Synthesis and Biological Evaluation of Novel Bouchardatine

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Synthesis and Biological Evaluation of Novel Bouchardatine Derivatives as Potential Adipogenesis/Lipogenesis Inhibitors for Anti-obesity Treatment Yong Rao, Hong Liu, Lin Gao, Hong Yu, Tian-Miao Ou, Jia-Heng Tan, Shi-liang Huang, Honggen Wang, Ding Li, Lian-Quan Gu, Ji-Ming Ye, and Zhi-Shu Huang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01566 • Publication Date (Web): 17 Nov 2015 Downloaded from http://pubs.acs.org on November 19, 2015

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Journal of Medicinal Chemistry

Synthesis and Biological Evaluation of Novel Bouchardatine Derivatives as Potential Adipogenesis/Lipogenesis Inhibitors for Anti-obesity Treatment

Yong Raoa, †, Hong Liua, †, Lin Gaoa, Hong Yua, Tian-Miao Oua, Jia-Heng Tana, Shi-Liang Huanga, Hong-Gen Wanga, Ding Lia, Lian-Quan Gua, Ji-Ming Yeb, and Zhi-Shu Huanga,*

a

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People′s Republic

of China b

Molecular Pharmacology for Diabetes Group, Health Innovations Research Institute and School of

Health Sciences, RMIT University, Melbourne, VIC 3083, Australia

1

ACS Paragon Plus Environment


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ABSTRACT

Our recent study has shown that the natural product bouchardatine (1) can reduce the triglyceride (TG) contents in 3T3-L1 adipocytes (EC50 ≈ 25 µM). Here, we synthesized two series of compounds by introducing amine side chains at the 5 or 8 position of 1 and evaluated the lipid-lowering activity of derivatives. It was found that some of the compounds had significant lipid-lowering effects, and the most active compound 3d showed better activity (EC50 = 0.017 µM) than 2 (EC50 = 0.086 µM), a reported compound by us. Further the mechanism studies revealed that 3d blocked TG accumulation via activating LKB1-AMPK signaling pathway, efficiently down-regulated the expression of key regulators of adipogenesis/lipogenesis. Cell uptake assay and confocal imaging of 3d in cells indicated that compound 3d had a favorable cell permeability. Our results suggest that 3d may be a promising agent for the treatment of obesity and related metabolic disorders.

KEYWORDS:

Bouchardatine

derivatives;

3T3-L1

adipocytes;

adipogenesis/lipogenesis;

lipid-lowering; obesity

2


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INTRODUCTION Obesity is a chronic metabolic disease with increasing rates worldwide and is associated with increased disability, morbidity and mortality. Obesity is characterized by increased adipose tissue due to an imbalance between energy intake and energy expenditure and this excess energy is stored in the form of triglycerides (TGs).1 Many studies have demonstrated that obesity is associated with many health problems, including hyperlipidemia, hypertension, type II diabetes, respiratory disorders, and non-alcoholic fatty liver disease (NAFLD).2 Some studies have reported that approximately 60% of the liver TG content is derived from free fatty acids (FFAs) excreted from adipose tissue. Increased FFA levels result in intracellular lipid accumulation in the liver, causing the development of NAFLD.3,4 In light of the widespread occurrence of obesity and the tremendous threat of obesity to public health, prevention or treatment options for obesity has become one of the most attractive research areas. Currently, there are only four agents approved by FDA used for clinical treatment of obesity, including orlistat, lorcaserin, phentermine/topiramate, bupropion/naltrexone, and liraglutide, however, they all are accompanied by complications and their effects diminish with increasing treatment, which limits their clinical use.5-8 Therefore, discovering and identifying novel anti-obesity agents has become an attractive area of research.5 Recent studies have demonstrated that drugs characterized by improving energy metabolism showed potential for the treatment of obesity in vivo.9-11 3T3-L1 cell line is a well-established in vitro metabolic cell model for obesity.12 In addition, 3T3-L1 cell-based method has been shown to be an efficacious tool for screening beneficial compounds with lipid-lowering effects.13 Several stages are involved in the differentiation of 3T3-L1 preadipocytes into mature adipocytes, such as adipogenesis and lipogenesis. Adipogenesis involves two aspects: cell proliferation progress and adipocyte differentiation.12,14 Adipogenesis is regulated by some cell cycle regulators and differentiating factors, 3



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such as the peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer binding proteins α (C/EBPα) and sterol regulated element binding protein-1 (SREBP-1).15-17 These adipogenic factors co-regulate the expression of lipogenic proteins and initiate fatty acid synthesis, such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD-1).18,19 Moreover, adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) is a key participant in energy metabolism, and it has been reported that the activation of AMPK leads to the inhibition of adipogenesis and lipogenesis and may be a potential target for obesity treatment.20-22 Several studies have demonstrated the effectiveness of some natural products in controlling obesity. The anti-obesity effects of these compounds are mediated by various pathways, including lipid absorption,23 energy intake and expenditure,24,25 adipogenesis and lipogenesis.26 1 (Bouchardatine , Figure 1) is a naturally occurring alkaloid with C-ring opening that belongs to the rutaecarpine family based on its biosynthetic pathway.27 The rutaecarpine family exhibits several biological activities, including anti-cancer, anti-inflammatory, and anti-tuberculosis effects.28,29 More importantly, although the anti-obesity effect of rutaecarpine (Figure 1) has been reported,30 studies on the biological activities of 1 have not been reported until our recent studies. In our study, we found 1 had a moderate inhibition (EC50 ≈ 25 µM) of adipogenesis/lipogenesis without cytotoxicity in 3T3-L1.31 Compound 2 (R17, Figure 1), a derivative with a N,N-dimethylethylenediamine side chain at the 5-position of 1 synthesized by us, was efficient in reducinglipid accumulation in 3T3-L1 adipocytes.32 These results suggest that the introduction of a suitable side chain might be important for improving the lipid-lowering activity of its derivatives. In this paper, in order to develop this type of novel anti-obesity drug candidate, a series of analogues of 2 (Series I) were synthesized by introducing different amine side chains at the 5-position. To explore the effects of aldehyde groups and the position of the side chain on the lipid-lowering activity, a series of imine side chain 4


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substituted derivatives with different amines (Series II) was also synthesized via reactions of aldehydes at the 8-position of 1 (Figure 1). Biological evaluation revealed that most of these new compounds showed better lipid-lowering effects in 3T3-L1 and HepG-2 cell models than their parent compound 1, and furthermore, compound 3d, which is the most active compound, was further evaluated at the cell and molecular levels.

RESULTS AND DISCUSSION Chemistry. The synthetic routes to the target compounds are shown in Scheme 1. The key intermediate a was prepared according to a literature procedure.33 The intermediate a reacted with ammonium acetate and DMSO-water to give natural product 1 according to the literature.34 Compound 1 was treated with SOCl2 to provide the chloride 1a. Compound 1a was then allowed to react with desired amines to provide series I compounds (3a ~3u). In another approach, 1 was directly reacted with a series of amines to give the Series II compounds (4a ~ 4y). To investigate the effects of the aldehyde groups and imine groups of the compounds on the lipid lowering activity, 1 was reduced to 5 with NaBH4. 3c and 4b were also reduced to 6 and 7, respectively, with NaBH4. The chemical structures of all 49 synthesized compounds are listed in Figure 2. The single-crystal structure of 4t was shown in Figure S1 and the result further confirmed the proposed structures of the Series II compounds.35

Evaluation of the lipid-lowering activities of the derivatives using the 3T3-L1 cell model. The 3T3-L1 cell line is a well-established metabolic in vitro cell model for obesity.12 3T3-L1 cell-based screening of compounds with lipid-lowering effects has been shown to be an effective tool for the identification of anti-obesity compounds.13 Using this approach, the efficacy of all the new 5



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analogs were evaluated, and the structure-activity relationship (SAR) was explored. As shown in Figure 3A and Table S2, the natural product 1 had a weak lipid-lowering effect with 14.8 ± 1.8% and 20.1 ± 3.3% decreases in TG accumulation at 1 and 10 µM, respectively. Compound 2 decreased the TG content by 82.0 ± 0.1% and 89.6 ± 2.5% at 1 and 10 µM, respectively. Most of the Series I and Series II compounds showed better lipid-lowering activity than natural product 1, especially at higher concentration of 10 µM. Totally, 14 of the 49 new derivatives had strong efficacies with greater than 50% reductions in triglyceride accumulation at 1 µM. In addition, representative microscope images were captured (×40) after staining with Oil Red O (Figure S2A). The compound with the greatest activity, 3d, could reduce the TG level by approximately 87.6 ± 0.1% and 94.9 ± 2.6% at 1 and 10 µM in 3T3-L1 cells, respectively, which was more potent than 2. The results further confirmed that the lipid-lowering effects of the derivatives could be significantly improved after the introduction of side chains. The impact of the end group of the amine side chain on the lipid-lowering activity was also investigated. The Series I compounds 3a ~ 3g (except 3f), with side chain end groups containing nitrogen atoms, showed relatively better lipid-lowering activity than 3h ~ 3j, which had alkyl end groups, and 3k ~ 3o, which had secondary amine side chains. 3p ~ 3t (except 3t), which had aromatic amine side chains, displayed relatively weak lipid-lowering activity compared with the other compounds in Series I. However, for the Series II compounds, an obvious SAR similar to that in Serious I was not found. Five of six compounds in Series I had inhibitory activity that were greater than 80% at 10 µM, including 3a (80.1 ± 1.1%), 3b (87.3 ± 1.6%), 3c (87.2 ± 4.5%), 3d (94.9 ± 2.6%), 3k (81.6 ± 3.1%), and 3l (82.2 ± 5.6%). This result indicated that the Series I compounds were better at reducing the TG level than Series II. 6


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In addition, by comparing the inhibitory activities of compounds from different series but with the same side chain, it was found that the Series I compounds were better than the Series II compounds when the ends of their side chains were tertiary amine or cyclic amine groups, but the Series II compounds were better when the ends of their side chains were other groups, such as branched alkyl group, phenyl group, or hydroxyl group (Figure S3). The lengths, the degrees of freedom and the steric hindrances of the end of the amine side chains were also explored. However, we did not find an obvious relationship between these structural factors and the lipid-lowering activities.

The effects of the derivatives on the cholesterol and triglyceride levels based on the HepG-2 cell model. The liver is an important organ. Its function is to maintain lipid and glucose homeostasis in vivo.36,37 To investigate whether 1 and its derivatives affect lipid absorption and accumulation in the liver, we treated human hepatoma HepG-2 cells with 0.5 mM oleic acid sodium in the presence or absence of the compounds for triglyceride and cholesterol content analysis, respectively. As shown in Figure 4, Figure S2B, and Table S3, compound 1 weakly reduced the triglyceride (16.7 ± 4.0%) and cholesterol (24.1 ± 4.2%) contents at 1 µM in HepG-2 cells, and 2 approximately decreased TG accumulation by 53.3 ± 0.7% and cholesterol content by 46.2 ± 0.7% in HepG-2 cells. Similar to the results in 3T3-L1 cells, most of the compounds in Series I showed better TG-lowering activities than natural product 1 in the HepG-2 model and had a similar SAR, i.e., 3a, 3b, 3c, and 3d, displayed the greatest activity for TG reduction. However, for cholesterol-lowering activity, many derivatives showed a similar ability to natural product 1, and only three compounds (3b, 3c, and 3t) exerted more than 50% inhibitory activity. Notably, the compound with the most lipid-lowering effect in 3T3-L1 (3d) cells exerted the higher TG reduction (~70%), but a less than 7



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10% decrease in the cholesterol content in HepG-2 cells, suggesting that these compounds mainly affected triglyceride metabolism. Based on the results in 3T3-L1 adipocytes and HepG-2 cells, it could be assumed that 1 and its derivatives had the ability to influence lipid metabolism without cell selectivity.

Dynamic changes in triglyceride contents and cell number. The differentiation of 3T3-L1 preadipocytes into mature adipocytes is initiated by mitotic clonal expansion (MCE), which means that, during the first 16-20 h after the initiation of differentiation, the cells would undergo almost two rounds of mitosis, until cell contact inhibition began again.38,39 Considering that both MCE and cell proliferation activity are essential to the differentiation process, we sought to determine whether these compounds’ lipid-lowering effect are associated with MCE and cell proliferation during differentiation. Thus, 3T3-L1 adipocytes were treated with some of the compounds, i.e., 3c, 3d, 3e, 4b, 4l, and 2 at 0.5 µM, as well as 1 at 5 µM. As shown in Figure 5A, after induction, the cells had an exponential increase in cell numbers in the early stages of differentiation until contact inhibition began again at 72 h (Day 3) as shown in the DMSO group. Natural product 1 mildly inhibited cell proliferation progress and delayed the time point of cell contact inhibition to Day 5, and compound 2 significantly inhibited cell proliferation progress and delayed the time point of cell contact inhibition to Day 6. Compared with 2, all synthesized compounds could inhibit cell proliferation activity with similar tendencies and delay the time point of contact inhibition to Day 6. This result suggested that the lipid-lowering effect of the compounds might be related to cell proliferation inhibition. Meanwhile, we also examined the dynamic changes in the TG content during adipocytes differentiation. As shown in Figure 5B, treatment with the tested compounds significantly reduced triglyceride accumulation during all of the developmental stages of the preadipocytes, and 3d also 8


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showed the greatest activity for decreasing the TG content in 3T3-L1 cells out of all tested compounds. Based on the lipid-lowering ability in 3T3-L1 cells and the effect on cell viability (Table S2) of the derivatives, compound 3d was found to be the most potent compound for further studies.

3d had a higher lipid-lowering effect and lower cytotoxicity in 3T3-L1 adipocytes. To further evaluate the inhibitory effects of 3d on lipid accumulation, 2-day post-confluent 3T3-L1 preadipocytes were treated with 3d or 2 at the indicated concentrations for 6 days after the initiation of differentiation. On day 6, intracellular lipid accumulation was determined via a TG assay. As shown in Figure 6A, both 3d and 2 decreased intracellular lipid accumulation in a dose-dependent manner in the 3T3-L1 adipocytes, and these lipid-lowering effects were further confirmed by Oil Red O staining (Figure S2A). Compared with 2 (EC50 = 0.086 µM), 3d reduced the TG content more efficiently in 3T3-L1 adipocytes, as indicated by its EC50 value of 0.017 µM through a TG assay, nearly a 5-fold increase in the lipid-lowering effect. Meanwhile, the lipid-lowering effect of 3d was also observed in the HepG-2 cell model. As shown in Figure 6B and Figure S2B, 3d decreased the triglyceride content in a dose dependent manner in the HepG-2 model and showed a similar activity to 2, which reduced the lipid content by up to ~32% for 3d and ~30% for 2, respectively (relative to the vehicle-treated control cells), at the maximum dosage used (10 µM), suggesting that the lipid-lowering effect of 3d was not highly selective for both cell models. To investigate whether the reduction in lipid accumulation is caused by cytotoxicity, an LDH release/cytotoxicity assay was performed during 3T3-L1 preadipocytes differentiation in the presence of 3d or 2 for 24 h, 48 h, and 72 h, respectively. As shown in Figure 6C, at a concentration of 10 µM, 2 had a moderate cytotoxicity effect on cell viability (~58% cell viability relative to vehicle-treated control cells) within the first 24 h of differentiation, and the longer the drug treatment, the greater the 9



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decrease in the cell viability. However, 3d showed no cytotoxicity to 3T3-L1 adipocytes, even for a longer incubated time, approximately 83%~87% cell viability relative to vehicle-treated control cells, suggesting that 3d has potential as an anti-obesity agent.

3d inhibited TG accumulation in the early stages of cell differentiation in 3T3-L1 adipocytes. From preadipocytes to mature adipocytes, 3T3-L1 cells undergo multiple stages of differentiation, such as adipogenesis, lipogenesis, and terminal differentiation stage.40 To determine whether 3d reduces lipid accumulation by blocking adipogenesis and/or lipogenesis, we treated 3T3-L1 cells with 3d (1 µM) at various periods, namely, day 0-3, 0-6, 0-9, 3-6, 3-9, and 6-9, which represent different stages of 3T3-L1 adipocyte differentiation (Figure 6D). On day 9, the intracellular lipid content was determined via TG assays and Oil Red O staining, respectively, and representative microscopy images were captured. As shown in Figure 6E and Figure S4, 3d treatment during day 0-3, 3-6, and 6-9 resulted in ~85%, ~80%, and 60% reductions in lipid accumulation, respectively. Consistent with TG content results, incubation of 3T3-L1 with 3d for various periods led to reductions of mature adipocytes number and lipid droplets accumulation in a similar trend as shown in the representative microscopy images (Figure S4). Notably, unlike 2 treatment, which had a time-dependent lipid-lowering effect,29 the lipid-lowering effect of 3d treatment did not show an obvious time dependence, as the 3-, 6-, and 9-day treatments (Days 0-3, 0-6, 0-9) led to similar reductions in the lipid accumulation. These data suggest that 3d reduced TG accumulation in 3T3-L1 adipocytes mainly during the early stages of cell differentiation by inhibiting adipogenesis.

3d treatment resulted in cell cycle arrest but did not induce cell apoptosis in 3T3-L1 adipocytes. To further confirm the effect of 3d on cell mitosis after adipogenic induction, cells treated with 3d were analyzed by using flow cytometry and 2 was employed as a control. As shown 10


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in Figure 7A and Figure 7B, before differentiation, 3T3-L1 cells were retained in the phase of cell contact inhibition as indicated by ~80.4% of the cells in G0/G1 phase, ~2.4% in S phase, and ~14.3% in G2/M phase. After incubation with differentiation cocktail for 24 h, a significant increase was observed in the ratio of the S phase in the control group (~18.1%) accompanied by a decrease in the G0/G1 phase (~37.6%) and an increase in the G2/M phase (~37.2%), which indicated that cell proliferation progress was initiated after adipogenic cocktail induction during 3T3-L1 differentiation. In addition, 2 treatment (3 µM) resulted in a significant reduction in the ratio in the S phase, which was accompanied by an increase in the G0/G1 ratio from 37.6% to 53.4%, suggesting that the lipid-lowering effect of 2 is closely related to the inhibition of cell proliferation progress during 3T3-L1 differentiation. Compared with 2, 3d showed a stronger effect on inhibition of cell proliferation progress. 3d treatment resulted in a significant dose dependent decrease in the S phase ratio (~6.4% at 0.3 µM, ~5.2% at 1.0 µM, ~3.3% at 3.0 µM, respectively), accompanied by an increase in the G2/M ratio (~40.2% at 0.3 µM, ~46.9% at 1.0 µM, and ~57.9% at 3.0 µM, respectively) and a decrease in the G0/G1 phase (~51.8% at 0.3 µM, ~42.9% at 1.0 µM, and 33.5% at 3.0 µM, respectively). These results indicated that 3d might inhibit cell proliferation progress by arresting the cell cycle mainly in the G2/M phase, unlike 2, which occurs mainly in the G0/G1 phase. It has been reported that apoptosis is closely related to inhibition of adipogenesis and cell cycle progress.41-43 Thus, we also examined whether treatment of 3T3-L1 adipocytes with 3d could lead to cell apoptosis. As shown in Figure 7C, no major effect was observed in the ratio of cell death and apoptosis, which indicated that the lipid-lowering effect of 3d in 3T3-L1 adipocytes might correlate with the inhibition of cell mitosis during the early stages of differentiation. This result is consistent with the inhibition of cell proliferation by 3d during adipogenesis of 3T3-L1.

11



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3d inhibited expression of adipogenesis-related markers in 3T3-L1 adipocytes. 3T3-L1 preadipocyte differentiation into mature adipocytes is regulated by several transcription factors (such as C/EBPα, PPARγ, and SREBP-1) during adipogenesis and some enzymes (such as FAS, ACC, and SCD-1), which are involved in regulating fatty acid synthesis.44,45 To determine whether the lipid-lowering effect of 3d was association with its effect on the expression of these transcription factors and enzymes. We next examined the effect of 3d on the expression of these adipogenesis-related markers and fatty acid synthesis proteins by RT-PCR and Western blot analysis with 3d at several periods. After 3d treatment, a decrease was observed in transcriptional level of early differentiation factors at first 24 hr of differentiation, such as C/EBPβ, C/EBPδ (Figure 8A). We also examined the expression of C/EBPα, PPARγ, and SREBP-1c, which are the downstream targets of C/EBPβ, C/EBPδ. As expected, after 3d administrated, a significant reduction was observed in the expression of C/EBPα, PPARγ, and SREBP-1c compared with DMSO group at both transcriptional and translational level after 3d treatment (Figure 8B and 8C). And 3d at concentration of 1 µM decreased the expression of these adipogenic factors to a level similar to a 3 µM 2 treatment, which is consistent with its more potent lipid-lowering effect. PPARγ, SREBP-1c and C/EBPα are known to be regulated in the early change stage during adipocyte differentiation, which can activate lipogenic markers such as ACC, FAS and SCD-1.18,19 These three markers are critical enzymes involved in lipogenesis. Given the reduction in lipid content and the downregulation of PPARγ, C/EBPα and SREBP-1c, it is hypothesized that the activities of these lipogenic enzymes may be suppressed. As expected, the protein levels of lipogenic enzymes ACC, FAS and SCD-1 were significantly decreased in a dose-dependent manner after the treatment of 3d for 6 days (Figure 8D). These results suggested that 3d blocked lipid accumulation by

12


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decreasing the expression of transcription factors and enzymes that were critical for adipogenesis/lipogenesis in 3T3-L1 adipocytes.

3d activated AMPK signaling pathway distinguished from AICAR. To investigate whether the activation of AMPK is involved in the lipid-lowering effect of 3d, the effect of 3d on AMPK signaling pathway in 3T3-L1 adipocytes was examined. Western blot analysis showed that 3d could activate AMPK signaling pathway as evidenced by increasing the phosphorylated level of AMPKα (Thr-172) and its downstream target ACC after 3 days treatment of 3d (Figure 9A). Key modulators of AMPK are the upstream regulators, such as liver kinase B1 (LKB1), calmodulin-dependent protein kinase kinase (CaMKK).46-48 Thus we further analyzed the expression of these two regulators in 3T3-L1 adipocytes. After 3d treatment, a significant increase was observed in the expression of protein LKB1, whereas a decrease was observed in the expression of protein CaMKKα (Figure 9B), suggesting that activation of AMPK signaling pathway mediated by 3d might be associated with LKB1. To verify our hypothesis, we selected AICAR as a control, which is an agonist of AMPK signaling pathway. As shown in Figure 9C, AICAR treatment resulted in activation of AMPK signaling pathway, and a significant decrease in the expression of protein C/EBPα, PPARγ, whereas no major effect was observed in the expression of protein LKB1. The results suggest that 3d share an important and different mechanism from AICAR on activation of AMPK signaling pathway.

3d inhibited adipogenesis/lipogenesis through activating LKB1-AMPK pathway as an upstream mechanism. To analyze the relationship between the lipid-lowering effect of 3d and its effect on AMPK signaling pathway, we first determined the uptake of 3d in cellular within 1 h. The 13



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fluorescence of 3d was observed in cellular as early as 5 min, and the fluorescence intensity of 3d was kept increasing within 1h, suggesting that 3d had a favorable cell permeability (Figure S5). In light of above data, the effect of 3d (3 µM) on AMPK signaling pathway was detected in a time-dependent manner. As shown in Figure 10A and Figure 10F, after 10 min treatment of 3d, both increase in the protein expression level of LKB1 and in the phosphorylated level of AMPKα was observed. And after 2 h treatment of 3d, the protein expression level of LKB1 and pAMPKα increased more obviously (Figure 10B and 10F), and a moderate decrease was observed in the transcriptional level of C/EBPβ, C/EBPδ (Figure 10C and 10F). Moreover, after 24 h treatment of 3d, the mRNA level of adipogenic markers showed a significant decrease, in cooperated with the increase of the levels of LKB1, pAMPKα, and pACC (Figure 10D, 10E, and 10F). On the other hand, the similar results for all markers except LKB1 were also observed in AICAR (0.2 mM) treatment group, which further confirmed a different action mechanism of 3d from that of AICAR. These data indicated that 3d inhibited adipogenesis/lipogenesis through activation of the LKB1-AMPK pathway as an upstream mechanism.

CONCLUSIONS

In this study, based on our previous research results that the natural product bouchardatine and its derivative 2 show beneficial lipid-lowering activity in 3T3-L1 cells,31 we designed and synthesized two series of bouchardatine derivatives by introducing different amine side chains at the 5- or 8position of 1 and performed systematic biological evaluations in cellular. Based on the results of the lipid-lowering effects of the derivatives on 3T3-L1 cells, the SAR was discussed and summarized as follows: (1) The Series I compounds reduced the TG content more effectively than the Series II 14


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compounds; (2) for Series I, the compounds with primary amine side chains showed better lipid-lowering effects than those with secondary amine side chains, and compounds with primary amine end groups on the side chains had stronger lipid-lowering activity than those with other types of end groups (such as alkyl groups or aromatic groups); (3) the length, degrees of freedom and steric hindrance of the amine side chain also seems to affect the lipid-lowering activity, but we did not find a clear relationship. Moreover, in HepG-2 cells, most of derivatives reduced TG level more than 1, but for the cholesterol level, no obvious differences were observed between the derivatives and 1. These results suggest that the derivatives mainly affected triglyceride metabolism. Based on the results in cellular, we selected 3d as a representative compound to perform further studies. Compared with the hit compound 2 (EC50 = 0.086 µM), 3d (EC50 = 0.017 µM) showed a better lipid-lowering effect and lower cytotoxicity to cells. In HepG-2 cells, 3d efficiently reduced the triglyceride content more than cholesterol. Further mechanistic studies demonstrated that 3d inhibited TG accumulation in 3T3-L1 adipocytes via a quick and significant effect on LKB1-AMPK signaling pathway for 9 days, and thus blocked cell proliferation progress and down-regulated the mRNA and protein levels of adipogenic factors and its downstream targets, such as ACC, FAS, and SCD-1. The results suggest that LKB1-AMPK signaling pathway plays a pivotal role for lipid-lowering effect of 3d, which is consistent with latest research that activation of AMPK could be a potential target for treatment of metabolic disease, such as obesity.22, 49-50 AMPK activation is regulated by LKB1, a major upstream protein kinase of AMPK, through phosphorylating the α subunit of AMPK at Thr-172.51 In our study, it was found that 3d could activate LKB1 in a dose- and time-dependent manner different from AICAR, suggesting that 3d shared a novel mechanism on activation of AMPK signaling pathway. Whether the effect of 3d on increased expression of LKB1 is associated with improving energy state requires a further study. 15



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In addition, our previous studies demonstrated that 2 has a ~43.8% bioavailability in SD mice and the value of MRT, t1/2 was 3.56 h, and 3.14 h, respectively.32 In this study, although we didn’t perform the pharmacokinetics study of 3d in vivo, uptake analysis and fluorescence assay revealed that 3d had a favorable cell permeability (Figure S5), and the amount of 3d in cells was up to ~38% after 24 h treatment (Figure S6), indicate that 3d has potential as an excellent anti-obesity agent. Further studies are required to confirm the hypothesis that 3d can effectively reduce the lipid content in vivo. Moreover, the identification of the target of 2 or 3d in vivo would help to determine a useful target for obesity treatment and aid in drug discovery. This work is ongoing in our group.

EXPERIMENTAL SECTION 1. Chemistry General Methods. The reagents and anhydrous solvents were used as received. 1H and

13

C

NMR spectra were recorded using tetramethylsilane (TMS) as an internal standard in DMSO-d6, MeOD or CDCl3 and a Bruker BioSpin GmbH spectrometer at 400 MHz. Mass spectra (MS) were recorded on a Shimadzu LCMS-2010A instrument with an ESI-ACPI mass selective detector, and high-resolution mass spectra (HRMS) were recorded on a Shimadzu LCMS-IT-TOF. Flash column chromatography was performed with silica gel (200-300 mesh) or Al2O3 (200-300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. Melting points (m.p.) were determined using an SRS-OptiMelt automated melting point instrument without correction. The purities of the synthesized compounds were confirmed to be higher than 95% using analytical HPLC performed with a Shimadzu LC-20AB system equipped with an Ultimate XB-C18 column (4.6 × 250 mm, 5 µm) and eluted with methanol–water (35:65 to 50:50) at a flow rate of less than 0.5 mL·min-1.

16


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Synthesis of the intermediates. The intermediates a and 1 were prepared following the process shown in Scheme 1 using the method in a literature report.33, 34 2-(4-chloroquinazolin-2-yl)-1H-indole-3-carbaldehyde (1a). 1 (1.5 g, 5.2 mmol) was dissolved in SOCl2, and two drops of DMF were added. Then, it was heated at 80 °C for 6 h. Next, most of the SOCl2 was removed by using a rotary evaporator. The residual solution was poured into ice water, and the pH was adjusted to 7.0. The residue was filtered, washed with water, and desiccated under vacuum to give the crude product, which was then purified by using column chromatography with dichloromethane, resulting in a pale yellow solid with a 57% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.79 (s, 1H), 11.22 (s, 1H), 8.35 (d, J = 8.1 Hz, 1H), 8.30 (d, J = 7.8 Hz, 1H), 8.20 (q, J = 8.0 Hz, 2H), 7.93 (t, J = 6.8 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.28 (t, J = 7.3 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 189.6, 162.3, 153.7, 151.2, 140.4, 136.9, 136.7, 130.6, 129.0, 126.5, 126.3, 125.6, 123.6, 122.6, 122.6, 117.7, 113.5. Purity: 98.5% by HPLC. MS (ESI + APCI) m/z: 308.2 [M+H]+, 306.1 [M-H]-.

General procedure for the preparation of the Series I compounds. A mixture of intermediate 1a (0.15 g, 0.49 mmol), triethylamine (30 µL), and the respective amine (1.5 mmol) in toluene (8 mL) was refluxed for 10 h, cooled to room temperature, and extracted with saturated salt water. The organic phase was collected, concentrated, and purified via flash chromatography on a silica gel to give the desired product. 2-(4-((2-(dimethylamino)ethyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde

(3a):

Following the general procedure, compound 1a (0.15 g, 0.49 mmol) and N, N-dimethylethane-1, 2-diamine (0.15 mL, 1.5 mmol) were used, and the desired product was obtained as a light yellow

17



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solid (0.10 g, 59%). m.p. 203.8-205.3 oC; 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H), 11.32 (s, 1H), 8.53 (t, J = 5.3 Hz, 1H), 8.30 (t, J = 8.7 Hz, 2H), 7.85 (d, J = 3.8 Hz, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.61-7.55 (m, 1H), 7.31 (t, J = 7.1 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 3.83 (dd, J = 12.3, 6.3 Hz, 2H), 2.68 (t, J = 6.5 Hz, 2H), 2.30 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 190.1, 160.1, 154.8, 149.6, 143.7, 136.1, 133.8, 128.2, 126.8, 126.7, 124.8, 123.5, 123.1, 122.4, 116.9, 114.6, 113.3, 57.9, 45.6, 39.2. Purity: 97.1% by HPLC. HRMS (ESI) m/z: calcd for C21H21N5O, [M+H]+ 360.1819, found 360.1817. 2-(4-((2-(diethylamino)ethyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3b): Following the general procedure, compound 1a (0.15 g, 0.49 mmol) and N, N-diethylethane-1, 2-diamine (0.16 mL, 1.5 mmol) were used, and the desired product was obtained as a light red solid (0.11 g, 57%). m.p. 174.2-176.8 oC; 1H NMR (400 MHz, CDCl3) δ11.45 (s, 1H), 10.56 (s, 1H), 8.51 (d, J = 5.1 Hz, 1H), 7.77 (t, J = 9.8 Hz, 2H), 7.71-7.65 (m, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.41-7.37 (m, 1H), 7.27 (dd, J = 5.3, 1.9 Hz, 2H), 7.14 (s, 1H), 3.66 (d, J = 4.1 Hz, 2H), 2.76 (t, J = 5.7 Hz, 2H), 2.63 (dd, J = 14.4, 7.2 Hz, 4H), 1.08 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 191.1, 159.3, 154.3, 149.3, 142.9, 135.1, 133.0, 128.3, 127.0, 126.5, 124.9, 123.2, 123.0, 121.4, 117.5, 114.3, 111.6, 50.9, 46.8, 38.3, 11.4. Purity: 95.7% by HPLC. HRMS (ESI) m/z: calcd for C23H25N5O, [M+H]+ 388.2132, found 388.2119. 2-(4-((2-(piperidin-1-yl)ethyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde

(3c):

Following the general procedure compound 1a (0.15 g, 0.49 mmol) and 2-(piperidin-1-yl)ethanamine (0.21 mL, 1.5 mmol) were used, and the desired product was obtained as a light red solid (0.10 g, 53%). m.p. 178.3-179.2 oC; 1H NMR (400 MHz, CDCl3) δ 11.49 (s, 1H), 10.16 (s, 1H), 8.53 (d, J= 8.4 Hz, 1H), 7.86 (d, J= 9.5 Hz, 1H), 7.80-7.71 (m, 2H), 7.51 (d, J= 8.2 Hz, 1H), 7.49-7.44 (m, 1H), 18


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7.35 (d, J= 5.7 Hz, 1H), 7.32-7.28 (m, 1H), 7.05 (s, 1H), 3.75 (dd, J= 10.5, 5.6 Hz, 2H), 2.73 (t, J= 5.9 Hz, 2H), 2.52 (s, 4H), 1.72-1.62 (m, 4H), 1.53 (d, J= 4.8 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 191.0, 159.4, 154.4, 149.4, 142.7, 134.9, 132.9, 128.4, 127.1, 126.4, 124.9, 123.3, 123.0, 121.2, 117.6, 114.3, 111.4, 56.3, 54.1, 37.9, 26.1, 24.3. Purity: 98.6% by HPLC. HRMS (ESI) m/z: calcd for C24H25N5O, [M+H]+ 400.2132, found 400.2131. 2-(4-((2-(pyrrolidin-1-yl)ethyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde Following

the

general

procedure

compound

1a

(0.15

g,

0.49

(3d): mmol)

and

2-(pyrrolidin-1-yl)ethanamine (0.20 mL, 1.5 mmol) were used, and the desired product was obtained as a light yellow solid (0.12 g, 64%), m.p. 203.9-205.0 oC; 1H NMR (400 MHz, CDCl3) δ 11.47 (s, 1H), 10.27 (s, 1H), 8.53 (dd, J = 6.7, 1.8 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.80-7.76 (m, 1H), 7.73 (dd, J = 8.3, 1.2 Hz, 1H), 7.48-7.46 (m, 1H), 7.44 (dd, J = 3.2, 1.4 Hz, 1H), 7.35-7.28 (m, 2H), 6.92 (s, 1H), 3.78 (dd, J = 10.7, 5.4 Hz, 2H), 2.90 (t, J = 5.9 Hz, 3H), 2.67 (s, 5H), 1.86 (s, 4H). 13C NMR (101 MHz, CDCl3) δ 190.9, 159.5, 154.3, 149.4, 142.7, 134.9, 133.1, 128.4, 127.1, 126.5, 125.0, 123.4, 121.4, 117.6, 114.3, 111.4, 53.9, 39.9, 29.7, 23.6. Purity: 98.6% by HPLC. HRMS (ESI) m/z: calcd for C23H23N5O, [M+H]+ 386.1975, found 386.1967. 2-(4-((3-morpholinopropyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3e): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and 3-morpholinopropan-1-amine (0.22 mL, 1.5 mmol) were used, and the desired product was obtained as a light yellow solid (0.10 g, 51%). m.p. 195.7-197.5 oC; 1H NMR (400 MHz, CDCl3) δ 11.48 (s, 1H), 10.09 (s, 1H), 8.53 (d, J = 8.2 Hz, 1H), 8.30 (s, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.76 (t, J = 7.1 Hz, 1H), 7.48 (dd, J = 13.4, 7.1 Hz, 2H), 7.35 (d, J = 7.1 Hz, 1H), 7.30 (t, J = 8.5 Hz, 1H), 3.87 (t, J = 7.8 Hz, 4H), 3.82 (dd, J = 10.3, 5.7 Hz, 2H), 2.69 (t, J = 7.9 Hz, 2H), 2.62 (s, 4H), 1.97 (dd, J = 10.8, 5.6 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 19



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191.0, 159.7, 154.5, 149.3, 142.7, 134.8, 132.9, 128.5, 127.1, 126.1, 124.9, 123.3, 123.0, 121.4, 117.6, 114.4, 111.3, 67.0, 59.3, 54.0, 43.1, 23.1. Purity: 98.1% by HPLC. HRMS (ESI) m/z: calcd for C24H25N5O2, [M+H]+ 416.2081, found 416.2080. 2-(4-((3-(piperidin-1-yl)propyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde Following

the

general

procedure

compound

1a

(0.15

g,

0.49

(3f): mmol)

and

3-(piperidin-1-yl)propan-1-amine (0.22 mL, 1.5 mmol) were used, and the desired product was obtained as a light red solid (0.12 g, 58%). m.p. 183.7-184.9 oC; 1H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.18 (d, J = 7.4 Hz, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.84 (dd, J = 14.4, 7.5 Hz, 2H), 7.70 (d, J = 7.8 Hz, 1H), 7.53 (t, J = 8. 2 Hz, 1H), 7.37 (t, J = 8. 0 Hz, 1H), 7.25 (t, J = 7.1 Hz, 1H), 3.86 (s, 2H), 2.43 (s, 4H), 2.03 (s, 2H), 1.50 (s, 4H), 1.37 (s, 2H), 1.21 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 188.17, 162.2, 156.4, 135.9, 134.3, 133.9, 128.7, 127.2, 126.2, 126.0, 124.9, 122.8, 121.9, 121.5, 120.4, 119.0, 113.2, 56.8, 56.1, 53.8, 27.2, 25.3, 23.9. Purity: 98.9% by HPLC. HRMS (ESI) m/z: calcd for C25H27N5O, [M+H]+ 414.2288, found 414.2274. 2-(4-((3-(4-methylpiperazin-1-yl)propyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3g): Following

the

general

procedure

compound

1a

(0.15

g,

0.49

mmol)

and

3-(4-methylpiperazin-1-yl)propan-1-amine (0.20 mL, 1.5 mmol) were used, and the desired product was obtained as a light yellow solid (0.14 g, 68%). m.p. 212.0-213.3 oC; 1H NMR (400 MHz, CDCl3) δ 11.49 (s, 1H), 10.11 (s, 1H), 8.65 (s, 1H), 8.53 (d, J = 7.3 Hz, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.85 (d, J = 8.1 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.46 (dt, J = 6.9, 3.3 Hz, 2H), 7.33 (dt, J = 8.0, 6.1 Hz, 2H), 3.82 (dd, J = 9.8, 5.3 Hz, 2H), 2.76-2.69 (m, 4H), 2.64 (s, 4H), 1.96 (dd, J = 9.6, 4.8 Hz, 2H), 1.25 (s, 2H).

13

C NMR (101 MHz, CDCl3) δ 191.1, 159.7, 154.5, 149.3, 142.8, 134.8, 132.8, 128.3, 127.1,

125.8, 124.8, 123.3, 122.9, 122.1, 117.5, 114.4, 111.4, 58.8, 55.1, 53.4, 46.2, 43.2, 23.1. Purity: 20


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98.5% by HPLC. HRMS (ESI) m/z: calcd for C25H28N6O, [M+H]+ 429.2397, found 429.2402. 2-(4-((4-methylpentyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3h): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and 4-methylpentan-1-amine (0.08 mL, 1.5 mmol) were used, and the desired product was obtained as a light yellow solid (0.09 g, 49%). m.p. 199.6-201.2 oC; 1H NMR (400 MHz, DMSO-d6) δ 13.63 (s, 1H), 13.12 (s, 1H), 10.47 (s, 1H), 8.24 (d, J = 15.9 Hz, 2H), 7.88 (d, J = 17.5 Hz, 2H), 7.64 (d, J = 31.4 Hz, 2H), 7.38 (d, J = 26.5 Hz, 2H), 4.12 (s, 1H), 3.34 (s, 4H), 1.24 (d, J = 19.1 Hz, 6H), 0.86 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 188.0, 161.6, 148.8, 145.7, 136.3, 135.4, 132.06, 129.1, 128.1, 127.8, 126.6, 125.8, 123.7, 122.3, 120.6, 115.5, 113.7, 67.8, 30.2, 28.8, 22.9, 14.4. Purity: 98.9% by HPLC. HRMS (ESI) m/z: calcd for C23H24N4O, [M+H]+ 373.2023, found 373.2021. 2-(4-(isobutylamino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3i): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and isobutylamine (0.15 mL, 1.5 mmol) were used, and the desired product was obtained as a light brown solid (0.10 g, 57%). m.p. 194.2-195.6 oC; 1H NMR (400 MHz, DMSO-d6) δ 12.32 (s, 1H), 11.34 (s, 1H), 8.61 (t, J = 5.4 Hz, 1H), 8.37 (d, J = 8.2 Hz, 1H), 8.29 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 3.7 Hz, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.65-7.52 (m, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.23 (t, J = 7.4 Hz, 1H), 3.50 (t, J = 6.2 Hz, 2H), 2.16-2.05 (m, 1H), 1.00 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 189.7, 159.7, 154.3, 149.2, 143.3, 135.6, 133.2, 127.7, 126.2, 126.2, 124.3, 123.0, 122.5, 121.9, 116.5, 114.1, 112.7, 48.4, 27.5, 20.3. Purity: 95.5% by HPLC. HRMS (ESI) m/z: calcd for C21H20N4O, [M+H]+ 345.1710, found 345.1719. 2-(4-(tert-butylamino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3j): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and tert-Butylamine (0.15 mL, 1.5 mmol) were used, and the desired product was obtained as a light brown solid (0.10 g, 59%). m.p. 198.0-199.7 oC; 1H 21



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Page 22 of 65

NMR (400 MHz, DMSO-d6) δ 12.21 (s, 1H), 11.22 (s, 1H), 8.48 (d, J = 8.2 Hz, 1H), 8.29 (d, J = 7.8 Hz, 1H), 7.87-7.83 (m, 2H), 7.69 (d, J = 8.1 Hz, 1H), 7.61-7.54 (m, 2H), 7.32 (t, J = 7.6 Hz, 1H), 7.25 (t, J = 7.4 Hz, 1H), 1.63 (s, 9H).

13

C NMR (101 MHz, CDCl3) δ 190.4, 158.8, 153.6, 149.2,

142.6, 134.8, 133.0, 128.8, 127.1, 126.6, 125.0, 123.4, 123.1, 120.6, 117.5, 114.4, 111.4, 52.9, 28.9. Purity: 97.1% by HPLC. HRMS (ESI) m/z: calcd for C21H20N4O, [M+H]+ 345.1710, found 345.1707. 2-(4-(4-methylpiperazin-1-yl)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3k): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and N-methyl piperazine (0.17 mL, 1.5 mmol) were used, and the desired product was obtained as a light yellow powder (0.11 g, 58%). m.p. 147.8-149.1 oC; 1H NMR (400 MHz, CDCl3) δ 11.41 (s, 1H), 10.08 (s, 1H), 8.53 (d, J = 7.7 Hz, 1H), 7.91 (t, J = 7.4 Hz, 2H), 7.77 (t, J = 7.7 Hz, 1H), 7.48 (t, J = 7.5 Hz, 2H), 7.37-7.28 (m, 2H), 3.89 (s, 4H), 2.69 (s, 4H), 2.41 (s, 3H).

13

C NMR (101 MHz, CDCl3) δ 190.4, 164.7, 153.2, 152.0, 142.0,

135.0, 133.1, 128.8, 127.1, 125.9, 125.2, 125.1, 123.5, 123.2, 117.7, 115.6, 111.3, 54.8, 49.9, 46.1. Purity: 98.7% by HPLC. HRMS (ESI) m/z: calcd for C22H21N5O, [M+H]+ 372.1819, found 372.1823. 2-(4-(diethylamino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3l):

Following the general

procedure compound 1a (0.15 g, 0.49 mmol) and diethylamine (0.08 mL, 1.5 mmol) were used, and the desired product was obtained as a light brown solid (0.11 g, 58%). m.p. 183.8-184.3 oC; 1H NMR (400 MHz, CDCl3) δ 11.36 (s, 1H), 10.33 (s, 1H), 8.51 (dd, J = 6.0, 2.7 Hz, 1H), 8.51 (dd, J = 6.0, 2.7 Hz, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.40-7.36 (m, 2H), 7.29 (dt, J = 6.3, 4.3 Hz, 2H), 3.76 (q, J = 7.0 Hz, 4H), 1.42 (t, J = 7.0 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 190.5, 162.2, 153.0, 151.9, 142.7, 135.0, 132.5, 128.4, 127.0, 125.2, 124.9, 124.8, 123.3, 123.0, 117.5, 115.1, 111.5, 45.3, 12.9. Purity: 98.7% by HPLC. HRMS (ESI) m/z: calcd for C21H20N4O, [M+H]+ 345.1710, found 345.1715. 22


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2-(4-(pyrrolidin-1-yl)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3m): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and pyrrole (0.10 mL, 1.5 mmol) were used, and the desired product was obtained as a light yellow solid (0.08 g, 52%). m.p. 184.3-185.1 oC; 1H NMR (400 MHz, CDCl3) δ 11.32 (s, 1H), 8.46 (d, J = 6.8 Hz, 1H), 8.12 (d, J = 8.3 Hz, 1H), 7.87 (d, J = 6.8 Hz, 1H), 7.70 (t, J = 7.4 Hz, 1H), 7.49 (d, J = 6.9 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.31 (dd, J = 17.1, 7.5 Hz, 2H), 3.96 (s, 4H), 2.08 (s, 4H).

13

C NMR (101 MHz, CDCl3) δ 190.86, 159.56, 153.39,

151.54, 142.75, 134.80, 132.41, 128.17, 127.07, 125.49, 124.96, 124.82, 123.30, 122.95, 121.69, 115.67, 111.35, 51.38, 29.70. Purity: 98.9% by HPLC. HRMS (ESI) m/z: calcd for C22H21N5O, [M+H]+ 343.1553, found 343.1545. 2-(4-(piperidin-1-yl)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3n): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and piperidine (0.15 mL, 1.5 mmol) were used, and the desired product was obtained as a light yellow solid (0.09 g, 52%). m.p. 181.7-183.0 oC; 1H NMR (400 MHz, CDCl3) δ 11.42 (s, 1H), 10.22 (s, 1H), 8.52 (d, J = 7.2 Hz, 1H), 7.90 (d, J = 2.4 Hz, 1H), 7.88 (d, J = 2.5 Hz, 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.47 (d, J = 2.2 Hz, 1H), 7.45 (t, J=4.0 Hz, 1H), 7.35-7.31 (m, 1H), 3.78 (d, J = 5.5 Hz, 4H), 1.89-1.78 (m, 4H), 1.56 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 190.7, 165.0, 153.3, 151.8, 142.3, 135.0, 133.0, 128.5, 127.1, 125.7, 125.4, 125.0, 123.4, 123.1, 117.7, 115.8, 111.4, 51.3, 26.0 24.7. Purity: 97.5% by HPLC. HRMS (ESI) m/z: calcd for C22H20N4O, [M+H]+ 357.1710, found 357.1711. 2-(4-morpholinoquinazolin-2-yl)-1H-indole-3-carbaldehyde

(3o):

Following

the

general

procedure compound 1a (0.15 g, 0.49 mmol) and morpholine (0.13 mL, 1.5 mmol) were used, and the desired product was obtained as a light yellow powder (0.08 g, 48%). m.p. 178.4-179.3 oC; 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H), 11.25 (s, 1H), 8.29 (d, J = 7.2 Hz, 1H), 8.09 (d, J = 7.4 23



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Page 24 of 65

Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.89 (t, J = 8.1 Hz, 1H), 7.66 (d, J = 7.4 Hz, 1H), 7.59 (t, J=4.1 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.25 (t, J = 4.2 Hz, 1H), 3.87 (s, 8H). 13C NMR (101 MHz, DMSO-d6) δ 189.40, 163.80, 152.96, 151.53, 142.51, 135.68, 133.45, 128.24, 127.92, 126.20, 125.60, 124.49, 122.67, 121.98, 116.51, 114.70, 112.74, 66.02, 49.77. Purity: 98.9% by HPLC. HRMS (ESI) m/z: calcd for C21H18N4O2, [M+H]+ 359.1503, found 359.1497. 2-(4-((4-nitrophenyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3p): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and p-nitroaniline (0.21 g, 1.5 mmol) were used, and the desired product was obtained as a light yellow solid (0.10 g, 50%). m.p. 237.6-239.8 oC; 1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.27 (d, J = 7.9 Hz, 1H), 8.21 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 2.0 Hz, 1H), 7.93 (d, J = 1.8 Hz, 1H), 7.92-7.89 (m, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.61 (t, J = 6.9 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 6.70 (s, 1H), 6.61 (t, J =4.1 Hz, 1H), 6.59 (t, J = 3.2 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 187.5, 161.7, 156.8, 153.9, 145.3, 135.8, 134.9, 134.6, 128.8, 127.6, 127.4, 126.8, 126.3, 126.1, 125.3, 125.0, 123.2, 122.4, 122.2, 121.7, 120.1, 113.2, 112.3. Purity: 96.8% by HPLC. HRMS (ESI) m/z: calcd for C23H15N5O3, [M+H]+ 410.3857, found 410.3846. 2-(4-((4-fluorophenyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3q): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and 4-Fluoroaniline (0.17 g, 1.5 mmol) were used, and the desired product was obtained as a light yellow solid (0.11 g, 57%). m.p. 248.1-251.1 oC; 1

H NMR (400 MHz, DMSO-d6): δ 12.80 (s, 1H), 9.48 (s, 1H), 8.34 (d, J= 7.8 Hz, 1H), 8.20 (d, J=

7.6 Hz, 1H), 7.98-7.90 (m, 2H), 7.87 (dd, J= 13.9, 7.4 Hz, 2H), 7.71 (d, J= 8.1 Hz, 1H), 7.56 (t, J= 7.2 Hz, 1H), 7.39 (dd, J= 15.2, 7.1 Hz, 3H), 7.30 (t, J= 7.3 Hz, 1H). 13C NMR (101 MHz, DMSO-d6): δ 187.4, 162.0, 159.9, 152.7, 149.0, 146.3, 143.8, 135.9, 134.5, 131.6, 129.0, 127.3, 126.5, 125.9, 24


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125.2, 123.4, 123.3, 121.8, 119.7, 116.2, 116.0, 113.0, 112.9; Purity: 95.3% by HPLC. HRMS (ESI) m/z: calcd for C23H15FN4O, [M+H]+ 383.1303, found 383.1287. 2-(4-((4-methoxyphenyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3r): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and p-Anisidine (0.18 g, 1.5 mmol) were used, and the desired product was obtained as a light brown solid (0.10 g, 54%). m.p. 201.2-203.4 oC; 1H NMR (400 MHz, DMSO-d6) δ 12.72 (s, 1H), 10.48 (s, 1H), 9.45 (s, 1H), 8.33 (d, J = 8.1 Hz, 1H), 8.20 (d, J = 6.8 Hz, 1H), 7.91 (d, J = 8.9 Hz, 2H), 7.85 (d, J = 7.9 Hz, 2H), 7.70 (t, J = 8.2 Hz, 1H), 7.55 (t, J = 7.3 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 7.10 (d, J = 8.9 Hz, 2H), 3.84 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 187.4, 162.1, 158.7, 150.2, 149.1, 146.6, 140.3, 135.9, 134.8, 134.5, 131.2, 129.0, 127.6, 127.2, 126.4, 125.9, 125.1, 123.2, 122.8, 121.8, 121.6, 120.1, 119.8, 114.6, 113.2, 55.4. Purity: 96.7% by HPLC. HRMS (ESI) m/z: calcd for C24H18N4O2, [M+H]+ 395.1503, found 395.1491. 2-(4-((3-chlorophenyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3s): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and 3-chloroaniline (0.15 mL, 1.5 mmol) were used, and the desired product was obtained as a light yellow solid (0.10 g, 52%). m.p. 190.4-191.6 oC; 1

H NMR (400 MHz, DMSO-d6) δ 12.43 (s, 1H), 10.93 (s, 1H), 10.19 (s, 1H), 8.60 (d, J = 8.2 Hz,

1H), 8.27 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 3.5 Hz, 2H), 7.92 (s, 1H), 7.80-7.71 (m, 2H), 7.65 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.31 (dd, J = 15.2, 7.7 Hz, 2H), 7.24 (t, J = 7.4 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 189.3, 158.2, 153.9, 149.9, 142.7, 140.2, 135.8, 134.0, 133.0, 130.2, 127.9, 127.1, 126.0, 124.5, 124.1, 123.3, 122.7, 122.6, 122.0, 121.6, 116.5, 114.2, 112.8. Purity: 97.4% by HPLC. HRMS (ESI) m/z: calcd for C23H15N4OCl, [M+H]+ 399.1007, found 399.1009. 2-(4-(phenethylamino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3t): Following the general 25



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Page 26 of 65

procedure compound 1a (0.15 g, 0.49 mmol) and β-phenethylamine (0.2 mL, 1.5 mmol) were used, and the desired product was obtained as a light yellow solid (0.11 g, 57%). m.p. 211.3-213.0 oC; 1H NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H), 11.38 (s, 1H), 8.71 (t, J = 5.2 Hz, 1H), 8.30 (d, J = 8.0 Hz, 2H), 7.86 (d, J = 3.8 Hz, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.58 (dt, J = 8.0, 4.0 Hz, 1H), 7.34 (t, J = 5.3 Hz, 2H), 7.31 (d, J = 4.7 Hz, 2H), 7.27-7.19 (m, 2H), 3.90 (dd, J = 13.9, 6.5 Hz, 2H), 3.04 (t, J = 7.5 Hz, 2H).

13

C NMR (101 MHz, DMSO-d6) δ 189.6, 159.6, 154.4, 149.1, 143.31, 139.3, 135.6,

133.2, 128.8, 128.4, 127.7, 126.3, 126.2, 124.3, 122.9, 122.6, 121.9, 116.4, 114.1, 112.8, 42.6, 34.7. Purity: 97.7% by HPLC.

HRMS (ESI) m/z: calcd for C25H20N4O, [M+H]+ 393.1710, found

393.1710. 2-(4-((2-hydroxyethyl)amino)quinazolin-2-yl)-1H-indole-3-carbaldehyde (3u): Following the general procedure compound 1a (0.15 g, 0.49 mmol) and 2-aminoethanol (0.1 mL, 1.5 mmol) were used, and the desired product was obtained as a light brown solid (0.07 g, 46%). m.p. 196.7-198.2 oC; 1

H NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H), 11.29 (s, 1H), 8.53 (s, 1H), 8.36 (d, J = 7.7 Hz, 1H),

8.29 (d, J = 7.4 Hz, 1H), 7.85 (s, 2H), 7.66 (d, J = 7.6 Hz, 1H), 7.57 (t, J=4.1 Hz, 1H), 7.31 (t, J=7.8 Hz, 1H), 7.24 (t, J=8.1 Hz, 1H), 4.86 (s, 1H), 3.77 (d, J = 16.6 Hz, 4H).

13

C NMR (101 MHz,

DMSO-d6) δ 189.7, 159.8, 154.3, 149.1, 143.3, 135.5, 133.2, 129.6, 127.7, 126.2, 124.3, 123.1, 122.5, 121.9, 116.4, 114.2, 112.7, 59.2, 43.7. Purity: 97.2% by HPLC. HRMS (ESI) m/z: calcd for C19H16N4O2, [M+H]+ 333.1346, found 333.1346.

General procedure for the preparation of the Series II compounds. A mixture of intermediate 1 (0.15 g, 0.52 mmol), anhydrous MgSO4 (60 mg, 0.49 mmol), FeCl3 (32 mg, 0.2 mmol), and the respective amine (0.6 mmol) in CHCl3 (10 mL) was reacted at room temperature for 6 h. Then, the

26


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solid was filtered, and the filtrate was extracted with saturated salt water. The organic phase was collected, concentrated, and purified by flash chromatography on Al2O3 or recrystallized with methanol to furnish the desired product. 2-(3-(((2-morpholinoethyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4a): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and 2-morpholinoethanamine (0.08 mL, 0.6 mmol) were used, and the desired product was obtained as a light yellow solid (0.09 g, 49%). m.p. 165.8-167.1 oC; 1H NMR (400 MHz, DMSO-d6) δ 12.62 (s, 1H), 9.12 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.85 (dd, J = 14.0, 7.5 Hz, 2H), 7.70 (d, J = 8.1 Hz, 1H), 7.53 (t, J = 7.1 Hz, 1H), 7.38 (t, J = 7.1 Hz, 1H), 7.27 (t, J = 7.4 Hz, 1H), 3.98 (s, 2H), 3.57 (s, 4H), 2.88 (s, 2H), 1.23 (s, 4H). 13C NMR (101 MHz, CDCl3) δ 163.0, 155.6, 149.2, 146.4, 135.0, 134.2, 131.4, 129.0, 127.2, 126.6, 126.4, 125.6, 122.6, 122.0, 118.8, 112.8, 112.2, 67.0, 58.8, 53.8, 29.7. Purity: 99.6% by HPLC. HRMS (ESI) m/z: calcd for C23H23N5O2, [M+H]+ 402.1925, found 402.1912. 2-(3-(((2-(pyrrolidin-1-yl)ethyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one

(4b):

Following the general procedure compound 1 (0.15 g, 0.52 mmol) and 2-(pyrrolidin-1-yl)ethanamine (0.08 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.11 g, 54%). m.p. 163.1-164.6 oC; 1H NMR (400 MHz, CDCl3) δ 8.86 (s, 1H), 8.28 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.72 (s, 2H), 7.50 (d, J = 8.2 Hz, 1H), 7.43 (t, J = 4.1 Hz, 1H), 7.36 (t, J = 4.2 Hz, 1H), 7.23 (t, J = 7.9 Hz, 1H), 4.12 (t, J = 6.0 Hz, 2H), 3.31 (t, J = 5.7 Hz, 2H), 2.88 (s, 4H), 1.87 (s, 4H). 13C NMR (101 MHz, CDCl3) δ 163.1, 156.0, 149.2, 146.3, 135.0, 134.2, 131.3, 129.0, 127.2, 126.5, 126.3, 125.6, 122.5, 122.0, 118.8, 112.7, 112.1, 56.2, 54.5, 29.7, 23.5. Purity: 98.9% by HPLC. HRMS (ESI) m/z: calcd for C23H16N4O, [M+H]+ 386.1975, found 386.1960. 2-(3-(((2-(4-methylpiperazin-1-yl)ethyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4c): 27



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Page 28 of 65

Following the general procedure compound 1 (0.15 g, 0.52 mmol) and 2-(4-methylpiperazin-1-yl) ethanamine (0.09 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.12 g, 57%). m.p. 178.2-180.1 oC; 1H NMR (400 MHz, CDCl3) δ 8.80 (s, 1H), 8.30 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.72 (s, 2H), 7.51 (d, J = 7.8 Hz, 1H), 7.43 (t, J = 4.1 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.26 (t, J=8.0 Hz,1H), 3.99 (t, J = 6.5 Hz, 2H), 3.00 (t, J = 6.6 Hz, 2H), 2.66 (s, 4H), 2.46 (s, 4H), 2.26 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 163.1, 155.5, 149.1, 146.5, 135.0, 134.1, 131.4, 129.0, 127.1, 126.6, 126.3, 125.5, 122.6, 121.9, 118.8, 112.8, 112.1, 58.3, 57.5, 55.1, 53.3, 46.0. Purity: 96.8% by HPLC. HRMS (ESI) m/z: calcd for C24H26N6O, [M+H]+ 415.2241, found 415.2225. 2-(3-(((2-(dimethylamino)ethyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one Following

the

general

procedure

compound

1

(0.15

g,

0.52

mmol)

(4d): and

N,N-dimethylethane-1,2-diamine (0.06 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.10 g, 51%). m.p. 175.4-178.0 oC; 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 8.18 (d, J = 7.5 Hz, 1H), 7.67-7.54 (m, 3H), 7.33 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 7.7 Hz, 1H), 7.11 (t, J = 8.3 Hz, 1H), 3.86 (s, 2H), 2.90 (s, 2H), 2.32 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 163.1, 155.4, 149.1, 146.4, 135.0, 134.0, 131.3, 128.9, 127.1, 126.5, 126.2, 125.4, 122.5, 121.8, 118.7, 112.7, 112.1, 59.7, 58.1, 45.8. Purity: 95.4% by HPLC. HRMS (ESI) m/z: calcd for C21H21N5O, [M+H]+ 360.1819, found 360.1803. 2-(3-(((2-(diethylamino)ethyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one Following

the

general

procedure

compound

1

(0.15

g,

0.52

(4e): mmol)

and

N,N-diethylethane-1,2-diamine (0.06 mL, 0.6 mmol) were used, and the desired product was obtained as a light red solid (0.10 g, 48%). m.p. 162.7-163.6 oC; 1H NMR (400 MHz, CDCl3) δ 8.68 28


Page 29 of 65

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(s, 1H), 8.29 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.63 (d, J = 3.5 Hz, 2H), 7.37 (d, J = 7.6 Hz, 2H), 7.28 (d, J = 6.6 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 3.87 (t, J = 6.4 Hz, 2H), 3.02 (t, J = 6.6 Hz, 2H), 2.64 (dd, J = 14.1, 7.0 Hz, 4H), 1.06 (t, J = 7.0 Hz, 6H).

13

C NMR (101 MHz, CDCl3) δ

162.0, 154.1, 148.0, 145.5, 134.1, 132.8, 130.2, 127.7, 125.9, 125.4, 125.0, 124.1, 121.3, 120.6, 117.5, 111.5, 111.1, 57.2, 52.2, 46.4, 10.8. Purity: 96.9% by HPLC. HRMS (ESI) m/z: calcd for C23H25N5O, [M+H]+ 388.2132, found 388.2128. 2-(3-(((2-(piperidin-1-yl)ethyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one

(4f):

Following the general procedure compound 1 (0.15 g, 0.52 mmol) and 2-(piperidin-1-yl)ethanamine (0.08 mL, 0.6 mmol) were used, and the desired product was obtained as a light yellow solid (0.10 g, 48%). m.p. 170.3-171.7 oC; 1H NMR (400 MHz, MeOD) δ 8.88 (s, 1H), 8.17 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 8.1 Hz, 1H), 7.77 (q, J = 8.2 Hz, 2H), 7.57 (d, J = 8.2 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.20 (t, J = 7.2 Hz, 1H), 3.98 (t, J = 6.7 Hz, 2H), 2.95 (t, J = 6.9 Hz, 2H), 2.63 (s, 4H), 1.64 (dt, J = 11.0, 5.6 Hz, 4H), 1.50 (dd, J = 10.7, 5.6 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 163.06, 155.44, 149.15, 146.45, 134.99, 134.08, 131.31, 128.97, 127.11, 126.59, 126.26, 125.48, 122.60, 121.89, 118.75, 112.77, 112.06, 59.15, 57.60, 54.87, 25.87, 24.19. Purity: 98.2% by HPLC. HRMS (ESI) m/z: calcd for C23H25N5O, [M+H]+ 400.2132, found 400.2126. 2-(3-(((3-morpholinopropyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4g): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and 3-morpholinopropan-1-amine (0.08 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.11 g, 51%). m.p. 168.4-169.9 oC; 1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H), 8.23 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 3.8 Hz, 2H), 7.43 (d, J = 8.1 Hz, 1H), 7.35 (dd, J = 7.7, 3.6 Hz, 1H), 7.32-7.27 (m, 1H), 7.19 (t, J = 7.5 Hz, 1H), 3.82 (t, J = 6.6 Hz, 2H), 3.67-3.63 (m, 4H), 2.54-2.48 (m, 2H), 2.44 29



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Page 30 of 65

(s, 4H), 2.07 (dt, J = 13.8, 6.8 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 163.1, 154.9, 149.1, 146.47, 135.1, 134.14 131.3, 129.0, 127.1, 126.6, 126.3, 125.5, 122.6, 121.9, 118.7, 112.7, 112.1, 67.0, 58.3, 56.4, 53.7, 29.7. Purity: 98.5% by HPLC. HRMS (ESI) m/z: calcd for C24H25N5O2, [M+H]+ 416.2081, found 416.2083. 2-(3-(((3-(pyrrolidin-1-yl)propyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one Following

the

general

procedure

compound

(0.15

5

g,

0.52

(4h):

mmol)

and

3-(pyrrolidin-1-yl)propan-1-amine (0.12 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.11 g, 54%). m.p. 195.2-196.8 oC; 1H NMR (400 MHz, CDCl3) δ 9.07 (s, 1H), 8.14 (d, J = 7.7 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.80 (dd, J = 15.6, 7.5 Hz, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.49 (t, J = 7.1 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 7.22 (t, J = 7.4 Hz, 1H), 3.85 (t, J = 6.3 Hz, 2H), 2.63 (t, J =7.8 Hz, 2H), 2.47 (s, 4H), 2.00 (m, 2H), 1.66 (s, 4H).13C NMR (101 MHz, CDCl3) δ 163.0, 154.8, 149.1, 146.5, 135.1, 134.0, 131.3, 129.0, 127.1, 126.1, 126.2, 125.4, 122.5, 121.8, 118.7, 112.7, 112.1, 58.2, 54.1, 54.0, 29.7, 23.5. Purity: 99.1% by HPLC. HRMS (ESI) m/z: calcd for C24H25N5O, [M+H]+ 400.2132, found 400.2120. 2-(3-(((3-(4-methylpiperazin-1-yl)propyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4i):

Following

the

general

procedure

compound

1

(0.15

g,

0.52

mmol)

and

3-(4-methylpiperazin-1-yl)propan-1-amine (0.08 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.11 g, 51%). m.p. 187.9-189.3 oC; 1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 3.6 Hz, 2H), 7.48 (d, J = 8.0 Hz, 1H), 7.39 (dd, J = 7.9, 4.1 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.24 (t, J = 6.3 Hz, 1H), 3.85 (t, J = 6.3 Hz, 2H), 2.71 (d, J = 6.8 Hz, 4H), 2.61 (s, 4H), 2.34 (s, 3H), 2.21-2.12 (m, 2H), 0.92-0.78 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 163.0, 155.1, 149.1, 146.4, 30


Page 31 of 65

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135.1, 134.1, 131.2, 129.0, 127.1, 126.6, 126.3, 125.5, 122.4, 121.9, 118.7, 112.6, 112.2, 58.1, 55.8, 54.36, 52.4, 45.5, 29.7. Purity: 96.5% by HPLC. HRMS (ESI) m/z: calcd for C25H28N6O, [M+H]+ 429.2397, found 429.2380. 2-(3-(((4-(dimethylamino)butyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one Following

the

general

procedure

compound

1

(0.15

g,

0.52

(4j):

mmol)

and

N,N-dimethylbutane-1,4-diamine (0.09 mL, 0.6 mmol) were used, and the desired product was obtained as a light red solid (0.11 g, 54%). m.p. 184.0-185.4 oC; 1H NMR (400 MHz, CDCl3) δ 8.76 (s, 1H), 8.29 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 7.6 Hz,1H), 7.71 (s, 2H), 7.49 (d, J = 7.7 Hz, 1H), 7.43-7.33 (m, 2H), 7.25 (t, J = 7.6 Hz, 1H), 3.84 (t, J = 4.1 Hz, 2H), 2.44 (t, J = 7.8 Hz, 2H), 2.28 (s, 6H), 2.01-1.91 (m, 2H), 1.77-1.66 (m, 2H).

13

C NMR (101 MHz, CDCl3) δ 163.1, 154.6, 149.1,

146.5, 135.1, 134.0, 131.2, 128.9, 127.1, 126.5, 126.2, 125.4, 122.5, 121.8, 118.7, 112.7, 112.1, 60.1, 59.3, 45.3, 28.7, 25.3. Purity: 96.1% by HPLC. HRMS (ESI) m/z: calcd for C23H25N5O, [M+H]+ 388.2132, found 388.2117. 2-(3-(((4-(diethylamino)butyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one Following

the

general

procedure

compound

1

(0.15

g,

0.52

mmol)

(4k): and

N,

N-diethylbutane-1,4-diamine (0.09 mL, 0.6 mmol) were used, and the desired product was obtained as a light red solid (0.10 g, 48%). m.p. 197.2-199.1 oC; 1H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 1H), 817 (d, J = 8.2 Hz, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.88-7.80 (m, 2H), 7.69 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.36 (t, J = 7.4 Hz, 1H), 7.25 (t, J = 7.4 Hz, 1H), 3.85 (t, J = 7.4 Hz, ,2H), 2.51 (dt, J = 3.5, 1.7 Hz, 2H), 2.43 (m, 4H), 1.88– 1.80 (m, 2H), 1.59-1.52 (m, 2H), 0.91 (t, J = 7.1 Hz, 6H).13C NMR (101 MHz, CDCl3) δ 162.1 153.6, 148.1, 145.5, 134.1, 133.1, 130.3, 128.7, 128.0, 126.11, 125.6, 125.3, 124.5, 121.6, 120.9, 117.8, 111.1, 59.3, 51.6, 45.8, 27.9, 23.8, 10.4. Purity: 31



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96.0% by HPLC. HRMS (ESI) m/z: calcd for C25H29N5O, [M+H]+ 416.2445, found 416.2446. 2-(3-(((4-methylpentyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4l): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and 4-methylpentan-1-amine (0.03 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.11 g, 58%). m.p. 201.9-203.3 oC; 1H NMR (400 MHz, CDCl3) δ 8.79 (s, 1H), 8.33 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.73 (s, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.43 (s, 1H), 7.40-7.35 (m, 1H), 7.28 (d, J = 6.2 Hz, 1H), 3.83 (t, J = 6.8 Hz, 1H), 2.00-1.92 (m, 2H), 1.68-1.60(m, 1H), 1.34 (q, J =11.9 Hz, 1H), 0.93 (d, J = 5.6 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 163.1, 154.3, 149.1, 146.6, 135.0, 134.1, 131.3, 129.0, 127.1, 126.7, 126.3, 125.5, 122.7, 121.8, 118.8, 112.8, 112.1, 60.8, 36.6, 28.7, 27.8, 22.6. Purity: 95.3% by HPLC. HRMS (ESI) m/z: calcd for C23H24N4O, [M+H]+ 373.2023, found 373.2008. 2-(3-((ethylimino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4m): Foll-owing the general procedure compound 1 (0.15 g, 0.52 mmol) and ethanamine (0.03 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.10 g, 63%). m.p. 181.4-182.8 oC; 1H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 1H), 8.17 (d, J = 7.7 Hz, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.85 (m, J = 7.8 Hz, 2H), 7.69 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 8.0 Hz,1H), 7.37 (t, J = 8.0 Hz, 1H), 7.25 (t,, J = 8.0 Hz, 1H), 3.87 (q, J = 11.9 Hz, 2H), 1.46 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 163.2, 153.9, 149.2, 146.6, 135.0, 134.1, 131.3, 129.0., 127.1, 126.6, 126.2, 125.5, 122.6, 121.8, 118.8, 112.8, 112.1, 54.6, 16.2. Purity: 96.5% by HPLC. HRMS (ESI) m/z: calcd for C19H16N4O, [M+H]+ 317.1397, found 317.1385. 2-(3-((propylimino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4n): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and propylamine (0.05 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.11 g, 62%). m.p. 167.4-169.1 oC; 1H NMR 32


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(400 MHz, CDCl3) δ 8.82 (t, 1H), 8.33 (d, J = 7.9 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.74 (s, 2H), 7.53 (d, J = 7.9 Hz, 1H), 7.44 (t, J = 8.2 Hz, 1H), 7.39(t, J = 8.0 Hz, 1H)7.29 (d, J = 7.5 Hz, 1H), 3.83 (t, J = 6.7 Hz, 2H), 2.04-1.95 (m, 2H), 1.09 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 163.1, 154.4, 149.2, 146.6, 135.0, 134.1, 131.3, 129.0, 127.1, 126.7, 126.3, 125.5, 121.9, 118.81, 112.8, 112.1, 100.0, 62.2, 24.1, 11.9. Purity: 99.4% by HPLC. HRMS (ESI) m/z: calcd for C20H18N4O, [M+H]+ 331.1553, found 331.1540. 2-(3-((butylimino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4o): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and butylamine (0.06 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.10 g, 56%). m.p. 169.4-171.3 oC; 1H NMR (400 MHz, CDCl3) δ 8.63 (s, 1H), 8.22 (d, J= 7.8 Hz, 1H), 7.71 (d, J= 7.9 Hz, 1H), 7.58 (s, 2H), 7.31 (d, J= 7.9 Hz, 3H), 7.23 (t, J= 7.9 Hz, 1H), 7.14 (dd, J= 14.8, 7.2 Hz, 2H), 3.72 (t, J= 6.8 Hz, 2H), 1.87-1.80 (m, 2H), 1.47-1.38 (m, 2H), 0.92 (t, J= 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 162.3, 153.4, 148.3, 145.6, 134.0, 133.0, 130.2, 127.9, 126.0, 125.6, 125.2, 124.4, 121.6, 120.7, 117.9, 117.7, 111.7, 111.0, 59.1, 31.9, 19.5, 12.8. Purity: 99.9% by HPLC. HRMS (ESI) m/z: calcd for C21H20N4O, [M+H]+ 345.1710, found 345.1694. 2-(3-((isobutylimino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4p): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and isobutylamine (0.06 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.09 g, 49%). m.p. 181.1-182.9 oC; 1H NMR (400 MHz, MeOD) δ 8.87 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 7.7 Hz, 1H), 7.78 (s, 2H), 7.58 (d, J = 8.1 Hz, 1H), 7.46(t, J = 7.4 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.23 (t, J = 7.4 Hz, 1H), 3.68 (d, J = 6.4 Hz, 2H), 2.26-2.19 (m, 1H), 1.08 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 162.0, 153.6, 148.0, 145.6, 134.1, 133.0, 130.2, 127.9, 126.0, 125.5, 125.2, 124.4, 121.5, 120.7, 33



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117.7, 111.6, 111.1, 67.5, 28.6, 19.7. Purity: 97.1% by HPLC. HRMS (ESI) m/z: calcd for C21H20N4O, [M+H]+ 345.1710, found 345.1696. 2-(3-((tert-butylimino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4q): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and tert-Butylamine (0.06 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.09 g, 54%). m.p. 190.7-192.3 oC; 1H NMR (400 MHz, DMSO-d6) δ 12.63 (s, 1H), 8.97 (s, 1H), 8.17 (dd, J = 7.0, 3.6 Hz, 2H), 7.88-7.81 (m, 3H), 7.70 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 8.0 Hz, 2H), 7.37 (t, J = 7.8 Hz, 1H), 7.26 (t, J = 8.2 Hz,1H), 1.52 (s, 9H).

13

C NMR (101 MHz, DMSO-d6) δ 162.6, 150.7, 135.8, 134.3, 127.5, 127.1, 126.1,

125.9, 124.9, 123.1, 122.1, 121.4, 120.3, 119.3, 113.2, 113.0, 112.3, 29.3, 27.1. Purity: 96.2% by HPLC. HRMS (ESI) m/z: calcd for C21H20N4O, [M+H]+ 345.1710, found 345.1697. 2-(3-((cyclohexylimino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4r): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and cyclohexanamine (0.07 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.09 g, 48%). m.p. 186.8-189.2 oC; 1H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 8.06 (d, J = 7.1 Hz, 1H), 7.82 (s, 2H), 7.68 (d, J = 7.2 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.35 (t, J = 4.5 Hz, 1H), 7.23 (t, J = 6.5 Hz, 1H), 1.94-1.82 (m, 4H), 1.81-1.65 (m, 4H), 1.46-1.34 (m, 2H), 1.31-1.23 (m, 1H).

13

C NMR (101

MHz, DMSO-d6) δ 162.3, 154.1, 149.1, 147.0, 135.8, 134.3, 131.4, 128.6, 127.1, 126.2, 125.9, 124.9, 122.0, 121.4, 119.1, 113.0, 112.2, 66.9, 33.8, 25.1, 24.1. Purity: 99.5% by HPLC. HRMS (ESI) m/z: calcd for C23H22N4O, [M+H]+ 371.1866, found 371.1850. 2-(3-((phenylimino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4s): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and aniline (0.05 mL, 0.6 mmol) were used, and the desired product was obtained as a light yellow solid (0.11 g, 53%). m.p. 231.3-232.8 oC; 1H NMR 34


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(400 MHz, CDCl3) δ 10.39 (s, 1H), 9.20 (s, 1H), 8.35 (d, J= 7.9 Hz, 1H), 7.97 (d, J= 8.0 Hz, 1H), 7.77 (d, J= 4.8 Hz, 2H), 7.72 (d, J= 7.7 Hz, 2H), 7.57 (d, J= 8.1 Hz, 1H), 7.53 (s, 1H), 7.49(t, J = 87 Hz, 2H), 7.43 (dd, J= 13.7, 5.8 Hz, 2H), 7.34 (dd, J= 13.4, 6.4 Hz, 2H).

13

C NMR (101 MHz,

DMSO-d6) δ 162.0, 153.0, 149.0, 147.5, 146.5, 135.9, 134.5, 131.7, 129.4, 129.0, 127.2, 126.9, 126.5, 125.9, 125.2, 124.0, 121.5, 119.8, 113.1, 112.4, 104.95. Purity: 96.4% by HPLC. HRMS (ESI) m/z: calcd for C23H16N4O, [M+H]+ 365.1397, found 365.1386. 2-(3-(((4-(diethylamino)phenyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one

(4t):

Following the general procedure compound 1 (0.15 g, 0.52 mmol) and p-Amino-N, N-diethylaniline hydrochloride (0.12 g, 0.6 mmol) were used, and the desired product was obtained as a light red solid (0.14 g, 64%). m.p. 216.5-218.3 oC; 1H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 9.38 (s, 1H), 8.31 (d, J= 8.0 Hz, 1H), 8.20 (d, J= 7.8 Hz, 1H), 7.90-7.83 (m, 4H), 7.70 (d, J= 8.2 Hz, 1H), 7.55 (t, J= 7.3 Hz, 1H), 7.39 (t, J= 7.5 Hz, 1H), 7.27 (t, J= 7.3 Hz, 1H), 6.80 (d, J= 8.9 Hz, 2H), 3.43 (dd, J= 13.7, 6.7 Hz, 4H), 1.15 (t, J= 6.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 163.1, 147.5, 146.7, 143.9, 135.2, 133.9, 130.5, 129.1, 127.0, 126.5, 126.0, 125.4, 122.7, 122.4, 121.6, 118.9, 114.3, 112.1, 112.0, 44.6, 12.7. Purity: 98.8% by HPLC. HRMS (ESI) m/z: calcd for C27H25N5O, [M+H]+ 436.2132, found 436.2127. 2-(3-(((4-(dimethylamino)phenyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one Following

the

general

procedure

compound

1

(0.15

g,

0.52

mmol)

(4u): and

N,

N-Dimethyl-p-phenylenediamine monohydrochloride (0.10 g, 0.6 mmol) were used, and the desired product was obtained as a light red solid (0.11 g, 54%). m.p. 213.4-215.0 oC; 1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 7.9 Hz, 1H), 7.57 (d, J = 8.5 Hz, 4H), 7.33-7.28 (m, 2H), 7.19 (t, J =7.6Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H), 6.65 (d, J = 8.7 Hz, 2H), 2.90 (s, 35



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6H). 13C NMR (101 MHz, CDCl3) δ 163.1, 150.1, 146.6, 144.7, 135.17, 133.9, 130.7, 129.1, 127.0, 126.5, 126.1, 125.5, 122.4, 121.7, 118.9, 114.2, 112.8, 112.1, 40.4. Purity: 97.7% by HPLC. HRMS (ESI) m/z: calcd for C25H21N5O, [M+H]+ 408.1819, found 408.1801. 2-(3-((p-tolylimino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4v): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and p-toluidine (0.04 g, 0.6 mmol) were used, and the desired product was obtained as a light yellow solid (0.09 g, 49%). m.p. 229.4-231.1 oC; 1H NMR (500 MHz, DMSO-d6) δ 12.76 (s, 1H), 9.46 (s, 1H), 8.31 (d, J = 8.1 Hz, 1H), 8.19 (d, J = 7.9 Hz, 1H), 7.88 (t, J = 7.6 Hz, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.2 Hz, 1H), 7.54 (t, J = 7.3 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 8.1 Hz, 2H), 7.28 (t, J = 7.5 Hz, 1H), 2.37 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 162.0, 151.8, 149.0, 146.6, 144.9, 136.7, 136.0, 134.5, 131.6, 129.9, 129.1, 127.2, 126.5, 126.0, 125.2, 121.8, 121.7, 121.4, 119.8, 114.0, 113.1, 20.7. Purity: 98.9% by HPLC. HRMS (ESI) m/z: calcd for C24H18N4O, [M+H]+ 379.1553, found 379.1537. 2-(3-(((3-chlorophenyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4w): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and 3-chloroaniline (0.06 mL, 0.6 mmol) were used, and the desired product was obtained as a light yellow solid (0.12 g, 57%). m.p. 230.5-233.1 oC; 1

H NMR (400 MHz, CDCl3) δ 9.08 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H),

7.76-7.71 (m, 2H), 7.61 (s, 1H), 7.59 (d, J = 1.9 Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.48-7.43 (m, 1H), 7.42-7.39 (m, 1H), 7.38 (d, J = 4.5 Hz, 1H), 7.33-7.28 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 162.6, 151.6, 148.5, 145.8, 135.2, 135.1, 134.2, 131.6, 130.6, 129.3, 127.4, 126.9, 126.7, 126.6, 126.0, 122.6, 122.3, 120.9, 120.3, 118.7, 114.2, 112.9, 112.4. Purity: 95.3% by HPLC. HRMS (ESI) m/z: calcd for C23H15ClN4O, [M+H]+ 399.1007, found 399.0990. 2-(3-((phenethylimino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4x): Following the general 36


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procedure compound 1 (0.15 g, 0.52 mmol) and β-phenethylamine (0.08 mL, 0.6 mmol) were used, and the desired product was obtained as a light brown solid (0.10 g, 50%). m.p. 205.9-207.5 oC; 1H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 8.35 (d, J = 7.8 Hz, 1H), 7.76-7.70 (m, 3H), 7.45 (dd, J = 10.0, 6.7 Hz, 2H), 7.35 (t, , J = 7.8 Hz, 1H), 7.30 (t, J = 6.3 Hz, 3H), 7.25 (d, J = 5.0 Hz, 1H), 7.21 (d, J = 7.5 Hz, 1H), 7.17 (t, J = 7.1 Hz, 1H), 4.10 (t, J = 7.1 Hz, 2H), 3.32 (t, J = 7.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 163.1, 155.0, 149.2, 146.5, 144.3, 139.5, 135.0, 134.1, 131.3, 129.0, 128.5, 127.1, 126.7, 126.3, 126.3, 125.5, 122.7, 121.9, 118.8, 112.6, 112.0, 61.7, 37.1. Purity: 96.1% by HPLC. HRMS (ESI) m/z: calcd for C25H20N4O, [M+H]+ 393.1710, found 393.1692. 2-(3-(((2-hydroxyethyl)imino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one (4y): Following the general procedure compound 1 (0.15 g, 0.52 mmol) and 2-aminoethanol (0.04 mL, 0.6 mmol) were used, and the desired product was obtained as a light yellow solid (0.08 g, 50%). m.p. 193.6-194.7 oC; 1

H NMR (500 MHz, DMSO-d6) δ 9.06 (s, 1H), 8.16 (d, J = 7.7 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H),

7.85-7.83 (m, 2H), 7.68 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 7.3 Hz, 1H), 7.35 (t, J = 7.3 Hz, 1H), 7.24 (t, J = 7.3 Hz, 1H), 3.91 (s, 4H). 13C NMR (126 MHz, DMSO-d6) δ 162.7, 157.4, 149.2, 147.0, 135.8, 134.4, 131.8, 128.6, 127.2, 126.3, 126.0, 125.0, 121.8, 121.6, 119.1, 113.1, 112.1, 61.0, 60.6. Purity: 97.0% by HPLC. HRMS (ESI) m/z: calcd for C19H16N4O2, [M+H]+ 333.1346, found 333.1332.

General procedure for the preparation of 5, 6, and 7. A solution of sodium borohydride (0.045 g, 1.2 mmol) in methanol (2 mL) was added dropwise to a solution of 1 or 3c or 4b (0.4 mmol) in methanol (6 mL). The reaction mixture was stirred at room temperature for 2 h. Water (0.5 mL) was added to quench the reaction, and the solvent was evaporated. Then, 10 mL of a saturated sodium bicarbonate solution was added to the mixture, and the crude product was extracted with 37



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Page 38 of 65

dichloromethane. The combined organic layers were dried (Na2SO4), and the solvent was evaporated. The desired product was purified by flash chromatography on silica gel. 2-(3-(hydroxymethyl)-1H-indol-2-yl)quinazolin-4(3H)-one (5): Following the general procedure compound 1 (0.12 g, 0.4 mmol) was used, and the desired product was obtained as a light yellow solid (0.06 g, 56%). m.p. 276.0-278.3 oC; 1H NMR (400 MHz, MeOD) δ 8.31 (dd, J = 8.0, 1.3 Hz, 1H), 8.09 (s, 2H), 8.02 - 7.96 (m, 1H), 7.85 (d, J = 5.4 Hz, 1H), 7.83 (d, J = 5.3 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.25 (t, J = 7.4 Hz, 1H), 5.08 (s, 2H). 13

C NMR (101 MHz, MeOD) δ 168.8, 161.4, 150.3, 142.7, 139.5, 137.4, 135.9, 130.7, 129.6, 128.9,

128.3, 122.9, 121.4, 121.2, 120.9, 113.8, 65.3. Purity: 99.5% by HPLC. HRMS (ESI) m/z: calcd for C23H23N5O2, [M+H]+ 292.1041, found 292.1067. (2-(4-((2-(piperidin-1-yl)ethyl)amino)quinazolin-2-yl)-1H-indol-3-yl)methanol (6): Following the general procedure compound 3c (0.16 g, 0.4 mmol) was used, and the desired product was obtained as a light brown solid (0.08 g, 50%). m.p. 191.4-193.1 oC; 1H NMR (400 MHz, CDCl3) δ 10.10 (s, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.68 (dd, J = 7.3, 4.4 Hz, 2H), 7.55 (d, J = 8.1 Hz, 1H), 7.40 (t, J = 7.4 Hz, 1H), 7.22 (d, J = 7.9 Hz, 1H), 7.11 (t, J = 7.3 Hz, 1H), 5.14 (s, 2H), 3.82 (s, 2H), 2.78 (t, J = 6.0 Hz, 2H), 2.55 (d, J = 10.8 Hz, 4H), 1.70 (d, J = 4.9 Hz, 4H), 0.91-0.84 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 159.4, 155.2, 149.2, 135.3, 133.3, 133.1, 127.8, 127.5, 125.8, 123.9, 121.7, 119.8, 119.6, 119.5, 113.5, 111.8, 56.0, 55.6, 53.9, 36.9, 29.7, 25.0. Purity: 99.0% by HPLC. HRMS (ESI) m/z: calcd for C23H23N5O2, [M+H]+ 402.2249, found 402.2273. 2-(3-(((2-(pyrrolidin-1-yl)ethyl)amino)methyl)-1H-indol-2-yl)quinazolin-4(3H)-one

(7):

Following the general procedure compound 4b (0.15 g, 0.4 mmol) was used, and the desired product was obtained as a light brown solid (0.08 g, 54%). m.p. 182.7-183.8 oC 1H NMR (400 MHz, CDCl3) 38


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δ 9.75 (s, 1H), 8.27 (d, J = 8.0 Hz, 1H), 7.74-7.69 (m, 2H), 7.64 (d, J = 8.1 Hz, 1H), 7.44-7.40 (m, 2H), 7.30 (d, J = 7.7 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 4.26 (s, 2H), 3.03-2.98 (m, 2H), 2.95 (d, J = 5.0 Hz,2H), 2.66 (s, 4H), 1.79 (s, 4H).

13

C NMR (101 MHz, CDCl3) δ 163.6, 148.1, 135.5, 134.1,

129.0, 128.3, 126.7, 126.5, 126.0, 124.9, 121.5, 120.3, 119.2, 115.0, 111.7, 108.1, 54.6, 54.2, 46.5, 42.6, 23.5. Purity: 99.0% by HPLC. HRMS (ESI) m/z: calcd for C23H23N5O2, [M+H]+ 388.2093, found 388.2115.

2. Pharmacology Cell lines and cell culture. 3T3-L1 fibroblasts, HepG-2 and Hela cells were purchased from the American Type Culture Collection. The cells were maintained in DMEM (Geneon, USA) supplemented with 10% fetal bovine serum (BI, USA) and 1% penicillin and streptomycin (Gibco, USA) in a humidified atmosphere containing 5% CO2 in air at 37 °C.

3T3-L1 preadipocyte differentiation assay. Briefly, the 3T3-L1 fibroblast differentiation procedure was carried out as follows:29, 45 Two days after post-confluence (Day 0), the medium was changed to a differentiation cocktail mixture containing 0.5 mM 3-isobutyl-1-methylxanthine (Sigma, USA), 100 ng/mL Dexamethason (MP, USA) and 2 µg/mL insulin (Sigma, USA) in DMEM with 10% (v/v) FBS and 1% (v/v) penicillin and streptomycin for 3 days (Day 3). Thereafter, the medium was replaced by 10% FBS/DMEM containing 2 µg/mL insulin for another 3 days (Day 6). Stock solutions of the test compounds were made and stored at -20 °C. The compounds were added on Day 0 in the differentiation induction medium for 3 days and replaced with another aliquot in post-differentiation medium for another 3 days. The UND group and the differentiation control group were supplemented with the same volume of DMSO as a vehicle control for all experiments. 39



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HepG-2 cells were seeded in 48-well plates at a density of 5×104 cells / well. After 24 h, the cells were incubated with 0.5 mM oleic acid sodium (Sigma, USA) and test compounds at the indicated concentrations for 24 h. The control group was supplemented with the same volume of DMSO as a vehicle control for all experiments.

Red Oil O staining and measuring the lipid content. Oil Red O staining was performed using the following procedure.30 The cells were washed with ice-cold PBS buffer (0.2 M NaCl, 10 mM Na2HPO4, 3 mM KCl, and 2 mM KH2PO4, pH 7.4) and then fixed with 4% formaldehyde (v/v) for 1 h at room temperature followed by three washes with distilled water. The cells were stained with freshly prepared Oil Red O working solution for 30 min at room temperature and washed three times with distilled water. The plates were scanned using an OLYMPUS CKX41 microscope and camera (OLYMPUS). The Oil Red O working solution was prepared by mixing 6 mL of 0.5% Oil Red O (w/v, Sigma) in isopropanol with 4 mL of ddH2O followed by filtration through a 0.22-µm filter (Millipore). The lipid contents of the cells were measured by extracting the Oil Red O from the stained cells with 100% isopropyl alcohol and obtaining the optical density at 510 nm.

The total cholesterol content and triglyceride assay. After treatment, the cells were collected and washed three times with PBS (pH 7.4). Then, the cells were lysed with distilled water containing 0.2% Triton X-100 (MP, 194854) for 1 h at room temperature and then ultra-sonicated for 15 min. The lysates were collected and centrifuged at 4 °C, 12,000g for 15 min. The cholesterol and triglyceride contents of the cells were measured using GPO-POD Assay Kits and Cholesterol Analysis Kits (Jiancheng Bio, China), and the protein levels were determined using BCA protein assay kits (Pierce, USA). Then, the results were expressed as “mmol triglyceride (cholesterol) / g 40


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protein” as previously described.36

Cell viability analysis. The cell viability was determined using the MTT method (MP, USA), according to the manufacturer’s instructions. Briefly, 3T3-L1 preadipocytes, HepG-2 and Hela cells were seeded in four 96-wells plates at a density of 6×103 cells with 100 microliter culture medium per well. After 24 h, the cells were incubated in the presence or absence of the indicated concentrations of the compounds for 24 h; the control group was administered the same volume of DMSO as the test compounds. Next, the culture medium was replaced with media containing 0.5 mg/mL MTT solution and incubated for 4 h at 37 °C. Then, 150 µL of DMSO was added to each well to dissolve the formazan, and the absorbance was measured at 490 nm using a microplate reader (Biotek, USA). Each assay was carried out in triplicate.

Dehydrogenase (LDH) Assay. After the cells were treated with the compounds for the indicated times, the culture medium was carefully discarded, and 100 µL of 1×LDH releasing reagent was added (one volume of 10×LDH releasing reagent in 10 volumes of PBS (pH 7.4)), incubated at 37 °C for 1 h. The cell culture plate was centrifuged, and the supernatant was ready for use. The enzyme activity in the medium was determined using LDH Cytotoxicity Assay kits (Beyotime) according to the manufacturer’s protocol. The absorbance of the samples was read at 490 nm with a microplate reader (Biotek, USA). The LDH in the medium was expressed as a percentage of the maximum LDH activity. The equation used for the cell viability (%) calculation was as follows: Cell viability (%) = 100 - (LDH activity after compound supplementation - Blank group)/(the maximum LDH activity Blank group) × 100) as previously described. Blank group: without both the compound treatment and 41



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the LDH releasing reagent; Maximum LDH activity: without the compound treatment but with added LDH releasing reagent; Compound treatment: with both the compound treatment and the LDH releasing reagent.

Cell cycle and apoptosis analysis. The cells were carefully harvested and centrifuged. The cellular pellets were washed twice in ice-cold PBS, centrifuged and fixed gently with 70% ethanol overnight at -20 °C. The ethanol was removed by centrifugation (1,500 rpm, 5 min) and PBS containing propidium iodide (PI) (50 µg/mL, Sigma) and RNase A (100 µg/mL, AppliChem, Germany) were added and incubated for 30 min in the dark at room temperature. The cell cycle was analyzed using a FACS flow cytometer (Beckman, USA). The percentages of cells within the G0/G1, S and G2/M cell cycle phases after acquiring 10,000 cells were analyzed using Win-MDI 2.8 software (Joseph Trotter, Scripps, CA, USA).

RNA Extraction and RT-PCR. Total RNA was extracted from cells or tissue using RNAiso Plus according to the manufacturer’s instructions. cDNA was synthesized by using Oligo (1) from 1 µg of total RNA in a 20 µL reaction, and cDNA was used for amplication of specific target gene by using PCR. β-Actin was used for loading control primers and their sequences are listed in Table 1. The thermal cycle conditions were as follows: after heating at 95 °C for 10 min, PCR amplification was done with 35 cycles of 95 °C for 30 s, the respective annealing temperature for 45 s, 72 °C for 1 min, followed by a terminal extension at 72 °C for 10 min. The sequence of oligonucleotide primers are described in Table S1. The PCR products were separated on 1.2% (m/v) agarose gels and analyzed by Alpha Imaging System. The expression levels of genes were normalized to that of β-actin. Data were analyzed using Image J software. 42


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Western blotting. Cultured cells were washed with ice-cold PBS (pH 7.4), lysed with lysis buffer [1×RIPA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 1 mM NaF, 1 µg/mL aprotinin, 1 µg/mL pepstatin, and 1 µg/mL leupeptin] on ice for 0.5 h. Then, the lysates were centrifuged at 4 °C 12,000 g for 30 min to remove debris. The protein concentrations of the lysates were determined using a BCA protein assay kit. Protein samples denatured in SDS sample buffer (125 mM Tris-HCl, pH 6.8, 50% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, and 0.01% (w/v) bromophenol blue) were subjected to SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Millipore). The blotted membranes were blocked with 5% (w/v) bovine serum albumin in Tris-buffered saline containing 0.1% (v/v) Tween 20 for 1 h and then incubated with primary antibodies for 16 h at 4 °C. After three washes in Tris-buffered saline containing 0.1% (v/v) Tween 20, the membranes were incubated with anti-mouse or anti-rabbit IgG and horseradish peroxidase–linked antibodies for 1 h. Immunoreactive signals were detected with ChemiDoc™ XRS Imaging System (Bio-Rad) and quantified with Image Lab™ 3.0 software (Bio-Rad). The key adipogenic markers were examined by Western blotting using specific antibodies including ACC (cell signaling), FAS (cell signaling), SCD-1 (cell signaling), PPARγ (cell signaling), C/EBPα (Santa Cruz), and SREBP-1c (Santa Cruz). Immunolabeled bands were quantified by densitometry and representative blots are shown.

Statistical analysis. One-way ANONA followed by two-tailed unpaired Student’s t-tests were applied for multiple comparisons. The data were expressed as the means ± S.E.M. p < 0.05 was considered significant.

43



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ASSOCIATED CONTENT Supporting Information Additional experimental results, crystallographic data for SYS-S20, 1H and

13

C NMR spectra,

HRMS and HPLC assay data for final compounds are available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENTS We thank the Natural Science Foundation of China (81273433, 91213302, and 81330077), Specialized Research Fund for the Doctoral Program of Higher Education of China (20110171110051), and Guangdong Provincial Key Laboratory of Construction Foundation (Grant 2011A060901014) for financial support of this study.

AUTHOR INFORMATION Corresponding Author * For Zhi-Shu Huang: phone and fax: 020-39943056; E-mail: [email protected]. Author Contributions † These authors contributed equally.

ABBREVIATIONS ACC, acetyl-CoA carboxylase; AMPK, adenosine 5’-monophosphate (AMP)-activated protein kinase; C/EBP α, CCAAT-enhancer binding proteins α; FAS, fatty acid synthase; FFA, free fatty acid; IBMX, 3-isobutyl-1-methylxanthine; LDH, lactic dehydrogenase; LKB1, liver kinase B1; MCE,

44


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mitotic clonal expansion; NAFLD, non-alcoholic fatty liver disease; PPARγ, peroxisome proliferator-activated receptor γ; SCD-1, stearoyl-CoA desaturase 1; SREBP-1, sterol regulated element binding protein-1; TG, triglyceride.

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FIGURE LEGENDS

Figure 1. The structures of rutaecarpine, Bouchadatine (1), R17 (2), and the two series of compounds derived from 1.

Figure 2. Chemical structures of the new compounds.

Figure 3. Evaluation of the lipid-lowering effect of 1 and its derivatives in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were treated with the compounds on Day 0 for 3 days during differentiation at concentrations of 1 and 10 µM. Then, the triglyceride contents were determined via Oil Red O staining on Day 6. 2 was considered the control. A) Determination of the effect of 1 and its derivatives on the TG level in 3T3-L1 adipocytes. B) Representative microscopy images of the 3T3-L1 adipocytes were captured after Oil Red O staining (× 40). The concentration of the compounds was 1 µM; n = 3 independent experiments; values are expressed as the means ± S.D; UND: undifferentiated group; Control: differentiated group without treatment with the compounds.

Figure 4. Determination of the effect of 1 and its derivatives on lipid metabolism in HepG-2 cells. The cells were incubated with 0.5 mM oleic acid sodium in the presence or absence of the compounds at 1 µM for 24 h. The triglyceride/cholesterol contents were determined according to the method presented in the manuscript. n = 3 independent experiments; values are expressed as the means ± S.D.

Figure 5. Determination of the triglyceride content and change in the cell number during 3T3-L1 51



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differentiation. Two days after post-confluence, the 3T3-L1 preadipocytes were treated with the compounds at the indicated concentrations for 9 days during differentiation. Every day, cells were collected and counted, and then, the triglyceride content was evaluated via TG assay. (A) The number of cells was counted each day at the indicated time. (B) The lipid accumulation in the 3T3-L1 adipocytes was determined. DMSO: differentiated group without treatment with the compounds.

Figure 6. Evaluation of the lipid-lowering effect of 3d in 3T3-L1 adipocytes and HepG-2 cells. The 3T3-L1 preadipocytes were treated with 3d and 2 at the indicated concentrations for 6 days during differentiation. On day 6, the intracellular TG contents were determined via TG assay and Oil Red O staining. UND: undifferentiated group; Control: differentiated group without treatment of compounds. (A) The TG contents in the 3T3-L1 adipocytes were determined via TG assay after incubation with 3d and 2 at 0.01, 0.1, 1.0, 3.0, 5.0, 10.0 µM. n = 3 independent experiments; values are expressed as means ± S.D. *, p < 0.05; **, p < 0.01 compared with the control group. (B) HepG-2 cells were induced with 0.5 mM oleic acid sodium in the presence or absence of 3d for 24 h at the indicated concentrations. The intracellular TG contents were determined via TG assay. n = 3 independent experiments; values are expressed as means ± S.D. Blank: without oleic acid sodium induction. Control: induction by oleic acid sodium but without treatment with the compounds. *, p < 0.05: **, p < 0.01 compared with control group.

##

p < 0.01 compared with the Blank group. (C) 3d

is not cytotoxic to 3T3-L1 cells. Confluent 3T3-L1 cells were treated with 3d and 2 at 1.0, 2.5, 5.0, and 10.0 µM for 24 h, 48 h, and 72 h during the early stage of 3T3-L1 differentiation. Culture medium was collected each day and used for analysis as described in the Material and Methods section. (D) Schematic diagram of the 3d treatment of the 3T3-L1 adipocytes. The 3T3-L1 52


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preadipocytes were treated with 1 µM 3d for various periods. Control indicates differentiation adipocytes without 3d treatment. On Day 9, the intracellular TG content was evaluated via TG assay. (E) The TG contents in the 3T3-L1 adipocytes were determined via TG assay. n = 3 independent experiments; values are expressed as means ± S.D. ###, p < 0.001, vs UND group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs DMSO group.

Figure 7. Cell cycle progress and apoptosis analysis by FACS in 3T3-L1 adipocytes. Post-confluent 3T3-L1 preadipocytes were incubated with adipogenic cocktail in the presence or absence of 3d (0.3, 1.0, and 3.0 µM) for 24 h. After 24 h, the cells were collected, washed three times with PBS (pH 7.4) and analyzed by FACS. (A) The cells were fixed by 70% ethanol overnight at -20 °C and stained with propidium iodide (PI) for 30 min. The cell cycle distribution was determined by FACS. (B) The data refer to G0/G1, S, G2/M after 3d (0.3, 1.0, and 3.0 µM) exposure. n = 3 independent experiments; values are expressed as means ± S.D; ##, p < 0.01 vs UND group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs control group. (C) The 3T3-L1 preadipocytes were exposed to 3d for 24 h in the presence of adipogenic cocktail. The cells were collected and stained with Annexin V-FITC and PI for 10 min. The percentage of apoptotic cells was determined by FACS.

Figure 8. Effects of 3d on the expression of adipogenic markers and downstream lipogenic enzymes in 3T3-L1 adipocytes. The 3T3-L1 preadipocytes were incubated with adipogenic cocktail in the presence or absence of 3d (0.3, 1.0, and 3.0 µM) for the indicated periods during 3T3-L1 differentiation and then harvested. The protein level was determined through Western blot. (A) mRNA levels of early adipogenic factors C/EBPβ, C/EBPδ (24 h treatments). (B) mRNA levels of C/EBPα, PPARγ and SREBP-1c (24 h treatments). (C) Protein levels of C/EBPα, PPARγ and 53



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SREBP-1c (3-day treatments). (D) Protein levels of ACC, FAS, and SCD-1 (6-day treatments). GAPDH and Tubulin were viewed as loading controls. All data were normalized to GAPDH/Tubulin, and the percentage changes in the expression were calculated relative to the controls. n = 3 independent experiments; values are expressed as means ± S.D. **, p < 0.01; ***, p < 0.001 compared with the control groups; ##, p < 0.01; ###, p < 0.01 compared with the Blank group.

Figure 9. Effect of 3d on LKB1-AMPK signaling pathway in 3T3-L1 adipocytes. The 3T3-L1 preadipocytes were incubated with adipogenic cocktail in the presence or absence of 3d (0.3, 1.0, and 3.0 µM) for the indicated periods during 3T3-L1 differentiation and then harvested. The protein level was determined through Western blot. (A) Protein expression levels of AMPK signaling pathway related proteins (6 days treatments). (B) Protein expression levels of LKB1, CaMKKα in 3T3-L1 adipocytes (6 days treatments). (C) Expression of LKB1-AMPK signaling axis related protein in 3T3-L1 adipocytes after addition of 3d and AICAR followed with incubation for 6 days. All data were normalized to GAPDH/Tubulin, and the percentage changes in the expression were calculated relative to the controls. n = 3 independent experiments; values are expressed as means ± S.D. **, p < 0.01 compared with the control groups; #, p < 0.01; ##, p < 0.01; compared with the Blank group; @, p < 0.05; @@, p < 0.01 compared with AICAR treatment group.

Figure 10. A significant effect of 3d on LKB1-AMPK signaling pathway in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were incubated with adipogenic cocktail in the presence or absence of 3d (0.3, 1.0, and 3.0 µM) for the indicated periods during 3T3-L1 differentiation and then harvested. The mRNA and protein levels were determined by using RT-PCR and Western blot analysis. (A) Protein expression levels of LKB1-AMPK signaling axis related proteins after 10 minute treatments of 54


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AICAR and 3d. (B) Protein expression levels of LKB1-AMPK signaling axis related proteins after 2 h treatments of AICAR and 3d. (C) mRNA levels of C/EBPβ, C/EBPδ after 2 h treatments of AICAR and 3d. (D) Protein expression levels of LKB1-AMPK signaling axis related proteins after 24 h treatments of AICAR and 3d. (E) mRNA level of adipogenic factors after 24 h treatments of AICAR and 3d. (F) Data were normalized to GAPDH or Actin. Relative protein levels of LKB1, pAMPKα were normalized to GAPDH, viewed 10 min DMSO group as 100%, relative protein levels of other groups were compared with 10 min DMSO group, respectively. Relative mRNA levels of C/EBPβ, C/EBPδ were normalized to each time point of Actin level (n = 3 independent experiments).

Scheme 1. Synthesis of Series I and Series II compounds. Reagents and conditions: (i) NH4OAc, DMSO-H2O (19:1), 150 °C, 20 h; (ii) SOCl2, DMF, 80 °C, 6 h; (iii) amine, toluene, Et3N, reflux, 10 h. (iv) amine, CHCl3, FeCl3, MgSO4, r.t, 6 h.

55



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Figure 1

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NR1R2

Compds

N

Compds

NR1R2

3h

N H

3o

N

N H

N

3i

N H

3p

H N

NO2

3c

N H

N

3j

H N

3q

H N

F

3d

N H

N

3k

N

3r

H N

OCH3

3e

N H

N

3s

H N

3f

N H

N

3g

N H

N

N H

3b

N NH

NR1R2

N

3a

NR1R2 OHC

Compds

N

O

Series I

Cl

Compds

3l

O

3m

3n

N

Compds

R3

N

3t

N

N

3u

R3

Compds

N H OH

N H R3

O

O R3N

4a

N

4j

N

4r

4b

N

4k

N

4s

4c

N

4d

4e

N 4l

4t

N

N

4m

4u

N

N

4n

4v

HN N NH

Series II

Cl 4f

N

4o

4w

O 4g

N

4p

4x

4h

N

4q

4y

4i

N

OH

N

O HOH2C

HN

HN HOH2C

O HN

N NH

5

N HN

N

N NH

N

N NH

6

7

Figure 2 57



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Figure 3

Figure 4

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Figure 5

Figure 6 59



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Figure 7

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Figure 8

61



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Figure 9

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Figure 10

63



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Scheme 1

64


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Graphic Abstract

65


Synthesis and Biological Evaluation of Novel Bouchardatine

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