Dual structure-activity relationship of osteoclastogenesis inhibitor methyl gerfelin based on teg scanning

Article abstract of DOI:10.1021/bc3003666

Methyl gerfelin derivatives, each having an amine-terminated tri(ethylene glycol) linker at the peripheral position, were designed and systematically synthesized. These TEGylated derivatives were then subjected to a structure-activity relationship (SAR) study to examine their glyoxalase 1-inhibition activity and binding affinity toward the three binding proteins identified. Among the derivatives synthesized, that with a NH₂-TEG linker at the C6-methyl group showed the most potent glyoxalase 1-inhibiting activity and glyoxalase 1 selectivity. These results indicated that derivatization at the C6-methyl group would be suitable for the further development of selective glyoxalase 1 inhibitors.

Full text of DOI:10.1021/bc3003666

Article
pubs.acs.org/bc
Dual Structure−Activity Relationship of Osteoclastogenesis Inhibitor
Methyl Gerfelin Based on TEG Scanning
Naoki Kanoh,*,† Takahiro Suzuki,† Makoto Kawatani,‡ Yasuhiro Katou,† Hiroyuki Osada,‡
and Yoshiharu Iwabuchi†
†Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan
‡Chemical Biology Core Facility, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
S
* Supporting Information
ABSTRACT: Methyl gerfelin derivatives, each having an amine-terminated tri(ethylene glycol) linker at the peripheral position,
were designed and systematically synthesized. These “TEGylated” derivatives were then subjected to a structure−activity
relationship (SAR) study to examine their glyoxalase 1-inhibition activity and binding affinity toward the three binding proteins
identified. Among the derivatives synthesized, that with a NH2-TEG linker at the C6-methyl group showed the most potent
glyoxalase 1-inhibiting activity and glyoxalase 1 selectivity. These results indicated that derivatization at the C6-methyl group
would be suitable for the further development of selective glyoxalase 1 inhibitors.
Target protein “fishing” experiments using these methyl
gerfelin-photo-cross-linked beads and peptide mass finger-
printing (PMF) analysis8 identified three cellular proteins as
major methyl gerfelin-binding proteins: namely, glyoxalase 1
(GLO1),9,10 sterol binding protein 2 (SCP2),11,12 and small
glutamine-rich tetratricopeptide repeat containing protein A
(SGTA).13 On the basis of the subsequent biochemical and
biological analysis, it was concluded that methyl gerfelin (2)
inhibited GLO1 activity, resulting in an accumulation of methyl
glyoxal that led to the inhibition of osteoclastogenesis.5 We also
obtained the cocrystal structure of the methyl gerfelin (2)-
GLO1 complex, which showed that a catechol moiety on ring A
of methyl gerferin (2) coordinates to the active site zinc ion of
GLO1, while no significant interaction between ring B and
GLO1 was observed.
Although we have thus obtained a detailed picture of GLO1
inhibition by methyl gerfelin (2), we still do not understand the
binding mode between 2 and the other two binding proteins
and the consequences of the interaction. It is now believed that
small molecule drugs can act on multiple biomacromolecules,
which in some cases can lead to not only sequestration of the
drug from the main targets14 but also synergistic or adverse
drug reactions.15 Thus, to fully understand the biological effects
of methyl gerfelin (2) and to design more potent GLO1 and/or
INTRODUCTION
■
Osteoclast-targeting small molecule inhibitors are useful not
only for carrying out basic research on osteoclasts, but also for
treating bone-related diseases, including osteoporosis, rheuma-
toid arthritis, and cancer bone metastasis.1,2 To date, many
synthetic compounds and natural products have been reported
to inhibit the differentiation and function of osteoclasts, and
identification of the mechanisms underlying the inhibiting
activity have deepened our understanding of osteoclast
function.
In efforts to identify new small molecule inhibitors of
osteoclast function, we found in 2008 that a fungal-derived
natural product gerfelin (1)3,4 and its methyl ester methyl
gerfelin (2) inhibited osteoclast differentiation in vitro (Figure
1A).5 Methyl gerfelin (2) suppressed osteoclastogenesis with an
IC50 value of 2.8 μM without affecting the survival and function
of mature osteoclasts at the effective concentration.
To identify the cellular target(s) of methyl gerfelin (2) and
the underlying inhibition mechanism, we prepared methyl
gerfelin (2)-immobilized affinity beads by using a photo-cross-
linking protocol.6,7
Received: July 5, 2012
Revised: December 14, 2012
Published: December 27, 2012
Figure 1. Structure of gerfelin and methyl gerfelin.
© 2012 American Chemical Society
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osteoclastogenesis inhibitors, the relationship between the
gerfelin structure and GLO1-inhibiting activity as well as
GLO1 selectivity over SCP2 and SGTA are of particular
interest and remain to be clarified.
shifts (δ) are given from TMS (0.00 ppm) or CHCl3 (7.26
ppm) in CDCl3, or from CHD2OD (3.30 ppm) in CD3OD as
internal standards. For 13C NMR spectra, chemical shifts (δ)
are given from CDCl3 (77.0 ppm) or CD3OD (49.0 ppm) as
internal standards. The following abbreviations were used to
indicate the multiplicities: s = singlet, d = doublet, t = triplet, q
= quartet, m = multiplet, br = broad. Mass spectra were
recorded on JEOL JMS-DX303, JEOL JNM-AL500, and JEOL
JMS-700 mass spectrometers.
The first of these SAR studies (i.e., the study of the relation
between GLO1 inhibition and M-GFN structure) can be easily
performed by an enzyme inhibition assay of methyl gerfelin
derivatives. However, the latter SAR study (that examining the
relation between GLO1 selectivity and M-GFN structure) has
to be done through an assay other than an enzymatic one,
because the other two proteins are categorized in different
classes and are not related to each other or to GLO1. An in
vitro ligand-binding assay such as a pull-down experiment is a
candidate, but would require a common functional group on
every derivative in order to introduce the derivatives to affinity
matrices, or to introduce a reporter group on the derivatives.
To address this issue, we propose herein a TEG scanning
strategy, in which drug derivatives each having an amine-
terminated tri(ethylene glycol) (NH2-TEG) linker at a
peripheral position are systematically prepared and tested in
both functional and binding assays. The NH2-TEG linker is
designed to be used for two purposes: as a protruding area or a
“bump” to assist in identifying the regions of a molecule that
are important or unimportant for GLO1-inhibition activity, and
as a “handle” to be introduced on affinity matrices for the pull-
down experiments.
Synthesis of TMG Compounds. Synthesis of TMG1 (3).
To a solution of ester 10 (88.0 mg, 0.256 mmol) in MeCN (2.1
mL) were added bromide 11 (66.5 mg, 0.213 mmol) and
K2CO3 (162 mg, 1.17 mmol) at room temperature. After being
stirred at reflux temperature for 13.5 h, the reaction mixture was
filtered through a pad of Celite. The filtrate was concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography (EtOAc/Hexane = 1/2 to 1/1) to give
protected TMG1 (122 mg, 0.212 mmol, 100%) as a yellow oil.
1
Protected TMG1: IR (neat): 3383, 1715 cm−1; H NMR
(400 MHz, CDCl3): δ 6.45 (d, J = 2.0 Hz, 1H), 6.44 (d, J = 1.2
Hz, 1H), 6.39 (d, J = 2.0 Hz, 1H), 6.32 (d, J = 1.2 Hz, 1H),
5.04 (br s, 1H), 4.08 (t, J = 5.0 Hz, 2H), 3.87 (s, 3H), 3.81 (t, J
= 5.0 Hz, 2H), 3.69 (t, J = 4.8 Hz, 2H), 3.60 (t, J = 4.6 Hz, 2H),
3.53 (t, J = 5.0 Hz, 2H), 3.31 (br q, J = 4.8 Hz, 2H), 2.24 (s,
3H), 2.23 (s, 3H), 1.66 (s, 6H), 1.44 (s, 9H); 13C NMR (100
MHz, CDCl3): δ 168.2, 158.5, 157.1, 155.8, 149.1, 138.1, 137.4,
135.6, 131.6, 118.9, 118.5, 113.8, 110.7, 105.9, 99.8, 79.1, 70.9,
70.3, 70.3, 69.4, 68.8, 52.0, 40.4, 28.4, 25.8, 21.3, 19.7; HRMS
(EI): calcd for C30H41O10N (M+): 575.2730, found: 575.2753.
To a mixture of protected TMG1 (36 mg, 62.5 μmol) in
H2O (0.25 mL) was added trifluoroacetic acid (TFA) (1.25
mL) at 0 °C. After being stirred at room temperature for 1 h,
the reaction mixture was concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(MeOH/CHCl3 = 1/9) to give TMG1 (3) (27.2 mg, 62.5
μmol, 100%) as a yellow solid.
TMG1 (3): Mp. 43−44 °C; IR (neat): 3359, 3186, 1722
cm−1; 1H NMR (400 MHz, CDCl3): δ 6.75 (d, J = 1.6 Hz, 1H),
6.57 (s, 1H), 6.28 (s, 1H), 6.24 (d, J = 1.6 Hz, 1H), 5.75−5.40
(br s, 4H), 4.21 (t, J = 4.4 Hz, 2H), 3.87 (s, 3H), 3.76 (t, J = 4.4
Hz, 2H), 3.63 (t, J = 4.0 Hz, 2H), 3.53 (t, J = 4.0 Hz, 2H), 3.45
(s, 2H), 2.78 (s, 2H), 2.19 (s, 6H); 13C NMR (100 MHz,
CDCl3): δ 168.7, 159.7, 158.1, 147.4, 142.4, 138.0, 135.2, 128.7,
118.4, 112.9, 112.0, 109.8, 101.2, 71.1, 70.6, 70.4, 70.4, 69.7,
52.0, 40.6, 20.8, 19.7; HRMS (EI): calcd for C22H29O8N (M+):
435.1893, found: 435.1881.
Thus, to gain insights into the SAR between GLO1-
inhibition activity and GLO1-binding selectivity, we planned
to apply this TEGylation strategy to methyl gerfelin (2). To
this end, we designed and synthesized TEGylated methyl
gerfelin (TMG) derivatives, and subjected them to GLO1-
inhibition and protein pull-down assays. In addition, some of
the TMG derivatives were subjected to an in vitro osteoclasto-
genesis inhibition assay. The results are reported in this article.
EXPERIMENTAL PROCEDURES
■
Materials and Methods. All reactions were carried out
under an argon atmosphere with dehydrated solvents under
anhydrous conditions, unless otherwise noted. Dehydrated
THF and CH2Cl2 were purchased from Kanto Chemical Co.,
Inc. Other solvents were dehydrated and distilled according to
standard protocols. Reagents were obtained from commercial
suppliers and used without further purification, unless
otherwise noted. The preparation of compounds 1, 2, 9, 11,
14, 17, 21, 25, and 39 was performed as described in the
literatures those mentioned in the Results section. Compounds
26 and 36 are commercially available. The syntheses of
compounds 10, 12, 15, 16, 18−20, 22−24, 27−35, 37, and 38
are described in the Supporting Information. Reactions were
monitored by thin-layer chromatography (TLC) carried out on
silica gel plates (Merck Kieselgel 60 F254) or NH-silica gel
plates (Fuji Silysia Chemical Co., Ltd.). Column chromatog-
raphy was performed on silica gel 60N (spherical, neutral, 63-
210 μm; Kanto Chemical Co., Inc.). Flash column chromatog-
raphy was performed on silica gel 60N (spherical, neutral, 40−
50 μm; Kanto Chemical Co., Inc.) or Chromatorex NHDM
1020 (NH2-coated silica gel, 100−200 mesh; Fuji Silysia
Chemical Co., Ltd.). All melting points were determined with a
Yazawa Micro Melting Point BY-2 apparatus and were
uncorrected. IR spectra were recorded on a JASCO FT/IR-
Synthesis of TMG2 (4). To a mixture of amide 16 (6.45 mg,
11.5 μmol) in H2O (0.33 mL) was added TFA (1.0 mL) at 0
°C. After being stirred at room temperature for 3.5 h, the
reaction mixture was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
(MeOH/CHCl3 = 1/9 to 1/4) to give TMG2 (4) (4.80 mg,
11.4 μmol, 99%) as a red solid.
TMG2 (4): Mp. 90−92 °C; IR (neat): 3065, 1577 cm−1; 1H
NMR (400 MHz, CD3OD): δ 7.87 (s, 1H), 6.47 (s, 1H), 6.25
(s, 1H), 6.22 (s, 2H), 3.62 (s, 2H), 3.64 (s, 4H), 3.54 (m, 4H),
2.84 (d, J = 4.0 Hz, 2H), 2.28 (s, 3H), 2.14 (s, 3H); 13C NMR
(100 MHz, CD3OD): δ 171.6, 161.0, 158.9, 148.1, 144.6, 139.7,
136.4, 129.9, 119.4, 113.8, 113.5, 110.6, 103.2, 79.4, 71.5, 71.3,
70.6, 41.5, 40.5, 20.9, 20.5; HRMS (EI): calcd for C21H28O7N2
(M+): 420.1897, found: 420.1890.
1
410 spectrophotometer. H NMR (400 MHz) and 13C NMR
spectra (100 MHz) were recorded on JEOL JNM-AL-400
spectrometers, respectively. For H NMR spectra, chemical
Synthesis of TMG3 (5). A solution of biaryl ether 27 (2.85
mg, 4.46 μmol) in 1.25 M HCl/MeOH (1.0 mL) was stirred
1
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Article
under reflux for 18 h. The reaction mixture was concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography (MeOH/CHCl3 = 0/1 to 1/4) to give
TMG3 (5) (2.0 mg, 4.46 μmol, 99%) as an orange oil.
Preparation of TMG-Immobilized Beads. Commercially
available Affi-Gel 10 (20 μmol NHS/mL of gel; BIO-RAD)
beads were washed twice with Milli-Q (volume of Milli-Q to
volume of beads = 4:1) at room temperature. To the swelled
beads (200 μL) were added a solution of TMG compound (20
μmol) in coupling solution (1:1 dioxane/0.1 M aq. NaHCO3,
300 μL) and MeOH (2 × 120 μL) at room temperature. The
mixture was shaken at the same temperature for 2 h. The
resultant beads were washed with MeOH (5 × 900 μL). To the
beads was added a 1 M solution of ethanolamine in Tris-HCl
buffer (0.1 M Tris-HCl, pH 8.0, 1 mL), and the mixture was
shaken for an another 1 h. The beads were transferred into a
spin column using Milli-Q (4 × 400 μL). The beads were
washed successively with MeOH, DMSO, MeOH, and PBS
buffer (137 mM NaCl, 3 mM KCl, 9 mM NaHPO4, 1.5 mM
KH2PO4, pH 7.4, 3 × 400 μL each). The beads were suspended
in PBS buffer containing 1 mM sodium azide, and stored at 4
°C. The beads were washed with PBS prior to use.
Expression and Purification of His-Tagged Proteins. BL21
(DE3) pLysS E. coli cells expressing each of the His-tagged
proteins were grown in LB media (0.5% Bacto-Yeast Extract,
1% Bacto-Tryptone, 1% NaCl) containing 100 mg/L ampicillin
with shaking at 37 °C until the OD600 reached 0.2. The cells
were then incubated with 0.1 mM IPTG for 4 h and harvested
by centrifugation at 2000g at 4 °C for 5 min. After cell lysis by
homogenization with a syringe and sonication, the insoluble
material was removed by centrifugation at 22 000g at 4 °C for
30 min, and the supernatant was collected as a cell lysate. Each
His-tagged protein was purified on a Ni Sepharose 6 Fast Flow
column (GE Healthcare) according to the manufacturer’s
instructions.
1
TMG3 (5): IR (neat): 3420, 1655 cm−1; H NMR (400
MHz, CDCl3): δ 7.15 (s, 1H), 6.62 (s, 1H), 6.36 (s, 1H), 6.24
(s, 1H), 4.80 (s, 2H), 3.92 (s, 3H), 3.77−3.60 (m, 8H), 3.54
(br s, 2H), 2.84 (br s, 2H), 2.22 (s, 3H); 13C NMR (100 MHz,
CDCl3): δ 171.2, 165.4, 163.9, 147.7, 144.4, 141.7, 129.2, 113.8,
112.7, 107.5 103.9, 101.8, 71.3, 70.7, 70.4, 70.2, 70.1, 52.0, 29.7,
20.9; HRMS (EI): calcd for C22H29NO9 (M+): 451.1842,
found: 451.1844.
Synthesis of TMG4 (6). To a solution of carbamate 28 (15.6
mg, 31.0 μmol) in CH2Cl2 (1 mL) was added TFA (2.0 mL) at
0 °C. After being stirred at room temperature for 1 h, the
reaction mixture was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
(MeOH/CHCl3 = 1/20) to give TMG4 (6) (13.5 mg, 31.0
μmol, 100%) as a red solid.
TMG4 (6): Mp. 98−99 °C; IR (neat): 3366, 1718, 1654
cm−1; 1H NMR (400 MHz, CDCl3): δ 6.63 (d, J = 1.4 Hz, 1H),
6.36 (d, J = 1.4 Hz, 1H), 6.33 (d, J = 2.2 Hz, 1H), 6.31 (d, J =
2.2 Hz, 1H), 4.02 (t, J = 4.2 Hz, 2H), 3.93 (s, 3H), 3.69−3.62
(m, 6H), 3.54 (t, J = 4.8 Hz, 2H), 2.88 (t, J = 5.0 Hz, 2H), 2.49
(s, 3H), 2.23 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 172.0,
165.0, 162.6, 151.5, 147.1, 143.5, 136.3, 134.9, 114.6, 113.5,
111.7, 106.7, 102.0, 73.0, 73.0, 70.3, 69.8, 69.6, 51.9, 41.3, 24.3,
21.1; HRMS (EI): calcd for C22H29O8N (M+): 435.1893,
found: 435.1848.
Synthesis of TMG5 (7). To a solution of carbamate 29 (9.0
mg, 16.8 μmol) in CH2Cl2 (1 mL) was added TFA (1 mL) at 0
°C. After being stirred at room temperature for 1 h, the
reaction mixture was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
(MeOH/CHCl3 = 1/20) to give TMG5 (7) (7.3 mg, 16.8
μmol, 100%) as a red solid.
TMG5 (7): Mp. 39−40 °C; IR (neat): 3363, 1719, 1652
cm−1; 1H NMR (400 MHz, CDCl3): δ 6.55 (d, J = 1.2 Hz, 1H),
6.50 (d, J = 1.2 Hz, 1H), 6.41 (d, J = 2.2 Hz, 1H), 6.24 (d, J =
2.2 Hz, 1H), 4.15 (t, J = 4.4 Hz, 2H), 3.91 (s, 3H), 3.86 (t, J =
4.4 Hz, 2H), 3.67 (s, 4H), 2.81 (t, J = 5.0 Hz, 2H), 2.48 (s,
3H), 2.24 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 172.1,
165.1, 163.1, 148.3, 143.2, 142.2, 138.1, 127.9, 116.1, 112.1,
111.1, 106.1, 101.5, 72.4, 70.7, 69.7, 69.5, 68.6, 51.8, 40.8, 24.3,
21.0; HRMS (EI): calcd for C22H29O8N (M+): 435.1893,
found: 435.1877.
Synthesis of TMG6 (8). To a solution of phenol 35 (15.4 mg,
48.4 μmol) in MeOH (484 μL) were added amine (36) (21.5
mg, 145 μmol), AcOH (22 μL, 387 μmol), and NaBH3CN
(12.2 mg, 194 μmol) at room temperature. After being stirred
at room temperature for 39 h, the reaction mixture was
concentrated and purified by silica gel column chromatography
(MeOH/CHCl3 = 0/1 to 1/9 to 1/4) to give TMG6 (8) (19.7
mg, 43.7 μmol, 90%) as a brown solid.
In Vitro GLO1-Inhibition Assay. Kinetic measurements were
carried out by using a Microplate Reader (BioTek Synergy Mx)
to monitor the increase in absorbance at 240 nm originating
from the formation of S-D-lactoylglutathione (ε240 = 3.37
mM−1cm−1) at 30 °C. After the reaction buffer (0.1 M sodium
phosphate, 14.6 mM MgSO4, 1 mM glutathione, 8 mM
methylglyoxal, pH 7.0) was preincubated at 30 °C for 5 min,
the reaction was initiated by the addition of His-GLO1 (24 ng)
with or without compound.
Detection of His-SGTA, His-GLO1, and His-SCP2 by
Using TMG Beads. The TMG beads were incubated in
binding buffer (10 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2, 1
mM EDTA, pH 7.6) containing His-SGTA (68 μg), His-GLO1
(40 μg), His-SCP2 (30 μg), 0.1% Triton X-100, and a protease
inhibitor mixture (Roche) (total 700 μL) at 4 °C for 4.5 h on a
rotator. The reacted TMG beads were washed with binding
buffer containing 0.1% Triton X-100 (7 × 1 mL) followed by
binding buffer alone (3 × 1 mL). Twenty microliters of the
SDS sample buffer (125 mM Tris-HCl, 10% 2-mercaptoetha-
nol, 4% SDS, 20% glycerol, 0.004% bromophenol blue, pH 6.8)
was added to the washed beads and the bound proteins were
eluted by boiling at 110 °C for 5 min. The eluted proteins were
separated by SDS-PAGE and visualized by CBB staining.
Detection of Endogenous SGTA, GLO1, and SCP2
Using TMG Beads. RAW264.7 cells were harvested and
washed twice with PBS buffer (137 mM NaCl, 3 mM KCl, 9
mM NaHPO4, 1.5 mM KH2PO4, pH 7.4), then resuspended in
binding buffer containing 0.1% Tween 20 and protease
inhibitor mixture (Roche). The washed cells were homogenized
with a syringe and sonicated. Insoluble materials were removed
by centrifugation at 22 000g at 4 °C for 30 min, and the
supernatant was collected as cell lysate. After the cell lysate (4
TMG6 (8): Mp. 63−64 °C; IR (neat): 3023, 1651 cm−1; 1H
NMR (400 MHz, CD3OD): δ 6.70 (s, 1H), 6.50 (s, 1H), 6.36
(d, J = 2.4 Hz, 1H), 6.17 (d, J = 2.4 Hz, 1H), 3.91 (s, 3H), 3.66
(br s, 1H), 3.61 (br s, 1H), 3.56 (t, J = 5.0 Hz, 2H), 2.89 (t, J =
5.2 Hz, 2H), 2.79 (t, J = 5.0 Hz, 2H), 2.44 (s, 3H); 13C NMR
(100 MHz, CD3OD): δ 173.0, 165.2, 164.0, 149.6, 143.9, 143.5,
139.7, 130.3, 113.8, 113.7, 112.6, 108.7, 102.6, 72.3, 71.2, 70.5,
58.3, 53.7, 52.3, 41.7, 23.9, 18.4; HRMS (FAB): calcd for
C22H31O8N2 (M++H): 451.2080, found: 451.2087.
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mg total protein) was precleared by incubation with control
beads (20 μL) at 4 °C for 1 h, it was incubated with TMG
beads (20 μL) at 4 °C for 12.5 h. The reacted TMG beads were
washed with binding buffer containing 0.1% Triton X-100 (7 ×
1 mL) followed by binding buffer alone (3 × 1 mL). The
bound proteins were eluted with SDS sample buffer, separated
by SDS-PAGE, and transferred to PVDF membrane. After the
membrane was treated with monoclonal primary antibodies
against SGTA (sc-100875, Lot# K1710; Santa Cruz Biotech
Inc.) followed by a HRP-conjugated antimouse IgG polyclonal
secondary antibody (Santa Cruz Biotech Inc.), or polyclonal
primary antibodies against GLO1 (sc-67351, Lot# G0208;
Santa Cruz Biotech Inc.) followed by a HRP-conjugated
antirabbit IgG polyclonal secondary antibody (Zymed) or
polyclonal primary antibodies against SCP2 (sc-32835, Lot#
D1510; Santa Cruz Biotech Inc.) followed by a HRP-
conjugated antigoat IgG polyclonal secondary antibody
(Santa Cruz Biotech Inc.), the immune complexes were
detected with SuperSignal West Dura Extended Duration
substrate (Thermo Scientific) according to the manufacturer’s
instructions.
tion at the C5′-methyl group adjacent to the electron-rich A
ring. In contrast, we considered that the selective halogenation
of the C6-methyl group in the presence of the C5′-methyl
group would not be straightforward; we therefore planned to
derivatize the C6-methyl group before the coupling of the A
and B rings.
Synthesis of TEGylated Methyl Gerfelin Derivatives.
Synthesis of TMG1 (3) began with gerfelin diacetonide (9), a
synthetic intermediate of Watanabe’s first total synthesis17 of
methyl gerfelin (2) (Scheme 1A). After trans-esterification of 9,
the resulting phenol 10 was coupled with a known bromide
11,18 and global deprotection using TFA provided TMG1 (3)
in 97% yield in three steps.
Scheme 1. Synthesis of TMG1 (3) and TMG2 (4)
Osteoclastogenesis Suppression Assay. The osteoclas-
togenesis suppression assay was performed as described in the
literature.5 Briefly, mouse bone marrow-derived macrophages
(BMM) were treated with various concentrations of TMG
compound in the presence of the receptor activator of the NF-
kB ligand (RANKL) and macrophage colony stimulating factor
(M-CSF) for 72 h. Cells were fixed and stained for tartrate-
resistant acid phosphatase (TRAP), and TRAP-positive multi-
nucleated cells were counted.
RESULTS AND DISCUSSION
■
Design and Synthesis of TEGylated Methyl Gerfelin
Derivatives. We designed six TEGylated methyl gerfelin
derivatives (TMG1−6: 3−8) with an NH2-TEG linker on the
peripheral groups of the methyl gerfelin molecule: i.e., on the
C1-carboxyl group, C6- and C5′-methyl groups, and C2-, C2′-,
C3′-hydroxyl groups (Figure 2).16 The introduction of an NH2-
TEG unit at each of the above-mentioned positions except for
C6-methyl was thought to be possible via an appropriately
protected methyl gerfelin. In particular, we expected that
benzylic radical halogenation would be suitable for derivatiza-
Reagents and conditions: (a) MeONa, MeOH, rt, 10 h, 97%; (b) 11,
K2CO3, MeCN, reflux, 13.5 h, 100%; (c) TFA-H2O (5:1), rt, 1 h,
100%; (d) KOH, THF-H2O (1:1), reflux, 6 h, 100%; (e) 12, EDCI,
DMAP, CH2Cl2, rt, 24 h, 55% from 14; (f) KOH, THF-H2O (1:1), 50
°C, 1 h, 95%; (g) TFA-H2O (3:1), rt, 1 h, 99%. TFA = trifluoroacetic
acid, THF
= tetrahydrofuran, EDCI = 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride, DMAP = 4-
(dimethylamino)pyridine.
For the synthesis of TMG2 (4), gerfelin diacetonide (9) was
first converted to carboxylic acid 12 via alkali hydrolysis in
quantitative yield (Scheme 1B). Acid hydrolysis of 9 (TFA,
H2O, 95 °C) quantitatively led to the decarboxylated product
13 instead. Treatment of carboxylic acid 12 with amine 14 in
the presence of EDCI and DMAP afforded the pseudodimeric
amide 15 in 55% yield from 14, which was then hydrolyzed
Figure 2. Design of TEGylated methyl gerfelin derivatives.
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with potassium hydroxide to give amide 16 in 95% yield. Direct
coupling of amine 14 with gerfelin acetonide (9) was not found
to be efficient. Deprotection of acid-labile protecting groups
with TFA provided TMG2 (4) in 99% yield.
Scheme 3. Synthesis of TMG4 and TMG5
TMG3 (5) was synthesized as follows (Scheme 2). TBS
ether 18, which was prepared from the known phenol 17,17 was
Scheme 2. Synthesis of TMG3 (5)
Reagents and conditions: (a) 11, NaI, K2CO3, MeCN, reflux, 9 h, 41%
for 28, 10% for 29; (b) TFA-CH2Cl2 (2:1), rt, 1 h, quant.
HMBC experiments. The higher reactivity of C2′−OH over
C3′−OH was also observed when gerfelin (1) was treated with
diazomethane: methyl gerfelin C2′-O-methyl ether (30) and
methyl C3′-O-methyl ether (31) were obtained in 38% and
14% yields, respectively, along with methyl gerfelin (2, 44%).
Deprotection using TFA provided TMG4 (6) and TMG5 (7)
in quantitative yields.
TMG6 (8) was synthesized as follows (Scheme 4). Starting
from methyl gerfelin (2), all the phenolic hydroxyl groups were
Scheme 4. Synthesis of TMG6
Reagents and conditions: (a) TBSCl, imidazole, CH2Cl2, rt, 2.5 h,
89%; (b) NBS, BPO, CCl4, reflux, 5 h; (c) 21, NaH, DMF, 0 °C to rt,
2.5 h; (d) MeONa, MeOH, 50 °C, 16 h, 20% for 3 steps; (e) PhNTf2,
K2CO3, acetone, rt, 8 h, 65%; (f) MOMCl, iPr2NEt, CH2Cl2, rt, 5 h,
99%; (g) 25, 26, Pd(OAc)2, K3PO4, toluene, 95 °C, 18 h, 39%; (h)
HCl, MeOH, reflux, 18 h, 99%. TBS = t-butyldimethylsilyl, NBS = N-
bromosuccinimide, BPO = benzoyl peroxide, Tf = trifluoromethane-
sulfonyl, MOM = metoxymethyl.
subjected to a radical bromination condition to give benzyl
bromide 19. Without protection of the phenolic hydroxyl
group, we could not obtain the desired bromide 19, and
dibromobenzene derivative 20 was obtained instead. Sub-
sequent coupling of 19 with alcohol 2118 followed by
methanolysis of the resulting ester 22 produced 23 in 20%
yield in 3 steps. After conversion of 23 to MOM-protected
triflate 24, 24 was coupled with the known phenol 2517 by
using palladium acetate and phosphine ligand 26 to afford
diphenyl ether 27. Finally, the removal of acid-labile protecting
groups with methanolic HCl provided TMG3 (5) in 99% yield.
TMG4 (6) and TMG5 (7) were synthesized from methyl
gerfelin (2) (Scheme 3). Coupling of 2 with the aforemen-
tioned bromide 11 provided ethers 28 and 29 in moderate but
reproducible yield. The structure of 28 was determined by
Reagents and conditions: (a) TBSCl, imidazole, DMAP, CH2Cl2, rt,
6.5 h, 99%; (b) NBS, BPO, CCl4, reflux, 20 h; (c) CaCO3, H2O-
dioxane (1:1), reflux, 12.5 h, 52% in 2 steps; (d) MnO2, CH2Cl2, rt, 24
h, 91%; (e) TBAF, THF, rt, 0.5 h, 94%; (f) 36, NaBH3CN, AcOH,
MeOH, rt, 39 h, 90%. TBAF = tetra-n-butylammonium fluoride.
protected as its TBS ether to give triTBS ether 32 in almost
quantitative yield. Radical bromination at the C5′ methyl group
in 32 proceeded as expected to afford unstable benzyl bromide
33, which was converted to benzyl alcohol 34 in 52% yield in 2
steps. Because the coupling of crude 33 and alcohol 21, or that
of alcohol 34 and bromide 11 did not work well, we decided to
utilize a reductive coupling strategy: that is, alcohol 34 was
oxidized with MnO2, and the resulting aldehyde was treated
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with TBAF to give aldehyde 35. Reductive coupling of 35 and
diamine 36 using NaBH3CN and AcOH proceeded smoothly
to give the desired TMG6 (8) in 90% yield.
Synthesis of the Gerfelin A Ring Derivative. In addition
to TMG1−6, a methyl gerfelin A ring derivative was designed
and synthesized to verify the importance of the B ring for the
GLO1-inhibiting activity. To this end, the protected phenol 25
was coupled with bromide 11 and the following global
deprotection gave the A ring derivative 37 in 88% yield in 2
steps (Scheme 5).
Scheme 5. Synthesis of the Gerfelin A Ring Derivative
Reagents and conditions: (a) 11, NaI, K2CO3, MeCN, reflux, 24 h,
95%; (b) TFA-H2O (4:1), rt, 2 h, 93%.
In Vitro GLO1 Inhibition of TMG Compounds and
Other Derivatives. With the desired TMG compounds and
their synthetic intermediates in hand, the in vitro GLO1-
inhibitory activities of these compounds were determined
according to the protocol reported by Oray et al.19 The
structures of the compounds and their IC50 values are
summarized in Figure 3.
Among the TMG compounds, TMG1, TMG2, and TMG3
were found to retain GLO1-inhibitory activity. TMG3 showed
inhibitory activity (0.78 0.17 μM) comparable to those of
methyl gerfelin (2) and gerfelin (1). This result indicates that
the functionalization at the C6-methyl group had almost no
effect on the interaction with, and thus the inhibition of, GLO1.
As seen in TMG1 and TMG2, introduction of an NH2-TEG
linker at the C1 carbonyl and C2 hydroxyl groups had some
effect, increasing the IC50 value up to 2-fold that of the parent
molecule. In contrast, TMG4 and TMG5, each of which had a
linker at the C2′ or C3′ hydroxyl groups, respectively, almost
lost their GLO1-inhibitory activity. Introduction of a methyl
group or acetonide at the same position also had the same
effect (see compounds 9, 30, and 31). TMG6 having a linker at
the C5′ methyl also lost its GLO1-inhibitory activity. Indeed,
modification of the C5′ methyl group tends to lower the
biological activity. The observed rank order of C5′-methyl
modification for GLO1-inhibitory activity was methyl (parent
compound 2) > aldehyde (35) > hydroxyl (38) > TEG-NH2
linker (8).
Overall, these results are mostly consistent with the X-ray
structure of GLO1 complexed with methyl gerfelin: the
methoxycarbonyl group in the B ring of 2 points outward
from the substrate-binding pocket of GLO1, whereas the C5′-
methyl group is placed in the deep pocket of the substrate-
binding site with the C2′- and C3′-hydroxyl groups bound to
the Zn catalytic center of GLO1. However, it should be
emphasized that the GLO1-inhibitory activity of decarboxylated
compound 13, as well as that of the A ring derivatives 37 and
39, was quite low. This means that, although no interaction
between the B ring unit and GLO1 was observed in the
cocrystal structure,5 the presence of the B ring unit is quite
important for the biological activity of these derivatives. Also,
the loss of biological activity in the C2′- and C3′-O-TEGylated
and methylated derivatives (TMG4, TMG5, 30 and 31)
Figure 3. In vitro GLO1-inhibition activity of TMG compounds and
other derivatives. IC50 values are reported in μM. The compounds are
placed in a left-to-right direction in order of decreasing GLO1-
inhibition activity.
indicated that both phenolic hydroxyl groups have to be in an
unprotected form to exert GLO1 inhibition.
Preparation of TMG Beads. Next, to estimate the
selectivity of TMG compounds toward the three binding
proteins, the compounds were introduced on affinity matrices
and subjected to competitive pull-down experiments. The
TMG compound-introduced affinity beads (TMG beads) were
prepared as follows: Swelled NHS-activated Affi-Gel 10 beads
were treated with each TMG compound in 0.1 M aq.
NaHCO3-dioxane (1:1), and the resultant beads were blocked
with ethanolamine and washed successively to afford TMG1−6
beads (Figure 4A). The amount of TMG molecules introduced
on the affinity beads was estimated to be 3.69 0.11 nmol/
μL.20 Control affinity beads were also prepared in the same
manner.
Protein-Binding Property of Each TMG. Each TMG
bead was independently combined with a mixed solution of
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assumed from this result that SGTA loosely recognizes and
binds to aromatic ring(s) of methyl gerfelin (2). On the other
hand, the (His)6-GLO1 protein was found to bind selectively to
TMG1-, TMG2-, TMG3-, and TMG6 beads. Among these
beads, the TMG3 beads exhibited the best binding activity
toward the (His)6-GLO1 protein. The binding profile of TMG
beads to the (His)6-SCP2 protein was quite similar to that to
the (His)6-GLO1 protein, suggesting that GLO1 and SCP2
recognize and bind to almost the same region of the surface of
methyl gerfelin (2). However, in contrast to (His)6-GLO1, the
amount of (His)6-SCP2 protein bound to TMG3 beads did not
increase. These results indicate that TMG3 had better
selectivity toward GLO1 than did SGTA and SCP2. We
performed the binding experiments with each of the three
purified proteins, and almost the same results were obtained
(data not shown).
Pull-down experiments utilizing cell lysate of RAW264.7 cells
instead of purified proteins were also carried out (Figure 3C).
Each TMG bead was independently incubated with the cell
lysate of RAW264.7 cells, and the bound proteins were resolved
with SDS-PAGE and detected by Western blot analysis. Again,
TMG3 beads precipitated endogenous GLO1 more efficiently
than other TMG beads. Only low levels of binding with
endogenous SGTA and SCP2 were detected in these cases,
suggesting the superior selectivity of TMG3. From these
experiments, we concluded that TMG3 is a selective binder and
inhibitor of GLO1.
Osteoclastogenesis Suppression of TMG Compounds.
The levels of in vitro osteoclastogenesis inhibition activity of
TMG1, TMG3, and TMG4 were determined by cellular
phenotype-based screening using mouse bone marrow-derived
macrophages (BMMs). Briefly, BMMs were treated with test
compounds for three days in the presence of the receptor
activator of the NF-kB ligand (RANKL) and macrophage
colony stimulating factor (M-CSF), and differentiated cells
were stained by tartrate-resistant acid phosphatase (TRAP) and
counted. As a result, TMG1 and TMG3 were found to suppress
osteoclastogenesis with IC50 values of 16 and 12 μM,
respectively, whereas TMG4 exhibited no inhibition at 10
μM, but showed cellular toxicity at 30 μM concentration
(Figure 5).
Figure 4. Protein-binding property of each TMG bead. (A) Structure
of TMG compounds and TMG beads. (B,C) Pull-down assay using
TMG beads. Control beads or each type of TMG bead was
independently combined with a mixed solution of His-SGTA, His-
GLO1, and His-SCP2 (B), or with RAW264.7 cell lysate (C). The
separated bead-bound proteins were visualized by CBB staining (B) or
Western blotting (C). C: control.
three purified (His)6-tagged recombinant proteins, and the
bead-bound proteins were separated using SDS-PAGE and
visualized using CBB staining (Figure 4B).
All three proteins were found to bind to the TMG beads but
not to the control beads, indicating that the proteins bind to the
methyl gerfelin portion. The (His)6-SGTA protein was found
to bind to all TMG compounds to a similar extent. It was
Figure 5. Osteoclastogenesis suppression of methyl gerfelin and TMG
compounds.
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and the results of the osteoclastogenesis suppression assay. This
material is available free of charge via the Internet at http://
pubs.acs.org.
CONCLUSION
■
We have prepared six TMG compounds and tested them for
GLO1-inhibition activity and GLO1-binding selectivity. From
the GLO1-inhibition assay, it was found that each of TMG 1−3
retained the inhibitory activity, with the activity of TMG3 being
the strongest. Additional SAR experiments using synthetic
intermediates confirmed the importance of the B-ring and C1-
carbonyl group for GLO1 inhibition. We could conclude from
these experiments that derivatization at the C6-methyl group is
most suitable for use in subsequent experiments, including
fluorescent labeling studies.
AUTHOR INFORMATION
Corresponding Author
*E-mail: nkanoh@m.tohoku.ac.jp. Phone & Fax: +81-22-795-
6847.
■
Notes
The authors declare no competing financial interest.
On the other hand, information about the binding mode of
methyl gerfelin (2) toward SCP2 and SGTA proteins and
information about the GLO1 selective-binding property of
TMG compounds were obtained from pull-down experiments
using TMG beads. That is, (1) SGTA recognizes aromatic
ring(s) of methyl gerfelin (2), and (2) GLO1 and SCP2 share
almost the same but not the identical area of methyl gerfelin
(2).
TMG3 (5) is found to selectively bind to and inhibit GLO1.
From the osteoclastogenesis inhibition assay, TMG3 (5)
inhibited osteoclastogenesis comparable with, but a little
stronger than, TMG1 (3). This indicated that binding of
methyl gerfelin (2) to SCP2 and SGTA does not show any
synergistic effects. However, a sequestration effect by either
SCP2 or SGTA is sufficient to explain the results of the cell-
based assay.
The TEG-scanning strategy demonstrated here is based on
two different methodologies, i.e., methyl scanning and
PEGylation. Methyl scanning is a method developed by Pirrung
et al.,21 in which methylated drug derivatives are systematically
synthesized and used to determine the sites of interaction
between drugs and their macromolecular target(s). Although
the methyl scanning strategy has been shown to work well in
the design of biotinylated affinity probe for a small-molecule
insulin mimic demethylasterriquinone B1,21,22 the probe had to
be resynthesized from the beginning, so the introduction of a
terminal-functionalized linker is thought to be more valuable.
PEGylation23 is originally defined as the modification of a
bioactive molecule by the linking of poly(ethylene glycol) for a
prolonged resistance in body and a decreased degradation by
metabolic enzymes, but here we utilize oligo(ethylene glycol) as
a hydrophilic linker to connect a drug and a polymer support to
be used for the binding assay. Although the length and
molecular weight of PEG are important for the original
PEGylation strategy due to the toxicity of oligo(ethylene
glycol), our cell-based assay showed that not all TMG
compounds exerted cytotoxicity at the effective concentration.
This indicates that a NH2-TEG linker can be used for our
purposes without any significant problem.
ACKNOWLEDGMENTS
■
We thank Prof. Yoshiteru Oshima (Tohoku University) for his
support, and Dr. Hiroshi Takayama (Tohoku University) for
his technical assistance. This work was partially supported by a
Grant-in-Aid for Scientific Research on the Innovative Area
“Chemical Biology of Natural Products” from The Ministry of
Education, Culture, Sports, Science and Technology, Japan
(No. 23102013 to N.K.).
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