Development of novel DIF-1 derivatives that selectively suppress innate immune responses

Article abstract of DOI:10.1016/j.bmc.2015.06.027

The multiple pharmacological activities of differentiation-inducing factor-1 (DIF-1) of the cellular slime mold Dictyostelium discoideum led us to examine the use of DIF-1 as a 'drug template' to develop promising seed compounds for drug discovery. DIF-1 and its derivatives were synthesized and evaluated for their regulatory activities in innate immune responses. We found two new derivatives (4d and 5e) with highly selective inhibitory activities against production of the antimicrobial peptide attacin in Drosophila S2 cells and against production of interleukin-2 in Jurkat cells.

Full text of DOI:10.1016/j.bmc.2015.06.027

Bioorganic & Medicinal Chemistry 23 (2015) 4311–4315
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of novel DIF-1 derivatives that selectively suppress
innate immune responses
Van Hai Nguyen a, Haruhisa Kikuchi a, , Yuzuru Kubohara b,c, Katsunori Takahashi d, Yasuhiro Katou a,
⇑
Yoshiteru Oshima a,
⇑
a Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku, Sendai 980-8578, Japan
b Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan
c Graduate School of Health and Sports Science, Juntendo University, 1-1 Hiraga-gakuendai, Inzai, Chiba 270-1695, Japan
d Department of Medical Technology, Faculty of Health Science, Gunma Paz College, Takasaki 370-0006, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The multiple pharmacological activities of differentiation-inducing factor-1 (DIF-1) of the cellular slime
mold Dictyostelium discoideum led us to examine the use of DIF-1 as a ‘drug template’ to develop promis-
ing seed compounds for drug discovery. DIF-1 and its derivatives were synthesized and evaluated for
their regulatory activities in innate immune responses. We found two new derivatives (4d and 5e) with
highly selective inhibitory activities against production of the antimicrobial peptide attacin in Drosophila
S2 cells and against production of interleukin-2 in Jurkat cells.
Received 16 May 2015
Revised 10 June 2015
Accepted 11 June 2015
Available online 17 June 2015
Keywords:
Innate immunity
Drosophila
Ó 2015 Elsevier Ltd. All rights reserved.
Immunosuppressor
Cellular slime mold
Structure–activity relationship
1. Introduction
Recently, we screened for innate immune regulators by using
transgenic Drosophila.12–14 Because of the utility of DIF-1 (1c) as
The cellular slime mold Dictyostelium discoideum is an excellent
model organism for the study of cellular and developmental biol-
ogy.1 Differentiation-inducing factor-1 (DIF-1) (1c) was previously
identified as a signal molecule that induces stalk cell differentia-
tion in Dictyostelium discoideum in the presence of cAMP.2–4 In
our previous studies, we showed that DIF-1 (1c) possesses multiple
pharmacological activities in mammalian cells, such as anti-leuke-
mic activity, acceleration of differentiation,5–7 and promotion of
glucose consumption.8 Some DIF-1 (1c) derivatives showed novel
pharmacological activities. Specifically, DIF-3 (2) suppressed the
infection rate and growth of Trypanosoma cruzi,9 and Br-DIF-1 (3)
accelerated the differentiation of P19CL6 cells into spontaneously
beating cardiomyocyte-like cells (Fig. 1).10 These results suggest
that DIF-1 (1c) may be considered as a promising ‘drug template’
that may be used to develop seed compounds with multiple phar-
macological activities.
a drug template, we decided to evaluate the effects of DIF-1 (1c)
and its derivatives on the innate immune system. We found that
the novel derivatives 4d and 5e selectively inhibited production
of the antimicrobial peptide attacin in Drosophila S2 cells. These
compounds also selectively suppressed interleukin-2 (IL-2) pro-
duction in Jurkat cells.15
2. Results
2.1. Effects of DIF-1 and benzene derivatives on the innate
immunity of Drosophila
Using att-luc assay, we investigated the effect of DIF-1 (1c) on
the innate immune system of Drosophila.16 In this assay, we used
transgenic Drosophila S2 cells carrying the att-luc reporter gene,
which was activated by addition of peptidoglycan (PGN).17 As
shown in Figure 2A, DIF-1 (1c) inhibited attacin production in S2
Innate immunity is the front line of self defense against micro-
bial infections.11 Innate immune regulators have potential as ther-
apeutic medications for sepsis or opportunistic infective diseases.
cells in
However, because it also inhibited the proliferation of S2 cells
(CC50 22.5 M), its selectivity to inhibition of attacin production
was low, having a selective index (SI) of 4.9 (Table 1).
a concentration-dependent manner (IC50 4.6 lM).
l
⇑
Corresponding authors. Tel.: +81 22 795 6824; fax: +81 22 795 6821.
E-mail address: hal@mail.pharm.tohoku.ac.jp (H. Kikuchi).
http://dx.doi.org/10.1016/j.bmc.2015.06.027
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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V. H. Nguyen et al. / Bioorg. Med. Chem. 23 (2015) 4311–4315
TH-DIF-1 (5c), were synthesized according to our previous work.7
We could obtain the deoxo-derivative 6 by reductive removal of
the carbonyl group of DIF-1 (1c) using triethylsilane in trifluo-
roacetic acid.18 Compound 7, which has a methyl group instead of
a
methoxy group, was synthesized from orcinol instead of
5-methoxyresorcinol through the same synthetic route for DIF-1
(1c).
Evaluation of the activities of the aforementioned compounds
showed that DIF-3 (2), Br-DIF-1 (3), and 7 inhibited both attacin
production and S2 cell proliferation, resulting in low SI values
(Table 1). Derivative 6 showed weak inhibitory activity, indicating
the role of the carbonyl group in the inhibition. Although TM-DIF-1
(4c) and TH-DIF-1 (5c) inhibited attacin production more weakly
than did DIF-1 (1c), they almost did not influence the proliferation
Figure 1. Structures of DIF-1 (1c), DIF-3 (2), and Br-DIF-1 (3).
of S2 cells (CC50 > 100 lM). Therefore, these compounds show pro-
mise because of their high selectivity to inhibition of attacin pro-
duction (having SI values of 7.8 and 10.3, respectively;
Fig. 2B and C and Table 1).
2.2. Synthesis of TM-DIF-1 and TH-DIF-1 derivatives and their
effects on Drosophila innate immunity
To investigate in detail the structure–activity relationships for
TM-DIF-1 (4c) and TH-DIF-1 (5c), we synthesized their derivatives,
which have varied acyl chain lengths, and examined their effects
on the innate immune response. Derivatives 4a, 4b, 4d, 4e, 5a,
5e, and 5f, which, respectively, have 4, 5, 7, 8, 4, 8, and 9 carbons
in the acyl chain, were synthesized from their corresponding acyl
chlorides according to the synthetic routes for TM-DIF-1 (4c) and
TH-DIF-1 (5c) (Scheme 1). Their inhibitory activities on the innate
immune response suggest that derivatives with longer acyl chains
tend to show higher activity and high selectivity to innate immu-
nity (Table 2). However, the selectivities of 4e and 5f (respectively,
having eight and nine carbons in the acyl chain) were relatively
low. Overall, 4d (SI 8.3) and 5e (SI 23.9) showed promise.
The acyl chain length of the most selective inhibitors of attacin
production among TM-DIF-1 (4c) and TH-DIF-1 (5c) derivatives is
related to lipophilicity, which influences their cell-membrane per-
meability. As TM-DIF-1 (4c) derivatives possess three methoxy
groups, they are more liposoluble than are TH-DIF-1 (5c) deriva-
tives, which have three hydroxyl groups. The former derivatives
thus have greater lipophilicity for permeation of cell membranes
with shorter acyl chains.
Figure 2. Effects of DIF-1 (1c) (A), TM-DIF-1 (4c) (B), and TH-DIF-1 (5c) (C) on
peptidoglycan (PGN)-induced attacin production (att-luc assay) in S2 cells and cell
viability. Cells were incubated for 8 h with PGN at the indicated concentrations of
DIF-1 derivatives and then assayed for luciferase production (black triangles). Cell
viability (black circles) was determined by incubating cells with the indicated
concentrations of DIF-1 derivatives for 24 h. Results are presented as mean values
( SD) of four independent experiments (n = 4).
2.3. Effects of compounds 4d and 5e on IL-2 production in
Jurkat cells
Table 1
Suppressive effects of DIF-1 derivatives on innate immunity
Compound
Immunosuppressive effect Cytotoxicity Selective indexc
Compounds that are effective against Drosophila innate immu-
nity may regulate mammal immunity12–14 because of the promi-
nent conservation of innate immunity between Drosophila and
mammals.19,20 In our previous study,21 TM-DIF-1 (4c) and TH-
DIF-1 (5c) showed inhibitory activity against IL-2 production15 in
Jurkat cells. Thus, we evaluated the inhibitory activity of 4d and
5e against IL-2 production. Concanavalin A (ConA) was added to
a culture of Jurkat cells, in which the compounds had been added
previously.22,23 Compounds 4d and 5e showed selective inhibition
against ConA-induced IL-2 production without suppressing cell
proliferation (Fig. 3). Although 4d showed weaker activity than
a
b
IC50
(lM)
CC50 (lM)
DIF-1 (1c)
DIF-3 (2)
Br-DIF-1 (3)
4.6
4.6
3.7
7
22.5
8.7
12.9
4.9
1.9
3.5
TM-DIF-1 (4c) 18.9
TH-DIF-1 (5c)
6
7
148
102
>100
13.8
7.8
9.9
58.6
3.8
10.3
>1.7
3.6
a
IC50 value is the concentration of compound that suppressed attacin production
to 50% of the control (peptidoglycan only) level. It was determined by assessing the
effects of various concentrations of the compound.
b
did 5e in the att-luc assay (IC50 values of 16.8
lM and 3.7 lM,
CC50 value is the concentration of compound that suppressed the proliferation
of S2 cells to 50% of the control (DMSO) level. It was determined by assessing the
effects of various concentrations of the compound.
respectively), they showed very similar inhibitory activities against
IL-2 production at the same concentration (5 lM).
c
Selective index (SI) is the CC50/IC50 ratio.
3. Conclusion
To discover more inhibitors with high selectivity, we evaluated
the derivatives with varied substituents on the benzene ring in
DIF-1 (1c) (Scheme 1). DIF-3 (2), Br-DIF-1 (3), TM-DIF-1 (4c), and
We synthesized and evaluated the effects of DIF-1 (1c) deriva-
tives on the production of attacin in Drosophila S2 cells to test their

V. H. Nguyen et al. / Bioorg. Med. Chem. 23 (2015) 4311–4315
4313
Scheme 1. General procedure for the synthesis of DIF-1 derivatives. Reagents and conditions: (a) acyl chloride, AlCl3, CH2Cl2 (44–57%), rt; (b) SO2Cl2 (2.2 equiv), CHCl3–EtOH,
rt (74–93%); (c) SO2Cl2 (1.1 equiv), CHCl3–EtOH, rt (85%); (d) PyÁHBr3, pyridine, rt (62%); (e) p-TsOMe, K2CO3, DMF, rt (80–95%); (f) BBr3, CH2Cl2, À78 °C, then rt (40%); (g)
Et3SiH, CF3COOH, rt (96%).
Table 2
Suppressive effects of TH-DIF-1 and TM-DIF-1 derivatives on innate immunity
Compound
Immunosuppressive effect Cytotoxicity Selective indexc
a
b
IC50
(l
M)
CC50 (lM)
4a
4b
20.8
23.5
144
149
148
6.9
6.3
7.8
TM-DIF-1 (4c) 18.9
7
4d
4e
5a
16.8
15.3
21.5
9.9
3.7
6.5
140
51.4
>100
102
88.5
82.8
8.3
3.4
>4.7
10.3
23.9
12.7
TH-DIF-1 (5c)
5e
5f
Figure 3. (A) Effects of derivatives 4d and 5e on concanavalin A (ConA)-induced
interleukin-2 (IL-2) production in Jurkat cells. Cells were pre-incubated for 30 min
a
IC50 value is the concentration of compound that suppressed attacin production
with 0.1% DMSO (control), 1 lM cyclosporine A (CsA), or 5 lM of each DIF-1
derivative. After addition of ConA, cells were further incubated for 12 h and assayed
for IL-2 protein production. (B) Cell viability of Jurkat cells. Cells were pre-incubated
to 50% of the control (peptidoglycan only) level. It was determined by assessing the
effects of various concentrations of the compound.
b
CC50 value is the concentration of compound that suppressed the proliferation
for 30 min with 0.1% DMSO (control) or 5 lM of each DIF-1 derivative. Results are
presented as mean values ( SD) of three independent experiments (n = 3).
of S2 cells to 50% of the control (DMSO) level. It was determined by assessing the
effects of various concentrations of the compound.
c
Selective index (SI) is the CC50/IC50 ratio.
4. Experiment section
4.1. General methods
regulatory effects on innate immunity. We found two novel deriva-
tives, 4d and 5e, which selectively suppressed attacin production
with only weak inhibition of the proliferation of S2 cells.
Compound 4d possesses a benzene ring with three methoxy
groups and an acyl chain with seven carbons, whereas 5e possesses
three phenolic hydroxyl groups and an acyl chain with eight car-
bons. As 4d and 5e selectively suppressed IL-2 production in
Jurkat cells, they are also effective in the human immune system.
Our results suggest that 4d and 5e are promising candidates for
development into novel immunosuppressive drugs.
Analytical TLC was performed on silica gel 60 F254 (Merck).
Column chromatography was carried out on silica gel 60
(70–230 mesh, Merck). NMR spectra were recorded on JEOL JNM
ECA-600 and AL-400. Chemical shifts for 1H and 13C NMR were
given in parts per million (d) relative to tetramethylsilane (dH
0.00) and residual solvent signals (dC 77.0) as internal standards.
Mass spectra were measured on JEOL JMS-700 and JMS-DX303.

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V. H. Nguyen et al. / Bioorg. Med. Chem. 23 (2015) 4311–4315
HPLC was carried on LC-908 (Japan Analytical Industry Co. Ltd),
using the column YMC-GPC-T2000 (/ 20 mm  600 mm) (YMC
Co. Ltd).
(2C), 23.3, 22.6, 14.0; EIMS m/z 366 [M+4]+, 364 [M+2]+, 362
[M]+, 263 (base); HREIMS m/z 362.1040 [M]+ (362.1050 calculated
for C17H24O435Cl2).
4.2. Synthesis of DIF-1 (1c), DIF-3 (2) and their derivatives
4.4. Synthesis of TH-DIF-1 (5c) and its derivatives
DIF-1 (1c) and DIF-3 (2) were synthesized according to previous
1 M Solution of boron tribromide in dichloromethane (520 lL,
study.7 Sulfuryl chloride (64.0 mg, 0.474 mmol) and ethanol
0.520 mmol) was added to a solution of DIF-1 (1c) (20.0 mg,
0.065 mmol) in dichloromethane (2 mL) at 0 °C. After being stirred
for 16 h at 0 °C, the mixture was poured into water (30 mL) and
extracted with ethyl acetate (30 mL) three times. The organic layer
was washed with brine, dried over anhydrous sodium sulfate, and
evaporated. The residue was chromatographed over silica gel
eluted by hexane–ethyl acetate (9:1) to give TH-DIF-1 (5c)
(7.5 mg, 0.026 mmol (yield 40%)). This compound has been charac-
terized before.7
(40 lL) were added to a solution of 8c (51.4 mg, 0.216 mmol) in
chloroform (2.5 mL) at room temperature. After being stirred for
1 h, the mixture was evaporated. The residue was chro-
matographed over silica gel eluted by hexane–ethyl acetate (9:1)
to give DIF-1 (1c) (61.6 mg, 0.201 mmol (yield 93%)). In a similar
procedure, DIF-3 (2) (85%) was synthesized from 8c with 1.1 equiv
of sulfuryl chloride. These compounds have been characterized
before.7
In the similar procedure, compounds 1a (yield 87%), 1b (78%),
1d (83%), 1e (74%) and 1f (81%) were prepared from 8a, 8b, 8d,
8e and 8f, respectively. 1a, 1b, 1d and 1e have been characterized
before.7 Data for 1f: Colorless amorphous solid; 1H NMR (400 MHz,
CDCl3) d 10.34 (2H, br s), 3.98 (3H, s), 3.13 (2H, t, J = 7.3 Hz), 1.70
(2H, quin, J = 7.3 Hz), 1.23–1.40 (10H, m), 0.89 (3H, t, J = 7.0 Hz);
13C NMR (100 MHz, CDCl3) d 206.4, 166.5, 163.9 (2C), 105.2, 94.3
(2C), 61.4, 44.1, 31.8, 29.4, 29.2, 29.0, 24.9, 22.7, 14.3; EIMS m/z
352 [M+4]+, 350 [M+2]+, 348 [M]+, 330, 263, 235 (base); HREIMS
m/z 348.0886 [M]+ (348.0895 calculated for C16H22O435Cl2).
In the similar procedure, compounds 5a (yield 45%), 5e (48%)
and 5f (40%) were prepared from 1a, 1e and 1f, respectively.
Data for 5a: Yellow amorphous solid; 1H NMR (400 MHz, CDCl3)
d 10.50 (2H, br s), 6.49 (1H, br s), 3.10 (2H, t, J = 7.2 Hz), 1.59
(2H, sext, J = 7.3 Hz), 1.00 (3H, t, J = 7.6 Hz); 13C NMR (100 MHz,
CDCl3) d 206.0, 156.4, 153.0 (2C), 104.7 (2C), 99.8, 46.1, 17.7,
14.3; EIMS m/z 268 [M+4]+, 266 [M+2]+, 264 [M]+, 221 (base);
HREIMS m/z 263.9961 [M]+ (263.9956 calculated for
C
10H10O435Cl2). Data for 5e: Yellow amorphous solid; 1H NMR
(400 MHz, CDCl3) d 10.50 (2H, br s), 6.49 (1H, br s), 3.11 (2H, t,
J = 7.3 Hz), 1.70 (2H, quin, J = 7.7 Hz), 1.25–1.40 (8H, m), 0.89
(3H, t, J = 7.1 Hz); 13C NMR (100 MHz, CDCl3) d 206.2, 156.4,
153.0 (2C), 104.7 (2C), 99.8, 44.2, 31.7, 29.3, 29.1, 24.3, 22.6,
14.1; EIMS m/z 324 [M+4]+, 322 [M+2]+, 320 [M]+, 302, 249, 221
(base); HREIMS m/z 320.0570 [M]+ (320.0581 calculated for
4.3. Synthesis of TM-DIF-1 (4c) and its derivatives
TM-DIF-1 (4c) was synthesized according to previous study.7
Potassium carbonate (20.8 mg, 0.750 mmol) and methyl p-toluene-
sulfonate (70.0 mg, 0.461 mmol) were added to a solution of DIF-1
(1c) (70.5 mg, 0.230 mmol) in acetone (2 mL) at room temperature.
After being stirred for 16 h, the mixture was poured into water
(30 mL) and extracted with ethyl acetate (30 mL) three times.
The organic layer was washed with brine, dried over anhydrous
sodium sulfate and evaporated. The residue was chromatographed
over silica gel eluted by hexane–ethyl acetate (19:1) to give TM-
DIF-1 (4c) (73.2 mg, 0.218 mmol (yield 95%)). This compound has
been characterized before.7
C
14H18O435Cl2). Data for 5f: Yellow amorphous solid; 1H NMR
(400 MHz, CDCl3) d 10.49 (2H, br s), 6.50 (1H, br s), 3.11 (2H, t,
J = 7.3 Hz), 1.70 (2H, quin, J = 7.6 Hz), 1.21–1.40 (10H, m), 0.88
(3H, t, J = 7.1 Hz); 13C NMR (100 MHz, CDCl3) d 206.2, 156.4,
153.0 (2C), 104.7 (2C), 99.8, 44.3, 31.9, 29.5, 29.3, 29.2, 24.2,
22.7, 14.1; EIMS m/z 338 [M+4]+, 336 [M+2]+, 334 [M]+, 316, 249,
221 (base); HREIMS m/z 334.0744 [M]+ (334.0737 calculated for
C
15H20O345Cl2).
In the similar procedure, compounds 4a (yield 90%), 4b (88%),
4d (83%) and 4e (80%) were prepared from 1a, 1b, 1d and 1e,
respectively. Data for 4a: Colorless oil; 1H NMR (400 MHz, CDCl3)
d 3.92 (3H, s), 3.83 (6H, s), 2.75 (2H, t, J = 7.6 Hz), 1.71 (2H, sext,
J = 7.4 Hz), 0.98 (3H, t, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d
202.5, 154.5, 151.9 (2C), 129.2, 119.57 (2C), 62.7 (2C), 60.9, 46.8,
16.9, 13.5; EIMS m/z 310 [M+4]+, 308 [M+2]+, 306 [M]+, 263 (base);
HREIMS m/z 306.0420 [M]+ (306.0426 calculated for
4.5. Synthesis of 4,6-dichloro-2-hexyl-5-methoxybenzene-1,3-
diol (6)
Triethylsilane (41 lL, 0.260 mmol) was added to a solution of
DIF-1 (20.0 mg, 0.065 mmol) in trifluoroacetic acid (1 mL) at room
temperature. After being stirred for 8 h, the mixture was evapo-
rated. The residue was purified by GPC HPLC (column, YMC-GPC
T-2000 (
u
20 mm  600 mm, TMC Co., Ltd); solvent, ethyl acetate)
C
13H16O435Cl2). Data for 4b: Colorless oil; 1H NMR (400 MHz,
to give 6 (18.2 mg, 0.063 mmol (yield 96%)). Data for 6: Yellow oil;
1H NMR (400 MHz, CDCl3) d 5.65 (2H, br s), 3.90 (3H, s), 2.60 (2H, t,
J = 7.9 Hz), 1.47 (2H, quin, J = 7.6 Hz), 1.19–1.32 (6H, m), 0.91 (3H,
t, J = 6.6 Hz); 13C NMR (100 MHz, CDCl3) d 149.7, 149.4 (2C), 113.6
(2C), 106.9, 60.8, 31.7, 29.3, 28.7, 24.3, 22.6, 14.1; EIMS m/z 296
[M+4]+, 294 [M+2]+, 292 [M]+, 221 (base); HREIMS m/z 292.0617
[M]+ (292.0632 calculated for C13H18O335Cl2).
CDCl3) d 3.92 (3H, s), 3.83 (6H, s), 2.77 (2H, t, J = 7.4 Hz), 1.66
(2H, quin, J = 7.8 Hz), 1.38 (2H, sext, J = 7.2 Hz), 0.93 (3H, t,
J = 7.7 Hz); 13C NMR (100 MHz, CDCl3) d 202.6, 154.4, 151.9 (2C),
129.2, 119.7 (2C), 62.7 (2C), 60.9, 44.7, 25.4, 22.1, 13.8; EIMS m/z
324 [M+4]+, 322 [M+2]+, 320 [M]+, 263 (base); HREIMS m/z
306.0570 [M]+ (320.0582 calculated for C14H18O435Cl2). Data for
4d: Colorless oil; 1H NMR (400 MHz, CDCl3) d 3.92 (3H, s), 3.82
(6H, s), 2.76 (2H, t, J = 7.4 Hz), 1.67 (2H, quin, J = 7.0 Hz), 1.25–
1.39 (6H, m), 0.89 (3H, t, J = 6.6 Hz); 13C NMR (100 MHz, CDCl3) d
202.6, 154.4, 151.9 (2C), 129.2, 119.7 (2C), 62.7 (2C), 60.9, 45.0,
31.5, 28.7, 23.3, 22.5, 14.0; EIMS m/z 352 [M+4]+, 350 [M+2]+,
348 [M]+, 263 (base); HREIMS m/z 348.0912 [M]+ (348.0896 calcu-
lated for C16H22O435Cl2). Data for 4e: Colorless oil; 1H NMR
4.6. Synthesis of 1-(2,6-dihydroxy-4-methylphenyl)hexan-1-one
(9)
Aluminum chloride (215.7 mg, 1.62 mmol) was added to a solu-
tion of orcinol (100.0 mg, 0.81 mmol) in dichloromethane (5 mL) at
room temperature. After 15 min, hexanoyl chloride (170 lL,
(400 MHz, CDCl3)
d
3.92 (3H, s), 3.82 (6H, s), 2.76 (2H, t,
1.21 mmol) was added. The mixture was stirred for 3 h, poured
into water (25 mL), and extracted with ethyl acetate (30 mL) three
times. The organic layer was washed with brine, dried over anhy-
drous sodium sulfate, and evaporated. The residue was
J = 7.7 Hz), 1.67 (2H, quin, J = 7.2 Hz), 1.24–1.37 (8H, m), 0.88
(3H, t, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d 202.6, 154.4,
151.9 (2C), 129.2, 119.7 (2C), 62.7 (2C), 60.9, 45.0, 31.7, 29.0

V. H. Nguyen et al. / Bioorg. Med. Chem. 23 (2015) 4311–4315
4315
chromatographed over silica gel eluted by hexane–ethyl acetate
(9:1) to give 9 (126.5 mg, 0.57 mmol (yield 70%)). Data for 9:
Colorless amorphous solid; 1H NMR (400 MHz, acetone-d6) d
11.84 (2H, br s), 6.61 (2H, s), 3.06 (2H, t, J = 7.4 Hz), 2.34 (3H, s),
1.68 (2H, quin, J = 7.0 Hz), 1.30–1.35 (4H, m), 0.91 (3H, t,
J = 6.9 Hz); 13C NMR (100 MHz, acetone-d6) d 205.5, 162.9 (2C),
147.3, 105.5, 91.1 (2C), 44.4, 31.4, 23.6, 23.4, 18.9, 14.3; EIMS
m/z 222 [M]+, 204, 151 (base); HREIMS m/z 222.1245 [M]+
(222.1256 calculated for C13H18O3).
human IL-2 and then with streptavidin-horseradish peroxidase,
color was developed and the levels of IL-2 were quantified by mea-
suring the absorbance of each sample at 450 nm and 550 nm. The
readings at 550 nm were subtracted from the readings at 450 nm.
To determine cell viability, cells were incubated under the same
conditions, and the percentage of viable cells compared with
controls was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,
5-diphenyl tetrazolium bromide (MTT) assay according to standard
procedures.
4.7. Synthesis of 1-(3,5-dichloro-2,6-dihydroxy-4-methylphenyl)
hexan-1-one (7)
Acknowledgements
We thank Dr. J.L. Imler for att-luc reporter construct. This work
was supported in part by Grant-in-Aid for Scientific Research (Nos.
23710247, 24590110 and 25350959) from the Ministry of
Education, Culture, Sports, Science, and Technology, Japan; Drug
Discovery, Informatics, and Structural Life Science from the
Ministry of Education, Culture, Sports, Science, and Technology,
Japan; Kobayashi International Scholarship Foundation; the
SUNBOR GRANT from the Suntory Institute for Bioorganic
Research; and the Astellas Foundation for Research on Metabolic
Disorders.
Sulfuryl chloride (31.0 mg, 0.23 mmol) and ethanol (20 lL)
were added to a solution of 9 (25.0 mg, 0.11 mmol) in chloroform
(1 mL) at room temperature. After being stirred for 1 h, the mixture
was evaporated. The residue was chromatographed over silica gel
eluted by hexane–ethyl acetate (9:1) to give 7 (26.1 mg, 0.09 mmol
(yield 85%)). Data for 7: Yellow amorphous solid; 1H NMR
(400 MHz, CDCl3) d 10.21 (2H, br s), 3.14 (2H, t, J = 7.7 Hz), 2.52
(3H, s), 1.71 (2H, quin, J = 7.0 Hz), 1.33–1.38 (4H, m), 0.92 (3H, t,
J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d 206.9, 154.7 (2C), 141.7,
113.4 (2C), 108.4, 44.6, 31.4, 23.9, 23.5, 18.9, 14.0; EIMS m/z 294
[M+4]+, 292 [M+2]+, 290 [M]+, 272, 219 (base); HREIMS m/z
290.0458 [M]+ (290.0476 calculated for C13H16O335Cl2).
References and notes
1. Dictyostelium. A Model System for Cell and Developmental Biology; Maeda, Y.,
Inoue, K., Takeuchi, I., Eds.; Universal Academy: Tokyo, 1997.
2. Town, C. D.; Gross, J. D.; Kay, R. R. Nature 1976, 262, 717.
3. Morris, H. R.; Taylor, G. W.; Masento, M. S.; Jermyn, K. A.; Kay, R. R. Nature 1987,
328, 811.
4.8. Measurement of cytotoxicity and luciferase activity (att-luc
assay)
4. Kay, R. R.; Berks, M.; Traynor, D. Dev. Suppl. 1989, 107, 81.
5. Kubohara, Y. Eur. J. Pharmacol. 1999, 381, 57.
6. Kubohara, Y. Biochem. Biophys. Res. Commun. 1997, 236, 418.
7. Gokan, N.; Kikuchi, H.; Nakamura, K.; Oshima, Y.; Hosaka, K.; Kubohara, Y.
Biochem. Pharmacol. 2005, 70, 676.
8. Omata, W.; Shibata, H.; Nagasawa, M.; Kojima, I.; Kikuchi, H.; Oshima, Y.;
Hosaka, K.; Kubohara, Y. FEBS J. 2007, 274, 3392.
9. Nakajima-Shimada, J.; Hatabu, T.; Hosoi, Y.; Onizuka, Y.; Kikuchi, H.; Oshima,
Y.; Kubohara, Y. Biochem. Pharmacol. 2013, 85, 1603.
10. Seya, K.; Kanemaru, K.; Matsuki, M.; Hongo, K.; Kitahara, H.; Kikuchi, H.;
Oshima, Y.; Kubohara, Y.; Okumura, K.; Motomura, S.; Furukawa, K.-I. Br. J.
Pharmacol. 2012, 165, 870.
11. Janeway, C., Jr. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7461.
12. Sekiya, M.; Ueda, K.; Fujita, T.; Kitayama, M.; Kikuchi, H.; Oshima, Y.; Kurata, S.
Life Sci. 2006, 80, 113.
Drosophila S2 cells and S2att-luc cells, harboring luciferase repor-
ter gene driven by attacin promoter,16 were cultured in Schneider’s
Drosophila medium (Gibco-BRL) supplemented with 10% FBS and
1% antibiotics/antimycotics at 25 °C. Cytotoxicity was measured
using the colorimetric thiazoyl blue conversion assay using WST-
8 solution (nacalai tesque) as described previously.12 To determine
the effect of compounds on attacin production, S2att-luc cells were
pre-treated with compounds for 1.5 h and stimulated by
100 ng/mL peptidoglycan. At 8 h after stimulation, cells were lysed
with Glo-lysis buffer (Promega), and luciferase activities were
measured by One-Glo (Promega).
13. Sekiya, M.; Ueda, K.; Okazaki, K.; Kikuchi, H.; Kurata, S.; Oshima, Y. Biochem.
Pharmacol. 2008, 75, 2165.
14. Kikuchi, H.; Isobe, M.; Sekiya, M.; Abe, Y.; Hoshikawa, T.; Ueda, K.; Kurata, S.;
Katou, Y.; Oshima, Y. Org. Lett. 2011, 13, 4624.
4.9. Assay for IL-2 production by Jurkat cells and determination
of cell viability
15. Gaffen, S. L.; Liu, K. D. Cytokine 2004, 28, 109.
16. Goto, A.; Matsushita, K.; Gesellchen, V.; Chamy, L. E.; Kuttenkeuler, D.;
Takeuchi, O.; Hoffmann, J. A.; Akira, S.; Boutros, M.; Reichhart, J. M. Nat.
Immunol. 2008, 9, 97.
17. Leulier, F.; Parquet, C.; Pili-Floury, S.; Ryu, J. H.; Caroff, M.; Lee, W. J.; Mengin-
Lecreulx, D.; Lemaitre, B. Nat. Immunol. 2003, 4, 478.
18. Rao, J. A.; Ravichandran, K.; O’Malley, G. J.; Cava, M. P. Can. J. Chem. 1987, 65, 31.
19. Hoffmann, J. A.; Reichhart, J. M. Nat. Immunol. 2002, 3, 121.
20. Akira, S.; Takeda, K.; Kaisho, T. Nat. Immunol. 2001, 2, 675.
21. Takahashi, K.; Murakami, M.; Hosaka, K.; Kikuchi, H.; Oshima, Y.; Kubohara, Y.
Life Sci. 2009, 85, 438.
Jurkat cells were pre-incubated for 30 min in 90 mm culture
dishes filled with 10 mL RPMI (at 1 Â 106 cells/mL) in the presence
of the test compounds (5 lM) or 0.1% DMSO (vehicle). After the
addition of ConA to a final concentration of 25 lg/mL, the cells
were further incubated for 12 h. Aliquots of the culture media were
collected, and the levels of IL-2 were assessed by using immunoas-
say kits (ENDOGEN, Rockford, IL). Briefly, 50 lL aliquots (in dupli-
cate) of the culture media or standards for IL-2 from the kits were
added to the wells of 96-well plates pre-coated with anti-human
IL-2 antibody. After incubation with biotinylated antibodies to
22. Tanaka, S.; Akaishi, E.; Hosaka, H.; Okamura, S.; Kubohara, Y. Biochem. Biophys.
Res. Commun. 2005, 335, 162.
23. Aherne, S. A.; O’Brien, N. M. Mol. Nutr. Food. Res. 2008, 52, 664.