Revised structure and synthesis of celastramycin A, A potent innate immune suppressor

Article abstract of DOI:10.1021/ol9002306

After searching for natural substances that regulate Innate Immunity using the ex vivo Drosophila culture system, a benzoyl pyrrole-type compound, celastramycin A, was Identified and Isolated as a potent suppressor. By synthesizing the previously reported

Full text of DOI:10.1021/ol9002306

ORGANIC
LETTERS
2009
Vol. 11, No. 8
1693-1695
Revised Structure and Synthesis of
Celastramycin A, A Potent Innate
Immune Suppressor
Haruhisa Kikuchi,* Mizuki Sekiya, Yasuhiro Katou, Kazunori Ueda,
Takahiro Kabeya, Shoichiro Kurata, and Yoshiteru Oshima*
Graduate School of Pharmaceutical Sciences, Tohoku UniVersity, Aoba-yama,
Aoba-ku, Sendai 980-8578, Japan
hal@mail.pharm.tohoku.ac.jp; oshima@mail.pharm.tohoku.ac.jp
Received February 3, 2009
ABSTRACT
After searching for natural substances that regulate innate immunity using the ex vivo Drosophila culture system, a benzoyl pyrrole-type
compound, celastramycin A, was identified and isolated as a potent suppressor. By synthesizing the previously reported structure 1 and
another benzoyl pyrrole-type compound 2 reported in a Japanese patent, the correct structure of celastramycin A was confirmed to be 2.
Compound 2 suppressed the production of IL-8 (IC50 0.06 µg/mL) in human umbilical vein endothelial cells (HUVECs).
Innate immunity is the first line of defense against infectious
microorganisms,1,2 and the basic mechanisms of this process,
including pathogen recognition and immune response activa-
tion, are evolutionarily conserved.3 In mammals, innate
immunity interacts with adaptive immunity and has a key
role in regulating the immune response.4 Therefore, innate
immunity is a good target for the development of immune
regulators that suppress unwanted immune responses, such
as septic shock, inflammatory diseases, and autoimmunity.
For example, eritoran, an LPS (lipopolysaccharide) antago-
nist,5 and TAK-242, an inhibitor of the TLR4 (Toll-like
receptor 4)-induced signaling pathway,6 are in clinical trials
for treatment of severe sepsis.
To screen pharmaceuticals that target innate immunity, we
established an ex vivo culture system based on the innate
immune response of Drosophila, which is highly useful for
identifying immune regulators that act on human innate
immunity.7 We used this system to search for natural
substances that regulate innate immunity and identified and
isolated a benzoyl pyrrole-type compound from Streptomyces
sp. as a potent suppressor. Interestingly, our isolated com-
pound showed the same 1H and 13C NMR and mass spectra
as those of both celastramycin A (1)8 and another benzoyl
pyrrole-type compound 2 reported in a Japanese patent.9 It
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Oshima, Y.; Kurata, S. Life Sci. 2006, 80, 113–119. (b) Sekiya, M.; Ueda,
K.; Okazaki, K.; Kikuchi, H.; Kurata, S.; Oshima, Y. Biochem. Pharmacol.
2008, 75, 2165–2174.
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Res. 2006, 55, 511–515. (b) Kim, H. M.; Park, B. S.; Kim, J.-I.; Kim, S. E.;
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Lee, J.-O. Cell 2007, 130, 906–917.
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S.; De Castro Franc¸a, S.; Groth, I.; Schlegel, B.; Mo¨llmann, U.; Gollmick,
F.; Gra¨fe, U.; Leistner, E. Planta 2002, 216, 162–167.
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Seiyaku KK, Japan). Jpn. Kokai Tokkyo Koho JP 09059249 A 19970304
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Tamura, N.; Kitazaki, T. J. Med. Chem. 2005, 48, 7457–7467. (b)
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10.1021/ol9002306 CCC: $40.75
Published on Web 03/24/2009
2009 American Chemical Society

Scheme 1. Synthesis of Compound 1
was difficult to confirm the structure of our compound by
derivatization because we isolated a limited amount of the
compound. Therefore, we decided to determine which
structure, either 1 or 2, was correct by synthesizing both
compounds.
NMR spectra of synthetic 1 were different from those of
the compound we isolated and the reported spectra of 18
(Table 1).
The synthesis of 1 is illustrated in Scheme 1. After
O-methylation of commercially available 4-n-hexylresorcinol
(3), ortho-lithiation followed by carboxylation gave com-
pound 4.10 Treating 4 with sulfuryl chloride afforded 3-chloro
derivative 5 as the sole product. In regard to the pyrrole
moiety, pyrrole-2-carboxylic acid (6) was chlorinated with
sulfuryl chloride to produce 4,5-dichloro compound 7.
Decarboxylation of 7 with heat in ethanolamine11 and
subsequent N-silylation gave the N-TIPS protected pyrrole
8, which underwent Friedel-Crafts acylation with an acyl
chloride derived from 5 to produce ꢀ-benzoylpyrrole 9.
ꢀ-Acylation of 8 was induced by its N-TIPS group,12 which
was cleaved during the reaction. In the HMBC spectrum of
10, an N-methyl derivative of 9, the correlation peak for H-2′
to the N-methyl carbon atom, and the N-methyl proton to
C-2′ confirmed the position of a benzoyl group at C-3′
(Figure 1). Finally, demethylation of 9 with BBr3 allowed
Table 1. 13C NMR Spectral Data of Synthetic and Reported 1
and 2a
synthetic 1
synthetic 2
reported 18
reported 29
1
2
3
4
5
6
7
2′
3′
4′
5′
1′′
2′′
3′′
4′′
5′′
6′′
111.6
149.6
110.3
134.2
124.3
157.2
190.2
122.9
123.9
109.8
114.7
29.4b
29.1b
29.1b
31.7
112.6
147.9
110.4c
133.7
124.8
157.3
182.8
129.0
119.7
110.3c
121.6
29.4d
29.2d
29.1d
31.7
112.6
148.0
110.3e
133.7
124.8
157.3
182.8
129.0
119.6
110.3e
121.4
29.4f
29.4f
29.1f
31.7
112.6
147.9
110.3g
133.8
124.8
157.3
182.8
128.9
119.8
110.3g
121.6
29.4h
29.3h
29.1h
31.7
us to complete the synthesis of 1. However, the 1H and 13
C
22.6
14.1
22.6
14.1
22.6
14.1
22.7
14.1
a 600 MHz for 1H and 150 MHz for 13C in pyridine-d5. b These signals
were indistinguishable. c These signals were indistinguishable. d These
signals were indistinguishable. e These signals were indistinguishable. f These
signals were indistinguishable. g These signals were indistinguishable.
h These signals were indistinguishable.
The synthesis of 2 is illustrated in Scheme 2. Carboxylic
acid 5 was converted into its acid chloride, and then a
Friedel-Crafts reaction with pyrrole gave R-benzoylpyrrole
11. Chlorination of 11 with sulfuryl chloride afforded 4,5-
Figure 1. Selected HMBC correlations of 10 and 13.
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Org. Lett., Vol. 11, No. 8, 2009

immunity were evaluated using the ex vivo Drosophila
culture system.7 Compound 2 showed a potent immunosup-
pressive effect (IC50 0.008 µg/mL), while 1, 12, and 14 had
no effect. These results indicated that the R-benzoylpyrrole
moiety, two phenolic hydroxyl groups, and the imino proton
in the structure of 2 are crucial for the biological activity.
Scheme 2. Synthesis of Compound 2
The TNF-R signaling pathway in humans plays a critical
role in the inflammatory response, sepsis, and rheumatoid
arthritis by producing costimulatory molecules, cytokines,
chemokines, and adhesion molecules through the activation
of NF-κB,13 which shares some similarity with the imd
pathway in Drosophila innate immunity. To examine whether
compound 2 suppresses the mammalian TNF-R signaling
pathway as well as the Drosophila imd pathway, we
investigated the effect of 2 on TNF-R-stimulated production
of IL-8, a neutrophil chemotactic factor, in human umbilical
vein endothelial cells (HUVECs). Compound 2 showed a
potent suppressive effect (IC50 0.06 µg/mL) on the production
of IL-8, like LL-Z-1640-214 (5Z-7-oxozeaenol) (IC50 0.01
µg/mL). LL-Z-1640-2 is a highly potent inhibitor of TAK1,15
which regulates the TNF-R signaling pathway.16 This result
indicates that compound 2 can be used as a lead compound
for novel immunosuppressive agents. Further investigations
on the structure-activity relationship of this compound and
its mechanism of action are in progress.
dichloro compound 12 selectively. No correlation peak for
H-3′ to the N-methyl carbon atom and the N-methyl proton
to C-3′ in the HMBC spectrum of 13, an N-methyl derivative
of 12, indicated the position of a benzoyl group at C-2′
(Figure 1). Finally, compound 2 was synthesized by treating
Acknowledgment. This work was supported in part by a
Grant-in-AidforScientificResearch(No.18032013,19310135,
18•5061) from the Ministry of Education, Science, Sports
and Culture of Japan; Takeda Science Foundation; The
Uehara Memorial Foundation; Program for Promotion of
Basic Research Activities for Innovative BioSciences; and
Chugai Pharmaceutical Co., Ltd.
1
12 with BBr3. The H and 13C NMR spectra of synthetic 2
were identical to those of our compound and to the spectra
of 18 and 29 reported in the literature (Table 1). Therefore,
the reported structure of celastramycin A (1) is incorrect,
and the correct structure for 1 and the compound that we
isolated is 2. The chemical shift (δc 182.8) of a carbonyl
carbon in synthesized 2 was significantly different from that
in synthesized 1 (δc 190.2). This fact may be useful to
distinguish between R-benzoylpyrrole and ꢀ-benzoylpyrrole.
In addition, compound 14, an N-methyl derivative of 2, was
synthesized by treating 13 with BBr3.
Supporting Information Available: Experimental meth-
ods and NMR spectra of new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL9002306
The immunosuppressive effects of 1, 2, 12, and 14 on the
imd (immune deficiency) pathway in Drosophila innate
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