Pseurotin A and its analogues as inhibitors of immunoglobuline E production

Article abstract of DOI:10.1016/j.bmcl.2009.01.029

A natural product, pseurotin A inhibits IgE production in vitro. Wide variety of chemical modification of pseurotin A was performed. Structure-activity relationship studies of pseurotin analogues elucidated that 10-deoxypseurotin A strongly inhibits IgE p

Full text of DOI:10.1016/j.bmcl.2009.01.029

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1457–1460
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pseurotin A and its analogues as inhibitors of immunoglobuline E production
*
Minoru Ishikawa , Tomohisa Ninomiya, Hirotomo Akabane, Nobuaki Kushida, Go Tsujiuchi,
Makoto Ohyama, Shuichi Gomi, Keiko Shito, Takashi Murata
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A natural product, pseurotin A inhibits IgE production in vitro. Wide variety of chemical modification of
pseurotin A was performed. Structure–activity relationship studies of pseurotin analogues elucidated
Received 28 October 2008
Revised 8 January 2009
Accepted 12 January 2009
Available online 15 January 2009
that 10-deoxypseurotin A strongly inhibits IgE production with IC50 of 0.066
activity of another natural product, synerazol was also found.
lM. An immunosuppressive
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Pseurotin A
Inhibitor of IgE production
Structure–activity relationship
10-Deoxypseurotin A
Synerazol
Immunosuppressive activity
Atopy, characterized by raised immunoglobulin E (IgE) levels,
underlies allergic diseases such as asthma, rhinitis, and dermatitis.
Asthma is defined as a chronic inflammatory disorder of the airway
in which many cells play a role, particularly mast cells, eosinophils
and T lymphocytes. The resulting inflammation causes episodes of
wheezing, breathlessness, chest tightness, and cough. About the
medications, inhaled corticosteroids acting in a multifunctional
manner are very effective at suppressing allergic symptoms, and
remain the mainstay for treating serious asthma.1 However, corti-
costeroids are rather non-specific in their action and their use
raises concerns regarding side effects and compliance particularly
in children and adolescents. Further, a recent clinical study showed
that 51% of patients, who were prescribed with inhaled corticoste-
roids, were classified as having uncontrolled asthma.2 For the oral
treatments, drugs such as histamine and leukotriene antagonists
target single effector molecules and provide some relief, but the
heterogeneous nature of asthma makes this approach unsuccessful
for most patients.3
itors as oral, disease modifying agents may be seen as a major ther-
apeutic advance. So far, among the several classes of small
molecule inhibitors of IgE production have been identified,6–11
none is on the market or in clinical development. Herein, we de-
scribe the novel IgE production inhibitor, pseurotin A, and struc-
ture–activity relationship of pseurotin analogues.
The inhibitory activity of IgE production was evaluated as pre-
viously described manner.12 Briefly, IgE responses were elicited
from B-cells that were isolated from spleens of naive BALB/c mice
and cultured 6 days with interleukin-4 and lipopolysaccharide
(LPS). IgE levels were determined by enzyme-linked immunosor-
bent assay (ELISA). As a result of screening, an inhibitor of specific
IgE production with IC50 of 3.6 lM was isolated from the fermen-
tation broth of Aspergillus sp. This compound was identical to pseu-
rotin A. The cytotoxicity, inhibition of phytohemagglutinin (PHA)-
induced T-cell proliferation13 or a B-cell viability of pseurotin A
were far from the concentration that inhibited IgE production,
which indicates that observed inhibition was not due to cytotoxic-
ity or a general antiproliferative effect. Furthermore, pseurotin A
IgE levels are elevated in allergic diseases and correlate well
with severity of symptom.4 Further, humanized anti-IgE monoclo-
nal antibody omalizumab, which binds to free circulating IgE, has
clinical efficacy in allergic asthma.5 However, high cost or insuffi-
cient compliance with injection may impact on the active usage
of this therapy. On the basis of this evidence, IgE production inhib-
did not inhibit mixed-lymphocyte reaction (MLR) 14 at 10
lM indi-
cated that the observed inhibition was not due to non-specific
immunosuppressive activity, but was effected on the B-cell. In con-
trast, a corticosteroid prednisolone inhibited IgE production with
IC50 of 5.9 nM. But prednisolone also inhibited PHA-induced T-cell
proliferation (84% at 10 lM) and MLR (IC50: 78 nM). Therefore, the
basic concept could be verified in relevant target cells.
* Corresponding author. Present address: Institute of Molecular and Cellular
Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032,
Japan.
Pseurotin A (1) is a microbial secondary metabolite isolated
from the fermentation broth of Pseudeurotium ovalis STORK (Asco-
mycetes) in 1976,15 and possesses a highly substituted 1-oxa-7-
E-mail address: m-ishikawa@iam.u-tokyo.ac.jp (M. Ishikawa).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.029

1458
M. Ishikawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1457–1460
azaspiro[4,4]non-2-ene-4,6-dione skeleton. The core structure of 1
is found in other natural products, too (Fig. 1). These pseurotin-re-
lated natural products also possess interesting biological activities,
including apomorphine-antagonistic activity of pseurotin A,16a
D16a and F,16b chitin synthase inhibitory activity17 of pseurotin A
and F, monoamine oxidase inhibitory activity of pseurotin A,18
induction of cell differentiation of pseurotin A,19 antifungal activity
and synergistic activity with azole-type antifungal agents of syner-
azol (4) isolated from the fermentation broth of Aspergillus fumiga-
tus SANK 10588 in 1991,20 and anti-angiogenic activity of
azaspirene21a and synerazol.21b But immunomodulate activity of
these natural products has not been reported.
The novel pharmacological activity of 1 and limited number of
its analogues reported prompted us to modify 1 obtained from fer-
mentation. We envisioned that 5d obtained via oxidative cleavage
of 1 could be used for the modification of the C10–C15 side chain of
1 with structural diversity (Scheme 1). First of all, Wittig reaction
of stabilized ylides or Horner–Wadsworth–Emmons (HWE) reac-
tion29 with 5d afforded (E)-olefin 9a–d. Consecutive Wittig reac-
tion with 9c provided (E, E)-diene 9e which resembled
azaspirene. (Z)-olefin 9f was also synthesized under HWE reaction
with the Ando reagent.30 Reductive amination of 5d with a variety
of amines afforded 10a–f. Next, the reported olefin cross metathe-
sis23 method was applied to substrates and gave 11a–f. Hydroge-
nation of 11b–e gave various saturated side chain 12b–e. As
results, we efficiently synthesized pseurotin analogues which pos-
sesses variously modified the side chain.
Hydroxyl groups, methoxyl group and lactam moiety were
modified as following. Selectively methylated analogues 13–15
were synthesized with MeI and K2CO3, MeI and Ag2O,31 or
Me3OBF4.32 Ethyl acetalization of 1 in acidic condition26 provided
16. 9-Deoxypseurotin A (18) was synthesized by Robins’s deoxy-
genation condition.33 In detail, treatment of 1 with thioacyl chlo-
ride and DMAP in CH3CN gave 9-O-phenoxythiocarbonyl
derivative 17a. While investigating the thiacylation conditions,
10-O-thioacylated 17b was obtained in 46% yield when pyridine
as base and DCM as solvent were used. Reductive cleavage of
17a–b gave 18 and 10-deoxypseurotin A (19), respectively.
12, 13-epoxypseurotin A (20) and 12,13-cyclopropylpseurotin A
(21) were synthesized from 1 via epoxidation or modified Sim-
These important biological properties of pseurotins and related
natural products have attracted attention to structure–activity
relationship (SAR) studies of their analogues. But only limited
number of chemical modifications of pseurotin-related natural
products obtained from fermentation have been reported as shown
in Figure 2, that is, 12,13-dihydroxypseurotin A (5a),22 12,13-dib-
romopseurotin
A
(5b),15a 10,11-acetonide (5c),15a aldehyde
5d,15a,22b (10R, 11S)-diastereomer of 4 (5f),23 triacetylpseurotin A
(6a),15a tetraacetylpseurotin A (6b),15a 7,9-N,O-diacetylsynerazol,24
17-dihydropseurotin A (7).22a Synthesis of 1 from 4,24 and synthe-
ses of 4 and pseurotin E (2) from 123 were also reported. Fluori-
nated 1 (8a, 8b) and 4 were produced by the precursor-directed
biosynthesis using fluorophenylalanine.21a Total syntheses of
1,25,26 pseurotin F2 (3),25,26 427 and azaspirene28,26 were reported
by Hayashi et al. and Tadano et al. But total syntheses of pseurotin
analogues27 were reported few.
Figure 1. Structure of pseurotin-related natural products.
Figure 2. Reported pseurotin analogues.

M. Ishikawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1457–1460
1459
Scheme 1. Chemical modification of pseurotin A. Reagents and conditions: (a) Ph3P = CR1R2, THF, 4 °C, 50% (9a), 72% (9b), 85% (9c), 55% (9e); (b) (EtO)2POCH2CN, i-Pr2EtN, LiCl,
CH3CN, À30 °C, 9% (9d);(c) (PhO)2POCH2CO2Et, Triton B, THF, À78 °C, 22%(9f); (d)R1R2 NH, NaBH3CN, AcOH, MeOH, 4 °C–rt, 28%(10a), 14%(10b), 11%(10c), 19%(10d), 19%(10e),
10% (10f); (e) Grubbs second generation, DCM, 40 °C, (E)-hex-3-ene, 70% (11a), but-2-ene, 64% (11b), hex-1-ene, 61% (11c), methyl acrylate, 80% (11d), but-3-enylbenzene, 91%
(11e), methylenecyclohexane, 25% (11f); (f) (PPh3)3RhCl, H2, toluene, EtOH, rt, 53% (12b); (g) Pd/C, H2, EtOH, rt, 53% (12c), 30% (12d), 70% (12e); (h) MeI, K2CO3, DMF, 4 °C, 60%
(13); (i) MeI, Ag2O, CH3CN, 100 °C (lW), 3% (14); (j) Me3OBF4, K2CO3, DCM, rt, 32% (15); (k) AcCl, EtOH, 30 °C, 28% (16); (l) PhO(CS)Cl, DMAP, CH3CN, rt, 19% (17a); (m) PhO(CS)Cl,
pyridine, DCM, 7 °C, 46% (17b); (n) n-Bu3SnH, AIBN, toluene, 75 °C, 26% (18), 46% (19) (o) mCPBA, DCM, rt, 55% (20); (p) ZnEt2, ClCH2I, DCE, 4 °C, 26% (21).
mons–Smith reaction.34 Stereochemistry of 20 and 21 have not
Table 1
been determined.
IgE inhibitory activity of pseurotin analogs
Both known and newly synthesized pseurotin analogues were
evaluated for the inhibitory activity of IgE production. First of
SAR studies, available pseurotin related natural products (2,23
3,35 and 423 in Fig. 1) were evaluated for IgE production inhibition
(Table 1). Among them, 4 showed ten times stronger IgE inhibitory
Compound
IgE production
IC50 (lM)
1
2
3
4
5a–f
6a–b
7
8a–b
9a–f
10a
10b
10c
10d
10e
10f
11a–f
12b–e
13
3.6
>10
>10
0.26
>10
>10
>10
>10
>10
>10
>10
3.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
activity compared with 1 (IC50: 0.26 lM).
(10R, 11S)-Diastereomer 5f23,27 was inactive, suggesting that
stereochemistry of the epoxide is important for the activity. C12–
C13 (E)-olefin 2 and 11a–e, trisubstituted olefin 11f, 12,13-dihydro
derivatives 5a and 12b–e, 12,13-epoxide 20 and 12,13-cyclopro-
pane 21 did not inhibit IgE production, suggesting that C12–C13
(Z)-olefin is essential for the activity. 11-O-methyl analog 15 lost
the activity indicated that 11-hydroxy group is also important for
the activity. 10-Deoxy analog 19 showed fifty times more potent
activity than 1 with IC50 of 0.066
lM. This result suggested that
10-hydroxy group is unnecessary for the activity. n-Butyl amino
derivative 10c was equipotent to 1 (IC50: 3.1 lM). The amino group
might be mimic to 11-hydroxy group.
14
15
16
18
19
20
21
Modification of the spiro skeleton (3, 13, 14, 16 and 18) or the
benzoyl moiety (7 and 8a–b) was almost inactive. These results
indicated that the spiro skeleton and the benzoyl moiety are
important for the activity.
0.066
>10
>10
Potent inhibitors36 of IgE production were further investigated
for the specificity (Table 2). The K562 cytotoxicity, or the B-cell via-
bility of these inhibitors were far from the concentration that
inhibited IgE production. In addition, the most potent inhibitor
thermore, the specificity of 19 for other immunoglobulin were
investigated. The IC50s of immunoglobulin37 were IgE selective
19 did not inhibit PHA-induced T-cell proliferation (10 lM). Fur-

1460
M. Ishikawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1457–1460
Canonica, G. W.; Hedgecock, S.; Fox, H.; Blogg, M.; Surrey, K. Allergy 2005, 60,
Table 2
309.
Specificity of IgE production inhibitors
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Compound
IgE
K562
B-cell
viability
IC50 (lM)
T-cell
proliferation
% inhibition at
MLR
IC50
production cytotoxicity
IC50
(
l
M)
IC50
(
l
M)
7. Kawada, K.; Arimura, A.; Tsuri, T.; Fuji, M.; Komurasaki, T.; Yonezawa, S.;
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10
l
M
(lM)
1
4
10c
19
3.6
0.26
3.1
>10
>10
>10
>10
nta
>10
>10
>10
4.4
0%
54%
2%
0%
84%
>10
3.1
nta
0.066
nta
Prednisolone 0.0059
>10
0.078
a
Not tested.
(IgM/IgE: 44, IgG2a/IgE: 23, IgG1/IgE: 3.5). In contrast, predniso-
lone inhibited IgE, IgG and IgM production with no selectivity
(IgM/IgE: 0.4, IgG2a/IgE: 0.8, IgG1/IgE: 1.0). These data suggested
that 19 might be potent and specific inhibitor of IgE production.
On the other hand, surprisingly, 4 showed T cell proliferation-
14. Soulillou, J.-P.; Carpenter, C. B.; Lundin, A. P.; Strom, T. B. J. Immunol. 1975, 115,
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inhibitory activity of 54% inhibition at 10
l
M. Furthermore, 4
inhibited MLR with IC50 of 3.1
suppressive activity.
lM, indicating 4 has also immuno-
In summary, we found the inhibitory activity of the IgE produc-
tion of the natural product, pseurotin A (1). Wide variety of chem-
ical modification of 1, especially the C10–C15 side chain was
performed by, that is, Wittig olefination, reductive amination, ole-
fin cross metathesis. Structure–activity relationship of pseurotin
analogues revealed the pharmacophore and elucidated that 10-
deoxypseurotin
A (19) inhibits IgE production with IC50 of
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0.066 M. We also showed the immunosuppressive activity of
l
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the pseurotin A analogue, synerazol (4). Other biological activities
of synthesized analogues, including reported activities of pseurotin
related natural products, are also interesting.
23. Ishikawa, M.; Ninomiya, T. J. Antibiot. 2008, 61, 692.
Acknowledgments
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The authors thank Ms. Shigeko Miki and Ms. Takako Miyara
(Meiji Seika Kaisha, LTD.) for mass spectral analysis, Dr. Yuji Tabata
(Meiji Seika Kaisha, LTD.) for evaluation of cytotoxicity, and Dr.
Satoshi Yoshida, Mr. Makoto Ishikawa and Dr. Takeshi Furuuchi
(Meiji Seika Kaisha, LTD.) for helpful discussion for chemical
modification.
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36. 10c: 1H NMR (400 MHz, CDCl3) d 0.59 (3H, t, J = 7.3 Hz), 1.04 (2H, tq, J = 7.6,
7.3 Hz), 1.24 (2H, tt, J = 7.6, 7.3 Hz), 1.43 (3H, s), 2.39–2.54 (2H, m), 3.07 (3H, s),
3.56 (2H, d, J = 4.1 Hz), 4.37 (1H, s), 7.17 (2H, dd, J = 7.6, 8.2 Hz), 7.33 (1H, br t,
J = 7.6 Hz), 8.00 (2H, br d, J = 8.2 Hz); FABMS m/z 403 (M+H)+.19: 1H NMR
(400 MHz, CDCl3) d 0.98 (3H, t, J = 7.6 Hz), 1.57 (3H, s), 2.04–2.18 (2H, m), 2.61
(1H, dd, J = 3.9, 10.2 Hz), 2.91 (1H, dd, J = 5.1, 9.0 Hz), 3.14 (1H, d, J = 4.4 Hz),
3.39 (3H, s), 4.16 (1H, d, J = 12.7 Hz), 4.64 (1H, d, J = 12.7 Hz), 4.92–4.99 (1H,
m), 5.44–5.56 (2H, m), 7.37 (1H, br s), 7.50 (2H, br dd, J = 7.3, 8.4 Hz), 7.66 (1H,
br t, J = 7.3 Hz), 8.33 (2H, dd, J = 1.2, 8.4 Hz); ESIMS m/z 416 (M+H)+.
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