Design, synthesis, and biological activities of madindoline analogues

Article abstract of DOI:10.1016/j.bmcl.2006.01.107

A research program is under way to develop a series of madindoline-based inhibitors targeting interleukin 6. Such inhibitors will have potential use in fighting a variety of diseases for which no effective therapeutic drugs currently exist. Madindoline is

Full text of DOI:10.1016/j.bmcl.2006.01.107

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2807–2811
Design, synthesis, and biological activities of madindoline analogues
Daisuke Yamamoto, Toshiaki Sunazuka, Tomoyasu Hirose, Naoto Kojima,
Eisuke Kaji and Satoshi Omura*
The Kitasato Institute, and Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, School
of Pharmaceutical Sciences, Kitasato University, Japan
CREST, The Japan Science and Technology Corporation (JST), 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan
Received 11 November 2005; revised 30 January 2006; accepted 31 January 2006
Available online 14 February 2006
Abstract—A research program is under way to develop a series of madindoline-based inhibitors targeting interleukin 6. Such inhib-
itors will have potential use in fighting a variety of diseases for which no effective therapeutic drugs currently exist. Madindoline is
no longer available from natural sources. Consequently, we have developed a purely synthetic route to ensure a supply of the com-
pound. The synthesis of a range of analogues is described, all of which were evaluated for their inhibitory activity against the growth
of IL-6-dependent 7TDI cells. From these assays, several synthetic madindoline analogues were identified as highly promising can-
didates for further development.
Ó 2006 Published by Elsevier Ltd.
Interleukin 6 (IL-6) is a multifunctional cytokine in-
volved in control of antibody production, T cell activa-
tion, hematopoiesis, and acute responses. Moreover,
uncontrolled IL-6 activity is known to cause various
serious diseases.1 It has been reported that excess IL-6
production is closely associated with cancer cachexia,2
Castleman’s disease,3 rheumatoid arthritis,4 hypercalce-
mia,5 and multiple myeloma.6 No effective therapeutic
drugs for these diseases have been developed, but our re-
search shows that a low molecular weight compound
could be developed for therapeutic use that modulates
function of IL-6 via a new mode of action.
Figure 1. Structures of (+)-madindolines A ((+)-1) and B ((+)-2).
In 1996, we reported the isolation of the two novel
indole alkaloids from a culture broth of Streptomyces
nitrosporeus K93-0711, madindolines A (+)-1 and B
(+)-2 (Fig. 1). Both are selective inhibitors of IL-6.7,8
The biological activity profiles of (+)-1 and (+)-2 were
exceptional. Both (+)-1 and (+)-2 specifically inhibited
the growth of the IL-6-dependent MH60 cell line (IC50
values of 8 and 30 lM, respectively), but they did not af-
fect the IL-6-independent MH60 cell line.
does not inhibit formation of the IL-6/IL-6R/gp130
complex.
Furthermore, oral administration of (+)-1 to ovariecto-
mized (OVX) mice significantly suppressed the decrease
in bone mass and increase in serum Ca2+ level after
ovariectomy. This suppression mechanism was distinct
from that of 17b-estradiol.9
Detailed biological studies of the compound have
showed that (+)-1 competitively binds to gp130, but
Unfortunately, the scarcity of natural material has pre-
cluded further evaluation. Madindolines are no longer
available from natural sources due to mutation of the
originating bacterial strain.
Keywords: Natural Products; Total Synthesis; Structure–activity
relationships.
*
Intrigued by the novel architecture, the significant IL-6
inhibitory activity, and the scarcity of these natural
Corresponding author. Tel.: +81 3 5791 6101; fax: +81 3 5791
6380; e-mail: omura-s@kitasato.or.jp
0960-894X/$ - see front matter Ó 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2006.01.107

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D. Yamamoto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2807–2811
products, we devised a process which achieved their to-
tal synthesis in 2000.10 Although the synthetic route de-
fined for the first time their relative and absolute
configurations, it did not produce enough material to al-
low extensive biological studies. We therefore developed
a more practical (i.e., scalable) total synthesis to produce
(+)-madindolines, thereby generating a source of materi-
al for further studies and biological development. Our
initial approach to (+)-madindoline synthesis has been
refined into a practical and effective route for total syn-
thesis11 (Scheme 1). The process has so far led to the
generation of over 1 g of the synthetic natural product.
the aldehyde, as depicted in Scheme 2. Thus, Wittig–
Horner reaction12 of the appropriate aldehyde with 9
produced moderate yields of the methyl allylsilane
(10a), ethyl allylsilane (10b), heptyl allylsilane (10c),
nonyl allylsilane (10d), undecyl allylsilane (10e), henei-
cosyl allylsilane (10f), and benzyl allylsilane (10g). All
were used in the synthesis of analogues (15a–g).
Although Z/E mixtures were obtained, these diastereo-
mers generated (Z)-a,b-unsaturated acyl chlorides as a
single isomer (12a–g). Finally, the desired (Z)-a,b-unsat-
urated acyl chlorides (12a–g) were incorporated into our
efficient and shorter synthesis system to produce the
side-chain modified analogues (15a–g) with sufficient
quantities for in vitro studies.
In parallel with our efforts to develop a reliable source of
product for testing, we broadened our interest to include
the production of analogues. This allowed us to investi-
gate structure–activity relationship, as well as to identify
more stable madindolines which retained their ability to
inhibit IL-6.
The growth inhibition activity of the three series (two of
them: vide infra) of analogues investigated was assayed
against IL-6-dependent 7TDI cells and the bioactivity of
the analogues compared against the natural compound.
The degree of cell growth inhibition was measured in the
presence of 10 lM of each of the tested analogues. The
level of inhibition using 10 lM madindoline A was de-
fined as 1.0. Tables 1–4 show the relative impact of the
analogues compared with madindoline A.
Our strategy for synthesis of madindoline analogues fo-
cused on two distinct aspects: (1) length of the side
chains of the cyclopentene ring and (2) functionalization
of the 3a-hydroxyfuroindoline ring.
Access to the side-chain modified madindoline ana-
logues was secured by modifying the alkyl group to
It is interesting to note that while the shorter and the
longest side chain analogues (Table 1, compounds 15a,
Scheme 1. Summary of short total synthesis of (+)-1 and (+)-2.
Scheme 2. Reagents and conditions: (a) NaH, DME, 0 °C; (b) KOH, EtOH–H2O, rt; (c) SOCl2, D.

D. Yamamoto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2807–2811
Table 1. Activities of the side-chain modified analogues
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Table 2. Activities of the 5-halo sector analogues
Table 4. Activities of madindoline analogues
Given the structural information (Fig. 1), this seems to
identify an oxidation at the 5-position. Indeed, it
appears that (+)-1 and (+)-2 are easily metabolized
and subsequently produce 5-hydroxy madindolines in
plasma. Consequently, we were concerned that the 5-ha-
lo analogues might increase tolerances of oxidation in a
living body. We therefore focused on a careful assess-
ment of the action of the drug. The more efficient sec-
ond-generation synthesis system we had developed
caused us to examine whether we could access clinical
candidate madindolines by this new method. Attain-
ment of this goal would involve a functionalization of
the 3a-hydroxyfuroindoline moiety (see Scheme 3).
Table 3. Activities of the acyl sector analogues
Syntheses of the 5-halo madindolines were achieved,
accompanied by each N-halo succinimide reagent
(Scheme 4). Moreover, esterification of the 3a-hydroxyl
group was achieved by treatment with several acid anhy-
drides, DMAP, and pyridine, leading to the desired acyl
derivatives (Scheme 5).
15b, and 15f) possess no activity as compared to mad-
indoline A, the appropriately longer side-chain ana-
logues (Table 1, compounds 15c, 15d, and 15e)
exhibited significantly greater potency.
Table 2 and 3 depict the 5-halo and the acyl sector
analogues in order of potency in comparison with the
natural compound. Although increased activity of

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D. Yamamoto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2807–2811
Scheme 3. Reagents and conditions: (a) LDA, THF, À78 °C; (b) TBAT, DMF, D.
would be continued in the future. Interestingly, hexano-
ate and benzoate analogues (compounds 17b and 17d)
exhibited much higher efficacy than the natural com-
pound, however, decanoate analogue (compound 17c)
possessed no activity.
Finally, our investigation of the madindoline series
(Fig. 2) examined the effect of stereochemistry and
parts of the system on a compound’s IL-6 inhibitory
properties. Although (À)-3, cyclopentendione 18,13
and the inversion of stereochemistry of the 3a-furoind-
oline moiety (À)-2 do not invoke the activities as
compared to madindoline A, the reductive 3a-hydroxy-
furoindoline moiety 19 appeared to retain its
potency.
Scheme 4. Reagents: (a) NCS or NBS or NIS, CH2Cl2.
In summary, preliminary studies of the totally synthet-
ic madindoline analogues indicated enhanced activi-
ties, reinforcing initial chemical biology studies that
revealed a number of unexpected structure–activity
relationships within the madindoline class. Notably,
promising candidates 15c, 15d, and 15e demonstrated
the importance of side chain length. The 5-halo and
some of the acyl sector analogues also exhibit en-
hanced activities. Using madindolines as a lead struc-
ture, the collection of synthetic analogues we have
produced offer promising leads to the discovery of a
series of potent IL-6 inhibitors. Further in vitro and
in vivo studies on madindoline derivatives are in
progress.
Scheme 5. Reagents: (a) (R3CO)2O, DMAP, pyridine.
5-chloro madindoline 16a was not observed, 5-bromo
and iodo madindolines (compounds 16b and 16c)
showed moderate levels of activity. The unprecedented
result would mean that enhanced activities of the 5-halo
sector analogues depend on their hydrophobicity rather
than improvement of stabilities against oxidation for
metabolism. Metabolic studies of 16a, 16b, and 16c
Figure 2. Structures of madindoline analogues.

D. Yamamoto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2807–2811
2811
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Acknowledgments
This work was supported by a Grant of the 21st Century
COE Program, Ministry of Education, Culture, Sports,
Science and Technology (MEXT), The Japan Science
and Technology Corporation (JST), a Grant-in-Aid
for Scientific Research from the Ministry of Education,
Science, Sports and Culture, Japan, and the Japan Kei-
rin Association.
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