Synthesis and Anticancer Evaluation of Benzyloxyurea Derivatives

Abstract

A series of novel benzyloxyurea derivatives was designed, synthesized by substituting different benzyls or phenyls on N,N′-positions of the hydroxyurea (HU). These target compounds were evaluated for their anticancer activity in vitro against human leukemia cell line K562 and murine leukemia cell line L1210 in comparison with HU by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Some of the compounds showed promising anticancer activity against the cells. Molecular docking experiments with Saccharomyces cerevisiae R1 domain indicated that 4a and 4f′ have stronger affinity than 4m and 4n. Flow cytometry study showed that compound 4g exerted greater apoptotic activity against K562 cells line than HU.

As we all know, because of its low cure rate and high mortality, tumor has become one of the most terrible diseases around the world. Hydroxyurea (HU), as the preferred treatment drug of chronic myelogenous leukemia, could also be used to treat melanoma, tumor of the ovary, human immunodeficiency virus (HIV) infection, β-mediterranean disease and so on.^1–5) But the use of HU is limited by its metabolic and inherent cytotoxicity.^6–9)

Eukaryotic ribonucleoside reductase (RR) catalyzes nucleoside diphosphate conversion to deoxynucleoside diphosphate. Crucial for rapidly dividing cells, RR is an attractive therapeutic target of cancer, and over the past years, many chemotherapeutics inhibiting different subunits of RR have been developed and tested clinically for their anticancer activities and anti-HIV activities.^10–13) HU is the first RR inhibitor applied to the clinic, and is utilized for the therapeutic of neoplasms because of its influences on the DNA replication of cancer cells.^1,14) To overcome the drawbacks of HU, numerous medicinal chemistry efforts have been made to design and synthesize novel HU derivatives.^9,15–18) In previous paper, we reported HU monosubstituents and disubstituents with benzyls at N-positions of HU.^6,19) The results indicated that the stronger hydrophobic nature of the HU derivatives might favor the cytotoxic activity and benzyl groups at the N-position of HU were associated with enhanced cytotoxic activity. The disadvantages associated with HU’s physicochemical properties, e.g., very high hydrophilicity (log P: −1.80) and small molecular size, may be overcomed by the structural modification. This background prompted us to further explore more novel benzyloxyurea derivatives with different substituents at N,N′-positions of HU. In this study, a series of novel benzyloxyurea derivatives substituted with different benzyls or phenyls at N,N′-position of HU were synthesized. The target compounds were evaluated for their anticancer activity in vitro against human leukemia cell line K562 and murine leukemia cell line L1210 in comparison with HU by the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Meanwhile, the technology of molecular docking and flow cytometry were used to some of the high active compounds, which revealed many useful information.

Results and Discussion

Chemistry

The carbamate intermediates 3a–g were synthesized via three steps. In the presence of CH₃CH₂ONa, acetoxime and various substituted benzyl chloride were added in anhydrous CH₃CH₂OH at room temperature to give 1a–g, which were then treated with concentrated HCl to afford 2a–g. In the presence of pyridine, compounds 2a–g were treated with 4-nitrophenyl chloroformate in CH₂Cl₂ to give 3a–g,^19–22) which were converted to target compounds 4a–u in the presence of Et₃N in CH₂Cl₂ with substituted anilines or p-phenylenediamine at room temperature (Chart 1). Target compounds 4a′–g′²³⁾ could be provided in the presence of Na₂CO₃ with hydroxylamine hydrochloride in CH₃OH and CH₂Cl₂.

Chart 1. Synthesis of Target Compounds

Reagents and conditions: i) CH₃CH₂ONa, CH₃CH₂OH, room temp. ii) condensed HCl, room temp. iii) 4-nitrophenyl chloroformate, pyridine, CH₂Cl₂, reflux. iv) substituted anilines, Et₃N, CH₂Cl₂, room temp. v) p-phenylenediamine, Et₃N, CH₂Cl₂, room temp. vi) hydroxylamine hydrochloride, Na₂CO₃, methanol, CH₂Cl₂.

Anticancer Activity

The prepared compounds 4a–u and 4a′–g′ were evaluated for cytotoxic properties on human leukemia cell line K562 and murine leukemia cell line L1210 with HU as positive control. Inhibition of cell-proliferation was measured by the improved MTT assay.^6,24) The inhibitory potency (IC₅₀) of tested compounds are given in Table 1. In general, some target compounds showed good inhibitory effects on L1210 and K562, especially 4a, 4b, 4d, 4e, 4g, 4q, 4c′ and 4f′. Among these compounds, 4a–g, 4j, 4k, 4p, 4q, 4t, 4c′ and 4f′ exhibited stronger inhibitory effects on L1210 than HU, wherein the anticancer activity of 4g (IC₅₀: 15.31 µM) and 4b (IC₅₀: 22.61 µM) were the highest two compounds. 4a, 4b, 4d, 4e, 4g, 4l, 4o, 4q, 4s and 4a′–g′ showed higher activity on K562 than HU, wherein 4f′ (IC₅₀: 1.32 µM), 4g′ (IC₅₀: 3.84 µM) revealed the highest inhibitory effect on K562. Nevertheless, the inhibitory activities of 4h, 4i, 4m, 4n, 4r and 4u were lower than HU no matter on K562 or L1210.

Table 1. IC₅₀ Values of Test Compounds against L1210 and K562

Compd.	IC50 (μM)a)
	L1210	K562
HU	201.05	73.60
4a	64.92	17.70
4b	22.61	21.00
4c	50.44	>160
4d	35.94	57.00
4e	54.98	21.38
4f	64.37	101.00
4g	15.31	60.30
4h	>160	>160
4i	>160	>160
4j	117.56	>160
4k	131.12	>160
4l	>160	54.56
4m	>160	>160
4n	>160	>160
4o	>160	31.93
4p	106.49	>160
4q	34.35	41.54
4r	>160	>160
4s	>160	58.61
4t	64.77	>160
4u	>160	>160
4a′	>160	28.77
4b′	>160	31.17
4c′	78.40	17.98
4d′	n.d. b)	34.76
4e′	>160	13.19
4f′	115.50	1.32
4g′	n.d.	3.84

a) IC₅₀ is the drug concentration effective in inhibiting 50% of the cell growth measured by MTT method. b) n.d. means not determined.

Apoptosis Assay for Compound 4g by Flow Cytometry

Among the twenty-eight compounds tested for anti-proliferative effect, nine compounds 4a, 4b, 4d, 4e, 4f, 4g, 4q, 4c′, 4f′ showed a significant inhibition of proliferation for the two cancer cell lines, among these 4g exhibited the best activity to L1210 cells and the nearly equipotent activity to K562 cells with HU. In order to investigate whether the potent compounds exhibited their anti-proliferative effect on K562 cancer cell through induction of apoptosis, the compound 4g was selected for further apoptosis assay in comparison with HU. The studies of flow cytometry were undertaken on compound 4g and HU in five different concentrations. The results shown in Fig. 1 and Table 2 revealed that compound 4g exhibited higher population of apoptotic cells than HU at the same concentration. The results indicated 4g could induce apoptosis of K562 cancer cells.

Fig. 1. Flow Cytometric Results after the Exposure of K562 Cell Line for 24 h to the Active Compound 4g and HU

Four areas in the diagrams represent four different cell states: necrotic cells (Q1), late apoptotic or necrotic cells (Q2), apoptotic cells (Q3) and living cells (Q4).

Table 2. Apoptosis of K562 Cell Line in Different Concentrations of Compound 4g and HU

Concentration (mol/L)	Apoptosis of K562 cell line (%)
	4g	HU
10−3	68.1	28.0
10−4	31.6	26.0
10−5	22.7	18.3
10−6	17.5	11.9
10−7	13.6	3.06

Molecular Docking Study

In order to determine the interactions between target compounds and RR. The potent compounds 4a and 4f′, as well as the least active compounds 4m and 4n were docked into the active site of RR by autodock tool 1.5.2 version. RR activity requires formation of a complex between subunits R1 and R2 in which the R2 C-terminal peptide binds to R1. The subsite A and subsite B in Saccharomyces cerevisiae R1 (ScR1) domain of RR are the active site in which R2 C-terminal heptapeptide, P7 and its peptidomimetic P6 bound to ScR1. P7 and P6, both of which inhibit ScRR.²⁵⁾ So the subsite B (PDB id-2ZLF) was selected as the receptor. The interior of the cavity in subsite B was narrow and consisted of two pockets accommodating the hydrophobic residues. These residues were on the pocket A LEU716, ARG717, GLN692, and on the pocket B LYS723, SER726, MET727, LYS693, ILE696 and TYR730.

The compounds 4a and 4f′, which displayed the high anticancer activity, were embedded into the cleft of two pockets in the active site (Fig. 2). Good complementarities were observed between the docked ligand and the hydrophobic subsites of the enzymatic cavity. In the pocket A, the benzyl group of 4a and the hydroxylamine group of 4f′ were stabilized by LEU716, ARG717 and GLN692, while the phenyl group of 4a and the benzyl group of 4f′ interacted with the residues LYS723, MET727, LYS693 and TYR730 of the pocket B. The hydrogen bonds were identified between GLN 692 and LEU716 and the acyl group of the ligand (Fig. 3). The docking conformation of compounds 4m and 4n, superposed to the one of compounds 4a and 4f′ in Fig. 2, showed the different docking poses and hydrogen bond interactions. Because of the bulky substituent structures, 4m and 4n could not be embed the pocket A, and they formed hydrogen bonds by the acyl group with residue LYS693 on the pocket B. The compound 4m interacted with the residues LYS723, SER726, MET727, LYS693, ILE696, TYR730 of the pocket B and GLN 692 of the pocket A (Fig. 4). Because compound 4m could not interacte with residues LEU716 and ARG717 in the interior of the pocket A, the binding of 4m to the active site was decreased. This molecular modeling study into the active site of RR allowed to provide a rationale to the increasing activity measured for 4a and 4f′ compared to 4m and 4n. Building up a p-phenylene bis benzyloxyurea structures had a negative effect on the activity. This suggested that the smaller substituent groups were preferred on the nitrogen atom of HU, giving a moderate increase of binding affinity.

Fig. 2. The Docking Modes of Compounds 4a, 4f′, 4m and 4n at ScR1

The compounds are color coded as carbon green (4m), pink (4a), nattier blue (4f′) and beige (4n), nitrogen blue, oxygen red and hydrogen white. The hydrogen bonds are drawn in red lines. Protein is represented by the molecular surface.

Fig. 3. Plausible Binding Modes for Compounds 4a and 4f′ Superposed in the Binding Site of ScR1

The compounds are color coded as carbon nattier blue (4a), beige (4f′), nitrogen blue, oxygen red and hydrogen purple. The hydrogen bonds are drawn in nattier blue lines.

Fig. 4. Plausible Binding Modes for Compound 4m in the Binding Site of ScR1

The compound is color coded as carbon black, nitrogen blue, oxygen red and hydrogen white. The hydrogen bonds are drawn in nattier blue lines.

Conclusion

The anticancer activity of the newly synthesized benzyloxyurea derivatives 4a–u and 4a′–g′ was estimated by studying their inhibitory effects on human leukemia cell line K562 and murine leukemia cell line L1210. Some compounds showed improved anticancer activity on L1210 and K562 cell lines compared to HU. Among them, the most promising compounds were 4a, 4b, 4d, 4e, 4g, 4q, 4c′ and 4f′, showed stronger effect than that of HU in both L1210 and K562 cell lines. Among the 1,4-phenylene bis benzyloxyurea derivatives 4h–n, 4h, 4i, 4m and 4n were inactive no matter on K562 or L1210 cell line, indicating the bulky substituents on the HU nitrogen had a negative effect on the activity. Then, a molecular docking study of the four compounds revealed potential binding mode into the RR receptor, and explained the main structure–activity relationships. Moreover, flow cytometry analysis of compound 4g showed that it could induce apoptosis of K562 significantly than HU.

Experimental

Materials

Substituted benzyl chloride and acetoxime, as well as substituted anilines and 4-nitrophenyl chloroformate were purchased from Shanghai Da Rei Finechemical Co., Ltd., other commercial solvents were purchased from Sinopharm Group Co., Ltd. All reagents were used without further purification unless stated.

Apparatus

Infrared spectra were measured on KBr pellets on a Shimadzu FTIR-8400 spectrometer in the range of 4000–400 cm⁻¹. ¹H- and ¹³C-NMR spectra were performed on Brucker AV 600 spectrometer. Chemical shifts (δ values) and coupling constants (J values) were reported in parts per million (ppm) and hertz (Hz), respectively. Chemical shifts were relative to tetramethylsilane (TMS) except for solvents that were used, and the signals were quoted as s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). The mass spectra (electrospray ionization (ESI)) were recorded on Agilent 1100 MSD/TOF and Waters 2695 LC-ZQ4000. Melting points were obtained on a XA-4 instrument which was uncorrected.

Synthesis of Compounds 2a–g General Procedure

Intermediates 1a–g were prepared following our previous paper.¹⁹⁾ The obtained yellow oily liquid 1a–g were added dropwise to concentrated hydrochloric acid. The mixture was stirred at room temperature for 5 h and was distilled under reduced pressure. The formed white flake solid product was filtered off, washed with ethyl acetate to afford compounds 2a–g.

O-Benzyl-hydroxylamine Hydrochloride (2a)

Yield 59.2%, white solid, mp 231–233°C. IR (KBr) cm⁻¹: 2812, 2671, 1599, 1539, 1498, 1454, 897, 694. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 4.80 (s, 2H, CH₂), 5.46 (s, 2H, NH₂), 6.80 (s, 1H, ArH), 7.27 (s, 2H, ArH), 7.35 (d, J=9.6 Hz, 2H, ArH).

O-(4-Methylbenzyl)hydroxylamine Hydrochloride (2b)

Yield 49.9%, white solid, mp 224–225°C. IR (KBr) cm⁻¹: 2914, 2667, 1597, 1508, 1458, 851, 806. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 2.35 (s, 3H, CH₃), 4.91 (s, 2H, CH₂), 5.15 (s, 2H, NH₂), 7.17 (dd, J=7.2 Hz, J=4.2 Hz, 2H, ArH), 7.31 (d, J=7.8 Hz, 1H, ArH), 7.46 (d, J=7.8 Hz, 1H, ArH).

O-(4-Bromobenzyl)hydroxylamine Hydrochloride (2c)

Yield 55.0%, white solid, mp 202–204°C. IR (KBr) cm⁻¹: 2839, 2665, 1605, 1593, 1541, 1458, 810. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 4.93 (s, 2H, CH₂), 7.32 (dd, J=9.0 Hz, J=7.2 Hz, 3H, ArH), 7.54 (d, J=8.4 Hz, 1H, ArH).

O-(4-Fluorobenzyl)hydroxylamine Hydrochloride (2d)

Yield 40.5%, white solid, mp 221–223°C. IR (KBr) cm⁻¹: 2812, 2660, 1605, 1508, 1489, 1456, 864. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 4.80 (s, 2H, CH₂), 7.08 (m, 2H, ArH), 7.35 (dd, J=13.2 Hz, J=7.8 Hz, 1H, ArH), 7.53 (s, 1H, ArH).

O-(3-Fluorobenzyl)hydroxylamine Hydrochloride (2e)

Yield 36.7%, white solid, mp 197–199°C. IR (KBr) cm⁻¹: 2810, 2664, 1597, 1508, 1489, 1448, 787, 762. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 5.19 (s, 2H, CH₂), 7.12 (d, J=9.6 Hz, 2H, ArH), 7.17 (d, J=7.8 Hz, 2H, ArH).

O-(4-Chlorobenzyl)hydroxylamine Hydrochloride (2f)

Yield 43.8%, white solid, mp 224–226°C. IR (KBr) cm⁻¹: 2810, 2665, 1605, 1508, 1491, 1458, 814. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 4.93 (s, 2H, CH₂), 7.34 (t, J=7.9 Hz, 2H, ArH), 7.39 (d, J=8.4 Hz, 1H, ArH), 7.43 (s, 1H, ArH).

O-(3-Chlorobenzyl)hydroxylamine Hydrochloride (2g)

Yield 67.5%, white solid, mp 200–202°C. IR (KBr) cm⁻¹: 2806, 2665, 1599, 1576, 1510, 1493, 814. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 4.93 (s, 2H, CH₂), 7.36 (m, 4H, ArH).

Synthesis of Compounds 3a–g General Procedure

To a stirring suspension of 2a–g (0.1 mol) and pyridine (0.2 mol) in anhydrous CH₂Cl₂ (150 mL), 4-nitrophenyl chloroformate (0.1 mol) was added dropwise and reflux for 7 h. The resulting mixture was filtered and the filtrate was diluted with CH₂Cl₂ (100 mL) and washed with H₂O (200 mL×3). Then the organic phase was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure, the residue was recrystallized from chloroform : petroleum ether (5 : 1) to give compounds 3a–g.

1-(4-Nitrophenyl)-N-benzyloxy Carbamate (3a)

Yield 74.7%, white solid, mp 95–97°C. IR (KBr) cm⁻¹: 3275, 3082, 2943, 1724, 1524, 1481, 841. Matrix assisted laser desorption/ionization (MALDI)-MS m/z: 311.0639 (M+Na⁺, Calcd for C₁₄H₁₂N₂O₅Na: 311.0644).

1-(4-Nitrophenyl)-N-[O-(4-methylbenzyl)]carbamate (3b)

Yield 63.1%, white solid, mp 103–105°C. IR (KBr) cm⁻¹: 3271, 2936, 1728, 1543, 1481, 810. MALDI-MS m/z: 325.0794 (M+Na⁺, Calcd for C₁₅H₁₄N₂O₅Na: 325.0800).

1-(4-Nitrophenyl)-N–[O-(4-bromobenzyl)]carbamate (3c)

Yield 81.2%, white solid, mp 152–153°C. IR (KBr) cm⁻¹: 3271, 3074, 2936, 1728, 1543, 1481, 810. MS (ESI) m/z: 388.71 (M+Na⁺).

1-(4-Nitrophenyl)-N-[O-(4-fluorobenzyl)]carbamate (3d)

Yield 79.0%, white solid, mp 88–90°C. IR (KBr) cm⁻¹: 3302, 3082, 1736, 1524, 1481, 856. MS (ESI) m/z: 306.61 (M⁺).

1-(4-Nitrophenyl)-N-[O-(3-fluorobenzyl)]carbamate (3e)

Yield 83.3%, white solid, mp 88–90°C. IR (KBr) cm⁻¹: 3267, 3121, 3082, 2947, 1728, 1598, 1485, 817. MALDI-MS m/z: 329.0545 (M+Na⁺, Calcd for C₁₄H₁₁FN₂O₅Na: 329.0550).

1-(4-Nitrophenyl)-N-[O-(4-chlorobenzyl)]carbamate (3f)

Yield 61.3%, white solid, mp 137–139°C. IR (KBr) cm⁻¹: 3267, 3074, 2939, 1728, 1543, 1481, 814. MALDI-MS m/z: 345.0265 (M+Na⁺, Calcd for C₁₄H₁₁ClN₂O₅Na: 345.0254).

1-(4-Nitrophenyl)-N-[O-(3-chlorobenzyl)]carbamate (3g)

Yield 78.8%, white solid, mp 88–90°C. IR (KBr) cm⁻¹: 3271, 3074, 2928, 1724, 1543, 1477, 876, 795. MALDI-MS m/z: 345.0249 (M+Na⁺, Calcd for C₁₄H₁₁ClN₂O₅Na: 345.0254).

Synthesis of Compounds 4a–g General Procedure

To a stirring solution of compound 3a–g (6.2 mmol) and aniline (7.7 mmol) in CH₂Cl₂ (40 mL), anhydrous Et₃N (7.2 mmol) was added and stirred for 12 h at room temperature. The resulting mixture was washed successively with 1 mol/L NaHCO₃ (30 mL×3), 1 mol/L HCl (30 mL×2), H₂O (30 mL×2). The organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure, the residue was washed with anhydrous Et₂O (6 mL) and recrystallized from AcOEt and petroleum ether to afford target compounds 4a–g.

N-Phenyl-N′-benzyloxyurea (4a)

Yield, 34.4%. mp 106–107°C. IR (KBr) cm⁻¹: 3325, 3202, 3028, 2916, 2858, 1659, 1593, 1496, 1450, 1072, 949. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 4.90 (s, 2H, CH₂), 7.29 (t, J=7.9 Hz, 2H, ArH), 7.33 (m, 2H, ArH), 7.41 (m, 2H, ArH), 7.44 (dd, J=11.5, 4.0 Hz, 4H, ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 79.18, 119.60, 123.96, 129.24, 129.31, 129.39, 137.15, 144.51, 156.76. MALDI-MS m/z: 243.1129 (M+H⁺, Calcd for C₁₄H₁₅N₂O₂: 243.1134).

N-Phenyl-N′-(4-methylbenzyloxy)urea (4b)

Yield, 45.1%. mp 140–142°C. IR (KBr) cm⁻¹: 3333, 3202, 3028, 2916, 1858, 1663, 1593, 1535, 1447, 1072, 949. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 2.39 (s, 3H, CH₃), 4.85 (s, 2H, CH₂), 7.08 (d, J=7.3 Hz, 1H, ArH), 7.23 (d, J=7.7 Hz, 2H, ArH), 7.29 (m, 2H, ArH), 7.33 (m, 2H, ArH), 7.39 (s, 1H, NH), 7.44 (m, 2H ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 21.26, 79.02, 119.51, 123.87, 128.98, 129.47, 129.66, 132.07, 137.25, 139.25. 156.74. MALDI-MS m/z: 257.1285 (M+H⁺, Calcd for C₁₅H₁₇N₂O₂: 257.1290).

N-Phenyl-N′-(4-bromobenzyloxy)urea (4c)

Yield, 4.0%. mp 146–147°C. IR (KBr) cm⁻¹: 3356, 3179, 3086, 2924, 2862, 1666, 1593, 1535, 1446, 1069, 952, 934, 799. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 4.86 (s, 2H, CH₂), 7.03 (s, 1H, NH), 7.09 (s, 1H, ArH), 7.32 (dd, J=7.9, 2.8 Hz, 4H, ArH), 7.36 (d, J=7.6 Hz, 2H, ArH), 7.40 (s, 1H, NH), 7.56 (d, J=8.3 Hz, 2H, ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 78.24, 119.62, 123.44, 124.10, 129.07, 130.95, 132.15, 134.01, 137.04, 156.76. MALDI-MS m/z: 321.0242 (M+H⁺, Calcd for C₁₄H₁₄BrN₂O₂: 321.0239).

N-Phenyl-N′-(4-fluorobenzyloxy)urea (4d)

Yield, 5.2%. mp 127–128°C. IR (KBr) cm⁻¹: 3344, 3194, 3082, 2920, 2866, 1663, 1597, 1535, 1446, 1218, 1068, 948, 822. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 4.87 (s, 2H, CH₂), 7.10 (m, 3H, ArH), 7.31 (t, J=7.9 Hz, 2H, ArH), 7.36 (d, J=7.6 Hz, 2H, ArH), 7.41 (s, 1H, NH), 7.42 (m, 2H, ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 78.22, 115.94, 119.49, 124.03, 129.06, 130.99, 131.34, 137.10, 156.86, 162.40. MALDI-MS m/z: 261.1028 (M+H⁺, Calcd for C₁₄H₁₄FN₂O₂: 261.1039).

N-Phenyl-N′-(3-fluorobenzyloxy)urea (4e)

Yield, 40.8%. mp 105–107°C. IR (KBr) cm⁻¹: 3321, 3202, 3078, 2866, 1659, 1597, 1535, 1446, 1254, 1072, 956, 914. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 4.89 (s, 2H, CH₂), 7.08 (dd, J=12.5, 4.9 Hz, 2H, ArH), 7.15 (d, J=9.2 Hz,1H, ArH), 7.21 (d, J=7.6 Hz, 1H, ArH), 7.30 (t, J=7.9 Hz, 2H, ArH), 7.37 (m, 3H, ArH), 7.39 (s, 1H, NH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 78.19, 116.06, 119.60, 124.74, 129.05, 130.57, 137.08, 137.53, 156.84, 162.13. MALDI-MS m/z: 261.1028 (M+H⁺, Calcd for C₁₄H₁₄FN₂O₂: 261.1039).

N-Phenyl-N′-(4-chlorobenzyloxy)urea (4f)

Yield, 13.3%. mp 144–146°C. IR (KBr) cm⁻¹: 3356, 3182, 3086, 2957, 2866, 1666, 1597, 1575, 1446, 1207, 1087, 952. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 4.86 (s, 2H, CH₂), 7.09 (t, J=7.3 Hz, 1H, ArH), 7.31 (t, J=7.9 Hz, 2H, ArH), 7.37 (m, 4H, ArH), 7.39 (m, 2H, ArH), 7.40 (s, 1H, NH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 78.22, 119.63, 124.09, 129.07, 129.17, 130.69, 133.54, 135.26, 137.06, 156.86. MALDI-MS m/z: 277.0752 (M+H⁺, Calcd for C₁₄H₁₄ClN₂O₂: 277.0744). In addition, the exact stereostructure of 4f have been determined by X-ray crystal structure analysis in our previous paper.²⁶⁾

N-Phenyl-N′-(3-chlorobenzyloxy)urea (4g)

Yield, 23.2%. mp 117–119°C. IR (KBr) cm⁻¹: 3325, 3229, 3059, 2936, 2835, 1659, 1596, 1528, 1447, 1211, 1061, 937, 810. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 4.87 (s, 2H, CH₂), 7.09 (t, J=7.4 Hz, 1H, ArH), 7.17 (s, 1H, NH), 7.31 (dd, J=10.6, 5.1 Hz, 3H, ArH), 7.35 (d, J=7.9 Hz, 1H, ArH), 7.37 (m, 2H, ArH), 7.40 (s, 1H, NH), 7.43 (d, J=8.0 Hz, 2H, ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 78.17, 119.63, 124.07, 127.30, 129.15, 129.36, 130.24, 134.85, 137.08, 156.83. MALDI-MS m/z: 277.0741 (M+H⁺, Calcd for C₁₄H₁₄ClN₂O₂: 277.0744).

Synthesis of Compounds 4h–n General Procedure

To a stirring solution of compound 3a–g (2.4 mmol) and anhydrous Et₃N (7.2 mmol) in CH₂Cl₂ (15 mL), p-phenylenediamine (1.2 mmol) was added and stirred for 8 h at room temperature. The resulting mixture was filtered and the solid was recrystallized from DMF and anhydrous Et₂O to afford target compounds 4h–n.

N,N′-(1,4-Phenylene)-bis-(N″-benzyloxy)urea (4h)

Yield, 43.9%. mp 224–225°C. IR (KBr) cm⁻¹: 3302, 3221, 3067, 3032, 2912, 1858, 1655, 1535, 1443, 1219, 1083, 949, 820. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.82 (s, 4H, CH₂), 7.34 (m, 2H, ArH), 7.38 (m, 8H, ArH), 7.46 (m, 4H, ArH), 8.63 (s 2H, NH), 9.41 (s, 2H, NH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 77.41, 119.79, 127.95, 128.12, 128.72, 133.71, 136.41, 157.08. MALDI-MS m/z: 407.1729 (M+H⁺, Calcd for C₂₂H₂₃N₄O₄: 407.1719).

N,N′-(1,4-Phenylene)-bis-[N″-(4′-methylbenzyloxy)]urea (4i)

Yield, 50.4%. mp 231–232°C. IR (KBr) cm⁻¹: 3321, 3213, 3074, 2916, 1858, 1659, 1520, 1416, 1238, 1072, 952, 806. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 9.36 (s, 2H, NH), 8.58 (s 2H, NH), 7.38 (s, 4H, ArH), 7.34 (s, 4H, ArH), 7.20 (d, 4H, ArH), 4.76 (s, 4H, CH₂), 2.30 (s, 6H, CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 20.70, 77.28, 119.76, 128.67, 128.87, 133.34, 137.23, 133.70, 157.05. MALDI-MS m/z: 435.2027 (M+H⁺, Calcd for C₂₄H₂₇N₄O₄: 435.2032).

N,N′-(1,4-Phenylene)-bis-[N″-(4′-bromobenzyloxy)]urea (4j)

Yield, 32.7%. mp 279–281°C. IR (KBr) cm⁻¹: 3314, 3213, 3082, 2920, 2862, 1659, 1520, 1416, 1072, 953, 795. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.79 (s, 4H, CH₂), 7.39 (s, 4H, ArH), 7.43 (d, J=8.4 Hz, 4H, ArH), 7.58 (m, 4H, ArH), 8.68 (s 2H, NH), 9.40 (s, 2H, NH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 76.49, 119.87, 121.15, 130.89, 133.01, 133.71, 135.91, 157.06. MALDI-MS m/z: 564.9891 (M+H⁺, Calcd for C₂₂H₂₁Br₂N₄O₄: 564.9909).

N,N′-(1,4-Phenylene)-bis-[N″-(4′-fluorobenzyloxy)]urea (4k)

Yield, 62.2%. mp 241–242°C. IR (KBr) cm⁻¹: 3306, 3217, 3078, 2912, 2862, 1655, 1512, 1416, 1227, 1080, 949, 818. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.79 (s, 4H, CH₂), 7.21 (m, 4H, ArH), 7.39 (s, 4H, ArH), 7.51 (m, 4H, ArH), 8.65 (s 2H, NH), 9.40 (s, 2H, NH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 76.53, 114.88, 119.87, 131.03, 132.67, 133.71, 157.06, 161.06. MALDI-MS m/z: 443.1524 (M+H⁺, Calcd for C₂₂H₂₁F₂N₄O₄: 443.1531).

N,N′-(1,4-Phenylene)-bis-[N″-(3′-fluorobenzyloxy)]urea (4l)

Yield, 25.7%. mp 216–217°C. IR (KBr) cm⁻¹: 3310, 3221, 3082, 2912, 2878, 1663, 1593, 1450, 1223, 1092, 1072, 960, 941, 864. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.84 (s, 4H, CH₂), 7.16 (td, J=8.6, 2.4 Hz, 2H, ArH), 7.28 (d, J=7.6 Hz, 2H, ArH), 7.33 (d, J=9.6 Hz, 2H, ArH), 7.42 (m, 6H, ArH), 8.71 (s 2H, NH), 9.44 (s, 2H, NH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS): δ 77.06, 115.20, 115.68, 120.48, 125.06, 130.63, 134.31, 140.05, 157.68, 161.77. MALDI-MS m/z: 443.1530 (M+H⁺, Calcd for C₂₂H₂₁F₂N₄O₄: 443.1531).

N,N′-(1,4-Phenylene)-bis-[N″-(4′-chlorobenzyloxy)]urea (4m)

Yield, 33.1%. mp 236–238°C. IR (KBr) cm⁻¹: 3314, 3198, 3078, 2920, 2862, 1659, 1519, 1415, 1238, 1088, 953, 799. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.81 (s, 4H, CH₂), 7.39 (s, 4H, ArH), 7.44 (m, 4H, ArH), 7.49 (d, J=8.4 Hz, 4H, ArH), 8.66 (s, 2H, NH), 9.39 (s, 2H, NH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 76.44, 119.86, 128.08, 130.59, 132.56, 133.71, 135.49, 157.06. MALDI-MS m/z: 475.0932 (M+H⁺, Calcd for C₂₂H₂₁Cl₂N₄O₄: 475.0940).

N,N′-(1,4-Phenylene)-bis-[N″-(3′-chlorobenzyloxy)]urea (4n)

Yield, 28.4%. mp 237–239°C. IR (KBr) cm⁻¹: 3318, 3202, 3082, 2920, 2866, 1659, 1543, 1416, 1219, 1076, 953, 783. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.83 (s, 4H, CH₂), 7.41 (m, 10H, ArH), 7.57 (s, 2H, ArH), 8.74 (s, 2H, NH), 9.45 (s, 2H, NH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ:76.98, 120.49, 127.76, 128.36, 128.92, 130.54, 133.38, 134.29, 139.66, 157.67. MALDI-MS m/z: 475.0937 (M+H⁺, Calcd for C₂₂H₂₁Cl₂N₄O₄: 475.0940).

Synthesis of Compounds 4o–u General Procedure

To a stirring solution of 4-NH₂ substituted aniline (3.0 mmol) and anhydrous Et₃N (7.2 mmol) in CH₂Cl₂ (25 mL), compounds 3a–g (3.0 mmol) were added and stirred for 18 h at room temperature. The resulting mixture was filtered and the filtrate was adjusted to pH=1 with 1 mol/L HCl, diluted with CH₂Cl₂ (30 mL) and extracted with H₂O (40 mL×3). The water phases were combined and adjusted to pH >7 with Na₂CO₃. Subsequently, the solution was extracted with CH₂Cl₂ (40 mL×3), the combined extracts were dried over anhydrous Na₂SO₄, filtered and concentrated to afford oil liquid, which was recrystallized from CH₂Cl₂ and petroleum ether to give target compounds 4o–u.

N-(4-Aminophenyl)-N′-benzyloxyurea (4o)

Yield 8.79%. mp 115–117°C. IR (KBr) cm⁻¹: 3410, 3325, 3179, 3051, 2916, 2858, 1659, 1512, 1439, 1234, 1072, 960, 848. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 3.57 (s, 2H, NH₂), 4.88 (s, 2H, CH₂), 6.63 (d, J=8.7 Hz, 2H, ArH), 7.04 (s, 1H, NH), 7.10 (d, J=8.7 Hz, 2H, ArH), 7.22 (s, 1H, NH), 7.41 (m, 5H, ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 78.97, 115.51, 122.09, 128.32, 128.91, 129.10, 129.24, 129.33, 135.24, 143.18, 157.41. MALDI-MS m/z: 258.1233 (M+H⁺, Calcd for C₁₄H₁₆N₃O₂: 258.1243).

N-(4-Aminophenyl)-N′-(4-methylbenzyloxy)urea (4p)

Yield, 17.0%. mp 99–101°C. IR (KBr) cm⁻¹: 3410, 3333, 3190, 3074, 2924, 2851, 1659, 1512, 1435, 1230, 1072, 960, 833, 806, 756. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 2.37 (s, 3H,CH₃), 3.56 (s, 2H, NH₂), 4.83 (s, 2H, CH₂), 6.63 (m, 2H, ArH), 7.00 (s, 1H, NH), 7.10 (m, 2H, ArH), 7.22 (s, 1H, NH), 7.26 (d, J=7.8 Hz, 2H, ArH), 7.32 (d, J=7.9 Hz, 2H, ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 21.25, 78.83, 115.50, 122.04, 128.41, 129.42, 129.57, 132.23, 139.06, 143.13, 157.46. MALDI-MS m/z: 272.1407 (M+H⁺, Calcd for C₁₅H₁₈N₃O₂: 272.1399).

N-(4-Aminophenyl)-N′-(4-bromobenzyloxy)urea (4q)

Yield, 8.0%. mp 137–139°C. IR (KBr) cm⁻¹: 3406, 3333, 3202, 3071, 2924, 2858, 1655, 1528, 1435, 1269, 1234, 1069, 930, 829, 799. ¹H-NMR(600 MHz, CDCl₃, TMS) δ: 3.58 (s, 2H, NH₂), 4.83 (s, 2H, CH₂), 6.64 (m, 2H, ArH), 6.97 (s, 1H, NH), 7.12 (m, 2H, ArH), 7.20 (s, 1H, NH), 7.30 (d, J=8.3 Hz, 2H, ArH), 7.55 (d, J=8.3 Hz, 2H, ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 78.08, 115.55, 122.2, 123.30, 128.12, 130.93, 132.08, 134.19, 143.35, 157.24. MALDI-MS m/z: 336.0356 (M+H⁺, Calcd for C₁₄H₁₅BrN₃O₂: 336.0348).

N-(4-Aminophenyl)-N′-(4-fluorobenzyloxy)urea (4r)

Yield, 10.8%. mp 132–134°C. IR (KBr) cm⁻¹: 3406, 3325, 3190, 3071, 2928, 2855, 1658, 1512, 1439, 1269, 1219, 1072, 960, 833, 793. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 3.58 (s, 2H, NH₂), 4.85 (s, 2H, CH₂), 6.64 (m, 2H, ArH), 6.97 (s, 1H, NH), 7.11 (m, 4H, ArH), 7.21 (s, 1H, NH), 7.41 (dd, J=8.5, 5.4 Hz, 2H, ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 78.07, 115.67, 115.87, 122.13, 128.19, 131.13, 131.20, 143.30, 157.31, 162.35. MALDI-MS m/z: 276.1139 (M+H⁺, Calcd for C₁₄H₁₅FN₃O₂: 276.1148).

N-(4-Aminophenyl)-N′-(3-fluorobenzyloxy)urea (4s)

Yield, 9.6%. mp 120–122°C. IR (KBr) cm⁻¹: 3414, 3325, 3163, 3078, 2932, 2858, 1658, 1550, 1512, 1438, 1242, 1065, 960, 833. ¹H-NMR(600 MHz, CDCl₃, TMS) δ: 3.58 (s, 2H, NH₂), 4.87 (s, 2H, CH₂), 6.64 (m, 2H, ArH), 7.08 (m, 2H, ArH), 7.10 (s, 1H, NH), 7.13 (d, J=8.7 Hz, 2H, ArH), 7.20 (d, J=7.6 Hz, 1H, ArH), 7.24 (s, 1H, NH), 7.38 (td, J=7.9, 5.9 Hz, 1H, ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 78.06, 115.72, 115.99, 122.17, 124.70, 128.15, 130.52, 137.68, 143.32, 157.28, 162.12. MALDI-MS m/z: 276.1142 (M+H⁺, Calcd for C₁₄H₁₅FN₃O₂: 276.1148).

N-(4-Aminophenyl)-N′-(4-chlorobenzyloxy)urea (4t)

Yield, 14.1%. mp 135–136°C. IR (KBr) cm⁻¹: 3414, 3337, 3202, 3071, 2928, 2858, 1655, 1528, 1435, 1269, 1234, 1087, 930, 829, 799,. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 3.58 (s, 2H, NH₂), 4.85 (s, 2H, CH₂), 6.64 (m, 2H, ArH), 6.69 (s, 1H, NH), 7.12 (m, 2H, ArH), 7.21 (s, 1H, NH), 7.38 (m, 4H, ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 78.03, 115.54, 122.18, 129.10, 130.66, 133.68, 135.13, 143.33, 157.25. MALDI-MS m/z: 292.0859 (M+H⁺, Calcd for C₁₄H₁₅ClN₃O₂: 292.0853).

N-(4-Aminophenyl)-N′-(3-chlorobenzyloxy)urea (4u)

Yield, 11.1%. mp 138–139.5°C. IR (KBr) cm⁻¹: 3406, 3329, 3167, 3082, 2920, 2858, 1659, 1543, 1439, 1269, 1234, 1072, 961, 790. ¹H-NMR (600 MHz, CDCl₃, TMS) δ: 3.58 (s, 2H, NH₂), 4.85 (s, 2H, CH₂), 6.64 (m, 2H, ArH), 7.04 (s, 1H, NH), 7.14 (m, 2H, ArH), 7.24 (s, 1H, NH), 7.30 (dt, J=7.1, 1.5 Hz,1H, ArH), 7.36 (dt, J=15.2, 4.8 Hz,2H, ArH), 7.42 (d, J=1.6 Hz,1H, ArH). ¹³C-NMR (150 MHz, CDCl₃, TMS) δ: 78.02, 115.54, 122.18, 127.27, 128.13, 129.20, 129.22, 130.19, 134.79, 137.22, 143.33, 157.22. MALDI-MS m/z: 292.0862 (M+H⁺, Calcd for C₁₄H₁₅ClN₃O₂: 292.0853).

Synthesis of Compounds 4a′–g′ General Procedure

A suspension of hydroxylamine hydrochloride (0.02 mol) and Na₂CO₃ (0.02 mol) in anhydrous CH₃OH (70 mL) was stirred at room temperature for 0.5 h. Compounds 3a–g were dissolved in anhydrous CH₂Cl₂ (140 mL) previously, and were added to the corresponding mixture and stirred for another 4 h. The reaction mixture was filtered, and the filtrate was evaporated, the residue was subjected to column chromatography (EtOAc/petroleum ether: 1/2) to give target compounds 4a′–g′.

N-Benzyloxy-N-hydroxyurea (4a′)

Yield 28.0%. mp 98–99°C. IR (KBr) cm⁻¹: 3329, 3053, 2941, 2876, 1676, 1455, 1073, 912, 755, 700. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.74 (s, 2H, CH), 7.32 (m, 1H, ArH), 7.36 (m, 2H, ArH), 7.42 (m, 2H, ArH), 8.51 (s, 1H, NH), 9.05 (s, 1H, NH), 9.66 (s, 1H, OH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 77.69, 128.47, 128.70, 129.09, 137.07, 161.57. MALDI-MS m/z: 182.0694 (M⁺, Calcd for C₈H₁₀N₂O₃:182.0691).

N-(4-Methylbenzyloxy)-N′-hydroxyurea (4b′)

Yield, 30.0%. mp 130–133°C. IR (KBr) cm⁻¹: 3414, 3315, 3212, 3061, 2924, 2863, 1647, 1516, 1427, 804. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 2.30 (s, 3H, CH₃), 4.68 (s, 2H, CH₂), 7.17 (d, J=7.8 Hz, 2H, ArH), 7.28 (d, J=7.8 Hz, 2H, ArH), 8.50 (s, 1H, NH), 9.01 (s, 1H, NH), 9.62 (s, 1H, OH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 21.34, 77.57, 129.25, 133.98, 137.7, 161.55. MALDI-MS m/z: 196.0845 (M⁺, Calcd for C₉H₁₂N₂O₃:196.0848).

N-(4-Bromobenzyloxy)-N′-hydroxyurea (4c′)

Yield, 22.0%. mp 133–136°C. IR (KBr) cm⁻¹: 3311, 3203, 2946, 2873, 1663, 1591, 1447, 1400, 788. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.71 (s, 2H, CH), 7.37 (d, J=8.3 Hz, 2H, ArH), 7.56 (d, J=8.3 Hz, 2H, ArH), 8.53 (s, 1H, NH), 9.10 (s, 1H, NH), 9.70 (s, 1H, OH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 76.77, 121.63, 131.25, 131.60, 136.59, 161.53. MALDI-MS m/z: 260.9874 (M+H⁺, Calcd for C₈H₁₀BrN₂O₃: 260.9875).

N-(4-Fluorobenzyloxy)-N′-hydroxyurea (4d′)

Yield, 20.1%. mp 112–114°C. IR (KBr) cm⁻¹: 3410, 3317, 3228, 2878, 1639, 1616, 1508, 1443, 860. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.71 (s, 2H, CH), 7.19 (m, 2H, ArH), 7.46 (dd, J=8.6, 5.7 Hz, 2H, ArH), 8.53 (s, 1H, NH), 9.08 (s, 1H, NH), 9.68 (s, 1H, OH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 76.85, 115.47, 131.35, 133.34, 161.58, 163.23. MALDI-MS m/z: 200.0589 (M⁺, Calcd for C₈H₉FN₂O₃: 200.0597).

N-(3-Fluorobenzyloxy)-N′-hydroxyurea (4e′)

Yield, 18.0%. mp 98–100°C. IR (KBr) cm⁻¹: 3403, 3311, 3227, 3059, 2932, 2855, 1631, 1454, 870, 785. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.76 (s, 2H, CH), 7.14 (td, J=8.4, 2.4 Hz, 1H, ArH), 7.23 (d, J=7.6 Hz, 1H, ArH), 7.28 (m, 1H, ArH), 7.40 (td, J=7.9, 6.1 Hz, 1H, ArH), 8.52 (s, 1H, NH), 9.12 (s, 1H, NH), 9.70 (s, 1H, OH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 76.17, 114.56, 114.88, 124.19, 130.08, 139.66, 161.01, 161.26, 162.87. MALDI-MS m/z: 200.0595 (M⁺, Calcd for C₈H₉FN₂O₃: 200.0597).

N-(4-Chlorobenzyloxy)-N′-hydroxyurea (4f′)

Yield, 32.0%. mp 138–139°C. IR (KBr) cm⁻¹: 3436, 3313, 3205, 2946, 2874, 1662, 1597, 1498, 1448, 803. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.72 (s, 2H, CH), 7.43 (m, 4H, ArH), 8.51 (s, 1H, NH), 9.08 (s, 1H, NH), 9.68 (s, 1H, OH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 76.73, 128.67, 130.93, 133.05, 136.18, 161.53. MALDI-MS m/z: 216.0297 (M⁺, Calcd for C₈H₉ClN₂O₃: 216.0302).

N-(3-Chlorobenzyloxy)-N′-hydroxyurea (4g′)

Yield, 25.0%. mp 102–103°C. IR (KBr) cm⁻¹: 3415, 3317, 3220, 3058, 1645, 1600, 1521, 1081, 912, 765. ¹H-NMR (600 MHz, DMSO-d₆, TMS) δ: 4.74 (s, 2H, CH), 7.3 (m, 3H, ArH), 7.51 (s, 1H, ArH), 8.53 (s, 1H, NH), 9.13 (s, 1H, NH), 9.71 (s, 1H, OH). ¹³C-NMR (150 MHz, DMSO-d₆, TMS) δ: 76.68, 127.46, 128.30, 128.67, 130.57, 133.41, 139.80, 161.55. MALDI-MS m/z: 216.0308 (M⁺, Calcd for C₈H₉ClN₂O₃: 216.0302).

Cytotoxicity Assay in Vitro

The murine leukemia cell line L1210 and human K562 leukemia cell line were purchased from Nanjing Keygen Biotech. Co., Ltd., China. The cell lines were cultured in improved RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin G and 100 IU/mL streptomycin sulfate in a humidified, 5% CO₂ atmosphere at 37°C. According to the growth situation cell lines, the culture solution was centrifuged every two to three days with low-speed. The logarithmic growth phase cells were collected for experiments. The cytotoxicities of the target compounds against L1210 and K562 cell lines were examined by the MTT assay. Tumor cells (1×10⁶ cells/mL) were inoculated in 96-well culture plates (90 µL/well). Then 10 µL of culture medium containing synthetic compound of various concentrations was added to the wells, then the cells were incubated for 48 h at 37°C in 5% CO₂ atmosphere. Twenty microliters of MTT was added at a final concentration of 5 mg/mL and after 4 h incubation, 100 µL Triple solution (10% SDS, 5% isobutanol, 0.01 mol/L HCl) were added. The suspension was placed in the dark incubator at 37°C overnight and the optical density was measured at 570 nm.

Apoptosis Assay

Flow cytometry was undertaken on the instrument of FACS Calibur (Beckman Coulter Co., U.S.A.) with Annexin V-fluorescein isothiocyanate (FITC)/PI, Nanjing Keygen Technology Development Co., China) as detection kits, and hydroxyurea as positive control drug. The logarithmic growth phase of K562 cell line was washed with Phosphate Buffered Saline (PBS, Nanjing Keygen Technology Development Co., China) and centrifuged for 5 min. The cells were seeded at a density of 5×10⁵ cells/well. After 24 h at 37°C in 5%CO₂ atmosphere, l–5×10⁵ K562 cells were collected and suspended with Binding Buffer (500 µL), then Annexin V-FITC (5 µL) and PI (5 µL) were added and incubated 10 min at room temperature. After that, the fluorescence of K562 cells were measured no more than 1 h using a flow cytometer.

Acknowledgment

This work was supported by the National Nature Science Fundation of China (Grant No. 81360469) and the program of Nanchang Department of Science and Technology (No. 2008368). The authors also thank Center of Analysis and Testing of Nanchang University for assistance with the MS and NMR testing of compounds.

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