Synthesis and pharmacological activity of triazolo[1,5-α]triazine derivatives inhibiting eosinophilia

Article abstract of DOI:10.1021/jm970759u

In continuation of our previous work on eosinophilia inhibitors, we synthesized an additional series of inhibitors, which consisted of 5-amino- 1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole derivatives and a newly developed series of 1,2,4-triazolo[1,5-

Full text of DOI:10.1021/jm970759u

J . Med. Chem. 1998, 41, 2985-2993
2985
Syn th esis a n d P h a r m a cologica l Activity of Tr ia zolo[1,5-a ]tr ia zin e Der iva tives
In h ibitin g Eosin op h ilia
Fumihiko Akahoshi, Shinji Takeda, Takehiro Okada,† Masahiko Kajii, Hiroko Nishimura, Masanori Sugiura,‡
Yoshihisa Inoue, Chikara Fukaya, Youichiro Naito,* Takashi Imagawa, and Norifumi Nakamura
Pharmaceutical Research Division, Yoshitomi Pharmaceutical Industries, Ltd., 2-25-1, Shodai-Ohtani,
Hirakata, Osaka 573-1153, J apan
Received November 10, 1997
In continuation of our previous work on eosinophilia inhibitors, we synthesized an additional
series of inhibitors, which consisted of 5-amino-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole
derivatives and a newly developed series of 1,2,4-triazolo[1,5-a]-1,3,5-triazine derivatives. We
evaluated their inhibitory activity on the airway eosinophilia model, which was induced by
the intravenous (iv) injection of Sephadex particles. In the 1,2,4-triazole series with various
substituents at the 3 position of the triazole ring such as 2-furyl, pyridyl, and phenoxy, none
of derivatives had comparable activity to the previously reported compound GCC-AP0341,
5-amino-3-(4-chlorophenyl)-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole. In the triazolo-
[1,5-a]triazine series, 2-(4-chlorophenyl)-6-methyl-1,2,4-triazolo[1,5-a]-1,3,5-triazine-7(6H)-
thione (3h ) was highly potent, and when given orally it had an ID50 value of 0.3 mg/kg, which
is comparable to that of GCC-AP0341. The fact that the structure-activity relationship of
these two series was quite similar suggests that a common substructure, such as the 1,2,4-
triazole ring with a substituted phenyl ring at the 3 position and a thiocarbonyl moiety at the
1 position, could contribute to the activity. Our selected compound 3h was less active than
GCC-AP0341 in the antigen-induced hyper-responsiveness model in guinea pigs; however, we
plan to carry out further studies on eosinophil functions, especially on their activation, using
our two compounds, 3h and GCC-AP0341.
In tr od u ction
shown in Schemes 1 and 2. The compounds synthesized
are listed in Tables 1-4. In the 3-substituted triazole
derivatives 2, most of the compounds, i.e., 2a ,b,d ,e,g-
j, were prepared according to the reported method
shown in Scheme 1 (route a f f).3 For synthesis of the
phenoxy derivative 1f (R1 ) OPh), the cyclization of
diphenyl cyanocarbonimidate with hydrazine was used
(Scheme 1, route e).4 The triazoles obtained were
treated with methyl isothiocyanate to obtain good yields
of the target compounds.
The synthesis of triazolo[1,5-a]triazine derivatives is
shown in Scheme 2. The triazolo[1,5-a]triazine-7(6H)-
thione derivatives 3 were prepared from the corre-
sponding triazole compounds 2 by the reaction with
diethoxymethyl acetate and good yields obtained.5 The
compounds that had methyl or phenyl groups at the 5
position of the triazolo[1,5-a]triazine ring, 4a ,b, 5a ,b,
were prepared by the reaction of 5-amino-1-[(methyl-
amino)thiocarbonyl]-1H-1,2,4-triazole or 5-amino-3-(4-
chlorophenyl)-1-[(methylamino)thiocarbonyl]-1H-1,2,4-
triazole with triethyl orthoacetate or triethyl ortho-
benzoate. As the synthesis of the triazolo[1,5-a]triazin-
7(6H)-one 8 from 5-amino-1-[(methylamino)carbonyl]-
1H-1,2,4-triazole gave a low yield, an alternative syn-
thetic route reported by Hirata et al. was used.5
Compound 8 was prepared by the reaction of diaz-
omethane with triazolo[1,5-a]triazin-7(6H)-one (7), which
was synthesized from the 5-amino-1-(aminocarbonyl)-
1H-1,2,4-triazole (6). The synthesis of 7-ethoxy-1,2,4-
triazolo[1,5-a]-1,3,5-triazine (9) was produced using 6
with triethyl orthoformate. The isomers 10a ,b were
synthesized from 3-amino-1H-1,2,4-triazole and di-
In a previous study, we described the synthesis and
pharmacological activity of GCC-AP0341 [5-amino-3-(4-
chlorophenyl)-1-[(methylamino)thiocarbonyl]-1H-1,2,4-
triazole], which was very potent in the Sephadex-
induced lung eosinophilia model in rats.1,2 GCC-
AP0341 blocked the allergen-induced airway hyper-
responsiveness in guinea pigs.1 To the best of our
knowledge, this was the first evidence that an eosino-
philia inhibitor alone could be a potent agent to treat
the asthmatic response. We previously demonstrated
that GCC-AP0341 was a well-defined agent for the
treatment of asthma;1 therefore we have attempted to
identify other potent compounds based on the GCC-
AP0341 molecule in order to confirm that eosinophilia
is necessary for chronic asthma and airway hyper-
responsiveness and that an inhibitor of eosinophilia
could be a novel agent for the treatment of asthma.
Ch em istr y
Our synthetic designs based on the structure of GCC-
AP0341 mainly involved 5-amino-1-[(methylamino)-
thiocarbonyl]-1H-1,2,4-triazole derivatives 2, which had
various substituents at the 3 position of the triazole
ring, such as 2-furyl, 2-thienyl, pyridyl, phenoxy, and
benzyl, and triazolo[1,5-a]triazine derivatives 3-5, as
* Address correspondence to this author. Phone: +81-720-56-9319.
Fax: +81-720-57-5020. E-mail: ynaito@yoshitomi.co.jp.
† Present address: Nippon Roche Research Center, 200, Kajiwara,
Kamakura, Kanagawa 247, J apan.
‡
Present address: Astra J apan Ltd., Midosuji Daiwa Bldg, 6-8,
Kyutaromachi 3-chome, Chuo-ku, Osaka 541, J apan.
S0022-2623(97)00759-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/11/1998

2986 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Akahoshi et al.
Sch em e 1a
a
Reagents: (a) aminoguanidine hydrochloride or nitrate, NaOMe, MeOH; (b) SOCl2, DMF (cat.), CH2Cl2, reflux; (c) aminoguanidine
hydrochloride, NaOH, H2O, 0 °C; (d) DMSO, 180 °C; (e) hydrazine, MeOH, 0 °C to room temperature; (f) MeNCS, 1 N NaOH, THF.
Sch em e 2a
a
Reagents: (a) (EtO)2CHOAc, room temperature to 90 °C; (b) MeC(OEt)3, 145 °C; (c) PhC(OMe)3, 150 °C; (d) KOCN, HCl (aq), room
temperature; (e) (EtO)2CHOAc, 100 °C; (f) CH2N2, ether, 0 °C to room temperature; (g) (EtO)3CH, 140 °C; (h) pyridine, 50 °C; (i) pyridine,
reflux; (j) RNH2, 90-125 °C.

Triazolo[1,5-a]triazines Inhibiting Eosinophilia
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 2987
Ta ble 1. Physical Data and Biological Activity for 3-Substituted 5-Amino-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole
Derivatives
no.
R1
mpa (°C)
formulab
C8H9N5OS
C8H9N5S2
C9H9ClN6S
inhibitionc (%)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2-furyl
173-175
170-172
208-211
215-225
270-272
184-186
209-210
173-175
275-277
212-214
183-185
18
-7
-9
-2
14
-5
25
-24
58
2-thienyl
6-chloro-3-pyridyl
5-chloro-2-pyridyl
4-chloro-1-naphthyl
PhO
4-Cl-Ph-CHdCH
Ph-CH2
C9H9ClN6S‚0.1H2O
C
C
C
14H12ClN5S
10H11N5OS
12H12ClN5S
C11H13N5S
4-Ph-Ph
Ph-CtC
C
C
C
16H15N5S
12H11N5S
11H12N6S
2j
2k
12
0
37
Ph-CHdN
SMe
(54)d
88e
(95)f
GCC-AP0341
dexamethasone
a
b
Uncorrected. Compounds were analyzed for C, H, N (0.4% using the formula indicated. c Percent inhibition value on the Sephadex-
d
induced lung eosinophilia model at an intraperitoneal (ip) dose of 3 mg/kg. Value in parentheses is percent inhibition at an ip dose of
30 mg/kg. e SE value for GCC-AP0341 was determined to be (4.2% from six experiments. f Value in parentheses is percent inhibition at
an ip dose of 0.1 mg/kg.
Ch a r t 1. Structure of GCC-AP0341
methyl N-cyanodithioiminocarbonate in pyridine by
controlling the reaction temperature at 50 °C and reflux,
but a structural determination of these two isomers
could not be obtained by spectroscopy. The amination
of 10a or 10b with methylamine or cyclohexylamine,
respectively, produced 11a or 11b.
Resu lts a n d Discu ssion
In continuation of our previous work on eosinophilia
inhibitors,1,2a-d we modified various compounds based
on GCC-AP0341 and investigated their effect on a
Sephadex-induced lung eosinophilia model, in which the
eosinophilia was induced in the airway by intravenous
injection of Sephadex particles on days 0, 2, and 5.
In a previous study,1 we reported that GCC-AP0341
[5-amino-3-(4-chlorophenyl)-1-[(methylamino)thio-
carbonyl]-1H-1,2,4-triazole] (Chart 1) is a highly potent
eosinophilia inhibitor and showed its structure-activity
relationship (SAR). Typical characteristics of the SAR
are as follows. (1) The amino function at the 5 position
of the triazole ring is necessary for the high potency.
(2) For the 3 position of the triazole ring, a phenyl ring
that is substituted by a halogen at the 4 position
facilitates the activity. (3) Concerning the (methyl-
amino)thiocarbonyl moiety at the 1 position, the thio-
carbonyl moiety and methylamino substitute facilitate
the activity. Considering these results, we initiated a
program to investigate other potent molecules based on
the GCC-AP0341 structure.
We performed a screening test using the Sephadex
model with the compounds injected intraperitoneally (ip)
at doses of 30 or 3 mg/kg. The percent inhibition values
obtained in this model are summarized in Tables 1-4.
Since the 4-chlorophenyl moiety in GCC-AP0341
contributes to the high potent activity, we determined
whether another ring system, such as pyridine, thio-
phene, or naphthalene, in place of the phenyl ring had
an effect, and the results are shown in Table 1 (2a -e).
For this set of compounds, we prepared 2-furyl (2a ),
2-thienyl (2b ), 6-chloro-3-pyridinyl (2c), 5-chloro-2-
pyridinyl (2d ), and 4-chloro-1-naphthyl (2e) derivatives.
Although compounds 2c,d were expected to have similar
activity to GCC-AP0341, they in fact showed less
activity at a dose of 3 mg/kg. These results, especially
the low activity of 2c,d , indicate that the electronic and
not the steric factor may determine the activity of GCC-
AP0341. The result1 that 5-amino-1-[(methylamino)-
thiocarbonyl]-3-(methylthio)-1H-1,2,4-triazole (12) had
an inhibition of 54% at a dose of 30 mg/kg ip prompted
us to study the effect of introducing a junctional moiety
between the triazole ring and the phenyl ring of GCC-
AP0341 as shown in Table 1 (2f-k ). This series of
compounds has oxygen (2f), vinylene (2g), methylene
(2h ), phenylene (2i), acetylene (2j), and imine (2k ) as
junctional moieties. None of the derivatives was more
active than GCC-AP0341, although the phenylene de-
rivative (2i) showed comparable activity with an inhibi-
tion of 58%. This was the first evidence that a substit-
uent with a moiety sterically larger than 4-(trifluoro-
methyl)phenyl1 at the 3 position of the triazole ring had
potent activity.
Aminophylline2a was reported to have an inhibitory
effect in the Sephadex model, and this prompted us to
study a series of fused bicyclic derivatives incorporating
the 5-amino-1-[(methylamino)thiocarbonyl]-1H-1,2,4-
triazole function. Typical examples were triazolo[1,5-
a]triazine molecules as shown in Tables 2 and 3. Table
2 lists 6-methyl-1,2,4-triazolo[1,5-a]-1,3,5-triazine-7(6H)-
thione (3a ) which had an inhibition of 69% at a dose of
30 mg/kg. Analogous compounds, which included pu-
rine 3b, pyrazolo[3,4-d]pyrimidine 3c, and quinazoline
3d , were ineffective. For 1,2,4-triazolo[1,5-a]-1,3,5-

2988 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Akahoshi et al.
Ta ble 2. Physical Data and Biological Activity of the
1,2,4-Triazolo[1,5-a]-1,3,5-triazine-7(6H)-thione Derivatives
less than GCC-AP0341, and although the reason for this
is not clearly understood, absorption and/or metabolism
might be a cause. We have firmly believed that eosi-
nophilia is a critical phenomenon for airway hyper-
responsiveness and that an inhibitor of eosinophilia
should be a potent inhibitor of airway hyper-responsive-
ness.2e,7 However, compound 3h showed lower activity
compared to that of GCC-AP0341 in the ascaris-induced
hyper-responsiveness model despite having highly po-
tent activity for eosinophilia inhibition. Initially we
could not interpret this result; however recent reports
may offer some explanations. A report that multiple
antigen challenge produces pulmonary eosinophilia
without any pulmonary hyper-responsiveness in actively
sensitized guinea pigs shows that eosinophilia is not
always a necessary factor in the airway hyper-respon-
siveness model.8 Also, we have noticed that it is not
eosinophil recruitment (eosinophilia) but eosinophil
activation that is linked to airway hyper-responsive-
ness.9 On the basis of these results, we plan to conduct
further examinations on the eosinophil functions, such
as their activation, with compound 3h and GCC-
AP0341. We have examined the effect of 3h on IL-5-
mediated human eosinophil survival, and 3h manifested
less activity compared to GCC-AP0341 as shown in
Figure 3.10 Further research based on these results is
being conducted.
a
b
Uncorrected. Compounds were analyzed for C, H, N (0.4%
using the formula indicated. c Percent inhibition value on the
In conclusion, we screened several compounds modi-
fied from our previously reported compound GCC-
AP0341, using the Sephadex-induced lung eosinophilia
model, and found a highly potent eosinophilia inhibitor,
2-(4-chlorophenyl)-6-methyl-1,2,4-triazolo[1,5-a]-1,3,5-
triazine-7(6H)-thione (3h ). In the structure 3h , we
found that a thiocarbonyl moiety in the 7 position and
a 4-chlorophenyl moiety in the 3 position of the triazolo-
[1,5-a]-1,3,5-triazine molecule play an important role in
the inhibitory activity on the eosinophilia model. Since
the structure-activity relationships described in our
present study and those obtained for GCC-AP0341 seem
to be quite similar, we suggest that these two com-
pounds should have a common substructure for exerting
the highly potent eosinophilia inhibition. Compound 3h
showed highly potent activity in the Sephadex model
but was less effective on antigen-induced airway hyper-
responsiveness model compared to GCC-AP0341. In
further examinations on the eosinophil functions, such
as their activation, using both 3h and GCC-AP0341 will
be valuable.
Sephadex-induced lung eosinophilia model at an ip dose of 30 mg/
kg.
triazin-7(6H)-one (8), where its thiocarbonyl moiety was
replaced with a carbonyl one in 3a , the inhibitory
activity was decreased. The introduction of methylthio,
amino, or ethoxy substituents at the 5 and/or 7 position
of the triazolo[1,5-a]triazine ring (9, 10a ,b, 11a ,b)
reduced the activity, which indicated that the 1,2,4-
triazole[1,5-a]-1,3,5-triazine-7(6H)-thione moiety is es-
sential for the activity.
Other structure-activity relationships for the 1,2,4-
triazolo[1,5-a]-1,3,5-triazine-7(6H)-thione are shown in
Table 4, and their effects follow. (1) A shorter length
substituent at the 6 position, such as methyl, contrib-
utes to the activity (3a > 3e > 3f). (2) At the 5 position,
introduction of substituents, such as methyl 4a and
phenyl 5a , reduces the activity. (3) Substituents at the
2 position, which are a 4-substituted phenyl moiety,
such as 4-chlorophenyl 3h , 4-trifluoromethyl 3l, 2,4-
dichlorophenyl 3m , and 3,4-dichlorophenyl 3n , contrib-
ute to the activity, and the activity is comparable to that
of GCC-AP0341, whereas the nonsubstituted phenyl
ring 3g completely abolished the activity. The introduc-
tion of an ethoxalyl moiety at the 4 position of the
phenyl ring reduced the activity, which was also ob-
served in the derivatives of GCC-AP0341.1 On the basis
of these results, we selected compound 3h , and a dose-
response study on the Sephadex-induced lung eosino-
philia model was conducted (Figure 1).
Exp er im en ta l Section
Ch em istr y. Melting points were determined with a Yanaco
melting point apparatus and are uncorrected. 1H NMR spectra
were measured on Bruker AC-200 and AMX-500 NMR spec-
trometers with tetramethylsilane as the internal standard;
chemical shifts are given on the δ (ppm) scale. Infrared (IR)
spectra were obtained on a Shimazu IR-420 instrument.
5-Am in o-3-(2-fu r yl)-1-[(m et h yla m in o)t h ioca r b on yl]-
1H-1,2,4-tr ia zole (2a ). To a cooled (0 °C) solution of NaOMe
prepared from sodium (10.9 g, 0.476 mol) and MeOH (150 mL),
was added aminoguanidine hydrochloride (52.6 g, 0.476 mol).
Then methyl 4-furoate (15.0 g, 0.119 mol) in MeOH (100 mL)
was added dropwise to the resultant mixture. The reaction
mixture was stirred at reflux for 24 h and poured into ice-
water. The pH value of the water layer was adjusted to 3-4
with 3 N HCl. The solvent was distilled away under reduced
pressure. MeOH was added to the residue for extraction. The
Like GCC-AP0341, the oral administration of 3h
potently suppressed the eosinophilia with an ID50 value
of 0.3 mg/kg (Figure 1). The efficacy of 3h against
bronchial asthma in antigen (ascaris)-induced hyper-
responsiveness in guinea pigs was performed at the ip
dose of 1 mg/kg as shown in Figure 2.6 The result
showed that 3h suppressed the hyper-responsiveness

Triazolo[1,5-a]triazines Inhibiting Eosinophilia
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 2989
Ta ble 3. Physical Data and Biological Activity of the 1,2,4-Triazolo[1,5-a]-1,3,5-triazine Derivatives
no.
R1
R2
mpa (°C)
formulab
C6H7N5O
C5H6N6S‚0.5H2O
C5H6N6S
inhibitionc (%)
9
H
OEt
108-109
228-233
264-266
248-250
194-195
6
-29
-7
38
10a d
10bd
11a
11b
NH2 (SMe)
SMe (NH2)
NH-Me
SMe (NH2)
NH2 (SMe)
NH-Me
C6H9N7‚0.2H2O
NH-cyclohexyl
NH-cyclohexyl
C
16H25N7
1
a
b
Uncorrected. Compounds were analyzed for C, H, N (0.4% using the formula indicated. c Percent inhibition value on the Sephadex-
d
induced lung eosinophilia model at an ip dose of 30 mg/kg. Structural determination of these isomers (10a ,b) could not be obtained by
spectroscopy.
Ta ble 4. Physical Data and Biological Activity of the 1,2,4-Triazolo[1,5-a]-1,3,5-triazine-7(6H)-thione Derivatives
no.
3a
3e
3f
4a
5a
3g
3h
R1
Me
n-Pro
n-Bu
Me
Me
Me
Me
R2
R3
mpa (°C)
formulab
C5H5N5S
C7H9N5S
C8H11N5S
C6H7N5S
inhibitionc (%)
H
H
H
Me
Ph
H
H
H
H
H
H
Ph
238-240
168-169
132-134
147-148
246-248
>300
69
49
23
18
29
-1
92
C
C
C
11H9N5S
11H9N5S
11H8ClN5S
H
Ph(4-Cl)
>300
(90)d
(38)d
(84)d
79
3i
3j
3k
3l
3m
3n
3o
4b
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
Me
Ph
Ph(4-F)
Ph(4-Br)
>300
C11H8FN5S
C11H8BrN5S
C12H8N6S
C12H8F3N5S
C11H7Cl2N5S
C11H7Cl2N5S
C9H10N6O3S
>300
Ph(4-CN)
Ph(4-CF3)
Ph(2,4-Cl2)
Ph(3,4-Cl2)
NHCOCO2Et
Ph(4-Cl)
>300
270-272
277-279
282-284
280-282
>300
(87)d
(90)d
(89)d
-2
C
12H10ClN5S‚0.2H2O
(60)d
(5)d
(88)d
5b
Ph(4-Cl)
267-270
C17H12ClN5S
GCC-AP0341
a
b
Uncorrected. Compounds were analyzed for C, H, N (0.4% using the formula indicated. c Percent inhibition value on the Sephadex-
d
induced lung eosinophilia model at an ip dose of 30 mg/kg. Value in parentheses is percent inhibition at an ip dose of 3 mg/kg.
F igu r e 1. Dose-response curve of 3h and GCC-AP0341 (ip
or po) on the Sephadex-induced lung eosinophilia model. Each
point was calculated using the same procedure as described
in the Experimental Section.
F igu r e 2. Effect of 3h on the ascaris-induced hyper-
responsiveness model in guinea pigs. Values are the mean (
SE. Statistical analysis was performed using Bonferroni’s
insoluble inorganic salt was removed by filtration. The filtrate
was concentrated under reduced pressure to give 3-amino-5-
(2-furyl)-1H-1,2,4-triazole which was used without further
method. *P < 0.05 vs asthma control. **P < 0.01 vs asthma
purification. To a mixture of the compound obtained above
control.
in 1 N NaOH (30.7 mL) and THF (60 mL) was added methyl
isothiocyanate (11.0 g, 0.15 mol). The reaction mixture was
stirred at room temperature for 1 h and neutralized with 3 N
HCl, and the precipitated solid was collected by filtration. The
solid obtained was chromatographed on silica gel (CHCl3) and
recrystallized from CHCl3 to give white crystals (3.68 g,
16%): mp 173-175 °C; IR (KBr, cm-1) 3300, 3050, 1650, 1620;

2990 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Akahoshi et al.
room temperature for 2 h, MeOH was removed under reduced
pressure and the residue was chromatographed on silica gel
(CHCl3/MeOH, 7/1) to give 3-amino-5-phenoxy-1H-1,2,4-tri-
azole as white crystals (6.84 g, 100%). The procedure used
for the preparation of 2a was repeated with this compound as
a starting material. The crude product obtained was chro-
matographed on silica gel (CHCl3/MeOH, 30/1) and recrystal-
lized from hexane and CHCl3 to give white crystals (41%): mp
184-186 °C; IR (KBr, cm-1) 3280, 3120, 3060, 1660, 1570,
1
1515; H NMR (DMSO-d6) δ 3.00 (s, 3H, CH3), 7.21 (t, 1H, J
) 7.7 Hz, CH), 7.27 (d, 2H, J ) 7.7 Hz, CH), 7.41 (t, 2H, J )
7.7 Hz, CH), 8.48 (brs, 2H, NH2), 9.67 (brs, 1H, NH).
5-Am in o-3-(4-ch lor ost yr yl)-1-[(m et h yla m in o)t h ioca r -
bon yl]-1H-1,2,4-tr ia zole (2g). The procedure used for the
preparation of 2a was repeated with methyl 4-chlorocinnamate
as a starting material. After the neutralization, the precipi-
tated solid was collected by filtration to give white crystals
(32%): mp 209-210 °C; IR (KBr, cm-1) 3330, 3270, 3010, 1655,
F igu r e 3. Effect of 3h and dexamethasone on IL-5-mediated
human eosinophil viability. Each point represents the mean
(SD (n ) 4).
1H NMR (DMSO-d6) δ 3.07 (s, 3H, CH3), 6.67 (d d, 1H, J )
3.4, 1.8 Hz, CH), 7.00 (d, 1H, J ) 3.4 Hz, CH), 7.86 (d, 1H, J
) 1.8 Hz, CH), 8.35 (brs, 2H, NH2), 9.96 (brs, 1H, NH).
5-Am in o-1-[(m eth yla m in o)th ioca r bon yl]-3-(2-th ien yl)-
1H-1,2,4-tr ia zole (2b). The procedure used for the prepara-
tion of 2a was repeated with methyl 2-thiophenecarboxylate
as a starting material. The crude product obtained was
chromatographed on silica gel (CHCl3) to give white crystals
1
1520; H NMR (DMSO-d6) δ 3.06 (s, 3H, CH3), 6.93 (d, 1H, J
) 16.1 Hz, CH), 7.46 (d, 2H, J ) 8.5 Hz, CH), 7.49 (d, 1H, J )
16.1 Hz, CH), 7.68 (d, 2H, J ) 8.5 Hz, CH), 8.25 (brs, 2H, NH2),
9.93 (brs, 1H, NH).
5-Am in o-3-ben zyl-1-[(m eth yla m in o)th ioca r bon yl]-1H-
1,2,4-tr ia zole (2h ). The procedure used for the preparation
of 2a was repeated with methyl phenylacetate as a starting
material. After the neutralization, the precipitated solid was
collected by filtration to give white powdery crystals (24%):
mp 173-175 °C; IR (KBr, cm-1) 3300, 3050, 1635, 1505; 1H
NMR (DMSO-d6) δ 3.03 (s, 3H, CH3), 3.81 (s, 2H, CH2), 7.22
(m, 1H, CH), 7.27-7.32 (m, 4H, CH), 8.14 (brs, 2H, NH2), 9.88
(brs, 1H, NH).
1
(13%): mp 170-172 °C; IR (KBr, cm-1) 3300, 1650, 1520; H
NMR (DMSO-d6) δ 3.08 (s, 3H, CH3), 7.2 (m, 1H, CH), 7.66 (d,
1H, J ) 3.4 Hz, CH), 7.71 (d, 1H, J ) 5.0 Hz, CH), 8.36 (brs,
2H, NH2), 9.91 (brs, 1H, NH).
5-Am in o-3-(6-ch lor o-3-p yr id yl)-1-[(m eth yla m in o)th io-
ca r bon yl]-1H-1,2,4-tr ia zole (2c). To a solution of 6-chlo-
ronicotinic acid (10.0 g, 63.5 mmol) in CH2Cl2 (100 mL) were
added dropwise DMF (10 mL) and thionyl chloride (5.1 mL,
70 mmol). The reaction mixture was stirred at reflux for 1 h
and poured into a solution of aminoguanidine hydrochloride
(24.6 g, 222 mmol) in 2.2 N NaOH (100 mL) under ice cooling.
The precipitated solid was collected by filtration to give
6-chloronicotinic acid 2-amidinohydrazide as a pale-brown
solid (6.87 g, 51%). A mixture of this material (5.40 g, 25.3
mmol) in DMSO (50 mL) was stirred at 180 °C for 1 h. After
the reaction, to this reaction mixture were added 1 N NaOH
(20 mL) and methyl isothiocyanate (1.5 g, 20 mmol). The
reaction mixture was stirred at room temperature for 1 h and
neutralized with 3 N HCl, and the precipitated solid was
collected by filtration. The solid obtained was chromato-
graphed on silica gel (CHCl3) and recrystallized from CHCl3
to give white crystals (2.9 g, 29%): mp 208-211 °C; IR (KBr,
5-Am in o-1-[(m eth yla m in o)th ioca r bon yl]-3-(4-p h en yl-
p h en yl)-1H-1,2,4-tr ia zole (2i). The procedure used for the
preparation of 2a was repeated with methyl 4-phenylbenzoate
as a starting material. The crude product obtained was
chromatographed on silica gel (CHCl3) and recrystallized from
CHCl3 to give white crystals (24%): mp 275-277 °C; IR (KBr,
1
cm-1) 3250, 3000, 1725, 1645, 1520, 1500; H NMR (DMSO-
d6) δ 3.10 (s, 3H, CH3), 7.30-7.58 (m, 3H, CH), 7.75 (d, 2H, J
) 7.0 Hz, CH), 7.82 (d, 2H, J ) 8.4 Hz, CH), 8.14 (d, 2H, J )
8.4 Hz, CH), 8.36 (brs, 2H, NH2), 10.1 (brs, 1H, NH).
5-Am in o-1-[(m eth yla m in o)th ioca r bon yl]-3-(2-p h en yl-
eth yn yl)-1H-1,2,4-tr ia zole (2j). The procedure used for the
preparation of 2a was repeated with methyl phenylpropiolate
as a starting material. The crude product obtained was
chromatographed on silica gel (CHCl3/MeOH, 50/1) and re-
crystallized from CHCl3 to give white crystals (4%): mp 212-
214 °C; IR (KBr, cm-1) 3220, 2220, 1630, 1540, 1500; 1H NMR
(DMSO-d6) δ 3.04 (s, 3H, CH3), 7.44-7.54 (m, 3H, CH), 7.60
(d, 2H, J ) 6.7 Hz, CH), 8.33 (brs, 2H, NH2), 10.1 (brs, 1H,
NH).
1
cm-1) 3300, 3050, 1640, 1520; H NMR (DMSO-d6) δ 3.09 (s,
3H, CH3), 7.69 (d, 1H, J ) 8.0 Hz, CH), 8.37 (d d, 1H, J ) 8.0,
2.0 Hz, CH), 8.42 (brs, 2H, NH2), 8.99 (d, 1H, J ) 2.0 Hz, CH),
10.1 (brs, 1H, NH).
5-Am in o-3-(N-b en zylid en ea m in o)-1-[(m et h yla m in o)-
th ioca r bon yl]-1H-1,2,4-tr ia zole (2k ). A mixture of 3,5-
diamino-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole1 (3.00
g, 17.4 mmol), benzaldehyde (1.99 g, 18.8 mmol), and DL-
camphor-10-sulfonic acid (110 mg, 0.474 mmol) in EtOH (100
mL) was stirred at 60 °C for 5 h. After cooling to room
temperature, the precipitated solid was collected by filtration
to give pale-yellow crystals (2.13 g, 47%): mp 183-185 °C; IR
(KBr, cm-1) 3320, 3180, 1635, 1500; 1H NMR (DMSO-d6) δ 3.05
(s, 3H, CH3), 7.56 (t, 2H, J ) 7.2 Hz, CH), 7.62 (t, 1H, J ) 7.2
Hz, CH), 7.99 (d, 2H, J ) 7.2 Hz, CH), 8.36 (brs, 2H, NH2),
9.16 (s, 1H, CH), 10.1 (brs, 1H, NH).
6-Me t h yl-1,2,4-t r ia zolo[1,5-a ]-1,3,5-t r ia zin e -7(6H )-
th ion e (3a ). A mixture of 5-amino-1-[(methylamino)thiocar-
bonyl]-1H-1,2,4-triazole1 (1.49 g, 9.48 mmol) and diethoxy-
methyl acetate (12 mL) was stirred at 80 °C for 2 h. After
cooling to room temperature, the precipitated solid was col-
lected by filtration to give white crystals (1.39 g, 88%): mp
238-240 °C; IR (KBr, cm-1) 2300, 1585, 1560; 1H NMR
(DMSO-d6) δ 3.35 (s, 3H, CH3), 8.57 (s, 1H, CH), 8.86 (s, 1H,
CH).
5-Am in o-3-(5-ch lor o-2-p yr id yl)-1-[(m eth yla m in o)th io-
ca r bon yl]-1H-1,2,4-tr ia zole (2d ). The procedure used for the
preparation of 2a was repeated with ethyl 5-chloro-2-pyridi-
necarboxylate as a starting material. The crude product
obtained was chromatographed on silica gel (CHCl3/MeOH,
250/1) and recrystallized from CHCl3 to give white crystals
(16%): mp 215-225 °C; IR (KBr, cm-1) 3300, 3050, 1635, 1520;
1H NMR (DMSO-d6) δ 3.09 (s, 3H, CH3), 8.09 (s, 2H, CH), 8.34
(brs, 2H, NH2), 8.72 (s, 1H, CH), 10.1 (brs, 1H, NH).
5-Am in o-3-(4-ch lor o-1-n aph th yl)-1-[(m eth ylam in o)th io-
ca r bon yl]-1H-1,2,4-tr ia zole (2e). The procedure used for the
preparation of 2a was repeated with methyl 4-chloro-1-
naphthoate as a starting material. The crude product obtained
was chromatographed on silica gel (CHCl3) and recrystallized
from CHCl3 to give white crystals (5%): mp 270-272 °C; IR
1
(KBr, cm-1) 3300, 3080, 1660, 1570, 1515; H NMR (DMSO-
d6) δ 3.13 (s, 3H, CH3), 7.62-7.90 (m, 3H, CH), 8.20 (d, 1H, J
) 7.9 Hz, CH), 8.25-8.50 (m, 3H, CH, NH2), 9.21 (m, 1H, CH),
10.1 (brs, 1H, NH).
5-Am in o-1-[(m et h yla m in o)t h ioca r b on yl]-3-p h en oxy-
1H-1,2,4-tr ia zole (2f). To a solution of diphenyl cyanocar-
bonimidate (9.53 g, 38.8 mmol) in MeOH (150 mL) was added
hydrazine (1.5 mL, 48 mmol) at 0 °C. After stirring at 0 °C to
1-Meth ylpu r in e-6(1H)-th ion e (3b). A mixture of 4-amino-
N-methylimidazole-5-carbothioamide1 (1.2 g, 7.7 mmol) and

Triazolo[1,5-a]triazines Inhibiting Eosinophilia
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 2991
triethyl orthoformate (15 mL, 90 mmol) was stirred at 140 °C
for 4 h. After cooling to room temperature, the precipitated
solid was collected by filtration and recrystallized from CHCl3
and MeOH to give gray crystals (340 mg, 27%): mp >285 °C;
IR (KBr, cm-1) 3400, 3000, 1600, 1565; 1H NMR (DMSO-d6) δ
3.90 (s, 3H, CH3), 8.39 (s, 1H, CH), 8.73 (s, 1H, CH), 13.6 (s,
1H, NH).
A solution of 3-amino-1H-1,2,4-triazole (1.61 g, 19.2 mmol)
and dimethyl N-cyanodithioiminocarbonate (3.30 g, 22.6 mmol)
in pyridine (65 mL) was stirred at reflux for 2.5 h. After
removal of the solvent, the crude product obtained was
chromatographed on silica gel (CHCl3/MeOH, 10/1) and re-
crystallized from AcOEt to give yellow crystals (909 mg,
26%): mp 264-266 °C; IR (KBr, cm-1) 3850, 3350, 3100, 2300,
1860, 1650, 1550; 1H NMR (DMSO-d6) δ 2.50 (s, 3H, CH3), 8.34
(s, 1H, CH), 8.84 (s, 2H, NH2).
5-Meth ylp yr a zolo[3,4-d ]p yr im id in e-4(5H)-th ion e (3c).
The procedure used for the preparation of 3a was repeated
with 3-amino-N-methylpyrazole-4-carbothioamide1 as a start-
ing material at 140 °C. After the reaction, the precipitated
solid was collected by filtration to give white crystals (64%):
mp >235 °C; IR (KBr, cm-1) 3400, 3150, 3100, 1605, 1565,
1505; 1H NMR (DMSO-d6) δ 3.83 (s, 3H, CH3), 8.26 (s, 1H,
CH), 8.66 (s, 1H, CH), 14.0 (brs, 1H, NH).
5,7-Bis(m e t h yla m in o)-1,2,4-t r ia zolo[1,5-a ]-1,3,5-t r i-
a zin e (11a ). A mixture of 10a or 10b (928 mg, 5.09 mmol)
and methylamine (30 wt % solution in EtOH, 30 mL) was
stirred in a sealed tube at 125 °C for 20 h. After cooling to
room temperature, the solvent was removed under reduced
pressure. Recrystallization from EtOH gave white crystals
(560 mg, 61%): mp 248-250 °C; IR (KBr, cm-1) 3300, 1640,
1605, 1515; 1H NMR (DMSO-d6, 60 °C) δ 2.83 (brs, 3H, CH3),
2.95 (brs, 3H, CH3), 7.18 (brs, 1H, NH), 8.00 (s, 1H, CH), 8.14
(s, 1H, NH).
3-Met h ylqu in a zolin e-4(3H)-t h ion e (3d ). A mixture of
2-amino-N-methylbenzenecarbothioamide1 (500 mg, 3.01 mmol)
and diethoxymethyl acetate (5 mL) was stirred at room
temperature for 2 h. The reaction mixture was chromato-
graphed on silica gel (hexane/AcOEt, 3/1) and recrystallized
from benzene to give yellow crystals (208 mg, 59%): mp 142-
143 °C; IR (KBr, cm-1) 1600, 1550; 1H NMR (DMSO-d6) δ 3.90
(s, 3H, CH3), 7.64 (d t, 1H, J ) 1.3, 7.5 Hz, CH), 7.75 (d, 1H,
J ) 7.5 Hz, CH), 7.90 (d t, 1H, J ) 1.5, 7.5 Hz, CH), 8.70 (d d,
1H, J ) 1.5, 7.5 Hz, CH), 8.71 (s, 1H, CH).
5-Am in o-1-(a m in oca r bon yl)-1H-1,2,4-tr ia zole (6). A so-
lution of 3-amino-1H-1,2,4-triazole (8.41 g, 0.100 mol) and
potassium cyanate (8.11 g, 0.100 mol) in 0.64 N HCl (159 mL)
was stirred at room temperature for 2 h. After the reaction,
the precipitated crystals were collected by filtration to give a
mixture of 6 and 3-amino-1-(aminocarbonyl)-1H-1,2,4-triazole.
Recrystallization from hot water gave 6 as white crystals (2.95
g, 23%): mp 160-163 °C; IR (KBr, cm-1) 3440, 3360, 3170,
3090, 1740, 1645, 1615, 1515; 1H NMR (DMSO-d6) δ 7.15 (brs,
2H, NH2), 7.51 (s, 1H, CH), 7.56 (brs, 1H, NH), 7.73 (brs, 1H,
NH).
5,7-Bis(cycloh exyla m in o)-1,2,4-tr ia zolo[1,5-a ]-1,3,5-tr i-
a zin e (11b). A mixture of 10a or 10b (2.2 g, 12 mmol) and
cyclohexylamine (12 mL, 105 mmol) was stirred at 90 °C for
19 h. The reaction mixture was chromatographed on silica
gel (hexane/AcOEt, 1/3) and recrystallized from AcOEt to give
white crystals (594 mg, 9.3%): mp 194-195 °C; IR (KBr, cm-1
3230, 2910, 2850, 1640, 1600, 1570; H NMR (DMSO-d6, 60
°C) δ 1.18-1.90 (m, 20H, CH2), 3.95 (m, 2H, CH), 5.12 (brs,
1H, NH), 5.94 (brs, 1H, NH), 7.92 (s, 1H, CH).
)
1
6-n -P r op yl-1,2,4-t r ia zolo[1,5-a ]-1,3,5-t r ia zin e-7(6H )-
th ion e (3e). The procedure used for the preparation of 3a
was repeated with 5-amino-1-[(n-propylamino)(thiocarbonyl)]-
1H-1,2,4-triazole1 as a starting material at room temperature.
After the reaction, the precipitated solid was collected by
filtration to give white crystals (90%): mp 168-169 °C; IR
(KBr, cm-1) 3050, 2970, 1585, 1560; 1H NMR (DMSO-d6) δ 0.94
(t, 3H, J ) 7.5 Hz, CH3), 1.85 (sext, 2H, J ) 7.5 Hz, CH2),
4.39 (d d, 2H, J ) 6.3, 7.4 Hz, CH2), 8.57 (s, 1H, CH), 8.88 (s,
1H, CH).
1,2,4-Tr ia zolo[1,5-a ]-1,3,5-t r ia zin -7(6H)-on e (7). The
procedure used for the preparation of 3a was repeated with 6
as a starting material at 100 °C. After the reaction, the
precipitated solid was collected by filtration and recrystallized
from MeOH to give white crystals (57%): mp 247 °C dec; IR
(KBr, cm-1) 3370, 3200, 2620, 1765, 1600, 1560; 1H NMR
(DMSO-d6) δ 8.39 (s, 1H, CH), 8.41 (s, 1H, CH), 13.3 (brs, 1H,
NH).
6-n -Bu t yl-1,2,4-t r ia zolo[1,5-a ]-1,3,5-t r ia zin e -7(6H )-
th ion e (3f). The procedure used for the preparation of 3a was
repeated with 5-amino-1-[(n-butylamino)(thiocarbonyl)]-1H-
1,2,4-triazole1 as a starting material at room temperature.
After the reaction, the precipitated solid was collected by
filtration to give white crystals (66%): mp 132-134 °C; IR
1
(KBr, cm-1) 3080, 3040, 2970, 1585, 1560; H NMR (DMSO-
6-Meth yl-1,2,4-tr iazolo[1,5-a ]-1,3,5-tr iazin -7(6H)-on e (8).
To a cooled (0 °C) solution of 7 (637 mg, 4.65 mmol) in Et2O
(10 mL) was added an ethereal solution of diazomethane (0.6
M, 26 mL). The mixture was stirred at 0 °C to room
temperature for 17 h. The precipitated solid was collected by
filtration to give yellow crystals (592 mg, 84%): mp 223-226
°C dec; IR (KBr, cm-1) 3090, 3060, 1760, 1600, 1555; 1H NMR
(DMSO-d6) δ 3.54 (s, 3H, CH3), 8.42 (s, 1H, CH), 8.61 (s, 1H,
CH).
d6) δ 0.93 (t, 3H, J ) 7.4 Hz, CH3), 1.38 (sext, 2H, J ) 7.4 Hz,
CH2), 1.80 (m, 2H, CH2), 4.43 (t, 2H, J ) 7.6 Hz, CH2), 8.57 (s,
1H, CH), 8.88 (s, 1H, CH).
5,6-Dim eth yl-1,2,4-tr ia zolo[1,5-a ]-1,3,5-tr ia zin e-7(6H)-
th ion e (4a ). A mixture of 5-amino-1-[(methylamino)thiocar-
bonyl]-1H-1,2,4-triazole1 (2.00 g, 12.7 mmol) and triethyl
orthoacetate (13 mL, 71 mmol) was stirred at 140 °C for 3 h.
After cooling to room temperature, the precipitated solid was
collected by filtration to give pale-brown crystals (497 mg,
7-Eth oxy-1,2,4-tr ia zolo[1,5-a ]-1,3,5-tr ia zin e (9). A mix-
ture of 6 (1.69 g, 13.3 mmol) and triethyl orthoformate (20 mL,
0.12 mol) was stirred at 140 °C for 20 h. After cooling to room
temperature, the reaction mixture was concentrated under
reduced pressure. The crude product obtained was chromato-
graphed on silica gel (CHCl3/MeOH, 30/1) and recrystallized
from CHCl3 to give white crystals (1.44 g, 60%): mp 108-109
1
22%): mp 147-148 °C; IR (KBr, cm-1) 3530, 3430, 1585; H
NMR (DMSO-d6) δ 2.71 (s, 3H, CH3), 3.96 (s, 3H, CH3), 8.51
(s, 1H, CH).
6-Met h yl-5-p h en yl-1,2,4-t r ia zolo[1,5-a ]-1,3,5-t r ia zin e-
7(6H)-th ion e (5a ). The procedure used for the preparation
of 4a was repeated with 5-amino-1-[(methylamino)thio-
carbonyl]-1H-1,2,4-triazole1 and trimethyl orthobenzoate as a
starting material. After the reaction, the precipitated solid
was collected by filtration and recrystallized from MeOH to
1
°C; IR (KBr, cm-1) 2990, 1740, 1600, 1550; H NMR (DMSO-
d6) δ 1.33 (t, 3H, J ) 7.2 Hz, CH3), 4.04 (q, 2H, J ) 7.2 Hz,
CH2), 8.42 (s, 1H, CH), 8.66 (s, 1H, CH).
give white crystals (40%): mp 246-248 °C; IR (KBr, cm-1
)
5-Am in o-7-(m eth ylth io)-1,2,4-tr ia zolo[1,5-a ]-1,3,5-tr ia z-
in e (10a ) or 7-Am in o-5-(m eth ylth io)-1,2,4-tr ia zolo[1,5-a ]-
1,3,5-tr ia zin e (10b). A solution of 3-amino-1H-1,2,4-triazole
(1.55 g, 18.5 mmol) and dimethyl N-cyanodithioiminocarbonate
(3.61 g, 24.7 mmol) in pyridine (75 mL) was stirred at 50 °C
for 36 h. After cooling to room temperature, the precipitated
solid was collected by filtration to give white crystals (211 mg,
8.4%): mp 228-233 °C; IR (KBr, cm-1) 3060, 1690, 1665, 1590,
1540; 1H NMR (DMSO-d6) δ 2.49 (s, 3H, CH3), 8.89 (s, 2H,
NH2), 9.01 (s, 1H, CH).
3410, 1590; 1H NMR (DMSO-d6) δ 3.73 (s, 3H, CH3), 7.57-
7.66 (m, 3H, CH), 7.72 (m, 2H, CH), 8.61 (s, 1H, CH).
6-Met h yl-2-p h en yl-1,2,4-t r ia zolo[1,5-a ]-1,3,5-t r ia zin e-
7(6H)-th ion e (3g). The procedure used for the preparation
of 3a was repeated with 5-amino-1-[(methylamino)thiocarbo-
nyl]-3-phenyl-1H-1,2,4-triazole1 as a starting material at room
temperature. After the reaction, the precipitated solid was
collected by filtration to give white crystals (92%): mp >300
1
°C; IR (KBr, cm-1) 1595, 1585, 1560; H NMR (DMSO-d6) δ

2992 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Akahoshi et al.
3.85 (s, 3H, CH3), 7.55-7.59 (m, 3H, CH), 8.19 (m, 2H, CH),
8.88 (s, 1H, CH).
mp 280-282 °C; IR (KBr, cm-1) 3400, 3300, 1725, 1600, 1580,
1545; 1H NMR (DMSO-d6) δ 1.31 (t, 3H, J ) 7.1 Hz, CH3),
3.82 (s, 3H, CH3), 4.32 (q, 2H, J ) 7.1 Hz, CH), 8.86 (s, 1H,
CH), 11.9 (brs, 1H, NH).
2-(4-Ch lor op h en yl)-6-m eth yl-1,2,4-tr ia zolo[1,5-a ]-1,3,5-
tr ia zin e-7(6H)-th ion e (3h ). The procedure used for the
preparation of 3a was repeated with 5-amino-3-(4-chloro-
phenyl)-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole1 as a
starting material at 90 °C. After the reaction, the precipitated
solid was collected by filtration to give white crystals (96%):
mp >300 °C; IR (KBr, cm-1) 3040, 1585, 1555; 1H NMR
(DMSO-d6) δ 3.85 (s, 3H, CH3), 7.64 (d, 2H, J ) 8.5 Hz, CH),
8.19 (d, 2H, J ) 8.5 Hz, CH), 8.89 (s, 1H, CH).
2-(4-F lu or op h en yl)-6-m eth yl-1,2,4-tr ia zolo[1,5-a ]-1,3,5-
tr ia zin e-7(6H)-th ion e (3i). The procedure used for the
preparation of 3a was repeated with 5-amino-3-(4-fluoro-
phenyl)-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole1 as a
starting material at 90 °C. After the reaction, the precipitated
solid was collected by filtration to give white crystals (96%):
mp >300 °C; IR (KBr, cm-1) 1600, 1565; 1H NMR (DMSO-d6)
δ 3.84 (s, 3H, CH3), 7.41 (t, 2H, J ) 8.5 Hz, CH), 8.23 (d d,
2H, J ) 6.0, 8.5 Hz, CH), 8.90 (s, 1H, CH).
2-(4-Br om op h en yl)-6-m eth yl-1,2,4-tr ia zolo[1,5-a ]-1,3,5-
tr ia zin e-7(6H)-th ion e (3j). The procedure used for the
preparation of 3a was repeated with 5-amino-3-(4-bromo-
phenyl)-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole1 as a
starting material. After the reaction, the precipitated solid
was collected by filtration to give white crystals (97%): mp
>300 °C; IR (KBr, cm-1) 3400, 3010, 1600, 1550, 1505; 1H NMR
(DMSO-d6) δ 3.82 (s, 3H, CH3), 7.78 (d, 2H, J ) 8.5 Hz, CH),
8.12 (d, 2H, J ) 8.5 Hz, CH), 8.90 (s, 1H, CH).
2-(4-Cya n op h en yl)-6-m eth yl-1,2,4-tr ia zolo[1,5-a ]-1,3,5-
tr ia zin e-7(6H)-th ion e (3k ). The procedure used for the
preparation of 3a was repeated with 5-amino-3-(4-cyano-
phenyl)-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole1 as a
starting material. After the reaction, the precipitated solid
was collected by filtration to give white crystals (90%): mp
>300 °C; IR (KBr, cm-1) 3400, 2210, 1600, 1550; 1H NMR
(DMSO-d6) δ 3.85 (s, 3H, CH3), 8.05 (d, 2H, J ) 8.5 Hz, CH),
8.35 (d, 2H, J ) 8.5 Hz, CH), 8.93 (s, 1H, CH).
2-(4-Ch lor op h en yl)-5,6-d im et h yl-1,2,4-t r ia zolo[1,5-a ]-
1,3,5-tr ia zin e-7(6H)-th ion e (4b). A mixture of 5-amino-3-
(4-chlorophenyl)-1-[(methylamino)thiocarbonyl]-1H-1,2,4-tri-
azole1 (1.50 g, 5.60 mmol), triethyl orthoacetate (10 mL, 53
mmol), and acetic acid (0.5 mL) was stirred at 140 °C for 5 h.
After cooling to room temperature, the precipitated solid was
collected by filtration and chromatographed on silica gel
(CHCl3/MeOH, 100/1) to give white crystals (910 mg, 56%):
mp >300 °C; IR (KBr, cm-1) 1590; 1H NMR (TFA-d) δ 2.96 (s,
3H, CH3), 4.19 (s, 3H, CH3), 7.66 (d, 2H, J ) 8.4 Hz, CH), 8.10
(d, 2H, J ) 8.4 Hz, CH).
2-(4-Ch lor op h en yl)-6-m et h yl-5-p h en yl-1,2,4-t r ia zolo-
[1,5-a ]-1,3,5-tr iazin e-7(6H)-th ion e (5b). The procedure used
for the preparation of 4b was repeated with trimethyl orthoben-
zoate as a starting material. The precipitated solid was
collected by filtration, chromatographed on silica gel (CHCl3/
MeOH, 100/1), and recrystallized from CHCl3 to give white
crystals (1.26 g, 59%): mp 267-270 °C; IR (KBr, cm-1) 1580,
1
1575; H NMR (DMSO-d6) δ 3.75 (s, 3H, CH3), 7.59-7.67 (m,
5H, CH), 7.74 (d, 2H, J ) 8.4 Hz, CH), 8.22 (d, 2H, J ) 8.4
Hz, CH).
Sep h a d ex-In d u ced Lu n g Eosin op h ilia . Sephadex G-200
(fine) particles (Pharmacia) in sterilized saline (0.5 mg/mL)
were fully swollen by boiling in water for 5 h, and 1 mL of the
suspension was injected into each rat (Male, Wistar strain,
five rats in each group) through a tail vein on days 0, 3, and
5. A drug or vehicle was ip administered 10 min before
Sephadex injections. On day 7 each rat was sacrificed by CO2
gas, and the trachea were cannulated. Three to four milliliters
of phosphate-buffered saline (PBS) containing 6 units/mL
heparin prewarmed to 37 °C was injected into the airways from
a syringe connected to the cannula, and the solution was
collected into a conical tube put on ice. The same procedure
was repeated twice, and the solution was combined together
followed by centrifugation at 150g for 10 min. The cell pellet
thus obtained was suspended in 0.5 mL of RPMI-1640, and
the suspension was diluted 10-fold with Hinkelman reagent.
The number of eosinophils and the number of total cells were
counted under a microscope. The number of rats used for a
compound or vehicle group was five, and a mean value in each
group was applied to the equation to calculate inhibitory
activity. The inhibitory activity of a compound was calculated
from the following equation: inhibition (%) ) 100 - [(no. of
eosinophils/no. of total cells)compound/(no. of eosinophils/no.
of total cells)vehicle] × 100.
2-(4-(Tr iflu or om eth yl)p h en yl)-6-m eth yl-1,2,4-tr ia zolo-
[1,5-a ]-1,3,5-tr ia zin e-7(6H)-th ion e (3l). The procedure used
for the preparation of 3a was repeated with 5-amino-3-(4-
(trifluoromethyl)phenyl)-1-[(methylamino)thiocarbonyl]-1H-
1,2,4-triazole as a starting material at 90 °C. After the
reaction, the precipitated solid was collected by filtration to
give white crystals (93%): mp 270-272 °C; IR (KBr, cm-1
)
1600, 1580, 1530; 1H NMR (DMSO-d6) δ 3.85 (s, 3H, CH3), 7.95
(d, 2H, J ) 8.0 Hz, CH), 8.39 (d, 2H, J ) 8.0 Hz, CH), 8.92 (s,
1H, CH).
2-(2,4-Dich lor op h en yl)-6-m et h yl-1,2,4-t r ia zolo[1,5-a ]-
1,3,5-tr ia zin e-7(6H)-th ion e (3m ). The procedure used for
the preparation of 3a was repeated with 5-amino-3-
(2,4-dichlorophenyl)-1-[(methylamino)thiocarbonyl]-1H-1,2,4-tri-
azole1 as a starting material. After the reaction, the precipi-
tated solid was collected by filtration to give white crystals
(84%): mp 277-279 °C; IR (KBr, cm-1) 3350, 3005, 1730, 1600,
Air way Hyper sen sitivity in Actively Sen sitized Gu in ea
P igs. Male guinea pigs (Hartley strain) were actively sensi-
tized by the ip injection of ascaris antigen (20 µg) suspended
in a silica gel on days 0 and 14. Three weeks after the first
sensitization, ascaris antigen (0.25 µg/mL) was inhaled into
the airway of guinea pigs by the ultrasonic neblizar, and 1
week later a similar procedure was carried out. 3h or vehicle
was ip injected for 4 consecutive days. Metpyron (50 mg/kg,
ip) was injected 24 and 4 h before carbachol inhalation;24 h
after the second inhalation, carbachol at a concentration of
40, 80, or 120 µg/mL was inhaled in a chamber to induce
bronchoconstriction, which was monitored for the detection of
airway resistance (Rrs). The concentration of the constrictor
to be inhaled was increased step by step from 40 to 120 µg/
mL after checking that Rrs had returned to baseline level. As
a control animal, a carbachol-inhaled animal with no sensiti-
zation was used.
Eosin op h il Su r viva l Assa y. Human eosinophils obtained
from heparinized peripheral blood were purified by centrifuga-
tion through six discontinuous Metrizamide gradients as
described before,8 and the purity was more than 90%. The
purified cells were then transferred to a 96-well microplate
after washing them thoroughly with Tyrode solution contain-
ing 0.1% gelatin followed by suspension in RPMI-1640 supple-
mented with 10% fetal calf serum and 25 mM HEPES (1.25 ×
1
1540, 1500; H NMR (DMSO-d6) δ 3.85 (s, 3H, CH3), 7.64 (d
d, 1H, J ) 8.4, 2.0 Hz, CH), 7.86 (d, 1H, J ) 2.0 Hz, CH), 8.02
(d, 1H, J ) 8.4 Hz, CH), 8.92 (s, 1H, CH).
2-(3,4-Dich lor op h en yl)-6-m et h yl-1,2,4-t r ia zolo[1,5-a ]-
1,3,5-tr ia zin e-7(6H)-th ion e (3n ). The procedure used for the
preparation of 3a was repeated with 5-amino-3-(3,4-dichlo-
rophenyl)-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole1 as
a starting material. After the reaction, the precipitated solid
was collected by filtration to give white crystals (80%): mp
282-284 °C; IR (KBr, cm-1) 3400, 3050, 1600, 1565, 1550,
1
1500; H NMR (DMSO-d6) δ 3.85 (s, 3H, CH3), 7.85 (d, 1H, J
) 8.4 Hz, CH), 8.13 (d d, 1H, J ) 8.4, 1.9 Hz, CH), 8.28 (d,
1H, J ) 1.9 Hz, CH), 8.92 (s, 1H, CH).
2-(Eth oxa lyla m in o)-6-m eth yl-1,2,4-tr ia zolo[1,5-a ]-1,3,5-
tr ia zin e-7(6H)-th ion e (3o). The procedure used for the
preparation of 3a was repeated with 5-amino-3-(ethoxal-
ylamino)-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole as a
starting material at 90 °C. After the reaction, the precipitated
solid was collected by filtration to give white crystals (78%):

Triazolo[1,5-a]triazines Inhibiting Eosinophilia
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 2993
105 cells/mL). The inoculated cells were maintained in the
presence or absence of recombinant human IL-5 (Genzyme)
for 4 days in an atmosphere of 5% CO2 in air. The ratio of
viable cells was determined by the trypan blue exclusion test.
3h or dexamethasone dissolved in DMSO was added to the
microplate at the time of inoculation, and care was taken so
that the concentration of DMSO might not exceed 0.5%.
(3) Mullican, M. D.; Wilson, M. W.; Connor, D. T.; Kostlan, C. R.;
Schrier, D. J .; Dyer, R. D. Design of 5-(3,5-di-tert-butyl-4-
hydroxyphenyl)-1,3,4-thiadiazoles, -1,3,4-oxadiazoles, and -1,2,4-
triazoles as orally active, nonulcerogenic antiinflammatory
agents. J . Med. Chem. 1993, 36, 1090-1099.
(4) Webb, R. L.; Labaw, C. S. Diphenyl cyanocarbonimidate. A
versatile synthon for the construction of heterocyclic ststems.
J . Heterocycl. Chem. 1982, 19, 1205-1206.
(5) Hirata, T.; Twanmoh, L.; Wood, H. B.; Goldin, A.; Driscoll, J . S.
5-Azapurines and the structures of sym-triazole intermediates.
J . Heterocycl. Chem. 1972, 9, 99-106.
(6) Inoue, H.; Aratani, H.; Takishima, T.; Kagoshima, M.; Iwahisa,
Y.; Terasawa, M. Effect of a potent PAF antagonist, Y-24180,
on an allergen-induced late asthmatic response in guinea-pigs.
J . Lipid Mediat. 1992, 5, 173-176.
(7) Teixeira, M. M.; Williams, T. J .; Hellewell, P. G. Mechanisms
and pharmacological manipulation of eosinophil accumulation
in vivo. Trends Pharmacol. Sci. 1995, 16, 418-423.
(8) Lefort, J .; Bachelet, C.-M.; Leduc, D.; Vargaftig, B. B. Effects of
antigen provocation of IL-5 transgenic mice on eosinophil
mobilization and bronchial hyperresponsiveness J . Allergy Clin.
Immunol. 1996, 97, 788-799.
(9) Watson, J . W.; Cohan, V. L.; Shay, A. K.; Freiert, K. W. Multiple
antigen challenge produces pulmonary eosinophilia but not
pulmonary hyperresponsiveness in actively sensitized guinea
pigs. Agents Actions 1992, 37, 174-177.
(10) Wallen, N.; Kita, H.; Weiler, D.; Gleich, G. J . Glucocorticoids
inhibit cytokine-mediated eosinophil survival. J . Immunol. 1991,
147, 3490-3495.
Refer en ces
(1) Naito, Y.; Akahoshi, F.; Takeda, S.; Okada, T.; Kajii, M.;
Nishimura, H.; Sugiura, M.; Fukaya, C.; Kagitani, Y. Synthesis
and pharmacological activity of triazole derivatives inhibiting
eosinophilia. J . Med. Chem. 1996, 39, 3019-3029.
(2) (a) Spicer, B. A.; Baker, R. C.; Hatt, P. A.; Laycock, S. M.; Smith,
H. The effects of drugs on Sephadex-induced eosinophilia and
lung hyper-responsiveness in the rat. Br. J . Pharmacol. 1990,
101, 821-828. (b) Laycock, S. M.; Smith, H.; Spicer, B. A. Airway
hyper-reactivity and blood, lung and airway eosinophilia in rats
treated with Sephadex particles. Int. Arch. Allergy Appl. Im-
munol. 1986, 81, 363-367. (c) Spicer, B. A.; Baker, R.; Laycock,
S. M.; Smith, H. Correlation between blood eosinophilia and
airways hyper-responsiveness in rats. Agents Actions 1989, 26,
63-65. (d) Cook, R. M.; Musgrove, N. R.; Smith, H. Eosinophils
and the granulomatous reaction in rats injected with Sephadex
particles. Pulm. Pharmacol. 1989, 2, 185-190. (e) Desreumaux,
P.; Capron, M. Eosinophils in allergic reactions. Curr. Opin.
Immunol. 1996, 8, 790-795.
J M970759U