Abstract
Novel sulfaguanidines incorporating acridine moiety were synthesized by the reaction of cyclohexane-1,3-dione, sulfaguanidine, and aromatic aldehydes. Synthesis of these compounds was performed in water at room temperature, and their structures were confirmed by using spectral analysis (IR, 1H-NMR, 13C-NMR, and HRMS). Human carbonic anhydrase isoenzymes (hCA I and II) were purified from erythrocyte cells with affinity chromatography. hCA I was purified 83.40-fold with a specific activity, 1060.9 EU mg protein−1, and hCA II was purified 262.32-fold with a specific activity, 3336.8 EU mg protein−1. The inhibitory effects of newly synthesized sulfaguanidines and acetazolamide, (AAZ) as a control compound, on hydratase and esterase activities of these isoenzymes have been studied in vitro. Synthesized compounds have moderate inhibition potentials on hCA I and hCA II isoenzymes. IC50 values of compounds for esterase activity are in the range of 118.4 ± 7.0 μM–257.5 ± 5.2 μM for hCA I and 86.7 ± 3.0 μM–249.4 ± 10.2 μM for hCA II, respectively.
Introduction
Acridine molecules were discovered in the nineteenth century, and derivatives of acridine are still being exploredCitation1. Acridine derivatives have been classified as octahydroacridines, tetrahydroacridines, dihydroacridines, and acridonesCitation1. This molecule group has important biological actives as anticancerCitation2, antiglaucomaCitation3,Citation4, antimicrobialCitation5, calcium channel blockingCitation6, and potassium cannel openingCitation7. Herein, we report the synthesis of novel sulfaguanidines incorporating acridine moiety as carbonic anhydrase inhibitors.
Carbonic anhydrases (CAs) (EC 4.2.1.1) are one of the most important enzymes in the metabolisms of living organisms. These enzymes catalyze reversible hydration of carbon dioxide in a two-step reaction to yield bicarbonate and proton in the cellsCitation8–14. Sixteen different CA isoenzymes are expressed in mammalsCitation15. hCA I is major CA isoenzyme in humans, and hCA II is present in the eyesCitation16. hCA II plays a role in the secretion of aqueous humor because of the hydration of carbon dioxide affects the ion balanceCitation17. Glaucoma is a group of diseases characterized by an elevation in intraocular pressure (IOP) that results in visual field loss and irreversible blindnessCitation18. The risk factors for glaucoma disease include age, race, ocular hypertension, severe myopia, and a family history of glaucomaCitation16.
Sulfonamides are very important compounds for the medicine industry and they are now extensively used drugs for the treatment or conservation of different illnessesCitation11,19–21. In clinical medicine, they have been used as anticancer, anti-obesity, antibacterial, antifungal, diuretics, carbonic anhydrase inhibitors, and anticonvulsant agentsCitation12,Citation22,Citation23. Carbonic anhydrase inhibitors are used for the treatment of glaucoma because these agents reduce IOP. Some systemic and topical sulfonamide drugs were mainly used clinically as antiglaucoma agents for a long time. Acetazolamide (AAZ), the systemic compound, was used as a medicine clinically to treat the disease. Then, it was suggested that reducing aqueous humor secretion might provide an effective means of lowering IOP to treat the diseaseCitation24. But, the orally consumed drugs led to different side effects because they also inhibit other CA isoenzymes in the body. Almost 20 years ago, two topical CA inhibitors, dorzolamide (DZA) and brinzolamide (BRZ), have been developedCitation16. These inhibitors have been used to treat the disease and they have fewer side effects than othersCitation25. As a result, these drugs, AAZ, DZA, and BRZ, may be an effective antiglaucoma agent, but they tend to pose tolerability problems in many patients because of local and systemic side effects. Thus, it is necessary to develop new agents.
Investigation of inhibitory effects of acridine sulfonamide compounds on hCA I and hCA II has limited number of studies in literatureCitation26,Citation27. In the point of view of the results obtained from these studies, acridine sulfonamides compounds are very effective on these isoenzymes. In this study, synthesis of novel sulfaguanidineCitation28,Citation29 derivatives was performed by using a facile synthesis method in water at room temperature. Then, these compounds were characterized by IR, 1H-NMR, and 13C-NMR data and satisfactory mass spectral analyses, respectively. The inhibition effects of newly synthesized compounds on hCA I and hCA II isoenzymes were investigated under in vitro conditions.
Methods
Chemistry
The chemicals used in the synthesis of acridine sulfonamide derivatives were provided by the Merck and Aldrich Chemical Company, and cyanogen bromide-activated Sepharose®4B for affinity column and electrophoresis reagents were obtained from the Sigma Chem. Co. All chemicals and solvents used for the synthesis were spectroscopic reagent grade. Melting points were measured on a Bibby Stuart Scientific apparatus. Fourier Transform Infrared (FT-IR) spectra were recorded on Bruker Optics, Andrtex 70 FT-IR spectrometer using ATR diamond crystal. The 1H-NMR and 13C-NMR spectra were obtained with a Bruker DPX-300 FT-NMR instrument in DMSO-d6 as solvent with trimethylsilane as the internal reference, at 300 and 75 MHz, respectively. Chemical shifts are expressed in δ units (ppm). The mass analyses were performed on Agilent Tecnologies 6530 Accurate-Mass Q-TOF LC/MS as high-resolution mass spectrometry. Inhibition data were recorded by using a spectrophotometer (SHIMADZU UV 1700 PharmaSpec).
General procedure for preparation for acridine sulfonamide derivatives 4–18
A mixture of a cyclohexane-1,3-dione 1 (0.224 g, 2 mmol), sulfaguanidine 2 (0.214 g, 1 mmol), benzaldehyde derivatives 3a–i (0.106 g, 1 mmol), and DBSA (0.420 g) in H2O (40 mL) was stirred at room temperature for 48 h. The progress of the reaction was monitored by thin layer chromatography. Once the reaction was completed, the mixture was cooled to room temperature, and the solids were filtered out and washed with H2O3. The sulfaguanidine products recrystallized from the following solvent mixture (ethanol-water 90%) for each compound (61–87%).
N-(Diaminomethylene)-4-(1,8-dioxo-9-phenyl-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)benzenesulfonamide (4)
As yellow solid, 0.355 g, 72%, mp 251–253 °C. IR (cm−1): 3437 and 3331 (NH2), 3090 (Ar-H), 2951 and 2887 (C-H), 1637 (C=O), 1616 (C=N), 1537 and 1516 (C=C), 1359 and 1130 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.62–1.65 (m, 4H, -CH2), 1.81–1.99 (m, 4H, -CH2), 2.20–2.31 (m, 4H, -CH2), 5.15 (s, 1H, CH), 6.84 (br, 4H, 2 × NH2), 7.09–7.13 (m, 2H, Ar-H), 7.19–7.31 (m, 3H, Ar-H), 7.58 (br, 2H, Ar-H), 7.93 (d, 2H, J = 8.69 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 20.32 (CH2), 26.90 (CH2), 31.31 (CH), 36.86 (CH2), 114.45 (C), 126.24 (CH), 126.59 (CH), 128.56 (CH), 129.03 (CH), 129.72 (CH), 141.12 (C), 145.02 (C), 152.39 (C), 158.66 (C), 165.32 (C), 195.93 (C=O). HRMS (QTOF-ESI) Found, m/z: 525.1454 [M + Cl]−. C26H26ClN4O4S. Calculated, m/z: 525.1363.
4-(9-(4-Cyanophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N (diaminomethylene)benzenesulfonamide (5)
As yellow solid, 0.430 g, 78%, mp 325–326 °C. IR (cm−1): 3399 and 3338 (NH2), 3060 (Ar-H), 2947 and 2888 (C-H), 2222 (CN), 1627 (C=O), 1594 and 1537 (C=C), 1360 and 1135 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.50–1.70 (m, 2H, -CH2), 1.75–2.35 (m, 4H, -CH2), 2.22 (m, 6H, -CH2) 5.20 (s, 1H, -CH), 6.70–6.95 (br, 4H, 2 × NH2), 7.50 (d, 2H, J = 7.9 Hz, Ar-H), 7.66 (m, 2H, Ar-H), 7.70 (d, 2H, J = 7.6 Hz, Ar-H), 7.92 (d, 2H, J = 7.6 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 21.07 (CH2), 28.28 (CH2), 32.69 (CH), 36.56 (CH2), 109.10 (CN), 113.44 (C), 119.50 (C), 127.40 (CH), 129.13 (CH), 130.80 (CH), 132.58 (CH), 140.89 (C), 145.79 (C), 152.33 (C), 153.07 (C), 158.67 (C), 195.93 (C=O); HRMS (QTOF-ESI) Found, m/z 550.1412 [M + Cl]−. C27H25ClN5O4S. Calculated, m/z: 550.1316.
4-(9-(3-Cyanophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(diaminomethylene)benzenesulfonamide (6)
As yellow solid, 0.441 g, 80%, mp 300–302 °C. IR (cm−1): 3453 and 3351 (NH2), 3101 (Ar-H), 2952 and 2890 (C-H), 2229 (CN), 1623 (C=O), 1541 (C=C), 1359 and 1131 (SO2); 1H NMR (300 MHz, DMSO-d6) δ, ppm (J, Hz): 1.65–1.68 (m, 2H, -CH2), 1.80–1.84 (m, 4H, -CH2), 2.19–2.23 (m, 6H, -CH2), 5.14 (s, 1H, -CH), 6.84 (br, 4H, 2 × NH2), 7.47 (t, 1H, J = 8.29, Ar-H), 7.63–7.67 (m, 5H, Ar-H), 7.93 (d, 2H, J = 8.60 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ, ppm): 21.07 (CH2), 28.33 (CH2), 32.45 (CH), 36.55 (CH2), 111.35 (CN), 113.49 (C), 119.60 (C), 127.35 (CH), 129.94 (CH), 130.29 (CH), 130.82 (CH), 131.63 (CH), 133.17 (CH), 140.93 (C), 145.77 (C), 148.41 (C), 153.07 (C), 158.67 (C), 196.01 (C=O); HRMS (QTOF-ESI) Found, m/z 550.1423 [M + Cl]−. C27H25ClN5O4S. Calculated, m/z: 550.1316.
N-(Diaminomethylene)-4-(9-(4-nitrophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8 octahydroacridin-10(9H)-yl)benzenesulfonamide (7)
As yellow solid, 0.418 g, 78%, mp 316–317 °C. IR (cm−1): 3437 and 3331 (NH2), 3090 (Ar-H), 2951 and 2887 (C-H), 1637 (C=O), 1616 (C=N), 1537 and 1516 (C=C), 1361 and 1137 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.55–1.73 (m, 2H, -CH2), 1.82–1.96 (m, 4H, -CH2), 2.25–2.34 (m, 6H, -CH2), 5.25 (s, 1H, -CH), 6.60–7.00 (br, 4H, 2 × NH2), 7.58 (d, 2H, J = 8.7 Hz, Ar-H), 7.65 (m, 2H, Ar-H), 7.93 (d, 2H, J = 8.6 Hz, Ar-H), 8.12 (d, 2H, J = 8.6 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 21.06 (CH2), 28.29 (CH2), 32.61 (CH), 36.55 (CH2), 113.38 (C), 123.88 (CH), 127.37 (CH), 129.32 (CH), 130.89 (CH), 140.87 (C), 145.82 (C), 146.17 (C), 153.17 (C), 154.40 (C), 158.66 (C), 195.92 (C=O); HRMS (QTOF-ESI) Found, m/z 570.1311 [M + Cl]−. C26H25ClN5O6S. Calculated, m/z: 570.1214.
N-(Diaminomethylene)-4-(9-(3-nitrophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)benzenesulfonamide (8)
As yellow solid, 0.440 g, 82%, mp 290–291 °C. IR (cm−1): 3478 and 3363 (NH2), 3096 (Ar-H), 2946 and 2889 (C-H), 1639 (C=O), 1548 and 1521 (C=C), 1355 and 1143 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.50–1.70 (m, 2H, -CH2), 1.82–1.98 (m, 4H, -CH2), 2.15–2.35 (m, 6H, -CH2), 5.25 (s, 1H, -CH), 6.60–6.90 (br, 4H, 2 × NH2), 7.54–7.65 (m, 2H, Ar-H), 7.63 (m, 1H, Ar-H), 7.76 (d, 1H, J = 7.6 Hz, Ar-H), 7.94 (d, 2H, J = 8.6 Hz, Ar-H), 8.01 (d, 1H, J = 8.1 Hz, Ar-H) 8.13 (m, 1H, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 21.09 (CH2), 28.25 (CH2), 32.34 (CH), 36.52 (CH2), 113.58 (C), 121.47 (CH), 122.69 (CH), 127.46 (CH), 128.00 (CH), 130.32 (CH), 134.63 (CH), 140.86 (C), 145.81 (C), 147.97 (C), 148.94 (C), 153.18 (C), 158.64 (C), 196.01 (C=O); HRMS (QTOF-ESI) Found, m/z 570.1312 [M + Cl]−. C26H25ClN5O6S. Calculated, m/z: 570.1214.
N-(Diaminomethylene)-4-(9-(4-fluorophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)benzenesulfonamide (9)
As yellow solid, 0.372 g, 73%r, mp 210 °C. IR (cm−1): 3433 and 3340 (NH2), 3070 (Ar-H), 2951 and 2874 (C-H), 1622 (C=O), 1537 (C=C), 1361 and 1133 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.60–1.66 (m, 2H, -CH2), 1.81–1.94 (m, 4H, -CH2), 2.20–2.28 (m, 6H, -CH2), 5.15 (s, 1H, -CH), 6.75–6.90 (br, 4H, 2 × NH2), 7.01–7.07 (m, 2H, Ar-H), 7.29–7.34 (m, 2H, Ar-H), 7.55–7.65 (br, 2H, Ar-H), 7.92 (d, 2H, J = 8.7 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 21.12 (CH2), 28.22 (CH2), 31.19 (CH), 36.66 (CH2), 114.39 (C), 114.98 (CH), 115.26 (CH), 127.40 (CH), 129.62 (CH), 130.74 (C), 141.07 (C), 143.18 (C), 145.75 (C), 152.47 (C), 158.68 (C), 195.96 (C=O); HRMS (QTOF-ESI) Found, m/z 543.1377 [M + Cl]−. C26H25ClFN4O4S. Calculated, m/z: 543.1269.
4-(9-(4-Chlorophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(diaminomethylene)benzenesulfonamide (10)
As yellow solid, 0.436 g, 83%, mp 285 °C. IR (ATR cm−1): 3433 and 3341 (NH2), 2951 (C-H), 1622 (C=O), 1538 (C=C), 1360 and 1132 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.60–1.66 (m, 2H, -CH2), 1.81–1.94 (m, 4H, -CH2), 2.20–2.29 (m, 6H, -CH2), 5.12 (s, 1H, -CH), 6.65–6.95 (br, 4H, 2 × NH2), 7.33–7.87 (m, 4H, Ar-H), 7.55–7.65 (br, 2H, Ar-H), 7.92 (d, 2H, J = 8.8 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 21.12 (CH2), 28.24 (CH2), 31.47 (CH), 36.65 (CH2), 113.25 (C), 127.41 (CH), 128.46 (CH), 129.99 (CH), 130.80 (CH), 141.02 (C), 145.52 (C), 145.92 (C), 150.42 (C), 152.62 (C), 158.69 (C), 196.91 (C=O); HRMS (QTOF-ESI) Found, m/z 559.1085 [M + Cl]−. C26H25Cl2N4O4S. Calculated, m/z: 559.0974.
4-(9-(4-Bromophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(diaminomethylene)benzenesulfonamide (11)
As yellow solid, 0.450 g, 79%, mp 288 °C. IR (ATR cm−1): 3428 and 3335 (NH2), 2949 (C-H), 1622 (C=O), 1538 (C=C), 1360 and 1132 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.60–1.66 (m, 2H, -CH2), 1.81–1.94 (m, 4H, -CH2), 2.19–2.30 (m, 6H, -CH2), 5.10 (s, 1H, -CH), 6.75–6.85 (br, 4H, 2 × NH2), 7.26 (d, 2H, J = 8.4 Hz, Ar-H), 7.42 (d, 2H, J = 8,4 Hz, Ar-H), 7.55–7.63 (br, 2H, Ar-H), 7.92 (d, 2H, J = 8.7 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) (ppm): 21.11 (CH2), 28.23 (CH2), 31.55 (CH), 36.65 (CH2), 114.02 (C), 119.318 (C), 127.41 (CH), 130.25 (CH), 130.79 (CH), 131.39 (CH), 141.01 (C), 145.77 (C), 146.34 (C), 152.64 (C), 158.68 (C), 195.92 (C=O); HRMS (QTOF-ESI) Found, m/z 603.0574 [M + Cl]−. C26H25BrClN4O4S. Calculated, m/z: 603.0468.
N-(Biaminomethylene)-4-(9-(2,4-dichlorophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)benzenesulfonamide (12)
As yellow solid, 0.392 g, 70%, mp 265–266 °C. IR (cm−1): 3425 and 3330 (NH2), 3100 (Ar-H), 2946 and 2869 (C-H), 1621 (C=O), 1537 (C=C), 1359 and 1136 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.50–1.65 (m, 2H, -CH2), 1.77–1.94 (m, 4H, -CH2), 2.05–2.25 (m, 6H, -CH2), 5.25 (s, 1H, -CH), 6.50–7.00 (br, 4H, 2 × NH2), 7.31 (d, 1H, J = 8.3 Hz, Ar-H), 7.37 (s, 1H, Ar-H), 7.50 (d, 1H, J = 8.4 Hz, Ar-H), 7.64 (m, 2H, Ar-H), 7.93 (m, 2H, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 21.17 (CH2), 28.46 (CH2), 32.90 (CH), 36.61 (CH2), 113.31 (C), 127.24 (CH), 128.94 (CH), 130.63 (CH), 131.12 (CH), 131.36 (C), 134.03 (C), 134.24 (CH), 141.11 (C), 143.16 (C), 145.76 (C), 152.95 (C), 158.66 (C), 195.61 (C=O); HRMS (QTOF-ESI) Found, m/z 593.0594 [M + Cl]−. C26H24Cl3N4O4S. Calculated, m/z: 593.0584.
N-(Diaminomethylene)-4-(9-(4-methoxyphenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)benzenesulfonamide (13)
As yellow solid, 0.316 g, 67%, mp 222–225 °C. IR (cm−1): 3428 and 3330 (NH2), 2946 and 2835 (C-H), 1628 (C=O), 1538 and 1505 (C=C), 1361 and 1133 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.61–1.64 (m, 2H, -CH2), 1.80–1.93 (m, 4H, -CH2), 2.20–2.27 (m, 6H, -CH2), 3.70 ppm (s, 3H, -OCH3) 5.05 (s, 1H, -CH), 6.80 (d, 2H, J = 8.7 Hz, Ar-H), 6.8 (br, 4H, 2 × NH2), 7.20 (d, 2H, J = 8.7, Ar-H), 7.58 (br, 2H, Ar-H), 7.92 (d, 2H, J = 8.8 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 21.17 (CH2), 28.21 (CH2), 30.75 (CH), 36.72 (CH2), 55.35 (CH3), 113.90 (CH), 114.77 (C), 127.43 (CH), 128.85 (CH), 130.74 (CH), 139.34 (C), 141.18 (C), 145.67 (C), 152.10 (C), 157.81 (C), 158.65 (C), 195.95 (C=O); MS(CI) m/z 520 [M]− (100%). HRMS (QTOF-ESI) Found, m/z 555.1578 [M + Cl]−. C27H28ClN4O5S. Calculated, m/z: 555.1469.
N-(Diaminomethylene)-4-(9-(2,4-dimethoxyphenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)benzenesulfonamide (14)
As yellow solid, 0.413 g, 75%, mp 220 °C. IR (cm−1): 3429 and 3331 (NH2), 3058 (Ar-H), 2944 and 2887 (C-H), 1620 (C=O), 1536 (C=C), 1360 and 1136 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.56 (m, 2H, -CH2), 1.76 –1.92 (m, 4H, -CH2), 2.10–2.18 (m, 6H, -CH2), 3.70 (s, 3H, -OCH3), 3.77 (s, 3H, -OCH3), 5.05 (s, 1H, -CH), 6.39–6.43 (m, 1H, Ar-H) 6.46 (s, 1H, Ar-H), 6.83 (br, 4H, 2 × NH2), 7.20 (d, 1H, J = 8.3, Ar-H), 7.59 (br, 2H, Ar-H), 7.94 (d, 2H, J = 7.8 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 21.31 (CH2), 28.50 (CH2), 30.00 (CH), 36.85 (CH2), 55.45 (CH3), 56.71 (CH3), 99.93 (CH), 105.16 (CH), 113.83 (C), 127.10 (C), 127.48 (CH), 130.77 (CH), 132.45 (CH), 141.65 (C), 145.51 (C), 152.03 (C), 158.67 (C), 159.16 (C), 159.42 (C), 195.57 (C=O); HRMS (QTOF-ESI) Found, m/z 585.1681 [M + Cl]−. C28H30ClN4O6S. Calculated, m/z: 585.1575.
N-(Diaminomethylene)-4-(9-(4-hydroxyphenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)benzenesulfonamide (15)
As yellow solid, 0.310 g, 61%, mp 225–227 °C. IR (cm−1): 3473 and 3364 (NH2), 2949 and 2888 (C-H), 1611 (C=O), 1586 (C=N), 1548 (C=C), 1363 and 1143 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.62–1.65 (m, 2H, -CH2), 1.80–1.90 (m, 4H, -CH2), 2.20– 2.27 (m, 6H, -CH2), 5.00 (s, 1H, -CH), 6.62 (d, 2H, J = 8.4 Hz, Ar-H), 6.83 (br, 4H, 2 × NH2), 7.07 (d, 2H, J = 8.4, Ar-H), 7.57 (br, 2H, Ar-H), 7.91 (d, 2H, J = 8.6 Hz, Ar-H), 9.10 (s, 1H, OH); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 21.17 (CH2), 28.20 (CH2), 30.55 (CH), 36.77 (CH2), 114.97 (C), 115.25 (CH), 127.42 (CH), 128.74 (CH), 130.74 (CH), 137.67 (C), 141.23 (C), 145.68 (C), 151.94 (C), 155.83 (C), 158.69 (C), 195.93 (C=O); HRMS (QTOF-ESI) Found, m/z 541.1417 [M + Cl]−. C26H26ClN4O5S. Calculated, m/z: 541.1312.
N-(Diaminomethylene)-4-(1,8-dioxo-9-(p-tolyl)-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)benzenesulfonamide (16)
As yellow solid, 0.409 g, 81%, mp 227 °C. IR (cm−1): 3439 and 3336 (NH2), 3031 (Ar-H), 2953 and 2890 (C-H), 1654 (C=O), 1616 (C=N), 1542 and 1510 (C=C), 1357 and 1123 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.58–1.64 (m, 2H, -CH2), 1.80–1.93 (m, 4H, -CH2), 2.20–2.28 (m, 6H, -CH2), 2.22 (s, 3H, -CH3), 5.05 (s, 1H, -CH), 6.75–6.90 (br, 4H, 2 × NH2), 7.01 (d, 2H, J = 8.0 Hz, Ar-H), 7.18 (d, 2H, J = 8.0 Hz, Ar-H), 7.52–7.60 (br, 2H, Ar-H), 7.93 (d, 2H, J = 8.7 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 21.06 (CH3), 21.17 (CH2), 28.29 (CH2), 31.17 (CH), 36.74 (CH2), 114.65 (C), 126.13 (CH), 128.32 (CH), 129.13 (CH), 130.76 (CH), 135.12 (C), 141.17 (C), 144.12 (C), 145.72 (C), 152.21 (C), 158.68 (C), 195.89 (C=O); HRMS (QTOF-ESI) Found, m/z 539.1520 [M + Cl]−. C27H28ClN4O4S. Calculated, m/z: 539.1624.
N-(Diaminomethylene)-4-(9-(4-ethylphenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)benzenesulfonamide (17)
As yellow solid, 0.410 g, 79%, mp 305 °C. IR (cm−1): 3442 and 3343 (NH2), 2951 and 2875 (C-H), 1615 (C=O), 1540 (C=C), 1360 and 1133 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1,14 (t, 3H, J = 7,6 Hz, -CH3) 1.59–1.65 (m, 2H, -CH2), 1.81–1.94 (m, 4H, -CH2), 2.20–2.28 (m, 6H, -CH2), 2.50–2.56 (m, 2H, -CH2), 5.10 (s, 1H, -CH), 6.70–6.80 (br, 4H, 2 × NH2), 7.07 (d, 2H, J = 8.9 Hz, Ar-H), 7.20 (d, 2H, J = 8,0 Hz, Ar-H), 7.52–7.62 (br, 2H, Ar-H), 7.92 (d, 2H, J = 8.0 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 16.01 (CH3), 21.15 (CH2), 21.16 (CH2), 28.21 (CH2), 31.24 (CH), 36.73 (CH2), 114.66 (C), 127.44 (CH), 127.83 (CH), 127.94 (CH), 130.73 (CH), 141.18 (C), 141.46 (C), 144.39 (C), 145.71 (C), 152.22 (C), 158.68 (C), 195.91 (C=O); HRMS (QTOF-ESI) Found, m/z 553.1778 [M + Cl]−. C28H30ClN4O4S. Calculated, m/z: 553.1676.
4-(9-([1,1′-Biphenyl]-4-yl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(diaminomethylene)benzenesulfonamide (18)
As yellow solid, 0.493 g, 87%, mp 320–321 °C. IR (cm−1): 3424 and 3327 (NH2), 3028 (Ar-H), 2942 and 2879 (C-H), 1614 (C=O), 1543 (C=C), 1361 and 1136 (SO2); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.65–1.68 (m, 2H, -CH2), 1.83–1.97 (m, 4H, -CH2), 2.20–2.25 (m, 6H, -CH2), 5.05 (s, 1H, -CH), 6.84 (br, 4H, 2 × NH2), 7.26–7.47 (m, 7H, Ar-H), 7.53 (d, 2H, J = 8.2 Hz, Ar-H), 7.61 (d, 2H, J = 7.3, Ar-H), 7.93 (d, 2H, J = 8.8 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 21.16 (CH2), 28.27 (CH2), 31.55 (CH), 36.43 (CH2), 114.40 (C), 126.98 (CH), 127.57 (CH), 127.59 (CH), 128.55 (CH), 129.35 (CH), 130.08 (CH), 130.81 (CH), 138.27 (C), 140.73 (C), 141.14 (C), 145.76 (C), 146.29 (C), 152.47 (C), 158.69 (C), 195.97 (C=O); HRMS (QTOF-ESI) Found, m/z 601.1555 [M + Cl]−. C32H30ClN4O4S. Calculated, m/z: 601.1676.
Biochemistry
Purification of carbonic anhydrase I and II isoenzymes from human erythrocytes
Erythrocytes were purified from human blood. The blood samples were centrifuged at 1500 rpm for 20 min, and plasma was removed. Later, red cells were washed with isotonic solution (0.9% NaCl), and the erythrocytes were hemolyzed with 1.5 volumes of ice-cold water. Cell membranes were removed by centrifugation at 4 °C, 20 000 rpm for 30 min. The pH of hemolysate was adjusted to 8.7 with solid TRIS (tris(hydroxymethyl)aminomethane). The hemolysate was applied to affinity column (Sepharose®4B-L-tyrosine-p-aminobenzene sulfonamide) pre-equilibrated with 25.0-mM TRIS-HCl/0.1M Na2SO4 (pH 8.7). After extensive washing with a solution of 25.0-mM TRIS-HCl/22.0 mM Na2SO4 (pH 8.7), the hCA I and hCA II isoenzymes were eluted with the solution of 1.0 M NaCl/25.0 mM Na2HPO4 (pH 6.3) and 0.1 M NaCH3COO/0.5 M NaClO4 (pH 5.6), respectivelyCitation30. For quantitative protein determination, the Bradford method was used with bovine serum albumin as a standardCitation31. Also purity control of the isoenzymes was performed with SDS-PAGE after the purificationCitation32.
Determination of hydratase and esterase activities of hCA I and hCA II
The CO2 hydratase activity of the enzyme was determined at 0 °C in a veronal buffer (pH 8.15) with the pH-stat method as indicator and saturated carbon dioxide solution as substrate in a final volume of 4.2 mL. The time (in seconds) taken for the solution to change from pH 8.15 to pH 6.50 was measured. The enzyme unit (EU) is the enzyme amount that reduces the nonenzymatic reaction time by 50%. The activity of an enzyme unit was calculated by using the equation ((t0–tc)/tc) where t0 and tc are times for pH change of the non-enzymatic and enzymatic reactions, respectivelyCitation33.
Esterase activity was assayed by following the change in the absorbance at 348 nm of 4-nitrophenylacetate to 4-nitrophenylate ion over a period of 3 min at 25 °C using a spectrophotometer according to the method described in the literatureCitation34. The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL of 0.05 M TRIS–SO4 buffer (pH 7.4), 1.0 mL of 3.0 mM 4-nitrophenylacetate, 0.5 mL H2O, and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution.
Determination of inhibition percentages, and IC50 values of the compounds
Inhibition percentages were determined by measuring hydratase and esterase activities of the isoenzymes in the presence of inhibitorsCitation35. Also IC50 values of inhibitors having the 50% and more inhibition effect were calculated.
To determine the IC50 values of the inhibitors, hydratase and esterase activities of hCA I and II were assayed in the presence of various inhibitor concentrations as mentioned above. Regression analysis graphs were drawn by plotting percent enzyme activity versus inhibitor concentration and determined IC50 valuesCitation36–38.
Statistical analysis
All the presented data were confirmed in at least three independent experiments and are expressed as the mean ± standard deviation (SD). Data were analyzed by using a one-way analysis of variance for multiple comparisons (SPSS 13.0, SPSS Inc., Chicago, IL). p < 0.0001 was considered to be statistically significant.
Results and discussion
The general synthetic method shown was employed to prepare sulfaguanidine derivatives 4–18. All spectral data were in agreement with the assigned structures. All novel sulfaguanidine compounds were synthesized in water in a single process through two successive reactions (Aldol condensation and Michael addition) and using three-component reaction methodCitation4,Citation5,Citation27. There-component reactions, which combine in one pot at least three simple building blocks, provide a most powerful platform to access diversity as well as complexity in a limited number of reaction steps. Moreover, these reactions were realized with the aim of a phase-transfer catalyst-Bronsted acid as p-dodecylbenzenesulfonic acid (DBSA). In recent years, using DBSA as a combine catalyst (phase-transfer catalyst-Bronsted acid) has been a popular application in organic chemistryCitation39. Novel sulfaguanidine compounds were prepared by one-pot reaction in processing high yields, room temperature, and simple work-up procedure (Scheme 1).
Scheme 1. Synthesis of acridine sulphaguanidine derivative compounds (4–18).
The infrared (IR) spectra of all the sulfaguanidine compounds showed sharp peaks for the carbonyl groups in the region of 1611 and 1654 cm−l. The compounds 4, 7, 15, and 16 produced peaks that belong to imine group 1616 and 1586 cm−1. The compounds 5 and 6 exhibited peaks that belong to CN group 2229 and 2222 cm−1, respectively. Moreover, in the IR spectra of the compounds, aliphatic C–H stretching bands at 2953–2835 cm−1 and aromatic C–H stretching bands at 3101–3028 cm−1 were observedCitation40. The NH2 vibrations of acridine sulfonamide compounds were observed in the region of 3478 and 3327 cm−1. The 1H-NMR spectra of compounds showed the CH2 group protons of the cyclohexene rings of the compounds showing multiple peaks in the 1.50–2.56 ppm range. The Compound 16 showed a singlet peak that belongs to protons of the methyl group 2.22 ppm. The Compound 17 that belongs to protons of the ethyl group exhibited a triplet peak (3H) at 1.14 ppm and multiple peaks (2H) at 2.50–2.56 ppm. Signals for the methoxy group of protons for compounds 13 and 14 were shown in the range of 3.70–3.77 ppm. The signals for the CH protons were observed at 5.00–5.25 ppm, and the signals for the aromatic protons were observed in the range of 7.01–8.13 ppm. The hydroxyl group proton of Compound 15 was observed as a broad signal at 9.10 ppm. The broad peaks between 6.50 and 7.00 ppm were assigned to the sulfaguanidine (−SO2NC(NH2)2) groups of protons of all the compounds 4–1841. The 13C-NMR (APT) spectra of the compounds 5 and 6 displayed signals for the cyano group carbons for 109.10 and 111.35 ppm, respectively. All the compounds 4–18 showed carbonyl carbons peaks at 195.57–196.91 ppm. The mass spectra of all the sulfaguanidine compounds demonstrated molecular ion and molecular ion isotope peaks successfully.
To investigate the inhibition effects of sulfaguanidine derivatives on carbonic anhydrase isoenzymes, first, hCA I and hCA II were purified from human erythrocytes. After affinity purification, qualitative protein assay was performed at 280 nm. hCA I was purified 83.40-fold with a specific activity, 1060.9 EU mg protein−1, and hCA II was purified 262.32-fold with a specific activity, 3336.8 EU mg protein−1 ().
Table 1. Summary of purification procedure for hCA I and hCA II.
Purity of the isoenzymes was controlled with SDS-PAGE. Following the purification step, the inhibitory effects of novel compounds (4–18) and AAZ (as a control compound) on the activities of hCA I and hCA II isoenzymes were studied as in vitro. CA inhibition percentages and IC50 values were listed in . Inhibition percentages of the compounds were given in , and also percent enzyme activity versus inhibitor concentration graphics of compounds 7 and 8, were given in
Figure 1. Inhibition percentages of 4–18 on hCA I and hCA II isoenzymes, as graphical presentation.
Figure 2. Percent enzyme activity versus inhibitor concentration graphics of the inhibitory compounds. (a) Effect of 7 on the esterase activity of hCA II. (b) Effect of 8 on the esterase activity of hCA I.
Table 2. Inhibition percentages and IC50 values of synthesized compounds on hCA I and hCA II isoenzymes.
Activity study results show that all of the novel compounds exhibited no inhibition effects on the hydratase activities of hCA I and hCA II. On the other hand, all synthesized compounds have inhibition potential on the esterase activities of hCA I and II (, ). Inhibition percentages of the compounds were in the range of %73.91 ± 0.73–%22.17 ± 0.22 for hCA I, and %76.47 ± 0.77–%20.45 ± 0.21 for hCA II. Some of the synthesized compounds (4, 5, 7, 8, 10, 11, 13, 16, and 17) have 50% or higher inhibition effects on the esterase activities of the isoenzymes. So IC50 values of these compounds were calculated. But newly sulfaguanidines have weak inhibition effects for Ki determination. The inhibition potentials of other compounds (6, 9, 12, 14, 15, and 18) are lower than 50%. These differences in the inhibitory effects are formed by different substituents in the compounds or the presence of same substituents in different locations. Even if the structure has small changes, as discussed below, the effect of inhibition can change to a large extent.
Against to the slow cytosolic isoform hCA I, 4, 5, 7, 8, 10, 11, 13, 16, and 17 have moderate inhibitory potentials with IC50 values in the range of 118.4 ± 7.0 μM–257.5 ± 5.2 μM. Among these compounds, 8 showed the most powerful inhibition potential on the enzyme (IC50 value 118.4 ± 7.0 μM). Also similar to this compound, 7 and 11 have more powerful inhibitor effects than other compounds on hCA I (IC50 values 145.7 ± 4.2 μM and 123.1 ± 3.1 μM, respectively). These results show that substituents such as − NO2 and − Br by interaction with the hCA I isoenzyme might increase inhibition potentials of the compounds. The position of the substituents might affect the inhibition effects of compounds. IC50 values of the compounds containing − NO2 group in the para (7) and meta (8) positions are 145.7 ± 4.2 μM and 118.4 ± 7.0 μM, respectively. So we conclude that the − NO2 group in the meta position of 8 interacts with the enzyme better.
Against the rapid cytosolic isoform hCA II, all of the compounds show similar inhibition effects to hCA I with IC50 values in the range of 86.7 ± 3.0 μM − 249.4 ± 10.2 μM. The Compound 7 has the most effective inhibition potential on the enzyme (IC50 value 86.7 ± 3.0 μM). Similarly 5 (IC50 value 114.3 ± 3.1 μM) and 10 (IC50 value 138.2 ± 4.3 μM) are more potent inhibitors than other compounds on hCA II. Like to hCA I, nitro substituents in the compounds 7 and 8 have assumed an increasing role to inhibition effects on also hCA II. The position of –CN substituents in the compounds 5 and 6 has changed the inhibition effects greatly. IC50 values of the compound 5 are 204.8 ± 6.1 μM for hCA I and 114.3 ± 3.1 μM for hCA II. But the Compound 6 has weaker inhibition potential on hCA I and II, %inhibition percentages are 35.01 ± 0.35 and 32.14 ± 0.32, respectively. While the Compound 9, containing fluorine substituent in para position, has weaker inhibition potential on both hCA I and hCA II (%inhibition percentages are 24.19 ± 0.25 and 20.45 ± 0.21, respectively); the compounds 10 and 11, containing chlorine and bromine substituents in para positions, respectively, have moderate inhibitory effects on these isoenzymes (). Hence it is apparent that the size of the halogen substituent changed the inhibitory effects in a positive direction. However, the compound having multiple halogen substituents, 12, has less potent inhibitor on the isoenzymes. A similar situation is observed between the compounds 13 and 14. Differences in steric effects could be shown as the cause of these great changes in the inhibitory effects ().
Conclusion
Sulfaguanidine derivatives were synthesized a facile and effective method, and the inhibition effects of them on hCA I and hCA II isoenzymes were investigated as in vitro. According to inhibition studies, these new compounds have moderate inhibitory effects (p < 0.0001) on the isoenzymes. In summary, carbonic anhydrase inhibition profiles of new acridine derivatives were imparted to literature with this study. Supplementary data associated with this study can be found, in the online version.
Declaration of interest
The authors are very grateful to Dumlupınar University Research Fund for providing financial support for this project (Grant No. 2011-16 and Grant No. 2012-38).
Supplementary material available online
IENZ_1187605_Supplementary_Material.pdf
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