Synthesis and pharmacological evaluation of some 8- cyanopyrido[3',2':4,5]thieno[3,2-d]triazine derivatives as inhibitors of nitric oxide and eicosanoid biosynthesis

Article abstract of DOI:10.1021/jm991085l

A series of 8-cyanopyrido[3',2':4,5]thieno[3,2-d]-1,2,3-triazines, substituted at C-4 and C-7, were synthesized and evaluated as nitric oxide and prostaglandin E2 inhibitors in murine peritoneal macrophages stimulated with bacterial endotoxin. Several compounds exhibited considerable activity, compounds 10 and 13 being the most interesting ones with IC₅₀ values of 11.2 and 3.4 μM on nitrites and 0.9 and 0.6 μM on prostaglandin E₂ production, respectively. None of the examples of pyridothienotriazines that were active at 10 μM showed any effect on inducible nitric oxide synthase, cyclooxygenase-2, and cyclooxygenase-1 enzymes, suggesting that they act by modifying the level of expression of these inducible enzymes.

Full text of DOI:10.1021/jm991085l

4720
J . Med. Chem. 1999, 42, 4720-4724
Brief Articles
Syn th esis a n d P h a r m a cologica l Eva lu a tion of Som e
8-Cya n op yr id o[3′,2′:4,5]th ien o[3,2-d ]tr ia zin e Der iva tives a s In h ibitor s of
Nitr ic Oxid e a n d Eicosa n oid Biosyn th esis
J ose´ Ma Quintela,‡ Carlos Peinador,‡ Liliana Ma Gonza´lez,‡ Ricardo Riguera,*,† Inmaculada Rioja,§
Ma Carmen Terencio,§ Amalia Ubeda,§ and Ma J ose´ Alcaraz§
Departamento de Quı´mica Fundamental, Universidad de La Corun˜a, La Corun˜a, Spain,
Departamento de Quı´mica Orga´nica, Universidad de Santiago, Santiago de Compostela, Spain, and
Departamento de Farmacologı´a, Universidad de Valencia, Valencia, Spain
Received May 27, 1999
A series of 8-cyanopyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazines, substituted at C-4 and C-7, were
synthesized and evaluated as nitric oxide and prostaglandin E2 inhibitors in murine peritoneal
macrophages stimulated with bacterial endotoxin. Several compounds exhibited considerable
activity, compounds 10 and 13 being the most interesting ones with IC50 values of 11.2 and 3.4
µM on nitrites and 0.9 and 0.6 µM on prostaglandin E2 production, respectively. None of the
examples of pyridothienotriazines that were active at 10 µM showed any effect on inducible
nitric oxide synthase, cyclooxygenase-2, and cyclooxygenase-1 enzymes, suggesting that they
act by modifiying the level of expression of these inducible enzymes.
In tr od u ction
the constitutive isoenzymes in normal physiology, the
selective modulation of NO and PGE2 overproduction
by iNOS and COX-2 might represent an important
therapeutic goal in different inflammatory pathologies.
Nitric oxide (NO) is a key mediator in various physi-
ological and pathological processes. NO is formed by the
enzyme NO synthase (NOS), which exists as three
distinct isoforms: namely (i) endothelial NOS, (ii)
neuronal NOS, and (iii) inducible NOS (iNOS).1 It has
been reported that expression is induced in macro-
phages by a variety of agents, such as cytokines and
bacterial endotoxins, during the inflammatory process.
This enzyme is capable of producing high concentrations
of NO, which contributes to the antimicrobial and
antitumor action of macrophages.2 However, NO pro-
duction during inflammatory response may become
“self-destructive” owing to its oxidative properties, as
occurs in chronic inflammatory diseases.3,4 Further-
more, NO contributes significantly to the circulatory
failure associated with endotoxic shock.5
Prostanoids regulate a great number of physiological
processes and are synthesized by two cyclooxygenase
(COX) isoforms. COX-1 synthesizes different pros-
tanoids involved in homeostatic functions, whereas
COX-2, whose expression is restricted under basal
conditions, is upregulated by inflammatory stimuli
resulting in increased PGE2 production, which has been
suggested to play an important role in the pathophysi-
ology of inflammation and arthritis.6,7
Many compounds described as inhibitors of NOS
contain heterocyclic structures such as imidazole, tria-
zole, or triazine.9 During a project directed toward the
identification of new inhibitors, we selected for study
the pyridothieno-1,2,3-triazine nucleus and, after some
screening, centered our efforts on the 8-cyanopyrido-
thieno-1,2,3-triazine derivatives substituted at C-4 and
C-7. In this paper we describe the synthesis of a series
of these derivatives and their capability to inhibit the
generation of NO and PGE2 by murine macrophages.
Although some 1,2,3-triazine derivatives have been
described as potential antifungal drugs10 and others
possess antianaphylactic activity,11 this is the first time
pyridothieno-1,2,3-triazine derivatives have been identi-
fied as effective in vitro inhibitors of nitric oxide and
eicosanoid biosynthesis.
Ch em istr y
The preparation of pyridothienotriazines 7-22 was
accomplished in good yields by intramolecular conden-
sation of a diazonium ion with an adjacent nucleophilic
function. This procedure has proven useful in the
synthesis of various five- and six-membered ring nitro-
gen heterocycles, including 1,2,3-triazines,12 and we
found in fact that the diazotization of 3-amino-2,5-
dicyano-6-substituted thieno[2,3-b]pyridines 6 consti-
tutes a direct and very convenient route to the title ring
system. The synthetic route is represented in Scheme
1 along with the target compounds showing the substi-
tution pattern.
The co-induction of iNOS and COX-2 in cells stimu-
lated with bacterial endotoxins and other inflammatory
stimuli has been described.8 Due to the importance of
* Corresponding author. Phone: +34-981-591091. Fax: +34-981-
591091. E-mail: ricardo@usc.es.
‡ Universidad de La Corun˜a.
† Universidad de Santiago.
§ Universidad de Valencia.
10.1021/jm991085l CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/09/1999

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4721
Sch em e 1a
To evaluate the structure-activity relationship, dif-
ferent substituents (R1 and R2) were placed in two
positions (C-4 and C-7) of the pyridothienotriazine
skeleton. Comparative analysis of the data shown in
Table 1 indicates some trends: The 8-cyanopyridothieno-
1,2,3-triazines 7-10 with a chloro substituent (R2 ) Cl)
at position C-4 of the triazine nucleus and a nitrogen
heterocycle at C-7 (R1) were found to be good inhibitors
of PGE2 production, and reductions of about 50% of the
control value were obtained in most cases. Compound
10, with an N′-(p-acetylphenyl)piperazine group as R1,
was found to be much more effective (95% inhibition),
practically suppressing the generation of PGE2. In
contrast, the presence of an amino group or a linear
amine at C-7 (R1) and a bromo instead of a chloro
substituent atom at C-4 (11, 12) is associated with a
total loss of activity on PGE2 production.
As for the inhibition of NO formation, only three
chlorine-containing compounds (7, 9, and 10) are active,
suppressing around 40-50% of the nitrite accumulated.
In a similar way to previous observations in the PGE2
test, compounds 11 and 12 do not inhibit the production
of NO at all. From these data, compound 10 emerges
as the most interesting example within this series
because it is effective in inhibiting the production of both
metabolites.
When we consider the compounds carrying a mor-
pholine substituent at C-7 (R1) and different hetero-
cycles at C-4 (R2), i.e., compounds 13-16, a moderate
decrease (30-40%) in accumulation of both metabolites
is observed in 14-16 at 10 µM (Table 1), with compound
13 (R1 ) morpholine, R2 ) 4′-methylpiperidine) being
much more active, producing around 90% inhibition on
production of NO and PGE2 at that concentration.
When heterocycles other than morpholine are incor-
porated as R1 substituents (compounds 17-20), inhibi-
tory action on both metabolites of around 50% is
obtained. Once again, the presence of acyclic amines in
position R1 (21, 22) does not improve the effectiveness
of these compounds.
Table 2 shows the IC50 values for the inhibition of
nitrite and PGE2 accumulation of the two most active
pyridothienotriazines examined, 10 and 13. Comparison
of these data with the reference values of aminoguani-
dine (AG) (an inhibitor of NOS activity), NS 398 (a
selective inhibitor of COX-2), and the corticosteroid
dexamethasone, shown in Table 2, illustrates the po-
tential interest of these two pyridothienotriazines as
inhibitors of the excess production of NO and prosta-
glandins. Compounds that inhibit excess production of
NO and prostaglandins by macrophages might be of
benefit for the prevention and treatment of autoimmune
diseases, septic shock, and different inflammatory pa-
thologies.1
Finally, to assess whether the inhibition observed in
cultured cells is related, or not, to a direct action on
enzymatic activities, we decided to assay the effect of
the 8-cyanopyridothieno-1,2,3-triazines on iNOS, COX-
2, and COX-1 enzymes and to compare them with
aminoguanidine (AG), a known iNOS inhibitor that
reduces the production of nitrites and citrulline forma-
tion, and with NS 398 and indomethacin, two COX
inhibitors that are very effective in reducing eicosanoid
synthesis in vitro. The results are shown in Table 3 and
The introduction of substituents R1 and R2 is carried
out using the same reaction: aromatic nucleophilic
substitution of an aromatic halide, thus simplifying the
process. The key â-enaminonitrile intermediates 6a -g
were synthesized from 2-amino-6-chloro-3,5-dicyano-
pyridine (1), as previously reported.13 Nitrosation of 6
with sodium nitrite in HCl or HBr (6f to give 12)
afforded the halo-substituted triazines 7-12, which
produced the pyridothienotriazine derivatives 13-22 in
good yields by halide displacement with the appropriate
secondary amine as a nucleophile. All compounds
prepared in this work gave satisfactory elemental
analyses and spectral data (IR, 1H NMR, 13C NMR, and
MS) that are consistent with the structures proposed.
Biologica l Resu lts a n d Discu ssion
Stimulation of macrophages with bacterial lipopolysac-
charide (LPS) causes expression of iNOS and COX-2
with the consequent generation of large quantities of
NO and PGs.8 To establish the activity of the title
compounds, we determined the accumulation of nitrite
and PGE2 as an index of enzyme expression/activity;
24-h LPS-stimulated peritoneal macrophages produced
1018 ng of nitrite/mL and 3.5 ng of PGE2/mL with
respect to 90.5 ng of nitrite/mL and 0.4 ng of PGE2/mL
in untreated cells.
As shown in Table 1, the production of these media-
tors was attenuated by most of the cyanopyridothieno-
triazines at 10 µM. At that concentration, these com-
pounds did not exert cytotoxic effects during a 24-h
incubation period as indicated by MTT reduction (data
not shown).

4722 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Brief Articles
Ta ble 1. Structure and Chemical Data of 8-Cyanopyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazines 7-22 and Their Inhibitory Action (%)
on the Accumulation of Nitrites and PGE2 in Stimulated Peritoneal Macrophages
a
b
Compounds were assayed at 10 µM. All elemental analyses for C, H, and N agree within (0.4% of calculated values. Data shown
are mean ( SEM (n ) 6-9). **pe0.01.
Ta ble 2. IC50 Values of Selected 8-Cyanopyrido[3′,2′:4,5]-
thieno[3,2-d]-1,2,3-triazines for the Inhibition of Nitrite and
PGE2 Accumulation in Stimulated Macrophages
Ta ble 3. Effect of a Selection of 8-Cyanopyridothieno-
1,2,3-triazines on iNOS, COX-1, and COX-2 Activities in Vitroa
iNOS
COX-1
COX-2
a
IC50 (µM)
(pmol citrulline‚ (ng TXB2/mg (ng PGE2/mg
compd
control
7
8
9
10
13
15
16
17
18
19
mg protein/min)
protein
protein)
compd
nitrites
PGE2
14.1 ( 1.0
14.0 ( 0.4
ND
128.1 ( 5.1
16.6 ( 0.9
10
13
11.2 (10.5-12.0)
3.4 (1.0-5.2)
25.1 (22.9-28.8)
ND
0.9 (0.4-1.7)
0.6 (0.3-1.0)
ND
3.1 (1.3-5.4) nM
1.0 (0.7-2.4) nM
112.5 ( 11.3 16.5 ( 1.8
130.5 ( 10.8 16.2 ( 1.5
127.6 ( 23.1 13.8 ( 1.2
aminoguanidine
NS 398
dexamethasone
13.1 ( 0.2
14.1 ( 0.4
13.1 ( 0.6
15.1 ( 0.9
14.2 ( 0.6
12.5 ( 0.6
12.4 ( 0.5
13.5 ( 0.9
13.1 ( 1.0
ND
104.1 ( 2.1
130.3 ( 5.9
15.9 ( 1.3
13.0 ( 1.1
35.8 (12.2-89.1) nM
a
149.3 ( 31.0 16.5 ( 1.9
141.5 ( 20.8 16.6 ( 1.4
127.2 ( 33.7 14.3 ( 0.9
124.1 ( 13.5 13.9 ( 2.0
134.1 ( 15.3 15.8 ( 1.4
123.1 ( 11.2 14.6 ( 1.2
Values represent the concentration required to produce 50%
inhibition of the response, along with the 95% confidence limits.
ND, not determined.
indicate that none of the compounds active at 10 µM
significantly modified the enzymatic activities in vitro.
This fact suggests that the effect of these compounds
could be due to their action on the degree of induction/
expression of NOS and COX proteins caused by LPS in
macrophages.
In summary, we report here that 8-cyanopyridothieno-
1,2,3-triazines 10 and 13 are potent inhibitors of NO
and PGE2 generation on murine macrophages and that
this action is not related to a direct effect on NOS or
COX activities. Further studies are underway to eluci-
date the mechanisms of action and to optimize the
inhibitory effect.
20
NS 398
indomethacin
aminoguanidine
ND
8.5 ( 0.8**
8.6 ( 1.0**
ND
ND
5.4 ( 0.3**
63.8 ( 4.8**
ND
a
Data shown are mean ( SEM (n ) 6-9). **pe0.01. ND., not
determined. All compounds were assayed at a concentration of 10
µM, except aminoguanidine which was assayed at 100 µM.
Amersham Iberica, (Madrid, Spain). NS 398 was purchased
from Universal Biologicals Ltd. (London, U.K.). The rest of the
reagents were obtained from Sigma Chemical Co. (MO).
Mou se Ma cr op h a ges Cu ltu r e. Highly purified peritoneal
macrophages were harvested by peritoneal lavage 4 days after
ip injection of 1 mL of 10% thioglycolate broth. Cells were
resuspended in culture medium (120 nM NaCl, 4.7 mM KCl,
1.2 mM CaCl2‚7H2O, 1.2 mM KH2PO4, 25 mM NaHCO3, 10
mM HEPES, 1 mM arginine, and 10 mM glucose) supple-
mented with 10% fetal bovine serum, 2 mM glutamine, 100
Exp er im en ta l Section
P h a r m a cology. [5,6,8,11,12,14,15(n)-3H]PGE2, [5,6,8,9,11,-
12,14,15(n)-3H]TXB2, and l-[3H]arginine were obtained from

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4723
IU mL-1 penicillin, 100 µg mL-1 streptomycin and incubated
at 37 °C for 2 h. The adherent cells were incubated with E.
coli lipopolysaccharide (10 µg/mL) at 37 °C for 24 h in the
presence of test compounds or vehicle. Nitrite and PGE2 levels
were assayed in culture supernatants by a fluorimetric method14
and by radioimmunoassay,15 respectively. The mitochondrial-
dependent reduction of 3-(4,5-dimethylthioxol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) to formazan was used to
assess the possible cytotoxic effects of compounds.
Assa y of Cyclooxygen a se-1 Activity. Human platelets
were sonicated at 4 °C in an ultrasonicator at maximum
potency. Microsomes were prepared by centrifugation at 2000g
for 5 min at 4 °C followed by centrifugation of the supernatant
at 100000g for 100 min at 4 °C. Microsomes (20 µg of protein/
tube) were incubated with arachidonic acid and test com-
pounds or vehicle.16 TXB2 levels were determined by radio-
immunoassay.15
Assa y of Cyclooxygen a se-2 Activity. Murine peritoneal
macrophages incubated with E. coli lipopolysaccharide (10 µg/
mL) at 37 °C for 24 h were collected and sonicated at 4 °C in
an ultrasonicator at maximum potency, and microsomes were
prepared as above.16 Microsomes (40 µg of protein/tube) were
used as a source of cyclooxygenase-2, and reactions were
carried out in the same conditions as above. PGE2 synthesis
was determined by radioimmunoassay.15
Assa y of iNOS Activity. High-speed (100000g) superna-
tants from sonicated peritoneal macrophages were obtained
as described above. NOS activity was determined in superna-
tants by monitoring the conversion of L-[3H]arginine to L-[3H]-
citrulline.17
NMR (CDCl3) 21.5 (CH3), 30.8 (CH), 34.1 (CH2), 48.7 (CH2N),
93.2 (C-8), 114.2 (C-4a), 117.2 (CN), 129.2 (C-9a), 141.5 (C-9),
151.1, 152.1, 159.4, 165.8; MS (EI) m/e 344 (M+), 346 (M+
+
2). Anal. (C15H13ClN6S) C, H, N.
7-(4-Ben zylp ip er id in o)-4-ch lor o-8-cya n op yr id o[3′,2′:
4,5]th ien o[3,2-d ]-1,2,3-tr ia zin e (9): purified by flash chro-
matography using CH2Cl2/AcOEt (20:1) as eluent; mp 218-
1
220 °C; H NMR (CDCl3) 1.45 (m, 2H), 1.93 (m, 3H), 2.63 (d,
2H, J ) 6.7 Hz), 3.17 (m, 2H), 4.81 (m, 2H), 7.14-7.37 (m,
5H), 8.87 (s, 1H); 13C NMR (CDCl3) 32.0 (CH2), 37.9 (CH), 42.7
(CH2Ph), 48.7 (NCH2), 93.3 (C-8), 114.4 (C-4a), 117.2 (CN),
129.2 (C-9a), 126.1, 128.3, 129.0, 139.5 (C6H5), 141.5 (C-9),
151.1, 152.2, 159.4, 165.8; MS (FAB) m/e 421 (MH)+, 423
[(MH)+ + 2]. Anal. (C21H17ClN6S) C, H, N.
7-(N -4′-Ace t ylp h e n ylp ip e r a zin o)-8-cya n o-4-ch lor o-
p yr id o[3′,2′:4,5]th ien o[3,2-d ]-1,2,3-tr ia zin e (10): purified by
flash chromatography using AcOEt/hexanes (2:1) as eluent;
1
mp 236-238 °C; H NMR (CDCl3) 2.55 (s, 3H), 3.64 (t, 4H, J
) 5.3 Hz), 4.25 (t, 4H, J ) 5.3 Hz), 6.91 (d, 2H, J ) 9.0 Hz),
7.94 (d, 2H, J ) 9.0 Hz), 8.97 (s, 1H); MS (FAB) m/e 450 (MH)+,
452 [(MH)+ + 2]. Anal. (C21H16ClN7OS) C, H, N.
7-Am in o-4-ch lor o-8-cya n op yr id o[3′,2′:4,5]th ien o[3,2-d ]-
1,2,3-tr ia zin e (11): purified by flash chromatography using
CH2Cl2/AcOEt (10:1) as eluent; mp 254-256 °C; 1H NMR
(DMSO-d6) 8.25 (s, 2H), 9.13 (s, 1H); 13C NMR (DMSO-d6) 91.7
(C-8), 113.4 (C-4a), 115.6 (CN), 128.4 (C-9a), 139.9 (C-9), 151.5,
151.7, 161.1, 166.5; MS (FAB) m/e 263 (MH)+, 265 [(MH)+
2]. Anal. (C9H3ClN6S) C, H, N.
+
7-Ben zylam in o-4-br om o-8-cyan opyr ido[3′,2′:4,5]th ien o-
[3,2-d ]-1,2,3-tr ia zin e (12): purified by flash chromatography
using CH2Cl2/AcOEt (20:1) as eluent; mp 208-210 °C; 1H NMR
(CD3COCD3) 4.92 (d, 2H, J ) 6.1 Hz), 7.22-7.38 (m, 3H), 7.46-
7.50 (m, 2H), 8.81 (s, 1H), 9.02 (s, 1H); MS (EI) m/e 396 (M+),
398 [(M)+ + 2]. Anal. (C16H9BrN6S) C, H, N.
Syn t h esis of P yr id o[3′,2′:4,5]t h ien o[3,2-d ]-1,2,3-t r i-
a zin es 13-22. Gen er a l P r oced u r e. A solution of the corre-
sponding halogenated derivative (7-12) (0.60 mmol) and the
appropriate secondary amine (0.72 mmol) in 3:1 (v/v) THF/
EtOH (15 mL) was refluxed until the starting material
disappeared (TLC). After cooling, the solid was filtered off and
recrystallized from EtOH/acetone.
Sta tistica l An a lysis. The results are presented as mean
( SEM; n represents the number of experiments. Inhibitory
concentration 50% (IC50) values were calculated from at least
four significant concentrations (n ) 6). The level of statistical
significance was determined by analysis of variance (ANOVA)
followed by Dunnett’s t-test for multiple comparisons.
Ch em ica l Meth od s. All reagents used were commercial
grade chemicals from freshly opened containers. Melting points
were determined on a Bu¨chi 510 apparatus and are uncor-
rected. IR spectra were recorded as potassium bromide disks
1
on a Perkin-Elmer 783 spectrophotometer. H and 13C NMR
8-Cya n o-4-(4-m eth ylp ip er id in o)-7-m or p h olin op yr id o-
[3′,2′:4,5]th ien o[3,2-d ]-1,2,3-tr ia zin e (13): mp 210-212 °C;
1H NMR (CDCl3) 1.01 (d, 3H, J ) 6.1 Hz), 1.34 (m, 2H), 1.85
(m, 3H), 3.24 (m, 2H), 3.89 (m, 8H), 4.81 (m, 2H), 8.81 (s, 1H);
13C NMR (CDCl3) 21.6 (CH3), 31.0 (CH), 34.1 (CH2), 46.6
(NCH2), 48.5 (NCH2), 66.8 (OCH2), 93.7 (C-8), 111.2 (C-4a),
117.1 (C-9a), 117.4 (CN), 140.4 (C-9), 148.6, 152.4, 160.1, 164.1;
MS (EI) m/e 395 (M+). Anal. (C19H21N7OS) C, H, N.
8-C y a n o -7-m o r p h o lin o -4-t h io m o r p h o lin o p y r id o -
[3′,2′:4,5]th ien o[3,2-d ]-1,2,3-tr ia zin e (14): mp 235-237 °C;
1H NMR (CDCl3) 2.83 (t, 4H, J ) 5.1 Hz), 3.91 (m, 8H), 4.37
(t, 4H, J ) 5.1 Hz), 8.82 (s, 1H); 13C NMR (CDCl3) 27.1 (SCH2),
48.4 (NCH2), 49.1 (NCH2), 66.5 (OCH2), 93.8 (C-8), 111.6 (C-
4a), 116.8 (C-9a), 117.3 (CN), 140.3 (C-9), 148.9, 152.3, 160.1,
164.1; MS (FAB) m/e 400 (MH)+. Anal. (C17H17N7OS2) C, H,
N.
spectra were obtained on a Bruker AC 200F instrument at
room temperature, and values are reported as δ units. Mass
spectra were obtained on a VG QUATTRO spectrometer in
electron impact (EI) or fast atom bombardment (FAB) mode
using thioglycerol as the matrix. The silica gel 60 HF254+366
used for analytical thin-layer chromatography and the silica
gel 60 (230-400 mesh) employed for flash chromatography
were purchased from Merck. Microanalyses for C, H, and N
were performed by the Elemental Analysis General Service
of the University of La Corun˜a. Compounds 2a -f, 3a -f, 4a -
g, 5a -g, and 6a -g were prepared according to modified
literature procedures.13
Syn th esis of P yr id o[3′,2′:4,5]th ien o[3,2-d ]-1,2,3-tr ia z-
in es 7-12. Gen er a l P r oced u r e. To an ice-cooled solution of
the corresponding aminonitrile 6 (6.73 mmol) in 1:1 (v/v) HCl/
AcOH or HBr/AcOH (50 mL) was added dropwise a solution
of sodium nitrite (10.1 mmol) in water (10 mL). The solution
was stirred at room temperature for 3 h, and then the mixture
was poured into water (200 mL). The resulting solid was
filtered off and purified by flash chromatography on silica gel.
8-Cya n o-4-ch lor o-7-m or p h olin op yr id o[3′,2′:4,5]th ien o-
[3,2-d ]-1,2,3-tr ia zin e (7): purified by flash chromatography
using CH2Cl2/AcOEt (25:1) as eluent; mp 201-203 °C; 1H NMR
(CDCl3) 3.90 (t, 4H, J ) 4.6 Hz), 4.07 (t, 4H, J ) 4.6 Hz), 8.94
(s, 1H); 13C NMR (CDCl3) 48.2 (CH2N), 66.4 (CH2O), 93.7 (C-
8), 115.3 (C-4a), 116.9 (CN), 129.5 (C-9a), 141.5 (C-9), 150.9,
152.4, 159.7, 166.5; MS (EI) m/e 332 (M+), 334 (M+ + 2). Anal.
(C13H9ClN6OS) C, H, N.
8-Cya n o-7-m or p h olin o-4-p ip er a zin op yr id o[3′,2′:4,5]-
1
th ien o[3,2d ]-1,2,3-tr ia zin e (15): mp 295-297 °C; H NMR
(DMSO-d6) 3.27 (t, 4H, J ) 4.6 Hz), 3.79 (m, 8H), 4.15 (t, 4H,
J ) 4.6 Hz), 9.04 (s, 1H); 13C NMR (DMSO-d6) 42.6 (NCH2),
42.9 (NCH2), 48.2 (NCH2), 65.8 (OCH2), 93.7 (C-8), 111.5 (C-
4a), 115.9 (C-9a), 117.4 (CN), 141.5 (C-9), 148.9, 152.5, 159.9,
163.7; MS (FAB) m/e 383 (MH)+. Anal. (C17H18N8OS) C, H, N.
4-(N-4′-Acetylp h en ylp ip er a zin o)-8-cya n o-7-m or p h oli-
n opyr ido[3′,2′:4,5]th ien o[3,2-d]-1,2,3 tr iazin e (16): mp 179-
181 °C; 1H NMR (CDCl3) 2.55 (s, 3H), 3.62 (t, 4H, J ) 5.2 Hz),
3.92 (m, 8H), 4.28 (t, 4H, J ) 5.2 Hz), 6.92 (d, 2H, J ) 9.0
Hz), 7.93 (d, 2H, J ) 9.0 Hz), 8.88 (s, 1H); MS (FAB) m/e 501
(MH)+. Anal. (C25H24N8O2S) C, H, N.
8-Cya n o-4-ch lor o-7-(4-m et h ylp ip er id in o)p yr id o[3′,2′:
4,5]th ien o[3,2-d ]-1,2,3-tr ia zin e (8): purified by flash chro-
matography using CH2Cl2/AcOEt (40:1) as eluent; mp 190-
192 °C; 1H NMR (CDCl3) 1.02 (d, 3H, J ) 6.2 Hz), 1.40 (m,
4-(N-4′-Acetylp h en ylp ip er a zin o)-8-cya n o-7-(4-m eth yl-
piper idin o)pyr ido[3′,2′:4,5]th ien o[3,2-d]-1,2,3-tr iazin e (17):
1
mp 246-248 °C; H NMR (CDCl3) 1.02 (d, 3H, J ) 6.2 Hz),
2H), 1.83 (m, 3H), 3.21 (m, 2H), 4.75 (m, 2H), 8.84 (s, 1H); 13
C
1.41 (m, 2H), 1.85 (m, 3H), 2.55 (s, 3H), 3.16 (m, 2H), 3.61 (t,

4724 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Brief Articles
(2) MacMicking, J .; Xie, Q.; Nathan, C. Nitric oxide and macrophage
function. Annu. Rev. Immunol. 1997, 15, 323-350.
(3) Kaur, H.; Halliwell, B. Evidence for nitric oxide-mediated
oxidative damage in chronic inflammation. Nitrotyrosine in
serum and synovial fluid from rheumatoid patients. FEBS Lett.
1994, 350, 9-12.
4H, J ) 5.2 Hz), 4.27 (t, 4H, J ) 5.2 Hz), 4.67 (m, 2H), 6.93
(d, 2H, J ) 8.9 Hz), 7.93 (d, 2H, J ) 8.9 Hz), 8.83 (s, 1H); MS
(FAB) m/e 513 (MH)+. Anal. (C27H28N8OS) C, H, N.
8-Cya n o-4,7-b is(4-m et h ylp ip er id in o)p yr id o[3′,2′:4,5]-
th ien o[3,2-d ]-1,2,3-tr ia zin e (18): mp 186-188 °C; 1H NMR
(CDCl3) 0.99 (d, 3H, J ) 6.1 Hz), 1.00 (d, 3H, J ) 6.1 Hz),
1.36 (m, 4H), 1.78 (m, 6H), 3.14 (m, 4H), 4.60 (m, 2H), 4.79
(m, 2H), 8.75 (s, 1H); 13C NMR (CDCl3) 21.6 (CH3), 30.8 (CH),
30.9 (CH), 33.9 (CH2), 34.0 (CH2), 46.5 (NCH2), 48.7 (NCH2),
93.0 (C-8), 110.8 (C-4a), 115.9 (C-9a), 117.7 (CN), 140.0 (C-9),
148.8, 152.4, 159.8, 164.3; MS (EI) m/e 407 (M+). Anal.
(C21H25N7S) C, H, N.
(4) Mo´ilanen, E.; Vapaatalo, H. Nitric oxide in inflammation and
immune response. Ann. Med. 1995, 27, 359-367.
(5) Thiemermann, C. The role of arginine: nitric oxide pathways
in circulatory shock. Adv. Pharmacol. 1994, 28, 45-79.
(6) Vane, J . R.; Mitchell, J . A.; Appleton, I.; Tomlison, A.; Bishop-
Bailey, D.; Croxtall, J .; Willoughby, D. A. Inducible isoforms of
cyclooxygenase and nitric-oxide synthase in inflammation. Proc.
Natl. Acad. Sci. U.S.A. 1994, 91, 2046-2050.
(7) Kang, R. Y.; Freire Moar, J .; Sigal, E.; Chu, C. Q. Expression of
cyclooxygenase-2 in human and an animal model of rheumatoid
arthritis. Br. J . Rheumatol. 1996, 35, 711-718.
(8) Habib, A.; Bernard, C.; Lebret, M.; Cre´minon, C.; Esposito, B.;
Tedgui, A.; Maclouf, J . Regulation of the expression of cyclooxy-
genase-2 by nitric oxide in rat peritoneal macrophages. J .
Immunol. 1997, 158, 3845-3851.
(9) Fukuto, J . M.; Chaudhuri, G. Inhibition of constitutive and
inducible nitric oxide synthase: potential selective inhibition.
Annu. Rev. Pharmacol. Toxicol. 1995, 35, 165-194.
(10) Guerrera, F.; Salerno, L.; Sarva`, M. C.; Siracusa, M. A. Synthesis
and antifungal activity of pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-
triazine derivatives. Farmaco 1993, 48, 1725-1733.
(11) Wagner, G.; Leistner, S.; Viewer, H.; Krasselt, U.; Prantz, J .
Synthese neuer pyrido[3′,2′:4,5]thieno[3,2-d]1,2,3-triazin-deri-
vate als antianaphylaktika. Pharmazie 1993, 48, 514-518.
(12) Beck, J . R.; Yahner, J . A. Synthesis of [1]Benzothieno[3,2-d]-v-
triazine derivatives. A unique diazonium ion cyclization. J . Org.
Chem. 1976, 41, 1733-1734.
4-(N-4′-Acetylph en ylpiper azin o)-7-(4-ben zylpiper azin o)-
8-cya n op yr id o[3′,2′:4,5]th ien o[3,2-d ]-1,2,3-tr ia zin e (19):
1
mp 270-272 °C; H NMR (DMSO-d6) 2.45 (s, 3H), 3.50-3.62
(m, 10H), 4.10-4.62 (m, 8H), 6.98 (d, 2H, J ) 8.8 Hz), 7.34-
7.63 (m, 5H), 7.84 (d, 2H, J ) 8.8 Hz), 9.11 (s, 1H); MS (FAB)
m/e 590 (MH)+. Anal. (C32H31N9OS) C, H, N.
7-(4-Ben zylpiper azin o)-8-cyan o-4-(4-m eth ylpiper azin o)-
p yr id o[3′,2′:4,5]th ien o[3,2-d ]-1,2,3-tr ia zin e (20): mp 203-
1
205 °C; H NMR (CDCl3) 2.38 (s, 3H), 2.63 (m, 8H), 3.60 (s,
2H), 3.95 (t, 4H, J ) 4.9 Hz), 4.09 (t, 4H, J ) 4.9 Hz), 7.30-
7.37 (m, 5H), 8.81 (s, 1H); 13C NMR (CDCl3) 45.8 (NCH2), 45.9
(CH3), 48.2 (NCH2), 52.7 (NCH2), 54.7 (NCH2), 62.8 (CH2C6H5),
93.5 (C-8), 111.4 (C-4a), 116.5 (C-9a), 117.0 (CN), 127.3, 128.3,
129.2, 137.5 (C6H5), 140.4 (C-9), 149.2, 152.8, 159.9, 164.3; MS
(FAB) m/e 486 (MH)+. Anal. (C25H27N9S) C, H, N.
7-Ben zyla m in o-8-cya n o-4-(4-m eth ylp ip er a zin o)p yr id o-
[3′,2′:4,5]th ien o[3,2-d ]-1,2,3-tr ia zin e (21): mp 265-267 °C;
1H NMR (DMSO-d6) 2.58 (s, 3H), 3.35 (m, 4H), 4.08 (m, 4H),
4.64 (d, 2H), 7.27-7.35 (m, 5H), 8.72 (s, 1H), 8.90 (s, 1H); 13
C
NMR (DMSO-d6) 42.8 (CH3), 43.1 (NCH2), 44.6 (CH2C6H5), 52.1
(NCH2), 91.9 (C-8), 110.2 (C-4a), 113.9 (C-9a), 115.9 (CN),
126.8, 127.4, 128.3, 138.9 (C6H5), 138.4 (C-9), 149.3, 152.2,
158.5, 165.0; MS (FAB) m/e 417 (MH)+. Anal. (C21H20N8S) C,
H, N.
7-Ben zyla m in o-8-cya n o-4-m or p h olin op yr id o[3′,2′:4,5]-
th ien o[3,2-d ]-1,2,3-tr ia zin e (22): mp 279-281 °C; 1H NMR
(DMSO-d6) 3.77 (m, 4H), 3.87 (m, 4H), 4.64 (d, 2H, J ) 5.8
Hz), 7.18-7.39 (m, 5H), 8.67 (t, 1H, J ) 5.8 Hz), 8.85 (s, 1H);
13C NMR (DMSO-d6) 44.5 (NHCH2), 45.6 (NCH2), 65.8 (OCH2),
91.9 (C-8), 110.3 (C-4a), 114.2 (C-9a), 115.9 (CN), 126.9, 127.5,
128.3, 138.9 (C6H5), 138.6 (C-9), 149.3, 152.7, 158.5; MS (FAB)
m/e 404 (MH)+. Anal. (C20H17N7OS) C, H, N.
(13) Quintela, J . M.; Peinador, C.; Veiga, C.; Gonza´lez, L.; Botana,
L. M.; Alfonso, A.; Riguera, R. Synthesis and antiallergic activity
of pyridothienopyrimidines. Bioorg. Med. Chem. 1998, 6, 1911-
1925.
(14) Misko, T. P.; Schilling, R. J .; Salvemini, D.; Moore, W. M.; Currie,
M. G. A fluorometric assay for the measurement of nitrite in
biological samples. Anal. Biochem. 1993, 214, 11-16.
(15) Moroney, M. A.; Alcaraz, M. J .; Forder, R. A.; Carey, F.; Hoult,
J . R. S. Selectivity of neutrophil 5-lipoxygenase and cyclooxy-
genase inhibition by an antiinflammatory flavonoid glycoside
and related aglycone flavonoids. J . Pharm. Pharmacol. 1988,
40, 787-792.
(16) Escrig, V.; Ubeda, A.; Ferra´ndiz, M. L.; Darias, J .; Sa´nchez, J .
M.; Alcaraz, M. J .; Paya´, M. Variabilin: a dual inhibitor of
human secretory and cytosolic phospholipase A2 with antiin-
flammatory activity. J . Pharmacol. Exp. Ther. 1997, 282, 123-
131.
(17) Mitchell, J . A.; Sheng, H.; Fo¨rstermann, U.; Murad, F. Charac-
terization of nitric oxide synthases in nonadrenergic noncholin-
ergic nerve-containing tissue from the rat anococcygeus muscle.
Br. J . Pharmacol. 1991, 104, 289-291.
Ack n ow led gm en t. This work was supported by
XUGA 10308B95 and 20908B97 and CICYT PM98-0227
and SAF 97-0249.
Refer en ces
(1) Moncada, S.; Higgs, A. The L-arginine-nitric oxide pathway. N.
Engl. J . Med. 1993, 329, 2002-2012.
J M991085L