2-Iminopyrrolidines as potent and selective inhibitors of human inducible nitric oxide synthase

Article abstract of DOI:10.1021/jm970840x

A series of substituted 2-iminopyrrolidines has been prepared and shown to be potent and selective inhibitors of the human inducible nitric oxide synthase (hiNOS) isoform versus the human endothelial nitric oxide synthase (heNOS) and the human neuronal ni

Full text of DOI:10.1021/jm970840x

J . Med. Chem. 1998, 41, 3675-3683
3675
2-Im in op yr r olid in es a s P oten t a n d Selective In h ibitor s of Hu m a n In d u cible
Nitr ic Oxid e Syn th a se|
Timothy J . Hagen,*,† Arija A. Bergmanis,† Steven W. Kramer,† Kam F. Fok,† Albert E. Schmelzer,†
Barnett S. Pitzele,† Lydia Swenton,§ Gina M. J erome,‡ Christine M. Kornmeier,‡ William M. Moore,‡
Linda F. Branson,‡ J ane R. Connor,‡ Pamela T. Manning,‡ Mark G. Currie,‡ and E. Ann Hallinan†
Discovery Medicinal Chemistry, Searle, Monsanto, 4901 Searle Parkway, Skokie, Illinois 60077, Discovery Pharmacology,
Searle, Monsanto, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167, and Physical Methodology, Searle, Monsanto,
4901 Searle Parkway, Skokie, Illinois 60077
Received December 15, 1997
A series of substituted 2-iminopyrrolidines has been prepared and shown to be potent and
selective inhibitors of the human inducible nitric oxide synthase (hiNOS) isoform versus the
human endothelial nitric oxide synthase (heNOS) and the human neuronal nitric oxide synthase
(hnNOS). Simple substitutions at the 3-, 4-, or 5-position afforded more potent analogues than
the parent 2-iminopyrrolidine 1. The effect of ring substitutions on both potency and selectivity
for the different NOS isoforms is described. Substitution at the 4- and 5-positions of the
2-iminopyrrolidine yielded both potent and selective inhibitors of hiNOS. In particular, (+)-
cis-4-methyl-5-pentylpyrrolidin-2-imine, monohydrochloride (20), displayed potent inhibition
of hiNOS (IC50 ) 0.25 µM) and selectivities of 897 (heNOS IC50/hiNOS IC50) and 13 (hnNOS
IC50/hiNOS IC50). Example 20 was shown to be an efficacious inhibitor of NO production in
the mouse endotoxin assay. Furthermore, 20 displayed in vivo selectivity, versus heNOS
isoform, by not elevating blood pressure at multiples of the effective dose in the mouse.
In tr od u ction
Nitric oxide (NO) is an endogenously produced inor-
ganic free radical that has been implicated in a number
of biological actions. Nitric oxide synthase (NOS)
F igu r e 1. Numbering system for 2-iminopyrrolidines.
catalyzes the oxidation of arginine to citrulline and
nitric oxide. There have been several recent reviews
on the subject of NOS and the biological activity of
NO.1-8 There are three isoforms of NOS: type I or
nNOS (neuronal), type II or iNOS (inducible), and type
III or eNOS (endothelial). Both the endothelial and
neuronal isoforms are constitutive. The endothelial
isoform is responsible for blood pressure regulation and
platelet aggregation. The induced isoform is found
predominately in activated macrophages as well as
other cell types in which nitric oxide plays a role in host
defense mechanisms. Selective inhibitors of the induced
isoform of NOS are postulated to be useful in the
treatment of numerous disease states that are mediated
by the overproduction of NO.1-8
It was recently reported that 2-iminoazaheterocycles
are potent inhibitors of human NOS.9 The ring sizes
ranged from five- to nine-membered rings, with the most
potent compounds being iminopiperidine and imino-
homopiperidine, 1.0 and 2.0 µM for hiNOS inhibition,
respectively. The he/hi selectivity10 was reported to be
8 and the hn/hi selectivity10 was reported to be 1.8 for
the iminohomopiperidine ring. The parent iminopyr-
rolidine ring was a modest (26 µM) inhibitor of hiNOS
with the he/hi selectivity being 3.6 and the hn/hi
selectivity being 0.8. It seemed apparent that the
potency and selectivity, as the ring size progressed from
five to seven, depended on increasing steric bulk. If the
iminopyrrolidine ring was used as a scaffold, functional-
ity could be directed into the desired spatial and steric
regions, realizing potent and selective iminopyrrolidine
inhibitors of the isoforms of human NOS.
Ch em istr y
Several synthetic methods were used to synthesize
the iminopyrrolidine rings. Most analogues were syn-
thesized from the corresponding lactams (Scheme 3).
There are numerous synthetic routes to form pyrrolidin-
2-ones. The methods used in this paper can be divided
into two categories: the radical ring cyclization of Itoh
that produced predominately the trans isomers11 and
reduction/cyclization of a γ-nitro ester. The γ-nitro
esters could be readily prepared from the Michael
addition of a nitro alkyl derivative and an acrylate. The
routes are represented in Schemes 1 and 2.
| Abbreviations: NO, nitric oxide; NOS, nitric oxide synthase;
hiNOS, human inducible NOS; heNOS, human endothelial NOS;
hnNOS, human neuronal NOS; LPS, lipopolysaccharide; L-NMA, NG-
monomethyl-L-arginine; L-NNA, NG-nitro-L-arginine.
* Author for correspondence. Tel: (847) 982-4778. Fax: (847) 982-
4714.
The disubstituted lactams prepared according to
Scheme 1 were predominately the trans diastereomers,
while the lactams prepared according to the method
described in Scheme 2 were nearly equimolar mixtures
of cis and trans diastereomers. In most cases, the cis
and trans diastereomers were separated by column
chromatography. The lactams were converted to the
† Discovery Medicinal Chemistry.
‡ Discovery Pharmacology.
§ Physical Methodology.
S0022-2623(97)00840-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/19/1998

3676 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hagen et al.
Ta ble 1. Comparison of IC50 Values for Inhibition of Human
NOS Isoforms of Monosubstituted Iminopyrrolidines
Sch em e 1
IC50 (µM)
SEL
compd
R
hiNOS heNOS hnNOS heNOSa hnNOSb
1
2
3
4
none
3-Me
4-Me
5-Me
4-Et
26
93
9.1
14.2
24.9
29.8
5.9
20
3.0
4.7
9.8
5.46
3.65
0.81
11
2.44
8.91
0.4
0.78
0.26
4
0.96
1.6
11.3
1.4
10.2
3.4
14
5
Sch em e 2
L-NMA9
L-NNA9
10
0.5
0.7
0.066
7.6
0.5
0.066
a
b
heNOS selectivity ) IC50 heNOS/IC50 hiNOS. hnNOS selec-
tivity ) IC50 hnNOS/IC50 hiNOS.
while the 3- or 5-methyl compounds 2 and 4 (IC50’s )
7.0 and 8.8 µM, respectively) were more potent than the
parent compound 1 (IC50 ) 26 µM). The 4-ethyl
compound 5 was also more potent at inhibiting hiNOS
(3.35 µM) than 1 but less potent than the 4-methyl
analogue 3. In addition, IC50 values for two commonly
used inhibitors, NG-monomethyl-L-arginine (L-NMA)
and NG-nitro-L-arginine (L-NNA), are included in Table
1 to facilitate comparison of the data in this paper with
our earlier published work and with other work in the
literature.
Sch em e 3
Disubstitution at the 4- and 5-positions resulted in
very potent inhibitors of hiNOS; the results are sum-
marized in Table 2. The 4-methyl-5-ethyl analogue 6
was the most potent inhibitor of hiNOS, with IC50
)
0.17 µM. Increasing the steric bulk in the 5-position
resulted in compounds that retained potency and in-
creased selectivity as the size of the substituent in the
5-position increased. The heNOS/hiNOS selectivity
progressed from 10 to 153 in the series of trans-
iminopyrrolidines as the substituent increased from
ethyl (6) to n-pentyl (9) and benzyl (11). A similar
increase in selectivity for 7-substituted iminohomopi-
peridines and 6-substituted iminopiperidines has been
reported.13,14
There is a dramatic increase in potency and selectivity
in going from the trans-4-methyl-5-pentyliminopyrroli-
dine 9 to the cis-4-methyl-5-pentyliminopyrrolidine 10,
with hiNOS IC50’s of 0.9 versus 0.36 µM, respectively.
The potency for the cis analogues 10 and 14 is 12-15-
fold greater than the potency of the corresponding trans
analogues 9 and 13. There is a corresponding 14-fold
increase in selectivity for the cis analogues 10 and 14
versus the corresponding trans analogues 9 and 13. The
heNOS/hiNOS selectivity of the trans analogue 9 is 53,
while that of the cis analogue 10 is 750. This trend is
reversed in the 5-benzyliminopyrrolidines; the trans
analogue 11 is both more potent and selective than the
cis analogue 16.
amidine via the iminoether, as illustrated in Scheme 3.
The lactam was reacted with trimethyloxonium tet-
rafluoroborate (Me3O+BF4-) in dichloromethane to form
the iminoether, which was reacted with ammonium
chloride in refluxing methanol to prepare the desired
iminopyrrolidines. The single enantiomers were iso-
lated by chiral HPLC chromatography of an intermedi-
ate, by chromatographic separation of a diastereomeric
ureide,12 or by resolution as a diastereomeric salt.
The relative stereochemistry of the pyrrolidin-2-ones
was determined by NMR spectroscopy. When possible,
comparisons were made to examples or intermediates
from the literature.11 The stereochemistry of the ring
substituents was determined by a comparison of the 13
C
NMR chemical shifts of methyl and methylene groups
attached to the ring in the lactam precursors of 9-11
and 16. In the more crowded cis isomers, the 13C NMR
shifts are 5-6 ppm upfield relative to the corresponding
shifts of the trans isomers. The assignment of the 13C
NMR shifts of the methylenes was determined from
13C-1H heterocorrelation spectra. The stereochemical
assignments of related compounds were based on a
comparison of the coupling patterns and chemical shifts
of the ring methine protons.
The 4-position could also be substituted with a tri-
fluoromethyl group. The potent and most selective
analogues are 14 and 10, with IC50’s of 0.81 and 0.36
µM for hiNOS inhibition and selectivities of 484 he/hi
and 6.7 hn/hi and 671 he/hi and14.8 hn/hi. The results
of the 5-benzyl-substituted iminopyrrolidines are sum-
marized in Table 3. The 5-benzyliminopyrrolidine 15,
which lacks a substituent in the 4-position, does not
Resu lts a n d Discu ssion
Str u ctu r e-Activity Rela tion sh ip s. Substitution
of a simple methyl group at the 3-, 4-, or 5-position of
the iminopyrrolidine ring resulted in increased potency
compared to the parent compound 1; the results are
summarized in Table 1. The 4-methyl analogue 3 was
the most potent inhibitor of hiNOS (IC50 ) 1.4 µM),

2-Iminopyrrolidines as Inhibitors of hiNOS
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3677
Ta ble 2. Comparison of IC50 Values for Inhibition of Human NOS Isoforms of Substitued Iminopyrrolidines
IC50 (µM)
SEL
compd
R4
Me
R5
hiNOS
0.17
heNOS
1.6
hnNOS
0.17
heNOSa
10
hNNOSb
1
6
ethyl
(trans)
propyl
(trans)
butyl
(trans)
pentyl
(trans)
pentyl
(cis)
7
8
Me
0.30
0.66
0.91
0.36
3.0
3.5
0.39
0.85
1.31
5.37
8.29
12
1
Me
23.6
47.5
243
458
36
2
9
Me
53
1
10
11
12
13
14
Me
671
153
ND
32.3
484
15
3
Me
benzyl
(trans)
pentyl
(Me)2
CF3
CF3
64% @
100 µMc
10
17% @
100 µMc
336
46% @
100 µMc
9.05
ND
1
pentyl
(trans)
pentyl
(cis)
0.81
392
5.44
7
a
b
heNOS selectivity ) IC50 heNOS/IC50 hiNOS. hnNOS selectivity ) IC50 hnNOS/IC50 hiNOS. c Percent inhibition of nitrite formation
at indicated concentration. ND, not done.
Ta ble 3. Comparison of IC50 Values for Inhibition of Human NOS Isoforms of Benzyl-Substituted Iminopyrrolidines
IC50 (µM)
SEL
compd
R4
R5
hiNOS
heNOS
hnNOS
heNOSa
ND
hnNOSb
ND
15
H
benzyl
26% @
100 µMc
3
16% @
100 µMc
458
47% @
100 µMc
8.29
11
16
17
18
19
Me
benzyl
(trans)
benzyl
(cis)
benzyl
(trans)
benzyl
(cis)
153
46
3
Me
14
22
74
639
11.2
12.1
38.3
0.8
0.5
0.5
ND
CF3
CF3
(Me)2
1010
2920
45
39
benzyl
12% @
3% @
17% @
ND
100 µMc
100 µMc
100 µMc
a
b
heNOS selectivity ) IC50 heNOS/IC50 hiNOS. hnNOS selectivity ) IC50 hnNOS/IC50 hiNOS. c Percent inhibition of nitrite formation
at indicated concentration. ND, not done.
retain the potency that is observed in the 4,5-disub-
stitued analogues 11 and 16. In addition, the analogues
that have a 4,4-dimethyl substition, 19 and 12, are far
less potent than the corresponding 4-methyl analogues
3, 10, 11, and 16. These results stress the importance
of 4,5-disubstituted iminopyrrrolidines as NOS inhibi-
tors.
Furthermore, the enantiomers of 14 were separated
via chiral HPLC to yield the (-)-isomer 23 and the (+)-
isomer 22. The results obtained for the enantiomers
are summarized in Table 4. The (+)-isomer 22 was both
more potent and more selective; hiNOS IC50 ) 0.23 µM
and selectivity of 916 he/hi and 10.9 hnc/hi. The
enantiomers of 10 were also separated into their single
enantiomers to give the (-)-isomer 21 and the (+)-
isomer 20. Again the most active analogue was the (+)-
isomer 20 with hiNOS IC50 ) 0.25 µM and selectivities
of 897 he/hi and 12.7 hnc/hi. In addition, the single
enantiomer of 20 could be prepared on a large scale via
resolution as its L-tartrate salt as illustrated in Scheme
4. The racemic iminoether was reacted with diammo-
nium L-tartrate in methanol. Selective crystallization
from ethanol yielded 20B, while recrystallization from
water yielded the other diasteromeric salt 21B.
In Vivo Resu lts. Compound 20 is a selective, potent
inhibitor of iNOS in vivo. It inhibits LPS-induced
plasma nitrite/nitrate 75% at 10 mg/kg and has an ED50
of approximately 3 mg/kg following oral administration
in the mouse low-endotoxin assay. Following iv admin-
istration of up to 10 mg/kg, it had no effect on mean
arterial pressure in the conscious, restrained mouse
(Figure 2).
Con clu sion s
The synthesis of substituted iminopyrrolidines has led
to potent inhibitors of the human inducible isoform of
NOS. The most potent analogue is 6 with an IC50 of
0.17 µM. Analogues in this series also possess potency

3678 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hagen et al.
Ta ble 4. Comparison of IC50 Values for Inhibition of Human NOS Isoforms of Enantiomers of Selected Substituted
Iminopyrrolidines
IC50 (µM)
heNOS
226
SEL
compd
R4
R5
hiNOS
0.25
hnNOS
3.2
heNOSa
897
hnNOSb
13
(+)-isomer 20
Me
pentyl
(cis)
(-)-isomer 21
(+)-isomer 22
(-)-isomer 23
Me
pentyl
(cis)
pentyl
(cis)
pentyl
(cis)
4.5
578
212
20.6
2.53
45.7
130
916
226
5
11
6
CF3
CF3
0.23
7.5
1680
a
b
heNOS selectivity ) IC50 heNOS/IC50 hiNOS. hnNOS selectivity ) IC50 hnNOS/IC50 hiNOS.
Exp er im en ta l Section
Chemicals and reagents were purchased from Aldrich
Chemical Co. (Milwaukee, WI) or Sigma (St. Louis, MO) unless
otherwise noted. L-[2,3-3H]arginine was purchased from Du-
Pont NEN (Boston, MA); (6R)-tetrahydro-L-biopterin was from
Research Biochemicals, Inc. (Natick, MA); frozen rat cerebella
were from Pel-Freez (Rogers, AR). Purification of synthesized
compounds was performed using a Delta Pak C-18 column or
crystallization. 1H NMR spectra were obtained at 300 or 400
MHz on a QE300 or Varian VXR-400 spectrometer in D2O,
DMSO-d6, or CDCl3 (δ units). 13C NMR spectra were obtained
at 125 MHz on a Varian VXR-500 spectrometer in D2O, DMSO-
d6, or CDCl3. Mass spectra were obtained on either a VG
model 250 or a Finnigan MAT 90 spectrometer. Elemental
analyses were performed on a Carlo Erba model 1106 elemen-
tal analyzer. Male Lewis rats (150-200 g) were purchased
from Harlan Sprague-Dawley (Indianapolis, IN) and were
housed and cared for in accordance with the guidelines of the
Institutional Animal Care and Use Committee and in ac-
cordance with NIH guidelines on laboratory animal welfare.
Escherichia coli lipopolysaccharide (serotype 0111:B4, West-
phal extract) (LPS, endotoxin) was purchased from Sigma
Chemical Co. (St. Louis, MO). Ultrafree-MC filter units were
from Millipore (Bedford, MA).
F igu r e 2. In vivo selectivity of 20 in the mouse.
Sch em e 4
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.14,16 Human inducible NOS (hiNOS), human endo-
thelial constitutive NOS (heNOS), and human neuronal
constitutive NOS (hnNOS) were each cloned from RNA
extracted from human tissue. The cDNA for hiNOS was
isolated from a λcDNA library made from RNA extracted from
a colon sample from a patient with ulcerative colitis; heNOS
was isolated from a λcDNA library made from RNA extracted
from human umbilical vein endothelial cells (HUVEC); and
hnNOS 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. Enzyme activity was isolated from
soluble cell extracts and partially purified by DEAE-Sepharose
chromatography.14 The Km values for L-arginine for hiNOS,
heNOS, and hnNOS were 7, 4, and 6 µM, respectively.9 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 reaction 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 mCi of L-[2,3-
3H]arginine. For constitutive NOS, calmodulin was included
at a final concentration of 40 nM. Following incubation 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
and selectivity for hiNOS. The 4-methyl-5-pentyl ana-
logue 10 has a selectivity of 671 versus heNOS and 15
versus hnNOS, while retaining potency for hiNOS with
an IC50 ) 0.36 µM. The relative stereochemistry is
important, with the cis diastereomers generally being
more selective and potent than the trans diastereomers.
The absolute stereochemistry is critical with the (+)-
isomer (20) being more active and selective than the (-)-
isomer (21). The analogues 20 and 22 are highly potent
and selective inhibitors of hiNOS, with IC50’s of 0.25 and
0.23 µM, selectivities versus heNOS greater than 800,
and selectivities versus hnNOS greater than 10. Fur-
thermore, 20 is potent in vivo in mouse models of nitric
oxide synthase inhibition and exhibits marked selectiv-
ity as demonstrated by the lack of an elevation in blood
pressure. The use of 20 and other analogues will be
pivotal in determining the effectiveness of potent and
selective inhibitors of hiNOS in human diseases in
which hiNOS is theorized to play a role.

2-Iminopyrrolidines as Inhibitors of hiNOS
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3679
scintillation counter. All assays were performed at least in
duplicate; standard deviations were 10% or less. Production
of [3H]citrulline was linear with time over the course of the
assay.
500 mg (70%, from the lactam). HRMS: m/z M+ 98.0844;
C5H10N2 requires 98.0844. 1H NMR (D2O, 400 MHz): 1.13 (d,
J ) 7 Hz, 3H), 2.53 (dd, 1H), 2.71 (m, 1H), 3.03 (dd, 1H), 3.28
(dd, 1H), 3.80 (dd, 1H). Anal. (C5H10N2‚HCl‚ 0.25H2O‚0.75NH4-
Cl) C, H, N, Cl.
In Vivo Selectivity. Male BALB/c mice (BALB/cAnHsd,
Harlan Sprague-Dawley, Inc., Indianapolis, IN), weighing
28-32 g, were anesthetized with metofane (Mallinckrodt
Veterinary Inc., Mundelein, IL), and the femoral artery and
vein were cannulated using PE-10 intramedic polyethylene
tubing (Becton Dickinson and Co., Sparks, MD). The mice
were allowed to recover from the anesthesia and restrained.
The arterial cannula was connected to a Cobe disposable
pressure transducer (Cobe Laboratories, Inc., Lakewood, CO),
and hemodynamic parameters (i.e., mean arterial, systolic, and
diastolic pressures, as well as heart rate) were measured using
a Gould transducer signal conditioner (model 13-6615-50) and
a Gould TA recording system (Gould Instrument Systems Inc.,
Valley View, OH). Baseline data were collected for 30 min
prior to administration of iNOS inhibitors dissolved in PBS,
administered as iv bolus injections. For 5 h following the oral
dose hemodynamic parameters were recorded. The iv bolus
injections were given at regular intervals through the venous
cannula such that doses were cumulative. Parameters were
recorded during the injections and for a period of time following
the last dose (∼20 min). The dose of compound resulting in a
>10% change from baseline mean arterial pressure was
considered to be a nonselective effect of the compound on eNOS
in the conscious BALB/c mouse.
5-Meth ylp yr r olid in -2-im in e Hyd r och lor id e (4). The
title compound was prepared according to literature method.18
HRMS: m/z M+ 98.0844; C5H10N2 requires 98.0844. 1H NMR
(D2O, 300 MHz): 1.13 (d, J ) 7 Hz, 3H), 1.63 (m, 1H), 2.22
(m, 1H), 2.69 (m, 1H), 2.77 (m, 1H), 3.97 (hextet, 1H).
4-Eth yl-4-pyr r olidin -2-im in e Hydr och lor ide (5). 4-Ethyl-
4-pyrrolidin-2-one was prepared from 2-buten-1-ol and trichlo-
roacetonitrile10 in a manner similar to the method described
for the lactam of 3. A solution of 3-buten-2-ol (8.7 mL, 100
mmol) in THF (50 mL) was added to potassium hydride (KH;
600 mg, 15 mmol) over 15 min. The resulting alkoxide solution
was added to a stirred solution of trichloroacetonitrile (10 mL,
100 mmol) in ether (100 mL) at -10 °C. The solution was
stirred at 0 °C for 3 h, followed by removal of solvent under
reduced pressure (temperature < 25 °C); pentane (400 mL)
and methanol (1 mL) were added, and the mixture was filtered.
Concentration afforded a yellow oil (17.4 g). The oil was
dissolved in xylene (450 mL) and refluxed for 2.5 h. The
solvent was removed to yield 16.8 g of N-(2-butenyl)-2,2,2-
trichloroacetamide as a white solid. The N-(2-butenyl)-2,2,2-
trichloroacetamide was converted to 4-ethylpyrrolidin-2-one by
the method described for the lactam of 3. 1H NMR (CDCl3,
300 MHz): 0.93 (t, J ) 7 Hz, 3H), 1.50 (p, J ) 7 Hz, 2H), 2.02
(m, 1H), 2.31-2.50 (m, 2H), 3.03 (dd, J ) 9, 7 Hz, 1H), 3.50
(dd, J ) 9, 8 Hz, 1H), 5.84 (br s, 1H).
4-Ethylpyrrolidin-2-one (750 mg, 6.6 mmol) was reacted
with trimethyloxonium tetrafluoroborate (1.18 g, 8 mmol)
according to general procedure A to yield, after chromatogra-
phy, the iminoether. The iminoether was reacted with am-
monium chloride (394 mg, 16 mmol) according to general
procedure B to yield the title material 5, 535 mg (64%, from
the lactam). 1H NMR (CDCl3, 300 MHz): 0.95 (t, J ) 7.5 Hz,
3H), 1.53 (pentet, J ) 7.5 Hz, 2H), 2.49 (heptet, J ) 7.5 Hz,
1H), 2.60 (dd, J ) 18, 7.5 Hz, 1H), 3.12 (dd, J ) 18, 8 Hz, 1H),
3.28 (dd, J ) 11, 7 Hz, 1H), 3.75 (dd, J ) 11, 8 Hz, 1H), 8.78
(br s, 1H), 9.36 (br s, 2H). Anal. (C6H12N2‚HCl‚0.8H2O) C, H,
N, Cl.
tr a n s-5-Eth yl-4-m eth ylp yr r olid in -2-im in e Hyd r och lo-
r id e (6). 5-Ethyl-4-methylpyrrolidin-2-one was prepared from
cis-2-penten-1-ol and trichloroacetonitrile10 in a manner simi-
lar to the method described for lactam 3. The 5-ethyl-4-
methylpyrrolidin-2-one was flash chromatographed (EA) and
then recrystallized (n-hexane). 1H NMR (CDCl3, 300 MHz):
0.97 (t, J ) 7 Hz, 3H), 1.13 (d, J ) 7 Hz, 3H), 1.45 (m, 1H),
1.62 (m, 1H), 1.99 (dd, J ) 17, 8 Hz, 1H), 2.11 (m, 1H), 2.53
(dd, J ) 17, 8 Hz, 1H), 3.12 (dt, J ) 8, 6 Hz, 1H), 6.23 (br s,
1H).
5-Ethyl-4-methylpyrrolidin-2-one (625 mg, 5 mmol) was
reacted with trimethyloxonium tetrafluoroborate (800 mg, 5.5
mmol) according to general procedure A to yield, after chro-
matography, 704 mg of the iminoether. The iminoether (704
mg, 5 mmol) was reacted with ammonium chloride (279 mg,
5.6 mmol) according to general procedure B to yield the title
material 6, 560 mg (59%, from the lactam). 1H NMR (CDCl3,
300 MHz): 1.02 (t, J ) 7 Hz, 3H), 1.18 (d, J ) 7 Hz, 3H), 1.57
(m, 1H), 1.70 (m, 1H), 2.22 (heptet, J ) 7 Hz, 1H), 2.57 (dd, J
) 18, 7.5 Hz, 1H), 3.21 (dd, J ) 18, 8 Hz, 1H), 3.42 (q, J ) 7
Hz, 1H), 8.51 (br s, 1H), 9.53 (br s, 1H), 9.76 (br s, 1H). Anal.
(C7H14N2‚HCl‚H2O) C, H, N, Cl.
Low -En d otoxin Assa y. Male BALB/c mice were treated
with an intraperitoneal injection of 12.5 mg/kg endotoxin (LPS)
to induce systemic expression of inducible nitric oxide syn-
thase, resulting in markedly elevated plasma nitrite/nitrate
levels. Compounds were administered orally by gavage 30 min
prior to LPS administration, and plasma nitrite/nitrate levels
were determined 5 h following LPS administration using a
fluorescence assay.16 Data are expressed as the percent
inhibition of the induced nitrite/nitrate production.
Gen er a l P r oced u r e A: Syn th esis of Im in oeth er s fr om
La cta m s. To a stirred solution of the lactam (1 equiv) in CH2-
Cl2 was added trimethyloxonium tetrafluoroborate (Me3O+BF4
)
-
(1.2 equiv, Lancaster) under argon. The mixture was stirred
for 12 h, diluted with CH2Cl2, extracted with KHCO3 or
NaHCO3 and brine, dried (Na2SO4), filtered, and stripped of
all solvent to provide the crude iminoether.
Gen er a l P r oced u r e B: Syn th esis of Im in op yr r olid in es
fr om Im in oeth er s. To a stirred solution of the iminoether
in MeOH was added ammonium chloride (NH4Cl), and the
resulting mixture was refluxed for 4-12 h. After cooling to
room temperature the solution was filtered and stripped of all
solvent to provide the crude iminopyrrolidine hydrochloride.
The material was dissolved in water and extracted with
EtOAc, and the aqueous phase was chromatographed via
RPHPLC eluting with ACN/H2O (gradient). The iminopyrro-
lidine product was lyophilized.
2-Im in op yr r olid in e Hyd r och lor id e (1). The title com-
pound was prepared according to literature method.18 Anal.
(C4H8N2‚HCl‚0.25H2O) C, H, N.
3-Meth ylp yr r olid in -2-im in e Hyd r och lor id e (2). The
title compound was prepared according to literature method.18
1H NMR (D2O, 300 MHz): 1.15 (d, J ) 7 Hz, 3H), 1.71 (m,
1H), 2.25 (m, 1H), 3.00 (m, 1H), 3.42 (m, 1H), 3.51 (m, 1H).
4-Meth ylp yr r olid in -2-im in e Hyd r och lor id e (3). 4-Me-
thylpyrrolidin-2-one was prepared from 3,3-dichloro-4-(chlo-
romethyl)pyrrolidin-2-one.11 A mixture of 3,3-dichloro-4-
(chloromethyl)pyrrolidin-2-one (3 g, 15 mmol), tributyltin
hydride (14.3 mL), and AIBN (25 mg) was heated at 140 °C
for 8 h. The product was chromatographed to yield 900 mg
(61%) of 4-methylpyrrolidin-2-one as a solid.
The 4-methylpyrrolidin-2-one (500 mg, 5 mmol) was reacted
with trimethyloxonium tetrafluoroborate (890 mg, 6 mmol)
according to general procedure A to yield, after chromatogra-
phy, 610 mg of the iminoether. The iminoether (610 mg, 5
mmol) was reacted with ammonium chloride (200 mg, 4 mmol)
according to general procedure B to yield the title material 3,
tr a n s-5-P r opyl-4-m eth ylpyr r olidin -2-im in e Hydr och lo-
r id e (7). 5-Propyl-4-methylpyrrolidin-2-one was prepared
from trans-2-hexen-1-ol and trichloroacetonitrile11 in a manner
similar to the method described for the lactam in 3. 1H NMR
(CDCl3, 300 MHz): 0.96 (t, J ) 7 Hz, 3H), 1.12 (d, J ) 7 Hz,
3H), 1.23-1.65 (m, 4H), 1.98 (dd, J ) 16, 7.5 Hz, 1H), 2.08
(m, 1H), 2.52 (dd, J ) 16, 8 Hz, 1H), 3.18 (m, 1H), 6.91 (br s,
1H).
5-Propyl-4-methylpyrrolidin-2-one (700 mg, 5 mmol) was
reacted with trimethyloxonium tetrafluoroborate (800 mg, 5.5

3680 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hagen et al.
mmol) according to general procedure A to yield, after chro-
matography, 760 mg of the iminoether. The iminoether (760
mg, 5 mmol) was reacted with ammonium chloride (290 mg,
5.4 mmol) according to general procedure B to yield the title
material 7, 500 mg (57%, from the lactam). 1H NMR (DMSO-
d6, 400 MHz): 0.92 (t, J ) 7 Hz, 3H), 1.07 (d, J ) 7 Hz, 3H),
1.20-1.63 (m, 4H), 2.17 (septet, J ) 7 Hz, 1H), 2.43 (dd, J )
17, 7 Hz, 1H), 2.97 (dd, J ) 17, 9 Hz, 1H), 3.44 (q, J ) 6 Hz,
1H). Anal. (C8H10N2‚0.33H2O‚1.1HCl) C, H, N, Cl.
tr a n s-5-Bu tyl-4-m eth ylp yr r olid in -2-im in e Hyd r och lo-
r id e (8). 5-Butyl-4-methylpyrrolidin-2-one was prepared from
trans-2-hepten-1-ol and trichloroacetonitrile11 in a manner
similar to the method described for the lactam in 3. 1H NMR
(CDCl3, 300 MHz): 0.92 (t, J ) 7 Hz, 3H), 1.13 (d, J ) 7 Hz,
3H), 1.25-1.7 (m, 6H), 1.97 (dd, J ) 16, 7.5 Hz, 1H), 2.08 (m,
1H), 2.52 (dd, J ) 16, 8 Hz, 1H), 3.16 (m, 1H), 7.05 (br s, 1H).
5-Butyl-4-methylpyrrolidin-2-one (1.0 g, 6.43 mmol) was
reacted with trimethyloxonium tetrafluoroborate (1.15 g, 7.78
mmol) according to general procedure A to yield, after chro-
matography, 660 mg of the iminoether. The iminoether (660
mg, 3.9 mmol) was reacted with ammonium chloride (231 mg,
4.3 mmol) according to general procedure B to yield the title
material 8, 432 mg (35%, from the lactam). 1H NMR (DMSO-
d6, 400 MHz): 0.89 (t, J ) 6.7 Hz, 3H), 1.06 (d, J ) 6.8 Hz,
3H), 1.22-1.60 (m, 6H), 2.13 (heptet, J ) 7 Hz, 1H), 2.41 (dd,
J ) 17.5, 7.5 Hz, 1H), 2.98 (dd, J ) 17.5, 8 Hz, 1H), 3.76 (q, J
) 7 Hz, 1H), 9.3 (br s, 3H). 13C NMR (DMSO-d6, 125 MHz):
169.6 (C-2), 66.3 (C-5), 37.2 (C-3), 35.0 (C-4), 33.2 (C-7), 27.4
(C-8), 21.9 (C-9), 17.9 (C-6), 13.7 (C-10). Anal. (C9H18N2‚HCl)
C, H, N.
Hz, 1H), 9.3 (br s, 3H). 13C NMR (DMSO-d6, 125 MHz): 13.7,
17.8 (CH3’s); 21.8, 24.6, 30.9, 33.2, 37.1 (CH2’s); 34.9, 66.4
(CH’s); 169.3 (CdN). Anal. (C10H18N2‚HCl) C, H, N, Cl.
cis-5-P en t yl-4-m et h ylp yr r olid in -2-im in e H yd r och lo-
r id e (10). cis-5-Pentyl-4-methylpyrrolidin-2-one (700 mg, 4.5
mmol) was reacted with trimethyloxonium tetrafluoroborate
(806 mg, 5.5 mmol) according to general procedure A to yield,
after chromatography, 610 mg of the iminoether. The imino-
ether (610 mg, 4.5 mmol) was reacted with ammonium chloride
(200 mg, 4 mmol) according to general procedure B to yield
the title material 10, 441 mg (70%, from the lactam). 1H NMR
(D2O, 400 MHz): 0.88 (distorted t, J ) 7 Hz, 3H), 1.02 (d, J )
7.1 Hz, 3H), 1.3-1.6 (m, 8H), 2.54 (dd, J ) 17.1, 6.8 Hz, 1H),
2.70 (apparent sept., J ) 7 Hz, 1H), 2.97 (dd, J ) 17.2, 7.9
Hz, 1H), 3.92 (dt, J ) 7.9, 6.8 Hz, 1H). 13C NMR (D2O, 125
MHz): 15.8, 16.2, 24.7, 28.0, 31.5, 33.9, 35.7, 40.2, 66.0, 178.3.
Anal. (C10H18N2‚HCl) C, H, N, Cl.
tr a n s-5-Ben zyl-4-m eth ylpyr r olidin -2-im in e Hydr och lo-
r id e (11) a n d cis-5-Ben zyl-4-m eth ylp yr r olid in -2-im in e
Hyd r och lor id e (16). Methyl crotonate (6.4 g, 64 mmol) was
mixed with â-nitroethylbenzene19 (9.7 g, 64 mmol), potassium
carbonate (8.9 g, 64 mmol), and Aliquat 336 (20 drops). The
mixture was sonicated at room temperature. When the
reaction, monitored by GC, was complete, the mixture was
acidified with HCl (1 N) and the aqueous phase extracted with
ether. Purification by chromatography on silica gel yielded
methyl 3-methyl-4-nitrobenzenepentanoate (14.7 g, 91%). The
methyl 3-methyl-4-nitrobenzenepentanoate (5 g, 20 mmol) in
absolute MeOH was hydrogenated over RaNi at 55 °C and 60
psi for 24 h. The reaction product was purified by column
chromatography to yield cis,trans-5-benzyl-4-methylpyrrolidin-
2-one (2.35 g, 62%) as a mixture of diastereomers.
tr a n s-5-P en tyl-4-m eth ylp yr r olid in -2-im in e Hyd r och lo-
r id e (9). cis- and trans-5-pentyl-4-methylpyrrolidin-2-one
The material cis,trans-5-benzyl-4-methylpyrrolidin-2-one
(1.35 g, 7 mmol) was reacted with trimethyloxonium tetrafluo-
roborate according to general procedure A to yield, after
chromatography, 610 mg of the iminoether as a mixture of
diastereomers. The iminoether (610 mg, 5 mmol) was reacted
with ammonium chloride (200 mg, 4 mmol) according to
general procedure B to yield the title materials 11 and 16,
followed by chromatography on reverse-phase HPLC to gener-
ate the cis and trans title materials, 11 (300 mg) and 16 (220
mg).
were prepared from nitrohexane and methyl crotonate.
A
suspension of methyl crotonate (7.6 g, 76 mmol), 1-nitrohexane
(10.0 g, 76 mmol), K2CO3 (10.5 g), and Aliquat 336 (10 drops)
was sonicated for 6 h. To the reaction was added Et2O (200
mL). The reaction mixture was filtered, acidified with 0.5 N
HCl, extracted with brine, dried over Na2SO4 (anhydrous),
filtered, and concentrated under reduced pressure to give 13
g of yellow liquid. The product was purified by column
chromatography to give 6.0 g (34%) of methyl 3-methyl-4-
nitrononanoate.
11: 1H NMR (D2O, 400 MHz) 1.05 (d, J ) 6.7 Hz, 3H), 2.40
(m, 1H), 2.45 (dd, J ) 17.5, 6.4 Hz, 1H), 2.85 (dd, J ) 17.5,
8.2 Hz, 1H), 2.90 (dd, J ) 14.1, 6.8 Hz, 1H), 3.01 (dd, J ) 14.1,
5.7 Hz, 1H), 3.89 (apparent q, J ) 6 Hz, 1H), 7.25-7.5 (m,
1H), 7.32 (complex d, J ) 8 Hz, 2H), 7.35 (complex t, J ) 8
Hz, 1H), 7.42 (complex t, J ) 8 Hz, 2H). Anal. (C10H18N2‚
HCl).
16: DSC 142.08 °C (sharp). 1H NMR (D2O, 400 MHz): 1.17
(d, J ) 7.0 Hz, 3H), 2.54 (dd, J ) 17, 8 Hz, 1H), 2.74 (dd, J )
14, 10 Hz, 1H), 2.81 (septet, J ) 7 Hz, 1H), 2.99 (dd, J ) 14,
3 Hz, 1H), 2.99 (dd, J ) 17, 8 Hz, 1H), 4.25 (m, 1H), 7.29-
7.45 (m, 5H). Anal. (C10H18N2‚HCl) C, H, N, Cl.
5-P en tyl-4,4-d im eth ylp yr r olid in -2-im in e Hyd r och lo-
r id e (12). The ethyl 3,3-dimethylacrylate (4.9 g, 38 mmol)
was mixed with nitrohexane (5.0 g, 38 mmol) and 1 M
tetrabutylammonium fluoride (38 mL) and heated at 40 °C
for 24 h. The reaction mixture was diluted with diethyl ether
and washed with brine, followed by water. Purification by
chromatography on silica gel yielded methyl 3,3-dimethyl-4-
nitrononanoate (6.6 g, 67%).
The methyl 3,3-dimethyl-4-nitrononanoate (5.6 g, 24 mmol)
in absolute MeOH was hydrogenated over RaNi at 55 °C and
60 psi for 24 h. The reaction product was purified by column
chromatography to yield 5-pentyl-4,4-dimethylpyrrolidin-2-one
(2.63 g, 60%).
A solution of methyl 3-methyl-4-nitrononanoate (5.0 g) in
MeOH was reduced under catalytic hydrogenation conditions
(60 psi, 55 °C) using Raney nickel. The reaction was heated
for 16 h to effect cyclization after reduction of the nitro group.
After concentration of the reaction mixture under reduced
pressure, the residue was purified by column chromatography
to give 3.3 g of a light-yellow liquid. A second column was
run to separate the cis- and trans-5-pentyl-4-methylpyrrolidin-
2-one.
tr a n s-5-P en tyl-4-m eth ylp yr r olid in -2-on e: GC tR ) 14.7
min.20 1H NMR (CDCl3, 400 MHz): 0.89 (t, J ) 6 Hz, 3H),
1.11 (d, J ) 7 Hz, 3H), 1.22-1.60 (m, 6H), 1.95 (dd, J ) 16,
7.5 Hz,1H), 2.07 (heptet, J ) 7 Hz,1H), 2.50 (dd, J ) 16, 8
Hz,1H), 3.14 (q, J ) 6 Hz,1H), 6.43 (br s, 1H). 13C NMR
(CDCl3, 125 MHz): 13.9, 19.3, 22.5, 25.9, 31.7, 35.5, 35.6, 38.7,
62.0, 177.5.
cis-5-P en tyl-4-m eth ylp yr r olid in -2-on e: GC tR ) 15.15
min.20 1H NMR (CDCl3, 400 MHz): 0.89 (t, J ) 6 Hz, 3H),
1.00 (d, J ) 7 Hz, 3H), 1.20-1.50 (m, 6H), 1.98 (dd, 1H), 2.54
(heptet, 1H), 2.43 (dd, 1H), 3.55 (m, 1H), 6.37 (br s, 1H). 13C
NMR (CDCl3, 125 MHz): 13.8, 14.4, 22.3, 26.0, 30.2, 31.6, 32.5,
38.5, 57.5, 177.6.
trans-5-Pentyl-4-methylpyrrolidin-2-one (1.0 g, 5.9 mmol)
was reacted with trimethyloxonium tetrafluoroborate (1.05 g,
7.14 mmol) according to general procedure A to yield, after
chromatography, 790 mg of the iminoether. The iminoether
(740 mg, 4 mmol) was reacted with ammonium chloride (238
mg, 4.45 mmol) according to general procedure B to yield the
title material 9, 650 mg (66%, from the lactam). 1H NMR
(DMSO-d6, 400 MHz): 0.88 (t, J ) 6.8 Hz, 3H), 1.06 (d, J )
6.8 Hz, 3H), 1.2-1.6 (m, 8H), 2.13 (heptet, J ) 7 Hz, 1H), 2.41
(dd, J ) 17.5, 7.5 Hz), 2.97 (dd, J ) 17.5, 8.5), 3.39 (q, J ) 8
The 5-pentyl-4,4-dimethylpyrrolidin-2-one (2.6 g, 14.3 mmol)
was reacted with trimethyloxonium tetrafluoroborate accord-
ing to general procedure A to yield, after chromatography, 2.0
g of the iminoether. The iminoether (2.0 g, 10 mmol) was
reacted with ammonium chloride (529 mg, 10 mmol) according
to general procedure B to yield 12 (1.67 g). 1H NMR (D2O,
400 MHz): 0.88 (distorted t, J ) 7 Hz, 3H), 1.04 (s, 3H), 1.17

2-Iminopyrrolidines as Inhibitors of hiNOS
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3681
(s, 3H), 1.27-1.63 (m, 8H), 2.64 (d, J ) 17.3 Hz, 1H), 2.70 (d,
J ) 17.3 Hz, 1H), 3.57 (dd, J ) 9.4, 4.0 Hz, 1H). 13C NMR
(D2O, 125 MHz): 16.2, 23.8, 28.6 (CH3’s); 24.7, 28.6, 31.8, 33.8,
46.9 (CH2’s); 42.5 (C), 71.8 (CH), 172.7 (CdNH). Anal.
(C11H20N2‚HCl) C, H, N, Cl.
tr a n s-5-P en tyl-4-(tr iflu or om eth yl)p yr r olid in -2-im in e
Hyd r och lor id e (13). A suspension of ethyl 4,4,4-trifluoro-
crotonate (10.0 g, 59 mmol), 1-nitrohexane (7.86 g, 60 mmol),
K2CO3 (4.1 g), and Aliquat 336 (6 drops) was sonicated for 5
h. To the reaction was added Et2O (200 mL). The reaction
mixture was filtered, extracted with brine, dried over Na2SO4
(anhydrous), filtered, and concentrated under reduced pressure
to give a yellow liquid. The product was purified by column
chromatography to give 13.8 g (77%) of methyl 3-(trifluorom-
ethyl)-4-nitrononanoate.
A solution of methyl 3-(trifluoromethyl)-4-nitrononanoate
(13.0 g) in MeOH was reduced under catalytic hydrogenation
conditions (60 psi, 55 °C) using Raney nickel. The reaction
was heated for 8 h to effect cyclization after reduction of the
nitro group. After concentration of the reaction mixture under
reduced pressure, the residue was purified by column chro-
matography to give 9.0 g of a light-yellow liquid. A second
column was run to separate the cis- and trans-5-pentyl-4-
(trifluoromethyl)pyrrolidin-2-one.
tr a n s-5-P en tyl-4-(tr iflu or om eth yl)p yr r olid in -2-on e: Rf
) 0.5 (silica gel; EA). 1H NMR (CDCl3, 400 MHz): 0.90 (t, J
) 6 Hz, 3H), 1.22-1.41 (m, 6H), 1.53 (m, 1H), 1.65 (m, 1H),
2.49 (dd, J ) 17.5, 6.1 Hz, 1H), 2.62 (dd, J ) 17.5, 10.0 Hz,
1H), 2.75 (dqdd, J ) 10.0, 8.8, 6.1, 4.4 Hz, 1H), 3.75 (dt, J )
7.5, 4.4 Hz, 1H) 6.50 (br s, 1H). 13C NMR (CDCl3, 125 MHz,
was hydrogenated over Pd/C (4%) at 55 °C and 5 psi for 40 h.
The reaction product was purified by column chromatography
to yield 5-benzylpyrrolidin-2-one (0.61 g, 29%).
The 5-benzylpyrrolidin-2-one (0.54 g, 3.1 mmol) was reacted
with trimethyloxonium tetrafluoroborate (550 mg, 3.7 mmol)
according to general procedure A to yield, after chromatogra-
phy, 460 mg of the iminoether. The iminoether (460 mg, 2.4
mmol) was reacted with ammonium chloride (144 mg, 2.7
mmol) according to general procedure B to yield the title
material 15, 310 mg (48%, from the lactam).
1H NMR (D2O,
400 MHz): 1.96 (dddd, J ) 13.2, 9.6, 6.3, 5.1 Hz, 1H), 2.29
(dddd, J ) 13.2, 10.0, 7.9, 6.4 Hz, 1H), 2.63 (ddd, J ) 17.9,
9.6, 6.4 Hz, 1H), 2.77 (ddd, J ) 17.9, 10.0, 6.3 Hz, 1H), 2.91
(dd, J ) 13.9, 6.5 Hz, 1H), 2.95 (dd, J ) 13.9, 6.2 Hz, 1H),
4.32 (dq, J ) 8, 6 Hz, 1H), 7.31 (complex d, J ) 7.5 Hz, 2H),
7.36 (tt, J ) 7, 2 Hz, 1H), 7.42 (complex t, J ) 7.5 Hz, 2H). 13
C
NMR (D2O, 125 MHz): 28.4, 32.5, 43.0 (CH2’s); 64.3 (CH);
129.9, 131.7, 132.5 (dCH’s); 139.9 (dC); 174 (CdN). Anal.
(C11H14N2‚HCl) C, H, N, Cl.
t r a n s-5-Be n zyl-4-(t r iflu or om e t h yl)p yr r olid in -2-im -
in e Hyd r och lor id e (17) a n d cis-5-Ben zyl-4-(tr iflu or om -
eth yl)p yr r olid in -2-im in e Hyd r och lor id e (18). The ethyl
4,4,4-(trifluoromethyl)crotonate (5.5 g, 33 mmol) and â-nitro-
ethylbenzene19 (5.0 g, 33 mmol) were reacted with potassium
carbonate (4.6 g, 33 mmol) and Aliquat 336 (10 drops), in a
manner similar to the method described in 11. Purification
by chromatography on silica gel yielded the methyl 3-(trifluo-
romethyl)-4-nitrobenzenepentanoate (4.4 g, 42%).
The methyl 3-(trifluoromethyl)-4-nitrobenzenepentanoate
(4.3 g, 13.5 mmol) in absolute MeOH was hydrogenated over
RaNi at 55 °C and 60 psi for 16 h. The reaction product was
purified by column chromatography to yield cis,trans-5-benzyl-
4-(trifluoromethyl)pyrrolidin-2-one (2.33 g, 71%) as a mixture
of diastereomers.
HETCOR): 13.8 (C-11), 22.3 (C-10), 25.0 (C-8), 30.3 (q, J CF
)
3 Hz, C-3), 31.3 (C-9), 36.7 (C-7), 43.6 (q, J CF ) 29 Hz, C-4),
54.2 (C-5), 126.7 (q, J CF ) 277 Hz, C-6), 174.8 (C-2). Anal.
(C10H16NOF3‚0.1EA) C, H, N.
The cis,trans-5-benzyl-4-(trifluoromethyl)pyrrolidin-2-one
(0.74 g, 3 mmol) was treated with trimethyloxonium tetrafluo-
roborate (0.54 g, 3.7 mmol) in DCM (20 mL) by general method
A, to yield the iminoether (0.58 g, 76%) as a mixture of
diastereomers. A solution of the iminoethers (0.58 g, 2.3 mmol)
in MeOH (20 mL) was reacted with ammonium chloride (134
mg, 2.3 mmol) by general method B, followed by chromatog-
raphy on reverse-phase HPLC to generate the cis and trans
title materials, trans-5-benzyl-4-(trifluoromethyl)pyrrolidin-2-
imine (240 mg) and cis-5-benzyl-4-(trifluoromethyl)pyrrolidin-
2-imine (250 mg).
tr a n s-5-Ben zyl-4-(t r iflu or om et h yl)p yr r olid in -2-im i-
n e (17): 1H NMR (D2O, 400 MHz) 2.93 (dd, J ) 18.6, 9.6 Hz,
1H), 3.00 (dd, J ) 14.0, 6.6 Hz, 1H), 3.02 (dd, J ) 18.6, 4.5
Hz, 1H), 3.12 (dd, J ) 14.0, 5.3 Hz, 1H), 3.28 (m, 1H), 4.58
(m, 1H), 7.32 (complex d, 2H), 7.38 (complex t, 1H), 7.43
(complex t, 2H). 13C NMR (D2O, 125 MHz): 32.8, 42.4, 45.0
(q, J CF ) 29 Hz), 63.9, 129.1 (q, J CF ) 277 Hz), 130.4, 131.9,
132.7, 138.2, 171.3. Anal. (C12H13N2F3‚1HCl‚1.1NH4Cl‚
0.67H2O) C, H, N.
cis-5-P en tyl-4-(tr iflu or om eth yl)p yr r olid in -2-on e: Rf )
0.4 (silica gel; EA). 1H NMR (CDCl3, 400 MHz): 0.90 (t, J )
6, 3H), 1.22-1.45 (m, 6H), 1.54 (m, 1H), 1.66 (m, 1H), 2.50
(dd, J ) 17.0, 9.1 Hz, 1H), 2.57 (dd, J ) 17.0, 9.1 Hz, 1H),
3.20 (qtd, J ) 9.4, 9.1, 7.6 Hz, 1H), 3.79 (ddd, J ) 10.7, 7.6,
3.4 Hz, 1H) 6.48 (br s, 1H). 13C NMR (CDCl3, 125 MHz,
HETCOR): 13.9 (C-11), 22.4 (C-10), 26.2 (C-8), 30.2 (q, J CF
2 Hz, C-3), 30.7 (q, J CF ) 2 Hz, C-7), 31.5 (C-9), 42.1 (q, J CF
)
)
29 Hz, C-4), 54.5 (C-5), 126.0 (q, J CF ) 278 Hz, C-6), 174.7
(C-2). Anal. (C10H16NOF3) C, H, N.
trans-5-Pentyl-4-methylpyrrolidin-2-one (1.05 g, 4.7 mmol)
was reacted with trimethyloxonium tetrafluoroborate (770 mg,
5.2 mmol) according to general procedure A to yield, after
chromatography, 1.12 g of the iminoether. The iminoether
(1.12 g, 4.64 mmol) was reacted with ammonium chloride (250
mg, 4.64 mmol) according to general procedure B to yield the
title material 13, 410 mg (34%, from the lactam). 1H NMR
(D2O, 300 MHz): 0.9 (t, J ) 6 Hz, 3H), 1.15-1.7 (m, 6H), 1.95
(dd, J ) 16, 7.5 Hz, 1H), 2.40-2.83 (m, 3H), 3.72 (m, 1H). Anal.
(C10H16NOF3‚HCl) C, H, N, Cl.
cis-5-Be n zyl-4-(t r iflu or om e t h yl)p yr r olid in -2-im in e
(18): 1H NMR (D2O, 400 MHz) 2.91 (br t, J ) 13 Hz, 1H),
3.22 (br dd, J ) 14, 2 Hz, 1H), 3.28 (dd, J ) 18, 9 Hz, 1H),
3.36 (dd, J ) 18, 8.5 Hz, 1H), 3.72 (heptet, J ) 8 Hz, 1H), 4.60
(m, 1H), 7.34 (complex d, J ) 8 Hz, 2H), 7.37 (complex t, J )
7 Hz, 1H), 7.44 (complex t, J ) 8.5 Hz, 2H). 13C NMR (D2O,
125 MHz): 33.0, 38.4, 44.4 (q, J CF ) 29 Hz), 63.9, 128.6 (q,
J CF ) 278 Hz), 131.9, 132.0, 132.2, 139.7, 171. Anal.
(C12H13N2F3‚1HCl‚0.1AcOH) C, H, N, Cl.
4,4-Dim eth yl-5-ben zylp yr r olid in -2-im in e Hyd r och lo-
r id e (19). Ethyl dimethyl acrylate (10.75 g, 84 mmol) was
mixed with â-nitroethylbenzene19 (12.68 g, 84 mmol) in tetra-
n-butylammonium fluoride in THF (84 mL, 1 M) and heated
at 40 °C. When the reaction, monitored by GC, was complete,
the mixture was treated with brine (saturated) and the
aqueous phase was extracted with ether. Purification by
chromatography on silica gel yielded the product ethyl 3,3-
dimethyl-4-nitrobenzenepentanoate (9.04 g, 34%). Anal.
(C15H21NO4).
cis-5-P en tyl-4-(tr iflu or om eth yl)p yr r olid in -2-im in e Hy-
d r och lor id e (14). cis-5-Pentyl-4-(trifluoromethyl)pyrrolidin-
2-one (500 mg, 5 mmol) was reacted with trimethyloxonium
tetrafluoroborate (890 mg, 6 mmol) according to general
procedure A to yield, after chromatography, 610 mg of the
iminoether. The iminoether (610 mg, 5 mmol) was reacted
with ammonium chloride (200 mg, 4 mmol) according to
general procedure B to yield the title material 14, 500 mg (70%,
from the lactam). 1H NMR (DMSO-d6, 400 MHz): 0.88 (t, J
) 6 Hz, 3H), 1.20-1.65 (m, 6H), 3.01 (dd, J ) 17, 7 Hz, 1H),
3.17 (dd, J ) 17, 9 Hz, 1H), 3.74 (m, 1H), 4.12 (apparent q, J
) 7 Hz, 1H). Anal. (C10H17N2F3‚1HCl‚0.15NH4Cl) C, H, N,
Cl.
5-Ben zylp yr r olid in -2-im in e Hyd r och lor id e (15). The
methyl acrylate (2.84 g, 33 mmol) was mixed with â-nitroeth-
ylbenzene19 (5.0 g, 33 mmol), potassium carbonate (4.6 g, 33
mmol), and Aliquat 336 (10 drops), in a manner similar to the
method described in 11. Purification by chromatography on
silica gel yielded methyl 4-nitrobenzenepentanoate (4.0 g,
55%). The methyl 4-nitrobenzenepentanoate (4.0 g, 18 mmol)
The above product (3.5 g, 12.5 mmol) in absolute MeOH was

3682 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hagen et al.
hydrogenated over Ra Ni at 55 °C and 60 psi for 6 h. The
reaction product was purified by column chromatography to
yield 4,4-dimethyl-5-benzylpyrrolidin-2-one (2.41 g, 95%).
(CH’s); 173.2 (CdN). [R]D ) -34.7, CHCl3. Anal. (C10H18N2‚
HCl) C, H, N.
(+)-cis-5-P en t yl-4-(t r iflu or om et h yl)p yr r olid in -2-im i-
n e, Mon oh yd r och lor id e (22). The title compound was
isolated from 14 by chiral HPLC on a Chirobiotic V column
with 20:80:0.1 EtOH/HEP/TFA as the mobile phase. 1H NMR
(400 MHz, DMSO-d6): 0.89 (t, 3H), 1.2-1.7 (m, 8H), 3.02 (dd,
1H), 3.17 (dd, 1H), 3.63 (m, 1H), 4.13 (m, 1H), 8.8 (s, 1H), 9.4
(s, 1H), 10.3 (s, 1H). [R]D ) +30.0, MeOH. Anal. (C10H17N2F3‚
1HCl‚1.5H2O‚0.025TFA) C, H, N.
(-)-cis-5-P en t yl-4-(t r iflu or om et h yl)p yr r olid in -2-im i-
n e, Mon oh yd r och lor id e (23). The title compound was
isolated from 14 by chiral HPLC on a Chirobiotic V column
with 20:80:0.1 EtOH/HEP/TFA as the mobile phase. 1H NMR
(400 MHz, DMSO-d6): 0.89 (t, 3H), 1.2-1.7 (m, 8H), 3.02 (dd,
1H), 3.17 (dd, 1H), 3.63 (m, 1H), 4.13 (m, 1H), 8.8 (s, 1H), 9.4
(s, 1H), 10.3 (s, 1H). [R]D ) -33.8, MeOH. Anal. (C10H17N2F3‚
1HCl‚0.9H2O) C, H, N.
The above product (1.04 g, 5.1 mmol) was treated with
trimethyloxonium tetrafluoroborate (0.91 g, 6.2 mmol) in DCM
(25 mL) by general method A, to yield the iminoether (0.83 g,
75%). A solution of the iminoether (0.8 g, 3.5 mmol) in MeOH
(60 mL) was reacted with ammonium chloride (187 mg, 3.5
mmol) by general method B followed by chromatography on
reverse-phase HPLC to generate the title material 19 (570 mg,
68%). 1H NMR (DMSO, 400 MHz): 1.06 (s, 3H), 1.07 (s, 3H),
2.66 (s, 2H), 2.70 (dd, J ) 14, 10 Hz, 1H), 2.88 (dd, J ) 14, 5
Hz, 1H), 3.92 (dd, J ) 10, 5 Hz, 1H), 7.27 (t, J ) 8 Hz, 1H),
7.29 (d, J ) 8 Hz, 2H), 7.35 (t, J ) 8 Hz, 2H), 8.30 (br s, 1H),
9.12 (br s, 2H). Anal. (C13H18N2‚1HCl) C, H, N, Cl.
(+)-cis-4-Meth yl-5-p en tylp yr r olid in -2-im in e, Mon oh y-
d r och lor id e (20). A solution of 5-pentyl-4-methylpyrrolidin-
2-one (4.29 g, 25.0 mmol) and (R)-(-)-1-(1-naphthyl)ethyl
isocyanate (5.00 g, 25.3 mmol) in 200 mL of benzene was
heated at reflux for 24 h. Additional isocyanate (5.00 g, 25.3
mmol) was added. After an additional 24 h of heating, the
reaction mixture was concentrated under reduced pressure.
The resulting diastereomeric ureide mixture was separated
by column chromatography to yield 2.0 g of ureide A (Rf )
0.5) and 2.1 g of ureide B (Rf ) 0.33) [TLC solvent system (9:1
EA/MeOH), EM silica gel 60 F254 plates].
cis-5-P en tyl-4-m eth ylp yr r olid in -2-im in e, Sem ita r tr a te
(20B). cis-5-Pentyl-4-methylpyrrolidin-2-one (19.0 g, 111
mmol) was reacted with trimethyloxonium tetrafluoroborate
(35.2 g, 166 mmol) according to general procedure A to yield,
after chromatography, 21.7 g of the iminoether. To a suspen-
sion of diammonium L-tartrate (14.40 g, 78 mmol) in 110 mL
of dry methanol was added 16.8 g (70.6 mmol, by GC analysis)
of the crude methyl iminoether. The mixture was refluxed for
5 h, the reaction was cooled to room temperature, 50 mL of
MTBE was added, and the mixture was stirred for an ad-
ditional 15 min. The excess diammonium L-tartrate was
filtered, washed with two portions of 1:1 MTBE-MeOH (50
mL total), and dried.
The solvent was removed from the mother liquor via rotary
evaporation and azeotroping with hexane. The resulting solids
were triturated with MTBE (250 mL) for 90 min, filtered, and
washed with 2 × 50 mL of MTBE. The solids were filtered to
yield 17.3 g, 101%, of a white solid. Elemental analysis and
1H NMR (CDCl3) show this salt to be the bis-amidine tartrate.
Crude tartrate (14.3 g) was then dissolved in 118 mL of hot
absolute ethanol (100%), giving a cloudy solution which was
filtered while hot, rinsed down with approximately 30 mL of
ethanol, and the now clear solution concentrated to ap-
proximately 100-mL volume, cooled to room temperature,
seeded, and cooled in the refrigerator (2-4 °C) overnight. The
product was then collected in a fritted glass filter funnel,
washed with approximately 60 mL of 3:1 MTBE-EtOH, and
filtered to yield 8.6 g of fluffy, white solid. HPLC analysis
showed approximately 29% of the opposite isomer to be
present. These solids were then redissolved in 68 mL of hot
absolute ethanol (100%) and cooled to room temperature, at
which point crystals began to form without additional seeding.
The mixture solidified, necessitating the addition of 10 mL of
ethanol. After standing at room temperature for a total of 90
min, the slushy mixture was filtered, and the solids were
washed with approximately 30 mL of 3:1 MTBE-EtOH,
yielding 3.50 g of white crystals. DSC 205.28 °C (sharp). 1H
NMR (D2O, 400 MHz): 0.88 (t, J ) 6.4 Hz, 3H), 1.02 (d, J )
7.0 Hz, 3H), 1.24-1.44 (m, 6H), 1.44-1.52 (m, 2H), 2.53 (dd,
J ) 17.1, 6.7 Hz, 1H), 2.70 (sep., J ) 7 Hz, 1H), 2.96 (dd, J )
17.1, 7.9 Hz, 1H), 3.92 (q, J ) 7 Hz,1H), 4.32 (s, 1H). 13C NMR
(D2O, 125 MHz): 18.7, 19.1, 27.6, 30.9, 34.4, 36.7, 38.5, 43.1,
68.8,79.6,176.0,184.1. [R]D )+42.6,MeOH. Anal. (C24H46N4O6)
C, H, N.
To NaH (0.28 g, 11.9 mmol) was added n-BuOH (40 mL).
After stirring for 10 min, a solution of ureide B (1.87 g, 5.1
mmol) in 10 mL of n-BuOH was added to n-BuONa. After
heating for 18 h, the reaction mixture was concentrated under
reduced pressure. The residue was dissolved in EtOAc. The
organic solution was washed with brine, dried (MgSO4),
filtered, and concentrated under reduced pressure. The crude
product was purified by column chromatography to give 0.23
g of (+)-cis-5-pentyl-4-methylpyrrolidin-2-one ([R]D ) +29.6,
CHCl3).
(+)-cis-5-Pentyl-4-methylpyrrolidin-2-one (210 mg, 1.2 mmol)
was reacted with trimethyloxonium tetrafluoroborate (220 mg,
1.5 mmol) according to general procedure A to yield, after
chromatography, 180 mg of the iminoether. The iminoether
(180 mg) was reacted with ammonium chloride (74 mg, 1.4
mmol) according to general procedure B to yield the title
material 20, 102 mg (54%, from the lactam). 1H NMR (D2O,
400 MHz): 0.88 (t, J ) 7 Hz, 3H), 1.02 (d, J ) 7.2 Hz, 3H),
1.23-1.43 (m, 6H), 1.43-1.60 (m, 2H), 2.13 (m, 1H), 2.53 (dd,
J ) 17.2, 6.7 Hz, 1H), 2.70 (sep., J ) 7.2 Hz, 1H), 2.96 (dd, J
) 17.2, 7.9 Hz, 1H), 3.92 (td, J ) 7.8, 6.4 Hz, 1H). 13C NMR
(D2O, 125 MHz): 15.8, 16.2 (CH3’s); 24.7, 28.0, 31.5, 33.8, 40.2
(CH2’s); 35.7, 66.0 (CH’s); 173.2 (CdN). [R]D ) +42.6, MeOH.
Anal. (C10H18N2‚HCl) C, H, N.
(-)-cis-4-Meth yl-5-p en tylp yr r olid in -2-im in e, Mon oh y-
d r och lor id e (21). To NaH (0.28 g, 11.9 mmol) was added
n-BuOH (40 mL). After stirring for 10 min, a solution of ureide
A (1.87 g, 5.1 mmol) in 10 mL of n-BuOH was added to
n-BuONa. After heating for 18 h, the reaction mixture was
concentrated under reduced pressure. The residue was dis-
solved in EtOAc. The organic solution was washed with brine,
dried (MgSO4), filtered, and concentrated under reduced
pressure. The crude product was purified by column chroma-
tography to give 0.23 g of (-)-cis-5-pentyl-4-methylpyrrolidin-
2-one ([R]D ) -36.5, CDCl3).
(-)-cis-5-Pentyl-4-methylpyrrolidin-2-one (210 mg, 1.2 mmol)
was reacted with trimethyloxonium tetrafluoroborate (220 mg,
1.5 mmol) according to general procedure A to yield, after
chromatography, 180 mg of the iminoether. The iminoether
(180 mg) was reacted with ammonium chloride (74 mg, 1.4
mmol) according to general procedure B to yield the title
material 21, 102 mg (54%, from the lactam). 1H NMR (D2O,
400 MHz): 0.88 (t, J ) 6 Hz, 3H), 1.02 (d, J ) 7.0 Hz, 3H),
1.23-1.44 (m, 6H), 1.44-1.62 (m, 2H), 2.53 (dd, J ) 17.1, 6.7
Hz, 1H), 2.70 (septet, J ) 7 Hz, 1H), 2.97 (dd, J ) 17.1, 7.9
Hz, 1H), 3.92 (q, J ) 7 Hz, 1H). 13C NMR (D2O, 125 MHz):
15.8, 16.2 (CH3’s); 24.7, 28.0, 31.5, 33.8, 40.2 (CH2’s); 35.7, 66.0
Ack n ow led gm en t. Cloning of human NOS isoforms
was performed by Charles P. Rodi, Patrick M. Sullivan,
and J oseph F. Wiese. Expression in insect cells was
performed by Shaukat H. Rangwala, Rodney G. Combs,
and J ennifer L. Pierce. Isolation of NOS isoforms was
performed by Christine M. Kornmeier, Michael S.
Davies, Harold D. Woodward, and Margaret H. Marino.
NMR spectra were run by Lilian Garcia and Flordeliza
Mamaril. NMR spectra were analyzed by Lydia Swen-
ton, David Lankin, and Patricia Finnegan. Mass spec-

2-Iminopyrrolidines as Inhibitors of hiNOS
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3683
Alcohols via the [3.3]-Sigmatropic Rearrangement of Allylic
Trichloroacetimidates and the Subsequent Ruthenium-Catalyzed
Cyclization of N-Allyltrichloroacetamides. J . Org. Chem. 1992,
57, 1682-1689.
tral analyses were done by Norman Liu. Microanalyses
were performed by E. Zielinski and associates. HPLC
analyses were performed by Randy Fronek, Carlos
Orihuela, Dorothy Honda, Yuri Zelechenok, and Scott
Cassady. The authors would also like to thank Dr.
Robert Manning, Dr. Donald Hansen, J r., Ms. Sofya
Tsymbalov, and Mr. Keith Webber for helpful discus-
sions.
(12) Pirkle, W. S.; Robertson, M. R.; Hyun, M. H. Optical Resolution
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B. S. 2-Iminohomopiperidine Salts as Selective Inhibitors of
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J M970840X