Design, Synthesis, Biological Evaluation, and Molecular Docking Studies of Quinolone Derivatives as Potential Antitumor Topoisomerase I Inhibitors

Abstract

A novel series of quinolone derivatives (6a–n) were designed and synthesized, and their biological activities were evaluated as potential antitumor topoisomerase I (Top I) inhibitors. Among these compounds, 6j exhibited the most potent antitumor activities against multiple cancer cell lines. Docking simulation was performed to insert compound 6j into the crystal structure of DNA-Top I to determine the probable binding model.

DNA-topoisomerase I (Top I) is an important enzyme to relax supercoiled DNA for transcription, replication, and mitosis.¹⁾ Top I relaxes DNA by producing reversible single-strand DNA breaks. The generally accepted mechanism of Top I action involves a nucleophilic attack by the catalytic tyrosine 723 residue of Top I on the phosphate group at 3′-end of the broken DNA strand, which leads to the break of the DNA phosphodiester bond and the formation of a binary Top I-DNA covalent complex (Top I-DNAcc). The rotation of the 5′-end around the intact strand allows for relaxation of the supercoil.^1,2) As it plays a pivotal role in cellular proliferation, Top I is often overexpressed in human tumors. Thus, DNA-Top I has been identified as a promising cancer therapeutic target.³⁾ Camptothecin (CPT) (1)⁴⁾ and its clinically used analogues, topotecan (2) and irinotecan (3)^5,6) (Fig. 1), were found to inhibit Top I activity by intercalating into the cleavage complex and preventing the relegation step of the catalytic cycle. As a result, the covalent Top I-DNA adduct produces collisions with advancing replication forks and transcription complexes, which triggers irreversible DNA damage and apoptosis.^7,8)

Fig. 1. Structures of 1 and Its Derivatives

Although the camptothecins possess potent antitumor activity, they also suffer from well-identified drawbacks, including solubility and bioactivity, dose-limiting toxicity.^7–10) Additionally, the E-ring lactone of camptothecin is readily opened to its hydroxycarboxylate form in vivo.¹¹⁾ This form is less active and binds strongly to human blood proteins.¹²⁾ Thus, these problems recommended further development of other non-CPT Top I inhibitors with better pharmacokinetic features.

The non-CPT Top I inhibitors include indenoisoquinolines,^13–15) indolocarbazones,¹⁶⁾ saintopin,¹⁷⁾ benzophenazines,¹⁸⁾ terpyridines,¹⁹⁾ and 3-arylisoquinolines.²⁰⁾ The successful clinical cases of the non-CPT derivatives were attributed to the better chemical stability at longer lifetimes of the trapped cleavage complex of the non-CPT Top I inhibitors at the absence of a lactone ring in their skeleton.²¹⁾

Quinolones are among the most common frameworks present in bioactive molecules and hence represent an attractive starting point for the design of combinatorial libraries.²²⁾ Recently, You and co-workers²³⁾ discovered series of quinolone derivatives as potential Top I inhibitors by scaffold modification. In addition, Al-Trawneh et al.²⁴⁾ also reported that tetracyclic fluoroquinolones exhibited high antiproliferative activity against breast MCF-7 and lung A549 tumor cell lines.

We attempted to design quinolone derivatives by scaffold modification (Fig. 2). Fragment A was designed based on Top I inhibitors CPUY013²⁵⁾ and A-62176.²⁶⁾ Fragment B was designed on the basis of Top I-DNA and CPT binding mode¹⁾ which showed that the amino acid residues Asn⁷²² provides interactions with the A-ring of CPT. The target scaffold was a combination of fragment A and B, with water soluble sulfonyl guanidine as a linker. Herein, we report the synthesis and in vitro evaluation of the antitumor properties of novel N-substituted piperazinylquinolone derivatives (6a–n). The probable mechanism of the active compound (6j) was also examined by docking studies.

Fig. 2. Compound Generation by Scaffold Modification

Results and Discussion

Chemistry

N-Substituted piperazinylquinolone derivatives (6a–n) were prepared via a two-step one-pot tandem process. The general synthetic pathway of target compounds is outlined in Chart 1. The description of substituents and yields are listed in Table 1.

Chart 1. General Synthesis of Compounds (6a–n)

Reagents and conditions: (a) NH₂CN (50%), butanone, 40–60°C, 3 h; (b) norfloxacin or ciprofloxacin, 80°C, 3 h.

Table 1. Structure and Synthetic Yield of the Target Compounds

Compound	R1	R2	Yield (%)
6a	H	Et	55
6b	H	c-C3H5	70
6c	4-Me	Et	76
6d	4-Me	c-C3H5	85
6e	4-Cl	Et	75
6f	4-Cl	c-C3H5	84
6g	4-CF3	Et	76
6h	4-CF3	c-C3H5	83
6i	2-Cl	Et	74
6j	2-Cl	c-C3H5	58
6k	2-CF3	Et	71
6l	2-CF3	c-C3H5	76
6m	3-Cl	Et	58
6n	3-Cl	c-C3H5	42

The reaction of arylsulfonyl chloride (4) with cyanamide to form intermediates 5 is a typical nucleophilic substitution reaction. The relatively weak nucleophilicity of the cyanamide and the presence of dicyanamide may lead to byproducts. The product arylsulfonyl cyanamide (5) can also react with cyanamide to form byproducts. Therefore the reaction conditions have to be carefully controlled. The target compound 6 was prepared by a subsequent nucleophilic addition of commercially available norfloxacin hydrochloride or ciprofloxacin hydrochloride with 5 in one-pot with yields ranging from 42% to 85% (Table 1). The intermediates 5 are soluble in aqueous medium, while the target compounds are not very soluble in the reaction medium. Thus the pH of each reaction mixture has to be well adjusted to precipitate the product.

Biological Activity and Discussion

Evaluation of the synthetic N-substituted piperazinylquinolone derivatives (6a–n) for antiproliferative activity was performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using three different human cancer cell lines, human lung carcinoma cell (A549), human promyelocytic leukemia cell (HL-60) and human cervical cancer cell (Hela). The assessments of antiproliferative activities were expressed as the concentration inhibiting 50% of cancer cell growth (IC₅₀). The Top I inhibitory activity assays was carried out using a topoisomerase I drug screening kit. The results were summarized in Table 2.

Table 2. IC₅₀ Cytotoxicity and Top I Inhibitory of Target Compounds

Compd.	IC50(μm)			Top Ia)
	A549	HL-60	Hela
6a	0.161	0.104	0.093	+
6b	0.153	0.099	0.087	+
6c	>0.193	0.146	0.122	−
6d	>0.189	0.139	0.118	−
6e	0.130	0.084	0.063	++
6f	0.123	0.079	0.068	++
6g	0.101	0.065	0.057	++
6h	0.096	0.062	0.055	++
6i	0.079	0.052	0.045	++++
6j	0.071	0.043	0.032	++++
6k	0.101	0.066	0.058	+++
6l	0.098	0.063	0.056	+++
6m	0.089	0.059	0.052	+++
6n	0.085	0.056	0.048	+++
Irinotecan	0.032	0.044	0.038	++++
Norfloxacin	>0.313	>0.313	>0.313	−
Ciprofloxacin	>0.302	>0.302	>0.302	−

a) Activity is expressed semi-quantitatively as follows: − no activity, + very weak activity, ++ weak activity, +++ lower activity than Irinotecan, ++++ similar or greater activity than Irinotecan.

As shown in Table 2, these N-substituted piperazinylquinolone derivatives bearing the fluoro atom and piperazine on the skeleton exhibited remarkable antiproliferative effects. Among them, compound 6j displayed the most potent inhibitory activity (IC₅₀=0.072 µm for A549, IC₅₀=0.043 µm for HL-60 and IC₅₀=0.039 µm for Hela), similar to the positive control Irinotecan (IC₅₀=0.032 µm for A549, IC₅₀=0.044 µm for HL-60 and IC₅₀=0.038 µm for Hela, respectively). As expected, compound 6j showed excellent Top I inhibitory activity (++++) comparable to Irinotecan. Consistency between cytotoxicities and Top I inhibitory activities was observed in these series, suggesting that the target of N-substituted piperazinylquinolones is limited to Top I.

Subsequently, structure–activity relationships (SAR) studies were inferred from Table 2. In general, target compounds with R²=cyclopropyl shown more potent activities than those with R²=ethyl at N-1 position. And the substituent group on the benzene ring had decisive affect on the IC₅₀ value. Compounds with electron-donating group on the benzene ring, such as 6c and d, shown weaker cytotoxic activities, and electron-withdrawing group such as chlorine or trifluoromethyl on the benzene ring would increase the activities. Moreover, the position of substituent (R¹) on the benzene ring also influenced the activities. Compounds with ortho substituent (6i–l) shown more potent activities than those with meta (6m, n) or para (1e–h) substituent.

Docking Study

Molecular docking is an application wherein molecular modeling techniques are used to predict how a protein interacts with small molecules.²⁷⁾ In the present study, to gain better understanding on the interactions between target compound and Top I (PDB code: 1T8I) and to explore their binding mode, a docking study was performed using the CDOCKER protocol in Discovery Studio 2.1 (Discovery Studio 2.1, Accelrys, Inc., San Diego, CA, U.S.A.).

Docking model illustrates that the quinolone derivatives of 6j was well positioned in the binding sites of DNA-Top I complex (Fig. 3). The structural model suggested that one of the two sulfonyl oxygens, fluoro atom on the quinolone skeleton and quinolone ring, interacted with enzyme (Fig. 3a). One of the sulfonyl oxygen formed one hydrogen bond with the amino hydrogen of Lys425. And the fluoro atom on the quinolone ring of compound 6j associates with Arg364 by two hydrogen bonds. The quinolone skeleton of compound 6j is calculated to interact with Arg364 and is likely stabilized by π-stacking interactions.

Fig. 3a. Compound 6j Bound into Top I

The dotted lines show the hydrogen bonds and the solid lines show the π-cation interactions.

Fig. 3b. 2D Ligand Interaction Diagram of Compound 6j with Top I Using Discovery Studio Program with the Essential Amino Acid Residues at the Binding Site Are Tagged in Circles

Fig. 3c. 3D Model of the Interaction between Compound 6j and Top I Bonding Site

The protein is represented by molecular surface. Compound 6j is depicted by balls.

All the amino acid residues which had interactions with compound 6j were displayed in Fig. 3b. The enzyme surface model was shown in Fig. 3c, which revealed that the molecule was well embedded in the active pocket. This molecular docking result, along with the biological assays, suggested that N-substituted piperazinylquinolone derivatives were potential Top I inhibitors as anti-cancer agents.

Conclusion

In our present work, a novel series of quinolone derivatives (6a–n) have been synthesized and evaluated. These compounds exhibited potent antiproliferative activities against A549, HL-60, and Hela cells. Among them, compound 6j demonstrated strong cytotoxicity as well as potent Top I inhibitory activity. Consistency between cytotoxicities and Top I inhibitory activities suggesting that the target of N-substituted piperazinylquinolones is limited to Top I. Molecular docking studies shown that compound 6j bound to the DNA-Top I binding site by three hydrogen bonds and two π-cation interactions which might play crucial roles in its antiproliferative activities. Further discovery of more enhanced N-substituted piperazinylquinolone derivatives with better anticancer activity and lower toxicity and side-effects is under the way.

Experimental

Melting points were determined on Büchi B-540 melting point apparatus and are uncorrected. ¹H-NMR (300 MHz) spectra were recorded on a Bruker AV 300 MHz spectrometer. Mass spectra were obtained on a Thermo Finnigan LCQ-Advantage spectrometer (electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI)). High resolution (HR)-MS was carried out on an APEX (Bruker) mass III spectrometer. The compounds were dissolved in DMSO-d₆. Chemical shifts, δ, are given in ppm relative to tetramethylsilane (TMS) (δ=0) and are referenced by using the residual undeuterated solvent signal. Coupling constants, J, are reported in Hz, multiplicities being marked as: singlet (s), broad singlet (br s), doublet (d), triplet (t), quartet (q), multiplet (m).

General Experimental Procedure

Benzenesulfonyl chloride (0.01 mol) in butanone (30 mL) was heated with stirring to 40°C, and cyanamide solution (50%) was added dropwise during 30 min. The temperature was raised to 60°C and stirring continued for 3 h. After cooling the reaction mixture to below 30°C, norfloxacin hydrochloride or ciprofloxacin hydrochloride monohydrate (0.008 mol) was added. The mixture was heated to 80°C and maintained for 3 h. After cooling to below 40°C, the reaction mixture was poured into cold water while stirring, white crystals or powders were precipitated, filtered, washed with water, and dried at 80°C. Analytically pure samples were obtained by recrystallization from aqueous ethanol.

1-Ethyl-6-fluoro-4-oxo-7-(4-(N-(phenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic Acid (6a): White crystals, mp 221–225°C; 55% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.41 (t, 3H, J=4.5 Hz), 3.19 (m, 4H), 3.43 (m, 4H), 4.58 (q, 2H, J=4.5 Hz), 7.17 (d, 1H, J=3.0 Hz), 7.35 (m, 2H), 7.58 (m, 3H), 7.86 (d, 1H, J=9.0 Hz), 8.82 (s, 1H), 8.91 (br s, 2H), 15.05 (br s, 1H); HR-MS Calcd for C₂₃H₂₅FN₅O₅S [M+H⁺]: 502.1560. Found: 502.1554.

1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(N-(phenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic Acid (6b): White crystals, mp 295–298°C; 70% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 3.33 (m, 4H), 3.53 (m, 4H), 3.83 (m, 1H), 7.28 (d, 1H, J=3.0 Hz), 7.33–7.42 (m, 2H), 7.48–7.65 (m, 3H), 7.90 (d, 1H, J=9.0 Hz), 8.63 (s, 1H), 9.23 (br s, 2H), 15.05 (br s, 1H); HR-MS Calcd for C₂₄H₂₅FN₅O₅S [M+H⁺]: 514.1560. Found: 514.1553.

1-Ethyl-6-fluoro-4-oxo-7-(4-(N-tosylcarbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic Acid (6c): White crystals, mp 257–260°C; 76% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.41 (t, 3H, J=4.5 Hz), 2.45 (s, 3H), 3.19 (m, 4H), 3.43 (m, 4H), 4.58 (q, 2H, J=4.5 Hz), 7.17 (d, 1H, J=3.0 Hz), 7.35 (m, 2H), 7.58 (m, 3H), 7.86 (d, 1H, J=9.0 Hz), 8.82 (s, 1H), 8.91 (br s, 2H), 15.05 (br s, 1H); HR-MS Calcd for C₂₄H₂₇FN₅O₅S [M+H⁺]: 516.1717. Found: 516.1724.

1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(N-tosylcarbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic Acid (6d): White crystals, mp 262–265°C; 85% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 2.45 (s, 3H), 3.33 (m, 4H), 3.53 (m, 4H), 3.83 (m, 1H), 7.22 (d, 1H, J=3.0 Hz), 7.38 (m, 2H), 7.58 (m, 2H), 7.86 (d, 1H, J=9.0 Hz), 8.63 (s, 1H), 9.23 (br s, 2H), 15.05 (br s, 1H); HR-MS Calcd for C₂₅H₂₇FN₅O₅S [M+H⁺]: 528.1717. Found: 528.1723.

7-(4-(N-(4-Chlorophenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (6e): Light yellow crystals, mp 297–299°C; 75% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.41 (t, 3H, J=4.5 Hz), 3.19 (m, 4H), 3.43 (m, 4H), 4.58 (q, 2H, J=4.5 Hz), 7.17 (d, 1H, J=3.0 Hz), 7.46 (m, 2H), 7.66 (m, 2H), 7.90 (d, 1H, J=9.0 Hz), 8.95 (s, 1H), 9.11 (br s, 2H), 15.10 (br s, 1H); HR-MS Calcd for C₂₃H₂₄ClFN₅O₅S [M+H⁺]: 536.1171. Found: 536.1164.

7-(4-(N-(4-Chlorophenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (6f): Light yellow crystals, mp 288–291°C; 84% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 3.33 (m, 4H), 3.53 (m, 4H), 3.83 (m, 1H), 7.28 (d, 1H, J=3.0 Hz), 7.36 (m, 2H), 7.60 (m, 2H), 7.90 (d, 1H, J=9.0 Hz), 8.63 (s, 1H), 9.23 (br s, 2H), 15.05 (br s, 1H); HR-MS Calcd for C₂₄H₂₄ClFN₅O₅S [M+H⁺]: 548.1171. Found: 548.1179.

1-Ethyl-6-fluoro-4-oxo-7-(4-(N-(4-(trifluoromethyl)phenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic Acid (6g): White-like crystals, mp 217–220°C; 76% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.41 (t, 3H, J=4.5 Hz), 3.19 (m, 4H, –CH₂), 3.43 (m, 4H), 4.58 (q, 2H, J=4.5 Hz), 7.17 (d, 1H, J=3.0 Hz), 7.56 (m, 2H), 7.90 (d, 1H, J=9.0 Hz), 8.22 (m, 2H), 8.86 (s, 1H), 9.15 (br s, 2H), 15.08 (br s, 1H); HR-MS Calcd for C₂₄H₂₄F₄N₅O₅S [M+H⁺]: 570.1434. Found: 570.1427.

1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(N-(4-(trifluoromethyl)phenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic Acid (6h): White-like crystals, mp 248–251°C; 83% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 3.33 (m, 4H), 3.53 (m, 4H), 3.83 (m, 1H), 7.28 (d, 1H, J=3.0 Hz), 7.47 (m, 2H), 7.90 (d, 1H, J=9.0 Hz), 8.06 (m, 2H), 8.63 (s, 1H), 9.23 (br s, 2H), 15.05 (br s, 1H); HR-MS Calcd for C₂₅H₂₄F₄N₅O₅S [M+H⁺]: 582.1434. Found: 582.1440.

7-(4-(N-(2-Chlorophenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (6i): Pale yellow crystals, mp 255–258°C; 74% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.41 (t, 3H, J=4.5 Hz), 3.19 (m, 4H), 3.43 (m, 4H), 4.58 (q, 2H, J=4.5 Hz), 7.17 (d, 1H, J=3.0 Hz), 7.46 (m, 3H), 7.90 (d, 1H, J=9.0 Hz), 8.46 (m, 1H), 8.95 (s, 1H), 9.11 (br s, 2H), 15.10 (br s, 1H); HR-MS Calcd for C₂₃H₂₄ClFN₅O₅S [M+H⁺]: 536.1171. Found: 536.1166.

7-(4-(N-(2-Chlorophenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (6j): Pale yellow crystals, mp 286–289°C; 58% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 3.33 (m, 4H), 3.53 (m, 4H), 3.83 (m, 1H), 7.28 (d, 1H, J=3.0 Hz), 7.46 (m, 3H), 7.90 (d, 1H, J=9.0 Hz), 8.46 (m, 1H), 8.63 (s, 1H), 9.23 (br s, 2H), 15.05 (br s, 1H); HR-MS Calcd for C₂₄H₂₄ClFN₅O₅S [M+H⁺]: 548.1171. Found: 548.1169.

1-Ethyl-6-fluoro-4-oxo-7-(4-(N-(2-(trifluoromethyl)phenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic Acid (6k): White-like crystals, mp 218–221°C; 71% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.41 (t, 3H, J=4.5 Hz), 3.19 (m, 4H), 3.43 (m, 4H), 4.58 (q, 2H, J=4.5 Hz), 7.17 (d, 1H, J=3.0 Hz), 7.64–7.85 (m, 3H), 7.90 (d, 1H, J=9.0 Hz), 8.50 (m, 1H), 8.95 (s, 1H), 9.11 (br s, 2H), 15.10 (br s, 1H); HR-MS Calcd for C₂₄H₂₄F₄N₅O₅S [M+H⁺]: 570.1434. Found: 570.1437.

1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(N-(2-(trifluoromethyl)phenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic Acid (6l): White-like crystals, mp 237–240°C; 76% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 3.33 (m, 4H), 3.53 (m, 4H), 3.83 (m, 1H), 7.28 (d, 1H, J=3.0 Hz), 7.64–7.85 (m, 3H), 7.90 (d, 1H, J=9.0 Hz), 8.50 (m, 1H), 8.73 (s, 1H), 9.18 (br s, 2H), 15.05 (br s, 1H); HR-MS Calcd for C₂₅H₂₄F₄N₅O₅S [M+H⁺]: 582.1434. Found: 582.1427.

7-(4-(N-(3-Chlorophenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (6m): White-like crystals, mp 222–225°C; 58% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.41 (t, 3H, J=4.5 Hz), 3.19 (m, 4H), 3.43 (m, 4H), 4.58 (q, 2H, J=4.5 Hz), 7.17 (d, 1H, J=3.0 Hz), 7.35–7.60 (m, 3H), 7.86 (s, 1H), 7.95 (d, 1H, J=9.0 Hz), 8.86 (s, 1H), 9.16 (br s, 2H), 15.00 (br s, 1H); HR-MS Calcd for C₂₃H₂₄ClFN₅O₅S [M+H⁺]: 536.1171. Found: 536.1178.

7-(4-(N-(3-Chlorophenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (6n): White-like crystals, mp 228–232°C; 42% yield; ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 3.33 (m, 4H), 3.53 (m, 4H), 3.83 (m, 1H), 7.28 (d, 1H, J=3.0 Hz), 7.35–7.60 (m, 3H), 7.86 (s, 1H), 7.90 (d, 1H, J=9.0 Hz), 8.63 (s, 1H), 9.23 (br s, 2H), 15.05 (br s, 1H); HR-MS Calcd for C₂₄H₂₄ClFN₅O₅S [M+H⁺]: 548.1171. Found: 548.1177.

Anti-proliferation Assay

The antiproliferative activities of the prepared compounds against A549, HL-60 and Hala cells were evaluated using a standard MTT-based colorimetric assay. Target tumor cell lines were grown to log phase in RPMI 1640 medium supplemented with 10% fetal bovine serum. After diluting to 1×10⁶ cells mL⁻¹ with the complete medium, 100 µL of the obtained cell suspension was added to each well of 96-well culture plates. The subsequent incubation was permitted at 37°C, 5% CO₂ atmosphere for 24 h before the cytotoxicity assessments. Each concentration was in triplicate, and Irinotecan was used as the positive control. After 72 h incubation at 37°C, 5% CO₂ atmosphere, 10 µL of MTT solution in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, U.S.A.) was added to each well. After three hours incubation at 37°C, 150 µL DMSO was added to each well. The plates were then vibrated for 10 min for complete dissolution. The optical absorbance was measured at 570 nm on an automated microplate spectrophotometer (Bio-Rad, U.S.A.). In all experiments three replicate wells were used for each drug concentration. The IC₅₀ value was defined as the concentration at which 50% of the cells could survive. The results were summarized in Table 2.

Docking Simulations

The pdb file about the crystal structure of DNA-Top I bound to SA315F (PDB code: 1T8I)^28,29) was obtained from the RCSB Protein Data Bank (http://www.pdb.org). The molecular docking procedure was performed by using CDOCKER protocol for receptor-ligand interactions of Discovery Studio 2.5. For ligand preparation, the 3D structures of 6j were generated and minimized using Discovery Studio 2.5. For protein preparation, the hydrogen atoms were added. The whole DNA-Top I domain defined as a receptor and the site sphere was selected based on the ligand binding location of SA315F, then the SA315F removed and the ligands prepared by us was placed during the molecular docking procedure. CHARMm was selected as the force field. The molecular docking was performed with a simulated annealing method.

Acknowledgment

We are grateful to the Natural Science Foundation of Zhejiang Province (No. Y2100908) for financial support.

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