1,5-benzoxazepines vs 1,5-benzodiazepines. one-pot microwave-assisted synthesis and evaluation for antioxidant activity and lipid peroxidation inhibition

Article abstract of DOI:10.1021/jm100739n

Amino-1,5-benzoxazepines 2 and 5 and hydroxyl-1,5-benzodiazepines 3 and 6 have been synthesized in one-pot solvent-free conditions from 2,3-diaminophenol and ketones through microwave assisted acid catalysis, the benzoxazepine/ benzodiazepine ratio depend

Full text of DOI:10.1021/jm100739n

J. Med. Chem. 2010, 53, 8409–8420 8409
DOI: 10.1021/jm100739n
1,5-Benzoxazepines vs 1,5-Benzodiazepines. One-Pot Microwave-Assisted Synthesis and Evaluation
for Antioxidant Activity and Lipid Peroxidation Inhibition
Constantinos G. Neochoritis,† Constantinos A. Tsoleridis,*,† Julia Stephanidou-Stephanatou,*,† Christos A. Kontogiorgis,‡
and Dimitra J. Hadjipavlou-Litina‡
†Laboratory of Organic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Macedonia, Greece, and
‡Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Macedonia, Greece
Received June 18, 2010
Amino-1,5-benzoxazepines 2 and 5 and hydroxyl-1,5-benzodiazepines 3 and 6 have been synthesized in one-
pot solvent-free conditions from 2,3-diaminophenol and ketones through microwave assisted acid catalysis,
the benzoxazepine/benzodiazepine ratio depending on the R1 and R3 aryl substituents. The otherwise
inaccessible and unknown 2,2-dimethyl-4-aryl-1,5-benzodiazepines 8 were also prepared in an analogous
manner. The reaction mechanism was investigated by means of DFT calculations. Structural assignments of
the new compounds as well as complete assignment of 1H and 13C NMR signals have been unambiguously
achieved on the basis of the analysis of their 1H and 13C NMR (1D and 2D), IR, MS, and elemental analysis
data, whereas the presence of an amino group in 5 and of a hydroxyl in 6 was confirmed by derivatization.
Compounds 2, 3, 5f, 6a, 6c, 6d, 6f, 6h, 8c, and 12 were evaluated as antioxidants and lipid peroxidation
inhibitors in vitro. Compound 6f was also evaluated as anti-inflammatory agent in vivo. Compounds 2 and
6f were found to be the most potent as inhibitors of lipoxygenase and of lipid peroxidation, respectively.
Introduction
Some time ago, we described a facile synthesis of 2,3-dihydro-
1H-1,5-benzodiazepines by condensation of ketones with o-phe-
nylenediamines by application of microwave irradiation.7 In
the meantime, although many methods for the preparation of
benzodiazepines using o-phenylenediamines as starting materi-
als appeared in the literature,8 the possibility of benzodiazepine
formation bearing a hydroxyl in the phenylenediamine moiety
was never investigated. The use of 2,3-diaminophenol, instead
of o-phenylenediamine, could lead to the formation of either
hydroxybenzodiazepines or aminobenzoxazepines, so the whole
project looked very interesting.
Heterocyclic compounds are highly ranked among phar-
maceutically important natural and synthetic materials. The
remarkable ability of heterocyclic nuclei to serve as both
biomimetics and active pharmacophores has largely contrib-
uted to their unique value as traditional key elements of
numerous drugs. Heterocyclic derivatives such as morphine
alkaloids, β-lactam antibiotics, and benzodiazepines are just a
few examples from various pharmaceuticals featuring a het-
erocyclic component.1
The benzodiazepine nucleus is a pharmacophoric scaffold,
and many benzodiazepines have recently received great atten-
tion because of their wide range of therapeutic and pharma-
cological properties. Many members of the benzodiazepine
family are nowadays widely used as antianxiety, antidepres-
sant, sedative, hypnotic, tranquilizing, anticonvulsant, anti-
histaminic, analgesic, and anti-inflammatory agents.2,3
Because of their wide range of biological applications, the
development of mild, efficient, and environmentally friendly
protocols continues to be a challenging endeavor in synthetic
organicchemistry. Asa result, considerable attention has been
drawn recently to new improved methods for the preparation
of 1,5-benzodiazepines4 also by means of three component
reactions.5
Moreover, taking into consideration that diazepam causes
a decrease in peroxidative decomposition of polyunsaturated
fatty acids of membranes on withdrawal, which could be due
to stabilization of membranes after long-term binding of dia-
zepam,9 it looked promising to examine the possibility of
hydroxybenzodiazepines and/or aminobenzoxazepines to act as
inhibitors of lipoxygenase (LOXa) activity and of peroxidation
a Abbreviations: AAPH, 2,20-azobis(2-amidinopropane) hydrochloride;
AM1, Austin model 1; au, atomic unit of energy (1 au = 627.51 kcal/mol);
B3LYP, Becke three-parameter Lee-Yang-Parr hybrid functional;
6-31G*, a type of valence double-ζ polarized basis set; ClogP, calculated
logarithm of partition coeficient log(Coctanol/Cwater); COLOC, two-
2
3
dimensional C-H correlation via long-range coupling JCH or JCH
;
;
H-H COSY, two-dimensional H-H correlation spectroscopy via nJHH
Concerning their counterparts, the one nitrogen benzoan-
nelated seven-membered ring heterocycles (the oxaza derivatives),
although much less studied than benzodiazepines, became
increasingly interesting because of their biological activity.6
C-H COSY or HETCOR, two-dimentional heteronuclear correlation
via 1JCH; DFT, density functional theory; DPPH, 1,l-diphenyl-2-picryl-
hydrazyl radical; ICPE, Inhibition of carrageenin-induced rat paw
edema; IMA, indomethacin; LOX, lipoxygenase; LPO, lipid peroxida-
tion; MW, microwave irradiation; NDGA, nordihydroguaiaretic acid;
NOESY, two-dimensional H-H nuclear Overhauser effect correlation
spectroscopy; NSAIDs, nonsteroidal anti-inflammatory drugs; Trolox,
trade name for 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid; TS, transition state; p-TsA, p-toluenesulfonic acid; ZPE, zero point
energy.
*To whom correspondence should be addressed. For C.A.T.: phone,
+30 2310 997865; fax, +30 2310 997679; e-mail, tsolerid@chem.auth.gr.
For J.S.-S.: phone, þ30 2310 997831; fax, þ30 2310 997679; e-mail,
ioulia@chem.auth.gr.
r
2010 American Chemical Society
Published on Web 11/04/2010
pubs.acs.org/jmc

8410 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Neochoritis et al.
of biological membranes as well as to present anti-inflamma-
tory activity.
From Scheme 2 and Table 1 it can be concluded that the
presence of electron-withdrawing substituents in the aceto-
phenone moiety stabilizes the initially formed 1,5-benzo-
xazepines 5 and consequently renders their isolation more
possible. It is characteristic that in the case of the p-nitro-
substituted acetophenone 4g only the benzoxazepine deriva-
tive 5g was formed, which could not be completely trans-
formed to benzodiazepine 6g even after prolonged refluxing
with acetic acid (after 5 days of reflux 5g/6g = 1:5 and then
decomposition had occurred). To the contrary, in the pre-
sence of electron-releasing substituents, the electron rich
oxygen is easily protonated resulting in spontaneous trans-
formation of benzoxazepines 5 to benzodiazepines 6. As a
result, in the case of the methoxy-substituted acetophenones
4b-d only the corresponding benzodiazepine derivatives
6b-d were isolated. Moreover, it should also be noticed that
the 9-hydroxybenzodiazepines 7 were never traced.
Results and Discussion
Chemistry. Very recently, we have investigated the synthe-
sis and antioxidant as well as the aldose reductase inhibition
of some 1,5-benzodiazepine derivatives10 and also their cyto-
genetic activity in vitro in normal lymphocyte cultures.11 In
continuation of our previous work concerning benzodia-
zepines7,12 this paper describes a study of the synthesis of
6-hydroxy-2,3-dihydro-1H-1,5-benzodiazepines versus 6-amino-
2,3-dihydro-1,5-benzoxazepines by condensation of ketones
with 2,3-diaminophenol through one-pot microwave assis-
ted acid catalyzed reaction without solvent and the testing of
the novel compounds with regard to their antioxidant ability
as well as to their potent lipid peroxidation (LPO) inhibitory
activity.
The possibility of formation of the otherwise inaccessible and
unknown 2,2-dimethyl-4-arylbenzodiazepines 8 was also exam-
ined (Scheme 3 and Table 2). Thus, when o-phenylenediamine
1b was mixed with acetophenone 4a and acetone in a 1:2:0.5
molar ratio in the presence of catalytic amount of acetic acid
and then irradiated with microwaves, 2,2-dimethyl-4-phenyl-
benzodiazepine 8c was formed in 92% yield. This reaction
seems to be very sensitive to the molar ratio of the starting
materials. Indeed, when the amount of acetone was increased
to 1 molar ratio, a mixture of all three possible products,
namely, 8c and the known10 9 and 10, was formed in 45%,
16%, and 31% yield, respectively. Analogously, from 4f the
product 8d was isolated in 93% yield. 2,3-Diaminophenol
behaved analogously, affording with acetophenones 4b and 4e
the corresponding 6-hydroxy-2,2-dimethyl substituted benzo-
diazepines 8a and 8b in 56% and 95% yield, respectively.
Initially, the reaction was attempted simply by mixing 2,3-
diaminophenol 1 with acetone, in a 1.5:2 molar ratio, in the
presence of a catalytic amount of acetic acid and irradiating
in a Biotage Initiator 2.0 microwave oven, whereupon a mix-
ture of aminobenzoxazepine 2 and hydroxybenzodiazepine 3
in a 3:4 ratio was formed (Scheme 1). Separation between
2 and 3 became feasible, when immediately after irradiation
the reaction mixture was quenched with a 10% sodium bi-
carbonate solution. Otherwise, complete transformation of
2 to 3 was observed by the traces of acetic acid, which re-
mained in the reaction mixture after microwave irradiation.
The reaction proceeded analogously to give a mixture of
2 and 3 (7:3 ratio), when p-TsA was used instead of acetic
acid. These results indicated that the oxazepine derivative
2 constitutes the kinetic product, whereas the benzodiaze-
pine derivative 3 the thermodynamic one.
After the selectivity of the reaction with acetone was
established, in order to investigate the possibility of a more
general application of the method, the reaction was repeated
with a number of substituted acetophenones 4, and the re-
sults are shown in Scheme 2 and Table 1.
Table 1. Reaction Conditions and Products for the Reaction of Scheme 2
time
(min)
power
(W)
5
(%)
6
(%)
5/6a
ratio
total yield
(%)
ketone
4a
4b
4bb
4c
2
3
2
2
2
3
2
2
3
5
80
240
80
35
59
88
37:63
0:100
0:100
0:100
0:100
29:71
100:0
42:58
100:0
61:39
94
88
80
90
93
86
81
97
81
77
trace
80
90
80
80
4d
4e
4eb
4f
93
61
Scheme 1. Reaction of 2,3-Diaminophenol with Acetone
240
80
25
81
41
81
47
trace
56
80
4g
4h
240
80
trace
30
a Quenched immediately after irradiation. b With p-TsA as catalyst.
Scheme 2. Reaction of 2,3-Diaminophenol with Substituted Acetophenones

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8411
Scheme 3. Reaction of Diamines 1 with a Mixture of Acetone and Acetophenone
Table 2. Reaction Conditions and Products for the Reaction of Scheme 3
products (%)
amine
acetophenone
molar ratioa
time (min)
power (W)
1a
1a
1a
1b
1b
1b
4e
4e
4b
4a
4a
4f
1:2:1
2
2
2
2
2
2
80
80
80
80
80
80
3 (10)
6e (21)
8b (51)
8b (95)
8a (56)
10 (31)
8c (92)
8d (93)
1:2:0.5
1:2:0.5
1:2:1
6b (20)
9 (16)
8c (45)
1:2:0.5
1:2:0.5
a Amine/acetophenone/acetone.
Scheme 4. Derivatization of 5a and 6a by Reacting with p-Tolyl
Isocyanate
of aryl substituents, the unsubstituted and the p-OMe and
p-NO2 substituted aryl derivatives were examined, and the
results are presented in Tables 3 and 4.
As already mentioned, according to the proposed plausible
mechanism shown in Scheme 5, after the first aldol conden-
sation of the ketone moiety, intermediate 13 is formed. The
more electronic charge computed on N-5 of diaminophenol 1
suggests that most possibly this is the favored amino group
that reacts with the carbonyl carbon of 13 to afford the
intermediate imine 14. In the next step, the seven-membered
ring closes by reaction of the hydroxyl or the amino group of
the intermediate conformer 14 or 16, respectively, leading to
the protonated intermediate 15 or 17. From the calculated
values of total energies (Etotal, Table 4) it is concluded that
benzodiazepines 6 are slightly more stable than benzoxaze-
pines 5, constituting thus the thermodynamic products. Even
though compounds 6 are thermodynamically favored over 5,
in most cases a mixture of both products with varied ratios is
obtained. This fact can be attributed to the slow interconver-
sion of the intermediate conformers 14 and 16 via the hin-
dered rotation of the aromatic ring around the Ar-N bond,
due to the two substituents ortho to the imino nitrogen.
Moreover, the formation of compounds 5 can be explained
by studying the thermochemical data of the transition state.
According to these results (Table 4) considering the inter-
mediate conformers 14 and 16 as transition states to 15 and
17, respectively, the relative activation energy of 14 was
found slightly lower than that of 16 (1.3-3.2 kcal/mol), thus
favoring the formation of the protonated intermediate 15
over that of 17; therefore, intermediate 15 can be considered
as the kinetic product, whereas intermediate 17 is the ther-
modynamic one. Although this energy difference of 1.3-3.2
kcal/mol seems to be rather small in absolute value, it always
points toward the same direction and is decisive for the
kinetic/thermodynamic pathway.
Finally, the differentiation between aminobenzoxazoles 5
and hydroxybenzodiazepines 6 was also established beyond
doubt by their derivatization. For this reason, 5a was trans-
formed to the arylurea derivative 11, whereas 6a was trans-
formed to the carbamate one 12 (Scheme 4), easily differ-
entiated from their carbonyl IR absorptions13,14 at 1665 and
1738 cm-1, respectively.
For the formation of benzoxazepines 5and benzodiazepines 6
a plausible mechanistic scheme (Scheme 5) involving aldol
condensation of two acetophenone molecules to 13 could be
implicated, whereupon the initially formed conformers 14
and 16 could finally cyclize to the seven-membered ring pro-
ducts 5 and 6, respectively. The fact that 9-hydroxybenzodia-
zepines 7 (Scheme 1) were never traced offers decisive proof
in favor of the proposed mechanism.
To further support the above mechanism, some DFT cal-
culations have been carried out on the first protonation site
of aminophenol 1 and on oxa- or aza-protonation selectivity
of 5 or 6 as well as on the stability of intermediates 15 and 17.
For this purpose, in order to investigate the possible influence
On the other hand, in the exploration of the mechanism of
conversion of 5 to 6, the traces of acetic acid from the initial
condensation reaction were implicated. Hence, the proton-
ation site of 5 and 6 and the stability of the protonated

8412 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Neochoritis et al.
Scheme 5. Plausible Mechanism Leading to Benzoxazepine 5 and Benzodiazepine Derivatives 6, as Well as for Conversion of 5 to 6a
a These intermediates do not depict the absolute configuration of the transition state and the final products 5 and 6 are mixtures of their R and S
enantiomers.
Table 3. Net Atomic Charges (Mulliken) on Heteroatoms for the Neutral Species 1, 2, 3, and Some Selected Substituted 5 and 6
atoms
1a
2
5a
5b
5g
atoms
3
6a
6b
6g
Qnet Atomic Charges (Electrons) in Compounds
O1
-0.6565
-0.8393
-0.7913
-0.5619
-0.5187
-0.7946
-0.5675
-0.5873
-0.7925
-0.5686
-0.5964
-0.7627
-0.5639
-0.5813
-0.7923
N1
N5
6-O
-0.6609
-0.5463
-0.6546
-0.6962
-0.6193
-0.6558
-0.7007
-0.6345
-0.6549
-0.7091
-0.6186
-0.6431
N5
6-N
Hydrogen Charges Summed up on Heteroatoms
O1
-0.2369
-0.1946
-0.1680
-0.5619
-0.5187
-0.1421
-0.5675
-0.5873
-0.1294
-0.5686
-0.5964
-0.1322
-0.5639
-0.5813
-0.1173
N1
N5
6-O
-0.3538
-0.5463
-0.2384
-0.3764
-0.6193
-0.2323
-0.3845
-0.6345
-0.2338
-0.3443
-0.6186
-0.2202
N5
6-N
a The atom numbering in 1 is arbitrary, analogous as in the rest of compounds, for simplicity reasons.
intermediates 15 and 17 were investigated. The computa-
tional results indicate that the favored protonation sites
of 5 and 6 are the heteroatoms in position 1 of the seven-
membered ring, since these atoms have more electronic
charge than the exocyclic amino nitrogen and hydroxyl
group, respectively (Table 3). After protonation, the result-
ing intermediates 17 are computed to be more stable than 15
by 7.3-24.2 kcal/mol. Protonated intermediates 15 are
computed to have the tendency to open the benzoxazepine
ring, since the final O1-C2 bond length is computed to vary
IR, MS, NMR (1H, 13C, H-H COSY, C-H COSY, and C-H
COLOC), and elemental analysis data.
The 3-methylene protons of compounds 5 and 6 constitute
an AB spin system giving two distinct doublets with J ≈
2
13-14 Hz at about 2.9-3.1 δ and 3.2-3.5 δ, respectively.
The endo-proton being in pseudoaxial configuration in the
diazepine ring is shielded by the CdN anisotropy field,
whereas the exo-proton being in the pseudoequatorial con-
figuration is deshielded by the two adjacent phenyls.12c In the
COSY spectrum of 6f there is a weak cross-peak between the
3-Hexo and the N1-H via a 4J coupling through a W, almost
coplanar conformation. In most 1H NMR spectra this
3-Hexo proton signal is split by a small coupling of ∼1.2 Hz or
is slightly broadened. In compound 6f the above 3-Hendo has
˚
between 1.70 and 1.80 A. Consequently, the protonated
compounds 5 are gradually converted again to the open
form 14, which may recyclize to 17 and finally transformed
to 6.
Structure Assignment of the New Compounds. The assigned
molecular structures of all new compounds 2, 3, 5, 6, 8, 11,
and 12 were based on rigorous spectroscopic analysis including
3
COLOC correlation via JCH with the C-10 of the 2-phenyl
group. In practice, this finding led us to the acceptance that
in solution this proton has a significant coupling with C-10

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8413
Table 4. Total DFT Energies of Formation (B3LYP/6-31G*) for the Neutral Compounds 2 and 3 and for Their Protonated Intermediates 2H and 3H
for Some Substituted Derivatives of 5 and 6, Their Protonated Intermediates 15 and 17, and Their Transition Intermediates 14 and 16
2
3
2H
3H
a
a
a
Etotal
-651.373833
-651.373265
1.52
-651.697902
-651.727397
-18.51
ΔEb
5a
6a
5b
6b
5g
6g
Etotal
ΔEb
-1034.735771
-1034.738193
-1.52
17a
-1263.716398
-1263.719183
-1.75
17b
-1443.734070
-1443.735835
-1.11
17g
15a
15b
15g
Etotal
ΔEb
-1035.061874
-1035.100457
-24.21
-1264.078311
-1264.089926
-7.29
-1444.043362
-1444.074656
-19.63
14a
16a
14b
16b
14g
16g
a
Etotal
ΔEb
-1035.071508
-1035.068739
1.74
3.148
-1264.069061
-1264.067026
1.28
3.481
-1444.043340
-1444.038226
3.21
3.142
TS bondc
2.786
-59.01
2.919
-89.32
2.635
-18.14
νd
-70.68
-25.18
-118.40
a Sum of electronic energy and zero-point energy (ZPE, the oscilator’s vibrational energy at absolute zero) correction (au); 1 au = 627.51 kcal/mol.
b Relative stabilization energy: ΔE = Ebenzodiazepine - Ebenzoxazepine (kcal/mol). c Interatomic distance for the new forming bond in TS (A). d Imaginary
˚
frequency (cm-1).
Figure 1. Global lower energy conformation of compound 6a (from two different views) that fits COLOC and NOESY data (by DFT).
carbon; hence, the dihedral angle Hendo-C3-C2-C10 is near
180° or 0° in almost coplanar configuration. In Figure 1, the
favored conformation calculated by DFT that fits with the
previous observations is shown, where the two phenyl
groups are arranged on the same side of diazepine ring
and in almost parallel configuration.
In 1H NMR of many members of 6-aminobenzoxazepines
the two amino protons are differentiated, giving two absorp-
tions. One signal at ∼7-8 ppm is assigned to a proton form-
ing a hydrogen bond with N-5, whereas the remaining proton
is resonating at ∼4-5 ppm.
and 3JCH in compounds 5a and 6f are depicted. Some impor-
tant NOESY correlations in compound 6f are also included,
confirming the proposed structure.
Biological Evaluation. In the present investigation, com-
pounds 2, 3, 5f, 6a,c,d,f,h, 8c, and 12 were studied in vitro
with regard to their antioxidant ability in comparison to
well-known antioxidant agents such as nordihydroguaiaretic
acid (NDGA) and 6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid (Trolox). For estimation of the antioxidative
potential of chemical components, different experimental
approaches were used.15 To evaluate the in vitro anti-
oxidant activity of the synthesized compounds two different
2
3
Figure 2. COLOC correlations via JCH and JCH observed in
compounds 5a and 6f.
2
In Figure 2 the COLOC correlations observed via JCH
antioxidant assays have been used: (a) Interaction with the
stable free radical 1,l-diphenyl-2-picrylhydrazyl radical (DPPH)
and (b) Interaction with the water-soluble azo compound
2,20-azobis(2-amidinopropane) hydrochloride (AAPH), used
as a source of peroxyl radicals (ROO ). Both methods require
3
a spectrophotometric measurement and a certain reaction
time in order to obtain reproducible results.16 The DPPH
method is describedasa simple, rapid, and convenient method
independent of sample polarity for screening many samples
for radical scavenging activity.17 These advantages made the
DPPH method interesting for testing our compounds.

8414 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Neochoritis et al.
Table 5. % Interaction of the Tested Compounds (at 100 and 50 μM) with DPPH (DPPH %) after 20 and 60 min, Inhibition Values (IC50) of Lipid
Peroxidation (LPO) Induced by AAPH, in Vitro Inhibition of Soybean Lipoxygenase (LOX), in Vivo % Inhibition of Carrageenin-Induced Rat Paw
Edema (ICPE %) at 0.01 mmol/kg Body Weighta
DPPH %, 20 min
100 μM 50 μM
86 87
DPPH %, 60 min
100 μM 50 μM
86 88
compds
ClogP
LPO inhib IC50 (μM)
% LOX inhib 100 μM
ICPE
2
1.75
2.08
6.69
4.91
4.65
4.51
6.37
4.39
4.05
7.39
70
77
98
3
66
86
30
27
36
54
32
15
10
93
44
87
65
86
29
27
36
53
32
14
9
67
86
NAb
NAb
28
5f
6a
47.5
50
6c
6d
60
46
37
3
6f
6h
2.5
46
NAb
NAb
6
35*
8c
12
45
50
3
84
NDGA
Trolox
IMAc
96
63
47**
a Each in vitro experiment was performed at least in triplicate, and the standard deviation of absorbance was less than 10% of the mean. Each in vivo
result represents the mean obtained from 6-15 animals in two independent experiments. In all cases, significant difference from control was as follows:
(/) p < 0.1, (//) p < 0.01 (Student’s t test). b NA: no action detectable under the reported experimental conditions. c IMA: indomethacin.
The use of the AAPH peroxyl radical is recommended as
more appropriate for measuring radical-scavenging activity
in vitro because the activity of the AAPH peroxyl radical
shows a greater similarity to cellular activities, such as lipid
peroxidation (LPO).18 The water-soluble azo compound
AAPH has been extensively used as a clean and controllable
source of thermally produced alkylperoxyl free radicals.19
The interaction of the examined compounds with the stable
free radical DPPH is shown in Table 5. This interaction indi-
cates their radical scavenging ability in an iron-free system.
The above-mentioned compounds were examined for their
DPPH interaction at 100 and 50 μM after 20 and 60 min.
Compounds 2, 3, 5f, and 6f, showed the best DPPH interac-
tion percentage values, some of them displaying similar
values (86%) to that of the reference compound NDGA
(93%) at the same concentration. These compounds have
either a phenolic group or a free aromatic amino group in
their structure, so they are able to donate a hydrogen atom.
The presence of substituents with low lipophilicity, expressed
as π values (π value expresses the hydrophobic contribution
of a substituent), like OCH3 (π = -0.02) or NO2 group (π =
-0.28) diminishes reducing ability (6c, 6d, 6h). On the
contrary, a substituent with high π value, e.g., Cl (0.71), as
in compound 6f, increases the scavenging result. The role of
the electronic effect of the substituents is not well-defined,
and it looks contradictory. Thus, compound 6h with a R2 =
NO2 group with a negative inductive effect (-I) presents
similar results with the unsubstituted derivative 6a, whereas
6f with a chloro substituent, with a negative inductive effect
(-I) too, is more potent than 6a. The most interesting deriv-
ative is the unsubstituted compound 2 combining an aro-
matic amino moiety and an oxazepine ring. The second more
significant is the benzoxazepine 5f with a chloro substituent.
The unsubstituted diazepine 3 with a phenylhydroxyl group
instead of a free aromatic amino group is less potent than 2.
Lower calculated20 lipophilicity values seem to be important,
since 2 is less lipophilic than 3 (ClogP for 2 is 1.75, and for 3 it
is 2.08). For the sake of structure comparison, benzoxaze-
pine 5f presents higher interaction with DPPH compared to
benzodiazepine 6f, indicating that the derivative with a free
aromatic amino group in its structure is more able to donate
a hydrogen atom and to act as an antioxidant. For com-
pound 3 the interaction values were found to be time and
concentration dependent, whereas for compounds 6a, 6c, 6d,
6f, 6h, 8c, and 12 no results were detectable at 50 μM. The
disappearance of the phenolic hydroxyl in compound 12, due
to the transformation of 6a to the carbamate 12, supports the
decrease of the reducing ability. Each in vitro experiment was
performed at least in triplicate, and the standard deviation of
absorbance was less than 10% of the mean.
In our studies, AAPH was used as a free radical initiator to
follow oxidative changes of linoleic acid to conjugated diene
hydroperoxide. Azo compounds generating free radicals
through spontaneous thermal decomposition are useful for
free radical production studies in vitro.19 The results (Table 5)
showed that benzodiazepine 6f, having a free phenolic group,
a benzodiazepine ring, and lipophilic (R1 = Cl) substituents,
is the best inhibitor of lipid peroxidation, displaying an IC50
value of 2.5 μM. No significant differences are observed
between the oxazepine 2 and diazepine 3. Benzoxazepine 5f
also presents lower anti-lipid peroxidation (anti-LPO) activity
compared to benzodiazepine 6f. Compounds 6d, 5f, and 6h
presented similar IC50 values (46-47.5 μM), whereas com-
pounds 6a and 12 follow with IC50 values of 50 μM. It seems
that a free phenolic hydroxyl group in combination with a
benzodiazepine ring leads to better inhibition of lipid perox-
idation. The transformation of 6a to carbamate 12 did not
offer any advantage in anti-LPO activity (both present similar
IC50 values). Between the methoxybenzodiazepines, 6d is a
more potent inhibitor than 6c, indicating that R3 = OCH3 >
R2 = OCH3. From our results it seems that higher inhibition
of lipid peroxidation (lower IC50 values) is correlated with
higher lipophilicity values (ClogP values, Table 5). These data
are supported by literature findings, which revealed that
lipophilicity (ClogP) is the main physicochemical parameter
influencing the anti-LPO activity.21
Lipoxygenases (LOXs) play a significant role in mem-
brane lipid peroxidation by forming hydroperoxides in the
lipid bilayer22 from the biotransformation of arachidonic
acid catalyzed by LOX. Inhibitors of LOX have attracted
attention initially as potential agents for the treatment of
inflammatory and allergic diseases, certain types of cancer,
and cardiovascular diseases.23
In this context, we decided to further evaluate the syn-
thesized derivatives for their ability to inhibit soybean LOX
by the UV absorbance based enzyme assay.24 It has been

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8415
Conclusions
shown that inhibition of plant LOX activity by nonsteroidal
anti-inflammatory drugs (NSAIDs) is qualitatively similar
to their inhibition of the rat mast cell LOX and may be used
as a simple qualitative screen for such activity. Most of the
LOX inhibitors are antioxidants or free radical scavengers.25
LOXs contain a “non-heme” iron per molecule in the enzyme
active site as high-spin Fe2þ in the native state and the high
spin Fe3þ in the activated state. Some studies suggest a re-
lationship between LOX inhibition and the ability of the
inhibitors to reduce Fe3þ at the active site to the catalytically
inactive Fe2þ. This inhibition is related to their ability to
reduce the iron species in the active site to the catalytically
inactive ferrous form,25 whereas several LOX inhibitors are
excellent ligands for Fe3þ. From the tested derivatives only
compound 2 presented 98% inhibition at 100 μM, a value
considerable better than that of the reference compound
NDGA (Table 5). Although lipophilicity is referred to as an
important physicochemical property for LOX inhibitors,26
herein the most potent compound, namely, the oxazepine
analogue 2 (98%) with a ClogP of 1.75, does not follow this
concept. For the sake of comparison oxazepine 2 is potent
whereas the diazepine 3 is inactive, under our experimental
conditions. A methoxy substitution on 6a leading to 6c does
not increase significantly the inhibition in 6c. The transfor-
mation also of 6a to the carbamate derivative 12 diminishes
the LOX inhibition.
Our study indicates that lipid peroxidation inhibitory
activity is not always accompanied by DPPH radical scaveng-
ing activity. Thus, although compounds such as 6f, 5f, 6d,
6a, 6h, and 12 inhibit lipid peroxidation potently, they
present low DPPH scavenging activity. This is in accordance
with the finding of Curini et al.,27 who have studied the
antioxidant and LOX inhibitory activity of five natural
prenyloxycarboxylic acids. They showed that boropinic
acid was the most efficient lipid peroxidation inhibitor
(LOX), although it was not the most active DPPH radical
scavenger.
Compound 6f, which was the most potent lipid perox-
idation inhibitor, was tested as an in vivo anti-inflamma-
tory agent. In acute toxicity experiments, the in vivo
examined compound did not present toxic effects in doses
up to 0.2 mmol/kg body weight. Ulcerogenicity was not
found. The in vivo anti-inflammarory effect of compound
6f was assessed by using the carrageenin-induced rat paw
edema (ICPE) model. Acute inflammation is due to the
release of chemical mediators, which cause edema as a
result of extravasations of fluid and proteins from the local
microvasculature and accumulation of polymorphonuclear
leukocytes at the inflammatory site. The induced edema is a
nonspecific inflammation highly sensitive to NSAIDs.
Thus, it has been accepted as a useful tool for studying
new anti-inflammatory agents.28 It reliably predicts the
anti-inflammatory potency of the NSAIDs, as a result of
inhibition of prostaglandin amplification. Compound 6f
showed 35% percentage of protection, while the reference
drug indomethacin (IMA) induced 47% protection at an
equivalent dose.
In the present study several new benzodiazepines and
benzoxazepines have been synthesized with the aim of study-
ing their antioxidant and anti-inflammatory activities. The
synthesis of 6-hydroxybenzodiazepines versus their 6-amino-
benzoxazepine counterparts was investigated, and it was
established that benzoxazepines constitute the kinetic prod-
ucts, whereas benzodiazepines constitute the thermodynamic
ones. In addition, the otherwise inaccessible and unknown
2,2-dimethyl-4-arylbenzodiazepines were also synthesized.
In terms of biological activity, the most interesting antiox-
idant derivatives were those with the benzoxazepine moiety
and the amino group substituent. Improved inhibitory activ-
ities on lipid peroxidation were observed by retaining a phe-
nolic group and a benzodiazepine ring. Benzodiazepine 6f was
the best inhibitor of lipid peroxidation, displaying an IC50
value of 2.5 μM against the value of 63 μM of the reference
compound Trolox, whereas the trimethyl-substituted benzo
xazepine 2 showed the best LOX inhibition (98%), much better
than the reference compound NDGA (84%). Compounds 2
and 6f can be used as lead molecules for the design of agents
with excellent LOX and LPO inhibitory activities.
Experimental Section
General. DPPH and NDGA were purchased from Aldrich
Chemical Co. (Milwaukee, WI). Soybean LOX, linoleic acid
sodium salt, and indomethacin were obtained from Sigma
Chemical Co. (St. Louis, MO), and carrageenin, type K, was
commercially available. For the in vivo experiments male and
female Fischer-344 rats (180-240 g) were used. For the in vitro
tests a Lambda 20 (Perkin-Elmer) UV-visible double beam
spectrophotometer was used. Melting points were measured on
a Kofler hot stage and are uncorrected. Column chromatog-
raphy was carried out using Merck silica gel (70-230 mesh). Thin
layer chromatography (TLC) was performed using precoated
silica gel 0.25 mm glass plates containing fluorescent indicator
UV254 (Macherey-Nagel) using a 3:1 mixture of petroleum
ether-ethyl acetate. Petroleum ether refers to the fraction boil-
ing between 60 and 80 °C. NMR spectra were recorded at room
temperature on a Bruker AM 300 or AVANCE III 300 spectrom-
1
eter at 300 MHz for H and 75 MHz for 13C, using CDCl3 as
solvent, unless otherwise is indicated. The chemical shifts are
expressed in δ values (ppm) relative to TMS as internal standard
for 1H and relative to TMS (0.00 ppm) or to CDCl3 (77.05 ppm)
for 13C NMR spectra. Coupling constants nJ are reported in Hz.
Second order 1H spectra, where it was possible, were analyzed
by simulation.29 IR spectra were recorded on a Perkin-Elmer
1600 series FTIR spectrometer and are reported in wave num-
bers (cm-1). LC-MS (ESI, 1.65 eV) spectra were recorded on
LCMS-2010 EV system (Shimadzu). Low-resolution electron
impact mass spectra (EIMS) were obtained on a 6890N GC/MS
instrument (Agilent Technology); results are reported as m/z (rel
intensity in %) at ionization energy of 70 eV. The purity of all
novel compounds was confirmed to exceed 95% by elemental
analysis performed with a Perkin-Elmer 2400-II CHN analyzer.
Structural assignments of the derived compounds were estab-
lished by analysis of their IR, MS, and NMR spectra (1H, 13C,
COSY, NOESY, HETCOR, and COLOC). For the computa-
tional analysis, transition states and products were built by
ChemDraw (ChemDraw7) and optimized by AM1 as imple-
mented in MOPAC package30 and subsequently optimized by
density functional theory (DFT) using B3LYP level with 6-31G-
(d) basis set as implemented in the Gaussian 03 package.31,32 The
corresponding transition states of the reactions were located as
described in our previous work.33
Physicochemical Studies. Determination of Lipophilicity
as ClogP Values. Since lipophilicity is a significant physico-
chemical property determining distribution, bioavailability,
metabolic activity, and elimination, we tried to calculate
theoretically the lipophilicity values of benzoxazepines/ben-
zodiazepines as ClogP values in n-octanol-buffer by the
ClogP programme of Biobyte Corp.20
Reaction of 2,3-Diaminophenol with Acetone under Microwave
Irradiation. Acetone (2.0 mmol), 2,3-diaminophenol 1 (1.5 mmol),

8416 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Neochoritis et al.
and a drop of acetic acid were mixed thoroughly and were
irradiated in a Biotage Initiator 2.0 microwave oven at a power of
80 W for 2 min. The crude product was dissolved in dichloro-
methane (30 mL) and washed with 5% aqueous NaHCO3 and
with water. The organic layer was dried over Na2SO4, and after
filtration the solvent was evaporated and the residue was purified
by column chromatography on silica gel using petroleum ether-
ethyl acetate (10:1) of slowly increasing polarity to afford in elu-
tion order compounds 3 and 2.
6-Hydroxy-2,2,4-trimethyl-2,3-dihydro-1H-1,5-benzodiazepine
(3). Light yellow crystals (0.114 g, 56%), mp 145-147 °C. IR
(KBr) νmax: 3448, 3345, 1639, 1584 cm-1. 1H NMR: 1.28 (s, 6H,
2-CH3), 2.29 (s, 3H, 4-CH3), 2.52 (s, 2H, 3-H), 3.69 (br s, 1H,
NH), 6.10 (dd, J = 8.1, 1.1 Hz, 1H, 9-H), 6.43 (dd, J = 8.0,
1.4 Hz, 1H, 7-H), 6.89 (dd, J = 8.1, 8.0 Hz, 1H, 8-H), 8.10 (br s,
1H, OH). 13C NMR: 30.1 (2-CH3), 30.7 (4-CH3), 49.4 (C-3), 57.0
(C-2), 103.9 (C-7), 109.6 (C-9), 121.6 (C-6a), 128.2 (C-8), 139.6
(C-9a), 154.2 (C-6), 167.0 (C-4). MS (LCMS) m/z (%) 205 (100,
Mþ þ H). Anal. Calcd for C12H16N2O (204.27): C, 70.56; H,
7.90; N, 13.71. Found: C, 70.51; H, 7.81; N, 13.68.
crystals (0.342 g, 88%), mp 193-195 °C. IR (KBr) νmax: 3360,
3210, 1627, 1610 cm-1. 1HNMR:1.68 (s, 3H, 2-CH3), 3.07 (d, J =
14.0 Hz, 1H, 3-Hendo), 3.39 (d, J = 14.0 Hz, 1H, 3-Hexo), 3.74 (s,
3H, 400-OCH3), 3.83 (s, 3H, 40-OCH3), 4.16 (br s, 1H, NH), 6.28
(dd, J = 8.1, 1.2 Hz, 1H, 9-H), 6.49 (dd, J = 7.9, 1.2 Hz, 1H, 7-H),
6.76 (d, J = 8.7 Hz, 2H, 30,50-H), 6.84 (d, J = 8.7 Hz, 2H, 300,500-
H), 6.98 (dd, J = 7.9, 8.1 Hz, 1H, 8-H), 7.33 (d, J = 8.7 Hz, 2H,
20,60-H), 7.63 (d, J = 8.7 Hz, 2H, 200,600-H), 7.90 (br s, 1H, OH).
13C NMR (/ indicates that the assignments may be interchanged):
30.6 (2-CH3), 45.7 (C-3), 55.3 (40-OCH3), 55.4 (400-OCH3), 65.6
(C-2), 104.0 (C-7), 109.8 (C-9), 113.6 (C-300,500),* 113.7 (C-30,50),*
122.8 (C-5a), 126.4 (C-20,60), 128.5 (C-8), 128.6 (C-200,600), 133.1
(C-100), 139.5 (C-9a),* 139.7 (C-10),* 154.6 (C-6), 158.5 (C-40),
161.1 (C-400), 163.1 (C-4). MS (LCMS) m/z (%) 389 (100, Mþ þ
H). Anal. Calcd for C24H24N2O3 (388.46): C, 74.21; H, 6.23; N,
7.21. Found: C, 74.09; H, 6.15; N, 7.30.
From 3-Methoxyacetophenone. 6-Hydroxy-2,4-bis(3-methoxy-
phenyl)-2-methyl-2,3-dihydro-1H-1,5-benzodiazepine (6c). Light
yellow crystals (0.350 g, 90%), mp 165-167 °C. IR (KBr) νmax
:
3363, 3221, 1629, 1598 cm-1. 1H NMR: 1.68 (s, 3H, 2-CH3), 3.07
(d, J = 14.2 Hz, 1H, 3-Hendo), 3.49 (d, J = 14.2 Hz, 1H, 3-Hexo),
3.62 (s, 3H, 30-OCH3), 3.77 (s, 3H, 300-OCH3), 4.31 (br s, 1H,
NH), 6.30 (dd, J = 8.3, 1.4 Hz, 1H, 9-H), 6.48 (dd, J = 7.6,
1.4 Hz, 1H, 7-H), 6.69 (dd, J = 8.2, 1.5 Hz, 1H, 40-H), 6.88-6.96
(m, 3H, 20,60,400-H), 7.00 (dd, J = 7.6, 8.3 Hz, 1H, 8-H),
7.10-7.17 (m, 2H, 200,600-H), 7.17-7.25 (m, 2H, 50,500-H), 7.90
(br s, 1H, OH). 13C NMR (/ indicates that the assignments may
be interchanged): 30.8 (2-CH3), 46.2 (C-3), 55.1 (400-OCH3), 55.3
(40-OCH3), 65.1 (C-2), 103.7 (C-7), 109.6 (C-9), 111.4 (C-20),
112.1 (C-200), 112.4 (C-40), 115.7 (C-400), 119.6 (C-600), 122.1 (C-
5a), 129.2 (C-50), 129.2 (C-500), 129.5 (C-8), 139.5 (C-9a), 142.1
(C-100), 148.8 (C-10), 154.9 (C-6), 159.49 (C-300),* 159.55 (C-30),*
162.9 (C-4). MS (LCMS) m/z (%) 389 (100, Mþ þ H). Anal.
Calcd for C24H24N2O3 (388.46): C, 74.21; H, 6.23; N, 7.21.
Found: C, 74.29; H, 6.30; N, 7.15.
6-Amino-2,2,4-trimethyl-2,3-dihydro-1,5-benzoxazepine (2).
Brown crystals (0.086 g, 42%), mp 135-136 °C. IR (KBr)
ν
max: 3437, 3358, 3333, 1632, 1578 cm-1. 1H NMR: 1.35 (s, 6H,
2-CH3), 2.28 (s, 2H, 3-H), 2.38 (s, 3H, 4-CH3), 3.7 (br s, 1H,
NH2), 6.56 (dd, J = 7.9, 1.1 Hz, 1H, 9-H), 6.70 (dd, J = 8.0,
1.2 Hz, 1H, 7-H), 6.87 (dd, J = 8.0, 7.9 Hz, 1H, 8-H), 6.9 (br,
1H, NH2). 13C NMR: 29.3 (4-CH3), 30.1 (2-CH3), 46.3 (C-3),
68.2 (C-2), 111.1 (C-7), 116.9 (C-7), 123.5 (C-8), 125.4 (C-6a),
143.3 (C-9a), 150.9 (C-6), 170.6 (C-4). MS (LCMS) m/z (%)
205 (100, Mþ þ H). Anal. Calcd for C12H16N2O (204.27):
C, 70.56; H, 7.90; N, 13.71. Found: C, 70.49; H, 7.95; N,
13.64.
General Procedure for the Reaction of 2,3-Diaminophenol with
Acetophenones under Microwave Irradiation. Acetophenone 4
(2.0 mmol), 2,3-diaminophenol 1 (1.5 mmol), and a drop of
acetic acid were mixed thoroughly and were irradiated in the
microwave oven at a power and time indicated in Table 1. The
crude product was worked up as above to afford in elution order
compounds 6 and 5.
From 2-Methoxyacetophenone. 6-Hydroxy-2,4-bis(2-methoxy-
phenyl)-2-methyl-2,3-dihydro-1H-1,5-benzodiazepine (6d). Light
yellow crystals (0.361 g, 93%), mp 64-66 °C. IR (KBr) νmax
:
3393, 3200, 1625, 1596, 1578 cm-1. 1H NMR: 1.68 (s, 3H, 2-CH3),
2.77 (d, J = 13.9 Hz, 1H, 3-Hendo), 3.07 (s, 3H, 40-OCH3), 3.83
(s, 3H, 400-OCH3), 4.39 (d, J = 13.9 Hz, 1H, 3-Hexo), 4.64 (br s,
1H, NH), 6.25-6.32 (m, 2H, 30,300-H), 6.34 (dd, J = 7.8, 1.1 Hz,
1H, 9-H), 6.44 (dd, J = 8.0, 1.1 Hz, 1H, 7-H), 6.66 (ddd, J = 7.0,
7.0, 1.1 Hz, 1H, 50-H), 6.75 (ddd, J = 7.0, 7.0, 1.1 Hz, 1H, 500-H),
6.85 (dd, J = 7.0, 1.2 Hz, 1H, 60-H), 6.96 (dd, J = 8.0, 7.8 Hz,
1H, 8-H), 7.08 (ddd, J = 7.1, 7.0, 1.1 Hz, 1H, 40-H), 7.13 (dd,
J = 7.0, 1.1 Hz, 1H, 600-H), 7.21 (ddd, J = 7.5, 7.0, 1.1 Hz, 1H,
400-H), 8.10 (br s, 1H, OH). 13C NMR (/ indicates that the
assignments may be interchanged): 30.0 (2-CH3), 46.9 (C-3),
53.8 (40-OCH3), 55.5 (400-OCH3), 62.5 (C-2), 101.4 (C-7), 108.1
(C-9), 110.2 (C-300),* 110.7 (C-30),* 120.13 (C-500),* 120.15 (C-
50),* 120.9 (C-5a), 127.9 (C-600),* 128.0 (C-60),* 128.7 (C-8),
128.7 (C-40),* 129.8 (C-400),* 132.5 (C-100),* 133.0 (C-10),* 139.2
(C-9a), 155.2 (C-6),* 155.3 (C-20),* 157.4 (C-200), 165.3 (C-4).
MS (LCMS) m/z (%) 389 (100, Mþ þ H). Anal. Calcd for C24-
H24N2O3 (388.46): C, 74.21; H, 6.23; N, 7.21. Found: C, 74.30;
H, 6.19; N, 7.14.
From Acetophenone. 6-Hydroxy-2-methyl-2,4-diphenyl-2,3-
dihydro-1H-1,5-benzodiazepine (6a). Yellow crystals (0.194 g, 59%),
mp 88-90 °C. IR (KBr) νmax: 3446, 3360, 1610, 1578 cm-1
.
1H NMR: 1.60 (s, 3H, 2-CH3), 2.98 (d, J = 14.3 Hz, 1H, 3-Hendo),
3.45 (d, J=14.6 Hz, 1H, 3-Hexo), 4.32(brs, 1H, NH), 6.22(dd, J=
7.9, 1.1 Hz, 1H, 9-H), 6.45 (dd, J = 7.9, 1.1 Hz, 1H, 7-H), 6.94 (dd,
J = 7.9, 7.9 Hz, 1H, 8-H), 7.04-7.09 (m, 3H, 20,40,60-H), 7.20-7.30
(m,5H,30,50,300,400,500-H), 7.53 (dd, J=7.5, 1.4, 2H, 200,600-H), 8.07,
(br s, 1H, OH). 13C NMR: 30.6 (2-CH3), 45.9 (C-3), 64.6 (C-2),
103.4 (C-7), 109.4 (C-9), 121.9 (C-5a), 125.1 (C-20,60), 126.7 (C-
200,600), 126.9 (C-40), 128.1 (C-30,50), 128.2 (C-300,500), 129.0 (C-400),
129.5 (C-8), 139.5 (C-100), 140.4 (C-9a), 146.9 (C-10), 154.9 (C-6),
162.8 (C-4). MS (LCMS) m/z (%) 329 (100, Mþ þ H). Anal. Calcd
for C22H20N2O (328.41): C, 80.46; H, 6.14; N, 8.53. Found: C,
80.61; H, 6.23; N, 8.65.
6-Amino-2-methyl-2,4-diphenyl-2,3-dihydro-1,5-benzoxazepine
(5a). Green crystals (0.115 g, 35%), mp 155-157 °C. IR (KBr)
ν
max: 3360, 1627, 1610 cm-1. 1H NMR: 1.72 (s, 3H, 2-CH3), 3.02
(d, J = 13.0 Hz, 1H, 3-Hendo), 3.20 (d, J = 13.0 Hz, 1H, 3-Hexo),
6.58 (d, J = 7.8, 1.4 Hz, 1H, 9-H), 6.83 (dd, J = 7.8, 8.1 Hz, 1H,
8-H), 6.90 (dd, J = 8.1, 1.4 Hz, 1H, 7-H), 7.15-7.35 (m, 8H, 20,
30,40,50,60,300,400,500-H), 7.61 (dd, J = 6.9, 2.2 Hz, 2H, 200,600-H),
7.65 (br s, 2H, NH2). 13C NMR: 30.5 (2-CH3), 43.3 (C-3), 73.2
(C-2), 111.4 (C-9), 119.9 (C-7), 122.0 (C-8), 125.4 (C-20,60), 125.9
(C-5a), 127.1 (C-40), 127.2 (C-200,600), 128.1 (C-300,500), 128.3 (C-
30,50), 130.1 (C-400), 139.5 (C-100), 142.8 (C-9a), 147.3 (C-10),
148.6 (C-6), 168.8 (C-4). MS (LCMS) m/z (%) 329 (91, Mþ þ H),
301 (100). Anal. Calcd for C22H20N2O (328.41): C, 80.46; H,
6.14; N, 8.53. Found: C, 80.30; H, 6.05; N, 8.61.
From 4-Methylacetophenone. 6-Hydroxy-2-methyl-2,4-bis-
(4-methylphenyl)-2,3-dihydro-1H-1,5-benzodiazepine (6e). Green
crystals (0.217 g, 61%), mp 68-70 °C. IR (KBr) νmax: 3379 (NH),
3210 (br, OH), 1626, 1583 cm-1. 1H NMR: 1.67 (s, 3H, 2-CH3),
2.28 (s, 3H, 40-CH3), 2.37 (s, 3H, 400-CH3), 3.14 (d, J = 14.3 Hz,
1H, 3-Hendo), 3.44 (d, J = 14.3 Hz, 1H, 3-Hexo), 4.41 (br s, 1H,
NH), 6.27 (dd, J = 8.2, 1.4 Hz, 1H, 9-H), 6.48 (dd, J = 7.9, 1.4
Hz, 1H, 7-H), 7.00 (dd, J = 8.2, 7.9 Hz, 1H, 8-H), 7.05 (d, J = 7.9
Hz, 2H, 30,50-H), 7.15 (d, J = 7.9 Hz, 2H, 300,500-H), 7.28 (d, J =
7.9 Hz, 2H, 20,60-H), 7.58 (d, J = 7.9 Hz, 2H, 200,600-H), 8.23 (br s,
1H, OH). 13C NMR (/ indicates that the assignments may be
interchanged): 20.7 (40-CH3), 21.1 (400-CH3), 30.4 (2-CH3), 45.8
(C-3), 64.2 (C-2), 103.4 (C-7), 109.5 (C-9), 122.1 (C-5a), 124.9
From 4-Methoxyacetophenone. 6-Hydroxy-2,4-bis(4-methoxy-
phenyl)-2-methyl-2,3-dihydro-1H-1,5-benzodiazepine (6b). Brown

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8417
(C-20,60), 126.8 (C-200,600), 128.7 (C-8), 128.86 (C-30,50),* 128.86
(C-300,500),* 137.0 (C-40), 137.8 (C-100), 139.7 (C-9a), 139.7 (C-400),
144.3 (C-10), 154.8 (C-6), 162.7 (C-4). MS (LCMS) m/z (%) 379
(100, Mþ þ Na), 357 (70, Mþ þ H). Anal. Calcd for C24H24N2O
(356.46): C, 80.87; H, 6.79; N, 7.86. Found: C, 80.95; H, 6.75; N,
7.80.
387 (90). Anal. Calcd for C22H18N4O5 (418.40): C, 63.15; H, 4.34;
N, 13.39. Found: C, 63.05; H, 4.29; N, 13.47.
6-Hydroxy-2-methyl-2,4-bis(4-nitrophenyl)-2,3-dihydro-1H-
1,5-benzodiazepine (6g). This compound was isolated from 5g
after acid catalysis. Red crystals, mp 235-237 °C. IR (KBr)
ν
max: 3450 br, 3399, 1624, 1597, 1518, 1346 cm-1. 1H NMR: 1.81
6-Amino-2-methyl-2,4-bis(4-methylphenyl)-2,3-dihydro-1,5-
benzoxazepine (5e). Oil (0.089 g, 25%. IR (KBr) νmax: 3350,
3207, 1679, 1607, 1581 cm-1. 1H NMR: 1.67 (s, 3H, 2-CH3), 2.29
(s, 3H, 40-CH3), 2.34 (s, 3H, 400-CH3), 3.03 (d, J = 14.3 Hz, 1H,
3-Hendo), 3.16 (d, J = 14.3 Hz, 1H, 3-Hexo), 6.65 (dd, J = 7.7, 1.6
Hz, 1H, 9-H), 6.88 (dd, J = 8.0, 1.6 Hz, 1H, 7-H), 6.95 (dd, J =
8.0, 7.7 Hz, 1H, 8-H), 7.09 (d, J = 8.3 Hz, 2H, 30,50-H), 7.11 (d,
J = 8.3 Hz, 2H, 300,500-H), 7.1 (br s, 2H, NH2), 7.47 (d, J = 8.3 Hz,
2H, 20,60-H), 7.63 (d, J = 8.3 Hz, 2H, 200,600-H). 13C NMR-
(/ indicates that the assignments may be interchanged): 20.9
(40-CH3), 21.4 (400-CH3), 30.4 (2-CH3), 42.6 (C-3), 72.6 (C-2),
111.0 (C-9), 119.4 (C-7), 123.1 (C-8), 125.1 (C-5a), 125.2 (C-
20,60), 127.3 (C-200,600), 128.9 (C-300,500),* 129.1 (C-30,50),* 136.76
(C-100),* 136.81 (C-40),* 140.5 (C-400), 144.4 (C-10),* 144.5 (C-
9a),* 149.5 (C-6), 168.8 (C-4). MS (LCMS) m/z (%) 357 (100,
Mþ þ H). Anal. Calcd for C24H24N2O (356.46): C, 80.87; H,
6.79; N, 7.86. Found: C, 80.80; H, 6.72; N, 7.94.
From 4-Chloroacetophenone. 6-Hydroxy-2-methyl-2,4-bis(4-
chlorophenyl)-2,3-dihydro-1H-1,5-benzodiazepine (6f). Green
crystals (0.222 g, 56%), mp 118-120 °C. IR (KBr) νmax: 3381,
3375, 1624, 1584 cm-1. 1H NMR: 1.67 (s, 3H, 2-CH3), 2.95 (d,
J = 14.2 Hz, 1H, 3-Hendo), 3.52 (d, J = 14.2 Hz, 1H, 3-Hexo),
4.29 (br s, 1H, NH), 6.28 (dd, J = 8.2, 1.2 Hz, 1H, 9-H), 6.48
(dd, J = 8.0, 1.2 Hz, 1H, 7-H), 7.00 (dd, J = 8.2, 8.0 Hz, 1H,
8-H), 7.11 (d, J = 8.9 Hz, 2H, 30,50-H), 7.20 (d, J = 8.7 Hz, 2H,
20,60-H), 7.26 (d, J = 8.7 Hz, 2H, 300,500-H), 7.47 (d, J = 8.9 Hz,
2H, 200,600-H), 7.86 (br s, 1H, OH). 13C NMR: 31.3 (2-CH3),
45.7 (C-3), 64.9 (C-2), 104.0 (C-7), 109.5 (C-9), 122.0 (C-5a),
126.7 (C-20,60), 128.1 (C-200,600), 128.4 (C-30,50), 128.5 (C-
300,500), 129.5 (C-8), 132.8 (C-40), 136.0 (C-400), 138.7 (C-100),
139.2 (C-9a), 145.2 (C-10), 154.9 (C-6), 161.5 (C-4). MS
(LCMS) m/z (%) 397/399/401 (100, Mþ þ H). Anal. Calcd
for C22H18Cl2N2O (397.30): C, 66.51; H, 4.57; N, 7.05. Found:
C, 66.41; H, 4.27; N, 6.98.
(s, 3H, 2-CH3), 3.09 (d, J = 14.4 Hz, 1H, 3-Hendo), 3.82 (d, J =
14.4 Hz, 1H, 3-Hexo), 4.48 (br s, 1H, NH), 6.35 (dd, J = 8.2, 1.3
Hz, 1H, 9-H), 6.52 (dd, J = 8.0, 1.3 Hz, 1H, 7-H), 7.00 (dd, J =
8.2, 8.0 Hz, 1H, 8-H), 7.43 (d, J = 8.8 Hz, 2H, 20,60-H), 7.67 (d,
J = 8.7 Hz, 2H, 200,600-H), 7.69 (s, 1H, OH), 8.01 (d, J = 8.8 Hz,
2H, 30,50-H), 8.16 (d, J = 8.7 Hz, 2H, 300,500-H). 13C NMR: 32.0
(2-CH3), 45.9 (C-3), 64.8 (C-2), 104.4 (C-7), 109.3 (C-9), 121.5
(C-5a), 123.8 (C-30,50,300,500), 126.4 (C-20,60), 127.4 (C-200,600),
130.9 (C-8), 139.0 (C-9a), 145.7 (C-100), 146.9 (C-40), 148.4 (C-
400), 153.2 (C-10), 155.5 (C-6), 159.2 (C-4). MS (LCMS) m/z (%)
417 (100, Mþ - H). Anal. Calcd for C22H18N4O5 (418.40): C,
63.15; H, 4.34; N, 13.39. Found: C, 63.28; H, 4.21; N, 13.17.
From 3-Nitroacetophenone. 6-Hydroxy-2-methyl-2,4-bis(3-
nitrophenyl)-2,3-dihydro-1H-1,5-benzodiazepine (6h). Yellow
1
crystals (0.126 g, 30%), mp 152-154 °C. H NMR: 1.85 (s,
3H, 2-CH3), 3.08 (d, J = 14.0 Hz, 1H, 3-Hendo), 3.77 (d, J =
14.0 Hz, 1H, 3-Hexo), 4.37 (br s, 1H, NH), 6.41 (dd, J = 8.0, 1.1
Hz, 1H, 9-H), 6.53 (dd, J = 8.0, 1.1 Hz, 1H, 7-H), 7.09 (t, J =
8.0 Hz, 1H, 8-H), 7.35 (dd, J = 8.0, 7.6 Hz, 1H, 50-H), 7.47 (dd,
J = 7.6, 7.2 Hz, 1H, 500-H), 7.58 (br s, 1H, OH), 7.68 (d, J = 7.6
Hz, 1H, 60-H), 7.89 (d, J = 7.6 Hz, 1H, 600-H), 7.96 (d, J = 8.3
Hz, 1H, 40-H), 8.16 (s, 1H, 20-H), 8.17 (d, J = 7.2 Hz, 1H, 400-
H), 8.28 (s, 1H, 200-H). 13C NMR: 31.6 (2-CH3), 45.4 (C-3),
66.6 (C-2), 104.9 (C-7), 109.9 (C-9), 120.6 (C-200), 122.2 (C-20),
124.4 (C-8), 127.5 (C-5a), 129.5 (C-40), 129.5 (C-400), 129.6 (C-
50), 130.4 (C-500), 131.7 (C-600), 132.3 (C-60), 138.0 (C-10), 141.4
(C-9a), 148.1 (C-300), 148.1 (C-100), 148.2 (C-30), 154.9 (C-6),
161.2 (C-4). MS (LCMS) m/z (%) 441 (100, Mþ þ Na). Anal.
Calcd for C22H18N4O5 (418.40): C, 63.15; H, 4.34; N, 13.39.
Found: C, 63.08; H, 4.40; N, 13.19.
6-Amino-2-methyl-2,4-bis(3-nitrophenyl)-2,3-dihydro-1,5-benzo-
xazepine (5h). Yellow crystals (0.197 g, 47%), mp 193-195 °C.
1H NMR: 1.83 (s, 3H, 2-CH3), 3.05 (d, J = 13.2 Hz, 1H,
3-Hendo), 3.35 (d, J = 13.2 Hz, 1H, 3-Hexo), 6.73 (dd, J = 7.2,
1.5 Hz, 1H, 9-H), 6.91 (dd, J = 7.9, 7.2 Hz, 1H, 8-H), 6.97 (dd,
J = 7.9, 1.5 Hz, 1H, 7-H), 7.39-7.50 (m, 3H, 50,500-H, NH),
7.94-8.05 (m, 3H, 40,60,600-H), 8.14 (dd, J = 9.1, 1.9 Hz, 1H, 400-
H), 8.26 (t, J = 1.9 Hz, 1H, 20-H), 8.52 (t, J = 1.9 Hz, 1H, 200-H).
13C NMR: 30.8 (2-CH3), 43.2 (C-3), 73.8 (C-2), 112.2 (C-9),
120.9 (C-7), 121.2 (C-8), 121.3 (C-20), 121.8 (C-40), 122.2 (C-200),
124.5 (C-400), 126.2 (C-5a), 129.3 (C-50), 129.5 (C-500), 132.0 (C-
600), 132.7 (C-60), 140.5 (C-100), 140.7 (C-9a), 146.7 (2 ꢀ C-30,300),
148.3 (C-10), 149.4 (C-6), 164.5 (C-4). MS (LCMS) m/z (%) 441
(100, Mþ þ Na). Anal. Calcd for C22H18N4O5 (418.40): C,
63.15; H, 4.34; N, 13.39. Found: C, 63.28; H, 4.46; N, 13.24.
General Procedure for the Reaction of 2,3-Diaminophenol with
Acetophenones and Acetone under Microwave Irradiation. Aceto-
phenone 4 (2.0 mmol), 2,3-diaminophenol 1 (1.0 mmol), acetone
(0.5 mmol), and four drops of acetic acid were mixed thoroughly
and were irradiated in the microwave oven at a power and time
indicated in Table 2. The crude product was worked up as above
to afford compounds 8, 9, and 10.
6-Amino-2-methyl-2,4-bis(4-chlorophenyl)-2,3-dihydro-1,5-
benzoxazepine (5f). Green crystals (0.163 g, 41%), mp 187-189 °C.
IR (KBr) νmax: 3450, 3340, 1576 cm-1
.
1H NMR (CDCl3
þ
DMSO-d6): 1.73 (s, 3H, 2-CH3), 2.90 (d, J=13.4 Hz, 1H, 3-Hendo),
3.13 (d, J = 13.4 Hz, 1H, 3-Hexo), 4.40 (br s, 1H, NH2), 6.73-6.80
(m, 3H, 7,8,9-H), 7.17 (d, J = 8.8 Hz, 2H, 30,50-H), 7.20 (d, J =
8.4 Hz, 2H, 300,500-H), 7.48 (d, J = 8.8 Hz, 2H, 20,60-H), 7.54 (d,
J=8.4 Hz, 2H, 200,600-H), 9.26 (br s, 1H, NH2).13CNMR(CDCl3 þ
DMSO-d6) (/ indicates that the assignments may be interchanged):
30.3 (2-CH3), 43.1 (C-3), 72.6 (C-2), 111.2 (C-9), 119.2 (C-7),*
119.6 (C-8),* 126.7 (C-5a), 126.8 (C-20,60), 127.6 (C-200,600),* 127.7
(C-300,500),* 127.9 (C-30,50), 132.0 (C-40), 135.2 (C-400), 137.6 (C-100),
139.2 (C-9a), 146.0 (C-10), 147.3 (C-6), 165.2 (C-4). MS (LCMS)
m/z (%) 397/399/401 (100, Mþ þ H). Anal. Calcd for C22H18-
Cl2N2O (397.30): C, 66.51; H, 4.57; N, 7.05. Found: C, 66.33; H,
4.40; N, 6. 97.
From 4-Nitroacetophenone. 6-Amino-2-methyl-2,4-bis(4-nitro-
phenyl)-2,3-dihydro-1,5-benzoxazepine (5g). Red crystals (0.339 g,
81%), mp 180-182 °C. IR (KBr) νmax: 3423, 1597, 1514, 1349
cm-1. 1H NMR: 1.83 (s, 3H, 2-CH3), 3.05 (d, J = 13.6 Hz, 1H,
3-Hendo), 3.35 (dd, J = 13.6, 1.3 Hz, 1H, 3-Hexo), 5.19 (br s, 1H,
NH2), 6.73 (dd, J = 7.9, 1.5 Hz, 1H, 9-H), 6.91 (t, J = 7.9 Hz, 1H,
8-H), 6.97 (dd, J = 7.9, 1.5 Hz, 1H, 7-H), 7.72 (d, J = 9.0 Hz, 2H,
20,60-H), 7.75 (d, J = 9.0 Hz, 2H, 200,600-H), 7.70-7.78 (br, 1H,
NH2), 8.08 (d, J = 9.0 Hz, 2H, 30,50-H), 8.09 (d, J = 9.0 Hz, 2H,
300,500-H). 13C NMR: 31.0 (2-CH3), 43.4 (C-3), 73.0 (C-2), 112.5
(C-9), 120.9 (C-7), 121.8 (C-8), 123.5 (C-300,500), 123.6 (C-30,50),
126.4 (C-5a), 126.8 (C-200,600), 127.7 (C-20,60), 139.7 (C-6), 144.8
(C-10), 146.4(C-100), 146.9 (C-400), 148.5(C-40), 154.3(C-9a), 164.5
(C-4). MS (LCMS) m/z (negative polarity, %) 417 (100, Mþ - H),
From 2,3-Diaminophenol and 4-Methoxyacetophenone. 6-
Hydroxy-4-(4-methoxyphenyl-2,2-dimethyl-2,3-dihydro-1H-
1,5-benzodiazepine (8a). Green crystals (0.166 g, 56%), mp
1
75-77 °C. H NMR: 1.34 (s, 6H, 2 ꢀ 2-CH3), 2.96 (s, 2H,
3-CH2), 3.80 (br s, 1H, NH), 3.89 (s, 3H, OCH3), 6.21 (dd, J =
8.0, 1.0 Hz, 1H, 9-H), 6.53 (dd, J = 8.0, 1.0 Hz, 1H, 7-H), 6.96
(t, J = 8.0 Hz, 1H, 8-H), 6.98 (d, J = 7.0 Hz, 2H, 30,50-H), 7.70
(br s, 1H, OH), 7.92 (d, J = 7.0 Hz, 2H, 20,60-H). 13C NMR: 30.7
(2 ꢀ 2-CH3), 44.1 (C-3), 55.4 (40-OCH3), 60.5 (C-2), 104.1 (C-7),
110.1 (C-9), 113.8 (C-30,50), 123.1 (C-5a), 128.3 (C-8), 128.7
(C-20,60), 133.1 (C-10), 139.3 (C-9a), 154.4 (C-6), 161.3 (C-40),
163.3 (C-4). MS (LCMS) m/z (%) 319 (65, Mþ þ Na), 297

8418 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Neochoritis et al.
(100, Mþ þ H). Anal. Calcd for C18H20N2O2 (296.36): C,
72.95; H, 6.80; N, 9.45. Found: C, 73.07; H, 6.91; N, 9.32.
From 2,3-Diaminophenol and 4-Methylacetophenone. 6-Hy-
droxy-4-(4-methylphenyl-2,2-dimethyl-2,3-dihydro-1H-1,5-ben-
zodiazepine (8b). Oil (0.266 g, 95%). IR (KBr) νmax: 3383,
154-156 °C. IR (KBr) νmax: 3303, 1738 cm-1. 1H NMR: 1.74 (s,
3H, 2-CH3), 2.21 (s, 3H, 4000-CH3), 2.99 (d, J = 13.4 Hz, 1H,
3-Hendo), 3.07 (d, J = 13.4 Hz, 1H, 3-Hexo), 3.53 (br s, 1H, 1-H),
6.71 (dd, J = 8.0, 1.1 Hz, 1H, 9-H), 6.85 (dd, J = 9.0, 1.1 Hz, 1H,
7-H), 6.95 (d, J = 7.6 Hz, 2H, 3000,5000-H), 7.00-7.08 (m, 1H,
8-H), 7.00-7.08 (m, 2H, 20,60-H), 7.12-7.28 (m, 6H,
30,40,50,300,400,500-H), 7.41 (d, J = 7.6, 2H, 2000,6000-H), 7.64 (d,
J = 8.3, 2H, 200,600-H), 7.8 (br s, 1H, 6-CONH). 13C NMR
(/ indicates that the assignments may be interchanged): 21.2
(4000-CH3), 27.3 (2-CH3), 40.4 (C-3), 60.4 (C-2), 118.5 (C-7),
121.9 (C-9), 124.7 (C-200,600),* 125.3 (C-20,60),* 126.4 (C-5a),
126.8 (C-40), 128.3 (C-30,50,300,500), 128.4 (C-8), 129.1 (C-
2000,3000,5000,6000), 130.6 (C-400), 133.8 (C-4000), 134.3 (C-1000),
137.9 (C-100), 146.4 (C-9a), 146.9 (C-10), 151.9 (br, NHCdO),
155.7 (C-6), 164.8 (C-4). MS (LCMS) m/z (%) 484 (100, Mþ þ
Na). Anal. Calcd for C30H27N3O2 (461.55): C, 78.07; H, 5.90; N,
9.10. Found: C, 78.20; H, 6.13; N, 8.97.
Biological Assays. Each in vitro experiment was performed at
least in triplicate. The results were averaged, and the standard
deviation of absorbance was less than 10% of the mean. The
results are presented in Table 5.
In Vitro Assays. Determination of the Reducing Activity of the
Stable Radical DPPH.34 To an ethanolic solution of DPPH
(0.05 mM) in absolute ethanol an equal volume of the com-
pounds (final concentrations of 50 and 100 μM) dissolved in
DMSO was added. The mixture was shaken vigorously and
allowed to stand for 20 or 60 min. Absorbance at 517 nm was
determined spectrophotometrically, and the percentage of ac-
tivity was calculated. All tests were undertaken on three repli-
cates, and the results were averaged (Table 5).
1
3400-3200 (br), 1626, 1211 cm-1. H NMR: 1.31 (s, 6H, 2 ꢀ
2-CH3), 2.40 (s, 3H, 40-CH3), 2.96 (s, 2H, 3-CH2), 3.00 (br s, 1H,
NH), 6.17 (dd, J = 7.9, 1.4 Hz, 1H, 9-H), 6.48 (dd, J = 7.9, 1.4 Hz,
1H,7-H),6.94(t,J=7.9 Hz, 1H, 8-H), 7.24 (d, J=8.0 Hz, 2H, 30,50-
H), 7.60 (br s, 1H, OH), 7.81 (d, J=8.0 Hz, 2H, 20,60-H). 13CNMR:
21.3 (40-CH3), 30.6 (2 ꢀ 2-CH3), 44.7 (C-3), 59.3 (C-2), 103.8 (C-7),
109.8 (C-9), 122.5 (C-5a), 127.0 (C-30,50), 128.7 (C-8), 129.2 (C-20,60),
137.9 (C-10), 139.4 (C-9a), 140.3 (C-40), 154.7 (C-6), 163.2 (C-4). MS
(LCMS) m/z(%) 281(35, Mþ þ H), 266(10), 171(22), 155(25), 153
(100). Anal. Calcd for C18H20N2O (280.36): C, 77.11; H, 7.19; N,
9.99. Found: C, 77.07; H, 7.26; N, 10.08.
From o-Phenylenediamine and Acetophenone. 2,2-Dimethyl-
4-phenyl-2,3-dihydro-1H-1,5-benzodiazepine (8c). Yellow crys-
tals (0.230 g, 92%), mp 110-112 °C. 1H NMR: 1.34 (s, 6H, 2 ꢀ
2-CH3), 2.74 (s, 2H, 3-CH2), 3.00 (br s, 1H, NH), 6.77-6.82 (m,
1H, 9-H), 7.01-7.08 (m, 2H, 7,8-H), 7.28-7.31 (m, 1H, 6-H),
7.45-7.50 (m, 3H, 30,40,50-H), 8.01-8.06 (m, 2H, 20,60-H). 13C
NMR: 30.8 (2 ꢀ 2-CH3), 40.8 (C-3), 69.4 (C-2), 121.7 (C-9),
122.1 (C-7), 126.0 (C-8), 127.3 (C-20,60), 128.0 (C-6), 128.5 (C-
30,50), 130.2 (C-40), 137.7 (C-9a), 139.9 (C-10), 141.2 (C-5a),
168.6 (C-4). MS (GCMS) m/z (%) 250 (41, Mþ), 235 (100), 194
(30), 133 (38). Anal. Calcd for C17H18N2 (250.34): C, 81.56; H,
7.25; N, 11.19. Found: C, 81.41; H, 7.17; N, 11.09.
From o-Phenylenediamine and 4-Chloroacetophenone. 2,2-
Dimethyl-4-(4-chlorophenyl)-2,3-dihydro-1H-1,5-benzodiazepine
(8d). Yellow crystals (0.265 g, 93%), mp 77-79 °C. 1H NMR:
1.32 (s, 6H, 2 ꢀ 2-CH3), 2.69 (s, 2H, 3-H), 3.10 (br s, 1H, NH),
6.75-6.80 (m, 1H, 9-H), 7.00-7.15 (m, 2H, 7,8-H), 7.25-7.30
(m, 1H, 6-H), 7.41 (d, J = 8.8 Hz, 2H, 30,50-H), 7.96 (d, J = 8.8 Hz,
2H, 20,60-H). 13C NMR (/ indicates that the assignments may
be interchanged): 30.7 (2 ꢀ 2-CH3), 40.7 (C-3), 69.3 (C-2),
121.7 (C-9), 122.2 (C-7), 126.2 (C-8), 127.9 (C-6), 128.6 (C-
20,60),* 128.7 (C-30,50),* 136.4 (C-40), 137.7 (C-9a), 138.2 (C-10),
140.9 (C-5a), 167.2 (C-4). MS (LCMS) m/z (%) 284/286 (100,
Mþ). Anal. Calcd for C17H17ClN2 (284.78): C, 71.70; H, 6.02;
N, 9.84. Found: C, 71.51; H, 6.17; N, 10.01.
Reaction of 5a and 6a with p-Tolyl Isocyanate. A mixture of 5a
(or 6a) (1 mmol) and p-tolyl isocyanate (0.146 g, 1.1 mmol) was
refluxed in dry toluene (10 mL) for 10 h. The solvent was evap-
orated from the crude reaction mixture and the remainder was
purified by column chromatography on silica gel using petro-
leum ether-ethyl acetate (5:1) of slowly increasing polarity to
afford 1-(2-methyl-2,4-diphenyl-2,3-dihydro-1,5-benzoxazepin-
6-yl)-3-(4-tolyl)urea (11) [or 2-methyl-2,4-diphenyl-2,3-dihy-
dro-1H-1,5-benzodiazepin-6-yl(4-tolyl)carbamate (12)].
1-(2-Methyl-2,4-diphenyl-2,3-dihydro-1,5-benzoxazepin-6-yl)-3-
(4-tolyl)urea (11). Yellow crystals (0.355 g, 77%), mp 117-
118 °C. IR (KBr) νmax: 3272, 3196, 1654 cm-1. 1H NMR: 1.62
(s, 3H, 2-CH3), 2.36 (s, 3H, 4000-CH3), 2.79 (d, J = 16.3 Hz, 1H,
3-Hendo), 3.36 (d, J = 16.3 Hz, 1H, 3-Hexo), 6.82 (d, J = 8.1 Hz,
1H, 9-H), 6.91 (d, J = 8.6 Hz, 1H, 7-H), 7.10-7.23 (m, 10H, aro-
matics), 7.28-7.31 (m, 2H, aromatics), 7.41 (tt, J = 7.6, 1.4 Hz,
1H, 400-H), 7.50-7.54 (m, 2H, 200,600-H), 9.01 (br s, 1H, NH),
10.70 (br s, 1H, NH). 13C NMR (/ indicates that the assignments
may be interchanged): 20.7 (4000-CH3), 29.6 (2-CH3), 43.9 (C-3),
74.3 (C-2), 115.4 (C-9), 119.0 (C-8), 119.2 (C-7), 125.5 (C-20,60),
126.4 (C-400)0 , 126.5 (C-5a), 127.2 (C-200,600),* 127.4 (C-2000,6000),*
127.9 (C-3 ,500), 128.4 (C-30,50), 129.2 (C-3000,5000), 130.0 (C-400),
133.3 (C-4000), 135.1 (C-1000), 139.1 (C-9a), 139.3 (C-100), 146.1
(C-10), 147.5 (C-6), 152.0 (NHCONH), 169.5 (C-4). MS (LCMS)
m/z (%) 484 (30, Mþ þ Na), 459 (70), 333 (75), 301 (100). Anal.
Calcd for C30H27N3O2 (461.55): C, 78.07; H, 5.90; N, 9.10.
Found: C, 78.30; H, 6.05; N, 8.99.
Inhibition of Linoleic Acid Lipid Peroxidation.19 The water-
soluble azo compound AAPH is used as a free radical initiator
for in vitro studies of free radical production. Production of
conjugated diene hydroperoxide by oxidation of linoleic acid
sodium salt in an aqueous solution is monitored at 234 nm. This
assay can be used to follow oxidative changes and to understand
the contribution of each tested compound.
An amount of 10 μL of the 16 mM linoleic acid sodium salt
solution was added to the UV cuvette containing 0.93 mL of
0.05 M phosphate buffer, pH 7.4, prethermostated at 37 °C. The
oxidation reaction was initiated at 37 °C under air by the
addition of 50 μL of 40 mM AAPH solution. Oxidation was car-
ried out in the presence of aliquots (10 μL) in the assay without
antioxidant, and lipid oxidation was measured in the presence of
the same level of DMSO. The rate of oxidation at 37 °C was
monitored by recording the increase in absorption at 234 nm
caused by conjugated diene hydroperoxides.
Soybean LOX Inhibition Study in Vitro.34 The tested com-
pounds dissolved in DMSO were incubated at room tempera-
ture with sodium linoleate (0.1 mL) and 0.2 mL of enzyme
solution (1 part of enzyme 1 ꢀ 10-4 w/v in saline, and 9 parts of
saline). The conversion of sodium linoleate to 13-hydroperox-
ylinoleic acid at 234 nm was recorded and compared with the
appropriate standard inhibitor.
In Vivo Assays: Inhibition of the Carrageenin-Induced Edema..34
Edema was induced in the right hind paw of Fisher 344 rats
(150-200 g) by the intradermal injection of 0.1 mL of 2% car-
rageenin in water. Both sexes were used. Females pregnant were
excluded. Each group was composed of 6-15 animals. The
animals, which have been bred in our laboratory, were housed
under standard conditions and received a diet of commercial
food pellets and water ad libitum during the maintenance, but
they were entirely fasted during the experiment period. Our
studies were in accordance with recognized guidelines on animal
experimentation. The tested compound, 0.01 mmol/kg body
weight, was diluted in water with a few drops of Tween 80 and
ground in a mortar before use, and it was given intraperitoneally
simultaneously with the carrageenin injection. The rats were
euthanized 3.5 h after carrageenin administration. The differ-
ence between the weight of the injected and uninjected paws was
2-Methyl 2,4-Diphenyl-2,3-dihydro-1H-1,5-benzodiazepin-6-
yl(4-tolyl)carbamate (12). Yellow crystals (0.318 g, 69%), mp

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8419
calculated for each animal. The change in paw weight was com-
pared with that in control animals (treated with water) and
expressed as a percent inhibition of the edema (% ICPE values).
Indomethacin was tested as a reference compound in 0.01 mmol/kg
(47%). Values of ICPE (%) are the mean from two different
experiments with a standard error of the mean of less than 10%.
phosphanylidene or phosphono-succinate substituent. Tetrahedron
2009, 65, 2684–2688. (d) Shaabani, A.; Rezayan, A. H.; Keshipour,
S.; Sarvary, A.; Ng, S. W. A novel one-pot three-(in situ five-)com-
ponent condensation reaction: an unexpected approach for the synthesis
of tetrahydro-2,4-dioxo-1H-benzo[b][1,5]diazepine-3-yl-2-methylpro-
~
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Acknowledgment. We thank the Research Committee of
the Aristotle University of Thessaloniki, Greece, for granting
a distinction scholarship to Constantinos G. Neochoritis. We
also thank Dr. C. Hansch, Dr. A. Leo, and Biobyte Corp. (201
West 4th Street, Suite 204, Claremont, California 91711) for
free access to the C-QSAR program.20
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