Inhibitors of adhesion molecules expression; The synthesis and pharmacological properties of 10H-pyrazino[2,3-b][1,4]benzothiazine derivatives

Article abstract of DOI:10.1248/cpb.50.922

During a search for novel, orally-active inhibitors of upregulation of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), we found a new series of 10H-pyrazino[2,3-b][1,4]benzothiazine derivatives to be potent ICAM-1 inhibitors. Of the

Full text of DOI:10.1248/cpb.50.922

922
Chem. Pharm. Bull. 50(7) 922—929 (2002)
Vol. 50, No. 7
Inhibitors of Adhesion Molecules Expression; The Synthesis and
Pharmacological Properties of 10H-Pyrazino[2,3-b][1,4]benzothiazine
Derivatives
Toshihiko KANEKO, ,a Richard S. J. CLARK,a Norihito OHI,a Tetsuya KAWAHARA,a Hiroshi AKAMATSU,a
*
Fumihiro OZAKI,a Atsushi KAMADA,a Kazuo OKANO,a Hiromitsu YOKOHAMA,a Kenzo MURAMOTO,a
b
Masayoshi OHKURO,a 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 February 5, 2002; accepted April 11, 2002
During a search for novel, orally-active inhibitors of upregulation of adhesion molecules such as intercellu-
lar adhesion molecule-1 (ICAM-1), we found a new series of 10H-pyrazino[2,3-b][1,4]benzothiazine derivatives to
be potent ICAM-1 inhibitors. Of these compounds, N-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-
piperidin-4-yl]-N؅,N؅-dimethylsulfamide 7p showed the potent oral inhibitory activities against neutrophil mi-
gration in a murine interleukin-1 (IL-1) induced paw inflammation model. The synthesis and structure–activity
relationships of these amide derivatives are described.
Key words adhesion molecule inhibitor; rheumatoid arthritis; 10H-pyrazino[2,3-b][1,4]benzothiazine
In various inflammatory and immune diseases leukocytes cules, especially ICAM-1, have been disclosed in the litera-
migrate from the vasculature into surrounding tissue, where ture, however to date no such low-molecular weight com-
they participate in the inflammatory response, resulting in pounds have been clinically evaluated.19—23) Recently, antag-
tissue damage, swelling, pain and loss of function. This onists of the ICAM-1/LFA-1 interaction and of VLA-4 have
process is a cascade of events wherein the leukocytes adhere also been reported.24—40)
first transiently, then firmly to the endothelial cells lining
During the course of our search for new orally-active
blood vessels, then infiltrate through the vessel wall. Adhe- adhesion molecules inhibitors, we discovered the 10H-
sion molecules such as intercellular adhesion molecule-1 pyrazino[2,3-b][1,4]benzothiazine derivative 7b that inhibits
(ICAM-1),1) E-selectin2) and vascular cell adhesion mole- ICAM-1 expression on human umbilical vein endothelial
cule-1 (VCAM-1)3,4) are upregulated on the endothelium by cells (HUVEC) with an IC50 value of 0.32 mM. In this report
interleukin-1 (IL-1) or tumor necrosis factor-a (TNF-a) and we describe our initial structure activity studies which focus
mediate steps in this cascade of events.5,6)
on modifications to the piperidine ring of 7b.
It has also been established that activated lymphocytes
Chemistry The 10H-pyrazino[2,3-b][1,4]benzothiazine
play a pivotal role in the development and progression of in- derivatives shown in Tables 1—4 were prepared as shown in
flammatory diseases such as rheumatoid arthritis (RA), coli- Charts 1—3. Compound 2 was synthesized from 4-chloro-3-
tis, psoriasis, multiple sclerosis etc., in which immunological nitrobenzoic acid 1 by displacement of chloride with sodium
mechanisms are involved. For T cell activation and clonal ex- disulfide (Na2S2), reduction of the nitro group and esterifica-
pansion to occur, in addition to the stimulation of T cell re- tion.41,42) These intermediates, including aminothiophenol 2,
ceptor (TCR) with antigen-MHC complex expressed on anti- were unstable to purification. The 10H-pyrazino[2,3-b][1,4]-
gen presenting cells, a costimulatory signal mediated by ac- benzothiazine ring was constructed by condensation of 2
cessory molecules is also required, and TCR-mediated acti- with 2,3-dichloropyrazine. The reduction of ester 3 with
vation in the absence of such a signal can lead to T cell unre- lithium aluminum hydride led to alcohol 4, which on chlori-
sponsiveness. The interactions of adhesion molecules such as nation with methanesufonyl chloride afforded the key 10H-
lymphocyte function associated antigen-1 (LFA-1)/ICAM- pyrazino[2,3-b][1,4]benzothiazine intermediate 5. The ben-
17—10) and very late antigen-4 (VLA-4)/VCAM-111,12) have
been demonstrated to transmit this costimulatory signal, and
hence it is expected that interfering with these interactions
will lead not only to the inhibition of leukocyte infiltration
outlined above, but also to unresponsiveness to antigen.
Hence inhibitors of adhesion molecules appear to be an at-
tractive means for both ameliorating the inflammatory re-
Fig. 1. Structures of the Known Small Molecule Cell Adhesion Molecule
sponse and achieving the long-term modulation of the im-
mune response in autoimmune diseases.
Expression Inhibitor (PD144795 and A205804) and ICAM-1/LFA-1 Bind-
ing Inhibitor (RWJ-50271)
It has been reported that anti-adhesion molecule antibodies
are able to ameliorate the inflammatory reaction and im-
munological parameters in various animal models.13,14) In
clinical trials a monoclonal antibody to ICAM-1 and an
ICAM-1 antisense oligonucleotide showed beneficial
effects.15—18) A number of inhibitors of cell adhesion mole-
To whom correspondence should be addressed. e-mail: t3-kaneko@hhc.eisai.co.jp
© 2002 Pharmaceutical Society of Japan

July 2002
923
Chart 1
Chart 2
Chart 3
famoyl chloride51) or sulfamide, followed by deprotection
using 10% palladium on carbon (Pd/C). Sulfamides 6s and 6t
were synthesized using the method of G. E. DuBois as shown
in Chart 3.52,53) Catechol sulfate 10 was condensed with N-
benzyl piperidine to obtain sulfamate ester 11. The key sec-
ondary amines 6s and 6t were obtained by coupling of this
sulfamate ester 11 with piperidine or morpholine respec-
tively, followed by debenzylation using Pd/C.
zyl chloride 5 was condensed with the amines 6a—x, to ob-
tain the target molecules 7a—x.
With the exceptions of 6n and 6p—t, the secondary
amines 6a—x are either commercially available or known in
the literature.43—50) Sulfonamide 6n and sulfamides 6p—r
were prepared as shown in Chart 2 by condensation of 4-
amino-1-benzylpiperidine with trifluoromethanesulfonic an-
hydride, N,N-dimethylsulfamoyl chloride or N-methylsul-

924
Vol. 50, No. 7
Table 1. ICAM-1 Inhibitory Activity of Simple Ring Derivatives (7a—f)
Biological Assay The 10H-pyrazino[2,3-b][1,4]benzoth-
iazine derivatives were first evaluated for their inhibitory ac-
tivity against the expression of adhesion molecules such as
ICAM-1 on TNF-a-stimulated HUVEC in vitro. A solution
of the test compound was added to HUVEC, which was then
stimulated with TNF-a (1 ng/ml) for 4 h. After fixing the
HUVEC with glutaraldehyde, ICAM-1 expression on the cell
surface was evaluated using an ELISA.54) For in vitro studies
the relative potencies are expressed as IC50 values. Tests were
run in duplicate, and the IC50 value determinations were per-
formed by the least-squares method using four concentra-
tions of compound.
The effect of the compounds on neutrophil accumulation
was next evaluated in a mouse IL-1-induced paw inflamma-
tion model. Briefly, the compounds to be tested were orally
administered to BALB/c mice 30 min prior to a rat rIL-1 in-
jection. Two hours after this injection, the injected paws were
removed and myeloperoxidase (MPO) was measured spec-
trophotometrically as a marker enzyme for neutrophil content
according to a literature method.55)
Compound
No.
ICAM-1a)
IC50 (mM)
NR1R2
7a
7b
7c
7d
0.49
0.27
0.83
1.1
7e
7f
2.0
5.0
a) Concentration of compound inhibiting ICAM-1 up-regulation by 50% of control
value.
Table 2. ICAM-1 Inhibitory Activity of Piperidine Amide Derivatives
(7g—i)
Results and Discussion
Initially, the effect of ring size of the cyclic amine in com-
pound 7 was investigated as shown in Table 1. The azetidine
derivative 7a, and 7- or 8-membered-ring (7c, d) compounds
were 2—4-fold less active than piperidine 7b. The morpho-
line derivative 7e and piperazine derivative 7f were also pre-
pared to examine the possibility of introducing a heteroatom
at the 4-position of piperidine, but were less active than the
parent structure. These results suggest that the 6-membered-
ring is favorable for activity.
Compound
No.
ICAM-1a)
IC50 (mM)
X
Position
7g
7h
7i
CONH2
CONH2
CONH2
2
3
4
11.7
1.02
0.69
As shown in Table 2 we next turned our attention to substi-
tuted piperidine derivatives, examining the optimal position
for substitution using the carboxamide group as a probe. The
4-carboxamide 7i showed good activity, with an IC50 value of
0.69 mM, while the 3-derivative 7h showed a slight loss of ac-
tivity, with an IC50 value of 1.02 mM. On the other hand, the
2-carboxamide 7g showed a 17-fold loss in inhibitory activ-
ity compared to 7i. These results suggest that steric hin-
drance around the benzylamine nitrogen is unfavorable. We
decided to optimize further at the 4-position of the piperi-
dine.
Next, modification of the carboxamide moiety of 7i was
investigated with results shown in Table 3. Retro-amide 7j
was twice as active in vitro as amide 7i, while methanesul-
fonamide 7k, used as a bioisostere of carboxamide, also
showed 2-fold greater inhibitory activity than 7i. We evalu-
ated the inhibitory activity of 7k against neutrophil infiltra-
tion in a mouse IL-1-induced paw inflammatory model at an
oral dose of 10 mg/kg. In this model, antibody to ICAM-1
(KAT-1) has been found effective in reducing neutrophil infil-
tration in the IL-1 injected paw (data not shown). It showed
significant inhibition of neutrophil migration in this model
with an inhibition value of 85.9Ϯ4.0%. Hence, we next fo-
cused on sulfonamide derivatives in order to obtain stronger
orally-active compounds. Compounds with an alkylene
spacer between the piperidine and methanesulfonamide
(7l, m) retained potency both in vitro and in vivo. Replace-
ment of the methyl group of compound 7k with a trifluo-
romethyl group 7n, or phenyl group 7o also led to retention
of in vitro potency, but to loss in potency in the IL-1-induced
a) Concentration of compound inhibiting ICAM-1 up-regulation by 50% of control
value.
Table 3. Biological Activities of Piperidine Sulfonamides and Sulfamides
(7j—t)
Compound
No.
ICAM-1a)
IC50 (mM) (10 mg/kg, p.o.)
IL-1 pawb)
X
ClogP
7j
7k
7l
7m
7n
7o
7p
7q
7r
NHCOMe
NHSO2Me
CH2NHSO2Me
CH2CH2NHSO2Me 0.36
NHSO2CF3
NHSO2Ph
NHSO2NMe2
NHSO2NHMe
NHSO2NH2
0.34
0.32
0.36
N.T.
—
85.9Ϯ4.0
77.6Ϯ13.0
89.7Ϯ4.0
14.2Ϯ3.2
11.6Ϯ3.2
83.4Ϯ7.4
47.1Ϯ15.3
10.1Ϯ8.0
0.67
1.29
1.82
2.78
2.46
0.53
1.40
0.25
0.30
0.37
0.32
0.32
0.26
7s
0.32
0 (30)c)
1.72
7t
0.30
66.1Ϯ11.9 (30)c) 0.87
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. c) Percentage inhibition at a dose of 30 mg/kg p.o. N.T.: not tested.
paw model in mice at a dose of 10 mg/kg. These results sug-
gest that the degree of lipophilicity of this terminal portion of
compound 7k may be critical for in vivo activity. To evaluate
the lipophilicity of our compounds, we calculated their

July 2002
925
ClogP, the values for 7k, 7n and 7o being 0.67, 2.78 and 2.46 7p were also evaluated in fed male rats as shown in Table 5
respectively.56) We speculated that the preferred ClogP value (10 mg/kg p.o., 3 mg/kg i.v., nϭ3). Compound 7p has a Cmax
is around 0.6 for these 4-aminopiperidine derivatives and of 0.86Ϯ0.17 mM, a mean residence time (MRT) of
went on to prepare and examine the low ClogP sulfamide de- 11.4Ϯ2.1 h, and oral bioavailability of 69.0Ϯ1.9%. In this
rivatives 7p—t shown in Table 3. In vitro, all these deriva- study, the demethyl metabolite, shown as 7q, was also ob-
tives showed equivalent potency. Of these compounds, the served with a Cmax of 0.52Ϯ0.05 mM, and a MRT of 11.4Ϯ
N,N-dimethyl derivative 7p was as active in the IL-1 paw 1.7 h.
model as the methanesulfonamide 7k. The ClogP value of 7p
is also around 0.6, the value suggested above. Furthermore, Conclusion
the morpholinesulfamide 7t showed 66.1Ϯ11.9% inhibition
In order to develop an orally-active inhibitor of adhesion
in the IL-1 paw model at 30 mg/kg, while the activity of the molecules expression, 10H-pyrazino[2,3-b][1,4]benzoth-
equivalent piperidine sulfamide 7s was low. Taken together iazine derivatives were synthesized and their pharmacologi-
these results suggest that introduction of a “carrier” sulfon- cal properties were evaluated. We found 8-methylpiperidine
amide or sulfamide moiety into these types of compounds to to be a prefered substituent, and that 4-sulfonamide- and 4-
bring their ClogP to around 0.6 can be an important factor in sulfamide-substituted piperidine derivatives showed espe-
achieving in vivo activity in the IL-1 paw model.
cially potent in vivo activities. In particular, N-[1-(10H-
We next reexamined the piperazine scaffold, preparing pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-yl]-
amides 7u—x. Carboxamide 7u showed weak activity in NЈ,NЈ-dimethylsulfamide 7p significantly inhibited neu-
vitro compared with the piperidine carboxamide 7j, the same trophil migration in the mouse IL-1-induced paw inflamma-
trend as observed in the simple ring compounds 7b and 7f. tion model after oral administration at a dose of 10 mg/kg.
However, both sulfonamide 7v and sulfamide 7w showed This compound has also good oral bioavailability. This may
good in vitro activities. These compounds also suppressed prove to be valuable therapeutic agent in the treatment of
neutrophil migration moderately in the IL-1-induced paw in- chronic disorders such as rheumatoid arthritis. Efforts to dis-
flammation model. 7x, the analog of 7p, showed no in- cover novel adhesion molecule inhibitors possessing more
hibitory activity in this model even at a dose of 30 mg/kg. potent oral activity are ongoing.
The activities of all the piperazine series were less potent
Experimental
than those of the corresponding piperidine analogues.
The inhibitory activity on other adhesion molecules was
studied. The compound 7p suppressed the up-regulation of
E-selectin with an IC50 value of 0.55 mM and that of VCAM-
1 with an IC50 value of 0.36 mM.
Melting points were measured using a Yanako melting-point apparatus
and are uncorrected. The 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:
sϭsinglet, dϭdoublet, tϭtriplet, qϭquartet, mϭmultiplet, brϭbroad. Mass
spectra (MS) were obtained on a JEOL JMS-HX100 mass spectrometer.
High resolution mass spectra (HR-MS) were obtained on a JEOL JMS-
SX102AQQ mass spectrometer. Elemental analysis was performed with a
Heraeus Elemental Analyzer CHN-O-RAPID. Materials were used as
bought without 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 organic extracts were dried over anhydrous MgSO4, and solvents were
removed with a rotary evaporator under reduced pressure.
Methyl 10H-Pyrazino[2,3-b][1,4]benzothiazine-8-carboxylate (3) To
a suspension of 4-chloro-3-nitrobenzoic acid 1 (500 g, 2.46 mol) in ethanol
(2250 ml) was added dropwise a solution of sodium hydroxide (67 g,
1.7 mol) in water (125 ml), followed by portion-wise addition of a solution
of Na2S2 (prepared from sodium sulfide/9H2O (600 g, 2.50 mol) and sulfur
(80 g, 2.5 mol)). The resulting mixture was refluxed for 30 min, cooled to
room temperature (rt), then filtered to obtain 450 g of a dark-green solid pre-
cipitate.
To a suspension of this crude compound (450 g) and tin (1098 g, 9.25 mol)
in ethanol (2500 ml) was added dropwise conc.HCl until reaction was com-
plete by TLC. The tin residue was removed by filtration, and the filtrate was
evaporated. Conc.HCl and ethanol were added to the residue and filtration of
the precipitate gave 270 g of a pale yellow powder.
This powder (270 g) was added to a hydrogen chloride saturated methanol
solution (900 ml). The resulting mixture was refluxed for 7 h, then was con-
centrated in vacuo to give the crude pale yellow solid (320 g).
To a suspension of this solid (320 g, 1.72 mol) in N,N-dimethylformamide
(DMF, 320 ml) was added dropwise 2,3-dichloropyrazine (256 g, 1.72 mol).
The resulting mixture was stirred at 100 °C for 30 min, cooled to rt, and
added to 640 ml of water, then the precipitate was filtered off and washed
with water and ether to give 128 g of 3 as a yellow powder (20%, from 1).
mp 265—268 °C. 1H-NMR (DMSO-d6) d: 3.78 (3H, s), 7.02 (1H, d,
Jϭ8.2 Hz), 7.29 (1H, dd, Jϭ1.9, 8.2 Hz), 7.31 (1H, d, Jϭ1.9 Hz), 7.64 (1H,
d, Jϭ2.9 Hz), 7.65 (1H, d, Jϭ2.9 Hz), 9.63 (1H, s). FAB-MS m/z: 259 (M)ϩ.
10H-Pyrazino[2,3-b][1,4]benzothiazine-8-methanol (4) To a stirred
suspension of lithium aluminum hydride (40 g, 1.1 mol) in tetrahydrofuran
(THF, 1 l) was added dropwise a solution of 3 (200 g, 771 mmol) in THF
The pharmacokinetic characteristics of dimethylsulfamide
Table 4. Biological Activities of Piperazine Amide Derivatives (7u—x)
Compound
No.
ICAM-1a)
IC50 (mM)
IL-1 pawb)
(10 mg/kg)
R
ClogP
7u
7v
7w
7x
–COMe
1.4
18.6Ϯ5.9
53.4Ϯ11.8
41.8Ϯ22.4
0 (30)c)
1.16
1.58
1.46
1.61
–SO2Me
–SO2NH2
–SO2NMe2
0.39
0.58
1.0
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. c) Percentage inhibition at a dose of 30 mg/kg p.o.
Table 5. Pharmacokinetic Characteristics of 7p
Tmax
Cmax
AUC0—24 h
MRT0—24 h
B.A.0—24 h
(%)
(h)
(mM)
(mM ·h)
(h)
10 mg/kg, p.o. 9.00Ϯ7.51 0.86Ϯ0.17
11.5Ϯ0.3
11.4Ϯ2.1
69.0Ϯ1.9
T1/2(a)
T1/2(b)
CLt
Vdss
(h)
(h)
(ml/h/kg)
(ml/kg)
3 mg/kg, i.v. 0.33Ϯ0.26 2.30Ϯ0.37
MRT is mean residence time.
1335Ϯ26
3566Ϯ198

926
Vol. 50, No. 7
(2.5 l) over 1 h at below 15 °C under a nitrogen atmosphere. After comple-
tion of addition, the resulting mixture was stirred for 1 h at this temperature,
then was cooled with ice-water, and water was added dropwise (40 ml), fol-
lowed by 15% aqueous sodium hydroxide solution (40 ml), and finally water
(120 ml), all at under 20 °C. Stirring was continued for a further 30 min, then
the reaction mixture were filtered. The aluminum residue was washed well
with 4ϫ1 l of THF, then the organic phases were evaporated to give 125 g
(70%) of 4 as a yellow crystalline solid. mp 187—189 °C. 1H-NMR
(DMSO-d6) d: 4.30 (2H, d, Jϭ6.0 Hz), 5.17 (1H, t, Jϭ6.0 Hz), 6.70 (1H, d,
Jϭ7.9 Hz), 6.75 (1H, s), 6.83 (1H, d, Jϭ7.9 Hz), 7.61 (1H, d, Jϭ2.6 Hz),
7.63 (1H, d, Jϭ2.6 Hz), 9.50 (1H, s). FAB-MS m/z: 231 (M)ϩ.
8-Chloromethyl-10H-pyrazino[2,3-b][1,4]benzothiazine (5) To a so-
lution of 4 (7.0 g, 30 mmol) and pyridine (6.1 ml, 76 mmol) in DMF (50 ml)
was added dropwise methanesulfonyl chloride (5.9 ml, 76 mmol) under ni-
trogen at 0 °C. The resulting mixture was stirred for 1 h at rt, then was
poured into a mixture of NaHCO3–water–CH2Cl2, and extracted with ethyl
acetate (AcOEt). The extract was washed with brine, dried, and evaporated.
The precipitate was washed with ether to give 4.7 g (62%) of 5 as a yellow
powder. mp 161—162 °C. 1H-NMR (DMSO-d6) d: 4.58 (2H, s), 6.78—6.80
(1H, m), 6.80—6.84 (1H, m), 6.90 (1H, dd, Jϭ1.7, 7.9 Hz), 7.63—7.66 (2H,
m), 9.58 (s, 1H). FAB-MS m/z: 249 (M)ϩ.
4.95 (2H, br s), 6.83 (1H, dt, Jϭ2.0, 8.0 Hz), 6.91 (1H, dd, Jϭ2.0, 8.0 Hz),
7.10 (1H, dt, Jϭ2.0, 8.0 Hz), 7.22 (1H, dd, Jϭ2.0, 8.0 Hz), 7.24—7.32 (5H,
m).
N-(1-Benzylpiperidin-4-yl)-N؅,N؅-pentamethylenesulfamide
(12s)
This sulfamide was prepared according to the procedure of DuBois.53) A so-
lution of 11 (8.7 g, 24 mmol) and piperidine (2.4 g, 29 mmol) in 1,4-dioxane
(50 ml) was refluxed for 4 h under a nitrogen atmosphere. The reaction mix-
ture was allowed to cool and then poured into water, and extracted with
AcOEt. The extract was washed with brine, dried, and evaporated. The
residue was chromatographed on silica gel (4% MeOH–CH2Cl2) to afford
12s (4.9 g, 60%) as a pale red solid. mp 82—83 °C. 1H-NMR (CDCl3) d:
1.47—1.60 (4H, m), 1.55—1.70 (4H, m), 1.90—2.05 (2H, m), 2.10 (2H,
br t, Jϭ10.8 Hz), 2.79 (2H, d, Jϭ11.6 Hz), 3.15 (4H, t, Jϭ5.6 Hz), 3.10—
3.30 (1H, m), 3.48 (2H, s), 4.00—4.15 (1H, m), 7.22—7.40 (5H, m).
N-(1-Benzylpiperidin-4-yl)-N؅,N؅-(3-oxapentamethylene)sulfamide
(12t) In the same manner as described for the preparation of 12s, 12t was
obtained as a pale red solid (62%). mp 99—100 °C. 1H-NMR (CDCl3) d:
1.48—1.60 (2H, m), 1.98 (2H, br d, Jϭ12 Hz), 2.10 (2H, br t, Jϭ11.6 Hz),
2.80 (2H, br d, Jϭ12 Hz), 3.17 (4H, t, Jϭ4.8 Hz), 3.15—3.30 (1H, m), 3.49
(2H, s), 3.74 (4H, t, Jϭ4.8 Hz), 4.06—4.16 (1H, m), 7.20—7.35 (5H, m).
8-(Piperidin-1-ylmethyl)-10H-pyrazino[2,3-b][1,4]benzothiazine (7b)
A mixture of 5 (0.50 g, 2.0 mmol), piperidine (0.20 g, 2.3 mmol), K2CO3
(0.83 g, 6.0 mmol) and DMF (20 ml) was stirred at 60 °C for 3 h. The result-
ing mixture was poured into water, and was then extracted with AcOEt. The
extract was washed with brine, dried, and evaporated. The residue was chro-
matographed on silica gel (5% MeOH–CH2Cl2) to give the product (0.45 g,
75%) as a yellow solid. mp 178.5—179.5 °C. 1H-NMR (DMSO-d6) d:
1.30—1.43 (2H, m), 1.35—1.53 (4H, m), 2.17—2.35 (4H, m), 3.20 (2H, s),
6.67 (1H, d, Jϭ8.0 Hz), 6.74 (1H, s), 6.81 (1H, d, Jϭ8.0 Hz), 7.58—7.66
(2H, m), 9.42 (1H, s). FAB-MS m/z: 299 (MϩH)ϩ. Anal. Calcd for
C16H18N4S: C, 64.40; H, 6.08; N, 18.78. Found: C, 64.21; H, 5.90; N, 18.56.
8-(Azetidin-1-ylmethyl)-10H-pyrazino[2,3-b][1,4]benzothiazine (7a)
In the same manner as described for the preparation of 7b, 7a was obtained
as yellow solid (68%). mp 158—161 °C. 1H-NMR (DMSO-d6) d: 1.96 (2H,
quint., Jϭ6.8 Hz), 3.08 (4H, t, Jϭ6.8 Hz), 3.32 (2H, s), 6.67 (1H, dd, Jϭ1.6,
7.6 Hz), 6.72 (1H, d, Jϭ2.0 Hz), 6.82 (1H, d, Jϭ7.6 Hz), 7.62—7.66 (2H,
m), 9.45 (1H, s). FAB-MS m/z: 271 (MϩH)ϩ. Anal. Calcd for C14H14N4S: C,
62.20; H, 5.22; N, 20.72. Found: C, 62.04; H, 5.14; N, 20.85.
N-(1-Benzylpiperidin-4-yl)trifluoromethanesulfonamide (9n) To
a
solution of 4-amino-1-benzylpiperidine (6.8 g, 36 mmol) in CH2Cl2 (100 ml)
was added triethylamine (NEt3, 7.4 ml, 53 mmol) and trifluoromethanesul-
fonic anhydride (10 g, 36 mmol) at Ϫ78 °C. The reaction mixture was stirred
for 2 h at Ϫ78 °C—rt, water 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 (40% AcOEt–hexane) to give
1
7.2 g (63%) of 9n as a colorless solid. mp 131—132 °C. H-NMR (DMSO-
d6) d: 1.46—1.60 (2H, m), 1.75 (2H, br d, Jϭ10 Hz), 1.90—2.10 (2H, m),
2.76 (2H, br d, Jϭ11.6 Hz), 3.18—3.38 (1H, m), 3.44 (2H, s), 7.20—7.34
(5H, m), 9.41 (1H, s).
N-(1-Benzylpiperidin-4-yl)-N؅,N؅-dimethylsulfamide (9p) To a solu-
tion of 4-amino-1-benzylpiperidine (3.0 g, 16 mmol) in CH2Cl2 (100 ml) was
added pyridine (4.0 ml) and N,N-dimethylsulfamoyl chloride (2.1 ml,
19 mmol) at 0 °C. The reaction mixture was stirred for 12 h at rt, water was
added, and then the mixture was extracted with AcOEt. The extract was
washed with brine, dried, and evaporated. The residue was chromatographed
on NH silica gel (4% MeOH–CH2Cl2) to afford 9p (4.5 g, 94%) as a pale
yellow solid. mp 105—106 °C. 1H-NMR (CDCl3) d: 1.65—1.85 (2H, m),
2.07 (2H, br d, Jϭ11.2 Hz), 2.33 (2H, br s), 2.78 (6H, s), 3.00 (2H, br d,
Jϭ11.2 Hz), 3.30 (1H, br s), 3.70 (2H, s), 4.50—4.80 (1H, m), 7.26—7.44
(5H, m).
N-(1-Benzylpiperidin-4-yl)-N؅-methylsulfamide (9q) To a solution of
N-methylsulfamoyl chloride51) (5.8 g, 45 mmol) and NEt3 (9.5 ml, 68 mmol)
in toluene (50 ml) was added 4-amino-1-benzylpiperidine (7.7 g, 41 mmol)
at 0 °C. The reaction mixture was stirred for 12 h at rt, an aqueous solution
of K2CO3 was added, and then the mixture was extracted with AcOEt. The
extract was washed with brine, dried, and evaporated. The residue was chro-
matographed on NH silica gel (70% AcOEt–hexane) to afford 9q (3.5 g,
30%) as a colorless solid. mp 135.5—136.5 °C. 1H-NMR (DMSO-d6) d:
1.34—1.50 (2H, m), 1.77 (2H, br d, Jϭ11.2 Hz), 1.91 (2H, br t, Jϭ11.2 Hz),
2.40 (3H, d, Jϭ4.8 Hz), 2.71 (2H, br d, Jϭ11.2 Hz), 2.84—2.96 (1H, m),
3.40 (2H, s), 6.57 (1H, q, Jϭ5.2 Hz), 6.85 (1H, d, Jϭ7.6 Hz), 7.18—7.33
(5H, m).
8-(Hexamethyleniminomethyl)-10H-pyrazino[2,3-b][1,4]benzothiazine
(7c) In the same manner as described for the preparation of 7b, 7c was ob-
tained as yellow solid (69%). mp 161—163 °C. 1H-NMR (DMSO-d6) d:
1.54 (8H, s), 2.43—2.55 (4H, m), 3.38 (2H, s), 6.70 (1H, dd, Jϭ1.6, 8.0 Hz),
6.80 (1H, s), 6.81 (1H, d, Jϭ8.0 Hz), 7.61 (1H, d, Jϭ2.8 Hz), 7.62 (1H, d,
Jϭ2.8 Hz), 9.45 (1H, s). HR-MS (FAB) m/z: Calcd for C17H21N4S (MϩH)ϩ:
313.1487. Found: 313.1494.
8-(Heptamethylenimnomethyl)-10H-pyrazino[2,3-b][1,4]benzothi-
azine (7d) In the same manner as described for the preparation of 7b, 7d
was obtained as yellow solid (66%). mp 133—135 °C. 1H-NMR (DMSO-d6)
d: 1.45—1.66 (10H, m), 2.46 (4H, t, Jϭ5.6 Hz), 3.38 (2H, s), 6.74 (1H, d,
Jϭ8.0 Hz), 6.83 (1H, d, Jϭ1.6 Hz), 6.84 (1H, d, Jϭ7.6 Hz), 7.60—7.68 (2H,
m), 9.54 (1H, s). HR-MS (FAB) m/z: Calcd for C18H23N4S (MϩH)ϩ:
327.1643. Found: 327.1609.
8-[(Morpholin-4-yl)methyl]-10H-pyrazino[2,3-b][1,4]benzothiazine
(7e) In the same manner as described for the preparation of 7b, 7e was ob-
tained as yellow solid (67%). mp 165—166 °C. 1H-NMR (DMSO-d6) d:
2.30 (4H, br s), 3.25 (2H, s), 3.54 (4H, br t, Jϭ4.4 Hz), 6.70 (1H, d,
Jϭ7.5 Hz), 6.75 (1H, s), 6.84 (1H, d, Jϭ7.5 Hz), 7.56—7.66 (2H, m), 9.45
(1H, s). ESI-MS m/z: 301 (MϩH)ϩ. Anal. Calcd for C15H16N4OS·0.1H2O:
C, 59.62; H, 5.40; N, 18.54. Found: C, 59.58; H, 5.26; N, 18.48.
8-[(4-Methylpiperazin-1-yl)methyl]-10H-pyrazino[2,3-b][1,4]benzothi-
azine (7f) In the same manner as described for the preparation of 7b, 7f
was obtained as yellow solid (63%). mp 191—192 °C. 1H-NMR (DMSO-d6)
d: 2.12 (3H, s), 2.10—2.50 (8H, br s), 3.23 (2H, s), 6.68 (1H, d, Jϭ8.0 Hz),
6.74 (1H, s), 6.82 (1H, d, Jϭ7.6 Hz), 7.62 (2H, s), 9.43 (1H, s). ESI-MS m/z:
314 (MϩH)ϩ. Anal. Calcd for C16H19N5S·0.51H2O: C, 59.57; H, 6.25; N,
21.71. Found: C, 59.24; H, 5.87; N, 21.52.
N-(1-Benzylpiperidin-4-yl)sulfamide (9r) To a solution of 4-amino-1-
benzylpiperidine (9.9 g, 52 mmol) in 1,2-dimethoxyethane (50 ml) was
added sulfamide (5.0 g, 52 mmol) at rt. The reaction mixture was stirred at
100 °C for 10 min and at 130 °C for 2 h, then concentrated in vacuo. The
residue was chromatographed on NH silica gel (60% AcOEt–hexane) to af-
1
ford 9r (3.8 g, 27%) as a colorless solid. mp 90—91 °C. H-NMR (DMSO-
d6) d: 1.34—1.46 (2H, m), 1.81 (2H, br d, Jϭ10.8 Hz), 1.92 (2H, br t,
Jϭ11.6 Hz), 2.72 (2H, br d, Jϭ11.6 Hz), 2.96—3.08 (1H, m), 3.40 (2H, s),
6.45 (2H, s), 6.51 (1H, d, Jϭ7.6 Hz), 7.18—7.32 (5H, m).
2-Hydroxyphenyl N-(1-Benzylpiperidin-4-yl)sulfamate (11) This sul-
famate was prepared according to the procedure of DuBois.53) To a solution
of 1-benzyl-4-aminopiperidine (9.1 g, 48 mmol) and NEt3 (5.6 ml, 40 mmol)
in DMF (120 ml) was added a solution of catecol sulfate52) (9.0 g, 52 mmol)
in CH2Cl2 (20 ml) at 0 °C. The reaction mixture was stirred for 2.5 h at 0 °C,
poured into a 1% NaCl solution (500 ml), then extracted with Et2O. The ex-
tract was washed with brine, dried, and evaporated. The residue was chro-
matographed on silica gel (5% MeOH–CH2Cl2) to afford 11 (17 g, 99%) as a
colorless oil. 1H-NMR (CDCl3) d: 1.58 (2H, dq, Jϭ4.0, 12 Hz), 1.96 (2H, d,
Jϭ12 Hz), 2.08 (2H, t, Jϭ12 Hz), 2.81 (2H, m), 3.44 (1H, m), 3.48 (2H, s),
1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidine-2-car-
boxamide (7g) In the same manner as described for the preparation of 7b,
7g was obtained as yellow solid (44%). mp 209—211 °C. 1H-NMR (DMSO-
d6) d: 1.14—1.28 (1H, m), 1.24—1.44 (1H, m), 1.44—1.60 (2H, m), 1.54—
1.70 (1H, m), 1.64—1.76 (1H, m), 1.80—1.92 (1H, m), 2.60—2.70 (1H, m),
2.68—2.78 (1H, m), 2.91 (1H, d, Jϭ13.6 Hz), 3.56 (1H, d, Jϭ13.6 Hz), 6.77
(1H, s), 6.74—6.84 (1H, m), 6.83 (1H, d, Jϭ8.4 Hz), 7.04 (1H, s), 7.09 (1H,
s), 7.62 (1H, d, Jϭ2.8 Hz), 7.63 (1H, d, Jϭ2.4 Hz), 9.40 (1H, s). FAB-MS

July 2002
927
m/z: 342 (MϩH)ϩ. Anal. Calcd for C17H19N5OS·0.5H2O: C, 58.26; H, 5.75;
N, 19.98. Found: C, 58.17; H, 5.77; N, 19.91.
(MϩH)ϩ. Anal. Calcd for C17H18F3N5O2S2·1.7H2O: C, 42.89; H, 4.53; N,
14.71. Found: C, 42.93; H, 4.28; N, 14.70.
1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidine-3-car-
N-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
boxamide (7h) In the same manner as described for the preparation of 7b, yl]benzenesulfonamide (7o) In the same manner as described for the
7h was obtained as brown oil (59%). 1H-NMR (DMSO-d6) d: 1.20—1.40 preparation of 7b, 7o was obtained as yellow solid (55%). mp 157—158 °C.
(1H, m), 1.30—1.50 (1H, m), 1.54—1.65 (1H, m), 1.65—1.78 (1H, m), 1H-NMR (DMSO-d6) d: 1.25—1.48 (2H, m), 1.40—1.60 (2H, m), 1.74—
1.78—1.94 (1H, m), 1.93 (1H, t, Jϭ10.8 Hz), 2.22—2.34 (1H, m), 2.62— 1.96 (2H, m), 2.50—2.70 (2H, m), 2.85—3.00 (1H, m), 3.20 (2H, s), 6.65
2.80 (2H, m), 3.24 (1H, d, Jϭ13.6 Hz), 3.28 (1H, d, Jϭ13.2 Hz), 6.70 (1H, (1H, d, Jϭ7.6 Hz), 6.71 (1H, s), 6.82 (1H, d, Jϭ8.0 Hz), 7.53—7.70 (5H,
dd, Jϭ1.6, 8.0 Hz), 6.74 (1H, d, Jϭ1.6 Hz), 6.76 (1H, s), 6.85 (1H, d, m), 7.70—7.83 (1H, m), 7.75—7.90 (2H, m), 9.44 (1H, s). ESI-MS m/z: 454
Jϭ8.0 Hz), 7.27 (1H, s), 7.64 (1H, d, Jϭ3.2 Hz), 7.65 (1H, d, Jϭ2.8 Hz), (MϩH)ϩ. Anal. Calcd for C22H23N5O2S2·0.3H2O: C, 57.57; H, 5.18; N,
9.46 (1H, s). HR-MS (FAB) m/z: Calcd for C17H20N5OS (MϩH)ϩ: 15.26. Found: C, 57.56; H, 4.94; N, 15.22.
342.1389. Found: 342.1346.
N-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]-N؅,N؅-dimethylsulfamide (7p) In the same manner as described for
1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidine-4-car-
boxamide (7i) In the same manner as described for the preparation of 7b,
the preparation of 7n, 7p was obtained as yellow solid (50%). mp 202—
7i was obtained as yellow solid (56%). mp 225—228 °C. 1H-NMR (DMSO- 203 °C. H-NMR (DMSO-d6) d: 1.35—1.50 (2H, m), 1.70—1.80 (2H, m),
1
d6) d: 1.53 (2H, br t, Jϭ10.8 Hz), 1.50—1.70 (2H, m), 1.84 (2H, br t,
Jϭ10.8 Hz), 1.94—2.10 (1H, m), 2.66—2.84 (2H, m), 3.23 (2H, s), 6.68
(1H, d, Jϭ8.0 Hz), 6.60—6.75 (1H, br s), 6.76 (1H, s), 6.82 (1H, d,
1.84—1.96 (2H, m), 2.61 (6H, s), 2.64—2.74 (2H, m), 2.90—3.00 (1H, m),
3.22 (2H, s), 6.66 (1H, d, Jϭ8.0 Hz), 6.73 (1H, s), 6.82 (1H, d, Jϭ8.0 Hz),
7.19 (1H, d, Jϭ8.0 Hz), 7.62 (2H, s), 9.44 (1H, s). ESI-MS m/z: 421
Jϭ7.6 Hz), 7.19 (1H, br s), 7.62 (2H, s), 9.44 (1H, br s). FAB-MS m/z: 342 (MϩH)ϩ. Anal. Calcd for C18H24N6O2S2: C, 51.41; H, 5.75; N, 19.98.
(MϩH)ϩ. Anal. Calcd for C17H19N5OS·0.3H2O: C, 58.87; H, 5.70; N, 20.19. Found: C, 51.29; H, 5.64; N, 20.05.
Found: C, 58.89; H, 5.49; N, 20.00.
N-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]-N؅-methylsulfamide (7q) In the same manner as described for the
preparation of 7n, 7q was obtained as yellow solid (47%). mp 209—210 °C.
N-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]acetamide (7j) In the same manner as described for the preparation of
7b, 7j was obtained as yellow solid (61%). mp 212—214 °C. 1H-NMR 1H-NMR (DMSO-d6) d: 1.34—1.48 (2H, m), 1.77 (2H, br d, Jϭ10 Hz),
(DMSO-d6) d: 1.25—1.40 (2H, m), 1.66 (2H, br d, Jϭ10.4 Hz), 1.75 (3H, s), 1.82—1.94 (2H, m), 2.41 (3H, d, Jϭ4.8 Hz), 2.70 (2H, br d, Jϭ12 Hz),
1.86—2.00 (2H, m), 2.69 (2H, br d, Jϭ10.4 Hz), 3.23 (2H, s), 3.40—3.54
(1H, m), 6.68 (1H, d, Jϭ7.6 Hz), 6.73 (1H, s), 6.82 (1H, d, Jϭ7.6 Hz), 7.62 Jϭ8.4 Hz), 6.73 (1H, s), 6.82 (1H, d, Jϭ7.6 Hz), 6.87 (1H, d, Jϭ8.0 Hz),
(2H, s), 7.73 (1H, d, Jϭ6.8 Hz), 9.44 (1H, s). FAB-MS m/z: 356 (MϩH)ϩ.
7.62 (1H, d, Jϭ2.8 Hz), 7.63 (1H, d, Jϭ2.4 Hz), 9.44 (1H, s). ESI-MS m/z:
Anal. Calcd for C18H21N5OS·0.35H2O: C, 59.76; H, 6.05; N, 19.36. Found: 407 (MϩH)ϩ. Anal. Calcd for C17H22N6O2S2: C, 50.23; H, 5.45; N, 20.67.
2.84—2.96 (1H, m), 3.22 (2H, s), 6.58 (1H, q, Jϭ4.8 Hz), 6.67 (1H, d,
C, 59.46; H, 5.68; N, 19.17.
Found: C, 50.04; H, 5.30; N, 20.68.
N-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
N-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]methanesulfonamide (7k) In the same manner as described for the yl]sulfamide (7r) In the same manner as described for the preparation of
preparation of 7b, 7k was obtained as yellow solid (65%). mp 220—223 °C. 7n, 7r was obtained as yellow solid (44%). mp 196—199 °C. 1H-NMR
1H-NMR (DMSO-d6) d: 1.34—1.52 (2H, m), 1.79 (2H, br d, Jϭ11.6 Hz), (DMSO-d6) d: 1.32—1.48 (2H, m), 1.81 (2H, br d, Jϭ11.6 Hz), 1.90 (2H,
1.88—2.04 (2H, m), 2.71 (2H, br d, Jϭ11.2 Hz), 2.90 (3H, s), 3.02—3.18
(1H, m), 3.25 (2H, s), 6.70 (1H, d, Jϭ7.6 Hz), 6.76 (1H, s), 6.85 (1H, d, s), 6.45 (2H, s), 6.53 (1H, d, Jϭ7.2 Hz), 6.67 (1H, d, Jϭ8.0 Hz), 6.73 (1H,
Jϭ7.6 Hz), 7.07 (1H, d, Jϭ7.2 Hz), 7.61—7.69 (2H, m), 9.46 (1H, s). ESI- s), 6.82 (1H, d, Jϭ8.0 Hz), 7.62 (2H, s), 9.44 (1H, s). ESI-MS m/z: 393
MS m/z: 392 (MϩH)ϩ. Anal. Calcd for C17H21N5O2S2·0.1H2O: C, 51.91; H, (MϩH)ϩ. Anal. Calcd for C16H20N6O2S2·0.3H2O: C, 48.30; H, 5.22; N,
br t, Jϭ11.2 Hz), 2.70 (2H, br d, Jϭ11.2 Hz), 2.94—3.10 (1H, m), 3.22 (2H,
5.43; N, 17.81. Found: C, 51.89; H, 5.32; N, 17.59.
21.12. Found: C, 48.27; H, 5.09; N, 21.23.
N-[[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
N-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]methyl]methanesulfonamide (7l) In the same manner as described for yl]-N؅,N؅-pentamethylenesulfamide (7s) In the same manner as de-
the preparation of 7b, 7l was obtained as yellow solid (53%). mp 218— scribed for the preparation of 7n, 7s was obtained as yellow solid (45%). mp
1
221 °C. H-NMR (DMSO-d6) d: 1.04—1.18 (2H, m), 1.30—1.44 (1H, m), 56—57 °C. 1H-NMR (DMSO-d6) d: 1.35—1.54 (4H, m), 1.44—1.56 (4H,
1.65 (2H, br d, Jϭ11.2 Hz), 1.85 (2H, br t, Jϭ11.6 Hz), 2.76 (2H, br d,
Jϭ11.2 Hz), 2.80 (2H, t, Jϭ6.0 Hz), 2.86 (3H, s), 3.25 (2H, s), 6.70 (1H, dd,
m), 1.77 (2H, br d, Jϭ11.6 Hz), 1.92 (2H, br d, Jϭ11.6 Hz), 2.71 (2H, br d,
Jϭ11.6 Hz), 2.98 (5H, t, Jϭ5.6 Hz), 3.24 (2H, s), 6.69 (1H, d, Jϭ7.6 Hz),
Jϭ1.6, 8.0 Hz), 6.76 (1H, d, Jϭ2.0 Hz), 6.84 (1H, d, Jϭ8.0 Hz), 6.98 (1H, t, 6.75 (1H, d, Jϭ1.6 Hz), 6.84 (1H, d, Jϭ8.0 Hz), 7.20 (1H, d, Jϭ7.6 Hz),
Jϭ6.4 Hz), 7.62—7.66 (2H, m), 9.45 (1H, s). ESI-MS m/z: 406 (MϩH)ϩ.
Anal. Calcd for C18H23N5O2S2·0.2H2O: C, 52.84; H, 5.76; N, 17.12. Found:
C, 52.86; H, 5.56; N, 17.44.
7.64 (1H, d, Jϭ2.8 Hz), 7.65 (1H, d, Jϭ2.8 Hz), 9.46 (1H, s). FAB-MS m/z:
461 (MϩH)ϩ. Anal. Calcd for C21H28N6O2S2: C, 54.76; H, 6.13; N, 18.25.
Found: C, 54.74; H, 5.82; N, 18.51.
N-[2-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-
N-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
4-yl]ethyl]methanesulfonamide (7m) In the same manner as described yl]-N؅,N؅-(3-oxapentamethylene)sulfamide (7t) In the same manner as
for the preparation of 7b, 7m was obtained as yellow solid (46%). mp 168—
described for the preparation of 7n, 7t was obtained as yellow solid (45%).
1
170 °C. H-NMR (DMSO-d6) d: 1.03—1.18 (2H, m), 1.20—1.40 (1H, m), mp 189—190 °C. 1H-NMR (DMSO-d6) d: 1.36—1.48 (2H, m), 1.77 (2H,
1.38 (2H, q, Jϭ7.2 Hz), 1.61 (2H, br d, Jϭ12.0 Hz), 1.85 (2H, br t, br d, Jϭ10.8 Hz), 1.91 (2H, br t, Jϭ10.8 Hz), 2.70 (2H, br d, Jϭ11.6 Hz),
Jϭ10.8 Hz), 2.75 (2H, br d, Jϭ10.4 Hz), 2.87 (3H, s), 2.94 (2H, q, 2.94 (4H, t, Jϭ4.4 Hz), 2.90—3.06 (1H, m), 3.22 (2H, s), 3.60 (4H, t,
Jϭ6.8 Hz), 3.24 (2H, s), 6.70 (1H, d, Jϭ8.0 Hz), 6.76 (1H, d, Jϭ1.6 Hz), Jϭ4.4 Hz), 6.67 (1H, d, Jϭ7.6 Hz), 6.73 (1H, s), 6.82 (1H, d, Jϭ7.6 Hz),
6.84 (1H, d, Jϭ8.0 Hz), 6.91 (1H, t, Jϭ6.0 Hz), 7.62—7.66 (2H, m), 9.45 7.36 (1H, d, Jϭ8.0 Hz), 7.62 (2H, s), 9.44 (1H, s). FAB-MS m/z: 463
(1H, s). ESI-MS m/z: 420 (MϩH)ϩ. Anal. Calcd for C19H25N5O2S2: C,
54.39; H, 6.01; N, 16.69. Found: C, 54.25; H, 5.79; N, 16.66.
(MϩH)ϩ. Anal. Calcd for C20H26N6O3S2: C, 51.93; H, 5.67; N, 18.17.
Found: C, 51.67; H, 5.47; N, 18.19.
N-[1-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)piperidin-4-
yl]trifluoromethanesulfonamide (7n) To a solution of 9n (7.0 g, 22
mmol) in ethanol (100 ml), 10% Pd/C (1.0 g) was added. The flask was then
N,N-[3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-azapen-
tamethylene]acetamide (7u) In the same manner as described for the
preparation of 7b, 7u was obtained as yellow solid (62%). mp 195—196 °C.
placed under a hydrogen atmosphere and stirred at rt for 12 h. The catalyst 1H-NMR (DMSO-d6) d: 1.95 (3H, s), 2.25 (2H, t, Jϭ4.8 Hz), 2.31 (2H, t,
was removed by filtration and the filtrate was concentrated to give 6n as a
Jϭ4.8 Hz), 3.29 (2H, s), 3.34—3.43 (4H, m), 6.70 (1H, d, Jϭ8.4 Hz), 6.75
colorless solid (0.9 g, 18%). A mixture of 5 (0.5 g, 2.0 mmol), 6n (0.56 g, (1H, s), 6.84 (1H, d, Jϭ8.0 Hz), 7.60—7.66 (2H, m), 9.45 (1H, s). FAB-MS
2.4 mmol) and K2CO3 (0.83 g, 6.0 mmol) in DMF (30 ml) was stirred at m/z: 341 (M)ϩ. Anal. Calcd for C17H19N5OS·0.7H2O: C, 57.67; H, 5.81; N,
60 °C for 3 h. The resulting mixture was poured into water, and then ex- 19.78. Found: C, 57.59; H, 5.72; N, 19.87.
tracted with AcOEt. The extract was washed with brine, dried, and evapo-
N,N-[3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-azapen-
rated. The residue was chromatographed on silica gel (2% MeOH–CH2Cl2) tamethylene]methanesulfonamide (7v) In the same manner as described
to give 7n (0.35 g, 39%) as a yellow solid. mp 199—201 °C. 1H-NMR for the preparation of 7b, 7v was obtained as yellow solid (54%). mp 221—
1
(DMSO-d6) d: 1.43—1.60 (2H, m), 1.75 (2H, br d, Jϭ10.4 Hz), 1.96 (2H,
222 °C. H-NMR (DMSO-d6) d: 2.38—2.45 (4H, m), 2.85 (3H, s), 3.04—
br t, Jϭ10.4 Hz), 2.73 (2H, br d, Jϭ11.2 Hz), 3.25 (2H, s), 3.15—3.40 (1H, 3.12 (4H, m), 3.32 (2H, s), 6.71 (1H, dd, Jϭ1.6, 7.6 Hz), 6.74 (1H, d,
m), 6.67 (1H, d, Jϭ7.6 Hz), 6.73 (1H, s), 6.83 (1H, d, Jϭ7.6 Hz), 7.62 (1H, Jϭ1.6 Hz), 6.84 (1H, d, Jϭ7.6 Hz), 7.62 (1H, d, Jϭ2.8 Hz), 7.63 (1H, d,
d, Jϭ3.2 Hz), 7.63 (1H, d, Jϭ3.2 Hz), 9.45 (2H, s). ESI-MS m/z: 446
Jϭ2.4 Hz), 9.45 (1H, s). FAB-MS m/z: 377 (M)ϩ. Anal. Calcd for

928
Vol. 50, No. 7
C16H19N5O2S2: C, 50.91; H, 5.07; N, 18.55. Found: C, 50.78; H, 5.06; N,
18.41.
N,N-[3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-azapen-
10) Kim J. J., Tsai A., Nottingham L. K., Morrison L., Cunning D. M., Oh
J., Lee D. J., Dang K., Dentchev T., Chalian A. A., Agadjanyan M. G.,
Weiner D. B., J. Clin. Invest., 103, 869—877 (1999).
tamethylene]sulfamide (7w) In the same manner as described for the 11) Damle N. K., Aruffo A., Proc. Natl. Acad. Sci. U.S.A., 88, 6403—6407
preparation of 7b, 7w was obtained as yellow solid (50%). mp 215—217 °C.
1H-NMR (DMSO-d6) d: 2.36—2.46 (4H, m), 2.90—3.00 (4H, m), 3.32 (2H,
s), 6.72 (1H, dd, Jϭ1.6, 8.0 Hz), 6.78 (3H, s), 6.86 (1H, d, Jϭ8.0 Hz),
(1991).
12) Udagawa T., Woodside D. G., Mclntyre B. W., J. Immunol., 157,
1965—1972 (1996).
7.62—7.68 (2H, m), 9.48 (1H, s). HR-MS (FAB) m/z: Calcd for 13) Rothlein R., Mainolfi E. A., Kishimoto T. K., Res. Immunol., 144,
C15H19N6O2S2 (MϩH)ϩ: 379.1011. Found: 379.0991.
735—739 (1993).
N,N-[3-(10H-Pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-3-azapen- 14) Cornejo C. J., Winn R. K., Harlan J. M., Adv. Pharmacol., 39, 99—
tamethylene]-N؅,N؅-dimethylsulfamide (7x) In the same manner as de- 142 (1997).
scribed for the preparation of 7b, 7x was obtained as yellow solid (55%). mp 15) Kavanaugh A. F., Davis L. S., Nichols L. A., Norris S. H., Rothlein R.,
171—173 °C. 1H-NMR (DMSO-d6) d: 2.33—2.40 (4H, m), 2.73 (6H, s),
3.08—3.16 (4H, m), 3.30 (2H, s), 6.70 (1H, d, Jϭ8.0 Hz), 6.75 (1H, m),
Scharchmidt L. A., Lipsky P. E., Arthritis Rheum., 37, 992—999
(1994).
6.84 (1H, d, Jϭ8.0 Hz), 7.62 (1H, d, Jϭ2.8 Hz), 7.63 (1H, d, Jϭ2.8 Hz), 16) Kavanaugh A. F., Schulze-Koops H., Davis L. S., Lipsky P. E., Arthri-
9.45 (1H, s). ESI-MS m/z: 407 (MϩH)ϩ. Anal. Calcd for C17H22N6O2S2: C,
tis Rheum., 40, 849—853 (1997).
50.23; H, 5.45; N, 20.67. Found: C, 50.21; H, 5.36; N, 20.78.
17) Davis L. S., Kavanaugh A. F., Nichols L. A., Lipsky P. E., J. Immunol.,
154, 3525—3537 (1995).
Pharmacology
18) Yacyshyn B. R., Bowen-Yacyshyn M. B., Jewell L., Tami J. A., Bennett
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1133—1142 (1998).
19) Boschelli D. H., Kramer J. B., Connor D. T., Lesch M. E., Schrier D. J.,
Ferin M. A., Wright C. D., J. Med. Chem., 37, 717—718 (1994).
Quantification of Adhesion Molecule Expression on HUVEC by
ELISA The quantification of adhesion molecule expression on HUVEC
by ELISA was performed according to the methods previously described.54)
The test compounds (or vehicle) were added to confluent HUVEC (Sanko
Junyaku, Tokyo, Japan) monolayer cultures in 96 well-plates, and 0.25 h 20) Boschelli D. H., Kramer J. B., Khatana S. S., Sorenson R. J., Connor
later, the cells were stimulated by the addition of TNF-a (final concentra-
tion: 1 ng/ml) or vehicle. After an incubation period of 4 h at 37 °C, HUVEC
were washed twice with 100 ml of PBS (phosphate-buffered saline), and then
D. T., Ferin M. A., Wright C. D., Lesch M. E., Imre K., Okonkwo G.
C., Schrier D. J., Conroy M. C., Ferguson E., Woelle J., Saxena U., J.
Med. Chem., 38, 4597—4614 (1995).
fixed with 100 ml of PBS containing 0.025% glutaraldehyde for 5 min at 21) Boschelli D. H., Connor D. T., Lesch M. E., Schrier D. J., Bioorg. Med.
room temperature. After the fixation, the cells were washed three times with
200 ml of PBS containing 0.1% BSA (PBSA). An aliquot of 100 ml of wash-
ing buffer containing 1 mg/ml of either mouse anti-human ICAM-1 (R&D,
Minneapolis, MN, U.S.A.), mouse anti-human E-selectin (R&D, Minneapo-
lis, MN, U.S.A.), or mouse anti-human VCAM-1 antibody (R&D, Min-
neapolis, MN, U.S.A.) was added to each well, and then the cells were stored
Chem., 4, 557—562 (1996).
22) Stewart A. O., Bhatia P. A., McCarty C. M., Patel M. V., Staeger M.
A., Arendsen D. L., Gunawardana I. W., Melcher L. M., Zhu G., Boyd
S. A., Fry D. G., Cool B. L., Kifle L., Lartey K., Marsh K. C., Kempf-
Grote A. J., Kilgannon P., Wisdom W., Meyer J., Gallatin W. M.,
Okasinski G. F., J. Med. Chem., 44, 988—1002 (2001).
at room temperature. Thirty minutes later, the cells were washed twice with 23) Zhu G., Arendsen D. L., Gunawardana I. W., Boyd S. A., Stewart A.
200 ml of PBSA. An aliquot of 100 ml of peroxidase-conjugated goat anti-
mouse IgG (Fc) polyclonal antibody solution (Cosmo Bio Co., Ltd., Tokyo,
Japan) which was diluted (1 : 1000) with washing PBSA, was added to each
O., Fry D. G., Cool B. L., Kifle L., Schaefer V., Meuth J., Marsh K. C.,
Kempf-Grote A. J., Kilgannon P., Gallatin W. M., Okasinski G. F., J.
Med. Chem., 44, 3469—3487 (2001).
well. After an incubation period of 30 min at room temperature, the cells 24) Sanfilippo P. J., Jetter M. C., Cordova R., Noe R. A., Chourmouzis E.,
were washed three times with 200 ml of washing buffer. An aliquot of 100 ml Lau C. Y., Wang E., J. Med. Chem., 38, 1057—1059 (1995).
of TMB substrate solution was applied to each well and, after adequate color 25) Burch R. M., Weitzberg M., Blok N., Muhlhauser R., Martin D.,
development, the reaction was quenched by the addition of 100 ml of 1 M
phosphoric acid. The absorbance at wavelength 450 nm was measured using
an automated microplate reader. (nϭ2).
Farmer S. G., Bator J. M., Connor J. R., Ko C., Kuhn W., McMillan B.
A., Raynor M., Shearer B. G., Tiffany C., Wilkins D. E., Proc. Natl.
Acad. Sci. U.S.A., 88, 355—359 (1991).
IL-1-Induced Paw Inflammation Model in the Mouse Pathogen-free 26) Hamilton G. S., Mewshaw R. E., Bryant C. M., Feng Y., Endemann
Balb/c mice (6 week old, Charles River laboratories, Astugi, Japan) were
orally dosed with vehicle (0.5% hydroxypropyl methyl cellulose) or the test
compounds dissolved or suspended in vehicle. Thirty minutes later, the left
G., Madden K. S., Janczak J. E., Perumattam J., Stanton L. W., Yang
X., Yin Z., Venkataramen B., Liu D. Y., J. Med. Chem., 38, 1650—
1656 (1995).
hind paw of each mouse was injected subcutaneously with rat recombinant 27) Liu G., Link J. T., Pei Z., Reilly E. B., Leitza S., Nguyen B., Marsh K.
IL-1a (rrIL-1a, obtained in our laboratories) (5 ng/50 ml/site). Two hours
post rrIL-1a injection, the animals were sacrificed and the injected paws
C., Okasinski G. F., von Geldern T. W., Ormes M., Fowler K., Gallatin
M., J. Med. Chem., 43, 4025—4040 (2000).
were removed with scissors. The paws were homogenized with 0.5% hexa- 28) Pei Z., Xin Z., Liu G., Li Y., Reilly E. B., Lubbers N. L., Huth J. R.,
decyl trimethylammonium bromide (HTAB) in potassium phosphate buffer
(pH 6), and the MPO activities of the homogenates were evaluated using o-
dianisidine–H2O2, as a marker enzyme for neutrophil content. MPO activity
Link J. T., von Geldern T. W., Cox B. F., Leitza S., Gao Y., Marsh K.
C., DeVries P., Okasinski G. F., J. Med. Chem., 44, 2913—2920
(2001).
in the inflamed paw tissue was measured by the spectrophotometric method 29) Kelly T. A., Jeanfavre D. D., McNeil D. W., Woska J. R., Jr., Reilly P.
reported previously.55) (nϭ3).
L., Mainolfi E. A., Kishimoto K. M., Nabozny G. H., Zinter R., Bor-
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