Synthesis and biological evaluation of molecular probes based on the 9-methylstreptimidone derivative DTCM-glutarimide

Article abstract of DOI:10.1016/j.bmcl.2011.11.045

Molecular probes based on 3-[(dodecylthiocarbonyl)methyl]glutarimide (DTCM-glutarimide) were synthesized and assessed for inhibitory activity against LPS-induced NO production. Among the probes examined, several derivatives exhibited potential for use in determining the target proteins of DTCM-glutarimide.

Full text of DOI:10.1016/j.bmcl.2011.11.045

Bioorganic & Medicinal Chemistry Letters 22 (2012) 164–167
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of molecular probes based
on the 9-methylstreptimidone derivative DTCM-glutarimide
Eisuke Ota a, Masatoshi Takeiri b, Miyuki Tachibana b, Yuichi Ishikawa a, Kazuo Umezawa b,
Shigeru Nishiyama a,
⇑
a Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522, Japan
b Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Molecular probes based on 3-[(dodecylthiocarbonyl)methyl]glutarimide (DTCM-glutarimide) were syn-
thesized and assessed for inhibitory activity against LPS-induced NO production. Among the probes
examined, several derivatives exhibited potential for use in determining the target proteins of DTCM-
glutarimide.
Received 3 August 2011
Revised 9 November 2011
Accepted 11 November 2011
Available online 18 November 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Molecular probe
NF-jB
9-Methylstreptimidone
Streptomyces sp.
NF-
j
B is a transcription factor involved in the regulation of im-
showed antiinflammatory activity in vivo.7 Comparison with 1 sug-
gested that 2 may have a different mode of action involving other
cascades rather than inhibition of NF-jB signaling. Analysis of the
mune and inflammatory responses by induction of interleukins
and iNOS,1–3 and is constitutively active in cancer and inflamma-
tory cells (i.e., macrophages). Activated NF-
ity and metastatic activity of cancer cells along with their
malignant character. Therefore, NF- B is considered an effective
molecular target for antiinflammatory or anticancer agents.
9-Methylstreptimidone (1), a glutarimide antibiotic, was first
isolated in 19744 and was reported to show antiinflammatory
activity.5 The natural product 1 inhibited NO production and iNOS
expression in LPS-stimulated RAW264.7 cells, and induced apopto-
sis in Jurkat cells and adult T-cell leukemia cells, similar to other
j
B can increase viabil-
differences in inhibitory mechanisms generated by small structural
differences between 1 and 2 will yield interesting information from
the viewpoint of drug discovery. Therefore, we examined the target
protein of 2 with the biotin-avidin method or photolabeling meth-
od. Biotin-tagged molecules are widely utilized in bioanalytical
applications to detect biomolecules due to the strong binding
interaction with avidin. Many techniques for biotin-avidin assays
have uncovered relationships between target molecules and re-
agents.8,9 On the other hand, photoaffinity labeling reveals the
structure and function of protein by utilizing aryl azide as a photo-
reactive agent,10 photolysis of which provided nitrenes that form
covalent bonds with target molecules, and capture even reversible
binding interaction in the nonpolar moiety. Thus, biologically ac-
tive molecular probes of 2, possessing biotin and photoaffinity la-
bel, were examined in this study.
j
NF-jB inhibitors. Low fermentation yield of 1 interfered with de-
tailed investigation of the inhibitory mechanism.
Here, we designed a new candidate inhibitor of NF-
j
B based on
1 (Fig. 1). Based on the design involving retention of glutarimide
and replacement of the hydrophobic moiety with linear alkyl
chains, simplified analogs of 1 were synthesized. Assessment of
LPS-induced NO production, indicated that 3-[(dodecylthiocarbon-
yl)methyl]glutarimide (DTCM-glutarimide; 2) was the most plau-
sible candidate for the inhibitor.6 In contrast to the remarkable
inhibitory activity of 2 against LPS-induced expression of iNOS in
RAW264.3 cells with low cytotoxicity similar to 1, neither direct
Molecular probes possessing a biotin or photoreactive group
were designed and synthesized as shown in Figure 2.
Initially, two biotinylated probes, 3 and 4, carrying different car-
bon chain lengths, were prepared to investigate the influence of
the alkyl chain on inhibitory activity. In our previous study, deriv-
atives possessing an amide instead of a thioester exhibited no
inhibitory activity.6 Therefore, we synthesized 5 as a negative con-
trol against 3 and 4. Furthermore, photoaffinity probe 6 was
synthesized to examine a different approach for determination of
target protein.
inhibition of LPS-induced nuclear translocation of NF-
j
B nor bind-
ing between NF-
j
B and DNA were observed.7 Compound 2 also
⇑
Corresponding author. Tel./fax: +81 45 566 1717.
E-mail address: nisiyama@chem.keio.ac.jp (S. Nishiyama).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.045

E. Ota et al. / Bioorg. Med. Chem. Lett. 22 (2012) 164–167
165
glutarimide
alkyl chain
O
O
O
OH
NH
O
O
NH
S
O
1
2
Figure 1. Structure of 9-methylstreptimidone (1) and DTCM-glutarimide (2).
O
Table 1
Inhibitory activity (IC50) for LPS-induced NO production and cytotoxicity (ED50) of 3–6
H
S
H
O
NH
H
N
HN
Compound
IC50
(lg/mL)
ED50 (lg/mL)
S
O
NH
3
4
5
6
>30
5
>30
4.5
>30
>30
>30
>30
O
O
3
O
O
H
S
H
O
O
NH
H
N
HN
S
O
Although synthesis of 4 may proceed in a similar way as in the
case of 3, the coupling reaction with 13 did not proceed smoothly.
Thus, thiol 9 was synthesized from an alternative starting material.
Thiol 9, prepared by the known procedure from commercially
available 12-aminododecanoic acid in six steps,15,16 was success-
fully transformed into thioester 11. Cleavage of a Boc group, fol-
lowed by coupling with biotin under the same conditions as that
of 3 afforded the probe 4.20
NH
O
O
O
4
5
6
H
NH
S
H
H
N
HN
N
H
O
NH
N3
O
Treatment of the intermediate 11 with N-succinimidyl 4-azi-
dobenzoate 14,17 instead of biotin succinimide ester, effected
production of 6,20 although 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide yielded no desired product.18
O
O
NH
H
N
S
O
O
Subsequently, amide 5 was synthesized from the mono-Boc
dodecanediamine (10).19 Coupling of amine 10 with 13, provided
the corresponding amide 12, which on deprotection followed by
condensation with the biotin succinimide derivative yielded 520
as a negative control.
To understand the validity of the synthetic probes, the synthe-
sized derivatives 3–6 were examined by LPS-induced NO produc-
tion in RAW264.7 cells, using DTCM-glutarimide as a positive
control (Table 1).21 Probes 3 and 5 have no inhibitory effect of
Figure 2. Synthetic probes possessing biotin (3, 4, and 5) or aryl azide (6).
Probes of 2 were synthesized as outlined in Scheme 1. Protected
cystamine 711 was treated with biotin succinimide ester12 and tri-
ethylamine in DMF to afford the biotinylated derivative 8. After
cleavage of a trityl group, condensation of acid 13, readily available
from diethyl 1,3-acetonedicarboxylate,13,14 afforded 3.20
O
H
HN
1) TFA,iPr3SiH, CH2Cl2
H
S
H
biotin-OSn, Et3N
H
N
S
H
O
NH
H
N
H2N
STr
HN
STr
S
X
O
13
DMF
2) , EDCI, DMF
NH
O
NH
NH
O
O
O
(79%)
(8.6% in 2 steps)
7
8
3
O
O
H
S
O
NH
O
NH
13
, EDCI
1) TFA, CH2Cl2
H
N
HN
BocHN
BocHN
O
X
O
11
X
11
2) biotin-OSn, Et3N, DMF
H
CH3CN
11
O
O
9
X = SH
(44%)
(73%)
11 X = S
12 X = NH
(60% in 2 steps)
(34% in 2 steps)
4
5
X = S
X = NH
10 X = NH2
O
N3
O
NH
1) TFA, CH2Cl2
O
N3
H
N
O
14
S
O
2) , Et3N, DMF
O
NH
O
N
11
O
HO
O
O
(17% in 2 steps)
O
6
13
14
Scheme 1. Synthesis of compounds 3, 4, 5, and 6.

166
E. Ota et al. / Bioorg. Med. Chem. Lett. 22 (2012) 164–167
Figure 3. Inhibition of iNOS expression by synthetic probes 4 and 6. Cells were incubated with or without indicated chemicals for 2 h, thereafter stimulated 1 lg/ml LPS for
24 h. Total cell lysates were subjected by Western blotting.
NO production. As expected, the length of the alkyl chain and the
thioester moiety were crucial factors for the inhibitory effect. In
contrast, the biotinylated probe 4 and the photoaffinity probe 6
inhibited NO production with low toxicity (ED50 > 30); their IC50
showed a different mode of action from that of 1. Biotinylated
probe 4 and photoaffinity probe 6 were potent inhibitors of LPS-in-
duced NO production, and will be available to search for the target
molecule of DTCM-glutarimide. Further investigations are cur-
rently underway in our laboratory.
values were 5 and 4.5 lg/mL, respectively, comparable to that of
2. On the basis of their activities against NO production, the probes
4 and 6 were evaluated by LPS-induced iNOS expression in RAW
264.7 cells. In Figure 3, the probes inhibited LPS-induced iNOS
expression. These results indicated that alterations of NO produc-
tion were caused by that of iNOS expression, and the effective
probes 4 and 6 were not NO scavengers or influential factors to
absorbance spectrum, but genuine inhibitors against signaling
pathways induced by LPS stimulation. In addition, though quite a
few biotinylated probes and photoaffinity probes extremely de-
crease their inhibitory activity within cells compared to original
inhibitors, our synthetic probes 4 and 6 exhibited respectable po-
tential against LPS-induced iNOS expression. Subsequently, 4 and
6 were submitted to electrophoretic mobility shift assay (EMSA)
to confirm the same action as that of 2. As expected (Fig. 4), 4
Acknowledgements
This work was financially supported in part by a High-Tech Re-
search Center Project for Private Universities: matching fund sub-
sidy from MEXT, 2006–2011, and by the Science Research
Promotion Fund from the Promotion and Mutual Aid Corporation
for Private School of Japan from MEXT.
References and notes
1. Hayden, M. S.; Ghosh, S. Genes Dev. 2004, 18, 2195.
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6. Ishikawa, Y.; Tachibana, M.; Matsui, C.; Obata, R.; Umezawa, K.; Nishiyama, S.
Bioorg. Med. Chem. Lett. 2009, 19, 1726.
and 6 did not inhibit NF-j
B induced by LPS as in the case of 2.7
From these results, synthetic probes will be utilized as powerful
tools for activity-based protein profiling in future studies.
In conclusion, we have designed and synthesized biologically
active probes to elucidate the inhibitory mechanism of 2, which
7. Takeiri, M.; Tachibana, M.; Kaneda, A.; Ito, A.; Ishikawa, Y.; Nishiyama, S.; Goto,
R.; Yamashita, K.; Shibasaki, S.; Hirokata, G.; Ozaki, M.; Todo, S.; Umezawa, K.
Inflammation Res. 2011, 60, 879.
8. Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1.
9. Sin, N.; Meng, L.; Wang, M. Q. W.; Wen, J. J.; Bornmann, W. G.; Crews, C. M. Proc.
Natl. Acad. Sci. U.S.A. 1997, 94, 6099.
10. Hua, S.; Inesi, G. Biochemistry 1997, 36, 11865.
11. Ruggles, E. L.; Deker, P. B.; Hondal, R. J. Tetrahedron 2009, 65, 1257.
12. Kottani, R.; Valiulin, R. A.; Kutateladze, A. G. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 13917.
13. Egawa, Y.; Suzuki, M.; Okuda, T. Chem. Pharm. Bull. 1963, 11, 589.
14. Gupta, K. A.; Saxena, A. K.; Jain, P. C.; Anand, N. Indian J. Chem., Sect. B 1987, 26,
341.
15. Usuki, Y.; Matsumoto, K.; Inoue, T.; Yoshioka, K.; Iioa, H.; Tanaka, T. Bioorg.
Med. Chem. Lett. 2006, 16, 1553.
16. Suzuki, T.; Hisakawa, S.; Itoh, Y.; Suzuki, N.; Takahashi, K.; Kawahata, M.;
Yamaguchi, K.; Nakagawa, H.; Miyata, N. Bioorg. Med. Chem. Lett. 2007, 17,
4208.
17. Kim, K. I.; Lee, J. W.; Ito, Y.; Kang, J. H.; Song, K. S.; Jang, E. C.; Son, T. I. J. Appl.
Polym. Sci. 2010, 117, 3029.
18. In order to prevent photolysis, the benzoate 6 was purified in a darkroom.
19. Morrell, A.; Placzek, M. S.; Steffen, J. D.; Antony, S.; Agama, K.; Pommier, Y.;
Cushman, M. J. Med. Chem. 2007, 50, 2040.
20. Spectroscopic data for 3: IR (KBr)
m 3282, 3080, 2926, 2858, 1697, 1645,
1265 cmÀ1 1H NMR (400 MHz, CD3OD) d 1.44 (2H, m), 1.62 (4H, m), 2.19 (2H,
;
t, J = 7.3 Hz), 2.38 (2H, m), 2.70 (5H, m), 2.71 (1H, d, J = 12.7 Hz), 2.92 (1H, dd,
J = 4.9,12.7 Hz), 3.06 (2H, br t, J = 6.3 Hz), 3.21 (1H, ddd, J = 4.4, 5.6, 8.8 Hz), 3.36
(1H, t, J = 6.3 Hz), 3.37 (1H, t, J = 6.8 Hz), 4.31 (1H, dd, J = 4.4, 7.8 Hz), 4.48 (1H,
ddd, J = 1.0, 4.9, 7.8 Hz); 13C NMR (100 MHz, CD3OD) d 26.8, 29.1, 29.51, 29.55,
29.8, 36.8, 37.8, 39.8, 41.1, 57.0, 61.6, 63.4, 166.1, 174.7, 176.2, 198.3. HRMS
(FAB) calcd for
Spectroscopic data for 4: IR (KBr)
1265 cmÀ1 1H NMR (400 MHz, CDCl3) d 1.22–1.78 (26H, m), 2.19 (1H, t,
C
19H29N4O5S2 [M+H]+: 457.1579. Found: m/z 457.1571.
m
3308, 2922, 2851, 1686, 1632,
;
J = 6.9 Hz), 2.20 (1H, t, J = 7.4 Hz), 2.36 (2H,m), 2.63 (2H, br d, J = 6.5 Hz), 2.74
(1H, d, J = 12.5 Hz), 2.75 (2H, m, overlapped with 1H signal), 2.90 (2H, br t,
J = 7.4 Hz), 2.93 (1H, dd, J = 5.2, 12.5 Hz), 3.17 (1H, ddd, J = 4.7, 7.0, 7.8 Hz), 3.23
(1H, t, J = 6.5 Hz), 3.24 (1H, t, J = 7.0 Hz), 4.33 (1H, m), 4.52 (1H, m), 4.98 (1H, s),
5.59 (1H, s), 5.63 (1H, s), 8.64 (1H, s); 13C NMR (100 MHz, DMSO-d6) d 25.4,
Figure 4. Effects of probes 4 and 6 on NF-
were treated with or without indicated chemicals for 2 h, and thereafter stimulated
g/ml LPS for 30 min. The nuclear proteins were then extracted and used for
EMSA.
jB activation in RAW264.7 cells. Cells
1
l

E. Ota et al. / Bioorg. Med. Chem. Lett. 22 (2012) 164–167
167
26.4, 27.3, 28.1, 28.2, 28.5, 28.8, 28.9, 29.00, 29.02, 29.1, 29.2, 35.2, 36.6, 38.4,
47.6, 55.5, 59.2, 61.1, 162.7, 171.8, 172.5, 197.1. HRMS (ESI) calcd for
29H49N4O5S2 [M+H]+: 597.3144. Found: m/z 597.3146. Spectroscopic data
for 5: IR (KBr)
3302, 3076, 2923, 2850, 1698, 1640, 1543, 1267 cmÀ1 1H NMR
NO production assay
Cells in complete medium (1 Â 105 cells/ml) were seeded in a 96-well plate
C
(Corning Inc., Corning, NY, USA), with each well receiving 100 ll of the cell
suspension. On the next day, the cells were treated with each chemical for 2 h
and then stimulated with 1 g/ml LPS for 20 h. Then 100 l Griess reagent
m
;
(400 MHz, DMSO-d6) d 1.24–1.61 (26H, m), 2,04 (2H, t, J = 7.4 Hz), 2.10 (2H, d,
J = 6.7 Hz), 2.27 (2H, m), 2.44 (2H, m, overlapped with 1H signal), 2.58 (1H, d,
J = 12.5 Hz), 2.81 (1H, dd, J = 5.0, 12.5 Hz), 3.01 (4H, br t, J = 6.7 Hz), 3.09 (1H,
ddd, J = 4.5, 6.1, 8.5 Hz), 4.12 (1H, m), 4.30 (1H, m), 6.35 (1H, s), 6.42 (1H, s),
7.72 (1H, t, J = 5.4 Hz), 7.87 (1H, t, J = 5.4 Hz), 10.70 (1H, s); 13C NMR (100 MHz,
DMSO-d6) d 25.4, 26.42, 26.44, 27.2, 28.77, 28.80, 29.05, 29.10, 29.2, 35.3, 37.0,
38.4, 39.1, 55.5, 59.2, 61.1, 162.7, 169.7, 171.8, 172.9. HRMS (ESI) calcd for
l
l
solution was added to each well. The concentration of NO was obtained by
measuring the absorbance at 570 nm with a microplate reader.
MTT assay
Cells in complete medium (1 Â 105 cells/ml) were seeded in a 96-well plate,
with each well receiving 100 ll of the cell suspension. On the next day, the
cells were treated with each chemical for 2 h and then stimulated with 1
LPS for 20 h. Then, 10 of MTT (3-[4,5-dimethylthiazol-2–yl]-2,5-
diphenyltetrazoliumbromide) solution was added to each well, and the cells
were incubated for 4 h at 37 °C under 5% CO2 plus air. Subsequently, the
lg/ml
C
29H50N5O5S [M+H]+: 580.3533. Found: m/z 580.3524. Spectroscopic data for
6: IR (KBr) 3343, 3090, 2919, 2851, 2124, 1677, 1629, 1605, 1536, 1500,
1280 cmÀ1 1H NMR (400 MHz, CDCl3) d 1.31 (18H, m), 1.60 (2H, m), 2.36 (2H,
ll
m
;
m), 2.63 (2H, d, J = 6.1 Hz), 2.76 (2H, m, overlapped with 1H signal), 2.90 (2H,
br t, J = 7.4 Hz), 3.43 (1H, t, J = 7.2 Hz), 3.45 (1H, t, J = 7.0 Hz), 6.06 (1H, s), 7.06
(2H, d, J = 8.3 Hz), 7.76 (2H, d, J = 8.3 Hz), 7.88 (1H, s); 13C NMR (100 MHz,
CDCl3) d 27.1, 27.8, 28.9, 29.2, 29.37, 29.44, 29.5, 29.6, 29.8, 29.9, 37.3, 40.3,
48.0, 119,1, 128.8, 131.5, 143.3, 166.6, 171.1, 196.7. HRMS (ESI) calcd for
culture supernatant was replaced with 100 ll DMSO to dissolve the formazan
crystals made from MTT by the enzymatic action of succinic dehydrogenase in
the mitochondria of live cells. The absorbance at 570 nm was measured with a
microplate reader.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared according to the method of Andrews and
C
26H38N5O4S [M+H]+: 516.2645. Found: m/z 516.2642.
21. The assays to determine inhibition of LPS-induced NO production, iNOS
Faller. The binding reaction mixture contained nuclear extract (5
protein), 1
g poly(dI-dC), and 10,000 cpm 32P-labeled probe (oligonucleotide
containing NF- B) in binding buffer (15 mM Tris–HCl[pH 7.0], 75 mM NaCl,
lg of
expression, NF-
j
B activation were performed as described below.
l
Cell culture
j
Mouse macrophage RAW264.7 cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine
1.5 mM EDTA, 1 mM DTT, 7.5% glycerol, and 1.5% NP-40). Samples were
incubated for 20 min at room temperature (RT) in this mixture. DNA/protein
complexes were separated from free DNA on 4% native polyacrylamide gel in
TBE buffer (22.5 mM Tris–HCl[pH8.3] and 0.5 mM EDTA). The DNA probes used
serum (JRH Biosciences, Lenexa, KS, USA), 200
Louis, MO, USA), 100 units/ml penicillin G (Sigma), 600
(Sigma), and 2.25 g/L NaHCO3 at 37 °C under 5% CO2 plus air.
l
g/ml kanamycin (Sigma, St.
l
g/ml -glutamine
L
for NF-jB binding were purchased from Promega (Madison, WI, USA).