Design and Synthesis of Structure-Simplified Derivatives of Gonytolide for the Promotion of Innate Immune Responses

Article abstract of DOI:10.1021/acs.jnatprod.5b00829

Gonytolide A (1), a dimeric chromanone substituted with the-lactone, shows promoting activity of innate immune responses. However, biological studies on this compound have been limited by the low amounts of 1 available from natural resources and the difficulty of its synthesis. In this study, we designed and synthesized structure-simplified gonytolide derivatives. Bischromone 10 and biflavone 13 both promoted the mammalian TNF-α signaling pathway and Drosophila innate immunity. They did not contain a chiral center and were easy to synthesize. Hence, they can be used as lead compounds for a new type of immunostimulating drugs and as research reagents.

Full text of DOI:10.1021/acs.jnatprod.5b00829

Article
pubs.acs.org/jnp
Design and Synthesis of Structure-Simplified Derivatives of
Gonytolide for the Promotion of Innate Immune Responses
Haruhisa Kikuchi,* Tsuyoshi Hoshikawa, Shoichiro Kurata, Yasuhiro Katou, and Yoshiteru Oshima
Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku, Sendai 980-8578, Japan
S
* Supporting Information
ABSTRACT: Gonytolide A (1), a dimeric chromanone substituted
with the γ-lactone, shows promoting activity of innate immune
responses. However, biological studies on this compound have been
limited by the low amounts of 1 available from natural resources
and the difficulty of its synthesis. In this study, we designed and
synthesized structure-simplified gonytolide derivatives. Bischro-
mone 10 and biflavone 13 both promoted the mammalian TNF-α
signaling pathway and Drosophila innate immunity. They did not
contain a chiral center and were easy to synthesize. Hence, they can
be used as lead compounds for a new type of immunostimulating
drugs and as research reagents.
everal dimeric and monomeric chromanones substituted
Swith the γ-lactone moiety have been isolated as fungal
metabolites.1 They exhibit interesting biological activities such
as cytotoxicity,1j,l antibacterial1d,g,k and antifungal properties,1g
and anticoccidial activities1c,c as well as inhibitory activities
against cytochrome P450 enzymes.1h,i However, only a few
synthetic studies on monomeric-type chromanones have been
reported.2 Moreover, dimeric chromanones containing γ-
lactones have not been synthesized because of their structural
complexity. Recently, the total synthesis of secalonic acids3 and
rugulotrosin A,4 biogenetically related dimeric tetrahydroxan-
thones, has been reported.
We have focused on regulators of innate immunity by
developing an ex vivo culture system based on the Drosophila
IMD signaling pathway to permit the screening of compounds
that target the innate immune system.5,6 During screening for
innate immune regulators from natural resources, we isolated
and identified a dimeric chromanone, gonytolide A (1), as an
innate immune promoter from the fungus Gonytrichum sp.,
along with its inactive derivatives, gonytolides B−G (2−7)
(Figure 1).6 Gonytolide A (1) also increased TNF-α-stimulated
production of IL-8 in human umbilical vein endothelial cells
and represents a lead compound for novel immunostimulating
agents against bacterial infections and tumors. However,
biological studies on this compound are limited because of
the low amounts of 1 available naturally. In addition, chemical
synthesis of 1 is difficult because of its complex dimeric
chromanone moiety, which includes four chiral centers and a
stereogenic axis. In this study, we synthesized structure-
simplified gonytolide derivatives to promote innate immune
responses.
RESULTS AND DISCUSSION
■
Design and Synthesis of Simplified Derivatives of
Gonytolide A. Structural simplification has been applied to
several biologically and pharmacologically active natural
products with the aim of developing novel drugs such as
Halaven (eribulin).7 Moreover, the structural simplification of
the highly potent protein kinase C activators, bryostatin 1 and
aplysiatoxin, provided a promising HIV eradication drug8 and
an anticancer drug candidate,9 respectively. Wender demon-
strated the principle of function-oriented synthesis,10 in which
the function of a biologically active lead structure can be
emulated by replacement with simpler scaffolds designed to
incorporate the activity-determining structural features of the
lead compound. Thus, we applied this concept to 1 to obtain a
biologically active and simplified derivative.
In our recent study,6 we revealed the structural requirements
of gonytolide A (1) for innate-immunity-promoting activity by
using natural gonytolides and their derivatives. As a result, we
noted the following observation: (i) two symmetrical
chromanone moieties through C-8 and C-8′ are crucial for
the activity; (ii) the opening of γ-lactone moieties does not
affect the activity; (iii) 5O,5′O-dimethylation does not affect
the activity. In fact, the γ-lactone ring-opened derivative 8
exhibits innate-immunity-promoting activity (Figure 2). To
increase synthetic accessibility, we then planned to remove
chiral centers from 8. Specifically, removal of methoxycarbonyl
groups and dehydrogenation at C-2 and C-2′ afforded
bischromone-type compound 9; dehydroxylation of 9 at C-9
and C-9′ affords compound 10. Moreover, reduction of a
Received: September 15, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. Structures of gonytolide A−G (1−7).
First, we attempted to synthesize methyl ester 10.
Compound 10 was synthesized by cyclization of the α,β-
unsaturated ketone 18, which was prepared by chain extension
of 3,3′-biphenyldicarboxylate 15 (Scheme 1). Although there
are limited studies on the dimerization of 2,5-dioxybenzoate
derivatives, the phenol oxidative dimerization of o-hydrox-
yacetophenone in a solid phase (FeCl3 on silica gel) is a
method that is reported to be useful.13 We adopted the same
reaction conditions and obtained 15 from monomeric benzoate
14, which was synthesized by the O-methylation of 2,6-
dihydroxy-4-methylbenzoic acid. HREIMS (m/z 390.1296
[M+]) of 15 indicated that it was a dimer of 14. HMBC
correlation of 15 between H-5 and C-4 and between H3-10 and
C-4 indicated that dimerization occurs at the ortho position of
hydroxy groups and not of methoxy groups. In this
dimerization reation, no isomeric dimer was obtained. After
the methoxymethylation of 15, Claisen condensation with
methyl dimethylphosphonate14 afforded ketophosphonate
dimer 17. Horner−Wadsworth−Emmons reaction of 17 with
methyl 5-oxopentanoate15 afforded α,β-unsaturated ketone 18,
which was cyclized into bischromone 19 under acidic
conditions. Addition and elimination of the bromine atom at
C-3 and C-3′ then afforded the desired bischromone 10.
Compounds 9, 11, 12, and 13 were synthesized in a route
similar to that utilized to prepare 10 (Scheme 2). Compounds
9 and 11 were obtained by debenzylation and dehydration of
22 and 25, respectively, which were synthesized from the
corresponding O-benzylated aldehydes, methyl (S)-4-(benzyl-
oxy)-5-oxopentanoate (20) and 5-(benzyloxy)pentanal.16
Compound 9 existed as a diastereomeric mixture because of
its 8,8′-chiral axis. Biflavone 13 was obtained from
ketophosphonate dimer 17 and p-anisaldehyde.
Figure 2. Outline of the structural simplification of gonytolide A (1).
Innate-Immunity-Promoting Activities of Simplified
Derivatives of Gonytolide A. We evaluated the innate-
immunity-promoting activities of 1 and structure-simplified
gonytolide derivatives 9−13 by using the ex vivo Drosophila
culture system (Figure 3).5 As previously reported,6a gonytolide
A (1) exhibited innate-immunity-promoting activity in a
concentration-dependent manner, and 1 at a concentration of
10 μg/mL led to an increase of the innate immune response to
greater than three times. The structure-simplified derivatives 9,
10, 12, and 13 at a concentration of 10 μg/mL also exhibited
innate-immune-promoting activity without cytotoxicity. These
methoxycarbonyl group afforded bis(2-(5-hydroxypentyl)-
chromone) 11, and acetylation of 11 afforded 12. However,
many biflavones have been isolated from plants.11 However,
only a few 8,8′-biflavone(cuppressuflavone)-type compounds
have been reported.12 When three methylene carbons and one
carbonyl carbon in 10 are replaced with a para-substituted
benzene ring, 8,8′-biflavone 13 is obtained. Thus, we decided to
synthesize 9−13 as simplified derivatives of gonytolide A (1).
B
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a
Scheme 1. Synthesis of bischromone 10
a
Reagents and conditions: (a) pTsOMe, K2CO3, DMF, rt; (b) BCl3, CH2Cl2, −80 °C→ 0 °C (71% (two steps)); (c) FeCl3−silica gel, 40 °C (32%);
(d) MOMCl, NaH, DMF, rt (91%); (e) methyl dimethylphosphonate, LHMDS, THF, rt (81%); (f) methyl 5-oxopentanoate, NaH, THF, rt (59%);
(g) HCl, MeOH, reflux (43%); (h) Py·HBr3, CH2Cl2, rt; (i) DBU, benzene, reflux (55% (two steps)).
a
Scheme 2. Synthesis of compounds 9 and 11−13
a
Reagents and conditions: (a) DIBAL, CH2Cl2, −78 °C (37%) ; (b) corresponding aldehydes (20, 5-(benzyloxy)pentanal or p-anisaldehyde), NaH,
THF, rt (22% (for 21), 27% (for 24)); (c) HCl, MeOH, reflux (50% (for 22), 49%(for 25), 15% (for 28 (two steps from 17)); (d) H2 (baloon),
Pd/C, MeOH, rt (88% (for 23), 90% (for 26)); (e) Py·HBr3, CH2Cl2, rt; (f) DBU, benzene, reflux (23% (for 9), 59% (for 11), 58% (for 13) (two
steps)) ; (g) Ac2O, pyridine rt (78%).
results indicate that the structure of 8,8′-bischromone bearing
appropriate side chains at C-2 and C-2′ is important for innate-
immunity promoting activity, whereas some substituents
producing chiral centers of 1 are not crucial. Surprisingly, the
activity of the biflavone-type compound 13 was comparable to
that of 1. However, 11 exhibited no innate-immunity-
promoting activity, indicating that the existence of a hydrophilic
hydroxy group in a side chain would not be appropriate. In a
similar case, gonytolide F (6) bearing a carboxyl group in a side
chain did not exhibit any innate-immunity-promoting activity,
as previously reported.6b
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Figure 3. Effects of structure-simplified gonytolide derivatives on the Drosophila innate immune response. (A) Effects of compounds on the DAP-
type peptidoglycan-mediated activation of Drosophila Dpt-lacZ. Activation is presented as the percentage of activity relative to the control (DMSO).
(B) Drosophila S2 cell viability in the presence of 10 μg/mL of compounds. Bars indicate standard errors of three independent measurements.
Symbols: * indicates p < 0.05, and ** indicates p < 0.01 vs DMSO.
Promoting Effects of Simplified Derivatives of
Gonytolide A on TNF-α-Stimulated Production of IL-8
in HUVECs. The Drosophila IMD signaling pathway resembles
the mammalian TNF-α signaling pathway.18 The TNF-α
signaling pathway plays a critical role in host defense against
several pathogens, in intrinsic tumor suppression, and in
inflammatory response by NF-κB-activated production of
costimulatory molecules, cytokines, chemokines, and adhesion
molecules. We investigated the effect of gonytolide A (1), 10
and 13 on the TNF-α-stimulated production of IL-8, a
neutrophil chemotactic factor in HUVECs. As shown in Figure
4, 10 μg/mL of 10 and 13 increased the production of IL-8,
although their activities were slightly weaker than that of 1.
be used as leads for a new type of immunostimulating drugs or
as research reagents.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Starting materials
were either commercially available or prepared as reported
previously in the literature. Analytical thin layer chromatog-
raphy was performed on silica gel 60 F254 (Merck). Column
chromatography was carried out on Silica Gel 60 (70−230
mesh, Merck). Nuclear magnetic resonance spectra were
recorded on JEOL JNM ECA-600 and AL-400. Mass spectra
were measured on JEOL JMS AX-500 and AX-700.
Methyl 2-hydroxy-6-methoxy-4-methylbenzoate (14). To
a solution of 2,6-dihydroxy-4-methylbenzoic acid (5.36 g, 31.8
mmol) in DMF (40 mL), we added potassium carbonate (15.4
g, 111 mmol) and methyl p-toluenesulfonate (14.4 mL, 95.4
mmol) at room temperature. After being stirred for 10 h, the
mixture was poured into 1 M hydrochloric acid and extracted
with ethyl acetate three times. The combined organic layer was
washed with saturated sodium bicarbonate solution and brine,
dried over anhydrous sodium sulfate, and concentrated in vacuo
to afford crude methyl 2,6-dimethoxybenzoate (5.88 g). Then,
1.0 M solution of boron trichloride in pantane (33.5 mL) was
added slowly to a solution of crude benzoate in dichloro-
methane (60 mL) at −80 °C. After being stirred for 2 h, the
mixture was poured into water and extracted with ethyl acetate
three times. The combined organic layer was washed with
brine, dried over anhydrous sodium sulfate, and concentrated in
vacuo. The residue was chromatographed over silica gel eluted
by hexane-ethyl acetate (19:1) to afford 14 (4.45 g, 22.7 mmol,
Figure 4. Effects of structure-simplified gonytolide derivatives on IL-8
production induced by TNF-α in HUVECs. HUVECs were treated
with 10 μg/mL of each compound for 3 h prior to stimulation with
TNF-α (1 ng/mL). Bars indicate standard errors of three independent
measurements. Symbols: * indicates p < 0.05, and ** indicates p <
0.01 vs control (DMSO).
1
71% (two steps)). Data for 14: colorless amorphous solid; H
NMR (400 MHz, CDCl3, δ): 11.54 (s, 1H), 6.43 (s, 1H), 6.22
(s, 1H), 3.93 (s, 3H), 3.84 (s, 3H), 2.30 (s, 3H); 13C NMR
(100 MHz, CDCl3, δ): 171.6, 163.5, 160.6, 146.5, 110.5, 103.4,
100.4, 56.1, 52.3, 22.2; LREIMS m/z 196 [M]+ (56%), 164
(100%), 136 (19%), 121 (11%); HREIMS m/z 196.0721 [M]+
(196.0736, calcd for C10H12O4).
Dimethyl 2,2′-dihydroxy-4,4′-dimethoxy-6,6′-dimethylbi-
phenyl-3,3′-dicarboxylate (15). A solution of iron(III)
chloride (10.6 g, 65.4 mmol) in diethyl ether (150 mL) and
ethanol (10 mL) was added to silica gel (37.1 g). The solvent
was removed to give a yellow solid, which was dried at 90 °C in
In conclusion, we designed and synthesized structure-
simplified gonytolide derivatives, and we found that bischro-
mone 10 and biflavone 13 promote the mammalian TNF-α
signaling pathway and Drosophila innate immunity. Although
the chemical synthesis of 1 is difficult because of its complex
dimeric chromanone, which includes four chiral centers and a
chiral axis, these structure-simplified derivatives without chiral
centers can be easily synthesized and obtained. Hence, they can
D
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vacuo. Then, a solution of 14 (4.60 g, 23.4 mmol) in
dichloromethane (50 mL) was added dropwisely to the silica
gel-bound iron(III) chloride. After well-mixing by rotation, the
solvent was removed in vacuo, and the resultant solid was kept
at 40 °C for 48 h. The solid was directly subjected to silica gel
column, and compound 15 (1.46 g, 3.74 mmol, 32%) was
afforded from the hexane−ethyl acetate (4:1) elutant. Data for
with ethyl acetate three times. The combined organic layer was
washed with saturated sodium bicarbonate solution and brine,
dried over anhydrous sodium sulfate, and concentrated in
vacuo. The residue was chromatographed over silica gel eluted
by hexane−ethyl acetate (2:1) to afford 18 (121 mg, 0.180
1
mmol, 59%). Data for 18: colorless oil; H NMR (400 MHz,
CDCl3, δ): 6.65 (s, 1H), 6.57 (dt, 1H, J = 16.0, 6.8 Hz), 6.33
(d, 1H, J = 16.0 Hz), 4.67 (d, 1H, J = 5.6 Hz), 4.63 (d, 1H, J =
5.6 Hz), 3.79 (s, 3H), 3.66 (s, 3H), 3.00 (s, 3H), 2.32 (t, 2H, J
= 7.6 Hz), 2.25−2.28 (m, 2H), 2.07 (s, 3H) 1.74−1.81 (m,
2H); 13C NMR (100 MHz, CDCl3, δ): 194.8, 173.5, 156.5,
153.0, 149.1, 141.0, 133.0, 123.3, 121.5, 108.5, 99.4, 56.5, 55.8,
51.5, 33.1, 31.6, 23.2, 20.7; LREIMS m/z 670 [M]+ (5%), 625
(100%), 569 (51%), 493 (79%); HREIMS m/z 670.2972 [M]+
(670.2989, calcd for C36H46O12).
1
15: yellowish amorphous solid; H NMR (600 MHz, CDCl3,
δ): 11.80 (s, 1H), 6.41 (s, 1H), 3.93 (s, 3H), 3.89 (s, 3H), 2.04
(s, 3H); 13C NMR (150 MHz, CDCl3, δ): 172.0, 161.0, 160.0,
146.1, 117.0, 104.0, 100.8, 56.1, 52.4, 20.7; LREIMS m/z 390
[M]+ (82%), 358 (42%), 343 (26%), 311 (100%); HREIMS m/
z 390.1296 [M]+ (390.1315, calcd for C20H22O8).
Dimethyl 4,4′-dimethoxy-2,2′-bis(methoxymethoxy)-6,6′-
dimethylbiphenyl-3,3′-dicarboxylate (16). To a solution of 15
(2.18 g, 5.58 mmol) in DMF (40 mL), we added sodium
hydride (60% oil suspension) (715 mg, 17.9 mmol) and
chloromethyl methyl ether (1.36 mL, 17.9 mmol) at 0 °C. After
being stirred for 3 h at room temperature, the mixture was
poured into 0.5 M hydrochloric acid and extracted with ethyl
acetate three times. The combined organic layer was washed
with saturated sodium bicarbonate solution and brine, dried
over anhydrous sodium sulfate, and concentrated in vacuo. The
residue was chromatographed over silica gel eluted by hexane-
ethyl acetate (4:1) to afford 16 (2.42 g, 5.06 mmol, 91%). Data
Dimethyl 4,4′-(5,5′-dimethoxy-7,7′-dimethyl-4,4′-dioxo-
8,8′-bichroman-2,2′-diyl)dibutanoate (19). Compound 18
(94.0 mg, 0.140 mmol) was dissolved in 10% hydrogen
chloride in methanol (2 mL). After being stirred for 18 h at 60
°C, the solution was concentrated in vacuo. The residue was
chromatographed over silica gel eluted by hexane−ethyl acetate
(1:1) to afford 19 (35.0 mg, 0.060 mmol, 59%) as a
1
diastereomeric mixture. Data for 19: colorless oil; H NMR
(400 MHz, CDCl3) 6.47−6.48 (m 1H), 4.23−4.28 (m, 1H),
3.94 (s, 3H), 3.59−3.62 (m, 3H), 2.51−2.64 (m, 2H), 2.10−
2.18 (m, 2H), 1.98−2.01 (m, 3H), 1.48−1.60 (m, 4H);
LREIMS m/z 582 [M]+ (100%), 567 (10%), 481 (18%);
HREIMS m/z 582.2448 [M]+ (582.2465, calcd for C32H38O10).
Dimethyl 4,4′-(5,5′-dimethoxy-7,7′-dimethyl-4,4′-dioxo-
4H,4′H-8,8′-bichromene-2,2′-diyl)dibutanoate (10). To a
solution of 19 (22.0 mg, 0.038 mmol) in dichloromethane (2
mL), we added pyridinium bromide perbromide (24.3 mg,
0.076 mmol) at 0 °C. After being stirred for 1 h, the mixture
was poured into water and extracted with ethyl acetate three
times. The combined organic layer was washed with saturated
sodium bicarbonate solution and brine, dried over anhydrous
sodium sulfate, and concentrated in vacuo. Then, the residue
was dissolved in benzene (1 mL), and DBU (20 μL, 0.134
mmol) was added to the solution at room temperature. After
being stirred for 1 h, the mixture was poured into 0.5 M
hydrochloric acid and extracted with ethyl acetate three times.
The combined organic layer was washed with brine, dried over
anhydrous sodium sulfate, and concentrated in vacuo. The
residue was chromatographed over silica gel eluted by ethyl
acetate-methanol (19:1) to afford 10 (12.2 mg, 0.021 mmol,
1
for 16: colorless oil; H NMR (400 MHz, CDCl3, δ): 6.65 (s,
1H), 4.75 (d, 1H, J = 5.6 Hz), 4.72 (d, 1H,J = 5.6 Hz), 3.87 (s,
3H), 3.84 (s, 3H), 3.00 (s, 3H), 2.05 (s, 3H); 13C NMR (100
MHz, CDCl3, δ): 166.9, 156.4, 153.4, 141.6, 123.2, 116.3,
108.5, 99.5, 56.5, 56.0, 52.3, 20.8; LREIMS m/z 478 [M]+
(15%), 402 (79%), 372 (100%); HREIMS m/z 478.1823 [M]+
(478.1839, calcd for C24H30O10).
Tetramethyl 2, 2′-(4, 4′-dimethoxy-2, 2′- bi s-
(methoxymethoxy)-6,6′-dimethylbiphenyl-3,3′-diyl)bis(2-
oxoethane-2,1-diyl)diphosphonate (17). To a solution of 16
(510 mg, 1.07 mmol) in THF (20 mL), we added dimethyl
methylphosphonate (0.60 mL, 5.52 mmol) and lithium
bis(trimethylsilyl)amide (1.0 M solution in THF) (11.0 mL,
11.0 mmol) at 0 °C. After being stirred for 12 h at room
temperature, the mixture was poured into 0.5 M hydrochloric
acid and extracted with ethyl acetate three times. The combined
organic layer was washed with saturated sodium bicarbonate
solution and brine, dried over anhydrous sodium sulfate, and
concentrated in vacuo. The residue was chromatographed over
silica gel eluted by ethyl acetate−methanol (9:1) to afford 17
1
1
(578 mg, 0.872 mmol, 81%). Data for 17: colorless oil; H
55% (2 steps)). Data for 10: colorless amorphous solid; H
NMR (400 MHz, CDCl3, δ): 6.66 (s, 1H), 4.72 (d, 1H, J = 6.0
Hz), 4.69 (d, 1H, J = 6.0 Hz), 3.86 (s, 3H), 3.79 (d, 3H, J = 2.0
Hz), 3.75 (d, 3H, J = 2.0 Hz), 3.61 (dd, 2H, J = 20.8, 3.6 Hz),
3.01 (s, 3H), 2.06 (s, 3H); 13C NMR (100 MHz, CDCl3, δ):
194.4 (d, J = 6.6 Hz), 156.4, 153.4, 142.4, 123.5, 122.4 (d, J =
4.6 Hz), 108.6, 100.1, 56.4, 55.8, 52.8 (d, J = 6.6 Hz), 52.7 (d, J
= 6.0 Hz), 42.7 (d, J = 131.0 Hz), 20.8; LREIMS m/z 662 [M]+
(13%), 617 (18%), 586 (57%), 556 (100%); HREIMS m/z
662.1878 [M]+ (662.1893, calcd for C28H40O14P2).
Dimethyl (5E,5′E)-7,7′-(4,4′-dimethoxy-2,2′-bis-
(methoxymethoxy)-6,6′-dimethylbiphenyl-3,3′-diyl)bis(7-ox-
ohept-5-enoate) (18). To a solution of 17 (200 mg, 0.302
mmol) in THF (8 mL), we added sodium hydride (60% in oil
dispersion) (28.8 mg, 0.72 mmol) at 0 °C. After 30 min, methyl
5-oxopentanoate15 (156 mg, 1.20 mmol) was added to the
mixture. After being stirred for 12 h at room temperature, the
mixture was poured into 0.5 M hydrochloric acid and extracted
NMR (400 MHz, CDCl3, δ): 6.80 (s, 1H), 6.04 (s, 1H), 4.04
(s, 3H), 3.62 (s, 3H), 2.34 (td, 2H, J = 7.2, 1.6 Hz), 2.14 (t, 2H,
J = 7.2 Hz), 2.10 (s, 3H) 1.63−1.67 (m, 2H); 13C NMR (100
MHz, CDCl3, δ): 178.1, 172.8, 165.4, 158.9, 155.8, 144.2,
115.9, 112.4, 111.4, 108.1, 56.3, 51.6, 32.5, 32.4, 21.3, 20.4;
LREIMS m/z 578 [M]+ (100%), 563 (21%), 549 (21%);
HREIMS m/z 578.2145 [M]+ (578.2152, calcd for C32H34O10).
Methyl (S)-4-(benzyloxy)-5-oxopentanoate (20). To a
solution of dimethyl (S)-2-(benzyloxy)pentanedioate17 (1.03
g, 3.87 mmol) in dichloromethane (14 mL), we added
diisobutylaluminum hydride (1.0 M in toluene) (4.2 mL, 4.2
mmol) at −80 °C. After 1 h, methanol (6 mL) was added to
the mixture, which was then warmed up to room temperature.
Saturated potassium sodium tartarate solution (25 mL) was
added, and the mixture stirred for 30 min. The mixture was
extracted with ethyl acetate three times. The combined organic
layer was washed with saturated sodium bicarbonate solution
E
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and brine, dried over anhydrous sodium sulfate, and
concentrated in vacuo. The residue was chromatographed
over silica gel eluted by hexane−ethyl acetate (9:1) to afford 20
(314 mg, 1.33 mmol, 37%). Data for 20: colorless oil; 1H NMR
(400 MHz, CDCl3, δ): 9.70 (s, 1H), 7.29−7.35 (s, 5H), 4.70
(d, 1H, J = 11.6 Hz), 4.39 (d, 1H, J = 11.6 Hz), 3.99 (dd, 1H, J
= 8.0, 4.8 Hz), 3.75 (s, 3H), 2.56 (t, 2H, J = 7.2 Hz), 2.02−2.16
(m, 2H); 13C NMR (100 MHz, CDCl3) δ 201.2, 172.5, 137.1,
128.4 (2C), 128.1 (2C), 128.0, 72.5, 72.4, 52.0, 29.2, 27.6;
LREIMS m/z 218 [M-H2O]+ (4%), 193 (3%), 177 (10%), 146
(22%), 130 (43%), 91 (100%); HREIMS m/z 218.0931 [M-
H2O]+ (218.0943, calcd for C13H14O3).
3H), 2.38−2.49 (m, 4H), 2.11 (s, 6H), 1.77−1.85 (m, 2H),
1.57−1.64 (m, 2H); 13C NMR (100 MHz, CDCl3, δ): 178.13,
178.10, 174.69, 174.62, 165.4 (0.5C), 165.37 (0.5C), 165.36
(0.5C), 165.34 (0.5C), 159.0, 158.9, 155.93 (0.5C), 155.919
(0.5C), 155.911 (0.5C), 155.8 (0.5C), 144.28, 144.25 (0.5C),
144.24 (0.5C), 115.96, 115.94, 112.5, 112.4, 111.38, 111.34
(0.5C), 111.33 (0.5C), 108.07, 108.03, 68.99 (0.5C), 68.97
(0.5C), 68.90, 56.3 (2C), 52.89 (0.5C), 52.87 (0.5C), 52.82
(0.5C), 52.81 (0.5C), 30.4 (0.5C), 30.3 (0.5C), 30.2 (0.5C),
30.1 (0.5C), 28.92 (0.5C), 28.91 (0.5C), 28.7, 20.4, 20.3;
LREIMS m/z 610 [M]+ (100%), 551 (36%); HREIMS m/z
610.2029 [M]+ (610.2050, calcd for C32H34O12).
Dimethyl (4S,4′S,5E,5′E)-7,7′-(4,4′-dimethoxy-2,2′-bis-
(methoxymethoxy)-6,6′-dimethylbiphenyl-3,3′-diyl)bis(4-
(benzyloxy)-7-oxohept-5-enoate) (21). A similar procedure to
that for the synthesis of 18 was used. From compounds 17
(240 mg, 0.362 mmol) and 20 (363 mg, 1.54 mmol),
compound 21 (70.0 mg, 0.079 mmol, 22%) was afforded as a
(2E,2′E)-1,1′-(4,4′-Dimethoxy-2,2′-bis(methoxymethoxy)-
6,6′-dimethylbiphenyl-3,3′-diyl)bis(7-(benzyloxy)hept-2-en-
1-one) (24). A similar procedure to the synthesis of 18 was
used. From compounds 17 (272 mg, 0.411 mmol) and 5-
(benzyloxy)pentanal (315 mg, 1.64 mmol), compound 24
(87.2 mg, 0.110 mmol, 27%) was obtained. Data for 24:
1
1
diastereomeric mixture. Data for 21: colorless oil; H NMR
colorless oil; H NMR (400 MHz, CDCl3, δ): 7.25−7.33 (m,
(400 MHz, CDCl3, δ): 7.23−7.34 (m, 5H), 6.64 (br. s, 1H),
6.53−6.60 (m, 1H), 6.31 (dd, 1H, J = 15.6, 3.2 Hz), 4.69 (d,
1H, J = 10.8 Hz), 4.60−4.66 (m, 2H), 4.39 (d, 1H, J = 10.8
Hz), 3.93−3.96 (m, 1H), 3.76 (s, 3H), 3.74 (s, 3H), 2.97 (s,
3H), 2.32−2.38 (s, 2H), 2.06 (s, 1.5H), 2.05 (s, 1.5H), 1.86−
1.91 (m, 2H); LREIMS m/z 882 [M]+ (14%), 837 (88%), 675
(100%), 631 (14%), 599 (34%), 383 (42%), 91 (56%);
HREIMS m/z 882.3817 [M]+ (882.3827, calcd for C50H58O14).
Dimethyl (4S,4′S)-4,4′-(5,5′-dimethoxy-7,7′-dimethyl-4,4′-
dioxo-8,8′-bichroman-2,2′-diyl)bis(4-(benzyloxy)butanoate)
(22). A similar procedure to the synthesis of 19 was used. From
compound 21 (65.4 mg, 0.074 mmol), compound 22 (28.9 mg,
0.036 mmol, 49%) was afforded as a diastereomeric mixture.
5H), 6.64 (s, 1H), 6.60 (dt, 1H, J = 16.0, 7.2 Hz), 6.32 (d, 1H, J
= 16.0 Hz), 4.66 (d, 1H, J = 5.6 Hz), 4.62 (d, 1H, J = 5.6 Hz),
4.48 (s, 2H), 3.77 (s, 3H), 3.45 (t, 2H, J = 6.4 Hz), 2.98 (s,
3H), 2.24 (dt, 2H, J = 7.2, 6.8 Hz), 2.07 (s, 3H), 1.53−1.69 (m,
4H); 13C NMR (100 MHz, CDCl3, δ): 195.0, 156.4, 153.0,
150.5, 140.8, 138.4, 132.5, 128.5 (2C), 127.5 (2C), 127.4,
123.3, 121.6, 108.5, 99.4, 72.8, 69.8, 56.4, 55.7, 32.2, 29.2, 24.6,
20.7; LREIMS m/z 794 [M]+ (5%), 749 (100%), 631 (41%),
279 (44%), 261 (40%), 91 (79%); HREIMS m/z 794.4016
[M]+ (794.4030, calcd for C48H58O10).
2,2′-Bis(4-(benzyloxy)butyl)-5,5′-dimethoxy-7,7′-dimethyl-
8,8′-bichroman-4,4′-dione (25). A similar procedure to the
synthesis of 19 was used. From compound 24 (56.0 mg, 0.070
mmol), compound 25 (24.8 mg, 0.035 mmol, 49%) was
afforded as a diastereomeric mixture. Data for 25: colorless oil;
1H NMR (400 MHz, CDCl3, δ): 7.25−7.38 (m, 10H), 6.45 (s,
1H), 6.38 (s, 1H), 4.41−4.52 (m, 4H), 4.18−4.27 (m, 2H),
3.86−3.95 (s, 6H), 3.25−3.37 (m, 4H), 2.50−2.59 (m, 4H),
1.96−2.04 (m, 6H), 1.44−1.67 (m, 8H), 1.23−1.31 (m, 4H);
LREIMS m/z 706 [M]+ (100%), 616 (28%), 543 (86%), 437
(86%); HREIMS m/z 706.3471 [M]+ (706.3506, calcd for
C44H50O8).
1
Data for 22: colorless oil; H NMR (400 MHz, CDCl3, δ):
7.28−7.31 (m, 5H), 6.44 (br. s, 1H), 4.56−4.61 (m, 1H),
4.21−4.28 (m, 1H), 4.09−4.22 (m, 1H), 3.90−3.93 (m, 3H),
3.77−3.81 (m, 1H), 3.66−3.69 (m, 3H), 2.48−2.60 (m, 2H),
1.97−2.02 (m, 3H), 1.54−1.73 (m, 4H); LREIMS m/z 794
[M]+ (95%), 688 (22%), 673 (17%), 602 (26%), 597 (32%),
587 (57%), 481 (100%); HREIMS m/z 794.3297 [M]+
(794.3302, calcd for C46H50O12).
Dimethyl (4S,4′S)-4,4′-(5,5′-dimethoxy-7,7′-dimethyl-4,4′-
dioxo-8,8′-bichroman-2,2′-diyl)bis(4-hydroxybutanoate)
(23). Under hydrogen atmosphere, compound 22 (30.0 mg,
0.038 mmol) and 20% palladium hydroxide on carbon (6.0 mg)
in methanol (1.0 mL) was stirred at room temperature for 2 h.
After filtration through a Celite pad, the filtrate was
concentrated in vacuo. The residue was chromatographed
over silica gel eluted by hexane−ethyl acetate (1:4) to afford 23
(20.0 mg, 0.033 mmol, 88%) as a diastereomeric mixture. Data
for 23: colorless oil; 1H NMR (400 MHz, CDCl3, δ): 6.43 (m,
1H), 4.13−4.28 (m, 1H), 3.75−4.00 (m, 1H), 3.88−3.91 (m,
3H), 3.64−3.68 (m, 3H), 2.47−2.62 (m, 2H), 1.91−1.98 (m,
3H), 1.58−1.70 (m, 4H); LREIMS m/z 614 [M]+ (100%), 497
(34%), 481 (33%); HREIMS m/z 614.2382 [M]+ (614.2363,
calcd for C32H38O12).
2,2′-Bis(4-hydroxybutyl)-5,5′-dimethoxy-7,7′-dimethyl-
8,8′-bichroman-4,4′-dione (26). A similar procedure to the
synthesis of 23 was used. From compound 25 (14.8 mg, 0.021
mmol), compound 26 (10.2 mg, 0.019 mmol, 90%) was
afforded as a diastereomeric mixture. Data for 26: colorless oil;
1H NMR (400 MHz, CDCl3, δ): 6.46 (s, 1H), 4.20−4.31 (m,
1H), 3.93 (s, 3H), 3.44−3.53 (m, 2H), 2.55 (m, 2H), 1.98−
2.05 (s, 3H), 1.58−1.65 (m, 2H), 1.34−1.47 (m, 2H), 1.22−
1.29 (m, 2H); LREIMS m/z 526 [M]+ (100%), 453 (40%);
HREIMS m/z 526.2564 [M]+ (526.2567, calcd for C30H38O8).
Dimethyl (4S,4′S)-4,4′-(5,5′-dimethoxy-7,7′-dimethyl-4,4′-
dioxo-4H,4′H-8,8′-bichromene-2,2′-diyl)bis(4-hydroxybuta-
noate) (11). A similar procedure to the synthesis of 10 was
used. From compound 26 (7.4 mg, 0.014 mmol), compound
11 (4.3 mg, 0.008 mmol, 59%) was obtained. Data for 11:
Dimethyl (4S,4′S)-4,4′-(5,5′-dimethoxy-7,7′-dimethyl-4,4′-
dioxo-4H,4′H-8,8′-bichromene-2,2′-diyl)bis(4-hydroxybuta-
noate) (9). A similar procedure to the synthesis of 10 was used.
From compound 23 (21.2 mg, 0.034 mmol), compound 9 (4.9
mg, 0.008 mmol, 23%) was afforded as a diastereomeric
mixture. Data for 9: colorless amourphous solid; 1H NMR (400
MHz, CDCl3, δ): 6.79 (s, 1H), 6.78 (s, 1H), 6.06 (s, 1H), 6.05
(s, 1H), 4.03 (s, 6H), 3.95−4.02 (m, 2H), 3.73 (s, 3H), 3.71 (s,
1
colorless amourphous solid; H NMR (400 MHz, CDCl3, δ):
6.78 (s, 1H), 6.04 (s, 1H), 4.03 (s, 3H), 3.46 (br. s, 2H), 2.31
(br. s, 2H), 2.12 (s, 3H), 1.60−1.69 (m, 2H), 1.32−1.39 (m,
2H); 13C NMR (100 MHz, CDCl3, δ): 178.4, 166.4, 158.9,
156.0, 144.1, 116.1, 112.5, 111.2, 107.9, 62.0, 56.3, 33.0, 31.4,
22.5, 20.4; LREIMS m/z 522 [M]+ (100%), 507 (19%), 425,
F
DOI: 10.1021/acs.jnatprod.5b00829
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(12%) 355 (26%); HREIMS m/z 522.2237 [M]+ (522.2254,
calcd for C30H34O8).
BRL) in each well of a 96 well plate at 25 °C. For each
condition, six females were cultured to produce six replicates.
The test compounds were dissolved in DMSO and added to
the culture medium. To determine the effects of the test
compounds on the innate immune response, we cultured Dpt-
lacZ larvae in the presence of 100 ng/mL peptidoglycans from
Escherichia coli (InvivoGen, San Diego, CA) and the compound
at 25 °C for 12 h. The cultured individual larvae were sonicated
with 200 μL of reaction buffer (60 mM Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, and 1 mM MgCl2) using an ultrasonic
processor (Misonix, New York, NY). After centrifugation
(10000g) at 4 °C for 10 min, supernatant was harvested, and β-
galactosidase activity and total protein amount of supernatant
were determined as previously described.5a β-Galactosidase
activity was normalized to total protein amount.
Measurement of Cytotoxicity. Drosophila S2 cells were
cultured in Schneider’s medium (Gibco-BRL) supplemented
with 10% FBS and 1% antibiotics and antimycotics at 25 °C.
Cytotoxicity was measured using the colorimetric thiazoyl blue
coversion assay using WST-8 solution (Nacalai Tesque) as
described previously.5a
Chemokine Assay. The detailed procedure was described
previously.5a Briefly, human umbilical vein endothelial cells
(HUVECs) were purchased from Lonza (Walkersville, MA),
cultured in 25 cm2 culture flasks, and maintained in EGM-2
medium (Lonza)) at 5% CO2 and 37 °C. The cells were
incubated in the presence or absence of the compound in a final
volume of 100 μL for 3 h with 5% CO2 at 37 °C, and then 11
μL of 10 ng/mL hTNF-α was added to each well. After 12 h of
incubation with 5% CO2 at 37 °C, the HUVEC culture
supernatants were harvested for enzyme-linked immunosorbent
assay. Commercial enzyme-linked immunosorbent assay kits
were used for immunologic quantification of hIL-8 (Biosource,
Invitrogen).
4,4′-(5,5′-Dimethoxy-7,7′-dimethyl-4,4′-dioxo-4H,4′H-
8,8′-bichromene-2,2′-diyl)bis(butane-4,1-diyl) diethanoate
(12). To a solution of 11 (2.5 mg, 0.005 mmol) in pyridine
(0.5 mL), we added acetic anhydride (10 μL) at room
temperature. After being stirred for 15 h, the mixture was
concentrated in vacuo. The residue was chromatographed over
silica gel eluted by ethyl acetate to afford 12 (2.2 mg, 0.004
1
mmol, 78%). Data for 12: colorless amourphous solid; H
NMR (400 MHz, CDCl3, δ): 6.79 (s, 1H), 6.04 (s, 1H), 4.03
(s, 1H), 3.91 (t, 2H, J = 6.0 Hz), 2.30 (t, 2H, J = 7.2 Hz), 2.09
(s, 3H), 2.01 (s, 3H), 1.37−1.47 (m, 4H); 13C NMR (100
MHz, CDCl3, δ): 178.1, 170.9, 165.8, 159.0, 155.9, 144.1,
115.9, 112.5, 111.3, 107.9, 63.5, 56.3, 32.7, 27.5, 22.7, 20.8,
20.4; LREIMS m/z 606 [M]+ (100%), 591 (25%), 549 (25%),
429 (28%), 355 (33%); HREIMS m/z 606.2472 [M]+
(606.2465, calcd for C34H38O10).
5,5′-Dimethoxy-2,2′-bis(4-methoxyphenyl)-7,7′-dimethyl-
8,8′-bichroman-4,4′-dione (28). To a solution of 17 (187 mg,
0.282 mmol) in THF (8 mL), we added sodium hydride (60%
in oil dispersion) (27.6 mg, 0.691 mmol) at 0 °C. After 30 min,
p-anisaldehyde (230 mg, 1.69 mmol) was added to the mixture.
After being stirred for 8 h at room temperature, the mixture was
poured into 0.5 M hydrochloric acid and extracted with ethyl
acetate three times. The combined organic layer was washed
with saturated sodium bicarbonate solution and brine, dried
over anhydrous sodium sulfate, and concentrated in vacuo to
give crude 27. This crude was dissolved in 10% hydrogen
chloride in methanol (4 mL). After being stirred for 15 h at 60
°C, the solution was concentrated in vacuo. The residue was
chromatographed over silica gel eluted by hexane−ethyl acetate
(1:3) to afford 19 (25.0 mg, 0.042 mmol, 15% (2 steps)) as a
diastereomeric mixture. Data for 28: yellowish amourphous
solid; 1H NMR (400 MHz, CDCl3, δ): 7.00 (d, 2H, J = 8.8 Hz),
6.92−6.97 (m, 2H), 6.73 (d, 2H, J = 8.8 Hz), 6.65 (d, 1.1H, J =
8.8 Hz), 6.62 (d, 0.9H, J = 8.8 Hz), 6.43 (s, 0.6H), 6.42 (s,
0.4H), 6.41 (s, 0.6H), 6.40 (s, 0.4H), 5.25−5.32 (m, 1.1H),
5.21 (dd, 0.5H, J = 7.6, 7.6 Hz), 5.01 (dd, 0.4H, J = 12.0, 3.6
Hz), 3.89 (s, 1.5H), 3.88 (s, 2.5H), 3.87 (s, 2.0H), 3.71 (s,
1.4H), 3.69 (s, 1.6H), 3.64 (s, 1.7H), 3.63 (s, 1.3H), 2.64−2.81
(m, 4H), 1.95−2.04 (m, 6H); LREIMS m/z 594 [M]+ (100%),
431 (40%), 311 (19%); HREIMS m/z 594.2255 [M]+
(594.2254, calcd for C36H34O8).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00829.
Figures showing the NMR and HMQC spectra of the
new compounds. (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: hal@mail.pharm.tohoku.ac.jp. Phone: +81-22-795-
6824. Fax: +81-22-795-6821.
■
5,5′-Dimethoxy-2,2′-bis(4-methoxyphenyl)-7,7′-dimethyl-
4H,4′H-8,8′-bichromene-4,4′-dione (11). A similar procedure
to the synthesis of 10 was used. From compound 28 (10.0 mg,
0.017 mmol), compound 13 (5.5 mg, 0.009 mmol, 58%) was
1
Notes
obtained. Data for 13: colorless amourphous solid; H NMR
The authors declare no competing financial interest.
(400 MHz, CDCl3, δ): 7.17 (d, 2H, J = 9.2 Hz), 6.81 (s, 1H),
6.71 (d, 2H, J = 9.2 Hz), 6.57 (s, 1H), 4.04 (s, 3H), 3.72 (s,
3H), 2.12 (s, 3H); 13C NMR (100 MHz, CDCl3, δ): 178.6,
162.1, 160.8, 159.0, 155.6, 144.6, 127.2 (2C), 123.2, 116.3,
114.5 (2C), 112.7, 108.1, 107.0, 56.5, 55.4, 20.6; LREIMS m/z
590 [M]+ (100%), 575 (11%), 561 (16%), 543 (6%); HREIMS
m/z 590.1942 [M]+ (590.1941, calcd for C36H30O8).
Ex vivo Drosophila Culture Assay. The detailed
procedure was described previously.5a Briefly, the abdominal
cavity of third-instar larva was opened using fine pincettes.
Individual whole larval tissues were cultured in Schneider’s
Drosophila medium (Gibco-BRL, Invitrogen, Carlsbad, CA)
containing 20% fetal bovine serum (Valley Biomedical,
Winchester, VA) and 1% antibiotics and antimycotics (Gibco-
ACKNOWLEDGMENTS
■
This work was supported in part by Grant-in-Aid for Scientific
Research (no. 23710247 and no. 25350959) from the Ministry
of Education, Culture, Sports, Science, and Technology, Japan;
the Platform Project for Supporting in Drug Discovery and Life
Science Research from the Ministry of Education, Culture,
Sports, Science, and Technology (MEXT) and the Japan
Agency for Medical Research and development (AMED); the
Program for the Promotion of Basic Research Activities for
Innovative Biosciences (PROBRAIN); the SUNBOR GRANT
from the Suntory Institute for Bioorganic Research; and the
Astellas Foundation for Research on Metabolic Disorders.
G
DOI: 10.1021/acs.jnatprod.5b00829
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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