http://informahealthcare.com/enz ISSN: 1475-6366 (print), 1475-6374 (electronic) J Enzyme Inhib Med Chem, Early Online: 1–5 ! 2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.928704
Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by University of Ulster at Jordanstown on 12/15/14 For personal use only.
RESEARCH ARTICLE
Carbonic anhydrase and acetylcholinesterase inhibitory effects of carbamates and sulfamoylcarbamates Hu¨lya Go¨c¸er1,2, Akın Akinciog˘lu1,2, Su¨leyman Go¨ksu2, _Ilhami Gu¨lc¸in2,3, and Claudiu T. Supuran4 1
Central Researching Laboratory, Agri Ibrahim Cecen University, Agri, Turkey, 2Faculty of Science, Department of Chemistry, Atatu¨rk University, Erzurum, Turkey, 3Department Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia, and 4Laboratorio di Chimica Bioinorganica, Universita` degli Studi di Firenze, Via della Lastruccia, Sesto Fiorentino, Firenze, Italy Abstract
Keywords
Carbonic anhydrases (CA), as a family of metalloenzymes, are found in almost every type of tissue and play an important role in catalyzing the equilibration of carbon dioxide and carbonic acid. In this study, a series of carbamate derivative was synthesized, and their inhibition effects on hCA I, hCA II and acetylcholinesterase (AChE) enzymes were investigated. They were determined to be very good inhibitor against for both isoenzymes (hCA I and hCA II) and AChE. The hCA I and hCA II were effectively inhibited by the carbamate derivatives, with inhibition constants (Ki) in the range of 194.4–893.5 nM (for hCA I) and 103.9–835.7 nM (for hCA II). On the other hand, Ki parameters of these compounds for AChE enzyme inhibition were determined in the range of 12.0–61.3 nM. The results clearly showed that both CA isoenzymes and AChE were inhibited by carbamate derivatives at the nM levels.
Acetylcholinesterase, AChE, CA, carbamates, carbonic anhydrase, enzyme inhibition
Introduction 1
Carbamates are beneficial biologically active compounds . There are some drugs in the markets containing the carbamate functional group. Neostigmine and eserine drugs are inhibitors of acetylcholinesterase (AChE) and they are used in the treatment of myasthenia gravis disease2. A drug felbamate, commercially known as Felbatol, is an anticonvulsant drug used in the treatment of epilepsy3. In addition, there are some other beneficial biologically active carbamates and sulfamoylcarbamate derivatives used in treatment of sedative-hypnotic medication, insomnia, hypnotics, anxiolytic and muscle relaxants4. In addition, carbonic anhydrase (CA)5 and Hepatitis C virus inhibitory properties of carbamates have also been addressed6. Enzymes are synthesized by living cells and speed up chemical reactions during the metabolism of living organisms7. Enzymes, named biological catalysts, do not produce any by-product, which is a major advantage in synthesis. Isoenzymes have different amino acid sequence but they catalyze the same biochemical reactions. Isoenzymes usually display different kinetic parameters such as KM, and they have different regulatory properties. They are different in their interests against substrates, cofactors and inhibitors. Furthermore, each isoenzyme has different isoelectric points and electrophoretic mobility. It was reported that CA has 16 different isoenzyme located in different tissues8,9. CA provides overall metabolic CO2 transport. This enzyme also plays a role in the accumulation of H+ and HCO 3 in many tissues. Isoenzymes _ Address for correspondence: Prof. Dr. Ilhami Gu¨lc¸in, Faculty of Science, Department of Chemistry, Atatu¨rk University, 25240 Erzurum, Turkey. Tel: +90 4422314389. Fax: +90 442 2360948. E-mail: igulcin@ atauni.edu.tr; [email protected]
History Received 8 May 2014 Revised 23 May 2014 Accepted 23 May 2014 Published online 20 June 2014
have some important functions in kidney, gastric mucosa and eye lens10. Both isoenzymes were purified and characterized from many sources like human erythrocytes, fish erythrocytes, rat erythrocytes, rat saliva, bovine bone and bovine leukocytes, salivary glands, muscles, brain, nerve myelin sheath, pancreas, prostate, endometrial tissues, bacteria and a variety of plant sources. The molecular mass of the enzyme was found to be approximately 30 KDa for mammals11–14. CA isoenzymes are physiologically important enzymes for human body. CA plays an important role in many critical physiological events such as carbon dioxide (CO2)/ bicarbonate (HCO 3 ) respiration, pH and CO2 homeostasis, electrolyte secretion, biosynthetic reactions like gluconeogenesis, lipogenesis and urea synthesis, bone resorption, calcification and tumor formation9,15,16. In plants, CA is mainly found in the thylakoid membrane chloroplasts and cytosol. In the literature, there are many inhibition studies for CA isoenzymes. Generally mononegatif anions and some of the neutral organic compounds like sulfamides and sulfonamides cause recycled inhibition by binding tightly to the enzyme. Acetazolamide is the most commonly used inhibitor for CA isoenzymes17. Due to their pharmacokinetic properties, CA isoenzyme inhibitors (CAIs) were used in treating diseases such as epilepsy and glaucoma. Sulfonamide derivatives are CA’s specific and potent inhibitors16. Alcohols, organic solvents, amide compounds and other neutral loosely inhibitors constitute a weak inhibition in binding the enzyme18,19. CAIs are reserved typically for patients at high risk of vision loss at long-term use. When combined with mitotic, they exhibit stronger effects20. Cholinesterases (ChEs) are special carboxylic ester hydrolases that knocks down the esters of choline and they separated into two classes, AChE (EC3.1.1.7) and butyrylcholinesterase21,22 (BChE,
Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by University of Ulster at Jordanstown on 12/15/14 For personal use only.
2
H. Go¨c¸er et al.
EC 3.1.1.8). AChE is situated in the group of ChEs and hydrolyses the neurotransmitter acetylcholine (ACh) to acetic acid and choline23–26. AChE is known as real ChE, while BuChE is known as non-specific ChE or pseudocholinesterase27. AChE is found in high concentrations in the brain and in red blood cells. BuChE is found in serum, pancreas, liver and central nervous system28. These two enzymes have different optimum substrate concentrations. AChE is a necessary enzyme for the nervous system. AChE inhibitors are used in treatment of several neuromuscular diseases and were studied for treatment of Alzheimer’s disease21,22,29. Organophosphorus and carbamate derivatives are known as the best inhibitors for AChE catalytic activity30. Carbamate pesticides show their toxicity by modifying irreversibly the catalytic serine residue in AChE and the inhibition of the AChE31. As carbamates show important biological activities; CA and ACh esterase inhibitory properties of some carbamate derivatives will be useful for further biological studies. In this research, we aimed to investigate CA and ACh inhibitory properties of two carbamates and six sulfamoylcarbamates for the first time.
Experimental section General information Sulfamoylcarbamates (1, 3 and 4)32, sulfamoylcarbamates (5, 7 and 8)22, carbamate (2)33 and carbamate (6)34 were synthesized as described in the literature. Purification of CA isoenzymes from human erythrocytes with affinity chromatography The fresh human erythrocytes were centrifuged at 10 000 rpm for 30 min, and precipitate was removed and the serum was separated, and the pH was tuned to 8.7 with solid Tris35,36. Before doing so, sepharose-4B-tirozyne-sulfanylamide affinity column balanced with Tris-HCl/Na2SO4 (25 mM/0.1 M, pH 8.7) and tuned human erythrocyte sample was applied to the column. The affinity gel was washed with Tris-HCl/Na2SO4 (25 mM/22 mM, pH: 8.7). hCA I and II isoenzyme were separated with H2NSO3H/Na2HPO4 (0.25 M/0.25 M, pH: 6.7). These studies were performed at 4 C8,37,38. SDS polyacrylamide gel electrophoresis The purity of both hCA I and II was identified using SDS polyacrylamide gel electrophoresis, performed according to the Laemmli’s procedure39 and described in our previous study40. The running and separating gels contained 10% and 3% acrylamide and SDS (0.1%), respectively41,42. A 20-mg sample was administered to the electrophoresis. Gels were stained with some difference of the same solvent without the dye. These solvents are prepared as 1.5 h in Coomassie Brilliant Blue R-250 (0.1%) in methanol (50%) and 10% acetic acid43. Protein identification The content protein during the purification steps was identified with spectrophotometer at 595 nm according to the Bradford method44 explained in previous studies45–49. As in other studies, bovine serum albumin was used as standard in this study50–53. Esterase activity assay This study was made according to the method described by Verpoorte et al.54 described previously55,56. Both CA isoenzymes activities were assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate to 4-nitrophenylate ion over a period of 3 min at 25 C using a spectrophotometer (Shimadzu,
J Enzyme Inhib Med Chem, Early Online: 1–5
UVmini-1240 UV-VIS spectrophotometer, Kyoto, Japan)57. The Ki values were calculated from experiments using three different carbamate derivatives 1–8 concentrations and PNF as the substrate at five different concentrations to create Lineweaver– Burk curves reported previously58–60. Determination of anticholinesterase activity The inhibitory effects of carbamate derivatives 1–8 on AChE activities were measured by slightly modifying the spectrophotometric method of Ellman et al.61. Acetylthiocholine iodide (AChI) was used as substrate of the reaction. 5,50 -dithio-bis(2-nitrobenzoic acid) (DTNB) (product no: D8130-1G, Sigma-Aldrich Co., St. Louis, MO) was used for the determination of the AChE activity. Namely, 100 mL of Tris/HCl buffer (1 M, pH 8.0), 10 mL of sample solution dissolved in ultra pure water at different concentrations and 50 mL AChE from electric eel (Electrophorus electricus) (518 units/mg, product no: C3389-500UN, SigmaAldrich) solution were mixed, incubated for 10 min at 25 C. Then 50 mL of DTNB (0.5 mM) was added. The reaction was then started by the addition of 50 mL of AChI (10 mM, product no: 01480-1G, Sigma-Aldrich). The hydrolysis of these substrates was observed spectrophotometrically by the formation of yellow 5thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, determined by the enzymatic hydrolysis of AChI, at a wavelength of 412 nm. In order to determine the effect of carbamate derivatives 1–8 on AchE, different carbamate derivatives were added to the reaction mixture. The enzyme activity was measured. The IC50 values were obtained from activity (%) versus compounds plots21,62,63.
Results and discussion CA purification and activity assay The purity of the enzymes was confirmed using SDS polyacrylamide gel electrophoresis by hCA I and II isoenzymes were purified separately from human erythrocytes with Sepharose-4BL-Thyrosine-sulfanilamide affinity column chromatography14,64. After hCA purification, we investigated the inhibition effects of carbamate and sulfamoylcarbamate derivatives on hCA I and II. The inhibition measurement results were performed with esterase activity method35,65. In this method, when the CA enzyme hydrolyses the phenyl acetate, the resultant final product shows absorption at 348 nm19,66. CA isoenzymes inhibition effects In recent years, the CA isoenzymes have become an interesting target for the design of inhibitors or activators for biomedical applications. With this purpose, we have investigated the inhibitory effects of the carbamate derivatives and sulfamoylcarbamate on the CA isoenzymes hCA I and II. Lineweaver–Burk graphs were drawn for the Ki values determination. Then the average of Ki and IC50 values were calculated from these graphs (Table 1). At first step, we report the inhibitory effects of carbamate derivatives (2 and 6) and sulfamoylcarbamate (1, 3, 4, 5, 7 and 8) on the esterase activity of hCA I and II under in vitro conditions. The results are listed in Table 1. These compounds were shown to be effective inhibitors of cytosolic isoenzymes, hCA I and II (Table 1). Carbamates (2 and 6) and sulfamoylcarbamate (1, 3, 4, 5, 7 and 8) were all found to inhibit hCA I, with Ki values ranging from 264.16–586.50 nM for carbamates (2 and 6) to 194.41–893.50 nM for sulfamoylcarbamate (1, 3, 4, 5, 7 and 8). On the other hand, AZA, which used as positive standard inhibitor for CA isoenzymes, showed less inhibition with Ki values of 36.20 mM. Carbamates and sulfamoylcarbamate (1–8) were all found to inhibit hCA II, with Ki
Inhibitory effects of some carbamates
DOI: 10.3109/14756366.2014.928704
values ranging from 103.90–835.74 nM for carbamates (2 and 6) to 167.51–482.00 nM for sulfamoylcarbamate (1, 3, 4, 5, 7 and 8) with a comparable potency as the reference compound AZA with Ki value of 3.70 mM.
Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by University of Ulster at Jordanstown on 12/15/14 For personal use only.
ACh esterase enzyme inhibition effects Different types of AChE inhibitors have been studied for the treatment of Alzheimer’s disease67. These inhibitors are rapidly causes inhibition on the AChE and BChE alone68. Results obtained from these studies showed that an investigation into the mechanism of action of AChE might cause the design of inhibitors, which can be used as a therapeutic in the future. In addition, some studies showed that the AChE inhibitors have different properties in terms of action mechanism, metabolism and brain selectivity. The structures of the investigated carbamate derivatives were shown in the Figure 1. This study focused on the influence of carbamates on the inhibition kinetics of AChE and recovery of corresponding carbamylated enzymes. In our study, a series of carbamates was investigated for their ability to inhibit AChE. Carbamate derivatives 1–8 were evaluated in two groups as carbamate (2 and 6) and
3
sulfamoylcarbamate (1, 3, 4, 5, 7 and 8). These both groups were investigated in more details. According to our data, inhibitory effects these carbamates revealed a significant elevation in the case of AChE. Generally, these compounds showed higher inhibition and higher lipophilicity. Considering the results, all compounds expressed significantly higher inhibition activity. All of carbamate derivatives studied significantly higher AChE inhibitory activity than that of standard AChE inhibitors such as rivastigmine and galanthamine68. Furthermore, the IC50 values of these both standard compounds are summarized in Table 2. As can be seen in the results obtained from Figure 1, AChE was effectively inhibited by carbamates derivatives 1–8, with Ki values in the range of 12.0 ± 2.01 to 61.3 ± 2.59 nM. Furthermore, all of carbamate derivatives (1–8) had almost similar Ki values. The differences between the highest and the lowest Ki values of carbamate derivatives 1–8 were only four-fold. The most active carbamate derivative is compound 2 and showed a Ki value of 12.0 ± 2.01 nM and IC50 value calculated as 29.9 nM. As a result, carbamates and sulfamoylcarbamate (1–8) were all found to inhibit AChE, with Ki values ranging from 12.06 to 14.40 nM for carbamates (2 and 6) to 15.52–61.38 nM for sulfamoylcarbamate (1, 3, 4, 5, 7 and 8).
Table 1. Human carbonic anhydrase isoenzymes (hCA I and II) inhibition value with carbamate derivatives by an esterase assay with NPA. IC50 (nM)
Table 2. IC50 values of acetylcholinesterase enzyme (AChE) inhibition parameters of carbamate derivatives comparison with standards rivastigmine (RIV) and galanthamine (GLT).
KI (nM)
Compounds
hCA I
hCA II
hCA I
hCA II
1* 2y 3* 4* 5* 6y 7* 8* AZAz
319.6 388.0 236.0 244.0 686.0 324.0 300.2 226.2
251.9 524.2 259.6 134.2 184.8 251.5 191.0 194.6
387.3 264.2 295.7 245.6 893.5 586.5 194.4 207.5 36.20
196.2 835.7 167.5 378.5 342.8 103.9 176.8 482.0 3.70
*Sulfamoylcarbamates. yCarbamate derivatives. zAZA was used as positive standard inhibitor for both CA isoenzymes (CA I and II). Ki values belonging to AZA was expressed as micromolar levels.
Compounds 1 2 3 4 5 6 7 8 RIV* GLT*
MeO
2
MeO
27.6 ± 12.9 12.0 ± 2.0 15.5 ± 1.8 31.4 ± 7.3 61.3 ± 25.9 14.4 ± 3.3 38.4 ± 5.1 57.6 ± 6.9 – –
3
NHCO2CH2Ph 6
NHSO2NHCO2CH2Ph 5 NHSO2NHCO2CH2Ph
MeO MeO
7
NHSO2NHCO2CH2Ph
MeO MeO
MeO
MeO
0.9754 0.9914 0.9877 0.9776 0.9855 0.9622 0.9819 0.9565 – –
NHCO2CH2Ph
4
NHSO2NHCO2CH2Ph
50.20 29.90 105.40 66.91 69.92 95.78 104.10 94.86 501.00 4.00
MeO
NHSO2NHCO2CH2Ph
MeO
KI (nM)
MeO
MeO MeO
MeO
r2
*Rivastigmine (RIV) and galanthamine (GLT) were used as positive standards and obtained from the reference of 37 and expressed as micromolar (mM).
NHSO2NHCO2CH2Ph
1
IC50 (nM)
8
Figure 1. The chemical structures of synthesized carbamate derivatives.
4
H. Go¨c¸er et al.
Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by University of Ulster at Jordanstown on 12/15/14 For personal use only.
Conclusion ACh is one of the key neurotransmitter for peripheral nervous system, and therefore the inhibition of AChE has been proposed as a drug for the neurotoxicity69. Carbamate derivatives (1–8) were effective of AChE inhibition properties. In addition, CA inhibitory properties of 1–8 have also been evaluated. Carbamate derivatives 1–8 were evaluated in two groups as carbamate (2 and 6) and sulfamoylcarbamate (1, 3, 4, 5, 7 and 8). The results of the two groups were generally the same. These carbamates can be important lead compounds for greater synthetic and biological objectives. Most importantly, carbamate derivatives 1–8 may be used in generating potent hCA I and II inhibitors. Referring to the literature, there are studies about the inhibition effect of carbamate derivatives on these enzymes70. Specifically, carbamate derivatives are numerous works on AChE inhibitory effect. Furthermore, these studies may play an important role in drug design for Alzheimer’s disease.
Declaration of interest The authors declared that there is no conflict of interests.
References 1. Vacondio F, Silva C, Mor M, Testa B. Qualitative structuremetabolism relationships in the hydrolysis of carbamates. Drug Metab Rev 2010;42:551–89. 2. Milkani E, Lambert CR, McGimpsey GW. Direct detection of acetylcholinesterase inhibitor binding with an enzyme-based surface plasmon resonance sensor. Anal Biochem 2011;408:212–19. 3. Kung CH, Kwon CH. Carbamate derivatives of felbamate as potential anticonvulsant agents. Med Chem Res 2010;19:498–513. 4. Gilman AG, Goodman LS, Rall TW, Murad F, eds. Goodman and Gilman’s the pharmacological basis of therapeutics. 7th ed. New York: Macmillan Publishing Co; 1985. 5. Gavernet L, Funes JLG, Palestro PH, et al. Inhibition pattern of sulfamide-related compounds in binding to carbonic anhydrase isoforms I, II, VII, XII and XIV. Bioorg Med Chem 2013;21:1410–18. 6. Su L, Li L, Li Y, et al. Simple and accurate approaches to predict the activity of benzothiadiazine derivatives as HCV inhibitors. Med Chem Res 2012;21:2079–96. 7. Shahwar D, Raza MA, Khan T. Synthesis and evaluation of enzyme inhibitory potential of some derivatives of scopolamine. Turk J Chem 2012;36:257–66. _ Supuran CT. Carbonic anhydrase 8. Oztu¨rk Sarikaya SB, Gu¨lc¸in I, inhibitors. Inhibition of human erythrocyte isoenzymes I and II with a series of phenolic acids. Chem Biol Drug Des 2010;75:515–20. _ Scozzafava A, Supuran CT. Carbonic 9. Innocenti A, Gu¨lc¸in I, anhydrase inhibitors. Antioxidant polyphenol natural products effectively inhibit mammalian isoforms I-XV. Bioorg Med Chem Lett 2010;20:5050–3. _ Beydemir S 10. Sentu¨rk M, Gu¨lc¸in I, ¸ , et al. In vitro inhibition of human carbonic anhydrase I and II isoenzymes with natural phenolic compounds. Chem Biol Drug Des 2011;77:494–9. 11. Feldstein JB, Silverman DN. Purification and characterization of carbonic anhydrase from the saliva of the rat. J Biol Chem 1984;259: 5447–53. 12. Krungkrai SR, Suraveratum N, Rochanakij S, Krungkrai J. Characterization of carbonic anhydrase in Plasmodium falciparium. Int J Parasitol 2001;31:661–8. ¨ I. _ Effects of gentamicine on 13. Beydemir S ¸ , C¸iftc¸i M, Ku¨freviog˘lu O enzyme activities of carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo. Biol Pharm Bull 2002;25: 966–9. _ Effect of melatonin on carbonic anhydrase 14. Beydemir S ¸ , Gu¨lc¸in I. from human erythrocyte in vitro and from rat erythrocyte in vivo. J Enzyme Inhib Med Chem 2004;19:193–7. _ Rosmarinic acid: a potent carbonic anhydrase 15. Topal M, Gu¨lc¸in I. isoenzymes inhibitor. Turk J Chem 2014. [Epub ahead of print]. DOI: 10.3906/kim-1403–5. 16. Supuran CT, Scozzafava A. Carbonic anhydrase inhibitors part 94. 1,3,4-thiadiazole-2-sulfonamide derivatives as antitumor agents? Eur J Med Chem 2000;35:867–74.
J Enzyme Inhib Med Chem, Early Online: 1–5
_ Beydemir S. Phenolic compounds as antioxidants: carbonic 17. Gu¨lc¸in I, anhydrase isoenzymes inhibitors. Mini Rev Med Chem 2013;13: 408–30. 18. Liang YJ, Lipscom WN. Substrate and inhibitor binding to human carbonic anhydrase II: a theoretical study. In: Botre´ F, ed. Inhibitors in the carbonic anhydrase from biochemistry and physiology and clinical medicine. Weinheim: VCH Publishers; 1991:50–64. _ Ekinci D. The inhibitory effect of 19. Coban TA, Beydemir S ¸ , Gu¨lc¸in I, ethanol on carbonic anhydrase isoenzymes: in vivo and in vitro studies. J Enzyme Inhib Med Chem 2008;23:266–70. _ 20. Oragli KM. Klinik Oftalmolji. 4. Baskı. Istanbul: Nobel Tıp Kitabevi; 2001. ¨ zta¸skın N, et al. Synthesis, antioxidant 21. Go¨c¸er H, Akıncıog˘lu A, O and antiacetylcholinesterase activities of sulfonamide derivatives of dopamine related compounds. Arch Pharm 2013;346:783–92. _ Go¨ksu S. Novel sulfamides and 22. Akıncıog˘lu A, Nar M, Gu¨lc¸in I, sulfonamides incorporating tetralin scaffold as carbonic anhydrase and acetylcholine esterase inhibitors. Arch Pharm 2014;347:68–76. 23. Taylor P, Radic Z. The cholinesterases: from genes to proteins. Annu Rev Pharmacol Toxicol 1994;34:281–320. 24. Dembele K, Haubruge E, Gaspar C. Concentration effects of selected insecticides on brain acetylcholinesterase in the common carp (Cyprinus carpio). Ecotoxicol Environ Saf 2000;45:49–54. 25. Fulton MH, Key PB. Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorus insecticide exposure and effects. Environ Toxicol Chem 2001;20:37–45. 26. Vale JA. Toxicokinetic and toxicodynamic aspects of organophosphorus (OP) insecticide poisoning. Toxicol Lett 1998;102–103: 649–52. 27. Nese C¸. Butrylcholinesterase: structure and physiological importance. Turk J Biochem 2003;28:54–61. 28. Allam AR, Sridhar GR, Das UN. Elevated butrylcholinesterase and acetylcholinesterase may predict the development of type 2 diabetes mellitus and Alzheimer’s disease. Med Hypothesis 2007;69:1272–6. 29. Greenblatt HM, Dvir H, Silman I, Sussman JL. Acetylcholinesterase: a multifaceted target for structure-based drug design of anticholinesterase agents for the treatment of Alzheimer’s disease. J Mol Neurosci 2003;20:369–83. 30. Hobbiger F. The inhibition of acetylcholinesterase by organophosphorus compounds and its reversal. Proc R Soc Med 1961;54:403–5. 31. Massoulie J, Pezzementi L, Bon S, et al. Molecular and cellular biology of cholinesterases. Prog Neurobiol 1993;41:31–91. 32. Akıncıog˘lu A, Akbaba Y, Go¨c¸er, H, et al. Novel sulfamides as potential carbonic anhydrase isoenzymes inhibitors. Bioorg Med Chem 2013;21:1379–85. 33. Akbaba Y, Akıncıog˘lu A, Go¨c¸er H, et al. Carbonic anhydrase inhibitory properties of novel sulfonamide derivatives of aminoindanes and aminotetralins. J Enzyme Inhib Med Chem 2014;29: 35–42. 34. Go¨ksu S, Kazaz C, Su¨tbeyaz Y, Sec¸en H. A concise synthesis of 2amino-1,2,3,4-tetrahydronaphthalene-6,7-diol (6,7-ADTN) from naphthalene-2,3-diol. Helv Chim Acta 2003;86:3310–13. _ Beydemir S 35. Gu¨lc¸in I, ¸ , Bu¨yu¨kokurog˘lu ME. In vitro and in vivo effects of dantrolene on carbonic anhydrase enzyme activities. Biol Pharm Bull 2004;27:613–16. _ Beydemir S 36. Gu¨lc¸in I, ¸ , C¸oban TA, Ekinci D. The inhibitory effect of dantrolene sodium and propofol on 6-phosphogluconate dehydrogenase from rat erythrocyte. Fresen Environ Bull 2008;17: 1283–7. _ Da¸stan A, et al. Carbonic anhydrase inhibitors. 37. Sentu¨rk M, Gu¨lc¸in I, Inhibition of human erythrocyte isozymes I and II with a series of antioxidant phenols. Bioorg Med Chem 2009;17:3207–11. _ et al. Sildenafil is a strong 38. Coban TA, Beydemir S, Gu¨lc¸in I, activator of mammalian carbonic anhydrase isoforms I-XIV. Bioorg Med Chem 2009;17:5791–5. 39. Laemmli DK. Cleavage of structural proteins during in assembly of the head of bacteriophage T4. Nature 1970;227:680–5. _ Purification and characterization of peroxidase 40. Ko¨ksal E, Gu¨lc¸in I. from cauliflower (Brassica oleracea L.) buds. Protein Peptide Lett 2008;15:320–6. ¨ I. _ C¸iftci M, Ku¨freviog˘lu O _ Dantrolene inhibits 41. Sentu¨rk M, Gu¨lc¸in I, human erythrocyte glutathione reductase. Biol Pharm Bull 2008;31: 2036–9. _ O ¨ zdemir M. The inhibitory 42. Si¸seciog˘lu M, C¸ankaya M, Gu¨lc¸in I, effect of propofol on lactoperoxidase. Protein Peptide Lett 2009;16: 46–9.
Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by University of Ulster at Jordanstown on 12/15/14 For personal use only.
DOI: 10.3109/14756366.2014.928704
¨ zdemir M. Interactions of _ O 43. Si¸seciog˘lu M, C¸ankaya M, Gu¨lc¸in I, melatonin and serotonin to lactoperoxidase enzyme. J Enzyme Inhib Med Chem 2010;25:779–83. 44. Bradford MM. A rapid and sensitive method for the quantitation of protein utilizing the principle of protein dye binding. Ana Biochem 1976;72:248–54. 45. Gu¨ney M, Co¸skun A, Topal F, et al. Oxidation of cyanobenzocycloheptatrienes: synthesis, photooxygenation reaction and carbonic anhydrase isoenzymes inhibition properties of some new benzotropone derivatives. Bioorg Med Chem 2014;22:2537–43. _ Menzek A. Synthesis and carbonic 46. C¸etinkaya Y, Go¨c¸er H, Gu¨lc¸in I, anhydrase isoenzymes inhibitory effects of brominated diphenylmethanone and its derivatives. Arch Pharm 2014;347:354–9. _ Ku¨freviog˘lu O ¨ I, _ C¸iftc¸i M. Glucose 647. Beydemir S ¸ , Gu¨lc¸in I, phosphate dehydrogenase: in vitro and in vivo effects of dantrolene sodium. Pol J Pharmacol 2003;355:787–92. _ Beydemir S 48. Gu¨lc¸in I, ¸ , Hisar O. The effect of a-tocopherol on the antioxidant enzymes activities and lipid peroxidation of rainbow trout (Oncorhynchus mykiss). Acta Vet Hung 2005;53:425–33. _ Hisar O, et al. Effect of melatonin on glucose49. Beydemir S ¸ , Gu¨lc¸in I, 6-phospate dehydrogenase from rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo. J Appl Anim Res 2005; 28:65–8. 50. Iga DP, Schmidt R, Iga S, et al. Synthesis and characterization of new chromogenic substrates for exoglycosidases: alpha glucosidase, alpha-mannosidase, and beta galactosidase. Turk J Chem 2013;37: 299–307. _ Ku¨freviog˘lu O ¨ I, _ Oktay M. Purification and characteriza51. Gu¨lc¸in I, tion of polyphenol oxidase from nettle (Urtica dioica L.) and inhibition effects of some chemicals on the enzyme activity. J Enzyme Inhib Med Chem 2005;20:297–302. _ et al. Effect of low molecular weight 52. Hisar O, Beydemir S ¸ , Gu¨lc¸in I, plasma inhibitors of rainbow trout (Oncorhyncytes mykiss) on human erythrocytes carbonic anhydrase-II isozyme activity in vitro and rat erythrocytes in vivo. J Enzyme Inhib Med Chem 2005;20: 35–9. _ Beydemir S 53. Gu¨lc¸in I, ¸ , Hisar O, et al. Melatonin administration increases antioxidant enzymes activities and reduce lipid peroxidation in the rainbow trout (Oncorhynchus mykiss, Walbaum) erythrocytes. Turk J Vet Anim Sci 2009;33:241–5. 54. Verpoorte JA, Mehta S, Edsall JT. Esterase activities of human carbonic anhydrase. J Biol Chem 1967;242:4221–9. 55. Balaydın HT, Sentu¨rk M, Go¨ksu S, Menzek A. Synthesis and carbonic anhydrase inhibitory properties of novel bromophenols and their derivatives including natural products. Vidalol B Eur J Med Chem 2012;54:423–8. 56. Akbaba Y, Balaydın HT, Menzek A, et al. Synthesis and biological evaluation of novel bromophenol derivatives as carbonic anhydrase inhibitors. Arch Pharm 2013;346:447–54.
Inhibitory effects of some carbamates
5
_ 57. Nar M, C¸etinkaya Y, Gu¨lc¸in I, Menzek A. (3,4Dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its derivatives as carbonic anhydrase isoenzymes inhibitors. J Enzyme Inhib Med Chem 2013;28:402–6. 58. Oztu¨rk Sarıkaya SB, Topal F, S ¸ entu¨rk M, et al. In vitro inhibition of a-carbonic anhydrase isozymes by some phenolic compounds. Bioorg Med Chem Lett 2011;21:4259–62. _ Da¸stan A, et al. Carbonic anhydrase inhibitors. 59. S ¸ entu¨rk M, Gu¨lc¸in I, Inhibition of human erythrocyte isozymes I and II with a series of antioxidant phenols. Bioorg Med Chem 2009;17:3207–11. ¨ zdemir H, Gu¨lc¸in I, _ Ku¨freviog˘lu O ¨ I. _ One-step 60. Atasaver A, O purification of lactoperoxidase from bovine milk by affinity chromatography. Food Chem 2013;136:864–70. 61. Ellman GL, Courtney KD, Andres V, Featherston RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88–95. 62. Aksu K, Nar M, Tanc¸ M, et al. The synthesis of sulfamide analogues of dopamine related compounds and their carbonic anhydrase inhibitory properties. Bioorg Med Chem 2013;21:2925–31. ¨ ztu¨rk Sarıkaya SB, Gu¨lc¸in I, _ Supuran CT. Carbonic 63. Innocenti A, O anhydrase inhibitors. Inhibition of mammalian isoforms I-XIV with a series of natural product polyphenols and phenolic acids. Bioorg Med Chem 2010;18:2159–64. 64. ArasHisar S ¸ , Hisar O, Beydemir S ¸ , et al. Effect of vitamin E on carbonic anhydrase enzyme activity in rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo. Acta Vet Hung 2004;52: 413–22. _ et al. The effect of melatonin 65. Hisar O, Beydemir S ¸ , Gu¨lc¸in I, hormone on carbonic anhydrase enzyme activity in rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo. Turk J Vet Anim Sci 2005;29:841–5. _ Ekinci D. Morphine inhibits 66. Coban TA, Beydemir S ¸ , Gu¨lc¸in I, erythrocyte carbonic anhydrase in vitro and in vivo. Biol Pharm Bull 2007;30:2257–61. 67. Raschetti R, Albanese E, Vanacore N, Maggini M. Cholinesterase inhibitors in mild cognitive impairment: a systematic review of randomised trials. PLoS Med 2007;4:1818–28. 68. Imramovsky A, Stepankova S, Vanco J, et al. Acetylcholinesteraseinhibiting activity of salicylanilide N-alkylcarbamates and their molecular docking. Molecules 2012;17:10142–58. 69. Rickwood CJ, Galloway TS. Acetylcholinesterase inhibition as a biomarker of adverse effect: a study of Mytilus edulis exposed to the priority pollutant chlorfenvinphos. Aquatic Toxicol 2004;67: 45–56. 70. Alterio V, Fiore DA, D’Ambrosio K, et al. Multiple binding modes of inhibitors to carbonic anhydrases: How to design specific drugs targeting 15 different isoforms? Chem Rev 2012;112: 4421–68.
Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by University of Ulster at Jordanstown on 12/15/14 For personal use only.
RESEARCH ARTICLE
Carbonic anhydrase and acetylcholinesterase inhibitory effects of carbamates and sulfamoylcarbamates Hu¨lya Go¨c¸er1,2, Akın Akinciog˘lu1,2, Su¨leyman Go¨ksu2, _Ilhami Gu¨lc¸in2,3, and Claudiu T. Supuran4 1
Central Researching Laboratory, Agri Ibrahim Cecen University, Agri, Turkey, 2Faculty of Science, Department of Chemistry, Atatu¨rk University, Erzurum, Turkey, 3Department Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia, and 4Laboratorio di Chimica Bioinorganica, Universita` degli Studi di Firenze, Via della Lastruccia, Sesto Fiorentino, Firenze, Italy Abstract
Keywords
Carbonic anhydrases (CA), as a family of metalloenzymes, are found in almost every type of tissue and play an important role in catalyzing the equilibration of carbon dioxide and carbonic acid. In this study, a series of carbamate derivative was synthesized, and their inhibition effects on hCA I, hCA II and acetylcholinesterase (AChE) enzymes were investigated. They were determined to be very good inhibitor against for both isoenzymes (hCA I and hCA II) and AChE. The hCA I and hCA II were effectively inhibited by the carbamate derivatives, with inhibition constants (Ki) in the range of 194.4–893.5 nM (for hCA I) and 103.9–835.7 nM (for hCA II). On the other hand, Ki parameters of these compounds for AChE enzyme inhibition were determined in the range of 12.0–61.3 nM. The results clearly showed that both CA isoenzymes and AChE were inhibited by carbamate derivatives at the nM levels.
Acetylcholinesterase, AChE, CA, carbamates, carbonic anhydrase, enzyme inhibition
Introduction 1
Carbamates are beneficial biologically active compounds . There are some drugs in the markets containing the carbamate functional group. Neostigmine and eserine drugs are inhibitors of acetylcholinesterase (AChE) and they are used in the treatment of myasthenia gravis disease2. A drug felbamate, commercially known as Felbatol, is an anticonvulsant drug used in the treatment of epilepsy3. In addition, there are some other beneficial biologically active carbamates and sulfamoylcarbamate derivatives used in treatment of sedative-hypnotic medication, insomnia, hypnotics, anxiolytic and muscle relaxants4. In addition, carbonic anhydrase (CA)5 and Hepatitis C virus inhibitory properties of carbamates have also been addressed6. Enzymes are synthesized by living cells and speed up chemical reactions during the metabolism of living organisms7. Enzymes, named biological catalysts, do not produce any by-product, which is a major advantage in synthesis. Isoenzymes have different amino acid sequence but they catalyze the same biochemical reactions. Isoenzymes usually display different kinetic parameters such as KM, and they have different regulatory properties. They are different in their interests against substrates, cofactors and inhibitors. Furthermore, each isoenzyme has different isoelectric points and electrophoretic mobility. It was reported that CA has 16 different isoenzyme located in different tissues8,9. CA provides overall metabolic CO2 transport. This enzyme also plays a role in the accumulation of H+ and HCO 3 in many tissues. Isoenzymes _ Address for correspondence: Prof. Dr. Ilhami Gu¨lc¸in, Faculty of Science, Department of Chemistry, Atatu¨rk University, 25240 Erzurum, Turkey. Tel: +90 4422314389. Fax: +90 442 2360948. E-mail: igulcin@ atauni.edu.tr; [email protected]
History Received 8 May 2014 Revised 23 May 2014 Accepted 23 May 2014 Published online 20 June 2014
have some important functions in kidney, gastric mucosa and eye lens10. Both isoenzymes were purified and characterized from many sources like human erythrocytes, fish erythrocytes, rat erythrocytes, rat saliva, bovine bone and bovine leukocytes, salivary glands, muscles, brain, nerve myelin sheath, pancreas, prostate, endometrial tissues, bacteria and a variety of plant sources. The molecular mass of the enzyme was found to be approximately 30 KDa for mammals11–14. CA isoenzymes are physiologically important enzymes for human body. CA plays an important role in many critical physiological events such as carbon dioxide (CO2)/ bicarbonate (HCO 3 ) respiration, pH and CO2 homeostasis, electrolyte secretion, biosynthetic reactions like gluconeogenesis, lipogenesis and urea synthesis, bone resorption, calcification and tumor formation9,15,16. In plants, CA is mainly found in the thylakoid membrane chloroplasts and cytosol. In the literature, there are many inhibition studies for CA isoenzymes. Generally mononegatif anions and some of the neutral organic compounds like sulfamides and sulfonamides cause recycled inhibition by binding tightly to the enzyme. Acetazolamide is the most commonly used inhibitor for CA isoenzymes17. Due to their pharmacokinetic properties, CA isoenzyme inhibitors (CAIs) were used in treating diseases such as epilepsy and glaucoma. Sulfonamide derivatives are CA’s specific and potent inhibitors16. Alcohols, organic solvents, amide compounds and other neutral loosely inhibitors constitute a weak inhibition in binding the enzyme18,19. CAIs are reserved typically for patients at high risk of vision loss at long-term use. When combined with mitotic, they exhibit stronger effects20. Cholinesterases (ChEs) are special carboxylic ester hydrolases that knocks down the esters of choline and they separated into two classes, AChE (EC3.1.1.7) and butyrylcholinesterase21,22 (BChE,
Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by University of Ulster at Jordanstown on 12/15/14 For personal use only.
2
H. Go¨c¸er et al.
EC 3.1.1.8). AChE is situated in the group of ChEs and hydrolyses the neurotransmitter acetylcholine (ACh) to acetic acid and choline23–26. AChE is known as real ChE, while BuChE is known as non-specific ChE or pseudocholinesterase27. AChE is found in high concentrations in the brain and in red blood cells. BuChE is found in serum, pancreas, liver and central nervous system28. These two enzymes have different optimum substrate concentrations. AChE is a necessary enzyme for the nervous system. AChE inhibitors are used in treatment of several neuromuscular diseases and were studied for treatment of Alzheimer’s disease21,22,29. Organophosphorus and carbamate derivatives are known as the best inhibitors for AChE catalytic activity30. Carbamate pesticides show their toxicity by modifying irreversibly the catalytic serine residue in AChE and the inhibition of the AChE31. As carbamates show important biological activities; CA and ACh esterase inhibitory properties of some carbamate derivatives will be useful for further biological studies. In this research, we aimed to investigate CA and ACh inhibitory properties of two carbamates and six sulfamoylcarbamates for the first time.
Experimental section General information Sulfamoylcarbamates (1, 3 and 4)32, sulfamoylcarbamates (5, 7 and 8)22, carbamate (2)33 and carbamate (6)34 were synthesized as described in the literature. Purification of CA isoenzymes from human erythrocytes with affinity chromatography The fresh human erythrocytes were centrifuged at 10 000 rpm for 30 min, and precipitate was removed and the serum was separated, and the pH was tuned to 8.7 with solid Tris35,36. Before doing so, sepharose-4B-tirozyne-sulfanylamide affinity column balanced with Tris-HCl/Na2SO4 (25 mM/0.1 M, pH 8.7) and tuned human erythrocyte sample was applied to the column. The affinity gel was washed with Tris-HCl/Na2SO4 (25 mM/22 mM, pH: 8.7). hCA I and II isoenzyme were separated with H2NSO3H/Na2HPO4 (0.25 M/0.25 M, pH: 6.7). These studies were performed at 4 C8,37,38. SDS polyacrylamide gel electrophoresis The purity of both hCA I and II was identified using SDS polyacrylamide gel electrophoresis, performed according to the Laemmli’s procedure39 and described in our previous study40. The running and separating gels contained 10% and 3% acrylamide and SDS (0.1%), respectively41,42. A 20-mg sample was administered to the electrophoresis. Gels were stained with some difference of the same solvent without the dye. These solvents are prepared as 1.5 h in Coomassie Brilliant Blue R-250 (0.1%) in methanol (50%) and 10% acetic acid43. Protein identification The content protein during the purification steps was identified with spectrophotometer at 595 nm according to the Bradford method44 explained in previous studies45–49. As in other studies, bovine serum albumin was used as standard in this study50–53. Esterase activity assay This study was made according to the method described by Verpoorte et al.54 described previously55,56. Both CA isoenzymes activities were assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate to 4-nitrophenylate ion over a period of 3 min at 25 C using a spectrophotometer (Shimadzu,
J Enzyme Inhib Med Chem, Early Online: 1–5
UVmini-1240 UV-VIS spectrophotometer, Kyoto, Japan)57. The Ki values were calculated from experiments using three different carbamate derivatives 1–8 concentrations and PNF as the substrate at five different concentrations to create Lineweaver– Burk curves reported previously58–60. Determination of anticholinesterase activity The inhibitory effects of carbamate derivatives 1–8 on AChE activities were measured by slightly modifying the spectrophotometric method of Ellman et al.61. Acetylthiocholine iodide (AChI) was used as substrate of the reaction. 5,50 -dithio-bis(2-nitrobenzoic acid) (DTNB) (product no: D8130-1G, Sigma-Aldrich Co., St. Louis, MO) was used for the determination of the AChE activity. Namely, 100 mL of Tris/HCl buffer (1 M, pH 8.0), 10 mL of sample solution dissolved in ultra pure water at different concentrations and 50 mL AChE from electric eel (Electrophorus electricus) (518 units/mg, product no: C3389-500UN, SigmaAldrich) solution were mixed, incubated for 10 min at 25 C. Then 50 mL of DTNB (0.5 mM) was added. The reaction was then started by the addition of 50 mL of AChI (10 mM, product no: 01480-1G, Sigma-Aldrich). The hydrolysis of these substrates was observed spectrophotometrically by the formation of yellow 5thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, determined by the enzymatic hydrolysis of AChI, at a wavelength of 412 nm. In order to determine the effect of carbamate derivatives 1–8 on AchE, different carbamate derivatives were added to the reaction mixture. The enzyme activity was measured. The IC50 values were obtained from activity (%) versus compounds plots21,62,63.
Results and discussion CA purification and activity assay The purity of the enzymes was confirmed using SDS polyacrylamide gel electrophoresis by hCA I and II isoenzymes were purified separately from human erythrocytes with Sepharose-4BL-Thyrosine-sulfanilamide affinity column chromatography14,64. After hCA purification, we investigated the inhibition effects of carbamate and sulfamoylcarbamate derivatives on hCA I and II. The inhibition measurement results were performed with esterase activity method35,65. In this method, when the CA enzyme hydrolyses the phenyl acetate, the resultant final product shows absorption at 348 nm19,66. CA isoenzymes inhibition effects In recent years, the CA isoenzymes have become an interesting target for the design of inhibitors or activators for biomedical applications. With this purpose, we have investigated the inhibitory effects of the carbamate derivatives and sulfamoylcarbamate on the CA isoenzymes hCA I and II. Lineweaver–Burk graphs were drawn for the Ki values determination. Then the average of Ki and IC50 values were calculated from these graphs (Table 1). At first step, we report the inhibitory effects of carbamate derivatives (2 and 6) and sulfamoylcarbamate (1, 3, 4, 5, 7 and 8) on the esterase activity of hCA I and II under in vitro conditions. The results are listed in Table 1. These compounds were shown to be effective inhibitors of cytosolic isoenzymes, hCA I and II (Table 1). Carbamates (2 and 6) and sulfamoylcarbamate (1, 3, 4, 5, 7 and 8) were all found to inhibit hCA I, with Ki values ranging from 264.16–586.50 nM for carbamates (2 and 6) to 194.41–893.50 nM for sulfamoylcarbamate (1, 3, 4, 5, 7 and 8). On the other hand, AZA, which used as positive standard inhibitor for CA isoenzymes, showed less inhibition with Ki values of 36.20 mM. Carbamates and sulfamoylcarbamate (1–8) were all found to inhibit hCA II, with Ki
Inhibitory effects of some carbamates
DOI: 10.3109/14756366.2014.928704
values ranging from 103.90–835.74 nM for carbamates (2 and 6) to 167.51–482.00 nM for sulfamoylcarbamate (1, 3, 4, 5, 7 and 8) with a comparable potency as the reference compound AZA with Ki value of 3.70 mM.
Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by University of Ulster at Jordanstown on 12/15/14 For personal use only.
ACh esterase enzyme inhibition effects Different types of AChE inhibitors have been studied for the treatment of Alzheimer’s disease67. These inhibitors are rapidly causes inhibition on the AChE and BChE alone68. Results obtained from these studies showed that an investigation into the mechanism of action of AChE might cause the design of inhibitors, which can be used as a therapeutic in the future. In addition, some studies showed that the AChE inhibitors have different properties in terms of action mechanism, metabolism and brain selectivity. The structures of the investigated carbamate derivatives were shown in the Figure 1. This study focused on the influence of carbamates on the inhibition kinetics of AChE and recovery of corresponding carbamylated enzymes. In our study, a series of carbamates was investigated for their ability to inhibit AChE. Carbamate derivatives 1–8 were evaluated in two groups as carbamate (2 and 6) and
3
sulfamoylcarbamate (1, 3, 4, 5, 7 and 8). These both groups were investigated in more details. According to our data, inhibitory effects these carbamates revealed a significant elevation in the case of AChE. Generally, these compounds showed higher inhibition and higher lipophilicity. Considering the results, all compounds expressed significantly higher inhibition activity. All of carbamate derivatives studied significantly higher AChE inhibitory activity than that of standard AChE inhibitors such as rivastigmine and galanthamine68. Furthermore, the IC50 values of these both standard compounds are summarized in Table 2. As can be seen in the results obtained from Figure 1, AChE was effectively inhibited by carbamates derivatives 1–8, with Ki values in the range of 12.0 ± 2.01 to 61.3 ± 2.59 nM. Furthermore, all of carbamate derivatives (1–8) had almost similar Ki values. The differences between the highest and the lowest Ki values of carbamate derivatives 1–8 were only four-fold. The most active carbamate derivative is compound 2 and showed a Ki value of 12.0 ± 2.01 nM and IC50 value calculated as 29.9 nM. As a result, carbamates and sulfamoylcarbamate (1–8) were all found to inhibit AChE, with Ki values ranging from 12.06 to 14.40 nM for carbamates (2 and 6) to 15.52–61.38 nM for sulfamoylcarbamate (1, 3, 4, 5, 7 and 8).
Table 1. Human carbonic anhydrase isoenzymes (hCA I and II) inhibition value with carbamate derivatives by an esterase assay with NPA. IC50 (nM)
Table 2. IC50 values of acetylcholinesterase enzyme (AChE) inhibition parameters of carbamate derivatives comparison with standards rivastigmine (RIV) and galanthamine (GLT).
KI (nM)
Compounds
hCA I
hCA II
hCA I
hCA II
1* 2y 3* 4* 5* 6y 7* 8* AZAz
319.6 388.0 236.0 244.0 686.0 324.0 300.2 226.2
251.9 524.2 259.6 134.2 184.8 251.5 191.0 194.6
387.3 264.2 295.7 245.6 893.5 586.5 194.4 207.5 36.20
196.2 835.7 167.5 378.5 342.8 103.9 176.8 482.0 3.70
*Sulfamoylcarbamates. yCarbamate derivatives. zAZA was used as positive standard inhibitor for both CA isoenzymes (CA I and II). Ki values belonging to AZA was expressed as micromolar levels.
Compounds 1 2 3 4 5 6 7 8 RIV* GLT*
MeO
2
MeO
27.6 ± 12.9 12.0 ± 2.0 15.5 ± 1.8 31.4 ± 7.3 61.3 ± 25.9 14.4 ± 3.3 38.4 ± 5.1 57.6 ± 6.9 – –
3
NHCO2CH2Ph 6
NHSO2NHCO2CH2Ph 5 NHSO2NHCO2CH2Ph
MeO MeO
7
NHSO2NHCO2CH2Ph
MeO MeO
MeO
MeO
0.9754 0.9914 0.9877 0.9776 0.9855 0.9622 0.9819 0.9565 – –
NHCO2CH2Ph
4
NHSO2NHCO2CH2Ph
50.20 29.90 105.40 66.91 69.92 95.78 104.10 94.86 501.00 4.00
MeO
NHSO2NHCO2CH2Ph
MeO
KI (nM)
MeO
MeO MeO
MeO
r2
*Rivastigmine (RIV) and galanthamine (GLT) were used as positive standards and obtained from the reference of 37 and expressed as micromolar (mM).
NHSO2NHCO2CH2Ph
1
IC50 (nM)
8
Figure 1. The chemical structures of synthesized carbamate derivatives.
4
H. Go¨c¸er et al.
Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by University of Ulster at Jordanstown on 12/15/14 For personal use only.
Conclusion ACh is one of the key neurotransmitter for peripheral nervous system, and therefore the inhibition of AChE has been proposed as a drug for the neurotoxicity69. Carbamate derivatives (1–8) were effective of AChE inhibition properties. In addition, CA inhibitory properties of 1–8 have also been evaluated. Carbamate derivatives 1–8 were evaluated in two groups as carbamate (2 and 6) and sulfamoylcarbamate (1, 3, 4, 5, 7 and 8). The results of the two groups were generally the same. These carbamates can be important lead compounds for greater synthetic and biological objectives. Most importantly, carbamate derivatives 1–8 may be used in generating potent hCA I and II inhibitors. Referring to the literature, there are studies about the inhibition effect of carbamate derivatives on these enzymes70. Specifically, carbamate derivatives are numerous works on AChE inhibitory effect. Furthermore, these studies may play an important role in drug design for Alzheimer’s disease.
Declaration of interest The authors declared that there is no conflict of interests.
References 1. Vacondio F, Silva C, Mor M, Testa B. Qualitative structuremetabolism relationships in the hydrolysis of carbamates. Drug Metab Rev 2010;42:551–89. 2. Milkani E, Lambert CR, McGimpsey GW. Direct detection of acetylcholinesterase inhibitor binding with an enzyme-based surface plasmon resonance sensor. Anal Biochem 2011;408:212–19. 3. Kung CH, Kwon CH. Carbamate derivatives of felbamate as potential anticonvulsant agents. Med Chem Res 2010;19:498–513. 4. Gilman AG, Goodman LS, Rall TW, Murad F, eds. Goodman and Gilman’s the pharmacological basis of therapeutics. 7th ed. New York: Macmillan Publishing Co; 1985. 5. Gavernet L, Funes JLG, Palestro PH, et al. Inhibition pattern of sulfamide-related compounds in binding to carbonic anhydrase isoforms I, II, VII, XII and XIV. Bioorg Med Chem 2013;21:1410–18. 6. Su L, Li L, Li Y, et al. Simple and accurate approaches to predict the activity of benzothiadiazine derivatives as HCV inhibitors. Med Chem Res 2012;21:2079–96. 7. Shahwar D, Raza MA, Khan T. Synthesis and evaluation of enzyme inhibitory potential of some derivatives of scopolamine. Turk J Chem 2012;36:257–66. _ Supuran CT. Carbonic anhydrase 8. Oztu¨rk Sarikaya SB, Gu¨lc¸in I, inhibitors. Inhibition of human erythrocyte isoenzymes I and II with a series of phenolic acids. Chem Biol Drug Des 2010;75:515–20. _ Scozzafava A, Supuran CT. Carbonic 9. Innocenti A, Gu¨lc¸in I, anhydrase inhibitors. Antioxidant polyphenol natural products effectively inhibit mammalian isoforms I-XV. Bioorg Med Chem Lett 2010;20:5050–3. _ Beydemir S 10. Sentu¨rk M, Gu¨lc¸in I, ¸ , et al. In vitro inhibition of human carbonic anhydrase I and II isoenzymes with natural phenolic compounds. Chem Biol Drug Des 2011;77:494–9. 11. Feldstein JB, Silverman DN. Purification and characterization of carbonic anhydrase from the saliva of the rat. J Biol Chem 1984;259: 5447–53. 12. Krungkrai SR, Suraveratum N, Rochanakij S, Krungkrai J. Characterization of carbonic anhydrase in Plasmodium falciparium. Int J Parasitol 2001;31:661–8. ¨ I. _ Effects of gentamicine on 13. Beydemir S ¸ , C¸iftc¸i M, Ku¨freviog˘lu O enzyme activities of carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo. Biol Pharm Bull 2002;25: 966–9. _ Effect of melatonin on carbonic anhydrase 14. Beydemir S ¸ , Gu¨lc¸in I. from human erythrocyte in vitro and from rat erythrocyte in vivo. J Enzyme Inhib Med Chem 2004;19:193–7. _ Rosmarinic acid: a potent carbonic anhydrase 15. Topal M, Gu¨lc¸in I. isoenzymes inhibitor. Turk J Chem 2014. [Epub ahead of print]. DOI: 10.3906/kim-1403–5. 16. Supuran CT, Scozzafava A. Carbonic anhydrase inhibitors part 94. 1,3,4-thiadiazole-2-sulfonamide derivatives as antitumor agents? Eur J Med Chem 2000;35:867–74.
J Enzyme Inhib Med Chem, Early Online: 1–5
_ Beydemir S. Phenolic compounds as antioxidants: carbonic 17. Gu¨lc¸in I, anhydrase isoenzymes inhibitors. Mini Rev Med Chem 2013;13: 408–30. 18. Liang YJ, Lipscom WN. Substrate and inhibitor binding to human carbonic anhydrase II: a theoretical study. In: Botre´ F, ed. Inhibitors in the carbonic anhydrase from biochemistry and physiology and clinical medicine. Weinheim: VCH Publishers; 1991:50–64. _ Ekinci D. The inhibitory effect of 19. Coban TA, Beydemir S ¸ , Gu¨lc¸in I, ethanol on carbonic anhydrase isoenzymes: in vivo and in vitro studies. J Enzyme Inhib Med Chem 2008;23:266–70. _ 20. Oragli KM. Klinik Oftalmolji. 4. Baskı. Istanbul: Nobel Tıp Kitabevi; 2001. ¨ zta¸skın N, et al. Synthesis, antioxidant 21. Go¨c¸er H, Akıncıog˘lu A, O and antiacetylcholinesterase activities of sulfonamide derivatives of dopamine related compounds. Arch Pharm 2013;346:783–92. _ Go¨ksu S. Novel sulfamides and 22. Akıncıog˘lu A, Nar M, Gu¨lc¸in I, sulfonamides incorporating tetralin scaffold as carbonic anhydrase and acetylcholine esterase inhibitors. Arch Pharm 2014;347:68–76. 23. Taylor P, Radic Z. The cholinesterases: from genes to proteins. Annu Rev Pharmacol Toxicol 1994;34:281–320. 24. Dembele K, Haubruge E, Gaspar C. Concentration effects of selected insecticides on brain acetylcholinesterase in the common carp (Cyprinus carpio). Ecotoxicol Environ Saf 2000;45:49–54. 25. Fulton MH, Key PB. Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorus insecticide exposure and effects. Environ Toxicol Chem 2001;20:37–45. 26. Vale JA. Toxicokinetic and toxicodynamic aspects of organophosphorus (OP) insecticide poisoning. Toxicol Lett 1998;102–103: 649–52. 27. Nese C¸. Butrylcholinesterase: structure and physiological importance. Turk J Biochem 2003;28:54–61. 28. Allam AR, Sridhar GR, Das UN. Elevated butrylcholinesterase and acetylcholinesterase may predict the development of type 2 diabetes mellitus and Alzheimer’s disease. Med Hypothesis 2007;69:1272–6. 29. Greenblatt HM, Dvir H, Silman I, Sussman JL. Acetylcholinesterase: a multifaceted target for structure-based drug design of anticholinesterase agents for the treatment of Alzheimer’s disease. J Mol Neurosci 2003;20:369–83. 30. Hobbiger F. The inhibition of acetylcholinesterase by organophosphorus compounds and its reversal. Proc R Soc Med 1961;54:403–5. 31. Massoulie J, Pezzementi L, Bon S, et al. Molecular and cellular biology of cholinesterases. Prog Neurobiol 1993;41:31–91. 32. Akıncıog˘lu A, Akbaba Y, Go¨c¸er, H, et al. Novel sulfamides as potential carbonic anhydrase isoenzymes inhibitors. Bioorg Med Chem 2013;21:1379–85. 33. Akbaba Y, Akıncıog˘lu A, Go¨c¸er H, et al. Carbonic anhydrase inhibitory properties of novel sulfonamide derivatives of aminoindanes and aminotetralins. J Enzyme Inhib Med Chem 2014;29: 35–42. 34. Go¨ksu S, Kazaz C, Su¨tbeyaz Y, Sec¸en H. A concise synthesis of 2amino-1,2,3,4-tetrahydronaphthalene-6,7-diol (6,7-ADTN) from naphthalene-2,3-diol. Helv Chim Acta 2003;86:3310–13. _ Beydemir S 35. Gu¨lc¸in I, ¸ , Bu¨yu¨kokurog˘lu ME. In vitro and in vivo effects of dantrolene on carbonic anhydrase enzyme activities. Biol Pharm Bull 2004;27:613–16. _ Beydemir S 36. Gu¨lc¸in I, ¸ , C¸oban TA, Ekinci D. The inhibitory effect of dantrolene sodium and propofol on 6-phosphogluconate dehydrogenase from rat erythrocyte. Fresen Environ Bull 2008;17: 1283–7. _ Da¸stan A, et al. Carbonic anhydrase inhibitors. 37. Sentu¨rk M, Gu¨lc¸in I, Inhibition of human erythrocyte isozymes I and II with a series of antioxidant phenols. Bioorg Med Chem 2009;17:3207–11. _ et al. Sildenafil is a strong 38. Coban TA, Beydemir S, Gu¨lc¸in I, activator of mammalian carbonic anhydrase isoforms I-XIV. Bioorg Med Chem 2009;17:5791–5. 39. Laemmli DK. Cleavage of structural proteins during in assembly of the head of bacteriophage T4. Nature 1970;227:680–5. _ Purification and characterization of peroxidase 40. Ko¨ksal E, Gu¨lc¸in I. from cauliflower (Brassica oleracea L.) buds. Protein Peptide Lett 2008;15:320–6. ¨ I. _ C¸iftci M, Ku¨freviog˘lu O _ Dantrolene inhibits 41. Sentu¨rk M, Gu¨lc¸in I, human erythrocyte glutathione reductase. Biol Pharm Bull 2008;31: 2036–9. _ O ¨ zdemir M. The inhibitory 42. Si¸seciog˘lu M, C¸ankaya M, Gu¨lc¸in I, effect of propofol on lactoperoxidase. Protein Peptide Lett 2009;16: 46–9.
Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by University of Ulster at Jordanstown on 12/15/14 For personal use only.
DOI: 10.3109/14756366.2014.928704
¨ zdemir M. Interactions of _ O 43. Si¸seciog˘lu M, C¸ankaya M, Gu¨lc¸in I, melatonin and serotonin to lactoperoxidase enzyme. J Enzyme Inhib Med Chem 2010;25:779–83. 44. Bradford MM. A rapid and sensitive method for the quantitation of protein utilizing the principle of protein dye binding. Ana Biochem 1976;72:248–54. 45. Gu¨ney M, Co¸skun A, Topal F, et al. Oxidation of cyanobenzocycloheptatrienes: synthesis, photooxygenation reaction and carbonic anhydrase isoenzymes inhibition properties of some new benzotropone derivatives. Bioorg Med Chem 2014;22:2537–43. _ Menzek A. Synthesis and carbonic 46. C¸etinkaya Y, Go¨c¸er H, Gu¨lc¸in I, anhydrase isoenzymes inhibitory effects of brominated diphenylmethanone and its derivatives. Arch Pharm 2014;347:354–9. _ Ku¨freviog˘lu O ¨ I, _ C¸iftc¸i M. Glucose 647. Beydemir S ¸ , Gu¨lc¸in I, phosphate dehydrogenase: in vitro and in vivo effects of dantrolene sodium. Pol J Pharmacol 2003;355:787–92. _ Beydemir S 48. Gu¨lc¸in I, ¸ , Hisar O. The effect of a-tocopherol on the antioxidant enzymes activities and lipid peroxidation of rainbow trout (Oncorhynchus mykiss). Acta Vet Hung 2005;53:425–33. _ Hisar O, et al. Effect of melatonin on glucose49. Beydemir S ¸ , Gu¨lc¸in I, 6-phospate dehydrogenase from rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo. J Appl Anim Res 2005; 28:65–8. 50. Iga DP, Schmidt R, Iga S, et al. Synthesis and characterization of new chromogenic substrates for exoglycosidases: alpha glucosidase, alpha-mannosidase, and beta galactosidase. Turk J Chem 2013;37: 299–307. _ Ku¨freviog˘lu O ¨ I, _ Oktay M. Purification and characteriza51. Gu¨lc¸in I, tion of polyphenol oxidase from nettle (Urtica dioica L.) and inhibition effects of some chemicals on the enzyme activity. J Enzyme Inhib Med Chem 2005;20:297–302. _ et al. Effect of low molecular weight 52. Hisar O, Beydemir S ¸ , Gu¨lc¸in I, plasma inhibitors of rainbow trout (Oncorhyncytes mykiss) on human erythrocytes carbonic anhydrase-II isozyme activity in vitro and rat erythrocytes in vivo. J Enzyme Inhib Med Chem 2005;20: 35–9. _ Beydemir S 53. Gu¨lc¸in I, ¸ , Hisar O, et al. Melatonin administration increases antioxidant enzymes activities and reduce lipid peroxidation in the rainbow trout (Oncorhynchus mykiss, Walbaum) erythrocytes. Turk J Vet Anim Sci 2009;33:241–5. 54. Verpoorte JA, Mehta S, Edsall JT. Esterase activities of human carbonic anhydrase. J Biol Chem 1967;242:4221–9. 55. Balaydın HT, Sentu¨rk M, Go¨ksu S, Menzek A. Synthesis and carbonic anhydrase inhibitory properties of novel bromophenols and their derivatives including natural products. Vidalol B Eur J Med Chem 2012;54:423–8. 56. Akbaba Y, Balaydın HT, Menzek A, et al. Synthesis and biological evaluation of novel bromophenol derivatives as carbonic anhydrase inhibitors. Arch Pharm 2013;346:447–54.
Inhibitory effects of some carbamates
5
_ 57. Nar M, C¸etinkaya Y, Gu¨lc¸in I, Menzek A. (3,4Dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its derivatives as carbonic anhydrase isoenzymes inhibitors. J Enzyme Inhib Med Chem 2013;28:402–6. 58. Oztu¨rk Sarıkaya SB, Topal F, S ¸ entu¨rk M, et al. In vitro inhibition of a-carbonic anhydrase isozymes by some phenolic compounds. Bioorg Med Chem Lett 2011;21:4259–62. _ Da¸stan A, et al. Carbonic anhydrase inhibitors. 59. S ¸ entu¨rk M, Gu¨lc¸in I, Inhibition of human erythrocyte isozymes I and II with a series of antioxidant phenols. Bioorg Med Chem 2009;17:3207–11. ¨ zdemir H, Gu¨lc¸in I, _ Ku¨freviog˘lu O ¨ I. _ One-step 60. Atasaver A, O purification of lactoperoxidase from bovine milk by affinity chromatography. Food Chem 2013;136:864–70. 61. Ellman GL, Courtney KD, Andres V, Featherston RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88–95. 62. Aksu K, Nar M, Tanc¸ M, et al. The synthesis of sulfamide analogues of dopamine related compounds and their carbonic anhydrase inhibitory properties. Bioorg Med Chem 2013;21:2925–31. ¨ ztu¨rk Sarıkaya SB, Gu¨lc¸in I, _ Supuran CT. Carbonic 63. Innocenti A, O anhydrase inhibitors. Inhibition of mammalian isoforms I-XIV with a series of natural product polyphenols and phenolic acids. Bioorg Med Chem 2010;18:2159–64. 64. ArasHisar S ¸ , Hisar O, Beydemir S ¸ , et al. Effect of vitamin E on carbonic anhydrase enzyme activity in rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo. Acta Vet Hung 2004;52: 413–22. _ et al. The effect of melatonin 65. Hisar O, Beydemir S ¸ , Gu¨lc¸in I, hormone on carbonic anhydrase enzyme activity in rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo. Turk J Vet Anim Sci 2005;29:841–5. _ Ekinci D. Morphine inhibits 66. Coban TA, Beydemir S ¸ , Gu¨lc¸in I, erythrocyte carbonic anhydrase in vitro and in vivo. Biol Pharm Bull 2007;30:2257–61. 67. Raschetti R, Albanese E, Vanacore N, Maggini M. Cholinesterase inhibitors in mild cognitive impairment: a systematic review of randomised trials. PLoS Med 2007;4:1818–28. 68. Imramovsky A, Stepankova S, Vanco J, et al. Acetylcholinesteraseinhibiting activity of salicylanilide N-alkylcarbamates and their molecular docking. Molecules 2012;17:10142–58. 69. Rickwood CJ, Galloway TS. Acetylcholinesterase inhibition as a biomarker of adverse effect: a study of Mytilus edulis exposed to the priority pollutant chlorfenvinphos. Aquatic Toxicol 2004;67: 45–56. 70. Alterio V, Fiore DA, D’Ambrosio K, et al. Multiple binding modes of inhibitors to carbonic anhydrases: How to design specific drugs targeting 15 different isoforms? Chem Rev 2012;112: 4421–68.
