What Is the True Structure of D609, a Widely Used Lipid Related Enzyme Inhibitor?

Article abstract of DOI:10.1021/acs.orglett.6b00025

(Chemical Equation Presented) D609 (1) has been used as a lipid-related enzyme inhibitor during the past three decades. Although it has eight possible stereoisomers, no systematic research considering its chirality has been performed. In this paper, eight possible chiral alcohols as direct precursors of D609 were synthesized, and their stereochemistries were elucidated by a vibrational circular dichroism (VCD) technique. Phosphatidylcholine-specific phospholipase C and sphingomyelin synthase inhibition assays of these isomers showed considerable differences in their activities.

Full text of DOI:10.1021/acs.orglett.6b00025

Letter
pubs.acs.org/OrgLett
What Is the True Structure of D609, a Widely Used Lipid Related
Enzyme Inhibitor?
Mikako Kato,†,§ Mostafa A. S. Hammam,‡,§ Tohru Taniguchi,‡ Yoshiko Suga,‡ and Kenji Monde*,‡
‡
†Graduate School of Life Science and Faculty of Advanced Life Science, Frontier Research Center for Post-Genome Science and
Technology, Hokkaido University, Kita 21 Nishi 11, Sapporo 001-0021, Japan
S
* Supporting Information
ABSTRACT: D609 (1) has been used as a lipid-related enzyme inhibitor
during the past three decades. Although it has eight possible stereoisomers, no
systematic research considering its chirality has been performed. In this paper,
eight possible chiral alcohols as direct precursors of D609 were synthesized,
and their stereochemistries were elucidated by a vibrational circular dichroism
(VCD) technique. Phosphatidylcholine-specific phospholipase C and
sphingomyelin synthase inhibition assays of these isomers showed
considerable differences in their activities.
ricyclodecan-9-yl xanthogenate (D609, 1, Figure 1) is a
synthetic tricyclic compound having a xanthate group,
T
known as a phosphate group analogue. Though the first
synthesis was reported a half century ago,1 the first biological
study of it as antiviral activity started in 1984.2 After this
finding, numerous biological activities, such as antiviral,
antitumor, anti-inflammatory, and antiapoptosis properties,
have been reported.3,4 Most of these activities have been
attributed to the characteristic competitive inhibitory effect of
the D609 on phosphatidylcholine-specific phospholipase C
(PC−PLC)5−7 and indirectly on acidic sphingomyelinase
(SMase).8−10 D609 has also been found to be a sole inhibitor
of a sphingomyelin synthase (SMS) whose relationship to
metabolic syndrome and amyloid-β peptide clearance was
recently reported.11−16 During the last three decades, more
than 700 reports concerning D609’s biological activity,
especially related to lipid biology, have been published.
Currently, D609 is an extremely significant inhibitor for lipid
biology and lipid chemical biology.
Figure 2. Structures of all possible D609 stereoisomers (6−9) and
their corresponding precursor alcohols (2−5). Stereochemistry was
defined using the C-, O-, exo-, and endo-notational system.
analytical methods such as NMR techniques.18 Thus, we
attempted to reveal the stereochemistry of the D609 using the
emerging vibrational circular dichroism (VCD) technique.
VCD measures the differential absorption of left versus right
circularly polarized IR radiation on a chiral molecule by a
molecular vibrational transition. In the past decade, VCD
spectroscopy with ab initio theoretical calculations has been
established as a reliable and convenient method to determine
its absolute configuration.19,20 VCD has a great advantage to
distinguish between these simple and rigid stereo isomers for
hydrocarbons whose NMR signals are severely overlapped, and
thus, the assignment of peaks is extremely difficult.
Figure 1. Structure of D609 (1) (potassium tricyclodecan-9-yl
xanthogenate).
In the present paper, we report the first systematic chiral
preparation of all possible stereoisomers of D609 and
unambiguous determination of their enigmatic absolute
configurations as well as their relative stereochemistries by
VCD spectroscopy with the aid of their theoretical calculations.
In addition, we describe inhibition experiments of PC−PLC
D609 has three asymmetric centers, which lead to eight
stereoisomers including their enantiomers (Figure 2). However,
to the best of our knowledge, no systematic research has been
conducted to investigate the effect the chirality of D609s on
their biological activity so far.17 Furthermore, the commercially
available D609s commonly do not show any chiral information
and relative stereochemistry because of the enormous difficulty
of the assignment of their accurate stereochemistry by standard
Received: January 4, 2016
© XXXX American Chemical Society
A
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Letter
and SMS for all chiral isomers and the determination of
stereochemistry of commercial available D609s by utilizing
these authentic standard isomers.
A relatively degradable xanthate function should be
introduced in the final step to each corresponding alcohol;
therefore, four diastereomeric alcohols were planned to be
synthesized and followed by its chiral separation leading to
totally eight chiral stereoisomers. Each disastereoisomer is
defined using the C-, O-, exo-, and endo-notational system as
shown in Figure 2. First, the synthesis of C-exo- and O-endo-
type diastereomers 2 was conducted via the reduction of the
commercially available tricyclo[5.2.1.02,6]decan-8-one (10)
exhibiting well-confirmed stereochemistry by sodium borohy-
dride in methanol to afford a diastereomeric mixture of alcohols
2 and 3 (ratio = 10:1, respectively) in 97% yield. The resulted
alcohols were treated with 4-nitrobenzoyl chloride in THF in
the presence of pyridine to give the corresponding ester, which
was recrystallized to give the pure C-exo and O-endo
diastereomer 11 in 21% yield (Scheme 1). The chiral
Scheme 1. Synthesis of Chiral C-exo- and O-endo-Alcohol 2
Figure 3. Comparison of VCD (upper frame) and IR (lower frame)
spectra of (a) 2, (b) 3, (c) 4, and (d) 5. Measurement conditions:
CDCl3, l = 100 μm, c = 0.3 M, corrected by solvent spectra obtained
under the same measurement conditions. Calculation conditions:
DFT/B3LYP/6-311G(d,p) without considering solvent effects.
two to six stable conformers that differ in the orientation of the
hydroxyl group and the puckering of the terminal 5-membered
ring (Figure S2).21 The theoretical VCD and IR spectra of
these conformers were calculated and then averaged on the
basis of their Boltzmann populations and compared with the
experimental ones (Figure 3). All of the theoretical VCD
spectra for each predicted structure showed good agreement
with those measured for the isomers with the predicted relative
configuration; for example, the negative VCD signals
experimentally observed for (+)-2 at around 1350, 1290,
1000 cm−1 were well reproduced in the calculated spectrum for
(R)-2. The theoretical IR spectral features of each isomer also
agreed well with the experimental ones, except the sharp
intense IR signal predicted at ca. 1230 cm−1 (for 2, 4, and 5)
ascribed to a C−O−H bending transition, which is easily
perturbed by intermolecular interactions and proton exchange
under experimental conditions. Overall, the agreement between
the theoretical spectra and the experimental spectra not only
confirmed the relative configuration of these isomers but also
determined their absolute configurations as (R)-(+)-2, (S)-
(−)-3, (S)-(+)-4, and (R)-(−)-5.
To investigate their biological activities in terms of chirality,
potassium xanthates of all D609 isomers were synthesized from
alcohols in 50−70% yields by treating them with potassium tert-
butoxide and carbon disulfide. The resulting chiral D609s were
subjected to the PC-PLC and SMS inhibition assay. In vitro
inhibition assay of the phosphatidylcholine phospholipase C
from Bacillus cereus of all D609s showed that (R)-8 inhibited
the strongest IC50 (9 μM), while (R)-6 inhibited the weakest
IC50 (37 μM), demonstrating a maximum four times difference
separation was achieved by using a recently developed
middle-pressure chiral liquid chromatography separation
system (MPLC, CHIRALFLASH-IF) on relatively medium
scale to give enough enantiomers to perform two more
reactions toward the D609s. Enantiomerically pure esters (S)-
11 and (R)-11 were hydrolyzed using 4 M aqueous sodium
hydroxide in methanol to give the corresponding chiral alcohols
(C-exo, O-endo) (S)-2 and (R)-2 in 86% and 90% yields,
respectively. The alcohols (S)-3 and (R)-3 of (C-exo, O-exo)
were prepared via stereochemical conversion by applying
Mitsunobu reaction of 2 with 1-naphthaleneeacetic acid to
form racemic esters followed by chiral MPLC separation. Other
alcohols (S)-4, (R)-4 (C-endo, O-exo) and (S)-5, (R)-5 (C-endo,
O-endo) were prepared by the same strategy.21
In order to verify their relative configuration and to
determine their absolute configuration, we then applied VCD
spectroscopy to these enantiomerically pure diastereomers.
Figure 3 shows the VCD and IR spectra of (+)-2, (−)-3, (+)-4,
and (−)-5 measured in deuterated chloroform (CDCl3) with a
concentration of 0.3 M. The VCD spectra of these
diastereomers were sufficiently different to allow distinction
by theoretical calculations as described below. Meanwhile, the
enantiomers of each isomer exhibited virtually mirror-image
VCD and superimposable IR spectra, suggesting that the
enantioseparations of these isomers were indeed achieved
successfully.21
Prior to the theoretical calculations of their VCD and IR
spectra, a MMFF conformational search was conducted for
each isomer. The following DFT optimization resulted in only
B
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depending upon their stereochemistries.22 It is also a significant
point that this enzyme could recognize these simple
enantiomers of hydrocarbons ((S)- and (R)-8) that are
extremely difficult to distinguish by the standard methods.
Recent interest in the sphingomyelin synthase (SMS) for drug
discovery toward metabolic syndrome prompted us to
investigate another inhibition assay of the D609s by using
mouse cell lysate system overexpressed SMS1 and SMS2,
respectively.23 The D609 was known as the sole inhibitor of
SMS in relatively high concentration. All D609s showed almost
the same moderate inhibition (IC50 = 70−150 μM) toward
SMS1, while toward its isozyme SMS2, a significant difference
was observed between the stereoisomers (i.e., (S)-7; IC50 = 150
μM, (R)-9; IC50 = 840 μM) in their IC50.
Most biological studies were performed by using commer-
cially available D609s; therefore, their stereochemistries were
studied. An effective GC-analysis system (Zebron ZB-WAX 30
m × 0.25 mm × 0.25 μm) was created by utilizing four standard
alcohols whose stereochemistries were unambiguously con-
firmed by the previously described method. After removal of its
xanthate group from the sample D609, a resulting alcohol was
submitted to the GC analysis system. Two commercial samples
of D609 from two different resources (Sigma-Aldrich and Enzo
Life Science) were examined. Surprisingly, it was revealed that
these two samples have completely different stereochemistries.
In the Sigma case, the sample showed that it contains 84% of
the C-exo,O-exo isomer (7), 3% of the C-endo,O-exo isomer (8),
and unknown impurities, while in the Enzo case, it showed the
existence of 92% of the C-exo,O-endo isomer (6) and 8% of the
C-exo,O-exo isomer (7) in stark contrast with the Sigma case.
All possible stereoisomers of D609s were systematically
prepared and enantiometically separated as alcohol precursors
for the first time. The stereochemistries of all possible chiral
D609s were unambiguously determined by the VCD technique
with ab initio calculations. It is also a significant point that VCD
can distinguish the possible relative stereochemistries of D609s
as well as their absolute configurations, which is extremely
difficult to determine by normal methods. These isomers
showed significantly different biological activity toward
inhibition assay by using the PC−PLC and SMS system.
Finally, two D609s from commercial resource showed different
stereochemistries, which demonstrated different biological
activity. The control and unambiguous characterization of the
stereochemistry of D609s was successfully achieved. Our work
will contribute to the emerging lipid biology and lipid chemical
biology fields.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Dr. Kohei Yuyama, Prof. Susumu Mitsutake,
and Prof. Yasuyuki Igarashi at Hokkaido University for helpful
advice and discussions of the SMS inhibition assay. This work
was partially supported by the Innovation COE Project for
Future Medicine and Medicinal Research’ and a Grant in Aid
for Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
DEDICATION
■
This paper is dedicated to Prof. Koji Nakanishi at Columbia
University on the occasion of his 90th birthday.
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AUTHOR INFORMATION
Corresponding Author
■
*E-mail: kmonde@sci.hokudai.ac.jp.
Author Contributions
§M.K. and M.A.S.H. contributed equally.
C
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(22) The assay kit was obtained from InvitrogenTM EnzChek direct
phospholipase C assay kit, which was originally used by Eli Lilly & Co.
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