English

• 1.   1 Introduction

  The chromenopyridine and its derivatives have cha- racteristic drug-related properties. Compounds which contain the framework have some significant bioactivities including glucocorticoid receptor agonism, ^[1] anti-tumor, ^[2] anti-proliferation, ^[3] anti-myopia, ^[4] anti-hypertensive, ^[5] anti-histamine, ^[6] anti-rheumatic, ^[7] anti-asthma^[8] and so on.

  As an important part of the chromenopyridine family, 4H-chromenopyridine derivatives have also a variety of bioactivities such as anti-cancer, ^[9] anti-allergy, ^[10] anti- inflammatory, ^[11] resistance proliferation, ^[12] anti-bacte- rial^[13] and anti-tuberculosis, ^[14] etc. A classic example is ibuprofen, which acts as a non-steroidal anti-inflammatory and analgesic medicine by inhibiting the synthesis of prostaglandins. Ibuprofen has been used to treat conjunctivitis and relieve pain and inflammation.^{[15, 16]} Kemnitzer found that many 4-aryl-4H-chromenes could inhibit the proliferation of cancer cell by inducing apoptosis.^[17] Therefore, it is of theoretical significance and practical value to synthesize more chromeno[2, 3-b]pyridine derivatives with new structure.

  The 4H-chromeno[2, 3-b]pyridine derivatives were usually obtained by the condensation of aromatic aldehydes, malononitrile and phenols in the presence of K₂CO₃, ^[18] Et₃N^[19] or chitosan, ^{[20, 21]} etc. However, the range of catalyst and substrate is still a major problem. Therefore, it is still necessary to develop a new type of catalyst for their preparation.

  Trögerʼs base (TB, Figure 1), which was synthesized in 1887, ^[22] has caused great attention in molecular recognition, ^{[23, 24]} bioorganic chemistry, ^[25~27] supramolecular che- mistry^{[28, 29]} and so on due to its particular structure, ^[27] which enables TB and its derivatives to capture appropriate molecules and then to show catalytic activities.^[27] However, the weak alkalescence, limited active sites and simple hydrogen bond acceptor limit their application in organocatalysis.

  Figure 1

  

  Figure 1.  Structure of Trögerʼs base

  下载: 全尺寸图片 幻灯片

  To make full use of the structural advantages of TB and explore a new catalyst to synthesize chromeno[2, 3-b]- pyridine derivatives with potential bioactivity, herein we designed and synthesized a series of Schiff bases by the reaction of TB-NH₂ with heterocyclic aldehydes to increase the number and type of catalytic sites. Then they were used as efficient catalyst for the multicomponent reaction of substituted salicylaldehyde, malononitrile and substituted 2-mercaptoimidazole to afford 5-(1H-imidazol-2-ylthio)- 2, 4-diamino-5H-chromeno[2, 3-b]pyridine-3-carbonitriles in mild condition with high yield.

  2.   Results and discussion

  Firstly, a series of catalysts based on TB derivatives were synthesized (Scheme 1). Because p-nitroaniline could not react, 2-methyl-4-nitroaniline was used to obtain corresponding TB derivatives successfully due to the electro- donating effect of methyl.

  Figure 1

  

  Figure 1.  Synthesized Schiff bases

  下载: 全尺寸图片 幻灯片

  Then, the pH and calculated the pK_a value of catakyst 4 were tested. It can be seen from Table 1 that the basicity of 4 has been enhanced compared with that of TB by introducing heterocycles. 4f had the strongest basicity, which may be come from the pyridine ring. The basicity of 4g was weaker than that of 4f, probably because the ortho-methyl increased the steric hindrance of the N atom and weakened its ability to bind with hydrogen ions. The data in Table 1 also indicated that the methyl and its position on the heterocyclic ring had a significant effect on the melting point of 4.

  Table 1

  

  Table 1.  pK_a values and yields of 4^a

  下载: 导出CSV

  Compd.	pKa	R	yield/%
  TB	9.05	—
  1	9.13	—
  2	9.28	—
  4a	9.26	Thiophene-2-yl	93
  4b	9.20	3-Methyl-thiophene-2-yl	91
  4c	9.21	5-Methyl-thiophene-2-yl	88
  4d	9.11	Furan-2-yl	89
  4e	9.13	5-Methyl-furan-2-yl	86
  4f	9.35	Pyridine-2-yl	85
  4g	9.06	5-Methyl-pyridine-2-yl	81
  a Calculated from the pH value tested by dissolving 0.01 mmol of compounds in appropriate amount of ethanol and diluting to 10.0 mL with ethanol.

  The catalytic activity of 4 to the synthesis of 8h was evaluated firstly. As expected, the reaction of salicylaldehyde (1 mmol), malononitrile (2 mmol) and 1-methyl- 1H-imidazole-2-thiol (1 mmol) could be catalyzed by 4 (20 mol%) successfully by refluxing for 6 h in EtOH to give 8h in 58%~89% yields.

  The reaction conditions were then optimized based on the yield of 8h. As shown in Table 2, this reaction did not occur without catalyst (Entry 1). Neither the common organic base such as pyridine (Entry 2, pK_a=5.14^[31]) and 2, 2'-bipyridyl (Entry 3, pK_a=4.23^[31]) nor inorganic base such as NaHCO₃ (Entry 4, pK_a=10.25^[32]), CsCO₃ (Entry 5, pK_a=10.25^[32]), K₃PO₄ (Entry 6, pK_a=2.12^[32]), NaOH (Entry 7) and EtONa (Entry 8) could catalyze this reaction. Schiff base N-(pyridin-3-ylmethylene)aniline could also not catalyze the reaction (Entry 9). The intermediate 2 could catalyze this reaction, but the low yield of 8h was obtained (16%, Entry 10). Heterocyclic aldehydes 3a~3g were also not able to catalyze the reaction (Entries 11~17). The catalyst 4h which was synthesized by the condensation of intermediate 2 with benzaldehyde 3h could also catalyze the reaction, but the yield of 8h was also very low (15%, Entry 18). When seven Schiff base 4a~4g were used as catalyst, 8h could be obtained with 58%~89% yields (Entries 19~25), among which 4f gave the best result. The results showed that the electron effect and steric hindrance of the heterocyclic aldehyde have a great influence on the yield of 8h and were in accordance with the relevant laws. These results also indicated that the high catalytic function of 4 was not from their appropriate basicity or the C=N double bond separately, but from their whole structure.

  Table 2

  

  Table 2.  Synthesis of 8h under different reaction conditions^a

  下载: 导出CSV

  Entry	Cat.	Cat./mol%	Solvent	t/h	T/℃	Yieldb/%
  1	None	—	EtOH	6	Reflux	—
  2	Pyridine	20	EtOH	6	Reflux	—
  3	2, 2'-Dipyridyl	20	EtOH	6	reflux	—
  4	NaHCO3	20	EtOH	6	Reflux	—
  5	CsCO3	20	EtOH	6	Reflux	—
  6	K3PO4	20	EtOH	6	Reflux	—
  7	NaOH	20	EtOH	6	Reflux	—
  8	EtONa	20	EtOH	6	Reflux	—
  9		20	EtOH	6	Reflux	—
  10	2	20	EtOH	6	Reflux	16
  11	3a	20	EtOH	6	Reflux	—
  12	3b	20	EtOH	6	Reflux	—
  13	3c	20	EtOH	6	Reflux	—
  14	3d	20	EtOH	6	Reflux	—
  15	3e	20	EtOH	6	Reflux	—
  16	3f	20	EtOH	6	Reflux	—
  17	3g	20	EtOH	6	Reflux	—
  18	3h	20	EtOH	6	Reflux	15
  19	4a	20	EtOH	6	Reflux	71
  20	4b	20	EtOH	6	Reflux	63
  21	4c	20	EtOH	6	Reflux	58
  22	4d	20	EtOH	6	Reflux	78
  23	4e	20	EtOH	6	Reflux	70
  24	4f	20	EtOH	6	Reflux	89
  25	4g	20	EtOH	6	Reflux	85
  26	4f	5	EtOH	6	Reflux	35
  27	4f	10	EtOH	6	Reflux	51
  28	4f	15	EtOH	6	Reflux	62
  29	4f	25	EtOH	6	Reflux	91
  30	4f	20	MeCN	6	Reflux	35
  31	4f	20	DCM	6	Reflux	38
  32	4f	20	H2O	6	Reflux	46
  33	4f	20	EtOH	6	r.t.	—
  34	4f	20	EtOH	6	40	53
  aUnless otherwise noted, the reactions were carried out using salicylaldehyde (1 mmol), malononitrile (2 mmol) and 1-methyl-1H-imidazole-2-thiol (1 mmol).

  The loading of catalyst was then tested (Entries 24, 26~29). As it was increased, the yield increased obviously. When it was increased to 20 mol%, the yield increased significantly (89%). But the yield did not increase obviously (91%) after the loading of catalyst reached 25 mol%. Therefore, 20 mol% was the optimal one. Subsequently, the optimum solvent (Entries 24, 30~32) and temperature (Entries 24, 33, 34) was studied. To sum up, the optimal condition was using 20 mol% 4f as catalyst, the reaction system was stirred in ethanol at 80 ℃ for 6 h.

  With the optimized conditions in hand, we proceeded to explore the substrate scope of this transformation with a variety of salicylaldehydes 5 and substituted 2-mercaptoi- midazoles 7 (Table 3). As can be seen from Table 3, 4f has a good catalytic effect on this reaction, the reaction afforded medium to high yields (61%~89%) of the desired products with wide substrate scope. Electronic effects and steric hindrance of the substituents attached to salicylaldehyde and 2-mercaptoimidazole has little effect on the reaction yield.

  Table 3

  

  Table 3.  Synthesis of 8 under the optimal conditions

  下载: 导出CSV

  Entry	R1	R2	Product	Yield/%
  1	3-OH	H	8a	86
  2	3-OH	CH3	8b	83
  3	4-OH	H	8c	76
  4	5-Cl	H	8d	61
  5	3-CH3O	CH3	8e	79
  6	5-CH3	CH3	8f	87
  7	5-Cl	CH3	8g	85
  8	H	CH3	8h	89
  9	3-CH3	CH3	8i	80
  10	3-CH3O	H	8j	77
  11	5- CH3	H	8k	80
  12	3-F	H	8l	81
  13	5-OH	CH3	8m	79
  14	5-OH	H	8n	83
  15	5-NO2	H	8o	88
  16	5-NO2	CH3	8p	84
  17	5-Br	CH3	8q	87
  18	5-Br	H	8r	86
  19	5-F	H	8s	78
  As the signals in the aromatic hydrogen area merged together, it is hard to figure out the characteristic peaks in the 1H NMR spectrum. Thus, the research was focus on the signals in δ 4.00~4.65 (Figure 2).

  2.1   Study on the reaction mechanism

  In order to understand the catalytic function of catalyst 4f, ¹H NMR analysis (400 MHz) was applied to monitor the reaction process. The specific steps were as follows: catalyst 4f (20 mol%), 2-mercaptobenzimidazole (1 mmol), malononitrile (2 mmol), 3-methyl salicylaldehyde (1 mmol) and ethanol (10 mL) were added in a 25 mL round bottom flask and refluxed. 0.20 mL of the mixture was taken out and the solvent was removed under vaccum. The residue was then dissolved in d₆-DMSO for NMR analysis. The reaction process was monitored by repeating the operation at five-minute intervals.

  Figure 2

  

  Figure 2.  Changes of the signals as the reaction proceeded

  下载: 全尺寸图片 幻灯片

  From Figure 2, as the reaction proceeded, the signals of the methylene H atoms on TB backbone of 4f in δ 4.45 and 4.54 decreased gradually and disappeared at 20 and 25 min respectively. And a new signal (δ 4.35) appeared at 10 min. The results indicated that the TB framework participated in the catalytic process. More specifically, it can be speculated that the bridge N atoms react with special substrates or intermediates and then cause the shift of the adjacent H.

  Based on the results mentioned above, a possible mechanism was put forward (Scheme 3). In the presence of 4f, malononitrile changed as carbanion and condensated with the formyl group of the salicylaldehyde to produce the intermediate I. Intermediate I underwent intramolecular cyclization to form intermediate II. The sulfhydryl anion formed by 2-mercapto-1-methylimidazole under basic conditions attacked II to give intermediate III by 1, 4-addi- tion. III attacked another malononitrile via cycloaddition to give intermediate IV which was then aromatized to give the final product 8.

  Scheme 3

  

  Scheme 3.  A supposed mechanism

  下载: 全尺寸图片 幻灯片

  As mentioned above, the catalyst promotes the reaction by providing two active sites and alkaline environment.

  We then tested their anti-tumor activity on human breast adenocarcinoma cell (MCF-7), adenocarcinomic human alveolarbasal epithelial cell (A549) and cytotoxicity on human bronchial epithelial cell (HBE) with methyl thiazolyl tetrazolium (MTT) method (Table 4).

  Table 4

  

  Table 4.  IC₅₀ value of compound 8 against antitumor and normal cells^a

  下载: 导出CSV

  Entry	A549	MCF-7	HBE
  8a	—	12.2	16.3
  8b	—	12.2	15.3
  8d	—	17.9	15.7
  8f	—	15.1	17.4
  8h	39.7	8.4	4.9
  8i	—	16.4	11.5
  8j	—	15.5	26.7
  8m	—	16.2	15.8
  8n	—	13.8	15.1
  8r	40.9	—	11.3
  8s	—	11.1	7.2
  Adriamycin	4.05	0.027	7.01
  Cisplatin	27.59	66.61	7.77
  a IC50 > 64 μg/mL were marked with "—".

  From Table 4, compounds 8a, 8b, 8d, 8f, 8i, 8j, 8m, 8n and 8s had inhibitory effects on MCF-7 while 8r had inhibitory effect on A549. 8h had better inhibitory effect in lower concentrations on the two tumor cells. In terms of structure, it is the only one no substituents on the phenyl ring. Probably, its spatial structure is the most appropriate one to the two tumor cells. However, it also had cytoxicity on HBE. Therefore, it would be necessary to reduce its toxicity by structure modification. The compound with a fluorine atom was more effective than that bearing a bromine atom, probably due to the special properties of fluorine atom such as inductive effect.

  In the last, the anti-bacterial activities of product 8 on Staphylococcus aureus (wild type), Bacillus subtilis (wild type) and Escherichia coli (wild type) were also evaluated (Table 5). Only 8s was screened out due to its inhibition on Staphylococcus aureus and Escherichia coli. Further researches are necessary to study the specific reason.

  Table 5

  

  Table 5.  Inhibition ratio of 8s on two bacteria

  下载: 导出CSV

  Bacterium	Inhibition ratio/%
  	16	32	64	128
  Staphylococcus aureus	-	—	84.82	90.84
  Escherichia coli	-	94.56	100.00	100.00
  a The concentration (µg/mL) of 8s; bNo inhibition.

  3.   Conclusions

  In summary, a new series of Schiff base catalyst derived from Trögerʼs base and heterocyclic aldehydes was synthesized and used to promote the multi-component reaction of substituted salicylaldehyde, malononitrile and substituted 2-mercaptoimidazole to afford 5-(1H-imidazol-2- ylthio)-2, 4-diamino-5H-chromeno[2, 3-b]pyridine-3-carbo-nitrile derivatives. The mechanism study via ¹H NMR analysis indicated that the catalyst promotes the reaction by providing two active sites and alkaline environment. Some products were screened out due to their high antitumor and anti-bacterial activities in vitro. This study extended the application range of Trögerʼs base derivatives as catalysts.

  4.   Experimental section

  4.1   General

  All the reagents were purchased commercially. Tetrahydrofuran (THF) and toluene were dried and distilled from sodium. Ethanol was dried and distilled from magnesium. All reactions involving air- or moisture-sensitive materials were carried out under an argon atmosphere. All cancer cells, normal cell and bacterial were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China).

  All melting points were determined with an electrothermal digital melting point apparatus and were uncorrected. pHs were measured using a pH-3c digital pH meter (Shanghai Leici Device Works, Shanghai, China). NMR spectra were measured using a Bruker 400 MHz spectrometer. Mass spectra were recorded on a Micro TOF-Q (ESI) instrument. The MTT assay was performed on a microplate reader (SpectraMax M2).

  4.2   Synthesis of compound 1

  4-Nitro-2-toluidine (5.52 g, 40 mmol) and paraformaldehyde (7.2 g, 80 mmol) were added to a dry 50 mL two-necked flask, 80 mL of trifluoroacetic acid was then added to the flask dropwise at -15 ℃. After 3 h, the reaction was moved to room temperature for 48 h (TLC) in the dark. The reaction mixture was poured into ice water. When pH was adjusted with sodium hydroxide solution to 9, the yellow solid precipitated out. The filtered solid was dissolved in acetone and refluxed for about 20 min, cooled and recrystallized to give 4, 10-dimethyl-2, 8-dinitro-6H, 12H-5, 11-methanodibenzo[b, f][1, 5]diazocine (1), yellow solid, yield 68%. m.p. > 300 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.96 (s, 2H, ArH), 7.72 (s, 2H, ArH), 4.71 (d, J=17.2 Hz, 2H, CH₂×2), 4.33 (s, 2H, CH₂-Bridge), 4.11 (d, J=17.2 Hz, 2H, CH₂*2), 2.50 (s, 6H, CH₃×2); ¹³C NMR (100 MHz, CDCl₃) δ: 134.7, 128.3, 124.3, 120.2, 67.0, 54.7, 17.3. HRMS (ESI) calcd for C₁₇H₁₇N₄O₄ [M+H]⁺ 341.1250; found 341.1291.

  4.3   Synthesis of compound 2

  Compound 1 (0.34 g, 1 mmol), iron powder (0.84 g, 15 mmol) and glacial acetic acid (1.2 g, 20 mmol) were sequentially added to a 50 mL dry round bottom flask containing 10 mL of absolute ethanol. The mixture was refluxed for 16 h. After the reaction completion (TLC), 30 mL of distilled water was added, and the mixture was filtered with suction. The filtrate was extracted with dichloromethane (50 mL×3). The organic layer was washed with saturated sodium hydrogen carbonate until the pH value was 7. The organic layer was then dried over sodium sulfate and evaporated under reduced pressure. The crude product was purified by column chromatography (V_{petroleum ether}:V_{ethyl acetate}=1:9 gradient elution) to give 4, 10-dimethyl-2, 8-diamine-6H, 12H-5, 11-methanodibenzo-[b, f][1, 5]diazocine (2), gray solid, yield 83%. m.p. 209.6~211.2 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 6.28 (s, 2H, ArH), 5.96 (s, 2H, ArH), 4.59 (s, 4H, NH₂×2), 4.27 (d, J=17.2 Hz, 2H, CH₂×2), 4.07 (s, 2H, CH₂- Bridge), 3.62 (d, J=17.2 Hz, 2H, CH₂×2), 2.19 (s, 6H, CH₃×2); ¹³C NMR (100 MHz, CDCl₃) δ: 142.3, 133.7, 129.0, 116.3, 110.4, 68.0, 55.3, 17.0. HRMS (ESI) calcd for C₁₇H₁₇N₄ [M+H]⁺ 281.1766; found 281.1786.

  4.4   Synthesis of compound 4

  Compound 2 (0.28 g, 1.0 mmol), aldehyde 3 (1.2 mmol), triethylamine (1 mol%) and ethanol (5 mL) were added to a 50 mL dry round bottom flask and refluxed for 12 h. When the reaction finished (TLC), excess solvent was evaporated under reduced pressure, and the crude product was separated by appropriate column chromatography to afford compound 4.

  N, N'-(4, 10-Dimethyl-6H, 12H-5, 11-methanodibenzo[b, f]- [1, 5]diazocine-2, 8-diyl)bis(1-(thiophen-2-yl)methanimine) (4a): Yellow solid, yield 93%. m.p. 212.5~214.2 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.50 (s, 2H, N=CH), 7.47 (d, J=5.2 Hz, 2H, Thiophene-H), 7.42 (d, J=2.8 Hz, 2H, Thiophene-H), 7.11~7.09 (m, 2H, Thiophene-H), 6.98 (d, J=1.6 Hz, 2H, ArH), 6.69 (d, J=1.6 Hz, 2H, ArH), 4.62 (d, J=16.8 Hz, 2H, CH₂×2), 4.36 (s, 2H, CH₂-Bridge), 4.01 (d, J=16.8 Hz, 2H, CH₂×2), 2.44 (s, 6H, CH₃×2); ¹³C NMR (100 MHz, CDCl₃) δ: 152.3, 145.8, 144.1, 142.7, 133.1, 132.9, 130.6, 128.8, 128.1, 121.6, 116.7, 54.5, 30.6, 16.8. HRMS (ESI) calcd for C₂₇H₂₅N₄S₂ [M+H]⁺ 469.1521; found 469.1506.

  N, N'-(4, 10-Dimethyl-6H, 12H-5, 11-methanodibenzo[b, f]-[1, 5]diazocine-2, 8-diyl)bis(1-(3-methyl-thiophen-2-yl)me- thanimine) (4b): Yellow solid, yield 91%. m.p. 231.6~233.8 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.58 (s, 2H, N=C-H), 7.39 (d, J=3.2 Hz, 2H, Thiophene-H), 6.98 (d, J=2.0 Hz, 2H, Thiophene-H), 6.89 (dd, J=3.6, 1.2 Hz, 2H, ArH), 6.74 (d, J=3.2 Hz, 2H, ArH), 4.52 (d, J=17.2 Hz, 2H, CH₂×2), 4.26 (s, 2H, CH₂-Bridge), 3.99 (d, J=17.2 Hz, 2H, CH₂×2), 2.48 (s, 2H, ArCH₃×2), 2.37 (s, 2H, Thiophene CH₃×2); ¹³C NMR (100 MHz, DMSO-d₆) δ: 193.1, 170.1, 150.9, 142.3, 136.3, 133.0, 131.3, 129.8, 128.8, 122.0, 116.5, 30.7, 16.8, 13.6. HRMS (ESI) calcd for C₂₉H₂₉N₄S₂ [M+H]⁺ 497.1834; found 497.1829.

  N, N'-(4, 10-Dimethyl-6H, 12H-5, 11-methanodibenzo[b, f]- [1, 5]diazocine-2, 8-diyl)bis(1-(5-methyl-thiophen-2-yl)me- thanimine) (4c): Yellow solid, yield 88%. m.p. 178.3~179.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.55 (s, 2H, N=CH), 7.39 (d, J=4.8 Hz, 2H, Thiophene-H), 6.99 (s, 2H, ArH), 6.91 (d, J=4.8 Hz, 2H, Thiophene-H), 6.68 (s, 2H, ArH), 4.65 (d, J=17.2 Hz, 2H, CH₂×2), 4.39 (s, 2H, CH₂-Bridge), 4.04 (d, J=16.8 Hz, 2H, CH₂×2), 2.46 (s, 2H, ArCH₃×2), 2.45(s, 2H, Thiophene CH₃×2); ¹³C NMR (100 MHz, DMSO-d₆) δ: 152.2, 145.9, 144.8, 143.8, 140.5, 140.5, 133.4, 133.0, 128.7, 126.6, 121.5, 54.5, 30.6, 16.8, 15.5. HRMS (ESI) calcd for C₂₉H₂₉N₄S₂ [M+H]⁺ 497.1834; found 497.1848.

  N, N'-(4, 10-Dimethyl-6H, 12H-5, 11-methanodibenzo[b, f]-[1, 5]diazocine-2, 8-diyl)bis(1-(furan-2-yl)methanimine)(4d): Yellow solid, yield 89%. m.p. 197.3~200.9 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.22 (s, 2H, N=CH), 7.58 (s, 2H, Oxole-H), 7.01 (s, 2H, ArH), 6.88 (d, J=3.2 Hz, 2H, Oxole-H), 6.72 (d, J=1.6 Hz, 2H, ArH), 6.54~6.52 (m, 2H, Oxole-H), 4.62 (d, J=16.8 Hz, 2H, CH₂×2), 4.34 (s, 2H, CH₂-Bridge), 4.01 (d, J=16.8 Hz, 2H, CH₂×2), 2.44 (s, 6H, CH₃×2); ¹³C NMR (100 MHz, CDCl₃) δ: 152.2, 146.8, 146.5, 145.4, 133.9, 128.7, 121.9, 116.6, 115.8, 112.1, 55.2, 17.2. HRMS (ESI) calcd for C₂₇H₂₅N₄O₂ [M+H]⁺ 437.1978; found 437.1967.

  N, N'-(4, 10-Dimethyl-6H, 12H-5, 11-methanodibenzo[b, f]-[1, 5]diazocine-2, 8-diyl)bis(1-(5-methylfuran-2-yl)methani-mine) (4e): Yellow solid, yield 86%. m.p. 120.6~124.5 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.24 (s, 2H, N=CH), 6.98 (d, J=1.6 Hz, 2H, Oxole-H), 6.95 (d, J=3.2 Hz, 2H, ArH), 6.73 (s, 2H, Oxole-H), 6.31 (d, J=2.4 Hz, 2H, ArH), 4.53 (d, J=16.8 Hz, 2H, CH₂×2), 4.25 (s, 2H, CH₂-Bridge), 3.98 (d, J=16.8 Hz, 2H, CH₂×2), 2.37 (s, 6H, Ar-CH₃), 2.36 (s, 6H, Oxole-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 178.2, 169.8, 155.6, 150.6, 146.4, 144.0, 133.0, 128.8, 118.3, 116.4, 108.9, 67.0, 54.5, 16.8, 13.5. HRMS (ESI) calcd for C₂₉H₂₉N₄O₂ [M+H]⁺ 465.2291; found 465.2260.

  N, N'-(4, 10-Dimethyl-6H, 12H-5, 11-methanodibenzo[b, f]-[1, 5]diazocine-2, 8-diyl)bis(1-(pyridin-2-yl)methanimine) (4f): Yellow solid, yield 85%. m.p. 193.4~195.8 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.69 (d, J=8 Hz, 2H, pyridine-H), 8.54 (s, 2H, N=CH), 8.14 (d, J=8 Hz, 2H, pyridine-H), 7.78 (t, J=7.7, 4.4 Hz, 2H, pyridine-H), 7.35~7.33 (m, 2H, pyridine-H), 7.07 (d, J=1.6 Hz, 2H, ArH), 6.79 (d, J=1.6 Hz, 2H, ArH), 4.65 (d, J=16.8 Hz, 2H, CH₂×2), 4.37 (s, 2H, CH₂-Bridge), 4.05 (d, J=16.8 Hz, 2H, CH₂×2), 2.46 (s, 6H, Ar-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ: 159.3, 154.7, 149.6, 146.4, 145.0, 136.6, 134.0, 128.7, 124.9, 122.0, 121.7, 117.1, 67.6, 55.2, 17.2. HRMS (ESI) calcd for C₂₉H₂₇N₆ [M+H]⁺ 459.2297; found 459.2248.

  N, N'-(4, 10-Dimethyl-6H, 12H-5, 11-methanodibenzo[b, f]- [1, 5]diazocine-2, 8-diyl)bis(1-(6-methylpyridin-2-yl)me-

  thanimine) (4g): Yellow solid, yield 81%. m.p. 113.1~115.2 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.54 (s, 2H, C=NH), 7.96 (d, J=7.6 Hz, 2H, pydirine-H), 7.67 (t, J=7.6 Hz, 2H, pydirine-H), 7.20 (d, J=7.2 Hz, 2H, pydirine-H), 7.08 (s, 2H, ArH), 6.79 (s, 2H, ArH), 4.64 (d, J=16.8 Hz, 2H, CH₂×2), 4.36 (s, 2H, CH₂-Bridge), 4.04 (d, J=16.8 Hz, 2H, CH₂×2), 2.62 (s, 3H, pydirine-CH₃), 2.45 (s, 3H, pydirine-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ: 159.6, 158.3, 154.1, 146.4, 144.9, 136.8, 133.9, 128.6, 124.6, 121.9, 118.7, 117.3, 67.6, 55.1, 24.3, 17.2. HRMS (ESI) calcd for C₃₁H₃₁N₆ [M+H]⁺ 487.2610; found 487.2592.

  4.5   Synthesis of compound 8

  Salicylaldehydes (5) (1 mmol), malononitrile (6) (0.66 g, 2 mmol), 2-mercaptoimidazole (7) (1 mmol), 4f (20 mol%) and ethanol (5 mL) were added to a 50 mL round bottom flask and reacted to completion at 80 ℃ (TLC). After reaction was cooled to room temperature, the solid was filtered and washed with a small amount of water, then dissolved in N, N-dimethylformamide. Removed the insoluble substances via filtering under vacuum and distilled. Solid was precipitated out by adding water to the filtrate, then the mixture was filtered and recrystallized in DMSO to give compound 8.

  5-(1H-Imidazol-2-ylthio)-2, 4-diamino-9-hydroxy-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8a): Yellow solid, yield 86%. m.p. 286.2~289.5 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.43 (s, 1H, NH), 9.96 (s, 1H, OH), 7.10 (s, 1H, imidazole-H), 6.95 (s, 1H, imidazole-H), 6.88~6.87 (m, 2H, ArH), 6.78 (s, 1H, ArH), 6.70~6.68 (m, 2H, NH₂), 6.66~6.64 (m, 2H, NH₂), 6.59 (s, 1H, methine-H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 160.3, 159.1, 157.2, 145.5, 139.0, 135.9, 124.3, 120.2, 117.8, 116.4, 116.1, 115.6, 115.5, 86.0, 70.3, 47.3. HRMS (ESI) calcd for C₁₆H₁₁N₆O₂S [M-H]^- 351.0664; found 351.0689.

  2, 4-Diamino-9-hydroxy-5-(1-methyl-1H-imidazol-2-ylthio)-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8b): Yellow solid, yield 83%. m.p. 253.6~256.2 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.97 (s, 1H, OH), 7.11 (s, 1H, imidazole-H), 7.08 (d, J=2.4 Hz, 1H, imidazole-H), 6.91 (d, J=7.6 Hz, 1H, ArH), 6.88~6.86 (m, 2H, ArH), 6.71 (s, 2H, NH₂), 6.65 (d, J=2.8 Hz, 2H, NH₂), 6.63 (s, 1H, methine-H), 3.52 (s, 3H, OCH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 167.0, 166.5, 154.7, 153.5, 152.4, 134.6, 134.4, 128.6, 123.4, 122.0, 121.6, 114.6, 114.2, 88.8, 69.0, 43.8, 32.9. HRMS (ESI) calcd for C₁₇H₁₃N₆O₂S [M-H]^- 365.0821; found 365.0845.

  5-(1H-Imidazol-2-ylthio)-2, 4-diamino-8-hydroxy-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8c): Yellow solid, yield 76%. m.p. > 300 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.96 (s, 1H, OH), 8.48 (s, 1H, NH), 7.85 (s, 1H, imidazole-H), 7.31 (d, J=5.6 Hz, 1H, imidazole-H), 7.21 (d, J=8.4 Hz, 1H, ArH), 7.05 (s, 2H, ArH), 6.67 (s, 2H, NH₂), 6.55 (d, J=2.0 Hz, 1H, NH₂), 6.40 (d, J=7.2 Hz, 1H, NH₂), 6.06 (s, 1H, methine-H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 166.4, 162.5, 160.5, 149.0, 147.6, 144.7, 144.1, 133.7, 129.3, 124.7, 119.5, 117.9, 110.9, 95.4, 71.3, 44.0. HRMS (ESI) calcd for C₁₆H₁₁N₆O₂S [M-H]^- 351.0664; found 351.0684.

  5-(1H-Imidazol-2-ylthio)-2, 4-diamino-7-chloro-5H-chro-meno[2, 3-b]pyridine-3-carbonitrile (8d): Yellow solid, yield 61%. m.p. 267.6~270.5 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.52 (s, 1H, NH), 7.54 (dd, J=8.8, 2.8 Hz, 1H, imidazole-H), 7.40 (d, J=2.4 Hz, 1H, imidazole-H), 7.29 (dd, J=5.6, 4.0 Hz, 1H, ArH), 7.18 (t, 1H, J=3.0 Hz, ArH), 7.14 (s, 1H, ArH), 6.92 (s, 1H, NH₂), 6.77 (t, J=13.4 Hz, 2H, NH₂), 6.62 (s, 1H, NH₂), 4.92 (s, 1H, methine-H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 163.1, 162.5, 159.5, 135.7, 134.4, 133.8, 131.9, 131.3, 131.2, 130.2, 129.3, 120.3, 119.8, 97.9, 73.1, 44.4. HRMS (ESI) calcd for C₁₆H₁₀ClN₆OS [M-H]^- 369.0325; found 369.0310.

  2, 4-Diamino-9-methoxy-5-(1-methyl-1H-imidazol-2-yl-thio)-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8e): Ye- llow solid, yield 79%. m.p. 258.6.2~261.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.14 (s, 1H, imidazole-H), 7.08 (d, J=2.0 Hz, 2H, ArH), 7.07 (s, 1H, imidazole-H), 6.78~6.76 (m, 1H, ArH), 6.75 (s, 2H, NH₂), 6.69 (d, J=2.4 Hz, 1H, methine-H), 6.62 (s, 2H, NH₂), 3.86 (s, 3H, OCH₃), 3.52 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 162.7, 162.0, 157.2, 144.6, 139.7, 138.8, 126.8, 125.9, 124.3, 123.8, 120.5, 114.8, 111.0, 85.8, 73.4, 55.9, 48.5, 34.3. HRMS (ESI) calcd for C₁₈H₁₅N₆O₂S [M-H]^- 379.0977; found 379.0969.

  2, 4-Diamino-7-methyl-5-(1-methyl-1H-imidazol-2-yl-thio)-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8f): Yel- low solid, yield 87%. m.p. 295.4~296.1 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.22 (d, J=8.0 Hz, 1H, imidazole-H), 7.14 (s, 1H, ArH), 7.11 (s, 1H, ArH), 7.09 (d, J=2.4 Hz, 1H, ArH), 7.01 (s, 1H, imidazole-H), 6.74 (s, 2H, NH₂), 6.67 (d, J=2.4 Hz, 1H, methane-H), 6.64 (s, 2H, NH₂), 3.53 (s, 3H, CH₃), 2.23 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.5, 163.9, 157.4, 139.3, 138.8, 136.7, 136.1, 134.3, 131.5, 125.6, 124.2, 123.3, 113.9, 99.3, 77.1, 43.5, 35.2, 27.1. HRMS (ESI) calcd for C₁₈H₁₅- N₆OS [M-H]^- 363.1028; found 363.1059.

  2, 4-Diamino-7-chloro-5-(1-methyl-1H-imidazol-2-yl-thio)-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8g): Ye- llow solid, yield 85%. m.p. 281.1~283.4 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.48 (dd, J=7.2, 2.4 Hz, 1H, imidazole-H), 7.29 (d, J=4.8 Hz, 1H, ArH), 7.19~7.18 (m, 2H, ArH), 7.12 (d, J=2.4 Hz, 1H, imidazole-H), 6.82 (s, 2H, NH₂), 6.79 (d, J=2.4 Hz, 1H, methine-H), 6.56 (s, 2H, NH₂), 3.54 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 166.9, 160.4, 156.0, 149.7, 148.7, 130.8, 130.6, 129.1, 128.2, 127.6, 126.0, 113.7, 102.9, 96.5, 71.7, 42.8, 33.0. HRMS (ESI) calcd for C₁₇H₁₂ClN₆OS [M-H]^- 383.0482; found 383.0463.

  2, 4-Diamino-5-(1-methyl-1H-imidazol-2-ylthio)-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8h): Orange solid, yield 89%. m.p. 257.6~259.4 ℃; ¹H NMR (400 MHz, DMSO) δ: 7.43~7.39 (m, 1H, imidazole-H), 7.23 (d, J=8.4 Hz, 2H, ArH), 7.16 (d, J=4.4 Hz, 2H, ArH), 7.09 (d, J=2.4 Hz, 1H, imidazole-H), 6.77 (s, 2H, NH₂), 6.71 (d, J=2.4 Hz, 1H, methane-H), 6.62 (s, 2H, NH₂), 3.52 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 160.4, 157.2, 149.9, 148.6, 143.1, 138.6, 130.2, 127.3, 126.6, 125.9, 119.4, 116.9, 114.8, 105.6, 78.0, 43.5, 34.3. HRMS (ESI) calcd for C₁₇H₁₃N₆OS [M-H]^- 349.0872; found 349.0874.

  2, 4-Diamino-9-methyl-5-(1-methyl-1H-imidazol-2-yl-thio)-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8i): Ye- llow solid, yield 80%. m.p. 266.5~270.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.28~7.26 (m, 1H, imidazole-H), 7.15 (s, 1H, ArH), 7.08 (d, J=2.4 Hz, 1H, imidazole-H), 7.05~7.04 (m, 2H, ArH), 6.82 (s, 2H, NH₂), 6.69 (d, J=2.4 Hz, 1H, methane-H), 6.63 (s, 2H, NH₂), 3.52 (s, 3H, CH₃), 2.34 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 160.4, 159.2, 157.2, 152.4, 148.2, 145.9, 125.7, 120.5, 119.4, 119.3, 118.9, 114.8, 112.7, 85.9, 70.3, 43.9, 34.3, 15.6. HRMS (ESI) calcd for C₁₈H₁₅N₆OS [M-H]^- 363.1028; found 363.1063.

  5-(1H-Imidazol-2-ylthio)-2, 4-diamino-9-methoxy-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8j): Yellow solid, yield 77%. m.p. 272.4~274.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.45 (s, 1H, NH), 7.14 (s, 1H, imidazole-H), 7.09~7.08 (m, 3H, ArH), 6.87 (t, J=2.2 Hz, 1H, imidazole-H), 6.74 (s, 2H, NH₂), 6.70 (s, 2H, NH₂), 6.64 (t, J=1.6 Hz, 1H, methane-H), 3.87 (s, 3H, OCH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 160.3, 159.2, 157.2, 149.7, 147.4, 139.7, 131.6, 126.6, 125.0, 119.2, 115.9, 115.5, 112.2, 88.2, 70.3, 55.8, 47.0. HRMS (ESI) calcd for C₁₇H₁₃N₆O₂S [M-H]^- 365.0821; found 365.0865.

  5-(1H-Imidazol-2-ylthio)-2, 4-diamino-7-methyl-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8k): Yellow solid, yield 80%. m.p. 295.4~296.1 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.96 (s, 1H, NH), 7.26 (d, J=8.4 Hz, 1H, imidazole-H), 7.20 (s, 1H, ArH), 7.12 (s, 1H, imidazole-H), 7.10 (s, 2H, ArH), 6.82~6.73 (m, 1H, NH₂), 6.69 (s, 2H, NH₂), 6.62 (d, J=1.8 Hz, 1H, methane-H), 2.33 (s, 3H, CH₃); ¹³C NMR (100MHz, DMSO-d₆) δ: 162.3, 160.4, 156.9, 149.4, 142.7, 133.1, 130.6, 129.0, 126.1, 117.5, 116.5, 112.9, 98.6, 83.6, 70.3, 42.9, 20.3. HRMS (ESI) calcd for C₁₇H₁₃N₆OS [M-H]^- 349.0872; found 349.0841.

  5-(1H-Imidazol-2-ylthio)-2, 4-diamino-9-fluoro-5H-chro-meno[2, 3-b]pyridine-3-carbonitrile (8l): Yellow solid, yield 81%. m.p. 289.1~291.7 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.49 (s, 1H, NH), 7.37 (s, 1H, imidazole- H), 7.20 (s, 1H, ArH), 7.17~7.13 (s, 1H, ArH), 7.03 (d, J=8.0 Hz, 1H, imidazole-H), 6.90 (s, 1H, ArH), 6.84 (s, 2H, NH₂), 6.81 (s, 1H, methane-H), 6.72 (s, 2H, NH₂); ¹³C NMR (100 MHz, DMSO-d₆) δ: 162.5, 160.8, 156.1, 155.4 (d, J=240 Hz), 141.9, 129.8, 127.8 (d, J=20 Hz), 127.7, 127.6 (d, J=8 Hz), 127.1, 124.5, 121.8 (d, J=10 Hz), 117.4 (d, J=20 Hz), 93.3, 77.9, 43.1. HRMS (ESI) calcd for C₁₆H₁₀FN₆OS [M-H]^- 353.0621; found 353.0639.

  2, 4-Diamino-7-hydroxy-5-(1-methyl-1H-imidazol-2-yl-thio)-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8m): Yellow solid, yield 79%. m.p. 255.6~258.2 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.48 (s, 1H, OH), 7.10 (d, J=2.4 Hz, 1H, imidazole-H), 7.07~7.05 (m, 2H, ArH), 6.92 (dd, J=8.8, 2.8 Hz, 1H, imidazole-H), 6.70 (s, 2H, NH₂), 6.67 (d, J=2.4 Hz, 1H, ArH), 6.66 (d, J=3.2 Hz, 1H, methane-H), 6.58 (s, 2H, NH₂), 3.53 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 169.5, 168.3, 166.4, 153.7, 146.8, 142.6, 141.6, 140.9, 139.4, 135.0, 117.0, 114.0, 112.7, 95.6, 54.7, 44.3, 34.3. HRMS (ESI) calcd for C₁₇H₁₃N₆O₂S [M-H]^- 365.0821; found 365.0838.

  5-(1H-Imidazol-2-ylthio)-2, 4-diamino-7-hydroxy-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8n): Brick red solid, yield 83%. m.p. 260..3~261.5 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.46 (s, 1H, NH), 9.49 (s, 1H, OH), 7.06 (d, J=3.2 Hz, 1H, imidazole-H), 7.05 (s, 1H, ArH), 6.89 (t, 1H, J=2.4 Hz, imidazole-H), 6.80 (d, J=2.8 Hz, 1H, ArH), 6.77 (d, J=2.4 Hz, 1H, ArH), 6.69 (s, 2H, NH₂), 6.65~6.63 (m, 2H, NH₂), 6.62 (t, J=6.0 Hz, 1H, methine-H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 166.2, 166.0, 156.1, 134.2, 133.2, 133.0, 132.6, 131.8, 131.6, 130.1, 129.2, 128.5, 128.0, 89.8, 72.9, 45.54. HRMS (ESI) calcd for C₁₆H₁₁N₆O₂S [M-H]^- 351.0664; found 351.0680.

  5-(1H-Imidazol-2-ylthio)-2, 4-diamino-7-nitro-5H-chro-meno[2, 3-b]pyridine-3-carbonitrile (8o): Yellow solid, yield 88%. m.p. 233.5~235.5 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.46 (s, 1H, NH), 7.23 (dd, J=8.8, 2.8 Hz, 1H, imidazole-H), 7.14 (s, 1H, ArH), 7.06 (d, J=3.2 Hz, 1H, imidazole-H), 6.80~6.78 (m, 2H, ArH), 6.69 (s, 2H, NH₂), 6.65~6.64 (m, 1H, methane-H), 6.43 (s, 2H, NH₂); ¹³C NMR (100 MHz, DMSO-d₆) δ: 162.2, 159.4, 154.3, 153.3, 144.4, 142.8, 141.7, 137.7, 133.6, 130.1, 128.0, 125.9, 124.5, 108.5, 75.7, 43.3. HRMS (ESI) calcd for C₁₆H₁₀N₇O₃S [M-H]^- 380.0566; found 380.0557.

  2, 4-Diamino-5-(1-methyl-1H-imidazol-2-ylthio)-7-nitro-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8p): Orange solid, yield 84%. m.p. 279.9~282.4 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.27 (dd, J=9.2, 2.8 Hz, 1H, imidazole-H), 8.06 (d, J=2.8 Hz, 1H, ArH), 7.49 (d, J=5.2 Hz, 1H, imidazole-H), 7.31 (s, 1H, ArH), 7.14 (d, J=2.4 Hz, 1H, ArH), 6.91~6.90 (m, 2H, NH₂), 6.87 (s, 1H, methine-H), 6.61 (s, 2H, NH₂), 3.54 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.8, 162.3, 158.5, 156.6, 135.9, 135.4, 126.1, 124.5, 124.1, 118.5, 109.3, 106.3, 98.3, 93.7, 73.8, 42.0, 34.5. HRMS (ESI) calcd for C₁₇H₁₂N₇O₃S [M-H]^- 394.0722; found 394.0719.

  2, 4-Diamino-7-bromo-5-(1-methyl-1H-imidazol-2-yl-thio)-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8q): Yellow solid, yield 87%. m.p. 286.3~288.1 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.59 (dd, J=8.8, 2.0 Hz, 1H, imidazole-H), 7.32 (d, J=2.0 Hz, 1H, ArH), 7.24 (d, J=8.8 Hz, 1H, imidazole-H), 7.18 (s, 1H, ArH), 7.12 (d, J=2.0 Hz, 1H, ArH), 6.81 (s, 2H, NH₂), 6.79 (d, J=2.4 Hz, , 1H, methine-H), 6.57 (s, 2H, NH₂), 3.54 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 162.4, 162.3, 160.4, 157.1, 152.4, 152.1, 148.1, 142.9, 135.5, 124.6, 119.5, 114.2, 112.7, 94.6, 77.3, 41.6, 34.4. HRMS (ESI) calcd for C₁₇H₁₂BrN₆OS [M-H]^- 426.9977; found 426.9993.

  5-(1H-Imidazol-2-ylthio)-2, 4-diamino-7-bromo-5H-chro-meno[2, 3-b]pyridine-3-carbonitrile (8r): Yellow solid, yield 86%. m.p. 279.4~282.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.52 (s, 1H, NH), 7.61~7.57 (m, 1H, imidazole-H), 7.32 (d, J=2.0 Hz, 1H, ArH), 7.23 (d, J=8.8 Hz, 1H, imidazole-H), 7.17 (s, 1H, ArH), 6.92 (s, 1H, ArH), 6.81 (s, 2H, NH₂), 6.74 (s, 1H, methane-H), 6.63 (s, 2H, NH₂); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.7, 161.5, 154.9, 151.5, 150.8, 143.7, 137.4, 136.6, 136.2, 116.6, 113.1, 112.7, 94.3, 93.7, 75.5, 45.0. HRMS (ESI) calcd for C₁₆H₁₀BrN₆OS [M-H]^-: 412.9820; found 412.9862.

  5-(1H-Imidazol-2-ylthio)-2, 4-diamino-7-fluoro-5H-chromeno[2, 3-b]pyridine-3-carbonitrile (8s): Yellow solid, yield 78%. m.p. 276.9~280.1 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.51 (s, 1H, NH), 7.33~7.29 (m, 3H, ArH), 7.19~7.16 (m, 1H, imidazole-H), 7.12 (s, 2H, NH₂), 6.97~6.91 (m, 1H, imidazole-H), 6.75 (s, 1H, NH₂), 6.62 (s, 1H, methine-H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 160.4, 160.2, 157.0 (d, J=240 Hz), 148.0, 128.7, 119.2 (d, J=7 Hz), 118.6, 116.2, 115.4, 115.1, 114.9, 113.6, 113.3 (d, J=23 Hz), 112.7, 82.8, 70.5. HRMS (ESI) calcd for C₁₆H₁₀FN₆OS [M-H]^- 353.0621; found 353.0612.

  4.6   Materials and methods of bioactivity in vitro

  Human breast adenocarcinoma cell line (MCF-7), adenocarcinomic human alveolarbasal epithelial cells (A549) and human bronchial epithelial cells (HBE) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 ℃ in 5% CO₂ atmosphere. Cell viability was determined using the MTT assay (Sigma). In brief, cancer cells with same number were inoculated into each well in 96-well plates (Costar, Charlotte, NC) in 100 μL culture medium. After an overnight incubation, various concentrations of compounds were added for 48 h, and then MTT (1 mg/mL) was added to each well and incubated for 3 h at 37 ℃. Then, the medium was removed and MTT-formazan complex was dissolved in DMSO. The plates were then placed on a shaking table for 10 min to solubilize crystals adequately. The absorbance was measured using a SpectraMax M2 (Molecular Devices) at λ_ex=570 nm and λ_em=630 nm. The experiment was repeated 3 times. The 50% inhibition concentration (IC₅₀) was calculated by the following formula:

  $\lg \mathrm{IC}_{50}=X_{\mathrm{m}}-I\left(P-\left(3-P_{\mathrm{m}}-P_{\mathrm{n}}\right) / 4\right) $

  X_m is lg(maximum dose), I is lg(maximum dose/adjacent dose), P is sum of the positive response rates, P_m is maximum positive response rate, P_n is minimum positive response rate, determination of antibacterial activities

  4.7   Antitumor activity in vitro of the products

  In the determination of antibacterial activities, the stock solution (10 mg/mL) of every product was prepared by dissolving relative compound in DMSO. The solution was diluted to 1, 10 or 100 g/mL, respectively, and then the solutions were mixed with bacterial solution at ratio of 1:99 (the solution was diluted 100 times with the bacterial solution), and the final concentrations of the compound were 0.01, 0.1 and 1 g/mL, and the content of DMSO was 1%. The same concentrations (1, 10 and 100 g/mL, respectively) of ampicillin (AP), kanamycin (KAN) and meropenem were used as the controls. The experiment was performed in triplicate.

  Supporting Information  NMR of synthetic compounds. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn/.

  
  
• 1. [1]

     Weinstein, D. S.; Gong, H.; Doweyko, A. M. J. Med. Chem. 2011, 54, 7318. doi: 10.1021/jm200879j

  2. [2]

     Kolokythas, G.; Pouli, N.; Marakos, P.; Pratsinis, H.; Kletsas, D. Eur. J. Med. Chem. 2006, 41, 71. doi: 10.1016/j.ejmech.2005.10.011

  3. [3]

     Azuine, M. A.; Tokuda, H.; Takayasu, J. J. Pharm. Pharmacol. 2004, 49, 161. doi: 10.1016/j.phrs.2003.07.014

  4. [4]

     Srivastava, S.; Tripathi, K. R. P.; Ramachandran, R. J. Biol. Chem. 2005, 280, 30273. doi: 10.1074/jbc.M503780200

  5. [5]

     Evdokimov, N. M.; Kireev, A. S.; Yakovenko, A. A. J. Org. Chem. 2007, 72, 3443. doi: 10.1021/jo070114u

  6. [6]

     Anderson, D. R.; Hegde, S.; Reinhard, E.; Bioorg. Med. Chem. Lett. 2005, 15, 1587. doi: 10.1016/j.bmcl.2005.01.067

  7. [7]

     Bristol, J. A.; Gold, E. H.; Gross, I.; Lovey, R. G.; Long, J. J. Med. Chem. 1981, 24, 1010. doi: 10.1021/jm00140a020

  8. [8]

     Venkati, M.; Krupadananm, G. L. D. Synth. Commun. 2001, 31, 2589. doi: 10.1081/SCC-100105383

  9. [9]

     Nohara, A.; Ishiguro, T.; Ukawa, K.; Sugihara, H.; Maki, Y.; Sanno, Y. J. Med. Chem. 1985, 28, 559. doi: 10.1021/jm50001a005

  10. [10]

      Martin, M. J.; La-casa, C.; Alarcon-de-la-Lastra, C.; Cabeza, J.; Villegas, I.; Motilva, V. J. Med. Chem. 1998, 53, 82. doi: 10.1515/znc-1998-1-215

  11. [11]

      Kemnitzer, W.; Drewe, J.; Jiang, S.; Zhang, H.; Labreque, D.; Bubenick, M.; Denis, R.; Lamothe, S.; Gourdeau, H.; Tseng, B.; Kasibhatla, S.; Cai, S. X. J. Med. Chem. 2008, 51, 417. doi: 10.1021/jm7010657

  12. [12]

      Raju, R. R.; Mohan, S. K.; Reddy, S. J. J. Sci. Ind. Res. 2003, 62, 334. https://www.researchgate.net/publication/286588124_Novel_synthesis_of_non-steroidal_anti-inflammatory_drugs_by_electrocatalytic_hydrogenation

  13. [13]

      Panda, D.; Singh, J. P.; Wilson, L. J. Biol. Chem. 1997, 272, 7681. doi: 10.1074/jbc.272.12.7681

  14. [14]

      Haveliwala, D. D.; Kamdar, N. R.; Mistry, P. T.; Patel, S. K. Helv. Chim. Acta 2013, 96, 897. doi: 10.1002/hlca.201200121

  15. [15]

      Kamdar, N. R.; Haveliwala, D. D.; Mistry, P. T.; Patel, S. K. Eur. J. Med. Chem. 2010, 45, 5056. doi: 10.1016/j.ejmech.2010.08.014

  16. [16]

      Lee, X. P.; Kumazawa, T.; Hasegawa, C.; Arinobi, T.; Kato, A.; Seno, H.; Sato, K. Forensic Toxicol. 2010, 28, 96. doi: 10.1007/s11419-010-0096-8

  17. [17]

      Kemnitzer, W.; Drewe, J.; Jiang, S.; Zhang, H.; Zhao, J.; Crogan-Grundy, C.; Xu, L.; Lamothe, S.; Gourdeau, H.; Denis, R.; Tseng, B.; Kasibhatla, S.; Cai, S. X. J. Med. Chem. 2007, 50, 2858. doi: 10.1021/jm070216c

  18. [18]

      Akyol-Salman, I.; Lece-Sertoz, D.; Baykal, O. J. Ocul. Pharmacol. Ther. 2007, 23, 280. doi: 10.1089/jop.2006.108

  19. [19]

      Mishra, S.; Ghosh, R. Synth. Commun. 2012, 42, 2229. doi: 10.1080/00397911.2011.555284

  20. [20]

      Evdokimov, N. M.; Kireev, A. S.; Yakovenko, A. A.; Antipin, M. Y.; Magedov, I. V.; Kornienko, A. Tetrahedron Lett. 2006, 47, 9309. doi: 10.1016/j.tetlet.2006.10.110

  21. [21]

      Siddiqui, Z. N.; Khan, K. New J. Chem. 2013, 37, 1595. doi: 10.1039/c3nj00069a

  22. [22]

      Tröger, J. J. Prak. Chem. 1887, 36, 225. doi: 10.1002/prac.18870360123

  23. [23]

      Satishkumar, S.; Periasamy, M.; Tetrahedron:Asymmetry 2009, 20, 2257. doi: 10.1016/j.tetasy.2009.08.030

  24. [24]

      Kejík, Z.; Bříza, T.; Havlík, M.; Dolenský, B.; Kaplánek, R.; Králová, J.; Mikula, I.; Martásek, P. Dyes Pigm. 2016, 134, 212. doi: 10.1016/j.dyepig.2016.07.002

  25. [25]

      Veale, E. B.; Frimannsson, D. O.; Lawler, M.; Gunnlaugsson, T. Org. Lett. 2009, 11, 4040. doi: 10.1021/ol9013602

  26. [26]

      Paul, A.; Maji, B.; Misra, S. K.; A. Jain, K.; Muniyappa, K.; Bhattacharya, S. J. Med. Chem. 2012, 55, 7460. doi: 10.1021/jm300442r

  27. [27]

      Yuan, R.; Li, M. Q.; Xu, J. B.; Huang, S. Y.; Zhou, S. L.; Zhang, P.; Liu, J. J.; Wu, H. Tetrahedron 2016, 72, 4081. doi: 10.1016/j.tet.2016.05.042

  28. [28]

      Xiao, Y. C.; Zhang, L. L.; Xu, L.; Chung, T. S. J. Membr. Sci. 2017, 521, 65. doi: 10.1016/j.memsci.2016.08.052

  29. [29]

      Ishiwari, F.; Takeuchi, N.; Sato, T.; Yamazaki, H.; Osuga, R.; Kondo, J. N.; Fukushima, T. ACS Macro Lett. 2017, 6, 775. doi: 10.1021/acsmacrolett.7b00385

  30. [30]

      Yuan, R.; Wang, Y. J.; Fang, Y.; Ge, W. H.; Lin, W.; Li, M. Q.; Xu, J. B.; Wan, Y.; Liu, Y.; Wu, H. Chem. Eng. J. 2017, 316, 1026. doi: 10.1016/j.cej.2017.02.026

  31. [31]

      Brown, H. C.; McDaniel, D. H.; Hafliger, O. In Determination of Organic Structures by Physical Methods, Eds.: Braude, E. A.; Nachod, F. C., Academic Press, New York, 1955, 9, p. 22.

  32. [32]

      Parsons, R. Handbook of Electrochemical Constants, Butterworths Scientific Publications, London, 1959, p. 35.

• 

  Figure 1  Structure of Trögerʼs base

  下载: 全尺寸图片 幻灯片
  

  Figure 1  Synthesized Schiff bases

  下载: 全尺寸图片 幻灯片
  

  Figure 2  Changes of the signals as the reaction proceeded

  下载: 全尺寸图片 幻灯片
  

  Scheme 3  A supposed mechanism

  下载: 全尺寸图片 幻灯片

  Table 1.  pK_a values and yields of 4^a

  Compd.	pKa	R	yield/%
  TB	9.05	—
  1	9.13	—
  2	9.28	—
  4a	9.26	Thiophene-2-yl	93
  4b	9.20	3-Methyl-thiophene-2-yl	91
  4c	9.21	5-Methyl-thiophene-2-yl	88
  4d	9.11	Furan-2-yl	89
  4e	9.13	5-Methyl-furan-2-yl	86
  4f	9.35	Pyridine-2-yl	85
  4g	9.06	5-Methyl-pyridine-2-yl	81
  a Calculated from the pH value tested by dissolving 0.01 mmol of compounds in appropriate amount of ethanol and diluting to 10.0 mL with ethanol.

  下载: 导出CSV

  Table 2.  Synthesis of 8h under different reaction conditions^a

  Entry	Cat.	Cat./mol%	Solvent	t/h	T/℃	Yieldb/%
  1	None	—	EtOH	6	Reflux	—
  2	Pyridine	20	EtOH	6	Reflux	—
  3	2, 2'-Dipyridyl	20	EtOH	6	reflux	—
  4	NaHCO3	20	EtOH	6	Reflux	—
  5	CsCO3	20	EtOH	6	Reflux	—
  6	K3PO4	20	EtOH	6	Reflux	—
  7	NaOH	20	EtOH	6	Reflux	—
  8	EtONa	20	EtOH	6	Reflux	—
  9		20	EtOH	6	Reflux	—
  10	2	20	EtOH	6	Reflux	16
  11	3a	20	EtOH	6	Reflux	—
  12	3b	20	EtOH	6	Reflux	—
  13	3c	20	EtOH	6	Reflux	—
  14	3d	20	EtOH	6	Reflux	—
  15	3e	20	EtOH	6	Reflux	—
  16	3f	20	EtOH	6	Reflux	—
  17	3g	20	EtOH	6	Reflux	—
  18	3h	20	EtOH	6	Reflux	15
  19	4a	20	EtOH	6	Reflux	71
  20	4b	20	EtOH	6	Reflux	63
  21	4c	20	EtOH	6	Reflux	58
  22	4d	20	EtOH	6	Reflux	78
  23	4e	20	EtOH	6	Reflux	70
  24	4f	20	EtOH	6	Reflux	89
  25	4g	20	EtOH	6	Reflux	85
  26	4f	5	EtOH	6	Reflux	35
  27	4f	10	EtOH	6	Reflux	51
  28	4f	15	EtOH	6	Reflux	62
  29	4f	25	EtOH	6	Reflux	91
  30	4f	20	MeCN	6	Reflux	35
  31	4f	20	DCM	6	Reflux	38
  32	4f	20	H2O	6	Reflux	46
  33	4f	20	EtOH	6	r.t.	—
  34	4f	20	EtOH	6	40	53
  aUnless otherwise noted, the reactions were carried out using salicylaldehyde (1 mmol), malononitrile (2 mmol) and 1-methyl-1H-imidazole-2-thiol (1 mmol).

  下载: 导出CSV

  Table 3.  Synthesis of 8 under the optimal conditions

  Entry	R1	R2	Product	Yield/%
  1	3-OH	H	8a	86
  2	3-OH	CH3	8b	83
  3	4-OH	H	8c	76
  4	5-Cl	H	8d	61
  5	3-CH3O	CH3	8e	79
  6	5-CH3	CH3	8f	87
  7	5-Cl	CH3	8g	85
  8	H	CH3	8h	89
  9	3-CH3	CH3	8i	80
  10	3-CH3O	H	8j	77
  11	5- CH3	H	8k	80
  12	3-F	H	8l	81
  13	5-OH	CH3	8m	79
  14	5-OH	H	8n	83
  15	5-NO2	H	8o	88
  16	5-NO2	CH3	8p	84
  17	5-Br	CH3	8q	87
  18	5-Br	H	8r	86
  19	5-F	H	8s	78
  As the signals in the aromatic hydrogen area merged together, it is hard to figure out the characteristic peaks in the 1H NMR spectrum. Thus, the research was focus on the signals in δ 4.00~4.65 (Figure 2).

  下载: 导出CSV

  Table 4.  IC₅₀ value of compound 8 against antitumor and normal cells^a

  Entry	A549	MCF-7	HBE
  8a	—	12.2	16.3
  8b	—	12.2	15.3
  8d	—	17.9	15.7
  8f	—	15.1	17.4
  8h	39.7	8.4	4.9
  8i	—	16.4	11.5
  8j	—	15.5	26.7
  8m	—	16.2	15.8
  8n	—	13.8	15.1
  8r	40.9	—	11.3
  8s	—	11.1	7.2
  Adriamycin	4.05	0.027	7.01
  Cisplatin	27.59	66.61	7.77
  a IC50 > 64 μg/mL were marked with "—".

  下载: 导出CSV

  Table 5.  Inhibition ratio of 8s on two bacteria

  Bacterium	Inhibition ratio/%
  	16	32	64	128
  Staphylococcus aureus	-	—	84.82	90.84
  Escherichia coli	-	94.56	100.00	100.00
  a The concentration (µg/mL) of 8s; bNo inhibition.

  下载: 导出CSV
•