New amide derivatives of Probenecid as selective inhibitors of carbonic anhydrase IX and XII: Biological evaluation and molecular modelling studies Simone Carradori a,⇑ , Adriano Mollica a , Mariangela Ceruso b , Melissa D’Ascenzio c , Celeste De Monte c , Paola Chimenti c , Rocchina Sabia c , Atilla Akdemir d , Claudiu T. Supuran b,e,⇑ a Department of Pharmacy, ‘G. D’Annunzio’ University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy b Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy c Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy d Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Vatan Caddesi, 34093 Fatih, Istanbul, Turkey e Università degli Studi di Firenze, Neurofarba Dept., Section of Pharmaceutical and Nutraceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy article info Article history: Received 25 February 2015 Revised 5 May 2015 Accepted 6 May 2015 Available online 14 May 2015 Keywords: Probenecid Selective carbonic anhydrase IX inhibitors Selective carbonic anhydrase XII inhibitors Tertiary sulfonamides Molecular modelling abstract Novel amide derivatives of Probenecid were synthesized and discovered to act as potent and selective inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) transmembrane isoforms hCA IX and XII. The proposed chemical transformation of the carboxylic acid into an amide group led to a complete loss of hCA I and II inhibition (K i s >10,000 nM) and enhanced the inhibitory activity against hCA IX and XII, with respect to the parent compound (incorporating a COOH function). These promising biological results have been corroborated by molecular modelling studies within the active sites of the four studied human carbonic anhydrases, which enabled us to rationalize both the isoform selectivity and high activity against the tumor-associated isoforms hCA IX/XII. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Human carbonic anhydrases (hCAs, EC 4.2.1.1) are recognized to be highly conserved and widespread metalloenzymes that effi- ciently catalyze the reversible hydratation of CO 2 to bicarbonate and proton. 1 Sixteen isoforms (I–XV, with VA and VB being mito- chondrial isoforms) are involved in a wide range of physiological processes and their malfunctioning or altered expression could promote pathological situations. For this reason, selective isoform inhibition or activation is known to be an effective strategy in the treatment of important diseases. 2–4 Over the past few years, a specific interest has been oriented toward the two hCA trans- membrane isoforms (hCA IX and XII), 5–9 almost exclusively overex- pressed in hypoxic tumors, which represent a main player of growth and survival of malignancies since they regulate extracellu- lar and intracellular pH, control cell adhesion, and contribute to many adaptive changes in solid tumors. After their activation in a reduced oxygen environment, tumor cells reprogram their meta- bolism toward the glycolytic pathway. hCA IX overexpression is also involved in tumor resistance to radio- and chemotherapy and metastasis invasion. 10–12 The rational design of hCA inhibitors usually takes advantage of the introduction of a deprotonable zinc binding group (primary or secondary sulfonamide, sulfamate, sulfamide, hydroxamic acid, benzoic acid, phenol) which, despite its strong hCA inhibitory activity in the nanomolar range, often lacks isoform selectivity. 13,14 To overcome this problem, novel scaffolds were screened exploring even non-ionizable chemical functionalities (i.e., tertiary sulfon- amides) with the aim at unraveling the differences among these isoforms in terms of structure, tissue distribution and cellular localization. 15–19 Recently, we proposed a simple chemical modification of the well known uricosuric agent and CA inhibitor Probenecid, 20 lead- ing to the synthesis of a small series of amide derivatives with good inhibition profiles against hCA IX and XII (nanomolar range) in terms of isoform selectivity (K i CA II >10,000 nM) but retaining residual hCA I inhibition in the micromolar range. We kept constant the tertiary sulfonamide group of the parent compound, involved in important interactions with specific amino http://dx.doi.org/10.1016/j.bmc.2015.05.013 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding authors. Tel./fax: +39 0871 3554583 (S.C.); tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.). E-mail addresses: [email protected](S. Carradori), claudiu.supuran@ unifi.it (C.T. Supuran). Bioorganic & Medicinal Chemistry 23 (2015) 2975–2981 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Simone Carradori a,⇑, Adriano Mollica a, Mariangela Ceruso b, Melissa D’Ascenzio c, Celeste De Monte c,Paola Chimenti c, Rocchina Sabia c, Atilla Akdemir d, Claudiu T. Supuran b,e,⇑a Department of Pharmacy, ‘G. D’Annunzio’ University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italyb Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italyc Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italyd Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Vatan Caddesi, 34093 Fatih, Istanbul, Turkeye Università degli Studi di Firenze, Neurofarba Dept., Section of Pharmaceutical and Nutraceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
a r t i c l e i n f o a b s t r a c t
Article history:Received 25 February 2015Revised 5 May 2015Accepted 6 May 2015Available online 14 May 2015
Keywords:ProbenecidSelective carbonic anhydrase IX inhibitorsSelective carbonic anhydrase XII inhibitorsTertiary sulfonamidesMolecular modelling
Novel amide derivatives of Probenecid were synthesized and discovered to act as potent and selectiveinhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) transmembrane isoforms hCA IX and XII.The proposed chemical transformation of the carboxylic acid into an amide group led to a complete lossof hCA I and II inhibition (Kis >10,000 nM) and enhanced the inhibitory activity against hCA IX and XII,with respect to the parent compound (incorporating a COOH function). These promising biological resultshave been corroborated by molecular modelling studies within the active sites of the four studied humancarbonic anhydrases, which enabled us to rationalize both the isoform selectivity and high activityagainst the tumor-associated isoforms hCA IX/XII.
� 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Human carbonic anhydrases (hCAs, EC 4.2.1.1) are recognized tobe highly conserved and widespread metalloenzymes that effi-ciently catalyze the reversible hydratation of CO2 to bicarbonateand proton.1 Sixteen isoforms (I–XV, with VA and VB being mito-chondrial isoforms) are involved in a wide range of physiologicalprocesses and their malfunctioning or altered expression couldpromote pathological situations. For this reason, selective isoforminhibition or activation is known to be an effective strategy inthe treatment of important diseases.2–4 Over the past few years,a specific interest has been oriented toward the two hCA trans-membrane isoforms (hCA IX and XII),5–9 almost exclusively overex-pressed in hypoxic tumors, which represent a main player ofgrowth and survival of malignancies since they regulate extracellu-lar and intracellular pH, control cell adhesion, and contribute tomany adaptive changes in solid tumors. After their activation in a
reduced oxygen environment, tumor cells reprogram their meta-bolism toward the glycolytic pathway. hCA IX overexpression isalso involved in tumor resistance to radio- and chemotherapyand metastasis invasion.10–12
The rational design of hCA inhibitors usually takes advantage ofthe introduction of a deprotonable zinc binding group (primary orsecondary sulfonamide, sulfamate, sulfamide, hydroxamic acid,benzoic acid, phenol) which, despite its strong hCA inhibitoryactivity in the nanomolar range, often lacks isoform selectivity.13,14
To overcome this problem, novel scaffolds were screened exploringeven non-ionizable chemical functionalities (i.e., tertiary sulfon-amides) with the aim at unraveling the differences among theseisoforms in terms of structure, tissue distribution and cellularlocalization.15–19
Recently, we proposed a simple chemical modification of thewell known uricosuric agent and CA inhibitor Probenecid,20 lead-ing to the synthesis of a small series of amide derivatives with goodinhibition profiles against hCA IX and XII (nanomolar range) interms of isoform selectivity (Ki CA II >10,000 nM) but retainingresidual hCA I inhibition in the micromolar range.
We kept constant the tertiary sulfonamide group of the parentcompound, involved in important interactions with specific amino
2976 S. Carradori et al. / Bioorg. Med. Chem. 23 (2015) 2975–2981
acids in the CA active site, exploring the chemical variability linkedto the opposite portion of the lead compound (carboxylic acid func-tion). Only one series of Probenecid derivatives was reported andinvestigated as CA inhibitors so far, therefore we decided to extendhere our earlier studies by incorporating small aliphatic pendantssuch as ethyl, 3-/4-methylcyclohexyl, N-alkylpiperidine, N-alkyl-morpholine, benzyl, N,N-diethylethylendiamine and aromatic moi-eties (pyridine, substituted aryl rings) as outlined in Table 1 toenhance isoform selectivity and pursuing the aims of the ‘‘tail’’approach.21–23 In this context, the sulfonamide moiety was furtherfunctionalized by substitution modulating the interactions withthe enzyme active site or/and the physicochemical properties ofthe inhibitor. A more or less flexible hydrophobic/hydrophilic tailgroup could favourably adopt a proper conformation to interactwithin the hydrophobic/hydrophilic half of the active site.
2. Chemistry
Derivatives 1-11 were synthesized by reacting 4-(N,N-dipropy-lsulfamoyl)benzoic acid (Probenecid) with the corresponding
Table 1Inhibitory activity of derivatives 1–12 and reference compounds (Probenecid andAcetazolamide) against four selected hCA isoforms by stopped-flow CO2 hydraseassay
SN
O O
NH
O
CDI or SOCl2
DMF or DCMrt or 50 °C
SN
O O
O
OH+ H2N R
R
Probenecid
Compound R Ki (nM)
hCA I hCA II hCAIX
hCAXII
1 CH2CH3 >10,000 >10,000 151 965
2CH3
>10,000 >10,000 188 798
3CH3
>10,000 >10,000 2024 256
4 NO
>10,000 >10,000 213 419
5 N >10,000 >10,000 1256 66.0
6 N >10,000 >10,000 110 300
7 >10,000 >10,000 20.6 70.7
8
CH3
>10,000 >10,000 61.3 105
9 N >10,000 >10,000 199 9.9
10N
>10,000 >10,000 2037 9.9
11
Cl
>10,000 >10,000 23.6 34.7
12NO2
>10,000 >10,000 246 206
Probenecid >10,000 431 360 1245Acetazolamide
(AAZ)250 12 25 5.7
amine in presence of 1,10-carbonyldiimidazole (CDI) in N,N-dimethylformamide at room temperature or at 50 �C. Compound12 was obtained by converting Probenecid into its correspondingreactive acyl chloride using thionyl chloride in dry dichloro-methane, then the resulting intermediate reacted with the lessreactive 4-nitroaniline (Table 1). Purification via column chro-matography on silica gel afforded title compounds in discreteyields. All synthesized compounds were fully characterized by ana-lytical and spectral data (see Experimental section).
3. Biological evaluation
All the synthesized compounds (1–12) were tested against thetwo cancer-related isoforms of hCA (CA IX and XII) and their corre-sponding off-targets (CA I and II) in order to evaluate their biolog-ical activity and selectivity. On the basis of the obtained results,SAR for this class of Probenecid analogues as promising hCA inhi-bitors can be extrapolated. Unlike the known CA inhibitorAcetazolamide, all the tested compounds proved to be inactiveagainst the ubiquitously expressed isoforms of hCA I and II (off-tar-get dominant isoforms) at concentrations higher than 10 lM(Table 1). This behavior is better than that of the parent compoundProbenecid (with a carboxylic function) which retains a potentinhibitory activity (Ki = 431 nm) against hCA II (limited selectivity).This is a positive feature for a putative CA inhibitor since hCA I andII inhibition is often associated to unwanted side effects. This bio-logical behavior confirmed our choice of specific substituents withrespect to the previously published series.
In general, we found that the conversion of the carboxylic groupof Probenecid into its corresponding amide derivatives results in amarked inhibitory activity against the tumor-associated isoformhCA IX, with Ki values ranging from 2037 nM to 20.6 nM.Similarly, the activity against hCA IX was maintained, if not slightlyincreased, in both cycloaliphatic and (hetero)aromatic derivativesthroughout the series. The best hCA IX inhibitors were obtainedwith the substitution of the amidic nitrogen with a benzyl or 2-chloro/2-methylaryl group. As regards hCA XII inhibition, thesenew derivatives enhance their activity (all in the nanomolar range)and selectivity, especially when a pyridine ring is present at theamidic portion (Ki = 9.9 nM for compounds 9 and 10). They are verypotent inhibitors of this isoform and could be interesting pharma-cological tools for further experiments keeping in mind that thisisoform has been less investigated so far.
Collectively, the presence of a non-classical tertiary sulfon-amide zinc binding moiety in this series has contributed to thehypothesis that these inhibitors could display a non-classical bind-ing mode (via direct interaction of the tertiary sulfonamide groupwith the metal ion).
4. Docking studies
Compounds 7, 8, and 11 show Ki values in the range of 20.6–61.3 nM for hCA IX (Table 1). The docked poses of the Probenecidanalogs into hCA IX without zinc-bound water revealed that thetwo propyl groups prohibit the sulfonamide group of approachingthe Zn2+-ion and therefore no interaction occurs as with the classi-cal sulfonamides. This allowed a water molecule to bind to theZn2+-ion. Therefore, the dockings were repeated with a zinc-boundwater molecule. For compound 7, the two oxygen atoms of the sul-fonamide group form hydrogen bonds with the side chains ofAsn62 and Gln92 (Fig. 1A). The phenyl group that is located nextto the sulfonamide moiety could form hydrophobic contacts withthe side chains of Trp5 and His64. The terminal phenyl group isflexible and is able to interact with Trp5, His64 and Pro202. Theamide bond does not form hydrogen interactions with the protein
B
H64
W5
N62A
H64
W5
N62
S20
P202
T199
Q67
T200
L91
Q92
H119
H96
S20P202
T199
T200
Q67
L91
Q92
H94H96
H119
C D
H64
H64
W5
W5 N62
N62
S20P202
T199
T200
Q67
L91
Q92
H94
H119
H96T20
T200
T199
H94
Q92
T91S132
H119
K67 H96
E F
H64 H64
W5 W5
N62 N62
T20
T20
S132
T200Q92
T91
T199
K67H96
H94
H119
T91S132
K67
Q92
H119
H94
H96
T200
T199
S135
Figure 1. Docked poses of compounds 7 (A), 8 (B) and 11 (C) with hCA IX and compounds 9 (D), 10 (E) and 5 (F) in complex with hCA XII. The Zn2+-ion is indicated with a bluesphere, the zinc-bound water molecule (if present) is indicated with a red sphere, docked ligands are indicated in turquoise and magenta, hydrogen bonds between ligand andenzyme are indicated in thick red dashed lines, and bonds between the protein and the Zn2+-ion, the Zn2+-ion and intraprotein hydrogen bonds are indicated with thin dashedred lines.
S. Carradori et al. / Bioorg. Med. Chem. 23 (2015) 2975–2981 2977
but it is water exposed and so it could form interactions with thesolvent. None of the other compounds in this series (see Table 1)nor in our previously synthesized series were able to form similarinteractions as obtained for compound 7 with hCA IX (Fig. 1A),which could explain the higher measured Ki values.20
Compound 8 differs in two aspects from compound 7. The ter-minal phenyl group has an ortho-methyl substituent and it isdirectly bound to the amide bond, while compound 7 has a methylspacer and an unsubstituted phenyl ring. This influences the bind-ing modes and increases the Ki value by approximately 3-fold. Twobinding modes have been obtained (Fig. 1B). In the first docked
pose, one of the sulfonamide oxygen atoms forms a hydrogen bondto the side chain of His64, which is a neighboring residue to Asn62.The phenyl groups seem to form hydrophobic interactions withTrp5. In the second binding mode, the terminal phenyl moiety islocated close to His94 and forms hydrophobic interactions withthis residue. The amide bond is hydrogen bonded to both His64and Gln67. Compound 11 is related to 8 and contains a 2-chlorosubstituent instead of a 2-methyl substituent. Two docked poseshave been obtained (Fig. 1C) that are similar to compound 8(Fig. 1B). In the first docked pose, a hydrogen bond between thesulfonamide oxygen and the side chain of His64 is formed. The
2978 S. Carradori et al. / Bioorg. Med. Chem. 23 (2015) 2975–2981
chlorine substituent points toward Trp5. A second docked pose, inwhich the substituted terminal phenyl ring forms hydrophobicinteractions with the zinc-binding His94, was obtained. The amidebond of the ligand forms hydrogen bonds with the side chains ofHis64 and Gln67, while the aromatic ring interacts with His94and Trp5. The sulfonamide group is water exposed and most likelyis able to form hydrogen bonds with water molecules. The two pro-pyl substituents point toward the hydrophobic parts of Gln67 andLeu91. The chlorine substitution at the ortho position seems to befavorable. We suggest that this electronegative chlorine atominteracts with the side chain of His94 as observed in the seconddocked pose. Replacement of the chlorine substituent with amethyl group or moving it to a meta or para position (see previousseries20) results in an increased Ki value.
A different enzyme inhibition profile was observed for hCA XII.Compounds 9 and 10 showed the lowest measured Ki values(9.9 nM), while compound 11 showed a slightly higher Ki value(34.7 nM). Compounds 5 (66.0 nM) and 7 (70.7 nM) also showed Ki
values lower than 100 nM. Docking studies again revealed that nodirect interactions with the Zn2+-ion or the zinc-bound water mole-cule could be formed except for compounds 9 and 10. An interactionmay occur between the nitrogen atom of the pyridine and the Zn2+-ion. Compound 9 could form a hydrogen bond with the side chain ofLys67, while the sulfonamide of compound 10 and the side chain ofThr91 could form a hydrogen bond (Fig. 1D and E). While notobserved in the docking poses, the side chains of Asn62 andThr200 may adjust their conformation to form an additional hydro-gen bond to the sulfonamide of compounds 9 and 10, respectively.
Compound 5 does not form an interaction with the Zn2+-ion andmost likely a water molecule is able to bind to the zinc (Fig. 1F).The piperidine group is probably positively charged at physiologi-cal pH values and could form cation-p interactions with His94(shortest distance: 3.9 Å) and hydrophobic interactions with theside chain of Thr200. One of the sulfonamide oxygen atoms formsa hydrogen bond to the side chain of Thr91. Side chain reorienta-tions of Gln92 and Ser132 may result in additional hydrogen bondsto the amide bond and the other sulfonamide oxygen, respectively.In our previous series, we synthesized and tested an analog of com-pound 5 in which the piperidine ring was replaced by a morpholinering.20 The oxygen atom of the latter compound formed a hydrogenbond to the zinc-ion and the measured Ki value was 15.3 nM.20 Inour new series we have another analogue with a morpholine moi-ety but with a longer spacer (compound 4) that is not favorable andtherefore a higher Ki value.
For the other compounds, the obtained binding poses for hCAXII are different compared to hCA IX due to divergences in thebinding pockets of both enzymes (hCA IX: Ser20, Gln67, Leu91;hCA XII: Tyr20, Lys67 and Thr91). Especially, the large Tyr20located close to Trp5 makes it difficult for compounds 7, 8 and11 to adopt similar binding poses as found in hCA IX.
5. Conclusion
In the present paper we extended the scaffold of Probenecidanalogues previously investigated including new substituents viaamidic bond to explore the chemical space at this position. A smalllibrary of new amide derivatives of Probenecid were easily synthe-sized and evaluated as potent and selective inhibitors of the humantransmembrane carbonic anhydrase isoforms (hCA IX and XII).Compounds 1–12 showed an inversion of selectivity against CA Iand II compared to the parent drug Probenecid which was not ableto inhibit isoform I and was mildly active against isoform II.Docking studies of the most active compounds suggested enzymerecognition pattern for the design of novel selective hCA isoforminhibitors highlighting the differences with the parent compound.
6. Experimental protocols
6.1. General
Solvents were used as supplied without further purification.‘Petroleum ether’ refers to the fraction of petroleum ether boilingin the range 40–60 �C. Where mixtures of solvents are specified,the stated ratios are volume:volume. Unless otherwise indicated,all aqueous solutions used were saturated. Reagents were useddirectly as supplied by Sigma Aldrich� Italy. All reactions involvingair- or moisture-sensitive compounds were performed under anitrogen atmosphere using dried glassware and syringes tech-niques to transfer solutions. Column chromatography was carriedout using Sigma-Aldrich� silica gel (high purity grade, pore size60 Å, 200–425 mesh particle size). Analytical thin-layer chro-matography was carried out on Sigma–Aldrich� silica gel on TLAaluminum foils with fluorescent indicator 254 nm. Visualizationwas carried out under ultra-violet irradiation (254 nm). NMR spec-tra were recorded on a Bruker AV400 (1H: 400 MHz, 13C: 101 MHz).Chemical shifts are quoted in ppm, based on appearance ratherthan interpretation, and are referenced to the residual non deuter-ated solvent peak. The assignment of exchangeable protons (NH)was confirmed by the addition of D2O. Infra-red spectra wererecorded on a Bruker Tensor 27 FTIR spectrometer equipped withan attenuated total reflectance attachment with internal calibra-tion. Absorption maxima (mmax) are reported in wavenumbers(cm�1). Elemental analyses for C, H, and N were recorded on aPerkin-Elmer 240 B microanalyzer and the analytical results werewithin ±0.4% of the theoretical values for all compounds(Supporting information). All melting points were measured on aStuart� melting point apparatus SMP1, and are uncorrected.Temperatures are reported in �C. Where given, systematic com-pound names are those generated by ChemBioDraw Ultra� 12.0following IUPAC conventions.
6.2. Chemistry
General procedure for the synthesis of derivatives 1–11: 1,10-carbonyldiimidazole (CDI, 1.5 equiv) and the appropriate amine(1.5 equiv) were added to a stirring solution of Probenecid(1.0 equiv) in dry N,N-dimethylformamide. The reaction was stir-red for 4–24 h. Eventually, an additional equivalent of CDI wasadded. The mixture was diluted with dichloromethane, washedwith saturated NaHCO3, dried over anhydrous sodium sulfate, fil-tered and concentrated in vacuo. Procedure for the synthesis ofcompound 12: Probenecid (1.0 equiv) was dissolved in dichloro-methane and thionyl chloride was added dropwise. 1 drop of dryN,N-dimethylformamide was added and the reaction stirred for24 h. In a separate flask, 4-nitroaniline was suspended in drydichloromethane and triethylamine (2.75 equiv) and was addedunder nitrogen atmosphere. The solution of Probenecid chloridein dry dichloromethane was added to the solution of 4-nitroanilineand the reaction stirred for 24 h at 50 �C. The reaction was evapo-rated in vacuo. Purification via column chromatography on silicagel afforded title compounds in discrete yields (Table 1).
6.2.1. 4-(N,N-Dipropylsulfamoyl)-N-ethylbenzamide (1)1,10-Carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 equiv) and
1.31 mL of a 2 M solution of ethylamine in THF (2.63 mmol,1.5 equiv) were added to a stirring solution of Probenecid (1 g,3.50 mmol, 1.0 equiv) in 10.0 mL of dry N,N-dimethylformamideat room temperature. After 3 h the reaction was diluted withdichloromethane (20 mL) and washed with saturated NaHCO3
(20 mL). The organics were dried over anhydrous sodium sulfate,filtered and concentrated in vacuo. Purification by column
S. Carradori et al. / Bioorg. Med. Chem. 23 (2015) 2975–2981 2979
chromatography on silica gel (ethyl acetate/n-hexane 2:1) gavetitle compound as a white solid (0.64 g, 59% yield); mp 93–95 �C;IR mmax 3315 (m N–H), 2966 (m Csp2–H), 1633 (m C@O), 1343 (mas
1,10-Carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 equiv) and0.77 ml of N-3-(aminopropyl)morpholine (5.25 mmol, 1.5 equiv)were added to a stirring solution of Probenecid (1.0 g, 3.50 mmol,1.0 equiv) in 10.0 mL of dry N,N-dimethylformamide at room tem-perature. After four hours the reaction was diluted with dichloro-methane (20 mL) and washed with saturated NaHCO3 (20 mL).The organics were dried over anhydrous sodium sulfate, filteredand concentrated in vacuo. Purification by column
chromatography on silica gel (ethyl acetate/hexane 15:1) gave titlecompound as a white solid (0.91 g, 63% yield); mp 92–94 �C; IRmmax 3355 (m N–H), 2967 (m Csp2–H), 1634 (m C@O), 1347 (mas
1,10-Carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 equiv) and0.75 ml of 1-(2-aminoethyl)piperidine (5.25 mmol, 1.5 equiv) wereadded to a stirring solution of Probenecid (1.0 g, 3.50 mmol,1.0 equiv) in 10.0 mL of dry N,N-dimethylformamide at room tem-perature. After one day the reaction was diluted with dichloro-methane (20 mL) and washed with saturated NaHCO3 (20 mL).The organics were dried over anhydrous sodium sulfate, filteredand concentrated in vacuo. Purification by column chromatogra-phy on silica gel (ethyl acetate/petroleum ether 5:1) gave titlecompound as a yellow oil (1.02 g, 73% yield); IR mmax 3323 (m N–H), 2934 (m Csp2–H), 1644 (m C@O), 1337 (mas S@O), 1148 (ms
1,10-Carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 equiv) and0.74 ml of N,N-diethylethylendiamine (5.25 mmol, 1.5 equiv) wereadded to a stirring solution of Probenecid (1.0 g, 3.50 mmol,1.0 equiv) in 10.0 mL of dry N,N-dimethylformamide at room tem-perature. After one day the reaction was diluted with dichloro-methane (20 mL) and washed with saturated NaHCO3 (20 mL).The organics were dried over anhydrous sodium sulfate, filteredand concentrated in vacuo. Purification by column chromatogra-phy on silica gel (ethyl acetate/hexane 5:1) gave title compoundas a yellow oil (0.95 g, 71% yield); IR mmax 3334 (m N–H), 2967 (mCsp2–H), 1645 (m C@O), 1336 (mas S@O), 1148 (ms S@O), 739 (dCsp2–H) cm�1; 1H NMR (400 MHz, CDCl3) d 0.78 (6H, t, J = 7.4 Hz,2 � CH3), 0.96 (6H, t, J = 7.2 Hz, 2 � CH3), 1.46 (4H, sext,J = 7.4 Hz, 2 � CH2), 2.50 (4H, q, J = 7.6, 2 � CH2), 2.59 (2H, t,J = 6.0 Hz, CH2), 3.00 (4H, t, J = 7.6 Hz, 2 � CH2), 3.42 (2H, t,J = 6.1 Hz, CH2), 7.31 (1H, bs, NH), 7.75 (2H, d, J = 8.4 Hz, CH-Ar),7.84 (2H, d, J = 8.4 Hz, CH-Ar); 13C NMR (101 MHz, CDCl3) d 11.1(CH3), 11.7 (CH3), 21.9 (CH2), 37.5 (CH2), 46.7 (CH2), 49.9 (CH2),51.2 (CH2), 127.1 (Ar), 127.7 (Ar), 138.2 (Ar), 142.5 (Ar), 165.8(C@O).
6.2.7. 4-(N,N-Dipropylsulfamoyl)-N-(benzylbenzamide) (7)1,10-Carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 equiv) and
0.56 g of benzylamine (5.25 mmol, 1.5 equiv) were added to a stir-ring solution of Probenecid (1.0 g, 3.50 mmol, 1.0 equiv) in 10.0 mLof dry N,N-dimethylformamide at room temperature. The reactionwas stirred overnight, diluted with dichloromethane (20 mL) andwashed with saturated NaHCO3 (20 mL). The organics were driedover anhydrous sodium sulfate, filtered and concentrated in vacuo.Purification by column chromatography on silica gel (hexane/ethylacetate 1:2) gave the title compound as a white solid (0.89 g, 68%yield); mp 108–110 �C; IR mmax 3340 (m N–H), 2966 (m Csp2–H),
2980 S. Carradori et al. / Bioorg. Med. Chem. 23 (2015) 2975–2981
1,10-Carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 equiv) and0.49 g of 3-aminopyridine (5.25 mmol, 1.5 equiv) were added toa stirring solution of Probenecid (1.0 g, 3.50 mmol, 1.0 equiv) in10.0 mL of dry N,N-dimethylformamide at 50 �C. The reactionwas stirred overnight. The reaction was diluted with dichloro-methane (20 mL) and washed with saturated NaHCO3 (20 mL).The organics were dried over anhydrous sodium sulfate, filteredand concentrated in vacuo. Purification by column chromatogra-phy on silica gel (ethyl acetate:hexane 5:1) gave the title com-pound as a white solid (0.62 g, 49% yield); mp 134–136 �C; IRmmax 3323 (m N–H), 2967 (m Csp2–H), 1671 (m C@O), 1329 (mas
1,10-Carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 equiv) and0.49 g of 4-aminopyridine (5.25 mmol, 1.5 equiv) were added toa stirring solution of Probenecid (1.0 g, 3.50 mmol, 1.0 equiv) in10.0 mL of dry N,N-dimethylformamide at 50 �C. The reactionwas stirred overnight. The reaction was diluted with dichloro-methane (20 mL) and washed with saturated NaHCO3 (20 mL).The organics were dried over anhydrous sodium sulfate, filteredand concentrated in vacuo. Purification by column chromatogra-phy on silica gel (hexane/ethyl acetate 1:5) gave the title com-pound as a white solid (0.61 g, 48% yield); mp 131–136 �C; IRmmax 3364 (m N–H), 2963 (m Csp2–H), 1690 (m C@O), 1326 (mas
1.00 g of Probenecid (3.50 mmol, 1.0 equiv) was dissolved in15 mL of dichloromethane. 0.63 mL of thionyl chloride was addeddropwise. 1 drop of dry N,N-dimethylformamide was added andthe reaction refluxed for 24 h. In a separate flask, 4-nitroanilinewas suspended in 10 mL of dry dichloromethane. 0.7 mL of triethy-lamine (9.6 mmol, 2.75 equiv) was added under nitrogen atmo-sphere. The solution of acyl chloride in dry dichloromethane wasadded to the solution of 4-nitroaniline and the reaction stirredfor 24 h at 50 �C. The reaction was evaporated in vacuo and theresidue purified on silica gel (hexane/ethyl acetate 4:1) to give titlecompound as a yellow solid (1.77 g, 83% yield); mp 61–64 �C; IRmmax 3390 (m N–H), 2975 (m Csp2–H), 1696 (m C@O), 1505 (m N–O),1335 (mas S@O), 1247 (m N–O), 1149 (ms S@O), 740 (d Csp2–H)cm�1; 1H NMR (400 MHz, CDCl3) d 0.90 (6H, t, J = 7.2 Hz,2 � CH3), 1.57 (4H, sext, J = 7.2 Hz, 2 � CH2), 3.11 (4H, t,J = 7.6 Hz, 2 � CH2), 7.73 (2H, d, J = 8.0 Hz, CH-Ar), 7.94-7.98 (4H,m, CH-Ar), 8.30 (2H, d, J = 9.2 Hz, 2 � CH-Ar), 8.88 (1H, bs, NH);13C NMR (101 MHz, CDCl3) d 11.1 (CH3), 21.9 (CH2), 50.0 (CH2),119.7 (Ar), 125.1 (Ar), 127.3 (Ar), 128.2 (Ar), 138.2 (Ar), 142.9(Ar), 143.8 (Ar), 143.9 (Ar), 164.9 (C@O).
6.3. In vitro enzyme inhibition
An Applied Photophysics stopped-flow instrument has beenused for assaying the CA catalyzed CO2 hydration activity. Phenolred (0.2 mM) has been used as indicator, working at the absor-bance maximum of 557 nm, with 20 mM Hepes (pH 7.5, for a-CAs) as buffer, and 20 mM NaClO4 (for maintaining constant theionic strength), following the initial rates of the CA-catalyzed CO2
hydration reaction for a period of 10–100 s. The CO2 concentrationsranged from 1.7 to 17 mM for the determination of the kineticparameters and inhibition constants. For each inhibitor at leastsix traces of the initial 5–10% of the reaction have been used fordetermining the initial velocity. The uncatalyzed rates were deter-mined in the same manner and subtracted from the total observed
S. Carradori et al. / Bioorg. Med. Chem. 23 (2015) 2975–2981 2981
rates. Stock solutions of inhibitor (1 lM) were prepared in dis-tilled-deionized water and dilutions up to 0.1 nM were done there-after with the assay buffer. Inhibitor and enzyme solutions werepreincubated together for 15 min at room temperature prior toassay, in order to allow for the formation of the E–I complex orfor the eventual active site mediated hydrolysis of the inhibitor.The inhibition constants were obtained by non-linear least-squaresmethods using PRISM 3 and the Cheng–Prusoff equation,24 andrepresent the average from at least three different determinations.All recombinant CA isoforms were obtained in-house as previouslyreported.25,26
6.4. Molecular modelling studies
6.4.1. Preparation of Probenecid analog structuresThe Probenecid analogs 1–12 were prepared in 3D with the
MOE software package (v2013.08.02, Chemical Computing GroupInc., Montreal, Canada). All possible structural isomers of com-pounds 2 and 3 were constructed. Strong acids were deprotonatedand strong bases were protonated. Finally, the ligands were energyminimized using a steepest-descent protocol (MMFF94x forcefield).
6.4.2. Preparation of hCA crystal structures for docking studiesThe structures of hCA I (PDB: 3LXE, 1.90 Å), hCA II (PDB: 4E3D,
1.60 Å), hCA IX (PDB: 3IAI; 2.20 Å) and hCA XII (PDB: 1JD0; 1.50 Å)were obtained from the protein databank. The protein atoms, theactive site zinc ions and the zinc-bound water molecule of hCA IIwere retained and all other atoms were omitted. The remainingstructure was protonated using the MOE software package andsubsequently the obtained structure was energy-minimized(AMBER99 force field). Finally, the obtained protein models weresuperposed on the hCA I structure using the backbone Ca-atoms.The zinc-bound water molecule of hCA II coordinated well to thezinc ions of the other hCAs.
and the GoldScore scoring function were used to dock theProbenecid analogs into the hCA structures with and without thezinc-bound water molecule (25 dockings per ligand). The bindingpocket was defined as all residues within 13 Å of a centroid (x:�17.071, y: 35.081, 43.681; corresponding approximately to theposition of the thiadiazole ring of Acetazolamide in the 1JD0structure).
Acknowledgments
This work was financed by two FP7 EU projects (Metoxia andDynano) to C.T.S.
Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmc.2015.05.013.
References and notes
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