Total Synthesis of Hispidulin and the Structural Basis for Its Inhibition of Proto-oncogene Kinase Pim-1

Article abstract of DOI:10.1021/acs.jnatprod.5b00324

(Figure Presented). A new method is applied to synthesize hispidulin, a natural flavone with a broad spectrum of biological activities. Hispidulin exhibits inhibitory activity against the oncogenic protein kinase Pim-1. Crystallographic analysis of Pim-1 bound to hispidulin reveals a binding mode distinct from that of quercetin, suggesting that the binding potency of flavonoids is determined by their hydrogen-bonding interactions with the hinge region of the kinase. Overall, this work may facilitate construction of a library of hispidulin-derived compounds for investigating the structure-activity relationship of flavone-based Pim-1 inhibitors.

Full text of DOI:10.1021/acs.jnatprod.5b00324

Article
pubs.acs.org/jnp
Total Synthesis of Hispidulin and the Structural Basis for Its
Inhibition of Proto-oncogene Kinase Pim‑1
Shi-Wei Chao,†,‡ Ming-Yuan Su,§,⊥ Lih-Chu Chiou,∥,∇,○ Liang-Chieh Chen,†,‡ Chung-I Chang,*,§,⊥
and Wei-Jan Huang*,‡,⊗,#
‡
†School of Pharmacy and Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan
§Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan
⊥Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 106, Taiwan
∥Graduate Institute of Pharmacology, ∇Department of Pediatrics, and ○Graduate Institute of Brain and Mind Sciences, College of
Medicine, National Taiwan University, Taipei 100, Taiwan
⊗Ph.D. Program for the Clinical Drug Discovery from Botanical Herbs, Taipei 110, Taiwan
#School of Pharmacy, National Defense Medical Center, Taipei 114, Taiwan
S
* Supporting Information
ABSTRACT: A new method is applied to synthesize hispidulin, a natural flavone with a broad spectrum of biological activities.
Hispidulin exhibits inhibitory activity against the oncogenic protein kinase Pim-1. Crystallographic analysis of Pim-1 bound to
hispidulin reveals a binding mode distinct from that of quercetin, suggesting that the binding potency of flavonoids is determined
by their hydrogen-bonding interactions with the hinge region of the kinase. Overall, this work may facilitate construction of a
library of hispidulin-derived compounds for investigating the structure−activity relationship of flavone-based Pim-1 inhibitors.
tic disorders.18 Despite its various pharmacological properties,
ispidulin, a naturally occurring flavone, is widely
1−5
Hdistributed in plants of the Asteraceae.
few studies have attempted to synthesize hispidulin. Previously,
Kavvadias and co-workers developed a nine-step synthesis
toward hispidulin (Scheme 1).12 In our hands, the synthesis of
compounds 5 and 6 failed (Scheme 1).12 Additionally, this
approach indicated that the yields of the debenzylation (C-7, C-
4′) and selective demethylation (C-5) of compound 11 using
BCl3 were poor.
It reportedly
has antifungal,6 antiplatelet,7 anti-inflammatory,8 antiosteopor-
otic,9,10 anticonvulsant,11,12 and, in particular, anticancer
activity.1,13−17 Studies indicated that hispidulin induces growth
inhibition and apoptosis in various human cancer cell lines by
stimulating AMP-activated protein kinase (AMPK) to inhibit
the mammalian target of rapamycin (mTOR).16 Hispidulin also
triggers the vascular endothelial growth factor (VEGF)-related
PI3K/Akt/mTOR signaling pathway and suppresses angio-
genesis and cell growth in pancreatic cancer cells as well as in
an in vivo model.14 Additionally, hispidulin permeates the
blood−brain barrier (BBB) and acts at a benzodiazepine-
binding site of the human γ-aminobutyric acid (GABA)A
receptor, thus enhancing the GABA-binding affinity in a
positive allosteric manner.12 Recently, we used a bioassay-
guided approach to identify hispidulin as the active compound
in Clerodendrum inerme that can alleviate methamphetamine-
induced hyperlocomotion in mice, an animal model mimicking
Shen and co-workers also reported a seven-step semisyn-
thesis of hispidulin (Scheme 2).19 This approach starts with the
flavonoid glucuronide scutellarin isolated from Chinese
herbs.20−22 Despite a satisfactory overall yield, the H NMR
1
data for the synthesized compound using this method were not
consistent with the reported data for hispidulin,22,23 suggesting
an incorrect chemical structure for the synthesized compound.
Furthermore, commercial scutellarin is available only in
Received: April 12, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00324
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
a
Scheme 1. Synthesis of Hispidulin by Kavvadias and Co-workers
a
Reagents and conditions: (a) Me2SO4, K2CO3, acetone, Δ; (b) AlCl3, chlorobenzene, Δ; (c) BnBr, K2CO3, acetone, Δ; (d) (1) K2S2O8 pyridine,
10% NaOH(aq), RT; (2) 37% HCl(aq), Δ; (e) Me2SO4, K2CO3, acetone, Δ; (f) pyridine, RT; (g) KOH, pyridine, 60 °C; (h) HOAc, H2SO4, 60 °C;
(i) BCl3, CH2Cl2, −65 °C.
Scheme 2. Synthetic Route for Hispidulin by Shen and Co-workers
Figure 1. Examples of flavonols as Pim-1 kinase inhibitors.
milligram quantities, which hampers large-scale production of
hispidulin. These issues motivated us to develop a feasible
synthesis route toward hispidulin.
2D NMR spectroscopic techniques. An assay of enzyme
inhibitory activity against the Pim-1 kinase shows an IC50 value
of 2.71 μM. The specific binding mode of hispidulin was
determined using a cocrystal structure analysis of hispidulin
complexed to Pim-1. The results from this study would be
helpful for developing Pim-1-specific inhibitors based on a
flavonoid scaffold.
The A-, B-, and C-rings of hispidulin adopt a planar
conformation; such a structure is compatible with one of the
known chemical features of ATP-competitive kinase inhibitors.
Indeed, several flavonols such as quercetin, quercetagetin, and
myricetin have been identified to inhibit proto-oncogene kinase
Pim-1 (Figure 1).23 Among those flavonols, quercetagetin
displayed a high degree of selectivity for Pim-1 over a panel of
kinases. However, analysis of the crystal structure of Pim-1 in
complex with these flavonols revealed two different binding
poses,23 which have made prediction of the binding mode of
hispidulin to Pim-1 difficult. In this study, a new feasible
method was developed for synthesizing hispidulin. The
structure of the synthesized product was determined using
RESULTS AND DISCUSSION
■
Synthesis of Hispidulin. 2,4,6-Trihydroxybenzaldehyde
(12) was chosen as the starting material with a view to
transform the formyl group into a hydroxy group (Scheme 3).
Treatment of 2,4,6-trihydroxybenzaldehyde (12) with chlor-
odimethyl ether (MOMCl) gave the tris(methoxymethoxy)-
protected compound 13.24 Baeyer−Villiger oxidation followed
by basic hydrolysis25 yielded phenol 14. Methylation of
B
DOI: 10.1021/acs.jnatprod.5b00324
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
a
Scheme 3. Synthesis of Hispidulin
a
Reagents and conditions: (a) MOMCl, DIPEA, CH2Cl2, 0 °C, 82%; (b) (1) MCPBA, dry CH2Cl2; (2) K2CO3, MeOH, 65%; (c) Mel, K2CO3,
acetone, Δ, 93%; (d) HCl, MeOH, RT, 92%; (e) BF3−Et2O, Ac2O, RT, 32%; (f) MOMCl, K2CO3, acetone, Δ, 18a: 30%, 18b: 29%; (g) 18a, 4-
BnOPhCHO, KOH, EtOH, H2O, 0 °C, 86%; (h) NaOAc, EtOH, Δ, 83%; (i) I2, pyridine, 90 °C, 65%; (j) BCl3, CH2Cl2, 0 °C, 80%.
compound 14 with MeI afforded methyl ether 15 in a
quantitative yield. Demethoxymethylation of compound 15
with HCl gave triphenol 16.26 Friedel−Crafts acetylation of
compound 16 using Ac2O and BF3−Et2O provided compound
17.27 At room temperature, the reaction of compound 17 with
MOMCl preferentially afforded 4,6-bis(methoxymethoxy)-
protected 18b over the desired 2,4-bis(methoxymethoxy)-
protected 18a due to steric hindrance. The two compounds
18a and 18b could be distinguished based on NOESY
correlations of H-5 and C3-OMe (Figure 2) and their
HI. Debenzylation of compound 21 with BCl3 yielded
hispidulin. The 1H and 13C NMR spectra of hispidulin
resembled the reported data.30,31 Its structure was also
confirmed by 2D NMR techniques such as NOESY, HSQC,
and HMBC (Supporting Information). The purity of all
synthesized compounds was estimated to be at least 97% as
determined by HPLC analyses (Supporting Information).
Structure of Hispidulin Bound to Pim-1. The enzyme
inhibitory activity of hispidulin against Pim-1 was assessed
using quercetin as the reference (Figure 3). Hispidulin
Figure 2. NOESY correlations of compounds 18a and 18b.
HMBC data (Supporting Information). The yield of compound
18a, however, was improved by increasing the reaction
temperature and reducing the quantity of MOMCl to 2.5
equiv of compound 17. Aldol condensation of compound 18a
with 4-benzyloxybenzaldehyde yielded chalcone 19. Oxidative
cyclization28 of compound 19 using catalytic I2 did not provide
flavone 21, but afforded product 18a presumably via a retro-
Aldol-type reaction. Base-catalyzed cyclization of compound 19
with NaOAc29 gave flavanone 20. Compound 20 was converted
to flavone 21 through oxidative dehydrogenation with I2 and
simultaneous methoxymethoxy deprotection via the release of
Figure 3. Dose−response curve of hispidulin and quercetin for the
Pim-1 kinase.
exhibited activity with an IC50 value of 2.71 μM. There are
two reported binding poses of a flavonoid to Pim-1. In order to
establish the correct binding pose, the crystal structure of Pim-1
in complex with hispidulin at a 2.04 Å resolution (Table 1) was
generated. In the hispidulin-bound Pim-1 structure, the binding
orientation of hispidulin resembles that of myricetin and
5,7,3′,4′,5′-pentahydroxyflavone in Pim-1 (PDB code: 2O63
C
DOI: 10.1021/acs.jnatprod.5b00324
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
would make stronger hydrogen bonding than their A-rings
(Figure 4B and C). Moreover, the hinge interaction with
Glu121 is mediated by the 3-OH groups of quercetin and
quercetagetin, which is lacking in hispidulin (Figure 4B and C).
Hence, the presence of more hydrogen bond donors in the C-
ring of quercetin for interacting with the hinge may explain why
it has stronger inhibitory potency toward Pim-1 than hispidulin.
Further insights were obtained based on a comparison of
Pim-1 cocrystal structures with hispidulin and other reported
flavone compounds.23 Both hispidulin- and 5,7,3′,4′,5′-
pentahydroxyflavone-bound Pim-1 show an almost identical
binding mode (Figure 4D). Interestingly, the pentahydroxy-
flavone binds in the A-ring-in mode despite three hydroxy
groups in its B-ring; it has been suggested that the trisubstituted
B-ring may be too hydrophilic to be buried in the cleft.23
Although hispidulin and myricetin bind in the same orientation,
there are differences between the binding poses of these two
inhibitors (Figure 4E). Hispidulin moves inward by ∼1.5 Å to
form an interaction of its A-ring with Lys67. Its lack of a 3-OH
group prevents strong hydrogen bonding to the hinge residue
Glu121. An additional hydrogen-bonding interaction with the
backbone carbonyl of the unique hinge residue Pro123 is
absent for hispidulin due to the lack of a 5′-OH group. These
two differences lead to hispidulin moving away from the hinge
region by ∼2.5 Å.
Although hispidulin exists in many plants and has a wide
range of biological activities, isolating the compound from plant
sources needs tedious extraction, purification, and chromatog-
raphy. These time-consuming processes limit its availability in
large quantities for mechanistic evaluation of the in vivo
activities and for preparation of hispidulin derivatives for
structure−activity relationship (SAR) studies. The chemical
synthesis of hispidulin was reported previously;12,18 however,
these methods have several drawbacks, including poor
reproducibility and low yields in key reaction steps during
scale-up synthesis. In the current study, we synthesized
hispidulin via a different route and used the synthesized
compound to determine the binding mode of hispidulin as a
Pim-1 kinase inhibitor by a cocrystallographic analysis.
Although the synthetic route has more steps than previous
methods, it is highly feasible and reproducible, as confirmed by
detailed 1D NMR and 2D NMR analyses. Importantly, this
new synthetic protocol can be applied to synthesize 6-OMe-
retaining hispidulin-derived compounds with a modified B-ring,
which may enrich the compound’s diversity to permit
identification of specific Pim-1 inhibitors by exploring the
SAR of hispidulin derivatives.
Table 1. Data Collection and Structural Refinement
Statistics
Pim-1 bound to hispidulin
Data Collection
space group
cell dimens
a, b, c (Å)
P65
98.00, 98.00, 80.60
90.00, 90.00, 120.00
0.9
α, β, γ (deg)
wavelength (Å)
resolution (Å)
total observns
unique reflns
multiplicity
I/σI
50−2.04 (2.11−2.04)
160 436
28 099
5.7 (5.7)
25.07 (2.13)
0.068 (0.819)
100 (100)
Rmerge
completeness (%)
Refinement
resolution (Å)
no. reflns
50−2.04 (2.11−2.04)
26 911
R
work/Rfree
0.200/0.226
no. of atoms
protein
2224
22
compound
water
65
Wilson B factor (Å2)
average B factor (Å2)
protein
25.1
31.53
40.58
29.61
compound
water
rms deviations
bond lengths (Å)
bond angles (deg)
Ramachandran statistics (%)
most favored
additionally allowed
generously allowed
disallowed
0.009
1.33
93.7
6.3
0
0
PDB code
4XH6
and 2O65) with the A-ring bound deeply inside the ATP-
binding cleft, where the methoxy group makes a van der Waals
contact with a hydrophobic pocket guarded by the gate keeper
residue Leu120 of Pim-1 (Figure 4A). The activation loop for
hispidulin-bound Pim-1 adopts an active “DFG-in” conforma-
tion. Both the 7-OH group and the oxygen of the 6-OMe group
form hydrogen bonds with the side chain ε-amino group of the
absolutely conserved residue Lys67. Additionally, the oxygen of
the 6-OMe group indirectly interacts with the backbone of
Phe187 and the side chain of Glu89 through a water molecule.
Notably, no hydrogen-bonding interaction to the hinge
backbone of the kinase is found, due to the absence of a C-
ring hydroxy group.
The binding mode of hispidulin is significantly different from
that of quercetin or quercetagetin.23 The latter compounds
have been shown to bind deeply to the ATP-binding cleft of
Pim-1 via the B- rather than the A-ring. Comparing the
contrasting binding poses between hispidulin and the two
flavonols suggests that the hydrogen-bonding potential to the
conserved Lys67 determines whether the A- or the B-ring of
the compound binds to the interior portion of the ATP cleft. In
hispidulin, the A-ring has more hydrogen donors than the B-
ring; on the contrary, the B-ring of quercetin and quercetagetin
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
recorded with a Fisher-Johns apparatus (Fisher 12-144). IR spectra
(KBr disk) were recorded using a Jasco Fourier transform infrared
spectrometer (Jasco FT/IR-410). The NMR spectra (1H and 13C
NMR, HMBC, HSQC, and NOESY) were obtained with a Bruker
AV500 or AV600 using standard pulse programs. The MS data were
obtained with a Finnigan Mat TSQ-7000 mass spectrometer
(HRESIMS). HPLC was performed on an Ascentis C18 column
(150 × 4.6 mm) by using an L-2130 pump (Hitachi) and a UV/vis L-
2420 detector (Hitachi). All TLC analyses were performed on silica
gel plates (KG60-F254, Merck). Reagents and materials were used
without further purification, and chemicals were purchased from
ACROS (Geel, Belgium). Dichloromethane was distilled from CaH2
under a nitrogen atmosphere. MOMCl was purchased from TCI
D
DOI: 10.1021/acs.jnatprod.5b00324
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 4. Structural comparison of Pim-1 bound to hispidulin and other flavone compounds. (A) Stereoview of hispidulin bound to the ATP-
binding site of Pim-1 (this work). Bound hispidulin is shown as stick model with a simulated annealing 2Fo−Fc composite omit map contoured at the
1σ level. The compound is colored by atom type: green, carbon atoms; red, oxygen atoms. Hydrogen bonds are indicated by gray dashed lines, and
the water molecule is shown as a purple sphere. Binding residues are shown as red sticks and labeled. (B−E) Comparison of hispidulin (blue sticks)
with quercetin (B), quercetagetin (C), 5,7,3′,4′,5′-pentahydroxyflavone (D), and myricetin (E). The protein ribbon model is Pim-1 in the hispidulin-
bound structure. Residues that form hydrogen bonds (in dashed lines) with each of the superimposed compounds (in gray sticks) are shown in sticks
and labeled.
(Tokyo, Japan), and 2,4,6-trihydroxybenzaldehyde was acquired from
Alfa Aesar (Heysham, UK).
mL) and washed with 0.5 N HCl (2 × 50 mL) and 10% NaOH (2 ×
50 mL). The organic layer was dried over Na2SO4 and filtered, and the
solvent was removed in vacuo. The residue was purified by silica gel
chromatography (EtOAc−n-hexane, 1:2) to give 13 (15.23 g, 82%) as
a yellow, microcrystalline powder: mp 55−57 °C; IR (KBr) νmax 2907,
2826, 1678, 1604, 1573, 1442, 1386, 1213, 1144, 1045, 1020 cm−1; 1H
NMR (CDCl3, 600 MHz) δ 10.39 (1H, s), 6.49 (2H, s), 5.22 (4H, s),
5.17 (2H, s), 3.49 (6H, s), 3.46 (3H, s); 13C NMR (CDCl3, 150 MHz)
Synthesis of 2,4,6-Tris(methoxymethoxy)benzaldehyde (13). To a
solution of 12 (10 g, 64.9 mmol) in CH2Cl2 (100 mL) was added
DIPEA (56.5 mL, 324.4 mmol). The resulting mixture was stirred for
10 min in an ice-bath under N2. MOMCl (19.7 mL, 324.4 mmol) was
added dropwise to the reaction mixture. The mixture was warmed to
RT and stirred overnight. The solution was diluted with CH2Cl2 (100
E
DOI: 10.1021/acs.jnatprod.5b00324
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
δ 187.7, 163.3, 161.2, 111.1, 96.8, 94.9, 94.2, 56.5; HRESIMS m/z
309.0973 [M + Na]+ (calcd for C13H18NaO7, 309.0950).
chromatography (EtOAc−n-hexane, 1:8) to give 18a (476 mg, 30%)
and 18b (471 mg, 29%).
Synthesis of 2,4,6-Tris(methoxymethoxy)phenol (14). To a
solution of 13 (15 g, 52.4 mmol) in CH2Cl2 (100 mL) was added
70% MCPBA (19.37 g, 78.6 mmol) in portions. The resulting solution
was stirred at RT overnight, and then 10% NaOH(aq) (60 mL) and
MeOH (60 mL) were added. The mixture was stirred at RT for 3 h,
acidified with 2 N HCl(aq), and extracted with EtOAc (3 × 100 mL).
The combined organic layer was dried over Na2SO4 and filtered, and
the solvent was removed in vacuo. The residue was purified by silica
gel chromatography (EtOAc−n-hexane, 1:4) to give 14 (9.33 g, 65%)
as a brown oil: IR (KBr) νmax 3385, 2895, 2820, 1640, 1498, 1436,
1393, 1206, 1144, 1076, 1020 cm−1; 1H NMR (DMSO-d6, 600 MHz)
δ 8.15 (1H, s), 6.45 (2H, s), 5.10 (4H, s), 5.03 (2H, s), 3.39 (6H, s),
3.35 (3H, s); 13C NMR (DMSO-d6, 150 MHz) δ 149.2, 145.9, 133.4,
100.6, 95.4, 94.9, 55.9, 55.7; HRESIMS m/z 297.0968 [M + Na]+
(calcd for C12H18NaO7, 297.0950).
6-Hydroxy-3-methoxy-2,4-bis(methoxymethoxy)acetophenone
(18a): yellow oil; IR (KBr) νmax 3410, 2926, 2833, 1741, 1622, 1598,
1362, 1306, 1281, 1157, 1051 cm−1; 1H NMR (DMSO-d6, 600 MHz)
δ 11.89 (1H, s), 6.40 (1H, s), 5.22 (2H, s), 5.12 (2H, s), 3.66 (3H, s),
3.41 (3H, s), 3.39 (3H, s), 2.54 (3H, s); 13C NMR (DMSO-d6, 150
MHz) δ 203.0, 157.0, 155.5, 150.6, 135.1, 113.1, 100.0, 99.5, 94.8,
61.1, 57.8, 56.5, 32.5; HRESIMS m/z 309.0949 [M + Na]+ (calcd for
C13H18NaO7, 309.0950).
2-Hydroxy-3-methoxy-4,6-bis(methoxymethoxy)acetophenone
(18b): light yellow oil; IR (KBr) νmax 3410, 2926, 2833, 1736, 1697,
1616, 1591, 1486, 1411, 1275, 1151, 1070 cm−1; 1H NMR (DMSO-d6,
600 MHz) δ 12.96 (1H, s), 6.39 (1H, s), 5.26 (2H, s), 5.25 (2H, s),
3.65 (3H, s), 3.41 (3H, s), 3.40 (3H, s), 2.58 (3H, s); 13C NMR
(DMSO-d6, 150 MHz) δ 203.8, 156.7, 155.4, 155.0, 131.7, 108.4, 95.0,
94.6, 93.3, 60.4, 56.7, 56.4, 33.1; HRESIMS m/z 309.0951 [M + Na]+
(calcd for C13H18NaO7, 309.0950).
Synthesis of 2-Methoxy-1,3,5-tris(methoxymethoxy)benzene
(15). To a mixture of 14 (8.5 g, 31 mmol) and K2CO3 (34.25 g,
248.2 mmol) in acetone (60 mL) was added CH3I (7.7 mL, 124.1
mmol). The mixture was heated to 56 °C under N2. After filtration to
remove K2CO3, the filtrate was concentrated in vacuo, diluted with
H2O (30 mL), and extracted with EtOAc (3 × 60 mL). The combined
organic layer was dried over Na2SO4 and filtered, and the solvent was
removed in vacuo. The residue was purified by silica gel
chromatography (EtOAc−n-hexane, 1:5) to give 15 (8.31 g, 93%) as
a yellow oil; IR (KBr) νmax 2938, 2901, 2826, 1591, 1492, 1430, 1393,
1225, 1039, 1020 cm−1; 1H NMR (CDCl3, 600 MHz) δ 6.54 (2H, s),
5.17 (4H, s), 5.07 (2H, s), 3.80 (3H, s), 3.49 (6H, s), 3.45 (3H, s); 13C
NMR (CDCl3, 150 MHz) δ 153.6, 151.2, 135.2, 99.5, 95.4, 94.9, 61.1,
56.2, 56.0; HRESIMS m/z 311.1124 [M + Na]+ (calcd for
C13H20NaO7, 311.1107).
Synthesis of (E)-1-(6-Hydroxy-3-methoxy-2,4-bis-
(methoxymethoxy)phenyl)-3-(4-benzyloxyphenyl)prop-2-en-1-one
(19). To a mixture of 18 (450 mg, 1.57 mmol) and 4-
benzyloxybenzaldehyde (367 mg, 1.73 mmol) in EtOH (4 mL) was
added a solution of KOH (1.09 g) in EtOH−H2O (2 mL:1 mL)
dropwise by syringe. The resulting solution was cooled to 0 °C and
stirred for 3 h under N2. The mixture was warmed to RT overnight.
The reaction mixture was poured into ice water, acidified to pH 3−4
with 1 N HCl(aq), and extracted with EtOAc (3 × 30 mL). The
combined organic layer was dried over Na2SO4 and filtered, and the
solvent was removed in vacuo. The residue was purified by silica gel
chromatography (EtOAc−n-hexane, 1:6) to give 19 (650 mg, 86%) as
a yellow, microcrystalline powder; mp 61−63 °C; IR (KBr) νmax 3398,
2920, 2851, 1736, 1622, 1554, 1504, 1411, 1374, 1343 cm−1; 1H NMR
(CDCl3, 600 MHz) δ 13.25 (1H, s), 7.91 (1H, d, J = 15.5 Hz), 7.81
(1H, d, J = 15.5 Hz), 7.60 (2H, d, J = 8.7 Hz), 7.42 (2H, d, J = 7.2
Hz), 7.37 (2H, t, J = 7.2 Hz), 7.32 (1H, t, J = 7.2 Hz), 6.99 (2H, d, J =
8.7 Hz), 6.52 (1H, s), 5.25 (2H, s), 5.19 (2H, s), 5.10 (2H, s), 3.81
(3H, s), 3.50 (3H, s), 3.47 (3H, s); 13C NMR (CDCl3, 150 MHz) δ
192.9, 161.7, 160.8, 157.1, 151.9, 143.3, 136.5, 135.2, 130.4, 128.7,
128.3, 128.2, 127.5, 124.6, 115.3, 110.5, 100.8, 99.7, 94.7, 70.1, 61.2,
58.2, 56.6; HRESIMS m/z 503.1681 [M + Na]+ (calcd for
C27H28NaO8, 503.1682).
Synthesis of 4′-Benzyloxy-6-methoxy-5,7-bis(methoxymethoxy)-
flavanone (20). To a solution of 19 (600 mg, 1.25 mmol) in EtOH−
H2O (6 mL: 0.5 mL) was added NaOAc (410 mg, 5.00 mmol). The
reaction mixture was heated to 78 °C under N2 overnight. The mixture
was diluted with EtOAc (60 mL) and washed with distilled H2O (3 ×
30 mL). The organic layer was dried over Na2SO4 and filtered, and the
solvent was removed in vacuo. The residue was purified by silica gel
chromatography (EtOAc−n-hexane, 1:5) to give 20 (498 mg, 83%) as
a yellow, microcrystalline powder; mp 121−123 °C; IR (KBr) νmax
3404, 2920, 2845, 1734, 1684, 1604, 1517, 1467, 1418, 1380 cm−1; 1H
NMR (CDCl3, 600 MHz) δ 7.41 (2H, d, J = 7.2 Hz), 7.38 (2H, t, J =
7.2 Hz), 7.35 (2H, d, J = 8.6 Hz), 7.31 (1H, t, J = 7.2 Hz), 7.00 (2H, d,
J = 8.6 Hz), 6.60 (1H, s), 5.32 (1H, dd, J = 2.7, 13.4 Hz), 5.22 (2H, s),
5.20 (1H, d, J = 6.1 Hz), 5.16 (1H, d, J = 6.1 Hz), 5.07 (2H, s), 3.82
(3H, s), 3.62 (3H, s), 3.48 (3H, s), 2.99 (1H, dd, J = 13.4, 16.6 Hz),
2.73 (1H, dd, J = 2.6, 16.6 Hz); 13C NMR (CDCl3, 150 MHz) δ 189.7,
159.7, 159.2, 157.1, 151.3, 138.1, 136.8, 130.9, 128.6, 128.0, 127.7,
127.4, 115.1, 109.8, 100.3, 99.9, 94.7, 78.9, 70.1, 61.3, 57.5, 56.6, 45.3;
HRESIMS m/z 503.1697 [M + Na]+ (calcd for C27H28NaO8,
503.1682).
Synthesis of 1,3,5-Trihydroxy-2-methoxybenzene (16). To a
solution of 15 (8.32 g, 28.9 mmol) in MeOH (300 mL) was added
12 N HCl(aq) (21.6 mL) dropwise by addition funnel. The reaction
mixture was stirred at RT overnight. The mixture was concentrated in
vacuo, diluted with H2O (30 mL), and extracted with EtOAc (3 × 60
mL). The combined organic layer was dried over Na2SO4 and filtered,
and the solvent was removed in vacuo. The residue was purified by
silica gel chromatography (EtOAc−n-hexane, 1:4) to give 16 (4.15g,
92%) as a brown, microcrystalline powder: mp 180−184 °C; IR (KBr)
νmax 3342, 2938, 2839, 1616, 1517, 1480, 1374, 1275, 1144, 1051
1
cm−1; H NMR (DMSO-d6, 600 MHz) δ 8.76 (2H, s), 8.69 (1H, s),
5.72 (2H, s), 3.55 (3H, s); 13C NMR (DMSO-d6, 150 MHz) δ 153.5,
151.1, 128.8, 94.8, 60.1; HRESIMS m/z 157.0507 [M + H]+ (calcd for
C7H9O4, 157.0501).
Synthesis of 2,4,6-Trihydroxy-3-methoxyacetophenone (17). To a
solution of 16 (3 g, 19.2 mmol) in Ac2O (30 mL) was added BF3-Et2O
(2.6 mL, 1.1 mmol) dropwise by syringe in an ice-bath. The reaction
mixture was warmed to RT and stirred under N2 overnight. The
mixture was cooled to 0 °C, adjusted to pH 3−4 with 10% NaOH(aq)
,
and extracted with EtOAc (3 × 50 mL). The combined organic layer
was dried over Na2SO4 and filtered, and the solvent was removed in
vacuo. The residue was purified by silica gel chromatography (EtOAc−
n-hexane, 1:4) to give 17 (1.22 g, 32%) as a yellow, microcrystalline
powder: mp 156−159 °C; IR (KBr) νmax 3329, 2920, 2845, 1728,
1
1629, 1591, 1504, 1442, 1355, 1293 cm−1; H NMR (DMSO-d6, 600
MHz) δ 12.43 (1H, s), 11.51 (1H, s), 10.31 (1H, s), 5.89 (1H, s), 3.59
(3H, s), 2.55 (3H, s); 13C NMR (DMSO-d6, 150 MHz) δ 203.1, 159.0,
157.8, 156.7, 128.1, 104.1, 94.5, 60.1, 32.5; HRESIMS m/z 221.0422
[M + Na]+ (calcd for C9H10NaO5, 221.0426).
Preparation of Compounds 18a and 18b. To a mixture of 17
(1.1 g, 5.6 mmol) and K2CO3 (5.37 g, 38.9 mmol) in acetone (30 mL)
was added MOMCl (1.1 mL, 13.9 mmol) dropwise by syringe. The
reaction mixture was heated to 56 °C under N2 overnight. After
filtration to remove K2CO3, the filtrate was concentrated in vacuo,
diluted with distilled H2O (30 mL), and extracted with EtOAc (3 × 60
mL). The organic layer was dried over Na2SO4 and filtered, and the
solvent was removed in vacuo. The residue was purified by silica gel
Synthesis of 4′-Benzyloxy-6-methoxy-5,7-dihydroxyflavone (21).
To a solution of 20 (300 mg, 0.63 mmol) in pyridine (10 mL) was
added I2 (159 mg, 0.63 mmol). The mixture was heated at 90 °C for 3
h. The reaction mixture was diluted with EtOAc (30 mL) and washed
with distilled H2O (3 × 30 mL). The organic layer was dried over
Na2SO4 and filtered, and the solvent was removed in vacuo. The
residue was purified by silica gel chromatography (EtOAc−n-hexane,
1:2) to give 21 (159 mg, 65%) as a yellow, microcrystalline powder;
mp 152−153 °C; IR (KBr) νmax 3360, 2920, 2845, 1728, 1647, 1610,
F
DOI: 10.1021/acs.jnatprod.5b00324
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1498, 1455, 1368 cm−1; 1H NMR (DMSO-d6, 600 MHz) δ 13.01 (1H,
s), 10.71 (1H, s), 8.02 (2H, dd, J = 2.0, 9.0 Hz), 7.46 (2H, d, J = 7.3
Hz), 7.40 (2H, t, J = 7.3 Hz), 7.34 (1H, t, J = 7.3 Hz), 7.17 (2H, dd, J
= 2.0, 9.0 Hz), 6.85 (1H, s), 6.60 (1H, s), 5.22 (2H, s), 3.74 (3H, s);
13C NMR (DMSO-d6, 150 MHz) δ 182.3, 163.5, 161.5, 157.5, 152.9,
152.6, 136.7, 131.6, 128.7, 128.5, 128.2, 127.9, 123.2, 115.6, 104.3,
103.3, 94.5, 69.7, 60.1; HRESIMS m/z 413.1002 [M + Na]+ (calcd for
C23H18NaO6, 413.1001).
Synthesis of Hispidulin. To a solution of 21 (150 mg, 0.13
mmol) in dry CH2Cl2 (10 mL) at −78 °C was added 1 M BCl3 (69
μL) in CH2Cl2 (2 mL) dropwise by syringe under N2. The resulting
solution was warmed to RT over 1 h. The reaction mixture was
adjusted to pH 2−3 with saturated NaHCO3(aq) and extracted with
EtOAc (3 × 30 mL). The combined organic layer was dried over
Na2SO4 and filtered, and the solvent was removed in vacuo. The
residue was purified by silica gel chromatography (EtOAc−n-hexane,
1:1) to give hispidulin (89 mg, 80%) as a yellow microcrystalline
powder; mp 275−277 °C (lit.32 mp 271−273 °C); IR (KBr) νmax
3323, 2920, 2851, 1734, 1647, 1566, 1486, 1461, 1362 cm−1; 1H NMR
(DMSO-d6, 500 MHz) δ 13.07 (1H, s, OH), 7.92 (2H, d, J = 8.8 Hz,
H-2′, H-6′), 6.93 (2H, d, J = 8.8 Hz, H-3′, H-5′), 6.77 (1H, s, H-3),
6.59 (1H, s, H-8), 3.75 (3H, s, 6-OMe); 13C NMR (DMSO-d6, 125
MHz) δ 182.1 (C-4), 163.8 (C-2), 161.2 (C-4′), 157.3 (C-7), 152.8
(C-5), 152.4 (C-9), 131.4 (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-
OCH3); HRESIMS m/z 323.0515 [M + Na]+ (calcd for C16H12NaO6,
323.0532).
Structure Determination. All images were processed using the
HKL-2000 package. The crystal belongs to the hexagonal space group
P65. The structure was solved by the molecular replacement using the
program Phaser in the CCP4 suite by employing a Pim-1 model (PDB
code 1XWS) for searches. The models were manually rebuilt using the
program Coot and refined using the program refmac5. The model of
hispidulin was built using the program PRODRG and fitted to the
electron density map after the protein model has been completed. The
atomic coordinates and structure factors for the complex were
deposited in the Protein Data Bank (http://www.rcsb.org/) with the
accession number 4XH6.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00324.
1H and 13C NMR spectra and HPLC chromatogram of
all compounds synthesized; comparison of experimental
and reported hispidulin data; and NOESY, HMQC, and
HMBC spectra of compounds 18a,b and final product
hispidulin (PDF)
AUTHOR INFORMATION
■
Pim-1 Kinase Inhibition Assay. The Pim-1 kinase reaction was
performed in 20 mmol/L HEPES (pH 7.5), 10 mmol/L MgCl2, 1
mmol/L EGTA, 0.02% Brij 35, 0.02 mg/mL bovine serum albumin, 2
mmol/L DTT, and 1% DMSO. The final ATP concentration was 10
μm/L. The IC50 values of the tested compounds were determined by
Reaction Biology Corp. The purified recombinant Pim-1 kinase was
incubated with serial 2-fold dilutions of the tested compounds starting
at a concentration of 30 μM. The dose−response curves were obtained
using Prism 5.0 from GraphPad software.
Corresponding Authors
*Tel/Fax (C.-I. Chang): +886 2 27889759. E-mail: chungi@
gate.sinica.edu.tw.
*Tel (W.-J. Huang): +886 2 27361661, ext. 6152. Fax: +886 2
27355276. E-mail: wjhuang@tmu.edu.tw.
Notes
The authors declare no competing financial interest.
Expression and Purification of Pim-1. The production and
purification of the recombinant His6-tagged Pim-1 for crystallography
have been described previously.33 The cDNA fragment encoding Pim-
1 (residues 29−313) was synthesized (GenScript) and cloned into an
expression vector, in frame with a carboxyl-terminal His6 tag. The
recombinant protein was expressed in Escherichia coli BL21 (DE3)
cells using Terrific-Broth medium. Cells were cultured to an OD600 of
1.2 and induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside
(IPTG) at 15 °C for 16 h. Harvested bacteria were resuspended in
lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol, and
Roche EDTA-free protease inhibitor cocktail) and ruptured in a high-
pressure homogenizer (Avestin). The crude lysate was centrifuged at
35000 g for 45 min, and the supernatant was applied to a nickel-
nitrilotriacetic acid (Qiagen) column and washed with 20−100 mM
imidazole. The protein fraction, eluted with 250 mM imidazole, was
loaded onto a Mono Q column (GE Healthcare) with 25 mM Tris-
HCl pH 8.0, 100 mM NaCl, and 2 mM DTT and eluted with a linear
gradient of 0.1−1 M NaCl. The pooled Pim-1 fractions were
combined and passed on a Superdex 75 column (GE Healthcare)
equilibrated in 25 mM Tris-HCl pH 8.0, 200 mM NaCl, 2 mM MgCl2,
2 mM CaCl2, and 4 mM DTT.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support from the
Ministry of Science and Technology (NSC102-2320-B-001-016
to C.-I.C. and NSC102-2320-B-038-019-MY3 to W.-J.H.) and
from the Academia Sinica in Taiwan (C.-I.C.).
REFERENCES
■
(1) Cui, B. L.; Lee, Y. H.; Chai, H.; Tucker, J. C.; Fairchild, C. R.;
Raventos-Suarez, C.; Long, B.; Lane, K. E.; Menendez, A. T.; Beecher,
C. W. W.; Cordell, G. A.; Pezzuto, J. M.; Kinghorn, A. D. J. Nat. Prod.
1999, 62, 1545−1550.
(2) Flamini, G.; Antognoli, E.; Morelli, I. Phytochemistry 2001, 57,
559−564.
(3) Fullas, F.; Hussain, R. A.; Chai, H. B.; Pezzuto, J. M.; Soejarto, D.
D.; Kinghorn, A. D. J. Nat. Prod. 1994, 57, 801−807.
(4) Iwashina, T.; Kamenosono, T.; Ueno, T. Phytochemistry 1999, 51,
1109−1111.
(5) Rojas, A.; Villena, R.; Jimen
1279−1282.
́
ez, A.; Mata, R. J. Nat. Prod. 1991, 54,
Protein Crystallization. Pim-1 was crystallized under the reported
conditions.34 The protein was crystallized by the hanging-drop vapor-
diffusion method in which equal volumes of protein (7 mg/mL) and
reservoir solution (0.4−1.4 M NaOAc, 0.1 M imidazole, pH 6.5−7.1)
were mixed and allowed to equilibrate against the reservoir at 4 °C.
The rod-shaped crystals grew in about 2 days. Complexes of Pim-1
with hispidulin were obtained by soaking apo crystals in mother liquid
solutions with the compound, dissolved in DMSO, at a concentration
of 1 mM overnight. The crystals were harvested and cryoprotected in a
well solution containing 20% glycerol prior to data collection. The X-
ray diffraction data for the Pim-1−hispidulin complex were collected at
the SP44XU beamline at SPring-8, Japan.
(6) Tan, R. X.; Lu, H.; Wolfender, J. L.; Yu, T. T.; Zheng, W. F.;
Yang, L.; Gafner, S.; Hostettmann, K. Planta Med. 1999, 65, 64−67.
(7) Bourdillat, B.; Delautier, D.; Labat, C.; Benveniste, J.; Potier, P.;
Brink, C. Eur. J. Pharmacol. 1988, 147, 1−6.
(8) Clavin, M.; Gorzalczany, S.; Macho, A.; Munoz, E.; Ferraro, G.;
̃
Acevedo, C.; Martino, V. J. Ethnopharmacol. 2007, 112, 585−589.
(9) Nepal, M.; Choi, H. J.; Choi, B. Y.; Yang, M. S.; Chae, J. I.; Li, L.;
Soh, Y. Eur. J. Pharmacol. 2013, 715, 96−104.
(10) Zhou, R. K.; Wang, Z. H.; Ma, C. Cell Biochem. Biophys. 2014,
69, 311−317.
(11) Kavvadias, D.; Monschein, V.; Sand, P.; Riederer, P.; Schreier, P.
Planta Med. 2003, 69, 113−117.
G
DOI: 10.1021/acs.jnatprod.5b00324
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(12) Kavvadias, D.; Sand, P.; Youdim, K. A.; Qaiser, M. Z.; Rice-
Evans, C.; Baur, R.; Sigel, E.; Rausch, W. D.; Riederer, P.; Schreier, P.
Br. J. Pharmacol. 2004, 142, 811−820.
(13) Gao, H.; Wang, H.; Peng, J. J. Cell Biochem. Biophys. 2014, 69,
27−34.
(14) He, L. J.; Wu, Y. Y.; Lin, L.; Wang, J. Q.; Wu, Y. G.; Chen, Y. H.;
Yi, Z. F.; Liu, M. Y.; Pang, X. F. Cancer Sci. 2011, 102, 219−225.
(15) Lin, Y. C.; Hung, C. M.; Tsai, J. C.; Lee, J. C.; Chen, Y. L. S.;
Wei, C. W.; Kao, J. Y.; Way, T. D. J. Agric. Food Chem. 2010, 58,
9511−9517.
(16) Yang, J. M.; Hung, C. M.; Fu, C. N.; Lee, J. C.; Huang, C. H.;
Yang, M. H.; Lin, C. L.; Kao, J. Y.; Way, T. D. J. Agric. Food Chem.
2010, 58, 10020−10026.
(17) Yu, J.; Liu, H.; Lei, J.; Tan, W.; Hu, X.; Zou, G. Phytother. Res.
2007, 21, 817−822.
(18) Huang, W. J.; Lee, H. J.; Chen, H. L.; Fan, P. C.; Ku, Y. L.;
Chiou, L. C. J. Ethnopharmacol. 2015, 166, 18−22.
(19) Shen, M. Z.; Shi, Z. H.; Li, N. G.; Hao-Tang; Shi, Q. P.; Tang, Y.
P.; Yang, J. P.; Duan, J. A. Lett. Org. Chem. 2013, 10, 733−737.
(20) Chen, X. Y.; Cui, L.; Duan, X. T.; Ma, B.; Zhong, D. F. Drug
Metab. Dispos. 2006, 34, 1345−1352.
(21) Gafner, S.; Bergeron, C.; Batcha, L. L.; Reich, J.; Arnason, J. T.;
Burdette, J. E.; Pezzuto, J. M.; Angerhofer, C. K. J. Nat. Prod. 2003, 66,
535−537.
(22) Grayer, R. J.; Veitch, N. C.; Kite, G. C.; Paton, A. J.; Garnock-
Jones, P. J. Phytochemistry 2002, 60, 727−731.
(23) Holder, S.; Zemskova, M.; Zhang, C.; Tabrizizad, M.; Bremer,
R.; Neidigh, J. W.; Lilly, M. B. Mol. Cancer Ther. 2007, 6, 163−172.
(24) Anwar, H. F.; Hansen, T. V. Org. Lett. 2009, 11, 587−588.
(25) Nicolaou, K. C.; Sasmal, P. K.; Xu, H. J. Am. Chem. Soc. 2004,
126, 5493−5501.
(26) McInnes, A. G.; Ragan, M. A.; Smith, D. G.; Walter, J. A. Can. J.
Chem. 1985, 63, 304−313.
(27) Shi, L. S.; Kuo, S. C.; Sun, H. D.; Morris-Natschke, S. L.; Lee, K.
H.; Wu, T. S. Bioorg. Med. Chem. 2014, 22, 1889−1898.
(28) Bernard-Gauthier, V.; Boudjemeline, M.; Rosa-Neto, P.; Thiel,
A.; Schirrmacher, R. Bioorg. Med. Chem. 2013, 21, 7816−7829.
(29) Mavel, S.; Dikic, B.; Palakas, S.; Emond, P.; Greguric, I.; de
Gracia, A. G.; Mattner, F.; Garrigos, M.; Guilloteau, D.; Katsifis, A.
Bioorg. Med. Chem. 2006, 14, 1599−1607.
(30) Hase, T.; Ohtani, K.; Kasai, R.; Yamasaki, K.; Picheansoonthon,
C. Phytochemistry 1995, 40, 287−290.
(31) Nakasugi, T.; Komai, K. J. Agric. Food Chem. 1998, 46, 2560−
2564.
́
(32) Marques, M. R.; Stuker, C.; Kichik, N.; Tarrago, T.; Giralt, E.;
̈
Morel, A. F.; Dalcol, I. I. Fitoterapia 2010, 81, 552−556.
(33) Qian, K. C.; Studts, J.; Wang, L.; Barringer, K.; Kronkaitis, A.;
Peng, C.; Baptiste, A.; LaFrance, R.; Mische, S.; Farmer, B. Acta
Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2005, 61, 96−99.
(34) Kumar, A.; Mandiyan, V.; Suzuki, Y.; Zhang, C.; Rice, J.; Tsai, J.;
Artis, D. R.; Ibrahim, P.; Bremer, R. J. Mol. Biol. 2005, 348, 183−193.
H
DOI: 10.1021/acs.jnatprod.5b00324
J. Nat. Prod. XXXX, XXX, XXX−XXX