Design, Synthesis and Evaluation of Fe-S Targeted Adenosine 5 -Phosphosulfate Reductase

SUPPORTING INFORMATION

Design, Synthesis and Evaluation of Fe-S Targeted Adenosine 5’-Phosphosulfate Reductase Inhibitors

Hanumantharao Paritalaa, Yuta Suzukib, Kate S. Carrolla,*

aDepartment of Chemistry, The Scripps Research Institute, Jupiter, Florida, 33458, USA

bDepartment of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109, USA

*Corresponding author

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Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida, 33458, USA

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Phone: 561-228-2460

Fax: 561-228-2919

Synthesis of intermediates for 4a-4h and 8a-8h.

Synthesis of hydroxamates

General procedure for synthesis of hydroxamate derivatives

Bromoalkyl carboxylates (1 eq, 2mmol) was solubilized in 7ml of dichloromethane and added oxalylchloride (2 eq, 4mmol) at 0oC. After five minutes of stirring added 10µL of dimethylformamide as catalyst. The reaction was slowly brought to rt and the reaction progress was monitored by LC/MS by adding a small fraction of reaction mixture to ethanol and by monitoring the formation of subsequent ethyl ester. The reaction was concentrated up on completion and dried under high vacuum without any further purification to remove volatile organic materials. The dried material was solubilized in 10ml of dimethylformamide and charged with hydroxylamine hydrochloride (2eq, 2mmol) at rt and added diisopropylethylamine (2.5 eq, 2.5mmol) at rt and continued the reaction towards completion. The reaction was monitored on LC/MS and quenched after the reaction making no further progress. The contents were dried and purified using silica gel column chromatography using 20% ethylacetate in hexane as mobile phase. c: 1H NMR (CDCl3, 400 MHz): d = 8.0 (s, 1H), 3.51 (t, J = 6.0, 2H), 2.34 (t, J = 7.0, 2H), 2.07 (m, 3H); 13C NMR (CDCl3, 125 MHz): d = 170.1, 32.0, 30.3, 28.5, Mass calculated for C4H8BrNO2 182.0158. Observed (M+H) = 183.65; (M-H) = 181.21. d: 1H NMR (CDCl3, 400 MHz): d = 8.0 (s, 1H), 4.19 (s, 1H), 2.01(s, 1H). 13C NMR (CDCl3, 125 MHz): d = 168.2, 24.5, Mass calculated for C2H2BrNO2 153.9627. Observed (M+H) = 154.24; (M-H) = 152.76.

Synthesis of alkylthioates

Synthesis of e

1,5-dibromopentane (2eq, 3mmol) was solubilized in 10 ml of dimethylformamide and treated with potassium thioacetate (1eq, 1.5mmol) at rt. To the stirred suspension added sodium hydride (2eq, 2mmol) at 0 oC and allowed the reaction to occur at 0 oC for 30 minutes and slowly brought the reaction to rt. The reaction was monitored by LC/MS till no further progress was found. The reaction was quenched by adding 10 ml of water to the reaction mixture and the contents were extracted with ethyl acetate. The combined ethyl acetate fractions were concentrated and purified using flash chromatography using 10% ethyl acetate in hexanes. 1H NMR (CDCl3, 400 MHz): d = 3.51 (t, J = 6.8, 2H), 3.24 (t, J = 6.6, 2H), 2.34 (s, 3H), 1.83 (m, 4H), 1.29 (q, J = 6.1, 2H); 13C NMR (CDCl3, 125 MHz): d = 193.1, 33.7, 32.4, 30.3, 29.2, 28.5, 27.2. Mass calculated for C7H13BrOS 225.1465. Observed (M+H) = 226.52; (M-H) = 224.54.

Synthesis of f

To a well-stirred solution of the corresponding carboxylic acid (2.1 mmol), PPh3(2.2 mmol), and NBS (2.2 mmol) in CHCl3(5 mL) (stirred for 10 min) was added benzyltriethylammonium tetrathiomolybdate1(3 mmol) followed by dichloroethane (1 mmol). The reaction was complete after 12 h at rt (28° C). Diethyl ether (20 mL) was added to the reaction mixture, which was then filtered through a Celite pad. The residue was again extracted with DCM(5 mL) followed by extraction with diethyl ether (20 mL) and filtered again through a Celite pad. The combined extract was evaporated and the residue was purified by column chromatography on silica gel to give f [35].

Synthesis of alkyltriazole derivatives

General procedure for synthesis of g and h

1,4-dibromobutane (2eq, 4 mmol) was solubilized in 7ml of dimethylformamide and added triazole (1eq, 2mmol) was and stirred for 10 minutes at rt and the reaction mixture was cooled to 0o C and added sodium hydride (1eq, 2mmol). The reaction was monitored till no further progress was found on LC/MS. The reaction was quenched by adding water and by washing with saturated ammonium chloride solution. The aqueous liquor was then extracted with ethyl acetate and the combined ethyl acetate extracts were dried, concentrated and purified by silica gel column using 25% ethyl acetate in hexanes as mobile phase. g: 1H NMR (CDCl3, 400 MHz): d = 7.63 (q, J = 5.5, 2H), 4.46 (t, J = 6.8, 2H), 3.51 (t, J = 6.6, 2H), 1.82 (m, 4H), 13C NMR (CDCl3, 125 MHz): d = 130.1, 128.1, 51.2, 33.7, 32.4, 30.2, 26.8. Mass calculated for C6H10BrN3 204.0677. Observed (M+H) = 205.52; (M-H) = 203.42. h: 1H NMR (CDCl3, 400 MHz): d = 8.77 (s, 1H), 8.06 (s, 1H), 4.46 (t, J = 6.8, 2H), 3.51 (t, J = 6.6, 2H), 1.82 (m, 4H). 13C NMR (CDCl3, 125 MHz): d = 151.1, 147.2, 50.1, 33.4, 32.4, 29.2, 26.8. Mass calculated for C6H10BrN3 204.0677. Observed (M+H) = 205.52; (M-H) = 203.12.; (M-H) = 224.24.

Synthesis of intermediates for 13-16

Synthesis of hydroxy alkyl carboxylates

Synthesis of compound S1 (n=2 – 3). A solution of NaOH (1.2 eq) was added to lactone (1 equiv) at rt, and it was stirred for 24 h [52]. The solution was concentrated in vacuo, and the product was used for solid phase synthesis without purification.

S1 (n=2): The general procedure described for the synthesis of S1 (n=2) was followed using g-butyrolactone (824 uL; 10.8 mmol) to afford 1.24 g (9.8 mmol, 91 %) of product. Rf: 0.0 (ethyl acetate). 1H NMR (DMSO-d6, 400 MHz): d = 6.22 (t, J = 4.0, 1H), 3.39 (m, 2H), 2.02 (t, J = 6.0, 2H), 1.57 (m, 2H); ESI-LRMS calcd. For C4H7Na2O3 (M + Na+) 149.0, found 149.0.

S1 (n=3): The general procedure described for the synthesis of S1 (n=3) was followed using d(delta)-valerolactone (1.08 g; 10.8 mmol) to afford 1.50 g (10.7 mmol, 99 %) of product. The analytical data matched previously reported data.[52]

Synthesis of hydroxyl alkyl triazole analog moieties

Synthesis of compound S2 (n=1 – 4). To solution of alcohol (1 equiv) in CHCl3 (100 mL) was added TEA (1.2 equiv) followed by tert-butyldimethysilyl chloride (TBMSCl) (1.1 equiv) and DMAP (0.3 equiv) at rt under Ar.[53] The reaction mixture was stirred at rt for 10 h. The reaction mixture was then diluted with Et2O (30 mL), washed with 10% aqueous HCl solution (3 ´ 30 mL) followed by brine (30 mL), dried over Na2SO3, and concentrated in vacuo. The resulting residue was purified by silica gel chromatography eluting with 3:1 hexanes/ethyl acetate to provide the compound S2.

S2 (n=1): The general procedure described for the synthesis of S2 (n=1) was followed using 3-bromo-1-propanol (1.50 mL, 17.2 mmol) to afford 3.70 g (14.6 mmol, 85 %) of product. Rf: 0.73 (3:1 hexanes/ethyl acetate); 1H NMR (CDCl3, 500 MHz): d = 3.73 (t, J = 5.5, 2H), 3.51 (t, J = 6.5, 2H), 2.03 (m, 2H), 0.89 (s, 9H), 0.06 (s, 6H); 13C NMR (CDCl3, 125 MHz): d = 60.4, 35.5, 30.6, 25.9, 18.3, -5.4.

S2 (n=2): The general procedure described for the synthesis of S2 (n=2) was followed using 4-bromo-1-butanol (2.75 mL, 19.2 mmol) to afford 3.72 g (13.9 mmol, 73 %) of product. Rf: 0.74 (3:1 hexanes/ethyl acetate); 1H NMR (CDCl3, 500 MHz): d = 3.64 (t, J = 6.0, 2H), 3.45 (t, J = 7.0, 2H), 1.95 (m, 2H), 1.65 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H); 13C NMR (CDCl3, 125 MHz): d = 62.1, 34.0, 31.3, 29.5, 25.9, 18.3, -5.3.

S2 (n=3): The general procedure described for the synthesis of S2 (n=3) was followed 5-bromo-1-pentanol (1.45 mL, 12.0 mmol) to afford 2.19 g (8.62 mmol, 72 %) of product. Rf: 0.73 (3:1 hexanes/ethyl acetate); 1H NMR (CDCl3, 300 MHz): d = 3.61 (t, J = 6.3, 2H), 3.40 (t, J = 6.9, 2H), 1.87 (m, 2H), 1.50 (m, 4H), 0.89 (s, 9H), 0.04 (s, 6H).

S2 (n=4): The general procedure described for the synthesis of S2 (n=4) was followed using 6-bromo-1-hexanol (1.51 mL, 11.0 mmol) to afford 1.92 g (6.5 mmol, 59 %) of product. The analytical data matched previously reported data [53].

Synthesis of compound S3 (n=1 – 4). To a suspension of NaH (1.2 equiv) in DMF (30 mL) was added 1H-1,2,3-triazole (1.1 equiv) at rt under N2, and stirred for 1.5 h. Compound S2 (1 equiv) was added to the reaction mixture and stirred 15 h. The reaction was completed with the ratio of 1:2 product/side product due to the nucleophilicity of 2-N on 1H-1,2,3-triazole. The reaction mixture was concentrated in vacuo, and then diluted with EtOAc (30 mL), washed with water (3 ´ 30 mL), and dried over Na2SO3, and concentrated in vacuo. The resulting residue was purified by silica chromatography, eluting with 3:1 hexanes:ethyl acetate to 1:1 hexanes:ethyl acetate, to provide the compound S3.

S3 (n=1): The general procedure described for the synthesis of S3 (n=1) was followed using compound S2 (n=1) (3.20 g, 12.6 mmol) to afford 849 mg (3.52 mmol, 28 %) of product. Rf: 0.14 (3:1 hexanes/ethyl acetate); 1H NMR (CDCl3, 400 MHz): d = 7.69 (s, 1H), 7.57 (s, 1H), 4.51 (t, J = 7.2, 2H), 3.60 (t, J = 6.0, 2H), 2.11 (m, 2H), 0.90 (s, 9H), 0.05 (s, 6H); 13C NMR (CDCl3, 100 MHz): d = 133.5, 123.7, 59.0, 46.7, 32.9, 25.8, 18.1, -5.5. ESI-LRMS calcd. For C11H24N3OSi (M + H+) 242.2, found 242.1.

S3 (n=2): The general procedure described for the synthesis of S3 (n=2) was followed using compound S2 (n=2) (3.50 g, 13.1 mmol) to afford 861 mg (26 %) of product. Rf: 0.13 (3:1 hexanes/ethyl acetate); 1H NMR (CDCl3, 400 MHz): d = 7.65 (s, 1H), 7.53 (s, 1H), 4.39 (t, J = 7.2, 2H), 3.59 (t, J = 6.0, 2H), 1.95 (m, 2H), 1.49 (m, 2H), 0.84 (s, 9H), -0.01 (s, 6H); 13C NMR (CDCl3, 100 MHz): d = 133.6, 123.1, 62.1, 49.9, 29.3, 27.0, 25.8, 18.2, -5.5. ESI-LRMS calcd. For C12H25N3NaOSi (M + Na+) 278.2, found 278.1.

S3 (n=3): The general procedure described for the synthesis of S3 (n=3) was followed using compound S1 (n=3) (2.18 g, 7.75 mmol) to afford 492 mg (25 %) of product. Rf: 0.19 (3:1 hexanes/ethyl acetate); 1H NMR (CDCl3, 300 MHz): d = 7.66 (s, 1H), 7.52 (s, 1H), 4.36 (t, J = 7.2, 2H), 3.57 (t, J = 6.0, 2H), 1.91 (m, 2H), 1.55 (m, 2H), 1.35 (m, 2H), 0.85 (s, 9H), 0.01 (s, 6H). ESI-LRMS calcd. For C13H28N3OSi (M + H+) 270.2, found 270.2.

S3 (n=4): The general procedure described for the synthesis of S3 (n=4) was followed using compound S1 (n=4) (1.92 g, 6.52 mmol) to afford 655 mg (35 %) of product. Rf: 0.11 (3:1 hexanes/ethyl acetate); 1H NMR (CDCl3, 400 MHz): d = 7.66 (s, 1H), 7.55 (s, 1H), 4.36 (t, J = 7.6, 2H), 3.56 (t, J = 6.4, 2H), 1.90 (m, 2H), 1.46 (m, 2H), 1.34 (m, 4H), 0.85 (s, 9H), 0.01 (s, 6H). ESI-LRMS calcd. For C13H28N3OSi (M + H+) 284.2, found 284.2.

Synthesis of compound S4 (n=1 – 4). To solution of compound S3 (1 equiv) in THF was added 1M TBAF in THF (1.3 equiv) and stirred at rt for 10 h. The solution was concentrated in vacuo and purified by silica chromatography eluting with 9:1 ethyl acetate/methanol to provide the compound S4.

S4 (n=1): Procedure was followed using compound S3 (n=1) (547 mg, 2.27 mmol) to afford 272 mg (2.14 mmol, 94 %) of product. Rf: 0.38 (9:1 ethyl acetate/methanol); 1H NMR (CDCl3, 300 MHz): d = 7.61 (s, 1H), 7.60 (m, 1H), 4.48 (m, 2H), 3.57 (m, 2H), 2.07 (m, 2H); 13C NMR (CDCl3, 100 MHz): d = 133.4, 124.0, 58.1, 46.8, 32.6. ESI-LRMS calcd. For C5H10N3O (M + H+) 128.1, found 128.1.

S4 (n=2): The general procedure described for the synthesis of S4 (n=1) was followed using compound S3 (n=2) (834 mg, 3.37 mmol) to afford 424 mg (3.01 mmol, 95 %) of product. Rf: 0.41 (9:1 ethyl acetate/methanol); 1H NMR (DMSO-d6, 400 MHz): d = 7.99 (s, 1H), 7.72 (s, 1H), 4.48 (t, J = 7.2, 2H), 3.58 (t, J = 6.8, 2H), 1.99 (m, 2H), 1.52 (m, 2H). ESI-LRMS calcd. For C6H12N3O (M + H+) 142.1, found 142.1.

S4 (n=3): The general procedure described for the synthesis of S4 (n=3) was followed using compound S3 (n=3) (492 mg, 1.83 mmol) to afford 270 mg (1.74 mmol, 95 %) of product. Rf: 0.34 (9:1 ethyl acetate/methanol); 1H NMR (CDCl3, 300 MHz): d = 7.69 (s, 1H), 7.55 (s, 1H) 4.40 (t, J = 7.2, 2H), 3.58 (t, J = 3.9, 2H), 1.95 (m, 2H), 1.60 (m, 2H) (m, 2H), 1.40 (m, 2H). ESI-LRMS calcd. For C8H16N3O (M + H+) 156.1, found 156.1.

S4 (n=4): The general procedure described for the synthesis of S4 (n=4) was followed using compound S3 (n=4) (1.92 g, 6.52 mmol) to afford 655 mg (35 %) of product. Rf: 0.38 (9:1 ethyl acetate/methanol); 1H NMR (CDCl3, 300 MHz): d = 7.64 (s, 1H), 7.53 (s, 1H) 4.35 (t, J = 6.9, 2H), 3.58 (t, J = 6.3, 2H), 2.41 (s, 1H), 1.88 (m, 2H), 1.51 (m, 2H), 1.34 (m, 4H). ESI-LRMS calcd. For C8H16N3O (M + H+) 170.1, found 170.1.