Accepted Manuscript Selective inhibition of human carbonic anhydrases by novel amide derivatives of probenecid: synthesis, biological evaluation and molecular modelling studies Melissa D’Ascenzio, Simone Carradori, Daniela Secci, Daniela Vullo, Mariangela Ceruso, Atilla Akdemir, Claudiu T. Supuran PII: DOI: Reference:
S0968-0896(14)00446-5 http://dx.doi.org/10.1016/j.bmc.2014.06.003 BMC 11637
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
1 April 2014 31 May 2014 2 June 2014
Please cite this article as: D’Ascenzio, M., Carradori, S., Secci, D., Vullo, D., Ceruso, M., Akdemir, A., Supuran, C.T., Selective inhibition of human carbonic anhydrases by novel amide derivatives of probenecid: synthesis, biological evaluation and molecular modelling studies, Bioorganic & Medicinal Chemistry (2014), doi: http:// dx.doi.org/10.1016/j.bmc.2014.06.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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
Selective inhibition of human carbonic anhydrases by novel amide derivatives of probenecid: synthesis, biological evaluation and molecular modelling studies Melissa D’Ascenzioa,∗, Simone Carradoria, Daniela Seccia, Daniela Vullob, Mariangela Cerusob, Atilla Akdemird, Claudiu T. Supuranb,c,* a
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy. c Università degli Studi di Firenze, Neurofarba Dept., Section of Pharmaceutical and Nutriceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy. d Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Vatan Caddesi, 34093 Fatih, Istanbul, Turkey. 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, a well-known uricosuric agent, were synthesized and evaluated as inhibitors of human carbonic anhydrases (hCAs, EC 4.2.1.1). The transmembrane isoforms (hCA IX and XII) were potently and selectively inhibited by some of them. The proposed chemical modification led to a complete loss of hCA II inhibition (Ki s>10000 nM) and enhaced the inhibitory activity against the tumour-associated hCA XII (compound 4 showed a Ki value of 15.3 nM). The enzyme inhibitory data have also been validated by docking studies of the compounds within the active site of hCA XII.
Keywords: Probenecid Selective carbonic anhydrase IX inhibitors Selective carbonic anhydrase XII inhibitors Tertiary sulfonamides
1. Introduction Carbonic anhydrases (CA, EC 4.2.1.1) are a widely spread superfamily of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. 1 To date, fifteen human isoforms (hCA I-XV) have been isolated and classified on the basis of their cellular localization, tissue distribution, kinetic properties, and sensitivity to different classes of inhibitors.2,3 They are involved in a wide range of physiological processes and the selective inhibition of specific isoforms has been proved to be effective in the treatment of several pathological conditions such as hypertension, epilepsy, altitude sickness, glaucoma, obesity, osteoporosis, gastric and duodenal ulcers.4–8 More recently, it has been discovered that two CA transmembrane isoforms (CA IX and XII) are overexpressed in hypoxic tumors as part of the complex machinery that cancer cell use to compensate the changes in pH, nutrient and oxygen levels which characterize the microenvironment of solid tumors.9–13 As these adaptive changes are believed to contribute to the development of invasive and metastatic phenotypes, 14–16 an extensive effort has been made in the last few years to disrupt such pathological mechanisms by selectively targeting these two pH regulating enzymes: CA IX and XII. 17–23
———
2009 Elsevier Ltd. All rights reserved.
The zinc binding sulfonamide moiety has been widely used in the past to design carbonic anhydrase inhibitors.24–26 Although it is believed that the key interaction between these compounds and the active site of CAs is mediated by the formation of a coordinative bond between the positively charged zinc and the deprotonated sulfonamide,27,28 there is growing evidence that even non-ionizable tertiary substituted sulfonamides can efficiently inhibit different CA isoforms. 29–31 In particular, researchers have recently found that various fluorinated/non-fluorinated tertiary substituted benzenesulfonamides and other atypical chelating compounds were able to selectively inhibit one of the cancer related isoforms of CA (CA IX) in the low nanomolar range (Chart 1).30,31
Chart 1. Rational design of Probenecid derivatives
Corresponding authors: Dr. Melissa D’Ascenzio: Tel/Fax: +39 06 49693242; e-mail: [email protected]; Prof. Claudiu T. Supuran: Tel: 39-055-4573005; Fax: 39-055-4573385; e-mail: [email protected]. ∗
The similarity of these compounds to one of the best known uricosuric agents (probenecid), whose ability to interfere with CA catalytic activity was preliminary reported in the past,32 inspired the synthesis of a small series of probenecid derivatives which are reported herein. We wanted to explore whether derivatization of the COOH moiety of probenecid to amides has a consequence of the interaction of these compounds with the CA active site. 2. Chemistry Derivatives 1-10 were synthesized by reacting 4-(N,N-dipropylsulfamoyl)benzoic acid (probenecid) with the corresponding amine in the presence of 1,1’-carbonyldiimidazole (CDI) in N,N-dimethylformamide at room temperature (Table 1). Purification via column chromatography on silica gel afforded title compounds in satisfying yields. Compound 11 was obtained by treating probenecid with thionyl chloride in dichloromethane and then reacting the obtained acyl chloride with the strongly deactivated 2-nitroaniline. The reduction of compound 11 using hydrogen and palladium on activated carbon gave thereafter derivative 12 in high yield. All synthesized compounds were fully characterized by analytical and spectral data (see Section 7.2 for details). 3. Results and Discussion All the synthesized compounds (1-12) were tested against the two cancer-related isoforms of CA (CA IX and XII) and their corresponding off-targets (CA I and II) in order to evaluate their biological activity and selectivity. 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 for the determination of the kinetic parameters and inhibition constants ranged from 1.7 to 17 mM. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (1 µM) were prepared in distilled-deionized 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 non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation,33 and represent the mean from at least three different determinations. All recombinant CA isoforms were obtained in-house as previously reported. On the basis of the obtained results, SAR for this class of probenecid analogs as promising hCA inhibitors can be extrapolated as follows: (i) All tested compounds proved to be inactive against the ubiquitously expressed isoform of hCA II at concentrations higher than 10 µM. Conversely, no selectivity was observed against hCA I. (ii) All screened compounds showed to inhibit hCA IX and hCA XII in the nanomolar range (165 nM < Ki hCA IX < 265 nM and 15.3 nM < Ki hCA XII < 430 nM).
(iii) In general, we found that the conversion of the carboxylic group of probenecid into its corresponding amide resulted in a significative gain of activity of the obtained derivatives against the tumor-associated isoform CA XII, with Ki values dropping down from 1245 nM to 15.3 nM (compare probenecid and compound 4 in Table 1). Similarly, the activity against CA IX was maintained throughout the series, if not slightly increased, in both alicyclic and (hetero)aromatic derivatives. On the other hand, compounds 1-12 showed an inversion of selectivity against CA I and II if compared to the parent drug: while probenecid was not able to inhibit isoform I and was mildly active against isoform II, the newly synthesized amides show a consistent lack of activity against CA II and an ability to inhibit CA I in the nanomolar range (307 nM < Ki hCA I < 1980 nM). The introduction of a renowned zinc binding moiety (o-phenylendiamine) on derivative 12 did not change the pattern of inhibition or the potency of the obtained compound, thus supporting the hypothesis that these inhibitors could display a new binding mode which does not include a direct interaction with the metal ion. 4. Docking studies into hCA XII Lastly, docking studies were performed in order to better understand the enzyme-inhibitor recognition pattern and the isoform selectivity which characterize these probenecid analogs. Derivatives 1–12 are characterized by Ki values at least 3-fold lower than probenecid, with compounds 3 (32-fold) and 4 (81fold) showing the lowest Ki values (Table 1). The high number of interactions that compound 4 establishes within the active site of hCA XII might explain the low Ki value observed for this compound (Figure 1A). This compound has a morpholine ring with an alkaline nitrogen which is most likely partly protonated at physiological pH values and can form a cation-π adduct with His94. The oxygen atom of the morpholine ring directly interacts with the catalytic zinc ion although no Hbonds with Thr199 are observed. The oxygen and nitrogen atoms of the amide bond do not form any interactions with the enzyme and are located close to the solvent. The phenyl group of the ligand is believed form cation-π interactions with Lys67. One of the sulfonamide oxygens is hydrogen bonded to Ser132, while the other oxygen and the non-protonated tertiary nitrogen atom point out of the catalytic site, towards the protein surface. When the morpholine nucleus is replaced by a cyclohexyl ring (compound 2) neither binding to the zinc ion nor cation-π interactions with His94 are possible. Although the addition of a methyl substituent to the 2-position of the cyclohexyl moiety (compound 3) lowers the Ki value of about 10-folds, none of the enantiomers of compound 3 interact with the zinc ion. Nevertheless, the S-isomer forms various interactions within the active site of the enzyme (Figure 1B). In particular, both the 2methylcyclohexyl and the phenyl group are involved in hydrophobic stacking interactions with His94 and Trp5, respectively. The oxygen atoms of the sulfonamide moiety are hydrogen bonded to Trp5 and His64 side chain, while the nitrogen atom points out towards the solvent. This pose is not observed for compound 2 or for the R-isomer of compound 3. Compound 5, which has a phenyl ring in place of the cyclohexyl group of compounds 2 and 3, might interact with the zinc ion via the aromatic moiety (electrostatic interactions). The sulfonamide oxygen atoms form H-bonds to Thr91 and Gln92 (Figure 1C). Compounds 6-9, 11 and 12 are likely to form similar interactions within the active site of CA as shown for compound 5. In addition, the meta-nitro substituted aromatic ring
of compound 10 interacts with the backbone nitrogen of Thr199 and the zinc ion via the two oxygen atoms of the nitro group (Figure 1D). The phenyl group next to the sulfonamide moiety could form cation-π interactions with Lys67, while the sulfonamide oxygen and nitrogen atoms point towards the solvent. The ortho-nitro group of compound 11, the ortho-amine group of compound 12 and the chlorine atoms of compounds 8 and 9 are not able to directly interact with the zinc-ion and therefore, they are most likely to bind in a similar fashion to compound 5. This assumption seems to be consistent with the enzyme inhibition data which report similar Ki values for compounds 5-9, 11 and 12 and lower Ki values for compound 10. There is only a 4-fold difference in Ki values between probenecid and the closely related compound 1. Probenecid interacts with the zinc ion and the backbone of Thr199 via its carboxylic group and hydrogen bonds are formed between Asn62, Gln92 and the oxygen atoms of the sulfonamide moiety (Figure 1E). Finally, the phenyl group is involved in π-π stacking interactions with the imidazole side chain of His94. Similarly, compound 1 is allocated inside the active site of CA with a comparable orientation: one of its sulfonamide oxygens forms a hydrogen bond with Asn62 and its phenyl ring interacts with His94 via π-π stacking. However, its methylamide substituent points toward the side chains of Val143, Val207 and Trp209, and although its carbonyl group still interacts with the zinc ion, the interaction with Thr199 is lost (Figure 1F). The docking pose of compound 4 (Figure 1A) provides clues for obtaining derivatives with even higher affinity. The dipropylamine group points into the solvent and does not form any interactions with the hCA XII binding pocket. As such, this moiety does not contribute to binding interactions. In addition, only one of the sulfonamide oxygen groups forms a hydrogen bond (with Ser132). This group might be replaced by a –CH3OH group. Substitutions on this position could be explored to obtain higher affinity and probably also subtype-selectivity amongst the hCA isoforms. 5. Conclusion Novel amide derivatives of the well-known uricosuric agent probenecid were easily synthesized and evaluated as potent and selective inhibitors of the human carbonic anhydrase transmembrane isoforms (hCA IX and XII). The introduction of an alkyl/aryl amide portion led to a complete loss of hCA II inhibition (Ki > 10000 nM) and enhanced the inhibitory activity against hCA XII (see compound 4 with Ki value of 15.3 nM). Docking studies of the most active compounds explained the enzyme recognition pattern and highlighted the path towards the design of novel selective hCA isoform inhibitors. 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. 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 SigmaAldrich® 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, 13C: 101 MHz). Chemical shifts are quoted in ppm, based on appearance rather than interpretation, and are referenced to the residual non deuterated solvent peak. 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). 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 1-10: 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. After the reaction went to completion, the organics were diluted with dichloromethane, washed with saturated NaHCO3, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel gave title compounds in satisfying yields. 4 - (di pr op yl s ulf a m oyl )- N- m et hyl b enz am i de (1 ): 1,1’carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 1.31 mL of a 2M solution of methylamine in THF (2.63 mmol, 1.5 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. After four hours the reaction was diluted with dichloromethane and washed with saturated NaHCO3. 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 (0.21 g, 40% yield); mp 127-129 °C; IR νmax 3289 (ν N-H), 2960 (ν Csp2-H), 1639 (ν C=O), 1341 (νas S=O), 1163 (νs S=O), 856 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.81 (6H, t, J = 7.6 Hz, 2 x CH3), 1.46 (4H, sext, J = 7.6 Hz, 2 x CH2), 2.80 (3H, d, J = 4.4 Hz, CH3), 3.05 (4H, t, J = 7.6 Hz, 2 x CH2), 7.88 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 8.00 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 8.67 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.0 (2 x CH2), 26.8 (CH3 ), 50.0 (2 x CH2), 127.3 (2 x CH-Ar), 128.5 (2 x CH-Ar), 138.4 (C-Ar), 142.0 (C-Ar), 165.9 (C=O).
Table 1. Inhibitory activity of derivatives 1-12 and reference compounds (probenecid and acetazolamide) against selected hCA isoforms by stopped-flow CO2 hydrase assay.
Compound
R
hCA I
Ki (nM) hCA II hCA IX
hCA XII
1980
> 10 000
165
289
2
307
> 10 000
234
398
3
319
> 10 000
260
42.5
4
347
> 10 000
251
15.3
5
324
> 10 000
171
422
6
324
> 10 000
228
418
346
> 10 000
216
410
10
341
> 10 000
198
428
9
364
> 10 000
243
430
10
344
> 10 000
265
201
11
327
> 10 000
192
352
12
354
> 10 000
223
407
CH3
1
S
N
7 N
NH2
Probenecid
> 10 000
431
360
1245
Acetazolamide (AAZ)
250
12
25
5.7
N -cy cl oh exy l- 4-(N , N- di pr opy ls ulf a m o yl )b enz a mi de (2 ): 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.18 g of cyclohexylamine (2.62 mmol, 1.5 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 2:1) gave the title compound as a white solid (0.31 g, 53% yield); mp 127-129 °C; IR νmax 3291 (ν N-H), 2936 (ν Csp2-H), 1627 (ν C=O), 1330 (νas S=O), 1146 (νs S=O), 736 (δ Csp2 -H), cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.81 (6H, t, J = 7.6 Hz, 2 x CH3), 1.14 (1H, m, CH-cy), 1.29 (4H, m, 2 x CH2-cy), 1.46 (4H, sext, J = 7.2 Hz, 2 x CH2), 1.62 (1H, m, CH-cy), 1.75 (2H, m, 2 x CH-cy), 1.83 (2H, m, CH-cy), 3.04 (4H, t, J = 7.6 Hz, 2 x CH2), 3.78 (1H, m, CH-cy), 7.86 (2H, d, J = 6.8 Hz, 2 x CH-Ar), 8.00 (2H, d, J = 6.8 Hz, 2 x CH-Ar), 8.43 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.0 (2 x CH2), 25.3 (2 x CH2 -cy), 25.7 (CH2-cy), 32.8 (2 x CH2 cy), 49.0 (CH-cy), 50.0 (2 x CH2), 127.1 (2 x CH-Ar), 128.7 (2 x CH-Ar), 138.9 (C-Ar), 142.0 (C-Ar), 165.0 (C=O). 4 - (N, N- di p ro py ls ulf am o yl )-N - (2- m et h ylc y cl oh ex yl ) b enz a m i de (3 ): 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.30 g of 2-methylcyclohexylamine (2.62 mmol, 1.5 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 2:1) gave the title compound as yellow gum. Crystallization from n-hexane/ethyl acetate (1:1) gave title compound as a white solid (0.085 g, 13% yield); mp 93-94 °C; IR νmax 3315 (ν N-H), 2932 (ν Csp2-H), 1633 (ν C=O), 1332 (νas S=O), 1159 (νs S=O), 741 (δ Csp2-H) cm-1 ; 1 H-NMR (400 MHz, CDCl3) δ 0.86 (6H, t, J = 7.2 Hz, 2 x CH3), 0.98 (3H, d, J = 6.4 Hz, CH3), 1.16 (2H, m, CH2-cy), 1.39 (2H, m, 2 x CH-cy), 1.53 (4H, sext, J = 7.2 Hz, 2 x CH2), 1.76 (4H, m, 4 x CH-cy), 2.03 (1H, d, J = 10.8 Hz, CH-cy) 3.06 (4H, t, J = 7.6 Hz, 2 x CH2 ), 3.70 (1H, m, CH-cy), 6.17 (1H, bs, NH), 7.79 (2H, d, J = 8.0 Hz, 2 x CH-Ar), 7.84 (2H, d, J = 8.0 Hz, 2 x CH-Ar); 13 C-NMR (101 MHz, CDCl 3) δ 11.2 (2 x CH3), 19.2 (CH3), 22.0 (2 x CH2), 25.4 (CH2 -cy), 25.7 (CH2-cy), 33.6 (CH2-cy), 34.3 (CH2 -cy), 38.5 (CH-cy), 50.0 (2 x CH2), 54.9 (CH-cy), 127.2 (2 x CH-Ar), 127.6 (2 x CH-Ar), 138.9 (C-Ar), 142.5 (C-Ar), 165.8 (C=O). 4 - (N, N- di p ro py ls ulf am o yl )-N - (2- m o rph ol i no et hyl ) b enz a m i de (4 ): 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.35 ml of N-2-(aminoethyl)morpholine (2.63 mmol, 1.5 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. After four hours the reaction was diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (ethyl acetate:petroleum ether 10:1) gave title compound as a white solid (0.32 g, 46% yield); mp 90-93 °C; IR
νmax 3265 (ν N-H), 2960 (ν Csp2-H), 1639 (ν C=O), 1340 (νas S=O), 1150 (νs S=O), 743 (δ Csp2 -H) cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.86 (6H, t, J = 7.6 Hz, 2 x CH3), 1.53 (4H, sext, J = 7.6 Hz, 2 x CH2), 2.51 (4H, m, 2 x CH2-cy), 2.61 (2H, t, J = 5.6 Hz, CH2), 3.07 (4H, t, J = 7.2 Hz, 2 x CH2), 3.56 (2H, t, J = 4.8 Hz, CH2), 3.72 (4H, m, 2 x CH2-cy), 6.95 (1H, bs, NH), 7.84 (4H, m, 4 x CH-Ar); 13C-NMR (101 MHz, DMSO-d6) δ 11.2 (2 x CH3), 22.0 (2 x CH2), 36.2 (CH2), 50.0 (2 x CH2), 53.3 (CH2), 56.9 (2 x CH2 -cy), 66.9 (2 x CH2-cy), 127.3 (2 x CH-Ar), 127.7 (2 x CH-Ar), 138.2 (C-Ar), 143.0 (C-Ar), 166.0 (C=O). 4 - (di pr op yl s ulf a m oyl )- N- p hen yl b enz am i de) (5) : 1,1’carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.18 g of aniline (1.93 mmol, 1.1 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,Ndimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 2:1) gave the title compound as a white solid (0.41 g, 65% yield); mp 113-114 °C; IR νmax 3382 (ν N-H), 2964 (ν Csp2 H), 1678 (ν C=O), 1326 (νas S=O), 1153 (νs S=O), 855 (δ Csp2 -H), cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.79 (6H, t, J = 7.2 Hz, 2 x CH3), 1.46 (4H, sext, J = 7.2 Hz, 2 x CH2), 3.05 (4H, t, J = 6.8 Hz, 2 x CH2), 7.10 (1H, t, J = 7.2 Hz, CH-Ar), 7.34 (2H, t, J = 7.6 Hz, 2 x CH-Ar), 7.76 (2H, d, J = 8.0 Hz, 2 x CH-Ar), 7.92 (2H, d, J = 8.0 Hz, 2 x CH-Ar), 8.00 (2H, d, J = 8.0 Hz, 2 x CH-Ar), 10.46 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.1 (2 x CH2), 50.1 (2 x CH2), 120.9 (2 x CH-Ar), 124.5 (CH-Ar), 127.3 (2 x CH-Ar), 129.1 (2 x CH-Ar), 139.0 (2 x CHAr), 139.3 (C-Ar) 142.4 (C-Ar), 164.9 (C=O), (1C peak missing because of overlapping signals). 4 - (N, N- di pr o py ls ulf am o yl )-N - (pyri di n- 2- yl ) b enz a m i de (6) : 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.18 g of 2-aminopyridine (1.92 mmol, 1.1 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight After 24 h, additional 0.28 g of CDI (1.75 mmol, 1.0 eq) was added and the reaction was stirred overnight. After 24 h, additional 0.08 g of 2aminopyridine (0.875 mmol, 0.5 eq) were added and the reaction stirred for 24 h. The reaction was diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 1:1) gave the title compound as a white solid (0.16 g, 24% yield); mp 112-115 °C; IR νmax 3310 (ν N-H), 2965 (ν Csp2-H), 1529 (ν C=O), 1300 (νas S=O), 1156 (νs S=O), 870 (ν C=N), 784 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.83 (6H, t, J = 7.2 Hz, 2 x CH3), 1.51 (4H, sext, J = 7.6 Hz, 2 x CH2), 3.06 (4H, t, J = 7.6 Hz, 2 x CH2), 7.00 (1H, t, J = 6.0 Hz, 1 x CH-pyr), 7.70 (1H, t, J = 7.6 Hz, 1 x CH-pyr), 7.85 (2H, d, J = 7.2 Hz, 2 x CH-Ar), 8.01 (3H, m, 2 x CH-Ar + CHpyr), 8.34 (1H, d, J = 8.4 Hz, CH-pyr), 9.65 (1H, bs, NH); 13CNMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 21.9 (2 x CH2), 49.3 (2 x CH2), 114.7 (CH-Ar), 120.3 (CH-Ar), 127.3 (2 x CHAr), 128.2 (2 x CH-Ar), 137.8 (CH-Ar), 138.7 (C-Ar), 143.6 (CH-Ar), 147.7 (C-Ar), 151.5 (C-Ar), 167.8 (C=O).
A
B
His64
Trp5
C His64
His64
Asn62
Trp5
Trp5 Asn62
Asn62
Thr200 Thr200
His94
His94
2+
Zn
Zn2+
His94
Zn2+
Thr199
Lys67
Thr199
Lys67 Gln92
Lys67 Gln92
Gln92
Thr91
Ser135
Ser135
Thr91 Ser132
Thr91
Ser132
D
E
His64
F Thr199
Thr200 Thr199
2+
Zn
Thr200
Zn2+ Thr199
His94
His94 Trp5
Lys67
Thr200
Gln92
Trp5
Gln92 Ser135
Gln92
His94 His64
Lys67
Thr91
Asn62
Ser132
Lys67 Asn62
His64
Figure 1. Docked poses of compound 4 (A), compound 3 (B), compound 5 (C), compound 10 (D), probenecid (E) and compound 1 (F) with the active site of tumour-associated hCA XII. Hydrogen bonds are indicated with red dotted lines, binding interactions to the Zn2+ ion are indicated with blue dotted lines, cation-π interactions are indicated with red double-headed arrows, and major hydrophobic stackings are indicated with green double-headed arrows.
4 - (N, N- di p ro py ls ulf am o yl )-N - (1, 3, 4-t hi ad i az ol - 2- yl) b enz a m i de (7 ): 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.2 g of 2-amino-1,3,4-thiadiazole (1.92 mmol, 1.1 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. After 48 h, additional 0.28 g of CDI (1.75 mmol, 1.0 eq) were added and the reaction stirred overnight. The reaction was diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 1:2) gave title compound as a white solid (0.26 g, 40% yield); mp 180-182 °C; IR νmax 3139 (ν N-H), 2961 (ν Csp2 -H), 1536 (ν C=O), 1338 (νas S=O), 1152 (νs S=O), 735 (δ Csp2 -H), 681 (ν C-S) cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.79 (6H, t, J = 7.4 Hz, 2 x CH3), 1.46 (4H, sext, J = 7.2 Hz, 2 x CH2), 3.05 (4H, t, J = 7.6 Hz, 2 x CH2), 7.95 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 8.26 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 9.24 (1H, s, CH-Ar), 13.35 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.0 (2 x CH2), 50.0 (2 x CH2), 127.4 (2 x CH-Ar), 129.9 (2 x CH-Ar), 135.8 (C-Ar), 143.6 (CH-Ar), 149.7 (C-Ar), 166.0 (C=O), (1C peak missing because of overlapping signals). N -(3 -c hl o r op hen yl )- 4- (di pr op yl s ulf a m oyl )b enz am i de (8 ) : 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and
0.25 g of 3-chloroaniline (1.93 mmol, 1.1 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 3:1) gave title compound as a white solid (0.31 g, 45% yield); mp 117-119 °C; IR νmax 3289 (ν N-H), 2968 (ν Csp2 -H), 1668 (ν C=O), 1334 (νas S=O), 1153 (νs S=O), 835 (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 = 7.6 Hz, 2 x CH2), 3.07 (4H, t, J = 7.2 Hz, 2 x CH2), 7.20 (1H, m, J1 = 8.0 Hz, J2 = 2.2 Hz, J3 = 1.2 Hz, CH-Ar), 7.41 (1H, t, J = 8.0 Hz, CH-Ar), 7.71 (1H, m, J1 = 8.0 Hz, J2 = 2.0 Hz, J3 = 0.8 Hz, CH-Ar), 7.96 (2H, d, J = 8.8 Hz, 2 x CH-Ar), 7.97 (1H, s, CH-Ar), 8.13 (2H, d, J = 8.8 Hz, 2 x CH-Ar), 10.62 (1H, s, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.0 (2 x CH2), 50.1 (2 x CH2), 119.2 (CH-Ar), 120.3 (CH-Ar), 124.2 (CH-Ar), 127.4 (2 x CH-Ar), 129.2 (2 x CH-Ar), 130.9 (CH-Ar), 133.5 (C-Ar), 138.6 (C-Ar), 140.8 (CAr), 142.7 (C-Ar), 165.1 (C=O). N - (4 -c hl o r op hen yl )- 4- (di pr op yl s ulf a m oyl )b en z ami de (9 ) : 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.25 g of 4-chloroaniline (1.93 mmol, 1.1 eq) were added to a
stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 1:1) gave title compound as a white solid (0.27 g, 46% yield); mp 164-166 °C; IR νmax 3382 (ν N-H), 2319 (ν Csp2 -H), 1668 (ν C=O), 1336 (νas S=O), 1155 (νs S=O), 836 (δ Csp2 -H), cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.82 (6H, t, J = 7.6 Hz, 2 x CH3), 1.50 (4H, sext, J = 7.6 Hz, 2 x CH2), 3.07 (4H, t, J = 7.6 Hz, 2 x CH2 ), 7.43 (2H, d, J = 8.8 Hz, 2 x CH-Ar), 7.82 (2H, d, J = 8.8 Hz, 2 x CH-Ar), 7.96 (2H, d J = 8.4 ,2 x CH-Ar), 8.12 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 10.61 (1H, bs, NH); 13CNMR (101 MHz, DMSO-d6) δ 11.5 (2 x CH3), 22.0 (2 x CH2), 50.1 (2 x CH2), 122.4 (2 x CH-Ar),127.4 (2 x CH-Ar), 129.1 (2 x CH-Ar), 129.2 (2 x CH-Ar), 138.3 (C-Ar) 138.7 (C-Ar), 141.7 (C-Ar), 142.5 (C-Ar), 165.0 (C=O). 4 - (N, N- di p ro py ls ulf am o yl )-N - (3- ni tr op h en y l ) b enz a m i de (10 ) : 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.27 g of 3-nitroaniline (1.93 mmol, 1.1 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. After four hours the reaction was diluted with dichloromethane and washed with saturated NaHCO3. 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 title compound as a white solid (0.083 g, 10% yield); mp 185-188 °C; IR νmax 3382 (ν N-H), 2969 (ν Csp2-H), 1672 (ν C=O), 1534 (ν NO), 1329 (νas S=O), 1256 (ν N-O), 1160 (νs S=O), 740 (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 = 7.6 Hz, 2 x CH2), 3.08 (4H, t, J = 7.2 Hz, 2 x CH2), 7.68 (1H, t, J = 8.0 Hz, CH-Ar), 7.99 (3H, m, 3 x CH-Ar), 8.18 (3H, m, 3 x CH-Ar), 8.80 (1H, s, CH-Ar), 10.92 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.1 (2 x CH2), 50.1 (2 x CH2), 119.0 (CH-Ar), 126.7 (CH-Ar), 127.4 (2 x CH-Ar), 129.3 (2 x CH-Ar), 130.6 (CH-Ar), 138.2 (CH-Ar), 140.5 (C-Ar), 142.9 (C-Ar), 148.4 (C-Ar), 152.7 (CAr), 165.3 (C=O). 4- (N, N -di p ro pyls ulf a m oyl )-N - (2-ni tr o ph en y l ) b enz a m i de (11 ) : 1.00 g of probenecid (3.5 mmol, 1.00 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, 2-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 2-nitroaniline and the reaction stirred for 24 h at room temperature. The reaction was evaporated in vacuo and the residue purified on silica gel (petroleum ether:ethyl acetate 10:1) to give title compound as a yellow solid (0.71 g, 50% yield); mp 127-129 °C; IR νmax 3340 (ν N-H), 2964 (ν Csp2 H), 1695 (ν C=O), 1514 (ν N-O), 1336 (νas S=O), 1272 (ν N-O), 1152 (νs S=O), 1272 (ν N-O), 741 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, CDCl3) δ 0.88 (6H, t, J = 7.4 Hz, 2 x CH3), 1.57 (4H, sext, J = 7.6 Hz, 2 x CH2), 3.13 (4H, t, J = 7.6 Hz, 2 x CH2), 7.27 (1H, m, CH-Ar), 7.74 (1H, t, J = 7.8 Hz, CH-Ar), 7.97 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 8.10 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 8.30 (1H, d, J = 8.4 Hz, CH-Ar), 8.96 (1H, d, J = 8.4 Hz, CH-Ar), 11.40 (1H, bs, NH); 13C-NMR (101 MHz, CDCl3) δ 11.2 (2 x CH3), 22.0 (2 x CH2), 50.0 (2 x CH2), 117.7 (CH-Ar), 122.2 (CH-Ar), 123.9 (CH-Ar), 126.0 (CH-Ar), 127.7 (2 x CH-Ar), 128.1 (2 x
CH-Ar), 134.7 (C-Ar), 136.3 (C-Ar), 136.6 (C-Ar), 137.3 (C-Ar), 164.2 (C=O). 4 - (N, N- di pr o py ls ulf am o yl )-N - (2- a mi no p heny l ) b enz a m i de (12 ) : 0.200 g of 4-(N,N-dipropylsulfamoyl)-N-(2nitrophenyl)benzamide (10) (0.5 mmol, 1.00 eq) was dissolved in 50 mL of methanol. Palladium on carbon (10% Pd basis, 20 mg) was added and stirred under hydrogen pressure (1.85 bar) for 4 h. The palladium was filtered off through Celite® and the filtrate was evaporated in vacuo to give title compound as a white solid (0.140 g, 75% yield); mp: 165-167°C; IR νmax 3397 (ν N-H), 3270 (ν N-H(H)), 2967 (ν Csp2-H), 1530 (ν C=O), 1339 (νas S=O), 1152 (νs S=O), 741 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, DMSOd6) δ 0.84 (6H, t, J = 6.8 Hz, 2 x CH3), 1.49 (4H, sext, J = 6.8 Hz, 2 x CH2), 3.06 (4H, t, J = 6.4 Hz, 2 x CH2), 4.98 (2H, bs, NH2), 6.59 (1H, t, J = 6.4 Hz, CH-Ar), 6.79 (1H, d, J= 7.2 Hz, CH-Ar), 6.98 (1H, t, J = 7.2 Hz, CH-Ar), 7.17 (1H, d, J = 7.2 Hz, CH-Ar), 7.93 (2H, d, J = 6.8 Hz, 2 x CH-Ar), 8.17 (2H, d, J = 7.2 Hz, 2 x CH-Ar), 9.88 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.1 (2 x CH2), 50.1 (2 x CH2), 116.5 (CH-Ar), 116.6 (CH-Ar), 123.2 (CH-Ar), 127.1 (CH-Ar), 127.2 (CH-Ar), 127.3 (CH-Ar), 129.3 (2 x CH-Ar), 138.7 (2 x C-Ar), 142.3 (C-Ar), 143.8 (C-Ar), 164.7 (C=O). 6.3. Molecular modelling studies 6 . 3. 1. Pr ep a ra ti o n of li g an d fil es Compounds were built in 3D using MOE (version 2013.0801, Chemical Computing Group Inc., Montreal, Canada). All strong bases were protonated and all strong acids were deprotonated using and a subsequent steepest-descent energy minimization was performed using the MMFF94x forcefield. The ligand files were saved as multi-mol2 files. 6 . 3. 2. Pr ep a ra ti o n of p r ot ei n fi l es The crystal structures of hCA XII in complex with acetazolamide (1JD0; 1.50 Å) was obtained from the Protein Data Bank (PDB server). Chain A was retained when more than one chain was present. The cocrystallized inhibitor and the Zn2+ ion were retained and all other water molecules, buffers and ions were deleted. Hydrogen atoms were added using the “protonate 3D” tool of MOE and the system was energy minimized using the AMBER99 forcefield. 6 . 3. 3. D oc ki ng st udi es Docking studies were performed using the GOLD Suite package (version 5.2, Cambridge Crystallographic Data Centre, Cambridge, UK). Probenecid and its analogs 1–12 were docked into all protein structures (25 dockings per ligand) and scored with the GoldScore scoring function. The binding pocket was defined as all residues within 13 Å of a centroid (X: -17.071, Y: 35.081, Z: 43.681; this point corresponds approximately to the location of the thiadiazole ring of acetazolamide in the 1JDO structure).
Acknowledgments This work was financed by two FP7 EU projects (Metoxia and Dynano) to CTS. References and notes (1) (2) (3) (4)
Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem. Rev. 2012, 112, 4421. Supuran, C.T. Nat. Rev. Drug Discov. 2008, 7, 168. Hilvo, M.; Innocenti, A.; Monti, S. M.; De Simone, G.; Supuran, C. T.; Parkkila, S. Curr. Pharm. Des. 2008, 14, 672. (a) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146; (b) Supuran, C. T.; Scozzafava, A. Expert Opin. Ther. Pat.
(5)
(9) (10) (11) (12) (13) (14) (15) (16) (17)
(18)
(19) (20)
2000, 10, 575; (c) Swietach, P.; Wigfield, S.; Cobden, P.; Supuran, C. T.; Harris, A. L.; Vaughan-Jones, R. D. J. Biol. Chem. 2008, 283, 20473. (a) Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 677; (b) Supuran, C. T. Future Med. Chem. 2011, 3, 1165. (c) Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2004, 19, 199. Weber, C. E.; Kuo, P. C. Surg. Oncol. 2012, 21, 172. Warburg, O. Science 1956, 124, 269. Warburg, O.; Wind, F.; Negelein, E. J. Gen. Physiol. 1927, 8, 519. Gatenby, R. A.; Gillies, R. J. Nat. Rev. Cancer 2004, 4, 891. Webb, B. A.; Chimenti, M.; Jacobson, M. P.; Barber, D. L. Nat. Rev. Cancer 2011, 11, 671. Gatenby, R. A.; Smallbone, K.; Maini, P. K.; Rose, F.; Averill, J.; Nagle, R. B.; Worrall, L.; Gillies, R. J. Br. J. Cancer 2007, 97, 646. Dang, C. V.; Semenza, G. L. Trends Biochem. Sci. 1999, 24, 68. Semenza, G. L. Crit. Rev. Biochem. Mol. Biol. 2000, 35, 71. (a) Parks, S. K.; Chiche, J.; Pouysségur, J. Nat. Rev. Cancer 2013, 13, 611; (b) Lock, F. E.; McDonald, P. C.; Lou, Y.; Serrano, I.; Chafe, S. C.; Ostlund, C.; Aparicio, S.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Oncogene, 2013, 32, 5210; (c) Ward, C.; Langdon, S. P.; Mullen, P.; Harris, A. L.; Harrison, D. J.; Supuran, C. T.; Kunkler, I. H. Cancer Treat. Rev. 2013, 39, 171; d) Potter, C. P. S.; Harris, A. L. Br. J. Cancer 2003, 89, 2. (a) McDonald, P. C.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Oncotarget 2012, 3, 84; (b) Lou, Y.; McDonald, P. C.; Oloumi, A.; Chia, S. K.; Ostlund, C.; Ahmadi, A.; Kyle, A.; Auf dem Keller, U.; Leung, S.; Huntsman, D. G.; Clarke, B.; Sutherland, B. W.; Waterhouse, D.; Bally, M. B.; Roskelley, C. D.; Overall, C. M.; Minchinton, A.; Pacchiano, F.; Carta, F.; Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Cancer Res. 2011, 71, 3364; (c) Lounnas, N.; Rosilio, C.; Nebout, M.; Mary, D.; Griessinger, E.; Neffati, Z.; Spits, H.; Hagenbeek, T.; Asnafi, V.; Poulsen, S. A.; Supuran, C. T.; Peyron, J. F.; Imbert, V. Cancer Lett. 2013, 333, 76. Neri, D.; Supuran, C. T.; Nat. Rev. Drug Discov. 2011, 10, 767. (a) Winum, J. Y.; Carta, F.; Ward, C.; Mullen, P.; Harrison, D.; Langdon, S. P.; Cecchi, A.; Scozzafava, A.; Kunkler, I.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2012, 22, 4681; (b) Ebbesen, P.; Pettersen, E. O.; Gorr, T. A.; Jobst, G.; Williams, K.; Kieninger, J.; Wenger, R. H.; Pastorekova, S.; Dubois, L.; Lambin, P.; Wouters, B. G.; Van Den Beucken, T.; Supuran, C. T.; Poellinger, L.; Ratcliffe, P.; Kanopka, A.; Görlach, A.; Gasmann, M.; Harris, A. L.; Maxwell, P.; Scozzafava, A. J. Enzyme Inhib. Med. Chem. 2009, 24, 1.
(21)
(22)
(23) (24) (25)
(26)
(27) (28)
(29) (30)
(31)
(32) (33)
Tafreshi, N. K.; Bui, M. M.; Bishop, K.; Lloyd, M. C.; Enkemann, S. A.; Lopez, A. S.; Abrahams, D.; Carter, B. W.; Vagner, J.; Grobmyer, S. R.; Gillies, R. T.; Morse, D. C. Clin. Cancer Res. 2012, 18, 207. a) Gieling, R. G.; Williams, K. J. Bioorg. Med. Chem. 2013, 21, 1470; b) Borras, J.; Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Mincione, G.; Supuran, C.T. Bioorg. Med. Chem. 1999, 7, 2397. Said, H. M.; Supuran, C. T.; Hageman, C.; Staab, A.; Polat, B.; Katzer, A.; Scozzafava, A.; Anacker, J.; Flentje, M.; Vordermark, D. Curr. Pharm. Des. 2010, 16, 3288. Supuran, C.T.; Mincione, F.; Scozzafava, A.; Briganti, F.: Mincione, G.; Ilies, M.A. Eur. J. Med. Chem. 1998, 33, 247. a) Demirdağ, R.; Çomaklı, V.; Şentürk, M.; Ekinci, D.; İrfan Küfrevioğlu, Ö.; Supuran, C. T. Bioorg. Med. Chem. 2013, 21, 1522; b) Briganti, F.; Pierattelli, R.; Scozzafava, A.; Supuran, C.T. Eur. J. Med. Chem. 1996, 31, 1001. Rogez-Florent, T.; Meignan, S.; Foulon, C.; Six, P.; Gros, A.;BalMahieu, C.; Supuran, C. T.; Scozzafava, A.; Frédérick, R.; Masereel, B.; Depreuxa, P.; Lansiauxa, A.; Goossensa, J. F.; Gluszoka, S.; Goossens, L. Bioorg. Med. Chem. 2013, 21, 1451. Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. In Drug Design of Zinc-Enzyme Inhibitors; Supuran, C. T.; Winum, J.-Y., Eds.; John Wiley & Sons, Inc., 2009; pp. 138. Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.; Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti, S. M.; De Simone, G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233. Alp, C.; Maresca, A.; Alp, N. A.; Gültekin, M. S.; Ekinci, D.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 294. (a) Métayer, B.; Martin-Mingot, A.; Vullo, D.; Supuran, C. T.; Thibaudeau, S. Org. Biomol. Chem. 2013, 11, 7540; (b) Métayer, B.; Mingot, A.; Vullo, D.; Supuran, C. T.; Thibaudeau, S. Chem. Commun. 2013, 49, 6015. (a) D’Ascenzio, M.; Carradori, S.; De Monte, C.; Secci, D.; Ceruso, M.; Supuran, C. T. Bioorg. Med. Chem. 2014, 22, 1821; (b) Akdemir, A.; De Monte, C.; Carradori, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, DOI: 10.3109/14756366.2014.892936. (c) Carradori, S.; De Monte, C.; D’Ascenzio, M.; Secci, D.; Celik, G.; Ceruso, M.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2013, 23, 6759. Puscas, I.; Coltau, M.; Pasca, R. J. Pharmacol. Exp. Ther. 1996, 277, 1464. Cheng, H. C. J. Pharmacol. Toxicol. Methods 2001, 46, 61.
Selective inhibition of human carbonic anhydrases by novel amide derivatives of probenecid: synthesis, biological evaluation and molecular modelling studies Melissa D’Ascenzio, Simone Carradori, Daniela Secci, Daniela Vullo, Mariangela Ceruso, Atilla Akdemir, Claudiu T. Supuran
S0968-0896(14)00446-5 http://dx.doi.org/10.1016/j.bmc.2014.06.003 BMC 11637
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
1 April 2014 31 May 2014 2 June 2014
Please cite this article as: D’Ascenzio, M., Carradori, S., Secci, D., Vullo, D., Ceruso, M., Akdemir, A., Supuran, C.T., Selective inhibition of human carbonic anhydrases by novel amide derivatives of probenecid: synthesis, biological evaluation and molecular modelling studies, Bioorganic & Medicinal Chemistry (2014), doi: http:// dx.doi.org/10.1016/j.bmc.2014.06.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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
Selective inhibition of human carbonic anhydrases by novel amide derivatives of probenecid: synthesis, biological evaluation and molecular modelling studies Melissa D’Ascenzioa,∗, Simone Carradoria, Daniela Seccia, Daniela Vullob, Mariangela Cerusob, Atilla Akdemird, Claudiu T. Supuranb,c,* a
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy. c Università degli Studi di Firenze, Neurofarba Dept., Section of Pharmaceutical and Nutriceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy. d Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Vatan Caddesi, 34093 Fatih, Istanbul, Turkey. 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, a well-known uricosuric agent, were synthesized and evaluated as inhibitors of human carbonic anhydrases (hCAs, EC 4.2.1.1). The transmembrane isoforms (hCA IX and XII) were potently and selectively inhibited by some of them. The proposed chemical modification led to a complete loss of hCA II inhibition (Ki s>10000 nM) and enhaced the inhibitory activity against the tumour-associated hCA XII (compound 4 showed a Ki value of 15.3 nM). The enzyme inhibitory data have also been validated by docking studies of the compounds within the active site of hCA XII.
Keywords: Probenecid Selective carbonic anhydrase IX inhibitors Selective carbonic anhydrase XII inhibitors Tertiary sulfonamides
1. Introduction Carbonic anhydrases (CA, EC 4.2.1.1) are a widely spread superfamily of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. 1 To date, fifteen human isoforms (hCA I-XV) have been isolated and classified on the basis of their cellular localization, tissue distribution, kinetic properties, and sensitivity to different classes of inhibitors.2,3 They are involved in a wide range of physiological processes and the selective inhibition of specific isoforms has been proved to be effective in the treatment of several pathological conditions such as hypertension, epilepsy, altitude sickness, glaucoma, obesity, osteoporosis, gastric and duodenal ulcers.4–8 More recently, it has been discovered that two CA transmembrane isoforms (CA IX and XII) are overexpressed in hypoxic tumors as part of the complex machinery that cancer cell use to compensate the changes in pH, nutrient and oxygen levels which characterize the microenvironment of solid tumors.9–13 As these adaptive changes are believed to contribute to the development of invasive and metastatic phenotypes, 14–16 an extensive effort has been made in the last few years to disrupt such pathological mechanisms by selectively targeting these two pH regulating enzymes: CA IX and XII. 17–23
———
2009 Elsevier Ltd. All rights reserved.
The zinc binding sulfonamide moiety has been widely used in the past to design carbonic anhydrase inhibitors.24–26 Although it is believed that the key interaction between these compounds and the active site of CAs is mediated by the formation of a coordinative bond between the positively charged zinc and the deprotonated sulfonamide,27,28 there is growing evidence that even non-ionizable tertiary substituted sulfonamides can efficiently inhibit different CA isoforms. 29–31 In particular, researchers have recently found that various fluorinated/non-fluorinated tertiary substituted benzenesulfonamides and other atypical chelating compounds were able to selectively inhibit one of the cancer related isoforms of CA (CA IX) in the low nanomolar range (Chart 1).30,31
Chart 1. Rational design of Probenecid derivatives
Corresponding authors: Dr. Melissa D’Ascenzio: Tel/Fax: +39 06 49693242; e-mail: [email protected]; Prof. Claudiu T. Supuran: Tel: 39-055-4573005; Fax: 39-055-4573385; e-mail: [email protected]. ∗
The similarity of these compounds to one of the best known uricosuric agents (probenecid), whose ability to interfere with CA catalytic activity was preliminary reported in the past,32 inspired the synthesis of a small series of probenecid derivatives which are reported herein. We wanted to explore whether derivatization of the COOH moiety of probenecid to amides has a consequence of the interaction of these compounds with the CA active site. 2. Chemistry Derivatives 1-10 were synthesized by reacting 4-(N,N-dipropylsulfamoyl)benzoic acid (probenecid) with the corresponding amine in the presence of 1,1’-carbonyldiimidazole (CDI) in N,N-dimethylformamide at room temperature (Table 1). Purification via column chromatography on silica gel afforded title compounds in satisfying yields. Compound 11 was obtained by treating probenecid with thionyl chloride in dichloromethane and then reacting the obtained acyl chloride with the strongly deactivated 2-nitroaniline. The reduction of compound 11 using hydrogen and palladium on activated carbon gave thereafter derivative 12 in high yield. All synthesized compounds were fully characterized by analytical and spectral data (see Section 7.2 for details). 3. Results and Discussion All the synthesized compounds (1-12) were tested against the two cancer-related isoforms of CA (CA IX and XII) and their corresponding off-targets (CA I and II) in order to evaluate their biological activity and selectivity. 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 for the determination of the kinetic parameters and inhibition constants ranged from 1.7 to 17 mM. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (1 µM) were prepared in distilled-deionized 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 non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation,33 and represent the mean from at least three different determinations. All recombinant CA isoforms were obtained in-house as previously reported. On the basis of the obtained results, SAR for this class of probenecid analogs as promising hCA inhibitors can be extrapolated as follows: (i) All tested compounds proved to be inactive against the ubiquitously expressed isoform of hCA II at concentrations higher than 10 µM. Conversely, no selectivity was observed against hCA I. (ii) All screened compounds showed to inhibit hCA IX and hCA XII in the nanomolar range (165 nM < Ki hCA IX < 265 nM and 15.3 nM < Ki hCA XII < 430 nM).
(iii) In general, we found that the conversion of the carboxylic group of probenecid into its corresponding amide resulted in a significative gain of activity of the obtained derivatives against the tumor-associated isoform CA XII, with Ki values dropping down from 1245 nM to 15.3 nM (compare probenecid and compound 4 in Table 1). Similarly, the activity against CA IX was maintained throughout the series, if not slightly increased, in both alicyclic and (hetero)aromatic derivatives. On the other hand, compounds 1-12 showed an inversion of selectivity against CA I and II if compared to the parent drug: while probenecid was not able to inhibit isoform I and was mildly active against isoform II, the newly synthesized amides show a consistent lack of activity against CA II and an ability to inhibit CA I in the nanomolar range (307 nM < Ki hCA I < 1980 nM). The introduction of a renowned zinc binding moiety (o-phenylendiamine) on derivative 12 did not change the pattern of inhibition or the potency of the obtained compound, thus supporting the hypothesis that these inhibitors could display a new binding mode which does not include a direct interaction with the metal ion. 4. Docking studies into hCA XII Lastly, docking studies were performed in order to better understand the enzyme-inhibitor recognition pattern and the isoform selectivity which characterize these probenecid analogs. Derivatives 1–12 are characterized by Ki values at least 3-fold lower than probenecid, with compounds 3 (32-fold) and 4 (81fold) showing the lowest Ki values (Table 1). The high number of interactions that compound 4 establishes within the active site of hCA XII might explain the low Ki value observed for this compound (Figure 1A). This compound has a morpholine ring with an alkaline nitrogen which is most likely partly protonated at physiological pH values and can form a cation-π adduct with His94. The oxygen atom of the morpholine ring directly interacts with the catalytic zinc ion although no Hbonds with Thr199 are observed. The oxygen and nitrogen atoms of the amide bond do not form any interactions with the enzyme and are located close to the solvent. The phenyl group of the ligand is believed form cation-π interactions with Lys67. One of the sulfonamide oxygens is hydrogen bonded to Ser132, while the other oxygen and the non-protonated tertiary nitrogen atom point out of the catalytic site, towards the protein surface. When the morpholine nucleus is replaced by a cyclohexyl ring (compound 2) neither binding to the zinc ion nor cation-π interactions with His94 are possible. Although the addition of a methyl substituent to the 2-position of the cyclohexyl moiety (compound 3) lowers the Ki value of about 10-folds, none of the enantiomers of compound 3 interact with the zinc ion. Nevertheless, the S-isomer forms various interactions within the active site of the enzyme (Figure 1B). In particular, both the 2methylcyclohexyl and the phenyl group are involved in hydrophobic stacking interactions with His94 and Trp5, respectively. The oxygen atoms of the sulfonamide moiety are hydrogen bonded to Trp5 and His64 side chain, while the nitrogen atom points out towards the solvent. This pose is not observed for compound 2 or for the R-isomer of compound 3. Compound 5, which has a phenyl ring in place of the cyclohexyl group of compounds 2 and 3, might interact with the zinc ion via the aromatic moiety (electrostatic interactions). The sulfonamide oxygen atoms form H-bonds to Thr91 and Gln92 (Figure 1C). Compounds 6-9, 11 and 12 are likely to form similar interactions within the active site of CA as shown for compound 5. In addition, the meta-nitro substituted aromatic ring
of compound 10 interacts with the backbone nitrogen of Thr199 and the zinc ion via the two oxygen atoms of the nitro group (Figure 1D). The phenyl group next to the sulfonamide moiety could form cation-π interactions with Lys67, while the sulfonamide oxygen and nitrogen atoms point towards the solvent. The ortho-nitro group of compound 11, the ortho-amine group of compound 12 and the chlorine atoms of compounds 8 and 9 are not able to directly interact with the zinc-ion and therefore, they are most likely to bind in a similar fashion to compound 5. This assumption seems to be consistent with the enzyme inhibition data which report similar Ki values for compounds 5-9, 11 and 12 and lower Ki values for compound 10. There is only a 4-fold difference in Ki values between probenecid and the closely related compound 1. Probenecid interacts with the zinc ion and the backbone of Thr199 via its carboxylic group and hydrogen bonds are formed between Asn62, Gln92 and the oxygen atoms of the sulfonamide moiety (Figure 1E). Finally, the phenyl group is involved in π-π stacking interactions with the imidazole side chain of His94. Similarly, compound 1 is allocated inside the active site of CA with a comparable orientation: one of its sulfonamide oxygens forms a hydrogen bond with Asn62 and its phenyl ring interacts with His94 via π-π stacking. However, its methylamide substituent points toward the side chains of Val143, Val207 and Trp209, and although its carbonyl group still interacts with the zinc ion, the interaction with Thr199 is lost (Figure 1F). The docking pose of compound 4 (Figure 1A) provides clues for obtaining derivatives with even higher affinity. The dipropylamine group points into the solvent and does not form any interactions with the hCA XII binding pocket. As such, this moiety does not contribute to binding interactions. In addition, only one of the sulfonamide oxygen groups forms a hydrogen bond (with Ser132). This group might be replaced by a –CH3OH group. Substitutions on this position could be explored to obtain higher affinity and probably also subtype-selectivity amongst the hCA isoforms. 5. Conclusion Novel amide derivatives of the well-known uricosuric agent probenecid were easily synthesized and evaluated as potent and selective inhibitors of the human carbonic anhydrase transmembrane isoforms (hCA IX and XII). The introduction of an alkyl/aryl amide portion led to a complete loss of hCA II inhibition (Ki > 10000 nM) and enhanced the inhibitory activity against hCA XII (see compound 4 with Ki value of 15.3 nM). Docking studies of the most active compounds explained the enzyme recognition pattern and highlighted the path towards the design of novel selective hCA isoform inhibitors. 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. 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 SigmaAldrich® 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, 13C: 101 MHz). Chemical shifts are quoted in ppm, based on appearance rather than interpretation, and are referenced to the residual non deuterated solvent peak. 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). 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 1-10: 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. After the reaction went to completion, the organics were diluted with dichloromethane, washed with saturated NaHCO3, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel gave title compounds in satisfying yields. 4 - (di pr op yl s ulf a m oyl )- N- m et hyl b enz am i de (1 ): 1,1’carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 1.31 mL of a 2M solution of methylamine in THF (2.63 mmol, 1.5 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. After four hours the reaction was diluted with dichloromethane and washed with saturated NaHCO3. 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 (0.21 g, 40% yield); mp 127-129 °C; IR νmax 3289 (ν N-H), 2960 (ν Csp2-H), 1639 (ν C=O), 1341 (νas S=O), 1163 (νs S=O), 856 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.81 (6H, t, J = 7.6 Hz, 2 x CH3), 1.46 (4H, sext, J = 7.6 Hz, 2 x CH2), 2.80 (3H, d, J = 4.4 Hz, CH3), 3.05 (4H, t, J = 7.6 Hz, 2 x CH2), 7.88 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 8.00 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 8.67 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.0 (2 x CH2), 26.8 (CH3 ), 50.0 (2 x CH2), 127.3 (2 x CH-Ar), 128.5 (2 x CH-Ar), 138.4 (C-Ar), 142.0 (C-Ar), 165.9 (C=O).
Table 1. Inhibitory activity of derivatives 1-12 and reference compounds (probenecid and acetazolamide) against selected hCA isoforms by stopped-flow CO2 hydrase assay.
Compound
R
hCA I
Ki (nM) hCA II hCA IX
hCA XII
1980
> 10 000
165
289
2
307
> 10 000
234
398
3
319
> 10 000
260
42.5
4
347
> 10 000
251
15.3
5
324
> 10 000
171
422
6
324
> 10 000
228
418
346
> 10 000
216
410
10
341
> 10 000
198
428
9
364
> 10 000
243
430
10
344
> 10 000
265
201
11
327
> 10 000
192
352
12
354
> 10 000
223
407
CH3
1
S
N
7 N
NH2
Probenecid
> 10 000
431
360
1245
Acetazolamide (AAZ)
250
12
25
5.7
N -cy cl oh exy l- 4-(N , N- di pr opy ls ulf a m o yl )b enz a mi de (2 ): 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.18 g of cyclohexylamine (2.62 mmol, 1.5 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 2:1) gave the title compound as a white solid (0.31 g, 53% yield); mp 127-129 °C; IR νmax 3291 (ν N-H), 2936 (ν Csp2-H), 1627 (ν C=O), 1330 (νas S=O), 1146 (νs S=O), 736 (δ Csp2 -H), cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.81 (6H, t, J = 7.6 Hz, 2 x CH3), 1.14 (1H, m, CH-cy), 1.29 (4H, m, 2 x CH2-cy), 1.46 (4H, sext, J = 7.2 Hz, 2 x CH2), 1.62 (1H, m, CH-cy), 1.75 (2H, m, 2 x CH-cy), 1.83 (2H, m, CH-cy), 3.04 (4H, t, J = 7.6 Hz, 2 x CH2), 3.78 (1H, m, CH-cy), 7.86 (2H, d, J = 6.8 Hz, 2 x CH-Ar), 8.00 (2H, d, J = 6.8 Hz, 2 x CH-Ar), 8.43 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.0 (2 x CH2), 25.3 (2 x CH2 -cy), 25.7 (CH2-cy), 32.8 (2 x CH2 cy), 49.0 (CH-cy), 50.0 (2 x CH2), 127.1 (2 x CH-Ar), 128.7 (2 x CH-Ar), 138.9 (C-Ar), 142.0 (C-Ar), 165.0 (C=O). 4 - (N, N- di p ro py ls ulf am o yl )-N - (2- m et h ylc y cl oh ex yl ) b enz a m i de (3 ): 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.30 g of 2-methylcyclohexylamine (2.62 mmol, 1.5 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 2:1) gave the title compound as yellow gum. Crystallization from n-hexane/ethyl acetate (1:1) gave title compound as a white solid (0.085 g, 13% yield); mp 93-94 °C; IR νmax 3315 (ν N-H), 2932 (ν Csp2-H), 1633 (ν C=O), 1332 (νas S=O), 1159 (νs S=O), 741 (δ Csp2-H) cm-1 ; 1 H-NMR (400 MHz, CDCl3) δ 0.86 (6H, t, J = 7.2 Hz, 2 x CH3), 0.98 (3H, d, J = 6.4 Hz, CH3), 1.16 (2H, m, CH2-cy), 1.39 (2H, m, 2 x CH-cy), 1.53 (4H, sext, J = 7.2 Hz, 2 x CH2), 1.76 (4H, m, 4 x CH-cy), 2.03 (1H, d, J = 10.8 Hz, CH-cy) 3.06 (4H, t, J = 7.6 Hz, 2 x CH2 ), 3.70 (1H, m, CH-cy), 6.17 (1H, bs, NH), 7.79 (2H, d, J = 8.0 Hz, 2 x CH-Ar), 7.84 (2H, d, J = 8.0 Hz, 2 x CH-Ar); 13 C-NMR (101 MHz, CDCl 3) δ 11.2 (2 x CH3), 19.2 (CH3), 22.0 (2 x CH2), 25.4 (CH2 -cy), 25.7 (CH2-cy), 33.6 (CH2-cy), 34.3 (CH2 -cy), 38.5 (CH-cy), 50.0 (2 x CH2), 54.9 (CH-cy), 127.2 (2 x CH-Ar), 127.6 (2 x CH-Ar), 138.9 (C-Ar), 142.5 (C-Ar), 165.8 (C=O). 4 - (N, N- di p ro py ls ulf am o yl )-N - (2- m o rph ol i no et hyl ) b enz a m i de (4 ): 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.35 ml of N-2-(aminoethyl)morpholine (2.63 mmol, 1.5 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. After four hours the reaction was diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (ethyl acetate:petroleum ether 10:1) gave title compound as a white solid (0.32 g, 46% yield); mp 90-93 °C; IR
νmax 3265 (ν N-H), 2960 (ν Csp2-H), 1639 (ν C=O), 1340 (νas S=O), 1150 (νs S=O), 743 (δ Csp2 -H) cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.86 (6H, t, J = 7.6 Hz, 2 x CH3), 1.53 (4H, sext, J = 7.6 Hz, 2 x CH2), 2.51 (4H, m, 2 x CH2-cy), 2.61 (2H, t, J = 5.6 Hz, CH2), 3.07 (4H, t, J = 7.2 Hz, 2 x CH2), 3.56 (2H, t, J = 4.8 Hz, CH2), 3.72 (4H, m, 2 x CH2-cy), 6.95 (1H, bs, NH), 7.84 (4H, m, 4 x CH-Ar); 13C-NMR (101 MHz, DMSO-d6) δ 11.2 (2 x CH3), 22.0 (2 x CH2), 36.2 (CH2), 50.0 (2 x CH2), 53.3 (CH2), 56.9 (2 x CH2 -cy), 66.9 (2 x CH2-cy), 127.3 (2 x CH-Ar), 127.7 (2 x CH-Ar), 138.2 (C-Ar), 143.0 (C-Ar), 166.0 (C=O). 4 - (di pr op yl s ulf a m oyl )- N- p hen yl b enz am i de) (5) : 1,1’carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.18 g of aniline (1.93 mmol, 1.1 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,Ndimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 2:1) gave the title compound as a white solid (0.41 g, 65% yield); mp 113-114 °C; IR νmax 3382 (ν N-H), 2964 (ν Csp2 H), 1678 (ν C=O), 1326 (νas S=O), 1153 (νs S=O), 855 (δ Csp2 -H), cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.79 (6H, t, J = 7.2 Hz, 2 x CH3), 1.46 (4H, sext, J = 7.2 Hz, 2 x CH2), 3.05 (4H, t, J = 6.8 Hz, 2 x CH2), 7.10 (1H, t, J = 7.2 Hz, CH-Ar), 7.34 (2H, t, J = 7.6 Hz, 2 x CH-Ar), 7.76 (2H, d, J = 8.0 Hz, 2 x CH-Ar), 7.92 (2H, d, J = 8.0 Hz, 2 x CH-Ar), 8.00 (2H, d, J = 8.0 Hz, 2 x CH-Ar), 10.46 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.1 (2 x CH2), 50.1 (2 x CH2), 120.9 (2 x CH-Ar), 124.5 (CH-Ar), 127.3 (2 x CH-Ar), 129.1 (2 x CH-Ar), 139.0 (2 x CHAr), 139.3 (C-Ar) 142.4 (C-Ar), 164.9 (C=O), (1C peak missing because of overlapping signals). 4 - (N, N- di pr o py ls ulf am o yl )-N - (pyri di n- 2- yl ) b enz a m i de (6) : 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.18 g of 2-aminopyridine (1.92 mmol, 1.1 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight After 24 h, additional 0.28 g of CDI (1.75 mmol, 1.0 eq) was added and the reaction was stirred overnight. After 24 h, additional 0.08 g of 2aminopyridine (0.875 mmol, 0.5 eq) were added and the reaction stirred for 24 h. The reaction was diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 1:1) gave the title compound as a white solid (0.16 g, 24% yield); mp 112-115 °C; IR νmax 3310 (ν N-H), 2965 (ν Csp2-H), 1529 (ν C=O), 1300 (νas S=O), 1156 (νs S=O), 870 (ν C=N), 784 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.83 (6H, t, J = 7.2 Hz, 2 x CH3), 1.51 (4H, sext, J = 7.6 Hz, 2 x CH2), 3.06 (4H, t, J = 7.6 Hz, 2 x CH2), 7.00 (1H, t, J = 6.0 Hz, 1 x CH-pyr), 7.70 (1H, t, J = 7.6 Hz, 1 x CH-pyr), 7.85 (2H, d, J = 7.2 Hz, 2 x CH-Ar), 8.01 (3H, m, 2 x CH-Ar + CHpyr), 8.34 (1H, d, J = 8.4 Hz, CH-pyr), 9.65 (1H, bs, NH); 13CNMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 21.9 (2 x CH2), 49.3 (2 x CH2), 114.7 (CH-Ar), 120.3 (CH-Ar), 127.3 (2 x CHAr), 128.2 (2 x CH-Ar), 137.8 (CH-Ar), 138.7 (C-Ar), 143.6 (CH-Ar), 147.7 (C-Ar), 151.5 (C-Ar), 167.8 (C=O).
A
B
His64
Trp5
C His64
His64
Asn62
Trp5
Trp5 Asn62
Asn62
Thr200 Thr200
His94
His94
2+
Zn
Zn2+
His94
Zn2+
Thr199
Lys67
Thr199
Lys67 Gln92
Lys67 Gln92
Gln92
Thr91
Ser135
Ser135
Thr91 Ser132
Thr91
Ser132
D
E
His64
F Thr199
Thr200 Thr199
2+
Zn
Thr200
Zn2+ Thr199
His94
His94 Trp5
Lys67
Thr200
Gln92
Trp5
Gln92 Ser135
Gln92
His94 His64
Lys67
Thr91
Asn62
Ser132
Lys67 Asn62
His64
Figure 1. Docked poses of compound 4 (A), compound 3 (B), compound 5 (C), compound 10 (D), probenecid (E) and compound 1 (F) with the active site of tumour-associated hCA XII. Hydrogen bonds are indicated with red dotted lines, binding interactions to the Zn2+ ion are indicated with blue dotted lines, cation-π interactions are indicated with red double-headed arrows, and major hydrophobic stackings are indicated with green double-headed arrows.
4 - (N, N- di p ro py ls ulf am o yl )-N - (1, 3, 4-t hi ad i az ol - 2- yl) b enz a m i de (7 ): 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.2 g of 2-amino-1,3,4-thiadiazole (1.92 mmol, 1.1 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. After 48 h, additional 0.28 g of CDI (1.75 mmol, 1.0 eq) were added and the reaction stirred overnight. The reaction was diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 1:2) gave title compound as a white solid (0.26 g, 40% yield); mp 180-182 °C; IR νmax 3139 (ν N-H), 2961 (ν Csp2 -H), 1536 (ν C=O), 1338 (νas S=O), 1152 (νs S=O), 735 (δ Csp2 -H), 681 (ν C-S) cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.79 (6H, t, J = 7.4 Hz, 2 x CH3), 1.46 (4H, sext, J = 7.2 Hz, 2 x CH2), 3.05 (4H, t, J = 7.6 Hz, 2 x CH2), 7.95 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 8.26 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 9.24 (1H, s, CH-Ar), 13.35 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.0 (2 x CH2), 50.0 (2 x CH2), 127.4 (2 x CH-Ar), 129.9 (2 x CH-Ar), 135.8 (C-Ar), 143.6 (CH-Ar), 149.7 (C-Ar), 166.0 (C=O), (1C peak missing because of overlapping signals). N -(3 -c hl o r op hen yl )- 4- (di pr op yl s ulf a m oyl )b enz am i de (8 ) : 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and
0.25 g of 3-chloroaniline (1.93 mmol, 1.1 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 3:1) gave title compound as a white solid (0.31 g, 45% yield); mp 117-119 °C; IR νmax 3289 (ν N-H), 2968 (ν Csp2 -H), 1668 (ν C=O), 1334 (νas S=O), 1153 (νs S=O), 835 (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 = 7.6 Hz, 2 x CH2), 3.07 (4H, t, J = 7.2 Hz, 2 x CH2), 7.20 (1H, m, J1 = 8.0 Hz, J2 = 2.2 Hz, J3 = 1.2 Hz, CH-Ar), 7.41 (1H, t, J = 8.0 Hz, CH-Ar), 7.71 (1H, m, J1 = 8.0 Hz, J2 = 2.0 Hz, J3 = 0.8 Hz, CH-Ar), 7.96 (2H, d, J = 8.8 Hz, 2 x CH-Ar), 7.97 (1H, s, CH-Ar), 8.13 (2H, d, J = 8.8 Hz, 2 x CH-Ar), 10.62 (1H, s, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.0 (2 x CH2), 50.1 (2 x CH2), 119.2 (CH-Ar), 120.3 (CH-Ar), 124.2 (CH-Ar), 127.4 (2 x CH-Ar), 129.2 (2 x CH-Ar), 130.9 (CH-Ar), 133.5 (C-Ar), 138.6 (C-Ar), 140.8 (CAr), 142.7 (C-Ar), 165.1 (C=O). N - (4 -c hl o r op hen yl )- 4- (di pr op yl s ulf a m oyl )b en z ami de (9 ) : 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.25 g of 4-chloroaniline (1.93 mmol, 1.1 eq) were added to a
stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. The reaction was stirred overnight, diluted with dichloromethane and washed with saturated NaHCO3. The organics were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel (petroleum ether:ethyl acetate 1:1) gave title compound as a white solid (0.27 g, 46% yield); mp 164-166 °C; IR νmax 3382 (ν N-H), 2319 (ν Csp2 -H), 1668 (ν C=O), 1336 (νas S=O), 1155 (νs S=O), 836 (δ Csp2 -H), cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 0.82 (6H, t, J = 7.6 Hz, 2 x CH3), 1.50 (4H, sext, J = 7.6 Hz, 2 x CH2), 3.07 (4H, t, J = 7.6 Hz, 2 x CH2 ), 7.43 (2H, d, J = 8.8 Hz, 2 x CH-Ar), 7.82 (2H, d, J = 8.8 Hz, 2 x CH-Ar), 7.96 (2H, d J = 8.4 ,2 x CH-Ar), 8.12 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 10.61 (1H, bs, NH); 13CNMR (101 MHz, DMSO-d6) δ 11.5 (2 x CH3), 22.0 (2 x CH2), 50.1 (2 x CH2), 122.4 (2 x CH-Ar),127.4 (2 x CH-Ar), 129.1 (2 x CH-Ar), 129.2 (2 x CH-Ar), 138.3 (C-Ar) 138.7 (C-Ar), 141.7 (C-Ar), 142.5 (C-Ar), 165.0 (C=O). 4 - (N, N- di p ro py ls ulf am o yl )-N - (3- ni tr op h en y l ) b enz a m i de (10 ) : 1,1’-carbonyldiimidazole (0.43 g, 2.63 mmol, 1.5 eq) and 0.27 g of 3-nitroaniline (1.93 mmol, 1.1 eq) were added to a stirring solution of probenecid (0.5 g, 1.75 mmol, 1.0 eq) in 5.0 mL of dry N,N-dimethylformamide at room temperature. After four hours the reaction was diluted with dichloromethane and washed with saturated NaHCO3. 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 title compound as a white solid (0.083 g, 10% yield); mp 185-188 °C; IR νmax 3382 (ν N-H), 2969 (ν Csp2-H), 1672 (ν C=O), 1534 (ν NO), 1329 (νas S=O), 1256 (ν N-O), 1160 (νs S=O), 740 (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 = 7.6 Hz, 2 x CH2), 3.08 (4H, t, J = 7.2 Hz, 2 x CH2), 7.68 (1H, t, J = 8.0 Hz, CH-Ar), 7.99 (3H, m, 3 x CH-Ar), 8.18 (3H, m, 3 x CH-Ar), 8.80 (1H, s, CH-Ar), 10.92 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.1 (2 x CH2), 50.1 (2 x CH2), 119.0 (CH-Ar), 126.7 (CH-Ar), 127.4 (2 x CH-Ar), 129.3 (2 x CH-Ar), 130.6 (CH-Ar), 138.2 (CH-Ar), 140.5 (C-Ar), 142.9 (C-Ar), 148.4 (C-Ar), 152.7 (CAr), 165.3 (C=O). 4- (N, N -di p ro pyls ulf a m oyl )-N - (2-ni tr o ph en y l ) b enz a m i de (11 ) : 1.00 g of probenecid (3.5 mmol, 1.00 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, 2-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 2-nitroaniline and the reaction stirred for 24 h at room temperature. The reaction was evaporated in vacuo and the residue purified on silica gel (petroleum ether:ethyl acetate 10:1) to give title compound as a yellow solid (0.71 g, 50% yield); mp 127-129 °C; IR νmax 3340 (ν N-H), 2964 (ν Csp2 H), 1695 (ν C=O), 1514 (ν N-O), 1336 (νas S=O), 1272 (ν N-O), 1152 (νs S=O), 1272 (ν N-O), 741 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, CDCl3) δ 0.88 (6H, t, J = 7.4 Hz, 2 x CH3), 1.57 (4H, sext, J = 7.6 Hz, 2 x CH2), 3.13 (4H, t, J = 7.6 Hz, 2 x CH2), 7.27 (1H, m, CH-Ar), 7.74 (1H, t, J = 7.8 Hz, CH-Ar), 7.97 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 8.10 (2H, d, J = 8.4 Hz, 2 x CH-Ar), 8.30 (1H, d, J = 8.4 Hz, CH-Ar), 8.96 (1H, d, J = 8.4 Hz, CH-Ar), 11.40 (1H, bs, NH); 13C-NMR (101 MHz, CDCl3) δ 11.2 (2 x CH3), 22.0 (2 x CH2), 50.0 (2 x CH2), 117.7 (CH-Ar), 122.2 (CH-Ar), 123.9 (CH-Ar), 126.0 (CH-Ar), 127.7 (2 x CH-Ar), 128.1 (2 x
CH-Ar), 134.7 (C-Ar), 136.3 (C-Ar), 136.6 (C-Ar), 137.3 (C-Ar), 164.2 (C=O). 4 - (N, N- di pr o py ls ulf am o yl )-N - (2- a mi no p heny l ) b enz a m i de (12 ) : 0.200 g of 4-(N,N-dipropylsulfamoyl)-N-(2nitrophenyl)benzamide (10) (0.5 mmol, 1.00 eq) was dissolved in 50 mL of methanol. Palladium on carbon (10% Pd basis, 20 mg) was added and stirred under hydrogen pressure (1.85 bar) for 4 h. The palladium was filtered off through Celite® and the filtrate was evaporated in vacuo to give title compound as a white solid (0.140 g, 75% yield); mp: 165-167°C; IR νmax 3397 (ν N-H), 3270 (ν N-H(H)), 2967 (ν Csp2-H), 1530 (ν C=O), 1339 (νas S=O), 1152 (νs S=O), 741 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, DMSOd6) δ 0.84 (6H, t, J = 6.8 Hz, 2 x CH3), 1.49 (4H, sext, J = 6.8 Hz, 2 x CH2), 3.06 (4H, t, J = 6.4 Hz, 2 x CH2), 4.98 (2H, bs, NH2), 6.59 (1H, t, J = 6.4 Hz, CH-Ar), 6.79 (1H, d, J= 7.2 Hz, CH-Ar), 6.98 (1H, t, J = 7.2 Hz, CH-Ar), 7.17 (1H, d, J = 7.2 Hz, CH-Ar), 7.93 (2H, d, J = 6.8 Hz, 2 x CH-Ar), 8.17 (2H, d, J = 7.2 Hz, 2 x CH-Ar), 9.88 (1H, bs, NH); 13C-NMR (101 MHz, DMSO-d6) δ 11.4 (2 x CH3), 22.1 (2 x CH2), 50.1 (2 x CH2), 116.5 (CH-Ar), 116.6 (CH-Ar), 123.2 (CH-Ar), 127.1 (CH-Ar), 127.2 (CH-Ar), 127.3 (CH-Ar), 129.3 (2 x CH-Ar), 138.7 (2 x C-Ar), 142.3 (C-Ar), 143.8 (C-Ar), 164.7 (C=O). 6.3. Molecular modelling studies 6 . 3. 1. Pr ep a ra ti o n of li g an d fil es Compounds were built in 3D using MOE (version 2013.0801, Chemical Computing Group Inc., Montreal, Canada). All strong bases were protonated and all strong acids were deprotonated using and a subsequent steepest-descent energy minimization was performed using the MMFF94x forcefield. The ligand files were saved as multi-mol2 files. 6 . 3. 2. Pr ep a ra ti o n of p r ot ei n fi l es The crystal structures of hCA XII in complex with acetazolamide (1JD0; 1.50 Å) was obtained from the Protein Data Bank (PDB server). Chain A was retained when more than one chain was present. The cocrystallized inhibitor and the Zn2+ ion were retained and all other water molecules, buffers and ions were deleted. Hydrogen atoms were added using the “protonate 3D” tool of MOE and the system was energy minimized using the AMBER99 forcefield. 6 . 3. 3. D oc ki ng st udi es Docking studies were performed using the GOLD Suite package (version 5.2, Cambridge Crystallographic Data Centre, Cambridge, UK). Probenecid and its analogs 1–12 were docked into all protein structures (25 dockings per ligand) and scored with the GoldScore scoring function. The binding pocket was defined as all residues within 13 Å of a centroid (X: -17.071, Y: 35.081, Z: 43.681; this point corresponds approximately to the location of the thiadiazole ring of acetazolamide in the 1JDO structure).
Acknowledgments This work was financed by two FP7 EU projects (Metoxia and Dynano) to CTS. References and notes (1) (2) (3) (4)
Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem. Rev. 2012, 112, 4421. Supuran, C.T. Nat. Rev. Drug Discov. 2008, 7, 168. Hilvo, M.; Innocenti, A.; Monti, S. M.; De Simone, G.; Supuran, C. T.; Parkkila, S. Curr. Pharm. Des. 2008, 14, 672. (a) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146; (b) Supuran, C. T.; Scozzafava, A. Expert Opin. Ther. Pat.
(5)
(9) (10) (11) (12) (13) (14) (15) (16) (17)
(18)
(19) (20)
2000, 10, 575; (c) Swietach, P.; Wigfield, S.; Cobden, P.; Supuran, C. T.; Harris, A. L.; Vaughan-Jones, R. D. J. Biol. Chem. 2008, 283, 20473. (a) Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 677; (b) Supuran, C. T. Future Med. Chem. 2011, 3, 1165. (c) Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2004, 19, 199. Weber, C. E.; Kuo, P. C. Surg. Oncol. 2012, 21, 172. Warburg, O. Science 1956, 124, 269. Warburg, O.; Wind, F.; Negelein, E. J. Gen. Physiol. 1927, 8, 519. Gatenby, R. A.; Gillies, R. J. Nat. Rev. Cancer 2004, 4, 891. Webb, B. A.; Chimenti, M.; Jacobson, M. P.; Barber, D. L. Nat. Rev. Cancer 2011, 11, 671. Gatenby, R. A.; Smallbone, K.; Maini, P. K.; Rose, F.; Averill, J.; Nagle, R. B.; Worrall, L.; Gillies, R. J. Br. J. Cancer 2007, 97, 646. Dang, C. V.; Semenza, G. L. Trends Biochem. Sci. 1999, 24, 68. Semenza, G. L. Crit. Rev. Biochem. Mol. Biol. 2000, 35, 71. (a) Parks, S. K.; Chiche, J.; Pouysségur, J. Nat. Rev. Cancer 2013, 13, 611; (b) Lock, F. E.; McDonald, P. C.; Lou, Y.; Serrano, I.; Chafe, S. C.; Ostlund, C.; Aparicio, S.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Oncogene, 2013, 32, 5210; (c) Ward, C.; Langdon, S. P.; Mullen, P.; Harris, A. L.; Harrison, D. J.; Supuran, C. T.; Kunkler, I. H. Cancer Treat. Rev. 2013, 39, 171; d) Potter, C. P. S.; Harris, A. L. Br. J. Cancer 2003, 89, 2. (a) McDonald, P. C.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Oncotarget 2012, 3, 84; (b) Lou, Y.; McDonald, P. C.; Oloumi, A.; Chia, S. K.; Ostlund, C.; Ahmadi, A.; Kyle, A.; Auf dem Keller, U.; Leung, S.; Huntsman, D. G.; Clarke, B.; Sutherland, B. W.; Waterhouse, D.; Bally, M. B.; Roskelley, C. D.; Overall, C. M.; Minchinton, A.; Pacchiano, F.; Carta, F.; Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Cancer Res. 2011, 71, 3364; (c) Lounnas, N.; Rosilio, C.; Nebout, M.; Mary, D.; Griessinger, E.; Neffati, Z.; Spits, H.; Hagenbeek, T.; Asnafi, V.; Poulsen, S. A.; Supuran, C. T.; Peyron, J. F.; Imbert, V. Cancer Lett. 2013, 333, 76. Neri, D.; Supuran, C. T.; Nat. Rev. Drug Discov. 2011, 10, 767. (a) Winum, J. Y.; Carta, F.; Ward, C.; Mullen, P.; Harrison, D.; Langdon, S. P.; Cecchi, A.; Scozzafava, A.; Kunkler, I.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2012, 22, 4681; (b) Ebbesen, P.; Pettersen, E. O.; Gorr, T. A.; Jobst, G.; Williams, K.; Kieninger, J.; Wenger, R. H.; Pastorekova, S.; Dubois, L.; Lambin, P.; Wouters, B. G.; Van Den Beucken, T.; Supuran, C. T.; Poellinger, L.; Ratcliffe, P.; Kanopka, A.; Görlach, A.; Gasmann, M.; Harris, A. L.; Maxwell, P.; Scozzafava, A. J. Enzyme Inhib. Med. Chem. 2009, 24, 1.
(21)
(22)
(23) (24) (25)
(26)
(27) (28)
(29) (30)
(31)
(32) (33)
Tafreshi, N. K.; Bui, M. M.; Bishop, K.; Lloyd, M. C.; Enkemann, S. A.; Lopez, A. S.; Abrahams, D.; Carter, B. W.; Vagner, J.; Grobmyer, S. R.; Gillies, R. T.; Morse, D. C. Clin. Cancer Res. 2012, 18, 207. a) Gieling, R. G.; Williams, K. J. Bioorg. Med. Chem. 2013, 21, 1470; b) Borras, J.; Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Mincione, G.; Supuran, C.T. Bioorg. Med. Chem. 1999, 7, 2397. Said, H. M.; Supuran, C. T.; Hageman, C.; Staab, A.; Polat, B.; Katzer, A.; Scozzafava, A.; Anacker, J.; Flentje, M.; Vordermark, D. Curr. Pharm. Des. 2010, 16, 3288. Supuran, C.T.; Mincione, F.; Scozzafava, A.; Briganti, F.: Mincione, G.; Ilies, M.A. Eur. J. Med. Chem. 1998, 33, 247. a) Demirdağ, R.; Çomaklı, V.; Şentürk, M.; Ekinci, D.; İrfan Küfrevioğlu, Ö.; Supuran, C. T. Bioorg. Med. Chem. 2013, 21, 1522; b) Briganti, F.; Pierattelli, R.; Scozzafava, A.; Supuran, C.T. Eur. J. Med. Chem. 1996, 31, 1001. Rogez-Florent, T.; Meignan, S.; Foulon, C.; Six, P.; Gros, A.;BalMahieu, C.; Supuran, C. T.; Scozzafava, A.; Frédérick, R.; Masereel, B.; Depreuxa, P.; Lansiauxa, A.; Goossensa, J. F.; Gluszoka, S.; Goossens, L. Bioorg. Med. Chem. 2013, 21, 1451. Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. In Drug Design of Zinc-Enzyme Inhibitors; Supuran, C. T.; Winum, J.-Y., Eds.; John Wiley & Sons, Inc., 2009; pp. 138. Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.; Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti, S. M.; De Simone, G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233. Alp, C.; Maresca, A.; Alp, N. A.; Gültekin, M. S.; Ekinci, D.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 294. (a) Métayer, B.; Martin-Mingot, A.; Vullo, D.; Supuran, C. T.; Thibaudeau, S. Org. Biomol. Chem. 2013, 11, 7540; (b) Métayer, B.; Mingot, A.; Vullo, D.; Supuran, C. T.; Thibaudeau, S. Chem. Commun. 2013, 49, 6015. (a) D’Ascenzio, M.; Carradori, S.; De Monte, C.; Secci, D.; Ceruso, M.; Supuran, C. T. Bioorg. Med. Chem. 2014, 22, 1821; (b) Akdemir, A.; De Monte, C.; Carradori, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, DOI: 10.3109/14756366.2014.892936. (c) Carradori, S.; De Monte, C.; D’Ascenzio, M.; Secci, D.; Celik, G.; Ceruso, M.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2013, 23, 6759. Puscas, I.; Coltau, M.; Pasca, R. J. Pharmacol. Exp. Ther. 1996, 277, 1464. Cheng, H. C. J. Pharmacol. Toxicol. Methods 2001, 46, 61.
Selective inhibition of human carbonic anhydrases by novel amide derivatives of probenecid: synthesis, biological evaluation and molecular modelling studies Melissa D’Ascenzio, Simone Carradori, Daniela Secci, Daniela Vullo, Mariangela Ceruso, Atilla Akdemir, Claudiu T. Supuran
