Conjugation of Toll-like receptor 7 agonist to ethacrynic acid
9-
(4-
(aminomethyl)benzyl)-
8-
methoxy-
2-
(2-
methoxyethoxy)-
9H-
purin-
6-
amine (compound2): 4-((6-amino-8-methoxy-2-(2-methoxyethoxy)-9H-purin-9-yl) methyl) benzonitrile
(compound1) was prepared following previously reported methods [
19]. Raney Ni (500 mg) and ammonium hydroxide (3 mL) were added to a solution of compound
1 (1.5 g, 4.23 mmol) in 1,4-dioxane (40 mL). The reaction mixture was subjected to hydrogenation at 3.5 atm, at 50 °C for 7 h. After reaction completion, the mixture was filtered and the filtrate was concentrated under reduced pressure to afford compound
2 (1.38 g, 91%) as a white solid. ESI–MS (positive ion mode): calculated for C
17H
23N
6O
3 m/z [M + 1] 359.2; found 359.0.
6-amino-9-(4-(aminomethyl)benzyl)-2-(2-methoxyethoxy)-9H-purin-8-ol (compound3): A suspension of compound 2 (358 mg, 1 mmol) in concentrated hydrochloric acid (10 mL) was placed in a round-bottomed flask. The reaction was stirred at room temperature overnight. The mixture was adjusted to pH 9 with 2 N NaOH and further purified by precipitating and washing in water to obtain a white solid (317 mg, 93%). ESI–MS (positive ion mode): calculated for C16H21N6O3 m/z [M + 1] 345.2; found 345.0.
N-(4-((6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)benzyl)-2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)acetamide (compound4, T7-EA): To a solution of ethacrynic acid (EA, 341 mg, 1.125 mmol), O-Benzotriazole-N,N,N’,N’-tetramethyl- uronium-hexafluorophosphate (HBTU) (427 mg, 1.125 mmol) dissolved in anhydrous DMF (8 mL) was added triethylamine (TEA) (0.42 mL, 3 mmol) and a catalytic amount of 4-dimethylaminopyridine. A solution of compound 3 (344 mg, 1 mmol) in DMF (2 mL) was added dropwise to the reaction mixture and stirred at room temperature for 4 h. After reaction completion, the reaction content was diluted with water (100 mL), then filtered and washed with ice-cold water. The crude material was purified by flash chromatography (5% MeOH/CH2Cl2) to afford compound 4 (T7-EA) as a white solid (475 mg, 76%). 1H-NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.54 (t, J = 6.0 Hz, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.25 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.3 Hz, 2H), 7.10 (d, J = 8.6 Hz, 1H), 6.48 (s, 2H), 6.07 (s, 1H), 5.55 (s, 1H), 4.83 (s, 2H), 4.79 (s, 2H), 4.30 (d, J = 6.0 Hz, 2H), 4.28-4.22 (m, 2H), 3.62-3.55 (m, 2H), 3.27 (s, 3H), 2.38 (q, J = 7.4 Hz, 2H), 1.09 (t, J = 7.4 Hz, 3H). 13C-NMR (100 MHz, DMSO-d6) δ 195.59, 167.14, 160.31, 155.97, 152.71, 149.88, 149.64, 148.18, 144.42, 138.65, 136.28, 132.97, 129.86, 129.81, 127.98, 127.93, 121.73, 112.39, 98.80, 70.70, 68.40, 65.75, 58.53, 42.62, 42.19, 23.41, 12.84. ESI–MS (positive ion mode): calculated for C29H31Cl2N6O6 m/z [M + 1] 629.2; found 629.1.
In vitro measurements of cytotoxicity and cytokines induction
Human monocyte cell line THP-1 cells (0.5 × 104) were incubated for 24 h with T7-EA at concentrations ranging from 0.01 to 10 μM, cell viability was detected with a Cell Counting Kit-8 (CCK8) (Beyotime, China).
Human peripheral blood mononuclear cells (PBMCs) were isolated from human buffy coats obtained from 3 healthy volunteers, as described previously [
20]. PBMCs (2 × 10
6/mL) were incubated with various compounds for 18 h at 37 °C and 5% CO
2, and then the culture supernatants were collected. The levels of cytokines (IL-6, IFN-α) in the supernatants were determined by ELISA (BD Biosciences Pharmingen, La Jolla, CA). The minimum cytokine detection level was 15 pg/mL.
Bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs) were isolated from C57BL/6 mice and grown as previously described [
21,
22]. Then, the cells were seeded in 96-well plates at a density of 5 × 10
4 cells per well. Compounds were added to the cultures at a final concentration ranging from 0.01 to 10 μM. After 22 h of incubation, the culture supernatants were collected and assayed by ELISA for IL-6 induction (BD Biosciences Pharmingen, La Jolla, USA). The minimum IL-6 detection level was 15 pg/mL.
RNA isolation and real-time PCR
Balb/c mice (n = 3) were injected in the gastrocnemius muscles with 35 nmol T7, T7-EA, R848 or vehicle (10% DMSO in saline) in a 50μL volume. After 1, 3 and 7 days of injection, the muscles were harvested and immediately frozen in liquid nitrogen and stored at − 80 °C. Total RNA was isolated from cells or tissues using TRIzol reagent (Invitrogen, Carlsbad, USA). The RNA samples were further purified using a Qiagen RNeasy Protect Kit (Qiagen, Valencia, USA). Reverse transcription was carried out with 1 μg total RNA using SuperScript III (Invitrogen, Carlsbad, USA). Serum HBV DNA was extracted from 100 uL of serum and measured following the manufacturer’s instructions (Qiagen, Hilden, Germany). The cDNA and HBV DNA samples were run in duplicate and quantified by real-time PCR (Bio-rad CFX96, Hercules, USA) using the following primer sets: HBV forward 5′CACATCAGGATTCCTAGGACC3′, reverse 5′GGTGAGTGATTGGAGGTTG3′; β-actin forward 5′ GGCTGTATTCCCCTCCATCG3′, reverse 5′ CCAGTTGGTAACAATGCCATGT3′; IL-4 forward 5′ATGGATGTGCCAAACGTCCT3′, reverse 5′AAGCACCTTGGAAGCCCTAC3′, CCL2 forward 5′TCAGCCAGATGCAGTTAACG3′, reverse 5′CTCTCTTGAGCTTGGTGACA3′; CCL4 forward 5′GCAACACCATGAAGCTCTGC3′, reverse 5′CCATTGGTGCTGAGAACCCT3′; TNF-α forward 5′CAAAATTCGAGTGACAAGCCTG3′, reverse 5′GAGATCCATGCCGTTGGC3′. β-actin was used as an internal control for sample loading and normalization. The data were analyzed using the comparative Ct method, where Ct is the cycle number at which the fluorescence first exceeds the threshold. The ΔCt values from each cell line were obtained by subtracting the Ct values for β-actin from the Ct values for the sample. One difference of Ct value represents a 2-fold difference in the level of mRNA. The mRNA level was expressed as a percentage with respect to the control.