Development of a Water Soluble Self-assembling Analogue of Vizantin

Abstract

Vizantin, 6,6′-bis-O-(3-nonyldodecanoyl)-α,α′-trehalose, has been developed as a safe immunostimulator on the basis of a structure–activity relationship study with trehalose 6,6′-dicorynomycolate. Our recent study indicated that vizantin acts as an effective Toll-like receptor-4 (TLR4) partial agonist to reduce the lethality of an immune shock caused by lipopolysaccharide (LPS). However, because vizantin has low solubility in water, the aqueous solution used in in vivo assay systems settles out in tens of minutes. Here, vizantin was chemically modified in an attempt to facilitate the preparation of an aqueous solution of the drug. This paper describes the concise synthesis of a water-soluble vizantin analogue in which all the hydroxyl groups of the sugar unit were replaced by sulfates. The vizantin derivative displayed micelle-forming ability in water and potent TLR-4 partial agonist activity.

Introduction

Septic shock is a severe condition caused by lipopolysaccharide (LPS), which is an outer membrane component of Gram-negative bacteria.^1–5) LPS interacts with the Toll-like receptor-4 (TLR-4)/MD2 protein complex on immune cells, and triggers immune responses through the release of various cytokines and chemokines.^6–10) In particular, LPS strongly induces tumor necrosis factor-α (TNF-α) that causes a cytokine storm,^11,12) which is related to septic shock. Because septic shock and/or Gram-negative sepsis are fatal in approximately 30–40% of patients, TLR antagonists for LPS have thus far attracted considerable interest as potential treatments for these methods of sepsis.^13–16)

Recently, we have developed vizantin (1),^17,18) 6,6′-bis-O-(3-nonyldodecanoyl)-α,α′-trehalose, as a safe immunostimulator on the basis of a structure–activity relationship study with trehalose 6,6′-dicorynomycolate (TDCM) (2)^19–24) that was first characterized as a structural component of the outer surface membrane of Corynebacterium diphtheriae (Fig. 1). Like LPS, vizantin also interacts with the TLR4/MD2 protein complex on immune cells. Unlike LPS, however, vizantin induces little TNF-α. Focusing on the difference between LPS and vizantin, our research demonstrated that vizantin represses TNF-α production from THP-1 cells triggered by Escherichia (E.) coli LPS in a dose-dependent manner.²⁵⁾ Furthermore, the simultaneous administration of vizantin and LPS to C57BL/6 mice significantly increased survival rate by comparison to a single administration of LPS. These promising experimental findings based on in vivo assays with mice exceeded our expectations.

Fig. 1. Vizantin (1) and Trehalose 6,6′-Dicorynomycolate (TDCM)

However, special attention was needed to prepare the injection of vizantin, which displays poor solubility in water. The vizantin component in the aqueous solution readily precipitated within several tens of minutes even when using permitted emulsifiers such as Tween 80 and 5% mannitol. Accordingly, the injection solution of vizantin needed to be prepared immediately prior to administration. In this study, we attempted to develop a readily dissolvable vizantin derivative to simplify its therapeutic application for injection.

Results and Discussion

We reasoned that replacing all the hydroxyl groups in the sugar unit of 1 to sulfates would improve the solubility of vizantin in water (Fig. 2). The Clog P value of the designed 2,2′,3,3′,4,4′-hexakis-O-sulfated vizantin derivative 3 was 15.66, which suggested that 3 may still be difficult to dissolve in the monomer state. However, we anticipated that 3 might act as an ionic surfactant, which would homogeneously disperse in water by micelle formation.

Fig. 2. Plan to Improve the Solubility of Sulfated Vizantin Derivative 3 by Self-assembly (Micelle Formation)

Initially, the sulfation of vizantin was carried out in an arbitrary manner with a large excess amount of pyridine-sulfur trioxide complex (SO₃·Py) in N,N-dimethylformamide (DMF) for 4 h at room temperature (Chart 1). Triethylamine and methanol were added to the reaction mixture, which was then subject to size-exclusion chromatography (SEC) with Sephadex^® LH-20 to remove excessive SO₃·Py. After freeze-drying, the resulting crude product of 4 was treated with an ion-exchange resin (Na⁺ form) in H₂O for 12 h to convert the substituted ammonium sulfate groups (−OSO₃H·NEt₃) to the sodium salt isomer (−OSO₃Na). Finally, after removing the ion-exchange resin, the mixture was subjected to SEC with Sephadex^® G-25 to give the desired 3.^26–31) However, the overall yield of 3 from vizantin was only 29%. Thus, we embarked on improving the synthesis method in more detail (Table 1).

Chart 1. Previous Experimental Methods for Preparing Sulfated Vizantin Derivative 3

Table 1. Optimization of After-Treatment for the Sulfation Reaction of 1

Entry	SO3·NMe3 (eq)	Time (h)	Treatment	Yield (%)a)
1	24	48	Method A	17
2	30	12	Method A	19
3	30	12	Method B (MeOH)b)	14
4	30	12	Method B (EtOH)c)	16
5	30	12	Method C	39

a) Isolated yield of 3. b) MeOH was used to precipitate the product. c) EtOH was used to precipitate the product. d) Each reaction was performed on a 100 mg scale.

Based on reports describing the sulfation of trehalose 6,6′-diesters resembling vizantin,³²⁾ we initially used 24 eq of SO₃·NMe₃ in DMF (entry 1). The sulfation reaction gradually proceeded at 50 °C leading to the disappearance of starting material within 48 h. The product was subsequently purified according to a previous report (Method A in Chart 2). Specifically, 10% Na₂CO₃ aqueous solution and then MeOH were added to the reaction mixture and resulting precipitate (a mixture of sulfated vizantin and inorganic salts) was recovered by filtration. The precipitate was washed with MeOH and then purified by SEC. However, the isolated yield of 3 was only 17%. Even using 30 eq of SO₃·NMe₃ gave a similar result as that of the 24 eq of SO₃·NMe₃, although the reaction time was reduced to 12 h (entry 2). The low yield could have resulted from poor extraction of product from the reaction mixture. In an attempt to improve the efficiency of extraction, ion-exchange resin was used instead of 10% Na₂CO₃ aqueous solution (Method B). Ion-exchange resin and distilled water were added to the reaction mixture, which was stirred for 12 h. The resulting mixture was filtered to remove the ion-exchange resin, and MeOH slowly added to the filtrate to precipitate the product.

Chart 2. Optimization of the Reaction Conditions of 1

However, this procedure only gave 3 in 14% yield (entry 3). Replacing MeOH with EtOH made no significant difference (entry 3 vs. 4). After several trials of different extraction procedures, we found that the most suitable protocol involved direct addition of AcONa-saturated EtOH solution into the reaction mixture (Method C). This procedure gave the highest yield of isolated 3 (entry 5). The addition of AcONa-saturated EtOH promoted the sedimentation of a byproduct Na₂SO₄ along with 3, but this mixture could be readily separated by SEC with Sephadex^® G-25 to give 3 as a pure product in 39% yield. Moreover, by comparison to the alternative methodologies the addition of AcONa-saturated EtOH accelerated the precipitation of 3.

Next, the optimal condition for the sulfation of 1 to 3 was investigated (Table 2). AcONa-saturated EtOH was used for after-treatment in all cases. Use of SO₃·NEt₃ (30 equivalent (equiv.)) in DMF at 50 °C furnished 3 in moderate yield (entry 1). Application of pyridine complex (SO₃·Py) gave similar results to SO₃·NEt₃ (entry 2). Among the surveyed SO₃ reagents, SO₃·DMF complex afforded the best result with an isolated yield for 3 of 70% (entry 3). The reaction time was also shortened to 4 h. Indeed, using 40 eq of SO₃·DMF complex allowed the reaction to reach completion within 2 h (entry 4). However, the final yield of 3 was no different from that of entry 3. Conducting the reaction at elevated temperature resulted in a slightly reduced yield (entry 5).

Table 2. The Screening of SO₃ Reagents for the Sulfation Reaction of 1

Entry	SO3 reagent (equiv.)	Temp. (°C)	Time (h)	Yield (%)a)
1	SO3·Et3N (30)	50 °C	7	38
2	SO3·Py (30)	50 °C	6	44
3	SO3·DMF (30)	50 °C	4	70
4	SO3·DMF (40)	50 °C	2	71
5	SO3·DMF (30)	100 °C	2	67

a) Isolated yield of 3. b) Each reaction was performed on a 100 mg scale.

Using the SO₃·DMF complex under the best conditions we attempted to synthesize 3 from naked trehalose in a one-pot reaction. However, there was a lack of regioselectivity for the primary alcohols of 5 in the esterification of 7 (Chart 3). The existence of the desired product 3 was confirmed by TLC, but the product was difficult to isolate. After carefully surveying the combination of 7 with trehalose derivatives, the synthesis of 3 was achieved by using a trimethylsilyl (TMS)-protected derivative 6.³³⁾ The overall yield was 53% from 6 without isolation of 1 until the final stage. Having established a concise synthesis of 3 from 6, we prepared a test sample on a scale of tens of grams.

Chart 3. The Synthesis of 3 from a Trehalose Derivative

Next, the solubility of 3 was investigated. In the previous in vivo assay, 0.1–1.0 mM suspensions of vizantin were used for injection.²⁵⁾ Accordingly, 3 was dissolved in distilled water (DW) and saline over a wide range of concentrations from 3 µM. Figure 3 shows the solution states of 3 at concentrations >5.0 mM. Solubility was assessed by optical density measurements using a wavelength of 620 nm. 3 showed good solubility in DW, and no significant change was observed up to 25.0 mM by comparison with a blank sample. When saline was used as the aqueous medium, the solubility of 3 was slightly decreased and 3 began to precipitate at 17.5 mM. Nonetheless, taking into consideration the salting-out effect, compound 3 showed good solubility in saline.

Fig. 3. The Solution States of 3 in Distilled Water and Saline

Optical density (OD) was measured at a wavelength of 620 nm.

As shown in Figs. 4A and 4C, Tyndall scattering was clearly observed for 5.0 mM solutions of 3 in both DW and saline, respectively. These findings supported our hypothesis that 3 can act as an ionic surfactant to form a molecular aggregate such as a micelle. Therefore, the critical micelle concentration (CMC) values of 3 in DW (0.32 mM) and saline (0.03 mM) were determined using a CMC assay kit.

Fig. 4. A and C: Tyndall Scattering Displayed by 3 at a Concentration of 5.0 mM in Distilled Water and Saline, Respectively; B and D: Size Distribution of 3 at a Concentration of 5.0 mM in Distilled Water and Saline, Respectively

Next, these apparent sizes at 5 mM in each solvent were measured as a hard sphere by a dynamic light scattering method. Figures 4B and 4D show the apparent size distributions in DW and saline. In DW, three peaks were detected in the size range of 2 to 1000 nm. The major components comprised it with an average diameter of 507.3 nm (peak III), which accounted for 64% of the total signal. In saline, two peaks appeared under the same conditions as that observed in DW. The main distribution (peak I) was in the range of 10 to 100 nm and occupied 81% of the total signal. This result indicated that the saline solution of 3 showed a greater degree of uniformity in terms of size than the corresponding solution in DW.

Transmission electron microscopy (TEM) was used to examine the shape of the formed molecular aggregates (Fig. 5). In both methods of medium, 3 was found to form string-like micelles. Moreover, the micelles tended to stack together to generate multilayered structures, particularly in DW. These TEM images were consistent with the experimentally determined micelle size distributions in DW and saline. Taken together, these findings demonstrated that saline is a more suitable aqueous medium than DW for preparing a finer colloidal solution of 3.

Fig. 5. A and B Show TEM Images of the Molecular Aggregates at 5.0 mM of 3 in Distilled Water and Saline, Respectively

Because 3 showed desirable solubility in saline at concentrations below 15 mM (Fig. 3), its efficacy was also evaluated by focusing on the inhibitory effect of LPS-induced release of TNF-α. To best compare the effects of 3 and vizantin, the expression level of mRNA related to TNF-α production was measured (Fig. 6). When THP-1 cells exposed to LPS were treated with a 10 µM saline solution of 3 the mRNA expression level of TNF-α was significantly suppressed. Under the same experimental conditions, vizantin showed a weaker effect than that of 3.

Fig. 6. Inhibitory Effect of LPS-Induced TNF-α with Vizantin (1) and 3

Vizantin was dissolved in DMSO and then diluted with distilled water. THP-1 cells were incubated at 37 °C for 1 h. mRNA levels were measured using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Data represent mean ± standard error (S.E.) (n = 3). ^††p < 0.01, compared to control (no LPS). ** p < 0.01, compared to vehicle (DMSO only).

Sulfated vizantin 3 showed no cytotoxicity against THP-1 cells even at a concentration of 50 µM (Fig. 7). Therefore, the findings in Fig. 6 suggested that 3 inhibited the binding of LPS to the TLR4/MD2 protein complex more effectively than vizantin (1). To further investigate the potent inhibitory effect of 3 compared to vizantin, docking simulations of 3 with TLR4/MD2 protein complex were conducted.^12–16) These results are shown in Fig. 8. A is the simulation result under normal conditions. One of the fatty acid side chains of 3 projected outside the cavity of the MD2 protein, while the remaining three side chains were located inside the cavity. The stabilizing energy of the formed complex of 3 and MD2 protein was −105.45 kcal/mol, which was significantly greater than that of the complex of vizantin and MD2 protein (−31.13 kcal/mol).²⁵⁾ By limiting the target site of 3 to the cavity on the MD2 protein, all of the side chains fitted inside the cavity (B). However, the stabilizing energy of the resulting complex decreased to −41.18 kcal/mol. Although we do not consider that these simulations accurately reflect the real effects, the simulations correlated well with the experimental results of the inhibitory activity of LPS-induced TNF-α release.

Fig. 7. Cytotoxicity of 3 to THP-1 Monocytes and THP-1 Macrophages

THP-1 monocytes and THP-1 macrophages were exposed for 24 h to different concentrations of 3 (1–50 µM), and then respective cell viability was measured by MTT assay. For controls, saline was used. Data represent mean ± S.E. (n = 4).

Fig. 8. Docking Simulation Analysis of 3 with MD2 Using MOE Software

The purple and dark green areas indicate hydrophilic and hydrophobic surfaces, respectively. The light gray, gray, red, and yellow parts of the compound indicate hydrogen, carbon, oxygen, and sulfur atoms, respectively.

Conclusion

A derivative of vizantin with hexasulfate groups (3) was developed as an TLR-4 partial agonist to prevent septic shock. TEM images as well as Tyndall scattering suggests 3 acts as an anionic surfactant to dissolve in water by forming string-like micelles. A detailed investigation of the mechanism of micelle formation and its adjuvant properties are currently underway. The present findings indicate that 3 is a promising candidate therapeutic drug for treating septic shock and/or Gram-negative sepsis.

Experimental

General Information

Reactions were performed under argon unless otherwise stated. Commercially available chemicals were used as purchased without further purification. Specialty grade and dehydrated solvents were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Progress of the reactions was monitored using thin layer chromatography with precoated silica gel 60 F254 plates (Merck, Darmstadt, Germany). Spots were visualized under UV illumination at 254 nm after immersion in 2% p-anisaldehyde and 5% H₂SO₄ in EtOH, followed by heating on a hot plate. Solvents were removed under reduced pressure using a standard rotary evaporator. Flash column chromatography was performed using 63–210 mesh silica gel 60 (Kanto Chemical Co., Inc.). Fourier transform (FT)-IR spectra were measured using a JASCO FT/IR-410 infrared spectrophotometer (JASCO, Tokyo, Japan) and significant peaks reported in wavenumbers (cm⁻¹). ¹H-NMR and ¹³C-NMR spectra were recorded on a 300-MR spectrometer, 500-MR spectrometer (Varian, Palo Alto, CA, U.S.A.). The ¹H chemical shifts are reported in parts per million (ppm) from an internal standard of TMS (0.00 ppm), residual CHCl₃ (7.26 ppm), d₆-dimethylsulfoxide (DMSO) (2.50 ppm), and the ¹³C chemical shifts are reported using an internal standard of TMS (0.00 ppm), CDCl₃ (77.03 ppm, central peak), d₆-DMSO (39.50 ppm, central peak). The ¹H-NMR spectra are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet br = broad), relative integral, coupling constants J (Hz). High-resolution mass spectra (HRMS) were recorded on a JEOL M-station JMS-700 (JEOL, Tokyo, Japan). Specific rotations were determined using the sodium D line (λ = 589 nm) at specific temperatures and are reported as follows: [α]_D^temp., concentration (c = g/100 mL), and solvent. The mean secondary particle size of dynamic light scattering was recorded on Zetasizer Nano ZS (Malvern Panalytical Ltd., Malvern, U.K.) and was obtained by cumulant analysis. Samples for transmission electron microscopy were prepared using the freeze-fracture replica method³⁴⁾ and then images recorded on a JEM-1400 Flash (JEOL) instrument with an acceleration voltage of 140 kV. The docking simulation study of 2,2′,3,3′4,4′-hexakis-O-sulfaneted vizantin 3 was carried out using Molecular Operating Environment software (MOE 2020.09, Chemical Computing Group, Montreal, Canada). The X-ray crystallographic structures of MD2 (PDB ID: 2E59) and TLR4/MD2 complex (PDB ID: 3FXI) were used. The minimizations of MD2 and TLR4/MD2 complex were performed using MMFF94s force-field. The active sites of MD2 and TLR4/MD2 complex were identified using MOE a Site Finder. The docking simulations with compound 3 and these proteins (Fig. 7) were performed using ASE Dock program (MOLSIS lnc., Tokyo, Japan).

The critical micelle concentration (CMC) value in DW and saline were determined by CMC assay kit (ProFoldin, Hudson, U.S.A.).

The Preparation of 2,2′,3,3′4,4′-Hexakis-O-sulfaneted Vizantin 3 and 2,2′,3,3′,4,4′-Hexakis-O-(trimethylsilyl)-vizantin S1 from 2,2′,3,3′4,4′-Hexakis-O-(trimethylsilyl)-α, α-Trehalose (6)

3-Nonyldodecanoic acid (7)¹⁸⁾ (10.0 g, 30.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI·HCl) (12.2 g, 63.6 mmol) and 4-dimethylamino pyridine (DMAP) (311 mg, 2.55 mmol) were dissolved in CH₂Cl₂ (43 mL) at room temperature. After the mixture was stirred for 1 h at room temperature, 2,2′,3,3′,4,4′-hexakis-O-(trimethylsilyl)-α,α-trehalose³³⁾ (6) (9.89 g, 12.8 mmol) was added to the mixture at room temperature. The resulting mixture was stirred for 19 h at room temperature and then concentrated with CH₂Cl₂ under reduced pressure. The residue was dissolved with Et₂O and then distilled water was added. The organic phase was separated and washed three times with a 1.0 M NaOH aqueous solution and three times with a saturated NaCl aqueous solution. The separated organic phase was dried over MgSO₄ and concentrated in vacuo to give 2,2′,3,3′,4,4′-hexakis-O-(trimethylsilyl)-vizantin S1 as a yellow syrup (21 g) (Step A).

Next, the syrup obtained in step A was dissolved in MeOH/CH₂Cl₂ (1 : 1, 120 mL). Amberlite^® IR120 (H⁺ form) was added at room temperature until the mixture reached pH 2. The resulting suspension was stirred for 2 h at room temperature and then filtered. The filtrate was concentrated in vacuo to give vizantin (1) as a yellow syrup (10 g) (Step B).

Next, the residue obtained in step B was mixed with sulfur trioxide N,N-dimethylformamide complex (SO₃·DMF) (58.8 g, 384 mmol) and dissolved in DMF (40 mL) at room temperature. The mixture was stirred for 4 h at 50 °C and then added dropwise to saturated sodium acetate in EtOH at room temperature. The suspension was centrifuged, and the resulting residue washed three times with saturated sodium acetate in EtOH and clarified by a second centrifugation step. The resulting precipitate was dissolved in a small amount of distilled water and then separated by size-exclusion chromatography using Sephadex^® G-25 (distilled water only). The purified fraction was lyophilized to give 3 (10.7 g, 6.82 mmol, 53% by 3 steps) as a white powder. 3: [α]_D²¹ = +57.8° (c = 0.6 H₂O); FT IR (neat) 3484, 2960, 2923, 2853, 1717 cm⁻¹; ¹H-NMR (500 MHz in d₆-DMSO) δ: 0.84 (12H, t, J = 8.0 Hz), 1.25 (64H, m), 1.73 (2H, m), 2.14 (4H, d, J = 6.5 Hz), 4.03 (2H, dd, J = 10.0, 3.0 Hz), 4.09 (2H, d, J = 10.5 Hz), 4.20 (4H, m), 4.31 (2H, d, J = 10.5 Hz), 4.48 (2H, t, J = 10.0 Hz), 5.33 (2H, d, J = 3.5 Hz); ¹³C-NMR (125 MHz in d₆-DMSO) δ: 13.95, 22.10, 25.75, 25.86, 28.71, 28.96, 29.27, 29.31, 31.30, 32.98, 33.05, 33.90, 38.56, 67.64, 73.74, 74.05, 75.09, 79.19, 91.51, 172.48; MS (MALDI⁺) m/z 1571 [M + H]⁺ HRMS (MALDI⁺) m/z Calcd for C₅₄H₉₇O₃₁S₆Na₆ 1571.3724. Found 1571.3733.

Optional purification for S1: 3-Nonyldodecanoic acid (2.0 g, 6.12 mmol), compound 6 (1.98 g, 2.56 mmol), EDCI·HCl (2.44 g, 12.7 mmol), DMAP (62.2 mg, 0.51 mmol) and CH₂Cl₂ (10 mL) were used according to step A. The obtained yellow syrup was purified by silica gel chromatography (Hexane/AcOEt = 20 : 1 to 10 : 1) to give compound S1 (3.43 g, 2.46 mmol, 96%) as a colorless syrup. S1: [α]_D¹⁹ = +67.3° (c = 0.4 CHCl₃); FT IR (neat) 1742, 2854, 2928, 2956 cm⁻¹; ¹H-NMR (300 MHz in CDCl₃) δ: 0.13 (36H, s), 0.15 (18H, s), 0.87 (12H, t, J = 7.2 Hz), 1.25 (64H, m), 1.84 (2H, m), 2.27 (4H, t, J = 6.6 Hz), 3.41 (2H, t, J = 9.9 Hz), 3.46 (2H, dd, J = 10.5, 9.0 Hz), 3.89 (2H, t, J = 8.7 Hz), 3.97 (2H, m), 4.02 (2H, dd, J = 14.7, 4.2 Hz), 4.29 (2H, d, J = 9.9 Hz), 4.90 (2H, d, J = 3.0 Hz); ¹³C-NMR (75 MHz in CDCl₃) δ: 0.20, 0.93, 1.07, 14.15, 22.71, 26.55, 29.38, 29.63, 29.93, 31.93, 33.65, 33.77, 34.65, 38.93, 63.08, 70.68, 71.87, 72.63, 73.51, 94.39, 173.51; MS (FAB⁺) m/z 1413 [M + Na]⁺; HRMS (FAB⁺) m/z Calcd for C₇₂H₁₅₀O₁₃Si₆Na 1413.9590. Found 1413.9608.

Measurement of TNF-α mRNA Expression Levels

THP-1 cells were cultured in RPMI-1640 medium (Sigma Life Science, St. Louis, MO, U.S.A.) containing 10% fetal bovine serum (Sigma Life Science) and 1% penicillin–streptomycin (Nacalai Tesque, Kyoto, Japan) at 37 °C/5% CO₂. The THP-1 cells were stimulated with 1.0 µg/mL LPS and co-supplemented with 10 µM vizantin or compound 3 for 1 h. Total RNA was extracted using a NucleoSpin RNA kit (TaKaRa Bio Inc., Shiga, Japan) according to the manufacturer’s protocol. cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. Quantitative reverse-transcription PCR was performed on a StepOne Plus Real-Time PCR System (Applied Biosystems) using the SYBR-Green PCR kit (TaKaRa Bio Inc.). Sequences of the respective oligonucleotide primer pairs were as follows: GAPDH, 5′-GGGAAACTGTGGCGTGAT-3′ and 5′-GTCCACCACTGACACGTTG-3′; TNF-α, 5′-CCCAGGGACCTCTCTCTAATCA-3′ and 5′-CCTCAGCTTGAGGGTTTGCTA-3′. The relative TNF-α mRNA expression levels were normalized against that of GAPDH using the ∆∆CT method.

Measurement of Cytotoxicity

The cytotoxicity of 2,2′,3,3′4,4′-hexakis-O-sulfaneted vizantin 3 was determined by the 3-(4,5-dimethyl-thiazol)-2,5-diphenyl-tetrazolium bromide (MTT) assays. In brief, the undifferentiated THP-1 cells (monocytes) and phorbol 12-myristate 13-acetate (PMA; Sigma Life Science)-differentiated THP-1 macrophages were plated in 96-well plate format at 1 × 10⁵ cells per well (N = 4). These cells were exposed for 24 h to different concentrations of compound 3 (1–50 µM). After compound 3 exposure, 10 µL of MTT (5 mg/mL in phosphate buffered saline, Sigma Life Science) was added to every single well in the plate which was incubated for 3 h at 37 °C. Then 100 µL of solubilization buffer (10% sodium dodecyl sulfate in 0.01 N HCl) was added to all wells and thoroughly mixed. After overnight incubation at 37 °C, the absorbance was measured at 570 nm by using a microplate reader (Spectra MAX 340, Molecular Devices, Sunnyvale, CA, U.S.A.).

Acknowledgments

This study was financially supported by Grant-in-Aid of the feasibility study (FS) stage, seeds validation program (No. AS2531332Q) form Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-STEP) of Japan Science and Technology Agency (JST). The authors thank Dr. Yasuko Okamoto, Tokushima Bunri University, for determining the HRMS, and appreciate the kind help from Dr. Masami Tanaka, Tokushima Bunri University, on the NMR analysis.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials. ¹H- and ¹³C-NMR spectra of 3 and S1 to this article can be found online.

References

• 1) Annane D., Bellissant E., Cavaillon J.-M., Lancet, 365, 63–78 (2005).
• 2) Parrillo J. E., N. Engl. J. Med., 358, 954–956 (2008).
• 3) Kulp A., Kuehn M. J., Annu. Rev. Microbiol., 64, 163–184 (2010).
• 4) Hotchkiss R. S., Moldawer L. L., Opal S. M., Reinhart K., Turnbull I. R., Vincent J.-L., Nat. Rev. Dis. Primers., 2, 16045 (2016).
• 5) Cecconi M., Evans L., Levy M., Rhodes A., Lancet, 392, 75–87 (2018).
• 6) Billiau A., Vandekerckhove F., Eur. J. Clin. Invest., 21, 559–573 (1991).
• 7) Poltorak A., Smirnova I., Clisch R., Beutler B., J. Endotoxin Res., 6, 51–56 (2000).
• 8) Thomson A. W., Lotze M. T., “The Cytokine Handbook, Two-Volume Set,” Elsevier, Amsterdam, 2003.
• 9) Park B. S., Song D. H., Kim H. M., Choi B.-S., Lee H., Lee J.-O., Nature (London), 458, 1191–1195 (2009).
• 10) Ohto U., Chem. Pharm. Bull., 65, 697–705 (2017).
• 11) Fowler D. H., Foley J., Whit-Shan H. J., Odom J., Castro K., Steinberg S. M., Gea-Banacloche J., Kasten-Sportes C., Gress R. E., Bishop M. R., Clin. Gastroenterol. Hepatol., 2, 237–245 (2004).
• 12) Martin G. S., Expert Rev. Anti Infect. Ther., 10, 701–706 (2012).
• 13) Opal S. M., Laterre P.-F., Francois B., et al., JAMA, 309, 1154–1162 (2013).
• 14) Yamamoto H., Oda M., Kanno M., Tamashiro S., Tamura I., Yoneda T., Yamasaki N., Domon H., Nakano M., Takahashi H., Terao Y., Kasai Y., Imagawa H., Chem. Pharm. Bull., 64, 246–257 (2015).
• 15) Liu Z., Chen L., Yu P., Zhang Y., Fang B., Wu C., Luo W., Chen X., Li C., Liang G., J. Med. Chem., 62, 5453–5469 (2019).
• 16) Romerio A., Gotri N., Franco A. R., Artusa V., Shaik M. M., Pasco S. T., Atxabal U., Matamoros-Recio A., Mínguez-Toral M., Zalamea J. D., Franconetti A., Abrescia N. G. A., Jimenez-Barbero J., Anguita J., Martín-Santamaría S., Peri F., J. Med. Chem., 66, 3010–3029 (2023).
• 17) Yamamoto H., Oda M., Nakano M., Yabiku K., Shibutani M., Nakanishi T., Suenaga M., Inoue M., Imagawa H., Nagahama M., Matsunaga Y., Himeno S., Setsu K., Sakurai J., Nishizawa M., Chem. Pharm. Bull., 61, 452–459 (2013).
• 18) Yamamoto H., Oda M., Nakano M., Watanabe N., Yabiku K., Shibutani M., Inoue M., Imagawa H., Nagahama M., Himeno S., Setsu K., Sakurai J., Nishizawa M., J. Med. Chem., 56, 381–385 (2013).
• 19) Ioneda T., Lenz M., Pudles J., Biochem. Biophys. Res. Commun., 13, 110–114 (1963).
• 20) Senn M., Ioneda T., Pudles J., Lederer E., Eur. J. Biochem., 1, 353–356 (1967).
• 21) Datta A. K., Takayama K., Carbohydr. Res., 245, 151–158 (1993).
• 22) Nishizawa M., Minagawa R., Garcia D. M., Hatakeyama S., Yamada H., Tetrahedron Lett., 35, 5891–5894 (1994).
• 23) Nishizawa M., García D. M., Minagawa R., Noguchi Y., Imagawa H., Yamada H., Watanabe R., Yoo Y. C., Azuma I., Synlett, 1996, 452–454 (1996).
• 24) Nishizawa M., Yamamoto H., Imagawa H., Barbier-Chassefière V., Petit E., Azuma I., Papy-Garcia D., J. Org. Chem., 72, 1627–1633 (2007).
• 25) Oda M., Yamamoto H., Shibutani M., Nakano M., Yabiku K., Tarui T., Kameyama N., Shirakawa D., Obayashi S., Watanabe N., Nakase H., Suenaga M., Matsunaga Y., Nagahama M., Takahashi H., Imagawa H., Kurosawa M., Terao Y., Nishizawa M., Sakurai J., J. Immunol., 193, 4507–4514 (2014).
• 26) Oda M., Kurosawa M., Yamamoto H., Domon H., Takenaka S., Ohsumi T., Maekawa T., Yamasaki N., Furue Y., Terao Y., Microbiol. Immunol., 64, 493–501 (2020).
• 27) Hasegawa T., Takenaka S., Oda M., Domon H., Hiyoshi T., Sasagawa K., Ohsumi T., Hayashi N., Okamoto Y., Yamamoto H., Ohshima H., Terao Y., Noiri Y., BMC Microbiol., 20, 361 (2020).
• 28) Takenaka S., Hasegawa T., Oda M., Yamamoto H., Naksagoon T., Nagata R., Suzuki Y., Ohsumi T., Noiri Y., Jpn. J. Conserv. Dent., 63, 173–180 (2020).
• 29) Takenaka S., Hasegawa T., Oda M., Takahashi N., Isono T., Ohkura N., Yamamoto H., Tabeta K., Noiri Y., Jpn. J. Conserv. Dent., 63, 61–72 (2020).
• 30) Hayashi N., Furue Y., Kai D., Yamada N., Yamamoto H., Nakano T., Oda M., PLOS ONE, 13, e0206696 (2018).
• 31) Oda M., Kurosawa M., Yamamoto H., Domon H., Kimura T., Isono T., Maekawa T., Hayashi N., Yamada N., Furue Y., Kai D., Terao Y., Microbiol. Immunol., 62, 310–316 (2018).
• 32) Liu J., Park S. K., Moore J. A., Cramer S. M., Ind. Eng. Chem. Res., 45, 9107–9114 (2006).
• 33) Khan A. A., Chee S. H., McLaughlin R. J., Harper J. L., Kamena F., Timmer M. S. M., Stocker B. L., ChemBioChem, 12, 2572–2576 (2011).
• 34) Heuser J. E., J. Mol. Biol., 169, 155–195 (1983).