Discovery of Benzoazepinequinoline (BAQ) Derivatives as Novel

Article Cite This: J. Med. Chem. 2018, 61, 10228−10241

pubs.acs.org/jmc

Discovery of Benzoazepinequinoline (BAQ) Derivatives as Novel, Potent, Orally Bioavailable Respiratory Syncytial Virus Fusion Inhibitors Xiufang Zheng,† Chungen Liang,† Lisha Wang,† Baoxia Wang,† Yongfu Liu,† Song Feng,† Jim Zhen Wu,† Lu Gao,† Lichun Feng,† Li Chen,† Tao Guo,‡ Hong C. Shen,† and Hongying Yun*,†

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†

Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Building 5, 720 Cailun Road, Shanghai 201203, China ‡ International Discovery Service Unit, Research Service Division, WuXi AppTec (Shanghai) Co., Ltd., Lane 31, Yiwei Road, Waigaoqiao, Shanghai 200131 China S Supporting Information *

ABSTRACT: A novel benzoazepinequnoline (BAQ) series was discovered as RSV fusion inhibitors. BAQ series originated from compound 2, a hit from similarity-based virtual screening. In SAR exploration, benzoazepine allowed modifications in the head moiety. Benzylic sulfonyl on benzoazepine and 6-Me on quinoline were crucial for good anti-RSV activity. Although the basic amine in the head portion was crucial for anti-RSV activity, the attenuated basicity was required to reduce Vss. Introducing oxetane to the head portion led to discovery of compound 1, which demonstrated single-digit nM anti-RSV activity against different RSV strains, reasonable oral exposure in plasma, and 78-fold higher exposure in lung. Compound 1 also displayed 1 log viral reduction in a female BALB/c mice RSV model by b.i.d. oral dosing at 12.5 mg/kg. A single resistant mutant at L138F in fusion protein proved compound 1 to be a RSV fusion inhibitor.

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INTRODUCTION As a negative-sense single-stranded RNA virus, human respiratory syncytial virus (RSV), belongs to the family of Paramyxoviridae.1 The most common cause of human RSV infection is acute upper and lower respiratory tract infection in infants and young children. Almost all children by age of 3 can be infected by RSV at least once.2 RSV infection is normally mild and associated with upper respiratory tract symptoms in normal adults and elder children. However, populations associated with high-risk factors, such as premature birth, congenital heart disease, chronic pulmonary diseases, and immunocompromised conditions, are more prone to mortality and morbidity due to lower respiratory tract infections. Severe RSV infection often leads to bronchiolitis and pneumonia with an increased chance of morbidity or mortality in young children and immunocompromised adults.2,3 Furthermore, the sequela of severe RSV infection at young age is recurrent wheezing or asthma,4 and RSV-related mortality rate becomes higher in high-risk populations.5,6 Although numerous attempts in inactivated subunits and live-attenuated vaccination are under development, there is no © 2018 American Chemical Society

available vaccine for RSV infection. Palivizumab, a humanized monoclonal antibody against RSV fusion protein, was approved for prophylaxis in high-risk infants in 1998. However, palivizumab showed no efficacy in the treatment of established RSV infection.7 Ribavirin, an aerosol formulation comprising ribavirin, is the only approved antiviral therapy for RSV infection. Due to limited efficacy and side effects, it is rarely used in clinic.8 As such, safe and effective therapy for RSV infection is a high unmet medical need. RSV fusion (F) protein is a surface glycoprotein on the viral envelope. The F protein plays a major role for the virus entry into host cells together with the G surface glycoprotein.9 Depletion of the RSV G protein can only reduce the efficiency of entry process but cannot terminate the entry process. Moreover, the RSV F protein also promotes syncytia formation between infected cell and adjacent cells. Inhibition of viral entry and spread by targeting RSV F protein has emerged as a promising treatment for RSV infected patients. Small molecule Received: September 7, 2018 Published: October 19, 2018 10228

DOI: 10.1021/acs.jmedchem.8b01394 J. Med. Chem. 2018, 61, 10228−10241

Journal of Medicinal Chemistry

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Figure 1. Selected RSV fusion inhibitors under development.

from the similarity-based virtual screening approach (Figure 2).

RSV F protein inhibitors have great potential to decrease the duration and severity of respiratory symptoms and the subsequent risk of prolonged hospitalization and complications. In the past, several structurally different and potent RSV F protein inhibitors were reported.10−13 A number of RSV F inhibitors had successfully progressed to preclinical stage, but only a few had entered clinical development (Figure 1).14 JNJ2408068 was a picomolar RSV F inhibitor (EC50 = 0.16 nM) with low cytotoxicity against several lab strains. However, its long tissue retention time in the tested species hindered its further development.11 Another picomolar RSV F inhibitor, TMC-353121, exhibited anti-RSV efficacy in inhibiting RSV replication prophylactically and therapeutically.12 BMS-433771 was the first RSV F inhibitor to demonstrate oral bioavailability13 but was terminated for further development likely due to business reasons. VP-14637, a nanomolar RSV F inhibitor, was formulated and used as an inhaled dry powder product called MDT-637 in phase I trials. This molecule demonstrated good tolerance in healthy human subjects.15 Most recently, GS-5806 achieved proof-of-concept in human RSV challenge studies and showed a significant viral load reduction (4.2 log10) and a significant disease severity reduction.16 In a phase IIa trial with the RSV-A Memphis 37b virus inoculated healthy adults, daily oral dosing JNJ53718678, a picomolar RSV F inhibitor, reduced viral load and clinical severity, which established clinical proof of concept of RSV treatment by using RSV F inhibitor.17 In our endeavors to develop highly potent RSV F inhibitors, we used a similarity-based virtual screening approach and generated several hits as chemistry starting points. In our similarity-based virtual screening, MOS (2D similarity) and ROCS (3D similarity) were used in parallel to screen the Roche Smart library. JNJ-2408068, TMC-353121, and BMS433771 were chosen as reference compounds for the virtual screening. In our previous publication, we reported compound 7 as a validated hit as RSV F inhibitor by this approach.18 In this article, we describe the discovery of benzoazepinequnoline (BAQ) series derived from compound 2, another hit

Figure 2. BAQ series originating from ring expansion of compound 2.

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RESULTS AND DISCUSSION Author: As aforementioned, BAQ series originated from compound 2. We were delighted to see the reasonable antiRSV activity of compound 2 in CPE assay (EC50 = 0.22 μM; Table 1). Further exploration disclosed that ring size reduction to five-membered ring 3 resulted in a total loss of anti-RSV activity and strong cytotoxicity. The anti-RSV activity remained the same with ring expansion to a seven-membered ring (4, EC50 = 0.22 μM).18 To explain the correlation of ring size to anti-RSV activity, we did the conformation analysis (Figure 3). Compound 3 (a five-membered ring analogue) adopted almost a planar conformation. Compound 2 (a six-membered ring analogue) adopted a conformation with nearly 40° dihedral angle between quinoline and tetrahydroisoquinoline. Finally, compound 4 (a seven-membered ring analogue) adopted a conformation with almost 90° dihedral angle between quinoline and benzoazepine. These observations gave us a hint that the active RSV inhibitors may prefer a nonplanar scaffold with a dihedral angle between two ring systems up to 90°. An exploration on the head portion (Rh) of compound 2 revealed a flat SAR (Table 2). All the modifications on Rh resulted in a significant loss of antiviral activity. Furthermore, derivatives with basic terminals (2b, 2d, and 2f) demonstrated better antiviral activity than the nonbasic terminal derivatives (2a, 2c, and 2g). The chain length between the two amino 10229



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Table 1. SAR Results of R2 of the Scaffold Derived from Compound 2a

Table 2. SAR Exploration of the Head Portion (Rh) of the Scaffold Derived from Compound 2a

a

EC50: the concentration of compound that reduced 50% cytopathic effect of RSV Long strain infected HEp-2 cells. CC50: the concentration of compound that manifests cytotoxicity toward 50% of uninfected HEp-2 cells. bTherapeutic index (TI) is the ratio CC50/ EC50.

a

Figure 3. Conformations of 2, 3, and 4 at the lowest energy. Compound 2 is in orange, compound 3 in pink, and compound 4 in green.


groups of Rh also impacted on activity. For example, twocarbon linker derivatives (2d and 2f) provided better anti-RSV activity than three- and four-carbon linker derivatives (2b and 2e). Further profiling of compound 2 revealed a very low permeability (0 × 10−6 cm/s) in the PAMPA assay, which indicated a very low cellular permeability potential. Thus, compound 2 was not suitable for oral dosing. Considering the flat SAR of the derivatives of compound 2 on Rh and poor permeability of compound 2, we turned our attention to compound 4 (BAQ). Compound 4 showed identical activity and TI to compound 2 (Table 3). However, different from compound 2, compound 4 allowed further modification on Rh without sacrificing potency. For example, compound 5, a close analogue to 2d, showed the identical good activity to compound 4. Moreover, the PAMPA of compound 5 was 0.99, which was categorized as medium to high cell permeability. Subsequently, the rat single-dose pharmacokinetics (SDPK) of compound 5 showed reasonable oral exposure with moderate oral bioavailability (Table 4). Both in vitro rat liver microsomal clearance and in vivo clearance were medium

to high, which needed to be further optimized. The high volume of distribution (Vss) was most likely due to the lipophilic basic nature of compound 5. In the resistant mutation selection study, a single mutant at D331N in RSV F protein was identified in compound 5 resistant virus at passage 7. The anti-RSV activity was dropped from 0.20 μM to 2.3 μM in resistant virus. It proved that compound 5 was targeting on RSV fusion protein. Encouraged by the good potency, reasonable oral absorption, and modeling understanding, we selected compound 5 as a new chemistry starting point for further optimization. Chemistry modification was initiated at R1 on quinoline. Selected data are summarized in Table 5. A Cl-walk on quinolone revealed that 6-position was critical for antiviral potency. SAR exploration at the 6-position disclosed that the 6-Me analogue (5f) demonstrated the best potency and the largest TI, although it still showed strong cytotoxicity like the other R1 derivatives. The cytotoxicity cannot be addressed by optimizing R1 only but was eventually addressed by the combined optimization on both R1 and X, which was described in Table 6. 10230



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Table 3. Head Portion (Rh) Exploration on the Scaffold Derived from Compound 4a

a EC50: the concentration of compound that reduced 50% cytopathic effect of RSV Long strain infected HEp-2 cells. CC50: the concentration of compound that manifests cytotoxicity toward 50% of uninfected HEp-2 cells. bTherapeutic index (TI) is the ratio CC50/EC50.

Table 4. DMPK Profile of Compound 5a compd RLM (mL min−1 kg−1) 5

AUC0−INF (iv 2 mg/kg) (h·ng/mL)

AUC0−INF (po 10 mg/kg) (h·ng/mL)

324

510

23 (medium)

in vivo CL (mL min−1 kg−1) Vss (L/kg) 89 (high)

106

F (%) 32

a

The SDPK study in male Wistar rats was carried out according to the standard procedures described in the Supporting Information. Listed are plasma clearance (CL), volume of distribution at steady state (Vss), area under the curve (AUC), oral bioavailability (F), rat liver microsomal clearance (RLM).

Table 5. SAR Exploration of R1 of the Quinolone Scaffold Derived from Compound 5a

compd

R1

EC50 (μM)a

CC50 (μM)

TIb

5 5a 5b 5c 5d 5e 5f 5g 5h 5i

H 5-Cl 6-Cl 7-Cl 8-Cl 6-OMe 6-Me 6-F 6-CF3 6-SO2Me

0.20 6.6 0.12 >3.2 1.1 0.32 0.073 1.5 1.1 5.6

23 23 >3.2 7.5 25 4.8 9.4 8.0 6.7 13

115 3 >27 2 23 15 129 5 6 2

the carbon adjacent to the terminal amine. All these Met ID results cast lights on how to further optimize compound 5. Our optimization strategy focused on reducing the electronic density of benzene (Table 6). Two approaches were explored. The first was to introduce fluorine to benzene to block a metabolic “hot spot”, which unfortunately failed (6f and 6g). The other approach was to replace the benzyl carbon (X in Figure 2) with heteroatoms, which was proven to be very successful. In particular, introducing the electron withdrawing group SO2 led to discovery of compounds 6a and 6h (R1 = H or Me and X = SO2), which led to significantly improved metabolic stability. The liver microsomal clearance in tested species was low. In addition, compounds 6a and 6h also demonstrated superior anti-RSV activity and good TI compared to other modifications. Compound 6h demonstrated the best anti-RSV activity (0.004 μM) and TI (16,500), as shown in Table 6. In the next round of optimization, the head portion (Rh) was re-explored with the optimal 6-Me and X as SO2 (Table 7). We observed a much broader tolerance of substitution on Rh. The length of alkyl chain between two amino groups was identified to be crucial for antiviral activity. Compound 6h and compound 7a were optimal for excellent anti-RSV activity (EC50 = 0.004 μM and 0.001 μM, respectively). Methyl substitution on either terminal amine (7d and 7e) or alkyl chain (7f−i) reduced the ant-RSV activity. Replacing the terminal amine by heterocycles (from 7j to 7m) significantly reduced the anti-RSV activity. Oxetanylation of terminal amine (7s) led to much reduced anti-RSV activity. Acetylation of the terminal amine (7p) totally removed the anti-RSV activity. Clearly introducing electron withdrawing group at the terminal amine would most likely reduce anti-RSV activity. In addition, replacing terminal amine with hydroxyl group (7q) resulted in a 10-fold decrease in anti-RSV activity compared to compound 6h.

a


As described in Table 4, compound 5 showed medium to high in vitro and in vivo clearance. On the basis of the metabolic identification (Met ID) study in mouse and rat liver microsomes (Figure 4), the benzoazepine moiety was identified as a metabolic “hot spot” generating 90% of metabolites in rat liver microsomes. In particular, the benzylic position (X in Figure 2) and benzene ring of benzoazepine were highly likely to be accountable for metabolism. In the head moiety, amide and acid were generated by oxidation of 10231



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Table 6. X Optimization Impacts on Both Potency and XLM

a EC50: the concentration of compound that reduced 50% cytopathic effect of RSV Long strain infected HEp-2 cells. CC50: the concentration of compound that manifests cytotoxicity toward 50% of uninfected HEp-2 cells. bTherapeutic index (TI) is the ratio CC50/EC50. cScaled intrinsic clearance of compounds in human, rat, and mouse liver microsomes (HLM, RLM, and MLM).

Figure 4. Met ID of compound 5 in rat and mouse liver microsomes.

7q, the one without ionizable head amine, showed high lipophilicity (mlLogD), good permeability, but low aqueous solubility. On the contrary, all the other terminal amino analogues demonstrated good solubility. In terms of pKb, it was known that oxetane as a carbonyl surrogate can reduce pKb of adjacent amine.20 Introducing oxetane next to the terminal amine in compound 1 led to the reduction of the pKb more than 2 log units compared to any other analogues containing a terminal amino group. The impact of oxetane on the pKb was quickly diminished with the elongated distance to terminal amine such as compound 7r. Compounds 1 and 7r demonstrated high clearance in liver microsomal clearance assay. Except for compound 6h, all the tested analogues showed good permeability in PAMPA assay. In addition, the plasma protein binding of tested compounds was medium to high. In the SDPK study in male Wistar rats (Table 10), compounds 1, 5, 7q, and 7r all demonstrated reasonable oral

It was noteworthy that oxetane analogues (7r and 1) both demonstrated excellent anti-RSV activity at single-digit nM level (EC50 = 0.005 μM and 0.002 μM, respectively). Compound 1 also demonstrated the highest therapeutic index (TI > 50 000) throughout the whole chemical series in the CPE assay. Since compound 1 was identified with the best potency and therapeutic index, it was further profiled against different RSV strains in CPE assay. To our delight, compound 1 demonstrated consistent and high anti-RSV activity against different RSV strains in CPE and plaque reduction assays. Regarding selectivity, compound 1 showed no activity to influenza H1N1 in CPE assay (Table 8), which indicated that it did not target on a broad-antiviral host target. ADME and in Vivo Efficacy Assessment of Selected BAQs. The physicochemical properties and ADME of selected BAQs were listed in Table 9. The mlLogD had correlation with pKb of the head amine except for compound 7q. Compound 10232



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Table 7. SAR Exploration at the Rh Position with Fixed 6-Me and X = SO2a

a

EC50: the concentration of compound that reduced 50% cytopathic effect of RSV Long strain infected HEp-2 cells. CC50: the concentration of compound that manifests cytotoxicity toward 50% of uninfected HEp-2 cells.

Table 8. Anti-RSV Activity and Selectivity of Compound 1a RSV Long, EC50 compd (μM) 1

0.002

RSV A2, EC50 (μM)

RSV B, EC50 (μM)

plaque reduction, EC90 (μM)

influenza H1N1, EC50 (μM)

0.004

0.002

0.003

100

exposure. Compound 6h did not show any oral exposure, which could be due to its low cellular permeability. The most important finding was that the Vss of compound 1 was as low as that of compound 7q, which did not contain a basic head group. It is well-known that basic lipophilic molecules typically lead to a high volume of distribution, which may cause unexpected accumulation of molecule in undesired tissues. Hence the project team made the decision to choose compound 1 for further characterization.

a

EC50: the concentration of compound that reduced 50% cytopathic effect of RSV Long, A2, and B strains. Also listed is plaque reduction in long strain and influenza H1N1 strain infected HEp-2 cells.

Table 9. Physicochemical and ADME Properties of Compounds 1, 5, 6h, 7q, and 7r compd

mlLogDa

pKb,b head amine

LYSAc (μg/mL)

HLM/RLM/MLMd (mL min−1 kg−1)

PAMPAe (10−6 cm/s)

PPBf (human/mouse)

1 5 6h 7q 7r

2.20 0.73 0.88 2.57 1.12

8.0 10.4 10.4 none 9.9

482 >460 >466 9 >480

17/95/204 10/23/19 5/8/1 36/81/120 12/37/23

0.73 0.99 0.12 1.53 0.59

2.4/11 NA 7.8/8 NA 3/10

a

Machine learning log D. bPredicted by MoKa v2.0. cLyophilization solubility assay (LYSA) (μg/mL). dScaled intrinsic clearance of compounds in human, rat, and mouse liver microsomes (HLM, RLM, and MLM). eParallel artificial membrane permeability assay (10−6 cm/s). fPlasma protein binding assay (% unbound fraction). 10233



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Table 10. Rat SDPK Profiles of Selected BAQ Compoundsa

most convincing explanation of high exposure of compound 1 in lung. To our RSV fusion inhibitor project, high exposure in lung was an advantage. As we previously mentioned, an overly high Vss might lead to unexpected accumulation and potential toxicity risk for basic molecule. We utilized oxetane to control pKb of head amine and minimized the potential risk of toxicity. Finally, compound 1 was tested in a mouse RSV model (Figure 5). This compound demonstrated 1 log unit of viral titer reduction at 12.5 mg/kg by oral dosing twice a day (b.i.d.). The anti-RSV efficacy was dose dependent. The RSV titer was below detectable limit at 50 mg/kg and 150 mg/kg, po, b.i.d. Typically, an antiviral drug needs to have enough exposure to maintain antiviral activity, and one criterion is that the plasma Cmin is higher than IC90. In our case, the oral T1/2 of 50 mg/kg was only 1.25 h, and the total plasma drug concentration was below IC90 after 8 h. It could be assumed that at 12.5 mg/kg, po, b.i.d. dosing, the in vivo efficacy was not likely to be observed. However, considering the high lung exposure of compound 1, we speculated that the observed good anti-RSV efficacy at such a low dose was a result of the high lung exposure and longer lung T1/2 of compound 1. Unfortunately, we were unable to test the drug concentration in lung in RSV mouse model due to the limited amount of samples. We plan to provide more evidence on the relationship of pKb and lung distribution in our succeeding manuscript. There were more examples carrying oxetane with diverse modifications on 6-position and SO2 replacement in our previous publication.19 Among those modification, compound 1 remained the top molecule considering anti-RSV activity, TI, DMPK, and toxicity profiles. In the resistant mutation selection study, a single mutant at L138F in RSV F protein was identified in compound 1 resistant virus at the passage 7. The anti-RSV activity of compound 1 was dropped from 2 nM to 2.56 μM, which proved that the target of compound 1 was indeed RSV fusion protein. The single resistant mutation study provided us the opportunity to seek the potential binding site of compound 1 in RSV F protein. Actually, L138 located quite close to the binding pocket of RSV prefusion protein with JNJ-2408068 reported by McLellan et al.22 We docked compound 1 into the same 3-fold-symmetric cavity in profusion RSV F-protein (Figure 6). We observed that the 6-Me entered into a small hydrophobic pocket and picked more hydrophobic interaction with protein, such as Met 396. The oxygen atoms of sulfone formed strong hydrophobic interaction with Phe 140. The model suggested that the oxetane moiety did not form any interaction with the protein, and it mainly controlled the conformation. All these three factors possibly accounted for 100-fold potency increase compared to original hit, compound

compd parameter plasma

t1/2 (iv, h) Vss (iv, L/kg) CL (iv) (mL min−1 kg−1) AUC(0−∞) (iv, (μg/L)·h) AUC(0−∞) (po, (μg/L)·h) F (%)

1

5

6h

7q

7r

1.7 4.1 51

12 73 106

4.5 17 97

1.0 2.5 33

4.1 20 53

686

324

346

717

466

558

510

0

1640

1267

16

32

0

59

29

a

The SDPK study in male Wistar rats was carried out according to the standard procedures (iv, 2 mg/kg; po, 10 mg/kg). Major parameters, including plasma clearance (CL), volume of distribution at steady state (Vss), T1/2 (iv), area under the cure (AUC), and oral bioavailability (F), are reported.

Since the in vivo anti-RSV model was run in mice, the additional SDPK in IRC mice was run with compound 1 at different doses (Table 11). The Vss in mouse SDPK was very Table 11. Mouse SDPK Profiles of Compound 1a dose (mg/kg) parameter t1/2 (po, h) Vss (iv, L/kg) CL (iv) (mL min−1 kg−1) AUC(0−∞) (iv, (μg/L)·h) AUC(0−∞) (po, (μg/L)·h) F (%) lung AUC(0−∞) (po, (μg/L)·h) AUC(0−∞),lung/AUC(0−∞),plasma (po) plasma

50

200

450

1.3

2.3

2.9

1352 36 NA NA

4.7 222 150 9884 66 769 641 78

44 532 132 NA NA

a

The SDPK study in male ICR mice was carried out according to the standard procedures (iv, 2 mg/kg; po, 50, 150, 450 mg/kg). Major parameters, including plasma clearance (CL), volume of distribution at steady state (Vss), T1/2 (iv), area under the cure (AUC), and oral bioavailability (F), are reported.

close to the one measured in rat SDPK. The oral exposure was over dose proportional, which could be due to the saturation of metabolic enzymes at high doses. In the medium dose (200 mg/kg), the lung exposure was also analyzed. It was very interesting to see the exposure of lung was 78-fold higher than plasma. It is known that basic drugs are bound to certain binding sites containing phosphatidylserine.21 Phosphatidylserine is highly expressed in the surface of epithelium cells in lung. Compound 1 could accumulate in lung as a counterion to phosphatidylserine, which is quite acidic. This is perhaps the

Figure 5. In vivo reduction of RSV titer in mouse lung with compound 1 (po, b.i.d.). Female BALB/c mice were used for in vivo efficacy assay. Animals were anesthetized, and to the animals were intranasally administrated RSV Long strain (5 × 105 plaque-forming units [PFU]). After oral administration of compound 1 for 4 days, the mice were euthanized with CO2, and lungs were harvested and analyzed for viral titer. 10234



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Figure 6. Docking model of compound 1 binding to a 3-fold symmetric cavity in prefusion RSV F glycoprotein (the docking procedure is described in the Supporting Information). (a) Side view of the compound 1 in the binding pocket of prefusion FSV F glycoprotein (shown in white). Leu138 is shown as a stick with color in orange. Compound 1 is shown as ball-and-stick, with carbon atoms colored in green, nitrogen atoms in blue, and oxygen atoms in red. (b) 2D ligand-interaction diagram was generated in MOE. The interactions of compound 1 with RSV F main-chain and sidechain atoms are shown as blue and green dotted lines, respectively, and the arrowhead points to the acceptor.

Scheme 1a

a Reagents and conditions: (a) H2SO4, MeOH, reflux, 18 h, 91% (crude); (b) 2-chloroethanamine hydrochloride, NaH, DMF, rt, overnight, 47%; (c) LiAlH4, THF, reflux, 18 h, 90%; (d) Ac2O, Et3N, DCM, 0 °C to rt, 1 h; (e) mCPBA, DCM, 0 °C to rt, 1 h; (f) NaOH, EtOH/H2O, reflux, overnight, 77%; (g) 2,4-dichloro-6-methylquinoline, 1-butanol, microwave, 160 °C, 2 h; (h) 3-(aminomethyl)-N,N-dibenzyloxetan-3-amine, PdCl2(dppf), dppf, t-BuONa, dioxane, microwave, 120 °C, 1 h, 65%; (i) Pd(OH)2, H2, MeOH, rt, 16 h, 17%.

to the procedure shown in Scheme 1. Starting with 2sulfanylbenzoic acid 8, esterification with methanol gives methyl 2-sulfanylbenzoate 9, which was followed by annulation with 2-chloroethylamine to afford 3,4-dihydro-1,4-benzothiazepin-5(2H)-one 10. Reduction of compound 10 with LiAlH4 provided 2,3,4,5-tetrahydro-1,4-benzothiazepine 11, which was converted to 13 via acetylation and oxidation. The subsequent deacylation afforded the desired building block 14, which underwent a coupling reaction with 2,4-dichloro-6-methylquinoline to generate the key intermediate 15. Another

2. The docking model also demonstrated that the Leu 138 was very close to the binding pocket, and L138F mutation may change the conformation of the binding pocket. This appeared to elucidate the huge drop of the antiviral activity of compound 1 to this resistant virus.

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CHEMISTRY

As exemplified by the synthesis of compound 1, BAQ deriatives described herein were mostly prepared according 10235



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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 BAQ Series with N-[(3-Aminooxetan-3-yl)methyl]-2-(1,1-dioxido2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-6-methylquinolin4-amine (1). Exemplified. To a cooled solution of concentrated sulfuric acid (72 g) in methanol (1.5 L) at 0 °C was added 2sulfanylbenzoic acid (8, 300 g, 1.95 mol) in portions under argon atmosphere. After being refluxed with stirring for 18 h, the reaction mixture was concentrated in vacuo. The residue was diluted with water (800 mL), basified with a saturated aqueous solution of sodium bicarbonate to about pH 7, and extracted with dichloromethane (600 mL × 3). The combined organic layers were washed with brine (800 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to afford methyl 2-sulfanylbenzoate (9, 300 g, yield 91%) as a light yellow oil, which was used for the next step without further purification. To a cooled solution of methyl 2-sulfanylbenzoate (9, 200 g, 1.19 mol) in tetrahydrofuran and N,N-dimethylformamide (2 L, V/V = 1/ 1) was added 2-chloroethanamine hydrochloride (138 g, 1.19 mol) at 0 °C followed by sodium hydride (143 g, 3.57 mol, 60% in mineral oil) in portions. After being stirred at room temperature overnight, the reaction mixture was poured into ice−water and extracted with ethyl acetate (900 mL × 4). The organic layers were combined, washed with brine (900 mL × 3), dried over sodium sulfate, and concentrated in vacuo. The residue was stirred in a mixture of ethyl acetate and petroleum ether (300 mL, V/V = 1/1) for 1 h. The solid was collected by filtration and dried in vacuo to afford 3,4-dihydro1,4-benzothiazepin-5(2H)-one (10, 100 g, yield 47%). To a bottle containing a cooled suspension of lithium aluminum hydride (44 g, 1.17 mol) in dry tetrahydrofuran (1.5 L) was added 3,4-dihydro-1,4-benzothiazepin-5(2H)-one (10, 150 g, 0.84 mol) in portions at 0 °C. After being refluxed for 18 h, the reaction mixture was cooled to 0 °C, followed by addition of water (25 mL) dropwise. The reaction mixture was then filtered through a pad of Celite and washed with dichloromethane. The filtrate was dried over sodium sulfate and evaporated in vacuo to afford 2,3,4,5-tetrahydro-1,4benzothiazepine (11, 125 g, yield 90%), which was used for the next step without further purification. To a solution of 2,3,4,5-tetrahydro-1,4-benzothiazepine (11, 5 g, 30.3 mmol) in dry dichloromethane (100 mL) was added triethylamine (5.06 mL, 36.3 mmol) at room temperature, followed by the dropwise addition of acetic anhydride (3.43 mL, 36.3 mmol) at 0 °C under nitrogen. The resulting solution was stirred for 1 h while allowing the temperature to rise slowly to room temperature. The mixture was washed with brine (50 mL × 2), dried over sodium sulfate, filtered, and concentrated in vacuo to afford 1-(2,3-dihydro1,4-benzothiazepin-4(5H)-yl)ethanone (12, 6.28 g, quantitative yield) as a yellow oil, which was used for next step without further purification. To a cooled solution of 1-(2,3-dihydro-1,4-benzothiazepin-4(5H)yl)ethanone (12, 6.27 g, 30.2 mmol) in dichloromethane (100 mL) was added a suspension of 3-chloroperoxybenzoic acid (20.9 g, 90.8 mmol, 75% purity) in dichloromethane (50 mL) at 10 °C. After the addition, the resulting mixture was stirred for 1 h while allowing the temperature to rise slowly to room temperature. The mixture was washed with a saturated aqueous solution of sodium carbonate (100 mL × 2), a saturated aqueous solution of sodium sulfite (100 mL × 2) and brine (100 mL) in sequence. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was stirred in diethyl ether (50 mL) and the solid was collected by filtration and dried in vacuo to afford 1-(1,1-dioxido-2,3-dihydro-1,4benzothiazepin-4(5H)-yl)ethanone (13, 6 g, yield 83%) as a white powder. To a solution of 1-(1,1-dioxido-2,3-dihydro-1,4-benzothiazepin4(5H)-yl)ethanone (13, 6 g, 25 mmol) in ethanol (25 mL) were added sodium hydroxide (5 g, 125 mmol) and water (28 mL). The mixture was refluxed overnight and then concentrated in vacuo. The residue was extracted by ethyl acetate (60 mL × 4). The combined organic layers were extracted by hydrochloric acid (80 mL, 3 N). The

coupling with 3-(aminomethyl)-N,N-dibenzyloxetan-3-amine yielded compound 16, which was finally debenzylated to afford 1 after HPLC purification. The structure of compound 1 could be successfully assigned by spectroscopic analysis on the basis of 1 H, 13C, H−H COSY, H−C HSQC, and H−C HMBC NMR experiments. This efficient synthetic route was successfully applied to the synthesis of the BAQ analogues described in this paper.

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CONCLUSIONS In summary, we have discovered a novel series of BAQ derivatives as RSV fusion inhibitors derived from compound 2, a similarity-based virtual screening hit. In the SAR exploration of the BAQ series, the combination of sulfonyl group at the benzylic position of benzoazepine and 6-Me on quinoline led to single-digit nM anti-RSV compounds (1, 6h, 7a, 7b, and 7r). Sulfonyl group replacement at the benzylic position of benzoazepine also significantly improved the metabolic stability (compound 6a vs compound 5). Although the basic terminal amine on the head region was crucial for anti-RSV activity, it was discovered that reduced basicity was important for reducing Vss. Oxetane was introduced adjacent to the terminal NH2, which led to discovery of compound 1 with much reduced Vss and intact anti-RSV activity. Compound 1 was the most balanced molecule in the BAQ series. It demonstrated good anti-RSV activity against different RSV strains in CPE assays (IC50: 2 nM in Long strain, 4 nM in A2 strain, and 2 nM in B strain) and reasonable oral exposure in plasma and 78-fold higher exposure in lung. It also demonstrated good anti-RSV activity (1 log unit) in a female BALB/c mice model by b.i.d. oral dosing at 12.5 mg/kg. In the resistant mutation selection study, a single mutant at L138F in RSV F protein was identified in compound 1 resistant virus, which confirmed compound 1 as a RSV fusion inhibitor. On the basis of its favorable in vitro and in vivo properties, compound 1 was considered as a promising clinical candidate for RSV treatment.

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EXPERIMENTAL SECTION

Synthetic Chemistry General Comments. Intermediates and final compounds were purified by flash chromatography using one of the following instruments: (i) Biotage SP1 system and the Quad 12/ 25 cartridge module; (ii) ISCO Combi-flash chromatography instrument. Silica gel brand and pore size: (i) KP-SIL 60 Å, particle size 40−60 μm; (ii) CAS registry no., silica gel, 63231-67-4, particle size 47−60 μm silica gel; (iii) ZCX from Qingdao Haiyang Chemical Co., Ltd., pore 200−300 or 300−400. Intermediates and final compounds were purified by preparative HPLC on reversed phase column using XBridge Perp C18 (5 μm, OBD 30 mm × 100 mm) column or SunFire Perp C18 (5 μm, OBD 30 mm × 100 mm) column. LC/MS spectra were obtained using a MicroMass Plateform LC (Waters Alliance 2795-ZQ2000). Standard LC/MS conditions were as follows (running time 6 min). Acidic condition: A, 0.1% formic acid in H2O; B, 0.1% formic acid in acetonitrile. Basic condition: A, 0.01% NH3·H2O in H2O; B, acetonitrile. Neutral condition: A, H2O; B, acetonitrile. Mass spectra (MS): Generally only ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the positive mass ion (M + H)+. The microwave assisted reactions were carried out in a Biotage Initiator Sixty. NMR spectra were obtained using Bruker Avance 400 MHz. All reactions involving air-sensitive reagents were performed under an argon atmosphere. Reagents were used as received from commercial suppliers without further purification unless otherwise noted. All of the final compounds had purities greater than 95% based 10236



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acidic aqueous layer was washed with ethyl acetate (60 mL × 2), then basified with a saturated aqueous solution of sodium bicarbonate to pH > 7, and extracted with ethyl acetate (60 mL × 4). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to afford 2,3,4,5-tetrahydro-1,4-benzothiazepine 1,1-dioxide (14, 3.8 g, yield 77%). MS obsd (ESI+) [(M + H)+] 198. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.89 (dd, J = 1.2, 7.6 Hz, 1 H), 7.56 (t, J = 7.6 Hz, 1 H), 7.47 (t, J = 7.6 Hz, 1 H), 7.42 (d, J = 7.6 Hz, 1 H), 4.04 (s, 2 H), 3.32−3.30 (m, 2 H), 3.30−3.25 (m, 2 H), 2.64 (s, 1 H). To a solution of 2,4-dichloro-6-methylquinoline (2.12 g, 10 mmol) in 20 mL of 1-butanol was added 2,3,4,5-tetrahydro-1,4-benzothiazepine 1,1-dioxide (14, 2.16 g, 11 mmol). The mixture was stirred at 160 °C under microwave irradiation for 2 h. After being cooled to room temperature, the mixture was diluted with dichloromethane, washed with brine, dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by chromatography to give 8-(4-chloro-6-methylquinolin-2-yl)6,7,8,9-tetrahydro-5-thia-8-azabenzocycloheptene 5,5-dioxide (15, 3.7 g, yield 99%), which was used for next step without further purification. MS obsd (ESI+) [(M + H)+] 373. A mixture of 4-(4-chloro-6-methylquinolin-2-yl)-8-methoxy2,3,4,5-tetrahydro-1,4-benzothiazepine 1,1-dioxide (15, 100 mg, 0.27 mmol), sodium tert-butoxide (52 mg, 0.54 mmol), 1,1′-bis(diphenylphosphino)ferrocene−palladium(II) dichloride (22 mg, 0.027 mmol), 1,1′-bis(diphenylphosphino)ferrocene (15 mg, 0.027 mmol), and 3-(aminomethyl)-N,N-dibenzyloxetan-3-amine (76 mg, 0.27 mmol) in 1,4-dioxane (4 mL) was stirred at 120 °C under microwave irradiation for 1 h. The resulting mixture was concentrated in vacuo. The residue was purified by preparative HPLC to afford N[[3-(dibenzylamino)oxetan-3-yl]methyl]-2-(1,1-dioxo-3,5-dihydro2H-1lambda6,4-benzothiazepin-4-yl)-6-methylquinolin-4-amine (16, 108 mg, yield 65%) as a white powder. MS obsd (ESI+) [(M + H)+] 619. A mixture of N-[[3-(dibenzylamino)oxetan-3-yl]methyl]-2-(1,1dioxo-3,5-dihydro-2H-1lambda6,4-benzothiazepin-4-yl)-6-methylquinolin-4-amine (108 mg, 0.17 mmol), 10% palladium hydroxide on active carbon (14 mg) in methanol (6 mL) was stirred for 16 h at room temperature under hydrogen atmosphere (1 bar). The resulting mixture was concentrated in vacuo. The residue was purified by preparative HPLC to afford N-[(3-aminooxetan-3-yl)methyl]-2-(1,1dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-6-methylquinolin4-amine (1, 13 mg, yield 17%). Mp 229.3−229.7 °C. MS obsd (ESI+) [(M + H)+] 439. HRMS calcd [(M + H)+] 439.17984, measured [(M + H)+] 439.17988. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.96 (d, J = 7.07 Hz, 1 H), 7.88 (dd, J = 1.26, 7.83 Hz, 1 H), 7.69 (s, 1H), 7.62 (dt, J = 1.14, 7.52 Hz, 1 H), 7.47 (t, J = 7.67 Hz, 1 H), 7.34 (d, J = 8.34 Hz, 1 H), 7.25 (dd, J = 1.52, 8.59 Hz, 1 H), 6.45 (br t, J = 5.56 Hz, 1 H), 6.20 (s, 1 H), 5.09 (br s, 2 H), 4.45 (d, J = 6.06 Hz, 2 H), 4.32−4.42 (m, 2 H), 3.63 (br t, J = 4.55 Hz, 2 H), 3.55 (d, J = 5.31 Hz, 2 H), 2.53−2.81 (m, 2 H), 2.36 (s, 3 H). 13C NMR (101 MHz, DMSO-d6) δ 155.6, 151.6, 146.5, 141.1, 137.7, 133.5, 133.3, 131.2, 129.9, 128.2, 127.5, 127.0, 120.5, 115.5, 85.5, 82.2, 57.0, 55.3, 51.9, 49.7, 45.0, 41.0, 40.9, 40.8, 21.4. For the following BAQ series, except those described specifically, all the analogues are prepared in analogy to 1 from the commercially available building blocks. 1-Amino-3[[2-(3,4-dihydro-1H-isoquinolin-2-yl)-4-quinolyl]amino]propan-2-ol (2). MS obsd (ESI+) [(M + H)+] 349. 1H NMR (400 MHz, CD3OD) δ ppm 8.21 (d, J = 8.08 Hz, 1 H), 7.88 (d, J = 8.34 Hz, 1 H), 7.78 (t, J = 7.71 Hz, 1 H), 7.50 (t, J = 7.71 Hz, 1 H), 7.34− 7.41 (m, 1 H), 7.28−7.34 (m, 3 H), 6.23 (s, 1 H), 4.88−4.93 (m, 2 H), 4.29 (br d, J = 6.06 Hz, 1 H), 3.96 (t, J = 5.81 Hz, 2 H), 3.60− 3.78 (m, 2 H), 3.23−3.31 (m, 1 H), 3.16 (t, J = 5.81 Hz, 2H), 3.04 (br dd, J = 9.98, 12.25 Hz, 1 H). 3-[[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]amino]propane-1,2-diol (2a). MS obsd (ESI+) [(M + H)+] 350. 1H NMR (400 MHz, CDCl3) δ ppm 7.75 (br d, J = 8.31 Hz, 1 H), 7.60 (br d, J = 8.19 Hz, 1 H), 7.37 (br t, J = 7.70 Hz, 1 H), 7.27−7.31 (m, 1 H), 7.18−7.25 (m, 3 H), 7.11 (t, J = 7.57 Hz, 1 H), 5.69 (br s, 1 H), 4.77

(s, 2 H), 4.09−4.18 (m, 1 H), 3.84−3.95 (m, 3 H), 3.78 (dd, J = 5.26, 11.37 Hz, 1 H), 3.24−3.51 (m, 2 H), 3.01 (t, J = 5.75 Hz, 2 H). N′-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]propane-1,3-diamine (2b). MS obsd (ESI+) [(M + H)+] 333. 1H NMR (400 MHz, CD3OD) δ ppm 8.22 (d, J = 8.38 Hz, 1 H), 7.88 (d, J = 7.82 Hz, 1 H), 7.77 (t, J = 7.76 Hz, 1 H), 7.49 (t, J = 7.44 Hz, 1 H), 7.29−7.40 (m, 4 H), 6.11 (s, 1 H), 4.90−4.94 (m, 2 H), 3.96 (t, J = 5.93 Hz, 2 H), 3.71 (t, J = 7.03 Hz, 2 H), 3.10−3.31 (m, 4 H), 2.22 (quin, J = 7.37 Hz, 2 H). 1-[[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]amino]propan-2-ol (2c). MS obsd (ESI+) [(M + H)+] 334. 1H NMR (400 MHz, CD3OD) δ ppm 7.97−8.13 (m, 1 H), 7.60−7.85 (m, 2 H), 7.45 (ddd, J = 1.10, 7.11, 8.32 Hz, 1 H), 7.18−7.33 (m, 4 H), 6.12 (s, 1 H), 4.80−4.86 (m, 2 H), 4.15 (dt, J = 4.74, 6.67 Hz, 1 H), 3.89 (t, J = 5.95 Hz, 2 H), 3.41−3.55 (m, 2 H), 3.10 (t, J = 5.95 Hz, 2 H), 1.30 (d, J = 6.39 Hz, 3 H). N′-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]ethane1,2-diamine (2d). MS obsd (ESI+) [(M + H)+] 319. 1H NMR (400 MHz, CD3OD) δ ppm 8.04−8.28 (m, 1 H), 7.84−7.99 (m, 1 H), 7.79 (dt, J = 1.16, 7.73 Hz, 1 H), 7.51 (ddd, J = 1.16, 7.09, 8.25 Hz, 1 H), 7.29−7.37 (m, 4 H), 6.16 (s, 1 H), 4.90−4.93 (m, 2 H), 3.84−4.03 (m, 4 H), 3.35−3.43 (m, 2 H), 3.04−3.31 (m, 2 H). N′-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]butane1,4-diamine (2e). MS obsd (ESI+) [(M + H)+] 347. 1H NMR (400 MHz, CD3OD) δ ppm 8.22 (d, J = 7.91 Hz, 1 H), 7.87 (d, J = 8.19 Hz, 1 H), 7.77 (t, J = 7.78 Hz, 1 H), 7.42−7.56 (m, 1 H), 7.29−7.39 (m, 4 H), 6.07 (s, 1 H), 4.90−4.92 (m, 2 H), 3.95 (t, J = 5.93 Hz, 2 H), 3.63 (t, J = 6.60 Hz, 2 H), 3.12−3.31 (m, 2 H), 3.06 (br t, J = 7.27 Hz, 2 H), 1.83−1.99 (m, 4 H). 1-[2-[[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]amino]ethyl]guanidine (2f). The synthetic procedure was described in the Supporting Information. MS obsd (ESI+) [(M + H)+] 361. 1H NMR (400 MHz, CD3OD) δ ppm 8.12 (d, J = 8.31 Hz, 1 H), 7.82 (d, J = 8.31 Hz, 1 H), 7.72 (t, J = 7.64 Hz, 1 H), 7.44 (t, J = 7.58 Hz, 1 H), 7.25−7.34 (m, 4 H), 6.09 (s, 1 H), 4.86 (s, 2 H), 3.92 (t, J = 5.81 Hz, 2 H), 3.75 (br t, J = 5.69 Hz, 2 H), 3.62 (br t, J = 5.62 Hz, 2 H), 3.11 (t, J = 5.75 Hz, 2 H). 2-[[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-4-quinolyl]amino]ethylurea (2g). The synthetic procedure was described in the Supporting Information. MS obsd (ESI+) [(M + H)+] 362. 1H NMR (400 MHz, CD3OD) δ ppm 8.00 (d, J = 8.19 Hz, 1 H), 7.76 (d, J = 8.31 Hz, 1 H), 7.67 (t, J = 7.70 Hz, 1 H), 7.19−7.41 (m, 5 H), 6.16 (s, 1 H), 4.86 (s, 2 H), 3.92 (t, J = 5.87 Hz, 2 H), 3.35−3.58 (m, 4 H), 3.09 (t, J = 5.81 Hz, 2 H). 1-Amino-3-[(2-isoindolin-2-yl-4-quinolyl)amino]propan-2ol (3). MS obsd (ESI+) [(M + H)+] 335. 1H NMR (400 MHz, CD3OD) δ ppm 7.86−8.06 (m, 1 H), 7.68−7.77 (m, 1 H), 7.58 (s, 1 H), 7.38−7.46 (m, 2 H), 7.31−7.38 (m, 2 H), 7.23−7.29 (m, 1 H), 5.90 (s, 1 H), 4.89−4.97 (m, 4 H), 3.98−4.18 (m, 1 H), 3.43−3.58 (m, 2 H), 3.01 (dd, J = 3.73, 13.02 Hz, 1 H), 2.85 (dd, J = 7.95, 12.96 Hz, 1 H). 1-Amino-3-[2-(1,3,4,5-tetrahydrobenzo[c]azepin-2-yl)quinolin-4-ylamino]propan-2-ol (4). MS obsd (ESI+) [(M + H)+] 363. 1H NMR (400 MHz, CD3OD) δ ppm 8.05−8.03 (d, J = 7.6 Hz, 1 H), 7.78−7.76 (d, J = 7.6 Hz, 1 H), 7.72−7.68 (m, 1 H), 7.52−7.50 (m, 1 H), 7.44−7.40 (m, 1 H), 7.23−7.19 (m, 3 H), 6.12 (s, 1 H), 4.97 (s, 2 H), 4.16−4.11 (m, 3 H), 3.56−3.55 (d, J = 6 Hz, 2 H), 3.20−3.16 (dd, J = 2.8, 12.8 Hz,1 H), 3.11−3.08 (m, 2 H), 2.99−2.94 (m, 1 H), 1.99−1.98 (m, 2 H). N-[2-(1,3,4,5-Tetrahydro-2H-2-benzazepin-2-yl)quinolin-4yl]ethane-1,2-diamine (5). MS obsd (ESI+) [(M + H)+] 333. HRMS calcd [(M + H)+] 333.20737, measured [(M + H)+] 333.20677. 1H NMR (400 MHz, CD3OD) δ ppm 7.91−7.89 (m, 1 H), 7.62−7.60 (m, 1 H), 7.52−7.45 (m, 2 H), 7.21−7.12 (m, 3 H), 5.96 (s, 1 H), 4.10 (s, 2 H), 3.51−3.47 (t, J = 6.0 Hz, 1 H), 3.04−2.98 (m, 5 H), 2.84 (s, 2 H), 1.94−1.91 (m, 2 H). N-[5-Chloro-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine (5a). MS obsd (ESI+) [(M + H)+] 367. 1H NMR (400 MHz, CD3OD) δ ppm 7.46−7.42 (m, 2 H), 7.27−7.23 (m, 1 H), 7.16−7.11 (m, 3 H), 7.02 (66, J = 7.6, 1.2 Hz, 1 10237



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N-[2-(5,5-Difluoro-1,3,4,5-tetrahydro-2H-2-benzazepin-2yl)-6-methylquinolin-4-yl]ethane-1,2-diamine (6c). Compound 6c was prepared in analogy to 1 by using 5,5-difluoro-2,3,4,5tetrahydro-1H-benzazepine (6c-c; see Supporting Information for its synthesis) instead of 2,3,4,5-tetrahydro-1,4-benzothiazepine 1,1dioxide 14. MS obsd (ESI+) [(M + H)+] 383. 1H NMR (400 MHz, CD3OD) δ ppm 7.89 (s, 1 H), 7.72−7.70 (d, J = 6.8 Hz, 1 H), 7.70−7.67 (d, J = 6.8 Hz, 2 H), 7.65−7.42 (m, 3 H), 6.00 (s, 1 H), 5.02 (s, 2 H), 4.23−4.20 (t, 2 H), 3.88−3.78 (t, 2 H), 3.58−3.55 (t, 2 H), 2.71−2.61 (m, 2 H), 2.46 (s, 3 H). N-[6-Methyl-2-(1,2,3,5-tetrahydro-4H-1,4-benzodiazepin-4yl)quinolin-4-yl]ethane-1,2-diamine (6d). MS obsd (ESI+) [(M + H)+] 348. 1H NMR (400 MHz, CD3OD) δ ppm 7.90 (s, 1 H), 7.67 (d, J = 8.4 Hz, 1 H), 7.58 (d, J = 8.4 Hz, 1 H), 7.42 (d, J = 7.2 Hz, 1 H), 7.12 (t, J = 8.0 Hz, 1 H), 6.88 (t, J = 7.2 Hz, 1 H), 6.82 (d, J = 8.0 Hz, 1 H), 5.95 (s, 1 H), 4.87 (s, 2 H), 4.00 (t, J = 4.8 Hz, 2 H), 3.72 (t, J = 6.0 Hz, 2 H), 3.46 (t, J = 4.8 Hz, 2 H), 3.18 (t, J = 6.0 Hz, 2 H), 2.49 (s, 3 H). 4-{4-[(2-Aminoethyl)amino]-6-methylquinolin-2-yl}-1,3,4,5tetrahydro-2H-1,4-benzodiazepin-2-one (6e). MS obsd (ESI+) [(M + H)+] 362. 1H NMR (400 MHz, CD3OD) δ ppm 7.64 (s, 1 H), 7.44−7.40 (m, 2 H), 7.28 (dd, J = 1.6, 8.4 Hz, 1 H), 7.18 (dd, J = 1.6, 8.0 Hz, 1 H), 7.09 (dd, J = 0.8, 8.0 Hz, 1 H), 6.98 (dd, J = 0.8, 8.0 Hz, 1 H), 6.00 (s, 1 H), 4.87 (s, 2 H), 4.72 (s, 2 H), 3.40 (t, J = 6.4 Hz, 2 H), 2.95 (t, J = 6.4 Hz, 2 H), 2.40 (s, 3 H). N-[2-(7-Fluoro-1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)-6methylquinolin-4-yl]ethane-1,2-diamine (6f). MS obsd (ESI+) [(M + H)+] 365. 1H NMR (400 MHz, CD3OD) δ ppm 7.82 (s, 1 H), 7.58 (d, J = 8.4 Hz, 1 H), 7.45 (m, 1 H), 7.17 (m, 1 H), 6.94 (m, 1 H), 6.84 (m, 1 H), 6.03 (d, J = 4.0 Hz, 1 H), 4.87 (s, 2 H), 3.95 (m, 4 H), 3.56 (t, J = 6.4 Hz, 2 H), 3.10 (m, 4 H), 2.48 (s, 3 H). N-[2-(8-Fluoro-1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)-6methylquinolin-4-yl]ethane-1,2-diamine (6g). MS obsd (ESI+) [(M + H)+] 365. 1H NMR (400 MHz, CD3OD) δ ppm 7.68 (s, 1 H), 7.48 (d, J = 8.4 Hz, 1 H), 7.28 (m, 2 H), 7.12 (m, 1 H), 6.83 (m, 1 H), 5.93 (s, 1 H), 4.80 (s, 2 H), 4.09 (s, 2 H), 3.46 (t, J = 6.0 Hz, 2 H), 3.00 (m, 4 H), 2.40 (s, 3 H), 1.90 (m, 2 H). N-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)yl)-6-methylquinolin-4-yl]ethane-1,2-diamine (6h). MS obsd (ESI+) [(M + H)+] 397. 1H NMR (400 MHz, CD3OD) δ ppm 7.96 (d, J = 7.58 Hz, 1 H), 7.86 (br d, J = 7.33 Hz, 1 H), 7.67 (s, 1 H), 7.62 (t, J = 7.08 Hz, 1 H), 7.39−7.46 (m, 2 H), 7.28 (d, J = 8.69 Hz, 1 H), 6.06 (s, 1 H), 5.15 (s, 2 H), 4.53 (br s, 2 H), 3.49−3.66 (m, 4 H), 3.06−3.17 (m, 2 H), 2.41 (s, 3 H). N-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)yl)-6-methylquinolin-4-yl]propane-1,3-diamine (7a). MS obsd (ESI+) [(M + H)+] 411. 1H NMR (400 MHz, CD3OD) δ ppm 7.98 (dd, J = 1.01, 7.83 Hz, 1 H), 7.83 (d, J = 7.07 Hz, 1 H), 7.56−7.64 (m, 2 H), 7.39−7.47 (m, 2 H), 7.27 (dd, J = 1.77, 8.59 Hz, 1 H), 6.00 (s, 1 H), 5.14 (s, 2 H), 4.40−4.67 (m, 2 H), 3.58 (t, J = 4.93 Hz, 2 H), 3.41 (t, J = 6.82 Hz, 2 H), 2.84 (t, J = 6.95 Hz, 2 H), 2.41 (s, 3 H), 1.91 (quin, J = 6.88 Hz, 2 H). N-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)yl)-6-methylquinolin-4-yl]butane-1,4-diamine (7b). MS obsd (ESI+) [(M + H)+] 425. 1H NMR (400 MHz, CD3OD) δ ppm 8.01− 7.95 (m, 1 H), 7.92−7.86 (m, 1 H), 7.83−7.76 (m, 1 H), 7.69−7.61 (m, 1 H), 7.61−7.54 (m, 1 H), 7.50−7.42 (m, 1 H), 7.39−7.31 (m, 1 H), 5.96 (s, 1 H), 5.20 (br s, 2 H), 4.54 (br s, 2 H), 3.63 (br s, 2 H), 3.47 (br s, 2 H), 3.37 (s, 2 H), 2.42 (s, 3 H), 2.27−2.24 (m, 3 H), 1.86 (d, J = 3.28 Hz, 3 H). N-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)yl)-6-methylquinolin-4-yl]pentane-1,5-diamine (7c). MS obsd (ESI+) [(M + H)+] 439. 1H NMR (400 MHz, CD3OD) δ ppm 7.91− 8.07 (m, 1 H), 7.78−7.89 (m, 1 H), 7.74 (s, 1 H), 7.62 (q, J = 7.33 Hz, 1 H), 7.41−7.55 (m, 2 H), 7.29−7.36 (m, 1 H), 6.00 (s, 1 H), 5.16 (s, 2 H), 4.37−4.71 (m, 2 H), 3.52−3.70 (m, 2 H), 3.39−3.44 (m, 2 H), 3.28−3.37 (m, 2 H), 2.24 (s, 3 H), 1.71−1.84 (m, 4 H), 1.54−1.64 (m, 2 H). N-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)yl)-6-methylquinolin-4-yl]-N′-methylethane-1,2-diamine (7d). MS obsd (ESI+) [(M + H)+] 411. 1H NMR (400 MHz, CD3OD) δ

H), 5.97 (s, 1 H), 4.79 (s, 2 H), 4.13 (br s, 2 H), 3.38−3.21 (m, 2 H), 3.04−3.02 (m, 2 H), 2.96 (t, J = 6.0 Hz, 2 H), 1.91−1.86 (m, 2 H). N-[6-Chloro-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine (5b). MS obsd (ESI+) [(M + H)+] 367. 1H NMR (400 MHz, CD3OD) δ ppm 7.87 (d, J = 2.4 Hz, 1 H), 7.47−7.42 (m, 2 H), 7.34 (dd, J = 9.0, 2.2 Hz, 1 H), 7.15−7.09 (m, 3 H), 5.99 (s, 1 H), 4.76 (s, 2 H), 4.11 (br s, 2 H), 3.38−3.32 (m, 2 H), 3.00−2.93 (m, 4 H), 1.87 (t, J = 5.2 Hz, 2 H). N-[7-Chloro-2-(1,3,4,5-tetrahydro-2-benzazepin-2-yl)-4quinolyl]ethane-1,2-diamine (5c). MS obsd (ESI+) [(M + H)+] 367. 1H NMR (400 MHz, CD3OD) δ ppm 7.71 (d, J = 8.84 Hz, 1 H), 7.48 (d, J = 2.02 Hz, 1 H), 7.38 (d, J = 7.07 Hz, 1 H), 7.09−7.13 (m, 1 H), 7.02−7.07 (m, 2 H), 6.97 (dd, J = 2.02, 8.84 Hz, 1 H), 5.88 (s, 1 H), 4.66 (s, 2 H), 4.04 (br s, 2 H), 3.37 (s, 1 H), 3.29−3.31 (m, 1 H), 2.85−2.94 (m, 4 H), 1.80 (br s, 2 H). N-[8-Chloro-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine (5d). MS obsd (ESI+) [(M + H)+] 367. 1H NMR (400 MHz, CD3OD) δ ppm 7.24 (d, J = 8.0 Hz, 1 H), 7.60 (d, J = 6.8 Hz, 1 H), 7.55 (d, J = 7.6 Hz, 1 H), 7.13−7.09 (m, 3 H), 6.98 (t, J = 8.0 Hz, 1 H), 6.03 (s, 1 H), 4.89 (s, 2 H), 4.20 (br s, 2 H), 3.46 (t, J = 6.0 Hz, 2 H), 3.07−3.02 (m, 4 H), 1.92 (t, J = 5.2 Hz, 2 H). N-[6-Methoxy-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine (5e). MS obsd (ESI+) [(M + H)+] 363. 1H NMR (400 MHz, CD3OD) δ ppm 7.60 (d, J = 9.2 Hz, 1 H), 7.50 (d, J = 6.4 Hz, 1 H), 7.42 (d, J = 2.4 Hz, 1 H), 7.25−7.18 (m, 4 H), 6.01 (s, 1 H), 4.93 (s, 2 H), 4.13 (br s, 2 H), 3.89 (s, 3 H), 3.53 (t, J = 6.4 Hz, 2 H), 3.10−3.01 (m, 4 H), 1.96 (s, 2 H). N-[6-Methyl-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2 diamine (5f). MS obsd (ESI+) [(M + H)+] 347. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.68 (s, 1 H), 7.55 (d, J = 6.8 Hz, 1 H), 7.29 (d, J = 8.4 Hz, 1 H), 7.21 (d, J = 6.8 Hz, 1 H), 7.13−7.05 (m, 3 H), 6.67 (br s, 1 H), 6.00 (s, 1 H), 4.79 (s, 2 H), 4.11 (br s, 2 H), 3.52 (s, 2 H), 3.05−2.99 (m, 4 H), 2.35 (s, 3 H), 1.77 (s, 2 H). N-[6-Fluoro-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine (5g). MS obsd (ESI+) [(M + H)+] 351. 1H NMR (400 MHz, CD3OD) δ ppm 7.60−7.52 (m, 2 H), 7.47 (d, J = 6.8 Hz, 1 H), 7.26−7.21 (m, 1 H), 7.17−7.12 (m, 3 H), 6.04 (s, 1 H), 4.82 (s, 2 H), 4.15 (br s, 2 H), 3.45 (t, J = 6.4 Hz, 2 H), 3.06−2.99 (m, 4 H), 1.91 (t, J = 5.4 Hz, 2 H). N-[2-(1,3,4,5-Tetrahydro-2H-2-benzazepin-2-yl)-6(trifluoromethyl)quinolin-4-yl]ethane-1,2-diamine (5h). MS obsd (ESI+) [(M + H)+] 401. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.36 (s, 1 H), 7.57−7.45 (m, 3 H), 7.12 (br s, 3 H), 6.99 (s, 1 H), 6.03 (s, 1 H), 4.81 (s, 2 H), 4.15 (br s, 2 H), 3.29 (br s, 2 H), 3.01 (s, 2 H), 2.82 (s, 2 H), 1.77 (s, 2 H). N-[6-(Methylsulfonyl)-2-(1,3,4,5-tetrahydro-2H-2-benzazepin-2-yl)quinolin-4-yl]ethane-1,2-diamine (5i). MS obsd (ESI+) [(M + H)+] 411. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.518 (s, 0.5 H), 8.514 (s, 0.5 H), 7.76−7.73 (m, 1 H), 7.61 (d, J = 6.4 Hz, 0.5 H), 7.52 (d, J = 6.8 Hz, 0.5 H), 7.47−7.34 (m, 1 H), 7.13−7.07 (m, 3 H), 6.23 (s, 0.5 H), 6.04 (s, 0.5 H), 4.83 (d, J = 6.4 Hz, 2 H), 4.34 (br s, 2 H), 3.15−3.28 (m, 6 H), 3.00 (s, 2 H), 2.81 (t, J = 6.4 Hz, 1 H), 1.76 (br s, 2 H). N-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)yl)quinolin-4-yl]ethane-1,2-diamine (6a). MS obsd (ESI+) [(M + H)+] 383. 1H NMR (400 MHz, CD3OD) δ ppm 7.87 (m, 1 H), 7.81 (d, J = 7.83 Hz, 2 H), 7.55 (m, 1 H), 7.47 (m, 1 H), 7.33 (m, 2 H), 7.04 (m, 1 H), 5.94 (m,1 H), 5.12 (s, 2 H), 3.57 (t, J = 4.6 Hz, 2 H), 3.43 (t, J = 6.3 Hz, 2 H), 3.33 (m, 2 H), 2.97 (t, J = 6.4 Hz, 2 H). N-[2-(2,3-Dihydro-1,4-benzoxazepin-4(5H)-yl)quinolin-4yl]ethane-1,2-diamine (6b). Compound 6b was prepared in analogy to 1 by using 2,3,4,5-tetrahydro-1,4-benzoxazepine hydrochloride (6b-c; see Supporting Information for its synthesis) instead of 2,3,4,5-tetrahydro-1,4-benzothiazepine 1,1-dioxide 14. MS obsd (ESI+) [(M + H)+] 335. 1H NMR (400 MHz, CD3OD) δ ppm 8.61 (s, 1 H), 7.90 (d, J = 7.6 Hz, 1 H), 7.60−7.51 (m, 3 H), 7.23 (br s, 2 H), 7.09−6.98 (m, 2 H), 6.05 (s, 1 H), 4.89 (s, 2 H), 4.27 (s, 2 H), 4.25 (s, 2 H), 3.61 (s, 2 H), 3.15 (s, 2 H). 10238



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ppm 8.03−8.0 (m, 3 H), 7.87−7.84 (d, J = 8.8 Hz, 1 H), 7.71−7.70 (d, J = 1.2 Hz, 1 H), 7.57−7.53 (m, 2 H), 6.07 (s, 1 H), 5.40 (s, 2 H), 4.56 (s, 2 H), 3.96−3.93 (dd, J = 6.0, 6.4 Hz, 2 H), 3.75−3.73 (q, J = 4.4 Hz, 2 H), 3.43−3.40 (q, J = 6 Hz, 2 H), 2.80 (s, 3 H), 2.46 (s, 3 H). N′-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)yl)-6-methylquinolin-4-yl]-N,N-dimethylethane-1,2-diamine (7e). MS obsd (ESI+) [(M + H)+] 425. 1H NMR (400 MHz, CD3OD) δ ppm 8.05−8.01 (m, 3 H), 7.88−7.86 (d, J = 8.8 Hz, 1 H), 7.70−7.68 (d, J = 1.2 Hz, 1 H), 7.56−7.52 (m, 2 H), 6.05 (s, 1 H), 5.41 (s, 2 H), 4.56 (s, 2 H), 3.96−3.93 (dd, J = 6.0, 6.4 Hz, 2 H), 3.75−3.73 (q, J = 4.4 Hz, 2 H), 3.43−3.40 (q, J = 6.0 Hz, 2 H), 3.01 (s, 6 H), 2.45 (s, 3 H). N∼2∼-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin4(5H)-yl)-6-methylquinolin-4-yl]propane-1,2-diamine (7f). MS obsd (ESI+) [(M + H)+] 411. 1H NMR (400 MHz, CD3OD) δ ppm 7.97 (dd, J = 7.83, 1.01 Hz, 1 H), 7.82 (d, J = 7.33 Hz, 1 H), 7.69− 7.56 (m, 2 H), 7.46−7.38 (m, 2 H), 7.28 (dd, J = 8.46, 1.64 Hz, 1 H), 6.05 (s, 1 H), 5.13 (s, 2 H), 4.48 (br s, 2 H), 3.56 (m, 2 H), 3.42− 3.49 (m, 3 H), 2.41 (s, 3 H), 1.38 (d, J = 5.81 Hz, 3 H). N∼1∼-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin4(5H)-yl)-6-methylquinolin-4-yl]propane-1,2-diamine (7g). MS obsd (ESI+) [(M + H)+] 411. 1H NMR (400 MHz, CD3OD) δ ppm 7.91 (dd, J = 7.83, 1.01 Hz, 1 H), 7.83 (d, J = 7.33 Hz, 1 H), 7.69 (s, 1 H), 7.58 (d, J = 1.26 Hz, 1 H), 7.44 (d, J = 8.59 Hz, 1 H), 7.38−7.31 (m, 1 H), 7.28 (dd, J = 8.59, 1.77 Hz, 1 H), 6.04 (s, 1 H), 5.13 (br s, 2 H), 4.48 (br s, 2 H), 3.56 (t, J = 4.67 Hz, 2 H), 3.52−3.41 (m, 3 H), 2.41 (s, 3 H), 1.38 (d, J = 6.06 Hz, 3 H). N∼2∼-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin4(5H)-yl)-6-methylquinolin-4-yl]-2-methylpropane-1,2-diamine (7h). MS obsd (ESI+) [(M + H)+] 425. 1H NMR (400 MHz, CD3OD) δ ppm 7.98 (dd, J = 7.83, 1.26 Hz, 1 H), 7.86 (d, J = 6.82 Hz, 1 H), 7.70 (s, 1 H), 7.61 (td, J = 7.45, 1.26 Hz, 1 H), 7.47−7.42 (m, 2 H), 7.29 (dd, J = 8.59, 1.77 Hz, 1 H), 6.09 (s, 1 H), 5.16 (s, 2 H), 4.53 (br s, 2 H), 3.58 (t, J = 4.80 Hz, 2 H), 3.27 (s, 2 H), 2.43 (s, 3 H), 1.27 (s, 6 H). N∼1∼-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin4(5H)-yl)-6-methylquinolin-4-yl]-2-methylpropane-1,2-diamine (7i). MS obsd (ESI+) [(M + H)+] 425. 1H NMR (400 MHz, CD3OD) δ ppm 7.97 (d, J = 7.83 Hz, 1 H), 7.85 (d, J = 7.58 Hz, 1 H), 7.70 (s, 1 H), 7.60 (t, J = 7.33 Hz, 1 H), 7.48−7.38 (m, 2 H), 7.29−7.27 (m, 1 H), 6.09 (s, 1 H), 5.15 (br s, 2 H), 4.52 (br s, 2 H), 3.57 (br s, 2 H), 3.26 (s, 2 H), 2.43 (s, 3 H), 1.34−1.20 (m, 6 H). 2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-6methyl-N-[2-(piperazin-1-yl)ethyl]quinolin-4-amine (7j). MS obsd (ESI+) [(M + H)+] 466. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.14−7.18 (m, 1 H), 7.04 (d, J = 7.58 Hz, 1 H), 6.74−6.82 (m, 2 H), 6.58−6.65 (m, 2 H), 6.47 (dd, J = 1.52, 8.59 Hz, 1 H), 5.22 (s, 1 H), 4.33 (s, 2 H), 3.72 (br s, 2 H), 2.77 (br t, J = 4.67 Hz, 2 H), 2.67 (t, J = 6.57 Hz, 2 H), 2.55−2.57 (m, 2 H), 2.13 (t, J = 4.80 Hz, 4 H), 1.94 (t, J = 6.57 Hz, 2 H), 1.79 (br s, 2 H), 1.60 (s, 3 H). 2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-6methyl-N-[2-(morpholin-4-yl)ethyl]quinolin-4-amine (7k). MS obsd (ESI+) [(M + H)+] 467. 1H NMR (400 MHz, CDCl3) δ ppm 8.05 (dd, J = 1.01, 7.83 Hz, 1 H), 7.66 (d, J = 7.33 Hz, 1 H), 7.51 (dt, J = 1.14, 7.52 Hz, 2 H), 7.38 (t, J = 7.66 Hz, 1 H), 7.33 (d, J = 8.63 Hz, 1 H), 7.28 (br s, 1 H), 5.88 (s, 1 H), 5.62 (br s, 1 H), 5.13 (s, 2 H), 4.6 (br s, 2 H), 3.74−3.81 (m, 4 H), 3.58 (br s, 2 H), 3.31 (q, J = 5.31 Hz, 2 H), 2.82 (t, J = 5.94 Hz, 2 H), 2.50−2.61 (m, 4 H), 2.46 (s, 3 H). 2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-6methyl-N-[2-(piperidin-1-yl)ethyl]quinolin-4-amine (7l). MS obsd (ESI+) [(M + H)+] 465. 1H NMR (400 MHz, CDCl3) δ ppm 8.04 (d, J = 7.6 Hz, 1 H), 7.67 (d, J = 7.6 Hz, 1 H), 7.51 (m, 2 H), 7.38 (t, J = 7.6 Hz, 1 H), 7.30 (m, 2 H), 5.88 (s, 1 H), 5.74 (m, 1 H), 5.12 (s, 2 H), 4.6 (br s, 2 H), 3.58 (s, 2 H), 3.28 (m, 2 H), 2.75 (t, J = 8.4 Hz, 2 H), 2.46 (m, 7 H), 1.62 (m, 4 H), 1.52 (m, 2 H). 2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-6methyl-N-(piperidin-2-ylmethyl)quinolin-4-amine (7m). MS obsd (ESI+) [(M + H)+] 451. 1H NMR (400 MHz, CD3OD) δ ppm 8.07 (d, J = 7.6 Hz, 1 H), 7.93 (s, 1 H), 7.85 (d, J = 7.6 Hz, 1

H), 7.73−7.70 (m, 2 H), 7.62−7.58 (m, 2 H), 6.02 (s, 1 H), 5.35 (s, 2 H), 4.51 (s, 2 H), 3.75−3.71 (m, 4 H), 3.51−3.40 (m, 2 H), 2.98− 2.92 (m, 1 H), 2.46 (s, 3 H), 2.11−2.05 (m, 1 H), 1.96−1.90 (m, 2 H), 1.78−1.56 (m, 3 H). N-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)yl)-6-methylquinolin-4-yl]glycinamide (7n). The synthetic procedure was described in the Supporting Information. MS obsd (ESI+) [(M + H)+] 411. 1H NMR (400 MHz, CD3OD) δ ppm 8.51 (s, 1 H), 8.12 (s, 1 H), 8.07 (d, J = 7.6 Hz, 1 H), 8.00 (d, J = 7.6 Hz, 1 H), 7.87 (d, J = 8.8 Hz, 1 H), 7.67−7.12 (m, 2 H), 7.57 (t, J = 8.0 Hz, 1 H), 5.31 (s, 2 H), 4.66 (br s, 2 H), 4.25 (s, 2 H), 3.78 (s, 2 H), 2.52 (s, 3 H). N∼2∼-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin4(5H)-yl)-6-methylquinolin-4-yl]glycinamide (7o). MS obsd (ESI+) [(M + H)+] 411. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.87 (d, J = 7.83 Hz, 1 H), 7.82 (d, J = 7.33 Hz, 1 H), 7.74−7.64 (m, 2 H), 7.52−7.41 (m, 2 H), 7.36−7.30 (m, 1 H), 7.30−7.19 (m, 2 H), 7.10 (t, J = 5.56 Hz, 1 H), 5.87 (s, 1 H), 5.00 (br s, 2 H), 4.40 (br s, 2 H), 3.86 (d, J = 5.81 Hz, 2 H), 3.59 (br s, 2 H), 3.32 (s, 1 H), 2.36 (s, 3 H). N-(2-{[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin4(5H)-yl)-6-methylquinolin-4-yl]amino}ethyl)acetamide (7p). MS obsd (ESI+) [(M + H)+] 439. 1H NMR (400 MHz, CD3OD) δ ppm 8.16 (s, 1 H), 7.95 (d, J = 1.8 Hz, 1 H), 7.86 (d, J = 1.9 Hz, 1 H), 7.62 (t, J = 3.6 Hz, 2 H), 7.46 (t, J = 3.8 Hz, 1 H), 7.31 (d, J = 2.1 Hz, 1 H), 7.22 (d, J = 2.1 Hz, 1 H), 6.84 (s, 1 H), 6.20 (s, 1 H), 5.10 (s, 2 H), 4.43 (br s, 2 H), 3.62 (s, 2 H), 3.32 (m, 4 H), 2.34 (s, 3 H), 1.89 (s, 3 H). 2-{[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)yl)-6-methylquinolin-4-yl]amino}ethanol (7q). MS obsd (ESI+) [(M + H)+] 398. HRMS calcd [(M + H)+] 398.15329, measured [(M + H)+] 398.15313. 1H NMR (400 MHz, CD3OD) δ ppm 8.08 (d, J = 7.6 Hz, 1 H), 7.91 (s, 1 H), 7.85 (d, J = 7.6 Hz, 1 H), 7.74−7.67 (m, 2 H), 7.59−7.56 (m, 2 H), 6.09 (s, 1 H), 5.29 (s, 2 H), 4.51 (s, 2 H), 3.83 (t, J = 5.6 Hz, 2 H), 3.74−3.72 (m, 2 H), 3.62 (t, J = 5.6 Hz, 2 H), 2.47 (s, 3 H). N-[3-(Aminomethyl)oxetan-3-yl]-2-(1,1-dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)-yl)-6-methylquinolin-4-amine (7r). MS obsd (ESI+) [(M + H)+] 439. HRMS calcd [(M + H)+] 439.17984, measured [(M + H)+] 439.17975. 1H NMR (400 MHz, CD3OD) δ ppm 7.98−7.96 (m, 1 H), 7.58−7.56 (d, J = 6.8 Hz, 1 H), 7.44−7.40 (m, 2 H), 7.32−7.30 (t, 1 H), 7.19 (m, 2 H), 5.99 (s, 1 H), 5.54 (s, 2 H), 4.98−4.97 (d, J = 6.0, 4 H), 4.64−4.63 (m, 2 H), 3.51 (s, 2 H), 3.30 (s, 2 H), 2.34 (s, 3 H). N-[2-(1,1-Dioxido-2,3-dihydro-1,4-benzothiazepin-4(5H)yl)-6-methylquinolin-4-yl]-N′-(oxetan-3-yl)ethane-1,2-diamine (7s). MS obsd (ESI+) [(M + H)+] 453, 1H NMR (400 MHz, CD3OD) δ ppm 8.10 (dd, J = 1.01, 7.83 Hz, 1 H), 7.88 (br d, J = 6.57 Hz, 2 H), 7.51−7.75 (m, 4 H), 6.03 (s, 1 H), 5.30 (s, 2 H), 4.76−4.87 (m, 2 H), 4.42−4.65 (m, 4 H), 3.99−4.22 (m, 1 H), 3.66−3.90 (m, 2 H), 3.48−3.66 (m, 2 H), 2.94 (br s, 2 H), 2.48 (s, 3 H). CPE Assay. CPE assay was performed to assess the protective effects of compounds on cell viability. Plates (96-well) were seeded with 6000 Hep-2 cells per well. Cells were infected the next day with RSV at MOI 0.02 to produce an approximately 90% cytopathic effect after 5 days. Cells were incubated during this period in the presence or absence of serial dilutions of compounds. The viability of cells was assessed after 5 days by CCK-8 (Dojindo Molecular Technologies, Inc.). Results were expressed as 50% effective concentrations (EC50) and 50% cell cytotoxicity (CC50) values. Plaque reduction assays were carried out by infecting Hep-2 cell monolayers with 0.5 mL of 200 PFU/mL of RSV Long strain per well of a 12-well plate with or without the presence of serial diluted compounds. After 2 h, cells were overlaid with DMEM/F12 containing 4% FBS and 0.55% agarose and compounds. Plates were incubated for 3 days, and cells were then fixed with 4% paraformaldehyde for 6 h. The agarose plugs were removed, and viral plaques were visualized by immunostaining. Cells were blocked with 1× TBS buffer with 1% BSA−0.5% Triton X-100. Plates were then incubated in the presence of a mouse anti-RSV monoclonal antibody (NCL-RSV3; Novocastra) at 1:300 dilution followed by a rabbit anti-mouse horseradish peroxidase-labeled 10239



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secondary antibody. The plaque staining was developed with 4chloro-1-naphthol in the presence of hydrogen peroxide, and plaques were counted. In Vivo Efficacy Study. Six-week-old female BALB/c mice were purchased from Jackson Laboratories and fed a standard diet and water ad libitum. Animals were housed in specific-pathogen-free conditions. The Animal Care Committee of Roche Innovation Center China approved the protocol. Animals were anesthetized intraperitoneally with ketamine/xylazine before any intranasal administration. RSV Long strain (5 × 105 plaque-forming units [PFU] for all animal experiments), drugs, and controls were given in 100 μL volumes. Animals were euthanized with CO2, and lungs were harvested. For histopathologic analysis, the left lower lobe of the lung was removed and inflated with 10% formalin. Specimens were fixed, paraffin embedded, stained, and analyzed.

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petroleum ether; EA, ethyl acetate; HPLC, high performance liquid chromatography; IPA, isopropanol; PD, pharmacodynamics; PK, pharmacokinetics; prep-HPLC, preparative high performance liquid chromatography; SD, standard deviation

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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.jmedchem.8b01394. Coordinates information for structure representation (PDB) Detailed experimental procedures for the synthesis of analogues 2f, 2g, and 7n and intermediates 6b-c and 6cc; 1H NMR, 13C NMR, DEPT-135, H−H COSY, HSQC, and HMBC spectra of compound 1; and pharmacokinetic studies (PDF) Molecular formula strings of all the compounds in this article and some data (CSV)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 21 28946727. Fax: +86 21 50790293. E-mail: [email protected]. ORCID

Song Feng: 0000-0003-4170-4058 Tao Guo: 0000-0001-6055-0985 Hong C. Shen: 0000-0002-3477-8932 Hongying Yun: 0000-0003-0518-2221 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Qingshan Gao, Ying Ji, Wei Li, Wenzhi Chen, Yang Lu, Yongguo Li, Liqin Chen, Peilan Ding, Wei Zhang, Hongxia Qiu, Yi Zhang, Yuxia Zhang, Sheng Zhong, Ki Sun, Rong Zhao, Shuang Ren, Jian Xin, and Rong Zhao for purification of the final compounds and the analytical assistance. We thank the process group, including Jin She and Yi Ren, for their assistance with large scale synthesis campaigns. We also thank Kunlun Xiang for CPE, plaque reduction, and resistant mutation selection assays. In addition, we thank Wuxi and ChemPartner chemists for working together with us in some building block synthesis.

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ABBREVIATIONS USELD RSV, respiratory syncytial virus; RSV F, respiratory fusion; BAQ, benzoazepinequinoline; PAMPA, parallel artificial membrane permeability assay; LYSA, lyophilization solubility assay; ADME, absorption, distribution, metabolism, and excretion; mlLogD, machine learning log D; PPB, plasma protein binding; SDPK, single-dose pharmacokinetics; PE, 10240



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(17) Stevens, M.; Rusch, S.; DeVincenzo, J.; Kim, Y.-I.; Harrison, L.; Meals, E. A.; Boyers, A.; Fok-Seang, J.; Huntjens, D.; Lounis, N.; Mariën, K.; Remmerie, B.; Roymans, D.; Koul, A.; Verloes, R. Antiviral activity of oral JNJ-53718678 in healthy adult volunteers challenged with respiratory syncytial virus: a placebo-controlled study. J. Infect. Dis. 2018, 218 (5), 748−756. (18) Zheng, X.; Wang, L.; Wang, B.; Miao, K.; Xiang, K.; Feng, S.; Gao, L.; Shen, H. C.; Yun, H. Discovery of piperazinylquinoline derivatives as novel respiratory syncytial virus fusion inhibitors. ACS Med. Chem. Lett. 2016, 7, 558−562. (19) Liang, C.; Yun, H.; Wang, L.; Gao, L.; Feng, S.; Wong, J. C.; Wu, J. Z.; Liu, Y.; Feng, L.; Chen, L.; Huang, M.; Guo, T.; Wu, X.; Zheng, X. Compounds for the Treatment and Prophylaxis of Respiratory Syncytial Virus Disease. WO2013/020993A1, Feb 14, 2013. (20) Burkhard, J. A.; Wuitschik, G.; Rogers-Evans, M.; Muller, K.; Carreira, E. M. Oxetanes as versatile elements in drug discovery and synthesis. Angew. Chem., Int. Ed. 2010, 49, 9052. (21) Hanada, K.; Akimoto, S.; Mitsui, K.; Mihara, K.; Ogata, H. Enantioselective tissue distribution of the basic drugs disopyramide, flecainide and verapamil in rats: role of plasma protein and tissue phosphatidylserine binding. Pharm. Res. 1998, 15, 1250−1256. (22) Battles, M. B.; Langedijk, J. P.; Furmanova-Hollenstein, P.; Chaiwatpongsakorn, S.; Costello, H. M.; Kwanten, L.; Vranckx, L.; Vink, P.; Jaensch, S.; Jonckers, T. H. M.; Koul, A.; Arnoult, E.; Peeples, M. E.; Roymans, D.; McLellan, J. S. Molecular mechanism of respiratory syncytial virus fusion inhibitors. Nat. Chem. Biol. 2016, 12, 87−93.

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Discovery of Benzoazepinequinoline (BAQ) Derivatives as Novel

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