2-amino-4H-3,1-benzoxazin-4-ones as inhibitors of C1r serine protease

Article abstract of DOI:10.1021/jm970394d

A series of 2-amino-4H-3,1-benzoxazin-4-ones have been synthesized and evaluated as inhibitors of the complement enzyme C1r. C1r is a serine protease at the beginning of the complement cascade, and complement activation by β-amyloid may represent a major contributing pathway to the neuropathology of Alzheimer's disease. Compounds such as 7-chloro-2-[(2- iodophenyl)amino]benz[d][1,3]oxazin-4-one (32) and 7-methyl-2-[(2- iodophenyl)amino]benz[d][1,3]oxazin-4-one (37) show improved potency compared to the reference compound FUT-175. Many of these active compounds also possess increased selectivity for C1r compared to trypsin and enhanced hydrolytic stability relative to 2-(2-iodophenyl)-4H-3,1-benzoxazin-4-one (1).

Full text of DOI:10.1021/jm970394d

1060
J . Med. Chem. 1998, 41, 1060-1067
2-Am in o-4H-3,1-ben zoxa zin -4-on es a s In h ibitor s of C1r Ser in e P r otea se
Sheryl J . Hays,*,‡ Bradley W. Caprathe,‡ J ohn L. Gilmore,‡ Nilam Amin,† Mark R. Emmerling,† Walter Michael,†
Ravi Nadimpalli,† Rathna Nath,† Kadee J . Raser,† Daniel Stafford,† Desiree Watson,† Kevin Wang,† and
J uan C. J aen‡
Departments of Chemistry and Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert
Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received J une 13, 1997
A series of 2-amino-4H-3,1-benzoxazin-4-ones have been synthesized and evaluated as inhibitors
of the complement enzyme C1r. C1r is a serine protease at the beginning of the complement
cascade, and complement activation by â-amyloid may represent a major contributing pathway
to the neuropathology of Alzheimer’s disease. Compounds such as 7-chloro-2-[(2-iodophenyl)-
amino]benz[d][1,3]oxazin-4-one (32) and 7-methyl-2-[(2-iodophenyl)amino]benz[d][1,3]oxazin-
4-one (37) show improved potency compared to the reference compound FUT-175. Many of
these active compounds also possess increased selectivity for C1r compared to trypsin and
enhanced hydrolytic stability relative to 2-(2-iodophenyl)-4H-3,1-benzoxazin-4-one (1).
In tr od u ction
C1r is a trypsin-like serine protease that is part of
the C1 complex (one C1q, two C1r, and two C1s
molecules) and is located at the beginning of the
classical complement cascade. C1r is responsible for the
proteolytic cleavage of C1s, the next protein in the
pathway.9 By selecting a target that is early in the
cascade, it should be possible to prevent many of the
amplification events that occur once the cascade is fully
activated.
6-Amidino-2-naphthyl-4-guanidinobenzoate (FUT-
175)10 and 2-(2-iodophenyl)-4H-3,1-benzoxazin-4-one (1)11
have been shown to act as inhibitors of C1r. FUT-175
has demonstrated little selectivity for C1r over other
serine proteases, while compound 1 suffers from a lack
of chemical stability. A series of 2-amino-benzo[d][3,1]-
oxazin-4-ones have been targeted to overcome these
difficulties. The synthesis and C1r inhibition for this
series of compounds are reported here.
The complement system is an important part of the
host’s immune response to foreign antigens.1 The
classical complement pathway involves the sequential
activation of nine major proteins or protein complexes
designated as C1-C9 (Figure 1). This activation re-
quires the interaction of the C1q subunit of C1 with an
antigen-antibody complex. Following activation, a
highly regulated cascade of events occurs in which
discrete interactions of soluble proteins catalyze sub-
sequent steps. The final step in the pathway is the
formation of a membrane attack complex which creates
a pore in any accessible cellular membrane causing
lethal and sublethal damage. The complement cascade
can additionally increase vascular permeability and
promote phagocytic recognition by chemotaxis.
Alzheimer’s disease (AD) is the most common degen-
erative dementia affecting primarily the elderly popula-
tion. The disease is characterized by the decline of
multiple cognitive functions and a progressive loss of
neurons in the central nervous system. Deposition of
â-amyloid peptide in the form of plaques is a classical
feature of the disease, and these deposits have recently
been shown to activate the classical complement system
(Figure 1).2,3 This activation by â-amyloid may repre-
sent a major contributing event to the neuropathology
of AD.4-6 These initial observations which suggest the
existence of an inflammatory component in the neuro-
degeneration observed in AD have been extended to the
clinic. A small clinical study using the nonsteroidal
antiinflammatory drug indomethacin indicated that
indomethacin significantly slowed the progression of the
disease.7 Additional studies with various antiinflam-
matory drugs have confirmed this initial observation.8
Specific inhibition of the complement system may
represent another approach to modulate the neuroin-
flammatory component of AD. While there are many
potential targets for the prevention of complement
activation, we have chosen to explore C1r inhibition.
Ch em istr y
2-Aminobenz[d][1,3]oxazin-4-one (2) was synthesized
from anthranilic acid and cyanogen bromide as de-
scribed previously (see Scheme 1).12 Treatment of 2
with sodium hydride and trifluoroethyl iodide afforded
the alkylated product 3.
The syntheses of the majority of the benzoxazinones
are shown in Scheme 2. An appropriately substituted
methyl anthranilate I was treated with an isocyanate
to produce the urea III in moderate to good yield.
Alternatively, the methyl anthranilate isocyanate II
(purchased or prepared from the methyl anthranilate I
and phosgene) was reacted with an amine to form the
‡ Department of Chemistry.
† Department of Neuroscience Therapeutics.
S0022-2623(97)00394-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/05/1998

Inhibitors of C1r Serine Protease
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1061
F igu r e 1. Activation of the classical complement pathway by â-amyloid.
Sch em e 1a
a
(a) CNBr, NaOH, H2O; (b) CF3CH2I, NaH, DMF, 150 °C.
Sch em e 2a
a
(a) HNR2R3, pyridine or THF or toluene; (b) R3 ) H, R2-NdCdO, pyridine or THF or toluene; (c) H2SO4, rt, N2; (d) aq NaOH, EtOH,
rt; (e) EDCI‚HCl, CH2Cl2, rt; (f) NaH, R3I, THF, 60 °C; (g) phosgene, HCl, toluene, EtOAc, reflux; (h) H2NR2, NaH, THF or DMF, 50 °C.
Method A: steps a, c. Method B: steps b, c. Method C: step h.
intermediate urea III. When R3 was hydrogen, the urea
III was cyclized with concentrated sulfuric acid to
produce the requisite benzoxazinones IV. This product
IV could be further reacted by treatment with sodium
hydride and methyl or ethyl iodide to obtain the alky-
lated derivative V. The urea III could also be cyclized
to the final heterocyclic target by a two-step procedure
using sodium hydroxide to hydrolyze the ester, followed
by ring closure with 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (EDCI) to produce the
desired benzoxazinone V. Physical data and method of
synthesis for the benzoxazinones and intermediate
ureas are summarized in Table 1.
monitored by the production of benzyl mercaptan using
standard protocols.13 All assays were performed in
triplicate at each inhibitor concentration, and the results
are expressed as the mean IC50. In all cases, standard
errors are e10% of the mean value. To determine a
compound’s stability in aqueous buffer, the benzoxazi-
none was allowed to incubate in assay buffer for 60 min,
and the enzyme assay was repeated. The loss of enzyme
inhibition from the “initial” value to the “60-min” value
is assumed to reflect the aqueous hydrolysis of the
benzoxazinone ring to its inactive acyclic form. The
results of the enzyme assays can be seen in Table 2.
To explore the benzoxazinone’s selectivity for C1r
versus other serine proteases, trypsin inhibition was
examined for the most potent of the C1r inhibitors. A
functional test for complement inhibition was performed
on selected inhibitors of C1r. Complement hemolytic
activity was measured by the hemolysis of sheep
erythrocytes that have been sensitized by specific
antibodies against the erythrocytes. The degree of
hemolysis is directly correlated to the total complement
activity. The results of the trypsin and complement
inhibition studies are shown in Table 3.
Biologica l Testin g
Benzoxazinones were assayed for inhibition of puri-
fied human C1r using CbzGly-Arg-S-Bzl as a substrate.
This substrate was chosen as a result of the more
comprehensive substrate specificity study using human
C1r described previously by McRae and co-workers.13
The compounds were dissolved in DMSO, diluted to
concentrations ranging from 1 µM to 0.01 mM with Tris
buffer, and assayed immediately. Enzyme activity was

1062 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Ta ble 1. Synthetic Data
Hays et al.
corresponding urea starting
2-amino-4H-3,1-benzoxazin-4-one product (V)
material (III)
no.
R1
R2
R3
method (yield)
mp, °C
no.
mp, °C (yield)
2
3
4
5
6
7
8
9
H
H
H
5-Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
a (92%)
b (11%)
A (80%)
Ba (35%)
c
200-202
244-245
202-203
194-197
190-191
140-143
237-239
190-224
104-108
129-130
202-203
151-153
152-155
146-149
153-155
157-159
168-169
256-258
261-263
176-179
198.5-200
251-254
276-277
326-329 dec
288 dec
290-293
287-289
231-232
187-189
188-191
225-227
179-180
235-236
203-204
275-277
174-176
156-157
214-218
214-216
237-239
186-189
173-174
177-179
121-123
162-164
190-194
209.5-211
237-238
225-229
153-156
243-246
208-211
CF3CH2
Me
(CH3)2CH
C6H5
2-I-C6H4
3-I-C6H4
4-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4CH2
2-I-C6H4CH2CH2
2-F-C6H4
54
55
56
57
58
59
60
120-121 (63%)
not taken (52%)
141-142 (85%)
162-164 (78%)
167-168 (60%)
190-192 (71%)
144-148 (80%)
Bb,d (86%)
A (81%)
A (67%)
Ab
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
b (24%)
A (92%)
Ab (94%)
A (99%)
A (79%)
A (91%)
c (34%)
A (60%)
C (11%)
A (55%)
c (62%)
B (84%)
C (57%)
A (31%)
C (15%)
C (24%)
C (18%)
C (71%)
B (46%)
B (51%)
B (63%)
B (77%)
B (34%)
B (55%)
B (56%)
C (4%)
B (79%)
B (40%)
C (15%)
B (59%)
Bd (44%)
B (72%)
B (58%)
B (37%)
B (71%)
B (63%)
Bd (64%)
B (43%)
B (47%)
Bd (53%)
B (69%)
B (56%)
B (15%)
61
62
63
64
65
66
67
169-170 (60%)
132-135 (86%)
178-179 (56%)
179-180 (25%)
not taken (27%)
201-202 (92%)
140-142 (75%)
2-Cl-C6H4
2-Br-C6H4
2-Me-C6H4
2-(OCF3)-C6H4
2-(SMe)-C6H4
2,6-diCl-C6H3
2,5-diCl-C6H3
2,6-diMe-C6H3
3-thiophenylmethyl
2-(1,3,4-thiadiazolyl)
2-(1,3,4-triazolyl)
2-thiazolidinyl
2-thiazolyl
3-tetrahydrothiophenyl
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-Cl-C6H4
2-Cl-C6H4
2,6-diCl-C6H3
2-Cl-C6H4
2-Cl-C6H4
68
69
70
205-206 (33%)
219-222 (72%)
237-239 (94%)
71
not taken (95%)
H
H
H
7-NMe2
5-Cl
6-Cl
7-Cl
6,7-diCl
5,7-diCl
5-Me
6-Me
7-Me
6-OMe
7-OMe
6,7-diOMe
7-NO2
7-F
6,7-diF
7-CF3
7-(CH3)2CH
7-cyclohexyl
6,7-CHdCH-CHdCH-
7-Cl
72
73
74
75
76
77
78
not taken (70%)
183-185 (80%)
226-227 (53%)
187-188 (25%)
212-214 (33%)
220-221 (30%)
185-187 (53%)
79
80
189-190 (64%)
not taken (46%)
81
82
83
84
85
86
87
88
89
90
91
92
93
94
178-180 (70%)
178-181 (87%)
186-187 (29%)
241-243 (19%)
177-178 (30%)
93-95 (84%)
194-198 (79%)
210-211 (36%)
207-209 (27%)
198-200 (82%)
180-183 (40%)
183-187 (43%)
186-188 (72%)
160-162 (74%)
6,7-diCl
7-Cl
7-Me
7-NO2
7-CF3
2,6-diCl-C6H3
a
b
d
Reference 12. Procedure is described in the Experimental Section. c Reference 14. Ethyl ester of the anthranilic acid.
Resu lts a n d Discu ssion
(IC50 ) 16.7 µM) are potent inhibitors of C1r as
described previously but are hydrolytically labile.11
Replacement of the aryl substituent by primary or
aliphatic amines has been used as a successful strategy
to inhibit the serine protease human elastase and
improve benzoxazinone stability12 but was unsuccessful
for inhibiting C1r (compounds 2-6, IC50 > 62.5 µM).
Additionally, benzyl- and phenethylamine derivatives
12 and 13 were also inactive.
Active benzoxazinones have been shown to exhibit a
time-dependent inhibition of C1r. This is believed to
occur via a nucleophilic attack of the active site serine
of the enzyme on the carbonyl of the heterocycle to
produce acyl enzyme intermediates. Substitution at the
2-position of the benzoxazinone is a sensitive parameter
that affects enzyme activity as well as chemical stability.
Directly bonded aryl derivatives such as compound 1

Inhibitors of C1r Serine Protease
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1063
Ta ble 2. C1r Inhibition by 2-Amino-4H-3,1-benzoxazinones
C1r inhibition (IC50, µM)
no.
R1
R2
R3
initial
60 min
FUT-175
1
2
3
4
5
6
7
8
12
16.7
>62.5
>62.5
>62.5
>62.5
>62.5
3.5
>62.5
>62.5
1.4
>62.5
>62.6
>62.5
50
12
50
H
H
H
5-Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3CH2
Me
(CH3)2CH
C6H5
2-I-C6H4
3-I-C6H4
4-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4CH2
2-I-C6H4CH2CH2
2-F-C6H4
14.7
16.6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
>62.5
8.0
15.2
38
2-Cl-C6H4
2-Br-C6H4
6.5
5.1
31
4.0
2-Me-C6H4
2-OCF3-C6H4
2-SMe-C6H4
2,6-diCl-C6H3
2,5-diCl-C6H3
2,6-diMe-C6H3
3-thiophenylmethyl
2-(1,3,4-thiadiazolyl)
2-(1,3,4-triazolyl)
2-thiazolidinyl
2-thiazolyl
3-tetrahydrothiophenyl
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-I-C6H4
2-Cl-C6H4
2-Cl-C6H4
2,6-diCl-C6H4
2-Cl-C6H4
2-Cl-C6H4
>62.5
>62.5
0.9
2.0
0.9
2.0
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
14.5
4.0
0.7
0.5
>62.5
>62.5
>62.5
0.8
H
H
H
7-N(CH3)2
5-Cl
6-Cl
7-Cl
6,7-diCl
5,7-diCl
5-Me
6-Me
7-Me
6-OMe
7-OMe
6,7-diOMe
7-NO2
7-F
6,7-diF
7-CF3
7-(CH3)2CH
7-cyclohexyl
6,7-CHdCH-CHdCH-
7-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>62.5
>62.5
0.9
>62.5
1.5
>62.5
55
>62.5
47
47
7.8
2.8
0.8
1.7
7.0
0.4
>62.5
4.0
>62.5
>62.5
0.2
16.6
0.8
0.8
2.0
0.4
1.4
1.4
1.5
6,7-diCl
7-Cl
7-Me
7-NO2
7-CF3
>62.5
0.9
0.9
>62.5
2,6-diCl-C6H4
4.2
Appropriately substituted 2-anilinobenzoxazinones
have demonstrated good activity as inhibitors of C1r.
While no inhibition was observed for the unsubstituted
aniline 6, derivatives which contain at least one ortho
substituent (compounds 7, 15, 16, 18, 20, and 21) have
demonstrated improved potency relative to the reference
agent FUT-175 (IC50 ) 12 µM).11 The best anilino
substituents included the o-iodo (7, IC50 ) 1.4 µM) and
2,6-dichloro (20, IC50 ) 0.9 µM) analogues, while the
m- and p-iodoanilino derivatives (compounds 8 and 9)
were inactive. Several ortho substituents which were
small such as fluoro or electronically more neutral such
as methyl and thiomethyl analogues (compounds 14, 17,
and 19, respectively) produced poor enzyme inhibition.
Derivatives which replaced the ortho-substituted aniline
group with a heterocycle (compounds 23-28) demon-
strated no enzyme inhibition. Changing R3 from hy-
drogen to methyl produced an equipotent derivative (10,
IC50 ) 1.4 µM), while the slightly larger ethyl replace-
ment abolished activity (11, IC50 > 62.5 µM).

1064 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Hays et al.
Ta ble 3. Biological Activity of Benzoxazinones
IC50, µM
conformation strain than the larger iodo and bromo
substituents, thereby increasing the compounds’ aque-
ous stability.
no.
initial C1r
trypsin
hemolysis
Phenyl substitution of the benzoxazinone’s benzene
ring also profoundly influences the compounds’ hydro-
lytic stability. The presence of a chloro, methyl, or
fluoro group in the 7-position greatly stabilizes the
heterocyclic ring structure to aqueous addition (com-
pounds 32, 37, and 42), while substitution at the
6-position has a deleterious effect (compounds 31, 33,
43, and 47). It is difficult to determine the influence of
substitution at the 5-position since only one analogue
FUT-175
1
12
0.017
15.3
4.5
16.6
16.7
1.4
5.1
2.0
0.8
0.75
1.7
0.4
0.8
1.5
(61%)a
(72%)a
>200
10
16
21
37
41
42
44
48
53
>100
>100
>100
>100
>100
>100
>100
87.1
(131)a
(114)a
(126%)a
NDb
(146%)a
(138%)a
(120%)a
exhibited enzymatic inhibition. This result (30, IC50
)
a
Represents percent of control at 200 µM. b ND, not determined.
14.5 µM) suggests that this substitution pattern nega-
tively influences hydrolytic stability.
In an effort to maximize C1r inhibition and to increase
inhibitor stability, a series of 2-o-chloroanilino deriva-
tives with electron-withdrawing groups in the 7-position
were synthesized. Several of these compounds substi-
tuted with either a 7-chloro (48, IC50 ) 0.8 µM; 50, IC50
) 0.4 µM), 7-methyl (51, IC50 ) 1.4 µM), or 7-trifluo-
romethyl (53, IC50 ) 1.5 µM) are among the most stable
and potent benzoxazinones tested for C1r activity. The
6,7-dichloro derivative 49 and 7-nitro analogue 52 are
potent C1r inhibitors but are relatively unstable in
aqueous media.
The benzoxazinones with the most potent C1r inhibi-
tory activity and hydrolytic stability were examined for
trypsin inhibition. This serves as a quick determination
of the selectivity of the compound as an inhibitor of C1r
versus other serine proteases. The reference compounds
FUT-175 and 1 were better or equipotent inhibitors of
trypsin compared to C1r, and it was one of the goals of
the project to improve the compounds’ selectivity. While
the N-methylated analogue 10 shows no selectivity, all
the other derivatives demonstrated increased inhibition
of C1r when compared to trypsin (see Table 3).
These same compounds were also examined in a
functional assay using erythrocyte hemolysis as a
functional end point of complement inhibition. FUT-
175 showed good activity in this model with an IC50 of
16.6 µM. The benzoxazinones, however, were margin-
ally active at 200 µM (compound 1 or 10) or showed no
activity in this model. Several possible explanations
exist for this lack of functional activity. Although these
aminobenzoxazinones showed good aqueous stability,
one of the components of this assay is human serum
and these compounds may be less stable to enzymatic
degradation. Alternatively, the inhibitors may be tightly
binding to plasma proteins. In conclusion, while the
initial goals of enhanced C1r inhibition, improved serine
protease selectivity, and increased aqueous stability
over the 2-arylbenzoxazinone series were achieved, the
compounds proved to be inactive in the functional
hemolysis assay. Additional series of benzoxazinones
are under investigation to address this problem.
Substitution of the benzoxazinone ring also greatly
affects enzyme activity. Chloro or methyl substitution
of the 5-position (30, 5-chloro, IC50 ) 14.5 µM; 34, 5,7-
dichloro, IC50 > 62.5 µM; 35, 5-methyl, IC50 > 62.5 µM)
reduced or abolished enzyme activity. This result was
in contrast with the 5-substituted benzoxazinones pre-
pared as human elastase inhibitors where 5-alkyl
substitution was highly advantageous for enzyme inhi-
bition.12 Compounds that are substituted in the 6-posi-
tion with a neutral or electron-donating group (36,
6-methyl, IC50 > 62.5 µM; 38, 6-methoxy, IC50 ) 55 µM;
40, 6,7-dimethoxy, IC50 ) 47 µM) showed little or no
activity, while electron-withdrawing groups in this
position (31, 6-chloro, IC50 ) 4.0 µM; 33, 6,7-dichloro,
IC50 ) 0.5 µM; 43, 6,7-difluoro, IC50 ) 7.0 µM) main-
tained or improved initial activity. Electron-donating
groups in the 7-position of the benzoxazinone ring
showed no activity (29, 7-dimethylamino; 39, 7-meth-
oxy), while neutral (37, 7-methyl, IC50 ) 0.8 µM) or
electron-withdrawing (32, 7-chloro, IC50 ) 0.7 µM; 41,
7-nitro, IC50 ) 0.8 µM; 42, 7-fluoro, IC50 ) 1.7 µM; 44,
7-trifluoromethyl, IC50 ) 0.4 µM) groups demonstrated
improved activity relative to the unsubstituted parent
7. Neutral substituents in the 7-position that are
relatively bulky compared to the methyl group (45,
7-isopropyl; 46, 7-cyclohexyl) demonstrated no appre-
ciable enzyme inhibition.
Stability of the compounds was assessed using the
enzyme assay as described above. The advantage of this
technique is that it provides a fast and easy method to
determine a derivative’s aqueous stability with the
disadvantage that a compound must be biologically
active for a “60-min” value to be obtained. Attempts to
determine the half-life of a compound in aqueous buffer
using ultraviolet spectroscopy were unsuccessful due to
the multiple chromophores in the molecule. Although
aliphatic amino substitution has been reported to
increase benzoxazinone stability, anilino substitution
does not necessarily improve compound stability relative
to the 2-iodophenyl derivative 1. Comparison of 1 with
the 2-substituted iodoaniline derivative 7 shows a
comparable decrease in enzyme inhibition after the 60-
min incubation. Additionally, methyl substitution of the
anilino nitrogen (compound 10) further destabilizes the
benzoxazinone ring structure. While these o-iodo de-
rivatives do not improve stability, the o-chloro analogues
do show increased hydrolytic stability with little or no
benzoxazinone breakdown after 60 min (compounds 15,
20, and 21). The smaller chloro group may provide less
Exp er im en ta l Section
Ch em istr y. All melting points were determined on a
MELT-TEMP II capillary melting point apparatus and are
uncorrected. Infrared (IR) spectra were determined as KBr
pellets on a Mattson Cygnus 100 or a Nicolet MX1 instrument.
Proton magnetic resonance (NMR) was recorded on either a
Varian XL-300 or a Bruker AM250 spectrometer; chemical
shifts are reported in δ units relative to internal tetrameth-

Inhibitors of C1r Serine Protease
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1065
ylsilane. All mass spectra were obtained on a Finnigan 4500
GCMS or a VG analytical 7070 E/F spectrometer. Elemental
analyses were performed on a CEC model 240 elemental
analyzer and are within 0.4% of the theoretical values unless
otherwise indicated. Medium-pressure liquid chromatography
utilized E. Merck silica gel, 230-400 mesh. All reactions were
run under N2, unless indicated otherwise. All analytical
(C,H,N) and spectroscopic (1H NMR, IR, MS) data are in
agreement with the proposed structures.
of 1 M HCl in ether solution) in 500 mL of toluene was added
dropwise a 12.5% solution of phosgene in toluene (26 mL, 32.8
mmol) at room temperature. The mixture was refluxed for 1
h, cooled to room temperature, treated with another portion
of phosgene in toluene solution (26 mL), and refluxed for an
additional 12 h. The cloudy solution was concentrated,
suspended into 250 mL of hexanes, and filtered through a pad
of Celite. The filtrate was concentrated and distilled (bp 50-
60 °C, 0.50-0.25 mmHg) to give 5.0 g (78%) of 2-iodophenyl
isocyanate as a colorless liquid: 1H NMR (CDCl3) δ 6.88-6.92
(m, 1H), 7.13 (dd, 1H, J ) 1.4, 8.0 Hz), 7.27-7.31 (m, 1H),
7.79 (dd, 1H, J ) 1.4, 8.0 Hz). Anal. Calcd for C7H4INO: C,
34.32; H, 1.65; N, 5.72. Found: C, 34.38; H, 1.68; N, 5.67.
Meth od A. 2-[[2-(2-Iod op h en yl)eth yl]a m in o]ben z[d ]-
[1,3]oxa zin -4-on e (13). Thionyl chloride (13.9 mL, 190.6
mmol) was added dropwise to a stirring solution of 2-iodophen-
ylacetic acid (5.0 g, 19.1 mmol) in 500 mL of methylene
chloride. The solution was refluxed for 4 h, cooled, and
concentrated to a light-brown oil. Cold concentrated am-
monium hydroxide (100 mL) was carefully added dropwise.
Solid formed, and the mixture was filtered, washed with water,
and vacuum-dried to yield 4.18 g (84%) of 2-(2-iodophenyl)-
A solution of 2-iodophenyl isocyanate (3.36 g, 13.7 mmol)
and ethyl 2-aminobenzoate (5.1 mL, 34.5 mmol) in 250 mL of
THF was stirred at room temperature for 24 h. The solution
was concentrated and then partitioned between ethyl acetate
and 10% HCl. The ethyl acetate extract was washed with
brine, dried (MgSO4), and concentrated to an oily white solid.
The sample was suspended in ether and the insoluble white
solid that resulted from filtration yielded 4.38 g (78%) of ethyl
1
acetamide as a tan solid: mp 180-182 °C; H NMR (DMSO-
d6) δ 3.56 (s, 2H), 6.96-7.00 (m, 2H), 7.30-7.36 (m, 2H), 7,43
(bs, 1H), 7.82 (dd, 1H, J ) 0.8, 8.0 Hz). Anal. Calcd for C8H8-
INO: C, 36.81; H, 3.09; N, 5.37. Found: C, 36.90; H, 3.09; N,
5.22.
1
2-[3-(2-iodophenyl)ureido]benzoate (57): mp 162-164 °C; H
NMR (CDCl3) δ 1.41 (t, 3H, J ) 7.2 Hz), 4.37 (q, 2H, J ) 7.2
Hz), 6.82-6.87 (m, 2H), 7.01-7.05 (m, 1H), 7.34-7.38 (m, 1H),
7.51-7.55 (m, 1H), 7.80 (dd, 1H, J ) 1.4, 8.0 Hz), 7.99 (dd,
1H, J ) 1.7, 8.2 Hz), 8.03 (dd, 1H, J ) 1.7, 8.0 Hz), 8.51 (dd,
Boron trifluoride etherate (9.2 mL, 74.8 mmol) was added
dropwise to a stirring suspension of 2-(2-iodophenyl)acetamide
(3.9 g, 15.0 mmol) and sodium borohydride (2.1 g, 56.0 mmol)
in 500 mL of THF maintained at 0 °C. The mixture was
allowed to warm to room temperature overnight. The mixture
was cooled to 0 °C, carefully quenched by dropwise addition
of water (50 mL), concentrated to remove the THF, basified
with concentrated ammonium hydroxide to a pH of 9-10, and
then extracted into ethyl acetate. The ethyl acetate extract
was washed with brine, dried (MgSO4), filtered, and concen-
trated to a hygroscopic white solid. The HCl salt was made
and crystallized from ethanol to give 3.1 g (73%) of 2-(2-
iodophenyl)ethylamine hydrochloride as a white solid: mp
251-255 °C; 1H NMR (DMSO-d6) δ 2.96-3.04 (m, 4H), 7.01-
7.05 (m, 1H), 7.34-7.41 (m, 2H), 7.87 (dd, 1H, J ) 0.8, 7.8
Hz), 8.24 (bs, 3H). Anal. Calcd for C8H10IN‚HCl: C, 33.89;
H, 3.91; N, 4.94. Found: C, 34.05; H, 3.83; N, 4.88.
A solution of 2-carbomethoxyphenyl isocyanate (0.55 g, 3.10
mmol), 2-(2-iodophenyl)ethylamine hydrochloride (0.80 g, 2.82
mmol), and N,N-diisopropylethylamine (0.75 mL, 4.30 mmol)
in 100 mL of THF was stirred at room temperature for 24 h.
The solution was concentrated and then partitioned between
ethyl acetate and 10% HCl. The ethyl acetate extract was
washed with brine, dried (MgSO4), and concentrated. The
residue was chromatographed (silica gel, 20% ethyl acetate
in hexanes) to yield 1.02 g (86%) of methyl 2-[3-[2-(2-iodophen-
yl)ethyl]ureido]benzoate (62) as a white solid: mp 132-135
°C; 1H NMR (DMSO-d6) δ 2.88 (t, 2H, J ) 7.4 Hz), 3.28-3.30
(m, 2H), 3.86 (s, 3H), 6.96-7.01 (m, 2H), 7.31-7.38 (m, 2H),
7.48-7.53 (m, 1H), 7.66 (bs, 1H), 7.84 (dd, 1H, J ) 0.8, 7.8
Hz), 7.89 (dd, 1H, J ) 1.6, 8.1 Hz), 8.36 (dd, 1H, J ) 0.8, 8.6
Hz), 9.75 (bs, 1H). Anal. Calcd for C17H17IN2O3: C, 48.13; H,
4.04; N, 6.60. Found: C, 48.16; H, 4.09; N, 6.64.
1H, J ) 1.0, 8.4 Hz), 10.76 (s, 1H). Anal. Calcd for C16H15
-
IN2O3: C, 46.85; H, 3.69; N, 6.83. Found: C, 46.75; H, 3.62;
N, 6.56.
A solution of ethyl 2-[3-(2-iodophenyl)ureido]benzoate (57;
0.50 g, 1.22 mmol) in concentrated sulfuric acid (10 mL, 181.0
mmol) was stirred at 50 °C for 1 h. The solution was cooled
to room temperature and poured into ice-water. After
neutralization with saturated NaHCO3 solution, the product
was collected by filtration. The solid was washed with water
and vacuum-dried to yield 0.38 g (86%) of 7 as an off-white
solid: mp 140-143 °C; FT-IR (KBr) 1763 cm-1 1H NMR
;
(CDCl3) δ 6.85-6.90 (m, 1H), 7.26-7.32 (m, 2H), 7.41-7.45
(m, 1H), 7.68-7.73 (m, 1H), 7.82 (dd, 1H, J ) 1.4, 8.0 Hz),
8.11 (dd, 1H, J ) 1.6, 7.8 Hz), 8.44 (dd, 1H, J ) 1.6, 8.1 Hz).
Anal. Calcd for C14H9IN2O2: C, 46.18; H, 2.49; N, 7.69.
Found: C, 46.13; H, 2.43; N, 7.51.
2-[N-(2-Iodoph en yl)-N-m eth ylam in o]ben z[d][1,3]oxazin -
4-on e (10). A solution of 2-iodoaniline (6.0 g, 27.4 mmol) and
ethyl formate (11.1 mL, 137.4 mmol) in 200 mL of THF was
added dropwise to a stirring suspension of sodium hydride (1.4
g, 34.2 mmol, 60% dispersion in mineral oil washed twice with
50 mL of hexanes) in 250 mL of THF at room temperature.
The mixture was stirred for 24 h, cooled in an ice bath, and
quenched by dropwise addition of water. The sample was
concentrated (to remove the THF) and then partitioned
between ethyl acetate and saturated KH2PO4 solution. The
ethyl acetate extract was washed with brine, dried (MgSO4),
filtered, and concentrated to give 6.3 g (93%) of N-(2-iodophen-
yl)formamide as an off-white solid: mp 118-119 °C; 1H NMR
(DMSO-d6) δ 6.93-7.02 (m, 1H), 7.33-7.41 (m, 1H), 7.78 (dd,
1H, J ) 1.1, 8.1 Hz), 7.88 (dd, 1H, J ) 1.1, 7.8 Hz), 8.34 (s,
1H), 9.54 (bs, 1H). Anal. Calcd for C7H6INO: C, 34.03; H,
2.45; N, 5.67. Found: C, 34.16; H, 2.47; N, 5.67.
A suspension of sodium borohydride (3.6 g, 95.7 mmol) and
N-(2-iodophenyl)formamide (6.3 g, 25.5 mmol) in 500 mL of
THF was treated dropwise with boron trifluoride etherate (15.7
mL, 127.6 mmol) at 0 °C. The mixture was allowed to warm
to room temperature overnight. The mixture was cooled to 0
°C and carefully quenched by dropwise addition of water (125
mL). The THF was removed under vacuum, and the residue
was basified with concentrated ammonium hydroxide to a pH
of 9-10 and then extracted with ethyl acetate. The organic
extract was washed with brine, dried (MgSO4), filtered, and
concentrated to a tan oil. The residue was treated with HCl
and crystallized from ethanol to give 5.1 g (74%) of N-methyl-
2-iodoaniline hydrochloride as a white solid: mp 154-155 °C
dec; 1H NMR (DMSO-d6) δ 2.77 (s, 3H), 6.48-6.52 (m, 1H),
6.70 (d, 1H, J ) 8.0 Hz), 7.24-7.28 (m, 1H), 7.66 (dd, 1H, J )
Concentrated sulfuric acid (3.0 mL, 54.3 mmol) was added
to 62 (0.60 g, 1.41 mmol), and the solid slowly dissolved. The
sample was stirred at room temperature for 1.5 h and then
carefully poured into a vigorously stirred mixture of sodium
bicarbonate (14.0 g, 166.6 mmol, ca. 3 equiv vs H2SO4 used)
in water (250 mL) and ethyl acetate (250 mL). The ethyl
acetate extract was washed with brine, dried (MgSO4), filtered,
and concentrated to afford 0.52 g (94%) of the title compound
as a white solid: mp 151-153 °C; FT-IR (KBr) 3298, 1746,
1632, 1476 cm-1; 1H NMR (CDCl3) δ 3.09-3.13 (m, 2H), 3.68-
3.73 (m, 2H), 4.97 (bs, 1H), 6.91-6.95 (m, 1H), 7.16-7.24 (m,
1H), 7.24-7.32 (m, 3H), 7.62-7.66 (m, 1H), 7.83 (d, 1H, J )
8.0 Hz), 8.02 (dd, 1H, J ) 1.5, 7.8 Hz). Anal. Calcd for C16H13
-
IN2O2: C, 49.00; H, 3.34; N, 7.14. Found: C, 48.93; H, 3.25;
N, 7.07.
Meth od B. 2-[(2-Iodoph en yl)am in o]ben z[d][1,3]oxazin -
4-on e (7). To a suspension of 2-iodoaniline hydrochloride
(prepared from 5.7 g, 26.0 mmol, of 2-iodoaniline and 33 mL

1066 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Hays et al.
1.4, 7.7 Hz), 7.80 (bs, 2H). Anal. Calcd for C7H8IN‚HCl: C,
1H), 7.93-7.96 (dd, 1H, J ) 1.4, 8.4 Hz), 11.5 (s, 1H). Anal.
Calcd for C13H10N2O2S: C, 60.45; H, 3.90; N, 10.85. Found:
C, 60.24; H, 3.92; N, 10.72.
31.20; H, 3.37; N, 5.20. Found: C, 30.90; H, 3.20; N, 5.17.
A solution of 2-carbomethoxyphenyl isocyanate (0.93 g, 5.25
mmol), N-methyl-2-iodoaniline hydrochloride (1.56 g, 5.79
mmol), and N,N-diisopropylethylamine (1.1 mL, 6.32 mmol)
in 100 mL of toluene was refluxed for 36 h. The solution was
concentrated and then partitioned between chloroform and
saturated KH2PO4 solution. The chloroform extract was
washed with brine, dried (MgSO4), and concentrated. The
residue was chromatographed (silica gel, CH2Cl2) to yield 1.18
g (55%) of methyl 2-[3-(2-iodophenyl)-3-methylureido]benzoate
2-[(2,2,2-Tr iflu or oet h yl)a m in o]b en z[d ][1,3]oxa zin -4-
on e (3). 2-Aminobenz[d][1,3]oxazin-4-one (2; 1.0 g, 6.2 mmol),
trifluoroethyl iodide (2.6 g, 12.4 mmol), and sodium hydride
(0.32 g of a 60% oil dispersion, 8.0 mmol) were combined with
DMF (25 mL) in a rocking autoclave and heated to 150 °C for
4 h. The reaction mixture was cooled and partitioned between
CH2Cl2 (200 mL) and water (2 × 200 mL). The organic layer
was dried (MgSO4), and the volatiles were removed under
reduced pressure. The crude residue was chromatographed
(silica gel, 25% ethyl acetate in hexanes). The product was
then recrystallized from ethyl acetate-hexanes to afford 170
mg (11%) of 2-[(2,2,2-trifluoroethyl)amino]benz[d][1,3]oxazin-
4-one as a solid: mp 244-245 °C; FT-IR (KBr) 3075, 2946,
1
as a light-yellow solid: mp 144-148 °C; H NMR (CDCl3) δ
3.29 (s, 3H), 3.67 (s, 3H), 6.91-6.95 (m, 1H), 7.12-7.16 (m,
1H), 7.41 (dd, 1H, J ) 1.6, 7.8 Hz), 7.46-7.52 (m, 2H), 7.87
(dd, 1H, J ) 1.7, 8.0 Hz), 7.99 (dd, 1H, J ) 1.4, 8.0 Hz), 8.60
(dd, 1H, J ) 1.0, 8.7 Hz), 9.91 (bs, 1H). Anal. Calcd for
1
1728, 1669, 1453 cm-1; H NMR (DMSO-d6) δ 4.68-4.78 (q,
C
16H15IN2O3: C, 46.85; H, 3.69; N, 6.83. Found: C, 47.15; H,
2H, J ) 9.2, 18.4 Hz), 7.20-7.28 (m, 2H), 7.69-7.75 (m, 1H),
7.95-7.98 (d, 1H, J ) 7.9 Hz), 11.70 (bs, 1H). Anal. Calcd
for C10H7F3N2O2: C, 49.19; H, 2.89; N, 11.47. Found: C, 49.11;
H, 3.07; N, 11.50.
3.77; N, 6.86.
A mixture of methyl 2-[3-(2-iodophenyl)-3-methylureido]-
benzoate (0.50 g, 1.22 mmol) in 20 mL of ethanol and 0.1 N
NaOH (17 mL, 1.70 mmol) was refluxed for 30 min. The
solution was concentrated to remove the ethanol, acidified with
10% HCl to pH 1-2, and then extracted into ethyl acetate.
The organic extract was washed with brine, dried (MgSO4),
filtered, and concentrated to give 2-[3-(2-iodophenyl)-3-methyl-
ureido]benzoic acid as a light-yellow solid. The solid was
dissolved into 150 mL of chloroform (ethanol-free) and treated
with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride (0.26 g, 1.36 mmol). The solution was stirred at room
temperature for 2 h and washed with saturated NaHCO3
solution. The organic extract was dried (MgSO4) and concen-
trated to give a colorless oil, which upon crystallization from
ether-hexanes afforded 0.42 g (91%) of 2-[N-(2-iodophenyl)-
2-[N-Eth yl-N-(2-iod op h en yl)a m in o]ben z[d ][1,3]oxa zin -
4-on e (11). 2-[(2-Iodophenyl)amino]benz[d][1,3]oxazin-4-one
(7; 0.308 g, 0.85 mmol) was dissolved in THF (5 mL), and NaH
(0.040 g of a 60% oil dispersion, 1.0 mmol) was added. The
reaction mixture was stirred for 30 min, and ethyl iodide (0.312
g, 2.0 mol) was added. The reaction mixture was heated to
60 °C and stirred for 24 h. The reaction mixture was
partitioned between water (200 mL) and ethyl acetate (200
mL). The ethyl acetate layer was dried (MgSO4), and the
volatiles were removed under reduced pressure. The crude
residue was chromatographed (silica gel, 30% ethyl acetate
in petroleum ether). This residue was then recrystallized from
ether-hexanes to afford 80 mg (25%) of the desired product
(11) as a white solid: mp 129-130 °C; FT-IR (KBr) 3422, 2980,
1713, 1664, 1609, 1481 cm-1; 1H NMR (CDCl3) δ 1.39-1.43 (t,
3H, J ) 7.0 Hz), 4.17-4.35 (m, 2H), 7.14-7.19 (m, 1H), 7.28-
7.34 (m, 2H), 7.48-7.53 (m, 1H), 7.72-7.77 (m, 1H), 7.96-
7.98 (dd, 1H, J ) 1.4, 8.2 Hz), 8.28-8.31 (dd, 1H, J ) 1.4, 8.0
Hz). Anal. Calcd for C16H13IN2O2: C, 49.00; H, 3.34; N, 7.14.
Found: C, 49.24; H, 3.52; N, 7.03.
N-methylamino]benz[d][1,3]oxazin-4-one as
a light-yellow
solid: mp 104-108 °C dec; FT-IR (KBr) 1749, 1620, 1603 cm-1
;
1H NMR (CDCl3) δ 3.44 (s, 3H), 7.08-7.12 (m, 1H), 7.17-7.24
(m, 1H), 7.31-7.46 (m, 3H), 7.62-7.65 (m, 1H), 7.93 (dd, 1H,
J ) 1.2, 8.0 Hz), 8.02 (dd, 1H, J ) 1.2, 8.0 Hz). Anal. Calcd
for C15H11IN2O2: C, 47.64; H, 2.93; N, 7.41. Found: C, 47.50;
H, 3.00; N, 7.37.
Meth od C. 2-[[(3-Th iop h en eyl)m eth yl]a m in o]ben z[d ]-
[1,3]oxa zin -4-on e (23). 3-Thiophenecarboxaldehyde (10.0 g,
89 mmol), hydroxylamine hydrochloride (18.8 g, 270 mmol),
and sodium acetate (22.1 g, 270 mmol) were combined in
ethanol (175 mL) and water (175 mL) and refluxed overnight.
The reaction mixture was cooled, and most of the ethanol was
removed in vacuo. The sample was extracted into ethyl acetate
(200 mL). The organic layer was dried (MgSO4), and the
volatiles were removed under reduced pressure. Water (400
mL) was added to the residue, and a crystalline product was
obtained. The product was filtered and recrystallized from
ethyl acetate-hexanes to afford 3.0 g (27%) of the oxime of
3-thiophenecarboxaldehyde. This oxime (1.03 g, 8.1 mmol) was
dissolved in methanol saturated with ammonia (100 mL) and
hydrogenated with Raney nickel (1.0 g). The reaction mixture
was filtered, and the volatiles were removed under reduced
pressure. A 1 N solution of HCl in ether (150 mL) was added
to the crude residue, and a precipitate formed. The solid was
filtered and washed well with ether to afford 0.582 g (49%) of
(3-thienyl)methylamine hydrochloride as a solid: 1H NMR
(DMSO-d6) δ 3.95-4.05 (s, 2H), 7.2-7.3 (m, 1H), 7.5-7.7 (m,
2H), 8.3-8.7 (bs, 1H).
P r ep a r a tion of Sta r tin g Ma ter ia ls. Meth yl 2-Am in o-
4-isop r op ylben zoa te (Sta r tin g Ma ter ia l for 45 a n d 86).
Nitric acid (63 g, 1 mol) was added to a solution of 4-isopro-
pylbromobenzene (50 g, 0.25 mol) dropwise at 0 °C. The
reaction mixture was stirred for 3 h, poured onto ice, and
extracted with ether (2 × 500 mL). The organic layer was
dried (MgSO4), and the volatiles were removed under reduced
pressure. The crude residue was distilled (1 mmHg, 105-129
°C) to afford 19.5 g (32%) of 2-nitro-4-isopropylbromoben-
1
zene: FT-IR (LF) 2966, 1535, 1358 cm-1; H NMR (CDCl3) δ
1.26-1.28 (d, 6H, J ) 7.0 Hz), 2.93-3.00 (m, 1H), 7.28-7.31
(dd, 1H, J ) 2.2, 8.2 Hz), 7.62-7.64 (d, 1H, J ) 8.4 Hz), 7.68-
7.69 (d, 1H, J ) 2.2 Hz). Anal. Calcd for C9H10BrNO2: C,
44.29; H, 4.13; N, 5.74. Found: C, 43.91; H, 4.10; N, 5.48.
2-Nitro-4-isopropylbromobenzene (19.5 g, 80 mmol) and
copper(I) cyanide (14.3 g, 160 mmol) were refluxed in DMF
(250 mL) for 24 h. The reaction mixture was poured into water
(500 mL) and extracted with ether (3 × 500 mL). The
combined organic layers were filtered through Celite and
washed with water (3 × 500 mL). The organic layer was dried
(MgSO4), and the volatiles were removed under reduced
pressure. The crude residue was chromatographed (silica gel,
To a suspension of (3-thienyl)methylamine hydrochloride
(0.57 g, 3.80 mmol) in 10 mL of DMF was added sodium
hydride (0.34 g of 60% dispersion in mineral oil, 8.50 mmol)
at room temperature. After bubbling ceased, 2-carbomethox-
yphenyl isocyanate (0.34 g, 8.4 mmol) was added. The sample
was heated at 75 °C for 5 days, cooled to room temperature,
and poured into water. A precipitate formed which was
recrystallized from methanol-ethyl acetate to give 0.56 g
(57%) of 2-[[(3-thiopheneyl)methyl]amino]benz[d][1,3]oxazin-
CH2Cl2-petroluem ether, 1:1) to afford 4.3
g (28%) of
2-nitro-4-isopropylbenzonitrile: FT-IR (KBr) 2969, 1539, 1344
cm-1; 1H NMR (CDCl3) δ 1.32-1.34 (d, 6H, J ) 7.0 Hz), 3.08-
3.13 (m, 1H), 7.65-7.68 (dd, 1H, J ) 1.7, 8.0 Hz), 7.82-7.84
(d, 1H, J ) 8.0 Hz), 8.18-8.19 (d, 1H, J ) 1.7 Hz). Anal. Calcd
for C10H10N2O2: C, 63.15; H, 5.30; N, 14.73. Found: C, 63.13;
H, 5.22; N, 14.90.
2-Nitro-4-isopropylbenzonitrile (4.3 g, 22.6 mmol) was re-
fluxed overnight in a mixture of water-acetic acid-H2SO4 (2:
1:1, 60 mL). The reaction mixture was poured onto ice water
(200 mL), basified with 1 N NaOH, and extracted with ether
1
4-one (23) as a solid: mp 251-254 °C; H NMR (DMSO-d6) δ
5.07 (s, 2H), 7.09-7.11 (dd, 1H, J ) 1.2, 4.8 Hz), 7.18-7.23
(m, 2H), 7.37-7.38 (m, 1H), 7.45-7.47 (m, 1H), 7.63-7.68 (m,

Inhibitors of C1r Serine Protease
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1067
(250 mL). The aqueous layer was separated, acidifed with
concentrated HCl, and extracted with ether (250 mL). The
organic layer was dried (MgSO4), and the volatiles were
removed under reduced pressure to afford 4.8 g (68%) of
2-nitro-4-isopropylbenzoic acid as a solid: FT-IR (KBr) 2968,
2663, 1703, 1535, 1356 cm-1; 1H NMR (DMSO-d6) δ 1.22-1.25
(d, 6H, J ) 7.0 Hz), 3.02-3.10 (m, 1H), 7.65-7.68 (dd, 1H, J
of 595 nm. Based on a plot of percent inhibition of trypsin
against log [inhibitor], an IC50 was generated using Sigma plot.
Data are the average of two to four determinations with data
ranges within 20% of the average values.
Hem olysis Assa y: The hemolysis assay was performed
using a DiaMedix EZ Complement kit. Compounds (5 µL) to
be tested were added to a test tube containing a standardized
suspension of sheep erythrocytes sensitized with antibodies
to sheep erythrocytes. The mixture was incubated for 60 min
at 25 °C and then centrifuged. The absorbance of the super-
nate was read at 405 nm. The results are expressed as percent
of control at 200 µM.
) 1.4, 8.0 Hz), 7.79-7.83 (m, 2H). Anal. Calcd for C10H11
-
NO4: C, 57.41; H, 5.30; N, 6.70. Found: C, 57.78; H, 5.46; N,
6.84.
2-Nitro-4-isopropylbenzoic acid (3.09 g, 14.8 mmol) was
hydrogenated with Raney nickel in water (100 mL) and NH4-
OH (10 mL). The reaction mixture was filtered and acidified
with concentrated HCl. A precipitate formed and was collected
by filtration. The solid was heated in MeOH and filtered again
to afford 1.2 g (45%) of 2-amino-4-isopropylbenzoic acid
hydrochloride, which was carried on without further purifica-
tion: 1H NMR (DMSO-d6) δ 1.05-1.20 (d, 6H, J ) 7.0 Hz),
2.6-2.8 (m, 1H), 6.3-6.5 (m, 2H), 7.6-7.7 (m, 3H).
Refer en ces
(1) For reviews see: Tomlinson, S. Complement defense mecha-
nisms. Curr. Opin. Immunol. 1993, 5, 83-89. (b) Benjamini, E.;
Leskowitz, S. Immunology: A Short Course; Wiley-Liss: New
York, 1991; pp 135-150.
(2) Rogers, J .; Cooper, N. R.; Webster, S.; Schultz, J .; McGeer, P.
L.; Styren, S. D.; Civin, W. H.; Brachova, L.; Bradt, B.; Ward,
P.; Lieberburg, I. Complement activation by â-amyloid in Alzhe-
imer’s disease. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 10016-
10020.
(3) J iang, H.; Burdick, D.; Glabe, C. G.; Cotman, C. W.; Tenner, A.
â-Amyloid activates complement by binding to a specific region
of the collagen-like domain of the C1q A chain. J . Immunol.
1994, 152, 5050-5059.
(4) Kalaria, R. N. The immunopathology of Alzheimer’s disease and
some related disorders. Brain Pathol. 1993, 3, 333-347.
(5) McGeer, P. L.; McGeer, E. G. The inflammatory response system
of brain: Implications for therapy of Alzheimer and other
neurodegenerative diseases. Brain Res. Rev. 1995, 21, 195-218.
(6) Eikelenboom, P.; Zhan, S.-S.; van Gool, W. A.; Allsop, D.
Inflammatory mechanisms in Alzheimer’s disease. Trends Phar-
macol. Sci. 1994, 15, 447-450.
(7) Rogers, J .; Kirby, L. C.; Hempelman, S. R.; Berry, D. L.; McGeer,
P. L.; Kaszniak, A. W.; Zalinski, J .; Cofield, M.; Mansukhani,
L.; Willson, P.; Kogan, F. Clinical trial of indomethacin in
Alzheimer’s disease. Neurology 1993, 43, 1609-1611.
(8) McGeer, P. L.; Schulzer, M.; McGeer, E. G. Arthritis and
antiinflammatory agents as possible protective factors for Alzhe-
imer’s disease: A review of 17 epidemiologic studies. Neurology
1996, 47, 425-432.
(9) Fothergill, J .; Kemp, G.; Paton, N.; Carter, P.; Gray, P. The
structures of human C1r and C1s and their relationship to other
serine proteases. Behring Inst. Mitt. 1989, 84, 72-79.
(10) Aoyama, T.; Ino, Y.; Ozeki, M.; Oda, M.; Sato, T.; Koshiyama,
Y.; Suzuki, S.; Fujita, M. Pharmacological studies of FUT-175,
nafamstat mesilate I. Inhibition of protease activity in in vitro
and in vivo experiments. J pn. J . Pharmacol. 1984, 35, 203-
227.
(11) Gilmore, J . L.; Hays, S. J .; Caprathe, B. W.; Lee, C.; Emmerling,
M. R.; Michael, W.; J aen, J . C. Synthesis and evaluation of
2-aryl-4H-3,1-benzoxazin-4-ones as C1r serine protease inhibi-
tors. Bioorg. Med. Chem. Lett. 1996, 6, 679-682.
(12) Krantz, A.; Spencer, R. W.: Tam, T. F.; Liak, T. J .; Copp, L. J .;
Thomas, E. M.; Tafferty, S. P. Design and synthesis of 4H-3,1-
benzoxazin-4-ones as potent alternate substrate inhibitors of
human leukocyte elastase. J . Med. Chem. 1990, 33, 464-479.
(13) McRae, B. J .; Lin, T. Y.; Powers, J . C. Mapping the substrate
binding site of human C1r and C1s with peptide thioesters.
Development of new sensitive substrates. J . Biol. Chem. 1981,
256, 12362-12366.
2-Amino-4-isopropylbenzoic acid hydrochloride was refluxed
in H2SO4 (20 mL) and methanol (80 mL) overnight. The
reaction mixture was poured onto ice water (400 mL) and
extracted with methylene chloride (200 mL). The organic layer
was washed with saturated NaHCO3 (200 mL) and dried
(MgSO4), and the volatiles were removed under reduced
pressure to afford 0.805 g (62%) of methyl 2-amino-4-isopro-
pylbenzoate as a solid: mp 40-41 °C; FT-IR (KBr) 3472, 3367,
1
2960, 1691, 1622, 1437, 1307 cm-1; H NMR (CDCl3) δ 1.1-
1.34 (d, 6H, J ) 7.0 Hz), 2.7-2.9 (m, 1H), 3.85 (s, 3H), 6.5-
6.6 (m, 2H), 7.7-7.87 (d, 1H). Anal. Calcd for C11H15NO2: C,
72.07; H, 8.21; N, 6.00. Found: C, 71.87; H, 8.04; N, 5.97.
Biologica l Meth od s. In h ibition of C1r (In itia l): Com-
pounds to be tested were dissolved in DMSO at concentrations
ranging from 1 mM to 0.01 mM, and a 10-µL aliquot of each
concentration was deposited into a well of a 96-well microtiter
plate; 50 µL of Cbz-Gly-Arg-S-Bzl (1.5 mM in DMSO/H2O, 3:7)
and 50 µL of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) solution
(0.75 mM in 150 mM Tris buffer) were dispensed in the wells
of the microtiter plate, followed by the immediate addition of
50 µL of the C1r enzyme solution (20 µg/mL purified human
C1r in 10 mM Tris buffer (pH 7.4)). After a 30-s incubation,
the hydrolyzed substrate was read in the kinetic mode using
a Molecular Devices Thermomax microplate reader set to a
wavelength of 405 nm. The assay background was followed
by monitoring wells that contain substrate, buffer, and DTNB
solutions but no enzyme. All assays were performed in
triplicate at each inhibitor concentration tested. The results
are expressed in IC50 values.
In h ibition of C1r (60 m in ): A 50-µL aliquot of 150 mM
Tris buffer (pH 7.5) was added to wells containing 10 µL of
inhibitor in DMSO. The mixture sat at room temperature for
60 min; then 50 µL of substrate solution, 50 µL of DTNB, and
50 µL of enzyme solution were added to the wells to start the
reaction. The plate was read as described above.
In h ibition of Tr yp sin : A microplate caseinolysis assay
was carried out as previously described.15 Briefly, 0.5 mg/mL
casein (sodium salt; Sigma Co.), 20 mM dithiothreitol, and 50
mM Tris-HCl (pH 7.4) were mixed in the presence of various
concentrations of an inhibitor. After 1.25 µg of trypsin (bovine
pancreas; Sigma Co.) was added, the microtiter plate (250 µL)
was incubated for 60 min at 25 °C. The samples were
processed for colorimetric development and read on a Molec-
ular Devices Thermomax microplate reader at an absorbance
(14) Papadopoulos, E. P.; Torres, C. D. Convenient preparation of
N-substituted 2- amino-4H-3,1-benzoxazin-4-ones and 3-substi-
tuted 2, 4(1H,3H)-quinazoline-diones. J . Heterocycl. Chem. 1982,
19, 269-272.
(15) Buroker-Kilgore, M.; Wang, K. K. W. A Coomassie brilliant blue
G-250-based colorimetric assay for measuring activity of calpain
and other proteases. Anal. Biochem. 1993, 208, 389-392.
J M970394D