Design, combinatorial synthesis and biological evaluations of novel 3-amino-1'-((1-aryl-1H-1,2,3-triazol-5-yl)methyl)-2'-oxospiro[benzo[a] pyrano[2,3-c]phenazine-1,3'-indoline]-2-carbonitrile antitumor hybrid molecules

European Journal of Medicinal Chemistry 135 (2017) 125e141

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Design, combinatorial synthesis and biological evaluations of novel

0

3

[

-amino-1 -((1-aryl-1H-1,2,3-triazol-5-yl)methyl)-2 -oxospiro[benzo

0

a] pyrano[2,3-c]phenazine-1,3 -indoline]-2-carbonitrile antitumor

hybrid molecules

Yuanyuan Lu ^a

,

b

,

*

, Linlin Wang , Xiaobing Wang ^a^d, Tao Xi ^a^b, Jianmin Liao ^a^b,

a

,

b

,

a

,

c

a, c, **

Zhixiang Wang , Feng Jiang

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China

School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China

School of Engineering, China Pharmaceutical University, Nanjing 210009, China

a

b

c

d

Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China

a r t i c l e i n f o

a b s t r a c t

0

A combinatorial chemical library of ﬁfty-nine novel 3-amino-1 -((1-aryl-1H-1,2,3-triazol-5-yl)methyl)-

Article history:

Received 12 February 2017

Received in revised form

0

2

-oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -indoline]-2-carbonitrile, designed as hybrid molecules

of phenazine, pyran, indole and 1,2,3-triazole pharmacophores, were constructed in this study. Cytotoxic

evaluation indicated that some compounds exhibited moderate cytotoxicity against HCT116, MCF7,

HepG2 and A549 cancer cell lines in vitro, in which compound 36 was found to have best antiproliferative

12 April 2017

Accepted 14 April 2017

Available online 18 April 2017

activity against the A549 cancer cell line with IC50 value of 5.4 mM. All compounds had low or no effect

against L02 and HUVEC non-cancer cell lines. Compound 36 was further conﬁrmed to mainly locate

mitochondria in A549 cancer cells via laser-scanning confocal microscopy. Moreover, compound 36 was

proved to increase ROS production and induce cell cycle arrest in S phase. Western blot analysis illus-

trated Bax/Bcl-2 ratio was increased at dose-dependent manner, and both cleaved caspase-3 and cleaved

caspase-9 was enhanced by treated with compound 36. All the above evidences in vitro indicated that

compound 36 might induce the apoptosis of A549 cancer cells via a mitochondria-dependent pathway.

Keywords:

Hybrid molecules

Antiproliferative activity

ROS

Mitochondria

Cell cycle

Apoptosis

©

1. Introduction

importance of molecular hybridization in drug discovery, pyrano

3,2-a]phenazine hybrid molecules containing phenazine and py-

[

Diversity-oriented synthesis (DOS) is a powerful tool to design

and synthesize novel molecular skeletons of diverse polycyclic

heterocycles with potential medicinal and biological activities [1,2].

Molecular hybridization, as a rational and well-established syn-

thetic strategy deriving from DOS chemistry, can generate new

chemical libraries of diverse hybrid molecules used for drug

screening [3e5]. A number of molecular hybrids based on natural

product skeletons or pharmacophores have been designed, syn-

thesized and biologically evaluated [6e19]. In consideration of the

ran pharmacophores were successfully designed and synthesized,

which displayed excellent antitumor activity against human HepG2

cancer cell line [20]. These ﬁndings straightly inspired us to utilize

molecular hybridization strategy to produce potential bioactive

compounds.

It is well-known that the indole ring is a widely distributed

framework that is present in many pharmaceuticals and natural

compounds [21,22]. Triazoles, especially 1,2,3-triazole, and their

derivatives have been proved a pharmacologically important het-

erocyclic nucleus. Potential pharmaceuticals based on 1,2,3-

triazoles include the anticancer compound carboxyamidotriazole

(

CAI) [23], the nucleoside derivative non-nucleoside reverse tran-

*

Corresponding author. State Key Laboratory of Natural Medicines, China Phar-

maceutical University, Nanjing 210009, China.

* Corresponding author. State Key Laboratory of Natural Medicines, China Phar-

scriptase inhibitor tert-butyldimethylsilylspiroaminooxathioledi-

oxide (TSAO) [24] and so on. Click chemistry, mainly the copper(I)-

catalyzed azide-alkyne cycloaddition (CuAAC), offers a robust

*

maceutical University, Nanjing 210009, China.

E-mail addresses: luyy@cpu.edu.cn (Y. Lu), daniel_chem@126.com (F. Jiang).

http://dx.doi.org/10.1016/j.ejmech.2017.04.040

126

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

approach to construct 1,2,3-triazole moieties, which are attractive

connecting units because they are stable to metabolic degradation

and capable of hydrogen bonding, which can well bind biomole-

cular targets and improve compounds' solubility [25]. The rapid

construction and screening of focused combinatorial fragment li-

braries using CuAAC click chemistry is a highly efﬁcient way for

establishing structure-activity relationship (SAR) and for discov-

ering bioactive molecules [26,27].

Although phenazines, pyrans, indoles and 1,2,3-triazole are

attracting great attention in academic and industrial areas, there

are not literature concerning the preparation of compounds

incorporating all of these motifs. As a part of ongoing efforts to

establish antitumor compound libraries and discover new anti-

tumor agents, we demonstrated the design of novel hybrids

incorporating phenazine, pyran, indole and 1,2,3-triazole motifs,

and the construction of a new compound library for biological

screening. We anticipate that the novel hybrids may display potent

antitumor activity and demonstrate interesting mechanism of ac-

tion. Here, we report the design and construction of combinatorial

chemical library of ﬁfty-nine novel compounds, cytotoxic evalua-

tion and antitumor mechanism of these compounds were investi-

gated in details.

to have the best cell proliferation inhibitory activities against A549

cancer cell line with the IC50 values of 5.4 M. Obviously, com-

m

pounds 28~59 containing amine substituted phenazine skeleton

own better antiproliferative effect than compounds 1~27 contain-

ing benzophenazine skeleton. Considering the substituent effects of

these compounds, it seemed that the compounds with electron-

withdrawing groups such as chloro and bromo, especially tri-

ﬂuoromethyl group, demonstrated much potent antiproliferative

activities. In order to investigate the detailed antitumor mecha-

nism, we chose compound 36 for further mechanism study.

2.3. Compound 36 mainly located mitochondria in A549 cancer

cells

In order to ﬁgure out where compound 36 located, a laser-

scanning confocal microscopy was conducted to detect where

compound 36 sited in A549 cancer cells. The living cancer cells

were respectively incubated with either of our compound (0, 5,

10 mM), Mito Tracker Red CMXROS (0.1 mM) in culture medium for

30 min [32,33]. As shown in Fig. 3, the merged picture of compound

36 could overlay with Mito Tracker Red CMXROS very well.

2.4. Compound 36 increased ROS production in A549 cancer cells

2

. Results and discussion

Reactive oxygen species (ROS) can inﬂuence cell states,

2

.1. Chemistry

including cell survival, proliferation, differentiation, apoptosis, and

cell death [34e36]. In order to detect whether induced cytotoxic

effect of compound 36 is related with ROS level, we measured total

ROS levels in compound 36 treated A549 cancer cells. As shown in

Fig. 4, compared with that of the control group, compound 36

increased apoptosis in A549 cancer cells (P < 0.01) at dose-

dependent manner.

0

3

-amino-1 -((1-aryl-1H-1,2,3-triazol-5-yl)methyl)-2 -oxospiro

0

[

(

benzo[a]pyrano[2,3-c]phenazine-1,3 -indoline]-2-carbonitrile

1~59) were synthesized following the general methods as shown

in Scheme 1. Phenazine derivatives c and d was easily prepared

according to the procedure described in the literature [28,29]. As

shown in Scheme 2 1-(prop-2-yn-1-yl)indoline-2,3-dione (f) was

prepared by substitution reaction with propargyl bromide. The

synthesis of aromatic azide (i) was carried out by azidation reaction

of aromatic amines. Subsequently, we performed multi-component

one-pot reaction to get compound g or h using compound c or d,

Malononitrile (e), 1-(prop-2-yn-1-yl)indoline-2,3-dione (f) and 1,4-

diazabicyclo [2.2.2]octane (DABCO). Finally, target compounds

2.5. Compound 36 induced S phase arrest in A549 cancer cells

We investigated the effect of compound 36 on the cell pro-

gression of A549 cancer cells using ﬂuorescence activated cell

sorter (FACS) by staining with propidium iodide. The cell cycle

proﬁle of A549 cancer cells were shown in Fig. 5a and b after

1

~59 were synthesized using compound g or h, and aromatic azide

incubated at different doses of compound 36 (0, 3, 6, and 12

mM) for

(i) with the method of classic CuAAC click reaction. The structures

24 h. Treatment of compound 36 at 0, 3, 6, 12 M for 24 h increased

m

of compounds 1~27 and 28~59 were shown in Fig. 1 and Fig. 2,

respectively.

S phase population to 17.8%, 30.5%, 41.7%, and 51.1%, respectively. All

the above results indicated that compound 36 can induce S phase

arrest in A549 cancer cells.

2

.2. Cell proliferation inhibitory activities of compounds 1~59

To further conﬁrm the S phase arrest effects of compound 36,

changes in the expression levels of S checkpoint related proteins

were investigated by western blot analysis, which included Cyclin

B, Cyclin D, Cyclin E, CDK1, CDK2, p21, and p53. These results were

shown in Fig. 5c and d. After treatment by compound 36 for 24 h,

the expression of p53 and p21 were increased. Meanwhile, cyclin E

and CDK2 were inhibited in a dose-dependent manner. However,

the expression of cyclin B, cyclin D and CDK1 showed no signiﬁcant

difference between control groups. Therefore, these results

conﬁrmed that compound 36 induced S phase arrest by up-

regulating the expression of both p53 and p21, concomitant with

a decrease of both cyclin E and CDK2 in A549 cancer cells.

The target compounds (1~59) were examined for their effects on

four cancer cell viability (Human hepatocellular carcinoma cell line

HepG2), Human lung adenocarcinoma cell line (A549), Human

breast cancer cell line (MCF7) and Human colon cancer cell line

HCT116)), as well as Human normal liver cell (L02) and Human

(

umbilical vein endothelial cells (HUVEC) non-cancer cell lines,

respectively, using HCPT and cisplatin as the reference drugs

[30,31].

As reported in Table 1, the majority of these compounds man-

ifested weak or less antiproliferative effect on the four cancer cell

lines. Meanwhile, all compounds had low or no effect against L02

and HUVEC non-cancer cell lines. All compounds showed less

antiproliferative activities against HepG2 and MCF7 cancer cell

2.6. Compound 36 induced apoptosis in A549 cancer cells

lines (IC50 > 15

erate proliferative inhibitory activities against HCT116 cancer cell

line with the IC50 values of around 10 M. Furthermore, several

m

M). Compounds 18, 30, 35 and 58 showed mod-

Since A549 cancer cells treated with compound 36 showed a

profound S phase arrest accompanied by activation of ROS level, we

wanted to investigate whether the inhibition effect leads to

apoptosis. As shown in Fig. 6a and b, more than 40% cancer cells

m

compounds such as 29, 50, 52 and 57 showed moderate prolifera-

tive inhibitory activities against A549 cancer cell line with the IC50

were detected apoptosis at the concentration of 12

mM. We further

values of around 10 mM. Delightedly, one compound, 36 was found

conﬁrmed expression levels of Bax, BCl-2, cleaved caspase-3 and

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

127

Scheme 1. Synthetic routes of target compounds 1~59. Reagents and conditions: (I) DABCO, EtOH, 91%; (II) Conc. HCl, FeCl

Sodium ascorbate, CuSO , THF/H O, 55e82%.

3

, 35%; (III) e, f, DABCO, EtOH, 72% (g), 76% (h); (IV) i,

4

2

cleaved caspase-9 by subjecting the whole cell lysates of treated

A549 cancer cells with compound 36 to western blot analysis. As

shown in Fig. 6c and d, Bax/Bcl-2 ratio was increased at dose-

dependent manner, and both cleaved caspase-3 and cleaved

caspase-9 were enhanced by treating with compound 36. Overall,

our results conﬁrmed that compound 36 might induce the

apoptosis of A549 cancer cells via a mitochondrial-dependent

pathway.

designed as hybrid molecules of phenazine, pyran, indole and 1,2,3-

triazole pharmacophores, were constructed. Cytotoxic evaluation

indicated that some compounds exhibited moderate cytotoxicity

against HCT116, MCF7, HepG2 and A549 cancer cell lines in vitro, in

which compound 36 was found to have best antiproliferative ac-

tivity against the A549 cancer cell line with IC50 value of 5.4 mM. All

compounds had low or no effect against L02 and HUVEC non-

cancer cell lines. Compound 36 was further conﬁrmed to mainly

locate mitochondria in A549 cancer cells via laser-scanning

confocal microscopy. Moreover, compound 36 was proved to in-

crease ROS production and induce cell cycle arrest in S phase.

Western blot analysis illustrated Bax/Bcl-2 ratio was increased at

dose-dependent manner, and both cleaved caspase-3 and cleaved

caspase-9 was enhanced by treating with compound 36, which

3

. Conclusions

In this study, a combinatorial chemical library of ﬁfty-nine novel

0

3

[

-amino-1 -((1-aryl-1H-1,2,3-triazol-5-yl)methyl)-2 -oxospiro

0

benzo[a]pyrano[2,3-c]phenazine-1,3 -indoline]-2-carbonitrile,

128

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

consideration of the interesting antitumor mechanism of com-

pound 36, it will be promising to proceed to optimize the structure

of this type of hybrids based on compound 36 as lead compound.

Further work will be published in the near future.

4. Experimental section

4.1. 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 (100e300 mesh). TLC was conducted on silica gel

2

50 micron, GF254 plates with short-wavelength UV light for

1

13

visualization. H NMR and C NMR spectroscopic measurements

were performed on a Bruker AV-500 NMR spectrometer, using TMS

as internal references at 298 K, respectively. High-resolution mass

spectra (HRMS) were recorded on an Agilent 6520B UPLC-Q-TOF

mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).

The melting points (Mp) were determined on an X4-type apparatus

Scheme 2. Synthesis of compounds f and i. Reagents and conditions: (I) propargyl

bromide, K CO , DMF, 95%; (II) NaN , NaNO , 1 M HCl, 67e84%.

2

3

2

indicated that compound 36 might induce the apoptosis of A549

cancer cells via mitochondrial-dependent pathway. In

1

without correction. All of the products were identiﬁed by H and

a

13

C NMR and HRMS spectrometry.

Fig. 1. Structures of compounds 1~27.

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

129

Fig. 2. Structures of compounds 28~59.

4

i

.2. General procedure for the synthesis of intermediates c, d, f and

mixture was heated to reﬂux under stirring. After completion of the

reaction (0.5 h), as monitored by TLC, the reaction mixture was

allowed to cool to room temperature and then washed with satu-

rated sodium carbonate solution for three times. The solid product

was ﬁltered off, washed with deionized water for three times, dried

in vacuo, and subsequently recrystallized from hot ethanol to give

compound g.

Compounds c, d, f and i were prepared according to the methods

described in the literature [28,29,37] and [38], respectively.

4

.3. General procedure for the synthesis of intermediates g and h

Compound h was prepared with the similar method of prepar-

ing compound g.

Compound g was prepared according to the method described

as below. Compounds c (1 mmol), Malononitrile e (1 mmol), 1-

prop-2-yn-1-yl)indoline-2,3-dione f (1 mmol) were added to a 25-

mL round-bottomed ﬂask containing DABCO (0.2 mmol) in ethanol

15 mL). The ﬂask was ﬁtted with a condenser, and the resulting

(

4.4. General procedure for the synthesis of compounds 1~59

Compounds 1~59 were prepared according to the method

(

130

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

Table 1

Cell proliferation inhibitory activities of Compounds, 1~59 (48 h).

compd

IC50 (m

M)^a

HepG2^b

A549^c

MCF7^d

HCT116^e

L02^f

HUVEC^g

1

2

3

4

5

6

7

8

9

>50

39.6 ± 4.5

>50

35.2 ± 5.4

>50

42.6 ± 3.4

32.6 ± 3.1

44.3 ± 4.2

>50

43.6 ± 3.3

36.6 ± 3.5

>50

1

2

3

4

5

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

>50

42.2 ± 5.4

21.5 ± 4.8

>50

17.9 ± 1.8

>50

33.5 ± 2.7

>50

38.0 ± 5.5

15.8 ± 1.9

>50

30.5 ± 3.6

>50

25.2 ± 4.1

>50

44.2 ± 5.2

>50

42.5 ± 5.1

>50

24.6 ± 2.4

>50

34.4 ± 2.5

>50

30.7 ± 7.1

31.6 ± 2.2

>50

45.2 ± 4.3

>50

31.2 ± 4.2

11.3 ± 2.5

>50

22.9 ± 3.1

>50

29.2 ± 2.3

38.1 ± 2.2

>50

22.8 ± 2.4

>50

20.4 ± 3.2

17.2 ± 2.6

>50

38.6 ± 4.2

>50

34.2 ± 1.7

>50

18.3 ± 2.2

>50

18.2 ± 2.8

22.1 ± 2.5

31.7 ± 3.01

12.3 ± 0.74

24.6 ± 2.56

>50

10.3 ± 2.3

>50

29.8 ± 4.4

18.9 ± 2.1

27.4 ± 2.1

5.4 ± 0.7

>50

37.5 ± 5.1

>50

31.2 ± 1.7

>50

10.9 ± 1.9

28.1 ± 2.4

11.8 ± 2.2

>50

43.4 ± 3.5

>50

12.7 ± 2.0

>50

26.1 ± 2.1

12.4 ± 1.5

18.9 ± 2.1

>50

37.6 ± 2.5

>50

37.2 ± 4.1

>50

23.9 ± 2.3

28.7 ± 2.4

>50

12.1 ± 2.6

>50

3.2 ± 1.6

1.2 ± 0.7

>50

28.9 ± 2.5

26.8 ± 0.3

>50

16.5 ± 2.7

>50

33.1 ± 2.8

45.1 ± 3.9

>50

17.3 ± 2.5

>50

18.4 ± 2.7

>50

48.1 ± 3.2

37.8 ± 3.8

>50

33.2 ± 2.2

28.1 ± 2.8

>50

43.5 ± 3.5

17.8 ± 3.6

20.9 ± 2.9

>50

37.8 ± 2.3

>50

0.8 ± 0.2

1.3 ± 0.3

h

i

HCPT

CDDP

10.3 ± 1.4

0.4 ± 0.1

2.8 ± 1.1

4.8 ± 1.4

a

Each data point represents the mean ± SD from three different experiments performed in triplicate. Cancer cell lines used.

Human hepatocellular carcinoma cell line (HepG2).

Human lung adenocarcinomacell line (A549).

Human breast cancer cell line (MCF7).

Human colon cancer cell line (HCT116).

Human normal liver cell (L02).

Human umbilical vein endothelial cells (HUVEC).

Hydroxycamptothecine for cytotoxicity.

b

c

d

e

f

g

h

i

cis-dichlorodiamine platinum for cytotoxicity.

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

131

Fig. 3. A549 cancer cells were stained with compound 36 (0, 5, and 10

mM) and Mito Tracker Red CMXROS (0.1

mM) for 30 min (a) 0

m

M compound 36; (b) 0

m

M CMXROS; (c) merged

images of (a) and (b); (d) 5 M compound 36; (e) 0.1 M CMXROS; (f) merged images of (d) and (e); (g) 10

m

mM compound 36; (h) 0.1 mM CMXROS; (i) merged images of (g) and (h).

Ex@580 nm for the red channel (570e750 nm), and Ex@488 nm for green channel (470e520 nm). (For interpretation of the references to colour in this ﬁgure legend, the reader is

referred to the web version of this article.)

described in the literature [37]. Compound g or h, and aromatic

azide (i) reacted under the classic CuAAC click reaction conditions

to produce the ﬁnal products.

8.21e8.19 (m, 1H), 8.08e8.02 (m, 2H), 7.79e7.76 (m, 1H), 7.65 (s,

2H), 7.57e7.54 (m, 2H), 7.51 (s,1H), 7.40 (t, J ¼ 8.0 Hz, 2H), 7.29e7.26

(m, 2H), 7.10 (t, J ¼ 8.0 Hz, 2H), 6.86 (t, J ¼ 7.0 Hz, 1H), 5.33 (s, 2H),

13

2

6

.34 (s, 3H); C NMR (125 MHz, DMSO-d ) d 176.6, 163.3, 159.4,

0

143.8, 142.8, 140.5, 139.4, 139.2, 134.3, 132.7, 130.6, 130.3, 130.2,

130.0, 129.7, 129.2, 128.6, 128.3, 127.4, 125.6, 124.5, 123.3, 122.9,

4

.4.1. 3-amino-2 -oxo-1 -((1-(o-tolyl)-1H-1,2,3-triazol-5-yl)

0

methyl)spiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -indoline]-2-

1

22.7, 122.3, 122.1, 122.0, 121.5, 121.2, 118.5, 116.9, 116.5, 116.4, 116.2,

carbonitrile 1

þ

Yield 68%; Yellow solid; Mp > 250 ꢀ_C_;₁H NMR (500 MHz,

109.3, 35.8, 21.4; HR ESI-MS [M þ H]

m/z ¼ 613.2106 (calcd for

þ

C

37

25

H N

8

O

2

, 613.2100).

DMSO-d

.28e8.25 (m, 2H), 8.09e8.06 (m,1H), 8.04e8.01 (m,1H), 7.92e7.81

m, 2H), 7.77e7.74 (m, 1H), 7.61e7.57 (m, 2H), 7.46e7.42 (m, 2H),

.39e7.23 (m, 4H), 7.09e7.06 (m,1H), 6.85 (t, J ¼ 7.5 Hz,1H), 5.42 (d,

6

)

d

9.28 (d, J ¼ 7.8 Hz, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H),

8

(

7

0

4

.4.3. 3-amino-1 -((1-(4-ﬂuorophenyl)-1H-1,2,3-triazol-5-yl)

0

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

1

3

J ¼ 15.6 Hz, 1H), 5.29 (d, J ¼ 15.6 Hz, 1H), 2.08 (s, 3H); C NMR

125 MHz, DMSO-d 177.2, 163.5, 158.9, 143.1, 142.3, 140.4, 138.3,

38.1, 134.2, 133.5, 130.7, 130.6, 130.5, 130.3, 129.5, 129.4, 128.5,

28.2,127.3,125.7,125.1,123.1,122.8,122.6,122.3,122.2,122.1,121.9,

21.7, 118.3, 116.5, 116.4, 116.3, 116.0, 108.4, 35.7, 17.6; HR ESI-MS

indoline]-2-carbonitrile 3

(

1

6

)

d

ꢀ

1

Yield 73%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

9.28 (d, J ¼ 7.6 Hz, 1H), 8.57 (s, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 8.20

d, J ¼ 8.5 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H), 7.96 (d,

J ¼ 8.5 Hz, 1H), 7.92e7.90 (m, 1H), 7.80e7.75 (m, 2H), 7.64 (s, 2H),

.59e7.55 (m, 1H), 7.42e7.36 (m, 2H), 7.28 (td, J ¼ 7.6, 1.0 Hz, 1H),

6

d ) d

(

þ

2

[

M þ H] m/z ¼ 613.2104 (calcd for C37

25 8

H N O , 613.2100).

7

13

7

.09e7.07 (m, 2H), 6.85 (t, J ¼ 7.5 Hz, 1H), 5,32 (s, 2H); C NMR

0

4

.4.2. 3-amino-2 -oxo-1 -((1-(m-tolyl)-1H-1,2,3-triazol-5-yl)

(125 MHz, DMSO-d 176.8, 163.1, 159.5, 143.6, 142.7, 140.8, 139.9,

6

) d

0

methyl)spiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -indoline]-2-

139.4, 134.7, 133.0, 130.8, 130.5, 130.3, 130.2, 129.8, 129.2, 128.8,

128.4, 127.7, 125.3, 124.8, 123.3, 122.8, 122.6, 122.5, 122.0, 122.0,

carbonitrile 2

Yield 66%; Yellow solid; Mp > 250 C; ¹H NMR (500 MHz,

ꢀ

121.9, 121.9, 118.0, 116.7, 116.7, 116.4, 116.4, 108.7, 35.5; HR ESI-MS

[M þ H]^þm/z ¼ 617.1841 (calcd for C36

H22FN

O

þ

, 617.1850).

DMSO-d

6

)

d

9.28 (d, J ¼ 7.0 Hz, 1H), 8.54 (d, J ¼ 8.0 Hz, 2H),

8

2

132

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

Fig. 4. Effect of compound 36 on ROS levels. A549 cancer cells were treated with compound 36 (0, 3, 6, and 12

compound 36; (d) 12

M compound 36. The histogram showed population of ROS (ꢁ) cells.

mM). (a) 0 mM compound 36; (b) 3 mM compound 36; (c) 6 mM

m

0

4

.4.4. 3-amino-1 -((1-(4-chlorophenyl)-1H-1,2,3-triazol-5-yl)

DMSO-d

6

)

d

9.27 (d, J ¼ 7.7 Hz, 1H), 8.54 (d, J ¼ 8.1 Hz, 1H), 8.40 (s,

0

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

indoline]-2-carbonitrile 4

1H), 8.24 (d, J ¼ 8.5 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H),

7.95 (s, 1H), 7.87e7.84 (m, 1H), 7.75e7.71 (m, 2H), 7.62 (s, 2H),

7.55e7.49 (m, 2H), 7.39 (d, J ¼ 8.5 Hz, 2H), 7.28 (td, J ¼ 7.8, 1.0 Hz,

1H), 7.08 (d, J ¼ 7.5 Hz, 1H), 6.85 (t, J ¼ 7.5 Hz, 1H), 5.40 (d,

ꢀ

1

Yield 76%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

9.27 (d, J ¼ 8.0 Hz,1H), 8.62 (s,1H), 8.54 (d, J ¼ 8.0 Hz,1H), 8.20

d, J ¼ 8.5 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H), 7.95 (s,

H), 7.78 (d, J ¼ 9.0 Hz, 2H), 7.66 (s, 2H), 7.59 (d, J ¼ 9.0 Hz, 2H),

.58e7.54 (m, 1H), 7.42 (d, J ¼ 8.0 Hz, 1H), 7.28 (td, J ¼ 7.8, 1.1 Hz,

6

d ) d

(

1

7

1

0

1

13

J ¼ 15.6 Hz,1H), 5.32 (d, J ¼ 15.6 Hz,1H); C NMR (125 MHz, DMSO-

6

d ) d 176.8, 165.3, 159.2, 144.1, 142.6, 140.5, 139.3, 139.0, 133.5, 133.3,

130.6, 130.4, 130.1, 130.0, 129.7, 129.6, 129.2, 129.0, 126.7, 125.1,

125.0, 124.6, 122.8, 122.5, 122.3, 122.1, 121.8, 121.5, 121.2, 117.0, 116.8,

H), 7.09 (d, J ¼ 7.5 Hz, 1H), 7.04 (d, J ¼ 8.5 Hz, 1H), 6.85 (td, J ¼ 7.5,

13

þ

.8 Hz, 1H), 5.33 (s, 2H); C NMR (125 MHz, DMSO-d

6

)

d

177.3,

116.6, 116.4, 116.3, 108.6, 35.5; HR ESI-MS [M þ H] m/z ¼ 633.1559

þ

64.8, 159.7, 144.3, 142.2, 140.9, 139.7, 139.2, 133.8, 133.6, 130.6,

30.5, 130.2, 130.0, 129.7, 129.1, 128.6, 128.2, 126.1, 125.9, 124.3,

23.8, 122.9, 122.7, 122.3, 122.1, 122.0, 121.6, 121.4, 117.4, 116.5, 116.3,

(calcd for C36

H22ClN

8

O

2

, 633.1554).

1

0

4.4.6. 3-amino-1 -((1-(3-chlorophenyl)-1H-1,2,3-triazol-5-yl)

þ

16.2, 116.0, 108.9, 35.1; HR ESI-MS [M þ H] m/z ¼ 633.1551 (calcd

0

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

indoline]-2-carbonitrile 6

þ

for C36

H22ClN

8

O

2

, 633.1554).

ꢀ

1

Yield 65%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

0

4

.4.5. 3-amino-1 -((1-(2-chlorophenyl)-1H-1,2,3-triazol-5-yl)

d

6

)

d

9.28 (d, J ¼ 8.0 Hz, 1H), 8.65 (s, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 8.19

0

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

indoline]-2-carbonitrile 5

(d, J ¼ 8.5 Hz,1H), 8.10e8.06 (m,1H), 8.04e8.01 (m,1H), 7.95 (s,1H),

7.80e7.72 (m, 3H), 7.66 (s, 2H), 7.55e7.51 (m, 2H), 7.41 (d, J ¼ 8.0 Hz,

1H), 7.29 (td, J ¼ 8.0, 1.1 Hz, 1H), 7.09 (d, J ¼ 7.5 Hz, 1H), 7.01 (d,

Yield 68%; Yellow solid; Mp > 250 C; ¹H NMR (500 MHz,

ꢀ

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

133

Fig. 5. Treatment of compound 36 induced S phase arrest along with an increase of p53, p21 and a decrease of cyclin E, CDK2 in a dose-dependent manner in A549 cancer cells. (a)

FACS analysis. A549 cancer cells were treated with various concentrations (0, 3, 6, and 12 M) of compound 36 for 24 h. (b) Quantiﬁcation of cells' distribution per phase. Values

represent the mean ± SD obtained from three different experiments. (c) A549 cancer cells treated 24 h with compound 36 were subjected to western blot analysis. -actin was used

as the loading control. (d) The levels of proteins in (c) were normalized to -actin. Values represent the mean ± SD obtained from three different experiments. *P < 0.05, **P < 0.01,

**P < 0.001 signiﬁcantly different from the value of control (untreated).

m

b

*

0

4.4.8. 3-amino-1 -((1-(2-bromophenyl)-1H-1,2,3-triazol-5-yl)

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

J ¼ 8.5 Hz, 1H), 6.85 (td, J ¼ 7.5, 0.7 Hz, 1H), 5.35 (d, J ¼ 15.7 Hz, 1H),

13

0

5

.31 (d, J ¼ 15.7 Hz, 1H); C NMR (125 MHz, DMSO-d

63.2,159.3,143.9, 141.8, 140.7, 140.3,140.1, 134.3, 134.1, 131.6, 130.8,

30.6, 129.9, 129.5, 129.4, 128.9, 128.6, 126.5, 126.3, 124.6, 123.4,

23.2, 122.5, 122.1, 121.7, 121.4, 121.3, 121.0, 117.7, 116.8, 116.5, 116.3,

6

)

d

177.8,

1

indoline]-2-carbonitrile 8

ꢀ

1

Yield 78%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

1

d

6

)

d

9.28 (d, J ¼ 7.8 Hz, 1H), 8.54 (d, J ¼ 7.5 Hz, 1H), 8.37 (s, 1H), 8.25

þ

116.1, 108.7, 35.3; HR ESI-MS [M þ H] m/z ¼ 633.1561 (calcd for

(d, J ¼ 8.0 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H), 7.89e7.86

(m, 2H), 7.77e7.74 (m, 1H), 7.60 (s, 2H), 7.56 (td, J ¼ 7.5, 1.5 Hz, 1H),

þ

C

36

H22ClN

8

O

2

, 633.1554).

7

.52 (td, J ¼ 7.5, 1.8 Hz, 1H), 7.45e7.41 (m, 2H), 7.37 (d, J ¼ 8.0 Hz,

0

4

.4.7. 3-amino-1 -((1-(4-bromophenyl)-1H-1,2,3-triazol-5-yl)

1H), 7.28 (td, J ¼ 7.5, 0.8 Hz, 1H), 7.08 (d, J ¼ 7.0 Hz, 1H), 6.85 (t,

13

0

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

J ¼ 7.5 Hz, 1H), 5.41 (d, J ¼ 15.6 Hz, 1H), 5.31 (d, J ¼ 15.6 Hz, 1H);

C

indoline]-2-carbonitrile 7

NMR (125 MHz, DMSO-d 162.7, 159.9, 148.1, 147.8, 145.6, 144.2,

6

) d

ꢀ

1

Yield 72%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

9.27 (dd, J ¼ 8.0, 0.8 Hz, 1H), 8.62 (s, 1H), 8.54 (d, J ¼ 8.0 Hz,

H), 8.20 (d, J ¼ 8.5 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H),

143.1, 141.3, 135.1, 133.0, 133.0, 131.9, 131.2, 131.0, 130.7, 130.6, 130.3,

129.6, 129.2, 129.1, 128.8, 128.5, 128.1, 125.8, 125.3, 123.8, 123.1,

d

1

7

(

6

) d

122.9, 122.3, 122.1, 122.1, 121.6, 118.4, 109.1, 67.4, 25.5; HR ESI-MS

[M þ H]^þm/z ¼ 677.1058 (calcd for C36

H22BrN

O

þ

, 677.1049).

.78e7.74 (m, 1H), 7.72 (s, 4H), 7.66 (s, 2H), 7.57e7.54 (m, 1H), 7.42

d, J ¼ 8.0 Hz, 1H), 7.28 (td, J ¼ 7.5, 1.1 Hz, 1H), 7.09 (dd, J ¼ 7.5,

.8 Hz, 1H), 7.04 (d, J ¼ 8.5 Hz, 1H), 6.85 (td, J ¼ 7.5, 0.8 Hz, 1H), 5.32

s, 2H); ¹³C NMR (125 MHz, DMSO-d

177.5, 163.7, 158.5, 143.5,

41.7, 140.3, 140.2, 139.7, 134.7, 134.3, 131.5, 131.3, 131.2, 129.9, 129.7,

29.3, 128.6, 128.2, 126.5, 126.1, 124.6, 123.8, 123.5, 122.6, 122.3,

21.7, 121.5, 121.2, 120.9, 117.5, 116.4, 116.2, 115.6, 115.2, 107.9, 35.8;

8

2

0

4.4.9. 3-amino-1 -((1-(3-bromophenyl)-1H-1,2,3-triazol-5-yl)

0

(

1

6

)

d

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

indoline]-2-carbonitrile 9

ꢀ

1

Yield 76%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

d

6

)

d

9.28 (d, J ¼ 7.6 Hz, 1H), 8.64 (s, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 8.19

þ

2

,

HR ESI-MS [M þ H] m/z ¼ 677.1055 (calcd for C36

H22BrN

8

O

(d, J ¼ 8.2 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H), 7.87 (t,

6

77.1049).

J ¼ 2.0 Hz, 1H), 7.82 (dd, J ¼ 8.1, 1.2 Hz, 1H), 7.75e7.72 (m, 1H),

134

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

Fig. 6. Treatment of compound 36 induced apoptosis along with effect of compound 36 on the relative protein expression levels of Bcl-2, Bax, cleaved caspase-3, and cleaved

caspase-9 in A549 cancer cells. (a) FACS analysis. A549 cancer cells were treated with various concentrations (0, 3, 6, and 12 M) of compound 36 for 24 h. (b) Ratio of apoptosis

A549 cancer cells after treated with compound 36. (c) A549 cancer cells treated 24 h with compound 36 were subjected to western blot analysis. -actin was used as the loading

m

b

control. (d) The levels of proteins in (c) were normalized to

b-actin. Values represent the mean ± SD obtained from three different experiments. *P < 0.05, **P < 0.01, ***P < 0.001

signiﬁcantly different from the value of control (untreated).

0

7

.65e7.64 (m, 3H), 7.54e7.51 (m, 1H), 7.48 (t, J ¼ 8.1 Hz, 1H), 7.41 (d,

4.4.11. 3-amino-1 -((1-(2-methoxyphenyl)-1H-1,2,3-triazol-5-yl)

0

J ¼ 8.0 Hz, 1H), 7.29 (td, J ¼ 7.8, 1.0 Hz, 1H), 7.09 (d, J ¼ 7.3 Hz, 1H),

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

indoline]-2-carbonitrile 11

7

1

.01 (d, J ¼ 8.4 Hz, 1H), 6.85 (t, J ¼ 7.5 Hz, 1H), 5.35 (d, J ¼ 15.7 Hz,

13

ꢀ

1

H), 5.30 (d, J ¼ 15.7 Hz, 1H); C NMR (125 MHz, DMSO-d

6

)

d

177.3,

Yield 63%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

1

60.1, 148.2, 144.3, 143.2, 141.4, 140.6, 140.4, 139.9, 138.0, 135.3,

32.2, 132.2, 131.8, 131.3, 131.0, 130.8, 130.7, 130.4, 129.3, 128.9,

28.2, 125.9, 125.4, 123.9, 123.2, 123.0, 122.8, 122.8, 122.7, 119.2,

d

6

)

d

9.28 (d, J ¼ 7.9 Hz, 1H), 8.54 (d, J ¼ 8.0 Hz,1H), 8.32 (s, 1H), 8.22

(d, J ¼ 8.5 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H), 7.84e7.80

(m, 1H), 7.67e7.60 (m, 4H), 7.50e7.47 (m, 1H), 7.36 (d, J ¼ 8.0 Hz,

1H), 7.28 (td, J ¼ 7.7, 1.0 Hz, 1H), 7.23 (d, J ¼ 8.5 Hz, 2H), 7.12 (td,

J ¼ 7.5, 1.0 Hz, 1H), 7.07 (d, J ¼ 7.4 Hz, 1H), 6.84 (td, J ¼ 7.5, 0.5 Hz,

þ

1

19.2, 118.5, 110.4, 109.2, 35.9; HR ESI-MS [M þ H] m/z ¼ 677.1035

þ

(

calcd for C36

H22BrN

8

O

2

, 677.1049).

1

H), 5.37 (d, J ¼ 15.6 Hz, 1H), 5.29 (d, J ¼ 15.6 Hz, 1H), 3.64 (s, 3H);

1

3

6

C NMR (125 MHz, DMSO-d ) d 177.3, 163.6, 149.2, 145.0, 142.9,

0

142.1,141.8,141.2,139.8,139.6,135.5,133.9,133.7,133.2,132.5,132.3,

132.0, 131.3, 130.5, 129.4, 129.1, 128.9, 127.3, 126.8, 124.5, 123.7,

4

.4.10. 3-amino-1 -((1-(4-methoxyphenyl)-1H-1,2,3-triazol-5-yl)

0

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

1

23.3, 122.0, 121.8, 121.5, 119.1, 118.6, 118.3, 111.4, 109.2, 56.2, 35.6;

indoline]-2-carbonitrile 10

þ

3

,

Yield 64%; Yellow solid; Mp > 250 ꢀ_C_;₁H NMR (500 MHz,

HR ESI-MS [M þ H] m/z ¼ 629.2054 (calcd for C37

25

H N

8

O

629.2050).

DMSO-d 9.27 (d, J ¼ 7.8 Hz, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 8.49 (s,

H), 8.21 (d, J ¼ 8.5 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H),

.94e7.90 (m, 1H), 7.80e7.77 (m, 1H), 7.66e7.65 (m, 3H), 7.61e7.58

6

)

d

1

7

(

1

0

4.4.12. 3-amino-1 -((1-(2-ethoxyphenyl)-1H-1,2,3-triazol-5-yl)

0

m, 1H), 7.40 (d, J ¼ 8.0 Hz, 1H), 7.29e7.26 (m,1H), 7.13 (d, J ¼ 8.5 Hz,

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

H), 7.09e7.05 (m, 3H), 6.84 (t, J ¼ 7.5 Hz, 1H), 5.32 (s, 2H), 3.81 (s,

indoline]-2-carbonitrile 12

13

ꢀ

1

3

H); C NMR (125 MHz, DMSO-d

6

)

d

177.6, 162.3, 148.3, 144.6,

Yield 74%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

1

42.6, 141.8, 141.4, 140.5, 139.2, 139.0, 134.7, 133.5, 133.2, 132.6,

d

6

)

d

9.27 (d, J ¼ 7.9 Hz, 1H), 8.55 (d, J ¼ 8.0 Hz, 1H), 8.40 (s, 1H), 8.19

132.5, 131.5, 131.3, 131.0, 129.8, 129.6, 129.1, 129.0, 126.6, 125.3,

(d, J ¼ 8.5 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H), 7.76e7.74

(m, 1H), 7.69 (dd, J ¼ 7.9, 1.6 Hz, 1H), 7.60 (s, 2H), 7.55e7.52 (m, 1H),

7.46e7.43 (m,1H), 7.35 (d, J ¼ 7.8 Hz,1H), 7.27 (td, J ¼ 7.7,1.0 Hz,1H),

7.17 (d, J ¼ 7.8 Hz, 1H), 7.13e7.07 (m, 3H), 6.84 (td, J ¼ 7.4, 0.5 Hz,

123.7,123.5,123.4,122.7, 122.6,122.4,119.5,119.3, 118.4,111.0, 109.5,

þ

5

C

6.6, 35.2; HR ESI-MS [M þ H] m/z ¼ 629.2057 (calcd for

þ

37

25

H N

8

O

3

, 629.2050).

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

135

1

H), 5.35 (d, J ¼ 15.6 Hz, 1H), 5.30 (d, J ¼ 15.7 Hz, 1H), 3.99e3.93 (m,

148.2, 144.0, 143.3, 141.4, 140.6, 140.5, 140.0, 139.9, 135.3, 131.9,

131.3, 131.1, 130.8, 130.4, 129.3, 128.9, 128.3, 125.9, 125.4, 123.9,

123.2, 123.0, 122.1, 120.9, 120.8, 120.8, 120.8, 120.0, 120.0, 119.8,

13

1

H), 3.78e3.72 (m, 1H), 0.88 (t, J ¼ 7.0 Hz, 3H); C NMR (125 MHz,

176.9,164.3,149.1,146.7,143.4,143.2,142.3,141.5,140.6,

39.9,137.3, 135.6,134.3,133.7, 133.5,133.1,132.6,131.6,130.4,129.7,

28.6, 128.3, 128.1, 127.4, 125.3, 123.8, 122.4, 122.2, 122.5, 121.0,

6

DMSO-d ) d

1

þ

118.6, 110.4, 109.3, 36.1, 24.5; HR ESI-MS [M þ H] m/z ¼ 656.2138

þ

(calcd for C38H

26

N

9

O

4

, 656.2159).

þ

1

19.7, 118.3, 118.0, 110.6, 108.6, 59.1, 35.6, 15.6; HR ESI-MS [M þ H]

þ

0

m/z ¼ 643.2212 (calcd for C38

H

27

N

8

O

3

, 643.2206).

4.4.17. 4-(5-((3-amino-2-cyano-2 -oxospiro[benzo[a]pyrano[2,3-c]

0

phenazine-1,3 -indolin]-1 -yl)methyl)-1H-1,2,3-triazol-1-yl)

0

4.4.13. 3-amino-1 -((1-(4-ethoxyphenyl)-1H-1,2,3-triazol-5-yl)

benzenesulfonamide 17

0

ꢀ

1

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

Yield 60%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

indoline]-2-carbonitrile 13

d

6

)

d

9.27 (d, J ¼ 7.8 Hz, 1H), 8.68 (s, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 8.18

ꢀ

1

Yield 77%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

(d, J ¼ 8.5 Hz, 1H), 8.08 (t, J ¼ 7.5 Hz, 1H), 8.02 (t, J ¼ 7.5 Hz, 1H),

7.96e7.91 (m, 4H), 7.74e7.71 (m, 1H), 7.66 (s, 2H), 7.53e7.51 (m,

3H), 7.44 (d, J ¼ 7.8 Hz, 1H), 7.29 (t, J ¼ 7.5 Hz, 1H), 7.09 (d, J ¼ 7.3 Hz,

1H), 7.00 (d, J ¼ 8.5 Hz, 1H), 6.85 (t, J ¼ 7.5 Hz, 1H), 5.36 (d,

d

6

)

d

9.27 (d, J ¼ 7.8 Hz, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 8.48 (s, 1H), 8.21

(

d, J ¼ 8.5 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H), 7.80e7.77

(

m,1H), 7.70e7.58 (m, 5H), 7.39 (d, J ¼ 7.9 Hz,1H), 7.28 (d, J ¼ 7.8 Hz,

13

1

H), 7.13 (d, J ¼ 8.5 Hz,1H), 7.08 (d, J ¼ 7.4 Hz,1H), 7.04 (d, J ¼ 9.0 Hz,

J ¼ 15.6 Hz,1H), 5.32 (d, J ¼ 15.6 Hz,1H); C NMR (125 MHz, DMSO-

2

H), 6.84 (t, J ¼ 7.5 Hz, 1H), 5.33 (d, J ¼ 15.7 Hz, 1H), 5.30 (d,

6

d ) d 176.8, 159.5, 147.7, 142.8, 140.9, 140.0, 139.9, 139.4, 134.7, 130.8,

1

3

J ¼ 15.7 Hz, 1H), 4.08 (q, J ¼ 7.0 Hz, 2H), 1.34 (t, J ¼ 7.0 Hz, 3H);

C

130.7, 130.3, 130.3, 129.8, 129.7, 128.8, 128.4, 127.9, 127.4, 125.8,

125.3, 124.8, 123.3, 122.6, 122.5, 121.2, 121.1, 121.1, 118.0, 113.7, 113.7,

6

NMR (125 MHz, DMSO-d ) d 177.5, 163.4, 148.5, 146.2, 143.9, 143.2,

þ

1

42.7, 141.2, 141.1, 139.5, 138.9, 136.6, 134.6, 134.1, 133.8,133.6,132.6,

31.3, 129.9, 129.6, 128.8, 128.6, 128.3, 127.2, 125.6, 124.9, 122.7,

22.4,122.1,121.0,119.3,118.6,118.3,111.3,109.8, 60.4, 35.3,15.8; HR

111.8, 109.9, 108.7, 48.7, 35.7; HR ESI-MS [M þ H] m/z ¼ 678.1674

þ

(calcd for C36

H N

24 9

O

4

S , 678.1672).

þ

0

ESI-MS [M þ H] m/z ¼ 643.2215 (calcd for C38

H N

27 8

O

3

, 643.2206).

4.4.18. 3-(5-((3-amino-2-cyano-2 -oxospiro[benzo[a]pyrano[2,3-c]

phenazine-1,3 -indolin]-1 -yl)methyl)-1H-1,2,3-triazol-1-yl)

benzenesulfonamide 18

0

4

.4.14. 3-amino-1 -((1-(4-nitrophenyl)-1H-1,2,3-triazol-5-yl)

0

ꢀ

1

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

Yield 81%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

indoline]-2-carbonitrile 14

d

6

)

d

9.26 (d, J ¼ 7.8 Hz,1H), 8.66 (s, 1H), 8.54 (d, J ¼ 8.0 Hz,1H), 8.32

ꢀ

1

Yield 80%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

9.28 (d, J ¼ 7.6 Hz,1H), 8.79 (s, 1H), 8.54 (d, J ¼ 8.5 Hz,1H), 8.36

(s, 1H), 8.17 (d, J ¼ 8.5 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m,

1H), 7.86 (d, J ¼ 8.0 Hz, 2H), 7.73e7.65 (m, 4H), 7.55 (s, 2H),

7.54e7.50 (m,1H), 7.45 (d, J ¼ 8.0 Hz,1H), 7.30 (t, J ¼ 7.5 Hz,1H), 7.09

(d, J ¼ 7.2 Hz,1H), 6.97 (d, J ¼ 8.5 Hz,1H), 6.85 (t, J ¼ 7.5 Hz,1H), 5.36

6

d ) d

(

d, J ¼ 9.0 Hz, 2H), 8.24 (d, J ¼ 8.0 Hz, 1H), 8.18 (d, J ¼ 8.5 Hz, 1H),

8

1

.10e8.01 (m, 4H), 7.64 (s, 2H), 7.52e7.49 (m, 1H), 7.45 (d, J ¼ 7.8 Hz,

H), 7.31e7.28 (m, 1H), 7.09 (d, J ¼ 7.0 Hz, 1H), 6.96 (d, J ¼ 8.7 Hz,

H), 6.85 (t, J ¼ 7.5 Hz, 1H), 5.37 (d, J ¼ 15.8 Hz, 1H), 5.32 (d,

13

(d, J ¼ 15.5 Hz, 1H), 5.32 (d, J ¼ 15.5 Hz, 1H); C NMR (125 MHz,

6

DMSO-d ) d 176.8, 159.5, 147.7, 145.7, 143.8, 142.6, 140.8, 140.0,

13

J ¼ 15.5 Hz, 1H); C NMR (125 MHz, DMSO-d

6

)

d

176.5, 163.6, 160.5,

139.8, 139.4, 138.7, 136.5, 134.7, 130.8, 130.6, 130.5, 130.3, 130.2,

129.8, 128.8, 128.4, 128.3, 127.5, 125.3, 124.8, 123.3, 122.6, 122.6,

122.5, 122.2, 117.9, 117.0, 109.8, 108.7, 48.7, 35.4; HR ESI-MS [M þ

1

47.4, 144.6, 144.3, 142.3, 141.9, 141.4, 139.6, 139.2, 139.0, 134.2,

31.8, 131.7, 131.3, 130.1, 129.6, 128.4, 128.1, 127.9, 127.6, 127.2, 125.7,

þ

24 9 4

H N O S , 678.1672).

1

3

6

24.4,123.8,123.3,123.2,121.5,119.7,119.3,118.2,109.5,108.3,107.9,

H] m/z ¼ 678.1679 (calcd for C36

þ

4

5.5; HR ESI-MS [M þ H] m/z ¼ 644.1799 (calcd for C36

22 9

H N O ,

0

44.1795).

4.4.19. 3-amino-1 -((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-5-yl)

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

indoline]-2-carbonitrile 19

0

4

.4.15. 3-amino-1 -((1-(4-aminophenyl)-1H-1,2,3-triazol-5-yl)

0

ꢀ

1

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

Yield 72%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

indoline]-2-carbonitrile 15

d

6

)

d

9.28 (d, J ¼ 7.7 Hz, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 8.49 (s, 1H), 8.21

Yield 68%; Yellow solid; Mp > 250 C; ¹H NMR (500 MHz,

ꢀ

(d, J ¼ 8.6 Hz, 1H), 8.08 (t, J ¼ 7.5 Hz, 1H), 8.03 (t, J ¼ 7.5 Hz, 1H),

7.96e7.89 (m,1H), 7.80e7.76 (m,1H), 7.65 (s,1H), 7.60e7.57 (m,1H),

7.47e7.45 (m, 2H), 7.39 (d, J ¼ 7.9 Hz,1H), 7.29e7.26 (m, 2H), 7.12 (d,

J ¼ 7.2 Hz, 1H), 7.09 (t, J ¼ 7.3 Hz, 1H), 6.84 (t, J ¼ 7.5 Hz, 1H), 5.32 (s,

DMSO-d

1

6

)

d

9.26 (d, J ¼ 7.2 Hz, 1H), 8.54 (d, J ¼ 7.8 Hz, 1H), 8.32 (s,

H), 8.21 (d, J ¼ 8.0 Hz, 1H), 8.07 (t, J ¼ 7.2 Hz, 1H), 8.02 (t, J ¼ 7.0 Hz,

H), 7.82e7.79 (m, 1H), 7.66e7.61 (m, 3H), 7.38e7.33 (m, 3H), 7.27

13

(

t, J ¼ 7.2 Hz, 1H), 7.16 (d, J ¼ 8.3 Hz, 1H), 7.08 (d, J ¼ 7.0 Hz, 1H), 6.84

6

2H), 2.26 (s, 3H), 2.25 (s, 3H); C NMR (125 MHz, DMSO-d ) d 177.8,

t, J ¼ 7.0 Hz, 1H), 6.62 (d, J ¼ 8.2 Hz, 2H), 5.45 (s, 2H), 5.31 (d,

161.5, 148.7, 143.5, 143.1, 141.9, 140.3, 140.1, 139.8, 139.6, 139.3, 136.1,

135.1,132.8,131.7,131.4,130.5,130.1,129.8,129.5,128.6,128.2,126.5,

126.3, 123.6, 123.1, 122.6, 122.4, 122.1, 118.6, 117.5, 117.2, 116.9, 110.7,

13

J ¼ 15.5 Hz,1H), 5.27 (d, J ¼ 15.5 Hz,1H); C NMR (125 MHz, DMSO-

d

1

6

)

d

176.8, 162.2, 159.5, 147.7, 143.9, 143.7, 142.7, 140.8, 140.0, 139.9,

39.4, 138.3, 134.7, 130.7, 130.4, 130.3, 130.2, 129.8, 128.8, 128.4,

27.6, 127.3, 127.3, 125.3, 124.8, 123.3, 122.6, 122.5, 122.2, 119.9,

þ

109.9, 36.5, 21.6, 21.3; HR ESI-MS [M þ H] m/z ¼ 627.2265 (calcd

þ

for C38

H N

27 8

O

2

, 627.2257).

þ

1

19.9, 117.9, 109.8, 108.7, 48.7, 35.7; HR ESI-MS [M þ H] m/

þ

0

z ¼ 614.2062 (calcd for C36

24

H N

9

O

2

, 614.2053).

4.4.20. 3-amino-1 -((1-(2,5-dimethylphenyl)-1H-1,2,3-triazol-5-yl)

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

indoline]-2-carbonitrile 20

0

4

.4.16. N-(4-(5-((3-amino-2-cyano-2 -oxospiro[benzo[a]pyrano

0

ꢀ

1

[

2,3-c]phenazine-1,3 -indolin]-1 -yl)methyl)-1H-1,2,3-triazol-1-yl)

phenyl)acetamide 16

Yield 63%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

Yield 69%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

d

6

)

9.28 (d, J ¼ 7.8 Hz, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 8.26 (d,

ꢀ

1

J ¼ 8.5 Hz, 1H), 8.22 (s, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H),

7.88e7.85 (m, 1H), 7.74e7.71 (m, 1H), 7.61 (s, 2H), 7.39 (d, J ¼ 7.9 Hz,

1H), 7.36 (d, J ¼ 8.5 Hz,1H), 7.29 (d, J ¼ 7.8 Hz, 2H), 7.25 (t, J ¼ 8.0 Hz,

1H), 7.08 (d, J ¼ 7.4 Hz, 1H), 6.93 (s, 1H), 6.85 (t, J ¼ 7.5 Hz, 1H), 5.39

d

8

1

6

)

d

10.15 (s, 1H), 9.27 (d, J ¼ 7.8 Hz, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H),

.50 (s, 1H), 8.28e8.17 (m, 2H), 8.09e8.06 (m, 1H), 8.04e8.01 (m,

H), 7.79e7.76 (m, 1H), 7.71e7.58 (m, 6H), 7.40 (d, J ¼ 7.8 Hz, 1H),

7

.29e7.26 (m, 1H), 7.10e7.04 (m, 2H), 6.84 (t, J ¼ 7.5 Hz, 1H), 5.32 (s,

(d, J ¼ 15.6 Hz,1H), 5.29 (d, J ¼ 15.6 Hz,1H), 2.27 (s, 3H), 2.02 (s, 3H);

H), 2.07 (s, 3H); ¹³C NMR (125 MHz, DMSO-d

)

d

177.4, 169.0, 160.1,

13

2

6

C NMR (125 MHz, DMSO-d ) d 177.9, 160.6, 148.2, 143.6, 143.1,

136

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

141.7, 140.9, 140.4, 139.3, 135.9, 135.5, 133.5, 131.8, 130.4, 130.2,

148.2, 143.3, 143.2, 141.4, 140.6, 140.5, 139.9, 137.4, 135.3, 133.5,

1

29.7, 129.4, 129.2, 128.8, 128.6, 128.2, 128.0, 127.7, 127.3, 127.2,

133.5, 132.4, 131.3, 131.3, 131.2, 130.9, 130.8, 130.4, 130.0, 129.5,

128.9, 128.6, 125.9, 125.9, 125.8, 125.7, 125.4, 123.9, 123.1, 123.0,

127.0, 126.5, 126.4, 126.2, 123.9, 123.5, 123.2, 118.8, 111.5, 109.7, 67.8,

þ

3

C

6.5, 25.1; HR ESI-MS [M þ H] m/z ¼ 627.2261 (calcd for

118.3, 110.4, 109.3, 36.1, 17.5; HR ESI-MS [M þ H] m/z ¼ 647.1693

þ

38 27

H N

8

O

2

, 627.2257).

(calcd for C37

H24ClN

8

O

2

, 647.1711).

0

4

.4.21. 3-amino-1 -((1-(3,5-dimethylphenyl)-1H-1,2,3-triazol-5-yl)

4.4.25. 3-amino-1 -((1-(3-chloro-4-ﬂuorophenyl)-1H-1,2,3-triazol-

0

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

5-yl)methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

indoline]-2-carbonitrile 21

indoline]-2-carbonitrile 25

Yield 66%; Yellow solid; Mp > 250 C; ¹H NMR (500 MHz,

ꢀ

Yield 82%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

ꢀ

1

DMSO-d

H), 8.20 (d, J ¼ 8.4 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H),

.78e7.75 (m, 1H), 7.67 (s, 2H), 7.57e7.54 (m, 1H), 7.38 (d, J ¼ 7.8 Hz,

6

)

d

9.28 (d, J ¼ 7.8 Hz, 1H), 8.56 (d, J ¼ 8.0 Hz, 1H), 8.47 (s,

d

6

)

d

9.27 (d, J ¼ 7.8 Hz, 1H), 8.60 (s, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 8.19

1

7

1

(d, J ¼ 8.2 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.02 (m, 1H), 7.88 (dd,

J ¼ 6.4, 2.6 Hz, 1H), 7.84e7.81 (m, 1H), 7.74e7.72 (m, 1H), 7.65 (s,

2H), 7.59 (t, J ¼ 9.0 Hz, 1H), 7.53e7.50 (m, 1H), 7.42 (d, J ¼ 7.8 Hz,

1H), 7.29 (t, J ¼ 7.7 Hz,1H), 7.09 (d, J ¼ 7.0 Hz,1H), 6.95 (d, J ¼ 8.5 Hz,

1H), 6.85 (t, J ¼ 7.5 Hz, 1H), 5.34 (d, J ¼ 15.6 Hz, 1H), 5.29 (d,

H), 7.31e7.26 (m, 3H), 7.09e7.06 (m, 3H), 6.85 (d, J ¼ 7.5 Hz, 1H),

13

5

6

.31 (s, 2H), 2.30 (s, 6H); C NMR (125 MHz, DMSO-d ) d 177.4,

1

60.1, 148.2, 143.9, 143.3, 141.4, 140.6, 140.5, 139.9, 139.7, 139.7,

36.8, 135.3, 131.3, 131.1, 130.8, 130.8, 130.4, 130.3, 129.3, 128.9,

28.3, 125.9, 125.3, 123.9, 123.2, 123.2, 122.2, 122.2, 118.6, 117.9,

13

J ¼ 15.6 Hz, 1H); C NMR (125 MHz, DMSO-d

6

) d 177.7, 160.8, 148.6,

144.6, 143.1, 142.7, 141.9, 141.5, 139.5,136.1, 135.3, 132.5,132.3, 132.0,

131.8, 131.4, 130.9, 130.4, 130.2, 130.1, 128.5, 128.3, 126.6, 126.4,

þ

1

17.9, 117.9, 110.4, 109.2, 36.1, 21.2, 21.2; HR ESI-MS [M þ H] m/

þ

z ¼ 627.2247 (calcd for C38

27

H N

8

O

2

, 627.2257).

123.5, 123.2, 122.9, 122.8, 122.3, 122.2, 121.6, 121.5, 118.5, 110.7,

þ

1

C

09.7, 35.6; HR ESI-MS [M þ H] m/z ¼ 651.1468 (calcd for

0

þ

2

4

.4.22. 3-amino-1 -((1-(2,4-dimethylphenyl)-1H-1,2,3-triazol-5-yl)

36

H

21ClFN

8

O

, 651.1460).

0

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

0

indoline]-2-carbonitrile 22

4.4.26. 3-amino-1 -((1-(2,5-dichlorophenyl)-1H-1,2,3-triazol-5-yl)

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

Yield 64%; Yellow solid; Mp > 250 C; ¹H NMR (500 MHz,

ꢀ

0

DMSO-d

H), 8.25 (d, J ¼ 8.4 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.02 (m, 1H),

.93 (d, J ¼ 2.2 Hz, 1H), 7.88e7.85 (m, 1H), 7.75e7.72 (m, 1H),

.64e7.60 (m, 3H), 7.52 (d, J ¼ 8.5 Hz, 1H), 7.39 (d, J ¼ 7.9 Hz, 1H),

.36 (d, J ¼ 8.5 Hz, 1H), 7.28 (t, J ¼ 7.7 Hz, 1H), 7.08 (d, J ¼ 7.4 Hz, 1H),

6

)

d

9.28 (d, J ¼ 7.8 Hz, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 8.42 (s,

indoline]-2-carbonitrile 26

ꢀ

1

7

6

1

d

Yield 59%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

d

6

)

d

9.28 (d, J ¼ 7.8 Hz, 1H), 8.54 (d, J ¼ 7.8 Hz, 1H), 8.42 (s, 1H), 8.25

(d, J ¼ 8.5 Hz, 1H), 8.16 (d, J ¼ 8.5 Hz, 1H), 8.09e8.06 (t, J ¼ 7.0 Hz,

1H), 8.03 (t, J ¼ 7.5 Hz, 1H), 7.93e7.89 (m, 1H), 7.86 (d, J ¼ 7.5 Hz,

1H), 7.76e7.72 (m, 1H), 7.70e7.67 (m, 1H), 7.61 (s, 1H), 7.49 (d,

J ¼ 2.3 Hz, 1H), 7.41 (d, J ¼ 8.0 Hz, 1H), 7.32e7.28 (m, 1H), 7.21 (s,

1H), 7.08 (d, J ¼ 7.2 Hz, 1H), 6.85 (t, J ¼ 7.5 Hz, 1H), 5.39 (d,

.85 (t, J ¼ 7.4 Hz, 1H), 5.39 (d, J ¼ 15.6 Hz, 1H), 5.31 (d, J ¼ 15.6 Hz,

13

H), 3.27 (s, 3H), 1.77e1.75 (m, 3H); C NMR (125 MHz, DMSO-d

177.5, 160.0, 148.2, 143.3, 143.3, 141.4, 140.6, 140.5, 139.9, 135.7,

35.3, 133.8, 131.3, 130.9, 130.8, 130.7, 130.7, 130.4, 129.8, 129.6,

29.6, 129.4, 129.0, 129.0, 128.9, 128.6, 126.2, 125.9, 125.4, 123.9,

6

)

13

1

J ¼ 15.5 Hz,1H), 5.31 (d, J ¼ 15.5 Hz,1H); C NMR (125 MHz, DMSO-

6

d ) d 176.9, 160.4, 149.6, 144.8, 144.6, 143.8,142.9,142.7, 140.6, 136.5,

þ

1

23.2, 123.0, 118.3, 110.4, 109.3, 67.5, 36.1, 25.6; HR ESI-MS [M þ H]

135.4, 132.8, 132.7, 132.4, 131.8, 131.5, 130.7, 130.5, 129.7, 129.3,

128.8, 128.6, 127.7, 127.0, 123.5, 123.3, 122.9, 122.5, 122.3, 122.0,

þ

m/z ¼ 627.2262 (calcd for C38

H

27

N

8

O

2

, 627.2257).

þ

1

21.4, 121.2, 118.6, 110.3, 109.3, 35.3; HR ESI-MS [M þ H] m/

0

þ

2

, 667.1165).

4

.4.23. 3-amino-1 -((1-(3-chloro-4-methylphenyl)-1H-1,2,3-

z ¼ 667.1173 (calcd for C36

H21Cl

2

N

8

O

0

triazol-5-yl)methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-

0

1

,3 -indoline]-2-carbonitrile 23

4.4.27. 3-amino-1 -((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-5-yl)

Yield 68%; Yellow solid; Mp > 250 C; ¹H NMR (500 MHz,

DMSO-d

9.28 (d, J ¼ 7.8 Hz, 1H), 8.60 (s, 1H), 8.54 (d, J ¼ 8.2 Hz,

H), 8.27e8.17 (m, 2H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H),

ꢀ

methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-1,3 -

0

6

)

d

indoline]-2-carbonitrile 27

ꢀ

1

Yield 67%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

7

.77e7.74 (m, 2H), 7.68e7.66 (m, 2H), 7.55 (t, J ¼ 7.1 Hz, 1H), 7.51 (d,

d

6

)

d

9.28 (d, J ¼ 7.9 Hz, 1H), 8.65 (s, 1H), 8.54 (d, J ¼ 8.1 Hz, 1H), 8.19

J ¼ 8.5 Hz, 1H), 7.41 (d, J ¼ 7.8 Hz, 1H), 7.30e7.27 (m, 1H), 7.09 (d,

J ¼ 7.3 Hz, 1H), 7.04 (d, J ¼ 8.5 Hz, 1H), 6.85 (t, J ¼ 7.3 Hz, 1H), 5.32 (s,

(d, J ¼ 8.5 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.01 (m, 1H), 7.93 (d,

J ¼ 2.2 Hz, 1H), 7.84e7.78 (m, 2H), 7.74e7.70 (m, 1H), 7.65 (s, 2H),

7.53e7.49 (m, 1H), 7.42 (d, J ¼ 7.4 Hz, 1H), 7.29 (td, J ¼ 7.8, 1.0 Hz,

1H), 7.09 (d, J ¼ 7.4 Hz,1H), 6.94 (d, J ¼ 8.5 Hz,1H), 6.85 (t, J ¼ 7.5 Hz,

2

H), 2.37 (s, 3H); ¹³C NMR (125 MHz, DMSO-d

) d 177.4, 160.1, 148.2,

6

1

44.2, 143.2, 141.4, 140.6, 140.5, 139.9, 136.4, 135.8, 135.3, 134.5,

32.6, 131.3, 131.1, 130.8, 130.7, 130.4, 129.3, 128.9, 128.2, 125.9,

25.4, 123.9, 123.2, 123.0, 122.5, 120.4, 120.4, 118.8, 118.8, 118.6,

13

1H), 5.34 (d, J ¼ 15.6 Hz, 1H), 5.30 (d, J ¼ 15.6 Hz, 1H); C NMR

6

(125 MHz, DMSO-d ) d 177.3, 160.1, 148.2, 144.4, 143.2, 141.3, 140.6,

þ

1

10.4, 109.2, 36.0, 19.6; HR ESI-MS [M þ H] m/z ¼ 647.1701 (calcd

140.0,139.9,136.4,135.3,132.7, 132.1,131.4, 131.3, 131.0, 130.9, 130.6,

130.4, 129.3, 128.9, 128.1, 125.9, 125.4, 123.9, 123.2, 123.0, 122.8,

þ

for C37

H24ClN

8

O

2

, 647.1711).

1

22.0, 122.0, 120.3, 120.3, 118.5, 110.4, 109.2, 35.9; HR ESI-MS [M þ

0

þ

8 2

O

4

.4.24. 3-amino-1 -((1-(3-chloro-6-methylphenyl)-1H-1,2,3-

H] m/z ¼ 667.1140 (calcd for C36

H21Cl

2

N

, 667.1165).

0

triazol-5-yl)methyl)-2 -oxospiro[benzo[a]pyrano[2,3-c]phenazine-

,3 -indoline]-2-carbonitrile 24

0

1

4.4.28. 3 ,5 -diamino-2-oxo-1-((1-(o-tolyl)-1H-1,2,3-triazol-5-yl)

methyl)spiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -carbonitrile

ꢀ

1

0

Yield 75%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

d

6

)

d

9.28 (d, J ¼ 7.9 Hz, 1H), 8.54 (d, J ¼ 7.8 Hz, 1H), 8.33 (s, 1H), 8.26

28

Yield 66%; Yellow solid; Mp > 250 C; ¹H NMR (500 MHz,

ꢀ

(

d, J ¼ 8.4 Hz, 1H), 8.09e8.06 (m, 1H), 8.04e8.02 (m, 1H), 7.88e7.85

(

7

m, 1H), 7.74e7.71 (m, 1H), 7.62 (s, 2H), 7.52 (dd, J ¼ 8.2, 2.2 Hz, 1H),

DMSO-d

6

)

d

8.25 (s, 1H), 7.90 (d, J ¼ 8.6 Hz, 1H), 7.67e7.64 (m, 1H),

.46 (d, J ¼ 8.3 Hz,1H), 7.41 (d, J ¼ 8.0 Hz,1H), 7.35 (d, J ¼ 8.1 Hz,1H),

7.47e7.42 (m, 3H), 7.35e7.32 (m, 2H), 7.28e7.22 (m, 5H), 7.07 (s,

1H), 7.01 (d, J ¼ 6.6 Hz, 1H), 6.85 (td, J ¼ 7.5, 0.8 Hz, 1H), 6.59 (s, 2H),

7

6

.30e7.28 (m, 1H), 7.21 (d, J ¼ 2.1 Hz, 1H), 7.08 (d, J ¼ 7.4 Hz, 1H),

13

.85 (t, J ¼ 7.4 Hz, 1H), 5.39 (d, J ¼ 15.6 Hz, 1H), 5.29 (d, J ¼ 15.6 Hz,

5.36 (d, J ¼ 15.6 Hz, 1H), 5.24 (d, J ¼ 15.6 Hz, 1H), 2.07 (s, 3H);

C

1

H), 2.06 (s, 3H); ¹³C NMR (125 MHz, DMSO-d

6

)

d

177.4, 160.0,

6

NMR (125 MHz, DMSO-d ) d 177.3, 159.8, 143.6, 143.3, 143.1, 142.5,

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

137

1

42.4, 139.0, 136.6, 136.0, 135.5, 133.3, 131.8, 131.8, 130.3, 130.2,

d

6

)

d

8.36 (s, 1H), 7.89 (d, J ¼ 8.6 Hz, 1H), 7.72 (d, J ¼ 6.8 Hz, 1H),

128.9, 128.8, 128.2, 128.0, 127.3, 127.3, 126.1, 126.1, 125.5, 123.8,

7.66e7.64 (m, 1H), 7.61e7.58 (m, 1H), 7.54e7.51 (m, 2H), 7.48 (dd,

J ¼ 8.0, 1.7 Hz, 1H), 7.45e7.42 (m, 1H), 7.35 (d, J ¼ 7.7 Hz, 1H),

7.28e7.19 (m, 4H), 7.08 (s, 1H), 7.01 (d, J ¼ 7.3 Hz, 1H), 6.87e6.84 (m,

þ

1

23.1, 118.3, 112.8, 109.2, 102.6, 36.1, 17.8; HR ESI-MS [M þ H] m/

þ

z ¼ 578.2058 (calcd for C33

H N

24 9

O

2

, 578.2053).

1

H), 6.56 (s, 1H), 5.35 (d, J ¼ 15.6 Hz, 1H), 5.26 (d, J ¼ 15.6 Hz, 1H);

0

13

4.4.29. 3 ,5 -diamino-2-oxo-1-((1-(m-tolyl)-1H-1,2,3-triazol-5-yl)

6

C NMR (125 MHz, DMSO-d ) d 176.8, 159.1, 143.0, 142.8, 142.7,

0

methyl)spiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -carbonitrile

142.6, 141.9, 141.7, 138.5, 135.5, 135.0, 134.3, 131.5, 130.6, 129.9,

129.8, 128.4, 128.4, 128.3, 128.3, 128.1, 127.9, 127.9, 127.7, 125.5,

2

9

ꢀ

1

þ

Yield 69%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.52 (s, 1H), 7.95 (s, 1H), 7.85 (d, J ¼ 8.6 Hz, 1H), 7.58e7.54 (m,

H), 7.52 (s, 1H), 7.42e7.36 (m, 2H), 7.29e7.25 (m, 4H), 7.07 (s, 1H),

123.3, 122.6, 117.6, 112.4, 108.7, 102.4, 35.6; HR ESI-MS [M þ H] m/

þ

d

6

)

d

z ¼ 598.1498 (calcd for C32

H21ClN

9

O

2

, 598.1507).

2

0 0

4.4.34. 3 ,5 -diamino-1-((1-(3-bromophenyl)-1H-1,2,3-triazol-5-

yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 34

7

6

d

.02 (d, J ¼ 7.3 Hz,1H), 6.92 (d, J ¼ 8.6 Hz,1H), 6.85 (t, J ¼ 7.5 Hz,1H),

13

0

.56 (s, 2H), 5.28 (s, 2H), 2.34 (s, 3H); C NMR (125 MHz, DMSO-d

177.2, 162.8, 159.7, 143.6, 143.2, 143.1, 142.4, 142.2, 140.0, 139.0,

36.9, 136.1, 135.5, 130.2, 130.0, 130.0, 129.7, 128.9, 128.6, 128.1,

6

)

ꢀ

1

Yield 55%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

1

3

C

28.0, 123.8, 123.1, 122.2, 120.7, 120.7, 118.4, 117.4, 117.4, 109.2, 102.9,

d

6

)

d

8.64 (s, 1H), 7.89 (t, J ¼ 1.8 Hz, 1H), 7.85e7.82 (m, 2H), 7.65 (d,

þ

6.1, 21.3; HR ESI-MS [M þ H] m/z ¼ 578.2057 (calcd for

J ¼ 8.0 Hz, 1H), 7.54 (t, J ¼ 7.0 Hz, 1H), 7.49 (t, J ¼ 8.0 Hz, 1H), 7.39 (d,

J ¼ 8.0 Hz, 1H), 7.29e7.21 (m, 4H), 7.07 (s, 1H), 7.02 (d, J ¼ 7.2 Hz,

þ

H N

33 24 9

O

2

, 578.2053).

13

1

H), 6.87e6.82 (m, 2H), 6.56 (s, 2H), 5.28 (s, 2H); C NMR

0

4.4.30. 3 ,5 -diamino-1-((1-(2-ethylphenyl)-1H-1,2,3-triazol-5-yl)

6

(125 MHz, DMSO-d ) d 176.7, 159.2, 143.7, 143.1, 142.7, 142.5, 141.9,

141.7, 138.5,137.5, 135.5, 135.0,131.6, 131.6, 131.3, 129.6, 128.3, 128.0,

127.6, 127.4, 123.3, 122.6, 122.4, 122.4, 122.2, 122.1, 118.8, 117.7, 112.4,

0

methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 30

ꢀ

1

þ

Yield 60%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.22 (s, 1H), 7.90 (d, J ¼ 8.6 Hz, 1H), 7.70e7.65 (m, 1H),

.51e7.45 (m, 3H), 7.33e7.22 (m, 6H), 7.18 (d, J ¼ 7.8 Hz, 1H), 7.08 (s,

H), 7.01 (d, J ¼ 7.3 Hz, 1H), 6.85 (t, J ¼ 7.5 Hz, 1H), 6.56 (s, 2H), 5.38

108.7, 102.5, 35.4; HR ESI-MS [M þ H] m/z ¼ 642.0983 (calcd for

þ

d

7

1

6

)

d

C

32

H

21BrN

9

O

2

, 642.1002).

0

4.4.35. 3 ,5 -diamino-1-((1-(2-bromophenyl)-1H-1,2,3-triazol-5-

yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 35

Yield 74%; Yellow solid; Mp > 250 C; Yellow solid. Mp C. H

NMR (500 MHz, DMSO-d 8.33 (s, 1H), 7.91e7.86 (m, 2H),

0

(

d, J ¼ 15.6 Hz, 1H), 5.23 (d, J ¼ 15.6 Hz, 1H), 2.36 (q, J ¼ 7.5 Hz, 2H),

13

0

.93 (t, J ¼ 7.5 Hz, 3H); C NMR (125 MHz, DMSO-d

6

)

d

176.6, 159.0,

ꢀ

1

43.0, 142.6, 142.5, 141.8, 141.6, 139.0, 138.4, 135.4, 134.9, 129.9,

29.7, 129.6, 128.3, 128.2, 128.1, 127.6, 127.5, 126.6, 126.6, 125.9,

25.9, 125.1, 123.2, 123.1, 122.4, 117.4, 112.4, 108.6, 102.3, 35.6, 23.4,

1

6

) d

7.68e7.64 (m, 1H), 7.56e7.50 (m, 2H), 7.48e7.44 (m, 1H), 7.42e7.40

(m, 1H), 7.34 (d, J ¼ 7.8 Hz, 1H), 7.28e7.24 (m, 4H), 7.08 (s, 1H), 7.01

(d, J ¼ 7.4 Hz, 1H), 6.86 (t, J ¼ 7.5 Hz, 1H), 6.56 (s, 2H), 5.36 (d,

þ

2

1

4.5; HR ESI-MS [M þ H] m/z ¼ 592.2218 (calcd for C34

26 9

H N O ,

5

92.2209).

13

J ¼ 15.6 Hz,1H), 5.26 (d, J ¼ 15.6 Hz,1H); C NMR (125 MHz, DMSO-

0

4.4.31. 3 ,5 -diamino-1-((1-(4-ﬂuorophenyl)-1H-1,2,3-triazol-5-yl)

6

d ) d 177.3, 159.7, 143.6, 143.2, 143.1, 143.1, 142.4, 142.3, 139.0, 136.5,

0

methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 31

136.0, 135.5, 134.2, 134.2, 132.3, 130.3, 129.3, 129.3, 129.0, 128.9,

128.8, 128.8, 128.2, 128.2, 126.0, 123.8, 123.1, 118.9, 118.1, 109.3,

ꢀ

1

þ

Yield 80%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.56 (s, 1H), 7.84 (d, J ¼ 8.0 Hz, 1H), 7.80e7.78 (m, 2H),

.58e7.55 (m, 1H), 7.40e7.36 (m, 3H), 7.28e7.25 (m, 4H), 7.07 (s,

102.9, 36.2; HR ESI-MS [M þ H] m/z ¼ 642.0995 (calcd for

þ

d

6

)

d

C

32

H

21BrN

9

O

2

, 642.1002).

7

1

0

H), 7.01 (d, J ¼ 7.5 Hz,1H), 6.89 (d, J ¼ 8.5 Hz,1H), 6.85 (t, J ¼ 7.5 Hz,

4.4.36. 3 ,5 -diamino-2-oxo-1-((1-(3-(triﬂuoromethyl)phenyl)-1H-

1,2,3-triazol-5-yl)methyl)spiro[indoline-3,1 -pyrano[3,2-a]

phenazine]-2 -carbonitrile 36

13

0

H), 6.56 (s, 2H), 5.27 (s, 2H); C NMR (125 MHz, DMSO-d

6

)

d

162.3,

0

1

59.2,143.6,143.1,142.7,142.5,141.9,141.8,138.6,138.5,138.4,138.4,

36.6, 135.3, 135.3, 135.0, 129.8, 129.7, 128.4, 128.1, 127.6, 127.4,

23.3, 122.6, 122.1, 122.0, 117.8, 116.7, 116.4, 108.7, 102.4, 35.7; HR

Yield 68%; Yellow solid; Mp > 250 C; ¹H NMR (500 MHz,

ꢀ

DMSO-d

6

)

d

8.74 (s, 1H), 8.13 (d, J ¼ 7.6 Hz, 1H), 8.00 (s, 1H),

þ

2

ESI-MS [M þ H] m/z ¼ 582.1786 (calcd for C32

H21FN

9

O

, 582.1802).

7.82e7.76 (m, 3H), 7.50e7.47 (m, 1H), 7.41 (d, J ¼ 8.0 Hz, 1H),

7

.29e7.26 (m, 3H), 7.19e7.16 (m, 1H), 7.07 (s, 1H), 7.02 (d, J ¼ 7.3 Hz,

0

4.4.32. 3 ,5 -diamino-1-((1-(4-chlorophenyl)-1H-1,2,3-triazol-5-yl)

1H), 6.86 (t, J ¼ 7.5 Hz, 1H), 6.77 (d, J ¼ 8.5 Hz, 1H), 6.55 (s, 2H), 5.31

0

13

methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 32

(d, J ¼ 15.6 Hz, 1H), 5.26 (d, J ¼ 15.6 Hz, 1H); C NMR (125 MHz,

6

DMSO-d ) d 177.2, 159.7, 144.3, 143.6, 143.3, 143.0, 142.4, 142.2,

ꢀ

1

Yield 67%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.61 (s, 1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 7.84 (d, J ¼ 9.0 Hz, 1H), 7.79

139.9, 139.2,139.0, 137.4, 136.2, 136.1, 135.5, 131.6, 131.1, 130.0, 128.9,

128.4,128.1,127.8,125.6,124.2,123.8,123.2,122.9,118.3,117.0,112.9,

6

d ) d

þ

(

7

2

d, J ¼ 8.9 Hz, 1H), 7.63e7.54 (m, 4H), 7.39 (d, J ¼ 8.0 Hz, 1H),

109.2, 102.9, 35.8; HR ESI-MS [M þ H] m/z ¼ 632.1783 (calcd for

þ

.28e7.24 (m, 3H), 7.07 (s,1H), 7.01 (d, J ¼ 7.2 Hz,1H), 6.88e6.84 (m,

C

33

H

21

F

3

N

9

O

2

, 632.1770).

13

6

H), 6.56 (s, 2H), 5.27 (s, 2H); C NMR (125 MHz, DMSO-d ) d 159.2,

0

1

58.1, 157.9, 156.0, 141.7, 137.0, 132.8, 129.9, 129.7, 129.7, 129.7, 129.6,

28.3, 128.0, 127.7, 127.6, 127.4, 126.5, 124.5, 124.4, 124.4, 123.3,

22.7, 122.6, 121.4, 121.4, 121.4, 118.6, 117.8, 108.7, 102.4, 25.4; HR

4.4.37. N-(4-(5-((3 ,5 -diamino-2 -cyano-2-oxospiro[indoline-3,1 -

pyrano[3,2-a]phenazin]-1-yl)methyl)-1H-1,2,3-triazol-1-yl)phenyl)

acetamide 37

þ

9 2

H21ClN O ,

Yield 66%; Yellow solid; Mp > 250 C; ¹H NMR (500 MHz,

ꢀ

ESI-MS [M þ H] m/z ¼ 598.1503(calcd for C32

5

98.1507).

DMSO-d

6

)

d

10.14 (s,1H), 8.48 (s, 1H), 7.85 (d, J ¼ 8.5 Hz,1H), 7.70 (d,

J ¼ 9.0 Hz, 2H), 7.66 (d, J ¼ 9.0 Hz, 2H), 7.59e7.56 (m, 1H), 7.38 (d,

J ¼ 8.0 Hz,1H), 7.31e7.25 (m, 4H), 7.07 (s,1H), 7.01 (d, J ¼ 7.5 Hz,1H),

6.93 (d, J ¼ 8.5 Hz, 1H), 6.85 (t, J ¼ 7.5 Hz, 1H), 6.55 (s, 2H), 5.26 (s,

0

4.4.33. 3 ,5 -diamino-1-((1-(2-chlorophenyl)-1H-1,2,3-triazol-5-yl)

0

methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 33

13

6

2H), 2.07 (s, 3H); C NMR (125 MHz, DMSO-d ) d 177.2, 169.0,159.7,

ꢀ

1

Yield 59%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

143.9, 143.6, 143.2, 143.0, 142.4, 142.2, 140.0, 139.0, 136.0, 135.5,

138

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

0 0

4.4.42. 3 ,5 -diamino-1-((1-(4-nitrophenyl)-1H-1,2,3-triazol-5-yl)

methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

1

31.9, 130.2, 128.9, 128.6, 128.1, 128.0, 123.8, 123.1, 122.1, 120.8,

0

120.8, 120.0, 120.0, 119.8, 118.4, 114.6, 112.9, 109.2, 102.9, 36.0, 24.5;

þ

3

HR ESI-MS [M þ H] m/z ¼ 621.2117 (calcd for C34

H

25

N

10

O

,

carbonitrile 42

ꢀ

1

6

21.2111).

Yield 67%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

d

6

)

d

8.80 (s, 1H), 8.36 (d, J ¼ 9.0 Hz, 2H), 8.07 (d, J ¼ 9.0 Hz, 2H), 7.82

0

4

.4.38. 3 ,5 -diamino-1-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-5-

(d, J ¼ 8.5 Hz, 1H), 7.53e7.50 (m, 1H), 7.43 (d, J ¼ 8.0 Hz, 1H),

7.30e7.27 (m, 2H), 7.22e7.19 (m, 1H), 7.07 (s, 1H), 7.02 (d, J ¼ 7.5 Hz,

1H), 6.86 (t, J ¼ 7.5 Hz, 1H), 6.78 (d, J ¼ 8.5 Hz, 1H), 6.56 (s, 2H), 6.24

0

yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 38

ꢀ

1

13

Yield 81%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.47 (s, 1H), 7.86 (d, J ¼ 8.5 Hz, 1H), 7.66 (d, J ¼ 9.0 Hz, 2H),

.60e7.57 (m,1H), 7.37 (d, J ¼ 8.0 Hz,1H), 7.32e7.24 (m, 4H), 7.06 (d,

(s, 1H), 5.32 (d, J ¼ 15.6 Hz, 1H), 5.28 (d, J ¼ 15.6 Hz, 1H); C NMR

d

6

)

d

6

(125 MHz, DMSO-d ) d 177.2, 159.7, 147.1, 144.7, 143.6, 143.2, 143.0,

7

142.3, 142.2, 141.1, 135.5, 130.2, 128.9, 128.8, 128.4, 128.2, 128.1,

127.9, 125.9, 125.9, 125.9, 123.8, 123.2, 123.0, 120.9, 120.9, 120.9,

J ¼ 9.0 Hz, 3H), 7.01 (dd, J ¼ 7.5, 0.7 Hz, 1H), 6.95 (d, J ¼ 8.5 Hz, 1H),

þ

6

5

d

1

.85 (td, J ¼ 7.5, 0.7 Hz, 1H), 6.56 (s, 2H), 5.28 (d, J ¼ 15.8 Hz, 1H),

118.2, 112.9, 109.2, 102.9, 35.8; HR ESI-MS [M þ H] m/z ¼ 609.1748

13

þ

.25 (d, J ¼ 15.8 Hz, 1H), 3.81 (s, 3H); C NMR (125 MHz, DMSO-d

177.2, 159.7, 143.5, 143.2, 143.0, 142.4, 142.2, 139.0, 135.5, 130.4,

30.2, 128.9, 128.6, 128.1, 128.0, 128.0, 123.8, 123.8, 123.1, 123.1,

6

)

(calcd for C32

H

21

N

10

O

4

, 609.1747).

0 0

4.4.43. 3 ,5 -diamino-1-((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-

5-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 43

0

22.2, 121.9, 121.9, 121.9, 118.3, 115.2, 115.2, 115.2, 112.9, 109.2, 102.9,

þ

5

C

6.0, 36.0; HR ESI-MS [M þ H] m/z ¼ 594.2004 (calcd for

þ

ꢀ

1

33 24

H N

9

O

3

, 594.2002).

Yield 61%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

d

6

)

d

8.47 (s, 1H), 7.85 (d, J ¼ 8.5 Hz, 1H), 7.59e7.56 (m, 1H),

0

4

.4.39. 3 ,5 -diamino-1-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-5-

7.48e7.44 (m, 2H), 7.36 (d, J ¼ 8.0 Hz,1H), 7.30e7.22 (m, 5H), 7.07 (s,

1H), 7.01 (d, J ¼ 7.0 Hz, 1H), 6.97e6.94 (m, 1H), 6.85 (t, J ¼ 7.4 Hz,

0

yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 39

1H), 6.56 (s, 2H), 5.29 (d, J ¼ 15.7 Hz, 1H), 5.25 (d, J ¼ 15.7 Hz, 1H),

ꢀ

1

13

Yield 69%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.29 (s, 1H), 7.86 (d, J ¼ 8.5 Hz, 1H), 7.62e7.59 (m, 2H), 7.48 (t,

J ¼ 7.5 Hz, 1H), 7.36 (t, J ¼ 7.5 Hz, 1H), 7.32 (d, J ¼ 7.8 Hz, 1H),

.27e7.22 (m, 4H), 7.11 (t, J ¼ 7.5 Hz, 1H), 7.07e7.05 (m, 2H), 7.00 (d,

J ¼ 7.3 Hz, 1H), 6.85 (t, J ¼ 7.5 Hz, 1H), 6.56 (s, 2H), 5.33 (d,

2.25 (d, J ¼ 5.3 Hz, 6H); C NMR (125 MHz, DMSO-d

6

) d 177.2, 159.7,

d

6

)

d

143.6,143.3,143.1,142.4,142.3,139.1,139.0,138.5,137.5,137.4,135.5,

134.9, 130.9, 130.2, 128.9, 128.8, 128.7, 128.2, 128.1, 128.0, 123.9,

7

123.2, 122.1, 121.2, 118.4, 117.5, 114.6, 109.3, 102.9, 36.1, 19.8, 19.4; HR

ESI-MS [M þ H]^þm/z ¼ 592.2211 (calcd for C34

H

26

N

9

O

þ

, 592.2209).

2

13

J ¼ 15.6 Hz, 1H), 5.23 (d, J ¼ 15.6 Hz, 1H), 3.65 (s, 3H); C NMR

125 MHz, DMSO-d 177.3, 159.7, 151.5, 143.6, 143.2, 142.4, 142.2,

39.0, 136.0, 135.5, 130.9, 130.4, 130.3, 128.8, 128.8, 128.2, 128.1,

28.0, 126.4, 126.0, 125.9, 125.7, 125.6, 123.8, 123.1, 121.3, 118.0,

0

(

1

6

)

d

4.4.44. 3 ,5 -diamino-1-((1-(2-ethyl-6-methylphenyl)-1H-1,2,3-

triazol-5-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]

phenazine]-2 -carbonitrile 44

Yield 58%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

6

d ) d 8.13 (s, 1H), 7.92e7.89 (m, 1H), 7.72e7.67 (m, 2H), 7.55e7.52

0

þ

ꢀ

1

13.5, 113.0, 109.3, 102.9, 56.4, 36.3; HR ESI-MS [M þ H] m/

þ

z ¼ 594.2008 (calcd for C33

H N

24 9

O

3

, 594.2002).

(

m, 2H), 7.41 (t, J ¼ 7.5 Hz, 1H), 7.29e7.21 (m, 6H), 7.08 (d, J ¼ 1.2 Hz,

0

4

.4.40. 3 ,5 -diamino-1-((1-(4-ethoxyphenyl)-1H-1,2,3-triazol-5-

1H), 7.00 (d, J ¼ 7.5 Hz, 1H), 6.55 (s, 2H), 5.47 (d, J ¼ 15.5 Hz, 1H),

5.20 (d, J ¼ 15.8 Hz, 1H), 2.12e2.07 (m, 2H), 1.82 (s, 3H), 0.88 (t,

0

yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 40

13

J ¼ 7.5 Hz, 3H); C NMR (125 MHz, DMSO-d

6

) d 177.3, 159.7, 143.6,

ꢀ

1

Yield 75%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.47 (s, 1H), 7.85 (d, J ¼ 8.4 Hz, 1H), 7.64 (d, J ¼ 9.0 Hz, 2H),

.60e7.57 (m, 1H), 7.36 (d, J ¼ 8.0 Hz, 1H), 7.32e7.24 (m, 4H),

.07e7.00 (m, 4H), 6.96 (d, J ¼ 8.5 Hz, 1H), 6.85 (t, J ¼ 7.5 Hz, 1H),

.56 (s, 2H), 5.28 (d, J ¼ 15.7 Hz, 1H), 5.24 (d, J ¼ 15.7 Hz, 1H), 4.08

143.3, 143.1, 142.5, 142.3, 139.2, 135.5, 135.4, 130.7, 130.4, 130.4,

129.5, 129.3, 129.3, 128.8, 128.8, 128.2, 128.2, 128.0, 127.4, 123.8,

123.8123.2, 123.1, 123.0, 118.1, 117.5, 109.3, 102.9, 36.4, 24.1, 17.2,

6

d ) d

7

6

þ

2

15.5; HR ESI-MS [M þ H] m/z ¼ 606.2374 (calcd for C35

28 9

H N O ,

606.2366).

13

(

q, J ¼ 7.0 Hz, 2H),1.34 (t, J ¼ 7.0 Hz, 3H); C NMR (125 MHz, DMSO-

177.2, 159.7, 159.0, 143.9, 143.5, 143.2, 143.0, 142.4, 142.2, 139.0,

36.0, 135.5, 130.3, 130.2, 128.9, 128.7, 128.1, 128.0, 123.8, 123.1,

0 0

4.4.45. 3 ,5 -diamino-1-((1-(2,5-dimethylphenyl)-1H-1,2,3-triazol-

5-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

d

6

)

d

0

1

6

C

22.1, 121.9, 121.9, 121.9, 118.3, 115.7, 115.7, 115.7, 112.9, 109.2, 102.9,

carbonitrile 45

þ

ꢀ

1

4.0, 36.0, 15.0; HR ESI-MS [M þ H] m/z ¼ 608.2154 (calcd for

Yield 69%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.18 (s, 1H), 7.90 (d, J ¼ 8.5 Hz, 1H), 7.67e7.64 (m, 1H),

þ

H

34 26

N

9

O

3

, 608.2159).

6

d ) d

7.44e7.41 (m, 1H), 7.36 (d, J ¼ 8.0 Hz,1H), 7.30e7.23 (m, 5H), 7.16 (d,

0

4.4.41. 3 ,5 -diamino-1-((1-(2-ethoxyphenyl)-1H-1,2,3-triazol-5-yl)

J ¼ 8.5 Hz, 1H), 7.08 (s, 1H), 7.01 (d, J ¼ 7.3 Hz, 1H), 6.93 (s, 1H), 6.85

0

methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 41

(t, J ¼ 7.5 Hz, 1H), 6.56 (s, 2H), 5.34 (d, J ¼ 15.6 Hz, 1H), 5.23 (d,

13

J ¼ 15.6 Hz, 1H), 2.26 (s, 3H), 2.01 (s, 3H); C NMR (125 MHz,

ꢀ

1

Yield 58%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.37 (s, 1H), 7.84 (d, J ¼ 8.5 Hz, 1H), 7.68 (dd, J ¼ 7.5, 1.0 Hz, 1H),

.54 (t, J ¼ 7.5 Hz, 1H), 7.43 (t, J ¼ 7.5 Hz, 1H), 7.30 (d, J ¼ 8.0 Hz, 1H),

.26e7.22 (m, 4H), 7.16 (d, J ¼ 8.5 Hz,1H), 7.10 (t, J ¼ 7.5 Hz,1H), 7.07

s, 1H), 7.01 (d, J ¼ 7.5 Hz, 1H), 6.94 (d, J ¼ 8.5 Hz, 1H), 6.85 (t,

J ¼ 7.5 Hz, 1H), 6.55 (s, 2H), 5.30 (d, J ¼ 15.7 Hz, 1H), 5.25 (d,

6

DMSO-d ) d 177.3, 159.7, 143.6, 143.6, 143.3, 143.2, 142.4, 142.3,

d

6

)

d

139.1, 136.8, 136.3, 135.5, 131.6, 131.6, 130.7, 130.3, 130.0, 128.9,

128.9, 128.8, 128.2, 128.2, 126.5, 126.5, 125.5, 123.8, 123.1, 118.2,

7

(

þ

113.0, 109.2, 102.9, 36.1, 20.6, 17.4; HR ESI-MS [M þ H] m/

þ

z ¼ 592.2208 (calcd for C34

26

H N

9

O

2

, 592.2209).

13

0 0

4.4.46. 3 ,5 -diamino-1-((1-(3,5-dimethylphenyl)-1H-1,2,3-triazol-

5-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

carbonitrile 46

J ¼ 15.7 Hz, 1H), 3.60 (t, J ¼ 6.5 Hz, 2H), 0.90 (t, J ¼ 7.0 Hz, 3H);

NMR (125 MHz, DMSO-d 177.3, 159.6, 150.4, 143.6, 143.2, 143.2,

42.4, 142.2, 139.0, 136.0, 135.5, 130.7, 130.2, 128.8, 128.8, 128.0,

27.8, 126.1, 125.8, 125.1, 125.1, 123.8, 123.1, 121.3, 121.3, 118.1, 114.2,

C

0

6

)

d

1

ꢀ

1

Yield 75%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.45 (s, 1H), 7.85 (d, J ¼ 8.8 Hz, 1H), 7.57 (t, J ¼ 7.5 Hz, 1H), 7.36

(d, J ¼ 7.8 Hz, 1H), 7.31e7.25 (m, 5H), 7.07 (d, J ¼ 4.0 Hz, 2H), 7.02 (d,

þ

1

14.2, 113.0, 109.2, 102.9, 64.7, 36.3, 14.3; HR ESI-MS [M þ H] m/

6

d ) d

þ

z ¼ 608.2157 (calcd for C34

H

26

N

9

O

3

, 608.2159).

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

139

0

J ¼ 7.3 Hz,1H), 6.90 (d, J ¼ 8.4 Hz, 1H), 6.85 (t, J ¼ 7.3 Hz, 1H), 6.56 (s,

4.4.51. 3 ,5 -diamino-1-((1-(2,5-dimethoxyphenyl)-1H-1,2,3-

13

0

2

H), 6.24 (br s, 1H), 5.27 (s, 2H), 2.30 (s, 6H); C NMR (125 MHz,

DMSO-d 176.6, 159.1,142.9,142.6,142.5,141.8, 141.6,139.0,139.0,

36.2, 134.9, 129.7, 129.7, 129.5, 129.5, 128.2, 128.2, 128.0, 127.5,

27.4, 127.4, 123.2, 123.2, 122.5, 122.5, 121.5, 117.3, 117.3, 117.3, 108.5,

triazol-5-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]

phenazine]-2 -carbonitrile 51

0

6

)

d

ꢀ

1

Yield 71%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

1

d

6

)

d

8.31 (s, 1H), 7.86 (d, J ¼ 8.5 Hz, 1H), 7.63e7.60 (m, 1H),

þ

102.4, 35.5, 20.6, 20.6; HR ESI-MS [M þ H] m/z ¼ 592.2218(calcd

7.38e7.35 (m, 1H), 7.31 (d, J ¼ 7.8 Hz, 1H), 7.27e7.25 (m, 3H), 7.19 (d,

J ¼ 3.0 Hz, 1H), 7.16 (d, J ¼ 9.0 Hz, 1H), 7.07e7.04 (m, 3H), 7.00 (d,

J ¼ 7.2 Hz, 1H), 6.85 (t, J ¼ 7.4 Hz, 1H), 6.56 (s, 2H), 5.33 (d,

þ

for C34

26

H N

9

O

2

, 592.2209).

0

4

.4.47. 3 ,5 -diamino-1-((1-(2,6-diethylphenyl)-1H-1,2,3-triazol-5-

J ¼ 15.6 Hz, 1H), 5.23 (d, J ¼ 15.6 Hz, 1H), 3.77 (s, 3H), 3.59 (s, 3H);

0

13

yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

6

C NMR (125 MHz, DMSO-d ) d 176.8, 159.1, 153.0, 144.8, 143.0,

carbonitrile 47

142.6,142.6,141.8,141.7, 141.7,138.5,135.4, 134.9,129.7,128.3,128.3,

127.5, 127.4, 125.7, 125.2, 123.3, 122.5, 117.4, 115.5, 114.2, 114.2, 112.4,

ꢀ

1

Yield 77%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.15 (s, 1H), 7.92e7.90 (m, 1H), 7.55e7.54 (m, 2H), 7.46 (t,

þ

d

6

)

d

110.2, 110.2, 108.7, 102.3, 56.2, 55.7, 35.7; HR ESI-MS [M þ H] m/

þ

J ¼ 7.7 Hz,1H), 7.29e7.17 (m, 6H), 7.08 (s,1H), 7.01 (d, J ¼ 7.7 Hz,1H),

z ¼ 624.2106 (calcd for C34

H

26

N

9

O

4

, 624.2108).

6

1

6

.85 (t, J ¼ 7.3 Hz, 1H), 6.56 (s, 2H), 6.25 (s, 1H), 5.49 (d, J ¼ 15.6 Hz,

0

H), 5.20 (d, J ¼ 15.6 Hz, 1H), 2.07e2.02 (m, 4H), 0.87 (t, J ¼ 7.5 Hz,

4.4.52. 3 ,5 -diamino-1-((1-(3-chloro-4-methylphenyl)-1H-1,2,3-

13

0

H); C NMR (125 MHz, DMSO-d

6

)

d

177.2,159.6,143.5,143.3,143.1,

triazol-5-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]

0

1

42.5, 142.3, 141.3, 139.1, 136.0, 135.4, 134.9, 130.9, 130.4, 129.5,

29.3, 128.8, 128.8, 128.2, 128.2, 128.0, 127.3, 127.3, 126.5, 123.8,

23.8, 123.2, 123.1, 118.1, 109.3, 102.9, 36.5, 24.0, 24.0, 15.5, 15.5; HR

phenazine]-2 -carbonitrile 52

ꢀ

1

Yield 74%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.60 (s, 1H), 7.84 (d, J ¼ 8.5 Hz, 1H), 7.76 (d, J ¼ 1.9 Hz, 1H), 7.68

(dd, J ¼ 7.2, 2.0 Hz, 1H), 7.57e7.54 (m, 1H), 7.51 (d, J ¼ 8.5 Hz, 1H),

.38 (d, J ¼ 7.8 Hz, 1H), 7.28e7.22 (m, 4H), 7.07 (s, 1H), 7.02 (d,

J ¼ 7.2 Hz, 1H), 6.88e6.84 (m, 2H), 6.56 (s, 2H), 5.27 (s, 2H), 2.37 (s,

6

d ) d

þ

2

ESI-MS [M þ H] m/z ¼ 620.2528 (calcd for C36

30

H N

9

O

, 620.2522).

7

0

4

5

.4.48. 3 ,5 -diamino-1-((1-(2,6-diﬂuorophenyl)-1H-1,2,3-triazol-

0

13

-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

6

3H); C NMR (125 MHz, DMSO-d ) d 177.2,159.7,144.1,143.6,143.2,

carbonitrile 48

Yield 63%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.45 (s, 1H), 7.88 (d, J ¼ 8.5 Hz, 1H), 7.72e7.66 (m, 1H),

.64e7.62 (m, 1H), 7.43e7.38 (m, 4H), 7.27 (t, J ¼ 7.5 Hz, 1H), 7.23 (s,

H), 7.12 (d, J ¼ 8.5 Hz, 1H), 7.08 (s, 1H), 7.01 (d, J ¼ 7.5 Hz, 1H), 6.86

t, J ¼ 7.5 Hz, 1H), 6.55 (s, 2H), 5.33 (d, J ¼ 15.6 Hz, 1H), 5.29 (d,

143.0, 142.4, 142.2, 139.0, 136.4, 135.8, 135.5, 134.5, 132.6, 132.6,

130.1, 128.9, 128.6, 128.1, 127.9, 123.8, 123.1, 122.5, 120.4, 120.4,

118.9, 118.9, 118.3, 112.9, 109.2, 102.9, 35.9, 19.6; HR ESI-MS [M þ

ꢀ

1

6

d ) d

7

2

(

þ

2

H] m/z ¼ 612.1649 (calcd for C33

9

H23ClN O , 612.1663).

0

4.4.53. 3 ,5 -diamino-1-((1-(3-chloro-6-methylphenyl)-1H-1,2,3-

13

0

J ¼ 15.6 Hz, 1H); C NMR (125 MHz, DMSO-d

54.3, 143.0, 142.6, 142.6, 141.9, 141.7, 138.5, 135.4, 134.9, 132.5,

32.4, 132.2, 129.7, 128.3, 128.3, 128.3, 127.6, 127.4, 126.5, 123.3,

22.6, 117.5, 112.9, 112.6, 112.6, 112.4, 108.7, 102.4, 35.5; HR ESI-MS

6

)

d

176.8, 159.1, 157.7,

triazol-5-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]

0

1

phenazine]-2 -carbonitrile 53

ꢀ

1

Yield 76%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.30 (s, 1H), 7.90 (d, J ¼ 8.5 Hz,1H), 7.66e7.63 (m, 1H), 7.52 (dd,

J ¼ 8.4, 2.2 Hz, 1H), 7.46e7.41 (m, 2H), 7.38 (d, J ¼ 8.0 Hz, 1H), 7.27

1

6

d ) d

þ

2

[M þ H] m/z ¼ 600.1716 (calcd for C32

H

20

F

2

N

9

O

, 600.1708).

(

td, J ¼ 7.8, 1.0 Hz, 1H), 7.24 (s, 2H), 7.21 (d, J ¼ 2.2 Hz, 1H), 7.16 (d,

0

4

5

.4.49. 3 ,5 -diamino-1-((1-(2,4-dichlorophenyl)-1H-1,2,3-triazol-

J ¼ 8.5 Hz, 1H), 7.08 (s, 1H), 7.01 (d, J ¼ 7.4 Hz, 1H), 6.86 (t, J ¼ 7.5 Hz,

0

-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

1H), 6.55 (s, 2H), 5.34 (d, J ¼ 15.6 Hz, 1H), 5.24 (d, J ¼ 15.6 Hz, 1H),

13

carbonitrile 49

Yield 64%; Yellow solid; Mp > 250 C; ¹H NMR (500 MHz,

DMSO-d

8.39 (s, 1H), 7.89 (d, J ¼ 8.5 Hz, 1H), 7.68e7.60 (m, 3H),

.51 (d, J ¼ 8.5 Hz, 1H), 7.45e7.42 (m, 1H), 7.36 (d, J ¼ 8.5 Hz, 1H),

.28e7.24 (m, 3H), 7.17 (d, J ¼ 8.5 Hz, 1H), 7.07 (s, 1H), 7.01 (d,

J ¼ 7.5 Hz, 1H), 6.86 (t, J ¼ 7.5 Hz, 1H), 6.56 (s, 2H), 5.35 (d,

6

2.05 (s, 3H); C NMR (125 MHz, DMSO-d ) d 177.3, 159.7, 143.6,

ꢀ

143.3, 143.2, 143.1, 142.4, 142.3, 139.0, 137.4, 136.1, 135.5, 133.5,

133.5, 132.5, 131.2, 130.3, 130.0, 128.9, 128.9, 128.3, 128.1, 125.9,

6

) d

7

125.9, 125.7, 123.8, 123.1, 118.1, 113.0, 109.2, 102.9, 36.0, 17.5; HR ESI-

MS [M þ H]^þm/z ¼ 612.1646 (calcd for C33

H23ClN

O

þ

, 612.1663).

9

2

13

0

J ¼ 15.6 Hz,1H), 5.26 (d, J ¼ 15.6 Hz,1H); C NMR (125 MHz, DMSO-

177.3, 159.7, 143.7, 143.6, 143.2, 143.1, 142.4, 142.3, 139.1, 135.7,

35.5, 133.8, 130.7, 130.7, 130.3, 129.8, 129.6, 129.6, 129.0, 129.0,

28.9, 128.9, 128.2, 128.1, 126.2, 124.9, 123.8, 123.1, 118.0, 109.2,

4.4.54. 3 ,5 -diamino-1-((1-(3-chloro-4-ﬂuorophenyl)-1H-1,2,3-

0

d

6

)

d

triazol-5-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]

0

1

phenazine]-2 -carbonitrile 54

ꢀ

1

Yield 73%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.60 (s, 1H), 7.91 (dd, J ¼ 6.4, 2.7 Hz,1H), 7.85e7.81 (m, 2H),

7.60 (t, J ¼ 9.0 Hz, 1H), 7.55e7.52 (m, 1H), 7.39 (d, J ¼ 8.0 Hz, 1H),

þ

102.9, 36.1; HR ESI-MS [M þ H] m/z ¼ 632.1121 (calcd for

6

d ) d

þ

C

32

H

20Cl

2

N

9

O

2

, 632.1117).

7

.29e7.21 (m, 4H), 7.07 (s, 1H), 7.02 (d, J ¼ 7.3 Hz, 1H), 6.86 (t,

0

4

5

.4.50. 3 ,5 -diamino-1-((1-(2,5-dichlorophenyl)-1H-1,2,3-triazol-

J ¼ 7.5 Hz, 1H), 6.80 (d, J ¼ 8.5 Hz, 1H), 6.55 (s, 2H), 5.29 (d,

0

13

-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

J ¼ 15.6 Hz,1H), 5.25 (d, J ¼ 15.6 Hz,1H); C NMR (125 MHz, DMSO-

carbonitrile 50

Yield 67%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.39 (s, 1H), 7.89 (d, J ¼ 8.5 Hz, 1H), 7.74 (d, J ¼ 8.5 Hz, 1H),

.70e7.63 (m, 3H), 7.74e7.71 (m, 1H), 7.37 (d, J ¼ 8.0 Hz, 1H),

.29e7.25 (m, 3H), 7.11 (d, J ¼ 8.5 Hz, 1H), 7.07 (s, 1H), 7.01 (d,

J ¼ 7.5 Hz, 1H), 6.86 (t, J ¼ 7.5 Hz, 1H), 6.56 (s, 2H), 5.34 (d,

6

d ) d 177.2, 159.7, 155.6, 144.3, 143.6, 143.2, 143.0, 142.4, 142.2, 139.0,

ꢀ

1

136.1, 135.5, 133.9, 130.1, 128.9, 128.5, 128.1, 127.9, 123.8, 123.2,

122.9, 122.6, 122.6, 121.1, 121.0, 118.6, 118.3, 118.3, 112.9, 109.2, 102.9,

6

d ) d

7

þ

2

35.8; HR ESI-MS [M þ H] m/z ¼ 616.1412 (calcd for C32

9

H20ClFN O ,

616.1413).

13

0 0

4.4.55. 3 ,5 -diamino-1-((1-(2,4-dinitrophenyl)-1H-1,2,3-triazol-5-

J ¼ 15.6 Hz,1H), 5.26 (d, J ¼ 15.6 Hz,1H); C NMR (125 MHz, DMSO-

177.3, 159.7, 143.6, 143.3, 143.1, 142.4, 142.3, 139.1, 135.6, 135.5,

32.8, 132.5, 132.5, 131.8, 130.3, 128.9, 128.9, 128.2, 128.1, 128.1,

0

d

6

)

d

yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -

1

carbonitrile 55

ꢀ

1

3

6

28.1, 127.6, 126.2,123.8,123.8,123.1, 123.1, 118.0, 112.9,109.2,102.9,

Yield 65%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.97 (d, J ¼ 2.5 Hz, 1H), 8.70 (s, 1H), 8.58 (dd, J ¼ 8.8, 2.5 Hz,

1H), 7.88 (d, J ¼ 8.6 Hz, 1H), 7.74 (d, J ¼ 8.8 Hz, 1H), 7.60e7.57 (m,

þ

2

,

6.0; HR ESI-MS [M þ H] m/z ¼ 632.1124 (calcd for C32

H20Cl

2

N

9

O

6

d ) d

32.1117).

140

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

1

7

H), 7.36 (d, J ¼ 7.7 Hz, 1H), 7.34e7.26 (m, 4H), 7.08 (s, 1H),

.04e7.01 (m, 2H), 6.86 (t, J ¼ 7.4 Hz, 1H), 6.56 (s, 2H), 5.32 (s, 2H);

3

658.1718).

1

C NMR (125 MHz, DMSO-d

43.6, 143.3, 143.0, 142.4, 142.3, 139.0, 135.5, 133.0, 130.3, 128.9,

28.9, 128.8, 128.8, 128.7, 128.2, 128.0, 127.9, 127.9, 125.4, 123.9,

6

)

d

177.3, 159.7, 147.8, 144.4, 143.7,

4.5. Cell culture and reagents

1

HCT116, MCF7, HepG2 and A549 cancer cells were purchased

from the Cell Bank of the Shanghai Institute of Cell Biology. MCF7

and HepG2 were cultured in DMEM medium (Gibco/BRL, Grand

Island, New York, USA) supplemented with 10% fetal bovine serum

(FBS, Gibco/BRL, v/v), penicillin (100 units/mL), and streptomycin

(100 mg/L). HCT116 and A549 cells were cultured in RPMI 1640

medium (Gibco/BRL, Grand Island, New York, USA) supplemented

with 10% fetal FBS (v/v), penicillin (100 units/mL), and streptomycin

(100 mg/L). Cells were maintained at 37 C in a humidiﬁed atmo-

2

sphere of 5% CO . Compounds were dissolved in DMSO as a

100 mmol/L stock solution was used for the treatment of cells. In

solvent control group, cells were treated with 0.1% DMSO.

þ

1

23.2, 121.8, 118.1, 112.9, 109.2, 102.9, 35.8; HR ESI-MS [M þ H] m/

þ

20 11 6

H N O , 654.1598).

z ¼ 654.1607 (calcd for C32

0

4.4.56. 3 ,5 -diamino-1-((1-(4-chloro-6-nitrophenyl)-1H-1,2,3-

0

triazol-5-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]

phenazine]-2 -carbonitrile 56

0

ꢀ

1

Yield 62%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.53 (s,1H), 8.38 (d, J ¼ 2.4 Hz,1H), 7.92 (dd, J ¼ 8.6, 2.3 Hz,1H),

.89 (d, J ¼ 8.5 Hz, 1H), 7.63e7.60 (m, 1H), 7.45 (d, J ¼ 8.6 Hz, 1H),

.37e7.33 (m, 2H), 7.28e7.26 (m, 3H), 7.08 (s, 1H), 7.06 (s, 1H), 7.01

d, J ¼ 7.4 Hz, 1H), 6.87e6.84 (m, 1H), 6.56 (s, 2H), 5.32 (d,

ꢀ

d

7

(

6

)

d

13

J ¼ 15.7 Hz,1H), 5.28 (d, J ¼ 15.7 Hz,1H); C NMR (125 MHz, DMSO-

177.6, 161.4, 143.6, 143.3, 143.2, 143.0, 142.8, 142.6, 141.2, 136.7,

36.5,132.3,131.7,131.4,131.1,129.8,129.7,129.4,129.2,129.0,128.9,

28.5, 128.2, 127.8, 126.7, 125.6, 123.8, 123.5, 118.4, 109.1, 102.7, 36.7;

d

6

)

d

4.6. Cell viability assay

1

4

Cells (4 ꢂ 10 per well) were treated with the compound at

þ

4

,

HR ESI-MS [M þ H] m/z ¼ 643.1365 (calcd for C32

H20ClN10

O

concentrations (0e50 mM) in 96-well plates for 48 h. Cell viability

6

43.1358).

was analyzed by MTT (SunshineBio, Nanjing, Jiangsu, China) assay.

MTT (20

kept at 37 C in 5% CO

sample was recorded at 570 nm on automatic plate reader (Bio Rad,

Hercules, California, USA).

m

L, 5 mg/mL) was added to each well, and the plates were

0

ꢀ

4

2

5

.4.57. 3 ,5 -diamino-1-((1-mesityl-1H-1,2,3-triazol-5-yl)methyl)-

2

for 4 h. The optical density (OD) of each

0

-oxospiro[indoline-3,1 -pyrano[3,2-a]phenazine]-2 -carbonitrile

7

ꢀ

1

Yield 61%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.08 (s, 1H), 7.95 (s, 1H), 7.91 (d, J ¼ 8.5 Hz, 1H), 7.72e7.67 (m,

H), 7.55e7.50 (m, 2H), 7.25e7.22 (m, 3H), 7.08 (s, 1H), 7.04 (s, 2H),

d

1

6

)

d

4.7. Laser-scanning confocal microscopy

7

.00 (d, J ¼ 7.2 Hz, 1H), 6.86e6.83 (m, 1H), 6.55 (s, 2H), 5.44 (d,

A549 cancer cells were separately cultured by using the same

13

ꢀ

J ¼ 15.6 Hz,1H), 5.19 (d, J ¼ 15.6 Hz,1H), 2.30 (s, 3H),1.79 (s, 6H);

C

medium at 37 C 20% CO

2

. They were cultured 2 days. Then, the

NMR (125 MHz, DMSO-d

6

)

d

176.6, 159.0, 143.0, 142.6, 142.6, 141.9,

living A549 cancer cells were incubated in a confocal dishes, with

either of the compound 36 (2, 4, and 8 M), Mito Tracker Red

CMXROS (0.1 M, Yeasen, Shanghai, China) in culture medium for

1

41.6, 139.3, 138.5, 135.4, 134.8, 134.3, 134.3, 133.1, 129.7, 128.6,

28.6, 128.6, 128.4, 128.2, 127.6, 127.6, 125.3, 125.2, 123.2, 122.6,

m

1

[

22.4, 117.4, 112.4, 108.6, 102.4, 35.8, 20.4, 16.5, 16.5; HR ESI-MS

30 min, respectively. Finally, these cells were used for cell imaging

using confocal microscopy.

M þ H]^þm/z ¼ 606.2366 (calcd for C35

þ

2

28 9

H N O , 606.2366).

0

4

1

.4.58. 3 ,5 -diamino-2-oxo-1-((1-(2,4,6-trichlorophenyl)-1H-

4.8. Measurement of reactive oxygen species generation

0

,2,3-triazol-5-yl)methyl)spiro[indoline-3,1 -pyrano[3,2-a]

0

phenazine]-2 -carbonitrile 58

Yield 68%; Yellow solid; Mp > 250 C; ¹H NMR (500 MHz,

DMSO-d

8.38 (s, 1H), 8.00 (s, 2H), 7.89 (d, J ¼ 7.2 Hz, 1H),

.69e7.65 (m, 1H), 7.51e7.48 (m, 1H), 7.42 (d, J ¼ 7.8 Hz, 1H), 7.32 (d,

J ¼ 7.7 Hz, 1H), 7.26 (td, J ¼ 7.5, 1.0 Hz, 1H), 7.21 (s, 2H), 7.08 (s, 1H),

.01 (d, J ¼ 7.5 Hz, 1H), 6.86 (t, J ¼ 7.5 Hz, 1H), 6.55 (s, 2H), 5.39 (d,

Measurement of total ROS levels in A549 cancer cells, treat with

compound 36, was performed by using the MitoSOXTM Red

mitochondrial superoxide indicator (M36008, ThermoFisher Sci-

entiﬁc). Once in mitochondria, MitoSOXTM Red reagent is readily

oxidized by superoxide. The oxidation product becomes highly

ﬂuorescent upon binding to nucleic acids. The kit identiﬁed two

different cell population of ROS(ꢁ) and ROS(þ) cells. Percentages of

cells population were measured using ﬂuorescence-activated cell

sorting (FACS) caliber ﬂow cytometry (BD Bioscience, USA).

ꢀ

6

) d

7

13

J ¼ 15.8 Hz,1H), 5.24 (d, J ¼ 15.6 Hz,1H); C NMR (125 MHz, DMSO-

177.3, 159.7, 143.5, 143.2, 143.2, 142.4, 142.2, 139.0, 136.9, 136.0,

35.5, 133.9, 132.2, 130.3, 129.5, 129.5, 129.5, 129.3, 128.8, 128.1,

28.1, 126.7, 126.7, 123.8, 123.8, 123.1, 123.1, 117.9, 113.0, 109.3, 102.9,

6

d ) d

1

3

þ

6.2; HR ESI-MS [M

þ

H] m/z

¼

666.0727 (calcd for

4.9. Cell apoptosis and cell cycle analysis

þ

C

32

H19Cl

3

N

9

O

2

, 666.0727).

5

A549 cancer cells (3 ꢂ 10 cells/dish) were treated with com-

0

4

1

.4.59. 3 ,5 -diamino-1-((1-(4-chloro-2,5-dimethoxyphenyl)-1H-

pound 36 in the concentration of 2, 4, and 8 mM for 24 h. The cells

0

,2,3-triazol-5-yl)methyl)-2-oxospiro[indoline-3,1 -pyrano[3,2-a]

were then harvested with centrifugation at 1800 rpm for 5 min and

0

ꢀ

phenazine]-2 -carbonitrile 59

Yield 70%; Yellow solid; Mp > 250 C; H NMR (500 MHz, DMSO-

8.31 (d, J ¼ 3.9 Hz, 1H), 7.86 (d, J ¼ 8.6 Hz, 1H), 7.63e7.60 (m,

H), 7.40e7.37 (m, 3H), 7.31 (d, J ¼ 7.8 Hz, 1H), 7.27e7.24 (m, 3H),

.07e7.05 (m, 2H), 7.01 (d, J ¼ 7.3 Hz, 1H), 6.85 (t, J ¼ 7.5 Hz, 1H),

ﬁxed with 70% ethanol for 12 h at ꢁ20 C. The cells pellets were

ꢀ

1

washed with PBS (phosphate-buffered saline), and incubated with

d

6

)

d

RNase A (Sigma, China, 2.5

mg/mL) and propidium (Sigma, China,

1

7

6

3

50 g/mL). The samples was determined by ﬂuorescence-activated

m

cell sorting (FACS) caliber ﬂow cytometry (BD Bioscience, USA) [20].

.56 (s, 2H), 5.34 (d, J ¼ 15.6 Hz,1H), 5.24 (d, J ¼ 15.6 Hz,1H), 3.85 (s,

H), 3.61 (s, 3H); ¹³C NMR (125 MHz, DMSO-d

)

d

177.4, 159.7, 149.1,

4.10. Western blotting analysis

6

1

45.4, 143.5, 143.2, 143.2, 143.0, 142.9, 142.4, 142.3, 139.0, 139.0,

36.0, 135.5, 130.0, 128.8, 128.8, 128.1, 128.0, 125.8, 125.0, 123.9,

23.1, 122.6, 118.0, 115.7, 113.0, 110.0, 109.2, 102.9, 57.3, 57.3, 36.3;

5

A549 cancer cells (3 ꢂ 10 cells/dish) treated with compound 36

were lysed and collected using RIPA lysis buffer. BCA protein assay

kit was used to determine concentrations of protein samples.

þ

4

HR ESI-MS [M þ H] m/z ¼ 658.1717 (calcd for C34

9

H25ClN O ,

Y. Lu et al. / European Journal of Medicinal Chemistry 135 (2017) 125e141

141

Proteins were resolved by 12% SDS-PAGE and electroblotted onto

nitrocellulose membranes. The blots were blocked with 5% non-fat

dry milk for 2 h at room temperature and then incubated overnight

with speciﬁc antibodies in TBST overnight at 4 C. After washing,

chemiluminescent detection was recorded using ECL kit (Bio-Rad,

Hercules, CA). Primary rabbit monoclonal antibodies of cleaved-

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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), State Key Laboratory

of Natural Medicines, China Pharmaceutical University (grant No.

JKGQ201112 and 201102), the Fundamental Research Funds for the

Central Universities (grant No. 2013017), National Found for

Fostering Talents of Basic Science(NFFTBS) (grant No. J1030830),

National College Students Innovation and Entrepreneurship

Training Program (grant No. 201410316046, G14046 and SY13100),

the Project Program of National Nature Science Foundation of China

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Appendix A. Supplementary data

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Article Doi

Design, combinatorial synthesis and biological evaluations of novel 3-amino-1'-((1-aryl-1H-1,2,3-triazol-5-yl)methyl)-2'-oxospiro[benzo[a] pyrano[2,3-c]phenazine-1,3'-indoline]-2-carbonitrile antitumor hybrid molecules

DOI: 10.1016/j.ejmech.2017.04.040

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