Absolute structure of prunustatin A, a novel GRP78 molecular chaperone down-regulator

Article abstract of DOI:10.1021/ol7017856

In the course of our screening program for regulators of a molecular chaperone GRP78 expression, we isolated a novel inhibitor of GRP78 expression, designated as prunustatin A, from Streptomyces violaceoniger 4521-SVS3. The planar structure of prunustatin

Full text of DOI:10.1021/ol7017856

ORGANIC
LETTERS
2007
Vol. 9, No. 21
4239-4242
Absolute Structure of Prunustatin A, a
Novel GRP78 Molecular Chaperone
Down-Regulator
Yukiko Umeda,† Kazuo Furihata,† Shohei Sakuda,† Hiromichi Nagasawa,†
Ken Ishigami,† Hidenori Watanabe,† Miho Izumikawa,§ Motoki Takagi,§
Takayuki Doi,‡ Yoichi Nakao, and Kazuo Shin-ya*,§
Graduate School of Agricultural and Life Sciences, The UniVersity of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan, Biological Information Research Center,
National Institute of AdVanced Industrial Science and Technology, Koto-ku,
Tokyo 135-0064, Japan, Department of Applied Chemistry, Tokyo Institute of
Technology, Meguro-ku, Tokyo 152-8552, Japan, and School of AdVanced Science and
Engineering, Waseda UniVersity, Shinjuku-ku, Tokyo 169-8555, Japan
kshinya@jbirc.aist.go.jp
Received July 26, 2007
ABSTRACT
In the course of our screening program for regulators of a molecular chaperone GRP78 expression, we isolated a novel inhibitor of GRP78
expression, designated as prunustatin A, from Streptomyces violaceoniger 4521-SVS3. The planar structure of prunustatin A was determined
to be an oxidized type of the neoantimycin family. Its absolute stereochemistry was established to be 2R, 4S, 6S, 7R, 9S, and 29S by analyzing
chemically degraded components obtained from the derivative of prunustatin A.
GRP78 acts as a molecular chaperone in the endoplasmic
reticulum (ER) to promote protein folding. The enhancement
of ER stress response has been proven to play a role in
mechanisms of resistance to chemotherapy and hypoglycemic
stress in solid tumors.1 The ER stress response causes an
increase in gene expression of a number of ER chaperones
such as GRP78/Bip and GRP94.2 Thus, substances that
directly down-regulate grp78 transcription are expected to
be promising drugs for the treatment of cancer. In the course
of our screening program for regulators of a molecular
chaperone GRP78 expression, we isolated a novel inhibitor
of GRP78 expression, designated as prunustatin A (1), from
Streptomyces Violaceoniger 4521-SVS3 (Figure 1). The
planar structure of prunustatin A has been elucidated by a
variety of NMR spectral analyses.3 1 is a member of
neoantimycin family,4,5 but the absolute stereochemistry of
these analogues was not fully investigated. To the best of
† The University of Tokyo.
‡ Tokyo Institute of Technology.
§ National Institute of Advanced Industrial Science and Technology.
Waseda University.
(3) Umeda, Y.; Chijiwa, S.; Furihata, K.; Furihata, K.; Sakuda, S.;
Nagasawa, H.; Watanabe, H.; Shin-ya. K. J. Antibiot. 2005, 58, 206-209.
(4) Cagliote, L.; Misiti, D.; Selva, A.; Arcamone, F.; Cassinelli, G.
Tetrahedron 1969, 25, 2193-2221.
(1) Kaufman, R. J. Genes DeV. 1999, 13, 1211-1233.
(2) Katschinski, D. M.; Jacobson, E. L.; Wiedmann, G. J.; Robins,
H. I. J. Cancer Res. Clin. Oncol. 2001, 127, 425-435.
(5) Hayashi, K.; Nozaki, H. J. Antibiot. 1999, 52, 325-328.
10.1021/ol7017856 CCC: $37.00
© 2007 American Chemical Society
Published on Web 09/21/2007

prunustatin-producing strain and is considered to be a reduced
type of 1 at the C-1 position.
2 also consists of an amino acid and amino acid derived
R-hydroxy acid residues classified into a 3,4-dihydroxy-2,2-
dimethyl-5-phenylpentanoic acid, a lactic acid, a 2-hydroxy-
3-methylpentanoic acid, a threonine, and a 3-formylamino-
2-hydroxybenzoic acid moieties. 2 (10 mg) was hydrolyzed
with 3 N NaOH for 10 min at 95 °C. Then, 6 N HCl was
added to this reaction mixture to adjusted the pH to 2-3
followed by the extraction with ethyl acetate. The aqueous
layer was further subjected to acid hydrolysis for 12 h to
obtain threonine (3) and lactic acid (4) residues. The organic
layer from alkaline hydrolysis was concentrated to dryness,
and the dried residue was dissolved in saturated NaHCO3
aqueous solution followed by the extraction with diethyl ether
to provide 5-benzyl-4-hydroxy-3,3-dimethyldihydrofuran-2-
one (5, 3.2 mg, 99% purity), which was spontaneously
Figure 1. Structure of prunustatin A (1) and SW-163A (2).
our knowledge, few compounds such as 1, verrucosidins,6,7
and versipelostatin8-10 were reported to inhibit the expression
of GRP78 elicited by glucose deprivation. Since verucoccidin
and the formylaminosalicylate moiety of antimycins were
reported as the respiratory inhibitor, the mode of action
mechanisms of 1 is attributed to respiratory inhibition.
Recently, we succeeded in isolating the derivative of 1, of
which the formylaminosalicylate moiety was replaced by
benzoate moiety.11 Contrary to our speculation, the activity
to suppress the expression of GRP78 still remained in this
derivative, which suggested the exsistance of other target
molecules. In addition, the exact target molecules of ver-
sipelostatin have not been proven yet. Since the stereochem-
istry of active substances frequently contributes to their mode
of action mechanisms, the elucidation of the absolute
configuration of 1 is strongly desired, which will provide
the three-dimensional structural information between 1 and
versipelostatin.10 In this paper, we report herein the absolute
stereochemistry of 1.
Preparation of Hydrolyzed Components. Despite recent
progress in the NMR-based configuration analysis of natural
products,10,12,13 chemical degradation in combination with
partial synthesis is still considered the sole reliable method
for determining the stereochemistry. As 1 mainly consists
of cyclized amino and R-hydroxy acid moieties, the deter-
mination of the stereochemistry of 1 was performed by
analyzing the absolute configuration of each component
obtained by alkaline and acidic hydrolysis. However, the
racemization could occur at the C-2 position in 1 during the
reaction of alkaline hydrolysis, so that we employed SW-
163A (2)14 instead of 1, which was also produced by the
Figure 2. MTPA ester of 5-benzyl-4-hydroxy-3,3-dimethyldihy-
drofuran-2-one (5) and application of modified Mosher’s method.
converted from 3,4-dihydroxy-2,2-dimethyl-5-phenylpen-
tanoic acid in acidic condition.15 The residual aqueous layer
was adjusted to pH 2-3 with 6 N HCl and re-extracted with
EtOAc to afford 2-hydroxy-3-methylpentanoic acid (6,
1.3 mg). This protocol seemed to afford 6 with no major
hydrolysis byproducts (more than 90% purity) according to
1
the H NMR spectrum of 6.
Absolute Stereochemistry of the Threonine (3) and
Lactic Acid (4) Residues. The absolute configuration at C-6
and C-7 in 3 was determined by Marfey’s method,16 which
is applicable for the determination of the stereochemistry of
amino acids. After acid hydrolysis, the reaction solution was
adjusted to neutral and evaporated in vacuo. The residue was
dissolved in 0.1 M NaHCO3 and successively 10 mM NR-
(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (FDAA) in ac-
etone was added. The mixture was kept at 70 °C for 10 min
with frequent shaking. After workup with the addition of
0.2 N HCl, the filtered reaction mixture was subjected to
HPLC (Cosmosil ODS 5C18-MS, 4.6 i.d. × 250 mm, 15%
aq CH3CN with 0.05% TFA). The standard D- and L-
threonine and D- and L-allo-threonine were reacted with
FDAA in the same manner as described above. The threonine
residue (3) obtained from 2 was determined to be L-threonine
(retention time: FDAA conjugated 3, 42.5 min; L-Thr,
42.5 min; D-Thr, 83.0 min; L-allo-Thr, 46.2 min; D-allo-Thr,
57.5 min).
(6) Choo, S.-J.; Park, H.-R.; Ryoo, I.-J.; Kim, J.-P.; Yun, B.-S.; Kim,
C.-J.; Shin-ya, K.; Yoo, I.-D. J. Antibiot. 2005, 58, 210-213.
(7) Park, H.-R.; Ryoo, I.-J.; Choo, S.-J.; Hwang, J.-H.; Kim, J.-Y.; Cha,
M.-R.; Shin-ya, K.; Yoo, I.-D. Toxicology 2007, 229, 253-261.
(8) Park, H.-R.; Furihata, K.; Hayakawa, Y.; Shin-ya, K. Tetrahedron
Lett. 2002, 43, 6941-6945.
(9) Park, H.-R.; Tomida, A.; Sato, S.; Tsukumo, Y.; Yun, J.; Yamori,
T.; Hayakawa, Y.; Tsuruo, T.; Shin-ya, K. J. Natl. Cancer Inst. 2004, 96,
1300-1310.
(10) Park, H.-R.; Chijiwa, S.; Furihata, K.; Hayakawa, Y.; Shin-ya, K.
Org. Lett. 2007, 9, 1457-1460.
(11) Izumikawa, M.; Ueda, J.; Chijiwa, S.; Takagi, M.; Shin-ya, K. J.
Antibiot. In press.
(12) Kobayashi, H.; Shin-ya, K.; Furihata, K.; Hayakawa, Y.; Seto, H.
Tetrahedron Lett. 2001, 42, 4021-4032.
(13) Hayakawa, Y.; Kim, J. W.; Adachi, H.; Shin-ya, K.; Fujita, K.; Seto,
H. J. Am. Chem. Soc. 1998, 120, 3524-3525.
(15) Benedetti, E.; Ganis, P.; Bombieri, G.; Caglioti, L.; Germain, G.
Acta. Crystallogr. 1975, B31, 1097-1100.
(16) Adamson, J. G.; Hoang, T.; Grivici, A.; Lajoie, G. A. Anal. Biochem.
1992, 202, 210-214.
(14) Takahashi, K.; Tsuda, E.; Kurosawa, K. J. Antibiot. 2001, 54,
867∼873.
4240
Org. Lett., Vol. 9, No. 21, 2007

Conclusively, the stereochemistry at the C-4 position in 2
was established as S.
Scheme 1. Degradation of 2
Determination of Stereochemistry of the Lactone
Moiety (5). The absolute configuration at the C-1 position
in 2 was established by modified Mosher’s method18 as
follows. The lactone compound (5), which was obtained from
the ether layer of alkaline hydrolysis of 2, was reacted with
(+)-MTPA or (-)-MTPA chloride to give (R)- and (S)-
MTPA ester, respectively (Table 1). The proton chemical
Table 1. 1H (500 MHz) NMR Data for the (S)- and (R)-MTPA
Esters of 5
S-MTPA ester δH
(multiplicity, J ) Hz)
R-MTPA ester δH
(multiplicity, J ) Hz)
no.
2-Me
1.33
1.32
1.11
1.06
4
5
4.77
4.78
2.785 (dd, J ) 14.5, 9.5)
2.66 (dd, J ) 14.5, 4.0)
7.115 (dd, J ) 7.5, 1.5)
7.425 (m)
2.825 (dd, J ) 14.5, 9.5)
2.71 (dd, J ) 14.5, 4.0)
7.158 (dd, J ) 7.5, 1.5)
7.425 (m)
6
7
8
7.54 (m)
7.53 (m)
shifts at the C-2 and C-12 positions in the (R)-MTPA ester
derivative of 5 appeared at a lower field than those of the
(S)-MTPA ester derivative. On the other hand, the proton
chemical shifts of both singlet methyl moieties located at
the C-11 position in the (R)-MTPA ester derivative were
observed at a higher field than those of the (S)-MTPA ester
derivative. Thus, the absolute stereochemistry at the C-1
position in 5 was deduced to be the R configuration.
The stereochemistry at the C-2 position in 2 was confirmed
by the determination of the relative configuration between
C-1 and C-2 employing the J-based method.19 The small
The stereochemistry of the C-4 position which was
included in the lactate moiety was determined by utilizing a
chiral column according to the method described previously
as follows.17 The acid hydrolysate, which was independently
prepared, was neutralized with 5 N NaOH and evaporated
to dryness, and the residue was taken up in DMF and treated
with p-bromophenacyl bromide in the presence of KF at
50 °C for 12 h. The reaction mixture was concentrated to
dryness and separated by preparative silica gel-TLC (n-
hexane:EtOAc ) 3:1) to give p-bromophenacyl lactate.
According to the retention time in HPLC on a chiral
stationary phase (Chiralcel OJ, 4.6 i.d. × 250 mm; n-hexane:
2-propanol ) 9:1), the lactate derived from 2 was elucidated
to be L-lactate (observed, 46.4 min; standard L-lactate
derivative, 46.4 min; standard D-lactate derivative, 49.6 min).
3
coupling constant values for J6H-7H revealed that these
protons are in the gauche orientation. Moreover, the small
2
coupling constants (2J1C-2H and J2C-1H) indicate that H-1
and an oxygen atom at C-2, and H-2 and an oxygen atom at
C-1 are both in the anti location as shown in Figure 3.
Figure 3. The absolute stereochemistry of C-1 and C-2 in 2.
Scheme 2. Synthesis of the FDAA Derivative of 3
Considering these relative stereochemistries, the C-2 position
was assigned as R by comparing with the absolute config-
uration of C-1 (R).
(17) Matsunaga, S.; Liu, P. C.; Celatka, A.; Panek, J. S.; Fusetani, N. J.
Am. Chem. Soc. 1999, 121, 5605-5606.
(18) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092-4096.
(19) Murata, M.; Matsuoka, S.; Matsumori, N.; Paul, G. K.; Tachibana,
K. J. Am. Chem. Soc. 1999, 121, 870-871.
Org. Lett., Vol. 9, No. 21, 2007
4241

ester derivative of 6 and the authentic sample of (2S,3S)-
methyl 2-hydroxy-3-methylpentanoate, which was derived
from L-isoleucine, also supported the stereochemistry of 6.
Absolute Stereochemistry of 1. To determine the final
stereochemical structure of 1, we applied the Dess-Martin
Periodinane (DMP)22 to obtain the selective oxidized type
of 2 at the C-1 position. The optical rotation of the oxidized
type of 2 [+22.8 (c 0.24, CHCl3)] was almost the same as
Scheme 3. Preparation of the FDAA Derivative of 3
1
that of 1 [+21.2 (c 0.01, CHCl3)]. The H NMR data and
MS spectrum (m/z) and retention time of analyzed HPLC
supported that the oxidative type of 2 possesses the same
stereochemistry as 1. The reduction of the ketone moiety in
1 with NaBH4 unexpectedly afforded only one product. The
1H NMR spectrum of this reduced product from 1 completely
corresponded with that of 2. Conclusively, the absolute
stereochemistry of prunustatin A was determined as 2R, 4S,
6S, 7R, 9S, and 29S as shown in Figure 1. Although the
planar structures of 1 and 2 are closely similar to that of
neoantimycin, it is interesting that the absolute stereochem-
istry at C-1 and C-2 of 1 and 2 is opposite that reported for
neoantimycin (1R and 2R).23
1 showed more potent inhibitory activity than 2 against
GRP78 expression induced by 2-deoxyglucose stimulation
in HT1080 cells which were transformed by the luciferase
reporter gene inserted under GRP78 promoter. Thus, the
redox state at the C-1 position is considered to be essential
for exhibiting stronger biological activity. 1 belongs to the
neoantimycin family, which is well-known as a respiratory
inhibitor. In our preliminary biological studies, mitochondrial
respiratory inhibitors specifically suppressed the induction
of GRP78 expression elicited by glucose starvation, but not
by chemically inducing ER stresses such as tunicamycin
treatment. It showed that mitochondrial function plays an
significant role in exerting ER stress responces, and it also
gave us an new idea for targeting a solid tumor. Studies on
detailed biological activities of 1 are now underway.
Determination of the Stereochemistry of the 2-Hy-
droxy-3-methylpentanoic Acid Moiety (6). The absolute
configuration of the stereogenic center at C-29 and C-9 can
be determined by using NOE analysis combined with J
coupling analysis. The relative configuration between C-29
and C-9 was determined as follows. A large 3JH-H coupling
constant (10 Hz) between 9-H and 29-H suggested that these
protons were in the anti orientation. In the same way, small
3JC-H coupling constants (∼2 Hz) between 9-H and C-30
(3J30C-9H ≈ 2 Hz) and 9-H and C-31 (3J31C-9H ≈ 2 Hz)
suggested that C-30 and C-31 were both in the gauche
orientation and two possible relative structures were con-
structed as shown in Figure 4 A,B. An NOE between the
Acknowledgment. This work was supported by the
Industrial Technology Research Grant Program in 01A04006b
from the New Energy and Industrial Technology Develop-
ment Organization (NEDO) of Japan.
Figure 4. Relative stereochemistry of C-9 and C-29 in 2.
doublet methyl signal of 28-H (δH 1.31) and the doublet
methyl signal of 30-H (δH 0.87) in the high-resolution
NOESY spectrum reasonably explains the relative config-
uration at C-9 and C-29 to be S* and S*, respectively.
An optical rotation (-33.8, c 0.18, in CHCl3) of 2-hy-
droxy-3-methylpentanoic acid (6) obtained from the organic
layer of the alkaline hydrolysis of 2 established the absolute
structure unambiguously as 9S and 29S, respectively (refer-
ence data for (2S,3S)-2-hydroxy-3-methylpentanoic acid:
Supporting Information Available: Selected spectra
1
used for configuration assignments of 1 and 2; H NMR of
1, 2, 5, 6, R- and S-MTPA esters of 5, standard (2S,3S)-2-
hydroxy-3-methyl pentanoic acid, the oxidized derivative of
2, and reduced derivative of 1, NOESY, CT-HMBC, J-
2,3
resolved HMBC for measurement of
J
of 2, MS
C-H
spectrum of 5, 6, and the MTPA ester of 5. This material is
available free of charge via the Internet at http://pubs.acs.org.
[R]D -21.6 (c 1.0, in CHCl3)).20,21 The corresponding H
NMR spectrum and HPLC retention time between the methyl
1
OL7017856
(22) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277-
7287.
(23) Takeda, Y.; Masuda, T.; Matsumoto, T.; Takechi, Y.; Shingu, T.;
Flos, H. G. J. Nat. Prod. 1998, 61, 978-981.
(20) Murai, A.; Amino, Y.; Ando, T. J. Antibiot. 1985, 38, 1610-1612.
(21) Shin, I.; Lee, M.-R.; Lee, J.; Jung, M.; Lee, W.; Jongbok Yoon, J.
J. Org. Chem. 2000, 65, 7667-7675.
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