2-Iminopiperidine and other 2-iminoazaheterocycles as potent inhibitors of human nitric oxide synthase isoforms.

Article abstract of DOI:10.1021/jm950766n

A series of 2-iminoazaheterocycles have been prepared and shown to be potent inhibitors of human nitric oxide synthase (NOS) isoforms. This series includes cyclic amidines ranging from five- to nine-membered rings, of which 2-iminopiperidine and 2-iminohomopiperidine were the most potent inhibitors, with IC50 values of 1.0 and 2.0 microM, respectively, for human inducible nitric oxide synthase. This series of cyclic inhibitors was further expanded to include analogs with heteroatoms in the 3-position of the six-membered ring. This modification was tolerated for sulfur and oxygen, but nitrogen reduced the inhibitory potency. The oral administration of 2-iminopiperidine in lipopolysaccharide (LPS)-treated rats inhibited the LPS-induced increase in plasma nitrite/nitrate levels in a dose-dependent manner, demonstrating its ability to inhibit inducible NOS activity in vivo. These cyclic amidines represent a new class of potent NOS inhibitors and the foundation for potential therapeutic agents.

Full text of DOI:10.1021/jm950766n

J . Med. Chem. 1996, 39, 669-672
669
Expedited Articles
2-Im in op ip er id in e a n d Oth er 2-Im in oa za h eter ocycles a s P oten t In h ibitor s of
Hu m a n Nitr ic Oxid e Syn th a se Isofor m s†
William M. Moore,*,‡ R. Keith Webber,§ Kam F. Fok,§ Gina M. J erome,‡ J ane R. Connor,‡ Pamela T. Manning,‡
Pamela S. Wyatt,‡ Thomas P. Misko,‡ Foe S. Tjoeng,§ and Mark G. Currie‡
Departments of Molecular Pharmacology/ Inflammatory Diseases Research and Medicinal Chemistry, G. D. Searle Research
and Development, Monsanto Company, Mail Zone T3G, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167
Received October 13, 1995X
A series of 2-iminoazaheterocycles have been prepared and shown to be potent inhibitors of
human nitric oxide synthase (NOS) isoforms. This series includes cyclic amidines ranging
from five- to nine-membered rings, of which 2-iminopiperidine and 2-iminohomopiperidine were
the most potent inhibitors, with IC50 values of 1.0 and 2.0 µM, respectively, for human inducible
nitric oxide synthase. This series of cyclic inhibitors was further expanded to include analogs
with heteroatoms in the 3-position of the six-membered ring. This modification was tolerated
for sulfur and oxygen, but nitrogen reduced the inhibitory potency. The oral administration
of 2-iminopiperidine in lipopolysaccharide (LPS)-treated rats inhibited the LPS-induced increase
in plasma nitrite/nitrate levels in a dose-dependent manner, demonstrating its ability to inhibit
inducible NOS activity in vivo. These cyclic amidines represent a new class of potent NOS
inhibitors and the foundation for potential therapeutic agents.
In tr od u ction
Nitric oxide synthase (NOS) catalyzes the oxidation
of L-arginine to produce L-citrulline and the biologically
active free radical nitric oxide. Several recent reviews
describe the progress that has been made in the study
of NOS and the biological activity of nitric oxide.1-5 In
general there are two major classes of NOS: constitutive
and inducible. The constitutive enzymes require Ca2+
/
F igu r e 1. Structures of compounds I-VIII.
calmodulin for activity and are further divided into
neuronal and endothelial isoforms. The endothelial
isoform is found predominantly in the vascular endo-
thelium and generates low concentrations of nitric oxide
which lowers blood pressure and inhibits platelet ag-
gregation. Nitric oxide generated by the neuronal
enzyme appears to function as a neurotransmitter
regulating neuronal transmission. The inducible iso-
form is found in activated macrophages as well as many
other cell types and produces nitric oxide which plays
a role in host defense. It is this form of the enzyme that
is implicated in the excessive production of nitric oxide
that destroys functional tissue during acute and chronic
inflammation.
Identification of potent and selective inhibitors of the
inducible form of NOS has been the subject of intense
interest because of their therapeutic potential for the
treatment of diseases mediated by excessive production
of nitric oxide.6,7 Structural analogs of L-arginine such
as NG-monomethyl-L-arginine (L-NMA),8,9 NG-nitro-L-
arginine (L-NNA),10 L-N5-(1-iminoethyl)ornithine (L-
NIO),11 and L-thiocitrulline12 have been shown to be
inhibitors of the various forms of NOS but lack selectiv-
ity for the inducible isoform. We have recently reported
that L-N6-(1-iminoethyl)lysine (L-NIL) is a potent and
selective inhibitor of mouse inducible NOS and sup-
presses the increase in plasma nitrite/nitrate levels and
joint inflammation associated with adjuvant arthri-
tis.13,14 Non-amino acid NOS inhibitors have been
described and include aminoguanidine,15 isothioureas,16
and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.17 Here
we report for the first time that 2-iminopiperidine (II)
(Figure 1) and the homologous series of cyclic amidines
are potent non-amino acid inhibitors of human NOS
with modest selectivity for inducible NOS over the
endothelial constitutive isoform.
In evaluating compounds containing the amidino
group as potential NOS inhibitors, we found that
2-iminopiperidine (II) was a potent inhibitor of human
NOS isoforms, with IC50 values of 1.0 µM for hiNOS,
4.7 µM for hecNOS, and 1.1 µM for hncNOS (Table 1).
In order to investigate the effect of ring size on potency
and selectivity, we synthesized the corresponding five-,
seven-, eight-, and nine-membered ring analogs and
determined their potency and selectivity as NOS inhibi-
tors. As shown in Table 1, 2-iminopiperidine (II) was
the most potent of this series. The seven-membered
ring compound, 2-iminohomopiperidine (III), had simi-
† Abbreviations: NOS, nitric oxide synthase; hiNOS, recombinant
human inducible NOS; hecNOS, recombinant human endothelial
constitutive NOS; hncNOS, recombinant human neuronal constitutive
NOS; L-NIL, L-N6-(1-iminoethyl)lysine; L-NIO, L-N5-(1-iminoethyl)-
ornithine; L-NNA, NG-nitro-L-arginine; L-NMA, NG-monomethyl-L-
arginine; LPS, lipopolysaccharide.
* Author for correspondence. Tel: (314) 694-8951. Fax: (314) 694-
8949.
‡ Molecular Pharmacology/Inflammatory Diseases Research.
§ Medicinal Chemistry.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1996.
0022-2623/96/1839-0669$12.00/0 © 1996 American Chemical Society

670 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
Moore et al.
Ta ble 1. Comparison of IC50 Values for Inhibition of Human
NOS Isoforms
selectivityb
a
IC50 (µM)
compd
n
X
hiNOS hecNOS hncNOS hec/hi hnc/hi
I
II
III
IV
V
VI
VII
VIII
L-NIL
L-NMA
L-NNA
0
1
2
3
4
1
1
1
CH2
CH2
CH2
CH2
CH2
S
26
1.0
2.0
10
9.8
2.9
1.8
25
4.6
14
7.6
93
4.7
16
59
90
7.1
8.2
87
138
5.9
0.5
20
1.1
3.5
10
6.8
3.2
3.7
17
61
10
3.6
4.7
8.0
5.9
9.2
2.4
4.6
3.5
0.8
1.1
1.8
1.0
0.7
1.1
2.1
0.7
F igu r e 2. Plot of the reciprocal of hiNOS velocity (in units of
min/pmol) versus the reciprocal of L-arginine concentration (in
units of µM-1) at varying concentrations of 2-iminopiperidine
(II). NOS activity was determined by measuring the conver-
sion of L-[2,3-3H]arginine to L-[2,3-3H]citrulline as described
in the Experimental Section except that the reaction was
initiated by the addition of enzyme and the incubation at 37
°C was for 10 min. Enzyme activity was linear over this time
period for each concentration of L-arginine. Each point is the
average of duplicate determinations.
O
NH
30
0.4
0.066
13
0.7
0.066
0.5
a
IC50 values were determined with hiNOS, hecNOS, and
hncNOS by testing each compound at eight concentrations. NOS
activity was measured in the presence of a final L-arginine
concentration of 30 µM by monitoring the conversion of L-[2,3-
3H]arginine to L-[2,3-3H]citrulline as described in the Experimental
b
Section. Selectivity is defined as the ratio of the IC50(hecNOS)
or IC50(hncNOS) to IC50(hiNOS).
in Table 1, compounds VI and VII are potent inhibitors
of each of the human isoforms, with IC50 values very
similar to that of 2-iminopiperidine. Compound VIII
was substantially less potent with an IC50 of 25 µM for
hiNOS.
2-Iminopiperidine (II), 2-iminohomopiperidine (III),
and 2-imino-5,6-dihydro-1,3-thiazine (VI) were evalu-
ated in a mouse RAW cell assay to determine their effect
on the inhibition of cellular mouse inducible NOS
activity. RAW cells were stimulated with lipopolysac-
charide (LPS), and the inhibition of nitrite accumulation
in the medium was determined. RAW cell mouse
inducible NOS IC50 values for compounds II, III, and
VI were 21, 54, and 14 µM, respectively, demonstrating
the ability of these compounds to inhibit the intracel-
lular enzyme.
The ability of 2-iminopiperidine to inhibit inducible
NOS in vivo was also determined. Oral bioavailability
and efficacy were assessed by determining the ability
of either 2-iminopiperidine or L-NIL, a previously
described inhibitor of inducible NOS, to inhibit the
increase in plasma nitrite/nitrate levels generated from
inducible NOS following the administration of LPS.
Compounds were administered orally by gavage 1 h
prior to LPS administration. Plasma nitrite/nitrate
levels were measured 5 h following LPS administration
and were elevated >15-fold compared to saline-treated
animals (428 ( 43 vs 23.2 ( 3.3 µM). Both 2-iminopi-
peridine and L-NIL inhibited the increase in plasma
nitrite/nitrate levels in a dose-dependent manner, dem-
onstrating the ability of both compounds to inhibit
inducible NOS activity in vivo (Figure 3).
lar potency with an IC50 of 2.0 µM for hiNOS. In
comparison, potency for hiNOS was substantially less
for the smaller five-membered ring 2-iminopyrrolidine
(I) and slightly less with the larger eight (IV)- and nine
(V)-membered ring analogs (Table 1). Both 2-iminopi-
peridine (II) and 2-iminohomopiperidine (III) were more
potent for hiNOS than L-NMA, L-NNA, and L-NIL.
Selectivity for hiNOS as compared to hecNOS is indi-
cated by the ratio of IC50(hecNOS) to IC50(hiNOS). As
seen in Table 1, the hecNOS/hiNOS selectivity ratios
for compounds I-V range from 3.6 to 9.2 and generally
tend to increase with increasing ring size. 2-Imino-
homopiperidine (III) and 2-azacyclononanone imine (V)
were the most selective with hecNOS to hiNOS IC50
ratios of 8 and 9.2, respectively. Both 2-iminopiperidine
(II) and 2-iminohomopiperidine (III) were substantially
more selective for hiNOS over hecNOS than L-NMA and
L-NNA. Selectivity for hiNOS compared to hncNOS for
compounds I-V, given in Table 1 as the ratio of IC50-
(hncNOS) to IC50(hiNOS), ranged from 0.7 to 1.8,
indicating essentially little or no selectivity for hiNOS
over the neuronal isoform.
In order to investigate the nature of inhibition of
hiNOS by 2-iminopiperidine, a Lineweaver-Burk double-
reciprocal plot analysis of the kinetics of hiNOS in the
presence of different fixed concentrations of 2-iminopi-
peridine was performed. As shown in Figure 2, the
double-reciprocal plots resulted in lines intersecting at
the same point on the y-axis, indicating that 2-iminopi-
peridine is a competitive inhibitor of L-arginine binding
to hiNOS. A Ki of 0.36 µM for 2-iminopiperidine was
determined from a replot of the slope of each double-
reciprocal plot versus the concentration of 2-iminopi-
peridine (data not shown).
2-Iminopiperidine and the series of cyclic amidines
described here represent a new class of potent, non-
amino-acid-based NOS inhibitors and the foundation for
potential therapeutic agents. Further investigation of
this class of inhibitors is in progress.
We expanded this series of compounds with the
preparation of analogs of 2-iminopiperidine with N, S,
or O in the 3-position to afford 2-imino-5,6-dihydro-1,3-
thiazine (VI), 2-imino-5,6-dihydro-1,3-oxazine (VII), and
2-iminotetrahydropyrimidine (VIII) (Figure 1). As shown
Exp er im en ta l Section
L-[2,3-3H]Arginine was purchased from DuPont NEN (Bos-
ton, MA); (6R)-tetrahydro-L-biopterin was from Research Bio-
chemicals, Inc. (Natick, MA); L-NMA and L-NNA were from

2-Iminoazaheterocycles as Inhibitors of NOS Isoforms
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 671
in duplicate; standard deviations were 10% or less. Production
of [3H]citrulline was linear with time for hiNOS, hecNOS, and
hncNOS over the time period utilized for the assay.
Mea su r em en t of Cellu la r NOS Activity. The mouse
macrophage cell line RAW 264.7 was maintained in Dulbecco’s
MEM (DMEM) containing 10% fetal bovine serum in 5% CO2
at 37 °C. Prior to assay, cells were plated at 2 × 105 cells/
well onto a 96-well plate in a total volume of 100 µL. The
cells were induced to express iNOS with 10 µg/mL LPS in 10%
fetal bovine serum containing Earle’s salts (without phenol
red) and 2 mM L-glutamine for 17 h. Cells were then washed
twice with Krebs-Ringer-HEPES (25 mM, pH 7.5) buffer
containing 2 mg/mL D-glucose followed by a 1 h preincubation
on ice (50 µL total volume) in buffer containing five different
concentrations of the inhibitor and 30 µM L-arginine. The
assay was initiated by warming the plate to 37 °C. After 120
min, the plate was cooled on ice and 30 µL aliquots were
removed for the fluorometric determination of nitrite as
previously described.15,19 The generation of nitrite by the cells
was linear for 2 h and the nitrite produced was ca. 100 pmol/
well without inhibitor after subtracting out background from
uninduced cells. IC50 values were the mean of three wells with
a SEM of 10-15%.
In Vivo Effica cy Deter m in a tion . To induce the systemic
expression of inducible NOS resulting in markedly elevated
(>15-fold) plasma nitrite/nitrate levels,14,20-22 rats were treated
with an intraperitoneal injection of 10 mg/kg LPS. Compounds
were administered orally (0.1-100 mg/kg) 1 h prior to LPS
administration, and blood was collected 5 h following LPS
administration. Plasma was separated and then filtered
through a 10 000 MW cutoff Ultrafree microcentrifuge filter
unit at 14 000 rpm for 15 min. Plasma nitrite/nitrate concen-
trations, following conversion of nitrate to nitrite by nitrate
reductase, were determined using a fluorometric assay for the
measurement of nitrite in biological samples, as described
previously.14,20
F igu r e 3. In vivo efficacy of 2-iminopiperidine (II) and L-NIL
in LPS-treated rats. Male Lewis rats were treated with
various doses of either 2-iminopiperidine (9) or L-NIL (b) orally
1 h prior to the intraperitoneal administration of 10 mg/kg
LPS. Plasma nitrite/nitrate levels were measured 5 h follow-
ing LPS administration using a fluorometric assay as described
in the Experimental Section. Values are means ( SEM for n
) 6 animals/dose and represent the percent inhibition of
animals treated with LPS alone (428 ( 43 µM) compared to
saline-treated controls (23.2 ( 3.3 µM).
Sigma (St. Louis, MO); 2-iminopiperidine hydrochloride as well
as other chemicals and reagents were purchased from Aldrich
Chemical Co. (Milwaukee, WI) or Sigma. L-NIL was prepared
and homogeneity assessed as previously described.13 Purifica-
tion of synthesized compounds was performed by crystalliza-
tion or HPLC chromatography on a Waters Prep LC2000
instrument using a Delta Pak C-18 column. 1H- and 13C-NMR
spectra were obtained at 300 MHz on a Varian VXR300
spectrometer in D2O. Mass spectra were obtained on either a
VG Model 250 or a Finnigan MAT 90 spectrometer. Elemental
analyses were performed by Atlantic Microlabs, Inc. (Norcross,
GA). Male Lewis rats (150-200 g) were purchased from
Harlan Sprague-Dawley (Indianapolis, IN) and housed and
cared for in accordance with the guidelines of the Institutional
Animal Care and Use Committee and in accordance with NIH
guidelines on laboratory animal welfare. Escherichia coli
lipopolysaccharide (serotype 0111:B4, Westphal extract) was
purchased from Sigma (St. Louis, MO). Ultrafree-MC filter
units were from Millipore (Bedford, MA).
Syn th esis of In h ibitor s. 2-Im in op yr r olid in e Hyd r o-
ch lor id e (I). 2-Iminopyrrolidine hydrochloride was prepared
by the literature method23 via reaction of 4-chlorobutyronitrile
with ammonia in ethanol in a sealed pressure vessel at 135
°C for 9 h. Filtration of the reaction mixture through Norit,
evaporation of solvent, and crystallization from ethanol-ethyl
ether afforded the title product in 62% yield as a white solid,
mp 149-150 °C. Anal. (C4H9N2Cl1‚1/4H2O) C, H, N.
2-Im in oh om op ip er id in e Hyd r oiod id e (III).24 To a solu-
tion of 5 g (0.039 mol) of thiocaprolactam in 100 mL of acetone
was added 6.4 g (0.045 mol) of iodomethane. This mixture
was stirred for 48 h at 25 °C. Filtration afforded 9.5 g of the
thio iminoether hydroiodide salt as a white solid, mp 177-
181 °C; 3 g of this iminoether was dissolved in 80 mL of ethanol
saturated with anhydrous ammonia, sealed, and stirred at 25
°C for 3 days. Concentration to a reduced volume followed by
ethyl ether trituration afforded 2.2 g (39%) of the title product
as a white solid mp 135-141 °C. 1H-NMR (D2O): δ 1.45-
1.55 (m, 2H), 1.55-1.7 (m, 4H), 2.5 (m, 2H), 3.28 (m, 2H).
MS: 113 (MH+). Anal. (C6H13N2I1) C, H, N.
2-Aza cycloocta n on e Im in e Hyd r och lor id e (IV). A solu-
tion of 5 g (0.04 mol) of 2-azacyclooctanone in 15 mL of benzene
was stirred at reflux while 4.8 g (0.038 mol) of dimethyl sulfate
was added dropwise. After the addition was complete, stirring
was continued at reflux for 18 h. The heat was then removed;
the reaction mixture was diluted with ethyl acetate and
washed with two 100 mL portions of aqueous potassium
carbonate. The organic layer was dried (MgSO4), filtered, and
concentrated to afford 4.2 g of the corresponding iminoether
as a yellow oil; 2.2 g (0.016 mol) of the iminoether was
dissolved in 50 mL of anhydrous ethanol, and 0.85 g (0.016
mol) of ammonium chloride was added. This mixture was
stirred at 25 °C for 3 days. Removal of the solvent in vacuo
afforded 1.7 g (48%) of the title product as a white solid, mp
166-175 °C. 1H-NMR (D2O): δ 1.3-1.45 (m, 4H), 1.45-1.55
(m, 2H), 1.6-1.7 (m, 2H), 2.45 (m, 2H), 3.3 (m, 2H). MS: 127
(MH+). Anal. (C7H15N2Cl1‚1/4H2O) C, H, N.
Assa y of NOS Activity. NOS activity was measured by
monitoring the conversion of L-[2,3-3H]arginine to L-[2,3-3H]-
citrulline.13,18 Human inducible NOS, human endothelial
constitutive NOS, and human neuronal constitutive NOS were
each cloned from RNA extracted from human tissue.19 The
cDNA for hiNOS was isolated from a λcDNA library made from
RNA extracted from a colon sample from a patient with
ulcerative colitis; the cDNA for hecNOS was isolated from a
λcDNA library made from RNA extracted from human
unbilical vein endothelial cells (HUVEC); and the cDNA for
hncNOS was isolated from a λcDNA library made from RNA
extracted from human cerebellum obtained from a cadaver.
The recombinant enzymes were expressed in Sf9 insect cells
using a baculovirus vector.19 Enzyme activity was isolated
from soluble cell extracts and partially purified by DEAE-
Sepharose chromatography.15,19 The Km values for L-arginine
for hiNOS, hecNOS, and hncNOS were 7, 4, and 6 µM,
respectively. To measure NOS activity, 10 µL of enzyme was
added to 40 µL of 50 mM Tris (pH 7.6) and the reaction
initiated by the addition of 50 µL of a mixture containing 50
mM Tris (pH 7.6), 2.0 mg/mL bovine serum albumin, 2.0 mM
DTT, 4.0 mM CaCl2, 20 µM FAD, 100 µM tetrahydrobiopterin,
0.4 mM NADPH, and 60 µM L-arginine containing 0.9 µCi of
L-[2,3-3H]arginine. The final concentration of L-arginine in the
assay was 30 µM. For hecNOS and hncNOS, calmodulin was
included at a final concentration of 40 nM. Following incuba-
tion at 37 °C for 15 min, the reaction was terminated by
addition of 300 µL of cold buffer containing 10 mM EGTA, 100
mM HEPES (pH 5.5), and 1.0 mM L-citrulline. The [3H]-
citrulline was separated by chromatography on Dowex 50W
X-8 cation exchange resin and radioactivity quantified with a
liquid scintillation counter. All assays were performed at least
2-Aza cyclon on a n on e Im in e Hyd r och lor id e (V). 2-Aza-
cyclononanone imine hydrochloride was prepared analogously
to 2-azacyclooctanone imine hydrochloride from 2-azacy-

672 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
Moore et al.
(10) Furfine, E. S.; Harmon, M. F.; Paith, J . E.; Garvey, E. P.
Selective inhibition of constitutive nitric oxide synthase by NG-
nitroarginine. Biochemistry 1993, 32, 8512-8517.
(11) Rees, D. D.; Palmer, R. M. J .; Schulz, R.; Hodson, H. F.; Moncada,
S. Characterization of three inhibitors of endothelial nitric oxide
synthase in vitro and in vivo. Br. J . Pharmacol. 1990, 101, 746-
752.
clononanone to afford 2.75 g (45%) of the 2-azacyclononanone
imine as a white solid, mp 108-128 °C. 1H-NMR (D2O): δ
1.3-1.6 (m, 8H), 1.55-1.65 (m, 2H), 2.45 (m, 2H), 3.35 (m,
2H). MS: 141 (MH+). Anal. (C8H17N2Cl1‚1/3H2O) C, H, N.
2-Im in otetr ah ydr opyr im idin e Hem isu lfate (VIII). 2-Im-
inotetrahydropyrimidine hemisulfate was prepared by the
literature method25 via reaction of 1,3-diaminopropane with
methyl isothiourea hemisulfate in water at reflux for 3 h.
Evaporation of solvent and preparative HPLC chromatography
(using a linear gradient over 30 min from 0% to 40% aceto-
nitrile in 0.1% trifluoroacetic acid in water) afforded the title
product as a white solid in 61% yield, mp 304 °C dec. 1H-
NMR (D2O): δ 1.75 (p, 2H), 3.13 (t, 4H). Anal. (C4H9N3‚
1/2H2SO4) C, H, N.
(12) Narayanan, K.; Griffith, O. W. Synthesis of L-Thiocitrulline,
L-Homothiocitrulline, and S-Methyl-L-Thiocitrulline: A New
Class of Potent Nitric Oxide Synthase Inhibitors. J . Med. Chem.
1994, 37, 885-887.
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T. P.; Currie, M. G. L-N6-(1-iminoethyl)lysine:
a selective
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2-Im in o-5,6-d ih yd r o-1,3-th ia zin e Hyd r och lor id e (VI).26
A solution of 4 g (0.03 mol) 3-chloropropylamine hydrochloride
and 3 g (0.03 mol) of potassium thiocyanate in 10 mL of water
was stirred at 90 °C for 2 h. Evaporation of solvent afforded
a semisolid residue which was extracted with boiling ethanol.
The title product precipitated from the ethanol upon cooling
to afford a white crystalline solid, 2.5 g (55%). Recrystalliza-
tion from 2-propanol afforded a white solid, mp 110-111 °C.
1H-NMR (D2O): δ 2.05 (p, 2H), 3.0 (dt, 4H). Anal. (C4H9N2S1-
Cl1) C, H, N.
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J erome, G. M.; Webber, R. K.; Tjoeng, F. S.; Currie, M. G.
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2-Im in o-5,6-d ih yd r o-1,3-oxa zin e Hyd r och lor id e (VII).27
A solution of 4 g (0.03 mol) of 3-chloropropylamine hydrochlo-
ride and 2 g (0.03 mol) of sodium cyanate in 10 mL of water
was stirred at 90 °C for 2 h. Evaporation of solvent afforded
a semisolid residue which was extracted with boiling ethanol.
The title product precipitated from the ethanol upon cooling
and addition of ethyl ether to afford a white crystalline solid,
2.5 g (61%), mp 123-124.5 °C. 1H-NMR (D2O): δ 2.0 (p, 2H),
3.38 (t, 2H), 4.37 (t, 2H).
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J erome, G. M.; Rangwala, S. H.; Misko, T. P.; Colburn, S. M.;
Ornberg, R. L.; Wiese, J . F.; Currie, M. G. Isolation from
inflamed colon of two sequences for human inducible nitric oxide
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