Selective heterocyclic amidine inhibitors of human inducible nitric oxide synthase

Article abstract of DOI:10.1016/S0960-894X(01)00523-6

The potency and selectivity of a series of 5-hetero-2-iminohexahydroazepines were examined as inhibitors of the three human NOS isoforms. The effect of ring substitution of the 5-carbon for a heteroatom is presented. Potencies (IC₅₀'s) for these inhibitors are in the low micromolar range for hi-NOS with some examples exhibiting a 500× selectivity versus hec-NOS.

Full text of DOI:10.1016/S0960-894X(01)00523-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2651–2653
Selective Heterocyclic Amidine Inhibitors of Human Inducible
Nitric Oxide Synthase
Alan E. Moormann,a,* Sue Metz,a Mihaly V. Toth,a William M. Moore,b Gina Jerome,b
Christine Kornmeier,b Pamela Manning,b Donald W. Hansen, Jr.,c Barnett S. Pitzelec
and R. K. Webbera
aPharmacia, 700 Chesterfield Parkway North, St. Louis, MO 63198, USA
bPharmacia, 800 N. Lindberg, St. Louis, MO 63167, USA
cPharmacia, 4901 Searle Parkway, Skokie, IL 60676, USA
Received 8 June 2001; accepted 25 July 2001
Abstract—The potency and selectivity of a series of 5-hetero-2-iminohexahydroazepines were examined as inhibitors of the three
human NOS isoforms. The effect of ring substitution of the 5-carbon for a heteroatom is presented. Potencies (IC50’s) for these
inhibitors are in the low micromolar range for hi-NOS with some examples exhibiting a 500Â selectivity versus hec-NOS. # 2001
Elsevier Science Ltd. All rights reserved.
The production of nitric oxide (NO) from the induced
expression of i-NOS has been shown to play a role in
many pathological processes, while the constitutive
production of NO from endothelial NOS (e-NOS) plays
a role in the regulation of vascular smooth muscle tone.
Therefore, the selective inhibition of i-NOS versus e-
NOS is desirable and such a selective inhibitor could
find utility for controlling many pathological states.1À3
the corresponding analogue with the heteroatom incor-
porated into the 5-position of the ring.
The compounds were screened for binding against the
human isoforms of NOS.8
The compounds where X=CH2, O, and NMe were
synthesized, as shown in Scheme 1. The ketone 5 was
converted to the oxime 6. Beckmann rearrangement of
oxime 6 produced a 3:7 mixture of lactams 7 and 8.
When the desired lactam 8 did not crystallize, the lac-
tams were isolated using silica chromatography, eluting
with MeCN/EtOAc, lactam 7 eluting first. The lactam 8
was converted first to the iminoether 9 using Meerwein’s
reagent, then to the amidines 1–3, 10–15 with NH4Cl in
refluxing EtOH. Compounds 16 and 17 were synthe-
sized by reduction of the olefins 12 and 13, respectively.
Compounds 18 and 19 were synthesized by reduction of
the nitro group of compounds 14 and 15, respectively.
The cyclic amidine, 1 (Fig. 1) and its carbon homologues
exhibit binding selectivity in favor of i-NOS.4À7 Mole-
cular modeling of 1 in the active site of murine i-NOS,
suggested that the 5-carbon of the amidine ring is in
close proximity to the heme iron of the enzyme. Hetero-
atoms were incorporated into the ring at the 5-position
in an attempt to increase potency and selectivity. The 5-
carbon was replaced by heteroatoms, compounds 2–4,
in an attempt to interact with the enzyme’s heme iron.
The heteroatom can alter the sterics, bond lengths,
angles, and solvation.
Because Meerwein’s reagent was found to alkylate the
sulfur, the sulfur heterocycles were synthesized as shown
The carbon analogues chosen for this study were easily
synthesized cyclic amidines with good potency and
selectivity. These carbon analogues were compared to
*Corresponding author. Fax: +1-636-737-7425; e-mail: alan.e.
moormann@pharmacia.com
Figure 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00523-6

2652
A. E. Moormann et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2651–2653
in Scheme 2. The seven-member ring was synthesized as
follows: a Michael addition of l-cysteine ethyl ester 20
to acrylonitrile in DMF produced the nitrile 21. This
nitrile was stirred with EtOH saturated with HCl at 0 ꢀC
to form the iminoether 22, which cyclized to the cyclic
amidine 23 upon treatment with Amberlyst A-21 ion
exchange resin. Compound 24 was synthesized in like
manner from d-cysteine ethyl ester. The seven-member
ring 4 was synthesized in like manner starting with 2-
aminoethanethiol. The eight-member ring 28 was syn-
thesized as follows: bromide 26 was alkylated with the
sodium salt of 3-thiopropionitrile9 then refluxed with
hydrazine hydrate in EtOH to produce the aminonitrile
27. This nitrile was converted with HCl/EtOH to the
iminoether followed by treatment with Amberlyst A-21
ion exchange resin to produce the cyclic amidine 28.
Incorporation of oxygen (3, 13, 15, 17, and 19) did not
increase potency, but did increase the selectivity.
Compound 17 is noteworthy in that the increase in
selectivity of i-NOS versus e-NOS is 560 times, and
versus n-NOS is 10.6 times.
Incorporation of sulfur (4, 23, 24, 25, and 28) produced
mixed results. Compound 4, when compared to the carbon
analogue 1, exhibited no difference in i-NOS potency, but
a modest increase in selectivity versus e-NOS was
observed. The 7-ethyl carboxylate analogues, 23 and 24,
produced compounds comparable in potency to the
carbon analogue, 10. The enantiomers were compared:
the S-isomer (23) exhibited potency comparable to the
R-isomer (24) and exhibited slightly better selectivity for
i-NOS versus e-NOS.
As shown in Table 1, nitrogen substitution, 2, produced
a compound with greatly diminished activity. The
energy to desolvate the amine and the steric bulk of the
methyl probably prevents the molecule from entering
the active site.
The eight-member ring analogue 11, was modestly
active. The sulfur analogue, 28, lost some potency, but
did exhibit an increased selectivity for i-NOS versus e-
NOS. The 8-ethyl carboxylate analogue, 25, lost
potency.
Incorporation of the heteroatom did not increase the
potency toward i-NOS when compared to the
Scheme 1. (a) NH2OH*HCl/NaHCO in EtOH; (b) benzene sulfo-
nylchloride (1.1 equiv)/2.5 N NaOH (1.1 equiv) in acetone at 0 ꢀC
warming to ambient temperature for 18 h. Column eluting with
MeCN/EtOAc; (c) Me3OBF4/CH2Cl2 18 h at ambient temperature;
quenched with satd NaHCO3; (d) 1.0 equiv NH4Cl refluxed in EtOH
for 18 h; (e) 10% Pd/C–H2 in EtOH.
Scheme 2. (a) Ethyl l-cysteine (1 equiv)/acrylonitrile (2 equiv)/K2CO3
(1.1 equiv) in DMF; (b) EtOH saturated with HCl at 0 ꢀC; 21 added
and warmed to ambient temperature; concentrated to dryness; (c)
Amberlyst A-21 (3 equiv) in EtOH; (d) sodium-3-thiopropionitrile9 in
EtOH; (e) hydrazine hydrate (1.1 equiv) refluxed in EtOH.
Table 1. SAR for the heterocyclic amidinesa
Compd
i-NOS IC50, mM
e-NOS IC50, mM (Selectivity)
n-NOS IC50, mM (Selectivity)
1
2
3
4
2.06
15.25 (7.4)
6.42% @100 mM
72.68 (5.2)
3.5 (1.7)
32.1% @100 mM
14.03
2.7
25.89% @100 mM
17.74 (1.2)
6.8 (2.5)
43.6 (16.1)
10
11
12
13
14
15
16
17
18
19
23
24
25
28
74.9% @100 mM
9.98
0.89
9.26
0.152
0.979
0.517
2.43
0.701
6.66
70.9% @100 mM
70.9% @100 mM
55.7% @100 mM
13.1
11.45% @100 mM
58.9 (5.9)
205.9 (230)
709 (76)
2.77 (18.2)
49.3 (50.3)
71.4% @100 mM
10.15 (1.01)
3.39 (3.8)
108 (11.6)
0.339 (2.2)
6.24 (6.4)
0.85 (1.6)
25.9 (10.6)
0.932 (1.3)
8.39 (1.2)
102.5 (198)
1363 (561)
39.07 (55.7)
247.8 (37.2)
13.9% @100 mM
8.66% @100 mM
2.4% @100 mM
186.3 (14.2)
82.6% @100 mM
84.78% @100 mM
13.9% @100 mM
21.0 (1.6)
aThe enzyme binding was performed as described in ref 8.

A. E. Moormann et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2651–2653
2653
corresponding carbon analogue. The lack of enhanced
potency implies that the heme iron does not interact
with the heteroatom.
Metz, S.; Moore, W. M.; Peterson, K. B.; Pitzele, B. S.;
Spangler, D. P.; Webber, R. K.; Toth, M. V.; Trivedi, M.;
Tjoeng, F. S. U.S. Patent 5 854 234, 1998; Chem. Abstr. 1999,
130, 95489
6. Hansen, D. W., Jr.; Hagen, T. J.; Kramer, S. W.; Metz, S.;
Peterson, K. B.; Spangler, D. P.; Toth, M. V.; Fok, K. F.;
Webber, R. K.; Tjoeng, F. S.; Pitzele, B. S. International WO
96/35677, 1996; Chem. Abstr. 1997, 126, 59880
7. Caldwell, C. G.; Hagmann, W. K.; Maccoss, M.; Shah, S.
K.; Shankaran, K.; Furman, K. L. U.S. Patent 6 043 358,
1997; Chem. Abstr. 1997, 127, 34255
The incorporation of the oxygen or sulfur atom, in
some cases, increased the selectivity of i-NOS versus
e-NOS when compared to the corresponding carbon
analogue. In the one occurrence for a direct compar-
ison, sulfur 3 was more selective than oxygen 4 and
carbon 1. One interpretation of this increase in selectiv-
ity is an overall conformational or steric change caused
by the sulfur atom containing analogue, which favors
the i-NOS enzyme versus the e-NOS enzyme.
8. 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. Synthesis of the sodium salt of 3-thiopropionitrile: 3-bro-
mopropionitrile (13.3 g, 0.1 mol) and potassium thioacetate
(14.25 g, 0.125 mol) was dissolved in DMF (125 mL) and stirred
for 16 h at ambient temperature. The reaction mixture was
quenched with brine (700 mL) and the product extracted 3Â
Et2O (200 mL) then hexane (200 mL). The combined organic
extracts were washed with brine, dried over MgSO4, and
concentrated. The residue was chromatographed over silica,
eluting with EtOAc/hexane to yield 10.8 g of the S-3-acetyl-
thiopropionitrile. C5H7NOS (MW 129.17); MS: M+NH4 @
147. S-3-Acetylthiopropionitrile (5.8 g; 0.045 mol) was dis-
solved in MeOH (100 mL) and H2SO4 (10 drops) was stirred
for 2 days. The reaction mixture was quenched at 0 ꢀC with
NaOMe prepared from Na metal (1.03 g; 0.45 mol) and
MeOH (30 mL). The resulting mixture was concentrated and
used as is.
References and Notes
1. Rees, D. D.; Palmer, R. M. J.; Moncada, S. Proc. Natl.
Acad. Sci. U.S.A. 1989, 86, 3375.
2. Marletta, M. A. J. Med. Chem. 1994, 37, 1899.
3. Moncada, S. J. Royal Soc. Med. 1999, 92, 164.
4. Hansen, D. W., Jr.; Peterson, K. B.; Trivedi, M.; Kramer,
S. W.; Webber, R. K.; Tjoeng, F. S.; Moore, W. M.; Jerome,
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.
5. Hansen, D. W., Jr.; Currie, M. G.; Hallinan, E. A.; Fok, K.
F.; Hagen, T. J.; Bergmanis, A. A.; Kramer, S. W.; Lee, L. F.;