4.1 General

A Waters Acquity UPLC Class I/Xevo G2 QTOF mass spectrometer (Milford, MA, USA) was used to obtain the high-resolution ESI–MS (HR-ESI–MS) data. 1H-NMR and 13C-NMR spectra were acquired on a Bruker Avance III 400 spectrometer spectrometer (Karlsruhe, Germany) with TMS as an internal standard. Melting points were determined using an X-4 digital micro-melting tester (Beijing Teck Instrument Co., Ltd.). Chromatographic analysis for purity determination (HPLC) was achieved on a Ultimate®XB-C18; 4.6 × 250 mm; 5 μm; column (Welch Materials, Inc. Shanghai, China). Gradient elution was performed starting at 5% MeOH increasing to 100% MeOH at 20 min, and detection was conducted at 254 nm. The ratio of peak areas in the chromatograms was used to express the purity in percentage.

4.2 Synthetic procedures4.2.1 General procedure for the synthesis of intermediates 2

To a 100 mL flask, hydroxybenzoic acids 1 (0.1 mmol) and acetic anhydride (10 mL) were added. While the mixture was stirred, concentrated sulfuric acid (0.15 mL) was drip in. The reaction mixture was refluxed at 80 °C for 2 h. By adding 60 mL distilled water, the white precipitate was formed, and then was filtered and dried to obtain acetoxybenzoic acid intermediate 2.

4.2.2 General procedure for the synthesis of intermediates 4

Methyl isothioureide sulfate (3, 0.025 mmol), 30% K2CO3 solution (12.5 mL), and di-tert-butyl dicarbonate (0.1 mol) were dissolved in CH2Cl2 (25 mL) and stirred for 24 h at room temperature. The crude product was washed with water, and was purified by silica gel column chromatography using petroleum ether/ethyl acetate (4:1, v/v) as eluent to give compound 4 (Yield 98%) as an off- white solid.

4.2.3 General procedure for the synthesis of intermediates 5 and 10

A solution of 4 (7.5 mmo1) in CH2Cl2 (20 mL) was added in batches to a alkyl diamine (1, 3-propylene diamine, or 1, 4-butyric diamine, or 1, 5-pentylenediamine) (0.03 mol). The reaction mixture was stirred at 50 °C for 4 h. The crude product was washed with water to give series compounds 5. Series compounds 10 was prepared following the method described for the preparation of series compounds 5, employing 3-amino-propanol or 4-amino-1-butanol instead of alkyl diamine.

4.2.4 General procedure for the synthesis of intermediates 6a–6c, 8a–8u, and 11a–11e

Intermediates 2 (0.04 mol, 1.0 eq) and 5 (1.0 eq) were dissolved in DCM (20 mL) or DMF (20 mL) solution. To this solution, DCC (1.2 eq) and DMAP (0.1 eq) were added. The resulting reaction mixture was stirred for 24 h at room temperature. The reaction progress was detected by TLC. After completion of the reaction, a large amount of by-product DCU would be precipitated in the solution and filtered. Excess solvent of filtrate was removed by evaporation under reduced pressure to obtain the corresponding amide products 8a–8u. The method described above was used to synthesize amide products 11a–11e, employing series compounds 10 instead of series compounds 5. Similarly, amide products 6a–6c were also synthesized using series compounds 5 with 2-picolinic acid, nicotinic acid, isonicotinic acid, respectively.

4.2.5 Procedure for the synthesis of 7a–7c

A mixture of compounds 6a–6c (0.41 mmol, 1.0 eq) and zinc bromide (2.1 mmol, 5.0 eq) was stirred in DCM at room temperature for 10 h. The reaction progress was detected by TLC. After the completion of the reaction, the organic phase was washed with water, and further purified over Sephadex LH-20 eluted with MeOH to obtain target compounds 7a–7c.

N-(4-guanidinobutyl)-2-pyridinecarboxamide (7a). Yield 72%, white solid. Mp: 171–173 °C; 1H NMR (D2O, 400 MHz) δ 8.55 (m, 1H, ArH), 7.95 (m, 2H, ArH), 7.56 (m, 1H, ArH), 3.41 (t, J = 6.4 Hz, 2H), 3.17 (t, J = 6.6 Hz, 2H), 1.62 (m, 4H); 13C NMR (100 MHz, D2O) δ 167.2, 156.6, 149.0, 148.7, 138.4, 127.1, 122.3, 40.6, 38.9, 25.6, 25.2; HR-ESI-MS (positive mode) m/z: 236.1515 [M + H]+ (calculated for C11H18N5O, 236.1511); Purity (HPLC): 95.1%.

N-(4-guanidinobutyl)-3-pyridinecarboxamide (7b). Yield 85%, white solid. Mp: 190–192 °C; 1H NMR (D2O, 400 MHz) δ 8.76 (d, J = 1.7 Hz, 1H, ArH), 8.60 (dd, J = 1.7, 5.0 Hz, 1H, ArH), 8.10 (m, 1H, ArH), 7.51 (dd, J = 7.7, 5.0 Hz, 1H, ArH), 3.34 (t, J = 6.1 Hz, 2H), 3.14 (t, J = 6.4 Hz, 2H), 1.59 (m, 4H); 13C NMR (100 MHz, D2O) δ 168.0, 156.6, 150.8, 146.7, 136.3, 130.2, 124.3, 40.6, 39.3, 25.5, 25.2; HR-ESI-MS (positive mode) m/z: 236.1517 [M + H]+ (calculated for C11H18N5O, 236.1511); Purity (HPLC): 97.7%.

N-(4-guanidinobutyl)-4-pyridinecarboxamide (7c). Yield 71%, white solid. Mp: 108–110 °C; 1H NMR (D2O, 400 MHz) δ 8.62 (d, J = 6.2 Hz, 2H, ArH), 7.64 (d, J = 6.2 Hz, 2H, ArH), 3.38 (t, J = 6.3 Hz, 2H), 3.17 (t, J = 6.5 Hz, 2H), 1.62 (m, 4H); 13C NMR (100 MHz, D2O) δ 168.6, 156.6, 149.4 (2C), 142.2, 121.5 (2C), 40.6, 39.3, 25.4, 25.2; HR-ESI-MS (positive mode) m/z: 236.1524 [M+H]+ (calculated for C11H18N5O, 236.1511); Purity (HPLC): 99.7%.

4.2.6 Procedure for the synthesis of 9a–9u

Compounds 8a–8u (1.16 mmol) and trifluoroacetic acid (1.16 mmol) were dissolved in Dichloromethane (10 mL). The resulting reaction mixture was heated at 50 °C for 24 h. After completion of the reaction, allowed it to room temperature and filtered. The crude product was recrystallized from MeOH to yield the final compounds 9a–9u.

N-(3-guanidinobutyl)-2-hydroxybenzamide (9a). Yield 75%, white solid. Mp: 118–120 °C; 1H NMR (D2O, 400 MHz) δ 7.66 (d, J = 7.9 Hz, 1H, ArH), 7.43 (t, J = 7.6 Hz, 1H, ArH), 6.97 (m, 2H, ArH), 3.45 (t, J = 6.6 Hz, 2H), 3.24 (t, J = 6.6 Hz, 2H), 1.87 (qui, J = 6.6 Hz, 2H); 13C NMR (100 MHz, D2O) δ 170.8, 158.0, 157.6, 134.9, 129.1, 120.9, 117.9, 117.4, 39.6, 37.5, 28.5; HR-ESI-MS (positive mode) m/z: 237.1378 [M + H]+ (calculated for C11H17N4O2, 237.1352); Purity (HPLC): 98.6%.

N-(3-guanidinobutyl)-3-hydroxybenzamide (9b). Yield 82%, white solid. Mp: 130–132 °C; 1H NMR (D2O, 400 MHz) δ 7.35 (t, J = 7.8 Hz, 1H, ArH), 7.25 (d, J = 7.8 Hz, 1H, ArH), 7.17 (s, 1H, ArH), 7.05 (d, J = 7.8 Hz, 1H, ArH), 3.43 (t, J = 6.7 Hz, 2H), 3.24 (t, J = 6.7 Hz, 2H), 1.87 (qui, J = 6.7 Hz, 2H); 13C NMR (100 MHz, D2O) δ 171.9, 158.0, 157.0, 136.5, 131.5, 120.24, 120.20, 115.1, 39.9, 38.2, 28.8; HR-ESI-MS (positive mode) m/z: 237.1378 [M + H]+ (calculated for C11H17N4O2, 237.1352); Purity (HPLC): 99.2%.

N-(3-guanidinobutyl)-4-hydroxybenzamide (9c). Yield 80%, white oil. 1H NMR (D2O, 400 MHz) δ 7.63 (d, J = 8.6 Hz, 2H, ArH), 6.90 (d, J = 8.6 Hz, 2H, ArH), 3.39 (t, J = 6.8 Hz, 2H), 3.21 (t, J = 6.8 Hz, 2H), 1.84 (m, 2H); 13C NMR (100 MHz, D2O) δ 170.4, 159.1, 156.7, 129.1 (2C), 125.3, 115.3 (2C), 38.6, 36.8, 27.5; HR-ESI-MS (positive mode) m/z: 237.1374 [M + H]+ (calculated for C11H17N4O2, 237.1352); Purity (HPLC): 97.8%.

N-(4-guanidinobutyl)-3-methoxy-4-hydroxybenzamide (9d). Yield 77%, white oil. 1H NMR (D2O, 400 MHz) δ 7.20 (m, 2H, ArH), 6.86 (d, J = 8.2 Hz, 1H, ArH), 3.81 (s, 3H), 3.36 (t, J = 6.7 Hz, 2H), 3.19 (t, J = 6.7 Hz, 2H), 1.82 (qui, J = 6.7 Hz, 2H); 13C NMR (100 MHz, D2O) δ 170.8, 158.0, 149.8, 148.3, 126.6, 122.1, 116.1, 111.9, 56.9, 40.1, 38.3, 29.1; HR-ESI-MS (positive mode) m/z: 267.1474 [M + H]+ (calculated for C12H19N4O3, 267.1457); Purity (HPLC): 99.4%.

N-(3-guanidinobutyl)-2,4-dihydroxybenzamide (9e). Yield 83%, white solid. Mp: 178–180 °C; 1H NMR (D2O, 400 MHz) δ 7.58 (d, J = 8.7 Hz, 1H, ArH), 6.43 (dd, J = 2.4, 8.7 Hz, 1H, ArH), 6.37 (d, J = 2.4 Hz, ArH), 3.41 (t, J = 6.7 Hz, 2H) 3.22 (t, J = 6.6 Hz, 2H), 1.85 (m, 2H); 13C NMR (100 MHz, D2O) δ 169.6, 160.6, 160.0, 156.5, 129.3, 107.8, 107.6, 102.6, 38.6, 36.3, 27.6; HR-ESI-MS (positive mode) m/z: 253.1297 [M + H]+ (calculated for C11H17N4O3, 253.1301); Purity (HPLC): 99.8%.

N-(3-guanidinobutyl)-3,4-dihydroxybenzamide (9f). Yield 74%, white oil. 1H NMR (D2O, 400 MHz) δ 7.10 (br.s, 1H, ArH), 7.03 (d, J = 7.0 Hz, 1H, ArH), 6.76 (d, J = 7.0 Hz, 1H, ArH), 3.22 (br.s, 2H), 3.05 (br.s, 2H), 1.69 (br.s, 2H); 13C NMR (100 MHz, D2O) δ 169.4, 156.4, 147.7, 143.6, 125.2, 120.1, 115.3, 114.5, 38.6, 36.8, 27.6; HR-ESI-MS (positive mode) m/z: 253.1331 [M + H]+ (calculated for C11H17N4O3, 253.1301); Purity (HPLC): 97.8%.

N-(3-guanidinobutyl)-3,5-dihydroxybenzamide (9g). Yield 72%, white solid. Mp: 68–70 °C; 1H NMR (D2O, 400 MHz) δ 6.68 (d, J = 2.2 Hz, 2H, ArH), 6.48 (t, J = 2.2 Hz, 1H, ArH), 3.35 (t, J = 6.8 Hz, 2H), 3.18 (t, J = 6.7 Hz, 2H), 1.81 (m, 2H); 13C NMR (100 MHz, D2O) δ 170.2, 157.0 (2C), 156.7, 136.2, 106.2 (2C), 105.8, 38.6, 36.9, 27.4; HR-ESI-MS (positive mode) m/z: 253.1348 [M + H]+ (calculated for C11H17N4O3, 253.1301); Purity (HPLC): 99.3%.

N-(3-Guanidinobutyl)-3,4,5-trihydroxybenzamide (9h). Yield 73%, white solid. Mp: 77–79 °C; 1H NMR (D2O, 400 MHz) δ 6.74 (s, 2H, ArH), 3.23 (t, J = 6.8 Hz, 2H), 3.08 (t, J = 6.8 Hz, 2H), 1.71 (qui, J = 6.8 Hz, 2H); 13C NMR (100 MHz, D2O) δ 169.6, 156.6, 144.6 (2C), 136.2, 124.8, 107.3 (2C), 38.7, 36.9, 27.6; HR-ESI-MS (positive mode) m/z: 269.1272 [M + H]+ (calculated for C11H17N4O4, 269.1250); Purity (HPLC): 92.8%.

N-(4-guanidinobutyl)-2-hydroxybenzamide (9i). Yield 80%, white oil. 1H NMR (400 MHz, CD3OD) δ 7.60 (d, J = 7.4 Hz, 1H, ArH), 7.20 (t, J = 8.2 Hz, 1H, ArH), 6.73 (m, 2H, ArH), 3.27 (t, J = 6.2 Hz, 2H), 3.06 (t, J = 6.3 Hz, 2H), 1.51 (m, 4H); 13C NMR (100 MHz, CD3OD) δ 171.2, 161.3, 158.8, 134.8, 129.0, 120.2, 118.6, 117.2, 42.2, 39.9, 27.8, 27.4; HR-ESI-MS (positive mode) m/z: 251.1523 [M + H]+ (calculated for C12H19N4O2, 251.1508); Purity (HPLC): 96.1%.

N-(4-guanidinobutyl)-3-hydroxybenzamide (9j). Yield 83%, white solid. Mp: 84–86 °C; 1H NMR (400 MHz, CD3OD) δ 7.17 (m, 3H, ArH), 6.87 (br.s, 1H, ArH), 3.32 (br.s, 2H), 3.15 (br.s, 2H), 1.58 (br.s, 4H); 13C NMR (100 MHz, CD3OD) δ 170.6, 158.9, 158.7, 137.1, 130.7, 119.6, 119.1, 115.3, 42.1, 40.2, 27.8, 27.3; HR-ESI-MS (positive mode) m/z: 251.1530 [M + H]+ (calculated for C12H19N4O2, 251.1508); Purity (HPLC): 96.0%.

N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide (9k). Yield 85%, white solid. Mp: 105–107 °C; 1H NMR (400 MHz, D2O) δ 7.28 (d, J = 2.0 Hz, 1H, ArH), 7.24 (dd, J = 2.0, 8.2 Hz, 1H, ArH), 6.89 (d, J = 8.2 Hz, 1H, ArH), 3.83 (s, 3H), 3.32 (d, J = 6.2 Hz, 2H), 3.15 (d, J = 6.4 Hz, 2H), 1.59 (br.s, 4H); 13C NMR (100 MHz, D2O) δ 169.9, 156.6, 148.4, 147.1, 125.7, 120.8, 115.0, 110.9, 55.7, 40.6, 39.2, 25.6, 25.2; HR-ESI-MS (positive mode) m/z: 281.1634 [M + H]+ (calculated for C13H21N4O3, 281.1614); Purity (HPLC): 95.4%.

N-(4-guanidinobutyl)-2,4-dihydroxybenzamide (9l). Yield 76%, white solid. Mp: 74–76 °C; 1H NMR (400 MHz, D2O) δ 7.45 (d, J = 8.8 Hz, 1H, ArH), 6.36 (dd, J = 2.4, 8.8 Hz, 1H, ArH), 6.27 (d, J = 2.4 Hz, 1H, ArH), 3.24 (br.s, 2H) 3.09 (d, J = 5.7 Hz, 2H), 1.53 (br.s, 4H); 13C NMR (100 MHz, D2O) δ 169.6, 160.7, 159.8, 156.6, 129.6, 108.5, 107.8, 102.8, 40.7, 38.7, 25.6, 25.2; HR-ESI-MS (positive mode) m/z: 267.1454 [M + H]+ (calculated for C12H19N4O3, 267.1457); Purity (HPLC): 95.1%.

N-(4-guanidinobutyl)-3,4-dihydroxybenzamide (9m). Yield 73%, white solid. Mp: 157–159 °C; 1H NMR (400 MHz, CD3OD) δ 7.21 (d, J = 2.0 Hz, 1H, ArH), 7.12 (dd, J = 8.3, 2,0 Hz, 1H, ArH), 6.73 (d, J = 8.3 Hz, 1H, ArH), 3.24 (br.s, 2H) 3.10 (br.s, 2H), 1.51 (m, 4H); 13C NMR (100 MHz, CD3OD) δ 170.5, 158.7, 150.1, 146.2, 127.1, 120.8, 116.1, 115.9, 42.1, 40.2, 27.8, 27.2; HR-ESI-MS (positive mode) m/z: 267.1450 [M + H]+ (calculated for C12H19N4O3, 267.1457); Purity (HPLC): 95.2%.

N-(4-guanidinobutyl)-3,5-dihydroxybenzamide (9n). Yield 83%, white solid. Mp: 81–83 °C; 1H NMR (400 MHz, D2O) δ 6.68 (d, J = 2.2 Hz, 2H, ArH), 6.49 (t, J = 2.2 Hz, 1H, ArH), 3.30 (t, J = 6.2 Hz, 2H), 3.14 (t, J = 6.3 Hz, 2H), 1.57 (br.s, 4H); 13C NMR (100 MHz, D2O) δ 170.1, 157.0 (2C), 156.6, 136.4, 106.1 (2C), 105.8, 40.6, 39.2, 25.5, 25.2; HR-ESI-MS (positive mode) m/z: 267.1451 [M + H]+ (calculated for C12H19N4O3, 267.1457); Purity (HPLC): 98.9%.

N-(4-Guanidinobutyl)-3,4,5-trihydroxybenzamide (9o). Yield 71%, white solid. Mp: 102–104 °C; 1H NMR (400 MHz, D2O) δ 6.77 (s, 2H, ArH), 3.18 (br.s, 2H), 3.02 (br.s, 2H), 1.46 (br.s, 4H); 13C NMR (100 MHz, D2O) δ 169.5, 156.5, 144.6 (2C), 136.1, 124.9, 107.2 (2C), 40.6, 39.2, 25.6, 25.2; HR-ESI-MS (positive mode) m/z: 283.1425 [M + H]+ (calculated for C12H19N4O4, 283.1406); Purity (HPLC): 96.0%.

N-(5-guanidinopentyl)-2-hydroxybenzamide (9p). Yield 82%, white oil. 1H NMR (400 MHz, D2O) δ 7.79 (d, J = 7.4 Hz, 1H, ArH), 7.38 (t, 1H, J = 7.4 Hz, ArH), 7.11 (m, 2H, ArH), 3.51 (t, J = 6.5 Hz, 2H), 3.12 (t, J = 7.4 Hz, 2H), 1.81 (m, 4H), 1.57 (m, 2H); 13C NMR (100 MHz, D2O) δ 169.7, 156.9 (2C), 133.8, 128.1, 119.9, 116.9, 116.7, 39.2, 39.0, 27.8, 26.3, 22.9; HR-ESI-MS (positive mode) m/z: 265.1678 [M + H]+ (calculated for C13H21N4O2, 265.1665); Purity (HPLC): 97.6%.

N-(5-guanidinopentyl)-3-hydroxybenzamide (9q). Yield 86%, white oil. 1H NMR (400 MHz, CD3OD) δ 7.26 (m, 3H, ArH), 6.95 (d, J = 7.0 Hz, 1H, ArH), 3.39 (t, J = 7.0 Hz, 2H), 3.20 (t, J = 7.0 Hz, 2H), 1.66 (m, 4H), 1.46 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 170.5, 158.9, 158.7, 137.3, 130.1, 119.5, 119.0, 115.3, 42.4, 40.6, 30.2, 29.6, 25.0; HR-ESI-MS (positive mode) m/z: 265.1668 [M + H]+ (calculated for C13H21N4O2, 265.1665); Purity (HPLC): 99.9%.

N-(5-guanidinopentyl)-4-hydroxybenzamide (9r). Yield 79%, white oil. 1H NMR (400 MHz, CD3OD) δ 7.72 (d, J = 8.8 Hz, 2H, ArH), 6.84 (t, J = 8.8 Hz, 2H, ArH), 3.39 (t, J = 7.0 Hz, 1H), 2.94 (t, J = 7.6 Hz, 2H), 1.69 (m, 4H), 1.46 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 170.2, 162.1, 158.8, 130.2 (2C), 126.5, 116.1 (2C), 40.6, 40.4, 30.1, 28.2, 24.8; HR-ESI-MS (positive mode) m/z: 265.1672 [M + H]+ (calculated for C13H21N4O2, 265.1665); Purity (HPLC): 96.7%.

N-(5-guanidinopentyl)-4-hydroxy-3-methoxybenzamide (9s). Yield 85%, white oil. 1H NMR (400 MHz, CD3OD) δ 7.45 (d, J = 2.0 Hz, 1H, ArH), 7.37 (dd, J = 2.0, 8.2 Hz, 1H, ArH), 6.85 (d, J = 8.2 Hz, 1H, ArH), 3.91 (s, 3H), 3.39 (t, J = 7.0 Hz, 2H), 3.19 (t, J = 7.1 Hz, 2H), 1.65 (m, 4H), 1.45 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 170.0, 158.7, 151.2, 148.8, 126.9, 122.0, 115.9, 111.9, 56.5, 42.4, 40.7, 30.3, 29.6, 25.1; HR-ESI-MS (positive mode) m/z: 295.1788 [M + H]+ (calculated for C14H23N4O3, 295.1770); Purity (HPLC): 95.4%.

N-(5-guanidinopentyl)-3,5-dihydroxybenzamide (9t). Yield 78%, white solid. Mp: 88–90 °C; 1H NMR (400 MHz, CD3OD) δ 6.72 (d, J = 2.2 Hz, 2H, ArH), 6.44 (t, J = 2.2 Hz, 1H, ArH), 3.36 (t, J = 7.1 Hz, 2H), 3.19 (t, J = 7.1 Hz, 2H), 1.65 (m, 4H), 1.44 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 170.7, 159.9(2C), 158.7, 138.0, 106.7(2C), 106.5, 42.4, 40.6, 30.1, 29.5, 25.0; HR-ESI-MS (positive mode) m/z: 281.1626 [M + H]+ (calculated for C13H21N4O3,281.1614); Purity (HPLC): 99.8%.

N-(5-guanidinobutyl)-3,4,5-trihydroxybenzamide (9u). Yield 72%, white solid. Mp: 93–95 °C; 1H NMR (400 MHz, D2O) δ 6.80 (s, 2H, ArH), 3.22 (t, J = 6.9 Hz, 2H), 3.05 (t, J = 6.9 Hz, 2H), 1.49 (m, 4H), 1.28 (m, 2H); 13C NMR (100 MHz, D2O) δ 169.7, 156.5, 144.7 (2C), 136.1, 125.2, 107.3 (2C), 40.8, 39.5, 27.8, 27.4, 23.0; HR-ESI-MS (positive mode) m/z: 297.1602 [M+H]+ (calculated for C13H21N4O4, 297.1563); Purity (HPLC): 96.6%.

4.2.7 General procedure for the synthesis of intermediates 12a–12e

To a solution of compounds 11a–11e in MeOH (10 mL), 12% NaOH (10 mL) solution was added. The resulting mixture was stirred at room temperature for 10 min. The reaction progress was detected by TLC. After concentration, the crude product was obtained and purified by silica gel column chromatography using ethyl acetate as eluent to give compounds 12a–12e.

4.2.8 Procedure for the synthesis of 13a–13e

Compounds 13a–13e were synthesized by using the same way for 7a–7c. Nevertheless, compounds 12a–2e was used instead of compounds 6a–6c.

3-Guanidinopropyl 3-hydroxybenzoate (13a). Yield 77%, white solid. Mp: 140–142 °C; 1H NMR (400 MHz, CD3OD) δ 7.52 (dt, J = 7.9, 1.4 Hz, 1H, ArH), 7.45 (dd, J = 2.4, 1.4 Hz, 1H, ArH), 7.31 (t, J = 7.9 Hz, 1H, ArH), 7.05 (ddd, J = 7.9, 2.4, 1.4 Hz, 1H, ArH), 4.40 (t, J = 6.2 Hz, 2H), 3.39 (t, J = 6.9 Hz, 2H), 2.08 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 168.0, 158.9, 158.8, 132.5, 130.7, 121.6, 121.4, 117.1, 63.1, 39.5, 29.2; HR-ESI-MS (positive mode) m/z: 238.1196 [M + H]+ (calculated for C11H16N3O3, 238.1192); Purity (HPLC): 99.8%.

3-Guanidinopropyl 4-hydroxybenzoate (13b). Yield 73%, white oil. 1H NMR (400 MHz, CD3OD) δ 7.90 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 4.36 (t, J = 5.9 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 2.08 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 168.0, 163.0, 158.1, 132.6 (2C), 121.7, 116.0 (2C), 62.7, 39.4, 28.8; HR-ESI-MS (positive mode) m/z: 238.1189 [M + H]+ (calculated for C11H15N3O3, 238.1192); Purity (HPLC): 99.8%.

3-Guanidinopropyl 4-hydroxy-3-methoxybenzoate (13c). Yield 88%, white oil. 1H NMR (400 MHz, CD3OD) δ 7.58 (dd, J = 8.3, 1.9 Hz, 1H, ArH), 7.53 (d, J = 1.9 Hz, 1H, ArH), 6.86 (d, J = 8.3 Hz, 1H, ArH), 4.37 (t, J = 6.1 Hz, 2H), 3.91 (s, 3H), 3.40 (t, J = 6.9 Hz, 2H), 2.09 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 168.1, 158.1, 154.4, 148.4, 125.0 (2C), 115.8, 113.1, 62.8, 56.5, 39.4, 28.9; HR-ESI-MS (positive mode) m/z: 268.1302 [M + H]+ (calculated for C12H18N3O4, 268.1297); Purity (HPLC): 99.8%.

3-Guanidinopropyl 3,5-dihydroxybenzoate (13d). Yield 79%, white solid. Mp: 70–72 °C; 1H NMR (400 MHz, CD3OD) δ 6.95 (d, J = 2.2 Hz, 2H, ArH), 6.50 (t, J = 2.2 Hz, 1H, ArH), 4.37 (t, J = 6.2 Hz, 2H), 3.38 (t, J = 6.9 Hz, 2H), 2.07 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 168.0, 159.7 (2C), 158.5, 132.9, 108.8 (2C), 108.3, 63.0, 39.4, 29.1; HR-ESI-MS (positive mode) m/z: 254.1147 [M + H]+ (calculated for C11H16N3O4, 254.1141); Purity (HPLC): 98.3%.

4-Guanidinobutyl 4-hydroxybenzoate (13e). Yield 78%, white solid. Mp: 79–81 °C; 1H NMR (400 MHz, CD3OD) δ 7.88 (d, J = 8.8 Hz, 2H, ArH), 6.85 (d, J = 8.8 Hz, 2H, ArH), 4.31 (t, J = 6.1 Hz, 2H), 3.29 (t, J = 7.0 Hz, 2H), 1.85 (m, 2H), 1.76 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 168.2, 163.1, 158.1, 132.6 (2C), 121.9, 116.0 (2C), 65.1, 42.0, 26.8, 26.3; HR-ESI-MS (positive mode) m/z: 252.1362 [M + H]+ (calculated for C12H18N3O3, 252.1348); Purity (HPLC): 99.7%.

4.3 Molecular docking

Molecular docking was carried out using Discovery Studio 2017 R2 (Accelrys, San Diego, USA). Docking studies were conducted with DS CDOCKER program. The cryoelectron microscopy (cryo-EM) structure of NHE-1 from Homo sapiens (PBD ID: 7DSX) was obtained from the Protein Data Bank. All water molecules and allied ligand were removed followed by protein preparation protocol with the CHARMm force field. The simulated annealing parameters were set as follows: heating steps and cooling steps were set to 2000 and 5000, respectively, while heating and cooling temperatures were set to 700 and 300, respectively. Other parameters were kept as default. Ten top-ranked conformations for each docked compound were retained and visually inspected for binding pattern analysis using Discovery Studio Visualizer.

4.4 Biological activity4.4.1 Cell culture and cell viability assay

The H9c2 cardiomyocyte (rat myocardial cell line) and HEK 293 cell (Human Embryonic Kidney 293 cells) were obtained from ATCC (American type culture collection) and grown in Dulbecco’s modifed Eagle’s medium (DMEM) (Wuhan Pricella Biotechnology Co., Ltd.) with 10% fetal bovine serum (FBS, Wuhan Pricella Biotechnology Co., Ltd.) and 1% penicillin–streptomycin (Beijing Solarbio Science & Technology Co.,Ltd.) at 37 ℃ in a humidified incubator consisting of 5% CO2 and 95% air. After reaching about 70–80% confluence, the H9c2 cardiomyocyte was digested by trypsinization (0.25% Trypsin–EDTA solution, Beijing Solarbio Science&Technology Co.,Ltd) every three days. After seeded into the 96-well culture plates for 24 h with 3000 cells/well, the cells were divided into control group, Dox treated group, Dex treated group, buthutin A treated group, and compounds-treated group. The cells in Dox-treated group were treated with 1 μM Dox for 24 h, while the Dex-treated group, buthutin A treated group, and compounds-treated groups were treated with 20 μM Dex and 1 μM Dox, 1 μM buthutin A and 1 μM Dox, and 1 μM target compound and 1 μM Dox for 24 h, respectively. At the same time, control group cells were incubated continuously with the normal medium. After treatment, the MTT solution (Beijing Solarbio Science&Technology Co., Ltd.) with the final concentration of 0.5 mg/ml was added into each well and incubated for 4 h at 37 ℃. After formazan product in each well was dissolved, the absorbance of each well was measured at 570 nm by using a microplate reader (BioTek Instruments, Inc.) [21]. The protection ratio of each compound was calculated as (Acompound–Ablank)/(Acontrol–Ablank) × 100%.

4.4.2 Measurement of intracellular pH (pHi)

The pHi of cells was measured by the pH-sensitive fluorescent probe [2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM, Beyotime)] [29]. After seeded into the 96-well plate for 24 h with 10,000 cells/well, H9c2 cardiomyocyte was incubated with Krebs Solution for 30 min, and then the diluted BCECF-AM with the final concentration of 1 μM was added into each well for another 30 min. The BCECF-AM was washed up with Krebs Solution and incubated for 30 min, the fluorescence value was measured with Spectra Max iD3 (Molecular Devices) at 480 nm and 440 nm for excitation and 530 nm for emission, then fluorescence intensity ratio (FIR) was calculated as FIR480 nm/FIR440 nm × 100%. The pHi standard curve was made as follows: five wells with almost the same values of FIR were selected, and added the high-potassium solution containing 4 mg/L nigericin sodium salt (Beijing psaitong Biotechnology Co., Ltd) with different pH gradients (6.5, 6.8, 7.1, 7.4 and 7.7), and incubated for 8 min; then the pHi standard curve was derived based on the pH value and corresponding FIR value. Then, the H9c2 cardiomyocyte was divided into control group, model group, cariporide treated group (1 μM), buthutin A treated group (1 μM), and compounds treated group (1 μM). Except the control group, the cells of other groups were incubated with 25 mM NH4Cl diluted with Krebs Solution for 3 min, and then the cells were washed with sodium-free Krebs Solution to clean NH4Cl. Target compound diluted with sodium-free Krebs Solution was added to the cells and cultured for 10 min. Then the target compound was washed with sodium-containing Krebs Solution, and the FIR value of each well was detected. The pHi values of each group were calculated according to the standard pHi curve. The activity of NHE-1 is expressed as the rate of pHi change per minute (dpHi/min) from the sodium-free to sodium-containing phase. In addition, the HEK293 cells were divided into control group, model group, cariporide (3 µM)-treated group, cariporide (3 µM) and buthutin A (1 µM)-treated group, cariporide (3 µM) and 9k (1 µM)-treated group, cariporide (3 µM) and 9m (1 µM)-treated group, and cariporide (3 µM) and 9o (1 µM)-treated group. All these groups received the intracellular pH (pHi) measurement to estimate the NHEs activity.

4.5 Statistical analysis

The data of this research were expressed as mean ± SEM, which were analyzed with SPSS 19.0 and GraphPad Prism 7.0. All tests were carried out at least in quintuplicate. The statistical comparisons in the different group were performed using one-way ANOVA followed by Tukey's post hoc test. Differences were considered to have statistical significance at p < 0.05.