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Abstract

Seven novel N-alkyl-plinabulin derivatives with aryl groups moieties (nitroquinoline, 1,4-dihydroquinoline, 4-methoxybenzene, and 4-chlorobenzene) have been synthesized via aldol condensation and alkylation in one-pot, and tested for their cytotoxicity against 4 cancer cell lines (KB, HepG2, Lu, and MCF7). Compounds (Z)−3-((6,8-dimethyl-4-oxo-1,4-dihydroquinolin-2-yl)methylene)−6-((Z)−4-methoxybenzylidene)−1-(prop-2-yn-1-yl)piperazine-2,5-dione (5a), (Z)−6-((Z)−4-methoxybenzylidene)−1-(prop-2-yn-1-yl)−3-((1,6,8-trimethyl-4-oxo-1,4-dihydroquinolin-2-yl)methylene)piperazine-2,5-dione (5b), and (Z)−3-((Z)−4-chlorobenzylidene)−1,4-dimethyl-6-((8-methyl-4-nitroquinolin-2-yl)methylene)piperazine-2,5-dione (8) showed strong cytotoxicity against 3 of the cancer cells lines (KB, HepG2 and Lu) with IC₅₀ values ranging from 3.04 to 10.62 µM. The quinoline-derived compounds had higher cytotoxic activity than the benzaldehyde derivatives. The successful synthesis of these derivatives offers useful information for the development of more potent vascular disrupting agents based on plinabulin.

Keywords

anticancer cytotoxicity pliabulin VAC quinoline

In recent years, plinabulin (NPI-2358), a synthetic analog of 2,5-diketopiperazine, has been reported to be a promising vascular disrupting agent (VDA) thus serving as an anti-cancer drug. VDAs target microtubules—the essential filamentous structure of eukaryotic cells, particularly by inducing rapid depolymerization of microtubules in highly proliferating tumor selective vascular endothelial cells, thus causing tumor vascular collapse.¹ Historically, notable VDAs, such as colchicine and combretastatin, have been found to be effective anti-cancer drugs.² On the other hand, plinabulin has a relatively favorable safety profile while still exhibiting colchicine-like tubulin depolymerizing activity. Several plinabulin derivatives showed high cytotoxicity against different cancer cell lines, for example, benzophenone derivatives were evaluated against human HT-29 colorectal cancer cells, while 5-tert-butyl-substituted imidazole analogs were not highly active.^3,4 The benzene ring of the benzoyl group could induce additional π–π interaction, which could be beneficial to antiproliferation.^5,6 Furan-containing derivatives tested against the human lung cancer NCI-H460 cell line exhibited potent cytotoxic activity at the nanomolar level.⁷ Of special interest is a novel colchicine-type anti-microtubule compound: KPU-300, an agent endowed with a 2-pyridyl substituent capable of acting as a potent radiosensitizer.⁸ Currently, plinabulin is being evaluated in Phase III clinical trial in combination with docetaxel and in phase I/II clinical trial in combination with nivolumab for stage IIIb/IV non-small cell lung cancer (NCT02504489 and NCT02812667).⁹ At the same time, there has also been strong interest in the synthesis of plinabulin derivatives to explore options for heightened effectiveness and new applications. This paper reports 7 novel N-alkyl-plinabulin derivatives with aryl group moieties and their cytotoxic activity against some cancer cell lines in vitro.

Results and Discussion

The development of plinabulin derivatives with aryl moieties is a research direction of interest due to the ability of these moieties to alter significantly the hydrophobicity and π-π interaction capacity of the compound.^10,11 In this study, the moieties of choice were nitroquinoline, 1,4-dihydroquinoline, 4-methoxybenzene, and 4-chlorobenzene. The quinoline ring is commonly acknowledged for its antimalarial potency, and the quinine derivative, Chloroquine, has been the most widely used anti-malarial drug since the 1940s.¹² The versatility of this ring system has enabled the synthesis of a large array of derivatives with diverse biological activities, notably the derivatives of quinoline-2-carbaldehyde.^13,14 Promising quinoline-2-carbaldehyde and 1,4-dihydroquinoline-2-carbaldehyde derivatives previously reported in another study were applied in this work.¹⁵ 4-Methoxybenzene and 4-chlorobenzene, 2 of the most prevalent substructures in anticancer molecules as scanned against the National Cancer Institute’s 60 human tumor cell lines, were also utilized.¹⁶ The successful synthesis of these derivatives offers useful information for the development of more potent VDAs based on plinabulin. Thus, in continuation of our previous work, we have prepared new biological active plinabulin analogs.¹⁷ The general procedures for the synthesis of the 7 novel N-alkyl-plinabulin derivatives with different aryl moieties is outlined in 1 -4.

Scheme 1

Reagent and conditions: (i) 1.5 equiv. R¹CHO (2) 1.5 equiv. K₂CO₃, 1.5 equiv. propagyl bromide, DMF, r.t, 24 hours; (ii) 1.5 equiv. R¹CHO (2) 1.5 equiv. K₂CO₃, 1.5 equiv. MeI, DMF, r.t, 24 hours.

Scheme 2

Reagent and conditions: (i) 1.5 equiv. R²CHO (4), 1.5 equiv. K₂CO₃, DMF, reflux, 24 hours; (ii) 1.5 equiv. R²CHO (4) 1.5 equiv. K₂CO₃, 1.5 equiv. MeI, DMF, 0→80 °C, 24 hours.

Scheme 3

Reagent and conditions: (i) 1.5 equiv. R²CHO (4) 1.5 equiv. K₂CO₃, DMF, reflux, 24 hours; (ii) 1.5 equiv. R²CHO (4) 1.5 equiv. K₂CO₃, 1.5 equiv. MeI, DMF, 0→80 °C, 24 hours.

Scheme 4

Reagent and conditions: 1.5 equiv. 4-methoxybenzaldehyde (4) 1.5 equiv. K₂CO₃, 1.5 equiv. MeI, DMF, 0→80 °C, 24 hours.

The N-propargyl-plinabulin derivatives 5 and 6 were synthesized from 1,4-diacetyl-2,5-piperazinedione 1 through 2 steps^{1

-8,11,17} (Schemes 1 and 2, Supplemental Figures S6-S13). The first was aldol condensation between aldehyde derivatives and 1,4-diacetyl-2,5-piperazinedione via K₂CO₃-catalysis and methylation with propargyl bromide in one-pot⁴ to give the N-propargyl-piperidinedione derivatives 3 in high yield 91% (Supplemental Figures S1 and 2). In this reaction, K₂CO₃ behaved as a Lewis base to act both for proton promotion and deprotonation.¹⁸ The hydrolysis of the N-acetyl group leads to parallel N-propargylation (Scheme 1). In the second step (Scheme 2), the N-propargyl-plinabulin derivatives 5 were prepared via continuous aldol reaction of aldehyde derivatives 4 with N-propargyl-piperidinedione 3. These reactions were carried out in refluxing DMF.

Similarly, the N-propagyl-N-methyl-plinabulin derivatives 6 were synthesized from starting material 3 via continuous aldol reaction with 4-chlorobenzaldehyde and N -alkylation with methyl iodide in one-pot. This reaction was carried out in refluxing DMF in the presence of K₂CO₃ (Scheme 2).

Reaction of 1,4-diacetyl-2,5-diketopiperazine 1 with 4-chlorobenzaldehyde, methyl iodide, and potassium carbonate in DMF at room temperature gave product 4a (Scheme 2) in 86% yield (Supplemental Figure S3). Finally, compound 4a was reacted with 4-chlorobenzaldehyde under aldol reaction conditions to provide N-alkylproduct 7 in moderate yields (Supplemental Figures S14 and S15). N-propagyl-N-methyl-plinabulin (8) was obtained under aldol reaction conditions of 4a with 8-methyl-4-nitroquinoline-2-carbaldehyde in the presence of methyl iodide (Scheme 3, Supplemental Figures S16 and S17). The moving from starting 4a to 4b in the reaction with 4-methoxylbenzaldehyde provides symmetrical compound 9 (Scheme 4, Supplemental Figures S18 and 19).

The cytotoxicity evaluation results of the novel synthesized plinabulin derivatives against the epidermoid carcinoma cell line (KB), hepatoma carcinoma cell line (HepG2), lung cancer cell line (Lu), and breast carcinoma cell line (MCF-7) are presented in Table 1.

Table 1

Cytotoxicity Evaluation of Novel N-Alkyl-Plinabulin Derivatives.

Compounds	Cytotoxicity (IC₅₀, µM)
Compounds	KB	HepG2	Lu	MCF7
5a	3.04 ± 0.12	5.86 ± 0.27	10.62 ± 0.51	>100
5b	3.87 ± 0.14	6.41 ± 0.32	6.84 ± 0.35	>100
5c	>100	>100	>100	>100
6	>100	>100	>100	>100
7	>100	>100	>100	>100
8	4.85 ± 0.21	5.14 ± 0.23	7.48 ± 0.36	>100
9	>100	>100	>100	>100
Ellipticine	1.30 ± 0.08	1.30 ± 0.08	1.79 ± 0.12	2.11 ± 0.20

Ellipticine was used as a positive control.

As the results shown in Table 1, compounds 5a, 5b, and 8 showed strong cytotoxicity against 3 of the cancer cells lines (KB, HepG2 and Lu). The structure–activity relationship between N-alkyl-plinabulin and cytotoxic activity was comprehensible because of the higher activity shown by compounds 5a, 5b, and 8 (one aryl = quinoline substituents) as compared to that of compounds 5 c, 6, 7, and 9 (bis aryl = benzene substituents). The quinoline-derived compounds had higher cytotoxic activity than the benzene derivatives, but substitution on the quinoline ring had no clear effect on cytotoxicity, therefore further investigations are required. Moreover, the 1,4-dihydroquinoline derivatives 5a, 5b) demonstrated higher potencies against the epidermoid carcinoma cell line (KB), while quinoline derivative 8 exhibited strong cytotoxicity against hepatoma carcinoma (HepG2).

Experimental

General

All reactions were performed in appropriate oven-dried glass apparatus and under a nitrogen atmosphere. Unless otherwise stated, solvents and chemicals were obtained from commercial sources and used without further purification. Column chromatography was performed using silica gel (60Å, particle size 40‐60 μm). NMR spectra were recorded on a Bruker Advance I spectrophotometer (500 MHz). Chemical shifts (δ) are given in parts per million (ppm) and coupling constants (J) in Hertz (Hz). High resolution mass spectra (HRMS) were recorded on either a Q-executive or a Q-TOF₂ instrument, and IR spectra on a Perkin Elmer Spectrum Two.

General Procedure for the Synthesis of Compounds 3, 4a, and 4b

A solution of 1,4-diacetyl-2,5-piperazinedione (1) (1.0 equiv.), 4-benzaldehyde derivatives (1.5 equiv.) and propargyl bromide (1.5 equiv.) or methyl iodide (1.5 equiv.) in dry DMF (10 ml) was cooled to 0 °C. A solution of K₂CO₃ (1.5 equiv.) in dry DMF (10 ml) was then added dropwise over 30 minutes. After the addition was complete, the mixture was allowed to reach room temperature and then stirred for 24 hours. Afterwards, the reaction mixture was added to water (50 ml) and extracted with ethyl acetate. The organic phase was washed with water and saturated bine. Drying of the organic phase (MgSO₄), filtration of the drying agent, and evaporation of the solvent in vacuo afforded crude compounds 3 or 4, which were then purified by silica gel column chromatography (n-hexane-EtOAc, 8:2) to obtain pure compound 3, 4a, and 4b.

(Z)-1-Acetyl-3-(4-Methoxybenzylidene)-4-(Prop-2-Yn-1-Yl)piperazine-2,5-Dione (3)

91% yield, yellow solid.¹H NMR (CDCl₃, 500 MHz): 7.38 (1H, s); 7.33 (2H, dd, J = 2.0; 7.0 Hz); 6.94 (2H, dd, J = 2.0; 7.0 Hz); 4.53 (2H, s); 4.31 (2H, d, J = 2.5 Hz); 3.85 (3H, s); 2.62 (3H, s); 2.13 (1H, t, J = 2.5 Hz). ¹³C NMR (CDCl₃, 125 MHz): 171.4; 164.8; 164.3; 160.9; 131.4 (2xC); 129.2; 127.7; 127.1; 124.5; 114.4 (2xC); 72.7; 55.4; 45.1; 33.6; 26.5. HR-ESI-MS: Found m/z 313.1179; calcd. for C₁₇H₁₇N₂O₄: 313.1183 [M + H]⁺.

(Z)-1-Acetyl-3-(4-Chlorobenzylidene)-4-Methylpiperazine-2,5-Dione (4a)

86% yield, yellow solid. ¹H NMR (CDCl₃, 500 MHz), δ (ppm): 7.40 (2H, d, J = 8.5 Hz), 7.38 (1H, s), 7.31 (2H, d, J = 8.5 Hz), 4.54 (2H, s), 2.96 (3H, s), 2.62 (3H, s). HR-ESI-MS: Found m/z 293.0687; Calcd. for C₁₄H₁₄ClN₂O₃: 293.0687 [M + H]⁺.

(Z)-1-Acetyl-3-(4-Methoxybenzylidene)-4-Methylpiperazine-2,5-Dione (4b)

88% yield, yellow solid.¹H NMR (CDCl₃, 500 MHz), δ (ppm): 7.30 (1H, s), 7.28 (2H, dd, J = 2.0, 7.0 Hz), 6.92 (2H, dd, J = 2.0, 7.0 Hz), 4.51 (2H, s), 3.85 (3H, s), 2.96 (3H, s), 2.62 (3H, s). ¹³C NMR (CDCl₃, 125 MHz): 171.5, 165.1, 164.1, 160.6, 131.5 (2xC), 129.8, 125.8, 124.8, 114.1 (2xC), 55.4, 45.3, 34.2, 26.6. HR-ESI-MS: Found m/z 289.1191; Calcd. for C₁₅H₁₇N₂O₄: 289.1183 [M + H]⁺.

General Procedure for the Synthesis of Compounds 5a-C and 7

A solution of compound 3 (1.0 equiv.) or compound 4a (1.0 equiv.) and 4-benzaldehyde derivatives (1.5 equiv.) in dry DMF (5 ml) was cooled to 0 °C. A solution of K₂CO₃ (1.5 equiv.) in dry DMF (5 ml) was then added dropwise over 30 minutes. After the addition was complete, the mixture was heated to 80 °C and stirred for 24 hours. Afterwards, the reaction mixture was added to water (50 ml) and extracted with ethyl acetate. The organic phase was washed with water and saturated bine. Drying of the organic phase (MgSO₄), filtration of the drying agent, and evaporation of the solvent in vacuo afforded crude compounds 5 or 7, which were then purified by silica gel column chromatography (n-hexane-EtOAc, 8:2 or 85:15, v/v) to obtain pure compound 5a, 5b, 5 c and 7.

(Z)-3-((6,8-Dimethyl-4-Oxo-1,4-Dihydroquinolin-2-Yl)methylene)-6-((Z)-4-Methoxybenzylidene)-1-(Prop-2-Yn-1-Yl)piperazine-2,5-Dione (5a)

38% yield, yellow solid.¹HNMR (CDCl₃, 500 MHz), δ (ppm): 8.00 (2H, d, J = 8.5 Hz), 7.70 (1H, s), 7.37 (1H, s), 7.30 (2H, d, J = 8.5 Hz), 7.29 (1H, s), 6.63 (1H, s), 6.38 (1H, s), 4.96 (2H, d, J = 2.5 Hz), 3.97 (3H, s), 2.77 (3H, s), 2.52 (1H, t, J = 2.5 Hz), 2.42 (3H, s). ¹³C NMR (CDCl₃, 125 MHz), δ (ppm): 162.2, 159.7, 154.4, 152.8, 145.6, 135.8, 135.6, 134.9, 133.5, 133.1, 133.0 (2xC), 131.9, 129.1, 128.5 (2xC), 128.2, 119.7, 118.4, 105.8, 101.9, 77.9, 75.2, 55.7, 54.5, 21.7, 19.0. HR-ESI-MS: Found m/z 454.1753; Calcd. for C₂₇H₂₄N₃O₄: 454.1761 [M + H]⁺.

(Z)-6-((Z)-4-Methoxybenzylidene)-1-(Prop-2-Yn-1-Yl)-3-((1,6,8-Trimethyl-4-Oxo-1,4-Dihydroquinolin-2-Yl)methylene)piperazine-2,5-Dione (5b)

43% yield, yellow solid.¹H NMR (CDCl₃, 500 MHz), δ (ppm): 7.79 (1H, s), 7.44 (1H, s), 7.42 (1H, s), 7.29 (2H, d, J = 8.5 Hz), 6.91 (2H, d, J = 8.5 Hz), 6.87 (1H, s), 6.76 (1H, s), 4.48 (2H, d, J = 2.5 Hz), 4.05 (3H, s), 3.84 (3H, s), 2.83 (3H, s), 2.49 (3H, s), 2.14 (1H, t, J = 2.5 Hz). ¹³C NMR (CDCl₃, 125 MHz), δ (ppm): 162.4, 160.0, 159.1, 145.1, 135.9, 135.7, 133.1, 131.8, 130.9 (2xC), 127.1, 126.0, 122.3, 119.9, 118.4, 114.0 (2xC), 113.2, 102.2, 101.1, 77.7, 72.4, 55.7, 55.3, 35.6, 21.7, 19.1. HR-ESI-MS: Found m/z 468.1947. Calcd. for C₂₈H₂₆N₃O₄: 468.1918 [M + H]⁺.

3-((Z)-4-Chlorobenzylidene)-6-((Z)-4-Methoxybenzylidene)-1-(Prop-2-Yn-1-Yl)piperazine-2,5-Dione (5c)

35% yield, yellow solid. IR (KBr) ν_max (cm^-1): 3336, 3072, 2926, 2851, 2132, 1734, 1683, 1700, 1613, 1583, 1558, 1336, 1179, 1088, 845. ¹H NMR (DMSO-d₆ , 500 MHz), δ (ppm): 7.43 (2H, d, J = 7.5 Hz), 7.36 (2H, d, J = 7.5 Hz), 7.27 (1H, s), 7.24 (2H, d, J = 8.5 Hz), 7.00 (2H, d, J = 8.5 Hz), 6.98 (1H, s), 4.72 (2H, d, J = 2.5 Hz), 3.84 (3H, s), 2.53 (1H, t, J = 2.5 Hz).¹³C NMR (DMSO-d₆ , 125 MHz), δ (ppm): 159.6, 159.4, 157.8, 134.7, 131.4, 131.0 (2xC), 129.8 (2xC), 129.7 (2xC), 129.2, 126.8, 126.4, 121.3, 115.5, 114.8 (2xC), 78.1, 75.9, 55.8, 46.7. HR-ESI-MS: Found m/z 393.1021. Calc. for C₂₂H₁₈ClN₂O₃: 393.1000 [M + H]⁺.

3,6-Bis((Z)-4-Chlorobenzylidene)-1-Methylpiperazine-2,5-Dione (7)

36% yield, yellow solid.¹H NMR (CDCl₃, 500 MHz), δ (ppm): 7.42‐7.36 (6H, overlapped), 7.23 (1H, s), 7.22 (2H, d, J = 8.5 Hz), 7.00 (1H, s), 2.99 (3H, s).¹³C NMR (CDCl₃, 125 MHz), δ (ppm): 159.2 (2xC = O amide), 134.8, 134.6, 132.2, 131.1, 130.9, 129.9 (2xC), 129.8 (2xC), 128.6 (2xC), 128.0 (2xC), 126.1, 119.7, 116.2, 36.7. HR-ESI-MS: Found m/z 373.0521; Calcd. for: C₁₉H₁₅Cl₂N₂O₂: 373.0505 [M + H]⁺.

General Procedure for the Synthesis of Compounds 6, 8, and 9

A solution of compound 3 (1.0 equiv.) or compound 4a,b (1.0 equiv.), 4-benzaldehyde derivatives (1.5 equiv.) or 8-methyl-4-nitroquinoline-2-carbaldehyde (1.5 equiv.) and methyl iodide (1.5 equiv.) in dry DMF (10 ml) was cooled to 0 °C. A solution of K₂CO₃ (1.5 equiv.) in dry DMF (5 ml) was then added dropwise over 30 minutes. After the addition was complete, the mixture was heated to 80 °C and stirred for 24 hours. Afterwards, the reaction mixture was added to water (50 ml) and extracted with ethyl acetate. The organic phase was washed with water and saturated bine. Drying of the organic phase (MgSO₄), filtration of the drying agent, and evaporation of the solvent in vacuo afforded the crude compounds, which were then purified by silica gel column chromatography (n-hexane-EtOAc, 8:2 or 85:15, v/v) to obtain pure compound 6, 8, and 9.

3-((Z)-4-Chlorobenzylidene)-6-((Z)-4-Methoxybenzylidene)-4-Methyl-1-(Prop-2-Yn-1-Yl)piperazine-2,5-Dione (6)

33% yield, yellow solid. IR (KBr) ν_max (cm^-1): 3330, 3076, 2926, 2855, 2135, 1715, 1680, 1668, 1600, 1580, 1551, 1333, 1169, 1076, 845. ¹H NMR (DMSO-d₆ , 500 MHz), δ (ppm): 7.39 (2H, dd, J = 2.0, 8.0 Hz), 7.37 (1H, s), 7.23 (2H, d, J = 8.0 Hz), 7.00 (2H, dd, J = 2.0, 8.0 Hz), 6.97 (2H, d, J = 8.0 Hz), 6.96 (1H, s), 4.75 (2H, d, J = 2.5 Hz), 3.84 (3H, s), 3.03 (3H, s), 2.55 (1H, t, J = 2.5 Hz).¹³C NMR (DMSO-d₆ , 125 MHz), δ (ppm): 159.9, 159.7, 157.7, 131.0 (2xC), 130.5, 129.5 (2xC), 127.0, 124.6, 120.6, 119.9, 117.2, 116.0, 114.9 (2xC), 114.7 (2xC), 78.1, 75.9, 55.8, 46.6, 36.6. HR-ESI-MS: Found m/z 407.1173. Calcd. for C₂₃H₂₀ClN₂O₃: 407.

(Z)-3-((Z)-4-Chlorobenzylidene)-1,4-Dimethyl-6-((8-Methyl-4-Nitroquinolin-2-Yl)methylene)piperazine-2,5-Dione (8)

29% yield, yellow solid. IR (KBr) ν_max (cm^-1): 3047, 2924, 2855, 1735, 1680, 1616, 1583, 1487, 1334, 1165, 1089, 819, 756. ¹H NMR (DMSO-d₆ , 500 MHz), δ (ppm): 8.02 (1H, s), 7.45 (1H, d, J = 8.0 Hz), 7.63 (1H, dd, J = 1.0, 8.0 Hz), 7.38‐7.50 (5H, m, overlap), 7.21 (1H, s), 7.11 (1H, s), 3.02 (3H, s), 2.88 (3H, s), 2.67 (3H, s). ¹³C NMR (DMSO-d₆ , 125 MHz), δ (ppm): 160.8, 160.6, 151.8, 146.7, 141.3, 137.1, 134.7, 132.2, 132.2, 131.6 (2xC), 131.2, 128.3 (2xC), 128.0, 124.4, 123.7, 121.4, 119.1, 118.6, 117.2, 36.0, 35.3, 17.7. HR-ESI-MS: Found m/z 463.1175. Calcd. for C₂₄H₂₀ClN₄O₄: 463.1168 [M + H]⁺.

3-((Z)-4-Chlorobenzylidene)-6-((Z)-4-Methoxybenzylidene)-1,4-Dimethylpiperazine-2,5-Dione (9)

37% yield, yellow solid. ¹H NMR (CDCl₃, 500 MHz), δ (ppm): 7.28 (4H, d, J = 7.0 Hz), 7.15 (2H, s), 6.90 (4H, d, J = 7.0 Hz), 3.84 (6H, s), 3.00 (6H, s).¹³C NMR (CDCl₃, 125 MHz), δ (ppm): 162.9 (2xCO amide), 159.9 (2xC), 131.1 (4xC), 130.1 (2xC), 125 (2xC), 121.7 (2xC), 113.9 (4xC), 55.3 (2xC), 35.1 (2xC). HR-ESI-MS: Found m/z 378.1579, calcd. for C₂₂H₂₂N₂O₄: 378.1574 [M + H]⁺.

Cell culture and cell viability assay

The synthesized compounds were evaluated for their cytotoxicity against the 4 hoursuman cancer cell lines, epidermoid carcinoma (KB), hepatoma carcinoma (HepG2), lung cancer cell line (Lu) and breast carcinoma (MCF₇) obtained from the American Type Culture Collection (USA). The cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere (95% air and 5% CO2). Exponentially growing cells were used throughout the experiments. The inhibitory effects of the compounds on the growth of the cancer cell lines were determined by measuring their metabolic activity using a 3-[4,5-dimethylthiazol-2-yl]−2,5-diphenyl-trazolium bromide (MTT) assay. Briefly, the cell lines (1 × 105 cells/mL) were treated for 3 days with a series of concentrations of the compounds (in DMSO): 0.125, 0.5, 2.0, 8.0, 32.0, and 128.0 µg/mL. After incubation, 0.1 mg MTT solution (50 µL of a 2 mg/mL solution) was added to each well, and the cells were then incubated at 37 °C for 4 hours. The plates were centrifuged at 1000 rpm for 10 minutes at room temperature, and the medium was then carefully aspirated. Dimethyl sulfoxide (150 µL) was added to each well to dissolve the formazan crystals. The plates were read immediately at 540 nm on a microplate reader (TECAN GENIOUS). All the experiments were performed 3 times, and the mean absorbance values were calculated. The results are expressed as the percentage of inhibition that produced a reduction in the absorbance by the treatment of the compounds compared to the untreated controls. A dose-response curve was generated, and the inhibitory concentration of 50% (IC50) was determined for each compound, as well as for each cell line.

Conclusions

In conclusion, 7 novel N-alkyl-plinabulin derivatives with aryl group moieties (nitroquinoline, 1,4-dihydroquinoline, 4-methoxybenzene, and 4-chlorobenzene) were successfully synthesized via aldol condensation alkylation in one-pot. Preliminary cytotoxicity evaluation of these compounds against the KB, HepG2, Lu, and MCF7 cell lines demonstrated diverse levels of effectiveness and selectivity, thus providing useful information for the development of more potent VDAs based on plinabulin. Compounds 5a, 5b, and 8 showed strong cytotoxicity against three of the cancer cells lines (KB, HepG2 and Lu), and the quinoline-derived compounds have higher cytotoxic activity than the benzaldehyde derivatives.

Supplemental Material

Online supplementary file 1 - Supplemental material for Synthesis and Cytotoxic Activity of Several Novel N-Alkyl-Plinabulin Derivatives With Aryl Group Moieties

Supplemental material, Online supplementary file 1, for Synthesis and Cytotoxic Activity of Several Novel N-Alkyl-Plinabulin Derivatives With Aryl Group Moieties by Pham The Chinh, Pham Thi Tham, Duong Huong Quynh, Nguyen Van Tuyen, Dinh Thuy Van, Phan Thanh Phuong, Tran Thi Thu Hang and Phan Van Kiem in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors are indebted to the Vietnamese National Foundation for Science and Technology Development (NAFOSTED), code 104.01-2016.18 for financial support.

ORCID iD

Phan Van Kiem

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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Synthesis and Cytotoxic Activity of Several Novel N -Alkyl-Plinabulin Derivatives With Aryl Group Moieties

Abstract

Keywords

Results and Discussion

Experimental

General

General Procedure for the Synthesis of Compounds 3, 4a, and 4b

(Z)-1-Acetyl-3-(4-Methoxybenzylidene)-4-(Prop-2-Yn-1-Yl)piperazine-2,5-Dione (3)

(Z)-1-Acetyl-3-(4-Chlorobenzylidene)-4-Methylpiperazine-2,5-Dione (4a)

(Z)-1-Acetyl-3-(4-Methoxybenzylidene)-4-Methylpiperazine-2,5-Dione (4b)

General Procedure for the Synthesis of Compounds 5a-C and 7

(Z)-3-((6,8-Dimethyl-4-Oxo-1,4-Dihydroquinolin-2-Yl)methylene)-6-((Z)-4-Methoxybenzylidene)-1-(Prop-2-Yn-1-Yl)piperazine-2,5-Dione (5a)

(Z)-6-((Z)-4-Methoxybenzylidene)-1-(Prop-2-Yn-1-Yl)-3-((1,6,8-Trimethyl-4-Oxo-1,4-Dihydroquinolin-2-Yl)methylene)piperazine-2,5-Dione (5b)

3-((Z)-4-Chlorobenzylidene)-6-((Z)-4-Methoxybenzylidene)-1-(Prop-2-Yn-1-Yl)piperazine-2,5-Dione (5c)

3,6-Bis((Z)-4-Chlorobenzylidene)-1-Methylpiperazine-2,5-Dione (7)

General Procedure for the Synthesis of Compounds 6, 8, and 9

3-((Z)-4-Chlorobenzylidene)-6-((Z)-4-Methoxybenzylidene)-4-Methyl-1-(Prop-2-Yn-1-Yl)piperazine-2,5-Dione (6)

(Z)-3-((Z)-4-Chlorobenzylidene)-1,4-Dimethyl-6-((8-Methyl-4-Nitroquinolin-2-Yl)methylene)piperazine-2,5-Dione (8)

3-((Z)-4-Chlorobenzylidene)-6-((Z)-4-Methoxybenzylidene)-1,4-Dimethylpiperazine-2,5-Dione (9)

Cell culture and cell viability assay

Conclusions

Supplemental Material

Online supplementary file 1 - Supplemental material for Synthesis and Cytotoxic Activity of Several Novel N-Alkyl-Plinabulin Derivatives With Aryl Group Moieties

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iD

Supplemental Material

References

Supplementary Material