Inhibitor for FcεRI expression on mast cell from Verbasucum thapsus L.

Article abstract of DOI:10.1016/j.bmcl.2018.09.007

We found out 2′,3′-dihydroxypuberulin from South American medicinal plant, V. thapsus L., as a candidate of an anti-allergic lead which inhibits the expression of high-affinity receptor of IgE (FcεRI) on the surface of mast cells. Furthermore, the analysis of structure-activity relationship by using synthesized 2′,3′-dihydroxypuberulin analogs revealed that both hydroxy groups in the side chain and both of methyl moieties on phenolic hydroxy groups were crucial for potent activity, but absolute configuration of C-3′ position wasn't. The active principle, 2′,3′-dihydroxypuberulin, was disclosed to down-regulate the mRNA level of β-chain of FcεRI, different from previous reported active natural product reducing γ-chain level.

Full text of DOI:10.1016/j.bmcl.2018.09.007

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Bioorganic & Medicinal Chemistry Letters
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Inhibitor for FcεRI expression on mast cell from Verbasucum thapsus L.
⁎
Satoru Tamuraa,b, , Kunichika Yoshihiraa, Tomikazu Kawanob, Nobutoshi Murakamia
a
Graduate School of Pharmaceutical Sciences, Osaka University, Japan
School of Pharmacy, Iwate Medical University, Japan
b
A B S T R A C T
We found out 2′,3′-dihydroxypuberulin from South American medicinal plant, V. thapsus L., as a candidate of an anti-allergic lead which inhibits the expression of
high-affinity receptor of IgE (FcεRI) on the surface of mast cells. Furthermore, the analysis of structure-activity relationship by using synthesized 2′,3′-dihydrox-
ypuberulin analogs revealed that both hydroxy groups in the side chain and both of methyl moieties on phenolic hydroxy groups were crucial for potent activity, but
absolute configuration of C-3′ position wasn’t. The active principle, 2′,3′-dihydroxypuberulin, was disclosed to down-regulate the mRNA level of β-chain of FcεRI,
different from previous reported active natural product reducing γ-chain level.
Allergic disease type-I such as hay fever, atopic dermatitis, allergic
asthma, and anaphylaxis, was known to be directly caused by chemical
mediators degranulated from mast cells. This degranulation is triggered
by binding of allergens to IgEs on the surface of mast cells through the
medium of high-affinity receptor of IgE (FcεRI). Thus, some of anti-
allergic agents have targets on the sequential system, for example the
mast cell stabilizers and the inhibitors for biosynthesis of chemical
mediators. We've been attracted to the interaction between IgE and
FcεRI expressed on the surface of mast cells and reported several nat-
ural products as anti-allergic seeds repressing the FcεRI expression on
mast cells.1,2 FcεRI is a key molecule, as well as IgE, for secretion of
chemical mediators inducing allergic symptoms, but FcεRI knock-out
mice were reported to survive normally other than resistant to ana-
phylaxis.3 Therefore, the compounds reducing the expression level of
FcεRI on mast cells are considered to be excellent candidates for de-
velopment of anti-allergic drugs preventing IgE from the binding to
mast cell without occurring serious side effects.
We've been engaged in search for the active principles inhibiting
FcεRI expression on mast cells from medicinal plants and the catechins
bearing the pyrogallol moiety1 and isoflavones isolated from Puerariae
Flos2 were disclosed to show the inhibitory activity. Further exploration
of the active principles from natural resources allowed us to find out
2′,3′-dihydroxypuberulin (1) from Verbasucum thapsus L., a South
American medicinal plant. The present study showed that bioassay-
guided isolation, identification, structure-activity relationship (SAR)
analysis by using synthesized analogs and the biological property of 1
comparing the mechanism with those of previous active principles.
We already reported the bioassay system for the evaluation of the
expression level of FcεRI on the surface of mast cells.1,2 In brief, the
human mast cells, HMC-14, were incubated with each test sample for
72 h, then the harvested cells were treated with anti-FcεRI α-chain
antibody followed by FITC-labeled secondary antibody. The expression
level of FcεRI on the cell surface was detected as fluorescence of FITC
with flow cytometer. Through the screening for active extracts down-
regulating FcεRI on the human mast cells by use of this assay from
about 300 kinds of South American medicinal plants, the MeOH extract
of V. thapsus L. was disclosed to show the substantial inhibitory activity
for FcεRI expression on HMC-1 cells. This MeOH extract was subjected
to partition between EtOAc and H2O, and the resulting EtOAc extract
displayed the more potent activity than H2O extracts. Subsequently,
bioassay-guided separation from the EtOAc extract through SiO2 and
ODS column chromatography led us to isolate 1.3 g of the active prin-
ciple 1 from 100 g of the crude drug, which showed 49.0% inhibition at
the 30 μg/mL concentration.
The structure elucidation of 1 on the basis of the spectroscopic
analysis such as FAB-HRMS, IR, and NMR, directed us to identify the
active principle 1 as 2′,3′-dihydroxypuberulin by the comparison with
the previous reported data.5 (Fig. 1) In order to examine the absolute
configuration at C-2′ position of 1, the HPLC with chiral stationary
phase was applied to analysis and separation for 1.6 As a result, it was
revealed that 1 was the racemic mixture and both enantiomers ex-
hibited the similar activity to that of 1, 45.1% and 44.3% inhibition at
30 μg/mL concentration, respectively. Namely, the absolute configura-
tion of 1 was suggested to have little participation for its activity.
The related compounds to 1 were reported to be isolated from
natural resources and show the inhibition against reverse transcriptase
and Taq DNA polymerase.7 Thus, we tried SAR analysis so as to clarify
the further possibility of relative coumarins to exhibit bioactivity and
the crucial partial structure for inhibition of FcεRI expression. As shown
in Fig. 2, for the assessment of the contribution of 2′-OH, 3′-OH, 6-O-Me
⁎
Corresponding author at: School of Pharmacy, Iwate Medical University, Japan
E-mail address: satamura@iwate-med.ac.jp (S. Tamura).
https://doi.org/10.1016/j.bmcl.2018.09.007
Received 2 April 2018; Received in revised form 29 August 2018; Accepted 6 September 2018
0960-894X/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:TAMURA,S.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2018.09.007

S. Tamura et al.
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First of all, commercially available 7,8-dihydroxy-6-methox-
ycoumarin 8 was utilized for synthesis of 2–6 as shown in Scheme 1.
The induction of methyl group regio-selectively to 8-hydroxy group of 8
was accomplished by methyl iodide in the presence of K2CO3 at 60 °C to
afford common intermediate 9,8 which was subjected to following al-
kylation of 7-hydroxy moiety by use of the corresponding alkyl bro-
mides, 1-bromo-3-methylbutane (for 2), 4-bromo-2-methyl-2-butanol9
(for 3), 1-bromo-3-methyl-2-butanol10 (for 4), to give the desired ana-
logs 2, 3 and 4 in moderate yield. The structure of 2, 3, and 4 were
identified by the analysis of 1H, 13C and 2D NMR spectra. In brief, HH
COSY and HMBC showed us the corresponding side chain sub-structures
which were consistent with the coupling patterns and the coupling
constants of the signals observed in 1H NMR. Additionally, the cross
peaks between 1′-H and 7-C in HMBC were detected, respectively.
Epoxyanalog 5 was also prepared from 9 through the prenylation and
the successive epoxydation by m-chloroperbenzoic acid (mCPBA) in
Fig. 1. Structure of 2′,3′-dihydroxypuberulin (1), an FcεRI expression inhibitor
from V. thapsus.
and 8-O-Me moieties toward the biological potency, the analogs lacking
these functional groups (2–4, 6 and 7) and epoxide congener 5 were
prepared. Since both the enantiomers of natural product 1 showed the
similar activity each other, 3′-dehydroxyanalog 4, epoxyanalog 5, 8-O-
demethylanalog 6 and 6-O-demethylanalog 7 were designed as racemic.
Fig. 2. Structure of synthesized analogs of 1.
Scheme 1. Synthesis of 2′,3′-dihydroxypuberulin
analogs (2–6). Reagents and conditions: (a) MeI,
K2CO3, acetone, 60 °C, 82%; (b) 1-bromo-3-methyl-
butane (for 2), 4-bromo-2-methyl-2-butanol (for 3),
1-bromo-3-methyl-2-butanol (for 4), K2CO3, acetone,
82% for 2, 73% for 3, 67% for 4; (c) prenyl bromide,
K2CO3, THF-acetone (for 10), acetone (for 11); (d)
mCPBA, CH2Cl2, 84%
acetone, 40% 2 steps.
2 steps; (e) OsO4, NMO,
2

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Scheme 2. Synthesis of 6-O-demethylanalog (7). Reagents and conditions: (a) Ph2CCl2, pyridine, 55%; (b) 1,3-dibromo-5,5,-dimethylhydantoin, CHCl3; (c) MeI,
K2CO3, DMF, 60 °C, 73% 2 steps; (d) CuCl2, NaOMe, DMF-H2O, 100 °C; (e) EDCI·HCl, DMPA, MeOH, 70% 2 steps; (f) TBS-Cl, imidazole, CH2Cl2, 50 °C, 80%; (g)
DIBAL, THF, 0 °C, 88%; (h) Dess-Martin periodinane, CH2Cl2; (i) Tebbe reagent, THF, 0 °C, 64% 2 steps; (j) TBAF, THF, 97%; (k) acryloyl chloride, Et3N, CH2Cl2,
quant.; (l) Grubbs 2nd generation catalyst, CH2Cl2, reflux, 79%; (m) Amberlyst 15E, MeOH, 60 °C, 84%; (n) AcCl, Et3N, CH2Cl2, 65%; (o) prenyl bromide, K2CO3,
acetone-THF, 99%; (p) K2CO3, MeOH; (q) OsO4, NMO, acetone, 49% 2 steps.
84% yield for 2 steps. For the synthesis of 8-O-demethylanalog 6, after
prenylation of 7-hydroxy group of 8, resulting 11 was dihydroxylated
reduction followed by Dess-Martin oxidation. Resulted aldehyde was
treated with Tebbe reagent to form exo-methylene 16. The deprotection
of TBS group by tetra-n-butylammonium fluoride (TBAF) followed by
acylation of resultant hydroxy group by acryloyl chloride furnished
cyclization precursor 17 in 97% yield for two steps. The NOE experi-
ment on 17 led us observe the cross peaks between acrylate-olefin and
methoxy group and between styrene-olefin and aromatic proton which
were consistent with the desired substituted position on benzene-ring.
The ring-closing metathesis of 17 was smoothly performed by Grubbs
2nd generation catalyst13 to give 6,7,8-trioxygenated coumarin in 79%
yield, then the removal of diphenyl acetal moiety by acidic resin Am-
berlyst 15E furnished 18. After the acetylation of 6-hydroxy group, the
induction of prenyl moiety at 7-hydroxy group by prenyl bromide in the
presence of K2CO3 to yield 19. Finally, deacetylation in basic condition
followed by dihydroxylation by OsO4 with NMO to afford 6-O-de-
methylanalog 7. (Scheme 2)
Synthesized analogs (2–7) were evaluated for their inhibitory ac-
tivity against FcεRI expression on HMC-1 cells. As illustrated in Fig. 3,
neither demethylanalogs 6 and 7 showed little activity and the analogs
modified in the side chain (2–5) exhibited weaker activity than natural
product 2′,3′-dihydroxypuberulin (1). Thus, both 6- and 8-O-methyl
moieties as well as a dihydroxy partial structure in the side chain were
revealed to be important for potent activity of 1.
by OsO4 with N-methylmorpholine N-oxide (NMO) to furnish
6
(Scheme 1). The regio-selectivity of the prenylation in first step was
previously reported.11 The different alkylated position between me-
thylation for 9 and prenylation for 11 was assumed to arise from the
reaction temperature; higher temperature led the 6-O-alkylation to es-
cape from steric hindrance and more acidic 7-OH group according to
the carbonyl moiety was preferred to react at room temperature.
In case of 7, 6,7,8-trioxygenated coumarin skeleton was constructed
from methyl gallate (12) as depicted in Scheme 2, because of the dif-
ficulty of obtaining 6,7-dihydroxy-8-methoxycoumarin by regio-selec-
tive demethylation of 1. The 1,2-diol protection of 12 by di-
chlorodiphenylmethane afforded diphenyl acetal, which was subjected
to the regio-selective bromination at C-4 by 1,3,-dibromo-5,5-di-
methylhydantoin12 to give 13. Then, methyl moiety was induced into 3-
hydroxy group of 13 to provide 14 in moderate yield from 12. After
conversion of bromine atom of 14 to hydroxy moiety by CuCl2 in the
presence of sodium methoxide, carboxylic group resulted by hydrolysis
of methyl ester was re-constructed to afford 15 in 70% yield for two
steps. The free phenolic hydroxy group of 15 was protected as a t-bu-
tyldimethylsilyl (TBS) ether, then carbomethoxy portion was changed
to formyl group through the diisobutylaluminium hydride (DIBAL)
Next, we analyzed which subunit of FcεRI was responsible for down-
regulation of FcεRI expression due to 1 by measuring the mRNA level of
each subunit. FcεRI generally consists of four subunits, one α -chain,
one β -chain and two disulfide-linked γ-chains. Previously, we clarified
the active flavonoids epigallocatechin gallate and tectorigenin to re-
duce the mRNA level of γ-chain specifically and down-regulate FcεRI on
HMC-1.1,2 Therefore, the effect on the mRNA expression of each subunit
was compared among 1 and previous active compounds by the same
procedure. In brief, after cultivation of HMC-1 cells with 1 for 36 h,
total RNA was collected in the usual manner. The mRNA level of each
subunit was determined by reverse transcription PCR (RT-PCR) fol-
lowed by staining the provided PCR product with ethidium bromide. As
a result, 1 was revealed to suppress the expression of β -chain mRNA
significantly, but not α- nor γ-chains (Fig. 4). Thus, 1 was disclosed to
down-regulate FcεRI in a different way from previous active principles
and suggested to induce the fault in construction of FcεRI by lack of β
-chain.
In conclusion, we found out 2′,3′-dihydroxypuberulin (1) as an in-
hibitor for FcεRI expression on human mast cells from South American
medicinal plant V. thapsus L. and 1 was clarified to suppress the mRNA
level of β -chain of FcεRI. Additionally, through the SAR analysis by use
of synthesized analogs, not only 6- and 8-O-methyl moieties but also
diol structure in side chain were revealed to be crucial for potent ac-
tivity of 1. Up to now, no anti-allergic agents based on the down-
Fig. 3. Inhibitory activity of 1 and its analogs 2–7 for FcεRI expression. Bar
graphs represent mean
SD for triplicate experiments.
3

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Acknowledgements
This work was supported by JSPS KAKENHI Grant Number
JP24790109, Grant-in-Aid for Young Scientists (B) and Research funds
from San-Ei Gen F. F. I. Inc.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.bmcl.2018.09.007.
References
1. Tamura S, Yoshihira K, Fujiwara K, Murakami N. Bioorg Med Chem Lett.
2010;20:2299.
2. Tamura S, Yoshihira K, Tokumaru M, Zisheng X, Murakami N. Bioorg Med Chem Lett.
2010;20:3872.
3. Dombrowicz D, Flamand V, Brigman K, Koller BH, Kinet JP. Cell. 1933;75:969.
4. Butterfield JH, Wailer D, Dewald G, Gleich GJ. Leuk Res. 1988;12:345.
5. Kanlayavattanakul M, Ruangrungsi N, Watanabe T, Ishikawa T. Heterocycles.
2003;61:183.
6. HPLC conditions: Chiralpak AS (4.6 × 250 mm), n-hexane:iPrOH:trifluoroacetic acid
= 80:20:0.1, detection: UV λ = 365 nm, 1.5 mL/min. Rt:18.3 min, 22.6 min.
7. Hugo AG, Imena MM, Gladys MC, Carlos ET, Carlos RP. Bioorg Med Chem Lett.
2014;24:760.
8. The structure of 9 including the position of methyl group was identified with re-
ference to the following reports: Hu H-B, Zheng X-D, Jian Y-F, Liu J-X, Zhu J-H. Arch.
Pharm. Res. 2011;34:1097.
9. The preparation of 4-bromo-2-methyl-2-butanol was performed by the reaction of
ethyl 3-bromopropionate with 2.5 eq. of MeMgBr.
Fig. 4. Expression level of mRNA of each FcεRI subunit after treatment of 1.
The amount of mRNA was normalized against g3pdh, the compared with ve-
hicle. Bar graphs represent mean
SD for triplicate experiments.
10. Dalton DR, Dutta VP. The preparation of 1-bromo-3-methyl-2-butanol was performed
by the reaction of 3-methyl-1-butene with N-bromosuccinimide in aqueous dimethyl
sulfoxide. J Chem Soc B. 1971:85.
11. Maes D, Syngel KV, Debenedetti S, Kimpe ND. Tetrahedron. 2006;62:4426.
12. Azarifar D, Bosra HG, Tajbaksh MJ. Het Chem. 2007;44:467.
13. Hodgson DM, Angrish D. Chem Commun. 2005;39:4902.
regulation of FcεRI expression on mast cells have been developed, yet.
Although we have some inhibitors for FcεRI expression, the biggest
problem that the suppression of FcεRI truly leads anti-allergic effects as
phenotype still remains for exploitation of them as novel anti-allergic
agents. In order to get over this issue, further investigation including in
vivo test are in progress.
4