The authors are grateful to the Wellcome Trust (Catalyst Biomedica Development Award 063729/Z/01/Z) for financial support.This communication describes the synthesis of a new class of N-hydroxyguanidine (NHG) pro-drugs which release nitric oxide (NO), triggered by the action of gamma-glutamyl transpeptidase (gamma-GT), and have potential for the treatment of acute renal injury/failure (ARI/ARF).Publisher PDFPeer reviewe

Botting, Nigel P.

Kulczynska, Agnieszka

Megson, Ian L.

Webb, David J.

Zhang, Qingzhi

Chemical Communications

English

PubMed

The authors are grateful to the Wellcome Trust (Catalyst Biomedica Development Award 063729/Z/01/Z) for financial support.This communication describes the synthesis of a new class of N-hydroxyguanidine (NHG) pro-drugs which release nitric oxide (NO), triggered by the action of gamma-glutamyl transpeptidase (gamma-GT), and have potential for the treatment of acute renal injury/failure (ARI/ARF).Peer reviewe

St Andrews Research Repository

This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 1389--1391 1389Cite this: Chem. Commun.,2013,49, 1389A new class of NO-donor pro-drugs triggered byc-glutamyl transpeptidase with potential forreno-selective vasodilatation†Qingzhi Zhang,*a Agnieszka Kulczynska,a David J. Webb,b Ian L. Megson*c andNigel P. Bottingz*aThis communication describes the synthesis of a new class ofN-hydroxyguanidine (NHG) pro-drugs which release nitric oxide(NO), triggered by the action of c-glutamyl transpeptidase (c-GT),and have potential for the treatment of acute renal injury/failure(ARI/ARF).Acute renal injury (AKI), or failure (ARF), is a common compli-cation that aﬀects millions of people worldwide, particularly inintensive care units, where it is associated with a mortality rateof between 50% and 80%.1 There is no eﬀective pharmaceuticaltherapy to date. One of the major causes of AKI is ischemia-reperfusion injury,2,3 following aortic ring cross-clampingduring by-pass surgery, which can lead to renal ischemia.4Reperfusion of ischemic renal tissue causes the generation ofreactive oxygen species which induce renal cell injury5 andpromote impairment of renal perfusion at least in part viainactivation of the vasodilator, nitric oxide (NO).6–8 Thus, akidney selective vasodilator with antioxidant properties isattractive to maintain blood flow to oﬀset AKI and scavengethe reactive oxygen species. Localisation of activity to thekidney would avoid a systemic reduction in blood pressure.Dopamine and fenoldopam, specific agonists of the dopamine-1 receptor, have been used clinically in an eﬀort to reduce therisk of perioperative renal dysfunction, but the eﬀectivenessof these agents is not clear.9,10 We hypothesised that aneﬀective exogenous NO-donor, which selectively increases renalvasodilatation, would oﬀer an alternative.There are a wide range of NO-donor drugs in existence,11including conventional organic nitrates and nitrites, S-nitrothiols,NONOates and N-hydroxyguanidines (NHGs).12–16 The NHGs1 are analogues of No-hydroxy-L-arginine (NOHA), a biosyn-thetic intermediate involved in the generation of NO fromL-arginine.11 Several enzymatically activated NHG pro-drugshave been reported such as peptidylglycine a-amidating mono-oxygenase (PAM)-active O-carboxymethyl N-hydroxyguanidines17and N-b-galactosidases-active (b-D-galactopyranos-1-yl)oxyguani-dine.18 Our approach aimed to mask the NO generating N-OHgroup with a g-glutamyl residue to facilitate activation by theenzyme, g-glutamyl transpeptidase (g-GT). Given that g-GT isprimarily expressed in the kidney (5–10 fold higher than in theliver and pancreas),19 it was envisaged that this enzyme could beused to trigger reno-selective release of an NHG and subsequentin situ generation of NO (Scheme 1). A similar strategy hasbeen described for reno-selective L-3,4-dihydroxyphenylalanine(L-DOPA), the Glu-DOPA.20,21However, the direct coupling of NHGs with a g-glutamylresidue was hampered by intramolecular cyclization anddehydration leading to a 1,2,4-oxadiazole ring; or alternativelylactamization and release of a pyroglutamic acid (Scheme 2,data not included).In an eﬀort to prevent these modes of cyclization, weinvestigated the use of a bridge between the NHG and theg-glutamyl group. Both g-glutamyl itself and g-aminobutanoyl(GABA)22 were explored as linkers. Thus 2a and 2b becamesynthesis targets (Scheme 3) and they were prepared viaappropriately protected dipeptide intermediates (ESI;† SchemeS1). Unfortunately 2a gradually decomposed presumably dueto the carboxylic acid moieties promoting autodegradation.Scheme 1 Approach to g-GT triggered release of NHG 1 and the reno-selectiverelease of nitric oxide.a University of St Andrews, EaStChem School of Chemistry and Centre forBiomolecular Sciences, North Haugh, St Andrews, Fife KY16 9ST, UK.E-mail: qz@st-andrews.ac.uk; Fax: +44 (0)1334 463808; Tel: +44 (0)1334 467274b Centre for Cardiovascular Science, The Queen’s Medical Research Institute, TheUniversity of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UKc Free Radical Research Facility, Department of Diabetes and CardiovascularScience, The University of The Highlands & Islands, Centre for Health Science,Old Perth Road, Inverness, IV2 3JH, UK. E-mail: ian.megson@uhi.ac.uk;Fax: +44 (0)1463 711245; Tel: +44 (0)1463 279562† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2cc38382a‡ N. P. Botting died on 4th June 2011.Received 21st November 2012,Accepted 20th December 2012DOI: 10.1039/c2cc38382awww.rsc.org/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 21 December 2012. Downloaded on 05/08/2013 16:18:16. View Article OnlineView Journal  | View Issue1390 Chem. Commun., 2013, 49, 1389--1391 This journal is c The Royal Society of Chemistry 2013On the other hand, 2b could be purified by preparative HPLCbut was found to be resistant to g-GT-mediated cleavage in vitroand was considered not to be a useful pro-drug. This promptedthe preparation of 3 (Scheme 3), involving the conjugation ofonly one GABA-Glu dipeptide onto a hydroxamic acid, analternative NO-donor.11 Compound 3 too, unfortunately, wasfound to be resistant to g-GT mediated deacylation, suggestingthat the GABA-Glu peptide linker is not suitable for g-GTcleavage in this setting.g-Glutamyl anilines are known substrates for g-GT23 andpresented an alternative linker option. The success of such anapproach would involve a 1,6-elimination following the actionof g-GT on N-g-glutamylaminobenzyloxy-guanidine 4a–c, asillustrated in Scheme 4. Similar spacers have been employedpreviously in anticancer pro-drug design.24In the event, the synthesis of 4a–c was successfully accom-plished through a six-step reaction sequence (Scheme 4).Firstly, g-glutamylation of 4-aminobenzylalcohol with Alloc-L-glutamic acid 1-allyl ester (Alloc-Glu-OAll) (ESI;† Scheme S1)gave benzyl alcohol 5. Conversion of the benzylalcohol moietyto the corresponding bromide 6 followed by nucleophilic dis-placement with BocNHOH generated aminooxide 7, and thentreatment with CF3COOH–DCM, gave the key intermediate 8which was coupled with the required amino(alkyl/aryliminio)-methanesulfonate 9a–c to generate 10a–c. Finally the All/Allocgroups were removed under neutral conditions with([Pd(PPh3)4]/PhSiH3) to give 4a–c.The same aminobenzyl linker was also used for theg-glutamylation of N-hydroxyformamidines (NHFs) (Scheme 5).N0-Hydroxy-N-(4-butyl-2-methylphenyl)formamidine25 and N0-hydroxy-N-(3-chloro-4-morpholin-4-ylphenyl)formamide26 have been docu-mented as 20-hydroxyeicosatetraenoic acid (20-HETE) inhibitors.20-HETE is a major metabolite of arachidonic acid and is a potentvasoconstrictor; localisation of an NHF would counter the eﬀect of20-HETE and induce a synergic vasodilation eﬀect mediated byNO. Thus N0-hydroxyphenylethylformamidine 12 was prepared inthis study and converted to pro-drug 14.Pro-drugs 4a–c and 14 were rapidly cleaved by g-GT and theywere completely deacylated after 1 h, as judged by LC-MS. Fig. 1(a)and (b) illustrates the LCMS trace of 4b and the conversion of 4b todeacylated intermediate 15 [M-Glu]+ by g-GT. This was in clearcontrast to the GABA-linked candidates 2b and 3, which proved tobe resistant to the action of g-GT. 1,6-Elimination and loss of thelinker from 15 to generate the parent NHG 1b is significantly slower(trace amount of parent 1b was detected by selective ion monitoringatm/z 180) than the cleavage of the g-glutamylmoiety. In preliminaryexperiments with animal tissue, LC-MS analysis revealed B90%conversion of 4b (100 mM) to 1b in a rat renal homogenate (37 1C;45 min). In addition, 4b was found to induce substantial vaso-dilatation in rat isolated perfused kidney preparations (50% ofmaximum vasodilatation induced by B40 mM 4b). Details of thebioactivity of these pro-drugs will be reported elsewhere.In summary, several candidate NO-donor pro-drugs havebeen prepared, designed for activation by g-GT. The pro-drugsScheme 2 Cyclization of direct coupling of NHGs with g-glutamyl residue(s).Scheme 3 Design of Glu/Gaba linked g-glutamyl NO-donor pro-drugs of NHGand hydroxamic acid.Scheme 4 Design and synthesis of aminobenzyl linked g-glutamyl NO-donorpro-drugs of NHG: (i) 4-aminobenzylalcohol, EEDQ, DCM, rt, 12 h, 85%; (ii) PBr3,THF, 0 1C, 2 h, 87%; (iii) BocNHOH, NaH, THF, 0 1C, 4 h, 83%; (iv) CF3CO2H, DCM,92%; (v) 9a R = Ph or 9b R = PhCH2CH2 or 9c R = furfuryl, Et3N, DMAP, DCM,38–53%; (vi) [Pd(PPh3)4], PhSiH3, DCM, 37–89%.Scheme 5 Synthesis of N-hydroxyformamidine and its glutamyl pro-drug:(i) Me2NCH(OMe2), reflux, 2 h, quantitative; (ii) NH2OHHCl, MeOH, 63%;(iii) 8, THF, reflux, 29%; (iv) [Pd(PPh3)4], PhSiH3, DCM, rt, 6 h, 53%.Communication ChemCommOpen Access Article. Published on 21 December 2012. Downloaded on 05/08/2013 16:18:16. View Article OnlineThis journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 1389--1391 1391comprise the parent NO-donor, a linker and a g-glutamylmoiety. GABA-linked pro-drugs are not suitable substrates forg-GT, but those linked by the aminobenzyl moiety proved to begood substrates for the enzyme. The g-glutamyl group is cleavedrapidly, with a slower decomposition of the aminobenzyl linker.Improved design is now focussed on tuning the spacer toencourage a more rapid release of the parent NHG drug.The authors are grateful to the Wellcome Trust (CatalystBiomedica Development Award 063729/Z/01/Z) for financial support.Thanks go to Prof. David O’Hagan (University of St Andrews) forhis input into manuscript preparation.References1 R. W. Schrier, W. Wang, B. Poole and A. Mitra, J. Clin. Invest., 2004,114, 5–14.2 J. V. Bonvebtre and J. M. Weinberg, J. Am. Soc. Nephrol., 2003, 14,2199–2210.3 T. A. Sutton, C. J. Fisher and B. A. Molitoris, Kidney Int., 2002, 62,1539–1549.4 G. Mariscalco, R. Lorusso, C. Dominici, A. Renzulli and A. Sala, Ann.Thorac. Surg., 2011, 92, 1539–1547.5 P. A. Grace, Br. J. Surg., 1994, 81, 637–647.6 M. L. Brocq, S. J. Leslie, P. Milliken and I. L. Megson, Antioxid.Redox Signaling, 2008, 10, 1631–1674.7 A. MacKenzie and W. Martin, Br. J. Pharmacol., 1998, 124, 710–728.8 M. Saran, C. Michel and W. Bors, Free Radical Res. Commun., 1990,10, 221–226.9 M. D. Denton, G. M. Chertow and H. R. Brady, Kidney Int., 1996, 49,4–14.10 T. Bove, G. Landoni, M. G. Calabro`, G. Aletti, G. Marino,E. Cerchierini, G. Crescenzi and A. Zangrillo, Circulation, 2005,111, 3230–3235.11 P. G. Wang, T. B. Cai and N. Taniguchi, Nitric Oxide Donors: ForPharmaceutical and Biological Applications, Wiley-VCH, 2005, ISBN,3-527-31015-0.12 A. Renodon-Cornie`re, S. Dijols, C. Perollier, D. Lefevre-Groboillot,J. L. Boucher, R. Attias, M. A. Sari, D. Stuehr and D. Mansuy, J. Med.Chem., 2002, 45, 944–954.13 D. Mansuy and J. L. Boucher, Drug Metab. Rev., 2002, 34, 593–606.14 M. Xian, X. P. Li, X. P. Tang, X. C. Chen, X. L. Zheng, J. J. Galligan,D. L. Kreulen and P. G. Wang, Bioorg. Med. Chem. Lett., 2001, 11,2377–2380.15 M. Xian, N. Fujiwara, T. Cai, Z. Wen, S. Kazuma, A. Janczuk, X. Tang,V. Telyatnikov, Y. Miyamoto, N. Taniguchi and P. G. Wang, Bioorg.Med. Chem., 2002, 10, 3049–3055.16 Q. Jia, T. Cai, M. Huang, H. Li, M. Xian, T. L. Poulos and P. G. Wang,J. Med. Chem., 2003, 46, 2271–2274.17 D. Schade, J. Kotthaus, H. Hungeling, J. Kotthaus and B. Clement,ChemMedChem, 2009, 4, 1595–1599.18 D. Schade, J. Kotthaus, N. Klein, J. Kotthaus and B. Clement, Org.Biomol. Chem., 2011, 9, 5249–5259.19 J. P. Ward, Ann. R. Coll. Surg. Engl., 1975, 57, 248–261, AcademicPress.20 S. Wilk, H. Mizoguchi and M. Orlowski, J. Pharmacol. Exp. Ther.,1978, 206, 227–232.21 M. Barthelmebs, A. Caillette, J. D. Ehrhardt, J. Velly and J. L. Imbs,Kidney Int., 1990, 37, 1414–1422.22 R. G. G. Leenders, K. A. A. Gerrits, R. Ruijtenbeek andH. W. Scheeren, Tetrahedron Lett., 1995, 36, 1701–1704.23 A. Me´nard, R. Castonguay, C. Lherbet, C. Rivard, Y. Roupioz andJ. W. Keillor, Biochemistry, 2001, 40, 12678–12685.24 B. E. Toki, C. G. Cerveny, A. F. Wahl and P. D. Senter, J. Org. Chem.,2002, 67, 1866–1872.25 M. Sato, T. Ishii, Y. Kobayashi-Matsunaga, H. Amada, K. Taniguchi,N. Miyata and K. Kameo, Bioorg. Med. Chem. Lett., 2001, 11,2993–2995.26 N. Miyata, T. Seki, Y. Tanaka, T. Omura, K. Taniguchi, M. Doi,K. Bandou, S. Kametani, M. Sato, S. Okuyama, L. Cambj-Sapunar,D. R. Harder and R. J. Roman, J. Pharmacol. Exp. Ther., 2005, 314,77–85.Fig. 1 LCMS trace of 4b incubated in Krebs buﬀer at 37 1C for 1 h (a) without g-GT and glutamyl acceptor Gly–gly, 4b is intact; (b) with g-GT (100 mU mL1) andglutamyl acceptor Gly–gly (5 mM), 4b is deglutamylated to give the species 15.ChemComm CommunicationOpen Access Article. Published on 21 December 2012. Downloaded on 05/08/2013 16:18:16. View Article Online

Antioxid. Redox Signaling,

Free Radical Res.

M a n s u ya n dJ .L .B o u c h e r ,Drug Metab. Rev.,

Nitric Oxide Donors: For Pharmaceutical and Biological Applications,

A new class of NO-donor pro-drugs triggered by γ-glutamyl transpeptidase with potential for reno-selective vasodilatation

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      A new class of NO-donor pro-drugs triggered by gamma-glutamyl transpeptidase with potential for reno-selectivevasodilatationCitation for published version:Zhang, Q, Kulczynska, A, Webb, DJ, Megson, IL & Botting, NP 2013, 'A new class of NO-donor pro-drugstriggered by gamma-glutamyl transpeptidase with potential for reno-selective vasodilatation' ChemicalCommunications, vol 49, no. 14, pp. 1389-1391. DOI: 10.1039/C2CC38382A, 10.1039/c2cc38382aDigital Object Identifier (DOI):10.1039/C2CC38382A10.1039/c2cc38382aLink:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher's PDF, also known as Version of recordPublished In:Chemical CommunicationsPublisher Rights Statement:Gold Open Access paidGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 28. Apr. 2017This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 1389--1391 1389Cite this: Chem. Commun.,2013,49, 1389A new class of NO-donor pro-drugs triggered byc-glutamyl transpeptidase with potential forreno-selective vasodilatation†Qingzhi Zhang,*a Agnieszka Kulczynska,a David J. Webb,b Ian L. Megson*c andNigel P. Bottingz*aThis communication describes the synthesis of a new class ofN-hydroxyguanidine (NHG) pro-drugs which release nitric oxide(NO), triggered by the action of c-glutamyl transpeptidase (c-GT),and have potential for the treatment of acute renal injury/failure(ARI/ARF).Acute renal injury (AKI), or failure (ARF), is a common compli-cation that aﬀects millions of people worldwide, particularly inintensive care units, where it is associated with a mortality rateof between 50% and 80%.1 There is no eﬀective pharmaceuticaltherapy to date. One of the major causes of AKI is ischemia-reperfusion injury,2,3 following aortic ring cross-clampingduring by-pass surgery, which can lead to renal ischemia.4Reperfusion of ischemic renal tissue causes the generation ofreactive oxygen species which induce renal cell injury5 andpromote impairment of renal perfusion at least in part viainactivation of the vasodilator, nitric oxide (NO).6–8 Thus, akidney selective vasodilator with antioxidant properties isattractive to maintain blood flow to oﬀset AKI and scavengethe reactive oxygen species. Localisation of activity to thekidney would avoid a systemic reduction in blood pressure.Dopamine and fenoldopam, specific agonists of the dopamine-1 receptor, have been used clinically in an eﬀort to reduce therisk of perioperative renal dysfunction, but the eﬀectivenessof these agents is not clear.9,10 We hypothesised that aneﬀective exogenous NO-donor, which selectively increases renalvasodilatation, would oﬀer an alternative.There are a wide range of NO-donor drugs in existence,11including conventional organic nitrates and nitrites, S-nitrothiols,NONOates and N-hydroxyguanidines (NHGs).12–16 The NHGs1 are analogues of No-hydroxy-L-arginine (NOHA), a biosyn-thetic intermediate involved in the generation of NO fromL-arginine.11 Several enzymatically activated NHG pro-drugshave been reported such as peptidylglycine a-amidating mono-oxygenase (PAM)-active O-carboxymethyl N-hydroxyguanidines17and N-b-galactosidases-active (b-D-galactopyranos-1-yl)oxyguani-dine.18 Our approach aimed to mask the NO generating N-OHgroup with a g-glutamyl residue to facilitate activation by theenzyme, g-glutamyl transpeptidase (g-GT). Given that g-GT isprimarily expressed in the kidney (5–10 fold higher than in theliver and pancreas),19 it was envisaged that this enzyme could beused to trigger reno-selective release of an NHG and subsequentin situ generation of NO (Scheme 1). A similar strategy hasbeen described for reno-selective L-3,4-dihydroxyphenylalanine(L-DOPA), the Glu-DOPA.20,21However, the direct coupling of NHGs with a g-glutamylresidue was hampered by intramolecular cyclization anddehydration leading to a 1,2,4-oxadiazole ring; or alternativelylactamization and release of a pyroglutamic acid (Scheme 2,data not included).In an eﬀort to prevent these modes of cyclization, weinvestigated the use of a bridge between the NHG and theg-glutamyl group. Both g-glutamyl itself and g-aminobutanoyl(GABA)22 were explored as linkers. Thus 2a and 2b becamesynthesis targets (Scheme 3) and they were prepared viaappropriately protected dipeptide intermediates (ESI;† SchemeS1). Unfortunately 2a gradually decomposed presumably dueto the carboxylic acid moieties promoting autodegradation.Scheme 1 Approach to g-GT triggered release of NHG 1 and the reno-selectiverelease of nitric oxide.a University of St Andrews, EaStChem School of Chemistry and Centre forBiomolecular Sciences, North Haugh, St Andrews, Fife KY16 9ST, UK.E-mail: qz@st-andrews.ac.uk; Fax: +44 (0)1334 463808; Tel: +44 (0)1334 467274b Centre for Cardiovascular Science, The Queen’s Medical Research Institute, TheUniversity of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UKc Free Radical Research Facility, Department of Diabetes and CardiovascularScience, The University of The Highlands & Islands, Centre for Health Science,Old Perth Road, Inverness, IV2 3JH, UK. E-mail: ian.megson@uhi.ac.uk;Fax: +44 (0)1463 711245; Tel: +44 (0)1463 279562† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2cc38382a‡ N. P. Botting died on 4th June 2011.Received 21st November 2012,Accepted 20th December 2012DOI: 10.1039/c2cc38382awww.rsc.org/chemcommChemCommCOMMUNICATIONDownloaded by University of Edinburgh on 20 February 2013Published on 21 December 2012 on http://pubs.rsc.org | doi:10.1039/C2CC38382AView Article OnlineView Journal  | View Issue1390 Chem. Commun., 2013, 49, 1389--1391 This journal is c The Royal Society of Chemistry 2013On the other hand, 2b could be purified by preparative HPLCbut was found to be resistant to g-GT-mediated cleavage in vitroand was considered not to be a useful pro-drug. This promptedthe preparation of 3 (Scheme 3), involving the conjugation ofonly one GABA-Glu dipeptide onto a hydroxamic acid, analternative NO-donor.11 Compound 3 too, unfortunately, wasfound to be resistant to g-GT mediated deacylation, suggestingthat the GABA-Glu peptide linker is not suitable for g-GTcleavage in this setting.g-Glutamyl anilines are known substrates for g-GT23 andpresented an alternative linker option. The success of such anapproach would involve a 1,6-elimination following the actionof g-GT on N-g-glutamylaminobenzyloxy-guanidine 4a–c, asillustrated in Scheme 4. Similar spacers have been employedpreviously in anticancer pro-drug design.24In the event, the synthesis of 4a–c was successfully accom-plished through a six-step reaction sequence (Scheme 4).Firstly, g-glutamylation of 4-aminobenzylalcohol with Alloc-L-glutamic acid 1-allyl ester (Alloc-Glu-OAll) (ESI;† Scheme S1)gave benzyl alcohol 5. Conversion of the benzylalcohol moietyto the corresponding bromide 6 followed by nucleophilic dis-placement with BocNHOH generated aminooxide 7, and thentreatment with CF3COOH–DCM, gave the key intermediate 8which was coupled with the required amino(alkyl/aryliminio)-methanesulfonate 9a–c to generate 10a–c. Finally the All/Allocgroups were removed under neutral conditions with([Pd(PPh3)4]/PhSiH3) to give 4a–c.The same aminobenzyl linker was also used for theg-glutamylation of N-hydroxyformamidines (NHFs) (Scheme 5).N0-Hydroxy-N-(4-butyl-2-methylphenyl)formamidine25 and N0-hydroxy-N-(3-chloro-4-morpholin-4-ylphenyl)formamide26 have been docu-mented as 20-hydroxyeicosatetraenoic acid (20-HETE) inhibitors.20-HETE is a major metabolite of arachidonic acid and is a potentvasoconstrictor; localisation of an NHF would counter the eﬀect of20-HETE and induce a synergic vasodilation eﬀect mediated byNO. Thus N0-hydroxyphenylethylformamidine 12 was prepared inthis study and converted to pro-drug 14.Pro-drugs 4a–c and 14 were rapidly cleaved by g-GT and theywere completely deacylated after 1 h, as judged by LC-MS. Fig. 1(a)and (b) illustrates the LCMS trace of 4b and the conversion of 4b todeacylated intermediate 15 [M-Glu]+ by g-GT. This was in clearcontrast to the GABA-linked candidates 2b and 3, which proved tobe resistant to the action of g-GT. 1,6-Elimination and loss of thelinker from 15 to generate the parent NHG 1b is significantly slower(trace amount of parent 1b was detected by selective ion monitoringatm/z 180) than the cleavage of the g-glutamylmoiety. In preliminaryexperiments with animal tissue, LC-MS analysis revealed B90%conversion of 4b (100 mM) to 1b in a rat renal homogenate (37 1C;45 min). In addition, 4b was found to induce substantial vaso-dilatation in rat isolated perfused kidney preparations (50% ofmaximum vasodilatation induced by B40 mM 4b). Details of thebioactivity of these pro-drugs will be reported elsewhere.In summary, several candidate NO-donor pro-drugs havebeen prepared, designed for activation by g-GT. The pro-drugsScheme 2 Cyclization of direct coupling of NHGs with g-glutamyl residue(s).Scheme 3 Design of Glu/Gaba linked g-glutamyl NO-donor pro-drugs of NHGand hydroxamic acid.Scheme 4 Design and synthesis of aminobenzyl linked g-glutamyl NO-donorpro-drugs of NHG: (i) 4-aminobenzylalcohol, EEDQ, DCM, rt, 12 h, 85%; (ii) PBr3,THF, 0 1C, 2 h, 87%; (iii) BocNHOH, NaH, THF, 0 1C, 4 h, 83%; (iv) CF3CO2H, DCM,92%; (v) 9a R = Ph or 9b R = PhCH2CH2 or 9c R = furfuryl, Et3N, DMAP, DCM,38–53%; (vi) [Pd(PPh3)4], PhSiH3, DCM, 37–89%.Scheme 5 Synthesis of N-hydroxyformamidine and its glutamyl pro-drug:(i) Me2NCH(OMe2), reflux, 2 h, quantitative; (ii) NH2OHHCl, MeOH, 63%;(iii) 8, THF, reflux, 29%; (iv) [Pd(PPh3)4], PhSiH3, DCM, rt, 6 h, 53%.Communication ChemCommDownloaded by University of Edinburgh on 20 February 2013Published on 21 December 2012 on http://pubs.rsc.org | doi:10.1039/C2CC38382AView Article OnlineThis journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 1389--1391 1391comprise the parent NO-donor, a linker and a g-glutamylmoiety. GABA-linked pro-drugs are not suitable substrates forg-GT, but those linked by the aminobenzyl moiety proved to begood substrates for the enzyme. The g-glutamyl group is cleavedrapidly, with a slower decomposition of the aminobenzyl linker.Improved design is now focussed on tuning the spacer toencourage a more rapid release of the parent NHG drug.The authors are grateful to the Wellcome Trust (CatalystBiomedica Development Award 063729/Z/01/Z) for financial support.Thanks go to Prof. David O’Hagan (University of St Andrews) forhis input into manuscript preparation.References1 R. W. Schrier, W. Wang, B. Poole and A. Mitra, J. Clin. Invest., 2004,114, 5–14.2 J. V. Bonvebtre and J. M. Weinberg, J. Am. Soc. Nephrol., 2003, 14,2199–2210.3 T. A. Sutton, C. J. Fisher and B. A. Molitoris, Kidney Int., 2002, 62,1539–1549.4 G. Mariscalco, R. Lorusso, C. Dominici, A. Renzulli and A. Sala, Ann.Thorac. Surg., 2011, 92, 1539–1547.5 P. A. Grace, Br. J. Surg., 1994, 81, 637–647.6 M. L. Brocq, S. J. Leslie, P. Milliken and I. L. Megson, Antioxid.Redox Signaling, 2008, 10, 1631–1674.7 A. MacKenzie and W. Martin, Br. J. Pharmacol., 1998, 124, 710–728.8 M. Saran, C. Michel and W. Bors, Free Radical Res. Commun., 1990,10, 221–226.9 M. D. Denton, G. M. Chertow and H. R. Brady, Kidney Int., 1996, 49,4–14.10 T. Bove, G. Landoni, M. G. Calabro`, G. Aletti, G. Marino,E. Cerchierini, G. Crescenzi and A. Zangrillo, Circulation, 2005,111, 3230–3235.11 P. G. Wang, T. B. Cai and N. Taniguchi, Nitric Oxide Donors: ForPharmaceutical and Biological Applications, Wiley-VCH, 2005, ISBN,3-527-31015-0.12 A. Renodon-Cornie`re, S. Dijols, C. Perollier, D. Lefevre-Groboillot,J. L. Boucher, R. Attias, M. A. Sari, D. Stuehr and D. Mansuy, J. Med.Chem., 2002, 45, 944–954.13 D. Mansuy and J. L. Boucher, Drug Metab. Rev., 2002, 34, 593–606.14 M. Xian, X. P. Li, X. P. Tang, X. C. Chen, X. L. Zheng, J. J. Galligan,D. 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Webb, David J

Megson, Ian L

Botting, Nigel P

University of the Highlands and Islands Institutional Repository

A new class of NO-donor pro-drugs triggered by ¿-glutamyl transpeptidase with potential for reno-selective vasodilatation

This communication describes the synthesis of a new class of N-hydroxyguanidine (NHG) pro-drugs which release nitric oxide (NO), triggered by the action of gamma-glutamyl transpeptidase (gamma-GT), and have potential for the treatment of acute renal injury/failure (ARI/ARF).</p

A new class of NO-donor pro-drugs triggered by γ-glutamyl transpeptidase with potential for reno-selective vasodilatation

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