Biological activities of novel derivatives of differentiation-inducing factor 3 from Dictyostelium discoideum

Article abstract of DOI:10.1248/bpb.b17-00484

Differentiation-inducing factor-3 (DIF-3; 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), which is found in the cellular slime mold Dictyostelium discoideum, is a potential candidate compound for the development of new medicines; DIF-3 and its der

Full text of DOI:10.1248/bpb.b17-00484

Vol. 40, No. 11
Biol. Pharm. Bull. 40, 1941–1947 (2017)
1941
Regular Article
Biological Activities of Novel Derivatives of Differentiation-Inducing
Factor 3 from Dictyostelium discoideum
,b
Katsunori Takahashi,a Haruhisa Kikuchi,* Van Hai Nguyen,b Yoshiteru Oshima,b
,d
Hirotaka Ishigaki,a Junko Nakajima-Shimada,c and Yuzuru Kubohara*
a Department of Medical Technology, Faculty of Health Science, Gunma Paz College; Takasaki 370–0006, Japan:
b Laboratory of Natural Product Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University; Sendai
c
980–8578, Japan: Department of Molecular and Cellular Parasitology, Graduate School of Health Sciences, Gunma
d
University; Maebashi 371–8514, Japan: and Laboratory of Health and Life Science, Graduate School of Heath and
Sports Science, Juntendo University; Inzai, Chiba 270–1695, Japan.
Received June 15, 2017; accepted August 21, 2017
Differentiation-inducing factor-3 (DIF-3; 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one),
which is found in the cellular slime mold Dictyostelium discoideum, is a potential candidate compound for
the development of new medicines; DIF-3 and its derivatives possess several beneficial biological activities,
including anti-tumor, anti-Trypanosoma cruzi, and immunoregulatory effects. To assess the relationship be-
tween the biological activities of DIF-3 and its chemical structure, particularly in regard to its alkoxy group
and the length of the alkyl chains at the acyl group, we synthesized two derivatives of DIF-3, 1-(3-chloro-2,6-
dihydroxy-4-methoxyphenyl)octan-1-one (DIF-3(+3)) and 1-(3-chloro-2,6-dihydroxy-4-butoxyphenyl)-hexan-
1-one (Hex-DIF-3), and investigated their biological activities in vitro. At micro-molar levels, DIF-3(+3) and
Hex-DIF-3 exhibited strong anti-proliferative effects in tumor cell cultures, but their anti-T. cruzi activities
at 1µM in vitro were not as strong as those of other known DIF derivatives. In addition, Hex-DIF-3 at 5µM
significantly suppressed mitogen-induced interleukin-2 production in vitro in Jurkat T cells. These results
suggest that DIF-3(+3) and Hex-DIF-3 are promising leads for the development of anti-cancer and immuno-
suppressive agents.
Key words anti-tumor agent; Dictyostelium discoideum; interleukin-2; Jurkat cell; Trypanosoma cruzi
The characteristic chlorinated alkylphenones differentiation- tumor, anti-T. cruzi, and anti-inflammatory drugs.
inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-
In the present study, to further assess the relationship be-
methoxyphenyl)hexan-1-one) and DIF-3 (1-(3-chloro-2,6- tween the biological effects of DIF-3 derivatives and their
dihydroxy-4-methoxyphenyl)hexan-1-one) (Fig. 1A) were chemical structures, particularly in regard to the alkoxy
originally identified as stalk-cell differentiation-inducing group and the length of the alkyl chains at the acyl group,
factors in the cellular slime mold Dictyostelium discoi- we synthesized two new derivatives, namely 1-(3-chloro-2,6-
deum,1–4) an excellent model organism for studying cell and dihydroxy-4-methoxyphenyl)octan-1-one (DIF-3(+3)) and Hex-
developmental biology.5) Recently, cellular slime molds have DIF-3 (Figs. 1B, C). Here we show how the chemical structure
attracted interest as potential novel drug resources. To date, of each DIF derivative affects its biological activity in vitro;
we and others have shown that DIF-1, DIF-3, and their deriva- these findings inform the effective design of DIF derivatives
tives suppress cell growth and occasionally induce or promote as novel drugs.
cell differentiation in both cancer cells and non-transformed
(normal) mammalian cells.6–15) In addition, some DIF de-
MATERIALS AND METHODS
rivatives promote glucose consumption in mammalian cells
in vitro and in vivo16–18); others regulate interleukin 2 (IL-2)
production in human Jurkat T cells19–21) and suppress innate
immune responses in Drosophila S2 cells.21) Furthermore,
Synthesis of DIF-3(+3)
Synthesis of 1-(2,6-Dihydroxy-4-methoxyphenyl)nonan-1-one
Aluminum (III) chloride (95mg, 0.714mmol) was added to
some derivatives of DIF-3, specifically 1-(3-chloro-2,6- a solution of 5-methoxyresorcinol (50mg, 0.357mmol) in di-
dihydroxy-4-methoxyphenyl)-heptan-1-one (DIF-3(+2)) and chloromethane (2mL) at room temperature. After 15min, non-
1-(3-chloro-2,6-dihydroxy-4-ethoxyphenyl)hexan-1-one (Bu- anyl chloride (100µL, 0.555mmol) was added. The mixture
DIF-3) (Fig. 1A), have potent biological effects against tumors, was stirred for 3h, poured into water (20mL), and extracted
Trypanosoma cruzi, and inflammation9,12,15,19,20,22,23); the pro- three times with ethyl acetate (20mL). The organic layer was
tozoan T. cruzi causes Chagas disease (American trypanoso- washed with saturated NaCl solution, dried over anhydrous
miasis) in humans. The differing pharmacological activities sodium sulfate, and evaporated to dryness. The residue was
of various DIF derivatives might reflect specific modifications chromatographed over silica gel and eluted by hexane–ethyl
of their side chains. Taken together, these findings highlight acetate (19:1) to give 1-(2,6-dihydroxy-4-methoxyphenyl)-
the potential of DIF derivatives in the treatment of several nonan-1-one (70mg, 0.250mmol [yield, 70%]). The spectral
diseases, including cancer, diabetes, inflammation, and try- data of the eluate were identical to those reported previously
panosomiasis. In particular, DIF-3(+2) and Bu-DIF-3 may be for 1-(2,6-dihydroxy-4-methoxyphenyl)nonan-1-one.21)
advantageous lead compounds for the development of anti-
*To whom correspondence should be addressed. e-mail: hal@mail.pharm.tohoku.ac.jp; ykuboha@juntendo-u.ac.jp
© 2017 The Pharmaceutical Society of Japan

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Biol. Pharm. Bull.
Vol. 40, No. 11 (2017)
Fig. 1. Synthetic Routs to DIF Derivatives
(A) Chemical structures of DIF-1, DIF-3, and the DIF-3 derivatives reported previously. (B, C) Synthetic routes of DIF-3(+3) and Hex-DIF-3. The DIF derivatives were
synthesized as described in Materials and Methods. (D) Chemical structures of potentially promising DIF derivatives that have not yet been synthesized.
Synthesis of DIF-3(+3)
washed with saturated NaCl solution, dried over anhydrous
Sulfuryl chloride (22µL, 0.267mmol) and ethanol sodium sulfate, and evaporated to dryness. The residue was
(60 µL) were added to a solution of 1-(2,6-dihydroxy-4- chromatographed over silica gel and eluted by using hex-
methoxyphenyl)nonan-1-one (50mg, 0.178mmol) in chlo- ane–ethyl acetate (3:1) to give 5-butoxyresorcinol (53.6mg,
roform (3mL) at room temperature. After being stirred for 0.255mmol [yield, 32%]). Data for 5-hexyloxyresorcinol:
4h, the mixture was evaporated to dryness. The residue was colorless oil; 1H-NMR (600MHz, acetone-d6) δ: 8.17 (2H,
chromatographed over silica gel and eluted by using hexane– brs), 5.95 (1H, t, J=2.2Hz), 5.92 (2H, d, J=2.2Hz), 3.86 (2H,
ethyl acetate (9:1) to generate DIF-3(+3) (23mg, 0.072mmol t, J=6.3Hz), 1.66–1.73 (2H, m), 1.40–1.48 (2H, m), 1.29–1.37
[yield, 41%]). Data for DIF-3(+3): yellowish amorphous solid; (4H, m), 0.89 (3H, t, J=7.1Hz); 13C-NMR (150MHz, acetone-
1H-NMR (600MHz, CDCl3), δ 13.66 (1H, brs), 6.75 (1H, brs), d6) δ: 162.1, 160.0 (2C), 96.2, 94.4 (2C), 68.2, 32.3, 30.0, 26.4,
6.13 (1H, s), 3.90 (3H, s), 3.08 (2H, t, J=7.5Hz), 1.69 (2H, 23.2, 14.3; EI-MS m/z 210 [M+] (33%), and 126 (100%); and
quint, J=7.5Hz), 1.25–1.40 (10H, m), 0.88 (3H, t, J=6.9Hz); HR-EI-MS m/z 210.1245 [M]+ (210.1256 Calcd. for C12H18O3).
13C-NMR (150MHz, CDCl3) δ: 206.0, 164.9, 160.1, 154.1,
104.7, 99.5, 93.5, 56.4, 44.0, 31.8, 29.5, 29.4, 29.2, 24.5, 22.7,
Synthesis of 1-(2,6-Dihydroxy-4-hexyloxyphenyl)hexan-1-one
Aluminum (III) chloride (38mg, 0.286mmol) was added to
14.1; electron ionization (EI)-MS m/z 316 [M+2]+ (13%), 314 a solution of 5-hexyloxyresorcinol (30mg, 0.143mmol) in di-
[M+] (37%), 296 (32%), 229 (39%), 216 (56%), 201 (100%); chloromethane (2mL) at room temperature. After 15min, hex-
high resolution (HR)-EI-MS m/z 314.1294 [M]+ (314.1285 anoyl chloride (30µL, 0.215mmol) was added. The mixture
Calcd for C16H23O435Cl).
Synthesis of Hex-DIF-3
Synthesis of 5-Hexyloxyresorcinol
was stirred for 3h, poured into water (10mL), and extracted
three times with ethyl acetate (10mL). The organic layer was
washed with saturated NaCl solution, dried over anhydrous
1-Bromohexane (190µL, 1.35mmol) and potassium carbon- sodium sulfate, and evaporated. The residue was chroma-
ate (701mg, 5.08mmol) were added to a solution of phloro- tographed over silica gel and eluted by using hexane–ethyl
glucinol (100mg, 0.793mmol) in dimethyl formamide (4mL). acetate (9:1) to give 1-(2,6-dihydroxy-4-hexyloxyphenyl)-
After being stirred for 20h at room temperature, the mixture hexan-1-one (26mg, 0.084mmol [yield, 59%]). Data
was poured into 0.5M hydrochloric acid (20mL) and extracted for 1-(2,6-dihydroxy-4-hexyloxyphenyl)hexan-1-one: color-
three times with ethyl acetate (20mL). The organic layer was less amorphous solid; 1H-NMR (600MHz, CDCl3) δ: 10.05

Vol. 40, No. 11 (2017)
Biol. Pharm. Bull.
1943
(2H, brs), 5.91 (2H, s), 3.93 (2H, t, J=6.6Hz), 3.05 (2H, t, well containing 1mL of MEM-α-FBS (LM8) or DMEM-FBS
J=7.5Hz), 1.71–1.78 (2H, m), 1.66–1.71 (2H, m), 1.40–1.46 (3T3-L1 and HeLa) and 10µM of one of the DIF derivatives
(2H, m), 1.31–1.39 (8H, m), 0.91 (3H, t, J=7.1Hz), 0.90 (3H, or 0.2% (v/v) dimethyl sulfoxide (DMSO). After the 3-d in-
t, J=7.1Hz); 13C-NMR (150MHz, CDCl3) δ: 206.2, 165.0 cubation, the spent medium was discarded and replaced with
(2C), 163.2, 104.7, 94.8 (2C), 68.3, 43.9, 31.6, 31.5, 28.9, 25.6, 1mL of fresh medium without the derivative or DMSA but
24.4, 22.6, 22.5, 14.0 (2C); EI-MS m/z 308 [M+] (51%), 290 containing 5% (v⁄v) Alamar blue (a vital cell indicator; Wako
(19%), 265 (40%), 252 (20%), 237 (100%), 168 (52%), and 153 Pure Chemical Industries, Ltd.) until the color of the medium
(50%); and HR-EI-MS m/z 308.2004 [M]+ (308.1988 Calcd. changed. Relative live cell number was assessed by measuring
for C18H28O4).
absorbance at a wavelength of 570nm (reference, 595nm), as
Synthesis of Hex-DIF-3
described previously.9,23)
Sulfuryl chloride (2µL, 0.023mmol) and ethanol
To determine the IC50 of each compound in regard to cell
(20µL) were added to a solution of 1-(2,6-dihydroxy-4- proliferation, cells were cultured for 3d in the presence of
hexyloxyphenyl)hexan-1-one (14mg, 0.045mmol) in chloro- various concentrations of each compound. Relative live cell
form (1mL) at room temperature.
numbers were determined by using Alamar blue, and the IC50
After being stirred for 2h, the mixture was evaporated. value was determined from the dose–response curve drawn
The residue was chromatographed over silica gel eluted by by using the average values of three independent experiments.
using hexane–ethyl acetate (9:1) to give Hex-DIF-3 (6.3mg,
Infection and Growth of T. cruzi in Vitro Anti-T. cruzi
0.018mmol [yield, 41%]). Data for Hex-DIF-3: yellowish amor- activities of DIF derivatives were investigated as described.22)
1
phous solid; H-NMR (600MHz, CDCl3) δ: 13.65 (1H, brs), Briefly, a round coverslip (12mm) was placed in each well
6.66 (1H, brs), 6.09 (1H, s), 4.02 (2H, t, J=6.6Hz), 3.06 (2H, of a 24-well plate (Corning, Corning, NY, U.S.A.); exponen-
t, J=7.4Hz), 1.77–1.84 (2H, m), 1.65–1.72 (2H, m), 1.42–1.49 tially growing HT1080 cells (5×103 cells) were added to each
(2H, m), 1.30–1.37 (8H, m), 0.92 (3H, t, J=7.2Hz), 0.91 (3H, t, well and incubated at 37°C (5% CO2) for 1d. The cells were
J=7.2Hz); 13C-NMR (150MHz, CDCl3) δ: 205.9, 164.9, 159.7, infected with T. cruzi trypomastigotes (2×105 per well) as
154.1, 104.5, 99.8, 94.1, 69.4, 43.9, 31.6, 31.4, 28.7, 28.6, 24.2, described,28) and BZL or a DIF derivative (final concentra-
22.6, 22.5, 13.99, 13.97; EI-MS m/z 344 [M+2]+ (33%), 342 tion: 0.1 or 1µM) was added immediately. After 3d, the rate
[M+] (94%), 324 (36%), 299 (63%), 286 (45%), 271 (90%), 240 of infection of host cells with T. cruzi was assessed as de-
(66%), 202 (65%), 187 (100%); HR-EI-MS m/z 342.1608 [M]+ scribed.22,30) The percentage of infected host cells (i.e., cells
(342.1598 Calcd. for C18H27O435Cl).
containing multiple amastigotes) and the mean number of
Hydrophobic Index and Molecular Volume (M.V.) To amastigotes per infected cell were determined by assessing at
estimate the membrane permeability of each compound, its hy- least 200 host cells.
drophobic index (ClogP) was calculated by using ChemDraw
10.0 software (CambridgeSoft, Cambridge, MA, U.S.A.). M.V. kat cells (1×106 cells/mL) were pre-incubated (37°C, 5% CO2
was calculated by using the website Molinspiration.24)
in air) for 0.5h in 12-well culture plates containing 1mL
IL-2 Production and Cell Viability in Jurkat Cells Jur-
Reagents and Cells In addition to DIF-3(+3) and RPMI medium and 5µM DIF derivative or 0.1% DMSO (ve-
Hex-DIF-3, several DIF derivatives were synthesized as hicle). After the 0.5-h preincubation, ConA (final concentra-
described.15) The anti-T. cruzi drug benznidazole (BZL: N- tion, 25µg/mL) was added to each culture, and the cells were
benzyl-2-nitro-1H-imidazole-1-acetamide; Sigma-Aldrich, St. further incubated for 12h. Aliquots of the culture media were
Louis, MO, U.S.A.) was kindly provided by Dr. Yutaka Suto collected, and the level of IL-2 was assessed by using im-
(Takasaki University of Health and Welfare, Japan). FK506 munoassay kits (Endogen, Rockford, IL, U.S.A.), as described
(tacrolimus) was purchased from Calbiochem (San Diego, CA, previously.19,20) In addition, cell viability was evaluated
U.S.A.), and concanavalin A (ConA) was from Seikagaku by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Corporation (Tokyo, Japan).
bromide.19,20)
Human cervical cancer HeLa cells and murine fibroblast
To determine the IC50 of each compound in regard to the
3T3-L1 cells were grown and maintained at 37°C (5% CO2 in ConA-induced IL-2 production, cells were cultured for 12h
air) in Dulbecco’s modified Eagle’s medium (DMEM) (cata- in the presence of various concentrations of each compound.
log no. D5796, Sigma-Aldrich) supplemented with 10% (v/v) Relative IL-2 production was determined by using immunoas-
fetal bovine serum (FBS).16,23) Murine osteosarcoma LM8 say kits, and the IC50 value was determined from the dose–
cells25) were grown and maintained at 37°C (5% CO2 in air) in response curve drawn by using the average values of three
MEM-α (Wako Pure Chemical Industries, Ltd., Osaka, Japan) independent experiments.
supplemented with 10% FBS.26) Human T-lymphocyte Jurkat
Glucose Consumption-Promoting Activity of DIF De-
cells were maintained at 37°C (5% CO2) in RPMI-1640 me- rivatives in 3T3-L1 Cells Confluent 3T3-L1 cells in 12-well
dium (Sigma-Aldrich) supplemented with 10% (v/v) FBS.19,20) plates were incubated for 10–15h with 10µM DIF derivatives,
Human fibrosarcoma HT1080 cells (Japan Health Sciences and glucose consumption was measured as described previ-
Foundation, Tokyo, Japan) were used as the in vitro hosts for T. ously.16)
cruzi. The Tulahuen strain of T. cruzi27) and HT1080 cells were
Statistical Analysis Welch’s t-test (two-tailed) was per-
formed for the statistical analyses. Values were considered to
maintained and passaged in DMEM-FBS as described.22,28,29)
All media also contained 75µg/mL penicillin and 50µg/mL be significantly different when the p value was less than 0.05.
streptomycin.
Mammalian Cell Proliferation Assay LM8 (2.5×103
RESULTS AND DISCUSSION
cells/well), 3T3-L1 (5×103 cells/well), or HeLa cells (5×103
cells/well) were incubated for 3d in 12-well plates, with each
Synthesis of DIF-3(+3) and Hex-DIF-3 We chemically

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Vol. 40, No. 11 (2017)
Fig. 2. Effects of DIF Derivatives on Cell Growth in HeLa, LM8, and 3T3-L1 Cells
HeLa (A), LM8 (B), or 3T3-L1 (C) cells were incubated for 3d with 0.1% DMSO (Control) or 10µM of DIF derivatives, and relative live cell numbers were assessed.
Results are presented as the means and S.D. (bars) of three independent experiments. * p<0.05; **p<0.01; ***p<0.001 versus Control.
Table 1. Comparison of IC50 Values
IC50 (µM) for cell growth in
IC50 (µM) for CIIP in
3
Compound
M.W.
clogP
M.V. (Å)
HeLa
>20
16.2
11.5
8.3
LM8
3T3L1
Jurkat
DIF-1
18.2
15.5
7.8
>20
>20
16
—
—
—
—
—
7.0
4.0
2.5
307.17
272.73
286.75
300.78
314.81
286.75
314.81
342.86
3.46
2.90
3.32
3.74
4.16
3.24
4.15
4.98
255.45
241.91
258.71
275.52
292.32
258.71
292.32
325.92
DIF-3
DIF-3(+1)
DIF-3(+2)
DIF-3(+3)
Et-DIF-3
Bu-DIF-3
Hex-DIF-3
7.2
14.8
12.0
9.5
11.8
7.4
14.7
11.2
4.3
3.2
2.0
2.5
3.2
3.9
M.W., molecular weight. IC50 values in regard to cell growth in HeLa, LM8, and 3T3L1 cells and to ConA-induced IL-2 production (CIIP) in Jurkat cells, as well as the
hydrophobic index (clogP value) and molecular volume (M.V.) of each compound, were determined as described in Materials and Methods.
synthesized the novel DIF-3 derivatives DIF-3(+3) and Hex- tumor-growth-suppressive among the DIF-3 derivatives with
DIF-3 as described in Materials and Methods (Figs. 1B, C). alkoxy group modifications. Table 1 also shows the hydrophobic
We designed these derivatives to explore the chemical struc- index (clogP) and calculated M.V. of each compound; although
ture–biological activity relationship (particularly the impor- there was no strong relationship between antiproliferative activ-
tance of the alkyl chains) of DIF-3 derivatives.
ity and the values, the appropriate number of carbons at the
Effects of DIF Derivatives on Cell Growth in HeLa, LM8, acyl group and alkoxy group seemed to be important for activ-
and 3T3-L1 Cells We first evaluated the effects of various ity. Building on the current results, we might next synthesize
DIF derivatives on cell growth in human cervical cancer HeLa additional DIF-3 derivatives, such as Bu-DIF-3(+1) and Hex-
cells, mouse osteosarcoma LM8 cells, and mouse fibroblast DIF-3(+2) (Fig. 1D), which might be even more potent anti-
3T3-L1 cells (as a model for non-transformed cells) (Fig. 2) tumor agents for clinical use in cancer therapy.
and determined the IC50 for cell growth effects (Table 1). Both
Effects of DIF Derivatives on the Infection and Growth
DIF-3(+3) and Hex-DIF-3 significantly (p<0.01) suppressed cell of T. cruzi We next examined the effects of DIF derivatives
growth in all three cell lines. In addition, the growth-suppress- on the infection and growth of T. cruzi in HT1080 cells (Fig.
ing effects of DIF-3(+2) were greater than those of DIF-3(+3), 3). When added to the culture medium at 1µM, the clinical
whereas Hex-DIF-3 and Bu-DIF-3 did not differ in this regard. anti-T. cruzi agent BZL significantly (p<0.05) suppressed both
Although we need to more precisely assess the effects of these the infection and growth of T. cruzi. In comparison, DIF-3
derivatives on non-transformed cells, our current results suggest and Bu-DIF-3-rather than DIF-3(+3) or Hex-DIF-3—strongly
that, among DIF-3 derivatives with acyl group modifications, suppressed T. cruzi infection and growth at concentrations of
DIF-3(+1) or DIF-3(+2) might most strongly suppress tumor 0.1 to 1µM (p<0.05). Among the DIF derivatives tested, Bu-
cell growth and that Bu-DIF-3 or Hex-DIF-3 might be the most DIF-3 suppressed T. cruzi infection and growth most strongly,

Vol. 40, No. 11 (2017)
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1945
Fig. 3. Effects of DIF Derivatives on Infection and Growth of T. cruzi
Trypanosoma cruzi trypomastigotes were incubated for 3d with HT1080 cells in the presence of 0.1% DMSO (Control) or the indicated concentrations of BZL or DIF
derivatives, after which the infection rate (A) and number of amastigotes in the HT1080 cells (B) were assessed microscopically. Results are presented as the means and
S.D. (bars) of four independent experiments. *p<0.05 versus Control.
Fig. 4. Effects of DIF Derivatives on IL-2 Production in Jurkat T Cells
Jurkat cells were pre-incubated for 0.5h with 0.1% DMSO (Control) or 5µM DIF derivatives (A: DIF-3, DIF-3(+1), DIF-3(+2) and DIF-3(+3). B: Et-DIF-3, Bu-DIF-3
and Hex-DIF-3). After the pre-incubation, ConA (25µg/mL) was added and the cells were incubated for another 12h. IL-2 production and relative live cell number were
assessed as described in Materials and Methods. Results are presented as the means and S.D. (bars) of three independent experiments. * p<0.05; **p<0.01; ***p<0.001
versus Control.

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Biol. Pharm. Bull.
Vol. 40, No. 11 (2017)
suggesting that it, rather than Hex-DIF-3 or DIF-3(+3), should described previously.16–18)
be pursued as a lead compound for the development of anti-T.
DIF Derivatives as New Leads for Drug Development
In D. discoideum, DIF-1 accomplishes at least two physiologi-
cruzi agents, as described previously.22)
Effects of DIF Derivatives on IL-2 Production in Jurkat cal functions: the regulation of cell differentiation1,3,4) and the
T Cells We then examined the effects of DIF derivatives on modulation of chemotactic cell movement.31) DIF-3 was first
ConA-induced IL-2 production (CIIP) in Jurkat cells (Fig. 4) identified as a differentiation-inducing factor with modest
and determined the IC50 of Et-DIF-3, Bu-DIF-3 and Hex-DIF-3 activity2,32) and was later shown to be the initial product dur-
for CIIP (Table 1). At 5µM, both DIF-3(+1) and DIF-3(+2) sig- ing DIF-1 breakdown in D. discoideum.3,4,33) In comparison,
nificantly (p<0.05 and p<0.01, respectively) promoted CIIP, DIF-1, DIF-3, and their derivatives exert multiple biological
as described previously,19) whereas DIF-3(+3) at the same activities, including anti-tumor,6–9,11,12) immunoregulatory,19–21)
concentration had no effect on CIIP (Fig. 4A). In contrast, Et- and glucose-uptake promoting effects16–18) in mammalian cells.
DIF-3 and Bu-DIF-3 at 5µM significantly (p<0.05 and p<0.01, In addition, some derivatives of DIF-3 exhibit anti-T. cruzi
respectively) suppressed CIIP, as described previously,19,20) and activity.22) It is noteworthy that DIF derivatives with different
Hex-DIF-3 suppressed CIIP more strongly than did Bu-DIF-3 side chains often have different biological activities, suggest-
(Fig. 4B, Table 1). Overall, there was no strong relationship ing that, as a group, DIF derivatives offer many opportunities
between the CIIP suppressive activity and clogP or M.V. of for the development of novel medicines for diverse diseases.
each DIF derivative. However, the number of carbons at the
In this study, we investigated the biological activities of the
alkoxy group seemed to be important for biological activ- novel DIF derivatives DIF-3(+3) and Hex-DIF-3 and found
ity; among the compounds tested, the more carbons present, that both derivatives are promising lead compounds for the
the higher the compound’s activity. It is noteworthy that, at development of anti-cancer agents; Hex-DIF-3 may also be an
the concentration tested (5µM), the DIF derivatives had little effective lead for novel immunosuppressive drugs. For these
effect or, if any, a little effect on cell viability during the 12-h purposes, the relationship between the chemical structure and
incubation (Fig. 4B). Given that Hex-DIF-3 significantly sup- biological activities of likely compounds that have not yet
pressed the growth of 3T3-L1 cells (non-transformed cells) at been synthesized (such as those in Fig. 1D) should be ana-
an IC50 of 3.9µM after a 3-d incubation (Table 1), the thera- lyzed in detail by using various in vitro and in vivo systems.
peutic use of Hex-DIF-3 as an immunosuppressive drug might
lead to some adverse effects. Regardless, these results provide
insight into the development of novel drugs for the treatment
of inflammatory diseases.
CONCLUSION
In summery, we synthesized two derivatives of DIF-3,
Effects of DIF Derivatives on Glucose Consumption in DIF-3(+3) and Hex-DIF-3, and investigated their biological ac-
3T3-L1 Cells Given that DIF-1 and some of its derivatives tivities in vitro. The results revealed that DIF-3(+3) and Hex-
promote glucose consumption in mammalian cells,16–18) we DIF-3 are promising leads for the development of anti-cancer
examined the effects of DIF-1, DIF-3(+3), and Hex-DIF-3 on and immunosuppressive agents.
glucose consumption in confluent 3T3-L1 cells (Fig. 5). DIF-1
at 10µM significantly (p<0.01) promoted glucose consump-
Acknowledgments This work was supported in part
tion, as previously reported,16,17) whereas the two derivatives by Grants from the Japan Society for the Promotion of Sci-
of DIF-3 were not as potent as DIF-1. These results suggest ence (JSPS) KAKENHI (no. 15K07964 to YO and YK; no.
that DIF-1-rather than any DIF-3 derivative to date-may be a 16H03279 to HK); the Platform Project for Support in Drug
promising lead compound for a novel anti-diabetic agent, as Discovery and Life Science Research from the Japan Agency
for Medical Research and Development (AMED) (to HK and
YO); the Shorai Foundation for Science and Technology (to
HK); the Kobayashi International Scholarship Foundation (to
HK); and the Takeda Science Foundation (to HK).
Conflict of Interest Patents related to this article were
issued on 12 September 2014 (No. 5610433) and 17 October
2014 (No. 5630751) in Japan. Gunma University holds pat-
ent No. 5610433; YK and JNS are the inventors of the patent
(regarding anti-Trypanosoma agents). Gunma University and
Tohoku University hold patent No. 5630751; YK, HK, KT,
and YO are the inventors of the patent (regarding inhibitors of
IL-2 production in T cells).
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