2-Iminohomopiperidinium salts as selective inhibitors of inducible nitric oxide synthase (iNOS)

Article abstract of DOI:10.1021/jm9704715

An attractive approach to the treatment of inflammatory conditions such as osteo- and rheumatoid arthritis, inflammatory bowel disease, and sepsis is through the selective inhibition of human inducible nitric oxide synthase (hiNOS) since localized excess

Full text of DOI:10.1021/jm9704715

J . Med. Chem. 1998, 41, 1361-1366
1361
Expedited Articles
2-Im in oh om op ip er id in iu m Sa lts a s Selective In h ibitor s of In d u cible Nitr ic
Oxid e Syn th a se (iNOS)†
Donald W. Hansen, J r.,*,‡ Karen B. Peterson,‡ Mahima Trivedi,‡ Steven W. Kramer,‡ Ronald K. Webber,§
Foe S. Tjoeng,§ William M. Moore,| Gina M. J erome,| Christine M. Kornmeier,| Pamela T. Manning,|
J ane R. Connor,| Thomas P. Misko,| Mark G. Currie,| and Barnett S. Pitzele‡
Department of Discovery Medicinal Chemistry, G. D. Searle Research and Development, 4901 Searle Parkway,
Skokie, Illinois 60077, G. D. Searle Research and Development, 700 Chesterfield Parkway North,
Chesterfield, Missouri 63198, and Department of Discovery Pharmacology, Searle Research and Development,
800 North Lindbergh Boulevard, St. Louis, Missouri 63167
Received February 24, 1998
An attractive approach to the treatment of inflammatory conditions such as osteo- and
rheumatoid arthritis, inflammatory bowel disease, and sepsis is through the selective inhibition
of human inducible nitric oxide synthase (hiNOS) since localized excess nitric oxide (NO) release
has been implicated in the pathology of these diseases. A series of monosubstituted
iminohomopiperidinium salts possessing lipophilic functionality at ring positions 3, 5, 6, and
7 has been synthesized, and series members have demonstrated the ability to inhibit the hiNOS
isoform with an IC50 as low as 160 nM (7). Compounds were found that selectively inhibit
hiNOS over the human endothelial constitutive enzyme (heNOS) with a heNOS/hiNOS IC50
ratio in excess of 100 and as high as 314 (9). Potencies for inhibition of hiNOS and the human
neuronal constitutive enzyme (hnNOS) are comparable. Substitution in the 3 and 7 positions
provides compounds that exhibit the greatest degree of selectivity for hiNOS and hnNOS over
heNOS. Submicromolar potencies for 6 and 7 in a mouse RAW cell assay demonstrated the
ability of these compounds to inhibit iNOS in a cellular environment. These latter compounds
were also found to be orally bioavailable and efficacious due to their ability to inhibit the increase
in plasma nitrite/nitrate levels in a rat LPS model.
In tr od u ction
three principal sites of NO formation are paralleled by
the three different forms of NO synthase (NOS) derived
from distinct genes. The endothelial (eNOS) and neu-
ronal (nNOS) enzymes are generally regarded as con-
stitutive and are Ca2+- and calmodulin-dependent, while
the inducible (iNOS) isoform characterized in macroph-
ages and various other cells is usually Ca2+- and
calmodulin-independent.
The current intense nitric oxide (NO) research effort
in both academic and industrial institutions is due to
the accumulating data suggesting that this free radical
is a messenger molecule with extremely diverse
functions.1-7 NO, characterized initially as an endo-
thelium-derived releasing factor in blood vessels, is
considered a main determinant of blood pressure. More
recently, NO has been proposed as a neurotransmitter
or neuromodulator in the brain and peripheral auto-
nomic nervous system. NO has now been linked to the
regulation of a number of biological functions such as
memory and learning, peristalsis, and penile erection.
It has also been demonstrated that NO is synthesized
by immune system cells such as macrophages and
neutrophils in response to various types of infection and
tumor cells. The immune system appears to use NO to
eliminate or control the invading pathogens. These
Imbalances in finely tuned NO synthesis appear to
lead to serious consequences.7 It is, therefore, apparent
that significant pharmaceutical potential exists for
selective agents that normalize NO levels in disease
states where these levels are either depressed or
elevated. For situations involving low levels of NO, the
practical approach of using drugs that release NO has
already found application in areas such as treatment
of angina. The reduction of NO by selective inhibition
of specific isoenzymes is expected to be of value in
disease states such as endotoxic shock where dramatic
hypotension has been linked to high levels of NO.
Elevated levels of NO have also been related to inflam-
mation from a variety of causes. In addition, there is
accumulating evidence that increased levels of NO may
play a role in postischemic stroke damage. The NO
generated in all the latter aforementioned conditions
appears to be due to stimulation of iNOS. Its inhibition
may be a key and effective route to ameliorating these
† Abbreviations: NOS, nitric oxide synthase; hiNOS, recombinant
human inducible nitric oxide synthase; heNOS, recombinant human
endothelial constitutive enzyme; hnNOS, recombinant human neuronal
constitutive enzyme; L-NMA, NG-monomethyl-L-arginine; L-NAME, NG-
nitro-L-arginine methyl ester; L-NIO, L-N5-(1-iminoethyl)ornithine;
L-NIL, L-N6-(1-iminoethyl)lysine.
* Author for correspondence: tel, (847) 982-7739; fax, (847) 982-
4714.
‡ G. D. Searle Research and Development, IL.
§ G. D. Searle Research and Development, Chesterfield, MO.
| G. D. Searle Research and Development, St. Louis, MO.
S0022-2623(97)00471-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/28/1998

1362 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Hansen et al.
Sch em e 1a
Ta ble 1. Enzyme Inhibition of Substituted
Iminohomopiperidinium Salts
a
(a) NH2OH‚HCl, NaOAc, EtOH; (b) 80% H2SO4, 120 °C, or
benzenesulfonyl chloride, acetone; (c) HPLC isomeric separation
when necessary; (d) Me3O+BF4-, CH2Cl2, rt, or Me2SO4, benzene,
Dean-Stark trap; (e) NH4Cl, MeOH reflux.
oximes (B) and subsequently subjected to a variety of
Beckmann rearrangement conditions to provide sepa-
rable regioisomeric mixtures of substituted caprolac-
tams (C). In the case of 2-substituted cyclohexanones,
the 7-substituted caprolactams predominated in the
mixture. The caprolactam derivative was subsequently
treated with trimethyloxonium tetrafluoroborate to
generate the respective imino ethers (D). Reaction of
these materials with ammonium chloride provided our
target 2-iminohomopiperidinium hydrochlorides (E).
The iminohomopiperidinium salts indicated in the
accompanying table were evaluated, as detailed in the
description of the citrulline assay, for their ability to
inhibit each of the three human isoforms of NOS. The
parent iminohomopiperidinium chloride 1, reported
previously,19 has micromolar potency for hiNOS, 7-fold
selectivity over heNOS, and only 2-fold selectivity over
hnNOS. When an ethyl function is inserted into the 3
position, the potency of resulting compound 2 is slightly
enhanced and its selectivity over heNOS is more than
tripled. Analogue 3, containing an allyl group at
position 3, has comparable potency for hiNOS but is 65-
fold selective over heNOS. All of these three substituted
salts are nearly equipotent for hiNOS and hnNOS.
Analogues that are methyl-substituted at positions 5
and 6, 4 and 5, have enzyme inhibition profiles less
potent but similar to that of their parent, 1.
In contrast, compounds possessing a substituent at
position 7, compounds 6-9, exhibit dramatic improve-
ment in potency, selectivity, or both as compared to 1.
The analogue containing an n-propyl function, 7, has a
hiNOS IC50 of 160 nM and selectivity of nearly 60. Its
unsaturated relative, 6, has similar potency for hiNOS
(190 nM) but is 110-fold selective over the heNOS
isoform and 6-fold over hnNOS. Figuratively inserting
a methylene unit into the side chain of 7 gives rise to
the n-butyl analogue 8 which has reduced hiNOS
potency (500 nM) but dramatically enhanced selectivity
over heNOS (>200-fold). Aliphatic branching added at
the 2′ carbon of 8 gives rise to 9. This analogue, while
having a hiNOS IC50 of only 2.1 µM, is greater than 300-
fold selective for inhibition of hiNOS over heNOS.
It is interesting to note that introduction of π char-
acter into substituents at either the 3 or 7 position of
these iminohomopiperidinium salts gives rise to ana-
logues, 3 and 6, that exhibit reduced potency but
disease states. Our focus has been on the design and
synthesis of novel inhibitors selective for human induc-
ible nitric oxide synthase (hiNOS).
The natural substrate for NOS, arginine, has been
the obvious basis of inhibitor design. Much pharmacol-
ogy and biology have been determined utilizing some
of the early relatively nonspecific inhibitor derivatives
of Arg such as NG-monomethyl-L-arginine (L-NMA),8,9
NG-nitro-L-arginine methyl ester (L-NAME),10 and L-N5-
(1-iminoethyl)ornithine (L-NIO).11 More recently we
have reported that a lysine derivative, L-N6-(1-imino-
ethyl)lysine (L-NIL), is a potent and selective inhibitor
of mouse inducible NOS and suppresses both increases
in plasma nitrite/nitrate levels and joint inflammation
associated with adjuvant arthritis.12,13 A number of
non-amino-acid NOS inhibitors have also been described
and include such agents as aminoguanidine,14 isothio-
ureas,15,16 amidines,17 and 2-amino-5,6-dihydro-6-meth-
yl-4H-1,3-thiazine.18 We recently reported that 2-imi-
nopiperidinium hydrochloride and the homologous series
of amidinium salts are potent non-amino-acid inhibitors
of human NOS with modest selectivity for inducible
NOS over the endothelial constitutive isoform.19 We
now report a series of monosubstituted 2-iminohomopi-
peridinium salts, examples of which inhibit the hiNOS
isoform with a potency as low as 160 nM (7) and
selectively inhibit hiNOS over the human endothelial
constitutive enzyme (heNOS) with a hiNOS/heNOS IC50
ratio in excess of 100 and as high as 314 (9).
Resu lts a n d Discu ssion
The target molecules illustrated in Table 1 were
synthesized as diagrammed in Scheme 1. The well-
known alkyl-substituted cyclohexanone starting materi-
als (A) were obtained either commercially, by direct
alkylation of cyclohexanone, or by alkylation of 2-(car-
boalkoxy)cyclohexanone followed by decarboxylation.
These cyclohexanones (A) were then converted to their

2-Iminohomopiperidiniums as Inhibitors of iNOS
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1363
F igu r e 1. 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 7. NOS activity was determined by measuring the conversion 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.
Ta ble 2. In Vitro and in Vivo Efficacy of Substituted
Iminohomopiperidium Salts
significantly increased selectivity for hiNOS versus
heNOS over their respective aliphatic relatives, 2 and
raw cell
rat low endotoxin
7. However, when a π-rich phenyl group is appended
to the 7 position as illustrated by analogue 10, both
potency and selectivity are severely reduced. In fact,
compound 10 is one of only two analogues that inhibit
hnNOS to a greater degree than hiNOS.
compd
IC50 (µM)a
(% inhib at 10 mg/kg)b
1
2
3
4
6
7
8
9
10
54 ((22)
3.5 ((0.7)
19 ((6.8)
24 ((14)
0.6 ((0.1)
0.6 ((0.1)
1.6 ((1.1)
3.3 ((1.6)
31 ((38)
ndc
nd
nd
nd
80 ((3)
85 ((3)
60 ((1)
nd
In general, the substituted iminohomopiperidinium
salts described inhibit both hiNOS and hnNOS to a
similar degree and are far less effective at inhibiting
heNOS. Analogues containing substituents at positions
3, 5, and 6 have hiNOS IC50’s between about 2 and 8
µM and selectivities for hiNOS over heNOS of up to 65-
fold. Relatives with aliphatic functionality at ring
position 7 not only exhibit hiNOS inhibition potencies
as low as 160 nM but also show selectivities for hiNOS
over heNOS of greater than 300-fold and are, to our
knowledge, the most selective agents reported to date.
Since all the compounds discussed except 5 are racemic
mixtures of two enantiomers, it is also conceivable that
there is a single isomer that possesses better potency
and/or selectivity.
In order to investigate the nature of inhibition of
hiNOS by 7, the most potent iNOS inhibitor in the
series, a Lineweaver-Burk double-reciprocal plot analy-
sis of the kinetics of hiNOS in the presence of different
fixed concentrations of 7 was performed. As shown in
Figure 1, the double-reciprocal plots resulted in lines
intersecting at the same point on the y-axis, indicating
that 7 is a competitive inhibitor of L-arginine binding
to hiNOS. A Ki of 0.09 µM for 7 was determined from
a replot of the slope of each double-reciprocal plot versus
the concentration of 7 (data not shown).
nd
a
The ability of compounds to inhibit mouse iNOS in LPS-
stimulated mouse RAW cells was determined as described in the
b
Experimental Section. The ability of compounds to inhibit rat
iNOS in LPS-treated male Lewis rats was determined by measur-
ing plasma nitrite concentrations as described in the experimental
section. c nd, not determined.
The compounds were also evaluated in a cell-based
mouse RAW cell assay for their ability to inhibit murine
iNOS (see Table 2). Cells were stimulated with li-
popolysaccharide (LPS), and inhibition of nitrite produc-
tion in the medium was determined after 2 h of
treatment with and without inhibitor. The RAW cell
mouse iNOS IC50 values for compounds 6-8 were 0.6,
0.6, and 1.6 µM, respectively, demonstrating the ability
of these compounds to inhibit iNOS in a cellular milieu.
These three analogues were also the most potent at
inhibiting hiNOS, and in general, the RAW cell IC50
values parallel those for hiNOS inhibition.
The ability of compounds 6-8 to inhibit iNOS in vivo
was also evaluated. Oral bioavailability and efficacy
were assessed by determining their ability to inhibit the

1364 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Hansen et al.
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
in duplicate; standard deviations were 10% or less. Production
of [3H]citrulline was linear with time for hiNOS, heNOS, and
hnNOS over the time period utilized for the assay.
Mea su r em en t of Cellu la r NOS Activity. The ability of
compounds to inhibit mouse iNOS activity was determined
using LPS-stimulated RAW 264.7 mouse cells as described
previously.19 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 × 1005 cells/well in a 96-well plate and were
induced to express iNOS by incubating for 17 h with 10 ug/
mL LPS (Escherichia coli serotype 0111:B4). Cells were then
washed and preincubated for 1 h on ice in Krebs Ringer buffer
containing 25 mM HEPES, pH 7.5, 2 mg/mL D-glucose, 30 uM
L-arginine, and five different concentrations of the inhibitor.
The cells were then warmed to 37 °C for 2 h, and the amount
of nitrite generated was determined by a fluorometric assay.21
Each IC50 was a mean of at least six determinations.
In Vivo Effica cy Deter m in a tion . The ability of com-
pounds 6-8 to inhibit rodent iNOS in vivo was determined
using LPS-treated rats (to induce systemic iNOS expression)
as described previously.13,22 One hour prior to LPS adminis-
tration (10 mg/kg, intraperitoneally), compounds 6-8 were
administered orally to rats at a dose of 10 mg/kg. Blood was
collected 5 h following LPS administration, and plasma was
separated and filtered through 10 000 MW cutoff Ultrafree
microcentrifuge filter units. Plasma nitrite/nitrate concentra-
tions were determined using a fluorometric assay for the
measurement of nitrite/nitrate in biological samples.21 The
basal and stimulated values for plasma nitrite/nitrates are 15.7
((0.7) and 289 ((37) µM, respectively, for one experiment and
23.2 ((0.1) and 238 ((23) µM, respectively, for another. The
percent inhibition was calculated using values obtained from
rats treated with LPS alone in the absence of inhibitor.
Gen er a l Syn th etic P r oced u r e for Oxim e F or m a tion .
A substituted cyclohexanone (1 equiv) was combined with
hydroxylamine hydrochloride (NH2OH‚HCl; 1.5 equiv) and
sodium acetate (1.8 equiv) in a mixture of EtOH (1 mL/mmol
of ketone) and water (0.6 mL/mmol of ketone). The mixture
was refluxed for 5 h under a N2 atmosphere, cooled to room
temperature, and allowed to stir for an additional 24 h. All
solvent was removed from the reaction mixture under reduced
pressure. The residue was partitioned between EtOAc and
water, and the organic phase was washed with brine, dried
(Na2SO4), and stripped of all solvent under reduced pressure
to provided the oximes in yields of 84-99%. Each oxime,
without further purification, showed essentially one peak with
a 100% purity by peak area integration on a Shimadzu GC-
14A gas chromatograph (GC) with a 0.25-mm × 25 M methyl,
5% phenylsilicone column, and NMR and IR spectra were
consistent with the assigned structures.
increase in plasma nitrite/nitrate levels generated from
iNOS following the administration of LPS to rats. The
compounds were given at a dose of 10 mg/kg orally by
gavage 1 h prior to LPS administration. After 5 h, the
plasma nitrite/nitrate levels were elevated approxi-
mately 15-fold compared to saline-treated animals. The
basal and stimulated values for plasma nitrite/nitrates
are 15.7 ((0.7) and 289 ((37) µM, respectively, for one
experiment and 23.2 ((0.1) and 238 ((23) µM, respec-
tively, for another. Compounds 6-8 produced 80%,
85%, and 60% inhibition, respectively, of the plasma
nitrite/nitrate levels found in animals treated with LPS
without inhibitor, and this data correlates relatively
with their in vitro data (see Table 2). This convincingly
demonstrates their ability to inhibit inducible NOS
activity in vivo following oral administration.
In summary, these iminohomopiperidinium salts are
potent inhibitors of human NOS, and certain series
members are highy selective for the inducible isoform
over heNOS. The greatest selectivity resides in ana-
logues with substitution in the 7 position of the imino-
homopiperidine ring. It is also very significant that
these materials not only inhibit iNOS in a cellular
environment but are also orally bioavailable and effica-
cious in a model of inflammation.
Exp er im en ta l Section
Proton (1H) NMR spectra were recorded at 500, 400, 300,
and 80 MHz on Varian VXR-500, Varian VXR-400, General
Electric model QE-300, and Varian FT-80A instruments.
Infrared spectra were measured on Perkin-Elmer model 283B
and model 681 instruments. Specific optical rotations were
measured at the sodium D line at 21 °C on a Perkin-Elmer
model 241 digital polarimeter (1-dm cell). Mass spectra were
taken on a Finnigan-MAT model 8430 system with high
resolution, FAB, and chemical ionization capability. Elemen-
tal analyses were determined by the Searle Laboratories
Microanalytical Department under the direction of Mr. E.
Zielinski. Microanalyses were obtained for the stated elements
and were within 0.4% of the theoretical values for the formula
unless otherwise stated. Preparative high-performance liquid
chromatography (HPLC) was performed on a Rainin model
HPX system. Analytical HPLC was carried out on a Waters
model 45 instrument equipped with a Kratos Sprectroflow
model 773 detector and a Hewlett-Packard model 3390A
integrator. Reverse-phase HPLC was run on a YMC AQ ODS-
363-10P column, with eluants of aqueous acetonitrile-acetic
acid. The substituted cyclohexanones were purchased from
the following: Aldrich Chemical Co. (Milwaukee, WI), Pfaltz
& Bauer, Inc. (Waterbury, CT), Frinton Laboratories (Vine-
land, NJ ), and Farchan Laboratories, Inc. (Gainesville, FL).
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 as previously described.19 hiNOS, heNOS, and
hnNOS were each cloned from RNA extracted from human
tissue.20 The recombinant enzymes were expressed in Sf9
insect cells using a baculovirus vector.20 Enzyme activity was
isolated from soluble cell extracts and partially purified by
DEAE-Sepharose chromatography.14,20 The Km values for
L-arginine for hiNOS, heNOS, and hnNOS 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 heNOS and hnNOS, calmodulin was
included at a final concentration of 40 nM. Following incuba-
tion at 37 °C for 15 min, the reaction was terminated by
Gen er a l P r oced u r e A: Oxim es to La cta m s (Beck m a n
Rea r r a n gem en t). One equivalent of oxime suspended or
dissolved in 80% H2SO4 (0.16 mL/mmol of oxime) was added
dropwise (∼10 min) to 80% H2SO4 (0.13 mL/mmol of oxime)
stirred magnetically and maintained at 120 °C with an
external oil bath. An exotherm was noted within 5 min of the
start of addition, and the temperature of the reaction rose to
between 140 and 160 °C before cooling again to 120 °C. Ten
minutes after the flask had cooled to 120 °C, it was removed
from the bath and allowed to cool to room temperature. The
product mixture was diluted with water (0.5 mL/mmol of
oxime) and brought to pH 6 with concentrated NH4OH. This
solution was further diluted with water (0.5 mL/mmol of
oxime) and extracted with CH2Cl2. The combined organic
phase was washed with brine, dried (Na2SO4), filtered, and
stripped of all solvent under reduced pressure. The residues
were purified and isomers, when present, separated by HPLC
on silica gel to provide the lactams in 40-80% yield. Each

2-Iminohomopiperidiniums as Inhibitors of iNOS
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1365
chromatographed lactam showed essentially one peak with a
peak area of 100% on a Shimadzu GC-14A GC with a 0.25-
mm × 25 M methyl, 5% phenylsilicone column, and NMR
spectra were consistent with the assigned structures.
1H), 1.90-1.73 (m, 2H), 1.30-1.13 (m, 2H), 0.99 (d, 3H, J ) 7
Hz); 13C NMR (D2O) δ 174.3, 43.6, 37.1, 36.9, 32.4, 31.1, 22.9;
HRMS (M+) C7H14N2 requires m/e 126.116, found m/e 126.116.
Anal. (C7H14N2‚0.85HCl‚0.8H2O) C, H, N, Cl.
Hexa h yd r o-(6R)-m eth yl-1H-a zep in -2-im in e, m on eh y-
d r och lor id e (5): synthesized by general procedure A and its
iminoether by general procedure C; 1H NMR (500 MHz, D2O)
δ 3.34 (m, 1H), 3.24 (m, 1H), 2.68 (m, 1H), 2.62 (m, 1H), 1.94-
1.86 (m, 2H), 1.77 (m, 1H), 1.63 (m, 1H), 1.47 (m, 1H), 0.92 (d,
3H, J ) 7 Hz); 13C NMR (D2O) δ 175.6, 52.0, 39.6, 34.5, 33.9,
24.0, 20.7; HRMS (M+) C7H14N2 requires m/e 126.116, found
m/e 126.117; [R]D ) -12.7° ((3.5°) (MeOH, c ) 0.869). Anal.
(C7H14N2‚1.0HCl‚0.25H2O‚0.55NH4Cl) C, H, N, Cl.
Hexa h yd r o-3-(2-p r op en yl)-1H-a zep in -2-im in e, m on o-
h yd r och lor id e (6): synthesized by general procedure B and
its iminoether by general procedure C; 1H NMR (400 MHz,
D2O) δ 5.85 (m, 1H), 5.26-5.19 (m, 2H), 3.74 (m, 1H), 2.75
(ddd, 1H, J ) 14.5, 12.4, 1.7 Hz), 2.59 (dd, 1H, J ) 14.5, 6.6
Hz), 2.40 (t, 2H, J ) 7 Hz), 2.04-1.94 (m, 2H), 1.87 (m, 1H),
1.65 (m, 1H), 1.56-1.35 (m, 2H); 13C NMR (D2O) δ 175.1, 136.7,
121.5, 58.0, 41.6, 35.7, 34.4, 31.0, 25.6; HRMS (M+) C9H16N2
Gen er a l P r oced u r e B: Oxim es to La cta m s (Beck m a n
Rea r r a n gem en t). To 1 equiv of oxime dissolved in 50 mL of
acetone was added 1 N NaOH (1.1 equiv). After this mixture
cooled in an ice bath, benzenesulfonyl chloride (1.0 equiv) was
added dropwise (5 min) to the stirred reaction mixture
maintained under a N2 atmosphere. The reaction was allowed
to warm to room temperature and stir for 48 h. If the product
precipitated from the reaction mixture, it was filtered and
washed with acetone. The filtrate was concentrated and the
residue partitioned between EtOAc and brine. The organic
layer was dried (Na2SO4), filtered, and stripped of all solvent
under reduced pressure. The residue was treated as described
in General Procedure A, above.
Gen er a l P r oced u r e C: La cta m s to Im in oeth er s. To a
stirred slurry of trimethyloxonium tetrafluoroborate (Me3-
O+BF4-, Lancaster; 1.2 equiv) in CH2Cl2 (5 mL/equiv of lactam)
under Ar was added the lactam (1 equiv). This mixture was
stirred at room temperature for 12 h before it was diluted with
CH2Cl2 (5 mL/equiv of lactam) and partitioned between
saturated KHCO3 and EtOAc. The organic phase was sepa-
rated, dried (Na2SO4), filtered, and stripped of all solvent under
reduced pressure to provide the crude iminoether. This
material was chromatographed on a short-path Merck flash
silica column eluting with EtOAc/n-hexane (1:1). The lactam
product had a GC peak area of 100% and NMR and IR spectra
consistent with the indicated product.
Gen er a l P r oced u r e D: La cta m s to Im in oeth er s. One
equivalent of lactam (2.5 g, 19.7 mmol) dissolved in benzene
(2 mL/mmol of lactam) was dried for 30 min with a Dean-
Stark trap. To this mixture was added dimethyl sulfate (2
equiv), and heating was continued for an additional 17 h.
After cooling to room temperature, the reaction was diluted
with EtOAc (2.5 mL/mmol of lactam) and washed with
saturated NaHCO3. The aqueous layer was extracted with
EtOAc and the combined organic phase dried (Na2SO4),
filtered, and stripped of all solvent under reduced pressure to
yield a residue that was chromatographed and characterized
as described in General Procedure C, above.
requires m/e 152.131, found m/e 152.132. Anal. (C9H16
N2‚1.0HCl‚0.75H2O‚0.05NH4Cl) C, H, N, Cl.
-
Hexa h yd r o-7-p r op yl-1H-a zep in -2-im in e, m on oh yd r o-
ch lor id e (7): synthesized by general procedure B and its
iminoether by general procedure C; 1H NMR (400 MHz, CD3-
OD) δ 3.62 (m, 1H), 2.79 (ddd, 1H, J ) 14.3, 12.2, 1.9 Hz),
2.61 (dd, 1H, J ) 14.6, 6.6 Hz), 2.06-1.95 (m, 2H), 1.85 (m,
1H), 1.75-1.32 (m, 7H), 0.98 (t, 3H, J ) 7.2 Hz); 13C NMR
(CD3OD) δ 173.9, 57.1, 38.4, 35.0, 32.4, 29.7, 24.5, 20.1, 14.0;
HRMS (M+) C9H18N2 requires m/e 154.147, found m/e 154.147.
Anal. (C9H18N2‚1.0HCl‚0.4H2O‚0.05NH4Cl) C, H, N, Cl.
7-Bu tylh exah ydr o-1H-azepin -2-im in e, m on oh ydr och lo-
r id e (8): synthesized by general procedure B and its imino-
1
ether by general procedure C; H NMR (400 MHz, CD3OD) δ
3.60 (m, 1H), 2.79 (ddd, 1H, J ) 14.5, 12.4, 1.9 Hz), 2.61 (ddt,
1H, J ) 14.6, 6.7, 1.5 Hz), 2.06-1.95 (m, 2H), 1.86 (m, 1H),
1.76-1.32 (m, 9H), 0.95 (t, 3H, J ) 7 Hz); 13C NMR (CD3OD)
δ 173.9, 57.4, 36.0, 35.0, 32.4, 29.7, 29.1, 24.5, 23.5, 14.2;
HRMS (M+) C10H20N2 requires m/e 168.163, found m/e 168.163.
Anal. (C10H20N2‚1.0HCl‚0.4‚H2O‚0.05NH4Cl) C, H, N, Cl.
7-(2-Eth ylbu tyl)h exa h yd r o-1H-a zep in -2-im in e, m on o-
h yd r och lor id e (9): synthesized by general procedure B and
its iminoether by general procedure C; 1H NMR (400 MHz,
CD3OD) δ 3.66 (m, 1H), 2.82 (ddd, 1H, J ) 15.5, 12.5, 2.0 Hz),
2.62 (ddt, 1H, J ) 15.5, 6.5, 1.5 Hz), 2.06-1.95 (m, 2H), 1.86
(dt, 1H, J ) 15.5, 4.0 Hz), 1.78-1.28 (m, 10H), 0.91 (t, 3H, J
) 7.0 Hz), 0.89 (t, 3H, J ) 7.0 Hz); 13C NMR (CD3OD) δ 173.9,
55.1, 40.2, 37.8, 35.4, 32.4, 29.6, 26.4, 25.4, 24.5, 10.8, 10.6;
HRMS (EI) calcd for C12H24N2 m/e 196.194, found m/e 196.194.
Anal. (C12H24N2‚1.0HCl‚0.6H2O) C, H, N, Cl.
Gen er a l P r oced u r e: Am id in iu m Ch lor id e Syn th esis.
An equivalent of imino ether and NH4Cl (0.9 equiv) were
refluxed in MeOH (10 mL/mmol of iminoether) under a
nitrogen atmosphere from 3.5 to 24 h. After the reaction cooled
to room temperature, it was filtered, stripped of all solvent
under reduced pressure, and partitioned between water and
EtOAc. The organic and aqueous phases were separated, and
the aqueous phase was washed with another portion of EtOAc
before it was lyophilized to provide the product amidinium
salts in 44-93% yield. Each of the amidinium salts possessed
Hexa h yd r o-7-p h en yl-1H-a zep in -2-im in e, m on oh yd r o-
ch lor id e (10): synthesized by general procedure B and its
iminoether by general procedure C; 1H NMR (CD3OD) δ 7.50-
7.30 (m, 5H), 2.99 (ddd, 1H, J ) 15, 12, 2 Hz), 2.74 (dd, 1H, J
) 15, 7 Hz), 2.12-1.76 (m, 6H), 1.60 (m, 1H); 13C NMR (CD3-
OD) δ 172.1, 140.0, 129.0, 128.7, 128.1, 127.4, 126.3, 59.5, 35.0,
31.0, 28.2, 22.7; HRMS (EI) calcd for C12H16N2 m/e 188.131,
found m/e 188.131. Anal. (C12H16N2‚HCl‚0.5H2O) C, H, N, Cl
(N is off by 0.52).
a characteristic IR absorption between 1600 and 1733 cm-1
.
Compound 1 was prepared as previously described.19
3-Eth ylh exah ydr o-1H-azepin -2-im in e, m on oh ydr och lo-
r id e (2): synthesized by general procedure A and its imino-
1
ether by general procedure C; H NMR (400 MHz, CD3OD) δ
3.52-3.40 (m, 2H), 2.75 (m, 1H), 1.95-1.60 (m, 8H), 1.04 (t,
3H, J ) 7 Hz); 13C NMR (CD3OD) δ 176.3, 45.0, 44.3, 29.4,
28.9, 28.8, 23.1, 12.0; HRMS (EI) calcd for C8H16N2 m/e
140.131, found m/e 140.132. Anal. (C8H16N2‚HCl‚0.2H2O‚0.01-
NH4Cl) C, H, N, Cl (H is off by 0.74).
Ack n ow led gm en t. We wish to thank Dr. Ann
Hallinan for helpful discussions; Ms. Dorothy S. Honda,
Mr. Randy J . Fronek, and Ms. Sharon H. Kinder for
chromatographic purifications; and Ms. Patricia M.
Finnegan for aid in interpreting NMR spectra.
Hexa h yd r o-3-(2-p r op en yl)-1H-a zep in -2-im in e, m on o-
h yd r och lor id e (3): synthesized by general procedure B and
its iminoether by general procedure C; 1H NMR (400 MHz,
CD3OD) δ 5.80 (m, 1H), 5.25-5.13 (m, 2H), 3.53-3.45 (m, 2H),
2.95 (m, 1H), 2.60 (m, 1H), 2.38 (m, 1H), 2.0-1.6 (m, 6H); 13
C
Su p p or tin g In for m a tion Ava ila ble: Standard deviation
values for the compounds in Table 1 (1 page). Ordering
information is given on any current masthead page.
NMR (D2O) δ 177.0, 137.3, 120.8, 46.1, 43.9, 35.7, 34.4, 30.8,
29.9; HRMS (EI) calcd for C9H16N2 m/e 152.131, found m/e
152.132. Anal. (C9H16N2‚HCl‚0.4H2O) C, H, N, Cl.
Refer en ces
Hexa h yd r o-5-m eth yl-1H-a zep in -2-im in e, m on oh yd r o-
ch lor id e (4): synthesized by general procedure A and its
iminoether by general procedure D; 1H NMR (400 MHz, CD3-
OD) δ 3.50-3.37 (m, 2H), 2.75 (m, 1H), 2.63 (m, 1H), 1.95 (m,
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