Abstract
A series of novel 3-substituted amino-4-hydroxycoumarin derivatives have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their CHS inhibition activity and antimicrobial activity in vitro. The enzymatic assay indicated that most of the compounds have good inhibitory activity against CHS, in which compound 6o with IC50 of 0.10 mmol/L had stronger activity than that of polyoxins B, which acts as control drug with IC50 of 0.18 mmol/L. As far as the antifungal activity is concerned, most of the compounds possessed moderate to excellent activity against some representative pathogenic fungi. Especially, compound 6b was found to be the most potent agent against Cryptococcus neoformans with minimal inhibitory concentration (MIC) of 4 μg/mL. Moreover, the results of antibacterial screening showed that these compounds have negligible actions to some tested bacteria. Therefore, these compounds would be promising to develop selective antifungal agents.
Introduction
In the past decades, as the major causes of high morbidity and mortality rates in patients who received antineoplastic chemotherapy, organ transplants or suffered AIDS, the invasive fungal infections have become a more and more alarming problem to be settledCitation1. The treatment of fungal infections, particularly those caused by drug-resistant fungal pathogens, is often complicated by high toxicity, low tolerability or narrow spectrum of activityCitation2–4. Furthermore, the representative antifungal drugs which were used in clinic at present have certain limitation. For instance, azoles exhibit broad antifungal spectrum and are vulnerable to resistance; adverse side effects of amphotericin B are infusional toxicity, nephrotoxicity; flucytosine is restricted to pathogenic yeasts and fungal strains resistant to echinocandins have emergedCitation5–7. Consequently, antifungal agents of new molecular scaffolds with high efficiency, broad spectrum and low toxicity are highly desirable.
Chitin is widely distributed in invertebrates, the cuticles of arthropod exoskeletons and the fungal cell wallsCitation8, but it is absent in plants and humansCitation9,Citation10. It is a linear polysaccharide chain that is composed of thousands of N-acetyl-d-glucosamine (GlcNAc) residues joined by β-1,4-glycosidic bondsCitation11,Citation12. The biosynthesis of chitin is catalyzed by chitin synthase (CHS) which uses the uridine diphosphoryl-N-acetyl-d-glucosamine (UDP-GlcNAc) as the substrate donor to provide the GlcNAc ()Citation13,Citation14. Thus, the CHS signifies an ideal target for the development of pesticides and antifungal agentsCitation15. Although many efforts have been made to discover the CHS inhibitors in the past decades, not much progress has been made. Such inhibitors possess the potential to inhibit the biosynthesis of chitin efficiently. The nucleoside polyoxins and nikkomycins, the representative competitive CHS inhibitorsCitation16 isolated from culture filtrates of Streptomyces strains, possess some of the structural features of the natural substrate UDP-GlcNAc and can interfere with the synthesis of fungal cell wall and ecdysis of insect in vivo by inhibiting the CHS. The inhibition constants (Ki) of nucleoside polyoxins are in range of 0.1–1 μMCitation17,Citation18. The low inhibitory activity and weak efficacy of these compounds maybe due to the degradation of their dipeptide side chains in the organism. Many analogues of nikkomycins and polyoxins were designed and developed, but none of them has entered in clinical trialsCitation19–21. Therefore, new CHS inhibitors will be needed.
Figure 1. The biosynthesis of chitin.
Coumarin is chemically known as 2H-chromen-2-one heterocycle containing an oxygen atomCitation22. The structural type of coumarin enables its derivatives to interact readily with all kinds of enzymes and receptors in organisms through weak bond interactions. So they exhibit wide potentiality as medicinal drugsCitation23 with various biological and pharmacological activities such as antibacterial, antifungalCitation24, antioxidantCitation25, anti-inflammatoryCitation26, analgesicCitation27, anticancerCitation28, anthelminticCitation29, anti-HIVCitation30 and antiviralCitation31 activities. In recent studies, the coumarin heterocycles (), as antimicrobial agents, have attracted extensive interest. For instance, the 7-substituted coumarin (II) showed a moderate efficacy (minimal inhibitory concentration, MIC = 125 μg/mL) against Cryptococcus neoformansCitation32; the 4-chloro-3-phenylimino coumarin (III) exhibited moderate antifungal activity (MIC = 15 μg /mL) against Aspergillus niger and Candida albicans in comparison with fluconazoleCitation33; the Imidazolethione coumarin hydrazide derivatives (IV) had good antibacterial activity against Escherichia coli with inhibitory zone diameter of 32 mm in 25 μg/mLCitation34 and the 4-azidomethyl coumarin sulfonamides derivatives (V) showed excellent antifungal efficacies against C. albicans, A. niger and Fusarium oxysporum with MIC values of 1–4 μg/mL, which were 2–8 times more potent than fluconazole (MIC = 8 μg/mL)Citation35. Thus, the coumarin is a versatile fragment used to design the novel compounds with pharmacological activity.
Figure 2. Some reported antimicrobial agents containing coumarin moiety.
In continuation of previous study for developing new CHS inhibitorsCitation37, in this work we focused our attention on 3-substituted amino-4-hydroxycoumarin derivatives bearing an N-phenylpiperazine group on l-tartaric amide side chain. We expected that substituting the 3-position of the coumarin with a linear moiety which resembles roughly the side chain of polyoxins B and Nikkomycin Z () would lead to novel derivatives of coumarin which possess broad-spectrum antifungal activity and CHS inhibition activity. The flexible and different substituents were introduced to the coumarin ring or the aryl of phenylpiperazine in order to investigate their bioactivity and to draw the structure–activity relationship of these derivatives. Thus, we report herein synthesis and biological evaluation of the 3-substituted amino-4-hydroxycoumarin derivatives as novel CHS inhibitors.
Figure 3. The structures of polyoxin B, Nikkomycin Z and designed compounds.
Experimental protocols
Chemistry
All the reagents, solvents and instruments used in this article are produced in China unless indicated. All reactions were monitored by thin layer chromatography (TLC) on pre-coated silica gel plates. The melting points were determined by X-6 melting point apparatus without correction. 1H NMR and 13C NMR spectra were recorded on Bruker AV 300 MHz spectrometer (Switzerland) using CDCl3 or DMSO-d6 as solvents and tetramethylsilane (TMS) as an internal standard. The chemical shifts are expressed in δ, ppm. The mass spectra were recorded using Agilent single quadrupole liquid chromatogram–mass spectrograph (LC–MS) (Santa Clara, CA). Highresolution mass spectra were determined by Finnigan/AMT95 (San Jose, CA).
General procedure for the synthesis of compounds
Synthesis of diethyl-l-tartarate (1)
To a solution of l-tartaric acid (10.00 g, 66.67 mmol) and EtOH (100 mL), SOCl2 (10.7 mL, 146.7 mmol) was added dropwise in an ice bath. The mixture was stirred at room temperature for 24 h. Then the solvent was evaporated in vacuum and CH2Cl2 (30 mL) was added to the mixture. The resulting mixture was washed with saturated aqueous NaHCO3 and the aqueous layer was extracted with CH2Cl2 (30 mL). The combined organic extracts were washed with brine and dried by anhydrous Na2SO4. After filtering, the organic layer was concentrated in vacuum. The residue was purified by flash column chromatography on silica gel eluting with ethyl acetate–petroleum ether (P.E.) (1:5) to give the diethyl-l-tartarate (1) (12.6 g, yield, 91%) as a light yellow oil. 1H NMR (300 MHz, CDCl3) δ 4.54 (s, 2H, CH), 4.33 (q, J = 7.1 Hz, 4H, CH2), 3.07 (s, 2H, OH), 1.34 (t, J = 7.1 Hz, 6H, CH3).
Synthesis of l-(4R,5R)-2,2-dimethyl-1,3-dioxolane-4,5-dicarboxylic acid monoethyl ester (2)
To a solution of diethyl-l-tartrate (1) (26.4 g, 128 mmol) and p-TsOH·H2O (0.3 g) in dry benzene (300 mL) 2,2-dimethoxypropane (20 g, 192 mmol) was added and the mixture was stirred for 4 h at 80 °C. After cooling to room temperature, the resulting mixture was washed with saturated aqueous NaHCO3 and the aqueous layer was extracted with EtOAc. The combined organic extracts were washed with brine and water. Then solution was dried by anhydrous Na2SO4. After being filtered, the solution was concentrated in vacuum to give diethyl(2R,3R)-2,3-O-isopropylidenetartrate (29.8 g, yield, 95%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3) δ 4.81 (d, J = 4.2 Hz, 1H, CH), 4.79 (d, J = 4.8 Hz, 1H, CH), 4.29 (q, J = 7.1 Hz, 4H, CH2), 1.50 (s, 6H, C–CH3), 1.32 (t, J = 7.1 Hz, 6H, CH3). To a solution of diethyl(2R,3R)-2,3-O-isopropylidenetartrate (10 g, 40.5 mmol), distilled water (100 mL) and 1,4-dioxine (100 mL) the NaOH solution (1 mol/L, 43 mL) was added dropwise in half hour. The mixture was stirred at room temperature for 2–3 h and extracted with CH2Cl2 (200 mL). The aqueous layer was acidized with concentrated hydrogen chloride to pH 2 and extracted with CH2Cl2 (200 mL). The combining organic solution was dried by anhydrous Na2SO4. After being filtered, the solution was concentrated in vacuum. The residue was purified by flash column chromatography on silica gel eluting with ethyl acetate–P.E. (1:5, 1:1) to give the desired product 2 (7.3 g, yield, 83%) as a light yellow oil. 1H NMR (300 MHz, CDCl3) δ 10.07 (s, 1H, COOH), 4.88 (d, J = 5.2 Hz, 1H, CH), 4.81 (d, J = 5.4 Hz, 1H, CH), 4.31 (q, J = 7.1 Hz, 2H, CH3CH2O), 1.53 (s, 3H, C–CH3), 1.51 (s, 3H, C–CH3), 1.33 (t, J = 7.1 Hz, 3H, CH3CH2O).
General procedure for preparation of the 1-(4-substituted phenyl)piperazine (4a–e)
The mixture of 4-substituted aniline (10 g, 78.4 mmol), bis-(2-chloroethylamine)hydrochloride (14.7 g, 82.4 mmol) and para-toluenesulphonic acid (PTSA) (0.5, 3%) in xylene (44 mL) was heated to reflux at 140–145 °C for 12–24 h. When the reaction was completed, the mixture was cooled to room temperature to crystallize. The crystal was filtrated and recrystallized in the distilled water to give the desired product with yield 73–85%.
General procedure of synthesizing intermediate 5
l-(4R,5R)-2,2-dimethyl-1,3-dioxolane-4,5-dicarboxylic acid monoethyl ester (2) (5 g, 22.9 mmol) was dissolved in 30 mL CH2Cl2, then N-hydroxybenzotriazole (3.1 g, 22.9 mmol), 4-dimethylaminopyridine (0.25 g, 2.2 mmol) and N,N-dicyclohexylcarbodiimide (5.2 g, 25.2 mmol) were added in the solution. The mixture was stirred in room temperature for 0.5 h and 1-(4-subsituted-phenyl)piperazine (4) (18.4 mmol) was added in 15 mL CH2Cl2 and then stirred for 20 h in room temperature. After the reaction was completed, the mixture was filtered. The solution was washed with saturated aqueous NaHCO3 and 1 mol/L hydrochloric acid, and dried by anhydrous Mg2SO4. After being filtered, the solution was concentrated in vacuum. The residue was purified by flash column chromatography on silica gel eluting with ethyl acetate–P.E. (1:5) to give product 5.
l-(4R,5R)-5-(4-(4-fluorophenyl)piperazine-1-carbonyl)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid (5a)
Grey liquid; yield 54%; 1H NMR (300 MHz, CDCl3) δ 10.18 (s, 1H, COOH), 7.04 (d, J = 8.1 Hz, benzene-3,5-2H), 6.82 (d, J = 8.1 Hz, benzene-2.6-2H), 5.27 (d, J = 6.0 Hz, 1H, CH), 4.94 (d, J = 6.0 Hz, 1H, CH), 3.81 (s, 4H, piperazine), 3.27 (s, 4H, piperazine), 1.52 (s, 3H, CH3), 1.44 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) δ 172.48, 168.22, 155.83, 147.70, 126.75, 117.12, 112.65, 76.43, 75.65, 49.85, 49.37, 45.52, 42.78, 26.24.
l-(4R,5R)-5-(4-(4-chlorophenyl)piperazine-1-carbonyl)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid (5b)
White solid; yield 40%; mp 120–121 °C; 1H NMR (300 MHz, CDCl3) δ 10.27 (s, 1H, COOH), 7.24 (d, J = 8.9 Hz, benzene-3,5-2H), 6.86 (d, J = 8.9 Hz, benzene-2.6-2H), 5.25 (d, J = 6.0 Hz, 1H, CH), 4.90 (d, J = 6.0 Hz, 1H, CH), 3.96 (t, J = 12.1 Hz, 2H, piperazine), 3.73 (t, J = 18.0 Hz, 2H, piperazine), 3.30–3.17 (m, 2H, piperazine), 3.10 (dd, J = 19.6, 8.3 Hz, 2H, piperazine), 1.51 (s, 3H, CH3), 1.43 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) δ 172.28, 167.12, 149.20, 129.13, 125.75, 118.00, 112.97, 76.33, 75.65, 49.86, 49.27, 45.51, 42.58, 26.25.
l-(4R,5R)-5-(4-(4-bromophenyl)piperazine-1-carbonyl)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid (5c)
Yellow solid; yield 67%; mp 129–130 °C; 1H NMR (300 MHz, CDCl3) δ 10.15 (s, 1H, COOH), 7.31 (d, J = 7.8 Hz, benzene-3,5-2H), 6.75 (d, J = 7.8 Hz, benzene-2.6-2H), 5.20 (d, J = 6.0 Hz, 1H, CH), 4.99 (d, J = 6.0 Hz, 1H, CH), 3.89 (t, J = 13.6 Hz, 2H, piperazine), 3.68 (t, J = 18.5 Hz, 2H, piperazine), 3.27 (s, 2H, piperazine), 3.10 (t, J = 19.0 Hz, 2H, piperazine), 1.50 (s, 3H, CH3), 1.44 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) δ 172.27, 167.32, 150.12, 127.33, 118.75, 118.10, 112.96, 76.43, 75.65, 49.87, 49.26, 45.49, 42.57, 26.23.
l-(4R,5R)-5-(4-(4-methylphenyl)piperazine-1-carbonyl)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid (5d)
Light yellow solid; yield 45%; mp 125–127 °C; 1H NMR (300 MHz, CDCl3) δ 10.27 (s, 1H, COOH), 7.13 (d, J = 8.2 Hz, 2H, benzene), 6.95 (d, J = 8.2 Hz, 2H, benzene), 5.23 (d, J = 6.1 Hz, 1H, CH), 4.90 (d, J = 6.1 Hz, 1H, CH), 4.10–3.90 (m, 2H, piperazine), 3.90–3.77 (m, 2H, piperazine), 3.26 (m, 2H, piperazine), 3.21–3.05 (m, 2H, piperazine), 2.30 (s, 3H, benzene–CH3), 1.52 (s, 3H, C–CH3), 1.45 (s, 3H, C–CH3). 13C NMR (75 MHz, CDCl3) δ 172.78, 167.55, 149.22, 130.14, 125.75, 115.70, 112.67, 76.37, 75.65, 49.75, 49.32, 45.49, 42.54, 26.21.
l-(4R,5R)-5-(4-(4-methoxyphenyl)piperazine-1-carbonyl)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid (5e)
Light yellow solid; yield 52%; mp 136–137 °C; 1H NMR (300 MHz, CDCl3) δ 10.27 (s, 1H, COOH), 6.78 (d, J = 7.9 Hz, benzene-3,5-2H), 6.69 (d, J = 7.9 Hz, benzene-2.6-2H), 5.25 (d, J = 6.0 Hz, 1H, CH), 4.90 (d, J = 6.0 Hz, 1H, CH), 3.94 (m, 2H, piperazine), 3.85–3.63 (m, 2H, piperazine), 3.32–3.16 (m, 2H, piperazine), 3.10–2.96 (m, 2H, piperazine), 3.80 (s, 3H, OCH3), 1.51 (s, 3H, CH3), 1.43 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) δ 172.38, 167.22, 150.82, 149.20, 118.12, 115.75, 112.97, 76.34, 75.55, 49.87, 49.46, 45.75, 42.62, 26.32.
General procedure of synthesis of target compounds 6
The intermediate compounds 5 (1.35 mmol) were dissolved in 10 mL CH2Cl2 and HOBt (1.35 mmol), DMAP (1.4 mmol), DCC (1.45 mmol) were added. The mixture was stirred in room temperature for 0.5 h and then a 3-amino-4-hydroxycoumarin derivative (1.24 mmol) in 10 mL CH2Cl2 was added. The mixture was stirred for 20 h in 35 °C and filtered. The organic layer was washed with saturated NaHCO3 aqueous and 1 mol/L hydrochloric acid, and dried over anhydrous Mg2SO4. After being filtered, the solution was concentrated in vacuum to give a yellow solid. The solid was dissolved in 50% (TFA and water v/v) TFA (10 mL) and stirred for 24 h and then the solution was concentrated. The resulting oil was purified by silica gel chromatography eluting with ethyl acetate–P.E. (1:5) to give the target compound 6 ().
Scheme 1. Synthesis of compounds 6a–t. Reagents and conditions: (a) SOCl2, EtOH, rt, 12 h. (b) CH3C(OCH3)2CH3, p-TsOH, benzene, 80 °C; dioxine, H2O, NaOH. (c) SOCl2, CHCl3, 40–50 °C, 4 h. (d) xylol, p-TsOH, 145 °C. (e) DCC, HOBt, DMAP, CH2Cl2. (f) DCC, HOBt, DMAP, CH2Cl2;50%CF3COOH, 35 °C.
(2R,3R)-4-(4-(4-fluorophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxycoumarin-3-yl)-4-oxobutanamide (6a)
Yellow solid; yield 20%; [α]20D = +46 (c = 1, CH3OH). mp 218–219 °C; IR (KBr disk): ν 3446 (OH), 3398, 3340 (NH), 2940, 2828 (CH), 1690, 1638 (C = O), 1589, 1536, 1495, 1457 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.19 (s, 1H, coumarin-OH), 8.88 (s, 1H, NH), 7.90 (d, J = 7.4 Hz, 1H, coumarin-5-H), 7.83 (dd, J = 8.4, 5.1 Hz, 2H, coumarin-7,8-H), 7.56 (t, J = 7.4 Hz, 1H, coumarin-6-H), 7.33 (d, J = 7.6 Hz, 2H, benzene-3,5-H), 7.18 (d, J = 8.7 Hz, 2H, benzene-2,6-H), 4.55 (d, J = 27.7 Hz, 2H, CH), 4.25–3.93(m, 4H, piperazine-H), 3.56 (m, 4H, piperazine-H), 1.99 (s, 1H, OH), 1.92 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6) δ 173.65, 169.46, 160.23, 158.87, 154.75, 151.12, 148.17, 132.15, 125.45, 124.11, 118.16, 117.60, 116.95, 116.47, 99.89, 72.78, 71.63, 48.86, 48.07, 44.89, 42.23. LC–MS: m/z 472 [M + H]+, HRMS: calcd for C23H22FN3O7 [M + H]+, 472.1442; found, 472.1500.
(2R,3R)-4-(4-(4-chlorophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxycoumarin-3-yl)-4-oxobutanamide (6b)
Yellow solid; yield 15%; [α]20D = +52 (c = 1, CH3OH). mp 184–186 °C; IR (KBr disk): ν 3445 (OH), 3379, 3339 (NH), 2928, 2830 (CH), 1690, 1640 (C = O), 1599, 1538, 1493, 1455 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.21 (s, 1H, coumarin-OH), 9.02 (s, 1H, NH), 7.92 (d, J = 7.5 Hz, 1H, coumarin-5-H), 7.79 (d, 2H, coumarin-7,8-H), 7.54 (t, J = 7.5 Hz, 1H, coumarin-6-H), 7.35 (d, J = 7.9 Hz, 2H, benzene-3,5-H), 7.09 (d, J = 8.2 Hz, 2H, benzene-2,6-H), 4.55 (d, J = 27.7 Hz, 2H, CH), 3.89 (m, 4H, piperazine-H), 3.45 (m, 4H, piperazine-H), 2.47 (s, 1H, OH), 2.35 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6) δ 172.95, 169.62, 160.19, 155.13, 150.85, 148.32, 132.54, 126.87, 125.32, 124.01, 118.23, 117.15, 116.87, 116.42, 101.13, 72.42, 71.25, 48.78, 48.11, 44.81, 42.14. LC–MS: m/z 488 [M + H]+, HRMS: calcd for C23H22ClN3O7 [M + H]+, 488.1146; found, 488.1223.
(2R,3R)-4-(4-(4-bromophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxycoumarin-3-yl)-4-oxobutanamide (6c)
White solid; yield 34%; [α]20D = +22 (c = 1, CH3COOCH2CH3). mp 190–191 °C; IR (KBr disk): ν 3445 (OH), 3407, 3343 (NH), 2928, 2830 (CH), 1691, 1640 (C = O), 1606, 1540, 1492, 1455 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.22 (s, 1H, coumarin-OH), 9.29 (s, 1H, NH), 7.89 (d, J = 7.7 Hz, 1H, coumarin-5-H), 7.72 (t, J = 7.7 Hz, 1H, coumarin-7-H), 7.53 (d, 2H, coumarin-6,8-H), 7.35 (d, J = 7.9 Hz, 2H, benzene-3,5-H)), 6.72 (d, J = 8.1 Hz, 2H, benzene-2,6-H)), 4.62 (d, 2H, CH), 3.95–3.61 (m, 4H, piperazine-H), 3.21 (s, 4H, piperazine-H), 2.86 (s, 2H, OH). 13C NMR (75 MHz, DMSO-d6) δ 173.68, 169.59, 160.07, 153.82, 150.81, 150.27, 132.60, 131.95, 125.15, 124.08, 118.06, 116.60, 116.58, 110.87, 103.85, 72.52, 71.23, 48.66, 48.17, 44.85, 42.14. LC–MS: m/z 532 [M + H]+, HRMS: calcd for C23H22BrN3O7 [M + H]+, 532.0641; found, 532.0665.
(2R,3R)-4-(4-(4-methylphenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxycoumarin-3-yl)-4-oxobutanamide (6d)
Yellow solid; yield 19%; = +41 (c = 1.1, CH3OH). mp 195–197 °C; IR (KBr disk): ν 3446 (OH), 3405, 3344 (NH), 2959, 2931, 2829 (CH), 1690, 1639 (C = O), 1600, 1541, 1495, 1456 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.20 (s, 1H, coumarin-OH), 9.40 (s, 1H, NH), 7.91 (d, J = 7.8 Hz, 1H, coumarin-5-H), 7.68 (t, J = 7.8 Hz, 1H, coumarin-7-H), 7.44 (dd, J = 12.2, 7.9 Hz, 2H, coumarin-6,8-H), 7.06 (d, J = 8.2 Hz, 2H, benzene-3,5-H)), 6.89 (d, J = 8.3 Hz, 2H, benzene-2,6-H)), 4.82 (s, 1H, CH), 4.52 (s, 1H, CH), 4.03 (d, J = 7.1 Hz, 2H, OH), 3.85–3.51 (m, 4H, piperazine-H), 3.14 (s, 4H, piperazine-H), 2.21 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ 173.42, 169.81, 160.14, 155.82, 150.56, 148.35, 132.41, 128.15, 126.52, 125.42, 124.31, 118.07, 116.60, 112.87, 101.16, 72.78, 71.12, 48.63, 48.27, 44.92, 42.12. LC–MS: m/z 468 [M + H]+, HRMS: calcd for C24H25N3O7 [M + H]+, 468.1693; found, 468.1725.
(2R,3R)-4-(4-(4-methoxyphenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxycoumarin-3-yl)-4-oxobutanamide (6e)
Yellow solid; yield 15%; = +62 (c = 1, CH3OH). mp 222–224 °C; IR (KBr disk): ν 3445 (OH), 3404, 3340 (NH), 3000, 2930, 2829 (CH), 1690, 1640 (C = O), 1601, 1545, 1496, 1458 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.21 (s, 1H, coumarin-OH), 9.36 (s, 1H, NH), 7.98 (d, J = 7.5 Hz, 1H, coumarin-5-H), 7.54 (dd, J = 15.9, 8.1 Hz, 1H, coumarin-7-H), 7.35 (t, J = 7.8 Hz, 2H, coumarin-6,8-H), 6.93 (d, J = 9.0 Hz, 2H, benzene-3,5-H), 6.86 (d, J = 9.0 Hz, 2H, benzene-2,6-H), 4.81 (d, J = 4.3 Hz, 1H, CH), 4.12 (d, J = 4.4 Hz, 1H, CH), 3.97 (t, J = 14.9 Hz, 2H, piperazine-H), 3.78 (s, 3H, OCH3), 3.73 (s, 2H, piperazine-H), 3.16 (s, 2H, piperazine-H), 3.10–2.89 (m, 2H, piperazine-H), 2.63 (s, 2H, OH). 13C NMR (75 MHz, DMSO-d6) δ 173.45, 169.60, 160.02, 153.78, 150.52, 149.87, 148.27, 132.59, 125.41, 124.01, 117.96, 116.95, 116.42, 114.58, 99.89, 72.51, 71.32, 48.55, 48.16, 44.89, 42.09. LC–MS: m/z 484 [M + H]+, HRMS: calcd for C24H25N3O8 [M + H]+, 484.1642; found, 484.1692.
(2R,3R)-4-(4-(4-fluorophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxy-6-methylcoumarin-3-yl)-4-oxobutanamide (6f)
Yellow solid; yield 15%; = +55 (c = 1, CH3OH). mp 246–247 °C; IR (KBr disk): ν 3493 (OH), 3381, 3326 (NH), 2949, 2835 (CH), 1690, 1640 (C = O), 1588, 1541, 1495, 1450 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.21 (s, 1H, coumarin-OH), 9.36 (s, 1H, NH), 7.69 (s, 1H, coumarin-5-H), 7.45 (d, J = 8.4 Hz, 1H, coumarin-7-H), 7.36 (d, J = 8.5 Hz, 1H, coumarin-8-H), 7.02 (d, J = 8.8 Hz, 2H, benzene-3,5-H), 6.76 (d, J = 8.8 Hz, 2H, benzene-2,6-H), 4.72 (d, 2H, CH), 3.75 (d, 4H, piperazine-H), 3.56 (s, 2H, OH), 3.23 (s, 4H, piperazine-H), 2.42 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ 172.98, 169.59, 160.03, 158.82, 152.75, 150.12, 146.76, 135.45, 132.15, 128.11, 118.06, 117.60, 116.95, 115.97, 102.29, 72.58, 71.23, 48.75, 48.05, 44.91, 42.21, 22.21. LC–MS: m/z 486 [M + H]+, HRMS: calcd for C24H24FN3O7 [M + H]+, 486.1598; found, 486.1625.
(2R,3R)-4-(4-(4-chlorophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxy-6-methylcoumarin-3-yl)-4-oxobutanamide (6g)
Yellow solid; yield 11%; = +54 (c = 1.2, CH3OH). mp 195–197 °C; IR (KBr disk): ν 3494 (OH), 3382, 3330 (NH), 2947, 2831 (CH), 1691, 1639 (C = O), 1581, 1539, 1470, 1441 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.35 (s, 1H, coumarin-OH), 9.42 (s, 1H, NH), 7.62 (s, 1H, coumarin-5-H), 7.46 (d, J = 8.5 Hz, 1H, coumarin-7-H), 7.35 (d, J = 8.5 Hz, 1H, coumarin-8-H), 7.30 (d, J = 8.1 Hz, 2H, benzene-3,5-H), 6.65 (d, J = 8.1 Hz, 2H, benzene-2,6-H), 4.85 (s, 2H, CH), 3.82 (s, 4H, piperazine-H), 3.28 (s, 4H, piperazine-H), 2.91 (s, 2H, OH), 2.35 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ 173.52, 169.55, 160.09, 157.13, 151.35, 148.22, 135.87, 132.58, 129.87, 128.46, 127.13, 118.07, 117.25, 116.62, 101.25, 72.85, 71.21, 48.77, 48.12, 44.84, 42.14, 21.20. LC–MS: m/z 502 [M + H]+, HRMS: calcd for C24H24ClN3O7 [M + H]+, 502.1303; found, 502.1326.
(2R,3R)-4-(4-(4-bromophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxy-6-methylcoumarin-3-yl)-4-oxobutanamide (6h)
Light yellow solid; yield 33%; = +47 (c = 1, CH3OH). mp 199–200 °C; IR (KBr disk): ν 3495 (OH), 3378, 3329 (NH), 2946, 2830 (CH), 1690, 1638 (C = O), 1587, 1541, 1475, 1445 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.22 (s, 1H, coumarin-OH), 9.39 (s, 1H, NH), 7.70 (s, 1H, coumarin-5-H), 7.48 (d, J = 8.4 Hz, 1H, coumarin-7-H), 7.36 (t, J = 7.5 Hz, 3H, coumarin-8-H, benzene-3,5-H), 6.94 (d, J = 8.8 Hz, 2H, benzene-2,6-H), 4.82 (s, 1H, CH), 4.52 (s, 1H, CH), 3.72 (d, J = 17.0 Hz, 4H, piperazine-H), 3.60 (s, 2H, OH), 3.20 (s, 4H, piperazine-H), 2.41 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ 173.59, 169.61, 160.05, 153.85, 151.26, 150.19, 135.15, 132.60, 131.95, 127.12, 118.05, 116.90, 116.58, 115.21, 104.02, 72.56, 71.25, 48.69, 48.15, 44.79, 42.08, 21.23. LC–MS: m/z 546 [M + H]+, HRMS: calcd for C24H24BrN3O7 [M + H]+, 546.0798; found, 546.0822.
(2R,3R)-4-(4-(4-methylphenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxy-6-methylcoumarin-3-yl)-4-oxobutanamide (6i)
Yellow solid; yield 21%; = +43 (c = 1, CH3OH). mp 217–218 °C; IR (KBr disk): ν 3500 (OH), 3382, 3330 (NH), 2962, 2945, 2830 (CH), 1691, 1636 (C = O), 1581, 1540, 1474, 1441 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.19(s, 1H, coumarin-OH), 9.36 (s, 1H, NH), 7.53 (s, 1H, coumarin-5-H), 7.21 (s, 2H, benzene-3,5-H), 7.14 (s, 2H, benzene-2,6-H), 7.04 (d, J = 8.2 Hz, 2H, coumarin-7,8-H), 5.14 (s, 1H, CH), 4.68 (s, 1H, CH), 4.21–3.79 (m, 4H, piperazine-H), 3.47 (d, J = 22.0 Hz, 4H, piperazine-H), 3.25 (s, 2H, OH), 2.33 (s, 3H, CH3), 2.26 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ 173.62, 169.60, 160.04, 155.81, 150.06, 148.65, 135.42, 132.41, 128.15, 127.31, 126.52, 124.97, 118.07, 114.60, 102.25, 72.72, 71.13, 48.66, 48.20, 44.82, 42.12, 21.81, 21.25. LC–MS: m/z 482 [M + H]+, HRMS: calcd for C25H27N3O7 [M + H]+, 482.1849; found, 182.1867.
(2R,3R)-4-(4-(4-methoxyphenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxy-6-methylcoumarin-3-yl)-4-oxobutanamide (6j)
Yellow solid; yield 25%; = +65 (c = 1.1, CH3OH). mp 231–232 °C; IR (KBr disk): ν 3493 (OH), 3380, 3327 (NH), 3001, 2954, 2837 (CH), 1689, 1632 (C = O), 1579, 1536, 1467, 1440 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.25 (s, 1H, coumarin-OH), 9.37 (s, 1H, NH), 7.68 (s, 1H, coumarin-5-H), 7.39 (d, 2H, coumarin-7,8-H), 6.85 (d, J = 8.6 Hz, 2H, benzene-3,5-H), 6.58 (d, J = 8.6 Hz, 2H, benzene-2,6-H), 4.85 (d, J = 4.3 Hz, 1H, CH), 4.21 (d, J = 4.4 Hz, 1H, CH), 3.95 (t, 2H, piperazine-H), 3.76 (s, 3H, OCH3), 3.73 (s, 2H, piperazine-H), 3.15 (s, 2H, piperazine-H), 3.10 (s, 2H, piperazine-H), 2.84 (s, 2H, OH), 2.35(s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ 173.65, 169.62, 160.07, 155.78, 150.52, 149.87, 148.07, 135.41, 132.59, 126.91, 118.06, 117.35, 116.42, 114.58, 101.89, 72.55, 71.22, 55.81, 48.59, 48.18, 44.83, 42.19, 21.42. LC–MS: m/z 498 [M + H]+, HRMS: calcd for C25H27N3O8 [M + H]+, 498.1798; found, 498.1830.
(2R,3R)-4-(4-(4-fluorophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4,6-dihydroxycoumarin-3-yl)-4-oxobutanamide (6k)
Yellow solid; yield 30%; = +27 (c = 1, CH3COOCH2CH3). mp 198–200 °C; IR (KBr disk): ν 3445 (OH), 3398, 3327 (NH), 2932, 2826 (CH), 1695, 1640 (C = O), 1585, 1541, 1498, 1450 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.30 (s, 1H, coumarin-OH), 9.45 (s, 1H, NH), 7.85 (s, 1H, coumarin-5-H), 7.71 (d, J = 8.3 Hz, 1H, coumarin-7-H), 7.51 (d, J = 8.3 Hz, 1H, coumarin-8-H), 7.16 (d, J = 7.6 Hz, 2H, benzene-2,6-H), 7.02 (d, J = 7.7 Hz, 2H, benzene-3,5-H), 6.45 (s, 1H, coumarin-6-OH), 4.78 (s, 1H, CH), 4.49 (s, 1H, CH), 3.75 (s, 4H, piperazine-H), 3.24 (s, 4H, piperazine-H), 2.95 (s, 2H, OH). 13C NMR (75 MHz, DMSO-d6) δ 172.68, 169.60, 160.07, 158.85, 155.75, 154.45, 150.72, 148.71, 126.15, 118.06, 117.59, 116.95, 115.67, 112.11, 101.28, 72.52, 71.24, 48.76, 48.08, 44.91, 42.12. LC–MS: m/z 488 [M + H]+, HRMS: calcd for C23H22FN3O8 [M + H]+, 488.1391; found, 488.1426.
(2R,3R)-4-(4-(4-chlorophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4,6-dihydroxycoumarin-3-yl)-4-oxobutanamide (6l)
Light yellow solid; yield 35%; = +52 (c = 1, CH3OH). mp 170–172 °C; IR (KBr disk): ν 3446 (OH), 3340, 3328 (NH), 2932, 2826 (CH), 1691, 1639 (C = O), 1594, 1540, 1494, 1451 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.32 (s, 1H, coumarin-OH), 9.41 (s, 1H, NH), 7.82 (s, 1H, coumarin-5-H), 7.69 (d, J = 8.2 Hz, 1H, coumarin-7-H), 7.49 (d, J = 8.3 Hz, 1H, coumarin-8-H), 7.25 (d, J = 7.6 Hz, 2H, benzene-2,6-H), 6.98 (d, J = 7.7 Hz, 2H, benzene-3,5-H), 6.70 (s, 1H, coumarin-6-OH), 4.83 (s, 1H, CH), 4.54 (s, 1H, CH), 3.69 (d, J = 43.7 Hz, 4H, piperazine-H), 3.20 (s, 4H, piperazine-H), 2.87 (s, 2H, OH). 13C NMR (75 MHz, DMSO-d6) δ 173.58, 169.66, 160.09, 155.75, 153.82, 148.75, 147.55, 144.45, 128.59, 125.95, 118.11, 116.76, 115.55, 112.19, 101.01, 72.58, 71.14, 48.85, 48.15, 44.89, 42.14. LC–MS: m/z 504 [M + H] +, HRMS: calcd for C23H22ClN3O8 [M + H]+, 504.1095; found, 504.1123.
(2R,3R)-4-(4-(4-bromophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4,6-dihydroxycoumarin-3-yl)-4-oxobutanamide (6m)
Light yellow solid; yield 40%; = +67 (c = 1.2, CH3OH). mp 185–187 °C; IR (KBr disk): ν 3445 (OH), 3341, 3327 (NH), 2930, 2829 (CH), 1690, 1640 (C = O), 1597, 1546, 1498, 1451 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.36 (s, 1H, coumarin-OH), 9.39 (s, 1H, NH), 7.75 (d, J = 8.5 Hz, 1H, coumarin-8-H), 7.45 (d, J = 7.6 Hz, 2H, benzene-2,6-H), 7.19 (d, J = 8.4 Hz, 1H, coumarin-7-H), 7.05 (s, 1H, coumarin-5-H), 6.85 (d, J = 7.7 Hz, 2H, benzene-3,5-H), 6.36 (s, 1H, coumarin-6-OH), 4.76 (d, 2H, CH), 3.73 (d, J = 43.7 Hz, 4H, piperazine-H), 3.19 (s, 4H, piperazine-H), 3.05 (s, 2H, OH). 13C NMR (75 MHz, DMSO-d6) δ 173.68, 169.61, 160.07, 155.76, 153.89, 148.69, 146.46, 131.15, 125.89, 118.23, 116.76, 116.15, 115.49, 112.21, 101.32, 72.61, 71.11, 48.88, 48.20, 44.85, 42.12. LC–MS: m/z 548 [M + H]+, HRMS: calcd for C23H22BrN3O8 [M + H]+, 548.0590; found, 548.0612.
(2R,3R)-4-(4-(4-methylphenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4,6-dihydroxycoumarin-3-yl)-4-oxobutanamide (6n)
White solid; yield 33%; = +56 (c = 1, CH3OH). mp 189–190 °C; IR (KBr disk): ν 3445 (OH), 3398 (NH), 2926, 2825 (CH), 1695, 1640 (C = O), 1583, 1539, 1491, 1456 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.32 (s, 1H, coumarin-OH), 9.41 (s, 1H, NH), 7.85 (d, J = 8.1 Hz, 1H, coumarin-8-H), 7.39 (d, J = 7.9 Hz, 2H, benzene-2,6-H), 7.12 (d, J = 8.0 Hz, 1H, coumarin-7-H), 6.98 (s, 1H, coumarin-5-H), 6.81 (d, J = 7.6 Hz, 2H, benzene-3,5-H), 5.86 (s, 1H, coumarin-6-OH), 4.76 (s, 1H, CH), 4.52 (s, 1H, CH), 3.76 (s, 4H, piperazine-H), 3.25 (s, 4H, piperazine-H), 2.95 (s, 2H, OH), 2.36 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ 172.98, 169.59, 160.11, 155.88, 153.75, 148.58, 147.45, 129.36, 128.15, 125.63, 118.22, 115.51, 114.76, 112.53, 103.15, 72.69, 71.06, 48.79, 48.19, 44.83, 42.09, 21.45. LC–MS: m/z 484 [M + H]+, HRMS: calcd for C24H25N3O8 [M + H]+, 484.1642; found, 484.1651.
(2R,3R)-4-(4-(4-methoxyphenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4,6-dihydroxycoumarin-3-yl)-4-oxobutanamide (6o)
White solid; yield 26%; = +50 (c = 1, CH3OH). mp 201–202 °C; IR (KBr disk): ν 3445 (OH), 3399, 3329 (NH), 3001, 2928, 2827 (CH), 1698, 1640 (C = O), 1585, 1540, 1496, 1451 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.35 (s, 1H, coumarin-OH), 9.43 (s, 1H, NH), 7.79 (d, J = 8.6 Hz, 1H, coumarin-8-H), 7.14 (d, J = 8.7 Hz, 1H, coumarin-7-H), 6.95(t, 3H, J = 7.5 Hz benzene-2,6-H, coumarin-5-H), 6.81 (d, J = 7.4 Hz, 2H, benzene-3,5-H), 5.91 (s, 1H, coumarin-6-OH), 4.85 (s, 1H, CH), 4.58 (s, 1H, CH), 3.86 (s, 3H, OCH3), 3.81 (s, 4H, piperazine-H), 3.19 (s, 4H, piperazine-H), 2.86 (s, 2H, OH). 13C NMR (75 MHz, DMSO-d6) δ 173.36, 169.60, 160.06, 155.47, 153.26, 148.56, 146.25, 145.11, 125.58, 118.21, 116.25, 115.36, 114.78, 112.69, 103.26, 72.64, 71.03, 55.78, 48.74, 48.21, 44.90, 42.10. LC–MS: m/z 500 [M + H]+, HRMS: calcd for C24H25N3O9 [M + H]+, 500.1591; found, 500.1666.
(2R,3R)-4-(4-(4-fluorophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxy-6-chlorocoumarin-3-yl)-4-oxobutanamide (6p)
Light yellow solid; yield 41%; = +47 (c = 1.1, CH3OH). mp 266–267 °C; IR (KBr disk): ν 3452 (OH), 3369, 3339 (NH), 2941, 2830 (CH), 1664, 1632 (C = O), 1601, 1530, 1479, 1453 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.25 (s, 1H, coumarin-OH), 9.39 (s, 1H, NH), 7.34–7.29 (m, 3H, coumarin-7-H, benzene-2,6-H), 7.21 (s, 1H, coumarin-5-H), 7.09 (s, 1H, coumarin-8-H), 6.98 (d, J = 8.2 Hz, 2H, benzene-3,5-H), 4.80 (s, 1H, CH), 4.51 (s, 1H, CH), 3.67 (d, J = 44.5 Hz, 4H, piperazine-H), 3.19 (s, 4H, piperazine-H), 2.89 (s, 1H, OH), 2.73 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6) δ 172.69, 169.59, 160.03, 158.25, 155.75, 150.19, 147.32, 135.24, 129.43, 127.14, 122.47, 118.06, 117.53, 115.95, 101.89, 72.53, 71.21, 48.69, 48.11, 44.85, 42.25. LC–MS: m/z 506 [M + H]+, HRMS: calcd for C23H21ClFN3O7 [M + H]+, 506.1052; found, 506.1075.
(2R,3R)-4-(4-(4-chlorophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxy-6-chlorocoumarin-3-yl)-4-oxobutanamide (6q)
White solid; yield 29%; = +25 (c = 1, CH3COOCH2CH3). mp 189–192 °C; IR (KBr disk): ν 3451 (OH), 3370, 3340 (NH), 2940, 2828 (CH), 1671, 1630 (C = O), 1600, 1535, 1481, 1450 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.20 (s, 1H, coumarin-OH), 9.40 (s, 1H, NH), 7.26 (dd, J = 21.1, 12.3 Hz, 4H, coumarin-5,7-H, benzene-2,6-H), 7.07 (d, J = 7.5 Hz, 1H, coumarin-8-H), 6.98 (d, J = 8.2 Hz, 2H, benzene-3,5-H), 4.80 (s, 1H, CH), 4.51 (s, 1H, CH), 3.67 (d, J = 44.5 Hz, 4H, piperazine-H), 3.19 (s, 4H, piperazine-H), 2.89 (s, 1H, OH), 2.73 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6) δ 173.85, 169.55, 159.70, 152.70, 149.92, 149.42, 132.18, 129.24, 129.08, 123.21, 123.07, 118.79, 118.20, 117.62, 104.57, 72.52, 71.18, 48.79, 48.35, 44.87, 42.14. LC–MS: m/z 522 [M + H]+, HRMS: calcd for C23H21Cl2N3O7 [M + H]+, 522.0757; found, 522.0765.
(2R,3R)-4-(4-(4-bromophenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxy-6-chlorocoumarin-3-yl)-4-oxobutanamide (6r)
Yellow solid; yield 15%; = +64 (c = 1, CH3OH). mp 187–188 °C; IR (KBr disk): ν 3452 (OH), 3371, 3340 (NH), 2949, 2827 (CH), 1669, 1630 (C = O), 1600, 1539, 1481, 1451 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.19 (s, 1H, coumarin-OH), 9.42 (s, 1H, NH), 7.46 (s, 1H, coumarin-5-H), 7.35 (d, J = 7.9 Hz, 2H, benzene-2,6-H), 7.21–7.09 (m, 2H, coumarin-7,8-H), 6.89 (d, J = 7.9 Hz, 2H, benzene-3,5-H), 4.91 (s, 1H, CH), 4.49 (s, 1H, CH), 3.82 (d, J = 44.5 Hz, 4H, piperazine-H), 3.26 (s, 4H, piperazine-H), 3.02 (s, 2H, OH). 13C NMR (75 MHz, DMSO-d6) δ 173.79, 169.67, 159.91, 153.76, 149.96, 148.89, 132.26, 129.58, 129.24, 124.78, 120.20, 118.81, 118.12, 114.21, 103.98, 72.55, 71.21, 48.80, 48.41, 44.90, 42.15. LC-–MS: m/z 566 [M + H]+, HRMS: calcd for C23H21ClBrN3O7 [M + H]+, 566.0251; found, 566.0274.
(2R,3R)-4-(4-(4-methylphenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxy-6-chlorocoumarin-3-yl)-4-oxobutanamide (6s)
Yellow solid; yield 21%; = +53 (c = 1.1, CH3OH). mp 179–182 °C; IR (KBr disk): ν 3451 (OH), 3370, 3340 (NH), 2968, 2940, 2830 (CH), 1669, 1631 (C = O), 1601, 1536, 1480, 1453 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.35 (s, 1H, coumarin-OH), 9.46 (s, 1H, NH), 7.51–7.36 (m, 4H, coumarin-5,7-H, benzene-2,6-H), 7.11 (d, 1H, coumarin-8-H), 6.97 (d, J = 7.9 Hz, 2H, benzene-3,5-H), 4.79 (s, 1H, CH), 4.36 (s, 1H, CH), 3.78 (d, J = 44.5 Hz, 4H, piperazine-H), 3.23 (s, 4H, piperazine-H), 2.91 (s, 2H, OH), 2.36 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ 173.86, 169.55, 159.89, 152.96, 150.03, 149.39, 132.25, 129.36, 128.55, 126.21, 123.18, 120.03, 118.21, 115.12, 103.79, 72.58, 71.26, 48.79, 48.46, 44.92, 42.16, 21.36. LC–MS: m/z 502 [M + H]+, HRMS: calcd for C24H24ClN3O7 [M + H]+, 502.1303; found, 502.1321.
(2R,3R)-4-(4-(4-methoxyphenyl)piperazin-1-yl)-2,3-dihydroxy-N-(4-hydroxy-6-chlorocoumarin-3-yl)-4-oxobutanamide (6t)
Light yellow solid; yield 18%; = +66 (c = 1, CH3OH). mp 215–216 °C; IR (KBr disk): ν 3452 (OH), 3374, 3339 (NH), 3001, 2946, 2831 (CH), 1665, 1631 (C = O), 1600, 1539, 1480, 1450 (C = C). 1H NMR (300 MHz, DMSO-d6) δ 13.35 (s, 1H, coumarin-OH), 9.46 (s, 1H, NH), 7.48 (s, 1H, coumarin-5-H), 7.26 (d, J = 8.5 Hz, 1H, coumarin-7-H), 7.11 (d, J = 8.8 Hz,1H, coumarin-8-H), 6.98 (d, J = 7.5 Hz, 2H, benzene-2,6-H), 6.75 (d, J = 7.6 Hz, 2H, benzene-3,5-H), 4.85 (s, 1H, CH), 4.41 (s, 1H, CH), 3.86 (d, J = 44.5 Hz, 4H, piperazine-H), 3.76 (s, 3H, OCH3), 3.32 (s, 4H, piperazine-H), 2.99 (s, 2H, OH). 13C NMR (75 MHz, DMSO-d6) δ 173.78, 169.59, 159.99, 153.85, 151.21,150.09, 149.42, 132.19, 129.24, 123.18, 119.95, 118.46, 117.56, 114.49, 103.52, 72.61, 71.19, 55.36, 48.82, 48.45, 44.96, 42.11. LC–MS: m/z 518 [M + H]+, HRMS: calcd for C24H24ClN3O8 [M + H]+, 518.1252; found, 518.1269.
Biological activity assay
Inhibition of CHS assay
Yeast cells (Saccharomyces cerevisiae CGMCC2.145) were grown in 400 mL yeast extract peptone dextrose (YPD) medium to an OD 600 nm of 2–3 corresponding to about 2–3 g (wet weight) of cells and collected through centrifugation at 1500g for 15 min at 4 °C, then washed with water. The precipitates were suspended in 20 mL 50 mmol/L Tris-HCl solution at pH 7.0 which contains 40 μL fungal protease inhibitor cocktail and 50 μL solution of 200 mmol/L phenylmethanesulfonyl fluoride in DMSO, and were lysed by sonication treatment at 4 °C for 80 min. Insoluble materials were removed. One volume of supernatant was mixed with two volumes solution of 10% (w/w) sucrose dissolved in 100 mmol/L Tris-HCl buffer at pH 7.5, and centrifuged at 55 000g for 2 h at 4 °C. After centrifugation, the supernatant was discarded and the pellet was re-suspended in 50 mmol/L Tris-HCl at pH 7.5 and 33% glycerol to serve as CHS sample, and stored at −80 °C. The stock solutions of the candidate compounds were made by dissolving compounds with DMSO for no more than 10.0 g/L and diluting with sterile water for final concertration of DMSO below 3%. The solution of the candidate compounds was made by diluting the stock solution with 50 mM Tris-HCl buffer solution at pH 7.5Citation36,Citation37.
Two hundred microliters 30 μg/mL wheatgerm agglutinin (WGA) stock solutions in 50 mmol/L Tris-HCl at pH 7.5 were added to each well of the microplate and were incubated at room temperature for 16 h, then the solutions in the wells were removed and the plates were washed at least three times with distilled water. Three hundred microliters of 3 mg/mL bovine serum albumin in 50 mmol/L Tris-HCl buffer solution at pH 7.5 were added to each wells and incubated at 37 °C for 2 h. After removing the solution, the wells were washed by Tris-HCl solution for three times. Fifty microliters solution of 80 mmol/L GlcNAc plus 4 mmol/L UDP-GlcNAc, 50 μL solution of a candidate compound and 50 μL of CHS sample were added to a well and added 50 mM Tris-HCl buffer solution to a total volume of 200 μL. Microplates were incubated at 25 °C for 60 min. Then, the unbound components were removed and wells were washed with distilled water for three times.
To each well, 200 μL solution of 1 μg/ml wheatgerm agglutinin-Horse Reddish Peroxidase (WGA-HRP) in 50 mmol/L Tris-HCl at pH 7.5 was added. After being gently shaken for 6–15 min, the microplates were further kept at 37 °C for 15 min, and then washed five times with distilled water. Finally, 150 μL peroxidase substrate buffer solution (0.8 mmol/L TMB, 2 mmol/L H2O2, 50 mmol/L Na2HPO4–citric acid, pH3.7) was added and the mixtures reacted in lucifuge place for 30 min at 37 °C. The reaction was stopped with 50 μL 2 mol/L H2SO4 and measured with Biotek ELX 800 Microplate reader (Winooski, VT) at 450 nm. Standard chitin of 0.50 g/L was used to construct a response of absorbance at 450 nm with logarithmic quantities of chitin in wells. There was a linear response of absorbance at 450 nm to logarithmic quantities of chitin from 3.1 to 50 mg/L in a total volume of 200 μL solution in microplate wells. Parameters of such a linear response showed good consistency during independent repetitive assays. Chitin in each well was calculated accordingly to estimate half-inhibition concentration (IC50) of a test compound.
Antibacterial and antifungal assays
MIC (mg/mL) is defined as the lowest concentration of target compounds that completely inhibited the growth of microorganismCitation38. All the synthesized compounds 6a–t were tested for antimicrobial activity in vitro by the standard two-folds serial dilution method in 96-well microplates according to the National Committee for Clinical Laboratory Standards (NCCLS). DMSO was used as a solvent control to ensure that the solvent had no effect on microorganism growth. All the bacteria and fungi growth was monitored visually and spectrophotometrically, and the experiments were performed in triplicate.
Antifungal activity assay
Antifungal activity was screened against four main pathogenic fungal species (C. albicans CMCC 76615, Aspergillus fumigatus GIMCC 3.19, C. neoformans ATCC 32719 and Aspergillus flavus ATCC 16870) in clinic. Fluconazole and polyoxin B were used as standard antifungal drugs. DMSO was used as a solvent control. A spore suspension in sterile distilled water was prepared from 1-day old culture of the fungi growing on the media containing 1% peptone, 2% glucose and solid media as well as 15% agar. The final spore concentration was 1–5 × 103 spore mL−1. All target compounds were dissolved in DMSO to prepare the stock solutions. The tests were made resulting in 12 wanted concentrations (0.25–512 mg/mL). These dilutions were incubated at 37 °C for 24 h. The MIC values of antifungal activity in μg/mL are summarized in .
Table 1. The MIC values (μg/mL) of compounds 6a–t against fungi in vitro.
Antibacterial activity assays
Antibacterial activity was screened against three Gram positive (Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, Methicillin-resistant Staphylococcus aureus N 315) and three Gram negative (E. coli JM 109, Proteusbacillus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 9027) bacteria by using streptomycin and ofloxacin as a standard antibacterial drugs. The bacterial suspension was adjusted with culture medium to a concentration of 1 × 105 Colony Forming Unit (CFU). The culture medium consisted of 1% peptone, 0.3% beef extract, 0.5% sodium chloride in distilled water, the solid media as well as 15% agar. All compounds were dissolved in DMSO to prepare the stock solutions. The tests were carried out at a required concentration of 512, 256,128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μg/mL. These dilutions were incubated at 37 °C for 24 h. The MIC values of antibacterial activity in μg/mL are summarized in Table 2 (see the supplementary information).
Results and discussion
CHS inhibitory activity
All the synthesized compounds 6a–t showed the potency of inhibiting the CHS, but some compounds, such as compounds 6e, 6j, 6l, 6m, 6p, 6q, 6s and 6t were not further screened because their inhibition ratio were less than 15% at a concentration of 300 μg/mL. The inhibition ratios of other compounds are depicted in , and these derivatives with higher inhibition ratios were further screened for their IC50 values which were calculated and shown in . Among them, compounds 6b, 6c, 6d, 6h, 6i, 6n and 6r had IC50 values of 0.19, 0.17, 0.19, 0.21, 0.18, 0.20 and 0.16 mmol/L, respectively, which were almost equal to that of polyoxin B whose IC50 value was 0.18 mmol/L. Especially, compound 6o with IC50 of 0.10 mmol/L had the most potential CHS inhibitory activity in these compounds. Compounds 6a, 6f, 6g and 6k exerted slightly lower inhibitory activity.
Figure 4. The inhibition ratio of compounds at 300 μg/mL.
Figure 5. The IC50 values of the some compounds against CHS.
Antimicrobial activity
All target compounds 6a–t showed weak or no antibacterial efficacy against all the tested bacterial strains (the results are listed in Table 2 in the supplementary information). The strongest activity of these compounds against all tested bacteria is 6h with MIC of 32 μg/mL against E. coli, but it is 8-fold weaker than streptomycin and 64-fold weaker than ofloxacin both with MIC values of less than 4 μg/mL. Furthermore, none of the MIC values of other compounds against the six tested strains was less than 64 μg/mL. It turned out that these compounds 6a–t have no effect on the tested strains.
The results of antifungal activity in showed that target compounds 6a–t exhibited moderate even excellent efficacy against all the tested fungal strains. Compounds 6b, 6c, 6h, 6k, 6o, 6r and 6t against C. albicans had the MIC values of 32 μg/mL, which were equal to that of polyoxin B. Compounds 6b, 6c, 6e, 6i, 6k, 6o and 6r have good antifungal activity against A. flavus with the MIC values of 64 μg/mL, which were comparable with fluconazole and polyoxin B whose MIC values both were 64 μg/mL. Meanwhile, compounds 6d and 6n with MIC of 32 μg/mL exhibit better activity than fluconazole and polyoxin B. To Aspergillums fumigatus, compounds 6b, 6c, 6m, 6n and 6r were comparable with polyoxin B whose MIC value was 64 μg/mL. Especially, compound 6o with MIC values of 16 μg/mL exhibits better activity than fluconazole. Compound 6b has very high activity against C. neoformans with MIC value of 4 μg/mL which is twice more potent than fluconazole and four times more potent than polyoxin B.
From these assays data, we can see that these synthesized compounds have selective bioactivity against fungi, but they have no effects on bacteria. These results indicated that the design of these compounds as antifungal agents was rational. In general, the compounds which have good inhibitory activity against CHS showed good antifungal activity. To some extent, the antifungal activity of these compounds has positive correlation with their inhibitory activity against CHS.
In terms of the structural features of these compounds, it is seen that a suitable lipid–water partition coefficient could be beneficial to the bioactivity. For instance, compound 6o, in which the hydroxy group might increase the hydrophilicity of the molecule while the methoxy group could improve the lipophilicity of the compound, and the double effects resulted in a better lipid–water partition coefficient of this compound, exerted excellent CHS inhibitory activity. Meanwhile, the properties of substituted groups (R2) on coumarin ring greatly affected the inhibitory activity against CHS. The compounds with electron-donating group such as H, OH groups exhibit to be more active than these compounds with electron-withdrawing groups. For example, these compounds 6p, 6q, 6s and 6t bearing the Cl group in coumarin ring have little inhibitory activity against CHS with inhibition ratio less than 15% in 300 μg/mL. In contrast, the compounds which bear the electron-withdrawing groups, such as Cl, Br groups, in phenylpiperazine ring showed broad-spectrum antifungal activity. However, compounds 6d, 6i and 6n exhibited actively anti-fungal activity to all tested pathogenic fungi.
Conclusion
A series of novel 3-amino-4-hydroxycoumarin derivatives containing an N-phenylpiperazine moiety, a l-tartrate acid amide and 4-hydroxycoumarin scaffold in one molecule have been designed and synthesized in order to find new lead compounds that possess the excellent bioactivity against CHS and fungal activity. The enzymatic assay results showed that all these target compounds have CHS inhibitory activity. Among them, compounds 6a, 6b, 6c, 6d, 6i, 6n, 6o and 6r exhibited comparatively good activity against CHS; especially, 6o with IC50 value of 0.1 mmol/L is the strongest CHS inhibitor in these compounds. The antifungal assay showed that most of these compounds exhibit moderate even excellent activity against the tested strains which are the common pathogen in clinic. The microbiological results revealed that these compounds exhibited more significant antifungal activity than activity against bacteria. This indicated that it is possible to develop new selective CHS inhibitors from our series of compounds which may have potential for the treatment of fungal infections.
Supplementary material available online
Supplementary Table 2
Supplemental Material.pdf
Download PDF (160.7 KB)Declaration of interest
The authors report no conflicts of interest.
This work was supported by National Natural Science Foundation of China (no. 81071427), the Education Ministry of China (no. 20125503110007). The authors alone are responsible for the content and writing of this article.
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