WSS2220, a novel cyclic tetrapeptide with a new sulfonoamino acid, exhibits potent and selective inhibitory activity against GlyT1

Article abstract of DOI:10.1016/j.tetlet.2008.03.054

In the course of our screening program for novel glycine transporter inhibitors, we obtained a novel cyclic tetrapeptide (WSS2220) from the culture broth of Nonomuraea sp. The structure of WSS2220 was elucidated based on physicochemical data. WSS2220 contained a novel amino acid (4-oxo-3′-sulfoisoleucine), the absolute stereochemistry of which was determined by chemical modifications and the modified Mosher's method. WSS2220 selectively inhibited glycine transporter type1 with an IC₅₀ of 20 nM.

Full text of DOI:10.1016/j.tetlet.2008.03.054

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3067–3070
WSS2220, a novel cyclic tetrapeptide with a new sulfonoamino acid,
exhibits potent and selective inhibitory activity against GlyT1
a,
b
b
a
a
*
Yuichi Terui , Chu Yi-wen , Li Jun-ying , Osamu Nozawa , Tsutomu Ando ,
Takuya Fukunaga a, Takeshi Aoki a, Yoshihisa Toda a, Akira Kawashima a
a Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403, Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan
b Sichuan Industrial Institute of Antibiotics, 9 Shabanqiao Road, Chengdu, Sichuan 610051, China
Received 7 February 2008; revised 10 March 2008; accepted 11 March 2008
Available online 14 March 2008
Abstract
In the course of our screening program for novel glycine transporter inhibitors, we obtained a novel cyclic tetrapeptide (WSS2220)
from the culture broth of Nonomuraea sp. The structure of WSS2220 was elucidated based on physicochemical data. WSS2220 contained
a novel amino acid (4-oxo-30-sulfoisoleucine), the absolute stereochemistry of which was determined by chemical modifications and the
modified Mosher’s method. WSS2220 selectively inhibited glycine transporter type1 with an IC50 of 20 nM.
Ó 2008 Elsevier Ltd. All rights reserved.
N-Methyl-D-aspartate (NMDA) receptor blockers, such
as phencyclidine (PCP), are known to induce schizo-
phrenic-like symptoms (both positive and negative symp-
toms) in humans. Thus, a dysfunction in glutamatergic
transmission may play an important role in the pathophys-
iology of schizophrenia (hypoglutamatergic hypothesis).
Glycine is known to act as a requisite co-agonist of gluta-
mate for the activation of NMDA receptors, and its levels
at synaptic clefts are controlled by glycine transporter
type1 (GlyT1) on glial cells. Therefore, the blockers of
GlyT1 are expected to function as antipsychotics. Several
GlyT1 inhibitors have been reported, and some of these
agents are now being investigated in clinical trials.1
To identify new inhibitors of GlyT1 from natural
origins, we tested the inhibitory activities of thousands of
fermentation extracts on [3H]-glycine uptake in glial cells.
During this screening program, we obtained a novel cyclic
tetrapeptide, named WSS2220 (1), from a fermentation
broth of Nonomuraea sp. strain TA-0426.2 Compound 1
inhibited [3H]-glycine uptake in a human glial cell line
(T98G) with an IC50 of 12.5 nM. In this Letter, we report
the isolation, structure determination, and biological
activity of 1 (Fig. 1).
The WSS2220-producing organism, TA-0426, was iso-
lated from a soil sample collected in Henan Province,
China. The strain was taxonomically identified as genus
Nonomuraea sp. To produce 1, TA-0426 was cultured in
a seed medium for 3 days at 28 °C on a rotary shaker at
200 rpm.3 This seed medium was then inoculated into the
production medium and cultured for 7 days at 28 °C on
a rotary shaker at 200 rpm.4 The culture broth (10 L) was
extracted with n-BuOH and concentrated in vacuo to give
an oily residue (14.9 g). This n-BuOH extract was sus-
pended in distilled water (1 L) and extracted with EtOAc.
*
Corresponding author. Tel.: +81 48 669 3107; fax: +81 48 652 7254.
Fig. 1. Structure of WSS2220 (1).
E-mail address: yuichi.terui@po.rd.taisho.co.jp (Y. Terui).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.03.054

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Y. Terui et al. / Tetrahedron Letters 49 (2008) 3067–3070
The resulting aqueous layer was passed through a column
of Diaion HP-20. After being washed with 20% aqueous
acetone (1 L), the materials containing 1 were eluted with
40% and 60% acetone. These fractions were combined
and concentrated in vacuo. The resulting residue was then
purified by silica gel column chromatography, eluting with
CHCl3–MeOH 9:1 to give WSS2220 (1, 363.9 mg) as a
colorless powder.
(Phe), and five pairs of signals at 60.5 and 60.5 ppm (d),
33.3 and 34.0 ppm (d), 24.9 and 25.4 ppm (t), 15.3 and
15.4 ppm (q), and 9.8 and 10.0 ppm (q) suggested the pres-
ence of two isoleucine residues (Ile). The presence of these
amino acid residues was also supported by a detailed anal-
ysis of 2D NMR data (Fig. 2) and the advanced Marfey’s
method (vide infra). The structure of the remaining amino
acid residue was also determined by an analysis of the 2D
NMR data. Analysis of the COSY spectrum indicated con-
nectivity between the NH on C-200 to CH2-600. The HMBC
correlation between H-200 and C-100 indicated the connec-
tion of C-100/C-200. The HMBC correlations observed from
H-500 [d 1.82 (3H, s)] to C-300 and C-400 (d 207.0) allowed the
connection of an acetyl group at the C-300 position. The 13C
chemical shift value of the methylene carbon at position 600
(d 51.1) suggested that a sulfonate group was attached at
this position. Thus, this amino acid residue was confirmed
to be 4-oxo-30-sulfoisoleucine (Osi), which is a novel amino
acid.
Based on the degree of unsaturation, 1 was deduced to
be a cyclic tetrapeptide. The HMBC correlation observed
from the Phe a-proton (H-2000) to the Osi carbonyl carbon
(C-100) established the two amino acid-sequence of 1, as
shown in Figure 2. The HMBC correlations of NH-2/
C-1000, NH-20/C-1, NH-200/C-10, and NH-2000/C-100 and the
NOESY correlations of NH-2/H-3000, NH-20/H-3, NH-200/
H-30, and NH-2000/H-300 established all of the amino acid
sequences of 1. The configurations of Phe and Ile were
determined to be L-Phe and L-Ile by acid hydrolysis and
application of the advanced Marfey’s method using stan-
dard amino acids with known configurations.5 Since the
FDLA (1-fluoro-2,4-dinitrophenyl-5-leucineamide) deriva-
tive of Osi was not retained on an ODS column, its
absolute configuration at the a-position could not be deter-
mined by this method.
HRTOF-MS of 1 showed a quasi-molecular ion peak at
625.2302 [M+2Na]+, corresponding to the quasi-molecular
formula C27H39N4O8SNa2 (calcd for C27H39N4O8SNa2:
625.2284). IR absorptions at 1693 and 1533 cmꢀ1 sug-
gested the presence of amide functions. Absorptions at
1221, 1172, and 1044 cmꢀ1, together with the high water-
solubility of 1, suggested the presence of a sulfonate group.
1
The 13C and H NMR spectral data of 1 are shown in
Table 1. In the 13C NMR spectrum of 1, the signals of four
carbonyl carbons (d 169.7, 171.0, 171.5, and 172.0) and
four methines (d 56.1, 57.0, 60.5, and 60.5) were observed.
These data suggested that 1 was a tetrapeptide compound.
Another carbonyl signal at d 207.0 was assigned as a
ketone. Signals of sp2 carbon [d 126.6 (d), 128.4 (d) (2C),
128.8 (d) (2C), and 136.8 (s)] and a methylene carbon (d
35.8) suggested the presence of a phenylalanine residue
Table 1
1H NMR (500 MHz) and 13C NMR (125 MHz) data for 1 in DMSO-d6
Position
Ile-1
13C
1H
1
2
3
4
5
6
171.5
60.5
33.3
24.9
9.8
3.82 (dd, J = 11.0, 11.0)
2.14 (m)
1.46 (m), 1.08 (m)
0.79a
0.79a
15.3
2-NH
7.05 (br)
To determine the stereochemistry of Osi, 1 was chemi-
cally degraded as shown in Scheme 1. Compound 1 was
reduced by NaBH4 to give 2 as a diastereomeric mixture
(3:1). Acid hydrolysis of 2 yielded L-Phe, L-Ile, and cyclo-
4-hydroxy-30-sulfoisoleucine (3). Compound 3 was purified
using a strong cation exchange (SCX) resin. Compound 3
was a single isomer, and its diastereomer was not obtained.
The relative stereochemistry of 3 was determined by
Ile-2
10
172.0
60.5
34.0
25.4
10.0
15.4
20
3.82 (dd, J = 11.0, 11.0)
1.75 (m)
1.46 (m), 1.08 (m)
0.86 (d, J = 6.7 Hz)
0.79a
30
40
50
60
20-NH
7.72 (br)
Osi
100
169.7
56.1
49.0
207.0
30.6
51.1
200
4.18 (dd, J = 11.6, 11.6 Hz)
3.50 (m)
1
analyzing the H NMR spectrum in detail.6 The coupling
300
constants between 2-H/3-H and 3-H/4-H were 11.0 Hz
400
500
1.82 (s)
2.96 (m), 2.81 (br)
7.80 (br)
600
200-NH
Phe
1000
2000
3000
171.0
57.0
35.8
4.35 (m)
3.11 (dd, J = 7.3, 14.0)
2.99 (dd, J = 10.4, 14.0)
4000
136.8
128.4
128.8
126.6
5000, 9000
6000, 8000
7000
7.20–7.32a
7.20–7.32a
7.16 (t, J = 7.3 Hz)
7.92 (br)
2000-NH
a
Fig. 2. 2D NMR analysis of 1.
Not assigned precisely because of signal overlapping.

Y. Terui et al. / Tetrahedron Letters 49 (2008) 3067–3070
3069
Table 2
GlyT-inhibitory activities of 1 (nM)
Compound
T98G
rGlyT1
rGlyT2
1
12.5
4.8
NT
20.4
3.0
NT
2261
NT
21.1
ALX5407
ALX1393
ALX5407 (Tocris): selective inhibitor of GlyT1.
ALX1393 (Sigma–Aldrich): selective inhibitor of GlyT2.
Eagle Medium with 10% fetal bovine serum. 2 ꢁ 104
cells/well were plated in a 96-well plate and incubated at
37 °C in a humidified incubator (95% air, 5% CO2). After
24 h, the cells were washed and incubated at room temper-
ature with 250 nM [3H]-glycine and 1 in an assay buffer.9
After 15 min, the assay buffer was removed, and the cells
were washed twice with the assay buffer. The cells were
then lysed with 0.5 M NaOH, a scintillation cocktail was
added, and the cell lysates were counted using a scintilla-
tion counter. Compound 1 inhibited glycine uptake in
T98G with an IC50 of 12.5 nM. ALX5407, a selective inhi-
bitor of GlyT1, showed inhibitory activity in this assay
system with an IC50 of 4.8 nM.10
Next, we evaluated the selectivity of 1 against GlyT1
and GlyT2 subtypes. The selectivity of GlyT inhibitory
activity was determined by the ability of 1 to inhibit the
uptake of [3H]-glycine in COS7 cells that had been trans-
fected with rat GlyT1 or GlyT2. Compound 1 inhibited
glycine uptake in these cells with IC50 of 20.4 nM and
2261 nM, respectively. ALX1393, a selective inhibitor of
GlyT2, showed inhibitory activity in this assay system with
an IC50 of 21.1 nM.11 These data revealed that 1 was a
selective inhibitor of GlyT1 (Table 2).
In summary, we have isolated a novel cyclic tetrapeptide
with selective GlyT1-inhibitory activity. Compound 1 con-
tained a novel amino acid, 4-oxo-30-sulfoisoleucine (Osi).
While alkyl sulfonate groups have been found in some
natural products,12 a highly functionalized sulfonoamino
acid such as Osi has not been reported previously.
Scheme 1. Reagents and conditions: (a) NaBH4, MeOH, rt, 24 h; (b) 6 M
HCl, 110 °C, 12 h; (c) (R)-(ꢀ)- or (S)-(+)-MTPACl, CH2Cl2, pyridine, rt,
4 h; (d) LiBH4, THF, rt, 6 h.
and 9.6 Hz, respectively, which reflected an anti/anti-con-
figuration of 2-H/3-H/4-H. Thus, the relative configuration
of 3 was determined to be 2S*, 3S*, and 4R*. This configu-
ration was also supported by a comparison of the 1H NMR
data between 3 and (2S,3S,4R)-cyclo-4-hydroxyisoleucine
(8), which is contained in funebrine (9) isolated from Qua-
raribea funebris.7 The coupling constants between 2-H/3-H
and 3-H/4-H of 8 were reported to be 11.6 Hz and 9.6 Hz,
respectively, which agrees well with those of 3.
To elucidate the absolute stereochemistry of 3, 3 was
converted to (S)- and (R)-MTPA amides (4, 5).8 The modi-
fied Mosher’s method was applied to 4 and 5, but no sys-
tematic Dd shift values (d4 ꢀ d5) were observed. The
MTPA groups seemed to assume a conformation different
from the ideal one. Therefore, 4 and 5 were converted to
linear alcohols 6 and 7 by reduction with LiBH4. After
reduction, the Dd shift values (d6 ꢀ d7) indicated a 2S con-
figuration (Fig. 3). Thus, the absolute configuration of Osi
was established to be 2S, 3S, as shown in Figure 1.
Acknowledgments
We thank Dr. Yuriko Nozawa for performing the LC/
MS analyses, Dr. Atsushi Okada for performing the
NMR measurements, and Mr. Haruaki Yamamoto for
fermenting TA-0426.
To measure the inhibitory activity toward glycine
uptake, T98G cells were cultured in Dulbecco’s Modified
References and notes
`
1. (a) Depoortere, R.; Dargazanli, G.; Estenne-Bouhtou, G.; Coste, A.;
Lanneau, C.; Desvignes, C.; Poncelet, M.; Heaulme, M.; Santucci, V.;
Decobert, M.; Cudennec, A.; Voltz, C.; Boulay, D.; Terranova, J. P.;
´
Stemmelin, J.; Roger, P.; Marabout, B.; Sevrin, M.; Vige, X.; Biton,
B.; Steinberg, R.; Franßcon, D.; Alonso, R.; Avenet, P.; Oury-Donat,
´
F.; Perrault, G.; Griebel, G.; George, P.; Soubrie, P.; Scatton, B.
Neuropsychopharmacology 2005, 30, 1963–1985; (b) Lowe, J. A., III.
Expert Opin. Ther. Patents. 2005, 15, 1657–1662.
Fig. 3. Dd shift values (ppm, 500 MHz, CD3OD) obtained for (S)- and
(R)-MTPA amides (6, 7).
2. (a) Toda, Y.; Chu, Y.; Li, J.; Terui, Y., Fukunaga, T.
WO2003104265; (b) Chu, Y.; Li, J.; Toda, Y. CN1535980.

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Y. Terui et al. / Tetrahedron Letters 49 (2008) 3067–3070
3. The seed medium was composed of soluble starch 2.5%, glucose 1.0%,
fish meal 0.5%, pharma media 0.3%, NZ case 0.3%, yeast extract 0.2%
and CaCO3 0.2%.
J = 9.6, 6.3 Hz). 13C NMR (125 MHz, D2O): d 20.6 (q), 46.7 (d), 53.0
(t), 57.5 (d), 82.4 (d), 175.3 (s).
7. (a) Raffauf, R. F.; Zennie, T. M.; Onan, K. D.; Le Quesne, P. W. J.
Org. Chem. 1984, 49, 2714–2718; (b) Tamura, O.; Iyama, N.;
Ishibashi, H. J. Org. Chem. 2004, 69, 1475–1480.
4. The production medium was composed of corn starch 2.0%, glycerol
1.0%, glucose 0.5%, pharma media 0.5%, cottonseed flour 0.5%, malt
extract 0.5%, polypepton 0.3%, Mg2SO4 0.05% and CaCO3 0.3%. The
pH of the medium was adjusted to 7.0 before sterilization.
5. (a) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem.
1997, 69, 5146–5151; (b) FDLA-derivatives were analyzed using a
YMC-Pack Pro C18 (100 ꢁ 4.6 mm; i.d., 3 lm; YMC) column at
40 °C. Acetonitrile–water containing 5 mM of ammonium formate
and 5 mM of formic acid was used as the mobile phase under a linear-
gradient elution mode (acetonitrile, 15–85%, 20 min) at a flow rate of
1.0 mL/min with UV detection at 215 nm. L- and D-FDLA deriva-
tives of Phe from 1 were detected at 9.11 min and 14.93 min,
respectively. L- and D-FDLA derivatives of Ile from 1 were detected at
10.60 min and 14.93 min, respectively. L-FDLA derivatives of stan-
dard L-Ile and L-allo-Ile were detected at 10.62 min and 10.36 min,
respectively.
8. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092–4096.
9. The assay buffer was composed of 10 mM HEPES (pH 7.4), 150 mM
NaCl, 1 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 10 mM glucose.
10. Atkinson, B. N.; Bell, S. C.; De Vivo, M.; Kowalski, L. R.; Lechner,
S. M.; Ognyanov, V. I.; Tham, C.-S.; Tsai, C.; Jia, J.; Ashton, D.;
Klitenick, M. A. Mol. Pharmacol. 2001, 60, 1414–1420.
11. Xu, T. X.; Gong, N.; Xu, T. L. Neuroreport 2005, 16, 1227–1231.
12. (a) Matsunaga, S.; Fusetani, S.; Konosu, S. J. Nat. Prod. 1985, 48,
236–241; (b) Rinken, M.; Lehmann, W. D.; Ko¨nig, W. A. Liebigs
Ann. Chem. 1984, 10, 1672–1684; (c) Kamiyama, T.; Umino, T.;
Itezono, Y.; Nakamura, Y.; Sato, T.; Yokose, K. J. Antibiot. 1995, 48,
929–936; (d) Shinagawa, S.; Kasahara, S.; Wada, Y.; Harada, S.;
Asai, M. Tetrahedron 1984, 40, 3465–3470; (e) Ito, T.; Ezaki, N.;
Ohba, K. J. Antibiot. 1982, 35, 533–535; (f) Reshef, V.; Mizrachi, E.;
Maretzki, T.; Silberstein, C.; Loya, S.; Hizi, A.; Carmeli, S. J. Nat.
Prod. 1997, 60, 1251–1260.
6. NMR data for 3: 1H NMR (500 MHz, D2O): d 1.54 (3H, d,
J = 6.3 Hz), 2.78 (1H, m), 3.24 (1H, dd, J = 10.2, 15.0 Hz), 3.33
(1H, dd, J = 4.1, 15.0 Hz), 4.53 (1H, d, J = 11.0 Hz), 4.59 (1H, dq,