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

Article abstract of DOI:10.1039/c2cc38382a

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 γ-glutamyl transpeptidase (γ-GT), and have potential for the treatment of acute renal injury/failur

Full text of DOI:10.1039/c2cc38382a

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COMMUNICATION
A new class of NO-donor pro-drugs triggered by
c-glutamyl transpeptidase with potential for
reno-selective vasodilatation†
Cite this: Chem. Commun., 2013,
49, 1389
Received 21st November 2012,
Accepted 20th December 2012
Qingzhi Zhang,*a Agnieszka Kulczynska,a David J. Webb,b Ian L. Megson*c and
Nigel P. Bottingz*a
DOI: 10.1039/c2cc38382a
www.rsc.org/chemcomm
This communication describes the synthesis of a new class of
There are a wide range of NO-donor drugs in existence,11
N-hydroxyguanidine (NHG) pro-drugs which release nitric oxide including conventional organic nitrates and nitrites, S-nitrothiols,
(NO), triggered by the action of c-glutamyl transpeptidase (c-GT), NONOates and N-hydroxyguanidines (NHGs).12–16 The NHGs
and have potential for the treatment of acute renal injury/failure 1 are analogues of No-hydroxy-L-arginine (NOHA), a biosyn-
(ARI/ARF).
thetic intermediate involved in the generation of NO from
L-arginine.11 Several enzymatically activated NHG pro-drugs
Acute renal injury (AKI), or failure (ARF), is a common compli- have been reported such as peptidylglycine a-amidating mono-
cation that affects millions of people worldwide, particularly in oxygenase (PAM)-active O-carboxymethyl N-hydroxyguanidines17
intensive care units, where it is associated with a mortality rate and N-b-galactosidases-active (b-D-galactopyranos-1-yl)oxyguani-
of between 50% and 80%.1 There is no effective pharmaceutical dine.18 Our approach aimed to mask the NO generating N-OH
therapy to date. One of the major causes of AKI is ischemia- group with a g-glutamyl residue to facilitate activation by the
reperfusion injury,2,3 following aortic ring cross-clamping enzyme, g-glutamyl transpeptidase (g-GT). Given that g-GT is
during by-pass surgery, which can lead to renal ischemia.4 primarily expressed in the kidney (5–10 fold higher than in the
Reperfusion of ischemic renal tissue causes the generation of liver and pancreas),19 it was envisaged that this enzyme could be
reactive oxygen species which induce renal cell injury5 and used to trigger reno-selective release of an NHG and subsequent
promote impairment of renal perfusion at least in part via in situ generation of NO (Scheme 1). A similar strategy has
inactivation of the vasodilator, nitric oxide (NO).6–8 Thus, a been described for reno-selective L-3,4-dihydroxyphenylalanine
kidney selective vasodilator with antioxidant properties is (L-DOPA), the Glu-DOPA.20,21
attractive to maintain blood flow to offset AKI and scavenge
However, the direct coupling of NHGs with a g-glutamyl
the reactive oxygen species. Localisation of activity to the residue was hampered by intramolecular cyclization and
kidney would avoid a systemic reduction in blood pressure. dehydration leading to a 1,2,4-oxadiazole ring; or alternatively
Dopamine and fenoldopam, specific agonists of the dopamine- lactamization and release of a pyroglutamic acid (Scheme 2,
1 receptor, have been used clinically in an effort to reduce the data not included).
risk of perioperative renal dysfunction, but the effectiveness
In an effort to prevent these modes of cyclization, we
of these agents is not clear.9,10 We hypothesised that an investigated the use of a bridge between the NHG and the
effective exogenous NO-donor, which selectively increases renal g-glutamyl group. Both g-glutamyl itself and g-aminobutanoyl
vasodilatation, would offer an alternative.
(GABA)22 were explored as linkers. Thus 2a and 2b became
synthesis targets (Scheme 3) and they were prepared via
appropriately protected dipeptide intermediates (ESI;† Scheme
S1). Unfortunately 2a gradually decomposed presumably due
to the carboxylic acid moieties promoting autodegradation.
a University of St Andrews, EaStChem School of Chemistry and Centre for
Biomolecular 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 467274
b Centre for Cardiovascular Science, The Queen’s Medical Research Institute, The
University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK
c Free Radical Research Facility, Department of Diabetes and Cardiovascular
Science, 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
Scheme 1 Approach to g-GT triggered release of NHG 1 and the reno-selective
release of nitric oxide.
‡
N. P. Botting died on 4th June 2011.
c
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g-Glutamyl anilines are known substrates for g-GT23 and
presented an alternative linker option. The success of such an
approach would involve a 1,6-elimination following the action
of g-GT on N-g-glutamylaminobenzyloxy-guanidine 4a–c, as
illustrated in Scheme 4. Similar spacers have been employed
previously in anticancer pro-drug design.24
In 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 moiety
to the corresponding bromide 6 followed by nucleophilic dis-
placement with BocNHOH generated aminooxide 7, and then
treatment with CF3COOH–DCM, gave the key intermediate 8
which was coupled with the required amino(alkyl/aryliminio)-
methanesulfonate 9a–c to generate 10a–c. Finally the All/Alloc
groups were removed under neutral conditions with
([Pd(PPh3)4]/PhSiH3) to give 4a–c.
Scheme 2 Cyclization of direct coupling of NHGs with g-glutamyl residue(s).
The same aminobenzyl linker was also used for the
g-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 potent
vasoconstrictor; localisation of an NHF would counter the effect of
20-HETE and induce a synergic vasodilation effect mediated by
NO. Thus N0-hydroxyphenylethylformamidine 12 was prepared in
this study and converted to pro-drug 14.
Pro-drugs 4a–c and 14 were rapidly cleaved by g-GT and they
were 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 to
deacylated intermediate 15 [M-Glu]+ by g-GT. This was in clear
contrast to the GABA-linked candidates 2b and 3, which proved to
be resistant to the action of g-GT. 1,6-Elimination and loss of the
linker from 15 to generate the parent NHG 1b is significantly slower
(trace amount of parent 1b was detected by selective ion monitoring
at m/z 180) than the cleavage of the g-glutamyl moiety. In preliminary
experiments 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% of
maximum vasodilatation induced by B40 mM 4b). Details of the
bioactivity of these pro-drugs will be reported elsewhere.
Scheme 3 Design of Glu/Gaba linked g-glutamyl NO-donor pro-drugs of NHG
and hydroxamic acid.
On the other hand, 2b could be purified by preparative HPLC
but was found to be resistant to g-GT-mediated cleavage in vitro
and was considered not to be a useful pro-drug. This prompted
the preparation of 3 (Scheme 3), involving the conjugation of
only one GABA-Glu dipeptide onto a hydroxamic acid, an
alternative NO-donor.11 Compound 3 too, unfortunately, was
found to be resistant to g-GT mediated deacylation, suggesting
that the GABA-Glu peptide linker is not suitable for g-GT
cleavage in this setting.
In summary, several candidate NO-donor pro-drugs have
been prepared, designed for activation by g-GT. The pro-drugs
Scheme 4 Design and synthesis of aminobenzyl linked g-glutamyl NO-donor
pro-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%.
c
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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 mLꢀ1) and
glutamyl acceptor Gly–gly (5 mM), 4b is deglutamylated to give the species 15.
11 P. G. Wang, T. B. Cai and N. Taniguchi, Nitric Oxide Donors: For
Pharmaceutical and Biological Applications, Wiley-VCH, 2005, ISBN,
3-527-31015-0.
comprise the parent NO-donor, a linker and a g-glutamyl
moiety. GABA-linked pro-drugs are not suitable substrates for
g-GT, but those linked by the aminobenzyl moiety proved to be
good substrates for the enzyme. The g-glutamyl group is cleaved
rapidly, with a slower decomposition of the aminobenzyl linker.
Improved design is now focussed on tuning the spacer to
encourage a more rapid release of the parent NHG drug.
The authors are grateful to the Wellcome Trust (Catalyst
Biomedica Development Award 063729/Z/01/Z) for financial support.
Thanks go to Prof. David O’Hagan (University of St Andrews) for
his input into manuscript preparation.
`
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