Total synthesis and metabolic stability of hispidulin and its d-labelled derivative

Article abstract of DOI:10.3390/molecules22111897

Hispidulin is a naturally occurring flavone known to have various Central nervous system (CNS) activities. Proposed synthetic approaches to synthesizing hispidulin have proven unsatisfactory due to their low feasibility and poor overall yields. To solve these problems, this study developed a novel scheme for synthesizing hispidulin, which had an improved overall yield as well as more concise reaction steps compared to previous methods reported. Additionally, using the same synthetic strategy, d-labelled hispidulin was synthesized to investigate its metabolic stability against human liver microsome. This work may produce new chemical entities for enriching the library of hispidulin-derived compounds.

Full text of DOI:10.3390/molecules22111897

molecules
Article
Total Synthesis and Metabolic Stability of Hispidulin
and Its d-Labelled Derivative
Liang-Chieh Chen 1,2 ID , Kai-Cheng Hsu 3, Lih-Chu Chiou 4,5, Hui-Ju Tseng 1,6 and
Wei-Jan Huang 1,6,7,8,
*
1
Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan;
pigjay15@gmail.com (L.-C.C.); d343105007@tmu.edu.tw (H.-J.T.)
School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan
Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology,
Taipei Medical University, Taipei 110, Taiwan; piki@tmu.edu.tw
Graduate Institute of Brain and Mind Sciences, College of Medicine, National Taiwan University,
Taipei 110, Taiwan; lcchiou@ntu.edu.tw
Graduate Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei 100, Taiwan
Program in Biotechnology Research and Development, College of Pharmacy, Taipei Medical University,
Taipei 110, Taiwan
2
3
4
5
6
7
8
Program for the Clinical Drug Discovery from Botanical Herbs, Taipei 110, Taiwan
School of Pharmacy, National Defense Medical Center, Taipei 114, Taiwan
Correspondence: wjhuang@tmu.edu.tw; Tel.: +886-2-27361661 (ext. 6152)
*
Received: 18 October 2017; Accepted: 2 November 2017; Published: 4 November 2017
Abstract: Hispidulin is a naturally occurring flavone known to have various Central nervous
system (CNS) activities. Proposed synthetic approaches to synthesizing hispidulin have proven
unsatisfactory due to their low feasibility and poor overall yields. To solve these problems, this study
developed a novel scheme for synthesizing hispidulin, which had an improved overall yield as well
as more concise reaction steps compared to previous methods reported. Additionally, using the same
synthetic strategy, d-labelled hispidulin was synthesized to investigate its metabolic stability against
human liver microsome. This work may produce new chemical entities for enriching the library of
hispidulin-derived compounds.
Keywords: hispidulin; total synthesis; flavone; microsome stability; deuterium-labelled compound
1. Introduction
Flavonoids, a group of polyphenolic compounds, occur ubiquitously in plants. These compounds
have generally been categorized into several classes, including chalcones, flavonols, flavones,
isoflavones, flavan-3-ols, neoflavones, flavanones and anthocyanins, based on their chemical
structures [
antioxidative [
anticancer [7] activities. These facts indicate the important role of flavonoid compounds.
1
]. In addition to the diverse structures, they also have many biological properties, such as
2
], anti-inflammatory [ ], antimicrobial [ ], anticonvulsant [ ], antidepressant [ ] and
3
4
5
6
Molecules 2017, 22, 1897; doi:10.3390/molecules22111897
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1897
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Our recent clinical case report described a patient with a refractory chronic motor tic disorder
who dramatically improved after taking the leaf juice of a local herb, Clerodendrum inerme (L.) Gaertn
(CI) [
as a mouse model of motor tic [
model, we further isolated the constituents from CI leaf extract to identify a flavone hispidulin
(6-methoxy-40,5,7-trihydroxyflavone) to be the main active ingredient (Figure 1) [
]. Hispidulin
significantly attenuated MIH and even in amounts up to 100 mg/kg was incapable of affecting
spontaneous locomotor activity or performance in mice [ ]. Importantly, hispidulin had no hit on
8]. The ethanol extract of CI leaf alleviated methamphetamine-induced hyperlocomotion (MIH)
9]. Using a bioassay-guided purification procedure in this animal
9
9
human ether-à-go-go-related gene (hERG) channels, an undesirable target in drug development [10].
3'
OH
4'
2'
1
8
B
1'
HO
O
O
5'
7
2
A
C
O
6'
6
3
4
5
OH
Hispidulin
Figure 1. The chemical structure of hispidulin.
Hispidulin is widely distributed in Asteraceae [11
–14] and some of the Lamiaceae [15]. Further
assays for screening towards 92 target proteins associated with Central nervous system (CNS) diseases
indicated that hispidulin formed strong bonds with GABAA receptors (IC50 = 0.73
inhibited catecholamine-O-methyl-transferase (COMT) (IC50 1.32 M) [10]. By acting as a positive
allosteric modulator (PAM), it enhanced cerebellar α6GABAA receptor activity. Studies revealed
some C6-substituted flavones are identified to be PAMs of GABAA receptors [16 17]. The results of a
−
1.78 µM) and
µ
,
structure–activity relationship (SAR) study indicated the C6 substituent in flavones greatly contributes
to activity [10].
Because of its interesting biological activity, several research groups have developed synthetic
approaches to hispidulin [18–23]. For example, Shen and coworkers developed a strategy for
semisynthesis of hispidulin in seven reaction steps by using a naturally occurring scutellarin
(Scheme 1) [22]. Although this method is concise and has an overall yield as high as 10.7% [22],
the researchers showed that, upon the large-scale synthesis of compound
1
, the protection of the
catechol moiety of scutellarein using dichlorodiphenylmethane at 175 ◦C failed [20
,
23]. A seven-step
synthesis route developed by Lin and coworkers solved this problem but reduced the overall yield to
7.1% (Scheme 1) [20]. Zhang and coworkers then developed a scheme that only required four reaction
steps (Scheme 1). Nonselective MOM (methoxymethyl) protection of scutellarein caused the overall
yield of the synthesis of hispidulin to be decreased (6.3%) [23]. Despite the satisfactory overall yield of
these strategies, the tedious purification procedure required to isolate scutellarin from plants limits
their use for large-scale preparation of hispidulin.
Kavvadias and coworkers developed
a
method synthesizing hispidulin from
2,4,6-trihydroxyacetophenone compound in nine reaction steps (Scheme 2). However, the
6
overall yield of this method is very limited (1.1%) [18]. We recently developed a feasible and
reproducible approach for synthesizing hispidulin (Scheme 3) [21]. This method slightly improved the
overall yield due to the low yield of Friedel–Crafts acetylation of compound
8
as well as unsatisfactory
regioselective MOM protection of compound
and high-yield route to synthesize hispidulin.
9. These facts motivated us to investigate an efficient

Molecules 2017, 22, 1897
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OH
OH
O
O
HO
HO
O
O
O
OH
HO
OH
Scutellarin
OH
OBn
HO
HO
O
O
O
O
O
Ph
Ph
Shen and coworkers
OH
O
OH
Zhang and coworkers
Scutellarein
1
OMOM
Lin and coworkers
MOMO
HO
O
OAc
OBn
OH
O
4
BnO
AcO
O
BnO
O
O
O
OAc O
OH
3
2
OMOM
MOMO
O
O
O
OH
OH
HO
O
O
5
OH
O
Hispidulin
Scheme 1. Synthesis of hispidulin starting from scutellarin.
OH
HO
OH
Nine steps
HO
O
O
O
OH
O
OH
6
Hispidulin
Scheme 2. Synthesis of hispidulin by Kavvadias and coworkers.

Molecules 2017, 22, 1897
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HO
O
OH
O
HO
O
OH
HO
OH
MOMO
O
OR2
O
O
OH
OH
OH
OR1
9
8
7
10a: R1 = MOM, R2 = H
10b: R1 = H, R2 = MOM
OH
HO
O
O
O
OH
Hispidulin
Scheme 3. Synthesis of hispidulin in our previous study.
Deuterium is a stable isotope of hydrogen. Because deuterium has a stronger chemical bond
with carbon than hydrogen, deuterium-labelled compounds can affect the absorption, distribution,
metabolism and toxicology of their counterpart compound [24,25]. The Food and Drug Administration
(FDA) recently approved the first deuterated drug, deutetrabenazine, for treating involuntary writhing
movements or chorea in Huntington’s disease [26]. Deuterium incorporation of tetrabenazine markedly
increased its half-life and area under the curve (AUC) in plasma [27]. Study showed that the pig
caecal microflora metabolized hispidulin to scutellarein due to O-demethylation at the C6 position [28].
This result suggested that replacement of OCH3 of this position of hispidulin using OCD3 may
reduce its metabolic liability. Therefore, this study replaced C6-OCH3 in hispidulin with C6-OD3
to investigate whether such modification allows enhancement of the metabolic stability. Notably,
introducing deuterium into hispidulin leads to a new chemical entity (NCE) that meets the criteria for
a 505(b) (2) patent application [27].
The new hispidulin synthesis scheme developed in this study is more feasible compared to
all methods previously reported. In particular, it had the highest overall yield, which may help
to synthesize C6-OCH3-containing hispidulin derivatives. The same strategy can also be used to
synthesize d-labelled hispidulin. The microsome stability of hispidulin and its deuterium counterpart
were compared.
2. Results and Discussion
2.1. Retrosynthetic Analysis of Hispidulin
Figure 2 shows the retrosynthetic analysis to successfully synthesize hispidulin. Hispidulin
can be produced from compound
I via debenzylation and oxidative cyclization. Compound I is
derived from compound II and commercially available compound III, which are used to conduct
Claisen–Schmidt condensation and deprotection of MOM (methoxymethoxy). Compound II in turn
can be prepared from compound IV via methylation. Compound IV is considered to be the key
intermediate that possesses two different protecting groups as well as acetyl and hydroxy moieties.
Selective Bayer–Villiger reaction of compound
V provided compound IV. Compound V is prepared
from compound VI via Stille coupling. Benzylation and regioselective iodination of compound VII
gives compound VI. Starting from commercially available compound 7, Compound VII is synthesized
via selective MOM protection.

Molecules 2017, 22, 1897
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OH
HO
O
OH
O
OH
PG2O
O
OPG2
OH
HO
O
O
O
+
Oxidative
PG1O
PG1O
O
O
OH
cyclization,
Claisen-Schmidt
condensation,
deprotection
I
III
II
debenzylation
Hispidulin
Methylation
OPG2
PG2O
OPG2
PG2O
O
OPG2
O
PG2O
O
I
HO
PG1O
Stille coupling
Selective
Baeyer-Villiger
oxidation
PG1O
PG1O
O
VI
V
IV
Key intermediate
Regioselective iodination,
benzylation
5
PG2O
O
OPG2
HO
OH
6
1
4
O
3
2
HO
OH
Selective
protection
VII
7
Figure 2. Retrosynthetic analysis of hispidulin.
2.2. Synthesis of Hispidulin
Scheme 4 shows a synthetic approach originally developed for hispidulin. Using compound
as the starting material, selective MOM protection towards its C4- and C6-OH gave compound 11
6
.
First, N-bromosuccinimide (NBS) was used for bromination of C3 of compound 11; however, only
undesirable C3 and C5 dibromination occurred. Alternatively, compound 11, when reacted with I2 in
the presence of Lewis acid CF3CO2Ag, provided selectively iodinated product 12 in the high yield
(90%). The experimental result resulted from the difference of steric hindrance between C3 and C5 upon
using the more bulky reagent I2. Benzylation of compound 12 generated compound 13. Next, several
reported oxidation methods were used [29–31] in attempts to convert the iodide group of compound 13
into phenol. These oxidation catalysts included CuI, Cu and Pd, as shown in Table 1. Reaction using
Pd2dba3 coupled with the ligand 2-di-tert-butylphosphino-20,40,60-triisopropylbiphenyl (tBuXPhos)
gave compound 14, but its yield was poor (27%). Despite the use of microwave irradiation, the yields
were incapable of being improved. Instead, Claisen–Schmidt condensation of compound 13, reacted
with 4-(benzyloxy)benzaldehyde prior to selective deprotection of MOM group in the presence of HCl,
gave chalcone 15. The selective deprotection reaction can be manipulated by decreasing the reaction
time to avoid further MOM deprotection of C4. Oxidative cyclization of compound 15 in the presence of
catalytic I2 provided flavone 16. Notably, excess I2 caused an undesirable retro-aldol reaction that gave
C4- and C6-deprotected compound 13. Treatment of catalytic Pd(PPh3)4 and tributyl(1-ethoxyvinyl)tin
converted compound 16 to compound 17 through the Stille reaction. Baeyer–Villiger oxidation followed
by basic hydrolysis of compound 17 by using H2O2 or m-chloroperoxybenzoic acid (MCPBA) [32
]
failed to give expected product 18.

Molecules 2017, 22, 1897
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Table 1. Attempts towards the oxidation of aryl halide 13.
◦
Entry
Catalyst
Ligand
Base
Solvent
T ( C)
Yield (%)
1
2
3
4
5
6
7
8
CuI
Cu
Cu
Pd2dba3
Pd2dba3
Pd2dba3
Pd2dba3
Pd2dba3
1
1,10-Phenanthroline
-
KOH
NaOH
NaOH
KOH
KOH
KOH
KOH
KOH
DMSO–H2O
H2O
100
100
100
trace
trace
trace
27
14
26
-
DMSO–H2O
Dioxane–H2O
Dioxane–H2O
Dioxane–H2O
Dioxane–H2O
DMF
tBuXPhos
tBuXPhos
tBuXPhos
tBuXPhos
tBuXPhos
100
70
100 1
100 2
100 1
23
trace
Microwave reaction: 100 W, 20 min; 2 microwave reaction: 120 W, 20 min.
5
HO
OH
O
MOMO
OMOM
6
MOMO
I
OMOM
O
a
b
c
4
3
1
3
2
OH
OH
O
OH
6
11
12
OH
MOMO
I
OMOM
MOMO
HO
OMOM
HO
O
O
d
27%
O
OBn O
13
OBn O
14
OH
Hispidulin
e
OBn
5
MOMO
I
OH
6
OBn
MOMO
O
O
4
3
1
2
OBn O
OBn O
15
19
f
OBn
OBn
OBn
g
MOMO
O
O
MOMO
HO
O
MOMO
I
O
OBn O
OBn O
OBn O
17
18
16
Scheme 4. Initial attempt for the synthesis of hispidulin. Reagents and conditions: (
a
) MOMCl, DIPEA,
) K2CO3, BnBr, DMF, 0 ◦C, 99%;
) Pd2dba3, tBuXPhos, KOH, dioxane–H2O, 90 ◦C, 27%; (◦
) (1) BnOPhCHO, KOH, EtOH, H2O,
f) I2, DMSO, 120 C, 32%; (g) tributyl(1-ethoxyvinyl)tin,
CH2Cl2, 0 ◦C, 64%; ( ) CF3COOAg, I2, CH2Cl2, 0 ◦C, 90%; (
b c
(
d
e
◦
◦
0 C; (2) c-HCl, MeOH,◦THF, 0 C, 77%; (
Pd(PPh3)4, dioxane, 100 C, 61%.
Thus, we developed a new synthesis (Scheme 5) for hispidulin using the retrosynthetic analysis
as shown in Figure 2. First, 2,4,6-trihydroxybenzaldehyde reacted with MOMCl gave the
7
bis(methoxymethoxy)-protected compound 20. Regioselective iodination of compound 20 prior to
reaction with BnBr produced compound 21. The chemical structure of compound 21 was confirmed
by rotating frame nuclear Overhauser effect spectroscopy (ROESY) spectrum. Figure03000shows the key
000
0000
000
δH 3.54), H-1 (δH 5.29) and H-1
correlation of H-5 (δH 6.84) to H-2
(δH 3.52), H-2
(
(δH 5.32); and

Molecules 2017, 22, 1897
7 of 14
0
0000
00
0000
H-1 (δH 10.30) to H-2
(
δH 3.54), H-1 (δH 4.99) and H-1 (δH 5.32) (Supplementary Materials). Stille
coupling of compound 21 produced compound 22. Table 2 shows how tributyl(1-ethoxyvinyl)tin was
used to optimize Stille coupling. First, catalyst Pd(PPh3)4 was used in dioxane at 100 ◦C. The reaction
had a satisfactory yield (68%), but the reaction time was up to 30 h. Replacement of the solvent by
toluene led to the reaction time decreasing to 24 h. Further experiments using palladium catalysts
such as PdCl2(PPh3)2 and Pd(dppf)Cl2 in dioxane or toluene showed that PdCl2(PPh3)2 significantly
improved the yield and decreased the reaction time. In particular, PdCl2(PPh3)2 coupled with toluene
not only gave the highest yield, but also had the lowest reaction time. Baeyer–Villiger oxidation and
basic hydrolysis of compound 22 afforded compound 14. Methylation of compound 14 using CH3I gave
compound 23a. Claisen–Schmidt condensation of compound 23a with 4-(benzyloxy)benzaldehyde
prior to MOM deprotection produced chalcone 24a. Cyclization of compound 24a in the presence
of catalytic I2 generated flavone 25a. Although debenzylatio◦n of compound 25a catalyzed by 10%
Pd–C did not yield hispidulin, a reaction using BCl3 at
−
80 C successfully converted compound
25a into hispidulin. The chemical structure of hispidulin was identified as judged by 2D-NMR
analyses. Figure 4 shows the key correlation in the ROESY spectrum of hispidulin that 5-OH (δH 13.07)
(
was correlated to 6-OMe000 δH 3.74) and H-8 (δH 6.59) was correlated to H-20 and H-60 (δH 7.92).
Additionally, the HMBC spectrum showed that 5-OH (δH 13.07) correlated to C-5 (δC 152.8), C-6
δC 131.4), C-7 (δC 157.3), C-9 (δC 152.4) and C-10 (δC 104.1); H-8 (δH 13.07) correlated to C-6 (δC 131.4),
(
C-7 (δC 157.3), C-9 (δC 152.4) and C-10 (δC 104.1); 6-OMe-H (δH 3.74) correlated to C-6 (δC 131.4)
(Supplementary Materials). The 1H- and 13C-NMR data of synthesized hispidulin were similar to
those of hispidulin previously isolated (Supplementary Materials) [33].
5
HO
OH
4
MOMO
O
OMOM
MOMO
O
OMOM
I
MOMO
O
OMOM
6
1
a
b
c
O
3
2
OH
OH
OBn
OBn O
22
7
21
20
MOMO
HO
OMOM
MOMO
OMOM
HO
OH
OBn
d
e
f
R
R
O
O
OBn O
OBn O
OBn O
14
23a: R = CH3
23b: R = CD3
24a: R = CH3
24b: R = CD3
OBn
OH
HO
O
HO
O
g
h
R
O
R
O
OBn O
OH
O
25a: R = CH3
25b: R = CD3
Hispidulin: R = CH3
d-Hispidulin: R = CD3
Scheme 5. Synthesis of hispidulin and d-hispidulin. Reagents and conditions: (
CH2Cl2, 0 ◦C, 80%; ( ) (1) CF3COOAg, I2, CH2Cl2, 0 ◦C; (2) K2CO3, BnBr, DMF, 0 ◦C, 89%;
) tributyl(1-ethoxyvinyl)tin, PdCl2(PPh3)2, toluene, 100 ◦C, 83%; ( ) (1) MCPBA, CH2Cl2, 0 ◦C;
23a: CH3I, K2CO3, acetone, 56 C, 92%; 23b: CD3I, K2CO3, acetone,
) (1) BnOPhCHO, KOH, EtOH, H2O, 0 ◦C; (2) c-HCl, MeOH, THF, 0 ◦C; 24a: 92%, 24b
) I2, DMSO, 120 ◦C, 25a: 93%, 25b: 79%; ( 80 ◦C, hispidulin:
) 1M BCl3 in hexane; CH2Cl2,
85%, d-hispidulin: 80%.
a) MOMCl, DIPEA,
b
(
c
d
◦
(2) 10% NaOH(aq), MeOH, 68%; (
56 ◦C, 93%; (
81%; (
e)
f
:
g
h
−

Molecules 2017, 22, 1897
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Figure 3. Key rotating frame nuclear Overhauser effect spectroscopy (ROESY) correlations of
compound 21.
Table 2. Optimization of reaction condition for Stille coupling of compound 21.
Entry
Catalyst
Solvent
Yield (%)
Reaction Time (h)
1
2
3
4
5
6
Pd(PPh3)4
Pd(PPh3)4
PdCl2(PPh3)2
PdCl2(PPh3)2
Pd(dppf)Cl2
Pd(dppf)Cl2
Dioxane
Toluene
Dioxane
Toluene
Dioxane
Toluene
68
70
73
83
34
53
30
24
20
10
78
43
Figure 4. Key ROESY and HMBC correlations of hispidulin.
2.3. Synthesis of d-Hispidulin
The synthesis of d-hispidulin is described in Scheme 5. Methylation of compound 14 using CD3I
gave compound 23b. The same method to hispidulin was used to synthesize d-labelled hispidulin
starting from compound 23b. Due to the absence of a proton signal of the CD3O group in the 1H-NMR
spectra of the d-containing intermediate compounds 23b, 24b, 25b and d-hispidulin, these compound
structures were identified depending on the 13C-NMR spectra without 1H decoupling and the mass
technique. The 13C-NMR spectra revealed a characteristic multiplet splitting pattern of the 13C signal
for the CD3O group in compounds 23b, 24b, 25b and d-hispidulin. The mass spectra also supported
chemical structures of these d-labelled compounds. All synthesized compounds had an estimated
purity of at least 98% as determined by HPLC analysis (Supplementary Materials).
2.4. Comparison of Hispidulin Synthesis Methods
Strategies for synthesizing hispidulin can be classified as semisynthesis and total synthesis
strategies. The starting material used in most semisynthetic methods is scutellarin, which is a natural
product. Table 3 shows that the semisynthesis routes had fewer reaction steps compared to the total
synthesis methods; however, they need tedious isolation procedures for scutellarin, which limits the
scale for further chemical modification. Furthermore, their overall yields are only 6.3–10.7% [20,22,23].
For total synthesis, Kavvadias and coworkers developed a nine-step synthesis approach. The starting
material used in this method is commercially available 2,4,6-trihydroxyacetophenone. Although this
method solves the issue of the source for starting material, its drawback is low overall yield [18].
We previously developed a feasible route of hispidulin synthesis that has an overall yield comparable

Molecules 2017, 22, 1897
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to that of the method developed by Kavvadias and coworkers [21]. This present study further made
the reaction steps more concise. In particular, the synthetic scheme showed the highest overall yield of
all approaches to synthesize hispidulin.
Table 3. Comparison of hispidulin synthesis methods.
Research Group
Synthesis Route
Reaction Steps
Overall Yield (%)
Shen and coworkers
Lin and coworkers
Zhang and coworkers
Kavvadias and coworkers
Chao and coworkers
This study
Semisynthesis
Semisynthesis
Semisynthesis
Total synthesis
Total synthesis
Total synthesis
Seven
Seven
Four
Nine
Ten
10.7
7.1
6.3
1.1
1.6
Eight
26.9
2.5. Human Liver Microsome Stability
Metabolic stability is associated with susceptibility of compounds to biotransformation. Metabolic
half-life (t1/2) and intrinsic clearance (CLint) was compared between hispidulin and d-hispidulin by
testing these synthesized compounds in a human liver microsome stability assay. The study revealed
that FDA-approved deuterated agent deutetrabenazine had a t1/2 (8.6 h) superior to tetrabenazine
(4.8 h). In addition to t1/2, the AUC of deutetrabenazine (542 ng
·
hr/mg) was also higher than that of its
counterpart compound (261 ng hr/mg) [27]. In contrast, the experimental results indicated hispidulin
·
and d-hispidulin had no significant difference in t1/2 and CLint (Table 4), which suggested that the
C6-OMe of hispidulin is resistant to be modified by the human liver microsome. The metabolic site of
hispidulin in the human liver microsome is worthy of further study.
Table 4. Human liver microsome stability of hispidulin and d-hispidulin.
1
Compound
Half-Life (min)
CLint (mL/min/mg Protein)
Hispidulin
d-Hispidulin
Testosterone
46
43
19
0.0298
0.0325
0.0727
1
Intrinsic clearance (CLint) was calculated based on CLint = k/P, where k is the elimination rate constant and P is
the protein concentration in the incubation.
The experiments showed that this method of synthesizing hispidulin and its d-labelled derivative
is highly feasible. Specifically, it increases overall yield compared to previous methods. The t1/2 and
intrinsic clearance of these two compounds were identified as well. Overall, this synthetic route can be
applied to produce 6-OMe-containing hispidulin derivatives as new chemical entities for investigating
their biological activities.
3. Experimental Section
3.1. General Information
The NMR spectra (1H- and 13C-NMR, ROESY, HSQC and HMBC) were obtained with a Bruker
AV500 using standard pulse programs. The MS data were recorded with a Finnigan Mat TSQ-7000
mass spectrometer (HR–ESI–MS) (Thermo, Ringoes, NJ, USA). The HPLC was performed on a C18
column (150 mm
×
4.6 mm, Ascentis) by using an L-2130 pump (Hitachi, Ibaraki, Japan) and a
UV/vis L-2420 detector (Hitachi, Ibaraki, Japan). The column chromatography was performed on
silica gel (70-230 mesh, Merck, Darmstadt, Germany). All TLC analyses were performed on silica gel
plates (KG60-F254, Merck, Darmstadt, Germany). Reagents and materials were used without further
purification and chemicals were purchased from ACROS (Geel, Belgium). Dry dichloromethane was
distilled from CaH2 under nitrogen atmosphere. MOMCl was acquired from TCI (Tokyo, Japan), and
2,4,6-trihydroxybenzaldehyde was purchased from Alfa Aesar (Heydham, UK).

Molecules 2017, 22, 1897
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3.2. Chemistry
2-Hydroxy-4,6-bis(methoxymethoxy)benzaldehyde (20). To a solution of compound
7
(10 g, 64.9 mmol)
in CH2Cl2 (200 mL) was added DIPEA (28.3 mL, 162.2 mmol). The resulting mixture was stirred
for 10 mins in an ice-bath under N2. MOMCl (10.8 mL, 142.7 mmol) was added dropwise to the
reaction mixture by addition funnel. The reaction mixture was warmed to room temperature (rt) and
stirred for 3 h. The mixture was concentrated in vacuo. The residue was diluted with EtOAc (100 mL)
and washed with distilled H2O (3
×
80 mL). The organic layer was dried over Na2SO4, filtered and
removed in vacuo. The residue was purified by silica gel chromatography (EtOAc:n-hexane = 1:9) to
give compound 20 (12.3 g, 80%), a light-yellow microcrystalline powder; 1H-NMR (CDCl3, 300 MHz)
δ
12.29 (1H, s), 10.16 (1H, s), 6.25 (1H, d, J = 2.1 Hz), 6.23 (1H, d, J = 2.1 Hz), 5.24 (2H, s), 5.18 (2H, s),
3.51 (3H, s), 3.47 (3H, s); 13C-NMR (CDCl3, 125 MHz)
94.0, 56.6, 56.5; HR–ESI–MS m/z 243.0858 [M + H]+ (calcd. for C11H15O6, 243.0863).
δ 192.1, 165.6, 165.5, 161.2, 106.9, 96.6, 94.6, 94.1,
2-Benzyloxy-3-iodo-4,6-bis(methoxymethoxy)benzaldehyde (21). To a mixture ◦of compound 20 (7.2 g,
29.6 mmol) and CF3CO2Ag (7.8 g, 35.5 mmol) in CH2Cl2 (150 mL) at 0 C was added I2 (8.3 g,
32.5 mmol) in CH2Cl2 (200 mL) dropwise by an addition funnel over 2 h. The reaction mixture was
warmed to rt and stirred for 3 h. Then, saturated Na2S2O3(aq) (50 mL) was added and the mixture
was washed with distilled H2O (3
removed in vacuo. The residue was dissolved in DMF (100 mL) and then K2CO3 (7.2 g, 51.8 mmol) was
×
100 mL). The organic layer was dried over Na2SO4, filtered and
added. Benzyl bromide (460
0 C. The resulting solution was warmed to rt and stirred for 3 h. The mixture was diluted with EtOAc
µ
L, 3.9 mmol) was added dropwise to the mixture by an addition funnel at
◦
(300 mL) and washed with distilled H2O (3
and removed in vacuo. The residue was purified by silica gel chromatography (EtOAc:n-hexane = 1:5)
to give compound 21 (12.1 g, 89%), a white microcrystalline powder; 1H-NMR (CDCl3, 300 MHz)
10.30 (1H, s), 7.66 (2H, dd, J = 1.8, 8.1 Hz), 7.45–7.36 (3H, m), 6.84 (1H, s), 5.32 (2H, s), 5.29 (2H, s),
×
00 mL). The organic layer was dried over Na2SO4, filtered
δ
4.99 (2H, s), 3.54 (3H, s), 3.52 (3H, s); 13C-NMR (CDCl3, 125 MHz)
δ 187.2, 161.9, 161.8, 161.6, 136.1,
129.0, 128.5, 128.4, 115.5, 98.2, 95.2, 94.9, 78.5, 56.7; HR–ESI–MS m/z 459.0294 [M + H]+ (calcd. for
C18H20O6I, 459.0299).
3-Acetyl-2-benzyloxy-4,6-bis(methoxymethoxy)benzaldehyde (22). To a mixture of compound 21 (5 g,
10.9 mmol) and PdCl2(PPh3)2 (766 mg, 1.1 mmol) in toluene (200 mL) was added tributyl(1-ethoxyvinyl)tin
(5.5 mL, 16.4 mmol). The resulting solution was heated to 100 ◦C and stirred for 12 h. After cooling
to rt, the reaction mixture was acidified with 1 M HCl (50 mL) and stirred for 30 min. The mixture
was diluted with EtOAc (200 mL) and washed with distilled H2O (3
was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel
×
100 mL). The organic layer
chromatography (EtOAc:n-hexane = 1:4) to give compound 22 (3.5 g, 83%), a yellow microcrystalline
powder; 1H-NMR (CDCl3, 300 MHz)
δ
10.37 (1H, s), 7.48 (2H, dd, J = 1.8, 7.8 Hz), 7.41–7.33 (3H, m),
6.80 (1H, s), 5.29 (2H, s), 5.24 (2H, s), 4.96 (2H, s), 3.53 (3H, s), 3.49 (3H, s), 2.43 (3H, s); 13C-NMR
(CDCl3, 125 MHz)
δ 200.9, 187.3, 162.1, 159.3, 158.4, 136.1, 129.0, 128.8, 128.6, 128.5, 128.4, 121.8, 114.3,
97.5, 95.0, 94.5, 79.1, 56.8, 32.5; HR–ESI–MS m/z 375.1432 [M + H]+ (calcd. for C20H23O7, 375.1438).
2-Benzyloxy-3-hydroxy-4,6-bis(methoxymeth◦oxy)acetophenone (14). To a solution of 70% MCPBA (6.8 g,
27.6 mmol) in dry CH2Cl2 (100 mL) at 0 C was added compound 22 (3.4g, 9.2 mmol) in CH2Cl2
(100 mL) dropwise by an addition funnel over 1 h. The resulting solution was warmed to rt and
stirred for 8 h. Then, saturated Na2S2O3(aq) (30 mL) was added and the reaction mixture was washed
with distilled H2O (3
×
100 mL). The organic layer was dried over Na2SO4, filtered and concentrated
in vacuo. The residue was dissolved in MeOH (80 mL) and 10% NaOH(aq) (60 mL) was added.
The resulting solution was stirred at rt for 1.5 h. The mixture was diluted with EtOAc (250 mL) and
washed with distilled H2O (3
concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc:n-hexane = 1:4)
to give compound 14 (2.3 g, 68%), a light-yellow oil; 1H-NMR (CDCl3, 300 MHz)
7.44 (2H, dd, J = 1.8,
×
100 mL). The organic layer was dried over Na2SO4, filtered and
δ

Molecules 2017, 22, 1897
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8.1 Hz), 7.40–7.32 (3H, m), 6.76 (1H, s), 5.61 (1H, s), 5.21 (2H, s), 5.08, (2H, s), 5.06 (2H, s), 3.53 (3H, s),
3.46 (3H, s), 2.45 (3H, s); 13C-NMR (CDCl3, 125 MHz)
δ 201.3, 146.6, 146.2, 143.2, 136.9, 134.9, 128.5,
128.3, 122.1, 100.4, 96.0, 95.8, 76.3, 56.6, 56.3, 32.6; HR–ESI–MS m/z 363.1431 [M + H]+ (calcd. for
C19H23O7, 363.1438).
2-Benzyloxy-3-methoxy-4,6-bis(methoxymethoxy)acetophenone (23a). To a mixture of compound 14 (587 mg,
1.6 mmol) and K2CO3 (1.1 g, 8.1 mmol) in acetone (20 mL) was added CH3I (0.5 mL, 8.1 mmol).
The resulting solution was heated to 56 ◦C and stirred for 5 h. The reaction mixture was concentrated
in vacuo, diluted with EtOAc (100 mL) and washed with distilled H2O (3
was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel
×
50 mL). The organic layer
chromatography (EtOAc:n-hexane = 1:5) to give compound 23a (558 mg, 92%), a white microcrystalline
powder; 1H-NMR (CDCl3, 300 MHz)
δ
7.42 (2H, dd, J = 2.0, 8.3 Hz), 7.40–7.32 (3H, m), 6.76 (1H, s), 5.23
(2H, s), 5.11 (2H, s), 5.07 (2H, s), 3.85 (3H, s), 3.53 (3H, s), 3.46 (3H, s), 2.38 (3H, s); 13C-NMR (CDCl3,
125 MHz) δ 200.6, 151.8, 149.4, 149.2, 137.9, 136.7, 128.1, 128.0, 127.7, 121.4, 99.6, 95.0, 94.9, 76.0, 60.8,
56.0, 55.9, 32.1; HR–ESI–MS m/z 377.1587 [M + H]+ (calcd. for C20H25O7, 377.1595).
2-Benzyloxy-3-[2H3]-methoxy-4,6-bis(methoxymethoxy)acetophenone (23b). Following the procedure as
described for compound 23a, reaction of compound 14 (1.8 g, 4.8 mmol), K2CO3 (3.3 g, 24.2 mmol),
CD3I (1.5 mL, 24.2 mmol) in acetone (40 mL) gave compound 23b (1.7 g, 93%), a white microcrystalline
powder; 1H-NMR (CDCl3, 300 MHz)
δ
7.42 (2H, dd, J = 2.1, 8.4 Hz), 7.39–7.31 (3H, m), 6.76 (1H, s),
5.23 (2H, s), 5.11 (2H, s), 5.07 (2H, s), 3.53 (3H, s), 3.46 (3H, s), 2.38 (3H, s); 13C-NMR (CDCl3, 125 MHz)
δ
200.6, 151.8, 149.4, 149.2, 137.8, 136.7, 128.1, 128.0, 127.7, 121.4, 99.6, 95.0, 94.9, 76.0, 56.0, 55.9, 32.1;
HR–ESI–MS m/z 380.1775 [M + H]+ (calcd. for C20H22D3O7, 380.1783).
(E)-1-(2-Benzyloxy-4,6-dihydroxy-3-methoxyphenyl)-3-(4-benzyloxyphenyl)prop-2-en-1-one (24a). To a
solution of 23a (545 mg, 1.45 mmol) and 4-benzyloxybenzaldyde (615 mg, 2.9 mmol) in EtOH (20 mL)
◦
was added KOH (813 mg, 14.5 mmol) in EtOH–H2O (3 mL:3 mL) dropwise by addition funnel at 0 C
over 30 min. The resulting solution was warmed to rt and stirred for 24 h. The mixture was diluted
with distilled H2O (50 mL) and washed with EtOAc (3
×
50 mL). The organic layer was dried over
Na2SO4, filtered and concentrated in vacuo. MeOH–THF (14.5 mL:14.5 mL) and 12 M HCl (0.7 mL) was
◦
added to the residue at 0 C. The resulting solution was warmed to rt and stirred for 8 h. The reaction
mixture was diluted with distilled H2O (50 mL) and washed with EtOAc (3
layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica
×
50 mL). The organic
gel chromatography (EtOAc:n-hexane = 1:4) to give 24a (641 mg, 92%), a microcrystalline powder;
1H-NMR (DMSO-d6, 300 MHz)
δ
12.92 (1H, s), 10.52 (1H, s), 7.59 (2H, s), 7.48–7.45 (2H, m), 7.44–7.37
(5H, m), 7.32–7.29 (5H, m), 6.94 (2H, d, J = 8.8 Hz), 6.21 (1H, s), 5.16 (2H, s), 5.06 (2H, s), 3.77 (3H, s);
13C-NMR (DMSO-d6, 125 MHz)
192.0, 160.2, 159.6, 157.7, 153.1, 143.0, 136.7, 136.6, 134.4, 130.3, 128.5,
δ
128.4, 128.3, 128.2, 128.0, 127.8, 127.4, 124.6, 115.2, 109.0, 99.7, 75.7, 69.4, 60.7; HR–ESI–MS m/z 483.1795
[M + H]+ (calcd. for C30H27O6, 483.1802).
(E)-1-(2-Benzyloxy-4,6-dihydroxy-3-[2H3]methoxyphenyl)-3-(4-benzyloxyphenyl)prop-2-en-1-one
(24b).
According to the procedure as described for compound 24a, reaction of compound 23b (1.0 g,
2.6 mmol), 4-benzyloxybenzaldyde (1.1 g, 5.3 mmol) and KOH (1.5 g, 26.4 mmol) in EtOH (60 mL)
followed by treatment of 12 M HCl (1.3 mL) and MeOH–THF (26.4 mL:26.4 mL) gave compound 24b
(1.0 g, 81%), a yellow microcrystalline powder; 1H-NMR (DMSO-d6, 300 MHz)
δ
12.95 (1H, s), 10.54
(1H, s), 7.59 (2H, s), 7.48–7.45 (2H, m), 7.44–7.36 (5H, m), 7.33–7.29 (5H, m), 6.94 (2H, d, J = 8.8 Hz), 6.22
(1H, s), 5.16 (2H, s), 5.06 (2H, s); 13C-NMR (DMSO-d6, 125 MHz)
192.0, 160.2, 159.7, 157.7, 153.2,
δ
143.0, 136.7, 136.6, 134.4, 130.3, 128.5, 128.4, 128.3, 128.2, 128.0, 127.8, 127.4, 124.6, 115.2, 109.0, 99.7, 75.7,
69.4; HR–ESI–MS m/z 486.1983 [M + H]+ (calcd. for C30H24D3O6, 486.1990).
40-Benzyloxy-6-methoxy-5-benzyloxy-7-hydroxyflavone (25a). To a solution of compound 24a (598 mg,
1.2 mmol) in dry DMSO (100 mL) was added I2 (32 mg, 0.1 mmol) in DMSO (3 mL) dropwise by
syringe. The resulting solution was heated to 120 ◦C, and stirred for 2 h. After cooling to rt, saturated

Molecules 2017, 22, 1897
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Na2S2O3(aq) (10 mL) was added to the reaction mixture. The mixture was diluted with EtOAc (100 mL)
and wash with distilled H2O (3 50 mL). The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc:n-hexane = 1:3)
to give 25a (555 mg, 93%), a yellow microcrystalline powder; 1H-NMR (DMSO-d6, 300 MHz)
10.76
×
δ
(1H, s), 7.98 (2H, d, J = 8.9 Hz), 7.61 (2H, d, J = 7.0 Hz), 7.48 (2H, d, J = 7.0 Hz), 7.44–7.34 (6H, m),
7.18 (2H, d, J = 8.9 Hz), 6.91 (1H, s), 6.67 (1H, s), 5.22 (2H, s), 5.00 (2H, s), 3.75 (3H, s); 13C-NMR
(DMSO-d6, 125 MHz)
δ 175.8, 160.9, 160.1, 156.1, 153.8, 150.7, 139.6, 137.6, 136.6, 128.5, 128.3, 128.1,
128.0, 127.8, 123.4, 115.3, 111.4, 106.0, 100.1, 75.6, 69.5, 60.9; HR–ESI–MS m/z 481.1637 [M + H]+ (calcd.
for C30H25O6, 481.1646).
40-Benzyloxy-6-[2H3]methoxy-5-benzyloxy-7-hydroxyflavone (25b). Following the procedure as described
for compound 25a, reaction of compound 24b (849 mg, 1.7 mmol) with I2 (44 mg, 0.2 mmol) in DMSO
(120 mL) gave compound 25b (664 mg, 79%), a yellow microcrystalline powder; 1H-NMR (DMSO-d6,
300 MHz)
δ 10.75 (1H, s), 7.98 (2H, d, J = 8.9 Hz), 7.61 (2H, d, J = 6.8 Hz), 7.48 (2H, dd, J = 1.8, 8.4 Hz),
7.44–7.34 (6H, m), 7.18 (2H, d, J = 8.9 Hz), 6.91 (1H, s), 6.67 (1H, s), 5.22 (2H, s), 5.01 (2H, s); 13C-NMR
(DMSO-d6, 125 MHz)
δ 175.8, 160.9, 160.1, 156.1, 153.7, 150.7, 139.5, 137.6, 136.6, 128.5, 128.3, 128.1,
128.0, 127.8, 123.4, 115.3, 111.4, 106.0, 100.1, 75.6, 69.5; HR–ESI–MS m/z 484.1827 [M + H]+ (calcd. for
C30H22D3O6, 484.1834).
Hispidulin. To a solution of compound 25a (301 mg, 0.6 mmol) in dry CH2Cl2 (40 ◦mL) was added
1 M BCl3 (2.5 mL, 2.5 mmol) in dry CH2Cl2 (5 mL) dropwise by syringe at
−
78 C over 20 min.
The resulting solution was stirred for 1 h. The reaction mixture was diluted with distilled H2O
(50 mL) and washed with EtOAc (3 50 mL). The organic layer was dried over Na2SO4, filtered and
×
concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc:n-hexane = 1:2)
to give hispidulin (160 mg, 85%), a yellow microcrystalline powder; 1H-NMR (DMSO-d6, 500 MHz)
δ
13.07 (1H, s, 5-OH), 10.73 (1H, s, 7-OH), 10.38 (1H, s, 40-OH), 7.92 (2H, d, J = 8.9 Hz, H-20, H-60),
6.92 (2H, d, J = 8.9 Hz, H-30, H-50), 6.77 (1H, s, H-3), 6.59 (1H, s, H-8), 3.74 (3H, s, 6-OMe); 13C-NMR
(DMSO-d6, 125 MHz)0 δ 182.2 (C-4), 163.9 (C-2), 161.2 (C-40), 157.3 (C-7), 152.8 (C-5), 152.4 (C-9), 131.4
0
0
(C-6), 128.5 (C-2’, C-6 ), 121.2 (C-1), 116.0 (C-3 , C-5 ), 104.1 (C-10), 102.4 (C-3), 94.3 (C-8), 60.0 (6-OMe);
HR–ESI–MS m/z 301.0702 [M + H]+ (calcd. for C16H13O6, 301.0707).
d-Hispidulin. Following the procedure as described for hispidulin, reaction of compound 25b (536 mg,
1.1 mmol) in CH2Cl2 (75 mL) with 1 M BCl3 (4.4 mL, 4.4 mmol) in CH2Cl2 (8.8 mL) gave d-hispidulin
(268 mg, 80%), a yellow microcrystalline powder; 1H-NMR (DMSO-d6, 500 MHz)
δ
13.07 (1H, s, 5-OH),
10.70 (1H, s, 7-OH), 10.36 (1H, s, 40-OH), 7.91 (2H, d, J = 8.9 Hz, H-20, H-60), 6.92 (2H, d, J = 8.9 Hz,
H-30, H-50), 6.76 (1H, s, H-3), 6.58 (1H, s, H-8); 13C-NMR (DMSO-d6, 125 MHz)
δ 182.1 (C-4), 163.8
(C-2), 161.2 (C-4’), 157.2 (C-7), 152.8 (C-5), 152.4 (C-9), 131.3 (C-6), 128.5 (C-20, C-60), 121.2 (C-1),
116.0 (C-30, C-50), 104.1 (C-10), 102.4 (C-3), 94.2 (C-8); HR–ESI–MS m/z 304.0888 [M + H]+ (calcd. for
C16H10D3O6, 304.0895).
3.3. Human Liver Microsome Stability Assay
Mixed-gender human liver microsomes (Lot # 1210347) were purchased from XenoTech.
The reaction mixture minus NADPH was prepared as described below. The test compounds were
added into the reaction mixture at a final concentration of 1
µM. A separate reaction with the
control compound, testosterone, was run simultaneously with the reactions with the test compounds.
An◦aliquot of the reaction mixture (without cofactor) was equilibrated in a shaking water bath at
37 C for 3 min. After addition of cofactor to initiate the reaction, the mixture was incubated in
◦
a shaking water bath at 37 C. Aliquots (100
µL) were withdrawn at 0, 10, 20, 30 and 60 min for
the test compounds and testosterone. The reaction was terminated by immediately combining the
tested compounds and testosterone samples with 400 L of ice-cold 50/50 acetonitrile (ACN)/H2O
µ
containing 0.1% formic acid and internal standard. The samples were then mixed and centrifuged to
precipitate proteins. All samples were assayed by LC-MS/MS using electrospray ionization. The peak

Molecules 2017, 22, 1897
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area response ratio (PARR) to internal standard was compared to the PARR at time 0 to determine the
percent remaining at each time point. The values for half-life (t1/2) and intrinsic clearance (CLint) of the
tested compounds were determined by Absorption System Corp. Half-life calculated using GraphPad
software was fitted to a single-phase exponential decay equation.
Supplementary Materials: The following are available online. 1H- and 13C-NMR spectra and HPLC chromatogram
1
of all compounds synthesized; H- and 13C-NMR spectra comparison of experimental and reported hispidulin;
ROESY spectra of compound 21; and ROESY, HMQC and HMBC spectra of final products hispidulin and d-hispidulin.
Acknowledgments: We gratefully acknowledge the financial support from the Ministry of Science and
Technology (MOST105-2320-B-038-024-MY3 to W.-J. Huang, MOST 105-2311-B-038-001 to K.-C. Hsu and
MOST105-2325-B-002-004 to L.-C. Chiou).
Author Contributions: Liang-Chieh Chen, Kai-Cheng Hsu, Hui-Ju Tseng and Wei-Jan Huang conceived and
designed the experiments; Liang-Chieh Chen and Hui-Ju Tseng performed the experiments; Liang-Chieh Chen,
Kai-Cheng Hsu and Wei-Jan Huang analyzed the data; Liang-Chieh Chen, Lih-Chu Chiou, Kai-Cheng Hsu and
Wei-Jan Huang wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 12
, 32, 33, 34, 35, 36, 37, 38, 41, 42, 43, 44, 45a, 45b, 46a, 46b, 47a,
47b, hispidulin and d-hispidulin are available from the authors.
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