Design and synthesis of inhibitors of inducible nitric oxide synthase. Discovery of a new chemical lead with potential for oral bioavailability

Article abstract of DOI:10.1016/S0223-5234(03)00017-5

A series of 2-iminopiperidines fused to small-membered rings (Tables 1 and 2) were synthesised and biologically evaluated using an in vitro human nitric oxide synthase (NOS) inhibition assay. Fused bicyclic compounds 5-9 exhibited nearly the same potency as compound 1 in the hiNOS inhibition assay. Among these, the 1-methyl analogues 8 and 9 showed better isoform selectivity than their corresponding unsubstituted analogues 7 and 6, respectively. Compounds 5 and 6 were also evaluated by an in vivo NO accumulation assay in a mouse model. The discovery process of new chemical leads for an orally bioavailable inhibitor of human inducible NOS (iNOS) is reported. The structure-activity relationship (SAR) study and chemistry of these compounds are also reported.

Full text of DOI:10.1016/S0223-5234(03)00017-5

European Journal of Medicinal Chemistry 38 (2003) 277ꢀ288
/
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Laboratory note
Design and synthesis of inhibitors of inducible nitric oxide synthase.
Discovery of a new chemical lead with potential for oral
bioavailability
Yasufumi Kawanaka b,*, Kaoru Kobayashi a, Shinya Kusuda a, Tadashi Tatsumi a,
Masayuki Murota a, Toshihiko Nishiyama a, Katsuya Hisaichi a, Atsuko Fujii a,
Keisuke Hirai a, Masao Naka a, Masaharu Komeno a, Hisao Nakai a, Masaaki Toda a
a Minase Research Institute, Ono Pharmaceutical Co. Ltd., Shimamoto, Mishima, Osaka 618-8585, Japan
b Fukui Research Institute, Ono Pharmaceutical Co. Ltd., Technoport, Yamagishi, Mikuni, Sakai, Fukui 913-8538, Japan
Received 13 September 2002; received in revised form 7 January 2003; accepted 15 January 2003
Abstract
A series of 2-iminopiperidines fused to small-membered rings (Tables 1 and 2) were synthesised and biologically evaluated using
an in vitro human nitric oxide synthase (NOS) inhibition assay. Fused bicyclic compounds 5ꢀ9 exhibited nearly the same potency as
/
compound 1 in the hiNOS inhibition assay. Among these, the 1-methyl analogues 8 and 9 showed better isoform selectivity than
their corresponding unsubstituted analogues 7 and 6, respectively. Compounds 5 and 6 were also evaluated by an in vivo NO
accumulation assay in a mouse model. The discovery process of new chemical leads for an orally bioavailable inhibitor of human
inducible NOS (iNOS) is reported. The structureꢀ
/
activity relationship (SAR) study and chemistry of these compounds are also
reported.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Inhibitor of inducible nitric oxide synthase; 5-Methyl-2-azabicyclo[4.1.0]heptan-3-imines; Cyclopropanation; Dibromocarbene; Amidine
1. Introduction
found in brain cells and peripheral nerve cells, and the
endothelial enzyme (eNOS) found in vascular endothe-
lial cells [3]. Unlike iNOS, both nNOS and eNOS are
intermittently activated by transient elevations in the
intracellular calcium level leading to calmodulin binding
and stimulation of enzyme activity. Recent studies have
shown that overproduction of NO is associated with
In recent years, nitric oxide (NO) has been identified
as one of the important and unique mediators of diverse
physiological processes [1,2]. Endogenous NO is pro-
duced by nitric oxide synthase (NOS), which catalyses
the oxidation of
L-arginine to L-citrulline to yield NO
several pathophysiological states [6ꢀ8]. Overproduction
/
[3ꢀ5]. The NOS family of enzymes is composed of an
/
of NO by iNOS has been implicated in the tissue
destruction that occurs in states such as chronic
inflammation, septic shock, vascular dysfunction in
diabetes, and transplant rejection. Substrate analogue
inducible form and two constitutive forms. Inducible
NOS (iNOS) is produced by cells such as activated
macrophages, smooth muscle cells, and hepatocytes.
iNOS produces a large amount of NO, which is
sustained over a long period of time after the enzyme
induction by cytokines or endotoxins. The two consti-
tutive isoforms of NOS (cNOS) that have been eluci-
dated are as follows: the neuronal enzyme (nNOS),
inhibitors such as NG-methyl-
[9,10], NG-nitro-
-arginine -NNA) [11], N-imi-
noethyl- -ornitine ( -NIO) [12], and -thiocitrulline
L-arginine (L-NMA)
L
(L
L
L
L
[13] have been used in both animal models and clinical
trials to block iNOS-mediated synthesis of NO and to
treat hypotension associated with septic shock [14,15].
However, the patient’s blood pressure must be mon-
itored continuously during treatment since these com-
* Corresponding author.
E-mail address: kawanaka@ono.co.jp (Y. Kawanaka).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00017-5

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Y. Kawanaka et al. / European Journal of Medicinal Chemistry 38 (2003) 277ꢀ288
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Table 1
Inhibitory activities of fused- and spiro-bicyclic 2-iminopiperidines against hiNOS, heNOS and their isoform selectivitya
c
aAll compounds were prepared as a racemic mixture. bReference [16]. Reference [25].
pounds nonselectively inhibit both cNOS and iNOS.
Thus, the development of selective iNOS inhibitors is
currently of considerable interest in pharmaceutical
research, because of their predicted large therapeutic
potential.
amino acids have been reported as selective iNOS
inhibitors [16,17]. In our previous paper [18], we
reported on 5,6-dihyrdopyridin-2-imines as potent in-
hibitors of iNOS, with a heNOS/hiNOS selectivity of
0.6- to 11-fold. Since the discovery of 2-iminopiperidine
[16] as a potent non-amino acid inhibitor of iNOS, this
small molecule seems to be one of the most attractive
Many studies have focused on the design and synth-
esis of non-amino acid structures, and a number of non-
Fig. 1. Molecular design of a bicyclo[4.1.0] framework.

Y. Kawanaka et al. / European Journal of Medicinal Chemistry 38 (2003) 277ꢀ
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279
Table 2
Inhibitory activities of 2-aza-bicyclo[4.1.0]heptan-3-imines against hiNOS, heNOS and their isoform selectivitya
aAll compounds were prepared as a racemic mixture.
chemical leads because of its ability to accept a diversity
of chemical modifications. As described in Fig. 1, our
chemical modifications of 1 started with the preparation
of 2-iminopiperidines fused to a small-membered ring
and a spiro-derivative, such as the compounds listed in
Table 1. Here we report on the discovery process leading
to a series of bicyclic 2-iminopiperidines fused to small-
membered rings as new chemical leads for selective
inhibitors of iNOS.
bond in 11 by a modified Simmonsꢀ
/
Smith reaction [20]
afforded 12 at a 35% yield, which was converted to 2 by
the usual procedures to prepare an amidine from the
corresponding amide.
As described in Fig. 3, the spiro-derivative 3 was
prepared from dinitrile 13 [21]. Selective reduction of
one of the nitrile functions of 13 afforded an aminoni-
trile 14 at a 32% yield, which was transformed into an
ethyl imidate 15. Conversion of 15 to 3 was accom-
plished by the usual aminolysis, followed by treatment
with hydrogen chloride.
As described in Fig. 4, 4 was prepared from a
cyclopentanone fused to a cyclobutane, which was
synthesised from the cis-1,2-bis(iodomethyl)cyclobutane
(16) [22]. Direct Beckmann rearrangement of the bicyclic
cyclopentanone yielded 17, which was transformed to 4
by the usual procedure used to prepare an amidine from
the corresponding amide.
2. Chemistry
The test compounds 2ꢀ
were prepared as outlined in Figs. 2ꢀ
2, 2 was prepared from 10. Birch reduction of 10
provided 4-methyl-3,6-dihydropyridin-2-one (11) at a
good yield (84%) [19]. Cyclopropanation of the double
/
4 and 6ꢀ
/
9 in Tables 1 and 2
/5. As shown in Fig.
Fig. 2. Synthesis of 2. Reagents: (a) Li, liquid NH3, THFꢀ
/
EtOH, ꢁ ,
78 8C then H2O (84%); (b) CH2I2, AlMe3, CH2Cl2, 0 8C (35%); (c) Et3OꢂBF4ꢁ
/
CH2Cl2; (d) NH3, EtOH; (e) 4 M HCl dioxane (66% in three steps).

280
Y. Kawanaka et al. / European Journal of Medicinal Chemistry 38 (2003) 277ꢀ288
/
Fig. 3. Synthesis of 3. Reagents: (a) NaBH4, CoCl6×
/
6H2O, MeOH, ꢁ
/
15 8C (32%); (b) HCl (gas), MeOH, room temperature; (c) NH3, EtOH; (d) 4 M
HCl dioxane (78% in three steps).
Deprotection of the p-methoxybenzyl group of 24 and
25 resulted in 26 (26-anti: 36% yield, 26-syn: 61% yield)
and 28 (28-anti: 90% yield, 28-syn: 76% yield), respec-
tively. Reductive elimination of the gem-dibromine of
26 and 28 afforded 27 (27-anti: 90% yield, 27-syn: 91%
yield) and 29 (29-anti: 14% yield, 29-syn: 87% yield),
respectively.
The stereochemistry of 27-anti, 29-anti was deter-
mined based on the nuclear overhauser effects (NOEs)
between 5-CH3 and H6, and between H4?, H7endo, and H5
using NMR technique (Fig. 6a). The stereochemistry of
27-syn and 29-syn was also determined based on the
NOEs between 5-CH3, H7endo, and H7exo, and between
H5 and H6 (Fig. 6b).
Fig. 4. Synthesis of 4. Reagents: (a) KH, FAMSO, THF; (b) H2SO4
aq, acetone; (c) NH2OSO3H, HCO2H, reflux (9% in three steps); (d)
Et3OꢂBF4ꢁ, CH2Cl2; (e) NH3, EtOH; (f) 4 M HCl dioxane (33% in
three steps).
Compounds 6ꢀ9 were prepared from 18 as outlined in
/
Fig. 5. Aminolysis of 18 with p-methoxybenzylamine,
followed by formation of an imide with acetic anhy-
dride, resulted in 19 at a 95% yield. Partial reduction of
19 with sodium borohydride afforded 20 at a 95% yield.
Methylation of 19 with methyl lithium gave 21 at a 95%
yield. Dehydration of 20 and 21 with p-toluenesulfonic
acid resulted in 22 and 23 at a yield of 60 and 99%,
respectively. Insertion of a dibromocarbene into the
newly formed double bonds of 22 gave 24 (24-anti: 30%
yield, 24-syn: 12% yield). Cyclopropanation of 23 with
dibromocarbene gave 25 (25-anti: 26% yield, 25-syn: 7%
yield). Both of these cyclopropanations led to two
stereoisomers, the syn-isomer [23] (minor product) and
the anti-isomer [24] (major product), respectively, which
were separable by column chromatography.
Fig. 6. (a) NOE of 27-anti (Rꢃ
/
H) and 29-anti (Rꢃ/Me); (b) NOE of
27-syn (RꢃH) and 29-syn (Rꢃ
/
/Me).
Fig. 5. Synthesis of 6ꢀ
(95%); (d) MeLi, THF, ꢁ
syn: 12%, 25-anti: 26%, 25-syn: 7%); (g) BF3×
CH2Cl2, (j) NH3, EtOH, (k) 4 M HCl dioxane (16ꢀ
/
9. Reagents: (a) PMBNH2, toluene, room temperature; (b) Ac2O, Et3N, 80 8C (95% in two steps); (c) NaBH4, EtOH, 0 8C
78 8C (95%); (e) p-TsOH×H2O, toluene, 80 8C (22: 60%, 23: 99%); (f) CHBr3, 50% NaOH aq, aliquat-336 (24-anti: 30%, 24-
OEt2, anisole, 100 8C (36ꢀ90%); (h) n-Bu3SnH, AlBN, benzene, reflux (14ꢀ
91%); (i) Et3OꢂBF4ꢁ
61% in three steps).
/
/
/
/
/
,
/

Y. Kawanaka et al. / European Journal of Medicinal Chemistry 38 (2003) 277ꢀ
/288
281
Compounds 27 and 29 were converted to their
corresponding cyclic amidines 6ꢀ7 and 8ꢀ9, respec-
demonstrated more potent hiNOS inhibition than 1,
with 3.6-fold isoform selectivity.
/
/
tively, by the usual procedures. The anti-isomers 6 and 9
were prepared from the corresponding anti-isomers of
27 (27-anti) and 29 (29-anti), respectively, which were
obtained as major products of the dibromocyclopropa-
nation of 22 and 23, respectively. The syn-isomers 7 and
8 were prepared from the corresponding syn-isomers of
27 (27-syn) and 29 (29-syn), respectively, which were
obtained as minor products of the cyclopropanation of
22 and 23, respectively.
It has been reported that introduction of an alkyl
group at position-6 of 2-iminopiperidines increases the
potency of iNOS inhibition [16]. On the basis of this
information, a methyl group was introduced at the
corresponding position of the more active syn-isomer 7,
yielding 8 with reduced iNOS inhibition and increased
isoform selectivity. Introduction of a methyl group at
the corresponding position of the anti-isomer 6 afforded
9, which retained its potency for iNOS inhibition and
showed increased isoform selectivity. The introduction
of a methyl group at position-6 of 6 and 7 was effective
in increasing the isoform selectivity without any marked
loss of potency for iNOS inhibition.
3. Results and discussion
In order to evaluate the newly identified chemical
leads 5 and 6 for their ability to inhibit iNOS in vivo,
mice were subcutaneously (sc) injected with 5 and 6 at 3
h after lipopolysaccharide (LPS) treatment [26]. Then
the plasma NOx accumulation from 3 to 6 h after LPS
injection was determined. As described in Table 3, test
compounds 5 and 6 inhibited NOx accumulation in
plasma and their ID50 values were 0.036 and 0.013 mg
kgꢁ1, sc. To assess the acute toxicity of each compound,
the maximum tolerated dose (MTD) was determined. As
shown in Table 3, the MTD of 5 and 6 was 10 and 20 mg
kgꢁ1, respectively, when a single intravenous (iv) dose
was given to normal mice. Safety was assessed from the
ratio of the MTD and ID50 values for NOx accumula-
tion. According to the MTD/ID50 of NOx accumulation
data for the compounds, 6 was thought to be tolerable
enough to allow for an in vivo evaluation.
Test compounds were biologically evaluated for their
ability to inhibit the two isoforms of NOS, i.e. human
iNOS (hiNOS) and human eNOS (heNOS), and their
isoform selectivity was determined by the ratio of the
two IC50 values: heNOS/hiNOS. 2-Imino-4-methylpi-
peridine 1 [16] was prepared as a mixture of racemates
and biologically evaluated as a standard compound,
which demonstrates inhibitory activities against the two
isoforms of NOS, hiNOS (IC50ꢃ
/
0.14 mM) and heNOS
(IC50ꢃ1.1 mM), showing an isoform selectivity of
/
nearly 8-fold. Cyclopropanation of 4-methyl-3,6-dihy-
dropyridin-2-imine [18] yielded 2, which showed a
marked reduction in the potency of hiNOS and heNOS
inhibition as well as less isoform selectivity relative to 1.
The spiro analogue 3 showed a 1.4-fold reduction in
inhibitory activity and less isoform selectivity compared
with 1. Compound 4, a 2-iminopiperidine fused to a
cyclobutane in the cis-manner, demonstrated a 5-fold
reduction in the potency of hiNOS inhibition and a 4-
fold reduction in heNOS inhibition, so the isoform
selectivity was 6-fold. The cis-fused 5,6-bicyclic deriva-
tive 5 [25] was also synthesised and biologically eval-
uated. Compound 5 demonstrated nearly the same
potency and isoform selectivity as compound 1. Cyclo-
propanation of 4-methyl-3,4-dihydropyridin-2-imine
yielded 6 as the main product, in which the cis-fused
cyclopropane moiety showed anti-stereochemistry with
respect to the 4-methyl group. This compound showed a
1.6-fold increase in hiNOS inhibition relative to 1, with
2.1-fold isoform selectivity. The corresponding syn-
isomer 7, which was obtained as the minor product of
the above-mentioned cyclopropanation reaction, also
4. Conclusions
In conclusion, 2-iminopiperidines fused to small-
membered rings and a spiro analogue were synthesised
and biologically evaluated. Among these, compounds,
cis-fused cyclisation of position-5 and position-6 of 1
was effective in retaining the potency of iNOS inhibi-
tion, as illustrated in 5ꢀ7. Compounds 2 and 4, in which
/
cyclopropane and cyclobutane were fused to position-4
and -5, respectively, in a cis-manner, demonstrated
weaker inhibitory activity. A spiro analogue 3 also
retained the same potency for iNOS inhibition as 1, but
showed a loss of isoform selectivity. The cis-fused
Table 3
Pharmacological evaluation of 5 and 6 in mice
Compound
Mouse iNOS IC50 (mM)
NOx ID50 (mg kgꢁ1, sc)
MTD (mg kgꢁ1, iv)
MTD/NOx
5
6
0.030
0.007
3.5
0.036
0.013
26
10
20
280
1500
120
L
-NMMA
3000

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Y. Kawanaka et al. / European Journal of Medicinal Chemistry 38 (2003) 277ꢀ288
/
bicyclic analogue 5 retained both the potency and iNOS
selectivity of 1. Other cis-fused bicyclic analogues, 6 and
7, also retained the potency of 1, but showed reduced
isoform selectivity. Introduction of a methyl group at
position-1 of bicyclic analogues 6 and 7 was effective for
increasing isoform selectivity and retaining hiNOS
slowly warmed up to room temperature (r.t.) removing
the ammonia with a stream of nitrogen and then treated
with additional water (50 mL). The reaction mixture was
extracted with CHCl3 (50 mLꢄ3). The combined
/
organic layers were washed with 1 M HCl and brine,
dried over anhydrous sodium sulphate and evaporated
under reduced pressure. The residue was purified by
inhibitory activity, as illustrated in 8ꢀ9. Further opti-
/
misation of 2-imino-4-methylpiperidines fused to cyclo-
propanes and oral bioavailability of the optimised
inhibitors will be reported elsewhere.
column chromatography on silica gel (CHCl3ꢀ
5/1) to afford 11 as a white crystal (4.3 g, 84%). M.p.:
83ꢀ
85 8C. IR (KBr) (cmꢁ1) 3207, 3056, 2893, 1704,
/
MeOH,
/
1663, 1499, 1440, 1406, 1378, 1339, 1289, 1155, 1130,
1
1043, 1002, 949, 838, 805, 726, 536, 492, 464. H-NMR
5. Experimental
(200 MHz, CDCl3) d 7.60ꢀ
3.90 (m, 2H), 2.80 (m, 2H), 1.72 (s, 3H). MS (APCI,
Pos.) m/z 112 [Mꢂ
H]ꢂ; HRMS (MALDI-TOF, Pos.)
calc. for C6H9N1O1ꢂ
Hꢂ: 112.0762; found: 112.0742.
TLC Rf 0.51 (CHCl3ꢀMeOH, 10/1).
/
6.70 (brs, 1H), 5.43 (m, 1H),
5.1. Chemistry
/
/
Melting points (m.p.) were determined by Yanaco
micro m.p. apparatus MP-500D and are uncorrected.
Analytical samples were homogeneous as confirmed by
thin layer chromatography (TLC), and afforded spec-
troscopic results consistent with the assigned structures.
All 1H-NMR spectra were obtained on a Varian
Gemini-200, VXR-200s spectrometer. The chemical
shift values are reported in ppm (d) and coupling
constants (J) in Hertz (Hz). Fast atom bombardment
mass spectra (FABMS) and electron ionisation (EI)
were obtained on a JEOL JMS-DX303HF or PerSeptive
Voyager Elite spectrometer. Atmospheric pressure che-
mical ionisation (APCI) was determined on a Hitachi
M1200H spectrometer. Matrix assisted laser desorption
ionisation-time of flight high-resolution mass spectra
(MALDI-TOF HRMS) were obtained on a PerSeptive
Voyager Elite spectrometer. IR spectra were measured
/
5.1.2.2. 6-Methyl-3-azabicyclo[4.1.0]heptan-4-one (12).
To a stirred solution of 11 (2.0 g, 18.0 mmol) and CH2I2
(8.8 mL, 109 mmol) in CH2Cl2 (81 mL) was added
Me3Al (1 M in n-hexane, 72 mL, 72 mmol) at 0 8C
under an argon atmosphere and stirred for 3 days at r.t..
The reaction mixture was diluted with CH2Cl2 (60 mL)
and cooled to 0 8C. KF (12.1 g, 208 mmol) and water
(3.9 mL) were added carefully. The insoluble substances
were dissolved in water and extracted with CHCl3. The
organic layer was washed with saturated Na2S2O3 and
brine, dried over anhydrous magnesium sulphate and
evaporated under reduced pressure. The residue was
purified by column chromatography on silica gel (n-
hexaneꢀ
12 as a pale yellow oil (785 mg, 35%). H-NMR (200
MHz, CDCl3) d 6.00ꢀ5.65 (brs, 1H), 3.71 (dd, Jꢃ12.6,
2.7 Hz, 1H), 3.41 (ddd, Jꢃ12.6, 5.4, 1.8 Hz, 1H), 2.51
(d, Jꢃ17.2 Hz, 1H), 2.25 (d, Jꢃ17.2 Hz, 1H), 1.17 (s,
3H), 1.00 (m, 1H), 0.67 (t, Jꢃ5.0 Hz, 1H), 0.46ꢀ0.38
(m, 1H). MS (APCI, Pos.) m/z 126 [Mꢂ
H]ꢂ; HRMS
(MALDI-TOF, Pos.) calc. for C7H11N1O1ꢂ
Hꢂ:
126.0919; found: 126.0923. TLC Rf 0.53 (CHCl3ꢀ
/
EtOAc, 1/2 to CHCl3ꢀ
/
MeOH, 30/1) to afford
1
on a Perkinꢀ
spectrometer. Column chromatography was carried out
on silica gel [Merck silica gel 60 (0.063ꢀ0.200 mm) or
/Elmer FTIR 1760X or JASCO FTIR-430
/
/
/
/
/
/
Wako Gel C200 or Fuji Silysia FL60D]. TLC was
performed on silica gel (Merck TLC plate, silica gel 60
/
/
/
F254).
/
/
5.1.1. Starting materials
MeOH, 10/1).
2-Hydroxy-4-methylpyridine (10) and 3-methylgluta-
ric anhydride (18) are commercially available. Com-
pounds 13, 16 were synthesised according to the
literature [21,22a].
5.1.3. Preparation of compound 3
5.1.3.1. [1-(2-Aminoethyl)cyclopropyl]acetonitrile
(14). To a stirred solution of 13 [21] (5.0 g, 41.6 mmol)
5.1.2. Preparation of compound 12
in MeOH (200 mL) was added CoCl2×
/
6H2O (5.0 g, 21.0
mmol) and NaBH4 (4.2 g, 111 mmol) at ꢁ
/
15 8C under
5.1.2.1. 4-Methyl-3,6-dihydropyridin-2-one (11). To a
stirred solution of 2-hydroxy-4-methylpyridine (10) (5
g, 45.8 mmol) in THF (45 mL) and EtOH (4 mL) was
an argon atmosphere. The reaction mixture was stirred
for 15 min and was treated with 2 M HCl carefully. The
resulting solution was warmed up to r.t., stirred for 15
min, evaporated and diluted water. The resulting
mixture was washed with EtOAc and the aqueous layer
was treated with 5 M NaOH to adjust the pH value to 9.
After removing insoluble substance by filtration, the
aqueous layer was extracted with CHCl3. The organic
added liquid NH3 (150 mL) at ꢁ78 8C. Small pieces of
/
lithium metal (954 mg, 137.4 mmol) were portionwisely
added to the reaction mixture and the formed solution
was stirred for an additional 1.5 h at ꢁ
/
78 8C before
quenched with water (50 mL). The reaction mixture was

Y. Kawanaka et al. / European Journal of Medicinal Chemistry 38 (2003) 277ꢀ
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283
layer was washed with brine, dried over anhydrous
magnesium sulphate and evaporated under reduced
pressure. The residue was purified by column chroma-
and the reaction mixture was heated at reflux tempera-
ture for 3 h. After completing the reaction, the reaction
mixture was cooled to r.t. and evaporated under reduced
pressure. The residue was treated with water and
extracted with Et2O. The organic layer was washed
with saturated aqueous sodium bicarbonate, brine, dried
over anhydrous magnesium sulphate and evaporated to
afford the bicyclo[3.2.0]heptan-3-one (480 mg), which
was used for the subsequent reaction without further
purification.
tography on silica gel (CHCl3ꢀ
/
MeOH, 5/1ꢀ1/2) to
/
afford 14 as a pale yellow oil (1.7 g, 32%). IR (neat)
1
(cmꢁ1) 2245. H-NMR (200 MHz, CDCl3) d 2.85 (t,
Jꢃ
0.54 (s, 4H). MS (APCI, Pos.) m/z 125 [Mꢂ
Rf 0.17 (CHCl3ꢀMeOHꢀAcOH, 10/1/1).
/
7.6 Hz, 2H), 2.41 (s, 2H), 1.59 (t, Jꢃ
/
7.6 Hz, 2H),
/
H]ꢂ. TLC
/
/
5.1.3.2. 6-Azaspiro[2.5]octan-5-imine hydrochloride salt
(3). To a stirred solution of 14 (830 mg, 6.7 mmol) in
EtOH (13 mL) was bubbled with HCl gas at 0 8C and
stirred for 3 h at r.t.. The reaction mixture was
evaporated to give 15, which was used for the subse-
quent reaction without further purification.
To a stirred solution of 15 in EtOH (3 mL) was added
saturated ethanolic ammonia (12 mL) under an argon
atmosphere at r.t. and stirring was continued for an
additional 24 h. The reaction mixture was diluted with
CHCl3 (16 mL) and the resulting precipitates were
removed by filtration. The filtrate was concentrated
under reduced pressure. The reaction mixture was
treated with 2 M NaOH (19 mL) and extracted with
To a stirred solution of the bicyclo[3.2.0]heptan-3-one
(480 mg) obtained above in HCO2H (8 mL) was added
NH2OSO3H (725 mg, 6.4 mmol) and the reaction
mixture was heated at reflux temperature for 3 h. After
completing the reaction, the reaction mixture was cooled
to r.t. and treated with 5 M NaOH to adjust the pH
value to 7. The mixture was extracted with CHCl3 and
washed with brine, dried over anhydrous magnesium
sulphate, and evaporated under reduced pressure. The
residue was purified by column chromatography on
silica gel (EtOAc to EtOAcꢀ
a pale yellow oil (48 mg, 9%). 1H-NMR (200 MHz,
CDCl3) d 6.55 (brs, 1H), 3.39 (ddd, Jꢃ13.2, 4.2, 2.0 Hz,
1H), 3.09 (ddd, Jꢃ13.2, 5.6, 2.6 Hz, 1H), 2.89 (m, 1H),
2.70 (m, 1H), 2.40 (dd, Jꢃ15.4, 5.6 Hz, 1H), 2.32ꢀ2.10
(m, 3H), 1.90ꢀ1.76 (m, 2H). MS (APCI, Pos.) m/z 126
[Mꢂ
H]ꢂ; HRMS (MALDI-TOF, Pos.) calc. for
C7H11N1O1ꢂ
Hꢂ: 126.0919; found: 126.0893. TLC Rf
0.50 (CHCl3ꢀMeOH, 9/1).
/MeOH, 9/1) to afford 17 as
/
/
CHCl3 (30 mLꢄ/2). The combined organic layers were
/
/
dried over anhydrous sodium sulphate and evaporated
under reduced pressure. The residue was converted to its
/
/
hydrochloride with 4 M HClꢀ
and further purified by column chromatography on
MeOH, 5/1) to afford 3 as a pale
/
EtOAcꢀ
/
EtOH at 0 8C
/
/
silica gel (CHCl3ꢀ
/
yellow solid (839 mg, 78%). IR (KBr) (cmꢁ1) 3292,
3122, 3002, 2946, 2877, 1683, 1537, 1444, 1422, 1379,
1340, 1017, 815. 1H-NMR (200 MHz, DMSO-d6) d 9.75
(brs, 1H), 8.78 (brs, 1H), 8.53 (brs, 1H), 3.35 (m, 2H),
5.1.5. General procedure for preparation of the key
intermediate (22), (23)
5.1.5.1. 1-(4-Methoxybenzyl)-4-methylpiperidine-2,6-
dione (19). To a stirred solution of 3-methylglutaric
anhydride (18) (20 g, 156 mmol) in THF (300 mL) was
added p-methoxybenzylamine (23 g, 168 mmol) at r.t.
and stirred for 30 min. After completing the reaction,
the reaction mixture was evaporated and the residue was
diluted with EtOAc. The mixture was treated with 1 M
HCl and extracted with EtOAc. The organic layer was
washed with brine, dried over anhydrous magnesium
sulphate, and evaporated to afford the residue, which
was used for the subsequent reaction without further
purification.
To a stirred solution of the compound obtained above
in Ac2O (100 mL) was added Et3N (20 mL) at r.t. The
reaction mixture was heated at 80 8C for 1 h. After
completing the reaction, the mixture was cooled to r.t.
and evaporated under reduced pressure. The residue was
diluted with EtOAc and water. The organic layer was
washed with 1 M HCl, saturated aqueous sodium
bicarbonate, brine and dried over anhydrous magne-
sium sulphate and evaporated under reduced pressure.
The residue was purified by column chromatography on
2.45 (s, 2H), 1.56 (t, Jꢃ
103ꢀ105 8C; MS and HRMS analysis showed only the
ion from C7H12N2 corresponding to the loss of HCl, MS
(FAB, Pos.) m/z 125 [Mꢂ
H]ꢂ; HRMS (MALDI-TOF,
Pos.) calc. for C7H12N2ꢂ
Hꢂ: 125.1079; found:
125.1050. TLC Rf 0.33 (CHCl3ꢀMeOHꢀAcOH, 10/1/1).
/5.8 Hz, 2H), 0.45 (s, 4H). M.p.:
/
/
/
/
/
5.1.4. Preparation of compound 17
5.1.4.1. (DL)-(1S,6S)-3-azabicyclo[4.2.0]octan-4-one
(17). To a stirred suspension of KH (650 mg, 16.2
mmol) in THF (8 mL) was added methyl methylsulfi-
nylmethyl sulphide (FAMSO) (589 mg, 4.7 mmol) in
THF (2 mL) at 0 8C under an argon atmosphere. After
10 min, cis-1,2-bis(iodomethyl)cyclobutane (16) [22a]
(1.4 g, 4.3 mmol) was added to this suspension and
stirred for 4 h at r.t. The reaction mixture was diluted
with CH2Cl2 (7 mL) and the resulting precipitates were
removed by filtration. Then the filtrate was concentrated
under reduced pressure.
To a stirred solution of the compound obtained above
in acetone (5 mL) was added 9 M H2SO4 (0.3 mL) at r.t.
silica gel (EtOAcꢀ
/
n-hexane, 10/1ꢀ1/1) to afford 19 as a
/

284
Y. Kawanaka et al. / European Journal of Medicinal Chemistry 38 (2003) 277ꢀ288
/
pale yellow solid (37 g, 95%). M.p.: 50ꢀ
/
51 8C. IR (KBr)
TOF, Pos.) calc. for C14H17N1O2ꢂ
/
Hꢂ: 232.1337;
n-hexane, 1/1).
(cmꢁ1) 3368, 2968, 2871, 1719, 1671, 1611, 1514, 1469,
1428, 1387, 1339, 1291, 1347, 1218, 1175, 1142, 1109,
1062, 1029, 970, 937, 886, 832, 806, 776, 646, 628, 595,
found: 232.1313. TLC Rf 0.78 (EtOAcꢀ
/
5.1.5.4. 6-Hydroxy-1-(4-methoxybenzyl)-4,6-
dimethylpiperidin-2-one (21). To a stirred solution of 19
(20 g, 81 mmol) in THF (300 mL) was added MeLi (1 M
1
562, 518. H-NMR (200 MHz, CDCl3) d 7.32 (d, Jꢃ
/
9.0 Hz, 2H), 6.80 (d, Jꢃ
(s, 3H), 2.80ꢀ2.65 (m, 2H), 2.40ꢀ
Jꢃ6.2 Hz, 3H). MS (APCI, Pos.) m/z 248 [Mꢂ
HRMS (MALDI-TOF, Pos.) calc. for C14H17N1O3ꢂ
Hꢂ: 248.1287; found: 248.1259. TLC Rf 0.82 (EtOAcꢀ
/
9.0 Hz, 2H), 4.88 (s, 2H), 3.77
2.12 (m, 3H), 1.04 (d,
H]ꢂ;
/
/
in Et2O, 87 mL, 87 mmol) at ꢁ78 8C under an argon
/
/
/
atmosphere. After completing the reaction, the reaction
mixture was diluted with Et2O and saturated aqueous
ammonium chloride (100 mL) was added. The organic
layer was washed with brine, dried over magnesium
sulphate and evaporated under reduced pressure. The
residue was purified by column chromatography on
/
/
n-hexane, 1/1).
5.1.5.2. 6-Hydroxy-1-(4-methoxybenzyl)-4-
methylpiperidin-2-one (20). To a stirred solution of 19
(14 g, 56.6 mmol) in EtOH (300 mL) was added NaBH4
(4.2 g, 111 mmol) at 0 8C. The reaction mixture was
stirred for 2 h at r.t.. After completing the reaction, the
resulting mixture was cooled to 0 8C and treated with 1
M HCl to adjust the pH value to 7. After removal of the
solvent by evaporation, the resulting mixture was
extracted with EtOAc and washed with brine. The
combined organic layers were dried over anhydrous
sodium sulphate and evaporated under reduced pres-
sure. The residue was purified by column chromatogra-
silica gel (EtOAcꢀ
solid (20 g, 95%). M.p.: 66ꢀ
/
n-hexane, 1/1) to afford 21 as a white
)
/
68 8C. IR (KBr) (cmꢁ1
3309, 2963, 1704, 1633, 1548, 1515, 1459, 1370, 1304,
1
1252, 1173, 1160, 1036, 830, 696, 589. H-NMR (200
MHz, CDCl3) d 7.20 (d, Jꢃ
Hz, 2H), 5.85 (br, 1H), 4.36 (d, Jꢃ
3H), 2.63ꢀ2.00 (m, 5H), 2.13 (s, 3H), 1.00 (d, Jꢃ
3H). MS (APCI, Pos.) m/z 264 [Mꢂ
H]ꢂ; HRMS
(MALDI-TOF, Pos.) calc. for C15H21N1O3ꢂ
Hꢂ:
264.1600; found: 264.1585. TLC Rf 0.13 (EtOAcꢀn-
/
8.8 Hz, 2H), 6.86 (d, Jꢃ
5.6 Hz, 2H), 3.80 (s,
6.4 Hz,
/
8.8
/
/
/
/
/
/
hexane, 1/1).
phy on silica gel (EtOAcꢀn-hexane, 1/1) to afford 20 as
/
a white crystal (13 g, 95%). M.p.: 130ꢀ
/
131 8C. IR (KBr)
5.1.5.5. 1-(4-Methoxybenzyl)-4,6-dimethyl-3,4-
(cmꢁ1) 3259, 2952, 1626, 1514, 1479, 1413, 1299, 1249,
1170, 1101, 1065, 1025, 974, 902, 838, 819, 767, 742, 619,
dihydropyridin-2-one (23). Compound 23 was prepared
from 21 in 99% yield according to the same procedure as
described for the preparation of 22 from 20. Colourless
oil; IR (neat) (cmꢁ1) 2957, 2836, 1674, 1614, 1513, 1457,
1388, 1248, 1176, 1110, 1034, 959, 891, 819, 772, 659,
583, 528. 1H-NMR (200 MHz, CDCl3) d 7.31 (d, Jꢃ
/
9.0
4.70 (m, 2H),
2.05 (m, 3H),
13.6, 12.8, 3.6 Hz,
6.6 Hz, 3H). MS (APCI, Pos.) m/z 250
H]ꢂ; HRMS (MALDI-TOF, Pos.) calc. for
C14H19N1O3ꢂ
Hꢂ: 250.1443; found: 250.1437. TLC Rf
0.24 (EtOAcꢀn-hexane, 1/1).
Hz, 2H), 6.80 (d, Jꢃ
4.40ꢀ4.25 (m, 1H), 3.75 (s, 3H), 2.80ꢀ
1.95ꢀ1.60 (m, 2H), 1.47 (ddd, Jꢃ
1H), 1.04 (d, Jꢃ
[Mꢂ
/
9.0 Hz, 2H), 5.10ꢀ
/
/
/
/
/
548. 1H-NMR (200 MHz, CDCl3) d 7.11 (d, Jꢃ
2H), 6.84 (d, Jꢃ8.8 Hz, 2H), 4.92 (d, Jꢃ15.8 Hz, 1H),
4.89 (s, 1H), 4.70 (d, Jꢃ15.8 Hz, 1H), 3.78 (s, 3H),
2.80ꢀ2.20 (m, 3H), 1.85 (s, 3H), 1.04 (d, Jꢃ7.0 Hz, 3H).
MS (APCI, Pos.) m/z 246 [Mꢂ
H]ꢂ; HRMS (MALDI-
TOF, Pos.) calc. for C15H19N1O2ꢂ
Hꢂ: 246.1494;
found: 246.1477. TLC Rf 0.58 (EtOAcꢀn-hexane, 1/1).
/8.8 Hz,
/
/
/
/
/
/
/
/
/
/
/
5.1.5.3. 1-(4-Methoxybenzyl)-4-methyl-3,4-
/
dihydropyridin-2-one (22). A solution of 20 (3.4 g, 13.6
mmol) and p-toluenesulfonic acid monohydrate (150
mg, 0.8 mmol) in toluene (70 mL) was stirred for 1.5 h at
80 8C. After cooling at r.t., the reaction mixture was
diluted with EtOAc and washed sequentially with
saturated aqueous sodium bicarbonate and brine. The
organic layer was dried over magnesium sulphate and
evaporated under reduced pressure. The residue was
purified by column chromatography on silica gel
5.1.6. General procedure for preparation of compounds
27 and 29
5.1.6.1. (DL)-(1S,5S,6R)-7,7-dibromo-2-(4-
methoxybenzyl)-5-methyl-2-azabicyclo[4.1.0]heptan-3-
one (24-anti) and (DL)-(1R,5S,6S)-7,7-dibromo-2-(4-
methoxybenzyl)-5-methyl-2-azabicyclo[4.1.0]heptan-3-
one (24-syn). To a stirred solution of 22 (2 g, 8.7 mmol)
in CHBr3 (25 mL, 287 mmol) were added aliquat-336
(0.1 mL, 0.22 mmol) and 50% aqueous sodium hydrox-
ide (6.4 g) at 0 8C under an argon atmosphere. The
reaction mixture was stirred for 22 h at 0 8C. After
completing the reaction, the reaction mixture was
treated with saturated aqueous ammonium chloride
and extracted with Et2O. The organic layer was washed
with brine and dried over magnesium sulphate, and
evaporated under reduced pressure. The residue was
(EtOAcꢀ
/
n-hexane, 1/5ꢀ1/3) to afford 22 as a pale
/
yellow oil (1.9 g, 60%). IR (neat) (cmꢁ1) 3421, 2957,
2836, 1668, 1612, 1585, 1513, 1457, 1409, 1385, 1304,
1248, 1212, 1176, 1147, 1105, 1034, 946, 822, 717, 580,
518. 1H-NMR (200 MHz, CDCl3) d 7.22 (d, Jꢃ
2H), 6.85 (d, Jꢃ8.6 Hz, 2H), 5.96 (dd, Jꢃ
1H), 5.03 (dd, Jꢃ7.8, 3.3 Hz, 1H), 4.70ꢀ4.50 (m, 2H),
3.79 (s, 3H), 2.80ꢀ2.05 (m, 3H), 1.05 (d, Jꢃ6.8 Hz, 3H).
MS (APCI, Pos.) m/z 232 [Mꢂ
H]ꢂ; HRMS (MALDI-
/
8.6 Hz,
/
/
7.8, 1.5 Hz,
/
/
/
/
/

Y. Kawanaka et al. / European Journal of Medicinal Chemistry 38 (2003) 277ꢀ
/288
285
purified by column chromatography on silica gel
(EtOAcꢀn-hexane, 1/5ꢀ1/3) to afford 24-anti as a pale
yellow oil (1.1 g, 30%) and 24-syn as a pale yellow oil
1309, 1017, 932, 882, 763, 707, 522. 1H-NMR (200
MHz, CDCl3) d 6.86 (brs, 1H), 3.38 (dd, Jꢃ10.0, 4.0
Hz, 1H), 2.52ꢀ2.34 (m, 3H), 2.18 (dd, Jꢃ10.0, 6.2 Hz,
1H), 1.33 (d, Jꢃ6.2 Hz, 3H). MS (APCI, Pos.) m/z 286
[Mꢂ
H, 81Br, 81Br]ꢂ, 284 [MꢂH, 79Br, 81Br]ꢂ, 282
[Mꢂ
H, 79Br, 79Br]ꢂ; HRMS (MALDI-TOF, Pos.) calc.
for C7H9Br2N1O1ꢂ
Hꢂ: 281.9129; found: 281.9117.
TLC Rf 0.32 (EtOAcꢀn-hexane, 1/2).
/
/
/
/
/
(420 mg, 12%).
/
5.1.6.1.1. Compound 24-anti. IR (neat) (cmꢁ1) 2961,
2836, 1667, 1612, 1513, 1415, 1379, 1248, 1175, 1075,
1033, 845, 764, 721, 581, 503. 1H-NMR (200 MHz,
/
/
/
/
CDCl3) d 7.29 (d, Jꢃ
2H), 5.47 (d, Jꢃ14.8 Hz, 1H), 3.81 (d, Jꢃ
3.81 (s, 3H), 2.98 (d, Jꢃ9.8 Hz, 1H), 2.40ꢀ
1.77 (dd, Jꢃ9.8, 5.4 Hz, 1H), 1.26 (m, 3H). MS (APCI,
Pos.) m/z 404 [Mꢂ
H, 79Br, 81Br]ꢂ; HRMS (MALDI-
TOF, Pos.) calc. for C15H17Br2N1O2ꢂ
Hꢂ: 401.9704;
n-hexane, 1/2).
/
8.4 Hz, 2H), 6.89 (d, Jꢃ
14.8 Hz, 1H),
2.00 (m, 3H),
/
8.4 Hz,
/
/
/
/
/
5.1.6.4. (DL)-(1R,5S,6R)-5-methyl-2-
/
azabicyclo[4.1.0]heptan-3-one (27-anti). To a stirred
mixture of 26-anti (530 mg, 1.9 mmol) in benzene (1 mL)
were added n-Bu3SnH (4 mL, 14.9 mmol), azobisisobu-
tylonitrile (42 mg, 0.26 mmol) and the reaction mixture
was stirred with heating at reflux temperature for 2 h
under an argon atmosphere. After cooling in an ice
bath, the reaction mixture was diluted with EtOAc (10
mL) and washed with 10% aqueous potassium fluoride
(10 mL). Insoluble substance was removed by filtration.
The organic layer was washed with brine and dried over
magnesium sulphate, and evaporated under reduced
pressure. The residue was purified by column chroma-
/
/
found: 401.9717. TLC Rf 0.38 (EtOAcꢀ
/
5.1.6.1.2. Compound 24-syn. IR (neat) (cmꢁ1) 2962,
2836, 1652, 1513, 1456, 1415, 1389, 1341, 1303, 1249,
1176, 1108, 1033, 885, 849, 773, 582, 545. 1H-NMR (200
MHz, CDCl3) d 7.33 (d, Jꢃ
Hz, 2H), 4.93 (d, Jꢃ14.2 Hz, 1H), 4.54 (d, Jꢃ
1H), 3.82 (s, 3H), 3.27 (d, Jꢃ10.2 Hz, 1H), 2.50ꢀ
(m, 3H), 2.20ꢀ2.10 (m, 1H), 1.28 (d, Jꢃ6.2 Hz, 3H).
MS (APCI, Pos.) m/z 404 [Mꢂ
H, 79Br, 81Br]ꢂ; HRMS
(MALDI-TOF, Pos.) calc. for C15H17Br2N1O2ꢂ
Hꢂ:
401.9704; found: 401.9703. TLC Rf 0.18 (EtOAcꢀn-
/
8.6Hz, 2H), 6.91 (d, Jꢃ
14.2 Hz,
2.45
/
8.6
/
/
/
/
/
/
/
/
tography on silica gel (EtOAcꢀ
afford 27-anti as a white powder (214 mg, 90%). H-
NMR (200 MHz, CDCl3) d 6.50ꢀ6.05 (brs, 1H), 2.70ꢀ
2.56 (m, 1H), 2.24ꢀ1.76 (m, 3H), 1.19 (d, Jꢃ6.6 Hz,
3H), 1.05ꢀ0.84 (m, 2H), 0.60ꢀ0.40 (m, 1H). MS (APCI,
Pos.) m/z 126 [Mꢂ
H]ꢂ; HRMS (MALDI-TOF, Pos.)
calc. for C7H11N1O1ꢂ
Hꢂ: 126.0919; found: 126.0906.
TLC Rf 0.11 (EtOAcꢀn-hexane, 4/1).
/
n-hexane, 1/6ꢀ2/1) to
/
1
/
hexane, 1/2).
/
/
/
/
5.1.6.2. (DL)-(1S,5S,6R)-7,7-dibromo-5-methyl-2-
/
/
azabicyclo[4.1.0]heptan-3-one (26-anti). A solution of
24-anti (993 mg, 2.5 mmol), anisole (1.7 mL, 15.6 mmol)
/
/
in BF3×
/
OEt2 complex (4.0 mL) was stirred for 35 h at
/
100 8C under an argon atmosphere. After cooling in an
ice bath, water and 5 M NaOH were slowly added to the
reaction mixture. The resulting mixture was extracted
with CHCl3 and washed with saturated aqueous sodium
bicarbonate and then brine. The organic layer was dried
over magnesium sulphate and evaporated under reduced
pressure. The residue was purified by column chroma-
5.1.6.5. (DL)-(1S,5S,6S)-5-methyl-2-
azabicyclo[4.1.0]heptan-3-one (27-syn). Compound 27-
syn was prepared from 26-syn in 91% yield according to
the same procedure as described for the preparation of
27-anti from 26-anti. White powder; 1H-NMR (200
MHz, DMSO-d6) d 7.86 (brs, 1H), 2.60 (m, 1H), 2.22
tography on silica gel (EtOAcꢀ
afford 26-anti as a white powder (530 mg, 36%). M.p.:
106ꢀ
109 8C. IR (KBr) (cmꢁ1) 3205, 3099, 2960, 1656,
1474, 1452, 1407, 1365, 1352, 1286, 1200, 1121, 1096,
1
1072, 1015, 933, 884, 774, 717, 553, 497, 467. H-NMR
/
n-hexane, 1/2ꢀ
/
2/1) to
(m, 1H), 1.98 (dd, Jꢃ
16.2, 11.8 Hz, 1H), 1.17 (m, 1H), 1.01 (d, Jꢃ
3H), 0.58ꢀ0.44 (m, 2H). MS (APCI, Pos.) m/z 126 [Mꢂ
H]ꢂ; HRMS (MALDI-TOF, Pos.) calc. for
C7H11N1O1ꢂ
Hꢂ: 126.0919; found: 126.0933. TLC Rf
0.12 (EtOAcꢀn-hexane, 1/2).
/
16.2, 6.0 Hz, 1H), 1.64 (dd, Jꢃ
/
/6.4 Hz,
/
/
/
/
(200 MHz, CDCl3) d 7.40ꢀ
9.8 Hz, 1H), 2.30ꢀ1.95 (m, 3H), 1.80 (dd, Jꢃ
Hz, 1H), 1.29 (d, Jꢃ6.0 Hz, 3H). MS (APCI, Pos.) m/z
286 [Mꢂ
H, 81Br, 81Br]ꢂ, 284 [MꢂH, 79Br, 81Br]ꢂ, 282
[Mꢂ
H, 79Br, 79Br]ꢂ; HRMS (MALDI-TOF, Pos.) calc.
for C7H9Br2N1O1ꢂ
Hꢂ: 281.9129; found: 281.9130.
TLC Rf 0.75 (CHCl3ꢀMeOH, 10/1).
/
6.90 (brs, 1H), 3.20 (d, Jꢃ
/
/
/
/9.8, 4.4
/
5.1.6.6. (DL)-(1S,5S,6R)-7,7-dibromo-2-(4-
/
/
methoxybenzyl)-1,5-dimethyl-2-azabicyclo[4.1.0]heptan-
3-one (25-anti). Compounds 25-anti and 25-syn were
prepared from 23 in 26 and 7% yields, respectively,
according to the same procedure as described for the
preparation of 24-anti and 24-syn from 22.
/
/
/
5.1.6.3. (DL)-(1R,5S,6S)-7,7-dibromo-5-methyl-2-
azabicyclo[4.1.0]heptan-3-one (26-syn). Compound 26-
syn was prepared from 24-syn in 61% yield according to
the same procedure as described for the preparation of
Pale yellow solid; m.p.: 118ꢀ )
119 8C. IR (KBr) (cmꢁ1
3435, 2965, 1655, 1512, 1438, 1305, 1243, 1182, 1033,
/
1
856, 756, 588, 512. H-NMR (200 MHz, CDCl3) d 7.24
(d, Jꢃ
Jꢃ15.4 Hz, 1H), 3.83 (d, Jꢃ
2.40ꢀ2.00 (m, 3H), 1.61ꢀ1.40 (m, 1H), 1.50 (s, 3H), 1.31
/
8.8 Hz, 2H), 6.84 (d, Jꢃ/8.8 Hz, 2H), 5.37 (d,
26-anti from 24-anti. White powder; m.p.: 113ꢀ
/
116 8C.
/
/15.4 Hz, 1H), 3.79 (s, 3H),
IR (KBr) (cmꢁ1) 3196, 2967, 1658, 1475, 1385, 1343,
/
/

286
Y. Kawanaka et al. / European Journal of Medicinal Chemistry 38 (2003) 277ꢀ288
/
(d, Jꢃ
/
6.2 Hz, 3H). MS (APCI, Pos.) m/z 420 [Mꢂ
/
H,
NMR (200 MHz, CDCl3) d 6.21 (brs, 1H), 2.17 (dd, Jꢃ
6.6, 2.2 Hz, 1H), 2.08 (dd, Jꢃ6.6, 3.8 Hz, 1H), 1.93 (m,
1H), 1.36 (s, 3H), 1.17 (d, Jꢃ6.6 Hz, 3H), 0.86ꢀ0.79 (m,
2H), 0.66 (m, 1H). MS (FAB, Pos.) m/z 140 [Mꢂ
H]ꢂ;
HRMS (MALDI-TOF, Pos.) calc. for C8H13N1O1ꢂ
Hꢂ: 140.1075; found: 140.1049. TLC Rf 0.23 (EtOAcꢀ
/
81Br, 81Br]ꢂ, 418 [Mꢂ
/
H, 79Br, 81Br]ꢂ, 416 [Mꢂ
/
H,
79Br, 79Br]ꢂ; HRMS (MALDI-TOF, Pos.) calc. for
C16H19Br2N1O2ꢂ
/
/
/
/
Hꢂ: 415.9861; found: 415.9845. TLC
/
Rf 0.40 (EtOAcꢀn-hexane, 1/2).
/
/
/
5.1.6.7. (DL)-(1R,5S,6S)-7,7-dibromo-2-(4-
n-hexane, 4/1).
methoxybenzyl)-1,5-dimethyl-2-azabicyclo[4.1.0]heptan-
3-one (25-syn). Pale yellow oil; IR (neat) (cmꢁ1) 2961,
2835, 1652, 1514, 1455, 1404, 1304, 1248, 1177, 1108,
1034, 808, 759, 648, 540. 1H-NMR (200 MHz, CDCl3) d
5.1.6.11. (DL)-(1S,5S,6S)-1,5-dimethyl-2-
azabicyclo[4.1.0]heptan-3-one (29-syn). Compound 29-
syn was prepared from 28-syn in 87% yield according to
the same procedure as described for the preparation of
27-anti from 26-anti. Pale yellow powder; 1H-NMR
(200 MHz, CDCl3) d 6.30 (brs, 1H), 2.36 (m, 1H), 2.26
7.28 (d, Jꢃ
(d, Jꢃ15.6 Hz, 1H), 4.29 (d, Jꢃ
3H), 2.80ꢀ2.30 (m, 3H), 1.70ꢀ1.50 (m, 1H), 1.66 (s, 3H),
1.31 (d, Jꢃ
H, 81Br, 81Br]ꢂ, 418 [Mꢂ
/
8.8 Hz, 2H), 6.86 (d, Jꢃ/8.8 Hz, 2H), 5.17
/
/15.6 Hz, 1H), 3.80 (s,
/
/
/
6.4 Hz, 3H). MS (APCI, Pos.) m/z 420 [Mꢂ
/
(m, 1H), 1.66 (dd, Jꢃ
1.13 (m, 1H), 1.08 (d, Jꢃ
Hz, 1H), 0.58 (dd, Jꢃ9.2, 6.0 Hz, 1H). MS (APCI,
Pos.) m/z 140 [Mꢂ
H]ꢂ: HRMS (MALDI-TOF, Pos.)
calc. for C8H13N1O1ꢂ
Hꢂ: 140.1075; found: 140.1082.
TLC Rf 0.22 (EtOAcꢀn-hexane, 4/1).
/16.6, 11.6 Hz, 1H), 1.38 (s, 3H),
/
H, 79Br, 81Br]ꢂ, 416 [Mꢂ
/H,
/
6.4 Hz, 3H), 0.71 (t, Jꢃ6.0
/
79Br, 79Br]ꢂ; HRMS (MALDI-TOF, Pos.) calc. for
C16H19Br2N1O2ꢂ
/
/
Hꢂ: 415.9861; found: 415.9868. TLC
/
Rf 0.31 (EtOAcꢀn-hexane, 1/2).
/
/
/
5.1.6.8. (DL)-(1S,5S,6R)-7,7-dibromo-1,5-dimethyl-2-
azabicyclo[4.1.0]heptan-3-one (28-anti). Compound
28-anti was prepared from 25-anti in 90% yield accord-
ing to the same procedure as described for the prepara-
tion of 26-anti from 24-anti. Pale yellow powder; m.p.:
5.1.7. Genaral procedure for preparation of compounds 2,
4, 6, 7, 8 and 9
5.1.7.1. 6-Methyl-3-azabicyclo[4.1.0]heptan-4-imine
hydrochloride salt (2). To a stirred solution of 12 (781
mg, 6.2 mmol) in CH2Cl2 (6 mL) was added triethylox-
onium tetrafluoroborate (2 M in CH2Cl2, 3.5 mL, 7
mmol) under an argon atmosphere, and the reaction
mixture was stirred at r.t. for 5 h. Concentration of the
reaction mixture was done under reduced pressure, and
it was used for the subsequent reaction without further
purification.
To a stirred solution of the compound obtained above
in EtOH (3 mL) was added saturated ethanolic ammo-
nia (12 mL) under an argon atmosphere at r.t. and
stirring was continued for an additional 24 h. Then the
reaction mixture was diluted with CHCl3 (15 mL) and
the resulting precipitates were removed by filtration.
The filtrate was concentrated under reduced pressure.
The residue was treated with 2 M NaOH (17 mL) and
165ꢀ
1394, 1272, 1184, 1055, 992, 804, 767, 649, 556. 1H-
NMR (200 MHz, CDCl3) d 6.10 (brs, 1H), 2.27 (dd, Jꢃ
13.0, 2.8 Hz, 1H), 2.08ꢀ1.95 (m, 2H), 1.72 (s, 3H), 1.54
(dd, Jꢃ4.5, 1.1 Hz, 1H), 1.31 (d, Jꢃ6.4 Hz, 3H); MS
(APCI, Pos.) m/z 300 [Mꢂ H,
H, 81Br, 81Br]ꢂ, 298 [Mꢂ
79Br, 81Br]ꢂ, 296 [MꢂH, 79Br, 79Br]ꢂ; HRMS
(MALDI-TOF, Pos.) calc. for C8H11Br2N1O1ꢂ
Hꢂ:
295.9286; found: 295.9293. TLC Rf 0.37 (EtOAcꢀn-
hexane, 4/1).
/
167 8C. IR (KBr) (cmꢁ1) 3183, 3083, 2960, 1669,
/
/
/
/
/
/
/
/
/
5.1.6.9. (DL)-(1R,5S,6S)-7,7-dibromo-1,5-dimethyl-2-
azabicyclo[4.1.0]heptan-3-one (28-syn). Compound 28-
syn was prepared from 25-syn in 76% yield according to
the same procedure as described for the preparation of
26-anti from 24-anti. Pale yellow powder; m.p.: 128ꢀ
/
130 8C. IR (KBr) (cmꢁ1) 3434, 3190, 3090, 2961, 1656,
extracted with CHCl3 (30 mLꢄ2). The combined
/
1396, 1302, 1086, 923, 801, 751, 524. 1H-NMR (200
organic layers were dried over anhydrous sodium
sulphate and evaporated under reduced pressure. The
residue was converted to its hydrochloride with 4 M
MHz, CDCl3) d 6.50 (brs, 1H), 2.70ꢀ
(s, 3H), 1.80ꢀ1.50 (m, 1H), 1.34 (d, Jꢃ
(APCI, Pos.) m/z 300 [Mꢂ
H, 81Br, 81Br]ꢂ, 298 [Mꢂ
79Br, 81Br]ꢂ, 296 [MꢂH, 79Br, 79Br]ꢂ; HRMS
(MALDI-TOF, Pos.) calc. for C8H11Br2N1O1ꢂ
Hꢂ:
295.9286; found: 295.9298. TLC Rf 0.30 (EtOAcꢀn-
/
2.30 (m, 3H), 1.77
6.6 Hz, 3H). MS
H,
/
/
/
/
HClꢀ
/
EtOAcꢀ
/
EtOH at 0 8C and further purified by
/
column chromatography on silica gel (CHCl3ꢀ
/MeOH,
/
5/1) to afford 2 as a pale yellow powder (657 mg, 66%).
IR (KBr) (cmꢁ1) 3273, 3109, 2926, 1733, 1674, 1453,
/
1
hexane, 1/1).
1023, 750, 698. H-NMR (200 MHz, CDCl3) d 9.70ꢀ
/
9.50 (brs, 1H), 9.10ꢀ
3.75ꢀ3.50 (m, 2H), 3.13 (d, Jꢃ
Jꢃ17.5 Hz, 1H), 1.21 (s, 3H), 1.15ꢀ
0.47 (m, 2H). M.p.: 191ꢀ192 8C. MS and HRMS
analysis showed only the ion from C7H12N2 correspond-
ing to the loss of HCl, MS (APCI, Pos.) m/z 125 [Mꢂ
/
8.90 (brs, 1H), 8.75ꢀ
17.5 Hz, 1H), 2.66 (d,
1.02 (m, 1H), 0.67ꢀ
/
8.50 (brs, 1H),
5.1.6.10. (DL)-(1R,5S,6R)-1,5-dimethyl-2-
/
/
azabicyclo[4.1.0]heptan-3-one (29-anti). Compound
29-anti was prepared from 28-anti in 14% yield accord-
ing to the same procedure as described for the prepara-
/
/
/
/
1
tion of 27-anti from 26-anti. Pale yellow powder; H-
/

Y. Kawanaka et al. / European Journal of Medicinal Chemistry 38 (2003) 277ꢀ
/288
287
H]ꢂ; HRMS (MALDI-TOF, Pos.) calc. for C7H12N2ꢂ
Hꢂ: 125.1079; found: 125.1062. TLC Rf 0.29 (CHCl3ꢀ
MeOHꢀAcOH, 20/4/1).
/
Compound 8 was prepared from 29-syn in 16% yield
according to the same procedure as described for the
/
/
preparation of 2 from 12. Orange oil; IR (neat) (cmꢁ1
)
3404, 2976, 1673, 1459, 1212, 1111. 1H-NMR (200
MHz, CDCl3) d 10.18 (brs, 1H), 8.75 (brs, 1H), 8.57
5.1.7.2. (DL)-(1S,6S)-3-azabicyclo[4.2.0]octan-4-
imine hydrochloride salt (4). Compound 4 was prepared
from 17 in 33% yield according to the same procedure as
described for the preparation of 2 from 12. Beige
powder; IR (KBr) (cmꢁ1) 3317, 3104, 2975, 2933,
1677, 1520, 1435, 1418, 1361, 1329, 1197, 1158, 960,
814, 680, 604, 548, 491. 1H-NMR (200 MHz, DMSO-d6)
(brs, 1H), 2.80 (m, 1H), 2.38ꢀ
(m, 1H), 1.52 (s, 3H), 1.32ꢀ1.18 (m, 1H), 1.14 (3H, d,
Jꢃ6.4 Hz, 3H), 0.82 (m, 1H), 0.71 (m, 1H). MS and
/
2.16 (m, 1H), 2.00ꢀ
/
1.78
/
/
HRMS analysis showed only the ion from C8H14N2
corresponding to the loss of HCl, MS (APCI, Pos.) m/z
139 [Mꢂ
C8H14N2ꢂ
(CHCl3ꢀMeOHꢀ
/
H]ꢂ; HRMS (MALDI-TOF, Pos.) calc. for
d 9.74 (brs, 1H), 9.20 (brs, 1H), 8.83 (brs, 1H), 3.28ꢀ
3.12 (m, 2H), 2.94ꢀ2.42 (m, 4H), 2.28ꢀ2.02 (m, 2H),
1.67 (m, 1H), 1.46 (m, 1H). M.p.: 138ꢀ140 8C. MS and
/
/
Hꢂ: 139.1235; found: 139.1253. TLC Rf 0.43
AcOH, 10/1/1).
/
/
/
/
/
HRMS analysis showed only the ion from C7H12N2
corresponding to the loss of HCl, MS (APCI, Pos.) m/z
5.1.7.6. (DL)-(1R,5S,6R)-1,5-dimethyl-2-
azabicyclo[4.1.0]heptan-3-imine hydrochloride salt (9).
Compound 9 was prepared from 29-anti in 59% yield
according to the same procedure as described for the
125 [Mꢂ
C7H12N2ꢂ
(CHCl3ꢀMeOHꢀ
/
H]ꢂ; HRMS (MALDI-TOF, Pos.) calc. for
/
Hꢂ: 125.1079; found: 125.1097. TLC Rf 0.16
AcOH, 20/2/1).
/
/
preparation of 2 from 12. Yellow oil; IR (neat) (cmꢁ1
)
3370, 1673, 1522, 1458, 1158, 1114. 1H-NMR (200
MHz, DMSO-d6) d 10.12 (brs, 1H), 8.92 (brs, 1H),
5.1.7.3. (DL)-(1R,5S,6R)-5-methyl-2-
azabicyclo[4.1.0]heptan-3-imine hydrochloride salt (6).
Compound 6 was prepared from 27-anti in 34% yield
according to the same procedure as described for the
preparation of 2 from 12. White powder; IR (KBr)
(cmꢁ1) 3108, 1670, 1626, 1418, 1351, 1227, 1030, 894,
8.38 (brs, 1H), 2.44 (dd, Jꢃ
Jꢃ16.6, 7.0 Hz, 1H), 1.98 (m, 1H), 1.36 (s, 3H), 1.06 (d,
Jꢃ7.0 Hz, 3H), 1.00ꢀ0.90 (m, 1H), 0.85ꢀ0.81 (m, 2H).
MS and HRMS analysis showed only the ion from
C8H14N2 corresponding to the loss of HCl, MS (FAB,
/
16.6, 5.4 Hz, 1H), 2.28 (d,
/
/
/
/
1
796, 725. H-NMR (200 MHz, DMSO-d6) d 9.98 (brs,
1H), 8.95 (brs, 1H), 8.54 (brs, 1H), 2.82 (m, 1H), 2.46
Pos.) m/z 139 [Mꢂ
calc. for C8H14N2ꢂ
TLC Rf 0.41 (CHCl3ꢀ
/
H]ꢂ; HRMS (MALDI-TOF, Pos.)
/
Hꢂ: 139.1235; found: 139.1214.
MeOHꢀAcOH, 10/1/1).
(dd, Jꢃ
/
16.0, 5.0 Hz, 1H), 2.27 (dd, Jꢃ/16.0, 7.0 Hz,
/
/
1H), 2.02 (m, 1H), 1.2ꢀ
/
1.0 (m, 1H), 1.08 (d, Jꢃ/6.8 Hz,
3H), 0.88 (m, 1H), 0.73 (m, 1H). MS and HRMS
analysis showed only the ion from C7H12N2 correspond-
5.2. Biological assays
ing to the loss of HCl, MS (APCI, Pos.) m/z 125 [Mꢂ
H]ꢂ; HRMS (MALDI-TOF, Pos.) calc. for C7H12N2ꢂ
Hꢂ: 125.1079; found: 125.1054; TLC Rf 0.18 (CHCl3ꢀ
MeOHꢀAcOH, 15/2/1).
/
5.2.1. Preparation of partially purified enzyme and
determination of Ki values
/
/
Human eNOS was overexpressed in Sf-21 cells, by
infecting the cells with baculovirus carrying heNOS
cDNA. The hiNOS was overexpressed in A549 by
stimulation with LPS (10 mg mLꢁ1) plus cytokines (10
ng mLꢁ1 TNF-a, 5 ng mLꢁ1 IL-1b and 100 ng mLꢁ1
interferon-g). Human eNOS and iNOS were partially
purified by chromatography on 2?, 5?-ADP-Sepharose
gels. NOS activity was determined by the method for the
/
5.1.7.4. (DL)-(1S,5S,6S)-5-methyl-2-
azabicyclo[4.1.0]heptan-3-imine hydrochloride salt (7).
Compound 7 was prepared from 27-syn in 61% yield
according to the same procedure as described for the
preparation of 2 from 12. Pale yellow amorphous; IR
(KBr) (cmꢁ1) 2955, 1677, 1423, 1370, 1150, 1082, 1020,
973, 885, 841, 736, 457, 431. 1H-NMR (200 MHz,
DMSO-d6) d 9.98 (brs, 1H), 8.72 (brs, 1H), 8.30 (brs,
conversion of [14C]-
minor modification. The conversion rates for various
concentrations of the test compounds and -arginine
L-arginine to L-citrulline with a
L
1H), 2.90 (m, 1H), 2.36 (m, 1H), 2.20ꢀ
(m, 1H), 1.02 (d, Jꢃ6.2 Hz, 3H), 0.82 (dt, Jꢃ
Hz, 1H), 0.64 (dt, Jꢃ9.2, 6.4 Hz, 1H). M.p.: 112ꢀ
/
1.98 (m, 2H), 1.30
were measured. Dixon and lineweaver-Bulk plots were
constructed to determine the Ki values and the mode of
inhibition.
/
/6.4, 3.4
/
/
114 8C. MS and HRMS analysis showed only the ion
from C7H12N2 corresponding to the loss of HCl, MS
Selectivity was evaluated as the rate of the IC50 values
for heNOS and hiNOS.
(APCI, Pos.) m/z 125 [Mꢂ
TOF, Pos.) calc. for C7H12N2ꢂ
125.1087. TLC Rf 0.21 (CHCl3ꢀMeOHꢀ
/
H]ꢂ; HRMS (MALDI-
/
Hꢂ: 125.1079; found:
AcOH, 20/2/1).
5.2.2. Enzyme assay with recombinant mouse iNOS
Recombinant mouse iNOS was purchased from Cay-
man Chemical (Cat. No. 60862) and the inhibitory
activities of the test compounds were evaluated by
/
/
5.1.7.5. (DL)-(1S,5S,6S)-1,5-dimethyl-2-
azabicyclo[4.1.0]heptan-3-imine hydrochloride salt (8).
measuring the conversion rate from [14C]-
L-arginine to

288
Y. Kawanaka et al. / European Journal of Medicinal Chemistry 38 (2003) 277ꢀ288
/
[14C]-
L
-citrulline, and then the IC50 values were deter-
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38 (1995) 4343ꢀ4362.
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mined.
The ID50 value was determined from a logꢀ
transformation of the doseꢀresponse curves (5 and 6; 3,
10, 30 mg kgꢁ1, sc -NMMA; 10, 30 and 100 mg kgꢁ1
/
9685.
/logit
/
/
L
,
/
sc). The ID50 value was defined as the concentration of
test compound that produced a 50% inhibition in the
NOx accumulation induced by LPS treatment alone
[26]. The MTD was defined as the maximum dose at
which no death was observed within 24 h after an iv
injection administration. The doses used were 5, 10, 20,
30, 40 and 50 mg kgꢁ1 for 5 and 6 and 1000, 2000, 3000,
/
/
887.
[14] W.M. Moore, R.K. Webber, K.F. Fok, G.M. Jerome, J.R.
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672.
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/
481.
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Manning, B.S. Pitzele, M.V. Toth, M. Trivedi, M.E. Zupec, F.S.
4000 and 5000 mg kgꢁ1 for
L-NMMA.
Tjoeng, J. Med. Chem. 41 (1998) 96ꢀ101.
/
5.2.3. Inhibition of NOx accumulation and the maximum
tolerated dose (MTD) in mice
[17] W.K. Hagmann, C.G. Galdwell, P. Chen, P.L. Durette, C.K.
Esser, T.J. Lanza, I.E. Kopka, R. Guthikonda, S.K. Shah, M.
MacCoss, R.M. Chabin, D. Fletcher, S.K. Grant, B.G. Green,
J.L. Humes, T.M. Kelly, S. Luell, R. Meurer, V. Moore, S.G.
Pacholok, T. Pavia, H.R. Williams, K.K. Wong, Bioorg. Med.
The test compounds or saline were administered
subcutaneously at 3 h after the LPS (10 mg kgꢁ1, iv)
injection into 7 weeks old Balb/c mice (Charles River
Japan, Inc.). Blood was collected by venipuncture from
the abdominal aorta under light anaesthesia at 6 h after
LPS treatment. Plasma was obtained by centrifugation
and the concentration of accumulated NOx over 3 h was
determined by the method described in ref. [26]. To
evaluate the acute toxicity, the MTD (iv maximum dose
where no death was observed within 24 h after the
administration) of the test compound was determined.
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/
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/
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/
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/
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