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Synthesis of isoxazole-containing sulfonamides with potent carbonic anhydrase II and VII inhibitory properties
Accepted Manuscript Synthesis of isoxazole-containing sulfonamides with potent carbonic anhydrase II and VII inhibitory properties Cevher Altug, Hanife Güneş, Alessio Nocentini, Simona Maria Monti, Martina Buonanno, Claudiu T. Supuran PII: DOI: Reference:
S0968-0896(16)31415-8 http://dx.doi.org/10.1016/j.bmc.2017.01.008 BMC 13487
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Accepted Date:
12 December 2016 5 January 2017
Please cite this article as: Altug, C., Güneş, H., Nocentini, A., Maria Monti, S., Buonanno, M., Supuran, C.T., Synthesis of isoxazole-containing sulfonamides with potent carbonic anhydrase II and VII inhibitory properties, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.01.008
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Bioorganic & Medicinal Chemistry
Synthesis of isoxazole-containing sulfonamides with potent carbonic anhydrase II and VII inhibitory properties Cevher Altuga,*, Hanife Güneşa, Alessio Nocentinib, Simona Maria Montic, Martina Buonanno c and Claudiu T. Supuranb,* a
Department of Chemistry, Abant Izzet Baysal University, TR-14280 Bolu, Turkey Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy c Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Napoli, Italy b
A RT I C L E I N F O
A BS T RA C T
Article history:
Two series of benzenesulfonamide containing isoxazole compounds were prepared by using conventional and microwave (MW) methods. 5-Amino-3-aryl-N-(4-sulfamoylphenyl)isoxazole4-carboxamide derivatives were synthesized by the reaction of hydroxymoyl chlorides with 2cyano-N-(4-sulfamoylphenyl)acetamide in the presence of triethylamine. The synthesized 5amino isoxazoles were reacted with various benzoyl chlorides in order to obtain 5amidoisoxazoles. The novel compounds were screened in vitro as inhibitors of four human (h) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1): hCA I, hCA II, hCA IV and hCA VII. The derivatives of the first series were shown to possess excellent inhibitory activity against the cytosolic isoform hCA II, an antiglaucoma drug target, with K Is in the range of 0.5–49.3 nM and hCA VII, a recently validated anti-neuropathic pain target with KIs in the range of 4.3-51.9 nM.
Keywords: 5-amino isoxazole 5-amidoisoxazole Carbonic anhydrase sulfonamide amide
2009 Elsevier Ltd. All rights reserved.
1. Introduction Heterocyclic compounds play a fundamental role in life and biochemical processes. Among these, isoxazoles represent a versatile class of heterocycles due to the variety of biological and pharmacological applications they possess. Indeed, their derivatives exhibit ulcerogenic1, antifungal2, antimicrobial3, cyclooxygenase (COX-2) inhibitory4, antinociceptive5, 6 7 8 anticancer , hypoglycemic , analgesic anti-inflammatory9, and human immunodeficiency virus (HIV) inhibitory10 activities. Moreover, isoxazole derivatives are used as herbicides 11 and insecticides12. Depending on the position of the amino moiety at the isoxazole ring (3-, 4- or 5-), aminoisoxazoles13 are endowed with remarkable properties as active CNS (central nervous system) agents14, potent cholinergic channel activator used for treatment of Alzheimer disease15 and antimicrobial activity16. Sulfonamides gained significant importance in medicinal chemistry since their antibacterial properties were discovered by Gerhard Domagk. At a later time, it was proved that, in vivo, prontosil undergoes a metabolic reduction which forms paminobenzenesulfonamide that is the responsible of the antibacterial activity.17 Since then, sulfonamide derivatives became popular in the pharmaceutical area thanks to their biological activities as antibacterial, anticancer, antiviral, anticonvulsant, anti-inflammatory, and HIV protease
inhibitors.18,19,20 Moreover, sulfonamides are the most classical inhibitors of the zinc-enzymes carbonic anhydrases (CA, EC 4.2.1.1).21 Seven distinct genetic enzymatic families have been identified so far: the α-, β-, γ-, δ-, ζ-. η- and θ-CAs.21 The 15 human (h) isozymes, which all belong to the α-class, represent valuable biological targets for the design of CA inhibitors (CAIs) with many biomedical applications.21,22 In particular, CAI and CAII are ubiquitous isoforms, targets for some deaseases (such as cerebral and retinal edema, glaucoma, epilepsy, and probably altitude sickness), but often off-targets since they are the main responsible of the majority of side effects of non-selective inhibitors.21 The membrane-associated isoform CA IV is a drug target for retinitis pigmentosa and stroke, in addition to glaucoma, together with CA II and XII.23 CAVII has been shown to be involved in epileptiform activity together with CA II and XIV.24 Moreover hCA VII was recently validated as a new drug target for neuropathic pain.25 Many sulfonamides-CAIs are clinically used for decades to treat several diseases (e.g., glaucoma, edema, epilepsy).21b Unfortunately the lack in selectivity of sulfonamide-like CAIs against the different human isozymes is the main topic that prevents a wider pharmaceutical application to date. The “tail approach” is the mainly applied method for the research of isoform-selective sulfonamide-CAIs.26 Indeed, it consists in
appending different tails to the aromatic/heterocyclic ring bearing the sulfonamides in order to modulate the possible interactions the ligand may establish with the middle/outer parts of the active site. Over the years, a varied range of synthesis of isoxazoles has been documented. Up to know, the most widely used reaction path refers to Quilico’s studies on the synthesis of isoxazoles from nitrile oxides and unsaturated compounds.27 The previous approach used by the Altug group for the synthesis of 5aminoisoxazoles28 provided for the use of the key intermediate phenylsulfonyl acetonitrile as active methylene containing nitrile. Since those compounds showed promising in vitro anticancer activities, bearing in mind the two biologically active isoxazole and sulfonamide, we aimed to combine these two portions on a single molecule by applying the tail approach in order to obtain new CAIs. Firstly, a series of 5-amino-3-aryl-N-(4sulfamoylphenyl) isoxazole-4-carboxamides was synthesized and subsequently such derivatives were reacted with benzoyl chlorides to achieve a second series of 5-benzamido-3-phenyl-N(4-sulfamoylphenyl)isoxazole-4-carboxamides. All these novel compounds were screened for their carbonic anhydrase inhibitory properties against four human isoforms: hCA I, hCA II, hCA IV and hCA VII. 2. Result and Discussion 2.1 Chemistry The synthesis of 2-cyano-N-(4-sulfamoylphenyl)acetamide 1 was achieved according to the literature method.29 Sulfonamide-based 5-aminoisoxazole analogues were synthesized via reaction of the proper hydroxymoyl chloride with 2-cyano-N-(4sulfamoylphenyl)acetamide in presence of triethylamine to obtain the 5-amino-3-aryl-N-(4-sulfamoylphenyl)isoxazole-4carboxamide derivatives (3a – k).
Table 1. Synthesis of 5-amino-3-aryl-N-(4-sulfamoylphenyl)isoxazole-4carboxamides Compound
Ar
Yield (%)
Yield (%)
Microwave*
Conventional
3a
2,6-DiClC₆ H₃
65
52
3b 3c 3d 3e 3f 3g 3h 3i 3j 3k
2,4-DiClC₆ H₃ 4-ClC₆ H₄ 4-FC₆ H₄ 4-BrC₆ H₄ C₆ H₅ 4-O₂ NC₆ H₄ 2-O₂ NC₆ H₄
33 28 39 38 40 38 42 62 65 21
27 24 32 26 35 28 64 88 53 15
2-Cl-4,5-DiMeOC6H2
5-Cl-2-furyl 5-Cl-2-thienyl
The reactions were conducted in a Microwave: 90˚C, 300 psi, 200 W, 30 minute. *
The reactions proceeded with rather high yields, when electron withdrawing or electron releasing substituents containing hydroxymoyl chlorides reacted with active methylene containing compound 1. The same derivatives were also synthesized by means of a microwave though showing similar yields. Thereafter, the obtained 5-aminoisoxazoles were reacted with various benzoyl chlorides in the presence of Cs 2CO3 to form 5(4-substitutedbenzamido)-3-aryl-N-(4sulfamoylphenyl)isoxazole-4-carboxamide 5a – m with moderate yields.
Table 2. Synthesis of 5-amino-3-aryl-N-(4-sulfamoylphenyl) isoxazole-4-carboxamides
Compound
Ar
R
Yield (%)
5a
2,6-DiClC6H3
NO2
Conventional 92
5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m
2,4-DiClC6H3 4-FC6H4 C₆ H₅ 2-Cl-4,5-DiMeOC6H2 2,6-DiClC6H3 2,4-DiClC6H3 2,6-DiClC6H3 2,4-DiClC6H3 2,6-DiClC6H3 2,4-DiClC6H3 2,6-DiClC6H3 2,4-DiClC6H3
NO2 NO2 NO2 NO2 H H Cl Cl Br Br MeO MeO
79 39 37 18 62 62 84 61 79 62 41 40
In order to optimize the reaction conditions, we used different kind of bases, prolonged heating or microwave irradiation, but those conditions showed lower yields. All synthesized compounds were purified by column chromatography and characterized with spectroscopic measurements (FT-IR, 1H NMR, 13C NMR, HRMS) and physical data (m.p.). IR spectrum of compounds 3a – k showed characteristic peaks for the amino groups near the sulfonamide and isoxazole ring ones at 3425 and 3306 cm-1, amidic carbonyl showed a signal at 1658 cm-1, whereas asymmetric and symmetric vibrational frequencies for sulfonamides were observed at 1327 and 1157 cm-1 respectively. In addition, the amide groups of the 5-amidoisoxazoles were shown to aroused at 1676 and 1653 cm-1. The analysis of the 1H NMR spectra showed the sulfonamide peaks for compounds 3a – k at 7.2 ppm (SO2NH2) as broad singlets, whereas the remaining signals appeared in their usual ranges (see Experimental for details). Additional difficulties were observed for 13C NMR, cause of the low quality spectra obtained in DMSO-d6 for compounds 5f – m that required the use of acetone-d6 to see more efficiently the carbons signals. Finally, high resolution mass spectra (HRMS) was recorded for all the synthesized derivatived and molecules ion peaks were correctly determined. 2.2. Carbonic anhydrase inhibition We investigated the CA inhibitory activities of compounds 3a – k and 5a – m by applying the stopped flow carbon dioxide
hydrase assay30, in comparison to acetazolamide (AAZ) as standard CAI, against four physiologically significant isoforms hCA I, II, IV and VII. The following structure–activity relationship (SAR) can be extrapolated from the inhibition data shown in Table 3: Table 3. Inhibition data of human CA isoforms hCA I, II, IV and VII with sulfonamides 3a-k and 5a-m reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay30
Compound
hCA I
KI (nM) hCA II
hCA IV
hCA VII
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m AAZ
50.1 7469.6 293.8 404.8 774.7 473.3 4762.2 359.7 5910.6 286.8 402.0 2962.3 7295.2 1579.4 2868.4 9260.9 3654.6 >10000 6529.5 3941.1 2860.6 6376.6 2419.8 8704.7 250
8.2 6.3 0.6 6.6 8.6 4.6 0.8 0.5 4.4 49.3 5.4 426.1 1778.9 167.5 569.0 297.3 150.7 246.3 58.3 350.0 270.9 188.1 71.1 77.4 12
145.5 113.4 47.0 442.3 286.2 451.3 311.8 246.2 47.2 407.0 266.0 304.5 428.4 292.9 410.4 2396.8 443.8 4429.4 197.6 79.6 108.0 372.3 71.3 288.1 74
51.9 29.0 7.2 27.4 7.4 7.0 9.6 4.3 7.8 8.0 5.7 88.9 602.5 376.2 423.5 628.7 701.1 1000.0 62.0 54.6 26.8 65.3 67.1 86.3 2.5
(i) The cytosolic isoform hCA I was moderately inhibited by most of the sulfonamides of the first series with inhibition constants (KIs) ranging between 286.8 and 774.7 nM. Among these, instead compounds 3b, 3g and 3i were measured to inhibit CA I only at the micromolar concentration, whereas derivative 3a arose as the best inhibitor with a KI of 50.1 nM. The second series derivatives, that bear an amidic function at the 5 position of the isoxazole moiety were shown to act as weak CA I inhibitors with KIs ranging between 1579.4 and 9260.9 nM, wheareas compound 5g did not significantly the enzyme up to 10 µM. (ii) The physiologically dominant isoform hCA II was very potently inhibited by all the first series derivatives (KI values ranging between 0.5 and 49.3 nM, Table 3). It is worth stressing the subnanomolar inhibitory activity against CAII of compounds 3c, 3d, 3g and 3h which incorporate respectively a 4-Cl, 4-F, 4NO2 or 2-NO2 moiety on the aryl group. These values make such derivatives by far better inhibitors compared to the clinically used standard AAZ (KI value of 12.1 nM). Conversely, derivatives 5a-5m showed a diminished hCA II inhibitory effectiveness with KIs spanning in the medium-high nanomolar range (58.3-569.0 nM) with the exception of compound 5b, which was active only at a micromolar concentration (KI 1778.9 nM). (iii) Most of the reported sulfonamides showed comparable effectiveness in inhibiting the membrane-associated isoform hCA IV with KI values spanning in the medium nanomolar range (108.0- 451.3 nM). Derivatives 3c and 3i from the first series and
5i and 5l from the second one exhibited the best CAIV inhibitory activity (respectively KIs: 47.0, 47.2, 79.6 and 71.3 nM), comparable to the standard AAZ (KI value of 74 nM), whereas 5e and 5g inhibited the isozyme in the micromolar range (KI values of 2396.8 and 4429.4 nM). (iv) Finally the hCA VII inhibition data shown in Table 3 clearly highlight a quite similar tendency to one observed for hCA II. Indeed, the benzoil-substituted derivatives 5a-5m showed a diminished inhibitory efficacy in comparison to 3a-3k which incorporate a free amine at the 5 position of the isoxazole core: the KIs measured for the compounds of the first series span from 4.3 to 51.9 nM, whereas within the second series the values showed an interesting correlation to the substitution pattern on the aryl moiety. In fact, derivatives 5h-5m, which possess a halogen atom on the aryl moiety are endowed with the lowest inhibition constants ranging between 26.8 to 86.3 nM, while the remaining ones showed KIs measured from 376.2 to 1000.0 nM. (v) In general, the functionalization of 3a-3k amine moiety with benzoyl groups worsened the compounds inhibitory profiles against all the screened CA isoforms, with the exception of CAIV which was rather equally inhibited by the compounds of the first and second series. We may hypothesize the incorporation of bulky groups orthogonally to the benzensulfonamide moiety lead an excessive steric hyndrace that negatively affect the inhibitory effectiveness of the reported isoxazole derivatives against hCAI, II and VII. 3. Conclusions We reported two new series of benzenesulfonamides incorporating isoxazole moieties, which have been prepared by means of conventional and MW methods. Derivatives 3a-k and 5a-m were investigated for the inhibition of four physiologically relevant CA isoforms: hCA I, II, IV, and VII. The derivatives of the first series were shown to possess excellent inhibitory activity against the cytosolic isoform hCA II, an antiglaucoma drug target, with KIs in the range of 0.5–49.3 nM and hCA VII, a recently validated anti-neuropathic pain target with KIs in the range of 4.3-51.9 nM. The inhibition profiles reported for all the screened compounds highlighted an almost general dimished inhibitory effectiveness for the 5amidoisoxazole 5a-m in comparison to their synthetic precursor 5-amidoisoxazole 3a-k. 4. Experimental protocols 4.1. Chemistry Anhydrous solvents and all reagents were purchased from Sigma–Aldrich and Carlo Erba. Nuclear magnetic resonance (1H NMR, 13C NMR) spectra were recorded using Bruker 300 MHz and Jeol 400 MHz spectrometer in DMSO-d6 and Acetone- d6. Chemical shifts are reported in parts per million (ppm) and the coupling constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s (singlet); d (doublet); m (multiplet); broad s (broad singlet); dd (doublets of doublet); app s (apparently singlet); app d (apparently doublet); app t (apparently triplet). Fourier Transform Infrared (FT-IR) spectra were recorded on SHIMADZU FT-IR-8400S. The mass analyses were performed on Waters 2695 Alliance Micromass ZQ (LCMS) and Waters SYNAPT (HRMS). Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel F-254 plates. Flash chromatography purifications were performed on Merck Silica gel 60 (230–400 mesh ASTM) as the stationary phase and ethyl acetate/n-hexane were used as eluents. Melting points (mp) were measured in open capillary tubes with a MELTEMP apparatus and are uncorrected.
General synthetic procedure of isoxazole sulfonamides 3a – k A suspension of 2-cyano-N-(4-sulfamoylphenyl)acetamide 1 (1 equiv.) and triethylamine (TEA) (2.5 equiv.) in ethanol (15 mL) was heated under reflux until the reaction mixture became clear. Thus, a solution of α-chlorooxime 2 (2 equiv.) in ethanol (5 mL) was added to the mixture and the resulting solution was heated under reflux until the consumption of the starting material (TLC monitoring). The reaction mixture was cooled to r.t. and the formed precipitated was filtered, washed with ethanol and recrystallized from ethylacetate (compounds 3a, 3f, 3g, 3h and 3i). In case the precipitate did not form the reaction mixture was concentrated in vacuo and the obtained residue purified by silica gel coloumn chromatography eluting with 50 % EtOAc in nhexane (compounds 3b, 3c, 3d, 3e, 3j and 3k). 5-Amino-3-(2,6-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (3a)
5-Amino-3-(4-chlorophenyl)-N-(4-sulfamoylphenyl)isoxazole4-carboxamide (3c)
Obtained as white solid (160 mg, 24%), m.p. 221 – 222˚C, Rf: 0.51. max (KBr, cm-1) 3394 (NH₂ ), 3448 (NH), 3228 (NH₂ ), 1654 (HN-C=O), 1620 (NH bent), 1589 (C=C), 1327 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₃ ClN₄ O₄ S [M+H]+ 393.0424, found 393.0428. 1H NMR (400 MHz, DMSO-d6) δ 9.31 (1 H, broad s, NH), 7.74 (2 H, broad s, NH2), 7.71 (2 H, d, J 8.9, aromatic CH), 7.62 (2 H, d, J 8.7, aromatic CH – SO2NH2), 7.58 (2 H, d, J 8.9, aromatic CH), 7.52 (2 H, d, J 8.7, aromatic CH – SO2NH2), 7.21 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 171.7, 161.6, 160.2, 142.4, 138.6, 135.0, 130.7, 129.0, 128.3, 126.9, 119.5, 88.2. m/z (TOF ES+) 393 (M+H+, 100%), 434 (M + MeCN, 35%). 5-Amino-3-(4-fluorophenyl)-N-(4-sulfamoylphenyl)isoxazole4-carboxamide (3d)
Obtained as light pink solid (1.06 g, 52%), m.p. 252 – 253˚C, Rf: 0.53. max (KBr, cm-1) 3321 (NH₂ ), 3448 (NH), 1666 (HN-CO), 1627 (NH bent), 1589 (C=C) 1327 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₂ Cl₂ N₄ O₄ S [M+H]+ 427.0035, found 427.0055. 1H NMR (400 MHz, DMSO-d6) δ 8.88 (1 H, broad s, NH), 7.97 (2 H, broad s, NH2), 7.67 (2 H, d, J 8.7, aromatic CH – SO2NH2), 7.60 – 7.57 (2 H, m), 7.54 (2 H, d, J 8.6, aromatic CH – SO2NH2), 7.53 – 7.49 (1 H, m, aromatic CH), 7.20 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSOd6) δ 169.4, 159.8, 158.2, 141.8, 138.2, 134.5, 132.0, 128.3, 127.8, 126.3, 119.4, 89.1. m/z (TOF ES+) 427 (M+H+, 100%), 468 (M + MeCN, 45%). 5-Amino-3-(2,4-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (3b)
Obtained as white solid (435 mg, 27%), m.p. 218 – 219˚C, Rf: 0.53. max (KBr, cm-1) 3340 (NH₂ ), 3452 (NH), 3232 (NH₂ ), 1670 (HN-C=O), 1627 (NH bent), 1593 (C=C), 1319 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₂ Cl₂ N₄ O₄ S [M+H]+ 427.0035, found 427.0029. 1H NMR (400 MHz,DMSO-d6) δ 8.98 (1 H, broad s, NH), 7.83 (2 H, broad s, NH2), 7.72 (1 H, d, J 1.9, aromatic CH), 7.68 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.60 – 7.58 (1 H, m, aromatic CH), 7.56 (1 H, d, J 1.9, aromatic CH), 7.50 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.19 (2 H, broad s, SO2NH2).13C NMR (100 MHz, DMSO-d6) δ 170.7, 161.2, 159.3, 142.4, 138.5, 135.7, 134.2, 133.7, 129.6, 127.9, 127.6, 126.9, 119.6, 89.7. m/z (TOF ES+) 335 (19%), 427 (M+H+, 100%), 468 (M + MeCN, 45%).
Obtained as white solid (356 mg, 32%), m.p. 203 – 205˚C, Rf: 0.51. max (KBr, cm-1) 3483 (NH₂ ), 3340 (NH), 3236 (NH₂ ), 1674 (HN-C=O), 1627 (NH bent), 1593 (C=C), 1323 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₃ FN₄ O₄ S [M+H]+ 377.0720, found 377.0722. 1H NMR (400 MHz, DMSO-d6) δ 9.19 (1 H, broad s, NH), 7.73 (2 H, broad s, NH2), 7.70 (2 H, d, J 8.7, aromatic CH – SO2NH2), 7.68 – 7.63 (2 H, m, aromatic CH), 7.57 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.30 (2 H, app. t, J 8.8, aromatic CH), 7.21 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 171.6, 163.4 (d, 1JC-F 247.0, C–F), 161.6, 160.3, 142.3, 138.6, 131.2 (d, 3 JC-F 8.6, C–F), 126.9, 125.8 (d, 4JC-F 3.2, C–F), 119.4, 116.0 (d, 2 JC-F 21.8, C–F), 88.2. m/z (TOF ES+) 310 (17%), 377 (M+H+, 100%), 418 (M + MeCN, 28%). 5-Amino-3-(4-bromophenyl)-N-(4-sulfamoylphenyl)isoxazole4-carboxamide (3e)
Obtained as white solid (196 mg, 26%), m.p. 238 – 239˚C, Rf: 0.53. max (KBr, cm-1) 3441 (NH₂ ), 3306 (NH), 3151 (NH₂ ), 1658 (HN-C=O), 1620 (NH bent), 1589 (C=C), 1327 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated
C₁ ₆ H₁ ₃ BrN₄ O₄ S [M+H] + 436.9919, found 436.9926. 1H NMR (400 MHz, DMSO-d6) δ 9.33 (1 H, broad s, NH), 7.74 (2 H, broad s, NH2), 7.71 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.66 (2 H, d, J 8.5, aromatic CH), 7.59 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.55 (2 H, d, J 8.5, aromatic CH), 7.21 (2 H, broad s, NH2). 13C NMR (100 MHz, DMSO-d6) δ 171.6, 161.6, 160.3, 142.4, 138.6, 131.9, 130.9, 128.7, 126.9, 123.8, 119.5, 88.1. m/z (TOF ES+) 341 (20%), 436 (M+H+, 100%), 480 (M + MeCN, 50%). 5-Amino-3-phenyl-N-(4-sulfamoylphenyl)isoxazole-4carboxamide (3f)
Obtained as white solid (451 mg, 35%), m.p. 232 – 233˚C, Rf: 0.51. max (KBr, cm-1) 3387 (NH₂ ), 3479 (NH), 3228 (NH₂ ), 1678 (HN-C=O), 1631 (NH bent), 1593 (C=C), 1315 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₄ N₄ O₄ S [M+H]+ 359.0814, found 359.0818. 1H NMR (400 MHz, DMSOd6) δ 8.97 (1 H, broad s, NH), 7.73 (2 H, broad s, NH2), 7.69 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.61 (2 H, dd, J 7.5, 1.8, aromatic CH), 7.51 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.50 – 7.46 (3 H, m, aromatic CH), 7.20 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 171.2, 161.2, 160.6, 141.8, 138.1, 129.9, 128.8, 128.6, 128.5, 126.5, 118.8, 87.7. m/z (TOF ES+) 273 (17%), 281 (30%), 325 (80%), 359 (M+H+, 100%), 400 (M + MeCN, 10%). 5-Amino-3-(4-nitrophenyl)-N-(4-sulfamoylphenyl)isoxazole-4carboxamide (3g)
Obtained as brown solid (269 mg, 28%), m.p. 250-251˚C, Rf: 0.49. max (KBr, cm-1) 3302 (NH₂ ), 3425 (NH), 1654 (HNC=O), 1624 (NH bent), 1589 (C=C), 1519 (NO₂ asym.), 1350 (NO₂ sym.), 1327 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₃ N₅ O₆ S [M-H]+ 402.0508, found 402.0517. 1H NMR (400 MHz, DMSO-d6) δ 9.52 (1 H, broad s, NH), 8.29 (2 H, d, J 8.8, aromatic CH), 7.87 (2 H, d, J 8.8, aromatic CH), 7.83 (2 H, broad s, NH2), 7.70 (2 H, d, J 8.7, aromatic CH – SO2NH2), 7.60 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.21 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 171.2, 160.9, 159.5, 148.1, 141.9, 138.2, 135.5, 129.8, 126.4, 123.5, 119.2, 87.9. m/z (TOF ES+) 359 (23%), 402 (M-H+, 100%). 5-Amino-3-(2-nitrophenyl)-N-(4-sulfamoylphenyl)isoxazole-4carboxamide (3h)
Obtained as yellow solid (700 mg, 64%), m.p. 234˚C (decomp.), Rf: 0.2. max (KBr, cm-1) 3329 (NH₂ ), 3255 (NH₂ ), 1674 (HNC=O), 1593 (C=C), 1489 (NO₂ asym), 1369 (NO₂ sym.), 1323 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₃ N₅ O₆ S [M+H]+ 404.0665, found 404.0646. 1H NMR (400 MHz, DMSO-d6) δ 9.81 (1 H, broad s, NH), 8.06 (1 H, d, J 8.3, aromatic CH), 7.92 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.88 (1 H, d, J 1.4, aromatic CH), 7.73 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.47 – 7.41 (1 H m, aromatic CH), 7.20 (2 H, broad s, SO2NH2), 7.06 – 6.99 (1 H, m, aromatic CH), (NH2 peak obscured by broad water peak from DMSO). 13C NMR (100 MHz, DMSO-d6) δ 169.2, 162.3, 150.1, 142.4, 138.4, 132.8, 131.4, 126.8, 122.8, 120.5, 119.9, 115.9, 114.4, 106.3. m/z (TOF ES+) 275 (20%), 283 (43%), 303 (55%), 404 (M+H+, 100%), 445 (M + MeCN, 10%). 5-Amino-3-(2-chloro-4,5-dimethoxyphenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (3i)
Obtained as white solid (1.12 g, %88), m.p. 179 – 180˚C, Rf: 0.38. max (KBr, cm-1) 3468 (NH₂ ), 2947 (CH₃ ), 1662 (HNC=O), 1627 (NH bent), 1593 (C=C), 1330 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₁ ₈ H₁ ₇ ClN₄ O₆ S [M+H]+ 453.0636, found 453.0631. 1H NMR (400 MHz, DMSO-d6) δ 8.26 (1 H, broad s, NH), 7.83 (2 H, broad s, NH2), 7.68 (2 H, d, J 8.0, aromatic CH – SO2NH2), 7.41 (2 H, d, J 8.0, aromatic CH – SO2NH2), 7.20 (2 H, broad s, SO2NH2,), 7.16 (1 H, s, aromatic CH), 7.14 (1 H, s, aromatic CH), 3.83 (3 H, s, OCH3), 3.79 (3 H, s, OCH3). 13C NMR (100 MHz, DMSO-d6) δ 170.9, 160.9, 158.6, 150.7, 147.7, 141.6, 138.1, 126.6, 124.1, 118.9, 118.8, 114.4, 113.0, 88.6, 56.1, 55.9. m/z (TOF ES+) 453 (M+H+, 100%), 494 (M + MeCN, 20%). 5-Amino-3-(5-chlorofuran-2-yl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (3j)
Obtained as white solid (302 mg, 53%), m.p. 227˚C (decomp.), Rf: 0.53. max (KBr, cm-1) 3313 (NH₂ ), 3402 (NH), 3217 (NH₂ ), 1662 (HN-C=O), 1624 (NH bent), 1593 (C=C), 1334 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated
C₁ ₄ H₁ ₁ ClN₄ O₅ S [M+H]+ 383.0217, found 383.0225. 1H NMR (400 MHz, DMSO-d6) δ 9.78 (1 H, broad s, NH), 7.80 (2 H broad s, NH2), 7.75 (2 H, app. d, J 9.4, aromatic CH – SO2NH2), 7.72 (2 H, app. d, J 9.5, aromatic CH – SO2NH2), 7.23 (2 H, broad s, SO2NH2), 7.04 (1 H, d, J 3.5, aromatic CH), 6.67 (1 H, d, J 3.5, aromatic CH). 13C NMR (100 MHz, DMSO-d6) δ 171.2, 160.6, 150.8, 142.9, 142.0, 138.2, 136.6, 126.5, 119.0, 114.9, 108.9, 86.6. m/z (TOF ES+) 383 (M+H+, 100%), 424 (M + MeCN, 20%). 5-Amino-3-(5-chlorothiophen-2-yl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (3k)
NH), 9.17 (1 H, broad s, NH), 8.39 – 8.32 (4 H, m, aromatic CH), 7.73 (2 H, d, J 8.3, aromatic CH – SO2NH2), 7.66 (2 H, d, J 8.4, aromatic CH – SO2NH2), 7.54 (2 H, d, J 7.4, aromatic CH), 7.51 – 7.43 (1 H, m, aromatic CH), 7.14 (2 H, broad s, SO2NH2,).13C NMR (100 MHz, DMSO-d6) δ 167.1, 160.6, 158.4, 149.0, 142.6, 138.0, 134.9, 131.5, 130.7, 129.8, 128.8, 128.2, 127.4, 126.6, 123.8, 119.9, 118.4. m/z (TOF ES+) 389 (70%), 576 (M+H+, 100%). 3-(2,4-Dichlorophenyl)-5-(4-nitrobenzamido)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5b)
Obtained as orange solid (114 mg, 79%), m.p. 251 – 252˚C, Rf: Obtained as dark yellow solid (279 mg, 15%), m.p. 178-180˚C, Rf: 0.53. max (KBr, cm ) 3448 (NH₂ ), 3263 (NH), 1658 (HNC=O), 1620 (NH bent), 1589 (C=C), 1327 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C14H11ClN4O4S2 [M+H]+ 398.9989, found 389.9989. 1H NMR (400 MHz, DMSO-d6) δ 9.88 (1 H, broad s, NH), 7.76 (2 H, broad s, NH2), 7.71 (2 H, d, J 9.1, aromatic CH – SO2NH2), 7.67 (2 H, d, J 9.1, aromatic CH – SO2NH2), 7.43 (1 H, d, J 4.0, aromatic CH), 7.22 (2 H, broad s, SO2NH2), 7.14 (1 H, d, J 4.0, aromatic CH). 13C NMR (100 MHz, DMSO-d6) δ 171.3, 161.4, 155.1, 142.6, 138.8, 130.9, 129.8, 129.2, 128.1, 127.0, 119.8, 87.9. m/z (TOF ES+) 293 (20%), 301 (31%), 321 (40%), 398 (M+H+, 100%), 440 (M + MeCN, 48%). -1
General synthetic procedure of isoxazole sulfonamides 5a – k Cs₂ CO₃ (3 equiv.) and 5-aminoisoxazole 3 (1 equiv.) were added to a solution of the proper benzoyl chloride 4 (1 equiv.) in MeCN (5 mL) at ice-bath temperature and the obtained suspension was stirred at r.t. until the consumption of the starting material (TLC monitoring). Then, the reaction mixture was concentrated under vacuo and the obtained residue was treated with water and extracted with EtOAc (3x15 ml). The organic layers were dried over Na2SO4, filtered-off and concentrated under vacuo to give a residue that was purified by silica gel coloumn chromatography eluting with 75 % EtOAc in n-hexane. 3-(2,6-Dichlorophenyl)-5-(4-nitrobenzamido)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5a)
Obtained as yellow solid (265 mg, 92%), m.p. 208 – 209˚C, Rf:0.12. max (KBr, cm-1) 3321 (NH₂ ), 3232 (NH), 3167 (NH), 1685 (HN-C=O), 1593 (HN-C=O), 1527 (asym. NO₂ ), 1346 (sym. NO₂ ), 1315 (asym. SO₂ ), 1153 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ Cl₂ N₅ O₇ S [M+H]+ 576.0148, found 576.0142. 1H NMR (400 MHz, DMSO-d6) δ 11.58 (1 H, broad s,
0.26. max (KBr, cm-1) 3398 (NH₂ ), 3298 (NH), 3225 (NH), 1674 (HN-C=O), 1593 (HN-C=O), 1531 (asym. NO₂ ), 1346 (sym. NO₂ ), 1315 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ Cl₂ N₅ O₇ S [M+H]+ 576.0148, found 576.0146. 1H NMR (400 MHz, DMSO-d6) δ 11.64 (1 H broad s, NH), 9.06 (1 H, broad s, NH), 8.43 – 8.28 (4 H, m, aromatic CH), 7.73 (2 H d, J 8.8, aromatic CH – SO2NH2), 7.71 (1 H, d, J 1.9, aromatic CH), 7.66 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.50 (1 H, dd, J 8.2, 1.8, aromatic CH), 7.45 (1 H, d, J 8.1, aromatic CH), 7.17 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSOd6) δ 167.1, 160.7, 159.9, 157.2, 149.0, 145.9, 142.6, 139.5, 137.9, 134.8, 134.6, 132.7, 129.8, 129.0, 127.4, 127.3, 124.5, 123.8, 118.5. m/z (TOF ES+) 389 (75%), 576 (M+H+, 100%). 3-(4-Fluorophenyl)-5-(4-nitrobenzamido)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5c)
Obtained as orange solid (41 mg, 39%), m.p. 230 – 231˚C, Rf: 0.31. max (KBr, cm-1) 3267 (NH), 3244 (NH), 1674 (HN-C=O), 1589 (HN-C=O), 1523 (asym. NO₂ ), 1346(sym. NO₂ ), 1315 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₆ FN₅ O₇ S [M-H]+ 524.0676, found 524.0674. 1H NMR (400 MHz, DMSO-d6) δ 12.35 (1 H, broad s, NH), 11.89 (1 H, broad s, NH), 8.35 (2 H, d, J 8.9, aromatic CH), 8.31 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.77 – 7.73 (4 H, m, aromatic CH), 7.70 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.27 (2 H, app. t, J 8.9, aromatic CH), 7.18 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 163.9, 162.5 (d, 1JC-F 194.0, C–F), 161.4, 161.1, 160.8, 156.4, 148.7, 148.2, 142.3, 137.6, 131.4 (d, 3 JC-F 8.5, C–F), 129.4, 126.9, 123.4, 119.6, 118.3, 114.7 (d, 2JC-F 22.3, C–F). m/z (TOF ES+) 524 (M-H+, 100%). 5-(4-Nitrobenzamido)-3-phenyl-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5d)
Obtained as dark yellow solid (56 mg, 37%), m.p. 207 – 208˚C, Rf: 0.12. max (KBr, cm-1) 3448 (NH), 3383 (NH), 1670 (HNC=O), 1612 (HN-C=O), 1527 (asym. NO₂ ), 1346 (sym. NO₂ ), 1315 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₇ N₅ O₇ S [M+H]+ 508.0927, found 508.0926. 1H NMR (400 MHz, DMSO-d6) δ 12.32 (1 H, broad s, NH), 11.73 (1 H, broad s, NH), 8.35 (2 H, d, J 8.8, aromatic CH), 8.29 (2 H, d, J 8.4, aromatic CH), 7.74 (2 H, d, J 8.9, aromatic CH), 7.71 – 7.66 (4 H, m, aromatic CH), 7.46 – 7.43 (3 H, m, aromatic CH), 7.17 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 165.2, 161.8, 160.4, 148.8, 148.7, 142.2, 139.0, 137.6, 129.4, 129.1, 128.9, 128.7, 127.8, 126.8, 124.0, 123.4, 118.3. m/z (TOF ES+) 355 (70%), 508 (M+H+, 100%). 3-(2-Chloro-4,5-dimethoxyphenyl)-5-(4-nitrobenzamido)-N(4-sulfamoylphenyl) isoxazole-4-carboxamide (5e)
Obtained as yellow solid (26 mg, 18%), m.p. 250 – 251˚C, Rf: 0.19. max (KBr, cm-1) 3460 (NH), 3275 (NH), 2958 (CH3), 2854 (CH₃ ), 1674 (HN-C=O), 1616 (HN-C=O), 1523 (asym. NO₂ ), 1350 (sym. NO₂ ), 1330 (asym. SO₂ ), 1153 (sym. SO₂ ). HRMS: calculated C₂ ₅ H₂ ₀ ClN₅ O₉ S [M]+ 602.0749, found 602.0765. 1H NMR (400 MHz, DMSO-d6) δ 12.42 (1 H, broad s, NH), 11.71 (1 H, broad s, NH), 8.37 – 8.31 (4 H, m, aromatic CH), 7.72 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.67 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.17 (2 H, broad s, SO2NH2), 7.09 (1 H, s, aromatic CH), 6.97 (1 H, s, aromatic CH), 3.83 (3 H, s, OCH3), 3.76 (3 H, s, OCH3). 13C NMR (100 MHz, DMSO-d6) δ 172.9, 160.7, 152.5, 150.0, 149.0, 147.5, 146.2, 142.8, 139.5, 131.7, 129.8, 127.4, 124.9, 124.5, 123.8, 118.5, 118.4, 114.4, 112.9, 56.4, 56.3. m/z (TOF ES+) 271 (30%), 402 (48%), 602 (M+, 100%). 5-Benzamido-3-(2,6-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide(5f)
Obtained as white solid (329 mg, 62%), m.p. 214 – 215˚C, Rf: 0.48. max (KBr, cm-1) 3387 (NH₂ ), 3248 (NH), 3186 (NH), 1689 (HN-C=O), 1670 (HN-C=O), 1527 (C=C), 1315 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated
C₂ ₃ H₁ ₆ Cl₂ N₄ O₅ S [M+H]+ 531.0297, found 531.0296. 1H NMR (300 MHz, DMSO-d6) δ 11.91 (1 H, broad s, NH), 11.00 (1 H, broad s, NH), 8.08 (2 H, d, J 7.4, aromatic CH), 7.71 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.66 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.61 – 7.56 (3 H, m, aromatic CH), 7.55 – 7.49 (3 H, m, aromatic CH), 7.10 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, Acetone-d6) δ 169.6, 159.7, 158.8, 156.9, 142.4, 138.4, 135.3, 132.1, 132.0, 131.8, 131.7, 131.6, 128.6, 128.4, 128.2, 127.2, 118.8. m/z (TOF ES+) 531 (M+H+, 100%). 5-Benzamido-3-(2,4-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5g)
Obtained as white solid (79 mg, 62%), m.p. 211 – 212˚C, Rf: 0.51. max (KBr, cm-1) 3387 (NH₂ ), 3182 (NH), 3105 (NH), 1689 (HN-C=O), 1666 (HN-C=O), 1554 (C=C), 1303 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₆ Cl₂ N₄ O₅ S [M+H]+ 531.0297, found 531.0294. 1H NMR (400 MHz, Acetone-d6) δ 11.21 (1 H, broad s, NH), 11.06 (1 H, broad s, NH), 8.19 (2 H, d, J 8.0, aromatic CH), 7.75 (2 H, app. d, J 9.3, aromatic CH – SO2NH2), 7.72 (2 H, app. d, J 9.3, aromatic CH – SO2NH2), 7.59 (1 H, app. s, aromatic CH), 7.56 – 7.44 (5 H m, aromatic CH), 6.44 (2 H, broad s, SO2NH2,). 13C NMR (100 MHz Acetone-d6) δ 172.6, 160.4, 159.9, 152.3, 142.8, 138.0, 137.4, 135.3, 134.9, 132.7, 131.7, 131.5, 129.1, 128.5, 128.3, 127.2, 127.1, 124.6, 118.6. m/z (TOF ES+) 531 (M+H+, 100%). 5-(4-Chlorobenzamido)-3-(2,6-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5h)
Obtained as light yellow solid (142 mg, %84), m.p. 193 –194˚C, Rf: 0.37. max (KBr, cm-1) 3404 (NH₂ ), 3230 (NH), 3076 (NH), 1676 (HN-C=O), 1664 (HN-C=O), 1560 (C=C), 1319 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ Cl₃ N₄ O₅ S [M+H]+ 564.9907, found 564.9908. 1H NMR (300 MHz, DMSO-d6) δ 12.16 (1 H, broad s, NH), 11.76 (1 H, broad s, NH), 8.21 – 8.06 (2 H, m, aromatic CH), 7.74 (2 H, d, J 8.6, aromatic CH), 7.66 (2 H, d, J 8.8, aromatic CH), 7.62 – 7.48 (5 H, m, aromatic CH), 7.21 (2 H, broad s, SO2NH2). 13C NMR (100 MHz Acetone-d6) δ 168.5, 160.4, 158.7, 144.6, 143.2, 137.5, 136.1, 135.2, 130.8, 130.4, 129.0, 128.1, 127.8, 127.3, 124.6, 118.2, 118.0. m/z (TOF ES+) 151 (18%), 564 (M+H+, 100%). 5-(4-Chlorobenzamido)-3-(2,4-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5i)
Obtained as white solid (103 mg, 61%), m.p. 267 – 268˚C, Rf: 0.40. max (KBr, cm-1) 3398 (NH₂ ), 3259 (NH), 3095 (NH), 1676 (HN-C=O), 1653 (HN-C=O), 1560 (C=C), 1319 (asym. SO₂ ), 1159 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ Cl₃ N₄ O₅ S [M+H]+ 564.9907, found 564.9935. 1H NMR (300 MHz, DMSO-d6) δ 12.09 (1 H, broad s, NH), 11.75 (1 H, broad s, NH), 8.14 – 7.99 (2 H, m, aromatic CH), 7.75 (2 H, d, J 8.6, aromatic CH), 7.71 – 7.66 (3 H, m, aromatic CH), 7.61 (2 H, d, J 8.6, aromatic CH), 7.55 – 7.40 (2 H, m, aromatic CH), 7.21 (2 H, broad s, SO2NH2,). 13C NMR (100 MHz, Acetone-d6) δ 171.7, 168.2, 160.5, 160.0, 143.1, 137.6, 136.4, 135.0, 134.9, 132.4, 130.4, 130.0, 128.9, 128.2, 127.3, 126.8, 124.6, 118.3, 118.2. m/z (TOF ES+) 151 (13%), 564 (M+H+, 100%). 5-(4-Bromobenzamido)-3-(2,6-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5j)
Obtained as light yellow solid (144 mg, 79%), m.p. 266 –267˚C, Rf: 0.37. max (KBr, cm-1) 3398 (NH₂ ), 3240 (NH), 3076 (NH), 1680 (HN-C=O), 1653 (HN-C=O), 1560 (C=C), 1317 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ 79BrCl₂ N₄ O₅ S [M+H]+ 608.9402, found 608.9414. 1 H NMR (300 MHz, DMSO-d6) δ 12.16 (1 H, broad s, NH), 11.75 (1 H, broad s, NH), 8.16 – 7.99 (2 H, m, aromatic CH), 7.76 – 7.71 (4 H, m, aromatic CH), 7.66 (2 H d, J 8.8, aromatic CH), 7.58 (2 H, d, J 8.8, aromatic CH), 7.54 – 7.47 (1 H, m, aromatic CH), 7.21 (2 H, broad s, SO2NH2,). 13C NMR (100 MHz, Acetone-d6) δ 170.7, 168.1, 160.2, 158.8, 154.9, 150.3, 142.9, 141.1, 137.7, 135.2, 131.2, 131.0, 130.7, 127.9, 127.3, 125.1, 118.3. m/z (TOF ES+) 307 (16%), 324 (28%), 329 (46%), 608 (70%), 610 (M+H+, 100%).
5-(4-Bromobenzamido)-3-(2,4-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5k)
Obtained as white solid (150 mg, 62%), m.p. 232 – 233˚C, Rf: 0.74. max (KBr, cm-1) 3398 (NH₂ ), 3257 (NH), 3088 (NH), 1683 (HN-C=O), 1653 (HN-C=O), 1560 (C=C), 1319 (asym.
SO₂ ), 1159 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ 79BrCl₂ N₄ O₅ S [M+H]+ 608.9402, found 608.9404. 1 H NMR (400 MHz, Acetone-d6) δ 11.30 (1 H, broad s, NH), 10.62 (1 H, broad s, NH), 8.12 – 8.03 (2 H, m, aromatic CH), 7.76 – 7.73 (1 H, m, aromatic CH), 7.73 – 7.63 (4 H, m, aromatic CH), 7.64 – 7.57 (1 H, m, aromatic CH), 7.55 – 7.46 (3 H, m, aromatic CH), 6.43 (2 H, broad s, SO2NH2,). 13C NMR (100 MHz, Acetone-d6) δ 175.2, 168.0, 160.1, 159.9, 148.6, 142.5, 138.3, 135.7, 135.6, 134.7, 132.9, 132.8, 131.6, 130.4, 129.2, 127.3, 127.2, 126.0, 118.9. m/z (TOF ES+) 307 (13%), 329 (23%), 608 (62%), 610 (M+H+, 100%). 3-(2,6-Dichlorophenyl)-5-(4-methoxybenzamido)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5l)
Obtained as white solid (92 mg, 41%), m.p. 238 – 240˚C, Rf: 0.48. max (KBr, cm-1) 3400 (NH₂ ), 3240 (NH), 3088 (NH), 2929 (-CH), 2841 (-CH), 1683 (HN-C=O), 1653 (HN-C=O), 1560 (C=C), 1317 (asym. SO₂ ), 1159 (sym. SO₂ ). HRMS: calculated C₂ ₄ H₁ ₈ Cl₂ N₄ O₆ S [M+H]+ 561.0402, found 561.0419. 1H NMR (400 MHz, Acetone-d6) δ 11.20 (1 H, broad s, NH), 11.02 (1 H, broad s, NH), 8.18 (2 H, d, J 7.2, aromatic CH), 7.78 – 7.70 (4 H, m, aromatic CH), 7.55 – 7.46 (3 H, m, aromatic CH), 7.04 (2 H, d, J 7.2, aromatic CH), 6.44 (2 H, broad s, SO2NH2), 3.85 (3 H, s, OCH3). 13C NMR (100 MHz, Acetoned6) δ 162.7, 160.0, 158.7, 155.3, 150.5, 145.3, 143.9, 142.8, 137.9, 135.3, 132.1, 131.3, 130.5, 128.1, 127.2, 118.5, 113.5, 55.0. m/z (TOF ES+) 151 (21%), 561 (M+H+, 100%). 3-(2,4-Dichlorophenyl)-5-(4-methoxybenzamido)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5m)
Obtained as white solid (90 mg, 40%), m.p. 153 – 154˚C, Rf: 0.53. max (KBr, cm-1) 3398 (NH₂ ), 3257 (NH), 3103 (NH), 2933 (-CH), 2841 (-CH), 1680 (HN-C=O), 1653 (HN-C=O), 1560 (C=C), 1319 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₂ ₄ H₁ ₈ Cl₂ N₄ O₆ S [M+H]+ 561.0402, found 561.0395. 1H NMR (400 MHz, Acetone-d6) δ 11.12 (1 H, broad s, NH), 10.44 (1 H, broad s, NH), 8.12 (2 H, d, J 8.9, aromatic CH), 7.76 (2 H, d, J 8.6, aromatic CH), 7.69 (2 H, d, J 8.9, aromatic CH), 7.61 (1 H, d, J 2.0, aromatic CH), 7.57 (1 H, d, J 8.2, aromatic CH), 7.49 (1 H, dd, J 8.3, 2.0, aromatic CH), 7.04 (2 H, d, J 8.9, aromatic CH), 6.46 (2 H, broad s, SO2NH2,), 3.85 (3 H, s, OCH3). 13C NMR (100 MHz, Acetone-d6) δ 166.2, 162.9, 160.0, 159.7, 152.7, 148.3, 142.2, 138.3, 138.2, 135.7, 134.5, 132.8, 130.3, 129.2, 127.9, 127.2, 127.0, 118.9, 113.6, 54.9. m/z (TOF ES+) 173 (36%), 307 (40%), 324 (68%), 461 (20%), 561 (M+H+, 100%).
4.2. CA inhibition An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration activity.30 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CAcatalyzed CO2 hydration reaction for a period of 10-100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier, 26 and represent the mean from at least three different determinations. All CA isofoms were recombinant ones obtained in-house as reported earlier.31-32
Acknowledgments We are extremely grateful to TUBITAK (The Scientific and Technological Research Council of Turkey, Grant 114Z181) and AIBU BAP (Grant No: 2013.3.3.668) for financial support. References 1. Daidone, G.; Raffa, D.; Maggio, B.; Plescia, F.; Cutuli, V. M. C.; Mangano, N. G.; Caruso, A. Arch. Pharm. (Weinheim). 1999, 332, 50. 2. Santos, M. M. M.; Faria, N.; Iley, J.; Coles, S. J.; Hursthouse, M. B.; Martins, M. L.; Moreira, R. Bioorganic Med. Chem. Lett. 2010, 20, 193. 3. Velaparthi, S.; Brunsteiner, M.; Uddin, R. J. Med. Chem 2008, 51, 1999. 4. Selvam, C.; Jachak, S. M.; Thilagavathi, R.; Chakraborti, A. K. Bioorg. Med. Chem. Lett. 2005, 15, 1793. 5. Badio, B.; Garraffo, H. M.; Plummer, C. V.; Padgett, W. L.; Daly, J. W. Eur. J. Pharmacol. 1997, 321, 189. 6. Kamal, A.; Bharathi, E. V.; Reddy, J. S.; Ramaiah, M. J.; Dastagiri, D.; Reddy, M. K.; Viswanath, A.; Reddy, T. L.; Shaik, T. B.; Pushpavalli, S. N. C. V. L.; Pal, M. Eur. J. Med. Chem. 2011, 46, 691. 7. Nellgard, B.; Wieloch, T. Experimantal brain Res. 1992, 92, 259. 8. Gilron, I.; Max, M. B.; Lee, G.; Booher, S. L.; Sang, C. N.; Chappell, A. S.; Dionne, R. A. Clin. Pharmacol. Ther. 2000, 68, 320. 9. Kalgutkar, A. S.; Nguyen, H. T.; et al Drug Metab. Dispos. 2003, 31, 1240. 10. Gomha, S.; Badrey, M.; Abdalla, M.; Arafa, R. Med. Chem. Comm. 2014, 0, 1. 11. Crook, E. M.; Law, K. Biochem. J. 1950, 2, 37. 12. Upadhyay, A.; Gopal, M.; Srivastava, C.; Pandey, N. D. J. Pestic. Sci. 2010, 35, 464. 13. Hamama, W. S.; Ibrahim, M. E.; Zoorob, H. H. Synth. Commun. 2013, 43, 2393. 14. Moser, M.; Moser, E. I. J. Neurosci. 1998, 18, 7535.
15. Lin, N.; He, Y.; Kopecka, H. Tetrahedron Lett. 1995, 36, 2563 16. Thamizharasi, S.; Vasantha, J.; Reddy, B. S. R. Eur. Polym. J. 2002, 38, 551. 17. Bryant B; Knights K. Pharmacology Heal. Prof. 2011, 645. 18. Casini, A.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Curr. Cancer Drug Targets 2002, 2, 55. 19. Supuran, C. T.; Casini, A.; Scozzafava, A.; Chimica, D.; Bioinorganica, C. Med. Res. Rev. 2003, 23, 535. 20. Dai, H.; Stepan, A. F.; Plummer, M. S.; Zhang, Y.; Yu, J. J. Am. Chem. Soc. 2011, 133, 7222. 21. (a) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C.T.; De Simone, G. Chem. Rev. 2012, 112, 4421; (b) Supuran, C.T. Nat. Rev. Drug Discov. 2008, 7, 168; (c) Neri, D.; Supuran, C.T. Nat. Rev. Drug Discov. 2011, 10, 767; (d) Supuran CT. Expert Opin Drug Discov. 2016. http://dx.doi.org/10.1080/ 17460441.2017.1253677. in press. 22. (a) Supuran CT. J Enzyme Inhib Med Chem. 2016, 31, 345; (b) Carta, F.; Supuran, C.T.; Scozzafava, A. Future Med. Chem. 2014, 6,1149; (c) Supuran, C.T. Bioorg. Med. Chem. 2013, 21, 1377. 23. (a) Matsui, H.; Murakami, M.; Wynns, G. C.; Conroy, C. W.; Mead, A.; Maren, T. H.; Sears, M. L. Exp. Eye Res. 1996, 62, 409; (b) Datta, R.; Waheed, A.; Bonapace, G.; Shah, G. N.; Sly, W. S. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 3437; (c) Ochrietor, J. D.; Clamp, M. F.; Moroz, T. P.; Grubb, J. H.; Shah, G. N.; Waheed, A.; Sly, W. S.; Linser, P. J. Exp. Eye Res. 2005, 81, 492; (d) Tang, Y.; Xu, H.; Du, X.; Lit, L.; Walker, W.; Lu, A.; Ran, R.; Gregg, J. P.; Reilly, M.; Pancioli, A.; Khoury, J. C.; Sauerbeck, L. R.; Carrozzella, J. A.; Spilker, J.; Clark, J.; Wagner, K. R.; Jauch, E. C.; Chang, D. J.; Verro, P.; Broderick, J. P.; Sharp, F. R. J. Cereb. Blood Flow Metab. 2006, 26, 1089. 24. Hen, N.; Bialer, M.; Yagen, B.; Maresca, A.; Aggarwal, M.; Robbins, A. H.; McKenna, R.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2011, 54, 3977. 25. (a) Supuran CT. Expert Rev Neurother. 2016, 16, 961; (b) Carta F, Di Cesare Mannelli L, Pinard M, et al. Bioorg Med Chem. 2015, 23,1828. 26. (a) Bozdag, M.; Ferraroni, M.; Nuti, E.; Vullo, D.; Rossello, A.; Carta, F.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2014, 22, 334; (b) Lopez, M.; Salmon, A. J.; Supuran, C. T.; Poulsen, S. A. Curr. Pharm. Des. 2010, 16, 3277; Nocentini, A.; Carta, F.; Ceruso, M.; Bartolucci, G.; Supuran, C. T. Bioorg. Med. Chem. 2015, 23, 6955; (d) Nocentini, A.; Ferraroni, M.; Carta, F.; Ceruso, M.; Gratteri, P.; Lanzi, C.; Masini, E.; Supuran, C. T. J. Med. Chem. 2016 DOI 10.1021/acs.jmedchem.6b01389, in press. 27. Grünanger, P. and Vita-Finzi, P. (eds) (1999) Isoxazoles, in Chemistry of Heterocyclic Compounds: Isoxazoles, Part 1, Volume 49, John Wiley & Sons, Inc., Hoboken, NJ, USA. 28. (a) Altuǧ, C.; Büyükbayram, M.; Kavas, Ö.; Yavuz, M. Z. Tetrahedron 2014, 70, 3590; (b) Altuğ, C.; Dürüst, Y.; Elliot, M. C. Tetrahedron Lett. 2009, 50, 7392; (c) Altuğ, C.; Elliott, M. C.; Kariuki, B. M.; Zaal, M. Org. Biomol. Chem. 2010, 8, 4978. 29. Alafeefy, A. M.; Isik, S.; Al-jaber, N. A.; Vullo, D.; Abdelaziz, H. A.; Ashour, A. E.; Awaad, A. S.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2013, 23, 3570. 30. Khalifah, R.J. J. Biol. Chem. 1971, 246, 2561. 31. (a) De Luca V, Del Prete S, Supuran CT, Capasso C. J Enzyme Inhib Med Chem. 2015, 30, 277; (b) Vullo D, Del Prete S, Osman S.M et al. Bioorg Med Chem Lett. 2014, 24, 240; (c) Del Prete S, De Luca V, Scozzafava A, Carginale V, Supuran CT, Capasso C. J Enzyme Inhib Med Chem. 2014, 29, 23; (d) Nocentini A., Vullo D., Bartolucci G., Supuran C.T. Bioorg Med Chem. 2016, 24, 3612.
32. (a) Bozdag M, Pinard M, Carta F, et al. J Med Chem. 2014, 57, 9673; (b) Nocentini A., Ceruso M., Carta F., Supuran C.T. J Enzyme Inhib Med Chem. 2016, 31, 1226; (c) Bozdag M, Ferraroni M, Nuti E, et al. Bioorg Med Chem. 2014, 22, 334. .
Graphical Abstract
Synthesis of isoxazole-containing sulfonamides with potent carbonic anhydrase II and VII inhibitory properties Cevher Altug*, Hanife Güneş, Alessio Nocentini, Simona Maria Monti, Martina Buonanno, Claudiu T. Supuran*
S0968-0896(16)31415-8 http://dx.doi.org/10.1016/j.bmc.2017.01.008 BMC 13487
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Accepted Date:
12 December 2016 5 January 2017
Please cite this article as: Altug, C., Güneş, H., Nocentini, A., Maria Monti, S., Buonanno, M., Supuran, C.T., Synthesis of isoxazole-containing sulfonamides with potent carbonic anhydrase II and VII inhibitory properties, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.01.008
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Bioorganic & Medicinal Chemistry
Synthesis of isoxazole-containing sulfonamides with potent carbonic anhydrase II and VII inhibitory properties Cevher Altuga,*, Hanife Güneşa, Alessio Nocentinib, Simona Maria Montic, Martina Buonanno c and Claudiu T. Supuranb,* a
Department of Chemistry, Abant Izzet Baysal University, TR-14280 Bolu, Turkey Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy c Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Napoli, Italy b
A RT I C L E I N F O
A BS T RA C T
Article history:
Two series of benzenesulfonamide containing isoxazole compounds were prepared by using conventional and microwave (MW) methods. 5-Amino-3-aryl-N-(4-sulfamoylphenyl)isoxazole4-carboxamide derivatives were synthesized by the reaction of hydroxymoyl chlorides with 2cyano-N-(4-sulfamoylphenyl)acetamide in the presence of triethylamine. The synthesized 5amino isoxazoles were reacted with various benzoyl chlorides in order to obtain 5amidoisoxazoles. The novel compounds were screened in vitro as inhibitors of four human (h) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1): hCA I, hCA II, hCA IV and hCA VII. The derivatives of the first series were shown to possess excellent inhibitory activity against the cytosolic isoform hCA II, an antiglaucoma drug target, with K Is in the range of 0.5–49.3 nM and hCA VII, a recently validated anti-neuropathic pain target with KIs in the range of 4.3-51.9 nM.
Keywords: 5-amino isoxazole 5-amidoisoxazole Carbonic anhydrase sulfonamide amide
2009 Elsevier Ltd. All rights reserved.
1. Introduction Heterocyclic compounds play a fundamental role in life and biochemical processes. Among these, isoxazoles represent a versatile class of heterocycles due to the variety of biological and pharmacological applications they possess. Indeed, their derivatives exhibit ulcerogenic1, antifungal2, antimicrobial3, cyclooxygenase (COX-2) inhibitory4, antinociceptive5, 6 7 8 anticancer , hypoglycemic , analgesic anti-inflammatory9, and human immunodeficiency virus (HIV) inhibitory10 activities. Moreover, isoxazole derivatives are used as herbicides 11 and insecticides12. Depending on the position of the amino moiety at the isoxazole ring (3-, 4- or 5-), aminoisoxazoles13 are endowed with remarkable properties as active CNS (central nervous system) agents14, potent cholinergic channel activator used for treatment of Alzheimer disease15 and antimicrobial activity16. Sulfonamides gained significant importance in medicinal chemistry since their antibacterial properties were discovered by Gerhard Domagk. At a later time, it was proved that, in vivo, prontosil undergoes a metabolic reduction which forms paminobenzenesulfonamide that is the responsible of the antibacterial activity.17 Since then, sulfonamide derivatives became popular in the pharmaceutical area thanks to their biological activities as antibacterial, anticancer, antiviral, anticonvulsant, anti-inflammatory, and HIV protease
inhibitors.18,19,20 Moreover, sulfonamides are the most classical inhibitors of the zinc-enzymes carbonic anhydrases (CA, EC 4.2.1.1).21 Seven distinct genetic enzymatic families have been identified so far: the α-, β-, γ-, δ-, ζ-. η- and θ-CAs.21 The 15 human (h) isozymes, which all belong to the α-class, represent valuable biological targets for the design of CA inhibitors (CAIs) with many biomedical applications.21,22 In particular, CAI and CAII are ubiquitous isoforms, targets for some deaseases (such as cerebral and retinal edema, glaucoma, epilepsy, and probably altitude sickness), but often off-targets since they are the main responsible of the majority of side effects of non-selective inhibitors.21 The membrane-associated isoform CA IV is a drug target for retinitis pigmentosa and stroke, in addition to glaucoma, together with CA II and XII.23 CAVII has been shown to be involved in epileptiform activity together with CA II and XIV.24 Moreover hCA VII was recently validated as a new drug target for neuropathic pain.25 Many sulfonamides-CAIs are clinically used for decades to treat several diseases (e.g., glaucoma, edema, epilepsy).21b Unfortunately the lack in selectivity of sulfonamide-like CAIs against the different human isozymes is the main topic that prevents a wider pharmaceutical application to date. The “tail approach” is the mainly applied method for the research of isoform-selective sulfonamide-CAIs.26 Indeed, it consists in
appending different tails to the aromatic/heterocyclic ring bearing the sulfonamides in order to modulate the possible interactions the ligand may establish with the middle/outer parts of the active site. Over the years, a varied range of synthesis of isoxazoles has been documented. Up to know, the most widely used reaction path refers to Quilico’s studies on the synthesis of isoxazoles from nitrile oxides and unsaturated compounds.27 The previous approach used by the Altug group for the synthesis of 5aminoisoxazoles28 provided for the use of the key intermediate phenylsulfonyl acetonitrile as active methylene containing nitrile. Since those compounds showed promising in vitro anticancer activities, bearing in mind the two biologically active isoxazole and sulfonamide, we aimed to combine these two portions on a single molecule by applying the tail approach in order to obtain new CAIs. Firstly, a series of 5-amino-3-aryl-N-(4sulfamoylphenyl) isoxazole-4-carboxamides was synthesized and subsequently such derivatives were reacted with benzoyl chlorides to achieve a second series of 5-benzamido-3-phenyl-N(4-sulfamoylphenyl)isoxazole-4-carboxamides. All these novel compounds were screened for their carbonic anhydrase inhibitory properties against four human isoforms: hCA I, hCA II, hCA IV and hCA VII. 2. Result and Discussion 2.1 Chemistry The synthesis of 2-cyano-N-(4-sulfamoylphenyl)acetamide 1 was achieved according to the literature method.29 Sulfonamide-based 5-aminoisoxazole analogues were synthesized via reaction of the proper hydroxymoyl chloride with 2-cyano-N-(4sulfamoylphenyl)acetamide in presence of triethylamine to obtain the 5-amino-3-aryl-N-(4-sulfamoylphenyl)isoxazole-4carboxamide derivatives (3a – k).
Table 1. Synthesis of 5-amino-3-aryl-N-(4-sulfamoylphenyl)isoxazole-4carboxamides Compound
Ar
Yield (%)
Yield (%)
Microwave*
Conventional
3a
2,6-DiClC₆ H₃
65
52
3b 3c 3d 3e 3f 3g 3h 3i 3j 3k
2,4-DiClC₆ H₃ 4-ClC₆ H₄ 4-FC₆ H₄ 4-BrC₆ H₄ C₆ H₅ 4-O₂ NC₆ H₄ 2-O₂ NC₆ H₄
33 28 39 38 40 38 42 62 65 21
27 24 32 26 35 28 64 88 53 15
2-Cl-4,5-DiMeOC6H2
5-Cl-2-furyl 5-Cl-2-thienyl
The reactions were conducted in a Microwave: 90˚C, 300 psi, 200 W, 30 minute. *
The reactions proceeded with rather high yields, when electron withdrawing or electron releasing substituents containing hydroxymoyl chlorides reacted with active methylene containing compound 1. The same derivatives were also synthesized by means of a microwave though showing similar yields. Thereafter, the obtained 5-aminoisoxazoles were reacted with various benzoyl chlorides in the presence of Cs 2CO3 to form 5(4-substitutedbenzamido)-3-aryl-N-(4sulfamoylphenyl)isoxazole-4-carboxamide 5a – m with moderate yields.
Table 2. Synthesis of 5-amino-3-aryl-N-(4-sulfamoylphenyl) isoxazole-4-carboxamides
Compound
Ar
R
Yield (%)
5a
2,6-DiClC6H3
NO2
Conventional 92
5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m
2,4-DiClC6H3 4-FC6H4 C₆ H₅ 2-Cl-4,5-DiMeOC6H2 2,6-DiClC6H3 2,4-DiClC6H3 2,6-DiClC6H3 2,4-DiClC6H3 2,6-DiClC6H3 2,4-DiClC6H3 2,6-DiClC6H3 2,4-DiClC6H3
NO2 NO2 NO2 NO2 H H Cl Cl Br Br MeO MeO
79 39 37 18 62 62 84 61 79 62 41 40
In order to optimize the reaction conditions, we used different kind of bases, prolonged heating or microwave irradiation, but those conditions showed lower yields. All synthesized compounds were purified by column chromatography and characterized with spectroscopic measurements (FT-IR, 1H NMR, 13C NMR, HRMS) and physical data (m.p.). IR spectrum of compounds 3a – k showed characteristic peaks for the amino groups near the sulfonamide and isoxazole ring ones at 3425 and 3306 cm-1, amidic carbonyl showed a signal at 1658 cm-1, whereas asymmetric and symmetric vibrational frequencies for sulfonamides were observed at 1327 and 1157 cm-1 respectively. In addition, the amide groups of the 5-amidoisoxazoles were shown to aroused at 1676 and 1653 cm-1. The analysis of the 1H NMR spectra showed the sulfonamide peaks for compounds 3a – k at 7.2 ppm (SO2NH2) as broad singlets, whereas the remaining signals appeared in their usual ranges (see Experimental for details). Additional difficulties were observed for 13C NMR, cause of the low quality spectra obtained in DMSO-d6 for compounds 5f – m that required the use of acetone-d6 to see more efficiently the carbons signals. Finally, high resolution mass spectra (HRMS) was recorded for all the synthesized derivatived and molecules ion peaks were correctly determined. 2.2. Carbonic anhydrase inhibition We investigated the CA inhibitory activities of compounds 3a – k and 5a – m by applying the stopped flow carbon dioxide
hydrase assay30, in comparison to acetazolamide (AAZ) as standard CAI, against four physiologically significant isoforms hCA I, II, IV and VII. The following structure–activity relationship (SAR) can be extrapolated from the inhibition data shown in Table 3: Table 3. Inhibition data of human CA isoforms hCA I, II, IV and VII with sulfonamides 3a-k and 5a-m reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay30
Compound
hCA I
KI (nM) hCA II
hCA IV
hCA VII
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m AAZ
50.1 7469.6 293.8 404.8 774.7 473.3 4762.2 359.7 5910.6 286.8 402.0 2962.3 7295.2 1579.4 2868.4 9260.9 3654.6 >10000 6529.5 3941.1 2860.6 6376.6 2419.8 8704.7 250
8.2 6.3 0.6 6.6 8.6 4.6 0.8 0.5 4.4 49.3 5.4 426.1 1778.9 167.5 569.0 297.3 150.7 246.3 58.3 350.0 270.9 188.1 71.1 77.4 12
145.5 113.4 47.0 442.3 286.2 451.3 311.8 246.2 47.2 407.0 266.0 304.5 428.4 292.9 410.4 2396.8 443.8 4429.4 197.6 79.6 108.0 372.3 71.3 288.1 74
51.9 29.0 7.2 27.4 7.4 7.0 9.6 4.3 7.8 8.0 5.7 88.9 602.5 376.2 423.5 628.7 701.1 1000.0 62.0 54.6 26.8 65.3 67.1 86.3 2.5
(i) The cytosolic isoform hCA I was moderately inhibited by most of the sulfonamides of the first series with inhibition constants (KIs) ranging between 286.8 and 774.7 nM. Among these, instead compounds 3b, 3g and 3i were measured to inhibit CA I only at the micromolar concentration, whereas derivative 3a arose as the best inhibitor with a KI of 50.1 nM. The second series derivatives, that bear an amidic function at the 5 position of the isoxazole moiety were shown to act as weak CA I inhibitors with KIs ranging between 1579.4 and 9260.9 nM, wheareas compound 5g did not significantly the enzyme up to 10 µM. (ii) The physiologically dominant isoform hCA II was very potently inhibited by all the first series derivatives (KI values ranging between 0.5 and 49.3 nM, Table 3). It is worth stressing the subnanomolar inhibitory activity against CAII of compounds 3c, 3d, 3g and 3h which incorporate respectively a 4-Cl, 4-F, 4NO2 or 2-NO2 moiety on the aryl group. These values make such derivatives by far better inhibitors compared to the clinically used standard AAZ (KI value of 12.1 nM). Conversely, derivatives 5a-5m showed a diminished hCA II inhibitory effectiveness with KIs spanning in the medium-high nanomolar range (58.3-569.0 nM) with the exception of compound 5b, which was active only at a micromolar concentration (KI 1778.9 nM). (iii) Most of the reported sulfonamides showed comparable effectiveness in inhibiting the membrane-associated isoform hCA IV with KI values spanning in the medium nanomolar range (108.0- 451.3 nM). Derivatives 3c and 3i from the first series and
5i and 5l from the second one exhibited the best CAIV inhibitory activity (respectively KIs: 47.0, 47.2, 79.6 and 71.3 nM), comparable to the standard AAZ (KI value of 74 nM), whereas 5e and 5g inhibited the isozyme in the micromolar range (KI values of 2396.8 and 4429.4 nM). (iv) Finally the hCA VII inhibition data shown in Table 3 clearly highlight a quite similar tendency to one observed for hCA II. Indeed, the benzoil-substituted derivatives 5a-5m showed a diminished inhibitory efficacy in comparison to 3a-3k which incorporate a free amine at the 5 position of the isoxazole core: the KIs measured for the compounds of the first series span from 4.3 to 51.9 nM, whereas within the second series the values showed an interesting correlation to the substitution pattern on the aryl moiety. In fact, derivatives 5h-5m, which possess a halogen atom on the aryl moiety are endowed with the lowest inhibition constants ranging between 26.8 to 86.3 nM, while the remaining ones showed KIs measured from 376.2 to 1000.0 nM. (v) In general, the functionalization of 3a-3k amine moiety with benzoyl groups worsened the compounds inhibitory profiles against all the screened CA isoforms, with the exception of CAIV which was rather equally inhibited by the compounds of the first and second series. We may hypothesize the incorporation of bulky groups orthogonally to the benzensulfonamide moiety lead an excessive steric hyndrace that negatively affect the inhibitory effectiveness of the reported isoxazole derivatives against hCAI, II and VII. 3. Conclusions We reported two new series of benzenesulfonamides incorporating isoxazole moieties, which have been prepared by means of conventional and MW methods. Derivatives 3a-k and 5a-m were investigated for the inhibition of four physiologically relevant CA isoforms: hCA I, II, IV, and VII. The derivatives of the first series were shown to possess excellent inhibitory activity against the cytosolic isoform hCA II, an antiglaucoma drug target, with KIs in the range of 0.5–49.3 nM and hCA VII, a recently validated anti-neuropathic pain target with KIs in the range of 4.3-51.9 nM. The inhibition profiles reported for all the screened compounds highlighted an almost general dimished inhibitory effectiveness for the 5amidoisoxazole 5a-m in comparison to their synthetic precursor 5-amidoisoxazole 3a-k. 4. Experimental protocols 4.1. Chemistry Anhydrous solvents and all reagents were purchased from Sigma–Aldrich and Carlo Erba. Nuclear magnetic resonance (1H NMR, 13C NMR) spectra were recorded using Bruker 300 MHz and Jeol 400 MHz spectrometer in DMSO-d6 and Acetone- d6. Chemical shifts are reported in parts per million (ppm) and the coupling constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s (singlet); d (doublet); m (multiplet); broad s (broad singlet); dd (doublets of doublet); app s (apparently singlet); app d (apparently doublet); app t (apparently triplet). Fourier Transform Infrared (FT-IR) spectra were recorded on SHIMADZU FT-IR-8400S. The mass analyses were performed on Waters 2695 Alliance Micromass ZQ (LCMS) and Waters SYNAPT (HRMS). Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel F-254 plates. Flash chromatography purifications were performed on Merck Silica gel 60 (230–400 mesh ASTM) as the stationary phase and ethyl acetate/n-hexane were used as eluents. Melting points (mp) were measured in open capillary tubes with a MELTEMP apparatus and are uncorrected.
General synthetic procedure of isoxazole sulfonamides 3a – k A suspension of 2-cyano-N-(4-sulfamoylphenyl)acetamide 1 (1 equiv.) and triethylamine (TEA) (2.5 equiv.) in ethanol (15 mL) was heated under reflux until the reaction mixture became clear. Thus, a solution of α-chlorooxime 2 (2 equiv.) in ethanol (5 mL) was added to the mixture and the resulting solution was heated under reflux until the consumption of the starting material (TLC monitoring). The reaction mixture was cooled to r.t. and the formed precipitated was filtered, washed with ethanol and recrystallized from ethylacetate (compounds 3a, 3f, 3g, 3h and 3i). In case the precipitate did not form the reaction mixture was concentrated in vacuo and the obtained residue purified by silica gel coloumn chromatography eluting with 50 % EtOAc in nhexane (compounds 3b, 3c, 3d, 3e, 3j and 3k). 5-Amino-3-(2,6-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (3a)
5-Amino-3-(4-chlorophenyl)-N-(4-sulfamoylphenyl)isoxazole4-carboxamide (3c)
Obtained as white solid (160 mg, 24%), m.p. 221 – 222˚C, Rf: 0.51. max (KBr, cm-1) 3394 (NH₂ ), 3448 (NH), 3228 (NH₂ ), 1654 (HN-C=O), 1620 (NH bent), 1589 (C=C), 1327 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₃ ClN₄ O₄ S [M+H]+ 393.0424, found 393.0428. 1H NMR (400 MHz, DMSO-d6) δ 9.31 (1 H, broad s, NH), 7.74 (2 H, broad s, NH2), 7.71 (2 H, d, J 8.9, aromatic CH), 7.62 (2 H, d, J 8.7, aromatic CH – SO2NH2), 7.58 (2 H, d, J 8.9, aromatic CH), 7.52 (2 H, d, J 8.7, aromatic CH – SO2NH2), 7.21 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 171.7, 161.6, 160.2, 142.4, 138.6, 135.0, 130.7, 129.0, 128.3, 126.9, 119.5, 88.2. m/z (TOF ES+) 393 (M+H+, 100%), 434 (M + MeCN, 35%). 5-Amino-3-(4-fluorophenyl)-N-(4-sulfamoylphenyl)isoxazole4-carboxamide (3d)
Obtained as light pink solid (1.06 g, 52%), m.p. 252 – 253˚C, Rf: 0.53. max (KBr, cm-1) 3321 (NH₂ ), 3448 (NH), 1666 (HN-CO), 1627 (NH bent), 1589 (C=C) 1327 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₂ Cl₂ N₄ O₄ S [M+H]+ 427.0035, found 427.0055. 1H NMR (400 MHz, DMSO-d6) δ 8.88 (1 H, broad s, NH), 7.97 (2 H, broad s, NH2), 7.67 (2 H, d, J 8.7, aromatic CH – SO2NH2), 7.60 – 7.57 (2 H, m), 7.54 (2 H, d, J 8.6, aromatic CH – SO2NH2), 7.53 – 7.49 (1 H, m, aromatic CH), 7.20 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSOd6) δ 169.4, 159.8, 158.2, 141.8, 138.2, 134.5, 132.0, 128.3, 127.8, 126.3, 119.4, 89.1. m/z (TOF ES+) 427 (M+H+, 100%), 468 (M + MeCN, 45%). 5-Amino-3-(2,4-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (3b)
Obtained as white solid (435 mg, 27%), m.p. 218 – 219˚C, Rf: 0.53. max (KBr, cm-1) 3340 (NH₂ ), 3452 (NH), 3232 (NH₂ ), 1670 (HN-C=O), 1627 (NH bent), 1593 (C=C), 1319 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₂ Cl₂ N₄ O₄ S [M+H]+ 427.0035, found 427.0029. 1H NMR (400 MHz,DMSO-d6) δ 8.98 (1 H, broad s, NH), 7.83 (2 H, broad s, NH2), 7.72 (1 H, d, J 1.9, aromatic CH), 7.68 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.60 – 7.58 (1 H, m, aromatic CH), 7.56 (1 H, d, J 1.9, aromatic CH), 7.50 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.19 (2 H, broad s, SO2NH2).13C NMR (100 MHz, DMSO-d6) δ 170.7, 161.2, 159.3, 142.4, 138.5, 135.7, 134.2, 133.7, 129.6, 127.9, 127.6, 126.9, 119.6, 89.7. m/z (TOF ES+) 335 (19%), 427 (M+H+, 100%), 468 (M + MeCN, 45%).
Obtained as white solid (356 mg, 32%), m.p. 203 – 205˚C, Rf: 0.51. max (KBr, cm-1) 3483 (NH₂ ), 3340 (NH), 3236 (NH₂ ), 1674 (HN-C=O), 1627 (NH bent), 1593 (C=C), 1323 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₃ FN₄ O₄ S [M+H]+ 377.0720, found 377.0722. 1H NMR (400 MHz, DMSO-d6) δ 9.19 (1 H, broad s, NH), 7.73 (2 H, broad s, NH2), 7.70 (2 H, d, J 8.7, aromatic CH – SO2NH2), 7.68 – 7.63 (2 H, m, aromatic CH), 7.57 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.30 (2 H, app. t, J 8.8, aromatic CH), 7.21 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 171.6, 163.4 (d, 1JC-F 247.0, C–F), 161.6, 160.3, 142.3, 138.6, 131.2 (d, 3 JC-F 8.6, C–F), 126.9, 125.8 (d, 4JC-F 3.2, C–F), 119.4, 116.0 (d, 2 JC-F 21.8, C–F), 88.2. m/z (TOF ES+) 310 (17%), 377 (M+H+, 100%), 418 (M + MeCN, 28%). 5-Amino-3-(4-bromophenyl)-N-(4-sulfamoylphenyl)isoxazole4-carboxamide (3e)
Obtained as white solid (196 mg, 26%), m.p. 238 – 239˚C, Rf: 0.53. max (KBr, cm-1) 3441 (NH₂ ), 3306 (NH), 3151 (NH₂ ), 1658 (HN-C=O), 1620 (NH bent), 1589 (C=C), 1327 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated
C₁ ₆ H₁ ₃ BrN₄ O₄ S [M+H] + 436.9919, found 436.9926. 1H NMR (400 MHz, DMSO-d6) δ 9.33 (1 H, broad s, NH), 7.74 (2 H, broad s, NH2), 7.71 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.66 (2 H, d, J 8.5, aromatic CH), 7.59 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.55 (2 H, d, J 8.5, aromatic CH), 7.21 (2 H, broad s, NH2). 13C NMR (100 MHz, DMSO-d6) δ 171.6, 161.6, 160.3, 142.4, 138.6, 131.9, 130.9, 128.7, 126.9, 123.8, 119.5, 88.1. m/z (TOF ES+) 341 (20%), 436 (M+H+, 100%), 480 (M + MeCN, 50%). 5-Amino-3-phenyl-N-(4-sulfamoylphenyl)isoxazole-4carboxamide (3f)
Obtained as white solid (451 mg, 35%), m.p. 232 – 233˚C, Rf: 0.51. max (KBr, cm-1) 3387 (NH₂ ), 3479 (NH), 3228 (NH₂ ), 1678 (HN-C=O), 1631 (NH bent), 1593 (C=C), 1315 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₄ N₄ O₄ S [M+H]+ 359.0814, found 359.0818. 1H NMR (400 MHz, DMSOd6) δ 8.97 (1 H, broad s, NH), 7.73 (2 H, broad s, NH2), 7.69 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.61 (2 H, dd, J 7.5, 1.8, aromatic CH), 7.51 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.50 – 7.46 (3 H, m, aromatic CH), 7.20 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 171.2, 161.2, 160.6, 141.8, 138.1, 129.9, 128.8, 128.6, 128.5, 126.5, 118.8, 87.7. m/z (TOF ES+) 273 (17%), 281 (30%), 325 (80%), 359 (M+H+, 100%), 400 (M + MeCN, 10%). 5-Amino-3-(4-nitrophenyl)-N-(4-sulfamoylphenyl)isoxazole-4carboxamide (3g)
Obtained as brown solid (269 mg, 28%), m.p. 250-251˚C, Rf: 0.49. max (KBr, cm-1) 3302 (NH₂ ), 3425 (NH), 1654 (HNC=O), 1624 (NH bent), 1589 (C=C), 1519 (NO₂ asym.), 1350 (NO₂ sym.), 1327 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₃ N₅ O₆ S [M-H]+ 402.0508, found 402.0517. 1H NMR (400 MHz, DMSO-d6) δ 9.52 (1 H, broad s, NH), 8.29 (2 H, d, J 8.8, aromatic CH), 7.87 (2 H, d, J 8.8, aromatic CH), 7.83 (2 H, broad s, NH2), 7.70 (2 H, d, J 8.7, aromatic CH – SO2NH2), 7.60 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.21 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 171.2, 160.9, 159.5, 148.1, 141.9, 138.2, 135.5, 129.8, 126.4, 123.5, 119.2, 87.9. m/z (TOF ES+) 359 (23%), 402 (M-H+, 100%). 5-Amino-3-(2-nitrophenyl)-N-(4-sulfamoylphenyl)isoxazole-4carboxamide (3h)
Obtained as yellow solid (700 mg, 64%), m.p. 234˚C (decomp.), Rf: 0.2. max (KBr, cm-1) 3329 (NH₂ ), 3255 (NH₂ ), 1674 (HNC=O), 1593 (C=C), 1489 (NO₂ asym), 1369 (NO₂ sym.), 1323 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₁ ₆ H₁ ₃ N₅ O₆ S [M+H]+ 404.0665, found 404.0646. 1H NMR (400 MHz, DMSO-d6) δ 9.81 (1 H, broad s, NH), 8.06 (1 H, d, J 8.3, aromatic CH), 7.92 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.88 (1 H, d, J 1.4, aromatic CH), 7.73 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.47 – 7.41 (1 H m, aromatic CH), 7.20 (2 H, broad s, SO2NH2), 7.06 – 6.99 (1 H, m, aromatic CH), (NH2 peak obscured by broad water peak from DMSO). 13C NMR (100 MHz, DMSO-d6) δ 169.2, 162.3, 150.1, 142.4, 138.4, 132.8, 131.4, 126.8, 122.8, 120.5, 119.9, 115.9, 114.4, 106.3. m/z (TOF ES+) 275 (20%), 283 (43%), 303 (55%), 404 (M+H+, 100%), 445 (M + MeCN, 10%). 5-Amino-3-(2-chloro-4,5-dimethoxyphenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (3i)
Obtained as white solid (1.12 g, %88), m.p. 179 – 180˚C, Rf: 0.38. max (KBr, cm-1) 3468 (NH₂ ), 2947 (CH₃ ), 1662 (HNC=O), 1627 (NH bent), 1593 (C=C), 1330 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₁ ₈ H₁ ₇ ClN₄ O₆ S [M+H]+ 453.0636, found 453.0631. 1H NMR (400 MHz, DMSO-d6) δ 8.26 (1 H, broad s, NH), 7.83 (2 H, broad s, NH2), 7.68 (2 H, d, J 8.0, aromatic CH – SO2NH2), 7.41 (2 H, d, J 8.0, aromatic CH – SO2NH2), 7.20 (2 H, broad s, SO2NH2,), 7.16 (1 H, s, aromatic CH), 7.14 (1 H, s, aromatic CH), 3.83 (3 H, s, OCH3), 3.79 (3 H, s, OCH3). 13C NMR (100 MHz, DMSO-d6) δ 170.9, 160.9, 158.6, 150.7, 147.7, 141.6, 138.1, 126.6, 124.1, 118.9, 118.8, 114.4, 113.0, 88.6, 56.1, 55.9. m/z (TOF ES+) 453 (M+H+, 100%), 494 (M + MeCN, 20%). 5-Amino-3-(5-chlorofuran-2-yl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (3j)
Obtained as white solid (302 mg, 53%), m.p. 227˚C (decomp.), Rf: 0.53. max (KBr, cm-1) 3313 (NH₂ ), 3402 (NH), 3217 (NH₂ ), 1662 (HN-C=O), 1624 (NH bent), 1593 (C=C), 1334 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated
C₁ ₄ H₁ ₁ ClN₄ O₅ S [M+H]+ 383.0217, found 383.0225. 1H NMR (400 MHz, DMSO-d6) δ 9.78 (1 H, broad s, NH), 7.80 (2 H broad s, NH2), 7.75 (2 H, app. d, J 9.4, aromatic CH – SO2NH2), 7.72 (2 H, app. d, J 9.5, aromatic CH – SO2NH2), 7.23 (2 H, broad s, SO2NH2), 7.04 (1 H, d, J 3.5, aromatic CH), 6.67 (1 H, d, J 3.5, aromatic CH). 13C NMR (100 MHz, DMSO-d6) δ 171.2, 160.6, 150.8, 142.9, 142.0, 138.2, 136.6, 126.5, 119.0, 114.9, 108.9, 86.6. m/z (TOF ES+) 383 (M+H+, 100%), 424 (M + MeCN, 20%). 5-Amino-3-(5-chlorothiophen-2-yl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (3k)
NH), 9.17 (1 H, broad s, NH), 8.39 – 8.32 (4 H, m, aromatic CH), 7.73 (2 H, d, J 8.3, aromatic CH – SO2NH2), 7.66 (2 H, d, J 8.4, aromatic CH – SO2NH2), 7.54 (2 H, d, J 7.4, aromatic CH), 7.51 – 7.43 (1 H, m, aromatic CH), 7.14 (2 H, broad s, SO2NH2,).13C NMR (100 MHz, DMSO-d6) δ 167.1, 160.6, 158.4, 149.0, 142.6, 138.0, 134.9, 131.5, 130.7, 129.8, 128.8, 128.2, 127.4, 126.6, 123.8, 119.9, 118.4. m/z (TOF ES+) 389 (70%), 576 (M+H+, 100%). 3-(2,4-Dichlorophenyl)-5-(4-nitrobenzamido)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5b)
Obtained as orange solid (114 mg, 79%), m.p. 251 – 252˚C, Rf: Obtained as dark yellow solid (279 mg, 15%), m.p. 178-180˚C, Rf: 0.53. max (KBr, cm ) 3448 (NH₂ ), 3263 (NH), 1658 (HNC=O), 1620 (NH bent), 1589 (C=C), 1327 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C14H11ClN4O4S2 [M+H]+ 398.9989, found 389.9989. 1H NMR (400 MHz, DMSO-d6) δ 9.88 (1 H, broad s, NH), 7.76 (2 H, broad s, NH2), 7.71 (2 H, d, J 9.1, aromatic CH – SO2NH2), 7.67 (2 H, d, J 9.1, aromatic CH – SO2NH2), 7.43 (1 H, d, J 4.0, aromatic CH), 7.22 (2 H, broad s, SO2NH2), 7.14 (1 H, d, J 4.0, aromatic CH). 13C NMR (100 MHz, DMSO-d6) δ 171.3, 161.4, 155.1, 142.6, 138.8, 130.9, 129.8, 129.2, 128.1, 127.0, 119.8, 87.9. m/z (TOF ES+) 293 (20%), 301 (31%), 321 (40%), 398 (M+H+, 100%), 440 (M + MeCN, 48%). -1
General synthetic procedure of isoxazole sulfonamides 5a – k Cs₂ CO₃ (3 equiv.) and 5-aminoisoxazole 3 (1 equiv.) were added to a solution of the proper benzoyl chloride 4 (1 equiv.) in MeCN (5 mL) at ice-bath temperature and the obtained suspension was stirred at r.t. until the consumption of the starting material (TLC monitoring). Then, the reaction mixture was concentrated under vacuo and the obtained residue was treated with water and extracted with EtOAc (3x15 ml). The organic layers were dried over Na2SO4, filtered-off and concentrated under vacuo to give a residue that was purified by silica gel coloumn chromatography eluting with 75 % EtOAc in n-hexane. 3-(2,6-Dichlorophenyl)-5-(4-nitrobenzamido)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5a)
Obtained as yellow solid (265 mg, 92%), m.p. 208 – 209˚C, Rf:0.12. max (KBr, cm-1) 3321 (NH₂ ), 3232 (NH), 3167 (NH), 1685 (HN-C=O), 1593 (HN-C=O), 1527 (asym. NO₂ ), 1346 (sym. NO₂ ), 1315 (asym. SO₂ ), 1153 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ Cl₂ N₅ O₇ S [M+H]+ 576.0148, found 576.0142. 1H NMR (400 MHz, DMSO-d6) δ 11.58 (1 H, broad s,
0.26. max (KBr, cm-1) 3398 (NH₂ ), 3298 (NH), 3225 (NH), 1674 (HN-C=O), 1593 (HN-C=O), 1531 (asym. NO₂ ), 1346 (sym. NO₂ ), 1315 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ Cl₂ N₅ O₇ S [M+H]+ 576.0148, found 576.0146. 1H NMR (400 MHz, DMSO-d6) δ 11.64 (1 H broad s, NH), 9.06 (1 H, broad s, NH), 8.43 – 8.28 (4 H, m, aromatic CH), 7.73 (2 H d, J 8.8, aromatic CH – SO2NH2), 7.71 (1 H, d, J 1.9, aromatic CH), 7.66 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.50 (1 H, dd, J 8.2, 1.8, aromatic CH), 7.45 (1 H, d, J 8.1, aromatic CH), 7.17 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSOd6) δ 167.1, 160.7, 159.9, 157.2, 149.0, 145.9, 142.6, 139.5, 137.9, 134.8, 134.6, 132.7, 129.8, 129.0, 127.4, 127.3, 124.5, 123.8, 118.5. m/z (TOF ES+) 389 (75%), 576 (M+H+, 100%). 3-(4-Fluorophenyl)-5-(4-nitrobenzamido)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5c)
Obtained as orange solid (41 mg, 39%), m.p. 230 – 231˚C, Rf: 0.31. max (KBr, cm-1) 3267 (NH), 3244 (NH), 1674 (HN-C=O), 1589 (HN-C=O), 1523 (asym. NO₂ ), 1346(sym. NO₂ ), 1315 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₆ FN₅ O₇ S [M-H]+ 524.0676, found 524.0674. 1H NMR (400 MHz, DMSO-d6) δ 12.35 (1 H, broad s, NH), 11.89 (1 H, broad s, NH), 8.35 (2 H, d, J 8.9, aromatic CH), 8.31 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.77 – 7.73 (4 H, m, aromatic CH), 7.70 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.27 (2 H, app. t, J 8.9, aromatic CH), 7.18 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 163.9, 162.5 (d, 1JC-F 194.0, C–F), 161.4, 161.1, 160.8, 156.4, 148.7, 148.2, 142.3, 137.6, 131.4 (d, 3 JC-F 8.5, C–F), 129.4, 126.9, 123.4, 119.6, 118.3, 114.7 (d, 2JC-F 22.3, C–F). m/z (TOF ES+) 524 (M-H+, 100%). 5-(4-Nitrobenzamido)-3-phenyl-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5d)
Obtained as dark yellow solid (56 mg, 37%), m.p. 207 – 208˚C, Rf: 0.12. max (KBr, cm-1) 3448 (NH), 3383 (NH), 1670 (HNC=O), 1612 (HN-C=O), 1527 (asym. NO₂ ), 1346 (sym. NO₂ ), 1315 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₇ N₅ O₇ S [M+H]+ 508.0927, found 508.0926. 1H NMR (400 MHz, DMSO-d6) δ 12.32 (1 H, broad s, NH), 11.73 (1 H, broad s, NH), 8.35 (2 H, d, J 8.8, aromatic CH), 8.29 (2 H, d, J 8.4, aromatic CH), 7.74 (2 H, d, J 8.9, aromatic CH), 7.71 – 7.66 (4 H, m, aromatic CH), 7.46 – 7.43 (3 H, m, aromatic CH), 7.17 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, DMSO-d6) δ 165.2, 161.8, 160.4, 148.8, 148.7, 142.2, 139.0, 137.6, 129.4, 129.1, 128.9, 128.7, 127.8, 126.8, 124.0, 123.4, 118.3. m/z (TOF ES+) 355 (70%), 508 (M+H+, 100%). 3-(2-Chloro-4,5-dimethoxyphenyl)-5-(4-nitrobenzamido)-N(4-sulfamoylphenyl) isoxazole-4-carboxamide (5e)
Obtained as yellow solid (26 mg, 18%), m.p. 250 – 251˚C, Rf: 0.19. max (KBr, cm-1) 3460 (NH), 3275 (NH), 2958 (CH3), 2854 (CH₃ ), 1674 (HN-C=O), 1616 (HN-C=O), 1523 (asym. NO₂ ), 1350 (sym. NO₂ ), 1330 (asym. SO₂ ), 1153 (sym. SO₂ ). HRMS: calculated C₂ ₅ H₂ ₀ ClN₅ O₉ S [M]+ 602.0749, found 602.0765. 1H NMR (400 MHz, DMSO-d6) δ 12.42 (1 H, broad s, NH), 11.71 (1 H, broad s, NH), 8.37 – 8.31 (4 H, m, aromatic CH), 7.72 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.67 (2 H, d, J 8.8, aromatic CH – SO2NH2), 7.17 (2 H, broad s, SO2NH2), 7.09 (1 H, s, aromatic CH), 6.97 (1 H, s, aromatic CH), 3.83 (3 H, s, OCH3), 3.76 (3 H, s, OCH3). 13C NMR (100 MHz, DMSO-d6) δ 172.9, 160.7, 152.5, 150.0, 149.0, 147.5, 146.2, 142.8, 139.5, 131.7, 129.8, 127.4, 124.9, 124.5, 123.8, 118.5, 118.4, 114.4, 112.9, 56.4, 56.3. m/z (TOF ES+) 271 (30%), 402 (48%), 602 (M+, 100%). 5-Benzamido-3-(2,6-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide(5f)
Obtained as white solid (329 mg, 62%), m.p. 214 – 215˚C, Rf: 0.48. max (KBr, cm-1) 3387 (NH₂ ), 3248 (NH), 3186 (NH), 1689 (HN-C=O), 1670 (HN-C=O), 1527 (C=C), 1315 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated
C₂ ₃ H₁ ₆ Cl₂ N₄ O₅ S [M+H]+ 531.0297, found 531.0296. 1H NMR (300 MHz, DMSO-d6) δ 11.91 (1 H, broad s, NH), 11.00 (1 H, broad s, NH), 8.08 (2 H, d, J 7.4, aromatic CH), 7.71 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.66 (2 H, d, J 8.9, aromatic CH – SO2NH2), 7.61 – 7.56 (3 H, m, aromatic CH), 7.55 – 7.49 (3 H, m, aromatic CH), 7.10 (2 H, broad s, SO2NH2). 13C NMR (100 MHz, Acetone-d6) δ 169.6, 159.7, 158.8, 156.9, 142.4, 138.4, 135.3, 132.1, 132.0, 131.8, 131.7, 131.6, 128.6, 128.4, 128.2, 127.2, 118.8. m/z (TOF ES+) 531 (M+H+, 100%). 5-Benzamido-3-(2,4-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5g)
Obtained as white solid (79 mg, 62%), m.p. 211 – 212˚C, Rf: 0.51. max (KBr, cm-1) 3387 (NH₂ ), 3182 (NH), 3105 (NH), 1689 (HN-C=O), 1666 (HN-C=O), 1554 (C=C), 1303 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₆ Cl₂ N₄ O₅ S [M+H]+ 531.0297, found 531.0294. 1H NMR (400 MHz, Acetone-d6) δ 11.21 (1 H, broad s, NH), 11.06 (1 H, broad s, NH), 8.19 (2 H, d, J 8.0, aromatic CH), 7.75 (2 H, app. d, J 9.3, aromatic CH – SO2NH2), 7.72 (2 H, app. d, J 9.3, aromatic CH – SO2NH2), 7.59 (1 H, app. s, aromatic CH), 7.56 – 7.44 (5 H m, aromatic CH), 6.44 (2 H, broad s, SO2NH2,). 13C NMR (100 MHz Acetone-d6) δ 172.6, 160.4, 159.9, 152.3, 142.8, 138.0, 137.4, 135.3, 134.9, 132.7, 131.7, 131.5, 129.1, 128.5, 128.3, 127.2, 127.1, 124.6, 118.6. m/z (TOF ES+) 531 (M+H+, 100%). 5-(4-Chlorobenzamido)-3-(2,6-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5h)
Obtained as light yellow solid (142 mg, %84), m.p. 193 –194˚C, Rf: 0.37. max (KBr, cm-1) 3404 (NH₂ ), 3230 (NH), 3076 (NH), 1676 (HN-C=O), 1664 (HN-C=O), 1560 (C=C), 1319 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ Cl₃ N₄ O₅ S [M+H]+ 564.9907, found 564.9908. 1H NMR (300 MHz, DMSO-d6) δ 12.16 (1 H, broad s, NH), 11.76 (1 H, broad s, NH), 8.21 – 8.06 (2 H, m, aromatic CH), 7.74 (2 H, d, J 8.6, aromatic CH), 7.66 (2 H, d, J 8.8, aromatic CH), 7.62 – 7.48 (5 H, m, aromatic CH), 7.21 (2 H, broad s, SO2NH2). 13C NMR (100 MHz Acetone-d6) δ 168.5, 160.4, 158.7, 144.6, 143.2, 137.5, 136.1, 135.2, 130.8, 130.4, 129.0, 128.1, 127.8, 127.3, 124.6, 118.2, 118.0. m/z (TOF ES+) 151 (18%), 564 (M+H+, 100%). 5-(4-Chlorobenzamido)-3-(2,4-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5i)
Obtained as white solid (103 mg, 61%), m.p. 267 – 268˚C, Rf: 0.40. max (KBr, cm-1) 3398 (NH₂ ), 3259 (NH), 3095 (NH), 1676 (HN-C=O), 1653 (HN-C=O), 1560 (C=C), 1319 (asym. SO₂ ), 1159 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ Cl₃ N₄ O₅ S [M+H]+ 564.9907, found 564.9935. 1H NMR (300 MHz, DMSO-d6) δ 12.09 (1 H, broad s, NH), 11.75 (1 H, broad s, NH), 8.14 – 7.99 (2 H, m, aromatic CH), 7.75 (2 H, d, J 8.6, aromatic CH), 7.71 – 7.66 (3 H, m, aromatic CH), 7.61 (2 H, d, J 8.6, aromatic CH), 7.55 – 7.40 (2 H, m, aromatic CH), 7.21 (2 H, broad s, SO2NH2,). 13C NMR (100 MHz, Acetone-d6) δ 171.7, 168.2, 160.5, 160.0, 143.1, 137.6, 136.4, 135.0, 134.9, 132.4, 130.4, 130.0, 128.9, 128.2, 127.3, 126.8, 124.6, 118.3, 118.2. m/z (TOF ES+) 151 (13%), 564 (M+H+, 100%). 5-(4-Bromobenzamido)-3-(2,6-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5j)
Obtained as light yellow solid (144 mg, 79%), m.p. 266 –267˚C, Rf: 0.37. max (KBr, cm-1) 3398 (NH₂ ), 3240 (NH), 3076 (NH), 1680 (HN-C=O), 1653 (HN-C=O), 1560 (C=C), 1317 (asym. SO₂ ), 1157 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ 79BrCl₂ N₄ O₅ S [M+H]+ 608.9402, found 608.9414. 1 H NMR (300 MHz, DMSO-d6) δ 12.16 (1 H, broad s, NH), 11.75 (1 H, broad s, NH), 8.16 – 7.99 (2 H, m, aromatic CH), 7.76 – 7.71 (4 H, m, aromatic CH), 7.66 (2 H d, J 8.8, aromatic CH), 7.58 (2 H, d, J 8.8, aromatic CH), 7.54 – 7.47 (1 H, m, aromatic CH), 7.21 (2 H, broad s, SO2NH2,). 13C NMR (100 MHz, Acetone-d6) δ 170.7, 168.1, 160.2, 158.8, 154.9, 150.3, 142.9, 141.1, 137.7, 135.2, 131.2, 131.0, 130.7, 127.9, 127.3, 125.1, 118.3. m/z (TOF ES+) 307 (16%), 324 (28%), 329 (46%), 608 (70%), 610 (M+H+, 100%).
5-(4-Bromobenzamido)-3-(2,4-dichlorophenyl)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5k)
Obtained as white solid (150 mg, 62%), m.p. 232 – 233˚C, Rf: 0.74. max (KBr, cm-1) 3398 (NH₂ ), 3257 (NH), 3088 (NH), 1683 (HN-C=O), 1653 (HN-C=O), 1560 (C=C), 1319 (asym.
SO₂ ), 1159 (sym. SO₂ ). HRMS: calculated C₂ ₃ H₁ ₅ 79BrCl₂ N₄ O₅ S [M+H]+ 608.9402, found 608.9404. 1 H NMR (400 MHz, Acetone-d6) δ 11.30 (1 H, broad s, NH), 10.62 (1 H, broad s, NH), 8.12 – 8.03 (2 H, m, aromatic CH), 7.76 – 7.73 (1 H, m, aromatic CH), 7.73 – 7.63 (4 H, m, aromatic CH), 7.64 – 7.57 (1 H, m, aromatic CH), 7.55 – 7.46 (3 H, m, aromatic CH), 6.43 (2 H, broad s, SO2NH2,). 13C NMR (100 MHz, Acetone-d6) δ 175.2, 168.0, 160.1, 159.9, 148.6, 142.5, 138.3, 135.7, 135.6, 134.7, 132.9, 132.8, 131.6, 130.4, 129.2, 127.3, 127.2, 126.0, 118.9. m/z (TOF ES+) 307 (13%), 329 (23%), 608 (62%), 610 (M+H+, 100%). 3-(2,6-Dichlorophenyl)-5-(4-methoxybenzamido)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5l)
Obtained as white solid (92 mg, 41%), m.p. 238 – 240˚C, Rf: 0.48. max (KBr, cm-1) 3400 (NH₂ ), 3240 (NH), 3088 (NH), 2929 (-CH), 2841 (-CH), 1683 (HN-C=O), 1653 (HN-C=O), 1560 (C=C), 1317 (asym. SO₂ ), 1159 (sym. SO₂ ). HRMS: calculated C₂ ₄ H₁ ₈ Cl₂ N₄ O₆ S [M+H]+ 561.0402, found 561.0419. 1H NMR (400 MHz, Acetone-d6) δ 11.20 (1 H, broad s, NH), 11.02 (1 H, broad s, NH), 8.18 (2 H, d, J 7.2, aromatic CH), 7.78 – 7.70 (4 H, m, aromatic CH), 7.55 – 7.46 (3 H, m, aromatic CH), 7.04 (2 H, d, J 7.2, aromatic CH), 6.44 (2 H, broad s, SO2NH2), 3.85 (3 H, s, OCH3). 13C NMR (100 MHz, Acetoned6) δ 162.7, 160.0, 158.7, 155.3, 150.5, 145.3, 143.9, 142.8, 137.9, 135.3, 132.1, 131.3, 130.5, 128.1, 127.2, 118.5, 113.5, 55.0. m/z (TOF ES+) 151 (21%), 561 (M+H+, 100%). 3-(2,4-Dichlorophenyl)-5-(4-methoxybenzamido)-N-(4sulfamoylphenyl)isoxazole-4-carboxamide (5m)
Obtained as white solid (90 mg, 40%), m.p. 153 – 154˚C, Rf: 0.53. max (KBr, cm-1) 3398 (NH₂ ), 3257 (NH), 3103 (NH), 2933 (-CH), 2841 (-CH), 1680 (HN-C=O), 1653 (HN-C=O), 1560 (C=C), 1319 (asym. SO₂ ), 1161 (sym. SO₂ ). HRMS: calculated C₂ ₄ H₁ ₈ Cl₂ N₄ O₆ S [M+H]+ 561.0402, found 561.0395. 1H NMR (400 MHz, Acetone-d6) δ 11.12 (1 H, broad s, NH), 10.44 (1 H, broad s, NH), 8.12 (2 H, d, J 8.9, aromatic CH), 7.76 (2 H, d, J 8.6, aromatic CH), 7.69 (2 H, d, J 8.9, aromatic CH), 7.61 (1 H, d, J 2.0, aromatic CH), 7.57 (1 H, d, J 8.2, aromatic CH), 7.49 (1 H, dd, J 8.3, 2.0, aromatic CH), 7.04 (2 H, d, J 8.9, aromatic CH), 6.46 (2 H, broad s, SO2NH2,), 3.85 (3 H, s, OCH3). 13C NMR (100 MHz, Acetone-d6) δ 166.2, 162.9, 160.0, 159.7, 152.7, 148.3, 142.2, 138.3, 138.2, 135.7, 134.5, 132.8, 130.3, 129.2, 127.9, 127.2, 127.0, 118.9, 113.6, 54.9. m/z (TOF ES+) 173 (36%), 307 (40%), 324 (68%), 461 (20%), 561 (M+H+, 100%).
4.2. CA inhibition An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration activity.30 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CAcatalyzed CO2 hydration reaction for a period of 10-100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier, 26 and represent the mean from at least three different determinations. All CA isofoms were recombinant ones obtained in-house as reported earlier.31-32
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Graphical Abstract
Synthesis of isoxazole-containing sulfonamides with potent carbonic anhydrase II and VII inhibitory properties Cevher Altug*, Hanife Güneş, Alessio Nocentini, Simona Maria Monti, Martina Buonanno, Claudiu T. Supuran*