Discovery and Biological Evaluations of Halogenated 2,4-Diphenyl

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Discovery and Biological Evaluations of Halogenated 2,4-Diphenyl Indeno[1,2-b]pyridinol Derivatives as Potent Topoisomerase II#-targeted Chemotherapeutic Agents for Breast Cancer Tara Man Kadayat, Seojeong Park, Aarajana Shrestha, Hyunji Jo, Soo-Yeon Hwang, Pramila Katila, Ritina Shrestha, Mahesh Raj Nepal, Keumhan Noh, Sang Kyoon Kim, Woo-Suk Koh, Kil Soo Kim, Yong Hyun Jeon, Tae Cheon Jeong, Youngjoo Kwon, and Eung-Seok Lee J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00970 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 10, 2019

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Journal of Medicinal Chemistry

Discovery and Biological Evaluations of Halogenated 2,4-Diphenyl Indeno[1,2b]pyridinol Derivatives as Potent Topoisomerase II-targeted Chemotherapeutic Agents for Breast Cancer Tara Man Kadayat,†,#,⊥ Seojeong Park,‡,⊥ Aarajana Shrestha,†,⊥ Hyunji Jo,‡ Soo-Yeon Hwang,‡ Pramila Katila,† Ritina Shrestha,† Mahesh Raj Nepal,† Keumhan Noh,† Sang Kyoon Kim,§ Woo-Suk Koh,§ Kil Soo Kim,§,∥ Yong Hyun Jeon,§ Tae Cheon Jeong,† Youngjoo Kwon,‡,* Eung-Seok Lee†,*

†College ‡College

of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans

University, Seoul 120-750, Republic of Korea #New

Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu

41061, Republic of Korea §Laboratory

Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu

41061, Republic of Korea ∥ College

of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of

Korea ⊥

These authors contributed equally to this study.

*Corresponding Authors: *E-mail: [email protected]. Tel: +82 2 3277-4653. Fax: +82 2 3277-2851 (Y.K.) *E-mail: [email protected]. Tel: +82 53 810-2827. Fax: +82 53 810-4654 (E.-S.L.)

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Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract With the aim of developing new effective topoisomerase II-targeted anticancer agents, we synthesized a series of hydroxy- and halogenated 2,4-diphenyl indeno[1,2-b]pyridinols using a microwave-assisted single step synthetic method, and investigated structure-activity relationships. The majority of compounds with chlorophenyl group at 2-position and phenol group at 4-position of indeno[1,2-b]pyridinols exhibited potent anti-proliferative activity and topoisomerase II-selective inhibition. Of the one hundred seventy-two compounds tested, 89 showed highly potent and selective topoisomerase II inhibition and anti-proliferative activity in the nanomolar range against human T47D breast (2.6 nM) cancer cell lines. In addition, mechanistic studies revealed compound 89 is a non-intercalative topoisomerase II poison, and in vitro studies showed it had promising cytotoxic effects in diverse breast cancer cell lines and was particularly effective at inducing apoptosis in T47D cells. Furthermore, in vivo administration of compound 89 had significant antitumor effects in orthotopic mouse model of breast cancer.


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Introduction Cancers are heterogeneous malignant diseases caused by abnormal cell division, and cancer cells have the ability to disseminate through the blood or lymphatic systems and invade distant organs.1 DNA topoisomerase (topo) has the unique ability to alleviate the DNA torsional stress that occurs during replication, transcription, recombination, and chromosomal segregation and is an important clinical target for the treatment of cancer.2-4 There are many clinically approved topo inhibiting drugs, such as doxorubicin and teniposide, but due to the complexities of cancer, drug resistance, and systemic side-effects, the selectivity for topo is being scrutinized.5 Of the major types of clinically important human topo enzymes, topo I transiently breaks only one strand of DNA, whereas, topo II, (which has α and β isoforms) cuts both strands in the presence of magnesium and ATP binding.6-8 Despite similar catalytic properties and 70% similarly between the amino acid sequences of two different topo II isoforms, only the expression level of topo II varies significantly during cell growth.9-11 This unique characteristic makes topo II an efficient and safe target as its use minimizes topo IImediated toxicities such as cardiotoxicity and leukemia.12-14 Therefore, research efforts are being directed toward developing safer and more effective topo II -targeting cancer chemotherapies.15

Over the past few decades, several studies on drug discovery and development have revealed the importance of hydroxyl and halogen moieties on molecular scaffolds of potential interest.16-18 With the aim of identifying novel topo-targeted anticancer agents, our group previously designed and synthesized several flexible 2,4,6-trisubstituted pyridines with phenolic and chlorophenyl groups at the 2- and/or 4-positions, and thienyl, furyl, or phenyl groups at the 6-position.19-21 The promising results obtained using these flexible pyridine derivatives prompted us to design and synthesize rigid benzofuro[3,2-b]pyridines and 5H3 ACS Paragon Plus Environment


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indeno[1,2-b]pyridines incorporating phenolic, chlorophenyl, and other aryl groups at 2- and 4-positions (Fig. 1). Introductions of various functionalities in rigid benzofuro[3,2-b]pyridine and 5H-indeno[1,2-b]pyridine allowed us to explore structure-activity relationships based on their topo inhibitory and anticancer activities.22, 23 In particular, we found that a phenol or chlorophenyl ring at the 2- or 4-positions of the benzofuro[3,2-b]pyridine skeleton (Fig. 2, compounds A and B) were crucial for the dual inhibitions of topo I and II.22, 24 However, benzofuro[3,2-b]pyridin-7-ol (compound C) showed potent and selective topo II inhibition.25 On the other hand, 5H-indeno[1,2-b]pyridine compounds D and E with a phenol or chlorophenyl group at the 2- or 4-positions acted as a non-intercalative topo II catalytic inhibitor and as a strong cytotoxic agent, respectively.23, 26

R 2

R

6

N

R

Modification to rigid analogs

4

R

Flexible 2,4,6-trisubstituted pyridines

Y X

R

Z

R= Z

N

Where, Y= H, OH or Cl Z= S or O

Rigid analogs X= O: benzofuro[3,2-b]pyridine X= CH2: 5H-indeno[1,2-b]pyridine

Figure 1. Structures of previously reported rigid analogs of 2,4,6-trisubstituted pyridines.


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OH HO

benzofuro[3,2-b]pyridine 4 2

O

N

O

O HO

O HO

N

N

A Non-intercalative dual topo I and II catalytic inhibitor

N

B

Cl

OH

C

Non-intercalative dual topo I and II catalytic inhibitor

Potent and selective topo II inhibitor

OH HO

Hydroxylation at 6-, 7-, 8- & 9- position

4 2

N

Cl

N

5H-indeno[1,2-b]pyridine

6

N

E

D Non-intercalative topo II catalytic inhibitor

R

Strong cytotoxic for HCT15 colon cancers cell (IC50 =0.31 M)

R1

N

OH 7

9

8

R, R1= H, OH, Cl, F, CF3

5H-indeno[1,2-b]pyridinol scaffold (1-172)

Figure 2. Strategy for the design of 2,4-diphenyl-substituted-5H-indeno[1,2-b]pyridinols. Compounds A-E are previously synthesized active compounds, and compounds 1-172 are newly prepared in the present study. Based on these reported findings, our major focus was to maximize topo II-targeting anticancer activity by introducing a hydroxyl group at positions 6-, 7-, 8- or 9- of the 5Hindeno[1,2-b]pyridine skeleton. We designed 172 novel rigid 2,4-disubstituted 5Hindeno[1,2-b]pyridinol compounds incorporating moieties such as phenyl, phenolic, chlorophenyl, fluorophenyl and trifluoromethylphenyl at the 2- or 4-positions (Fig. 2). Here, for the first time we have introduced fluorine (-F) and trifluoromethyl (-CF3) functionalities at the 2- phenyl rings anticipating to discover the novel and more potent topo II inhibitors. Compounds were synthesized using a microwave-assisted single step, three-components strategy, which proved to be rapid, effective, and environmental-friendly. In addition, as compared to the previously reported Kröhnke synthetic method, the devised method does not involve additional synthetic steps for the preparations of intermediates and pyridinium iodide salts. Compound 89 exhibited highly potent topo IIselective inhibition and antiproliferative activity in the nanomolar range (~2.6 nM) against T47D human breast cancer cell lines and was selected for further mechanistic and in vivo ADME profile studies. In 5 ACS Paragon Plus Environment


addition, bioluminescent imaging and in vivo studies were performed to investigate the potential of compound 89 for breast cancer therapy.

Results and Discussion Chemistry. Initially, we utilized the previously reported three-step Kröhnke synthetic method23 (Scheme 1A) to prepare 2,4-diphenyl 5H-indeno[1,2-b]pyridinols (compounds 1 to 4) by adding hydroxy-indanone intermediates (Va-d) (1.0 equiv) and pyridinium iodide salt (II) (1.5 equiv) to glacial acetic acid and then adding anhydrous ammonium acetate (NH4OAc; 10.0 equiv). This mixture was then refluxed at 100 °C for 24 h, and compounds 1 to 4 were synthesized in yields of 35.7 to 51.6% as described in the Supporting Information. However, as shown in Scheme 1B and detailed in the Experimental Section the use of microwave radiation27 and three-component reactions between equivalent amounts of benzaldehyde, hydroxy-1-indanone, and acetophenone in the presence of NH4OAc (2.5 equiv.) in DMF at 120oC for 20-30 min resulted relatively higher yields (38.5 to 63.6%) of target compounds 1 to 4. Thus, to synthesize a series of 2,4-disubstituted 5H-indeno[1,2b]pyridinols (compounds 5-172), we utilized this microwave-assisted synthetic method (Scheme 2). The observed range of compounds yield (11.1 to 58.6%) depends on substitution of hydroxyl and halogen groups at 2- and 4-phenyl ring of 5H-indeno[1,2-b]pyridinols. Compounds 1-4 without any substitution at 2- and 4-phenyl ring have highest range of yield (38-63%), while on substituting hydroxyl group at 2- or 4-phenyl position, compounds 5-28 (except 18) resulted decrease in yield ranging from 23-58%. Similarly, on addition of halogen group, chlorinated compounds 29-100, most of the compounds have moderate yield ranging from 20-43%. However, most of fluorine containing compounds (101-172) have low yield (below 20%). Purity and characterization of all compounds synthesized were established by HPLC, MS, NMR and TLC. 6 ACS Paragon Plus Environment

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Scheme 1. Synthesis of compounds 1-4a, (A) Using conventional Kröhnke synthetic approach, and (B) Microwave-assisted single-step synthetic method. A

O

HO

CH3

O III (Hydroxy-indanone)

I (Acetophenone)

O

(ii)

(i)

R = 6-OH a

H

N

7-OH b

8-OH c

9-OH d

IV (Benzaldehyde)

O N I-

(iii)

HO

II (Pyridinium iodide Salt)

N

O Va-d (Hydroxy-indanone Intermediate)

R

1-4 (35.7-51.6%)

B

H O Benzaldehyde

+

HO

CH3

+ O

hydroxy-1-indanone

(iv)

O Acetophenone

aReagents

OH N 1-4 (38.5-63.6%)

and conditions: (i) iodine (1.0 equiv.), pyridine, 3 h at 140 °C, 66% yield; (ii) aq. NaOH, EtOH, 1-3 h at room temperature, 28.8- 81.7% yield; (iii) NH4OAc (10.0 equiv.), glacial acetic acid, 24 h at 100oC, 35.7-51.6% yield; (iv) NH4OAc (2.5 equiv.), DMF, 200 W, 20-30 min at 120 oC, 38.5-63.6% yield.



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Scheme 2. Synthesis of compounds 5-172a R R = H/OH R

H

+

HO

O

+

R

CH3

6

2

(i)

N

R

O

O

4

OH 7

9

5-28

8

(2,4-phenol series) Cl 4

Cl

H

+

HO

HO

O

+

CH3

N

HO

OH 7

9

29-64

O

O

6

2

(i)

8

(2-phenol-4-chlorophenyl series)

OH R1 = Cl, F, CF3

HO

H O

+

R1

HO

+ O

CH3

(i)

4 6

2

R1

N

65-172

O

OH 7

9

8

(2-halophenyl-4-phenol series)

aReagents

and conditions: (i) NH4OAc (2.5 equiv.), DMF, 200 W, 30-180 min at 120-150 oC, 11.1-58.6% yield.


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In vitro Cell Proliferation Inhibitory Activities. First, we evaluated the abilities of the 172 synthesized compounds to inhibit the four human cancer cell lines HCT15, T47D, DU145, and HeLa (IC50 values are provided in Tables 1-6). Most of the compounds potently inhibited cell proliferation as compared with etoposide (the reference compound). Camptothecin, etoposide and adriamycin were used as positive control for antiproliferative activity. In the 2,4-diphenyl series (compounds 1-4), all four compounds inhibited HCT15, T47D and HeLa proliferation more potently than etoposide. Compound 3 had greater antiproliferative activity (IC50 = 0.19 µM) than all three reference compounds in the T47D cell line. Interestingly, almost all the 2-phenyl, 4-phenol series (compounds 5-16) significantly inhibited the proliferations of HCT15, T47D, and DU145 cell lines, whereas half of the compounds inhibited HeLa proliferation more than etoposide. Compounds 5, 7-10, 12 inhibited DU145 proliferation significantly more (IC50 < 0.81 µM) than the three reference compounds. Similarly, most compounds in the 2-phenol, 4-phenyl series (compounds 17-28) inhibited the proliferation of DU145 (IC50 = 0.27-0.71 µM) significantly more than the three reference compounds. However, only compounds 21, 22, 25-27 potently inhibited the proliferation of HCT15 cells (IC50 = 1.02-1.58 µM) lines. Compounds 21, 24, 26, and 27 inhibited T47D cell proliferation, and compounds 17, 26-28 inhibited HeLa proliferation (IC50 = 1.83-2.74 µM) better than etoposide (Table 1). Likewise, in the 2-phenol, 4-chlorophenyl series (compounds 29-64), with the exceptions of compounds 30-32 in the HeLa cell line, compounds 29-40, which contained a chlorine group at the ortho-position of 4-phenyl ring, exhibited antiproliferative activities similar to/or greater than etoposide. However, when the chlorine group was in the meta- or para-positions, most compounds showed weak to moderate antiproliferative activity,



especially in the HCT15, T47D, and HeLa cell lines. Only compounds 44, 45, 49, and 57 inhibited HCT15 proliferation more than etoposide, whereas compounds 44, 49-51, 57, 60, 62, and 63 inhibited T47D proliferation more than etoposide. Similarly, only compounds 46, 47, 49-51, 57, and 62 inhibited HeLa proliferation more etoposide. Compounds, 44, 47, 4951, 56, 57, and 60-63, inhibited DU145 proliferation significantly more than etoposide (Table 2). Interestingly, interchanging the positions of chlorine and hydroxyl groups altered antiproliferative activities. For example, the 2-chlorophenyl, 4-phenol series (compounds 65100) had better antiproliferative activities than the 2-phenol, 4-chlorophenyl series in all four cell lines. With the exception of compound 100, all compounds in the 2-chlorophenyl, 4phenol series displayed greater antiproliferative activity (IC50 = 0.00267-3.5 µM) than etoposide (IC50 = 4.85 µM) in the T47D cell line. Similarly, in the DU145 cell line, almost all compounds in the 2-chlorophenyl, 4-phenol series showed greater antiproliferative activity (IC50 = 0.072-2.03 µM) than etoposide (IC50 = 2.19 µM). Likewise, with the exception of compound 100 in the HCT15 cell line and of compounds 69, 79, 95, and 100 in the HeLa cell line, all compounds displayed greater or antiproliferative activities similar to etoposide in these two cell lines. As shown in Table 3, compound 78 exhibited highly potent antiproliferative activity (IC50 = 1.33 nM) against HCT15 cells, whereas compound 89 showed IC50 value 2.67 nM against T47D cells. It is very interesting to note that both compounds (78 and 89) showed antiproliferative activity at nanomolar levels. In the 2-fluorophenyl, 4-phenol series (compounds 101-136) (Table 4), with the exceptions of compounds 104, 110, 127, 128, and 136, all compounds inhibited T47D proliferation more than or to extents similar (IC50 = 0.19 to 2.97 µM) as the three positive controls, and compound 111 had the greatest effect (IC50 = 0.19 µM). Moreover, half of the compounds in this series inhibited HeLa proliferation (IC50 = 2.25 µM to 4.94 µM) more than 10 ACS Paragon Plus Environment

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etoposide (IC50 = 5.76 µM). However, in DU145 cell line, only compounds 107, 115, 120, 121, and 123, which contained ortho- or meta-phenolic groups inhibited (IC50 = 0.40 µM to 1.06 µM) proliferation greater than etoposide (IC50 = 1.15 µM). The other compounds showed only moderate anti-proliferative activity. In the case of the HCT15 cell line, only compounds 119 (IC50 = 1.19 µM) and 123 (IC50 = 1.46 µM) exhibited greater anti-proliferative activity than etoposide (IC50 = 1.72 µM). The other compounds inhibited HCT15 less than or at levels similar to the three positive controls. The IC50 values of members of the 2-trifluorophenyl, 4-phenol series (compounds 137 - 172) for inhibitions of the proliferations of HCT15, T47D, DU145, and HeLa cell lines are provided in Table 5. With the exceptions of compounds 157, 158, 167, 168, and 172, all of these compounds inhibited T47D proliferation (IC50 = 0.51 µM to 4.2 µM) more than etoposide (IC50 = 4.26 µM). In HeLa cells, all compounds containing an ortho-phenolic group (137 to 148) displayed stronger anti-proliferative activity (IC50 = 1.99 µM to 7.87 µM) than etoposide (IC50 = 9.75 µM). Similarly, with the exceptions of six compounds (156, 158, 161, 164, 168, and 172), all members of the meta- and para- phenolic series inhibited HeLa cell proliferation more (IC50 = 2.03 µM to 6.66 µM) than etoposide. Likewise, with the exceptions of six compounds (148, 157, 161, 164, 168, and 172) all compounds inhibited DU145 proliferation (IC50 = 1.14 µM to 3.46 µM) more than etoposide (IC50 = 4.49 µM). Finally, in the case of the HCT15 cell line, with the exceptions of compounds (145, 146, 155 - 158, 160, 164, 168, 169, 171, and 172), all compounds inhibited proliferation (IC50 = 1.32 µM to 4.04 µM) more than etoposide (IC50 = 4.06 µM).


Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

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Table 1. Structures, topo I and II inhibitory activities, and IC50 values for compounds 1-28 in the four cancer cell lines. %Inhibition of Compound

R1

R2

Camptothecin

Topo I 100 µM

20 µM

71.6

NT

Etoposide

100 µM

20 µM

76.2

31.1

Adriamycin

R2

N

R1

N

R2

1

-

6’-OH

2

-

7’-OH

3

-

8’-OH

4

-

9’-OH

5

2-OH

6’-OH

6

2-OH

7’-OH

7

2-OH

8

*IC50 (µM)

Topo II HCT15

T47D

DU145

HeLa

1.6±0.03

1.98±0.01

0.81±0.02

1.24±0.02

6.97±0.17

4.02±0.04

1.93±0.03

4.96±0.03

1.78±0.06

0.34±0

1.04±0.01

0.82±0.01

4.6

NT

75.7

1.3

3.39±0.18

1.77±0.03

2.71±0.05

4.18±0.07

2.1

NT

54.2

2.1

3.37±0.03

0.7±0.01

3.82±0.02

4.94±0.1

2.6

NT

79.9

5.5

3.13±0.03

0.19±0.01

3.49±0.02

4.38±0.01

0.0

NT

57.5

0.0

2.07±0.05

0.74±0.01

1.4±0.02

3.08±0.08

0.0

NT

78.5

0.0

3.47±0.21

2.33±0.07

0.8±0.02

6.32±0.02

8’-OH

8.2 0.0

NT NT

74.8 97.4

0.0 0.0

2.08±0.08 3.01±0.04

2.44±0.05 2.33±0.07

1±0.16 0.15±0.01

27.4±0.06 3.74±0.07

2-OH

9’-OH

0.1

NT

95.8

0.1

1.36±0.02

2.44±0.05

0.17±0.03

2.95±0.07

9

3-OH

6’-OH

4.6

NT

95.4

0.0

2.52±0.08

2.89±0.1

0.19±0.02

6.74±0.35

10

3-OH

7’-OH

20.0

NT

96.8

0.0

1.48±0.01

2.66±0.12

0.6±0.01

5.64±0.05

11

3-OH

8’-OH

0.5

NT

1.2

NT

1.37±0.01

0.84±0.01

1.57±0.02

1.38±0.01

12

3-OH

9’-OH

1.6

NT

6.3

NT

1.48±0.03

2.01±0.06

0.72±0.01

2.03±0.08

13

4-OH

6’-OH

4.4

NT

1.5

NT

2.78±0.04

2.89±0.07

1.28±0.03

1.94±0.03

14

4-OH

7’-OH

11.1

NT

2.5

NT

>50

3.55±0.29

2.48±0.01

2.75±0.01

15

4-OH

8’-OH

6.0

NT

2.4

NT

>50

2.99±0.08

1.38±0.01

>50

16

4-OH

9’-OH

17

2-OH

6’-OH

10.2 6.6

NT NT

4.0 1.1

NT NT

>50 >50

>50 6.43±0.06

4.13±0.09 2.49±0.12

>50 1.83±0.01


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R1

N

R3

18

2-OH

7’-OH

19

2-OH

8’-OH

2.0 0.0

NT NT

19.0 2.8

NT NT

>50 >50

>50 >50

1.62±0.02 1.87±0.02

>50 >50

20

2-OH

9’-OH

1.0

NT

1.2

NT

>50

>50

9.61±0.33

>50

21

3-OH

6’-OH

0.4

NT

4.5

NT

1.58±0.05

3.92±0.05

0.52±0.03

6.2±0.02

22

3-OH

7’-OH

10.2

NT

36.0

3.3

1.3±0.03

4.12±0.02

0.27±0.09

5.7±0.02

23

3-OH

8’-OH

0.0

NT

3.5

NT

>50

>50

0.42±0

>50

24

3-OH

9’-OH

0.0

NT

0.0

NT

>50

3.68±0.03

0.43±0.02

>50

25

4-OH

6’-OH

5.3

NT

4.4

NT

1.02±0.05

6.23±0.02

0.37±0.03

5.74±0.24

26

4-OH

7’-OH

8.8

NT

42.8

8.3

1.27±0.04

0.73±0.01

0.71±0.01

2.67±0.12

27

4-OH

8’-OH

20.9

NT

54.8

9.3

1.43±0.04

3.43±0.05

0.57±0.02

2.74±0.09

28

4-OH

9’-OH

1.1

NT

3.7

NT

>50

11.56±0.45

0.66±0.06

2.06±0.02

NT: Not Tested; HCT-15: human colorectal adenocarcinoma; T47D: Human breast ductal carcinoma; DU145 (human prostate tumor cell line; HeLa: human cervix adenocarcinoma cell line; Adriamycin: positive control for antiproliferative activity; Etoposide: positive control for topo IIα and antiproliferative activity; Camptothecin: positive control for topo I and antiproliferative activity. *Results are presented as the means±SDs of three independent experiments performed in triplicate.



Page 14 of 130

Table 2. Structures, topo I and II inhibitory activities, and IC50 values of compounds 29-64 in the four cancer cell lines. %Inhibition Compound

R1

R2

Camptothecin

Topo I 100 µM

20 µM

78.6

38.2

Etoposide

100 µM

20 µM

77.9

38.3

Adriamycin

Cl

R1

N

R2

29

2-OH

6’-OH

30

2-OH

7’-OH

31

2-OH

32

*IC50 (µM)

Topo II HCT15

T47D

DU145

HeLa

0.16±0.001

1.64±0.07

0.05±0.004

1.24±0.02

5.25±0.03

5.54±0.21

4.13±0.46

8.09±0.12

2.02±0.01

1.12±0.07

2.96±0.01

1.89±0.01

23.7

NT

79.2

1.8

4.12±0.08

3.03±0.01

4.03±0.03

4.59±0.04

8’-OH

37.0 59.1

7.4 14.8

55.9 74.1

3.5 38.2

3.13±0.02 7.98±0.1

3.82±0.07 3.28±0.05

4.43±0.008 3.99±0.02

17.89±0.06 >50

2-OH

9’-OH

23.9

NT

56.5

1.6

2.03±0.02

2.93±0.05

5.67±0.02

>50

33

3-OH

6’-OH

28.1

NT

67.5

11.4

8.28±0.09

2.88±0.06

5.63±0.01

7.36±0.03

34

3-OH

7’-OH

77.4

6.3

91.3

20.0

3.82±0.02

2.84±0.29

2.35±0.02

6.48±0.04

35

3-OH

8’-OH

55.9

6.3

93.2

13.8

2.5±0.03

2.91±0.01

2.03±0.01

5±0.01

36

3-OH

9’-OH

79.3

0.2

80.2

3.6

2.43±0.01

2.51±0.04

3.21±0.01

3.64±0.02

37

4-OH

6’-OH

62.9

0

97.0

13.0

2.42±0.01

1.73±0.01

5.84±0.14

3.72±0.01

38

4-OH

7’-OH

11.9

NT

80.0

7.0

4.27±0.04

2.7±0.06

3.25±0.06

4.92±0.11

39

4-OH

8’-OH

4.0

NT

74.8

2.0

2.91±1.46

1.64±0.1

4.65±0.005

6.5±0.01

40

4-OH

9’-OH

41

2-OH

6’-OH

10.3 0

NT NT

66.9 75.4

0.8 1.5

1.05±0.1 2.45±0.03

0.96±0.03 12.03±0.1

0.86±0.002 6.01±0.02

2.79±0.01 20.53±0.95

42

2-OH

7’-OH

43

2-OH

8’-OH

1.6 1.9

NT NT

74.3 67.2

3.4 4.0

>50 29.77±0.44

>50 7.69±0.06

>50 37.14±0.59

>50 19.98±0.24

44

2-OH

9’-OH

0

NT

74.5

2.4

2.46±0.01

1.97±0.06

2.82±0.02

14.34±0.58

45

3-OH

6’-OH

7.4

NT

64.6

2.3

4.79±0.04

33.09±0.02

21.3±0.1

35.49±0.15


Page 15 of 130 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


Cl

R1

N

R2

Cl

R1

N

R2

46

3-OH

7’-OH

47

3-OH

8’-OH

48

3-OH

9’-OH

49

4-OH

6’-OH

50

4-OH

7’-OH

51

4-OH

8’-OH

52

4-OH

9’-OH

53

2-OH

6’-OH

54

2-OH

7’-OH

55

2-OH

56

3.7

NT

83.0

27.0

25.31±0.34

22.63±0.75

6.29±0.04

7.74±0.54

11.6

NT

76.3

3.5

6.6±0.01

7.2±0.03

3.22±0.02

6.24±0.04

8.9

NT

71.9

0.3

>50

>50

17.12±1.33

>50

6.1

NT

75.3

2.2

2.39±0.02

1.92±0.14

1.64±0.02

1.66±0.08

8.8

NT

73.9

3.3

5.69±0.15

1.86±0.01

2.25±0.02

5.94±0.03

8.8

NT

73.6

1.2

7.59±0.05

2.76±0.03

2.1±0.004

8.06±0.02

3.2 8.4

NT NT

71.6 71.6

13.3 1.5

>50 >50

19.03±0.71 >50

6.04±0.055 12.82±0.26

43.78±0.17 >50

8’-OH

21.4 2.9

NT NT

70.8 21.4

0 0

>50 >50

>50 >50

> 50 > 50

>50 >50

2-OH

9’-OH

0

NT

70.5

6.4

18.79±2.23

13.04±0.21

1.95±0.01

>50

57

3-OH

6’-OH

0

NT

86.3

4.4

2.58±0.04

2.11±0.03

3.94±0.004

4.63±0.05

58

3-OH

7’-OH

2.1

NT

74.3

6.2

>50

>50

> 50

>50

59

3-OH

8’-OH

7.3

NT

80.4

12.4

>50

>50

32.18±0.19

>50

60

3-OH

9’-OH

9.3

NT

95.7

8.5

14.67±0.13

2.22±0.01

4.07±0.02

9.01±0.01

61

4-OH

6’-OH

1.3

NT

97

7.5

9.1±0.03

12.75±0.16

1.81±0.12

18.32±0.19

62

4-OH

7’-OH

7.7

NT

98.1

8.9

18.48±0.41

1.51±0.02

1.43±0.01

6.83±0.09

63

4-OH

8’-OH

14.2

NT

95.3

10.8

12.01±0.21

2.67±0.03

1.74±0.01

10.05±0.02

64

4-OH

9’-OH

0

NT

96.8

9.3

>50

>50

> 50

>50

*Results are presented as the means±SDs of three independent experiments performed in triplicate.



Page 16 of 130

Table 3. Structures, topo I and II inhibitory activities, and IC50 values of 65-100 in the four cancer cell lines %Inhibition Compound

R1

R2

Camptothecin

Topo I

*IC50 (µM)

Topo II

100 µM

20 µM

72.5

NT

Etoposide

100 µM

20 µM

75.0

35.6

Adriamycin

OH

R1

N

R2

65

2-Cl

6’-OH

66

2-Cl

7’-OH

67

2-Cl

68

HCT15

T47D

DU145

HeLa

0.35±0.018

0.59±0.008

0.38±0.07

1.39±0.07

1.27±0.007

4.85±0.017

2.19±0.14

4.13±0.012

0.74±0.007

1.5±0.063

1.59±0.06

2.04±0.06

0

NT

82.0

0

0.77±0.001

0.92±0.002

0.83±0.01

0.68±0.001

8’-OH

11.7 0.0

NT NT

70.5 76.8

8.2 5.8

2.34±0.23 4.94±0.06

2.86±0.07 2.84±0.06

1.51±0.04 0.77±0.02

0.84±0.01 6.71±0.13

2-Cl

9’-OH

13.4

NT

80.9

3.7

3.17±0.22

0.87±0.01

0.29±0.01

1.12±0.04

69

3-Cl

6’-OH

0.0

NT

83.5

6.9

7.47±0.07

0.64±0.04

0.56±0.02

>50

70

3-Cl

7’-OH

14.6

NT

80.8

2.6

2.76±0.01

2.77±0.05

0.36±0.01

2.69±0.05

71

3-Cl

8’-OH

22.9

NT

87.9

10.8

1.76±0.03

1.16±0.01

0.32±0.02

2.77±0.05

72

3-Cl

9’-OH

13.1

NT

80.0

3.2

1.31±0.08

0.96±0

0.22±0.02

3.62±0.04

73

4-Cl

6’-OH

14.5

NT

80.3

7.0

2.31±0.14

1.25±0.07

0.61±0.01

1.96±0.01

74

4-Cl

7’-OH

26.8

NT

97.45

4.4

3.97±0.01

0.62±0.03

0.65±0.02

3.84±0.06

75

4-Cl

8’-OH

8.0

NT

86.6

25.7

1.78±0.02

0.72±0.04

0.49±0.03

4.61±0.03

76

4-Cl

9’-OH

77

2-Cl

6’-OH

0.0 0

NT NT

83.9 100.0

25.3 24.9

1.23±0.03 0.8±0.002

2.41±0.06 1.53±0.015

0.55±0.02 0.92±0.01

5.31±0.01 0.59±0.004

78

2-Cl

7’-OH 5.6

NT

100.0

32.3

0.78±0.002

0.81±0.002

0.91±0.03

0.55±0.02

0.57±0.01

>50

0.12±0.01

0.24±0.02

5.25±0.03

0.00133±0.0

79

2-Cl

8’-OH

3.9

NT

88.7

38.3

0023 3.04±0.01

80

2-Cl

9’-OH

6.3

NT

89.6

10.9

3.82±0.08


Page 17 of 130 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


OH

R1

N

R2

OH

R1

N

R2

81

3-Cl

6’-OH

82

3-Cl

7’-OH

83

3-Cl

8’-OH

84

3-Cl

9’-OH

85

4-Cl

6’-OH

86

4-Cl

7’-OH

87

4-Cl

8’-OH

88

4-Cl

9’-OH

89

2-Cl

6’-OH

90

2-Cl

7’-OH

91

2-Cl

92

0.5

NT

100.0

24.1

1.13±0.01

1.52±0.001

1.49±0.02

3.31±0.071

0

NT

80.2

37.8

1.62±0.01

0.86±0.004

3.21±0.037

5.99±0.04

1.5

NT

90.5

14.3

0.84±0.02

1.37±0.02

3.28±0.45

5.44±0.14

16.9

NT

88.6

3.4

2.49±0.02

1.63±0.02

3.48±0.62

4.59±0.03

18.6

NT

76.8

42

1.82±0.001

1.96±0.01

2.03±0.019

4.32±0.005

0

NT

95.0

11.2

0.85±0.002

1.5±0.01

1.28±0.026

1.09±0.02

16.7

NT

87.8

51.9

0.86±0.01

3.5±0.08

4.43±0.28

1.73±0.17

7.4

NT

85.2

26.5

3.05±0.06

0.36±0.01

6.74±0.02

0

NT

100.0

78.9

0.014±0.001

3.5±0.08 0.00267±0.0 0021

0.072±0.001

2.46±0.049

8’-OH

7.6 0

NT NT

86.6 80.9

53.6 19.2

1.85±0.07 3.04±0.08

1.87±0 0.95±0.02

0.43±0.02 0.54±0.03

0.7±0.01 3.22±0.04

2-Cl

9’-OH

0

NT

30.3

3.6

2.97±0.04

0.61±0

0.19±0.01

2.85±0.01

93

3-Cl

6’-OH

1.2

NT

72.8

36.9

1.84±0.01

0.62±0.01

0.22±0.01

3.56±0.14

94

3-Cl

7’-OH

0

NT

100.0

31.9

0.75±0.003

1.2±0.01

1.2±0.006

1.17±0.01

95

3-Cl

8’-OH

8.7

NT

71.3

56.4

2.36±0.05

0.6±0

0.48±0.07

>50

96

3-Cl

9’-OH

0.0

NT

24.4

NT

1.3±0.44

0.83±0

0.03±0.02

6.23±0.03

97

4-Cl

6’-OH

6.7

NT

59.9

0.0

3±0.13

1.15±0.01

1.88±0.01

4.08±0.01

98

4-Cl

7’-OH

0

NT

100.0

50.2

0.77±0.003

1.84±0.004

1.91±0.009

1.02±0.01

99

4-Cl

8’-OH

0

NT

74.5

0.0

3.22±0.06

1.15±0.02

2.25±0.06

1.73±0.03

100

4-Cl

9’-OH

0.2

NT

26.2

NT

>50

>50

2.8±0.11

>50




Page 18 of 130

Table 4. Structures, topo I and II inhibitory activities, and IC50 values for compounds 101-136 in the four cancer cell lines %Inhibition Compound

R1

R2

Camptothecin

Topo I 100 µM

20 µM

67.5

NT

Etoposide

100 µM

20 µM

R1

N

R2

HCT15

T47D

DU145

HeLa

0.049±0.03

0.33±0.02

0.18±0.01

0.74±0.05

1.72±0.11

1.64±0.12

1.15±0.08

5.76±0.55

82.1

30.5

0.94±0.03

0.74±0.07

0.87±0.05

0.8±0.05

3.39±0.18

1.77±0.03

2.71±0.05

4.18±0.07

Adriamycin

OH

*IC50 (µM)

Topo II

101

2-F

6’-OH

0.0

NT

24.1

NT

102

2-F

7’-OH

0.0

NT

13.4

NT

3.37±0.03

0.7±0.01

3.82±0.02

4.94±0.1

103

2-F

8’-OH

0.0

NT

27.0

NT

2.98±0.01

0.61±0.01

4.07±0.02

4.36±0.06

104

2-F

9’-OH

0.0

NT

0.6

NT

3.76±0.07

4.56±0.09

9.9±0.1

7.63±0.17

105

3-F

6’-OH

0.0

NT

80.7

2.35±0.21

1.25±0.02

3.94±0.04

3.92±0.05

106

3-F

7’-OH

0.0

NT

28.0

4.8 NT

4.23±0.21

0.86±0.05

3.13±0.01

2.43±0.05

107

3-F

8’-OH

0.0

NT

28.3

NT

2.9±0.02

0.68±0.01

0.86±0.01

4.22±0.01

108

3-F

9’-OH

0.0

NT

8.4

NT

5.03±0.06

0.74±0.01

9.97±0.17

7.9±0.07

109

4-F

6’-OH

0.0

NT

67.0

0.61±0.01

2.36±0.01

31.95±0.91

4-F

7’-OH

0.0

NT

6.0

0.0 NT

4.31±0.16

110

4.61±0.13

3.14±0.03

4.76±0.1

14.33±0.31

111

4-F

8’-OH

0.0

NT

23.3

NT

3.13±0.03

0.19±0.01

3.49±0.02

4.38±0.01

112

4-F

9’-OH

0.0

34.4

0.0

2.07±0.05

0.74±0.01

1.4±0.02

3.08±0.08

113

2-F

6’-OH

9.7

NT NT

22.4

NT

4.98±0.11

2.71±0.05

2.03±0.03

6.75±0.05

114

2-F

7’-OH

9.1

NT

44.6

2.24±0.08

1.62±0.03

3.98±0.11

2-F

8’-OH

16.3

NT

28.1

4.9 NT

3.58±0.04

115

2.96±0.04

2.97±0.02

0.4±0.02

3.74±0.24

116

2-F

9’-OH

4.0

NT

8.7

NT

8.33±0.32

0.83±0.02

1.88±0.08

12.12±0.02

117

3-F

6’-OH

9.8

NT

43.2

5.6

4.9±0.2

0.89±0.01

2.37±0.04

7.04±0.49


Page 19 of 130 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


OH

R1

N

R2

OH

R1

N

R2

118

3-F

7’-OH

17.5

NT

119

3-F

66.2

0.0

2.71±0.05

0.79±0.01

1.41±0.02

3.85±0.23

8’-OH

17.1

NT

44.2

1.19±0.03

0.54±0.02

2.17±0.01

3.45±0.12

3-F

9’-OH

14.0

NT

17.8

4.9 NT

120

>50

1.16±0.03

0.94±0.01

15±0.2

121

4-F

6’-OH

26.2

NT

27.8

NT

2.68±0.06

1.72±0.04

0.56±0.03

6.71±0.02

122

4-F

7’-OH

22.3

NT

65.1

28.7

1.92±0.03

1.72±0.04

2.94±0.02

3.74±0.05

123

4-F

8’-OH

11.7

NT

48.3

5.4

1.46±0.02

0.68±0.01

1.06±0.05

3.82±0.04

124

4-F

9’-OH

2.3

NT

27.3

NT

4.04±0.04

2.24±0.08

3.3±0.03

8.81±0.04

125

2-F

6’-OH

5.4

NT

15.3

NT

2.81±0.07

0.87±0.01

1.81±0.01

6.19±0.03

126

2-F

7’-OH

12.2

NT

10.6

NT

3.01±0.04

1.39±0.01

1.26±0.03

4.84±0.27

127

2-F

8’-OH

3.3

NT

6.4

NT

>50

29.25±0.88

14.12±0.18

>50

128

2-F

9’-OH

11.7

NT

47.1

>50

47.25±0.68

10.28±0.29

>50

129

3-F

6’-OH

8.6

NT

0.7

1.4 NT

4.75±0.05

0.57±0.01

2.04±0.04

7.3±0.06

130

3-F

7’-OH

8.7

NT

4.5

NT

3.57±0.02

1.34±0.03

1.44±0.03

6.764±0.02

131

3-F

8’-OH

3.0

NT

6.2

NT

4.49±0.2

0.7±0.02

4.83±0.26

8.63±0.03

132

3-F

9’-OH

1.1

NT

67.0

3.9

>50

0.75±0.04

5.78±0.07

7.2±0.53

133

4-F

6’-OH

14.7

NT

50.8

3.6

2.23±0.02

1.37±0.09

1.84±0.01

4.88±0.02

134

4-F

7’-OH

8.0

NT

17.1

NT

4.4±0.05

1.85±0.02

1.59±0.04

3.84±0.07

135

4-F

8’-OH

0.9

NT

47.6

1.4

2.54±0.05

1.14±0.01

2.34±0.03

2.25±0.05

136

4-F

9’-OH

1.2

NT

75.3

1.4

>50

24.48±0.35

>50

>50

*Results are presented as the means±SDs of independent experiments performed in triplicate.



Page 20 of 130

Table 5. Structures, topo I and II inhibitory activities, and IC50 values of compounds 137-172 in the four cancer cell lines %Inhibition Compound

R1

R2

Camptothecin

Topo I 100 µM

20 µM

73.0

NT

Etoposide

100 µM

72.9

20 µM

39.6

Adriamycin

OH

R1

N

R2

*IC50 (µM)

Topo II HCT15

T47D

DU145

HeLa

0.16±0.001

0.25±0.003

0.62±0.02

0.5±0.01

4.06±0.02

4.26±0.03

4.49±0.03

9.75±0.08

0.94±0.003

1.18±0.01

1.04±0.01

1.98±0.01

137

2-CF3

6’-OH

3.5

NT

83.7

1.8

1.74±0.01

3.02±0.02

1.38±0.03

3.95±0.01

138

2-CF3

7’-OH

1.6

NT

90.2

3.7

1.61±0.01

1.14±0.02

2.02±0.02

2.92±0.01

139

2-CF3

8’-OH

0.4

NT

86.1

3.4

4.04±0.004

1.41±0.02

2.86±0.02

2.96±0.02

140

2-CF3

9’-OH

0.0

NT

84.8

4.0

1.64±0.01

0.75±0.001

2±0.26

1.99±0.01

141

3-CF3

6’-OH

0.3

NT

94.2

5.3

3.34±0.01

0.77±0.005

1.87±0.01

4.25±0.03

142

3-CF3

7’-OH

0.0

NT

85.4

4.1

3.89±0.002

0.7±0.002

1.72±0.05

3.1±0.02

143

3-CF3

8’-OH

0.0

NT

98.9

2.8

2.95±0.02

0.51±0.001

1.63±0.02

5.25±0.04

144

3-CF3

9’-OH

2.7

NT

95.8

4.3

2.84±0.01

0.98±0.01

3.38±0.06

5.73±0.04

145

4-CF3

6’-OH

0.0

NT

98.9

2.4

4.54±0.02

1.33±0.01

3.46±0.04

6.14±0.02

146

4-CF3

7’-OH

0.0

NT

99.9

11.9

4.33±0.02

0.83±0.001

2.09±0.01

7.87±0.02

147

4-CF3

8’-OH

0.0

NT

100.0

3.0

3.25±0.07

0.67±0.01

2.8±0.02

2.23±0.01

148

4-CF3

9’-OH

13.6

NT

92.2

0.9

3.49±0.01

4.2±0.03

4.5±0.03

5.24±0.02

149

2-CF3

6’-OH

65.7

0.0

89.5

2.1

3.39±0.03

1.46±0.05

1.8±0.01

3.79±0.05

150

2-CF3

7’-OH

11.7

NT

80.6

2.4

2.82±0.02

1.25±0.01

2.5±0.41

4±0.26

151

2-CF3

8’-OH

1.8

NT

79

3.1

1.67±0.01

1.22±0.002

1.83±0.02

3.71±0.03

152

2-CF3

9’-OH

15.9

NT

65.2

3.3

2.1±0.01

1.75±0.02

1.14±0.01

4.69±0.01

153

3-CF3

6’-OH

5.6

NT

67.3

40.5

2.69±0.001

1.91±0.01

1.62±0.02

4.17±0.02


Page 21 of 130 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


OH

R1

N

R2

OH

R1

N

R2

154

3-CF3

7’-OH

10.2

NT

85.2

29.0

2.22±0.01

1.33±0.01

1.42±0.04

2.03±0.02

155

3-CF3

8’-OH

5.2

NT

90.7

10.5

8.61±0.02

1.33±0.002

1.54±0.01

4.7±0.09

156

3-CF3

9’-OH

0.2

NT

71.6

0.7

14.16±0.02

3.63±0.02

2.77±0.05

>50

157

4-CF3

6’-OH

0.7

NT

10.9

NT

6.98±0.34

6.15±0.05

5.01±0.04

6.3±0.17

158

4-CF3

7’-OH

2.8

NT

100

14.9

6.82±0.08

8.9±0.12

1.59±0.03

11.39±0.06

159

4-CF3

8’-OH

0.0

NT

100

5.1

2.95±0.04

1.8±0.004

3.18±0.04

3.63±0.01

160

4-CF3

9’-OH

0.0

NT

100

0.0

11.27±0.07

2.14±0.01

2.22±0.02

6.66±0.08

161

2-CF3

6’-OH

7.1

NT

30.1

0.6

3.82±0.09

2.66±0.1

5.34±0.03

>50

162

2-CF3

7’-OH

13.9

NT

85.9

0

2.43±0.02

1.05±0.004

2.31±0.01

2.07±0.02

163

2-CF3

8’-OH

6.7

NT

81.0

1.2

1.32±0.01

1.47±0.01

2.78±0.01

1.52±0.004

164

2-CF3

9’-OH

3.0

NT

2.9

NT

>50

1.44±0.01

>50

>50

165

3-CF3

6’-OH

9.4

NT

84.4

0

2.62±0.003

1.23±0.003

1.75±0.02

4.57±0.04

166

3-CF3

7’-OH

55.8

0.3

88.7

4.6

3.47±0.01

1.43±0.01

1.9±0.02

4.52±0.04

167

3-CF3

8’-OH

8.5

NT

87.1

11.1

2.77±0.02

4.69±0.05

2.15±0.02

3.06±0.03

168

3-CF3

9’-OH

2.3

NT

12.9

NT

4.77±0.04

9.57±0.21

44.14±0.2

16.8±0.02

169

4-CF3

6’-OH

32.8

0.0

91.3

11.1

>50

2.01±0.02

2.11±0.02

6.54±1.55

170

4-CF3

7’-OH

87.4

0.0

83.2

1.1

2.63±0.02

1.91±0.01

1.14±0.04

4.8±0.03

171

4-CF3

8’-OH

100

0.0

83.1

0.5

5.03±0.02

1.36±0.01

2.98±0.03

5.51±0.02

172

4-CF3

9’-OH

18.5

NT

82.3

0

>50

>50

13.16±0.1

>50




Page 22 of 130

In Vitro Topo Inhibitory Activities. In view of their potent antiproliferative activities, we investigated topoisomerase I and II inhibitory activities by detecting conversion of supercoiled pBR322 plasmid DNA to its relaxed form in the presence of the synthesized compounds 1-172. Camptothecin and etoposide were used as reference compounds for topo I and II inhibition, respectively. Topo inhibitory activities evaluated at 100 and 20 µM are listed in Tables 1-5 and depicted in Figures 3-7. Interestingly, almost all compounds showed weak topo I inhibitory activity. Only compounds 36, 170, and 171 exhibited greater topo I inhibitory activity than reference compound, camptothecin, whereas compounds 31, 34, 35, 37, 149, and 166 inhibited topo I moderately at 100 µM. As regards of topo II inhibition, only compound 3 (member of the 2,4-diphenyl series) inhibited topo II (79.9%) more than etoposide (76.2%) at 100 µM, but at 20 µM its inhibitory effect was weak (5.5%). The other compounds of this series exhibited moderate topo II inhibition (54.2-75.7%) at 100 µM and negligible inhibition at 20 µM (Figure 3). (A)

(B)

Figure 3. Topoisomerase I and IIα inhibitory activities of the compounds investigated. (A) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane C: pBR322 DNA + topoisomerase I + camptothecin; Lane 1-28: pBR322 DNA + topoisomerase I + compound 1 – 28; (B) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase IIα; Lane E: pBR322 DNA + topoisomerase IIα + etoposide; Lane 1-28 : pBR322 DNA + topoisomerase IIα + compound 1 – 28.


Page 23 of 130 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(A)

(B)

Figure 4. Topoisomerase I and IIα inhibitory activities of the investigated compounds. (A) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane C: pBR322 DNA + topoisomerase I + camptothecin; Lane 29-64: pBR322 DNA + topoisomerase I + compound 29 – 64; (B) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase IIα; Lane E: pBR322 DNA + topoisomerase IIα + etoposide; Lane 29-64 : pBR322 DNA + topoisomerase IIα + compound 29 – 64. In the 2-phenyl, 4-phenol series (5-16), compounds 5 and 7-10 inhibited topo II (78.5-97.4%) significantly more than etoposide at 100 µM. However, with the exception of compound 6, all compounds exhibited little topo II inhibitory activity at 100 µM, and no compound inhibited topo II at 20 µM. Likewise, no compound in the 2-phenol, 4-phenyl series inhibited topo II more than etoposide at 100 and 20 µM (Table 1). However, in the 2phenol, 4-chlorophenyl series (29-64), most of compounds inhibited better or considerable topo II activity than etoposide at 100 µM, whereas at 20 µM none of the compounds in this series inhibited better than etoposide (Figure 4). Of the compounds with a chlorine group at the ortho- and para-positions, a half (29, 34-38, 57, and 59-64) significantly inhibited topo II (79.2-98.1%) at 100 µM. However, only compound 46 with chlorine group at the metaposition inhibited topo II (83.0%) more than etoposide at 100 µM (Table 2). 23 ACS Paragon Plus Environment


(A)

Page 24 of 130

(B)

Figure 5. Topoisomerase I and IIα inhibitory activities of the investigated compounds. (A) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane C: pBR322 DNA + topoisomerase I + camptothecin; Lane 65-100: pBR322 DNA + topoisomerase I + compound 65 – 100; (B) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase IIα; Lane E: pBR322 DNA + topoisomerase IIα + etoposide; Lane 65-100 : pBR322 DNA + topoisomerase IIα + compound 65 – 100. As shown in Figure 5 and Table 3, it was interesting to note the topo II inhibitory results of 2-chlorophenyl, 4-phenol series (65-100). With the exception of compound 66, compounds with a hydroxyl at the ortho- or meta-positions of 4-phenyl ring (65-88) displayed significantly better topo II inhibition (76.8-100%) than etoposide (75.0%) at 100 µM, whereas at 20 µM concentration, only compounds 79, 82, 85, and 87 with a hydroxyl at the meta-position inhibited topo II (37.8-51.9%) more than etoposide (35.6%). In addition, half of the compounds (89-91, 94, 95, and 98) with a para-positioned hydroxyl inhibited topo II (80.9-100%) significantly more than etoposide at 100 µM, and compounds 89, 90, 93, 95, and 98 inhibited topo II (36.9-78.9%) more than etoposide at 20 µM. Among all, compound 89 was the most potent topo II inhibitor (at 100 µM: 100% inhibition; at 20 µM: 78.9% inhibition). 24 ACS Paragon Plus Environment

Page 25 of 130 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In the 2-fluorophenyl, 4-phenol series (101-136), none of the compounds exhibited better topo IIα inhibition than etoposide at 100 and 20 µM (Figure 6, Table 4). Interestingly, with the exception of compounds 152, 153, 156, 157, 161, 164, and 168, all compounds in the 2-trifluoromethyl, 4-phenol series inhibited topo IIα better than etoposide (72.9%) at 100 µM. However, at 20 µM, only 153 inhibited topo IIα (40.5% inhibition) greater than etoposide (Figure 7, Table 5). (A)

Figure 6. Topoisomerase I and IIα inhibitory activities of the investigated compounds. (A) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane C: pBR322 DNA + topoisomerase I + camptothecin; Lane 101-136: pBR322 DNA + topoisomerase I + compound 101 – 136; (B) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase IIα; Lane E: pBR322 DNA + topoisomerase IIα + etoposide; Lane 101-136 : pBR322 DNA + topoisomerase IIα + compound 101 – 136.



(A)

Page 26 of 130

(B)

Figure 7. Topoisomerase I and IIα inhibitory activities of the investigated compounds. (A) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane C: pBR322 DNA + topoisomerase I + camptothecin; Lane 137-172: pBR322 DNA + topoisomerase I + compound 137 – 172; (B) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase IIα; Lane E: pBR322 DNA + topoisomerase IIα + etoposide; Lane 137-172 : pBR322 DNA + topoisomerase IIα + compound 137 – 172.

Structure-Activity Relationship (SAR) Study. One of the major aims of this study was to investigate SARs among the synthesized compounds with respect to their topo inhibitory and antiproliferative activities. This investigation was conducted by exploring the effects of hydroxyls at C-(6, 7, 8, and 9) in the indenopyridine ring, and by assessing the biological effects caused by introducing phenyl, phenol, and halophenyl moieties at the 2- or 4-positions of the indenopyridine ring. Of the compounds containing phenyl and phenol moieties (compounds 1-28), only ortho- or meta-phenol at the 4-position showed selective, strong topo II inhibitory activity. Interestingly. all the compounds showed significant antiproliferative activity in DU145 cell 26 ACS Paragon Plus Environment

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line, suggesting their potential use in human prostate cancer. In addition, when we incorporated a chlorine functionality at 4-phenyl ring, we found that all the compounds in the 2-phenol, 4-chlorophenyl series (compounds 29-64) exhibited moderate to strong topo II inhibition. SAR study revealed a meta- or para- phenol group at position 2, and ortho- or para-chlorophenyl group at position 4, resulted in stronger topo II inhibition at 100 µM. Comparatively, compounds with ortho-chlorophenyl exhibited stronger antiproliferative activity in HCT15, T47D and DU145 cells. Interestingly, interconversion of chlorine and hydroxyl group at 2- and 4- phenyl ring respectively, improves potency and selectivity towards topo II enzyme. Moreover, most of the meta- and para-phenolic group at 4position showed potent topo II inhibitory activity at 20 μM. With few exceptions, interconversion results in remarkable improvement in antiproliferative activity in all the tested cancer cell lines (HCT15, T47D, DU145 and HeLa). Noticeably, compounds 78 and 89 exhibited strongest antiproliferative activity in HCT15 and T47D, respectively in nanomolar range. In case of the 2-fluorophenyl, 4-phenolic series almost all compounds (101 to 136) with a fluorine at 2-phenyl ring showed weak topo I and IIα inhibitory activities. This result indicates that unlike chlorine functionality, fluorine substitution at 2-phenyl ring does not favor topo IIα inhibition. However, compounds showed significant anti-proliferative activity in T47D and DU145 cell lines. Interestingly, replacement of fluorine group by trifluoromethyl group in the 2-phenyl ring enhanced topo IIα inhibitory activity at 100 μM. SAR study revealed comparable antiproliferative activity of 2-trifluoromethylphenyl, 4phenolic series with 2-fluorophenyl, 4-phenolic series. In summary, from SAR study we observed that most of the compounds with a chlorine group at 2-phenyl ring and a meta- and para-hydroxyl group in the 4-phenyl ring of indenopyridinol (78, 79, 82, 87, 89, 90, 93, 94, 95 and 98) displayed strong and potent topo II inhibitory activity at 20 μM. These results highlight the potencies and significance of 227 ACS Paragon Plus Environment


Page 28 of 130

chlorophenyl, 4-phenolic series with regards to position and functional groups as compared to 2-phenolic, 4-chlorophenyl series and other compounds containing phenyl, fluorine and trifluoromethyl groups (Figure 8). Observed increases in antiproliferative activity achieved by the substitution of chlorophenyl and phenolic moieties in the 2- and 4- positions with a hydroxyl group at the C-(6, 7, 8, and 9) positions of the indenopyridine ring encouraged us to investigate the mechanisms responsible. Compounds 78 and 89 potently inhibited the proliferations of HCT15 and T47D cells at nanomolar levels (IC50 = 1.33 and 2.67 nM, respectively). However, compound 89 was selected for the mechanistic study because it most potently inhibited topo II (100% inhibition at 100 µM and 78.9% at 20 µM), significantly inhibited T47D proliferation (IC50= 2.67 nM), and was cytotoxic to several breast cancer cell lines (Table 6).

(A) HO

HO

HO

N

F

F3C

OH

N

>

>

50 39.42±4.74 >50 Camptothecin 23.98±2.27 31.86±4.66 0.044±0.001 Compound 89 2.7±0.01 2.64±0.03 2.083±0.015 *Results are presented as the means±SDs of three independent experiments performed in triplicate.



Page 30 of 130

Compound 89 acted as a non-intercalative topo II poison. We used ethidium bromide (EtBr; a fluorophore and well-known DNA intercalator) to confirm the DNA intercalative property of compound 89. In the EtBr displacement assay, the ability of 89 to displace EtBr was determined by measuring EtBr fluorescence intensity and this was compared with that of amsacrine (m-AMSA), a well-known DNA intercalative topo II poison.28 We observed m-AMSA dose-dependently quenched the fluorescence generated by EtBr-ctDNA complex, whereas compound 89 did not, which revealed 89 to be a DNA nonintercalator (Figure 9). In addition, a cleavable complex assay and band depletion assay were performed to determine whether compound 89 acted as a topo catalytic inhibitor or poison using etoposide as a reference compound. As shown in Figure 10A, generation of a linear DNA band (indicated by an arrow) by etoposide (a topo II poison) appeared to be due to its ability to stabilize the DNA-topo cleavable complex. Similarly, the appearance of faint linear DNA band when treated with a high concentration (500 µM) of compound 89 also indicated 89 acted as a topo II poison, albeit a weaker poison than etoposide. Likewise, in the band depletion assay, depletion of free topo IIα in the presence of etoposide only and in combination with compound 89 was in-line with its ability to form a topo II cleavable complex with DNA. However, when treated with compound 89 alone free topo IIα was observed, which showed 89 was a weaker topo IIα poison than etoposide (Figure 10B). Thus, both assays indicated compound 89 acts as a topo II poison and that its ability to stabilize topo II-DNA transient cleavable complex is less than that of etoposide.


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Figure 9. Competitive EtBr displacement assay of compound 89. The intercalative effect of 89 was assessed by fluorescence spectroscopy. EtBr-ctDNA fluorescence was measure using emission and excitation wavelengths of 510-700 nm and 471 nm, respectively, in the presence of m-AMSA and compound 89 (0-40 μM).

(A)

(B)

Figure 10. Detection of topo II-DNA cleavable complex formation, which was used to determine the mode of action of compound 89. (A) Cleavable complex assay: pBR322 DNA (250 ng) was incubated with etoposide (100 μM) and compound 89 (at 100 or 500 μM). Compound-induced truncated DNA was detected on 1% agarose gel containing EtBr (0.5 μg/ml) under UV light. Arrows indicate truncated DNA (linear form). (B) Band depletion assay: Topo II-DNA cleavable complex formation was estimated using a band depletion assay. T47D cells were treated with etoposide (50 μM) and compound 89 (50 μM) for 2 hours. Topo II trapped in DNA complex was freed by alkaline lysis and detected by immunoblotting with anti-topo II antibody. 31 ACS Paragon Plus Environment


Page 32 of 130

Compound 89 induced less DNA damage than etoposide. Stabilization of DNA-topo cleavable complex by a topo II poison has been reported to lead to the prevention of relegation that induce DNA damage resulting several toxicity.29, 30 To examine the effect of compound 89 on DNA damage, an alkaline comet assay was performed using etoposide as a reference compound. As shown in Figure 11A, formation of a comet tail after treating with etoposide (at 5 or 10 µM) revealed the presence of severe DNA damage, whereas treatment with compound 89 produced significantly less DNA damage, indicating compound 89 is probably safer than etoposide.

(A)

(B)

Figure 11. Evaluation of compound-induced DNA damage in T47D cells. (A) Alkaline comet assay. Comet slides were electrophoresed (20 min, 15 V) at high pH (pH >13) and stained with a SYBR gold solution. The comet tails were observed by fluorescence microscopy. (B) Quantification DNA tails. Comet tail generation was assessed using KometTM 5.0 software. Intensities of comet tails versus comet heads reflected the extent of DNA breaks. (n=30) (***, P < 0.001). 32 ACS Paragon Plus Environment

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Compound 89 induced T47D apoptosis via PARP cleavage and expressions of bax and bcl-2. The apoptotic effect of compound 89 was determined by Western blot by monitoring the expressions of the apoptotic protein markers poly ADP-ribose polymerase (PARP), bax, and bcl-2.31 As shown in Figure 12, treatment of T47D cells with compound 89 at concentrations of 10 and 30 µM, increased the expressions of cleaved PARP and bax, and decreased the level of bcl-2 protein demonstrating 89 induced apoptosis.

Figure 12. Treatment with compound 89 induced T47D cell apoptosis. T47D cells were treated with compound 89 at different concentrations for 24 hours. PARP, cleaved PARP, bax, and bcl-2 levels in cell lysates were assessed by western blotting.



Page 34 of 130

In vivo ADME profiles of compound 89. Based on its promising in vitro results, we investigated the pharmacokinetic profile of compound 89 using an LC-MS/MS method. As shown in Figure 13A, the parent ion of compound 89 was detected in positive ion mode [M+H]+ at m/z 386.04 and a product ion was detected at m/z 189.2. Representative LCMS/MS chromatograms of plasma blank, compound 89 from in vitro plasma stability test and telmisartan, the internal standard are shown in Figure 13B.

A

B

a

b

c

Figure 13. Mass spectrum of compound 89 (A) representative LC-MS/MS chromatogram (B) of the plasma blank (a), compound 89 from plasma stability test at 30 min (b), and telmisartan the internal standard (c).


Page 35 of 130 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The plasma stability of compound 89 was assessed in vitro (Table 7). A loss of more than 25% of 89 within 60 min was used as a cut-off. The concentration of 89 remained unchanged after incubation in plasma for 4 h, indicating adequate stability.

Table 7. Results of plasma stability tests on compound 89. Plasma Stability (%)

Time (hr)

Mice

Rats

Human

0

100.0 ± 0.0

100.0 ± 0.0

100.0 ± 0.0

0.25

108.7 ± 23.4

102.1 ± 7.9

98.7 ± 2.9

0.5

117.1 ± 18.2

99.8 ± 13.3

99.3 ± 1.6

1

110.2 ± 15.2

111.0 ± 16.7

104.4 ± 11.8

2

116.3 ± 13.0

119.6 ± 21.6

100.4 ± 5.7

4

130.8 ± 23.3

130.4 ± 24.7

105.2 ± 1.9

The results indicate the mean percentages of test substances remaining ± SDs for triplicate determinations.

We also conducted a plasma protein binding study using a micro-centrifuge unit to assess the available amount of free compound 89 in plasma. As shown in Table 8, unbound fractions of compound 89 in plasma samples of mice, rats, and humans were high (99.9%, 100%, and 95.9%, respectively), indicating that compound 89 should be able to induce its pharmacological effects. Table 8. Plasma protein binding of compound 89 Percentage of compound 89 not bound by plasma proteins

Time (hr) Mice

Rats

Human

0

100.0 ± 0.0

100.0 ± 0.0

100.0 ± 0.0

1

99.9 ± 0.0

100.0 ± 0.0

95.9 ± 3.7

Results are presented as the means±SDs of triplicate determinations.



In addition, a microsomal stability test was conducted to observe the rate of disappearance of compound 89 following incubation with various microsomes for different times. A half-life cut-off of 20 min was used for all microsomes tested. Compound 89 was found to have a half-life of more than 20 min for all microsomes tested (Figure 14). The halflives of compound 89 in the presence of mouse, rat, and human liver microsomes were 156.4 ± 23 min, 1560.2 ± 156.8 min, and 392.0 ± 64.4 min, respectively. These results indicate compound 89 was stable and that its concentration in the systemic circulation is likely to be high enough for it to be pharmacologically effective.

120

Microsomal stability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 130

100 80 60 ICR mice

40

SD rats 20

Human

0 0

5

10

15

30

Time (minutes)

45

60

120

Figure 14. Microsomal stability test results conducted on compound 89. Compound 89 was incubated with liver microsomes prepared from mice, rats, or human for different times and its rate of disappearance was determined at each time point. Results are presented as the mean percentages of compound 89 remaining ± SDs for triplicate determinations.


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Evaluation of the single-dose toxicity of compound 89. Compound 89 was intravenously administered to mice to examine its toxicity profile and in vivo efficacy. Briefly, male and female ICR mice were administered 89 once intravenously at 50 or 100 mg/kg and mortalities and changes in physical condition were noted. No lethality or any physical abnormality was observed over a 7-day observation period (Table 9). Furthermore, body weights were similar as compared with those of controls. The results suggest single intravenous treatment of mice with up to 100 mg/kg of compound 89 is probably safe.

Table 9. Mortalities and body weight changes following a single intravenous injection of compound 89 into ICR mice with. Male

Female

Dose, BW

BW

mg/kg

BW

BW

(Day 0)

(Day 7)

Mortality

Mortality

(Day 0)

(Day 7)

0

31.8 ± 0.4

33.0 ± 1.0

0/5

25.3 ± 0.7

25.7 ± 1.0

0/5

50

32.9 ± 0.5

34.3 ± 0.8

0/5

27.0 ± 0.5

27.0 ± 0.8

0/5

100

34.8 ± 0.5

36.3 ± 1.3

0/5

28.8 ± 0.6

26.7 ± 0.7

0/5

Intravenous administration of compound 89 retarded orthotopic MDA-MB231 tumor growth. We attempted to determine whether compound 89 has an ability to induce the antitumor effect in orthotopic breast cancer model. Human breast cancer MDA-MB231 expressing firefly luciferase (MDA-MB231-Luc) as an optical reporter was used to conduct in vivo study. The MDA-MB231 is an aggressive, triple-negative breast cancer cell line that is resistant to hormone therapies. Thus, many attempts have been tried to develop novel agents for treatment of triple negative breast cancer. After tumor challenge, compound 89 was intravenously administered to tumor bearing mice once daily for 10 days and tumor progression was monitored using bioluminescent imaging (Figure 15A). In vivo 37 ACS Paragon Plus Environment


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bioluminescent imaging revealed that treatment with compound 89 significantly retarded tumor growth as compared with vehicle-treated controls (Figure 15B and C). Furthermore, in consistent with in vivo results, tumor weights were lower in the compound 89-treated group than in vehicle-treated controls (Figure 16A and B). These results suggested that compound 89 has the potential to treat breast cancer.

Figure 15. Treatment with compound 89 had antitumor effects in our murine orthotopic MDA-MB231-Luc breast cancer model. (A) Schematic of in vivo therapy. Orthotopic breast cancer models were challenged to mammary fat pad of mice and then compound 89 was treated i.v. into tumor bearing mice once daily for 10 days. To monitor the antitumor effects of compound 89, bioluminescent imaging was conducted at the indicated times. (B) In vivo bioluminescent imaging and tumor progression. (C) Quantification of bioluminescent signals from tumor lesions. The in vivo study was performed in duplicate on 10 mice per group. *, P

Discovery and Biological Evaluations of Halogenated 2,4-Diphenyl

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