Novel antiallergic and antiinflammatory agents. Part II: Synthesis and pharmacology of TYB-2285 and its related compounds

Article abstract of DOI:10.1016/S0968-0896(98)00066-2

A series of m-bis(glycoloylamino)benzene derivatives was synthesized by treatment of the corresponding m-diaminobenzene derivatives with glycoloyl chloride derivatives in pyridine. Hydrolysis of acetyl compounds gave hydroxy derivatives, from which other acyl derivatives could be synthesized. These compounds were tested in the rat PCA (passive cutaneous anaphylaxis) assay by oral administration. Benzonitrile derivatives (4c, 5c, 6c, 4h, 5h) exhibited notable inhibition in this assay. Compounds 5c and 6c also showed remarkable inhibition of eosinophil adhesion to TNF- (tumor necrosis factor) α-treated HUVEC (human umbilical vein endothelial cells) in the range of 10^-8-10^-5 M. Compound 5c is now under investigation in Japan as TYB-2285 (Structures of compounds 1 and 2, DSCG, Lodoxamide ethyl and TYB-2285.) for asthma and atopic dermatitis in phase II clinical studies. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00066-2

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1077±1087
Novel Antiallergic and Antiin¯ammatory Agents. Part II:
Synthesis and Pharmacology of TYB-2285 and its Related
Compounds
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, Ohstu, Shiga 520-0292, Japan
Received 2 February 1998; accepted 27 March 1998
AbstractÐA series of m-bis(glycoloylamino)benzene derivatives was synthesized by treatment of the corresponding m-
diaminobenzene derivatives with glycoloyl chloride derivatives in pyridine. Hydrolysis of acetyl compounds gave
hydroxy derivatives, from which other acyl derivatives could be synthesized. These compounds were tested in the rat
PCA (passive cutaneous anaphylaxis) assay by oral administration. Benzonitrile derivatives (4c, 5c, 6c, 4h, 5h) exhib-
ited notable inhibition in this assay. Compounds 5c and 6c also showed remarkable inhibition of eosinophil adhesion
8
5
to TNF- (tumor necrosis factor) a-treated HUVEC (human umbilical vein endothelial cells) in the range of 10
M. Compound 5c is now under investigation in Japan as TYB-2285 (Figure 1) for asthma and atopic dermatitis in
10
phase II clinical studies. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
In addition, our compounds have unique e€ects, and in
particular, compounds 4c, 5c and 6c, showed a more
marked inhibition of eosinophil adhesion to TNF-a-
treated HUVEC3 than did compounds 1 and 2. Com-
pound 5c causes very unique e€ects. It inhibits the acute
response (AR) and late response (LR) in naturally sen-
sitized sheep.12 In addition, it inhibits airway hyperre-
sponsiveness (AHR). TYB-2285 (Figure 1) is now under
investigation in Japan for asthma and atopic dermatitis
in phase II clinical studies. In this paper, the synthesis
structure±activity relationships and some pharmacolo-
gical evaluations of this series compounds are described.
In the previous paper1, we reported that compounds 1
and 2 (Figure 1) inhibited the rat PCA (passive cuta-
neous anaphylaxis) reaction2 (30 mg/kg, p.o.) and that
they showed remarkable inhibition of eosinophil adhe-
sion to TNF-(tumor necrosis factor) a-treated HUVEC
(human umbilical vein endothelial cells)3 in the con-
8
centration range of 10
10 5 M. Recently, eosinophils
have come to be thought of as contributing to the
pathogenesis of allergic diseases such as asthma and
atopic dermatitis. Eosinophils accumulate in the lungs
and nasal airway after allergic challenge.4±6 The initial
phase in the process of eosinophil accumulation is eosi-
nophil adhesion to endothelial cells.7,8 For the purpose
of the development of new antiallergic and antiin¯am-
matory drugs for asthma and atopic dermatitis, we
focused on eosinophil adhesion to the endothelial cell.
Therefore, we synthesized new m-bis(glycoloylamino)-
benzene derivatives9 which have two active sites as do
DSCG (disodium cromoglycate)10 and Lodoxamide
ethyl11 (Figure 1). These compounds exhibit notable
inhibition in the rat PCA assay2 by oral administration.
Chemistry
All the compounds (Table 1) were prepared from a
commercially available acid chloride and m-phenylene-
diamine derivatives which were either commercially
available or could be synthesized easily. Synthesis was
carried out by General method A (Scheme 1). Com-
pounds 4±13 (R1=R2) were synthesized from m-phenyl-
enediamine derivatives and 2.1±2.2 eq. acid chloride in
high yields. These are symmetrical compounds, which
are identi®ed by 1H NMR. Starting m-phenylenedi-
amines (3) which were not commercially available, were
*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)00066-2

1078
M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1077±1087
Figure 1. Structures of compounds 1 and 2, DSCG, Lodoxamide ethyl and TYB-2285.
obtained from the corresponding m-dinitrobenzene
derivatives by the method of Wright et al.11 Hydroxy-
acetylamino derivatives (6) were synthesized from the
corresponding acetoxyacetylamino derivatives (5) by
hydrolysis in aqueous ammonia and methanol solution
(General method B, Scheme 1). The methoxycarbonyl
group of 5g was not hydrolysed under these conditions.
Propionyl, butyryl, isobutyryl, 2-acetoxybenzoyl and
benzoyl derivatives (7, 9, 10, 11, 12) were synthesized by
esteri®cation of the corresponding hydroxy derivatives
(6) (General method C, Scheme 1). 1,3-Diamino-4,6-
dicarboxybenzene (2j) was synthesized by the method of
Bogert et al.13 from m-phenylenediamine. Compounds
14±20c were synthesized from mono amide derivatives
which were reported in the previous paper.1 Compound
18c was synthesized from silyl ether (17c) and acetox-
yacetyl chloride. Other compounds were synthesized by
General method D (Scheme 2). Piperidino, morpholino
and methylsulfonyl derivatives were synthesized using a
chloroacetyl derivative (21c). Chlorine atoms of its
chloromethyl groups were substituted by secondary
amine and thiomethyl groups easily (General method E,
Scheme 3). All the compounds were determined by ele-
mentary analysis.
compounds. The results are shown in Table 2. These
compounds in which R1 is di€erent from R2, also
inhibited the rat PCA reaction. In the series of benzo-
nitrile compounds shown in Tables 1 and 2, bigger R
groups reduced activity in the rat PCA assay. In addi-
tion, compound 26c, which has no oxygen atom at the
a-position of the amide carbonyl group did not inhibit
the rat PCA reaction. The result indicates that the oxy-
gen atom at the a-position of the amide carbonyl group
is important for inhibition of the rat PCA reaction. We
then synthesized piperidino, morpholino and methane-
sulfonyl derivatives. However, these compounds showed
no inhibition in the PCA assay by oral administration.
Table 3 shows a comparison of compounds 4c, 5c, 4h and
5h in the PCA assay at a lower dose. All the compounds
inhibit the PCA reaction in a dose dependent manner.
The ED50 value of 5c in this assay is 0.3 mg/kg p.o. It is
clear that the compounds 4c and 5c, which have a chlor-
ine atom at the 2-position, are more active than 4h and 5h.
Recently, it has been reported that eosinophil sig-
ni®cantly relates to airway in¯ammation in asthma14±16
and atopic dermatitis.17 Therefore, we investigated the
e€ect of these compounds on eosinophil adhesion to
TNF-a-treated HUVEC. The results are shown in
Table 4. Compounds 5c and 6c caused inhibition of
eosinophil adhesion to TNF-a-treated HUVEC in the
Results and Discussion
8
5
range of 10
same range. Ketotifen fumarate showed no inhibition at
10 M. DSCG have no e€ect in the
The e€ects of bis-amide derivatives in the rat PCA assay
are shown in Table 1. The results of other compounds
are listed in Table 2. In Table 1, where R is a hydrogen,
methyl or lower acyl group, a series of compounds
inhibited the PCA reaction by oral administration.
Among those compounds, benzonitrile derivatives
inhibited the PCA as strongly as ketotifen fumarate.
Other compounds, which have CONH2, COOMe, CF3,
or hydrogen atom as Y group, inhibited the PCA
reaction slightly. When R are ethyl, phenyl, benzoyl
groups, those compounds also showed a slight inhibi-
tion. Therefore, we investigated other benzonitrile
6
less than 10 M, and neither did tranilast at less than
5
10
M. These concentrations are higher than the
plasma concentrations of the drugs at their usual clinical
doses. In comparison with DSCG, ketotifen fumarate
and tranilast, compounds 5c and 6c inhibit at lower
concentrations. Eosinophil adhesion to endothelial cells
is the initial phase in the process of eosinophil accumu-
lation. At the site of in¯ammation, cytokines such as
TNF-a and interleukin-1 (IL-1), are released, and
endothelial cells are activated. Recently, Tominaga et
al.3 showed that compounds 5c and 6c did not inhibit

M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1077±1087
1079
Table 1. Physical and pharmaceutical data of bisglycolic amide derivatives
Compd
no.
X
Y
H
R
mp (ꢀC)
Formula
Yield Recryst. PCA inhibition (%)
(%) solventa 30 mg/kg p.o.
4a
4b
5b
6b
7b
8b
4c
13c
5c
6c
7c
9c
10c
11c
4d
4e
5e
6e
7e
8f
4-COOEt
2-Cl
2-Cl
Me
90±91
209
C
15H20N2O5
92
78
53
67
90
61
59
60
29
62
30
48
36
43
77
70
83
78
67
62
56
79
81
83
63
53
71
77
70
67
51
82
88
95
24
A
B
53
27
5-CONH2 Me
5-CONH2 Ac
C13H16N3O5Cl.1/2H2O
C15H16N3O7Cl
C11H12N3O5Cl
C17H20N3O7Cl
C23H20N3O5C
216±218
254±257
195±198
250±251
173±174
152±153
195
C
ND
ND
NA
ND
70
2-Cl
2-Cl
5-CONH2
H
C
5-CONH2 COEt
5-CONH2 Ph
D
C
2-Cl
2-Cl
5-CN
5-CN
5-CN
5-CN
5-CN
5-CN
5-CN
5-CN
Me
Et
C13H14N3O4Cl
B
2-Cl
2-Cl
C
C
C
15H18N3O4Cl
15H14N3O6Cl
11H10N3O4Cl
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
A
D
D
D
D
D
D
D
D
D
E
38
94
Ac
H
2-Cl
2-Cl
228 (Dec.)
77
83
COEt
COnPr
COiPr
COPh(2-OAc)
141 (Dec.) C17H18N3O5Cl
2-Cl
2-Cl
143±144
140±141
166 (Dec.)
162±164
160±162
154±155
285±287
116±118
140±141
140±141
195±197
222±225
167±168
144 (Dec.)
131 (Dec.)
226±228
151±153
188±189
C
C
C
19H22N3O6Cl
19H22N3O6Cl
29H22N3O10Cl
91
89
2-Cl
2-Cl
14
5-COOMe Me
C14H17N2O6Cl
NA
NA
ND
ND
49
2-Cl
2-Cl
5-CF3
5-CF3
Me
Ac
C13H14N2O4CIF3
C15H14N2O6CIF3
C11H10N2O4CIF3
C17H18N2O6Cl
2-Cl
2-Cl
5-CF3
5-CF3
H
COEt
Ph
5-COOEt
5-COOMe
5-COOMe
5-COOMe
5-COOMe
5-CN
5-CN
5-CN
2-CN
2-CN
5-COOH
H
H
C
C
C
C
25H24N2O6
14H18N2O6
NA
NA
ND
ND
NA
88
4g
5g
6g
7g
4h
5h
6h
5i
H
Me
Ac
H
16H18N2O8.1/4H2O
12H14N2O6.1/4H2O
H
H
H
COEt
Me
Ac
C18H22N2O8.1/4H2O
H
C
C
C
C
C
13H15N3O4
15H15N3O6
H
16
H
H
Ac
11H11N3O4.1/4H2O
15H15N3O6.1/4H2O
11H11N3O4
ND
73
H
6i
H
H
ND
25
4j
H
Me
Ac
229 (Dec.) C13H16N2O
5k
6k
7k
12k
41
H
150±151
139±143
130±132
193±197
281 (Dec.)
C
C
16H20N2O6
10H12N2O6.1/4H2O
ND
ND
ND
ND
NA
91
H
H
H
H
H
COEt
COPh
Me
C16H20N2O6
H
4-COOH
ketotifen fumarate
H
6-COOH
C
C
24H20N2O6
14H16N2O8.1/2H2O
aA: EtOAc/Hexane; B: EtOH/EtOAc/Hexane; C: MeOH; D: EtOH; E: EtOH/H2O.
ND, Not done.
NA, Not active.
8
the adhesion of neutrophil at the same range (10
±
VLA-4 inhibited the late response (LR) and airway
hyper-responsiveness (AHR). In fact, the compound 5c,
named TYB-2285, inhibits the acute response (AR) and
the LR in naturally-sensitized sheep.12 In addition, it
inhibits the AHR. Sagara et al.15 demonstrated that a
mAb against VLA-4 inhibited the LR in sensitized guinea
pigs by inhibiting migration of eosinophils into the air-
way. Bently et al.16 reported that VCAM-1 expression
increased in patients with asthma after antigen challenge.
10 5 M). Eosinophil adhesion to TNF-a-treated
HUVEC was blocked by mAb against VLA-4 (anti-
VLA-4), but not by that against Mac-1 (anti-Mac-1).
However neutrophil adhesion was blocked by anti-Mac-
1, but not by anti-VLA-4.3 These results suggest that
compounds 5c and 6c might block the VIA-4/VCAM-1
pathway selectively. Abraham et al.14 showed that
treatment with a monoclonal antibody (mAb) against

1080
M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1077±1087
Scheme 1. Synthesis of bisglycolic amide derivatives.
Scheme 2. Synthesis of compounds 14c, 15c, 16c, 18c, 19c and 20c.
Groves et al.17 showed that VCAM-1 was upregulated
on dermal endothelium and dendritic cells in allergic
contact dermatitis and atopic dermatitis patients. These
results suggest that TYB-2285 may be suitable as a new
type of antiallergic and antiin¯ammatory drug for treat-
ing allergic diseases such as asthma and atopic dermatitis.
Recently, Yasuda et al.18 reported that anti-VLA-4
antibody, in combination with other anti-adhesion
molecule antibodies, inhibited the PCA reaction. We
speculate that TYB-2285 and its derivatives might inhi-
bit the PCA reaction by a mechanism related with the
inhibition of the VLA-4/VCAM-1 pathway. Further

M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1077±1087
1081
Scheme 3. Synthesis of compounds 22c±26c.
Table 2. Physical and pharmaceutical data of benzonitrile derivatives
Compd no. R1
R2
mp (ꢀC)
Formula
Yield (%) Recryst. PCA inhibition (%)
solventa 30 mg/kg p.o.
14c
15c
16c
18c
19c
20c
22c
23c
25c
26c
OMe
OPh
OAc
OAc
159±160
212±213
154±157
213±216
160 (Dec.)
119±120
290 (Dec.)
C
14H14N3O5Cl
19H16N3O5Cl
85
78
70
38
86
72
84
79
44
53
A
A
A
Ðb
A
A
B
92
75
C
OAc
OH
OCOCH2OAc
OAc
OMe
C17H16N2O8Cl
C13H12N3O3Cl
81
95
OH
C
12H12N3O4Cl
C16H18N3O5Cl
21H28N5O2Cl
94
89
OMe
piperidino
morpholino morpholino
OCOCH(CH3)2
piperidino
C
ND
35
296 (Dec.) C19H24N5O4Cl
204 (Dec.) C13H14N3O6S2Cl
B
SO2Me
Et
SO2Me
Et
C
NA
NA
200±201
C15H18N3O2Cl
C
aA: EtOH; B: CH3Cl/Toluene; C: MeOH.
bNot recryst.
ND, Not done.
NA, Not active.

1082
M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1077±1087
Table 3. E€ect of compounds in rat PCA assay
using TMS as an internal standard. 13C NMR were
recorded on a Varian XL-300 spectrometer using TMS
as an internal standard. Elemental analyses were per-
formed at Kyoto University and TOYOBO analytical
center. All starting materials were commercially avail-
able unless otherwise noted.
Compd no.
Inhibition (%) of rat PCA
Dose (mg/kg p.o.)
30
10
3
1
(ED50)
4c
5c
91** 84** 75** 52** (0.3 mg/kg)
93** 76** 52**
Passive cutaneous anaphylaxs (PCA) assay. Male Wistar
rats (weighing about 200 g) were passively sensitized by
intradermal injection of 0.1 mL of a solution of rat anti-
serum to egg albumin in each of two sites (four sites in
total) at both sides of the dorsal median line. After 48 h,
each rat was challenged by injecting a mixture (1 mL) of
egg albumin and Evans blue solution via the tail vein to
induce passive cutaneous anaphylaxis (PCA). Thirty
minutes after the challenge, the rats were sacri®ced to
take the bluing region, and the amount of pigment from
the bluing region was measured by the method of
Katayama et al.19 Test compounds were orally admin-
istrered to the rats (six rats/group) at a dose of 3 mg/kg
30 min before the antigen challenge.
4h
5h
88** 79** 46*
16
6
ketotifena
91** 76** 35* 18
aketotifen fumarate.
*
p<0.05, **p<0.01 versus control.
Table 4. E€ect of compounds on eosinophil adhesion to TNF-
a-treated HUVEC
Compd no.
Percentage of cell adhesion
Concentration of test compounds
10 8 M
10 7 M
10 6 M
10 5 M
5c
78*
54**
58**
52**
16***
NA
32**
5***
49**
NA
38*
25***
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
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 incu-
bated 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 recoincu-
bated with HUVEC in the presence of compounds. For
the experiments evaluating the compounds, the percen-
tage of control was calculated according to the follow-
ing formula:
6c
ketotifena
DSCG
tranilast
NA
22**
NA
aketotifen fumarate.
NA, Not active.
*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.
studies are required to determine the precise mechanism
of these compounds.
Conclusion
We have synthesized a number of bis-glycolylacetyl-
aminobenzene derivatives and investigated their phar-
macological activity. Among them, 3,5-bis(glycolylacetyl-
amino)benzonitrile derivatives showed antiallergic
activity in the PCA assay at 30 mg/kg p.o. and marked
inhibition of eosinophil adhesion to TNF-a-treated
HUVEC in the range of 10
compounds, 3,5-bis(acetoxyacetylamino)-4-chlorobenzo-
nitrile (5c), named TYB-2285, is now being investigated
in Japan for asthma and atopic dermatitis in phase II
clinical studies.
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).
8
10 5 M. One of the
General method A. Ethyl 2,4-bis(methoxyacetylamino)
benzoate (4a). Methoxyacetyl chloride (4.0 mL) was
added dropwise to a solution of ethyl 2,4-diamino-
benzoate (3.6 g) in pyridine (80 mL) at room tempera-
ture. The mixture was stirred at room temperature for
3 h. Thereafter, pyridine was distilled o€ under reduced
pressure. Water was added to the residue, and the
resulting mixture was extracted with chloroform. The
organic layer was washed with water and saturated
Experimental
Melting points were determined with a Mettler capillary
melting-point apparatus (Model FP 61) and uncorrected.
1H NMR spectra were recorded on a Varian FT 80A
spectrometer or a Varian Gemini-200 spectrometer

M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1077±1087
1083
sodium chloride solution, and dried over anhydrous
sodium sulfate. The solvent was distilled o€ under
reduced pressure. The resulting solids were recrystallized
from ethyl acetate/hexane to give 6.0 g of 4a; mp 90±
91 ꢀC. 1H NMR (DMSO-d6) d: 11.50 (1H, s), 10.12 (1H,
s), 8.86 (1H, d, J=2 Hz), 7.91 (1H, d, J=6 Hz), 7.55
(1H, dd, J=6 Hz and J=2 Hz), 4.28 (2H, q, J=7 Hz),
4.00 (1H, s), 3.42 (1H, s), 3.34 (3H, s), 1.32 (3H, t,
J=7 Hz). Anal. calcd for C15H20N2O6: C, 55.55; H,
6.22; N, 8.64, found: C, 55.57; H, 6.24; N, 8.64.
3,5-Bis(acetoxyacetylamino)-4-chlorobenzonitrile (5c).
From 4-chloro-3,5-diaminobenzonitrile (67 g) and
acetoxyacetyl chloride (95 mL): 43 g of 5c; mp 195 ꢀC.
1H NMR (DMSO-d6) d: 9.94 (2H, s), 4.73 (4H, s), 2.10
(6H, s). 13C NMR (DMSO-d6) d: 170.09 (s), 166.73 (s),
136.42 (s), 125.90 (s), 125.07 (d), 117.69 (s), 109.89 (s),
62.47 (t), 20.58 (t). Anal. calcd for C15H14N3O6Cl: C,
48.99; H, 3.92; N, 11.43; Cl, 9.64, found: C, 49.05; H,
3.84; N, 11.49; Cl, 9.50.
Methyl 3,5-bis(methoxyacetylamino)-4-chlorobenzoate (4d).
From methyl 4-chloro-3,5-diaminobenzoate dihydro-
chloride (1.1 g) and methoxyacetyl chloride (0.8 mL):
1.1 g of 4d; mp 162±164 ꢀC. 1H NMR (DMSO-d6) d: 9.41
(2H, s), 8.28 (2H, s), 4.07 (4H, s), 3.85 (3H, s), 3.43 (6H, s).
Anal. calcd for C14H17N2O6Cl; C, 48.78; H, 4.97; N, 8.13;
Cl, 10.28, found: C, 48.73; H, 5.10; N, 8.13; Cl, 10.13.
Compounds 4b±e, g, h, j±l, 5b, c, e, g±i, 8b, 8f, 13c and
26c were prepared in the same manner as 4a (General
method A).
3,5-Bis(methoxyacetylamino)-4-chlorobenzamide hemi-
hydrate (4b). From 3,5-diamino-4-chlorobenzamide
(2.8 g) and methoxyacetyl chloride (3.0 mL): 4.0 g of 4b;
mp 209 ꢀC. H NMR (DMSO-d6) d: 9.43 (1H, s), 8.11
3,5-Bis(methoxyacetylamino)-4-chlorobenzotri¯uoride (4e).
From 4-chloro-3,5-diaminobenzotri¯uoride (4.2 g) and
methoxyacetyl chloride (4.0 mL): 5.0 g of 4e; mp 160±
162ꢀC. 1H NMR (DMSO-d6) d: 9.50 (2H, s), 8.08 (2H, s),
4.08 (4H, s), 3.41 (6H, s). Anal. calcd for C13H14
N2O4ClF3: C, 44.02; H, 3.98; N, 7.90; Cl, 9.99; F, 16.07,
found: C, 44.17; H, 3.97; N, 7.95; Cl, 9.87; F, 15.99.
1
(2H, s), 7.98 (1H, s), 7.40 (1H, s), 4.06 (4H, s), 3.42 (6H,
.
s). Anal. calcd for C13H16N3O5Cl 1/2H2O: C, 46.09; H,
5.06; N, 12.40; Cl, 10.47, found: C, 45.84; H, 5.16; N,
12.29; Cl, 10.47.
3,5-Bis(acetoxyacetylamino)-4-chlorobenzamide
(5b).
From 4-chloro-3,5-diaminobenzamide (5.6 g) and acet-
oxyacetyl chloride (7.2 mL): 6.2 g of 5b; mp 216±218 ꢀC;
1H NMR (DMSO-d6) d: 9.79 (2H, s), 7.98 (2H, s), 7.95
(2H, s), 7.35 (1H, s), 4.70 (4H, s), 2.11 (6H, s). Anal.
calcd for C15H16N3O7Cl: C, 46.70; H, 4.18; N, 10.89,
found: C, 46.65; H, 4.28; N, 10.71.
3,5-Bis(acetoxyacetylamino)-4-chlorobenzotri¯uoride (5e).
From 4-chloro-3,5-diaminobenzotri¯uoride (4.2 g) and
acetoxyacetyl chloride (5.6 mL): 6.8 g of 5e; mp 154±
155 ꢀC 1H NMR (DMSO-d6) d: 9.92 (2H, s), 7.94
(2H, s), 4.73 (4H, s), 2.12 (6H, s). Anal. calcd for
C15H14N2O6ClF3: C, 43.86; H, 3.44; N, 6.82, found: C,
43.77; H, 3.32; N, 6.82.
3,5-Bis(phenoxyacetylamino)-4-chlorobenzamide
(8b).
From 4-chloro-3,5-diaminobenzamide (2.4 g) and phen-
oxyacetyl chloride (4.0 mL): 3.6 g of 8b; mp 250±251 ꢀC.
1H NMR (DMSO-d6) d: 9.89 (2H, s), 8.08 (2H s), 7.99
(1H, s), 7.60±6.80 (11H, m), 4.78 (4H, s). Anal. calcd for
C23H20N3O5Cl: C, 60.86; H, 4.48; N, 9.26, found: C,
60.82; H, 4.50; N, 9.29.
Ethyl 3,5-bis(phenoxyacetylamino)benzoate (8f). From
ethyl 3,5-diaminobenzoate dihydrochloride (2.5 g) and
phenoxyacetyl chloride (3.0 mL): 2.1 g of 8f; mp 140±
141 ꢀC. 1H NMR (DMSO-d6) d: 10.32 (2H, s), 8.31 (1H,
dd, J=2 Hz and J=2 Hz), 8.01 (2H, d, J=2 Hz), 7.95±
6.75 (10H, m), 4.69 (4H, s), 4.29 (2H, q, J=7 Hz), 1.30
(3H, t, J=7 Hz). Anal. calcd for C25H24N2O6Cl: C,
66.95; H, 5.39; N, 6.25, found: C, 66.97; H, 5.38; N, 6.25.
3,5-Bis(methoxyacetylamino)-4-chlorobenzonitrile (4c).
From 4-chloro-3,5-diaminobenzonitrile (3.3 g) and
methoxyacetyl chloride (4.0 mL): 5.1 g of 4c; mp 173±
174 ꢀC. H NMR (DMSO-d6) d: 9.50 (2H, s), 8.11 (2H,
Methyl 3,5-bis(methoxyacetylamino)benzoate (4g). From
methyl 3,5-diaminobenzoate dihydrochloride (7.2 g) and
methoxyacetyl chloride (6.0 mL): 5.2 g of 4g; mp 140±
1
s), 4.07 (4H, s), 3.41 (6H, s). Anal. calcd for
C13H14N3O4Cl: C, 50.09; H, 4.53; N, 13.48; Cl, 11.37,
found: C, 50.11; H, 4.34; N, 13.50; Cl, 11.47.
141 ꢀC. H NMR (DMSO-d6) d: 10.06 (2H,s), 8.78 (1H,
1
dd, J=2 Hz and J=2 Hz), 8.00 (2H, d, J=2 Hz), 3.98
(4H, s), 3.82 (3H, s), 3.36 (6H, s). Anal. calcd for
C14H18N2O6: C, 54.19; H, 5.85; N, 9.03, found: C,
54.12; H, 5.85; N, 9.00.
3,5-Bis(ethoxyacetylamino)-4-chlorobenzonitrile (13c).
From 4-chloro-3,5-diaminobenzonitrile (3.3 g) and
ethoxyacetyl chloride (4.8 mL): 4.0 g of 13c; mp 152±
153 ꢀC; H NMR (DMSO-d6) d: 9.45 (2H, s), 8.15 (2H,
1
s), 4.10 (4H, s), 3.60 (4H, q, J=7 Hz), 1.21 (6H, t,
J=7 Hz). Anal. calcd for C15H18N3O4Cl: C, 53.02; H,
5.34; N, 12.37; Cl, 10.43, found: C, 53.15; H, 5.33; N,
12.51; Cl, 10.43.
Methyl 3,5-bis(acetoxyacetylamino)benzoate 1/4 hydrate
(5g). From methyl 3,5-diaminobenzoate (8.3 g) and
acetoxyaꢀcetyl chloride (11.9 mL): 14.6 g of 5g; mp
1
195±197 C. H NMR (DMSO-d6) d: 10.25 (2H, s), 8.14

1084
M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1077±1087
(1H, dd, J=2 Hz and J=2 Hz), 7.92 (2H, d, J=2 Hz),
4.16 (4H, s), 3.82 (3H, s), 2.11 (6H, s) Anal. calcd for
.
C16H18N2O8 1/4H2O: C, 51.82; H, 5.03; N, 7.55, found:
C, 51.98; H, 4.91; N, 7.61.
J=7 Hz). Anal. calcd for C15H18N3O2Cl: C, 58.54; H,
5.89; N, 13.65; Cl, 11.52, found: C, 58.37; H, 5.83; N,
13.54; Cl, 11.46.
General method B. 3,5-Bis(hydroxyacetylamino)-4-chloro-
benzamide (6b). To a solution of 5b (0.8 g) in methanol
(100 mL) was added dropwise 28% aqueous ammonia
(10 mL). The mixture was stirred at room temperature
for 2 h. Thereafter, the solvent was distilled o€ under
reduced pressure. The resulting solids were recrystallized
from ethanol to give 0.4 g of 6b; mp 254±257 ꢀC. 1H
NMR (DMSO-d6) d: 9.45 (2H, s), 8.30 (2H, s), 7.97 (1H,
s), 7.40 (1H, s), 6.10 (2H, t, J=4 Hz) 4.08 (4H, d,
J=4 Hz). Anal. calcd for C11H12N3O5Cl: C, 43.79; H,
4.01; N, 13.93, found: C, 43.89; H, 4.21; N, 13.85.
3,5-Bis(methoxyacetylamino)benzonitrile (4h). From 3,5-
diaminobenzonitrile (4.4 g) and methoxyacetyl chloride
(6.0 mL): 4.3 g of 4h; mp 144 ꢀC. H NMR (DMSO-d6)
1
d: 10.06 (2H, s), 8.30 (1H, dd, J=2 Hz and J=2 Hz),
7.75 (2H, d, J=2 Hz), 4.00 (4H, s), 3.36 (6H, s). Anal.
calcd for C13H15N3O4: C, 56.31; H, 5.45; N, 15.15,
found: C, 56.24; H, 5.54; N, 15.21.
3,5-Bis(acetoxyacetylamino)benzonitrile 1/4 hydrate (5h).
From 3,5-diaminobenzonitrile (5.9g) and acetoxyacetyl
chloride (9.5 mL): 7.2 g of 5h; mp 131 ꢀC (Dec.). 1H
NMR (DMSO-d6) d: 10.38 (2H, s), 8.10 (1H, dd, J=2 Hz
and J=2 Hz), 7.65 (2H, d, J=2 Hz), 4.62 (4H, s), 2.09
Compounds 6c, e, g±i and k were prepared in the same
manner as 6b (General method B).
.
(6H, s). Anal. calcd for C15H15N3O6 1/4H2O: C 53.33; H,
4.62; N, 12.44, found: C, 53.28; H, 4.55; N, 13.37.
3,5-Bis(hydroxyacetylamino)-4-chlorobenzonitrile (6c).
From 5c (27 g): 13 g of 6c; mp 228 ꢀC (Dec.). H NMR
1
2,6-Bis(acetoxyacetylamino)benzonitrile (5i). From 2,6-
diaminobenzonitrile (9.3 g) and acetoxyacetyl chloride
(24 mL): 18.0 g of 5i; mp 151±153 ꢀC. 1H NMR (DMSO-
d6) d: 10.25 (2H, s), 7.85±7.40 (3H, m), 4.66 (4H, s), 2.11
(6H, s). Anal. calcd for C15H15N3O6: C, 54.05; H, 4.54;
N, 12.61, found: C, 54.03; H, 4.56; N, 12.57.
(DMSO-d6) d: 8.27 (2H, s), 7.85 (2H, s), 4.70 (4H, s),
3.30 (2H, s). Anal. calcd for C11H10N3O4Cl: C, 46.58;
H, 3.55; N, 14.81; Cl, 12.50, found: C, 46.57; H, 3.73; N,
14.81; Cl, 12.24.
3,5-Bis(hydroxyacetylamino)-4-chlorobenzotri¯uoride (6e).
From 7c (4.1 g): 2.1 g of 6e; mp 285±287 ꢀC. H NMR
1
3,5-Bis(methoxyacetylamino)benzoic acid (4j). From 3,5-
diaminobenzoic acid (6.1 g) and methoxyacetyl chloride
(8.0 mL): 8.0 g of 4j; mp 229 ꢀC (Dec.). 1H NMR
(DMSO-d6) d: 12.87 (1H, s), 9.92 (2H, s), 8.32±8.17 (1H,
m), 8.04±7.90 (2H, m), 3.97 (4H, s), 3.35 (6H, s). Anal.
calcd for C13H16N2O6: C, 52.70; H, 5.44; N, 9.46; found:
C, 52.82; H, 5.45; N, 9.60.
(DMSO-d6) d: 7.94 (2H, s), 8.29 (2H, s), 4.07 (4H, s).
Anal. calcd for C11H10N2O4ClF3: C, 40.45; H, 3.09; N,
8.58, found: C, 40.50; H, 3.06; N, 8.55.
Methyl 3,5-bis(hydroxyacetylamino)benzoate 1/4 hydrate
(6g). From 5g (8.0 g): 5.0 g of 6g); mp 222±225 ꢀC. 1H
NMR (DMSO-d6) d: 9.86 (2H, s), 8.23 (1H, dd,
J=2 Hz), 8.02 (2H, d, J=2 Hz), 5.54 (2H, t, J=5 Hz),
3.96 (4H, d, J=5 Hz) 3.81 (3H, s) Anal. calcd for
.
C12H14N2O6 1/4H2O: C, 50.26; H, 5.10; N, 9.77, found:
C, 50.54; H, 4.95; N, 9.85.
1,3-Bis(acetoxyacetylamino)benzene (5k). From m-phenyl-
enediamine (21.6 g) and acetoxyacetyl chloride (56 mL):
36.1 g of 5k; mp 150±151 ꢀC. 1H NMR (DMSO-d6) d:
10.04 (2H, s), 7.93±7.80 (1H, m), 7.30±7.15 (3H, m), 4.61
(4H, s), 2.12 (6H, s). Anal. calcd for C14H16N2O6: C,
54.54; H, 5.23; N, 9.09, found: C, 54.52; H, 5.16; N,
9.08.
3,5-Bis(hydroxyacetylamino)benzonitrile 1/4 hydrate
(6h). From 5h (2.3 g): 1.2 g of 6h; mp 226±228 ꢀC. 1H
NMR (DMSO-d6) d: 9.94 (2H, s), 8.35 (1H, dd, J=2 Hz
and J=2 Hz), 7.79 (2H, d, J=2 Hz), 5.60 (2H, s), 3.99
.
(4H, s). Anal. calcd for C11H11N3O4 1/4H2O: C, 52.07;
H, 4.57; N, 16.56, found: C, 52.28; H, 4.50; N, 16.76.
4,6-Bis(methoxyacetylamino)-m-phthalic acid hemihydrate
(4l). From 4,6-diamino-m-phthalic acid (5.4 g) and
methoxyacetyl chloride (5.5 mL): 2.5 g of 4l; mp 277 ꢀC
(Dec.). 1H NMR (DMSO-d6) d: 12.00 (2H, s), 9.92 (1H,
s), 8.63 (1H, s), 4.02 (4H, s), 3.42 (6H, s). Anal. calcd for
.
C14H16N2O8 1/2H2O: C, 48.14; 4.91; N, 8.02, found: C,
48.12; H, 4.86; N, 8.02.
2,6-Bis(hydroxyacetylamino)benzonitrile (6i). From 5i
(6.7 g): 3.8 g of 6i; mp 188±189 ꢀC. 2H NMR (DMSO-d6)
d: 9.60 (2H, s), 7.61 (3H, m), 6.12 (2H, s), 4.02 (4H, s).
Anal. calcd for C11H11N3O4: C, 53.01; H, 4.45; N,
16.86, found: C, 53.69; H, 4.45; N, 16.85.
3,5-Bis(butyrylamino)-4-chlorobenzonitrile (26c). From
3c (3.4 g) and butyryl chloride (4.6 mL): 3.3 g of 26c; mp
1,3-Bis(hydroxyacetylamino)benzene 1/4 hydrate (6k).
From 5k (30.0 g): 18.1 g of 6k; mp 139±143 ꢀC. 1H
NMR (DMSO-d6) d: 10.54 (2H, s), 8.04±7.90 (1H, m),
200±201 ꢀC. H NMR (DMSO-d6) d: 9.67 (2H, s). 7.93
1
(2H, s), 2.37 (4H, t, J=7 Hz), 1.62 (4H, m), 0.94 (6H, t,

M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1077±1087
1085
7.45±7.00 (3H, m), 5.52 (2H, t, J=6 Hz), 3.94 (4H, d,
.
J=6 Hz). Anal. calcd for C10H12N2O4 1/4H2O: C, 52.51;
H, 5.55; N, 12.25, found: C, 52.62; H, 5.59; N, 12.29.
9.92 (2H, s), 7.93 (2H, s), 4.76 (4H, s), 2.44 (4H, q,
J=8 Hz), 1.09 (6H, t, J=8 Hz). Anal. calcd for
C17H18N2O6ClF3: C, 46.53; H, 4.13; N, 6.38, found: C,
46.79; H, 4.22; N, 6.38.
General method C. 3,5-Bis(propionyloxyacetylamino)-4-
chlorobenzamide (7b). Propionyl chloride (0.3 mL) was
added dropwise to a solution of the compound 6b
(0.56 g) in pyridine (15 mL) at room temperature. The
mixture was stirred at room temperature for 1 h. There-
after, pyridine, was distilled o€ reduced pressure to
remove the solvent. Water was added to the residue.
The resulting solids were ®ltered and recrystallized from
ethanol to give 0.6 g of 7b; mp 195±198 ꢀC. 1H NMR
(DMSO-d6) d: 9.79 (2H, s), 7.99 (1H, s), 7.96 (2H, s), 7.39
(1H, s), 4.72 (4H, s), 2.42 (4H, q, J=7 Hz), 1.08 (6H, t,
J=7 Hz). Anal. calcd for C17H20N3O7Cl: C, 49.34; H,
4.87; N, 10.15, found: C, 49.10; H, 5.00; N, 10.03.
Methyl 3,5-bis(propionyloxyacetylamino)benzoate 1/4
hydrate (7g). From 6g (2.1 g) and propionic anhydride
(5 mL): 2.5 g of 7g; mp 167±168 ꢀC. H NMR (DMSO-
1
d6) d: 10.26 (2H, s), 8.12 (1H, dd, J=2 Hz and J=2 Hz),
7.91 (2H, d, J=2 Hz), 4.72 (4H, s), 3.87 (3H, s), 2.41
(4H, q, J=8 Hz), 1.06 (6H, t, J=8 Hz). Anal. calcd for
C18H22N2O8.1/4H2O: C, 54.20; H, 5.68; N, 7.02, found:
C, 54.41; 5.47; N, 7.13.
1,3-Bis(propionyloxyacetylamino)benzene (7k). From 6k
(2.2 g) and propionyl chloride (1.9 m): 3.0 g of 7k; mp
130±132 ꢀC. H NMR (DMSO-d6) d: 10.01 (2H, s), 7.88
1
(1H, m), 7.35±7.15 (3H, m), 4.60 (4H, s), 2.40 (4H, q,
J=7 Hz), 1.05 (6H, t, J=7 Hz). Anal. calcd for
C16H20N2O6: C, 57.14; H, 5.99; N, 8.33, found: C,
57.04; H, 6.01; N, 8.35.
Compounds 7c, e, g, k, 9c, 10c, 11c and 12k were pre-
pared in the same manner as 7b (General method C).
3,5-Bis(propionyloxyacetylamino)-4-chlorobenzonitrile
(7c). From 6c (1.7 g) and proionyl chloride (1.3 mL):
1,3-Bis(benzoyloxyacetylamino)benzene 1/4 hydrate (12k).
From 6k (2.2 g) and benzoyl chloride (2.6 g): 4.2 g of
12k; mp 193±197 ꢀC. 1H NMR (DMSO-d6) d: 10.18
(2H, s), 8.22±7.05 (14H, m), 4.89 (4H, s). Anal. calcd for
C24H20N2O6 1/4H2O: C, 65.97; H; 4.73; N, 6.41, found:
C, 66.09; H, 4.71; N, 6.61.
1.2 g of 7c; mp 141 ꢀC (Dec.). H NMR (DMSO-d6) d:
1
9.93 (2H, s), 7.97 (2H s), 4.75 (4H, s), 2.43 (4H, q,
J=7 Hz), 1.09 (6H, t, J=7 Hz). Anal. calcd for
C17H18N3O6Cl: C, 51.59; H, 4.58; N, 10.62; Cl, 8.96,
found: C, 51.62; H, 4.64; N, 10.53; Cl, 8.82.
3,5-Bis(butyryloxyacetylamino)-4-chlorobenzonitrile (9c).
From 6c (2.8 g) and butyryl chloride (2.3 mL), 2.3 g of
9c; mp 143±144 ꢀC. 1H NMR (DMSO-d6) d: 9.93 (2H,s),
7.98 (2Hs), 4.75 (4H,s), 2.40 (4H, q, J=7 Hz), 1.60 (4H,
m), 0.94 (6H, t, J=7 Hz). Anal. calcd for
C19H22N3O6Cl: C, 53.84; H, 5.23; N, 9.91, found: C,
53.62; H, 5.28; N, 9.98.
General method D. 3-(Acetoxyacetylamino)-4-chloro-5-
(methoxyacetylamino)benzonitrile (14c). Methoxyacetyl
chloride (1.0 mL) was added dropwise to a solution
of 3-acetoxyacetylamino-5-amino-4-chlorobenzonitrile
(2.7 g) in pyridine (50 mL) at room temperature. The
mixture was stirred for 1 h at room temperature. There-
after, the solvent was distilled o€ under reduced pres-
sure. To the residue was added water and resulting
solids were washed with water. The solids were recrys-
tallized from ethanol to give 2.1 g of 14c; mp 159±
3,5-Bis(isobutyryloxyacetylamino)-4-chlorobenzonitrile
(10c). From 6c (2.8 g) and isobutyryl chloride (2.6 mL):
2.8 g of 10c; mp 140±141 ꢀC. 1H NMR (DMSO-d6) d:
9.92 (2H, s), 7.99 (2H, s), 4.75 (4H, s), 2.85±2.45 (2H,
m), 1.14 (12H, d, J=8 Hz),. Anal. calcd for C19H22
N3O6Cl: C, 53.84; H, 5.23; N, 9.91, found: C, 53.59; H,
5.29; N, 9.92.
160 ꢀC. H NMR (DMSO-d6) d: 9.85 (1H, s), 9.64 (1H,
1
s), 8.12 (1H, d, J=2 Hz), 7.97 (1H, d, J=2 Hz), 4.75
(2H, s), 4.08 (2H, s), 3.43 (3H, s), 2.13 (3H, s). Anal.
calcd. for C14H14N3O5Cl: C, 49.50; H, 4.15; N, 12.37,
found: C, 49.30; H, 4.12; N, 12.33.
3,5-Bis(2-acetoxybenzoyloxyacetylamino)-4-chlorobenzo-
nitrile (11c). From 6c (2.8 g) and 2-acetoxybenzoyl
chloride (4.4 g): 2.6 g of 11C; mp 166 ꢀC (Dec). 1H NMR
(DMSO-d6) d: 10.08 (2H, s), 8.25±7.10 (10H, m), 5.00
(4H, s), 2.27 (6H, s). Anal. calcd for C29H22 N3O10Cl: C,
57.29; H, 3.65; N, 6.91, found: C, 57.01; H, 3.63; N,
6.89.
Compounds 15c and 16c were prepared in the same
manner as 14c (General method D). In the case of 16c, 2
equiv. acid chloride were used.
3-(Acethoxyacetylamino)-4-chloro-5-phenoxyacetylamino-
benzonitrile (15c). From 3-acetoxyacetylamino-5-amino-
4-chlorobenzonitrile (2.7 g) and phenoxyacetyl chloride
(1.5 mL): 3.1 g of 15; mp 212±213 ꢀC. 1H NMR
(DMSO-d6) d: 9.96 (1H, s), 9.82 (1H, s), 8.09 (1H, d,
J=3 Hz), 7.99 (1H, d, J=3 Hz), 7.52±6.82 (5H, m), 4.80
(2H, s), 4.76 (3H, s), 2.12 (3H, s). Anal. calcd for
3,5-Bis(propionyloxyacetylamino)-4-chlorobenzotri¯uoride
(7e). From 7d (1.2 g) and propionyl chloride (0.67 mL):
1.0 g of 7e; mp 116±118 ꢀC. 1H NMR (DMSO-d6) d:

1086
M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1077±1087
C19H16N3O5Cl: C, 56.80; H, 4.01; N, 10.46, found: C,
56.73; H, 4.08; N, 10.47.
hydroxyacetylamino-5-methoxyacetylaminobenzonitrile
(0.9 g) and isobutyryl chloride (0.35 mL). The resulting
solids were recrystallized from ethanol to give 0.8 g of
20c; mp 119±120 ꢀC. H NMR (DMSO-d6) d: 9.19 (2H,
1
3-(Acetoxyacetylamino)-5-[(acetoxyacetoxy)acetylamino]-
4-chlorobenzonitrile (16c). From 3-amino-4-chloro-5-
(hydroxyacetylamino)benzonitrile (2.3 g) and acetoxy-
acetyl chloride (2.4 mL): 3.0 g of 16c; mp 154±157 ꢀC. 1H
MMR (DMSO-d6) d: 9.98 (1H, s), 7.97 (2H, s), 4.85
(2H, s), 4.77 (2H, s), 4.74 (2H, s), 2.13 (3H, s), 2.13 (3H,
s). Anal. calcd for C17H16N3O8Cl: C, 47.96; H, 3.79; N,
9.87, found: C, 47.75; H, 3.77; N, 9.90.
s), 8.09 (1H, d, J=2 Hz), 7.99 (1H, d, J=2 Hz), 4.75
(2H, s), 4.06 (2H, s), 3.90 (3H, s), 2.60 (1H, q, J=7 Hz),
1.14 (6H, d, J=7 Hz). Anal. calcd for C16H18N3O5 Cl:
C, 52.25; H, 4.93; N, 11.43, found: C, 52.33; H, 4.87; N,
11.44.
3,5-Bis(chloroacetylamino)-4-chlorobenzonitrile (21c). To
a solution of 3,5-diamino-4-chlorobenzonitrile (5.50 g)
in acetic acid was added chloroacetyl chloride (6.3 mL)
at room temperature. The mixture was stirred at room
temperature for 1 h. The resulting solids were ®ltered
and washed with water. The solid recrystallized from
ethyl acetate/hexane to give 5.3 g of 21c; mp 231 ꢀC
(Dec.). 1H NMR (DMSO-d6) d: 10.10 (2H, s), 7.89 (2H,
s), 4.36 (4H, s).
3-Amino-4-chloro-5-(trimethylsiloxyacetylamino)benzo-
nitrile (17c). Triethylamine (2.8 mL) was added to a
solution of 3-amino-4-chloro-5-(hydroxyacetylamino)
benzonitrile (2.3 g) in tetrahydrofuran (100 mL). Tri-
methylsilyl chloride (2.5 mL) was added dropwise to the
mixture at room temperature. The mixture was stirred
at room temperature for 20 h. Thereafter, the solvent
was distilled o€ reduced pressure. The resulting solids
were ®ltered and washed with water. The solids were
recrystallized from ethyl acetate/hexane to give 2.0 g of
General method E. 3,5-Bis(piperidinoacetylamino)-4-chloro-
benzonitrile (22c). The mixture of the compound 21c
(3.2 g) in piperidine (50 mL) was stirred at 100 ꢀC for
2 h. Thereafter, piperidine was distilled o€ under
reduced pressure. The residue was extracted with
chloroform and the organic layer was washed with
water. The organic layer was dried over anhydrous
sodium sulfate. The solvent was distilled under reduced
pressure. The resulting solids were recrystallized from
chloroform/methanol to give 2.6 g of 22c; mp 290 ꢀC
(Dec.). 1H NMR (DMSO-d6) d: 10.20 (1H, s), 8.55 (2H,
s), 3.15 (4H, s), 2.70±2.40 (8H, m), 1.80±1.40 (12H, m).
Anal. calcd for C21H28N5O2Cl: C, 60.35; H, 6.75; N,
16.76; Cl, 8.48, found: C, 60.35; H, 6.82; N, 16.83; Cl,
8.38.
17c; mp 132±133 ꢀC. H NMR (DMSO-d6) d: 9.22 (1H,
2
8), 7.68 (1H, d, J=2 Hz), 6.90 (1H, d, J=2 Hz), 5.97
(2H, s), 4.19 (2H, s), 0.20 (9H, s).
3-(Acetoxyacetylamino)-4-chloro-5-(hydroxyacetylamino)
benzonitrile (18c). Acetoxyacetyl chloride (0.5 mL) was
added dropwise to a solution of the compound 1 (1.5 g)
in dichloromethane (100 mL) at room temperature. The
mixture was stirred at room temperature for 2 h. There-
after, the solvent was distilled o€ under reduced pressure.
The residue was dissolved in a small amount of methanol,
and water (10 mL) and 1N-HCl aq (1 mL) were added to
the solution. The mixture was stirred at room tempera-
ture. The resulting solids were ®ltered and washed with
water, ethanol and then diethylether to give 0.6 g of 18c:
mp 213±216 ꢀC. 2H NMR (DMSO-d6) d: 9.97 (1H, s),
9.52 (2H, s), 8.29 (1H, d, J=2 Hz), 7.90 (1H, d, J=2 Hz),
6.16 (1H, t, J=5 Hz), 4.74 (2H, s), 4.04 (2H, d, J=5 Hz),
2.13 (3H, s). Anal. calcd for C13H12N3O2Cl: C, 47.94;
H, 3.71; N, 12.90, found: C, 47.74; H, 3.77; N, 12.98.
Compound 23c was prepared in the same manner as 22c
(General method E).
3,5-Bis(morpholinoacetylamino)-4-chlorobenzonitrile (23c).
From 21c (3.9 g) and morpholine (50 mL): 4.0 g of 23c;
mp 290 ꢀC (Dec.). 1H NMR (CDCl3) d: 10.05 (1H, s),
8.57 (2H, s), 3.60 (8H, m), 3.20 (4H, s), 2.66 (8H, m).
Anal. calcd for C19H24N5O4Cl: C, 54.09; H, 5.73; N,
16.60; Cl, 8.40, found: C, 53.83; H, 5.72; N, 16.71; Cl,
8.36.
4-Chloro-3-(hydroxyacetylamino)-5-(methoxyacetylamino)
benzonitrile (19c). The compound 19c was prepared in
the same manner as 6b from 14c (0.4 g). The resulting
solids were recrystallized from ethanol to give 0.3 g of
19c; mp 160 ꢀC (Dec.). 1H NMR (DMSO-d6) d: 9.48
(2H, s), 8.30 (1H, d, J=2 Hz), 8.03 (1H, d, J=2 Hz),
6.10 (1H, s), 4.06 (4H, s), 3.42 (3H, s). Anal. calcd for
C12H12N3O4CL: C, 48.42; H, 4.06; N, 14.12, found: C,
48.18; H, 4.08; N, 14.02.
3,5-Bis(methylthioacetylamino)-4-chlorobenzonitrile (24c).
To a solution of the compound 21c (3.2 g) and tri-
ethylamine (6.2 mL) in DMF (40 mL) was added drop-
wise 30% methylmercaptane methanol solution (3.0 mL)
at room temperature. Thereafter, the mixture was stir-
red at room temperature for 3 h. Thereafter, water
(150 mL) was added to the mixture. The resulting solids
were ®ltered and washed with water. The solids were
recrystallized from ethanol to give 2.7 g of 24c; mp
4-Chloro-3-(isobutylryloxyacetylamino)-5-(methoxyacetyl-
amino)benzonitrile (20c). The compound 20c was pre-
pared in the same manner as 7b from 4-chloro-3-

M. Ban et al./Bioorg. Med. Chem. 6 (1998) 1077±1087
1087
183±184 ꢀC. 1H NMR (CDCl3) d: 9.50 (2H, s), 8.50 (2H,
s), 3.40 (4H, s), 2.20 (6H, s).
7. Monterfort, S.; Holgate, S. T. Respir Med., 1991, 85, 91±
100.
8. Sprinnger, T. A. Nature, 1990, 346, 425±434.
3,5-Bis(methylsulfonylacetylamino)-4-chlorobenzonitrile
(25c). To a solution of the compound 24c (2.3 g) and in
chloroform (250 mL) was added dropwise m-chloro-
perbenzoic acid (5.1 g) in chloroform (150 mL) at room
temperature. The mixture was re¯uxed for 4 h. After
cooling, the precipitate was ®ltered o€. The organic
layer was washed with water and dried over anhydrous
sodium sulfate. Chloroform was distilled o€ under
reduced pressure. The resulting solids were recrystallized
from methanol to give 1.2 g of 25c; mp 204 ꢀC (Dec.).
1H NMR (DMSO-d6) d: 10.25 (2H, s), 8.05 (2H, s), 4.45
(4H, s), 3.13 (6H, s). Anal. calcd for C13H14N3O6S2Cl:
C, 38.28; H, 3.46 N, 10.30; Cl, 8.69; S, 15.72, found: C,
38.29; H, 3.46; N, 10.40; Cl, 8.67; S, 15.67.
9. Taguchi, H.; Katsushima, T.; Ban, M.; Watanabe, A. US
Patent 4912135.
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