Novel arylpyrazino[2,3-c][1,2,6]thiadiazine 2,2-dioxides as inhibitors of platelet aggregation. 1. Synthesis and pharmacological evaluation

Article abstract of DOI:10.1021/jm981103j

A series of N-1-substituted derivatives of pyrazino[2,3- c][1,2,6]thiadiazine 2,2-dioxides bearing aryl groups at the pyrazino moiety have been prepared. The synthesis involves ring formation between the diaminothiadiazine and suitable dicarbonyl compound

Full text of DOI:10.1021/jm981103j

1698
J . Med. Chem. 1999, 42, 1698-1704
Novel Ar ylp yr a zin o[2,3-c][1,2,6]th ia d ia zin e 2,2-Dioxid es a s In h ibitor s of P la telet
Aggr ega tion . 1. Syn th esis a n d P h a r m a cologica l Eva lu a tion
Nuria Campillo,† Concepcio´n Garc´ıa,† Pilar Goya,† J uan A. Pa´ez,*,† Emilio Carrasco,‡ and Manuel Grau‡
Instituto de Quı´mica Me´dica CSIC, J uan de la Cierva 3, 28006 Madrid, Spain, and Almirall Prodesfarma, S.A. Research
Centre, Barcelona, Spain
Received October 13, 1998
A series of N-1-substituted derivatives of pyrazino[2,3-c][1,2,6]thiadiazine 2,2-dioxides bearing
aryl groups at the pyrazino moiety have been prepared. The synthesis involves ring formation
between the diaminothiadiazine and suitable dicarbonyl compounds and subsequent introduc-
tion of the substituent at N-1. The compounds have been tested in vitro, as inhibitors of rabbit
and human platelet aggregation, and ex vivo against rat platelet aggregation induced by
arachidonic acid, ADP, collagen, U46619, and I-BOP. The results obtained indicate that some
pyrazino[2,3-c][1,2,6]thiadiazine derivatives show significant platelet aggregation inhibition
similar to other antithrombotic agents and that the antiplatelet properties may be mediated
by interference with the arachidonic acid pathway.
In tr od u ction
tion, induced by different agonists, both in vitro and ex
vivo of a new series of N-1-substituted derivatives of
pyrazino[2,3-c][1,2,6]thiadiazine 2,2-dioxide derivatives
with one or two aryl rests at the pyrazino moiety.
The basic molecular mechanisms involved in the
formation and dissolution of thrombus are the subject
of continuous research. Platelet aggregation is an
important part of the process, and so, special attention
is given to agents capable of interfering with platelet
recruitment into forming thrombus and which can,
therefore, play an important role in the treatment of
atherosclerosis, thrombosis, and acute coronary syn-
dromes.1
One approach for the development of novel agents to
antiplatelet therapy via the arachidonic acid cascade
consists of the inhibition of the biological effects of
thromboxane A2 (TxA2).2-4 Different lines of research
are currently under investigation in this area including
the development of thromboxane receptor antagonists
as well as compounds interfering with the biosynthesis
of TxA2 through inhibition of cyclooxygenase or thom-
boxane synthase. However, this last approach has not
yet demonstrated efficacy in the clinic2 due probably to
PGH2 accumulation. Therefore, compounds with TxA2/
endoperoxide receptor blocking activity or compounds
displaying dual properties such as TxA2 synthase
inhibition and TxA2/endoperoxide receptor blockage
could be useful in thrombotic disease.2,5
Within this context, we describe here a new class of
platelet aggregation inhibitors with no structural rela-
tion to other antiplatelet agents known and which are
aryl derivatives of the pyrazino[2,3-c][1,2,6]thiadiazine
2,2-dioxide system. This heterocyclic structure, which
was first synthesized in our group,6 has been the subject
of our research, and we have previously reported on its
particular structural characteristics7-9 and, also, on the
biological properties of some of its derivatives bearing
alkyl groups at positions 6 and 7.10 We now wish to
report the synthesis and effect on the platelet aggrega-
Ch em istr y
The compounds prepared for this work are gathered
in Table 1. Their synthetic route is a two-step procedure
involving, first, the preparation of the parent NH-
pyrazino[2,3-c][1,2,6]thiadiazines with the desired aryl
substituents at positions 6 and 7 and then introduction
of the substituent at position N-1 using different alky-
lating agents.
Thus, the first step is the condensation between 3,4,5-
triamino-2H-1,2,6-thiadiazine 1,1-dioxide (1)11 and ad-
equately functionalized dicarbonyl compounds. So, re-
action of 1 with symmetric 1,2-dicarbonyl compounds
such as benzil, 2,2′-thenil, 2,2′-furil, and 2,2′-pyridil
afforded the corresponding 6,7-diphenyl, 6,7-di(2-thie-
nyl), 6,7-di(2-furyl), and 6,7-di(2-pyridyl) compounds 2,6
3, 4, and 5, respectively (Scheme 1).
When the dicarbonyl compound used to build the
pyrazino moiety is not symmetrical, then, in principle,
the two possible isomers at positions 6 and 7 can be
obtained. We had already reported one case in which
by replacing one of the two carbonyl compounds with
hydroxyimino function it was possible to obtain only the
isomer bearing at position 7 the substituent linked to
it.12 Thus, by reaction of 1 with R-hydroxyiminoac-
etophenone and 1-phenyl-2-hydroxyimino-1-propanone,
it was possible to obtain, exclusively, the 6-phenyl-7H-
and 6-phenyl-7-methylpyrazinothiadiazines 612 and 7.
However, when the keto aldehyde compounds phenylg-
lyoxal and 1-phenyl-1,2-propanedione were used, the
corresponding 7-phenyl-6H and 7-phenyl-6-methyl de-
rivatives 812 and 9 were obtained. The 6-bromo- and
6-chloropyrazinothiadiazines 1012 and 11 were prepared
from the unsubstituted at position 6 derivative 8 by
reaction with NBS in methanol and with NCS in DMF,
respectively (Scheme 1).
* To whom correspondence should be addressed. Fax: 91 5644853.
E-mail: juan@suricata.iqm.csic.es.
† Instituto de Qu´ımica Me´dica CSIC.
‡ Almirall Prodesfarma.
10.1021/jm981103j CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/30/1999

Arylpyrazino[2,3-c][1,2,6]thiadiazine 2,2-Dioxides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1699
Ta ble 1. Physicochemical Data for 4-Aminopyrazino[2,3-c][1,2,6]thiadiazine 2,2-Dioxides 3-5, 7, 9, and 11-24
compd
R1
R2
R3
mp (°C)
recryst solv
formula
anal.a
3
4
5
7
9
2-thienyl
2-furyl
2-pyridyl
Ph
Me
Cl
Ph
Ph
Ph
Ph
2-thienyl
2-furyl
2-pyridyl
Me
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
H
Et
268-9
310-12
>350
274-6
274-6
263-5
260-1
258
EtOH/H2O
H2O/EtOH
AcOH/H2O
EtOH/H2O
H2O
MeOH/H2O
EtOH
EtOH
C13H9N5O2S3
C13H9N5O4S
C15H11N7O2S‚1.5H2O
C12H11N5O2S
C12H11N5O2S
C11H8ClN5O2S
C19H17N5O2S
C24H19N5O2S
C21H19N5O4S
C23H23N5O4S
C19H16N6O3S
C15H13N5O2S3
C14H11N5O4S
C16H13N7O2S
C14H15N5O2S
C14H15N5O2S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,Cl,N,S
C,H,N
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,Cl,N,S
C,H,N,S
C,H,N,S
11
12
13
14
15
16
17
18
19
20
21
22
23
24
CH2Ph
CH2CO2Et
CH2CO2 Bu
CH2CONH2
Et
Me
Me
Et
234
EtOH
t
204-5
293-5
244-5
232-4
255-6
161-3
206-7
214-6
295
Ph
Ph
2-thienyl
2-furyl
2-pyridyl
Ph
Me
Cl
2-thienyl
2-furyl
2-pyridyl
Me
Ph
Ph
Acetone/H2O
iPrOH
EtOH/Acetone
EtOH/H2O
H2O/EtOH
EtOH
Et
Et
C13H12ClN5O2S
C12H11N5O2S
C15H14BrN5O4S
Ph
Br
H
Ph
Me
CH2CO2Et
CH2Cl2/MeOH
EtOH/H2O
182-4
a
Elemental analyses were within (0.4% of the calculated values for the formulas given.
Sch em e 1
Different alkyl derivatives of the pyrazino[2,3-c][1,2,6]-
tive 14 by treatment with ammonia in methanol at room
temperature.
thiadiazines were prepared using the corresponding
halides in acetone and with either potassium carbonate
or triethylamine as base (Table 1, Scheme 2). Thus, 6,7-
diphenylpyrazinothiadiazine 2 with ethyl iodide, benzyl
bromide, and ethyl and tert-butyl bromoacetate in
acetone and potassium carbonate yielded the corre-
sponding N-1 ethyl, benzyl, ethoxycarbonylmethyl, and
tert-butoxycarbonylmethyl derivatives 12, 13, 14, and
15, respectively. The carbamoylmethyl compound 16
was prepared from the 1-ethoxycarbonylmethyl deriva-
Treatment with ethyl iodide of 6,7-di(2-thienyl)-1H-
pyrazinothiadiazine 3, in the presence of triethylamine,
afforded the corresponding 1-ethyl derivative 17, while
reaction of the 6,7-di(2-furyl) derivative 4 with methyl
iodide was performed using potassium carbonate to give
the 1-methyl derivative 18. In the case of the 6,7-di(2-
pyridyl)pyrazinothiadiazine 5 with methyl iodide and
triethylamine, it was possible to obtain the N-1 methyl
derivative 19 in a very major proportion in relation to

1700 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Campillo et al.
Sch em e 2
Ta ble 2. 13C NMR Data (δ, DMSO-d6) for Pyrazino[2,3-c][1,2,6]thiadiazine 2,2-Dioxides 3-5, 7, 9, and 11-24
compd
R1
2-thienyl 2-thienyl
2-furyl 2-furyl
2-pyridyl 2-pyridyl
R2
R3
C-4
C-7
C-8a
C-6
C-4a
other signals
3
4
5
7
9
11
12
13
H
H
H
H
H
H
Et
157.9 149.5 146.9 138.2 119.2 139.5, 138.6, 132.0, 130.6, 129.4, 128, 8, 128.2
157.9 147.1 144.4 134.1 119.4 149.6, 149.1, 146.5, 143.9, 115.5, 112.7, 112.0
158.3 155.5 147.4 144.5 120.1 155.7, 148.3, 147.8, 136.7, 124.0, 123.8, 123.1
158.8 156.9 147.1 146.5 119.4 137.0, 129.2, 128.6, 128.1, 23.2
158.5 157.1 146.9 144.6 119.8 136.9, 129.5, 128.9, 128.2, 22.2
157.4 156.5 147.5 135.1 119.9 137.3, 130. 4, 129.5, 128.5
158.5 155.3 146.5 144.4 120.8 137.4, 137.1, 129.8, 128.6, 128.3, 128.1, 37.9, 14.0
158.6 155.1 146.6 144.9 121.0 137.1, 137.0, 136.9, 129.8, 128.6, 128.2, 128.2, 128.1,
127.3, 37.9, 14.0
Ph
Me
Cl
Me
Ph
Ph
Ph
Ph
Ph
Ph
CH2Ph
14
15
Ph
Ph
Ph
Ph
Ph
Ph
CH2CO2Et
158.3 155.0 146.1 145.3 120.8 158.9, 158.8, 138.5, 138.3, 129.3, 129.0, 121.9, 121.9,
115.2, 114.2, 55.0, 28.1
t
CH2CO2 Bu 158.5 155.0 146.2 145.2 120.8 137.0, 136.9, 129.9, 129.8, 129.7, 128.8, 128.2, 167.6,
61.1, 43.5, 14.1
16
17
CH2CONH2 158.3 154.7 146.6 144.7 121.2 137.0, 129.7, 129.6, 128.6, 128.2, 128.1, 168.2, 43.9
2-thienyl 2-thienyl Et
2-furyl 2-furyl Me
2-pyridyl 2-pyridyl Me
157.8 148.9 146.4 136.7 119.9 140.2, 138.6, 132.6, 130.6, 129.4, 128.8, 128.4, 127.5,
38.1, 13.6
157.7 146.9 143.6 132.8 120.4 149.5, 149.3, 146.6, 143.9, 115.9, 112.7, 112.1,
111.9, 28.3
158.6 155.5 147.8 143.3 121.1 137.0, 136.9, 129.9, 129.8, 129.7, 128.8, 128.2, 167.6,
61.1, 43.5, 14.1
18
19
20
21
22
23
24
Ph
Me
Cl
Ph
Br
Me
Ph
Ph
H
Et
Et
Et
Me
158.8 156.4 146.6 145.6 120.3 136.9, 129.4, 128.7, 128.2, 23.9, 37.7, 14.0
158.6 156.7 147.1 145.6 120.4 136.8, 129.3, 128.7, 128.2, 23.6, 28.0
157.1 155.5 147.5 136.5 120.9 135.1, 130.5, 129.6, 128.2, 28.4
158.4 145.5 147.9 143.7 122.3 134.4, 129.6, 128.8, 126.6, 28.3
157.3 157.0 146.6 130.6 121.8 167.2, 136.3, 130.4, 129.6, 128.7, 61.1, 43.6, 13.7
Ph
CH2CO2Et
the1-methyl-7-(N-methylpyridinio)pyrazino[2,3-c][1,2,6]-
thiadiazine iodide, all other alkylating conditions used
giving rise also to the dimethyl compound as a result of
methylation at the pyridine nitrogen.
13C NMR spectra have been carried out according to the
chemical shifts and from the long-range coupling con-
stants.
P la telet Aggr ega tion Stu d ies
The 1-ethyl derivatives 20, 21, and 22 were obtained
by reaction of compounds 7, 9, and 11 with ethyl iodide,
in acetone, with potassium carbonate in the first two
cases and with triethylamine in the last. Treatment of
the 6-phenyl derivative 6 with methyl iodide and
potassium carbonate yielded 23, and finally, the reaction
of the 6-bromo derivative 10 with ethyl bromoacetate
and triethylamine afforded 24.
The new arylpyrazinothiadiazines obtained were
screened for their platelet aggregation inhibitory activ-
ity in vitro on citrated rabbit platelet-rich plasma in
which aggregation was induced with arachidonic acid
(AA) and adenosine diphosphate (ADP). Selected com-
pounds were further evaluated for their in vitro effect
on human platelet aggregation induced by arachidonic
acid (AA), collagen (COLL), or the TxA2 mimetics
U46619 and I-BOP. Finally, ex vivo activity of the most
promising compounds was evaluated in rats measuring
the platelet aggregation induced by the above-men-
The structures of the newly synthesized compounds
have been established by analytical and spectroscopic
data (Table 2). The assignments of all compounds by

Arylpyrazino[2,3-c][1,2,6]thiadiazine 2,2-Dioxides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1701
Ta ble 3. Effect of Pyrazinothiadiazine 2,2-Dioxides 12-28 on
in Vitro Rabbit Platelet Aggregation Induced by Arachidonic
Acid (AA) and Adenosine Diphosphate (ADP)
higher concentrations (100 µg/mL) against ADP-induced
aggregation.14
Compounds 12, 14, 20, 25, and 26 were further
evaluated in vitro at different concentrations for their
effects on platelet aggregation in human plasma induced
by collagen, arachidonic acid, U46619, and I-BOP (Table
4). All the compounds inhibited both collagen- and
arachidonic acid-induced human platelet aggregation
being more potent against collagen than arachidonic
acid. With the exception of compound 26, all derivatives
were also active against U46619- and I-BOP-induced
platelet aggregation. In addition, they were devoid of
significant activity when ADP was used as platelet
aggregation-inducing agent (results not shown). As a
whole, the results obtained in both in vitro tests support
the view that the antiplatelet activity observed in this
series of derivatives could be mediated by interference
with the platelet arachidonic acid pathway. Confirming
the findings obtained in rabbit platelets, compound 14
was very potent against human platelet aggregation
induced by collagen and arachidonic acid but less active
when U46619 was used as aggregating agent. According
to this pharmacological profile, derivative 14 seems to
act selectively in the arachidonic acid cascade by
inhibiting platelet cyclooxygenase with a potency greater
than ASA (acetylsalicylic acid). On the other hand,
compounds 12, 20, and 25 behave as antagonists of the
endoperoxide/TxA2 receptor, although derivative 25 has
a somewhat more selective action against I-BOP-
induced platelet aggregation than derivative 12 which
is more selective against U46619. The in vitro potency
of these compounds when compared with that of BAY-U
varies according to different agonists used, but it is
always lower.
platelet
aggregation
inhibition
compd
12
13
14
15
16
17
18
19
20
21
22
23
24
R1
R2
R3
AAa
ADPb
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Et
0.3
3
0.1
1
3
3
1.0
3
1
1
10
0.3
10
1
1
46
30
30
32
47
43
63c
43
47
59c
23
43
36
33
31
CH2Ph
CH2CO2Et
CH2CO2 Bu
CH2CONH2
t
2-thienyl 2-thienyl Et
2-furyl 2-furyl Me
2-pyridyl 2-pyridyl Me
Ph
Me
Cl
Ph
Br
Ph
Br
H
Me
Ph
Ph
H
Ph
H
Ph
Ph
Ph
Et
Et
Et
Me
CH2CO2Et
25d
26d
27d
28d
ASA
adenosine
Et
Et
Et
>10
>10
H
CH2CO2Et
2.5
64
a
Several concentrations of 0.1 and 1.0 mg/mL have been
assayed in triplicate for each compound against AA (1 mM)-
induced platelet aggregation. Activity is expressed as the EC100
(mg/mL), effective concentration which produced a 100% inhibitory
b
response. Test compounds have been assayed at the fixed
concentration of 100 mg/mL against ADP (1-2 µM)-induced
platelet aggregation. Activity is expressed as the percentage of
the inhibitory response when compared to controls. c Test com-
pound inhibition by more than 50% of maximum platelet aggrega-
tion induced by ADP was considered of pharmacological signifi-
d
cance. These compounds had previously been reported in ref 10.
tioned agonists at scheduled times after oral adminis-
tration of test compounds.
Once the in vitro antiaggregating effect on rabbit and
human platelets was confirmed, the active derivatives
12, 14, 20, 25, and 26 were subjected to ex vivo
evaluation (Table 5) to assess their in vivo potency.
Derivatives 12, 20, and 25 demonstrated potent ex vivo
activity against rat platelet aggregation induced by
collagen and I-BOP after 30 min of their oral adminis-
tration which declined 120 min thereafter. The inhibi-
tory activity of these compounds against collagen-
induced aggregation at 30 min postdosing was equipotent
to that of reference compounds at 120 min and better
than that of ASA and ticlopidine when I-BOP was used
as aggregation inducer. Compound 26 showed weaker
but long-lasting antiplatelet activity, with about 70%
inhibition of collagen-induced aggregation after 2 h
Discu ssion
All the final N-1-substituted pyrazino[2,3-c][1,2,6]-
thiadiazine 2,2-dioxides 12-28 were primarily screened
for their in vitro antiplatelet properties on rabbit
platelet rich-plasma,13 and the results are shown in
Table 3. Significant inhibition of arachidonic acid-
induced rabbit platelet aggregation was observed for
most of the compounds with EC100 values ranging from
0.1 to 10 µg/mL. The most active derivative in this series
was compound 14 (EC100, 0.1 µg/mL) while the 6H-7-
phenyl derivatives 27 and 28 were devoid of significant
activity. On the other hand, only derivatives 18 and 21
showed an inhibitory activity greater than 50% at much
Ta ble 4. Effect of Selected Pyrazinothiadiazine 2,2-Dioxides on in Vitro Human Platelet Aggregation Induced by Arachidonic Acid
(AA), Collagen (COLL), U-46619, and I-BOPa
AAb
inhib (%)
COLLb
inhib (%)
U-46619b
inhib (%)
I-BOPb
inhib (%)
compd
concn
concn
concn
concn
BAY-U
ASA
12
14
20
0.2
25
30
1
30
30
30
96.0 ( 0.7
96.0 ( 1.1
94.0 ( 0.5
82.9 ( 11.8
93.0 ( 2.2
95.0 ( 2.0
34.8 ( 25.9c
0.2
12.5
1
3
1
97.0 ( 1.8
94.0 ( 0.8
95.0 ( 0.4
88.6 ( 3.6
97.0 ( 0.4
97.0 ( 0.2
95.4 ( 0.6
0.2
50
1
30
3
98.0 ( 2.5
28.0 ( 11.5c
82.0 ( 8.9
75.7 ( 6.6
81.0 ( 0.4
100 ( 0
0.1
50
10
93.0 ( 0.7
43.0 ( 15.6c
80.0 ( 1.4
NDd
99.0 ( 0.8
84.0 ( 6.7
43.1 ( 22.1c
3
1
3
25
26
1
0.6
10
e
a
The evaluation method is described in the Experimental Section. The concentrations of the aggregating agents for inducing submaximal
b
platelet aggregation were as follows: AA, 1 mM; COLL, 1-2 µg/mL; U-46619, 1.4 µM; I-BOP, 225-250 nM. Results expressed as the
lowest concentration (µM) required to obtain a submaximal (80-90%) inhibitory response. Each value is the mean ( SEM of 3-4
determinations. c Concentration which produced the greatest response observed. ND, not determined. e No effect up to 30 µM.
d

1702 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Campillo et al.
Ta ble 5. Inhibitory Effects of Selected Pyrazino[2,3-c][1,2,6]thiadiazine 2,2-Dioxides 30 and 120 min after Oral Administration on
Platelet Aggregation Induced by Arachidonic Acid (AA), Collagen (COLL), and I-BOP in Rats ex Vivoa
platelet aggregation inhibitory activityb
time (min) after
compd
dose (mg/kg)
administration
AA
COLL
I-BOP
BAY-U
ASA
ticlopidine
12
25
100
100
100
120
120
120
30
120
30
120
30
120
30
74.2 ( 14.6
93.8 ( 4.4
0.9 ( 0.7
6.5 ( 2.4
1.0 ( 0.9
20.0 ( 17.9
48.8 ( 24.4
11.3 ( 7.6
50.9 ( 23.8
9.0 ( 4.3
0
95.3 ( 1.7
84.6 ( 10.1
98.7 ( 0.4
82.6 ( 12.8
26.3 ( 21.3
93.5 ( 2.3
47.2 ( 23.7
95.5 ( 0.7
59.6 ( 19.6
53.9 ( 21.2
69.5 ( 13.6
72.5 ( 16.6
0.9 ( 0.6
57.1 ( 18.7
100 ( 0
30.0 ( 20.3
59.6 ( 21.8
50.3 ( 24.9
100.0 ( 0
51.2 ( 24.4
9.1 ( 3.7
25.2 ( 21.7
20
25
26
100
100
100
120
a
The evaluation method is described in the Experimental Section. The concentrations of the aggregating agents for inducing submaximal
b
platelet aggregation were as follows: AA, 0.4 mM; COLL, 1-2 µg/mL; I-BOP, 225-250 nM. Activity expressed as percentage of inhibition;
values are the mean ( SEM of 3-5 determinations.
precipitate was filtered, washed with dichloromethane, and
recrystallized from the appropriate solvent (Table 1).
postdosing. All compounds tested were less potent ex
vivo against arachidonic acid-induced platelet aggrega-
tion.
Compound 14 which had shown potent activity in
both in vitro tests was devoid of significant antiplatelet
effects when administered orally to rats. On the other
hand, none of the compounds tested were able to induce
any biologically significant activity against rat platelet
ADP-induced aggregation ex vivo (results not shown).
4-Am in o-6,7-d i(2-t h ie n yl)-1H -p yr a zin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (3). From 1 (1.00 g, 5.6 mmol),
methanol (40 mL), concentrated hydrochloric acid (0.6 mL),
and 2,2′-thenil (1.40 g, 6.2 mmol). Reaction time 15 h.
Recrystallization from ethanol/water yielded 3 (0.52 g, 54%).
1H NMR (DMSO-d6): δ 12.38 (br s, 1H, NH), 8.66 (br s, 1H,
NH2), 8.32 (br s, 1H, NH2), 7.82 (dd, 1H, Hthienyl), 7.76 (dd, 1H,
Hthienyl), 7.39 (dd, 1H, Hthienyl), 7.15 (dd, 1H, Hthienyl), 7.15 (dd,
1H, Hthienyl), 7.07 (dd, 1H, Hthienyl). This compound had previ-
ously been reported from compound 1 and thiophene-2-
carboxaldehyde.15
Con clu sion s
Arylpyrazinothiadiazines have been synthesized and
identified as a new class of platelet aggregation inhibi-
tors. Preliminary structural requirements for activity
are the presence of one phenyl group in the 6,7-
disubstituted derivatives or one phenyl group at position
6 in monosubstituted derivatives (previous results
indicated that compounds unsubstituted at position 1
and N-1-substituted 6,7-alkyl derivatives were devoid
of antiplatelet activity). In relation to position 1, the
results obtained indicate that only the 1-ethyl deriva-
tives show in vivo activity. Also, the pharmacological
results obtained from platelet aggregation studies sup-
port the view that the antiplatelet activity observed in
this series of derivatives may be mediated by interfer-
ence with the platelet arachidonic acid pathway.
On the basis of these findings, studies to establish
quantitative structure-activity relationships in order
to optimize the antiplatelet activity observed in com-
pounds 12, 25, and 26 will be reported in part 2 of this
research.
4-Am in o -6,7-d i(2-fu r y l)-1H -p y r a zin o [2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (4). From 1 (3.00 g, 16.8 mmol),
methanol (100 mL), concentrated hydrochloric acid (2.0 mL),
and 2,2′-furil (3.60 g, 18.5 mmol). Reaction time 2 h. Recrys-
tallization from water/ethanol yielded 4 (3.85 g, 69%). 1H NMR
(DMSO-d6): δ 12.45 (br s, 1H, NH), 8.69 (br s, 1H, NH2), 8.50
(br s, 1H, NH2), 7.93 (m, 1H, Hfuryl), 7.81-7.80 (m, 2H, Hfuryl),
7.03 (d, 2H, Hfuryl), 6.71-6.68 (m, 1H, Hfuryl).
4-Am in o-6,7-d i(2-p yr id yl)-1H -p yr a zin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (5). From 1 (3.80 g, 20.0 mmol),
acetic acid (130 mL), and 2,2′-pyridil (5.00 g, 18.5 mmol).
Reaction time 2 h. Recrystallization from acetic acid/water
yielded 5 (1.69 g, 45%). 1H NMR (DMSO-d6): δ 11.94 (br s,
1H, NH), 8.73 (br s, 1H, NH2), 8.67 (br s, 1H, NH2), 8.33 (dd,
1H, Hpyridyl), 8.19 (m, 2H, Hpyridyl), 7.90 (t, 2H, Hpyridyl), 7.77 (dd,
1H, Hpyridyl), 7.40-7.20 (m, 2H, Hpyridyl).
4-Am in o-7-m eth yl-6-p h en yl-1H-p yr a zin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (7). From 1 (5.00 g, 28.0 mmol),
1-phenyl-2-hydroxyimino-1-propanone (5.45 g, 33.6 mmol), and
methanol (60 mL). Reaction time 24 h. Recrystallization from
acetic acid/water yielded 7 (3.75 g, 46%). 1H NMR (DMSO-
d6): δ 12.29 (br s, 1H, NH),8.60 (br s, 1H, NH2), 8.48 (br s,
1H, NH2), 7.67-7.53 (m, 5H, Ph), 2.54 (s, 3H, CH3).
4-Am in o-6-m et h yl-7-p h en yl-1H -p yr a zin o[2,3-c]1,2,6-
th ia d ia zin e 2,2-Dioxid e (9). From 1 (5.00 g, 28.2 mmol),
1-phenyl-1,2-propanedione (5.00 g, 33.7 mmol), and acetic acid
(100 mL). Reaction time 7 h. Recrystallization from ethanol/
water yielded 9 (5.80 g, 71%). 1H NMR (DMSO-d6): δ 12.24
(br s, 1H, NH), 8.58 (br s, 1H, NH2), 8.42 (br s, 1H, NH2), 7.74
(m, 3H, Ph), 7.50 (m, 2H, Ph), 2.53 (s, 3H, CH3).
Exp er im en ta l Section
Gen er a l. Melting points were determined with a Reichert-
J ung Thermovar micro melting point apparatus and are
uncorrected. 1H NMR spectra (300 MHz) and 13C NMR spectra
(75 MHz) were recorded on a Varian XL-300 spectrometer and
are reported in ppm on the δ scale. The signals of the solvent
were used as reference. Mass spectra (electron impact, 70 eV)
were obtained on a VG 12-250 (VG Masslab). Elemental
analyses were performed on a Heraeus CHN-O-Rapid ana-
lyzer. Column chromatography was carried out on silica gel
(Merck, particle size 70-230 mesh).
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
3-5, 7, a n d 9. To a suspension of 1 (1.0 mmol) in methanol
and either concentrated hydrochloric acid or acetic acid was
added the corresponding carbonyl compound (1.1 mmol) and
the mixture was refluxed. The reaction mixture was evapo-
rated to dryness, and water was added to the residue. The
4-Am in o-6-ch lor o-7-p h en yl-1H -p yr a zin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (11). To a solution of 8 (4.00 g, 14.5
mmol) in DMF (20 mL) was added N-chlorosuccinimide (1.97
g, 14.5 mmol). The reaction mixture was stirred at room
temperature for 24 h. Then, the solution was evaporated to
dryness, and water was added to the residue. The precipitate
was filtered and recrystallized from methanol/water to yield
1
11 (3.73 g, 73%). H NMR (DMSO-d6): δ12.45 (br s, 1H, NH),
8.74 (br s, 1H, NH2), 8.63 (br s, 1H, NH2), 7.79-7.74 (m, 2H,
Ph), 7.75-7.52 (m, 3H, Ph).

Arylpyrazino[2,3-c][1,2,6]thiadiazine 2,2-Dioxides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1703
Gen er a l P r oced u r e for th e Alk yla tion of P yr a zin o[2,3-
c][1,2,6]th ia d ia zin e 2,2-Dioxid es. To the corresponding
4-amino-1H-pyrazino[2,3-c][1,2,6]thiadiazine 2,2-dioxide de-
rivative in acetone and either potassium carbonate or triethy-
lamine was added the alkyl halide. The reaction mixture was
refluxed and evaporated to dryness, and water was added to
the residue. The precipitate was filtered and recrystallized
from the appropriate solvent (Table 1).
19 (0.89 g, 60%). 1H NMR (DMSO-d6): δ 9.01 (br s, 1H, NH2),
8.94 (br s, 1H, NH2), 8.30 (d, 1H, Hpyridyl), 8.28-8.21 (m, 2H,
Hpyridyl), 8.19-7.87 (m, 3H, Hpyridyl), 7.39-7.29 (m, 2H,Hpyridyl),
3.48 (s, 3H, CH3).
4-Am in o-1-eth yl-7-m eth yl-6-ph en ylpyr azin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (20). From 7 (0.35 g, 1.2 mmol),
acetone (150 mL), potassium carbonate (0.09 g, 0.6 mmol), and
ethyl iodide (0.2 mL, 2.4 mmol). Reaction time 10 h. Recrys-
1
4-Am in o-1-e t h yl-6,7-d ip h e n ylp yr a zin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (12). From 2 (1.00 g, 2.8 mmol),
acetone (80 mL), potassium carbonate (0.18 g, 1.3 mmol), and
ethyl iodide (0.3 mL, 3.5 mmol). Reaction time 24 h. Recrys-
tallization from ethanol yielded 12 (0.83 g, 78%). 1H NMR
(DMSO-d6): δ 8.89 (br s, 1H, NH2), 8.76 (br s, 1H, NH2), 7.53-
7.30 (m, 10H, Ph), 4.12 (q, 2H, CH2), 1.37 (t, 3H, CH3).
4-Am in o-1-b en zyl-6,7-d ip h en ylp yr a zin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (13). From 2 (1.00 g, 2.8 mmol),
acetone (130 mL), potassium carbonate (0.19 g, 1.4 mmol),
potassium iodide (250 mg, 1.5 mmol), and benzyl bromide (3.8
mL, 3.5 mmol). Reaction time 30 h. Recrystallization from
tallization from methanol/water yielded 20 (0.37 g, 75%). H
NMR (DMSO-d6): δ 8.77 (br s, 2H, NH2), 8.59 (br s, 2H, NH2),
7.74 (m, 2H, Ph), 7.49 (m, 3H, Ph), 4.07 (q, 2H, CH2), 2.63 (s,
3H, CH3), 1.33 (t, 3H, CH3).
4-Am in o-1-eth yl-6-m eth yl-7-ph en ylpyr azin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (21). From 9 (1.11 g, 3.8 mmol),
acetone (120 mL), potassium carbonate (0.26 g, 1.9 mmol), and
ethyl iodide (0.63 mL, 7.6 mmol). Reaction time 24 h. Recrys-
tallization from ethanol/water yielded 21 (0.82 g, 67%). 1H
NMR (DMSO-d6): δ 8.78 (br s, 1H, NH2), 8.67 (br s, 1H, NH2),
7.75-7.55 (m, 5H, Ph), 4.04 (q, 2H, CH2), 2.61 (s, 3H, CH3),
1.30 (t, 3H, CH3).
1
ethanol yielded 13 (0.98 g, 79%). H NMR (DMSO-d6): δ 8.99
4-Am in o-6-ch lor o-1-eth yl-7-ph en ylpyr azin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (22). From 11 (2.00 g, 6.4 mmol),
acetone (80 mL), triethylamine (0.9 mL, 6.5 mmol), and ethyl
iodide (0.8 mL, 9.7 mmol). Reaction time 96 h. Recrystalliza-
(br s, 1H, NH2), 8.84 (br s, 1H, NH2), 7.51-7.27 (m, 15H, Ph),
5.23 (s, 2H, CH2).
4-Am in o-1-[(e t h oxyca r b on yl)m e t h yl]-6,7-d ip h e n yl-
p yr a zin o[2,3-c][1,2,6]th ia d ia zin e 2,2-Dioxid e (14). From
2 (2.25 g, 6.4 mmol), acetone (475 mL), potassium carbonate
(0.19 g, 1.4 mmol), potassium iodide (250 mg, 1.5 mmol), and
ethyl bromoacetate (2.7 mL, 9.5 mmol). Reaction time 40 h.
Recrystallization from ethanol yielded 14 (2.36 g, 85%). 1H
NMR (DMSO-d6): δ 9.05 (br s, 1H, NH2), 8.93 (br s, 1H, NH2),
7.55-7.31 (m, 10H, Ph), 4.76 (s, 2H, N-CH2), 4.13 (q, 2H,
O-CH2), 1.21 (t, 3H, CH3).
4-Am in o-1-[(ter t-b u t oxyca r b on yl)m et h yl]-6,7-d ip h e-
n ylpyr azin o[2,3-c][1,2,6]th iadiazin e 2,2-Dioxide (15). From
2 (1.50 g, 4.2 mmol), acetone (60 mL), potassium carbonate
(0.29 g, 2.1 mmol), potassium iodide (0.25 g, 1.5 mmol), and
tert-buthyl bromoacetate (0.65 mL, 4.6 mmol). Reaction time
90 h. The residue was purified by column chromatography
using as eluent dichloromethane/methanol (50/1) to give 15
(1.24 g, 58%). 1H NMR (DMSO-d6): δ 9.05 (br s, 1H, NH2),
8.90 (br s, 1H, NH2), 7.54-7.29 (m, 10H, Ph), 4.64 (s, 2H, CH2),
1.31 (s, 9H, CH3).
1
tion from ethanol yielded 22 (1.56 g, 74%). H NMR (DMSO-
d6): δ 8.93 (br s, 1H, NH2), 8.83 (br s, 1H, NH2), 7.87-7.83
(m, 2H, Ph), 7.56-7.55 (m, 3H, Ph), 4.04 (q, 2H, N-CH2), 1.30
(t, 3H, CH3).
4-Am in o -1-m e t h y l-6-p h e n y lp y r a zin o [2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (23). From 6 (3.00 g, 11.0 mmol),
acetone (300 mL), potassium carbonate (0.71 g, 5.1 mmol), and
methyl iodide (3.4 mL, 5.5 mmol). Reaction time 24 h.
Recrystallization from ethanol/water yielded 23 (1.10 g, 38%).
1H NMR (DMSO-d6): δ 9.36 (s, 1H, CH), 9.03 (s.a., 2H, NH2),
8.36-7.50 (m, 5H, Ph), 3.43 (s, 3H, CH3).
4-Am in o-6-b r om o-1-[(et h oxyca r b on yl)m et h yl]-7-p h e-
n ylpyr azin o[2,3-c][1,2,6]th iadiazin e 2,2-Dioxide (24). From
10 (2.94 g, 8.3 mmol), acetone (250 mL), triethylamine (1.2
mL, 8.3 mmol), and ethyl bromoacetate (3.3 mL, 11.4 mmol).
Reaction time 96 h. Recrystallization from ethanol/water
yielded 24 (2.62 g, 74%). 1H NMR (DMSO-d6): δ 9.11 (br s,
1H, NH2), 9.10 (br s, 1H, NH2), 7.79-7.74 (m, 2H, Ph), 7.56-
7.53 (m, 3H, Ph), 4.67 (s, 2H, CH2), 4.10 (q, 2H, O-CH2), 1.09
(t, 3H, CH3).
4-Am in o-1-eth yl-6,7-d i(2-th ien yl)p yr a zin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (17). From 3 (1,50 g, 4.1 mmol),
acetone (150 mL), triethylamine (0.6 mL, 8.2 mmol), and ethyl
iodide (0.6 mL (8.2 mmol). Reaction time 72 h. Recrystalliza-
tion from acetone/water yielded 17 (1.10 g, 69%). 1H NMR
(DMSO-d6): δ 8.85 (br s, 1H, NH2), 8.58 (br s, 1H, NH2), 7.85-
7.78 (m, 2H, Hthienyl), 7.43 (dd, 1H, Hthienyl), 7.22 (dd, 1H,
4-Am in o-1-(ca r ba m oylm eth yl)-6,7-d ip h en ylp yr a zin o-
[2,3-c][1,2,6]th ia d ia zin e 2,2-Dioxid e (16). A solution of 14
(0.90 g, 2,5 mmol) in methanol (80 mL) at 0 °C was saturated
with a slow stream of ammonia for 1 h and stirred at room
temperature for 3 h. Then the solution was evaporated to
dryness, and water was added to the residue. The precipitate
H
thienyl), 7.20-7.09 (m, 2H, Hthienyl), 4.08 (c, 2H, CH2) 1.38 (t,
1
3H, CH3).
was filtered to give 16 (0.79 g, 77%). H NMR (DMSO-d6): δ
4-Am in o-6,7-d i(2-fu r yl)-1-m eth ylp yr a zin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (18). From 4 (1.70 g, 5.3 mmol),
acetone (475 mL), potassium carbonate (0.40 g, 5.3 mmol), and
methyl iodide (0.7 mL, 10.6 mmol). Reaction time 96 h.
Recrystallization from ethanol yielded 18 (1.30 g, 72%). 1H
NMR (DMSO-d6): δ 8.88 (br s, 1H, NH2), 7.93 (br s, 1H, NH2),
7.93-7.80 (m, 2H, Hfuryl), 7.06-6.80 (m, 2H, Hfuryl), 6.70 (s, 2H,
8.98 (br s, 1H, NH2), 8.87 (br s, 1H, NH2), 7.52-7.22 (m, 10
H, Ph and 2H, CONH2), 4.53 (s, 2H, CH2).
In Vitr o P la telet Aggr ega tion Stu d ies. Blood was col-
lected from male New Zealand white rabbits (2.5-3.5 kg) by
cardiac puncture or from healthy volunteers who gave in-
formed consent (Blood Bank, Hospital Sant Pau, Barcelona,
Spain). The blood was anticoagulated with 3.8% trisodium
citrate (9:1, v/v). Platelet-rich plasma (PRP) and platelet-poor
plasma (PPP) were prepared by sequential centrifugation of
citrated blood at room temperature, at 250g for 10 min and at
2000g for 20 min, respectively. The platelet counts of the PRP
were adjusted to 5 × 105 cells/µL (rabbits) or 3 × 105 cells/µL
(humans) by dilution with PPP. Platelet aggregation was
monitored according to Born’s method14 using a four-channel
aggregometer (Aggrecorder, Menarini, Italy). Test compounds
(0.1-30 µM) dissolved in DMSO (2.5 µL) were preincubated
at 37 °C for 10 min with PRP (500 µL). After this time platelet
aggregation was induced by addition of arachidonic acid (AA;
1 mM), adenosine diphosphate (ADP; 1-2 µM), collagen
(COLL; 1-2 µg/mL) or the TxA2 mimetics U46619 (1.4 µM)
and I-BOP (225-250 nM). These concentrations of the agonists
have been chosen in order to produce 80-90% of maximal
aggregation upon addition of PRP and therefore to minimize
H
furyl), 3.44 (s, 3H, CH3).
4-Am in o-1-m eth yl-6,7-di(2-pyr idyl)pyr azin o[2,3-c][1,2,6]-
th ia d ia zin e 2,2-Dioxid e (19). From 5 (1.50 g, 4.6 mmol),
acetone (150 mL), triethylamine (0.6 mL, 4.6 mmol), and
methyl iodide (0.9 mL, 13.8 mmol). Reaction time 72 h. The
precipitated was filtered and washed several times with
dichloromethane to give 0.3 g (20%) of 4-amino-1-methyl-6-
(2-pyridyl)-7-(N-methylpyridinio)pyrazino[2,3-c][1,2,6]thia-
1
diazine 2,2-dioxide iodide. H NMR (DMSO-d6): δ 9.04 (br s,
1H, NH2), 8.95 (br s, 1H, NH2), 9.31 (d, 1H, Hpyridyl), 8.60 (t,
1H, Hpyridyl), 8.49 (d, 1H, Hpyridyl), 8.26-8.01 (m, 4H, Hpyridyl),
7.99-7.46 (d, 1H, Hpyridyl), 4.24 (s, 3H, CH3), 3.61 (s, 3H, CH3).
13C NMR (DMSO-d6): δ 158.1, 155.5, 147.2, 143.9, 121.1, 137.0,
136.9, 129.9, 129.8, 129.7, 128.8, 128.2, 166.4, 81.6, 44.1, 27.5.
MS (m/e) 382 (M+ - 127), 127 (I-). The filtrate was evaporated
to dryness and recrystallized from ethanol-acetone to yield

1704 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Campillo et al.
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the individual variability of response. Acetylsalicylic acid (ASA;
12.5-50 µM), BAY-U (0.1-0.3 µM), and adenosine (100 µM)
were used as reference compounds.
The percent inhibition of platelet aggregation produced by
test compounds was calculated 5 min after the addition of the
aggregating agents, from the reduction of the maximal am-
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the paired vehicle-treated experiments. The lowest concentra-
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tion required to obtain
response was determined for each compound from the corre-
sponding concentration-response curves.
a maximal (80-100% inhibition)
Ex Vivo P la telet Aggr ega tion Stu d ies. Male Wistar rats
weighing 200-250 g were used after an overnight fasting. Test
compounds suspended in carboxymethylcellulose 0.25% (w/v)
in Tween 80 (10%) were administered orally by gavage at the
dose of 100 mg/kg in a volume of 10 mL/kg of body weight.
Blood was obtained by cardiac puncture under ether anesthe-
sia at 30 and 120 min after administration. BAY-U (25 mg/
kg), ASA (100 mg/kg), and ticlopidine (100 mg/kg) adminis-
tered orally were used as reference compounds, and their effect
was determined at 120 min postdosing. Sodium heparin at a
final concentration of 1 U/mL was used as anticoagulant.
Platelet aggregation was determined as described above for
in vitro study in PRP diluted to (4-5) × 105 cells/µL in PPP.
The aggregation inducers employed were arachidonic acid (0.4
mM), collagen (1-2 µg/mL), or I-BOP (225-250 nM). Drug
activity was expressed as percent inhibition of platelet ag-
gregation at 5 min after challenge with agonists.
Ack n ow led gm en t. We are grateful to C. de Cas-
tellarnau from the Fundacio´n Investigacio´n Sant Pau,
Barcelona, Spain, for biological assays.
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diphosphate and its reversal. Nature 1962, 194, 927-929.
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diazine 1,1-Dioxides and aldehydes to give fused imidazo and
Pyrazino Derivatives. J . Heterocycl. Chem. 1988, 25, 891-893.
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