Inhibition of inducible nitric oxide synthase by acetamidine derivatives of hetero-substituted lysine and homolysine

Article abstract of DOI:10.1016/S0960-894X(00)00055-X

The synthesis and in vitro evaluation of the acetamidine derivatives of hetero-substituted lysine and homolysine analogues have identified potent inhibitors of human nitric oxide synthase enzymes, including examples with marked selectivity for the inducible isoform. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00055-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 597±600
Inhibition of Inducible Nitric Oxide Synthase by Acetamidine
Derivatives of Hetero-Substituted Lysine and Homolysine
Robert J. Young,* Richard M. Beams, Keith Carter, Helen A. R. Clark, Diane M. Coe,
C. Lynn Chambers, P. Ifeyinwa Davies, John Dawson, Martin J. Drysdale,
Karl W. Franzman, Colin French, Simon T. Hodgson, Harold F. Hodson,
Savvas Kleanthous, Peter Rider, Daniela Sanders, David A. Sawyer, Keith J. Scott,
Barry G. Shearer, Richard Stocker, Steven Smith, Miriam C. Tackley
and Richard G. Knowles
Glaxo Wellcome Research and Development, Stevenage, Hertfordshire, SG1 2NY, UK
Received 20 December 1999; accepted 24 January 2000
AbstractÐThe synthesis and in vitro evaluation of the acetamidine derivatives of hetero-substituted lysine and homolysine analo-
gues have identi®ed potent inhibitors of human nitric oxide synthase enzymes, including examples with marked selectivity for the
inducible isoform. # 2000 Elsevier Science Ltd. All rights reserved.
There has been extensive scienti®c interest in the funda-
mental biochemistry1 and diverse physiological roles2
of nitric oxide (NO) since this simple radical gas was
®rst shown to have actions identical to those proposed
for endothelium-derived relaxing factor (EDRF).3 NO
is produced by the oxidation of arginine by one of three
isoforms of the enzyme nitric oxide synthase (NOS).4
The calcium-regulated constitutive isoforms, endothelial
(eNOS, NOS-3) and neuronal (nNOS, NOS-1) have
important roles in, inter alia, regulation of blood pres-
sure and neurotransmission respectively, through pro-
duction of low-level pulses of NO.5 Higher, sustained,
levels of NO are produced by the calcium-independent
inducible isoform (iNOS, NOS-2), which is thought to
play a role in host defence mechanisms but is also
implicated in a number of diseases such as shock
conditions6 and various in¯ammatory processes.7
Despite the identi®cation of various classes of selective
iNOS inhibitors, such as amino heterocycles,8 ami-
dines,9 and isothioureas,10 the therapeutic potential of
inhibiting this enzyme remains unresolved, largely due
to the lack of potent, non-toxic, inhibitors possessing
levels of isoform selectivity that preserve the bene®cial
e€ects of cNOSs in pathophysiological circumstances.11
The acetamidine derivatives of l-ornithine (l-N-imino-
ethylornithine, l-NIO)12 and l-lysine (l-NIL),13
analogues of the NOS substrate l-arginine, are potent
inhibitors of iNOS, with both compounds showing
some selectivity over the constitutive isoforms. In this
paper we report on the synthesis and SAR of hetero-
substituted analogues and homologues of l-NIL,
including the sulphone GW273629 and sulphide
GW274150, which have similar potency to l-NIL and
signi®cantly increased isoform selectivity.
Chemistry
The required orthogonally protected intermediates 1 for
the sulphur containing compounds were constructed by
*Corresponding author. Fax: +44-1438-763615; e-mail: rjy40958@
glaxowellcome.co.uk
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00055-X

598
R. J. Young et al. / Bioorg. Med. Chem. Lett. 10 (2000) 597±600
S-alkylation of cysteine or homocysteine derivatives,
using aryl sulphonate esters of Cbz ethanolamine or
propanolamine 2 (Scheme 1). Appropriate oxidation
and/or protecting group manipulation, notably includ-
ing Cbz removal using transfer hydrogenolysis condi-
tions with the thioethers, produced suitable amine
intermediates for further functionalisation. The amines
were treated, either as the formate salt or free base, with
thioacetimidate reagents 314 to give the acetamidines in
essentially quantitative yield, prior to deprotection
under acidic conditions to give the target molecules
4, 5 and 6.15 Chiral HPLC/CD analysis con®rmed the
enantiospeci®city of the process in both series - indeed
the key 5-thiohomolysine derivative GW274150 was
®rst obtained by separation of a racemate, derived from
d/l-homocystine, by preparative chiral HPLC.16
the literature route,18 but with modi®ed protection
(Scheme 2). The terminal amine was unmasked by
hydrogenolysis, to facilitate the regiospeci®c amidina-
tion reaction, prior to deprotection yielding the target
compounds 10.
The isomeric 5-oxahomolysine 11 was synthesised in
racemic form starting from 2-chloroethyl ether using
sequential alkylations with the potassium salts of
phthalimide and diethyl phthalimidomalonate (Scheme
3). Following deprotection and decarboxylation, con-
version of 11 to the target racemic acetamidine 12 was
achieved by reaction with bu€ered ethyl acetimidate in
water, a method latterly superseded by the thioimidate
chemistry, vide supra.
The oxygen-containing compounds were synthesised in
one of two ways, depending on substitution. Thus
homochiral l-4-oxa lysine (7, n=1) and l-4-oxa homo-
lysine (7, n=2) intermediates, with appropriate ortho-
gonal protection, were constructed by reaction of
aziridine 817 with protected amino alcohols 9 using
Biological Evaluation and Discussion
The compounds described were screened to establish
iNOS activity and selectivity over the constitutive iso-
forms using recombinant human NOS enzymes19 in the
oxyhaemoglobin assay.20 The IC50 values are reported
Scheme 1. Synthesis of sulphur-containing compounds. Generic reagents, conditions and typical yields: (i) Na/liquid NH3 (R=R0=H, 90%) or
DTT, Et3N, CHCl3 (R=tBu, R0=Boc, 95%); (ii) 2, in situ reaction (R=R0=H), then Boc2O, NaOH, aq dioxane (80%) or 2, DBU, PhMe 45 ꢀC
0
.
(R=tBu, R =Boc, 65%); (iii) HCO2H, Pd Black, MeOH, Á, (R=H, 85%) or HCO2NH4, Pd(OH)2-C, EtOH, Á (R=tBu, 80%); (iv) 3 HCl or
TM
.
3 HBr, EtOH, then HCl, dioxane or HBr AcOH (90%); (v) oxone
aq MeOH (80%); (vi) H2O2, H2O (40%; 1:1 mixture of sulphoxide diaster-
eomers). N.B. compounds drawn in l-formÐracemic homocystine and d-cystine were also employed in this scheme. Final compounds isolated as
HBr or HCl salts.
.
Scheme 2. Synthesis of 4-oxa compounds. Reagents and conditions: (i) BF3 OEt2, CHCl3 (40%); (ii) Pd(OH)2-C; HCO2NH4, MeOH (95%), Á; (iii)
.
3 HCl, EtOH (90%); (iv) HCl aq MeOH, Á (90%) to give HCl salt.

R. J. Young et al. / Bioorg. Med. Chem. Lett. 10 (2000) 597±600
599
Scheme 3. Synthesis of 5-oxa compound. Reagents: (i) KNPhth, Et2NH (77%), then PhthNC(CO2Et)2K, KI, DMF (66%); (ii) 5 N aq NaOH,
.
EtOH, then aq HCl, Á (34%); MeC(=NH)OEt HCl, pH 10.5, H2O (50%).
from measurements taken 15±30 min after the inhibitors
were added to the test wells, in order to permit devel-
opment of the inhibition by these compounds, many of
which appear to have time-dependant kinetics.
homochiral d-isomers we evaluated, which contrasts to
the apparent speci®city for l-arginine as substrate and
l-NMMA as an inhibitor.21 Determination of the
selectivity of these compounds for iNOS versus eNOS
and nNOS (Table 2) showed that it was dicult to dis-
cern any clear SAR. However, all compounds appeared
to show at least low or moderate selectivity for iNOS
over both constitutive isoforms, whilst two compounds
in particular had high selectivity, the sulphone in the 1,1
series (GW273629) and the sulphide in the 1,2 series
(GW274150).
The SAR shows in Table 1 that the presence of the
simple ether or thioether linkage shows a marginal
increase in potency in the lysine series (n=1, m=1) and
a more substantial e€ect in the homologues (n=1,
m=2). The sulphoxides or sulphones are marginally less
active in the lysine series and substantially so in the
homologues. Where isomers are possible, it is clear that
the position of the heteroatom linker is crucial, in that
activity is only retained with two methylenes between it
and the acetamidine (i.e. n=1; but not n=2) in all
series. Interestingly, whilst not as potent as the natural
l-enantiomers, some activity was retained by the
In the interpretation of these results it is dicult to
provide rational explanations for either potency or
selectivity changes with such conformationally ¯exible
molecules, especially when considering the strong active
site homology in the amino acid binding domains
Table 1. IC50 values (mM) against human iNOSa
b
Isomer\X
S
O
SO
SO2
CH2
n=1, m=1
n=1, m=2
n=2, m=1
0.7 (l) 5.4 (d)
1.4 (l) 5.9 (d)
62.0 (l)
1.2 (l)
1.4 (dl)
>100 (l)
8.0 (l)
>100 (dl)
Ð
8.0 (l)
>100 (dl)
>100 (l)
1.6 (l)
20.0 (dl)
aFigures shown are averaged ®gures with an n of at least two separate experiments in all cases, using assay conditions as published,20 with an argi-
nine concentration of 30 mM.
bComparative ®gures in our assays using compounds made by the published methods.13 Data is shown for single enantiomers (l or d) or racemate
(dl) as indicated in parentheses; sulphoxides are 1:1 or 1:1:1:1 mixtures of diastereomers.
Table 2. Selectivity data with IC50 values (mM) against human NOS isoformsa,c
Compound
Isomer
X
Stereo
iNOS
eNOS
e/i
57
>18
19
>125
11
333
>17
51
nNOS
n/i
4
ent-4
5
GW273629
10
n=1, m=1
S
S
SO
SO2
O
S
S
O
l
d
0.7
5.4
8.0
8.0
1.2
1.4
5.9
1.4
1.6
20.0
6.6
40
>100
155
13.2
53
68
19
10
9
78
1,1
1,1
1,1
1,1
l
l
>1000
630
11.6
145
>100
6.0
37
>100
4.9
l
13.0
10
GW274150
ent-274150
12
n=1, m=2
1,2
1,2
1,1
2,1 or 1,2
n=0, m=1
l
466
>100
72
78
>100
104
>17
5
23
>5
d
d/l
l
NILb
CH2
CH2
CH2
49
>5
0.5
hNILb
l-NMMAb
d/l
l
3.5
0.7
a and bSee equivalent footnotes to Table 1.
cCompounds shown in Table 1 but not Table 2 all showed IC50s of >100 mM on eNOS and nNOS.

600
R. J. Young et al. / Bioorg. Med. Chem. Lett. 10 (2000) 597±600
of iNOS and eNOS.22 The Asp382 to Asn368 di€erence
between iNOS and eNOS would seem to be unim-
portant in imparting selectivity in these molecules,23
given conservation of the a-amino acid recognition
motif. It has also been suggested that imposing con-
formational rigidity is not a favourable approach to
impart selectivity.24
V.; Trivedi, M.; Zupec, M. E.; Tjoeng, F. S. Ibid. 1998, 41, 96;
Moore, W. M.; Webber, R. K.; Fok, K. F.; Jerome, G. M.;
Connor, J. R.; Manning, P. T.; Wyatt, P. S.; Misko, T. P.;
Tjoeng, F. S.; Currie, M. G. J. Med. Chem. 1996, 39, 669.
9. Garvey, E. P.; Oplinger, J. A.; Fur®ne, E. S.; Ki€, R. J.;
Laszlo, F.; Whittle, B. J. R.; Knowles, R. G. J. Biol. Chem.
1997, 272, 4959; Moore, W. M.; Webber, R. K.; Fok, K. F.;
Jerome, G. M.; Kornmeier, C. M.; Tjoeng, F. S.; Currie, M.
G. Bioorg. Med. Chem. 1996, 4, 1559; Hallinan, E. A.; Tsym-
balov, S.; Finnegan, P. M.; Moore, W. M.; Jerome, G. M.;
Currie, M. G.; Pitzele, B. S. J. Med. Chem. 1998, 41, 775.
10. Shearer, B. G.; Lee, S.; Oplinger, J. A.; Frick, L. W.;
Garvey, E. P.; Fur®ne, E. S. J. Med. Chem. 1997, 40, 1901;
Garvey, E. P.; Oplinger, J. A.; Tanoury, G. J.; Sherman, P. A.;
Fowler, M.; Marshall, S.; Harmon, M. F.; Paith, J.; Fur®ne,
E. S. J. Biol. Chem. 1994, 269, 26669.
Conclusion
We have reported the synthesis and SAR studies that
have led to the identi®cation of GW273629 and
GW274150, as potent and selective inhibitors of the
inducible form of nitric oxide synthase. These com-
pounds, which have very di€erent pharmacokinetic
pro®les, have been used to help explore the potential
role of selective iNOS inhibitors in many disease models,
full details of which will be published in due course.25
11. Whittle, B. J. R. Histochem. J. 1995, 27, 727; Laslo, F.;
Whittle, B. J. R. Eur. J. Pharmacol. 1997, 334, 99; Liaudet, L.;
Rosselet, A.; Schaller, M.-D.; Markert, M.; Perret, C.; Feihl,
F. J. Infect. Dis. 1998, 177, 127.
12. Rees, D. D.; Palmer, R. M. J.; Schulz, R.; Hodson, H. F.;
Moncada, S. Br. J. Pharmacol. 1990, 101, 746.
13. Beams, R. M.; Hodson, H. F.; Palmer, R. M. J. Interna-
tional Patent WO 93/13055; Chem. Abstr. 1993, 119, 181232;
Moore, W. M.; Webber, K.; Jerome, G. M.; Tjoeng, F. S.;
Misko, T. P.; Currie, M. G. J. Med. Chem. 1994, 37, 3886.
14. Shearer, B. G.; Oplinger, J. A.; Lee, S. Tetrahedron Lett.
1997, 38, 179.
15. All new compounds gave satisfactory spectral and analy-
tical data. E.g. for compound GW274150: 1H NMR (D2O) dH
2.10±2.35, (5H, m, 3-H and CH3), 2.76 (2H, t, 4-H), 2.87 (2H,
t, 6-H), 3. 51 (2H, t, 7-H), 4.12 (1H, t, 2-H); m/z (electrospray)
MH+=220 (100%); microanalysis, found C, 31.06; H, 4.53;
Acknowledgements
We gratefully acknowledge the contributions of numer-
ous people who have worked on the NOS projects over
a number of years for help in many ways. In particular,
we thank Lisa Bridge, Rachel Russell and Neale Fox-
well for early evaluation of compounds, the develop-
ment chemistry teams led by John O'Callaghan and
Mike Loft for their e€orts and our research colleagues
at RTP, USA notably Je€ Oplinger, Ed Garvey and Jon
Collins for their helpful discussions.
.
.
N, 9.08; C8H17N3O2S (CF3CO2H)2 H2O requires C, 30.97; H,
4.55; N, 9.03%.
16. Compounds separated on Crownpak (+) column eluted
with aqueous TFA (pH 2) retention times 4.1 and 4.6 min; CD
spectra (0.1N aq HCl) L-210 (+0.80) and D-210 ( 0.65) nm.
17. Baldwin, J. E.; Farthing, C. N.; Russell, A. T.; Scho®eld,
C. J.; Spivey, A. C. Tetrahedron Lett. 1996, 37, 3761.
18. Morie, T.; Kato, S.; Harada, H.; Yoshida, N.; Fujiwara, I.;
Matsumoto, J.-I. Chem. Pharm. Bull. 1995, 43, 1137.
19. Expression of human nitric oxide synthase isozymes;
Charles, I. G.; Scorer, C. A.; Moro, M. A.; Fernandez, C.;
Chubb, A.; Dawson, J.; Foxwell, N.; Knowles, R. G.; Baylis,
S. A. In Methods in Enzymology, 1996; Vol. 268 (Nitric Oxide,
Part A), pp 449±460.
References and Notes
1. Pfei€er, S.; Mayer, B.; Hemmens, B. Angew. Chem., Int. Ed.
1999, 38, 1714.
2. Adams, D. R.; Brochwicz-Lewinski, M.; Butler, A. R. Prog.
Chem. Org. Nat. Prod. 1999, 76, 1; Kerwin, J. F.; Lancaster, J.
R.; Feldman, P. F. J. Med. Chem. 1995, 38, 4343.
3. Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature 1987,
327, 524.
4. Knowles, R. G.; Moncada, S. Biochem. J. 1994, 298, 249;
Stuehr, D. J. Biochim. Biophys. Acta 1999, 1411, 217.
5. Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Pharmacol.
Rev. 1991, 43, 109; Radomski, M. W. Annals. Med. 1995, 27,
321.
20. Dawson J.; Knowles R. G. Details of the Oxyhaemoglobin
Assay: a microtiter-plate assay of human NOS isoforms. In
Meth. Mol. Biology, Titheradge, M. A., Ed.; Humana Press,
Totowa NJ, 1998; Vol. 100, pp 237±242.
6. Vallance, P.; Moncada, S. New. Horiz. 1993, 1, 77; Thie-
mermann, C. Adv. Pharmacol. 1994, 28, 45; Rees, D. D. Bio-
chem. Soc. Trans. 1995, 23, 1025.
21. Knowles, R. G.; Palacios, M.; Palmer, R. M. J.; Moncada,
S. Proc. Natl. Acad. Sci. USA 1989, 86, 5159.
22. Fishmann, T. O.; Hruza, A.; Niu, X. D.; Fosetta, J. D.;
Lunn, C. A.; Dolphin, E.; Prongay, A. J.; Reichert, P.; Lundell,
D. J.; Narula, S. K.; Weber, P. C. Nat. Struct. Biol. 1999, 6, 233.
23. Li, H.; Raman, C. S.; Glaser, C. B.; Blasko, E.; Young, T.
A.; Parkinson, J. F.; Whitlow, M.; Poulos, T. L. J. Biol Chem.
1999, 274, 21276.
24. Lee, Y.; Marletta, M. A.; Martasek, P.; Roman, L. J.;
Masters, B. S. S.; Silverman, R. B. Bioorg. Med. Chem. 1999,
7, 1097.
25. Preliminary communications of this data were given at the
6th International Meeting on The Biology of Nitric Oxide,
Stockholm, Sweden, 5±8 September 1999; Alderton, W. et al.;
Knowles, R. et al.; Beswick, P. et al. Acta Physiologica Scand.
1999, 167, (Suppl. 645), O-36; O-37; P-197.
7. Salvemini, D.; Marino, M. H. Inducible nitric oxide syn-
thase and in¯ammation Exp. Opin. Invest. Drugs 1998, 7, 65.
8. Hagen, T. J.; Bergmanis, A. A.; Kramer, S. W.; Fok, K. F.;
Schmelzer, A. E.; Pitzele, B. S.; Swenton, L.; Jerome, G. M.;
Kornmeier, C. M.; Moore, W. M.; Branson, L. F.; Connor, J.
R.; Manning, P. T.; Currie, M. G.; Hallinan, E. A. J. Med.
Chem. 1998, 41, 3675; Hansen, D. W.; Peterson, K. B.; Tri-
vedi, M.; Webber, R. K.; Tjoeng, F. S.; Moore, W. M.; Jer-
ome, G. M.; Kornmeier, C. M.; Manning, P. T.; Connor, J.
R.; Misko, T. P.; Currie, M. G.; Pitzele, B. S. J. Med. Chem.
1998, 41, 1361; Webber, R. K.; Metz, S.; Moore, W. M.;
Connor, J. R.; Currie, M. G.; Fok, K. F.; Hagen, T. J.; Hansen,
D. W.; Jerome, G. M.; Manning, P. T.; Pitzele, B. S.; Toth, M.