Accepted Manuscript New amide derivatives of Probenecid as selective inhibitors of carbonic anhydrase IX and XII: Biological evaluation and molecular modelling studies Simone Carradori, Adriano Mollica, Mariangela Ceruso, Melissa D’Ascenzio, Celeste De Monte, Paola Chimenti, Rocchina Sabia, Atilla Akdemir, Claudiu T. Supuran PII: DOI: Reference:

S0968-0896(15)00413-7 http://dx.doi.org/10.1016/j.bmc.2015.05.013 BMC 12309

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Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

25 February 2015 5 May 2015 6 May 2015

Please cite this article as: Carradori, S., Mollica, A., Ceruso, M., D’Ascenzio, M., Monte, C.D., Chimenti, P., Sabia, R., Akdemir, A., Supuran, C.T., New amide derivatives of Probenecid as selective inhibitors of carbonic anhydrase IX and XII: Biological evaluation and molecular modelling studies, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.05.013

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Bioorganic & Medicinal Chemistry j o ur n al h om e p a g e : w w w . e l s e v i er . c o m

New amide derivatives of Probenecid as selective inhibitors of carbonic anhydrase IX and XII: biological evaluation and molecular modelling studies Simone Carradoria,∗, Adriano Mollicaa, Mariangela Ceruso b, Melissa D’Ascenzioc, Celeste De Montec, Paola Chimentic, Rocchina Sabiac, Atilla Akdemird, Claudiu T. Supuranb,e,* a

Department of Pharmacy, “G. D’Annunzio” University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy. 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 Nutriceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy. b

A R T IC LE IN F O

A B S TR A C T

Article history: Received Received in revised form Accepted Available online

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 (Ki s>10000 nM) and enhaced 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 tumorassociated isoforms hCA IX/XII.

Keywords: Probenecid Selective carbonic anhydrase IX inhibitors Selective carbonic anhydrase XII inhibitors Tertiary sulfonamides Molecular modelling

1. Introduction Human carbonic anhydrases (hCAs, EC 4.2.1.1) are recognized to be highly conserved and widespread metalloenzymes that efficiently catalyze the reversible hydratation of CO2 to bicarbonate and proton.1 Sixteen isoforms (I-XV, with VA and VB as mitochondrial 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 In the last years, a specific interest has been oriented towards the two hCA transmembrane isoforms (hCA IX and XII),5-9 almost exclusively overexpressed in hypoxic tumors, which represent a main player of growth and survival of malignancies because they regulate extracellular 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 metabolism towards the glycolytic pathway. hCA IX overexpression is also involved in tumor resistance to radio- and chemotherapy and metastasis invasion.10-12

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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 sulfonamides) 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 leading 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 (Ki CA II >10000 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 acids in the CA active site, exploring the chemical variability linked to the opposite portion of the lead compound (carboxylic acid function). Only one series of Probenecid

∗ Corresponding authors: Simone Carradori Ph.D.: Tel/Fax: +39 0871 3554583; e-mail: [email protected]. Prof. Claudiu T. Supuran: Tel: +39-055-4573005; Fax: +39-055-4573385; e-mail: [email protected].

derivatives was reported and investigated as CA inhibitors so far and we decided to extend here our earlier studies incorporating small aliphatic pendants such as ethyl, 3-/4-methylcyclohexyl, Nalkylpiperidine, N-alkylmorpholine, benzyl, N,Ndiethylethylendiamine and aromatic moieties (pyridine, substituted aryl rings) as outlined in Table 1 to enhance isoform selectivity and pursuing the aims of the “tail” approach.21-23 In this context, the sulfonamide moiety is further functionalized by substitution modulating its interactions with the enzyme active site or/and its physicochemical properties. A more or less flexible hydrophobic/hydrophilic tail group could favourably adopt a proper conformation to interact within the hydrophobic/hydrophilic half of the isoform active site. 2. Chemistry Derivatives 1-11 were synthesized by reacting 4-(N,N-dipropylsulfamoyl)benzoic acid (Probenecid) with the corresponding amine in presence of 1,1’-carbonyldiimidazole (CDI) in N,N-dimethylformamide at room temperature or at 50 °C. Compound 12 was obtained by converting Probenecid into its corresponding reactive acyl chloride using thionyl chloride in dry dichloromethane, then the resulting intermediate reacted with the less reactive 4-nitroaniline (Table 1). Purification via column chromatography on silica gel afforded title compounds in discrete yields. All synthesized compounds were fully characterized by analytical and spectral data (see Experimental section). 3. Biological evaluation All the synthesized compounds (1-12) were tested against the two cancer-related isoforms of hCA (CA IX and XII) and their corresponding off-targets (CA I and II) in order to evaluate their biological activity and selectivity. On the basis of the obtained results, SAR for this class of Probenecid analogues, as promising hCA inhibitors, can be extrapolated. Unlike the known CA inhibitor Acetazolamide, all the tested compounds proved to be inactive against the ubiquitously expressed isoforms of hCA I and II (off-target dominant isoforms) at concentrations higher than 10 µM (Table 1). This behavior is better than that of the parent compound Probenecid (with a carboxylic function) which retains a potent inhibitory activity (Ki= 431 nm) against hCA II (limited selectivity). This is a positive feature for a putative CA inhibitor since hCA I and II inhibition is often associated to unwanted side effects. This biological behavior confirmed our choice of specific substituents with respect to the previously published series. In general, we found that the conversion of the carboxylic group of Probenecid into its corresponding amide derivatives results in a marked inhibitory activity against the tumorassociated isoform hCA IX, with Ki values ranging from 2037 nM to 20.6 nM. Similarly, the activity against hCA IX was maintained, if not slightly increased, in both cycloaliphatic and (hetero)aromatic derivatives throughout the series. The best hCA IX inhibitors were obtained with the substitution of the amidic nitrogen with a benzyl or 2-chloro/2-methylaryl group. As regards hCA XII inhibition, these new derivatives enhance their activity (all in the nanomolar range) and selectivity, especially when a pyridine ring is present at the amidic portion (Ki= 9.9 nM for compounds 9 and 10). They are very potent inhibitors of this isoform and could be interesting pharmacological tools for further experiments keeping in mind that this isoform has been less investigated so far. Collectively, the presence of a non-classical tertiary sulfonamide zinc binding moiety in this series has contributed to the hypothesis that these inhibitors could display a non-classical

binding mode (via direct interaction of the tertiary sulfonamide group with 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 Probenecid analogs into hCA IX without zinc-bound water revealed that the two propyl groups prohibit the sulfonamide group of approaching the Zn2+-ion and therefore no interaction occurs as with the classical sulfonamides. This allowed a water molecule to bind to the Zn2+-ion. Therefore, the dockings were repeated with a zinc-bound water molecule. For compound 7, the two oxygen atoms of the sulfonamide group form hydrogen bonds with the side chains of Asn62 and Gln92 (Figure 1A). The phenyl group that is located next to the sulfonamide moiety could form hydrophobic contacts with the side chains of Trp5 and His64. The terminal phenyl group is flexible and is able to interact with Trp5, His64 and Pro202. The amide bond does not form hydrogen interactions with the protein but it is water exposed and so it could form interactions with the solvent. None of the other compounds in this series (see Table 1) nor in our previously synthesized series were able to form similar interactions as obtained for compound 7 with hCA IX (Figure 1A), which could explain the higher measured Ki values.20 Compound 8 differs in two respects from compound 7. The terminal phenyl group has an ortho-methyl substituent and it is directly bound to the amide bond, while compound 7 has a methyl spacer and an unsubstituted phenyl ring. This influences the binding modes and increases the Ki value by approximately 3fold. Two binding modes have been obtained (Figure 1B). In the first docked pose, one of the sulfonamide oxygen atoms forms a hydrogen bond to the side chain of His64, which is a neighboring residue to Asn62. The phenyl groups seem to form hydrophobic interactions with Trp5. In the second binding mode, the terminal phenyl moiety is located close to His94 and forms hydrophobic interactions with this residue. The amide bond is hydrogen bonded to both His64 and Gln67. Compound 11 is related to 8 and contains a 2-chloro substituent instead of a 2-methyl substituent. Two docked poses have been obtained (Figure 1C) that are similar to compound 8 (Figure 1B). In the first docked pose, a hydrogen bond between the sulfonamide oxygen and the side chain of His64 is formed. The chlorine substituent points towards Trp5. A second docked pose, in which the substituted terminal phenyl ring forms hydrophobic interactions with the zinc-binding His94, was obtained. The amide bond of the ligand forms hydrogen bonds with the side chains of His64 and Gln67, while the aromatic ring interacts with His94 and Trp5. The sulfonamide group is water exposed and most likely is able to form hydrogen bonds with water molecules. The two propyl substituents point towards the hydrophobic parts of Gln67 and Leu91. The chlorine substitution at the ortho position seems to be favorable. We suggest that this electronegative chlorine atom interacts with the side chain of His94 as observed in the second docked pose. Replacement of this chlorine substituent with a methyl group or changing it to a meta or para position (see previous series20) 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 no direct interactions with the Zn2+-ion or the zincbound water molecule could be formed except for compounds 9 and 10. An interaction may occur between the nitrogen atom of the pyridine and the Zn2+-ion. Compound 9 could form a

hydrogen bond with the side chain of Lys67, while the sulfonamide of compound 10 and the side chain of Thr91 could form a hydrogen bond (Figures 1D and 1E). While not observed in the docking poses, the side chains of Asn62 and Thr200 may adjust their conformation to form an additional hydrogen bond to the sulfonamide of compounds 9 and 10, respectively. Compound 5 does not form an interaction with the Zn2+-ion and most likely a water molecule is able to bind to the zinc (Figure 1F). The piperidine group is probably positively charged at physiological pH values and could form cation-π interactions with His94 (shortest distance: 3.9 Å) and hydrophobic interactions with the side chain of Thr200. One of the sulfonamide oxygen atoms forms a hydrogen bond to the side chain of Thr91. Side chain reorientations of Gln92 and Ser132 may result in additional hydrogen bonds to the amide bond and the other sulfonamide oxygen, respectively. In our previous series, we synthesized and tested an analog of compound 5 in which the piperidine ring was replaced by a morpholine ring.20 The oxygen atom of the latter compound formed a hydrogen bond to the zinc-ion and the measured Ki value was 15.3 nM.20 In our new series we have another analog with a morpholine moiety but with a longer spacer (compound 4) that is not favorable and therefore a higher Ki value. For the other compounds, the obtained binding poses for hCA XII are different compared to hCA IX due to divergences in the binding pockets of both enzymes (hCA IX: Ser20, Gln67, Leu91; hCA XII: Tyr20, Lys67 and Thr91). Especially, the large Tyr20 located close to Trp5 makes it difficult for compounds 7, 8 and 11 to adopt similar binding poses as found in hCA IX. 5. Conclusion In the present paper we extended the scaffold of Probenecid analogues previously investigated including new substituents via amidic bond to explore the chemical space at this position. A small library of new amide derivatives of probenecid were easily synthesized and evaluated as potent and selective inhibitors of the human transmembrane carbonic anhydrase isoforms (hCA IX and XII). Compounds 1-12 showed an inversion of selectivity against CA I and II compared to the parent drug Probenecid which was not able to inhibit isoform I and was mildly active against isoform II. Docking studies of the most active compounds suggested enzyme recognition pattern for the design of novel selective hCA isoform inhibitors 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 boiling in 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 used directly as supplied by Sigma Aldrich® Italy. All reactions involving air- or moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. Column chromatography was carried out using Sigma-Aldrich® silica gel (high purity grade, pore size 60 Å, 200-425 mesh particle size). Analytical thin-layer

chromatography was carried out on Sigma-Aldrich® silica gel on TLA aluminum foils with fluorescent indicator 254 nm. Visualization was carried out under ultra-violet irradiation (254 nm). NMR spectra were recorded on a Bruker AV400 (1H: 400 MHz, 13 C: 101 MHz). Chemical shifts are quoted in ppm, based on appearance rather than interpretation, and are referenced to the residual non deuterated solvent peak. The assignment of exchangeable protons (NH) was confirmed by the addition of D2O. Infra-red spectra were recorded on a Bruker Tensor 27 FTIR spectrometer equipped with an attenuated total reflectance attachment with internal calibration. Absorption maxima (νmax ) are reported in wavenumbers (cm-1). Elemental analyses for C, H, and N were recorded on a Perkin-Elmer 240 B microanalyzer and the analytical results were within ± 0.4% of the theoretical values for all compounds (Supporting Information). All melting points were measured on a Stuart® melting point apparatus SMP1, and are uncorrected. Temperatures are reported in °C. Where given, systematic compound names are those generated by ChemBioDraw Ultra® 12.0 following IUPAC conventions. 6.2. Chemistry General procedure for the synthesis of derivatives 1-11: 1,1’carbonyldiimidazole (CDI, 1.5 eq) and the appropriate amine (1.5 eq) were added to a stirring solution of probenecid (1.0 eq) in dry N,N-dimethylformamide. The reaction was stirred at room temperature for 4-24 h. Eventually, an additional equivalent of CDI was added. The mixture was diluted with dichloromethane, washed with saturated NaHCO3, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Procedure for the synthesis of compound 12: Probenecid (1.0 eq) was dissolved in dichloromethane and thionyl chloride was added dropwise. 1 drop of dry N,N-dimethylformamide was added and the reaction refluxed for 24 h. In a separate flask, 4-nitroaniline was suspended in dry dichloromethane and triethylamine (2.75 eq) and was added under nitrogen atmosphere. The solution of Probenecid chloride in dry dichloromethane was added to the solution of 4-nitroaniline and the reaction stirred for 24 h at 50 °C. The reaction was evaporated in vacuo. Purification via column chromatography on silica gel afforded title compounds in discrete yields (Table 1). 4 - (N, N- Di p r opyl sulf a m oyl )-N- et h yl b enz a mi de (1 ): 1,1’-carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 eq) and 1.31 mL of a 2M solution of ethylamine in THF (2.63 mmol, 1.5 eq) were added to a stirring solution of probenecid (1 g, 3.50 mmol, 1.0 eq) in 10.0 mL of dry N,N-dimethylformamide at room temperature. After 3 hours the reaction was diluted with dichloromethane (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 chromatography on silica gel (ethyl acetate:n-hexane 2:1) gave title compound as a white solid (0.64 g, 59% yield); mp 93-95 °C; IR νmax 3315 (ν N-H), 2966 (ν Csp2-H), 1633 (ν C=O), 1343 (νas S=O), 1161 (νs S=O), 865 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, CDCl 3) δ 0.87 (6H, t, J = 7.2 Hz, 2 x CH3), 1.27 (3H, t, J = 9.2 Hz, CH3), 1.54 (4H, sext, J = 7.6 Hz, 2 x CH2), 3.08 (4H, t, J = 7.6 Hz, 2 x CH2), 3.49-3.53 (2H, m, NHCH2), 6.50 (1H, bs, NH), 7.80 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 7.87 (2H, d, J = 8.4 Hz, 2 x CH-Ar); 13C-NMR (101 MHz, CDCl3) δ 11.1 (CH3), 14.7 (CH2), 21.9 (CH2), 35.2 (CH3), 49.9 (CH2), 127.2 (Ar), 127.7 (Ar), 138.4 (Ar), 142.5 (Ar), 166.2 (C=O).

Table 1. Inhibitory activity of derivatives 1-12 and reference compounds (Probenecid and Acetazolamide) against four selected hCA isoforms by stopped-flow CO2 hydrase assay. O

O CDI or SOCl2

OH N

H2N R

+

S O O

N H

N DMF or DCM rt or 50 °C

R

S O O

Probenecid

Compound

R

1

CH2CH3

hCA I

Ki (nM) hCA II hCA IX

hCA XII

> 10 000

> 10 000

151

965

2

> 10 000

> 10 000

188

798

3

> 10 000

> 10 000

2024

256

4

> 10 000

> 10 000

213

419

> 10 000

> 10 000

1256

66.0

> 10 000

> 10 000

110

300

7

> 10 000

> 10 000

20.6

70.7

8

> 10 000

> 10 000

61.3

105

9

> 10 000

> 10 000

199

9.9

10

> 10 000

> 10 000

2037

9.9

11

> 10 000

> 10 000

23.6

34.7

12

> 10 000

> 10 000

246

206

Probenecid

> 10 000

431

360

1245

Acetazolamide (AAZ)

250

12

25

5.7

5

6

N

N

4 - (N, N- di p ro py ls ulf am o yl )-N - (3m et h yl cy cl oh exyl ) ben z ami d e (2): 1,1’carbonyldiimidazole (1.43 g, 8.83 mmol, 1.5 eq) and 1.0 g of 3-methylcyclohexylamine (8.83 mmol, 1.5 eq) were added to a

stirring solution of probenecid (1.0 g, 5.89 mmol, 1.0 eq) in 10.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane (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 chromatography on silica gel (petroleum ether:ethyl acetate 4:1) gave the title compound as a white solid (0.52 g, 24% yield); mp 130-133 °C; IR νmax 3280 (ν N-H), 2927 (ν Csp2 -H), 1626 (ν C=O), 1339 (νas S=O), 1161 (νs S=O) cm-1 ; 1 H-NMR (400 MHz, CDCl3) δ 0.88 (6H, t, J = 7.2 Hz, 2 x CH3), 0.94 (3H, d, J = 6.4 Hz, CH3), 1.11-1.14 (1H, m, cy), 1.39-1.83 (10H, m, 2 x CH2 + cy), 2.07-2.09 (2H, m, cy), 3.09 (4H, m, 2 x CH2), 3.97-3.99 (1H, m, cy), 6.10 (1H, bs, NH), 7.84 (4H, bs, CH-Ar); 13C-NMR (101 MHz, CDCl3) δ 11.2 (CH3), 21.9 (CH3 ), 22.4 (CH2), 24.8 (cy), 31.8 (cy), 32.8 (cy), 34.2 (cy), 41.9 (cy), 49.4 (CH2), 49.9 (cy), 127.2 (Ar), 127.6 (Ar), 138.6 (Ar), 142.6 (Ar), 165.4 (C=O). 4 - (N, N- di p ro py ls ulf am o yl )-N - (4m et h yl cy cl oh exyl ) ben z ami d e (3): 1,1’carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 eq) and 0.6 g of 4-methylcyclohexylamine (5.25 mmol, 1.5 eq) were added to a stirring solution of probenecid (1.0 g, 3.50 mmol, 1.0 eq) in 10.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane (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 chromatography on silica gel (petroleum ether:ethyl acetate 4:1) gave the title compound as a white solid (0.26 g, 20% yield); mp 108-110 °C; IR νmax 3330 (ν N-H), 2946 (ν Csp2 -H), 1631 (ν C=O), 1340 (νas S=O), 1161 (νs S=O) cm-1 ; 1 H-NMR (400 MHz, CDCl3) δ 0.88 (6H, t, J = 7.2 Hz, 2 x CH3), 0.94 (3H, d, J = 6.4 Hz, CH3), 1.11-1.28 (1H, m, cy), 1.53-80 (10H, m, 2 x CH2 + cy), 2.08-2.11 (2H, m, cy), 3.073.11 (4H, m, 2 x CH2), 3.87-3.90 (1H, m, cy), 6.02 (1H, bs, NH), 7.85-7.87 (4H, m, CH-Ar); 13C-NMR (101 MHz, CDCl 3) δ 11.2 (CH3), 21.9 (CH3), 22.1 (CH2), 29.2 (cy), 30.1 (cy), 32.0 (cy), 33.1 (cy), 33.8 (cy), 49.3 (CH2), 49.9 (cy), 127.2 (Ar), 127.6 (Ar), 131.8 (Ar), 142.8 (Ar), 166.9 (C=O). 4 - (N, N- di p ro py ls ulf am o yl )-N - (3m o r ph oli no pr o pyl ) b enz am i de (4 ): 1,1’carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 eq) and 0.77 ml of N-3-(aminopropyl)morpholine (5.25 mmol, 1.5 eq) were added to a stirring solution of probenecid (1.0 g, 3.50 mmol, 1.0 eq) in 10.0 mL of dry N,N-dimethylformamide at room temperature. After four hours the reaction was diluted with dichloromethane (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 chromatography on silica gel (ethyl acetate:hexane 15:1) gave title compound as a white solid (0.91 g, 63% yield); mp 92-94 °C; IR νmax 3355 (ν N-H), 2967 (ν Csp2-H), 1634 (ν C=O), 1347 (νas S=O), 1114 (νs S=O), 739 (δ Csp2 -H) cm-1 ; 1HNMR (400 MHz, CDCl3) δ 0.88 (6H, s, 2 x CH3), 1.55 (4H, s, 2 x CH2 ), 1.81-1.82 (2H, m, 2 x CH2), 2.51-2.57 (2H, m, CH2), 3.11 (4H, s, 2 x CH2), 3.60-3.69 (6H, m, CH2), 7.88-7.91 (4H, m, 4 x CH-Ar), 8.20 (1H, bs, NH); 13C-NMR (101 MHz, CDCl3) δ 11.2 (CH3), 21.9 (CH2), 23.9 (CH2), 40.7 (CH2), 49.8 (CH2), 53.8 (CH2), 58.6 (cy), 67.0 (cy), 127.2 (Ar), 127.6 (Ar), 138.4 (Ar), 142.6 (Ar), 165.9 (C=O). 4 - (N, N- di p ro py ls ulf am o yl )-N - (2- (1 p i p er id i nyl )et h yl ) b en z ami d e (5) : 1,1’carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 eq) and 0.75 ml of 1-(2-aminoethyl)piperidine (5.25 mmol, 1.5 eq) were added to a stirring solution of probenecid (1.0 g, 3.50 mmol, 1.0 eq) in 10.0 mL of dry N,N-dimethylformamide at room temperature. After one day the reaction was diluted with

dichloromethane (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 chromatography on silica gel (ethyl acetate:petroleum ether 5:1) gave title compound as a yellow oil (1.02 g, 73% yield); IR νmax 3323 (ν N-H), 2934 (ν Csp2-H), 1644 (ν C=O), 1337 (νas S=O), 1148 (νs S=O), 750 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, CDCl3) δ 0.83 (6H, bs, 2 x CH3), 1.44-1.56 (10H, m, CH2), 2.41-2.53 (6H, m, CH2), 3.05 (4H, bs, CH2), 3.51 (2H, bs, CH2), 7.37 (1H, bs, NH), 7.81-7.90 (4H, m, CH-Ar); 13 C-NMR (101 MHz, CDCl 3) δ 11.1 (CH3), 21.9 (CH2), 24.2 (CH2), 25.9 (CH2), 36.6 (cy), 49.9 (CH2), 54.2 (cy), 57.1 (cy), 127.1 (Ar), 127.7 (Ar), 138.1 (Ar), 142.5 (Ar), 165.9 (C=O). 4 - (N, N- di pr o py ls ul f am o yl )-N - ((2- (N,N d i et hyl )am i no )et h yl ) ben za m i d e (6 ): 1,1’carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 eq) and 0.74 ml of N,N-diethylethylendiamine (5.25 mmol, 1.5 eq) were added to a stirring solution of probenecid (1.0 g, 3.50 mmol, 1.0 eq) in 10.0 mL of dry N,N-dimethylformamide at room temperature. After one day the reaction was diluted with dichloromethane (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 chromatography on silica gel (ethyl acetate:hexane 5:1) gave title compound as a yellow oil (0.95 g, 71% yield); IR νmax 3334 (ν N-H), 2967 (ν Csp2-H), 1645 (ν C=O), 1336 (νas S=O), 1148 (νs S=O), 739 (δ Csp2 -H) cm-1 ; 1 H-NMR (400 MHz, CDCl 3) δ 0.78 (6H, t, J = 7.4 Hz, 2 x CH3), 0.96 (6H, t, J = 7.2 Hz, 2 x CH3), 1.53 (4H, sext, J = 7.6 Hz, 2 x CH2), 1.46 (4H, sext, J = 7.4 Hz, 2 x CH2), 2.50 (4H, quad, J = 7.6, 2 x CH2), 2.59 (2H, t, J = 6.0 Hz, CH2), 3.00 (4H, t, J = 7.6 Hz, 2 x 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) δ 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). 4 - (N, N- di pr o py ls ul f am o yl )-N - (benz yl b en z ami d e) (7 ) : 1,1’-carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 eq) and 0.56 g of benzylamine (5.25 mmol, 1.5 eq) were added to a stirring solution of probenecid (1.0 g, 3.50 mmol, 1.0 eq) in 10.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane (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 chromatography on silica gel (hexane:ethyl acetate 1:2) gave the title compound as a white solid (0.89 g, 68% yield); mp 108-110 °C; IR νmax 3340 (ν N-H), 2966 (ν Csp2 -H), 1642 (ν C=O), 1338 (νas S=O), 1158 (νs S=O), 856 (δ Csp2 -H), cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.80 (6H, t, J = 7.4 Hz, 2 x CH3), 1.46 (4H, sext, J = 7.4 Hz, 2 x CH2), 3.04 (4H, t, J = 7.4 Hz, 2 x CH2), 4.50 (2H, d, J = 6 Hz, NHCH2), 7.25 (1H, m, CH-Ar), 7.33 (4H, m, CH-Ar), 7.90 (2H, d, J = 8.4 Hz, CHAr), 8.07 (2H, d, J = 8.0 Hz, CH-Ar), 9.30 (1H, bs, NH); 13CNMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.0 (2 x CH2), 43.2 (2 x CH2), 50.1 (NH-CH2) 127.3 (Ar), 127.7 (Ar), 128.7 (Ar), 128.8 (Ar), 138.3 (Ar), 139.7 (Ar) 142.2 (Ar), 165.6 (C=O). 4 - (N, N- di pr o py ls ul f am o yl )-N - (2- m et hyl b enz am i de) (8 ) : 1,1’-carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 eq) and 0.56 g of 2-methylaniline (5.25 mmol, 1.5 eq) were added to a stirring solution of probenecid (1.0 g, 3.50 mmol, 1.0 eq) in 10.0 mL of dry N,N-dimethylformamide at 50 °C. The reaction was stirred overnight, diluted with dichloromethane (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 chromatography on silica gel (hexane:ethyl acetate 1:5) gave title compound as a white solid (0.27 g, 21% yield); mp 198-200 °C; IR νmax 3302 (ν N-H), 2963 (ν Csp2-H), 1693 (ν C=O), 1343 (νas S=O), 1155 (νs S=O), 852 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.80 (6H, t, J = 7.2 Hz, 2 x CH3), 1.47 (4H, sext, J = 7.2 Hz, 2 x CH2), 2.50 (3H, s, CH3), 3.05 (4H, t, J = 7.4 Hz, 2 x CH2), 7.11-8.13 (8H, m, CH-Ar), 8.24 (1H, bs, NH); 13CNMR (101 MHz, DMSO-d6 ) δ 11.4 (CH3), 18.5 (CH2), 22.0 (CH2), 50.1 (CH3), 121.9 (Ar), 123.1 (Ar), 126.5 (Ar), 127.5 (Ar), 128.2 (Ar), 130.6 (Ar), 137.9 (Ar), 163.4 (C=O). 4 - (N, N- di p ro py ls ulf am o yl )-N - (pyri di n- 3- yl ) b enz a m i de (9 ): 1,1’-carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 eq) and 0.49 g of 3-aminopyridine (5.25 mmol, 1.5 eq) were added to a stirring solution of probenecid (1.0 g, 3.50 mmol, 1.0 eq) in 10.0 mL of dry N,N-dimethylformamide at 50 °C. The reaction was stirred overnight. The reaction was diluted with dichloromethane (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 chromatography on silica gel (hexane:ethyl acetate 1:5) gave the title compound as a white solid (0.62 g, 49% yield); mp 134-136 °C; IR νmax 3323 (ν NH), 2967 (ν Csp2-H), 1671 (ν C=O), 1329 (νas S=O), 1167 (νs S=O), 859 (ν C=N), 778 (δ Csp2 -H) cm-1; 1H-NMR (400 MHz, CDCl3) δ 0.86 (6H, t, J = 7.4 Hz, 2 x CH3), 1.53 (4H, sext, J = 7.4 Hz, 2 x CH2), 3.07 (4H, t, J = 7.6 Hz, 2 x CH2), 7.31-7.34 (1H, m, CH-Ar), 7.72 (2H, d, J = 8.4 Hz, CH-Ar), 7.96 (2H, t, J = 8.4 Hz, CH-Ar), 8.30 (1H, d, J = 8.4 Hz, CH-Ar), 8.36 (1H, d, J = 4.4 Hz, CH-Ar), 8.81 (1H, d, J = 2.4 Hz, CH-Ar), 9.24 (1H, bs, NH); 13C-NMR (101 MHz, CDCl3) δ 11.1 (CH3), 21.8 (CH2), 49.9 (CH2), 123.8 (Ar), 127.1 (Ar), 127.8 (Ar), 128.3 (Ar), 135.1 (Ar), 138.3 (Ar), 141.7 (Ar), 142.7 (Ar), 145.4 (Ar), 165.5 (C=O). 4 - (N, N- di p ro py ls ulf am o yl )-N - (pyri di n- 4- yl ) b enz a m i de (1 0): 1,1’-carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 eq) and 0.49 g of 4-aminopyridine (5.25 mmol, 1.5 eq) were added to a stirring solution of probenecid (1.0 g, 3.50 mmol, 1.0 eq) in 10.0 mL of dry N,N-dimethylformamide at 50 °C. The reaction was stirred overnight. The reaction was diluted with dichloromethane (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 chromatography on silica gel (hexane:ethyl acetate 1:5) gave the title compound as a white solid (0.61 g, 48% yield); mp 131-136 °C; IR νmax 3364 (ν NH), 2963 (ν Csp2-H), 1690 (ν C=O), 1326 (νas S=O), 1165 (νs S=O), 857 (ν C=N), 742 (δ Csp2 -H) cm-1; 1H-NMR (400 MHz, CDCl3) δ 0.87 (6H, t, J = 7.4 Hz, 2 x CH3), 1.55 (4H, sext, J = 7.4 Hz, 2 x CH2 ), 3.08 (4H, t, J = 7.6 Hz, 2 x CH2), 7.69 (2H, d, J = 8.4 Hz, CH-Ar), 7.75 (2H, d, J = 6.4 Hz, CH-Ar), 7.93 (2H, d, J = 8.4 Hz, CH-Ar), 8.54 (2H, d, J = 6.4 Hz, CH-Ar), 9.29 (1H, bs, NH); 13C-NMR (101 MHz, CDCl3) δ 11.1 (CH3), 21.9 (CH2), 49.9 (CH2), 114.2 (Ar), 127.2 (Ar), 128.4 (Ar), 138.2 (Ar), 142.8 (Ar), 145.7 (Ar), 150.2 (Ar), 165.7 (C=O). N -(2 -c hl o r op hen yl )- 4- (N, Nd i p ro pyl s ulf am o yl )ben za mi d e (11 ) : 1,1’carbonyldiimidazole (0.85 g, 5.25 mmol, 1.5 eq) and 0.67 g of 2-chloroaniline (5.25 mmol, 1.5 eq) were added to a stirring solution of probenecid (1.0 g, 3.50 mmol, 1.0 eq) in 10.0 mL of dry N,N-dimethylformamide at 50 °C. The reaction was stirred overnight, diluted with dichloromethane (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 chromatography on silica gel (hexane:ethyl acetate 4:1) gave title compound as a white solid (0.73 g, 53% yield); mp 114-116 °C; IR νmax 3288 (ν N-H), 2961 (ν Csp2 -H), 1680 (ν C=O), 1341 (νas S=O), 1156 (νs S=O), 857 (δ Csp2-H) cm-1 ; 1H-NMR (400 MHz, DMSO-d6) δ 0.83 (6H, t, J = 7.2 Hz, 2 x CH3 ), 1.49 (4H, sext, J = 7.2 Hz, 2 x CH2), 3.07 (4H, t, J = 7.6 Hz, 2 x CH2), 7.31-7.34 (2H, m, CHAr), 7.35-7.43 (2H, m, CH-Ar), 7.96 (2H, d, J = 8.4 Hz, CHAr), 8.16 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 10.35 (1H, s, NH); 13 C-NMR (101 MHz, DMSO-d6) δ 11.4 (CH3), 22.1 (CH2), 50.1 (CH2), 127.4 (Ar), 128.0 (Ar), 129.1 (Ar), 130.1 (Ar), 135.3 (Ar), 137.8 (Ar), 142.7 (Ar), 164.5 (C=O). 4 - (N, N- di pr o py ls ulf am o yl )-N - (4- ni tr op hen yl ) b enz am i d e (12 ) : 1.00 g of probenecid (3.50 mmol, 1.0 eq) was dissolved in 15 mL of dichloromethane. 0.63 mL of thionyl chloride was added dropwise. 1 drop of dry N,Ndimethylformamide was added and the reaction refluxed for 24 h. In a separate flask, 4-nitroaniline was suspended in 10 mL of dry dichloromethane. 0.7 mL of triethylamine (9.6 mmol, 2.75 eq) was added under nitrogen atmosphere. The solution of probenecid chloride (solution 1) in dry dichloromethane was added to the solution of 4-nitroaniline and the reaction stirred for 24 h at 50 °C. The reaction was evaporated in vacuo and the residue purified on silica gel (hexane:ethyl acetate 4:1) to give title compound as a yellow solid (1.77 g, 83% yield); mp 61-64 °C; IR νmax 3390 (ν N-H), 2975 (ν Csp2-H), 1696 (ν C=O), 1505 (ν N-O), 1335 (νas S=O), 1247 (ν N-O), 1149 (νs S=O), 740 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, CDCl3) δ 0.90 (6H, t, J = 7.2 Hz, 2 x CH3 ), 1.57 (4H, sext, J = 7.2 Hz, 2 x CH2), 3.11 (4H, t, J = 7.6 Hz, 2 x 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 x CH-Ar), 8.88 (1H, bs, NH); 13C-NMR (101 MHz, CDCl 3) δ 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 been used for assaying the CA catalyzed CO2 hydration activity. Phenol red (0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5, for α-CAs) as buffer, and 20 mM NaClO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed 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 510% 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 (1 µM) were prepared in distilleddeionized water and dilutions up to 0.1 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 or for the eventual active site mediated hydrolysis of the inhibitor. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the ChengPrusoff equation,24 and represent the average from at least three different determinations. All recombinant CA isoforms were obtained in-house as previously reported.25,26 6.4. Molecular modelling studies

6 . 4. 1. Pr epa ra ti o n of Pro b en eci d an al og s tr u ct u r es The Probenecid analogs 1-12 were prepared in 3D with the MOE software package (v2013.08.02, Chemical Computing Group Inc., Montreal, Canada). All possible structural isomers of compounds 2 and 3 were constructed. Strong acids were deprotonated and strong bases were protonated. Finally, the ligands were energy minimized using a steepest-descent protocol (MMFF94x force field). 6 . 4. 2. Pr epa ra ti o n of h C A c ry st al st r uct ur es f or d o ck in g st u di es The 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, the active site zinc ions and the zinc-bound water molecule of hCA II were retained and all other atoms were omitted. The remaining structure was protonated using the MOE software package and subsequently the obtained structure was energy-minimized (AMBER99 force field). Finally, the obtained protein models were superposed on the hCA I structure using the backbone Cαatoms. The zinc-bound water molecule of hCA II coordinated well to the zinc ions of the other hCAs. 6 . 4. 3. D oc ki ng st udi es The GOLD Suite software package (v5.2, CCDC, Cambridge, UK) and the GoldScore scoring function were used to dock the Probenecid analogs into the hCA structures with and without the zinc-bound water molecule (25 dockings per ligand). The binding pocket was defined as all residues within 13 Å of a centroid (x: -17.071, y: 35.081, 43.681; corresponding approximately to the position of the thiadiazole ring of acetazolamide in the 1JD0 structure).

11. 12.

13.

14. 15.

16.

17. 18. 19. 20. 21.

Acknowledgments This work was financed by two FP7 EU projects (Metoxia and Dynano) to CTS.

22. 23.

References and notes

24. 25.

1. 2.

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A

B Q67

N62

N62 H96 Q67

Q92 Q92

H64

H64 W5

W5

H94

L91

H119

H96

L91 H119 T200

T199

T200 P202

P202

T199

S20

S20

C

N62

D

H64

N62 W5 Q67 Q92

K67

H64 H94

T200

W5

L91

H96

H94 T20

Q92

H96

H119

T200

H119

T199

T199

S132

P202

T91 S20

F

E N62

N62 H64

H64

H96 K67

W5

W5

H94

K67

T199 H96

H119

H94 T199 Q92

T200

Q92 T200

T20 T20

H119 T91 S135 T91 S132

S132

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 blue sphere, 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 and enzyme 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 dashed red lines.

Graphical Abstract

New amide derivatives of Probenecid as selective inhibitors of carbonic anhydrase IX and XII: biological evaluation and molecular modelling studies Simone Carradori, Adriano Mollica, Mariangela Ceruso, Melissa D’Ascenzio, Celeste De Monte, Paola Chimenti, Rocchina Sabia, Atilla Akdemir, Claudiu T. Supuran O

O OH N S O O Probenecid

1) CDI or SOCl2 2) RNH2

N

N H

R

S O O 20.6 nM < K i CA IX < 2037 nM 9.9 nM < K i CA XII < 965 nM