Dual-Tail Approach to Discovery of Novel Carbonic Anhydrase IX Inhibitors by Simultaneously Matching the Hydrophobic and Hydrophilic Halves of the Active Site

Zhuang Hou a, § , Bin Lin a, §, Yu Bao b, Hai-ning Yan a, Miao Zhang a, Xiao-wei Chang a, Xin-xin Zhang a, Zi-jie Wang a, Gao-fei Wei a, Mao-sheng Cheng a, Yang Liu a, *, Chun Guo a, **

a Key Laboratory of Structure-Based Drugs Design and Discovery (Ministry of Education),

School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016 China

b School of Life Sciences and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China

* Corresponding author. ** Corresponding author.§ These two authors contributed equally to this work.E-mail addresses: y.liu@syphu.edu.cn (Y. Liu), gc_66888@163.com (C. Guo).

Reagents were used without further purification unless otherwise specified. Solvents were dried

and redistilled prior to use in the usual way. Analytical TLC was performed using silica gel HF254.

Preparative column chromatography was performed with silica gel H. Melting points were obtained

on a Büchi melting point B-540 apparatus.1H and 13C NMR spectra were recorded on a Bruker ARX

600 MHz spectrometer. ESI-MS were obtained on Agilent ESI-QTOF instrument. HR-MS were

obtained on a Bruker micrOTOF_Q spectrometer.

General procedure for the synthesis of compound 8

To a solution of glucosamine hydrochloride 1 (15 g, 69 mmol) in H2O (150 mL) was added

NaHCO3 （17.39 g, 207 mmol） and trichloroethyl chloroformate（11.45 mL, 83 mmol） . The

reaction mixture was stirred for 5 h. Subsequently, a solid separated out which was filtered, dried to

afford 2 (23 g, 95%).

To a solution of 2 (23 g, 65.6 mmol) in pyridine (50 mL) was added acetic anhydride （70

mL） . The reaction mixture was stirred for 5 h and then H2O (100 mL) was added and stirring

continued for 20 min. The reaction mixture was washed with 0.1 M HCl (30 mL), satd aq NaHCO 3

(30 mL), and dried (MgSO4), Filtration, evaporation of solvent under diminished pressure to dryness

to give 3.

To a solution of 3 in dichloromethane (70 mL) was added HBr-HOAc（70 mL）. The reaction

mixture was stirred for 5 h and then H2O (100 mL) was added and stirring continued for 20 min. The

reaction mixture was washed with satd aq NaHCO3 (50 mL), and dried (MgSO4). Filtration,

evaporation of solvent under diminished pressure and chromatography of the residue (EtOAc–

petroleum ether, 1:5) gave 4 as a white solid (26 g, 62%).

To a solution of 4 (1 g, 1.85 mmol) in CH3OH (150 mL) was added Ag2CO3（0.71 g, 2.59

mmol ） . The reaction mixture was stirred for 1 h. Filtration, evaporation of solvent under

diminished pressure and chromatography of the residue (EtOAc–petroleum ether, 1:5) gave 5 as a

white solid (0.78 g, 85%).

Deprotected compound 6 was prepared by dissolving 5 (0.78 g, 1.57 mmol) in glacial acetic

acid with zinc powder (3.37g, 51.9 mmol). The mixture was stired until full deprotection was

evident by TLC. Filtration, evaporation of solvent under diminished pressure and chromatography of

the residue (acetone–petroleum ether, 1:2) gave 6 as a white solid (0.48 g, 96%).

To a solution of compound 6 (100.0 mg, 0.087 mmol) in dry CH2Cl2 (10 mL), substituted

cinnamoyl chloride (0.11 mmol) and pyridine (0.2 mL) were added at room temperature and the

reaction mixture was stirred for 1 h. The mixture was quenched by addition of MeOH and

concentrated. Then, to a solution of residue in CH2Cl2-MeOH (1:1, 10 mL), freshly prepared NaOMe

in MeOH solution (1.0 mol/L, 1 mL) was added. After it was stirred overnight, the mixture was

neutralized with Dowex H+ resin to pH 7, then filtered. The filtrate was concentrated and purified by

a silica gel column chromatography (10:1, CH2Cl2-MeOH) to afford 8 (84.6%) as a white solid.

General procedure for the synthesis of compound 12, 14a-14g

To a solution of 4 (26 g, 35.76 mmol) and dry NH4SCN（2.7 g, 35.76 mmol）in acetone (50

mL) and dry acetonitrile(50 mL). The reaction mixture was stirred for 2 h. Filtration, evaporation of

solvent under diminished pressure and chromatography of the residue (EtOAc–petroleum ether, 1:5)

gave 9 as a white solid (14 g, 55%).

Compound 9（14 g, 26.92 mmol）and sulfanilamide (4.63g, 26.92 mmol) was dissolved in

pyridine and the mixture was stirred for 1 h. The reaction mixture was washed with 0.1 M HCl (30

mL), satd aq NaHCO3 (30 mL), and dried (MgSO4). The product was purified by column

chromatography (acetone–petroleum ether, 1:5) in 98% yield.

Deprotected compound 11 was prepared by dissolving 10 (3 g, 4.34 mmol) in glacial acetic acid

with zinc powder (9.3 g, 143 mmol). Filtration, evaporation of solvent under diminished pressure

and chromatography of the residue (EtOAc–petroleum ether, 1:5) gave 11 as a white solid (2.0 g,

90%).

To a solution of 11 (100 mg, 0.19 mmol) in CH2Cl2-MeOH (1:1, 10 mL), freshly prepared

NaOMe in MeOH solution (1.0 mol/L, 1 mL) was added. After it was stirred overnight, the mixture

was neutralized with Dowex H+ resin to pH 7, then filtered. The filtrate was concentrated and

purified by a silica gel column chromatography (10:1, CH2Cl2-MeOH) to afford 12 (64.8mg, 85.6%)

as a white solid.

To a solution of compound 11 (100.0 mg, 0.19 mmol) in dry CH2Cl2 (10 mL), substituted

cinnamoyl chlorides (0.11 mmol) and pyridine (0.2 mL) were added at room temperature and the

reaction mixture was stirred for 1 h. The mixture was quenched by addition of MeOH and

concentrated. Then, to a solution of residue in CH2Cl2-MeOH (1:1, 10 mL), freshly prepared NaOMe

in MeOH solution (1.0 mol/L, 1 mL) was added. After it was stirred overnight, the mixture was

neutralized with Dowex H+ resin to pH 7, then filtered. The filtrate was concentrated and purified by

a silica gel column chromatography (10:1, CH2Cl2-MeOH) to afford 14a-14g (76.8%-92.7%) as a

white solid.

2-deoxy-2-[[3-(4-methoxylphenyl)-1-oxo-2-propenyl] amino]-β-D-Glucopyranose (8)

N-[4-(Aminosulfonyl) phenyl]-N，-(2-amino-2-deoxy-β-D-glucopyranosyl) thiourea (12)

N-[4-(Aminosulfonyl) phenyl]-N，-[2-[(2E)-3-(4-methoxylphenyl)-1-oxo-2-propen-1-yl] amino]-2-deoxy-β-D-

glucopyranosyl] thiourea (14a)

N-[4-(Aminosulfonyl) phenyl]-N，-[2-[(2E)-3-(3-methoxylphenyl)-1-oxo-2-propen-1-yl] amino]-2-deoxy-β-D-

glucopyranosyl] thiourea (14b)

N-[4-(Aminosulfonyl) phenyl]-N，-[2-[(2E)-3-(2-methoxylphenyl)-1-oxo-2-propen-1-yl] amino]-2-deoxy-β-D-

glucopyranosyl] thiourea (14c)

N-[4-(Aminosulfonyl) phenyl]-N，-[2-[(2E)-3-(2,3-dimethoxylphenyl)-1-oxo-2-propen-1-yl] amino]-2-deoxy-β-

D-glucopyranosyl] thiourea (14d)

N-[4-(Aminosulfonyl) phenyl]-N，-[2-[(2E)-3-(2,4-dimethoxylphenyl)-1-oxo-2-propen-1-yl] amino]-2-deoxy-β-

D-glucopyranosyl] thiourea (14e)

N-[4-(Aminosulfonyl) phenyl]-N，-[2-[(2E)-3-(2,4,5-trimethoxylphenyl)-1-oxo-2-propen-1-yl] amino]-2-deoxy-

β-D-glucopyranosyl] thiourea (14f)

N-[4-(Aminosulfonyl) phenyl]-N，-[2-[(2E)-3-(3,4,5-trimethoxylphenyl)-1-oxo-2-propen-1-yl] amino]-2-deoxy-

β-D-glucopyranosyl] thiourea (14g)