Introduction

Anti-platelet drugs are typically used in the clinical treatment of anti-thrombosis and prevention of thrombosis. Thrombosis that is caused secondary to disrupted atherosclerotic plaques is the initiator of most cardiovascular diseases including heart attacks and strokes. It is well-known that platelets play a major role in the pathogenesis of atherothrombosis (Brito et al. 2010; Eskandariyan et al. 2014). Picotamide (Figs. 1–1) is an anti-platelet drug with a dual inhibitory action, which inhibits both thromboxane A2 (TXA2) receptors and TXA2 synthase (Balsano and Violi 1993; Berrettini et al. 1990; Violi et al. 1988; Yip and Benavente 2011). Meanwhile, compared with aspirin, picotamide has the benefit of not interfering with endothelial prostacyclin (PGI2) production (Celestini and Violi 2007; Sartori et al. 1993). Recent research had indicated that picotamide is more effective than aspirin in reducing total mortality in patients with type II diabetes associated with peripheral arterial disease (PAD) (Neri Serneri et al. 2005).

Fig. 1
figure 1

Structures of Picotamide (1), target compounds (2), compound 1l (3) and compound 1c (4)

Full size image

Since 2000, we have concerned on the anti-platelet aggregating activity studies of various series of 4-methoxyisophthalamides (Figs. 1–2) and stated the relevant report (Liu et al. 2006, 2011, 2012). Among those compounds, nearly 30% compounds exhibited significant anti-platelet aggregation activities than picotamide. In particularly, compound 1l (Figs. 1–3) with benzyl groups attached to the phenyl rings in the parent compound showed excellent activity against the platelet aggregation induced by ADP, and its IC50 value is as low as 0.02 μM/L and its LD50 is over 5000 mg/kg. It is 30 times as pictoamide’s (IC50 = 0.60 μM/L, LD50 = 2500 mg/kg) (Liu et al. 2012, 2017a). All the data justify that 4-methoxyisophthalamides have the potential of becoming new anti-platelet drugs. Nonetheless, researches of alkyl substituted 4-methoxyisophthalamide are insufficient. And compound 1c (Figs. 1–4) with methyl groups in the p-position of the side chain phenyl rings showed moderate anti-platelet activity, which aroused our interest to continue to research.

To search for the new anti-platelet agents and perfect the SAR of 4-methoxyisophthalamides, the depth and breadth of research for the 4-methoxyisophthalamides have been enhanced and expanded. Among which in the alkyl, the original limited to methyl research extended to ethyl, isopropyl, n-/t-butyl and for unsaturated ethynyl and cyano synthesis and research for the first time. Total 19 novel compounds (1d–1w) were designed and synthesized. Compounds 1d–1k with alkyl (–CH2CH3 –CH(CH3)2 –(CH2)3CH3 –C(CH3)3) in different positions were synthesized to investigate the effect of increasing the steric hindrance of the substitutions on anti-platelet aggregation activity. Compound 1m with ethynyl (–C≡CH) was synthesized to investigate the effect of different hybridization substituent having an unsaturated bond on anti-platelet activity. We also try to increase the electron-withdrawing of the substitutions, such as cyano (–C≡N) and trifluoromethyl (–CF3) to synthesize the compounds 1n, 1o, 1p, 1q, 1r, 1s to investigate the effect of their introducing on anti-platelet aggregation. In order to investigate the effect of the number of substitution on anti-platelet aggregation activity, compounds 1t and 1u were designed and synthesized by increasing the number of alkyl on the phenyl ring. In order to investigate the effect of introducing chlorine on anti-platelet aggregation, we try to introduce methyl and chlorine on the phenyl ring to synthesize the compounds 1v and 1w.

The chemical structures of target compounds were confirmed by IR, MS, 1H-NMR, and 13C-NMR. Their inhibitory effects on platelet aggregation were tested and assessed by Born method for ADP inducer. Ten compounds that were significantly more active in vitro than control drug picotamide were selected and continued to test and assess for two inducers (collagen and arachidonic acid). And the compounds with higher activities on anti-platelet aggregation were selected to continue research of their cytotoxicities.

Materials and methods

General experimental techniques

All reagents for the synthesis of 4-methoxyisophthalamides were purchased (purity > 99%) and used without further purification. The melting points were determined with X-4 digital micro melting point apparatus (thermometer was uncorrected); Mass spectra (MS) were measured on Xevo GZQ-Tof VHRS mass spectrometer; IR spectra were recorded on Avatar 370 spectrophotometer; 1H-NMR spectra were determined with AVANCE III (400 MHz) spectrometer (Tetramethyl silane as an internal standard); 13C-NMR spectra were determined with AVANCE III (101 MHz) spectrometer.

Chemistry

Synthetic routes of 4-methoxylisophthaloyl dichloride

Route a

The reaction of phenol 1 with formaldehyde and hydrochloric acid in benzene gave 2,4-bis (chloromethyl) phenol 2, which reacted with dimethyl sulfate in water gave 2,4-bis (chloromethyl) -1-methoxybenzene 3. Then compound 3 was oxidized with potassium permanganate, 4-methoxyisophthalic acid 4 was obtained. Finally, using compound 4 reacted with sulfoxide chloride, a white solid 4-methoxyisophthaloyl dichloride 5 was obtained.

Route b

4-Methylphenol 1’ was used as starting material. Acetylation of 1’ with acetic anhydride gave 4-methylphenyl acetate 2’. And then compound 2’ was subjected to Fries rearrangement to 1- (2-hydroxy-5-methylphenyl) ethan-1-one 3’ in the presence of AlCl3 (Shaikh et al. 2016). The reaction of compound 3’ with dimethyl sulfate gave 2,4-bis (chloromethyl) -1-methoxybenzene 4’. Then the methyl and acetyl of compound 4’ was oxidized with potassium permanganate, 4-methoxyisophthalic acid 4 was obtained (Liu et al. 2017b). At least, compound 4 reacted with sulfoxide chloride, a white solid 4-methoxyisophthaloyl dichloride 5 was obtained.

Synthetic route of 4-methoxyisophthalamides

N1,N3-bis(2-ethylphenyl)-4-methoxyisophthalamide (1d)

A round-bottomed flask (100 ml) equipped with a magnetic stirrers was charged with 2-ethylaniline (10.0 mmol, 1.2 g) in anhydrous tetrahydrofuran (15 ml). The fresh 4-methoxyisophthaloyl dichloride 5 (5.0 mmol, 1.2 g) in dry tetrahydrofuran (15 ml) was added drop-wise to the above solution at room temperature. Ten hours after the addition of compound 5, dry triethylamine (1 ml) was added drop-wise to the reaction mixture. The resultant mixture reaction was stirred for 24 h at room temperature. After the reaction completed as monitored by thin layer chromatography (TLC). The solvent was removed under reduced pressure to obtain the crude product. The crude product was further purified by recrystallization from anhydrous ethanol to yield the desired product 1d as white solid. Yield: 55.00%, m.p.: 175.0~176.0 °C.

Compounds 1e–1w were prepared in the similar manner.

N1,N3-bis(2-methylphenyl)-4-methoxyisophthalamide (1a)

White solid (The residue was recrystallized by acetone.) Yield: 41.00%; m.p.: 218.0–220.0 °C; IR (KBr) υmax 3347, 1677, 1605, 1551, 1319, 1091, 816 cm−1; 1H NMR (400 MHz, CDCl3, TMS): δ = 9.77 (1H, s, CONH), 8.80 (1H, d, J = 2.2 Hz, 2-H), 8.24 (2H, m, Ar–H), 7.99 (1H, s CONH), 7.82 (1H, s, 2a-H), 7.30 (1H, s, 2b-H), 7.19 (6H, m, Ar–H), 4.16 (3H, s, OCH3), 2.36 (3H, s, Ar–CH3), 2.38 (3H, s, Ar–CH3); 13C NMR (101 MHz, CDCl3): δ = 162.2 (C, C = O), 159.5 (C, 4-C), 136.1 (C, 1a-C), 135.5 (C, 1b-C), 134.8 (CH, 6-C), 133.9 (C, 2a-C), 133.8(C, 2b-C), 130.1 (C, 1-C), 128.6 (CH, 3a-C), 128.4 (CH, 3b-C), 128.0 (C, 3-C), 126.7 (CH, 4a-C), 126.5 (CH, 4b-C), 125.8 (CH, 2-C), 125.0(CH, 5a-C), 124.3 (CH, 5b-C), 122.9 (CH, 6a-C), 121.4 (CH, 6b-C), 112.1 (CH, 5-C), 58.3 (CH3, OCH3), 14.1 (CH3, Ar–CH3), 13.8 (CH3, Ar–CH3); MS (m/z): 375.1063 [M+H]+ (Liu et al. 2012).

N1,N3-bis(3-methylphenyl)-4-methoxyisophthalamide (1b)

White solid (The residue was recrystallized by acetone.) Yield: 51.00%; m.p.: 179.0–180.0 °C; IR (KBr) υmax 3264, 1618, 1593, 1521, 1487, 1236, 1070, 732 cm−1; 1H NMR (400 MHz, CDCl3, TMS): δ = 9.72 (1H, s, CONH), 8.71 (1H, d, J = 1.9 Hz, 2-H), 8.24 (1H, dd, J = 1.0, 4.0 Hz, 6-H), 7.99 (1H, d J = 1.0 Hz, 2-H), 7.26 (1H, d, J = 4.0 Hz, 5-H), 7.55 (4H, m, 2a-H, 2b-H, 6a-H, 6b-H), 7.20 (4H, m, 3a-H, 3b-H, 5a-H, 5b-H), 4.14 (3H, s, OCH3), 2.35 (6H, s, 2 × Ar–CH3); 13C NMR (101 MHz, CDCl3): δ = 164.2 (C, C = O), 162.3 (C, C = O), 159.4 (C, 4-C), 140.7 (C, 3a-C), 140.4 (C, 3b-C), 135.6 (C, 1a-C), 135.5 (C, 1b-C), 133.7 (CH, 6-C), 130.0 (C, 1-C), 128.3 (CH, 5a-C), 128.2 (CH, 5b-C), 128.1 (CH, 2-C), 121.2 (CH, 2a-C, 2b-C), 120.7 (CH, 4a-C, 4b-C), 120.4 (CH, 6a-C, 6b-C), 112.1 (C, 3-C), 58.4 (CH3, OCH3), 15.6 (CH3, Ar–CH3), 15.6 (CH3, Ar–CH3); MS (m/z): 375.1065 [M+H]+ (Liu et al. 2012).

N1,N3-bis(4-methylphenyl)-4-methoxyisophthalamide (1c)

White solid (The residue was recrystallized by acetone); Yield: 30.00%; m.p.: 246.0–247.0 °C; IR (KBr) υmax 3264, 1618, 1593, 1521, 1487, 1236, 1070, 732 cm−1; 1H NMR (400 MHz, CDCl3, TMS): δ = 9.68 (1H, s, CONH), 8.71 (1H, s, 2-H), 8.25 (1H, dd, J = 1.9, 3.8 Hz, 6-H), 7.99 (1H, s CONH), 7.53 (2H, s, 2a-H, 2b-H), 7.45 (2H, m, 6a-H, 6b-H), 7.30 (1H, d, J = 3.8 Hz, 5-H), 7.27 (1H, m, 5a-H), 7.18 (1H, m, 5b-H), 6.99 (2H, m, 4a-H, 4b-H), 4.16 (3H, s, OCH3), 2.39 (6H, s, 2 × Ar–CH3); 13C NMR (101 MHz, CDCl3): δ = 164.2 (C, C = O), 162.3 (C, C = O), 159.0 (C, 4-C), 140.5 (C, 4a-C), 140.4 (C, 4b-C), 135.8 (C, 1a-C), 135.6 (C, 1b-C), 133.7 (CH, 6-C), 129.9 (C, 1-C), 128.4 (2 × CH, 3a-C, 5a-C), 128.3 (2 × CH, 3b-C, 5b-C), 127.9 (CH, 2-C), 121.0 (C, 3-C), 120.4 (2 × CH, 2a-C, 6b-C), 120.2 (2 × CH, 2b-C, 6b-C), 112.9 (CH, 5-C), 65.5 (CH3, OCH3), 15.6 (2 × CH3, Ar–CH3); MS (m/z): 375.1064 [M+H]+ (Liu et al. 2012).

N1,N3-bis(2-ethylphenyl)-4-methoxyisophthalamide (1d)

White solid (The residue was recrystallized by anhydrous ethanol.) Yield: 55.00%; m.p.: 175.0–176.0 °C; IR (KBr) υmax 3354.80, 2960.69, 1659.50, 1597.94, 1530.14, 1463.90, 1256.00, 753.97, 627.80 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 9.76 (1H, s, CONH), 8.80 (1H, d, J = 2.4 Hz, 2-H), 8.24 (1H, dd, J = 8.7, 2.4 Hz, 6-H), 8.19 (1H, d, J = 8.0 Hz, 5-H), 7.95 (1H, s, CONH), 7.83 (1H, d, J = 7.8 Hz, Ar–H), 7.29–7.23 (3H, m, Ar–H), 7.21–7.10 (4H, m, Ar–H), 4.14 (3H, s, OCH3), 2.71 (4H, m, 2 × CH2CH3), 1.34–1.24 (6H, m, 2 × CH2CH3); 13C-NMR (101 MHz, CDCl3): δ = 164.7 (C, C = O), 162.5 (C, C = O), 159.8 (C, 4-C), 136.3 (C, 1a-C), 135.8 (C, 1b-C), 135.1 (CH, 6-C), 134.2 (2 × C, 2a-C, 2b-C), 130.4 (C, 1-C), 128.8 (CH, 4a-C), 128.2 (CH, 4b-C), 126.9 (CH, 5a-C), 126.1 (CH, 5b-C), 125.3 (2 × CH, 3a-C, 3b-C), 124.5 (CH, 2-C), 123.2 (CH, 5-C), 121.6 (C, 3-C), 112.4 (2 × CH, 6a-C, 6b-C), 56.8 (CH, O-CH3), 25.0 (CH2, Ar–CH2CH3), 24.5 (CH2, Ar–CH2CH3), 14.4 (CH3, Ar–CH2CH3), 14.1 (CH3, Ar–CH2CH3); MS (m/z): 403.2042 [M+H]+.

N1,N3-bis(3-ethylphenyl)-4-methoxyisophthalamide (1e)

White solid (The residue was recrystallized by 75% ethanol.) Yield: 82.85%; m.p.: 219.2–221.7 °C; IR (KBr) υmax 3368.70, 2958.00, 2863.60, 1665.00, 1553.00, 1441.09, 1262.70, 1013.88, 785.75 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 9.69 (1H, s, CONH), 8.70 (1H, d, J = 2.3 Hz, 2-H), 8.22 (1H, dd, J = 8.7, 2.3 Hz, 6-H), 8.17 (1H, s, CONH), 7.57 (4H, dd, J = 8.2, 3.4 Hz, Ar–H), 7.21 (4H, dd, J = 8.2, 3.2 Hz, Ar–H), 7.14 (1H, d, J = 8.7 Hz, 5-H), 4.12 (3H, s, OCH3), 2.69–2.61 (4H, m, 2 × CH2CH3), 1.25 (6H, td, J = 7.6, 2.7 Hz, 2 × CH2CH3); 13C-NMR (101 MHz, CDCl3): δ = 164.3 (C, C = O), 162.5 (C, C = O), 159.6 (C, 4-C), 140.8 (C, 1a-C), 140.6 (C, 1b-C), 135.8 (C, 3a-C), 135.7 (C, 3b-C), 133.9 (CH, 6-C), 130.1 (C, 1-C), 128.5 (2 × CH, 5a-C, 5b-C), 128.4 (2 × CH, 2a-C, 2b-C), 128.3 (CH, 2-C), 121.4 (2 × CH, 2a-C, 2b-C), 120.9 (2 × CH, 6a-C, 6b-C), 120.6 (C, 3-C), 112.3 (C,5-C), 56.7 (CH3, O-CH3), 28.4 (2 × CH2, ArCH2CH3), 15.7 (2 × CH3, ArCH2CH3); MS: (m/z): 403.2031 [M+H]+.

N1,N3-bis(4-ethylphenyl)-4-methoxyisophthalamide (1f)

Compound (1f): White solid (The residue was recrystallized by anhydrous ethanol.) Yield: 65.30%; m.p.: 221.0–222.5 °C; IR (KBr) υmax 3358.42, 2961.45, 2929.90, 2871.20, 1674.39, 1652.30, 1601.80, 1517.70, 1319.96, 1264.69, 1118.60 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 9.65 (1H, s, CONH), 8.65 (1H, d, J = 2.0 Hz, 2-H), 8.33 (1H, s, CONH), 8.15 (1H, dd, J = 8.6, 2.0 Hz, 6-H), 7.55 (4H, dd, J = 14.3, 8.3 Hz, 2a-H, 6a-H, 2b-H, 6b-H), 7.18 (4H, d, J = 8.2 Hz, 3a-H, 5a-H, 3b-H, 5b-H), 7.06 (1H, d, J = 8.7 Hz, 5-H), 4.05 (3H, s, OCH3), 2.64 (4H, q, J = 7.4 Hz, 2 × Ar–CH2CH3), 1.24 (6H, t, J = 7.6 Hz, 2 × CH2CH3); 13C-NMR (101 MHz, CDCl3): δ = 164.4 (C, C=O), 162.4 (C, C=O), 159.1 (C, 4-C), 140.6 (C, 1a-C), 140.6 (C, 1b-C), 135.9 (C, 4a-C), 135.8 (C, 4b-C), 133.9 (CH, 6-H), 130.1 (C, 1-C), 128.6 (2 × CH, 2a-C, 2b-C), 128.4 (2 × CH, 6a-C, 6b-C), 128.1 (CH, 2-H), 121.2 (C, 3-C), 120.6 (2 × CH, 3a-C, 3b-C), 120.4 (2 × CH, 5a-C, 5b-C), 113.0 (CH, 5-C), 65.7 (CH3, OCH3), 28.5 (CH2, Ar–CH2CH3), 28.5 (CH2, Ar–CH2CH3), 15.7 (CH3, Ar–CH2CH3), 14.9 (CH3, Ar–CH2CH3); MS (m/z): 403.2031 [M+H]+.

N1,N3-bis(2-isopropylphenyl)-4-methoxyisophthalamide (1g)

White needles (The residue was recrystallized by anhydrous ethanol.) Yield: 70.83%; m.p.: 154.8–156.0 °C; IR (KBr) υmax 3376.10, 3270.50, 2961.18, 1656.57, 1527.50, 1467.14, 1265.48 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 9.76 (1H, s, CONH), 8.82 (1H, d, J = 2.3 Hz, 2-H), 8.29 (1H, dd, J = 8.7, 2.1 Hz, 6-H), 8.05 (1H, d, J = 7.8 Hz, 5-H), 7.97 (1H, s, CONH), 7.73 (1H, m, 6a-H), 7.34 (2H, m, 3a-H, 3b-H), 7.29 (1H, dd, J = 7.6, 1.4 Hz, 6b-H), 7.27–7.19 (4H, m, 4a-H, 5a-H, 4b-H, 5b-H), 4.16 (3H, s, OCH3), 3.20 (2H, m, 2 × Ar-CH(CH3)2), 1.34 (6H, d, J = 6.8 Hz, Ar-CH(CH3)2), 1.29 (6H, d, J = 6.8 Hz, Ar-CH(CH3)2); 13C-NMR (101 MHz, CDCl3) δ (ppm): 162.7 (2 × C, C=O), 159.8 (C, 4-C), 141.8 (C, 1a-C), 139.6 (C, 1b-C), 134.8 (C, 1-C), 134.2 (C, 2a-C), 134.2 (C, 2b-C), 130.4 (CH, 6-C), 128.2 (CH, 2-C), 126.6 (CH, 3a-C), 126.6 (CH, 3b-C), 125.9 (CH, 5a-C), 125.8 (CH, 5b-C), 125.6 (2 × CH, 4a-C, 4b-C), 124.3 (CH, 2-C), 121.6 (CH, 5-C), 112.4 (2 × CH, 6a-C, 6b-C), 56.8 (CH3, O-CH3), 28.4 (CH, Ar–CH(CH3)2), 28.3 (CH, Ar–CH(CH3)2), 23.3 (2 × CH3, Ar–CH(CH3)2), 23.2 (2 × CH3, Ar–CH(CH3)2); MS (m/z): 431.2354 [M+H]+.

N1,N3-bis(3-isopropylphenyl)-4-methoxyisophthalamide (1h)

White solid (The residue was recrystallized by cyclohexane.) Yield: 61.58%; m.p.: 152.0–154.0 °C; IR (KBr) υmax 3248.30, 1647.45, 1610.23, 1548.99, 1487.90, 1463.93, 2958.72 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 9.74 (1H, s, CONH), 8.75 (1H, d, J = 2.1 Hz, 2-H), 8.26 (1H, dd, J = 2.2, 8.6 Hz, 6-H), 8.14 (1H, s, CONH), 7.61 (1H, s, Ar–H), 7.55 (2H, d, J = 11.0 Hz, Ar–H), 7.45 (1H, d, J = 8.0 Hz, Ar–H), 7.31 (2H, td, J = 7.8, 4.5 Hz, Ar–H), 7.19 (1H, d, J = 8.7 Hz, 5-H), 7.05 (2H, t, J = 8.4 Hz, Ar–H), 4.16 (3H, s, OCH3), 2.94 (2H, dq, J = 13.4, 6.8 Hz, 2 × Ar–CH(CH3)2), 1.32–1.26 (12H, m, 2 × Ar–CH(CH3)2); 13C-NMR (101 MHz, CDCl3): δ = 164.1 (C, C=O), 162.5 (C, C=O), 159.8 (C, 4-C), 150.2 (C, 3a-C), 150.1 (C, 3b-C), 138.1 (C, 1a-C), 138.0 (C, 1b-C), 134.2 (CH, 5-C), 129.9 (C, 1-C), 129.1 (CH, 5a-C), 129.1 (CH, 5b-C), 128.3 (CH, 2-C), 122.9 (CH, 4a-C), 122.9 (CH, 4b-C), 121.5 (C, 3-C), 119.0 (CH, 6a-C), 118.4 (CH, 6b-C), 118.3 (CH, 2a-C), 117.9 (CH, 2b-C), 112.5 (CH, 5-C), 56.8 (CH3, O-CH3), 34.4 (2 × CH, Ar–CH(CH3)2), 24.1 (2 × CH3, Ar–CH(CH3)2); MS (m/z): 431.2332 [M+H]+.

N1,N3-bis(4-isopropylphenyl)-4-methoxyisophthalamide (1l)

White flocculent (The residue was recrystallized by acetone.) Yield: 82.90%; m.p.: 221.0–224.0 °C; IR (KBr) υmax 3429.6, 2954.2, 1656.9, 1526.9, 1530.1, 1316.3, 1256.0, 1030.7 cm−1; 1H-NMR (400 MHz, CDCl3): δ = 9.67 (1H, s, CONH), 8.69 (1H, d, J = 2.3 Hz, 2-H), 8.24 (1H, s, CONH), 8.20 (1H, dd, J = 8.7, 2.3 Hz, 6-H), 7.58 (4H, t, J = 8.0 Hz, 2a-H, 2b-H, 6a-H, 6b-H), 7.23 (4H, dd, J = 8.4, 3.2 Hz, 3a-H, 3b-H, 5a-H, 5b-H), 7.12 (1H, d, J = 8.7 Hz, 5-H), 4.09 (3H, s, OCH3), 2.91 (2H, qd, J = 10.8, 6.8 Hz, Ar–CH(CH3)2), 1.27 (6H, d, J = 2.7 Hz, Ar–CH(CH3)2), 1.25 (6H, d, J = 2.7 Hz, Ar–CH(CH3)2); 13C-NMR (100 MHz, CDCl3): δ = 164.3 (C, C=O), 162.4 (C, C=O), 159.2 (C, 4-C), 145.3 (C, 4a-C), 145.3 (C, 4b-C), 136.1 (C, 1a-C), 135.8 (C, 1b-C), 134.0 (C, 6-C), 130.0 (C, 1-C), 128.1 (CH, 2-C), 127.2 (2 × CH, 2a-C, 6a-C), 127.1 (2 × CH, 2a-C, 6a-C), 121.2 (C, 3-C), 120.6 (2 × CH, 3a-C, 5a-C), 120.4 (2 × CH, 5b-C, 5b-C), 113.1 (CH, 5-C), 65.7 (CH3, O-CH3), 33.8 (2 × CH, Ar–CH(CH3)2), 24.2 (4 × CH3,Ar–CH(CH3)2); MS (m/z):431.2361 [M+H]+.

N1,N3-bis(4-butylphenyl)-4-methoxyisophthalamide (1j)

White solid (The residue was recrystallized by 75% ethanol.) Yield: 73.58%; m.p.: 254.0–257.0 °C; IR (KBr) υmax 3240.13, 2959.03, 1591.35, 1507.43, 1485.28, 1461.83, 2927.05 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 9.64 (1H, s, CONH), 8.65 (1H, d, J = 1.7 Hz, 2-H), 8.35 (1H, s, CONH), 8.14 (1H, dd, J = 8.5, 1.7 Hz, 6-H), 7.54 (4H, dd, J = 16.2, 8.1 Hz, 2a-H, 6a-H, 2b-H, 6b-H), 7.15 (4H, d, J = 8.1 Hz, 3a-H, 5a-H, 3b-H, 5b-H), 7.05 (1H, d, J = 8.7 Hz, 5-H), 4.04 (3H, s, OCH3), 2.59 (4H, t, J = 7.4 Hz, Ar–CH2CH2CH2CH3), 1.59 (4H, dt, J = 15.2, 7.6 Hz, Ar–CH2CH2CH2CH3), 1.36 (4H, dq, J = 14.5, 7.2 Hz, Ar–CH2CH2CH2CH3), 0.94 (6H, t, J = 7.2 Hz, Ar–CH2CH2CH2CH3); 13C-NMR (101 MHz, CDCl3): δ = 164.4 (C, C=O), 162.4 (C, C=O), 159.5 (C, 4-C), 139.4 (C, 4a-C), 139.2 (C, 4b-C), 135.7 (2 × C, 1a-C, 1b-C), 133.8 (CH, 6-C), 130.1 (C, 1-C), 130.0 (4 × CH, 2a-C, 6a-C, 2b-C, 6b-C), 128.2 (CH, 2-C), 121.3 (C, 3-C), 120.7 (2 × CH, 3a-C, 5a-C), 120.4 (2 × CH, 3b-C, 5b-C), 112.1 (CH, 5-C), 56.6 (CH3, O-CH3), 35.2 (2 × CH2, Ar–CH2CH2CH2CH3), 33.8 (2 × CH2, Ar–CH2CH2CH2CH3), 22.4 (2 × CH2, Ar–CH2CH2CH2CH3), 14.1 (2 × CH3, Ar–CH2CH2CH2CH3); MS (m/z): 459.2656 [M+H]+.

N1,N3-bis(4-(tert-butyl)phenyl)-4-methoxyisophthalamide (1k)

White solid (The residue was recrystallized by anhydrous ethanol.) Yield: 55.43%; m.p.: 259.5–261.0 °C; IR (KBr) υmax 3339.43, 2953.56, 1659.56, 1606.20, 1529.48, 1252.80, 1100.98, 952.2, 829.8 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 9.66 (1H, s, CONH), 8.69 (1H, s, CONH), 8.20 (2H, m, 6-H, 2-H), 7.58 (4H, m, 2a-H, 6a-H, 2b-H, 6b-H), 7.36 (4H, m, 3a-H, 5a-H, 3b-H, 5b-H), 7.13 (1H, s, 5-H), 4.10 (3H, s, OCH3), 1.32–1.33 (18H, s, 2 × Ar–C(CH3)3); 13C-NMR (101 MHz, CDCl3): δ = 164.3 (C, C=O), 162.4 (C, C=O), 159.2 (C, 4-C), 147.6 (C, 4a-C), 147.5 (C, 4b-C), 135.8 (C, 1a-C), 135.5 (C, 1b-C), 133.9 (CH, 6-H), 130.0 (C, 1-C), 128.1 (CH, 2-C), 126.1 (2 × CH, 3a-C, 5a-C), 126.0 (2 × CH, 3b-C, 5b-C), 121.2 (C, 3-C), 120.1 (2 × CH, 2a-C, 6a-C), 113.1 (2 × CH, 2b-C, 6b-C), 112.9 (CH, 5-C), 65.7 (CH3, O-CH3), 34.5 (2 × C, Ar–C(CH3)3), 31.5 (6 × CH3, Ar–C(CH3)3); MS (m/z): 459.2649 [M+H]+.

N1,N3-bis(2-benzylphenyl)-4-methoxyisophthalamide (1l)

White solid (The residue was recrystallized by acetone.) Yield: 59.00%; m.p.: 238.0–240.0 °C; IR (KBr) υmax 3301, 3025, 2922, 1653, 1606, 1587, 1493, 1451, 1300, 1262, 1027, 825 cm−1; 1H-NMR (400 MHz, DMSO-d6, TMS): δ = 9.96 (1H, s, CONH), 9.79 (1H, s, CONH), 8.40 (1H, dd, J = 4.0, 12.0 Hz, 6-H), 8.04 (1H, d, J = 12.0 Hz, 2-H), 7.75 (1H, d, J = 4.0 Hz, 5-H), 7.11–7.36 (18H, m, Ar–H), 4.07 (2H, s, Ar–CH2–Ph), 4.00 (2H, s, Ar–CH2–Ph), 3.85 (3H, s, OCH3); 13C-NMR (101 MHz, DMSO-d6): δ = 164.7 (C, C=O), 163.5 (C, C=O), 160.8 (C, 4-C), 140.2 (C, 1a-C), 140.0 (C, 1b-C), 138.3 (C, 2a-C), 138.2 (C, 2b-C), 131.9 (CH, 6-H), 131.0 (2 × C, 1a’-C, 1b’-C), 128.5 (2 × CH, 3a’-C, 5a’-C), 128.4 (2 × CH, 3b’-C, 5b’-C), 128.2 (2 × CH, 2a’-C, 6a’-C), 128.0 (4 × CH, 2b’-C, 6b’-C, 4a-C, 4b-C), 126.6 (C, 1-C), 126.0 (C, 2-C), 125.5 (2 × CH, 3a-C, 3b-C), 125.4 (2 × CH, 4a’-C, 4b’-C), 120.4 (2 × CH, 6a-C, 6b-C), 116.5 (C, 3-C), 112.3 (C, 5-C), 60.3 (CH3, OCH3), 34.5 (CH2, Ar–CH2–Ph), 32.7 (CH2, Ar–CH2–Ph); MS (m/z): 527.6063 [M+H]+ (Liu et al. 2012).

N1,N3-bis(3-ethynylphenyl)-4-methoxyisophthalamide (1m)

White needles (The residue was recrystallized by ethanol.) Yield: 67.40%; m.p.: 174.1–175.7 °C; IR (KBr) υmax 3346.23, 32837, 2955.33, 1671.50, 649.38, 1582.54, 1554.22, 1431.70, 2104.20, 1185.81, 1017.99, 840, 790 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 9.69 (1H, s, CONH), 8.63 (1H, d, J = 2.3 Hz, 2-H), 8.58 (1H, s, CONH), 8.16 (1H, dd, J = 8.7, 2.3 Hz, 6-H), 7.86 (1H, s, Ar–H), 7.73–7.63 (3H, m, Ar–H), 7.32–7.28 (2H, m, Ar–H), 7.25 (2H, d, J = 8.6 Hz, 5a-H, 5b-H), 7.08 (1H, d, J = 8.8 Hz, 5-H), 4.09 (3H, s, OCH3), 3.11 (1H, s, C≡CH), 3.09 (1H, s, C≡CH); 13C-NMR (101 MHz, CDCl3): δ = 164.4 (C, C = O), 162.5 (C, C = O), 159.8 (C, 4-C), 138.2 (C, 1a-C), 138.1 (C, 1b-C), 134.3 (CH, 6-C), 130.3 (C, 1-C), 129.3 (CH, 5a-C), 129.2 (CH, 5b-C), 128.4 (CH, 4a-C), 128.3 (CH, 4b-C), 128.0 (CH, 2-C), 124.2 (CH, 2a-C), 123.9 (CH, 2b-C), 123.0 (C, 4a-C), 130.0 (C, 4b-C), 121.4 (CH, 6a-C), 121.1 (CH, 6b-C), 120.9 (C, 3-C), 112.4 (C, 5-C), 83.4 (2 × C, Ar–C≡CH), 77.7 (2 × CH, Ar–C≡CH), 56.8 (CH3, O–CH3); MS (m/z): 395.1388 [M+H]+.

N1,N3-bis(3-cyanophenyl)-4-methoxyisophthalamide (1n)

Brown solid (The residue was recrystallized by DMF: H2O = 1.5: 1); Yield: 56.70%; m.p.: 263.6–265.2 °C; IR (KBr) υmax 3356.13, 2952.32, 1668.50, 1546.26, 1420.47, 2228.49, 1179.80, 1086.95, 830, 792, 680 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 9.85 (1H, s, CONH), 8.73 (1H, s, 2-H), 8.65 (1H, s, CONH), 8.23 (1H, d, J = 8.7 Hz, 6-H), 8.02 (1H, s, 3a-H), 7.91 (1H, s, 3b-H), 7.84 (1H, d, J = 8.0 Hz, 4a-H), 7.78 (1H, d, J = 7.8 Hz, 4b-H), 7.44 (2H, t, J = 6.7 Hz, 5a-H, 5b-H), 7.38 (2H, t, J = 7.5 Hz, 6a-H, 6b-H), 7.16 (1H, d, J = 8.8 Hz, 5-H), 4.14 (3H, s, OCH3); 13C-NMR (101 MHz, DMSO-d6) δ = 166.4 (C, C=O), 164.5 (C, C=O), 164.3 (C, 4-C), 159.9 (C, 1a-C), 159.2 (C, 1b-C), 140.0 (CH, 4a-C), 139.6 (CH, 4b-C), 133.6 (CH, 2a-C), 132.2 (CH, 2b-C), 131.0 (CH, 6-C), 130.2 (CH, 5a-C), 129.3 (CH, 5b-C), 127.2 (C, 1-C), 126.1 (CH, 2-C), 124.8 (CH, 6a-H), 124.5 (CH, 6b-H), 124.4 (CH, 3-C), 123.0 (C, Ar–C≡N), 122.5 (C, Ar–C≡N), 118.6 (CH, 5-C), 112.1 (C, 3a-C), 111.6 (C, 3b-C), 56.4 (CH3, O–CH3); MS (m/z): 419.1131 [M + Na]+.

N1,N3-bis(4-cyanophenyl)-4-methoxyisophthalamide (1o)

White solid (The residue was recrystallized by ethanol.) Yield: 62.51%; m.p.: 271.5–273.3 °C; IR (KBr) υmax 3339.20, 2997.36, 1678.58, 1592.21, 1493.40, 2224.40, 1176.00, 1014.90, 823, 753 cm−1; 1H-NMR (400 MHz, DMSO-d6, TMS): δ = 10.70 (1H, s, CONH), 10.62 (1H, s, CONH), 8.26 (1H, d, J = 2.2 Hz, 2-H), 8.18 (1H, dd, J = 8.7, 2.2 Hz, 6-H), 8.00 (2H, d, J = 8.7 Hz, 2a-H, 2b-H), 7.94 (2H, d, J = 8.7 Hz, 6a-H, 6b-H), 7.83 (4H, dd, J = 8.7, 4.7 Hz, 3a-H, 5a-H, 3b-H, 5b-H), 7.36 (1H, d, J = 8.9 Hz, 5-H), 3.96 (3H, s, OCH3); 13C-NMR (101 MHz, DMSO-d6): δ = 164.8 (C, C=O), 164.6 (C, C=O), 159.3 (C, 4-C), 143.5 (C, 1a-C), 143.1 (C, 1b-C), 133.3 (2 × CH, 3a-C, 5a-C), 133.1 (2 × CH, 3b-C, 5b-C), 132.3 (CH, 6-C), 129.2 (C, 1-C), 126.0 (CH, 2-C), 124.8 (C, 3-C), 120.2 (2 × CH, 2a-C, 6a-C), 119.6 (2 × CH, 2b-C, 6b-C), 119.0 (2 × C, Ar–C≡N), 112.0 (CH, 5-C), 105.4 (C, 4a-C), 105.3 (C, 4b-C), 56.4 (CH3, O-CH3); MS (m/z): 419.1129 [M + Na]+.

N1,N3-bis(2-(trifluoromethyl)phenyl)-4-methoxyisophthalamide (1p)

White solid (The residue was recrystallized by acetonitrile.) Yield: 74.71%; m.p.: 172.6–174.3 °C; IR (KBr) υmax 3354.20, 2957.38, 1679.53, 1645.22, 1535.50, 1134.10, 1058.90, 824, 764 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 10.27 (2H, s, CONH), 8.63 (1H, d, J = 1.7 Hz, 2-H), 8.23–8.14 (2H, m, Ar–H), 7.80 (2H, dd, J = 2.4, 10.8 Hz, 3a-H, 3b-H), 7.75 (2H, t, J = 7.7 Hz, 4a-H, 4b-H), 7.55 (2H, t, J = 6.5 Hz, 5a-H, 5b-H), 7.43 (2H, t, J = 8.5 Hz, 6a-H, 6b-H), 4.09 (3H, s, OCH3); 13C-NMR (101 MHz, DMSO-d6): δ = 165.2 (C, C=O), 162.9 (C, C=O), 159.6 (C, 4-C), 135.8 (CH, 5a-C), 135.3 (CH, 5b-C), 133.3 (C, 1a-C), 133.1 (C, 1b-C), 131.3 (CH, 6-C), 127.4 (2 × CH, 3a-C, 3b-C), 126.7 (C, 1-C), 126.6 (2 × C, Ar–CF3), 126.3 (2 × CH, 4a-C, 4b-C), 126.2 (C, 2-C), 125.4 (2 × C, 2a-C, 2b-C), 122.3 (C, 3-C), 121.0 (CH, 5-C), 112.6 (2 × CH, 6a-C, 6b-C), 56.7 (CH3, O-CH3); MS (m/z): 483.1153 [M+H]+.

N1,N3-bis(3-(trifluoromethyl)phenyl)-4-methoxyisophthalamide (1q)

White solid (The residue was recrystallized by ethanol.) Yield: 81.60%; m.p.: 201.1–202.6 °C; IR (KBr) υmax 3335.26, 2961.30, 1686.50, 1568.21, 1496.48, 1441.10, 1337.38, 1226.16, 1014.30, 829, 790, 698 cm−1; 1H-NMR (400 MHz, DMSO-d6, TMS): δ = 10.60 (1H, s, CONH), 10.54 (1H, s, CONH), 8.31 (1H, d, J = 2.1 Hz, 2-H), 8.25 (2H, s, 2a-H, 2b-H), 8.19 (1H, dd, J = 8.8, 2.1 Hz, 6-H), 8.09 (1H, d, J = 8.4 Hz, 4a-H), 7.97 (1H, d, J = 8.1 Hz, 4b-H), 7.60 (2H, td, J = 8.0, 2.7 Hz, 5a-H, 5b-H), 7.46 (2H, t, J = 7.4 Hz, 6a-H, 6b-H), 7.36 (1H, d, J = 8.8 Hz, 5-H), 3.97 (3H, s, OCH3); 13C-NMR (101 MHz, DMSO-d6): δ = 164.7 (C, C = O), 164.5 (C, C=O), 159.1 (C, 4-C), 140.0 (C, 1a-C), 139.7 (1, 1b-C), 132.1 (CH, 6-C), 130.0 (C, 3a-C), 129.8 (C, 3b-C), 129.5 (CH, 5a-C), 129.3 (CH, 5b-C), 129.1 (C, 1-C), 126.1 (CH, 2-C), 125.5 (2 × CH, 2a-C, 2b-C), 124.8 (2 × CH, 6a-C, 6b-C), 123.8 (2 × C, Ar–CF3), 123.2 (C, 3-C), 120.0 (CH, 4a-C), 116.4 (CH, 4b-C), 112.0 (CH, 5-H), 56.4 (CH3, O-CH3); MS (m/z): 483.1141 [M+H]+.

N1,N3-bis(4-(trifluoromethyl)phenyl)-4-methoxyisophthalamide (1r)

White solid (The residue was recrystallized by ethanol.) Yield: 45.37%; m.p.: 244.7–245.0 °C; IR (KBr) υmax 3339.20, 1661.96, 1605.88, 1549.44, 1493.29, 1408.30, 1321.85, 1260.93, 1067.12 cm−1; 1H-NMR (400 MHz, DMSO-d6, TMS): δ = 10.64 (1H, s, CONH), 10.58 (1H, s, CONH), 8.28 (1H, d, J = 1.2 Hz, 2-H), 8.19 (1H, d, J = 8.7 Hz, 6-H), 8.03 (2H, d, J = 8.5 Hz, 3a-H, 3b-H), 7.97 (2H, d, J = 8.3 Hz, 3b-H, 5b-H), 7.73 (4H, dd, J = 8.4, 2.6 Hz, 2a-H, 6a-H, 2b-H, 6b-H), 7.36 (1H, d, J = 8.8 Hz, 5-H), 3.96 (3H, s, OCH3); 13C-NMR (101 MHz, DMSO-d6): δ = 164.8 (C, C = O), 164.6 (C, C = O), 159.2 (C, 4-C), 142.9 (C, 1a-C), 142.6 (C. 1b-C), 132.3 (2 × CH, 4a-C, 4b-C), 129.2 (CH, 6-H), 126.2 (C, 1-C), 126.0 (2 × CH, 3a-C, 5a-C), 125.9 (2 × CH, 3b-C, 5b-C), 125.8 (CH, 2-C), 125.8 (C, Ar–CF3), 125.0 (C, Ar–CF3), 123.9 (C, 3-C), 123.6 (CH, 2a-C), 123.1 (CH, 2b-C), 120.2 (CH, 6a-C), 119.6 (CH, 6b-C), 112.0 (CH, 5-C), 56.4 (CH3, O-CH3); MS (m/z): 483.1152 [M+H]+.

N1,N3-bis(2-chloro-4-(trifluoromethyl)phenyl)-4-methoxyisophthalamide (1s)

White solid (The residue was recrystallized by acetone.) Yield: 63.21%; m.p.: 226.0–229.0 °C; IR (KBr) υmax 3421.20, 3312.13, 1685.96, 1595.88, 1542.74, 1500.79, 1477.30, 1324.85, 1261.93, 1081.12 cm−1; 1H-NMR (400 MHz, DMSO-d6, TMS): δ = 10.76 (1H, s, CONH), 10.43 (1H, s, CONH), 8.74 (1H, d, J = 2.1 Hz, 2-H), 8.71 (1H, d, J = 8.8 Hz, 6a-H), 8.26 (1H, dd, J = 8.7, 2.2 Hz, 6-H), 8.00 (2H, d, J = 6.0 Hz, 3a-H, 3b-H), 7.90 (1H, d, J = 8.4 Hz, 6b-H), 7.79 (2H, t, J = 7.3 Hz, 5a-H, 5b-H), 7.48 (1H, d, J = 8.8 Hz, 5-H), 4.18 (3H, s, OCH3); 13C-NMR (101 MHz, DMSO-d6): δ = 164.2 (C, C=O), 162.5 (C, C=O), 160.0 (C, 4-C), 139.1 (C, 1a-C), 138.7 (C, 1b-C), 133.8 (2 × C, 4a-C, 4b-C), 131.7 (CH, 6-C), 129.5 (2 × C, 2a-C, 2b-C), 128.3 (C, 1-C), 126.7 (CH, 3a-C), 126.4 (CH, 3b-C), 125.3 (C, 1-C), 124.8 (2 × CH, 6a-C, 6b-C), 124.6 (2 × CH, 5a-C, 5b-C), 123.3 (CH, 2-C), 122.0 (2 × C, Ar–CF3), 120.4 (C, 3-C), 113.0 (CH, 5-H), 57.4(CH3, O–CH3); MS (m/z): 551.0373 [M+H]+.

N1,N3-bis(2,3-dimethylphenyl)-4-methoxyisophthalamide (1t)

White solid (The residue was recrystallized by acetone.) Yield: 61.58%; m.p.: 173.0–174.0 °C; IR (KBr) υmax 3369.52, 3273.61, 1665.90, 1637.69, 1607.32, 1526.80, 2821.76 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 9.68 (1H, s, CONH), 8.81 (1H, d, J = 1.8 Hz, 2-H), 8.26 (1H, dd, J = 8.5, 1.4 Hz, 6-H), 7.98 (1H, s, CONH), 7.89 (1H, d, J = 8.0 Hz, 5-H), 7.49 (1H, d, J = 7.9 Hz, Ar–H), 7.17 (1H, ddd, J = 19.7, 10.2, 5.5 Hz, Ar–H), 7.06 (2H, t, J = 8.1 Hz, Ar–H), 4.15 (3H, s, OCH3), 2.35 (3H, s, Ar–CH3), 2.33 (3H, s, Ar–CH3), 2.27 (3H, s, Ar–CH3), 2.22 (3H, s, Ar–CH3); 13C-NMR (101 MHz, CDCl3): δ = 162.6 (2 × C, C=O), 159.8 (C, 4-C), 137.7 (C, 3a-C), 137.4 (C, 3b-C), 136.2 (C, 1a-C), 135.4 (C, 1b-C), 134.1 (CH, 6-C), 130.4 (2 × C, 2a-C, 2b-C), 128.1 (C, 1-C), 128.0 (CH, 2-C), 127.9 (CH, 4a-C), 127.1 (CH, 4b-C), 126.2 (CH, 5a-C), 126.0 (CH, 5b-C), 122.8 (C, 3-C), 121.6 (CH, 6a-C), 121.4 (CH, 6b-C), 112.3 (CH, 5-C), 56.8 (CH3, O–CH3), 20.8 (CH3, 3a-CH3), 20.7 (CH3, 3b-CH3), 14.2 (CH3, 2a-CH3), 13.9 (CH3, 2b-CH3); MS (m/z): 403.2015 [M+H]+.

N1,N3-bis(3,4-dimethylphenyl)-4-methoxyisophthalamide (1u)

White floccule (The residue was recrystallized by 70% ethanol.) Yield: 58.00%; m.p.: 172.0–173.0 °C; IR (KBr) υmax 3133.96, 1666.73, 1543.65, 1402.17, 923.32, 840.95, 727.79 cm−1; 1H-NMR (400 MHz, CDCl3, TMS): δ = 9.52 (1H, s, CONH), 8.60 (1H, s, CONH), 8.55 (1H, s, 2-H), 8.06 (1H, d, J = 7.9 Hz, 6-H), 7.44 (1H, s, Ar-H), 7.41–7.33 (2H, m, 2a-H, 2b-H), 7.30 (1H, d, J = 8.1 Hz, Ar–H), 7.04 (2H, dd, J = 8.0, 2.6 Hz, 5a-H, 5b-H), 6.95 (1H, d, J = 8.7 Hz, 5-H), 3.97 (3H, s, OCH3), 2.20 (12H, s, 4 × Ar–CH3); 13C-NMR (101 MHz, CDCl3): δ = 164.5 (2 × C, C=O), 159.3 (C, 4-C), 137.2 (C, 3a-C), 137.0 (C, 3b-C), 136.0 (C, 1a-C), 135.7 (C, 1b-C), 133.4 (CH, 6-C), 132.8 (C, 4a-C), 132.6 (4b-C), 130.3 (C, 1-C), 129.9 (CH, 5a-C), 129.9 (CH, 5b-C), 128.2 (CH, 2-C), 122.0 (CH, 2a-C), 121.8 (CH, 2b-C), 121.3 (C, 3-C), 118.2 (CH, 6a-C), 118.0 (CH, 6b-C), 111.8 (CH, 5-C), 56.5 (CH3, O–CH3), 19.9 (2 × CH3, 3a-CH3, 3b-CH3), 19.2 (2 × CH3, 4a-CH3, 4b-CH3); MS (m/z): 403.2039 [M+H]+.

N1,N3-bis(3-chloro-2-methylphenyl)-4-methoxyisophthalamide (1v)

White solid (The residue was recrystallized by dioxane.) Yield: 85.10%; m.p.: 232.7–234.3 °C; IR (KBr) υmax 3375.13, 3255.68, 1667.20, 1524.26, 1437.48, 1180.20, 1047.42, 2948.29, 821, 779 cm−1; 1H-NMR (400 MHz, DMSO-d6, TMS): δ = 10.27 (1H, s, CONH), 10.03 (1H, s, CONH), 8.45 (1H, d, J = 2.0 Hz, 2-H), 8.19 (1H, dd, J = 9.2, 1.5 Hz, 6-H), 7.68 (1H, d, J = 7.9 Hz, 4a-H), 7.55 (1H, d, J = 8.0 Hz, 4b-H), 7.50 (1H, d, J = 7.9 Hz, 5-H), 7.38 (1H, d, J = 8.8 Hz, 5a-H), 7.33 (1H, d, J = 8.2 Hz, 5b-H), 7.20 (2H, td, J = 7.8, 4.0 Hz, 6a-H, 6b-H), 4.05 (3H, s, OCH3), 2.40 (3H, s, 2a-CH3), 2.28 (3H, s, 2b-CH3); 13C-NMR (101 MHz, DMSO-d6): δ = 164.4 (C, C=O), 163.4 (C, C=O), 159.3 (C, 4-C), 138.0 (C, 1a-C), 137.8 (C, 1b-C), 134.0 (C, 2a-C), 132.2 (C, 2b-C), 131.3 (CH, 6-C), 130.0 (C, 3a-C), 129.2 (C, 3b-C), 127.4 (CH, 5a-C), 127.3 (CH, 5b-C), 126.6 (C, 1-C), 126.3 (CH, 2-C), 124.7 (CH, 4a-C), 124.6 (CH, 4b-C) 124.4 (C, 3-C), 123.3 (CH, 5-C), 112.1 (2 × CH, 6a-C, 6b-C), 56.6 (CH3, O–CH3), 18.4 (CH3, 2a-CH3), 18.0 (CH3, 2b-CH3); MS (m/z): 443.0937 [M+H]+.

N1,N3-bis(3-chloro-4-methylphenyl)-4-methoxyisophthalamide (1w)

White solid (The residue was recrystallized by ethanol.) Yield: 84.30%; m.p.: 195.8–197.6 °C; IR (KBr) υmax 3361.19, 3327.30, 1645.22, 1592.26, 1400.48, 1258.40, 1181.27, 2947.25, 817, 578 cm−1; 1H-NMR (400 MHz, DMSO-d6, TMS): δ = 10.32 (2H, s, 2 × CONH), 8.25 (1H, d, J = 2.2 Hz, 2-H), 8.26 (1H, dd, J = 2.4, 8.8 Hz, 6-H), 8.18–8.11 (1H, m, Ar-H), 7.99–7.90 (1H, m, Ar-H), 7.63 (1H, dd, J = 8.3, 1.9 Hz, Ar-H), 7.58–7.51 (1H, m, Ar-H), 7.32 (3H, dd, J = 8.3, 1.9 Hz, Ar-H), 7.19–7.24 (3H, m, Ar–H), 3.96 (3H, s, OCH3), 2.29 (6H, s, 2 × Ar-CH3); 13C-NMR (101 MHz, DMSO-d6): δ = 164.42 (2 × C, C = O), 159.0 (C, 4-C), 138.3 (C, 1a-C), 138.1 (C, 1b-C), 133.0 (C, 3a-C), 132.8 (C, 3b-C), 131.8 (C, 6-C), 131.2 (C, 4a-C), 131.0 (C, 4b-C), 130.3 (C, 5a-C), 130.1 (C, 5b-C), 129.0 (C, 1-C), 126.3 (CH, 2-C), 124.8 (C, 3-C), 120.2 (CH, 2a-C), 119.5 (CH, 2b-C), 118.8 (CH, 6a-C), 118.3 (CH, 6b-C), 111.9 (CH, 5-C), 56.3 (CH3, O–CH3), 19.0 (2 × CH3, Ar–CH3); MS (m/z): 443.0924 [M+H]+.

Pharmacology

In vitro anti-platelet aggregation activity

The in vitro activity studies on anti-platelet aggregation of the target compounds have been done by using turbidimetric test (Born 1962): fresh venous blood was taken from the ear vein of rabbits, with 3.8% sodium citrate as anticoagulant (blood in proportion to anticoagulant agent were 9: 1), and then centrifuged at room temperature for 10 min (500–800 r/min) to obtain the platelet-rich plasma (PRP). The platelet-poor plasma (PPP) was obtained by centrifuging of PRP at room temperature for 15 min (3000 r/min). The target compounds were dissolved in DMSO (5 μl), and then, the solution was added into PRP (20 μl), and the same volume of DMSO without target compounds was added as a control group. After 2 min incubation, using ADP as the active inducer, platelet aggregation rate was measured. Using the same test method, the platelet aggregation rates of eight compounds induced by collagen and AA were also tested. The inhibition rates for the compounds were calculated according to the following formulas:

$$\begin{array}{*{20}{l}} {{\mathrm{Aggregation}} {{\% }}} \hfill & = \hfill & {\frac{{{\mathrm{PPP}} - {\mathrm{PRP}}}}{{{\mathrm{PPP}}}} \times 100\% } \hfill \cr {{\mathrm{Inhibition}}\%} \hfill & = \hfill & {\frac{{{\mathrm{S}} - {\mathrm{D}}}}{{\mathrm{S}}} \times 100\% } \hfill \end{array}$$

S: the platelet aggregation in the presence of solvent.

D: the platelet aggregation in the presence of test compounds.

Cytotoxicity assay

Mouse fibroblast cells (L-929) was chosen to evaluate the in vitro cytotoxicity of the target compounds via Cell Counting Kit-8 (CCK-8) assays. Experiment is mainly divided into the following steps:

  1. 1.

    Preparation for experiment: through cell thawing, cell culture and cell passage cultivation to obtain healthy cells. Target compounds were diluted into 10 μM/L and 100 μM/L with DMSO, respectively.

  2. 2.

    Cell inoculation: L-929 was added into 96-well microplates (1 × 104 cell/well), cultivated in a humidified 5% CO2 atmosohere at 37 °C for 24 h to allow cells to attach.

  3. 3.

    Cell to medicine: cells were exposed to target compounds at the concentrations of 10 μM/L and 100 μM/L and incubated at 37 °C for 48 h.

  4. 4.

    Measuring absorbance: After incubation at 37 °C for 48 h, the medium was removed and replaced with fresh complete medium of RPMI-1640, then CCK-8 solution was added to the 96-well plates at 10 μL per well and the absorbance of the solution was monitored at 450 nm using a microplate reader (Bio-Tek Flx800 fluorescence microplate reader) after 2 h of incubation (Xu et al. 1991).

Their cytotoxicity effect on L-929 cells were calculated by the following formulas:

$${\mathrm{Cell}}{\kern 1pt} {\mathrm{viability}}\left( {\mathrm{\% }} \right) = \frac{{{\mathrm{Abs}}\left( {{\mathrm{test}}{\kern 1pt} {\mathrm{cell}}} \right) - {\mathrm{Abs}}\left( {{\mathrm{blank}}{\kern 1pt} {\mathrm{cell}}} \right)}}{{{\mathrm{Abs}}\left( {{\mathrm{controlled}}{\kern 1pt} {\mathrm{cell}}} \right) - {\mathrm{Abs}}\left( {{\mathrm{blank}}{\kern 1pt} {\mathrm{cell}}} \right)}} \times 100{\mathrm{\% }}$$

Results and discussion

Chemistry

The synthetic pathway was disclosed in Scheme 1 and the structure of target compounds were shown in Table 1. Key intermediate 4-methoxylisophthaloyl dichloride was synthesized in two routes (Route a and Route b). The intermediate of compounds (1m, 1n, 1o, 1p, 1q, 1r, 1s, 1v, 1w) were prepared by Route a and the intermediate of compounds (1d, 1e, 1f, 1g, 1h, 1i, 1j, 1k, 1t, 1u) were synthesized by Route b.

Scheme 1
scheme 1

Synthesis routes of intermediate (Route a and b) and target compounds (1d–1w)

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Table 1 Structures of 4-Methoxylisophthalamides (1a–1w)
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At the onset of the experiments, the laboratory’s traditional method (Route a) was used to synthesize the intermediates. The drawback of this method is the use of volatile hydrogen chloride, it is not only difficult to handle but it also corrode the equipment. Moreover, it is dangerous for an industrial process. Considering the above question, a safer method (Route b) was used to synthesize the intermediates in subsequent experiments. Route b use Fries rearrangement as the key step, avoiding the use of large doses of hydrochloric acid, which is beneficial for the environment and equipment.

Pharmacology

In vitro anti-platelet aggregation activity

Taking picotamide and aspirin as positive control drugs, in vitro activities on anti-platelet aggregation were tested and assessed by using Born test for ADP inducer (Born 1962). After that, eight compounds (1c, 1h, 1i, 1k, 1p, 1q, 1v, and 1w), they were significantly more active in vitro than control drug picotamide and their IC50 values for ADP were calculated, they were selected and continued to testing and assessing for two inducers both collagen and AA. The anti-platelet aggregation activity of the target compounds was listed in Table 2.

Table 2 The inhibition rate and IC50 of tested compounds
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These results analysis for ADP and for collagen and for AA were shown in Figs. 2 and 3, respectively.

Fig. 2
figure 2

The Inhibition Rate and IC50 of Compounds

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Fig. 3
figure 3

The Inhibition Rate and IC50 of Compounds for collagen and AA

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As were shown in Fig. 2, compared with positive control drugs picotamide and aspirin, 9 compounds 1h, 1i, 1n, 1o, 1p, 1q, 1s, 1v, and 1w had superior platelet aggregation inhibition rate induced by ADP. Among them, compounds 1d, 1j, and 1k were equivalent to the positive control drug picotamide, slightly lower than aspirin. Simultaneously, the IC50 values of compounds 1h, 1i, 1k, 1p, 1q, 1v, 1w were lower than the positive control drug picotamide and the in vitro activities of four compounds 1h, 1i, 1q, and 1v with IC50 values of 0.24 μM/L, 0.16 μM/L, 0.09 μM/L and 0.09 μM/L, respectively, they were higher than that of two control drugs picotamide and aspirin with IC50 values of 0.60 μM/L and 0.26 μM/L induced by ADP (1.3 μM). Of course, among them, compound 1q, that was a N1,N3-bis (3-(trifluoromethyl)phenyl) -4-methoxyisophthalamide, had the highest platelet aggregation inhibition rate with lowest IC50 value of 0.09 μM/L. The inhibition rate of compound 1v was not high, but its IC50 value was the same as that of compound 1q, which indicated that 1v had high platelet inhibition rate at lower concentration.

Eight compounds (1h, 1i, 1k, 1p, 1q, 1u, 1v, and 1w) which were significantly more active in vitro than control drug picotamide were selected and continued to test and assess for two inducers both collagen and AA. The analysis in Fig. 3 showed two compounds 1p and 1q showed higher in vitro activities of inhibition rates of 51.25% and 50.07% than that of the 46.67% of picotamide and 46.67% of aspirin induced by collagen, while 1p showed higher in vitro activity of inhibition rate of 31.57% than that of the 29.67% of picotamide induced by AA at the concentration of 1.3 μM. Compound 1p, that was a N1,N3-bis (2-(trifluoromethyl)phenyl) -4-methoxyisophthalamide, had similar structure with compound 1q and both of them had shown good results in the pharmacological tests. which is worth further researching.

Cytotoxicity assay

The effect of the compounds with higher activities on cytotoxicities was evaluated on mouse fibroblast cell (L-929) via CCK-8 assays. The survival rate of cells was set out in Table 3 and the cell morphology after exposed to target compounds were shown in Fig. 4 (Xiong et al. 2007; Abe et al. 2000).

Table 3 Cytotoxicity effect on L-929
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Fig. 4
figure 4

The cell morphology of L-929 following 48 h incubation

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The result analysis of cytotoxicities was shown in Fig. 5, among the eight compound, six compound (1h, 1n, 1o, 1p, 1q, 1v) had higher cell survival rate than picotamide at the dose of 10 μM/L. And the cell survival rate of three compound (1h, 1p, 1q) was higher than that of picotamide at the dose of 100 μM/L. Among them, compound 1h and 1q had the lowest cytotoxicities at the dose of 10 μM/L and 100 μM/L.

Fig. 5
figure 5

Cell Survival Rate of Compounds

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Preliminary structure activity relationship (SAR) exploration

1. The effect of anti-platelet aggregation activity when the substitutions were saturated hydrocarbons

The order of inhibition of platelet was: in o-position of the phenyl rings: 1l#a>1d > 1g > 1aa. in m-position: 1h > 1e > 1ba. in p-position: 1i > 1k > 1j. That meant the activity could be enhanced when the saturated hydrocarbons with bigger steric hindrance were introduced in the phenyl rings.

2. The effect of anti-platelet aggregation activity when the substitution were unsaturated hydrocarbons

The order of inhibition of platelet was: 1o > 1n > aspirin > picotamide > 1m. the result showed that cyano- substituted compounds were more potent than the alkynyl-substituted analogs. Therefore, the relatively strong electron-withdrawing substituent were introduced into the phenyl rings maybe could serve to promote the anti-platelet aggregation activity.

3. The effect of anti-platelet aggregation activity when the substitutions were trifluoromethyl

The order of inhibition of platelet was: 1q > 1p > 1s > aspirin > picotamide > 1r. The result indicated that when the trifluoromethyl was introduced into the o-position and m-position of phenyl rings, the anti-platelet aggregation activity increased significantly. When a electron-withdrawing group (Cl) was introduced into the o-position at the same time, Anti-platelet aggregation activities are significantly improved (the inhibition rate: 1s > 1r).

4. The effect of anti-platelet aggregation activity when two substitutions were introduced into the phenyl rings

Two methyl groups were introduced into the phenyl rings, compounds 1t and 1u were obtained. But compounds 1t and 1u almost had no anti-platelet aggregation activity.

Methyl and chloro groups were simultaneously introduced into the phenyl rings, compounds 1v and 1w were obtained. The order of inhibition of platelet was: 1v > 1w > aspirin > picotamide. Therefore, methyl and electron-withdrawing groups were simultaneously introduced into the phenyl rings might help to promote the anti-platelet aggregation activity.

To sum up, the anti-platelet aggregation activity will increase when the saturated hydrocarbons with bigger steric hindrance were introduced in the side chain phenyl rings. The introduction of cyano and trifluoromethyl could enhance the activity on anti-platelet aggregation. Simultaneous introduction of two methyl groups was unfavorable to the activity, while the introduction of methyl and chloro groups could significantly enhance the activity on anti-platelet aggregation.

Conclusion

In summary, 19 novel 4-methoxyisophthalamide analogs were synthesized and evaluated for anti-platelet activities, the compounds with higher activities were selected to continue research on cytotoxicities. The result exhibited that compounds 1h, 1p, 1q, 1v showed stronger anti-platelet activity and lower cytotoxicities. And, more remarkably, compound 1q, that was a N1,N3-bis (3-trifluoromethylphenyl)-4-methoxyisophthalamides, had significant anti-platelet activity when the inducers were ADP and collagen and it had lower cytotoxicity at the dose of 100 μM/L. Thus, 1q is potential to be studied and may help in developing new anti-platelet drug with elevated activity and lower toxicity.