(Chlorosulfonyl)benzenesulfonyl Fluorides—Versatile Building Blocks

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Research Article Cite This: ACS Comb. Sci. 2018, 20, 672−680

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(Chlorosulfonyl)benzenesulfonyl FluoridesVersatile Building Blocks for Combinatorial Chemistry: Design, Synthesis and Evaluation of a Covalent Inhibitor Library

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Kateryna A. Tolmachova,†,‡ Yurii S. Moroz,†,⊥ Angelika Konovets,†,§ Maxim O. Platonov,§ Oleksandr V. Vasylchenko,§ Petro Borysko,∥ Sergey Zozulya,∥ Anastasia Gryniukova,∥ Andrey V. Bogolubsky,§ Sergey Pipko,§ Pavel K. Mykhailiuk,† Volodymyr S. Brovarets,‡ and Oleksandr O. Grygorenko*,†,§ †

National Taras Shevchenko University of Kyiv, Volodymyrska Street 60, Kyiv 01601, Ukraine Institute of Bioorganic Chemistry & Petrochemistry, NAS of Ukraine, Murmanska Street 1, Kyiv 02660, Ukraine § Enamine Ltd., Chervonotkatska Street 78, Kyiv 02094, Ukraine ∥ Bienta/Enamine Ltd., Chervonotkatska Street 78, Kyiv 02094, Ukraine ⊥ Chemspace, Ilukstes iela 38-5, Riga, LV-1082, Latvia ‡

S Supporting Information *

ABSTRACT: Multigram synthesis of (chlorosulfonyl)benzenesulfonyl fluorides is described. Selective modification of these building blocks at the sulfonyl chloride function under parallel synthesis conditions is achieved. It is shown that the reaction scope includes the use of (hetero)aromatic and electron-poor aliphatic amines (e.g., amino nitriles). Utility of the method is demonstrated by preparation of the sulfonyl fluoride library for potential use as covalent fragments, which is demonstrated by a combination of in silico and in vitro screening against trypsin as a model enzyme. As a result, several inhibitors were identified with activity on par with that of the known inhibitor. KEYWORDS: sulfonyl halides, parallel synthesis, chemoselectivity, covalent fragments, sulfonamides, serine protease inhibitors



INTRODUCTION

Sulfonyl fluorides have gained much interest in recent years primarily due to their reasonable reactivity toward nucleophilic attack. In organic synthesis, the sulfur(VI)−fluoride exchange (SufFEx) reaction was named “another good reaction for click chemistry” due to its high efficiency and tolerance to a range of functional groups.1 In medicinal chemistry, sulfonyl fluorides have been considered “privileged warheads” for design of covalent modifiers, which is again due to their controllable reactivity in the biological systems.2 The best known representatives of this class are benzyl sulfonyl fluoride (PMSF) and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), widely used in biochemical studies to inhibit serine proteases in an irreversible manner (Figure 1).3 These compounds are also examples of covalent fragments, which have attracted much attention in recent years.4 Either the SufFEx methodology or a design of covalent inhibitors can benefit from functionalized sulfonyl fluorides, especially if the use of a parallel synthesis methodology is intended. Recent examples of building blocks illustrating this idea include sulfonyl fluorides bearing the moieties of heteroaliphatic amines,5 chalcones,6 aldehydes,7 carboxylic acids,8 organoboronates,9 as well as benzyl10,11 aryl9 or © 2018 American Chemical Society

Figure 1. Known sulfonyl fluoridescovalent serine protease inhibitors.

vinyl12 bromides. Meanwhile, it is widely accepted that sulfonyl fluorides and sulfonyl chlorides differ significantly in their reactivity.13 Therefore, chlorosulfonyl-substituted sulfonyl fluorides might be envisaged as useful building blocks for the parallel synthesis of libraries of sulfonyl fluorides bearing a sulfonamide moiety. Although examples of such building blocks have been known in the literature for more than nine decades (compounds 1−7, Figure 2),14−17 only a few isolated examples that demonstrate their selective modification at the sulfonyl chloride moiety have been reported to date.15−20 In this work, we describe synthesis of all three isomeric (chlorosulfonyl)benzenesulfonyl fluorides Received: August 22, 2018 Revised: October 22, 2018 Published: October 24, 2018 672

DOI: 10.1021/acscombsci.8b00130 ACS Comb. Sci. 2018, 20, 672−680

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alkyl, Figure 3) in the presence of pyridine as a base and CH2Cl2 as the solvent occurred slowly but more or less cleanly at room temperature; typically, the conversion was completed after 48 h. The products 8{1−3,1−102} were obtained with a 61% synthesis success rate and 45% average yield (Table 1). Increasing the temperature to 40 °C led to the loss of chemoselectivity, so that mixtures containing mono- and bissulfonamides, as well as unreacted starting material, were formed. Low chemoselectivity was also observed with primary amines 9{103−106}, including sterically hindered tert-butylamine 9{103}, even if the reaction was performed at room temperature (although we managed to isolate the library members 8{2,104} and 8{2,106}, both in 23% yield, after chromatographic purification). Nevertheless, the method could be extended to less nucleophilic aliphatic amines bearing electron-withdrawing groups (e.g., α-alkyl α-amino nitriles 9{107−109} or fluorinated amines 9{110,111}, as well as acyl hydrazides 9{112−114} and phenols 9{115}), although in these cases, isolated yields were slightly lower (average value is 38%). The procedure did not work with the parent N(cyanomethyl)amines 9{116−118} and α,α-dialkyl α-amino nitriles 9{119,120}: mixtures of unidentified products were obtained in these experiments. Therefore, the developed procedure for the synthesis of SO2F-containing sulfonamides 8{1−3,9} can be effective for (hetero)aromatic as well as aliphatic amines with low nucleophilicity, hydrazides, and phenols. The method tolerates azole, azinone, and carboxamide N−H bonds, hydroxyl, nitrile, and aromatic bromide functions, as well as 2- or 4chloropyridine moieties. A total of 92 of 150 library members 8{1−3,9} were prepared in parallel synthesis during the validation step (61% synthesis success rate, 43% average yield), although in most cases, chromatographic purification of the products was necessary. Analysis of unsuccessful results obtained with aromatic amines did not reveal any additional regularities; in our opinion, the reasons behind these failures might be related to isolation and/or purification issues and not chemical problems. The following order of efficiency in the synthesis of the library 8 was observed for the sulfonyl halides 1−3 according to the synthesis success rate: 2 (87%) > 3 (61%) > 1 (14%). Poor results obtained with o-isomer 1 might be attributed to the limited stability of the final products 8{1,9} toward hydrolysis (possibly due to the participation of the osulfonamide moiety) since in most cases, the corresponding sulfonic acid was observed as the main component in the crude products according to LCMS data. These conclusions are supported by the results obtained from stability of the compounds 8 in DMSO solutions: whereas in the case of the products obtained from the sulfonyl halides 2 and 3, the compounds were stable at room temperature for several days, the compounds 8{1,9} showed considerable degradation at these conditions after overnight storage. Therefore, it is preferable to use their freshly prepared solutions. Further extension of the library 8 was aimed at demonstrating its potential to inhibit serine proteases using the classical target of this classtrypsinas a model enzyme. The library enumeration followed the principles of the REAL concept published elsewhere.22 In particular, virtual coupling of sulfonyl halides 1−3 and 1224 (hetero)aromatic or fluorinated aliphatic amines or α-aminonitriles 9 with inhouse validated reactivity in the sulfonamide bond formation

Figure 2. Known examples of chlorosulfonyl-substituted sulfonyl fluorides.

1−3 as well as demonstrate their utility for the preparation of a library of sulfonamide-containing sulfonyl fluorides 8 (Scheme 1). In addition to that, their potential for the covalent inhibition of serine proteases was evaluated using the classical target of this classtrypsinas a model enzyme. Scheme 1



RESULTS AND DISCUSSION It should be noted that although all three sulfonyl fluorides 1− 3 have been mentioned in the literature, their syntheses were not described in the corresponding papers; preparation of compound 2 was mentioned only in a patent.21 We have prepared all three building blocks 1−3 using a modification of the above-mentioned method (Scheme 2).21 The synthesis Scheme 2. Synthesis of the Building Blocks 1−3

commenced from the commercially available nitrobenzenesulfonyl chlorides 10−12, which reacted with KF−Et3N to give the corresponding sulfonyl fluorides 13−15 (82−93% yield). Catalytic hydrogenation of 13−15 led to the formation of anilines 16−18 (40−95% yield), which were subjected to the diazotation, followed by the Sandmeyer reaction with in situ generated SO2 to give the target compounds 1−3 in 60−91% yield. The next part of our work included design and synthesis of the library 8{1−3,9} based on the building blocks 1−3. Initially, validation of the common sulfonamide synthesis protocol was performed for the sulfonyl halides 1−3. A sulfonamide set of 150 compounds was enumerated from 1−3 and nucleophiles 9{1−120}, followed by random selection (with at least one product per reagent included). It was found that reaction of 1−3 with most primary and secondary (hetero)aromatic amines 9{1−102} (R1 = (het)aryl; R2 = H, 673

DOI: 10.1021/acscombsci.8b00130 ACS Comb. Sci. 2018, 20, 672−680

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Figure 3. continued

674

DOI: 10.1021/acscombsci.8b00130 ACS Comb. Sci. 2018, 20, 672−680

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Figure 3. Structures of nucleophilic reagents 9{1−138}.

afforded 3 × 1224 = 3672 synthesizable library members. These were subjected to covalent docking into the trypsin S1 pocket that is quite deep and planar so that comfortable accommodation of aromatic cores is possible.23 The catalytically active Ser195 was responsible for the covalent bond formation; in addition to that, several hydrogen bond donors/ acceptors were also present in the binding site, including side chains of Asp189, Ser190, and Gln192, as well as backbone NH groups of Ser195, Gly193, and Gly216. As a result of the docking procedure, 100 compounds were selected as virtual hits according to the following criteria: the built-in QXP scoring function,24 the number of hydrogen bonds (was exposed to a minimum of four hydrogen bonds), the protein−ligand contact surface area, and the distances from

ligand to the key points of the corresponding pharmacophore model (Figure 4). The model was based on the limited ligand mobility due to the covalent bond with Ser195 and the structural features of the entire series of compounds. Formation of hydrogen bonds between oxygen atoms of the two sulfo groups and Gly193, as well as Gln192 and/or Gly216, was desirable. An additional hydrogen bond with Gly219 might be also present for the compounds bearing a sulfonamide NH group. This interaction fixed the (het)aryl fragment L2 in the S1 subpocket; filling the subpocket and approaching a part of the ligand with Asp 189 was considered as one of the selection criteria. A separate filter removed ligands strongly exposed to the solution. Further processing of 675

DOI: 10.1021/acscombsci.8b00130 ACS Comb. Sci. 2018, 20, 672−680

Research Article

ACS Combinatorial Science Table 1. Parallel Synthesis of Sulfonyl Fluorides 8 #

product

yield (%)

#

product

yield (%)

#

product

yield (%)

#

product

yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

8{1,8} 8{1,19} 8{1,24} 8{1,40} 8{1,45} 8{1,46} 8{1,50} 8{1,51} 8{1,51} 8{1,52} 8{1,53} 8{1,56} 8{1,57} 8{1,73} 8{1,76} 8{1,77} 8{1,85} 8{1,98} 8{1,99} 8{1,102} 8{1,116} 8{1,118} 8{1,122} 8{1,123} 8{1,133} 8{1,137} 8{1,138} 8{1,140} 8{2,3} 8{2,4} 8{2,8} 8{2,15} 8{2,18} 8{2,19} 8{2,22} 8{2,23} 8{2,28} 8{2,29} 8{2,30} 8{2,32} 8{2,34} 8{2,36} 8{2,38} 8{2,42} 8{2,43}

0 0 2 0 0 24 0 11 11 0 0 0 0 0 0 0 0 0 0 0 0 0 13 60 74 10 37 38 59 58 0 35 48 56 12 8 15 27 57 30 35 59 97 51 99

46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

8{2,46} 8{2,48} 8{2,62} 8{2,66} 8{2,70} 8{2,71} 8{2,72} 8{2,80} 8{2,82} 8{2,85} 8{2,87} 8{2,91} 8{2,93} 8{2,94} 8{2,97} 8{2,106} 8{2,108} 8{2,109} 8{2,110} 8{2,112} 8{2,113} 8{2,114} 8{2,115} 8{2,119} 8{2,134} 8{3,1} 8{3,2} 8{3,3} 8{3,4} 8{3,5} 8{3,6} 8{3,7} 8{3,8} 8{3,9} 8{3,10} 8{3,12} 8{3,13} 8{3,14} 8{3,15} 8{3,16} 8{3,17} 8{3,18} 8{3,19} 8{3,20} 8{3,21}

99 32 43 61 25 99 0 0 6 99 0 7 23 0 99 23 43 16 43 99 54 44 37 0 5 0 0 66 68 26 0 14 46 0 12 0 99 99 23 0 23 30 22 0 30

91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135

8{3,23} 8{3,25} 8{3,26} 8{3,27} 8{3,28} 8{3,29} 8{3,31} 8{3,32} 8{3,33} 8{3,34} 8{3,35} 8{3,36} 8{3,37} 8{3,39} 8{3,41} 8{3,44} 8{3,45} 8{3,46} 8{3,47} 8{3,48} 8{3,49} 8{3,54} 8{3,55} 8{3,56} 8{3,58} 8{3,59} 8{3,60} 8{3,61} 8{3,63} 8{3,64} 8{3,65} 8{3,67} 8{3,68} 8{3,69} 8{3,74} 8{3,75} 8{3,76} 8{3,76} 8{3,77} 8{3,78} 8{3,79} 8{3,81} 8{3,82} 8{3,83} 8{3,84}

8 0 0 0 28 33 43 30 99 17 0 56 23 19 0 0 99 99 99 26 0 0 0 35 0 0 43 0 0 0 0 0 99 28 46 52 55 55 99 3 38 34 0 0 27

136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179

8{3,85} 8{3,85} 8{3,86} 8{3,86} 8{3,87} 8{3,88} 8{3,89} 8{3,90} 8{3,91} 8{3,92} 8{3,95} 8{3,96} 8{3,97} 8{3,98} 8{3,98} 8{3,99} 8{3,100} 8{3,101} 8{3,104} 8{3,105} 8{3,107} 8{3,108} 8{3,108} 8{3,109} 8{3,110} 8{3,111} 8{3,112} 8{3,114} 8{3,117} 8{3,119} 8{3,120} 8{3,121} 8{3,124} 8{3,125} 8{3,126} 8{3,127} 8{3,128} 8{3,129} 8{3,130} 8{3,131} 8{3,132} 8{3,135} 8{3,136} 8{3,137}

99 99 0 9 75 10 0 10 9 0 0 26 99 99 99 21 33 0 24 0 32 36 38 30 20 38 53 31 0 0 0 75 99 99 8 42 72 99 61 23 7 10 74 46

the complexes was carried out by visual inspection according to the assumed pharmacophore interaction model. A total of 62 of 100 sulfonyl fluorides from the resulting set were synthesized using the developed procedure (Table 1). These products were subjected to in vitro screening against trypsin, including a protein thermal shift assay (TSA),25−27 followed by a BApNA (Nα-benzoyl-DL-arginine p-nitroanilide) enzymatic digestion assay28 (Table 2). It is interesting to note that most of the identified hits demonstrated a negative thermal shift. This indicates destabilization of the folded protein in the presence of the ligand29 and is sometimes perceived as an undesirable feature of the TSA hits.30,31 Nevertheless, three of these compounds had IC50 values lower than the standard (PMSF) in the enzymatic digestion assay,

compound 8{1,122} being 5-fold more active than PMSF (Figure 5); others showed activity comparable to that of PMSF. It should be noted that all the identified hits were rather polar (cLogP = 0.77−2.6032), which might be useful to satisfy the hydrogen-bonding potential of the compounds in the protein binding site. In particular, the ligand-lipophilicity efficiency (defined as LLE = pIC50 − cLogP33) for the most active compound 8{1,122} (3.28) was improved compared to that of PMSF (1.93). These data demonstrate that the library 8{1−3,9} can be a promising tool for the discovery of covalent modifiers. Analysis of the binding mode for the two most active compounds 8{1,122} and 8{3,56} showed that in the case of 8{3,56}, the ligand fit well with the pharmacophore model 676

DOI: 10.1021/acscombsci.8b00130 ACS Comb. Sci. 2018, 20, 672−680

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for 8{1,122}: while the key hydrogen bonds with Gln192 and Gly193 were retained, the sulfo group attached to Ser195 also formed a hydrogen bond with catalytic His57. In addition to that, the N-4 atom of the thiadiazole ring formed a hydrogen bond with Ser214 buried in the S1 subpocket. In both cases, the corresponding heteroaromatic moieties filled the S1 subpocket of trypsin, as suggested by the pharmacophore model.



CONCLUSIONS The difference in reactivity of sulfonyl halides can be used to selectively modify the sulfonyl chloride moiety in the presence of the sulfonyl fluoride one within the same molecule. We demonstrated this feature for three isomeric (chlorosulfonyl)benzenesulfonyl fluorides. Therefore, these bifunctional building blocks are convenient starting points to generate the libraries of sulfonyl fluorides. The scope of the developed parallel synthesis procedure includes various (hetero)aromatic as well as electron-poor aliphatic amines (i.e., α-amino nitriles or fluorinated amines). The library of the obtained sulfonyl fluorides is a promising tool for the design of covalent modifiers targeting serine/threonine and lysine residues in the protein molecules, which is validated by discovery of several trypsin inhibitors having 2- and 5-times higher activity compared with the known protease inhibitor PMSF.

Figure 4. Pharmacophore model of ligand binding in the S1 pocket of trypsin (red and blue spheres show hydrogen bond donors and acceptors, respectively; L1, L2 denote aromatic/hydrophobic moieties).

Table 2. Results of in Vitro Testing of the Sulfonyl Fluorides 8 against Trypsin



EXPERIMENTAL SECTION General. All chemicals and solvents were obtained from Enamine Ltd. and used without further purification. 1H and 13 C NMR spectra were acquired on Bruker Advance DRX 400 and Bruker Avance DRX 500 spectrometers using DMSO-d6 as a solvent and tetramethylsilane as an internal standard. Melting points were determined on a Buchi melting point apparatus. LC-MS data were recorded on an Agilent 1100 HPLC equipped with a diode-matrix and mass-selective detector Agilent LC/MSD SL. Column: Zorbax SB-C18, 4.6 mm × 15 mm. Eluent: A, acetonitrile−H2O with 0.1% of TFA (95:5); B, H2O with 0.1% of TFA. Flow rate: 1.8 mL/min. Elemental analyses were performed at the Laboratory of Organic Analysis, Department of Chemistry, Kyiv National Taras Shevchenko University. Preparative flash chromatography was performed on a Combiflash Companion chromarograph (12 g RediSep columns, gradient Hexanes−i-PrOH as eluent). Synthesis of Sulfonyl Fluorides 13−15. To a solution of sulfonyl chloride 10−12 (103 g, 0.467 mol) in CH2Cl2 (1000 mL), solutions of KF (81.3 g, 1.40 mol) in H2O (400 mL) and Et3N (78.1 mL, 0.560 mol) in CH2Cl2 (400 mL) were added. The mixture was stirred at room temperature for 4 h, then the organic phase was separated, washed with H2O (400 mL) and saturated aq. NaHCO3 (400 mL), and evaporated in vacuo. 2-Nitrobenzenesulfonyl Fluoride (13). Yield: 78.6 g, 82%. For spectral and physical data, see refs 8, 34. 3-Nitrobenzenesulfonyl Fluoride (14). Yield: 85.3 g, 89%. For spectral and physical data, see ref 35. 4-Nitrobenzenesulfonyl Fluoride (15). Yield: 89.1 g, 93%. For spectral and physical data, see refs 34, 35. Synthesis of Sulfonyl Fluorides 16−18. Nitrobenzene derivatives 16−18 (78.2 g, 0.381 mol) were dissolved in MeOH (750 mL), and 5% Pd−C (12.5 g) was added. The resulting mixture was hydrogenated in an autoclave at 50 °C for 72 h. The catalyst was filtered off, and the combined

shown in Figure 4. In particular, the sulfo groups form hydrogen bonds with Gln192, Gly216, and Gly193, and the hydrogen bond was also present between the sulfonamide NH and Gly219 (Figure 6). A different binding mode was observed 677

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Figure 5. Dose−response curves for the most active compounds 8{1,122} and 8{3,56} compared to those for PMSF (red curves are the same).

Figure 6. Binding poses of compounds 8{1,122} and 8{3,56} in the S1 pocket of trypsin according to the docking results.

filtrates were evaporated in vacuo. The residue was triturated with t-BuOMe (750 mL). The solution was decanted from the residue and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was dried in vacuo. 2-Aminobenzenesulfonyl Fluoride (16). Yield: 26.7 g, 40%. For spectral and physical data, see ref 36. 3-Aminobenzenesulfonyl Fluoride (17). Yield: 52.7 g, 79%. Colorless liquid. 1H NMR (400 MHz, CDCl3): δ 7.45−7.31 (m, 2H), 7.22 (s, 1H), 6.98 (d, J = 6.9 Hz, 1H), 4.03 (s, 2H). 13 C NMR (125 MHz, CDCl3): δ 147.6, 133.7 (d, J = 23.2 Hz), 130.5, 121.4, 117.6, 113.3. 19F NMR (376 MHz, CDCl3): δ 64.6. LC/MS (EI): m/z = 176 [M + H]+. Anal. Calcd for C6H6FNO2S: C, 41.14; H, 3.45; N, 8.00; S, 18.30. Found: C, 41.03; H, 3.49; N, 7.97; S, 17.95. 4-Aminobenzenesulfonyl Fluoride (18). Yield: 63.4 g, 95%. For spectral and physical data, see ref 37. Synthesis of Sulfonyl Fluorides 1−3. Thionyl chloride (41.0 mL, 0.564 mol) was added dropwise to ice (250 g). The mixture was left for 48 h and then cooled to −5 °C, and CuCl (0.574 g, 5.80 mmol) was added to give solution A. Sulfonyl fluoride 16−18 (25.6 g, 0.146 mol) was dissolved in 20% aq. HCl (250 mL). The solution was cooled to −10 °C, and a solution of NaNO2 (12.1 g, 0.175 mol) in H2O (29.0 mL) was added dropwise. The resulting solution was stirred at room temperature for 20 min, cooled to −5 °C, and added dropwise to solution A. The mixture was stirred at −5 °C for 3 h and then filtered. The filtrates were extracted with CH2Cl2 (3 × 250 mL); the combined extracts were dried over Na2SO4 and evaporated in vacuo. The residue was recrystallized from hexanes. 2-(Chlorosulfonyl)benzenesulfonyl Fluoride (1). Yield: 22.6 g, 60%. Orange crystals; mp = 88−90 °C. 1H NMR (400 MHz, CDCl3): δ 8.46 (d, J = 7.4 Hz, 1H), 8.41 (d, J = 7.7

Hz, 1H), 8.03 (quint, J = 2.7 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ 142.5, 136.3, 135.7, 133.3 (d, J = 1.9 Hz), 132.3, 131.3 (d, J = 28.6 Hz). 19F NMR (376 MHz, CDCl3): δ 66.0. GC/MS (EI): m/z = 258 [M]+. Anal. Calcd for C6H4ClFO4S2: C, 27.86; H, 1.56; S, 24.79; Cl, 13.71. Found: C, 28.24; H, 1.65; S, 24.67; Cl, 14.02. 3-(Chlorosulfonyl)benzenesulfonyl Fluoride (2). Yield: 32.1 g, 85%. Yellowish crystals; mp = 27−29 °C. 1H NMR (400 MHz, CDCl3): δ 8.64 (s, 1H), 8.43 (d, J = 8.6 Hz, 1H), 8.36 (d, J = 8.1 Hz, 1H), 7.96 (t, J = 8.1 Hz, 1H). 13C NMR (125 MHz, CDCl3): δ 145.8, 135.2 (d, J = 28.1 Hz), 134.3, 133.4, 131.7, 127.1. 19F NMR (470 MHz, DMSO-d6): δ 66.2. LC/MS (EI): m/z 258 [M]+. Anal. Calcd for C6H4ClFO4S2: C, 27.86; H, 1.56; S, 24.79; Cl, 13.71. Found: C, 27.96; H, 1.78; S, 24.82; Cl, 13.47. 4-(Chlorosulfonyl)benzenesulfonyl Fluoride (3). Yield: 34.4 g, 91%. Yellowish powder; mp = 109−111 °C. 1H NMR (400 MHz, CDCl3): δ 8.33 (d, J = 8.4 Hz, 2H), 8.29 (d, J = 8.4 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 149.6, 138.9 (d, J = 27.4 Hz), 130.0, 128.3. 19F NMR (376 MHz, CDCl3): δ 65.6. LC/MS (EI): m/z = 258 [M]+. Anal. Calcd for C6H4ClFO4S2: C, 27.86; H, 1.56; S, 24.79; Cl, 13.71. Found: C, 27.95; H, 1.75; S, 24.48; Cl, 13.69. Parallel Synthesis of Sulfonyl Fluorides 8. Amine 9 (1 mmol), pyridine (0.5 mL), CHCl3 (1 mL), and sulfonyl chloride 1−3 (258 mg, 1 mmol) were placed into a vial, and the mixture was shaken at room temperature for 48 h. Then it was evaporated in vacuo. CHCl3 (3 mL) was added; the solution was washed with H2O (1 mL) and evaporated in vacuo. The residue was purified by preparative flash chromatography. 3-(N-(Pyridin-4-yl)sulfamoyl)benzene-1-sulfonyl Fluoride (9{2,85}). Yield: 86 mg, quant. Yellowish solid; mp = 207− 678

DOI: 10.1021/acscombsci.8b00130 ACS Comb. Sci. 2018, 20, 672−680

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ACS Combinatorial Science

was performed by raising the temperature to 34 °C at 2.5 °C/ min without signal detection followed by a 34 to 75 °C temperature ramp at 0.1 °C/s with constant fluorescence intensity reading at 1 s intervals using an EX470/EM623 nm filter set. Screening of compounds was carried out in quadruplicate (n = 4). The raw data of dye fluorescence intensity change upon protein melt were exported using the ViiA7 RUO software 1.2 (Applied Biosystems/ThermoFischer Scientific). Further data visualization, curve fitting, and melting temperature calculations on the raw fluorescence data were performed using custom-made Microsoft Excel scripts. The peak of the first derivative for the fluorescence curve was used to define melting temperature (Tm). Averaged Tm values for the control wells (n = 64 per plate) were used as a reference point to determine melting temperature shifts of compounds (ΔTm). An enzymatic trypsin assay using chromogenic BApNA substrate (Sigma-Aldrich, Cat. B4875) was performed using the procedures reported previously28 and recommended by the manufacturer.

209 °C. 1H NMR (400 MHz, DMSO-d6): δ 12.98 (br s, 1H), 8.41−8.20 (m, 3H), 8.11−8.02 (m, 2H), 7.93 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 7.2 Hz, 2H). 13C NMR (125 MHz, DMSOd6): δ 162.5, 146.5, 139.8, 134.1, 132.6 (d, J = 24.2 Hz), 132.2, 131.4, 125.6, 115.5. 19F NMR (376 MHz, DMSO-d6): δ 66.1. LC/MS (CI): m/z = 317 [M + H]+. Anal. Calcd for C11H9FN2O4S2: C, 41.77; H, 2.87; N, 8.86; S, 20.27. Found: C, 41.69; H, 2.92; N, 9.14; S, 20.56. Virtual Screening. Molecular docking was performed using a flexible ligand and a fixed receptor model. We used an algorithm of MCDOCK (Monte Carlo search) implemented in QXP docking software, which had shown high reproducing ability of ligand conformation with minimum RMSD in comparison to the crystallographic data but was more timeconsuming than standard methods.38 The binding site model was created on the basis of PDB 2PLX.23 When converting the structure of the protein and creating a binding site model, the center was exposed on Ser 190. The sulfonyl fluorides database was prepared for the dock combinatorial library. The maximum number of MCDOCK routine steps was set to 500, and the 10 best structures (based on built-in QXP scoring function)24 were retained for each compound. The resulting protein−ligand complex structures had been filtered by intrinsic Flo+ filters and multiRMSD software package.39 In Vitro Screening. Reference compoundsPMSF (phenylmethyl sulfonyl fluoride) and benzamidinewere obtained from Enamine Ltd. (Kyiv, Ukraine). Stock solutions of the tested compounds were prepared at 20 mM in DMSO and were stored at −20 °C until use. All thermal shift assay (TSA) experiments with trypsin (Sigma, Cat. T8003) were performed using a ViiA7 real-time PCR System equipped with a 384-well heat block (Applied Biosystems, USA). A general TSA methodology was adopted from the literature24−26,31 and experimentally modified in order to optimize conditions for measuring trypsin melting temperature shifts upon interaction with small molecules. To define the optimal buffer composition for the TSA procedure, a matrix of common biological buffers combined on a 96-well microplate, including phosphate, acetate, TRIS, HEPES, and MES at different pH levels and inorganic salt contents, was tested in a thermal melt experiment on a 384-well microplate (each buffer composition in quadruplicate). In addition, each buffer composition was tested at two different buffering component concentrations, resulting in two 384-well plate buffer screening experiments and a total of 192 different buffer compositions tested. Selection of the optimal buffer composition was based on balancing two criteria including the maximized melting temperature (thermal stability) of trypsin and maximized melting temperature shift induced by the benzamidine at a concentration of 500 μM. As a result, a buffer consisting of 20 mM Tris-HCl at pH 7.0 and 1 mM CaCl2 was selected for the screening. Trypsin was premixed with SYPRO Orange dye (Thermo Fischer Scientific, Cat. S6650, 5000× stock) to prepare a master mix at a 345 μg/mL protein and 10× dye concentration. PMSF and benzamidine were used as reference compounds at final concentrations of 200 uM and 500 uM, respectively. Tested compounds were added to the protein-dye master mix at 200 uM (1% DMSO concentration) and incubated at room temperature for 15 min in MicroAmp optical 384-well reaction plates (ThermoFisher, Cat. 4309849) sealed with optical sealing film (ThermalSeal RT2, Excel Scientific, Cat. TS-RT2). The volumes of all reaction mixtures were 10 μL (3.45 μg of trypsin per well). Thermal scanning



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscombsci.8b00130. Table S1 and copies of NMR spectra (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Pavel K. Mykhailiuk: 0000-0003-1821-9011 Volodymyr S. Brovarets: 0000-0001-6668-3412 Oleksandr O. Grygorenko: 0000-0002-6036-5859 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The work was funded by Enamine Ltd. The authors thank Prof. Andrey A. Tolmachev for his encouragement and support and Mr. Bohdan Vashchenko and Ms. Yuliya Kuchkovska for their help with manuscript preparation.



REFERENCES

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Research Article

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