Time-dependence and preliminary SAR studies in inhibition of nitric oxide synthase isoforms by homologues of thiocitrulline

Article abstract of DOI:10.1016/j.bmcl.2003.08.018

Treatment of N^α-Cbz-N^ε -(2-hydroxyethylaminothiocarbonyl)-L-lysine N-(2-hydroxyethyl)amide with boiling hydrochloric acid gave N^ε -(4,5-dihydrothiazol-2-yl)-L-lysine. This was a weak and non-isoform selective inhibitor of NOS, whereas N^ε -aminothiocarbonyl-L-lysine and its methyl ester were potent, with IC ₅₀=13 and 18 μM, respectively, against human iNOS and IC ₅₀=3 and 8 μM, respectively, against rat nNOS. Time dependence was observed for inhibition of nNOS by the ester.

Full text of DOI:10.1016/j.bmcl.2003.08.018

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3679–3680
Time-Dependence and Preliminary SAR Studies in Inhibition of
Nitric Oxide Synthase Isoforms by Homologues of Thiocitrulline
Claire L. M. Goodyer,a Edwin C. Chinje,b Mohammed Jaffar,b
Ian J. Stratfordb and Michael D. Threadgilla,*
aDepartment of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
bSchool of Pharmacy andPharmaceutical Sciences, University of Manchester, OxfordRoad, Manchester M13 9PL, UK
Received 20 May 2003; revised 1 August 2003; accepted 11 August 2003
Abstract—Treatment of Na-Cbz-Ne-(2-hydroxyethylaminothiocarbonyl)-l-lysine N-(2-hydroxyethyl)amide with boiling hydro-
chloric acid gave Ne-(4,5-dihydrothiazol-2-yl)-l-lysine. This was a weak and non-isoform selective inhibitor of NOS, whereas Ne-
aminothiocarbonyl-l-lysine and its methyl ester were potent, with IC50=13 and 18 mM, respectively, against human iNOS and
IC50=3 and 8 mM, respectively, against rat nNOS. Time dependence was observed for inhibition of nNOS by the ester.
# 2003 Elsevier Ltd. All rights reserved.
.
Nitric oxide (NO ) is biosynthesised from l-arginine in
two steps catalysed by nitric oxide synthases (NOSs).
There are three isoforms, endothelial NOS (eNOS) and
neuronal NOS (nNOS) (constitutive Ca2+-dependent
forms) and an inducible form (iNOS). Selective inhibi-
tion of each of these has potential applications in ther-
apy several diseases, including cancer.1ꢀ3 Early NOS
inhibitors included the arginine analogues No-mono-
methyl-l-arginine (l-NMMA), Nd-(iminoethyl)-l-orni-
thine (l-NIO), No-nitro-l-arginine (l-NNA) and its
methyl ester (l-NAME). These bind to the guanidinium
region of the arginine-binding site4 and are competitive
reversible inhibitors, which show potency but little iso-
form selectivity.5 We reported3 that Nd-(4,5-dihy-
drothiazol-2-yl)-l-ornithine 2 was a potent inhibitor of
rat iNOS and rat nNOS. This is a close analogue of the
known inhibitor l-thiocitrulline 1, which is also rela-
tively non-isoform-selective (Fig. 1).1
Figure 1. Structures of NOS inhibitors l-thiocitrulline 1 and Nd-(4,5-
dihydrothiazol-2-yl)-l-lysine 2.
gave both 8 and its ester 7 for evaluation, by analogy with
l-NNA and l-NAME. Reaction of 5 with 2-aminoetha-
nol in boiling acetone not only formed the required N0-(2-
hydroxyethyl)thiourea but also displaced the ester OMe
group, giving 9. Boiling with hydrochloric acid not only
effected cyclisation to the 4,5-dihydrothiazole but also
hydrolysed the a-amide and Cbz protection, giving Ne-
(4,5-dihydrothiazol-2-yl)-l-lysine 10.8
Scheme 1 shows the syntheses of the target lysine deri-
vatives. CbzLysOMe 4 was converted to isothiocyanate
5 with thiophosgene. Addition of ammonia then gave
the thiourea 6 in high yield. Debenzylation (HBr/
AcOH) removed the Cbz to give compound 7.6 The
ester was hydrolysed with aqueous hydrobromic acid,
yielding e-aminothiocarbonyl-l-lysine 8.7 This sequence
Compounds 7, 8 and 10 were evaluated for inhibition of
rat brain nNOS and of human iNOS (hiNOS).9 l-
NMMA (an amino-acid inhibitor) and 7-nitroindazole
(7-NI; a non-amino acid inhibitor)10 were used for
comparison. The assay used the conversion of l-
[U-14C]-arginine to l-[U-14C]-citrulline.11 Compounds
were initially evaluated at 100 mM. Incubation of the
inhibitors with the enzyme for various periods before
addition of l-[U-14C]-Arg to start the reaction revealed
that the inhibition of nNOS by 7 increased markedly
*Corresponding author. Tel.: +44-1225-386840; Fax: +44-1225-
386114; e-mail: m.d.threadgill@bath.ac.uk
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.018

3680
C. L. M. Goodyer et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3679–3680
for time-dependence, IC50 values were determined for 7,
8 and 10 against both isoforms with 15-min pre-incu-
bation (Table 2).
None of the compounds shows useful isoform selec-
tivity. The potency of 1 is retained in the higher
homologue 7 but insertion of an additional CH2 into
the linker between the amino-acid and the dihy-
drothiazole decreased potency markedly (10 vs 2). 1400
W
(N-3-aminomethylbenzyl)acetamidine) is a slow-
binding inhibitor of rat iNOS, taking 10 min of pre-
incubation with the enzyme to exert full inhibition.12
However, slow binding to nNOS has not been repor-
ted; homothiocitrulline ester 7 is the first compound for
1
which this has been observed. The H NMR spectrum
of a solution of 7 in D2O was unchanged after 40 min;
thus, ester hydrolysis is highly unlikely under the con-
ditions of the enzyme inhibition assay. Such slow
binding may reflect the need for 7 to adjust its con-
formation during the binding process; the origin of
this effect will be the subject of future study.
Scheme 1. Synthesis of lysine-derived thioureas 7, 8 and 4,5-dihy-
drothiazole 10. Reagents and conditions: (i) MeOH, SOCl2, 4 days,
65%; (ii) CSCl2, CaCO3, H2O, CHCl3, 16 h, 52%; (iii) NH3, CH2Cl2, 4
h, 59%; (iv) HBr, HOAc, 15 h, 100%; (v) aq HBr (48%), 16 h, 100%;
(vi) H2N(CH2)2OH, acetone, reflux, 4 h, 53%; (viii) aq HCl (6 M),
reflux, 36 h, 76%.
Acknowledgements
Table 1. Time-dependence of inhibition of NOS isoform activity by
known inhibitors l-NMMA, 7-nitroindazole (7NI) and by 7, 8
We thank AICR for financial support.
Compd
Isoform
Percentage inhibitiona
of NOS activity by
References and Notes
compounds (100 mM) with
pre-incubation (t min)b
1. Marletta, M. A. J. Med. Chem. 1994, 37, 1899.
2. Schmidt, H. H. H. W.; Walter, U. Cell 1994, 78, 919.
3. Ulhaq, S.; Chinje, E. C.; Naylor, M. A.; Jaffar, M.; Strat-
ford, I. J.; Threadgill, M. D. Bioorg. Med. Chem. 1999, 7,
1787.
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F. S.; Misko, T. P.; Currie, M. G. J. Med. Chem. 1994, 37,
3886.
5. Ogden, J. E.; Moore, P. K. Trends Biotech. 1995, 13, 70.
6. Data for 7: IR nmax 1240, 1742, 3030 cmꢀ1; NMR
((CD3)2SO) dH 1.43–1.90 (6H, m, b,g,d-H6), 3.67 (3H, s, CH3),
3.29 (2H, t, J=7.8 Hz, Lys e-H2), 4.01 (1H, m, a-H), 7.52 (1H,
br NH), 8.34 (1H, s, NH); MS m/z 220.1119 (M+H)
(C8H18N3O2S requires 220.1120).
t=0
t=5
t=10
t=15
t=20
l-NMMA
7-NI
nNOS
nNOS
94ꢂ1
59ꢂ1
77ꢂ2
14ꢂ1
77ꢂ5
97ꢂ4
97ꢂ2
99ꢂ2
96ꢂ1
74ꢂ3
68ꢂ2
90ꢂ2
67ꢂ1
98ꢂ5
97ꢂ3
98ꢂ1
75ꢂ1
58ꢂ2
86ꢂ1
73ꢂ2
97ꢂ1
97ꢂ2
97ꢂ2
74ꢂ1
56ꢂ2
85ꢂ1
70ꢂ3
97ꢂ1
97ꢂ3
hiNOS
nNOSc
hiNOSd
nNOSc
hiNOSd
61ꢂ5
83ꢂ11
79ꢂ1
97ꢂ3
97ꢂ1
7
8
aValues are means of three experimentsꢂstandard deviation.
bPre-incubation of the inhibitor with the enzyme preparation, prior to
initiating NOS activity by addition of l-[14C]-arginine.
cRat brain nNOS.
dRecombinant human iNOS.
7. Data for 8: IR nmax 1194, 1739, 3429 cmꢀ1; NMR (CDCl3)
dH 1.5–2.0 (6H, m, b,g,d-H6), 2.96 (2H, t, J=7.8 Hz, e-H2),
4.01 (1H, t, J=6.6 Hz, a-H); NMR (CD3OD) dC 22.3, 27.4,
30.1, 43.9, 52.7, 128.8, 170.4; MS m/z 206.0956 (M+H)
(C7H16N3O2S requires 206.0963).
Table 2. IC50 values for inhibition of NOS isoforms (mM)a
Compd
iNOSb
hiNOSc
nNOSd
8. Data for 10: IR nmax 1651, 1750, 3413 cmꢀ1; NMR
((CD3)2SO) dH 1.44 (6H, m, b,g,d-H6), 2.80 (2H, m, e-H2),
3.35 (2H, m, thiazole 5-H2), 3.49 (1H, m, a-H), 3.58 (2H, m,
thiazole 4-H2), 8.10 (3H, br, N+H3) 8.50 (2H, br, 2ꢁNH);
NMR ((CD3)2SO) dC 21.6, 29.5, 30.9, 38.6, 44.8, 51.8, 57.6,
169.6, 171.0; MS m/z 232.1115 (M+H) (C9H18N3O2S requires
232.1120).
9. Goodyer, C. L. M.; Chinje, E. C.; Jaffar, M.; Stratford, I.
J.; Threadgill, M. D. Bioorg. Med. Chem. 2003, 11, 4189.
10. Wolff, D. J.; Gribbin, B. J. Arch. Biochem. Biophys. 1994,
311, 300.
1
2
7
8
10
<5
17
1.3
3
8
>100
8.1e
13
18
>100
aIC50 values were measured using a 15-min pre-incubation of the
inhibitor with the enzyme.
bRat iNOS.
cRecombinant human iNOS.
dRat brain nNOS.
eData taken from ref 2. IC50 measured without pre-incubation.
11. Ulhaq, S.; Chinje, E. C.; Naylor, M. A.; Jaffar, M.; Strat-
ford, I. J.; Threadgill, M. D. Bioorg. Med. Chem. 1998, 6,
2139.
12. Garvey, E. P.; Oplinger, J. A.; Furfine, E. S.; Kiff, R. J.;
Laszlo, F.; Whittle, B. J. R.; Knowles, R. G. J. Biol. Chem.
1997, 272, 4959.
with increasing pre-incubation time (Table 1) but this
effect was not shown for hiNOS. No time-dependence
was seen for inhibition of either isoform by the
amino-acids l-NMMA or 8. Owing to the potential