Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx

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Inhibition of carbonic anhydrase isoforms I, II, IX and XII with novel Schiff bases: Identification of selective inhibitors for the tumorassociated isoforms over the cytosolic ones Busra Sarikaya, Mariangela Ceruso, Fabrizio Carta, Claudiu T. Supuran ⇑ Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm 188, Italy Neurofarba Department, Sezione di Scienze Farmaceutiche, Via U. Schiff 6, I-50019 Sesto Fiorentino (Firenze), Italy

a r t i c l e

i n f o

Article history: Received 2 August 2014 Revised 9 September 2014 Accepted 10 September 2014 Available online xxxx Keywords: Carbonic anhydrase Imine Sulfonamide Isoform-selective inhibitor Tumor-associated enzymes

a b s t r a c t A series of new Schiff bases was obtained from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)benzenesulfonamide and aromatic/heterocyclic aldehydes incorporating both hydrophobic and hydrophilic moieties. The obtained sulfonamides were investigated as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, as well as the transmembrane, tumor-associated CA IX and XII. Most derivatives were medium potency or weak hCA I/II inhibitors, but several of them showed nanomolar affinity for CA IX and/or XII, making them an interesting example of isoform-selective compounds. The nature of the aryl/hetaryl moiety present in the initial aldehyde was the main factor influencing potency and isoform selectivity. The best and most CA IX-selective compounds incorporated moieties such as 4-methylthiophenyl, 4-cyanophenyl-, 4-(2-pyridyl)-phenyl and the 4-aminoethylbenzenesulfonamide scaffold. The best hCA XII inhibitors, also showing selectivity for this isoform, incorporated 2-methoxy-4-nitrophenyl-, 2,3,5,6-tetrafluorophenyl and 4(2-pyridyl)-phenyl functionalities and were also derivatives of 4-aminoethylbenzenesulfonamide. The sulfanilamide and 3-fluorosulfanilamide derived Schiff bases were less active compared to the corresponding 4-aminoethyl-benzenesulfonamide derivatives. As hCA IX/XII selective inhibition is attractive for obtaining antitumor agents/diagnostic tools with a new mechanism of action, compounds of the type described here may be considered interesting preclinical candidates. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Schiff bases incorporating aromatic/heterocyclic sulfonamide moieties in their molecules have been extensively investigated as carbonic anhydrase (CA, EC 4.2.1.1)1–3 inhibitors (CAIs) over the last years.4–7 In fact, some of these derivatives were the first reported sulfonamides to possess some selectivity for the inhibition of some human (h) CA isoforms, such as hCA I, II or IV.4–7 Indeed, this superfamily of enzymes acting as catalysts for CO2 hydration to bicarbonate and protons, comprises a large number of genetic families (six, i.e., the a-, b-, c-, d-, f- and g-CAs), as well as numerous isoforms in most organisms investigated so far.1–3 For example, sixteen isozymes have been described in mammals. Some of them are cytosolic (CA I, CA II, CA III, CA VII and CA XIII), others are membrane bound (CA IV, CA IX, CA XII, CA XIV and CA XV), two are mitochondrial (CA VA, CA VB) and one is secreted in saliva and ⇑ Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385. E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).

milk (CA VI). Three of these proteins are catalytic (CA VIII, X and XI).1–3 CA I and CA II are the two major CA isozymes present at high concentrations in the cytosol of red blood cells of most vertebrates, and CA II is also one of the most active of all CAs, together with the tumor-associated, transmembrane isoforms CA IX.1–3 CA XII, another transmembrane isoform, as hCA IX, is also present in some tumors, but more diffuse in normal tissues compared to hCA IX.1 Many hCAs are therapeutic targets with the potential to be inhibited or activated, which elicits pharmacologic effects.8–10 For example, inhibitors targeting hCA II/IV/XII are used for the treatment of glaucoma, those targeting hCA VII/XIV for epilepsy management, whereas some hCA II/IV inhibitors are used as diuretics.8–10 hCA IX/XII inhibitors show applications as diagnostic tools for imaging hypoxic tumors, but several such agents are in early phase clinical development for the treatment of hypoxic tumors nonresponsive to the classical chemo- or radiotherapy.11–13 Inhibition of hCA IX/XII leads to the impairment of the growth of the primary tumor and metastases, and also to the depletion of the cancer stem

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B. Sarikaya et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

cell population, all these phenomena being useful in the management of hypoxic tumors, for which few therapeutic options are available nowadays.11–13 For such reasons, the design of new, potent and isoform-selective inhibitors of various CAs, may lead to clinical applications for treating a multitude of diseases.1–3 The Schiff bases containing sulfonamide inhibitors are in fact just one of the inhibitor classes investigated for such purposes. In addition to this, the reversible reaction between an amine and an aldehyde/ ketone has been employed to generate Dynamic Combinatorial Libraries-DCLs of sulfonamide CAIs, in which the enzyme acted as a template for the imine reaction formation, ‘selecting’ for the most effective inhibitors and avoiding thus time-consuming screening assays.6,7 Considering the fact that in most sulfonamide CAIs the tail functionalities are responsible for the potency and isoform-selectivity,14,15 we investigated a new series of Schiff bases incorporating aromatic sulfonamide and various aromatic/heterocyclic moieties (coming from the aldehyde component of the imine formation reaction) with the aim to obtain potent and selective inhibitors for the tumor-associated isoforms (hCA IX and XII) over the cytosolic, widespread ones hCA I and II. The facile synthesis of this class of CAIs as well as the availability of a large number of variously substituted amino-sulfonamides and aldehydes (as starting materials), make this class of compounds highly attractive for designing isoform-selective inhibitors. 2. Results and discussion 2.1. Chemistry Reaction of aromatic sulfonamides with aromatic/heterocyclic aldehydes in methanol allowed us to obtain the new Schiff bases 1–17 (Scheme 1) by the routine procedure of obtaining imines.4–7 The rationale for obtaining these new compounds is presented in the following. We have demonstrated earlier6,7,14,15 that the two main factors influencing the inhibition profile of sulfonamide CAIs against various mammalian isoforms are (i) the nature of the arylsulfonamide moiety which directly binds to the zinc ion from the enzyme active site (as sulfonamidate anion) and, (ii) the presence as well as chemical nature of the tails from the inhibitor molecule, which usually bind towards the middle part or exit of the active site cavity, contributing thus effectively to the selectivity of the inhibitors towards various isoforms, since those are the active site regions which are more variable among the 16 mammalian isoforms.1–3 By means of X-ray crystallography we and other researchers showed the contribution of these two factors in obtaining isoform-selective sulfonamide CAIs. For example, the presence of only hydrogens (as in compounds 1–3) or a 3-fluoro substituent (as in 4–6) on the phenylsulfamoyl ring which binds the metal ion may lead to different conformations of the arylsulfamoyl ring in the enzymeinhibitor adduct, which has as a consequence a very diverse inhibition profile against isoforms hCA I, II; VA, VB, VII, IX and XII.14a,b The same effect (a tilting of the arylsulfamoyl ring when bound to the active site metal ion) was observed for compound incorporating a 3-pyridylsulfamoyl instead of phenylsulfamoyl SO2NH2

SO2NH2 Ar-CHO X

dry MeOH n

NH2

n =0,2 X= H, F

X n

N Ar 1-17

Scheme 1. Preparation of Schiff bases 1–17.

moiety.14c This is the reason why we used three different arylsulfonamides for obtaining the new Schiff bases, sulfanilamide, 3-fluorosulfanilamide and 4-aminoethylbenzenesulfonamide (Scheme 1). In earlier work on Schiff bases as CAIs we showed4–7 that the nature of the tail, coming from the starting aldehyde/ketone employed in the synthesis, is one of the main factors governing the CA inhibitory efficacy as well as selectivity for the various isoforms. Except one study, in which some chalcones were employed in the synthesis,4a all other sulfonamide Schiff bases acting as CAIs investigated to date were prepared by using aldehydes as starting materials.4–7 Thus, we followed the same strategy for synthesizing the new compounds reported here but chose aromatic as well as heterocyclic aldehydes which possess a range of substituents in their molecules in order to investigate whether the presence of such moieties may lead to isoform-selective CAIs against the tumor-associated isoforms hCA IX and XII (Table 1). Indeed, both benzaldehyde as well as mono-, di- and tetrasubstituted benzaldehydes (in various position of the phenyl ring) with halogens (fluoro and bromo-substituted benzaldehydes were employed), hydroxyl, methoxy, pyridyl, nitro, methylthio and cyano moieties were included in the study. Among the heterocyclic derivatives used in the synthesis of the Schiff bases were benzothiophen-3-aldehyde, 2-furylaldehyde, furyl-2-aldehyde-4-sulfonic acid, and 5-bromopyrrole-3-aldehyde. It should be mentioned that as a large number of Schiff bases incorporating aromatic/heterocyclic aldehydes and aromatic sulfonamides were reported earlier,4–7 the choice of these synthons might seem random, but this is not the case. In fact we considered the earlier compounds which showed lower affinity for the widespread, off-target cytosolic isoforms hCA I and II as leads for the synthesis of compounds reported here.

Table 1 Inhibition of hCA isoforms I, II, IX and XII with sulfonamides 1–17 reported here and acetazolamide (AAZ; 5-acetamido-1,3,4-thiadiazole-2-sulfonamide) as standard, by a stopped-flow CO2 hydrase assay16 KIa (nM)

No

X

n

Ar

1

H

0

4-Br-2-OH-C6H3

hCA I

hCA II

hCA IX 28.1

hCA XII

4320

707

58.5

4372

682

162

617

3055

664

197

614

499 248 1511 7005

710 63.5 498 701

28.0 165 113 244

58.0 349 611 307

N

2

H

0

3

H

0 S

4 5 6 7

F F F H

0 0 0 2

Ph 2-Furyl Biphen-4-yl Biphen-4-yl

8

H

2

NaO3S

9 10 11 12

H H H H

2 2 2 2

3-Br-2-OH-C6H3 2-OH-4-MeO-C6H3 4-Br-2-OH-C6H3 2-MeO-4-O2N-C6H3

13

H

2

14 15

H H

2 2

16

H

2

O

35b

4.9b

5335 4400 4235 215

>10000 697 666 168

193

54.4

251 1870 106 154

498 438 143 4.5

N

4-MeS-C6H4 4-NC-C6H4

1345

403

27.9

2940 2190

709 713

23.6 18.5

35.5

1750

596

1880

55.5

2630 250

652 12

213 25

12.9 5.7

241 123

Br N H

17 AAZ

H —

2 —

2,3,5,6-C6HF4 —

a Errors within the range of ±5–10% of the reported values, from 3 different assays (data not shown). b From Ref. 6a.

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Compounds 1–17 were fully characterized by physico-chemical and spectral procedures which confirmed their structures (see Section 4 for details). 2.2. Carbonic anhydrase inhibition Compounds 1–17 were assayed16 as inhibitors of four physiologically important CA isoforms, that is, the cytosolic CA I and II, as well as the transmembrane, tumor-associated CA IX and XII, by a stopped-flow CO2 hydrase assay method (Table 1). The following should be noted regarding their inhibition profile: (i) The two cytosolic isoforms hCA I and II were weakly or even poorly inhibited by sulfonamides 1–17 reported here, except 8 reported earlier6a which showed an inhibition constant of 35 nM against hCA I and 4.9 nM against hCA II. For hCA I the inhibition constants ranged between 215 and 7005 nM. Only two compounds showed inhibitory properties in the same range as the clinically used agent acetazolamide AAZ, with inhibition constants in the range of 215–250 nM. They are 5 and 12, and incorporate fluorosulfanilamide and 2-furyl moieties in the case of 5, and the longer 4-aminoethyl-benzenesulfonamide scaffold and 2-methoxy-4-nitrophenylidene moieties (in the case of 12). In the case of hCA II, compound 9 was not inhibitory up to concentrations of 10 lM, one derivative, 5, showed a rather effective inhibition of hCA II (KI of 63.5 nM, around 5 times lower compared to AAZ) whereas the remaining derivatives were weak hCA II inhibitors with inhibition constants ranging between 168 and 713 nM (Table 1). (ii) hCA IX was weakly inhibited by two Schiff bases, 10 and 16 (KIs of 1870–1880 nM), and moderately inhibited by a large number of the new derivatives, such as 2, 3, 5–9, 11, 12, and 17, which had KIs in the range of 106–213 nM. It may be observed that these weakly effective hCA IX inhibitors incorporate all the three sulfonamide scaffolds present in the new compounds reported here, as well as a multitude of aldehyde fragments. However a number of the new sulfonamides, such as 1, 4, and 13–15 were effective hCA IX inhibitors, with KIs ranging from 18.5 to 27.9 nM, which is in the same range as the standard drug acetazolamide (KI of 25 nM) for which significant antitumor effects were reported in animal models of hypoxic tumors.17 Thus, these effective hCA IX inhibitors incorporated in their molecules again all three sulfonamide scaffolds, and phenyl, 4bromo-2-hydroxyphenyl-, 4-(2-pyridyl)-phenyl, 4-methylthiophenyl- and 4-cyanophenyl moieties. It is thus clear that both hydrophilic tails (such as in 1) as well as more lipophilic ones (as in 4, and 13–15) are beneficial for obtaining effective hCA IX inhibitors. It should be also observed that all these effective hCA IX inhibitors are also medium potency or ineffective hCA I and II inhibitors, making them selective for the tumor-associated isoform. (iii) The second transmembrane isoform, hCA XII, was moderately inhibited by the following Schiff bases: 2, 3, 5–7, 9– 11, 14 and 15, which showed KIs in the range of 123– 617 nM (Table 1). Again the nature of the aryl sulfonamide scaffold seems to be less influential for the biological activity compared to the tail. On the other hand, effective hCA XII inhibition was registered for the following sulfonamides: 1, 4, 8, 12, 13, 16 and 17, with KIs in the range of 4.5– 58.5 nM (AAZ has a KI of 5.7 nM against this isoform). The best, low nanomolar inhibitor was 12 (KI of 4.5 nM) which incorporated the longer sulfonamide scaffold (4-aminoethyl-benzenesulfonamide) as well as 2-methoxy-4-nitrobenzylidene moieties. It is interesting to note that this

3

compound is a hCA XII selective inhibitor over hCA I (selectivity ratio of 47.7), hCA II (selectivity ratio of 37.3), and hCA IX (selectivity ratio of 34.2). As far as we know this is the first time that a selectivity profile like this is reported in the literature for a sulfonamide CAI. The remaining effective inhibitors of hCA XII incorporated again the various sulfonamide scaffolds as well as the following tails: 4-bromo-2-hydroxyphenyl-, phenyl, 4-(2-pyridyl)-phenyl, 5-sulfonate-2-furyl, and 2,3,5,6-tetrafluorophenyl. It is thus apparent that in this case, the more hydrophilic tails led to the most effective inhibitors.

3. Conclusion A series of new Schiff bases was obtained from sulfanilamide, 3fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide and aromatic/heterocyclic aldehydes incorporating both hydrophobic and hydrophilic moieties. The obtained sulfonamides were investigated as inhibitors of four CA isoforms, the cytosolic CA I and II, as well as the transmembrane, tumor-associated CA IX and XII. Most derivatives were medium potency or weak hCA I/II inhibitors, but several of them showed nanomolar affinity for CA IX and/or XII, making them interesting examples of isoform-selective compounds. The nature of the aryl/hetaryl moiety present in the initial aldehyde was the main factor influencing potency and isoform selectivity. The best and most CA IX-selective compounds incorporated moieties such as 4-methylthiophenyl, 4-cyanophenyl-, 4-(2-pyridyl)-phenyl and the 4-aminoethylbenzenesulfonamide scaffold. The best hCA XII inhibitors, also showing selectivity for this isoform, incorporated 2-methoxy-4-nitrophenyl-, 2,3,5,6-tetrafluorophenyl and 4-(2-pyridyl)-phenyl functionalities and were also derivatives of 4-aminoethylbenzenesulfonamide. The sulfanilamide- and 3-fluorosulfanilamidederived Schiff bases were generally less active compared to the corresponding 4-aminoethyl-benzenesulfonamide derivatives. As hCA IX/XII selective inhibition is attractive for obtaining antitumor agents/diagnostic tools with a new mechanism of action, compounds of the type described here may be considered interesting preclinical candidates.

4. Experimental 4.1. Chemistry Anhydrous solvents and all reagents were purchased from Sigma–Aldrich, Alfa Aesar and TCI. All reactions involving airor moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringe techniques to transfer solutions. Nuclear magnetic resonance (1H NMR, 13C NMR, DEPT-135, HSQC, HMBC) spectra were recorded using a Bruker Advance III 400 MHz spectrometer in DMSO-d6 or acetone-d6. Chemical shifts are reported in parts per million (ppm) and the coupling constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; br s, broad singlet; dd, doublet of doublets, appt, apparent triplet. The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D2O. Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel F-254 plates. Flash chromatography purifications were performed on Merck Silica gel 60 (230–400 mesh ASTM) as the stationary phase and ethyl acetate/n-hexane were used as eluents. Melting points (mp) were determined using an electrothermal melting point or a Kofler apparatus and are uncorrected.

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4.1.1. General procedure for preparation of compounds 1– 176b,18 Sulfanilamide (1.0 equiv) was dissolved in dry MeOH and the appropriate aldehyde (1.0 equiv) was added to the reaction. The solution was stirred until the formation of a precipitate was completed which was collected by filtration, washed with ice-cold MeOH and dried under vacuo to afford the desired compounds 1–17. Synthesis of 4-[(4-bromo-2-hydroxy-benzylidene)-amino]-benzenesulfonamide 1

Br

OH O N

dry MeOH, 0°C 45 mins

NH2

OH

Br 1

Sulfanilamide (0.1 g 1.0 equiv) and 4-bromo-2-hydroxybenzaldehyde (0.12 g 1.0 equiv) in MeOH were treated according to the general procedure at 0 °C to afford the title compound 1 as a pale yellow solid in 30% yield.

4.1.1.1. 4-[(4-Bromo-2-hydroxy-benzylidene)-amino]-benzenesulfonamide 1. Mp 227–228 °C; dH (400 MHz, DMSO-d6) 7.25 (1H, dd, J = 8.4, 1.6), 7.26 (1H, d, J = 4.0), 7.43 (2H, s, exchange with D2O, ASO2NH2), 7.60 (2H, d, J = 8.4), 7.71 (1H, d, J = 8.4), 7.92 (2H, d, J = 8.4), 9.02 (1H, s, AN@CHA) 12.93 (1H, s, AOH); dC (101 MHz, DMSO-d6) 119.7, 120.5, 122.7, 123.5, 127.9, 128.0, 134.6, 143.1, 152.0, 161.8, 164.7; m/z (ESI-MS-negative) 355.08 (M H) . Synthesis of 4-[(4-pyridin-2-yl-benzylidene)-amino]-benzenesulfonamide 2 SO2NH2

N

O

N

H

NH2

O S N dry MeOH reflux, 24h

NH2

S 3

Sulfanilamide (0.2 g, 1.0 equiv) was dissolved in 5 ml dry MeOH, thianapthene-3-carboxaldehyde (0.19 g, 1.0 equiv) was added at rt. The solution was stirred for 24 h at 70 °C, then cooled down to rt, stirred for 1 h at 0 °C until the formation of a precipitate was complete which was collected by filtration, washed with ice-cold MeOH and dried under vacuo to afford the title compound 3 as a pale yellow solid in 25% yield.

SO2NH2 SO2NH2

SO2NH2 SO2NH2

4.1.1.3. 4-[(Benzo[b]thiophen-3-ylmethylene)-amino]-benzenesulfonamide 3. Mp 165–166 °C; dH (400 MHz, DMSO-d6) 7.40 (2H, s, exchange with D2O, ASO2NH2), 7.50 (2H, d, J = 8.4), 7.56 (2H, m), 7.92 (2H, d, J = 8.4), 8.15 (1H, d, J = 7.2), 8.64 (1H, s), 8.94 (1H, s), 8.96 (1H, m); dC (101 MHz, DMSO-d6) 122.2, 124.0, 125.8, 126.4, 126.5, 127.9, 134.0, 136.7, 139.9, 141.2, 142.0, 155.6, 158.6; m/z (ESI-MS-positive) 317.17 (M+H)+. Synthesis of 4-(benzylidene-amino)-3-fluoro-benzenesulfonamide 4 F

F NH2

H2NO2S

O

N H2NO2S

dry MeOH reflux, 48h

4

4-Amino-3-flourobenzenesulfonamide (0.05 g, 1.0 equiv) was dissolved in 4 ml dry MeOH, then benzaldehyde (0.048 g, 1.0 equiv) was added to the solution. The solution was stirred for 48 h at 70 °C, then was concentrated under vacuo, triturated by diethyl ether to afford the title compound 4 as a light brown solid in 26% yield.

N

dry MeOH, 0°C 1h

SO2NH2 2

Sulfanilamide (0.1 g 1.0 equiv) and 4-(2-pyridyl)-benzaldehyde (0.11 g 1.0 equiv) in MeOH were treated according to the general procedure at 0 °C to afford the title compound 2 as a white solid in 34% yield.

4.1.1.2. 4-[(4-Pyridin-2-yl-benzylidene)-amino]-benzenesulfonamide 2. Mp 254–255 °C; dH (400 MHz, Acetone-d6) 6.62 (2H, s, exchange with D2O, ASO2NH2), 7.42 (1H, m), 7.45 (2H, d, J = 8.8), 7.95 (1H, dd, J = 8.0, 2.0), 7.99 (2H, d, J = 8.4), 8.09 (1H, d, J = 8.0), 8.15 (2H, d, J = 8.4), 8.35 (2H, d, J = 8.4), 8.74 (1H, s), 8.76 (1H, m); dC (101 MHz, DMSO-d6) 121.7, 122.3, 124.2, 127.9, 127.9, 130.4, 137.0, 138.4, 142.2, 142.6, 150.7, 155.3, 155.9, 163.2; m/z (ESI-MS-positive) 338.17 (M+H)+. Synthesis of 4-[(benzo[b]thiophen-3-ylmethylene)-amino]benzenesulfonamide 3

4.1.1.4. 4-(Benzylidene-amino)-3-fluoro-benzenesulfonamide 4. Mp 146–147 °C; dH (400 MHz, DMSO-d6) 7.52 (2H, s, exchange with D2O, ASO2NH2), 7.53 (1H, d, J = 8.0), 7.60 (3H, m), 7.71 (1H, s), 7.73 (1H, m), 8.01 (2H, d, J = 8.0), 8.74 (1H, s); dC (101 MHz, DMSO-d6) 115.2 (d, J2C–F 23.06), 123.6, 124.1 (d, J3C–F 4.7), 130.5, 130.6, 134.0, 136.6, 143.2 (d, J3C–F 5.6), 144.2 (d, J2C–F 10.0) 155.3 (d, J1C–F 249.0) 166.7; dF (376.5 MHz, DMSO-d6) 127.37; m/z (ESI-MS-negative) 277.0 (M H) . Synthesis of 3-fluoro-4-[(furan-2-ylmethylene)-amino]-benzenesulfonamide 5 O

F

O

F

NH2 H2NO2S

O N

dry MeOH reflux, 24h

H2NO2S 5

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4-Amino-3-fluorobenzenesulfonamide (0.1 g, 1.0 equiv) was dissolved in 4 ml dry MeOH, then furfuraldehyde (0.05 g, 1.0 equiv) was added. The solution was stirred for 24 h at 70 °C, then it was cooled down to rt, stirred for an hour at 0 °C until a precipitate was formed, which was collected by filtration, washed with ice-cold MeOH and dried under vacuo to afford the title compound 5 as a brown solid in 43% yield. 4.1.1.5. 3-Fluoro-4-[(furan-2-ylmethylene)-amino]-benzenesulfonamide 5. Mp 198–199 °C: dH (400 MHz, DMSO-d6) 6.81 (1H, m), 7.33 (1H, d, J = 3.2), 7.50 (1H, dd, J = 8.8, 8.0), 7.50 (2H, s, exchange with D2O, ASO2NH2), 7.70 (2H, m), 8.08 (1H, s), 8.54 (1H, s); dC (101 MHz, DMSO-d6) 113.8, 114.6 (d, J2C–F 22.09), 120.3, 122.9, 123.4 (d, J3C–F 3.07), 143.0 (d, J3C–F 6.0) 143.2 (d, J2C–F 10.4), 148.5, 152.3, 153.1 (d, J4C–F 1.70), 155.0 (d, J1C–F 249.2); dF (376.5 MHz, DMSO-d6) 124.14; m/z (ESI-MS-negative) 267.0 (M H) . Synthesis of 4-[(biphenyl-4-ylmethylene)-amino]-3-fluorobenzenesulfonamide 6 SO2NH2

O H

F NH2

N

dry MeOH reflux, 24h

F

O O H

O S O Na O

O Na O S O

N

O

dry MeOH, 0°C 30 mins

8

SO2NH2

NH2

4-(2-Aminoethyl)benzenesulfonamide (0.5 g, 1.0 equiv) and 5-formyl-2-furansulfonic acid sodium salt (0.49 g, 1.0 equiv) in 5 ml MeOH were treated according to the general procedure at 0 °C. The solution was stirred at the same temperature for 30 min, then the solution was concentrated under vacuo to afford the title compound 8 as a yellow solid in 92% yield. 4.1.1.8. Sodium 5-{[2-(4-sulfamoyl-phenyl)-ethylimino]methyl}-furan-2-sulfonate 8. Mp 227–228 °C; dH (400 MHz, DMSO-d6) 3.04 (2H, t, J = 7.0), 3.85 (2H, t, J = 7.0), 6.51 (1H, d, J = 3.2), 6.81 (1H, d, J = 3.2), 7.29 (2H, s, exchange with D2O, ASO2NH2), 7.48 (2H, d, J = 8.4), 7.77 (2H, d, J = 8.4), 8.12 (1H, s); dC (101 MHz, DMSO-d6) 37.5, 62.5, 110.5, 115.3, 126.7, 130.3, 142.9, 145.2, 151.2, 151.5, 160.0; m/z (ESI-MS-positive) 381.2 (M+H)+. Experimental is in agreement with data reported in Ref. 6a. Synthesis of 4-{2-[(3-bromo-2-hydroxy-benzylidene)-amino]ethyl}-benzenesulfonamide 9 SO2NH2

4-Amino-3-fluorobenzenesulfonamide (0.1 g, 1.0 equiv) was dissolved in 4 ml dry MeOH, then biphenyl-4-carboxaldehyde (0.1 g 1.0 equiv) was added. The solution was stirred for 24 h at 70 °C, then it was cooled down to rt, stirred for 1 h at 0 °C until a precipitate was formed, which was collected by filtration, washed with ice-cold MeOH and dried under vacuo to afford the title compound 6 as a light pink solid in 43% yield. 4.1.1.6. 4-[(Biphenyl-4-ylmethylene)-amino]-3-fluoro-benzenesulfonamide 6. Mp 237–238 °C; dH (400 MHz, DMSO-d6) 7.48 (1H, appt, J = 7.2), 7.53 (2H, s, exchange with D2O, ASO2NH2), 7.56 (3H, m), 7.72 (1H, s), 7.74 (1H, m), 7.82 (2H, d, J = 7.2), 7.92 (2H, d, J = 8.4), 8.10 (2H, d, J = 8.4), 8.80 (1H, s); dC (101 MHz, DMSO-d6) 115.2 (d, J2C–F 23.0), 123.6, 124.2 (d, J3C–F 3.0), 128.4, 128.6, 129.9, 130.7, 131.4, 135.7, 140.4, 143.2 (d, J3C–F 6.0), 144.2 (d, J2C–F10.0), 145.4, 155.4 (d, J1C–F 249.2) 166.2; dF (376.5 MHz, DMSO-d6) 124.25; m/z (ESI-MS-negative) 353.1 (M H) . Synthesis of 4-{2-[(biphenyl-4-ylmethylene)-amino]-ethyl}benzenesulfonamide 7

Br

OH

O N

Br OH

dry MeOH, 0°C, 2h

SO2NH2

9

NH2

4-(2-Aminoethyl)benzenesulfonamide (0.15 g, 1.0 equiv) and 3bromo-2-hydroxy benzaldehyde (0.15 g, 1.0 equiv) in 10 ml MeOH were treated according to the general procedure at 0 °C to afford the title compound 9 as a pale yellow solid in 66% yield. 4.1.1.9. 4-{2-[(3-Bromo-2-hydroxy-benzylidene)-amino]-ethyl}benzenesulfonamide 9. Mp 197–198 °C; dH (400 MHz, DMSO-d6) 3.11 (2H, t, J = 7.0), 3.97 (2H, t, J = 7.0), 6.71 (1H, dd, J = 8.0, 7.6), 7.34 (2H, s, exchange with D2O, ASO2NH2), 7.39 (1H, d, J = 7.6), 7.51 (2H, d, J = 8.4), 7.66 (1H, d, J = 8.0), 7.80 (2H, d, J = 8.0), 8.56 (1H, s); dC (101 MHz, DMSO-d6) 36.7, 57.3, 112.9, 118.4, 118.9, 126.7, 130.2, 132.8, 137.0, 143.2, 144.0, 162.4, 167.1; m/z (ESI-MS-negative) 383.0 (M H) . Synthesis of 4-{2-[(2-hydroxy-4-methoxy-benzylidene)amino]-ethyl}-benzenesulfonamide 10

O H dry MeOH, 0°C 3 mins

NH2

SO2NH2

SO2NH2

6

SO2NH2

Synthesis of sodium 5-{[2-(4-sulfamoyl-phenyl)-ethylimino]methyl}-furan-2-sulfonate 86b

SO2NH2

N 7

HO

O

O

O

SO2NH2

N

NH2

4-(2-Aminoethyl)benzenesulfonamide (0.5 g, 1.0 equiv) and biphenyl-4-carboxaldehyde (0.45 g, 1.0 equiv) in 5 ml MeOH were treated according to the general procedure to afford the title compound 7 as a white solid in 95% yield. 4.1.1.7. 4-{2-[(Biphenyl-4-ylmethylene)-amino]-ethyl}-benzenesulfonamide 7. Mp 216–217 °C dH (400 MHz, DMSOd6) 3.07 (2H, t, J = 7.0), 3.90 (2H, t, J = 7.0), 7.33 (2H, s, exchange with D2O, ASO2NH2), 7.43 (1H, appt, J = 8.0), 7.52 (4H, m), 7.77 (8H, m), 8.38 (1H, s); dC (101 MHz, DMSO-d6) 37.5, 62.4, 126.2, 127.7, 127.9, 128.9, 129.4, 130.0, 130.3, 136.0, 140.3, 142.9, 143.1, 145.2, 161.9; m/z (ESI-MS-positive) 365.2 (M+H)+.

dry MeOH, 0°C, 2h

OH

SO2NH2 10

4-(2-Aminoethyl)benzenesulfonamide (0.16 g 1.0 equiv) and 2hydroxy-5-methoxy benzaldehyde (0.12 g 1.0 equiv) in 5 ml MeOH were treated according to the general procedure at 0 °C to afford the title compound 10 as a pale yellow solid in 78% yield. 4.1.1.10. 4-{2-[(2-Hydroxy-4-methoxy-benzylidene)-amino]ethyl}-benzenesulfonamide 10. Mp 150–151 °C; dH (400 MHz, DMSO-d6) 3.08 (2H, t, J = 7.0), 3.74 (3H, s), 3.91 (2H, t, J = 7.0), 6.84 (1H, d, J = 9.2), 6.97 (1H, dd, J = 9.2, 3.2), 7.03 (1H, d, J = 2.8), 7.33 (2H, s, exchange with D2O, ASO2NH2), 7.49 (2H, d, J = 8.0), 7.78 (2H, d, J = 8.0), 8.52 (1H, s); dC (101 MHz, DMSO-d6)

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6

B. Sarikaya et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

37.2, 56.5, 60.4, 115.8, 118.1, 119.3, 120.1, 126.6, 130.2, 143.0, 144.7, 152.5, 155.2, 166.7; m/z (ESI-MS-negative) 333.1 (M H) . Synthesis of 4-{2-[(4-bromo-2-hydroxy-benzylidene)-amino]ethyl}-benzenesulfonamide 11 SO2NH2

Br

OH

Br

O

N OH

dry MeOH, 0°C, 3 mins

NH2

d, J = 8.4), 7.86 (2H, d, J = 8.4), 7.94 (1H, dt, J = 8.0, 2.0), 8.05 (1H, d, J = 8.0), 8.20 (2H, d, J = 8.0), 8.40 (1H, s), 8.73 (1H, dd, J = 4.0, 0.8); dC (101 MHz, DMSO-d6) 37.5, 62.4, 121.5, 123.9, 126.5, 127.7, 129.2, 130.3, 137.4, 138.3, 141.5, 142.9, 145.2, 150.6, 156.2, 161.9; m/z (ESI-MS-positive) 366.25 (M+H)+. Synthesis of 4-{2-[(4-methylsulfanyl-benzylidene)-amino]ethyl}-benzenesulfonamide 14

SO2NH2 11

SO2NH2 S

4-(2-Aminoethyl)benzenesulfonamide (0.1 g, 1.0 equiv) and 4bromo-2-hydroxy benzaldehyde (0.1 g, 1.0 equiv) in 10 ml MeOH were treated according to the general procedure at 0 °C to afford the title compound 11 as a pale yellow solid in 94% yield.

N

NH2

4.1.1.11. 4-{2-[(4-Bromo-2-hydroxy-benzylidene)-amino]ethyl}-benzenesulfonamide 11. Mp 208–209 °C; dH (400 MHz, DMSO-d6) 3.08 (2H, t, J = 7.0), 3.91 (2H, t, J = 7.0), 7.03 (1H, dd, J = 8.0, 2.0), 7.08 (1H, d, J = 2.0), 7.33 (2H, s, exchange with D2O, –SO2NH2), 7.36 (1H, d, J = 8.0), 7.49 (2H, d, J = 8.0), 7.79 (2H, d, J = 8.4), 8.56 (1H, s), 13.97 (1H, br s, AOH); dC (101 MHz, DMSO-d6) 36.9, 58.9, 118.1, 121.0, 121.7, 126.6, 127.0, 130.2, 134.3, 143.1, 144.3, 164.3, 166.6; m/z (ESI-MS-negative) 383.0 (M H) . Synthesis of 4-{2-[(2-methoxy-4-nitro-benzylidene)-amino]ethyl}-benzenesulfonamide 12 SO2NH2

O2N

O O

O2N N

NH2

O

dry MeOH, 0°C, 45 mins

12

SO2NH2

4.1.1.12. 4-{2-[(2-Methoxy-4-nitro-benzylidene)-amino]-ethyl}benzenesulfonamide 12. Mp 185–186 °C; dH (400 MHz, DMSO-d6) 3.07 (2H, t, J = 7.0), 3.95 (2H, t, J = 7.0), 4.00 (3H, s), 7.29 (2H, s, exchange with D2O, ASO2NH2), 7.49 (2H, d, J = 8.4), 7.77 (2H, d, J = 8.4), 7.89 (1H, s), 7.90 (1H, d, J = 5.6), 8.06 (1H, d, J = 9.2), 8.64 (1H, s); dC (101 MHz, DMSO-d6) 37.3, 57.4, 62.8, 107.9, 116.6, 126.5, 128.6, 130.3, 130.6, 142.9, 144.9, 150.6, 156.3, 159.3; m/z (ESI-MS-positive) 364.17 (M+H)+. Synthesis of 4-{2-[(4-pyridin-2-yl-benzylidene)-amino]-ethyl}benzenesulfonamide 13 N

O

N

H N dry MeOH, 0°C, 5 mins NH2

13

dry MeOH, 0°C, 10 mins

SO2NH2 14

4-(2-Aminoethyl)benzenesulfonamide (0.2 g, 1.0 equiv) and 4(methylthio)benzaldehyde (0.15 g, 1.0 equiv) in 10 ml MeOH were treated according to the general procedure at 0 °C to afford the title compound 14 as a white solid in 69% yield. 4.1.1.14. 4-{2-[(4-Methylsulfanyl-benzylidene)-amino]-ethyl}benzenesulfonamide 14. Mp 200–201 °C; dH (400 MHz, Acetone-d6) 2.57 (3H, s), 3.10 (2H, t, J = 7.0), 3.90 (2H, t, J = 7.0), 6.53 (2H, s, exchange with D2O, ASO2NH2), 7.34 (2H, d, J = 8.3), 7.49 (2H, d, J = 8.3), 7.71 (2H, d, J = 8.3), 7.84 (2H, d, J = 8.3), 8.25 (1H, s); dC (101 MHz, DMSO-d6) 15.1, 37.5, 62.3, 126.3, 126.5, 129.2, 130.3, 133.4, 142.5, 142.8, 145.2, 161.6; m/z (ESI-MS-positive) 335.17 (M+H)+. Synthesis of 4-{2-[(4-cyano-benzylidene)-amino]-ethyl}-benzenesulfonamide 15 SO2NH2

4-(2-Aminoethyl)benzenesulfonamide (0.2 g, 1.0 equiv) and 2methoxy-4-nitrobenzaldehyde (0.18 g, 1.0 equiv) in 10 ml MeOH were treated according to the general procedure at 0 °C to afford the title compound 12 as a pale yellow solid in 63% yield.

SO2NH2

S

O

SO2NH2

4-(2-Aminoethyl)benzenesulfonamide (0.1 g, 1.0 equiv) and 4-(2pyridyl)-benzaldehyde (0.09 g, 1.0 equiv) in 10 ml MeOH were treated according to the general procedure at 0 °C to afford the title compound 13 as a white solid in 73% yield. 4.1.1.13. 4-{2-[(4-Pyridin-2-yl-benzylidene)-amino]-ethyl}-benzenesulfonamide 13. Mp 229–230 °C; dH (400 MHz, DMSOd6) 3.08 (2H, t, J = 7.0), 3.91 (2H, t, J = 7.0), 7.31 (2H, s, exchange with D2O, ASO2NH2), 7.43 (1H, m), 7.50 (2H, d, J = 8.4), 7.78 (2H,

O

N C

NH2

N

C N

dry MeOH, 0°C, 10 mins

SO2NH2

15

4-(2-Aminoethyl)benzenesulfonamide (0.15 g, 1.0 equiv) and 4cyanobenzaldehyde (0.10 g, 1.0 equiv) in 10 ml MeOH were treated according to the general procedure at 0 °C to afford the title compound 15 as a white solid in 43% yield. 4.1.1.15. 4-{2-[(4-Cyano-benzylidene)-amino]-ethyl}-benzenesulfonamide 15. Mp 168–169 °C; dH (400 MHz, Acetone-d6) 3.15 (2H, t, J = 7.0), 3.99 (2H, t, J = 7.0), 6.52 (2H, s, exchange with D2O, ASO2NH2), 7.50 (2H, d, J = 8.4), 7.84 (2H, d, J = 8.4), 7.88 (2H, d, J = 8.4), 7.98 (2H, d, J = 8.4), 8.42 (1H, s); dC (101 MHz, DMSOd6) 37.2, 62.2, 113.7, 119.5, 126.5, 129.3, 130.2, 133.6, 140.8, 142.9, 144.9, 161.2; m/z (ESI-MS-positive) 314.17 (M+H)+. Synthesis of 4-{2-[(5-bromo-1H-indol-3-ylmethylene)-amino]ethyl}-benzenesulfonamide 16 SO2NH2

O Br

HN N

N H

NH2

dry MeOH rt, 3h

Br

16

SO2NH2

4-(2-Aminoethyl)benzenesulfonamide (0.1 g, 1.0 equiv) was dissolved in 5 ml dry MeOH, then 5-bromoindole-3-carboxaldehyde (0.12 g, 1.0 equiv) was added to the solution at rt. The reaction was stirred for 3 h, concentrated under vacuo, triturated with

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B. Sarikaya et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

diethyl ether to afford the title compound 16 as a pale yellow solid in 43% yield. 4.1.1.16. 4-{2-[(5-Bromo-1H-indol-3-ylmethylene)-amino]ethyl}-benzenesulfonamide 16. Mp 184–185 °C; dH (400 MHz, DMSO-d6) 3.05 (2H, t, J = 7.0), 3.82 (2H, t, J = 7.0), 7.30 (2H, s, exchange with D2O, ASO2NH2), 7.33 (1H, dd, J = 8.0, 2.0), 7.43 (1H, d, J = 8.0), 7.52 (2H, d, J = 8.0), 7.78 (2H, d, J = 8.0), 7.83 (1H, s), 8.43 (2H, m, overlapping), 11.71 (1H, br s, @NH); dC (101 MHz, DMSO-d6) 38.5, 63.4, 114.6, 115.2, 115.3, 125.2, 126.5, 127.0, 128.0, 130.9, 133.3, 136.9, 142.8, 146.4, 157.61; m/z (ESIMS-positive) 406.0 (M+H)+. Synthesis of 4-{2-[(2,3,5,6-tetrafluoro-benzylidene)-amino]ethyl}-benzenesulfonamide 17 SO2NH2

NH2

O

F

F

F

F

F dry MeOH rt, 4h

F F F

N

17

SO2NH2

4-(2-Aminoethyl)benzenesulfonamide (0.42 g, 1.0 equiv) and 2,3,5,6-tetrafluorobenzaldehyde (0.37 g, 1.0 equiv) in 10 ml MeOH were treated according to the general procedure at 0 °C to afford the title compound 17 as a light brown solid in 20% yield. 4.1.1.17. 4-{2-[(2,3,5,6-Tetrafluoro-benzylidene)-amino]-ethyl}benzenesulfonamide 17. Mp 201–203 °C; dH (400 MHz, DMSO-d6) 3.07 (2H, t, J = 7.0), 3.97 (2H, t, J = 7.0), 7.32 (2H, s, exchange with D2O, ASO2NH2), 7.50 (2H, d, J = 8.0), 7.78 (2H, d, J = 8.0), 8.04 (1H, m), 8.48 (1H, s); dC (101 MHz, DMSO-d6) 37.5, 62.5, 109.3 (t, JC–F 24.0), 116.8 (t, JC–F 12.0), 126.6, 130.4, 143.1, 144.9, 145.7 (d, J1C–F 267.8), 146.6 (d, J1C–F 244.6), 152.2; dF (376.5 MHz, DMSO-d6) 139.36 (2F, s), 143.96 (2F, s); m/z (ESIMS-negative) 359.0 (M H) . 4.2. CA activity measurements and inhibition studies An SLX-Applied Photophysics stopped-flow instrument has been used for measuring the catalytic activity and inhibition with a CO2 hydration assay method.17 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) or 20 mM Tris (pH 8.3) as buffers, and 20 mM Na2SO4 or NaClO4 (for maintaining constant the ionic strength). The initial rates of the CA-catalyzed CO2 hydration reaction were followed for a period of 10–100 s. The concentrations of substrate (CO2) ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants, with at least six traces of the initial 5–10% of the reaction being used for determining the initial velocity, for each inhibitor. The uncatalyzed rates were determined subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done with the assay buffer. Enzyme and inhibitor solutions were preincubated prior to assay for 15 min (at room temperature), in order to allow for the formation of the E–I complex. The inhibition constants were obtained by non-linear leastsquares methods using PRISM 3 and the Cheng–Prusoff equation as reported earlier by our groups.3–7 The CAs were recombinant proteins obtained in-house as reported earlier.19,20 The concentrations of enzyme used in the assay were: 15.1 nM for hCAI, 8.7 nM for hCA II; 9.0 nM for hCA IX and 10.6 nM for hCA XII, respectively..

7

Acknowledgments This work was supported in part by an EU FP7 project (Dynano). B.S. thanks the Erasmus project for a mobility grant. References and notes 1. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) De Simone, G.; Alterio, V.; Supuran, C. T. Expert Opin. Drug Discov. 2013, 8, 793; (c) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759; (d) Aggarwal, M.; Kondeti, B.; McKenna, R. Expert Opin. Ther. Patents 2013, 23, 717; (e) Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 3467; (f) Harju, A. K.; Bootorabi, F.; Kuuslahti, M.; Supuran, C. T.; Parkkila, S. J. Enzyme Inhib. Med. Chem. 2013, 28, 231. 2. (a) Supuran, C. T. Bioorg. Med. Chem. 2013, 21, 1377; (b) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem. Rev. 2012, 112, 4421; (c) Supuran, C. T. Expert Opin. Ther. Patents 2013, 23, 677; (d) Pinard, M. A.; Boone, C. D.; Rife, B. D.; Supuran, C. T.; McKenna, R. Bioorg. Med. Chem. 2013, 21, 7210; (e) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229; (f) Capasso, C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, 29, 379. 3. (a) Innocenti, A.; Vullo, D.; Scozzafava, A.; Casey, J. R.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 573; (b) Bayram, E.; Senturk, M.; Kufrevioglu, I.; Supuran, C. T. Bioorg. Med. Chem. 2008, 16, 9101; (c) Winum, J. Y.; Innocenti, A.; Nasr, J.; Montero, J. L.; Scozzafava, A.; Vullo, D.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 2353; (d) Çavdar, H.; Ekinci, D.; Talaz, O.; Saraçog˘lu, N.; S ß entürk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 148; (e) Chohan, Z. H.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2003, 18, 259. 4. (a) Supuran, C. T.; Nicolae, A.; Popescu, A. Eur. J. Med. Chem. 1996, 31, 431; (b) Supuran, C. T.; Popescu, A.; Ilisiu, M.; Costandache, A.; Banciu, M. D. Eur. J. Med. Chem. 1996, 31, 439; (c) Puccetti, L.; Fasolis, G.; Vullo, D.; Chohan, Z. H.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 3096; (d) Luca, C.; Barboiu, M.; Supuran, C. T. Rev. Roum. Chim. 1991, 36, 1169. 5. (a) Supuran, C. T.; Scozzafava, A.; Popescu, A.; Bobes-Tureac, R.; Banciu, A.; Creanga, A.; Bobes-Tureac, G.; Banciu, M. D. Eur. J. Med. Chem. 1997, 32, 445; (b) Supuran, C. T.; Clare, B. W. Eur. J. Med. Chem. 1998, 33, 489; (c) Popescu, A.; Simion, A.; Scozzafava, A.; Briganti, F.; Supuran, C. T. J. Enzyme Inhib. 1999, 14, 407; (d) Scozzafava, A.; Banciu, M. D.; Popescu, A.; Supuran, C. T. J. Enzyme Inhib. 2000, 15, 533. 6. (a) Nasr, G.; Petit, E.; Supuran, C. T.; Winum, J. Y.; Barboiu, M. Bioorg. Med. Chem. Lett. 2009, 19, 6014; (b) Nasr, G.; Petit, E.; Vullo, D.; Winum, J. Y.; Supuran, C. T.; Barboiu, M. J. Med. Chem 2009, 52, 4853; (c) Supuran, C. T.; Mincione, F.; Scozzafava, A.; Briganti, F.; Mincione, G.; Ilies, M. A. Eur. J. Med. Chem. 1998, 33, 247. 7. (a) Nasr, G.; Cristian, A.; Barboiu, M.; Vullo, D.; Winum, J. Y.; Supuran, C. T. Bioorg. Med. 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