Volume 40 (2017) Issue 11 Pages 1941-1947
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 between 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 1 µM in vitro were not as strong as those of other known DIF derivatives. In addition, Hex-DIF-3 at 5 µM 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 immunosuppressive agents.
The characteristic chlorinated alkylphenones differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) and DIF-3 (1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) (Fig. 1A) were originally identified as stalk-cell differentiation-inducing factors in the cellular slime mold Dictyostelium discoideum,1–4) an excellent model organism for studying cell and developmental biology.5) Recently, cellular slime molds have attracted interest as potential novel drug resources. To date, we and others have shown that DIF-1, DIF-3, and their derivatives suppress cell growth and occasionally induce or promote cell differentiation in both cancer cells and non-transformed (normal) mammalian cells.6–15) In addition, some DIF derivatives 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, some derivatives of DIF-3, specifically 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)-heptan-1-one (DIF-3(+2)) and 1-(3-chloro-2,6-dihydroxy-4-ethoxyphenyl)hexan-1-one (Bu-DIF-3) (Fig. 1A), have potent biological effects against tumors, Trypanosoma cruzi, and inflammation9,12,15,19,20,22,23); the protozoan T. cruzi causes Chagas disease (American trypanosomiasis) in humans. The differing pharmacological activities of various DIF derivatives might reflect specific modifications of their side chains. Taken together, these findings highlight the potential of DIF derivatives in the treatment of several diseases, including cancer, diabetes, inflammation, and trypanosomiasis. In particular, DIF-3(+2) and Bu-DIF-3 may be advantageous lead compounds for the development of anti-tumor, anti-T. cruzi, and anti-inflammatory drugs.
(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.
In the present study, to further assess the relationship between the biological effects of DIF-3 derivatives and their chemical structures, particularly in regard to the alkoxy group and the length of the alkyl chains at the acyl group, we synthesized two new derivatives, namely 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)octan-1-one (DIF-3(+3)) and Hex-DIF-3 (Figs. 1B, C). Here we show how the chemical structure of each DIF derivative affects its biological activity in vitro; these findings inform the effective design of DIF derivatives as novel drugs.
Aluminum (III) chloride (95 mg, 0.714 mmol) was added to a solution of 5-methoxyresorcinol (50 mg, 0.357 mmol) in dichloromethane (2 mL) at room temperature. After 15 min, nonanyl chloride (100 µL, 0.555 mmol) was added. The mixture was stirred for 3 h, poured into water (20 mL), and extracted three times with ethyl acetate (20 mL). The organic layer was washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and evaporated to dryness. The residue was chromatographed over silica gel and eluted by hexane–ethyl acetate (19 : 1) to give 1-(2,6-dihydroxy-4-methoxyphenyl)nonan-1-one (70 mg, 0.250 mmol [yield, 70%]). The spectral data of the eluate were identical to those reported previously for 1-(2,6-dihydroxy-4-methoxyphenyl)nonan-1-one.21)
Sulfuryl chloride (22 µL, 0.267 mmol) and ethanol (60 µL) were added to a solution of 1-(2,6-dihydroxy-4-methoxyphenyl)nonan-1-one (50 mg, 0.178 mmol) in chloroform (3 mL) at room temperature. After being stirred for 4 h, the mixture was evaporated to dryness. The residue was chromatographed over silica gel and eluted by using hexane–ethyl acetate (9 : 1) to generate DIF-3(+3) (23 mg, 0.072 mmol [yield, 41%]). Data for DIF-3(+3): yellowish amorphous solid; 1H-NMR (600 MHz, CDCl3), δ 13.66 (1H, br s), 6.75 (1H, br s), 6.13 (1H, s), 3.90 (3H, s), 3.08 (2H, t, J=7.5 Hz), 1.69 (2H, quint, J=7.5 Hz), 1.25–1.40 (10H, m), 0.88 (3H, t, J=6.9 Hz); 13C-NMR (150 MHz, 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, 14.1; electron ionization (EI)-MS m/z 316 [M+2]+ (13%), 314 [M+] (37%), 296 (32%), 229 (39%), 216 (56%), 201 (100%); high resolution (HR)-EI-MS m/z 314.1294 [M]+ (314.1285 Calcd for C16H23O435Cl).
1-Bromohexane (190 µL, 1.35 mmol) and potassium carbonate (701 mg, 5.08 mmol) were added to a solution of phloroglucinol (100 mg, 0.793 mmol) in dimethyl formamide (4 mL). After being stirred for 20 h at room temperature, the mixture was poured into 0.5 M hydrochloric acid (20 mL) and extracted three times with ethyl acetate (20 mL). The organic layer was washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and evaporated to dryness. The residue was chromatographed over silica gel and eluted by using hexane–ethyl acetate (3 : 1) to give 5-butoxyresorcinol (53.6 mg, 0.255 mmol [yield, 32%]). Data for 5-hexyloxyresorcinol: colorless oil; 1H-NMR (600 MHz, acetone-d6) δ: 8.17 (2H, br s), 5.95 (1H, t, J=2.2 Hz), 5.92 (2H, d, J=2.2 Hz), 3.86 (2H, t, J=6.3 Hz), 1.66–1.73 (2H, m), 1.40–1.48 (2H, m), 1.29–1.37 (4H, m), 0.89 (3H, t, J=7.1 Hz); 13C-NMR (150 MHz, acetone-d6) δ: 162.1, 160.0 (2C), 96.2, 94.4 (2C), 68.2, 32.3, 30.0, 26.4, 23.2, 14.3; EI-MS m/z 210 [M+] (33%), and 126 (100%); and HR-EI-MS m/z 210.1245 [M]+ (210.1256 Calcd. for C12H18O3).
Aluminum (III) chloride (38 mg, 0.286 mmol) was added to a solution of 5-hexyloxyresorcinol (30 mg, 0.143 mmol) in dichloromethane (2 mL) at room temperature. After 15 min, hexanoyl chloride (30 µL, 0.215 mmol) was added. The mixture was stirred for 3 h, poured into water (10 mL), and extracted three times with ethyl acetate (10 mL). The organic layer was washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and evaporated. The residue was chromatographed over silica gel and eluted by using hexane–ethyl acetate (9 : 1) to give 1-(2,6-dihydroxy-4-hexyloxyphenyl)hexan-1-one (26 mg, 0.084 mmol [yield, 59%]). Data for 1-(2,6-dihydroxy-4-hexyloxyphenyl)hexan-1-one: colorless amorphous solid; 1H-NMR (600 MHz, CDCl3) δ: 10.05 (2H, br s), 5.91 (2H, s), 3.93 (2H, t, J=6.6 Hz), 3.05 (2H, t, J=7.5 Hz), 1.71–1.78 (2H, m), 1.66–1.71 (2H, m), 1.40–1.46 (2H, m), 1.31–1.39 (8H, m), 0.91 (3H, t, J=7.1 Hz), 0.90 (3H, t, J=7.1 Hz); 13C-NMR (150 MHz, CDCl3) δ: 206.2, 165.0 (2C), 163.2, 104.7, 94.8 (2C), 68.3, 43.9, 31.6, 31.5, 28.9, 25.6, 24.4, 22.6, 22.5, 14.0 (2C); EI-MS m/z 308 [M+] (51%), 290 (19%), 265 (40%), 252 (20%), 237 (100%), 168 (52%), and 153 (50%); and HR-EI-MS m/z 308.2004 [M]+ (308.1988 Calcd. for C18H28O4).
Sulfuryl chloride (2 µL, 0.023 mmol) and ethanol (20 µL) were added to a solution of 1-(2,6-dihydroxy-4-hexyloxyphenyl)hexan-1-one (14 mg, 0.045 mmol) in chloroform (1 mL) at room temperature.
After being stirred for 2 h, the mixture was evaporated. The residue was chromatographed over silica gel eluted by using hexane–ethyl acetate (9 : 1) to give Hex-DIF-3 (6.3 mg, 0.018 mmol [yield, 41%]). Data for Hex-DIF-3: yellowish amorphous solid; 1H-NMR (600 MHz, CDCl3) δ: 13.65 (1H, br s), 6.66 (1H, br s), 6.09 (1H, s), 4.02 (2H, t, J=6.6 Hz), 3.06 (2H, t, J=7.4 Hz), 1.77–1.84 (2H, m), 1.65–1.72 (2H, m), 1.42–1.49 (2H, m), 1.30–1.37 (8H, m), 0.92 (3H, t, J=7.2 Hz), 0.91 (3H, t, J=7.2 Hz); 13C-NMR (150 MHz, CDCl3) δ: 205.9, 164.9, 159.7, 154.1, 104.5, 99.8, 94.1, 69.4, 43.9, 31.6, 31.4, 28.7, 28.6, 24.2, 22.6, 22.5, 13.99, 13.97; EI-MS m/z 344 [M+2]+ (33%), 342 [M+] (94%), 324 (36%), 299 (63%), 286 (45%), 271 (90%), 240 (66%), 202 (65%), 187 (100%); HR-EI-MS m/z 342.1608 [M]+ (342.1598 Calcd. for C18H27O435Cl).
To estimate the membrane permeability of each compound, its hydrophobic index (C log P) was calculated by using ChemDraw 10.0 software (CambridgeSoft, Cambridge, MA, U.S.A.). M.V. was calculated by using the website Molinspiration.24)
In addition to DIF-3(+3) and Hex-DIF-3, several DIF derivatives were synthesized as described.15) The anti-T. cruzi drug benznidazole (BZL: N-benzyl-2-nitro-1H-imidazole-1-acetamide; Sigma-Aldrich, St. Louis, MO, U.S.A.) was kindly provided by Dr. Yutaka Suto (Takasaki University of Health and Welfare, Japan). FK506 (tacrolimus) was purchased from Calbiochem (San Diego, CA, U.S.A.), and concanavalin A (ConA) was from Seikagaku Corporation (Tokyo, Japan).
Human cervical cancer HeLa cells and murine fibroblast 3T3-L1 cells were grown and maintained at 37°C (5% CO2 in air) in Dulbecco’s modified Eagle’s medium (DMEM) (catalog no. D5796, Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS).16,23) Murine osteosarcoma LM8 cells25) were grown and maintained at 37°C (5% CO2 in air) in MEM-α (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% FBS.26) Human T-lymphocyte Jurkat cells were maintained at 37°C (5% CO2) in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% (v/v) FBS.19,20) Human fibrosarcoma HT1080 cells (Japan Health Sciences Foundation, Tokyo, Japan) were used as the in vitro hosts for T. cruzi. The Tulahuen strain of T. cruzi27) and HT1080 cells were maintained and passaged in DMEM-FBS as described.22,28,29)
All media also contained 75 µg/mL penicillin and 50 µg/mL streptomycin.
LM8 (2.5×103 cells/well), 3T3-L1 (5×103 cells/well), or HeLa cells (5×103 cells/well) were incubated for 3 d in 12-well plates, with each well containing 1 mL of MEM-α-FBS (LM8) or DMEM-FBS (3T3-L1 and HeLa) and 10 µM of one of the DIF derivatives or 0.2% (v/v) dimethyl sulfoxide (DMSO). After the 3-d incubation, the spent medium was discarded and replaced with 1 mL of fresh medium without the derivative or DMSA but containing 5% (v⁄v) Alamar blue (a vital cell indicator; Wako Pure Chemical Industries, Ltd.) until the color of the medium changed. Relative live cell number was assessed by measuring absorbance at a wavelength of 570 nm (reference, 595 nm), as described previously.9,23)
To determine the IC50 of each compound in regard to cell proliferation, cells were cultured for 3 d in the presence of various concentrations of each compound. Relative live cell numbers were determined by using Alamar blue, and the IC50 value was determined from the dose–response curve drawn by using the average values of three independent experiments.
Anti-T. cruzi activities of DIF derivatives were investigated as described.22) Briefly, a round coverslip (12 mm) was placed in each well of a 24-well plate (Corning, Corning, NY, U.S.A.); exponentially growing HT1080 cells (5×103 cells) were added to each well and incubated at 37°C (5% CO2) for 1 d. The cells were infected with T. cruzi trypomastigotes (2×105 per well) as described,28) and BZL or a DIF derivative (final concentration: 0.1 or 1 µM) was added immediately. After 3 d, the rate of infection of host cells with T. cruzi was assessed as described.22,30) The percentage of infected host cells (i.e., cells containing multiple amastigotes) and the mean number of amastigotes per infected cell were determined by assessing at least 200 host cells.
Jurkat cells (1×106 cells/mL) were pre-incubated (37°C, 5% CO2 in air) for 0.5 h in 12-well culture plates containing 1 mL RPMI medium and 5 µM DIF derivative or 0.1% DMSO (vehicle). After the 0.5-h preincubation, ConA (final concentration, 25 µg/mL) was added to each culture, and the cells were further incubated for 12 h. Aliquots of the culture media were collected, and the level of IL-2 was assessed by using immunoassay kits (Endogen, Rockford, IL, U.S.A.), as described previously.19,20) In addition, cell viability was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.19,20)
To determine the IC50 of each compound in regard to the ConA-induced IL-2 production, cells were cultured for 12 h in the presence of various concentrations of each compound. Relative IL-2 production was determined by using immunoassay kits, and the IC50 value was determined from the dose–response curve drawn by using the average values of three independent experiments.
Confluent 3T3-L1 cells in 12-well plates were incubated for 10–15 h with 10 µM DIF derivatives, and glucose consumption was measured as described previously.16)
Welch’s t-test (two-tailed) was performed for the statistical analyses. Values were considered to be significantly different when the p value was less than 0.05.
We chemically synthesized the novel DIF-3 derivatives DIF-3(+3) and Hex-DIF-3 as described in Materials and Methods (Figs. 1B, C). We designed these derivatives to explore the chemical structure–biological activity relationship (particularly the importance of the alkyl chains) of DIF-3 derivatives.
We first evaluated the effects of various DIF derivatives on cell growth in human cervical cancer HeLa cells, mouse osteosarcoma LM8 cells, and mouse fibroblast 3T3-L1 cells (as a model for non-transformed cells) (Fig. 2) and determined the IC50 for cell growth effects (Table 1). Both DIF-3(+3) and Hex-DIF-3 significantly (p<0.01) suppressed cell growth in all three cell lines. In addition, the growth-suppressing effects of DIF-3(+2) were greater than those of DIF-3(+3), whereas Hex-DIF-3 and Bu-DIF-3 did not differ in this regard. Although we need to more precisely assess the effects of these derivatives on non-transformed cells, our current results suggest that, among DIF-3 derivatives with acyl group modifications, DIF-3(+1) or DIF-3(+2) might most strongly suppress tumor cell growth and that Bu-DIF-3 or Hex-DIF-3 might be the most tumor-growth-suppressive among the DIF-3 derivatives with alkoxy group modifications. Table 1 also shows the hydrophobic index (c log P) and calculated M.V. of each compound; although there was no strong relationship between antiproliferative activity and the values, the appropriate number of carbons at the acyl group and alkoxy group seemed to be important for activity. Building on the current results, we might next synthesize additional DIF-3 derivatives, such as Bu-DIF-3(+1) and Hex-DIF-3(+2) (Fig. 1D), which might be even more potent anti-tumor agents for clinical use in cancer therapy.
HeLa (A), LM8 (B), or 3T3-L1 (C) cells were incubated for 3 d with 0.1% DMSO (Control) or 10 µM 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.
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 (c log P value) and molecular volume (M.V.) of each compound, were determined as described in Materials and Methods.
We next examined the effects of DIF derivatives on the infection and growth of T. cruzi in HT1080 cells (Fig. 3). When added to the culture medium at 1 µM, the clinical anti-T. cruzi agent BZL significantly (p<0.05) suppressed both the infection and growth of T. cruzi. In comparison, DIF-3 and Bu-DIF-3-rather than DIF-3(+3) or Hex-DIF-3—strongly suppressed T. cruzi infection and growth at concentrations of 0.1 to 1 µM (p<0.05). Among the DIF derivatives tested, Bu-DIF-3 suppressed T. cruzi infection and growth most strongly, suggesting that it, rather than Hex-DIF-3 or DIF-3(+3), should be pursued as a lead compound for the development of anti-T. cruzi agents, as described previously.22)
Trypanosoma cruzi trypomastigotes were incubated for 3 d 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.
We then examined the effects of DIF derivatives on ConA-induced IL-2 production (CIIP) in Jurkat cells (Fig. 4) and determined the IC50 of Et-DIF-3, Bu-DIF-3 and Hex-DIF-3 for CIIP (Table 1). At 5 µM, both DIF-3(+1) and DIF-3(+2) significantly (p<0.05 and p<0.01, respectively) promoted CIIP, as described previously,19) whereas DIF-3(+3) at the same concentration had no effect on CIIP (Fig. 4A). In contrast, Et-DIF-3 and Bu-DIF-3 at 5 µM significantly (p<0.05 and p<0.01, respectively) suppressed CIIP, as described previously,19,20) and Hex-DIF-3 suppressed CIIP more strongly than did Bu-DIF-3 (Fig. 4B, Table 1). Overall, there was no strong relationship between the CIIP suppressive activity and c log P or M.V. of each DIF derivative. However, the number of carbons at the alkoxy group seemed to be important for biological activity; among the compounds tested, the more carbons present, the higher the compound’s activity. It is noteworthy that, at the concentration tested (5 µM), the DIF derivatives had little effect or, if any, a little effect on cell viability during the 12-h incubation (Fig. 4B). Given that Hex-DIF-3 significantly suppressed the growth of 3T3-L1 cells (non-transformed cells) at an IC50 of 3.9 µM after a 3-d incubation (Table 1), the therapeutic 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.
Jurkat cells were pre-incubated for 0.5 h with 0.1% DMSO (Control) or 5 µM 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 (25 µg/mL) was added and the cells were incubated for another 12 h. 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.
Given that DIF-1 and some of its derivatives promote glucose consumption in mammalian cells,16–18) we examined the effects of DIF-1, DIF-3(+3), and Hex-DIF-3 on glucose consumption in confluent 3T3-L1 cells (Fig. 5). DIF-1 at 10 µM significantly (p<0.01) promoted glucose consumption, as previously reported,16,17) whereas the two derivatives of DIF-3 were not as potent as DIF-1. These results suggest that DIF-1-rather than any DIF-3 derivative to date-may be a promising lead compound for a novel anti-diabetic agent, as described previously.16–18)
Confluent 3T3-L1 cells were incubated for 10–15 h with 0.1% DMSO (Control) or 10 µM DIF derivatives, and the relative rates of glucose consumption were assessed. Results are presented as the mean values and S.D. (bars) of three independent experiments. * p<0.05; ** p<0.01 versus Control.
In D. discoideum, DIF-1 accomplishes at least two physiological functions: the regulation of cell differentiation1,3,4) and the modulation of chemotactic cell movement.31) DIF-3 was first identified as a differentiation-inducing factor with modest activity2,32) and was later shown to be the initial product during DIF-1 breakdown in D. discoideum.3,4,33) In comparison, DIF-1, DIF-3, and their derivatives exert multiple biological activities, including anti-tumor,6–9,11,12) immunoregulatory,19–21) and glucose-uptake promoting effects16–18) in mammalian cells. In addition, some derivatives of DIF-3 exhibit anti-T. cruzi activity.22) It is noteworthy that DIF derivatives with different side chains often have different biological activities, suggesting that, as a group, DIF derivatives offer many opportunities for the development of novel medicines for diverse diseases.
In this study, we investigated the biological activities of the novel DIF derivatives DIF-3(+3) and Hex-DIF-3 and found that both derivatives are promising lead compounds for the development of anti-cancer agents; Hex-DIF-3 may also be an effective lead for novel immunosuppressive drugs. For these purposes, the relationship between the chemical structure and biological activities of likely compounds that have not yet been synthesized (such as those in Fig. 1D) should be analyzed in detail by using various in vitro and in vivo systems.
In summery, we synthesized two derivatives of DIF-3, DIF-3(+3) and Hex-DIF-3, and investigated their biological activities in vitro. The results revealed that DIF-3(+3) and Hex-DIF-3 are promising leads for the development of anti-cancer and immunosuppressive agents.
This work was supported in part by Grants from the Japan Society for the Promotion of Science (JSPS) KAKENHI (no. 15K07964 to YO and YK; no. 16H03279 to HK); the Platform Project for Support in Drug 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).
Patents related to this article were issued on 12 September 2014 (No. 5610433) and 17 October 2014 (No. 5630751) in Japan. Gunma University holds patent 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).