l-Amino acid carbamate prodrugs of scutellarin - Springer Link

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Nov 12, 2014 - Feng-Jie Jiang • Yong-Long Zhao • Yong-Xi Dong • Min Luo •. Yong Huang ... dogs (Ge et al., 2003), metabolic instability in human plasma (e.g. ...
MEDICINAL CHEMISTRY RESEARCH

Med Chem Res (2015) 24:2238–2246 DOI 10.1007/s00044-014-1286-4

ORIGINAL RESEARCH

L-Amino

acid carbamate prodrugs of scutellarin: synthesis, physiochemical property, Caco-2 cell permeability, and in vitro anti-oxidative activity

Yu-Feng Cha • Shun Zhang • Hang Su • Yu Ou • Xiao-Zhong Fu Feng-Jie Jiang • Yong-Long Zhao • Yong-Xi Dong • Min Luo • Yong Huang • Yan-Yu Lan • Ai-Min Wang • Yong-Lin Wang



Received: 29 May 2014 / Accepted: 20 October 2014 / Published online: 12 November 2014 Ó Springer Science+Business Media New York 2014

Abstract A series of 40 -L-amino acid carbamate derivatives of scutellarin methyl ester were synthesized. Physiochemical properties evaluation results showed that designed target compounds have higher chemical and enzymatic stability, and aqueous solubility. The permeability (Papp AP to BL) of 4c, 4f, and 4g in Caco-2 cell was 8, 7, and 13 times higher than that of scutellarin, respectively, especially 4g had highest Papp AP to BL value (1.85 ± 0.29 9 10-6 cm/s) and lowest ER (Papp BL to AP/Papp AP to BL) value 0.56. In vitro anti-oxidative evaluation results revealed that 4g can protect against H2O2-induced PC12 cells oxidative damage by attenuating the MMP loss and decreasing H2O2-induced ROS production. Keywords Scutellarin  L-Amino acid carbamate  Caco-2 cell permeability  Anti-oxidative activity

Yu-Feng Cha and Shun Zhang have contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00044-014-1286-4) contains supplementary material, which is available to authorized users. Y.-F. Cha  S. Zhang  H. Su  X.-Z. Fu (&)  F.-J. Jiang  Y.-L. Zhao  Y.-X. Dong  M. Luo  Y.-Y. Lan  A.-M. Wang Engineering Research Center for the Development and Application of Ethnic Medicine and TCM (Ministry of Education), School of Pharmacy, Guiyang Medical College, Guiyang 550004, Guizhou, China e-mail: [email protected] Y. Ou Pharmacy Department, Guiyang Women and Children’s Hospital and Health Institute, Guiyang 550001, Guizhou, China Y. Huang  Y.-L. Wang Guizhou Provincial Key Laboratory of Pharmaceutics, Guiyang Medical College, Guiyang 550004, Guizhou, China

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Introduction The high rate of oxygen consumption per unit mass of tissue renders the brain especially vulnerably to the deleterious effects of oxidative stress, which can arise from the overproduction of reactive oxygen species (ROS). It is possible that oxidative stress is an important factor that may be involved in pathogenesis of neurodegenerative diseases such as Alzheimer’s and Parkinson’s (Huang et al., 2004; Leutner et al., 2005). So scavenging ROS has been recognized as an important therapeutic method in treating the diseases (Gackowski et al., 2008; Olanow, 1993). Scutellarin (40 ,5,6-trihydroxyflavone-7-glucuronide) is a primary active ingredient in breviscapine, which is extracted from the Chinese herb, Erigeron breviscapus (Zhang et al., 2000). It has been confirmed that scutellarin exhibits significant potential of attenuating H2O2-induced cytotoxicity and reducing intracellular accumulation of ROS, which may represent the cellular mechanisms for its neuroprotective action (Hong and Liu, 2000). Although scutellarin has been clinically used for a long time, some drawbacks limit its clinical application. Structurally, scutellarin contained glucuronic acid carboxyl and phenolic hydroxyl as hydrophilic groups, 2-phenyl-4Hchromen-4-one mother nucleus as hydrophobic moiety, as a result the compound had very poor ADME properties such as very low absolute bioavailability (0.4 %) in Beagle dogs (Ge et al., 2003), metabolic instability in human plasma (e.g., [30 % of scutellarin was degraded within 5 min in human plasma) (Chen et al., 2006), and low blood–brain barrier (BBB) permeability (Hu et al., 2005). These properties have been ascribed to its very low solubility in both water (0.056 mg/mL) and lipid (log p = -2.56) (Cao et al., 2006), intestinal instability and hepatic first-pass elimination (Wang et al., 2011).

Med Chem Res (2015) 24:2238–2246

Prodrug strategies such as PEG–scutellarin conjugates, ethyl, benzyl, and N,N-diethylglycolamide ester derivatives of scutellarin have been identified as effective methods to overcome the above disadvantages of scutellarin (Zhou et al., 2006; Cao et al., 2006; Ye et al., 2006). These studies revealed that glucuronic acid carboxyl and 40 hydroxyl moieties play important roles in the structural modification of scutellarin, because the above two moieties can be easily modified, which makes it possible to design and synthesize novel prodrugs with improved bioactivity and physiochemical properties using scutellarin as the lead compound (Lu et al., 2010; Zhou et al., 2006; Cao et al., 2006). An active transporting mechanism has been applied in designing flavonoids L-amino acid prodrugs such as quercetin–carbamate and tricin–carbamate conjugates (Kim et al., 2009; Ninomiya et al., 2011; Mulholland et al., 2001). These compounds were efficiently transported by the oligopeptide transporter 1 (PepT1), a membrane transport protein localized in the brush-border membranes of intestinal epithelial cells, as a result oral bioavailability of parent drugs could be significantly enhanced (RubioAliaga and Daniel, 2002). Moreover, studies revealed that in these compounds, L-amino acid carbamate structural fragments are crucial for enhanced water solubility and cell permeability. In addition, studies indicated that esterification of glucuronic acid carboxyl of scutellarin is a critical factor for improving its pharmacokinetic properties (Cao et al., 2006). Based upon above results, we designed and synthesized a series of carbamate prodrugs of scutellarin methyl ester via sub-structure combination methods, to preliminarily evaluate their physiochemical and biological properties. In this paper, eight novel carbamate prodrugs of scutellarin methyl ester (4a–4h) were obtained, and their physiochemical properties, Caco-2 cell permeability, and in vitro anti-oxidative activity were evaluated. It is expected that the results of the reported study might be able to afford some valuable information on new scutellarin prodrug design.

Experiment Chemistry Scutellarin (purity [95 %, HPLC) was provided by Feng shang jian Pharmaceutical Co. Ltd. (Yunnan, China). Scutellarin methyl ester was prepared according to the literature procedures (Lu et al., 2010). Other chemicals were 97–99 % pure and purchased from Sinopharm

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Chemical Reagent Co., Ltd. (SCRC). 1H NMR spectra (reference tetramethylsilane for dH, J values are given in Hz) were recorded on a Varian Mercury 400 spectrometer. Low-resolution mass spectra were obtained using ACQUITY TQD (triple quadrupole mass spectrometer) lowresolution mass spectrometer (Waters, America), and highresolution mass spectra were obtained using microTOFQ II ESI-Q-ToF LC/MS/MS (Bruker Daltonics). Flash chromatography was carried out on silica gel (200–300 mesh), and chromatographic solvent proportions are expressed on a volume:volume basis. Caco-2 cell permeability was evaluated using Agilent 1100 series HPLC system (Agilent Technologies, Palo Alto, CA, USA), reversed-phase chromatography used an analytical Agilent Eclipse XDB-C18 column (250 mm 9 4.6 mm i.d., 5 lm; Agilent, USA). All the anhydrous solvents were distilled over CaH2 or Na/benzophenone prior to use. Procedure for the preparation of scutellarin methyl ester 40 -L-amino acid carbamate conjugates (4a–h) is outlined in Scheme 1. A 250 mL, three-necked, round-bottomed flask, fitted with a nitrogen inlet adapter, was charged with 2.5 mmol of L-amino acid tert-butyl ester hydrochlorides, 50 mL of anhydrous CH2C12, and 0.8 mL (0.121 mol) of pyridine. The resulting solution was cooled at -10 °C for 30 min, then a solution of triphosgene [bis(trichloromethyl) carbonate] (BTC) (1.68 mmol in 3.0 mL of CH2C12) was added by syringe over 30 s, and the resulting light yellow solution was stirred at that temperature for 2 h. Then reaction mixture was washed twice with 50 mL of cold 0.1 M hydrochloric acid aqueous solution, ca. 30 mL of crushed ice and 20 mL of cold saturated NaCl aqueous solution, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to obtain the L-amino acid ester isocyanate (2a–h) quantitatively as light yellow oil. Under N2 atmosphere, scutellarin methyl ester (770 mg, 1.6 mmol) was dissolved in 15 mL anhydrous DMF, compounds (2a– h) solution (2.43 mmol in 10 mL anhydrous THF) and Et3N (34 lL, 0.24 mmol) was added to the mixture, then the reaction mixture stirred at 50 °C for 10 h. After that the solvent was evaporated in vacuo, and the residue was purified using routine flash column chromatography on silica gel (eluent:chloroform/methanol = 15:1–20:1 v/v) to obtained scutellarin methyl ester-40 -L-amino acid carbamate conjugates (3a–h). Compounds (3a–h) 0.22 mmol were dissolved in trifluoroacetic acid (TFA) (3 mL), mixture was stirred at 0 °C for 5–6 h, and the process was monitored by TLC assay, until reaction completed. Then TFA was evaporated in vacuo, and the residue was washed using chloroform and petroleum ether, centrifuge subside method to provide the target compounds 4a–h.

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O

O

a

H2N

N

O R

HO O O

HO O

C

.HCl

OH O OH O

O

2a-2h

1a-1h 1a: R = H 1b: R = -CH3 1c: R = -CH(CH3)2 1d: R = -CH2Ph 1e: R = -CH2COOBut 1f: R = -CH2CH2COOBut 1g: R = -CH2CH(CH3)2 1h: R = -CH(CH3)CH2CH3

OHO

b

R

O

OH HO O O

OH O OH O HO

O O

NH

O O

O R

OHO

3a-3h

2a: R = H 2b: R = -CH3 2c: R = -CH(CH3)2 2d: R = -CH2Ph 2e: R = -CH2COOBut 2f: R = -CH2CH2COOBut 2g: R = -CH2CH(CH3)2 2h: R = -CH(CH3)CH2CH3

3a: R = H 3b: R = -CH3 3c: R = -CH(CH3)2 3d: R = -CH2Ph 3e: R = -CH2COOBut 3f: R = -CH2CH2COOBut 3g: R = -CH2CH(CH3)2 3h: R = -CH(CH3)CH2CH3

OH

c

HO O O

O OH O

O O

O

NH O

HO

OH R1

OH O

4a-4h 4a: R1 = H 4b: R1 = -CH3 4c: R1 = -CH(CH3)2 4d: R1 = -CH2Ph 4e: R1 = -CH2COOH 4f: R1 = -CH2CH2COOH 4g: R1 = -CH2CH(CH3)2 4h: R1 = -CH(CH3)CH2CH3 Scheme 1 Reagents and conditions: (a) bis(trichloromethyl) carbonate (BTC), pyr, DCM, -10 °C, 2 h, 1 M HCl, yield 90–95 %; (b) anhydrous THF/DMF, Et3N, 50 °C, yield 40–55 %, 12 h; (c) TFA, 0 °C,5–6 h, total yields: 29.9–44.3 %

2-((4-(5,6-Dihydroxy-4-oxo-7-((2S,3S,4R,5R,6S)-3,4,5trihydroxy-6-(methoxy-carbonyl)tetrahydro-2H-pyran-2yloxy)-4H-chromen-2-yl)phenoxy)carbonylamino) acetic acid (4a) Compound 4a was light yellow powder, yield 22.57 %, (purity 99.1 %, HPLC). Rf = 0.13 (chloroform:methanol:acetic acid 7:1:1 (v/v/v)). 1H NMR(400 MHz, DMSOd6): d = 13.06 (brs, 1H, 5-OH), 10.46 (brs, 1H, 6-OH), 8.16 (t, 1H, J = 6.0 Hz, –NHCH2), 7.99 (d, 2H, J = 9.2 Hz, C20 60 -2H), 7.12 (s, 1H, C3-H), 6.97 (d, 2H, J = 8.8 Hz, C30 50 2H), 6.91 (s, 1H, C8-H), 5.37 (d, 1H, J = 7.6 Hz,C10 0 -H),

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4.23 (d, 1H, J = 9.6 Hz, C50 0 -H), 3.78 (d, 2H, J = 6.4 Hz, NHCH2), 3.67 (s, 3H, COOCH3), 3.41-3.29 (m, 3H, C20 0 30 00 4 -3H). 13C NMR (100 MHz, DMSO-d6) d = 182.3 (C, C-4), 171.4 (C, C-90 ), 168.9 (C, C-600 ), 164.2 (C, C-2), 161.2 (C, C-70 ), 155.8 (C, C-9), 150.6 (C, C-5), 148.9 (C, C-7), 147.1 (C, C-40 ), 130.8 (C, C-6), 128.5 (2 9 CH, C-20 , C-60 ), 121.3 (C, C-10 ), 116.0 (2 9 CH, C-30 , -50 ), 105.9 (CH, C-100 ), 102.5 (C, C-10), 97.5 (CH, C-3), 94.0 (CH, C-8), 75.2 (CH, C-200 ), 73.8 (CH, C-400 ), 73.3 (CH, C-500 ), 71.5 (CH, C-300 ), 52.1 (CH3, OCH3), 42.2 (CH, C-80 ). ESI–MS (m/z) [M?H]?: 578.63, [M-H]-:576.60; HRESIMS m/z (pos): 578.1141 C25H24NO15 (calcd. 578.1140).

Med Chem Res (2015) 24:2238–2246

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(R)-2-((4-(5,6-Dihydroxy-4-oxo-7-((2S,3S,4R,5R,6S)-3,4,5trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2yloxy)-4H-chromen-2yl)phenoxy)carbonylamino)propanoic acid (4b)

(R)-2-((4-(5,6-Dihydroxy-4-oxo-7-((2S,3S,4R,5R,6S)-3,4,5trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2yloxy)-4H-chromen-2-yl)phenoxy)carbonylamino)-3phenylpropanoic acid (4d)

Compound 4b was light yellow powder, yield 45.15 %, (purity 98.5 %, HPLC). Rf = 0.25 (chloroform:methanol:acetic acid 8:1:1 (v/v/v)). 1H NMR(400 MHz, DMSOd6): d = 13.06 (brs, 1H, 5-OH), 10.46 (brs, 1H, 6-OH), 8.23 (d, 1H, J = 7.6 Hz, –NHCH2), 7.99 (d, 2H, J = 8.8 Hz, C20 60 -2H), 7.13 (s, 1H, C3-H), 6.97 (d, 2H, J = 9.2 Hz, C30 50 -2H), 6.92 (s, 1H, C8-H), 5.37 (d, 1H, J = 7.2 Hz, C10 0 -H), 4.23 (d, 1H, J = 9.6 Hz, C50 0 -H), 3.67 (s, 3H, COOCH3), 3.53–3.34 (m, 3H, C200 30 0 400 -3H), 1.36 (d, 3H, J = 7.6 Hz, CHCH3). 13C NMR (100 MHz, DMSOd6) d = 182.2 (C, C-4), 174.3 (C, C-90 ), 169.2 (C, C-600 ), 164.6 (C, C-2), 161.5 (C, C-70 ), 155.4 (C, C-9), 153.5 (C, C-5), 153.2 (C, C-7), 152.6 (C, C-40 ), 128.7 (2 9 CH, C-20 , C-60 ), 123.5 (C, C-6), 121.0 (C, C-10 ), 116.0 (2 9 CH, C-30 , -50 ), 105.5 (CH, C-100 ), 102.9 (C, C-10), 99.5 (CH, C-3), 93.5 (CH, C-8), 75.4 (CH, C-200 ), 75.3 (CH, C-400 ), 72.7 (CH, C-500 ), 71.3 (CH, C-300 ), 52.0 (CH3, OCH3), 49.5 (CH, C-80 ), 17.2 (CH3, CHCH3). ESI–MS (m/z) [M?H]?: 592.63, [M-H]-: 590.57. HRESIMS m/z (pos): 592.1303 C26H26NO15 (calcd. 592.1297).

Compound 4d was gray-green-yellow powder, yield 41.35 %, (purity 99.2 %, HPLC). Rf = 0.55 (chloroform:methanol:acetic acid 10:1:1 (v/v/v)). 1H NMR (400 MHz, DMSO-d6): d = 13.01 (brs, 1H, 5-OH), 10.46 (brs, 1H, 6-OH), 8.24 (d, 1H, J = 8.4 Hz, –NHCH2), 7.98 (d, 2H, J = 8.8 Hz, C20 60 -2H), 7.32–7.24 (m, 5H, Ar–H), 7.12 (s, 1H, C3-H), 6.97 (d, 2H, J = 8.8 Hz, C30 50 -2H), 6.90 (s, 1H, C8-H), 5.37 (d, 1H, J = 7.2 Hz, C100 -H), 4.23 (d, 1H, J = 9.2 Hz, C50 0 -H), 3.67 (s, 3H, COOCH3), 3.36–3.26 (m, 3H, C20 0 300 40 0 -3H), 3.17-3.10 (m, 1H, NH2CHCH2[1H]Ar), 2.96–2.91 (m, 1H, NH2CHCH2[1H]Ar). 13C NMR (100 MHz, DMSO-d6) d = 182.2 (C, C-4), 173.1 (C, C-90 ), 169.1 (C, C-600 ), 164.6 (C, C-2), 161.4 (C, C-70 ), 155.3 (C, C-9), 153.5 (C, C-5), 153.3 (C, C-7), 152.6 (C, C-40 ), 137.6 (C, C-110 ), 129.2 (2 9 CH, C-130 ,C-150 ), 128.6 (2 9 CH, C-20 , C-60 ), 128.3 (2 9 CH, C-120 ,C-160 ), 126.5 (CH, C-140 ), 123.4 (C, C-6), 121.0 (C, C-10 ), 116.0 (2 9 CH, C-30 , -50 ), 105.5 (CH, C-100 ), 102.9 (C, C-10), 99.5 (CH, C-3), 93.4 (CH, C-8), 75.4 (CH, C-200 ), 75.3 (CH, C-400 ), 72.7 (CH, C-500 ), 71.2 (CH, C-300 ), 55.8 (CH, C-80 ), 52.0 (CH3, OCH3), 36.4 (CH2, C-100 ). ESI–MS (m/z) [M?H]?: 668.72, [MH]-: 666.57. HRESIMS m/z (pos): 668.1612 C32H30NO15 (calcd. 668.1610).

(R)-2-((4-(5,6-Dihydroxy-4-oxo-7-((2S,3S,4R,5R,6S)-3,4,5trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2yloxy)-4H-chromen-2-yl)phenoxy)carbonylamino)-3methylbutanoic acid (4c) Compound 4c was light yellow powder, yield 36.96 %, (purity 98.7 %, HPLC). Rf = 0.53 (chloroform:methanol:acetic acid 10:1:1 (v/v/v)). 1H NMR(400 MHz, DMSO-d6): d = 13.06 (brs, 1H, 5-OH), 10.46 (brs, 1H, 6-OH), 8.05 (d, 1H, J = 8.0 Hz, –NHCH2), 7.95 (d, 2H, J = 8.8 Hz, C20 60 -2H), 7.18 (s, 1H, C3-H), 6.97 (d, 2H, J = 8.8 Hz, C30 50 -2H),6.91 (s, 1H, C8-H), 5.29 (d, 1H, J = 7.6 Hz, C10 0 -H), 4.26 (d, 1H, J = 9.2 Hz, C50 0 -H), 3.93-3.90 (m, 1H, NHCH), 3.67 (s, 3H, COOCH3), 3.37–3.29 (m, 3H, C20 0 30 0 40 0 -3H), 2.15–2.10 (m, 1H, CH(CH3)2), 0.97–0.95 (m, 6H, 2 9 CH3). 13C NMR (100 MHz, DMSO-d6) d = 182.2 (C, C-4), 173.1 (C, C-90 ), 169.1 (C, C-600 ), 164.6 (C, C-2), 161.4 (C, C-70 ), 155.4 (C, C-9), 153.8 (C, C-5), 153.4 (C, C-7), 152.7 (C, C-40 ), 128.7 (2 9 CH, C-20 , C-60 ), 123.6 (C, C-6), 121.0 (C, C-10 ), 116.0 (2 9 CH, C-30 , -50 ), 105.5 (CH, C-100 ), 102.9 (C, C-10), 99.5 (CH, C-3), 93.5 (CH, C-8), 75.4 (CH, C-200 ), 75.3 (CH, C-400 ), 72.8 (CH, C-500 ), 71.2 (CH, C-300 ), 59.8 (CH, C-80 ), 52.0 (CH3, OCH3), 29.9 (CH, C-100 ), 18.0 (CH3, CHCH3),16.4 (CH3, CHCH3). ESI–MS (m/z) [M?H]?: 620.68, [M-H]-: 618.56. HRESIMS m/z (pos): 620.1607 C28H30NO15 (calcd. 620.1610).

(R)-2-((4-(5,6-Dihydroxy-4-oxo-7-((2S,3S,4R,5R,6S)-3,4,5trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2yloxy)-4H-chromen-2-yl)phenoxy)carbonylamino)succinic acid (4e) Compound 4e was gray-green-yellow powder, yield 29.52 %, (purity 98.9 %, HPLC). Rf = 0.53 (chloroform:acetic acid 10:1 (v/v)). 1H NMR (400 MHz, DMSOd6): d = 13.05 (brs, 1H, 5-OH), 10.45 (brs, 1H, 6-OH), 8.30 (d, 1H, J = 8.4 Hz, –NHCH2), 7.97 (d, 2H, J = 8.8 Hz, C20 60 -2H), 7.10 (s, 1H, C3-H), 6.96 (d, 2H, J = 8.8 Hz, C30 50 -2H), 6.90 (s, 1H, C8-H), 5.35 (d, 1H, J = 7.2 Hz, C100 -H), 4.36 (m, 1H,-NHCH), 4.22 (d, 1H, J = 9.6 Hz, C500 -H), 3.67 (s, 3H, COOCH3), 3.37–3.26 (m, 3H, C200 300 400 -3H), 2.79-2.74 (m, 1H, CH2[1H]COOH), 2.66–2.62 (m, 1H, CH2[1H]COOH). 13C NMR (100 MHz, DMSO-d6) d = 182.2 (C, C-4), 172.5 (C, C-110 ), 171.6 (C, C-90 ), 169.1 (C, C-600 ), 164.6 (C, C-2), 161.4 (C, C-70 ), 155.3 (C, C-9), 153.5 (C, C-5), 153.3 (C, C-7), 152.6 (C, C-40 ), 128.7 (2 9 CH, C-20 , C-60 ), 123.4 (C, C-6), 121.0 (C, C-10 ), 116.0 (2 9 CH, C-30 , -50 ), 105.5 (CH, C-100 ), 102.9 (C, C-10), 99.5 (CH, C-3), 93.5 (CH, C-8), 75.4 (CH, C-200 ), 75.3 (CH, C-400 ), 72.7 (CH, C-500 ), 71.2 (CH, C-300 ), 52.0 (CH3, OCH3), 50.7 (CH, C-80 ), 36.0 (CH2, C-100 ).

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ESI–MS (m/z) [M?H]?: 636.68, [M-H]-: 634.62. HRESIMS m/z (pos): 636.1196 C27H26NO17 (calcd. 636.1195). (R)-2-((4-(5,6-Dihydroxy-4-oxo-7-((2S,3S,4R,5R,6S)-3,4,5trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2yloxy)-4H-chromen-2yl)phenoxy)carbonylamino)pentanedioic acid (4f) Compound 4f was light yellow powder, yield 42.53 %, (purity 98.5 %, HPLC). Rf = 0.81 (chloroform:acetic acid 10:1 (v/v)). 1H NMR (400 MHz, DMSO-d6): d = 13.06 (brs, 1H, 5-OH), 10.46 (brs, 1H, 6-OH), 8.22 (d, 1H, J = 8.0 Hz, -NHCH2), 7.99 (d, 2H, J = 8.4 Hz, C20 60 -2H), 7.13 (s, 1H, C3-H), 6.97 (d, 2H, J = 8.8 Hz, C30 50 -2H), 6.90 (s, 1H, C8-H), 5.53 (d, 1H, J = 5.6 Hz, C100 -H), 4.23 (d, 1H, J = 9.6 Hz, C500 -H), 4.08-4.02 (m, 1H, –NHCH), 3.67 (s, 3H, COOCH3), 3.53-3.36 (m, 3H, C200 300 400 -3H), 2.422.36 (m, 2H, CH2COOH), 2.08–2.02 (m, 1H, CH2[1H]COOH), 1.89-1.82 (m, 1H, CH2[1H]COOH). 13C NMR (100 MHz, DMSO-d6) d = 182.2 (C, C-4), 173.8 (C, C-120 ), 173.4 (C, C-90 ), 169.1 (C, C-600 ), 164.6 (C, C-2), 161.4 (C, C-70 ), 155.3 (C, C-9), 153.5 (C, C-5), 153.4 (C, C-7), 152.6 (C, C-40 ), 128.7 (2 9 CH, C-20 , C-60 ), 123.5 (C, C-6), 121.0 (C, C-10 ), 116.0 (2 9 CH, C-30 , -50 ), 105.5 (CH, C-100 ), 102.9 (C, C-10), 99.5 (CH, C-3), 93.5 (CH, C-8), 75.4 (CH, C-200 ), 75.3 (CH, C-400 ), 72.7 (CH, C-500 ), 71.2 (CH, C-300 ), 53.3 (CH, C-80 ), 52.0 (CH3, OCH3), 30.0 (CH2, C-110 ), 26.3 (CH2, C-100 ). ESI–MS(m/z) [M?H]?:650.64, [M-H]-:648.65. HRESIMS m/z (pos): 650.1345 C28H28NO17 (calcd. 650.1352). (R)-2-((4-(5,6-Dihydroxy-4-oxo-7-((2S,3S,4R,5R,6S)-3,4,5trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2yloxy)-4H-chromen-2-yl)phenoxy)carbonylamino)-4methylpentanoic acid (4g) Compound 4g was gray-green-yellow powder, yield 43.72 %, (purity 99.1 %, HPLC). Rf = 0.50 (chloroform:methanol:acetic acid 7:1:1 (v/v/v)). 1H NMR (400 MHz, DMSO-d6): d = 13.05 (brs, 1H, 5-OH), 10.46 (brs, 1H, 6-OH), 8.16 (d, 1H, J = 8.0 Hz, –NHCH2), 7.99 (d, 2H, J = 9.2 Hz, C20 60 -2H), 7.13 (s, 1H, C3-H), 6.97 (d, 2H, J = 8.8 Hz, C30 50 -2H), 6.92 (s, 1H, C8-H), 5.38 (d, 2H, J = 7.2 Hz, C100 -H), 4.22 (d, 1H, J = 9.2 Hz, C500 -H), 4.06 (m, 1H, –NHCH), 3.66 (s, 3H, COOCH3), 3.53–3.36 (m, 3H, C200 300 400 -3H), 1.79–1.59 (m,1H, CHCH2[1H]), 1.57–1.51 (m, 1H, CHCH2[1H]), 0.95 (d, 3H, J = 6.8 Hz, CHCH3), 0. 91 (d, 3H, J = 6.4 Hz, CHCH3). 13C NMR (100 MHz, DMSO-d6) d = 182.4 (C, C-4), 173.1 (C, C-90 ), 169.0 (C, C-600 ), 164.3 (C, C-2), 161.3 (C, C-70 ), 155.7 (C, C-9), 150.6 (C, C-5), 149.0 (C, C-7), 147.1 (C, C-40 ), 130.8 (C, C-6), 128.5 (2 9 CH, C-20 , C-60 ), 121.3 (C, C-10 ), 116.0 (2 9 CH, C-30 , -50 ), 106.0 (CH, C-100 ), 102.6 (C, C-10),

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97.4 (CH, C-3), 94.2 (CH, C-8), 75.3 (CH, C-200 ), 73.9 (CH, C-400 ), 73.6 (CH, C-500 ), 71.5 (CH, C-300 ), 58.5 (CH, C-80 ), 52.1 (CH3, OCH3), 48.6 (CH2, C-100 ), 24.7 (CH, C-110 ), 15.5 (CH3, CHCH3), 11.4 (CH3, CHCH3). ESI–MS (m/z) [M?H]?:634.68, [M-H]-:632.75. HRESIMS m/z (pos): 634.1765 C29H32NO15(calcd. 634.1766). (2R,3S)-2-((4-(5,6-Dihydroxy-4-oxo-7-((2S,3S,4R,5R,6S)3,4,5-trihydroxy-6-(methoxycarbonyl)tetrahydro-2Hpyran-2-yloxy)-4H-chromen-2yl)phenoxy)carbonylamino)-3-methylpentanoic acid (4h) Compound 4h was light yellow powder, yield 36.06 %, (purity 98.2 %, HPLC). Rf = 0.52 (chloroform:methanol:acetic acid 7:1:1 (v/v/v)). 1H NMR (400 MHz, DMSOd6): d = 13.06 (brs, 1H, 5-OH), 10.44 (brs, 1H, 6-OH), 8.10 (d, 1H, J = 8.4 Hz, –NHCH2), 7.99 (d, 2H, J = 8.8 Hz, C20 60 -2H), 7.13 (s, 1H, C3-H), 6.97 (d, 2H, J = 8.8 Hz, C30 50 2H), 6.92 (s, 1H, C8-H), 5.38 (d, 1H, J = 7.2 Hz, C100 -H), 4.22 (d, 1H, J = 9.2 Hz, C500 -H), 3.97 (m, 1H, -NHCH), 3.66 (s, 3H, COOCH3), 3.42-3.27 (m, 3H, C200 300 400 -3H), 1.85 (m, 1H, CHCH3), 1.51–1.44 (m, 1H, CH2[1H]CH3), 1.31–1.23 (m, 1H, CH2[1H]CH3), 0.94 (d, 3H, J = 6.8 Hz, CHCH3), 0.88 (t, 3H, J = 7.6 Hz, CH2CH3). 13C NMR (100 MHz, DMSO-d6) d = 182.3 (C, C-4), 174.1 (C, C-90 ), 169.1 (C, C-600 ), 164.6 (C, C-2), 161.4 (C, C-70 ), 155.4 (C, C-9), 150.5 (C, C-5), 148.9 (C, C-7), 147.1 (C, C-40 ), 130.8 (C, C-6), 128.6 (2 9 CH, C-20 , C-60 ), 121.3 (C, C-10 ), 116.0 (2 9 CH, C-30 , -50 ), 105.9 (CH, C-100 ), 102.9 (C, C-10), 97.3 (CH, C-3), 94.0 (CH, C-8), 75.3 (CH, C-200 ), 73.8 (CH, C-400 ), 73.5 (CH, C-500 ), 71.5 (CH, C-300 ), 58.2 (CH, C-80 ), 52.5 (CH3, OCH3), 52.0 (CH, C-100 ), 24.2 (CH2, C-110 ), 23.0 (CH3, CHCH3), 21.2 (CH3, CH2CH3). ESI–MS (m/z) [M?H]?:634.68, [MH]-:632.81. HRESIMS m/z (pos): 634.1749 C29H32NO15 (calcd. 634.1766). Physiochemical property studies In vitro stability evaluation, 1 mg of target compounds (4a– h) was dissolved in 20 lL DMSO as stock solution, respectively, after that distilled water was added to above solutions to make final concentration for each compound was 0.2 mg/mL. After that 200 lL of diluted solution was added to 2 mL PBS (pH = 2.0 and 7.4) and plasma, which then were maintained at 37 ± 0.5 °C in screw-capped vials in a water bath. The samples were withdrawn at appropriate time intervals, and analyzed by UPLC–MS/MS protocols. In solubility test, 50 lL distilled water was added to 2–4 mg of target compounds (4a–h), the mixture was ultrasonic, then filtrated through microporous membrane filter (0.45 lm). After that the saturated solutions were diluted with distilled water to corresponding multiple, the solubility for each compound was calculated using standard curves.

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2243

Table 1 The physiochemical properties for scutellarin methyl ester carbamate derivatives Compounds

Stability in PBS (t1/2, h)

pH 2.0

Stability in plasma (t1/2, min)

pH 7.4

Table 2 Apparent permeability coefficients (Papp) of scutellarin methyl ester carbamate derivatives in the Caco-2 cells

Aqueous solubility

Compound

Papp A to B 9 10-6 (cm/s)a

(lg/mL)

Scutellarind

0.14 ± 0.24c

0.27 ± 0.15

1.93

4a

0

0



4b 4c

0.16 ± 0.25 1.39 ± 0.21

0.10 ± 0.34 2.14 ± 0.18

0.63 1.54

Scutellarin





3

15.24

4a

[72

1.0

5

65.78

4b

[72

2.5

5

365.80

4c

[72

5.0

5

22.54

4d

[72

9.0

5

4.73

4e

[72

4.0

30

426.51

4f

[72

4.0

30

123.10

4g

[72

4.2

20

127.26

4h

[72

3.8

25

32.87

Papp B to A 9 10-6 (cm/s)a

ER (Papp B to A/ Papp A to B)b

4e

0.31 ± 0.30

0.52 ± 0.25

1.68

4f

1.28 ± 0.27

1.57 ± 0.14

1.23

4g

1.85 ± 0.16

1.04 ± 0.28

0.56

4h

0.05 ± 0.04

0.10 ± 0.06

2.00

a

Papp Papp B

b c

A to B: to A:

transport of the compound from apical to basolateral; transport of the compound from basolateral to apical

ER (Papp B to A/Papp A to B): the ratio of Papp Data are mean ± SD (n = 3)

B to A

to Papp

A to B

d

UPLC–MS/MS analysis conditions for target compounds UPLC analyses were performed on a Waters ACQUITY UPLC instrument. The samples were separated on a BEH C l8 column (2.1 mm 9 50 mm, 1.7 lm). The mobile phase consisted of acetonitrile containing 0.1 % formic acid (A) and water containing 0.1 % formic acid (B). The elution gradient was as follows: 10 % A (0-2 min), 90 % A (2-3 min), and 10 % A (3 min). The mobile phase flow rate was 0.35 mL/min, and the column temperature was set at 45 °C. The injection volume was 1 lL. Mass spectrometry: All the mass experiments were carried out using a Waters ACQUITY TQD (triple quadrupole mass spectrometer) equipped with a Z-spray ESI source and connected to ACQUITY UPLC system. The acquisition parameters were collision gas, argon (Ar); nebulizing and drying gas, nitrogen (N2); source temperature, 120 °C; desolvation temperature, 350 °C; cone gas flow, 50 L/h; desolvation gas flow, 650 L/h; collision gas flow, 0.16 mL/min; capillary voltage, 3.0 kV; multi reaction monitor (MRM) mode was used; positive ions mode (ESI?), the confirmation ion pairs are (m/z) 578.1?287 (4a), 592.0?287 (4b), 620.1?287 (4c), 668.1?287 (4d), 636.1?287 (4e), 650.1?287 (4f), 634.1?287 (4g), 634.1?287 (4h), respectively; cone voltage and collision voltage were in the range of 30–40 V, respectively. The results are summarized in Table 1. Caco-2 cell permeability assay Caco-2 cell culture Caco-2 cells were obtained from Shanghai Institute of Material Medica (SIMM) and seeded onto MillicellTM

The concentration of test compounds was at 2.0 9 10-4 M for scutellarin; 1.5 9 10-4 M for compound 4b, 4c, 4e, 4f, 4g, and 4h. The incubation time was up to 120 min

Caco-2 plate at a density of 1.0 9 105 cells/cm2. Culture conditions were Caco-2 cells in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin and streptomycin, 3.7 g/L NaHCO3, and 1 % L- glutamine (Gln). Cells were grown at 37 °C in a humidified incubator with an atmosphere of 5 % CO2. Caco-2 cell-based permeability assay Caco-2 cells were seeded at a density of about 1 9 105 cells/cm2 on a 6 wells MillicellTM plate and left to grow for 21 days to reach confluence and differentiation. The integrity and transportation ability of the Caco-2 cell monolayer were examined by measuring the transepithelial electrical resistance (TEER) with an epithelial voltohmmeter (Millicell-ERS electrical resistance system (Millipore, Bedford, MA). Inserts with TEER values [300 X cm2 in culture medium were selected for transport experiments. On the 21st day, after washing the Caco-2 cell monolayer three times with prewarmed HBSS medium (pH = 7.4) and equilibrating in the same buffer. To determine the rate of drug transport in the apical to basolateral direction, 0.4 mL of target compounds (4a–h) with concentration of 1.5 9 10-4 M and scutellarin with concentration of 2.0 9 10-4 M was added to apical plate (AP), and the transport basolateral plate (BL) was filled with 0.6 mL of HBSS buffer. On the other hand, to determine transport rates from the basolateral to apical direction, 0.6 mL of target compounds (4a–h) with concentration of 1.5 9 10-4 M and scutellarin with concentration of 2.0 9 10-4 M was added to the BL, and the filter

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400

2.5

###

Papp× 10-6 cm/s

2.0

***

DCF fluorescence (% of Control)

*** ***

1.5

1.0

300

***

**

200

100

0.5

0

0.0 Scu

4b

4c

4e

4f

4g

4h

Control H2O2

VE

2.5

5 -6

Concentrations(10 M) Fig. 1 The apical-to-basolateral apparent permeability coefficients (Papp AP to BL) of scutellarin methyl ester 40 -L-amino acid carbamate conjugates (4b, 4c, 4e, 4f, 4g, and 4h) in Caco-2 cells (mean ± SD, n = 3). ***p \ 0.001, versus scutellarin

wells (apical compartment, AP) were filled with 0.4 mL of the HBSS buffer. To determine the target compounds 4a– h and scutellarin, 50 lL HBSS solution was taken from AP or BL side, 150 lL methanol was added to dilute the solution, then centrifuged at 15,000 r/min for 10 min. Aliquot of 10 lL of the supernatant solution was used for assay by HPLC method according to Huang et al. (2006). The results are summarized in Table 2 and Fig. 1. Statistical analysis All of the assays were carried out in at least three experiments with threefold sample. The results were expressed as the mean ± SEM, and the significance of the differences was analyzed by one-way analysis of variance (ANOVA) followed by Dunnetts’s multiple comparison test, with the scientific statistic software Origin, version 8.0.

Fig. 2 Effect of 4g on ROS production induced by H2O2 in PC12 cells. 4g was added to the culture medium 12 h prior to H2O2 addition. The ROS was quantified as described in ‘‘Measurement of intracellular ROS formation’’ section. Each vertical bar represents the mean ± SEM (n = 4). ###p \ 0.001 versus control; ***p \ 0.001 versus H2O2 group

Measurement of intracellular ROS formation The cells on 6-well plates were incubated with 10-5 M DCFH-DA (Sigma) in the DMEM in 5 % CO2/95 % air at 37 °C for 30 min. After the addition of 4g (2.5 and 5 lM) or 5 9 10-6 M vitamin E (purity 99 %), the cells were incubated at 37 °C for 12 h, followed by the addition of 800 lM H2O2. After 6 h of incubation, the cells were harvested and suspended in PBS. The fluorescence intensity was measured by a FACScan flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The result is illustrated in Fig. 2.

Anti-oxidative activity Measurement of mitochondrial membrane potential (MMP) PC12 cell culture and experimental protocols PC12 cells were purchased from Shanghai Institute of Biochemistry and Cell Biology (SIBCB). Cells were maintained in medium supplemented with 5 % heat-inactivated horse serum, 10 % FBS (Gibco), 100 U/mL penicillin and streptomycin, 3.7 g/L NaHCO3, and 1 % Lglutamine (Gln). Culture flasks were kept in humidified 5 % CO2/95 % air at 37 °C. The medium was changed every 3 days. PC12 cells were grown to 80–90 % confluence and then replanted at an appropriate density (according to the particular experiment) on culture plates. To induce oxidative stress, fresh H2O2 was prepared from a stock solution prior to each experiment.

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After the addition of 4g (2.5 and 5 9 10-6 M) or 5 9 10-6 M vitamin E (purity 99 %), the cells were incubated at 37 °C for 12 h, followed by the addition of 8 9 10-4 M H2O2. After 6 h of incubation, the medium was removed and about 1 9 106 cells were harvested by trypsinization. After the cells washed twice with PBS, they were incubated with Rh123 (10-5 M, Sigma) for 30 min at 37 °C in the dark. The cells were harvested and suspended in PBS. Then, the MMP was analyzed by a FACScan flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ) at an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The result is illustrated in Fig. 3.

Med Chem Res (2015) 24:2238–2246

2245

Rhodamine 123 fluorescence (% of Control)

120

***

100

***

80 60

###

40 20 0

Control H2O2

VE

2.5

5 -6

Concentrations(10 M) Fig. 3 Effect of 4g on loss of MMP induced by H2O2 in PC12 cells. 4g was added to the culture medium 12 h prior to H2O2 addition. The MMP was quantified as described in ‘‘Measurement of mitochondrial membrane potential (MMP)’’ section. Each vertical bar represents the mean ± SEM (n = 4). ###p \ 0.01 versus control; **p \ 0.01, ***P \ 0.001 versus H2O2 group

Statistical analysis All of the assays were carried out in at least three experiments with fourfold sample. The results were expressed as the mean ± SEM, and the significance of the differences was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test, with the scientific statistic software Origin, version 8.0.

Result and discussion In summary, L-amino acid carbamate conjugates of scutellarin methyl ester (4a–h) were obtained using facile synthetic procedures. Physiochemical properties evaluation results (Table 1) indicated that compounds with a free carboxyl group (4e–f) or with a larger steric hindrance alkyl group (4g–h) in their carbamate moiety are significantly more stable (t1/2 30 min) in plasma. These results would be attributed to the increasing steric hindrance of carbamate strand and the formation of inter molecular hydrogen bonds, which caused a decrease in the rate of hydrolysis. Except for compound 4d, aqueous solubility of all target compounds (22.54–426.51 lg/mL) was significantly higher than that of scutellarin (15.24 lg/ mL), especially the solubilities of compound 4b (365.80 lg/mL) and 4g (426.51 lg/mL), which were 24 and 28 times higher than that of scutellarin, respectively. Moreover, plasma metabolic studies revealed that compounds 4a–h were transferred into demethylation derivatives by esterase in the first place, which were further changed into scutellarin.

Caco-2 cell permeability assay (Table 2) showed that except for target compound 4h with ratio of Papp BL to AP/ Papp AP to BL(efflux rate, ER 2.00), which is higher than ER value (1.93) of scutellarin, the ratios of Papp BL to AP/Papp AP to BL (ER) of target compounds 4b–g were within the range of 0.56–1.68, which were less than ER value of scutellarin, suggesting that an efflux effect of scutellarin is higher than that of target compounds 4b–g. Among them, ER values of 4c, 4e, and 4f were higher than 1.0, while ER values of 4b and 4g were less than 1.0, indicating greater permeability in the BL to AP direction for compounds 4c, 4e, and 4f than that for compounds 4b and 4g. Moreover, compared with scutellarin, Caco-2 cell permeability of 4c, 4f, and 4g increased significantly, and their Papp AP to BL values were 8, 7, and 13 times higher than that of scutellarin, respectively, among them compound 4g had highest Papp AP to BL value (1.85 ± 0.16 9 10-6 cm/s) and lowest ER value 0.56 (Fig. 1). Comparison of Caco-2 cell permeability results of compounds 4g and 4h indicated that minor structural difference such as change in L-amino acid alkyl side chain can lead to great differences of Papp BL to AP/Papp AP to BL (ER value) and apical-to-basolateral apparent permeability coefficients (Papp AP to BL) between two compounds. The obtained results indicated that compound 4g have significantly higher Caco-2 cell permeability than that of scutellarin, which suggested that the human peptide transporter1 (hPepT1) might be at work in recognition as well as transport of the scutellarin L-amino acid carbamate conjugate 4g. Anti-oxidative activity assay in Fig. 2 showed that pretreatment with 2.5 and 5 9 10-6 M concentrations of 4g for 12 h reduce intensity of DCF-labeled cells when compared to H2O2-treated cultures; at a concentration of 5 9 10-6 M, 4g decreases DCF fluorescence to 191.02 ± 36.80 %, p \ 0.01 as compared with H2O2 group. The results in Fig. 3 suggested that pretreatment with 2.5 and 5 9 10-6 M concentrations of 4g for 12 h protect cells against the H2O2-induced lowering of MMP; at 5 9 10-6 M concentration of 4g, the MMP value was 86.08 ± 10.14 %, which was significantly different from that of H2O2 group (p \ 0.001). A significant decrease in MMP induced by ROS may release an apoptosis-inducing factor which activates caspase proteases, causes nuclear condensation, cytoplasmic fragmentation, and secondary generation of ROS (Zamzami et al., 1995; Petit et al., 1996). The present study revealed that H2O2 exposure induced oxidative stress characterized by potentially detrimental changes in intracellular ROS and MMP in PC12 cells. The result also showed that treatment with 4g may block these cellular events by attenuating intracellular accumulation of ROS and enhancing MMP, which may represent the cellular mechanisms for its neuroprotective action.

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2246 Acknowledgments This work was supported by the grants from National Natural Science Foundation of China (NSFC Nos. 81260473, 81460523), Projects of Guizhou Science and Technology Department (Nos. 2013-3031, 2012-3013, and 2013-4001), and Excellent Youth Scientific Talents Foundation of Guizhou Province (No. 2013-45).

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