Arch. Pharm. Res. DOI 10.1007/s12272-015-0619-2

RESEARCH ARTICLE

Synthesis and anticancer activity of 4-aza-daurinol derivatives Faisal Hayat1 • Seung-Hyuk Park1 • Nam-Song Choi2 • Juyeun Lee3 Sung Jean Park1 • Dongyun Shin1



Received: 6 January 2015 / Accepted: 25 May 2015 Ó The Pharmaceutical Society of Korea 2015

Abstract Daurinol, a natural aryl naphthalene lactone, has been reported to have antiproliferative activity against various cell lines, and has also been shown to be efficacious in an in vivo xenograft mouse model. In this study, we tried to discover a new scaffold that enables both rapid structure–activity relationship study of daurinol and scalable synthesis of active compounds. 4-Aza-daurinol, a bioisosterism-based scaffold of daurinol, was designed and 17 analogues were synthesized and evaluated against five representative cancer cell lines. Among them, the 2,3-dihydrobenzo[b][1,4]dioxinyl derivative was found to be the most potent and showed similar activity and tendency as daurinol. Keywords Daurinol  Anticancer  Bioisosteric  Aryltetrahydronaphthalene  Structure–activity relationship

Faisal Hayat and Seung-Hyuk Park have contributed equally to this study. & Sung Jean Park [email protected] & Dongyun Shin [email protected] 1

College of Pharmacy, Gachon University, 191 Hambakmoe-ro, Yeonsu-gu, Incheon 406-799, South Korea

2

College of Interdisciplinary & Creative Studies, Konyang University, 121 Daehak-ro, Nonsan-si, Chungchungnam-do 320-711, South Korea

3

College of Pharmacy, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu, Ansan, Geonggi-do 426-791, South Korea

Introduction Cancer is a leading cause of death. Cytotoxic anticancer drugs are still widely used to cure cancer, (Ferlay et al. 2015), while specific tumor-targeting agents or delivery systems are being actively studied and are now in the development stage (Faivre et al. 2006; Widmer et al. 2014). A major concern related to cytotoxic agents is their side effects on normal cells, which result in serious problems, such as myelosuppression and immunosuppression, which can cause discontinuation of therapy (Wang et al. 2006). For this reason, the discovery of structurally novel and effective agents with minimal toxicity towards normal cells is of interest for clinics as well as in the realm of medicinal chemistry (Jung et al. 2012; Park et al. 2014a). Daurinol is a naturally occurring aryl naphthalene lactone that has been isolated from several traditional medicinal plants (Batirov et al. 1981; Batsuren et al. 1981; AlAbed et al. 1990; Gozler et al. 1992) (Fig. 1). Recently, Nho’s group reported the isolation and anticancer activity of daurinol from Haplophyllum dauricum, which is found in eastern Asia, including China, Mongolia, and Russia, and has been used to treat tumors in Russia (Kang et al. 2011). Structurally, daurinol has an aryl naphthalene lactone skeleton and structural similarities with etoposide, a clinical anticancer drug, which consists of aryltetrahydronaphthalene lactone as the main scaffold and sugar (Hande 1998). Nho’s group suggested that daurinol’s anticancer activity is through the inhibition of topoisomerase IIa, and it’s in vitro and in vivo antitumor activity was demonstrated through the dramatic inhibition of the growth of colorectal tumors in a xenograft model. Moreover, daurinol presents a very different toxicological profile from that of etoposide, with very little loss of body weight and less

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Bruker AscendTM 600 and chemical shifts (d) are recorded in ppm on a d scale. Mass spectra were obtained using either a Waters Autopurification instrument with electron impact ionization (EI) or an AEI MS-9 using electrospray ionization (ES). All chemicals were obtained from TCI or Sigma-Aldrich. General procedure for reduction of nitro to amine Fig. 1 Structures of daurinol and 4-aza-daurinol

hematological toxicity. Based on the experimental data, daurinol is thought to be superior to etoposide in terms of side effects but with equipotent anticancer activity. More recently, daurinol has been proved to be active against ovarian, small-cell lung, and testicular cancer cells (Kang et al. 2014). It was proposed that daurinol could be part of a novel class of low-toxicity antitumor agents (Kang et al. 2011). We and other groups have already reported efficient synthetic methods for type-I and type-II aryl naphthalene lactones, including daurinol (Stevenson and Holmes 1971; Stevenson and Weber 1989, 1991; Park et al. 2014b). However, there still exists some difficulties in the largescale and versatile synthesis of analogues. To address these limitations, we searched for novel and easily synthesizable scaffolds with at least similar anticancer activity as daurinol. Bioisosteric replacement is a commonly adopted method in medicinal chemistry to discover new scaffolds for reasons of synthetic feasibility or patentability (Patani and LaVoie 1996; Lima and Barreiro 2005). In this study, we used classical bioisosteric replacement and designed 4-aza analogues of daurinol in which the C4-position of the naphthalene lactone was substituted with nitrogen (Fig. 1). Herein, we report the synthesis of 4-aza-daurinol derivatives and their anticancer activity against representative cancer cells.

Materials and methods The solvents used for the chemical synthesis were of reagent grade and were used without further purification. All reactions were carried out under nitrogen atmosphere or at designated conditions. The reactions were monitored by TLC analysis using Merck silica gel 60 F-254 thin layer plates. Flash column chromatography was carried out on Merck silica gel 60 (230–400 mesh). FT-IR spectra were obtained using a Shimadzu IR 435 spectrophotometer (KBr, cm-1). 1H-NMR spectra were recorded using a

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To a solution of nitro compound (10.0 mol) in MeOH, 10 % Pd on charcoal (10 mol%) was added, and the resulting suspension was then stirred under a hydrogen atmosphere (balloon) at room temperature for 2 h. After checking the disappearance of the starting material by thin layer chromatography, the palladium catalyst was filtered off through a Celite pad. The residue was concentrated under reduced pressure and used in the next step without purification. 3-Aminophenol (10) Grey oil; yield: 95 %; FT-IR (cm-1) 3652, 3504, 3420, 3043, 1630, 1173; 1H NMR (600 MHz, CDCl3) d (ppm) 7.01 (t, J = 8.4 Hz, 1H), 6.27 (d, J = 7.8 Hz, 1H), 6.23 (d, J = 7.8 Hz, 1H), 6.18 (t, J = 4.8 Hz, 1H), 4.64 (s, 1H), 3.65 (s, 2H); MS [ESI] m/z 110 [M?H]?. 5-Amino-2-methoxyphenol (11) Grey oil; yield: 96 %; FT-IR (cm-1) 3392, 3305, 2910, 2825, 1456; 1H NMR (600 MHz, CDCl3) d (ppm) 6.68 (d, J = 8.4 Hz, 1H), 6.35 (d, J = 2.4 Hz, 1H), 6.18 (dd, J = 2.4, 5.4 Hz, 1H), 3.82 (s, 3H); MS [ESI] m/z 140 [M?H]?. General procedure for the coupling of aniline and tetronic acid to anilinolactone The reaction mixture, consisting of aniline (5.0 mol) and tetronic acid (5.0 mol) in dioxane (50 mL), was stirred at room temperature for 2 days. After removal of the solvent, the residue was dissolved in ethyl acetate and resolidified to give anilinolactone as a grey solid. The solid was used in the next step without further purification. 4-(3-Hydroxyphenylamino)furan-2(5H)-one (12) Gray solid; yield: 83 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.12 (t, J = 8.4 Hz, 1H), 6.59 (dd, J = 2.4, 4.8 Hz, 2H), 6.45 (qd, J = 1.2, 0.6 Hz, 1H), 5.17 (s, 1H), 4.82 (s, 2H); MS [ESI] m/z 192 [M?H]?.

Synthesis and anticancer activity of 4-aza-daurinol derivatives

4-(3-Hydroxy-4-methoxyphenylamino)furan-2(5H)-one (13) Gray solid; yield: 78 %; 1H NMR (600 MHz, CDCl3) d (ppm) 6.87 (d, J = 9.0 Hz, 1H), 6.65 (d, J = 3.0 Hz, 1H), 6.56 (dd, J = 3.0, 9.0 Hz, 1H), 5.04 (s, 1H), 4.78 (s, 2H), 3.72 (s, 3H); MS [ESI] m/z 222 [M?H]?. General procedure for the synthesis of 4-aza-daurinol analogues Anilinolactone (0.5 mol) in trifluoroacetic acid (2 mL) was treated with p-chloranil (0.5 mol) and substituted benzaldehyde (0.5 mol), and the reaction mixture was then stirred at room temperature for 1 day. After removal of the volatile materials, the residue was purified by SiO2 column chromatography to afford the desired 4-aza-daurinol analogues. 9-(Benzo[d][1,3]dioxol-5-yl)-6-hydroxyfuro[3,4b]quinolin-1(3H)-one (14a) Gray solid; yield: 78 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.71 (d, J = 9.0 Hz, 1H), 7.35 (d, J = 2.4 Hz, 1H), 7.22 (dd, J = 2.4, 9.0 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 7.04 (d, J = 1.2 Hz, 1H), 6.90 (dd, J = 1.8, 7.8 Hz, 1H), 6.14 (d, J = 21.6 Hz, 2H), 5.40 (s, 2H); MS [ESI] m/z 322 [M?H]?; HR-MS calcd for C18H11NO5 321.0637; found 321.0642. 9-(3,4-Difluorophenyl)-6-hydroxyfuro[3,4-b]quinolin1(3H)-one (14e) Gray solid; yield: 73 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.79 (d, J = 9.0 Hz, 1H), 7.59 (d, J = 2.4 Hz, 1H), 7.38 (d, J = 12.0 Hz, 2H), 7.20–7.18 (m, 2H), 5.44 (s, 3H); MS [ESI] m/z 314 [M?H]?; HR-MS calcd for C17H9F2NO3 313.0550, found 313.0553. 9-(4-Chloro-3-fluorophenyl)-6-hydroxyfuro[3,4b]quinolin-1(3H)-one (14f) Gray solid; yield: 67 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.48 (d, J = 9.0 Hz, 1H), 7.60 (t, J = 7.2 Hz, 2H), 7.52 (d, J = 2.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 5.42 (s, 2H); MS [ESI] m/z 330 [M?H]?; HR-MS calcd for C17H9ClFNO3 329.0255, found 329.0248. 9-(3-Fluoro-4-hydroxyphenyl)-6-hydroxyfuro[3,4b]quinolin-1(3H)-one (14g) Gray solid; yield: 75 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.76 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 2.4 Hz, 1H),

7.33 (dd, J = 1.8, 11.4 Hz, 1H), 7.25 (dd, J = 2.4, 9.0 Hz, 1H), 7.15–7.09 (m, 2H); MS [ESI] m/z 312 [M?H]?; HRMS calcd for C17H10FNO4 311.0594; found 311.0596. 6-Hydroxy-9-(4-hydroxy-3-methoxyphenyl)furo[3,4b]quinolin-1(3H)-one (14h) Gray solid; yield: 78 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.79 (d, J = 9.0 Hz, 1H), 7.33 (d, J = 2.4 Hz, 1H), 7.21 (dd, J = 3.0, 9.6 Hz, 1H), 7.03 (d, J = 1.8 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.85 (dd, J = 1.8, 7.8 Hz, 1H), 5.38 (s, 1H), 3.75(s, 3H); MS [ESI] m/z 324 [M?H]?; HRMS calcd for C18H13NO5 323.0794, found 323.0798. 6-Hydroxy-9-(3-hydroxy-4-methoxyphenyl)furo[3,4b]quinolin-1(3H)-one (14i) Gray solid; yield: 69 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.93 (d, J = 9.6 Hz, 1H), 7.51 (d, J = 2.4 Hz, 1H), 7.20 (dd, J = 2.4, 9.0 Hz, 2H), 6.98 (dd, J = 1.8, 7.8 Hz, 2H), 5.40 (s, 2H), 4.12 (s, 3H); MS [ESI] m/z 324 [M?H]?; HR-MS: calcd for C18H13NO5 323.0794; found 323.0797. 9-(3,4-Dimethoxyphenyl)-6-hydroxyfuro[3,4-b]quinolin1(3H)-one (14j) Gray solid; yield: 78 %; 1H NMR (600 MHz, CDCl3) d (ppm) = 7.80 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 2.4 Hz, 1H), 7.25 (dd, J = 2.4, 9.0 Hz, 1H), 7.15 (d, J = 7.8 Hz, 1H), 7.10 (d, J = 1.8 Hz, 1H), 7.01 (dd, J = 1.8, 7.8 Hz, 1H), 5.43 (s, 2H), 3.88 (s, 3H), 3.76 (s, 3H); MS [ESI] m/z 338 [M?H]?; HR-MS calcd for C19H15NO5 337.0950, found 337.0946. 9-(Benzo[d][1,3]dioxol-5-yl)-6-hydroxy-7methoxyfuro[3,4-b]quinolin-1(3H)-one (15a) Gray solid; yield: 74 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.58 (s, 1H), 7.18 (s, 1H), 7.01 (d, J = 7.8 Hz, 1H), 6.93–6.90 (m, 2H), 5.36 (s, 2H), 3.92 (s, 3H); MS [ESI] m/z 352 [M?H]?; HR-MS calcd for C19H13NO6 351.0743, found 351.0746. 9-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-6-hydroxy-7methoxyfuro[3,4-b]quinolin-1(3H)-one (15b) Gray solid; yield: 81 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.58 (s, 1H), 7.21 (s, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 1.8 Hz, 1H), 6.92 (dd, J = 2.4, 8.4 Hz, 1H), 5.36 (s, 2H), 4.37 (q, J = 6.0 Hz, 4H), 3.92 (s, 3H); MS [ESI] m/z 366 [M?H]?; HR-MS: calcd for C20H15NO6 365.0899, found 365.0905.

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Scheme 1 Initial approach for the synthesis of 4-aza-daurinol. Reagents and conditions a PhCH2Br, K2CO3, EtOH, 50 °C, 1 day. b SnCl2 2H2O, EtOH, rt, 8 h. c Tetronic acid, dioxane, rt, 1 day. d Piperonal, chloranil, TFA, rt, 1 day

9-(3-Fluorophenyl)-6-hydroxy-7-methoxyfuro[3,4b]quinolin-1(3H)-one (15c)

6-Hydroxy-9-(4-hydroxy-3-methoxyphenyl)-7methoxyfuro[3,4-b]quinolin-1(3H)-one (15h)

Gray solid; yield: 76 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.61 (s, 1H), 7.55(q, J = 6.0 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.17 (d, J = 9.0 Hz, 1H), 7.03(s, 1H), 6.48 (s, 1H), 5.39 (s, 2H), 3.90 (s, 3H); MS [ESI] m/z 326 [M?H]?; HR-MS calcd for C18H12FNO4 325.0750, found 325.0754.

Gray solid; yield: 65 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.60 (s, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 1.8 Hz, 1H), 6.98 (dd, J = 1.8, 7.8 Hz, 2H), 5.37 (s, 2H), 3.93 (s, 3H), 3.91 (s, 3H); MS [ESI] m/z 354 [M?H]?; HR-MS calcd for C19H15NO6 353.0899, found 353.0803.

9-(3-Chlorophenyl)-6-hydroxy-7-methoxyfuro[3,4b]quinolin-1(3H)-one (15d) Gray solid; yield: 78 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.61 (s, 1H), 7.56–7.51 (m, 2H), 7.44 (t, J = 1.2 Hz, 1H), 7.34 (dt, J = 1.5, 1.5 Hz), 7.03 (s, 1H), 5.39 (s, 1H), 3.90 (s, 3H); MS [ESI] m/z [M?H]?; HR-MS: calcd for C18H12ClNO4 341.0455, found 341.0452. 9-(3,4-Difluorophenyl)-6-hydroxy-7-methoxyfuro[3,4b]quinolin-1(3H)-one (15e) Gray solid; yield: 62 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.61 (s, 1H), 7.37 (q, J = 8.4 Hz, 2H), 7.20–7.18 (m, 1H), 7.02 (s, 1H), 5.39 (s, 2H), 3.91 (s, 3H); MS [ESI] m/z 344 [M?H]?; HR-MS calcd for C18H11F2NO4 343.0656; found 343.0652. 9-(3-Fluoro-4-hydroxyphenyl)-6-hydroxy-7methoxyfuro[3,4-b]quinolin-1(3H)-one (15g) Gray solid; yield: 73 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.38 (s, 1H), 7.33 (s, 1H), 7.31 (d, J = 1.8 Hz, 1H), 7.11 (d, J = 1.8 Hz, 2H), 7.07 (s, 1H), 5.36 (s, 2H), 3.74 (s, 3H); MS [ESI] m/z 342 [M?H]?; HR-MS calcd for C18H12FNO5 341.0700, found 341.0708.

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6-Hydroxy-9-(3-hydroxy-4-methoxyphenyl)-7methoxyfuro[3,4-b]quinolin-1(3H)-one (15i) Gray solid; yield: 76 %; 1H NMR (600 MHz, CDCl3) d (ppm) = 7.08 (s, 1H), 7.03 (d, J = 7.8 Hz, 1H), 6.94 (d, J = 1.8 Hz, 1H), 6.84 (s, 1H), 6.78 (d, J = 2.4 Hz, 1H), 4.02 (s, 2H), 3.86 (s, 3H), 3.84 (d, J = 3.6 Hz, 3H); MS [ESI] m/z 354 [M?H]?; HR-MS: calcd for C19H15NO6 353.0899, found 353.0904. 9-(3,4-Dimethoxyphenyl)-6-hydroxy-7-methoxyfuro[3,4b]quinolin-1(3H)-one (15j) Gray solid; yield: 78 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.62 (s, 1H), 7.24 (s, 1H), 7.08–7.05 (m, 2H), 6.99 (d, J = 1.8 Hz, 1H), 5.40 (s, 2H), 4.00 (s, 3H), 3.90 (s, 3H), 3.89 (s, 3H); MS [ESI] m/z 368 [M?H]?; HR-MS calcd for C20H17NO6 367.1056, found 367.1058. 6-Hydroxy-7-methoxy-9-(3,4,5-trimethoxyphenyl)furo[3,4b]quinolin-1(3H)-one (15k) Gray solid; yield: 62 %; 1H NMR (600 MHz, CDCl3) d (ppm) 7.59 (s, 1H), 7.11 (s, 1H), 6.78 (s, 1H), 6.73 (s, 1H), 5.36 (q, J = 3.6 Hz, 2H), 4.02 (s, 3H), 3.88 (s, 3H), 3.83 (s,

Synthesis and anticancer activity of 4-aza-daurinol derivatives

Scheme 2 Synthesis of aza-daurinol derivatives. Reagents and conditions a H2, Pd/C, MeOH, rt, 1 h. b Tetronic acid, rt. c Substituted benzaldehydes, p-chloranil, TFA, rt

3H), 3.73 (s, 3H); MS [ESI] m/z 398 [M?H]?; HR-MS: calcd for C21H19NO7 397.1162, found 397.1165.

Measurement of anticancer activity To assess the antiproliferative activity of daurinol and 4-aza-daurinol analogues against various cancer cell lines, cell viability was determined by measurement of mitochondrial dehydrogenase activity using a Cell Counting Kit (CCK-8) purchased from Dojindo Laboratories (Tokyo, Japan). Briefly, cancer cells (5 9 103 cells per well) were plated in 96-well plates, incubated at 37 °C for 24 h, and then treated with 10 or 100 lM daurinol and 4-aza-daurinol analogues for 24 h.

Results and discussion Our work commenced with the establishment of a facile synthetic route to 4-aza-daurinol. In 1997, Itogawa’s group reported an efficient method for the synthesis of 4-aza-analogues of 1-aryl naphthalene lactones, exemplified by Taiwanin C, Justicidin B, Chinensin, and other analogues, all of which have alkoxy substituents on the A-ring (Hitotsuyanagi et al. 1997). Based on the procedure, we planned to synthesize 4-aza-daurinol (Scheme 1). 3-(Benzyloxy)-4methoxyaniline (5) was prepared from 5-nitroguaiacol (3) by the sequential benzylation of the phenolic hydroxyl group and the reduction of nitro with tin(II) chloride dihydrate (SnCl2 2H2O). The condensation of aniline 5 and tetronic acid was carried out using dioxane as the solvent at room

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F. Hayat et al. Table 1 Percent inhibition of antiproliferative activity of 4-aza-aryl naphthalene lactones

No.

R2

MTT assay (% inhibition)a HepG2

A431

A549

MCF7

HCT116

100

10

100

10

100

10

100

10

100

10

82

68

81

66

81

69

79

63

87

82

1

(Daurinol)

14a

3-OCH2O-4

18

6.1

32

10

33

5.5

46

5.5

46

6.8

14e

3-F, 4-F

1.7

–0.3

18

12

17

5.1

35

8.5

38

17

14f

3-F, 4-Cl

25

4.1

31

2.8

18

6.0

39

8.5

39

16

14g

3-F, 4-OH

12

7.1

22

17

20

4.7

57

7.1

48

12

14h

3-OMe, 4-OH

2.1

7.1

16

10

21

3.4

36

5.6

28

4.6

14i

3-OH, 4-OMe

22

8.3

43

13

44

16

54

18

64

31

14j

3,3-di-OMe,

46

11

72

19

55

3.9

61

39

77

12

15a

3-OCH2O-4

78

42

72

48

81

37

78

51

89

50

15b

3-O(CH2)2O4

81

56

84

61

80

51

78

63

91

81

15c

3-F

9.0

7.2

12

7.1

12

10

31

10

30

12

15d

3-Cl

16

8.0

73

18

52

11

75

28

79

51

15e

3-F, 4-F

38

7.0

42

23

30

2.6

54

12

53

13

15g

3-F, 4-OH

41

14

75

13

59

6.1

76

4.8

76

10

15h

3-OMe, 4-OH

30

–0.9

671

13

36

12

68

7.8

74

4.8

15i

3-OH, 4-OMe

77

42

69

51

76

33

73

55

89

64

15j

3,4-di-OMe,

27

5.3

46

11

23



45

14

57

14

15k

3,4,5-tri-OMe

20

10

16

4.9

11

4.8

15

9.4

24

16

a

Percent inhibitions were measured by MTT assay at 100 lM and 10 lM, respectively

Table 2 IC50 of daurinol and 15b for cell proliferation Entry

Cell line

Anticancer activity (IC50, lM) Daurinol (1)

15b

1

HepG2

8.40 ± 1.14

15.85 ± 0.77

2

A431

11.56 ± 1.85

11.03 ± 1.1

3

A549

4.33 ± 0.48

6.41 ± 0.84

4

MCF7

5.99 ± 0.24

7.88 ± 0.56

5

HCT116

3.48 ± 0.25

4.69 ± 0.40

temperature to afford vinylogous carbamate 6 in high yield. Finally, one-pot condensation and oxidative aromatization with carbamate 6 by reacting with piperonal in the presence of p-chloranil in trifluoroacetic acid was performed to give a mixture of products, including the desired benzylated 4-azadaurinol (7), benzyl-migrated compound (8), and others. Presumably, under acidic conditions, the benzyl group was

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dissociated and the resulting benzylic cation was captured by aromatic rings. Considering the mechanism of oxidative cyclization, we envisioned that non-alkylated phenolic carbamate (12 or 13) could serve as a precursor for aza-naphthalene lactone, which would avoid the use of a deprotection step (Scheme 2). To reveal the partial structure–activity relationship of the A-ring, two nitro compounds, 5-nitroguaiacol (9) and 3-nitrophenol (3), were chosen as the starting materials. They were reduced using H2 and 10 % palladium on carbon to produce the corresponding anilines 10 and 11, respectively, which, by treating with tetronic acid, were converted to vinylogous carbamates 12 and 13, respectively, at excellent yields. The reaction of vinylogous carbamates with various substituted benzaldehydes was accomplished to synthesize the desired 4-aza-daurinol and 4-aza-7-demethoxydaurinol analogues 14 and 15, respectively. Substituted benzaldehydes were

Synthesis and anticancer activity of 4-aza-daurinol derivatives

selected based on the substituents on daurinol, in which the aryl group has substituents at the C3 and C4 positions. The anticancer activity of 17 4-aza-daurinol analogues was evaluated. Representative cancer cell lines, such as liver cancer (HepG2), epidermal cancer (A431), lung cancer (A549), breast cancer (MCF7), and colon cancer (HCT116), were used, and daurinol was tested as a comparative compound. Initially, the two series of compounds, 6-hydroxy and 6-hydroxy-7-methoxy compounds, were screened at 100 and 10 lM using the MTT assay method in order to reveal the antiproliferative activities, and the results are presented in Table 1. Compounds 15a, 15b, and 15i displayed antiproliferative activities at 100 lM, and among them, 15b, a 1,4-dioxane analogue, showed almost equipotent activity as that of daurinol at 10 lM. Further evaluation of the IC50 of compound 15b in the selected cancer cell lines was carried out, as shown in Table 2. Daurinol and compound 15b exhibited slightly more potent activities against A549 (4.33 and 6.41 lM, respectively), MCF7 (5.99 and 7.88 lM, respectively), and HCT116 (3.48 and 4.69 lM, respectively) than that against HepG2 (8.40 and 15.85 lM, respectively) and A431 (11.56 and 11.03 lM, respectively). From the above data, some structural features of 4-azadaurinol that affect the anticancer activity are revealed. The series of 7-demethoxy analogues did not exhibit any notable antiproliferative activity. C-ring substituents had a significant effect on the anticancer activity and 1,4-dioxane analogues were found to be have a significant effect. 4-Azadaurinol (15a) exhibited a similar anticancer spectrum to that of daurinol, which implies that 4-aza-daurinol derivatives could lead to the future discovery of alternatives to daurinol.

Conclusion In summary, we designed a series of new 4-aza-aryl naphthalene lactones as bioisosterism-based daurinol analogues. Seventeen derivatives were synthesized and their antiproliferative activities against five representative cancer cell lines were evaluated. Changing the substituents on the C-ring of 4-aza-daurinol had a large influence on the anticancer activity and, among them, 4-aza-daurinol displayed similar anticancer activity to that of daurinol and 2,3-dihydrobenzo[b][1,4]dioxinyl derivatives were revealed to be the most potent. From this study, aryl-4-aza-naphthalene lactone, an isosterism-based scaffold of daurinol, is presented as a potential anticancer drug, and further structure–activity relationship study is ongoing.

Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A1007057).

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Synthesis and anticancer activity of 4-aza-daurinol derivatives.

Daurinol, a natural aryl naphthalene lactone, has been reported to have antiproliferative activity against various cell lines, and has also been shown...
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