3. Experimental Section
3.1. Materials
Normal whole blood was collected from adult donors at the Virginia Commonwealth University (Richmond, VA, USA) after informed consent. Hb was purified from discarded normal blood samples following a published procedure [
34]. The use of these human samples is in accordance with regulations of the IRB for Protection of Human Subjects. For the sickling assays, leftover blood samples from patients with homozygous SS were utilized, based on an approved IRB protocol at the Children’s Hospital of Philadelphia (Philadelphia, PA, USA), with informed consent.
3.2. Chemistry
Except as otherwise indicated, all reactions were carried out under a nitrogen atmosphere in flame- or oven-dried glassware. Reagents and solvents for chemical synthesis were purchased from Sigma-Aldrich (St. Louis, MO, USA; through Bayouni Imp. Inc., Dist., Jeddah, Saudi Arabia), Alfa Aesr (Haverhill, MA, USA; through Saggaf Co., Dist, Jeddah, Saudi Arabia) or Acros Organics (Geel, Belgium; through Abdullatif H. Abuljadayel Est., Dist, Jeddah, Saudi Arabia) as ACS-reagent grade; and used without further purification. Tetrahydrofuran (THF) was distilled from sodium/benzophenone-ketyl. Reactions were monitored by thin layer chromatography (TLC) with 0.25-mm pre-coated silica gel plates (E. Merck, Billerica, MA, USA). 1H-NMR spectra were recorded on an AV-300 NMR spectrometer (Bruker Billerica, MA, USA) equipped with the Top Spin software. Infrared spectra were recorded on a Bruker ATIR spectrometer. Melting points were recorded on a Buchi (Flawil, Switzerland; Bayouni Imp. Inc.) melting point apparatus and were uncorrected. LCMS were run on an Agilent 6130 Series (Santa Clara, CA, USA), single quad instrument. HPLC separation was run on 1200 SERIES HPLC using Chemstation Software (B.02, Agilent). Purities were assessed by HPLC and were confirmed to be >95% for all final compounds. Elemental analyses were performed using Vario, an Elementary apparatus (Shimadzu, Kyoto, Japan), located at the Organic Microanalysis Unit, Cairo University (Giza, Egypt). Column chromatography was performed on silica gel 60 (particle size 0.06–0.20 mm). Jones’ reagent was prepared by adding glacial acetic acid (6 mL) drop wise to an ice cold solution of CrO3 (6.7 g) in water (50 mL). The orange solution was stirred for 30 min at the same temperature. Freshly prepared solution was used for oxidation reactions.
3.3. General Procedure for the Synthesis of 2,3-Di/monosubstituted-4-[(1-trityl-1H-imidazol-2-yl)-hydroxymethyl]phenols 3a–c [40]
To a solution of 1-tritylimidazole (8.12 g, 26.160 mmol) in anhydrous THF (165 mL) was added n-BuLi (1.28 M in THF, 20.0 mL, 1.67 g, 13.08 mmol) at −20 °C over a period of 20 min under nitrogen atmosphere. The red solution was allowed to attain room temperature and stirred for 1 h, then cooled to −78 °C. In a separate flask the appropriate aldehyde 1a–c (10.47 mmol) was dissolved in anhydrous THF (4 mL) and added to the red solution dropwise at −78 °C. The reaction mixture was stirred at −78 °C for 1 h and slowly brought to room temperature during which red color tuned to yellow and then to colorless. After complete reaction, saturated NH4Cl (250 mL) was added to the reaction mixture at −78 °C. The resulting mixture was extracted with EtOAc (3 × 100 mL); the organic layer was separated, washed with water, saturated NaCl, and dried over anhydrous Na2SO4. The organic layer was evaporated in vacuo and the residue washed with cold CH2Cl2.
2,3-Dichloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenol (3a): white solid (3.99 g, 76%), m.p. 190.0–191.8 °C. 1H-NMR (DMSO-d6) δ 10.42 (s, 1H, OH), 7.25–7.21 (m, 9H, trityl-H), 7.08 (d, 2H, J = 8.7 Hz, Ar-H), 7.00–6.98 (m, 6H, trityl-H), 6.67 (d, 2H, J = 4.8 Hz, imidazole-H), 5.25 (d, 1H, J = 7.8 Hz, OH), 5.18 (d, 1H, J = 7.8 Hz, CH).
2-Chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenol (3b): white solid (2.76 g 93%), m.p. 165.2–166.5 °C. 1H-NMR (DMSO-d6) δ 9.83 (s, 1H, OH), 7.35–7.33 (m, 9H, trityl-H), 7.09–7.07 (m, 6H, trityl-H), 6.96 (s, 1H, Ar-H3), 6.57–6.39 (m, 3H, Ar-H and imidazole-H), 6.37 (d, 1H, J = 8.7 Hz, imidazole-H), 5.38 (d, 1H, J = 6.6 Hz, OH), 4.82 (d, 1H, J = 6.3 Hz, CH).
3-Chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenol (3c): white solid (2.1 g, 70.4%), m.p. 196.0–197.7 °C. 1H-NMR (DMSO-d6) δ 9.59 (s, 1H, OH), 7.24–7.00 (m, 10H, Ar-H and trityl-H), 6.99–6.65 (m, 6H, trityl-H), 6.64 (s, 1H, Ar-H2), 6.49–6.43 (m, 3H, Ar-H and imidazole-H), 5.23 (d, 1H, J = 6.6 Hz, OH), 4.90 (d, 1H, J = 6.6 Hz, CH).
3.4. General Procedure for the Synthesis of Ethyl {2,3-Di/monosubstituted-4-[(1-trityl-1H-imidazol-2-yl)-hydroxymethyl]phenoxy}esters 5a–f [41]
To a solution of the appropriate alcohol 3 (7.75 mmol) in anhydrous DMF (45 mL) was added K2CO3 (3.082 g, 22.300 mmol) at room temperature and stirred for 1 h. To the reaction mixture bromo ethyl esters 4 (10.70 mmol) was added and stirred for 5–6 h at room temperature. Water (450 mL) was added, and the precipitated solid was filtered and dried. The crude products were used as such in the next step without further purification.
Ethyl {2,3-dichloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}acetate (5a): white solid (4.82 g, 94%), m.p. 169.1–171.5 °C. The 1H-NMR (DMSO-d6) δ 7.39–7.10 (9H, m, trityl-H), 6.98–6.81 (m, 7H, trityl-H and Ar-H), 6.80 (d, 1H, J = 9 Hz, Ar-H), 5.56 (d, 1H, J = 6.9 Hz, OH), 4.80 (d,1H, J = 6.9 Hz, CH), 4.75 (s, 2H, OCH2), 4.18 (q, 2H, J = 6.9 Hz, CH2), 1.24 (t, 3H, J = 6.9 Hz, CH3).
Ethyl {2-chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}acetate (5b): white solid (2.0 g, 88.8%), m.p. 161.5–163.9 °C. 1H-NMR (DMSO-d6) δ 7.39–7.33 (m, 9H, trityl-H), 7.10–7.07 (m, 6H, trityl-H), 6.98 (s, 1H, Ar-H3), 6.66 (d, 1H, J = 8.7 Hz, Ar-H6), 6.57–6.49 (m, 3H, Ar-H5 and imidazole-H), 5.55 (s, 1H, OH), 4.87 (s, 1H, CH), 4.75 (s, 1H, OCH2), 4.16 (q, 2H, J = 7.1 Hz, CH2), 1.22 (t, 3H, J = 6.9 Hz, CH3).
Ethyl {3-chloro-4-[1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}acetate (5c): white solid (1.5 g, 84.4%), m.p. 189.3–192.3 °C. 1H-NMR (DMSO-d6) δ 7.30–7.22 (m, 10H, Ar-H and trityl-H), 7.08 (s, 1H, Ar-H2), 7.01–6.98 (m, 6H, trityl-H), 6.68–6.63 (m, 3H, Ar-H and imidazole-H), 5.25 (d, 1H, J = 6.9 Hz, OH), 5.12 (d, 1H, J = 6.9 Hz, CH), 4.75 (s, 2H, OCH2), 4.16 (q, 2H, J = 7.2 Hz, CH2), 1.23 (t, 3H, J = 6 Hz, CH3).
Ethyl 4-{[2,3-Dichloro-4-(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}butyrate (5d): white solid (3.0 g, 97.7%), m.p. 192.1–193.6 °C. 1H-NMR (DMSO-d6) δ 7.21–7.17 (m, 19H, trityl-H, Ar-H and imidazole-H), 5.8 (s, 1H, OH), 5.33 (t, 2H, J = 9 Hz, OCH2), 4.07 (m, 3H, CH and OCH2), 2.5 (2H, under DMSO, CH2), 1.98 (t, 2H, J = 6.6 Hz, CH2), 1.17 (t, 3H, J = 6.9 Hz, CH3).
Ethyl 4-{2-chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}butyrate (5e): white solid (4.1 g, 66%), m.p. 161.5–164.9 °C.1H-NMR (DMSO-d6) δH 7.33–7.20 (m, 9H, trityl-H), 7.09–7.08(m, 7H, trityl-H and Ar-H), 6.97 (s, 1H, Ar-H3), 6.73 (d, 1H, J = 8.7 Hz, Ar-H6), 6.56–6.54 (m, 2H, imidazole-H), 5.52 (s, 1H, OH), 4.86 (s, 1H, CH), 4.05 (q, 2H, J = 6.9 Hz, OCH2), 3.97 (t, 2H, J = 7.2, 6.9 Hz, OCH2), 2.45 (2H under DMSO, CH2), 1.93 (t, 2H, J = 6.6 Hz, CH2), 1.17 (t, 3H, J = 6. 9 Hz, CH3).
Ethyl 4-{3-Chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}butyrate (5f): white solid (3.7 g, 84.95%), m.p. 147.5–148.8 °C. 1H-NMR (DMSO-d6) δH 7.31–7.21 (m, 10H, trityl-H and ArH), 7.08 (s, 1H, Ar-H2), 7.01–6.99 (m, 6H, trityl-H), 6.66–6.61 (m, 3H, Ar-H and imidazole-H), 5.26 (s, 1H, OH), 5.06 (s, 1H, CH), 4.07 (q, 2H, J = 7.2, 6.9 Hz, CH2), 3.94(t, 2H, J = 6, 5.7 Hz, OCH2), 2.46 (under DMSO, CH2), 1.94(t, 2H, J = 6.6, 6.3 Hz, CH2), 1.19 (t, 3H, J = 6.9 Hz, CH3).
3.5. General Procedure for the Synthesis of Ethyl {[2,3-Di/monosubstituted-4-(1-trityl-1H-imidazol-2-yl)carbonyl]phenoxy}esters 6a–f [42]
To a solution of alcohol 5 (7.659 mmol) in acetone (45 mL), Jones reagent (30.6 mL) at 0 °C, was added. The reaction mixture was allowed to attain room temperature and stirred for 7–8 h. Then, water (50 mL) was added to the reaction mixture and extracted with CH2Cl2 (3 × 100 mL). The organic layer was separated, washed with water, followed by saturated NaCl. After drying over anhydrous Na2SO4, the organic layer was evaporated in vacuo; the obtained solid was collected and dried. The crude product was used as such in the next step without further purification.
Ethyl {[2,3-dichloro-4-(1-trityl-1H-imidazol-2-yl)carbonyl]phenoxy}acetate (6a): pale yellow solid (2.8 g, quantitative), m.p. 116.0–120.5 °C. 1H-NMR (DMSO-d6) δH 7.62 (d, 2H, J = 8.7 Hz, Ar-H), 7.30–7.19 (m, 15H, trityl-H), 6.45 (d, 2H, J = 1. 2 Hz, imidazole-H), 5.07 (s, 2H, OCH2), 4.19 (q, 2H, J = 7.2, 6.9 Hz, CH2), 1.23 (t, 3H, J = 7.2, 6.9 Hz, CH3).
Ethyl {[2-Chloro-4-(1-trityl-1H-imidazol-2-yl)carbonyl]phenoxy}acetate (6b): white solid (1.1 g, quantitative), m.p. 160.3–161.6 °C. 1H-NMR (DMSO-d6) δH 7.08–7.53 (m, 19H, trityl-H, Ar-H and imidazole-H), 6.46 (s, 1H, Ar-H3), 5.07 (s, 2H, OCH2), 4.20 (q, 2H, J = 7.2, 6.9 Hz, CH2), 1.23 (t, 3H, J = 7.2, 6.9 Hz, CH3).
Ethyl {[3-Chloro-4-(1H-imidazol-2-yl)carbonyl]phenoxy}acetate (6c): white solid (0.9 g, quantitative), m.p. 171.5–172.0 °C. 1H-NMR (DMSO-d6) δH 13.56 (s, 1H, NH), 7.74 (d, 1H, J = 6.9 Hz, Ar-H5), 7.54 (d, 1H, J = 1.2 Hz, Ar-H2), 7.22–7.01 (m, 3H, imidazole-H and Ar-H6), 4.95 (s, 2H, OCH2), 4.20 (q, 2H, J = 7.2, 6.9 Hz, CH2), 1.24 (t, 3H, J = 7.2, 6.9 Hz, CH3).
Ethyl 4-{[2,3-Dichloro-4-(1-trityl-1H-imidazol-2-yl)carbonyl]phenoxy}butyrate (6d): pale yellow solid (1.8 g, quantitative). The crude product was used as such in the next step without further purification. 1H-NMR (DMSO-d6) δH 7.33–7.19 (m, 19H, trityl-H, Ar-H and imidazole-H), 4.22 (t, 2H, J = 6.3, 6 Hz, OCH2), 4.07 (q, 2H, J = 7.2, 3.9 Hz, CH2), 3.55 (under DMSO, CH2), 3.05 (m, 2H,CH2), 1.18 (t, 3H, J = 7.2 Hz, CH3).
Ethyl 4-{[2-Chloro-4-(1-trityl-1H-imidazol-2-yl)carbonyl]phenoxy}butyrate (6e): white solid (3.7 g, quantitative), m.p. 169.1–171.5 °C. 1H-NMR (DMSO-d6) δH 7.30–7.19 (m, 19H, trityl-H, Ar-H and imidazole-H), 6.46 (s, 1H, Ar-H3), 4.22 (t, 2H, J = 6.3, 6 Hz, OCH2), 4.07 (q, 2H, J = 7.2, 6.9 Hz, OCH2), 3.55 (under DMSO, CH2), 2.07-2.03 (m, 2H,CH2), 1.18 (t, 3H, J = 7.2, 6.9 Hz, CH3).
Ethyl 4-{[3-Chloro-4-(1H-imidazol-2-yl)carbonyl]phenoxy}butyrate (6f): white solid (1.8 g, 89.74%), m.p. 112.1–114.0 °C. 1H-NMR (DMSO-d6) δH 13.52 (s, 1H, NH), 7.75 (d, 1H, J = 8.4 Hz, Ar-H5), 7.53 (s, 1H, Ar-H2), 7.22–6.99 (m, 3H, ArH6 and imidazole-H), 4.11–4.10 (m, 4H, two OCH2), 2.51 (under DMSO, CH2), 1.99 (t, 2H, J = 6.6 Hz, CH2), 1.19 (t, 3H, J = 6.9 Hz, CH3).
3.6. General Procedure for the Synthesis of {[2,3-Di/monosubstituted-4-(1H-imidazol-2yl)carbonyl]-phenoxy}acetic/butyric acids (7a–f) [43]
To solution of 6 (8.159 mmol) in aqueous THF (12 mL/ water 6 mL) was added LiOH·H2O (0.687 g, 16.37 mmol) at room temperature. The reaction mixture was stirred for 1 h during which the solution turned to green. The reaction was monitored with TLC. After complete reaction, the solvent was evaporated in vacuo. The crude material was then dissolved in minimum amount of water. The precipitated solid was filtered and the pH of the filtrate was adjusted to 6–7 using aqueous 1.5 N HCl, and the obtained solid product was collected by filtration and dried. The crude material was purified by column chromatography over silica gel (230–400 mesh) using 40 to 50% ethyl acetate in hexane as eluent to afford the desired final products 7a–i.
{[2,3-Dichloro-4-(1H-imidazol-2-yl)carbonyl]phenoxy}acetic acid, (7a): white solid (1.22 g, 51%), m.p. 240.2–243 °C. IR (KBr, νmax cm−1): 3527 (OH), 3200 (NH), 1662 (CO), 1269, 1077 (C-O-C); 1H-NMR (DMSO-d6) δH 13.64 (s, 1H, NH), 7.64–7.57 (m, 2H, Ar-H), 7.22–7.13 (m, 2H, imidazole-H), 4.95 (s, 2H, OCH2); LC/MS (ESI) m/z 315 (M + 1), 316 (M + 2), 317 (M + 3), 318 (M + 4).
{[2-Chloro-4-(1H-imidazol-2-yl)carbonyl]phenoxy}acetic acid (7b): pale yellow solid (1.04 g, 81.7%), m.p. 303–306 °C. 1H-NMR (DMSO-d6) δH 13.42 (brs, 1H, NH), 8.76 (d, 1H, J = 1.8 Hz, Ar-H3), 8.39 (t, 1H, J = 9, 1.8 Hz, Ar-H5), 7.38 (s, 2H, imidazole-H), 6.97 (d, 1H, J = 9 Hz, Ar-H6), 4.38 (s, 2H, OCH2); LC/MS (ESI) m/z 280.1 (M + ), 282.1 (M + 2).
{3-Chloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}acetic acid (7c): white solid (0.64 g, 64.0%), m.p. 230.8–233.9 °C. IR (KBr, νmax cm−1): 3497 (NH), 3306 (OH), 1647 (CO), 1269, 1085 (C-O-C); 1H-NMR (DMSO-d6) δH 13.56 (s, 1H, NH), 7.72 (d, 1H, J = 8.7 Hz, Ar-H5), 7.51 (s, 1H, imidazole-H), 7.20 (s, 1H, imidazole-H), 6.96 (d, 1H, J = 2.1 Hz, Ar-H2), 6.88 (t, 1H, J = 6.3, 2.4 Hz Ar-H6), 4.40 (s, 2H, OCH2); LC/MS (ESI) m/z 280.1 (M + )−, 282.1 (M + 2)−.
4-{2,3-Dichloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}butyric acid (7d): white solid (1.15 g, 69%), m.p. 167.5–169.4 °C. IR (KBr, νmax cm−1): 3527 (NH), 3247 (OH), 1643 (CO), 1274, 1041 (C-O-C). 1H-NMR (DMSO-d6) δH 13.61 (s, 1H, NH), 7.64 (d, 1H, J = 8.4 Hz, Ar-H5), 7.36 (s, 2H, imidazole-H), 7.29 (d, 1H, J = 8.7 Hz, Ar-H6), 4.19 (t, 2H, J = 6.6 Hz, OCH2), 2.13 (t, 2H, J = 6.6 Hz, OCH2), 1.94 (q, 2H, J = 6.6, 6.3 Hz, CH2). LC/MS (ESI) m/z 341 (M − 1)−, 343 (M + 1)−, 344 (M + 2)−, 346 (M + 4)−.
4-{2-Chloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}butyric acid (7e): white solid (2.4 g, 71%), m.p. 169.1–171.5 °C. 1H-NMR (DMSO-d6) δH 13.52 (brs, 1H, NH), 8.73 (s, 1H, Ar-H3), 8.52 (d, 1H, J = 7.8 Hz, Ar-H5), 7.41–7.32 (m, 3H, Ar-H6 and imidazole-H), 4.20 (t, 2H, J = 6 Hz, OCH2), 2.3 (t, 2H, J = 6 Hz, CH2), 1.99–1.97(m, 2H, CH2). LC/MS (ESI) m/z 307.1 (M − 1)−, 308 (M + )−, 309.1 (M + 1)−, 310 (M + 2)−, 312 (M + 4)−.
4-{3-Chloro-4-[(1H-imidazole-2-yl)carbonyl]phenoxy}butyric acid (7f): white solid (0.8 g, 48.5%), m.p. 188.4–191.1 °C. 1H-NMR (DMSO-d6) δH 13.53 (s, 1H, NH), 12.19 (s,1H,OH), 7.76 (d, 1H, J = 8.7 Hz, Ar-H5), 7.52 (s, 1H, Ar-H2), 7.23 (s, 1H, imidazole-H), 7.13(s, 1H, imidazole-H), 7.02 (d, 1H, J = 8.4 Hz, Ar-H6), 4.10 (t, 2H, J = 6.3, 6 Hz, OCH2), 2.40 (2H, under DMSO, CH2), 1.97 (m, 2H, CH2); LC/MS (ESI) m/z 307 (M − 1)−, 309.1 (M + 1)−, 310.1 (M + 2)−, 312 (M + 4)−.
3.7. General Procedure for the Synthesis of 3-{2,3-Di/monochloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxy-methyl]phenoxy}propanols 9a–c [41]
To a solution of the appropriate alcohol 3a–c (12.76 mmol) in anhydrous DMF (65 mL) was added K2CO3 (4.4 g, 31.84 mmol) at room temperature and stirred for 1 h. To the reaction mixture, 3-bromopropanol (2.12 g, 1.4 mL, 15.31 mmol) was added and stirred for 16 h at room temperature. The solid formed after addition of water (650 mL) was filtered, treated with diethyl ether and dried in vacuo.
3-{2,3-Dichloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}propanol (9a): white solid (6.34 g, 88.78%), m.p. 174.7–177.7 °C. 1H-NMR (DMSO-d6) δH 7.25–7.10 (m, 11H, trityl-H and Ar-H), 7.0–6.9 (m, 7H, trityl-H and imidazole-H), 6.87 (d, 1H, J = 8.4 Hz, imidazole-H), 5.29 (s, 2H, OH and CH), 4.6 (brs, 1H, OH), 4.09 (t, 2H, J = 6, 6.3 Hz, OCH2), 3.57 (t, 2H, J = 6, 6.3 Hz, OCH2), 1.83 (quint, 2H, J = 6.3 Hz, CH2).
3-{2-Chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}propanol (9b): white solid (3.9 g, 88.94%), m.p. 155.9–157.5 °C. 1H-NMR (DMSO-d6) δH 7.34–7.33 (m, 9H, trityl-H), 7.10–7.08 (m, 6H, trityl-H), 6.97 (s, 1H, Ar-H3), 6.74 (d, 1H, J = 8.4 Hz, Ar-H6), 6.57–6.51 (m, 3H, ArH and imidazole-H), 5.57 (brs, 1H, OH), 4.86 (s, 1H, CH), 4.59 (brs, 1H, OH), 4.01 (t, 2H, J = 6, 6.3 Hz, OCH2), 3.54 (t, 2H, J = 6, 6.3 Hz, OCH2), 1.83 (quint, 2H, J = 6.3 Hz, CH2).
3-{3-Chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}propanol (9c): white solid (4.5 g, 87.0%), m.p. 182.3–183.9 °C. 1H-NMR (DMSO-d6) δH 7.31–7.28 (m, 10H, Ar-H and trityl-H), 7.22 (s, 1H, Ar-H2), 7.08–7.00 (m, 6H, trityl-H), 6.66–6.60 (m, 3H, ArH and imidazole-H), 5.26 (s, 1H, CH), 5.04 (brs, 1H, OH), 4.56 (brs, 1H, OH), 3.97 (t, 2H, J = 6, 5.4 Hz, OCH2), 3. 49 (t, 2H, J = 6, 6.3 Hz, OCH2), 1.81 (quint, 2H, J = 6, 5.7 Hz, CH2).
3.8. General Procedure for the Synthesis of 3-{2,3-Di/monochloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}-propionic Acids 7g–i [42]
To a solution of alcohol 9a–c (10.72 mmol) in acetone (60 mL), Jones reagent (25 mL) was added at 0 °C. The reaction mixture was allowed to attain room temperature and stirred for 48 h and filtered to remove insoluble impurities. The filtrate was extracted with diethyl ether (6 × 50 mL). The organic layer was separated, dried over anhydrous Na2SO4 and evaporated in vacuo to get a pale yellow solid. The product was dissolved in 5% NaHCO3 solution (minimum amount) and washed with diethyl ether (2 × 50 mL) followed by ethyl acetate (2 × 50 mL). The pH of the aqueous layer was carefully adjusted to 6 using 1.5 N aqueous HCl. The solid product formed was filtered, washed with diethyl ether and dried.
3-{2,3-dichloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}propionic acid, 7g: white solid (0.45 g, 12.75%), m.p. 207 °C (decomposed). IR (KBr, νmax cm−1): 3497(NH), 3306 (OH), 1647(CO), 1600, 1409 (CO2−), 1270, 1035 (C-O-C). 1H-NMR (DMSO-d6) δ 13.64 (s, 1H, NH), 7.67 (d, 1H, J = 8.7 Hz, Ar-H5), 7.40 (s, 2H, imidazole-H), 7.28 (d, 1H, J = 8.7 Hz, Ar-H6), 4.36 (t, 2H, J = 6 Hz, OCH2), 2.68 (2H, J = 6.3, 5.7 Hz, CH2).
3-{2-Chloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}propionic acid (7h): white solid (1.0 g, 24.08%), m.p. 195.0–195.4 °C. IR (KBr, νmax cm−1): 3294 (NH), 3000–2800 (OH), 1703 (CO), 1613(CO), 1583 (C=N), 1254, 1037 (C-O-C). 1H-NMR (DMSO-d6) δH 13.47 (s, 1H, NH), 12.49(s, 1H, OH), 8.72 (d, 1H, J = 1.8 Hz, Ar-H3), 8.53 (t, 1H, J = 6.6, 2.1 Hz, Ar-H5), 7.52 (s, 1H, imidazole-H), 7.35(d, 1H, J = 8.7 Hz, Ar-H6), 7.31 (s, 1H, imidazole-H), 4.38 (t, 2H, J = 6, 5.7 Hz, OCH2), 2.78 (t, 2H, J = 6, 5.7 Hz, CH2). LC/MS (ESI) m/z 293 (M − 1), 295 (M + 1), 296 (M + 2).
3-{3-Chloro-4-(1H-imidazol-2-yl)carbonyl]phenoxy}propionic acid (7i): white solid (0.536 g, 21.22%), m.p. 193.0–194.2 °C. IR (KBr, νmax cm−1): 3301 (NH), 3100–2600 (OH), 1717 (CO), 1645 (CO), 1598 (C=N), 1294, 1033 (C-O-C). 1H-NMR (DMSO-d6) δH 13.53 (s, 1H, NH), 12.43 (s, 1H, OH), 7.75 (d, 1H, J = 8.7 Hz, Ar-H5), 7.53 (s, 1H, imidazole-H), 7.22 (s, 1H, imidazole-H) 7.14 (d, 1H, J = 2.1 Hz, Ar-H2), 7.02 (dd, 1H, J = 6.3, 2.4 Hz, Ar-H6), 4.28 (t, 2H, J = 6, 5.7 Hz, OCH2), 2.73 (t, 2H, J = 6, 5.7 Hz, CH2). LC/MS (ESI) m/z 293 (M − 1), 294 (M + ), 295 (M + 1), 296 (M + 2).
3.9. 3-Chloro-4-(1H-imidazol-2-yl)phenol (11) [30]
Glyoxal 10 (40% in H2O, 14 mL, 0.095 mol) was placed in water (20 mL), then added to a cooled solution (about 5 °C) of 2-chloro-4-hydroxybenzaldehyde 1c (5 g, 0.031 mmol) in methanol (10 mL) to afford a white turbid solution. Ammonium hydroxide (25% in H2O, 67 mL, 0.478 mmol) was added drop wise over a period of 1 h at 0–5 °C and the reaction mixture was stirred for 3 h at 0–5 °C. The mixture was allowed to warm to room temperature and stirred further for 24 h. The solution was concentrated in vacuo and the crude was purified by column chromatography using mixture of DCM/MeOH (95:5) as an eluent to obtain 11 as a beige solid (6.4 g, quantitative), m.p. 155–158 °C. 1H-NMR (300 MHz, DMSO-d6) δH 12.07 (brs, 1H, NH), 10.20 (brs, 1H, OH), 7.52–7.63 (m, 1H, Ar-H6), 7.10 (s, 1H, Ar-H2), 6.91 (s, 2H, imidazole-H), 6.83 (d, J = 8.50 Hz, 1H, Ar-H5).
3.10. Ethyl 2-[3-Chloro-4-(1H-imidazol-2-yl)phenoxy]acetate (12) [42]
To a cold (5–10 °C) solution of 11 (9.0 g, 0.049 mol) and K2CO3 (1 g, 0.070 mol), KI (0.37 g, 0.005 mol) in DMF (90 mL), was added ethyl bromoacetate (7 g, 0.049 mol) while stirring. The stirring was continued for 1 h at room temperature. Then the reaction was treated with water (150 mL) and extracted in DCM (2 × 100 mL). The combined organic layer was washed with water and brine, dried with anhydrous Na2SO4, and evaporated under vacuum to give the crude product. Purification was carried out using flash chromatography (hexane–EtOAC 3:7) to furnish a white solid of 12 (1.4 g, 11%), m.p. 131–133 °C. 1H-NMR (300 MHz, DMSO-d6) δH ppm 12.29 (brs, 1H, NH) 7.67 (d, J = 8.78 Hz, 1H, Ar-H5), 7.07–7.21 (m, 3H, Ar-H2 and imidazole-H), 7.03 (dd, J = 8.69, 2.55 Hz, 1H, Ar-H6), 4.90 (s, 2H, OCH2), 4.19 (q, J = 7.08 Hz, 2H, CH2), 1.22 (t, J = 7.08 Hz, 3H, CH3).
3.11. 2-[3-Chloro-4-(1H-imidazol-2-yl)phenoxy]acetic acid (13) [43]
To a solution of 12 (1.4 g, 0.005 mol) in THF (5.6 mL) and water (2.8 mL) was added LiOH·H2O (0.36 g, 0.015 mol) at room temperature and stirred for 30 min until consumption of the starting material. The reaction was treated with 1.5N HCl until the pH of the reaction mixture became 6.5. The precipitated product was filtered and dried under vacuum as an off-white solid, (1.15 g, 92%), m.p. 313–316 °C.1H-NMR (300 MHz, DMSO-d6) δH ppm 14.75 (brs,1H, NH), 7.81 (s, 2H, imidazole-H), 7.71 (d, J =8.69 Hz, 1H, Ar-H5), 7.34 (d, J = 2.46 Hz, 1H, Ar-H2), 7.18 (dd, J = 8.69, 2.46 Hz, 1H, Ar-H6), 4.88 (s, 2H, OCH2); LC-MS (ESI) m/z 251.9 (M + )−, 253.9 (M + 2)−; Anal. Calcd for (C11H9ClN2O3): C, 52.29; H, 3.59; Cl, 14.03; N, 11.09; Found: C, 52.15; H, 3.24; Cl, 14.40; N, 11.12.
3.12. General Procedure for the Synthesis of Ethyl [(2,3-Di/monochloro-4-formyl)phenoxy]acetates 14a,b [41]
To a cold mixture of 4-hydroxybenzaldehydes 1a,b (0.01 mol) and K2CO3 (0.015 mol) in DMF (20 mL), was added ethyl bromoacetate (0.012 mol) at 5–10 °C. The reaction mixture was stirred for 1hr then treated with water (20 mL) and extracted in DCM (2 × 25 mL). The combined organic extract was washed with water and brine then dried over anhydrous MgSO4. After evaporation in vacuo, the residue was treated with hexane to give the esters 14a,b.
Ethyl [(2,3-dichloro-4-formyl)phenoxy]acetate (14a): pale brown solid (2.4 g, 82%), m.p. 134–138 °C. 1H-NMR (300 MHz, CDCl3) δH ppm 10.39 (s, 1H, CHO); 7.87 (d, J = 8.78 Hz, 1H, Ar-H), 6.84 (d, J = 8.78 Hz, 1H, Ar-H), 4.83 (s, 2H, OCH2), 4.31 (q, J = 7.14 Hz, 2H, CH2), 1.32 (t, J = 7.13 Hz, 3H, CH3).
Ethyl [(2-chloro-4-formyl)phenoxy]acetate (14b): clear liquid (4.3 g, 94% yield); 1H-NMR (300 MHz, DMSO-d6) δH ppm 9.88 (s, 1H, CHO), 7.93–8.05 (m, 1H, Ar-H3), 7.86 (d, J = 8.50 Hz, 1H, Ar-H5), 7.30 (d, J = 8.50 Hz, 1H, Ar-H6), 5.09 (s, 2H, CH2), 4.25 (q, J = 7.04 Hz, 2H), 1.23 (t, J = 7.06 Hz, 3H, CH3).
3.13. General Procedure for the Synthesis of Ethyl 4-[(Di/monochloro-4-formyl)phenoxy]butanoates 14c–e [41]
To a solution of 1a–c (0.018 mol) in N-methylpyrrolidone (NMP, 30 mL) was added K2CO3 (0.0275 mol) and KI (1.8 mmol) at room temperature. After stirring for 30 min, ethyl 4-bromobutyrate (3.5 g, 0.0183 mol) was added, and the reaction was continued for 7 h at 60 °C. The reaction mixture was diluted with water (50 mL) and extracted with DCM (3 × 75 mL). The combined organic extract was washed with water then brine and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by column chromatography (6% EtOAc in hexane) to give the products 14c–e.
Ethyl 4-[(2,3-dichloro-4-formyl)phenoxy]butanoate (14c): white solid (4 g, 72%), m.p. 163–165 °C. 1H-NMR (300 MHz, DMSO-d6) δH ppm 10.19 (s, 1H, CHO), 7.84 (d, J = 8.88 Hz, 1H, Ar-H5), 7.32 (d, J = 8.78 Hz, 1H, Ar-H6), 4.25 (t, J = 6.23 Hz, 2H, CH2), 4.07 (q, J = 7.08 Hz, 2H, CH2), 2.51 (t, 2H, Under DMSO), 2.04 (quin, J = 6.77 Hz, 2H, CH2), 1.14 (q, J = 6.31, 3H, CH3).
Ethyl 4-[(2-chloro-4-formyl)phenoxy]butanoate (14d): clear liquid (7.1 g, 82%); 1H-NMR (300 MHz, DMSO-d6) δH ppm 9.86 (s, 1H, CHO), 7.94 (s, 1H, Ar-H3), 7.88 (d, J = 8.50 Hz, 1H, Ar-H6), 7.35 (d, J = 8.50 Hz, 1H, Ar-H5), 4.22 (t, J = 6.23 Hz, 2H, CH2), 4.07 (q, J = 7.08 Hz, 2H, CH2), 2.39 (2H, under DMSO), 1.96–2.12 (m, 2H, CH2), 1.18 (t, J = 7.13 Hz, 3H, CH3).
Ethyl 4-[(3-chloro-4-formyl)phenoxy]butanoate (14e): white solid, (5 g, 82%), m.p. 118–121 °C. 1H-NMR (300 MHz, DMSO-d6) δH 10.19 (s, 1H, CHO), 7.83 (d, J = 8.78 Hz, 1H, Ar-H5), 7.18 (d, J = 1.70 Hz, 1H, Ar-H3), 7.08 (dd, J = 8.78, 2.27 Hz, 1H, Ar-H6), 4.10–4.20 (m, 2H, CH2), 4.02–4.10 (m, 2H, CH2), 2.41–2.49 (2H, under DMSO, CH2), 1.99 (t, J = 6.80 Hz, 2H, CH2), 1.18 (t, J = 7.13 Hz, 3H, CH3)
3.14. General Procedure for the Synthesis of Ethyl [Di/monochloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]-acetate/butanoates 15a–e [44]
To a stirred solution of ethyl esters 14a–e (8.6 mmol) in chloroform (25 mL), was added ethylenediamine at 0 to 5 °C. After stirring for 30 min at room temperature, N-bromosuccinimide was added at 0 to 5 °C over 20 min. The reaction mixture was slowly warmed to room temperature and stirred for 16 h. NaHCO3 10% solution (25 mL) was added to quench the reaction, followed by extraction with chloroform (3 × 50 mL). The combined organic extract was concentrated to afford crude product 15a–e.
Ethyl [2,3-dichloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]acetate (15a): pale yellow solid (2.5 g, 91%), m.p. 132–134 °C. 1H-NMR (300 MHz, DMSO-d6) δH 7.45 (d, J = 8.69 Hz,1H, Ar-H5), 7.12 (d, J = 8.78 Hz, 1H, Ar-H6), 6.79 (brs, 1H, NH), 5.03 (s, 2H, OCH2), 4.18 (q, J = 7.08 Hz, 2H, CH2), 3.59 (brs, 4H, imidazoline-H), 1.22 (t, J = 7.03 Hz, 3H, CH3).
Ethyl [2-chloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]acetate (15b): greyish white solid (3.8 g, 66%), m.p. 124–126 °C. 1H-NMR (300 MHz, DMSO-d6) δH 7.89 (s, 1H, NH), 7.73 (d, J = 8.69 Hz, 1H, Ar-H6), 7.11 (brs, 1H, Ar-H3), 7.12 (d, J = 8.69 Hz, 1H, Ar-H5), 4.98 (s, 2H, OCH2) 4.18 (q, J = 7.02 Hz, 2H, CH2), 3.60 (s, 4H, imidazoline-H) 1.22 (t, J = 7.08 Hz, 3H, CH3).
Ethyl 4-[2,3-dichloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]butanoate (15c): pale greyish white solid (4 g, 89%); 1H-NMR (300 MHz, DMSO-d6) δH 7.48 (d, J = 8.59 Hz, 1H, Ar-H6), 7.19 (d, J = 8.88 Hz, 1H, Ar-H5) 4.13–4.25 (m, 2H, CH2) 4.07 (q, J = 6.89 Hz, 2H, CH2), 3.60 (m, 4H, imidazoline-H), 2.51 (t, 2H, under DMSO), 1.94–2.10 (m, 2H, CH2), 1.18 (t, J = 6.76 Hz, 3H, CH3).
Ethyl 4-[2-chloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]butanoate (15d): T pale greyish white solid (6.2 g, 76%), m.p. 138–140 °C.1H-NMR (300 MHz, DMSO-d6) δH 7.88 (d, J = 1.51 Hz, 1H, Ar-H3), 7.77 (dd, J = 8.64, 1.65 Hz, 1H, Ar-H5), 7.22(d, J = 8.59 Hz, 1H, Ar-H6), 4.14 (t, J = 6.98 Hz, 2H, CH2), 4.05 (q, J = 7.12 Hz, 2H,CH2), 3.62 (m, 4H, imidazoline-H), 2.47 (under DMSO, 2H, CH2), 2.01 (m, 2H, CH2), 1.18 (t, J = 7.08 Hz,3H, CH3).
Ethyl 4-[3-chloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]butanoate (15e): pale yellow solid (5 g, 87%), m.p. 137–139 °C. 1H-NMR (300 MHz, DMSO-d6) δH 7.52 (d, J = 8.69 Hz, 1H, Ar-H5), 7.07 (d, J = 2.27 Hz, 1H, Ar-H2), 6.95 (dd, J = 8.64, 2.31 Hz, 1H, Ar-H6), 3.99–4.10 (m, 4H, imidazoline-H), 3.60 (m, 4H, 2CH2), 2.45 (t, J = 7.32 Hz, 2H, CH2), 1.97 (t, J = 6.80 Hz, 2H, CH2),1.18 (t, J = 7.08 Hz, 3H, CH3).
3.15. General Procedure for the Synthesis of Ethyl [2,3-Dichloro-4-(1H-imidazol-2-yl)phenoxy]acetate/butanoates 16a–e [44]
To a solution of 15a–e (7.8 mmol) in DMSO (25 mL) was added K2CO3 (8.6 mmol) and diacetoxyiodobenzene (8.6 mmol) at room temperature. The reaction mixture was stirred for 60 min in a dark place, and then water was added followed by extraction with DCM (3 × 50 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was subjected to flash column chromatography (EtOAc 2:hexane 1) to afford the products 16a–e.
Ethyl [2,3-dichloro-4-(1H-imidazol-2-yl)phenoxy]acetate (16a): off-white solid (1.1g, 44%), m.p. 193–194 °C. 1H-NMR (300 MHz, DMSO-d6) δH 12.31 (brs, 1H, NH), 7.62 (d, J = 8.78 Hz, 1H, Ar-H6), 7.06–7.26 (m, 3H, Ar-H and imidazole-H), 5.04 (s, 2H, OCH2), 4.19 (q, J = 7.05 Hz, 2H, CH2), 1.22 (t, J = 7.08 Hz, 3H, CH3).
Ethyl [2-chloro-4-(1H-imidazol-2-yl)phenoxy]acetate (16b): greyish white solid (1.5 g, 43%), m.p. 164–166 °C. 1H-NMR (300 MHz, DMSO-d6) δH 12.47 (brs, 1H, NH), 8.00 (d, J = 2.08 Hz, 1H, Ar-H3), 7.82 (dd, J = 8.59, 2.17 Hz, 1H, Ar-H5), 4.96 (s, 2H, OCH2), 7.15 (m, 3H, Ar-H and imidazole-H), 4.19 (q, J = 7.08 Hz, 2H, CH2) 1.22 (t, J = 7.13 Hz, 3H, CH3).
Ethyl 4-[2,3-dichloro-4-(1H-imidazol-2-yl)phenoxy]butanoate (16c): off-white solid (2.3 g, 55.8%), m.p. 226–227 °C. 1H-NMR (300 MHz, CDCl3) δH 10.19 (brs, 1H, NH), 8.14 (d, J = 8.88 Hz, 1H, Ar-H6), 7.21 (s, 2H, imidazole-H), 6.98 (d, J = 8.97 Hz, 1H, Ar-H5), 4.00–4.34 (m, 4H, 2CH2), 2.61 (t, J = 7.18 Hz, 2H, CH2), 2.21 (quin, J = 6.61 Hz, 2H, CH2), 1.16–1.44 (m, 3H, CH3).
Ethyl 4-[2-chloro-4-(1H-imidazol-2-yl)phenoxy]butanoate (16d): greyish white solid (3.4 g, 57%), m.p. 179–182 °C. 1H-NMR (300 MHz, DMSO-d6) δH ppm 12.44 (brs, 1H, NH), 7.98 (d, J = 1.98 Hz, 1H, Ar-H3), 7.85 (dd, J = 8.59, 1.98 Hz, 1H, Ar-H5), 7.23(d, J = 8.69 Hz, 1H, Ar-H6), 7.10 (m, 2H, imidazole-H), 4.03–4.17 (m, 4H, 2CH2), 2.48 (under DMSO, 2H, CH2), 1.94–2.08 (m, 2H, CH2), 1.18 (t, J = 7.08 Hz, 3H, CH3).
Ethyl 4-[3-chloro-4-(1H-imidazol-2-yl)phenoxy]butanoate (16e): greyish white solid (2.58 g, 50%), m.p. 168–172 °C. 1H-NMR (300 MHz, DMSO-d6) δH 7.68 (d, J = 8.69 Hz, 1H, Ar-H5), 7.05–7.19 (m, 3H, Ar-H2 and imidazole-H), 7.00 (dd, J = 8.69, 2.55 Hz, 1H, Ar-H6), 3.99–4.15 (m, 4H, 2CH2), 2.43–2.50 (under DMSO, 2H, CH2), 1.99 (quin, J = 6.75 Hz, 2H, CH2) 1.19 (t, J = 7.13 Hz, 3H, CH3).
3.16. General Procedure for the Synthesis of 2-[Di/monochloro-4-(1H-imidazol-2-yl)phenoxy]acetic/butyric Acids 17a–e [43]
These compounds were prepared according to procedure described above for preparation of 7a–f, starting with esters 16a–e (3.2 mmol).
2-[2,3-Dichloro-4-(1H-imidazol-2-yl)phenoxy]acetic acid (17a): off-white solid (0.87 g, 82%), m.p. >300 °C (dec.). 1H-NMR (300 MHz, DMSO-d6) δH 15.08 (brs, 1H, OH), 7.81 (s, 1H, NH), 7.70 (d, J = 8.88 Hz, 1H, Ar-H6) 7.34 (d, J = 9.06 Hz, 1H, Ar-H5), 7.0–7.01 (m, 2H, imidazole-H), 5.00 (s, 2H,OCH2), LC/MS, m/z 286.9 (M + 1)+, 288.9 (M + 3)+, 291.1 (M + 5)+. Anal. Calcd for (C11H8Cl2N2O3): C, 46.02; H, 2.81; Cl, 24.70; N, 9.76; Found: C, 46.21; H, 3.02; Cl, 24.34; N, 9.49.
[2-Chloro-4-(1H-imidazol-2-yl)phenoxy]acetic acid (17b): off-white solid (1.2 g, 88.8%), m.p. >300 °C (dec.); 1H-NMR (300 MHz, DMSO-d6) δH 7.99 (d, J = 2.08 Hz, 1H, Ar-H3), 7.82 (dd, J = 8.69, 1.89 Hz, 1H, Ar-H5), 7.02–7.20 (m, 3H, Ar-H6 and imidazole-H), 4.85 (s, 2H, OCH2); LC/MS, m/z 251.0 (M−), 253.1 (M + 2)−.
4-[2,3-Dichloro-4-(1H-imidazol-2-yl)phenoxy]butanoic acid (17c): off-white solid (0.87 g, 82%), m.p. >300 °C (dec.); 1H-NMR (300 MHz, DMSO-d6) δH 12.23 (brs, 2H, NH and OH), 7.65 (d, J = 8.69 Hz, 1H, Ar-H6), 7.25 (d, J = 8.69 Hz, 1H, Ar-H5), 7.15 (s, 2H, imidazoline-H), 4.18 (t, J = 5.95 Hz, 2H, OCH2), 2.44 (t, J = 7.18 Hz, 2H, CH2), 1.93–2.08 (m, 2H, CH2); LC/MS, m/z 316.0 (M + 1)+, 318.0 (M + 3)+, 320.0 (M + 5)+.
4-[2-Chloro-4-(1H-imidazol-2-yl)phenoxy]butanoic acid (17d): pale brown solid (1.5 g, 83%), m.p. 219–220 °C; 1H-NMR (300 MHz, DMSO-d6) δH 12.47 (brs, 1H, OH), 12.20 (brs, 1H, NH), 7.98 (d, J = 1.89 Hz, 1H, Ar-H3), 7.85 (d, J = 8.59 Hz, 1H, Ar-H6), 7.23 (d, J = 8.69 Hz, 1H, Ar-H5), 7.11 (s, 2H, imidazole-H), 4.12 (t, J = 6.23 Hz, 2H, OCH2), 2.42 (t, J = 7.08 Hz, 2H, CH2), 1.95 (quin, J = 6.93 Hz, 2H, CH2); LC/MS (ESI), m/z 281.1 (M + 1)+, 283.1 (M + 3)+.
4-[3-Chloro-4-(1H-imidazol-2-yl)phenoxy]butanoic acid (17e): yellow solid (1.7 g, quantitative), m.p. 103–106 °C; 1H-NMR (300 MHz, DMSO-d6) δH 12.14 (brs, 1H, NH), 7.81 (d, J = 2.50 Hz, 1H, Ar-H2), 7.67 (d, J = 8.69 Hz, 1H, Ar-H5), 7.12 (d, J = 2.36 Hz, 2H, imidazole-H), 7.01 (dd, J = 8.73, 2.50 Hz, 1H, Ar-H6), 4.06 (t, J = 6.42 Hz, 2H, OCH2), 2.39 (t, J = 7.27 Hz, 2H, CH2), 1.95 (quin, J = 6.80 Hz, 2H, CH2); LC/MS, m/z 281.0 (M + 1)+, 283.1 (M + 3)+; Anal. Calcd for (C13H13ClN2O3): C, 55.62; H, 4.67; Cl, 12.63; N, 9.98; Found: C, 55.87; H, 5.01; Cl, 12.99; N, 9.88.
3.17. General Procedure for the Synthesis of 2,3-Di/monochloro-4-(3-hydroxypropoxy)benzaldehydes 18a–c [42]
To a cooled stirred solution of the appropriate 4-hydroxybenzaldehyde (1a–c) (0.031 mol) and K2CO3 (0.157 mol), KI (0.003 mol) in DMF (60 mL), was added 3-chloro-1-propanol (6 g, 0.062 mol) at 5–10 °C. The mixture was stirred for 16 h at 70 °C, then left to attain room temperature. Ice and water were added and the crude product was extracted with DCM (3 × 100 mL). The combined organic extract was washed with water and brine, dried over anhydrous MgSO4. The crude compound was purified by flash chromatography using (EtOAc 1:hexane 4) to afford ethers 18a–c.
2,3-Dichloro-4-(3-hydroxypropoxy)benzaldehyde (18a): pale yellow liquid (6.2 g, 79%); 1H-NMR (300 MHz, DMSO-d6) δH10.19 (brs, 1H, CHO), 7.95 (brs, 1H, OH), 7.32 (d, J = 8.14 Hz, 1H, Ar-H6), 4.59–4.70 (m, 1H, Ar-H5), 4.28 (m, 2H, OCH2), 3.60 (q, J = 5.19 Hz, 2H, CH2), 1.84–2.02 (m, 2H, CH2).
2-Chloro-4-(3-hydroxypropoxy)benzaldehyde (18b): pale yellow liquid (9.5 g, 86.6%); 1H-NMR (300 MHz, DMSO-d6) δH10.20 (s, 1H, CHO), 7.83 (d, J = 8.69 Hz, 1H, Ar-H6); 7.18 (d, J = 2.27 Hz, 1H, Ar-H3), 7.09 (dd, J = 8.73, 2.31 Hz, 1H, CH5), 4.61 (t, J = 5.15 Hz, 1H, OH), 4.19 (t, J = 6.37 Hz, 2H, OCH2), 3.55 (q, J = 5.89 Hz, 2H, CH2), 1.88 (quin, J = 6.26 Hz, 2H, CH2).
3-Chloro-4-(3-hydroxypropoxy)benzaldehyde (18c): pale yellow liquid (9.5 g, 86.6%). 1H-NMR (300 MHz, DMSO-d6) δH 9.85 (s, 1H, CHO), 7.70–8.08 (m, 2H, Ar-H2, Ar-H6), 7.34 (d, J = 8.50 Hz, 1H, Ar-H5), 4.55–4.69 (brs, 1H, OH), 4.24 (t, J = 6.18 Hz, 2H, OCH2), 3.53–3.68 (m, 2H, CH2), 1.92 (quin, J = 6.09 Hz, 2H, CH2).
3.18. General Procedure for the Synthesis of 3-[Di/monochloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]propan-1-ols 19a–c [44]
These intermediates were prepared according to procedure described above for the preparation of 15a–e starting with the appropriate 4-(3-hydroxypropoxy)benzaldehyde derivatives 18a–c (0.016 mol).
3-[2,3-Dichloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]propan-1-ol (19a): pale yellow liquid (4 g, 86%); 1H-NMR (300 MHz, DMSO-d6) δH 7.48 (d, J = 8.69 Hz, 1H, Ar-H6), 7.20 (d, J = 8.78 Hz, 1H, Ar-H5), 6.73 (brs, 1H, NH), 4.62 (brs, 1H, OH), 4.20 (t, J = 6.00 Hz, 2H, imidazoline-H), 3.78 (t, J = 8.31 Hz, 2H, imidazoline-H), 3.59 (brs, 2H, OCH2), 3.44 (t, J = 7.77 Hz, 2H, CH2),1.93 (quin, J = 6.72 Hz, 2H, CH2).
3-[2-Chloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]propan-1-ol (19b): pale yellow solid (7.74 g, 69.2%), m.p. 88–91 °C. 1H-NMR (300 MHz, DMSO-d6) δH 7.85 (d, J = 1.98 Hz, 1H, Ar-H3), 7.75 (dd, J = 8.59, 1.98 Hz, 1H, Ar-H5) 7.20 (d, J = 8.69 Hz, 1H, Ar-H6), 6.93 (brs, 1H, NH), 4.61 (brs, 1H, OH), 4.17 (t, J = 6.18 Hz, 2H, imidazoline-H), 3.75 (brs, 2H, imidazoline-H), 3.59 (t, J = 6.00 Hz, 2H, OCH2), 3.41 (brs, 2H, CH2), 1.90 (quin, J = 6.18 Hz, 2H, CH2).
3-[3-Chloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]propan-1-ol (19c): pale yellow liquid (10.3 g, 96.5%); 1H-NMR (300 MHz, DMSO-d6) δH 7.52 (d, J = 8.69 Hz, 1H, Ar-H5), 7.05 (d, J = 2.27 Hz, 1H, Ar-H2), 6.94 (dd, J = 8.64, 2.41 Hz, 1H, Ar-H6), 6.63 (brs, 1H, NH), 4.59 (d, J = 4.44 Hz, 1H, OH) 4.09 (t, J = 6.33 Hz, 2H, imidazoline-H), 3.54 (t, J = 6.18 Hz, 2H, imidazoline-H), 3.48 (t, J = 6.00 Hz, 2H, OCH2), 3.41 (brs, 2H, CH2), 1.85 (quin, J = 6.28 Hz, 2H, CH2).
3.19. General Procedure for the Synthesis of 3-[Di/monochloro-4-(1H-imidazol-2-yl)phenoxy]propan-1-ols 20a–c [44]
These intermediates were prepared following the same procedure described above for the preparation of 16a–c starting with the appropriate 19a–c (0.027 mol).
3-[2,3-Dichloro-4-(1H-imidazol-2-yl)phenoxy]propan-1-ol (20a): pale yellow liquid (3.8 g, 50 %); 1H-NMR (300 MHz, DMSO-d6) δH 12.29 (brs, 1H, NH) 7.66 (d, J = 8.78 Hz, 1H, Ar-H6), 7.26 (d, J = 8.88 Hz, 1H, Ar-H5), 7.15 (m, 2H, imidazole-H), 4.62 (brs, 1H, OH), 4.22 (t, J = 6.04 Hz, 2H, OCH2), 3.53–3.61 (m, 2H, CH2), 1.92 (t, J = 6.09 Hz, 2H, CH2).
3-[2-Chloro-4-(1H-imidazol-2-yl)phenoxy]propan-1-ol (20b): pale yellow liquid (3.2 g, 42%); 1H-NMR (300 MHz, DMSO-d6) δH 12.43 (brs, 1H, NH), 7.98 (d, J = 1.98 Hz, 1H, Ar-H3), 7.85 (dd, J = 8.59, 1.98 Hz, 1H, Ar-H5), 7.24 (d, J = 8.69 Hz, 1H, Ar-H6), 7.10 (brs, 2H, imidazole-H), 4.59 (t, J = 5.10 Hz, 1H, OH), 4.17 (t, J = 6.23 Hz, 2H, OCH2) 3.60 (q, J = 5.92 Hz, 2H, CH2), 1.90 (quin, J = 6.1 Hz, 2H, CH2).
3-[3-Chloro-4-(1H-imidazol-2-yl)phenoxy]propan-1-ol (20c): wine red liquid (7.1 g, 70%); 1H-NMR (300 MHz, DMSO-d6) δH 12.16 (brs, 1H, NH), 7.93 (d, J = 1.98 Hz, 1H, Ar-H2), 7.67 (d, J = 8.69 Hz, 1H, Ar-H5), 7.07–7.17 (m, 2H, imidazole-H), 7.01 (dd, J = 8.69, 2.36 Hz, 1H, Ar-H6), 4.60 (brs, 1H, OH), 4.11 (t, J = 6.28 Hz, 2H, OCH2), 3.56 (d, J = 4.25 Hz, 2H, CH2), 1.87 (quin, J = 6.2 Hz, 2H, CH2).
3.20. General Procedure for the Synthesis of 3-[2,3-Di/monochloro-4-(1H-imidazol-2-yl)phenoxy]propanoic Acids 21a–c [42]
To a stirred solution of the appropriate alcohol 20a–c (12.8 mmol) in acetone (40 mL), a solution of chromium trioxide (68 mmol) in H2O (50 mL) and AcOH (6 mL) was added while keeping the temperature between 5–10 °C. The mixture was stirred for 30 min at the same temperature then warmed up to room temperature for 16 h. The reaction was monitored by LC-MS till complete disappearance of the starting material. Then the reaction mixture was treated with NaHCO3 and the pH was adjusted to 8. The mixture was washed with DCM (3 × 50 mL) and the aqueous layer was concentrated under reduced pressure to 50 mL. Again, the pH was adjusted to pH 5 (1.5 N HCl). The precipitated solid product was collected by filtration, washed with water and dried. Purification by crystallization from MeOH gave the required products (21a–c).
3-[2,3-Dichloro-4-(1H-imidazol-2-yl)phenoxy]propanoic acids (21a): off-white solid (0.73 g, 18%), m.p. 193–194 °C. IR (KBr, νmax cm−1): 3585(NH), 3100 (OH), 1620 (CO), 1294, 1023 (C-O-C); 1H-NMR (300 MHz, DMSO-d6) δH 13.1(brs, 1H, OH), 12.4 (brs, 1H, NH), 7.64 (d, J = 8.50 Hz, 1H, Ar-H6), 7.27 (d, J = 8.69 Hz, 1H, Ar-H5), 7.15 (s, 2H, imidazole-H), 4.32 (t, J = 8.2 Hz, 2H, OCH2), 2.67 (t, J = 8.2 Hz, 2H, CH2); LC/MS: m/z 300.0 (M)−, 302.0 (M + 2)−, 304.0 (M + 4)−.
3-[2-Chloro-4-(1H-imidazol-2-yl)phenoxy]propanoic acid (21b): off-white solid (0.23 g, 44%), m.p. 210–213 °C. IR (KBr, νmax cm−1): 3443 (NH), 3142 (OH), 1687 (CO), 1269, 1065 (C-O-C); 1H-NMR (300 MHz, DMSO-d6) δH 12.35 (brs, 1H, OH), 7.97 (brs, 1H, NH), 7.86 (d, J = 8.59 Hz, 1H, Ar-H5), 7.27 (d, J = 8.40 Hz, 1H, Ar-H6), 7.11 (m, 3H, Ar-H3 and imidazole-H), 4.30 (t, J = 7.1 Hz, 2H, CH2), 2.74 (t, J = 6.2 Hz, 2H, CH2); LC/MS: m/z 267.0 (M + 1), 269.1 (M + 2); Anal. Calcd for (C13H11ClN2O3): C, 54.05; H, 4.16; Cl, 13.29; N, 10.50; Found: C, 53.79; H, 3.82; Cl, 13.61; N, 10.55.
3-[3-Chloro-4-(1H-imidazol-2-yl)phenoxy]propanoic acid (21c): off-white solid (0.6 g, 9.5%), m.p. 208–210 °C. IR (KBr, νmax cm−1): 3400 (NH), 3139 (OH), 1694 (CO), 1296, 1092 (C-O-C); 1H-NMR (300 MHz, DMSO-d6) δH 13.0, 12.21 (two brs, each 1H, OH and NH), 7.67 (d, J = 8.69 Hz, 1H, Ar-H5), 7.12 (m, 3H, Ar-H2 and imidazole-H), 7.01 (d, J = 7.08 Hz, 1H, Ar-H6), 4.24 (t, J = 5.17 Hz, 2H, OCH2), 2.70 (t, J = 5.43 Hz, 2H, CH2); LC/MS: m/z 267.0 (M + 1)−, 269.0 (M + 3)−.
3.21. RBC Morphological Antisickling Studies
KAUS-4, KAUS-23 and KAUS-24, and clofibrate were tested for their effect on sickle RBC morphology as previously reported [
18,
19,
22]. Briefly sickle RBCs were incubated under air in the absence or presence of 2 mM concentration of test compound (solubilized in DMSO) at room temperature for 1 h. Following, the suspension was incubated under hypoxic condition (4% oxygen/96% nitrogen) at 37 °C for 3 h. The suspension was fixed with 2% glutaraldehyde solution without exposure to air and then subjected to microscopic morphological analysis.
3.22. Oxygen Equilibrium Curve Studies
Normal blood samples (hematocrit 22%) in the absence (control) or presence of 2 mM concentration of the test KAUS compounds (solubilized in DMSO) were incubated at 37 °C for 1.5 h and then subjected to OEC analysis using tonometry as previously described [
18,
19,
22]. Briefly, the compound-treated blood samples is incubated in IL 237 tonometers (Instrumentation Laboratories, Inc. Lexington, MA, USA) for approximately 10 min at 37 °C, and allowed to equilibrate at oxygen tensions 7, 20, 40 and 60 mmHg. The samples were then aspirated into an ABL 700 Automated Blood Gas Analyzer (Radiometer) to determine the pH, partial pressure of CO
2 (pCO
2), partial pressure of oxygen (pO
2), and Hb oxygen saturation values (SO
2). The measured values of pO
2 (mmHg) and SO
2 at each pO
2 value were then subjected to a non-linear regression analysis using the program Scientist (Micromath, Salt Lake City, UT, USA) to estimate P
50 and Hill coefficient values (n
50). Clofibrate was tested as a positive control, while DMSO was tested as negative control.
3.23. Crystallization, Data Collection and Structure Determination of Deoxygenated Hb in Complex with KAUS-23
Freshly prepared solution of KAUS-23 in DMSO was incubated with deoxygenated Hb (40 mg/mL) for 30 min at Hb tetramer:KAUS-23 molar ratio of 1:10 at room temperature and then crystallized with 3.2 M sulfate/phosphate precipitant, pH 6.8 using the batch method as previously described [
18,
19,
22]. Diffraction data was collected at 100 K on an R-axis IV++ image plate detector using CuKα X-ray (λ = 1.5417) from a Rigaku Micro-Max™ -007 X-ray source equipped with Varimax confocal optics operating at 40 kV and 20 mA (Rigaku, The Woodlands, TX, USA). Crystals were cryoprotected with 15% glycerol. The dataset was processed with the d*trek software (v9.9.9.7, Rigaku Corporation, Tokyo, Japan) and the CCP4 suite of programs [
45].
The isomorphous native human deoxygenated Hb tetramer structure (PDB code 2DN2) was used as the starting model to refine the structure, using both Phenix and CNS refinement programs [
46,
47]. Model building and correction were carried out using COOT [
48,
49]. A round of refinement showed a bound KAUS-23 molecule at the central water cavity. The symmetry-related site was poorly defined. Therefore, only one KAUS-23 molecule was built into the model and refined. Also, included in the final model are two molecules of sulfate ions, and 400 water molecules. The structure refined to a final Rfactor/Rfree of 19.68/24.92% at a resolution of 2.15 Å. The atomic coordinate and structure factor files have been deposited in the RCSB Protein Data Bank with accession codes 5KDQ. Detailed crystallographic and structural analysis parameters are reported in
Table 3.