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Synthesis, preliminary biological evaluation and 3D-QSAR study of novel 1,5-disubstituted-2(1H)-pyridone derivatives as potential anti-lung cancer agents
⁎Corresponding author. Tel./fax: +86 731 82650370. qbli@csu.edu.cn (Qianbin Li)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Twenty-eight novel 1,5-disubstituted-2(1H)-pyridone derivatives were designed and synthesized for discovering more potent anti-lung cancer agents combined with anti-fibrotic profiles. The in vitro antiproliferative activities of the derivatives against A549 and NIH3T3 cell lines were tested by MTT assays. The majority of the tested analogues exhibited equivalent or an improved anti-lung cancer activity. Prominently, compound 4l displayed the best potency and selectivity toward A549 with an IC50 value of 20 μM, nearly comparable to the positive control cisplatin (IC50 = 10 μM) and even superior to the lead compound 22 (IC50 = 130 μM). Simultaneously, compound 4l showed significant inhibitory activity against NIH3T3 (IC50 = 55 μM), which may contribute to hindering the proliferation of lung cancer cells fundamentally. What is more, the 3D-QSAR models established on the activity data may provide new insights into the design of novel 2(1H)-pyridone derivatives and lay a theoretical foundation for further studies of promising anti-lung cancer activity with the maintenance of anti-fibrotic effect.
Keywords
2(1H)-pyridone derivatives
A549 cell line
Anti-lung cancer activity
3D-QSAR models
1 Introduction
Lung cancer is the markedly leading cause of cancer-related mortality worldwide, with over 228,000 new cases and more than 1159,000 deaths reported in 2014 in the United States (Ali et al., 2015). Approximately 85% of lung cancer patients can be histologically classified as non-small cell lung cancer (NSCLC) and the overall 5-year survival rate of this highly aggressive disease is only 15% (Roldan et al., 2014). The high prevalence and mortality rate are mainly attributable to the difficulty of early diagnosis and lung tumors’ elevated potential for local invasion as well as metastasis to distant tissues and organs (Moses et al., 2014). At present, treatments for lung cancer include surgery, radiation therapy, chemotherapy, and targeted therapies (Wei-Ting et al., 2015). Nevertheless, the outcomes are limited and do not show to be of sufficient benefit in reality despite advances in tumor biology research. The efficacy of conventional anti-lung cancer modalities has already encountered a plateau because of lacking selectivity or intrinsic and acquired resistance (Ying-Rui et al., 2012). On this account, it has become imperative for identifying more effective anti-lung cancer strategies with minimal toxicity. To our knowledge, the occurrence of fibrosis typically resulting from inflammation or damage is capable of obliterating architecture and function of the involved organs or tissues such as lung, liver and kidney (Krenning et al., 2010; Huang et al., 2011). Contemporarily, it is suggested that pulmonary fibrosis shares similar pathological ground to lung cancer, and certain tissues that undergo fibrosis subsequently are susceptible to carcinogenicity, thus aggravating the burden of lung cancer (Kenneth et al., 2006). Given that inhibiting the onset and progression of pulmonary fibrosis can ultimately reduce the formation of benign tumors and cancers, it makes great sense to develop specific anti-lung agents possessing anti-lung cancer property combined with an anti-fibrotic potential, which also seems to be of much interest as new therapeutic anti-lung cancer agents in future.
Pirfenidone (PFD, Fig. 1A) has emerged as a valuable agent for use in patients with idiopathic pulmonary fibrosis (IPF) (Azuma, 2010). This drug can inhibit the progression of fibrosis and collagen synthesis to ameliorate bleomycin-induced and cyclophosphamide-induced lung fibrosis (Iyer et al., 2000; Spond et al., 2003). Fluorofenidone (AKF-PFD, Fig. 1A), an analogue of pirfenidone, has been discovered by our group in programs engaged for anti-fibrosis agents. This compound shows comparable anti-fibrotic potency to pirfenidone, while it possesses a better pharmacokinetic profile (e.g. with a longer half-life) and less toxicity in rats (Yuan et al., 2011). In further optimization work, considerable efforts were undertaken by our research group to obtain more active and metabolically stable candidates in the past years. Accordingly, two series of novel 5-substituent-2(1H)-pyridone derivatives were synthesized and evaluated for the antiproliferative activity against NIH3T3 cells (Jun et al., 2012). As a result, compound 5b (Fig. 1) exhibited as highly potent anti-fibrosis agents with an IC50 of 80 μM about 34-fold of AKF-PFD (IC50 = 2750 μM). Besides, SAR analysis elucidated that introduction of a benzyl group at N-1 position of pyridone nucleus could enhance anti-fibrotic activity. Literature survey reveals that molecules containing a moiety of pyridone show a broad spectrum of promising pharmacological properties such as anti-cancer (Kyoung et al., 2008; Gretchen et al., 2009; Li et al., 2012; Jee et al., 2015), anti-diabetic (Dean et al., 2014), anti-bacterial (Manoranjan et al., 2014; Nisheeth et al., 2013), anti-pruritic (Masahide et al., 2012), anti-oxidant (Semple et al., 2003), anti-fungal (Najma et al., 2010) and anti-inflammatory (Hynes and Leftheri, 2005) activity. So far several pyridone derivatives have advanced into clinical trials or preclinical trials to combat cancers (Fig. 1). For instance, compound B (BMS-777607) (Weiqiang et al., 2015) demonstrates selective inhibition of proliferation in solid tumors by adopting an extended conformation binding to its protein counterpart. Compound C (Li et al., 2015) and D (Rongshi et al., 2006) exhibited potent cytotoxicity in vitro against human colon cancer cells HCT-116 and A549, respectively. Moreover, accumulating evidence indicates that PFD acts as a multi-targeted inhibitor of TGF-β (Burghardt et al., 2007), EGFR (Krishnan et al., 2007), TNF-α (Grattendick et al., 2008), and PFDGF (Levy et al., 2008). Thereinto, TGF-β and EGFR are also well-validated targets for anti-cancer agents. Subsequently, we proposed to develop analogues of pyridone as anti-cancer drugs with the maintenance of anti-fibrotic effect. As such, investigations in our group were aimed at designing new synthetic anti-lung cancer molecules along with anti-fibrotic activity prior to the present search. Recently, Zhu et al.’ (Weixing et al., 2013) systematic studies led to the identification of compound 22 (Fig. 1), which displayed both potency and selectivity (IC50 = 130 μM) toward the A549 cell lines. Importantly, it is discovered that the existence of aromatic ring on C-5 position of pyridin-2(1H)-one scaffold may lead to improvements in the sensitivity of most compounds toward lung cancer cell line. However, the activity of compound 22 is relatively weak, which inspired us to make further modification of pyridone moiety in the direction of developing novel potent molecules with equal or greater specificity for lung cancer than compound 22.
Chemical structures of substituted-2(1H)-pyridone derivatives as potent anti-cancer or anti-fibrosis agents.
For discovering more versatile substituted anti-lung cancer agents with the maintenance of anti-fibrotic effect, herein we disclosed design (Scheme 1) and synthesis of 28 novel 1,5-disubstituted-pyridone derivatives. With the pyridone moiety fixed, we introduced benzyl group instead of phenyl group at the N-1 position and different substituents on two aromatic rings for further optimization, based on the structure of compound 22. The antiproliferative profiles of the synthesized compounds were evaluated in vitro against human A549 and mice NIH3T3 cell lines by MTT assays. Additionally, 3D-QSAR studies were also carried out in the present work to define the structural determinants required to anti-lung cancer activity by means of MOE 2014 (Molecular Operating Environment 2014, Inc., Canada), which accounts for effect of the structural variability on the pharmacological properties and prospectively makes for the future design of novel potential anti-lung cancer drug candidates.
The ligand-based structural optimization on the 1,5-disubstituted pyridone scaffold to develop novel anti-lung cancer agents.
2 Results and discussion
2.1 Chemistry
The preparative route used to access the foregoing 1,5-disubstituted-2(1H)-pyridone analogues is illustrated in Scheme 2. In detail, twenty-eight compounds were synthesized starting from 5-methylpyridin-2(1H)-one. To a solution of starting material in anhydrous 1,4-dioxane, substituted benzyl bromide, potassium carbonate (K2CO3) and a catalytic amount of tetrabutylammonium iodide (TBAI) were added to produce compounds 1a–d in 70–89% yields (Marina et al., 2015). Alternatively, the reaction also proceeded at room temperature with N,N-dimethylformamide (DMF) as a solvent and using NaH or Cs2CO3 as base (Suresh and Tipparaju, 2008), though under such conditions the products are not easily separable from the solvent compared with the former. In the synthesis, the bromination reaction for intermediates 1a–d was the major challenge that we faced, while this synthetic approach is well-documented in the literature (Comins and Lyle, 1976; Morrow and Rapoport, 1974). Other than to afford the desired compounds 2a–d, it is observed that the radical reaction also easily led to dibromination of methyl and bromination of C-3 position of pyridone as well during the process of exploration. In order to avoid the occurrence of side reaction as far as possible, compounds 1a–d were treated with N-bromosuccinimide (NBS) to give common intermediates 2a–d for both series by means of using 2,2′-azobis(2-methylpropionitrile) (AIBN) as catalyst, according to previously reported methods (Weixing et al., 2013). Due to instability of bromination products during isolation, the corresponding crude products 2a–d were directly used for the following step instead of further purification. Subsequently, compounds 2a–d obtained from the above reaction and substituted aniline or sodium salt of substituted phenol were dissolved in acetonitrile and stirred at room temperature to yield the target compounds 3a–o, 4a–m (Table 1), respectively. All synthesized compounds gave satisfactory analytical and spectroscopic data, i.e. 1H NMR, 13C NMR, and EI-MS, which are in accordance with the assigned structures.
General synthesis of compounds 3a–o and 4a–m. Reagents and conditions: (i) K2CO3, TBAI, 1,4-dioxane, Reflux, 20 h, 70–89%; (ii) NBS, AIBN, CCl4, hv, 100 W, 2 h; (iii) acetonitrile, rt, 3 h, 25–57%; (iv) acetonitrile, rt, 3 h, 27–87%.
Compounds
R1
R2
Compounds
R1
R2
3a
2-Fluoro
H
4a
H
H
3b
4-Fluoro
H
4b
2-Fluoro
H
3c
3-Chloro
H
4c
2-Chloro
H
3d
4-Chloro
H
4d
2,4-Dichloro
H
3e
2,4-Dichloro
H
4e
3,4-Dichloro
H
3f
4-Methyl
H
4f
2-Methyl
H
3g
2,4-Dimethyl
H
4g
4-Methyl
H
3h
3,4-Dimethyl
H
4h
2-Methoxy
H
3i
3-Trifluoromethyl
H
4i
4-Methoxy
H
3j
2-Methoxy
H
4j
3,4-Dimethoxy
H
3k
3-Methoxy
H
4k
3-Trifluoromethyl
H
3l
3,4-Dimethoxy
H
4l
4-Fluoro
4-Methoxy
3m
4-Methyl
2-Fluoro
4m
4-Methyl
4-Methoxy
3n
4-Methyl
4-Fluoro
–
–
–
3o
4-Methyl
4-Methoxy
–
–
–
2.2 Biological activity
All the newly synthesized compounds were investigated for their cytotoxic activities against two cell lines, human lung adenocarcinoma A549 cells and mouse embryonic fibroblast NIH3T3 cells, by MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay in vitro, using Cisplatin (DDP) and PFD as reference compounds. The results expressed as inhibition constant (IC50) values are summarized in Table 2, which embodies the difference of activity among the compounds. The evidence of growth inhibition across two cell types suggested that most of these compounds bearing pyridone moiety inhibit cellular proliferation to some extent and the results revealed several interesting points including the broad range of potencies in the two series.
Compounds
IC50 ± SD (μM)a
NIAd
Compounds
IC50 ± SD (μM)a
NIAd
A549
NIH3T3
A549
NIH3T3
3a
185.00 ± 6.32
3.00 ± 0.12
0.016
4a
251.00 ± 9.01
59.00 ± 1.21
0.235
3b
148.0 ± 8.13
12.00 ± 0.34
0.081
4b
403.00 ± 8.45
62.00 ± 2.03
0.154
3c
103.0 ± 2.92
13.00 ± 0.63
0.126
4c
255.00 ± 9.43
11.00 ± 0.65
0.043
3d
80.00 ± 3.43
17.00 ± 0.48
0.213
4d
38.00 ± 2.17
18.00 ± 0.22
0.474
3e
86.00 ± 3.12
14.00 ± 0.79
0.163
4e
235.00 ± 3.95
45.00 ± 1.36
0.191
3f
153.00 ± 9.25
61.00 ± 3.63
0.399
4f
497.00 ± 15.93
39.00 ± 1.58
0.078
3g
119.00 ± 4.51
28.00 ± 1.58
0.235
4g
345.00 ± 7.98
41.00 ± 2.93
0.119
3h
424.00 ± 24.22
22.00 ± 0.92
0.052
4h
9.00 ± 0.94
49.00 ± 2.37
1.256
3i
40.00 ± 1.36
9.00 ± 0.24
0.225
4i
197.00 ± 6.32
30.00 ± 1.74
0.152
3j
265.00 ± 9.52
135.0 0 ± 4.95
0.509
4j
506.00 ± 20.81
27.00 ± 1.16
0.053
3k
219.0 ± 8.47
162.0 ± 3.12
0.740
4k
145.00 ± 5.67
20.00 ± 0.92
0.138
3l
568.0 ± 26.74
14.0 ± 0.57
0.025
4l
20.00 ± 0.75
55.00 ± 2.75
2.750
3m
163.00 ± 5.63
11.0 ± 0.23
0.067
4m
44.00 ± 1.26
40.00 ± 1.97
0.909
3n
89.00 ± 2.54
51.0 ± 1.79
0.573
DDPb
10.00 ± 0.29
3.00 ± 0.22
0.300
3o
81.00 ± 4.28
22.0 ± 1.16
0.272
PFDc
–
454.00 ± 10.45
–
22
130.00 ± 6.16
–
–
5b
–
80.00 ± 2.89
–
2.2.1 Anti-lung cancer activity
Taking compound 22 as the lead, we have carried out structural modification around the 2(1H)-pyridone series by focusing on the three diversification points at R1, R2, and the hydrogen bond donor on C-5 position attached to a phenyl ring. In order to get insight into the effect of different linkers between pyridone and aryl moiety on antiproliferative activity among compounds 3a–o and 4a–m, we first introduced electron-donating group at R1, such as methyl, methoxy group. On the basis of the data in Table 2, compound 3f with NH-phenyl linker displayed stronger activity than compound 4g with O-phenyl linker when the substitution is methyl group at R1 and the other parts are kept constant, whereas, the inhibitory activity of derivatives containing NH-phenyl linker such as compounds 3j and 3l showed lower activities than that of those containing O-phenyl linker such as 4h and 4j, regardless of whether it is monosubstituted or disubstituted by methoxy group at R1. In addition, it is observed that when substituted by strong electron-withdrawing group at R1, compounds bearing NH-phenyl linker such as compound 3i dramatically increased the anti-lung cancer activity compared with those bearing O-phenyl linker such as 4k. Analysis of substituent groups in the aromatic ring of the N-1 position indicated that without methoxy group at R2, the activity of compound with O-phenyl linker (4g) is inferior to that of compound with NH-phenyl linker (3f). However, once introduced methoxy group at R2, the activity of compounds with O-phenyl linker (4m) is superior to that of compounds with NH-phenyl linker (3o), which is just the opposite of the phenomenon found in derivatives without methoxy substitution at R2. These results above suggested that the divergence of substitution at R1 or R2 may account for the discrepancy of activity of compounds with different linker and they can serve as important active groups.
In the case of constant NH-phenyl linker, changing position of substituents or varying number of substituents at R1 on phenyl ring could also affect the activities of compounds. As depicted in Table 2, compound 3a (IC50 = 185 μM) with one fluorine atom on the 2-position of benzene ring showed weaker inhibitory activity than compound 3b (IC50 = 148 μM) with one fluorine atom on the 4-position of benzene ring. Besides, we found para-chloro derivative 3d to show a slight improvement in IC50 against A549 as compared to meta-chloro derivative 3c. Taken together, the activities of compounds with para position substituted on phenyl ring increased while those of compounds with ortho or meta position substituted brought reduced activity. Interestingly, compounds 3h and 3l (two methyl group at 3,4-position in benzene ring respectively) gave rise to a noteworthy loss in activity compared to the analogues bearing only one substituent (3f, 3k). Nevertheless, compound 3g with two methyl groups at 2,4-position displayed more potent inhibition than compound 3f. Compared to compounds 3d and 3e with two chlorine atoms at 3,4-position on phenyl ring did not exhibit big changes in activity, but the activity of 3e is even higher than 3c with only one chlorine atom. Additionally, when the linker is O-phenyl linker, compounds 4d and 4e containing two chlorine atoms at R1 had stronger inhibitory activity than 4c owning one chlorine atom substitution. From the analysis of test results, all the compounds with N-benzyl motif substituted were more active than those without substituents, suggesting that the presence of substituted N-benzyl moiety could improve anti-lung cancer activities to a certain extent.
2.2.2 Anti-fibrotic activity
Fibrosis is common in the establishment of benign tumors and cancers. Evidence has been provided that a close correlation of fibrosis of various tissues and their potential oncological transformation exists. Certain tissues, which undergo fibrosis, are subsequently susceptible to carcinogenicity and when tissue fibrosis in these precancerous tissues is halted, formation of cancer would be blocked (Kenneth et al., 2006). Taking this discovery into consideration, the anti-fibrotic activities of the synthetic derivatives in vitro are also assayed and the IC50 determined, with the purpose of obtaining some agents possessing anti-lung cancer property and at the same time having good inhibition of fibrosis.
Just as can been seen from Table 2, most of the novel derivatives exhibited good inhibitory activities to NIH3T3 cell lines, among which compound 3a displayed best inhibitory activity with an IC50 value of 3 μM, far superior to the lead compound 5b and PFD. Because the activities of compounds 3b, 3d, and 3f wore off gradually, it can be concluded that fluoro-substitution at para position was favorable to chloro-substitution and chloro-substitution was better than methyl-substitution. Additionally, what is most notable is that when the 3-position of phenyl ring was replaced by methoxy group (3k), chlorine (3c) and trifluoromethyl (3i) group respectively, the corresponding activities increased markedly, which indicated that strong electron-withdrawing group contributes to the improvement of activity against NIH3T3 cell lines most, followed by weaker electron-withdrawing group and then the electron-donating group. One reason might be that compounds bearing the lipophilic trifluoromethyl or chloro group could penetrate the cell membrane more easily. And the other could be that the trifluoromethyl group, a donor of hydrogen bond, could enhance the binding with target. Moreover, whether the hydrogen bond donor on C-5 position is NH group or oxygen group, compounds with dimethoxy-substitution at R1 showed more potent activity than compounds with mono-substitution. In addition, when the phenyl ring was substituted by two chlorine or methoxy group, derivatives 3e and 3l, which own NH group, had better inhibitory activity than derivatives 4d and 4j with oxygen group. While the para-substitution at R1 and R2 is methyl group and methoxy group separately, compound 3o had higher anti-fibrotic ability with an IC50 value of 22 μM than compound 4m with an IC50 value of 40 μM.
In summary, in order to have a good knowledge about the effect of the variations at R1, R2, and the hydrogen bond donor on antiproliferative activity, we made comprehensive assessments of the biological results. It is indicated that some of the tested compounds showed excellent inhibitory activity against A549 cell lines than the lead compound 5b. Besides, all of the tested compounds are far more excellent compared with PFD and even better than the lead compound 5b in anti-fibrotic activity. In accordance with the purpose of initial design, some compounds possessed both good anti-lung cancer activity and nice anti-fibrotic ability, such as compounds 3i, 4d, 4h, and 4l. After comparing the selectivity of 1,5-disubstituted-2(1H)-pyridone derivatives, we discovered that of all the derivatives, compound 4l displayed the best potency (IC50 = 20 μM) and selectivity (NIA = 2.750) toward the A549 cell line as well as showing good anti-fibrotic activity (IC50 = 55 μM).
2.3 3D-QSAR
For the sake of further acquiring a systematic structure and activity relationships (SAR) profile of the synthesized compounds and exploring a more powerful and selective anti-lung cancer agent, twenty-eight compounds with definite IC50 values against A549 and NIH3T3 cell lines were selected as the model dataset by means of the MOE 2014 software. By convention, the pIC50 scale (−log IC50), in which higher values indicated exponentially greater potency, was used as a method to measure inhibitory activity. Therefore, 3D-QSAR models were constructed on the pharmacophore-based molecular alignment to reasonably evaluate the designed molecules by using the corresponding pIC50 values which were converted from the obtained IC50 values. The way of this transformation was derived from an online calculator developed from an Indian medicinal chemistry laboratory (http://www.sanjeevslab.org/tools-IC50.htmL). The AutoGPA-based 3D-QSAR models obtained in the present study gave the observed and predicted pIC50 (μM) values along with their corresponding residual values against A549 and NIH3T3, which are presented in Tables 3 and 4, respectively. For a further step, models with good predictive ability were generated with the cross-validation correlation coefficient q2 values for A549 and NIH3T3 being 0.597 and 0.649. Statistically, the squared correlation coefficient R2 values were found to be 0.939 and 0.954. It apparently emerges the quite good agreement between predicted and observed pIC50 values, further highlighting the robustness of the present 3D-QSAR model. Their graphical relationships between predicted and experimental pIC50 values for both A549 and NIH3T3 are illustrated in Fig. 2. Notably, the activities of the prospectively tested compounds turned out to be in line with the predictions. As depicted in the figures, the models not only help to demonstrate the reliability of activity for new 1,5-disubstituted-2(1H)-pyridone derivatives as anti-lung cancer agents but also serve as a useful guide for the design of new inhibitors with better activities.
Compounds
Experimental pIC50a
Predicted pIC50
Residual error
3a
3.7330
3.6265
0.1065
3b
3.8300
3.9338
−0.1038
3c
3.9870
3.8816
0.1054
3d
4.0970
4.1054
−0.0084
3e
4.0660
4.1960
−0.1300
3f
3.8150
3.7499
0.0651
3g
3.9240
3.9213
0.0027
3h
3.3730
3.4965
−0.1235
3i
4.3800
4.4108
−0.0308
3j
3.5770
3.5544
0.0226
3k
3.6600
3.5810
0.0790
3l
3.2460
3.2097
0.0363
3m
3.7880
3.8510
−0.0630
3n
4.0510
3.9516
−0.0994
3o
4.0920
4.2051
−0.1131
4a
3.6000
3.4790
0.1210
4b
3.3950
3.5337
−0.1387
4c
3.5930
3.6910
−0.0980
4d
4.4200
4.2049
0.2151
4e
3.6290
3.6694
−0.0404
4f
3.3040
3.4602
−0.1562
4g
3.4620
3.4314
0.0306
4h
4.4090
4.4552
−0.0462
4i
3.7060
3.7479
−0.0419
4j
3.2960
3.2139
0.0821
4k
3.8390
3.8432
−0.0042
4l
4.6990
4.6665
0.0325
4m
4.3570
4.2572
0.0998
Compounds
Experimental pIC50a
Predicted pIC50
Residual error
3a
5.523
5.4342
0.0888
3b
4.921
4.8381
0.0829
3c
4.886
4.8295
0.0565
3d
4.770
4.8908
−0.1208
3e
4.854
4.9199
−0.0659
3f
4.215
4.2867
−0.0717
3g
4.553
4.5357
0.0173
3h
4.658
4.5263
−0.0764
3i
5.046
4.9250
0.1210
3j
3.870
3.8535
0.0165
3k
3.790
3.8656
−0.0756
3l
4.854
4.8886
0.0565
3m
4.959
5.0951
0.0864
3n
4.292
4.3479
−0.0559
3o
4.658
4.5263
0.1317
4a
4.229
4.3565
−0.1275
4b
4.208
4.1297
0.0783
4c
4.959
4.8726
0.0864
4d
4.745
4.7990
−0.0540
4e
4.347
4.3477
−0.0007
4f
4.409
4.2815
0.1275
4g
4.387
4.3248
0.0622
4h
4.310
4.3515
−0.0415
4i
4.523
4.5767
−0.0537
4j
4.569
4.5487
0.0203
4k
4.699
4.7543
−0.0553
4l
4.260
4.2543
0.0057
4m
4.398
4.3233
0.0747
Correlation plot of experimental versus predicted biological activity values against A549 (A) and NIH3T3 (B) by 3D-QSAR model.
Simultaneously, the molecules aligned with the iso-surfaces of the 3D-QSAR model coefficients on van der Waals grids and electrostatic potential grids are listed in Fig. 3. It needs to point out that negative charge is favoured near red contours and blue contours represent regions of desirable positive charge. Similarly, steric map indicated areas where steric bulk is predicted to increase (green) or decrease (yellow) activity (Dandan et al., 2013). It is widely acceptable that a better inhibitor based on the 3D-QSAR model should have strong Van der Waals attraction in the green areas and a polar group in the blue electrostatic potential areas.
3D-QSAR model coefficients on electrostatic potential grids and van der Waals grids for A549 (A, C) and NIH3T3 (B, D). Red contours represent negative electrostatic coefficients while blue contours represent positive electrostatic coefficients. The green contours indicate high steric tolerance, whereas yellow contours indicate denote regions of unfavorable steric effects at particular region around the ligand.
According to the maps (Fig. 3A), compounds with a high negative charge at para position on aromatic ring would possess higher activity, validating that methoxy group at para position on the aromatic ring is a better choice than methyl substitution and CH3 is worse than H, such as compounds 4i (IC50 = 197 μM), 4a (IC50 = 251 μM), and 4g (IC50 = 345 μM). It is also worthy to note that steric bulk is disfavored at the ortho- or meta-position in the aromatic ring (yellow contour), which complies with the biological results that compounds with methyl group at the ortho-position of N-1 benzyl ring showed lower levels of antiproliferative activity than compounds only with hydrogen at the same position, e.g. 4f (IC50 = 497 μM) and 4a (IC50 = 251 μM). In contrast, the use of bulkier and electronegative group at the para-position of N-1 benzyl ring appeared to be reasonably well tolerated. For instance, compound 4m (R1 = 4-CH3, R2 = 4-OCH3) and compound 3n (R1 = 4-CH3, R2 = 4-F) gave IC50 values of 44 μM and 89 μM, respectively, better than those of their counterpart compound 4g (IC50 = 345 μM) and compound 3f (IC50 = 153 μM). As a result, data summarized above demonstrated that compounds 4l, the most potent inhibitor (IC50 = 20 μM) with suitable substituent possessed an outstanding activity, whose 3D-QSAR model is presently developed as Fig. 3C. Aside from the above discussion, structural interpretation of the 3D-QSAR based on the inhibitory activity against NIH3T3 (Fig. 3B) also suggests there appear to be two major variations contributing to the differences of anti-fibrotic activity: (i) Electronegative substitution is favored at the meta-position of aromatic ring (red contour); (ii) steric bulk in the para-position of N-1 benzyl ring is disfavored (yellow contour). Evidently, meta-trifluoromethyl compound 3i (R1 = 3-CF3, R2 = H) showed the highest inhibitory ability among the compounds containing electronegative substitution at this position, with the order of anti-fibrotic activity being CF3 > Cl > OCH3. Likewise, the 3D-QSAR for the best molecule is shown in Fig. 3 D, confirming that compound 3a with small stereospecific blockade at R2 and electronegative at R1 has strongest inhibitory effect on NIH3T3 cells.
Fig. 4 indicates the best hypothesis with pharmacophoric features shared by all compounds in the dataset. F1 hydrophobic feature in Fig. 4A suggests the critical role in the anti-lung cancer profiles for F1 occupation, further validating the conclusion reported previously that F1 occupation can improve the sensitivity of most compounds to lung cancer cell line, whereas an aromatic (Aro/PiR) π-ring center contoured by orange contour in Fig. 4B illustrated that the benzene ring at N-1 benzyl group plays an important role in anti-fibrotic activity.
The 3D-QSAR pharmacophore model based aligned compounds for A549 (A) and NIH3T3 (B).
According to the results, the promising 3D-QSAR models fitted the inhibitory activity well thus provided us cogent foundation and new ideas about designing and optimizing more effective derivatives against anti-lung cancer agents, which paves the way for us in the further study. Besides, the common pharmacophore features further helped in screening out the potential ligand for anti-lung cancer activity.
3 Conclusions
In our present work, two series of 1,5-disubstituted-2(1H)-pyridone analogues were synthesized and evaluated for their biological activities. Preliminary results revealed that these compounds exhibited tolerated antiproliferative activities against A549 cells and NIH3T3 cells with micromolar potencies. Based on the preliminary results, compounds 3i, 4d, 4h, 4l and 4m displayed comparable or superior antiproliferative activities to those of the lead active compound 22. Most strikingly, compound 4l demonstrated the most selective activity which suppressed the growth of A549 cells with an IC50 value of 20 μM, comparable with the positive control DDP, and exhibited good inhibitory activity against fibroblasts with an IC50 of 55 μM. Importantly, the anti-fibrotic activities of the synthetic compound universally advantage over that of PFD, and out of the tested compounds, compound 3a possessed best anti-fibrotic activity with an IC50 value of 3 μM, 151 times as much as that of PFD. Simultaneously, QSAR models were also built with the activity data to check the previous work as well as to provide a reliable tool for design of anti-lung cancer agents in future. Therefore, it can be concluded that 1,5-disubstituted-2(1H)-pyridone derivatives are promising leads for further study as potential anti-lung cancer agents. The results of this study provided valuably theoretical basis for development of more potential therapeutic drugs possessing stronger anti-lung cancer activities with the maintenance of anti-fibrotic property to fight against lung cancer.
4 Experimental protocols
4.1 Materials and measurements
Unless otherwise indicated, all reagents and solvents used in current study were of analytical grade and were used as obtained from commercial suppliers without further purification. All reactions were monitored by Thin layer chromatography (TLC) on 0.25 mm glass-backed silica gel plates (silica GF-254), and visualized with ultraviolet (UV) light (254 nm). Column chromatography was carried out using glass columns with silica gel (200–300 mesh, Aldrich Chemical). All of the final compounds have purity greater than 95%, which were determined by analytical reverse-phase liquid chromatogram (Instrument model: Shimadzu LC-2010). A Welchrom® C18 (4.6 mm × 250 mm, 5 μm) was used at a temperature of 40 °C. The mobile phase consisted of methanol (60–85%) and water (40–15%). Analysis was conducted over 8.0 min run time at a flow rate of 1.0 mL/min. The Melting points were determined on a XT4MP apparatus (Taike Corp., Beijing, China) and are uncorrected. 1H NMR spectra and 13C NMR were recorded on either Varian INOV-400FT or INOV-500FT spectrophotometer operating at the indicated frequencies in DMSO-d6. Chemical shifts are expressed in parts per million (ppm) relative to an internal standard: tetramethylsilane (ppm = 0.00). Coupling constants are reported in units of hertz (Hz). Peak multiplicity abbreviations are as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), and m (multiplet).
4.2 Synthesis
4.2.1 General procedure for synthesis of 1-substituted benzyl-5-methylpyridin-2(1H)-one (1a–d)
A mixture of 5-methylpyridin-2(1H)-one (0.10 mol), substituted bromomethyl-benzene (0.12 mol), potassium carbonate (0.16 mol), TBAI (0.001 mol) was dissolved in 1,4-dioxane and then stirred under reflux condition for 10 h. After the solution was cooled to the room temperature, the formed precipitate was filtered and washed with 40 mL 1,4-dioxane. The filter liquor was concentrated in vacuo and then the residue was purified by column chromatography (petroleum ether/ethyl acetate = 2:1). Fractions containing product were concentrated in vacuo to afford 1a–d in 70–89% yields.
4.2.1.1 1-Benzyl-5-methylpyridin-2(1H)-one (1a)
Yellow solid, yield: 75%. M.p.: 66.7–67.5 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.69 (s, J = 1.6 Hz, 1H, CH⚌), 7.35–7.42 (d, J = 9.2 Hz, 1H, CH⚌), 7.17–7.20 (m, J = 4.8 Hz, 2H, Ar—H), 7.06–7.10 (m, 1H, Ar—H), 6.84–6.86 (m, J = 8.0 Hz, 2H, Ar—H), 6.39–6.42 (d, J = 9.2 Hz, 1H, CH⚌), 5.06 (s, 2H, —CH2—), 2.03 (s, 3H, —CH3). 13C NMR (100 MHz, DMSO-d6): δ 160.21 (C⚌O), 142.95 (C⚌C), 136.54 (Aromatic carbon), 129.78 (C⚌C), 128.45 (2 × Aromatic carbon), 127.69 (Aromatic carbon), 126.87 (2 × Aromatic carbon), 121.20 (C⚌C), 116.84 (C⚌C), 48.51 (—CH2—), 17.84 (—CH3). MS Calcd. for C13H13NO: 199.1. EI-MS m/z: 199.1 [M]+.
4.2.1.2 1-(2-Fluorobenzyl)-5-methylpyridin-2(1H)-one (1b)
White solid, yield: 70%. M.p.: 89.6–90.4 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.74 (s, 1H, CH⚌), 7.42–7.44 (d, J = 9.2 Hz, 1H, CH⚌), 7.31–7.34 (m, 1H, Ar—H), 7.24–7.28 (m, 1H, Ar—H), 7.06–7.12 (m, 2H, Ar—H), 6.39–6.42 (d, J = 9.2 Hz, 1H, CH⚌), 5.02 (s, 2H, —CH2—), 2.05 (s, 3H, —CH3). 13C NMR (100 MHz, DMSO-d6): δ 161.45 (C⚌O), 159.40, 156.38, 141.83(C⚌C), 137.24, 128.73 (C⚌C), 127.64, 126.95, 123.16, 120.13 (C⚌C), 117.52 (C⚌C), 115.28, 43.67 (—CH2—), 17.79 (—CH3). MS Calcd. for C13H12FNO: 217.1. EI-MS m/z: 217.1 [M]+.
4.2.1.3 1-(4-Methoxybenzyl)-5-methylpyridin-2(1H)-one (1c)
White solid, yield: 89%. M.p.: 85.7–67.6 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.75 (s, 1H, CH⚌), 7.39–7.42 (d, J = 9.2 Hz, 1H, CH⚌), 7.22–7.24 (m, J = 8.4 Hz, 2H, Ar—H), 6.84–6.87 (m, 4H, Ar—H), 6.36–6.39 (m, J = 9.2 Hz, 1H, CH⚌), 4.98 (s, 2H, —CH2—), 3.76 (s, 3H, —OCH3), 2.08 (s, 3H, —CH3). 13C NMR (100 MHz, DMSO-d6): δ 161.23 (C2), 158.94, 140.76 (C⚌C), 136.93, 129.75 (2 × Aromatic carbon), 124.69 (C⚌C), 119.94 (C⚌C), 117.28 (C⚌C), 114.13 (2 × Aromatic carbon), 50.69 (—OCH3), 43.62 (—CH2), 17.87 (—CH3). MS Calcd. for C14H15NO2: 229.1. EI-MS m/z: 229.1 [M]+.
4.2.1.4 1-(4-Fluorobenzyl)-5-methylpyridin-2(1H)-one (1d)
White crystal, yield: 84%. M.p.: 82.6–83.7 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.72 (s, 1H, CH⚌), 7.41–7.44 (d, J = 9.6Hz, 1H, CH⚌), 7.32–7.35 (m, 2H, Ar—H), 7.10–7.14 (m, J = 8.4 Hz, 2H, Ar—H), 6.37–6.41 (m, J = 9.2 Hz, 1H, CH⚌), 5.03 (s, 2H, —CH2—), 2.05 (s, 3H, —CH3). 13C NMR (100 MHz, DMSO-d6): δ 161.65 (C⚌O), 160.91, 158.43, 141.35 (C⚌C), 137.26, 129.75 (2 × Aromatic carbon), 125.07 (C⚌C), 120.16 (C⚌C), 117.89 (C⚌C), 115.36 (2 × Aromatic carbon), 43.72 (—CH2—), 17.63 (—CH3). MS Calcd. for C13H12FNO: 217.1. EI-M S m/z: 217.1 [M]+.
4.2.2 General procedure for synthesis of 1-substituted benzyl-5-(bromomethyl) pyridin-2(1H)-one (2a–d)
To a solution of 1a–d (5.0 mmol) in 75.0 mL carbon tetrachloride (CCl4) was added 0.4 mmol azodiisobutyronitrile (AIBN) followed by 5.0 mmol N-bromosuccinimide (NBS). The mixture was heated at reflux under the irradiation of 100 W light (Brand name: High Bay Light; Model: HL14600) for 2 h. After completion of the reaction, the solution was cooled to the room temperature and then the resulting solid was filtered. The filtrate solvent was evaporated under reduced pressure to obtain crude 1-substituted benzyl-5-(bromomethyl) pyridine-2(1H)-one (2a–d). Given the instability during isolation process, the crude product 2a–d was directly applied to the next step without further purification.
4.2.3 General procedure for synthesis of 1-substituted benzyl-5-substituted ((phenylamino)methyl)pyridin-2(1H)-one (3a–o)
To the above obtained crude product 2a–d (4.0 mmol) in 100 mL round-bottom flask, 35.0 mL acetonitrile and substituted aniline (4.0 mmol) were added. The reaction solution was allowed to stir at room temperature for 3 h, and monitored by TLC. After the reaction was completed, the precipitate was filtered off and washed with 5.0 mL of acetonitrile. The combined organic layers were then concentrated in vacuo. The residue was subjected to silica gel chromatography using 30% ethyl acetate in petroleum ether to afford the compounds 3a–o. The overall yields were 25–57% calculated with 2a–d as the initial materials.
4.2.4 General procedure for synthesis of 1-substituted benzyl-5-substituted (phenoxymethyl)pyridin-2(1H)-one (4a–m)
To a solution of sodium hydroxide (0.102 mol) in 20.0 mL anhydrous ethanol was added a phenol derivative (0.1 mol), and then the mixture was stirred for 0.5 h at 60 °C. The reaction solution was removed under reduced pressure after it cooled down. Upon concentration, the resulting mixture was dried in vacuum at 40 °C for 1 h to furnish sodium phenolate (0.1 mol). Subsequently, to a stirred solution of the crude 1-substituted benzyl-5-(bromomethyl)pyridin-2(1H)-one (2a–d) (0.10 mol) in 35.0 mL acetonitrile, sodium phenolate (0.1 mol) was added and the stirring was maintained at room temperature for 3 h (monitored by TLC). Filtrations and column chromatography (50% ethyl acetate in petroleum ether) were performed to provide the desired compounds (4a–m). The overall yields were 27–87% calculated with 2a–d as the initial materials.
4.3 Spectral properties of 1,5-disubstituted-2(1H)-pyridone derivatives
4.3.1 1-Benzyl-5-(((2-fluorophenyl)amino)methyl)pyridin-2(1H)-one (3a)
Light yellow oil, yield: 39%. 1H NMR (400 MHz, DMSO-d6) : δ 7.83–7.84 (s, J = 1.6 Hz, 1H, CH⚌), 7.48–7.51 (m, J = 9.2 Hz, 1H, CH⚌), 7.23–7.35 (m, 6H, Ar—H), 6.97–7.02 (t, J = 7.6 Hz, 1H, Ar—H), 6.83–6.90 (m, J = 7.2 Hz, 1H, Ar—H), 6.69–6.73 (t, J = 8.0 Hz, 1H, Ar—H), 6.51–6.56 (m, 1H, CH⚌), 6.40–6.42 (t, J = 5.2 Hz, 1H, —NH—), 5.06 (s, 2H, —CH2—), 4.05–4.06 (d, J = 6.0 Hz, 2H, —CH2—). 13C NMR (100 MHz, DMSO-d6) : δ 166.26 (C2), 154.92 (C4′), 157.43 (C4′), 145.77 (C4), 142.45 (C2″), 142.16 (C3′), 133.67 (C6), 132.71 (C4″, 6″), 132.65 (C3″, 7″), 129.74 (C5″), 125.01 (C7′), 121.91 (C5), 121.04 (C6′), 119.61 (C3), 119.43 (C4′), 117.69 (C8′), 56.34 (C1′), 47.51 (C1″). MS Calcd. for C19H17FN2O: 308.1. EI-MS m/z: 308.2 [M]+.
4.3.2 1-Benzyl-5-(((4-fluorophenyl)amino)methyl)pyridin-2(1H)-one (3b)
White powder, yield: 41%. M.p.: 105.3–106.2 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.79 (s, 1H, CH⚌), 7.43–7.46 (m, J = 4.2 Hz, 1H, CH⚌), 7.24–7.37 (m, 5H, Ar—H), 6.87–6.91 (m, J = 4.8 Hz, 2H, Ar—H), 6.56–6.59 (m, 2H, ArH), 6.41–6.43 (d, J = 5.6 Hz, 1H, CH⚌), 5.98–6.01 (t, J = 7.0 Hz, 1H, —NH—), 5.06 (s, 2H, —CH2—), 3.93–3.95 (d, J = 6.4 Hz, 2H, —CH2—). 13C NMR (100 MHz, DMSO-d6): δ 161.31 (C2), 155.89 (C6′), 153.59 (C6′), 145.26 (C4), 141.03 (C3′), 137.72 (C2″), 137.29 (C6), 128.79 (C4″,6″), 127.94 (C3″, 7″), 127.78 (C5″), 120.15 (C5), 116.97 (C3), 115.38 (C4′, 8′), 113.60 (C5′,7′), 51.42 (C1′), 43.92 (C1″). MS Calcd. for C19H17FN2O: 308.1. EI-MS m/z: 308.2 [M]+.
4.3.3 1-Benzyl-5-(((3-chlorophenyl)amino)methyl)pyridin-2(1H)-one (3c)
White powder, yield: 49%. M.p.: 129.1–130.1 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.81 (s, J = 2.0 Hz, 1H, CH⚌), 7.41–7.44 (m, J = 9.2 Hz, 1H, CH⚌), 7.25–7.33 (m, 5H, Ar—H), 7.02–7.06 (m, J = 8.0 Hz, 1H, Ar—H), 6.59–6.60 (m, 1H, Ar—H), 6.52–6.55 (m, J = 8.0 Hz, 2H, Ar—H), 6.43 (m, 1H, CH⚌), 6.38 (m, 1H, —NH—), 5.05 (s, 2H, —CH2—), 3.97–3.99 (d, J = 5.6 Hz, 2H, —CH2—). 13C NMR (100 MHz, DMSO-d6): δ 151.26 (C2), 140.04 (C3′), 130.87 (C4), 127.66 (C2″), 127.33 (C7′), 123.88 (C6), 120.59 (C5), 118.79 (C4″, 6″), 117.91 (C3″, 7″), 117.75 (C5″), 110.24 (C6′), 106.52 (C3), 105.59 (C5′), 101.48 (C4′), 99.62 (C8′), 41.46 (C1′), 33.11 (C1″). MS Calcd. for C19H17ClN2O: 324.1. EI-MS m/z: 324.2 [M]+.
4.3.4 1-Benzyl-5-(((4-chlorophenyl)amino)methyl)pyridin-2(1H)-one (3d)
Light yellow powder, yield: 45%. M.p.: 123.9–125.0 °C. 1H NMR (500 MHz, DMSO-d6): δ 7.77–7.78 (s, J = 2 Hz, 1H, CH⚌), 7.42–7.44 (m, J = 7.2 Hz, 1H, CH⚌), 7.24–7.33 (m, 5H, Ar—H), 7.05–7.07 (m, J = 7.2 Hz, 2H, Ar—H), 6.58–6.60 (m, J = 7.2 Hz, 2H, Ar—H), 6.41–6.43 (m, J = 7.2 Hz, 1H, CH⚌), 6.24–6.25 (m, J = 4.4 Hz, 1H, —NH—), 5.06 (s, 2H, —CH2—), 3.95–3.97 (d, J = 4.8 Hz, 2H, —CH2—). 13C NMR (125 MHz, DMSO-d6): δ 160.94 (C2), 147.13 (C3′), 140.61 (C4), 137.36 (C2″), 136.99 (C6), 128.47 (C5′, 7′), 128.45 (C4″, 6″), 127.60 (C3″, 7″), 127.43 (C5″), 119.85 (C5), 116.32 (C3), 113.84 (C4′, 8′), 51.08 (C1′), 43.06 (C1″). MS Calcd. for C19H17ClN2O: 324.1. EI-MS m/z: 324.1 [M]+.
4.3.5 1-Benzyl-5-(((2,4-dichlorophenyl)amino)methyl)pyridin-2(1H)-one (3e)
Light yellow crystal, yield: 52%. M.p.: 101.9–102.7 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.77–7.78 (s, J = 2.0 Hz, 1H, CH⚌), 7.45–7.48 (m, J = 9.2 Hz, 1H, CH⚌), 7.28–7.35 (m, 4H, Ar—H), 7.22–7.24 (m, 2H, Ar—H), 7.07–7.10 (m, J = 8.8 Hz, 1H, Ar—H), 6.67–6.69 (m, J = 9.4 Hz, 1H, Ar—H), 6.40–6.42 (m, J = 9.2 Hz, 1H, CH⚌), 6.17–6.20 (m, J = 6.0 Hz, 1H, —NH—), 5.05 (s, 2H, —CH2—), 4.11–4.13 (d, J = 6.0 Hz, 2H, —CH2—). 13C NMR (100 MHz, DMSO-d6): δ 161.78 (C2), 143.05 (C4), 141.03 (C3′), 137.73 (C2″), 137.50 (C5′), 129.11 (C6), 128.86 (C4″, 6″), 128.17 (C3″, 7″), 128.14 (C7′), 120.54 (C5″), 119.97 (C6′, C5), 119.19 (C4′), 116.87 (C3), 113.45 (C8′), 51.81 (C1′), 42.99 (C1″). MS Calcd. for C19H16Cl2N2O: 358.1. EI-MS m/z: 358.1 [M]+.
4.3.6 1-Benzyl-5-((p-tolylamino)methyl)pyridin-2(1H)-one (3f)
White crystal, yield: 38%. M.p.: 120.5–121.0 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.77–7.78 (s, J = 2.0 Hz, 1H, CH⚌), 7.43–7.46 (m, J = 9.2 Hz, 1H, CH⚌), 7.24–7.33 (m, 5H, Ar—H), 6.85–6.87 (m, J = 8.4 Hz, 2H, Ar—H), 6.49–6.51 (m, J = 8.4 Hz, 2H, Ar—H), 6.39–6.42 (d, J = 9.2 Hz, 1H, CH⚌), 5.81–5.84 (m, J = 6.0 Hz, 1H, —NH—), 5.05 (s, 2H, —CH2—), 3.93–3.94 (d, J = 5.6 Hz, 2H, —CH2—), 2.13 (s, 3H, —CH3). 13C NMR (100 MHz, DMSO-d6): δ 161.29 (C2), 146.29 (C3′), 140.99 (C4), 137.74 (C2″), 137.19 (C6′), 129.56 (C5′,7′), 128.78 (C4″, 6″), 127.92 (C3″, 7″), 127.75 (C6), 124.72 (C5″), 120.06 (C5), 117.29 (C3), 112.99 (C4″, 8″), 51.42 (C1′), 43.67 (C1″), 20.40 (—CH3). MS Calcd. for C20H20N2O: 304.2. EI-MS m/z: 304.2 [M]+.
4.3.7 1-Benzyl-5-(((2,4-dimethylphenyl)amino)methyl)pyridin-2(1H)-one (3g)
White powder, yield: 47%. M.p.: 154.5–156.1 °C. 1H NMR (400 MHz, DMSO-d6) : δ 7.75 (s, 1H, CH⚌), 7.46–7.49 (m, J = 9.6 Hz, 1H, CH⚌), 7.23–7.33 (m, 5H, Ar—H), 6.72–6.77 (m, 2H, Ar—H), 6.40–6.41 (m, 1H, Ar—H), 6.38–6.39 (m, 1H, CH⚌), 5.21 (s, 1H, —NH—), 5.05 (s, 2H, —CH2—), 4.02–4.04 (d, J = 6.0 Hz, 2H, —CH2—), 2.12 (s, 3H, —CH3), 2.06 (s, 3H, —CH3). 13C NMR (100 MHz, DMSO-d6): δ 161.62 (C2), 143.76 (C3′), 141.06 (C4), 137.78 (C2″), 137.24 (C6′), 131.12 (C5′), 128.96 (C4″, 6″), 128.05 (C3″, 7″), 127.23(C6), 124.91 (C7″), 122.67 (C5″), 120.15 (C5), 117.90 (C3), 110.63 (C8′), 51.67 (C1′), 43.55 (C1″), 20.49 (—CH3), 18.09 (—CH3). MS Calcd. for C21H22N2O: 318.2. EI-MS m/z: 318.2 [M]+.
4.3.8 1-Benzyl-5-(((3,4-dimethylphenyl)amino)methyl)pyridin-2(1H)-one (3h)
Light yellow solid, yield: 45%. M.p.: 112.4–114.3 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.77 (s, 1H, CH⚌), 7.42–7.44 (m, J = 9.6 Hz, CH⚌), 7.24–7.31 (m, 5H, Ar—H), 6.78–6.80 (m, J = 8 Hz, 1H, Ar—H), 6.38–6.41 (m, 2H, Ar—H), 6.30–6.32 (m, J = 8 Hz, 1H, CH⚌), 5.73–5.76 (m, J = 5.6 Hz, 1H, —NH—), 5.04 (s, 2H, —CH2—), 3.92–3.93 (d, J = 5.6 Hz, 2H), 2.06 (s, 3H, CH3), 2.04 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): δ 161.41 (C2), 146.57 (C3′), 141.03 (C4), 137.74 (C2″), 137.14 (C5′), 136.52 (C7′), 130.15 (C6), 128.83 (C4″, 6″), 127.95 (C3″, 7″), 127.81 (C5″), 123.77 (C6′), 120.09 (C5), 117.59 (C3), 114.65 (C4′), 110.53 (C8′), 51.53 (C1′), 43.58 (C1″), 20.09 (—CH3), 18.72 (—CH3). MS Calcd. for C21H22N2O: 318.2. EI-MS m/z: 318.2 [M]+.
4.3.9 1-Benzyl-5-(((3-(trifluoromethyl)phenyl)amino)methyl)pyridin-2(1H)-one (3i)
Light yellow powder, yield: 51%. M.p.: 108.5–110.6 °C. 1H NMR (500 MHz, DMSO-d6): δ 7.82 (s, 1H, CH⚌), 7.44–7.46 (m, J = 7.1 Hz, 1H, CH⚌), 7.24–7.32 (m, 6H, Ar—H), 6.82–6.84 (m, J = 7.2 Hz, 3H, Ar—H), 6.55–6.57 (m, J = 4.4 Hz, 1H, —NH—), 6.42–6.44 (m, J = 7.6 Hz, 1H, CH⚌), 5.06 (s, 2H, —CH2—), 4.03–4.04 (d, J = 4.4 Hz, 2H, —CH2—). 13C NMR (125 MHz, DMSO-d6): δ 161.45 (C2), 149.19 (C3′), 141.05 (C4), 137.82 (C2″), 137.52 (C5′), 130.33, 130.25 (C7′), 128.94 (C6, 4″, 6″), 128.04 (C3″, 5″, 7″), 127.92 (—CF3), 120.45 (C5), 116.57 (C3), 116.26 (C8′), 112.39 (C6′), 108.70 (C4′), 51.65 (C1′), 43.29 (C1″). MS Calcd. for C20H17F3N2O: 358.1. EI-MS m/z: 358.1 [M]+.
4.3.10 1-Benzyl-5-(((2-methoxyphenyl)amino)methyl)pyridin-2(1H)-one (3j)
Light yellow oil, yield: 57%. 1H NMR (500 MHz, DMSO-d6): δ 7.76 (s, 1H, CH⚌), 7.45–7.47 (m, J = 8.8 Hz, 1H, CH⚌), 7.23–7.30 (m, 5H, Ar—H), 6.77–6.79 (m, J = 7.6 Hz, 1H, Ar—H), 6.68–6.72 (m, J = 7.2 Hz, 1H, Ar—H), 6.52–6.56 (m, 2H, Ar—H), 6.38–6.40 (m, J = 8.8 Hz, 1H, CH⚌), 5.37 (s, 1H, —NH—), 5.05 (s, 2H, —CH2—), 4.03 (d, 2H, —CH2—), 3.75 (s, 3H, —CH3). 13C NMR (125 MHz, DMSO-d6): δ 161.75 (C2), 147.09 (C4′), 141.32 (C4), 137.85 (C3′), 137.72 (C2″), 137.49 (C5), 129.11 (C6, 4″, 6″), 128.13 (C3″, 5″, 7″), 121.42 (C7′), 120.35 (C6′), 117.89 (C3), 116.59 (C8′), 110.34 (C5′), 55.79 (C1′), 51.78 (—OCH3), 43.21 (C1″). MS Calcd. for C20H20N2O2: 320.2. EI-MS m/z: 320.2 [M]+.
4.3.11 1-Benzyl-5-(((3-methoxyphenyl)amino)methyl)pyridin-2(1H)-one (3k)
White powder, yield: 38%. M.p.: 151.3–152.9 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.79–7.80 (s, J = 2.4 Hz, 1H, CH⚌), 7.42–7.45 (m, J = 9.2 Hz, 1H, CH⚌), 7.24–7.33 (m, 5H, Ar—H), 6.91–6.95 (m, J = 8.0 Hz, 1H, Ar—H), 6.40–6.43 (m, J = 9.6 Hz, 1H, Ar—H), 6.17–6.20 (m, J = 9.2 Hz, 1H, CH⚌), 6.09–6.13 (m, 2H, Ar—H), 6.05–6.07 (m, 1H, —NH—), 5.05 (s, 2H, —CH2—), 3.95–3.96 (d, J = 6.0 Hz, 2H, —CH2—), 3.62 (s, 3H, —OCH3). 13C NMR (100 MHz, DMSO-d6): δ 161.31 (C2), 160.57 (C5′), 149.95 (C3′), 140.99 (C4), 137.75 (C2″), 137.26 (C6), 129.85 (C5), 128.81 (C4″, 6″), 127.94(C3″, 7″), 127.78 (C5″), 120.12 (C3), 117.17 (C7′), 105.89 (C6′), 101.91 (C8′), 98.40 (C4′), 54.86 (C1′), 51.46 (—OCH3), 43.39 (C1″). MS Calcd. for C20H20N2O2: 320.2. EI-MS m/z: 320.2 [M]+.
4.3.12 1-Benzyl-5-(((3,4-dimethoxyphenyl)amino)methyl)pyridin-2(1H)-one (3l)
Gray green crystal, yield: 43%. M.p.: 128.1–128.5 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.75 (s, 1H, CH⚌), 7.43–7.46 (m, J = 9.2 Hz, 1H, CH⚌), 7.24–7.32 (m, 5H, Ar—H), 6.66–6.68 (d, J = 8.8 Hz, 1H, Ar—H), 6.39–6.42 (m, J = 9.2 Hz, 1H, Ar—H), 6.27–6.28 (m, J = 1.6 Hz, 1H, Ar—H), 6.06–6.08 (m, J = 8.4 Hz, 1H, CH⚌), 5.63 (s, 1H,—NH—), 5.05 (s, 2H, —CH2—), 3.92–3.94 (d, J = 6.0 Hz, 2H, —CH2—), 3.62 (s, 3H, —OCH3), 3.60 (s, 3H, —OCH3). 13C NMR (100 MHz, DMSO-d6): δ 161.31 (C2), 150.10 (C5′), 143.67 (C4), 140.99 (C3′), 137.72 (C6′), 137.10 (C2″), 128.78 (C6, 4″, 6″), 127.89 (C3″, 7″), 127.75 (C5″), 120.06 (C5), 117.42 (C3), 114.57 (C7′), 103.57 (C8′), 99.29 (C4′), 56.84 (C1′), 55.45 (—OCH3), 51.41 (—OCH3), 44.10 (C1″). MS Calcd. for C21H22N2O3: 350.2. EI-MS m/z: 350.2 [M]+.
4.3.13 1-(2-Fluorobenzyl)-5-((p-tolylamino)methyl)pyridin-2(1H)-one (3m)
White solid, yield: 37%. M.p.: 124.7–125.9 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.77 (s, 1H, CH⚌), 7.42–7.44 (m, J = 9.2 Hz, 1H, CH⚌), 7.31–7.34 (m, 2H, Ar—H), 7.11–7.16 (m, 2H, Ar—H), 6.84–6.86 (m, J = 8.0 Hz, 2H, Ar—H), 6.48–6.50 (m, J = 8.0 Hz, 2H, Ar—H), 6.38–6.41 (m, J = 9.2 Hz, 1H, CH⚌), 5.79 (s, 1H, —NH—), 5.02 (s, 2H, —CH2—), 3.92–3.94 (m, J = 5.6 Hz, 2H, —CH2—), 2.13 (s, 3H, —CH3). 13C NMR (100 MHz, DMSO-d6): δ 161.33 (C2), 159.60 (C3″), 146.22 (C3′), 141.23 (C4), 137.26 (C2″), 129.96 (C7″), 129.88 (C5′, 7′), 129.59 (C6′), 124.84 (C6), 124.75 (C5″), 120.04 (C6″), 117.45 (C5), 115.75 (C3), 115.54 (C4″), 113.05 (C4′, 8′), 45.97 (C1′), 43.63 (C1″), 20.42 (—CH3). MS Calcd. for C20H19FN2O: 322.2. EI-MS m/z: 322.2 [M]+.
4.3.14 1-(4-Fluorobenzyl)-5-((p-tolylamino)methyl)pyridin-2(1H)-one (3n)
Light yellow crystal, yield: 33%. M.p.: 103.3–104.7 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.77 (s, 1H, CH⚌), 7.42–7.44 (m, J = 9.6 Hz, 1H, CH⚌), 7.31–7.34 (m, 2H, Ar—H), 7.11–7.16 (m, J = 8.4 Hz, 2H, Ar—H), 6.84–6.86 (m, J = 8.0 Hz, 2H, Ar—H), 6.48–6.50 (m, J = 8.0 Hz, 2H, Ar—H), 6.38–6.41 (m, J = 9.2 Hz, 1H, CH⚌), 5.79 (s, 1H, —NH—), 5.02 (s, 2H, —CH2—), 3.92–3.94 (m, J = 9.6 Hz, 2H, —CH2—), 2.13 (s, 3H, —CH3). 13C NMR (100 MHz, DMSO-d6): δ 163.29 (C5″), 161.67 (C2), 160.86 (C5″), 146.34 (C3′), 141.45 (C4), 137.28 (C2″), 134.08 (C6′), 130.52 (C5′, 7′), 129.85 (C3″, 7″), 125.15 (C6), 120.26 (C5), 117.92 (C3), 115.90 (C4″, 6″), 113.32 (C4′, 8′), 51.08 (C1′), 43.74 (C1″), 20.63 (—CH3). MS Calcd. for C20H19FN2O: 322.2. EI-MS m/z: 322.2 [M]+.
4.3.15 1-(4-Methoxybenzyl)-5-((p-tolylamino)methyl)pyridin-2(1H)-one (3o)
Light yellow crystal, yield: 25%. M.p.: 122.1–122.8 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.75 (s, 1H, CH⚌), 7.39–7.42 (m, J = 9.2 Hz, 1H, CH⚌), 7.22–7.24 (m, J = 8.4 Hz, 2H, Ar—H), 6.84–6.87 (m, J = 8.4 Hz, 4H, Ar—H), 6.48–6.50 (m, J = 8.0 Hz, 2H, Ar—H), 6.36–6.39 (m, J = 9.2 Hz, 1H, CH⚌), 5.78–5.81 (m, J = 6.0 Hz, 1H, —NH—), 4.96 (s, 2H, —CH2—), 3.91–3.92 (m, J = 5.6 Hz, 2H, —CH2—), 3.72 (s, 3H, —OCH3), 2.13 (s, 3H, —CH3). 13C NMR (100 MHz, DMSO-d6): δ 161.23 (C2), 158.97 (C5″), 146.28 (C3′), 140.82 (C4), 136.96 (C2″), 129.72 (C3″, 7″), 129.55 (C5′, 7′), 124.69 (C6), 119.99 (C5), 117.18 (C3), 114.13 (C4″, 6″), 112.98 (C4′, 8′), 55.37 (C1′), 50.79 (—OCH3), 43.66 (C1″), 20.38 (—CH3). MS Calcd. for C21H22N2O2: 334.1. EI-MS m/z: 334.1 [M]+.
4.3.16 1-Benzyl-5-(phenoxymethyl)pyridin-2(1H)-one (4a)
White crystal, yield: 46%. M.p.: 101.2–103.2 °C. 1H NMR (500 MHz, DMSO-d6): δ 7.95–7.97 (s, J = 8 Hz, 1H, CH⚌), 7.50–7.53 (m, J = 8 Hz, 1H, CH⚌), 7.27–7.33 (m, 7H, Ar—H), 6.92–6.99 (m, 3H, Ar—H), 6.44–6.47 (m, J = 8 Hz, 1H, CH⚌), 5.08–5.09 (m, J = 3 Hz, 2H, —CH2—), 4.79–4.81 (m, J = 4 Hz, 2H, —CH2—). 13C NMR (125 MHz, DMSO-d6): δ 161.51 (C2), 158.55 (C3′), 141.49 (C4), 139.32 (C2″), 137.76 (C6), 129.94 (C5′, 7′), 129.02 (C4″, 6″), 128.14 (C3″, 7″), 128.00 (C5″), 121.31 (C5), 120.37 (C6′), 115.35 (C4′, 8′), 114.76 (C3), 66.75 (C1′), 51.61 (C1″). MS Calcd. for C19H17NO2: 291.2. EI-MS m/z: 291.2 [M]+.
4.3.17 1-Benzyl-5-((2-fluorophenoxy)methyl)pyridin-2(1H)-one (4b)
White crystal, yield: 48%. M.p.: 103.1–104.3 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.97–7.98 (s, J = 1.6 Hz, 1H, CH⚌), 7.52–7.55 (m, J = 9.2 Hz, 1H, CH⚌), 7.10–7.35 (m, 8H, Ar—H), 6.95–6.98 (m, 1H, Ar—H), 6.46–6.48 (m, J = 9.2 Hz, 1H, CH⚌), 5.09 (s, 2H, —CH2—), 4.89 (s, 2H, —CH2—). 13C NMR (100 MHz, DMSO-d6): δ 161.68 (C2), 153.69 (C4′), 151.27 (C4′), 146.28 (C3′), 141.68 (C4), 139.71 (C2″), 137.59 (C6), 129.08 (C4″, 6″), 128.10 (C3″, 7″), 125.26 (C5″), 122.10 (C5), 122.03 (C7′), 120.44 (C6′), 116.64 (C3), 116.47 (C5′), 114.49 (C8′), 68.02 (C1′), 51.72 (C1″). MS Calcd. for C19H16FNO2: 309.2. EI-MS m/z: 309.2 [M]+.
4.3.18 1-Benzyl-5-((2-chlorophenoxy)methyl)pyridin-2(1H)-one (4c)
White crystal, yield: 87%. M.p.: 118.0–119.1 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.98 (s, 1H, CH⚌), 7.52–7.55 (m, J = 9.2 Hz, 1H, CH⚌), 7.41–7.43 (m, J = 8 Hz, 1H, Ar—H), 7.22–7.35 (m, 7H, Ar—H), 6.95–6.99 (m, J = 7.6 Hz, 1H, Ar—H), 6.48–6.50 (d, J = 9.2 Hz, 1H), 5.10 (s, 2H, —CH2—), 4.91 (s, 2H, —CH2—). 13C NMR (100 MHz, DMSO-d6): δ 161.34 (C2), 153.66 (C3′), 141.17 (C4), 139.30 (C2″), 137.51 (C5′), 130.29 (C6), 128.88 (C4″, 6″), 128.55 (C7′), 128.02 (C3″, 7″), 127.89 (C5″), 122.22 (C5), 122.08 (C4′), 120.29 (C6′), 115.09 (C3), 114.08 (C8′), 67.72 (C1′), 51.44 (C1″). MS Calcd. for C19H16ClNO2: 325.1. EI-MS m/z: 325.1 [M]+.
4.3.19 1-Benzyl-5-((2,4-dichlorophenoxy)methyl)pyridin-2(1H)-one (4d)
White crystal, yield: 39%. M.p.: 143.5–144.4 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.97 (s, 1H, CH⚌), 7.57 (m, 1H, Ar—H), 7.52–7.54 (m, J = 9.2 Hz, 1H, CH⚌), 7.26–7.36 (m, 7H, Ar—H), 6.47–6.49 (d, J = 9.2 Hz, 1H, CH⚌), 5.09 (s, 2H, —CH2—), 4.92 (s, 2H, —CH2—). 13C NMR (100 MHz, DMSO-d6): δ 161.3 (C2), 152.78 (C3′), 141.22 (C4), 139.50 (C2″), 137.49 (C6), 129.67 (C5′), 128.90 (C4″, 6″), 128.36 (C7′), 128.04 (C3″, 7″), 127.93 (C5″), 125.23 (C5), 123.19 (C4′), 120.35 (C6′), 116.39 (C3), 113.79 (C8′), 68.19 (C1′), 51.46 (C1″). MS Calcd. for C19H15Cl2NO2: 359.1. EI-MS m/z: 359.1 [M]+.
4.3.20 1-Benzyl-5-((3,4-dichlorophenoxy)methyl)pyridin-2(1H)-one (4e)
White crystal, yield: 30%. M.p.: 92.5–93.5 °C. 1H NMR (400 MHz DMSO-d6): δ 7.98–7.99 (s, J = 2.4 Hz, 1H, CH⚌), 7.50–7.53 (m, J = 9.6 Hz, 2H, CH⚌), 7.27–7.35 (m, 6H, Ar—H), 6.99–7.03 (m, J = 9.2 Hz, 1H, Ar—H), 6.45–6.48 (d, J = 9.6 Hz, 1H, CH⚌), 5.09 (s, 2H, —CH2—), 4.86 (s, 2H, —CH2—). 13C NMR (125 MHz, DMSO-d6): δ 161.27 (C2), 157.79 (C3′), 141.23 (C4), 139.45 (C2′), 137.47 (C5′), 131.86 (C7′), 131.21 (C6), 128.84 (C4″,6″), 127.94 (C3″, 7″), 127.84 (C5″), 122.98 (C6′), 120.24 (C5), 117.14 (C3), 116.26 (C4′), 113.81 (C8′), 67.45 (C1′), 51.43 (C1′). MS Calcd. for C19H15Cl2NO2: 359.1. EI-MS m/z: 359.1 [M]+.
4.3.21 1-Benzyl-5-((o-tolyloxy)methyl)pyridin-2(1H)-one (4f)
White crystal, yield: 51%. M.p.: 97.3–99.1 °C. 1H NMR (500 MHz DMSO-d6): δ 7.94 (s, 1H, CH⚌), 7.53–7.55 (m, J = 9 Hz, 1H, CH⚌), 7.27–7.35 (m, 5H, Ar—H), 7.12–7.15 (m, J = 7.5 Hz, 2H, Ar—H), 6.99–7.01 (m, J = 8 Hz, 1H, Ar—H), 6.83–6.86 (m, J = 7.5 Hz, 1H, Ar—H), 6.46–6.48 (m, J = 8 Hz, 1H, CH⚌), 5.09 (s, 2H, —CH2—), 4.82 (s, 2H, —CH2—), 2.11 (s, 3H, —CH3). 13C NMR (125 MHz, DMSO-d6): δ 161.52 (C2), 156.57 (C3′), 141.19 (C4′), 138.88 (C2″), 137.75 (C5′), 130.95 (C6), 129.03 (C4″, 6″), 128.19 (C3″, 7″), 128.03 (C5″), 127.34 (C7′), 126.61 (C4′), 121.07 (C5), 120.42 (C6′), 115.04 (C3), 112.64 (C8′), 66.94 (C1′), 51.59 (C1′), 16.52 (CH3). MS Calcd. for C20H19NO2: 305.1. EI-MS m/z: 305.1 [M]+.
4.3.22 1-Benzyl-5-((p-tolyloxy)methyl)pyridin-2(1H)-one (4g)
White crystal, yield: 52%. M.p.: 113.9–114.4 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.95–7.96 (d, J = 2 Hz, 1H, CH⚌), 7.49–7.52 (m, J = 9.2 Hz, 1H, CH⚌), 7.26–7.33 (m, 5H, ArH), 7.06–7.08 (m, J = 8.8 Hz, 2H, Ar—H), 6.85–6.87 (m, J = 8.4 Hz, 2H, Ar—H), 6.44–6.46 (m, J = 9.2 Hz, 1H, CH⚌), 5.09 (s, 2H, —CH2—), 4.77 (s, 2H, —CH2—), 2.23(s, 3H, —CH3). 13C NMR (125 MHz, DMSO-d6): δ 161.29 (C2), 156.20 (C3′), 141.26 (C4), 139.03 (C2″), 137.56 (C6′), 130.06 (C5′, 7′), 129.78 (C6), 128.81 (C4″, 6″), 127.93 (C3″, 7″), 127.79 (C5″), 120.14 (C5), 115.07 (C4′, 8′), 114.68 (C3), 66.53 (C1′), 51.38 (C1′), 20.35 (—CH3). MS Calcd. for C20H19NO2: 305.1. EI-MS m/z: 305.2 [M]+.
4.3.23 1-Benzyl-5-((2-methoxyphenoxy)methyl)pyridin-2(1H)-one (4h)
Light yellow crystal, yield: 69%. M.p.: 101.0–102.0 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.78–7.79 (s, J = 2.0 Hz, 1H, CH⚌), 7.45–7.48 (m, J = 9.2 Hz, 1H, CH⚌), 7.22–7.33 (m, 5H, Ar—H), 6.77–6.79 (m, J = 8.0 Hz, 1H, Ar—H), 6.68–6.72 (m, J = 7.6 Hz, 1H, Ar—H), 6.51–6.55 (m, J = 8.0 Hz, 2H, Ar—H), 6.38–6.40 (d, J = 8.8 Hz, 1H, CH⚌), 5.05 (s, 2H, —CH2—), 4.02–4.03 (m, J = 4.8 Hz, 2H, —CH2—), 3.75 (s, 3H, —CH3). 13C NMR (100 MHz, DMSO-d6): δ 161.27 (C2), 146.82 (C4′), 140.84 (C3′), 137.72 (C4), 137.61 (C2″), 137.19 (C6), 128.78 (C4″,6″), 127.86 (C3″, 5″, 7″), 127.71 (C5), 121.14 (C7′), 120.12 (C6′), 117.26 (C3), 116.19 (C8′), 110.09 (C5′), 55.57 (C1′), 51.37 (—OCH3), 43.11 (C1′). MS Calcd. for C20H19NO3: 321.2. EI-MS m/z: 321.2 [M]+.
4.3.24 1-Benzyl-5-((4-methoxyphenoxy)methyl)pyridin-2(1H)-one (4i)
Light yellow crystal, yield: 49%. M.p.: 100.9–101.9 °C. 1H NMR (500 MHz, DMSO-d6): δ 7.93–7.94 (s, J = 2 Hz, 1H, CH⚌), 7.50–7.52 (m, J = 9.5 Hz, 1H, CH⚌), 7.29–7.34 (m, 2H, Ar—H), 7.25–7.29 (m, 3H, Ar—H), 6.89–6.92 (m, 2H, Ar—H), 6.83–6.85 (m, 2H, Ar—H), 6.44–6.46 (m, J = 4.5 Hz, 1H, CH⚌), 5.08 (s, 2H, —CH2—), 4.74 (s, 2H, —CH2—), 3.61 (s, 3H, —CH3). 13C NMR (125 MHz, DMSO-d6): δ 161.49 (C2), 154.05 (C6′), 152.44 (C3′), 141.52 (C4), 139.24 (C2″), 137.77 (C6), 129.02 (C4″, 6″), 128.11 (C3″, 5″, 7″), 128.00 (C5), 120.33 (C3), 118.24 (C5′, 7′), 116.49 (C4′, 8′), 67.40 (C1′), 55.79 (—OCH3), 51.58 (C1′). MS Calcd. for C20H19NO3: 321.1. EI-MS m/z: 321.1 [M]+.
4.3.25 1-Benzyl-5-((3,4-dimethoxyphenoxy)methyl)pyridin-2(1H)-one (4j)
Light yellow crystal, yield: 39%. M.p.: 124.7–125.2 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.93 (s, 1H, CH⚌), 7.49–7.52 (m, J = 9.6 Hz, 1H, CH⚌), 7.26–7.33 (m, 5H, Ar—H), 6.82–6.84 (m, J = 8.8 Hz, 1H, Ar—H), 6.60–6.61 (m, J = 1.6 Hz, 1H, CH⚌), 6.45–6.50 (m, 2H, Ar—H), 5.09 (s, 2H, —CH2—), 4.75 (s, 2H, —CH2—), 3.70 (s, 3H, —OCH3), 3.68 (s, 3H, —OCH3). 13C NMR (100 MHz, DMSO-d6): δ 161.88 (C2), 153.00 (C3′), 150.15 (C5′), 143.90 (C6′), 141.81 (C4), 139.20 (C2″), 137.72 (C6), 129.18 (C4″, 6″), 128.21 (C3″, 5″, 7″), 120.42 (C5), 115.45 (C3), 113.08 (C7), 105.51 (C8′), 101.88 (C4′), 67.36 (C1′), 56.57 (—OCH3), 56.03 (—OCH3), 51.85 (C1′). MS Calcd. for C21H21NO4: 351.1. EI-MS m/z: 351.1 [M]+.
4.3.26 1-Benzyl-5-((3-(trifluoromethyl)phenoxy)methyl)pyridin-2(1H)-one (4k)
Light yellow crystal, yield: 48%. M.p.: 70.3–71.9 °C. 1H NMR (500 MHz DMSO-d6): δ 8.00 (s, 1H, CH⚌), 7.53–7.56 (m, 1H, CH⚌), 7.50–7.53 (m, 1H, Ar—H), 7.28–7.35 (m, 8H, Ar—H), 6.46–6.48 (m, J = 8.0 Hz, 1H, CH⚌), 5.10 (s, 2H, —CH2—), 4.92 (s, 2H, —CH2—). 13C NMR (125 MHz, DMSO-d6): δ 161.51 (C2), 158.87 (C3′), 141.45 (C4), 139.54 (C2″), 137.71 (C5′), 131.17 (C7′), 129.02 (C4″, 6″), 128.14 (C6), 128.03 (C3″, 7″), 125.55 (—CF3), 123.38 (C5″), 120.44 (C5), 119.68 (C3), 117.86 (C8′), 114.22 (C6′), 111.89 (C4′), 67.34, 51.64. MS Calcd. for C20H16F3NO2: 359.1. EI-MS m/z: 359.1 [M]+.
4.3.27 5-((4-Fluorophenoxy)methyl)-1-(3-methoxybenzyl)pyridin-2(1H)-one (4l)
White powder, yield: 41%. M.p.: 98.3–100.1 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.94–7.95 (s, J = 2.0 Hz, 1H, CH⚌), 7.48–7.51 (m, J = 9.2 Hz, 1H, CH⚌), 7.25–7.28 (m, J = 8.4 Hz, 2H, Ar—H), 7.09–7.14 (m, J = 8.8 Hz, 2H, Ar—H), 6.97–7.01 (m, 2H, Ar—H), 6.88–6.90 (d, J = 8.4 Hz, 2H, Ar—H), 6.43–6.45 (m, 1H, CH⚌), 5.01 (s, 2H, —CH2—), 4.77 (s, 2H, —CH2—), 3.72 (s, 3H, —OCH3). 13C NMR (100 MHz, DMSO-d6): δ 161.28 (C2), 159.06 (C5′), 158.10 (C6′), 155.75 (C6′), 154.67 (C3′), 141.20 (C4), 139.05 (C2″), 129.56 (C3″, 7″), 120.15 (C5), 116.58 (C3), 116.50 (C5′, 7′), 115.99 (C4′, 8′), 114.22 (C4″, 6″), 67.29 (C1′), 55.38 (—OCH3), 50.8 (C1′). MS Calcd. for C20H18FNO3: 339.1. EI-MS m/z: 339.1 [M]+.
4.3.28 1-(3-Methoxybenzyl)-5-((p-tolyloxy)methyl)pyridin-2(1H)-one (4m)
White powder, yield: 31%. M.p.: 118.6–120.4 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.93–7.93 (s, J = 2.0 Hz, 1H, CH⚌), 7.46–7.49 (m, J = 9.2 Hz, 1H, CH⚌), 7.25–7.27 (m, J = 8.8 Hz, 2H, Ar—H), 7.06–7.08 (m, J = 8.4 Hz, 2H, Ar—H), 6.85–6.89 (m, J = 10.0 Hz, 4H, Ar—H), 6.42–6.44 (m, J = 9.2 Hz, 1H, CH⚌), 5.00 (s, 2H, —CH2—), 4.75 (s, 2H, —CH2—), 3.72 (s, 3H, —OCH3), 2.22 (s, 3H, —CH3). 13C NMR (100 MHz, DMSO-d6): δ 161.31 (C2), 159.07 (C5′), 156.23 (C3′), 141.20 (C4), 138.92 (C2″), 130.11 (C3″, 7″), 129.78 (C6′), 129.70 (C5′, 7′), 129.59 (C6), 120.12 (C5), 115.05 (C4′, 8′), 114.65 (C3), 114.23 (C4″, 6″), 66.64 (C1′), 55.37 (—OCH3), 50.82 (C1′), 20.40 (—CH3). MS Calcd. for C21H21NO3: 335.2. EI-MS m/z: 335.2 [M]+.
4.4 Antiproliferative activity assay
The antiproliferative activity in vitro was determined using CellTiter 961 AQueous One Solution Cell Proliferation Assay Kit, according to operation instructions provided by the manufacturer (Promega, Madison, WI, USA). Briefly, A549 or NIH3T3 cell lines were cultured in 96-well plates and allowed to grow for 24 h and subsequently treated with all tested compounds at final concentrations of 1.5–500 μM for 72 h. Culture medium was then removed. 20 mL of MTT solution was added into each well of the 96-well assay plate containing the samples in 100 mL of culture medium. The subsequent incubation was performed in an atmosphere of humidity, 5% CO2 at 37 °C for 2 h before the cytotoxicity assessments. Optical absorbance was recorded at 490 nm using a Multiskan Ascent 354 microplate reader (Thermo Labsystems, Helsinki, Finland). The absorption value was determined by Ascent SoftwareTM (Thermo Labsystems, Helsinki, Finland). Survival ratios are expressed in percentages with respect to untreated cells and IC50 values were calculated by comparison with DMSO-treated control wells by creating the dose–response curves utilizing GraphPad PrismTM 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Each assay was carried out for at least three times.
4.5 3D-QSAR model
The 3D-DSAR models were performed by QSAR software of MOE 2014. In the present study, we have applied a novel approach of AutoGPA to develop a 3D-QSAR model on a series of 1,5-disubstituted-2(1H)-pyridone derivatives anti-lung cancer agents. The procedures followed for the development of AutoGPA models using MOE are as follows: (1) 3D structures of compounds were built using the builder tool of MOE 2014 and energy minimization was carried out using MMFF94x force field with generalized born solvation model; (2) The pIC50 values converted from the obtained IC50 (mM) and conformations of active compounds were stored in a database of the MOE; (3) Pharmacophore elucidation based on conformations of dataset; (4) Generation of 3D-QSAR model for all superimposed conformation by the AutoGPA module in MOE; (5) Selection of the best model based on q2 values; and (6) AutoGPA model: (Pharmacophore 3D-QSAR). The predictive ability of 3D-QSAR modeling can be evaluated based on the cross-validated correlation coefficient, which qualifies the predictive ability of the models. Golbrakh (Golbraikh and Tropsha, 2002) recommended that the criteria (i) r2 > 0.6; (ii) q2 > 0.5 should be fulfilled for a given 3D-QSAR model to be accepted, where r2 is the correlation coefficient between the predicted and observed activities. Usually, one can believe that the modeling complying with the above conditions is reliable.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No. 21172268).
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Appendix A
Supplementary material
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.arabjc.2015.08.001.
Appendix A
Supplementary material
