Synthesis, characterization and in vitro anti-invasive activity screening of polyphenolic and heterocyclic compounds

Article abstract of DOI:10.1016/S0968-0896(02)00539-4

Invasion is the hallmark of malignant tumors, and is responsible for the bad prognosis of the untreated cancer patients. The search for anti-invasive treatments led us to screen compounds of different classes for their effect in an assay for invasion. Thirty-nine new compounds synthesized in the present study along with 56 already reported compounds belonging mainly to the classes of lactones, pyrazoles, isoxazoles, coumarins, desoxybenzoins, aromatic ketones, chalcones, chromans, isoflavanones have been tested against organotypic confronting cultures of invasive human MCF-7/6 mammary carcinoma cells with embryonic chick heart fragments in vitro. Three of them (a pyrazole derivative, an isoxazolylcoumarin and a prenylated desoxybenzoin) inhibited invasion at concentrations as low as 1 μM; instead of occupying and replacing the heart tissue within 8 days, the MCF-7/6 cells grew around the heart fragments and left it intact, when treated with these compounds. At the anti-invasive concentration of 1 μM, the three compounds did not affect the growth of the MCF-7/6 cells, as shown in the sulforhodamine B assay. Aggregate formation on agar was not stimulated by any of the three anti-invasive compounds, making an effect on the E-cadherin/catenin complex improbable. This is an invasion suppressor that can be activated in MCF-7/6 cells by a number of other molecules. Our data indicate that some polyphenolic and heterocyclic compounds are anti-invasive without being cytotoxic for the cancer cells.

Full text of DOI:10.1016/S0968-0896(02)00539-4

Bioorganic & Medicinal Chemistry 11 (2003) 913–929
Synthesis, Characterization and In Vitro Anti-invasive Activity
Screening of Polyphenolic and Heterocyclic Compounds
Virinder S. Parmar,a,b,c,* Nawal K. Sharma,a,b Mofazzal Husain,a Arthur C. Watterson,c
Jayant Kumar,c Lynne A. Samuelson,c Ashok L. Cholli,c Ashok K. Prasad,a
Ajay Kumar,a,b Sanjay Malhotra,a Naresh Kumar,a Amitabh Jha,a Amarjit Singh,a
Ishwar Singh,a Himanshu,a,b Archana Vats,a Najam A. Shakil,a,c Smriti Trikha,a
Shubasish Mukherjee,a,b Sunil K. Sharma,a,c Sanjay K. Singh,a,b Ajay Kumar,a,d
Hriday N. Jha,d Carl E. Olsen,e Christophe P. Stove,f Marc E. Brackef
and Marc M. Mareelf,*
aBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi-110 007, India
bDepartment of Chemistry and Chemical Engineering, Polytechnic University, Six Metrotech Center, Brooklyn, NY 11201, USA
cInstitute for Nano Science Engineering and Technology, Department of Chemistry, University of Massachusetts,
One University Avenue, Lowell, MA 01854, USA
dDepartment of Chemistry, MMH College, Ghaziabad, UP, India
eChemistry Department, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, Frederiksberg C,
DK-1871 Copenhagen, Denmark
fLaboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, University Hospital,
De Pintelaan 185, B-9000 Gent, Belgium
Received 25 September 2002; accepted 21 October 2002
Dedicated to the cherished memory of our collaborator and friend (Late) Professor Sukant K. Tripathy
Abstract—Invasion is the hallmark of malignant tumors, and is responsible for the bad prognosis of the untreated cancer patients.
The search for anti-invasive treatments led us to screen compounds of different classes for their effect in an assay for invasion.
Thirty-nine new compounds synthesized in the present study along with 56 already reported compounds belonging mainly to the
classes of lactones, pyrazoles, isoxazoles, coumarins, desoxybenzoins, aromatic ketones, chalcones, chromans, isoflavanones have
been tested against organotypic confronting cultures of invasive human MCF-7/6 mammary carcinoma cells with embryonic chick
heart fragments in vitro. Three of them (a pyrazole derivative, an isoxazolylcoumarin and a prenylated desoxybenzoin) inhibited
invasion at concentrations as low as 1 mM; instead of occupying and replacing the heart tissue within 8 days, the MCF-7/6 cells
grew around the heart fragments and left it intact, when treated with these compounds. At the anti-invasive concentration of 1 mM,
the three compounds did not affect the growth of the MCF-7/6 cells, as shown in the sulforhodamine B assay. Aggregate formation
on agar was not stimulated by any of the three anti-invasive compounds, making an effect on the E-cadherin/catenin complex
improbable. This is an invasion suppressor that can be activated in MCF-7/6 cells by a number of other molecules. Our data indi-
cate that some polyphenolic and heterocyclic compounds are anti-invasive without being cytotoxic for the cancer cells.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
patients. Oncologists nowadays possess surgery, radio-
therapy, chemotherapy and immunotherapy as efficient
tools to tackle tumor growth. Invasion, however, is a
more resistant problem than growth, and anti-invasive
agents are sadly lacking in clinical practice.1 Progress in
this field can be expected from a better knowledge of the
molecular mechanisms of invasion, from the develop-
ment of relevant invasion models in vitro and from the
Growth and invasion are tumor activities that are
responsible for the fatal outcome of untreated cancer
*Corresponding author. Tel.: +91-11-27666555; fax: +91-11-
27667206; e-mail: virparmar@yahoo.co.in
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00539-4

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V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
synthesis or isolation of new compounds as candidates for
anti-invasive drugs. Among alkaloids and polyphenolics,
a number of compounds have already been detected that
could interfere with cellular protein complexes that are
implicated in invasion2ꢀ4 although the poor specificity of
their targets for tumor cells often limited their application
in medicine.
pounds 43–45, 49 and 50 were synthesised by the earlier
published procedure,11 the same procedure was fol-
lowed for the synthesis of 46–48 and 51. Of these, 46, 48
and 51 are new in literature, however, 477 has been
reported earlier.
The isoxazolylcoumarins 52–54 were synthesised by
treating the corresponding aryl-cyanomethylisoxazoles
with salicylaldehyde in ethanol in the presence of
sodium hydroxide, all the three are new compounds.
The coumarins 55,12 5613 and 5714 were synthesised by
the well known Pechmann condensation reaction.15 The
novel new coumarins 58–60 were prepared by butano-
ylation of 7,8-dihydroxy-4-methylcoumarin,14 7-
hydroxy-4-methylcoumarin16 and 5,7-dihydroxy-4-
methylcoumarin,17 respectively using butyric anhydride
and pyridine, while 61 was prepared by the methylation
of 5,7-dimethoxy-6-hydroxy-4-methylcoumarin (56)13
using dimethyl sulphate and potassium carbonate in
anhydrous acetone and is reported for the first time.
The amides 62,18 6318 and 64–69 were prepared by our
earlier published procedure.19
Because there is considerable need in oncology for anti-
invasive agents and very few agents of that kind are
available, no chemical rationale for directed screening
of compounds towards this goal is prevailing. As a part
of our continued search for new anti-invasive agents, we
tested ninety-five compounds belonging to lactones,
pyrazoles, isoxazoles, coumarins, desoxybenzoins, aro-
matic ketones, chalcones, chromans, isoflavanones and
other classes. These compounds were tested in an orga-
notypic assay for invasion. Here, a living host tissue
fragment was confronted with invasive cancer cells,
mimicking the elements of the micro-ecosystem
encountered in human tumors.5 Human MCF-7/6
mammary carcinoma cells6 were used in this assay,
because they can invade the normal tissue fragment
within 1 week, and also because their invasiveness was
proven to be sensitive to polyphenolics and hormones.
The compounds 70–72 and 7820 were prepared by pre-
nylation of 2,4-dihydroxyphenyl benzyl ketone and 4-
hydroxyacetophenone, respectively using 2-methylbut-
3-en-2-ol and BF3-etherate.21 The compounds 76 and 77
were obtained according to the method of Lars et al.22
Results
Preparation and characterization of compounds
Table 1. Effect of polyphenolic and heterocyclic compounds on
invasion of MCF-7/6 cells in vitro
The structural formulae of the compounds studied in
this report are shown in Figure 1. The lactones 1 and 2
were synthesised by our earlier published procedure by
condensing the corresponding acetophenone with ethyl
2-cyano-3,3-bis(thiomethyl)propenoate in the presence
of KOH in DMF.7 These lactones have been used as
starting materials for the synthesis of the pyrazolyl
acetonitriles 37 and 47 by treating the corresponding
lactone with hydrazine hydrate in methanol under reflux
conditions. However, the N-phenyl/aryl substituted
pyrazolyl acetonitriles 5–16 were obtained by conden-
sing phenyl/aryl hydrazine hydrate with lactones of the
type 1 and 2; the compounds 5 and 8–16 have been
prepared for the first time and characterized completely
from their spectral data, however the compounds 68,9
and 78 have been reported earlier. During the synthesis
of these pyrazolyl acetonitriles, we isolated in many
cases, the 3-amino-2,6-diaryl-4-oxo-4H-pyrano-[4,3-
c]pyrazoles 17–26 as co-products. Of these, 17 and 18
have been reported earlier,8 while 19–26 are not known
in literature and are being reported for the first time.
mM
mM
mM
Compound No. 10 1 Compound No. 10 1 Compound No. 10
1
1
2
3
4
5
6
7
8
ꢀ
ꢀ
0
0
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
+ ꢀ
+ ꢀ
+ ꢀ
+ ꢀ
+ ꢀ
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
9529
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0
0
0
0
0
+ ꢀ
ꢀ
ꢀ
0
0
+ ꢀ
ꢀ
0
+ ꢀ
+ +
+ ꢀ
+ ꢀ
+ +
0
ꢀ
ꢀ
ꢀ
0
0
0
ꢀ
9
+ ꢀ
+ ꢀ
+ ꢀ
ꢀ
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
+ ꢀ
+ ꢀ
+ ꢀ
+ ꢀ
ꢀ
0
ꢀ
ꢀ
0
0
+ ꢀ
ꢀ
ꢀ
ꢀ
0
0
0
0
0
0
0
0
0
0
ꢀ
ꢀ
0
0
0
0
0
0
0
0
0
0
0
0
+ ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0
0
0
0
0
0
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0
0
0
ꢀ
+ +
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0
0
0
0
0
+ ꢀ
The compounds 27–3710 were obtained by condensation
of pyrazolyl acetonitriles with pyrrole 2-carboxaldehyde
in the presence of sodium-t-butoxide in ethanol, both
the E and Z isomers could be obtained by varying the
reaction conditions.10 However, condensation of 3,4-
difluorobenzaldehyde with respective phenylpyrazolyl
acetonitriles under identical conditions afforded the
trans (E) condensation products 38–42, these com-
pounds have been prepared for the first time and were
well characterized from their spectral data. The com-
ꢀ
0
+ ꢀ
+ ꢀ
ꢀ
ꢀ
ꢀ
0
0
0
0
0
+ ꢀ
+ ꢀ
+ ꢀ
+ ꢀ
ꢀ ꢀ
+ ꢀ
+ ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0
0
0
0
0
+ ꢀ
ꢀ
0
+ ꢀ
+ ꢀ
+, anti-invasive; ꢀ, not anti-invasive; 0, not tested.

V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
915
The benzopyran 73 was obtained by cyclization of pre-
nyl group in 70 with adjacent hydroxyl group using
formic acid.23 The compound 79 was prepared by the
reaction of 4-hydroxyacetophenone with isoprene by the
method of Clemo and Ghatge.24 However, on subject-
ing the compound 70 to oxidative cyclisation using
m-chloroperoxybenzoic acid (m-CPBA),25 74 was
obtained, which on subsequent methylation using
dimethyl sulphate and potassium carbonate in anhy-
drous acetone yielded 75. The compounds 70–75 are
reported for the first time. However, the ketones 80–
85,25,26 the quinone 8627 and the chalcones 87–9228 were
synthesised by following the indicated literature meth-
ods, while the compounds 93–94 were obtained from the
collection of (Late) Professor T.R. Seshadri, FRS in
Delhi (India). The structures of all the thirty nine new
compounds synthesized in this study have been estab-
lished unambiguously on the basis of their spectral data,
1
that is H and 13C NMR, IR, UV, MS and HR-MS.
Invasion
Table 1 summarizes the results of the assays for inva-
sion. In control cultures treated with the solvent only,
the MCF-7/6 cells had invaded the PHF after 8 days of
incubation. In histological sections of these cultures,
only the remnants of the heart tissue could be discerned,
as visualized by the haematoxylin-eosin staining. Mas-
sive replacement of the heart fragment was evident from
selective immunohistochemistry of the MCF-7/6 cells
(Fig. 2A and B). Many compounds showed no inhibi-
tion of invasion, and histologically their cultures were
Figure 1. Structures of compounds tested as anti-invasive agents in vitro.

916
V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
Figure 1. (Continued).

V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
917
Figure 1. (Continued).

918
V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
Figure 1. (Continued).

V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
919
similar to controls. Other compounds were anti-invasive
Growth
at the concentration of 10 mM. Three out of the 95
compounds (39, 53 and 71) however, inhibited invasion
of MCF-7/6 cells at a concentration as low as 1 mM. At
0.1 mM, none of the compounds was active. As shown in
Figure 2D–F, cultures treated with anti-invasive com-
pounds showed an intact PHF surrounded by MCF-7/6
cells.
No growth inhibition of the MCF-7/6 cells could be
observed on histological sections from confronting cul-
tures treated with the anti-invasive compounds 39, 53 or
71. This was in contrast with the effect of the chalcone
95 (a typical anti-invasive compound studied by us
earlier29), which has now been seen to have in addition a
Figure 2. Light micrographs of sections from 8-day-old confronting cultures between precultured heart fragments (PHF) and MCF-7/6 cells. Sol-
vent-treated (0.1% DMSO) confrontations (A and B), that show invasion of MCF-7/6 cells, are compared with confrontations treated with 10 mM of
the compounds 39 (C and D), 53 (E and F) and 95 (G and H), that show absence of invasion. The sections on the left panels were stained with
haematoxylin-eosin; in the right panels MCF-7/6 antigens were revealed immunohistochemically and appear dark. Scale bare=50 mm.

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V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
remarkable growth inhibition activity on MCF-7/6 cells
(Fig. 2G and H). In the sulforhodamine B (SRB) assay,
no growth inhibition (not more than 20%) by the anti-
invasive compounds 39, 53 and 71 on MCF-7/6 cells
was noticed, neither at 10 mM nor at 1 mM concentration
(Fig. 3). The compound 95, however, which was used as
a positive control for growth inhibition (pos) on these
cells, reduced growth (by more than 70%) in this assay
at 10 mM concentration (Fig. 3). This control was
necessary because none of the present anti-invasive
compounds affected growth of MCF-7/6 cells at the
tested three concentrations.
activity at 10 mM concentration on MCF-7/6 cells in the
chick heart invasion assay (Table 1). A majority of
compounds found active at 10 mM concentration are
either pyrazole derivatives or aromatic ketones, mainly
desoxybenzoins and chalcones. Thirteen out of 16 pyr-
azolylacrylonitriles, namely compounds 27–30, 32–37,
39, 41 and 42 out of 27–42 exhibited anti-invasive
activity at 10 mM concentration on MCF-7/6 cells in the
chick heart invasion assay; one of them, that is (Z)-2-[3-
(4-chlorophenyl)-1-phenylpyrazol-5-yl)-3-(3,4-difluoro-
phenyl)acrylonitrile (39) was also active at 1 mM con-
centration. Out of the five pyrazolyl acrylonitriles 38–42
with 3,4-difluorophenyl substituent at C-3 position,
three compounds 39, 41 and 42 exhibited antiinvasive
activity at 10 mM concentration and among the three
compounds 39 was found active at 1 mM concentration.
This revealed that difluorophenyl moiety at the C-3
position together with the 4-chlorophenyl moiety at the
C-3 -position of pyrazole in compound 39 enhances the
anti-invasive activity of the acrylonitrile. Three com-
pounds, that is 43, 46 and 53 out of twelve isoxazole
derivatives 43–54 evaluated in this assay, exhibited anti-
invasive activity at 10 mM concentration; an iso-
xazolylcoumarin out of these three, that is 3-[3-(4-
methylphenyl)isoxazol-5-yl]-2H-1-benzopyran-2-one (53)
was also active at 1 mM concentration. Highest activity
of isoxazolylcoumarin 53 indicates that the construction
of a coumarin moiety involving the C-5 cyanomethyl
group of the isoxazole enhances the anti-invasive activ-
ity of the compound by manifold. Five prenylated des-
oxybenzoines, that is 70–72, 74 and 94 out of seven
compounds of this class, namely 70–75 and 94 evaluated
in this assay, exhibited activity at 10 mM concentration;
2,4-dihydroxy-3-prenyldesoxybenzoin (71) was also
active at 1 mM concentration. This indicates that
Cell aggregation
The poor capacity of MCF-7/6 cells to form cell aggre-
gates was evident from untreated and solvent treated
cultures; only small and irregular aggregates were
formed on semi-solid agar. Treatment with the anti-
invasive compounds 39, 53 or 71 at 10 or 1 mM con-
centrations did not alter the aggregation pattern of the
cells (data not shown). As a positive control for induc-
tion of cell aggregation, insulin-like growth factor-I
(IGF-I) at 500 ng/mL was applied; this treatment
induced the formation of large spheroidal aggregates.30
Relationship of anti-invasive activity of compounds with
their structures
All the compounds were first tested for anti-invasive
activity at concentration of 100 mM, those showing
positive results were further tested at lower concentra-
tions. Thirty-four compounds, that is 3, 6, 7, 11–13, 25,
27–30, 32–37, 39, 41–43, 46, 53, 59, 70–72, 74, 76, 86,
88, 92, 94 and 95 out of 95 exhibited anti-invasive
Figure 3. Effect of three anti-invasive compounds in the sulforhodamine B assay with MCF-7/6 cells grown in microtiter plates for 3 days. The cells
were treated with 0.1, 1 or 10 mM of each compound. Treatment with compound 95 (pos) is included as a positive control, and results are presented
as a percentage of solvent-treated controls (mean+standard deviation).

V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
921
prenylated desoxybenzoins are potential anti-invasive
compounds. The close inspection of the structures of
these desoxybenzoins also revealed that the compounds
with prenyl group present in the open form are more
active, particularly when the prenyl group is flanked by
two hydroxyl groups as in 71, which had the maximum
anti-invasive activity of the compounds of this class.
Together with these pyrazole and isoxazole derivatives,
and desoxybenzoins, one coumarin derivative 59 out of
seven (55–61) and three chalcones 88, 92 and 95 out of
seven (87–92 and 95) evaluated in this assay, showed
anti-invasive activity at 10 mM concentration. The three
most active compounds, that is 39, 53 and 71 that are
active at 1 mM concentration, do not show any anti-
invasive activity at 0.1 mM concentration.
(1H). The two ortho-coupled protons C-5H and C-6H
resonated at d 6.36 (d, J=8.85 Hz) and 7.64 (d,
J=8.88 Hz), respectively. The characteristic peaks for
the chelated hydroxyl group and the benzylic protons
appeared as two singlets at d 13.00 and d 4.20, respec-
tively, while the C-4 OH group appeared at d 6.28 as a
singlet, the five aromatic protons at C-20–C-60 appeared
as a multiplet between d 7.22 and 7.35. The 13C NMR
spectrum of 71 was in full agreement with the structure.
In additional experiments, we could demonstrate that
these compounds did not inhibit the growth of the
tumor cells, and that they did not exert their effect
through an activation of the MCF-7/6 E-cadherin/cat-
enin complex. In the same invasion assay, a number of
anti-invasive agents have previously been picked up that
inhibited both invasion and growth of the tumor cells.
One type of such compounds indeed interfered with a
molecular target that is common in both mitosis and
directional migration, such as the vinca alkaloids, that
bind to tubulin, and thus prevent the assembly of both
the mitotic spindle and of the cytoplasmic microtubule
complex.2 Another type of compounds was cytotoxic,
and the anti-invasive effect was the result of a reduced
number of intrinsically invasive cells in the confronting
cultures. These compounds were of interest to the
oncologist, provided the cytotoxicity was selective for
the tumor cells and not for the normal tissues.4,31 The
anti-invasive compounds in the present study (39, 53
and 71), however inhibited only invasion without
affecting growth, which excludes that they due their
activity to cytotoxicity. Such selective inhibitors of
invasion are interesting tools to dissect targets impli-
cated in the mechanisms of invasion from those impli-
cated in growth.
Discussion
From the 95 compounds, 39, 53 and 71 were found to
possess an anti-invasive activity on MCF-7/6 cells in the
chick heart invasion assay at a concentration as low as
1 mM. These three novel compounds have been fully
characterized. The molecular formula C24H14N3F2Cl of
(Z)-2-[3-(4-chlorophenyl)-1-phenylpyrazol-5-yl]-3-(3,4-
difluorophenyl) acrylonitrile (39) was determined on the
basis of its EI–MS, HR-MS and hydrogen and carbon
1
counts form its H and 13C NMR spectra. The absorp-
tion at 2215 cmꢀ1 in the IR spectrum of 39 revealed the
presence of –CꢁN group in the compound. Two charac-
teristic singlets, each for one proton appeared at d 6.93
and 7.03 for the C-3H and C-40H, respectively and the
12 aromatic protons present in compound 39 resonated
between d 7.20 to 7.82 as four multiplets and a doublet
1
in its H NMR spectrum. The 13C NMR spectrum of
compound 39 showing signals for all the 24 different
carbon atoms was in full agreement with its structure.
The molecular formula C19H13O3N of the iso-
xazolylcoumarin 53 was determined on the basis of its
The E-cadherin/catenin complex is located at the cell
membrane of normal epithelial cells, and this cell–cell
adhesion complex prevents epithelia to grow beyond
their normal tissue boundaries.32 In invasive tumors
derived from epithelial tissues, the expression or the
function of the E-cadherin/catenin complex are down-
regulated.33 The complex in MCF-7/6 cells is inactive,
but sensitive to functional upregulation by a number of
compounds that showed to possess an anti-invasive
activity.34 Tangeretin, a citrus methoxyflavone, for
instance was able to increase E-cadherin-dependent
cell–cell adhesion between MCF-7/6 cells and to inhibit
their invasion in the chick heart invasion assay.3 This
mechanism of action could be excluded for the three
anti-invasive compounds in this study (i.e., compounds
39, 53 and 71), since they did not stimulate cell aggre-
gation in vitro.
1
EI–MS and hydrogen and carbon counts from its H
and 13C NMR spectra. The absorption band at
1740 cmꢀ1 in the IR spectrum of 53 revealed the pre-
sence of a lactone carbonyl group of the coumarin
moiety. The singlet at d 2.40 integrating for three pro-
tons revealed the presence of the methyl group at the C-
400 position. The characteristic C-4H of the coumarin
moiety and the C-40H of the isoxazole moiety appeared
1
at d 8.01 (s) and 7.59 (s), respectively in the H NMR
spectrum of isoxazolylcoumarin 53, which also exhib-
ited resonances due to eight aromatic protons as two
multiplets for two protons each between d 7.15–7.21
and 7.39–7.49, and two doublets for two protons each
at d 7.28 and 7.78 (J=8 Hz each). The peaks in the 13C
NMR spectrum of the isoxazolylcoumarin 53 were in
full agreement with its structure. The molecular formula
C19H20O3 of 2,4-dihydroxy-3-prenyldesoxybenzoin (71)
was determined on the basis of its EI–MS, HR-MS and
A number of other flavonoids and polyphenolics were
shown to inhibit invasion in vitro35 and artificial meta-
stasis in vivo.36 Some anti-invasive flavonoids, such as
3,7-dimethoxyflavone37 and (+)-catechin38 appear to
act via targets in the normal tissues confronted by the
tumor cells. (+)-Catechin, for instance, binds to lami-
nin, an extracellular matrix protein.39 Among the pre-
nylatedchalcones, anti-invasive congeners were found
that were selectively cytotoxic for the MCF-7/6 cells,
1
hydrogen and carbon count from its H and 13C NMR
spectra. The IR and UV spectra of 71 revealed the pre-
sence of carbonyl and hydroxyl groups in the com-
pound. The 1H NMR spectrum of 71 exhibited the
characteristic resonances for the nine protons of the
prenyl group at d1.81 (6H), 3.42 (2H) and 5.24–5.27

922
V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
and did not affect the viability of the chick heart host
fragment.4
effusion of a breast adenocarcinoma patient.6 These
cells, whose identity was confirmed in our laboratory,40
are invasive both in vitro41 and in vivo.42 MCF-7/6 cells
were maintained in 25 cm2 Falcon tissue culture flasks
(Becton Dickinson, Europe, Meylan, France) in a mix-
ture of Dulbecco’s modification of Earle’s Medium and
Ham F12 (50:50; Flow, Irvine, UK), supplemented with
0.05% glutamine (w/v), 250 IU/mL penicillin, 100 mg/
mL streptomycin, 2.5 mg/mL amphotericine B and 10%
fetal bovine serum.
Our results with the anti-invasive compounds of this
study (39, 53 and 71) emphasize that, although the
mechanisms of action are not elucidated, tumor inva-
sion can be tackled via several targets that may serve as
many entries for therapeutic rationales. The present
study has revealed that compounds belonging to the
class of pyrazolylacrylonitriles, that is 27–42 are more
likely to exhibit anti-invasive activity against MCF 7/6
cells. The phenyl substituents at N-1 position in the
pyrazole and at C-3 position in the acrylonitrile moiety
enhance the anti-invasive activity of compounds of this
class as (Z)-2-[3-(4-chlorophenyl)-1-phenylpyrazol-5-yl]-
3-(3,4-difluorophenyl)acrylonitrile (39) was found active
even at 1 mM concentration. Similarly, presence of the
coumarin moiety at the C-5 position together with a C-4
substituted phenyl group at the C-3 position on an iso-
xazole nucleus enhance the anti-invasive activity of
compounds of this class, thus 3-[3-(4-methylphenyl)
isoxazol-5-yl]-2H-1-benzopyran-2-one (53) was found
active even at 1 mM concentration among the 12 iso-
xazole derivatives 43–54 evaluated for their anti-inva-
sive activity. The desoxybenzoins with C-prenyl group,
both in the open chain and cyclised form have been
identified as a potent class of anti-invasive compounds.
The presence of a prenyl group at the C-3 position on a
desoxybenzoin nucleus enhances the anti-invasive activ-
ity, thus 2,4-dihydroxy-3-prenyldesoxybenzoin (71)
exhibits activity at 1 mM concentration against MCF-7/
6 cells. Based upon these studies, further work is in
progress to identify a non-toxic, potent compound
active at very low concentrations to provide leads
towards new therapeutic agents in cancer research.
Assay for invasion
The assay consists of three-dimensional confrontations
between tumour cells and normal tissue.43 Fragments of
9-day-old embryonic chick heart were precultured and
selected having a diameter of 0.4 mm. These precultured
heart fragments (PHF) were confronted individually
with an aggregate (diameter of 0.2 mm) of MCF-7/6
cells, first on top of semi-solid agar overnight to allow
attachment, and subsequently in liquid medium for
eight days in suspension culture. The cultures were then
fixed and embedded individually in paraffin, and serially
sectioned for histological analysis. In order to evaluate
the interaction between the MCF-7/6 cells and PHF, the
sections were stained with haematoxylin-eosin or with
an immunohistochemical technique to reveal MCF-7
antigens with the 5D10 monoclonal antibody.44 Occu-
pation and destruction of the PHF was considered as
typical for invasion, while growth around the PHF was
scored as absence of invasion.
Treatments
All compounds were dissolved in dimethyl sulfoxide
(DMSO) to give a stock solution of 10ꢀ1 M from which
further dilutions in culture medium were prepared. For
each compound, at least two confrontations were trea-
ted in suspension with 10 mM for 8 days. If this concen-
tration appeared to be anti-invasive or cytotoxic, lower
concentrations (1 or 0.1 mM) were applied. Control cul-
tures were treated with solvent alone (DMSO at corre-
sponding concentrations).
Conclusion
Out of the 95 compounds belonging to the different
classes, which were evaluated for their anti-invasive
activity against invasive human MCF-7/6 mammary
carcinoma cells in confrontation with embryonic chick
heart fragment in vitro, three lead compounds, namely
39, 53 and 71 based upon the pyrazole, isoxazole and
desoxybenzoin nucleus, respectively were identified.
These inhibited the invasion of MCF-7/6 cells on
embryonic chick heart fragment at concentrations as
low as 1 mM. Further studies revealed that these com-
pounds are anti-invasive without being cytotoxic to
cancer cells. The results of the present study may find a
lead towards the development of new therapeutic agents
to fight cancer.
Assay for growth
MCF-7/6 cells were cultured and treated in Nunclon
microtitre plates (Nunc, Roskilde, Denmark) at an
initial concentration of 1.5 Â 106 cells/well for three
days. They were fixed with 50% trichloroacetic acid in
water at 2 ꢂC for 1 h, and stained with 0.4% sulforho-
damine B (SRB) in 1% glacial acetic acid at room tem-
perature for 30 min.45 Protein-bound stain was
solubilized and read at 490 nm with a nmax ELISA
reader (Molecular Devices, Palo Alto, CA, USA). The
correlation with cell number was initially confirmed by
cell counts using a Bruker counting chamber. At least
12 cultures were analysed for each treatment.
Experimental
Cells
MCF-7/6 cells, a variant of the MCF-7 cell family were
obtained from Dr. Henri Rochefort, Unite d’Endocrin-
ologie Cellulaire et Moleculaire, Montpellier, France.
MCF-7 cells were originally established from a pleural
Assay for cell aggregation
Confluent monolayers of MCF-7/6 cells were detached,
and 2 Â 104 cells were seeded in 200 mL medium on

V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
923
solidified agar (Difco, Detroit, MI, USA) in a microtiter
plate (Nunc), and treated with the test agents.30 Aggre-
gate formation was evaluated under an inverted micro-
scope after 24 h of incubation.
[1,3-Di(4-bromophenyl)pyrazol-5-yl]acetonitrile
(11).
White crystals, mp 112–113 ꢂC; IR (KBr): 2950, 2250,
1600, 1500, 1440, 1360, 1080, 1020, 960 and 840 cmꢀ1
;
UV (MeOH): 262 and 295 nm; HR-MS, C17H11N3Br2
(M+ 414.9330, calcd 414.9320); 1H NMR (250 MHz,
CDCl3): d 3.77 (2H, s, –CH2CN), 6.82 (1H, s, H-4), 7.35
(2H, d, J=8.8 Hz, H-200 and H-600), 7.54 (2H, d,
J=8.6 Hz, H-20 and H-60), 7.67 (2H, d, J=8.5 Hz, H-300
and H-500) and 7.70 (2H, d, J=8.5 Hz, H-30 and H-50);
13C NMR (62.89 MHz, CDCl3): d 16.18 (–CH2CN),
105.67 (C-4), 115.25 (–CH2CN), 122.50 (C-400), 122.95
(C-40), 126.62 (C-20 and C-60), 127.27 (C-200 and C-600),
131.06 (C-10), 131.88 (C-300 and C-500), 132.90 (C-30 and
C-50), 132.97 (C-100), 137.39 (C-3) and 151.46 (C-5); EI–
MS, m/z (% rel. int.): 415/419 [M]+/[M+4]+ (100.0/
52.5), 387/389/391(5.5/10.0/6.0), 335/337(3.12/4.22),
309/311(2.4/2.4), 257(10.3), 207/208/209 (2.12/4.3/2.2),
194/196(3.2/3.3), 155/156/157 (6.57/2.97/4.9), 114/
115(2.33/8.33) and 101/102(2.32/2.65) and 75(3.8).
Physical and spectral data of new compounds
[1-(4-Fluorophenyl)-3-(4-methylphenyl)pyrazol-5-yl]ace-
tonitrile (5). White solid, mp 118–119 ꢂC, IR (KBr):
3100, 2920, 2275, 1610, 1520, 1440, 1370, 1235, 1165,
1020, 970, 850 and 785 cmꢀ1; UV (MeOH): 274 and
292.5 nm; HRMS, C18H14 N3F (M+ 291.1161, calcd
1
291.1172); H NMR (250 MHz, DMSO-d6): d 2.33 (3H,
s, CH3), 4.29 (2H, s, –CH2CN), 6.96 (1H, s, H-4), 7.25
(2H, d, J=7.98 Hz, H-30 and H-50), 7.42–7.46 (2H, m,
H-20 and H-60), 7.63–7.69 (2H, m, H-200 and H-600) and
7.77 (2H, d, J=8.08 Hz, H-300 and H-500); 13C NMR
(62.89 MHz, DMSO-d6): d 15.48 (–CH2CN), 20.80
(CH3), 104.72 (C-4), 116.09 (–CH2CN), 116.46 (C-300
and C-500), 125.26 (C-30 and C-50), 126.95 (C-10), 127.09
(C-20 and C-60), 129.31 (C-200 and C-600), 134.51(C-100),
135.05 (C-3), 137.51 (C-40), 150.88 (C-5) and 164.50 (C-
400); EI–MS, m/z (% rel. int.): 291 [M]+ (10), 251(3),
130(6), 134(11), 91(43) and 95(100).
[3-(4-Bromophenyl)-1-(4-fluorophenyl)pyrazol-5-yl]aceto-
nitrile (12). White solid, mp 143–144 ꢂC; IR (KBr):
2950, 2250, 1600, 1520, 1440, 1230, 1080, 1020, 970, 860
and 810 cmꢀ1; UV (MeOH): 263 and 292.5 nm; HR-MS,
C17H11N3FBr (M+ 355.0117, calcd 355.0120); 1H NMR
(250 MHz, CDCl3): d 3.74 (2H, s, –CH2CN), 6.81 (1H,
s, H-4), 7.25 (2H, d, J=8.0 Hz, H-200 and H-600), 7.45
(2H, d, J=9.0 Hz, H-300 and H-500), 7.54 (2H, d,
J=8.6 Hz, H-20 and H-60) and 7.70 (2H, d, J=8.6 Hz,
H-30 and H-50); 13C NMR (62.8 MHz, CDCl3): d 16.09
(–CH2CN), 105.26 (C-4), 115.30 (–CH2CN), 116.93 (C-
300 and C-500), 122.42 (C-40), 127.27 (C-20 and C-60),
127.42 (C-200 and C-600), 131.17 (C-10), 131.87 (C-30 and
C-50), 133.14 (C-100), 134.50 (C-3), 151.24 (C-5) and
164.61 (C-400); EIMS, m/z (% rel. int.): 355/357 [M]+/
[M+2]+ (100.0/97.4), 327/329(12.3/14.0), 315/317(4.3/
4.4), 275/276(5.7/5.7), 249(6.4), 236(4.1), 194/196(2.0/
2.2), 194(2.0), 178/179(4.4/1.5), 155(2.3), 147(2.5), 134/
136(6.7/7.5), 111(2.7), 107(4.4) and 95(9.0).
[3-(4-Fluorophenyl)-1-phenylpyrazol-5-yl]acetonitrile (8).
White needles, mp 86–87 ꢂC; IR (KBr): 3120, 2940,
2220, 1680, 1570, 1520, 1470, 1420, 1250, 1180, 1060,
1020, 940 and 755 cmꢀ1; UV (MeOH): 258 and 296 nm;
1H NMR (300 MHz, CDCl3): d 3.79 (2H, s, –CH2CN),
6.82 (1H, s, H-4), 7.11 (2H, m, H-30 and H-50), 7.48–7.56
(5H, m, H-200, H-300, H-400, H-500 and H-600) and 7.85 (2H,
m, H-20 and H-60); EI–MS, m/z (% rel. int.): 277 [M]+
(100), 249(21), 155(22), 116(17), 95(26) and 77(94).
[3-(4-Methoxyphenyl)-1-phenylpyrazol-5-yl] acetonitrile
(9). White solid, mp 102–103 ꢂC; IR (KBr): 2240, 1620,
1510, 1440, 1260, 1180, 1040, 960, 840 and 770 cmꢀ1
;
UV (MeOH): 216 and 270 nm; HR-MS, C18H15 N3O
(M+ 289.1201, calcd 289.1215); 1H NMR (300 MHz,
CDCl3): d 3.79 (2H, s, –CH2CN), 3.86 (3H, s, –OCH3),
6.80 (1H, s, H-4), 6.97 (2H, d, J=8.7 Hz, H-30 and H-
50), 7.45–7.57 (5H, m, H-200, H-300, H-400, H-500 and H-600)
and 7.80 (2H, d, J=8.7 Hz, H-20 and H-60); EI–MS, m/z
(% rel. int.): 289 [M]+ (23), 288(100), 249(5), 146(5),
144(4) and 77(8).
[1-(4-Fluorophenyl)-3-(4-methoxyphenyl)pyrazol-5-
yl]acetonitrile (13). White crystals, mp 144–145 ꢂC; IR
(KBr): 3050, 2900, 2250, 1620, 1520, 1460, 1370, 1310,
1250, 1180, 1030, 970, 840 and 780 cmꢀ1; UV (MeOH):
1
237, 264 and 288 nm; H NMR (300 MHz, CDCl3): d
3.76 (2H, s, –CH2CN), 3.86 (3H, s, –OCH3), 6.79 (1H, s,
H-4), 6.97 (2H, d, J=8.7 Hz, H-30 and H-50), 7.26 (2H,
m, H-300 and H-500), 7.48 (2H, m, H-200 and H-600) and
7.78 (2H, d, J=8.7 Hz, H-20 and H-60); 13C NMR
(62.89 MHz, DMSO-d6): d 15.43 (–CH2CN), 55.06 (–
OCH3), 104.42 (C-4), 116.02 (C-300 and C-500), 116.39
(C-30 and C-50), 116.86 (–CH2CN), 124.82 (C-10), 126.88
(C-200 and C-600), 127.02 (C-20 and C-60), 134.39 (C-100),
135.09 (C-3), 150.73 (C-5), 159.27 (C-40) and 163.35 (C-
400); EIMS, m/z (% rel. int.): 307 [M]+ (25), 205(8),
169(3), 131(3), 146(1), 134(1), 95(1) and 18(100).
[1-(4-Fluorophenyl)-3-phenylpyrazol-5-yl]acetonitrile (10).
White solid, mp 126–127 ꢂC; IR (KBr): 3080, 2900,
2260, 1615, 1560, 1515, 1470, 1375, 1220, 1095, 965, 840
and 700 cmꢀ1; UV (MeOH): 261 and 282.5 nm; HR-MS,
1
C17H12N3F (M+ 277.1041, calcd 277.1015); H NMR
(300 MHz, CDCl3): d 3.76 (2H, s, –CH2CN), 6.86 (1H,
s, H-4), 7.26 (2H, m, H-300 and H-500), 7.37–7.51 (5H, m,
H-20, H-30, H-40, H-50 and H-60) and 7.18 (2H, d,
J=7.18 Hz, H-200 and H-600); 13C NMR (62.89 MHz,
CDCl3): d 16.09 (–CH2CN), 105.33 (C-4), 115.40 (–
CH2CN), 116.50 (C-300 and C-500), 125.73(C-40), 125.70
(C-20 and C-60), 125.93 (C-200 and C-600), 127.43 (C-100),
128.42 (C-10), 132.85 (C-30 and C-50), 133.63 (C-3),
150.56 (C-5) and 163.78 (C-400); EIMS, m/z (% rel. int.):
277 [M]+ (29), 249(59), 216(22), 137(32), 109(42),
95(100), 84(42), 75(34) and 51(20).
[3-(4-Chlorophenyl)-1-(4-fluorophenyl)pyrazol-5-yl]aceto-
nitrile (14). White solid, mp 130–131 ꢂC; IR (KBr):
3080, 2280, 1610, 1520, 1425, 1370, 1240, 1100, 1020,
850 and 740 cmꢀ1; UV (MeOH): 274 and 295 nm; HR-
MS, C17H11 N3FCl (M+ 311.0627, calcd 311.0629); H
1
NMR (60 MHz, CDCl3): d 3.78 (2H, s, –CH2CN), 6.88

924
V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
(1H, s, H-4), 7.10–7.35 (6H, m, H-20, H-200, H-30, H-50,
H-60 and H-600) and 7.47 (2H, d, J=8.0 Hz, H-300, H-500);
13C NMR (62.8 MHz, DMSO-d6): d 15.45 (–CH2CN),
105.00 (C-4), 116.08 (C-300 and C-500), 116.45 (C-200 and
C-600), 116.75 (–CH2CN), 127.02 (C-30 and C-50), 127.15
(C-10), 128.73 (C-20 and C-60), 131.08 (C-100), 132.66 (C-
3), 134.89 (C-40), 149.70 (C-5) and 163.51 (C-400); EI–
MS, m/z (% rel. int.): 311/313 [M]+/[M+2]+ (100.0/
34.0), 155.5/156.5(3.0/1.0), 271/273(7.0/2.0), 150/
152(4.0/1.0), 134(8.0), 111/113(4.0/1.0) and 95(16.0).
3-Amino-6-(3,4-methylenedioxyphenyl)-2-phenyl-4-oxo-
4H-pyrano[4,3-c]pyrazole (20). Brown solid, mp 259–
262 ꢂC; IR (KBr): 3392, 3282, 2901, 2791, 1722, 1637,
1494, 1443, 1355, 1271, 1037 and 950 cmꢀ1; UV
(MeOH): 235, 334 and 345 nm; HR-MS, C19H13 N3O4
(M+ 347.0898, calcd 347.0906); 1H NMR (60 MHz,
CDCl3+TFA): d 6.14 (2H, s, –OCH2O-), 6.80 (1H, s,
H-20), 7.04 (1H, s, H-7), 7.36–7.50 (2H, m, H-50 and H-
6000) and 7.70 (5H, brm, H-200, H-300, H-400, H-500 and H-
6 ); EI–MS, m/z (% rel. int.): 347 [M]+ (100.0),
319(34.2), 290(16.7), 226(5.4), 149(12.1), 121(4.2),
119(8.1) and 77(22.1).
[1,3-Di(4-fluorophenyl)pyrazol-5-yl]acetonitrile
(15).
White solid, mp 134–136 ꢂC; IR (KBr): 3080, 3000,
2260, 1615, 1520, 1450, 1360, 1230, 1150, 1100, 960 and
840 cmꢀ1; UV (MeOH): 225 and 287 nm; HR-MS,
3-Amino-2-(4-fluorophenyl)-6-(4-methylphenyl)-4-oxo-
4H-pyrano [4,3-c]pyrazole (21). White solid, mp
>300 ꢂC; IR (KBr): 3350, 2750, 1730, 1640, 1450, 1380,
1165, 1050, 840 and 740 cmꢀ1; UV (MeOH): 250, 260
C17H11 N3F2 (M+ 295.0930, calcd 295.0921); H NMR
1
(250 MHz, CDCl3): d 3.74 (2H, s, –CH2CN), 6.79 (1H,
s, H-4), 7.12 (2H, m, H-300 and H-500), 7.25 (2H, m, H-30
and H-50), 7.45 (2H, d, J=9.1 Hz, H-200 and H-600) and
7.81 (2H, d, J=8.9 Hz, H-20 and H-60); 13C NMR
(62.8 MHz, CDCl3): d 16.05 (–CH2CN), 105.11 (C-4),
115.35 (–CH2CN), 115.85 (C-300 and C-500), 116.88 (C-30
and C-50), 127.26 (C-200 and C-600), 127.40 (C-20 and C-
60), 128.41 (C-10), 133.02 (C-100), 134.55 (C-3), 151.42 (C-
5), 160.95 and 164.89 (C-400 and C-40); EIMS, m/z (%
rel. int.): 295 [M]+ (100), 294(27), 267(24), 255(7),
216(2), 173(5), 148(4), 138(5), 96(9) and 75(3).
1
and 275 nm; H NMR (90 MHz, CDCl3+TFA): d 2.38
(3H, s, CH3), 7.03 (1H, s, H-7), 7.15–7.42 (4H, brm, H-
30, H-300, H-50 and H-500), 7.50 (2H, d, J=8.0 Hz, H-200
and H-600) and 7.72 (2H, d, J=8.3 Hz, H-20 and H-60);
13C NMR (62.89 MHz, DMSO-d6): d 20.79 (CH3), 89.59
(C-7), 94.43 (C-9), 124.93 (C-30 and C-50), 126.68 (C-300
and C-500), 126.83 (C-200 and C-600), 129.34 (C-20 and C-
60), 129.48 (C-6), 133.82 (C-10), 139.46 (C-40), 148.47 (C-
100), 155.47 (C-8), 158.32 (C-3), 163.13 (C-400) and 173.90
(C-4); EI–MS, m/z (% rel. int.): 335 [M]+ (100.0),
307(17.0), 278(9.0), 244(4.0), 167.5(3.0), 153.5(4.0),
137(5.0), 119(10.0), 95(12.0) and 91(9.0).
[1-(4-Chlorophenyl)-3-(4-methoxyphenyl)pyrazol-5-
yl]acetonitrile (16). White fluffy solid, mp 142–143 ꢂC;
IR (KBr): 2980, 2900, 2280, 1620, 1535, 1500, 1450,
1370, 1250, 1180, 1090, 970 and 840 cmꢀ1; UV (MeOH):
236, 265 and 291 nm; HR-MS, C18H14N3OCl (M+
323.0834, calcd 323.0825); 1H NMR (250 MHz, CDCl3):
d 3.78 (2H, s, CH2CN), 3.86 (3H, s, OCH3), 6.80 (1H, s,
C-4H), 6.96 (2H, d, J=8.3 Hz, C-30H and C-50H), 7.44
(2H, d, J=8.4 Hz, C-200H and C-600H), 7.52 (2H, d,
J=8.3 Hz, C-20H and C-60H) and 7.78 (2H, d,
J=8.4 Hz, C-300H and C-500H); 13C NMR (62.5 MHz,
DMSO-d6): d 15.51 (CH2CN), 55.06 (OCH3), 104.35 (C-
4), 114.10 (C-300 and C-500), 116.84 (CH2CN), 124.68 (C-
30 and C-50), 124.80 (C-10), 126.68 (C-200 and C-600),
129.35 (C-20 and C-60), 132.53 (C-400), 134.41 (C-5),
137.51 (C-40), 150.98 (C-100) and 159.33 (C-3); EI–MS,
m/z (% rel. int.): 325 [M+2]+ (33), 323 [M]+ (100),
308(14), 280(4), 205(2), 111(9) and 90(2).
3-Amino-6-(4-chlorophenyl)-2-(4-fluorophenyl)-4-oxo-4H-
pyrano[4,3-c]pyrazole (22). Pale white solid, mp 242 ꢂC;
IR (KBr): 3345, 3122, 2929, 1720, 1637, 1509, 1404,
1254, 1095, 1037 and 809 cmꢀ1; UV (MeOH): 250, 300
1
and 312 nm; H NMR (60 MHz, CDCl3+TFA): d 7.16
(1H, s, H-7), 7.40 (4H, m, H-30, H-300, H-50 and H-500)
and 7.70 (4H, m, H-20, H-200, H-60 and H-600); 13C NMR
(62.8 MHz, DMSO-d6): d 89.57 (C-7), 95.76 (C-9),
116.03 (C-300 and C-500), 116.40 (C-200 and C-600), 126.71
(C-30 and C-50), 126.82 (C-10), 127.92 (C-20 and C-60),
131.05 (C-6), 134.22 (C-40), 148.51 (C-100), 154.11 (C-8),
157.96 (C-3), 159.24 (C-400) and 163.14 (C-4); EI–MS,
m/z (% rel. int.): 355/357 [M]+/[M+2]+ (11.0/4.0), 327/
329(1.0/1.0),
298/300(1.0/1.0),
274/277(11.0/4.0),
246(2.0), 203(1.0), 163(2.0), 139/141(16.0/5.0), 111/
115(5.0/2.0), 95(2.0), 73(11.0), 60(16.0), 43(10.0) and
18(100).
3-Amino-6-(4-fluorophenyl)-2-phenyl-4-oxo-4H-pyr-
ano[4,3-c] pyrazole (19). White solid, mp 255–257 ꢂC; IR
(KBr): 3470, 3360, 3100, 1735, 1645, 1570, 1510, 1370,
1230, 1170, 1040, 920 and 855 cmꢀ1; UV (MeOH): 250,
275 and 295 nm; HR-MS, C18H12 N3O2 F (M+
321.0932, calcd 321.0914); 1H NMR (60 MHz,
CDCl3+TFA): d 6.48 (1H, s, H-7), 7.30 (7H, brm, H-
200, H-30, H-300, H-400, H-50, H-500 and H-600) and 7.68
(2H, m, H-20 and H-60); 13C NMR (62.89 MHz, DMSO-
d6): d 89.71 (C-7), 95.21 (C-9), 115.57 (C-30 and C-50),
115.92 (C-20 and C-60), 124.02 (C-200 and C-600), 127.88
(C-10), 129.44 (C-300, C-400 and C-500), 137.44 (C-6),
148.26 (C-100), 154.37 (C-8), 158.19 (C-3), 160.83 (C-4)
and 164.77 (C-40); EI–MS, m/z (% rel. int.): 321 [M]+
(100.0), 293(31.9), 264(22.8), 226(7.4), 123(17.0),
119(7.4), 95(20.9) and 77(74.8).
3-Amino-2,6-di(4-fluorophenyl)-4-oxo-4H-pyrano[4,3-
c]pyrazole (23). White solid, mp >300 ꢂC, IR (KBr):
3440, 3080, 2900, 1725, 1645, 1515, 1420, 1225, 1165,
1100, 1050, 925 and 815 cmꢀ1; UV (MeOH): 286 and
296 nm; HR-MS, C18H11N3O2F2 (M+ 339.0821, calcd
1
339.0819); H NMR (250 MHz, CDCl3): d 7.08 (1H, s,
H-7), 7.17 (2H, d, J=8.5 Hz, H-300 and H-500), 7.34 (2H,
d0, J=8.5 Hz, H-30 and H-50), 7.76 (2H, d, J=8.5 Hz, H-
2 and H-60) and 7.87 (2H, d, J=8.8 Hz, H-200 and H-
600); 13C NMR (62.8 MHz, CDCl3): d 91.04 (C-9), 92.22
(C-7), 117.96 (C-300 and C-500), 118.33 (C-30 and C-50),
126.32 (C-200 and C-600), 128.40 (C-20 and C-60), 128.84
(C-6), 144.63 (C-100), 148.09 (C-8), 149.21 (C-10), 159.69
(C-3), 160.21 and 161.57 (C-40 and C-400) and 161.77 (C-
4); EI–MS, m/z (% rel. int.): 339 [M]+ (100), 311(29),

V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
925
282(15), 244(4.0), 188(3.2), 169(3.1), 155(2.4), 137(4.1),
123(9.7), 111(4.12), 109(2.8), 95(11.9) and 75(2.2).
NMR (62.8 MHz, CDCl3): d 101.79 (CN), 106.37 (C-40),
115.64 (C-2), 118.21 (C-5000), 125.54, 125.79 (C-200, C-300,
C-500 and C-600), 126.31 (C-2000), 128.47, 128.88 (C-40000, C-
400), 128.75, 129.70 (C-20000, C-30000, C-50000, C-60000), 129.76,
129.80 (C-6000, C-3), 132.11 (C-100), 137.79, 139.31 (C-10000,
C-1000), 143.76, 149.74, 152.49, 154.02 (C-30, C-3000, C-4000
and C-50); EI–MS, (rel. int.) m/z (%): 383 [M]+ (100),
382(4), 357(10), 279(5), 271(20), 243(7), 180(8), 167(7)
and 77(14).
3-Amino-2-(4-chlorophenyl)-6-(4-fluorophenyl)-4-oxo-4H-
pyrano[4,3-c]pyrazole (24). Pale yellow solid, mp
>300 ꢂC; IR (KBr): 3440, 3340, 2940, 1710, 1640, 1425,
1400, 1310, 1260, 1100, 1040, 920 and 840 cmꢀ1; UV
(MeOH): 250, 300 and 312 nm; HR-MS, C18H11 N3O2 F
Cl (M+ 355.0511, calcd 355.0524); 1H NMR (250 MHz,
DMSO-d6): d 6.90 (2H, brs, –NH2), 7.13 (1H, s, H-7),
7.35 (2H, m, H-30 and H-50), 7.61 (4H, bs, H-200, H-300,
H-500 and H-600) and 7.81 (2H, d, J=8.4 Hz, H-20 and H-
60); EI–MS, m/z (% rel. int.): 357 [M+2]+ (16.7), 355
[M]+ (46.2), 327(14.5), 153/154(9.0/18.5), 123(16.9),
113(7.9) and 95(12.6).
(Z)-2-[3-(4-Chlorophenyl)-1-phenylpyrazol-5-yl]-3-(3,4-
difluorophenyl) acrylonitrile (39). White solid, mp 137–
138 ꢂC; IR (KBr): 3436, 2925, 2215, 1596, 1522, 1496,
1434, 1285, 1161, 1088, 925, 818 cmꢀ1; UV (MeOH):
211, 263 and 307 nm; HR-MS, C24H14N3F2Cl (M+
417.0824, calcd 417.0844); 1H NMR (250 MHz, CDCl3);
d 6.93 (1H, s, H-3), 7.03 (1H, s, H-40), 7.20–7.27 (1H, m,
H-2000), 7.40 (2H, d, J=8.7 Hz, H-300 and H-500), 7.47–
7.54 (6H, m, H-200, H-30000, H-40000, H-5000, H-50000 and H-
600), 7.57–7.66 (1H, m, H-6000), 7.82 (2H, m, H-20000 and
H-60000); 13C NMR (62.8 MHz, CDCl3): d 101.61 (CN),
106.26 (C-4), 115.59 (C-2), 118.14 (C-5000), 125.52 (C-20000,
C-60000), 126.42 (C-2000), 127.04 (C-300 and C-500), 128.96
(C-30000 and C-50000), 129.03 (C-40000), 129.56 (C-200 and C-
600), 129.63, 129.73 (C-6000 and C-3), 130.07 (C-400),
134.29 (C-100), 138.00, 139.19 (C-1000 and C-10000), 143.95
(C-30), 149.80 (C-3000), 152.52 (C-50) and 153.88 (C-4000);
EI–MS, (rel. int.) m/z (%): 419 ([M+2]+, 85), 417
([M]+, 100), 416(35), 354(4), 306(28), 304(76), 242(9),
167(9) and 77(14).
3-Amino-2-(4-fluorophenyl)-6-(4-methoxyphenyl)-4-oxo-
4H-pyrano [4,3-c]pyrazole (25). Pale yellow solid, mp
178–180 ꢂC; IR (KBr): 3357, 2928, 1719, 1636, 1509,
1256, 1180, 1033 and 839 cmꢀ1; UV (MeOH): 254, 291
and 303 nm; 1H NMR (250 MHz, DMSO-d6): d 3.82
(3H, s, –OCH3), 6.77 (1H, s, H-7), 7.04 (2H, d,
J=8.96 Hz, H-30 and H-50), 7.40 (2H, m, H-300 and H-
500), 7.61 (2H, m, H-200 and H-600) and 7.82 (2H, d,
J=8.92 Hz, H-20 and H-60); 13C NMR (62.8 MHz,
DMSO-d6): d 55.22 (–OCH3), 89.47 (C-7), 93.41 (C-9),
116.02 (C-300 and C-500), 116.39 (C-200 and C-600), 126.58
(C-30 and C-50), 126.72 (C-20 and C-60), 130.74 (C-10),
133.82 (C-6), 148.41 (C-8), 149.12 (C-100), 155.42 (C-3),
158.35 (C-40), 160.42 (C-4) and 163.07 (C-400); EI–MS,
m/z (% rel. int.): 351 [M]+ (100.0), 323(13.0), 294(12.0),
244(3.0), 175.5(2.0), 161.5(5.0), 137(7.0), 135(17.0) and
95(22.0).
(Z)-2-[3-(4-Bromophenyl)-1-phenylpyrazol-5-yl]-3-(3,4-
difluorophenyl) acrylonitrile (40). White solid, mp 145–
146 ꢂC; IR (KBr): 3433, 2925, 2215, 2064, 1595, 1521,
1496, 1289, 1169, 1071, 925, 816, 758, 695 and 500 cmꢀ1
;
3-Amino-2-(4-chlorophenyl)-6-(4-methoxyphenyl)-4-oxo-
4H-pyrano[4,3-c] pyrazole (26). Buff coloured solid, mp
>300 ꢂC; IR (KBr): 3400, 1740, 1640, 1530, 1470, 1380,
1260, 1170, 1030 and 830 cmꢀ1; UV (MeOH): 242, 302
and 390 nm; HR-MS, C19H14N3O3Cl (M+ 367.0534,
calcd 367.0724); 1H NMR (250 MHz, DMSO-d6): d 3.82
(3H, s, OCH3), 6.89 (2H, brs, NH2), 7.04 (3H, m, C-
3000H, C-50H and C-7H), 7.59–7.62 (4H, m, C-200H, C-
6 H, C-20H and C-60H) and 7.82 (2H, d, J=8.4 Hz, C-
300H and C-500H); 13C NMR (62.5 MHz, DMSO-d6): d
55.36 (OCH3), 89.70 (C-9), 93.46 (C-7), 114.30 (C-300
and C-500), 124.66 (C-30 and C-50), 126.01 (C-200 and C-
600), 126.75 (C-20 and C-60), 129.52 (C-6), 132.25(C-100),
136.43 (C-400), 148.55 (C-8), 149.50 (C-10), 155.61 (C-40),
158.48 (C-3) and 160.55 (C-4); EI–MS, m/z (% rel. int.):
369 [M+2]+ (33), 367 [M]+ (100), 341(3), 339(10),
310(8), 272(2), 185(2), 135(17), 127(6), 111(13) and
77(10).
UV (MeOH): 208 and 270 nm; HR-MS, C24H14 N3F2Br
(M+ 461.0339, calcd 461.0339); 1H NMR (250 MHz,
CDCl3): d 6.94 (1H, s, H-3), 7.02 (1H, s, H-40), 7.19–
7.26 (1H, m, H-2000), 7.44–7.57 (8H, m, H-200, H-300, H-
30000, H-40000, H-50000, H-500, H-5000 and H-600), 7.59–7.65 (1H,
m, H-6000) and 7.54 (2H, m, H-20000 and H-60000); 13C NMR
(62.8 MHz, CDCl3): d 101.56 (CN), 106.22 (C-40),
115.56 (C-2), 118.23 (C-5000), 122.47 (C-400), 125.48 (C-
20000 and C-60000), 126.41 (C-2000), 127.29 (C-200 and C-600),
129.02 (C-40000), 129.60 (C-30000 and C-50000), 129.01, 129.70
(C-6000 and C-3), 131.09 (C-100), 131.87 (C-300 and C-500),
138.00, 139.15 (C-1000 and C-10000), 143.94 (C-30), 151.14
(C-50), 149.77 and 153.85 (C-3000 and C-4000); EI–MS, (rel.
int.) m/z (%): 463 ([M+2]+, 96), 461 ([M]+, 100),
460(30), 435(9), 289(6), 269(23), 242(8), 167(13) and
77(17).
(Z)-2-[3-(4-Methylphenyl)-1-phenylpyrazol-5-yl]-3-(3,4-
difluorophenyl) acrylonitrile (41). White solid mp 143-
144 ꢂC; IR (KBr): 3435, 2923, 2215, 1597, 1521, 1499,
1434, 1286, 1169, 1061, 921, 805 cmꢀ1; UV (MeOH):
207, 267 and 307 nm; HR-MS, C25H17N3F2 (M+
397.1379, calcd 397.1391); 1H NMR (250 MHz, CDCl3):
d 2.38 (3H, s, –CH3), 6.92 (1H, s, H-3), 7.02 (1H, s, H-
40), 7.15–7.18 (1H, m, H-2000), 7.23 (2H, d, J=7.98 Hz,
H-300 and H-500), 7.42–7.47 (2H, m, H-40000, H-5000), 7.40–
7.53 (4H, m, H-200, H-30000, H-50000 and H-600), 7.56–7.64
(1H, m, H-6000), 7.76 (2H, m, H-20000 and H-60000); 13C
(Z)-2-(1,3-Diphenylpyrazol-5-yl)-3-(3,4-difluorophenyl)
acrylonitrile (38). White solid, mp 154–155 ꢂC; IR
(KBr): 3447, 2924, 2363, 2215, 1598, 1502, 1429, 1281,
1152, 903, 816, 770, 690 and 526 cmꢀ1; UV (MeOH):
207, 261 and 310 nm; HR-MS, C24H15N3F2 (M+
383.1233, calcd 383.1234); 1H NMR (250 MHz, CDCl3);
d 6.96 (1H, s, H-3), 7.04 (1H, s, H-40), 7.22–7.26 (1H, m,
H-2000), 7.35–7.48 (5H, m, H-300, H-400, H-40000, H-500, H-
5000), 7.51–7.54 (4H, m, H-200, H-30000, H-50000, H-600), 7.56–
7.65 (1H, m, H-6000), 7.88 (2H, m, H-20000, H-60000); 13C

926
V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
NMR (62.8 MHz, CDCl3): d 21.27 (–CH3), 101.83
(CN), 106.21 (C-40), 115.66 (C-2), 118.08 (C-5000), 125.52,
125.68 (C-20000, C-200, C-60000 and C-600), 126.35 (C-2000),
128.78 (C-40000), 129.29 (C-400), 129.43, 129.53, 129.66 (C-
3000, C-30, C-5000, C-50 and C-b), 129.76 (C-6000), 137.68,
138.34, 139.35 (C-100, C-1000 and C-10000), 143.64 (C-30),
149.69 (C-50), 152.30 and 152.47 (C-3000 and C-4000); EI–
MS, (rel. int.) m/z (%): 397 ([M+], 100), 396(36), 371(8),
284(65), 257(3), 194(8), 167(4) and 77(8).
1720, 1600 and 1550 cmꢀ1; UV (MeOH): 310, 271, 259,
1
254 and 228 nm; H NMR (250 MHz, CDCl3+TFA): d
6.50 (1H, s, H-7), 7.15 (2H, m, H-30 and H-50), 7.80 (2H,
m, H-20 and H-60) and 9.20 (2H, brs, NH2); 13C NMR
(62.8 MHz, DMSO-d6): d 98.29, 127.31, 128.47, 128.55,
128.98, 129.28, 129.35, 136.05, 156.96, 161.95, 162.80
and 169.68; EI–MS, m/z (rel. int.%): 246 [M]+ (5),
230(10), 201(5), 187(8), 163(7), 135(6), 123(100),
107(12), 95(56), 75(24) and 42(24).
(Z)-2-[3-(4-Methoxyphenyl)-1-phenylpyrazol-5-yl]-3-
(3,4-difluorophenyl) acrylonitrile (42). Light yellow solid,
mp 135–137 ꢂC; IR (KBr): 3436, 2967, 2215, 1612, 1522,
1438, 1291, 1258, 1029, 840, 761, 696 and 522 cmꢀ1; UV
(MeOH): 271 and 209 nm; HR-MS, C25H17 N3OF2
(M+ 413.1325, calcd 413.1340); 1H NMR (250 MHz,
CDCl3): d 3.84 (3H, s, –OCH3), 6.88 (1H, s, H-3), 6.95
(2H, d, J=8.9 Hz, H-300 and H-500), 7.04 (1H, s, H-40),
7.16–7.25 (1H, m, H-2000), 7.46–7.53 (6H, m, H-200, H-
30000, H-40000, H-5000, H-50000 and H-600), 7.56–7.63 (1H, m,
H-6000) and 7.81 (2H, m, H-20000 and H-60000); 13C NMR
(62.8 MHz, CDCl3): d 55.28 (OCH3), 101.82 (CN),
105.94 (C-40), 114.13 (C-300 and C-500), 115.67 (C-2),
118.06 (C-5000), 124.83 (C-100), 126.49 (C-20000 and C-60000),
126.35 (C-2000), 127.07 (C-30000 and C-50000), 128.75 (C-40000),
129.66 (C-3), 129.53 (C-200 and C-600), 129.76 (C-6000),
137.67, 139.34 (C-1000 and C-10000), 143.62 (C-30), 149.68,
153.75 (C-3000 and C-4000), 152.07 (C-50) and 159.91 (C-
400); EI–MS, m/z (rel. int.): 413 [M]+ (100), 398(9),
370(4), 300(30), 210(7), 150(3) and 77(6).
[3-(4-Chlorophenyl)isoxazol-5-yl]-2H-1-benzopyran-2-one
(52). White solid, mp 242–244 ꢂC; IR (KBr): 1740,
1675, 1608, 1440, 1275, 1218, 1195, 1101, 817 and
1
752 cmꢀ1; UV (MeOH): 350, 320, 305 and 245 nm; H
NMR (300 MHz, CDCl3+ traces of TFA): d 7.30 (1H,
s, H-40), 7.45–7.47 (2H, m, H-7 and H-8), 7.70–7.90
(4H, m, H-300, H-500, H-5 and H-6), 7.93 (2H, d,
J=8.5 Hz, H-200 and H-600) and 8.73 (1H, s, H-4); 13C
NMR (75 MHz, CDCl3+1 drop of TFA): d 103.07,
117.39, 128.27, 128.55, 129.10, 129.38, 129.52, 130.27,
133.68, 137.31, 146.74, 151.01, 159.60, 160.15, 162.78
and 163.09; EI–MS, m/z (rel. int.): 323 [M]+ (20),
322(60), 294(10), 206(11), 198(30), 186(25), 173(100),
150(10), 129(25), 107(72) and 75(18).
3-[3-(4-Methylphenyl)isoxazol-5-yl]-2H-1-benzopyran-2-
one (53). White solid, mp 212 ꢂC; IR (Nujol): 1740,
1667, 1608, 1282, 1225, 952 and 882 cmꢀ1; UV (MeOH):
1
355, 345 and 325 nm; H NMR (250 MHz, CDCl3): d
2.40 (3H, s, CH3), 7.15–7.21 (2H, m, H-7 and H-8), 7.28
(2H, d, J=8.0 Hz, H-300 and H-500), 7.39–7.49 (2H, m,
H-5 and H-6), 7.59 (1H, s, H-40), 7.78 (2H, d, J=8.0 Hz,
H-200 and H-600) and 8.01 (1H, s, H-4); 13C NMR
(62.8 MHz, CDCl3): d 21.41 (CH3), 104.07 (C-40), 115.40
(C-8), 116.50 (C-10), 118.83 (C-400), 123.90 (C-7), 126.16
(C-3), 126.80 (C-300 and C-500), 128.69(C-100), 129.54 (C-
200 and C-600), 131.82 (C-6), 134.08 (C-5), 140.13 (C-4),
153.35, 163.11 and 163.25 (C-2, C-30, C-50 and C-9); EI–
MS, m/z (rel. int.): 303 [M+] (8), 302(35), 274(3), 211(4),
186(13), 185(100), 158(5), 130(8), 102(4), 91(10) and
65(5).
[3-(3,4-Methylenedioxyphenyl)isoxazol-5-yl] acetonitrile
(46). White solid, mp 161 ꢂC; IR (nujol): 2240, 2070,
1599, 1498, 1382, 1265, 1227, 1147, 942, 885 and
810 cmꢀ1; UV (MeOH): 295, 260 and 215 nm; 1H NMR
(250 MHz, CDCl3): d 2.24 (2H, s, CH2CN), 5.98 (2H, s,
OCH2O), 6.70 (1H, d, J=8.1 Hz, H-50), 6.82 (1H, s, H-
4), 7.00 (1H, d, J=2.0 Hz, H-20) and 7.10 (1H, m, H-60);
13C NMR (62.8 MHz, CDCl3): d 12.20 (–CH2CN),
101.29 (–OCH2O–), 106.23 (C-20), 108.05 (C-50), 109.50
(CN), 112.98 (C-4), 120.36 (C-60), 130.75 (C-10), 147.9
(C-40), 148.56 (C-30), 150.10 (C-5) and 155.56 (C-3); EI–
MS, m/z (rel. int.%): 228 [M+].
3-[3-(3,4-Methylenedioxyphenyl)isoxazol-5-yl]-2H-1-ben-
zopyran-2-one (54). White solid, mp 235–237 ꢂC; IR
(Nujol): 1720, 1663, 1604, 1462, 1282, 1251, 1213, 1048,
937 and 811 cmꢀ1; UV (MeOH): 345, 310 and 305 nm;
1H NMR (250 MHz, CDCl3+TFA): d 6.06 (2H, s, –
OCH2O–), 6.92 (1H, d, J=7.9 Hz, H-500), 7.34–7.48
(5H, m, H-40, H-200, H-600, H-7 and H-8), 7.66–7.74 (2H,
m, H-5 and H-6) and 8.55 (1H, s, H-4); 13C NMR
(62.8 MHz, CDCl3–TFA): d 101.73 (C-40), 103.98 (–
OCH2O–), 106.92 (C-200), 108.96 (C-500), 116.75 (C-3),
116.97 (C-8), 117.59 (C-100), 118.44 (C-10), 121.29,
122.04 (C-600 and C-7), 125.68 (C-6), 129.34 (C-5),
133.84 (C-4), 159.31, 159.99, 160.64, 162.88, 163.12 and
163.44 (C-2, C-30, C-300, C-400, C-50 and C-9); EI–MS, m/
z (rel. int.%): 333 [M]+ (100), 332(40), 305(33), 277(5),
249(6), 210(6), 188(15), 185(14), 173(54), 163(17),
160(18), 146(12), 130(7), 101(8) and 89(6).
3-(3,4-Methylenedioxyphenyl)-5-(methylthio)-4,5-dihydro-
isoxazol-5-yl] acetonitrile (48). White solid, mp 82 ꢂC;
IR (KBr): 2264, 1608, 1532, 1260, 1243, 1133, 1045, 935
and 912 cmꢀ1; UV (MeOH): 305, 270 and 219 nm; HR-
MS, C13H12 N2O3 S (M+ 276.0587, calcd 276.0569); 1H
NMR (250 MHz, CDCl3): d 2.25 (3H, s, SCH3), 3.20
(2H, s, CH2CN), 3.46 (1H, d, J=17.6 Hz, H-4a), 3.70
(1H, d, J=17.5 Hz, H-4b), 6.00 (2H, s, OCH2O), 6.70
(2H, m, H-20 and H-60) and 7.02 (1H, d, J=8.0 Hz, H-
50); 13C NMR (75 MHz, CDCl3): d 11.67 (CH2CN),
16.63 (SCH3), 28.74 (C-4), 46.73 (C-5), 101.00
(OCH2O), 104.00 (CN), 106.79, 108.26, 108.62, 121.85
and 122.12 (C-3, C-10, C-20, C-50 and C-60), and 148.23
and 149.92 (C-30 and C-40); EI–MS, m/z (rel. int.): 276
[M]+ (53), 229(100), 228(83), 201(17), 188(51), 171(21),
161(81), 160(36), 146(20), 121(22), 63(18) and 44(11).
7,8-Dibutanoyloxy-4-methyl-2H-1-benzopyran-2-one (58).
White solid mp 99–100 ꢂC; IR (KBr): 3084, 2972, 2935,
2877, 2363, 1764, 1738, 1612, 1575, 1443, 1372, 1268,
3-Amino-6-(4-fluorophenyl)pyrano[4,3-c]isoxazole-4-one
(51). White solid, mp 240–242 ꢂC; IR (Nujol): 3365,

V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
927
1235, 1137, 1096, 1051, 953, 869, 825, 745 and 589 cmꢀ1
;
153.52 and 156.19 (C-5, C-6 and C-7) and 160.80 (C-2);
EI–MS, m/z (rel. int.): 250 [M+] (100), 235(73), 208(13),
207(80), 179(32), 177(8), 164(28) and 149(18).
UV (MeOH): 308, 277 and 211 nm; HR-MS, C18H20O6
(M+ 332.1279, calcd 332.1260); 1H NMR (300 MHz,
CDCl3): d 1.10 (6H, m, 2ÂCH2CH2CH3), 1.85 (4H, m,
2ÂCH2CH2CH3), 2.47 (3H, s, C-4CH3), 2.60 (2H, t,
CH2–C¼O), 2.69 (2H, t, CH2–C¼O), 6.30 (1H, s, H-3),
7.19 (1H, d, J=8.7 Hz, H-6) and 7.53(1H, d, J=8.7 Hz,
H-5); 13C NMR (75 MHz, CDCl3): d 13.55 (2 Â
2,4-Dihydroxy-5-(3-methyl-2-butenyl)phenylbenzyl
ke-
tone (70). White solid, mp 105–106 ꢂC; IR (KBr): 3293,
3029, 2963, 2922, 1635, 1512, 1422, 1362, 1304, 1246,
1130, 1027, 850 and 695 cmꢀ1; UV (MeOH): 246, 313
and 320 nm; HR-MS, C19H20O3 (M+ 296.1418, calcd
.
CH2CH2CH3), 18.29 and 18.37 (2 CH2CH2CH3), 18.76
1
(C-4CH3), 35.45 and 35.77 (2 Â CH3CH2CH2–C¼O),
114.69 (C-10), 118.54 (C-6), 118.72 (C-3), 121.48 (C-5),
130.40 (C-4), 145.27 and 146.69 (C-7 and C-8), 151.87
(C-9), 159.18 (C-2), and 170.11 and 170.47 (2ÂC¼O);
CI-MS, m/z (rel. int.): 333 [M+1]+ (100).
296.1412); H NMR (250 MHz, CDCl3): d 1.79 (6H, s,
2ÂCH3), 3.26 (2H, d, J=6.95 Hz,-CH2–CH¼), 4.19
(2H, s,–COCH2–), 5.23–5.31 (1H, m, ¼CH–CH2–), 6.13
(1H, brs, C-4 OH), 6.34 (1H, s, H-3), 7.21–7.37 (5H, m,
H-20, H-30, H-40, H-50 and H-60), 7.64 (1H, s, H-6) and
12.51 (1H, s, C-2 OH); 13C NMR (62.8 MHz, CDCl3): d
17.85 and 25.83 (2ÂCH3), 29.51 (–CH2–CH¼), 45.09 (–
COCH2–), 103.78 (C-3), 113.27 [(CH3)2C¼CH–], 119.20
(C-5), 121.24 (C-6), 127.06 (C-40), 128.76 (C-30 and C-
50), 129.31 (C-20 and C-60), 131.93 [(¼ CH–CH2–],
134.57 (C-10), 135.38 (C-1), 161.59 (C-2), 164.08 (C-4)
and 202.16 (C¼O); EI–MS, m/z (% rel. int.): 296 [M]+
(15.5), 279(3.1), 221(2.9), 206(14.6), 205(100.0),
203(6.8), 163(2.6), 161(2.2), 150(2.8), 149(27.1),
121(2.6), 105(1.1), 91(6.1) and 77(1.6); FAB-MS, m/z
(% rel. int.): 297 [M+H]+ (100).
7-Butanoyloxy-4-methyl-2H-1-benzopyran-2-one
(59).
White solid, mp 92–93 ꢂC; IR (KBr): 2977, 2364, 1754,
1731, 1616, 1571, 1420, 1386, 1371, 1262, 1148, 1091,
1061, 983, 926, 873, 857, 817, 741, 614 and 454 cmꢀ1
;
UV (MeOH): 312, 274 and 210 nm; HR-MS, C14H14O4
(M+ 246.0879, calcd 246.0892); 1H NMR (300 MHz,
CDCl3): d 1.62 (3H, t, CH2CH2CH3), 1.85 (2H, m,
CH2CH2CH3), 2.48 (3H, s, C-4CH3), 2.64 (2H, t,
CH3CH2CH2C¼O), 6.28 (1H, s, H-3), 7.11 (2H, m, H-6
and H-8) and 7.65 (1H, d, J=8.5 Hz, H-5); 13C NMR
(75 MHz, CDCl3):
(CH2CH2CH3),
d
18.68 (C-4CH3), 36.06
13.53 (CH2CH2CH3), 18.24
2,4-Dihydroxy-3-(3-methyl-2-butenyl)phenyl benzyl ke-
tone (71). White solid, mp 103–104 ꢂC; IR (KBr): 3286,
2922, 1620, 1496, 1437, 1362, 1246, 1167, 1100, 1044,
848, 772 and 700 cmꢀ1; UV (MeOH): 215, 285, 318 and
326 nm; HR-MS, C19H20O3 (M+ 296.1418, calcd
(CH3CH2CH2C¼O), 110.38 (C-8), 114.37 (C-10),
117.67 (C-6), 118.07 (C-3), 125.27 (C-5), 151.89 (C-4),
153.08 (C-9), 154.07 (C-7), 160.48 (C-2) and 171.41
(C¼O); EI–MS, m/z (rel. int.): 246 [M]+ (15), 177(25),
176(100), 148(45), 147(12), 91(17), 86(38), 84(64),
71(88), 65(12), 51(36) and 49(83).
1
296.1412); H NMR (250 MHz, CDCl3): d 1.81 (6H, s,
2ÂCH3), 3.42 (2H, d, J=7.18 Hz, –CH2–CH¼), 4.20
(2H, s, –COCH2–), 5.24–5.27 (1H, m,=CH–CH2–),
6.28 (1H, s, C-4 OH), 6.36 (1H, d, J=8.85 Hz, H-5),
7.22–7.35 (5H, m, H-20, H-30, H-40, H-50 and H-60), 7.64
(1H, d, J=8.88 Hz, H-6) and 13.0 (1H, s, C-2 OH); 13C
NMR (62.8 MHz, CDCl3): d 17.88 and 25.77 (2ÂCH3),
29.88 (–CH2–CH¼), 44.78 (–COCH2–), 107.91 (C-5),
117.25 [(CH3)2C¼CH2–], 121.71 (C-3), 127.0 (C-40),
128.22 (C-30 and C-50), 128.70 (C-20 and C-60), 130.07
(C-6), 130.26 (–CH2–CH¼), 134.58 (C-10), 135.55 (C-1),
161.63 (C-2), 163.15 (C-4) and 202.18 (C¼O); EI–MS,
m/z (% rel. int.): 296 [M]+ (38.4), 279(2.5), 206(14.2),
205(97.7), 161(3.5), 150(10.0), 149(100.0), 105(2.0),
91(5.1) and 65(3.5).
5,7-Dibutanoyloxy-4-methyl-2H-1-benzopyran-2-one (60).
White solid, mp 79–80 ꢂC; IR (KBr): 3446, 3080, 2932,
1760, 1724, 1617, 1453, 1419, 1361, 1295, 1141, 1060,
1003, 925, 855, 747, 701 and 533 cmꢀ1; UV (MeOH):
284, 279 and 208 nm; HR-MS, C18H20O6 (M+
332.1252, calcd 332.1260); 1H NMR (300 MHz, CDCl3):
d
1.10 (6H, m, 2ÂCH2CH2CH3), 1.82 (4H, m,
2ÂCH2CH2CH3), 2.54 (3H, s, C-4CH3), 2.64 (4H, m,
2ÂCH3CH2CH2–C¼O), 6.20 (1H, s, H-3), 6.84 (1H, d,
J=2.0 Hz, H-6) and 7.12 (1H, d, J=2.0 Hz, H-8); 13C
NMR (75 MHz, CDCl3);
d
13.52 and 13.59
(2ÂCH2CH2CH3), 17.95 and 18.15 (2Â CH2CH2CH3),
22.82 (C-4CH3), 36.04 and 36.29 (2ÂCH3CH2CH2–
C¼O), 108.38 (C-8), 111.45 (C-10), 113.53 (C-6), 116.04
(C-3), 148.23 (C-4), 150.67 (C-9), 152.23 (C-5), 155.06
(C-7), 159.46 (C-2), 170.89 and 171.29 (2 Â C¼O); CI-
MS, m/z (rel. int.): 333 [M+1]+ (100), 263(9), 247(6),
193(2), 177(3), 105(19), 88(58), 86(6), 72(4) and 58(5).
2,4-Dihydroxy-3,5-di(3-methyl-2-butenyl)phenyl benzyl
ketone (72). Green needles, mp 102–103 ꢂC; IR (KBr):
3314, 2923, 1628, 1579, 1460, 1356, 1277, 1217, 1164,
1131, 1055, 962 and 727 cmꢀ1; UV (MeOH): 227, 289
and 336 nm; HR-MS, C24H28O3 (M+ 364.2029, calcd
1
364.2038); H NMR (250 MHz, CDCl3): d 1.73 (6H, 2s,
5,6,7-Trimethoxy-4-methyl-2H-1-benzopyran-2-one (61).
White solid, mp 116–117 ꢂC, IR (KBr): 2942, 1720,
1599, 1552, 1444, 1405, 1380, 1359, 1294, 1201, 1154,
2ÂCH3), 1.80 (6H, brs, 2ÂCH3), 3.24 and 3.41 (2d, 2H
each, J=7.25 and 7.13 Hz, 2–CH2–CH¼), 4.19 (2H, s,
–COCH2–), 5.19–5.31 (2H, m, 2Â=CH–CH2–), 6.21
(1H, s, C-4 OH), 7.24–7.36 (5H, m, H-20, H-30, H-40, H-
50 and H-60), 7.47 (1H, s, H-6) and 13.00 (1H, s, C-2
OH); 13C NMR (62.8 MHz, CDCl3): d 17.80, 17.88 and
21.82 (4ÂCH3), 25.80 and 28.53 (2–CH2–CH¼), 45.06
(–COCH2–), 112.59 and 114.10 [2Â(CH3)2C¼CH–],
118.99 (C-6), 121.24 (2¼CH–CH2–), 121.52 (C-3 and
C-5), 126.93 (C-40), 128.68 (C-30 and C-50), 129.22 (C-20
1107, 1076, 1017, 990, 916, 867, 819, 739 and 622 cmꢀ1
;
UV (MeOH): 320, 230 and 211 nm; 1H NMR
(300 MHz, CDCl3): d 2.87 (3H, s, CH3), 3.87, 3.94 and
3.98 (9H, 3s, 3Â-OCH3), 6.05 (1H, s, H-3) and 6.65 (1H,
s, H-8); 13C NMR (75 MHz, CDCl3): d 22.94 (CH3),
56.12, 60.90 and 61.27 (3ÂOCH3), 96.11 (C-8), 107.98
(C-10), 112.90 (C-3), 139.13 (C-4), 151.24 (C-9), 151.56,

928
V. S. Parmar et al. / Bioorg. Med. Chem. 11 (2003) 913–929
and C-60), 134.84 (C-10), 135.34 (C-1), 160.09 (C-2),
161.57 (C-4) and 202.12 (C¼O); EI–MS, m/z (% rel.
int.): 364 [M]+ (43.9), 347(2.7), 309(3.1), 274(20.7),
273(100.0), 271(4.9), 218(8.5), 217(55.6), 205(4.8),
162(4.7), 161(44.6), 149(8.0) and 91(6.2)
218(3.1), 217(20.5), 177(2.2), 175(3.2), 163(14.0),
138(2.5), 119(1.3), 118(1.2), 105(1.3), 91(5.6) and
77(1.2).
Acknowledgements
3,4-Dihydro-7-hydroxy-2,2-dimethyl-6-phenylacetyl-
2H-1-benzopyran (73). White solid, mp 110–112 ꢂC; IR
(Nujol): 2924, 1639, 1463, 1377, 1347, 1255, 1170, 1150,
1117, 984 and 736 cmꢀ1; UV (MeOH): 236, 285 and
327 nm; 1H NMR (250 MHz, CDCl3): d 1.34 (6H, s,
2ÂCH3), 1.81 (2H, t, J=6.7 Hz, H-3), 2.72 (2H, t,
J=6.7 Hz, H-4), 4.19 (2H, s, –COCH2–), 6.31 (1H, s, H-
8), 7.25–7.33 (5H, m, H-20, H-30, H-40, H-50 and H-60),
7.55 (1H, s, H-5) and 12.30 (1H, s, C-7 OH); 13C NMR
(62.8 MHz, CDCl3): d 21.75 and 26.95 (2ÂCH3), 32.68
(C-3 and C-4), 44.64 (COCH2), 77.51 (C-2), 104.81 (C-
8), 112.82 and 113.23 (C-10 and C-10), 126.97 (C-40),
128.67 (C-20 and C-60), 129.35 (C-30 and C-50), 131.90
(C-5) and 134.60 (C-6), 161.48 (C-9), 163.38 (C-7) and
201.62 (C¼O); EI–MS m/z (% rel. int.): 296 [M]+ (8),
204(100), 149(22), 121(4) and 44(12).
We thank Rita Colman and Arlette Verspeelt for tech-
nical assistance with the invasion assays, Georges De
Bruyne for immunohistochemistry, Chris Dragonetti
and Jean Roels Van Kerckvoorde for preparing the
illustrations and Dr. Oliver Seitz (Max-Planck-Institute,
Dortmund, Germany) for his assistance in getting the
HRMS of our compounds recorded. We also thank the
Danish International Development Agency (DANIDA,
Denmark), Council of Scientific and Industrial
Research (CSIR, New Delhi, India), Fonds voor
Wetenschappelijk Onderzoek and Sportvereniging tegen
Kanker (Brussels, Belgium) for financial assistance.
References and Notes
(ꢃ)-3,4-Dihydro-3,7-dihydroxy-2,2-dimethyl-6-phenyl-
acetyl-2H-1-benzopyran (74). White solid, mp 97–98 ꢂC;
IR (KBr): 3524, 2928, 1643, 1491, 1356, 1302, 1235,
1128, 1064, 950 and 728 cmꢀ1; UV (MeOH): 236, 284
and 395 nm; HR-MS, C19H20O4 (M+ 312.1349, calcd
312.1362); 1H NMR (300 MHz, CDCl3): d 1.35 and 1.37
(6H, 2s, 2ÂCH3), 2.74 (1H, dd, J=4.80 and 15.97 Hz,
H-4a), 3.03 (1H, dd, J=4.80 and 16.65 Hz, H-4b), 3.84
(2H, brs, C-3 OH and C-3H), 4.20 (2H, s, –COCH2–),
6.03 (1H, s, H-8), 7.27–7.32 (5H, m, H-20, H-30, H-40, H-
50 and H-60), 7.57 (1H, s, H-5) and 12.30 (1H, s, C-7
OH); 13C NMR (75.5 MHz, CDCl3): d 22.02 and 24.15
(2ÂCH3), 30.49 (C-4), 44.71 (–COCH2–), 69.28 (C-3),
78.31 (C-2), 98.18 (C-8), 110.72 (C-10), 126.36 (C-10),
126.99 (C-40), 128.68 (C-30 and C-50), 129.32 (C-20 and
C-60), 132.66 (C-5), 134.50 (C-6), 163.48 (C-7), 166.48
(C-9) and 201.73 (C¼O); EI–MS, m/z (% rel. int.): 312
[M]+ (15.3), 297(1.3), 279(1.1), 253(2.8), 222(13.8),
221(100.0), 204(2.4), 203(17.4), 163(3.9), 161(3.3),
150(1.6), 149(10.4), 122(1.9), 105(1.5), 91(4.9) and
77(1.1).
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