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Brian Safina

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Figure 1

Synthesis of T785

T785 was prepared via an eight step synthesis starting from commercially available starting materials.&nbsp;Commercially available 3-nitroquinoline-2,4-diol undergoes nitration and chlorination followed by a regioselective addition of tert-butyl N-(4-aminobutyl)carbamate.&nbsp;Aniline formation via hydrogenation and subsequent acylation undergoes imidazoquinoline ring formation under acidic conditions.&nbsp;Finally, displacement of the chloroquinoline by (2,4-dimethoxyphenyl)methanamine and acidic deptrotection yields T785 in ~36% overall yield.&nbsp;

Acetic acidNitric AcidPOCl3Pet etherethyl acetatewaterTHFTriethylaminePt/CNitrogen gaspentanoyl chlorideSodium sulfate (Na2SO4)MeODd-DMSONaHCO3HClMeOHquinoline-2,4-dioltert-butyl N-(4-aminobutyl)carbamate(2,4-dimethoxyphenyl)methanamine

RotoevaporaryFlaskSeperatory funnelStir plateNMRThin Layer ChromotagraphyHPLCLCMS

3-nitroquinoline-2,4-diol (1).&nbsp;To a solution of quinoline-2,4-diol (260 g, 1.61 mol, 1 eq) in AcOH (1.2 L) is added fuming nitric acid (152.49 g, 2.42 mol, 108.92 mL, 1.5 eq) dropwise at 0°C.&nbsp;The mixture is stirred at 65°C for 5 hours. The mixture is cooled to 25°C and quenched by the addition of ice-water (500 mL). The product is separated&nbsp;by&nbsp;filtration and washed with water (500 mL x 3), and dried to give 3-nitroquinoline-2,4-diol (320 g, 1.52 mol, 94.29%&nbsp;yield) as&nbsp;yellow solid.&nbsp;&nbsp;&nbsp;The crude product was used in the next step without further purification. LC/MS:&nbsp;&nbsp;m/z = 207.0 [M+H]+ 1H NMR (DMSO-d6, 400 MHz) δ 11.96 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 7.2 Hz,1H), 7.34 (d, J = 8.0 Hz, 1H), 7.27 (t, J = 7.2 Hz, 1H). 2,4-dichloro-3-nitroquinoline (2).&nbsp;3-nitroquinoline-2,4-diol (365 g, 1.77 mol, 1 eq) is added slowly to POCl3 (2.44 kg, 15.93 mol, 1.48 L, 9 eq) at 65°C and further heated to 90°C for 12 h. The mixture is concentrated under vacuum.&nbsp;The residue is then poured into ice-water (3 L) and stirred for an additional 0.5 h. The aqueous phase is filtered, washed by water (500 mL x 5) and the filter cake is collected and further purified by silica gel chromatography (Petroleum ether/Ethyl acetate=20/1, 3/1) to obtain 2,4-dichloro-3-nitro-quinoline (330 g, 1.32 mol, 74.39% yield, 97% purity) as yellow solid. LC/MS: m/z = 243.0 [M+H]+ 1H NMR (CDCl3, 400 MHz) δ 8.29 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.96 (t, J =8.4 Hz, 1H), 7.82 (t, J = 8.4 Hz, 1H). Tert-butyl N-[4-[(2-chloro-3-nitro-4-quinolyl) amino]butyl]carbamate (3).&nbsp;&nbsp;To a mixture of 2,4-dichloro-3-nitro-quinoline (330 g, 1.36 mol, 1 eq) and tert-butyl N-(4-aminobutyl)carbamate (281.18 g, 1.49 mol, 1.1 eq) in THF (1.5 L) is added Et3N (206.09 g, 2.04 mol, 283.48 mL, 1.5 eq) slowly at 0°C and stirred for 2 h. TLC (Petroleum ether: Ethyl acetate = 1:1, Rf = 0.43) showed the starting materials is consumed completely and one main spot is detected. The reaction is quenched by water (2 L) and extracted&nbsp;with EtOAc (800 mL x 3).&nbsp;The combined organic layer is washed by brine (1000 mL), dried over Na2SO4, filtered, and concentrated in vacuum. The crude product is washed by petroleum ether (1000 mL) and filtered to give tert-butyl N-[4-[(2-chloro-3-nitro-4-quinolyl) amino]butyl]carbamate (500 g, crude) as yellow solid. LC/MS:&nbsp;m/z = 395.2 [M+H]+ 1H NMR (CDCl3, 400 MHz) δ 8.12 (d, J = 7.2 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.75 (t, J =7.2 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 6.43 (s, 1H), 4.70 (s, 1H), 3.50-3.45 (m, 2H), 3.22-3.17(m, 2H), 1.80-1.75 (m, 2H), 1.67-1.61 (m, 2H), 1.44 (s, 9H). Tert-butyl N-[4-[(3-amino-2-chloro-4-quinolyl)amino]butyl]carbamate (4).&nbsp;To a solution of tert-butyl N-[4-[(2-chloro-3-nitro-4-quinolyl)amino]butyl]carbamate (150 g, 379.89 mmol, 1 eq) in EtOAc (800 mL) is added Pt/C (40 g, 10% purity) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (50 psi) at 25°C for 3 hours. LCMS and HPLC showed the starting material is consumed completely. The reaction mixture was filtered and the filtrate is concentrated to give tert-butyl N-[4-[(3-amino-2-chloro-4-quinolyl)amino]butyl]carbamate (110 g, crude) as an off-white solid. LC/MS:&nbsp;m/z = 365.2 [M+H]+ 1H NMR (CDCl3, 400 MHz) δ 7.90 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H) 7.49-7.45 (m,2H), 4.59 (s, 1H), 4.15-4.10 (m, 2H), 3.29-3.26 (m, 2H), 3.18-3.16 (m, 2H), 1.70-1.60 (m, 4H), 1.45 (s, 9H). Tert-butyl N-[4-[[2-chloro-3-(pentanoylamino)-4-quinolyl]amino]butyl] carbamate (5). To a mixture of tert-butyl N-[4-[(3-amino-2-chloro-4-quinolyl)amino]butyl] carbamate (500 g, 1.37 mol, 1 eq) and Et3N (208.00 g, 2.06 mol, 286.11 mL, 1.5 eq) in THF (1000 mL) is added&nbsp;pentanoyl&nbsp;chloride&nbsp;(247.85&nbsp;g,&nbsp;2.06&nbsp;mol,&nbsp;249.10&nbsp;mL,&nbsp;1.5&nbsp;eq)&nbsp;dropwise&nbsp;at&nbsp;0°C.&nbsp;&nbsp;&nbsp;The mixture is stirred at 0°C for 1 h. The mixture is poured into ice water (1000 mL) and stirred for 2 min. The aqueous phase is extracted with ethyl acetate (500 mL x 3).&nbsp;The combined organic phase was washed with brine (1000 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue is purified by re-crystallization from EtOAc/petroleum ether (1/1, 800 mL) to give the pure tert-butyl N-[4-[[2-chloro-3-(pentanoylamino)-4-quinolyl]amino] butyl] carbamate (550 g, 1.16 mol, 84.92% yield, 95% purity) as light yellow solid. LC/MS:&nbsp;m/z = 449.3 [M+H]+ 1H NMR (MeOD, 400 MHz) δ 8.14 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.46 (t, J = 6.8 Hz, 1H), 3.56 (t, J = 7.2 Hz, 2H), 3.07 (t, J = 7.2 Hz, 2H), 2.51 (t, J = 7.6 Hz, 2H), 1.77-1.66 (m, 4H), 1.54-1.41 (m, 4H), 1.41 (s, 9H), 1.01 (t, J = 7.2 Hz, 3H). Tert-butyl N-[4-(2-butyl-4-chloro-imidazo[4,5-c]quinolin-1-yl)butyl]carbamate (6).&nbsp;To a solution of tert-butyl N-[4-[[2-chloro-3-(pentanoylamino)-4-quinolyl]amino] butyl]carbamate (490 g, 1.09 mol, 1 eq) in toluene (1000 mL) is added AcOH (65.54 g, 1.09 mol, 62.42 mL, 1 eq) at 25°C. The mixture is stirred at&nbsp;100°C&nbsp;for&nbsp;15&nbsp;hours. The mixture is concentrated in vacuum.&nbsp;The residue is poured into ice water (1000 mL) and stirred for 5 min. The aqueous phase is extracted with ethyl acetate (500 mL x 3). The combined organic phase is washed with NaHCO3.aq (500 mL) and brine (800 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue is purified by re-crystallized from EtOAc/ petroleum ether (1/50, 500 mL) to give tert-butyl N-[4-(2-butyl-4-chloro-imidazo[4,5-c]quinolin-1- yl)butyl]carbamate (400 g, 928.14 mmol, 85.05% yield) as a white solid. LC/MS:&nbsp;m/z = 431.1 [M+H]+ 1H NMR (CDCl3, 400 MHz) δ 8.19 (d, J = 6.8 Hz, 1H), 8.09 (d, J = 7.6 Hz, 1H) 7.66-7.62 (m,2H), 4.61&nbsp;(s,&nbsp;1H),&nbsp;4.54&nbsp;(t,&nbsp;J = 7.6&nbsp;Hz,&nbsp;2H),&nbsp;3.21-2.20&nbsp;(m,&nbsp;2H),&nbsp;3.00&nbsp;(t,&nbsp;J = 8.0&nbsp;Hz, 2H), 1.97-1.88 (m, 4H), 1.71-1.69 (m, 2H), 1.55-1.49 (m, 2H), 1.42 (s, 9H), 1.01 (t, J = 7.2 Hz, 3H). Tert-butyl N-[4-[2-butyl-4-[(2,4-dimethoxyphenyl)methylamino]imidazo[4,5-c]quinolin-1-yl]butyl]carbamate (7).&nbsp;&nbsp;Tert-butyl N-[4-(2-butyl-4-chloro-imidazo[4,5-c]quinolin-1-yl)butyl] carbamate (62 g, 143.86mmol, 1 eq) in (2,4-dimethoxyphenyl)methanamine (120.27 g, 719.31 mmol, 108.35 mL, 5 eq) is stirred at 120°C for 3 hr.&nbsp;LCMS showed the reaction is completed.&nbsp;The reaction is quenched by addition of water (200 mL), acidified by diluted hydrochloride acid and extracted with&nbsp;EtOAc&nbsp;(300 mL x 3).&nbsp;The combined organic layers is washed by brine (500 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue is purified by re-crystallization from&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;EtOAc (200mL) to give tert-butyl N-[4-[2-butyl-4-[(2,4-dimethoxyphenyl)methylamino]imidazo[4,5-c]quinolin-1-yl]butyl]carba mate (70 g, 118.39 mmol, 82.29% yield, 95% purity) as white solid. LC/MS:&nbsp;m/z = 562.4 [M+H]+ 1H NMR (MeOD, 400 MHz) δ 8.22 (d, J = 8.4 Hz, 1H), 8.05 (s, 1H), 7.74 (t, J = 7.6 Hz, 1H),7.66 (t, J = 8.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 6.63-6.58 (m, 1H), 6.54 (d, J = 8.0 Hz, 1H) 4.62 (t, J = 7.2 Hz, 2H), 3.82 (s, 3H), 3.79 (s, 3H), 3.12 (t, J = 6.8 Hz, 2H), 3.00 (t, J = 7.6 Hz, 2H), 1.98-1.86 (m, 4H), 1.69-1.65 (m, 2H ), 1.53-1.50 (m, 2H), 1.37 (s, 9H), 1.01 (t, J = 7.6 Hz, 1-(4-aminobutyl)-2-butyl-imidazo[4,5-c]quinolin-4-amine (9).&nbsp;Tert-butyl N-[4-[2-butyl-4-[(2,4-dimethoxyphenyl)methylamino]imidazo[4,5-c] quinolin-1-yl] butyl]carbamate (30 g, 53.41 mmol, 1 eq) is added to HCl (12M, 200 mL) at 25°C. The mixture is stirred at 25°C for 12 hour. LCMS showed the starting material is consumed completely. The reaction mixture is filtered, washed with MeOH (500 mL) and the filtrate is concentrated to give the crude product, recrystallized from EtOAc (50 mL) to give the pure 1-(4-aminobutyl)-2-butyl-imidazo[4,5-c]quinolin-4-amine (17 g, 48.38 mmol, 90.58% yield, 99% purity, HCl) as white solid. 1H NMR (MeOD, 400 MHz) δ 8.28 (d, J = 7.6 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.75 (t, J =7.6 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 4.72 (t, J = 7.6 Hz, 2H), 3.08 (t, J = 7.6 Hz, 2H), 3.01 (t, J = 7.6 Hz, 2H), 2.07-1.85 (m, 6H), 1.62-1.53 (m, 2H), 1.05 (t, J = 7.6 Hz, 3H). LCMS (ESI): [M+H]+. calculated 312.21, [M+H]+ found 312.

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Method Article

Brian Safina, Art Lee

Brian Safina

Bolt Biotherapeutics

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Art Lee

DOI:

10.21203/rs.3.pex-1149/v1

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T785, chemical synthesis, small molecule, cancer

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Brian Safina, Art Lee

Brian Safina

Bolt Biotherapeutics

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Art Lee

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