile with a CC50 of 13 μM in HepDE19 cells. The C2 thiazol-2-yl HAP analogue 5 (Table 1), however, could lead to an improved CC50 and maintain a similar EC50 to that of 3. Subsequently, the 4-H HAP series of capsid inhibitors were evolved with the discovery of more potent second-generation analogues featuring the introduction of C6 morpholinyl group, which included 4a (example 31, WO 01/68640),19 4b (GLS-4),20,21 4c (NVR-010−001-E2),22 and an internal reference compound 6 (Table 1). Presumably, the C6 morpholinyl group brings additional interactions with HBV capsid proteins,22 which in turn contributes to the improved antiHBV activity as observed for analogue 6 compared to 5. The second-generation 4-H HAP 6 also demonstrated a more favorable cytotoxicity profile (CC50 of 6 is >100 μM) than that of the first-generation 4-H HAPs 3 and 5. However, compound 6 showed high liver microsome clearance. It also demonstrated a strong hERG inhibition with an IC20 of 0.48 μM, which was likely ascribed to dual effects of both the moderate basicity of morpholine “N” (pKa = 6.3) and the high lipophilicity of 6 (log P = 3.2) as documented.23 In our design of the third-generation 4-H HAPs, we would like to discover more potent and drug-like analogues by reducing both the basicity and lipophilicity of the second-generation 4-H HAPs through introduction of a polar substituent such as hydroxyl, sulfonamide, carboxamide, or carboxyl. However, it remains unclear whether a polar substituent as described above on 4-H HAPs can be tolerated in the largely hydrophobic binding pocket located at the interface of capsid dimers.22,24

normal capsid assembly process will block the HBV replication.6 There are several classes of HBV capsid assembly effectors reported to date with two distinct mechanism of actions. Most of the known capsid assembly effectors including compound 1 (AT-130),7 2 (DVR-23),8 and NVR 3-778 (structure unknown)9 act as HBV pgRNA encapsidation blockers by accelerating the assembly of normal-sized but empty capsids (Figure 1).10,11 By contrast, the heteroaryldihydropyrimidine (HAP) compounds represented by compound 3 (Bay 41-4109)6 is a unique chemical class of capsid assembly inhibitor, which can prevent the normal assembly of core proteins and lead to an aberrant formation of capsid. The misassembled capsid will be subsequently degraded by host proteasomes.6,12 Thus, the misassembly and depletion of HBV core proteins result in the inhibition of HBV replication. Moreover, it is hypothesized that the depletion of core proteins may suppress the function of viral covalently closed circular DNA (cccDNA)13 and help restore the innate immune response.14,15 HAPs thus represent a promising chemical class of anti-HBV agents with the potential to be the first new direct acting anti-HBV agent since the launch of nucleosides in 1998. However, the current HAPs have not been optimized in terms of drug-like properties for oral anti-HBV treatment and there is a significant need existing for the discovery of new HAPs with improved anti-HBV activity, pharmacokinetic, and safety profiles for clinical use. In this article, we describe the design and discovery of a new generation of 4-H HAPs featuring the introduction of a C6 carboxyl group with improved anti-HBV activity and better druglike properties. Furthermore, the elaboration of this chemical series culminates in a potential clinical candidate selection.

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RESULTS AND DISCUSSION Design and SAR. Compound 3 is the prototype of the firstgeneration HAP-based capsid assembly inhibitors. It showed moderate activity against HBV genome replication in HepG2.2.15 3353



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Table 1. Discovery of C6 Morpholine Carboxylic Acid 4-H HAP Analogues

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Table 1. continued

a

EC50 is the mean value for reduction of HBV DNA by 50% in HepG.2.2.15 cells. Experiments were run in duplicate, with variation 100 μM in HepDE19 cells, and the selectivity index (SI, defined by CC50/EC50) was >33333. In the preliminary ADME (absorption, distribution, metabolism, and excretion) and in vitro safety assessment, compound 10 showed significantly improved human/mouse liver microsome stability and no sign of hERG inhibition (IC20 > 10 μM) compared to that of the representative first- and the second-generation 4-H HAP analogues, likely as a result of both reduced lipophilicity and basicity (pKa is now 5.8). Moreover, compound 10 showed a moderate to high permeability (1.4 × 10−6 cm/s) in the 3357



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Table 3. Summary of SAR at the C4 Position of Third-Generation 4-H HAPs

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Table 3. continued

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Table 3. continued

a

Definitions as the same as that described before. bDiasteroisomer mixture at C4 position. cThe highest concentration tested.

lower lipophilicity than that of 6 as reflected by their log P values. This observation highlighted the unique profile of the

all of which maintained high anti-HBV activity. However, those analogues all showed high microsomal clearance despite the 3360



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Figure 2. Crystal structure of Cp149 Y132A in complex with 12. Compound 12 is highlighted in cyan sticks. The dimer forming the concave for the binding pocket is shown in light green. The dimer forming the cap for the binding pocket is colored in dark green. Black dash lines represent the key hydrogen bonds. PDB 5WRE.

previously.22,24 We decided to extensively study the substitution effect and bioisostere replacement of the C2 thiazolyl moiety. First, consistent with the previous observation for 3, difluoropyridinyl was well tolerated at the C2 position and the corresponding analogue 35 showed good anti-HBV activity but also a high cytotoxicity. Most of our attempts to replace the C2 thiazolyl with other bioisosteres did not lead to appreciable anti-HBV activity, as exemplified by C2 oxazolyl and pyrimidinyl analogues 36 and 37, which reflect a narrow SAR at the C2 position of 4-H HAP. Nevertheless, we did identify the N-methylimidazolyl and 5-methyloxazolyl analogues 38 and 39 with moderate anti-HBV activity. Meanwhile, small substituents on the C2 thiazole were well tolerated, as both the methyl and trifluoromethyl substituted thiazole analogues 40 and 42 showed comparable anti-HBV activity to that of 10. However, the cyclopropyl substituted thiazole analogue 41 was less active. During the SAR work, the collective information gained from in-house photoaffinity study,26 and the reported 5 Å cocrystal structure (2G34)24 indicated that the C4 position of 4-H HAP is located in a conservative, largely hydrophobic pocket, providing important interactions with capsid. As such, the SAR work at the C4 position of HAP became the chemistry focus in order to discover more potent analogues. The results are summarized in Table 3. First, bioisosteres of C4 phenyl, such as cyclohexyl, thiophenyl (substituted or unsubstituted), or pyridyl (substituted or unsubstituted), were not tolerated with a total loss of antiHBV activity for the respective analogues. Replacement of the C4 phenyl with small substituent such as methyl or cyclopropyl was not successful either. The SAR exploration was thus concentrated on the substituent fine-tunings on the C4 phenyl as shown in Table 3.

carboxyl group for the improved drug-like properties of 10. Meanwhile, bioisostere replacements of the C6 morpholine-3carboxylic acid were explored as well. Among all the attempts, the piperidine, piperazine, or N-acetylpiperazine analogue 23− 25 all led to a total loss of anti-HBV activity. The thiomorpholine analogue 26, however, maintained a moderate EC50 value of 0.038 μM. Compound 10 appeared to be a promising candidate for further ADME and in vitro safety assessment. In the meantime, we performed thorough SAR studies on C2, C4, and C5 positions of the third-generation 4-H HAPs (see structure of 3 for numbering convention) in order to identify a set of contenders of compound 10 for comparisons in further ADME and safety assessments. The results are shown in Tables 2 and 3. We first explored the SAR at the C5 position of 4-H HAP. The ethyl ester analogue 27 afforded a similar or slightly better EC50 than that of analogue 10. However, compound 27 was prone to metabolism in mouse liver microsome with a clearance of 54 mL min−1 kg−1. It was found that the size of C5 ester group had a significant impact on the anti-HBV activity as the n-propyl ester analogue 28, iso-propyl ester analogue 29, and trifluoroethyl ester analogue 30 all showed reduced anti-HBV activity compared to that of 10 and 27. The bulky tert-butyl ester analogue 31 had a much reduced anti-HBV activity. The polar C5 substituents (reflected by their log P values), including carboxylic acid (9 in Table 1), methoxyethyl ester (32), amide (33), and nitrile (34), were not tolerated with a dramatic drop of anti-HBV activities, which indicated a lipophilic environment at this binding position of capsid. Overall, the methyl ester and ethyl ester were well tolerated in terms of anti-HBV activity at the C5 position of HAP. The C2 substituent of the 4-H HAP series was indicated as an important moiety for capsid binding but was less explored 3361



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Table 4. Physicochemical and ADME Properties of 10, 60, and 68 compd

log Da

pKab (acid/base)

Lysac (μg/mL)

HLM/MLMc (mL min−1 kg−1)

PAMPAd (10−6 cm/s)

PPB (human/mouse)e

10 60 68

0.06 0.42 0.44

1.8/5.8 1.8/5.7 666 >679 >604

5/10 2/12 2/14

1.4 3.3 2.7

5.7/7.4 2.0/2.2 5.4/6.2

a Apparent partition coefficients at pH 7.4. bIonization constant. cDefinitions as the same as described before. dParallel artificial membrane permeability assay (10−6 cm/s). ePlasma protein binding assay (% unbound fraction).

We first investigated the o-substituent of the C4 phenyl group. Replacement of the o-Cl with o-H or o-F led to >5-fold potency drop (43 and 44 compared to 27). The o-Br and o-CH3 with a similar Van Der Walls radius27 as o-Cl were well tolerated, and the corresponding analogues 45−48 gave similar or slightly improved anti-HBV activity in HepG2.2.15 cells compared to that of compound 10 and 27. However, larger substituents like trifluoromethyl or cyclopropyl were not tolerated, with a significant potency reduction (analogues 49 and 50). This SAR observation indicated that the o-Cl of C4 phenyl of compound 10 might tightly fit into the capsid binding pocket. We subsequently explored the SAR of the p-substituent of the C4 phenyl. In general, the p-F of C4 phenyl could be successfully replaced with p-Cl, p-Br, and p-CH3 with equal or even better anti-HBV activity in some cases (52−55). However, the p-Cl, p-Br analogues 54 and 55 showed moderate cytotoxicity in HepDE19 cells. The p-H analogue 51 showed a 7-fold potency drop with an EC50 value of 0.014 μM. Analogue 56, bearing a bulky p-CF3, displayed an EC50 of >0.1 μM. The analogues containing polar substituents such as p-OCH3 and p-CN gave significantly reduced anti-HBV activities (57 and 58 vs 51). In the first- and the second-generation HAP series, the m-position of C4 phenyl has been barely explored. We have identified that the introduction of a m-F substituent on the C4 phenyl could further improve the anti-HBV activity, with analogues 60 and 61 showing sub-nM potency in HepG2.2.15 cells. However, m-Cl and m-CN substituents were not tolerated as analogues 62 and 63 showed EC50s > 0.1 μM. So far, the SAR studies suggest that the preferred substituents at each position of the C4 phenyl are (1) ortho position: −Cl, −Br, and methyl; (2) meta position: −H and −F; (3) para position: −F, −Cl, and −Br. With this SAR understanding in mind, several tri-substituted C4 phenyl analogues were designed and synthesized. As expected, all the analogues showed excellent anti-HBV activity, among which, analogues 65, 67, and 69 demonstrated sub-nM EC50 values in HepG2.2.15 cells. X-ray Crystal Structure. We have recently solved the cocrystal structure of Cp149 Y132A mutant hexamer in complex with analogue 12 (named as HAP_R01, PDB 5WRE),26 which nicely elucidated why the introduction of a carboxyl group on the C6 substituent (e.g., gem-difluoropyrrolidine and morpholine) of 4-H HAP can bring improved anti-HBV activity. As shown in Figure 2, the carboxylic acid moiety forms strong bidentate hydrogen bonds with the backbone NH and the side chain hydroxyl of S141. The high resolution structure (1.95 Å) also reveals the water network including a well-defined water molecule that coordinates the C6 carboxyl group and the N atom of C2 thiazolyl group (Figure 2) These hydrogen bonding interactions contribute to the increased binding affinity of the third-generation 4-H HAPs such as 10 and 12, as reflected by the improved anti-HBV activity compared to the corresponding noncarboxylic acid analogues (10 vs 6 as an

example). It was speculated that the reduced anti-HBV activity of the diastereoisomer 11, regioisomer 13, or the extended carboxylic acid analogues 14 and 15, relative to compound 10, could be ascribed to the complete or partial disruption of this H-bonding interaction network. On the other hand, the carboxylic acid derivatives 19−22 could at least partially maintain the H-bonding interactions and thus retain the good anti-HBV activity. Consistent with our SAR findings, the C2 thiazolyl, C4 phenyl, and C5 methyl ester of 12 bind to a largely hydrophobic pocket of capsid and make multiple van der Waals (VDW) contacts, contributing to additional binding affinity. This explains why the introduction of a polar substituent at those positions will lead to an anti-HBV activity drop. DMPK, in Vivo Efficacy, and Early Safety Assessment of the Selected Third-Generation 4-H HAPs. On the basis of anti-HBV activity, cytotoxicity, human/mouse metabolic stability, and structural diversity, analogues 10, 60, and 68 were selected for further physicochemical and ADME characterizations. The data are summarized in Table 4. High solubility is a critical factor for the development of oral anti-HBV agents to enable formulation development and maintain high plasma drug levels in vivo. It is also very important for HAP analogues to have a moderate or low liver clearance, as this often leads to a good oral bioavailability and high systemic exposure. As shown in Table 4, the thirdgeneration 4-H HAP analogues with an ionizable carboxylic acid group have demonstrated high solubility and low human/ mouse microsome clearance. In the cryopreserved human and mouse hepatocytes incubation studies of these analogues, the unchanged parent compounds are the major form and the detectable metabolites are mainly from the oxidation of C6-morpholine ring and a phase II glucuronide conjugation of carboxylic acid. The hydrolysis of C5-ester is almost negligible, especially in the human hepatocyte. Moreover, all three compounds show a medium to high permeability determined in the PAMPA assay and appreciable free fraction in human/ mouse plasma as reflected by their PPB (plasma protein binding) values. On the basis of the favorable physicochemical (log D and pKa) and in vitro ADME properties, these compounds were tested for in vivo PK studies in mice following single dose intravenous (IV) and oral (PO) administrations. The results are summarized in Table 5. 4-H HAP analogues such as 10 and 60 showed good oral absorption, as reflected by their respective values of oral Cmax, AUC, and F%. Analogue 68 showed relatively lower exposure as expressed by Cmax and AUC than that of compounds 10 and 68. Interestingly, all three compounds, especially compounds 10 and 68, demonstrated a higher liver exposure than their corresponding plasma levels, which is considered as a desirable attribute for liver targeting drugs such as HBV capsid inhibitors. The high liver-to-plasma exposure ratio (from 3.2 to 27) might point to a potential involvement of uptake hepatic transporters in governing the preferable liver disposition of these compounds 3362



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adduction), mutagenicity (Ames assay), or clastogenicity (micronucleus assay) liability. A 5-day in vivo toxicology assessment in rats supported further development of compound 10 that showed a supportive safety margin as defined by ratio of the plasma exposure (Cmax and AUC) in rat at NOAEL (no observed adverse effect level) versus the exposure at a projected human efficacious dose. In the in vitro DDI assessment, compound 10 displayed IC50 values of >50 μM for CYP inhibition in a panel of five enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2). Compound 10 had no time dependent inhibition (TDI) flags to CYP3A4 inhibition. However, this compound was identified as a strong CYP3A4 inducer in primary human hepatocytes as determined by the CYP3A4 mRNA levels (111% increase compared to the positive control rifampicin at 10 μM).29 This observation of CYP3A4 induction is likely related to 4-H HAP core as over 250 compounds of the thirdgeneration 4-H HAPs were screened and most of them demonstrated more or less CYP3A4 induction liabilities. The data of a few selected analogues is shown in Table 6.

Table 5. Single-Dose Pharmacokinetics of 10, 60 and 68 in Micea example Plasma t1/2 (IV, h) Vss (IV, L/kg) CL (IV) (mL min−1 kg−1) Cmax (PO, μg/L) AUC(0−∞) (IV, μg/L·h) AUC(0−∞) (PO, μg/L·h) F (%) Liver AUC(0−∞) (PO, μg/L·h) AUC(0−∞), liver/AUC(0−∞), plasma (PO)

10

60

68

1.5 2.5 19 413 878 962 37

3.0 4.1 66 783 253 624 82

1.4 1.9 44 283 379 344 30

11800 12.3

2005 3.2

9210 27

a

The single-dose pharmacokinetics (SDPK) study of selected compounds were carried out in mice as indicated according to the standard procedures (IV, 1 mg/kg; PO, 3 mg/kg). Major parameters, including plasma clearance (CL), volume of distribution at steady state (Vss), t1/2 (IV), maximal concentration (Cmax), area under the curve (AUC), and oral bioavailability (F) are reported.

Table 6. Relative Induction Values of CYP3A4 mRNA by Selected Analogues

and it could also explain the underestimation of the in vivo clearance from in vitro mouse microsomes. Compound 10 was the most advanced analogue and was further assessed against HBV infection in the hydrodynamic injection (HDI) mouse model.28 In this study, female BALB/c mice were hydrodynamically injected with replication-competent HBV DNA plasmid through the tail vein. Twenty hours post plasmid injection, mice were orally dosed by blank vehicle, entecavir (ETV, 0.1 mg/kg, once daily as the positive control), or different doses (3 and 12.5 mg/kg per dose, twice daily) of compound 10 for 5 days. Plasma and liver samples were collected at the indicated time points for HBV DNA quantification by real-time quantitative polymerase chain reaction (qPCR). As shown in Figure 3, with treatment of compound 10, HBV DNA copy numbers were rapidly and markedly reduced by about 100-fold in plasma and were no longer detectable in liver tissues, demonstrating a strong anti-HBV effect of 10 upon oral administration. The maximum efficacy of 10 was comparable to the maximum efficacy of ETV in this model. Compound 10 was further profiled in an effort to identify any early safety flags and to define DDI (drug−drug interaction) liabilities. The in vitro toxicology profiles of compound 10 were favorable with no signs of hERG, GSH (glutathione

compd

% of positive control (10 μM rifampicin)

10 12 17 27 42 60 68

111 109 81 183 72 144 56

Chemistry. As exemplified by the synthesis of compound 10, the third-generation 4-H HAP analogues described herein were mostly prepared according to the procedure shown in Scheme 1. First, amidine 10A-1 was prepared by reacting thiazole-2-carbonitrile with sodium methoxide followed by treatment with NH4Cl. Next, a three-component reaction of methyl acetoacetate, 2-chloro-4-fluorobenzaldehyde, and amidine 10A-1 was conducted to give dihydropyrimidine 10A-2. The racemic 10A-2 was then subjected to SFC (supercritical fluid chromatography) chiral separation to afford the enantiomerically pure (R)-10A-2 as the desired intermediate (compound 5 in Table 1) and its antipode (S)-10A-2. The absolute

Figure 3. Levels of HBV DNA in plasma (A) and liver (B) of HBV HDI mice upon treatment with the vehicle control, ETV control, and compound 10. The HBV DNA copy numbers were quantified by real-time qPCR. Error bars indicate standard error of the mean (six mice per group). 3363



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Scheme 1a

a

Reagents and conditions: (a) NaOMe, MeOH, rt, 3 h, then NH4Cl, overnight, 100% (crude); (b) KOAc, CF3CH2OH, reflux, 16 h, 60% for two steps from thiazole-2-carbonitrile; (c) SFC chiral separation; (d) NBS, CCl4, rt, 1 h, 77%; (e) DIPEA, DCM, ca. 1 h, 55%.

translated in vivo as demonstrated in mice PK and PD studies, where a low clearance, good oral bioavailability, preferential liver distribution, and strong anti-HBV effects were demonstrated. The favorable in vitro and short-term rat tox profiles also support the nomination of 10 (named as HAP_R10) for further development.

stereochemistry of 5 was unambiguously determined by X-ray diffraction study.30 Bromination of 5 gave 10A, which was finally reacted with (3S)-morpholine-3-carboxylic acid hydrochloride salt to provide 10 after HPLC purification. This short and concise synthetic route was successfully applied to the synthesis of the third-generation 4-H HAPs described in this paper, which greatly facilitated and expedited our SAR studies.

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CONCLUSIONS In summary, we have discovered the third-generation 4-H HAPs, which feature the introduction of a carboxyl group at the C6 position of 4-H HAP. This carboxyl group brings an increased binding affinity with capsid, which leads to an improved anti-HBV activity compared to that of the first- and second-generation 4-H HAPs. The high resolution X-ray crystal structure of analogue 12 bound into Cp149 Y132A mutant hexamer reveals that this carboxyl group facilitates the formation of a strong hydrogen bonding interaction network between 12, coordinated waters, and capsid. Besides the improved anti-HBV activity, compared to the first- and secondgeneration 4-H HAPs, the third-generation 4-H HAP compounds represented by 10 are more drug-like with an improved solubility and metabolic stability. The favorable in vitro biological activity and ADME properties of compound 10 are well

EXPERIMENTAL SECTION

Synthetic Chemistry General Comments. All of the intermediates were purified by silica gel chromatography using either a Biotage SP1 system or an ISCO CombiFlash chromatography instrument. All of the final compounds were purified by preparative HPLC (prep-HPLC) on a reversed-phase column using a Waters XBridge OBD Phenyl (30 mm × 100 mm, 5 μm) or OBD RP18 (30 mm × 100 mm, 5 μm) column under acidic conditions (A, 0.1% formic acid in H2O; B, 0.1% formic acid in acetonitrile) or basic conditions (A, 0.1% ammonia in H2O; B, acetonitrile). For SFC chiral separation, the intermediates were separated using a chiral column (Daicel Chiralpak IC, 30 mm × 250 mm, 5 μm) on a Mettler Teledo SFC-Multigram system (solvent system of 95% CO2 and 5% IPA (0.5% TEA in IPA), backpressure of 100 bar, UV detection at 254 nm). Optical rotation was measured using a Rudolph Autopol V automatic polarimeter at a wavelength of 589 nm. LC−MS spectra were obtained using a MicroMass Platform LC (Waters Alliance 2795-ZQ2000). NMR spectra were obtained using Bruker AVANCE 400 MHz spectrometer, operating at 400.13 MHz (1H) and 100.62 MHz (13C). 3364



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Melting point: 136−137 °C. MS: calcd (MH+) 495, measured (MH+) 495. HRMS: calcd (MH+) 495.0899, measured (MH+) 495.0893. 1H NMR (DMSO-d6, 400 MHz): δ ppm 12.88 (br. s, 1H), 9.87 (s, 1H), 8.04 (d, J = 4.0 Hz, 1H), 7.95 (d, J = 4.0 Hz, 1H), 7.44−7.39 (m, 2H), 7.15 (td, J = 8.76, 2.62 Hz, 1H), 6.04 (s, 1H), 4.25 (d, J = 20 Hz, 1H), 4.07−3.97 (m, 2H), 3.86−3.61 (m, 4H), 3.51 (s, 3H), 3.12−3.07 (m, 1H), 2.45−2.39 (m, 1H). 13C NMR (DMSO-d6, 100 MHz): δ ppm 172.8, 166.2, 162.5, 161.1 (d, 1JCF = 246.33 Hz, FC), 148.0, 144.8, 144.1, 138.7 (d, 4JCF = 2.83 Hz, FC), 133.1 (d, 3JCF = 10.52 Hz, FC), 131.4 (d, 3JCF = 8.56 Hz, FC), 125.1, 117.1 (d, 2JCF = 25.74 Hz, FC), 115.3 (d, 2JCF = 20.42 Hz, FC), 96.3, 69.4, 67.2, 62.0, 56.3, 54.8, 51.4, 48.7. For the Following Third-Generation 4-H HAPs, Except Described Specifically, All the Analogues Are Prepared in Analogy to 10 from the Commercially Available Building Blocks. ((3R)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine3-carboxylic Acid (11). MS: calcd (MH+) 495, measured (MH+) 495. 1 H NMR (DMSO-d6, 400 MHz): δ ppm 12.86 (br s, 1H), 9.88 (s, 1H), 8.07−7.85 (m, 2H), 7.47−7.29 (m, 2H), 7.16 (td, J = 8.5, 2.6 Hz, 1H), 6.03 (s, 1H), 4.18−4.02 (m, 2H), 3.93−3.80 (m, 2H), 3.77−3.58 (m, 2H), 3.47−3.24 (m, 4H), 3.14−2.99 (m, 1H), 1.38− 1.12 ppm (m, 1H). (2S)-1-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]-4,4-difluoro-pyrrolidine-2-carboxylic Acid (12). Melting point: 106−107 °C. MS: calcd (MH+) 515, measured (MH+) 515. HRMS: calcd (MH+) 515.0762, measured (MH+) 515.0770. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.21 (s, 2H), 7.63−7.60 (m, 1H), 7.38−7.35 (m, 1H), 7.22−7.18 (m, 1H), 6.29 (s, 1H), 4.57 (d, J = 16 Hz, 1H), 4.13−4.08 (m, 2H), 3.70−3.67 (m, 4H), 3.33−3.28 (m, 1H), 2.92−2.82 (m, 1H), 2.63− 2.56 (m, 1H). 4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-2-carboxylic Acid (13). MS: calcd (MH+) 495, measured (MH+) 495. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.02 (d, J = 3 Hz, 0.5H), 8.01 (d, J = 3 Hz, 0.5H), 7.91 (dd, J = 3 Hz, 0.5H), 7.90 (d, J = 3 Hz, 0.5H), 7.54 (dd, J = 6.0, 9.0 Hz, 0.5H), 7.53 (dd, J = 6.0, 9.0 Hz, 0.5H), 7.30 (dd, J = 3, 9 Hz, 1H), 7.15−7.09 (m, 1H), 6.21 (s, 1H), 4.82−4.55 (m, 3H), 4.31−4.24 (m, 1H), 4.12−3.91 (m, 2H), 3.74−3.65 (m, 1H), 3.66 (s, 3H), 3.55−3.37 (m, 2H). 2-[(3R)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholin-3-yl]acetic Acid (14). The synthetic procedure was described in the Supporting Information. MS: calcd (MH+) 509, measured (MH+) 509. 1 H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 3.01 Hz, 1H), 7.77 (d, J = 3.14 Hz, 1H), 7.44 (dd, J = 8.60, 6.21 Hz, 1H), 7.24 (dd, J = 8.78, 2.64 Hz, 1H), 7.06 (td, J = 8.38, 2.57 Hz, 1H), 6.17 (s, 1H), 4.32 (br d, J = 17.44 Hz, 1H), 4.07 (br s, 1H), 3.97 (br s, 1H), 3.67− 3.92 (m, 4H), 3.63 (s, 3H), 3.24 (br s, 1H), 2.85−3.09 (m, 1H), 2.60 (br d, J = 4.77 Hz, 2H). 3-[(3R)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholin-3-yl]propanoic Acid (15). Compound 15 was prepared in analogue to 10 by using 3-[(3R)-morpholin-3-yl]propanoic acid trifluoroacetic acid salt (15-a, see Supporting Information for its synthesis) instead of (3S)-morpholine-3-carboxylic acid hydrochloride salt. MS: calcd (MH+) 509, measured (MH+) 509. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.01 (d, J = 3.26 Hz, 1H), 7.90 (d, J = 3.01 Hz, 1H), 7.57 (dd, J = 8.78, 6.02 Hz, 1H), 7.29 (dd, J = 8.78, 2.51 Hz, 1H) 7.13 (td, J = 8.41, 2.51 Hz, 1H), 6.21 (s, 1H), 4.99 (br d, J = 18.32 Hz, 1H), 4.60 (br d, J = 15.31 Hz, 1H), 4.00−4.16 (m, 3H), 3.91 (br s, 1H), 3.67 (s, 5H), 3.45 (dt, J = 13.05, 4.77 Hz, 1H), 2.48−2.67 (m, 2H), 2.35 (br d, J = 7.03 Hz, 1H), 2.07−2.21 (m, 1 H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]-3-methyl-morpholine-3-carboxylic Acid (16). The synthetic procedure was described in the Supporting Information. MS: calcd (MH+) 509, measured (MH+) 509. 1H NMR (CDCl3, 400 MHz): δ ppm 8.06 (d, J = 3.0 Hz, 1H), 7.85 (d, J = 3.0 Hz, 1H), 7.36 (dd, J = 8.8, 5.8 Hz, 1H), 7.28 (s, 1H), 7.20 (dd, J = 8.3, 2.5 Hz, 1H), 6.30 (s, 1H), 4.46 (d, J = 17.1 Hz, 1H),

1

H NMR spectra were obtained using the single pulse zg30. 13C{1H} NMR spectra were obtained using composite pulse zgpg30 for proton decoupling. The chemical shifts were referenced against internal TMS(1H,13C), and the coupling constants were calculated as a whole of H,H and H,F. All of the starting materials were obtained commercially. All of the final compounds had purities greater than 95% based upon HPLC, LC−MS, and 1H NMR analyses. All of the reported yields are for isolated products and are not optimized. General Synthetic Procedures for the Synthesis of ThirdGeneration 4-H HAPs with (3S)-4-[[(4R)-4-(2-Chloro-4-fluorophenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (10) Exemplified. To a stirred solution of thiazole-2-carbonitrile (1.50 g, 14 mmol) in 5 mL of dry MeOH was added dropwise a solution of sodium methoxide (0.74 g, 14 mmol) in 10 mL of dry methanol. The reaction mixture was stirred at room temperature until the disappearance of starting material monitored by LC−MS. After that, ammonium chloride (1.50 g, 28 mmol) was added in one portion and the reaction mixture was stirred overnight. The undissolved material was removed by filtration, and the filtrate was concentrated in vacuo to afford the thiazole-2-carboxamidine hydrochloride (compound 10A-1) as a gray solid which was used directly in the next step without further purification. MS: calcd 128 (MH+), measured 128 (MH+). To a stirred solution of thiazole-2-carboxamidine hydrochloride (10A-1, 0.15 g, crude, ca. 1.0 mmol), methyl acetoacetate (0.12 g, 1.0 mmol), and 2-chloro-4-fluorobenzaldehyde (0.16 g, 1.0 mmol) in CF3CH2OH (8 mL) was added potassium acetate (0.20 g, 2.0 mmol). The reaction mixture was refluxed for 16 h in a nitrogen atmosphere. After it was cooled to room temperature, the reaction mixture was concentrated and the residue was dissolved in ethyl acetate and washed with brine. The organic layer was dried over anhydrous Na2SO4. The solvent was removed, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether: 1/4 to 1/2) to afford methyl 4-(2-chloro-4-fluoro-phenyl)-6-methyl-2thiazol-2-yl-1,4-dihydropyrimidine-5-carboxylate (compound 10A-2) as a yellow solid (219 mg, 60%). MS: calcd (MH+) 366, measured (MH+) 366. 1H NMR (DMSO-d6, 400 MHz): δ ppm 9.98 (s, 1H), 7.97 (d, J = 4.0 Hz, 1H), 7.90 (d, J = 4.0 Hz, 1H), 7.41 (dd, J = 8.0, 4.0 Hz, 1H), 7.35 (dd, J = 8.0, 8.0 Hz, 1H), 7.18 (td, J = 8.0, 4.0 Hz, 1H), 5.98 (s, 1H), 3.53 (s, 3H), 2.47 (s, 3H). The racemic compound 10A-2 was subjected to SFC chiral separation. The desired (−)-enantiomer methyl (4R)-4-(2-chloro-4fluoro-phenyl)-6-methyl-2-thiazol-2-yl-1,4-dihydropyrimidine-5-carboxylate ((R)-10A-2) has a relatively short retention time. The absolute stereochemistry of (R)-10A-2 was determined by X-ray diffraction study (see Supporting Information).

(R )‐10A‐2: [α]D20 − 55.0 (c 0.845, MeOH) (S)‐10A‐2: [α]D20 + 44.6 (c 0.175, MeOH) To a stirred solution of compound (R)-10A-2 (0.37 g, 1.0 mmol) in CCl4 (5 mL) was added NBS (0.20 g, 1.1 mmol) in portions. After the reaction mixture was stirred at room temperature for 1 h, the solvent was removed in vacuo and the residue was purified by column chromatography to give methyl (4R)-6-(bromomethyl)-4-(2-chloro-4fluoro-phenyl)-2-thiazol-2-yl-1,4-dihydropyrimidine-5-carboxylate (compound 10A) as a yellow solid (342 mg, 77%). MS: calcd 445 (MH+), measured 445 (MH+). To a stirred solution of compound 10A (0.049 g, 0.11 mmol) and (3S)-morpholine-3-carboxylic acid hydrochloride salt (CAS: 118792904-9, 0.029 g, 0.17 mmol) in dichloromethane (5 mL) was added dropwise DIPEA (0.078 mL, 0.45 mmol). The reaction mixture was stirred at room temperature until the disappearance of 10A. The mixture was diluted with EtOAc (10 mL) and washed successively with saturated aqueous NH4Cl solution and brine. The organic layer was separated and dried over anhydrous Na2SO4. The solvent was concentrated in vacuo, and the residue was purified by prep-HPLC to give (3S)-4-[[(4R)-4-(2-chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic acid (compound 10) as a light-yellow solid (30 mg, 55%). 3365



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2.64 Hz, 1H), 7.12 (td, J = 8.34, 2.64 Hz, 1H), 6.19 (s, 1H), 4.80 (d, J = 16.31 Hz, 1H), 4.61 (d, J = 16.31 Hz, 1H), 4.19 (br t, J = 5.77 Hz, 1H), 3.80 (ddd, J = 12.67, 8.41, 3.76 Hz, 1H), 3.65 (s, 3H), 3.35−3.42 (m, 1H), 2.16−2.35 (m, 2H), 1.86−2.07 (m, 2H), 1.77 (br t, J = 5.77 Hz, 2 H). (2S)-1-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]piperazine-2-carboxylic Acid (24). MS: calcd (MH+) 494, measured (MH+) 494. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.07−8.22 (m, 2H), 7.58 (dd, J = 8.8, 6.0 Hz, 1H), 7.34 (dd, J = 8.5, 2.5 Hz, 1H), 7.15 (td, J = 8.3, 2.6 Hz, 1H), 6.30 (s, 1H), 4.45 (d, J = 16.6 Hz, 1H), 4.20 (d, J = 16.8 Hz, 1H), 4.00 (t, J = 4.6 Hz, 1H), 3.54−3.69 (m, 5H), 3.16−3.41 (m, 3H), 2.95 (dt, J = 13.6, 4.4 Hz, 1H). (2S)-4-Acetyl-1-[[(4R)-4-(2-chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]piperazine-2-carboxylic Acid (25). MS: calcd (MH+) 536, measured (MH+) 536. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.98−8.15 (m, 2H), 7.58 (dd, J = 8.8, 6.0 Hz, 1H), 7.32 (dd, J = 8.5, 2.5 Hz, 1H), 7.14 (td, J = 8.4, 2.3 Hz, 1H), 6.26 (s, 1H), 4.65−4.81 (m, 1H), 4.51−4.61 (m, 1H), 4.19−4.46 (m, 2H), 3.75−4.17 (m, 2H), 3.66 (s, 3H), 3.38− 3.59 (m, 2H), 2.96−3.13 (m, 1H), 2.18 (d, J = 16.6 Hz, 3H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]thiomorpholine-3carboxylic Acid (26). The synthetic procedure was described in the Supporting Information. MS: calcd (MH+) 511, measured (MH+) 511. 1 H NMR (methanol-d4, 400 MHz): δ ppm 8.02−8.09 (m, 1H), 7.86− 8.00 (m, 1H), 7.46−7.60 (m, 1H), 7.24−7.36 (m, 1H), 6.99−7.19 (m, 1H), 6.12−6.31 (m, 1H), 4.80−4.87 (m, 1H), 4.38−4.52 (m, 2H), 3.80−3.99 (m, 1H), 3.66 (s, 3H), 3.36−3.51 (m, 2H), 3.10−3.29 (m, 2H), 2.76−2.89 (m, 1 H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-ethoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (27). MS: calcd (MH+) 509, measured (MH+) 509. HRMS: calcd (MH+) 509.1056, measured (MH+) 509.1049. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.08 (d, J = 3.0 Hz, 1H), 8.01 (d, J = 3.3 Hz, 1H), 7.60 (dd, J = 5.9, 8.7 Hz, 1H), 7.32 (dd, J = 2.5, 8.8 Hz, 1H), 7.15 (dt, J = 2.6, 8.3 Hz, 1H), 6.26 (s, 1H), 4.88 (d, J = 16.3 Hz, 1H), 4.59 (d, J = 16.6 Hz, 1H), 4.34−4.27 (m, 1H), 4.22 (d, J = 4.0 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 4.06−3.92 (m, 2H), 3.78 (ddd, J = 3.6, 8.4, 12.5 Hz, 1H), 3.27 (td, J = 3.7, 12.9 Hz, 1H), 1.16 (t, J = 7.2 Hz, 3H). (3S)-4-[[4-(2-Chloro-4-fluoro-phenyl)-5-propoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (28). MS: calcd (MH+) 523, measured (MH+) 523. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.98−7.96 (m, 1H), 7.79 (d, J = 3.0 Hz, 1H), 7.53−7.44 (m, 1H), 7.28−7.22 (m, 1H), 7.11−7.04 (m, 1H), 6.19 (s, 1H), 4.59−4.37 (m, 1H), 4.35−4.18 (m, 1H), 4.15−3.86 (m, 6H), 3.65 (br d, J = 19.3 Hz, 1H), 3.47−3.35 (m, 1H), 2.78 (br s, 1H), 1.63−1.51 (m, 2H), 0.84−0.77 (m, 3H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-isopropoxycarbonyl2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (29). MS: calcd (MH+) 523, measured (MH+) 523. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 3.0 Hz, 1H), 7.80 (d, J = 2.8 Hz, 1H), 7.49 (dd, J = 6.1, 8.7 Hz, 1H), 7.26 (dd, J = 2.6, 8.7 Hz, 1H), 7.08 (dt, J = 2.6, 8.3 Hz, 1H), 6.17 (s, 1H), 4.96−4.90 (m, 1H), 4.51 (br s, 1H), 4.39−4.19 (m, 1H), 4.19−4.04 (m, 2H), 3.88 (br s, 2H), 3.70 (br s, 1H), 3.36−3.34 (m, 1H), 2.72 (br s, 1H), 1.24 (d, J = 6.3 Hz, 3H), 0.99 (d, J = 6.3 Hz, 3H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-2-thiazol-2-yl-5-(2,2,2trifluoroethoxycarbonyl)-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (30). MS: calcd (MH+) 563, measured (MH+) 563. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.98 (d, J = 3.3 Hz, 1H), 7.77 (d, J = 3.0 Hz, 1H), 7.45 (dd, J = 6.0, 8.5 Hz, 1H), 7.24 (dd, J = 2.6, 8.7 Hz, 1H), 7.06 (dt, J = 2.8, 8.4 Hz, 1H), 6.20 (s, 1H), 4.61−4.44 (m, 2H), 4.35 (br s, 1H), 4.20 (br s, 1H), 4.06 (br d, J = 4.3 Hz, 1H), 4.09−4.01 (m, 1H), 3.93−3.79 (m, 2H), 3.67−3.51 (m, 2H), 2.66 (br d, J = 17.8 Hz, 1H). (3S)-4-[[5-tert-Butoxycarbonyl-4-(2-chloro-4-fluoro-phenyl)-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (31). MS: calcd (MH+) 537, measured (MH+) 537. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.96 (s, 1H), 7.79(s, 1H), 7.47

4.21 (br d, J = 17.1 Hz, 1H), 3.93−4.09 (m, 2H), 3.78−3.89 (m, 2H), 3.66 (s, 3H), 3.18−3.31 (m, 1H), 2.97−3.10 (m, 1H), 1.58 (s, 3H). (2R,3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]-2-methyl-morpholine-3-carboxylic Acid (17). The synthetic procedure was described in the Supporting Information. MS: calcd (MH+) 509, measured (MH+) 509. HRMS: calcd (MH+) 509.1056, measured (MH+) 509.1054. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.03 (d, J = 3.14 Hz, 1H), 7.90 (d, J = 3.14 Hz, 1H), 7.51 (dd, J = 5.96, 8.72 Hz, 1H), 7.28 (dd, J = 2.64, 8.66 Hz, 1H), 7.11 (dt, J = 2.64, 8.34 Hz,1H), 6.19 (s, 1H), 4.41 (d, J = 16.56 Hz, 1H), 4.20(d, J = 16.44 Hz, 1H), 4.00−4.13 (m, 1H), 3.75−3.98 (m, 3H), 3.63 (s, 3H), 3.51 (d, J = 8.91 Hz, 1H), 3.06 (dt, J = 3.51, 11.80 Hz, 1H), 1.36 (d, J = 6.27 Hz, 3H). (2R,3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]-2-isopropylmorpholine-3-carboxylic Acid (18). The synthetic procedure was described in the Supporting Information. MS: calcd (MH+) 537, measured (MH+) 537. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.01 (d, J = 3.14 Hz, 1H), 7.70−7.95 (m, 1H), 7.50 (dd, J = 5.96, 8.72 Hz, 1H), 7.28 (dd, J = 2.64, 8.78 Hz, 1H), 7.10 (dt, J = 2.70, 8.38 Hz, 1H), 6.02−6.27 (m, 1H), 4.35−4.61 (m, 1H), 4.21−4.35 (m, 1H), 4.11 (td, J = 3.00, 12.20 Hz, 1H), 3.72−3.97 (m, 2H), 3.67−3.71 (m, 1H), 3.65 (s,3H), 3.35−3.46 (m, 1H), 2.95−3.17 (m, 1H), 2.10 (m, 1H), 1.07 (dd, J = 4.83, 6.84 Hz, 6H). Methyl (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine3-carboxylate (19). MS: calcd (MH+) 509, measured (MH+) 509. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 3.0 Hz, 1H), 7.76 (d, J = 3.3 Hz, 1H), 7.42 (dd, J = 8.7, 6.1 Hz, 1H), 7.24 (dd, J = 8.8, 2.8 Hz, 1H), 7.04 (td, J = 8.4, 2.8 Hz, 1H), 6.17 (s, 1H), 4.40 (d, J = 17.8 Hz, 1H), 3.96−4.15 (m, 3H), 3.76−3.88 (m, 4H), 3.57−3.69 (m, 3H), 3.13−3.31 (m, 3H), 2.50 (dt, J = 11.9, 3.9 Hz, 1H). Methyl (4R)-6-[[(3S)-3-Carbamoylmorpholin-4-yl]methyl]-4-(2chloro-4-fluoro-phenyl)-2-thiazol-2-yl-1,4-dihydropyrimidine-5-carboxylate (20). The synthetic procedure was described in the Supporting Information. MS: calcd (MH+) 494, measured (MH+) 494. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 3.01 Hz, 1H), 7.71−7.81 (m, 1H), 7.42 (t, J = 6.76 Hz, 1H), 7.23 (dd, J = 2.32, 8.85 Hz, 1H), 7.06 (dt, J = 2.70, 8.38 Hz, 1H), 6.08−6.20 (m, 1H), 4.08−4.27 (m, 1H), 4.01 (dd, J = 3.26, 11.29 Hz, 1H), 3.67−3.92 (m, 4H), 3.57−3.64 (s, 3H), 3.35−3.43 (m, 1H), 3.00−3.09 (m, 1H), 2.50−2.68 (m, 1H). Methyl (4R)-4-(2-Chloro-4-fluoro-phenyl)-6-[[3-(hydroxymethyl)morpholin-4-yl]methyl]-2-thiazol-2-yl-1,4-dihydropyrimidine-5-carboxylate (21). MS: calcd (MH+) 481 measured (MH+) 481. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.98−8.02 (m, 1H), 7.91 (dd, J = 3.01, 0.75 Hz, 1H), 7.57 (dd, J = 8.66, 5.90 Hz, 1H), 7.30 (dd, J = 8.66, 2.64 Hz, 1H) 7.12 (td, J = 8.41, 2.76 Hz, 1H), 6.19−6.23 (m, 1H), 5.01 (br d, J = 15.56 Hz, 0.5H), 4.85 (d, J = 6.53 Hz, 1H), 4.70 (d, J = 15.56 Hz, 0.5H), 4.05−4.18 (m, 3H), 3.90−4.01 (m, 3H), 3.70−3.79 (m, 2H), 3.67 (d, J = 1.51 Hz, 3H), 3.43−3.53 (m, 1 H). Methyl (4R)-4-(2-Chloro-4- fluoro-phenyl)-6-[[(3R)-3(methanesulfonamidomethyl)morpholin-4-yl]methyl]-2-thiazol-2yl-1,4-dihydropyrimidine-5-carboxylate (22). Compound 22 was prepared in analogue to 10 by using N-[[(3R)-morpholin-3-yl]methyl]methanesulfonamide (22-a, see Supporting Information for its synthesis) instead of (3S)-morpholine-3-carboxylic acid hydrochloride salt. MS: calcd (MH+) 558, measured (MH+) 559. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.19 (br s, 1H), 7.96 (d, J = 3.01 Hz, 1H), 7.76 (d, J = 3.26 Hz, 1H), 7.48 (dd, J = 8.66, 6.15 Hz, 1H), 7.22 (dd, J = 8.78, 2.51 Hz, 1H), 7.04 (td, J = 8.47, 2.64 Hz, 1H), 6.18 (s, 1H), 4.13−4.34 (m, 2H), 3.93 (dd, J = 11.42, 2.89 Hz, 1H), 3.77− 3.83 (m, 2H), 3.72 (dd, J = 11.54, 6.78 Hz, 1H), 3.63 (s, 3H), 3.36− 3.46 (m, 2H), 2.98 (s, 3H), 2.85−2.92 (m, 1H), 2.80 (dq, J = 6.68, 3.38 Hz, 1H), 2.53−2.62 (m, 1 H). (2S)-1-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]piperidine-2-carboxylic Acid (23). MS: calcd (MH+) 493 measured (MH+) 493. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.99 (d, J = 3.26 Hz, 1H), 7.89 (d, J = 3.01 Hz, 1H), 7.55 (dd, J = 8.78, 6.02 Hz, 1H), 7.28 (dd, J = 8.66, 3366



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J = 8.66, 5.90 Hz, 1H), 7.39 (dd, J = 8.53, 2.51 Hz, 1H), 7.20 (td, J = 8.34, 2.64 Hz, 1H), 6.27 (s, 1H), 4.18−4.29 (m, 2H), 4.07 (dd, J = 11.42, 3.64 Hz, 1H), 3.85−3.94 (m, 2H), 3.73−3.81 (m, 1H), 3.63− 3.71 (m, 4H), 3.21 (ddd, J = 11.98, 5.84, 3.01 Hz, 1H), 2.67−2.79 (m, 4 H). (3S)-4-[(4R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2(4-methyl-thiazol-2-yl)-3,6-dihydro-pyrimidin-4-ylmethyl]-morpholine-3-carboxylic Acid (40). Compound 40 was prepared in analogy to 10 using 4-methyl-thiazole-2-carbonitrile instead of thiazole-2carbonitrile. MS: calcd (MH+) 509, measured (MH+) 509. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.51−7.59 (m, 2H), 7.28−7.34 (m, 1H), 7.09−7.18 (m, 1H), 6.19−6.26 (m, 1H), 4.71−4.79 (m, 1H), 4.44−4.53 (m, 1H), 4.08−4.23 (m, 3H), 3.90−4.04 (m, 2H), 3.66 (s, 4H), 3.07−3.18 (m, 1H), 2.51 (s, 3 H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-2-(4-cyclopropylthiazol-2-yl)-5-methoxycarbonyl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (41). Compound 41 was prepared in analogy to 10 by using 4-cyclopropyl-thiazole-2-carboxamidine (41-a, see Supporting Information for its synthesis) instead of thiazole-2carboxamidine 10A-1. MS: calcd (MH+) 535, measured (MH+) 535. 1 H NMR (methanol-d4, 400 MHz): δ ppm 7.61−7.64 (m, 1H), 7.53− 7.61 (m, 1H), 7.29−7.34 (m, 1H), 7.10−7.19 (m, 1H), 6.24 (s, 1H), 4.57−4.70 (m, 1H), 4.39−4.52 (m, 1H), 4.04−4.24 (m, 3H), 3.83− 4.04 (m, 2H), 3.66 (s, 3H), 3.51−3.63 (m, 1H), 3.01−3.15 (m, 1H), 2.12−2.27 (m, 1H), 0.90−1.11 (m, 4H). (4S)-4-[(4R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2(4-trifluoromethyl-thiazol-2-yl)-3,6-dihydropyrimidin-4-ylmethyl]morpholine-3-carboxylic Acid (42). Compound 42 was prepared in analogy to 41 by using 2-bromo-4-trifluoromethyl-thiazole (CAS: 41731-39-9) instead of 2-bromo-4-cyclopropyl-thiazole 41-c (see Supporting Information). Melting point: 122−125 °C. MS: calcd (MH+) 563, measured (MH+) 563. HRMS: calcd (MH+) 563.0774, measured (MH+) 563.0769. 1H NMR (methanol, 400 MHz): δ ppm 8.46 (s, 1H), 7.53 (dd, J = 8.5, 6.0 Hz, 1H), 7.28 (dd, J = 8.7, 2.6 Hz, 1H), 7.10 (td, J = 8.4, 2.8 Hz, 1H), 6.21 (s, 1H), 4.48 (d, J = 16.6 Hz, 1H), 4.11−4.30 (m, 3H), 3.90−4.05 (m, 2H), 3.73 (ddd, J = 12.7, 8.4, 3.8 Hz, 2H), 3.65 (s, 3H), 3.07−3.23 ppm (m, 1H). (3S)-4-[[(4S)-5-Ethoxycarbonyl-4-(4-fluorophenyl)-2-thiazol-2-yl1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (43). MS: calcd (MH+) 475, measured (MH+) 475. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.00 (d, J = 3.0 Hz, 1H), 7.84 (d, J = 3.0 Hz, 1H), 7.45−7.38 (m, 2H), 7.07 (t, J = 8.8 Hz, 2H), 5.71 (s, 1H), 4.58 (br d, J = 16.1 Hz, 1H), 4.31 (br s, 1H), 4.21−4.05 (m, 4H), 3.99−3.83 (m, 2H), 3.71 (br s, 1H), 3.54−3.41 (m, 1H), 2.85 (br s, 1H), 1.23 (t, J = 7.2 Hz, 3H). (3S)-4-[[(4R)-4-(2,4-Difluorophenyl)-5-ethoxycarbonyl-2-thiazol2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (44). MS: calcd 493 (MH+), exp 493 (MH+). 1H NMR (methanol-d4, 400 MHz): δ ppm 7.98 (d, J = 2.8 Hz, 1H), 7.79 (d, J = 2.8 Hz, 1H), 7.50−7.36 (m, 1H), 7.04−6.87 (m, 2H), 6.02−5.93 (m, 1H), 4.38−4.19 (m, 2H), 4.17−4.00 (m, 4H), 3.97−3.78 (m, 3H), 3.58 (br s, 1H), 2.71 (br s, 1H), 1.19 (t, J = 7.0 Hz, 3H). (3S)-4-[[(4R)-4-(2-Bromo-4-fluoro-phenyl)-5-ethoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (45). MS: calcd (MH+) 554, measured (MH+) 555. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.89 (d, J = 3.0 Hz, 1H), 7.55 (d, J = 3.0 Hz, 1H), 7.33−7.42 (m, 2H), 6.97−7.09 (m, 1H), 6.21 (s, 1H), 4.36 (d, J = 16.1 Hz, 1H), 3.97−4.18 (m, 5H), 3.92 (d, J = 6.0 Hz, 1H), 3.78−3.86 (m, 1H), 3.70 (br s, 1H), 3.26 (br s, 1H), 2.76 (br s, 1H), 1.16 (t, J = 7.0 Hz, 3H). (3S)-4-[[(4R)-4-(2-Bromo-4-fluoro-phenyl)-5-methoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (46). MS: calcd (MH+) 539 exp (MH+) 539. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 3.01 Hz, 1H), 7.78 (d, J = 3.01 Hz, 1H), 7.47−7.40 (m, 2H), 7.14−7.09 (m, 1H), 6.14 (s, 1H), 4.52 (d, J = 16.31 Hz, 1H), 4.15 (d, J = 16.06 Hz, 1H), 4.07 (t, J = 8.16 Hz, 1H), 3.61−3.73 (m, 4H), 3.21−3.31 (m, 1H), 2.78− 2.94 (m, 1H), 2.48−2.66 (m, 1H). (3S)-4-[[(4S)-5-Ethoxycarbonyl-4-(4-fluoro-2-methyl-phenyl)-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (47). MS: calcd (MH+) 489, measured (MH+) 489.

(m, 1H) 7.27 (m, 1H), 7.09 (m, 1H), 6.12 (s, 1H), 4.37−4.58 (m, 2 Hz), 3.99−4.23 (m, 2H), 3.80−3.97 (m, 2H), 3.68 (m, 1H), 3.45(m, 1H) 2.82 (m, 1H), 1.35 (s, 9H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-(2-methoxyethoxycarbonyl)-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (32). The synthetic procedure was described in the Supporting Information. MS: calcd (MH+) 539, measured (MH+) 539. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 3.3 Hz, 1H), 7.79 (d, J = 3.0 Hz, 1H), 7.48 (dd, J = 6.3, 8.8 Hz, 1H), 7.25 (dd, J = 2.5, 8.8 Hz, 1H), 7.07 (dt, J = 2.8, 8.4 Hz, 1H), 6.20 (s, 1H), 4.50 (br s, 1H), 4.39−4.22 (m, 1H), 4.22−4.06 (m, 4H), 3.97−3.81 (m, 2H), 3.68 (br s, 1H), 3.55−3.48 (m, 2H), 3.48−3.35 (m, 1H), 3.29 (s, 3H), 2.77 (br s, 1H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-(ethylcarbamoyl)-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (33). The synthetic procedure was described in the Supporting Information. MS: calcd (MH+) 508, measured (MH+) 508. 1 H NMR (methanol-d4, 400 MHz): δ ppm 7.96 (d, J = 3.14 Hz, 1H), 7.79 (br d, J = 2.89 Hz, 1H), 7.49 (dd, J = 6.15, 8.53 Hz, 1H), 7.25 (dd, J = 2.51, 8.66 Hz, 1H), 7.09 (dt, J = 2.64, 8.28 Hz, 1H), 6.13 (s, 1H), 4.05−4.31 (m, 2H), 4.01 (br d, J = 8.91 Hz, 1H), 3.81 (br s, 3H), 3.61 (br s, 1H), 3.02−3.29 (m, 3H), 2.70(m, 1H), 1.09 (t, J = 7.22 Hz, 3H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-cyano-2-thiazol-2yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (34). The synthetic procedure was described in the Supporting Information. MS: calcd (MH+) 462, measured (MH+) 462. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.01 (d, J = 4.0 Hz, 1H), 7.80 (br d, J = 3.01 Hz, 1H), 7.53 (br dd, J = 5.96, 8.60 Hz, 1H), 7.32 (br dd, J = 2.57, 8.72 Hz, 1H), 7.18−7.22 (m, 1H), 5.93 (s, 1H), 4.02(m, 2H), 3.71−3.90 (m, 3H), 3.44−3.51 (m, 2H), 2.97 (m, 1H), 2.51 (m, 1H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-2-(3,5-difluoro-2-pyridyl)-5-methoxycarbonyl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (35). MS: calcd (MH+) 525, measured (MH+) 525. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.51 (s, 1H), 8.47 (s, 2H), 7.73−7.72 (m, 1H), 7.50−7.46 (m, 1H), 7.25−7.22 (m, 1H), 7.08−7.04 (m, 1H), 6.22 (s, 1H), 4.24 (d, 1H, J = 16 Hz), 4.12 (d, 1H, J = 16 Hz), 4.07−4.04 (m, 1H), 3.89−3.79 (m, 3H), 3.62 (s, 3H), 3.47−3.44 (m, 1H), 3.25−3.19 (m, 1H), 2.67−2.65 (m, 1H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2oxazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (36). MS: calcd (MH+) 479 measured (MH+) 479. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.24 (s, 1H), 7.52−7.59 (m, 2H), 7.32 (dd, J = 8.53, 2.51 Hz, 1H), 7.15 (td, J = 8.28, 2.51 Hz, 1H), 6.25 (s, 1H), 4.40−4.51 (m, 2H), 4.15−4.21 (m, 1H), 3.85−4.09 (m, 4H), 3.66 (s, 3H), 3.50 (ddd, J = 12.23, 5.71, 3.39 Hz, 1H), 2.97 (ddd, J = 12.17, 6.53, 3.89 Hz, 1H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2pyrimidin-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (37). MS: calcd (MH+) 490, measured (MH+) 490. 1H NMR (methanol-d4, 400 MHz): δ ppm 9.14 (d, J = 4.8 Hz, 2H), 7.86 (t, J = 4.9 Hz, 1H), 7.67 (dd, J = 5.8, 8.8 Hz, 1H), 7.37 (dd, J = 2.5, 8.5 Hz, 1H), 7.20 (dt, J = 2.6, 8.3 Hz, 1H), 6.42 (s, 1H), 4.20 (br d, J = 4.3 Hz, 2H), 4.11 (dd, J = 3.3, 11.3 Hz, 1H), 4.05−3.95 (m, 1H), 3.91−3.85 (m, 1H), 3.79 (td, J = 3.4, 11.5 Hz, 1H), 3.70 (s, 3H), 3.37 (s, 1H), 3.18 (ddd, J = 2.8, 5.9, 8.9 Hz, 1H), 2.71−2.62 (m, 1H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2(1-methylimidazol-2-yl)-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (38). MS: calcd (MH+) 492, measured (MH+) 492. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.40 (dd, J = 6.1, 8.7 Hz, 1H), 7.31−7.24 (m, 2H), 7.12−7.02 (m, 2H), 6.19 (s, 1H), 4.55−4.44 (m, 1H), 4.37−4.25 (m, 1H), 4.10 (dd, J = 4.3, 7.5 Hz, 2H), 3.99 (s, 3H), 3.92−3.77 (m, 2H), 3.71 (br t, J = 4.0 Hz, 1H), 3.64 (s, 3H), 3.47−3.37 (m, 1H), 2.75 (br s, 1H). (3S)-4-[[(4R)-4-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2(5-methyloxazol-4-yl)-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (39). Compound 39 was prepared in analogy to 10 by using 5-methyl-oxazole-4-carboxamidine (39-a, see Supporting Information for its synthesis) instead of thiazole-2carboxamidine 10A-1. MS: calcd (MH+) 493, measured (MH+) 493. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.32 (s, 1H), 7.57 (dd, 3367



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H NMR (methanol-d4, 400 MHz): δ ppm 7.96 (d, J = 3.1 Hz, 1H), 7.78 (d, J = 3.0 Hz, 1H), 7.33 (dd, J = 8.6, 5.8 Hz, 1H), 6.77−6.97 (m, 2H), 5.94 (s, 1H), 4.50 (br d, J = 16.1 Hz, 1H), 4.31 (br s, 1H), 4.04−4.18 (m, 3H), 3.76−3.98 (m, 2H), 3.67 (br s, 1H), 2.62 (s, 3H), 1.16 ppm (t, J = 7.1 Hz, 3H). (3S)-4-[[(4S)-4-(4-Fluoro-2-methyl-phenyl)-5-methoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (48). MS: calcd (MH+) 475, measured (MH+) 475. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.96 (d, J = 3.3 Hz, 1H), 7.79 (br d, J = 2.8 Hz, 1H), 7.23−7.41 (m, 1H), 6.79−6.99 (m, 2H), 5.93 (s, 1H), 4.22−4.62 (m, 2H), 4.01−4.17 (m, 2H), 3.67−3.94 (m, 3H), 3.63 (s, 2H), 3.26−3.4 (m, 3H), 2.61 (m, 3H). (3S)-4-[[4-[4-Fluoro-2-(trifluoromethyl)phenyl]-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine3-carboxylic Acid (49). MS: calcd (MH+) 529, measured (MH+) 529. 1 H NMR (methanol-d4, 400 MHz): δ ppm 7.92−8.00 (m, 1H), 7.60− 7.82 (m, 2H), 7.49 (br d, J = 8.8 Hz, 1H), 7.37 (br s, 1H), 6.11 (s, 1H), 4.12 (br s, 2H), 3.87 (br s, 3H), 3.57 (s, 3H), 3.34 (m, 2H), 2.68 (br s, 2H). (3S)-4-[[(4S)-4-(2-Cyclopropyl-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine3-carboxylic Acid (50). MS: calcd (MH+) 501 measured (MH+) 501. 1 H NMR (methanol-d4, 400 MHz): δ ppm 8.01 (d, J = 3.26 Hz, 1H), 7.88 (d, J = 3.01 Hz, 1H), 7.36 (dd, J = 8.53, 5.77 Hz, 1H), 6.89 (td, J = 8.47, 2.64 Hz, 1H), 6.80 (dd, J = 10.67, 2.64 Hz, 1H), 6.36 (s, 1H), 4.67 (d, J = 16.81 Hz, 1H), 4.40 (br d, J = 16.81 Hz, 1H), 4.09−4.22 (m, 2H), 3.86−4.02 (m, 3H), 3.65 (s, 3H), 3.53−3.61 (m, 1H), 2.97 (br d, J = 11.29 Hz, 1H), 2.43−2.52 (m, 1H), 1.04−1.15 (m, 2H), 0.87−0.96 (m, 1H), 0.67−0.74 (m, 1H). (3S)-4-[[(4R)-4-(2-Chlorophenyl)-5-ethoxycarbonyl-2-thiazol-2-yl1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (51). MS: calcd (MH+) 491, measured (MH+) 491. 1H NMR (methanol-d4, 400 MHz): δ ppm 10.00 (br, 1H), 8.02 (d, J = 3.3 Hz, 1H), 7.93 (d, J = 3.0 Hz, 1H), 7.55−7.35 (m, 2H), 7.28 (dt, J = 2.0, 7.4 Hz, 2H), 6.09 (s, 1H), 4.32−3.77 (m, 6H), 3.77−3.45 (m, 3H), 3.14− 2.97 (m, 1H), 2.41 (d, J = 12.3 Hz, 1H), 1.05 (t, J = 7.2 Hz, 3H). (3S)-4-[[(4R)-4-(2-Chloro-4-methyl-phenyl)-5-ethoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (52). MS: calcd (MH+) 505, measured (MH+) 505. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.00 (d, J = 3.3 Hz, 1H), 7.86 (d, J = 3.3 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.28 (s, 1H), 7.13 (br d, J = 8.0 Hz, 1H), 6.19 (s, 1H), 4.63 (br d, J = 16.6 Hz, 1H), 4.39 (br d, J = 17.6 Hz, 1H), 4.00−4.20 (m, 4H), 3.80−3.98 (m, 3H), 3.47−3.63 (m, 1H), 2.97 (br s, 1H), 2.33 (s, 3H), 1.16 ppm (t, J = 7.2 Hz, 3H). (3S)-4-[[(4R)-4-(2-Chloro-4-methyl-phenyl)-5-methoxycarbonyl2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (53). MS: calcd (MH+) 491, measured (MH+) 491. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 3.3 Hz, 1H), 7.79 (d, J = 3.3 Hz, 1H), 7.22−7.35 (m, 2H), 7.10 (d, J = 8.0 Hz, 1H), 6.15 (s, 1H), 4.27−4.53 (m, 2H), 4.06−4.15 (m, 2H), 3.83−3.98 (m, 2H), 3.67−3.75 (m, 1H), 3.62 (s, 3H), 3.33 (m, 2H), 2.31 (s, 3H). (3S)-4-[[(4R)-4-(2,4-Dichlorophenyl)-5-ethoxycarbonyl-2-thiazol2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (54). MS: calcd (MH+) 525, measured (MH+) 525. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 3.01 Hz, 1H), 7.78 (d, J = 3.01 Hz, 1H), 7.49 (d, J = 2.01 Hz, 1H), 7.44 (d, J = 8.28 Hz, 1H), 7.32 (dd, J = 8.28, 2.01 Hz, 1H), 6.18 (s, 1H), 4.33−4.49 (m, 1H), 4.17−4.30 (m, 1H), 4.07 (d, J = 7.03 Hz, 5H), 3.78−3.96 (m, 2H), 3.61 (br s, 1H), 2.62−2.79 (m, 1H), 1.16 (t, J = 7.15 Hz, 3 H). (3S)-4-[[(4R)-4-(4-Bromo-2-chlorophenyl)-5-ethoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (55). MS: calcd (MH+) 569, measured (MH+) 569. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.98 (d, J = 3.0 Hz, 1H), 7.80 (br s, 1H), 7.64 (br d, J = 1.8 Hz, 1H), 7.23−7.52 (m, 2H), 6.18 (s, 1H), 4.82 (br, 1H), 3.97−4.27 (m, 4H), 3.61−3.96 (m, 3H), 3.17− 3.30 (m, 3H), 1.16 ppm (t, J = 7.2 Hz, 3H). (3S)-4-[[4-[2-Chloro-4-(trifluoromethyl)phenyl]-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine3-carboxylic Acid (56). MS: calcd (MH+) 545, measured (MH+) 545. 1 H NMR (methanol-d4, 400 MHz): δ ppm 8.01 (dd, J = 3.1, 2.1 Hz, 1H), 7.87 (dd, J = 3.1, 1.1 Hz, 1H), 7.78 (s, 1H), 7.55−7.73 (m, 2H), 1

6.29 (s, 1H), 4.69 (br d, J = 16.8 Hz, 1H), 4.33−4.56 (m, 2H), 4.06− 4.23 (m, 1H), 3.81−4.02 (m, 4H), 3.64 (d, J = 1.3 Hz, 2H), 2.99 ppm (br s, 2H). (3S)-4-[[(4S)-4-(4-Cyanophenyl)-5-ethoxycarbonyl-2-thiazol-2-yl1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (57). MS: calcd (MH+) 482, measured (MH+) 482. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.00 (d, J = 3.0 Hz, 1H), 7.82 (d, J = 3.0 Hz, 1H), 7.85−7.78 (m, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.58 (d, J = 8.3 Hz, 2H), 5.51 (s, 2H), 4.41 (br d, J = 11.5 Hz, 1H), 4.24−4.09 (m, 2H), 4.06 (br s, 2H), 3.95−3.78 (m, 2H), 3.64−3.54 (m, 1H), 2.75− 2.60 (m, 1H), 1.23 (t, J = 7.2 Hz, 3H). (3S)-4-[[(4S)-5-Ethoxycarbonyl-4-(4-methoxyphenyl)-2-thiazol-2yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (58). MS: calcd (MH+) 516, measured (MH+) 516. 1H NMR (CDCl3): δ ppm 7.90−7.94 (m, 1H), 7.58−7.69 (m, 3H), 7.34−7.43 (m, 1H), 6.29−6.37 (m, 1H), 4.30−4.58 (m, 7H), 4.18 (br s, 1H), 4.02−4.12 (m, 3H), 3.82−3.97 (m, 3H), 3.30−3.49 (m, 1H), 2.91 (d, J = 12.8 Hz, 1H), 1.13 (td, J = 7.0, 5.0 Hz, 3H). (S)-4-[(R)-6-(2-Chloro-3-fluorophenyl)-5-methoxycarbonyl-2thiazol-2-yl-3,6-dihydropyrimidin-4-ylmethyl]-morpholine-3-carboxylic Acid (59). MS: calcd (MH+) 495, measured (MH+) 495. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 2.76 Hz, 1H), 7.78 (br s, 1H), 7.24−7.38 (m, 2H), 7.10−7.21 (m, 1H), 6.23 (s, 1H), 4.40 (m, 1H), 4.25 (m, 1H), 4.07 (m, 2H), 3.87 (m, 2H), 3.62 (m, 4H), 2.70 (m, 2H). (3S)-4-[[(4R)-4-(2-Chloro-3-fluorophenyl)-5-ethoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (60). MS: calcd (MH+) 509, measured (MH+) 509. HRMS: calcd (MH+) 509.1056, measured (MH+) 509.1053. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.05 (d, J = 3.01 Hz, 1H), 7.95 (d, J = 3.26 Hz, 1H), 7.33−7.42 (m, 2H), 7.22−7.29 (m, 1H), 6.29 (s, 1H), 4.83 (d, J = 16.56 Hz, 1H), 4.52 (d, J = 16.56 Hz, 1H), 4.15− 4.27 (m, 3H), 4.10 (q, J = 6.94 Hz, 2H), 3.92−4.06 (m, 2H), 3.72 (ddd, J = 12.55, 8.28, 4.02 Hz, 1H), 3.11−3.21 (m, 1H), 1.15 (t, J = 7.15 Hz, 3H). (3S)-4-[[(4R)-4-(2-Bromo-3-fluorophenyl)-5-ethoxycarbonyl-2thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (61). MS: calcd (MH+) 554, measured (MH+) 553 and 555. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.93−8.00 (m, 1H), 7.78 (d, J = 3.26 Hz, 1H), 7.32−7.40 (m, 1H), 7.25−7.30 (m, 1H), 7.14 (td, J = 8.22, 1.38 Hz, 1H), 6.25 (s, 1H), 4.50 (d, J = 17.32 Hz, 1H), 4.26 (d, J = 11.54 Hz, 1H), 4.00−4.16 (m, 4H), 3.81−3.96 (m, 2H), 3.68 (br s, 1H), 3.39 (br s, 1H), 2.75 (br s, 1H), 1.14 (t, J = 7.03 Hz, 3H). (3S)-4-[[(4R)-4-(2,3-Dichlorophenyl)-5-ethoxycarbonyl-2-thiazol2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (62). MS: calcd (MH+) 525, measured (MH+) 525. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.98 (br s, 1H), 7.78 (br s, 1H), 7.53−7.36 (m, 2H), 7.34−7.21 (m, 1H), 6.26 (br s, 1H), 4.30−3.66 (m, 9H), 3.12−3.00 (m, 2H), 1.44−1.28 (m, 3H). (3S)-4-[[4-(2-Chloro-3-cyanophenyl)-5-ethoxycarbonyl-2-thiazol2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (63). MS: calcd (MH+) 516, measured (MH+) 516. 1H NMR (CDCl3, 400 MHz): δ ppm 7.90−7.94 (m, 1H), 7.58−7.69 (m, 3H), 7.34−7.43 (m, 1H), 6.29−6.37 (m, 1H), 4.30−4.58 (m, 7H), 4.18 (br s, 1H), 4.02−4.12 (m, 3H), 3.82−3.97 (m, 3H), 3.30−3.49 (m, 1H), 2.91 (d, J = 12.8 Hz, 1H), 1.13 (td, J = 7.0, 5.0 Hz, 3H). (3S)-4-[[(4R)-4-(2-Chloro-3,4-difluorophenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3carboxylic Acid (64). MS: calcd (MH+) 513, measured (MH+) 513. 1 H NMR (methanol-d4, 400 MHz): δ ppm 8.04 (d, J = 3.3 Hz, 1H), 7.93 (d, J = 3.0 Hz, 1H), 7.34−7.41 (m, 1H), 7.24−7.33 (m, 1H), 6.22 (s, 1H), 4.84 (d, J = 16.6 Hz, 1H), 4.51 (d, J = 16.6 Hz, 1H), 4.15− 4.26 (m, 3H), 3.91−4.05 (m, 2H), 3.72 (ddd, J = 12.6, 8.5, 3.8 Hz, 1H), 3.65 (s, 3H), 3.13−3.21 (m, 1H). (3S)-4-[[(4R)-4-(2-Chloro-3,4-difluoro-phenyl)-5-ethoxycarbonyl2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (65). MS: calcd 527 (MH+), exp 527 (MH+). 1H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 3.3 Hz, 1H), 7.78 (d, J = 3.0 Hz, 1H), 7.35−7.19 (m, 2H), 6.18 (s, 1H), 4.48 (d, J = 17.3 Hz, 1H), 4.31−4.19 (m, 1H), 4.16−4.01 (m, 4H), 3.97−3.82 (m, 2H), 3368



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3.67 (br s, 1H), 3.36 (d, J = 6.0 Hz, 1H), 2.80−2.63 (m, 1H), 1.16 (t, J = 7.0 Hz, 3H). (3S)-4-[[(4R)-4-(2-Bromo-3,4-difluorophenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3carboxylic Acid (66). Compound 66 was prepared in analogue to 10 by using 2-bromo-3,4-difluoro-benzaldehyde (66-a, see Supporting Information for its synthesis) instead of 2-chloro-4-fluorobenzaldehyde. MS: calcd (MH+) 557, measured (MH+) 557. 1H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 3.0 Hz, 1H), 7.70−7.83 (m, 1H), 7.17−7.37 (m, 2H), 6.18 (s, 1H), 4.76 (s, 2H), 4.48 (br s, 1H), 3.98−4.16 (m, 2H), 3.72−3.98 (m, 2H), 3.53−3.72 (m, 3H), 2.69 (br s, 2H). (3S)-4-[[(4R)-4-(2-Bromo-3,4-difluorophenyl)-5-ethoxycarbonyl2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (67). MS: calcd (MH+) 571 measured (MH+) 571. 1H NMR (methanol-d4, 400 MHz): δ ppm 10.01 (br s, 1H), 8.03 (d, J = 3.0 Hz, 1H), 7.94 (d, J = 3.3 Hz, 1H), 7.40−7.49 (m, 1H), 7.25 (ddd, J = 8.8, 5.3, 1.9 Hz, 1H), 6.05 (s, 1H), 4.17−4.26 (m, 1H), 4.05 (d, J = 17.3 Hz, 1H), 3.95 (q, J = 7.0 Hz, 3H), 3.79−3.86 (m, 1H), 3.68 (td, J = 7.8, 3.4 Hz, 2H), 3.56 (br s, 1H), 3.02−3.12 (m, 1H), 2.36− 2.45 (m, 1H), 1.05 (t, J = 7.0 Hz, 3H). (3S)-4-[[(4S)-4-(3,4-Difluoro-2-methyl-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3carboxylic Acid (68). Melting point: 138−140 °C. MS: calcd (MH+) 493, measured (MH+) 493. HRMS: calcd (MH+) 493.1352, measured (MH+) 493.1344. 1H NMR (methanol-d4, 400 MHz): δ ppm 8.05 (d, J = 3.0 Hz, 1H), 7.95 (d, J = 3.0 Hz, 1H), 7.23−7.32 (m, 1H), 7.03− 7.15 (m, 1H), 5.99 (s, 1H), 4.75 (d, J = 16.8 Hz, 1H), 4.46 (d, J = 16.6 Hz, 1H), 4.06−4.24 (m, 3H), 3.84−4.03 (m, 2H), 3.59−3.72 (m, 3H), 2.99−3.19 (m, 2H), 2.53 (d, J = 2.3 Hz, 3H). (3S)-4-[[(4S)-4-(3,4-Difluoro-2-methyl-phenyl)-5-ethoxycarbonyl2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic Acid (69). MS: calcd 507 (MH+), measured 507(MH+). 1H NMR (methanol-d4, 400 MHz): δ ppm 7.97 (d, J = 3.3 Hz, 1H), 7.79 (br s, 1H), 6.97−7.24 (m, 2H), 5.93 (s, 1H), 4.21−4.60 (m, 3H), 4.09 (q, J = 7.1 Hz, 3H), 3.87 (br s, 2H), 3.66 (br s, 1H), 2.79 (br s, 2H), 2.56 (d, J = 2.5 Hz, 3H), 1.16 (t, J = 7.0 Hz, 3H). Biology General Comments. Cells and Culture Conditions. HepG2.2.15 and HepDE19 are stably transfected cell lines containing the HBV genome. Both cell lines are derived from the hepatoblastoma cell line Hep G2 (American Type Culture Collection, ATCC HB-8065) by the published procedures.31,32 Both cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)-F12 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.5 mg/mL of G418. While HepG2.2.15 cells constitutively support HBV replication and production of virus particles, HepDE19 cells are inducible by tetracycline. Addition of 1 μg/mL tetracycline in culture medium suppresses HBV replication in HepDE19 cells, whereas switching to tetracycline-free medium resumes this process. Anti-HBV Activity in Vitro. HepG2.2.15 cells were seeded into 96-well plates (3 × 104 cells in 100 μL of media per well) and incubated overnight at 37 °C. The test compounds were serially halflog diluted in DMSO, then diluted 100 times in culture media. Then 100 μL of diluted compounds were added into the plates to reach 0.5% final concentration of DMSO in every well. Five days after compound treatment, culture supernatant was collected for further analysis. For quantitative PCR detection of extracellular HBV DNA, 100 μL of culture supernatant was collected and processed in MagNA Pure 96 nucleic acid purification system (Roche Applied Science) for viral DNA extraction. The extracted samples were subjected to HBV DNA quantification by qPCR. The effective compound concentration at which HBV replication is inhibited by 50% (EC50) was determined. Cytotoxicity and Selectivity Index. HepDE19 cells were seeded into 96-well plates (5 × 103 cells per well) and treated with compounds for CC50 determination. Five days after treatment, cell viability was measured by addition of 20 μL of CCK-8 reagent. Two hours after incubation at 37 °C, the absorbance at wavelengths of 450 and 630 nm (OD450 and OD630) was recorded by a plate reader. The concentration results in the death of 50% of the host cells (CC50) of each compound were determined.

The relative effectiveness of the compound in inhibiting viral replication compared to inducing cell death was defined as the selectivity index (CC50 value/EC50 value). On the basis of CC50 and EC50 data, selectivity indexes were determined. LYSA Solubility Assay. Samples are prepared in duplicate from 10 mM DMSO stock solution. After evaporation of DMSO with a centrifugal vacuum evaporator, compounds are dissolved in a 0.05 M phosphate buffer (pH 6.5), stirred for 1 h, and shaken for 2 h. After one night, the solutions are filtered using a microtiter filter plate. Then the filtrate and its 1/10 dilution are analyzed by HPLC-UV. In addition, a four-point calibration curve is prepared from the 10 mM stock solutions and used for the solubility determination of the compounds. The results are in μg/mL. In case the percentage of sample measured in solution after evaporation divided by the calculated maximum of sample amount is bigger than 80%, the solubility is reported as bigger than this value. Microsomal Stability Assay. Microsomes were preincubated with test compound for 10 min at 37 °C in 100 mM potassium phosphate buffer, pH 7.4. The reactions were initiated by adding NADPH regenerating system to give a final incubation volume of 400 μL. The incubations contained (finally) 1 μM test compound, 0.5 mg/mL liver microsomal protein, 3 mM glucose 6-phosphate, 1 mM NADP, 3 mM MgCl2, and 0.05 mg/mL glucose 6-phosphate dehydrogenase in 100 mM potassium phosphate buffer, pH 7.4. After incubation times of 0, 3, 6, 9, 15, and 30 min at 37 °C, 50 μL samples were removed and transferred to 150 μL of acetonitrile containing internal standard to terminating the reaction. Following precipitation and centrifugation, the amount of compound remaining in the samples were determined by LC-MS/MS. Parallel Artificial Membrane Permeability Assay. The permeation of drugs was measured using a “sandwich” construction. Sample stock solution (DMSO) was diluted to 150 μM (DMSO%

Discovery and Pre-Clinical Characterization of Third-Generation 4-H

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