Permeability of novel 4′-N-substituted (aminomethyl) benzoate-7-substituted nicotinic acid ester derivatives of scutellarein in Caco-2 cells and in an in vitro model of the blood-brain barrier

Article abstract of DOI:10.1007/s00044-016-1659-y

A series of 4′-N-substituted (aminomethyl) benzoate-7-substituted nicotinic acid ester derivatives of scutellarein was designed and synthesized. Evaluation of physiochemical properties showed that the newly designed compounds had greater chemical stability and aqueous solubility than scutellarin or scutellarein. The permeabilities (P_{app AP to BL}) of compounds 7b and 7e in Caco-2 cells were 5.9-fold and 3.7-fold higher than that of scutellarin, and 3.7-fold and 2.4-fold higher than that of scutellarein. The permeabilities (P_{app AP to BL}) of compounds 7b and 7e in an in vitro model of the blood–brain barrier were 9.7-fold and 5.9-fold higher than that of scutellarin, and 9.2-fold and 5.6-fold higher than that of scutellarein.

Full text of DOI:10.1007/s00044-016-1659-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1659-y
ORIGINAL RESEARCH
Permeability of novel 4′-N-substituted (aminomethyl) benzoate-7-
substituted nicotinic acid ester derivatives of scutellarein in
Caco-2 cells and in an in vitro model of the blood-brain barrier
Yu Ou1 Min Luo2 Yong-Xi Dong2 Hang Su2 Xiao-Zhong Fu2 Yu-Feng Cha2
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Shun Zhang2 Yong-Long Zhao2 Yong-Jun Li3 Yong-Lin Wang3
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Received: 24 July 2015 / Accepted: 4 July 2016
© Springer Science+Business Media New York 2016
Abstract A series of 4′-N-substituted (aminomethyl)
benzoate-7-substituted nicotinic acid ester derivatives of
scutellarein was designed and synthesized. Evaluation of
physiochemical properties showed that the newly designed
compounds had greater chemical stability and aqueous
solubility than scutellarin or scutellarein. The permeabilities
(Papp AP to BL) of compounds 7b and 7e in Caco-2 cells were
5.9-fold and 3.7-fold higher than that of scutellarin, and 3.7-
fold and 2.4-fold higher than that of scutellarein. The per-
meabilities (Papp AP to BL) of compounds 7b and 7e in an
in vitro model of the blood–brain barrier were 9.7-fold and
5.9-fold higher than that of scutellarin, and 9.2-fold and 5.6-
fold higher than that of scutellarein.
Introduction
Scutellarin (4′, 5, 6-trihydroxyflavone-7-glucuronide) is the
main active ingredient in the Chinese herb Erigeron bre-
viscapus (Vant.) Hand.-Mazz (Zhang et al., 2000), which
has been used in China since 1984 to treat acute cerebral
infarction as well as paralysis induced by cerebrovascular
diseases such as hypertension, cerebral thrombosis, and
cerebral hemorrhage (Pan et al., 2008; Zhou et al., 2006).
Scutellarin significantly attenuates the cytotoxicity of H2O2
and reduces the intracellular accumulation of reactive oxy-
gen species (ROS), cellular mechanisms that may account
for its neuroprotective effects (Hong and Liu, 2000).
Large amounts of the aglycone scutellarein have recently
been identified in urine and plasma samples following oral
administration of scutellarin to subjects in clinical trials. This
indicates that scutellarin is hydrolyzed in the colon to scu-
tellarein, which is then absorbed. Pharmacokinetic and phar-
macodynamic studies have shown that scutellarein has higher
relative bioavailability (301.8 %) and ROS-scavenging activ-
ity than scutellarin (Che et al., 2007; Chen et al., 2006),
suggesting that scutellarein might be the true bioactive com-
ponent of E. breviscapus (Zhang et al., 2003; Ju et al., 2005).
Although scutellarein has more potent antioxidant
activity and a higher absorption rate than scutellarin, its
clinical use is hindered by several factors. Structurally,
scutellarein contains four hydrophilic phenolic groups and
a hydrophobic 2-phenyl-4H-chromen-4-one core. These
structural features result in very poor absorption, distribu-
tion, metabolism, and excretion (ADME) properties,
including very low absolute bioavailability (7 %) in beagle
dogs (Ge et al., 2003) and low blood-brain barrier (BBB)
permeability (Hu et al., 2005). These shortcomings have
been attributed to its very low solubility in both water
(14 µg/mL) and lipid (log P = 0.96) (Dai et al., 2015) as
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Keywords Scutellarein Nicotinic acid Caco-2 cell
Permeability In vitro blood–brain barrier model
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Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1659-y) contains supplementary material,
which is available to authorized users.
Yu Ou and Min Luo contributed equally to this work
* Xiao-Zhong Fu
xiaozhong_fu@sohu.com
1
Pharmacy Department, Guiyang Women and Children’s Hospital
and Health Institute, Guiyang 550001 Guizhou, China
2
Engineering Research Center for the Development and
Application of Ethnic Medicine and TCM (Ministry of Education),
School of Pharmacy, Guizhou Medical University, Guiyang
550004 Guizhou, China
3
Guizhou Provincial Key Laboratory of Pharmaceutics, Guizhou
Medical University, Guiyang 550004 Guizhou, China

Med Chem Res
well as intestinal instability and rapid hepatic first-pass
elimination (Wang et al., 2011).
Pharmaceutical Co., Ltd. (Yunnan, China). Scutellarein (2),
6,7-ketal protected scutellarein (3), 4′-N-substituted (ami-
nomethyl) benzoic acids (4a–4d), and 4′-N-substituted
(aminomethyl) benzoate derivatives of scutellarein (5a–5d)
were synthesized as previously described (Fu et al., 2011;
Chen et al., 2005). Other chemicals were 97–99 % pure and
were purchased from Sinopharm Chemical Reagent Co.,
A number of prodrug strategies, including scutellarein
carbamates, scutellarein-O-alkylamines and scutellarein L-
amino acid esters, have been used successfully to improve
the ADME properties of scutellarein (Sang et al., 2015; Sang
et al., 2015; Fu et al., 2011). The phenolic hydroxyl groups
of scutellarein provide important ‘synthetic handles’ for the
structural modification of scutellarein and facilitate the
design and synthesis of novel prodrugs with the potential for
improved bioactivity and physiochemical properties.
A number of strategies have been proposed (Hansch
et al., 1987; Young et al., 1988; Bradbury, 1984) to improve
BBB penetration. These involve modification of the parent
structure to give prodrugs that are substrates for endogenous
BBB transporters, such as those for carboxylic acids, amino
acids, hexose derivatives, and small molecular weight
peptides (Zhang et al., 2011; Bonina et al., 2000;
Malakoutikhah et al., 2008).
Nicotinic acid esters, such as 10-O-nicotinate ginkgolide
B (Wu et al., 2012), 3-O-nicotinate penicillin V (Bartzatt,
2005), and nicotinic acid-mustard conjugate (Rajesh, 2014),
particularly, are transported across the BBB with high
efficiency. The efficient transport of nicotinic acid esters
across the BBB have been attributed to their ability to act as
substrates for the monocarboxylate transporter 1 (MCT1), a
proton-linked membrane transport protein that is expressed
on both luminal and abluminal membranes of brain capil-
lary endothelial cells (Enerson and Drewes, 2003). In a
previous study, we designed and synthesized a series of 4′-
N-substituted (aminomethyl) benzoate derivatives of scu-
tellarein that showed improved physiochemical properties
and enhanced in vitro antioxidant activity (Fu et al., 2011).
In our present search for scutellarein derivatives with high
BBB permeability, Caco-2 cell permeability and antioxidant
activity, we have adopted the substructure combination
principle to design compounds in which substituted nico-
tinic acid esters are attached to the 7-position of 4′-N-sub-
stituted (aminomethyl) benzoate derivatives of scutellarein.
We have prepared six novel 4′-N-substituted (amino-
methyl) benzoate-7-substituted nicotinic acid ester derivatives
of scutellarein (7a–7f) and evaluated their physiochemical
properties, Caco-2 cell permeability, and permeability in an
in vitro model of the BBB. Our results should afford valuable
information for the design of new scutellarin prodrugs.
1
Ltd. H-NMR spectra (reference tetramethylsilane for δH, J
values in Hz) were recorded using a Varian Mercury
400 spectrometer. Low-resolution mass spectra were
obtained using an ACQUITY TQD low-resolution mass
spectrometer (Waters, USA), and high-resolution mass
spectra were obtained using a microTOFQ II ESI-Q-ToF
LC/MS/MS instrument (Bruker Daltonics). Flash chroma-
tography was carried out on silica gel (200–300 mesh) and
chromatographic solvent proportions are expressed on a
volume:volume basis. Caco-2 cells were obtained from the
Shanghai Institute of Material Medica. Permeabilities in
Caco-2 cells and in the in vitro BBB model were evaluated
using an ACQUITY UPLC instrument (Waters, USA).
Samples were separated using a BEH C l8 column (2.1
mm × 50 mm, 1.7 μm), equipped with a BEH C18 Van-
Guard pre-column (2.1 mm × 5 mm, 1.7 μm). All anhydrous
solvents were distilled from CaH2 or Na/benzophenone
prior to use.
Preparation of 4′-N-substituted (aminomethyl) benzoate-7-
substituted nicotinic acid ester derivatives of scutellarein
(7a–7f)
The synthetic route is outlined in Scheme 1
A 100 mL, three-necked, round-bottomed flask, fitted
with
a
nitrogen inlet adapter, was charged with
6-chloronicotinic acid (500.0 mg, 3.18 mmol) and sub-
stituted amine (15.92 mmol). The resulting mixture was
stirred for 30 min until it became homogeneous and then
stirred at 120–130 °C for 6 h, when thin layer chromato-
graphy (TLC) (eluent:dichloromethane/methanol/acetic acid
(15:1:1, v/v/v)) indicated that the reaction was complete.
The resulting mixture was partially purified by flash column
chromatography on silica gel, using dichloromethane/
methanol/acetic acid (30:1:1, v/v/v) as the eluent. The
resulting crude product was dissolved in ethanol (10 mL)
and precipitated by the addition of acetic acid (0.5 mL).
This process was repeated three times and the suspension
was then centrifuged for 10 min at 4000 g. The sediment
was collected and washed with Et2O to obtain pure 6-
substituted nicotinic acid (6a–6c).
Experimental section
Materials and methods
A 50 mL, three-necked, round-bottomed flask, fitted with
a nitrogen inlet adapter, was charged with 4′-N-substituted
(aminomethyl) benzoate derivative of scutellarein (5a–d)
(0.21 mmol), 6-substituted nicotinic acid (6a–c) (0.27
mmol), 4-dimethylaminopyridine (0.21 mmol), CH2Cl2 (25
Scutellarin (purity > 95 % by high performance liquid
chromatography [HPLC]) was provided by Feng shang jian

Med Chem Res

Med Chem Res
Scheme 1 Reagents and conditions: a 8 % dilute sulfuric acid aqu-
eous, 90 °C, 20 h; b diphenyldichloromethane, DMAP, DEME, 170 °
C; c anhydrous EtOH, reflux 24 h; d DMAP, DCC, anhydrous THF, rt,
12 h; e CH3COCl/MeOH, EtOAc, −5–0 °C, 10 h; f N2 atmosphere,
120–130 °C, 12 h; g DMAP, DCC, CH2Cl2, rt, 20 h
6.99 (s, 1H, Ar–H8), 6.68 (s, 1H, Ar–H3), 6.52 (d, J = 8.0
Hz, 1H, pyr–H5), 3.54–3.45 (m, 6H, 3 × CH2N), 2.34–2.27
(m, 4H, 2 × CH2N), 1.50–1.47 (m, 6H, 3 × CH2), 1.37–1.35
(m, 4H, 2 × CH2). 13C NMR (100 MHz, DMSO-d6) δ =
182.6 (C, C-4), 164.6 (C, C-7′), 163.9 (C, C-2), 163.2 (C,
pyr–CO), 159.2(C, C-6), 158.2 (C, C-5), 154.6 (C, C-9),
153.9 (CH, pyr–C2), 153.0 (C, C-4′), 152.2 (C, C-7), 138.7
(C, C-4′′), 138.5 (CH, pyr–C4), 130.5 (2 × CH, C-2′′, C-6′
′), 129.9 (2 × CH, C-2′, C-6′), 128.9 (2 × CH, C-3′′, C-5′′),
128.6 (2 × CH, C-3′, C-5′), 127.7 (C, pyr–C3), 123.3 (C, C-
10), 123.1 (CH, pyr–C5), 111.7 (CH, C-3), 106.4 (CH, C-
8), 62.6 (CH2, CH2N), 54.3 (2 × CH2, 2 × CH2N), 47.3 (2 ×
CH2, 2 × CH2N), 26.0 (2 × CH2, 2 × CH2CH2N), 25.9 (2 ×
CH2, 2 × CH2CH2N), 24.2 (CH2, CH2CH2CH2). ESI-MS
(m/z): 662.3 [M+H]+. HRESIMS m/z (pos): 662.2478
C38H36N3O8 (calcd. 662.2497).
mL), and dimethyl sulphoxide (DMSO) (1 mL). The
resulting mixture was stirred for 30 min until it become
homogeneous and a solution of dicyclohexylcarbodiimide
(0.21 mmol) in CH2Cl2 (5.0 mL) was then added slowly to
the solution. After stirring at room temperature for 24 h, the
mixture was filtered and concentrated in vacuo. The residue
was purified by flash column chromatography on silica gel
using gradient elution with chloroform/methanol (100:1 to
30:1, v/v) to give the target compound. Products 7a–f were
obtained in this way as yellow solids in 36.4–45.1 % yield.
5,6-dihydroxy-4-oxo-2-(4-((4-(piperidin-1-ylmethyl)
benzoyl)oxy)phenyl)-4H-chromen-7-yl 6-(piperidin-1-yl)
nicotinate (7c)
5,6-dihydroxy-4-oxo-2-(4-((4-(pyrrolidin-1-ylmethyl)
benzoyl)oxy)phenyl)-4H-chromen-7-yl 6-(piperidin-1-yl)
nicotinate (7a)
Yellow solid, yield: 45.4 %, m.p. 264–266 °C.1H-NMR
(400 MHz, DMSO-d6): δ = 12.94(brs, 1H, 5-OH), 8.74 (s,
1H, pyr–H2), 8.17 (d, J = 8.0 Hz, 2H, Ar″–H2,6), 8.08 (d, J
= 8.0 Hz, 2H, Ar′–H2,6), 8.01 (dd, J1=8.0 Hz, J2 = 2.0 Hz,
1H, pyr–H4), 7.51–7.46 (m, 4H, Ar″-H3,5 and Ar′–H3,5),
7.00 (s, 1H, Ar–H8), 6.88 (d, J = 8.0 Hz, 1H, pyr–H5), 6.67
(s, 1H, Ar–H3), 3.68–3.65 (m, 6H, 3 × CH2N), 2.47–2.45
(m, 4H, 2 × CH2N), 1.61–1.59 (m, 4H, 2 × CH2), 1.51–1.45
(m, 6H, 3 × CH2). 13C NMR (100 MHz, DMSO-d6) δ =
182.6 (C, C-4), 164.6 (C, C-7′), 163.4 (C, C-2), 163.2 (C,
pyr–CO), 160.8 (C, C-6), 158.2 (C, C-5), 154.6 (C, C-9),
154.0 (CH, pyr–C2), 153.0 (C, C-4′), 151.9 (C, C-7), 139.0
(C, C-4′′), 138.7 (CH, pyr–C4), 130.5 (2 × CH, C-2′′, C-6′′),
129.8 (2 × CH, C-2′, C-6′), 129.0 (2 × CH, C-3′′, C-5′′),
128.6 (2 × CH, C-3′, C-5′), 127.8 (C, pyr–C3), 123.3 (C, C-
10), 123.1 (CH, pyr–C5), 112.0 (CH, C-3), 106.1 (CH, C-
8), 62.7 (CH2, CH2N), 54.4 (2 × CH2, 2 × CH2N), 45.8 (2 ×
CH2, 2 × CH2N), 25.9 (2 × CH2, 2 × CH2CH2N), 24.7 (2 ×
CH2, 2 × CH2CH2N), 24.3 (2 × CH2, 2 × CH2CH2CH2).
ESI-MS (m/z): 676.2 [M+H]+. HRESIMS m/z (pos):
676.2620 C39H38N3O8 (calcd. 676.2653).
Yellow solid, yield: 36.4 %, m.p. 242–244 °C.1H NMR
(400 MHz, DMSO-d6): δ = 12.95 (brs, 1H, 5-OH), 8.74(s,
1H, pyr–H2), 8.18(d, J = 8.0 Hz, 2H, Ar″–H2,6), 8.08 (d, J
= 8.0 Hz, 2H, Ar′–H2,6), 8.02 (dd, J1 = 8.0 Hz, J2 = 2.4 Hz,
1H, pyr–H4), 7.50–7.46 (m, 2H, Ar″–H3,5), 7.28–7.20 (m,
2H, Ar′–H3,5), 7.03 (s, 1H, Ar–H3), 6.69 (s, 1H, Ar–H8),
6.52(d, J = 8.0 Hz, 1H, pyr–H5), 3.55–3.44 (m, 6H, 3 ×
CH2N), 2.46–2.41(m, 4H, 2 × CH2N), 1.99–1.93 (m, 10H,
5 × CH2). 13C NMR (100 MHz, DMSO-d6) δ = 182.7 (C,
C-4), 164.6 (C, C-7′), 163.7 (C, C-2), 163.3 (C, pyr–CO),
159.1 (C, C-6), 157.8(C, C-5), 154.5 (C, C-9), 153.9 (CH,
pyr–C2), 153.0 (C, C-4′), 152.2 (C, C-7), 145.8(C, C-4′′),
138.4 (CH, pyr–C4), 130.4 (2 × CH, C-2′′, C-6′′), 129.7
(2 × CH, C-2′, C-6′), 129.4 (2 × CH, C-3′′, C-5′′), 128.7
(2 × CH, C-3′, C-5′), 127.5 (C, pyr–C3), 123.3 (C, C-10),
123.1 (CH, pyr–C5), 111.7 (CH, C-3), 106.5 (CH, C-8),
63.4 (CH2, CH2N), 53.0 (2 × CH2, 2 × CH2N), 47.2 (2 ×
CH2, 2 × CH2N), 25.7 (2 × CH2, 2 × CH2CH2N), 25.3 (2 ×
CH2, 2 × CH2CH2N), 24.1 (CH2, CH2CH2CH2). ESI-MS
(m/z): 662.3[M+H]+. HRESIMS m/z (pos): 662.2489
C38H36N3O8 (calcd. 662.2497).
2-(4-((4-((4-benzylpiperazin-1-yl)methyl)benzoyl)oxy)
phenyl)-5,6-dihydroxy-4-oxo-4H-chromen-7-yl 6-
(piperidin-1-yl)nicotinate (7d)
5,6-dihydroxy-4-oxo-2-(4-((4-(piperidin-1-ylmethyl)
benzoyl)oxy)phenyl)-4H-chromen-7-yl 6-(pyrrolidin-1-yl)
nicotinate (7b)
Yellow solid, yield: 62.2 %, m.p. 273–275 °C.1H-NMR
(400 MHz, DMSO-d6): δ = 12.94 (brs, 1H, 5-OH), 8.75 (s,
1H, pyr–H2), 8.18 (d, J = 8.8 Hz, 2H, Ar″–H2,6), 8.08 (d, J
= 8.0 Hz, 2H, Ar′–H2,6), 8.01 (dd, J1 = 9.2 Hz, J2 = 2.4 Hz,
1H, pyr–H4), 7.50–7.46 (m, 4H, Ar″–H3,5 and Ar′–H3,5),
7.29–7.18 (m, 5H, Ar–H), 7.02 (s, 1H, Ar–H8), 6.89 (d, 1H,
Yellow solid, yield: 45.1 %, m.p. 246–248 °C.1H-NMR
(400 MHz, DMSO-d6): δ = 12.93 (brs, 1H, 5-OH), 8.76 (s,
1H, pyr–H2), 8.17 (d, J = 8.4 Hz, 2H, Ar″–H2,6), 8.08 (d, J
= 8.0 Hz, 2H, Ar′-H2,6), 8.02 (dd, J1 = 8.8 Hz, J2 = 2.0 Hz,
1H, pyr–H4), 7.51–7.46 (m, 4H, Ar″–H3,5 and Ar′–H3,5),

Med Chem Res
J = 9.2 Hz, pyr–H5), 6.68 (s, 1H, Ar–H3), 3.68–3.66 (m,
4H, 2 × CH2N), 3.55 (s, 2H, CH2N), 3.44 (s, 2H, CH2N),
2.37 (m, 8H, 4 × CH2), 1.62–1.52 (2m, 6H, 3 × CH2). 13C
NMR (100 MHz, DMSO-d6) δ = 182.7 (C, C-4), 164.7 (C,
C-7′), 163.4 (C, C-2), 163.3 (C, pyr–CO), 160.8 (C, C-6),
157.9 (C, C-5), 154.6 (C, C-9), 154.0 (CH, pyr–C2), 153.1
(C, C-4′), 152.0 (C, C-7), 139.1 (C, C-4′′), 138.5 (CH,
pyr–C4), 130.5 (2 × CH, C-2′′, C-6′′), 129.7 (2 × CH, C-2′,
C-6′), 129.4 (2 × CH, Ph–C), 128.9 (2 × CH, C-3′′, C-5′′),
128.7 (2 × CH, C-3′, C-5′), 128.7 (2 × CH, Ph–C), 127.8 (C,
pyr–C3), 127.5 (CH, Ph–C), 123.3 (C, C-10), 123.0 (CH,
pyr–C5), 112.0 (CH, C-3), 106.1 (CH, C-8), 62.5 (CH2,
CH2N), 62.1 (CH2, PhCH2N), 53.2 (2 × CH2, 2 × CH2N),
53.0 (2×CH2, 2×CH2N), 45.9 (2×CH2, 2×CH2N), 25.7
(2×CH2, 2×CH2CH2N), 24.8 (CH2, CH2CH2CH2). ESI-MS
(m/z) 767.3 [M+H]+. HRESIMS m/z (pos): 767.3038
C45H43N4O8 (calcd. 767.3075).
pyr–H5), 6.78 (s, 1H, Ar–H3), 3.69–3.66 (m, 8H, 4 ×
CH2O), 3.33–3.25 (m, 6H, 3 × CH2N), 2.47–2.45 (m, 4H,
2 × CH2N). 13C NMR (100 MHz, DMSO-d6) δ = 182.7 (C,
C-4), 164.6 (C, C-7′), 163.4 (C, C-2), 163.1 (C, pyr–CO),
160.8 (C, C-6), 157.7 (C, C-5), 154.1 (C, C-9), 153.4 (CH,
pyr–C2), 152.6 (C, C-4′), 151.4 (C, C-7), 138.5 (C, C-4′′),
138.2 (CH, pyr–C4), 130.1 (2 × CH, C-2′′, C-6′′), 128.4
(2 × CH, C-2′, C-6′), 128.1 (2 × CH, C-3′′, C-5′′), 122.8
(2 × CH, C-3′, C-5′), 122.4 (C, pyr–C3), 111.4 (C, C-10),
105.6 (CH, pyr–C5), 104.9 (CH, C-3), 103.9 (CH, C-8),
72.5 (2 × CH2, 2 × CH2O), 72.3 (2 × CH2, 2 × CH2O), 63.1
(CH2, CH2N), 45.3 (4 × CH2, 4 × CH2N). ESI-MS (m/z):
680.5 [M+H]+. HRESIMS m/z (pos): 680.2243
C37H34N3O10 (calcd. 680.2239).
Evaluation of physiochemical properties of compounds
7a–f
5,6-dihydroxy-2-(4-((4-(morpholinomethyl)benzoyl)oxy)
phenyl)-4-oxo-4H-chromen-7-yl 6-(piperidin-1-yl)
nicotinate (7e)
In vitro stability testing was carried out as previously
described (Cao et al., 2006). Briefly, the test compound
(1 mg) was dissolved in DMSO (20 μL) to provide a stock
solution, which was then diluted with distilled water to a
concentration of 0.2 mg/mL. Aliquots (200 μL) of the
diluted solution were added to phosphate-buffered saline (2
mL, pH = 2.0 or 7.4), and 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
HPLC. For solubility testing, distilled water (50 μL) was
added to the test compound (3–5 mg). The mixture was
ultrasonicated and filtered through a microporous mem-
brane filter (0.45 μm). The saturated solution was then
serially diluted with distilled water and the solubility of the
compound was calculated using standard curves. HPLC
analyses were performed using a high performance liquid
chromatography instrument (LC-10AVP, Shimadzu,
Japan). Separation was carried out on an Agilent C18 col-
umn (4.6 mm × 150 mm, 5 µm), with gradient elution
(methanol/0.1 % H3PO4; 0–50 min, 40–60 %). The column
temperature was maintained at 40 °C and the flow rate was
1.0 mL/min. The results were summarized in Table 1.
Yellow solid, yield: 39.6 %, m.p. 247–249 °C. 1H-NMR
(400 MHz, DMSO-d6): δ 13.00 (brs, 1H, 5-OH), 8.94 (s,
1H, pyr–H2), 8.24 (d, J = 8.4 Hz, 2H, Ar″–H2,6), 8.15–8.07
(m, 3H, Ar′–H2,6, pyr–H4), 7.59–7.52 (m, 4H, Ar′-H3,5, Ar
″-H3,5), 7.08 (s, 1H, Ar–H8), 7.01–6.95 (m, 1H, pyr–H5),
6.75 (s, 1H, Ar–H3), 3.70–3.61 (m, 10H, 2 × CH2N, 2 ×
CH2O, ArCH2N), 2.51–2.49 (m, 4H, 2 × CH2N), 1.54–1.52
(m, 6H, 3 × CH2). 13C NMR (100 MHz, DMSO-d6) δ =
182.8 (C, C-4), 164.5 (C, C-7′), 163.9 (C, C-2), 163.2 (C,
pyr–CO), 160.6 (C, C-6), 158.1 (C, C-5), 154.8 (C, C-9),
154.1 (CH, pyr–C2), 153.4 (C, C-4′), 151.8 (C, C-7), 138.9
(C, C-4′′), 138.5 (CH, pyr–C4), 130.8 (2 × CH, C-2′′, C-6′′),
129.5 (2 × CH, C-2′, C-6′), 128.7 (2 × CH, C-3′, C-5′),
128.2 (2 × CH, C-3′′, C-5′′), 127.8 (C, pyr–C3), 123.4 (C,
C-10), 123.1 (CH, pyr–C5), 112.3 (CH, C-3), 103.9 (CH,
C-8), 72.7 (2 × CH2, 2 × CH2O), 63.1 (CH2, CH2N), 54.5
(2 × CH2, 2 × CH2N), 45.7 (2 × CH2, 2 × CH2N), 24.9 (2 ×
CH2, 2 × CH2CH2N), 24.2 (CH2, CH2CH2CH2). ESI-MS
(m/z): 678.2 [M+H]+. HRESIMS m/z (pos): 678.2420
C38H36N3O9 (calcd. 678.2446).
Caco-2 cell permeability assay
5,6-dihydroxy-2-(4-((4-(morpholinomethyl)benzoyl)oxy)
phenyl)-4-oxo-4H-chromen-7-yl 6-morpholinonicotinate
(7f)
Caco-2 cell culture
Caco-2 cells were seeded onto MillicellTM Caco-2 plates at
a density of 1.0 × 105 cells/cm2. The cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10 % fetal bovine serum and containing
penicillin (100 U/mL), streptomycin (100 U/mL), NaHCO3
(3.7 g/L) and L-glutamine (1 %). The cells were grown at
37 °C in a humidified incubator with an atmosphere of 5 %
CO2.
Yellow solid, yield: 43.1 %, m.p. 253–255 °C.1H-NMR
(400 MHz, DMSO-d6): δ = 12.94(s, 1H, 5-OH), 8.74 (s, 1H,
pyr–H2), 8.20 (d, J = 12.0Hz, 2H, Ar″–H2,6), 8.13 (d, J =
8.0Hz, 2H, Ar′–H2,6), 8.02 (dd, J1 = 9.2 Hz, J2 = 2.4 Hz,
1H, pyr–H4), 7.64 (m, 2H, Ar″–H3,5), 7.50 (d, J = 8.8Hz,
2H, Ar′–H3,5), 7.03 (s, 1H, Ar–H8), 6.90 (d, 1H, J = 9.6 Hz,

Med Chem Res
Table 1 The physiochemical properties for target compounds (7a–f)
and reference compounds
instrument. The samples were separated on a BEH C l8
column (2.1 mm × 50 mm, 1.7 μm), equipped with a Waters
BEH C18 VanGuard pre-column (2.1 mm × 5 mm, 1.7 μm).
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: 10–90 % A (0–3 min)
and 10 % A (3–4 min). The mobile phase flow rate was
0.35 mL/min, the column temperature was 45 °C and the
injection volume for the permeability assay was 5 μL. Mass
spectrometry identifications were carried out using a Waters
ACQUITY TQD (triple quadrupole mass spectrometer),
equipped with a Z-spray ESI source and connected to an
ACQUITY UPLC system. The acquisition parameters were
as follows: collision gas, argon; nebulizing, and drying gas,
nitrogen; source temperature, 120 °C; desolvation tem-
perature, 350 °C; cone gas flow rate, 50 L/h; desolvation gas
flow rate, 650 L/h; collision gas flow rate, 0.16 mL/min; and
capillary voltage, 3.0 kV. Selected ion monitoring mode
was used; in positive ion mode (ESI+), the confirmation ions
(m/z) were: 463.1 (scutellarin), 286.9 (scutellarein), 662.3
(7a), 662.3 (7b), 678.3 (7c), 767.0 (7d), 678.3 (7e), and
680.5 (7f). Cone voltages were 40, 45, 50, 40, 40, 45, 40,
and 45 V, respectively. All of the assays were carried out in
triplicate in at least three separate experiments. Results are
expressed as mean SEM and statistical significance was
analyzed by one-way analysis of variance (ANOVA) fol-
lowed by Dunnett’s multiple comparison test using Origin
scientific statistics software, version 8.0. The results are
summarized in Table 2 and Fig.1.
a
Compounds
Solubility
Stability in PBS (t1/2 h)
(µg/mL)
pH = 2.0
pH = 7.4
Scutellarin
65.9
—
—
Scutellarein
14.4
—
5.5
7a
7b
7c
7d
7e
7f
a
183.64
176.45
60.22
>72
>72
>72
>72
>72
>72
8.3
7.4
4.2
8.7
5.2
5.4
324.43
291.89
224.16
The solubility for target compounds were tested in pH = 2.0
phosphate buffer
Caco-2 cell-based permeability assay of compounds 7a–f
Caco-2 cells were seeded at a density of 1 × 105 cells/cm2
on a six-well MillicellTM plate and allowed to grow for
21 days to reach confluence and differentiate. The integrity
and transportation ability of the Caco-2 cell monolayer were
examined by measuring the transepithelial electrical resis-
tance (TEER) using an epithelial voltohmmeter (Millicell-
ERS electrical resistance system, Millipore, Bedford, MA,
USA). Inserts with TEER values > 300 Ω cm2 in the culture
medium were selected for transport experiments. On day 21,
the Caco-2 cell monolayer was washed three times with pre-
warmed (37 °C) serum-free DMEM and equilibrated using
the same medium. To determine the rate of drug transport in
the apical to basolateral direction, solutions of the test
compound (200 µM, 0.2 mL), scutellarin (200 µM, 0.2 mL)
and scutellarein (200 µM, 0.2 mL) were added to the apical
plate (AP). The transport basolateral plate (BL) was filled
with pre-warmed (37 °C) serum-free DMEM (1.2 mL). To
determine the transport rate in the basolateral to apical
direction, solutions of the test compound (200 µM, 1.2 mL),
scutellarin (200 µM, 1.2 mL) and scutellarein (200 µM, 1.2
mL) were added to the BL and the filter wells (apical plate,
AP) were filled with pre-warmed (37 °C) serum-free
DMEM (0.2 mL). For analysis of the test compound, scu-
tellarin and scutellarein, aliquots (200 μL) of the DMEM
solutions were taken from the AP or BL side, methanol (50
μL) was added and the mixtures were centrifuged at 13000
g for 10 min. Aliquots (10 μL) of the supernatant solutions
were analyzed by UPLC-MS/MS, using established meth-
ods, as described below.
In vitro BBB permeability assay
Primary culture of rat brain microvessel endothelial cells
(BMECs) and astrocytes
Brains were removed from five 2-weeks old Sprague-
Dawley rats, the hemispheres were washed, the cerebella
removed, and the meninges peeled off. The white matter
was removed and the gray matter was collected and stored
in cold DMEM before use. The gray matter was washed
three times with D-Hank solution, digested successively for
40 min with 0.1 % type II collagenase containing 0.005 %
DNase I and 0.1 % collagenase/dispase, and then cen-
trifuged at 800 g for 5 min at 4 °C. The pellets were resus-
pended in DMEM and filtered through nylon mesh (150
µm). The filtrate was collected, centrifuged at 800 g for 5
min at 4 °C and 20 % mass fraction of bovine serum albu-
min was then added to suspend the sediment. The resulting
suspension was centrifuged at 1000 g for 20 min at 4 °C.
The sediment was washed with DMEM and centrifuged at
800 g for 5 min at 4 °C. The resulting sediment was resus-
pended in 44 % mass fraction of Percoll and centrifuged at
1000 g for 10 min at 4 °C to provide high purity brain
UPLC-MS/MS conditions for analysis of test compounds
Ultra performance liquid chromatography (UPLC) analyses
were performed using
a Waters ACQUITY UPLC

Med Chem Res
Table 2 Apparent permeability coefficients (Papp) of target
compounds (7a–f) and reference compounds in Caco-2 cells
Establishment of an in vitro BBB model comprising a co-
culture of BMECs, astrocytes and brain microvascular
perithelial cells (BMPCs)
compounds
Papp AP to BL
×
Papp BL to AP
×
ER(Papp BL
to AP/Papp AP
10−6(cm/s)a
10−6(cm/s)a
b
)
to BL
The in vitro BBB model using primary BMECs, astrocytes
and BMPCs was established on rat tail collagen/fibronectin
(7.5 μg/mL)-coated 24-well polycarbonate Transwell filter
inserts (12 mm membrane diameter, 1.12 cm2 growth sur-
face area, 0.4 μm pore size) (Corning Costar Quality Bio-
logical, Gaithersburg, MD, USA). The astrocytes and
BMPCs were seeded onto one side of the filter at a density
of 1.5 × 104 cells per insert and allowed to adhere to the side
of the filter for 6 h. After 3 days the rat BMECs were seeded
onto the other side of the filter at a density of 2.0 × 104 cells
per filter and the Transwell plate was cultured in EGM-2
medium at 37 °C under an atmosphere containing 5 % CO2.
In vitro experiments using the BBB cell model were con-
ducted within 3–4 days of seeding the rat BMECs. TEER
was measured using a Millicell-ERS electrical resistance
system. Only cell layers with TEER values > 300 Ω/cm2
were used for the transport assays. Permeability of sodium
fluorescein, expression of alkaline phosphatase and γ-glu-
tamyl transpeptidase, and scanning electron microscopy
were used to confirm the integrity of the in vitro BBB
model.
Scutellarin(I)d 0.53 0.11c
Scutellarein(II) 0.83 0.21
1.05 0.21
1.46 0.24
2.30 0.47
2.94 0.38
2.91 0.67
1.26 0.22
1.91 0.35
1.69 0.44
1.98
1.76
1.62
0.94
1.51
1.80
0.97
1.01
7a
7b
7c
7d
7e
7f
1.42 0.33
3.11 0.87
1.93 0.54
0.70 0.13
1.97 0.38
1.67 0.42
a Papp A to B: transport of the compound from apical to basolateral; Papp
B to A: transport of the compound from basolateral to apical. b ER (Papp
c
B to A/Papp A to B): the ratio of Papp B to A to Papp A to B
.
Data were
mean SD (n = 3). d The concentration of test compounds was at 200
µM for scutellarin, scutellarein and compounds 7a–f. The incubation
time was up to 120 min
###
4
***
3
###
###
***
###
***
In vitro BBB permeability assay
***
###
2
***
Transport experiments were performed by simultaneously
adding final concentrations (25, 100, 400 μM) of test com-
pounds (7a–c, 7e and 7f), dissolved in serum-free DMEM,
to the apical side (AP, 0.2 mL) and serum-free DMEM to the
basolateral side (BL, 1.2 mL). For determination of time-
dependent transport of test compounds, aliquots (60 µL)
were taken from the BL medium after 20, 40, 60, 80, 100,
120, 140, 160, and 180 min and immediately replaced by an
equivalent volume of serum-free DMEM. Aliquots (60 µL)
were collected from the BL side after 90 min to determine
*
1
0
I
II 7a 7b 7c 7d 7e 7f
Fig. 1 The apical to basolateral apparent permeability coefficients
(Papp of 4′-N-substituted (aminomethyl) benzoate-7-
substituted nicotinic acid ester derivatives (7a–f), scutellarin and
scutellarein in Caco-2 cells (mean SD, n = 3). ***P < 0.001 as
compared with scutellarin(I) group. ###P < 0.001 as compared with
scutellarein (II) groups
)
AP to BL
the Papp AP
values of the test compounds. Results
to BL
obtained using UPLC-MS/MS detection were consistent
with those obtained in the Caco-2 cell-based permeability
assay. All of the assays were carried out in triplicate in at
least three separate experiments. Results are expressed as
mean SEM and statistical significance was analyzed by
ANOVA followed by Dunnetts’s multiple comparison test
using Origin scientific statistics software, version 8.0. The
results are summarized in Table 3, Fig. 2a and 2b.
microvascular section. The brain microvascular section was
then seeded in a culture bottle for primary culture. The
resulting rat BMECs were resuspended in EGM-2 endo-
thelium cell growth medium containing 5 % fetal bovine
serum, seeded into a rat tail collagen-coated flask containing
puromycin (4 mg/mL) and grown to confluence. Primary
cultures of rat BMECs were identified using an inverted
microscope and by rabbit anti-factor VIII fluorescence
immunocytochemistry. Astrocyte primary cultures were
prepared as previously described (Biegel et al., 1995) and
identified using rabbit anti-glial fibrillary acidic protein
(GFAP) in fluorescence immunocytochemistry.
Results and discussion
4′-N-substituted (aminomethyl) benzoate-7-substituted
nicotinic acid ester derivatives of scutellarein (7a–f) were

Med Chem Res
Table 3 Apparent permeability coefficients and efflux rate of target
compounds (7a–f) and reference compounds in BBB cell layer
a
2.5
2.0
1.5
1.0
0.5
0.0
###
Compound
Papp AP to BL
10−6(cm·s−1 a
×
Papp BL to AP
10−6(cm·s−1 a
×
ER(Papp BL
***
)
)
to AP/Papp
b
)
AP to BL
Scutellarin(I)d 0.17 0.08c
Scutellarein(II) 0.18 0.06
0.37 0.13
0.34 0.09
1.12 0.20
1.06 0.15
0.50 0.18
0.96 0.11
1.35 0.27
2.18
1.89
1.89
0.64
0.90
0.95
1.90
###
***
###
###
7a
7b
7c
7e
7f
0.59 0.11
1.65 0.37
0.56 0.12
1.01 0.13
0.71 0.16
###
***
***
***
I
II
7a 7b 7c 7e 7f
a Papp A to B: transport of the compound from apical to basolateral; Papp
b
B to A: transport of the compound from basolateral to apical. b ER (Papp
SC
c
SC-GA
7a
B to A/Papp A to B): the ratio of Papp B to A to Papp A to B
.
Data were
800
600
400
200
0
mean SD (n = 3). d The concentration of test compounds was at 200
µM for scutellarin, scutellarein and compounds 7a–f. The incubation
time was up to 120 min
7b
7c
7e
7f
obtained using straightforward synthetic methods. Evalua-
tion of physiochemical properties (Table 1) showed that
compounds 7a–f are more stable (t1/2 > 72 h) under acidic
conditions (pH = 2) than under neutral conditions (pH =
7.4, t1/2 4.2–8.7 h). Except for compound 7c, the solubilities
of the test compounds (7a–f) (176.45–324.43 μg/mL) were
significantly higher than those of scutellarein (14.4 μg/mL)
or scutellarin (65.9 μg/mL). The solubilities of compounds
7d, 7e, and 7f (324.43 μg/mL, 291.89 μg/mL and 224.16
μg/mL, respectively) are 22.5-fold, 20.2-fold, and 15.6-fold
higher than that of scutellarein.
0
20 40 60 80 100 120 140 160 180
Time(min)
Fig. 2 a Apparent permeability coefficients of compounds from apical
to basolatearl in BBB cell layer. The data were represented as mean
SE (n = 3). ***P < 0.001 vs. scutellarin(I); ###P < 0.001 vs. scutellarein
(II). b Apical to basolatearl cumulative transport flux of target com-
pounds (7a–f), scutellarin(I) and scutellarein(II) (200 µM) across the
BBB cell layer at different time intervals
In the Caco-2 cell permeability assay (Table 2), com-
pound 7d had an efflux ratio (ER, Papp BL to AP/Papp AP to BL
)
of 1.80, which is higher than the ER (1.76) of scutellarein.
The ERs of the other test compounds were in the range
0.94–1.62 and were thus lower than those of scutellarin and
scutellarein. The ER values of 7a, 7c, 7d, and 7f were >1.0,
whereas the ER values of 7b and 7e were <1.0, indicating
greater permeability in the BL to AP direction for com-
pounds 7a, 7c, 7d, and 7f than for compounds 7b or 7e. The
Caco-2 cell permeabilities of 7b, 7c, and 7e (Papp AP to BL
values of 3.11 0.87 × 10–6 cm/s, 1.93 0.54 × 10–6 cm/s
and 1.97 0.38 × 10–6 cm/s, respectively) were 5.9-fold,
3.6-fold, and 3.7-fold higher than that of scutellarin (0.53
0.01 × 10–6 cm/s) and 3.7-fold, 2.3-fold, and 2.4-fold higher
than that of scutellarein (0.83 0.21 × 10–6 cm/s). Com-
pound 7b had the highest Papp AP to BL value (3.11 0.87 ×
10–6 cm/s) and the lowest ER value (0.94) (Fig.1). Com-
pounds 7b, 7d, and 7e have significantly higher Caco-2 cell
permeability than scutellarin or scutellarein, indicating that
4′-N-substituted (aminomethyl) benzoic acid esterification
and 7-nicotinic acid esterification of scutellarein would
significantly enhance gut absorptive capacity.
The Papp AP to BL values across the BBB cell layer for
compounds (7a–c, 7e and 7f) are summarized in Table 3.
Permeabilities of all test compounds (Papp AP to BL values
0.56 0.12 × 10–6 cm/s to 1.65 0.37 × 10–6 cm/s) were
significantly higher than those of scutellarin (0.17 0.08 ×
10–6 cm/s) or scutellarein (0.18 0.08 × 10–6 cm/s). The
in vitro BBB permeability of 7b, 7e, and 7f (Papp AP to BL
values 1.65 0.37 × 10–6 cm/s, 1.01 0.13 × 10–6 cm/s and
0.71 0.16 × 10–6 cm/s, respectively) were 9.7-fold, 5.9-
fold, and 4.2-fold higher than that of scutellarin (0.17
0.08 × 10–6 cm/s) and 9.2-fold, 5.6-fold, and 3.9-fold higher
than that of scutellarein (0.18 0.06 × 10–6 cm/s). Com-
pound 7b had the highest Papp AP to BL value (1.65 0.37 ×
10–6 cm/s) and the lowest ER value (0.64) (Fig. 2a). Per-
meability of the test compounds 7a–f across the BBB cell
layer increased linearly with time (0–180 min) and the
permeabilities of compounds 7a–f were significantly higher
than those of scutellarin or scutellarein at all time points
(Fig. 2b). The obtained results confirm our hypothesis that
introduction of a nicotinic acid ester moiety at the 7-position

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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 Excellent Youth Scientific Talents
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