Highly Selective, Potent and Oral mTOR Inhibitor for Treatment of

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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, Jian-Yu Zheng, Shengyong Yang, Yan Fan, and Rong Xiang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01402 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

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Journal of Medicinal Chemistry

Highly Selective, Potent and Oral mTOR Inhibitor for Treatment of Cancer as Autophagy Inducer Qingxiang Guo, ⊥1 Chenhua Yu,⊥1 Chao Zhang,1 Yongtao Li ,1 Tianqi Wang,1 Zhi Huang,1 Xin Wang,1 Wei Zhou,1 Yao Li,1 Zhongxiang Qin,1 Cheng Wang,1 Ruifang Gao,1 Yongwei Nie, 1 Yakun Ma, 1Yi Shi, 1 Jianyu Zheng,4 Shengyong Yang,5 Yan Fan*1,2 and Rong Xiang*1,3 1

School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China

2

International Collaborative Laboratory of Biomedicine of the Ministry of Education,

94 Weijin Road, Tianjin 300071, China 3

2011 Project Collaborative Innovation Center for Biotherapy of Ministry of

Education, 94 Weijin Road, Tianjin 300071, China 4

State Key Laboratory and Institute of Elemento-Organic Chemistry, Collaborative

Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, China. 5

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,

Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu 610041, China

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Based on 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 mTOR1 (pS6, and p4E-BP1) and mTOR2 (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. Keywords: mTOR, Inhibitor, Cancer, Autophagy


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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

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



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(PIKK) family and share the highly conserved ATP binding pockets with sequence similarity of 25% in the kinase catalytic domain.12 Based on this knowledge, many of the previously reported ATP-competitive mTOR inhibitors (Figure S1) also inhibited PI3Ks, DNA-PK, ATM, and ATR.13-22

13, 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 offer an improved therapeutic potential compared to rapalogues as well as pan-mTOR inhibitors. In

our

study,

we

identified

novel

hit

compound

9a

bearing

pyrazino[2,3-c]quinolin-2(1H)-one scaffold through high-throughput screening (HTS) of our in-house compound library against mTOR based on the previously reported method.35 Compound 9a exhibit 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). Firstly, we synthesized the key intermediates 6a~j and the general synthetic routes are illustrated in Scheme 1. Briefly, commercially available 1 reacted


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with various aliphatic amines to provide 2a~h, yielded 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 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~9ag 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-2-oxoacetyl (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 as H, the acetonitrile group of R5 replaced by carboxamide (9ag), formyl (12a), hydroxyethyl (12b) acetamide (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-1-yl)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, five-membered 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) maintain or increase the activity, but it is decreased when R6 is naphthyl, phenyl group, 3-pyridyl, 4-pyridyl, 5- pyrimidyl, 1-methyl-6-oxo-1,6-dihydropyridin-3-yl (9o-9s, 9af), suggesting that nitrogen atom


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and it’s 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 group are in favor of the activities. In addition, compounds containing 1H-pyrazol-4-yl (9v), 1-methyl-1H-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 were 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

which

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 values in breast and cervical cell lines. It just exhibited weak inhibitory activity against human ovarian and lung cancer cells with over 1µM IC50 values. 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 activities and selectivity, kinase inhibition profiling assays with a fixed concentration of 1 µM of 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 1µM, 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


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(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 PIKK-related kinases ATR and PI3K. 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 increase 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. The 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 of 9m with GLU2190 inside inner hydrophobic pocket. It may explain when the amino or 2-aminopyridinyl-5-yl group was substituted by other group with different size (e.g., 9p-9u, 9y, 9ac, 9ad) decreased the bioactivity. Another important hydrogen bond is formed between the cyano group of 9m with LYS 2187 in 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-(piperidin-1-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 compound 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 result in cell cycle inhibition. We next investigated the effect of compound 9m treatment on breast cancer cells (T-47D, MCF7 and MDA-MB-231)


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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 were 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 non-apoptotic programmed cell 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 autophagy-initiating 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. 40

Since 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 0.5µM of 9m treatment for 48h 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 48h in comparison to rapamycin and non-treated 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 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 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 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 expected, treatment with 9m at 0.5 and 1


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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 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 60mg/Kg, 9m-treated 15 mg/Kg, 9m-treated 30 mg/Kg, 9m-treated 60 mg/Kg). After the tumor volume was 100-120mm3 in each group, the mice were given a gavage of 9m and rapamycin every day for the entire observation period. As shown in Figure 7A and 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 15mg/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(60mg/Kg) was 0.194g compared with 1.23g 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 blocked the mTOR activity in vivo,



immunohistochemical (IHC) analyses of T-47D tumors collected were carried out. As showed in Figure 7E, 9m caused a dose-dependent decrease of P-AKT (Ser473), P-4E-BP1(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 anti-tumor 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 an good oral bioavailability of 43.50% with an AUC(0−∞) = 2853.41 µg/L*h. The oral maximum plasma concentration (Cmax) was 807.24 µg/L and (Tmax) was 3h. 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/kg. 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 key pharmacokinetic property of the 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


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can be found in the Supporting Information. The moderate plasma protein binding of compound 9m is considered favorable from a drug development perspective. 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 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 mTOR1 and mTOR2. In vivo anti-tumor activity assays showed that an oral, 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. 4. EXPERIMENTAL SECTION 4.1 Chemistry Methods. 1H NMR (400 MHz) and

13

C 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.



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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 hours 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 (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) 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. 4.1.2.

tert-butyl

piperidine-1-carboxylate

(R)-3-((6-bromo-7-fluoro-3-nitroquinolin-4-yl) (2b).

The

desired

6-bromo-4-chloro-7-fluoro-3-nitroquinoline

(5

compound g,

was

16.37mmol)


amino)

prepared and

from

tert-butyl

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(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)pyrrolidine-1-carboxylate (1.9 g, 9.82 mmol) using the procedure



Page 18 of 97

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. 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 tert-butyl 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

piperidine-1-carboxylate

(S)-3-((6-bromo-3-nitroquinolin-4-yl) (2f).

The

6-bromo-4-chloro-3-nitroquinoline

desired (2

g,

compound 6.9

was

mmol)

amino)

prepared and

from

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. 1H NMR (400 MHz, Chloroform-d)


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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)； 13

C 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 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. 1

H 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-3-nitroquinoline (4 g, 19.23 mmol) and tert-butyl tert-butyl 3-aminopyrrolidine-1-carboxylate (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);

Page 20 of 97

13

C 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 intermediates 3a in 46% yield as 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, 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.


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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. 1

H 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-4-yl) 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) 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,



Page 22 of 97

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. 1

H 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.


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4.1.16 tert-butyl 4-((3-aminoquinolin-4-yl)amino)piperidine-1-carboxylate (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, 1.14mmol) and glyoxylic acid (115 mg, 1.25 mmol) monohydrate was dissoved in anhydrous tetrahydrofuran (60 mL)under inert atmosphere. After stirring 8 hours, HATU (475 mg, 1.25 mmol) and DIPEA (395 µL, 2.28 mmol) was 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 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. 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,


Page 24 of 97

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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]quinolin-1(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


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]

quinolin-1(2H)-yl)

pyrrolidine-1-carboxylate (4g). The desired compound was prepared from 3g (500 mg,



Page 26 of 97

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, Chloroform-d) 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 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]quinolin-1(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 hours. The reation mixture was concetrated under reduced pressure. The crude product was dissoved in DMF (40 mL), then DIPEA (8.79 mL)


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and bromoacetonitrile (890 µL) were added. After stirring overnight, water (400 mL) was added, 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). 13C 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


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]

quinolin-1(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. 1H 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)；13C 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),


Page 28 of 97

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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-1-yl)acetonitrile

(6h). The desired compound was prepared from 4h (40 mg, 0.11 mmol) using the



Page 30 of 97

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-1carboxamide

(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 oC. 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);

13

C 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-1-carboxamide (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,


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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)-8-fluoropyrazino

[2,3-c]quinolin-2(1H)-one (7a). To a two-necked flask, compound 5a (100 mg, 0.24 mmol), cyclopropanecarboxylic acid (21 mg, 0.24mmol), EDCI (70 mg, 0.36mmol), 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 hours, 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, dried by anhydrous magnesium sulphate. 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)；13C 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



Page 32 of 97

mmol) and commercially available propionic acid (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 suspeded in dichloromathane (15mL), the commercially available dimethylcarbamoyl chloride (30.8 mg, 0.29 mmol) DIPEA (82.4 mL, 0.48 mmol) and stirred 5 hours. The


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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


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



Page 34 of 97

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. 1H 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, 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)piperidin-4-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,


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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)；13C 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 suspeded in dichloromathane (15mL), the commercially available methyl 2-bromoacetate (43.8 mg, 0.29 mmol) DIPEA (82.4 mL, 0.48 mmol) and stirred 5 hours. 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-


Page 36 of 97

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 was subsequently added PdCl2(Ph3P)2 (8.8 mg, 0.1 equiv), Na2CO3 (378 µL, 0.39 mmol, 1M), and (6-aminopyridin-3-yl)boronic acid (34.7 mg, 0.25 mmol), then degassing with argon for 10 min, the resulting mixture was heated to 85 oC overenight. The reaction mixture was cooling to room temperature and filtering through Celite. Upon removal of the solvents, the residue was purified by silica gel column chromatography to afford 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-propionylpiperidin-4-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-3-yl)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


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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)-8-fluoropyrazino

[2,3-c] quinolin-2(1H)-one (8c). The desired compound was prepared from 7c (30 mg, 0.06 mmol) and (6-aminopyridin-3-yl)boronic acid (17.2 mg, 0.02 mmol) using the procedure described for 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)；13C 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)-



Page 38 of 97

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

[2,3-c]

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. 1H 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)；13C NMR (101 MHz, DMSO) δ 164.34, 163.20, 161.15, 159.76, 158.64, 157.42, 151.78, 151.49,


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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)-8fluoropyrazino[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


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-aminopyridin-3-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. 1H 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


Page 40 of 97

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purity 99%. HPLC: tR = 5.07 min. 4.1.52.

methyl

2-(4-(9-(6-aminopyridin-3-yl)-8-fluoro-2-oxopyrazino

[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. 1


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[2,3-c]

quinolin-

1(2H)-yl)piperidin-1-yl)acetonitrile (9a). The desired compound was prepared from 6a (38 mg, 0.09 mmol) and (6-aminopyridin-3-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-aminopyridin-3-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)；13C 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 (6-aminopyridin-3-yl)boronic acid (33 mg, 0.24 mmol) using the procedure described for compound 8a in 29% yield as a yellow solid. 1H 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 (d, J = 8.7 Hz, 1H), 6.62 (d, J =


Page 42 of 97

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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-3-ylboronic 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)；13C 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 (6-aminopyridin-3-yl)boronic acid (19 mg, 0.14 mmol) using the procedure described for compound 8a in 46% yield as a yellow solid. 1H 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-3-ylboronic 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,


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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 (6-aminopyridin-3-yl)boronic acid (11 mg, 0.07 mmol) using the procedure described for compound 8a in 96% yield as a yellow solid. 1H 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. 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-3-ylboronic 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)；13C 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-3-yl)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)； 13

C 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.


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4.1.62.

(S)-2-(3-(2-oxo-9-(quinolin-3-yl)pyrazino[2,3-c]

quinolin-1(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-aminopyridin-3-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]quinolin-1(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 (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


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mixture was heated to 85 oC overenight. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/methanol 100:1~30:1) to afford title compond in 82% yield as a yellow amorphous solid. mp 272-274 oC; 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, HRMS and HPLC Purity Analysis for compound 9m were in Supporting Information. 4.1.66.

2-(4-(2-oxo-9-(quinolin-3-yl)pyrazino[2,3-c]quinolin-1(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-2-ylboronic 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


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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-4-ylboronic 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-4-ylboronic 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]quinolin-1(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


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prepared

from

6a

(90

mg,

0.21

mmol)

and

1-methyl-4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-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)-2-oxopyrazino[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), 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,



Page 54 of 97

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-(4,4,5,5-tetramethyl-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)-2-oxopyrazino[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. 1

H 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


Page 55 of 97 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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-2-oxopyrazino[2,3-c] quinolin-1(2H)-yl)piperidin-1-yl)acetonitrile (9x). The desired compound was prepared

from

6a

(30

mg,

0.07

mmol)

1-(difluoromethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

and (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-1,2-dihydropyrazino

[2,3-c] quinolin-9-yl)picolinonitrile (9y). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and (6-cyanopyridin-3-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 (6-methoxypyridin-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 (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.


Page 56 of 97

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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-(4,4,5,5-tetramethyl-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. 1H 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[2,3-c]quinolin-1(2H)-yl) piperidin-1-yl)acetonitrile (9ab). The desired compound was prepared from 6a (30 mg, 0.07 mmol) and 2-(4-methoxyphenyl)-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 (6-methylpyridin-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


Page 58 of 97

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(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 procedure described for compound 8a in 55% yield as a white solid. 1H 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)； 13

C 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-3-yl)-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-2-yl)pyridin-2(1H)-one (25 mg,



Page 60 of 97

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)；13C 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]quinolin-1(2H)-yl)piperidine-1-carboxamide (9ag). The desired compound was prepared from 6i (50 mg, 0.12 mmol) and (6-aminopyridin-3-yl)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).

13

C 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-butyl4-(9-(6-aminopyridin-3-yl)-2-oxopyrazino[2,3-c]quinolin -1(2H)-yl)piperidine-1-carboxylate(10). The desired compound was prepared from 4e


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(600 mg, 1.31 mmol) and (6-aminopyridin-3-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]quinolin2(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 hours. 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]quinolin-1(2H)-yl) piperidine-1-carbaldehyde

(12a).

To

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), 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 hrs. 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. 1H 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,



Page 62 of 97

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);

13

C 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. 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-4-yl)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. 1H 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)


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piperidin-1-yl)acetamide

(12c).

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), 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. 1H 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); 13C 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[2,3-c]quinolin1(2H)-yl)piperidin-1-yl)acetate

(12d).

A

mixture

9-(6-aminopyridin-3-yl)-1-(piperidin-4-yl)pyrazino[2,3-c]quinolin-2(1H)-one

of 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. 1H 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);

13

C 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 guideline from ATCC. 4.2.2. Cytotoxicity Assays. The viability of cells as determined using the CCK8 assay method. Cells were seeded (2000-3000 cells per well) in 96-well plates. After incubation for 24h in serum-containing media, the cells were treated with drugs at different concentrations diluted with culture medium for 72h at 37℃ with a 5% CO2 atmosphere. Then, 10µL CCK8 regent (Promega, WI) were added to each well, and the plates were incubated for 1-4h at 37℃. Finally, absorbance values of test wells (AS), control wells (AC) and blank wells (Ab) at 450nm were read using the Microplate reader (Promega, WI). Inhibition ratio were calculated as followed: [(AC AS)/ (AC - Ab)] X 100%. IC50 values were calculated using GraphPad software


Page 64 of 97

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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 24h post plating. Cells were harvested 24h post-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 over night followed by incubation with peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG and developed with Pierce ECL reagent (cat. #17153, Millipore, Billerica, MA, USA). Antibody information: Phospho-AKT(Ser473) (Cell Signaling Technology; catalogue no.4060), Phospho-4E-BP1(Thr37/46) (Cell Signaling Technology; catalogue no.2855),



Phospho-4E-BP1(Ser65)

(Cell

Signaling

Technology;

Page 66 of 97

catalogue

no.9451),

Phospho-S6 (Ser240/244) (Cell Signaling Technology; catalogue no.5364), AKT antibody (Cell Signaling Technology; catalogue no.9272), 4E-BP1 antibody (Proteintech; catalogue no.60246), S6 antibody (Proteintech; catalogue no.14823), Anti-LC3B (SIGMA; catalogue no.L7543), β-actin (Santa Cruz; catalogue no.sc-47778). 4.2.6. Immunofluorescence staining. Briefly, cells obtained and seeded on glass coverslips in complete 1640 or DMEM in order to induce cell adhesion and then incubation with the inhibitors at different concentration. After 48h, the cells were fixed in 4% paraformaldehyde, permeabilized in 0.15% Triton X-100, saturated using 4% BSA in PBS at room temperature for 1h, and incubated overnight anti-LC3B 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,


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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 volume final

-tumor volume initial) for 9m-treated group]/ [(tumor volume

initial)

final

-tumor volume

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, paraffin-embedded 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 de-paraffinized and rehydrated through a graded series of ethano and incubated

with

primary

antibody

of

P-AKT(Ser473),

P-4E-BP1(Thr37/46),

P-4E-BP1(Ser65), P-S6 (Ser240/244), AKT, 4E-BP1, 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 hour. Sections were incubated with ABC-peroxidase and diaminobenzidine (DAB), counterstaining with hematoxylin and observed under a light microscopy (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 were analyzed by GraphPad Prism v5.0 software. Student t test and ANOVA. P

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