Novel antiallergic and antiinflammatory agents. Part I: Synthesis and pharmacology of glycolic amide derivatives

Article abstract of DOI:10.1016/S0968-0896(98)00065-0

A series of mono-glycoloylamino derivatives was synthesized by treatment of the corresponding aromatic monoamine derivatives with glycoloyl chloride derivatives in pyridine or dichloromethane, in the presence of a base such as triethylamine or pyridine. Hydrolysis of acetoxy compounds in aqueous ammonia and methanol solution produced hydroxy derivatives with ease. These compounds were tested in the rat PCA (passive cutaneous anaphylaxis) assay by oral administration. Thiazole and thiadiazole derivatives showed moderate inhibition in this assay. In contrast, benzothiazole and benzonitrile derivatives exhibited marked inhibition. In particular, compound 5t also showed marked inhibition of eosinophil adhesion to TNF (tumor necrosis factor) -α-treated HUVEC (human umbilical vein endothelial cells) in the range of 10^-8-10^-5M. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00065-0

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1069±1076
Novel Antiallergic and Antiin¯ammatory Agents. Part I:
Synthesis and Pharmacology of Glycolic Amide Derivatives
Masakazu Ban, Hiroaki Taguchi,* Takeo Katsushima, Mitsuru Takahashi,
Kiyotaka Shinoda, Akihiko Watanabe and Takanari Tominaga
Pharmaceuticals Research Center, TOYOBO Co., Ltd, 2-1-1 Katata, Ohtsu, Shiga 520-0292, Japan
Received 2 February 1998; accepted 27 March 1998
AbstractÐA series of mono-glycoloylamino derivatives was synthesized by treatment of the corresponding aromatic
monoamine derivatives with glycoloyl chloride derivatives in pyridine or dichloromethane, in the presence of a base
such as triethylamine or pyridine. Hydrolysis of acetoxy compounds in aqueous ammonia and methanol solution
produced hydroxy derivatives with ease. These compounds were tested in the rat PCA (passive cutaneous anaphylaxis)
assay by oral administration. Thiazole and thiadiazole derivatives showed moderate inhibition in this assay. In con-
trast, benzothiazole and benzonitrile derivatives exhibited marked inhibition. In particular, compound 5t also showed
marked inhibition of eosinophil adhesion to TNF (tumor necrosis factor) -a-treated HUVEC (human umbilical vein
8
endothelial cells) in the range of 10
10 5 M. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
compounds10,11 having other aromatic rings, and inves-
tigated these compounds in the PCA assay and for their
e€ects on eosinophil adhesion to TNF (tumor necrosis
factor)-a-treated HUVEC. In particular, 3-amino-4-
chloro-5-hydroxyacetylaminobenzonitrile (5t) inhibited
the PCA reaction as strongly as ketotifen fumarate and
Recently, eosinophils have come to be thought of as
contributing to the pathogenesis of allergic disease such
as asthma and atopic dermatitis. Eosinophils accumu-
late in the lungs and nasal airway after allergic chal-
lenges.1±3 The initial phase in the process of eosinophil
accumulation is the adhesion of eosinophils to endothe-
lial cells.4,5 Many antiallergic drugs have been developed
but few drugs causing the adhesion of eosinophil to
HUVEC (human umbilical vein endothelial cells) have
been investigated.6 To develop a new antiallergic and
antiin¯ammatory drug, we have investigated a series of
amide compounds. In a previous paper,7 we reported
that bis(glycoloylamino)pyrimidine derivatives showed
marked inhibition in the rat PCA (passive cutaneous
anaphylaxis) assay.8 These compounds possess two
active sites in their molecules as does DSCG (disodium
cromoglycate)9 (Figure 1). Though they have neither
anti-histamine nor histamine release inhibitory e€ects at
less than 10 4 M, they strongly inhibit the PCA reaction
at 30 mg/kg p.o. In order to search for a more active com-
pound in vivo, we synthesized new mono-glycoloylamino
showed marked inhibition of eosinophil adhesion to
8
TNF-a-treated HUVEC in the range of 10
10 5 M.
It is a metabolite of TYB-2285 (Figure 1), which is
under investigation in Japan for asthma and atopic
dermatitis in phase II clinical studies. In this paper, the
synthesis, structure±activity relationships and some
pharmacological evaluations of this series of com-
pounds are described.
Chemistry
The compounds shown in Table 1 were prepared by
treatment of ®ve-membered heterocyclic amines with
methoxyacetyl chloride or acetoxyacetyl chloride in
pyridine or dichloromethane in the presence of base
such as triethylamine or pyridine (General method A,
Scheme 1). Starting amines were commercially available.
Hydroxy compound 5h was prepared by hydrolysis of
acetoxyacetyl compound 4h in aqueous ammonia and
methanol solution (General method B, Scheme 1). The
*Corresponding author. Tel: 0775-21-1488; Fax: 0775-21-7132;
E-mail: taguchi@kt.toyobo.co.jp
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00065-0

1070
M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1069±1076
Figure 1. Structures of DSCG and TYB-2285.
Table 1. Physical and pharmacological data of thiazole, thiadiazole, and benzothiazole derivatives
Compd no.
X
Y
R
mp (ꢀC)
Formula
Yield (%) Recryst. Rat PCA inhibition
solventa
(%) 30 mg/kg p.o.
3a
3b
3c
3d
3e
3f
CH
CPh
CH Me
CH Me
107
115
116
220
100
261
111
215
208
C6H8N2O2S
73
79
58
24
78
44
65
77
90
A
B
B
A
B
C
C
A
A
50
72
C
12H12N2O2S
11H11N3O2S
CPh
N
N
Me
C
52
55
CCH3 Me
Me
C6H9N3O2S
C10H10N2O2S
C8H2O5S
C-CH=CH-CH=CH-C
CCOCOOH
96
N Me
CPh Me
NA
NA
84
3g
4h
5h
CNHCOCH2OMe
C-CH=CMe-CMe=CH-C
C-CH=CMe-CMe=CH-C
ketotifen fumarate
C15H17N3O4S
C13H14N3O3S
Ac
H
C
11H12N2O2S
80
95
aA: EtOH; B: EtOH/H2O; C: EtOAc/Hexane.
NA, Not active.
Scheme 1. Synthesis of thiazole, thiadiazole, and benzothiazole derivatives.
compounds shown in Table 2 were prepared in the same
manner as the ®ve-membered heterocyclic amides except
for compounds 3t, 4t and 5t (General method A,
Scheme 2). Starting amines were prepared by the
method of Klaubert et al.12 Compound 4k containing
an amino group was prepared by the reduction of the
corresponding nitro compound 4j. Hydroxyacetylamino
compounds 5m, 5n, 5o, 5q and 5r were easily synthesized
in the same manner as 5h (General method B, Scheme 2).
Compounds 3t and 4t were synthesized by the Schotten±
Baumann method (General method C, Scheme 3). The
monoamide compound was selectively obtained without
bisamide formation in a two phase system of ether and
1N NaOH(aq). In pyridine or dichloromethane in the
presence of triethylamine, a mixture of monoamide,
bisamide and starting amine was formed. Hydroxy
compound 5t was prepared in the same manner as 5h
(General method B, Scheme 3).

M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1069±1076
1071
Table 2. Physical and pharmacological data of benzene glycolic amides
Compd no.
X
Y
R
mp (ꢀC)
Formula
Yield (%) Recryst.
solventa
Rat PCA inhibition
(%) 30 mg/kg p.o.
4i
CONH2
CONH2
CONH2
CN
OMe
NH2
NHEt
Ac
Ac
Ac
163±165
181±184
162±163
C12H14N2O5
C11H13N3O4
C13H14N3O4
77
24
60
39
61
22
69
84
59
69
72
69
41
87
87
24
56
54
24
54
39
A
A
A
B
B
A
B
A
B
B
B
A
A
A
A
C
D
E
B
F
E
79
8
4k
4l
NA
91
3m
6m
7m
4m
5m
3n
6n
4n
5n
8n
4o
5o
4p
4q
5q
4r
NHMe
NHMe
NHMe
NHMe
NHMe
NHEt
Me 109±111
C
C
C
11H13N3O2
12H15N3O2
16H15N3O2
CN
CN
Et
Ph
Ac
H
133±134
201±202
144±145
160±162
ND
NA
ND
ND
ND
ND
95
CN
CN
C12H13N3O3
C10H11N2O2
C13H15N3O2
C13H17N3O2
CN
CN
Me 118±119
NHEt
NHEt
Et
Ac
H
84±87
110±113
111±112
CN
CN
C13H15N3O3
NHEt
NHEt
C
C
C
11H13N3O2
14HH N3O3
12H12N2O4
93
96
CN
CN
COEt 111-112
17
OMe
OMe
NEt2
Ac
H
131±132
142±145
50±51
131±132
126
30
CN
CN
C10H10N2O3
C15H19N3O3
ND
97
Ac
Ac
H
CN
CN
morpholino
morpholino
piperidino
piperidino
H
C15H17N3O4
C13H15N3O3
98
ND
ND
ND
NA
CN
CN
Ac
H
92
124
C16H19N3O3
C14H17N3O2
C11H13NO4
5r
3s
COOMe
Me
71±72
3t
4t
5t
163±165
185±188
203±205
C10H10N3O2Cl
C11H10N3O3Cl
C9H8N3O2Cl
48
45
83
G
A
A
90
92
93
95
ketotifen fumarate
aA: EtOH; B: EtOA/Hexane; C: Hexane; D: EtOH/Toluene/Hexane; E: EtOH/H2O; F: Toluene/Hexane; G: EtOH/EtOAc.
NA, Not active; ND, Not done.
Results and Discussion
that benzothiazoles (3e, 4h, 5h) inhibit the PCA more
strongly than thiazoles and thiadiazoles. Among these
compounds, 3e inhibited as strongly as ketotifen fuma-
rate. Compounds 3f and 3g were not active. Table 2
shows the activity of benzene derivatives having methoxy-
ethoxy-, phenoxy-, acetoxy-, propionyloxy and hydroxy-
acetylamino groups. In these cases, methoxy-, acetoxy-
E€ects of ®ve-membered heterocyclic methoxy-, acetoxy-
and hydroxyacetylamino compounds are shown in
Table 1. From the table it can be seen that thiazole
derivatives 3a and 3b and thiadiazole derivatives 3c
and 3d inhibit the PCA by oral administration, and

1072
M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1069±1076
Scheme 2. Synthesis of benzene glycolic amides.
Scheme 3. Synthesis of compounds 3t, 4t and 5t.
and hydroxyacetylamino compounds inhibited the PCA
reaction as strongly as ketotifen fumarate. But 2-carba-
moyl derivative 4i was less active in the rat PCA assay
than 2-cyano derivative 4n. Compound 3s, having a
methoxycarbonyl group in its benzene ring was not
active. In the cyano series, it was found that a bigger R
group (ethyl, phenyl) reduced activity in the rat PCA
assay. Hydrogen, methyl and acetyl derivatives have
marked or moderate e€ects. At the six-position, large
amino groups, such as diethylamino and morpholino,
are favored. Compound 4o is less active than its amino
compounds. Compounds 3t, 4t and 5t, m-glycoloylami-
nobenzonitriles also showed marked inhibition in the rat
PCA assay by oral administration.
that compound 5t did not inhibit the adhesion of neu-
8
5
trophil at the same range (10
10 M). Eosinophil
adhesion to TNF-a-treated HUVEC was blocked by
monoclonal antibody (mAb) against VLA-4 (anti-VLA-
4), but not by that against Mac-1 (anti-Mac-1). How-
ever, neutrophil adhesion was blocked by anti-Mac-1,
Table 3. The e€ect of compounds in eosinophil adhesion to
TNF-a-treated HUVEC
Compd
no.
Percentage of cell adhesion
Concentration of test compounds
10 5 M
10 6 M
10 7 M
10 8 M
4q
5o
86
90
NA
NA
NA
63
NA
NA
NA
66*
We investigated the e€ect of these compounds on eosino-
phil adhesion to TNF-a-treated HUVEC. The results
are shown in Table 3. Compounds 4h, 4q and 5o showed
a slight inhibition at 10 5 M and compound 5t showed a
4h
3t
70
60
5t
20***
NA
49**
38*
28***
NA
NA
22**
42***
61**
NA
DSCG
Ketotifena
Tranilast
NA
marked inhibition of eosinophil adhesion to TNF-a-
NA
8
treated HUVEC in the range 10
10 5 M in a con-
centration dependent manner. DSCG did not show an
e€ect at any concentration tested.13 These results indi-
cated that meta-substituted benzonitrile derivatives (3t,
5t) were more active than ortho-substituted ones (4h, 4q,
5o) in this assay. Recently, Tominaga et al.13 showed
NA, Not active.
aKetotifen fumarate.
*p<0.05, **p<0.01, ***p<0.001 versus control (%) adhesion of
eosinophils to TNF-a treated HUVEC in the absence of test
compounds.

M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1069±1076
1073
but not by anti-VLA-4. These results suggest that
compound 5t might block the VLA-4/VCAM-1 path-
way selectively. Compound 5t is a metabolite of TYB-
2285,14 which is being investigated in Japan for asthma
and atopic dermatitis in phase II clinical studies.
intensity of ¯uorescence was measured using an auto-
mated microplate ¯uorometer with 485/22 nM excitation
and a 530/25 nM emission ®lter. HUVEC were incubated
with each compound for 6 h during treatment with
100 U/mL TNF-a, and were washed before the adhesion
assay. Eosinophils were also preincubated for 20 min
with each compound, and were then recoincubated with
HUVEC in the presence of compounds. For the experi-
ments evaluating the compounds, the percentage of con-
trol was calculated according to the following formula:
Recently, Yasuda et al.15 reported that anti-VLA-4 anti-
bodies, in combination with other anti-adhesion molecule
antibodies, inhibited the PCA reaction. We speculate
that 5t and its derivatives might inhibit the PCA reac-
tion by a mechanism related to the inhibition of the
VLA-4/VCAM-1 pathway. Further studies are required
to determine the precise mechanism of these compounds.
Percentage of control=(the percentage adhering to
100 U/mL of TNF-a-treated HUVEC in the presence of
compounds the percentage adhering to untreated
HUVEC)/(the percentage adhering to 100 U/mL of
TNF-a-treated HUVEC in the absence of compounds
the percentage adhering to untreated HUVEC).
Conclusion
We have investigated a number of aromatic glycolic
amide derivatives to ®nd a unique type of antiallergic
and antiin¯ammatory drug. Compound 5t showed anti-
allergic activities in the PCA assay at 30 mg/kg p.o. and
marked inhibitory e€ects on eosinophil adhesion to
General method A. 2-(Methoxyacetylamino)thiazole (3a).
Methoxyacetyl chloride (5.0 mL) was added dropwise to
a solution of 2-aminothiazole (5 g) in pyridine (100 mL)
at room temperature. After the mixture was stirred at
room temperature for 3 h, the solvent was distilled o€
under reduced pressure. Chloroform and water were
added to the resulting oil. The organic layer was washed
with water and saturated sodium chloride solution.
Thereafter, the organic layer was dried over sodium
sulfate and the solvent was removed. The resulting
solids were washed with water. The solids were recrys-
tallized from ethanol to give 6.3 g of 3a; mp 105±
107 ꢀC; 1H NMR (DMSO-d6) d: 11.91 (1H, s), 7.44 (1H,
d, J=6 Hz), 7.18 (1H, d, J=6 Hz), 4.11 (2H, s), 3.33 (3H,
s). Anal. calcd for C6H8N2O2S: C, 41.85; H, 4.68; N, 16.27;
S, 18.62, found: C, 41.74; H, 4.62; N, 16.35; S, 18.68.
8
5
TNF-a-treated HUVEC at 10
10 M. These com-
pounds are anticipated to be a new type of antiallergic
and antiin¯ammatory drug for the treatment of asthma
and atopic dermatitis.
Experimental
Melting points were determined with a Mettler capillary
melting-point apparatus (Model FP 61) and uncor-
1
rected. H NMR spectra were recorded on a Varian FT
80A spectrometer or a Varian Gemini-200 spectrometer
using TMS as an internal standard. Elemental analyses
were performed at Kyoto University and TOYOBO
Analytical Center. All starting materials were commer-
cially available unless otherwise noted.
Compounds 3b±g, m, n, s, 4h±r, 6m, 7m and 8n were
prepared in the same manner as 3a (General method A).
Passive cutaneous anaphylaxs (PCA) assay. Male Wistar
rats (weighing about 200 g) were passively sensitized by
intradermal injection of rat anti-egg albumin antiserum
(100 mL/site) in the dorsal skin. After 48 h, each rat was
challenged by injecting a mixture (1 mL) of egg albumin
and Evans blue solution via the tail vein. Thirty minutes
after the challenge, the rats were sacri®ced to analyze
the bluing region, and the amount of pigment from the
bluing region was measured by the method of
Katayama et al.16 Test compounds were orally given at
30 mg/kg, 30 min before the antigen challenge.
2-(Methoxyacetylamino)-4-phenylthiazole (3b). From 2-
amino-4-phenylthiazole hydrobromide monohydrate
(5.5 g) and methoxyacetyl chloride (2.0 mL): 4.2 g; mp
113±115 ꢀC. 1H NMR (DMSO-d6) d: 12.05 (1H, s),
7.95±7.20 (6H, m), 4.15 (2H, s), 3.35 (3H, s). Anal. calcd
for C12H12N2O2S: C, 58.05; H 4.87; N, 11.28; S, 12.91,
found: C, 57.99; H, 4.89; N, 11.32; S, 12.65.
5-(Methoxyacetylamino)-3-phenyl-1,2,4-thiadiazole (3c).
From 5-amino-3-phenyl-1,2,4-thiadiazole (4.4 g) and
methoxyacetyl chloride (3.4 mL): 3.6 g; mp 113±116 ꢀC.
1H NMR (CDCl3) d: 10.15 (1H, s), 8.30±7.35 (5H, m),
4.15 (2H, s), 3.45 (3H, s). Anal. calcd for C11H11N3O2S:
C, 53.00; H, 4.45; N, 16.86; S, 12.86, found: C, 53.12; H,
4.72; N, 16.86; S, 12.81.
Eosinophil adhesion to TNF-ꢀ-treated HUVEC. CSFE-
labeled eosinophils (1Â105 cells/well) were layered over
HUVEC prestimulated with TNF-a. After 20 min of
incubation, unbound cells were removed by plate inver-
sion in RPMI-1640 for 30 min at room temperature.
After adding 0.3% Triton X to the adherent cells, the
2-(Methoxyacetylamino)-5-methyl-1,3,4-thiadiazole (3d).
From 2-amino-5-methyl-1,3,4-thiadiazole (9.2 g) and

1074
M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1069±1076
methoxyacetyl chloride (5.0 mL): 3.6 g; mp 219±220 ꢀC;
1H NMR (DMSO-d6) d: 12.21 (1H, s), 4.16 (2H, s), 3.35
(3H, s), 2.10 (3H, s). Anal. calcd for C6H9N3O2S: C,
38.49; H, 4.89; N, 22.44; S, 17.12, found: C, 38.34; H,
5.00; N, 22.75; S, 17.22.
(DMSO-d6) d: 10.52 (1H, s), 835±7.95 (3H, m), 4.72 (2H,
s), 2.13 (3H, s). Anal. Calcd for C11H9N3O5: C, 54.05; H,
4.54; N, 12.61, found: C, 54.03; H, 4.56; N, 12.57.
2-(Acetoxyacetylamino)-6-aminobenzamide (4k). Cyclo-
hexene (10.5 m) and 10% palladium-carbon (4.7 g) were
added to a mixture of 2-(acetoxyacetylamino)-6-nitro-
benzonitrile (5.7 g) and ethanol (50 mL). The mixture
was re¯uxed for 20 min, and the reaction mixture was
cooled to room temperature, and the solid materials
were ®ltered o€. The ®ltrate was distilled under reduced
pressure. The residual crude solids were recrystallized
from ethanol to give 1.2 g of 4k, mp 181±184 ꢀC. 1H
NMR (DMSO-d6) d: 10.00 (1H, s), 7.53 (2H, s), 7.03±
6.44 (3H, m), 5.26 (2H, s), 2.14 (3H, s). Anal. calcd for
C11H13N3O4: C, 52.59; H, 5.21; N, 16.73, found: C,
52.57; H, 5.22; N, 16.64.
2-(Methoxyacetylamino)benzothiazole (3e). From 2-ami-
nobenzothiazole(4.5 g) and methoxyacetyl chloride
(3.0 mL): 5.2 g; mp 99±100 ꢀC. H NMR (DMSO-d6) d:
1
11.20 (1H, s), 8.10±7.12 (4H, m), 4.18 (2H, s), 3.36 (1H,
s). Anal. calcd for C10H10N2O2S: C, 54.04; H, 4.53; N,
12.60; S, 14.42, found: C, 54.29; H, 4.48; N, 12.59; S,
14.27.
2-(Methoxyacetylamino)-4-thiazoleglyoxylic acid (3f).
From ethyl 2-amino-4-thiazoleglyoxylate (6.0 g) and
methoxyacetyl chloride (3.0 mL). The resulting oil was
dissolved 1N aqueous sodium hydroxide (30 mL), and
the solution was stirred at room temperature for 3 h.
Thereafter, to the solution was added conc. HCl to be
acidic. The resulting solids were ®ltered and washed
with water. The solids were recrystallized from ethyl
2-(Acetoxyacetylamino)-6-ethylaminobenzamide
(4l).
From 2-amino-6-ethylaminobenzamide (4.4 g) and acet-
oxyacetyl chloride (8.6 mL): 4.1 g; mp 124±125 ꢀC. 1H
NMR (DMSO-d6) d: 9.70 (1H, s), 7.52±7.11 (3H, m),
4.72 (2H, s), 3.36±3.05 (3H, m), 2.10 (3H, s), 1.09 (3H, t,
J=8 Hz). Anal. calcd for C13H14N3O4: C, 55.33; H,
4.93; N, 12.10, found: C, 55.34; H, 4.94; N, 12.09.
acetate/hexane to give 3.2 g of 3f; mp 178 ꢀC (Dec.). H
1
NMR (DMSO-d6) d: 12.46 (1H, s), 8.38 (1H, s), 4.13
(2H, s), 3.33 (3H, s). Anal. calcd for C8H8N2O5S: C,
39.34; H, 3.30; N, 11.47; S, 13.12, found: C, 39.31; H,
3.15; N, 11.61; S, 13.16.
2-(Methoxyacetylamino)-6-methylaminobenzonitrile (3m).
From 2-amino-6-methylaminobenzonitrile (3.3 g) and
methoxyacetyl chloride (4.2 mL): 1.9 g; mp 109±111 ꢀC.
1H NMR (DMSO-d6) d: 9.42 (1H, s), 7.40 (1H, dd,
J=8 Hz and J=8 Hz), 6.92 (1H, d, J=8 Hz), 6.48 (1H,
d, J=8 Hz), 6.20±6.01 (1H, m), 4.02 (2H, s), 3.45 (3H,
s), 2.75 (3H, d, J=5 Hz). Anal. calcd for C11H13N3O2:
C, 60.26; H, 5.98; N, 19.17, found: C, 60.09; H, 5.93; N,
19.46.
2,4-Bis(methoxyacetylamino)-5-phenylthiazole (3g). From
2,4-diamino-5-phenylthiazole monohydrogen bromide
(4.1 g) and methoxyacetyl chloride (3.0 mL): 3.3 g; mp
109±111 ꢀC. H NMR(DMSO-d6) d: 12.15 (1H, s), 9.19
1
(1H, s), 7.65±7.00 (5H, m), 4.14 (2H, s), 3.93 (1H, s),
3.34 (3H, s), 3.31 (3H, s). Anal. calcd for C15H17N3O4S:
C, 53.72; H, 5.12; N, 12.53; S, 9.56, found: C, 53.98; H,
5.15;N, 12.45; S, 9.57.
2-(Ethoxyacetylamino)-6-methylaminobenzonitrile (6m).
From 2-amino-6-methylaminobenzonitrile (3.3 g) and
ethoxyacetyl chloride (5.0 mL): 3.2 g; mp 133±134 ꢀC.
1H NMR (DMSO-d6) d: 9.32 (1H, s), 7.40 (1H, dd,
J=8 Hz and J=8 Hz), 7.14 (1H, d, J=8 Hz), 6.49 (1H,
d, J=8 Hz), 6.36±6.03 (1H, m), 4.03 (2H, s), 3.48 (2H, q,
J=7 Hz ), 2.25 (3H, d, J=5 Hz), 1.22 (3H, t, J=8 Hz).
Anal. calcd for C12H15N3O2: C, 61.79; H, 6.48; N,
18.01, found: C, 61.71; H, 6.37; N, 18.04.
2-(Acetoxyacetylamino)-5,6-dimethylbenzothiazole (4h).
From 2-amino-5,6-dimethylbenzothiazole (4.5 g) and
acetoxyacetyl chloride (3.0 mL): 5.4 g; mp 214±215 ꢀC.
1H NMR (DMSO-d6) d: 12.39 (1H, s), 7.67 (1H, s), 7.51
(1H, s), 4.78 (2H, s), 2.30 (6H, s), 2.13 (3H, s). Anal.
calcd for C13H14N2O3S: C, 56.10; H, 5.07; N, 10.06; S,
11.52, found: C, 56.06; H, 5.07; N, 10.06; S, 11.43.
2-(Acetoxyacetylamino)-6-methoxybenzamide (4i). From
2-amino-6-methoxybenzamide (1.7 g) and acetoxyacetyl
chloride (1.1 mL): 2.1 g; mp 163±165 ꢀC. 1H NMR
(DMSO-d6) d: 11.45 (1H, s), 8.05±6.84 (3H, m), 7.91
(2H, s), 4.60 (2H, s), 3.82 (3H, s), 2.20 (3H, s). Anal.
calcd for C12H14N2O5: C, 54.13; H, 5.30; N, 10.52,
found: C, 54.19; H, 5.36; N, 10.57.
6-Methylamino-2-(phenoxyacetylamino)benzonitrile (7m).
From 2-amino-6-methylaminobenzonitrile (3.3 g) and
phenoxyacetyl chloride (6.2 mL): 1.4 g; mp 201±202 ꢀC.
1H NMR (DMSO-d6) d: 9.76 (1H, s), 7.52±6.48 (8H, m),
6.12 (1H, q, J=5 Hz), 4.68 (2H, s), 2.74 (3H, d,
J=5 Hz). Anal. calcd, for C16H15N3O2: C, 68.31; H,
5.37; N, 14.94, found: C, 68.20; H, 5.38; N, 14.86.
2-(Acetoxyacetylamino)-6-nitrobenzonitrile (4j). From 2-
amino-6-nitrobenzonitrile (6.5 g) and acetoxyacetyl
chloride (4.4 mL): 5.8 g; mp 157±158 ꢀC. 1H NMR
2-(Acetoxyacetylamino)-6-methylaminobenzonitrile (4m).
From 2-amino-6-methylaminobenzonitrile (4.4 g) and

M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1069±1076
1075
1
acetoxyacetyl chloride (6.5 mL): 5.1 g; mp 144±145 ꢀC.
1H NMR (DMSO-d6) d: 9.82 (1H, s), 7.34±6.46 (3H, m),
6.06 (1H, q, J=5 Hz), 4.64 (2H, s), 2.74 (3H, d, J=5 Hz),
2.09 (3H, s). Anal. calcd for C12H13N3O3: C, 58.28; H,
5.30; N, 16.99, found: C, 58.21; H, 5.28; N, 17.08.
acetoxyacetyl chloride (1.8 mL): 1.1 g; mp 50±51 ꢀC. H
NMR (DMSO-d6) d: 9.91 (1H, s), 7.45 (1H, dd, J=8 Hz
and J=8 Hz), 7.06 (1H, d, J=8 Hz) 6.87 (1H, d,
J=8 Hz), 4.66 (2H, s), 3.29 (4H, q, J=8 Hz), 2.12 (3H,
s), 1.08 (6H, t, J=8 Hz). Anal. calcd for C15H19N3O3:
C, 62.27; H, 6.62; N, 14.52, found: C, 62.18; H, 6.63; N,
14.51.
2-Ethylamino-6-(methoxyacetylamino)benzonitrile (3n).
From 2-amino-6-(ethylamino)benzonitrile (3.7 g) and
methoxyacetyl chloride (2.8 mL): 3.2 g; mp 118±119 ꢀC.
1H NMR (DMSO-d6) d: 9.31 (1H, s), 7.35 (1H, dd,
J=8 Hz and J=8 Hz), 6.82 (1H, d, J=8 Hz), 6.54 (1H,
d, J=8 Hz), 5.86 (1H, t, J=5 Hz), 3.94 (2H, s), 3.44
(3H, s), 3.34±3.02 (3H, m), 1.13 (3H, t, J=8 Hz). Anal.
calcd for C12H15N3O2: C, 61.79; H, 6.48; N, 18.01,
found: C, 61.95; H, 6.49; N, 17.99.
2-(Acetoxyacetylamino)-6-morpholinobenzonitrile (4q).
From 2-amino-6-morpholinobenzonitrile (5.6 g) and
acetoxyacetyl chloride (3.3 mL): 4.7 g; mp 131±132 ꢀC.
1H NMR (DMSO-d6) d: 10.07 (1H, s), 7.55 (1H, d,
J=8 Hz), 7.21 (1H, d, J=8 Hz), 6.89 (1H, d, J=8 Hz),
4.68 (2H, s), 3.82±3.58 (4H, m), 3.22±3.00 (4H, m), 2.13
(3H, s). Anal. calcd for C15H17N3O4: C, 59.40; H, 5.65;
N, 13.85, found: C, 59.31; H, 5.60; N, 13.89.
6-(Ethoxyacetylamino)-2-(ethylamino)benzonitrile (6n).
From 2-amino-6-(ethylamino)benzonitrile (4.0 g) and
methoxyacetyl chloride (3.9 mL): 4.1 g; mp 84±87 ꢀC. 1H
NMR (DMSO-d6) d: 9.32 (1H, s), 7.45 (1H, dd, J=8 Hz
and J=8 Hz), 7.18 (1H, d, J=8 Hz), 6.61 (1H, d,
J=8 Hz), 5.97 (1H, m), 4.03 (2H, s), 3.38±3.05 (3H, m),
3.17 (2H, q, J=8 Hz), 1.22 (3H, t, J=8 Hz), 1.15 (3H, t,
J=8 Hz). Anal. calcd forC13H17N3O2: C, 63.14; H, 6.93;
N, 16.99, found: C, 63.10; H, 6.98; N, 16.97.
2-(Acetoxyacetylamino)-6-piperidinobenzonitrile (4r).
From 2-amino-6-piperidinobenzonitrile (8.8 g) and
acetoxyacetyl chloride (3.3 mL): 3.2 g; mp 90±92 ꢀC: H
1
NMR (DMSO-d6) d: 9.99 (1H, s), 7.50 (1H, dd, J=8 Hz
and J=8 Hz), 7.15 (1H, d, J=8 Hz), 6.85 (1H, d,
J=8 Hz), 4.68 (2H, s), 3.25±2.95 (4H, m), 1.91±1.48
(6H, m). Anal. calcd for C16H19N3O3: C, 63.77; H, 6.34;
N, 13.94, found: C, 63.99; H, 6.04; N, 13.99.
2-(Acetoxyacetylamino)-6-(ethylamino)benzonitrile (4n).
From 2-amino-6-(ethylamino)benzonitrile (6.7 g) and
acetoxyacetyl chloride (4.8 mL): 7.8 g; mp 110±113 ꢀC.
1H NMR (DMSO-d6) d: 9.69 (1H, s), 7.19±6.42 (3H, m),
6.02±5.87 (1H, m), 4.52 (2H, s), 3.14±3.01 (2H, m), 2.02
(3H, s), 1.08 (3H, t, J=8 Hz). Anal. calcd for
C13H15N3O3: C, 59.76; H, 5.79; N, 16.08, found: C,
59.79; H, 5.79; N, 16.07.
Methyl 2-(methoxyacetylamino)benzoate (3s). From
methyl anthranilate (4.5 g) and methoxyacetyl chloride
(5.2 mL): 2.6 g; mp 71±72 ꢀC. 1H NMR (DMSO-d6) d:
11.65 (1H, s), 8.85±6.95 (4H, m), 4.05 (2H, s), 3.90 (3H,
s), 3.45 (3H, s). Anal. calcd for C11H13NO4: C, 59.19; H,
5.87; N, 6.28, found: C, 59.11; H, 5.92; N, 6.25.
General method B. 6-Methylamino-2-(hydroxyacetyl-
amino)benzonitrile (5m). To a solution of 4m (2 g) in
methanol (250 mL) was added dropwise 28% aqueous
ammonia (5.5 mL). The mixture was stirred at room
temperature for 1 h. Thereafter, the solvent was distilled
o€ under reduced pressure. The resulting solids were
recrystallized from ethanol to give 1.4 g of 5m; mp 160±
2-Ethylamino-6-(propionyloxyacetylamino)benzonitrile
(8n). From 2-amino-6-(etylamino)benzonitrile (2.3 g)
and propionyloxyacetyl chloride (3.0 mL): 1.6 g; mp
103±105 ꢀC. H NMR (DMSO-d6) d: 9.69 (1H, s), 7.35
1
(1H, dd, J=8 Hz and J=8 Hz), 6.72 (1H, d, J=8 Hz),
6.51 (1H, d, J=8 Hz), 5.86 (1H, t, J=5 Hz), 4.64 (2H,
s), 3.35±2.98 (2H, m), 2.42 (2H, q, J=8 Hz), 1.14 (3H, t,
162 ꢀC. H NMR (DMSO-d6) d: 9.25 (1H, s), 7.50±7.05
1
(2H, m), 6.50±6.28 (1H, m), 6.06 (1H, q, J=4 Hz), 3.95
(2H, s), 2.72 (3H, d, J=4 Hz). Anal. calcd for
C10H11N2O2: C, 58.53; H, 5.40; N, 20.48, found: C,
58.28; H, 5.47; N, 20.43.
J=8 Hz), 1.06 (3H,
t J=8 Hz). Anal. calcd for
C14H17N3O3: C, 61.08; H, 622; N, 15.26, found: C,
61.06; H, 621; N, 15.28.
2-(Acetoxyacetylamino)-6-methoxybenzonitrile
(4o).
Compounds 5h and 5n±r were prepared in the same
manner as 5m (General method B).
From 2-amino-6-methoxybenzonitrile (3.7 g) and acet-
oxyacetyl chloride (2.7 mL): 5.4 g; mp 131±132 ꢀC. 1H
NMR (DMSO-d6) d: 9.94 (1H, s), 7.54±6.96 (3H, m),
4.65 (2H, s), 3.85 (3H, s), 2.10 (3H, s). Anal. calcd for
C12H12N2O4: C, 58.06; H, 4.87; N, 11.28, found: C,
58.02; H, 4.92; N, 11.29.
2-(Hydroxyacetylamido)-5,6-dimethylbenzothiazole (5h).
From the compound 4h (4.2 g): 3.2 g; mp 214±215 ꢀC.
1H NMR (DMSO-d6) d: 11.28 (1H, s), 7.66 (1H, s), 7.49
(1H, s), 5.49 (1H, t, J=7 Hz), 4.13 (2H, d, J=7 Hz),
2.31 (6H, s). Anal. calcd for C11H12N2O2S: C; 55.92; H,
5.12; N, 11.86; S, 13.57, found: C, 55.82; H, 5.19; N,
11.79; S, 13.50.
2-(Acetoxyacetylamino)-6-diethyaminobenzonitrile (4p).
From 2-amino-6-diethylaminobenzonitrile (3.0 g) and

1076
M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1069±1076
2-Ethylamino-6-(hydroxyacetylamino)benzonitrile (5n).
From the compound 4n (4.5 g): 2.6 g; mp 111±112 ꢀC.
1H NMR (DMSO-d6) d: 9.38 (1H, s), 7.19±6.42 (3H, m),
5.91 (1H, m), 3.94 (2H, s), 3.35±3.13 (3H, m), 1.14 (3H,
t, J=7 Hz). Anal. calcd for C11H13N3O2: C, 60.30; H,
5.98; N, 19.18, found: C, 60.19; H, 6.01; N, 19.17.
3-Amino-4-chloro-5-(acetoxyacetylamino)benzonitrile (4t).
From 4-chloro-3,5-diaminobenzonitrile (16.7 g) and
acetoxyacetyl chloride (10.8 mL): 12.1 g; mp 185±188 ꢀC.
1H NMR (DMSO-d6) d: 9.10 (1H, s), 7.27 (1H, d,
J=3 Hz), 6.92 (1H, d, J=3 Hz), 5.80 (2H, s), 4.18 (2H,
s), 2.08 (3H, s). Anal. calcd for C11H10N3O3Cl: C, 49.36;
H, 3.77; N, 15.70; Cl, 13.24, found: C, 49.20; H, 3.78; N,
15.52; Cl, 13.23.
2-(Hydroxyacetylamino)-6-methoxybenzonitrile (5o).
From the compound 4o (2.5 g): 1.8 g; mp 142±145 ꢀC;
1H NMR (DMSO-d6) d: 9.57 (1H, s), 7.72±7.62 (2H, m),
7.22±6.90 (1H, m), 6.06 (1H, s), 4.03 (2H, s), 3.90 (3H,
3-Amino-4-chloro-5-(hydroxyacetylamino)benzonitrile (5t).
From the compound 4t (2.7 g): 1.9 g; mp 203±205 ꢀC. 1H
NMR (DMSO-d6) d: 9.30 (1H, s), 7.72 (1H, d, J=3 Hz),
6.89 (1H, d, J=3 Hz), 6.23 (2H, s), 5.93 (2H, s), 4.02
(2H, s). Anal. calcd for C9H8N3O2Cl: C, 47.91; H, 3.57;
N, 18.62; Cl, 15.71, found: C, 47.67; H, 3.68; N, 18.57;
Cl, 15.78.
s). Anal. calcd for C10H N2O3: C, 58.25; H, 4.88; N,
13.58, found: C, 58.03; H, 4.91; N, 13.59.
10
2-(Hydroxyacetylamino)-6-morpholinobenzonitrile (5q).
From the compound 4q (3.0 g): 1.4 g; mp 126 ꢀC. 1H
NMR (DMSO-d6) d: 9.60 (1H, s), 7.74±7.48 (2H, m),
7.00±6.78 (1H, m), 6.08 (1H, t, J=6 Hz), 4.02 (2H, d,
J=6 Hz), 3.85±3.65 (4H, m), 3.27±3.00 (4H, m). Anal.
calcd for C13H15N3O3: C, 59.76; H, 5.79; N, 16.08,
found: C, 59.84; H, 5.78; N, 16.15.
References
1. Mullarkey, M. F.; Hill, J. S.; Webb, D. R. J. Allergy Clin.
Immunol., 1980, 65, 122±126.
2. De Monchy, J. G. R.; Kau€nan, H. R; Venge, P. Am. Rev.
Respir Dis. 1985, 131, 373±376.
2-(Hydroxyacetylamino)-6-piperidinobenzonitrile
(5r).
3. Burrows, B.; Hasan, F. M.; Barbee, R. A.; Halonen, M.;
Lebowitz, M. D. Ibid. 1980, 120, 709±720.
From the compound 4r (3.0 g): 1.4 g; mp 121±124 ꢀC.
1H NMR (DMSO-d6) d: 9.60 (1H, s), 7.65±7.48 (2H, m),
6.95±6.78 (1H, m), 6.08 (1H, t, J=6 Hz), 4.02 (2H, d,
J=6 Hz), 3.30±2.95 (4H, m), 1.98±1.48 (6H, m). Anal.
calcd for C14H17N3O2: C, 64.85; H, 6.61; N, 16.21,
found. C, 64.89; H, 6.62; N, 16.24.
4. Monterfort, S.; Holgate, S. T. Respir Med., 1991, 85, 91±
100.
5. Sprinnger, T. A. Nature, 1990, 346, 425±434.
6. Kyan-Aung, U.; Hallsworth, M.; Haskard D.; De Vos, C.;
Lee, T. H. J. Allergy Clin. Immunol., 1992, 90, 270±272.
7. submitted for publication.
General method C. 3-Amino-4-chloro-5-(methoxyacetyl-
amino)benzonitrile (3t). To a solution of 4-chloro-3,5-
diaminobenzonitrile (5.5 g) in diethylether (200 mL) was
added 1N sodium hydroxide solution (50 mL) and
thereto was added dropwise methoxyacetyl chloride
(3.0 mL) which was cooled in an ice bath. Thereafter, the
mixture was stirred at room temperature for 15 min. The
resulting solids were ®ltered and washed with water and
diethylether. The solids were recrystallized from etha-
8. Ovary, Z. Progress in Allergy, 1958, 5, 468±474.
9. Cox, J. S.; Beach, J. E; Blair, A. M. J. N.; Clarke, A. J.;
King, J.; Lee, T. B.; Loveday, D. E. E.; Moss, G. F.; Orr, T. S.
C.; Richie, J. T.; Sheard, P. Adv. Drug Res., 1970, 5, 115.
10. Taguchi, H.; Katsushima, T.; Ban, M.; Takahashi, M.;
Shinoda, K.; Watanabe, A. US Patent 5,041,462.
11. Taguchi, H.; Katsushima, T.; Ban, M.; Takahashi, M.;
Shinoda, K.; Watanabe, A. US Patent 5,036,086.
12. Klaubert, D. H.; Sellstedt, J. H.; Guinosso, C. L; Capetola,
R. L; Bell, S. C. J. Med. Chem., 1981, 24, 742±748.
nol/ethyl acetate to give 3.8 g of 3t; mp 163±165 ꢀC. H
1
NMR (DMSO-d6) d: 9.18 (1H, s), 7.54 (1H, d, J=3 Hz),
6.93 (1H, d, J=3 Hz), 5.95 (2H, s), 4.05 (2H, s), 3.44
(3H, s). Anal. Calcd for C10H10N3O2Cl: C, 50.12; H,
4.21; N, 17.53; Cl, 14.79, found: C, 50. 12; H, 4.3 1; N,
17.47; Cl, 14.82.
13. Tominaga, T.; Watanabe, A; Noma, S.; Tsuji, J; Koda, A.
Allergol. Int., 1996, 45, 91±96.
14. Taguchi, H.; Katsushima, T.; Ban, M.; Takahashi, M.;
Shinoda, K.; Watanabe, A. US Patent 4912135.
15. Yasuda, M.; Hasunuma, Y.; Adachi, H.; Sekine C.; Saka-
nishi, T.; Hashimoto, H.; Ra, C.; Yagita, H.; Okumura, K. Int.
Immunol., 1995, 7, 251±258.
Compound (4t) was prepared in the same manner as
general method C, compound (5t) was prepared in the
same manner as general method B.
16. Katayama, S.; Shionoyama, H.; Ohtake, S. Microbiol.
Immunol., 1978, 22, 89±101.