Synthesis and Characterization of 4,11-Diaminoanthra[2,3-b]furan-5

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Synthesis and characterization of 4,11-diaminoanthra[2,3-b]furan-5,10-diones: tumor cell apoptosis through tNOX-modulated NAD+/NADH ratio and SIRT1 Alexander S. Tikhomirov, Andrey E. Shchekotikhin, Yi-Hui Lee, Yi-Ann Chen, Chia-An Yeh, Victor V. Tatarskiy, Lyubov G. Dezhenkova, Valeria A. Glazunova, Jan Balzarini, Alexander A. Shtil, Maria N. Preobrazhenskaya, and Pin Ju Chueh J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00859 • Publication Date (Web): 03 Dec 2015 Downloaded from http://pubs.acs.org on December 5, 2015

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Journal of Medicinal Chemistry

For: Journal of Medicinal Chemistry

Synthesis and characterization of 4,11-diaminoanthra[2,3-b]furan-5,10-diones: tumor cell apoptosis through tNOX-modulated NAD+/NADH ratio and SIRT1

Alexander S. Tikhomirov a,b, Andrey E. Shchekotikhin a,b,*, Yi-Hui Lee c, Yi-Ann Chen c, Chia-An Yehc, Victor V. Tatarskiy Jr.d, Lyubov G. Dezhenkova a, Valeria A. Glazunova d, Jan Balzarini e, Alexander A. Shtil d,f, Maria N. Preobrazhenskaya a, and Pin Ju Chueh c,g,h,i,*

a

Gause Institute of New Antibiotics, 11B. Pirogovskaya Street, Moscow 119021, Russia

b

Mendeleyev University of Chemical Technology, 9 Miusskaya Square, Moscow 125190, Russia

c

Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan

d

Blokhin Cancer Center, 24 Kashirskoye shosse, Moscow 115478, Russia

e

Rega Institute for Medical Research, K.U. Leuven, 3000 Leuven, Belgium

f

National University of Science and Technology “MISIS”, 4 Leninsky Avenue, Moscow 119991,

Russia g

Graduate Institute of Basic Medicine, China Medical University, Taichung 40402, Taiwan

h

Department of Medical Research, China Medical University Hospital, Taichung 40402, Taiwan

i

Department of Biotechnology, Asia University, Taichung 41354, Taiwan

* Corresponding authors. Andrey E. Shchekotikhin: [email protected] Pin Ju Chueh: [email protected]

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Abstract A series of new 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives with different side chains were synthesized. Selected 2-unsubstituted derivatives 11-14 showed high antiproliferative potency on a panel of mammalian tumor cell lines including multidrug resistance variants. Compounds 11-14 utilized multiple mechanisms of cytotoxicity including inhibition of Top1/Top2mediated DNA relaxation, reduced NAD+/NADH ratio through tNOX inhibition, suppression of a NAD+-dependent Sirtuin 1 (SIRT1) deacetylase activity and activation of caspase-mediated apoptosis. Here, for the first time, we report that tumor-associated NADH oxidase (tNOX) and SIRT1 as important cellular targets of antitumor anthracene-9,10-diones.


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1.

Introduction

Derivatives of anthracene-9,10-dione represent a promising class of compounds as novel drug candidates with antitumor activity. Doxorubicin (Dox) (1, Fig. 1) is a semi-synthetic antitumor antibiotic bearing an anthracene-9,10-dione moiety. This drug has been successfully used in a wide range of cancer therapies for decades.1 The molecular mechanism of antitumor activity of anthracyclines is related to their ability to intercalate into the DNA duplex, leading to inhibition of topoisomerase 2 (Top2), blocking of replication and activation of apoptosis.2,3 Despite the high efficacy, the clinical use of these drugs can be limited by cardiotoxicity associated with generation of reactive oxygen species (ROS).4,5 Another prominent example is mitoxantrone (2, Fig. 1), which exhibited a high antitumor activity in patients with hematological and solid malignancies.6 Like anthracyclines, mitoxantrone is a potent DNA intercalator and Top2 poison.7 Reduced potential of diaminoanthracene-9,10-diones to generate ROS compared to doxorubicin may explain their less pronounced heart toxicity.8 Still, the efficacy of these drugs and anthracyclines is often hampered by the emergence of multidrug resistance (MDR).5 Thus, the search for new drug candidates with improved pharmacological properties based on the anthracene-9,10-dione core remains important.

Figure 1. Anticancer drugs with anthracene-9,10-dione moiety. The arene/hetarene-fused derivatives of anthracene-9,10-dione have a high potential for development as antitumor agents.9-20 Saintopin (3, Fig. 2A), a tetracyclic antibiotic, is a dual inhibitor of Top1/2 which blocks tumor cell growth (IC50 = 1 µM for HeLa cell line).11 Related topopyrones (e.g., 4, Fig. 2A) are also the derivatives of hydroxyanthraquinone fused with 1,43 ACS Paragon Plus Environment


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pyrone ring.12 These antibiotics have shown perspective antiproliferative (IC50 = 0.5-4.6 µM) and antiviral activities.13 In addition to being topoisomerase 1 (Top1) poisons, the topopyrones target and stabilize Top2-DNA complexes. Among the synthetic derivatives of linear pyrroloquinizarine the dual Top1/2 inhibitors have been identified with promising antitumor potency in cell culture and

in vitro (compound 5, Fig. 2A, IC50 = 0.07 µM for L1210 cell line) and the ability to circumvent MDR.14 Angular imidazole-fused anthracene-9,10-dione derivatives (compound 6, Fig. 2A) demonstrated a potent telomerase inhibition and hTERT repression activities with IC50 = 6.5 µM against PC-3 human prostate cancer cells.15 Among

heteroarene-fused

anthracenediones,

1,4-diaminoanthraquinone

is

especially

promising scaffold for the search of novel anticancer agents. Indeed, the 1,4-diaminoanthraquinone derivatives showed a good antiproliferative activity and, in contrary to hydroxyanthraquinones, did not generate ROS.8,16 Studies of structure-activity relationship (SAR) of heteroarene-fused derivatives

of

1,4-diaminoanthracenedione

with

one

heteroatom

revealed

that

linear

furanoanthraquinones (anthra[2,3-b]furan-5,10-diones) were more active than thiophene or pyrrole analogs.17-19 Derivatives of 4,11-diaminoanthra[2,3-b]furan-5,10-dione (7, Fig. 2B) potently inhibited Top1 and blocked the growth of pleiotropically drug resistant tumor cells. In addition, guanidination of the side chains of heteroarene-fused anthracenediones (8, 9, Fig. 1) increased their affinity to G-quadruplexes, thereby resulting in telomerase attenuation and down-regulation of transcription of several oncogenes.20 We have found that the substituents at the position 2 of the heterocyclic core of 4,11diaminoanthra[2,3-b]thiophene-5,10-diones

and

4,11-diaminoanthra[2,3-b]furan-5,10-diones

strongly influenced the antiproliferative and Top1 inhibitory potencies.18,19 In the present study we reported the synthesis and antitumor properties of a series of novel 4,11-diaminoanthra[2,3-b]furan5,10-diones and analyze SAR for this chemotype. In particular, we focused on the identification of new intracellular targets and molecular mechanisms of tumor cell death induced by anthra[2,3-

b]furan-5,10-diones. 4 ACS Paragon Plus Environment

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Figure 2. Potent antitumor arene/hetarene-fused analogs of anthracene-9,10-dione (A), linear 4,11diamino derivatives of heteroarene-fused anthracenediones (B). 2.

Chemistry

The alkoxy groups in the peri-positions of hetareneanthracenediones have been shown to be activated for nucleophilic substitution due to the electron-withdrawal effect of the quinone moiety.19 We took advantage of this property for the synthesis of the corresponding derivatives of 4,11diaminoanthra[2,3-b]furan-5,10-dione. For the introduction of the amino groups, we used 4,11dimethoxyanthra[2,3-b]furan-5,10-dione (10), which can be obtained via several alternative routes.21,22 Substitution of methoxy groups in 10 by treatment with ethylenediamine and its derivatives in THF at 50 °C yielded a series of furan-fused analogs 11-15 of the anticancer agent ametantrone with different side chains (Scheme 1).

Scheme 1a. Synthesis of 4,11-diaminoanthra[2,3-b]furan-5,10-diones 11-15.

a

Reagents and conditions: (a) diaminoalkanes, THF, 50 ºC, 1.5-2 h, yield 60-75%. 5 ACS Paragon Plus Environment


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To reveal the role of substituting groups at the position 2 of 4,11-diaminoanthra[2,3-b]furan5,10-diones, we synthesized the derivatives of 5,10-dioxoanthra[2,3-b]furan-2-carboxamide. 4,11Dimethoxy-5,10-dioxoanthra[2,3-b]furan-2-carboxylic acid (16)22 was transformed into the corresponding amides 17, 18 by treatment with N-methylpiperazine or Boc-ethylenediamine in the presence of PyBOP in DMSO (Scheme 2). The reaction with ethylenediamine in THF at 50 °C converted the amides 17, 18 into the 4,11-bis(2-aminoethylamino)derivatives 19, 20 (Scheme 2).

Scheme 2a. Synthesis of 4,11-diaminoanthra[2,3-b]furan-5,10-diones 19, 20 with an additional side chain in the furan core.

a

Reagents and conditions: (a) N-methylpiperazine, PyBOP, DMSO, rt, 1 h, yield 86%; (b) N-Boc-

ethylenediamine, PyBOP, DMSO, rt, 1 h, then DCM, HCl in MeOH, 2 h, yield 82%; (c) ethylendiamine, THF, 50 ºC, 1.5-2 h, yield 69-72%.

Anthra[2,3-b]thiophen-5,10-diones with the side chains containing guanidine groups have a high affinity to G-quadruplexes.18,20 Therefore, 11 and 19 were transformed into bisguanidines 21 and 22 by guanidination of amino groups in the side chains with pyrazole-1-carboxamidine (Scheme 3). New 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives 11-15 and 19-22 were evaluated for their antiproliferative activities and inhibition of intracellular enzymes crucial for tumor cell growth. 6 ACS Paragon Plus Environment

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Scheme 3a. Synthesis of bis(guanidino) derivatives 21, 22.

a

Reagents and conditions: (a) 1H-Pyrazole-1-carboxamidine hydrochloride, DIPEA, DMSO, 60 ºC,

5 h, yield 62-65%.

3.

Biological testing

3.1. Antiproliferative activity New anthra[2,3-b]furan-5,10-diones were tested against several mammalian tumor cell lines, such as murine leukemia L1210, human T-cell leukemia CEM, cervical carcinoma HeLa, and colon carcinoma HCT116. The antiproliferative activities of 11-15, 19-22, the reference compound 7, and Dox are summarized in Table 1. Compounds 11-15 demonstrated high antiproliferative potencies, close to that of the 2-methyl derivative 7 (Table 1). The derivative 12 bearing Nmethylethylenediamine side chains was the most potent in the series, even stronger than Dox for L1210 cell line. These data correlated with the antiproliferative activities of previously described 2methyl analogs.19 Introduction of 2-carboxamide group containing an additional basic center (compounds 19, 20 and 22) significantly decreased the antiproliferative activity compared to the 2unsubstituted compounds 11-15 and 21 or their 2-methyl analog 7 (Table 1). Transforming the terminal amino groups in the side chains into guanidines also decreased the cytotoxicity of 21 and 22.



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Table 1. Antiproliferative activities (IC50, µM) of anthra[2,3-b]furan-5,10-diones 11-15, 19-22 and reference compounds 7 and Dox for L1210, CEM, HeLa and HCT116 cell lines determined by MTT assay after 72 h incubation.

a

Compound 11

L1210 0.30 ± 0.11a

CEM 0.75 ± 0.01

HeLa 0.55 ± 0.21

HCT116 0.11± 0.07

12

0.10 ± 0.02

0.19 ± 0.06

0.17 ± 0.01

0.10± 0.09

13

0.23 ± 0.09

0.34 ± 0.17

0.39 ± 0.33

0.15± 0.01

14

0.14 ± 0.02

0.43 ± 0.04

0.46 ± 0.22

0.22± 0.08

15

0.73 ± 0.05

0.89 ± 0.02

0.87 ± 0.29

0.41± 0.16

19

26.12 ± 2.20

25.04 ± 3.31

61.30 ± 4.21

3.10± 0.50

20

26.41 ± 6.12

105.00 ± 25.19

83.75 ± 3.23

6.52± 0.71

21

5.54 ± 1.21

9.02 ± 2.80

3.27 ± 0.23

1.02± 0.09

22

79.32 ± 4.13

97.72 ± 12.12

38.37 ± 12.19

4.50± 0.90

7

0.15 ± 0.01

0.15 ± 0.02

0.19 ± 0.02

1.60± 0.04

Dox

0.37 ± 0.07

0.06± 0.01

0.20± 0.02

0.11 ± 0.03

Mean ± S.D. of 3 independent experiments.

Alternatively, growth profiles of AGS human gastric cancer cells were determined, with or without exposure to anthrafuranediones, by measuring cell impedance and displaying the values of cell index (CI).23,24 We showed that growth of AGS cells was inhibited by anthra[2,3-b]furan-5,10diones in a concentration- and time-dependent manner (Supporting Information, Fig. S1). Consistent with the results of MTT assays, 11-14 demonstrated higher antiproliferative activities than other compounds toward AGS cells. Interestingly, we also observed the effect of 12 on noncancerous lines and found that 12 exhibited less inhibition on cell growth (Supporting Information, Fig. S2) supported by the higher IC50 values of 0.78 µM for MRC-5 and 0.23 µM for NIH3T3 cells compared to the values of all cancer cell lines (Table 1). We next investigated the ability of 4,11-diaminoanthra[2,3-b]furan-5,10-diones to overcome MDR, a major obstacle to the therapeutic effectiveness of Dox and mitoxantrone.5 MTT assays were conducted in human myeloid leukemia cell line K562 and its isogenic P-glycoprotein (Pgp)8 ACS Paragon Plus Environment

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positive MDR subline K562/4 selected for resistance to Dox. Compounds 11-14 inhibited the proliferation of K562 cells at low micromolar concentrations. In striking contrast to Dox, compounds 11-13 exhibited similar potencies against K562 cell line and MDR K562/4 subline (Table 2). Indeed, a specific structure of the side chain is critical for overcoming Pgp-mediated MDR since compound 14 with its ethanolamino groups was less potent towards K562/4 subline than 11-13 with amino, methylamino and dimethylamino groups, respectively (Table 2). Thus, the resistance index (RI) for the ethanolamino derivative 14 was two orders of magnitude greater than that for 11-13. These results strongly suggested the potential of 11-13 in circumventing Pgpmediated MDR.

Table 2. Antiproliferative activities (IC50, µM) of 11-14, 19, 21, 22, reference compounds 7 and Dox for K562 cell line and K562/4 subline determined by MTT assay after 72 h incubation.

a

Compd 11

K562 0.11 ± 0.04a

K562/4 0.15 ± 0.05

RIb 1.36

12

0.21 ± 0.06

0.15 ± 0.02

0.71

13

0.12± 0.03

0.21 ± 0.06

1.75

14

0.11 ± 0.08

9.80 ± 2.01

89.09

19

25.17 ± 2.07

>100

>4

21

1.65 ± 0.54

6.53± 1.71

3.98

22

49.46 ± 4.21

>100

>2

7

0.12 ± 0.04

4.26 ± 0.33

35.50

Dox

0.12 ± 0.03

9.75 ± 0.92

81.25

b

Mean ± S.D. of 3 independent experiments. RI, resistance index = IC50(K562/4)/IC50(K562).

Compounds 12 and 14 exhibited strong antiproliferative activities against K562 cell line, whereas the 2-(hydroxyethyl)aminoethyl derivative 14 was 100-fold less active against the MDR subline K562/4 (Table 2). To explain this discrepancy, we investigated the ability of anthra[2,3b]furan-5,10-diones to be effluxed from the Pgp positive K562/4 cells. Flow cytometry analyses were performed after 2 h of incubation of K562 and K562/4 cells with 3 µM 12, 14 or Dox. 9 ACS Paragon Plus Environment


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Verapamil (Vp) was used as a blocker of Pgp mediated transport.25,26 As expected, K562/4 cells effluxed Dox; this effect was reversible by Vp (Fig. 3). In contrast, the accumulation of 12 in K562 cells was only slightly bigger than in Pgp-positive K562/4 cells. However, the accumulation of the 2-(hydroxyethyl)aminoethyl derivative 14 in K562/4 cells was significantly lower than in K562 cells. Thus, the antiproliferative potency of 12 and 14 against K562/4 cells (Table 2) was paralleled by the differential ability of these compounds to accumulate in Pgp-positive cells. One can conclude that anthra[2,3-b]furan-5,10-diones bearing 2-(hydroxyethyl)amino groups in their side chains are preferentially effluxed by Pgp compared with 12 containing the terminal methylamino groups.

Figure 3. Accumulation of 12, 14, and Dox in K562 and K562/4 cells after 2 h incubation. Cells were treated with 3 mM of 12, 14, and Dox. Shown are ratios (Au, arbitrary units) of mean fluorescence channels of respective histograms in K562 and K562/4 cells. Verapamil (Vp, 10 µM) was added 30 min. prior to the addition of other compounds. Data are mean ± S. E. obtained in 3 independent experiments.

3.2. Inhibition of Top1 and Top2 As mentioned above, intercalation into the DNA and the formation of ternary complexes between the drug, DNA and topoisomerases are the key prerequisites of the antitumor activity of anthraquinone-based drugs 1 and 2.27,28 The heterocyclic derivatives of anthraquinone bind to DNA 10 ACS Paragon Plus Environment

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and inhibit Top1 and Top2.14-19 Hence, we investigated our new 4,11-diaminoanthra[2,3-b]furan5,10-diones for the affinity to DNA and the ability to modulate Top1 and Top2 activity in vitro. Binding to DNA of the selected derivative 12 was measured by spectrophotometric titration. The increase of double stranded DNA concentration led to a decrease of extinction of absorption maximums of 12 at 540 and 585 nm accompanied with a 20 nm bathochromic shift (see Supporting Information, Fig. S3A). The observed spectral changes were similar for 229 and reflect the formation of drug-DNA complexes in which the ligand’s chromophore intercalated between DNA base pairs. With the binding isotherm generated in Scatchard coordinates (see Supporting Information, Fig. S3B) and the McGhee-von Hippel equation,30 we calculated major parameters of binding of 12 to DNA, that is, the binding constant (Ka=2.5 ± 0.2×106 M-1) and the number of base pairs covered by one molecule of 12 (L=3.0 ± 0.1 bp). Thus, the binding constant (Ka) of new anthrafurandione 12 was somewhat higher than the respective values for the conventional antitumor drugs 1 and 2 (Ka=2.0 and 1.0 ×106 M-1, respectively).29,31 Next, we compared the inhibition of Top1-mediated relaxation of supercoiled DNA by 12, 19, 21, 22 with previously developed Top1 inhibitors 8, 918,19 and camptothecin (Cpt).32 Fig. 4 shows that anthrafurandiones (0.5-10 µМ) exhibited differential ability to attenuate Top1. The 2unsubstituted compounds 12 and 21, at 2.5 µМ, completely blocked Top1-mediated relaxation, similarly to 8 and 9. The carboxamide group at the position 2 of the heterocycle (compound 19) significantly reduced the enzyme inhibition in comparison with the 2-unsubstituted analog 12. Guanidination of the terminal amino groups of the side chains of anthra[2,3-b]furan-5,10-diones notably increased the efficacy of Top1-mediated plasmid relaxation. The bisguanidine 21 was somewhat more active than the amino analog 12 at 0.5 µМ than carboxamide 22 (contains two guanidine groups) or its parent compound 19 at 2.5 µМ. The Top1 inhibitory potency of the furanfused 21 was similar to that of the thiophene analog 9, indicating that the structure of the heterocyclic moiety is not critical for enzyme inhibition.



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Figure 4. Inhibition of Top1 by anthra[2,3-b]furan-5,10-diones 12, 19, 21, 22. Electrophoretic mobility of supercoiled DNA after Top1-mediated relaxation of the DNA in the presence/absence of anthra[2,3-b]furan-5,10-diones 12, 19, 21, 22 reference compounds 8, 9 and camptothecin (Cpt). Lane 1: supercoiled plasmid DNA (рBR322); lane 2: the plasmid DNA treated with Top1 in the absence of drugs; lanes 3-20: the plasmid DNA treated with Top1 in the presence of 0.5, 2.5 or 10 µM of 8 (3-5), 9 (6-8), 12 (8-11), 19 (12-14), 21 (15-17), 22 (18-20) and Cpt (21).

We also investigated the ability of new 4,11-diaminoanthra[2,3-b]furan-5,10-diones to inhibit Top2-mediated

relaxation

of

supercoiled

DNA.

In

this

assay

the

4,11-bis(2-

(methylamino)ethylamino derivative 12 was more potent than the 4,11-bisguanidine 21 (Fig. 5, lanes 4, 8). Thus, we observed dual inhibition of Top1 and Top2 by selected anthrafurandiones, which may subsequently lead to DNA damage at multiple sites and activation of multiple mechanisms of tumor cell death.

Figure 5. Electrophoretic mobility of plasmid DNA after Top2-mediated relaxation in the presence/absence of anthra[2,3-b]furan-5,10-diones 12 and 21 and etoposide (VP-16). Lane 1: supercoiled plasmid DNA (рBR322); lane 2: the plasmid DNA treated with Top2 in the absence of 12 ACS Paragon Plus Environment

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drugs; lanes 3-11: the plasmid DNA treated with Top2 in the presence of 0.1, 0.5, 1.0 or 2.5 µM 12 (3-6), 21 (7-10) and10 µM VP-16 (11).

3.3. Induction of apoptosis In AGS cells a 24 h exposure to 12 induced a dose-dependent increase of the percentage of apoptotic cells at 1 µM as determined by Annexin V-FITC staining (Fig. 6A). Similar results were observed with 11-14 (see Supporting Information, Fig. S4). In contrast, no considerable apoptosis was found in AGS cells exposed to 10 µM of 19, 21, 22 for 24 h (Fig. 6B and Supporting Information Fig. S4). The superior apoptotic activities of 11-14 correlated with their high antiproliferative potency (Tables 1, 2).



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Figure 6. Apoptosis of AGS cells treated with indicated concentrations of 12 (A) and 21 (B) for 24 h. The percentage of apoptotic cells was determined by flow cytometry. Values are mean ± S. E. from at least three independent experiments (*p

Synthesis and Characterization of 4,11-Diaminoanthra[2,3-b]furan-5

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