Synthesis and biological evaluation of novel flavone-8-acetic acid derivatives as reversible inhibitors of aminopeptidase N/CD13

Article abstract of DOI:10.1021/jm021109f

The cell surface aminopeptidase N (APN/CD13), overexpressed in tumor cells, plays a critical role in angiogenesis. However, potent, selective, and, particularly, noncytotoxic inhibitors ot this protein are lacking, and the present work was undertaken with

Full text of DOI:10.1021/jm021109f

3900
J . Med. Chem. 2003, 46, 3900-3913
Syn th esis a n d Biologica l Eva lu a tion of Novel F la von e-8-a cetic Acid Der iva tives
a s Rever sible In h ibitor s of Am in op ep tid a se N/CD13
Brigitte Bauvois,† Marie-Line Puiffe,† J ean-Bernard Bongui,§ Sandrine Paillat,§ Claude Monneret,§ and
Daniel Dauzonne§,*
Unite´ 365 INSERM, Institut Curie, Section de Recherche, 26 rue d’Ulm, 75248 Paris Cedex 05, France and UMR 176 CNRS,
Institut Curie, Section de Recherche, 26 rue d’Ulm, 75248 Paris Cedex 05, France
Received December 9, 2002
The cell surface aminopeptidase N (APN/CD13), overexpressed in tumor cells, plays a critical
role in angiogenesis. However, potent, selective, and, particularly, noncytotoxic inhibitors ot
this protein are lacking, and the present work was undertaken with the aim of developing a
new generation of noncytotoxic inhibitors that bind to APN/CD13. In this context, we have
synthesized a series of novel flavone-8-acetic acid derivatives. Among the herein described and
evaluated compounds, the 2′,3-dinitroflavone-8-acetic acid (19b) proved to be the most efficient
and exhibited an IC50 of 25 µM which is 2.5 times higher than that of bestatin (1), the natural
known inhibitor of APN/CD13. However, in contrast to bestatin (1), the dinitroflavone 19b did
not induce any cytotoxicity to cultured human model cells. The presence of other substituents
such as NO2 or OCH3 groups at the 3′- or 4′-position of the B phenyl group, or the existence of
steric constraints (compounds 24 and 29), did not improve selectivity and potency. The flavone
19b affinity for APN/CD13 is not recovered with other proteases such as matrix metallopro-
teinase-9 (MMP-9), angiotensin converting enzyme (ACE/CD143), neutral endopeptidase (NEP/
CD10), γ-glutamyl transpeptidase (CD224), or the serine proteases dipeptidyl peptidase IV
(DPPIV/CD26) or cathepsin G.
In tr od u ction
1 has been shown to induce apoptosis of T and myeloid
cell lines.1,2 On the other hand, tumor cells which
overexpress APN/CD13 (such as melanoma cells, acute
lymphocytic leukemic cells, and urological cancer cells)
are highly motile and capable of migration through
extracellular matrix, and APN inhibition by 1 leads to
a loss of motility.2,4,7-9 Likewise, CD13 antibodies
inhibit cell growth1,2,4,10 and cell motility.11 Inhibition
of APN/CD13 could prevent the secretion of type IV
epithelial collagenase (MMP-9) implicated in collagen
degradation.9
Cell surface peptidases (ectopeptidases) constitute a
family of transmembrane enzymes present in a wide
variety of tissues and cell types.1,2 They have been
implicated in major biological processes such as me-
tabolism regulation, cell proliferation, survival, and
invasiveness.1,2 Aminopeptidase N (APN/CD13) (EC
3.4.11.2; CD13) is a widespread ectopeptidase which
preferentially releases neutral amino acids from the
N-terminal end of peptides.3,4 The function of APN/
CD13 depends on its location. In the intestinal brush
border, the enzyme is involved in the terminal degrada-
tion of small peptides.4,5 In synaptic membranes, APN/
CD13 inactivates neuropeptides (endorphins and en-
kephalins).4,5 Other biologically active peptides are
angiotensins and chemotactic peptides.4,5 For example,
inhibition of APN/CD13 enhances neutrophil chemo-
taxis in response to formyl methionyl leucine phenyl-
alanine (f-MLP) and substance P by preserving the
integrity of these inflammatory peptides.1 APN/CD13
could play a role in cell surface antigen processing
trimming peptides bound to HLA DR molecules.6
The HELAH motif of the Zn2+-binding active site of
APN/CD13 reveals its metallopeptidase nature.5 Thiol
groups are efficient zinc chelators for APN, and natural
inhibitors such as actinonin and bestatin (Ubenimex,
1) have been discovered and isolated.2 Several studies
have indicated that interaction of APN/CD13 with 1
blocks cell proliferation in various cell types including
leukocytes, epithelial and melanoma cells.1,2 In addition,
Angiogenesis is a cascade of processes emanating from
microvascular endothelial cells and plays a central role
in tumor growth and metastasis.12 Recently, APN/CD13
has received a great deal of detailed attention with
regard to angiogenesis.13,14 First, APN/CD13 is ex-
pressed exclusively within the endothelial vasculature
of mouse and human tumors but not in normal vascu-
lature.13 Second, APN antagonists (CD13 antibodies or
bestatin) significantly block induced-retinal neovascu-
larization in mice and in in vitro chorioallantoic mem-
brane angiogenesis.13-15 Although bestatin has shown
antitumor therapeutic effects in several clinical tri-
als,16,17 limitations to its use in vivo include a nonspecific
toxicity.18
* Corresponding author: Dr. Daniel Dauzonne, Phone: (33) 01 42
34 66 64. Fax: (33) 01 42 34 66 31. E-mail: daniel.dauzonne@curie.fr.
† Unite´ 365 INSERM.
A survey of the literature indicates that flavonoids
could play a prominent role in cancer prevention by
§ UMR 176 CNRS-Institut Curie.
10.1021/jm021109f CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/06/2003

Novel Flavone-8-acetic Acid Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3901
interfering with cell proliferation, survival, cell signal-
ing, and regulating the immune system.19-21 The anti-
angiogenic effects of some flavonoids have already been
described.22-26 In this context, the polyphenol epigallo-
catechin-3-gallate (EGCG, 2) is a direct inhibitor of
leukocyte elastase and matrix metalloproteinases (MMP)
2 and 9, enzymes often overexpressed in cancer and
degrading the extracellular matrix.27,28 Furthermore, 2
inhibits elastase at concentrations of 50, 150, and 2500
times lower than effective on MMP-2 and -9, thrombin
and cathepsin G, respectively.28 The inhibitory effects
on proteases of nonphenolic constituents have also been
detailed.29,30 In the family of flavones, nobiletin (3)
inhibits the enzymatic activity of MMP-930 whereas, at
0.3 × 10-3 M, baicalein (4) inhibits the ectopeptidases
APN/CD13 and neutral endopeptidase (NEP/CD10; EC
3.4.24.11) by 57% and 36%, respectively.29 At the same
concentration, two flavanol derivatives, quercetin (5)
and isorhamnetin (6), respectively, inhibit the activities
of NEP/CD10 (73%) and APN/CD13 (49%).29
In an attempt to discover novel noncytotoxic inhibitors
of APN/CD13, we became interested in exploring a
flavonoid track leading to the investigation of a series
of novel flavone-8-acetic acid derivatives. We report
herein the synthesis and biological data for compounds
belonging to this family which exhibit selective inhibi-
tion of APN/CD13 on a myeloid cell line from human
origin.
Ch em istr y
With regard to the chemistry, the already known
3-nitroflavone (10) and 2′,3-dinitroflavone (11),38 as well
as the new 8-substituted-3-nitroflavones 12, 13, and
19a -m described herein, were prepared by adapting a
methodology developed in our laboratory.39 Our ap-
proach required readily available substituted aromatic
aldehydes as starting materials and proved, in our
context, to be more versatile and more convenient than
the previously reported classical routes to flavones. The
general procedure for the preparation of the desired
flavone-8-acetic acid derivatives is depicted in Scheme
1.
The starting 2-hydroxy-3-allylbenzaldehyde (14) was
easily synthesized on a large scale from salicylaldehyde
via a Claisen rearrangement of the intermediate 2-al-
lyloxybenzaldehyde.40 The subsequent concerted Michael
condensation of 14 with the appropriate Z-(2-chloro-2-
nitroethenyl)benzenes 15a -m (prepared by reacting the
conveniently substituted aldehydes with bromoni-
tromethane in the presence of an excess of dimethyl-
ammonium chloride and a small amount of potassium
fluoride in refluxing xylene41) gave the 8-allyl-3-chloro-
3,4-dihydro-4-hydroxy-3-nitro-2-phenyl-2H-1-benzopy-
rans 16a -m in the exclusive 2S,3S,4S relative config-
uration. These intermediates were further oxidized in
dry dichloromethane using pyridinium chlorochromate
(PCC) in an ultrasonically assisted process to provide
the 8-allyl-3-chloro-2,3-dihydro-3-nitro-2-phenyl-4H-1-
benzopyran-4-ones 17a -m with the 2S,3R relative
configuration (it must be pointed out that these oxida-
tion reactions never go to completion under mechanical
stirring, even if a very large excess of PCC is used for a
prolonged period of time). The obtained oxo derivatives
17a -m , on treatment with 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) in anhydrous tetrahydrofuran, were then
easily converted, at room temperature, into the 8-allyl-
3-nitro-2-phenyl-4H-1-benzopyran-4-ones 18a -m by the
anti elimination of a molecule of hydrochloric acid.
Subsequent oxidative cleavage of the allylic double bond
of the nitroflavone derivatives 18a-m was next achieved
by adapting the procedure reported by Sharpless and
co-workers, using the ruthenium(III) chloride/sodium
periodate system in the acetonitrile/water/carbon tet-
rachloride ternary mixture as the solvent,42 to afford
the desired 3-nitro-4-oxo-2-phenyl-4H-1-benzopyran-8-
acetic acids 19a -m in moderate to good yields.
Additional studies have also indicated that some
flavonoids present antiangiogenic activities by sup-
pressing in vitro endothelial cell growth and the forma-
tion of new blood vessels in the chick embryo chorioal-
lantoic membrane.13 In this same context, flavone-8-
acetic acid (FAA, 7), a synthetic flavonoid known to
induce significant growth delay and regression of nu-
merous solid tumors subcutaneously implanted in
mice,31-35 has been shown to be an efficient antiangio-
genic agent.36 However, the promising activity observed
in murine models was not transposed in humans as
reflected by the disappointing results obtained in the
numerous clinical trials carried out with FAA (7).37
In addition, several conformationally constrained
tetracyclic analogues (24, 28, and 29) of the acids 19
were also prepared. Thus, as outlined in Scheme 2, the
cyclic carbamate 24 was obtained starting from the acid
19m via its methoxymethyl ester 20. Concomitant
catalytic reduction of both nitro and benzyl groups of
20 by hydrogen in the presence of palladium-on-charcoal
in ethyl acetate at room temperature gave the ami-

3902 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Bauvois et al.
Sch em e 1a
a
Reagents, conditions, and remarks: (a) Et3N, anhydrous THF, 20 °C, 24 h; (b) PCC, anhydrous CH2Cl2, )))), 19 h; (c) DBU, anhydrous
THF, 3 h; (d) RuCl3, NaIO4, CCl4, MeCN, H2O. Under certain conditions previously reported in the particular case of 18a , the related
aldehyde 12 can be obtained.39 The corresponding methyl ester 13 has also been prepared starting from the acid 19a using a standard
method.39
Sch em e 2a
a
Reagents and conditions: (a) MOMCl, Et3N, MeCN, 20 °C; (b) H2, Pd/C 10%, EtOAc, 20 °C, 16 h; (c) triphosgene, Et3N, anhydrous
CH2Cl2, 20 °C, 5 h; (d) MgBr2, anhydrous CH2Cl2, 20 °C, 16 h.
nophenol 21. Reaction of this intermediate with triphos-
gene in dry dichloromethane in the presence of trieth-
ylamine provided the tetracyclic ester 23, whose
subsequent hydrolysis under mild conditions using
magnesium bromide in the same solvent at room tem-
perature43 yielded the desired (6,8-dioxo-6,7-dihydro-8H-
5,13-dioxa-7-azabenzo[3,4]cyclohepta[1,2-b]naphthalen-
12-yl)acetic acid (24).
acetic acid (28) and (6,8-dioxo-5,6,7,8-tetrahydro-13-oxa-
5,7-diazabenzo[3,4]cyclohepta[1,2-b]naphthalen-12-yl)-
acetic acid (29), respectively. In this context, it is worth
mentioning that the choice of the methoxymethyl group
to form the intermediate esters was guided by the fact
that the use of more classical alkyl esters (e.g., methyl
esters) required too drastic hydrolytic conditions, in-
compatible with the stability of the molecules.
The synthesis of the cyclic urea 29 is depicted in
Scheme 3 starting from 19b via the preparation of its
methoxymethyl ester 22 followed by the catalytic reduc-
tion of the two nitro groups. This latter hydrogenation
afforded a mixture of the azabenzo[b]fluorene 25 (prob-
ably resulting from the nucleophilic attack of the in situ
formed anilino nitrogen atom on the carbon 3 bearing
partially reduced species of the nitro group of the
flavone) and of the expected diamine 26 in a 25/26 )
55/45 ratio. Further condensation of 26 with triphosgene
in anhydrous dichloromethane in the presence of tri-
ethylamine gave the tetracyclic diaza derivative 27.
Subsequent hydrolysis of the methoxymethyl esters 25
and 27 employing magnesium bromide in dry dichloro-
methane at room temperature yielded the corresponding
(10-oxo-10,11-dihydro-5-oxa-11-azabenzo[b]fluoren-6-yl)-
The hitherto unknown 2′-nitroflavone-8-acetic acid
(8), required for comparison, was synthesized via the
classical Baker-Venkataraman approach following a
procedure already described to obtain related com-
pounds (Scheme 4).44,45 The ammonium salt 9 was
prepared from the corresponding nitro derivative as
previously reported.39
Resu lts a n d Discu ssion
In Vitr o Assessm en t of Cytotoxicity. The effects
of the tested compounds on the U937 cell growth and
cytotoxicity were evaluated upon continuous exposure
at concentrations ranging from 10-7 to 10-3 M. A
treatment period of 3 days was selected since the control
cells were still in the exponential growth phase at this
time. At the highest concentration, cell mortality ex-

Novel Flavone-8-acetic Acid Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3903
Sch em e 3a
(concentration that caused 50% necrosis compared with
the control value) for the above-mentioned derivatives
were over 5 × 10-4 M (data not shown). In addition,
none of the products considered in the present study
induced apoptosis (data not shown).
In Vitr o In h ibition of AP N Activity. In a first set
of experiments, we assessed the ability of the tested
compounds to inhibit APN activity expressed by intact
U937 cells (Table 1). The potencies of these molecules
varied somewhat, depending upon the nature of their
substituent(s). Our results indicated that derivatives
19b-d , which bear a CH2COOH group in the 8-position
and two NO2 substituents in both 2′- and 3′-positions,
inhibited efficiently APN activity, and this to the same
extent as bestatin (1). Deletion (19a ) or replacement
(19i-m ) of the NO2 group in the 2′-position gave
compounds with a lesser degree of potency against APN
activity whereas the presence of an electron-donating
methoxy group in the ortho (19c) or para position (19d )
of the nitro substituent led to slightly lowered inhibitory
effects. We have also checked that the removal of the
nitro group in the 3-position (8), if it does not signifi-
cantly affect the inhibition, provided a much more toxic
compound (vide supra). A marked decreasing in potency
is observed when nitro or methoxy substituents are
located in 3′-and/or 4′-positions (19e-h ). The other
synthesized products exhibited less significant effects.
In this context, it is particularly worthy of note that the
flavone-8-acetic acid (7), the salt 9, the 3-nitroflavone
(10), the esters 13, 20-22, the allyl derivative 18b, the
amine 21, the tetracyclic derivatives 23, 24, 28, and 29,
and the flavone itself proved to unefficiently inhibit APN
activity.
a
Reagents, conditions, and remarks: (a) MOMCl, Et3N, MeCN,
20 °C; (b) H2, Pd/C 10%, EtOAc, 20 °C, 16 h. Compounds were
isolated in a 25/26 ) 55/45 ratio; (c) triphosgene, Et3N, anhydrous
CH2Cl2, 20 °C, 5 h; (d) MgBr2, anhydrous CH2Cl2, 20 °C, 16 h.
Sch em e 4a
From these results, we have logically selected 19b as
the best noncytotoxic inhibitor of APN activity.
We next examined the effects of 19b on a number of
other metalloproteinases (NEP, ACE, MMP-9) and the
unclassified γ-glutamyl transpeptidase (γ-GT) as well
as serine proteases (DPPIV, cathepsin G). These en-
zymes were chosen because of their major involvement
in different pathological conditions and thus represent
targets for therapeutic intervention.1,2,46,47 Proteolytic
activities of APN, NEP, ACE, γ-GT, DPPIV, and cathep-
sin G were measured using absorbance-based assays.
As expected, each protease activity was only down-
regulated by its specific inhibitor (Figure 1). The addi-
tion of 19b (10-3 M) to the incubation medium strongly
inhibited the proteolytic activity of APN, whereas the
other enzyme activities were not significantly affected
(Figure 1). The apparent affinity of APN present at the
surface of U937 cells for Ala-pNA (i.e., 1/Km) exhibited
a value of 103 M-1 and the apparent IC50 values of
bestatin and 19b were around 25 µM (data not shown).
Therefore, one could consider that 1 molecule of 19b
could compete with approximately 25 molecules of the
substrate Ala-pNA, thus indicating that 19b merely
exhibits a significant potent inhibitory effect toward
APN.
a
Reagents and conditions: (a) K2CO3, butanone reflux, 48 h;
(b) MeOH, H2SO4, 20 °C, 3 days, then H2O, 0 °C; (c) RuCl3, NaIO4,
CCl4, MeCN, H2O.
ceeded 40% with all the products tested (data not
shown). At 10-4 M, some derivatives exhibited cytotoxic
potency (8, 19m , 20-23 and to a lesser degree 11, 12,
19h , 19l, 24, 28) (Table 1) and markedly reduced cell
proliferation (over 30%). In contrast, the proliferation
of U937 cells was unaffected by the non cytotoxic 7, 9,
10, 13, 18b, 19a -g, 19i-k , and 29. In this context, it
is worth pointing out that bestatin (1) is much more
toxic for U937 cells than most of the herein studied
compounds. Moreover, we have assessed that the IC50
The effect of 19b toward the gelatinolytic activity of
MMP-9 was assessed by zymography (Figure 2). By this
technique, the gelatinolytic activity of MMP-9 was
revealed by the presence of a 92 kDa band in native
conditions (Figure 2a). Incubation of the zymograms
with EDTA or the specific inhibitor of MMP-9 resulted

3904 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Bauvois et al.
Ta ble 1
compound
R3
R8
R2′
R3′
R4′
R5′
cytotoxicity (%)a
inhibition APN (%)b
control
bestatin (1)
flavone
FAA (7)
8
9
10
11
12
<10
g40
<10
<10
g80
<10
<10
g40
g40
<10
<10
<10
<10
<10
<10
<10
<10
<10
g40
20-30
g20
<10
g40
g55
g95
g65
g85
g95
20-30
20-30
<10
0
85 ( 3
5 ( 9
30 ( 4
83 ( 2
33 ( 4
30 ( 9
e5
H
H
H
H
H
H
NO2
H
H
NO2
H
H
NO2
H
NO2
NO2
NO2
H
H
H
H
Cl
OCH3
CO2CH3
CO2Bn
OBn
OBn
OH
NO2
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
NO2
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
H
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
CH2CO2H
CH2CO2H
CH2CO2H
H
NH3+Cl-
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NH2
NO2
H
CH2CHO
CH2CO2CH3
CH2CHdCH2
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2MOMc
CH2CO2MOMc
CH2CO2MOMc
51 ( 3
e5
13
18b
19a
19b
19c
19d
19e
19f
19g
19h
19i
19j
19k
19l
19m
20
21
16 ( 2
68 ( 6
88 ( 3
75 ( 2
80 ( 2
63 ( 1
44 ( 3
61 ( 4
64 ( 1
76 ( 1
69 ( 1
66 ( 1
77 ( 1
76 ( 1
32 ( 1
19 ( 1
48 ( 1
25 ( 3
19 ( 3
10 ( 2
8 ( 4
22
23
24
28
29
a
b
Cytotoxicity was measured after 3 days of culture of U937 for a 10-4 M concentration of the tested compound. The reported inhibitions
of APN activity (measured in the presence of intact U937 cells and Ala-pNA for 15 min at 37 °C) in the presence of the tested compounds
(10-3 M) are the results of the mean of three to ten experiments ( SD. c MOM ) CH2OCH3.
in the inhibition of the gelatinolytic activity of MMP-9
by 90% (Figure 2b and 2c). In contrast, 19b did not alter
the gelatinolytic activity of MMP-9 (Figure 2d). Simi-
larly, the addition of bestatin (1), a noninhibitor of
MMP-9, had no effect on enzyme activity (Figure 2e).
Together, these data unambiguously demonstrated that
19b selectively inhibits APN activity.
Specific inhibition of APN/CD13 by 19b. Whole cell
lysates from U937 cells were incubated in the absence
or in the presence of MY7 antibody directed against
CD13 antigen or mIgG1 (control isotype), as described
in Experimental Procedures. The immunoprecipitates
bound to Protein G-Sepharose were tested for APN
activity. As shown in Figure 3, MY7 Ab was capable of
precipitating APN activity from U937 cells. Both be-
statin and 19b efficiently inhibited APN/CD13 (Figure
3). These results confirm that 19b, like bestatin (1),
inhibits APN activity endowed by the ectoprotease
CD13.
19b Rever sibly a n d Com p etitively In h ibits AP N/
CD13 of U937 Cells. To investigate the reversible
nature of the inhibition of APN/CD13 by 19b, U937 cells
were incubated with 10-3 M of 19b or bestatin (1, used
as a positive reversible inhibitor control) for 15 min
before extensive washing and further incubation in the
protease assay medium. As shown in Figure 4, APN
inhibition observed with 19b or bestatin was found to
be lost upon cell washing, indicating the reversible
nature of the inhibition by the dinitroflavone 19b. We
further examined the capacity of 19b to bind to the
catalytic site of APN/CD13 of intact U937 cells. For this
purpose, we used two mAbs directed against CD13:
WM15 Ab (which recognizes on CD13 the epitope
involved in the catalytic activity) and MY7 Ab (which
binds another epitope of CD13). Lineweaver-Burk plots
revealed that inhibition by 19b for WM15 Ab was
competitive (Figure 5A), whereas the inhibition by 19b
for MY7 Ab fit with an uncompetitive inhibition model
(Figure 5B). Together, our data indicate that 19b is a
reversible competitive inhibitor of APN/CD13.
Con clu sion s
We have herein reported on the capacity of some
flavone-8-acetic acid derivatives to inhibit the enzymatic
activity of the ectopeptidase APN/CD13. We have
synthesized and tested a set of variously substituted
compounds, and the best results were obtained with the
compound 19b bearing two NO2 groups in both 2′- and
3-positions. This selected 2′,3-dinitroflavone-8-acetic
acid (19b) proves to be noncytotoxic for the human
model U937 cell line and exhibits an exclusive inhibitory
APN/CD13 activity by reversibly binding to the catalytic
site of the enzyme. Furthermore, the comparison of the
apparent affinity of APN/CD13 of U937 cells for its

Novel Flavone-8-acetic Acid Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3905
F igu r e 2. Absence of effect of 19b on MMP-9 gelatinolytic
activity. The gelatinolytic activity of recombinant 92 kDa pro-
MMP-9 was analyzed using zymography performed with
increasing amounts of protein loaded (0.1, 1, and 10 ng).
Gelatinolytic activities are detected as clear bands in the gel.
(a) in the absence, and in the presence of (b) EDTA, (c) the
specific inhibitor of MMP-9 (2R)-2-[(4-biphenylsulfonyl)amino]3-
phenylpropionic acid, (d) 19b, (e) bestatin (1). All inhibitors
were tested at 10-3 M.
F igu r e 1. Effect of 19b on various protease activities. (A)
Intact U937 cells exhibit APN, γ-GT, and NEP activities at
their surface. Relative enzyme activity was expressed as the
percentage of the control activity without inhibitor: bestatin
(APN inhibitor), acivicin (γ-GT inhibitor), phosphoramidon
(NEP inhibitor), and 19b. (B) Purified soluble proteases
DPPIV, ACE, and cathepsin G were tested for their enzymatic
activities in the absence or presence of their respective
inhibitors (DFP for DPPIV and cathepsin G) (captopril for
ACE) or 19b. Values are expressed as percentages of controls
and represent the mean ( SD (n ) 3). All inhibitors were
tested at 10-3 M.
F igu r e 3. Immunoprecipitation of APN activity with MY-7
Ab directed against CD13, and inhibition by bestatin or 19b.
Whole cell lysates from U937 cells were immunoprecipitated
with mIgG1 (control isotype) or MY7/CD13 Ab. Material bound
to Protein G-Sepharose was tested for APN activity using Ala-
pNA (1 mg/mL) in the absence or presence of bestatin or 19b
(10-3 M).
synthetic substrate Ala-pNA (103 M-1) with the appar-
ent IC50 value of 19b (around 25 µM) indicates that the
inhibitory effect of 19b toward APN is strong.
Apart from APN/CD13, we have examined the effect
of 19b on other metalloproteinase activities including
MMP-9, NEP, and ACE, as well as on serine activities
(DPPIV and cathepsin G) and on γ-GT, all proteases
previously suggested to play critical roles in tumoral
processes.1,2,46,47 Our results indicate that 19b exclu-
sively inhibits the proteolytic activity of APN/CD13. The
results obtained in the U937 cell line were also observed
in other myeloid cell lines (HL-60, THP-1, Mono Mac6),
T (J urkat), B (Eskol, Ramos, RPMI 8266) and in normal
(T, B, monocytes) and leukemic (LLC, LMC) leukocytes
(data not shown). A growing accumulation of evidence
shows the crucial importance of APN/CD13 in tumor
metastasis and angiogenesis, both in vitro and in
vivo.2,4,7-9,11,13-15 When angiogenesis is blocked, tumor
growth is often suppressed, and tumor cell proliferation
is balanced by apoptosis. Modulation of growth arrest/
F igu r e 4. Reversibility of APN/CD13 inhibition by 19b. Intact
U937 cells were preincubated for 15 min in the absence
(control) or presence of 10-3 M bestatin (1) or 19b, washed
twice in protease buffer, and then assayed for APN activity.
Relative enzyme activity was expressed as the percentage of
the control activity expressed by U937 cells which have not
been washed and without inhibitor (mean ( SD, n ) 3).
apoptosis is associated with signaling through protein
phosphorylation.48-50 There is evidence that tyrosine
phosphorylation steps are involved in APN/CD13
signaling.51-54 Intracellular calcium flux and MAP
kinase phosphorylation are implicated in the activation
of monocytes and T cells through APN/CD13.52-54 Fla-
vone-8-acetic derivatives, and particularly 19b, may

3906 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Bauvois et al.
ratories (Vernaison, France) and, unless noted otherwise, the
results obtained are within 0.4% of the theoretical values.
However, it must be pointed out that the microanalyses have
not been carried out for compounds 16b, 16d -f, 16i, 16j, and
16m which have been otained as oily liquid after chromatog-
raphy and have been directly used in the subsequent step.
Compounds 9-13, 15a , 15b, 15e, 15f, 15g-j, 16a , 17a , 18a ,
and 19a were already prepared for a previous study. Synthetic
and analytical details for these derivatives have been pub-
lished elsewhere.39 The remaining novel 3-nitroflavones 19b-m
used in the present work and their precursors 16b-m , 17b-
m , and 18b-m were synthesized by adapting the methodology
developed in our laboratory starting from 2-hydroxy-3-allyl-
benzaldehyde (14) and appropriately substituted Z-2-chloro-
2-nitroethenylbenzenes (15a -m ).39
Gen er a l P r oced u r e for th e Syn th esis of th e Z-(2-
Ch lor o-2-n it r oet h en yl)b en zen es 15a -m . The relevant
benzaldehyde (0.3 mol, all of which are commercially available
except 2-formylbenzoic acid methyl ester,55 2-formylbenzoic
benzyl ester,56 and 2-benzyloxybenzaldehyde,57 which were
prepared according to previously reported procedures), xylene
(750 mL), dimethylammonium chloride (220.3 g, 2.7mol),
bromonitromethane (78.4 g, 39.12 mL, 0.56 mol), and potas-
sium fluoride (2.61 g, 45 mmol) were placed in a 2-L conical
flask fitted with a Dean-Stark apparatus (capacity about 20
mL). The mixture was vigorously refluxed with stirring for 3
h and then allowed to cool to room temperature. Removal of
the volatile materials under reduced pressure left a residue
which was taken up with H2O (300 mL) and CH2Cl2 (800 mL).
The organic layer was separated, and the aqueous phase was
extracted with CH2Cl2 (3 × 200 mL). The combined organic
extracts were dried (MgSO4), filtered and then evaporated in
vacuo to afford a crude product which was chromatographed
on a silica gel column (950 g, eluent CH2Cl2). Evaporation of
the solvent, followed by recrystallization, gave pure (2-chloro-
2-nitroethenyl)benzenes 15a -m in the exclusive Z configura-
tion. Compounds 15a , 15b, and 15e-j have been described in
a previous communication.39 Yields, recrystallization solvents,
physical constants, and spectral data for the hitherto unknown
derivatives 15c, 15d , and 15k -m are reported below:
F igu r e 5. Lineweaver-Burk plots for 19b and mAbs against
CD13. Representative plots of 1/bound CD13 Ab to U937 cells
vs 1/[CD13 Ab] in the presence or absence of 19b (10-3 M) are
shown. (A) WM15 Ab which recognizes on CD13 the epitope
involved in the catalytic activity, and (B) MY7 Ab which binds
another epitope of CD13. Staining of U937 cells with Abs in
the absence or presence of 19b was performed at 4 °C.
therefore represent valuable tools for investigating
mechanisms involved in these processes.
In conclusion, this novel series of noncytotoxic inhibi-
tors of APN bears the potential of producing selective
APN-mediated antiangiogenic inhibitors. We hope to
report some of these promising data soon.
Z-(2-Ch lor o-2-n it r oe t h e n yl)-3-m e t h oxy-2-n it r ob e n -
zen e (15c). Yield 65%; mp 127-128°C recrystallized from a
benzene/heptane mixture as pale yellow crystals; 1H NMR
(CDCl3 δ: 3.96 (s, 3H), 7.19 (dd, 1H, 8.2, 0.7), 7.48 (dd, 1H,
8.2, 0.7), 7.58 (t, 1H, 8.2), 8.20 (s, 1H); IR ν ) 1553, 1344 cm-1
;
MS m/z: 259, 261 (M + H)+, 276, 278 (M + NH4)+. Anal. (C9H7-
ClN2O5). C, H, N.
Exp er im en ta l Section
Z-(2-Ch lor o-2-n it r oe t h e n yl)-5-m e t h oxy-2-n it r ob e n -
zen e (15d ). Yield 60%; mp 94-95 °C recrystallized from a
benzene/heptane mixture as pale yellow crystals; 1H NMR
(CDCl3) δ: 3.94 (s, 3H), 7.07 (m, 2H), 8.30 (d, 1H, J ) 9.7),
8.69 (s, 1H); IR ν ) 1554, 1349 cm-1; MS m/z: 259, 261 (M +
H)+, 276, 278 (M + NH4)+. Anal. (C9H7ClN2O5). C, H, N.
Z-2-(2-Ch lor o-2-n itr oeth en yl)ben zoic Acid Meth yl Es-
ter (15k ). Yield 61%; mp 98-99 °C recrystallized from a
benzene/heptane mixture as pale yellow crystals; 1H NMR
(CDCl3) δ: 3.94 (s, 1H), 7.35 (dt, 1H, J ) 7.8, 1.3), 7.65 (m,
2H), 8.13 (d, 1H, J ) 7.8), 8.95 (s, 1H); IR ν ) 1718. 1542.
1342 cm-1; MS m/z: 242, 244 (M + H)+, 259, 261 (M + NH4)+.
Anal. (C10H8ClNO4). C, H, N.
Z-2-(2-Ch lor o-2-n itr oeth en yl)ben zoic Acid Ben zyl Es-
ter (15l). Yield 73%; amber-colored oily liquid; 1H NMR
(CDCl3) δ: 5.36 (s, 2H), 7.41 (m, 5H), 7.54 (dt, 1H, J ) 10.6,
1.1), 7.65 (m, 2H), 8.17 (dt, 1H, J ) 10.6. 1.1), 8.93 (s, 1H); IR
ν ) 1714. 1546. 1324. 1265 cm-1; MS m/z: 318. 320 (M + H)+,
335, 337 (M + NH4)+. Anal. (C16H12ClNO4). C, H, N.
Z-2-(2-Ch lor o-2-n itr oeth en yl)ben zyloxyben zen e (15m ).
Yield 80%; amber-colored oily liquid; 1H NMR (CDCl3) δ: 5.18
(s, 2H), 7.15 (m, 2H), 7.4 (m, 6H), 8.15 (dd, 1H, J ) 7.6, 0.5),
8.88 (s, 1H), IR ν ) 1556, 1350 cm-1; MS m/z: 290, 292 (M +
H)+, 307, 309 (M + NH4)+. Anal. (C15H12ClNO3). C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e 8-Allyl-
3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-3-n it r o-2-p h en yl-2H -1-
ben zop yr a n s 16a m . In a dry 1-L flask fitted with a drying
Ch em istr y. Starting materials and solvents were pur-
chased from Acros, Aldrich or Avocado. Reference bestatin (1)
and flavone were obtained from Sigma Chemical Co. A pure
sample of flavone-8-acetic acid (7) was kindly provided by
Merck (France). Melting points were measured on a Ko¨fler hot
stage apparatus and are uncorrected. Mass spectra were
obtained with a Nermag-Ribermag R10-10C spectrometer
applying a desorption chemical ionization (CI) technique using
ammonia as the reagent gas. Infrared spectra were obtained
with a Perkin-Elmer 1710 spectrophotometer for chloroform
1
solutions or KBr disks. The H NMR (300 MHz) spectra were
recorded on a Bruker AC 300 spectrometer. Chemical shifts
are expressed as parts per million downfield from tetrameth-
ylsilane. Splitting patterns have been designated as follows:
s (singlet), d (doublet), dd (doublet of doublet), ddd (doublet of
doublet of doublet), dt (double triplet), m (multiplet), and br
(broad signal). Coupling constants (J values) are listed in hertz
(Hz). Reactions were monitored by analytical thin-layer chro-
matography, and products were visualized by exposure to UV
light. Merck silica gel (230-400 Mesh ASTM) was used for
column chromatography. Acetone, methanol, and dichloro-
methane employed as eluents for column chromatography were
distilled on a rotary evaporator prior to use. Anhydrous
benzene was obtained by distillation from calcium hydride. Dry
THF was prepared by distillation from benzophenone/sodium.
All yields reported are unoptimized. Elemental analysis for
most of the new substances were performed by CNRS Labo-

Novel Flavone-8-acetic Acid Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3907
tube, the appropriate starting Z-(2-chloro-2-nitroethenyl)-
benzene 15a -m (80 mmol) and 3-allylslicylaldehyde (14,40 73
g, 0.45 mol) were dissolved in a minimal volume of anhydrous
THF (80 mL). Dry and freshly distilled triethylamine (4.36 g,
6.06 mL, 43 mmol) was quickly added via a syringe, and the
mixture was stirred at room temperature for 24-48 h under
inert atmosphere (monitoring by TLC). Acetic acid (about 1
mL) was then added, and the volatile materials were rotary-
evaporated in vacuo (0.6 mmHg) at 45-50 °C (excessive
heating and moisture must be avoided). The crude residue was
taken up with a lukewarm solution (50 °C) of Girard’s reagent
T [prepared by dissolving the commercial reagent (75 g, 0.45
mol) in a 8/1/1 mixture of CH3CH2OH, CH3COOH and H2O
(560 mL)]. The reaction medium was efficiently stirred for 2
h at room temperature and then diluted with CH2Cl2 (500 mL)
and water (400 mL). When an insoluble material remained, it
was filtered off on a sintered funnel and thoroughly rinsed with
CH2Cl2. The organic layer was separated, and the aqueous
phase was extracted with CH2Cl2 (3 × 250 mL). The combined
organic extracts were dried (MgSO4), filtered then concentrated
under reduced pressure to leave an aldehyde-free material.
Chromatography of the crude product on silica gel (500 g,
eluent CH2Cl2) afforded the desired derivatives 16a -m which,
after evaporation of the solvent, were recrystallized in ap-
propriate conditions. These benzopyrans were exclusively
obtained as a unique diastereomer with the relative configu-
ration 2R,3R,4R. Compound 16a has been described in a
former communication.39 Yields, recrystallization solvents,
physical constants, and spectral data for the novel derivatives
16b-m are reported below:
8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-3-n itr o-2-(2-n i-
tr op h en yl)-2H-1-ben zop yr a n (16b). Yield 63%; viscous yel-
low oil, 1H NMR (CDCl3) δ 2.64 (d, 1H, J ) 10.7, exchangeable
with D2O), 3.34-3.40 (m, 2H), 5.00 (dd, 1H, J ) 17.0, 1.7),
5.07 (dd, 1H, J ) 10.1, 1.7), 5.85-5.95 (m, 1H), 6.05 (d, 1H,
J ) 10.7), 6.72 (s, 1H), 7.12 (br t, 1H, J ) 8.0), 7.22 (br d, 1H,
J ) 7.5), 7.50 (br d, 1H, J ) 7.5), 7.64 (dt, 1H, J ) 8.3.1.2),
7.76 (dt, 1H, J ) 8.3. 1.2), 7.98 (dd, 1H, J ) 8.3, 1.2), 8.16 (dd,
1H, J ) 8.4, 1.3), IR ν ) 3560-3080, 1570, 1540, 1400, 1350
cm-1; MS m/z: 408, 410 (M + NH4)+.
8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-3-n it r o-2-(3-
m eth oxy-2-n itr oph en yl)-2H-1-ben zopyr an (16c). Yield 61%;
viscous amber-colored oil, 1H NMR (CDCl3) δ: 2.60 (d, 1H,
J ) 11.0, exchangeable with D2O), 3.42-3.55 (m, 2H), 3.90 (s,
3H), 4.90 (dd, 1H, J ) 17.1, 1.3), 5.15 (dd, 1H, J ) 10.1, 1.3),
5.60 (s, 1H), 5.83-5.98 (m, 2H), 7.10 (t, 1H, J ) 7.6), 7.13-
7.32 (m, 2H), 7.40 (d, 1H, J ) 7.3), 7.49-7.56 (m, 2H); IR ν )
3480, 1580, 1523, 1340 cm-1; MS m/z: 421, 423 (M + H)+, 438,
440 (M + NH4)+.
8.45 (d, 1H J ) 1.8). IR ν ) 3450, 1570, 1540, 1400, 1350 cm-1
MS m/z: 391, 393 (M + H)+, 408, 410 (M + NH4)+.
;
8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-2-(4-m et h oxy-
3-n itr op h en yl)-3-n itr o-2H-1-ben zop yr a n (16g). Yield 66%;
mp 134-135 °C, recrystallized from a benzene/heptane mix-
1
ture as pale yellow crystals; H NMR (CDCl3) δ: 2.50 (d, 1H,
J ) 11.8, exchangeable with D2O), 3.30-3.47 (m, 2H), 3.99 (s,
3H), 5.01 (dd, 1H, J ) 16.8, 1.3), 5.09 (dd, 1H, J ) 9.9, 1.3),
4.97-5.10 (m, 2H), 5.82 (s, 1H), 5.82-6.03 (m, 1H), 6.00 (d,
1H J ) 11.8), 7.08 (m, 2H), 7.24 (m, 1H), 7.45 (d, 1H, J ) 6.6),
7.54 (dd, 1H, J ) 8.7, 2.2), 8.06 (d, 1H, J ) 2.2), IR ν ) 3450.
1572. 1544. 1403. 1355 cm-1; MS m/z: 421, 423 (M + H)+, 438,
440 (M + NH4)+. Anal. (C19H17ClN2O7). C, H, N.
8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-2-(4-m et h oxy-
p h en yl)-3-n itr o-2H-1-ben zop yr a n (16h ). Yield 76%; mp
128-129 °C, recrystallized from a benzene/heptane mixture
as colorless crystals; 1H NMR (CDCl3) δ: 2.47 (d, 1H, J ) 11.9,
exchangeable with D2O), 3.29-3.51 (m, 2H), 3.82 (s, 3H), 5.02
(dd, 1H, J ) 17.0, 1.7), 5.09 (dd, 1H, J ) 9.4, 1.7), 5.75 (s, 1H),
5.89-6.01 (m, 1H), 6.03 (d, 1H, J ) 11.9), 6.91 (br d, 2H )
8.8), 7.07 (br t, 1H J ) 7.7), 7.22 (m, 1H), 7.39 (br d, 2H, J )
8.8), 7.43 (d, 1H, J ) 7.7). IR ν ) 3450. 1575. 1542. 1406. 1350
cm-1; MS m/z: 376. 378 (M + H)+, 393. 395 (M + NH4)+. Anal.
(C19H18ClNO5). C, H, N.
8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-2-(2-ch lor o-
p h en yl)-3-n itr o-2H-1-ben zop yr a n (16i). Yield 68%; viscous
1
amber-colored oil; H NMR (CDCl3) δ: 2.80 (d, 1H, J ) 11.2,
exchangeable with D2O), 3.28-3.51 (m, 2H), 5.02 (dd, 1H,
J ) 17.1, 1.3), 5.16 (dd, 1H, J ) 10.8, 1.3), 5.85-6.12 (m, 2H),
6.42 (s, 1H), 6.82 (t, 1H, J ) 7.6), 6.94 (d, 1H, J ) 7.6), 7.04-
7.21 (m, 2H), 8.01 (d, 1H, J ) 7.8); IR ν ) 3480, 1570, 1523
cm-1; MS m/z: 380, 382, 384 (M + H)+, 397, 399, 401 (M +
NH4)+. Anal. (C18H15Cl2NO4) C, H, N.
8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-2-(2-m et h oxy-
p h en yl)-3-n itr o-2H-1-ben zop yr a n (16j). Yield 42%; viscous
1
amber-colored oil; H NMR (CDCl3) δ: 2.53 (d, 1H, J ) 11.6,
exchangeable with D2O), 3.28-3.49 (m, 2H), 3.72 (s, 3H), 4.93
(dd, 1H, J ) 17.2, 1.6), 5.24 (dd, 1H, J ) 9.5, 1.6), 5.30 (s, 1H),
5.82-6.04 (m, 1H), 6.32 (d, 1H, J ) 11.6), 6.92 (dd, 1H, J )
8.3, 2.0), 7.00-7.13 (m, 2H), 7.20 (d, 1H, J ) 7.4), 7.32-7.51
(m, 2H), 7.83 (dd, 1H, J ) 8.3. 2.0), IR ν) 3480, 1570, 1544
cm-1; MS m/z: 376, 378 (M + H)+, 393, 395 (M + NH4)+.
2-[8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-3-n itr o-2H-1-
ben zop yr a n -2-yl]ben zoic Acid Meth yl Ester (16k ). Yield
51%; mp 155-156 °C, recrystallized from a benzene/heptane
mixture as white crystals; 1H NMR (CDCl3) δ: 2.62 (d, 2H,
J ) 11.1, exchangeable with D2O), 3.25-3.51 (m, 2H), 3.92 (s,
3H), 5.01 (dd, 1H, J ) 17.4, 1.2), 5.13 (dd, 1H, J ) 11.6, 1.2),
5.83-6.05 (m, 1H), 6.12 (d, 1H, J ) 11.1), 7.02-7.20 (m, 2H),
7.26 (d, 1H, J ) 7.4), 7.42-7.60 (m, 2H), 7.64 (dt, 1H, J ) 7.8,
0.8), 7.86 (dd, 1H, J ) 7.8, 0.8), 8.16 (dd, 1H, J ) 7.8, 0.8), IR
ν ) 3480, 1718, 1570, 1458, 1271 cm-1; MS m/z: 404, 406
(M + H)+, 421, 423 (M + NH4)+. Anal. (C20H18ClNO6) C, H, N.
2-[8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-3-n itr o-2H-1-
ben zop yr a n -2-yl]ben zoic Acid Ben zyl Ester (16l). Yield
73%; mp 122-123 °C, recrystallized from a benzene/heptane
mixture as white crystals; 1H NMR (CDCl3) δ: 2.77 (d, 2.H,
J ) 11.0, exchangeable with D2O), 3.28-3.54 (m, 2H), 5.02
(dd, 1H, J ) 18.2, 1.8), 5.13 (dd, 1H, J ) 10.8, 1.8), 5.28 (d,
1H, J ) 12.3), 5.40 (d, 1H, J ) 12.3), 5.85-6.00 (m, 1H), 6.01
(d, 1H, J ) 11.0), 7.02-7.13 (m, 3H), 7.28 (dd, 1H, J ) 7.1,
1.1), 7.40-7.59 (m, 6H), 7.64 (dt, 1H, J ) 7.1, 1.1), 7.92 (dd,
1H, J ) 7.8, 1.3), 8.10 (dd, 1H, J ) 7.1, 1.1); IR ν ) 3480,
1569, 1458, 1262 cm-1; MS m/z: 480, 482 (M + H)+, 497, 499
(M + NH4)+. Anal. (C26H22ClNO6) H, N; C, calcd, 65.07; found,
64.46.
8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-3-n it r o-2-(5-
m eth oxy-2-n itr oph en yl)-2H-1-ben zopyr an (16d). Yield 77%;
1
viscous amber-colored oil, H NMR (DMSO-d6) δ: 2.41 (d, 1H,
J ) 10.9, exchangeable with D2O), 3.20-3.42 (m, 2H), 3.90 (s,
3H), 4.93 (dd, 1H, J ) 16.9, 1.4), 5.05 (dd, 1H, J ) 10.3, 1.4),
5.70 (s, 1H), 5.82-6.01 (m, 2H), 7.00 (t, 1H, J ) 7.6), 7.18-
7.29 (m, 2H), 7.30 (d, 1H, J ) 8.7), 7.40 (d, 1H, J ) 7.6), 8.10
(d, 1H, J ) 8.7), IR ν ) 3480, 1582, 1526, 1341 cm-1; MS m/z:
421, 423 (M + H)+, 438, 440 (M + NH4)+.
8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-3-n itr o-2-(4-n i-
tr op h en yl)-2H-1-ben zop yr a n (16e). Yield 90%; viscous yel-
low oil; 1H NMR (CDCl3) δ: 2.70 (d, 1H, J ) 11.0, exchangeable
with D2O), 3.29-3.42 (m, 2H), 4.90 (dd, 1H, J ) 17.1, 1.6),
5.10 (dd, 1H, J ) 10.8, 1.6), 5.30 (s, 1H), 5.84-5.96 (m, 1H),
6.00 (d, 1H, J ) 11.0), 7.04-7.13 (m, 2H), 7.20 (d, 1H, J )
7.4), 7.60 (d, 1H, J ) 8.8), 8.30 (d, 2H, J ) 8.8), IR ν ) 3490,
1578, 1530, 1345 cm-1; MS m/z: 391, 393 (M + H)+, 408, 410
(M + NH4)+.
8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-3-n itr o-2-(3-n i-
tr op h en yl)-2H-1-ben zop yr a n (16f). Yield 95%; viscous yel-
low oil; 1H NMR (CDCl3) δ: 2.67 (d, 1H, J ) 11.9, exchangeable
with D2O), 3.30-3.49 (m, 2H), 5.02 (dd, 1H, J ) 17.1, 1.3),
5.12 (dd, 1H, J ) 10.1, 1.3), 5.90 (s, 1H), 5.91-6.00 (m, 1H),
6.03 (br d, 1H, J ) 11.9), 7.08-7.75 (m, 5H), 8.31 (m, 1H),
8-Allyl-3-ch lor o-3,4-d ih yd r o-4-h yd r oxy-2-(2-ben zyloxy-
p h en yl)-3-n itr o-2H-1-ben zop yr a n (16m ). Yield 83% from
chromatography as a viscous amber-colored oil. 1H NMR
(CDCl3) δ: 2.70 (d, 2.H, J ) 10.9, exchangeable with D2O), 3.42
(d, 1H, J ) 6.4), 4.90-5.22 (m, 5H), 5.88-6.12 (m, 1H), 6.31
(s, 1H), 6.82 (br d, 1H, J ) 7.8), 6.88 (dd, 1H, J ) 7.0, 1.0),
6.94 (dd, 1H, J ) 7.8, 1.1), 7.03-7.20 (m, 3H), 7.29-750 (m,
5H), 7.82 (dd, 1H, J ) 6.8, 1.4), IR ν ) 3460, 1558, 1461, 1273

3908 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Bauvois et al.
cm-1; MS m/z: 452, 454 (M + H)+, 469, 471(M + NH4)+. Anal.
(C25H22ClNO5) C, H, N.
122-124°C recrystallized from a benzene/hexane mixture as
pale yellow crystals; 1H NMR (CDCl3) δ: 3.46 (d, 2H, J ) 6.4),
4.00 (s, 3H), 5.06 (dd, 1H, J ) 17.5, 1.4), 5.13 (dd, 1H, J )
10.4, 1.4), 5.87-6.06 5.81-6.01(m, 1H), 6.29 (s, 1H), 7.12-
7.25 (m, 2H,), 7.53-7.59 (m, 2H), 7.92 (dd, 1H, J ) 8.9, 2.0),
8.10 (d, 1H, J ) 2.0), IR ν ) 1710, 1579, 1359 cm-1; MS m/z:
Gen er a l P r oced u r e for th e P r ep a r a tion of th e 8-Allyl-
3-ch lor o-2,3-d ih yd r o-3-n itr o-2-p h en yl-4H-1-ben zop yr a n -
4-on es 17a -m . In a previously dried 500-mL round-bottomed
flask equipped with a condenser surmounted by a drying tube,
the appropriate 3-chloro-3,4-dihydro-4-hydroxy-3-nitro-2-phen-
yl-2H-1-benzopyrans 16a -m (45 mmol) was dissolved in
anhydrous CH2Cl2 (200-350 mL). Pyridinium chlorochromate
(19.5 g, 90 mmol) was then added in one portion, and the flask
was immersed into an ultrasound bath for 24 h. The temper-
ature of the bath increased progressively from room temper-
ature to 55 °C. The reaction mixture was then suction-filtered
through a short pad of Celite, and the solid was exhaustively
rinsed with several portions of CH2Cl2. Evaporation of the
solvent under reduced pressure left a residue which was
directly chromatographed on a silica gel column (500 g, eluent
CH2Cl2). Removal of the solvent in vacuo, followed by recrys-
tallization in suitable solvents, afforded the desired derivatives
17a -m with the exclusive relative configuration 2R,3S. The
419, 421 (M + H)+, 436, 438 (M + NH4)+. Anal. (C19H15
-
ClN2O7): C, H, N.
8-Allyl-3-ch lor o-2,3-d ih yd r o-3-n itr o-2-(4-m eth oxyp h en -
yl)-4H-1-ben zop yr a n -4-on e (17h ). Yield 98% as a pale yellow
1
oil from chromatography; H NMR (CDCl3) δ: 3.35-3.55 (m,
2H), 3.84 (s, 3H), 5.06 (dd, 1H, J ) 17.1, 1.5), 5.11 (dd, 1H, J
) 10.7, 1.5), 5.88-6.02 (m, 1H), 6.23 (s, 1H), 6.96 (br d, 2H, J
) 8.8), 7.20 (t, 1H J ) 7.7), 7.43 (br d, 2H, J ) 8.8), 7.55 (dd,
1H, J ) 7.3, 1.5), 7.94 (dd, 1H, J ) 8.0, 1.6), IR ν ) 1711,
1570, 1342 cm-1 ; MS m/z: 374, 376 (M + H)+, 391, 393 (M +
NH4)+. Anal. (C19H16ClNO5): C, H, N.
8-Allyl-3-ch lor o-2,3-dih ydr o-3-n itr o-2-(2-ch lor oph en yl)-
4H-1-ben zop yr a n -4-on e (17i). Yield 89%; mp 91-92 °C
recrystallized from
a benzene/heptane mixture as yellow
1
benzopyran-4-one 17a has been described in
a previous
crystals; H NMR (CDCl3) δ: 3.55 (d, 2H, J ) 6.5), 4.92 (dd,
1H, J ) 17.3, 1.4), 5.16 (dd, 1H, J ) 10.6, 1.4), 5.79-6.00 (m,
1H), 6.73 (s, 1H), 7.24 (t, 1H, J ) 7.8), 7.42-7.48 (m, 3H), 7.65
(d, 1H, J ) 8), 7.89-8.04 (m, 2H); IR ν ) 1713, 1581, 1287
cm-1; MS m/z: 378, 380, 382 (M + H)+, 395, 397, 399 (M +
NH4)+. Anal. (C18H13Cl2NO4) C, H, N.
report.39 Yields, recrystallization solvents, physical constants,
and spectral data for the new compounds 17b-m are reported
below:
8-Allyl-3-ch lor o-2,3-d ih yd r o-3-n itr o-2-(2-n itr op h en yl)-
4H-1-ben zop yr a n -4-on e (17b). Yield 80%; mp 119-120 °C,
recrystallized from a benzene/heptane mixture as pale yellow
crystals; 1H NMR (CDCl3) δ: 3.43 (br d, 2H, J ) 5.6), 5.05 (dd,
1H, J ) 17.1, 1.4), 5.12 (dd, 1H, J ) 10.3, 1.4), 5.86-5.99 (m,
1H), 7.19-7.25 (m, 2H), 7.57 (br d, 1H J ) 7.8), 7.59-7.80
(m, 3H), 7.96 (dd, 1H, J ) 7.8, 1.3), 8.11 (br d, 1H, J ) 7.8),
IR ν ) 1714, 1538, 1356 cm-1; MS m/z: 389. 391 (M + H)+,
406. 408 (M + NH4)+. Anal. (C18H13ClN2O6) C, H, N.
8-Allyl-3-ch lor o-2,3-d ih yd r o-3-n itr o-2-(3-m eth oxy-2-n i-
tr op h en yl)-4H-1-ben zop yr a n -4-on e (17c). Yield 83%; mp
153-154 °C, recrystallized from a benzene/heptane mixture
8-Allyl-3-ch lor o-2,3-d ih yd r o-3-n itr o-2-(2-m eth oxyp h en -
yl)-4H-1-ben zop yr a n -4-on e (17j). Yield 93%; mp 113-114
°C recrystallized from a benzene/heptane mixture as yellow
crystals; 1H NMR (CDCl3) δ: 3.43 (d, 2H, J ) 6.5), 3.74 (s, 3H),
5.02 (dd, 1H, J ) 17.4, 1.4), 5.17 (dd, 1H, J ) 10.5, 1.4), 5.80-
6.02 (m, 1H), 6.73 (s, 1H), 6.94 (d, 1H, J ) 8), 7.03-7.29 (m,
2H), 7.40 (t, 1H, J ) 7.9), 7.52 (d, 1H, J ) 8), 7.83 (dd, 1H,
J ) 7.9. 1.6), 8.03 (dd, 1H, J ) 7.9. 1.6), IR ν ) 1711, 1583,
1290 cm-1; MS m/z: 374. 376 (M + H)+, 391. 393 (M + NH4)+.
Anal. (C19H16ClNO5) C, H, N.
1
as yellow crystals; H NMR (CDCl3) δ: 3.42 (d, 2H, J ) 6.5),
2-[8-Allyl-3-ch lor o-2,3-d ih yd r o-3-n itr o-4-oxo-4H-1-ben -
zop yr a n -2-yl]ben zoic Acid Meth yl Ester (17k ). Yield 97%;
mp 143-144 °C recrystallized from a benzene/heptane mixture
3.94 (s, 3H), 5.02 (dd, 1H, J ) 17.0, 1.6), 5.16 (dd, 1H, J )
10.1, 1.6), 5.80-6.01 (m, 1H), 6.52 (s, 1H), 7.09-7.30 (m, 2H),
7.44 (d, 1H, J ) 8.2), 7.56 (t, 1H, J ) 8.2), 7.63 (dd, 1H, J )
7.9. 1.6), 7.94 (dd, 1H, J ) 7.9. 1.6), IR ν ) 1715, 1584, 1529,
1352 cm-1; MS m/z: 419, 421 (M + H)+, 436, 438 (M + NH4)+.
Anal. (C19H15ClN2O7) C, H, N.
1
as white crystals; H NMR (CDCl3) δ: 3.52 (d, 2H, J ) 6.5),
3.81 (s, 3H), 4.92 (dd, 1H, J ) 16.8, 1.3), 5.16 (dd, 1H, J )
11.4, 1.3), 5.78-5.97 (m, 1H), 7.22 (s, 1H), 7.47 (t, 1H, J )
7.1), 7.49-7.68 (m, 2H), 7.68-7.76 (m, 2H), 8.12-8.30 (m, 2H),
IR ν ) 1718, 1579, 1476, 1289, 1272 cm-1; MS m/z: 402. 404
(M + H)+, 419. 421 (M + NH4)+. Anal. (C20H16ClNO6) H, N; C,
calcd, 59.78; found, 59.28.
8-Allyl-3-ch lor o-2,3-d ih yd r o-3-n itr o-2-(5-m eth oxy-2-n i-
tr op h en yl)-4H-1-ben zop yr a n -4-on e (17d ).Yield 62%; mp
161-162 °C recrystallized from a benzene/heptane mixture as
pale yellow crystals; 1H NMR (CD3OD) δ: 3.42 (d, 2H, J ) 6.5),
3.92 (s, 3H), 4.92 (dd, 1H, J ) 18.2, 1.6), 5.16 (dd, 1H, J )
10.0, 1.6), 5.87-6.06 (m, 1H), 7.24-7.38 (m, 2H), 7.39 (s, 1H),
7.54 (d, 1H, J ) 2.7), 7.76 (dd, 1H, J ) 7.9, 1.6), 7.93 (dd, 1H,
J ) 7.9, 1.6), 8.15 (d, 1H, J ) 9.1); IR ν) 1715, 1584, 1529,
1352 cm-1; MS m/z: 419, 421 (M + H)+, 436, 438 (M + NH4)+.
Anal. (C19H15ClN2O7) C, H, N.
2-[8-Allyl-3-ch lor o-2,3-d ih yd r o-3-n itr o-4-oxo-4H-1-ben -
zop yr a n -2-yl]ben zoic Acid Ben zyl Ester (17l). Yield 78%;
mp 95-97 °C recrystallized from a benzene/hexane mixture
1
as pale yellow crystals; H NMR (CDCl3) δ: 3.44 (d, 2H, J )
6.6), 5.03 (dd, 1H, J ) 18.3, 1.4), 5.16 (dd, 1H, J ) 10.1, 1.4),
5.34 (s, 2H), 5.84-6.03 (m, 1H), 7.22 (t, 1H, J ) 8), 7.28-7.40
(m, 3H), 7.39-7.49 (m, 2H), 7.51-7.59 (m, 2H), 7.60 (t, 1H,
J ) 8.1), 7.83 (dd, 1H, J ) 8.1, 1.5), 7.96 (dd, 1H, J ) 8.1,
1.5), 8.13 (d, 1H, J ) 8.1); IR ν ) 1714, 1578, 1476, 1289, 1266
cm-1; MS m/z: 478, 480 (M + H)+, 495, 497 (M + NH4)+. Anal.
(C26H20ClNO6) C, H, N.
8-Allyl-3-ch lor o-2,3-d ih yd r o-3-n itr o-2-(4-n itr op h en yl)-
4H-1-ben zop yr a n -4-on e (17e). Yield 50%; mp 112-113 °C
recrystallized from a benzene/heptane mixture as pale beige
1
crystals; H NMR (CDCl3) δ: 3.55 (d, 2H, J ) 6.5), 5.02 (dd,
1H, J ) 17.0, 1.5), 5.17 (dd, 1H, J ) 10.1, 1.5), 5.80-6.06 (m,
1H), 6.42 (s, 1H), 7.24 (t, 1H, J ) 7.8), 7.66 (d, 1H, J ) 7.8),
7.73 (d, 2H, J ) 8.3), 7.91 (d, 1H, J ) 7.8), 8.30 (d, 2H, J )
7.8); IR ν ) 1714, 1583, 1530, 1354 cm-1; MS m/z: 389, 391
(M + H)+, 406, 408 (M + NH4)+. Anal. (C18H13ClN2O6): C,
H, N.
8-Allyl-3-ch lor o-2,3-dih ydr o-3-n itr o-2-(2-ben zyloxyph en -
yl)-4H-1-ben zop yr a n -4-on e (17m ). Yield 61%; mp 161-162
°C recrystallized from a benzene/heptane mixture as pale
1
yellow crystals; H NMR (CDCl3) δ: 3.25-3.52 (m, 2H), 5.01
(dd, 1H, J ) 17.9, 1.5), 5.02 (s, 2H), 5.16 (dd, 1H, J ) 12.3,
1.5), 5.82-6.03 (m, 1H), 6.75 (s, 1H), 6.95 (br d 1H, J ) 8.2),
7.08-7.18 (m, 2H), 7.29-7.42 (m, 6H), 7.51 (dd, 1H, J ) 9.2,
1.3), 7.85 (dd, 1H, J ) 7.3, 1.8), 7.93 (dd, 1H, J ) 9.2, 1.3), IR
ν ) 1714, 1581, 1282 cm-1; MS m/z: 450, 452 (M + H)+, 467,
469 (M + NH4)+. Anal. (C25H20ClNO5) C, H, N.
8-Allyl-3-ch lor o-2,3-d ih yd r o-3-n itr o-2-(3-n itr op h en yl)-
4H-1-ben zop yr a n -4-on e (17f). Yield 68%; mp 110-112 °C
recrystallized from a benzene/heptane mixture as pale yellow
1
crystals; H NMR (CDCl3) δ: 3.49 (d, 2H, J ) 5.6), 5.09 (dd,
1H, J ) 17.0, 1.2), 5.16 (dd, 1H, J ) 10.1, 1.2), 5.85-6.02 (m,
1H), 6.43 (s, 1H), 7.24 (t, 1H, J ) 7.6), 7.60-7.74 (m, 3H), 7.97
(dd, 1H, J ) 7.9, 1.5), 8.36 (br d, J ) 8.1), 8.51 (s, 1H), IR ν )
1712, 1578, 1351 cm-1; MS m/z: 389, 391 (M + H)+, 406, 408
(M + NH4)+. Anal. (C18H13ClN2O6): C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e 8-Allyl-
3-n itr o-2-p h en yl-4H-1-ben zop yr a n -4-on es 18a -m . The re-
lated benzopyran-4-one 17a -m (30 mmol) was placed, under
argon atmosphere, in a 500-mL two-necked round-bottomed
flask fitted with a septum inlet. The compound was dissolved
in anhydrous THF (150 mL), and then 1,8-diazabicyclo[5.4.0]-
undec-7-ene (5.03 g, 4.94 mL, 33 mmol) was added in one
8-Allyl-3-ch lor o-2,3-d ih yd r o-3-n itr o-2-(4-m eth oxy-3-n i-
tr op h en yl)-4H-1-ben zop yr a n -4-on e (17g). Yield 62%; mp

Novel Flavone-8-acetic Acid Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3909
benzene/heptane mixture as yellow crystals; 1H NMR
(CDCl3) δ: 3.70 (d, 2H, J ) 6.3), 3.90 (s, 3H), 5.12 (dd, 1H,
J ) 17.0, 1.4), 5.19 (dd, 1H, J ) 10.1, 1.4), 5.95-6.11 (m, 1H),
7.03 (br d, 2H, J ) 9.0), 7.43 (t, 1H, J ) 7.6), 7.62 (dd, 1H,
J ) 7.3, 1.3), 7.72 (br d, 2H, J ) 9.0), 8.18 (dd, 1H, J ) 8.0,
1.6); IR ν ) 1662, 1541, 1376 cm-1; MS m/z: 338 (M + H)+,
355 (M + NH4)+. Anal. (C19H15NO5) C, H, N.
8-Allyl-3-n itr o-2-(2-ch lor op h en yl)-4H-1-ben zop yr a n -4-
on e (18i). Yield 58%; mp 103-104 °C recrystallized from a
benzene/heptane mixture as yellow crystals; 1H NMR (CDCl3)
δ: 3.60 (d, 2H, J ) 6.2), 5.01 (dd, 1H, J ) 17.1, 1.4), 5.16 (dd,
1H, J ) 10.1, 1.4), 5.85-6.04 (m, 1H), 7.32-7.60 (m, 5H), 7.57
(d, 1H, J ) 8.0), 8.22 (d, 1H, J ) 7.6); IR ν ) 1671, 1582, 1370
cm-1; MS m/z: 342, 344 (M + H)+, 359, 361 (M + NH4)+. Anal.
(C18H12ClNO4) C, H, N.
8-Allyl-3-n itr o-2-(2-m eth oxyp h en yl)-4H-1-ben zop yr a n -
4-on e (18j). Yield 87%; mp 141-143 °C recrystallized from a
benzene/heptane mixture as yellow crystals; 1H NMR (CDCl3)
δ: 3.60 (d, 2H J ) 6.4), 3.82 (s, 3H), 5.04 (dd, 1H, J ) 18.5,
1.2), 5.20 (dd, 1H, J ) 10.1, 1.2), 5.88-6.08 (m, 1H), 7.01 (d,
1H, J ) 7.8), 7.14 (t, 1H, J ) 7.8), 7.43 (t, 1H, J ) 7.8), 7.51-
7.69 (m, 3H), 8.21 (dd, 1H, J ) 7.9, 1.4); IR ν ) 1676, 1558,
1377 cm-1; MS m/z: 338 (M + H)+, 355 (M + NH4)+. Anal.
(C19H15NO5) C, H, N.
portion via a syringe. The reaction was slightly exothermic,
and its progress was monitored by thin-layer chromatography
(eluent CH2Cl2). When the starting material had completely
disappeared (reaction times: 1-3 h), aqueous HCl (0.5 N, 80
mL) and then CH2Cl2 (250 mL) were added. The organic layer
was separated, and the aqueous phase was extracted with CH2-
Cl2 (3 × 125 mL). The combined organic extracts were dried
(MgSO4), filtered and then evaporated under reduced pressure
to leave a residue which was chromatographed on a silica gel
column (320 g, eluent: CH2Cl2). Removal of the volatile
materials gave pure compounds 18a -m which were further
recrystallized in an appropriate solvent. The 8-allyl-3-nitrofla-
vone (18a ) has already been described in a previous com-
munication.39 Yields, physical constants, recrystallization
solvents, and spectral data for the novel derivatives 18b-m
are reported below:
8-Allyl-3-n it r o-2-(2-n it r op h en yl)-4H -1-b en zop yr a n -4-
on e (18b). Yield 63%; mp 162-163 °C recrystallized from a
benzene/heptane mixture as small yellow crystals; 1H NMR
(CDCl3) δ: 3.50 (d, 2H, J ) 6.5), 4.98 (dd, 1H, J ) 17.0, 1.5),
5.08 (dd, 1H, J ) 10.1, 1.5), 5.79-5.95 (m, 1H), 7.49 (t, 1H,
J ) 7.8), 7.60-7.72 (m, 2H), 7.81-7.91 (m, 2H), 8.24 (dd, 1H,
J ) 7.8, 1.4), 8.39 (dd, 1H, J ) 6.1, 3.5); IR ν ) 1672, 1589,
1536, 1348 cm-1; MS m/z: 353 (M + H)+, 370 (M + NH4)+.
Anal. (C18H12N2O6) C, H, N.
8-Allyl-3-n it r o-(3-m et h oxy-2-n it r op h en yl)-4H -1-b en -
zop yr a n -4-on e (18c). Yield 66%; mp 181-182 °C recrystal-
lized from a benzene/heptane mixture as pale beige needles;
1H NMR (CDCl3) δ: 3.53 (d, 2H, J ) 6.5), 4.00 (s, 3H), 5.01
(dd, 1H, J ) 17.0, 1.2), 5.16 (dd, 1H, J ) 10.1, 1.2), 5.79-6.04
(m, 1H), 7.22 (dd, 1H, J ) 8.6, 0.9), 7.38 (dd, 1H, J ) 8.6, 0.9),
7.42 (t, 1H, J ) 7.9), 7.56 (t, 1H, J ) 8.6), 7.67 (dd, 1H, J )
7.9, 1.5), 8.25 (dd, 1H, J ) 7.9, 1.5); IR ν ) 1671, 1582, 1381
cm-1; MS m/z: 383 (M + H)+, 400 (M + NH4)+. Anal.
(C19H14N2O7) C, H, N.
8-Allyl-3-n itr o-2-(5-m eth oxy-2-n itr op h en yl)-4H-1-ben -
zop yr a n -4-on e (18d ). Yield 70%; mp 172-173 °C recrystal-
lized from a benzene/heptane mixture as pale beige needles;
1H NMR (CD3OD) δ: 2.69 (d, 2H, J ) 6.5), 3.11 (s, 3H), 4.12
(dd, 1H, J ) 17.8, 1.5), 4.17 (dd, 1H, J ) 10.1, 1.5), 4.95-5.13
(m, 1H), 6.62 (dd, 1H, J ) 9.2, 2.8), 6.69-6.87 (m, 2H), 7.01
(dd, 1H, J ) 8.0, 1.5), 7.29 (dd, 1H, J ) 8.0, 1.5), 7.62 (d, 1H,
J ) 9.2); IR ν ) 1676, 1584, 1380 cm-1; MS m/z: 383 (M +
H)+, 400 (M + NH4)+. Anal. (C19H14N2O7) C, H, N.
a
2-[8-Allyl-3-n it r o-4-oxo-4H -1-b en zop yr a n -2-yl]b en zo-
ic Acid Meth yl Ester (18k ). Yield 70%; mp 119-121 °C
recrystallized from a benzene/heptane mixture as white crys-
tals; 1H NMR (CDCl3) δ: 3.34-3.54 (m, 2H), 3.91 (s, 3H), 5.04
(dd, 1H, J ) 17.1, 1.5), 5.20 (dd, 1H, J ) 10.1, 1.5), 5.82-6.03
(m, 1H), 7.18 (t, 1H, J ) 8.4), 7.46-7.57 (m, 3H), 7.65 (dt, 1H,
J ) 7.9, 1.5), 7.83 (dd, 1H, J ) 7.9, 1.5), 7.93 (dd, 1H, J ) 8.4,
1.6), 8.07 (dd, 1H, J ) 8.4, 1.6); IR ν ) 1719, 1668, 1537, 1381,
1284 cm-1; MS m/z: 366 (M + H)+, 383 (M + NH4)+. Anal.
(C20H15NO6) C, H, N.
2-[8-Allyl-3-n it r o-4-oxo-4H -1-b en zop yr a n -2-yl]b en zo-
ic Acid Ben zyl Ester (18l). Yield 60%; mp 103-104 °C
recrystallized from a benzene/heptane mixture as white crys-
tals; 1H NMR (CDCl3) δ: 3.39 (d, 2H, J ) 6.5), 5.01 (br d, 1H,
J ) 17.1), 5.06 (br d, 1H, J ) 10.1), 5.16 (s, 2H), 5.71-5.92
(m, 1H), 7.00-7.20 (m, 5H), 7.40 (t, 1H, J ) 8.7), 7.45-7.59
(m, 2H), 7.60-7.76 (m, 2H), 8.04 (d, 1H, J ) 7.9), 8.26 (dd,
1H, J ) 8.7, 3.2); IR ν ) 1718, 1669, 1537, 1381, 1284 cm-1
;
MS m/z: 442 (M + H)+, 459 (M + NH4)+. Anal. (C26H19NO6)
C, H, N.
8-Allyl-3-n it r o-2-(4-n it r op h en yl)-4H -1-b en zop yr a n -4-
on e (18e). Yield 79%; mp 159-160 °C recrystallized from a
benzene/heptane mixture as yellow needles; 1H NMR (CDCl3)
δ: 3.60 (d, 2H, J ) 6.5), 5.01 (d, 1H, J ) 17.0), 5.17 (d, 1H,
J ) 10.2), 5.00-5.28 (m, 1H), 6.64 (t, 1H, J ) 7.1), 6.85 (d,
1H, J ) 7.1), 6.96-7.19 (m, 2H), 7.23 (d, 1H, J ) 7.1), 7.43-
7.67 (m, 2H); IR ν ) 1676, 1580, 1379 cm-1; MS m/z: 353
(M + H)+, 370 (M + NH4)+. Anal. (C18H12N2O6) C, H, N.
8-Allyl-3-n it r o-2-(3-n it r op h en yl)-4H -1-b en zop yr a n -4-
on e (18f). Yield 80%; mp 140-142 °C recrystallized from a
benzene/heptane mixture as pale yellow crystals; 1H NMR
(CDCl3) δ: 3.71 (d, 2H, J ) 6.3), 5.13 (dd, 1H, J ) 17.1, 1.5),
5.24 (dd, 1H, J ) 10.2, 1.5), 5.90-6.11 (m, 1H); 7.51 (t, 1H,
J ) 7.7), 7.72 (dd, 1H, J ) 7.3, 1.9), 7.79 (t, 1H, J ) 8.1), 8.02
(dd, 1H, J ) 7.0, 1.0), 8.20 (dd, 1H, J ) 8.0, 1.6), 8.49 (dd, 1H,
J ) 8.4, 1.0), 8.66 (t, 1H, J ) 1.9); IR ν ) 1670, 1550, 1381
8-Allyl-3-n itr o-2-(2-ben zyloxyph en yl)-4H-1-ben zopyr an -
4-on e (18m ). Yield 87%; mp 147-148 °C recrystallized from
1
a benzene/heptane mixture as pale yellow crystals; H NMR
(DMSO-d6) δ: 3.58 (d, 2H, J ) 7.6), 5.04 (dd, 1H, J ) 16.9,
1.5), 5.06 (dd, 1H, J ) 10.2, 1.5), 5.20 (s, 2H), 5.90-6.04 (m,
1H), 7.19 (dt, 1H, J ) 7.8, 0.4), 7.25-7.37 (m, 6H), 7,57 (br t,
1H, J ) 7.8), 7.62 (dt, 1H, J ) 7.8, 1.7), 7.7 (dd, 1H, J ) 7.8,
1.7), 7,78 (dd, 1H, J ) 7.8, 1.4), 8,06 (dd, 1H, J ) 7.8, 1.4), IR
ν ) 1676, 1558, 1377 cm-1; MS m/z: 414 (M + H)+, 431 (M +
NH4)+. Anal. (C25H19NO5) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e 3-Nitr o-
4-oxo-2-p h en yl-4H -1-b en zop yr a n -8-a cet ic Acid 19a -m .
The relevant benzopyran-4-one 18a -m (2 mmol) was placed
in a 50 mL round-bottomed flask and then dissolved in a
mixture of CCl4 (4 mL), CH3CN (4 mL), and H2O (6 mL). The
biphasic solution was vigorously stirred at room temperature.
Sodium periodate (3.54 g, 15.3 mmoL) and ruthenium(III)
chloride hydrate (24.3 mg) were successively added. Stirring
was continued for 2 h before CH2Cl2 (20 mL) and H2O (20 mL)
were added. The reaction mixture was filtered through a
sintered funnel, and the insoluble material was rinsed with
CH2Cl2. The organic phase was decanted, and the aqueous
layer was extracted with a Et2O/THF 1/1 mixture (4 × 15 mL).
The combined organic extracts were dried (MgSO4) and then
evaporated under reduced pressure to leave a residue which
was flash-chromatographed on silica gel (45 g, eluent CH2Cl2/
CH3OH 9/1). Evaporation of the solvents gave the satisfactorily
pure acids 19a -m . It is worth mentioning that, in most cases,
these reactions also produced variable amounts (10-25%) of
the corresponding 3-nitro-4-oxo-2-phenyl-4H-1-benzopyran-8-
cm-1; MS m/z: 353(M + H)+, 370 (M + NH4)+. Anal. (C18H12
-
N2O6) C, H, N.
8-Allyl-3-n itr o-2-(4-m eth oxy-3-n itr op h en yl)-4H-1-ben -
zop yr a n -4-on e (18g). Yield 73%; mp 181-183 °C recrystal-
lized from a benzene/heptane mixture as pale yellow crystals;
1H NMR (CDCl3) δ: 3.69 (d, 2H, J ) 6.3), 4.08 (s, 3H), 5.13
(dd, 1H, J ) 17.4, 1.3), 5.21 (dd, 1H, J ) 12.8, 1.3), 5.95-6.02
(m, 1H), 7.24 (d, 1H, J ) 8.9), 7.47 (t, 1H, J ) 7.7), 7.68 (dd,
1H, J ) 8.6, 1.2), 7.85 (dd, 1H, J ) 8.8, 2.3), 8.20 (dd, 1H, J )
8.1, 1.4), 8.35 (d, 1H, J ) 2.3), IR ν ) 1672, 1545, 1380 cm-1
;
MS m/z: 383 (M + H)+, 400 (M + NH4)+. Anal. (C19H14N2O7)
C, H, N.
8-Allyl-3-n itr o-2-(4-m eth oxyp h en yl)-4H-1-ben zop yr a n -
4-on e (18h ). Yield 88%; mp 136-137 °C recrystallized from

3910 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Bauvois et al.
acetaldehyde as byproduct. 3-Nitroflavone-8-acetic acid (19a )
has been described in an earlier publication.39 Yields, physical
constants, recrystallization solvents, and spectral data for the
hitherto unknown 3-nitro-4-oxo-2-phenyl-4H-1-benzopyran-8-
acetic acid 19b-m are reported below:
3-Nitr o-2-(2-n itr oph en yl)-4-oxo-4H-1-ben zopyr an -8-ace-
tic Acid (19b). Yield 85%; mp 230-233 °C recrystallized from
a benzene/heptane mixture as a white powder; 1H NMR
(DMSO-d6) δ: 3,80 (s, 2H), 7.65 (br t, 1H, J ) 7.5), 7.92 (br d,
1H, J ) 7.5), 7.97-8.09 (m, 3H), 8.18 (br d, 1H, J ) 7.5), 8,48
(br d, 1H, J ) 7.6), 12.49 (br s, 1H, exchangeable with D2O);
IR ν ) 3010, 1713, 1662, 1530, 1347 cm-1; MS m/z: 371 (M +
H)+. Anal. (C17H10N2O8) C, H, N.
3-Nit r o-2-(3-m et h oxy-2-n it r op h en yl)-4-oxo-4H -1-b en -
zop yr a n -8-a cetic Acid (19c). Yield 52%; mp 213-214 °C
recrystallized from a benzene/heptane mixture as pale yellow
crystals; 1H NMR (DMSO-d6) δ: 3.83 (s, 2H), 4.02 (s, 3H), 7.58
(t, 1H, J ) 8.1), 7.69 (d, 1H, J ) 8.5), 7.81 (t, 1H, J ) 8.5),
7.95 (dd, 1H, J ) 8.1, 1.5), 8.11 (dd, 1H, J ) 8.1, 1.5), 12.73
(br s, 1H, exchangeable with D2O); IR ν ) 3028, 1718, 1669,
1582, 1381 cm-1; MS m/z: 401 (M + H)+, 418 (M + NH4)+.
Anal. (C18H12N2O9) C, H, N.
3-Nit r o-2-(5-m et h oxy-2-n it r op h en yl)-4-oxo-4H -1-b en -
zop yr a n -8-a cetic Acid (19d ). Yield 70%; mp 184-185 °C
recrystallized from a benzene/heptane mixture as pale yellow
crystals; 1H NMR (DMSO-d6) δ: 3.59 (s, 2H), 3.87 (s, 3H), 7.03
(d, 1H, J ) 2.6), 7.16 (dd, 1H, J ) 9.2, 2.6), 7.43 (t, 1H, J )
7.9), 7.58 (dd, 1H, J ) 7.9, 1.3), 8.11 (dd, 1H, J ) 7.9, 1.3),
8.32 (d, 1H, J ) 9.2), 12.73 (br s, 1H, exchangeable with D2O);
IR ν ) 3030, 1719, 1671, 1582, 1380 cm-1; MS m/z: 401 (M +
H)+, 418 (M + NH4)+. Anal. (C18H12N2O9) C, H, N.
3-Nitr o-2-(2-m eth oxyp h en yl)-4-oxo-4H-1-ben zop yr a n -
8-a cetic Acid (19j). Yield 68%; mp 212-213 °C recrystallized
from a benzene/heptane/acetonitrile mixture as pale yellow
crystals; 1H NMR (DMSO-d6) δ: 3.85 (s, 3H), 3.91 (s, 2H),
7.0.07-7.29 (m, 2H), 7.50-7.74 (m, 3H), 7.86 (dd, 1H, J ) 7.9,
1.3), 8.12 (dd, 1H, J ) 7.9, 1.3), 12.64 (br s, 1H, exchangeable
with D2O); IR ν ) 3030, 1720, 1667, 1585 cm-1; MS m/z: 356
(M + H)+, 373 (M + NH4)+. Anal. (C18H13NO7) C, H, N.
2-(8-Ca r b oxym et h yl-3-n it r o-4-oxo-4H -ch r om en -2-yl)-
ben zoic Acid Meth yl Ester (19k ). Yield 60%; mp 200-202
°C recrystallized from a benzene/heptane mixture as pale
1
yellow crystals; H NMR (DMSO-d6) δ: 3.87 (s, 2H), 4.01 (s,
3H), 7.67-7.86 (m, 2H), 7.88-8.02 (m, 3H), 8.42 (dd, 1H, J )
8.5, 1.1), 8.48 (dd, 1H, J ) 8.0, 1.3), 12.64 (br s, 1H,
exchangeable with D2O); IR ν) 3029, 1718, 1671, 1537, 1381,
1284 cm-1; MS m/z: 384 (M + H)+, 401 (M + NH4)+. Anal.
(C19H13NO8) H, N; C, calcd, 59.54; found, 59.11.
2-(8-Ca r b oxym et h yl-3-n it r o-4-oxo-4H -ch r om en -2-yl)-
ben zoic Acid Ben zyl Ester (19l). Yield 84%; mp 164-166
°C recrystallized from a benzene/heptane mixture as white
crystals; 1H NMR (DMSO-d6) δ: 3.87 (s, 2H), 5.31 (s, 2H), 6.92-
7.26 (m, 6H), 7.42 (dt, 1H, J ) 8.6, 1.9), 7.49 (dd, 1H, J ) 8.6,
1.9), 7.52-7.71 (m, 2H), 8.02 (dd, 1H, J ) 7.8, 1.1), 8.26 (dd,
1H, J ) 8.6, 1.9), 12.76 (br s, 1H, exchangeable with D2O); IR
ν ) 3028, 1719, 1669, 1536, 1380, 1284 cm-1; MS m/z: 460
(M + H)+, 477 (M + NH4)+. Anal. (C25H17NO8) C, H, N.
3-Nitr o-2-(2-ben zyloxyph en yl)-4-oxo-4H-1-ben zop yr a n -
8-a cetic Acid (19m ). Yield 62%; mp 155-157 °C recrystal-
1
lized from a benzene/heptane mixture as a white powder; H
NMR (DMSO-d6) δ: 3.90 (s, 2H), 5.19 (s, 2H), 7.19 (br t, 1H,
J ) 7.8), 7.22-7.38 (m, 6H), 7.59 (br t, 1H, J ) 7.7), 7.63 (dd,
1H, J ) 8.0, 1.6), 7.68 (dd, 1H, J ) 7.6, 1.6), 7.88 (dd, 1H, J )
7.7, 1.6), 8,10 (dd, 1H, J ) 7.7, 1.2); 12.64 (br s, 1H,
exchangeable with D2O); IR ν ) 3035, 1715, 1649, 1530, 1347
3-Nitr o-2-(4-n itr oph en yl)-4-oxo-4H-1-ben zopyr an -8-ace-
tic Acid (19e). Yield 53%; mp 243-244 °C recrystallized from
a benzene/heptane mixture as light beige crystals; 1H NMR
(DMSO-d6) δ: 3.86 (s, 2H), 7.49 (t, 1H, J ) 8), 7.84 (d, 1H, J )
8.0), 7.88 (d, 2H, J ) 8.6), 8.12 (d, 1H, J ) 8.0), 8.36 (d, 2H,
J ) 8.6), 12.89 ((br s, 1H, exchangeable with D2O); IR ν) 3028,
1714, 1669, 1578, 1376 cm-1; MS m/z: 371 (M + H)+, 388
(M + NH4)+. Anal. (C17H10N2O8) H, N; C: calcd, 55.14; found,
54.61.
cm-1; MS m/z: 432 (M + H)+, 449 (M + NH4)+. Anal. (C24H17
NO7) C, H, N.
-
3-Nitr o-2-(2-ben zyloxyp h en yl)-4-oxo-4H-1-ben zop yr a n -
8-a cetic Acid Meth oxym eth yl Ester (20). A stirred solution
of the above acid 19m (0.863 g, 2 mmol) in anhydrous
acetonitrile (18 mL) was placed under argon in a 50 mL round-
bottomed flask. Chloromethyl methyl ether (209 mg, 196 mL,
2.6 mmol) and triethylamine (263 mg, 362 mL, 2.6 mmol) were
then successively added at room temperature via a syringe.
The yellow solution became cloudy and then limpid while it
turned brown. The mixture (monitored by TLC) was stirred
for 5 additional hours. Evaporation of the solvent under
reduced pressure followed by chromatography (35 g SiO2,
eluent: CH2Cl2/CH3OH 95/5) gave pure ester 20. Yield 98%;
mp 144-146 °C recrystallized from a benzene/heptane mixture
as pale yellow crystals; 1H NMR (DMSO-d6) δ: 3.12 (s, 3H),
4.10 (s, 2H,), 5,12 (s, 2H), 5,21 (s, 2H), 7,18 (t, 1H, J ) 7.6),
7.20-7.41 (m, 6H), 7.55-7.65 (m, 3H), 7,92 (dd, 1H, J ) 7.8,
0.8), 8.13 (dd, 1H, J ) 7.8, 0.8); IR ν ) 1742, 1641, 1539, 1345
cm-1. MS m/z: 476 (M + H)+. Anal. (C26H21NO8) C, H, N.
3-Am in o-2-(2-h yd r oxyp h en yl)-4-oxo-4H-1-ben zop yr a n -
8-a cetic Acid Meth oxym eth yl Ester (21). The benzyl ether
20 (475 mg, 1 mmol) was dissolved in ethyl acetate (20 mL).
Palladium on charcoal (10%, 150 mg) was then added, and the
stirred mixture was maintened under hydrogen atmosphere
(1 atm) for 16 h. The suspension was filtered through a short
pad of Celite (thoroughly rinsing the solid with successively a
mixture CH2Cl2/CH3COCH3 9/1 and then a mixture CH2Cl2/
CH3OH 9/1). The obtained filtrate was evaporated in vacuo to
provide a crude residue which was further chromatographed
(35 g SiO2, eluent: CH2Cl2/CH3OH 97/3). Yield 89%; mp 172-
174 °C recrystallized from a benzene/heptane mixture as a pale
beige powder; 1H NMR (DMSO-d6) δ: 3.19 (s, 3H), 4.03 (s, 2H),
5.17 (s, 2H), 6.96-7.07 (m, 2H), 7.34-7.43 (m, 2H), 7.55 (dd,
1H, J ) 7.8, 1.5), 7.72 (dd, 1H, J ) 7.2, 1.1), 8,06 (dd, 1H, J )
8.0, 1.3); MS m/z: 356 (M + H)+. Anal. (C19H17NO6) C, H, N.
3-Nitr o-2-(3-n itr oph en yl)-4-oxo-4H-1-ben zopyr an -8-ace-
tic Acid (19f). Yield 87%; mp 240-242 °C recrystallized from
1
toluene as small beige crystals; H NMR (DMSO-d6) δ: 3.98
(s, 2H), 7.63 (br t, 1H, J ) 7.7), 7.92 (dd, 1H, J ) 7.7, 1.5),
7.94 (t, 1H, J ) 8.2), 8.14 (dd, 1H, J ) 8.0, 1.4), 8.18 (dd, 1H,
J ) 8.8, 1.0), 8.51-8.54 (m, 1H), 8.57 (dd, 1H, J ) 8.0, 1.5),
12.84 (br s, 1H, exchangeable with D2O); IR ν ) 3016, 1729,
1668, 1385, 1232 cm-1; MS m/z: 371 (M + H)+. Anal.
(C17H10N2O8) C, H, N.
3-Nit r o-2-(4-m et h oxy-3-n it r op h en yl)-4-oxo-4H -1-b en -
zop yr a n -8-a cetic Acid (19g). Yield 75%; mp 235-238 °C
(dec) pale yellow powder after washings in chloroform; 1H
NMR (CDCl3) δ: 3.86 (s, 2H), 4.02 (s, 3H), 7.52-7.62 (m, 2H),
7.86 (d, 1H, J ) 6.9), 8.06 (br d, 2H, J ) 7.4), 8,31 (br s, 1H),
12.96 (br s, 1H, exchangeable with D2O); IR ν ) 3100, 1720,
1670, 1530, 1375 cm-1; MS m/z: 401 (M + H))+, 418 (M +
NH4)+. Anal. (C18H12N2O9) C, H, N.
3-Nitr o-2-(4-m eth oxyp h en yl)-4-oxo-4H-1-ben zop yr a n -
8-a cetic Acid (19h ). Yield 76%; mp 157-160 °C recrystallized
from a toluene/acetonitrile mixture as small pale yellow
crystals; 1H NMR (DMSO-d6) δ: 3.80 (s, 3H), 3.94 (s, 2H), 7.15
(br d, 2H, J ) 8.9), 7.57 (t, 1H, J ) 8.8), 7.71 (br d, 2H, J )
8,9), 7.85 (dd,1H, J ) 7.3,1.0), 8.07 (dd,1H, J ) 8.0, 1.3), 13.28
(br s, 1H, exchangeable with D2O); IR ν ) 3050, 1712, 1668,
1532, 1378 cm-1; MS m/z: 356 (M + H))+, 373 (M + NH4)+.
Anal. (C18H13NO7) C, H, N.
3-Nit r o-2-(2-ch lor op h en yl)-4-oxo-4H -1-b en zop yr a n -8-
a cetic Acid (19i). Yield 45%; mp 229-230 °C recrystallized
from a benzene/heptane mixture as light beige crystals; 1H
NMR (DMSO-d6) δ: 3.86 (s, 2H), 7.69 (m, 5H), 7.51-7.80 (d,
1H, J ) 8.0), 8.23 (d, 1H, J ) 7.7), 12.71 (br s, 1H, exchange-
able with D2O); IR ν) 3028, 1718, 1671, 1582 cm-1; MS m/z:
360, 362 (M + H)+, 377, 379 (M + NH4)+. Anal. (C17H10ClNO6)
C, H, N.
3-Nitr o-2-(2-n itr oph en yl)-4-oxo-4H-1-ben zopyr an -8-ace-
tic Acid Meth oxym eth yl Ester (22). This ester was pre-
pared from 19b on a 3 mmol scale using the procedure above-
described for 20. Yield 98%; mp 130-132 °C recrystallized from

Novel Flavone-8-acetic Acid Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3911
a benzene/heptane mixture as a pale yellow powder; 1H NMR
(CDCl3) δ: 3.28 (s, 3H), 3.83 (s, 2H), 5.16 (s, 2H), 7.53 (t, 1H,
J ) 7.8), 7.66 (dd, 1H, J ) 6.1, 3.5), 7.73 (dd, 1H, H7, J ) 7.8,
1.6), 7.80-7.91 (m, 2H), 8.31 (dd, 1H, J ) 7.8, 1.6), 8.38 (dd,
1H, J ) 6.1, 2.5); IR ν ) 1745, 1639, 1537, 1348 cm-1; MS
m/z: 415 (M + H)+, 432 (M + NH4)+. Anal. (C19H14N2O9) C,
H, N.
described for 23. Yield 65%; mp 250-252 °C yellow powder;
1H NMR (DMSO d6) δ: 3.24 (s, 3H), 4.12 (s, 2H), 5.19 (s, 2H),
6,97 (t, 1H, J ) 7.4), 7.37 (d, 1H, J ) 7.4), 7.60-7.91 (m, 3H),
8.01 (d, 1H, J ) 8.2), 8.09 (d, 1H, J ) 8.2), 12.51 (s, 1H
exchangeable with D2O), 14,40 (s, 1H exchangeable with D2O).
MS m/z: 381 (M + H)+, 398 (M + NH4)+. Anal. (C20H16N2O6)
C, H, N.
(6,8-Dioxo-6,7-d ih yd r o-8H-5,13-d ioxa -7-a za ben zo[3,4]-
cycloh ep t a [1,2-b]n a p h t h a len -12-yl)a cet ic Acid Met h -
oxym eth yl Ester (23). A solution of the aminophenol 21 (355
mg, 1 mmol) in anhydrous CH2Cl2 (50 mL) was placed, under
inert atmosphere, in a 100 mL two-necked flask. Triphosgene
(111.4 mg, 0.375 mmol) dissolved in anhydrous CH2Cl2 (12 mL)
was added via a syringe at room temperature to the stirred
mixture. The yellow solution turned orange. After 3 h, a
precipitate appeared, and triethylamine was added dropwise
until the mixture became limpid again. Two hours later,
removal of the solvent under reduced pressure followed by
chromatography (40 g SiO2, eluent: CH2Cl2/CH3OH 98/2)
afforded pure tetracyclic ester 23. Yield 65%; mp 162-165 °C
recrystallized from a benzene/heptane mixture as an orange
powder; 1H NMR (DMSO-d6) δ: 3.25 (s, 3H), 4.19 (s, 2H), 5.21
(s, 2H), 7.43 (d, 1H, J ) 8.4), 7.53 (t, 1H, J ) 7.8), 7.57 (t, 1H,
J ) 8.4), 7.76 (dt, 1H, J ) 8.4, 1.2), 7.86 (dd, 1H, J ) 7.8, 1.2),
7.95 (dd, 1H, J ) 8.4, 1.2), 8.12 (dd, 1H, J ) 7.8, 1.2), 9.83 (s,
1H, exchangeable with D2O). MS m/z: 382 (M + H)+, 399
(M + NH4)+. Anal. (C20H15N O7) H, N; C, calcd, 62.99; found,
62.54.
(6,8-Dioxo-6,7-d ih yd r o-8H-5,13-d ioxa -7-a za ben zo[3,4]-
cycloh epta[1,2-b]n aph th alen -12-yl)acetic Acid (24). Adapt-
ing a previously reported method,43 dry magnesium bromide
(552 mg, 3 mmol) was added in one portion to a stirred solution
of the above methoxymethyl ester 23 (190 mg, 0.5 mmol) in
anhydrous CH2Cl2 (25 mL). The reaction medium became
cloudy and then yellow and was stirred overnight at room
temperature. Removal of the solvent under reduced pressure
followed by chromatography (90 g of SiO2, eluent: CH2Cl2/CH3-
OH 90/10) provided pure carboxylic acid 24. Yield 51%; mp >
260 °C yellow powder; 1H NMR (DMSO-d6) δ: 3.94 (s, 2H), 7.42
(d, 1H, J ) 7.0), 7.49 (br t, 1H, J ) 7.0), 7.57 (br t, 1H, J )
7.0), 7.69-7,85 (m, 2H), 7.98-8,10 (m, 2H). MS m/z: 338
(M + H)+, 355 (M + NH4)+. Anal. (C18H11N O6) C, H, N.
(10-Oxo-10,11-d ih yd r o-5-oxa -11-a za ben zo[b]flu or en -6-
yl)a cetic Acid Meth oxym eth yl Ester (25) a n d 3-Am in o-
2-(2-a m in op h en yl)-4-oxo-4H-1-ben zop yr a n -8-a cetic Acid
Meth oxym eth yl Ester (26). The dinitro derivative 22 (1.76
g, 4.24 mmol) was dissolved in EtOAc (160 mL). Palladium
on charcoal (10%, 450 mg) was then added, and the stirred
mixture was maintained under hydrogen atmosphere (1 atm)
for 16 h. The suspension was filtered through a short pad of
Celite (thoroughly rinsing the solid with successively a mixture
CH2Cl2/CH3COCH3 9/1 and then a mixture CH2Cl2/CH3OH
9/1). The obtained filtrate was evaporated in vacuo to give a
crude residue which was further chromatographed (200 g SiO2,
eluent: CH2Cl2/ CH3COCH3 in the successive proportions 9/1,
8/2, and, finally, 7/3) to provide 25 and then 26.
25: Yield 49%; mp 209-211 °C recrystallized from a ben-
zene/heptane mixture as flakey pale yellow crystals; 1H NMR
(CDCl3) δ: 3.38 (s, 3H, CH3), 4.17 (s, 2H), 5.30 (s, 2H), 7.29 (br
t, 1H, J ) 7.8), 7.46 (br t, 1H, J ) 7.5), 7.54 (dt, 1H, J ) 7.5,
0.9), 7,62 (dd, 1H, J ) 7.8, J ) 0,5), 7.69 (d, 1H, J ) 7.5), 8.01
(dd, 1H, J ) 7.8, 0.5), 8,47 (dd, 1H, J ) 7.5, J ) 0.9), 9.7 (br
s, 1H, exchangeable with D2O). MS m/z: 338 (M + H)+, 355
(M + NH4)+. Anal. (C19H15N O5) C, H, N.
(10-Oxo-10,11-d ih yd r o-5-oxa -11-a za ben zo[b]flu or en -6-
yl)a cetic Acid (28). This carboxylic acid was prepared from
25 (337 mg, 1 mmol) according to the procedure above-
described for 24. Yield 51%; mp > 260 °C pale yellow powder;
1H NMR (DMSO-d6) δ: 4.01 (s, 2H), 7.28 (ddd, 1H, J ) 8.2,
7.4, 1.3), 7.41-7.62 (m, 3H), 7.74 (dd, 1H, J ) 7.4, 1.3), 7.95
(dd, 1H, J ) 7.7, 0.9), 8.20 (d, 1H, J ) 8.2), 9.7 (br s, 1H
exchangeable with D2O). MS m/z: 294 (M + H)+, 311 (M +
NH4)+. Anal. (C17H11NO4) C, H, N.
(6,8-Dioxo-5,6,7,8-t et r a h yd r o-13-oxa -5,7-d ia za b en zo-
[3,4]cycloh ep ta [1,2-b]n a p h th a len - 12-yl)a cetic Acid (29).
This carboxylic acid was prepared from 27 (380 mg, 1 mmol)
according to the procedure above-described for 24. Yield 57%;
1
mp > 260 °C, yellow powder; H NMR (DMSO-d6) δ: 3.71 (s,
2H), 7.02 (t, 1H, J ) 7.6), 7.25-7.43 (m, 3H), 7.72 (br s, 2H),
8.18 (d, 1H, J ) 8.9), 12.29 (br s, 1H exchangeable with D2O).
MS m/z: 337 (M + H)+, 354 (M + NH4)+. Anal. (C18H12N2O5)
C, H, N.
1-(3-Allyl-2-h yd r oxyp h en yl)-3-h yd r oxy-3-(2-n itr op h en -
yl)p r op en on e (32). A mixture of 3-allyl-2-hydroxyacetophe-
none (30)45 (4.6 g, 26 mmol), commercial 2-nitrobenzoyl
chloride (31, 3.6 g, 19 mmol), and anhydrous K2CO3 (10 g) in
anhydrous butanone (150 mL) was heated under reflux for 48
h. The reaction mixture was allowed to cool to room temper-
ature, and the solid was filtered off. The filtrate was then
poured onto a mixture of ice and water (400 mL). The
precipitate formed was collected by filtration and recrystallized
to give pure 32. Yield 68%; mp 228-229 °C recrystallized from
methanol as small yellow crystals; 1H NMR (DMSO-d6) δ:
2.92-3.34 (m, 1H), 4.08 (s, 1H), 4.92 (dd, 1H, J ) 16.9, 1.2),
5.01 (dd, 1H, J ) 9.8, 1.2), 5.81-6.00 (m, 1H), 6.56 (t, 1H, J )
7.6), 6.98 (d, 1H, J ) 8.4), 7.26-7.38 (m, 1H), 7.38-7.47 (m,
2H), 7.58 (dt, 1H, J ) 8.4, 0.8), 7.87 (dd, 1H, J ) 8.4, 0.8),
16.43 (s, 1H exchangeable with D2O); IR ν ) 3560-3040, 1713,
1650, 1540 cm-1; MS m/z: 326 (M + H)+, 343 (M + NH4)+.
Anal. (C18H15NO5) H, N; C, calcd, 66.46; found, 65.95.
8-Allyl-2-(2-n itr op h en yl)-4H-1-ben zop yr a n -4-on e (33).
The preparation of this flavone was performed on a 1.2 mmol
scale starting from compound 32 by adapting a method
previously described for similar cases.44,58 Yield 97%; mp 129-
131 °C recrystallized from a benzene/heptane mixture as a
1
white powder; H NMR (CDCl3) δ: 3.49 (d, 2H, J ) 6.8), 5.01
(br d, 1H, J ) 16.6), 5.12 (d, 1H, J ) 8.9), 5.82-6.01 (m, 1H),
6.63 (s, 1H), 7.40 (t, 1H, J ) 7.8), 7.56 (d, 1H, J ) 7.1), 7.63-
7.86 (m, 3H), 8.08 (d, 1H, J ) 7.8), 8.14 (d, 1H, J ) 8); IR ν )
1670, 1582, 1381 cm-1; MS m/z: 308 (M + H)+, 325 (M +
NH4)+. Anal. (C18H13NO4) C, H, N.
2-(2-Nitr oph en yl)-4-oxo-4H-1-ben zopyr an -8-acetic Acid
(8). This carboxylic acid was prepared from the allyl derivative
33 (307 mg, 1 mmol) according to the procedure above-
described for compounds 19. Yield 48%; mp 186-188 °C
recrystallized from a benzene/heptane mixture as a pale yellow
powder; 1H NMR (DMSO-d6) δ: 3.87 (s, 2H), 6.63 (s, 1H), 7.40
(t, 1H, J ) 7.8), 7.56 (d, 1H, J ) 7.1), 7.67-7.83 (m, 3H), 8.08
(d, 1H, J ) 7.8), 8.14 (d, 1H, J ) 8.0); IR ν ) 1718, 1670, 1582,
1381 cm-1; MS m/z: 326 (M + H)+, 343 (M + NH4)+. Anal.
(C17H11NO6) C, H, N.
26 : Yield 40%; mp 190-192 °C orange clear powder; 1H
NMR (DMSO d6) δ: 3.34 (s, 3H), 3.79 (s, 2H), 5.19 (s, 2H), 7.07
(t, 1H, J ) 7.5), 7.27 (t, 1H, J ) 8.0), 7.42 (d, 1H, J ) 7.5),
7.50-7.61 (m, 2H), 7.69 (d, 1H, J ) 8.0), 8.13 (d, 1H, J ) 8.0).
MS m/z: 372 (M + H)+, 355 (M + NH4)+. Anal. (C19H18N2 O5)
C, H, N.
(6,8-Dioxo-5,6,7,8-t et r a h yd r o-13-oxa -5,7-d ia za b en zo-
[3,4]cycloh epta[1,2-b]n aph th alen - 12-yl)acetic Acid Meth -
oxym eth yl Ester (27). This tetracyclic ester was prepared
from 26 (354 mg, 1 mmol) according to the procedure above-
Ma ter ia ls for Biologica l Assa ys. The synthetic substrates
p-nitroanilide (pNA) derivatives, protease inhibitors, bovine
cathepsin G, and human angiotensin converting enzyme (ACE)
were provided by Sigma Chemical Co. (St Louis, MO). Human
recombinant pro-matrix metalloproteinase-9 (pro-MMP-9) with
a Mr 92 kDa was from R&D (UK). Purified human dipeptidyl
peptidase IV(DPPIV)/CD26 was a gift of Dr I De Meester.59
Goat F(ab′)2 fragment anti-mouse fluorescein-conjugated Ig,
monoclonal antibody (mAb) specific for CD13 (MY7, mIgG1)
and the control isotype mIgG1 were obtained from Coulter

3912 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Bauvois et al.
Immunotech (Coultronics, France). Monoclonal Ab specific for
CD13 (WM15, mIgG1) was from Pharmingen International
(Becton Dickinson Company). Protein G-Sepharose was from
Pharmacia (Uppsala, Sweden).
conjugated goat F(ab′)2 anti-mouse IgG were from Coulter
Beckman (Margency, France). Analysis was performed in a
FACS flow cytometer analyzer (Becton-Dickinson, Mountain
View, CA). Fluorescence data were expressed in relative
fluorescence intensity (%) and antigen relative density per cell.
Cell Cu ltu r e Con d ition s. Human monoblastic U937 cells
were maintained in RPMI 1640 medium supplemented with
10% heat-inactivated FCS (Gibco, Parsley, Scotland, LPS levels
< 0.1 ng/mL), 2 mM L-glutamine, 1 mM sodium pyruvate and
40 µg/mL gentamycin (Flow Laboratories, Rockville, MD) in
a 5% CO2 humidified atmosphere at 37 °C. Cells (0.5 × 105
cells/mL) were resuspended in fresh medium containing FCS
in tissue culture flasks and were grown for various periods of
time, in the absence or presence of different concentrations of
the tested compounds. In this context, it must be pointed out
that some of these derivatives (8, 20-23 and 29) precipitate
at high concentration (10-3 M). Cells were collected, washed
twice, and counted with a Coulter Counter ZM equipped with
a Coultronic 256 channelizer, and their viability was deter-
mined by trypan blue exclusion. Cell protease assays were
performed as described below. Apoptosis was assessed by flow
cytometry as described below by measuring the appearance
of Apo 2.7 antigen (Coulter Immunotech), a mitochondrial
membrane protein lately exposed on the surface of cells
undergoing programmed cell death.
Ack n ow led gm en t. We are grateful to Mrs. C.
Sylvestri for valuable technical assistance and to Mrs.
D. Rouillard for her dedicated helpfulness in FACS. This
work was supported by grants from the Institut Na-
tional de la Sante´ et de la Recherche Me´dicale
(I.N.S.E.R.M.), the Centre National de la Recherche
Scientifique (C.N.R.S.), the Ligue Nationale Franc¸aise
Contre le Cancer (Comite´ de Paris), and the Institut
Curie (Programme Incitatif et Coope´ratif: Angiogene`se
tumorale).
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