View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Florence Research Journal of Enzyme Inhibition and Medicinal Chemistry ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20 Dithiocarbamates with potent inhibitory activity against the Saccharomyces cerevisiae β-carbonic anhydrase Murat Bozdag, Fabrizio Carta, Daniela Vullo, Semra Isik, Zeid AlOthman, Sameh M. Osman, Andrea Scozzafava & Claudiu T. Supuran To cite this article: Murat Bozdag, Fabrizio Carta, Daniela Vullo, Semra Isik, Zeid AlOthman, Sameh M. Osman, Andrea Scozzafava & Claudiu T. Supuran (2016) Dithiocarbamates with potent inhibitory activity against the Saccharomyces cerevisiae β-carbonic anhydrase, Journal of Enzyme Inhibition and Medicinal Chemistry, 31:1, 132-136, DOI: 10.3109/14756366.2015.1010529 To link to this article: https://doi.org/10.3109/14756366.2015.1010529 Published online: 11 Feb 2015. Submit your article to this journal Article views: 201 View related articles View Crossmark data Citing articles: 8 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ienz20 http://informahealthcare.com/enz ISSN: 1475-6366 (print), 1475-6374 (electronic) J Enzyme Inhib Med Chem, 2016; 31(1): 132–136 ! 2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1010529 RESEARCH ARTICLE Dithiocarbamates with potent inhibitory activity against the Saccharomyces cerevisiae b-carbonic anhydrase Murat Bozdag1, Fabrizio Carta1, Daniela Vullo1, Semra Isik1, Zeid AlOthman2, Sameh M. Osman2, Andrea Scozzafava1, and Claudiu T. Supuran1,3 1Laboratorio di Chimica Bioinorganica, Dipartimento di Chimica, Universita` degli Studi di Firenze, Sesto Fiorentino, Florence, Italy, 2Department of Chemistry, College of Science, King Saud University, Riyadh, Saudi Arabia, and 3Sezione di Scienze Farmaceutiche, Dipartimento NEUROFARBA, Universita` degli Studi di Firenze, Sesto Fiorentino, Florence, Italy Abstract Keywords Dithiocarbamates (DTCs) prepared from primary or secondary amines, which incorporated b-Class enzyme, carbonic anhydrase, amino/hydroxyl-alkyl, mono-/bicyclic aliphatic/heterocyclic rings based on the quinuclidine, dithiocarbamate, inhibitor, piperidine, hydroxy-/carboxy-/amino-substituted piperidine, morpholine and piperazine scaf- Saccharomyces cerevisiae folds, were investigated for the inhibition of a- and b-carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, such as the human (h) isoform hCA I and II, as well as the History Saccharomyces cerevisiae b-CA, scCA. The yeast and its b-CA were shown earlier to be useful models of pathogenic fungal infections. The DTCs investigated here were medium potency hCA Received 16 December 2014 Revised 15 January 2015 I inhibitors (KIs of 66.5–910 nM), were more effective as hCA II inhibitors (KIs of 8.9–107 nM) and some of them showed excellent, low nanomolar activity against the yeast enzyme, Accepted 15 January 2015 with inhibition constants ranging between 6.4 and 259 nM. The detailed structure activity Published online 6 February 2015 relationship for inhibition of the yeast and human enzymes is discussed. Several of the investigated DTCs showed excellent selectivity ratios for inhibiting the yeast over the human cytosolic CA isoforms. Introduction CAI45. In the X-ray crystal structure of this anion bound to hCA II, a monodentate coordination of the inhibitor through one The primary sulfonamides possessing the general formula sulfur atom to the zinc ion from the enzyme active site has been RSO NH (where R may be an aromatic, heterocyclic or aliphatic 2 2 observed. Furthermore, a hydrogen bond in which another sulfur moiety, but also an even smaller groups such as OH and NH )1–7 2 of TTC and the OH of Thr199 (a crucial residue for the catalytic dominated the landscape of carbonic anhydrase (CA, EC 4.2.1.1) cycle of a-CAs)1–3 were involved, also stabilized the anion when inhibitors (CAIs) drug design for 70 years, since the discovery bound within the enzyme active site. Thus, the CSÀ moiety that such compounds inhibit the enzyme, in the 1940s8–15. 2 present in TTC was detected as a new zinc-binding group (ZBG) However, recently, new important chemotypes with such proper- for CA inhibition. As DTCs incorporate this ZBG, a first series of ties emerged, by combining structure-based drug discovery such compounds was prepared and evaluated for their inhibitory strategies (X-ray crystallography and fragment-based drug activity against several mammalian, fungal and bacterial CAs by design) with massive campaigns of screening vast library of this group40–44. Several low nanomolar and subnanomolar CAIs compounds for detecting such an activity16–38. The interest in were thus detected against all these enzymes. X-ray crystal CAIs is mainly motivated by their pharmacological properties and structures were also reported for three DTCs complexed to clinical use as diuretics, antiglaucoma, antiobesity or antiepileptic hCA II, i.e. compounds A–C (Figure 1)40,41. DTCs A–C inhibited agents, as well as anticancer agents/diagnostic tools1–3,29–39. hCA II with K s of 25, 41 and 0.95 nM, respectively, and hCA IX Among the new chemotypes with CA inhibitory properties I (an isoform involved in cancerogenesis)1,3,5 with K s of 53, 757 ultimately reported, the dithiocarbamates (DTCs) are undoubtedly I and 6.2 nM, respectively40,41. As seen from Figure 1, the binding among the most interesting ones40–44. mode of the new ZBG present in these compounds was identical These compounds have been rationally discovered as CAIs to that of TTC, with one sulfur coordinated to the metal ion, but after our report of trithiocarbonate (CS2À, TTC), an inorganic 3 the organic scaffold present in the DTC was observed to make anion similar to carbonate, as an interesting (milli–micromolar) extensive contacts with many amino acid residues from the active site. This explains the wide range of inhibitory power of these derivatives, from the subnanomolar to the micromolar range, for Address for correspondence: Daniela Vullo, Dipartimento di Chimica, 40–44 Laboratorio di Chimica Bioinorganica, Universita` degli Studi di Firenze, the entire series of around 30 DTC reported so far . Rm. 188, Via della Lastruccia 3, Sesto Fiorentino, Florence 50019, Italy. Interestingly, these compounds are highly water soluble (being Tel/Fax: +39 055 4573143. E-mail: [email protected] salts), and the DTC C was also effective in vivo as an DOI: 10.3109/14756366.2015.1010529 Dithiocarbamates with potent inhibitory activity 133 (A) R R S MOH, CS NH 2 N − + R1= H or Alkyl R1 R1 S M Solvent M = Na, K S S S 1a-21a 1b-21b N O N N CN − − S 1b–21b 1a–21a S− S Scheme 1. Preparation of DTCs , by reaction of amines with carbon disulfide in the presence of sodium/potassium hydroxide. A BC (B) the enzyme activity is at half maximal. All enzymes used were recombinant, produced in Escherichia coli as reported earlier13,47. The concentrations of enzymes used in the assay were: hCA I, 12.0 nM; hCA II, 9.2 nM and scCA, 15.3 nM. Results and discussion In the present article, we extended the series of DTCs investigated earlier40–44, including both primary as well as secondary deriva- tives, which were obtained in such a way as to explore novel chemical space. The starting amines (1a–21a) used to synthesize DTCs 1b–21b reported here (Scheme 1) included N,N-dimethy- laminoethylenediamine 1a, aminoalcohols with three to five carbon atoms in their molecule 2a–4a, the bicyclic quinuclidine- 3-amine (both the racemate as well as the R- and S-enantiomeric DTCs 5b–7b incorporating this scaffold were obtained), piperi- dine 8a and several of its derivatives with hydroxyl-, carboxy-, Figure 1. (A) Structure of DTCs A–C used as lead molecules in the acetamido- and boc-amido functionalities in various positions of present study. (B) Electronic density for the adduct of dithiocarbamate B the heterocyclic ring, of types 9a–16a; morpholine and piperazine 40,41 bound within the active site of hCA II . The zinc ion is shown as the derivatives 17a–19a, as well as phenethylamine 20a and its central sphere and the amino acid residues involved in the binding are sulfamoylated derivative, 4-aminoethylbenzenesulfonamide 21a, evidenced and numbered (hCA I numbering system)40. The zinc ligands a well know CAI which binds to the enzyme through the (except the sulfur from the DTC compound), His94, 96 and 119, are not 1,3 shown for the sake of simplicity. sulfamoyl moiety (Scheme 1). The choice of these scaffolds was motivated by the fact that the structure–activity relationship (SAR) for the inhibition of CAs with the DTCs reported earlier7,8 was primarily influenced by the organic scaffold of the inhibitor. antiglaucoma agent when administered topically directly to the In fact, important differences of activity were observed between eye of hypertensive rabbits, a widely used animal model of this primary and secondary DTCs and between aliphatic or aromatic/ disease41. However, for the moment only one series of DTCs was heterocyclic derivatives, respectively40–44. Here we investigated reported and investigated as CAIs, and this is the reason why we compounds belonging to these DTCs types, with a variety of extend here the earlier studies40,41, reporting the CA inhibitory substitution patterns, based on several scaffolds mentioned earlier properties of a new series of such compounds against another CA (Table 1) among which primary, aliphatic aminoalkyl- or of interest, the b-class enzyme from the yeast, Saccharomyces hydroxyalkyl derivatives (1b–4b); primary, bicyclic, bulky cerevisiae scCA46–48, a model organism of great relevance when DTCs (5b–7b), with various stereochemistries; secondary, piperi- investigating fungal CAs as drug targets49–51. dine, morpholine and piperazine-based DTCs, of types 8b–19b, for which the lead compound was the morpholine-DTC (com- Materials and methods pound C) reported earlier40.