3048 J. Med. Chem. 2010, 53, 3048–3064 DOI: 10.1021/jm9014394
A Series of r-Heterocyclic Carboxaldehyde Thiosemicarbazones Inhibit Topoisomerase IIr Catalytic Activity He Huang,†,§ Qin Chen,‡,§ Xin Ku,† Linghua Meng,*,‡ Liping Lin,‡ Xiang Wang,‡ Caihua Zhu,‡ Yi Wang,‡ Zhi Chen,† Ming Li,‡ Hualiang Jiang,† Kaixian Chen,† Jian Ding,‡ and Hong Liu*,† †
Drug Discovery and Design Center and ‡Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, P. R. China. § These two authors contributed equally to this work
Received September 29, 2009
A series of novel thiosemicarbazone derivatives bearing condensed heterocyclic carboxaldehyde moieties were designed and synthesized. Among them, TSC24 exhibited broad antiproliferative activity in a panel of human tumor cells and suppressed tumor growth in mice. The mechanism research revealed that TSC24 was not only an iron chelator but also a topoisomerase IIR catalytic inhibitor. Its inhibition on topoisomerase IIR was due to direct interaction with the ATPase domain of topoisomerase IIR which led to the block of ATP hydrolysis. Molecular docking predicted that TSC24 might bind at the ATP binding site, which was confirmed by the competitive inhibition assay. These results about the mechanisms involved in the anticancer activities of thiosemicarbazones will aid in the rational design of novel topoisomerase II-targeted drugs and will provide insights into the discovery and development of novel cancer therapeutics based on the dual activity to chelate iron and to inhibit the catalytic activity of topoisomerase IIR.
1. Introduction The thiosemicarbazone (TSC) is considered as one of the most important scaffolds and is embedded in many biologically active compounds.1-8 Brockman et al. first showed that 2-formylpyridine TSC possesses antileukemic activity in mice bearing leukemic cells.9 Following this report, various aliphatic, aromatic, and heteroaromatic carbaldehyde TSCs were synthesized and evaluated for antitumor activity against a wide spectrum of transplanted murine neoplasms.1,10-16 Examples of TSC derivatives demonstrating profound anticancer activities are presented in Figure 1. To date, the most significant progress in the treatment of cancer has been made with 3-aminopyridine-2-carboxaldehyde TSC (3-AP or triapine) which is currently in phase II clinical trials. Generally, the TSCs derived from five- or six-member heterocycle scaffolds possess potent anticancer activity, which is exemplified by 2-pyridine carbaldehyde derivatives, including 3-AP. However, the therapeutic potential of 3-AP was limited by its poor water solubility and toxicity profile.17 Therefore, there is increasing interest in structural modification of TSC derivatives with the aim of improving the pharmaceutical profile of existing candidates or of discovering novel derivatives. Up to now, most of the structure-activity relationship studies reported were focused on substitutions at the heterocycle core structure. Few data have been published describing the antitumor activity of heteroaromatic compounds that contain a condensed heterocycle. *To whom correspondence should be addressed. For L.M.: phone, þ86-21-50806722; fax, þ86-21-50806722; e-mail, lhmeng@ mail.shcnc.ac.cn. For H.L.: phone, þ86-21-50807042; fax, þ86-2150807088; e-mail,
[email protected].
pubs.acs.org/jmc
Published on Web 03/30/2010
TSCs were considered to suppress tumor growth by virtue of their ability to chelate iron (Fe).18-22 They are capable of removing Fe from biological systems and inhibit the activity of Fe-requiring proteins, including ribonucleotide reductase (RRa), a key enzyme involved in DNA synthesis and repair. Because neoplastic cells have a higher requirement for iron than healthy cells because of their rapid proliferation, TSCs selectively inhibit the proliferation of tumor cells. Meanwhile, several studies provided evidence indicating that TSCs exert their antitumor functions via more than Fe chelating. First, complexation to metal can potentiate rather than weaken the antitumor activities of some TSCs.23,24 Second, Fe chelation induces an arrest of the cell cycle at the S phase due to the inhibition of DNA synthesis. In contrast, an accumulation of cells at the G2/M phase or no change in cell cycle is often noticed after TSC treatment.1,11 Therefore, the mechanisms involved in the antitumor activities of TSCs are far from clear. In an effort to discover novel TSC derivatives with potent anticancer activity and to elucidate their mechanism of action, TSCs bearing condensed heterocyclic carboxaldehyde moieties were synthesized and their antitumor activities were evaluated. We found that several compounds exhibited potent cytotoxic activity in tumor cells. Among them, TSC24, which is the most active in the primary screening, displayed broad antiproliferative activity in a panel of human tumor cells including multidrug resistant cells. TSC24 displayed profound a Abbreviations: RR, ribonucleotide reductase; DSBs, double strand breaks; At-MDR, altered topoisomerase II-associated multidrug resistance; Topo, topoisomerase; VP16, etoposide; Acl, aclarubicin; ADR, adriamycin; CPT, camptothecin; EB, ethidium bromide; m-AMSA, amsacrine; HsATPase, human topoisomerase IIR ATPase domain; SPR, surface plasmon resonance spectroscopy.
r 2010 American Chemical Society
Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3049
Figure 1. Structures of some TSC derivatives demonstrating profound anticancer activities.
Scheme 1. Procedure for the Synthesis of R-Heterocyclic Carboxaldehyde Thiosemicarbazonesa
a Reagents and conditions: (a) hydrazine hydrate (85%), isopropanol, room temp; (b) quinoline-2-carbaldehyde, isopropanol, acetic acid (cat.), room temp; (c) R-heterocyclic carboxaldehyde, isopropanol, microwave irradiation, 100 °C, 30 min.
activity in mice bearing S-180 sarcoma. Importantly, the mechanistic studies revealed that TSC24 is a new topoisomerase IIR (Topo IIR) catalytic inhibitor in addition to its Fe chelating ability. By dissecting the effect of TSC24 on the individual steps of the catalytic cycle of Topo IIR, we found that TSC24 blocked Topo IIR-catalyzed ATP hydrolysis by competing with ATP for binding to the ATPase domain of Topo IIR. 2. Materials and Methods 2.1. Chemical Design and Synthesis. On the basis of the structural features of reported compounds, 36 new analogues (TSC1TSC36) were designed and synthesized. Keeping the quinoline ring at N1 position, we obtained compounds TSC1-TSC24 by introducing diverse groups to the N4 position. And by replacing the quinoline rings of TSC23 and TSC24 with the diverse condensed heteroaromatic rings, we got compounds TSC25-TSC36. Synthesis of these TSC derivatives was straightforward following the general procedures outlined in Scheme 1. The N4 substituted TSC derivatives B were prepared through the nucleophilic addition reaction of 85% hydrazine hydrate with various iso(thio)cyanates A at room temperature. Compounds B1-B22 were condensed with quinoline-2-carbaldehyde in isopropanol for 2 h, affording TSC1TSC22, respectively. Commercially available compounds C were successfully converted to target compounds TSC23-TSC36 by condensation of respective hydrazines to the appropriate aldehydes under microwave irradiation in isopropanol for 30 min. 2.2. In Vitro Cytotoxicity Assays. The cytotoxic activities of the compounds were evaluated using human acute leukemia HL60, human gastric cancer SGC7901, human cervical cancer HeLa, and human colon carcinoma HT-29 cells. Cells seeded in 96-well plates were treated in triplicate with gradient concentrations of compounds for 72 h. For suspended cells, cytotoxicity was assessed by measuring the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (Sigma, St. Louis, MO) to a colored
product.25 Sulforhodamine B assay (Sigma, St. Louis, MO) was applied to detect the cytotoxicity in adherent cells.26 The concentration required for 50% inhibition of cell growth compared with control cells (IC50) was determined by the Logit method. TSC24 was further evaluated for its antiproliferative activity against a panel of human tumor cells originated from different tissue types, including colon cancer, gastric cancer, breast cancer, lung cancer, hepatocellular cancer, leukemia, melanoma, rhabdomyosarcoma, cervical cancer, and oral squamous carcinoma. 2.3. In Vivo Antitumor Studies. In vivo antitumor studies were carried out with compound TSC24 in 7-week-old female Kunming mice (weight, 18-22 g) provided by the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. Mice were inoculated sc into the right armpit with S-180 sarcoma. On the next day, normal saline or 5-fluorouracil was administered by iv injection, and TSC24 was administered at indicated doses by ip injection for 7 days. After treatment, animals were killed by cervical dissociation, and solid tumors were removed and weighed. The inhibitory rate was calculated as the following formula: inhibitory rate (%) = {[(mean tumor weight of control group) (mean tumor weight of treatment group)]/(mean tumor weight of control group)}100%. 2.4. Fenton’s Reaction-Mediated Plasmid DNA Breaks. The effect of compounds on the integrity of plasmid DNA was analyzed as described previously.27 Briefly, pBR322 plasmid (0.2 μg) was incubated with reaction buffer containing 13 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes, pH 7.2), 1 mM H2O2, and 10 μM FeSO4 in the presence of indicated concentrations of desferrioxamine (DFO) or TSC24. After incubation at room temperature for 30 min, samples were immediately loaded with 5 μL of loading dye onto a 1% agarose gel containing 0.1 μg/mL ethidium bromide (EB). The gels were run at 90 V for 60 min, then visualized using a UV transilluminator and photographed. The intensity of DNA bands was quantified and the percentage of supercoiled plasmid DNA was calculated with the formula % SC = [1.4 SC/(L þ OC þ 1.4 SC)] 100.
3050 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
A correction factor of 1.4 was applied to count the relatively attenuated fluorescence of supercoiled (SC) DNA compared to the open-circular (OC) and linear (L) forms.27 2.5. Flow Cytometric Analysis of BrdU Incorporation and Cell Cycle Distribution. DNA synthesis was measured by BrdU incorporation assay using BrdU flow kit (BD Pharmingen, San Diego, CA) according to the manufacturer’s instructions. Cell cycle analyses were performed on a FACS Calibur cytometer (Becton Dickinson, Mountain View, CA) after cells were stained with propidium iodide (PI). Cell cycle distributions were analyzed using ModFit LT 3.0 software (Verity Software House Inc., Topsham, ME). 2.6. Topo I- and Topo IIr-Mediated Supercoiled pBR322 Relaxation Assay and Topo IIr-Mediated kDNA Decatenation Assay. Relaxation and decatenation assays were carried out as previously described.28 Briefly, reaction buffer contained 50 mM Tris-HCl (pH 8.0), 120 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 1 mM ATP (ATP was omitted in Topo I-mediated DNA relaxation), 0.25 μg of supercoiled pBR322 or 0.1 μg kDNA, 1 unit of Topo I or Topo IIR in a total volume of 20 μL. After incubation at 37 °C for 15 min, the reaction was terminated by the addition of 1 μL of SDS (10%). The DNA samples were mixed with loading buffer and subjected to electrophoresis in 1% agarose gel in TAE buffer at 90 V for 60 min. 2.7. Topo II-Mediated DNA Cleavage Assay. Prestrand passage DNA cleavage reactions were carried out in a final volume of 20 μL, containing 50 mM Tris-HCl (pH 8.0), 120 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.25 μg of supercoiled pBR322, and 10 units of Topo IIR. In the complete cleavage reactions, 1 mM ATP was included. After incubation at 37 °C for 15 min and the reaction stopped by the addition of 1 μL of SDS (10%), the reaction mixtures were incubated with 2 μL of proteinase K (0.8 mg/mL) at 45 °C for 30 min to digest the Topo IIR. Final products were mixed with loading buffer, heated at 70 °C for 2 min, and subjected to electrophoresis in 1% agarose. 2.8. Neutral Single-Cell Gel Electrophoresis Assay. HT-29 cells were pretreated with TSC24 or aclarubicin for 30 min, then exposed to 10 μM VP16 for 1 h. DNA double-strand breaks (DSBs) were evaluated by neutral single-cell gel electrophoresis assay as described.29,30 The images were captured with a fluorescence microscope (Olympus BX51, Olympus Co., Tokyo, Japan). 2.9. TSC24-DNA Interaction. A topoisomerase I unwinding assay and an ethidium bromide (EB) displacement fluorescence experiment were performed to examine the ability of TSC24 to intercalate into plasmid DNA as described.31,32 To determine whether TSC24 binds to the minor groove of DNA, a Hoechst 33258 displacement experiment was performed.32 2.10. Topo II-DNA Binding Assay. Topo II-DNA binding was evaluated by an electrophoretic mobility shift assay.33 Reactions were carried out in 20 μL of reaction buffer containing 0.2 μg of supercoiled pBR322 DNA and 200 nM human topo IIR in the presence or absence of TSC24. After incubation at 37 °C for 10 min, samples were separated on a 1% agarose gel containing 0.5 μg/mL EB by electrophoresis for 90 min in TAE buffer. 2.11. Purification of the Human Topoisomerase IIr ATPase Domain (HsATPase) and ATP Hydrolysis Assay. HsATPase was expressed and purified as described previously.34,35 The ATP hydrolysis catalyzed by HsATPase was coupled to the oxidation of NADH.17 The reactions were carried out in 96-well plates at 37 °C in a total volume of 100 μL containing 2 μg of HsATPase or 18 nM Topo IIR, 0.2 μg of pBR322 DNA, 1 mM ATP, 2 mM PEP, 0.16 mM NADH, 5 U of pyruvate kinase, and 8 U of lactate dehydrogenase. The OD values of NADH were monitored by a spectrophotometer (VERSAmax; Molecular Devices, Sunnyvale, CA), which was programmed to collect data every 20 s at 340 nm. 2.12. Surface Plasmon Resonance Spectroscopy (SPR). The SPR assay was performed at 25 °C using a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). HsATPase was immobilized onto CM5 sensor chips after activation of the surface carboxyl groups as previously reported.35 Serial dilutions of
Huang et al.
compounds were injected to flow across the chip surface, and sensorgrams were recorded. Equilibrium constants (KD) for evaluating protein-ligand binding affinity were determined using the steady-state affinity fitting analysis. 2.13. Molecular Docking. The crystal structure of HsATPase in complex with ADPNP was retrieved from the Protein Data Bank (PDB entry 1ZXM). DOCK 4.0 was used to predict the potential binding between TSC24 and HsATPase. For the target, the Connolly molecular surface was calculated with a probe radius of 1.4 A˚. The residues around the cocrystallized ligand within 7 A˚ were defined as binding site with Sybyl 6.8 (Tripos Associates Inc., St. Louis, MO). Within the binding site, spheres were generated therein with the DOCK program SPHGEN. SPHGEN outputted the spheres in clusters. The spheres within 5 A˚ radius of the natural ligand were selected for the target. The compound was docked into the binding site, allowing for ligand flexibility, using the grid-based energy scoring option for minimization after initial placement in the site. The box for the scoring grid was defined such that all spheres were enclosed with an extra 7 A˚ added in each dimension. Scoring grids for contact and energy scores were calculated with a grid spacing of 0.3 A˚. The bump check was set such that atoms closer than 75% of the van der Waals radii of the respective atoms were rejected. A 6-12 Lennard-Jones van der Waals potential was used along with a Coulomb potential using a distance-dependent dielectric coefficient of 4 to simulate salvation effects. The energy cutoff was set to 10 A˚. The radii used were those in the vdw.defn set. Ligand atoms were matched to receptor spheres using the anchor first search with the anchor size set to 10 atoms. The automatic matching option was used, and conformations were generated with the torsion drive option. 2.14. Statistical Analysis. All data were expressed as mean values ( SD. At least three independent experiments were conducted in triplicate. Statistical analysis was performed by unpaired Student’s t test. A p value less than 0.05 was considered to be statistically significant.
3. Results 3.1. Chemistry. The chemical structures of the 36 newly synthesized compounds (TSC1-TSC36) are shown in Table 1. These compounds were synthesized through the routes outlined in Scheme 1, and the details of the synthetic procedures and structural characterizations are described in the Experimental Section. 3.2. In Vitro Antitumor Studies. 3.2.1. Effects of TSCs on the Proliferation of Tumor Cells. All substances were evaluated for antiproliferative activites against the human acute leukemia (HL-60) cell line, human gastric cancer (SGC7901) cell line, human cervical cancer (Hela) cell line, and human colon carcinoma (HT-29) cell line. The data shown in Table 1 are IC50 values against tested tumor cells. The compounds bearing alkyl groups at the N4 position showed promising antiproliferative activity, while the unsubstituted and arylsubstituted derivatives at this position displayed low or moderate activity. Of all the compounds tested, TSC24 showed the best antitumor efficacy, with an IC50 ranging from 0.02 to 0.09 μM without distinct selectivity among the cell lines tested. TSC26, TSC28, TSC31, and TSC36 (average IC50 values on four cell lines are 0.36, 0.64, 1.71, and 0.56 μM, respectively) also demonstrated appreciable antiproliferative activities. 3.2.2. TSC24 Exhibits Broad Antiproliferative Activity against Tumor Cells. TSC24 showed potent antiproliferative activity with IC50 values in the nanomolar range against a panel of human tumor cells, including colon cancer, gastric cancer, breast cancer, lung cancer, hepatocellular cancer,
Article Table 1. Inhibition of Cell Proliferation of Thiosemicarbazones
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3051
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Huang et al.
Table 1. Continued
Table 2. Inhibition of Cell Proliferation of Compounds TSC24 against a Panel of Human Tumor Cells cell line colon IC50 (mean ( SD), μM
stomach
HT29
HCT116
Lovo
MKN45
MKN28
SGC-7901
0.02 ( 0.01
0.22 ( 0.10
0.06 ( 0.03
0.83 ( 0.14
0.17 ( 0.02
0.03 ( 0.00
cell line breast MDA-MB-231 IC50 (mean ( SD), μM
0.02 ( 0.02
lung
MCF-7
MDA-MB-435
0.34 ( 0.19
0.37 ( 0.17
NCI-H23
A549
0.08 ( 0.06
0.45 ( 0.35
cell line liver IC50 (mean ( SD), μM
blood
BEL-7402
SMMC-7721
0.18 ( 0.12
0.24 ( 0.01
HL-60
K562
U-937
0.05 ( 0.01
0.06 ( 0.05
0.03 ( 0.01
cell line other IC50 (mean ( SD), μM
A375
Rh30
Hela
KB
0.02 ( 0.01
0.002 ( 0.000
0.09 ( 0.02
0.77 ( 0.19
leukemia, melanoma, rhabdomyosarcoma, cervical cancer, and oral squamous carcinoma (Table 2). Meanwhile, TSC24 did not show obvious selectivity against these tumor cells. 3.2.3. TSC24 Overcomes Multidrug Resistance. MDR is considered to be an important impediment to the effective
chemotherapy of cancer. We next investigated the cytotoxicity of TSC24 in three MDR cell lines: K562/A02, MCF-7/ ADR, and KB/VCR. Resistance factor (RF), defined as the ratio of the IC50 against resistant cells to that against the corresponding parental cells (K562, MCF-7 or KB), was
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3053
Article Table 3. Resistance Factors of TSC24 and Reference Drug in MDR Sublinesa
IC50 (mean ( SD), μM compd
K562
K562/A02
KB
KB/VCR
MCF-7
MCF-7/ADR
TSC24 ADR VCR
0.16 ( 0.04 0.43 ( 0.17 NTb
0.06 ( 0.03 23.81 ( 2.99 NTb
0.78 ( 0.19 NTb 0.01 ( 0.00
0.30 ( 0.09 NTb 0.84 ( 0.08
0.34 ( 0.19 0.39 ( 0.12 NTb
0.20 ( 0.02 44.9 ( 1.27 NTb
resistance factor compd
K562/A02
KB/VCR
MCF-7/ADR
TSC24 0.37 0.39 0.57 ADR 55.93 NTb 115.13 VCR NTb 119.71 NTb a Cells were treated with indicated compounds for 72 h, and IC50 values were determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or sulforhodamin B assays. b NT: not tested. Table 4. TSC24 Inhibited Tumor Growth in Mice Implanted with S-180 Sarcoma Cellsa mice number
body weight (g)
treatment group
dosage ((mg/kg)/day) day
initial
end
initial
end
tumor weight, x ( SD (g)
inhibition rate (%)
NS TSC24 TSC24 TSC24 5-Fu
50 7 (ip) 25 7 (ip) 12.5 7 (ip) 50 2 (iv)
20 10 10 10 10
20 10 10 10 10
18.1 17.9 17.9 17.9 18.1
32.5 20.0 23.9 29.0 27.5
1.76 ( 0.36 0.44 ( 0.33 1.13 ( 0.55 1.45 ( 0.55 0.34 ( 0.22
76.7 35.8 17.6 80.7
P
