Highly Selective, Potent, and Oral mTOR Inhibitor for Treatment of

Article Cite This: J. Med. Chem. 2018, 61, 881−904

pubs.acs.org/jmc

Highly Selective, Potent, and Oral mTOR Inhibitor for Treatment of Cancer as Autophagy Inducer Qingxiang Guo,†,# Chenhua Yu,†,# Chao Zhang,† Yongtao Li,† Tianqi Wang,† Zhi Huang,† Xin Wang,† Wei Zhou,† Yao Li,† Zhongxiang Qin,† Cheng Wang,† Ruifang Gao,† Yongwei Nie,† Yakun Ma,† Yi Shi,† Jianyu Zheng,∥ Shengyong Yang,⊥ Yan Fan,*,†,‡ and Rong Xiang*,†,§ †

School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China International Collaborative Laboratory of Biomedicine of the Ministry of Education, 94 Weijin Road, Tianjin 300071, China § 2011 Project Collaborative Innovation Center for Biotherapy of Ministry of Education, 94 Weijin Road, Tianjin 300071, China ∥ State Key Laboratory and Institute of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, China ⊥ State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu 610041, China ‡

S Supporting Information *

ABSTRACT: On the basis of novel pyrazino[2,3-c]quinolin-2(1H)-one scaffold, we designed and identified a highly selective, potent and oral mTOR inhibitor, 9m. Compound 9m showed low nanomolar activity against mTOR (IC50 = 7 nM) and greater selectivity over the related PIKK family kinases, which demonstrated only modest activity against 3 out of the 409 protein kinases. In vitro assays, compound 9m exhibited high potency against human breast and cervical cancer cells and induced tumor cell cycle arrest and autophagy. 9m inhibited cellular phosphorylation of mTORC1 (pS6 and p4E-BP1) and mTORC2 (pAKT (S473)) substrates. In T-47D xenograft mouse model, oral administration of compound 9m led to significant tumor regression without obvious toxicity. In addition, this compound showed good pharmacokinetics. Collectively, due to its high potency and selectivity, compound 9m could be used as a mTOR drug candidate.

1. INTRODUCTION The mammalian target of rapamycin (mTOR) signaling pathway relevant to both extracellular and intracellular signals (convey hypoxic stress, energy, and nutrient status, etc.), regulate cellular growth, proliferation, and survival.1−3 mTOR kinase exists two distinct multiprotein signaling complexes, mTORC1 and mTORC2. mTORC1 is a key mediator for protein synthesis and growth through phosphorylation of S6K1 and 4E-BP1.4−6 mTORC2 complex is responsible for phosphorylating and activating AKT, a key kinase in the control of cell growth, metabolism, and survival.7 mTORC1 and mTORC2 serve as critical mediators of the PI3K/AKT and Ras/MAPK signaling pathways that are frequently dysregulated in many human cancers. Several rapamycin analogues (rapalogues) that are selective allosteric mTORC1 inhibitors have been extensively evaluated in a number of cancer clinical trials, and some of them have been approved for clinical use in certain cancer treatment.8,9 However, these rapalogues do not fully inhibit mTORC1 and are unable to directly inhibit mTORC2.6,10 Besides, upregulation of Akt through a negative feedback loop, which exists through S6K/ IRS1/PI3K, may also attribute to limit the efficacy of the rapalogues.11 Therefore, ATP-competitive mTOR kinase © 2018 American Chemical Society

inhibitors that simultaneously inhibit both mTORC1 and mTORC2 may expand therapeutic potential. mTOR, DNA-PK, ATM, and ATR as atypical class of protein kinases together with PI3Ks are key components of the phosphoinositide 3-kinase-related kinase (PIKK) family and share the highly conserved ATP binding pockets with sequence similarity of 25% in the kinase catalytic domain.12 On the basis of this knowledge, many of the previously reported ATP-competitive mTOR inhibitors (Figure S1 in Supporting Information) also inhibited PI3Ks, DNA-PK, ATM, and ATR.13−2213,16,17,23−33 PI3Ks regulated the production of 3-phosphoinositide lipid second messengers (PIP3), which are involved in cell proliferation, cell survival, angiogenesis, cell adhesion, and insulin signaling.13 ATR is responsible for a wide range of critical functions such as cell cycle checkpoint activation and DNA damage repair.34 Therefore, the development of ATP-competitive mTOR inhibitors that are selective over PI3Ks and ATR offers an improved therapeutic potential compared to rapalogues as well as panmTOR inhibitors. Received: September 21, 2017 Published: January 8, 2018 881

DOI: 10.1021/acs.jmedchem.7b01402 J. Med. Chem. 2018, 61, 881−904

Journal of Medicinal Chemistry

Article

(12c), methoxyacetyl (12d), the mTOR inhibitory activities were maintained or increased. In order to determine whether the orientation of acetonitrile group was responsible for the mTOR inhibitory activity, we introduced substituted five/six-membered N-heterocyclic ring to replace the piperidine. It turns out that compounds 9c−h bearing (S)-3-methylpiperidine, 4-methylpiperidine, (S)-3-methylpyrrolidine moiety had significantly decreased mTOR activity, which indicated that orientation of acetonitrile is critical for mTOR inhibitory activity. Interestingly, when the fluorine atom on quinoline scaffold was replaced by hydrogen (9i−l), the mTOR activity was recovered. Furthermore, we fixed R5 as the optimal 4-(piperidin-1yl)acetonitrile group, R1 = H (or F) and altered the R6 group with different subgroups, including hydrogen, quinolyl, naphthyl, phenyl, pyridyl, pyrimidyl, substituted phenyl and pyridyl, fivemembered heterocyclic rings. The chemical structures and bioactivities of compounds 6h, 6j, 9a,b, and 9m−af are shown in Table 2. When R1 = R6 = H (6h, 6j), the mTOR inhibitory activities were decreased distinctly. Replacement of the quinoline side chain at the R6 position with quinolyl group (9a, 9b, 9m, 9n) maintains or increases the activity, but it is decreased when R6 is naphthyl, phenyl group, 3-pyridyl, 4-pyridyl, 5- pyrimidyl, 1-methyl-6-oxo1,6-dihydropyridin-3-yl (9o−s, 9af), suggesting that nitrogen atom and its position are important to the activity. When the para position of phenyl group was substituted by methoxyl or amino group (9aa, 9ab), the mTOR activities are slightly recovered. We removed the amino group of 9a and found that the mTOR activity of 9p decreased. The inhibitory activity was not recovered when we changed the amino group to 4-methylpiperazin-1-yl (9t), morpholinyl (9u), methyl (9ac), trifluoromethyl (9ad), cyano group (9y), except the methoxyl group (9z), indicating that the amino and methoxyl groups are in favor of the activities. In addition, compounds containing 1H-pyrazol-4-yl (9v), 1-methyl1H-pyrazol-4-yl (9w), 1-(difluoromethyl)-1H-pyrazol-4-yl (9x) group at the R6 position had maintained the mTOR activities. Compound with furan-3-yl (9ae) group at the R6 position seemed to have decreased activity. Collectively, compounds 9a, 9b, 9m, 9n, 9i−l, 9v−x, 9z−ac, 9ag, 12a−d show considerable potency against mTOR in enzymatic assays. These compounds were chosen in further studies. 2.3. Cellular Assays. In order to find the most promising lead compound, 9a, 9b, 9m, 9n, 9i−l, 9v−x, 9z−ac, 9ag, 12a−d, with mTOR inhibitory activities over 95% in enzymatic assays, were chosen to test their celluar cytotoxic activity. Breast cancer cell line (T-47D) inhibition profiling assay with a fixed concentration of 5 μM and 1 μM was first carried out. We found that compounds 9a, 9b, 9m, 9i, 9j, 9x, 9z, 9aa, 9ac, 12d had significant T-47D inhibitory activities (Table S1). We further confirmed and detected the further dose that inhibits 50% of the cells present in the control wells (IC50) values of these compounds using standard CCK8 assay. Among these compounds, 9m, bearing hydrogen, 4-(piperidin-1-yl)acetonitrile, 6-aminopyridin-3-yl groups at the R1, R5, and R6 positions, respectively, showed the highest potency in breast cancer cell lines T-47D and MCF7 (Table S2). Subsequently, we performed all in vitro and vivo biological studies using this compound. The compound was next evaluated in a dose−response study against breast cancer cell lines (MCF7, T-47D, and MDA-MB-231), cervical cancer cell lines (HeLa and SiHa), ovarian cancer cell lines (Skov3 and OVCAR-5), and lung cancer cell line (H460) to test cellular cytotoxic activity (Table 3). The typical mTOR inhibitor rapamycin was used as a positive control. Compound 9m showed potent profile in vitro, with about 100 nM IC50 value in

In our study, we identified novel hit compound 9a bearing pyrazino[2,3-c]quinolin-2(1H)-one scaffold through highthroughput screening (HTS) of our in-house compound library against mTOR based on the previously reported method.35 Compound 9a exhibits a good IC50 value of 31 nM against mTOR but weak inhibitory activities against human cancer cells in vitro (Table S1), which required further optimization. Here, we conducted structural optimization and structure−activity relationship (SAR) analyses of compound 9a and explored highly selective, potent, and oral mTOR inhibitor for treatment of cancer as autophagy inducers.

2. RESULTS AND DISCUSSION 2.1. Chemistry. Structural optimization was focused on the R1, R5, and R6 regions of 9a (Figure 1). First, we synthesized the

Figure 1. Chemical structure of 9a (left) and the regions of structural optimization based on 9a (right).

key intermediates 6a−j and the general synthetic routes are illustrated in Scheme 1. Briefly, commercially available 1 reacted with various aliphatic amines to provide 2a−h, yielding from 65% to 96%. The nitro groups of 2a−h were reduced to produce and afford the corresponding amines 3a−h (36−76% yield). After that, 3a−h underwent two step condensations, aldimine condensation, and amide condensation, with glyoxylic acid monohydrate to yield intermediates 4a−h (26−82% yield). Intermediates 6a−j were obtained by deprotection of 4a−h followed by reacting with bromoacetonitrile or isocyanatotrimethylsilane (41−89% yield). Then, we synthesized the title compounds 8a−i, 9a−ag, 12a−d. The synthetic routes of these compounds are outlined in Scheme 2, Scheme 3, and Scheme 4. Amide condensation between 5a and diverse carboxylic acid was done to obtain 7a−h (34−76% yield). Nucleophilic substitution reaction between compound 5a and methyl 2-bromoacetate provided intermediate 7i. Final product compounds 8a−i, 9a−ag and intermediate 10 were readily obtained through the classic palladium-catalyzed Suzuki coupling between 4e, 7a−i, 6a−g, 6i, and relevant boronic acid/ester (22−96% yield). Title compounds 12a−d were obtained by deprotection of 10 followed by reacting with formaldehyde, 2-bromoethan-1-ol, 2-bromoacetamide, and methyl 2-bromoacetate. 2.2. Enzyme Inhibitory Activity and SAR. We initiated our SAR from R5 region. We fixed R6 as the original 2-aminopyridinyl-5-yl group and varied the R5 group substituent. As shown in Table 1, substitutions at the R5 position substantially impacted the activity. Compared with 9a, when R1 position is F, compounds with cyclopropanecarbonyl (8a), propionyl (8b), benzoyl (8c), N,N-dimethycarbonyl (8d), N,N-dimethyl-2oxoacetyl (8e), 4-(dimethylamino)benzoyl (8f), 3-methylbenzoyl (8g), or 2-hydroxypropanoyl (8h) groups at the piperidine nitrongen position of R5 all decreased mTOR inhibitory activities, revealing that the acetonitrile group at the piperidine nitrongen position of R5 may be optimal substituent. When R1 position is H and the acetonitrile group of R5 replaced by carboxamide (9ag), formyl (12a), hydroxyethyl (12b), acetamide 882



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Scheme 1a

Reagents and conditions: (A) R3-NH2, DIEA, DMF; (B) Fe/AcOH, 60 °C; (C) glyoxylic acid monohydrate, HATU, DIEA, THF; (D) HCl(conc), chloroform; (E) bromoacetonitrile, DIEA, DMF; (F) isocyanatotrimethylsilane, DIEA, DCM.

a

In this investigation, these data revealed that 9m is a highly selective and potent inhibitor against mTOR, which showed complete selectivity over the closely related ATR and PI3Ks and much more pronounced selectivity over other kinases in a preliminary screen. 2.5. Molecular Modeling of Compound 9m. The observed SAR demonstrates that a substituent with suitable hydrogen bond donor at the R6 position of the quinoline scaffold increases the binding affinity. Substitutions of the saturated nitrogen heterocyclic ring with suitable hydrogen acceptor at R5 position also increase the bioactivity. Molecular docking studies were carried out to rationalize the observed SAR and investigate the binding modes of compound 9m in mTOR. Compound 9m was docked into mTOR (PDB code 4jsx) by GOLD (version 5.0). Hydrogen atoms were added to the proteins by using Discovery Studio 3.1 (Accelrys Inc., San Diego, CA, USA). GoldScore was selected as the scoring function, and other parameters were set as default. The image was created using PyMOL.36 The proposed docking model was elucidated in Figure 3. Compound 9m is predicted to engage in a “hinge” hydrogen bond with VAL2240 using the quinoline nitrogen, and tricyclic quinolone core of the compound 9m makes key π-stacking with TRP 2239. The binding mode of compound 9m also reveals that a hydrogen bond is predicted to exist between the amino group

breast and cervical cell lines. It just exhibited weak inhibitory activity against human ovarian and lung cancer cells with over 1 μM IC50 value. The analyses above led to the discovery of a potent mTOR inhibitor that exhibited higher potency than rapamycin in vitro antiviability assays against human breast and cervical cancer. 2.4. Kinase Selectivity Profiling. To characterize the kinase inhibitory activity and selectivity, kinase inhibition profiling assays with a fixed concentration of 1 μM compound 9m were carried out against a series of 409 kinases through the Eurofins kinase profiling. The results are shown in Figure 2 and Table S3. Compound 9m displayed almost no inhibitory activity against most of human protein kinases. It only potently inhibits three kinds of kinases, mTOR, ATM, and DNA-PK. As shown in Table 4, further IC50 values of kinases with high inhibitory rate at 1 μM were examined. A panel of selected PIKK family related kinases are also presented in Table 4. Compound 9m is a highly potent inhibitor against mTOR kinase (mTOR, IC50 = 7 nM and mTOR/FKBP12, IC50 = 8 nM). Additional activity is only observed against the kinase associated with ATM (IC50 = 62 nM) and DNA-PK (IC50 = 26 nM). Compound 9m displayed almost no inhibitory activity against other PIKKrelated kinases ATR and PI3K. 883



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Scheme 2a

a Reagents and conditions: (A) R7-OH, EDCI, HOBt, DIEA, DMF; (B) methyl 2-bromoacetate, DIEA, DCM; (C) (6-aminopyridin-3-yl)boronic acid, Pd(PhP3)2Cl2, Na2CO3, 85 °C.

death that can be induced by many compounds related to mTOR inhibitors, which occurs in multiple processes to exert the effects of either cell death or cytoprotection. Three groups independently demonstrated that mTORC1 inhibits the autophagyinitiating UNC-5 like autophagy activating kinase (ULK) complex by phosphorylating complex components including autophagy related gene 13 (ATG13) and ULK1/2. Inhibition of mTORC1 results in increased ULK1/2 kinase activity. ULK1/2 then phosphorylates ATG13 and FIP200, which are critical subunits of the ULK1/2 kinase complex.38,39 Microtubule-associated protein 1 light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays a critical role in macroautophagy formation and is considered a suitable marker for this process.40Since the transition of LC3-I to LC3-II is an imperative step in autophagy and the amount of LC3-II puncta represents the number of autophagosomes, we utilized Western blot and immunofluorescent to analyze whether autophagy was involved in the process of cell death induced by 9m. The results showed that the protein levels of LC3-II revealed a large increase in breast cancer cells (T-47D, MCF7, and MDA-MB-231) with 0.25 and 0.5 μM 9m treatment for 48 h as compared with rapamycin or untreated controls (Figure 5A). The same phenomenon was observed in cervical cancer cells (SiHa and HeLa) (Figure 5B). Western-blotting findings demonstrated that autophagic cell death could be significantly induced by 9m treatment in breast cancer and cervical cancer cells, which might occur through different signal pathways. Furthermore, the effect of 9m on autophagosome formation in T-47D cells was assessed by measuring the formation of punctate acidic vesicles in the cytoplasm. Immunofluorescence staining for LC3-II further revealed that the number of LC3-II puncta remarkably increased in 9m treated cells for 48 h in comparison to rapamycin and nontreated controls (Figure 5C). A dose dependent increase of LC3-II autophagosome markers was also observed in other different cell lines including MCF7, MDA-MB-231, SiHa, and HeLa (Figure 5C and Figure 5D). 2.8. Effect of Compound 9m on mTOR Signaling Pathway. The ability of compound 9m to inhibit the mTOR downstream signaling proteins in breast and cervical cancer cells was assessed by Western blot analysis. We examined the ability of

of 9m with GLU2190 inside inner hydrophobic pocket. It may explain when the amino or 2-aminopyridinyl-5-yl group was substituted by another group with different size (e.g., 9p−u, 9y, 9ac, 9ad) decreased the bioactivity. Another important hydrogen bond is formed between the cyano group of 9m with LYS 2187 in the N-lobe pocket. This may provide a rationale for the potency decreased in mTOR inhibition compared to compounds 8a−h. This prediction is also consistent with our observations that the different orientation of cyano group (e.g., 9c−l) led to a considerable influence in the potency in comparison with the 4-(piperidin1-yl)acetonitrile group. In order to illustrate the SARs more rational, we select compound 9af as the representative inactive compound to do a molecular docking model with mTOR kinase domain (PDB code 4jsx) in Figure S2. Compared with 9m, the hydrogen bonding interaction between compound 9af and mTOR kinase was too weak, which may lead to the significant inhibition difference between compounds 9m and 9af. In addition, the docking scores of some our synthesized compounds are shown in Table S5. The SARs findings according to the docking pose and the docking scores are coordinating with our SAR results discussed above. 2.6. Effect of Compound 9m on Cell Cycle Progression. As reported previously, mTOR is a coordinator of cell fundamental biological processes, which regulates both cell growth and cell cycle progression.37 We therefore wondered whether our compound partly resulted in cell cycle inhibition. We next investigated the effect of compound 9m treatment on breast cancer cells (T-47D, MCF7, and MDA-MB-231) and cervical cancer cells (SiHa and HeLa) (Figure 4). Cells were treated with compound 9m with varying concentrations, rapamycin, and vehicle (DMSO) for 24 h according to IC50 values. Similar effects were observed in five type cell lines. Compared to the vehicle and rapamycin treated group control, cancer cells treated with compound 9m demonstrated a loss of S-phase cells and an increase in the percentage of cells in G0/G1 phase. It was evident that compound 9m blocked the tumor cells in G1-phase progression, which resulted in decreased S-phase populations. 2.7. Ability of Compound 9m To Induce Autophagy in Vitro. Autophagy is a kind of nonapoptotic programmed cell 884



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Scheme 3a

a

Reagents and conditions: (A) aryl/heteroaryl-boronic acid/ester, Pd(PhP3)2Cl2, Na2CO3, 85 °C.

Scheme 4a

Reagents and conditions: (A) (6-aminopyridin-3-yl)boronic acid, Pd(PhP3)2Cl2, Na2CO3, 85 °C; (B) HCl (conc), chloroform; (C) HCOOH, TBTU, DIEA, DMF; (D) 2-bromoethan-1-ol, NaHCO3, DMF, 70 °C; (E) 2-bromoacetamide, DIEA, DCM; (F) methyl 2-bromoacetate, DIEA, DMF, rt.

a

target of mTORC2. As shown in Figure 6, compound 9m potently blocks the phosphorylation of Akt at S473 site, 4E-BP1 at both Thr 37/46 and Ser65 sites, S6 at Ser240/244 site. As

9m to inhibit phosphorylation of Akt, 4E-BP1, and S6. As known, Ser240/244 of S6K and Thr 37/46 and Ser65 of 4E-BP1 are downstream targets of mTORC1. Ser473 of Akt is a downstream 885



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Table 1. Structures and Enzymatic Inhibition of mTOR at 1μM 8a−i, 9a, 9c−l, 9ag, 12a−da

a

Inhibition activities were determined using the KinaseProfiler of Eurofins. The data represent the mean values of two independent experiments.

expected, treatment with 9m at 0.5 and 1 μmol/L was able to maintain stronger suppression of both mTORC1 and mTORC2

than treatment with DMSO and rapamycin. From here, it is reasonable to conclude that one of the main causes that 886



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Table 2. Structures and Enzymatic Inhibition of mTOR at 1μM 6h, 6j, 9a,b, and 9m−afa

a

Inhibition activities were determined using the KinaseProfiler of Eurofins. The data represent the mean values of two independent experiments. 887



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Table 3. In Vitro Growth Inhibitory Activities (IC50) of Compound 9m and Rapamycin against Cancer Cell Lines

Table 4. Kinase Inhibition Profile of 9m against Selected Protein Kinasesa

IC50 a (nM)

kinase

cell line

9m

rapamycin

MCF7 T-47D MDA-MB-231 HeLa SiHa Skov3 OVCAR-5 H460

103 205 100 441 110 1850 2831 1775

4980 1576 >10000 >10000 1756 >10000 >10000 >10000

mTOR(h) mTOR/FKBP12(h) PIKK family ATM(h) DNA-PK(h)

a

IC50: the dose that inhibits 50% of the cells present in the control wells. The IC50 values are the average of at least three independent determinations.

IC50 (nM) 7 8

kinase

62 26 inhibition (%) at 1 μM

ATR/ATRIP(h) PI3 kinase (p110β/p85α)(h) PI3 kinase (p120γ)(h) PI3 kinase (p110δ/p85α)(h) PI3 kinase (p110α/p85α)(m) PI3 kinase (p110α/p65α)(m) PI3 kinase (p110α(E545 K)/p85α)(m) PI3 kinase (p110α(H1047R)/p85α)(m) PI3 kinase (p110β/p85β)(m) PI3 kinase (p110β/p85α)(m) PI3 kinase (p110δ/p85α)(m) PI3 kinase (p110α(E542 K)/p85α)(m) PI3 kinase (p110α/p85α)(h) PI3 kinase (p110α(E542 K)/p85α)(h) PI3 kinase (p110α(H1047R)/p85α)(h) PI3 kinase (p110α(E545 K)/p85α)(h) PI3 kinase (p110α/p65α)(h) PI3KC2α(h)

24 7 4 9 17 9 9 7 8 5 4 10 10 11 2 13 5 4

a

IC50 values were determined using KinaseProfiler by Eurofins. The data represent the mean values of two independent experiments

and rapamycin every day for the entire observation period. As shown in Figure 7A and Figure 7C, at the end of the observation period, the growth of xenograft tumors was significantly inhibited by 9m in a dose-dependent manner. Tumor growth inhibitions of 83.7%, 75.6%, 63.2% were observed in the T-47D model at doses of 60, 30, and 15 mg/kg, respectively. In contrast, rapamycin as the positive control was less potent (72.4%) than 9m at the same dose. The average tumor weight (Figure 7D) of the 9m-treated group(60 mg/kg) was 0.194 g compared with 1.23 g for control group, which was less than that of rapamycin-treated group (0.36g). Moreover, it also can be seen from Figure 7B that 9m did not cause significant weight loss and toxicity during the treatment period. To further confirm the ability of 9m to block the mTOR activity in vivo, immunohistochemical (IHC) analyses of T-47D tumors collected were carried out. As shown in Figure 7E, 9m caused a dose-dependent decrease of P-AKT (Ser473), P-4EBP1(Thr37/46), P-4E-BP1(Ser65), and P-S6(Ser240/244) in vivo. In addition, dose-dependent increases in staining for LC3B were observed, which confirmed a pronounced increase in tumor cell autophagy. 2.10. Pharmacokinetic Characteristics of Compound 9m in Rats. In light of the excellent antitumor activities of compound 9m both in vitro and in vivo, pharmacokinetic evaluation in rats following intravenous and oral administration of this compound was further evaluated (Table 5). Compound 9m showed a good oral bioavailability of 43.50% with AUC0−∞ = 2853.41 (μg/L)·h. The oral maximum plasma concentration (Cmax) was 807.24 μg/L, and (Tmax) was 3 h. The oral half-life (T1/2) was 9.98 h, and mean residence time (MRT0−∞) was 16.31 h. Besides, compound 9m showed oral apparent distribution volume (Vss) of 21.79 L/kg and a clearance of 3.51 L h−1 kg−1.

Figure 2. Kinase binding selectivity for compound 9m shown on the human kinome dendrogram. The inhibition rates were determined using the KinaseProfiler of Eurofins. The figure was generated by using an online KinMap program (http://kinhub.org/kinmap/).

compound 9m showed a higher antiproliferation activity against tumor cells than rapamycin could be due to it being able to inhibit both mTORC1 and mTORC2 signaling more efficiently. 2.9. In Vivo Effects of Compound 9m. To determine the in vivo antitumor effect of 9m, BALB/c nude mice bearing T-47D xenograft tumors were treated with 9m by intragastric administration per day for 3 weeks, respectively. The mice were randomly allocated to 5 groups with 5 mice in each groups (vehicle-treated, rapamycin-treated 60 mg/kg, 9m-treated 15 mg/kg, 9m-treated 30 mg/kg, 9m-treated 60 mg/kg). After the tumor volume was 100−120 mm3 in each group, the mice were given a gavage of 9m 888



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Figure 3. Representation of the predicted binding modes of compound 9m with mTOR kinase domain (PDB code 4jsx). (A) Proposed binding modes of compound 9m with mTOR. 9m is shown in orange, and mTOR backbone is shown in cyan. Hydrogen bond is shown in red. (B) Predicted key interactions of 9m with the ATP-binding pocket of mTOR kinase domain.

once-daily dose of compound 9m led to significant tumor regression in the T-47D xenograft model. In addition, this compound showed good pharmacokinetics. All of the data presented here support 9m as a novel and selective mTOR drug candidate and deserve further research and development.

All of these results indicated that 9m presented favorable pharmacokinetic properties which may be a promising lead compound for the treatment of tumor diseases. 2.11. Plasma Protein Binding of 9m in Rabbit and Human Plasma. We also tested plasma protein binding of 9m, which is also a key pharmacokinetic property. Compound 9m was tested for the ability to bind to plasma proteins over a 4 h time period (Table S4). At 4 μM concentration of 9m, 60.1% and 64.0% bound to rabbit and human plasma protein, respectively. Detailed experimental results and protocol can be found in the Supporting Information. The moderate plasma protein binding of compound 9m is considered favorable from a drug development perspective.

4. EXPERIMENTAL SECTION 4.1. Chemistry Methods. 1H NMR (400 MHz) and 13C NMR (101 MHz) spectra were taken on a Bruker AV-400 MHz spectrometer, and chemical shifts were reported in ppm downfield from internal Me4Si. High-resolution mass spectra (HRMS) were recorded on a VG ZAB-HS mass spectrometer under electron spray ionization (ESI). All of the solvents were purified and distilled according to the standard procedure. The commercially obtained materials were used directly without further purification unless otherwise noted. The purity of tested compound was assessed to be >95% by HPLC analysis on a Shimadzu Prominence-i LC-2030C 3D system (column, InertSustain C18, 4.6 mm × 250 mm, 5 μm; mobile phase, gradient elution of methanol/H2O; low rate, 1.0 mL/min; UV wavelength, 190−800 nm; temperature, 40 °C; injection volume, 10 μL). 4.1.1. tert-Butyl 4-((6-Bromo-7-fluoro-3-nitroquinolin-4-yl)amino)piperidine-1-carboxylate (2a).41 DIPEA (22 mL, 130.94 mmol) was added to a stirred solution of 6-bromo-4-chloro-7-fluoro-3-nitroquinoline (20 g, 65.47 mmol) and tert-butyl 4-aminopiperidine-1-carboxylate (14.4 g, 72.02 mmol) in DMF (150 mL), stirred 6 h at rt. After completion of reaction, diluted with water (150 mL), the precipitated solid was filtered off, washed with water, and dried uader vacuum. The crude product was purified by silica gel column chromatography to afford intermediates 2a in 88% yield as a yellow solid,. 1H NMR (400 MHz, chloroform-d) δ 9.38 (d, J = 8.6 Hz, 1H), 9.34 (s, 1H), 8.36

3. CONCLUSION Through a process of structural optimization toward hit compound 9a scaffold, we finally discovered a novel mTOR inhibitor, 9m. This compound is a 7 nM mTOR inhibitor with obvious selectivity over the related PIKK, which showed only modest activity against 3 out of the 409 protein kinases. In in vitro antiviability assays, 9m showed potent activities against human breast and cervical cancer cell lines. This compound also demonstrated an ability to induce tumor cell cycle G0/G1 phase arrest and autophagy in vitro. Western blot analysis demonstrated that compound 9m significantly inhibited the phosphorylation of Akt, 4E-BP1, and S6, which are downstream targets of mTORC1 and mTORC2. In vivo antitumor activity assays showed that an oral, 889



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Figure 4. 9m induces cell cycle arrest in breast cancer and cervical cancer cells. (A, B) Cell cycle analysis. Cells were seeded in 6-well plates at a density of 5 × 105 cells/mL and treated with increasing doses of 9m or rapamycin for 24 h. Then cells were fixed with 70% ethanol overnight at 4 °C and analyzed by flow cytometry after propidium iodide staining. (C, D) The percentage of cell cycle distribution was calculated by GraphPad software. 9m induced G1 arrest is remarkably accompanied by reduction of S phase. δ 162.08, 159.53, 154.55, 151.71, 151.59, 148.98, 148.67, 131.34, 131.31, 127.46, 117.52, 116.06, 115.85, 108.93, 108.70, 80.35, 55.72, 33.58, 28.50.

(d, J = 7.0 Hz, 1H), 7.68 (d, J = 9.0 Hz, 1H), 4.29−4.16 (m, 1H), 4.06 (d, J = 13.5 Hz, 2H), 3.06 (t, J = 12.1 Hz, 2H), 2.22−2.09 (m, 2H), 1.80−1.65 (m, 2H), 1.46 (s, 9H); 13C NMR (101 MHz, CDCl3) 890



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Figure 5. 9m induces cell autophagy in vitro. (A, B) Autophagy-related protein LC3B-II was detected in T-47D, MCF7, MDA-MB-231, SiHa, and HeLa cells by Western blot after treatment with different concentrations of 9m and rapamycin for 2 days. β-Actin was used as an internal control. (C, D) Immunofluorescence analysis for LC3B-II in T-47D, MCF7, MDA-MB-231, SiHa, and HeLa cells. Cells were exposed to 9m (0.25 μM, 0.5 nM, 1 μM, 2 μM) for 2 days and then fixed and immunofluorescence-stained using specific antibody against LC3B-II (green). DAPI (blue) was used for nuclear staining. Bars represent 20 μM. 4.1.2. tert-Butyl (R)-3-((6-Bromo-7-fluoro-3-nitroquinolin-4-yl)amino)piperidine-1-carboxylate (2b). The desired compound was prepared from 6-bromo-4-chloro-7-fluoro-3-nitroquinoline (5 g, 16.37 mmol) and tert-butyl (R)-3-aminopiperidine-1-carboxylate (3.6 g, 18.01 mmol) using the procedure described for compound 2a in 87% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.51 (d, J = 8.7 Hz, 1H), 9.34 (s, 1H), 8.42 (d, J = 7.0 Hz, 1H), 7.68 (d, J = 9.0 Hz, 1H), 4.26 (tp, J = 7.0, 3.3 Hz, 1H), 3.80 (d, J = 13.4 Hz, 1H), 3.46 (q, J = 6.2 Hz, 3H), 2.18−2.08 (m, 1H), 1.93−1.78 (m, 2H), 1.71−1.60 (m, 1H), 1.43 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 162.06, 159.50, 154.76, 151.66, 151.54, 149.03, 148.61, 131.25, 127.57, 117.63, 115.99, 115.77, 109.10, 108.86, 80.70, 53.72, 31.64, 28.39, 22.48. 4.1.3. tert-Butyl 4-(((6-Bromo-7-fluoro-3-nitroquinolin-4-yl)amino)methyl)piperidine-1-carboxylate (2c). The desired compound was prepared from 6-bromo-4-chloro-7-fluoro-3-nitroquinoline (3 g, 9.82 mmol) and tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (2.1 g, 9.82 mmol) using the procedure described for compound 2a in 65% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.73 (s, 1H), 9.30 (s, 1H), 8.46 (d, J = 6.6 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H),

4.38−4.00 (m, 2H), 3.91−3.67 (m, 2H), 2.91−2.58 (m, 2H), 1.88 (dd, J = 30.8, 11.0 Hz, 3H), 1.44 (s, 9H), 1.35−1.20 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 162.00, 159.45, 154.65, 151.82, 151.70, 149.88, 148.67, 131.89, 126.09, 117.19, 115.84, 115.63, 108.24, 108.01, 79.80, 55.04, 38.08, 29.81, 28.48. 4.1.4. tert-Butyl (S)-3-(((6-Bromo-7-fluoro-3-nitroquinolin-4-yl)amino)methyl)pyrrolidine-1-carboxylate (2d). The desired compound was prepared from 6-bromo-4-chloro-7-fluoro-3-nitroquinoline (3 g, 9.82 mmol) and tert-butyl (S)-3-(aminomethyl)pyrrolidine1-carboxylate (1.9 g, 9.82 mmol) using the procedure described for compound 2a in 73% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.68 (d, J = 5.0 Hz, 1H), 9.33 (s, 1H), 8.45 (d, J = 7.2 Hz, 1H), 7.67 (d, J = 9.3 Hz, 1H), 3.92 (d, J = 22.9 Hz, 2H), 3.67 (d, J = 9.1 Hz, 1H), 3.51 (d, J = 12.9 Hz, 1H), 3.45−3.31 (m, 1H), 3.22−3.09 (m, 1H), 2.65 (s, 1H), 2.21 (dq, J = 12.1, 6.2 Hz, 1H), 1.84−1.68 (m, 1H), 1.44 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 162.09, 159.55, 154.41, 151.87, 151.75, 149.69, 148.63, 131.71, 126.35, 117.23, 115.96, 115.74, 108.55, 108.31, 79.84, 51.98, 49.34, 49.01, 45.24, 45.01, 40.54, 39.60, 29.59, 28.94, 28.58. 891



Article

Figure 6. 9m is a selective inhibitor of mTOR. (A−C) Breast cancer cells were treated with the indicated concentrations of 9m and rapamycin, and then the whole cell lysates were analyzed by Western blot with the indicated antibodies. By use of immunoblotting, the inhibitory effect of 9m and rapamycin on mTORC1 and mTORC2 activity was assessed by monitoring S6 and 4E-BP1 phosphorylation (regarded as mTORC1) and AKT (S473) phosphorylation (regarded as mTORC2). (D, E) HeLa and SiHa cells were treated as in parts A−C. 4.1.5. tert-Butyl 4-((6-Bromo-3-nitroquinolin-4-yl)amino)piperidine-1-carboxylate (2e). The desired compound was prepared from 6-bromo-4-chloro-3-nitroquinoline (30 g, 104 mmol) and tertbutyl 4-aminopiperidine-1-carboxylate (23 g, 114 mmol) using the procedure described for compound 2a in 93% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.37−9.19 (m, 1H), 8.23 (s, 1H), 7.91−7.68 (m, 1H), 4.22 (tq, J = 12.7, 7.8, 5.8 Hz, 1H), 4.13−3.94 (m, 1H), 3.03 (t, J = 12.5 Hz, 1H), 2.21−2.04 (m, 2H), 1.76−1.62 (m, 1H), 1.44 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 154.48, 149.10, 149.01, 147.46, 135.78, 132.20, 128.52, 127.59, 120.93, 119.58, 80.15, 77.48, 77.16, 76.84, 55.54, 33.56, 28.43. 4.1.6. tert-Butyl (S)-3-((6-Bromo-3-nitroquinolin-4-yl)amino)piperidine-1-carboxylate (2f). The desired compound was prepared from 6-bromo-4-chloro-3-nitroquinoline (2 g, 6.9 mmol) and tert-butyl (S)-3-aminopiperidine-1-carboxylate (1.4 g, 6.9 mmol) using the procedure described for compound 2a in 96% yield as a yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 9.38 (d, J = 8.8 Hz, 1H), 9.27 (s, 1H), 8.25 (s, 1H), 7.80 (t, J = 7.3 Hz, 2H), 4.26 (qd, J = 7.3, 6.9, 3.3 Hz, 1H), 3.74 (dd, J = 13.5, 3.2 Hz, 1H), 3.54−3.35 (m, 3H), 2.09 (tt, J = 8.4, 4.2 Hz, 1H), 1.92−1.74 (m, 2H), 1.63 (tt, J = 13.1, 5.5 Hz, 1H), 1.40 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 154.64, 149.04, 147.39, 135.81, 132.12, 128.39, 127.69, 121.02, 119.69, 80.48, 31.48, 28.29, 22.31. 4.1.7. tert-Butyl 3-((6-Bromo-3-nitroquinolin-4-yl)amino)pyrrolidine-1-carboxylate (2g). The desired compound was prepared from 6-bromo-4-chloro-3-nitroquinoline (2 g, 6.9 mmol) and tert-butyl 3-aminopyrrolidine-1-carboxylate (1.3 g, 6.9 mmol) using the procedure described for compound 2a in 92% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.47 (dd, J = 41.3, 7.8 Hz, 1H), 9.19 (s, 1H), 8.20 (s, 1H), 7.74 (s, 2H), 4.84−4.66 (m, 1H), 3.78 (dd, J = 11.7, 5.7 Hz, 1H), 3.64−3.39 (m, 3H), 2.47−2.30 (m, 1H), 2.11 (dq, J = 12.0, 5.7 Hz, 1H), 1.40 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 154.14, 148.97,

148.62, 147.26, 135.71, 132.08, 128.34, 126.97, 120.39, 119.52, 80.07, 57.72, 56.74, 52.93, 52.60, 43.85, 43.51, 33.79, 32.98, 28.35. 4.1.8. tert-Butyl 4-((3-Nitroquinolin-4-yl)amino)piperidine-1-carboxylate (2h). The desired compound was prepared from 4-chloro-3nitroquinoline (4 g, 19.23 mmol) and tert-butyl 3-aminopyrrolidine-1carboxylate (4.22 g, 21.10 mmol) using the procedure described for compound 2a in 93% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.33 (d, J = 8.9 Hz, 1H), 9.30 (s, 1H), 8.10 (d, J = 8.5, 1.4 Hz, 1H), 7.96 (d, J = 8.4, 1.4 Hz, 1H), 7.75 (t, J = 8.3, 7.0, 1.4 Hz, 1H), 7.49 (t, J = 8.5, 6.9, 1.4 Hz, 1H), 4.38−4.25 (m, 1H), 4.09−3.95 (m, 2H), 3.01 (t, J = 12.2 Hz, 2H), 2.18−2.05 (m, 2H), 1.74−1.62 (m, 2H), 1.44 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 154.51, 150.39, 150.25, 147.20, 132.80, 130.66, 127.31, 126.11, 126.03, 119.58, 80.13, 55.49, 33.60, 28.44. 4.1.9. tert-Butyl 4-((3-Amino-6-bromo-7-fluoroquinolin-4-yl)amino)piperidine-1-carboxylate (3a). To a stirred solution of 2a (21 g, 44.75 mmol) in AcOH (150 mL), Fe powder (26.69 g, 447.50 mmol) was added in several portions at 60 °C. Upon completion of the reaction, the mixture was filtered off, washed with dichloromethane, concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to afford intermediate 3a in 46% yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 8.47 (s, 1H), 7.96 (d, J = 7.1, 1.3 Hz, 1H), 7.64 (d, J = 9.5, 1.3 Hz, 1H), 4.12 (s, 2H), 3.84 (s, 2H), 3.52−3.36 (m, 2H), 2.73 (t, J = 12.8 Hz, 2H), 1.89 (d, J = 12.5 Hz, 2H), 1.45 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 157.81, 155.35, 154.75, 144.87, 143.73, 143.62, 132.82, 132.52, 124.95, 123.07, 114.83, 114.62, 110.12, 109.89, 79.92, 53.27, 34.00, 28.55. 4.1.10. tert-Butyl (S)-3-((3-Amino-6-bromo-7-fluoroquinolin-4-yl)amino)piperidine-1-carboxylate (3b). The desired compound was prepared from 2b (1.8 g, 3.83 mmol) using the procedure described for compound 3a in 62% yield as a yellow foam. 1H NMR (400 MHz, 892



Article

Figure 7. Pharmacodynamic profile of 9m in vivo. (A) Growth inhibitory effect of 9m (15, 30, 60 mg/kg), rapamycin (60 mg/kg) or control on established T-47D xenografts in female BALB/C nude mice (N = 5 per group, mean ± SD). The p-values were determined using Student’s t test: (∗∗∗) p < 0.001. (B) Average body weight of xenograft tumor mice after treatment with different concentrations of 9m, rapamycin, or control. (C) Photograph of excised tumors with respective mice at day 18. Treating with 9m resulted in a decrease in tumor volume when compared with other groups (rapamycin 60 mg/kg or vehicle). (D) Tumor weight of mice in part C at day 18: (∗∗∗) p < 0.001. (E) Sections of tumors from mice treated with the indicated compounds for 3 weeks, analyzed by immunohistochemistry for the indicated proteins. Red bars represent 20 μM, and black bars represent 50 μM. chloroform-d) δ 8.36 (s, 1H), 7.90 (d, J = 7.2 Hz, 1H), 7.52 (d, J = 9.5 Hz, 1H), 3.96−3.87 (m, 1H), 3.60 (d, 1H), 3.45 (t, 3H), 1.95−1.77 (m, 1H), 1.75−1.54 (m, 2H), 1.53−1.44 (m, 1H), 1.36 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 157.50, 155.05, 144.84, 143.58, 143.47, 133.58, 131.78, 125.30, 122.28, 114.24, 114.03, 109.36, 109.12, 80.09, 53.49, 51.44, 31.68, 28.31, 22.92. 4.1.11. tert-Butyl 4-(((3-Amino-6-bromo-7-fluoroquinolin-4-yl)amino)methyl)piperidine-1-carboxylate (3c). The desired compound was prepared from 2c (3 g, 6.22 mmol) using the procedure described for compound 3a in 76% yield as a yellow foam. 1H NMR (400 MHz, DMSO-d6) δ 8.79 (d, J = 7.0 Hz, 1H), 8.27 (s, 1H), 7.99 (s, 1H), 7.86 (d, J = 9.2 Hz, 1H), 5.75 (s, 2H), 4.00−3.82 (m, 2H), 3.67 (d, J = 6.8 Hz,

2H), 2.79−2.53 (m, 2H), 2.03−1.84 (m, 1H), 1.72−1.60 (m, 2H), 1.35 (s, 9H), 1.03 (qd, J = 12.5, 4.2 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 158.53, 156.04, 153.79, 144.22, 135.19, 135.08, 130.26, 129.18, 128.36, 115.38, 107.27, 107.04, 106.03, 105.78, 78.48, 51.08, 36.93, 29.23, 28.10. 4.1.12. tert-Butyl (S)-3-(((3-Amino-6-bromo-7-fluoroquinolin-4yl)amino)methyl)pyrrolidine-1-carboxylate (3d). The desired compound was prepared from 2d (3 g, 6.41 mmol) using the procedure described for compound 3a in 36% yield as a yellow foam. 1H NMR (400 MHz, chloroform-d) δ 9.12 (s, 1H), 8.47 (d, 1H), 7.82 (d, J = 9.8 Hz, 1H), 4.01 (d, J = 26.1 Hz, 1H), 3.75 (dd, J = 38.1, 13.0 Hz, 3H), 3.61−3.43 (m, 1H), 3.40−3.18 (m, 1H), 2.69−2.31 (m, 2H), 1.79−1.62 (m, 1H), 1.49 (d, J = 17.4 Hz, 9H); 13C NMR (101 MHz, CDCl3) 893



Article

4.1.18. tert-Butyl (S)-3-(9-Bromo-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidine-1-carboxylate (4b). The desired compound was prepared from 3b (500 mg, 1.14 mmol) using the procedure described for compound 4a in 64% yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 9.16 (s, 1H), 8.50 (s, 1H), 8.25 (s, 1H), 7.91 (d, J = 8.8 Hz, 1H), 4.75−4.56 (m, 1H), 4.11 (t, J = 11.7 Hz, 2H), 3.71 (dq, J = 12.5, 6.5 Hz, 1H), 3.17 (dt, J = 12.0, 5.6 Hz, 1H), 3.04−2.77 (m, 2H), 2.01−1.83 (m, 2H), 1.44 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 160.81, 157.62, 154.84, 152.93, 151.83, 129.59, 127.65, 115.80, 80.54, 61.94, 55.94, 43.87, 28.53, 27.50, 18.70. 4.1.19. tert-Butyl 4-((9-Bromo-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)methyl)piperidine-1-carboxylate (4c). The desired compound was prepared from 3c (500 mg, 1.02 mmol) using the procedure described for compound 4a in 82% yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 9.23 (s, 1H), 8.72 (d, J = 6.9 Hz, 1H), 8.43 (s, 1H), 7.97 (d, J = 8.8 Hz, 1H), 4.89−4.55 (m, 2H), 4.28−4.01 (m, 2H), 2.63 (t, J = 12.7 Hz, 2H), 2.09 (dt, J = 7.4, 3.8 Hz, 1H), 1.74− 1.56 (m, 2H), 1.56−1.46 (m, 2H), 1.44 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 160.61, 158.04, 156.65, 154.68, 154.06, 150.40, 135.40, 135.35, 127.20, 118.10, 116.51, 116.30, 115.82, 110.67, 110.45, 103.48, 79.88, 50.38, 36.12, 29.63, 28.55. 4.1.20. tert-Butyl (S)-3-((9-Bromo-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)methyl)pyrrolidine-1-carboxylate (4d). The desired compound was prepared from 3d (500 mg, 1.14 mmol) using the procedure described for compound 4a in 50% yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 9.24 (s, 1H), 8.73 (d, J = 6.6 Hz, 1H), 8.44 (s, 1H), 8.02 (d, J = 8.7 Hz, 1H), 4.96−4.69 (m, 2H), 3.73−3.49 (m, 2H), 3.42−3.24 (m, 2H), 2.80−2.70 (m, 1H), 2.24−2.03 (m, 1H), 1.89 (p, J = 9.1 Hz, 1H), 1.45 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 160.67, 158.12, 156.56, 154.42, 153.96, 150.50, 135.35, 129.66, 127.13, 116.53, 116.33, 115.69, 90.96, 79.78, 49.90, 49.27, 48.02, 45.36, 45.03, 39.40, 38.14, 29.43, 28.62. 4.1.21. tert-Butyl 4-(9-Bromo-2-oxopyrazino[2,3-c]quinolin1(2H)-yl)piperidine-1-carboxylate (4e). The desired compound was prepared from 3e (500 mg, 1.19 mmol) using the procedure described for compound 4a in 28% yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 9.17 (s, 1H), 8.27 (s, 1H), 8.25 (d, J = 2.0 Hz, 1H), 8.12 (d, J = 8.9 Hz, 1H), 7.90 (dd, J = 8.9, 1.9 Hz, 1H), 4.78 (t, J = 11.8 Hz, 1H), 4.38 (d, J = 38.3 Hz, 2H), 3.07 (qd, J = 12.6, 4.4 Hz, 2H), 2.82 (s, 2H), 1.86 (d, J = 12.7 Hz, 2H), 1.49 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 157.52, 154.60, 152.41, 152.09, 147.67, 136.32, 133.98, 132.69, 127.88, 127.05, 120.62, 119.07, 114.18, 80.28, 63.31, 28.96, 28.57. 4.1.22. tert-Butyl (S)-3-(9-Bromo-2-oxopyrazino[2,3-c]quinolin1(2H)-yl)piperidine-1-carboxylate (4f). The desired compound was prepared from 3f (500 mg, 1.19 mmol) using the procedure described for compound 4a in 35% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 1H), 8.31 (s, 1H), 8.29 (s, 1H), 8.13−7.96 (m, 2H), 4.60 (t, 1H), 4.04−3.88 (m, 2H), 2.89−2.70 (m, 2H), 2.08−1.92 (m, 1H), 1.84 (d, J = 13.1 Hz, 1H), 1.55−1.17 (m, 11H); 13C NMR (101 MHz, DMSO) δ 157.20, 154.08, 152.18, 151.64, 147.21, 136.12, 133.48, 132.32, 127.46, 126.97, 119.39, 118.82, 79.25, 74.73, 60.51, 27.93, 26.60. 4.1.23. tert-Butyl 3-(9-Bromo-2-oxopyrazino[2,3-c]quinolin1(2H)-yl)pyrrolidine-1-carboxylate (4g). The desired compound was prepared from 3g (500 mg, 1.23 mmol) using the procedure described for compound 4a in 50% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 1H), 8.32 (s, 1H), 8.28 (s, 1H), 8.05 (s, 1H), 8.00 (d, J = 8.9, 1.8 Hz, 1H), 5.62−5.52 (m, 1H), 3.83−3.68 (m, 3H), 3.49 (td, J = 9.1, 5.5 Hz, 1H), 2.65−2.54 (m, 1H), 2.48−2.36 (m, 1H), 1.42 (s, 9H); 13C NMR (101 MHz, DMSO) δ 156.79, 153.16, 152.10, 151.44, 146.96, 136.36, 133.33, 131.93, 127.16, 119.19, 118.92, 78.49, 60.79, 60.08, 48.92, 48.38, 45.89, 45.38, 38.25, 29.86, 28.62, 28.20. 4.1.24. tert-Butyl 4-(2-Oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidine-1-carboxylate (4h). The desired compound was prepared from 3h (500 mg, 1.46 mmol) using the procedure described for compound 4a in 26% yield as a white solid. 1H NMR (400 MHz, chloroformd) δ 9.17 (s, 1H), 8.24 (d, J = 1.9 Hz, 1H), 8.22 (s, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.83 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.63 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 4.94−4.83 (m, 1H), 4.48−4.22 (m, 2H), 3.07 (qd, J = 12.3, 4.3 Hz, 2H), 2.91−2.67 (m, 2H), 1.96−1.73 (m, 2H), 1.48 (s, 9H); 13C

Table 5. Pharmacokinetic Characteristics of Compound 9m parameter

iv (20 mg/kg)

po (20 mg/kg)

AUC0−∞ ((μg/L)·h) Cmax (μg/L) Tmax F (%) MRT0−∞ (h) Vss (L/kg) CL (L h−1 kg−1) t1/2 (h)

6558.52 4473.47

2853.41 807.24 3.00 43.50 16.31 21.79 3.51 9.98

11.64 16.66 1.53 7.57

δ 158.81, 158.17, 156.35, 155.30, 143.93, 143.82, 128.92, 126.33, 125.43, 115.00, 109.96, 109.71, 80.77, 79.94, 68.39, 53.57, 50.74, 49.55, 46.02, 38.27, 30.96, 29.83, 28.72, 28.68. 4.1.13. tert-Butyl 4-((3-Amino-6-bromoquinolin-4-yl)amino)piperidine-1-carboxylate (3e). The desired compound was prepared from 2e (43 g, 95.56 mmol) using the procedure described for compound 3a in 64% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 8.29 (s, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.43 (dd, J = 8.8, 2.1 Hz, 1H), 5.33 (s, 2H), 4.73 (d, J = 10.5 Hz, 1H), 4.09−3.84 (m, 3H), 2.83−2.58 (m, 2H), 1.87−1.70 (m, 2H), 1.50 (td, J = 12.0, 4.2 Hz, 2H), 1.42 (s, 9H); 13C NMR (101 MHz, DMSO) δ 153.73, 143.65, 141.07, 134.82, 131.27, 130.32, 126.90, 126.56, 123.66, 118.76, 78.48, 59.75, 52.72, 32.76, 28.08. 4.1.14. tert-Butyl (S)-3-((3-Amino-6-bromoquinolin-4-yl)amino)piperidine-1-carboxylate (3f). The desired compound was prepared from 2f (2 g, 4.44 mmol) using the procedure described for compound 3a in 48% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 8.40 (s, 1H), 7.86 (s, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 8.9 Hz, 1H), 4.10−3.89 (m, 2H), 3.65 (dd, J = 13.1, 3.3 Hz, 1H), 3.54−3.40 (m, 2H), 3.30−3.04 (m, 2H), 1.88 (ddt, J = 12.0, 8.2, 4.1 Hz, 1H), 1.69 (ddt, J = 13.6, 10.0, 4.8 Hz, 1H), 1.38 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 155.23, 143.76, 142.49, 132.79, 132.59, 131.46, 128.91, 125.78, 122.79, 120.19, 80.06, 51.37, 31.82, 23.06. 4.1.15. tert-Butyl 3-((3-Amino-6-bromoquinolin-4-yl)amino)pyrrolidine-1-carboxylate (3g). The desired compound was prepared from 2g (2 g, 4.59 mmol) using the procedure described for compound 3a in 46% yield as a yellow foam. 1H NMR (400 MHz, chloroform-d) δ 8.47 (s, 1H), 7.86 (s, 1H), 7.79 (d, J = 8.9 Hz, 1H), 7.47 (d, J = 8.9 Hz, 1H), 4.16−4.08 (m, 1H), 4.07−3.96 (m, 1H), 3.69−3.59 (m, 1H), 3.47 (td, J = 16.8, 8.2 Hz, 1H), 3.39−3.21 (m, 1H), 2.12−2.01 (m, 1H), 1.94−1.82 (m, 1H), 1.45 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 154.78, 143.79, 142.40, 133.68, 131.75, 131.70, 129.06, 126.26, 122.33, 120.79, 79.77, 55.66, 54.91, 52.14, 51.70, 44.37, 44.01, 32.55, 31.96, 28.59. 4.1.16. tert-Butyl 4-((3-Aminoquinolin-4-yl)amino)piperidine-1carboxylate (3h). The desired compound was prepared from 2h (6.4 g, 17.2 mmol) using the procedure described for compound 3a in 43% yield as a white foam. 1H NMR (400 MHz, chloroform-d) δ 8.30 (s, 1H), 7.81−7.73 (m, 1H), 7.57 (dt, J = 7.9, 2.6 Hz, 1H), 7.29−7.21 (m, 2H), 3.92 (s, 2H), 3.77−3.60 (m, 1H), 3.45−3.32 (m, 1H), 3.31−3.17 (m, 1H), 2.54 (t, J = 12.8 Hz, 2H), 1.70 (d, J = 12.8 Hz, 2H), 1.26 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 154.73, 144.02, 143.45, 133.44, 132.42, 129.96, 126.28, 125.71, 124.88, 120.07, 120.04, 79.73, 53.12, 33.94, 28.50. 4.1.17. tert-Butyl 4-(9-Bromo-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidine-1-carboxylate (4a).42 Intermediate 3a (500 mg, 1.14 mmol) and glyoxylic acid (115 mg, 1.25 mmol) monohydrate were dissoved in anhydrous tetrahydrofuran (60 mL) under inert atmosphere. After stirring 8 h, HATU (475 mg, 1.25 mmol) and DIPEA (395 μL, 2.28 mmol) were added and stirred overnight. The reaction mixture was concetrated under reduced pressure. The crude products purified by silica gel column chromatography to afford compound 4a in 73% yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 9.18 (s, 1H), 8.34 (d, J = 6.9 Hz, 1H), 8.26 (s, 1H), 7.93 (d, J = 8.9 Hz, 1H), 4.79−4.67 (m, 1H), 4.40 (d, J = 28.5 Hz, 2H), 3.08 (qd, J = 12.5, 4.4 Hz, 2H), 2.82 (s, 2H), 1.87 (d, J = 12.7 Hz, 2H), 1.51 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 160.64, 158.10, 157.41, 154.55, 153.24, 152.06, 149.84, 149.73, 136.39, 129.51, 127.54, 116.24, 116.03, 115.71, 115.69, 110.06, 109.82, 80.30, 63.46, 28.93, 28.54. 894



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NMR (101 MHz, CDCl3) δ 157.91, 154.62, 151.95, 151.70, 149.41, 137.06, 131.39, 130.67, 127.67, 126.51, 124.23, 117.88, 80.19, 63.19, 28.80, 28.56. 4.1.25. 2-(4-(9-Bromo-8-fluoro-2-oxopyrazino[2,3-c]quinolin1(2H)-yl)piperidin-1-yl)acetonitrile (6a). To the suspension of compound 4a (3 g, 6.30 mmol) in ethyl acetate (45 mL) was added concentrated hydrochloric acid (1 mL), and the mixture was stirred 3 h. The reaction mixture was concetrated under reduced pressure. The crude product was dissoved in DMF (40 mL), then DIPEA (8.79 mL) and bromoacetonitrile (890 μL) were added. After stirring overnight, water (400 mL) was added, and the aqueous phase was extracted with ethyl acetate. The combined organic phase was separated, dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated to dryness under reduced pressure to yield a solid residue, which was purified by flash column chromatography to yield the desired product in 52% (two steps) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.34 (d, J = 7.3 Hz, 1H), 8.28 (s, 1H), 8.08 (d, J = 9.6 Hz, 1H), 4.65−4.55 (m, 1H), 3.79 (s, 2H), 3.04−2.86 (m, 4H), 2.30 (t, 2H), 2.01−1.89 (m, 2H). 13 C NMR (101 MHz, DMSO) δ 161.06, 158.53, 157.43, 153.45, 150.05, 143.80, 141.77, 131.97, 127.58, 116.58, 113.23, 111.64, 111.40, 110.75, 110.52, 59.27, 51.94, 43.07, 26.11. 4.1.26. (S)-2-(3-(9-Bromo-8-fluoro-2-oxopyrazino[2,3-c]quinolin1(2H)-yl)piperidin-1-yl)acetonitrile (6b). The desired compound was prepared from 4b (350 mg, 0.84 mmol) using the procedure described for compound 6a in 41% (two steps) yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 8.34 (d, J = 7.3 Hz, 1H), 8.31 (s, 1H), 8.09 (d, J = 9.5 Hz, 1H), 4.72 (t, J = 11.3 Hz, 1H), 3.82 (q, J = 17.2 Hz, 2H), 3.39 (t, J = 10.5 Hz, 1H), 3.16 (d, J = 10.6 Hz, 1H), 2.84 (d, J = 10.9 Hz, 1H), 2.72−2.55 (m, 1H), 2.24 (t, J = 11.5 Hz, 1H), 2.04−1.82 (m, 2H), 1.67−1.49 (m, 1H); 13C NMR (101 MHz, DMSO) δ 159.49, 157.10, 157.00, 152.75, 151.99, 149.16, 136.57, 130.20, 127.11, 115.88, 115.84, 115.82, 115.42, 115.21, 108.46, 108.22, 61.01, 53.33, 51.35, 45.27, 25.96, 24.32. 4.1.27. 2-(4-((9-Bromo-8-fluoro-2-oxopyrazino[2,3-c]quinolin1(2H)-yl)methyl)piperidin-1-yl)acetonitrile (6c). The desired compound was prepared from 4c (350 mg, 0.84 mmol) using the procedure described for compound 6a in 58% (two steps) yield as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.17 (s, 1H), 8.83 (d, J = 7.2 Hz, 1H), 8.46 (s, 1H), 8.08 (d, J = 9.4 Hz, 1H), 4.70 (s, 2H), 3.67 (s, 2H), 2.76 (d, J = 10.9 Hz, 2H), 2.03 (t, J = 11.1 Hz, 2H), 1.92−1.78 (m, 1H), 1.67 (d, J = 12.7 Hz, 2H), 1.41 (tt, J = 13.9, 7.0 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 159.38, 156.88, 156.27, 153.67, 150.40, 149.44, 149.33, 135.13, 130.71, 126.77, 115.82, 115.63, 115.42, 108.86, 108.63, 54.93, 51.03, 48.92, 45.32, 34.16, 28.99. 4.1.28. tert-Butyl (S)-3-((9-Bromo-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)methyl)pyrrolidine-1-carboxylate (6d). The desired compound was prepared from 4d (81 mg, 0.17 mmol) using the procedure described for compound 6a in 89% (two steps) yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 1H), 8.92 (d, J = 7.1 Hz, 1H), 8.44 (s, 1H), 8.04 (d, J = 9.4 Hz, 1H), 4.77 (dd, J = 15.1, 8.3 Hz, 1H), 4.60 (dd, J = 15.1, 5.8 Hz, 1H), 3.93−3.74 (m, 2H), 2.88−2.70 (m, 2H), 2.68−2.52 (m, 3H), 2.17−2.00 (m, 1H), 1.70 (dt, J = 13.7, 6.8 Hz, 1H); 13 C NMR (101 MHz, DMSO) δ 159.37, 156.87, 156.31, 153.46, 150.53, 149.38, 149.27, 135.34, 130.64, 126.65, 116.01, 115.71, 115.51, 115.30, 108.93, 108.71, 55.86, 51.02, 49.31, 41.00, 36.36, 27.72. 4.1.29. 2-(4-(9-Bromo-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (6e). The desired compound was prepared from 4e (140 mg, 0.31 mmol) using the procedure described for compound 6a in 85% (two steps) yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.27 (d, J = 19.9 Hz, 2H), 8.08 (d, 2H), 4.79− 4.42 (m, 1H), 3.79 (s, 2H), 3.07−2.84 (m, 4H), 2.36−2.22 (m, 2H), 2.04−1.86 (m, 2H); 13C NMR (101 MHz, DMSO) δ 157.22, 152.20, 151.63, 147.16, 136.46, 133.26, 132.26, 127.49, 119.16, 115.69, 61.44, 51.15, 45.02, 27.78. 4.1.30. (S)-2-(3-(9-Bromo-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (6f). The desired compound was prepared from 4f (150 mg, 0.33 mmol) using the procedure described for compound 6a in 75% (two steps) yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 1H), 8.32 (s, 1H), 8.23 (d, J = 1.9 Hz, 1H), 8.10−7.99 (m, 2H), 4.76 (t, J = 11.1 Hz, 1H), 3.82 (q, 2H), 3.40

(t, J = 10.4 Hz, 1H), 3.12 (d, J = 10.5 Hz, 1H), 2.84 (d, J = 11.1 Hz, 1H), 2.71−2.58 (m, 1H), 2.30−2.20 (m, 1H), 1.98 (d, J = 13.0 Hz, 1H), 1.89 (d, J = 13.3 Hz, 1H), 1.68−1.53 (m, 1H); 13C NMR (101 MHz, DMSO) δ 157.15, 152.20, 151.66, 147.19, 136.24, 133.30, 132.33, 127.34, 119.34, 118.88, 115.84, 60.83, 53.32, 51.32, 45.27, 25.98, 24.33. 4.1.31. 2-(3-(9-Bromo-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)pyrrolidin-1-yl)acetonitrile (6g). The desired compound was prepared from 4g (240 mg, 0.54 mmol) using the procedure described for compound 6a in 63% (two steps) yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 9.18 (s, 1H), 8.34 (s, 1H), 8.32 (s, 1H), 8.09 (d, J = 8.9 Hz, 1H), 7.89 (d, J = 8.9, 1.9 Hz, 1H), 5.51 (tt, J = 13.3, 6.4 Hz, 1H), 3.75 (s, 2H), 3.46 (t, J = 8.2 Hz, 1H), 3.39 (q, J = 8.1 Hz, 1H), 3.20 (t, J = 8.6 Hz, 1H), 3.10 (td, J = 7.9, 3.2 Hz, 1H), 2.65−2.44 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 157.04, 152.12, 152.04, 147.82, 136.12, 134.12, 132.70, 127.81, 126.38, 120.66, 118.96, 114.98, 61.33, 53.75, 52.55, 41.72, 30.13. 4.1.32. 2-(4-(2-Oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1yl)acetonitrile (6h). The desired compound was prepared from 4h (40 mg, 0.11 mmol) using the procedure described for compound 6a in 69% (two steps) yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.26 (s, 1H), 8.16 (t, J = 7.0 Hz, 2H), 7.90 (t, J = 7.7 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H), 4.74−4.62 (m, 1H), 3.78 (s, 2H), 3.02− 2.89 (m, 4H), 2.27 (t, J = 12.1 Hz, 2H), 1.97 (d, J = 11.8 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 157.16, 153.30, 146.53, 144.73, 139.16, 134.09, 129.26, 126.93, 126.75, 122.70, 118.06, 113.06, 59.50, 51.86, 42.63, 25.59. 4.1.33. 4-(9-Bromo-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidine-1-carboxamide (6i). A two-necked flask was charged with 9-bromo-1-(piperidin-4-yl)pyrazino[2,3-c]quinolin-2(1H)-one HCl salt (100 mg, 0.25 mmol), DIEA (358 μL, 2.02 mmol), and 15 mL of DCM under inert atmosphere, then trimethylsilylisocyanate (60 μL, 0.51 mmol) at 0 °C. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated and the title compound was purified by silica gel chromatography to yield the desired product in 78%. 1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.30 (s, 1H), 8.28 (s, 1H), 8.12−7.99 (m, 2H), 6.06 (s, 2H), 4.92−4.69 (m, 1H), 4.20−4.07 (m, 2H), 2.81−2.64 (m, 4H), 1.96−1.83 (m, 2H); 13C NMR (101 MHz, DMSO) δ 158.27, 157.08, 157.01, 153.56, 148.24, 142.07, 140.34, 135.99, 135.56, 131.82, 128.63, 127.26, 126.65, 121.15, 119.44, 65.15, 62.46, 56.28, 43.41, 28.37. 4.1.34. 4-(2-Oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidine-1carboxamide (6j). The desired compound was prepared from 5h (40 mg, 0.12 mmol) using the procedure described for compound 6i in 71% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 1H), 8.25 (s, 1H), 8.15 (dd, J = 15.4, 8.5 Hz, 2H), 7.89 (t, J = 7.7 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H), 6.05 (s, 2H), 4.93−4.80 (m, 1H), 4.17−4.03 (m, 2H), 2.82−2.64 (m, 4H), 1.96−1.83 (m, 2H); 13C NMR (101 MHz, DMSO) δ 157.16, 153.30, 146.53, 144.73, 139.16, 134.09, 129.26, 126.93, 126.75, 122.70, 118.06, 113.06, 59.50, 51.86, 42.63, 25.59. 4.1.35. 9-Bromo-1-(1-(cyclopropanecarbonyl)piperidin-4-yl)-8fluoropyrazino[2,3-c]quinolin-2(1H)-one (7a). To a two-necked flask, compound 5a (100 mg, 0.24 mmol), cyclopropanecarboxylic acid (21 mg, 0.24 mmol), EDCI (70 mg, 0.36 mmol), HOBt (49 mg, 0.36 mmol), DIEA (416 μL, 2.42 mmol), and DMF (8 mL) were charged. The mixture was stirred at room temperature for 12 h, then quenched by water. The mixture was extracted by ethyl acetate, and the combined organic layers were washed by saturated aqueous NaHCO3 solution, water, and brine and dried by anhydrous magnesium sulfate. The solvent was evaporated, and the residue was purified by silica gel column chromatography to afford title compond in 58% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.14 (s, 1H), 8.31 (d, J = 6.9 Hz, 1H), 8.23 (s, 1H), 7.89 (d, J = 8.9 Hz, 1H), 4.93−4.75 (m, 2H), 4.47 (d, J = 11.1 Hz, 1H), 3.27−2.92 (m, 3H), 2.73−2.58 (m, 1H), 1.99−1.87 (m, 2H), 1.77 (tt, J = 8.2, 4.7 Hz, 1H), 0.99 (d, J = 29.4 Hz, 2H), 0.86−0.71 (m, 2H); 13 C NMR (101 MHz, CDCl3) δ 172.10, 160.59, 158.05, 157.33, 153.26, 152.03, 149.87, 149.76, 136.18, 129.34, 127.51, 116.30, 116.09, 115.63, 110.07, 109.84, 63.19, 45.00, 41.78, 29.40, 28.71, 11.20, 7.68. 4.1.36. 9-Bromo-8-fluoro-1-(1-propionylpiperidin-4-yl)pyrazino[2,3-c]quinolin-2(1H)-one (7b). The desired compound was prepared from 5a (100 mg, 0.24 mmol) and commercially available propionic acid 895



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129.34, 128.53, 127.64, 127.58, 123.93, 116.37, 116.15, 115.65, 110.18, 109.95, 53.56, 41.84, 29.10, 28.92, 21.54. 4.1.42. (S)-9-Bromo-8-fluoro-1-(1-(2-hydroxypropanoyl)piperidin4-yl)pyrazino[2,3-c]quinolin-2(1H)-one (7h). The desired compound was prepared from 5a (100 mg, 0.24 mmol) and commercially available (S)-2-hydroxypropanoic acid (21.6 mg, 0.24 mmol) using the procedure described for compound 7a in 34% yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 9.16 (s, 1H), 8.28 (d, J = 6.8 Hz, 1H), 8.24 (s, 1H), 7.91 (d, J = 8.9 Hz, 1H), 4.97−4.75 (m, 2H), 4.52 (dq, J = 12.8, 6.5 Hz, 1H), 3.99 (d, J = 10.2 Hz, 1H), 3.23−2.97 (m, 3H), 2.74 (q, J = 12.1 Hz, 1H), 1.98 (d, J = 13.2 Hz, 2H), 1.38 (dd, J = 34.4, 6.5 Hz, 3H); 13 C NMR (101 MHz, CDCl3) δ 174.01, 173.75, 160.63, 158.09, 157.34, 157.27, 153.33, 152.06, 152.00, 149.96, 149.84, 136.00, 129.16, 127.56, 116.46, 116.24, 115.56, 110.21, 110.13, 109.97, 64.42, 64.29, 62.58, 44.41, 44.21, 42.26, 42.19, 29.22, 28.82, 28.66, 28.46, 21.87, 21.37. 4.1.43. Methyl 2-(4-(9-Bromo-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetate (7i). The 5a (100 mg, 0.24 mmol) was suspended in dichloromathane (15 mL), the commercially available methyl 2-bromoacetate (43.8 mg, 0.29 mmol), and DIPEA (82.4 mL, 0.48 mmol) was added then stirred 5 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to afford title compond in 72% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.15 (s, 1H), 8.33 (d, J = 6.9 Hz, 1H), 8.24 (s, 1H), 7.90 (d, J = 8.9 Hz, 1H), 4.59 (tt, J = 11.9, 3.5 Hz, 1H), 3.74 (d, J = 1.2 Hz, 3H), 3.36 (s, 2H), 3.28 (qd, J = 12.6, 3.7 Hz, 2H), 3.18 (d, J = 11.9 Hz, 2H), 2.47 (t, J = 11.8 Hz, 2H), 1.84 (d, J = 12.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 170.72, 160.60, 158.06, 157.44, 153.34, 152.07, 149.96, 149.85, 136.53, 129.67, 129.66, 127.54, 127.52, 116.25, 116.04, 115.79, 115.76, 109.94, 109.71, 63.31, 58.73, 54.14, 52.70, 51.88, 28.78. 4.1.44. 9-(6-Aminopyridin-3-yl)-1-(1-(cyclopropanecarbonyl)piperidin-4-yl)-8- fluoropyrazino[2,3-c]quinolin-2(1H)-one (8a).14 To a solution of compound 7a (55 mg, 0.13 mmol) in 1,4-dioxane (6 mL) at room temperature were subsequently added PdCl2(Ph3P)2 (8.8 mg, 0.1 equiv), Na2CO3 (378 μL, 0.39 mmol, 1M), and (6aminopyridin-3-yl)boronic acid (34.7 mg, 0.25 mmol), then degassing was with argon for 10 min, and the resulting mixture was heated to 85 °C overenight. The reaction mixture was cooled to room temperature and filtered through Celite. Upon removal of the solvents, the residue was purified by silica gel column chromatography to afford the title compond in 44% yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 9.17 (s, 1H), 8.31 (s, 1H), 8.25 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.96 (d, J = 11.0 Hz, 1H), 7.75 (d, J = 8.9 Hz, 1H), 6.70 (d, J = 8.6 Hz, 1H), 5.12− 4.79 (m, 4H), 4.55−4.38 (m, 1H), 3.29−2.99 (m, 3H), 2.69−2.53 (m, 1H), 1.96 (d, J = 11.9 Hz, 2H), 1.83−1.71 (m, 1H), 1.13−0.91 (m, 2H), 0.87−0.73 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 172.54, 162.01, 159.46, 158.77, 157.64, 152.39, 151.37, 149.53, 149.41, 147.16, 138.33, 138.29, 137.06, 127.53, 127.42, 127.36, 125.15, 125.11, 120.08, 115.56, 115.34, 115.08, 109.02, 62.93, 45.03, 41.82, 29.05, 28.34, 11.05, 7.62. HRMS(ESI) calcd for C25H24FN6O2+ 459.1939, found 459.1942 [M + H]+. HPLC purity 99%. HPLC: tR = 4.96 min. 4.1.45. 9-(6-Aminopyridin-3-yl)-8-fluoro-1-(1-propionylpiperidin4-yl)pyrazino[2,3-c]quinolin-2(1H)-one (8b). The desired compound was prepared from 7b (50 mg, 0.12 mmol) and (6-aminopyridin-3yl)boronic acid (33.1 mg, 0.24 mmol) using the procedure described for compound 8a in 47% yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 9.17 (s, 1H), 8.30 (s, 1H), 8.25 (s, 1H), 8.08 (d, J = 7.7 Hz, 1H), 7.95 (d, J = 11.1 Hz, 1H), 7.75 (dt, J = 8.6, 2.2 Hz, 1H), 6.72 (d, J = 8.6 Hz, 1H), 5.18−4.87 (m, 4H), 4.14−4.04 (m, 1H), 3.20−2.98 (m, 3H), 2.63−2.52 (m, 1H), 2.47−2.31 (m, 2H), 2.03−1.84 (m, 2H), 1.17 (t, J = 7.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 172.79, 161.96, 159.42, 158.80, 157.59, 152.33, 151.32, 149.47, 149.35, 147.17, 138.25, 136.99, 127.51, 127.36, 125.09, 125.04, 120.00, 115.51, 115.29, 115.02, 108.95, 62.79, 44.87, 41.25, 28.90, 28.30, 26.41, 9.31. HRMS(ESI) calcd for C24H24FN6O2+ 447.1939, found 447.1943 [M + H]+. HPLC purity 98%. HPLC: tR = 4.85 min. 4.1.46. 9-(6-Aminopyridin-3-yl)-1-(1-benzoylpiperidin-4-yl)-8fluoropyrazino[2,3-c]quinolin-2(1H)-one (8c). The desired compound was prepared from 7c (30 mg, 0.06 mmol) and (6-aminopyridin-3yl)boronic acid (17.2 mg, 0.02 mmol) using the procedure described for

(18 mg, 0.24 mmol) using the procedure described for compound 7a in 60% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.14 (s, 1H), 8.30 (d, J = 6.9 Hz, 1H), 8.23 (s, 1H), 7.89 (d, J = 8.9 Hz, 1H), 4.91 (d, 1H), 4.78 (tt, J = 11.4, 3.4 Hz, 1H), 4.10 (d, J = 10.1 Hz, 1H), 3.16−2.93 (m, 3H), 2.61 (td, J = 13.2, 12.8, 2.5 Hz, 1H), 2.48−2.31 (m, 2H), 1.92 (d, 2H), 1.16 (t, J = 7.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 172.31, 160.58, 158.05, 157.33, 153.31, 152.01, 149.93, 149.82, 136.11, 129.31, 127.52, 116.35, 116.14, 115.61, 110.06, 109.82, 63.10, 44.85, 41.24, 29.31, 28.70, 26.61, 9.56. 4.1.37. 1-(1-Benzoylpiperidin-4-yl)-9-bromo-8-fluoropyrazino[2,3-c]quinolin-2(1H)-one (7c). The desired compound was prepared from 5a (100 mg, 0.24 mmol) and commercially available benzoic acid (29.3 mg, 0.24 mmol) using the procedure described for compound 7a in 69% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.18 (s, 1H), 8.32 (d, J = 6.9 Hz, 1H), 8.28 (s, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.46 (q, J = 5.7, 4.7 Hz, 5H), 5.02 (s, 1H), 4.87 (t, 1H), 4.18−3.96 (m, 1H), 3.30−2.73 (m, 4H), 2.11−1.76 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 170.69, 160.66, 158.11, 157.39, 153.30, 152.05, 149.91, 149.80, 136.18, 135.61, 130.04, 129.32, 128.73, 127.57, 127.05, 116.38, 116.17, 115.64, 110.15, 109.91, 63.07, 47.41, 42.02, 40.88, 29.13, 29.06. 4.1.38. 4-(9-Bromo-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)N,N-dimethylpiperidine-1-carboxamide (7d). The 5a (100 mg, 0.24 mmol) was suspended in dichloromathane (15 mL), the commercially available dimethylcarbamoyl chloride (30.8 mg, 0.29 mmol), and DIPEA (82.4 mL, 0.48 mmol) and stirred 5 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to afford title compond in 76% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.16 (s, 1H), 8.33 (d, J = 6.9 Hz, 1H), 8.24 (s, 1H), 7.92 (d, J = 8.9 Hz, 1H), 4.71 (t, J = 11.8 Hz, 1H), 3.86 (d, J = 13.2 Hz, 2H), 3.14 (qd, J = 12.5, 3.9 Hz, 2H), 2.89 (s, 6H), 2.83 (t, J = 13.1 Hz, 2H), 1.89 (d, J = 12.7 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 164.79, 160.69, 158.14, 157.46, 153.21, 152.04, 149.84, 149.73, 136.39, 129.51, 127.55, 116.25, 116.04, 115.70, 110.15, 109.91, 63.57, 46.82, 38.60, 28.92. 4.1.39. 2-(4-(9-Bromo-8-fluoro-2-oxopyrazino[2,3-c]quinolin1(2H)-yl)piperidin-1-yl)-N,N-dimethyl-2-oxoacetamide (7e). The desired compound was prepared from 5a (100 mg, 0.24 mmol) and commercially available 2-(dimethylamino)-2-oxoacetic acid (28.1 mg, 0.24 mmol) using the procedure described for compound 7a in 68% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 13.65 (s, 2H), 9.15 (s, 1H), 8.35 (d, J = 7.2 Hz, 1H), 8.30 (s, 1H), 8.11 (d, J = 9.5 Hz, 1H), 4.95 (t, J = 11.5 Hz, 1H), 4.50 (d, J = 12.9 Hz, 1H), 3.63 (d, 1H), 3.40−3.22 (m, 2H), 2.95 (s, 3H), 2.89 (s, 3H), 2.76 (t, J = 12.3 Hz, 2H), 2.12 (d, J = 12.8 Hz, 1H), 2.01 (d, J = 13.0 Hz, 1H); 13C NMR (101 MHz, DMSO) δ 164.36, 163.28, 157.21, 152.67, 152.09, 130.54, 127.80, 127.35, 127.14, 124.50, 119.16, 115.91, 115.16, 114.95, 109.60, 108.22, 61.11, 53.59, 44.50, 41.84, 36.45, 32.89, 28.60, 27.88. 4.1.40. 9-Bromo-1-(1-(4-(dimethylamino)benzoyl)piperidin-4-yl)8-fluoropyrazino[2,3-c]quinolin-2(1H)-one (7f). The desired compound was prepared from 5a (100 mg, 0.24 mmol) and commercially available 4-(dimethylamino)benzoic acid (39.6 mg, 0.24 mmol) using the procedure described for compound 7a in 68% yield as a white solid. 1 H NMR (400 MHz, chloroform-d) δ 9.18 (s, 1H), 8.34 (d, J = 6.9 Hz, 1H), 8.27 (s, 1H), 7.92 (d, J = 8.9 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.3 Hz, 2H), 4.83 (dd, J = 13.4, 9.9 Hz, 1H), 4.59 (s, 2H), 3.17 (qd, J = 12.4, 4.2 Hz, 2H), 3.01 (s, 6H), 2.98−2.92 (m, 2H), 1.93 (d, J = 12.7 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 171.53, 160.66, 158.12, 157.43, 153.37, 152.06, 150.00, 149.89, 136.23, 129.40, 127.58, 116.40, 116.19, 115.69, 111.47, 110.10, 109.87, 63.41, 47.21, 40.46, 29.22. 4.1.41. 9-Bromo-8-fluoro-1-(1-(3-methylbenzoyl)piperidin-4-yl)pyrazino[2,3-c]quinolin-2(1H)-one (7g). The desired compound was prepared from 5a (100 mg, 0.24 mmol) and commercially available 3-methylbenzoic acid (33.6 mg, 0.24 mmol) using the procedure described for compound 7a in 60% yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 9.18 (s, 1H), 8.32 (d, J = 6.9 Hz, 1H), 8.28 (s, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.34−7.22 (m, 4H), 5.12−4.92 (m, 1H), 4.83 (t, 1H), 4.17−3.95 (m, 1H), 3.29−2.77 (m, 3H), 2.40 (s, 3H), 2.07− 1.78 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 170.90, 160.68, 158.14, 157.40, 153.27, 152.07, 149.88, 149.77, 138.71, 136.22, 135.59, 130.74, 896



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compound 8a in 55% yield as a white solid. 1H NMR (400 MHz, chloroform-d) δ 9.17 (s, 1H), 8.31 (d, J = 2.2 Hz, 1H), 8.26 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 11.0 Hz, 1H), 7.72 (dt, J = 8.6, 2.3 Hz, 1H), 7.51−7.39 (m, 5H), 6.66 (d, J = 8.6 Hz, 1H), 5.12−4.96 (m, 1H), 4.79 (s, 2H), 4.15−3.87 (m, 1H), 3.30−2.68 (m, 5H), 2.12−1.76 (m, 2H); 13 C NMR (101 MHz, CDCl3) δ 171.02, 162.04, 159.49, 158.57, 157.70, 152.51, 151.44, 149.65, 149.53, 146.75, 138.54, 138.50, 137.06, 135.11, 130.14, 128.68, 127.49, 127.30, 126.82, 125.15, 125.10, 120.14, 115.68, 115.46, 115.12, 109.27, 62.80, 47.29, 41.90, 28.96, 28.46. HRMS(ESI) calcd for C28H24FN6O2+ 495.1939, found 495.1943 [M + H]+. HPLC purity 97%. HPLC: tR = 6.08 min. 4.1.47. 4-(9-(6-Aminopyridin-3-yl)-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)-N,N-dimethylpiperidine-1-carboxamide (8d). The desired compound was prepared from 7d (50 mg, 0.11 mmol) and (6-aminopyridin-3-yl)boronic acid (30.1 mg, 0.22 mmol) using the procedure described for compound 8a in 49% yield as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.30 (s, 1H), 8.26 (s, 1H), 8.12 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 11.4 Hz, 1H), 7.74 (dt, J = 8.7, 2.2 Hz, 1H), 6.60 (d, J = 8.6 Hz, 1H), 6.38 (s, 2H), 5.10−4.93 (m, 1H), 3.74− 3.60 (m, 2H), 2.94−2.81 (m, 4H), 2.77 (s, 6H), 1.99−1.89 (m, 2H); 13C NMR (101 MHz, DMSO) δ 163.64, 160.94, 158.42, 157.36, 153.60, 152.21, 152.05, 148.56, 148.44, 144.13, 144.11, 138.23, 135.79, 135.76, 127.77, 127.72, 127.13, 123.35, 123.18, 119.05, 115.17, 114.18, 114.02, 113.96, 64.97, 62.35, 54.98, 45.62, 38.27, 28.09, 15.22. HRMS(ESI) calcd for C24H25FN7O2+ 462.2048, found 462.2051 [M + H]+. HPLC purity 96%. HPLC: tR = 5.42 min. 4.1.48. 2-(4-(9-(6-Aminopyridin-3-yl)-8-fluoro-2-oxopyrazino[2,3c]quinolin-1(2H)-yl)piperidin-1-yl)-N,N-dimethyl-2-oxoacetamide (8e). The desired compound was prepared from 7e (50 mg, 0.10 mmol) and (6-aminopyridin-3-yl)boronic acid (29 mg, 0.20 mmol) using the procedure described for compound 8a in 29% yield as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 1H), 8.33 (s, 1H), 8.26 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H), 7.98 (d, J = 11.3 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H), 6.62 (d, J = 8.6 Hz, 1H), 6.39 (s, 2H), 5.16 (s, 1H), 4.47 (d, J = 12.6 Hz, 1H), 3.60 (d, J = 13.5 Hz, 1H), 3.38−3.27 (m, 2H), 2.96 (s, 3H), 2.90 (s, 3H), 2.85−2.73 (m, 2H), 2.13 (d, J = 12.8 Hz, 1H), 2.03−1.96 (m, 1H); 13 C NMR (101 MHz, DMSO) δ 164.34, 163.20, 161.15, 159.76, 158.64, 157.42, 151.78, 151.49, 148.73, 148.60, 148.00, 137.57, 136.98, 128.75, 127.05, 126.68, 126.51, 126.16, 126.11, 118.31, 115.61, 115.17, 114.59, 114.37, 113.84, 107.87, 61.04, 44.47, 36.46, 32.90, 28.40, 27.71. HRMS(ESI) calcd for C25H25FN7O3+ 490.1997, found 490.1989 [M + H]+. HPLC purity 96%. HPLC: tR = 4.95 min. 4.1.49. 9-(6-Aminopyridin-3-yl)-1-(1-(4-(dimethylamino)benzoyl)piperidin-4-yl)-8-fluoropyrazino[2,3-c]quinolin-2(1H)-one (8f). The desired compound was prepared from 7f (30 mg, 0.06 mmol) and (6-aminopyridin-3-yl)boronic acid (16 mg, 0.11 mmol) using the procedure described for compound 8a in 62% yield as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 1H), 8.32 (s, 1H), 8.28 (s, 1H), 8.13 (d, J = 8.2 Hz, 1H), 8.01 (d, J = 11.5 Hz, 1H), 7.76 (dt, J = 8.6, 2.2 Hz, 1H), 7.35−7.26 (m, 2H), 6.77−6.69 (m, 2H), 6.61 (d, J = 8.6 Hz, 1H), 6.36 (s, 2H), 5.15 (s, 1H), 4.42−4.09 (m, 2H), 3.17−3.00 (m, 2H), 2.95 (s, 6H), 2.91−2.75 (m, 1H), 2.13−1.94 (m, 2H); 13C NMR (101 MHz, DMSO) δ 169.95, 161.19, 161.15, 159.90, 159.80, 158.64, 157.44, 151.82, 151.51, 151.23, 148.75, 148.62, 148.06, 148.03, 137.56, 137.10, 137.05, 128.82, 127.07, 126.65, 126.49, 126.22, 126.16, 122.01, 118.33, 115.21, 114.63, 114.40, 111.09, 107.86, 107.84, 61.79, 28.37, 26.34. HRMS(ESI) calcd for C30H29FN7O2+ 538.2361, found 538.2358 [M + H]+. HPLC purity 97%. HPLC: tR = 5.67 min. 4.1.50. 9-(6-Aminopyridin-3-yl)-8-fluoro-1-(1-(3-methylbenzoyl)piperidin-4-yl)pyrazino[2,3-c]quinolin-2(1H)-one (8g). The desired compound was prepared from 7g (30 mg, 0.06 mmol) and (6-aminopyridin3-yl)boronic acid (16 mg, 0.11 mmol) using the procedure described for compound 8a in 66% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.17 (s, 1H), 8.31 (s, 1H), 8.26 (s, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.95 (d, J = 11.0 Hz, 1H), 7.72 (dt, J = 8.7, 2.3 Hz, 1H), 7.32−7.26 (m, 2H), 7.25−7.21 (m, 2H), 6.65 (d, J = 8.6 Hz, 1H), 5.13−4.94 (m, 1H), 4.80 (s, 2H), 4.01 (s, 1H), 3.29−2.63 (m, 5H), 2.13−1.71 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 171.23, 162.05, 159.50, 158.79, 157.70, 152.43, 152.32, 151.37, 149.54, 149.42, 147.11, 138.62, 138.32, 137.06, 135.04, 130.80, 128.47, 127.61, 127.45, 127.27, 125.12, 125.07, 123.68,

120.03, 115.54, 115.32, 115.09, 109.07, 62.80, 47.26, 41.85, 28.97, 28.46, 21.21. HRMS(ESI) calcd for C29H26FN6O2+ 509.2096, found 509.2094 [M + H]+. HPLC purity 97%. HPLC: tR = 5.45 min. 4.1.51. (R)-9-(6-Aminopyridin-3-yl)-8-fluoro-1-(1-(2-hydroxypropanoyl)piperidin-4-yl)pyrazino[2,3-c]quinolin-2(1H)-one (8h). The desired compound was prepared from 7h (90 mg, 0.22 mmol) and (6-aminopyridin-3-yl)boronic acid (64 mg, 0.44 mmol) using the procedure described for compound 8a in 50% yield as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 1H), 8.32 (s, 1H), 8.27 (s, 1H), 8.13 (d, J = 8.2 Hz, 1H), 8.01 (d, J = 11.4 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 6.61 (d, J = 8.6 Hz, 1H), 6.38 (s, 2H), 5.14 (s, 1H), 4.98 (dd, J = 16.5, 6.9 Hz, 1H), 4.61−4.42 (m, 2H), 4.18 (t, J = 14.8 Hz, 1H), 3.17 (d, J = 11.8 Hz, 1H), 2.90−2.65 (m, 3H), 2.12−1.92 (m, 2H), 1.21 (dd, J = 15.6, 6.5 Hz, 3H); 13C NMR (101 MHz, DMSO) δ 172.15, 172.05, 161.12, 159.69, 158.61, 157.37, 151.78, 151.45, 148.73, 148.61, 147.86, 137.57, 137.00, 127.02, 126.60, 126.43, 126.18, 126.13, 118.31, 115.16, 114.56, 114.34, 107.89, 64.32, 64.13, 61.65, 43.68, 40.90, 28.72, 28.50, 28.02, 20.74, 20.59. HRMS(ESI) calcd for C24H24FN6O3+ 463.1888, found 463.1894 [M + H]+. HPLC purity 99%. HPLC: tR = 5.07 min. 4.1.52. Methyl 2-(4-(9-(6-Aminopyridin-3-yl)-8-fluoro-2oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetate (8i). The desired compound was prepared from 7i (50 mg, 0.11 mmol) and (6-aminopyridin-3-yl)boronic acid (32 mg, 0.22 mmol) using the procedure described for compound 8a in 35% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.29 (s, 1H), 8.27 (s, 1H), 8.10 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 11.5 Hz, 1H), 7.74 (dt, J = 8.8, 2.2 Hz, 1H), 6.60 (d, J = 8.7 Hz, 1H), 6.39 (s, 2H), 3.64 (s, 3H), 3.31 (s, 2H), 3.03−2.89 (m, 4H), 2.44−2.32 (m, 2H), 1.89 (d, J = 11.4 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 170.58, 161.12, 159.82, 158.61, 157.38, 151.84, 151.42, 148.78, 148.66, 147.94, 147.92, 137.57, 137.22, 127.06, 126.61, 126.44, 125.95, 125.89, 118.18, 115.25, 114.68, 114.46, 107.83, 62.12, 58.00, 51.73, 51.19, 27.81. HRMS(ESI) calcd for C24H24FN6O3+ 463.1888, found 463.1894 [M + H]+. HPLC purity 98%. HPLC: tR = 4.17 min. 4.1.53. 2-(4-(9-(6-Aminopyridin-3-yl)-8-fluoro-2-oxopyrazino[2,3c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9a). The desired compound was prepared from 6a (38 mg, 0.09 mmol) and (6-aminopyridin3-yl)boronic acid (25 mg, 0.18 mmol) using the procedure described for compound 8a in 75% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.29 (s, 1H), 8.27 (s, 1H), 8.09 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 11.5 Hz, 1H), 7.74 (d, J = 8.6 Hz, 1H), 6.62 (d, J = 8.6 Hz, 1H), 6.39 (s, 2H), 4.92−4.74 (m, 1H), 3.79 (s, 2H), 3.07−2.89 (m, 4H), 2.40−2.26 (m, 2H), 2.02−1.91 (m, 2H); 13C NMR (101 MHz, DMSO) δ 160.74, 158.22, 157.33, 153.64, 152.69, 151.76, 149.49, 149.36, 144.12, 137.45, 136.08, 127.55, 127.51, 127.19, 123.17, 123.00, 118.85, 115.09, 114.81, 114.61, 113.97, 59.93, 51.39, 43.99, 26.53. HRMS(ESI) calcd for C23H21FN7O+ 430.1786, found 430.1791 [M + H]+. HPLC purity 98%. HPLC: tR = 4.86 min. 4.1.54. 2-(4-(8-Fluoro-2-oxo-9-(quinolin-3-yl)pyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9b). The desired compound was prepared from 6a (50 mg, 0.11 mmol) and (6-aminopyridin3-yl)boronic acid (32 mg, 0.22 mmol) using the procedure described for compound 8a in 71% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 9.18 (s, 1H), 8.74 (s, 1H), 8.34 (d, J = 7.9 Hz, 1H), 8.30 (s, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.18−8.12 (m, 2H), 7.88 (t, J = 7.7 Hz, 1H), 7.72 (t, J = 7.5 Hz, 1H), 5.00−4.89 (m, 1H), 3.81 (s, 2H), 3.06−2.87 (m, 4H), 2.47−2.36 (m, 2H), 2.06−1.93 (m, 2H); 13 C NMR (101 MHz, DMSO) δ 160.98, 158.46, 157.46, 152.61, 152.06, 149.17, 149.04, 147.62, 142.60, 140.81, 138.28, 133.67, 129.36, 129.29, 129.03, 128.22, 128.09, 127.37, 124.01, 123.90, 123.84, 115.33, 114.51, 114.29, 113.27, 58.60, 51.52, 42.86, 25.52. HRMS(ESI) calcd for C27H22FN6O+ 465.1834, found 465.1835 [M + H]+. HPLC purity 98%. HPLC: tR = 6.36 min. 4.1.55. (S)-2-(3-(9-(6-Aminopyridin-3-yl)-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9c). The desired compound was prepared from 6b (50 mg, 0.12 mmol) and (6aminopyridin-3-yl)boronic acid (33 mg, 0.24 mmol) using the procedure described for compound 8a in 29% yield as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.34 (s, 1H), 8.30 (s, 1H), 8.16 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 11.7 Hz, 1H), 7.81 897



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HRMS(ESI) calcd for C23H21FN7O+ 430.1786, found 430.1783 [M + H]+. HPLC purity 96%. HPLC: tR = 4.73 min. 4.1.60. (S)-2-(3-((8-Fluoro-2-oxo-9-(quinolin-3-yl)pyrazino[2,3-c]quinolin-1(2H)-yl)methyl)pyrrolidin-1-yl)acetonitrile (9h). The desired compound was prepared from 6d (20 mg, 0.05 mmol) and quinolin-3ylboronic acid (12 mg, 0.07 mmol) using the procedure described for compound 8a in 56% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.28−9.17 (m, 2H), 8.92 (d, J = 7.9 Hz, 1H), 8.76 (s, 1H), 8.48 (s, 1H), 8.22−8.08 (m, 3H), 7.88 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.73 (t, J = 7.6 Hz, 1H), 5.03−4.90 (m, 1H), 4.86−4.70 (m, 1H), 3.65− 3.53 (m, 1H), 3.53−3.41 (m, 1H), 3.03−2.88 (m, 1H), 2.66−2.54 (m, 3H), 2.47−2.33 (m, 1H), 1.96−1.80 (m, 1H), 1.70−1.55 (m, 1H); 13 C NMR (101 MHz, DMSO) δ 160.99, 158.47, 156.56, 153.50, 150.56, 150.53, 150.27, 150.00, 149.87, 146.98, 136.37, 136.23, 130.44, 128.97, 128.93, 128.79, 128.47, 127.58, 127.42, 127.22, 126.76, 126.13, 125.95, 115.79, 115.26, 115.05, 55.58, 50.99, 49.22, 40.71, 36.36, 27.67. HRMS(ESI) calcd for C27H22FN6O+ 465.1834, found 465.1828 [M + H]+. HPLC purity 98%. HPLC: tR = 6.22 min. 4.1.61. (S)-2-(3-(9-(6-Aminopyridin-3-yl)-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9i). The desired compound was prepared from 6f (40 mg, 0.1 mmol) and (6-aminopyridin-3yl)boronic acid (28 mg, 0.2 mmol) using the procedure described for compound 8a in 58% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.47 (d, J = 2.5 Hz, 1H), 8.30 (s, 1H), 8.22 (s, 1H), 8.16 (s, 2H), 7.92 (dd, J = 8.7, 2.6 Hz, 1H), 6.61 (d, J = 8.6 Hz, 1H), 6.27 (s, 2H), 5.05−4.91 (m, 1H), 3.99−3.80 (m, 2H), 3.56−3.40 (m, 2H), 2.93−2.83 (m, 1H), 2.69−2.54 (m, 1H), 2.32−2.20 (m, 1H), 1.89−1.75 (m, 2H), 1.63−1.44 (m, 1H); 13C NMR (101 MHz, DMSO) δ 159.70, 157.34, 151.47, 150.42, 147.34, 146.65, 136.79, 135.84, 135.60, 130.79, 128.40, 127.38, 122.66, 120.32, 117.86, 115.72, 108.28, 61.21, 53.69, 51.35, 45.23, 25.60, 24.54. HRMS(ESI) calcd for C23H22N7O+ 412.1880, found 412.1882 [M + H]+. HPLC purity 98%. HPLC: tR = 4.28 min. 4.1.62. (S)-2-(3-(2-Oxo-9-(quinolin-3-yl)pyrazino[2,3-c]quinolin1(2H)-yl)piperidin-1-yl)acetonitrile (9j). The desired compound was prepared from 6f (40 mg, 0.1 mmol) and quinolin-3-ylboronic acid (35 mg, 0.2 mmol) using the procedure described for compound 8a in 44% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.46 (d, J = 2.4 Hz, 1H), 9.15 (s, 1H), 8.85 (d, J = 2.3 Hz, 1H), 8.56 (s, 1H), 8.49−8.41 (m, 1H), 8.38−8.25 (m, 2H), 8.15−8.07 (m, 2H), 7.84 (ddd, J = 8.3, 6.8, 1.4 Hz, 1H), 7.71 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H), 5.14−5.03 (m, 1H), 4.02−3.85 (m, 2H), 3.55 (dt, J = 21.3, 10.8 Hz, 2H), 2.91 (d, J = 10.8 Hz, 1H), 2.70−2.56 (m, 1H), 2.29 (t, J = 11.5 Hz, 1H), 1.83 (s, 2H), 1.68− 1.52 (m, 1H); 13C NMR (101 MHz, DMSO) δ 157.27, 151.76, 151.54, 149.50, 148.13, 147.07, 137.18, 134.67, 133.73, 131.80, 131.21, 130.04, 129.49, 128.72, 128.52, 127.62, 127.43, 127.40, 123.47, 117.85, 115.63, 61.36, 53.63, 51.39, 45.30, 25.60, 24.54. HRMS(ESI) calcd for C27H23N6O+ 447.1928, found 447.1925 [M + H]+. HPLC purity 97%. HPLC: tR = 5.95 min. 4.1.63. 2-(3-(9-(6-Aminopyridin-3-yl)-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)pyrrolidin-1-yl)acetonitrile (9k). The desired compound was prepared from 6g (40 mg, 0.1 mmol) and (6-aminopyridin3-yl)boronic acid (29 mg, 0.2 mmol) using the procedure described for compound 8a in 39% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.39 (d, J = 2.5 Hz, 1H), 8.32 (s, 1H), 8.28 (s, 1H), 8.19−8.10 (m, 2H), 7.83 (dd, J = 8.6, 2.6 Hz, 1H), 6.62 (d, J = 8.6 Hz, 1H), 6.27 (s, 2H), 5.69−5.57 (m, 1H), 3.92 (d, J = 2.2 Hz, 2H), 3.40−3.35 (m, 1H), 3.22 (t, J = 8.5 Hz, 1H), 3.18−3.09 (m, 1H), 2.99− 2.91 (m, 1H), 2.46−2.39 (m, 1H), 2.38−2.27 (m, 1H); 13C NMR (101 MHz, DMSO) δ 159.57, 156.93, 151.47, 150.33, 147.19, 146.45, 137.14, 135.79, 135.77, 130.57, 128.72, 127.28, 122.95, 120.23, 118.00, 116.19, 108.44, 60.74, 53.05, 51.66, 40.94, 29.18. HRMS(ESI) calcd for C22H20N7O+ 398.1724, found 398.1725 [M + H]+. HPLC purity 99%. HPLC: tR = 6.03 min. 4.1.64. 2-(3-(2-Oxo-9-(quinolin-3-yl)pyrazino[2,3-c]quinolin1(2H)-yl)pyrrolidin-1-yl)acetonitrile (9l). The desired compound was prepared from 6g (40 mg, 0.1 mmol) and quinolin-3-ylboronic acid (36 mg, 0.2 mmol) using the procedure described for compound 8a in 30% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.38 (d, J = 2.3 Hz, 1H), 9.15 (s, 1H), 8.81 (d, J = 2.4 Hz, 1H), 8.61 (s, 1H), 8.41 (dd, J = 8.8, 1.7 Hz, 1H), 8.36 (s, 1H), 8.31 (d, J = 8.6 Hz, 1H), 8.12

(d, J = 8.7 Hz, 1H), 6.62 (d, J = 8.7 Hz, 1H), 6.40 (s, 2H), 4.97−4.87 (m, 1H), 3.95−3.75 (m, 2H), 3.49−3.39 (m, 2H), 2.86 (d, J = 10.9 Hz, 1H), 2.68−2.56 (m, 1H), 2.25 (t, J = 11.5 Hz, 1H), 1.87−1.75 (m, 2H), 1.65−1.48 (m, 1H); 13C NMR (101 MHz, DMSO) δ 161.14, 159.72, 158.62, 157.25, 151.80, 151.38, 148.68, 148.55, 148.02, 147.99, 137.44, 137.42, 137.10, 127.07, 127.06, 126.44, 126.28, 125.60, 125.55, 118.08, 118.07, 115.67, 115.08, 115.07, 114.79, 114.57, 108.06, 61.32, 53.59, 51.33, 45.24, 25.65, 24.46. HRMS(ESI) calcd for C23H21FN7O+ 430.1786, found 430.1792 [M + H]+. HPLC purity 96%. HPLC: tR = 6.40 min. 4.1.56. (S)-2-(3-(8-Fluoro-2-oxo-9-(quinolin-3-yl)pyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9d). The desired compound was prepared from 6b (50 mg, 0.12 mmol) and quinolin-3ylboronic acid (41 mg, 0.24 mmol) using the procedure described for compound 8a in 55% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (t, J = 2.3 Hz, 1H), 9.17 (s, 1H), 8.76 (s, 1H), 8.42 (d, J = 7.9 Hz, 1H), 8.33 (s, 1H), 8.17−8.07 (m, 3H), 7.86 (t, J = 8.4, 6.9, 1.4 Hz, 1H), 7.72 (t, J = 7.4 Hz, 1H), 5.07−4.96 (m, 1H), 3.94−3.72 (m, 2H), 3.53−3.45 (m, 2H), 2.90 (d, J = 10.9 Hz, 1H), 2.65−2.54 (m, 1H), 2.26 (t, J = 11.4 Hz, 1H), 1.87−1.75 (m, 2H), 1.70−1.55 (m, 1H); 13 C NMR (101 MHz, DMSO) δ 161.24, 158.70, 157.06, 152.40, 152.06, 151.94, 147.70, 147.58, 146.28, 145.55, 145.41, 139.48, 138.26, 137.70, 135.12, 131.04, 130.32, 129.80, 129.72, 129.33, 128.50, 128.42, 127.72, 127.37, 127.22, 124.02, 123.85, 121.87, 120.56, 115.14, 113.57, 113.39, 58.01, 52.46, 43.50, 24.76, 22.17. HRMS(ESI) calcd for C27H22FN6O+ 465.1834, found 465.1840 [M + H]+. HPLC purity 99%. HPLC: tR = 6.29 min. 4.1.57. 2-(4-((9-(6-Aminopyridin-3-yl)-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)methyl)piperidin-1-yl)acetonitrile (9e). The desired compound was prepared from 6c (30 mg, 0.12 mmol) and (6aminopyridin-3-yl)boronic acid (19 mg, 0.14 mmol) using the procedure described for compound 8a in 46% yield as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 1H), 8.52 (d, J = 8.1 Hz, 1H), 8.45 (s, 1H), 8.24 (t, J = 1.9 Hz, 1H), 8.00 (d, J = 11.3 Hz, 1H), 7.73 (dt, J = 8.6, 2.3 Hz, 1H), 6.60 (d, J = 8.6 Hz, 1H), 6.38 (s, 2H), 4.81−4.71 (m, 2H), 3.66 (s, 2H), 2.78−2.69 (m, 2H), 2.06−1.96 (m, 3H), 1.63−1.54 (m, 2H), 1.44−1.31 (m, 2H); 13C NMR (101 MHz, DMSO) δ 161.08, 159.79, 158.56, 156.45, 152.83, 149.78, 149.26, 149.14, 148.05, 148.03, 137.63, 137.60, 135.78, 127.24, 127.08, 126.67, 126.62, 118.17, 115.86, 115.17, 115.16, 115.08, 114.86, 107.64, 50.98, 49.19, 45.27, 40.15, 34.30, 28.93. HRMS(ESI) calcd for C24H23FN7O+ 444.1943, found 444.1943 [M + H]+. HPLC purity 96%. HPLC: tR = 4.42 min. 4.1.58. 2-(4-((8-Fluoro-2-oxo-9-(quinolin-3-yl) pyrazino[2,3-c]quinolin-1(2H)-yl)methyl)piperidin-1-yl)acetonitrile (9f). The desired compound was prepared from 6c (20 mg, 0.05 mmol) and quinolin-3ylboronic acid (16 mg, 0.09 mmol) using the procedure described for compound 8a in 64% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.22 (d, J = 3.0 Hz, 2H), 8.79 (d, J = 7.9 Hz, 1H), 8.72 (s, 1H), 8.19−8.12 (m, 2H), 8.10 (d, J = 8.1 Hz, 1H), 7.88 (t, J = 8.4, 6.8, 1.5 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 4.79 (d, J = 7.0 Hz, 2H), 3.64 (s, 2H), 2.69 (d, J = 11.0 Hz, 2H), 2.15−1.92 (m, 3H), 1.71−1.48 (m, 2H), 1.41−1.27 (m, 2H); 13C NMR (101 MHz, DMSO) δ 160.98, 158.46, 156.47, 153.65, 150.47, 150.10, 149.98, 147.01, 136.19, 136.14, 130.48, 129.07, 129.03, 128.84, 128.36, 127.83, 127.48, 127.20, 126.80, 126.11, 125.94, 115.88, 115.34, 115.26, 115.14, 50.90, 49.23, 45.24, 34.11, 28.92. HRMS(ESI) calcd for C28H24FN6O+ 479.1990, found 479.1988 [M + H]+. HPLC purity 96%. HPLC: tR = 4.64 min. 4.1.59. (S)-2-(3-((9-(6-Aminopyridin-3-yl)-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)methyl)pyrrolidin-1-yl)acetonitrile (9g). The desired compound was prepared from 6d (20 mg, 0.05 mmol) and (6aminopyridin-3-yl)boronic acid (11 mg, 0.07 mmol) using the procedure described for compound 8a in 96% yield as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 8.64 (d, J = 8.1 Hz, 1H), 8.45 (s, 1H), 8.27 (s, 1H), 8.00 (d, J = 11.3 Hz, 1H), 7.84−7.63 (m, 1H), 6.61 (d, J = 8.6 Hz, 1H), 6.37 (s, 2H), 4.91 (dd, J = 15.0, 8.8 Hz, 1H), 4.74 (dd, J = 14.9, 5.7 Hz, 1H), 3.73−3.57 (m, 2H), 2.95−2.84 (m, 1H), 2.72−2.63 (m, 1H), 2.60−2.53 (m, 2H), 1.97−1.86 (m, 1H), 1.68−1.57 (m, 1H); 13C NMR (101 MHz, DMSO) δ 159.61, 158.58, 156.57, 152.69, 149.99, 149.18, 149.06, 147.98, 137.88, 135.91, 131.60, 128.78, 127.21, 127.04, 126.70, 126.66, 118.09, 115.90, 115.64, 115.19, 114.98, 114.77, 113.87, 107.73, 55.68, 50.88, 49.17, 40.78, 36.61, 27.66. 898



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(t, J = 8.4 Hz, 2H), 7.93−7.80 (m, 1H), 7.71 (t, J = 7.5 Hz, 1H), 5.79− 5.66 (m, 1H), 3.91 (s, 2H), 3.41−3.34 (m, 1H), 3.16 (q, J = 8.2 Hz, 1H), 3.01−2.91 (m, 1H), 2.43−2.29 (m, 2H); 13C NMR (101 MHz, DMSO) δ 156.91, 151.75, 151.38, 149.45, 147.93, 146.99, 137.53, 134.56, 133.87, 132.15, 130.92, 130.09, 129.81, 128.69, 128.63, 127.61, 127.36, 123.31, 118.01, 116.17, 60.92, 53.24, 51.73, 40.98, 29.36. HRMS(ESI) calcd for C26H21N6O+ 433.1771, found 433.1768 [M + H]+. HPLC purity 99%. HPLC: tR = 4.96 min. 4.1.65. 2-(4-(9-(6-Aminopyridin-3-yl)-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9m). To a solution of compound 6e (1 g, 2.53 mmol) in 1,4-dioxane (50 mL) at room temperature was subsequently added PdCl2(Ph3P)2 (88 mg, 0.1 equiv), Na2CO3 (7.6 mL, 7.54 mmol, 1M), and (6-aminopyridin-3-yl)boronic acid (208 mg, 3.02 mmol), then degassing with argon for 10 min, the resulting mixture was heated to 85 °C overenight. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/methanol 100:1 to 30:1) to afford title compond in 82% yield as a yellow amorphous solid. mp 272−274 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.42 (d, J = 2.5 Hz, 1H), 8.28 (s, 1H), 8.23−8.07 (m, 3H), 7.86 (dd, J = 8.6, 2.6 Hz, 1H), 6.61 (d, J = 8.6 Hz, 1H), 6.33 (s, 2H), 4.92−4.79 (m, 1H), 3.79 (s, 2H), 3.12−2.89 (m, 4H), 2.31 (t, J = 11.7 Hz, 2H), 2.01 (d, J = 11.9 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 157.38, 153.45, 151.91, 151.26, 147.52, 142.57, 137.41, 134.20, 132.46, 130.73, 128.95, 127.47, 123.78, 122.55, 117.90, 114.78, 114.19, 60.25, 51.39, 44.09, 26.74. HRMS(ESI) calcd for C23H22N7O+ 412.1880, found 412.1882 [M + H]+. HPLC purity 99%. HPLC: tR = 4.51 min. The spectra of 1H NMR, 13C NMR, and HRMS and HPLC purity analysis for compound 9m are in Supporting Information. 4.1.66. 2-(4-(2-Oxo-9-(quinolin-3-yl)pyrazino[2,3-c]quinolin1(2H)-yl)piperidin-1-yl)acetonitrile (9n). The desired compound was prepared from 6e (40 mg, 0.1 mmol) and quinolin-3-ylboronic acid (26 mg, 0.15 mmol) using the procedure described for compound 8a in 51% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.39 (d, J = 2.3 Hz, 1H), 9.15 (s, 1H), 8.81 (d, J = 2.3 Hz, 1H), 8.49 (s, 1H), 8.42 (dd, J = 8.8, 1.7 Hz, 1H), 8.34 (s, 1H), 8.33 (s, 1H), 8.20−8.09 (m, 2H), 7.84 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.71 (ddd, J = 8.1, 6.8, 1.3 Hz, 1H), 5.02−4.89 (m, 1H), 3.80 (s, 2H), 3.11−2.91 (m, 4H), 2.44−2.32 (m, 2H), 2.08 (d, J = 12.0 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 157.41, 152.71, 150.18, 145.93, 144.83, 142.76, 140.34, 138.52, 134.40, 133.45, 132.92, 131.36, 129.91, 129.62, 128.42, 127.56, 125.08, 122.09, 118.18, 113.12, 58.73, 51.54, 42.68, 25.47. HRMS(ESI) calcd for C27H23N6O+ 447.1928, found 447.1926 [M + H]+. HPLC purity 96%. HPLC: tR = 6.05 min. 4.1.67. 2-(4-(8-Fluoro-9-(naphthalen-2-yl)-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9o). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and naphthalen-2ylboronic acid (19 mg, 0.14 mmol) using the procedure described for compound 8a in 71% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 1H), 8.31 (s, 1H), 8.29 (s, 2H), 8.19−8.14 (m, 1H), 8.12 (s, 1H), 8.10 (d, J = 3.5 Hz, 1H), 8.06−8.00 (m, 1H), 7.85 (dt, J = 8.5, 2.0 Hz, 1H), 7.66−7.58 (m, 2H), 5.11−4.84 (m, 1H), 3.84 (s, 2H), 3.08−2.90 (m, 4H), 2.48−2.40 (m, 2H), 1.97 (d, J = 11.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 162.22, 159.66, 157.77, 152.62, 151.29, 149.81, 149.69, 137.42, 133.39, 133.18, 132.07, 130.96, 130.79, 128.81, 128.66, 128.64, 128.47, 127.77, 127.48, 127.47, 127.07, 126.97, 126.94, 126.70, 126.67, 115.60, 115.38, 115.05, 115.03, 114.41, 62.34, 51.87, 46.04, 28.04. HRMS(ESI) calcd for C28H23FN5O+ 464.1881, found 464.1881 [M + H]+. HPLC purity 99%. HPLC: tR = 7.82 min. 4.1.68. 2-(4-(8-Fluoro-2-oxo-9-(pyridin-3-yl)pyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9p). The desired compound was prepared from 6a (50 mg, 0.12 mmol) and pyridin-3-ylboronic acid (30 mg, 0.24 mmol) using the procedure described for compound 8a in 38% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 8.95 (d, J = 2.4 Hz, 1H), 8.72 (dd, J = 4.8, 1.6 Hz, 1H), 8.30 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.18 (dt, J = 7.9, 1.9 Hz, 1H), 8.11 (d, J = 11.2 Hz, 1H), 7.62 (dd, J = 7.9, 4.8 Hz, 1H), 4.94−4.80 (m, 1H), 3.77 (s, 2H), 2.96 (q, J = 11.8, 11.1 Hz, 4H), 2.35−2.22 (m, 2H), 2.03−1.91 (m, 2H); 13C NMR (101 MHz, DMSO) δ 160.87, 158.35, 157.26, 152.70, 151.60, 149.70, 149.55, 149.30, 149.27, 137.51, 136.71, 136.68, 130.50, 128.24, 128.20, 127.09, 125.39, 125.22, 123.92, 115.66, 115.22,

114.88, 114.67, 61.49, 51.08, 45.06, 27.57. HRMS(ESI) calcd for C23H20FN6O+ 415.1677, found 415.1677 [M + H]+. HPLC purity 96%. HPLC: tR = 6.81 min. 4.1.69. 2-(4-(8-Fluoro-2-oxo-9-(pyridin-4-yl)pyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9q). The desired compound was prepared from 6a (50 mg, 0.12 mmol) and pyridin-4ylboronic acid (30 mg, 0.24 mmol) using the procedure described for compound 8a in 63% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.81−8.72 (m, 2H), 8.30 (s, 1H), 8.28 (d, 1H), 8.12 (d, J = 11.4 Hz, 1H), 7.83−7.72 (m, 2H), 4.97−4.83 (m, 1H), 3.79 (s, 2H), 3.05−2.87 (m, 4H), 2.32 (t, J = 11.2 Hz, 2H), 1.98 (d, J = 12.0 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 160.47, 157.93, 157.27, 153.64, 152.00, 150.46, 150.33, 149.93, 143.78, 137.94, 129.67, 129.64, 127.29, 127.28, 126.61, 126.57, 123.85, 123.69, 115.18, 114.98, 113.88, 59.14, 51.42, 43.40, 25.96. HRMS(ESI) calcd for C23H20FN6O+ 415.1677, found 415.1676 [M + H]+. HPLC purity 99%. HPLC: tR = 3.33 min. 4.1.70. 2-(4-(8-Fluoro-2-oxo-9-(pyrimidin-5-yl)pyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9r). The desired compound was prepared from 6a (50 mg, 0.12 mmol) and pyridin-4ylboronic acid (30 mg, 0.24 mmol) using the procedure described for compound 8a in 34% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.33 (s, 1H), 9.21 (d, J = 1.5 Hz, 2H), 9.18 (s, 1H), 8.31 (s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 11.3 Hz, 1H), 4.87−4.77 (m, 1H), 3.74 (s, 2H), 3.01−2.87 (m, 4H), 2.26 (t, J = 11.8 Hz, 2H), 2.01 (d, J = 11.9 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 161.02, 158.48, 158.08, 157.40, 156.76, 152.37, 152.06, 148.89, 148.77, 138.42, 128.70, 128.39, 128.35, 127.30, 122.55, 122.38, 115.29, 114.25, 114.03, 113.03, 58.65, 51.44, 42.82, 25.51. HRMS(ESI) calcd for C22H19FN7O+ 416.1630, found 416.1630 [M + H]+. HPLC purity 96%. HPLC: tR = 5.33 min. 4.1.71. 2-(4-(8-Fluoro-2-oxo-9-phenylpyrazino[2,3-c]quinolin1(2H)-yl)piperidin-1-yl)acetonitrile (9s). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and phenylboronic acid (18 mg, 0.14 mmol) using the procedure described for compound 8a in 62% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 8.28 (s, 1H), 8.21 (d, J = 7.7 Hz, 1H), 8.06 (d, J = 11.3 Hz, 1H), 7.75 (d, J = 7.3 Hz, 2H), 7.66−7.48 (m, 3H), 5.00−4.88 (m, 1H), 3.81 (s, 2H), 3.06−2.86 (m, 4H), 2.43−2.32 (m, 2H), 1.95 (d, J = 12.0 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 162.02, 159.47, 157.69, 157.65, 152.63, 151.19, 149.77, 149.73, 149.64, 149.62, 137.32, 137.30, 134.58, 130.93, 130.92, 130.75, 130.74, 129.29, 129.09, 129.07, 129.03, 129.01, 127.40, 126.86, 126.81, 115.48, 115.26, 114.85, 114.17, 114.14, 62.14, 51.79, 46.01, 28.00. HRMS(ESI) calcd for C24H21FN5O+ 414.1725, found 414.1720 [M + H]+. HPLC purity 96%. HPLC purity 96%. HPLC: tR = 4.16 min. 4.1.72. 2-(4-(8-Fluoro-9-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9t). The desired compound was prepared from 6a (90 mg, 0.21 mmol) and 1-methyl-4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin2-yl)piperazine (98 mg, 0.32 mmol) using the procedure described for compound 8a in 45% yield as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ 9.16 (s, 1H), 8.46 (t, J = 2.3 Hz, 1H), 8.25 (s, 1H), 8.06 (d, J = 7.7 Hz, 1H), 7.94 (d, J = 10.9 Hz, 1H), 7.82 (d, J = 8.8 Hz, 1H), 6.87 (d, J = 8.9 Hz, 1H), 4.88 (t, J = 12.0 Hz, 1H), 3.83−3.68 (m, 4H), 3.64 (s, 2H), 3.31 (tt, J = 13.1, 6.6 Hz, 2H), 3.02 (d, J = 11.0 Hz, 2H), 2.73−2.36 (m, 9H), 1.92 (d, J = 12.8 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 161.87, 159.33, 157.36, 153.09, 152.76, 150.11, 144.83, 144.71, 143.47, 141.49, 139.69, 128.34, 128.30, 127.28, 124.78, 124.61, 120.26, 115.55, 113.08, 112.59, 111.71, 111.47, 58.82, 51.81, 51.73, 43.38, 42.67, 42.51, 25.51. HRMS(ESI) calcd for C28H30FN8O+ 513.2521, found 513.2525 [M + H]+. HPLC purity 99%. HPLC: tR = 6.28 min. 4.1.73. 2-(4-(8-Fluoro-9-(6-morpholinopyridin-3-yl)-2oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9u). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)morpholine (31 mg, 0.11 mmol) using the procedure described for compound 8a in 41% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 1H), 8.50 (d, J = 2.3 Hz, 1H), 8.28 (s, 1H), 8.15 (d, J = 8.1 Hz, 1H), 8.03 (d, J = 11.4 Hz, 1H), 7.93 (dt, J = 9.0, 1.9 Hz, 1H), 899



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(s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.13−8.03 (m, 2H), 7.03 (d, J = 8.6 Hz, 1H), 4.93−4.80 (m, 1H), 3.94 (s, 3H), 3.77 (s, 2H), 3.05−2.87 (m, 4H), 2.35−2.23 (m, 2H), 2.01−1.91 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 164.47, 161.96, 159.41, 157.71, 152.70, 151.38, 149.85, 149.73, 146.81, 146.78, 139.46, 137.34, 127.49, 127.38, 127.20, 126.14, 126.09, 123.73, 115.67, 115.45, 115.11, 114.30, 111.43, 111.21, 62.35, 53.87, 51.85, 45.88, 28.14. HRMS(ESI) m/z calcd for C24H22FN6O2+ 445.1783, found 445.1786 [M + H]+. HPLC purity 97%. HPLC: tR = 6.31 min. 4.1.79. 2-(4-(9-(4-Aminophenyl)-8-fluoro-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9aa). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and 4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (24 mg, 0.11 mmol) using the procedure described for compound 8a in 61% yield as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.06 (s, 1H), 8.26 (s, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 11.5 Hz, 1H), 7.48−7.33 (m, 2H), 6.73 (d, J = 8.3 Hz, 2H), 4.99−4.82 (m, 1H), 3.82 (s, 2H), 2.97 (t, J = 11.5 Hz, 4H), 2.46−2.27 (m, 2H), 1.94 (d, J = 11.9 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 161.19, 158.68, 157.31, 151.59, 151.19, 149.53, 148.52, 148.39, 137.05, 130.08, 130.05, 129.54, 129.37, 127.01, 126.10, 121.18, 115.54, 115.17, 114.58, 114.36, 113.91, 61.21, 51.14, 45.20, 27.42. HRMS(ESI) calcd for C24H22FN6O+ 429.1834, found 429.1830 [M + H]+. HPLC purity 98%. HPLC: tR = 5.39 min. 4.1.80. 2-(4-(8-Fluoro-9-(4-methoxyphenyl)-2-oxopyrazino[2,3c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9ab). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and 2-(4methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (25 mg, 0.11 mmol) using the procedure described for compound 8a in 56% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 1H), 8.28 (s, 1H), 8.17 (d, J = 8.1 Hz, 1H), 8.02 (d, J = 11.3 Hz, 1H), 7.74− 7.62 (m, 2H), 7.22−7.09 (m, 2H), 5.01−4.90 (m, 1H), 3.84 (s, 3H), 3.81 (s, 2H), 3.05−2.90 (m, 4H), 2.38 (t, J = 11.5 Hz, 2H), 1.94 (d, J = 11.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 162.10, 160.25, 159.55, 157.70, 152.32, 151.10, 149.44, 149.31, 137.22, 130.67, 130.58, 130.56, 130.50, 130.33, 127.38, 126.86, 126.32, 126.27, 115.34, 115.11, 114.89, 114.87, 114.56, 114.42, 114.21, 62.02, 55.39, 51.80, 46.02, 27.95. HRMS(ESI) calcd for C25H23FN5O2+ 444.1830, found 444.1827 [M + H]+. HPLC purity 97%. HPLC: tR = 6.98 min. 4.1.81. 2-(4-(8-Fluoro-9-(6-methylpyridin-3-yl)-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9ac). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and (6methylpyridin-3-yl)boronic acid (15 mg, 0.11 mmol) using the procedure described for compound 8a in 80% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 1H), 8.84 (d, J = 2.5 Hz, 1H), 8.29 (s, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.15−8.05 (m, 2H), 7.51 (d, J = 8.1 Hz, 1H), 4.94−4.81 (m, 1H), 3.79 (s, 2H), 3.07−2.87 (m, 5H), 2.60 (s, 3H), 2.33 (t, J = 12.2 Hz, 2H), 1.98 (d, J = 12.0 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 165.09, 161.77, 159.22, 158.77, 157.61, 153.01, 151.45, 150.09, 149.97, 148.02, 147.98, 137.68, 137.38, 128.08, 127.48, 126.95, 126.78, 126.62, 126.57, 124.20, 115.79, 115.57, 115.10, 114.36, 62.37, 51.86, 45.87, 28.11, 23.67. HRMS(ESI) calcd for C24H22FN6O+ 429.1834, found 429.1839 [M + H]+. HPLC purity 96%. HPLC: tR = 5.51 min. 4.1.82. 2-(4-(8-Fluoro-2-oxo-9-(6-(trifluoromethyl)pyridin-3-yl)pyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9ad). The desired compound was prepared from 6a (50 mg, 0.12 mmol) and (6-(trifluoromethyl)pyridin-3-yl)boronic acid (30 mg, 0.24 mmol) using the procedure described for compound 8a in 60% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 9.16 (s, 1H), 8.50 (d, J = 8.2 Hz, 1H), 8.31 (s, 1H), 8.29 (s, 1H), 8.13 (dd, J = 17.4, 9.7 Hz, 2H), 4.94−4.82 (m, 1H), 3.77 (s, 2H), 3.03−2.87 (m, 4H), 2.29 (t, J = 12.4 Hz, 2H), 1.99 (d, 2H); 13C NMR (101 MHz, DMSO) δ 160.77, 160.66, 158.26, 158.14, 157.23, 157.19, 153.05, 151.70, 149.98, 149.90, 146.32, 145.95, 138.90, 137.66, 137.57, 134.08, 133.96, 128.82, 127.22, 127.13, 123.92, 123.75, 122.94, 120.88, 120.21, 115.78, 115.26, 115.16, 114.97, 114.75, 61.50, 51.07, 45.07, 27.51. HRMS(ESI) calcd for C24H19F4N6O+ 483.1551, found 483.1547 [M + H]+. HPLC purity 96%. HPLC: tR = 4.99 min. 4.1.83. 2-(4-(8-Fluoro-9-(furan-3-yl)-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9ae). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and 2-(furan-3-yl)4,4,5,5-tetramethyl-1,3,2-dioxaborolane (21 mg, 0.11 mmol) using the

7.01 (d, J = 8.9 Hz, 1H), 4.91−4.80 (m, 1H), 3.78 (s, 2H), 3.73 (t, J = 4.9 Hz, 4H), 3.55 (t, J = 4.8 Hz, 4H), 3.04−2.90 (m, 4H), 2.36−2.26 (m, 2H), 1.96 (d, J = 11.9 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 161.13, 158.67, 158.61, 157.35, 152.02, 151.43, 149.02, 148.90, 147.66, 147.62, 138.11, 137.26, 127.08, 126.53, 126.50, 126.09, 125.92, 119.49, 115.79, 115.30, 114.72, 114.50, 106.69, 65.91, 61.46, 51.15, 45.05, 44.84, 27.55. HRMS(ESI) m/z calcd for C27H27FN7O2+ 500.2205, found 500.2207 [M + H]+. HPLC purity 96%. HPLC: tR = 6.19 min. 4.1.74. 2-(4-(8-Fluoro-2-oxo-9-(1H-pyrazol-4-yl)pyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9v). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and 4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (21 mg, 0.11 mmol) using the procedure described for compound 8a in 33% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 13.37 (s, 1H), 9.06 (s, 1H), 8.37−8.24 (m, 3H), 8.08−7.95 (m, 2H), 4.77−4.65 (m, 1H), 3.77 (s, 2H), 3.08−2.91 (m, 4H), 2.34−2.19 (m, 2H), 2.13−2.00 (m, 2H); 13C NMR (101 MHz, DMSO) δ 160.73, 158.22, 157.45, 151.49, 151.40, 148.25, 148.12, 137.21, 137.09, 127.94, 127.18, 123.37, 123.31, 120.93, 120.76, 115.83, 115.32, 114.65, 114.44, 113.97, 61.85, 51.26, 44.95, 27.71. HRMS(ESI) calcd for C21H19FN7O+ 404.1630, found 404.1630 [M + H]+. HPLC purity 98%. HPLC: tR = 4.71 min. 4.1.75. 2-(4-(8-Fluoro-9-(1-methyl-1H-pyrazol-4-yl)-2oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9w). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (22 mg, 0.11 mmol) using the procedure described for compound 8a in 74% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.06 (s, 1H), 8.32−8.24 (m, 3H), 8.00 (d, J = 11.9 Hz, 1H), 7.93 (s, 1H), 3.96 (s, 3H), 3.78 (s, 2H), 3.07−2.91 (m, 4H), 2.26 (t, J = 12.1 Hz, 2H), 2.04 (d, J = 11.8 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 161.03, 158.49, 157.39, 151.97, 150.11, 145.57, 145.44, 138.97, 137.47, 137.46, 130.72, 130.63, 127.17, 123.53, 123.46, 121.45, 121.28, 115.37, 114.29, 113.21, 112.92, 112.70, 58.90, 51.49, 42.81, 25.78. HRMS(ESI) calcd for C22H21FN7O+ 418.1786, found 418.1787 [M + H]+. HPLC purity 96%. HPLC: tR = 5.06 min. 4.1.76. 2-(4-(9-(1-(Difluoromethyl)-1H-pyrazol-4-yl)-8-fluoro-2oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9x). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and 1-(difluoromethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)1H-pyrazole (26 mg, 0.11 mmol) using the procedure described for compound 8a in 22% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 1H), 8.78 (d, J = 2.5 Hz, 1H), 8.36 (d, J = 7.8 Hz, 1H), 8.31 (s, 1H), 8.29 (s, 1H), 8.07 (d, J = 11.8 Hz, 1H), 7.95 (t, J = 60.0 Hz, 1H), 4.77−4.67 (m, 1H), 3.76 (s, 2H), 3.06−2.91 (m, 4H), 2.26 (t, J = 12.2 Hz, 3H), 2.08 (d, J = 11.9 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 160.64, 158.12, 157.40, 152.17, 151.62, 148.91, 148.78, 140.72, 137.32, 128.53, 128.45, 127.22, 124.73, 124.68, 118.88, 118.71, 116.91, 115.83, 115.25, 114.83, 114.62, 112.62, 110.14, 107.67, 61.90, 51.18, 44.96, 27.71. HRMS(ESI) calcd for C22H19F3N7O+ 454.1598, found 454.1596 [M + H]+. HPLC purity 96%. HPLC: tR = 5.38 min. 4.1.77. 5-(1-(1-(Cyanomethyl)piperidin-4-yl)-8-fluoro-2-oxo-1,2dihydropyrazino[2,3-c]quinolin-9-yl)picolinonitrile (9y). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and (6-cyanopyridin3-yl)boronic acid (16 mg, 0.11 mmol) using the procedure described for compound 8a in 51% yield as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 9.15 (s, 1H), 8.46 (dt, J = 8.0, 1.8 Hz, 1H), 8.31 (s, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.24 (d, J = 8.1 Hz, 1H), 8.16 (d, J = 11.4 Hz, 1H), 4.92−4.78 (m, 1H), 3.75 (s, 2H), 3.03−2.85 (m, 4H), 2.26 (t, J = 11.6 Hz, 2H), 1.99 (d, J = 12.2 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 160.75, 158.22, 157.35, 152.97, 151.91, 150.97, 149.75, 149.62, 138.40, 137.82, 134.17, 132.23, 131.54, 131.44, 129.13, 128.83, 128.71, 127.27, 123.90, 123.73, 117.40, 115.18, 114.84, 114.63, 113.58, 59.00, 51.44, 43.31, 25.85. HRMS(ESI) calcd for C24H19FN7O+ 440.1630, found 440.1624 [M + H]+. HPLC purity 96%. HPLC: tR = 4.94 min. 4.1.78. 2-(4-(8-Fluoro-9-(6-methoxypyridin-3-yl)-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9z). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and (6methoxypyridin-3-yl)boronic acid (17 mg, 0.11 mmol) using the procedure described for compound 8a in 84% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 8.54 (t, J = 2.2 Hz, 1H), 8.29 900



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HRMS(ESI) calcd for C22H21N6O2+ 401.1721, found 401.1725 [M + H]+. HPLC purity 98%. HPLC: tR = 4.29 min. 4.1.89. 9-(6-Aminopyridin-3-yl)-1-(1-(2-hydroxyethyl)piperidin-4yl)pyrazino[2,3-c]quinolin-2(1H)-one (12b). A mixture of 9-(6-aminopyridin-3-yl)-1-(piperidin-4-yl)pyrazino[2,3-c]quinolin-2(1H)-one HCl (30 mg, 0.07 mmol), sodium bicarbonate (34 mg, 0.4 mmol), 2-bromoethanol (9 μL, 0.12 mmol), and anhydrous DMF (5 mL) was stirred at 70 °C for 24 h under inert atmosphere.The reaction mixture was concentrated to dryness.The crude product was purified by silica gel chromatography to yield the title compound in 46% as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.43 (s, 1H), 8.28 (s, 1H), 8.23−8.09 (m, 3H), 7.84 (dd, J = 8.7, 2.6 Hz, 1H), 6.60 (d, J = 8.7 Hz, 1H), 6.32 (s, 2H), 4.86−4.76 (m, 1H), 4.46 (t, J = 5.3 Hz, 1H), 3.51 (q, J = 6.0 Hz, 2H), 3.10−2.92 (m, 4H), 2.39 (t, J = 6.3 Hz, 2H), 2.05 (t, J = 11.6 Hz, 2H), 1.91 (d, J = 12.5 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 159.73, 157.52, 151.56, 150.49, 147.33, 146.43, 137.00, 135.86, 135.66, 130.80, 128.67, 127.42, 122.83, 120.45, 118.11, 108.20, 62.57, 59.96, 58.86, 53.30, 27.93. HRMS(ESI) calcd for C23H25N6O2+ 417.2034, found 417.2038 [M + H]+. HPLC purity 97%. HPLC: tR = 4.35 min. 4.1.90. 2-(4-(9-(6-Aminopyridin-3-yl)-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetamide (12c). A mixture of 9-(6aminopyridin-3-yl)-1-(piperidin-4-yl)pyrazino[2,3-c]quinolin-2(1H)-one HCl (30 mg, 0.07 mmol), DIEA (86 μL, 0.4 mmol), 2-bromoacetamide (24 mg, 0.14 mmol), and anhydrous DCM (5 mL) was stirred at room temperature for 24 h under inert atmosphere.The reaction mixture was concentrated to dryness.The crude product was purified by silica gel chromatography to yield the title compound in 32% as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.42 (d, J = 2.5 Hz, 1H), 8.27 (s, 1H), 8.19−8.08 (m, 3H), 7.83 (dd, J = 8.6, 2.5 Hz, 1H), 7.26 (s, 1H), 7.18 (s, 1H), 6.60 (d, J = 8.7 Hz, 1H), 6.31 (s, 2H), 4.88−4.77 (m, 1H), 3.12−2.92 (m, 6H), 2.36−2.19 (m, 2H), 2.01−1.89 (m, 2H); 13 C NMR (101 MHz, DMSO) δ 159.69, 157.52, 151.54, 150.43, 147.30, 146.39, 136.95, 135.88, 135.67, 130.77, 128.62, 127.42, 122.80, 120.42, 118.09, 108.23, 99.51, 61.97, 60.48, 52.80, 27.69. ESI-HRMS m/z calcd for C23H23N7O2+ 430.1986, found 430.1988 [M + H]+. HPLC purity 99%. HPLC: tR = 5.02 min. 4.1.91. Methyl 2-(4-(9-(6-Aminopyridin-3-yl)-2-oxopyrazino[2,3c]quinolin-1(2H)-yl)piperidin-1-yl)acetate (12d). A mixture of 9-(6aminopyridin-3-yl)-1-(piperidin-4-yl)pyrazino[2,3-c]quinolin-2(1H)-one HCl (100 mg, 0.24 mmol), DIEA (251 μL, 1.5 mmol), 2-bromoacetamide (51 μL, 0.48 mmol), and anhydrous DMF (10 mL) was stirred at room temperature for 12 h under inert atmosphere.The reaction mixture was concentrated to dryness.The crude product was purified by silica gel chromatography to yield the title compound in 38% as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.41 (s, 1H), 8.27 (s, 1H), 8.21−8.07 (m, 3H), 7.83 (d, 1H), 6.60 (d, J = 8.6 Hz, 1H), 6.32 (s, 2H), 4.89−4.73 (m, 1H), 3.62 (s, 3H), 3.38−3.25 (m, 2H), 3.09− 2.89 (m, 4H), 2.36 (t, J = 12.2 Hz, 2H), 1.92 (d, J = 11.8 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 170.49, 159.63, 157.46, 151.49, 150.43, 147.30, 146.30, 136.90, 135.87, 135.69, 130.76, 128.63, 127.38, 122.85, 120.44, 118.04, 108.21, 62.07, 57.93, 51.83, 51.15, 27.83. HRMS(ESI) calcd for C24H25N6O3+ 445.1983, found 445.1986 [M + H]+. HPLC purity 98%. HPLC: tR = 4.15 min. 4.2. Biology Methods. 4.2.1. Materials. Human cancer cell lines were purchased from ATCC. All the cell lines were recently authenticated by cellular morphology and the short tandem repeat analysis at Microread Inc. (Beijing, China; May 2014) according to the guidelines from ATCC. 4.2.2. Cytotoxicity Assays. The viability of cells was determined using the CCK8 assay method. Cells were seeded (2000−3000 cells per well) in 96-well plates. After incubation for 24 h in serum-containing media, the cells were treated with drugs at different concentrations diluted with culture medium for 72 h at 37 °C with a 5% CO2 atmosphere. Then, an amount of 10 μL of CCK8 regent (Promega, WI) was added to each well, and the plates were incubated for 1−4 h at 37 °C. Finally, absorbance values of test wells (AS), control wells (AC), and blank wells (Ab) at 450 nm were read using the Microplate reader (Promega, WI). Inhibition ratio was calculated as follows: [(AC − AS)/(AC − Ab)] × 100%. IC50 values were calculated using GraphPad software.

procedure described for compound 8a in 55% yield as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.33−8.25 (m, 3H), 8.03 (d, J = 12.0 Hz, 1H), 7.96 (t, J = 1.7 Hz, 1H), 6.97 (d, J = 1.9 Hz, 1H), 4.75−4.65 (m, 1H), 3.76 (s, 2H), 3.05−2.92 (m, 4H), 2.26 (t, J = 12.0 Hz, 2H), 2.05 (d, J = 11.3 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 161.41, 158.87, 157.41, 151.99, 150.88, 146.66, 146.53, 144.80, 142.70, 142.59, 138.81, 127.24, 124.88, 124.82, 120.63, 120.46, 119.09, 119.07, 115.28, 113.29, 113.19, 113.06, 109.23, 58.88, 51.48, 42.82, 25.79. HRMS(ESI) calcd for C22H19FN5O2+ 404.1517, found 404.1519 [M + H]+. HPLC purity 97%. HPLC: tR = 6.12 min. 4.1.84. 2-(4-(8-Fluoro-9-(1-methyl-6-oxo-1,6-dihydropyridin-3yl)-2-oxopyrazino[2,3-c]quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9af). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)pyridin-2(1H)-one (25 mg, 0.11 mmol) using the procedure described for compound 8a in 34% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.28 (s, 1H), 8.18 (d, J = 2.6 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 11.3 Hz, 1H), 7.80 (dt, J = 9.4, 2.1 Hz, 1H), 6.58 (d, J = 9.4 Hz, 1H), 4.93−4.81 (m, 1H), 3.79 (s, 2H), 3.57 (s, 3H), 3.04−2.90 (m, 4H), 2.42−2.31 (m, 2H), 2.02−1.94 (m, 2H); 13 C NMR (101 MHz, DMSO) δ 161.37, 161.23, 158.85, 157.41, 152.15, 150.85, 146.53, 146.40, 140.65, 139.29, 127.20, 126.91, 126.86, 125.41, 125.25, 119.22, 115.29, 113.04, 112.83, 112.31, 99.61, 58.44, 51.52, 42.88, 40.15, 37.26, 25.51. HRMS(ESI) calcd for C24H22FN6O2+ 445.1783, found 445.1782 [M + H]+. HPLC purity 97%. HPLC: tR = 4.36 min. 4.1.85. 4-(9-(6-Aminopyridin-3-yl)-2-oxopyrazino[2,3-c]quinolin1(2H)-yl)piperidine-1-carboxamide (9ag). The desired compound was prepared from 6i (50 mg, 0.12 mmol) and (6-aminopyridin-3yl)boronic acid (34 mg, 0.25 mmol) using the procedure described for compound 8a in 46% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.40 (s, 1H), 8.26 (s, 1H), 8.20−8.05 (m, 3H), 7.83 (t, J = 9.4, 4.7 Hz, 1H), 6.60 (t, J = 8.3 Hz, 1H), 6.29 (s, 2H), 6.05 (s, 2H), 5.09−4.96 (m, 1H), 4.18−4.05 (m, 2H), 2.86−2.69 (m, 4H), 2.01−1.86 (m, 2H). 13C NMR (101 MHz, DMSO) δ 159.67, 157.82, 157.50, 151.60, 150.44, 147.31, 146.57, 136.80, 136.03, 135.70, 130.72, 128.72, 127.37, 123.06, 120.85, 118.05, 108.15, 62.15, 43.11, 28.16. HRMS(ESI) calcd for C22H22N7O2+ 416.1829, found 416.1833 [M + H]+. HPLC purity 98%. HPLC: tR = 4.55 min. 4.1.86. tert-Butyl 4-(9-(6-Aminopyridin-3-yl)-2-oxopyrazino[2,3c]quinolin-1(2H)-yl)piperidine-1-carboxylate (10). The desired compound was prepared from 4e (600 mg, 1.31 mmol) and (6-aminopyridin3-yl)boronic acid (360 mg, 2.61 mmol) using the procedure described for compound 8a in 52% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.40 (s, 1H), 8.25 (s, 1H), 8.20−8.03 (m, 3H), 7.82 (d, J = 8.8 Hz, 1H), 6.62 (d, J = 8.7 Hz, 1H), 6.29 (s, 2H), 5.09−4.97 (m, 1H), 4.20−4.01 (m, 2H), 2.97−2.69 (m, 4H), 2.04−1.89 (m, 2H), 1.42 (s, 9H); 13C NMR (101 MHz, DMSO) δ 159.62, 157.49, 153.81, 151.59, 150.42, 147.29, 146.46, 136.72, 136.03, 135.75, 130.72, 128.68, 127.36, 123.01, 120.73, 118.01, 108.24, 78.96, 61.67, 28.08. 4.1.87. 9-(6-Aminopyridin-3-yl)-1-(piperidin-4-yl)pyrazino[2,3-c]quinolin-2(1H)-one (11). To the suspension of compound 10 (320 mg, 0.68 mmol) in ethyl acetate (45 mL) was added concentrated hydrochloric acid (1 mL), and the mixture was stirred 3 h. The reation mixture was concetrated under reduced pressure to yield the crude product (without further purification). 4.1.88. 4-(9-(6-Aminopyridin-3-yl)-2-oxopyrazino[2,3-c]quinolin1(2H)-yl)piperidine-1-carbaldehyde (12a). To a mixture of 9-(6aminopyridin-3-yl)-1-(piperidin-4-yl)pyrazino[2,3-c]quinolin-2(1H)one HCl (30 mg, 0.07 mmol), formic acid (3.3 μL, 0.09 mmol), and DIPEA (644 μL, 0.11 mmol) in DMF (5 mL) was added TBTU (29 mg, 0.09 mmol). The reaction mixture was stirred at rt for 16 h. The mixture was concentrated to dryness. The residue was purified by silica gel chromatography to yield the title compound in 49% as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.43 (s, 1H), 8.28 (s, 1H), 8.22−8.10 (m, 3H), 8.06 (s, 1H), 7.85 (d, 1H), 6.61 (d, J = 8.6 Hz, 1H), 6.29 (s, 2H), 5.21−5.09 (m, 1H), 4.36 (d, J = 9.6 Hz, 1H), 3.87 (d, 1H), 3.20 (t, J = 12.8 Hz, 1H), 2.88−2.62 (m, 3H), 2.15−2.02 (m, 2H); 13C NMR (101 MHz, DMSO) δ 160.88, 159.64, 157.51, 151.58, 150.41, 147.32, 146.59, 136.69, 136.09, 135.70, 130.71, 128.72, 127.36, 123.06, 120.84, 118.04, 108.17, 61.49, 44.07, 38.03, 28.93, 27.63. 901



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S6, LC3B diluted 1:100 at 4 °C overnight, then tissues were incubated with biotin-labeled secondary antibody (Vector Laboratories, Inc., Burlingame, CA) at room temperature for 1 h. Sections were incubated with ABC-peroxidase and diaminobenzidine (DAB), counterstaining with hematoxylin and observed under a light microscope (Olympus). 4.2.9. Assessments of Pharmacokinetic Properties. The pharmacokinetics analysis of 9m was conducted in male Sprague-Dawley rats (Chinese Academy of Medical Science, Beijing, China). Briefly, catheters were surgically placed into the jugular veins of the rats to collect serial blood samples. 9m was dissolved in saline with 5% (v/v) DMSO. The animals were administered a single dose of 20 mg/kg 9m by iv and po after fasting overnight. Blood was collected and centrifuged immediately to isolate plasma. The plasma concentrations were determined using high performance liquid chromatography with HPLC analysis on a Shimadzu Prominence-i LC-2030C 3D system. 4.2.10. Statistical Analysis. Statistical analysis results were analyzed by GraphPad Prism version 5.0 software. For Student t test and ANOVA P < 0.05 was considered statistically significant. Values were expressed as means ± SEM. Significance was determined by χ2 test; others were determined by Student’s t test. A value of P < 0.05 was used as the criterion for statistical significance. ∗∗∗ indicates significant difference with P < 0.001.

4.2.3. Kinase Inhibition Assays. Kinase inhibition profiles were determined using KinaseProfiler services provided by Eurofins, and ATP concentrations used are the Km of corresponding kinases. The binding affinities of 9m projected on the human kinome tree were generated using the online Kinome Render program.43 4.2.4. Cell Cycle Assay. The different cell lines were seeded in 6-plates at a density of 5 × 105 cells/mL. All cells were treated with increasing concentrations of the indicated compounds 24 h after plating. Cells were harvested 24 h after treatment, washed in phosphate buffered saline (PBS), and fixed in ice cold 70% ethanol for at least 24 h. The fixed cells were then washed with room temperature PBS and stained with propidium iodide (50 mg/mL) in the presence of RNase A (0.5 mg) for 30 min at 37 °C. The stained cells were then analyzed using a flow cytometer and the resulting data analyzed with cell cycle analysis software (Modfit, BD). 4.2.5. Western Blotting. Cell lysates from different cell lines were prepared with RIPA buffer in the presence of protease inhibitor cocktails and phosphatase inhibitor cocktail 2 and 3 (P8340, P5726, and P0044, Sigma-Aldrich, St Louis, MO, USA). Protein (20−50 μg) was separated by 8−15% Tris-acrylamide gels and transferred onto PVDF membrane. The membrane was blocked in 5% skim milk, subsequently incubated with primary antibodies at 4 °C overnight followed by incubation with peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG and developed with Pierce ECL reagent (catalog no. 17153, Millipore, Billerica, MA, USA). Antibody information: Phospho-AKT(Ser473) (Cell Signaling Technology; catalog no. 4060), Phospho-4E-BP1(Thr37/46) (Cell Signaling Technology; catalog no. 2855), Phospho-4E-BP1(Ser65) (Cell Signaling Technology; catalog no. 9451), Phospho-S6 (Ser240/ 244) (Cell Signaling Technology; catalog no. 5364), AKT antibody (Cell Signaling Technology; catalog no. 9272), 4E-BP1 antibody (Proteintech; catalog no. 60246), S6 antibody (Proteintech; catalog no. 14823), Anti-LC3B (Sigma; catalog no. L7543), β-actin (Santa Cruz; catalogue no.sc-47778). 4.2.6. Immunofluorescence Staining. Briefly, cells were obtained and seeded on glass coverslips in complete 1640 or DMEM in order to induce cell adhesion and then incubation was done with the inhibitors at different concentration. After 48 h, the cells were fixed in 4% paraformaldehyde, permeabilized in 0.15% Triton X-100, saturated using 4% BSA in PBS at room temperature for 1 h, and incubated overnight antiLC3B at 1:200 dilutions in PBS-4% BSA and then with a FITC-conjugated secondary antibody. Slides were mounted in glycerol-DABCO and observed using a Nikon Eclipse E600 microscope equipped with a digital camera. Images were elaborated only for brightness and contrast using Adobe Photoshop 7. 4.2.7. In Vivo Assay. The animal studies were conducted under the approval of the Experimental Animal Management Committee of Nankai University. Human breast cancer cells T-47D were harvested during the exponential-growth phase, washed 3 times with serum-free medium, followed by resuspension at a concentration of 2 × 107 per mL. A total of 100 μL of cell suspension was injected into BALB/c nude mice (6−8 weeks) subcutaneously. After the tumors had grown to 100−120 mm3, all the mice were randomized into 5 groups (5 mice for each group) and dosed with 9m (15, 30, or 60 mg kg−1 d−1), rapamycin (60 mg kg−1 d−1), or vehicle. The compounds were dissolved in DMSO and administered orally. Mice were monitored for side effects every day. Body weights and tumor size were determined every other day. Tumor measurements were used using a digital vernier caliper, and the volumes were determined using the following calculation: (short2) × long × 0.5. Inhibition rate of tumor growth was calculated using the following formula: 100 × {1 − [(tumor volumefinal − tumor volumeinitial) for 9m-treated group]/ [(tumor volumefinal − tumor volumeinitial) for the vehicle-treated group]}. 4.2.8. Hematoxylin and Eosin (H&E) and Immunohistochemistry Staining. H&E staining was performed on the formalin-fixed, paraffinembedded orthotopic mice tumor tissues. Tumor tissue sections were deparaffinized, counterstained with hematoxylin and eosin, then observed under a light microscopy (Olympus). For immunohistochemistry assay, ormalin-fixed, paraffin-embedded 5 μm tumor tissue sections were deparaffinized and rehydrated through a graded series of ethanol and incubated with primary antibody of P-AKT(Ser473), P-4EBP1(Thr37/46), P-4E-BP1(Ser65), P-S6 (Ser240/244), AKT, 4E-BP1,

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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.7b01402. Literature reported dual PI3K/mTOR and selective ATPcompetitive mTOR inhibitors; cell inhibitory activities of some synthesized compounds; kinome profiling of 9m; molecular docking; protein plasma binding and stability of 9m; 1H NMR, 13C NMR, MS spectra and HPLC purity analysis for compound 9m (PDF) Molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*Y.F.: phone, +86 22 23509482; e-mail, [email protected]. *R.X.: phone, +86 22 23509482; e-mail, [email protected]. ORCID

Shengyong Yang: 0000-0001-5147-3746 Yan Fan: 0000-0002-8099-3203 Author Contributions #

Q.G. and C.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Project of Science and Technology Assistance in Developing Countries (Grant KY201501006) and the Natural Science Foundation of Tianjin (Grant 17JCQNJC13500) and the National Natural Science Foundation of China (Grant 81470354).

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ABBREVIATIONS USED mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; mTORC2, mTOR complex 2; ATP, adenosine triphosphate; PIKK, PI3K related kinases; Akt, v-akt murine thymoma viral oncogene homologue 1; pAKT, phosphorylated AKT; pAKT(S473), phosphorylated AKT at serine 473; DNAPK, DNA activated protein kinase; ATM, ataxia telangiectasia mutated kinase; ATR, ataxia telangiectasia and Rad-3-related kinase; 4E-BP1, eukaryotic translation initiation factor 4E 902



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binding protein 1; PK/PD, pharmacokinetics/pharmacodynamics; t1/2, half-life; AUC, area under the curve; Cmax, maximum concentration; F, % oral bioavailability; MRT, mean residence time; Vss, distribution volume; CL, clearance; DCM, dichloromethane; DMF, N,N-dimethylformamide; DIPEA, N,N-diisopropylethylamine; DMSO, dimethyl sulfoxide; LC3, microtubule-associated protein 1 light chain 3; tR, retention time

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Highly Selective, Potent, and Oral mTOR Inhibitor for Treatment of

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