Piperidine carboxylic acid derivatives of 10H-pyrazino[2,3-b][1,4] benzothiazine as orally-active adhesion molecule inhibitors

Article abstract of DOI:10.1248/cpb.52.675

Novel piperidine carboxylic acid derivatives of 10H-pyrazino[2,3-b][1,4] benzothiazine were prepared and evaluated for their inhibitory activity on the upregulation of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). Replacement of the methanesulfonyl group on the piperidine ring of previously prepared derivatives with a carboxylic acid-containing moiety resulted in a number of potent adhesion molecule inhibitors. Of these, (anti) [3-(10H-pyrazino[2,3-b][1,4]benzothiazin-8-yl)methyl-3-azabicyclo[3.3.1] non-9-yl]acetic acid 2q (ER-49890), showed the most potent oral inhibitory activities against neutrophil migration in an interleukin-1 (IL-1) induced paw inflammation model using mice, and leukocyte accumulation in a carrageenan pleurisy model in the rat, and therapeutic effect on collagen-induced arthritis in rats.

Full text of DOI:10.1248/cpb.52.675

June 2004
Chem. Pharm. Bull. 52(6) 675—687 (2004)
675
Piperidine Carboxylic Acid Derivatives of 10H-Pyrazino[2,3-
b][1,4]benzothiazine as Orally-Active Adhesion Molecule Inhibitors
Toshihiko KANEKO, ,a Richard S. J. CLARK,a Norihito OHI,a Fumihiro OZAKI,a Tetsuya KAWAHARA,a
*
Atsushi KAMADA,a Kazuo OKANO,a Hiromitsu YOKOHAMA,a Masayoshi OHKURO,a Kenzo MURAMOTO,a
b
Osamu TAKENAKA,a and Seiichi KOBAYASHI
a Tsukuba Research Laboratories, Eisai Co. Ltd.; 5–1–3, Tokodai, Tsukuba, Ibaraki 300–2635, Japan: and b Eisai Research
Institute of Boston, Inc.; 4 Corporate Drive, Andover, Massachusetts, 01810–2441, U.S.A.
Received December 18, 2003; accepted March 3, 2004
Novel piperidine carboxylic acid derivatives of 10H-pyrazino[2,3-b][1,4]benzothiazine were prepared and
evaluated for their inhibitory activity on the upregulation of adhesion molecules such as intercellular adhesion
molecule-1 (ICAM-1). Replacement of the methanesulfonyl group on the piperidine ring of previously prepared
derivatives with a carboxylic acid-containing moiety resulted in a number of potent adhesion molecule inhibitors.
Of these, (anti) [3-(10H-pyrazino[2,3-b][1,4]benzothiazin-8-yl)methyl-3-azabicyclo[3.3.1]non-9-yl]acetic acid 2q
(ER-49890), showed the most potent oral inhibitory activities against neutrophil migration in an interleukin-1
(IL-1) induced paw inflammation model using mice, and leukocyte accumulation in a carrageenan pleurisy
model in the rat, and therapeutic effect on collagen-induced arthritis in rats.
Key words ER-49890; adhesion molecule inhibitor; 10H-pyrazino[2,3-b][1,4]benzothiazine
Adhesion molecules, such as intercellular adhesion mole- ity and suppresses neutrophil infiltration in an IL-1-induced
cule-1 (ICAM-1), E-selectin and vascular cell adhesion mol- paw inflammation model.12) We report here the effect of re-
ecule-1 (VCAM-1), play a pivotal role in the inflammatory placing the piperidine sulfonamide moiety of 1a in order to
and immunological responses. They are initially upregulated investigate further the structure–activity relationships of this
on the endothelium by tumor necrosis factor-a (TNF-a) or family. Of particular interest to us was the introduction of a
interleukin-1 (IL-1) and go on to mediate the steps of leuko- carboxylic acid group, a bioisostere of the sulfonamide func-
cyte migration from the vasculature into the inflamed tissue, tion.
these leukocytes then promote the inflammatory response.1)
Chemistry We first prepared the right moieties of the
Also, the interaction of ICAM-1 and VCAM-1 expressed on compounds 2. The piperidine carboxylates 4a—c are either
antigen presenting cells with their ligands, such as lympho- commercially available or known in the literature.13,14) The
cyte function associated antigen-1 (LFA-1) and very late acti- other piperidine carboxylates 4d—i and 4l—v were prepared
vation antigen-4 (VLA-4), has been involved in activation of as shown in Charts 1—11. The syntheses of 4d and 4e are
T cells.2—5) Recently, the antigen recognition step has been shown in Chart 1. Coupling the aldehyde 615) with the appro-
visualized and ICAM-1 identified as one important compo- priate Horner–Emmons reagent gave the unsaturated ester 7.
nent of the immunological synapse.6) Hence, it is hoped that Hydrogenation of the resulting ester 7 using 10% palladium
interfering with these interactions will not only have an effect on carbon (Pd/C) followed by deprotection of the piperidine
on leukocyte infiltration, but also modulate T cell response. amine group with 4 N HCl/AcOEt provided saturated ester
Furthermore, it has been reported that anti-adhesion mole- 4d. The unsaturated ester 4e was also obtained by removal of
cule antibodies can ameliorate the inflammatory reaction and the Boc group of compound 7 using the same procedure.
immunological parameters in various animal models.7) For
The acetylenic ester 4f was prepared as shown in Chart 2.
these reasons the development of cell adhesion molecule in- Reaction of 4-piperidine aldehyde 9 with the Corey–Fuchs
hibitors as novel therapeutics for inflammatory and autoim-
mune diseases has been widely anticipated, and a consider-
able literature on such compounds has appeared.8—11)
We have reported that the piperidine sulfonamide and sul-
famide derivatives of 10H-pyrazino[2,3-b][1,4]benzothiazine
1a and 1b have potent ICAM-1 upregulation inhibitory activ-
Chart 1
Chart 2
© 2004 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: t3-kaneko@hhc.eisai.co.jp

676
Vol. 52, No. 6
Chart 6
Chart 3
Chart 4
Chart 7
Chart 8
Chart 5
reagent provided dibromo compound 10. This compound was hydrolysis using 10% HCl/MeOH. Treatment of 26 with p-
treated with n-butyllithium (n-BuLi) to afford the acetylide, TsOH, followed by hydrogenation using Pd(OH)2/C afforded
which was reacted with ethyl chloroformate.16) Deprotection the piperidine phenylacetate 4n.
of 11 provided acetylene intermediate 4f.
The (7-azaspiro[3.5]non-2-yl)carboxylate 4o was prepared
The amino-, oxy- and thio-acetic acid piperidine deriva- as shown in Chart 7. The cycloaddition reaction between
tives 4g—i were prepared as shown in Chart 3. The appropri- olefin 2818) and dichloroketene, generated in situ from
ate amino- or hydroxy-piperidine 12 or 14 was alkylated with trichloroacetyl chloride and zinc/copper, followed by reduc-
ethyl bromoacetate followed by deprotection to give com- tion with zinc afforded the 7-azaspiro[3.5]nonane core of 29.
pounds 4g or 4h respectively. Mercaptopiperidine 17 was Treatment of this ketone 29 with tosylmethyl isocyanide
synthesized using the method of Barrera as shown in Chart (TosMIC) in the presence of potassium tert-butoxide af-
3.17) Benzyl piperidone 16 was converted to its dithiol, which forded the cyanide 30. Deprotection of the piperidine amino
on reduction with sodium borohydride gave 17. Condensa- group with trifluoroacetic acid (TFA) followed by reprotec-
tion of 17 with ethyl bromoacetate followed by removal of tion furnished the benzyl amine 31. Hydrolysis of the cyano
the benzyl group using 1-chloroethyl chloroformate (ACE- moiety provided the carboxylic acid. Treatment with thionyl
Cl) afforded compound 4i.
chloride in MeOH, then debenzylation using Pd(OH)2/C gave
The a,a-dimethyl ester 4l was synthesized as shown in amine 4o.
Chart 4. Methylation of carboxylate 8 with methyl iodide fol-
lowed by deprotection using HCl in AcOEt gave 4l.
Compound 4p was synthesized as shown in Chart 8. Com-
pound 33, prepared by treatment of ketone 29 with the appro-
Compound 4m was prepared as shown in Chart 5. N-Alky- priate Horner–Emmons reagent, was hydrogenated over Pd/C
lation of hydroxypiperidine derivative 20 with benzyl bro- to give saturated ester 34. Deprotection of the piperidine
mide followed by dehydration using p-toluenesulfonic acid amino group using HCl/AcOEt gave amine 4p.
(p-TsOH) in toluene gave 1,2,5,6-tetrahydropyridine deriva-
The syntheses of bicyclic amines 4q—v are shown in
tive 21. This bromide 21 was treated with n-BuLi followed Charts 9—11. Compounds 4q and 4r were prepared as
by condensation with diethyl carbonate to furnish ester 22. shown in Chart 9. Hydrogenation of unsaturated ester 3519)
Hydrogenation of 22 using Pearlman’s catalyst yielded the using palladium on carbon provided a anti–syn mixture of 36
benzoate derivative of piperidine 4m.
in the ratio of 3 : 1. This ratio was determined by integration
1
The synthesis of the phenylacetic acid derivative of piperi- of the equatorial protons of C-2 and C-4 in the H-NMR
dine 4n was achieved as illustrated in Chart 6. Treatment of spectra of the 3-azabicyclo[3.3.1]nonanes at 2.90 ppm (anti)
benzyl piperidone 16 with phenyl Grignard reagent 23 fol- and 2.62 ppm (syn) respectively. To separate the isomers, the
lowed by deprotection furnished the benzaldehyde 24. The protective group on the nitrogen was exchanged using vinyl
phenylacetate 26 was obtained by condensation of aldehyde chloroformate. The anti- and syn-isomers were isolated from
24 with methyl methylsulfinylmethyl sulfide (FAMSO) and the mixture using column chromatography (seven times)

June 2004
677
Chart 9
Chart 10
Chart 12
(MOMCl) gave the compound 47, which was reduced with
lithium aluminum hydride (LiAlH4) and chlorinated to afford
the intermediate 49. Cyanation of 49 followed by hydrolysis
and esterification gave 51. Reduction of this ester and
methanesulfonylation followed by condensation with 4d gave
53. Deprotection then hydrolysis of 53 using 5 N HCl fol-
lowed by NaOH afforded target compound 2j.
The synthesis of amide junction derivative 2k is shown in
Chart 14. Hydrolysis of ester 47 and subsequent condensa-
tion with piperidine ester 4d via the acid anhydride gave
piperidine amide 56. Hydrolysis of ester 56 under alkaline
conditions followed by deprotection with 5 N HCl gave car-
boxylic acid 2k.
The synthesis of a,a-dimethyl carboxylic acid derivative
2l is shown in Chart 15. Condensation between 49 and
piperidine ester 4l gave compound 58. Hydrolysis of this
ester 58 under alkaline conditions followed by deprotection
of the N-methoxymethyl group of 59 with 5 N HCl gave car-
boxylic acid 2l.
Chart 11
with a yield of 29% (37) and 13% (38) respectively. Chlori-
nation of 37 or 38 with 4 N hydrochloric acid in dioxane, fol-
lowed by hydrolysis afforded the important intermediates 4q
or 4r respectively.
The syntheses of bicyclic intermediates 4s and 4v are
shown in Chart 10. N-Demethylation of 35 or the known
compound 3920) using ACE-Cl provided the corresponding
secondary amine.
Compounds 4t and 4u were synthesized as shown in Chart
11. Unsaturated esters 42 and 43, prepared by treatment of
the ketones 4021) and 4122) with the appropriate Horner–Em-
mons reagent, were hydrogenated over Pd/C to give saturated
esters 44 and 45. Deprotection of the amino group of the bi-
cyclic ring using ACE-Cl gave the corresponding amines 4t
and 4u.
With the exceptions of 2j, 2k and 2l, piperidine carboxylic
acid derivatives of 10H-pyrazino[2,3-b][1,4]benzothiazine
2a—v were synthesized as shown in Chart 12. Condensation
of readily available chloride 312) with the appropriate piperi-
dine carboxylates 4a—i and 4m—v in DMF using K2CO3
provided the esters 5a—i and 5m—v. Hydrolysis of these
esters under alkaline conditions provided the desired com-
pounds 2a—i and 2m—v.
Results and Discussion
The synthetic compounds were first evaluated for their in
vitro inhibitory activity on the expression of the adhesion
molecule ICAM-1 by TNF-a-stimulated human umbilical
vein endothelial cells (HUVEC). The effect of the com-
pounds on neutrophil accumulation after oral administration
was evaluated in a mouse IL-1-induced paw inflammation
model. In this model, myeloperoxidase activity was mea-
sured as the index of neutrophil infiltration.12)
Table 1 summarizes the results of our initial studies with
carboxylic acid derivatives. We first evaluated piperidine 4-
carboxylic acid 2a. The ICAM-1 inhibitory activity of this
compound was only 3-fold less than that of sulfonamide 1a.
However 2a showed only 61.5% inhibition of neutrophil
accumulation in the IL-1-induced paw model even at
100 mg/kg p.o. Simple replacement of the methansulfon-
The synthesis of 2j is shown in Chart 13. Protection of
readily available ester 4612) with methoxymethyl chloride

678
Vol. 52, No. 6
Chart 13
Table 1. Biological Data for Piperidine Carboxylic Acids (2a—i)
Compound
ICAM-1a)
IC50 (mM) (30 mg/kg, p.o.)
IL-1 pawb)
–X–CO2H
ClogP
0.40
No.
2a
1.0
61.5ꢀ4.4c)
2b
2c
0.53
0.47
71.9ꢀ11.7
69.2ꢀ3.5
1.02
1.55
Chart 14
2d
2e
2f
0.85
0.42
66.8ꢀ10.7
43.4ꢀ5.5
2.60
2.40
ꢁ10
N.T.
N.T.
N.T.
1.14
2g
ꢁ10
ꢁ10
ꢂ0.26
2h
2i
0.20
0.80
4.8
14.5 15.9
ꢀ
a) Concentration of compound inhibiting ICAM-1 up-regulation by 50% of control
value. b) Percentage inhibition of neutrophil infiltration in the mouse IL-1-induced
paw inflammation model at a dose of 30 mg/kg p.o. Values are the mean of three ani-
mals. c) Percentage inhibition of neutrophil infiltration in the mouse IL-1-induced
paw inflammation model at a dose of 100 mg/kg p.o. N.T.: not tested.
Chart 15
amide moiety of 1a by a carboxylic acid was shown not to be
suitable. We went on to look for suitable linkers between the
piperidine ring and carboxylic acid. Methylene-elongation
enhanced inhibitory activity both in vitro and in vivo by
about 2—3-fold (2b—d). Double bond insertion was also ac-
ceptable, but resulted in a slight loss of inhibitory activity in
the IL-1 paw model (2e). Triple bond insertion led to a loss
in potency of ICAM-1 inhibitory activity (2f). Introduction
of a heteroatom such as oxygen or nitrogen into this linker
resulted in loss of potency (2g, h). Thio derivative 2i also had
only weak ICAM-1 inhibitory activity and low potency in the
IL-1 paw model. Insertion of a heteroatom into this linker is
clearly unfavorable.
Next, we modified the benzyl position Y between the 10H-
pyrazino[2,3-b][1,4]benzothiazine core and the piperidine
ring. The ethylene compound 2j showed 5.6-fold lower in
vitro activity than 2d and had weak inhibitory activity in the
IL-1 paw model at an oral dose of 30 mg/kg. The amide type
2k exhibited complete loss of in vitro potency. Thus a single
methylene is the most suitable linker. All of the above piperi-
Table 2. Biological Data for Linker Y Modified Derivatives (2j, k)
Compound
No.
ICAM-1a)
IC50 (mM)
IL-1 pawb)
(30 mg/kg, p.o.)
–Y–
ClogP
2j
4.8
28.7ꢀ3.4
2.74
3.58
2k
ꢁ10
N.T.
a) Concentration of compound inhibiting ICAM-1 up-regulation by 50% of control
value. b) Percentage inhibition of neutrophil infiltration in the mouse IL-1-induced
paw inflammation model at a dose of 30 mg/kg p.o. Values are the mean of three ani-
mals. N.T.: not tested.
dine derivatives have only moderate potency even at a dose
of 30 mg/kg in IL-1-induced paw model.
We further examined the linker between piperidine and

June 2004
679
Table 4. Biological Data for Bicyclic Ring-Inserted Derivatives (2s—v)
Table 3. Biological Data for Ring-Inserted Derivatives (2l—r)
Compound
No.
ICAM-1a)
IC50 (mM)
IL-1 pawb)
(10 mg/kg, p.o.)
Compound
No.
ICAM-1a)
IC50 (mM)
IL-1 pawb)
(10 mg/kg, p.o.)
R
ClogP
3.00
R
ClogP
2l
0.88
1.0
21.7ꢀ16.1
2s
2t
9.4
9.6
N.T.
0
1.91
0.24
2m
0
3.07
2n
2o
0.71
0.93
88.0ꢀ0.9
82.8ꢀ6.8
2.59
1.27
2u
2v
0.81
83.2ꢀ1.2
1.33
1.33
ꢁ10
N.T.
a) Concentration of compound inhibiting ICAM-1 up-regulation by 50% of control
value. b) Percentage inhibition of neutrophil infiltration in the mouse IL-1-induced
paw inflammation model at a dose of 10 mg/kg p.o. Values are the mean of three ani-
mals.
2p
2q
0.90
3.0
88.8ꢀ2.0
91.5ꢀ4.5
1.89
1.89
1 paw model at a dose of 10 mg/kg. The potency of other
bicyclic derivatives was examined. 3-Azabicyclo[3.2.1]oc-
tane derivative 2u showed significant activity both with re-
spect to ICAM-1 inhibition and in the IL-1-induced paw in-
flammation model. But the tropane ring compound 2v lost
potency towards ICAM-1 inhibition.
2r
2.3
80.6ꢀ8.6
1.89
a) Concentration of compound inhibiting ICAM-1 up-regulation by 50% of control
value. b) Percentage inhibition of neutrophil infiltration in the mouse IL-1-induced
paw inflammation model at a dose of 10 mg/kg p.o. Values are the mean of three ani-
mals.
In our previous report,12) the preferable ClogP value of sul-
fonamide derivatives, 1a and 1b that were highly potent in
carboxylic acid. We wished to prepare symmetric compounds the IL-1 paw inflammation model, were stated to be around
that have significant activity in the IL-1 paw model as do 1a 0.6 as calculated using a previous version of ClogP software.
and 1b, but the a,a-dimethylcarboxylic acid derivative 2l We have also calculated and compared the values for car-
showed only 21.7% inhibition in this model at a dose of boxylic acid derivatives. The values of ClogP for 2n—r and
10 mg/kg p.o. Hence we investigated ring insertion. Introduc- 2u were within the range 1.27—2.59, and especially those of
tion of a phenyl ring, such as in benzoic acid 2m and phen- 2p—r were 1.9.23) To compare the values for the two com-
ylacetic acid 2n, gave compounds with the same in vitro po- pound classes, the ClogP of 1a and 1b were re-calculated
tency as 2d, though only 2n showed significant activity in the using the current version, and the results were 1.99 and 1.85,
IL-1 paw model at a dose of 10 mg/kg p.o. This in vivo activ- i.e. almost the same as the values of 2p—r in the carboxylic
ity of 2n is 3-fold stronger than that of 2d and is comparable acid series. These trends indicate that a value for the
to that of 1. We speculated that aliphatic carboxylic acids lipophilic parameter ClogPꢀ1.9 is a common important fac-
may be more effective in providing in vivo active compounds tor for activity for both types of compound in our in vivo
than aromatic acids, and so 7-azaspiro[3.5]nonane deriva- models.
tives such as 2o and 2p were prepared. These compounds re-
Of these compounds, we selected 2q for further evalua-
tained ICAM-1 inhibitory activity and inhibited significantly tion. The inhibitory activity of 2q on other adhesion mole-
neutrophil migration in the IL-1 paw model at a dose of cules was measured and the IC50 for inhibition of VCAM-1
10 mg/kg p.o. The insertion of a rigid ring between the found to be 2.5 mM, and for E-selectin found to be 4.5 mM.
piperidine and aliphatic carboxylic acid is preferable for in
Compared with 1b, 2q showed moderate in vitro ICAM-1
vivo activity, and provides a good carrier moiety. We next fo- inhibitory activity but strong potency in the IL-1-induced
cused on rigid ring systems such as bridged bicyclic deriva- paw model at a dose of 10 mg/kg. We examined the plasma
tives in place of the piperidine ring. 3-Azabicyclo- concentration of each compound at the time when in vivo po-
[3.3.1]nonane ring derivatives 2q and 2r showed moderate in tency was evaluated. As shown in Table 5, 2q showed about
vitro inhibitory activities. Surprisingly, both compounds 28-fold higher plasma concentration than 1b. Thus the strong
showed significant potency in the IL-1 induced paw model inhibitory activity of 2q likely results mainly from its much
equivalent to that of 1b and 2n.
higher plasma concentrations.
We further evaluated the potency of some carboxylic acid
Furthermore, the effects of 2q and 1b were evaluated in
derivatives of 3-azabicyclo[3.3.1]nonane-like skeletons as the carrageenan-induced pleurisy model in the rat. In-
shown in Table 4. a,b-Unsaturated carboxylic acid 2s had 3- trapleural injection of carrageenan induces leukocyte infiltra-
fold weaker ICAM-1 inhibitory activity compared to 2q. The tion into the pleural cavity. An antibody to ICAM-1 has been
7-oxo derivative of 3-azabicyclo[3.3.1]nonane 2t also had 3- reported effective in reducing leukocyte infiltration in the
fold weaker potency than 2q, and no inhibitory activity in IL- pleural cavity in this model.24) Compound 2q significantly in-

680
Vol. 52, No. 6
Table 5. Biological Activities and One Point Plasma Concentrations of 1b
and 2q
ICAM-1
IL-1 pawa)
Concentration in
IC50 (mM)
10 mg/kg p.o.
plasma (mM)
1b
2q
0.32
3.0
83.4ꢀ7.4
91.5ꢀ4.5
1.02ꢀ0.33
28.8ꢀ2.5
a) Percentage inhibition of neutrophil infiltration in the mouse IL-1-induced paw in-
flammation model at a dose of 10 mg/kg p.o.
Table 6. Effects of 1b and 2q on Carrageenan-Induced Pleurisy Model
Dose (mg/kg)
Leukocyte infiltrationa)
Fig. 1. Therapeutic Effect of 2q on Type II Collagen-Induced Arthritis in
Rats
2q
1b
1
3
10
30
3
36*ꢀ4
49*ꢀ3
67*ꢀ3
91*ꢀ3
35*ꢀ10
51*ꢀ11
Lewis female rats were immunized with an emulsion containing type II collagen and
muramyl dipeptide in Freund’s incomplete adjuvant. After 14 d, the rat were grouped
with the same mean volume of paws at 3.0 ml. Compound 2q was orally administered
daily for 10 d from day 14 to day 23. Each points show meanꢀS.E.M. (nꢃ8).
10
a) Percentage inhibition of leukocyte infiltration. Results were expressed as the
meanꢀstandard error of the mean (S.E.M.). Significant difference was obtained by one-
way ANOVA, ∗ pꢅ0.05. Values are the mean of six animals.
even at a dose of 100 mg/kg. By modifying the piperidine
moiety, we found that rigid ring insertion, an aliphatic car-
boxylic acid and ClogPꢀ1.9 are important for high in vivo
activities. In particular, 2q significantly inhibited neutrophil
migration in the mouse model at a dose of 10 mg/kg p.o. due
to its good pharmacokinetic properties. In carrageenan-in-
duced pleurisy model, 2q also exhibited preferable potency
in leukocyte infiltration. And furthermore, 2q showed signifi-
cant therapeutic effect in type II collagen-induced arthritis in
rats. 2q (ER-49890) was selected as a candidate for further
evaluation of its pharmacological properties. This compound
is expected to be a promising drug candidate for the treat-
ment of autoimmune inflammatory diseases such as rheuma-
toid arthritis.
Table 7. Pharmacokinetic Property of 2q and 1b12) in Rats
Tmax
Cmax
AUC0—24 h MRT0—24 h B.A.0—24 h
(h)
(mM)
(mM ·h)
(h)
(%)
2q, 3 mg/kg, p.o.
1b, 10 mg/kg, p.o.
1.0
9.0
13.6
0.86
90.1
11.5
5.14
11.4
98.5
69.0
T1/2(a) (h)
T1/2(b) (h) CLt (ml/h/kg) Vdss (ml/kg)
2q, 3 mg/kg, i.v.
1b, 3 mg/kg, i.v.
0.69
0.33
4.17
2.30
84.5
1335
287.4
3566
Experimental
Melting points were measured using a Yanako melting-point apparatus
and are uncorrected. Proton nuclear magnetic resonance (1H-NMR) spectra
were recorded on a Varian Unity 400 (400 MHz) spectrometer, and chemical
shifts are expressed in ppm downfield from tetramethylsilane (TMS) as an
internal reference. The following abbreviations are used: sꢃsinglet, dꢃdou-
blet, tꢃtriplet, qꢃquartet, quintꢃquintet, sextꢃsextet, septꢃseptet, mꢃ
multiplet, brꢃbroad. Mass spectra (MS) were obtained on a SSQ 7000 mass
spectrometer. High resolution mass spectra (HR-MS) were obtained on a Q-
Tof ultima global mass spectrometer. Elemental analysis was performed
with a Elemental Analyzer vario EL III. Materials were used as bought with-
out any special purification. Silica gel (Kieselgel 60, Merck) was used for
column chromatography, and silica gel (Kieselgel 60 F254, layer thickness
0.25 mm, Merck) for analytical thin layer chromatography (TLC). All or-
MRT is mean residence time.
hibited leukocyte infiltration from a dose of 1 mg/kg p.o. The
potency of compound 1b was 3-fold less than that of 2q.
The effect of compound 2q in the type II collagen-induced
arthritis model in the rat was examined (Fig. 1). In an experi-
ment examining therapeutic treatment, compound 2q at
doses of 2.5 to 20 mg/kg suppressed dose-dependently fur-
ther increase in paw swelling.
Pharmacokinetic studies of 2q were carried out in the fed ganic extracts were dried over anhydrous MgSO4, and solvents were re-
moved with a rotary evaporator under reduced pressure.
Ethyl (E)-4-[1-(tert-Butoxycarbonyl)piperidin-4-yl]-2-methyl-2-
butenoate (7) To a suspension of sodium hydride (NaH, 60% dispersion in
mineral oil, 5.2 g, 130 mmol) in N,N-dimethylformamide (DMF, 200 ml) was
male rat (3 mg/kg p.o., 3 mg/kg i.v., nꢃ2). This compound
has a Cmax of 13.6 mM, a mean residence time (MRT) of
5.14 h, and an oral bioavailability of 98.5%. Due to its better
pharmacokinetics in the rat, 2q also showed significant po-
tency in the carrageenan pleurisy model in the rat, greater
than might be predicted from its moderate in vitro ICAM-1
inhibition compared to that of 1b.
added triethyl 2-phosphonopropionate (31 g, 130 mmol) at room temperature
(rt), and the mixture was stirred at the same temperature for 30 min. To this
solution was added a solution of 615) (21 g, 92 mmol) in DMF (100 ml) at
0 °C, and the reaction mixture was stirred at rt for 12 h. The resulting mix-
ture was poured into ice-water, and extracted with ethyl acetate (AcOEt).
The extract was washed with brine, dried, and evaporated. The residue was
chromatographed on silica gel (10% AcOEt–hexane) to give 26 g (90%) of 7
Conclusion
1
as a colorless oil. H-NMR (CDCl3) d: 1.15 (2H, dq, Jꢃ3.6, 12.0 Hz), 1.30
We have studied derivatives of compound 1 in which a car-
boxylic acid-containing group has been used as a bioisostere
of the sulfonamide on the piperidine ring. Simple replace-
ment of the sulfonamide gave a derivative with only moder-
ate potency in the IL-1-induced paw inflammation model
(3H, t, Jꢃ7.2 Hz), 1.46 (9H, s), 1.50—1.70 (1H, m), 1.67 (2H, br d,
Jꢃ12.4 Hz), 1.83 (3H, s), 2.13 (2H, t, Jꢃ7.6 Hz), 2.68 (2H, br t,
Jꢃ12.0 Hz), 4.00—4.20 (2H, m), 4.19 (2H, q, Jꢃ7.2 Hz), 6.76 (1H, t,
Jꢃ7.6 Hz). ESI-MS m/z: 334 (MꢄNa)ꢄ.
Ethyl 4-[1-(tert-Butoxycarbonyl)piperidin-4-yl]-2-methylbutanoate (8)

June 2004
681
After cooling of the mixture to ꢂ78 °C, a solution of 8 (3.0 g, 9.6 mmol) in
THF (10 ml) was added at ꢂ78 °C, and the mixture was stirred at 0 °C for
30 min. After recooling of the mixture to ꢂ78 °C, methyl iodide (3.0 ml,
48 mmol) was added, and the mixture was allowed to warm to rt. The result-
ing mixture was poured into ice-water, and extracted with AcOEt. The ex-
tract was washed with brine, dried, and evaporated. The residue was chro-
matographed on silica gel (10% AcOEt–hexane) to give 1.5 g (48%) of 19 as
a colorless oil. 1H-NMR (CDCl3) d: 1.00—1.20 (1H, m), 1.06 (2H, dq,
Jꢃ4.0, 12.4 Hz), 1.15 (6H, s), 1.20—1.38 (2H, m), 1.24 (3H, t, Jꢃ7.2 Hz),
1.45 (9H, s), 1.48—1.60 (2H, m), 1.64 (2H, br d, Jꢃ13.2 Hz), 2.66 (2H, br t,
Jꢃ12.0 Hz), 4.00—4.16 (2H, m), 4.11 (2H, q, Jꢃ7.2 Hz). ESI-MS m/z: 350
(MꢄNa)ꢄ.
A suspension of 7 (13.8 g, 44.3 mmol) and 10% palladium on carbon (Pd/C,
0.95 g) in ethanol (EtOH, 100 ml) was stirred under a hydrogen atmosphere
at rt for 2 d. The catalyst was removed by filtration and the filtrate was con-
1
centrated to give 12.1 g (87%) of 8 as a colorless oil. H-NMR (CDCl3) d:
1.00—1.15 (2H, m), 1.14 (3H, d, Jꢃ6.8 Hz), 1.16—1.30 (2H, m), 1.25 (3H,
t, Jꢃ7.2 Hz), 1.30—1.50 (1H, m), 1.45 (9H, s), 1.50—1.74 (4H, m), 2.38
(1H, sext, Jꢃ6.8 Hz), 2.66 (2H, br t, Jꢃ12.0 Hz), 3.95—4.20 (2H, m), 4.13
(2H, q, Jꢃ7.2 Hz). ESI-MS m/z: 336 (MꢄNa)ꢄ.
Ethyl 4-[1-(tert-Butoxycarbonyl)piperidin-4-yl]propynate (11) To a
solution of carbon tetrabromide (13 g, 38 mmol) and triphenylphosphine
(20 g, 76 mmol) in dichloromethane (CH2Cl2, 200 ml) was added dropwise a
solution of 9 (4.0 g, 19 mmol) in CH2Cl2 (20 ml) at 0 °C, and the mixture
was stirred at 0 °C for 1 h. This solution was diluted with diethyl ether (Et2O,
200 ml), and the precipitate was filtered off. The filtrate was concentrated in
vacuo. The residue was chromatographed on silica gel (6% AcOEt–hexane)
to give 5.7 g (81%) of 10 as a colorless oil. To a solution of 10 (2.7 g,
7.3 mmol) in tetrahydrofuran (THF, 50 ml) was added n-butyllithium (n-
BuLi, 1.0 M solution in hexane, 15 ml, 15 mmol) at ꢂ78 °C under a nitrogen
atmosphere, and the mixture was stirred at ꢂ78 °C for 1 h and at 0 °C for
1 h. Ethyl chloroformate (2.5 ml, 26 mmol) was added at ꢂ78 °C, and the re-
action mixture was allowed to warm to rt. The resulting mixture was poured
into water, and extracted with AcOEt. The extract was washed with brine,
dried and evaporated. The residue was chromatographed on silica gel (10%
AcOEt–hexane) to give 2.1 g (quant.) of 11 as a colorless oil. 1H-NMR
(CDCl3) d: 1.31 (3H, t, Jꢃ7.2 Hz), 1.45 (9H, s), 1.59—1.70 (2H, m), 1.78—
1.87 (2H, m), 2.71 (1H, sept, Jꢃ4.0 Hz), 3.12—3.22 (2H, m), 3.64—3.78
(2H, m), 4.22 (2H, q, Jꢃ7.2 Hz). ESI-MS m/z: 304 (MꢄNa)ꢄ.
Ethyl [1-(Benzyl)piperidin-4-yl]aminoacetate (13) To a suspension of
1-benzyl-4-aminopiperidine 12 (10 g, 53 mmol) and potassium carbonate
(K2CO3, 11 g, 80 mmol) in DMF (50 ml) was added ethyl bromoacetate
(5.8 ml, 52 mmol) at 0 °C over 20 min, and the mixture was stirred at rt for
12 h. The resulting mixture was poured into ice-water, and extracted with
AcOEt. The extract was washed with brine, dried, and evaporated. The
residue was chromatographed on NH silica gel (Fuji Silysia, DW2035, 10%
AcOEt–hexane) to give 12 g (82%) of 13 as a colorless oil. 1H-NMR
(CDCl3) d: 1.27 (3H, t, Jꢃ6.8 Hz), 1.36—1.48 (2H, m), 1.76—1.84 (2H,
m), 2.01 (2H, dt, Jꢃ2.0, 12.0 Hz), 2.45 (1H, tt, Jꢃ4.0, 10.0 Hz), 2.80—2.88
(2H, m), 3.41 (2H, s), 3.49 (2H, s), 4.18 (2H, q, Jꢃ6.8 Hz), 7.20—7.34 (5H,
m). ESI-MS m/z: 277 (MꢄH)ꢄ.
Ethyl [1-(tert-Butoxycarbonyl)piperidin-4-yl]oxyacetate (15) To a so-
lution of 1-tert-butoxycarbonyl-4-piperidinol 14 (8.0 g, 40 mmol) in DMF
(100 ml) was added NaH (60% dispersion in mineral oil, 1.8 g, 45 mmol) at
rt, and the mixture was stirred at rt for 2 h. Ethyl bromoacetate (5.3 ml,
48 mmol) was added dropwise at 0 °C, and the reaction mixture was stirred
at rt for 2 h. The resulting mixture was poured into ice-water, and extracted
with AcOEt. The extract was washed with brine, dried, and evaporated. The
residue was chromatographed on silica gel (10% AcOEt–hexane) to give
4.1 g (36%) of 15 as a colorless oil. 1H-NMR (CDCl3) d: 1.29 (3H, t,
Jꢃ7.2 Hz), 1.45 (9H, s), 1.52—1.62 (2H, m), 1.80—1.92 (2H, m), 3.02—
3.12 (2H, m), 3.52—3.60 (1H, m), 3.70—3.86 (2H, m), 4.11 (2H, s), 4.22
(2H, q, Jꢃ7.2 Hz). ESI-MS m/z: 310 (MꢄNa)ꢄ.
Ethyl [1-(Benzyl)piperidin-4-yl]thioacetate (18) Hydrogen sulfide was
bubbled into a solution of 1-benzyl-4-piperidone 16 (18.9 g, 100 mmol) in
isopropyl alcohol (iPrOH, 100 ml) at rt for 4 h. After stirring of the mixture
at rt for 67 h, the solution was concentrated. To a solution of this residue in
iPrOH (100 ml) was added sodium borohydride (5.7 g, 150 mmol) in por-
tions at rt. After stirring of the mixture at 55 °C for 2 h, 1 N HCl was added
and the solution was stirred at 55 °C for 1 h. The resulting mixture was
poured into ice-water, and extracted with AcOEt. The extract was washed
with brine, dried, and evaporated to give 21.0 g (quant.) of 17 as a pale yel-
low oil. To a suspension of 17 (21.0 g, 100 mmol) and K2CO3 (13.8 g,
100 mmol) in acetonitrile (200 ml) was added a solution of ethyl bromoac-
etate (10 ml, 90 mmol) in acetonitrile (50 ml) at rt over 1 h. After stirring of
the mixture at rt for 16 h, the resulting mixture was poured into ice-water,
and extracted with AcOEt. The extract was washed with 1 N NaOH, brine,
dried, and evaporated. The residue was chromatographed on silica gel (20%
AcOEt–hexane) to give 20.3 g (69%) of 18 as a colorless oil. 1H-NMR
(CDCl3) d: 1.28 (3H, t, Jꢃ7.2 Hz), 1.55—1.70 (2H, m), 1.92—2.00 (2H,
m), 2.07 (2H, br t, Jꢃ11.2 Hz), 2.75—2.90 (3H, m), 3.24 (2H, s), 3.48 (2H,
s), 4.17 (2H, q, Jꢃ7.2 Hz), 7.20—7.34 (5H, m). ESI-MS m/z: 294 (MꢄH)ꢄ.
Ethyl 4-[1-(tert-Butoxycarbonyl)piperidin-4-yl]-2,2-dimethylbutanoate
(19) To a solution of diisopropylamine (5.4 ml, 39 mmol) in THF (50 ml)
was added n-BuLi (2.5 M solution in hexane, 15 ml, 38 mmol) at ꢂ10 °C
under a nitrogen atmosphere, and the mixture was stirred at 0 °C for 30 min.
4-(4-Bromophenyl)-1-benzyl-1,2,5,6-tetrahydropyridine (21) To
a
suspension of 20 (20 g, 78 mmol) and K2CO3 (16.2 g, 117 mmol) in DMF
(100 ml) was added benzyl bromide (11.1 ml, 93.3 mmol) dropwise at 0 °C
over 20 min, and the mixture was stirred at rt for 12 h. The reaction mixture
was poured into ice-water and extracted with AcOEt. The extract was
washed with brine, dried, and evaporated. The residue was chromatographed
on silica gel (50% AcOEt–hexane) to give 27 g (quant.) of the benzyl amine
as a colorless solid. A mixture of this amine (27 g, 78 mmol) and p-toluene-
sulfonic acid monohydrate (p-TsOH·H2O, 30.0 g, 158 mmol) in toluene
(100 ml) was refluxed under Dean–Stark conditions for 1 h. The resulting
mixture was cooled to 0 °C, then was poured into
a mixture of
K2CO3–water–AcOEt, and the precipitated solid was filtered off. The filtered
solution was extracted with AcOEt. The extract was washed with brine,
dried, and evaporated to give 8.7 g (34%) of 21 as a colorless solid. mp 81—
82 °C. 1H-NMR (CDCl3) d: 2.48—2.55 (2H, m), 2.71 (2H, t, Jꢃ5.6 Hz),
3.16 (2H, q, Jꢃ2.8 Hz), 3.63 (2H, s), 6.04—6.08 (1H, m), 7.11—7.45 (5H,
m), 7.24 (2H, d, Jꢃ8.4 Hz), 7.42 (2H, d, Jꢃ8.4 Hz). ESI-MS m/z: 328 (M)ꢄ.
Ethyl 4-(1-Benzyl-1,2,5,6-tetrahydropyridin-4-yl)benzoate (22) To a
solution of 21 (8.7 g, 27 mmol) in THF (200 ml) was added n-BuLi (2.6 M
solution in hexane, 13.5 ml, 35.1 mmol) at ꢂ78 °C under a nitrogen atmos-
phere, and the mixture was stirred at ꢂ78 °C for 15 min. To this solution
was added diethyl carbonate (34.0 ml, 281 mmol) at ꢂ78 °C, and the mixture
was stirred from ꢂ78 °C to rt over 3 h. The resulting mixture was poured
into ice-water, and extracted with AcOEt. The extract was washed with
brine, dried, and evaporated. The residue was chromatographed on silica gel
(10% AcOEt–hexane) to give 2.4 g (28%) of 22 as a yellow oil. 1H-NMR
(CDCl3) d: 1.39 (3H, t, Jꢃ7.2 Hz), 2.59 (2H, br s), 2.74 (2H, t, Jꢃ5.2 Hz),
3.14—3.28 (2H, m), 3.66 (2H, s), 4.37 (2H, q, Jꢃ7.2 Hz), 6.16—6.23 (1H,
m), 7.22—7.50 (5H, m), 7.44 (2H, d, Jꢃ8.8 Hz), 7.98 (2H, d, Jꢃ8.8 Hz).
ESI-MS m/z: 322 (MꢄH)ꢄ.
4-(1-Benzyl-4-hydroxypiperidin-4-yl)benzaldehyde (24) To a suspen-
sion of magnesium (0.74 g, 32 mmol) in THF (20 ml) was added a solution
of 4-bromobenzaldehyde diethyl acetal (5.0 ml, 25 mmol) in THF (10 ml) at
40 °C under a nitrogen atmosphere, and the mixture was stirred at rt for
30 min. After cooling of the mixture to 0 °C, a solution of 1-benzyl-4-piperi-
done 16 (5.6 g, 31 mmol) in THF (10 ml) was added at below 10 °C, and the
mixture was stirred at rt for 1 h. To this reaction mixture was added first an
aqueous solution of ammonium chloride (NH4Cl, 50 ml) and then 5 N HCl
until the pH 1 reached. The mixture was stirred at rt for 1 h, an aqueous so-
lution of sodium hydrogen carbonate (NaHCO3) was added, and then the
mixture was extracted with AcOEt. The extract was washed with brine,
dried, and evaporated. The residue was chromatographed on silica gel (70%
AcOEt–hexane) to give 4.0 g (54%) of 24 as a yellow oil. 1H-NMR (CDCl3)
d: 1.66—1.84 (3H, m), 2.18 (2H, dt, Jꢃ4.4, 13.2 Hz), 2.47 (2H, dt, Jꢃ2.4,
12.0 Hz), 2.78—2.90 (2H, m), 3.59 (2H, s), 7.20—7.44 (5H, m), 7.69 (2H,
d, Jꢃ8.4 Hz), 7.85 (2H, d, Jꢃ8.4 Hz), 9.98 (1H, s). ESI-MS m/z: 296
(MꢄH)ꢄ.
Methyl 4-(1-Benzyl-4-hydroxypiperidin-4-yl)phenylacetate (26) To a
solution of 24 (11.2 g, 38 mmol) in THF (100 ml) was added methyl methyl-
sulfinylmethyl sulfide (FAMSO, 39.6 g, 319 mmol) and 40% benzyltrimethyl-
ammonium hydroxide in methanol (40 ml) at rt, and the reaction mixture
was refluxed for 4 h. After cooling of the mixture to rt, the resulting mixture
was concentrated in vacuo. The residue was poured into water and extracted
with AcOEt. The extract was washed with brine, dried, and evaporated. The
residue was chromatographed on silica gel (20% MeOH–CH2Cl2) to give
26 g of crude 25 as a yellow oil. A solution of 25 (26 g, 38 mmol) in 10%
HCl–MeOH (400 ml) was refluxed for 6 h. After cooling and evaporation of
the solvent, the residue was poured into an aqueous solution of K2CO3 and
extracted with AcOEt. The extract was washed with brine, dried, and evapo-
rated. The residue was chromatographed on silica gel (70% AcOEt–hexane)
to give 5.6 g (43%) of 26 as a yellow oil. 1H-NMR (CDCl3) d: 1.63 (1H,
br s), 1.66—1.76 (2H, m), 2.15 (2H, dt, Jꢃ4.0, 12.8 Hz), 2.47 (2H, dt,

682
Vol. 52, No. 6
Jꢃ2.0, 12.0 Hz), 2.74—2.84 (2H, m), 3.58 (2H, s), 3.61 (2H, s), 3.69 (3H, 2.40 (4H, m), 3.05 (1H, quint, Jꢃ8.8 Hz), 3.45 (2H, s), 3.67 (3H, s), 7.19—
s), 7.23—7.37 (7H, m), 7.44—7.49 (2H, m). ESI-MS m/z: 340 (MꢄH)ꢄ.
Methyl 4-(1-Benzyl-1,2,5,6-tetrahydropyridin-4-yl)phenylacetate (27)
A mixture of 26 (5.8 g, 17 mmol) and p-TsOH·H2O (6.5 g, 34 mmol) in
toluene (100 ml) was refluxed under Dean–Stark conditions for 30 min.
After cooling to 0 °C, the mixture was poured into an aqueous solution of
NaHCO3 at 0 °C and the precipitate was filtered. The filtrate was extracted
with AcOEt. The extract was washed with brine, dried, and evaporated. A
solution of this residue and the filtrated precipitate in 10% HCl–MeOH
7.36 (5H, m). ESI-MS m/z: 274 (MꢄH)ꢄ.
Ethyl [7-(tert-Butoxycarbonyl)-7-azaspiro[3.5]non-2-ylidene]acetate
(33) To a suspension of NaH (60% dispersion in mineral oil, 0.59 g,
15 mmol) in DMF (50 ml) was added triethyl phosphonoacetate (3.3 g,
15 mmol) at rt, and the mixture was stirred at rt for 30 min. After cooling of
the mixture to 0 °C, a solution of 29 (2.5 g, 10 mmol) in DMF (10 ml) was
added, and the mixture was stirred at rt for 12 h. The resulting mixture was
poured into water, and extracted with AcOEt. The extract was washed with
(100 ml) was refluxed for 2 h. The reaction mixture was concentrated in brine, dried, and evaporated. The residue was chromatographed on silica gel
vacuo, and poured into an aqueous solution of NaHCO3 at 0 °C, then the
(10% AcOEt–hexane) to give 3.2 g (quant.) of 33 as a colorless oil. 1H-NMR
mixture was extracted with AcOEt. The extract was washed with brine, (CDCl3) d: 1.28 (3H, t, Jꢃ7.2 Hz), 1.46 (9H, s), 1.56—1.62 (4H, m), 2.57
dried, and evaporated. The residue was chromatographed on silica gel (30% (2H, br s), 2.85—2.90 (2H, m), 3.27—3.43 (4H, m), 4.15 (2H, q, Jꢃ7.2 Hz),
AcOEt–hexane) to give 3.4 g (62%) of 27 as a pale yellow solid. mp 58— 5.70 (1H, quint, Jꢃ2.4 Hz). ESI-MS m/z: 332 (MꢄNa)ꢄ.
59 °C. 1H-NMR (CDCl3) d: 2.51—2.58 (2H, m), 2.71 (2H, t, Jꢃ5.6 Hz),
Ethyl [7-(tert-Butoxycarbonyl)-7-azaspiro[3.5]non-2-yl]acetate (34)
3.17 (2H, q, Jꢃ2.8 Hz), 3.60 (2H, s), 3.64 (2H, s), 3.68 (3H, s), 6.02—6.07 A suspension of 33 (3.2 g, 10 mmol) and 10% Pd/C (0.9 g) in EtOH (50 ml)
(1H, m), 7.21 (2H, d, Jꢃ8.0 Hz), 7.23—7.40 (7H, m). ESI-MS m/z: 322
was stirred under a hydrogen atmosphere at rt for 48 h. The catalyst was re-
moved by filtration and the filtrate was concentrated to give 3.2 g (quant.) of
34 as a colorless oil. 1H-NMR (CDCl3) d: 1.25 (3H, t, Jꢃ7.2 Hz), 1.45 (9H,
s), 1.42—1.51 (3H, m), 1.52—1.65 (3H, m), 1.98—2.08 (2H, m), 2.41 (2H,
d, Jꢃ8.0 Hz), 2.63 (1H, sept, Jꢃ8.0 Hz), 3.25 (2H, t, Jꢃ5.6 Hz), 3.34 (2H, t,
Jꢃ5.6 Hz), 4.11 (2H, q, Jꢃ7.2 Hz). ESI-MS m/z: 334 (MꢄNa)ꢄ.
Ethyl (3-Methyl-3-azabicyclo[3.3.1]non-9-yl)acetate (36) A suspen-
sion of 3519) (0.30 g, 1.3 mmol) and Pd/C (0.10 g) in EtOH (30 ml) was
stirred under a hydrogen atmosphere at rt for 12 h. The catalyst was removed
by filtration and the filtrate was concentrated to give 0.30 g (quant.) of a
mixture of the anti and syn isomers of 36 (anti : synꢃ3 : 1) as a colorless oil.
1H-NMR (CDCl3) d: 1.25 (3H, t, Jꢃ7.2 Hz), 1.37—1.88 (7H, m), 1.92—
2.08 (1H, m), 2.13 (3H, s), 2.23 (3/2H, br d, Jꢃ11.2 Hz), 2.36 (1/2H, br d,
Jꢃ11.2 Hz), 2.32—2.58 (1H, m), 2.41 (1/2H, d, Jꢃ8.0 Hz), 2.48 (3/2H, d,
Jꢃ8.0 Hz), 2.62 (1/2H, br d, Jꢃ11.2 Hz), 2.90 (3/2H, br d, Jꢃ11.2 Hz), 4.13
(2H, q, Jꢃ7.2 Hz). ESI-MS m/z: 226 (MꢄH)ꢄ.
(MꢄH)ꢄ.
7-(tert-Butoxycarbonyl)-7-azaspiro[3.5]nonan-2-one (29) To a sus-
pension of zinc–copper couple (51.0 g, 781 mmol) and 2818) (11.4 g,
57.9 mmol) in Et2O (180 ml) was added a solution of trichloroacetyl chloride
(35.0 ml, 314 mmol) in 1,2-dimethoxyethane (DME, 100 ml) dropwise at rt
under a nitrogen atmosphere. After stirring of the mixture at rt for 12 h, the
reaction mixture was poured into an aqueous solution of NaHCO3 at 0 °C,
and filtrated. The filtrate was extracted with AcOEt. The extract was washed
with brine, dried, and evaporated. The residue was passed though silica gel
(20% AcOEt–hexane) to afford 29 g of the crude dichloroketone as a pale
brown oil. To a solution of this oil in saturated NH4Cl–MeOH (400 ml) was
added zinc (40.0 g, 612 mmol) portion wise at rt. The reaction mixture was
stirred at rt for 8 h, and then filtered. The filtrate was concentrated in vacuo.
The residue was chromatographed on silica gel (20% AcOEt–hexane) to
give 14 g (quant.) of 29 as a colorless solid. mp 55—56 °C. 1H-NMR
(CDCl3) d: 1.47 (9H, s), 1.70 (4H, t, Jꢃ5.2 Hz), 2.82 (4H, s), 3.41 (4H, t,
Jꢃ5.2 Hz). ESI-MS m/z: 262 (MꢄNa)ꢄ.
Ethyl (anti)-(3-Vinyloxycarbonyl-3-azabicyclo[3.3.1]non-9-yl)acetate
(37) and Ethyl (syn)-(3-Vinyloxycarbonyl-3-azabicyclo[3.3.1]non-9-yl)ac-
[7-(tert-Butoxycarbonyl)-7-azaspiro[3.5]non-2-yl]carbonitrile (30) etate (38) To a solution of 36 (2.2 g, 9.8 mmol) in 1,2-dichloroethane
To a suspension of 29 (6.0 g, 25 mmol) and p-tosylmethyl isocyanide (20 ml) was added vinyl chloroformate (4.2 ml, 49 mmol) at 0 °C. After stir-
(TosMIC, 5.4 g, 28 mmol) in DME (50 ml) was added a solution of potas-
ring of the mixture at 0 °C for 30 min, the mixture was refluxed for 3 h. After
sium tert-butoxide (5.6 g, 50 mmol) in tert-butanol (25 ml)-DME (25 ml) at cooling of the reaction mixture to rt, the volatiles were removed under re-
10 °C-rt over 1 h under a nitrogen atmosphere. After stirring of the mixture
at rt for 12 h, the reaction mixture was poured into ice-water, then extracted
duced pressure. The residue was poured into an aqueous solution of
NaHCO3 and extracted with AcOEt. The extract was washed with brine,
with AcOEt. The extract was washed with brine, dried, and evaporated. The dried and evaporated. The residue was chromatographed on silica gel (10%
residue was chromatographed on silica gel (20% AcOEt–hexane) to give
AcOEt–toluene) seven times to give 0.80 g (29%) of 37 and 0.35 g (13%) of
2.1 g (33%) of 30 as a colorless solid. mp 65—66.5 °C. 1H-NMR (CDCl3) d: 38 as a colorless oil. 37; 1H-NMR (CDCl3) d: 1.26 (3H, t, Jꢃ7.2 Hz),
1.45 (9H, s), 1.50—1.60 (2H, m), 1.60—1.66 (2H, m), 2.13—2.30 (4H, m), 1.42—1.52 (1H, m), 1.54—1.86 (7H, m), 2.20 (1H, t, Jꢃ7.2 Hz), 2.52 (2H,
3.07 (1H, quint, Jꢃ8.0 Hz), 3.25—3.36 (4H, m). ESI-MS m/z: 273 d, Jꢃ7.2 Hz), 3.14 (1H, br d, Jꢃ13.2 Hz), 3.22 (1H, br d, Jꢃ13.2 Hz), 4.08—
(MꢄNa)ꢄ.
4.26 (2H, m), 4.14 (2H, q, Jꢃ7.2 Hz), 4.44 (1H, dd, Jꢃ1.6, 6.4 Hz), 4.78
(1H, dd, Jꢃ1.6, 14.0 Hz), 7.23 (1H, dd, Jꢃ6.4, 14.0 Hz). ESI-MS m/z: 282
(MꢄH)ꢄ. 38; 1H-NMR (CDCl3) d: 1.26 (3H, t, Jꢃ7.2 Hz), 1.48—1.56 (1H,
m), 1.66—1.82 (5H, m), 1.84—1.93 (2H, m), 2.16 (1H, t, Jꢃ8.0 Hz), 2.46
(7-Benzyl-7-azaspiro[3.5]non-2-yl)carbonitrile (31) To a solution of
30 (6.4 g, 26 mmol) in CH2Cl2 (40 ml) was added trifluoroacetic acid (40 ml,
520 mmol) at 0 °C, and the reaction mixture was stirred at rt for 2.5 h. The
resulting mixture was poured into an aqueous solution of NaHCO3, and ex- (2H, d, Jꢃ8.0 Hz), 3.20—3.27 (1H, m), 3.28—3.36 (1H, m), 3.92 (2H, dd,
tracted with AcOEt. The extract was washed with brine, dried, and evapo-
rated to give 3.7 g (quant.) of crude secondary amine as a pale brown oil. To
a solution of this amine and triethylamine (8.2 ml, 59 mmol) in CH2Cl2
(50 ml) was added benzyl bromide (3.1 ml, 26 mmol) at 0 °C, and the mix-
ture was stirred at rt for 3 h. The resulting mixture was poured into water,
and extracted with AcOEt. The extract was washed with brine, dried, and
evaporated. The residue was chromatographed on NH silica gel (10%
AcOEt–hexane) to give 1.9 g (31%) of 31 as a colorless solid. mp 38—
Jꢃ6.8, 13.2 Hz), 4.15 (2H, q, Jꢃ7.2 Hz), 4.45 (1H, dd, Jꢃ1.6, 6.4 Hz), 4.79
(1H, dd, Jꢃ1.6, 14.4 Hz), 7.25 (1H, dd, Jꢃ6.4, 14.4 Hz). ESI-MS m/z: 282
(MꢄH)ꢄ.
Ethyl (3-Methyl-7-oxo-3-azabicyclo[3.3.1]non-9-ylidene)acetate (42)
In the same manner as described for the preparation of 33, 42 was obtained
1
from 4021) and triethyl phosphonoacetate as a colorless oil (59%). H-NMR
(CDCl3) d: 1.29 (3H, t, Jꢃ7.2 Hz), 2.26 (4H, s), 2.41—2.50 (2H, m), 3.14—
3.22 (2H, m), 3.72—3.82 (2H, m), 3.97 (1H, s), 4.17 (2H, q, Jꢃ7.2 Hz),
4.06—4.22 (2H, m), 5.74 (1H, s). ESI-MS m/z: 226 (MꢄH)ꢄ.
Ethyl (3-Methyl-3-azabicyclo[3.2.1]oct-8-ylidene)acetate (43) In the
same manner as described for the preparation of 33, 43 was obtained from
1
39 °C. H-NMR (CDCl3) d: 1.60 (2H, t, Jꢃ5.6 Hz), 1.69 (2H, t, Jꢃ5.6 Hz),
2.08—2.26 (4H, m), 2.20—2.40 (4H, m), 3.02 (1H, quint, Jꢃ8.8 Hz), 3.45
(2H, s), 7.12—7.34 (5H, m). ESI-MS m/z: 241 (MꢄH)ꢄ.
Methyl (7-Benzyl-7-azaspiro[3.5]non-2-yl)carboxylate (32) A mix- 4122) and triethyl phosphonoacetate as a colorless oil (42%). 1H-NMR
ture of 31 (3.8 g, 16 mmol) and KOH (7.0 g, 130 mmol) in ethylene glycol
(30 ml) was stirred at 180 °C for 10 h. After cooling of the mixture to rt,
sodium dihydrogenphosphate (NaH2PO4) was added to a suspension of the
residue in MeOH (500 ml), and the salt was filtrated off. The filtrate was
(CDCl3) d: 1.24—1.32 (3H, m), 1.60—1.77 (2H, m), 1.78—1.98 (2H, m),
2.14—2.32 (2H, m), 2.24 (3H, s), 2.47 (1H, br s), 2.80—2.92 (2H, m), 3.74
(1H, br s), 4.10—4.22 (2H, m), 5.63 (1H, s). ESI-MS m/z: 210 (MꢄH)ꢄ.
Ethyl (3-Methyl-7-oxo-3-azabicyclo[3.3.1]non-9-yl)acetate (44) In the
concentrated in vacuo. To a solution of this carboxylic acid in MeOH same manner as described for the preparation of 34, 44 (anti : synꢃ5 : 1) was
(500 ml) was added thionyl chloride (10 ml, 140 mmol) at 0 °C. The result-
ing mixture was stirred at rt for 3 d, then concentrated in vacuo. The residue
was poured into an aqueous solution of NaHCO3, and extracted with AcOEt.
The extract was washed with brine, dried, and evaporated. The residue was
obtained from 42 as a colorless oil (77%). 1H-NMR (CDCl3) d: 1.27 (3H, t,
Jꢃ7.2 Hz), 1.52—1.68 (2H, m), 2.06—2.15 (1H, m), 2.27 (3H, s), 2.33 (2H,
br d, Jꢃ11.2 Hz), 2.49 (1/3H, d, Jꢃ7.6 Hz), 2.63 (5/3H, d, Jꢃ7.6 Hz), 2.87
(1/3H, br d, Jꢃ11.6 Hz), 3.11 (5/3H, br d, Jꢃ11.6 Hz), 3.80 (2H, br d,
Jꢃ12.0 Hz), 3.95 (5/3H, br d, Jꢃ11.6 Hz), 4.07 (1/3H, br d, Jꢃ11.6 Hz),
chromatographed on NH silica gel (10% AcOEt–hexane) to give 3.7 g (84%)
1
of 32 as a pale brown solid. mp 41.5—42.5 °C. H-NMR (CDCl3) d: 1.59 4.15 (2H, q, Jꢃ7.2 Hz). ESI-MS m/z: 228 (MꢄH)ꢄ.
(2H, t, Jꢃ5.6 Hz), 1.63 (2H, t, Jꢃ5.6 Hz), 2.02 (4H, d, Jꢃ8.8 Hz), 2.20—
Ethyl (3-Methyl-3-azabicyclo[3.2.1]oct-8-yl)acetate (45) In the same

June 2004
683
manner as described for the preparation of 34, 45 (anti : synꢃ1 : 1) was ob-
tained from 43 as a colorless oil (90%). H-NMR (CDCl3) d: 1.26 (3H, t, Jꢃ11.2 Hz), 2.18 (2H, t, Jꢃ7.2 Hz), 2.72 (2H, br d, Jꢃ11.2 Hz), 3.22 (2H,
Jꢃ7.2 Hz), 1.66—2.10 (7H, m), 2.09 (1H, br d, Jꢃ11.2 Hz), 2.16 (1H, d, s), 6.67 (1H, d, Jꢃ8.0 Hz), 6.74 (1H, s), 6.81 (1H, d, Jꢃ8.0 Hz), 7.61 (1H, d,
1.41 (2H, q, Jꢃ7.2 Hz), 1.58 (2H, br d, Jꢃ11.2 Hz), 1.81 (2H, br t,
1
Jꢃ7.2 Hz), 2.23 (3/2H, s), 2.25 (3/2H, s), 2.34 (1H, br d, Jꢃ11.2 Hz), 2.49
(1H, dd, Jꢃ3.6, 10.8 Hz), 2.57 (1H, d, Jꢃ7.2 Hz), 2.72 (1H, dd, Jꢃ3.6,
Jꢃ2.8 Hz), 7.62 (1H, d, Jꢃ2.8 Hz), 9.41 (1H, s), 12.0 (1H, s). ESI-MS m/z:
371 (MꢄH)ꢄ. Anal. Calcd for C19H22N4O2S·1.5H2O: C, 57.41; H, 6.34; N,
10.8 Hz), 4.13 (1H, q, Jꢃ7.2 Hz), 4.14 (1H, q, Jꢃ7.2 Hz). ESI-MS m/z: 212 14.10. Found: C, 57.25; H, 6.55; N, 14.08.
(MꢄH)ꢄ.
Ethyl 4-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-
Ethyl [1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-
4-yl]carboxylate (5a) suspension of (1.0 g, 4.0 mmol), ethyl
4-yl]-2-methylbutanoate (5d) (1) To a solution of 8 (1.0 g, 3.2 mmol) in
AcOEt (10 ml) was added 4 N HCl–AcOEt (2.4 ml, 9.6 mmol) at 0 °C. The
resulting mixture was stirred at rt for 12 h, and concentrated in vacuo to give
0.80 g (quant.) of 4d as a colorless oil. 1H-NMR (DMSO-d6) d: 1.07 (3H, d,
A
3
isonipecotate (1.3 g, 8.3 mmol), and K2CO3 (1.7 g, 12 mmol) in DMF (30 ml)
was stirred at 60 °C for 3 h. After cooling of the mixture to rt, the resulting
mixture was poured into ice-water, and extracted with AcOEt. The extract Jꢃ6.8 Hz), 1.18 (3H, t, Jꢃ7.2 Hz), 1.10—1.60 (7H, m), 1.75 (2H, br d,
was washed with brine, dried, and evaporated. The residue was chro- Jꢃ13.2 Hz), 2.38 (1H, sext, Jꢃ6.8 Hz), 2.70—2.85 (2H, m), 3.19 (2H, br d,
matographed on silica gel (3% MeOH–CH2Cl2) to give 1.0 g (67%) of 5a as Jꢃ12.4 Hz), 4.06 (2H, q, Jꢃ7.2 Hz).
a yellow solid. mp 126—127 °C. 1H-NMR (CDCl3) d: 1.25 (3H, t,
Jꢃ7.2 Hz), 1.68—1.82 (2H, m), 1.87 (2H, dd, Jꢃ3.2, 13.2 Hz), 2.00 (2H, dt,
(2) A suspension of this oil 4d (0.80 g, 3.2 mmol), 3 (0.40 g, 1.6 mmol)
and K2CO3 (1.1 g, 8.0 mmol) in DMF (20 ml) was stirred at 60 °C for 3 h.
Jꢃ2.4, 11.2 Hz), 2.28 (1H, tt, Jꢃ4.0, 11.2 Hz), 2.81 (2H, br d, Jꢃ11.2 Hz), After cooling of the mixture to 0 °C, the resulting mixture was poured into
3.31 (2H, s), 4.13 (2H, q, Jꢃ7.2 Hz), 6.52 (1H, s), 6.53 (1H, d, Jꢃ1.2 Hz), ice-water, and extracted with AcOEt. The extract was washed with brine,
6.74 (1H, dd, Jꢃ1.2, 8.0 Hz), 6.81 (1H, d, Jꢃ8.0 Hz), 7.57 (1H, d, dried, and evaporated. The residue was chromatographed on silica gel (2%
Jꢃ2.8 Hz), 7.68 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 371 (MꢄH)ꢄ.
MeOH–CH2Cl2) to give 0.36 g (53%) of 5d as a yellow solid. mp 78—79 °C.
[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4- 1H-NMR (CDCl3) d: 1.14 (3H, d, Jꢃ6.8 Hz), 1.25 (3H, t, Jꢃ6.8 Hz), 1.10—
yl]carboxylic Acid (2a) To a solution of 5a (0.50 g, 1.3 mmol) in EtOH
(10 ml)–THF (10 ml) was added an aqueous solution (5 ml) of NaOH
(0.17 g, 4.3 mmol) under a nitrogen atmosphere, and the mixture was stirred
at 60 °C for 1 h. After cooling of the mixture to 0 °C, the reaction mixture
was poured slowly into an aqueous solution of NaH2PO4 and AcOEt at 0 °C.
The organic layer was separated, and the aqueous phase was concentrated in
vacuo to 1/2 volume. The residue was chromatographed on MCI GEL (MIT-
1.33 (5H, m), 1.35—1.50 (1H, m), 1.50—1.73 (3H, m), 1.83—2.00 (2H, m),
2.38 (1H, sext, Jꢃ6.8 Hz), 2.84 (2H, br d, Jꢃ10.0 Hz), 3.33 (2H, s), 4.13
(2H, q, Jꢃ6.8 Hz), 6.39 (1H, s), 6.56 (1H, s), 6.76 (1H, d, Jꢃ8.0 Hz), 6.83
(1H, d, Jꢃ8.0 Hz), 7.57 (1H, d, Jꢃ2.8 Hz), 7.69 (1H, d, Jꢃ2.8 Hz). ESI-MS
m/z: 427 (MꢄH)ꢄ.
4-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]-2-methylbutanoic Acid (2d) In the same manner as described for the
preparation of 2c, 2d was obtained from 5d as a yellow solid (79%). mp
SUBISHI CHEMICAL, CHP20P, water to MeOH) to give 0.35 g (78%) of
1
2a as a yellow solid. mp 257—258 °C. H-NMR (DMSO-d6) d: 1.44—1.58 187—188 °C. 1H-NMR (DMSO-d6) d: 0.96—1.24 (5H, m), 1.03 (3H, d,
(2H, m), 1.74 (2H, br d, Jꢃ11.2 Hz), 1.91 (2H, br t, Jꢃ11.2 Hz), 2.06—2.20 Jꢃ6.8 Hz), 1.26—1.40 (1H, m), 1.46—1.64 (1H, m), 1.59 (2H, br d,
(1H, m), 2.69 (2H, br d, Jꢃ12.0 Hz), 3.23 (2H, s), 6.67 (1H, d, Jꢃ8.0 Hz),
6.75 (1H, s), 6.82 (1H, d, Jꢃ8.0 Hz), 7.61 (1H, d, Jꢃ2.8 Hz), 7.63 (1H, d, (2H, br d, Jꢃ11.2 Hz), 3.23 (2H, s), 6.69 (1H, d, Jꢃ8.0 Hz), 6.76 (1H, s),
Jꢃ2.8 Hz), 9.42 (1H, s). FAB-MS m/z: 343 (MꢄH)ꢄ. Anal. Calcd for
6.83 (1H, d, Jꢃ8.0 Hz), 7.64 (2H, s), 9.43 (1H, s), 12.0 (1H, s). FAB-MS
C17H18N4O2S·1.3H2O: C, 55.81; H, 5.68; N, 15.31. Found: C, 55.89; H, m/z: 399 (MꢄH)ꢄ. Anal. Calcd for C21H26N4O2S·0.6H2O: C, 61.62; H,
Jꢃ10.4 Hz), 1.84 (2H, br t, Jꢃ10.4 Hz), 2.26 (1H, sext, Jꢃ6.8 Hz), 2.74
5.49; N, 15.34.
6.70; N, 13.69. Found: C, 61.46; H, 6.74; N, 13.68.
Ethyl [1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-
4-yl]acetate (5b) In the same manner as described for the preparation of
Ethyl (E)-4-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-
piperidin-4-yl]-2-methylbutenoate (5e) In the same manner as described
5a, 5b was obtained from 3 and ethyl (piperidin-4-yl)acetate13) as a yellow for the preparation of 5d, 5e was obtained from 7 and 3 as a yellow solid
solid (73%). mp 116—117 °C. 1H-NMR (CDCl3) d: 1.25 (3H, t, Jꢃ7.2 Hz),
1.22—1.36 (2H, m), 1.60—1.74 (2H, m), 1.70—1.86 (1H, m), 1.96 (2H, dt, (3H, t, Jꢃ7.2 Hz), 1.34—1.52 (1H, m), 1.56—1.72 (2H, m), 1.82 (3H, s),
Jꢃ2.4, 11.6 Hz), 2.22 (2H, d, Jꢃ6.8 Hz), 2.82 (2H, br d, Jꢃ11.6 Hz), 3.31 1.92 (2H, br t, Jꢃ10.0 Hz), 2.12 (2H, t, Jꢃ7.2 Hz), 2.84 (2H, br d,
(44%). mp 120—121 °C. 1H-NMR (CDCl3) d: 1.20—1.40 (2H, m), 1.29
(2H, s), 4.12 (2H, q, Jꢃ7.2 Hz), 6.47—6.57 (1H, m), 6.53 (1H, d, Jꢃ11.6 Hz), 3.32 (2H, s), 4.19 (2H, q, Jꢃ7.2 Hz), 6.47 (1H, s), 6.54 (1H, s),
Jꢃ1.6 Hz), 6.75 (1H, d, Jꢃ8.0 Hz), 6.82 (1H, d, Jꢃ8.0 Hz), 7.57 (1H, d, 6.72—6.80 (2H, m), 6.83 (1H, d, Jꢃ7.6 Hz), 7.57 (1H, d, Jꢃ2.8 Hz), 7.69
Jꢃ2.8 Hz), 7.69 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 385 (MꢄH)ꢄ.
(1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 425 (MꢄH)ꢄ.
[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]acetic Acid (2b) In the same manner as described for the preparation of
2a, 2b was obtained from 5b as a pale brown solid (86%). mp 127—128 °C.
1H-NMR (DMSO-d6) d: 1.05—1.25 (2H, m), 1.59 (3H, br d, Jꢃ11.2 Hz),
1.85 (2H, br t, Jꢃ11.2 Hz), 2.10 (2H, d, Jꢃ6.8 Hz), 2.71 (2H, br d,
(E)-4-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-
4-yl]-2-methylbutenoic Acid (2e) In the same manner as described for the
preparation of 2a, 2e was obtained from 5e as a yellow solid (50%). mp
136—137 °C. 1H-NMR (DMSO-d6) d: 1.19 (2H, dq, Jꢃ2.8, 12.0 Hz),
1.30—1.46 (1H, m), 1.59 (2H, br d, Jꢃ11.2 Hz), 1.72 (3H, d, Jꢃ1.6 Hz),
Jꢃ11.2 Hz), 3.22 (2H, s), 6.67 (1H, dd, Jꢃ1.6, 8.0 Hz), 6.74 (1H, d, 1.91 (2H, br t, Jꢃ10.8 Hz), 2.09 (2H, t, Jꢃ7.2 Hz), 2.77 (2H, br d,
Jꢃ1.6 Hz), 6.81 (1H, d, Jꢃ8.0 Hz), 7.61 (1H, d, Jꢃ2.8 Hz), 7.62 (1H, d, Jꢃ11.2 Hz), 3.28 (2H, s), 6.65 (1H, dt, Jꢃ1.6, 7.2 Hz), 6.69 (1H, dd, Jꢃ1.6,
Jꢃ2.8 Hz), 9.42 (1H, s). FAB-MS m/z: 357 (MꢄH)ꢄ. Anal. Calcd for
8.0 Hz), 6.76 (1H, d, Jꢃ1.6 Hz), 6.83 (1H, d, Jꢃ8.0 Hz), 7.62 (1H, d,
C18H20N4O2S·1.8H2O: C, 55.59; H, 6.12; N, 14.41. Found: C, 55.37; H, Jꢃ2.8 Hz), 7.63 (1H, d, Jꢃ2.8 Hz), 9.44 (1H, s). FAB-MS m/z: 397
6.11; N, 14.37.
(MꢄH)ꢄ. Anal. Calcd for C21H24N4O2S·0.8H2O: C, 61.38; H, 6.28; N,
13.63. Found: C, 61.60; H, 6.23; N, 13.64.
Ethyl 3-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-
4-yl]propynate (5f) To a solution of 11 (0.80 g, 2.8 mmol) and anisole
Ethyl 3-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-
4-yl]propionate (5c) In the same manner as described for the preparation
of 5a, 5c was obtained from 3 and ethyl 3-(piperidin-4-yl)propionate14) as a
yellow solid (63%). mp 111—112 °C. 1H-NMR (CDCl3) d: 1.25 (3H, t, (5 ml) in CH2Cl2 (5 ml) was added trifluoroacetic acid (5.0 ml, 65 mmol) at
Jꢃ6.8 Hz), 1.20—1.35 (3H, m), 1.53—1.65 (2H, m), 1.60—1.73 (2H, m), rt, and the mixture was stirred at rt for 30 min. The resulting mixture was
1.85—2.00 (2H, m), 2.31 (2H, t, Jꢃ8.0 Hz), 2.85 (2H, br d, Jꢃ10.0 Hz),
3.33 (2H, s), 4.12 (2H, q, Jꢃ6.8 Hz), 6.54 (1H, br s), 6.56 (1H, s), 6.75 (1H,
dd, Jꢃ1.0, 8.0 Hz), 6.82 (1H, d, Jꢃ8.0 Hz), 7.58 (1H, d, Jꢃ2.8 Hz), 7.69
(1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 399 (MꢄH)ꢄ.
poured into NaHCO3–ice-water, and extracted with AcOEt. The extract was
dried, and evaporated to give crude 4f as a yellow oil. A mixture of this oil, 3
(0.54 g, 2.2 mmol) and K2CO3 (0.91 g, 6.6 mmol) in DMF (20 ml) was
stirred at 60 °C for 3 h. After cooling to 0 °C, the mixture was poured into
3-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4- ice-water, and extracted with AcOEt. The extract was washed with brine,
yl]propionic Acid (2c) To a solution of 5c (0.50 g, 1.3 mmol) in EtOH
(10 ml)–THF (10 ml) was added 1 N NaOH (3.9 ml, 3.9 mmol) under a nitro-
gen atmosphere, and the mixture was stirred at 60 °C for 1 h. After cooling
dried, and evaporated. The residue was chromatographed on silica gel (60%
AcOEt–CH2Cl2) to give 0.53 g (61%) of 5f as a yellow solid. mp 129—
1
130 °C. H-NMR (CDCl3) d: 1.31 (3H, t, Jꢃ7.2 Hz), 1.68—1.80 (2H, m),
of the mixture to rt, the reaction mixture was concentrated in vacuo. The 1.84—1.94 (2H, m), 2.12—2.25 (2H, m), 2.50—2.60 (1H, m), 2.62—2.74
residue was diluted with water, and washed with AcOEt. The water phase (2H, m), 3.33 (2H, s), 4.22 (2H, q, Jꢃ7.2 Hz), 6.46 (1H, s), 6.53 (1H, s),
was concentrated to 1/2 volume. The residue was poured slowly into an 6.75 (1H, dd, Jꢃ1.2, 8.0 Hz), 6.83 (1H, d, Jꢃ8.0 Hz), 7.58 (1H, d,
aqueous solution of NaH2PO4. After stirring of the mixture at rt for 20 min,
Jꢃ2.8 Hz), 7.69 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 395 (MꢄH)ꢄ.
the precipitate was collected and dried to obtain 0.39 g (81%) of 2c as a yel-
3-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
low solid. mp 135—136 °C. 1H-NMR (DMSO-d6) d: 1.00—1.24 (3H, m), yl]propynoic Acid (2f) In the same manner as described for the prepara-

684
Vol. 52, No. 6
tion of 2a, 2f was obtained from 5f as a yellow solid (94%). mp 166—
167 °C. H-NMR (DMSO-d6) d: 1.48—1.64 (2H, m), 1.77—1.88 (2H, m),
2.22 (2H, br s), 2.50—2.75 (3H, m), 3.38 (2H, s), 6.73 (1H, dd, Jꢃ1.6,
8.0 Hz), 6.77 (1H, s), 6.87 (1H, d, Jꢃ8.0 Hz), 7.64 (1H, d, Jꢃ2.8 Hz), 7.65
(1H, d, Jꢃ2.8 Hz), 9.49 (1H, s). ESI-MS m/z: 367 (MꢄH)ꢄ. Anal. Calcd for
C19H18N4O2S·3.6H2O: C, 52.91; H, 5.89; N, 12.99. Found: C, 52.77; H,
5.79; N, 13.02.
Ethyl [1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-
4-yl]aminoacetate (5g) (1) A suspension of 13 (2.5 g, 9.0 mmol) and 20%
Pd(OH)2/C (0.9 g) in EtOH (30 ml) was stirred at rt for 6 h under a hydrogen
atmosphere (4 kg/cm2). The catalyst was removed by filtration and the fil-
trate was concentrated to give 1.7 g (quant.) of 4g as a colorless oil. 1H-
NMR (CDCl3) d: 1.28 (3H, t, Jꢃ7.2 Hz), 1.20—1.32 (2H, m), 1.67 (2H,
br s), 1.84 (2H, br d, Jꢃ11.6 Hz), 2.50—2.66 (3H, m), 3.09 (2H, br d,
Jꢃ12.8 Hz), 3.44 (2H, s), 4.20 (2H, q, Jꢃ7.2 Hz).
(2) In the same manner as described for the preparation of 5a, 5g was ob-
tained from 4g and 3 as yellow oil (74%). 1H-NMR (CDCl3) d: 1.28 (3H, t,
Jꢃ7.2 Hz), 1.37—1.50 (2H, m), 1.83 (2H, br d, Jꢃ11.2 Hz), 2.02 (2H, br t,
Jꢃ10.8 Hz), 2.44—2.54 (1H, m), 2.81 (2H, br d, Jꢃ11.6 Hz), 3.34 (2H, s),
3.42 (2H, s), 4.19 (2H, q, Jꢃ7.2 Hz), 6.55 (1H, s), 6.63 (1H, s), 6.75 (1H, d,
Jꢃ8.0 Hz), 6.82 (1H, d, Jꢃ8.0 Hz), 7.57 (1H, d, Jꢃ2.4 Hz), 7.69 (1H, d,
Jꢃ2.4 Hz). ESI-MS m/z: 400 (MꢄH)ꢄ.
Jꢃ12.0 Hz), 2.00 (2H, br t, Jꢃ11.2 Hz), 2.64—2.84 (1H, m), 2.72 (2H, br d,
Jꢃ10.8 Hz), 3.26 (2H, s), 3.27 (2H, s), 6.70 (1H, d, Jꢃ8.0 Hz), 6.77 (1H, s),
6.85 (1H, d, Jꢃ8.0 Hz), 7.64 (1H, d, Jꢃ2.8 Hz), 7.65 (1H, d, Jꢃ2.8 Hz),
9.45 (1H, s). ESI-MS m/z: 389 (MꢄH)ꢄ. Anal. Calcd for
C18H20N4O2S2·1.5H2O: C, 52.03; H, 5.58; N, 13.48. Found: C, 51.93; H,
5.69; N, 13.47.
Ethyl 4-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-
4-yl]benzoate (5m) (1) A suspension of 22 (1.8 g, 5.6 mmol) and 20%
Pd(OH)2/C (0.9 g) in EtOH (50 ml) was stirred at rt for 3 d under a hydrogen
atmosphere. The catalyst was removed by filtration and the filtrate was con-
centrated to give 1.3 g (quant.) of 4m as a colorless solid. 1H-NMR (CDCl3)
d: 1.39 (3H, t, Jꢃ7.2 Hz), 1.90—2.00 (4H, m), 2.67—2.82 (1H, m), 2.82—
2.96 (2H, m), 3.42 (2H, br d, Jꢃ12.8 Hz), 4.37 (2H, q, Jꢃ7.2 Hz), 7.30 (2H,
d, Jꢃ8.4 Hz), 8.00 (2H, d, Jꢃ8.4 Hz).
1
(2) A suspension of this amine 4m (1.3 g, 5.6 mmol), 3 (1.1 g, 4.4 mmol)
and K2CO3 (1.8 g, 13 mmol) in DMF (20 ml) was stirred at 60 °C for 3 h
under a nitrogen atmosphere. After cooling to rt, the mixture was poured
into ice-water and extracted with AcOEt. The extract was washed with brine,
dried and evaporated. The residue was chromatographed on silica gel (50%
AcOEt–CH2Cl2) to give 1.3 g (65%) of 5m as a yellow solid. mp 165—
1
166 °C. H-NMR (CDCl3) d: 1.38 (3H, t, Jꢃ6.8 Hz), 1.70—1.90 (4H, m),
2.00—2.15 (2H, m), 2.50—2.64 (1H, m), 3.00 (2H, br d, Jꢃ11.2 Hz), 3.40
(2H, s), 4.36 (2H, q, Jꢃ6.8 Hz), 6.42 (1H, s), 6.59 (1H, s), 6.79 (1H, d,
Jꢃ8.0 Hz), 6.85 (1H, d, Jꢃ8.0 Hz), 7.29 (2H, d, Jꢃ8.0 Hz), 7.58 (1H, d,
Jꢃ2.8 Hz), 7.70 (1H, d, Jꢃ2.8 Hz), 7.98 (2H, d, Jꢃ8.0 Hz). ESI-MS m/z:
447 (MꢄH)ꢄ.
[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]aminoacetic Acid (2g) In the same manner as described for the prepa-
ration of 2a, 2g was obtained from 5g as a yellow solid (85%). mp 234—
1
235 °C. H-NMR (DMSO-d6) d: 1.40—1.54 (2H, m), 1.78—1.92 (4H, m),
2.70—2.85 (3H, m), 3.10 (2H, s), 3.23 (2H, s), 6.67 (1H, dd, Jꢃ1.6, 8.0 Hz),
6.72 (1H, d, Jꢃ1.6 Hz), 6.83 (1H, d, Jꢃ8.0 Hz), 7.62 (1H, d, Jꢃ2.8 Hz),
7.63 (1H, d, Jꢃ2.8 Hz), 9.45 (1H, s). ESI-MS m/z: 372 (MꢄH)ꢄ. Anal.
Calcd for C18H21N5O2S·1.2H2O: C, 55.00; H, 6.00; N, 17.82. Found: C,
55.09; H, 6.06; N, 17.82.
Ethyl [1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-
4-yl]oxyacetate (5h) In the same manner as described for the preparation
of 5d, 5h was obtained from 15 and 3 as a yellow solid (90%). mp 85—
86 °C. 1H-NMR (CDCl3) d: 1.28 (3H, t, Jꢃ7.2 Hz), 1.55—1.75 (2H, m),
1.85—1.98 (2H, m), 2.05—2.20 (2H, m), 2.68—2.78 (2H, m), 3.33 (2H, s),
3.37—3.48 (1H, m), 4.10 (2H, s), 4.21 (2H, q, Jꢃ7.2 Hz), 6.48 (1H, s), 6.55
(1H, s), 6.75 (1H, dd, Jꢃ1.2, 8.0 Hz), 6.83 (1H, d, Jꢃ8.0 Hz), 7.58 (1H, d,
Jꢃ2.8 Hz), 7.69 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 401 (MꢄH)ꢄ.
[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]oxyacetic Acid (2h) In the same manner as described for the prepara-
tion of 2a, 2h was obtained from 5h as a yellow solid (62%). mp 137—
138 °C. 1H-NMR (DMSO-d6) d: 1.43 (2H, br q, Jꢃ9.6 Hz), 1.74—1.87 (2H,
m), 2.02 (2H, br t, Jꢃ9.6 Hz), 2.54—2.68 (2H, m), 3.25 (2H, s), 3.30—3.42
(1H, m), 3.97 (2H, s), 6.70 (1H, d, Jꢃ8.0 Hz), 6.77 (1H, s), 6.84 (1H, d,
Jꢃ8.0 Hz), 7.63 (1H, d, Jꢃ2.8 Hz), 7.65 (1H, d, Jꢃ2.8 Hz), 9.45 (1H, s).
ESI-MS m/z: 373 (MꢄH)ꢄ. Anal. Calcd for C18H20N4O3S·0.7H2O: C,
56.15; H, 5.60; N, 14.55. Found: C, 55.89; H, 5.70; N, 14.52.
Ethyl [1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-
4-yl]thioacetate (5i) (1) To a solution of 18 (3.0 g, 10 mmol) in 1,2-
dichloroethane (19 ml) was added 1-chloroethyl chloroformate (2.8 ml,
26 mmol) dropwise at 0 °C over 30 min under a nitrogen atmosphere. After
stirring at 0 °C for 15 min, the mixture was heated to 100 °C for 1 h, then
concentrated in vacuo. A solution of this residue in EtOH (300 ml) was re-
fluxed for 2 h, then the solvent was removed under reduced pressure to give
2.4 g (quant.) of secondary amine 4i as a pale brown solid. 1H-NMR
(DMSO-d6) d: 1.20 (3H, t, Jꢃ7.2 Hz), 1.56—1.72 (2H, m), 2.02—2.12 (2H,
m), 2.84—2.98 (2H, m), 3.00—3.12 (1H, m), 3.18—3.29 (2H, m), 3.43 (2H,
s), 4.10 (2H, q, Jꢃ7.2 Hz).
4-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]benzoic Acid (2m) In the same manner as described for the preparation
of 2c, 2m was obtained from 5m as a yellow solid (70%). mp 286—287 °C.
1H-NMR (DMSO-d6) d: 1.58—1.74 (2H, m), 1.76 (2H, br d, Jꢃ10.4 Hz),
2.04 (2H, br t, Jꢃ10.4 Hz), 2.50—2.64 (1H, m), 2.90 (2H, br d, Jꢃ11.2 Hz),
3.33 (2H, s), 6.74 (1H, d, Jꢃ8.0 Hz), 6.81 (1H, s), 6.86 (1H, d, Jꢃ8.0 Hz),
7.37 (2H, d, Jꢃ8.0 Hz), 7.64 (1H, d, Jꢃ2.8 Hz), 7.65 (1H, d, Jꢃ2.8 Hz),
7.87 (2H, d, Jꢃ8.0 Hz), 9.46 (1H, s). ESI-MS m/z: 419 (MꢄH)ꢄ. Anal.
Calcd for C23H22N4O2S·1.7H2O: C, 61.51; H, 5.70; N, 12.47. Found: C,
61.41; H, 5.78; N, 12.42.
Methyl 4-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-
piperidin-4-yl]phenylacetate (5n) (1) In the same manner as described
for the preparation of 5m, step 1, 4n was obtained from 27 as a colorless oil
(83%). 1H-NMR (CDCl3) d: 1.50—1.76 (3H, m), 1.81 (2H, br d,
Jꢃ12.8 Hz), 2.53—2.64 (1H, m), 2.66—2.78 (2H, m), 3.17 (2H, br d,
Jꢃ12.0 Hz), 3.59 (2H, s), 3.68 (3H, s), 7.17 (2H, d, Jꢃ8.0 Hz), 7.20 (2H, d,
Jꢃ8.0 Hz).
(2) In the same manner as described for the preparation of 5m, step 2, 5n
1
was obtained from 4n and 3 as a yellow solid (84%). mp 142—143 °C. H-
NMR (CDCl3) d: 1.68—1.84 (4H, m), 2.06 (2H, dt, Jꢃ3.2, 11.2 Hz), 2.43—
2.54 (1H, m), 2.97 (2H, br d, Jꢃ12.4 Hz), 3.38 (2H, s), 3.60 (2H, s), 3.69
(3H, s), 6.48—6.60 (1H, m), 6.58 (1H, s), 6.79 (1H, dd, Jꢃ1.6, 8.0 Hz), 6.85
(1H, d, Jꢃ8.0 Hz), 7.18 (2H, d, Jꢃ8.4 Hz), 7.22 (2H, d, Jꢃ8.4 Hz), 7.58
(1H, d, Jꢃ2.8 Hz), 7.69 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 447 (MꢄH)ꢄ.
4-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]phenylacetic Acid (2n) In the same manner as described for the prepa-
ration of 2c, 2n was obtained from 5n as a yellow solid (67%). mp 259—
1
260 °C. H-NMR (DMSO-d6) d: 1.62 (2H, dq, Jꢃ3.2, 12.0 Hz), 1.72 (2H,
br d, Jꢃ10.8 Hz), 1.96—2.06 (2H, m), 2.40—2.56 (1H, m), 2.88 (2H, br d,
Jꢃ11.2 Hz), 3.30 (2H, s), 3.49 (2H, s), 6.72 (1H, dd, Jꢃ1.6, 8.0 Hz), 6.80
(1H, d, Jꢃ1.6 Hz), 6.84 (1H, d, Jꢃ8.0 Hz), 7.17 (4H, s), 7.62 (1H, d,
Jꢃ2.8 Hz), 7.63 (1H, d, Jꢃ2.8 Hz), 9.44 (1H, s). ESI-MS m/z: 433 (MꢄH)ꢄ.
Anal. Calcd for C24H24N4O2S·0.3H2O: C, 65.82; H, 5.66; N, 12.79. Found:
C, 65.74; H, 5.65; N, 12.85.
(2) A suspension of this amine 4i (1.2 g, 5.0 mmol), 3 (1.0 g, 4.0 mmol)
and K2CO3 (2.2 g, 16 mmol) in DMF (20 ml) was stirred at 70 °C for 3 h.
After cooling to 0 °C, the mixture was poured into ice-water and extracted
with AcOEt. The extract was washed with brine, dried and evaporated. The
residue was chromatographed on silica gel (70% AcOEt–CH2Cl2) to give
Methyl [7-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-7-aza-
spiro[3.5]non-2-yl]carboxylate (5o) (1) In the same manner as described
for the preparation of 5m, step 1, 4o was obtained from 32 as a colorless oil
(88%). 1H-NMR (CDCl3) d: 1.53 (2H, t, Jꢃ5.2 Hz), 1.58 (2H, t, Jꢃ5.2 Hz),
1.87 (1H, br s), 2.04 (4H, d, Jꢃ8.8 Hz), 2.66—2.73 (2H, m), 2.74—2.80
(2H, m), 3.07 (1H, quint, Jꢃ8.8 Hz), 3.68 (3H, s).
1
0.86 g (51%) of 5i as a yellow solid. mp 87—88 °C. H-NMR (CDCl3) d:
1.28 (3H, t, Jꢃ7.2 Hz), 1.55—1.68 (2H, m), 1.98 (2H, br d, Jꢃ12.8 Hz),
2.07 (2H, br t, Jꢃ10.8 Hz), 2.77—2.87 (3H, m), 3.25 (2H, s), 3.32 (2H, s),
4.19 (2H, q, Jꢃ7.2 Hz), 6.42—6.58 (1H, m), 6.53 (1H, s), 6.75 (1H, dd,
Jꢃ1.2, 8.0 Hz), 6.83 (1H, d, Jꢃ8.0 Hz), 7.58 (1H, d, Jꢃ2.8 Hz), 7.69 (1H, d,
Jꢃ2.8 Hz). ESI-MS m/z: 417 (MꢄH)ꢄ.
(2) In the same manner as described for the preparation of 5m, step 2, 5o
1
was obtained from 4o and 3 as a yellow solid (80%). mp 183—184 °C. H-
NMR (CDCl3) d: 1.59 (2H, t, Jꢃ5.2 Hz), 1.64 (2H, t, Jꢃ5.2 Hz), 2.02 (4H,
d, Jꢃ8.8 Hz), 2.14—2.44 (4H, m), 3.05 (1H, quint, Jꢃ8.8 Hz), 3.29 (2H, s),
3.67 (3H, s), 6.43 (1H, br s), 6.54 (1H, s), 6.75 (1H, dd, Jꢃ1.6, 7.6 Hz), 6.82
(1H, d, Jꢃ7.6 Hz), 7.56—7.60 (1H, m), 7.67—7.72 (1H, m). ESI-MS m/z:
397 (MꢄH)ꢄ.
[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]thioacetic Acid (2i) In the same manner as described for the prepara-
tion of 2c, 2i was obtained from 5i as a yellow solid (83%). mp 137—
138 °C. 1H-NMR (DMSO-d6) d: 1.36—1.50 (2H, m), 1.90 (2H, br d,
[7-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-7-aza-

June 2004
685
1
spiro[3.5]non-2-yl]carboxylic Acid (2o) In the same manner as described
for the preparation of 2a, 2o was obtained from 5o as a yellow solid (64%).
mp 150—151 °C. 1H-NMR (DMSO-d6) d: 1.46 (2H, t, Jꢃ5.2 Hz), 1.50—
1.60 (2H, m), 1.80—1.90 (2H, m), 1.88—1.98 (2H, m), 2.06—2.34 (4H, m),
2.98 (1H, quint, Jꢃ8.4 Hz), 3.21 (2H, s), 6.69 (1H, d, Jꢃ8.0 Hz), 6.76 (1H,
d, Jꢃ1.6 Hz), 6.83 (1H, d, Jꢃ1.6, 8.0 Hz), 7.62—7.66 (2H, m), 9.44 (1H, s).
FAB-MS m/z: 383 (MꢄH)ꢄ. Anal. Calcd for C20H22N4O2S·0.7H2O: C,
60.80; H, 5.97; N, 14.18. Found: C, 60.61; H, 6.01; N, 14.18.
was obtained from 4r and 3 as a yellow solid (65%). mp 115—116 °C. H-
NMR (CDCl3) d: 1.25 (3H, t, Jꢃ7.2 Hz), 1.50—1.90 (7H, m), 2.00—2.10
(1H, m), 2.39 (2H, br d, Jꢃ12.0 Hz), 2.43 (2H, d, Jꢃ7.6 Hz), 2.50—2.70
(1H, m), 2.64 (2H, br d, Jꢃ10.4 Hz), 3.22 (2H, s), 4.12 (2H, q, Jꢃ7.2 Hz),
6.34—6.42 (1H, m), 6.48 (1H, d, Jꢃ1.6 Hz), 6.75 (1H, dd, Jꢃ1.6, 8.0 Hz),
6.81 (1H, d, Jꢃ8.0 Hz), 7.55 (1H, d, Jꢃ2.8 Hz), 7.67 (1H, d, Jꢃ2.8 Hz).
ESI-MS m/z: 425 (MꢄH)ꢄ.
(syn)-[3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-azabi-
cyclo[3.3.1]non-9-yl]acetic Acid (2r) In the same manner as described for
the preparation of 2c, 2r was obtained from 5r as a yellow solid (93%). mp
236—237 °C. 1H-NMR (DMSO-d6) d: 1.38—1.48 (1H, m), 1.54—1.68 (4H,
m), 1.68—1.80 (2H, m), 1.87 (1H, t, Jꢃ7.6 Hz), 2.30 (2H, d, Jꢃ7.6 Hz),
2.24—2.38 (2H, m), 2.55 (2H, br d, Jꢃ10.8 Hz), 2.50—2.70 (1H, m), 3.16
(2H, s), 6.67 (1H, d, Jꢃ7.6 Hz), 6.71 (1H, s), 6.82 (1H, d, Jꢃ7.6 Hz), 7.61
(1H, d, Jꢃ2.8 Hz), 7.62 (1H, d, Jꢃ2.8 Hz), 9.54 (1H, s). FAB-MS m/z: 397
(MꢄH)ꢄ. Anal. Calcd for C21H24N4O2S: C, 63.61; H, 6.10; N, 14.13. Found:
C, 63.24; H, 6.07; N, 14.09.
Ethyl
[7-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-7-aza-
spiro[3.5]non-2-yl]acetate (5p) (1) In the same manner as described for
the preparation of 5d, step 1, 4p was obtained from 34 as a colorless solid
1
(quant.). H-NMR (DMSO-d6) d: 1.16 (3H, t, Jꢃ7.2 Hz), 1.44—1.53 (2H,
m), 1.63 (2H, t, Jꢃ5.6 Hz), 1.73 (2H, t, Jꢃ5.6 Hz), 1.94—2.02 (2H, m),
2.41 (2H, d, Jꢃ8.0 Hz), 2.45—2.59 (1H, m), 2.86 (2H, t, Jꢃ5.6 Hz), 2.95
(2H, t, Jꢃ5.6 Hz), 4.02 (2H, q, Jꢃ7.2 Hz).
(2) In the same manner as described for the preparation of 5d, step 2, 5p
1
was obtained from 4p and 3 as a yellow solid (75%). mp 144—145 °C. H-
NMR (CDCl3) d: 1.24 (3H, t, Jꢃ7.2 Hz), 1.44 (2H, dd, Jꢃ8.4, 12.0 Hz),
1.52 (2H, t, Jꢃ5.2 Hz), 1.64 (2H, t, Jꢃ5.2 Hz), 1.96—2.06 (2H, m), 2.24
(2H, br s), 2.33 (2H, br s), 2.39 (2H, d, Jꢃ8.0 Hz), 2.60 (1H, sept,
Jꢃ8.0 Hz), 3.29 (2H, s), 4.10 (2H, q, Jꢃ7.2 Hz), 6.47 (1H, br s), 6.54 (1H,
s), 6.75 (1H, dd, Jꢃ1.2, 7.6 Hz), 6.82 (1H, d, Jꢃ7.6 Hz), 7.57 (1H, d,
Jꢃ2.8 Hz), 7.69 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 425 (MꢄH)ꢄ.
Ethyl [3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-azabi-
cyclo[3.3.1]non-9-ylidene]acetate (5s) To
a solution of 35 (2.0 g,
9.0 mmol) in 1,2-dichloroethane (8.4 ml) was added 1-chloroethyl chlorofor-
mate (2.9 ml, 27 mmol) dropwise at 0 °C for 30 min under a nitrogen atmos-
phere. After stirring at 0 °C for 15 min, the mixture was heated to 100 °C for
1 h, then concentrated in vacuo. A solution of this residue in EtOH (100 ml)
was refluxed for 2 h, then the solvent was removed under reduced pressure to
give 2.2 g (quant.) of secondary amine 4s as a pale yellow oil. A suspension
of this amine 4s (2.2 g, 9.0 mmol), 3 (1.5 g, 6.0 mmol) and K2CO3 (3.3 g,
24 mmol) in DMF (20 ml) was stirred at 80 °C for 3 h under a nitrogen at-
mosphere. After cooling to 0 °C, the mixture was poured into ice-water and
extracted with AcOEt. The extract was washed with brine, dried and evapo-
rated. The residue was chromatographed on silica gel (15% AcOEt–toluene)
to give 1.6 g (64%) of 5s as a yellow solid. mp 45—46 °C. 1H-NMR (CDCl3)
d: 1.28 (3H, t, Jꢃ7.2 Hz), 1.48—1.58 (1H, m), 1.75—1.90 (2H, m), 1.93—
2.03 (2H, m), 2.25—2.32 (3H, m), 2.71—2.86 (1H, m), 2.96—3.04 (2H, m),
3.19 (1H, d, Jꢃ13.2 Hz), 3.25 (1H, d, Jꢃ13.2 Hz), 3.94 (1H, s), 4.15 (2H, q,
Jꢃ7.2 Hz), 5.63 (1H, s), 6.39 (1H, s), 6.52 (1H, s), 6.79 (1H, dd, Jꢃ1.2,
8.0 Hz), 6.85 (1H, d, Jꢃ8.0 Hz), 7.56—7.59 (1H, m), 7.67—7.72 (1H, m).
ESI-MS m/z: 423 (MꢄH)ꢄ.
[7-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-7-aza-
spiro[3.5]non-2-yl]acetic Acid (2p) In the same manner as described for
the preparation of 2c, 2p was obtained from 5p as a yellow solid (84%). mp
1
124—125 °C. H-NMR (DMSO-d6) d: 1.38 (2H, dd, Jꢃ8.8, 12.0 Hz), 1.44
(2H, t, Jꢃ5.2 Hz), 1.50—1.60 (2H, m), 1.89 (2H, br t, Jꢃ10.8 Hz), 2.16
(2H, br s), 2.24 (2H, br s), 2.30 (2H, d, Jꢃ7.6 Hz), 2.40—2.56 (1H, m), 3.20
(2H, s), 6.69 (1H, dd, Jꢃ1.6, 8.0 Hz), 6.76 (1H, d, Jꢃ1.6 Hz), 6.83 (1H, d,
Jꢃ8.0 Hz), 7.63 (1H, d, Jꢃ2.8 Hz), 7.64 (1H, d, Jꢃ2.8 Hz), 9.43 (1H, s).
FAB-MS m/z: 397 (MꢄH)ꢄ. Anal. Calcd for C21H24N4O2S·1.5H2O: C,
59.55; H, 6.43; N, 13.23. Found: C, 59.66; H, 6.48; N, 13.33.
Ethyl (anti)-[3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-
azabicyclo[3.3.1]non-9-yl]acetate (5q) (1) A solution of 37 (0.50 g,
1.8 mmol) in 4 N HCl–dioxane (2.0 ml, 8.0 mmol) was stirred at rt for 1 h. To
this solution was added EtOH (30 ml) and the mixture was then refluxed for
1 h. After evaporating the volatiles at reduced pressure, the residue was
poured into ammonia–ice-water and extracted with CH2Cl2. The extract was
washed with brine, dried and evaporated to give 0.36 g (95%) of 4q as a col-
orless oil. 1H-NMR (CDCl3) d: 1.25 (3H, t, Jꢃ7.2 Hz), 1.48—1.65 (5H, m),
1.76—1.90 (3H, m), 2.06—2.23 (2H, m), 2.48 (2H, d, Jꢃ8.0 Hz), 3.00—
3.16 (4H, m), 4.13 (2H, q, Jꢃ7.2 Hz).
(2) A suspension of this amine 4q (0.36 g, 1.8 mmol), 3 (0.28 g, 1.1 mmol)
and K2CO3 (0.70 g, 5.1 mmol) in DMF (10 ml) was stirred at 80 °C for 3 h
under a nitrogen atmosphere. After cooling to 0 °C, the mixture was poured
into ice-water and extracted with AcOEt. The extract was washed with brine,
dried and evaporated. The residue was chromatographed on silica gel (15%
AcOEt–toluene) to give 0.27 g (57%) of 5q as a yellow solid. mp 142—
143 °C. 1H-NMR (DMSO-d6) d: 1.14 (3H, t, Jꢃ7.2 Hz), 1.34—1.48 (3H,
m), 1.56 (2H, br s), 1.60—1.75 (2H, m), 1.82—1.92 (1H, m), 2.15 (2H, br d,
Jꢃ11.2 Hz), 2.43 (2H, d, Jꢃ8.0 Hz), 2.40—2.60 (1H, m), 2.85 (2H, br d,
Jꢃ9.2 Hz), 3.15 (2H, s), 4.02 (2H, q, Jꢃ7.2 Hz), 6.67 (1H, dd, Jꢃ1.6,
8.0 Hz), 6.71 (1H, d, Jꢃ1.6 Hz), 6.82 (1H, d, Jꢃ8.0 Hz), 7.61 (1H, d,
Jꢃ2.8 Hz), 7.62 (1H, d, Jꢃ2.8 Hz), 9.55 (1H, s). FAB-MS m/z: 425
(MꢄH)ꢄ.
(anti)-[3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-azabi-
cyclo[3.3.1]non-9-yl]acetic Acid (2q) In the same manner as described
for the preparation of 2c, 2q was obtained from 5q as a yellow solid (64%).
mp 230—231 °C. 1H-NMR (DMSO-d6) d: 1.34—1.48 (3H, m), 1.58 (2H,
br s), 1.60—1.76 (2H, m), 1.80—1.90 (1H, m), 2.14 (2H, br d, Jꢃ10.0 Hz),
2.33 (2H, d, Jꢃ8.0 Hz), 2.44—2.60 (1H, m), 2.85 (2H, br d, Jꢃ10.0 Hz),
3.15 (2H, s), 6.67 (1H, d, Jꢃ8.0 Hz), 6.72 (1H, s), 6.82 (1H, d, Jꢃ8.0 Hz),
7.60 (1H, d, Jꢃ2.8 Hz), 7.62 (1H, d, Jꢃ2.8 Hz), 9.54 (1H, s). FAB-MS m/z:
397 (MꢄH)ꢄ. Anal. Calcd for C21H24N4O2S·0.1H2O: C, 63.32; H, 6.12; N,
14.07. Found: C, 63.19; H, 6.07; N, 13.90.
Ethyl (syn)-[3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-
azabicyclo[3.3.1]non-9-yl]acetate (5r) (1) In the same manner as de-
scribed for the preparation of 5q, step 1, 4r was obtained from 38 as a color-
less oil (quant.). 1H-NMR (CDCl3) d: 1.26 (3H, t, Jꢃ7.2 Hz), 1.54 (2H,
br s), 1.62—1.74 (1H, m), 1.76—1.94 (4H, m), 2.10—2.34 (3H, m), 2.55
(2H, d, Jꢃ8.0 Hz), 2.81—2.89 (2H, m), 3.14—3.24 (2H, m), 4.13 (2H, q,
Jꢃ7.2 Hz).
[3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-azabicy-
clo[3.3.1]non-9-ylidene]acetic Acid (2s) In the same manner as described
for the preparation of 2c, 2s was obtained from 5s as a yellow solid (1.7%).
1
mp 126—127 °C. H-NMR (DMSO-d6) d: 1.40—1.50 (1H, m), 1.62—1.78
(2H, m), 1.88—2.00 (2H, m), 2.17 (2H, br t, Jꢃ12.8 Hz), 2.33 (1H, s),
2.70—2.88 (1H, m), 2.93—3.02 (2H, m), 3.16 (1H, br d, Jꢃ13.2 Hz), 3.21
(1H, br d, Jꢃ13.2 Hz), 3.86 (1H, s), 5.57 (1H, s), 6.72 (1H, d, Jꢃ8.0 Hz),
6.77 (1H, s), 6.87 (1H, d, Jꢃ8.0 Hz), 7.60—7.68 (2H, m), 9.58 (1H, s),
11.98 (1H, s). HR-MS (ESI) m/z: Calcd for C21H23N4O2S (MꢄH)ꢄ:
395.1542. Found: 395.1551.
Ethyl [3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-7-oxo-3-
azabicyclo[3.3.1]non-9-yl]acetate (5t) In the same manner as described
for the preparation of 5s, 5t (anti : synꢃ5 : 1) was obtained from 44 and 3 as
1
a yellow oil (88%). H-NMR (CDCl3) d: 1.26 (3H, t, Jꢃ7.2 Hz), 1.70 (2H,
br s), 2.12 (1H, t, Jꢃ8.0 Hz), 2.39 (5/3H, br d, Jꢃ11.2 Hz), 2.50 (1/3H, d,
Jꢃ8.0 Hz), 2.54 (1/3H, br d, Jꢃ11.2 Hz), 2.65 (5/3H, d, Jꢃ8.0 Hz), 2.78
(1/3H, br d, Jꢃ11.6 Hz), 3.02 (5/3H, d, Jꢃ11.6 Hz), 3.35 (2H, s), 3.75
(5/3H, br d, Jꢃ11.6 Hz), 3.81 (1/3H, d, Jꢃ11.6 Hz), 3.96 (5/3H, br d,
Jꢃ11.6 Hz), 4.01 (1/3H, d, Jꢃ11.6 Hz), 4.14 (2H, q, Jꢃ7.2 Hz), 6.62 (1H,
br s), 6.69 (1H, s), 6.76—6.84 (1H, m), 6.81 (1H, s), 7.57 (1H, d, Jꢃ3.2 Hz),
7.67 (1H, d, Jꢃ3.2 Hz). ESI-MS m/z: 427 (MꢄH)ꢄ.
[3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-7-oxo-3-azabi-
cyclo[3.3.1]non-9-yl]acetic Acid (2t) In the same manner as described for
the preparation of 2a, 2t (anti : synꢃ5 : 1) was obtained from 5t as a yellow
solid (85%). mp 168—169 °C. 1H-NMR (DMSO-d6) d: 1.52 (2H, br s), 1.90
(1H, t, Jꢃ7.2 Hz), 2.20 (5/3H, br d, Jꢃ10.8 Hz), 2.40 (1/3H, br d,
Jꢃ10.8 Hz), 2.30—2.54 (2H, m), 2.58—2.94 (2H, m), 3.22 (2H, s), 3.56
(5/3H, br d, Jꢃ11.2 Hz), 3.60 (1/3H, br d, Jꢃ11.2 Hz), 3.77 (5/3H, br d,
Jꢃ10.0 Hz), 3.82 (1/3H, br d, Jꢃ10.0 Hz), 6.70—6.80 (2H, m), 6.82 (1H, d,
Jꢃ7.6 Hz), 7.60 (1H, d, Jꢃ2.8 Hz), 7.61 (1H, d, Jꢃ2.8 Hz), 9.50 (1H, s).
FAB-MS m/z: 399 (MꢄH)ꢄ. Anal. Calcd for C20H22N4O3S·1.8H2O: C,
55.75; H, 5.99; N, 13.00. Found: C, 55.64; H, 5.83; N, 12.76.
Ethyl [3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-azabi-
cyclo[3.2.1]oct-8-yl]acetate (5u) In the same manner as described for the
preparation of 5s, 5u (anti : synꢃ1 : 3) was obtained from 45 and 3 as a yel-
low solid (19%). mp 121—122 °C. 1H-NMR (CDCl3) d: 1.25 (3H, t,
Jꢃ7.2 Hz), 1.62—1.82 (4H, m), 1.92—2.18 (3H, m), 2.11 (1/2H, br d,
(2) In the same manner as described for the preparation of 5q, step 2, 5r

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Vol. 52, No. 6
Jꢃ10.4 Hz), 2.16 (1/2H, d, Jꢃ7.6 Hz), 2.36 (3/2H, br d, Jꢃ10.4 Hz), 2.42
(3/2H, dd, Jꢃ2.8, 10.8 Hz), 2.57 (3/2H, d, Jꢃ7.6 Hz), 2.65 (1/2H, dd, Jꢃ2.8,
10.8 Hz), 3.31 (1/2H, s), 3.33 (3/2H, s), 4.12 (1/2H, q, Jꢃ7.2 Hz), 4.13
(3/2H, q, Jꢃ7.2 Hz), 6.39 (1H, s), 6.51 (1H, s), 6.76 (1H, d, Jꢃ7.6 Hz), 6.81
6.94 (1H, dd, Jꢃ2.0, 8.0 Hz), 7.02 (1H, d, Jꢃ8.0 Hz), 7.10 (1H, d,
Jꢃ2.0 Hz), 7.85 (1H, d, Jꢃ2.8 Hz), 7.87 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z:
284 (M)ꢄ.
Methyl 10-Methoxymethyl-10H-pyrazino[2,3-b][1,4]benzothiazine-8-
(1H, d, Jꢃ7.6 Hz), 7.56 (1H, d, Jꢃ2.8 Hz), 7.67 (1H, d, Jꢃ2.8 Hz). ESI-MS acetate (51) A suspension of 50 (3.7 g, 13 mmol) and KOH (2.2 g,
m/z: 411 (MꢄH)ꢄ.
39 mmol) in EtOH (70 ml) was refluxed for 16 h. After cooling to rt, the
[3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-azabicy- mixture was poured into a cold aqueous solution of NaH2PO4, and extracted
clo[3.2.1]oct-8-yl]acetic Acid (2u) In the same manner as described for with AcOEt. The extract was washed with brine, dried, and evaporated to af-
the preparation of 2a, 2u (anti : synꢃ1 : 3) was obtained from 5u as a yellow ford 3.8 g (97%) of crude carboxylic acid as a black oil. A suspension of this
solid (60%). mp 134—135 °C. 1H-NMR (DMSO-d6) d: 1.54—2.10 (5H, m), oil (3.8 g, 13 mmol), methyl iodide (1.2 ml, 19 mmol) and K2CO3 (3.5 g,
2.24—2.56 (4H, m), 2.44 (2H, d, Jꢃ7.2 Hz), 2.40—2.64 (2H, m), 3.25 25 mmol) in DMF (50 ml) was stirred at rt for 12 h. The resulting mixture
(1/2H, s), 3.27 (3/2H, s), 6.68 (1H, d, Jꢃ8.0 Hz), 6.78 (1H, s), 6.81 (1H, d, was poured into water, and extracted with AcOEt. The extract was washed
Jꢃ8.0 Hz), 7.61 (1H, d, Jꢃ2.8 Hz), 7.62 (1H, d, Jꢃ2.8 Hz), 9.51 (1H, s).
FAB-MS m/z: 383 (MꢄH)ꢄ. Anal. Calcd for C20H22N4O2S·0.7H2O: C,
60.80; H, 5.97; N, 14.18. Found: C, 60.86; H, 5.81; N, 14.18.
with brine, dried, and evaporated. The residue was chromatographed on sil-
ica gel (15% AcOEt–hexane) to give 1.8 g (44%) of 51 as a yellow solid. mp
85—86 °C. 1H-NMR (CDCl3) d: 3.54 (3H, s), 3.57 (2H, s), 3.71 (3H, s),
Ethyl [8-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-8-azabi- 5.28 (2H, s), 6.89 (1H, dd, Jꢃ1.6, 7.6 Hz), 6.97 (1H, d, Jꢃ7.6 Hz), 7.07 (1H,
cyclo[3.2.1]oct-3-yl]acetate (5v) In the same manner as described for the d, Jꢃ1.6 Hz), 7.83 (1H, d, Jꢃ2.8 Hz), 7.84 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z:
preparation of 5s, 5v (exo : endoꢃ1 : 5) was obtained from 3920) and 3 as a
317 (M)ꢄ.
1
yellow oil (58%). H-NMR (CDCl3) d: 1.26 (3H, t, Jꢃ7.2 Hz), 1.20—1.33
10-Methoxymethyl-10H-pyrazino[2,3-b][1,4]benzothiazine-8-ethanol
(2H, m), 1.50—1.75 (2H, m), 1.95—2.08 (2H, m), 2.10—2.23 (2H, m), (52) To a suspension of LiAlH4 (76 mg, 2.0 mmol) in THF (20 ml) was
2.25—2.40 (1H, m), 2.45 (2H, d, Jꢃ8.0 Hz), 3.13 (2H, br s), 3.37 (5/3H, s),
added a solution of 51 (0.90 g, 2.8 mmol) in THF (10 ml) at 0 °C. The result-
3.40 (1/3H, s), 4.13 (2H, q, Jꢃ7.2 Hz), 6.48 (1H, s), 6.60 (1H, s), 6.79 (1H, ing mixture was stirred at 0 °C for 30 min, and water was added dropwise
d, Jꢃ8.0 Hz), 6.82 (1H, d, Jꢃ8.0 Hz), 7.54—7.60 (1H, m), 7.63—7.72 (1H, (0.08 ml), followed by 15% aqueous sodium hydroxide solution (0.08 ml),
m). ESI-MS m/z: 411 (MꢄH)ꢄ.
and finally water (0.24 ml). After stirring for 30 min, the reaction mixture
[8-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-8-azabicy- was filtered and the filtrate was evaporated. The residue was chro-
clo[3.2.1]oct-3-yl]acetic Acid (2v) In the same manner as described for matographed on silica gel (60% AcOEt–hexane) to give 0.70 g (86%) of 52
the preparation of 2a, 2v (exo : endoꢃ1 : 5) was obtained from 5v as a yel- as a yellow solid. mp 64—65 °C. 1H-NMR (CDCl3) d: 2.82 (2H, t,
1
low solid (63%). mp 148—149 °C. H-NMR (DMSO-d6) d: 1.21 (2H, br d,
Jꢃ6.4 Hz), 3.54 (3H, s), 3.85 (2H, t, Jꢃ6.4 Hz), 5.28 (2H, s), 6.86 (1H, dd,
Jꢃ13.6 Hz), 1.16—1.62 (2H, m), 1.88—2.09 (4H, m), 2.10—2.21 (1H, m), Jꢃ1.2, 8.0 Hz), 6.96 (1H, d, Jꢃ8.0 Hz), 7.03 (1H, d, Jꢃ1.2 Hz), 7.83 (1H, d,
2.34 (2H, d, Jꢃ8.4 Hz), 3.04 (2H, s), 3.29 (5/3H, s), 3.30 (1/3H, s), 6.74 Jꢃ2.8 Hz), 7.84 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 289 (M)ꢄ.
(1H, dd, Jꢃ1.6, 7.6 Hz), 6.82 (1H, d, Jꢃ7.6 Hz), 6.86 (1H, d, Jꢃ1.6 Hz),
7.63 (1H, d, Jꢃ2.8 Hz), 7.64 (1H, d, Jꢃ2.8 Hz), 9.47 (1H, s). FAB-MS m/z:
Ethyl 4-[1-[2-(10-Methoxymethyl-10H-pyrazino[2,3-b][1,4]benzothi-
azin-8-yl)ethyl]piperidin-4-yl]-2-methylbutanoate (53) To a solution of
383 (MꢄH)ꢄ. Anal. Calcd for C20H22N4O2S·0.8H2O: C, 60.52; H, 5.99; N, 52 (1.2 g, 4.1 mmol) and pyridine (0.73 ml, 9.0 mmol) in CH2Cl2 (30 ml) was
14.12. Found: C, 60.39; H, 6.10; N, 14.06.
added dropwise methanesulfonyl chloride (0.63 ml, 8.1 mmol) at 0 °C under
Methyl 10-Methoxymethyl-10H-pyrazino[2,3-b][1,4]benzothiazine-8- a nitrogen atmosphere. The resulting mixture was stirred at rt for 12 h, and
carboxylate (47) To a suspension of 4612) (415 g, 1.60 mol) in DMF (2.5 l)
was added NaH (60% dispersion in mineral oil, 66.0 g, 1.65 mol) portion
wise at ꢂ10—0 °C, and the mixture was stirred at 0 °C for 1 h. To this solu-
tion was added chloromethyl methyl ether (110 g, 1.37 mol) at below 5 °C, from this mesylate and 4d as a yellow oil (22%). H-NMR (CDCl3) d: 1.14
and the reaction mixture was stirred at rt for 2 h. The resulting mixture was
concentrated in vacuo. The residue was chromatographed on silica gel (50%
AcOEt–hexane) to give 1.5 g (quant.) of the mesylate as a yellow solid. In
the same manner as described for the preparation of 5d, 53 was obtained
1
(3H, d, Jꢃ7.2 Hz), 1.25 (3H, t, Jꢃ7.2 Hz), 1.20—1.34 (5H, m), 1.36—1.50
poured into ice-water, and extracted with CH2Cl2. The extract was washed (1H, m), 1.56—1.76 (3H, m), 1.97 (2H, br t, Jꢃ10.4 Hz), 2.39 (1H, sext,
with water, dried, and evaporated. The precipitate was washed with diiso- Jꢃ7.2 Hz), 2.48—2.60 (2H, m), 2.70—2.80 (2H, m), 2.97 (2H, br d,
propyl ether to give 335 g (69%) of 47 as a yellow solid. mp 151—152 °C. Jꢃ10.8 Hz), 3.53 (3H, s), 4.13 (2H, q, Jꢃ7.2 Hz), 5.26 (2H, s), 6.82 (1H, dd,
1H-NMR (CDCl3) d: 3.55 (3H, s), 3.91 (3H, s), 5.31 (2H, s), 7.06 (1H, d, Jꢃ1.2, 7.6 Hz), 6.92 (1H, d, Jꢃ7.6 Hz), 7.01 (1H, d, Jꢃ1.2 Hz), 7.82 (1H, d,
Jꢃ8.0 Hz), 7.62 (1H, dd, Jꢃ1.6, 8.0 Hz), 7.74 (1H, d, Jꢃ1.6 Hz), 7.85 (2H,
Jꢃ2.8 Hz), 7.83 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 485 (MꢄH)ꢄ.
s). FAB-MS m/z: 303 (M)ꢄ.
Ethyl 4-[1-[2-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-yl)ethyl]-
piperidin-4-yl]-2-methylbutanoate (54) To a solution of 53 (0.43 g,
0.89 mmol) in THF (10 ml) was added dropwise 5 N HCl (1.0 ml, 5.0 mmol)
at 0 °C. The resulting mixture was stirred at rt for 3 h, then was poured into a
mixture of NaHCO3–water, and extracted with AcOEt. The extract was
10-Methoxymethyl-10H-pyrazino[2,3-b][1,4]benzothiazine-8-
methanol (48) To a suspension of LiAlH4 (0.20 g, 5.3 mmol) in THF
(20 ml) was added a solution of 47 (2.0 g, 6.6 mmol) in THF (30 ml) at 0 °C.
The resulting mixture was stirred at 0 °C for 1 h, and water was added drop-
wise (0.2 ml), followed by 15% aqueous sodium hydroxide solution (0.2 ml), washed with brine, dried, and evaporated. The residue was chromatographed
and finally water (0.6 ml). After stirring for 30 min, the reaction mixture was
filtered and the filtrate was evaporated. The residue was chromatographed on
on silica gel (10% MeOH–AcOEt) to give 0.24 g (62%) of 54 as a pale
brown oil. 1H-NMR (CDCl3) d: 1.14 (3H, d, Jꢃ7.2 Hz), 1.10—1.35 (5H,
silica gel (60% AcOEt–hexane) to give 1.6 g (86%) of 48 as a yellow solid. m), 1.25 (3H, t, Jꢃ7.2 Hz), 1.35—1.50 (1H, m), 1.55—1.75 (3H, m), 1.95
mp 92—93 °C. 1H-NMR (CDCl3) d: 1.93 (1H, br s), 3.53 (3H, s), 4.62 (2H, (2H, br t, Jꢃ10.4 Hz), 2.38 (1H, sext, Jꢃ7.2 Hz), 2.45—2.55 (2H, m),
s), 5.29 (2H, s), 6.96 (1H, d, Jꢃ8.0 Hz), 6.99 (1H, d, Jꢃ8.0 Hz), 7.14 (1H,
s), 7.83 (2H, s). ESI-MS m/z: 275 (M)ꢄ.
2.60—2.70 (2H, m), 2.94 (2H, br d, Jꢃ10.8 Hz), 4.12 (2H, q, Jꢃ7.2 Hz),
6.33 (1H, br s), 6.35 (1H, d, Jꢃ1.6 Hz), 6.66 (1H, dd, Jꢃ1.6, 8.0 Hz), 6.79
(1H, d, Jꢃ8.0 Hz), 7.55 (1H, d, Jꢃ2.8 Hz), 7.67 (1H, d, Jꢃ2.8 Hz). ESI-MS
m/z: 441 (MꢄH)ꢄ.
8-Chloromethyl-10-methoxymethyl-10H-pyrazino[2,3-b][1,4]benzo-
thiazine (49) To a solution of 48 (10 g, 36 mmol) and pyridine (7.4 ml,
92 mmol) in DMF (70 ml) was added dropwise methanesulfonyl chloride
(7.0 ml, 90 mmol) at 0 °C under a nitrogen atmosphere. The resulting mix-
ture was stirred at rt for 1 h, then was poured into an aqueous solution of
NaHCO3 at 0 °C, and extracted with AcOEt. The extract was washed with
4-[1-[2-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-yl)ethyl]piperidin-4-
yl]-2-methylbutanoic Acid (2j) In the same manner as described for the
preparation of 2c, 2j was obtained from 54 as a yellow solid (64%). mp
1
239—240 °C. H-NMR (DMSO-d6) d: 1.01 (3H, d, Jꢃ6.8 Hz), 0.96—1.20
brine, dried, and evaporated. The precipitate was washed with ether to give (5H, m), 1.24—1.38 (1H, m), 1.46—1.60 (1H, m), 1.57 (2H, br d,
1
7.1 g (67%) of 49 as a yellow solid. mp 125—126 °C. H-NMR (CDCl3) d: Jꢃ10.8 Hz), 1.85 (2H, br t, Jꢃ10.4 Hz), 2.24 (1H, sext, Jꢃ6.8 Hz), 2.32—
3.55 (3H, s), 4.52 (2H, s), 5.29 (2H, s), 6.98—7.02 (2H, m), 7.17 (1H, s), 2.42 (2H, m), 2.42—2.56 (2H, m), 2.83 (2H, br d, Jꢃ11.2 Hz), 6.60 (1H, d,
7.84 (1H, d, Jꢃ2.8 Hz), 7.85 (1H, d, Jꢃ2.8 Hz). FAB-MS m/z: 293 (M)ꢄ.
10-Methoxymethyl-10H-pyrazino[2,3-b][1,4]benzothiazine-8-acetoni-
trile (50) A suspension of 49 (7.1 g, 24 mmol) and sodium cyanide (2.4 g,
49 mmol) in DMSO (50 ml) was stirred at rt for 12 h. The resulting mixture
was poured into water, and extracted with AcOEt. The extract was washed
with brine, dried, and evaporated. The residue was chromatographed on sil-
Jꢃ1.6 Hz), 6.63 (1H, dd, Jꢃ1.6, 7.6 Hz), 6.77 (1H, d, Jꢃ7.6 Hz), 7.60 (1H,
d, Jꢃ2.8 Hz), 7.62 (1H, d, Jꢃ2.8 Hz), 9.40 (1H, s). FAB-MS m/z: 413
(MꢄH)ꢄ. Anal. Calcd for C22H28N4O2S·0.8H2O: C, 61.89; H, 6.99; N,
13.12. Found: C, 61.78; H, 6.97; N, 13.13.
10-Methoxymethyl-10H-pyrazino[2,3-b][1,4]benzothiazine-8-car-
boxylic Acid (55) A suspension of 47 (3.0 g, 9.9 mmol) and NaOH (0.80 g,
ica gel (20% AcOEt–hexane) to give 5.2 g (76%) of 50 as a yellow solid. mp 20 mmol) in EtOH (20 ml)–water (15 ml) was stirred at rt for 12 h. After
1
121—122 °C. H-NMR (CDCl3) d: 3.55 (3H, s), 3.69 (2H, s), 5.28 (2H, s), evaporation of the solvent, the residue was diluted with an aqueous solution

June 2004
687
of NaH2PO4, and extracted with AcOEt. The extract was washed with brine,
dried, and evaporated to give 2.8 g (97%) of 55 as a yellow solid. mp 204—
205 °C. 1H-NMR (DMSO-d6) d: 3.40 (3H, s), 5.27 (2H, s), 7.22 (1H, d,
Jꢃ7.6 Hz), 7.52 (1H, dd, Jꢃ1.2, 7.6 Hz), 7.64 (1H, d, Jꢃ1.2 Hz), 7.93 (1H,
d, Jꢃ2.4 Hz), 7.97 (1H, d, Jꢃ2.4 Hz), 13.10 (1H, s). ESI-MS m/z: 289 (M)ꢄ.
Jꢃ2.8 Hz), 7.62 (1H, d, Jꢃ2.8 Hz), 9.45 (1H, s). FAB-MS m/z: 413
(MꢄH)ꢄ. Anal. Calcd for C22H28N4O2S·H2O: C, 61.37; H, 7.02; N, 13.01.
Found: C, 61.16; H, 6.93; N, 12.88.
Carrageenan-Induced Pleurisy in the Rat24) Male F344 Rats (6—7
week old, Charles River Japan Inc., Atsugi, Japan) were anesthetized with
Et2O, and l-carrageenan (0.2 ml/pleural cavity, 1% dissolved in saline,
Zushikagaku Laboratory, Inc., Kanagawa, Japan) was injected into the
pleural cavity of the rats. The test compound was orally administered 30 min
before the injection of carrageenan. After 5 h, the rats were sacrificed and
the exudate in the cavity was carefully collected by pipette and its weight
was measured. The numbers/volume of viable leukocytes in the exudate
fluid were counted with a hemocytometer. The numbers of infiltrated leuko-
cytes in the exudate fluid were calculated by the formula; (exudates
volume)ꢆ(numbers/volume of infiltrated leukocytes).
Ethyl
4-[1-[(10-Methoxymethyl-10H-pyrazino[2,3-b][1,4]benzothia-
zin-8-yl)carbonyl]piperidin-4-yl]-2-methylbutanate (56) To a solution
of 55 (1.0 g, 3.5 mmol), 4d (1.0 g, 4.0 mmol) and N,N-diisopropylethylamine
(iPr2EtN, 3.0 ml, 17 mmol) in CH2Cl2 (30 ml) was added benzotriazolyloxy-
tris[pyrrolidino]-phosphonium
hexafluorophosphate
(PyBop,
2.2 g,
4.2 mmol), and the mixture was stirred at rt for 12 h. The reaction mixture
was poured into water and extracted with AcOEt. The extract was washed
with brine, dried, and evaporated. The residue was chromatographed on sil-
1
ica gel (50% AcOEt–hexane) to give 1.2 g (71%) of 56 as a yellow oil. H-
NMR (CDCl3) d: 1.06—1.34 (4H, m), 1.15 (3H, d, Jꢃ7.2 Hz), 1.26 (3H, t,
Jꢃ7.2 Hz), 1.36—1.60 (2H, m), 1.60—1.90 (3H, m), 2.39 (1H, sext,
Jꢃ7.2 Hz), 2.75 (1H, br s), 2.97 (1H, br s), 3.51 (3H, s), 3.75 (1H, br s), 4.13
(2H, q, Jꢃ7.2 Hz), 4.65 (1H, br s), 5.26 (2H, s), 6.98 (1H, dd, Jꢃ1.2,
7.6 Hz), 7.03 (1H, d, Jꢃ7.6 Hz), 7.18 (1H, d, Jꢃ1.2 Hz), 7.85 (1H, d,
Jꢃ2.8 Hz), 7.86 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 485 (MꢄH)ꢄ.
Type II Collagen-Induced Arthritis in the Rat Lewis female rats were
supplied by Charles River Japan. Type II collagen solution (0.16%; Cosmo-
Bio, Japan) containing muramyl dipeptide (0.4 mg/ml) was emulsified with
an equal volume of Freund’s incomplete adjuvant (FIA; Difco Laboratories).
1 ml of this emulsion was injected intradermally at various sites in the back
of the animals. After 14 d, the rats were grouped with the same mean volume
of paws at 3.0 ml. Compound 2q was orally administered daily for 10 d from
day 14 to day 23 as a suspension in a 0.5% methylcellulose solution. The
severity of collagen-induced arthritis was assessed by quantification of hind
paw swelling (nꢃ8).
4-[1-[(10-Methoxymethyl-10H-pyrazino[2,3-b][1,4]benzothiazin-8-
yl)carbonyl]piperidin-4-yl]-2-methylbutanoic Acid (57) To a solution of
56 (0.70 g, 1.4 mmol) in EtOH (10 ml)–THF (10 ml) was added 5 N NaOH
(1.2 ml, 6.0 mmol) under a nitrogen atmosphere, and the mixture was stirred
at 80 °C for 2 h. After cooling of the mixture to 0 °C, the reaction mixture
was poured into an aqueous solution of NaH2PO4. The mixture was ex-
tracted with AcOEt. The extract was washed with brine, dried, and evapo-
rated. The residue was chromatographed on silica gel (80% AcOEt–hexane)
References
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1
to give 0.48 g (75%) of 57 as a yellow oil. H-NMR (CDCl3) d: 1.08—1.40
(4H, m), 1.20 (3H, d, Jꢃ7.2 Hz), 1.40—1.60 (2H, m), 1.60—1.90 (3H, m),
2.40—2.52 (1H, m), 2.74 (1H, br s), 2.98 (1H, br s), 3.51 (3H, s), 3.78 (1H,
br s), 4.65 (1H, br s), 5.26 (2H, s), 6.99 (1H, dd, Jꢃ1.6, 8.0 Hz), 7.03 (1H, d,
Jꢃ8.0 Hz), 7.18 (1H, d, Jꢃ1.6 Hz), 7.85 (1H, d, Jꢃ2.8 Hz), 7.87 (1H, d,
Jꢃ2.8 Hz). ESI-MS m/z: 456 (M)ꢄ.
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4-[1-[(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-yl)carbonyl]piperidin-
4-yl]-2-methylbutanoic Acid (2k) To a solution of 57 (0.13 g, 0.28 mmol)
in THF (10 ml) was added dropwise 5 N HCl (1.5 ml, 7.5 mmol) at 0 °C. The
resulting mixture was stirred at rt for 2 h, then was concentrated in vacuo.
The residue was diluted with water, and stirred at rt for 20 min. The precipi-
tate was collected to give 0.083 g (72%) of 2k as a yellow solid. mp 103—
104 °C. 1H-NMR (DMSO-d6) d: 0.96—1.10 (2H, m), 1.05 (3H, d,
Jꢃ6.8 Hz), 1.14—1.27 (2H, m), 1.31—1.42 (1H, m), 1.42—1.80 (4H, m),
2.29 (1H, sext, Jꢃ6.8 Hz), 2.68 (1H, br s), 2.96 (1H, br s), 3.59 (1H, br s),
4.37 (1H, br s), 6.73—6.77 (2H, m), 6.94—6.98 (1H, m), 7.66—7.68 (2H,
m), 9.59 (1H, s), 12.0 (1H, s). FAB-MS m/z: 413 (MꢄH)ꢄ. Anal. Calcd for
C21H24N4O3S·0.5H2O: C, 59.84; H, 5.98; N, 13.29. Found: C, 60.10; H,
5.96; N, 13.31.
Ethyl 4-[1-(10-Methoxymethyl-10H-pyrazino[2,3-b][1,4]benzo-
thiazin-8-ylmethyl)piperidin-4-yl]-2,2-dimethylbutanoate (58) In the
same manner as described for the preparation of 5d, 58 was obtained from
1
19 and 49 as a yellow oil (67%). H-NMR (CDCl3) d: 1.10—1.30 (5H, m),
1.14 (6H, s), 1.24 (3H, t, Jꢃ7.2 Hz), 1.46—1.56 (2H, m), 1.64 (2H, br d,
Jꢃ12.4 Hz), 1.98 (2H, br t, Jꢃ11.2 Hz), 2.84 (2H, br d, Jꢃ11.2 Hz), 3.41
(2H, s), 3.53 (3H, s), 4.10 (2H, q, Jꢃ7.2 Hz), 5.29 (2H, s), 6.93 (1H, dd,
Jꢃ1.2, 8.0 Hz), 6.96 (1H, d, Jꢃ8.0 Hz), 7.10 (1H, s), 7.83 (1H, d,
Jꢃ3.2 Hz), 7.83 (1H, d, Jꢃ3.2 Hz). ESI-MS m/z: 485 (MꢄH)ꢄ.
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4-[1-(10-Methoxymethyl-10H-pyrazino[2,3-b][1,4]benzothiazin-8-yl-
methyl)piperidin-4-yl]-2,2-dimethylbutanoic Acid (59) In the same
manner as described for the preparation of 57, 59 was obtained from 58 as a
yellow oil (72%). 1H-NMR (CDCl3) d: 1.10—1.25 (3H, m), 1.17 (6H, s),
1.25—1.40 (2H, m), 1.42—1.50 (2H, m), 1.74 (2H, br d, Jꢃ13.2 Hz), 2.16
(2H, br t, Jꢃ11.2 Hz), 3.22 (2H, br d, Jꢃ11.2 Hz), 3.51 (3H, s), 3.71 (2H, s),
5.27 (2H, s), 6.93 (1H, d, Jꢃ8.0 Hz), 6.97 (1H, d, Jꢃ8.0 Hz), 7.03 (1H, s),
7.83 (1H, d, Jꢃ2.8 Hz), 7.85 (1H, d, Jꢃ2.8 Hz). ESI-MS m/z: 457 (MꢄH)ꢄ.
4-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
20) Kato M., Ito K., Nishino S., Yamakuni H., Takasugi H., Chem. Pharm.
Bull., 43, 1351—1357 (1995).
yl]-2,2-dimethylbutanoic Acid (2l) To
a solution of 59 (0.14 g,
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20, 427—432 (1985) [Chem. Abstr., 105, 226282 (1986).]
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0.31 mmol) in THF (20 ml) was added dropwise 5 N HCl (1.0 ml, 5.0 mmol)
at 0 °C. The resulting mixture was stirred at rt for 2 h, then was concentrated
in vacuo. The residue was diluted with 1 N NaOH, and this aqueous phase
was chromatographed on MCI GEL (water to MeOH) to give 0.096 g (75%)
of 2l as a yellow solid. mp 247—248 °C. 1H-NMR (DMSO-d6) d: 0.92 (6H,
s), 0.98—1.14 (5H, m), 1.26—1.34 (2H, m), 1.55 (2H, br d, Jꢃ9.2 Hz), 1.80
(2H, br t, Jꢃ10.0 Hz), 2.71 (2H, br d, Jꢃ10.8 Hz), 3.20 (2H, s), 6.67 (1H, dd,
Jꢃ1.6, 8.0 Hz), 6.75 (1H, d, Jꢃ1.6 Hz), 6.80 (1H, d, Jꢃ8.0 Hz), 7.61 (1H, d,