Structures of the dimeric and monomeric chromanones, gonytolides A-C, isolated from the fungus Gonytrichum sp. and their promoting activities of innate immune responses

Article abstract of DOI:10.1021/ol2018449

Innate immunity is the front line of self-defense against microbial infection. After searching for natural substances that regulate innate immunity using an ex vivo Drosophila culture system, we identified a novel dimeric chromanone, gonytolide A, as an i

Full text of DOI:10.1021/ol2018449

ORGANIC
LETTERS
2011
Vol. 13, No. 17
4624–4627
Structures of the Dimeric and Monomeric
Chromanones, Gonytolides AꢀC, Isolated
from the Fungus Gonytrichum sp. and
Their Promoting Activities of Innate
Immune Responses
Haruhisa Kikuchi,* Masato Isobe, Mizuki Sekiya, Yuko Abe, Tsuyoshi Hoshikawa,
Kazunori Ueda, Shoichiro Kurata, Yasuhiro Katou, and Yoshiteru Oshima
Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku,
Sendai 980-8578, Japan
hal@mail.pharm.tohoku.ac.jp
Received July 9, 2011
ABSTRACT
Innate immunity is the front line of self-defense against microbial infection. After searching for natural substances that regulate innate immunity
using an ex vivo Drosophila culture system, we identified a novel dimeric chromanone, gonytolide A, as an innate immune promoter from the
fungus Gonytrichum sp. along with gonytolides B and C. Gonytolide A also increased TNF-R-stimulated production of IL-8 in human umbilical vein
endothelial cells.
Innate immunity is the front line of self-defense against
microbial infection,1,2 and the basic mechanisms of this
process, including pathogen recognition and immune re-
sponse activation, are evolutionarily conserved.3 In mam-
mals, innate immunity interacts with adaptive immunity
and plays a key role in regulating the immune response.4
Therefore, innate immunity is a good pharmaceutical
target for the development of immune regulators to sup-
press unwanted immune responses, such as septic shock,
inflammatory diseases, and autoimmunity. The innate
immune system also provides targets for the development
of agents that stimulate protective immune responses to-
ward some diseases, such as infectious diseases and cancer.
To screen pharmaceuticals that target innate immunity,
we established an ex vivo culture system based on the
DrosophilaIMD (immunedeficiency) signaling pathway.5,6
Because of the striking conservation between the mechan-
isms that regulate insect immunity and mammalian innate
immunity,2,3 Drosophila is a model organism for genetic
and molecular studies of innate immunity, and our culture
system has proven to be useful for identifying immune
regulators that act on human innate immunity. We used
this system to search for natural substances that regulate
(1) Takeda, K.; Akira, S. Int. Immunol. 2005, 17, 1–14.
(2) Hoffmann, J. A.; Reichhart, J. M. Nat. Immunol. 2002, 3, 121–
126.
(3) Hultmark, D. Curr. Opin. Immunol. 2004, 15, 12–19.
(4) Janeway, C. A., Jr. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7461–
7468.
(5) Yajima, M.; Takada, M.; Takahashi, N.; Kikuchi, H.; Natori, S.;
Oshima, Y.; Kurata, S. Biochem. J. 2003, 371, 205–210.
(6) Sekiya, M.; Ueda, K.; Fujita, T.; Kitayama, M.; Kikuchi, H.;
Oshima, Y.; Kurata, S. Life Sci. 2006, 80, 113–119.
r
10.1021/ol2018449
Published on Web 08/09/2011
2011 American Chemical Society

innate immunity.7ꢀ10 This paper describes the isolation and
structure elucidation of the novel dimeric and monomeric
chromanones, gonytolides AꢀC (1ꢀ3), from the fungus
Gonytrichum sp. The promoting activities of gonytolide A
(1) and its derivatives on innate immune responses are also
described.
Gonytrichum sp. was cultivated in production medium
containing cottonseed proteins. The culture broth (10 L)
was extracted with butanol at room temperature to yield
an extract (21.1 g). In the ex vivo Drosophila culture
system,6 100 μg/mL of the extract increased the innate
immune response by 176%. Then, bioactivity-guided frac-
tionation of the extract yielded gonytolide A (1) (87 mg).
Gonytolide B (2) (3 mg) and C (3) (2 mg) were also isolated
from the extract.
elucidated. Finally, taking into account the molecular
formula C32H30O14 and the structural symmetry, two
subunits I were proposed to be linked through the remain-
ing quaternary carbons C-8 and C-80 to give the planar
structure of gonytolide A (1).
The relative configuration of gonytolide A (1) was de-
termined by X-ray single-crystal analysis. The crystals of 1
were obtained by recrystallization from hexane/ethyl acet-
ate. The ORTEP diagram of 1 indicated that the relative
configuration was S*a, 2R*, 9S*, 20R*, 90S* (Figure 2).
HREIMS (m/z 638.1616 [Mþ]) indicated a molecular
formula of gonytolide A (1) as C32H30O14. The 13C NMR
spectrum of 1 showed the presence of the following carbon
atoms: one keto carbonyl, two ester carbonyl, five sp2
quaternary, one sp2 tertiary, one oxygenated quaternary,
one oxymethine, one methoxyl, three methylene, and one
methyl (Table S1 in Supporting Information). Because
only 16 carbons were observed in the 13C NMR spectrum
(150 MHz, CDCl3), compound 1 was deduced to have a
symmetrical structure. The signals of five sp2 quaternary
(δ 161.1, 156.1, 150.2, 113.7 and105.9) andonesp2 tertiary
carbons (δ 111.0) indicated the presence of a pentasub-
Figure 2. ORTEP stereo diagram of gonytolide A (1).
To determine the absolute configuration of 1, the fol-
lowing transformation was conducted (Scheme 1). γ-Lac-
tone ring opened compound 4 was obtained by the
treatment of 1 with HCl in methanol. Esterification of 4
with (R)-R-methoxyphenylacetic acid in the presence of
EDCI and DMAP yielded 9O,90O-(R)-MPA diester 5a.
In a similar manner, (S)-MPA diester 5b was afforded. The
ΔδRS value of each proton was calculated from the differ-
ence in the chemical shifts of 5a and 5b in 1H NMR spectra
(600 MHz, CDCl3), and the structure of 4 was fit into the
proposed model of R-methoxyphenylacetate11 in accor-
dance with the sign of ΔδRS. As a result, C-9 and C-90 had
S-configurations, and the absolute configuration of 1 was
determined to be Sa, 2R, 9S, 20R, 90S.
1
stituted benzene ring. The H NMR signal (Table S2,
Supporting Information) at δ 11.45 (1H, s) was assigned
to a phenolic proton hydrogen-bonded to a carbonyl
group, and the HMBC correlations of this phenolic pro-
ton to C-4a, C-5 and C-6 were observed. In addition, the
correlations of H-6 to C-5, C-8 and C-14, and of H3-14 to
C-6, C-7 and C-8 in the HMBC spectrum provided
evidence for the partial structure A (Figure 1). The
remaining sp2 carbon (δ 156.1) was attached to an oxygen
atom due to its chemical shift, and was assigned to C-8a.
1
1
On the other hand, Hꢀ H COSY revealed the connec-
tivity between C-9ꢀC-10, and the HMBC correlations for
H-9 to C-12; H-10 to C-11; and H-11 to C-10 and C-12
indicated a γ-lactone moiety. HMBC correlations were
observed for H3-15 to C-13; and H2-3 to C-2, C-4, C-9 and
C-13, which suggested the partial structure B. Due to the
HMBC correlation for H-3 to C-4a and the remaining
oxygen atom, the partial structures A and B were con-
nected through the C-4ꢀC-4a bond and the C-2ꢀOꢀC-8a
ether bond, respectively, and the structure of subunit I was
Scheme 1. Conversion of Gonytolide A (1) into MPA Diesters
5a and 5b
(7) Sekiya, M.; Ueda, K.; Okazaki, K.; Kikuchi, H.; Kurata, S.;
Oshima, Y. Biochem. Pharmacol. 2008, 75, 2165–2174.
(8) Kikuchi, H.; Okazaki, K.; Sekiya, M.; Uryu, Y.; Ueda, K.;
Katou, Y.; Kurata, S.; Oshima, Y. Eur. J. Med. Chem. 2011, 46, 1263–
1273.
Figure 1. Planar structure of gonytolide A (1).
Org. Lett., Vol. 13, No. 17, 2011
4625

The HREIMS of gonytolide B (2) (m/z 638.1613 [Mþ])
gave the molecular formula, C32H30O14, which was iden-
tical to that of 1. The 13C NMR spectrum (150 MHz,
CDCl3) of 2 showed two sets of 16 signals (C-2ꢀC-15 and
C-20ꢀC-150, respectively), and the chemical shifts of each
signal set were nearly identical to those of 1 (Table S1,
Supporting Information). The 1H NMR spectrum of 2 was
also similar to that of 1 (Table S2, Supporting Infor-
mation). As a result, compound 2 was composed of two
chromanone moieties, subunits I in the structure of 1,
which were bonded asymmetrically. The correlations of
C-2ꢀC-15 were the same as those observed for 1 in the
HMBC spectrum of 2, indicating that the subunit I was
linked to the other subunit through the quaternary carbon
C-8 (Figure 3). On the other hand, the HMBC correlations
of the C-50 phenolic proton (δH 11.72) to C-40a, C-50 and
C-60; H-80 to C-60 and C-80a; and H3-14 to C-60, C-70 and
C-80 suggested that another subunit I was connected to the
other one through the quaternary carbon C-60, and the
planner structure of 2 was thereby elucidated. Because
only a small amount of 2 was isolated, it was difficult
to carry out its chemical conversion and characterize
its stereochemistry. However, compounds 1 and 2 are
likely biosynthesized by the same pathway, suggesting
that the absolute configuration of 2 may be the same as
that of 1.
Information). Recently, racemic 3 was synthesized12 as the
intermediate compound in a process for the synthesis of
(()-blennolide C.13
We evaluated the promoting activities of 1ꢀ3 on Dro-
sophila innate immune response using the ex vivo Droso-
phila culture system.6 Gonytolide A (1), at up to 1 μg/mL
dosage, showed immune response-promoting activity, and
10 μg/mL of 1 increased to more than 4.5 times of the
Drosophila innate immune response (Figure 4). On the
other hand, compounds 2 and 3 showed no activity at 10
μg/mL (Figure S2, Supporting Information). These find-
ings indicated that the linkage of the two chromanone
moieties (e.g., subunit I) through C-8 and C-80 is important
for innate immune-promoting activity.
Figure 4. Effect of gonytolide A (1) on DAP-type peptidogly-
cans-mediated activation of Drosophila Dpt-lacZ. DAP-type
peptidoglycans-mediated activation of Dpt-lacZ (0) and Dro-
sophila S2 cell viability (b) are represented as the percent relative
to the control (DMSO). The bars indicate the standard errors of
three independent measurements. *p < 0.05 and **p < 0.01 vs
control (DMSO).
The Drosophila IMD signaling pathway resembles the
mammalian TNF-R signaling pathway.2,14 The TNF-R
signaling pathway plays a critical role in the host defense
against several pathogens, the intrinsic tumor suppression,
and the inflammatory response by producing costimula-
tory molecules, cytokines, chemokines, and adhesion mo-
lecules, through activation of NF-κB. We investigated the
effectof gonytolide A (1) on TNF-R-stimulatedproduction
of IL-8, a neutrophil chemotactic factor, in human umbi-
lical vein endothelial cells (HUVECs). As shown in Figure
5, 10μg/mL of compound1 increased the production of IL-
8 by 70%. Thus, compound 1 promoted the innate immune
response through the mammalian TNF-R signaling path-
way as well as through the Drosophila IMD pathway.
To reveal the structural requirements of gonytolide A (1)
for the innate immune-promoting activity, we synthesized
several derivatives by using natural gonytolide A (1).
Treatment of 1 with sulfuryl chloride produced 6,60-di-
chlorinated derivative 6 (Scheme 2). Methylation of 1 by
trimethylsilyldiazomethane in the presence of DIPEA
Figure 3. Planar structure of gonytolide B (2).
The HREIMS of gonytolide C (3) (m/z 320.0871 [Mþ])
gave the molecular formula, C16H16O7, which was equiva-
lent to a half of molecular formula of 1 (C32H30O14) plus
1
one hydrogen atom. The H and 13C NMR spectra of 3
were nearly identical to those of 1, although the signal of
H-8 (δ 6.38) emerged in the 1H NMR of 3 (Tables S1 and
S2, Supporting Information). These facts indicated that
compound 3 was a monomeric unit of 1. The absolute
configuration of 3 was determined by applying the same
transformation as was used to determine the absolute
configuration of 1 (Scheme S1 and Figure S1, Supporting
(9) Kikuchi, H.; Sekiya, M.; Katou, Y.; Ueda, K.; Kabeya, T.;
Kurata, S.; Oshima, Y. Org. Lett. 2009, 11, 1693–1695.
(10) Sekiya, M.; Ueda, K.; Okazaki, K.; Terashima, J.; Katou, Y.;
Kikuchi, H.; Kurata, S.; Oshima, Y Int. Immunopharmacol. 2011,
in press. doi:10.1016/j.intimp.2011.05.001.
€
(13) Zhang, W.; Krohn, K.; Zia-Ullah; Florke, U.; Pescitelli, G.; Di
(11) Trost, B. M.; Balletire, J. L.; Godleski, S.; McDougal, P. G.;
Balkovec, J. M. J. Org. Chem. 1986, 51, 2370–2374.
(12) Qin, T.; Johnson, P.; Porco, J. A., Jr. J. Am. Chem. Soc. 2011,
133, 1714–1717.
ꢀ
Bari, L.; Antus, S.; Kurtan, T.; Rheinheimer, J.; Draeger, S.; Schulz, B.
Chem.;Eur. J. 2008, 14, 4913–4923.
(14) Silverman, N.; Maniatis, T. Genes Dev. 2001, 15, 2321–42.
4626
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Figure 6. Effects of compounds (A) 6, (B) 7 and (C) 4 on DAP-
type peptidoglycans-mediated activation of Drosophila Dpt-
lacZ. DAP-type peptidoglycans-mediated activation of Dpt-
lacZ (0) and Drosophila S2 cell viability (b) are represented as
the percent relative to the control (DMSO). The bars indicate
the standard errors of three independent measurements. *p <
0.05 and **p < 0.01 vs control (DMSO).
Figure 5. Effect of gonytolide A (1) on IL-8 production induced
by TNF-R in HUVECs. HUVECs were treated with various
concentrations of 1 for 3 h prior to stimulation with 1 ng/mL of
TNF-R. The bars indicate the standard errors of four indepen-
dent measurements. **p < 0.01 vs absence of gonytolide A (1).
A few dimeric and monomeric chromanones substituted
with the γ-lactone moiety have been isolated from fila-
mentous fungi,13,15ꢀ23 and showed several biological ac-
tivities. However, this is the first report of the promoting
activity of an innate immune response by chromanone-
type compounds. Gonytolide A (1) and its biologically
active derivatives 4, 6 and 7 can be used as lead compounds
for novel immunostimulating agents against bacterial in-
fections and tumors, specific adjuvants and as probes for
innate immune system.
Scheme 2. Conversion of Gonytolide A (1) into 6 and 7
Acknowledgment. We are grateful to the Bloomington
Stock Center, Drosophila Genetic Resource Center at the
Kyoto Institute of Technology, and the Genetic Strain
Research Center of the National Institute of Genetics for
the fly stocks. This work was supported in part by a Grant-
in-Aid for Scientific Research (No. 21249004, 21117005,
21310134, 21117005 and 21710215) from the Ministry of
Education, Science, Sports and Culture of Japan; the Uehara
Memorial Foundation; the Takeda Science Foundation; the
Mitsubishi Foundation; and Program for Promotion of
Basic Research Activities for Innovative BioSciences.
afforded 5O,50O-dimethyl compound 7. The promoting
activities of 6, 7 and γ-lactone ring opened deivative 4
(Scheme 1) on Drosophila innate immune response were
evaluated (Figure 6). Because compound 6 showed almost
the same activity as 1, introduction of the substituents into
benzene rings is tolerant for the activity. Compound 4 also
maintained the activity, indicating that the γ-lactone
moiety is not crucial. The innate immune-promoting
activity of 7 was weaker than that of 1, because 10 μg/
mL of 7 increased the innate immune response by a
factor of only 2.5. Thus, the phenolic hydroxy groups at
C-5/50 and the hydrogen bonds between these groups
and the carbonyl groups at C-4/40 are not critical, but
may be effective in promoting activity.
Supporting Information Available. Tables, Figures,
Schemes, Experimental methods, NMR spectra of new
compounds and the cif file for gonytolide A (1). This
material is available free of charge via the Internet at
http://pubs.acs.org.
(19) Rukachaisirikul, V.; Chantaruk, S.; Pongcharoen, W.; Isaka,
M.; Lapanun, S. J. Nat. Prod. 2006, 69, 980–982.
(20) Guo, Z; She, Z.; Shao, C.; Wen, L.; Liu, F.; Zheng, Z.; Lin, Y.
Magn. Reson. Chem. 2007, 45, 777–780.
(21) Pontius, A.; Krick, A.; Kehraus, S.; Foegen, S. E.; Muller, M.;
(15) Franck, B.; Flasch, H. Fortschr. Chem. Org. Naturst. 1973, 30,
151–206.
€
€
€
Klimo, K.; Gerhauser, C.; Konig, G. M. Chem.;Eur. J. 2008, 14, 9860–
9863.
(22) Pontius, A.; Krick, A.; Mesry, R.; Kehraus, S.; Foegen, S. E.;
(16) Tabata, N.; Tomoda, H.; Matsuzaki, K.; Omura, S. J. Am.
Chem. Soc. 1993, 115, 8558–8564.
(17) Tabata, N.; Tomoda, H.; Iwai, Y.; Omura, S. J. Antibiot. 1996,
49, 267–271.
(18) Kanokmedhakul, S.; Kanokmedhakul, K.; Phonkerd, N.; Soy-
tong, K.; Kongsaeree, P.; Suksamrarn, A. Planta Med. 2002, 68, 834–
836.
€
€
€
Muller, M.; Klimo, K.; Gerhauser, C.; Konig, G. M. J. Nat. Prod. 2008,
71, 1793–1799.
(23) Yang, J.; Xu, F.; Huang, C.; Li, J.; She, Z.; Pei, Z.; Lin, Y. Eur. J.
Org. Chem. 2010, 3692–3695.
Org. Lett., Vol. 13, No. 17, 2011
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