Phenazine Derivatives - Ingenta Connect

Send Orders for Reprints to [email protected] Combinatorial Chemistry & High Throughput Screening, 2015, 18, 960-974

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Synthesis, Antitumor Activity, and Structure-Activity Relationship of Some Benzo[a]Pyrano[2,3-c]Phenazine Derivatives Jing Gao1, Ming Chen2, Xue Tong1, He Zhu1, Hongbin Yan2, Daichun Liu3, Wanjing Li3, Shengyu Qi3, Dake Xiao3, Yongzhi Wang3, Yuanyuan Lu1 and Feng Jiang*,3 1

School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, China

2

School of Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China

3

School of Engineering, China Pharmaceutical University, Nanjing, Jiangsu, China Abstract: A series of benzo[a]pyrano[2,3-c]phenazine derivatives with a wide range of substitutions at ring C of the benzophenazine were designed and synthesized using the one-pot, four-component domino reactions. The targeted compounds were evaluated for their antitumor activities against HCT116, MCF7, HepG2 and A549 cancer cell lines in vitro. The most active compound 6{1,2,1,9} Feng Jiang featured the CN and p-dimethylamino phenyl substituents on the γ-pyran structure on ring C. Significantly, compound 6{1,2,1,9} was found to have the highest growth inhibitory activity against the HepG2 cell line with the IC50 value of 6.71 µM, which was 1.6-fold more potent than positive control anticancer drug Hydroxycamptothecine (HCPT). Furthermore, structure-activity relationship (SAR) studies on the substitutions at ring C were discussed in details.

Keywords: Antitumor activity, benzo[a]pyrano[2,3-c]phenazine, SAR. 1. INTRODUCTION Novel diverse polycyclic heterocycles with potential medicinal and biological activity have been paid great attention at the interface of the fields of combinatorial chemistry, medicinal chemistry and chemical biology [1]. Diversity-oriented synthesis (DOS) has been becoming an important synthetic method to invent diverse small molecules, particularly those possessing skeletons resembling those found in natural products or drug-like molecules [2]. In DOS chemistry, multicomponent reactions (MCRs), which are the type of convergent, atom-economic, expedient, eco-friendly single-step chemical reactions, provide a powerful tool to produce diversely bioactive and regiochemical compound libraries [3]. Phenazines and pyrans are significant classes of heterocyclic compounds. A great number of natural and synthetic phenazines have been proved to possess various biological activities, including antimalarial [4], trypanocidal [5], fungicidal [6], antitumor [7] and antiplatelet [8] activities. The heterocyclic pyran structure is usually a functional framework which is present in a number of important pharmaceuticals and natural products such as carbohydrates, alkaloids, polyether antibiotics, pheromones and iridoids [9]. Owing to phenazine’s flat aromatic intercalative conjugated structures as well as structural characteristics of groove-binders or specific enzyme-binders, phenazine derivatives might potentially lead to selective and high*Address correspondence to this author at the School of Engineering, China Pharmaceutical University, Nanjing, Jiangsu 210009, China; Tel: 86 025 86185213; E-mail: [email protected] 1875-5402/15 $58.00+.00

affinity binders targeting DNA and DNA-enzyme complexes [7]. XR11576 (Fig. 1), a phenazine derivative which was modified on ring A and ring D of the benzophenazine, has been proved to be a potent antitumor compound [5, 10]. Although the synthesis of benzo[a]pyrano[2,3c]phenazine was reported in early times [11], to the best of our knowledge, the biological evaluation of benzo[a] pyrano[2,3-c]phenazine derivatives has not been reported. In our previous study [12], we found that substituted phenazines might have great cytotoxic activities against tumor cells. However, the synthesis of substituted phenazines is usually a challenge for the synthetic chemist because of the limited commercial availability. No efficient and generally applicable synthesis of substituted phenazines exists. Most of the reported synthetic methods have severe limitations to localization and electronic character of the substituents [7]. Considering synthetic accessibility, we aimed at designing a simple and feasible procedure to prepare substituted phenazines that contain substituent on ring C of the benzophenazine instead of the ring A and ring D of XR11576, as they could be readily accessible while maintaining potential antitumor activity. Meanwhile, in an effort to gain more insight into the effect of substituent groups on ring C, we intended to construct a structurally diverse compound library composed of substituted benzophenazines by introducing multisubstituted γ-pyran structure on ring C, and further investigated the antitumor activity and detailed structure-activity relationship (SAR) of these compounds. Accordingly, here we report the synthesis of a series of benzo[a]pyrano[2,3-c]phenazine derivatives (Fig. 1) via the one-pot, four-component domino reactions, and the biological evaluation of these compounds. Significantly, Compound 6{1,2,1,9} was found to possess a © 2015 Bentham Science Publishers

Benzo[a]Pyrano[2,3-c]Phenazine Derivatives

Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 10

961

N NH R2 A

O N

N

N

N

N

R3

R1

R4

B N R5 C

CN/COOEt

D

O NH2

O Benzophenazine

Benzo[a]pyrano[2,3-c]phenazine derivatives

XR 11576

Fig. (1). Bioactive heterocycle compounds based on benzophenazine structure.

better antitumor effect against HepG2 cancer cell in vitro compared to the clinical antitumor drug Hydroxycamptothecine (HCPT).

There is no obvious loss of catalytic activity to be observed after three cycles of the reaction. The synthetic pathway for the synthesis of the titled compounds consists of two steps. At first, benzo[a]phenazin5-ols are obtained from the condensation reaction of benzene-1, 2-diamine (1) and 2-hydroxy-1, 4naphthoquinone (2). Then, the resulting products are treated with alkylmalonates (4) and carbonyl compounds (5) to afford the related benzo[a]pyrano[2,3-c]phenazine derivatives as the desired products. This two-step procedure allows the one-pot four-component reaction to be controlled, avoiding the separation of intermediates as well as timeconsuming and costly purification processes.

2. RESULT AND DISCUSSION 2.1. Chemistry The synthesis of benzo[a]pyrano[2,3-c]phenazine derivatives was done according to following method [11e]. Firstly, benzene-1,2-diamine (1, 1 mmol) and 2-hydroxy-1,4naphthoquinone (2, 1 mmol) were added to a 25- mL roundbottomed flask containing DABCO (0.3 mmol) in ethanol under reflux conditions to form the corresponding phenazine (3). Subsequently, malono derivatives (4, 1 mmol, Fig. 2), and structurally diverse aromatic aldehydes (5, 1 mmol, Fig. 3) were added and the mixture proceeded under reflux conditions (Scheme 1). After completion of the reaction, as monitored by TLC, the reaction mixture was allowed to cool to room temperature. The solid product was filtered, dried, and subsequently recrystallized from hot ethanol. In order to recover the catalyst, ethanol was evaporated under reduced pressure, and the resulting solid was washed with ethyl acetate and was kept in refrigerator to separate DABCO as crystals. The recovered catalyst was reused three times.

Using the similar reaction conditions, the reaction scope was evaluated by using malononitrile 4{1} or ethyl cyanoacetate 4{2} (Fig. 2), and aromatic aldehydes 5{1}5{35} (Fig. 3). Commercially available aromatic aldehydes bearing either electron-withdrawing or electron-donating functional groups, such as fluoro (5{2}, 5{3}), chloro (5{4}, 5{5}, 5{23}, 5{24}, 5{25}, 5{26}), bromo (5{6}, 5{7}, 5{8}), nitro (5{12}, 5{13}, 5{14}), hydroxyl (5{11}), methyl (5{15}), ethyl (5{16}), isopropyl (5{17}), methoxy (5{18}, 5{19}, 5{20}, 5{21}, 5{22}, 5{27}, 5{28}, 5{29}, 5{30}, 5{31}, 5{32}, 5{33}, 5{34}, 5{35}), dimethylamino (5{9}) or R3 R2

R4

R1

R5

X H2 N

+

NH2

NC

1

N

N

OH

O

N

N

R4 R5

EtOH, Reflux

Reflux OH

O

R3

5

4

DABCO

+

CHO

R2 R1

X O NH2

3 6

2 X = CN or COOEt

Scheme 1. One-pot two-step tandem synthesis of benzo[a]pyrano[2,3-c]phenazine derivatives.

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Gaoet al.


diethylamino (5{10}) were all found to be suitable for the reaction to obtain benzo[a]pyrano[2,3-c]phenazine derivatives in good yields of 38-97% (Table 1). Given the numerous commercially available aromatic aldehydes, the present method could be applicable to the synthesis of phenazine-based compound libraries with high functional group diversity. CN

NC

COOEt

NC

1

Structures of these synthesized compounds were confirmed by FTIR, 1H NMR, 13C NMR spectroscopy and elemental analysis. In FTIR spectra of compounds 6 characteristic band of conjugated nitrile group (2184-2197 cm-1), as well as stretching bands of NH2 group (3113-3480 cm-1), bending bands of NH2 (1605-1664 cm-1) have been observed. The 1H NMR spectra of the title compounds featured chiral C(4)H proton singlet in the region 5.44-6.40 ppm and broad singlet of exchangeable NH2 group (7.07-7.56 ppm). 2.2. Biological Evaluation: SAR Studies

2

We synthesized fifty benzo[a]pyrano[2,3-c]phenazine derivatives in total. All of these compounds were further

Fig. (2). Malononitrile 4{1} and ethyl cyanoacetate 4{2}.

F

Cl

Br Br

Cl

F

Br

CHO

CHO

CHO

CHO

CHO

1

2

3

4

5

N

N

CHO

CHO

7

8

NO2

Me

Et

CHO

CHO

CHO

CHO 6

OH O2N

O2N CHO

CHO 9

10

CHO

CHO

CHO

12

11

13

15

14

OMe

OH

MeO

OMe

MeO

Cl

F

MeO

Cl

CHO

CHO

CHO

CHO

CHO 20

19

18

17

16

CHO

CHO

21

23

22

Cl

Cl

OMe

MeO Br

Cl Cl CHO 24

Cl

Cl CHO

CHO 25

OMe

MeO

MeO CHO

26

OMe

OMe

MeO CHO

CHO

CHO

27

28

30

29 Br

OMe

OMe

MeO

OMe

OMe

OMe

OMe OMe

MeO

OMe

Br

OMe

MeO

OMe

CHO

CHO

CHO

CHO

CHO

31

32

33

34

35

Fig. (3). Aromatic aldehyde components 5{1}-5{35}.


Table 1.


Synthesis of poly-substituted benzo[a]pyrano[2,3-c]phenazine derivatives 6.

Entry

Product

X

R1

R2

R3

R4

R5

Yield/%

1

6{1,2,1,1}

CN

H

H

2

6{1,2,1,2}

CN

F

H

H

H

H

94

H

H

H

3

6{1,2,1,3}

CN

H

H

F

91

H

H

90

4

6{1,2,1,4}

CN

Cl

H

5

6{1,2,1,5}

CN

H

H

H

H

H

92

Cl

H

H

6

6{1,2,1,6}

CN

Br

H

91

H

H

H

90

7

6{1,2,1,7}

CN

H

8

6{1,2,1,8}

CN

H

Br

H

H

H

88

H

Br

H

H

9

6{1,2,1,9}

CN

85

H

H

N(CH3)2

H

H

10

6{1,2,1,10}

CN

89

H

H

N(C2H5)2

H

H

86

11

6{1,2,1,11}

12

6{1,2,1,12}

CN

H

H

OH

H

H

84

CN

NO2

H

H

H

H

72

13 14

6{1,2,1,13}

CN

H

NO2

H

H

H

88

6{1,2,1,14}

CN

H

H

NO2

H

H

61

15

6{1,2,1,15}

CN

H

H

CH3

H

H

88

16

6{1,2,1,16}

CN

H

H

C2 H5

H

H

86

17

6{1,2,1,17}

CN

H

H

CH(CH3)2

H

H

87

18

6{1,2,1,18}

CN

OCH3

H

H

H

H

82

19

6{1,2,1,20}

CN

H

H

OCH3

H

H

97

20

6{1,2,1,21}

CN

H

H

OH

OCH3

H

93

21

6{1,2,1,22}

CN

H

H

OCH3

F

H

83

22

6{1,2,1,23}

CN

Cl

Cl

H

H

H

83

23

6{1,2,1,24}

CN

H

Cl

Cl

H

H

91

24

6{1,2,1,25}

CN

Cl

H

Cl

H

H

88

25

6{1,2,1,26}

CN

Cl

H

H

H

Cl

85

26

6{1,2,1,27}

CN

OCH3

H

H

Br

H

80

27

6{1,2,1,28}

CN

H

OCH3

OCH3

H

H

87

28

6{1,2,1,29}

CN

OCH3

H

H

OCH3

H

85

29

6{1,2,1,30}

CN

OCH3

H

OCH3

OCH3

H

79

30

6{1,2,1,31}

CN

H

OCH3

OCH3

OCH3

H

80

31

6{1,2,1,32}

CN

OCH3

H

OCH3

H

OCH3

83

32

6{1,2,1,33}

CN

OCH3

OCH3

OCH3

H

H

84

33

6{1,2,1,34}

CN

H

OCH3

Br

OCH3

H

88

34

6{1,2,1,35}

CN

H

Br

OCH3

OCH3

H

73

35

6{1,2,2,1}

COOEt

H

H

H

H

H

83

36

6{1,2,2,3}

COOEt

H

H

F

H

H

85

37

6{1,2,2,5}

COOEt

H

H

Cl

H

H

79

38

6{1,2,2,9}

COOEt

H

H

N(CH3)2

H

H

61

39

6{1,2,2,14}

COOEt

H

H

NO2

H

H

85

40

6{1,2,2,15}

COOEt

H

H

CH3

H

H

75

41

6{1,2,2,16}

COOEt

H

H

C2 H5

H

H

68

42

6{1,2,2,17}

COOEt

H

H

CH(CH3)2

H

H

82

43

6{1,2,2,18}

COOEt

OCH3

H

H

H

H

82

44

6{1,2,2,19}

COOEt

H

OCH3

H

H

H

82

45

6{1,2,2,21}

COOEt

H

H

OH

OCH3

H

69

46

6{1,2,2,23}

COOEt

Cl

Cl

H

H

H

71

47

6{1,2,2,28}

COOEt

H

OCH3

OCH3

H

H

60

48

6{1,2,2,29}

COOEt

OCH3

H

H

OCH3

H

59

49

6{1,2,2,30}

COOEt

OCH3

H

OCH3

OCH3

H

38

50

6{1,2,2,33}

COOEt

OCH3

OCH3

OCH3

H

H

43

963

964


investigated cytotoxicity evaluation in vitro against cultured HCT116 (human colorectal carcinoma), MCF7 (human breast adenocarcinoma), HepG2 (human hepatocellular carcinoma) and A549 (human lung adenocarcinoma) cell lines by the CCK8 (Cell Counting Kit-8) assay, respectively, using HCPT and Cisplatin as the reference drugs [13]. Since the tested compounds showed poor aqueous solubility, they were all dissolved in DMSO and then diluted in culture medium so that the effective DMSO concentration did not exceed 0.2%. The percent inhibition of viability for each concentration of the compounds was calculated with respect to the control and IC50 values were estimated with the software SPSS version 16.0 for Windows. Each experiment was repeated three times and the results were summarized in Table 2. Blank wells of all agents, containing the same concentrations of the tested compounds, had very low absorbance (data not shown). Therefore, interference of the color of the compounds in the assay seemed unlikely. The response parameter calculated was the IC50 value (Table 2), which corresponded to the compound concentration causing 50% mortality in net cells. As shown in Table 2, compound 6{1,2,1,9} exhibited the highest growth inhibitory activity against the HepG2 cell line with the IC50 value of 6.71 µM, which was 1.6-fold more potent than positive control HCPT with the IC50 value of 10.56 µM. However, it illustrated less potent against the HCT116, MCF7 and A549 cell lines than the positive controls in terms of IC50 values. Moreover, the other fortynine compounds illustrated poor or no activities against all of the four cell lines according to the data of Table 2. All compounds possessing COOEt substituent group in 3-position of γ-pyran ring showed weak or no activities against all of the four cell lines. While 3-position of γ-pyran ring was replaced by CN substituent group, some compounds exhibited excellent cytotoxic activities. For example, Compound 6{1,2,1,9} and 6{1,2,2,9} both possessed a pdimethylamino substituent group in benzene ring linking to chiral carbon atom of γ-pyran ring, and the only difference of their structure was the substituent group in 3-position of γpyran ring. When 3-position of γ-pyran ring was replaced by CN group rather than COOEt group, Compound 6{1,2,1,9} showed excellent cytotoxicity to HepG2 cell line, but Compound 6{1,2,2,9} illustrated no activity against all of the four cell lines. These results indicated that the CN substituent group played a significant role in antiproliferative activity. On the other hand, although the series 6{1,2,1,1}6{1,2,1,35} possessed CN substituent group in 3-position of γ-pyran ring, substituent groups’ variation in benzene ring linking to chiral carbon atom of γ-pyran ring could greatly affect their cytotoxic activities. For HCT116 cell line, compounds 6{1,2,1,9} and 6{1,2,1,11}, with respectively a pdimethylamino and p-hydroxyl substituent group, showed some cytotoxic activities with the IC50 values of 10.22 µM and 15.32 µM, respectively. For HepG2 cell line, only compound 6{1,2,1,9} with p-dimethylamino substituent group showed excellent cytotoxic activity with the IC50 value of 6.71 µM. But for MCF7 and A549 cell lines, all of these compounds showed weak or no activities no matter substituent groups in benzene ring linking to chiral carbon

Gaoet al.

atom of γ-pyran ring were electron-withdrawing or electrondonating functional groups, such as fluoro, chloro, bromo, nitro, hydroxyl, methyl, ethyl, isopropyl, methoxy, dimethylamino or diethylamino group. Therefore, we seemed to conclude that the CN and p-dimethylamino substituent groups played the crucial role in antiproliferative activity since omitting both of the two groups resulted in significant loss of the cytotoxic activity. 3. EXPERIMENTAL SECTION 3.1. General All commercially available chemicals and solvents were of analytical reagent grade and were used without further purification unless otherwise specified. Column chromatography was carried out on silica gel (100-300 mesh). TLC was conducted on silica gel 250 micron, GF254 plates with short-wavelength UV light for visualization. Melting points were measured on a SGWX-4 hot stage apparatus. Infrared spectra were measured on KBr pellets on a Nicolet IR200 FT-IR spectrometer in the range of 4000400 cm-1. 1H NMR and 13C NMR spectroscopic measurements were performed on a Bruker AV-500 NMR spectrometer, using TSP and TMS as internal references at 298 K, respectively. The elemental analysis was recorded on an Elementar Vario EL III analyzer. HCT116 (human colorectal carcinoma), MCF7 (human breast adenocarcinoma), HepG2 (human hepatocellular carcinoma) and A549 (human lung adenocarcinoma) cell lines were purchased from American type Cell Culture (ATCC, Shanghai, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and antibiotics. 3.2. General procedure for the Benzo[a]Pyrano[2,3-c]Phenazine (6)

Synthesis

of

Benzene-1,2-diamine (1, 1 mmol) and 2-hydroxy-1,4naphthoquinone (2, 1 mmol) were added to a 25- mL roundbottomed flask containing DABCO (0.3 mmol) in ethanol under reflux conditions to form the corresponding phenazine (3). Subsequently, malono derivatives (4, 1 mmol), and structurally diverse aromatic aldehydes (5, 1 mmol) were added and the mixture proceeded under reflux conditions. After completion of the reaction, as monitored by TLC, the reaction mixture was allowed to cool to room temperature. The solid product was filtered, dried, and subsequently recrystallized from hot ethanol. In order to recover the catalyst, ethanol was evaporated under reduced pressure, and the resulting solid was washed with ethyl acetate and was kept in refrigerator to separate DABCO as crystals. 3.2.1. Data for 3-Amino-1-phenyl-1H-benzo[a]pyrano[2,3c]phenazine-2-carbonitrile (6{1,2,1,1}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.19 (d, 1H, J = 8.0 Hz, Ar-H), 8.43 (d, 1H, J = 8.0 Hz, Ar-H), 8.25-8.22 (m, 1H, Ar-H), 8.13-8.11 (m, 1H, Ar-H), 8.00-7.97 (m, 1H, Ar-H), 7.93-7.89 (m, 3H, Ar-H), 7.40-7.38 (m, 4H, Ar-H), 7.21 (s, 2H, NH2), 7.09 (t, 1H, J = 7.0 Hz, Ar-H), 5.47 (s, 1H, CH); 13C NMR (125 MHz,


Table 2.


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Cytotoxicity (IC50, µM)a of the compounds against human tumor cell lines in vitro. IC50 (µM)

Compound No.

a

HCT116b

MCF7c

HepG2d

A549e

6{1,2,1,1} 6{1,2,1,2} 6{1,2,1,3}

86.52 160 79.23

160 160 160

65.45 160 58.68

160 160 160

6{1,2,1,4} 6{1,2,1,5} 6{1,2,1,6} 6{1,2,1,7} 6{1,2,1,8} 6{1,2,1,9}

74.45 160 160 83.12 102.34 10.22

67.85 78.43 160 160 67.22 51.02

160 160 160 75.13 45.46 6.71

160 160 53.23 66.45 56.15 25.18

6{1,2,1,10} 6{1,2,1,11} 6{1,2,1,12} 6{1,2,1,13} 6{1,2,1,14} 6{1,2,1,15}

89.46 15.32 160 86.57 59.55 160

66.43 62.32 160 160 160 160

45.08 41.25 76.45 160 160 160

160 53.16 160 160 160 160

6{1,2,1,16} 6{1,2,1,17} 6{1,2,1,18} 6{1,2,1,20} 6{1,2,1,21} 6{1,2,1,22}

160 160 160 83.16 141.23 160

160 56.34 76.46 160 160 160

160 160 61.42 55.38 160 160

160 67.78 160 73.62 160 160

6{1,2,1,23} 6{1,2,1,24} 6{1,2,1,25} 6{1,2,1,26} 6{1,2,1,27} 6{1,2,1,28}

58.33 76.68 160 85.56 52.13 76.77

132.12 82.24 160 160 160 95.56

72.28 160 160 50.22 68.34 64.38

67.44 135.86 160 160 160 160

6{1,2,1,29} 6{1,2,1,30} 6{1,2,1,31} 6{1,2,1,32} 6{1,2,1,33} 6{1,2,1,34}

53.14 83.22 84.12 160 160 160

160 160 122.36 59.43 75.37 160

160 160 160 51.25 43.16 74.87

85.34 51.36 160 160 160 55.38

6{1,2,1,35} 6{1,2,2,1} 6{1,2,2,3} 6{1,2,2,5} 6{1,2,2,9} 6{1,2,2,14}

50.12 62.15 160 160 160 160

160 160 160 160 160 160

160 160 52.12 44.58 160 160

160 160 160 160 48.96 135.87

6{1,2,2,15} 6{1,2,2,16} 6{1,2,2,17} 6{1,2,2,18} 6{1,2,2,19} 6{1,2,2,21}

87.56 58.98 160 160 76.46 53.23

160 88.42 160 160 160 160

55.76 160 160 160 65.24 160

160 160 160 160 160 73.24

6{1,2,2,23} 6{1,2,2,28} 6{1,2,2,29} 6{1,2,2,30} 6{1,2,2,33} HCPT

160 160 160 78.47 160 4.54

160 65.43 55.34 160 160 3.65

160 160 67.43 53.65 68.53 10.56

56.36 160 160 81.24 54.53 1.12

Cisplatin

1.86

6.58

0.47

2.78

IC50 is the drug concentration effective in inhibiting 50% of the cell growth measured by the CCK8 assay after 48 h drug exposure; bHuman colorectal carcinoma cell line; cHuman breast adenocarcinoma cell line; dHuman hepatocellular carcinoma cell line; eHuman lung adenocarcinoma cell line.

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DMSO-d6) (δ, ppm): 159.8, 146.2, 145.2, 141.5, 140.7, 140.1, 139.8, 130.7, 130.5, 130.2, 130.0, 129.1, 128.7, 128.3, 128.3, 127.5, 127.5, 126.5, 125.6, 124.9, 122.1, 113.9, 58.0, 37.4; IR (KBr, ν, cm-1): 3443, 3310, 3174, 2187, 1658, 1623, 1595, 1541, 1495, 1473, 1400, 1385, 1350, 1328, 1269, 1163, 1128, 1052, 1026, 760, 733, 702; Anal. Calcd. for C26H16N4O: C, 77.99; H, 4.03; N, 13.99%. Found: C, 77.86; H, 3.97; N, 13.87%. 3.2.2. Data for 3-Amino-1-(2-fluoro-phenyl)-1H-benzo[a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,2}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.26 (d, 1H, J = 8.0 Hz, Ar-H), 8.48 (d, 1H, J = 8.0 Hz, Ar-H), 8.29-8.28 (m, 1H, Ar-H), 8.07-7.92 (m, 5H, Ar-H), 7.36-7.32 (m, 3H, Ar-H and NH2), 7.16-7.00 (m, 3H, Ar-H), 5.77 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.6, 146.5, 145.2, 141.2, 140.7, 140.7, 139.8, 130.7, 130.5, 130.2, 130.1, 129.6, 128.7, 128.4, 128.4, 127.6, 127.5, 126.3, 125.6, 124.4, 122.1, 113.5, 58.0, 37.8; IR (KBr, ν, cm1 ): 3443, 3312, 3175, 2187, 1658, 1626, 1595, 1541, 1495, 1473, 1408, 1381, 1354, 1325, 1269, 1163, 1124, 1052, 1023, 760, 733, 701; Anal. Calcd. for C26H15FN4O: C, 74.63; H, 3.61; N, 13.39%. Found: C, 74.55; H, 3.57; N, 13.27%. 3.2.3. Data for 3-Amino-1-(4-fluoro-phenyl)-1H-benzo[a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,3}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.15 (t, 1H, J = 8.0 Hz, Ar-H), 8.41-8.39 (m, 1H, Ar-H), 8.30-8.21 (m, 1H, Ar-H), 8.14-8.08 (m, 1H, ArH), 7.98-7.95 (m, 1H, Ar-H), 7.90-7.81 (m, 3H, Ar-H), 7.447.40 (m, 4H, Ar-H and NH2), 7.04 (t, 2H, J = 9.0 Hz, Ar-H), 5.44 (d, 1H, J = 7.5 Hz, CH); 13C NMR (125 MHz, DMSOd6) (δ, ppm): 159.7, 146.1, 141.4, 140.0, 139.8, 139.4, 130.7, 130.4, 130.2, 130.0, 129.5, 129.4, 129.0, 129.0, 128.6, 125.5, 124.8, 122.1, 120.0, 115.0, 114.8, 113.6, 57.8, 36.7; IR (KBr, ν, cm-1): 3444, 3312, 3173, 2187, 1658, 1625, 1597, 1541, 1495, 1476, 1402, 1385, 1350, 1328, 1269, 1165, 1128, 1052, 1026, 763, 734, 705; Anal. Calcd. for C26H15FN4O: C, 74.63; H, 3.61; N, 13.39%. Found: C, 74.52; H, 3.59; N, 13.24%. 3.2.4. Data for 3-Amino-1-(2-chloro-phenyl)-1H-benzo[a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,4}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.29 (d, 1H, J = 7.5 Hz, Ar-H), 8.50 (d, 1H, J = 8.0 Hz, Ar-H), 8.32-8.30 (m, 1H, Ar-H), 8.08-7.93 (m, 5H, Ar-H), 7.43-7.42 (dd, 1H, J = 6.0, 3.5 Hz, Ar-H), 7.36 (s, 2H, NH2), 7.25-7.23 (dd, 1H, J = 5.5, 3.5 Hz, Ar-H), 7.147.11 (m, 2H, Ar-H), 6.03 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.4, 146.6, 141.3, 140.3, 139.5, 139.4, 130.7, 130.5, 130.2, 130.0, 129.5, 129.4, 129.3, 129.0, 128.6, 125.8, 124.8, 122.1, 120.0, 115.6, 114.8, 113.5, 57.8, 36.2; IR (KBr, ν, cm-1): 3448, 3311, 3173, 2187, 1658, 1623, 1593, 1542, 1495, 1473, 1402, 1385, 1353, 1328, 1269, 1166, 1128, 1052, 1021, 763, 732, 702; Anal. Calcd. for C26H15ClN4O: C, 71.81; H, 3.48; N, 12.88%. Found: C, 71.75; H, 3.47; N, 12.77%.

Gaoet al.

3.2.5. Data for 3-Amino-1-(4-chloro-phenyl)-1H-benzo [a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,5}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.29 (d, 1H, J = 8.0 Hz, Ar-H), 8.48 (d, 1H, J = 8.0 Hz, Ar-H), 8.33-8.31 (m, 1H, Ar-H), 8.21-8.19 (m, 1H, Ar-H), 8.05-8.02 (m, 1H, Ar-H), 8.00-7.94 (m, 3H, Ar-H), 7.46 (d, 2H, J = 8.5 Hz, Ar-H), 7.42 (s, 2H, NH2), 7.29 (d, 2H, J = 8.5 Hz, Ar-H), 5.56 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.7, 146.2, 144.2, 141.5, 140.6, 140.1, 139.8, 131.0, 130.8, 130.5, 130.3, 130.1, 129.5, 129.1, 129.0, 128.7, 128.2, 125.6, 124.8, 122.1, 119.9, 113.2, 57.5, 36.9; IR (KBr, ν, cm-1): 3444, 3315, 3176, 2185, 1658, 1625, 1599, 1541, 1495, 1473, 1401, 1385, 1355, 1328, 1269, 1161, 1125, 1052, 1025, 763, 734, 708; Anal. Calcd. for C26H15ClN4O: C, 71.81; H, 3.48; N, 12.88%. Found: C, 71.71; H, 3.37; N, 12.83%. 3.2.6. Data for 3-Amino-1-(2-bromo-phenyl)-1H-benzo[a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,6}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.28 (d, 1H, J = 7.5 Hz, Ar-H), 8.50 (d, 1H, J = 8.0 Hz, Ar-H), 8.30-8.28 (m, 1H, Ar-H), 8.12-8.10 (m, 1H, Ar-H), 8.06-7.92 (m, 4H, Ar-H), 7.60 (d, 1H, J = 7.5 Hz, ArH), 7.35 (s, 2H, NH2), 7.18-7.12 (m, 2H, Ar-H), 7.04-7.03 (m, 1H, Ar-H), 6.01 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.3, 146.3, 144.5, 141.6, 140.8, 140.1, 139.2, 131.4, 130.8, 130.6, 130.3, 130.1, 129.8, 129.1, 129.0, 128.9, 128.5, 125.6, 124.8, 122.1, 119.3, 113.2, 57.4, 36.7; IR (KBr, ν, cm-1): 3447, 3317, 3174, 2185, 1659, 1625, 1599, 1545, 1497, 1476, 1400, 1386, 1355, 1328, 1269, 1166, 1128, 1052, 1026, 763, 737, 708; Anal. Calcd. for C26H15BrN4O: C, 65.15; H, 3.15; N, 11.69%. Found: C, 65.03; H, 3.07; N, 11.59%. 3.2.7. Data for 3-Amino-1-(3-bromo-phenyl)-1H-benzo[a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,7}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.23 (d, 1H, J = 7.5 Hz, Ar-H), 8.45 (d, 1H, J = 8.0 Hz, Ar-H), 8.29-8.27 (m, 1H, Ar-H), 8.16-8.14 (m, 1H, Ar-H), 8.02-7.93 (m, 4H, Ar-H), 7.62 (d, 1H, J = 2.0 Hz, ArH), 7.44-7.42 (m, 3H, Ar-H and NH2), 7.31 (d, 1H, J = 8.5 Hz, Ar-H), 7.21 (t, 1H, J = 7.5 Hz, Ar-H), 5.52 (s, 1H, CH); 13 C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.6, 146.3, 144.3, 141.5, 140.8, 140.1, 139.2, 131.0, 130.8, 130.4, 130.3, 130.0, 129.8, 129.6, 129.3, 128.7, 128.2, 125.6, 124.8, 122.1, 119.6, 113.2, 57.5, 36.7; IR (KBr, ν, cm-1): 3444, 3315, 3176, 2185, 1658, 1625, 1599, 1541, 1495, 1473, 1401, 1385, 1355, 1328, 1269, 1161, 1125, 1052, 1025, 763, 734, 708; Anal. Calcd. for C26H15BrN4O: C, 65.15; H, 3.15; N, 11.69%. Found: C, 65.07; H, 3.11; N, 11.53%. 3.2.8. Data for 3-Amino-1-(4-bromo-phenyl)-1H-benzo[a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,8}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.23 (d, 1H, J = 1.0 Hz, Ar-H), 8.45 (d, 1H, J = 7.5 Hz, Ar-H), 8.30-8.28 (m, 1H, Ar-H), 8.16-8.14 (m, 1H,



Ar-H), 8.03-7.93 (m, 4H, Ar-H), 7.42-7.37 (m, 6H, Ar-H and NH2), 5.50 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.8, 146.9, 144.4, 141.6, 140.6, 140.5, 139.8, 131.0, 130.6, 130.5, 130.3, 130.0, 129.5, 129.1, 129.0, 128.7, 128.2, 125.6, 124.8, 122.4, 119.9, 113.6, 57.8, 36.2; IR (KBr, ν, cm1 ): 3444, 3315, 3176, 2185, 1658, 1625, 1599, 1541, 1495, 1473, 1401, 1385, 1355, 1328, 1269, 1161, 1125, 1052, 1025, 763, 734, 708; Anal. Calcd. for C26H15BrN4O: C, 65.15; H, 3.15; N, 11.69%. Found: C, 65.08; H, 3.01; N, 11.54%. 3.2.9. Data for 3-Amino-1-(4-dimethylamino-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,9}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.21 (d, 1H, J = 4.0 Hz, Ar-H), 8.74 (s, 1H, ArH), 8.42 (d, 1H, J = 7.0 Hz, Ar-H), 8.26-8.22 (m, 2H, Ar-H), 7.98-7.92 (m, 4H, Ar-H), 7.31 (s, 2H, NH2), 7.19 (s, 1H, ArH), 6.70 (d, 1H, J = 8.0 Hz, Ar-H), 6.60 (d, 1H, J = 8.5 Hz, Ar-H), 5.44 (d, 1H, J = 6.5 Hz, CH); 3.73 (s, 6H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.9, 146.9, 145.6, 145.1, 141.4, 140.6, 139.9, 139.7, 136.1, 130.6, 130.3, 130.0, 129.7, 129.0, 128.7, 128.5, 125.6, 124.7, 121.9, 120.3, 119.7, 115.4, 114.4, 112.6, 58.1, 55.6, 36.7; IR (KBr, ν, cm-1): 3444, 3318, 3176, 2188, 1658, 1629, 1601, 1544, 1497, 1473, 1405, 1385, 1358, 1328, 1269, 1166, 1125, 1052, 1022, 763, 736, 709; Anal. Calcd. for C28H21N5O: C, 75.83; H, 4.77; N, 15.79%. Found: C, 75.78; H, 4.71; N, 15.64%. 3.2.10. Data for 3-Amino-1-(4-diethylamino-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,10}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.28 (d, 2H, J = 7.5 Hz, Ar-H), 8.46 (d, 1H, J = 8.0 Hz, Ar-H), 8.34-8.26 (m, 2H, Ar-H), 8.16 (d, 1H, J = 8.0 Hz, Ar-H), 8.03-7.85 (m, 3H, Ar-H), 7.28 (s, 2H, NH2), 7.21 (d, 1H, J = 8.5 Hz, Ar-H), 6.49 (d, 2H, J = 8.5 Hz, Ar-H), 5.45 (s, 1H, CH); 3.20-3.15 (m, 4H, CH2), 0.98-0.84 (m, 6H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.9, 156.8, 145.2, 142.5, 139.4, 139.2, 130.9, 130.1, 129.9, 129.2, 129.0, 128.8, 128.7, 128.3, 128.0, 125.8, 124.8, 124.7, 122.8, 122.0, 120.4, 114.9, 111.1, 103.4, 58.5, 43.4, 36.1; IR (KBr, ν, cm-1): 3446, 3318, 3174, 2188, 1661, 1632, 1605, 1544, 1493, 1475, 1405, 1387, 1358, 1326, 1269, 1168, 1125, 1056, 1022, 767, 738, 709; Anal. Calcd. for C30H25N5O: C, 76.41; H, 5.34; N, 14.85%. Found: C, 76.32; H, 5.23; N, 14.74%. 3.2.11. Data for 3-Amino-1-(4-hydroxyl-phenyl)-1H-benzo [a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,11}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 11.49 (s, 1H, OH), 9.27 (d, 1H, J = 7.0 Hz, ArH), 8.46 (d, 1H, J = 8.0 Hz, Ar-H), 8.34-8.15 (m, 3H, Ar-H), 8.03-7.84 (m, 5H, Ar-H), 7.23-7.21 (m, 3H, Ar-H and NH2), 6.60 (d, 1H, J = 8.5 Hz, Ar-H), 5.46 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.7, 155.9, 145.8, 141.5, 139.8, 139.4, 135.6, 130.9, 130.6, 130.4, 130.1, 129.9, 129.2, 129.0, 128.8, 128.7, 128.6, 128.4, 128.0, 125.7, 124.8, 114.9, 114.5, 103.4, 58.4, 36.5; IR (KBr, ν, cm-1): 3442, 3314, 3178, 2188, 1666, 1638, 1605, 1545, 1498, 1475, 1409, 1386, 1358, 1323, 1269, 1163, 1127, 1056, 1022, 767, 738,

967

713; ; Anal. Calcd. for C26H16N4O2: C, 74.99; H, 3.87; N, 13.45%. Found: C, 74.82; H, 3.77; N, 13.34%. 3.2.12. Data for 3-Amino-1-(2-nitro-phenyl)-1H-benzo[a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,12}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.22 (d, 1H, J = 8.0 Hz, Ar-H), 8.47 (d, 1H, J = 8.0 Hz, Ar-H), 8.32-8.26 (m, 2H, Ar-H), 8.14-8.12 (m, 1H, Ar-H), 8.03-7.92 (m, 6H, Ar-H), 7.56-7.52 (m, 3H, Ar-H and NH2), 5.68 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.4, 146.5, 141.4, 140.6, 139.4, 139.4, 130.7, 130.3, 130.2, 130.0, 129.5, 129.4, 129.3, 129.0, 128.6, 125.8, 124.8, 122.1, 120.5, 115.0, 114.4, 113.6, 57.8, 36.6; IR (KBr, ν, cm1 ): 3446, 3310, 3169, 2184, 1654, 1629, 1597, 1545, 1492, 1475, 1406, 1386, 1350, 1328, 1265, 1165, 1128, 1056, 1026, 763, 737, 706; Anal. Calcd. for C26H15N5O3: C, 70.11; H, 3.39; N, 15.72%. Found: C, 70.02; H, 3.27; N, 15.64%. 3.2.13. Data for 3-Amino-1-(3-nitro-phenyl)-1H-benzo[a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,13}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.23 (d, 1H, J = 7.5 Hz, Ar-H), 8.47 (d, 1H, J = 7.5 Hz, Ar-H), 8.32-8.26 (m, 2H, Ar-H), 8.14-8.13 (m, 1H, Ar-H), 8.04-7.91 (m, 6H, Ar-H), 7.56-7.55 (m, 3H, Ar-H and NH2), 5.69 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.7, 146.3, 141.4, 140.5, 139.8, 139.4, 130.6, 130.4, 130.1, 130.0, 129.8, 129.4, 129.2, 129.0, 128.6, 125.5, 124.8, 122.6, 120.0, 115.3, 114.4, 113.6, 57.4, 36.7; IR (KBr, ν, cm1 ): 3446, 3313, 3169, 2186, 1653, 1629, 1598, 1542, 1492, 1475, 1402, 1383, 1354, 1328, 1262, 1165, 1128, 1054, 1026, 766, 739, 706; Anal. Calcd. for C26H15N5O3: C, 70.11; H, 3.39; N, 15.72%. Found: C, 70.05; H, 3.35; N, 15.68%. 3.2.14. Data for 3-Amino-1-(4-nitro-phenyl)-1H-benzo[a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,14}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.29-9.28 (m, 1H, Ar-H), 8.50 (d, 1H, J = 8.0 Hz, Ar-H), 8.32-8.30 (m, 1H, Ar-H), 8.18-8.16 (m, 1H, ArH), 8.11 (d, 2H, J = 9.0 Hz, Ar-H), 8.05-7.94 (m, 4H, Ar-H), 7.72 (d, 2H, J = 9.0 Hz, Ar-H), 7.52 (s, 2H, NH2), 5.68 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.5 146.5, 141.4, 140.3, 139.5, 139.4, 130.7, 130.6, 130.2, 130.1, 129.5, 129.4, 129.1, 129.0, 128.6, 125.4, 124.8, 122.6, 120.0, 115.6, 114.8, 113.6, 57.8, 36.3; IR (KBr, ν, cm-1): 3443, 3315, 3165, 2186, 1656, 1627, 1595, 1544, 1492, 1477, 1406, 1385, 1354, 1328, 1264, 1165, 1128, 1055, 1026, 764, 739, 702; Anal. Calcd. for C26H15N5O3: C, 70.11; H, 3.39; N, 15.72%. Found: C, 70.03; H, 3.31; N, 15.62%. 3.2.15. Data for 3-Amino-1-(4-methyl-phenyl)-1H-benzo[a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,15}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.27 (d, 1H, J = 8.0 Hz, Ar-H), 8.47 (d, 1H, J = 8.0 Hz, Ar-H), 8.32-8.30 (m, 1H, Ar-H), 8.21-8.19 (m, 1H, Ar-H), 8.04-7.93 (m, 5H, Ar-H), 7.34-7.29 (m, 3H, Ar-H and NH2), 7.03 (d, 2H, J = 8.0 Hz, Ar-H), 5.51 (s, 1H, CH), 2.15 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.8, 146.1, 142.3, 141.5, 140.7, 140.5, 139.8, 135.5, 130.7, 130.5, 130.2, 130.0, 129.0, 129.0, 128.8, 128.7, 127.4, 125.7, 124.9, 122.1, 114.1, 113.4, 58.1, 37.0, 20.4; IR (KBr, ν, cm1 ): 3443, 3318, 3168, 2186, 1654, 1624, 1596, 1544, 1497,

968


1475, 1406, 1386, 1356, 1328, 1268, 1168, 1128, 1058, 1026, 768, 739, 702; Anal. Calcd. for C27H18N4O: C, 78.24; H, 4.38; N, 13.52%. Found: C, 78.12; H, 4.27; N, 13.44%. 3.2.16. Data for 3-Amino-1-(4-ethyl-phenyl)-1H-benzo[a] pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,16}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.27 (d, 1H, J = 7.5 Hz, Ar-H), 8.47 (d, 1H, J = 7.5 Hz, Ar-H), 8.32-8.30 (m, 1H, Ar-H), 8.22-8.20 (m, 1H, Ar-H), 8.02-7.95 (m, 4H, Ar-H), 7.34-7.32 (m, 4H, Ar-H and NH2), 7.06 (d, 2H, J = 8.0 Hz, Ar-H), 5.53 (s, 1H, CH), 2.462.43 (m, 2H, CH2), 1.07 (t, 3H, J = 7.5 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.4, 146.4, 142.6, 141.6, 140.7, 140.4, 139.8, 135.6, 130.7, 130.5, 130.1, 130.0, 129.8, 129.0, 128.9, 128.5, 127.4, 125.7, 124.9, 122.1, 114.3, 113.4, 58.3, 37.0, 20.3; IR (KBr, ν, cm-1): 3443, 3315, 3168, 2185, 1658, 1621, 1596, 1543, 1493, 1473, 1408, 1383, 1356, 1324, 1268, 1164, 1128, 1055, 1026, 768, 735, 706; Anal. Calcd. for C28H20N4O: C, 78.49; H, 4.70; N, 13.08%. Found: C, 78.38; H, 4.67; N, 13.04%. 3.2.17. Data for 3-Amino-1-(4-isopropyl-phenyl)-1H-benzo [a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,17}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.28 (d, 1H, J = 8.0 Hz, Ar-H), 8.48 (d, 1H, J = 7.5 Hz, Ar-H), 8.33-8.31 (m, 1H, Ar-H), 8.24-8.22 (m, 1H, Ar-H), 8.04-7.94 (m, 4H, Ar-H), 7.36-7.33 (m, 4H, Ar-H and NH2), 7.10 (d, 2H, J = 8.0 Hz, Ar-H), 5.54 (s, 1H, CH), 2.762.71 (m, 1H, CH), 1.09-1.04 (m, 6H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 160.0, 146.5, 146.2, 142.6, 141.6, 140.8, 140.1, 139.9, 130.9, 130.5, 130.3, 130.0, 129.1, 129.1, 128.8, 127.4, 126.2, 125.7, 124.9, 122.1, 120.2, 114.3, 58.1, 56.0, 36.8, 32.8, 23.7, 23.6, 18.5; IR (KBr, ν, cm-1): 3444, 3314, 3165, 2184, 1654, 1626, 1595, 1543, 1498, 1477, 1406, 1383, 1356, 1326, 1268, 1166, 1126, 1055, 1026, 766, 735, 708; Anal. Calcd. for C29H22N4O: C, 78.71; H, 5.01; N, 12.66%. Found: C, 78.68; H, 4.92; N, 12.54%. 3.2.18. Data for 3-Amino-1-(2-methoxyl-phenyl)-1H-benzo [a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,18}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.29 (d, 1H, J = 8.0 Hz, Ar-H), 8.50 (d, 1H, J = 8.0 Hz, Ar-H), 8.31-8.29 (dd, 1H, J = 6.5, 3.5 Hz, Ar-H), 8.08-7.92 (m, 5H, Ar-H), 7.18-7.07 (m, 4H, Ar-H and NH2), 6.96 (d, 1H, J = 8.0 Hz, Ar-H), 6.76-6.74 (m, 1H, Ar-H), 5.88 (s, 1H, CH), 3.86 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.8, 156.6, 146.6, 141.2, 140.7, 139.6, 139.4, 132.7, 130.3, 130.1, 129.7, 129.6, 128.7, 128.5, 128.3, 127.3, 125.4, 124.5, 121.6, 120.0, 119.8, 112.9, 111.3, 56.8, 55.5, 31.7; IR (KBr, ν, cm-1): 3441, 3311, 3162, 2188, 1652, 1622, 1592, 1541, 1494, 1474, 1406, 1387, 1356, 1325, 1264, 1166, 1126, 1057, 1026, 766, 734, 708; Anal. Calcd. for C27H18N4O2: C, 75.34; H, 4.21; N, 13.02%. Found: C, 75.28; H, 4.16; N, 12.93%. 3.2.19. Data for 3-Amino-1-(4-methoxyl-phenyl)-1H-benzo [a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,20} Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.30-9.27 (m, 1H, Ar-H), 8.48 (d, 1H, J = 7.5 Hz, Ar-H), 8.34-8.29 (m, 1H, Ar-H), 8.25-8.16 (m, 2H, ArH), 8.03 (t, 1H, J = 7.5 Hz, Ar-H), 7.98-7.86 (m, 4H, Ar-H),

Gaoet al.

7.35 (s, 2H, NH2), 7.21 (s, 1H, Ar-H), 6.79 (d, 1H, J = 9.0 Hz, Ar-H), 5.53 (s, 1H, CH), 3.63 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.8, 156.6, 146.6, 141.2, 140.8, 139.5, 139.4, 132.6, 130.3, 130.1, 129.8, 129.6, 128.7, 128.6, 128.3, 127.3, 125.7, 124.5, 121.5, 120.6, 119.8, 112.4, 111.3, 56.4, 55.4, 31.6; IR (KBr, ν, cm-1): 3446, 3316, 3162, 2185, 1652, 1625, 1592, 1545, 1494, 1475, 1406, 1385, 1356, 1325, 1265, 1166, 1126, 1055, 1026, 765, 734, 705; Anal. Calcd. for C27H18N4O2: C, 75.34; H, 4.21; N, 13.02%. Found: C, 75.24; H, 4.12; N, 12.96%. 3.2.20. Data for 3-Amino-1-(4-hydroxyl-3-methoxyl-phenyl)1H-benzo[a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,21}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 11.48 (s, 1H, OH), 9.29 (d, 1H, J = 8.0 Hz, ArH), 8.53 (d, 1H, J = 8.0 Hz, Ar-H), 8.33-8.27 (dd, 1H, J = 6.5, 3.5 Hz, Ar-H), 8.08-7.94 (m, 5H, Ar-H), 7.18-7.03 (m, 4H, Ar-H and NH2), 6.97 (d, 1H, J = 8.0 Hz, Ar-H), 6.746.72 (m, 1H, Ar-H), 5.58 (s, 1H, CH), 3.85 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.8, 156.6, 146.6, 141.2, 140.7, 139.6, 139.3, 132.4, 130.6, 130.1, 129.7, 129.4, 128.7, 128.5, 128.1, 127.3, 125.4, 124.6, 121.6, 120.3, 119.8, 112.9, 111.2, 56.8, 55.6, 31.4; IR (KBr, ν, cm-1): 3441, 3311, 3162, 2188, 1652, 1622, 1592, 1541, 1494, 1474, 1406, 1387, 1356, 1325, 1264, 1166, 1126, 1057, 1026, 766, 734, 708; Anal. Calcd. for C27H18N4O3: C, 72.64; H, 4.06; N, 12.55%. Found: C, 72.55; H, 4.01; N, 12.46%. 3.2.21. Data for 3-Amino-1-(4-methoxyl-3-fluoro-phenyl)1H-benzo[a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2, 1,22}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.28 (d, 1H, J = 8.0 Hz, Ar-H), 8.48 (d, 1H, J = 7.5 Hz, Ar-H), 8.34-8.32 (m, 1H, Ar-H), 8.24-8.22 (m, 1H, Ar-H), 8.02-7.96 (m, 4H, Ar-H), 7.39 (s, 2H, NH2), 7.277.18 (m, 2H, Ar-H), 7.02 (t, 1H, J = 8.5 Hz, Ar-H), 5.53 (s, 1H, CH), 3.71 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.7, 156.7, 146.6, 141.5, 140.7, 139.3, 139.1, 132.7, 130.3, 130.1, 129.9, 129.6, 128.7, 128.5, 128.0, 127.6, 125.4, 124.5, 121.4, 120.0, 119.8, 112.9, 111.8, 56.8, 55.7, 31.5; IR (KBr, ν, cm-1): 3443, 3313, 3162, 2183, 1652, 1623, 1592, 1543, 1494, 1473, 1406, 1387, 1356, 1323, 1264, 1163, 1126, 1057, 1023, 766, 733, 708; Anal. Calcd. for C27H17FN4O2: C, 72.31; H, 3.82; N, 12.49%. Found: C, 72.25; H, 3.76; N, 12.46%. 3.2.22. Data for 3-Amino-1-(2-chloro-3-chloro-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,23}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.25 (d, 1H, J = 8.0 Hz, Ar-H), 8.47 (d, 1H, J = 8.0 Hz, Ar-H), 8.28-8.26 (m, 1H, Ar-H), 8.03-7.92 (m, 5H, Ar-H), 7.41-7.38 (m, 3H, Ar-H and NH2), 7.19-7.12 (m, 2H, Ar-H), 6.03 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.8, 156.5, 146.5, 141.2, 140.7, 139.5, 139.4, 132.7, 130.5, 130.1, 129.7, 129.5, 128.7, 128.5, 128.3, 127.5, 125.4, 124.5, 121.5, 120.0, 119.5, 112.9, 111.5, 56.8, 55.5, 31.5; IR (KBr, ν, cm-1): 3443, 3316, 3166, 2183, 1656, 1623, 1596, 1543, 1496, 1473, 1406, 1387, 1356, 1326, 1264, 1166, 1126, 1056, 1023, 766, 736, 707; Anal. Calcd. for C26H14Cl2N4O: C, 66.54; H, 3.01; N, 11.94%. Found: C, 66.45; H, 2.96; N, 11.81%.



3.2.23. Data for 3-Amino-1-(3-chloro-4-chloro-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,24}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.28-9.24 (m, 1H, Ar-H), 8.48-8.45 (m, 1H, ArH), 8.32-8.29 (m, 1H, Ar-H), 8.19-8.15 (m, 1H, Ar-H), 8.047.93 (m, 4H, Ar-H), 7.73-7.71 (m, 1H, Ar-H), 7.50-7.48 (m, 3H, Ar-H and NH2), 7.42-7.40 (m, 1H, Ar-H), 5.57 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.7, 146.3, 146.2, 141.4, 140.5, 140.1, 139.9, 130.8, 130.6, 130.5, 130.3, 130.2, 129.8, 129.2, 129.0, 128.5, 128.2, 125.5, 124.8, 122.2, 119.7, 112.4, 57.0, 36.9; IR (KBr, ν, cm-1): 3447, 3316, 3167, 2183, 1657, 1623, 1597, 1543, 1496, 1477, 1406, 1387, 1356, 1327, 1264, 1167, 1126, 1056, 1027, 766, 737, 707; Anal. Calcd. for C26H14Cl2N4O: C, 66.54; H, 3.01; N, 11.94%. Found: C, 66.41; H, 2.87; N, 11.86%. 3.2.24. Data for 3-Amino-1-(2-chloro-4-chloro-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,25}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.26-9.25 (m, 1H, Ar-H), 8.47 (d, 1H, J = 8.0 Hz, Ar-H), 8.29-8.27 (m, 1H, Ar-H), 8.05-7.92 (m, 5H, ArH), 7.57 (d, 1H, J = 1.5 Hz, Ar-H), 7.40 (s, 2H, NH2), 7.25 (d, 1H, J = 8.5 Hz, Ar-H), 7.18 (d, 1H, J = 8.5 Hz, Ar-H), 5.94 (s, 1H, CH); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.4, 146.7, 141.9, 141.5, 140.6, 140.1, 139.7, 133.2, 131.6, 131.5, 130.8, 130.5, 130.2, 130.2, 129.3, 129.0, 128.5, 128.4, 127.7, 125.5, 124.8, 122.2, 119.4, 112.2, 56.4, 34.3; IR (KBr, ν, cm-1): 3447, 3315, 3165, 2183, 1655, 1623, 1595, 1545, 1496, 1475, 1406, 1387, 1355, 1327, 1265, 1167, 1125, 1056, 1027, 765, 737, 705; Anal. Calcd. for C26H14Cl2N4O: C, 66.54; H, 3.01; N, 11.94%. Found: C, 66.46; H, 2.84; N, 11.82%. 3.2.25. Data for 3-Amino-1-(2-chloro-6-chloro-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,26}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.27 (d, 1H, J = 7.5 Hz, Ar-H), 8.46 (d, 1H, J = 7.5 Hz, Ar-H), 8.28 (s, 1H, Ar-H), 8.03-7.91 (m, 5H, Ar-H), 7.64-7.62 (m, 1H, Ar-H), 7.41 (s, 2H, NH2), 7.21-7.18 (m, 2H, Ar-H), 6.40 (s, 1H, CH); 13C NMR (125 MHz, DMSOd6) (δ, ppm): 159.6, 146.7, 141.6, 141.5, 140.6, 140.1, 139.6, 133.2, 131.6, 131.5, 130.6, 130.5, 130.2, 130.2, 129.6, 129.0, 128.6, 128.4, 127.6, 125.5, 124.6, 122.2, 119.6, 112.2, 56.4, 34.6; IR (KBr, ν, cm-1): 3447, 3316, 3166, 2183, 1656, 1626, 1595, 1545, 1496, 1475, 1406, 1386, 1355, 1323, 1263, 1167, 1123, 1056, 1023, 765, 733, 707; Anal. Calcd. for C26H14Cl2N4O: C, 66.54; H, 3.01; N, 11.94%. Found: C, 66.42; H, 2.85; N, 11.87%. 3.2.26. Data for 3-Amino-1-(2-methoxyl-5-bromo-phenyl)1H-benzo[a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,27}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.25 (d, 1H, J = 7.5 Hz, Ar-H), 8.47 (d, 1H, J = 8.0 Hz, Ar-H), 8.29-8.27 (m, 1H, Ar-H), 8.03-7.92 (m, 5H, Ar-H), 7.29-7.25 (m, 4H, Ar-H and NH2), 6.95 (d, 1H, J = 9.0 Hz, Ar-H), 5.78 (s, 1H, CH), 3.84 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.8, 146.7, 141.8, 141.5, 140.8, 140.1, 139.8, 133.2, 131.6, 131.5, 130.8, 130.5, 130.2, 130.2, 129.8, 129.6, 128.8, 128.4, 127.7, 125.8, 124.8,

969

122.2, 119.8, 112.2, 56.8, 34.2; IR (KBr, ν, cm-1): 3447, 3313, 3166, 2183, 1656, 1626, 1593, 1545, 1493, 1475, 1403, 1386, 1355, 1323, 1263, 1165, 1123, 1054, 1023, 765, 734, 704; Anal. Calcd. for C27H17BrN4O2: C, 63.67; H, 3.36; N, 11.00%. Found: C, 63.62; H, 3.25; N, 10.89%. 3.2.27. Data for 3-Amino-1-(3-methoxyl-4-methoxylphenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,28}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.29 (d, 1H, J = 8.0 Hz, Ar-H), 8.48 (d, 1H, J = 8.0 Hz, Ar-H), 8.35-8.33 (m, 1H, Ar-H), 8.30-8.28 (m, 1H, Ar-H), 8.02-7.96 (m, 4H, Ar-H), 7.36 (s, 2H, NH2), 7.24 (d, 1H, J = 2.0 Hz, Ar-H), 6.83 (d, 1H, J = 2.0 Hz, Ar-H), 6.79 (d, 1H, J = 8.5 Hz, Ar-H), 5.55 (s, 1H, CH), 3.73 (s, 3H, CH3), 3.61 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.9, 146.7, 141.9, 141.5, 140.9, 140.1, 139.9, 133.2, 131.9, 131.5, 130.9, 130.5, 130.2, 130.2, 129.3, 129.0, 128.9, 128.4, 127.9, 125.5, 124.8, 122.2, 119.9, 112.2, 56.5, 34.5; IR (KBr, ν, cm-1): 3445, 3313, 3165, 2183, 1656, 1625, 1593, 1545, 1494, 1475, 1403, 1384, 1355, 1324, 1263, 1165, 1124, 1054, 1023, 765, 735, 709; Anal. Calcd. for C28H20N4O3: C, 73.03; H, 4.38; N, 12.17%. Found: C, 72.92; H, 4.28; N, 12.05%. 3.2.28. Data for 3-Amino-1-(2-methoxyl-5-methoxylphenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2-carbonitrile (6{1,2,1,29}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.27 (d, 1H, J = 8.0 Hz, Ar-H), 8.48 (d, 1H, J = 8.0 Hz, Ar-H), 8.30-8.28 (m, 1H, Ar-H), 8.07-7.92 (m, 5H, Ar-H), 7.18 (s, 2H, NH2), 6.88 (d, 1H, J = 8.5 Hz, Ar-H), 6.71-6.65 (m, 2H, Ar-H), 5.77 (s, 1H, CH), 3.77 (s, 3H, CH3), 3.58 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.7, 146.7, 141.7, 141.5, 140.6, 140.1, 139.7, 133.7, 131.6, 131.5, 130.7, 130.7, 130.2, 130.2, 129.3, 129.0, 128.7, 128.4, 127.7, 125.5, 124.7, 122.2, 119.7, 112.2, 56.7, 34.6; IR (KBr, ν, cm-1): 3447, 3313, 3167, 2183, 1657, 1625, 1593, 1545, 1497, 1475, 1407, 1384, 1355, 1327, 1266, 1166, 1124, 1056, 1023, 765, 735, 706; Anal. Calcd. for C28H20N4O3: C, 73.03; H, 4.38; N, 12.17%. Found: C, 72.97; H, 4.23; N, 12.02%. 3.2.29. Data for 3-Amino-1-(2-methoxyl-4-methoxyl-5methoxyl-phenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2carbonitrile (6{1,2,1,30}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.27 (d, 1H, J = 8.0 Hz, Ar-H), 8.48 (d, 1H, J = 8.0 Hz, Ar-H), 8.30 (d, 1H, J = 7.5 Hz, Ar-H), 8.12 (d, 1H, J = 8.0 Hz, Ar-H), 8.03-7.92 (m, 4H, Ar-H), 7.13 (s, 2H, NH2), 6.93 (s, 1H, Ar-H), 6.59 (s, 1H, Ar-H), 5.65 (s, 1H, CH), 3.71 (s, 3H, CH3), 3.67 (s, 3H, CH3), 3.64 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.6, 146.7, 141.6, 141.5, 140.6, 140.6, 139.7, 133.6, 131.6, 131.5, 130.6, 130.5, 130.2, 130.2, 129.6, 129.0, 128.6, 128.4, 127.7, 125.6, 124.8, 122.6, 119.4, 112.2, 56.6, 34.6; IR (KBr, ν, cm-1): 3447, 3318, 3167, 2188, 1657, 1628, 1594, 1545, 1498, 1475, 1408, 1384, 1358, 1327, 1266, 1166, 1128, 1056, 1028, 766, 735, 708; Anal. Calcd. for C29H22N4O4: C, 71.01; H, 4.52; N, 11.42%. Found: C, 70.93; H, 4.43; N, 11.33%.

970


3.2.30. Data for 3-Amino-1-(3-methoxyl-4-methoxyl-5methoxyl-phenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2carbonitrile (6{1,2,1,31}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.20 (d, 1H, J = 8.0 Hz, Ar-H), 8.43 (d, 1H, J = 7.5 Hz, Ar-H), 8.27-8.23 (m, 2H, Ar-H), 7.98-7.91 (m, 4H, Ar-H), 7.39 (s, 2H, NH2), 6.78 (s, 2H, Ar-H), 5.52 (s, 1H, CH), 3.69 (s, 6H, CH3), 3.53 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.4, 146.7, 141.9, 141.5, 140.4, 140.1, 139.7, 133.4, 131.4, 131.3, 130.8, 130.4, 130.2, 130.2, 129.4, 129.0, 128.5, 128.4, 127.4, 125.5, 124.8, 122.2, 119.4, 112.2, 56.4, 34.4; IR (KBr, ν, cm-1): 3447, 3315, 3167, 2185, 1657, 1628, 1595, 1545, 1495, 1474, 1408, 1385, 1358, 1325, 1266, 1164, 1128, 1054, 1028, 764, 735, 704; Anal. Calcd. for C29H22N4O4: C, 71.01; H, 4.52; N, 11.42%. Found: C, 70.95; H, 4.47; N, 11.31%. 3.2.31. Data for 3-Amino-1-(2-methoxyl-4-methoxyl-6methoxyl-phenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2carbonitrile (6{1,2,1,32}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.22 (d, 1H, J = 8.0 Hz, Ar-H), 8.42 (d, 1H, J = 7.5 Hz, Ar-H), 8.25-8.21 (m, 2H, Ar-H), 7.97-7.91 (m, 4H, Ar-H), 7.37 (s, 2H, NH2), 6.78 (s, 2H, Ar-H), 5.53 (s, 1H, CH), 3.68 (s, 6H, CH3), 3.53 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.4, 146.6, 141.9, 141.6, 140.4, 140.1, 139.6, 133.4, 131.6, 131.5, 130.8, 130.6, 130.2, 130.2, 129.4, 129.0, 128.5, 128.4, 127.4, 125.6, 124.8, 122.2, 119.4, 112.2, 56.4, 34.6; IR (KBr, ν, cm-1): 3446, 3315, 3166, 2185, 1657, 1628, 1596, 1545, 1495, 1474, 1406, 1385, 1358, 1326, 1266, 1164, 1126, 1054, 1026, 764, 735, 706; Anal. Calcd. for C29H22N4O4: C, 71.01; H, 4.52; N, 11.42%. Found: C, 70.92; H, 4.43; N, 11.36%. 3.2.32. Data for 3-Amino-1-(2-methoxyl-3-methoxyl-4methoxyl-phenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2carbonitrile (6{1,2,1,33}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.21 (d, 1H, J = 8.0 Hz, Ar-H), 8.45 (d, 1H, J = 7.5 Hz, Ar-H), 8.28-8.21 (m, 2H, Ar-H), 7.95-7.90 (m, 4H, Ar-H), 7.35 (s, 2H, NH2), 6.77 (s, 2H, Ar-H), 5.54 (s, 1H, CH), 3.68 (s, 6H, CH3), 3.56 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.6, 146.6, 141.9, 141.6, 140.6, 140.1, 139.6, 133.6, 131.6, 131.5, 130.8, 130.6, 130.2, 130.2, 129.6, 129.0, 128.6, 128.4, 127.6, 125.6, 124.8, 122.2, 119.6, 112.2, 56.6, 34.6; IR (KBr, ν, cm-1): 3446, 3315, 3166, 2185, 1657, 1628, 1596, 1545, 1495, 1474, 1406, 1385, 1358, 1326, 1266, 1164, 1126, 1054, 1026, 764, 735, 706; Anal. Calcd. for C29H22N4O4: C, 71.01; H, 4.52; N, 11.42%. Found: C, 70.97; H, 4.49; N, 11.32%. 3.2.33. Data for 3-Amino-1-(3-methoxyl-5-methoxyl-4bromo-phenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2carbonitrile (6{1,2,1,34}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.24 (d, 1H, J = 8.0 Hz, Ar-H), 8.45 (d, 1H, J = 7.5 Hz, Ar-H), 8.28-8.20 (m, 2H, Ar-H), 7.95-7.91 (m, 4H, Ar-H), 7.35 (s, 2H, NH2), 6.74 (s, 2H, Ar-H), 5.54 (s, 1H, CH), 3.65 (s, 3H, CH3), 3.55 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.8, 146.6, 141.9, 141.8,

Gaoet al.

140.6, 140.1, 139.8, 133.6, 131.8, 131.5, 130.8, 130.6, 130.2, 130.2, 129.8, 129.0, 128.8, 128.4, 127.6, 125.8, 124.8, 122.2, 119.6, 112.2, 56.8, 34.8; IR (KBr, ν, cm-1): 3449, 3315, 3166, 2185, 1659, 1628, 1596, 1545, 1499, 1474, 1406, 1389, 1358, 1326, 1269, 1164, 1126, 1059, 1029, 769, 735, 709; Anal. Calcd. for C28H19BrN4O3: C, 62.35; H, 3.55; N, 10.39%. Found: C, 62.27; H, 3.44; N, 10.36%. 3.2.34. Data for 3-Amino-1-(3-methoxyl-4-methoxyl-5bromo-phenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2carbonitrile (6{1,2,1,35}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.20 (d, 1H, J = 8.0 Hz, Ar-H), 8.48 (d, 1H, J = 7.5 Hz, Ar-H), 8.27-8.20 (m, 2H, Ar-H), 7.98-7.90 (m, 4H, Ar-H), 7.38 (s, 2H, NH2), 6.77 (s, 2H, Ar-H), 5.54 (s, 1H, CH), 3.68 (s, 3H, CH3), 3.58 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.4, 146.6, 141.9, 141.4, 140.4, 140.1, 139.6, 133.4, 131.6, 131.5, 130.4, 130.6, 130.4, 130.2, 129.4, 129.0, 128.6, 128.4, 127.6, 125.6, 124.4, 122.2, 119.6, 112.4, 56.6, 34.4; IR (KBr, ν, cm-1): 3444, 3315, 3166, 2185, 1654, 1624, 1596, 1545, 1494, 1474, 1406, 1384, 1358, 1326, 1264, 1164, 1127, 1057, 1026, 767, 735, 707; Anal. Calcd. for C28H19BrN4O3: C, 62.35; H, 3.55; N, 10.39%. Found: C, 62.22; H, 3.42; N, 10.31%. 3.2.35. Data for Ethyl 3-amino-1-phenyl-1H-benzo[a] pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,1}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.20 (d, 1H, J = 8.0 Hz, Ar-H), 8.45 (d, 1H, J = 8.0 Hz, Ar-H), 8.27-8.21 (m, 1H, Ar-H), 8.15-8.11 (m, 1H, Ar-H), 8.01-7.95 (m, 1H, Ar-H), 7.95-7.87 (m, 3H, Ar-H), 7.40-7.36 (m, 4H, Ar-H), 7.22 (s, 2H, NH2), 7.09 (t, 1H, J = 7.0 Hz, Ar-H), 5.74 (s, 1H, CH), 4.11 (q, 2H, J = 7.0 Hz, CH2), 1.20 (t, 3H, J = 7.0 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.5, 146.5, 145.8, 141.5, 140.7, 140.3, 139.5, 130.9, 130.5, 130.1, 130.0, 129.1, 128.7, 128.5, 128.3, 127.8, 127.5, 126.5, 125.6, 124.9, 122.1, 113.3, 77.5, 58.2, 36.7, 14.4; IR (KBr, ν, cm-1): 3448, 3303, 3174, 2968, 2854, 2188, 1658, 1623, 1598, 1541, 1498, 1473, 1400, 1385, 1358, 1325, 1269, 1165, 1125, 1052, 1025, 765, 733, 705; Anal. Calcd. for C28H21N3O3: C, 75.15; H, 4.73; N, 9.39%. Found: C, 75.06; H, 4.60; N, 9.18%. 3.2.36. Data for Ethyl 3-amino-1-(4-fluoro-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,3}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.22 (d, 1H, J = 8.0 Hz, Ar-H), 8.43 (d, 1H, J = 8.0 Hz, Ar-H), 8.25-8.20 (m, 1H, Ar-H), 8.17-8.10 (m, 1H, Ar-H), 8.01-7.93 (m, 1H, Ar-H), 7.91-7.84 (m, 3H, Ar-H), 7.40-7.33 (m, 4H, Ar-H), 7.24 (s, 2H, NH2), 7.06 (t, 1H, J = 7.0 Hz, Ar-H), 5.70 (s, 1H, CH), 4.15 (q, 2H, J = 7.0 Hz, CH2), 1.27 (t, 3H, J = 7.0 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.1, 146.5, 145.1, 141.5, 140.7, 140.1, 139.5, 130.9, 130.5, 130.1, 130.0, 129.1, 128.7, 128.5, 128.1, 127.8, 127.5, 126.1, 125.6, 124.9, 122.1, 113.3, 77.1, 58.2, 36.7, 14.1; IR (KBr, ν, cm-1): 3441, 3303, 3171, 2961, 2850, 2188, 1658, 1623, 1598, 1543, 1498, 1473, 1403, 1385, 1358, 1323, 1269, 1163, 1125, 1052, 1023, 765, 733, 703; Anal. Calcd. for C28H20FN3O3: C, 72.25; H, 4.33; N, 9.03%. Found: C, 72.06; H, 4.20; N, 8.98%.



3.2.37. Data for Ethyl 3-amino-1-(4-chloro-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,5}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.24 (d, 1H, J = 8.0 Hz, Ar-H), 8.43 (d, 1H, J = 8.0 Hz, Ar-H), 8.24-8.20 (m, 1H, Ar-H), 8.18-8.12 (m, 1H, Ar-H), 8.01-7.91 (m, 1H, Ar-H), 7.88-7.82 (m, 3H, Ar-H), 7.42-7.32 (m, 4H, Ar-H), 7.24 (s, 2H, NH2), 7.03 (t, 1H, J = 7.0 Hz, Ar-H), 5.78 (s, 1H, CH), 4.18 (q, 2H, J = 7.0 Hz, CH2), 1.28 (t, 3H, J = 7.0 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 159.5, 146.5, 145.1, 141.5, 140.7, 140.1, 139.5, 130.9, 130.6, 130.1, 130.0, 129.1, 128.7, 128.5, 128.1, 127.8, 127.6, 126.7, 125.6, 124.9, 122.1, 113.6, 77.6, 58.2, 36.7, 14.6; IR (KBr, ν, cm-1): 3441, 3306, 3176, 2961, 2850, 2188, 1656, 1623, 1596, 1546, 1498, 1476, 1403, 1385, 1356, 1323, 1269, 1166, 1125, 1052, 1026, 765, 733, 706; Anal. Calcd. for C28H20ClN3O3: C, 69.78; H, 4.18; N, 8.72%. Found: C, 69.56; H, 4.02; N, 8.58%. 3.2.38. Data for Ethyl 3-amino-1-(4-dimethylaminophenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,9}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.21 (d, 1H, J = 8.0 Hz, Ar-H), 8.43 (d, 1H, J = 8.0 Hz, Ar-H), 8.27-8.23 (m, 1H, Ar-H), 8.16-8.11 (m, 1H, Ar-H), 8.05-7.93 (m, 1H, Ar-H), 7.86-7.81 (m, 3H, Ar-H), 7.44-7.31 (m, 4H, Ar-H), 7.25 (s, 2H, NH2), 7.08 (t, 1H, J = 7.0 Hz, Ar-H), 5.71 (s, 1H, CH), 4.13 (q, 2H, J = 7.0 Hz, CH2), 3.69 (s, 6H, CH3), 1.25 (t, 3H, J = 7.0 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.9, 146.6, 145.7, 141.5, 140.6, 140.2, 139.5, 130.7, 130.6, 130.3, 130.0, 129.1, 128.7, 128.5, 128.2, 127.8, 127.6, 126.9, 125.6, 124.9, 122.1, 113.3, 77.4, 58.2, 55.1, 36.4, 14.8; IR (KBr, ν, cm-1): 3446, 3301, 3171, 2961, 2850, 2181, 1656, 1623, 1591, 1546, 1498, 1476, 1401, 1385, 1351, 1323, 1262, 1166, 1125, 1052, 1022, 765, 732, 702; Anal. Calcd. for C30H26N4O3: C, 73.45; H, 5.34; N, 11.42%. Found: C, 73.26; H, 5.12; N, 11.24%. 3.2.39. Data for Ethyl 3-amino-1-(4-nitro-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,14}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.26 (d, 1H, J = 7.5 Hz, Ar-H), 8.46 (d, 1H, J = 8.5 Hz, Ar-H), 8.29-8.21 (m, 1H, Ar-H), 8.14-8.10 (m, 1H, Ar-H), 8.07-7.95 (m, 1H, Ar-H), 7.85-7.81 (m, 3H, Ar-H), 7.48-7.33 (m, 4H, Ar-H), 7.24 (s, 2H, NH2), 7.11 (t, 1H, J = 7.5 Hz, Ar-H), 5.78 (s, 1H, CH), 4.16 (q, 2H, J = 7.5 Hz, CH2), 1.22 (t, 3H, J = 7.5 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.7, 146.6, 145.7, 141.5, 140.7, 140.2, 139.7, 130.7, 130.6, 130.3, 130.0, 129.7, 128.8, 128.6, 128.2, 127.7, 127.4, 126.9, 125.4, 124.6, 122.1, 113.3, 77.8, 58.6, 36.5, 14.5; IR (KBr, ν, cm-1): 3446, 3306, 3171, 2961, 2850, 2186, 1656, 1623, 1596, 1546, 1498, 1476, 1401, 1386, 1351, 1326, 1262, 1166, 1125, 1056, 1022, 765, 736, 701; Anal. Calcd. for C28H20N4O5: C, 68.29; H, 4.09; N, 11.38%. Found: C, 68.06; H, 3.92; N, 11.02%. 3.2.40. Data for Ethyl 3-amino-1-(4-methyl-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,15}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.28 (d, 1H, J = 7.0 Hz, Ar-H), 8.44 (d, 1H, J =

971

8.0 Hz, Ar-H), 8.27-8.22 (m, 1H, Ar-H), 8.16-8.10 (m, 1H, Ar-H), 8.06-7.97 (m, 1H, Ar-H), 7.86-7.80 (m, 3H, Ar-H), 7.43-7.31 (m, 4H, Ar-H), 7.25 (s, 2H, NH2), 7.10 (t, 1H, J = 7.0 Hz, Ar-H), 5.72 (s, 1H, CH), 4.13 (q, 2H, J = 7.5 Hz, CH2), 2.12 (s, 3H, CH3), 1.26 (t, 3H, J = 7.0 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.9, 146.6, 145.9, 141.5, 140.9, 140.2, 139.9, 130.7, 130.6, 130.3, 130.0, 129.9, 128.8, 128.6, 128.2, 127.9, 127.4, 126.9, 125.4, 124.6, 122.9, 113.3, 77.9, 58.6, 36.9, 20.5, 14.2; IR (KBr, ν, cm-1): 3446, 3303, 3171, 2963, 2850, 2183, 1656, 1623, 1593, 1546, 1498, 1473, 1401, 1386, 1353, 1326, 1262, 1163, 1125, 1056, 1023, 765, 736, 703; Anal. Calcd. for C29H23N3O3: C, 75.47; H, 5.02; N, 9.10%. Found: C, 75.12; H, 4.91; N, 8.86%. 3.2.41. Data for Ethyl 3-amino-1-(4-ethyl-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,16}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.28 (d, 1H, J = 7.5 Hz, Ar-H), 8.41 (d, 1H, J = 8.5 Hz, Ar-H), 8.26-8.22 (m, 1H, Ar-H), 8.17-8.12 (m, 1H, Ar-H), 8.03-7.92 (m, 1H, Ar-H), 7.86-7.81 (m, 3H, Ar-H), 7.45-7.36 (m, 4H, Ar-H), 7.26 (s, 2H, NH2), 7.15 (t, 1H, J = 7.5 Hz, Ar-H), 5.76 (s, 1H, CH), 4.16 (q, 2H, J = 7.5 Hz, CH2), 2.45-2.41 (m, 2H, CH2), 1.26 (t, 3H, J = 7.0 Hz, CH3), 1.04 (t, 3H, J = 7.5 Hz, CH3); 13C NMR (125 MHz, DMSOd6) (δ, ppm): 158.5, 146.6, 145.5, 141.5, 140.9, 140.5, 139.9, 130.7, 130.5, 130.3, 130.0, 129.9, 128.8, 128.5, 128.2, 127.5, 127.4, 126.9, 125.5, 124.6, 122.5, 113.3, 77.9, 58.5, 36.9, 20.6, 14.5; IR (KBr, ν, cm-1): 3442, 3303, 3171, 2962, 2850, 2183, 1652, 1622, 1593, 1546, 1498, 1472, 1401, 1386, 1352, 1326, 1262, 1163, 1122, 1056, 1023, 762, 736, 702; Anal. Calcd. for C30H25N3O3: C, 75.77; H, 5.30; N, 8.84%. Found: C, 75.52; H, 4.99; N, 8.66%. 3.2.42. Data for Ethyl 3-amino-1-(4-isopropyl-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,17}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.25 (d, 1H, J = 7.5 Hz, Ar-H), 8.45 (d, 1H, J = 8.5 Hz, Ar-H), 8.25-8.21 (m, 1H, Ar-H), 8.17-8.11 (m, 1H, Ar-H), 8.06-7.91 (m, 1H, Ar-H), 7.88-7.81 (m, 3H, Ar-H), 7.44-7.33 (m, 4H, Ar-H), 7.25 (s, 2H, NH2), 7.12 (t, 1H, J = 7.0 Hz, Ar-H), 5.72 (s, 1H, CH), 4.13 (q, 2H, J = 7.0 Hz, CH2), 2.79-2.74 (m, 1H, CH), 1.28 (t, 3H, J = 7.5 Hz, CH3), 1.08-1.02 (m, 6H, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.7, 146.6, 145.7, 141.5, 140.9, 140.5, 139.7, 130.7, 130.5, 130.3, 130.0, 129.7, 128.8, 128.7, 128.2, 127.7, 127.4, 126.7, 125.5, 124.7, 122.5, 113.7, 77.9, 58.7, 36.9, 23.6, 23.5, 14.7; IR (KBr, ν, cm-1): 3442, 3308, 3177, 2962, 2850, 2188, 1652, 1628, 1593, 1546, 1498, 1478, 1407, 1388, 1357, 1328, 1262, 1167, 1128, 1058, 1023, 768, 737, 708; Anal. Calcd. for C31H27N3O3: C, 76.05; H, 5.56; N, 8.58%. Found: C, 75.82; H, 5.34; N, 8.22%. 3.2.43. Data for Ethyl 3-amino-1-(2-methoxyl-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,18}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.29 (d, 1H, J = 7.5 Hz, Ar-H), 8.46 (d, 1H, J = 8.5 Hz, Ar-H), 8.26-8.20 (m, 1H, Ar-H), 8.17-8.12 (m, 1H, Ar-H), 8.06-7.93 (m, 1H, Ar-H), 7.84-7.80 (m, 3H, Ar-H), 7.41-7.31 (m, 4H, Ar-H), 7.23 (s, 2H, NH2), 7.13 (t, 1H, J =

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7.0 Hz, Ar-H), 5.74 (s, 1H, CH), 4.11 (q, 2H, J = 7.5 Hz, CH2), 3.82 (s, 3H, CH3), 1.23 (t, 3H, J = 7.5 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.7, 146.4, 145.7, 141.4, 140.9, 140.5, 139.4, 130.7, 130.4, 130.3, 130.0, 129.7, 128.8, 128.4, 128.2, 127.4, 127.1, 126.7, 125.4, 124.7, 122.5, 113.4, 77.9, 57.4, 56.3, 14.4; IR (KBr, ν, cm-1): 3442, 3308, 3177, 2969, 2850, 2189, 1652, 1628, 1593, 1549, 1498, 1478, 1407, 1389, 1357, 1329, 1262, 1169, 1128, 1058, 1029, 769, 737, 709; Anal. Calcd. for C29H23N3O4: C, 72.94; H, 4.85; N, 8.80%. Found: C, 72.66; H, 4.54; N, 8.71%. 3.2.44. Data for Ethyl 3-amino-1-(3-methoxyl-phenyl)-1Hbenzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,19}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.27 (d, 1H, J = 7.0 Hz, Ar-H), 8.43 (d, 1H, J = 8.0 Hz, Ar-H), 8.29-8.20 (m, 1H, Ar-H), 8.15-8.11 (m, 1H, Ar-H), 8.02-7.94 (m, 1H, Ar-H), 7.87-7.82 (m, 3H, Ar-H), 7.44-7.36 (m, 4H, Ar-H), 7.26 (s, 2H, NH2), 7.15 (t, 1H, J = 7.5 Hz, Ar-H), 5.81 (s, 1H, CH), 4.16 (q, 2H, J = 7.0 Hz, CH2), 3.86 (s, 3H, CH3), 1.21 (t, 3H, J = 7.0 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.3, 146.4, 145.7, 141.3, 140.3, 140.1, 139.4, 130.6, 130.4, 130.3, 130.0, 129.6, 128.6, 128.4, 128.2, 127.6, 127.1, 126.7, 125.4, 124.6, 122.5, 113.6, 77.9, 57.4, 56.6, 14.6; IR (KBr, ν, cm-1): 3441, 3308, 3177, 2961, 2850, 2189, 1652, 1621, 1593, 1549, 1491, 1471, 1407, 1381, 1357, 1329, 1262, 1161, 1128, 1058, 1029, 761, 737, 701; Anal. Calcd. for C29H23N3O4: C, 72.94; H, 4.85; N, 8.80%. Found: C, 72.77; H, 4.64; N, 8.59%. 3.2.45. Data for Ethyl 3-amino-1-(4-hydroxyl-3-methoxylphenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,21}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 11.44 (s, 1H, OH), 9.23 (d, 1H, J = 7.5 Hz, ArH), 8.45 (d, 1H, J = 7.5 Hz, Ar-H), 8.29-8.22 (m, 1H, Ar-H), 8.17-8.12 (m, 1H, Ar-H), 8.07-7.98 (m, 1H, Ar-H), 7.87-7.81 (m, 3H, Ar-H), 7.47-7.39 (m, 4H, Ar-H), 7.24 (s, 2H, NH2), 7.15 (t, 1H, J = 7.0 Hz, Ar-H), 5.84 (s, 1H, CH), 4.18 (q, 2H, J = 7.5 Hz, CH2), 3.83 (s, 3H, CH3), 1.27 (t, 3H, J = 7.5 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.6, 146.4, 145.6, 141.6, 140.3, 140.1, 139.6, 130.6, 130.4, 130.3, 130.0, 129.6, 128.8, 128.4, 128.2, 127.8, 127.1, 126.8, 125.4, 124.8, 122.5, 113.8, 77.9, 57.4, 56.8, 14.1; IR (KBr, ν, cm-1): 3446, 3308, 3177, 2961, 2856, 2189, 1652, 1621, 1596, 1549, 1496, 1476, 1407, 1381, 1356, 1329, 1262, 1161, 1126, 1058, 1029, 761, 737, 706; Anal. Calcd. for C29H23N3O5: C, 70.58; H, 4.70; N, 8.51%. Found: C, 70.27; H, 4.45; N, 8.23%. 3.2.46. Data for Ethyl 3-amino-1-(2-chloro-3-chlorophenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,23}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.26 (d, 1H, J = 7.0 Hz, Ar-H), 8.42 (d, 1H, J = 7.0 Hz, Ar-H), 8.29-8.25 (m, 1H, Ar-H), 8.15-8.10 (m, 1H, Ar-H), 8.03-7.97 (m, 1H, Ar-H), 7.87-7.78 (m, 3H, Ar-H), 7.44-7.37 (m, 4H, Ar-H), 7.21 (s, 2H, NH2), 7.12 (t, 1H, J = 7.0 Hz, Ar-H), 5.88 (s, 1H, CH), 4.14 (q, 2H, J = 7.0 Hz, CH2), 1.21 (t, 3H, J = 7.5 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.1, 146.4, 145.1, 141.6, 140.3, 140.1, 139.6, 130.6, 130.4, 130.1, 130.0, 129.1, 128.8, 128.4,

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128.1, 127.8, 127.1, 126.1, 125.4, 124.8, 122.5, 113.1, 77.9, 57.4, 14.2; IR (KBr, ν, cm-1): 3446, 3308, 3175, 2961, 2856, 2185, 1655, 1621, 1596, 1545, 1496, 1476, 1405, 1381, 1356, 1325, 1265, 1161, 1126, 1055, 1029, 761, 735, 701; Anal. Calcd. for C28H19Cl2N3O3: C, 65.13; H, 3.71; N, 8.14%. Found: C, 65.01; H, 3.49; N, 7.95%. 3.2.47. Data for Ethyl 3-amino-1-(3-methoxyl-4-methoxyl phenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,28}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.24 (d, 1H, J = 7.5 Hz, Ar-H), 8.46 (d, 1H, J = 7.0 Hz, Ar-H), 8.29-8.22 (m, 1H, Ar-H), 8.17-8.13 (m, 1H, Ar-H), 8.06-7.95 (m, 1H, Ar-H), 7.85-7.80 (m, 3H, Ar-H), 7.44-7.34 (m, 4H, Ar-H), 7.25 (s, 2H, NH2), 7.19 (t, 1H, J = 7.0 Hz, Ar-H), 5.78 (s, 1H, CH), 4.12 (q, 2H, J = 7.5 Hz, CH2), 3.83 (s, 6H, CH3), 1.27 (t, 3H, J = 7.5 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.8, 146.4, 145.8, 141.3, 140.8, 140.1, 139.4, 130.8, 130.4, 130.3, 130.0, 129.8, 128.6, 128.4, 128.2, 127.8, 127.1, 126.8, 125.4, 124.6, 122.5, 113.6, 77.8, 57.8, 56.6, 56.1,14.8; IR (KBr, ν, cm-1): 3446, 3308, 3177, 2966, 2850, 2186, 1656, 1621, 1593, 1546, 1491, 1471, 1406, 1381, 1357, 1329, 1266, 1161, 1126, 1058, 1029, 766, 737, 702; Anal. Calcd. for C30H25N3O5: C, 70.99; H, 4.96; N, 8.28%. Found: C, 70.67; H, 4.62; N, 8.02%. 3.2.48. Data for Ethyl 3-amino-1-(2-methoxyl-5-methoxyl phenyl)-1H-benzo[a]pyrano[2,3-c]phenazine-2-carboxylate (6{1,2,2,29}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.19 (d, 1H, J = 7.5 Hz, Ar-H), 8.44 (d, 1H, J = 7.5 Hz, Ar-H), 8.29-8.24 (m, 1H, Ar-H), 8.17-8.11 (m, 1H, Ar-H), 8.06-7.98 (m, 1H, Ar-H), 7.87-7.83 (m, 3H, Ar-H), 7.48-7.32 (m, 4H, Ar-H), 7.29 (s, 2H, NH2), 7.12 (t, 1H, J = 7.5 Hz, Ar-H), 5.72 (s, 1H, CH), 4.13 (q, 2H, J = 7.0 Hz, CH2), 3.75 (s, 3H, CH3), 3.63 (s, 3H, CH3), 1.22 (t, 3H, J = 7.5 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.5, 146.4, 145.8, 141.5, 140.5, 140.1, 139.5, 130.8, 130.4, 130.3, 130.0, 129.5, 128.5, 128.4, 128.2, 127.5, 127.1, 126.8, 125.4, 124.5, 122.5, 113.6, 77.5, 57.8, 56.6, 56.5,14.2; IR (KBr, ν, cm-1): 3446, 3308, 3171, 2966, 2850, 2181, 1656, 1621, 1591, 1546, 1491, 1471, 1406, 1381, 1357, 1329, 1262, 1161, 1122, 1058, 1029, 762, 732, 705; Anal. Calcd. for C30H25N3O5: C, 70.99; H, 4.96; N, 8.28%. Found: C, 70.73; H, 4.69; N, 8.11%. 3.2.49. Data for Ethyl 3-amino-1-(2-methoxyl-4-methoxyl5-methoxyl -phenyl)-1H-benzo[a]pyrano[2,3-c]phenazine2-carboxylate (6{1,2,2,30}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.17 (d, 1H, J = 7.0 Hz, Ar-H), 8.49 (d, 1H, J = 7.0 Hz, Ar-H), 8.29-8.22 (m, 1H, Ar-H), 8.17-8.13 (m, 1H, Ar-H), 8.03-7.95 (m, 1H, Ar-H), 7.87-7.81 (m, 3H, Ar-H), 7.48-7.37 (m, 4H, Ar-H), 7.25 (s, 2H, NH2), 7.12 (t, 1H, J = 7.0 Hz, Ar-H), 5.79 (s, 1H, CH), 4.19 (q, 2H, J = 7.5 Hz, CH2), 3.75 (s, 3H, CH3), 3.63 (s, 3H, CH3), 3.57 (s, 3H, CH3),1.27 (t, 3H, J = 7.0 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.9, 146.4, 145.9, 141.5, 140.9, 140.4, 139.5, 130.9, 130.4, 130.3, 130.0, 129.9, 128.5, 128.4, 128.2, 127.9, 127.1, 126.8, 125.9, 124.5, 122.5, 113.9, 77.5,



57.8, 56.9, 56.5, 55.7, 14.2; IR (KBr, ν, cm-1): 3446, 3303, 3171, 2966, 2853, 2181, 1656, 1623, 1591, 1546, 1493, 1471, 1406, 1383, 1357, 1323, 1262, 1161, 1123, 1058, 1023, 762, 732, 703; Anal. Calcd. for C31H27N3O6: C, 69.26; H, 5.06; N, 7.82%. Found: C, 68.93; H, 4.84; N, 7.59%. 3.2.50. Data for Ethyl 3-amino-1-(2-methoxyl-3-methoxyl4-methoxyl -phenyl)-1H-benzo[a]pyrano[2,3-c]phenazine2-carboxylate (6{1,2,2,33}) Yellow solid, m.p. >250 ◦C; 1H NMR (500 MHz, DMSOd6) (δ, ppm): 9.14 (d, 1H, J = 7.5 Hz, Ar-H), 8.45 (d, 1H, J = 7.5 Hz, Ar-H), 8.27-8.21 (m, 1H, Ar-H), 8.17-8.11 (m, 1H, Ar-H), 8.01-7.96 (m, 1H, Ar-H), 7.87-7.83 (m, 3H, Ar-H), 7.42-7.34 (m, 4H, Ar-H), 7.23 (s, 2H, NH2), 7.18 (t, 1H, J = 7.5 Hz, Ar-H), 5.76 (s, 1H, CH), 4.16 (q, 2H, J = 7.0 Hz, CH2), 3.79 (s, 3H, CH3), 3.67 (s, 3H, CH3), 3.55 (s, 3H, CH3),1.25 (t, 3H, J = 7.0 Hz, CH3); 13C NMR (125 MHz, DMSO-d6) (δ, ppm): 158.4, 146.5, 145.9, 141.5, 140.9, 140.5, 139.5, 130.9, 130.5, 130.3, 130.1, 129.9, 128.7, 128.4, 128.2, 127.7, 127.1, 126.8, 125.7, 124.5, 122.7, 113.9, 77.7, 57.8, 56.7, 56.4, 55.3, 14.8; IR (KBr, ν, cm-1): 3448, 3303, 3171, 2968, 2853, 2188, 1656, 1623, 1598, 1548, 1493, 1471, 1406, 1388, 1357, 1328, 1262, 1161, 1128, 1058, 1028, 762, 738, 708; Anal. Calcd. for C31H27N3O6: C, 69.26; H, 5.06; N, 7.82%. Found: C, 68.89; H, 4.89; N, 7.67%. 3.3. Cytotoxicity and Proliferation Assay Four human cell lines were used in this study, human colorectal cancer cell line (HCT116), human lung adenocarcinoma epithelial cell line (A549), human hepatocellular carcinoma cell line (HepG2), and human breast adenocarcinoma cell line (MCF7). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; TBD Science), 100 U/mL penicillin and 100 μg/mL streptomycin (Yuanmu, China) at 37°C with 5% CO2. A CCK8 assay was used to measure cell proliferation and viability. Briefly, cells were plated in 96-well plates (1 ×104 cells/well) and routinely cultured for 24 h. Then, cells were treated with various concentrations of compounds or 1% dimethyl sulfoxide (DMSO) (vehicle) in DMEM supplemented with 3% FBS. After 44 h of treatment, 10 μL CCK8 solution (2.5 mg/mL) was added to each well, followed by incubation for 4 h at 37 °C with 5% CO2. Absorbance was measured at 450 nm using a microplate reader.

and A549 cell lines than the positive controls in terms of IC50 values. Moreover, the other forty-nine compounds illustrated poor or no activities against all of the four cell lines. SAR studies showed that the CN and p-dimethylamino phenyl substituents on the γ-pyran structure on ring C played the crucial role in antiproliferative activity since omitting both of the two groups resulted in significant loss of the cytotoxic activity. The results suggest that synthetically feasible benzo[a]pyrano[2,3-c]phenazine derivatives may prove to be advantageous for further design as antitumor agents. Additional optimum molecular design and SAR studies of substituted phenazines will be reported in future publications. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS We gratefully acknowledge financial support from the Project Program of Nature Science Foundation of Jiangsu Province of China for Young Scientists (grant No. BK20130648), College Students Innovation Project for the R&D of Novel Drugs (grant No. J1030830), National College Students Innovation and Entrepreneurship Training Program (grant No. G14046 and SY13100), State Key Laboratory of Natural Medicines, China Pharmaceutical University (grant No. JKGQ201112). REFERENCES [1]

[2]

CONCLUSION In summary, a series of benzo[a]pyrano[2,3-c]phenazine derivatives were synthesized via the one-pot, fourcomponent domino reactions, and their antitumor activities were evaluated. The in vitro biological evaluation of all compounds was examined against cultured HCT116, MCF7, HepG2 and A549 cell lines. The results of in vitro antitumor activity indicated that the most active compound 6{1,2,1,9} exhibited the highest growth inhibitory activity against the HepG2 cell line with the IC50 value of 6.71 µM, which was 1.6-fold more potent than positive control antitumor drug HCPT with the IC50 value of 10.56 µM. However, compound 6{1,2,1,9} illustrated less potent against the HCT116, MCF7

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[3]

(a) Thompson, L. A. Recent applications of polymer-supported reagents and scavengers in combinatorial, parallel, or multistep synthesis. Curr. Opin. Chem. Biol., 2000, 4, 324-337; (b) Trost, B. M. Atom economy-a challenge for organic synthesis: Homogeneous catalysis leads the way. Angew. Chem. Int. Ed., 1995, 34, 259-281; (c) Tietze, L. F. Domino reactions in organic synthesis. Chem. Rev., 1996, 96, 115-136; (d) Trost, B. M. The atom economy-a search for synthetic efficiency. Science, 1991, 254, 1471-1477; (e) Singh, M. S.; Nandi, G. C.; Samai, S. DABCO-Promoted three-component regioselective synthesis of functionalized chromen-5-ones and pyrano[3,2-c]chromen-5-ones via direct annulation of α-oxoketene-N,S-arylaminoacetals under solvent-free conditions. Green Chem., 2012, 14, 447-455; (f) Wen, L. R.; Li, Z. R.; Li, M.; Cao, H. Solvent-free and efficient synthesis of imidazo[1,2-a]pyridine derivatives via a one-pot threecomponent reaction. Green Chem., 2012, 14, 707-716. (a) Schreiber, S. L. Target-oriented and diversity-oriented organic synthesis in drug discovery. Science, 2000, 287, 1964-1969; (b) Strausberg, R. L.; Schreiber, S. L. From knowing to controlling: A path from Genomics to drugs using small molecule probes. Science, 2003, 300, 294-295; (c) Burke, M. D.; Schreiber, S. L. A planning strategy for diversity-oriented synthesis. Angew. Chem. Int. Ed., 2004, 43, 46-58; (d) Spring, D. R. Diversity-oriented synthesis: A challenge for synthetic chemists. Org. Biomol. Chem., 2003, 1, 3867-3870; (e) Horton, D. A.; Bourne, G. T.; Smythe, M. L. The combinatorial synthesis of bicyclic privileged structures or privileged substructures. Chem. Rev., 2003, 103, 893-930. (a) Marcaccini, S.; Torroba, T. Post-condensation modifications of the Passerini and Ugi reactions. Zhu, J.; Bienayme, H. Multicomponent Reactions, Wiley-VCH: Wienheim, Germany, 2005, p 33; (b) Domling, A. Recent developments in isocyanide based multicomponent reactions in applied chemistry. Chem. Rev., 2006, 106, 17-89; (c) Orru, R. V. A.; De Greef, M. Recent advances in solutionphase multicomponent methodology for the synthesis of heterocyclic compounds. Synthesis, 2003, 1471-1499; (d) Weber, L. The application of multicomponent reactions in drug

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[4]

[5]

[6]

[7] [8]

[9]

[10]

Gaoet al.

Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 10 discovery. Curr. Med. Chem., 2002, 9, 2085-2093; (e) Bienayme, H.; Hulme, C.; Oddon, G.; Schmitt, P. Maximizing synthetic efficiency: Multi-component transformations lead the way. Chem. Eur. J., 2000, 6, 3321-3329. (a) Makgatho, M.; Anderson, R.; O’Sullivan, J.; Egan, T.; Freese, J.; Cornelius, N.; Van Rensburg, C. Tetramethylpiperidinesubstituted phenazines as novel anti-plasmodial agents. Drug Dev. Res., 2000, 50, 195-202; (b) Andrade-Nieto, V.; Goulart, M.; da Silva, J. F.; da Silva, M. J.; Pinto, M.; Pinto, A.; Zalis, M.; Carvalho, L.; Krettli, A. Antimalarial activity of phenazines from lapachol, β-lapachone and its derivatives against Plasmodium falciparum in vitro and Plasmodium berghei in vivo. Bioorg. Med. Chem. Lett., 2004, 14, 1145-1149. Neves-Pinto, C.; Malta, V.; Pinto, M.; Santos, R.; Castro, S.; Pinto, A. Structure-activity relationships for pyrido-, imidazo-, pyrazolo-, pyrazino-, and pyrrolophenazinecarboxamides as topoisomerasetargeted anticancer agents. J. Med. Chem., 2002, 45, 740-743. (a) Cartwright, D.; Chilton, W.; Benson, D. Pyrrolnitrin and phenazine production by Pseudomonas cepacia, strain 5.5B, a biocontrol agent of Rhizoctonia solani. Appl. Microbiol. Biotechnol., 1995, 43, 211-216; (b) Ligon, J.; Dwight, S.; Hammer, P.; Torkewitz, N.; Hofmann, D.; Kempf, H.; Pee, K. Natural products with antifungal activity from Pseudomonas biocontrol bacteria. Pest. Manage. Sci., 2000, 56, 688-695. Laursen, J. B.; Nielsen, J. Phenazine natural products: Biosynthesis, synthetic analogues, and biological activity. Chem. Rev., 2004, 104, 1663-1685. Muller, M.; Sorrell, T. Inhibition of the human platelet cyclooxygenase response by the naturally occurring phenazine derivative, 1-hydroxyphenazine. Prostaglandins, 1995, 50, 301311. (a) Tietze, L. F.; Kettschau, G. Hetero Diels-Alder reactions in organic chemistry. Top. Curr. Chem., 1997, 189, 1-120; (b) Chaniyara, R.; Thakrar, S.; Kakadiya, R.; Marvania, B.; Detroja, D.; Vekariya, N.; Upadhyay, K.; Manvar, A.; Shah, A. DBUcatalyzed multicomponent synthesis: Facile access of 4,5,6,9tetrahydro-pyrido[3,2-c]quinolones. J. Hetercyclic Chem., 2014, 51, 466-474. (a) Wang, S.; Miller, W.; Milton, J.; Vicker, N.; Stewart, A.; Charlton, P.; Mistry, P.; Hardick, D.; Denny, W. Structure-activity relationships for analogues of the phenazine-based dual

Received: December 15, 2014

[11]

[12] [13]

topoisomerase I/II inhibitor XR11576. Bioorg. Med. Chem. Lett., 2002, 12, 415-418; (b) Vicker, N.; Burgess, L.; Chuckowree, I.; Dodd, R.; Folkes, A.; Hardick, D.; Hancox, T.; Miller, W.; Milton, J.; Sohal, S.; Wang, S.; Wren, S.; Charlton, P.; Dangerfield, W.; Liddle, C.; Mistry, P.; Stewart, A.; Denny, W. Novel angular benzophenazines: Dual topoisomerase I and topoisomerase II inhibitors as potential anticancer agents. J. Med. Chem., 2002, 45, 721-739. (a) Elisa, P. S.; Jorge, S. D.; Ana, E. B.; Angel, G. R. Synthesis of 9- and 10-membered macrolactones by selective ozonolysis of 1,4diazaphenanthrene derivatives. Tetrahedron, 2005, 61, 437-445; (b) Wang, S. L.; Wu, F. Y.; Cheng, C.; Zhang, G.; Liu, Y. P.; Jiang, B.; Shi, F.; Tu, S. J. Multicomponent synthesis of poly-substituted benzo[a]pyrano-[2,3-c]phenazine derivatives under microwave heating. ACS Comb. Sci., 2011, 13, 135-139; (c) Shaterian, H. R.; Moradi, F.; Mohammadnia, M. Nano copper(II) oxide catalyzed four-component synthesis of functionalized benzo[a]pyrano[2,3c]phenazine derivatives. C. R. Chimie, 2012, 15, 1055-1059; (d) Shaterian, H. R.; Mohammadnia, M. Mild basic ionic liquid catalyzed four component synthesis of functionalized benzo[a]pyrano[2,3-c]phenazine derivatives. J. Mol. Liquids, 2013, 177, 162-166; (e) Hasaninejad, A.; Firoozi, S. One-pot, sequential four-component synthesis of benzo[c]pyrano[3,2-a]phenazine, bisbenzo[c]pyrano[3,2-a]phenazine and oxospiro benzo[c]pyrano[3,2a]phenazine derivatives using 1,4-diazabicyclo[2.2.2]octane (DABCO) as an efficient and reusable solid base catalyst. Mol. Divers., 2013, 17, 499-513; (f) Hasaninejad, A.; Firoozi, S.; Mandegani, F. An efficient synthesis of novel spiro[benzo[c]pyrano[3,2-a]phenazines] via domino multicomponent reactions using L-proline as a bifunctional organocatalyst. Tetrahedron Letters, 2013, 54, 2791-2794. Gao, X.; Lu, Y.; Fang, L.; Fang, X.; Xing, Y.; Gou, S.; Xi, T. Synthesis and anticancer activity of some novel 2-phenazinamine derivatives. Eur. J. Med. Chem., 2013, 69, 1-9. (a) Mosmann, T. Rapid colorimetric assay for cellular growthandsurvival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 1983, 65, 55-63; (b) Miri, R.; Motamedi, R.; Rezaei, M. R.; Firuzi, O.; Javidnia, A.; Shafiee, A. Design, synthesis and evaluation of cytotoxicity of novel chromeno[4,3-b]quinolone derivatives. Arch. Pharm., 2011, 344, 111-118.

Revised: August 12, 2015

Accepted: September 9, 2015