Traversal of the Blood-Brain Barrier by Cleavable l -Lysine Conjugates of Apigenin

Article abstract of DOI:10.1021/acs.jafc.8b01187

Apigenin, a flavone abundant in parsley and celery, is known to act on several CNS receptors, but its very poor water solubility (<0.001 mg/mL) impedes its absorption in vivo and prevents clinical use. Herein, apigenin was directly conjugated with glycine, l-phenylalanine, and l-lysine to give the corresponding carbamate derivatives, all of which were much more soluble than apigenin itself (0.017, 0.018, and 0.13 mg/mL, respectively). The Lys-apigenin carbamate 10 had a temporary sedative effect on the mice within 5 min of intraperitoneal administration (single dose of 0.4 mg/g) and could be detected in the mice brain tissues at a concentration of 0.82 μg/g of intact Lys-apigenin carbamate 10 and 0.42 ug/g of apigenin at 1.5 h. This study accomplished the delivery of apigenin across the BBB in a manner that might be applicable to other congeners, which should inform the future development of BBB-crossing flavonoids.

Full text of DOI:10.1021/acs.jafc.8b01187

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Food and Beverage Chemistry/Biochemistry
Traversal of the Blood Brain Barrier by
Cleavable L-Lysine Conjugates of Apigenin
Tsung-Yun Wong, Ming-Shian Tsai, Lih-Ching Hsu, Shu-Wha Lin, and Pi-Hui Liang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01187 • Publication Date (Web): 20 Jun 2018
Downloaded from http://pubs.acs.org on June 20, 2018
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Journal of Agricultural and Food Chemistry
Traversal of the Blood Brain Barrier by Cleavable
Apigenin
L
-Lysine Conjugates of
TsungꢀYun Wong,1 MingꢀShian Tsai,2, 3 LihꢀChing Hsu,1 ShuꢀWha Lin,2, 4 PiꢀHui Liang1,5,*
T.ꢀW. Wong, Prof. Dr. L.ꢀC. Hsu, Prof. Dr. P.ꢀH. Liang
1. School of Pharmacy, College of Medicine, National Taiwan University, Taipei 100, Taiwan.
Dr. M.ꢀS. Tsai, Prof. Dr. S.ꢀW Lin
2. Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan
University Hospital, College of Medicine, National Taiwan University, Taipei 100, Taiwan
Dr. M.ꢀS. Tsai,
3. Present address: Department of Genetics, Stanford University School of Medicine, Stanford, CA
94305, USA
Prof. Dr. S.ꢀW Lin
4. Department of Clinical Medicine, National Taiwan University Hospital, National Taiwan
University
Prof. Dr. P.ꢀH. Liang
5. The Genomics Research Center, Academia Sinica, Taipei 128, Taiwan
AUTHOR INFORMATION
Corresponding Author
Eꢀmail: phliang@ntu.edu.tw
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Abstract
Apigenin, a flavone abundant in parsley and celery, is known to act on several CNS receptors,
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but its very poor water solubility (< 0.001 mg/mL) impedes its absorption in vivo and prevents
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clinical use. Herein, apigenin was directly conjugated with glycine,
L
ꢀphenylalanine, and ꢀlysine to
L
5
give the corresponding carbamate derivatives, all of which were much more soluble than apigenin
itself (0.017, 0.018, and 0.13 mg/mL, respectively). The Lysꢀapigenin carbamate 10 had a
temporary sedative effect on the mice within five minutes of intraperitoneal administration (single
dose of 0.4 mg/g), and could be detected in the mice brain tissues at a concentration of 0.82 ꢁg/g of
intact Lysꢀapigenin carbamate 10 and 0.42 ug/g of apigenin at 1.5 h. This study accomplished the
delivery of apigenin across the BBB in a manner that might be applicable to other congeners, which
should inform the future development of BBBꢀcrossing flavonoids.
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Key words: flavonoid; water solubility; blood brain barrier; amino acids; prodrug; apigenin
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INTRODUCTION
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Apigenin (Figure 1), 4’,5,7ꢀtrihydroxyflavone, is a flavonoid commonly found in various
plants, such as parsley, celery, and chamomile.1, 2 Several in vitro studies found it to affect various
receptors of the CNS. For instance, it inhibited adenosine receptors;3 acted as an opioid antagonist
(ꢀ = κ > δ in affinity);4 reduced glutamatergic transmission via inhibition of the
Nꢀmethylꢀ
ꢀasparate receptor when used to treat cultured cortical neurons;5 competed with
D
flunitrazepem for the benzodiazepine receptor, which is associated with anxiolytic and sedative
effects;6 and inhibited human monoamine oxidase – an enzyme targeted by antidepressants.7 A
series of in vitro studies relating to Alzheimer’s disease were also conducted, and its antioxidant
properties8 and ability to suppress proinflammatory mediators and protect against the toxicity
induced by Aβ25ꢀ35 in brain microvascular endothelial cells in rats assessed.9
Due to the very poor water solubility of apigenin, which can be ascribed to intramolecular
hydrogen bonding and molecular planarity,10 its therapeutic utility in vivo is negligible. From a
pharmacokinetic (PK) standpoint, the poor solubility of apigenin retards the process of dissolution,
resulting in a low and delayed absorption. Upon oral administration of a single dose of radiolabeled
apigenin to a rat, radioactivity could not be detected in the blood for 24 h, reflecting its poor
absorption profile and limited availability to the CNS.11 To date, efforts to improve the solubility of
apigenin have been largely focusing on its formulation. For instance, lipidꢀbased vehicles such as
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micelles and liposomes, which encapsulate apigenin in a hydrophilic sphere containing one or more
lipid layers, could enhance its oral bioavailability.12ꢁ14 Another, related tactic entailed the use of an
auxiliary such as 2ꢀhydroxypropylꢀβꢀcyclodextrin to form an inclusion complex.15 However, few
studies have sought to improve the solubility by way of chemical modifications.
The bloodꢀbrain barrier (BBB) is a selectively permeable membrane that segregates the
cerebral blood and the extracellular fluid in the CNS, and restricts the entry of more than 98% of all
smallꢀmolecule drugs. The endothelial cells of capillaries are sewed by numerous tight junctions
that render solutes either diffusing across lipid membranes (e.g., water, gases, and small lipophilic
molecules) or relying on specific transporters in capillary luminal membranes to enter brain tissues
(e.g., glucose and amino acids).16 Good BBB penetration can be achieved by two approaches:
nonꢀspecific interaction17 with the brain tissue; or through screening transporter substrates18 – but
nonꢀspecific binding to the brain could result in transporter accumulation in peripheral tissues. An
alternative approach to take advantages of transporter substrates is to utilize the prodrug approach,
in which a drug is conjugated with a BBB carrier by a cleavable linker.19
A carbamate bridge in quercetinꢀamino acid conjugates was reported to be soluble and stable
prodrug of quercetin, a flavonol.20, 21 To the best of our knowledge, no flavoneꢀamino acid
conjugate has been reported to enhance BBB uptake. In this work, apigenin was conjugated with
amino acids in order to enhance its solubility and ability to cross the BBB. There are three systems
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by which amino acids are transported across the BBB22ꢁ24: 1) system L1, which transports large,
neutral amino acids (LNAA), including phenylalanine, tryptophan, leucine, methionine, isoleucine,
tyrosine, histidine, valine, threonine and glutamine; 2) system y+, which transports cationic amino
acids (CAA), including lysine, arginine, and ornithine; and 3) system xꢀ, which transports anionic
amino acids (AAA) such as glutamate and aspartate. We reasoned that the amino acids with the
highest affinities for transporters in systems L1 and y+ would be most suitable for conjugation with
apigenin; and so the amino acid Km values (the concentration at which the transport rate is half of
the max transport rate Vmax) were compared. As a result, phenylalanine (system L1; Km = 170
ꢁmol/L; Vmax = 41 nmol/min/g ) and lysine (system y+; Km = 279 ꢁmol/L; = 22 nmol/min/g) were
selected.25 Their lowest Km values in the respective systems represent the highest affinities for their
transporters and advantages in BBBꢀcrossing transportation over their competitors. This choice of
phenylalanine and lysine is further validated by their categorization as essential amino acids,
reflecting the fact that brain tissues require a steady import of them from the blood.26 System xꢀ is
an inferior transportation mechanism to systems L1 and y+ (the uptake of glutamate and aspartate is
much lower ꢀ Vmax = 0.21 and 0.13 nmol/min/g for BBB, respectively)27 and so the conjugation of
apigenin with amino acids transported by system xꢀ was not investigated in this work. Glycine,
which is pumped out brain tissues by abluminal Na+ꢀdependent channels,28 without the need for a
specialized transportation system, was conjugated with apigenin to serve as a negative control.
Synthetic methods for amino acid and apigenin conjugations were developed and their
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physicalꢀchemical properties, in vitro assays for metabolic stability, and in vivo trials for PK profiles
were evaluated.
MATERIALS AND METHODS
General. Reagents and solvents for synthesis were reagent grade, and used without further
purification. 1H and 13C nuclear magnetic resonance (NMR) spectra were acquired on a
BrukerꢀAVꢀ400 (400 MHz). Chemical shifts are referenced to residual solvent peaks in parts per
1
million (δ) H δ 2.54, 13C δ 40.5 for d6ꢀDMSO. Coupling constants (J) are given in Hertz (Hz).
Splitting patterns are denoted as s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet),
m (multiplet). Mass spectra were acquired using a Burker bioTOF III and reported in m/z. HPLC
analyses were performed on Shimadzu LCꢀ20AT with a reverse phase Kanto PPC MightsilꢀRP18
(250 mm × 4.6 mm, 5 ꢁm) column and UVꢀVis detector SPDꢀM20A (General conditions for
chemical synthesis are in the Supplemental).
Synthesis of tert-Butyl
((((5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)carbonyl)amino)-ethanoate
(3). Bisꢀ(4ꢀnitrophenyl) carbonate (608 mg, 2.00 mmol) and DIPEA (0.70 mL, 4.0 mmol) were
added to a solution of tertꢀbutyl glycinate (2, 334 mg, 2.00 mmol) in DMF (3 mL). After being
stirred for 12 h at rt, the mixture was concentrated in vacuo to give a pale yellow oil, which was
added without further purification to a solution of apigenin (540 mg, 2.00 mmol) and DIPEA (1.57
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mL, 9.00 mmol) in THF (40 mL). The mixture was stirred for another 12 h and concentrated in
vacuo to give a residue which was purified by column chromatography (silica gel,
tetrahydrofuran/hexane = 4/5, Rf = 0.44) to give 3 (383 mg, 45%) as a white solid; 1HꢀNMR
(d6ꢀDMSO, 200 MHz) δ 12.87 (s, 1H), 8.31 (t, 1H, J = 6.0 Hz), 8.14 (d, 2H, J = 8.6 Hz), 7.35 (d, 2H,
J = 8.6 Hz), 6.99 (s, 1H), 6.55 (d, 1H, J = 1.7 Hz), 6.25 (d, 1H, J = 1.7 Hz), 3.80 (d, 2H, J = 6.0 Hz),
1.47 (s, 9H); 13CꢀNMR (d6ꢀDMSO, 50 MHz) δ 128.8, 169.8, 165.4, 163.6, 162.4, 158.4, 155.2,
154.7, 128.9, 128.5, 123.1, 106.0, 104.9, 100.0, 95.1, 82.0, 43.9, 28.7; HRMS (ESIꢀTOF) calcd. for
C22H22NO8 [M+H]+: 428.1345, found m/z 428.1355.
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Synthesis of
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((((5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)carbonyl)amino)ethanoic acid
(4). 3 (250 mg, 0.58 mmol) was dissolved in TFA (6 mL) at 0 °C, and the mixture was stirred for 3
h. After concentration in vacuo, the residue was recrystallized from acetone to give 4 (198 mg, 88%;
Rf value = 0.32, THF/hexane = 4/5) as a pale yellow solid. 1HꢀNMR (d6ꢀDMSO, 200 MHz) δ 12.88
(s, 1H), 8.31 (t, 1H, J = 6.0 Hz), 8.14 (d, 2H, J = 8.6 Hz), 7.35 (d, 2H, J = 8.6 Hz), 6.99 (s, 1H),
6.56 (d, 1H), 6.26 (d, 1H), 3.83 (d, 2H, J = 6.0 Hz); 13CꢀNMR (d6ꢀDMSO, 50 MHz) δ 182.9, 172.2,
165.4, 163.7, 162.5, 158.5, 155.2, 154.8, 128.9, 128.5, 123.2, 106.0, 104.9, 100.1, 95.1, 43.3;
HRMS (ESIꢀTOF) calcd. for C18H13NO8 [M+H]+: 372.0719, found m/z 372.0732. The HPLC purity
of 4 was 96.4% (tR =5.97 min, eluted with 0.5% formic acid aqueous solution and methanol, 0 min
35/65 → 9 min 5/95 → 15 min 5/95 → 18 min 50/50).
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Synthesis of (S)-tert-Butyl
2-((((5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)carbonyl)amino)-3-phenylp
ropanoate (6). A solution of ꢀphenylalanine tertꢀbutyl ester hydrochloride (5, 2.89 g, 11.2 mmol)
L
and DIPEA (19.5 mL, 11.2 mmol) in DMF (10 mL) was stirred for 10 min at r.t., and then added via
cannula into solution of bisꢀ(4ꢀnitrophenyl) carbonate (3.40 g, 16.7 mmol) in THF (50 mL) at 0 °C.
An additional equivalent of DIPEA (19.5 mL, 11.2 mmol) was added dropwise to the mixture over
2 min. The mixture was stirred at 0 °C for 2 h. TLC analysis showed a major product with an Rf
value of 0.44 (THF/hexane = 1/2). The resulting mixture was transferred to a dropping funnel and
added to a solution of apigenin (3.02 g, 11.2 mmol) and DIPEA (8.80 mL, 50.4 mmol) in DMF/THF
(7 mL/38mL) dropwise, over 1 h. After being stirred for 6 h at rt, the mixture was concentrated in
vacuo to give a residue which was purified by column chromatography (silica gel, THF/hexane =
4/7, with 0.5% acetic acid, Rf value = 0.32) followed to afford 6 (2.94 g, 51%; 92 % yield based on
recovered starting material, 1.36 g of 1) as a pale yellow solid. 1HꢀNMR (d6ꢀDMSO, 400 MHz) δ
12.87 (s, 1H), 8.67 (d, 1H, J = 8.0 Hz), 8.12 (d, 2H, J = 8.8 Hz), 7.38ꢀ7.26 (m, 5H), 7.25 (d, 2H, J =
8.8 Hz), 6.98 (s, 1H), 6.55 (d, 1H, J = 2.0 Hz), 6.26 (d, 1H, J = 2.0 Hz), 4.29ꢀ4.22 (m, 1H, J1 =9.6
Hz, J2 = 8.0 Hz, J3 = 5.8 Hz), 3.13ꢀ3.08 (dd, 1H, J1 = 13.7 Hz, J2 = 5.8 Hz), 3.01ꢀ2.96 (dd, 1H, J1 =
13.7 Hz, J2 = 9.6 Hz), 1.40 (s, 9H); 13CꢀNMR (d6ꢀacetone, 100 MHz) δ 183.8, 172.0, 165.9, 164.8,
164.1, 159.6, 155.8, 155.2, 138.9, 131.0, 130.0, 129.6, 129.3, 128.3, 127.6, 123.7, 117.3, 109.1,
106.7, 106.7, 106.2, 100.6, 95.7, 95.6, 82.9, 68.3, 58.0, 57.9, 39.1, 28.8, 25.3; HRMS (ESIꢀTOF)
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calcd. for C29H28NO8 [M+H]+: 518.1809, found m/z 518.1809.
Synthesis of
(S)-2-((((5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)carbonyl)amino)-3-phen
ylpropanoic acid (7). 6 (268 mg, 0.518 mmol) was dissolved in TFA (5 mL) at 0 °C, and the
mixture was stirred at 0 °C for 3 h. The mixture was diluted with toluene (1 mL), then concentrated
in vacuo to give a residue which was purified by column chromatography (silica gel, THF/hexane =
5/7, 0.5% acetic acid, Rf value = 0.25) to afford 7 (215 mg, 90 %) as a pale yellow solid. 1HꢀNMR
(d6ꢀDMSO, 400 MHz) δ12.88 (s, 1H), 8.42 (d, 1H, J = 8.4 Hz), 8.10 (d, 2H, J = 8.8 Hz), 7.39ꢀ7.28
(m, 5H), 7.22 (d, 2H, J = 8.8 Hz), 6.98 (s, 1H), 6.55 (d, 1H, J = 2.0 Hz), 6.25 (d, 1H, J = 2.0 Hz),
4.33ꢀ4.27 (m, 1H, J 1 = 10.7 Hz, J2 = 8.4 Hz, J3 = 4.3 Hz), 3.13ꢀ3.08 (m, 1H, J 1 = 13.8 Hz, J2 = 4.3
Hz), 3.01ꢀ2.96 (m, 1H, J1 = 13.8 Hz, J2 = 10.7 Hz), 1.40 (s, 9H); 13CꢀNMR (d6ꢀDMSO, 100MHz) δ
182.9, 173.9, 165.4, 163.6, 162.5, 158.4, 154.7, 138.7, 130.3, 130.2, 129.3, 128.9, 128.8, 128.4,
127.6, 123.0, 106.0, 104.9, 100.0, 95.1, 56.7, 37.5; HRMS (ESIꢀTOF) calcd. for C25H18NO8 [MꢀH]+:
460.1027, found m/z 460.1025. The HPLC purity of 7 was 99.4% (tR = 4.22 min, eluted with 0.5%
formic acid aqueous solution and methanol, 10/90 for 18 min).
Synthesis of
(S)-6-((tert-Butyloxycarbonyl)amino)-2-((((5-hydroxy-2-(4-hydroxyphenyl)-4H-chrome-7yl)ox
y)carbonyl)amino)hexanoic acid (9). A solution of NeꢀBocꢀLꢀlysine tertꢀbutyl ester hydrochloride
(8, 332 mg, 0.987 mmol) and DIPEA (181 ꢁL, 1.03 mmol) in DMF (4 mL) was stirred for 10 min,
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then added via cannula into a cold (0 °C) solution of bisꢀ(4ꢀnitrophenyl) carbonate (313 mg, 1.03
mmol) in THF (10 mL). An additional equivalent of DIPEA (181 ꢁL, 1.03 mmol) was added
dropwise to the mixture over 2 min. The mixture was stirred at 0 °C for 2 h. The resulting mixture
was transferred to a dropping funnel and added to a solution of apigenin (266 mg, 0.987 mmol) and
DIPEA (773 ꢁL, 4.44 mmol) in DMF/THF (3 mL/15mL) dropwise, over 1 h. After being stirred for
6 h at rt, the mixture was concentrated in vacuo to give a residue which was purified by column
chromatography (silica gel, THF/hexane = 1/2, then DCM/MeOH = 100/2) followed to afford the
apigeninꢀlysine(Boc)ꢀOBn conjugate 11 (Rf value = 0.27, THF/hexane = 2/3) as pale yellow liquid.
Then, 20% Pd/C (60 mg) was added to a solution of 11 in THF (15 mL), and the resulting slurry
was stirred under positive pressure of H2 for 30 h. After the filtration through a pad of Celite with
THF) and purification by column chromatography (silica gel, THF/hexane = 1/1, 1% AcOH), 9
(282 mg, 0.520 mmol, 53% overall) was obtained as clear liquid. 1HꢀNMR (d6ꢀDMSO, 400 MHz) δ
12.89 (s, 1H), 8.30 (d,1H, J = 7.8 Hz), 8.14 (d, 2H, J = 8.8 Hz), 7.35 (d, 2H, J = 8.7 Hz), 7.00 (s,
1H), 6.87 (t, 1H, J = 5.4 Hz), 6.56 (d, 1H, J = 1.9 Hz), 6.26 (d, 1H, J = 2.0 Hz), 4.03ꢀ3.98 (m, 1H),
3.87ꢀ3.77 (m, 1H), 3.63 (t, 3H, J = 6.7 Hz), 2.96 (d, 2H, J = 4.6 Hz), 1.82ꢀ1.68 (m, 6H), 1.41 (s,
9H) ; 13CꢀNMR (d6ꢀDMSO, 100 MHz) δ 182.9, 174.5, 165.4, 163.7, 162.5, 158.4, 156.6, 154.8,
129.5, 128.9, 128.4, 123.1, 117.0, 108.0, 106.0, 104.9, 100.0, 95.1, 78.4, 68.0, 67.62, 55.1, 31.4,
30.1, 29.9, 26.1, 24.6, 23.9; HRMS (ESIꢀTOF) calcd. for C27H31N2O10 [M+H]+: 543.1973, found
m/z 543.1982.
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Synthesis of
(S)-6-amino-2-((((5-hydroxy-2-(4-hydroxyphenyl)-4H-chrome-7yl)oxy)carbonyl)amino)
hexanoic acid hydrochloride (10). The mixture was diluted with toluene (1 mL), then concentrated
in vacuo to give a residue which was purified by column chromatography (silica gel, THF/hexane =
5/7, 0.5% acetic acid, Rf value = 0.25) to afford 7 (215 mg, 90 %) as a pale yellow solid. 9 (190 mg,
0.35 mmol) was dissolved in TFA (3 mL) at 0 oC, and the mixture was stirred for 3 h. The mixture
was diluted with toluene (1 mL) and saturated ammonium chloride solution (0.5 mL), then
concentrated in vacuo to give a residue which was recrystallized under methanol to afford 10 (139
mg, 90 %) as a pale brown solid. NMR data refers to the TFA salt. 1HꢀNMR (d6ꢀDMSO/dꢀTFA =
10/1, 400 MHz) δ 8.31 (d,1H, J = 7.8 Hz), 8.13 (d, 2H, J = 8.7 Hz), 7.34 (d, 2H, J = 8.7 Hz), 7.00 (s,
1H), 6.55 (s, 1H), 6.27 (s, 1H), 4.06ꢀ4.03 (m, 1H), 3.87ꢀ3.78 (m, 3H), 2.92ꢀ2.76 (m, 2H), 2.17ꢀ1.18
(m, 6H); 13CꢀNMR (d6ꢀDMSO/dꢀTFA = 10/1, 100 MHz) δ 182.9, 174.3, 165.3, 163.6, 162.3, 158.5,
154.8, 128.9, 128.5, 123.1, 108.0, 106.8, 106.0, 104.9, 67.6, 54.8, 31.1, 29.7, 27.4, 24.3, 24.2, 23.5;
HRMS (ESIꢀTOF) calcd. for C22H23N2O8 [MꢀCl]+: 443.1449, found m/z 443.1448. The HPLC
purity of 10 was 97.2% (tR = 4.43 min, eluted with 0.5% formic acid aqueous solution and methanol,
0 min 40/60 → 18 min 50/50).
Measurement of solubility. Saturated solutions of apigenin derivatives in 1.0 mL ddH2O were
equilibrated by shaking for 24 h and then centrifuged at 14000 g for 10 min. The supernatants were
filtered through 0.22 ꢁm PTFE membranes into 12 × 32 mm vials. Standard curves were established
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starting from 5 mg/mL stock solutions of Glyꢀapigenin carbamate 4, Pheꢀapigenin carbamate 7, and
Lysꢀapigenin carbamate 10 in THF, which diluted into concentrations of 0.5, 0.1, 0.05, 0.025,
0.0125, and 0.00625 mg/mL. An aliquot of 1 ꢁL sample solution was injected to HPLC each time.
Areas of corresponding peaks (λ = 254 nm) were plotted against concentrations.
Measurement of log P. The oilꢀwater partition coefficient P (log P) is the ratio of the equilibrium
concentration of a compound dissolved in a twoꢀphase system consisting of two immiscible
solvents. The log P value is related to the log k value in a reverse phase chromatography by the
following equation:29
log ꢀ = ꢁ log ꢂ + ꢃ
194
(ꢄꢅ − ꢄꢆ)
ꢂ =
ꢄꢆ
195
196
197
198
199
200
201
202
where k is the capacity factor, t0 is the dead time of NaNO2, which is unretained by the stationary
phase, and tR is the retention time of the corresponding compound. The analytical standards of
reference compounds, including benzyl alcohol, phenol, thiophene, benzene, and toluene, were
prepared at a concentration of 1 mg/mL in methanol. An aliquot of 1 ꢁL was injected each time,
isocratically eluted with 0.5% formic acid aqueous solution and methanol (30/70, v/v). Generally,
the log P is linearly related to log k. By plotting log P values of the reference standards versus their
log k, the regression equation was acquired as: log P = 1.6712 log k + 1.7904 (R2 = 0.9621).
Human Liver S9 Stability Assay. The suspension for the S9 assay was prepared as follows: 2.2
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mg/mL human liver S9 fraction (20ꢀdonor pool, mixed gender, Corning® Gentest™), 1.1 mM
EDTA, 3.67 mM MgCl2 and NADPH regenerating system (1.43 mM NADP, 3.67 mM
glucoseꢀ6ꢀphosphate and 0.44 U/mL glucoseꢀ6ꢀphosphate dehydrogenase) at a total volume of 90
ꢁL. After the suspension was preꢀincubated at 37 °C for 10 min, the substrate (Glyꢀapigenin
carbamate 4, Pheꢀapigenin carbamate 7, or Lysꢀapigenin carbamate 10, 0.5 mM in 10 ꢁL PBS) was
added to the suspension and incubated for 0, 5, 30, and 60 min, after which experiments were halted
by dilution with 100 ꢁL of a mixture of methanol/acetonitrile (25/75, v/v) at 0 °C. Followed by
vortexing and centrifugation at 14000 g for 10 min, supernatants were filtered through 0.22 ꢁm
PTFE membranes into 12x32 mm vials. An aliquot of 10 ꢁL the sample solution was injected to
HPLC each time, isocratically eluted with 0.5% formic acid aqueous solution and methanol (30/70,
v/v). Areas of corresponding peaks (λ = 254 nm) were plotted against concentrations.
Drug administration, sampling, extraction, and sample preparation. Eightꢀweekꢀold male,
wildꢀtype C56BL/6 (C57BL/6JNarl) mice were purchased from the National Laboratory Animal
Center (Nangang, Taiwan), kept at the laboratory animal center of National Taiwan University with
ad libitum access to water and food, and acclimated for at least 3ꢀ7 days. All animal protocols were
approved by the Institutional Animal Care and Use Committee (IACUC no. 20150014). In
following PK experiments, all suspensions were injected intraperitoneally. Mice were sacrificed
under anesthesia using 2,2,2ꢀtribromoethanol (Avertin®, MilliporeSigma, Brulington, MA, USA,
0.25 mg/g body weight by 2 min in advance) at the following time points: 15 min and 1.5 h after
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one bolus of Glyꢀapigenin carbamate 4, Pheꢀapigenin carbamate 7, and Lysꢀapigenin carbamate 10
(0.4 mg/g wt.; 0.2 mg/ꢁL in PBS); 15 min, 0.5, 1, 1.5, 2, 4, 6, and 12 h after one bolus of apigenin 1
(0.23 mg/g wt.; 0.115 mg/ꢁL in PBS) or Lysꢀapigenin carbamate 10 (0.4 mg/g wt.; 0.2 mg/ꢁL in
PBS). Blood samples were collected via cardiac puncture by syringes preloaded with 100 ꢁL 20%
EDTAꢀK2 solution, followed by centrifugation at 6000 g for 10 min. The entire dissected
intracranial contents were placed into round bottom, two milliliter microcentrifuge tubes with 1 mL
extraction buffer (TꢀPER® ThermoFisher Scientific, Waltham, MA, USA) and 7 mm stainless steel
,
beads, then homogenized by Tissuelyser II® (QIAGEN, Hilden, Germany) at 30 Hz for 3 min. All of
the containers were maintained at 0ꢀ5 °C throughout the processes. All samples were stored at ꢀ30
°C and analyzed within 12 h. Samples of 100 ꢁL plasma and 200 ꢁL brain crude suspension were
extracted with 200 ꢁL and 600 ꢁL of a 5 mM solution of ammonium acetate in a mixture of
THF/methanol, respectively. After centrifugation at 14000 g for 15 min, supernatants were filtered
through 0.22 ꢁm PTFE membranes into 12 × 32 mm vials for UPLCꢀMS/MS analyses.
UPLC-MS/MS analyses. Analyses were performed on a Waters ACQUITY UPLC system and a
Waters Quattro Premier XE tripleꢀquadrupole mass spectrometer (Waters, Milford, MA, USA). The
reverse phase BEH C18 (100 mm × 2.1 mm, 1.7 ꢁm, Waters) and VanGuard BEH C18 (5 mm×2.1
mm, 1.7 ꢁm, Waters) preꢀcolumns were used to separate the analytes. All data were acquired by
MassLynx V4.1. The mobile phase consisted of a 5 mM aqueous solution of ammonium acetate (A)
and methanol (B), set as follows: 0.00 min 70% A → 0.50 min 70% A → 4.00 min 10% A → 5.00
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min 10% A → 5.50 min 70% A → 8.50 min 70% A; at a rate of 0.3 mL/min for 8.50 min with 10
ꢁL per injection. The column oven was maintained at 60 °C. Multiple reaction monitoring (MRM)
method was applied in quantification. Mass spectrometer parameters were: capillary voltage, 3.0
kV/ 3.0 kV respectively for positive/negative ion mode; ion source temperature, 120 °C; desolvation
temperature, 450 °C; cone gas flow (N2), 50 L/h; desolvation gas flow (N2), 700 L/h; multiplier, 650
V; collision gas pressure (Ar), 3ꢀ4 × 10ꢀ3 mbar. Other parameters are listed in Table 1.
Calculation of the concentration in brain tissues. Brains were not perfused to wash out the blood
because this procedure would have altered the chemical equilibrium between the vascular
compartment and the whole brain.30 The concentration of compound in brain tissues was calculated
by the literature reported equation with modification.31
250
251
(ꢇꢈꢇꢉꢊꢋꢇꢌꢍꢋ ꢎꢍꢏꢈꢐꢊ × ꢇꢈꢇꢉꢊꢋꢇꢌꢍꢋ ꢑꢍꢋꢑꢊꢋꢄꢒꢁꢄꢌꢍꢋ) − (ꢓꢔꢕ × ꢃꢏꢍꢍꢖ ꢑꢍꢋꢑꢊꢋꢄꢒꢁꢄꢌꢍꢋ)
ꢗꢊꢌꢘℎꢄ ꢍꢙ ꢊꢋꢄꢌꢒꢊ ꢌꢋꢄꢒꢁꢑꢒꢁꢋꢌꢁꢏ ꢑꢍꢋꢄꢊꢋꢄ
252
253
The ‘suspension’ refers to the entire intracranial contents, homogenized in extraction buffer; the
average volume of which was 1.8 mL. The concentration in the numerator was determined by the
peak area of the m/z transition from a precursor ion to its corresponding product ion for
quantification (quantifiers, as shown in Table 1). The cerebral blood volume (CBV) was 35 ꢁL per
gram of brain.32 The concentration in brain tissues was obtained by subtracting the amount of
compound in the cerebral blood vessels from that in the suspension, then divided by the weight of
entire intracranial content, expressed in ꢁg/g. The contribution of cerebral blood to the weight of the
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whole brain is neglected in the calculation.
PK and statistical analyses. WinNonlinTM (version 5.2, Pharsight, MO, USA) was used to process
the plasma concentration versus time data. Cmax was the observed maximum concentration, and Tmax
corresponding to the time when acquired. AUC0→t represented the area under the plasma
concentration versus time curve from time zero to the time of the last quantifiable concentration,
which was calculated by trapezoidal rule. λz was estimated by linear regression with the last three
points on the semiꢀlog plot of the plasma concentration versus time curve. Terminal halfꢀlife (t1/2)
was derived from ln 2 over λz. AUC0→∞ was the sum of AUC0→t and the extrapolation term (as
calculated by the last quantifiable concentration over λz). Apparent clearance (CL/F) was derived
from the dose over AUC0→∞, further divided by λz for apparent volume of distribution (V/F). Mean
residue time (MRT) was acquired by using a nonꢀcompartment method.33
RESULTS AND DISCUSSION
In our synthetic strategy, a carbamate linkage is used to bridge the amino groups of the amino
acids and the 7ꢀOH of apigenin 1 (Figure 2). The conjugation process entails formation of the
corresponding pꢀnitrophenyl carbamates of the amino acids using bisꢀ(pꢀnitrophenyl) carbonate
under basic conditions, followed by substitution of apigenin 1 for the remaining pꢀnitrophenyl
group. Bisꢀ(pꢀnitrophenyl) carbonate is a relatively mild, selective coupling reagent compared with
triphosgen or pꢀnitrophenyl chloroformate, which are more reactive and possess higher reactivity
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precipitating the formation of ureas via double nucleophilic substitution of amino acids. The
pꢀnitrophenyl carbamates, in contrast, undergo only a single nucleophilic substitution of amino
acids. These carbamates are stable, and the introduction of the pꢀphenyl moiety permits the use of
THF
/
DMF in the subsequent step. By performing the reaction in THF DMF, complete conversion to
/
the pꢀnitrophenyl carbamate intermediate could be observed via TLC. In contrast, the use of aprotic
solvents such as DCM, dioxane, THF and acetonitrile resulted in much lower conversion, perhaps
due to the incompatibility of these solvents with the hydrochloride salts and freebases of the amino
acids.
In the second step, N,Nꢀdiisopropylethylamine deprotonates the 7ꢀOH of apigenin 1 (the most
acidic phenol hydroxyl group of apigenin 1 with an estimated pKa of 6.6),34 to give the conjugate
base of apigenin 1, which reacts with the pꢀnitrophenyl carbamate to afford the corresponding
amino acid conjugate of apigenin 1. The fair yield (45ꢀ51% over two steps) could not be improved
by increasing the ratio of pꢀnitrophenyl carbamates to apigenin 1 from 1:1 to 3:1, but no 7,4′ꢀadduct
sideꢀproducts were observed, and up to 90% of the unconverted apigenin 1 could be recycled.
Palladiumꢀcatalyzed hydrogenation to deprotect the benzyl group (Bn) of 8 gave 9, very cleanly, by
TLC. The yield was 53% over three steps, much similar to those of 2 → 3 (45%) and 5 → 6 (51%).
Removal of tertꢀbutyloxycarbonyl (Boc) group proceeded smoothly using TFA and three desired
productsꢀ Glyꢀapigenin carbamate 4, Pheꢀapigenin carbamate 7, Lysꢀapigenin carbamate 10 were
obtained. This route obviates the need to protect and deprotect the 4’ꢀ and 5ꢀ OH groups, or
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deprotect the 7ꢀOH group prior to coupling, saving several steps (Figure 2).
To evaluate the metabolic stability of amino acid conjugates of apigenin 1 in vitro, a human
liver S9 stability assay containing phase I and phase II enzymes was conducted. As shown in Figure
3, the remaining ratios after 12 h are at least 90%. After 24 h, the conversion of Glyꢀapigenin
carbamate 4 and Lysꢀapigenin carbamate 10 was 23%; and that of Pheꢀapigenin carbamate 7 was
7%. This result suggests that the carbamate groups of these conjugates can largely withstand
hydrolysis, and that the apigenin moiety is modestly susceptible to oxidation in phase I (e.g., at C6,
C8, or C3’) and to conjugation in phase II (e.g., glucuronidation at 5ꢀ or 4’ꢀOH). Gastrointestinal
stability, however, cannot be inferred from the liver S9 assay, and so the effect of these carbamates
when administered orally is not known; however, it has been reported that flavonoids undergo ring
fission into phenolic acids after oral administration because of the susceptibility to intestinal
microflora.35
In an effort to facilitate entire analyses and to optimize the qualities of signals, conditions of
UPLCꢀMS/MS and sample preparation were tuned in advance. For UPLCꢀMS/MS analysis, a
gradient elution of methanol and water from 3:7 to 9:1 within 4 min was found able to distinctly
separate apigenin 1, Glyꢀapigenin carbamate 4, Pheꢀapigenin carbamate 7, and Lysꢀapigenin
carbamate 10. A 5 mM aqueous solution of ammonium acetate was added in the mobile phase, to
suppress the formation of metal ion salt adducts (e.g. with NaCl), which resulted in unidentifiable
collision fragmentations. For sample preparation, a combination of THF and methanol (1:1,
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optimized) was found to ensure complete extraction: the capability of the former to solvate apigenin
1 and its amino acid conjugates far exceeds that of other organic solvents; the latter could
compensate for the mediocre ability of the former to precipitate proteins, which facilitates the
filtration of supernatant after samples centrifuged and diminishes matrix effects.
A preliminary in vivo assay with two time points (15 min and 90 min, n = 1) was performed
prior to a multiple time point assay. Both Glyꢀapigenin carbamate 4 and Pheꢀapigenin carbamate 7
were barely detectable in blood at 15 min after intraperitoneal injection; the signalꢀtoꢀnoise ratios
were lower than 10:1 for quantification. Neither were detected in blood samples at 90 min and in
brain samples at any time point. However, significant amounts of Lysꢀapigenin carbamate 10 was in
blood samples at both time points and in brain samples at 90 min. These results can be explained by
reference to its physicalꢀchemical properties. Firstly, the presumed superior solubility of the
Lysꢀapigenin carbamate 10 salt compared to those of the other two nonꢀsalt carbamates contributed
to the absorption of 10 in the peritoneal cavity, enabling its detection. As shown in Table 2, the
solubility of Lysꢀapigenin carbamate 10 is 0.13 mg/mL, 8ꢀfold and 7ꢀfold higher that of
Glyꢀapigenin carbamate 4 and Pheꢀapigenin carbamate 7, respectively; and over 100ꢀfold that of
apigenin 1. This trend is consistent with the aqueous solubility of unconjugated lysine, alanine,
glycine and apigenin 1, which is lysine > alanine > glycine > apigenin 1, and can rationalize our
observation that traces of aggregation of Glyꢀapigenin carbamate 4 and Pheꢀapigenin carbamate 7
were observed on the fringes of the visceral organs during dissection. Secondly, log P values of
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these carbamates were compared (Table 2). The higher the value, the higher the lipophilicity, which
would be expected to endow a compound with a superior passive permeability across the BBB.
Since the optimal value of log P for BBB penetration was suggested to be 2ꢀ5,36 Glyꢀapigenin
carbamate 4, Pheꢀapigenin carbamate 7, and Lysꢀapigenin carbamate 10 were predicted to be less
capable of diffusing across the BBB. Although Lysꢀapigenin carbamate 10 had an even lower log P
value (= 0.96), suggesting its passage through the BBB by diffusion to be unlikely, it was
nonetheless detected in the brain sample. Thus, the alternative mechanism – transportation via an
amino acid transporter – was assumed to account for its presence in brain.37,38 Given results in this
preliminary in vivo assay showing that only Lysꢀapigenin carbamate 10 could be detected at a later
time point, we therefore chose to further explore its PK and distribution in mice brain.
A PK profile of Lysꢀapigenin carbamate 10 was determined at a single dose of 0.4 mg per
gram of body weight of mouse (Figure 4). The parameters acquired are listed in Table 3 as mean ±
S.D.; the apparent volume of distribution (V/F) of Lysꢀapigenin carbamate 10 is 0.41 ± 0.13 (L/kg),
higher than circulating blood volume (0.06ꢀ0.08 L/kg), implying that a portion of Lysꢀapigenin
carbamate 10 was distributed into the peripheral tissues. The maximum observed blood
concentration (Cmax) is 49.44 ± 8.74 ꢁg/mL at 1.33 ± 0.29 h (Tmax), where an equilibrating process
across the BBB leads to a delay of 1ꢀ2 h between the Cmax in the blood and the brain. Therefore, the
actual highest brain concentration would be slightly higher than that at 1.5ꢀh as illustrated in Figure
5. The estimate of the concentration of Lysꢀapigenin carbamate 10 in mice brain using the entire
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dissected intracranial contents may be significantly influenced by the presence of residual blood.
We applied a correction by subtracting the amount of Lysꢀapigenin carbamate 10 in the
intravascular blood present in the brain.30 The peak concentration in brain of Lysꢀapigenin
carbamate 10 was 0.82 ± 0.18 ꢁg/g of tissue at 1.5 h (Figure 5, Table S13 in the supplemental).
Since the carbamate moiety of Lysꢀapigenin carbamate 10 was cleavable, serum and brain
concentrations of apigenin 1 were also detected, and found to peak at concentrations of 2.63ꢀ2.81
ug/mL and 0.42±0.11 ug/g, respectively at 1ꢀ1.5 h (Figure 4 & 5, and Table S9 & S14 in the
supplemental). The 10/1 ratio of peak concentration in blood and brain were around 17 and 2,
respectively. It seemed that the cleavage apigenin 1 from Lysꢀapigenin carbamate 10 was
immediately distributed to brain tissue, due to higher log P of apigenin 1. In an attempt to study the
PK profile of apigenin 1, IP injection of suspension apigenin 1 in a concentration of 0.23 mg per
gram of body weight of mouse (which equal to molar concentration of Lysꢀapigenin carbamate 10
in above mentioned assay) in PBS was conducted. The maximal serum concentration of apigenin 1
analyzed was 0.12 ± 0.04 ug/mL at 1 h (see Table S12 in the supplemental), and the concentration
of 1 after 4 h were under detection of limit (6.3 ng/mL). Due to the low concentration of apigenin 1
detected in serum, its brain concentration was below the detection of limit. The mean residue time
(MRT) and the systematic clearance (CL/F) of Lysꢀapigenin carbamate 10 were 4.87 ± 2.42(h) and
0.072 ± 0.012 (L/h/kg), respectively (Table 3).
Lysꢀapigenin carbamate 10 was observed to have a sedative effect on all of the mice who were
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given it –within 5 mins after its administration, their activity began to decrease, and they tended to
curl up along the boundary of cage. All recovered from this state after 4ꢀ6 h. The sedative effect of
Lysꢀapigenin carbamate 10 will be subjected to further investigation in due course.
In conclusion, amino acid conjugates of apigenin have been designed and synthesized;
concisely, and with acceptable yields. Optimization of UPLC elution and MS/MS conditions were
found to be necessary, especially for the salt Lysꢀapigenin carbamate 10, the ionization of which
was hampered by chloride ions. In a human liver S9 stability assay, the conjugates were at least
77% intact after incubation with the S9 fraction for 24 h. An in vivo assay showed that only
Lysꢀapigenin carbamate 10 could access brain tissues, presumably due to its superior solubility and
the assistance of cationic amino acid transporters. The brain concentration of Lysꢀapigenin
carbamate 10 in mice was 0.82 ± 0.18 microgram per gram tissues at 1.5 h after administration, and
a sedative effect was observed within 5 min. The development of apigenin conjugates that can cross
the BBB and desirably perturb the CNS can inform the future development of BBBꢀcrossing
flavonoids.
ASSOCIATED CONTENT
Supplementing Information
The Supplementary is available free of charge on the ACS Publications website at DOI:
The detailed UPLCꢀMass analyses to quantify serum and brain concentrations of compound 1 and
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10, UPLCꢀMass spectra, and 1D NMR spectra for compounds 3-4, 6-10 are provided. (PDF)
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: phliang@ntu.edu.tw phone:+886ꢀ2ꢀ33668694
ORCID
PiꢀHui Liang: 0000ꢀ0001ꢀ6668ꢀ4642
Author Contributions
Design of the experiments, T.ꢀY. W, P.ꢀH. L.; synthesis of compounds, T.ꢀT. W.; animal experiments,
T.ꢀY. W., M.ꢀS. T., L.ꢀC. H., S.ꢀW. L.; HPLC analysis and PK analysis, T.ꢀY. W; wrote, read, and
approved the manuscript, T.ꢀY. W, M.ꢀS. T., L.ꢀC. H., S.ꢀW. L., P.ꢀH. L.
Notes
The authors declare no competing financial interest.
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Funding
This research was supported by Academia Sinica, Ministry of Science and Technology [MOST
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104ꢀ2320ꢀBꢀ002ꢀ008ꢀMY3; 106ꢀ2627ꢀMꢀ0020001ꢀ; 107ꢀ0210ꢀ01ꢀ19ꢀ01; MOST ] (to P. H L.),
[MOST 103ꢀ2321ꢀBꢀ002 ꢀ018 ꢀ] (to S. W. L.), and Ministry of Health and Welfare [DOH
102ꢀTDꢀPBꢀ111ꢀTM029; MOHW 104ꢀTDUꢀPBꢀ211ꢀ000010] (to S. W. L.).
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