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Vehicle (without LPS)
Contol (LPS 0.6 mg/mouse)
ER-803064; 0.3 mg/mouse
ER-803064; 0.9 mg/mouse
f152A1; 0.3 mg/mouse
*
5. Goto, M.; Chow, J.; Muramoto, K.; Chiba, K. I.; Yamamoto, S.; Fujita, M.; Obaishi,
H.; Tai, K.; Mizui, Y.; Tanaka, I.; Young, D.; Yang, H.; Wang, Y. J.; Shirota, H.;
Gusovsky, F. J. Pharmacol. Exp. Ther. 2009 [Epub ahead of print].
*
6. For a recent review: Winssinger, N.; Barluenga, S. Chem. Commun. 2007, 1, 22.
7. (a) Fürstner, A.; Thiel, O. R.; Kindler, N.; Bartkowska, B. J. Org. Chem. 2000, 65,
7990; (b) Tatsuta, K.; Takano, S.; Sato, T.; Nakano, S. Chem. Lett. 2001, 2, 172; (c)
Selles, P.; Lett, R. Tetrahedron Lett. 2002, 43, 4627; (d) Henry, N.; Robertson, M.
N.; Marquez, R. Tetrahedron Lett. 2007, 48, 6088; (e) Dakas, P.-Y.; Barluenga, S.;
Totzke, F.; Zirrgiebel, U.; Winssinger, N. Angew. Chem., Int. Ed. 2007, 46, 6899;
(f) Krohn, K.; Shuklov, I. A. J. Carbohydr. Chem. 2007, 26, 419.
0
25
50
75
100 125 150
IL-6 (ng/ml)
Figure 8. Effects of ER-803064 and f152A1 on LPS-induced IL-6 Production in Mice
(for detailed description see Supplementary data note 14).
8. A number of syntheses on zearalenone 6 were known: See: (a) Vlattas, I. T.;
Harrison, L.; Tokes, J. H.; Freid, J. H.; Cross, A. D. J. Org. Chem. 1968, 33, 4176; (b)
Girotra, N. N.; Wendler, N. L. J. Org. Chem. 1969, 34, 3193; (c) Takahashi, T.;
Kasuga, K.; Takahashi, M.; Tsuji, J. J. Am. Chem. Soc. 1979, 101, 5072; (d) Rao, A.
V. R.; Deshmukh, M. N.; Sharma, G. V. M. Tetrahedron 1987, 43, 779; (e)
Kalivretenos, A.; Stille, J. K.; Hegedus, L. S. J. Org. Chem. 1991, 56, 2883; (f)
Solladie, G.; Maestro, M. C.; Rubio, A.; Pedregal, C.; Carreno, M. C.; Ruano, J. L. G.
J. Org. Chem. 1991, 56, 2317.
9. All routes for analog preparations were detailed in this publication: Boivin, R.;
Chiba, K.; Davis, H. A.; Diepitro, L.; Du, H.; Eguchi, Y.; Fujita, M.; Gilbert, S.;
Goto, M.; Harmange, J. C.; Inoue, A.; Jiang, Y.; Kawada, M.; Kawai, T.; Kawakami,
Y.; Kimura, A.; Kotake, M.; Kuboi, Y.; Lemelin, C.; Li, X. Y.; Matsushima, T.;
Mizui, Y.; Sakurai, H.; Schiller, S.; Shen, Y.; Spyvee, M.; Tanaka, I.; Wang, Y.;
Yamamoto, S.; Yoneda, N.; Kobayashi, S. (Eisai Co. Ltd., Japan; et al.) PCT Int.
Appl. WO 2003076424 A1 20030918, 2003.
11 with C4 (S)-Me group should result in a release in ring-strain of
the macrolactone ring, thereby making 15 a mimic conformation-
ally closer to the parent f152A1.
With these predictions, we synthesized analog 15 (Fig. 7). To
our delight, 15 (ER-803064) indeed restored the potency
(IC50 = 136 nM), with the excellent metabolic stability (>80% in
2 h vs ꢀ2% for f152A1). Further expanding SAR on C4 substitutions,
it showed that C4 (S)-Me analog was the most potent among the
series (Table S4). With all these properties, 15 (ER-803064) now
serves us as the new lead for further optimization and also as the
pharmacological benchmark.
In comparison to f152A1, mouse PK parameters of ER-803064
were then studied, thereby demonstrating clear improvements;
in particular, clearance improved from 42.6 L h/kg to 5.51. The
overall exposure increased approximately six-fold, normalized by
dose (Table 1). Although ER-803064 lost in vitro potency by nearly
10-fold in suppression of LPS-stimulated cytokine production from
macrophages, the short term LPS challenge in vivo model showed
much stronger suppression of cytokine production (IL-6) in mice
than f152A1 (1) at similar dosing (Fig. 8).14
10. Bürgi, H. B.; Dunitz, J. D.; Shefter, E. J. Am. Chem. Soc. 1973, 95, 5065.
11. a) The experimental details for mouse stability is summarized as following:
The test compound was either diluted in blood to the final concentration of 100
lM or 20 lM depending on the concentration of DMSO stock solution, or in
pooled mouse plasma containing EDTA obtained from Cocalico Biologicals, Inc.,
or in PBS (1X) either made in the lab or purchased from Gibcol BRL. The spiked
body fluids and PBS were incubated for different time period, usually 0, 15, 30,
60, 120, and 960 min at 37 °C with gentle shaking. 250
lL out of the spiking
fluids was mixed with 100% MeOH at the end of each time point. HPLC
analysis: First Waters Nova-Pak C18 column (80 Â 100 mm) was applied. The
HPLC running condition was as follows: Flow rate: 2 ml/min; Mobile phases: A:
250 mM NH4Ac, pH 4.9, B: 100% AcCN; Gradients (B): 30% (0-18 min), 50% (19-
24 min), 95% (25-30 min), and 30% (31 min and after); Detection: monitor at
235 nm with UV scanning between 220-400 nm; b) Sample (80
to PBS containing GSH (4 mM) alone, or GSH (4 mM) and glutathione S-
transferase (GST; 0.5 U/ L). The total reaction volume was 250 L. The reaction
lM) was added
In conclusion, our medicinal chemistry study has led to the dis-
covery of ER-803064 (15), a serum- and plasma-stable resorcylic
acid analog of ER-803064 (1). Addition of the (S)-Me group at C4
has resulted in a dramatic improvement in metabolic stability with
retention of bioactivity. Based on these findings, we make efforts
for development of an optimum drug candidate.
l
l
mixture without GSH and GST was applied as control. The reaction was carried
out for 40 min. The sample was prepared and analyzed as described in (a).
12. The experimental description of assays: TNF-PLAP(TNF
cells,THP-1-33 cells): A TNF -PLAP plasmid (TNF -promoter + 5’-UTR (1.4
kb) + PLAP + SV40 polyA + PGK-neo, Goto, M. et al. Mol. Pharmacol. 1996, 49,
860) was constructed with slight modification in which TNF -3’-UTR (772 b.p.)
was inserted between PLAP and SV40 polyA (TNF -promoter + 5’-UTR (1.4 kb)
+ PLAP + TNF -3’-UTR + SV40 polyA + PGKneo). Then the TNF -PLAP reporter
cells were established by stably transfecting the modified TNF -PLAP plasmid
a-PLAP reporter
a
a
a
a
Acknowledgments
a
a
a
into THP-1 cells. Actin-PLAP (b-actin-PLAP reporter cells,B164 cells): In order
to simultaneously evaluate non-specific effects of test compounds on
transcription, b-actin-PLAP reporter cells were also established by stably
transfecting b-actin-PLAP plasmid (b-actin-promoter (4.3 kb) + PLAP + SV40
Fermentation broth of a fungus, Curvularia verruculosa, was of-
fered from Biosource Laboratories, Mercian Corporation. We thank
Drs. Seiichi Kobayashi (Eisai, Japan) and Ieharu Hishinuma (Eisai
Research Institute of Boston Inc.) for discussion and helpful
advices.
polyA + PGKneo) into B164 cells. Cell-based assays with TNFa-PLAP and b-
actin-PLAP reporter cells: cells were maintained in RPMI1640 containing 10%
heat-inactivated endotoxin-free fetal bovine serum (FBS) and G418 (1 mg/mL).
These cells were seeded at a density of 1.0 Â 104 cells/well onto 96-well plate,
then were cultured in the presence or absence of test compounds for 30 min,
followed by stimulation with 100 ng/ml of lipopolysaccharide (LPS; E.coli
Supplementary data
0127:B08 or 011:B4). Total volume of the reaction mixture was 200 lL. After
the cultivation for 24-48 hrs, culture supernatant was harvested and alkaline
phosphatase activity in the supernatant was measured.
13. Experimental description for mouse plasma stability: Each compound was
dissolved in freshly prepared heparinized mouse blood at a concentration of 50
Supplementary data associated with this article can be found, in
l
g/ml or 50 lM, and incubated at 37 C for 0, 30 and 120 min. For HPLC analysis,
References and notes
the sample of each time point was directly extracted with MeOH containing 0.2
% perchloric acid immediately after the incubation. The HPLC column is YMC
J’sphere ODS H80 75x4.6 mm. The solvents is 30% acetonitrile in 0.15% KH2PO4
water solution buffered with phosphoric acid to pH=3.5. The pump speed is 1.1
mL/min. The amount of unchanged form was estimated from the peak area,
and the remaining % at each time point of the incubation was calculated by
comparing with the area at 0 min.
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14. The test compounds were co-injected with LPS through tail vain in the
formulation with 10
peripheral blood was harvested and plasma was separated for the
determination of IL-6 by ELISA. Each column represents mean with SEM (n =
6 - 8). P<0.05; statistically significant as compared with the LPS-treated
control group in Dunnett’s multiple comparison post hoc analysis.
% cyclodextrin. At 3 hours after the injection, the
*