In vitro antifungal evaluation and structure-activity relationships of a new series of chalcone derivatives and synthetic analogues, with inhibitory properties against polymers of the fungal cell wall

Article abstract of DOI:10.1016/S0968-0896(01)00116-X

Here we report the synthesis, in vitro antifungal evaluation and SAR study of 41 chalcones and analogues. In addition, all active structures were tested for their capacity of inhibiting Saccharomyces cerevisiae β(1,3)-glucan synthase and chitin synthase,

Full text of DOI:10.1016/S0968-0896(01)00116-X

Bioorganic & Medicinal Chemistry 9 (2001) 1999–2013
In Vitro Antifungal Evaluation and Structure–Activity
Relationships of a New Series of Chalcone Derivatives and
Synthetic Analogues, with Inhibitory Properties Against
Polymers of the Fungal Cell Wall
a
a
a,
b
Silvia N. Lo
´
pez, Marı
´
a V. Castelli, Susana A. Zacchino, * Jose
´
c
N. Domı
´
nguez,
s, Juan C. Ribas,
d
b
b
c
Gricela Lobo, Jaime Charris-Charris, Juan C. G. Corte
´
d
d
Cristina Devia, Ana M. Rodrıguez and Ricardo D. Enriz
´
aFarmacognosia, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario,
Suipacha 531, (2000) Rosario, Argentina
b
Laboratorio de Sı´ntesis Org a´ nica, Facultad de Farmacia, Universidad Central de Venezuela,
Apartado 40109, Nva. Granada, (1040) Caracas, Venezuela
c
Instituto de Microbiologı´a Bioquı´mica, Campus ‘Miguel de Unamuno’, Edificio Departamental #222,
C.S.I.C/Universidad de Salamanca, (37007) Salamanca, Spain
Quı´mica General, Facultad de Quı´mica, Bioquı´mica y Farmacia, Universidad Nacional de San Luis,
Chacabuco y Pedernera, (5700) San Luis, Argentina
d
Received 29 November 2000; accepted 5 March 2001
Abstract—Here we report the synthesis, in vitro antifungal evaluation and SAR study of 41 chalcones and analogues. In addition,
all active structures were tested for their capacity of inhibiting Saccharomyces cerevisiae b(1,3)-glucan synthase and chitin synthase,
enzymes that catalyze the synthesis of the major polymers of the fungal cell wall. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
agents, which overcome the above disadvantages. The
search is oriented to find new antifungal drugs, which
may selectively attack the fungi without inhibiting any
biochemical system of the host. Since fungal but not
mammalian cells possess a cell wall, its inhibition
represents an ideal mode of action for antifungal
Modern medicine has greatly increased the number of
immunocompromised patients. Chemotherapy, par-
enteral nutrition, transplantation surgery and the use of
broad-spectrum antimicrobial agents, added to the
occurrence of the acquired immunodeficiency syndrome
5ꢀ8
drugs.
(
who are highly susceptible to opportunistic infections.
AIDS), render true ‘livingPetri dishes’ individuals,
1
Duringthe course of our screeningpro gr am aimed to
detect selective antifungal compounds,
9ꢀ13
Systemic and dermic fungal infections are the cause of
great morbi-mortality in this type of patients, being
dermatomycoses a serious problem for children of the
Third World Nations as a consequence of poor sanita-
we report
here the antifungal activities, structure–activity rela-
tionships (SARs) and capacity of inhibitingthe in vitro
synthesis of the major fungal cell wall polymers, for a
series of chalcone derivatives, most of them not descri-
bed to date in the literature.
2
,3
tion care, too.
Since many of the currently available drugs have unde-
sirable side effects, are ineffective against new or re-
emerging fungi, or develop a rapid resistance, there is
an urgent need of a next generation of new antifungal
Previous studies on the antifungal properties for these
14,15
4
compounds, have shown
that some phenolic syn-
thetic chalcones, possess moderate antifungal proper-
ties. Conversely, two phenolic chalcones isolated from
1
6
*Correspondingauthor. E-mail: szaab gi l@citynet.net.ar
Myrica serrata did not display any activity against
0
968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00116-X

2000
S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
Candida albicans. In addition, a short series of chalcone
derivatives showed marginal antifungal activities not
improved with structural modifications.
Results and Discussion
Chemistry
1
7
We prepared a number of known and novel chalcone
derivatives by a Claisen–Schmidt condensation2 of the
appropriate aldehyde and acetophenone derivatives
(Schemes 1–3). After purification, chalcones were
obtained in 60–95%. Ketones 23–33 (Table 3) were
prepared in turn, by means of the 1,4 addition of an
anion enolate to the conjugated system as it is shown in
Scheme 3. This method allows us to prepare a wide
variety of substituted compounds on one or both rings.
0
Additional study of the antifungal properties of chal-
cone derivatives usinga lar ge number of compounds
seems to be necessary, and will provide information on
the SARs of this kind of compounds. Forty-one samples
(1–41, Tables 1–4) were tested against a panel of human
opportunistic pathogenic fungi, using the agar dilution
method. Based on the results obtained in this screen, a
SAR study includingconformational analysis, was per-
formed with the aim of explainingthe influence of
chalcone structural changes on the antifungal activity.
2
1ꢀ28
Some of them had been reported in the literature.
Their structures were established usingIR, NMR spec-
troscopy and mass spectrometry. NMR analysis of all
the chalcones obtained, showed that the trans isomer
In addition, the mechanism of action of most active
chalcones was assayed for their capacity of inhibitingin
vitro b(1,3)-glucan and chitin synthases, enzymes that
catalyze the biosynthesis of b(1,3)-glucan and chitin
2
9
was formed.
All the startingmaterials are commercially available
except 2-chloro-3-formyl-6,7-dimethoxyquinoline. It was
1
8,19
polymers of the fungal cell wall,
respectively.
Table 1. MIC values of chalcone derivatives actinga ga inst dermatophytes
MIC (mg/mL)
0
M. c.i
M. g.j
T. m.k
T. r.l
E. f.m
Compd
R
R
Mp
ꢁ
(
C)
1
2
3
4
5
6
7
8
9
H
4-NO
2-NO
2,4-Cl
3-CH
4-CH
H
H
H
H
H
H
H
H
H
55–57a
165–166
1.5
0.75
12.5
12.5
3
1.5
1.5
6.25
>50
3
1.5
1.5
6.25
50
3
3
1.5
3
3
6.25
>50
>50
50
>50
25
25
25
3
3
0.75
12.5
12.5
6.25
12.5
6.25
3
6.25
6.25
0.75
0.25
3
12.5
1.5
1.5
0.75
3
b
2
2
111–112
210
54–56
2
3
O
O
115–116c
84–86
3
3
12.5
1.5
3
4-CH
3
d
3,4-(CH
2,4-(CH
2,3-(CH
2,4-Cl
4-CH
3,4-(CH
3
O)
2
2
2
85–87
71–72
Oile
12.5
3
6.25
>50
>50
25
>50
3
25
50
12.5
6.25
6.25
>50
12.5
>50
>50
50
25
50
1.5
6.25
6.25
3
3
O)
O)
3
10
11
12
13
14
15
16
17
18
H
6.25
>50
12.5
25
>50
0.5
3
12.5
0.5
0.3
25
0
0
2
2 ,4 -Cl
0
2
215–216
95–96
102–104
>50
3
4 -CH
0
3
>50
50
>50
50
3
50
3
25
15
0
3
O)
2
3 ,4 -(CH
0
3
O)
O)
2
0
0
0
f
3,4,5-(CH
3
O)
3
3 ,4 ,5 -(CH
3
3
130–131
g
4-CH
2,3-(CH
4-N(CH3)
3-CH
3
O
4 -CH
0
3
93–94
110–112
3
O)
2
4 -Br
3 ,4 -(CH
0
0
h
2
3
O)
2
83–85
84–85
0
3
O
4 -Br
25
>50
15
n
Amp
o
6.25
12.5
Ket
a
Lit. 56–57.3
b
4
Lit. 164–165.
Lit. 114–116.
c
4
d
5
Lit. 88.
.
Lit. oil.
.
h
e
f
6
g 7
Lit. 83–84.8
i
j
Microsporum canis C 112.
Microsporum gypseum C 115.
k
l
Trichophyton mentagrophytes ATCC 9972.
Trichophyton rubrum C 113.
m
Epidermophyton floccosum C 114.
Amp, Amphotericin B.
Ket, ketoconazole.
n
o

S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
2001
obtained by the acetylation of 3,4-dimethoxyaniline in
pyridine with acetyl chloride at room temperature, to
give 3,4-dimethoxyanilide, which in turn was made to
trast, strongantifun ag l effects were obtained for the
compounds of the series against dermatophytes. These
results are shown in Tables 1 and 2. In particular com-
pounds 1–3, 5–10, and 18, showed strongantifun ag l
activities comparable to those of Amphotericin B and
Ketoconazole.
2
1
react with the Vilsmeier reagent (Scheme 2).
Antifungal assays
To carry out the antifungal evaluation, concentrations
of chalcones up to 50 mg/mL were incorporated to
growth media according with reported procedures.
The agar dilution method showed than none of the
compounds tested was active against the yeasts C. albi-
cans, Saccharomyces cerevisiae or Cryptococcus neofor-
mans nor against the filamentous fungi Aspergillus niger,
A. fumigatus or A. flavus (results not shown). In con-
As main observations, it can be stated that all the tested
dermatophytes were inhibited at 50 mg/mL and some of
them at lower concentrations. The most sensitive species
was Epidermophyton floccosum. Since dermatophytes
are a group of fungi which characteristically infect the
keratinized areas of the body and dermatomycoses are
very difficult to eradicate, it is very interestingto note
that chalcone derivatives showed activity against der-
matophytes and not against another type of fungi.
1
0
Table 2. MICs values of analogues of chalcone derivatives acting
against dermatophytes
To evaluate the SARs, the effects of structural changes
in three regions of the molecule of the non-substituted
chalcone 1, were considered: variation in the A and B
rings, and in the enone linkage (see general structure in
Table 1).
Regarding the influence of the substituents on the B
ring, an interesting structure–activity correlation can be
observed: electron-donating gr oups tended to weaken
the antifungal activity (compare MICs of compounds 5,
MIC (mg/mL)
Compd
A
R
M.c.a M.g.b T.m.c T.r.d
E.f.e
6
, 8–10 with MICs of chalcone 1). Electron-withdrawing
0
0
1
9
0
2 ,4 -Cl
12.5 12.5 12.5 6.25 12.5
groups in the para-position increased the potency: a
nitro substituent (compound 2) enhance 2–4 times the
activities in three of the fungi tested (compare results for
compounds 2 and 1 in Microsporum canis, Trichophyton
rubrum and E. floccosum). Nevertheless, when the NO2
group is in position 2 (chalcone 3), a decrease of activity
is observed with respect to compounds 1 and 2 (4–8 and
2
H
>50 >50 >50 >50
>50 >50 >50 >50
>50 >50 >50 >50
12.5
0
0
2
1
2
2 ,4 -OCH
3
>50
4–16 times, respectively), suggesting that the presence of
0
2
3 -OCH
3
>50
a group in the o-position of the B ringcould introduce
important steric effects. The low activity showed by
compound 4 is in agreement with this assumption.
However, when an OCH is added to C-2 of compound
3
a
Microsporum canis C 112.
M. gypseum C 115.
Trichophyton mentagrophytes ATCC 9972.
6
(!9), the activity did not change significatively (com-
b
c
pare MICs of compounds 6 and 9). Molecular model-
lingstudies performed with these compounds, are
described in the section below and provide some expla-
nations for these last findings.
d
T. rubrum C 113.
Epidermophyton floccosum C 114.
e
Table 3. Ketones tested in vitro for antifungal properties
Compda
X
Y
R
mp ( C)
ꢁ
23
24
25
26
27
28
29
30
31
32
33
C
2,3-(OCH
6
H
5
C
C
C
C
C
C
6
6
6
6
6
6
H
5
5
5
5
5
5
CH(CO
CH(CO
CH(CO
2
2
2
CH
CH
CH
3
3
3
)
2
)
2
)
2
95–96
3
)
2
C
6
H
H
3
3
H
H
H
H
H
135–136
129–130
75–77
133–135
154–156
130–131
125–127
115–116
128–130
110–112
Naphthyl
2,3-(OCH
2,4-(Cl)
3
)
2
C
6
–CH(CN)
–CH(CN)
–CH(CN)
–CH(CN)
–CH(CN)
–CH(CN)
–CH(CN)
–CH(CN)
2
2
C
6
H
3
2
2
2
2
2
2
2
2-Naphthyl
2-Naphthyl
2-Naphthyl
0
0
3 ,4 -(OCH
2 ,4 -(OCH
3
)
)
2
2
C
C
6
H
H
3
3
0
0
3
6
2-Chloro-3-quinolinyl
2-Chloro-3-quinolinyl
3,4,5-(OCH
6 5
C H
0
0
3 ,4 -(OCH
0
3
)
2
C
6
H
3
0
0
3
)
3
C
6
H
2
3 ,4 ,5 -(OCH
3
)
3
C
6
H
2
a
Compounds 23–33 inactive in all of the fungi tested up to 50 mg/mL.

2002
S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
We investigated the role played by ring B in the anti-
fungal activity of chalcone 1 by replacingbenzene with
alternative ringsystems. We found that the replacement
of benzene in compound 1 by naphtalene (!20) and by
substituted quinolines (!34 and 37) resulted in an
almost complete loss of activity.
The lack of activity of compound 11, possessingCl in
the ortho positions in both A and B rings (compare with
compound 4), suggested that the steric factors play a
crucial role in the antifungal properties of substituted
chalcones.
It is interestingto note that compound 14 do not inhibit
the fungal cell growth up to 50 mg/mL, while other tri-
methoxy A substituted ringchalcones were reported as
Several compounds were prepared in which both the B
and A rings of chalcone 1 were altered. From the var-
iation of the A ringin compounds 5 (!18), 6 (!15), 7
3
0
inhibitors of the human cell growth in vitro.
(
!12), 8 (!13), and 10 (!16), we can state that: elec-
tron-donating gr oups on ringB such as –OCH
or-CH
3 3
New analogues with furane, naphtalene and quinoline
as ringB (compounds 19, 21, 22, 35, 36, and 38–41)
were tested in order to add more information to the
structural requirements for activities. All of them were
inactive except compound 19. This structure possessing
a furane ring, showed a moderate but significant anti-
fungal activity against dermatophytes with MICs
between 6.25 and 12.5 mg/mL.
diminished the activity (compare MICs of compounds 7
and 12, 8 and 13); the same tendence is also observed
for compound 15 (compare its MICs with those of
chalcone 6 for Microsporum gypseum, Trichophyton
mentagrophytes, and T. rubrum). In addition, structure
1
7 possessed very low activity and compound 14 was
devoid of antifungal properties. The introduction of an
electron-withdrawingr go up on ringA (compounds
5
!18 and 10!16) does not introduce clear changes in
To gain insight into the importance of the enone linkage
for the antifungal activity, we broke the double bond a,
b to the carbonyl group, obtaining compounds 23–33
the antifungal effects, although they still possess sig-
nificant antifungal activities.
(Table 3). Since these ketones are devoid of antifungal
properties, the enone appears to be required for activity.
However, the lack of activity obtained for compounds
Table 4. Analogues of chalcone derivatives tested for antifungal
properties
11, 14, 20–22, 34–41, clearly indicates that the presence
of a enone linkage would be a structural requirement
necessary but not by itself sufficient for the antifungal
activity.
Compda
ꢁ
Conformational and electronic study
X
Y
R
mp ( C)
In order to understand the experimental results
obtained in agar dilution assays, a computer-assisted
conformational study were made on some selected
chalcones (compounds 1–3, 4, and 11). Calculations
reveal that the energies of rotamers depend essentially
on the torsional angles f and y (Fig. 1).
34
35
36
37
38
39
40
41
H
H
H
OCH
OCH
OCH
OCH
OCH
H
H
H
OCH
OCH
OCH
OCH
OCH
C
6
H
5
200–201
163–165
181–182c
0
0
b
2 ,4 -(OCH C
2 ,4 -Dimethyl-furyl
3
)
2
6
H
3
0 0
d
3
3
3
3
3
3
3
3
3
3
C
6
H
5
129–131
0
0
6 4
4 -BrC H
0
6 4
4 - ClC H
0
0
0
0
3 ,4 -(OCH
2 ,5 -(OCH
3
)
)
2
C
C
6
H
H
3
3
214–215
>150 desc.
3
2
6
Energy profiles of chalcone 1 in two different levels of
theory (AM1 and RHF/3-21G) are given in Figures 2
and 3. They show the influence of ringorientations on
the potential energy of the rotamers. Figure 2 shows
a
Compounds 34–41 inactive in all of the fungi tested at 50 mg/mL.
b
9
Lit. 163–165.
c
9
9
Lit. 181–182.
d
Lit. 129–131.
Scheme 1. Synthesis of chalcones and related compounds: (a) NaOH, MeOH, rt; (b) MeONa, MeOH, rt.

S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
2003
that for f, both levels of theory predict two preferred
the most noticeable feature is the different conforma-
tional behavior obtained for compounds possessingan
ortho-substituent of significant size on ring B (com-
pound 3) with respect to that with the same substituent
in the para position (chalcone 2) and without sub-
stituents (compound 1). The presence of the nitro sub-
stituent in the ortho position, introduces an important
ꢁ
conformations. Those with fffi0 and 180 are the lowest
in energy, confirming that planar forms are favoured in
comparison with other conformations. It is clear that
the energy barriers between the conformers, are com-
pletely different in function of the levels of theory used.
When the minima obtained by the scans, were opti-
mized at three different levels of theory (AM1, RHF/3-
ꢁ
steric hindrance (see the high energetic barrier at 180
for compound 3), suggesting a non-planar structure
2
1G and RHF-6-31G), AM1 calculations predict
f=18.65; RHF/3-21G: f=0.07 and RHF-6-31G:
f=6.99, respectively (Table 5). This difference is even
more noticeable when observingthe results obtained for
the torsional angle y (Fig. 3 and Table 5). Semi-empiri-
cal calculations indicate that the rotation of the A ring
is poorly restricted (low energy barrier).
In contrast RHF/3-21G calculations predict a very high
energy barrier suggesting that the planar forms are
ꢁ
highly preferred (y=0.02 ). RHF/6-31G calculations in
ꢁ
turn, predicts y=19.99 . From the above results with all
Figure 1. General structure of the chalcones showingthe torsional
angles f and y.
levels of theory, it is evident that we are dealingwith a p
electron delocalized system, therefore with a highly
conjugated molecule, and hence with planar forms.
Nevertheless, it seems that while AM1 calculations
undervalue the single-double character of several C–C
bonds in chalcones, RHF/3-21G calculations over-
stimate the delocalization in this structure. In turn,
RHF/6-31G calculations which give intermediate values
(
X-ray results reportinga planar conformation too,
Table 5), are in complete agreement with experimental
3
1
ꢁ
with f and y in ffi7 and ffi20 , respectively.
Different AM1 energy profiles obtained by rotating
angles f or y in compounds 1–3, 4, and 11 are described
in the followingpara gr aph. In order to obtain a better
degree of accuracy, the minima obtained for each com-
pound which the AM1 scan were optimized at RHF/3-
2
1G and RHF/6-31G levels of theory.
Figure 4 compares the different scans of compounds 1–4
when the B ringis rotated (modification of f). Herein,
Figure 2. Conformation potential energy curves of compound 1
obtained from AM1 and RHF/3-21G relaxed single scan computa-
tions. Torsional angle f measures the rotation about the B-ring.
Scheme 2. Synthesis of 2-chloro-3-formyl-6,7-dimethoxyquinoline: (a)
Py, AcCl, rt; (b) Vilsmeier reaction.
Figure 3. Conformation potential energy curves of compound 1
obtained from AM1 and RHF/3-21G relaxed single scan computa-
tions. Torsional angle y measures the rotation about the A-ring.
Scheme 3. Preparation of ketones: (a) CH
2 2 3 2
(CO CH ) , MeONa,
MeOH, rt; (b) CH (CN) , MeONa, MeOH, rt.
2
2

2004
S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
which would account for its lower antifungal activity
respective of chalcone 1. Compound 4, with a chloro
atom in the o-position show the same profile as com-
pound 3 confirmingthe steric hindrance produced by a
bulky substituent. Nevertheless, the presence of a meth-
oxy group in position 2 of ring B (compounds 9 and 10
energy profiles not shown) do not show a steric hin-
drance but a closely related conformational behavior to
those of compounds 1 and 2 (Table 5), indicatingthat
planar forms are the lowest energy conformations for
chalcones 9 and 10 in contrast to compound 3. This can
be explained by the fact that the methoxy group pos-
sesses a very high molecular flexibility and therefore it
can adopt an adequate spatial orderingwithout losing
the planarity.
regions with the lowest values are indicated in ‘red’.
Our results indicate that the derivatives taken as
models for this study (chalcones 1, 2, and 6) have
two highly positive blue areas (b- and carbonyl car-
bons), which provide rich sites for nucleophilic
attacks.
The MEP energy isosurfaces of derivatives 1, 2, and 6
are shown color-coded in the range from ꢀ60 (deepest
red) to 40 kcal/mol (deepest blue) in Figure 7. The
highest electrostatic potentials also reveal that the most
positive centers (blue areas) lie near to the b- and car-
bonyl carbons. The decrease or enhancement of anti-
fungal activity of chalcones when they possess donating
or attracting groups respectively, are in agreement with
these electronic studies.
Figure 5 compares the AM1 scans obtained for com-
pounds 4 and 11 when A ringis rotated. While the
conformational behavior of compound 4 (without sub-
stituents on ringA) is very similar to that of chalcone 1
The general picture emerging from the data reported
here, is that planar forms in chalcones, are the most
adequate spatial orderingneeded to produce the anti-
fungal response, being b- and carbonyl carbons the
most susceptible sites toward a possible nucleophilic
attack of the enzyme or receptor.
(
of compound 11 (possessinga chloro atom in the o-
Fig. 3) when the angle y is modified, the conformations
ꢁ
energy, suggesting the loss of planarity.
position of ringA) at yffi0 and 180 are very high in
Regarding the correlation of the antifungal activity of
compounds 1–3, 4, and 11 with the planarity of their
molecules, a decrease of activity was observed for these
compounds with a bulky ortho substituent on ringB
(
compounds 3 and 4) respective to the non-substituted
chalcone 1. The additional presence of ortho sub-
stituents on ringA, which affect the planarity too, con-
ducts to a total loss of activity (compare MICs of
chalcones 1, 4, and 11 against the fungi tested). The lack
of antifungal activity of compound 11 add new evidence
to the importance of planar forms as a conformational
requeriment of chalcones for their antifungal effects.
To rationalize a possible site of action at the electronic
level for these chalcones, we have performed an
electronic analysis. We used the Lowest Unoccupied
Molecular Orbital (LUMO) and Molecular Electro-
static Potential (MEP) which delineate electron deficient
areas which could be critical to the possible mechanism
of action.
Figure 4. A comparison of potential energy curves (PEC) for the tor-
sional angle f of compounds 1, 2, 3, and 4 each calculated from AM1
calculations.
On the map in Figure 6, regions with the highest abso-
lute value of LUMO are indicated in ‘blue’, while
Table 5. Torsional angles obtained for compounds 1–4, 9, 11 by
usingdifferent levels of theory
Compd
AM1
HF/3-21 G
Torsional
HF/6-31 G
Torsional
Torsional
ꢁ
ꢁ
ꢁ
angles ( )
angles ( )
angles ( )
f
y
f
y
f
y
1
2
3
4
9
18.65
0.39
43.74
34.54
17.54
31.98
33.49
0.43
26.85
31.46
33.97
119.03
0.07
0.46
47.13
34.01
10.28
31.06
0.02
0.75
7.67
6.19
0.10
6.99
16.21
42.11
33.30
17.24
30.7
19.99
18.34
18.80
8.63
20.33
132.89
Figure 5. A comparison of potential energy curves (PEC) for the tor-
sional angle f of compounds 4 and 11 each calculated from AM1
calculations.
11
138.9

S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
2005
Mode of action studies
and 18) are very weak inhibitors of this enzyme
IC >0.5 mg/mL). Nevertheless, chalcones 1 and 10 and
(
5
0
To gain insight into the mode of action of chalcone
derivatives, we tested the most active antifungal com-
pounds (structures 1–3, 5–10, 18 and 19) for their capa-
city of inhibitingin vitro the S. cerevisiae b(1,3)-glucan
synthase or chitin synthase, enzymes that catalyzes
the synthesis of the major polymers of the fungal cell-
wall, b(1,3)-glucan and chitin respectively. Results on
the in vitro assays are listed in Table 6.
furane derivative 19, showed interestinginhibitory
activities (0.144IC 40.44 mg/mL), beingchalcone
1
5
0
(IC =0.14 mg/mL) a stronginhibitor of b(1,3)-glucan
5
0
synthase, comparable to the known b(1,3)-glucan syn-
thase inhibitors Papulacandin B (IC =0.10 mg/mL) and
3
2
33
5
0
Aculeacin A (IC >0.50 mg/mL).
5
0
Figure 10 shows the comparative inhibitory activities of
the most active compounds against b(1,3)-glucan and
chitin synthases as a measure of percent of residual
activities for each enzyme. An analysis of this graphic
clearly shows that chalcones are much better inhibitors
of chitin synthase than of glucan synthase, suggesting
that they could be selective inhibitors of that enzyme.
This could be a very interestingfeature in the possible
development of these compounds as antifungal agents.
Regarding activity against chitin synthase-1, all the
compounds tested exhibited inhibitory activities at
2
0 mg/assay (% of inhibition ranging from 15 to 95%).
Since compounds 1, 5–10, 18, and 19 showed inhibitions
50%, serial dilutions of them (1, 2, 5, 10, and 20 mg/
5
assay) were tested for enzyme inhibition and their aver-
age inhibitory effect was calculated (Figs 8 and 9). The
IC50 values were all 40.22 mg/mL, beingcompound
the most active one with 94% inhibition at 20 mg
IC =0.02 mg/mL).
1
Although very active in cellular assays, chalcones 2 and
3 seem not to act by inhibitingthe synthesis of the major
polymers of the fungal cell wall (Table 6 and Fig. 9).
(
5
0
Results obtained from glucan synthase assays, showed
that most of chalcone derivatives (compounds 2, 3, 5–9,
Alternatively, since the crude enzymes were prepared
from S. cerevisiae, the species difference could account
for a lack of a strict correlation between the activities
shown by the compounds in the cellular and enzymatic
assays.
Conclusions
In an attempt to generate novel antifungal structures
with significant activities against human opportunistic
fungi but less toxic than those in use nowadays, we have
tested a series of chalcone derivatives and analogues, most
of them new compounds, with agar dilution methods.
Accordingwith the results obtained in these assays,
chalcones with different substituents on rings A and B,
showed to be active against dermatophytes and not
against another group of fungi. This type of fungi are
responsible for dermatomycoses, a type of infections
very difficult to eradicate.
Figure 6. The calculated LUMO for compounds 1, 6, and 2.
A detailed SAR study supported by theoretical calcula-
tions, aided to identify and understand the structural
requirements for the antifungal action of chalcones and
its derivatives.
Regarding their mode of action, active compounds were
assayed for their in vitro capacity of inhibiting b(1,3)-
glucan and chitin synthases, enzymes that catalyze the
synthesis of the two major polymers of the fungal cell wall,
b(1,3)-glucan and chitin. Results reported here allow us to
infer that some of the assayed chalcone derivatives and
analogues, might act at least in part, by inhibiting the bio-
synthesis of one or both polymers of the fungal cell wall.
The antifungal activity of these compounds against der-
matophytes, combined with their useful mode of action,
make these compounds attractive leaders for the develop-
ment of more potent and mainly safer antifungal drugs.
Figure 7. Molecular ectrostatic potential (MEP) maps obtained for
compounds 1, 6, and 2. The MEP were calculated from AM1 wave
function.

2006
S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
Experimental
determined on a Thomas micro hot stage apparatus.
Column chromatography was performed on silica gel
Macherey–Nagel, 0.063–0.2 mm) using a mixture of
Chemistry
(
1H NMR and 13C NMR spectra were recorded on a
JEOL GSX (270 MHz) spectrometer. Splittingpatterns
are described as singlet (s), doublet (d), triplet (t), quar-
tet (q) and multiplet (m). NMR values are given in d
hexane and ethyl acetate as eluent. Elemental analyses
were performed by Atlantic Microlab Inc., Norcross,
GA (USA); results were within Æ0.4% of predicted
values for all compounds.
units relative at CDCl . Mass spectrometry (MS) spec-
3
tra were obtained usinga Hewlett-Packard HP 5971A
Mass Selective Detector with EI (70 eV). IR spectra
were recorded as KBr pellets usinga Shimadzu model
Preparation of the followingcompounds were repor-
2
2,23
23
ted previously: chalcone (1),
3
4-nitrochalcone (2),
4-methoxychalcone (6), 3,4-dimethoxychalcone (8),
2
24
2
5
0
4
70 spectrometer. The uncorrected meltingpoints were
2,3-dimethoxychalcone (10),
6
4-methoxy-4 -methyl-
2
chalcone (15), 1-phenyl-3-(2-chloro-3-quinolinyl)-2-
propenone (35),28 1-(3 ,5 -dimethyl-2-furyl)-3-[3-(2-
1-phenyl-3-(2-
28
chloro-6,7-dimethoxy-3-quinolinyl)-2-propenone (37).
0
chloroquinolinyl)]-2-propenone (36),
0
2
8
Table 6. Capacity of inhibiting b(1,3)-glucan and chitin synthases
expressed in% of inhibition and IC50 values (mg/mL)
Chalcones 1–18 were prepared usinga sodium hydrox-
ide catalyzed condensation, whereas the remaining
compounds 19–22 and 34–41 were prepared using
sodium methoxide.
Compd
b(1,3)-Glucan synthase assay
Chitin synthase assay
%
Ia
IC50b
% Ic
IC50b
1
2
3
5
6
7
8
9
69.78Æ0.52
14.17Æ1.39
20.62Æ0.68
32.54Æ3.57
14.09Æ0.64
8.72Æ1.94
0.14
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
0.44
>0.50
>0.50
0.33
0.10
>0.50
93.70Æ0.09
0.02
15.47Æ0.66 >0.50
32.74Æ0.11 >0.50
67.38Æ0.92
61.71Æ0.46
57.31Æ0.62
70.12Æ1.11
78.78Æ0.02
76.28Æ1.79
52.94Æ0.04
57.45Æ0.93
94.64Æ0.12
0.13
0.07
0.22
0.10
0.10
0.08
0.06
0.22
0.03
42.06Æ0.15
39.81Æ1.87
54.15Æ0.91
14.55Æ0.45
24.58Æ0.67
54.20Æ1.21
10
18
19
21
Pap
Ac
Nik
0.0006d
a
Percent of inhibition at 20 mg/assay (total volume: 40 mL): mean
ÆSEM.
b
Concentration (mg/mL) that produces 50% of Inhibition.
c
Percent of inhibition at 20 mg/assay (total volume: 50 mL): mean
ÆSEM.
d
Value obtained from ref 29; Pap, papulacandin B; Ac, Aculeacin A;
Nik, Nikkomicin.
Figure 9. Effect of different concentrations of chalcones and a related
14
compound on the in vitro incorporation of [ C] glucose into insoluble
b(1,3)-glucan expressed as residual activity of the enzyme b(1,3)-glu-
can synthase.
Figure 8. Effect of different concentrations of chalcones and a related
compound on the in vitro incorporation of [14C] N-acetylglucosamine
into insoluble chitin, expressed as residual activity of the enzyme chitin
synthase-1.
Figure 10. Comparative values of the inhibitory activities of anti-
fungal chalcones and a related compound, against b(1,3)-glucan and
chitin synthase-1, expressed as percent of residual activity.

S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
2007
0
0
0
0
0
General procedures to prepare different substituted
chalcone derivatives
(C1), 132.7 (C4 ), 129.8 (C5, C2 ; C3 ; C5 ; C6 ), 122.3
(Ca), 121.0 (C6), 116.3 (C2), 113 (C4), 55.7 (CH ). MS
3
+
+
+
m/z [M ] 238, [Mꢀ1] 277, [Mꢀ29] 209. Anal.
A solution of an appropriately substituted methyl ketone
(5 mmol), an adecuated substituted aldehyde (5 mmol),
(C H O ) C, H; C: calcd, 80.64; found, 80.63; H:
1
6
14
2
calcd, 5.93; found, 5.94.
NaOH (0.5 g, 12.5 mmol) or NaOMe (5.4 mg, 0.1 mmol) in
MeOH or EtOH (10 mL) was stirred at room temperature
for 1–24h. Formation of a precipitate, generally accom-
panied by a color change in the reaction mixture, is indi-
cative for product formation. The solid product was
recrystallized from a minimum amount of solvent (EtOH/
4-Methylchalcone (7). This was synthesized using4-
methylbenzaldehyde (0.12 g, 1 mmol) and acetophenone
(0.12 g, 1 mmol) as starting materials: yellow solid
(1.05 g, 95%). IR (KBr): 1666 (C¼O), 1596 (C¼C)
ꢀ
1
1
0
0
cm
.
H NMR (CDCl ) d 8.03 (dd, 2H, H2 , 6
3
H O, 1:1 v/v). Other cases, the reaction were monitored for
2
J=8.50, J=1.90 Hz), 7.79 (d, 1H, Hb J=15.83 Hz),
0
TLC. The solvent was evaporated under reduced pressure.
Water was added and the product was extracted four times
with dichloromethane. The organic extract was con-
centrated in vacuum, and the residue was purified by
column chromatography to give the expected com-
pounds as white or yellow solids. On the other hand, if
the reaction yielded an oil instead of a solid, column
chromatography was used to purify the product.
7.45–7.57 (m, 7H, Ar, Ha), 7.21 (d, 1H, H4 J=9.95,
1
3
J=2.01 Hz), 2.38 (s, 3H, CH3); C NMR (CDCl ) d
3
0
(C4 ), 132.1 (C1), 129.6 C3; C5, C2; C6, C2 , C3 , C5 ;
191.4 (C¼O), 144.7 (Cb), 140.9 (C4), 138.3 (C1 ), 132.5
0
0
0
0
0
+
C6 ), 121.0 (Ca), 21.5 (–CH ). MS m/z [M ] 222,
3
+
+
[Mꢀ1] 221, [Mꢀ29] 194. Anal. (C H O) C, H; C:
1
6
14
calcd, 86.45; found, 86.42; H: calcd, 6.35; found, 6.36.
2,4-Dimethoxychalcone (9). This was synthesized using
2
-Nitrochalcone (3). This was synthesized using2-nitro-
benzaldehyde (0.14 g, 1 mmol) and acetophenone
2,4-dimethoxybenzaldehyde (0.17 g, 1 mmol) and aceto-
phenone (0.12 g, 1 mmol) as starting materials: yellow
solid (1, 14 g, 85%). IR (KBr) 1654 (C¼O), 1595 (C¼C)
(
(
0.12 g, 1 mmol) as starting materials: white solid
0.89 g, 70%). IR (KBr): 1654 (C¼O), 1592 (C¼C)
ꢀ
1 1
cm . H NMR (CDCl ) d 7.98 (dd, 2H, Ar, J=8.41,
3
ꢀ1
1
0
.56–7.53 (m, 3H, H3 , H4 , H5 ), 7.52 (d, 1H,
0
cm . H NMR (CDCl ) d 7.99–7.98 (m, 2H, H2 , H6 ),
J=1.48 Hz), 7, 54 (d, 1H, Hb J=15.89 Hz), 7.38 (dd,
1H, H6 J=1.98, J=8.16 Hz), 7.15 (d, 1H, H2
J=1.98 Hz), 6.89 (d, 1H, H5 J=8.16 Hz); 3.94 (s, 3H,
3
0
J=8.4 Hz, H6), 7.51 (m, 3H, H3, H4, H5), 6.89 (d, 1H,
0
0
7
1
3
13
J=15.38 Hz, Hb), 6.38 (d, 1H, J=15.38 Hz, Ha);
C
OCH ), 3.97 (s, 3H, OCH ); C NMR (CDCl ) d
3
3
3
NMR (CDCl ) d 189.9.0 (C¼O), 132.7 (C1), 149.4 (C2),
289.90 (C¼O), 162.75 (C2), 160.16 (C4), 139.67 (Cb),
3
0 0 0 0
138.1 (C1 ), 132.68 (C4 ), 130.79 (C6), 128.60 (C2 , C6 ,
1
32.5 (C4), 148.3 (C3), 129.3 (C5), 132.3 (C6), 141.7
0
0
0
0
0
0
0
(
Cb), 123.3 (Ca), 135.9 (C1 ), 128.7 (C2 , C3 , C5 , C6 ),
C3 , C5 ). 120.61 (Ca), 105.20 (C5), 98.32 (C3), 55.60
0
24. Anal. (C H NO ) C, H, N; C: calcd 71.14; found,
+
+
+
+
1
2
7
5
32.9 (C4 ). MS m/z [M ] 253, [Mꢀ1] 252, [Mꢀ29]
(OCH ), 55.33 (OCH ). MS m/z [Mꢀ31]
237,
205, [Mꢀ92] 176. Anal.
3
3
+
+
[Mꢀ62]
206, [Mꢀ63]
1
5
11
3
1.19; H: calcd, 4.38; found, 4.39; N: calcd 5.53; found
.54.
(C H O ) C, H; C: calcd 76.09; found 76.08; H: calcd,
17 16 3
6.01; found, 6.02.
0
thesized using2,4-dichlorobenzaldehyde (0. 1, 8 g
0
2
,4-Dichlorochalcone (4). This was synthesized using
,4-dichlorobenzaldehyde (0.18 g, 1 mmol) and aceto-
2,4-Dichloro-2 ,4 -dichlorochalcone (11). This was syn-
2
phenone (0.12 g, 1 mmol) as starting materials: beige
0
0
1 mmol) and 2 ,4 -dichloroacetophenone (0.19 g,
1 mmol) as startingmaterials: white solid (0.33 g, 95%).
solid (1.20 g, 87%); IR (KBr) 1658 (C¼O), 1596 (C¼C)
ꢀ1
1
ꢀ1
1
cm ; H NMR (CDCl ) d 8.51 (s, 1H, H3), 8.16 (dd,
IR (KBr) 1680 (C¼O), 1592 (C¼C) cm . H NMR
3
0
1
H, J=7.6, 1.99 Hz, H5), 8.02 (d, 1H, H J=8.4 Hz,
(CDCl ) d 7.87 (d, 1H, J=1.50 Hz, H6 ), 7.61 (d, 1H,
3
0
H6), 7.65 (d, 1H, J=15.83 Hz Ha), 7.60–7.50 (m, 5H,
H1 –H6 ), 7.80 (d, 1H, J=15.83 Hz, Hb), 7.6–7.70 (m,
J=15.83 Hz, Hb), 7.52 (s, 1H, H3), 7.49 (s, 1H, H3 ),
0
0
7.43 (d, 1H, J=15.83 Hz, Ha) 7.40 (d, 1H, J=1.50 Hz,
H6 ) 7.30 (d, 1H, J=2.2 Hz, H5), 6.90 (d, 1H,
0
0
13
0
5
H, H2 –H6 ); C NMR (CDCl ) d 189.9 (C¼O), 137.9
3
0
0
0
13
(
C1 ), 136.9 (Cb), 134.8 (C2, C4), 133.0 (C1, C4 ), 128.0
J=8.20 Hz, H6); C NMR (CDCl ) d 187.51 (C¼O),
3
0
0
+
0
134.83 (C2), 132.77 (C1), 130.72 (C3 ;C3), 129.28 (C6),
0
0
(
C2 , C3 , C5 , C5, C6), 123.3 (Ca). MS m/z [M ] 277,
138.51 (C4, C4 ), 137.21(C2 ), 136.89 (Cb), 135.13 (C1 ),
+
+
0
[Mꢀ1] 276, [Mꢀ29] 248. Anal. (C H Cl O) C, H,
Cl; C: calcd 65.21; found, 65.23; Cl: calcd 25.34; found
1
5
10
2
0
0
128.80 (C6 ), 128.01 (C5 ), 127.89 (C5), 123.93 (Ca). MS
+
+
+
2
5.35; H: calcd, 3.65; found, 3.64.
m/z [Mꢀ1] 345, [Mꢀ29] 317, [346ꢀ100] . Anal.
(C H Cl O) C, H, Cl; C: calcd 52.34; found 52.22; H:
calcd 2.34, found: 2.33, Cl: calcd, 40.67, found, 40.80.
1
5
8
4
3
-Methoxychalcone (5). This was synthesized using3-
methoxybenzaldehyde (0.14 g, 1 mmol) and aceto-
phenone (0.12 g, 1 mmol) as starting materials: yellow
crystals (1.13 g, 95%). IR (KBr): 1665 (C¼O), 1595
0
using4-methylbenzaldehyde (0.12 ,g 1 mmol) and 4
4-Methyl-4 -methylchalcone (12). This was synthesized
-
0
methylacetophenone (0.134 g, 1 mmol) as starting mate-
ꢀ
1 1
(
C¼C) cm . H NMR (CDCl ) d 8.18 (dd, 1H, J=8.9,
3
0
0
1
7
2
(
7
.9 Hz, H4), 7.98 (dd, 2H, J=9.0, 2.2 Hz, H2 , H6 ),
.43 (d, 1H, J=1.9 Hz, H2), 7.43 (dd, 1H J=8.2,
.2 Hz, H6), 7.82 (dd, 1H, J=8.9, 6.5 Hz, H5), 7.56–7.53
rials: yellow solid (0.189 g, 80%). IR (KBr) 1654 (C¼O),
ꢀ
1 1
1596 (C¼C) cm . H NMR (CDCl ) d 7.92 (d, 2H,
3
0
0
J=8.16 Hz, H2 , H6 ), 7.78 (d, 1H, J=15.83 Hz, Hb),
7.54–7.45 (m, 3H, Ar, Ha), 7.38 (d, 2H, J=7.91, H3,
H5), 7.20(d, 2H, J=7.91 Hz, H3, H5), 2, 42 (s, 3H,–
0
0
0
m, 3H, H3 , H4 , H5 ). 7.40 (d, 1H, J=15.38 Hz, Hb),
.27 (d, 1H, J=15.38 Hz, Ha); C NMR (CDCl ):
1
3
3
0
13
1
90.2 (C¼O), 161.9 (C3), 144.6 (Cb), 138.1 (C1 ), 136.1
CH ), 2.37 (s, 3H,–CH ); C NMR (CDCl ) d 187.0
3
3
3

2008
S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
0
0
(
1
C¼O), 142.1 (C4 ), 140.5 (Cb), 136.9 (C4), 131.9 (C1 ),
as startingmaterials: yellow solid (0.20 ,g 75%). IR
1
ꢀ
1
29.1, 128.5, 126.1, 121.0 (Ca), 22 (CH ). MS m/z
[Mꢀ1] 235, [Mꢀ29] 206. Anal. (C H O). C, H; C:
calcd 86.40; found, 86.40; H: calcd, 6.83; found, 6.84.
(KBr) 1683 (C¼O), 1590 (C¼C) cm
.
H NMR
3
+
+
0
J=1.73 Hz, H3 ), 7.51 (d, 1H, J=4 Hz, H5), 7.49 (d,
(CDCl ) d 7.85 (d, 1H, J=8.9 Hz, H6 ), 7.80 (d, 1H,
1
7
16
3
0
0
1H, J=16.0 Hz, Hb), 7.42 (dd, 1H, J=(.0, 4 Hz, H5 ),
0
synthesized using3,4-dimethoxybenzaldehyde (0.17,g
0
3
,4-Dimethoxy-3 ,4 -dimethoxychalcone (13). This was
7.00 (d, 1H, J=16 Hz, Ha), 6.47 (dd, 1H, J=8.0, 4 Hz,
H4), 6.39 (dd, 1H, J=8.0, 4.0 Hz, H3); C NMR
13
1
1
9
mmol) and 3,4-dimethoxyacetophenone (0.18 g,
mmol) as startingmaterials: yellow solid (0.30,g
(CDCl ) d 187.0 (C¼O), 155.3 (C2), 145.0 (C5), 141.6
3
0
0
0
0
(Cb), 139.6 (C4 ), 135.9 (C2 ), 135.3 (C1 ), 131.4 (C6 ),
ꢀ
1
1
0
0
+
5%). IR (KBr) 1680 (C¼O), 1592 (C¼C) cm . H
130.6 (Ca), 129.2 (C3 , C5 ). MS m/z: [MꢀH] 266,
13 8 2 2
0
+
+
NMR (CDCl ) d 7.68 (dd, 1H, J=8.0, 1.9 Hz, H6 ), 7.74
[Mꢀ29] 238, [Mꢀ118] 121. Anal. (C H Cl O ) C:
3
0
(
d, J=1.9 Hz, H2 ), 7.26 (d, J=15.38 Hz, Hb), 7.25 (d,
calcd: 58.65, found: 58.71 H: calcd 3.03, found 3.04; Cl:
calcd, 26.29, found, 26.24.
J=15.38 Hz, Ha), 7.06 (dd, 1H, J=8.2, 2.1 Hz, H6),
7
H5 ), 6.70 (d, 1H, J=8.2 Hz, H5), 3.88 [s, 6H,
.01 (d, 1H, J=1.91 Hz, H2), 6.92 (d, 1H. J=8.9 Hz,
0
1-(Phenyl)-3-(2-napthyl)-2-propen-1-one (20). This was
synthesized using2-naphtylaldehyde (0.16 ,g 1 mmol)
and acetophenone (0.12 g, 1 mmol) as starting materials:
yellow solid (0.21 g, 80%). IR (KBr) 1681 (C¼O), 1592
1
3
2
1
(OCH )], 3.87 [s, 3H, 2(OCH )]; C NMR (CDCl ) d
3
3
3
0
0
94.4 (C¼O), 151.3 (C4), 150.8 (C4 ), 149.2 (C3 ), 147.8
0 0
12.3 (C6), 120.7 (C6), 114.9 (C5 , C2), 112.8 (C2 , C5),
(
C3 ), 131.8 (C1 ), 128.0 (Cl), 144.1 (Cb), 122.9 (Ca),
0
0
ꢀ1
1
2
5
2
(C¼C) cm . H NMR (CDCl ) d 8.11–8.09 (m, 2H,
3
+
9.2; 55.3; 55.1 and 52.0 (OCH ). MS m/z [Mꢀ31]
H2, H3), 8.04 (d, 2H, J=8.0 Hz, H7, H8), 8.03–7.98 (m,
5H, Ar), 7.76 (d, 1H, J=15.38 Hz, Hb), 7.75–7.43 (m,
3
+
+
97, [Mꢀ62] 266, [Mꢀ162] 166. Anal. (C H O ) C,
1
9
20
5
1
3
H; C: calcd, 69.48; found, 69.50 H: calcd 6.14; found
6
3H, Ar), 7.34 (d, 1H, J=15.38 Hz, Ha); C NMR
0
.16.
(CDCl ) d 188.9 (C¼O), 140.5 (Cb), 137.4 (C1 ), 133.5
3
0
0
(
C9), 132.9 (C4 ), 132.8 (C5), 132.3 (C2), 128.4 (C2 ,
0
0 0 0
C3 , C5 , C6 ), 128.0 (C3, C6), 127.9 (C4), 126.6 (Ca),
+ + +
2
,3-Dimethoxy-4 -bromochalcone (16). This was synthe-
sized using2,3-dimethoxybenzaldehyde (0.16,6 g
mmol) and 4-bromoacetophenone (0.199 g, 1 mmol) as
startingmaterials: yellow solid (0.29 g, 85%). IR (KBr)
125.9 (C7, C8, C1). [M] 258, [Mꢀ1] 257, [Mꢀ29]
1
229. Anal. (C H O): C: calcd, 88.34, found, 88.35 H:
1
9
14
calcd, 5.47, found, 5.46.
ꢀ
1 1
1
660 (C¼O), 1594 (C¼C) cm . H NMR (CDCl ) d
3
0
(21). This was synthesized using2-naphtylaldehyde
0
8.08 (d, 1H, J=15.83 Hz, Hb), 7.86 (d, 1H, J=8.66 Hz,
H3 , H5 ), 7.75 (d, 2H, J=8.66 Hz, H5 , H6 ), 7.52 (d,
1-(2 ,4 -Dimethoxyphenyl)-3-(2-napthyl)-2-propen-1-one
0
0
0
0
0
0
1H, J=15.83 Hz, Ha), 7.25 (d, 1H, J=7.42 Hz, H2,
H6), 7.08 (t, 1H, J=7.91 Hz, H5), 6.96 (d, 1H,
J=7.17 Hz, H4); 3.87 (s, 3H, OCH ), 3.80 (s, 3H,
3
(0.14 g, 1 mmol) and 2 ,4 -dimethoxyacetophenone
(0.18 g, 1 mmol) as starting materials: yellow solid
ꢀ
1
(0.22 g, 85%). IR (KBr) 1680 (C¼O), 1590 (C¼C) cm
.
1
3
1
OCH3);
1
1
C NMR (CDCl ): 188.9 (C¼O), 152.9,
H NMR (CDCl ) d 8.01 (d, 1H, J=8.0 Hz, H4), 8.00
3
3
0
47.9(C3), 139.0 (C2), 138.6 (Cb), 136.7 (C1 ), 130.5,
31, 127.7 (C4 ), 124.7, 123.9, 123.0 (Ca), 114.0, 61.40
(d, 1H, J=8.0 Hz, H8), 7.85 (d, 1H, 8.02 Hz, H3), 7.84
(d, 1H, J=8.0 Hz, H5), 7.75 (s, 1H, H1), 7.74 (d, 1H,
0
+
+
0
(
Anal. (C H BrO ). C, H; C: calcd, 58.96; found,
CH ), 56.0 (CH ). MS m/z [Mꢀ1] 346, [Mꢀ29] 318.
J=8.0 Hz, H6 ), 7.51–7.49 (m, 2H, H6, H7), 7.44 (d,
3
3
1H, J=16.0 Hz, Hb), 7.34 (d, 1H, J=16.0, Ha), 6.53
(dd, 1H, J=8.0, 4.0 Hz, H5 ), 6.17 (d, 1H, J=4.0 Hz,
1
7
15
3
0
H3 ), 3.92 (s, 3H, OCH ), 3.89 (s, 3H, OCH );
58.97; H: calcd, 4.37; found 4.36, Br: calcd, 22.81;
found, 22.78.
0
13
C
3
3
0
0
0
NMR (CDCl ) d 188.9 (C¼O), 167.4 (C4 ), 163.1 (C2 ),
3
0
using3-methoxybenzaldehyde (0.14 g, 1 mmol) and 4
3
-Methoxy-4 -bromochalcone (18). This was synthesized
-
140.5 (Cb), 133.5 (C3, C4), 132.6 (C1), 130.6 (C6 ),
0
bromoacetophenone (0.20 g, 1 mmol) as starting mate-
127.9 (C5), 127.9 (C5), 126.6 (Ca), 125.7 (C2),
0
+
+
106.3 (C5 ), 56.0 (2 OCH ). [Mꢀ1] 317, [Mꢀ29]
3
+
+
rials: yellow solid (1.49 g, 94%) IR (KBr) 1654 (C¼O),
289,
[Mꢀ32]
286,
[Mꢀ137]
181.
Anal.
ꢀ
1 1
1
595 (C¼C) cm . H NMR (CDCl ) d 7.88 (d, 2H,
(C H O ): C: calcd 79.21, found 79.19, H: calcd
21 18 3
3
0
J=8.39 Hz, H5 ), 7.76 (d, 1H, Hb J=15.35 Hz), 7.64 (d,
5.70, found 5.71.
2
(
H, J=8.39 Hz Ar), 7.4 (d, 1H, J=15.35 Hz, Ha), 7.33
dd, 1H, J=8.9 Hz, H5); 7.21 (dd, 1H, J=2.02,
J=8.9 Hz, H4), 7.13 (d, 1H, J=1.98 Hz, H2); 6.97 (dd,
0
none (22). This was synthesized using3-methoxy-2-
naphtylaldehyde (0.19 g, 1 mmol) and 3 -methoxy-
1-(3 -Methoxyphenyl)-3-(3-methoxy-2-napthyl)-2-prope-
1
3
0
acetophenone (0.12 g, 1 mmol) as starting materials:
1
H, J=1.98, J=8.41 Hz, H6), 3.84 (s, 3H, OCH3);
C
NMR (CDCl ) d 188.9 (C¼O), 150.0 (C3), 144.6 (Cb),
3
0
0
0
0
1
1
36.8 (C1 ), 131.8 (C3 , C5 ), 129.9 (C5), 127.8 (C4 ),
orange solid (0.19 g, 68%). 1662 (C¼O), 1590 (C¼C)
ꢀ
1
1
22.3 (Ca), 116.3 (C2), 113.4 (C4). 55.1 (OCH ). MS
cm . H NMR (CDCl ) d 8.12 (d, 1H, J=16.0 Hz,
3
+
3
+
+
m/z [Mꢀ31]
286, [Mꢀ32]
285, [Mꢀ61]
256.
Hb), 8.01 (d, 1H, J=8.2 Hz, H8), 7.99 (s, 1H, H4), 7.97
(d, 1H, J=8.1 Hz, H4 ), 7.77 (s, 1H, H1), 7.85 (d, 1H,
0
J=8.1 Hz, H5), 7.51–7.49 (m, 4H, H6, H7, H5 , H6 ),
Anal. (C H BrO ) C, H; C: calcd 60.76; found
13
1
6
2
0
0
60.73 H: calcd, 4.15; found, 4.15; Br: calcd, 24.97;
found, 25.04.
0
.83 (s, 3H, OCH ), 3.75 (s, 3H, OCH ); C NMR
7.45 (d, 1H, J=16.0 Hz, Ha), 7.42 (d, 1H, 1.89 Hz, H2 ),
1
3
8
3 3
0
0
This was synthesized using2-furylaldehyde (0.096,g
0
1
-(2 ,4 -Dichlorophenyl)-3-(2-furyl)-2-propen-1-one (19).
(CDCl ) d 187.0 (C¼O), 161.9 (C3 ), 155.4 (C3), 140.5
3
0
0
(Cb), 138.4 (C1 ), 129.4 (C5 , C8a), 127.2 (C1, C8),
126.4 (Ca, C5, C6), 125.2 (C2), 123.7 (C7), 120.9 (C6 ),
0
0
0
1
mmol) and 2 ,4 -dichloroacetophenone (0.1 g, 1 mmol)

S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
2009
0
0
+
1
3
18.5 (C4 ), 114.2 (C2 ), 56.0 (2 OCH ). MS m/z [M]
3
124.8 (C6), 52, 9 (CH), 50.4 (2 OCH ), 46.7 (CH2), 24.6
3
+
+
+
+
18 (30), [Mꢀ1] 317(90), [Mꢀ29] 289 (100). Anal.
(CH). MS m/z [Mꢀ132]
258, [258ꢀH]
257,
[257ꢀCO] 229. Anal. (C H O ) C: calcd 73.82,
+
(
found 5.69.
C H O ): C: calcd 79.21, found 79.21 H: calcd 5.70,
21 18 3
24 22
5
found 73.85, H: calcd 5.68, found 5.68.
1
-(Phenyl)-3-(2,3-dimethoxyphenyl)-3-malonomethyl-pro-
General procedure for preparation of ketones (23–26)
panone (26). This was synthesized usingcompound 10
(74 mg, 0.5 mmol), dimethyl malonate (66 mg, 0.5 mmol)
and sodium methoxide (0.27 mg, 0.05 mmol) as starting
materials: pale-yellow solid (0.18 g, 70%). IR (KBr)
To a solution of dimethylmalonate (0.5 mmol) and a
chalcone derivative (0.5 mmol) in methanol (5 mL),
sodium methoxide (0.05 mmol) was added and warmed
until complete solubility. The mixture was stirred at
room temperature between 4 and 6 h or until a solid
appeared, indicatingthe formation of the product. The
ꢀ
1 1
1740 (COOR), 1660 (C¼O) cm . H NMR (CDCl ) d
3
0
0
8.11 (dd, 2H, J=8.1, 1.98 Hz, H2 , H6 ), 7.49 (m, 3H,
0
0
0
H3 , H4 , H5 ), 7.37 (d, 1H, J=8.0 Hz, H6), 7.35–6.8
(m, 2H, H4, H5), 4.73 (d, 1H, J=8.0 Hz, CH), 4.41 (d,
products were filtered and recristallized in EtOH–H O.
2
1
H, J=8 Hz, CH), 3.82 (t, 1H, J=16.0 Hz), 3.46 (t, 1H,
1
3
1
,3-Diphenyl-3-methylmalonyl-propanone (23). This was
J=16.0), 3.79 (s, 6H, OCH3); C NMR (CDCl ) d
3
0
128.4 (C2 , C3 , C5 , C6 ), 126.0 (C1), 121.6 (C4, C5, C6),
0
synthesized usingcompound 13 (104 mg, 0.5 mmol),
dimethyl malonate (66 mg, 0.5 mmol) and sodium
methoxide (0.27 mg, 0.05 mmol) as starting materials:
197.6 (C¼O), 147.4 (C2, C3), 137.9 (C1 ), 132.9 (C4 ),
0 0 0 0
107.2 (2 CꢂN), 56.6 (2 OCH ), 46.0 (CH ), 35.1 (2 CH).
3
2
+
+
white solid (0.162 g, 95%), (MeOH–H O). IR (KBr)
2
MS m/z [MꢀCH OH] 368, [MꢀCH (CO CH ) ] 268.
3
2
2
3 2
ꢀ
1
1
1
(
731.2 (C¼O, ester), 1664.0 (C¼O), cm . H NMR
Anal. (C H N O ) C: calcd 71.83, found 71.89, H:
20 18 2 3
calcd 5.43, found 5.43, N: calcd 8.38, found 8.36.
CDCl ) d 7.99–796 (m, 2H, Ar), 7.50–7.48 (m, 3H, Ar),
3
7
J=4 Hz), 3.83 (m, 1H), 3.75 (s, 6H, 2OCH ), 3.62–3.27
.42 (m, 2H, Ar), 7.38–7.34 (m, 3H, Ar), 4, 27 (d, 1H,
1-(Phenyl)-3-(2,4-dichlorophenyl)-1,1-dicyane-methyl-
propanone (27). This was synthesized usingcompound
5 (139 mg, 0.5 mmol), malononitrile (33 mg, 0.5 mmol)
and sodium methoxide (0.27 mg, 0.05 mmol) as starting
materials: beige solid IR (KBr) 2208 (CN), 1680
3
1
3
(
(
(
(
m, 1H); C NMR (CDCl ) d 197.6 (C¼O), 174.5
3
0
0
C10a, C10b), 139.4 (C1), 137.2 (C1 ), 132.7 (C4 ), 128.6
0
C10), 50.4 (2 OCH ), 46.7 (C8), 24.6 (C9). MS m/z
0
0
0
C2, C3, C5, C6, C2 , C3 , C5 , C6 ), 125.7 (C4), 52.9
3
+
+
ꢀ1
1
[
Mꢀ132] 229,
Mꢀ73] 258. Anal. (C H O ), C: calcd 70.56, found
258, [258ꢀH]+ 257 [257ꢀCO]
(C¼O) cm . H NMR (CDCl ) d 8.08 (m, 2H, Ar),
3
+
[
7.60 (d, 1H, J=8.2 Hz, H6), 7.59–7.56 (m, 3H, Ar), 7.52
(d, 1H, J=1.9 Hz, H3), 7, 35 (d, 1H, J=8.2 Hz, H5),
2
0
20
5
7
0.58, H: calcd 5.93, found 5.94.
4
.44 (d, 1H, J=8.0 Hz), 3.72 (t, 1H, J=16.0 Hz), 3.37 (t,
1
3
1
-(2,4-Dimethoxyphenyl)-3-(phenyl)-3-methylmalonyl-
1H, J=16.0 Hz); C NMR (CDCl ) d 196.5 (C¼O),
3
0
0
propanone (24). This was synthesized usingcompound 9
0.14 g, 0.5 mmol), dimethylmalonate (66 mg, 0.5 mmol
and sodium methoxide (0.27 mg, 0.05 mmol) in methanol
5 mL). Beige solid, (0.06 g, 60%). IR (KBr): 1731 (C¼O,
137.3 (C1, C1 ), 135.2 (C2), 134.9 (C6), 133.4 (C4 , C6),
0
0
0
(
132.4 (C2 , C6 ), 129.6 (C3), 126.8 (C3 , C5), 126.2 (C5),
107.0 (2 CꢂN), 45.0 (CH ), 35.10 (2 CH). MS m/z
2
+
+
(
[MꢀC H N ]
277,
[277ꢀH]
276.
Anal.
3
2
2
ꢀ
1 1
esther), 1664 (C¼O) cm . H NMR (CDCl ) d 7.99 (dd,
(C H Cl N O) C: calcd 63.15, found 63.10, H: calcd
18 12 2 2
3.54, found 3.53, N: calcd 8.19, found 8.19, Cl: calcd,
20.45, found, 20.51.
3
0
.0 Hz, H4 , H5 ), 7.31 (dd, 1H, J=8.0, 4.0 Hz, H5), 7.49
0
2
4
H, J=8.0, 2.1 Hz, H2 , H6 ), 7.50 (dd, 2H, J=8.0,
0
0
0
(
J=8.2 Hz, H4), 4.48–4.43 (m, 2H), 3.79 (s, 6H, 2OCH ),
m, 1H, H3 ), 7.05 (d, J=8.2 Hz, H6), 6.81 (d, 1H
1-(Phenyl)-3-(2-napthyl)-1,1-dicyane-methyl)-1-propanone
(28). This was synthesized usingcompound 22 (129 mg,
0.5 mmol), malononitrile (33 mg, 0.5 mmol) and sodium
methoxide (0.27 mg, 0.05 mmol) as starting materials:
orange solid (0.24 g, 75%). IR (KBr) 2208 (CN), 1680
3
1
3
3
.75 (s, 6H, 2CO CH ), 3.68–3.33 (m, 1H); C NMR
2 3
(
C3), 137.4 (C1 ), 132.9 (C4 ), 126.0 (C1), 128.6 (C2 , C3 ,
CDCl ) d 197.6 (C¼O), 174.5 (2CO CH ), 147.4 (C2,
3
2
3
0 0 0 0
C5 , C6 ), 121.6 (C5, C6), 56.6 (2 OCH ), 53.2 (CH),
0
0.4 (2 CO CH ), 47.0 (CH ), 19.0 (CH). MS m/z
0
3
ꢀ
1 1
5
(C¼O) cm . H NMR (CDCl ) d 8.10 (dd, 2H, J=7.5,
2
3
2
3
+
+
0 0
[
calcd 65.97, found 65.96, H: 6.04, found 6.02.
Mꢀ132] 268, [268ꢀH] 267. Anal. (C H O ), C:
1.3 Hz, H2 , H6 ), 8.03 (d, 1H, J=9.0 Hz, H4), 7.99 (m,
2H, Ar), 7.77 (dd, 1H, J=7.6, 1.3 Hz, H5), 7.63 (dd, 1H,
J=9.0, 2.0=Hz, H3), 7.49 (m, 3H, Ar, 7.43 (m, 2H, Ar),
2
2
24
7
1
-(2-Napthyl)-3-(phenyl)-3-methylmalonyl-propanone (25).
This was synthesized usingcompound 22 (129 mg,
.5 mmol), dimethyl malonate (66 mg, 0.5 mmol) and
4.43 (d, J=8.0 Hz, CH(CN) ), 4.16 (m, 1H,–CH–), 3.73
2
(dd, J=12, 8 Hz,–HCH–), 3.38 (dd, 12, 8 Hz, –HCH–);
0
1
3
0
C NMR (CDCl ) d 196.5 (C¼O), 137.5 (C1 ), 137.1
3
0
0
(C6), 127.6 (C4), 126.8 (C3 , C5 ), 126.0 (C5), 125.0 (C7),
0
sodium methoxide (0.27 mg, 0.05 mmol) as starting
materials: white solid (0.27 g, 68%). IR (KBr) 1731
(C8a), 135.2 (C2), 133.4 (C4 ), 132.4 (C2 , C6 ), 128.3
0
0
ꢀ
1 1
(
(
COOR), 1660 (C¼O) cm . H NMR (CDCl ) d 7.98
0
.45–4.43 (m, 2H), 3.75 (s, 6H, OCH ), 3, 65–3.30 (m,
107.0 (2 CꢂN), 44.8 (CH ), 37.8 (CH), 33.9 (CH). MS m/
3
2
0
0
0
+
+
+
dd, 2H J=8.0, 2.1 Hz, H2 , H6 ), 7.49 (m, 3H, H3 ,
z [MꢀC H N ] 258, [258ꢀH] 257, [257ꢀCO] 229.
3
2
2
0
H4 , H5 ), 7.45–7.33 (m, 7H, H5, H6, H7, H8, H9),
Anal. (C H N O) C: calcd 81.45, found 81.49, H: calcd
22 16 2
4
2
1
4.97, found 4.96, N: calcd, 8.64, found, 8.63.
3
1
3
H, CH2); C NMR (CDCl ) d 197.6 (C¼O, ketone),
3
0
0
0
74.5 (2 C¼O, esther), 137.4 (C1 ), 135.2 (C2), 133.5
1-(3 ,4 -Dimethoxylphenyl)-3-(2-napthyl)-1,1-dicyane-
methyl-1-propanone (29). This was synthesized using 24
(159 mg, 0.5 mmol), malononitrile (33 mg, 0.5 mmol)
0
0
0
0
(C8a), 132.9 (C4 ), 131.6 (C4a), 128.4 (C2 , C3 , C5 ,
C6 ), 127.5 (C3, C4, C5, C8), 126.7 (C1), 125.7 (C7),
0

2010
S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
0
H5 ), 4.6–4.3 (m, 2H), 3.90 (s, 6H, OCH ), 3.67–3.30 (m,
and sodium methoxide (0.27 mg, 0.05 mmol) as starting
materials: yellow solid (227 mg, 70%). IR (KBr) 2208
7.70 (d, 1H, J=1, 9 Hz, H1 ), 6.89 (d, 1H, J=7.0 Hz,
0
0
147.3 (C3 ), 145.3 (C2), 141.9 (C8a), 134.1 (C4), 132.8
3
ꢀ
1
1
13
(
CN), 1660 (C¼O) cm . H NMR (CDCl ) d 8.03 (d,
0
d, 1H, J=2.1 Hz, H1 ), 7.43 (m, 2H, Ar), 6.88 (dd, 1H,
2H); C NMR (CDCl ) d 195.6 (C¼O), 148.6 (C4 ),
3
3
0
(C1), 130.6 (C7), 127.8 (C8), 126.5 (C6), 122.1 (C6 ),
1
J=8.1 Hz, H5), 7.77 (dd, 1H, J=8.0, 2.1 Hz; H6 ), 7.70
H, J=8.1 Hz, H4), 7.99 (m, 2H, Ar), 7.78 (d, 1H,
0
116.6 (C3), 113.5 (C2 ), 107.0 (CN), 107.5 (CN), 55.9
0
J=8.0, 2.1 Hz, H3), 4.20–4.13 (m, 2H, CH(CN) ,–CH–),
0
(OCH ), 55.7 (OCH ), 45.1 (CH ), 35.2 (CH), 35.1
(
2
3
3
2
+
+
3
–
.90 (s, 6H, OCH ), 3.76 (m, 1H,–HCH–), 3.40 (m, 1H,
3
(CH). MS m/z [Mꢀ31]
388, [Mꢀ72]
357,
[MꢀC H N ] 353. Anal. (C H ClN O ) C: calcd
1
3
+
HCH–); C NMR (CDCl ) d 195.6 (C¼O), 148.8
3
3
2
2
23 18
3
3
0
C5), 125.2 (C5 ), 114.5 (C5 ), 113.5 (C2 ), 107.0 (2
0
(
(
C4 ), 147.3 (C3 ), 137.0 (C8a, C8), 127.6 (C4, C3), 126.1
65.85, found 65.96 H: calcd 4.33, found 4.31, N: calcd
10.02, found 9.98, Cl: calcd 8.34, found 8.32.
0
0
0
CꢂN), 55.9 (OCH ), 55.7 (OCH ), 44.8 (CH ), 37.8
3
3
2
+
0
0
0
(
CH), 33.9 (CH). MS m/z [MꢀC H N ]
318,
1-(3 ,4 ,5 -Trimethoxyphenyl)-3-(3,4,5-trimethoxiphenyl)-
1,1-dicyane-methyl-1-propanone (33). This was synthe-
sized usingcompound 14 (194 mg, 0.5 mmol), mal-
ononitrile (33 mg, 0.5 mmol) and sodium methoxide
3
2
2
+
+
[
318ꢀH] 317 [317ꢀCO] 289. Anal. (C H N O ) C:
2
4
20
2
3
calcd 74.97, found 74.95, H: calcd 5.25, found 5.24, N:
calcd 7.29, found 7.30.
(
0.27 mg, 0.05 mmol) as starting materials: yellow solid
ꢀ1
0
0
1
-(2 ,4 -Dimethoxyylphenyl)-3-(2-napthyl)-1,1-dicyane-
(295 mg, 65%). IR (KBr) 2208 (CN), 1660 (C¼O) cm
.
1
0
H2, H6), 4.37–4.23 (m, 2H), 3.98 (s, 6H, OCH ), 3.90 (s,
0
methyl-1-propanone (30). This was synthesized using
compound 23 (159 mg, 0.5 mmol), malononitrile (33 mg,
0.5 mmol) and sodium methoxide (0.27 mg, 0.05 mmol)
as starting materials: orange solid (230 mg, 60%). IR
H NMR (CDCl ) d 7.20 (s, 2H, H2 , H6 ), 5.92 (s, 2H,
3
3
3
1
6H OCH ), 3.79–3.40 (m, 2H,–CH –);
2
C NMR
(CDCl ) d 157.9 (C3, C5), 153.6 (C3 , C5 ), 140.08 (C4 ,
3
0
C4), 131.0 (C1, C1 ), 113.4 (C2 , C6 ), 105.9 (C2, C6),
0
0
3
ꢀ
1
1
0
104.9 (2 CN), 61.1 (4 OCH ), 56.5 (OCH ), 56.4
0
0
(
KBr) 2208 (CN), 1680 (C¼O) cm . H NMR (CDCl )
3
d 8.06 (d, 1H J=7.8 Hz, H4), 8.00 (m, 2H, Ar), 7.78 (d,
3
3
0
d, 1H, J=7.8 Hz, H3), 7.43 (m, 2H, Ar), 6.49–6.48 (m,
+
+
1
(
2
3
H, J=7.9 Hz, H5), 7.76 (d, 1H, J=8.0 Hz, H6 ), 7.63
(OCH ), 54.6 (CH ). MS m/z [Mꢀ66] 388, [388ꢀH]
3
2
+
387, [387ꢀCO] 283. Anal. (C H N O ) C: calcd,
24 26 2 7
H, Ar), 4.24–4.13 (m, 2H, CH(CN) ,–CH–), 3.92 (s,
2
63.41, found, 63.36 H: calcd, 5.77, found, 5.78, N: calcd,
6.17, found, 6.16.
H, OCH ), 3.89 (s, 3H, OCH ), 3.82–3.46 (m, 2H,
3
3
1
3
0
61.7 (C4 ), 137.0 (C8a, C8), 127.6 (C4, C3), 126.1 (C5),
CH2); C NMR (CDCl ) d 196.0 (C¼O), 165.3 (C2 ),
3
0
1
1
9
3
General procedure to prepare 2-chloro-6,7-dimethoxy-
quinolynilchalcones (34–41)
0
9.6 (C3 , C6 ), 55.9 (2 OCH ), 44.8 (CH ), 37.8 (CH),
0
0
17.8 (C1 ), 107.0 (2 CꢂN), 114.5 (C5 ), 111.0 (C5 ),
0
0
3
+
2
+
3.9 (CH). MS m/z: [Mꢀ32] 353 [MꢀC H N ] 318.
A solution of the acetophenone respective (1 mmol), 2-
chloro-6,7-dimethoxy-3-formyl (1 mmol) and sodium
methoxide (0.01 mmol) in methanol (5 mL) were stirred
at room temperature. After 24 h, a yellow solid was
isolated by filtration.
3
2
2
Anal. (C H N O ) C: calcd 74.97, found 74.99 H:
3
2
4
20
2
calcd 5.25, found 5.24, N: calcd 7.29, found 7.30.
1
1
-(Phenyl)-3-(2-chloro-3-quinolinyl)-1,1-dicyane-methyl-
-propanone (31). This was synthesized usingcom-
pound 36 (147 mg, 0.5 mmol), malononitrile (33 mg,
.5 mmol) and sodium methoxide (0.27 mg, 0.05 mmol)
as starting materials: orange solid (225 mg, 60%). IR
1-Phenyl-3-(2-chloro-3-quinolinyl)-2-propen-1-one (34).
(0.25 g, 85%). IR (KBr) 1651 (C¼O), 1595 (C¼C)
0
ꢀ
1
1
cm . H NMR (CDCl ) d 8.31 (d, 1H, J=8.30 Hz,
3
ꢀ
1 1
(
KBr) 2208 (CN), 1660 (C¼O) cm . H NMR (CDCl )
H5), 8.25 (s, 1H, H4), 8.11 (d, 1H, J=8.41 Hz, H8), 7.98
0 0
(d, 2H, 8.91 Hz, H2 , H6 ), 7.74 (d, 1H, J=15.83 Hz,
3
0
.03–7.69 (m, 3H, H6, H7, H8), 7.49–7.50 (m, 3H, H3 ,
0
d 9.00 (s, 1H, H2), 8.16–8.14 (m, 4H, H4, H5, H2 , H6 ),
0
H4 , H5 ), 4.44 (d, 1H, J=8 Hz, CH(CN) ), 4.16 (m,
0
0
0
8
Hb), 7.63–7.53 (m, 4H, H5 , H4 , H3 , H7), 7.34 (d, 1H,
0
0
13
J=15.83 Hz); C NMR (CDCl ) d 190 (C¼O), 144.1
2
3
1
3
0
132.7 (C4 ), 131.1 (C4a), 130.9 (C5), 129.7 (C6, C7),
1
1
(
H, CH), 3.73–3.37 (m, 1H, CH2); C NMR (CDCl ) d
(C2), 139.9 (C8a), 139.6 (C1 ), 136.9 (Cb), 135.5 (C4),
3
0
C4), 132.9 (C4 ), 128.6 (C2 , C3 , C5 , C6 ), 128.1 (C6,
0
0
80.0 (C¼O), 150.8 (C2), 147.4 (C8a), 137.4 (C1 ), 136.9
0
0
0
0
0
0
0
0
128.6 (C2 , C3 , C5 , C6 ). 129.5 (C7), 125.8 (C3), 121.7
+
+
+
C7), 127.9 (C4a), 127.0 (C5), 119.2 (CN), 119.0 (CN),
(Ca). MS m/z [M] 293, [Mꢀ1] , [Mꢀ36] 257,
18 12
+
+
+
4
5.7 (CH ), 24.8 (CH), 22.8 (CH). MS m/z [MꢀHCl]
[Mꢀ28] 265, [Mꢀ77] 216. Anal. (C H ClNO). C:
2
+
3
38, [MꢀC H N ]
309. Anal. (C H ClN O) C:
21 14
calcd, 70.18, found, 70.16 H: calcd, 3.93, found, 3.93, N:
calcd, 11.70, found, 11.71, Cl: calcd, 9.74, found, 9.76.
calcd 73.70; found 73.72; H: calcd 4.13; found 4.14; N:
calcd 4.78, found 4.77 Cl: calcd 11.93 found 11.92.
3
2
2
3
0
nyl)-2-propen-1-one (38). (0.41 g, 95%). IR (KBr) 1638
1-(4 -Bromo-phenyl)-3-(2-chloro-6,7-dimethoxy-3-quinoli-
0
dicyane-methyl)-propanone (32). This was synthesized
0
1
-(3 ,4 -Dimethoxyphenyl)-3-(2-chloro-3-quinolinyl)-1,1-
1
(C¼O), 1577 (C¼C). H NMR (CDCl ) d 8.52 (d, 2H,
3
0
0
0
0
using3-(2-chloro-3-quinolinyl)-1-(3 ,4 -dimethoxyphenyl)-
propenone (177 mg, 0.5 mmol), malononitrile (33 mg,
J=2.1 Hz, H2 , H6 ), 8.32 (s, 1H, H4), 8.29 (d, 1H,
J=15.70 Hz, Hb), 7.96 (d, 2H, J=8.42 Hz, Ar), 7.63 (d,
2H, J=8.42 Hz, Ar), 7.52 (d, 1H, J=15.70 Hz, Ha),
0
.5 mmol) and sodium methoxide (0.27 mg, 0.05 mmol)
as startingmaterials: yellow solid (269 m ,g 75%). IR
7.33 (s, 1H, H8), 7.06 (s, 1H, H5), 4.02 (s, 6H, OCH );
3
ꢀ
1
1
13
(
KBr) 2208 (CN), 1660 (C¼O) cm . H NMR (CDCl )
C NMR (CDCl ) d 189.7 (C¼O), 155.2 (C6, C7),
3
3
0
0
0
d 8.28 (s, 1H, H3), 8.23 (d, 1H, J=8.0 Hz, H4), 8.05–
145.9 (C2), 140.6 (C1 , C8a), 136.7 (Cb), 130.0 (C2 , C3 ,
0
0
0
0
7
.78 (m, 3H, Ar), 7.76 (dd, 1H, J=8.0, 2.0 Hz, H6 ),
C5 , C6 , C4, C4a), 128.2 (C4 ), 125.0 (C3), 122.6 (Ca),

S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
2011
1
18.0 (C8), 105.1 (C5), 56.8 (OCH ), 56.7 (OCH ). MS
3
Collection (Rockville, MD, USA): C. albicans ATCC
10231, S. cerevisiae ATCC 9763, C. neoformans ATCC
32264, A. flavus ATCC 9170, A. fumigatus ATCC
26934, A. niger ATCC 9029 and T. mentagrophytes
ATCC 9972. Strains were grown on Sabouraud-chlor-
3
+
+
+
+
m/z: [M] 353 [Mꢀ1] 352, [Mꢀ31] 322, [Mꢀ29]
+
3
5
3
1
24, [Mꢀ77] 276. Anal. (C H ClBrNO ), C: calcd
2
0
15
3
5.69, found 55.75 H: calcd 3.51, found 3.51; N: calcd
.25, found 3.24; Cl: calcd 8.11, found 8.09; Br: calcd
8.31, found 18.30.
ꢁ
amphenicol agar slants for 48 h at 30 C. Cell suspen-
sions in sterile distilled water were adjusted to give a
0
nyl)-2-propen-1-one (39). (0.29 g, 72%). IR (KBr) 1657
6
34
1
-(4 -Chloro-phenyl)-3-(2-chloro-6,7-dimethoxy-3-quinoli-
final concentration of 10 viable yeast cells/mL. Der-
matophytes: M. canis C 112, T. rubrum C 113, E. floc-
cosum C 114 and M. gypseum C 115 as well as Candida
tropicalis C131 are clinical isolates and were kindly
1
(
C¼O), 1574 (C¼C). H NMR (CDCl ) d 8.55 (d, 2H,
3
0 0
J=2.1 Hz, H2 , H6 ), 8.36 (s, 1H, H4), 8.30 (d, 1H,
J=15.70 Hz, Hb), 7.90 (d, 2H, J=8.0 Hz, Ar), 7.59 (d,
provided by CEREMIC, Centro de Referencia Micolo
gica, Facultad de Ciencias Bioquımicas y Farm-
aceuticas, Suipacha 531-(2000)-Rosario, Argentina.
´
-
2
H, J=8.42 Hz, Ar), 7.55 (d, 1H, J=15.70 Hz, Ha),
´
7
.35 (s, 1H, H8), 7.10 (s, 1H, H5), 3.99 (s, 6H, OCH3);
C NMR (CDCl ) d 189.7 (C¼O), 156.2 (C6, C7),
´
1
3
3
0
0
1
45.8 (C2), 140.5 (C1 , C8a), 139.2 (C4 ), 136.7
0
The strains were maintained on slopes of Sabouraud-dex-
trose agar (SDA, Oxoid) and subcultured every 15 days to
prevent pleomorphic transformations. Spore suspensions
0
0
0
(
(
(
Cb), 129.9 (C2 , C3 , C5 , C6 , C4, C4a), 124.0
C3), 122.9 (Ca), 118.1 (C8), 105.0 (C5), 56.9
OCH3), 56.8 (OCH3). MS m/z: [MꢀH]
+
34
387,
were obtained accordingto reported procedures
6
and
+
+
+
[
MꢀOCH ] 357, [Mꢀ29] 356, [Mꢀ77] 311. Anal.
adjusted to 10 spores with colony formingability/mL.
3
(
C H Cl NO ), C: calcd 62.01; found 62.02; H: calcd
20 15 2 3
3
1
.91; found 3.90; N: calcd 3.62, found 3.61; Cl: calcd
8.07, found 18.12.
Antifungal assays
The antifungal activity of chalcones was evaluated with
the agar dilution method by using Sabouraud-chlor-
amphenicol agar for both yeast and dermatophyte spe-
0 0
-(3 ,4 -Dimethoxy-phenyl)-3-(2-chloro-6,7-dimethoxy-3-
quinolinyl)-2-propen-1-one (40). (042 g, 92%). IR (KBr)
1
1
11,12
1
1
648 (C¼O), 1574 (C¼C). H NMR (CDCl ) d 8.30 (s,
cies as previously described.
Stock solutions of
3
H, H4), 8.18 (d, 1H, J=16.0 Hz, Hb), 7.91 (d, 2H,
compounds (10 mg/mL in DMSO) were diluted to give
serial two-fold dilutions that were added to each med-
ium resultingin concentrations ran ig ngfrom 0.10 to
50 mg/mL MIC for each compound was defined as the
lowest concentration that produces no visible fungal
growth after the incubation time.
0
.35 (s, 1H, H8), 7.10 (s, 1H, H5), 4.0 (s 6H, OCH ),
0
J=2.1 Hz, H2 , H6 ), 7.55 (d, 1H, J=15.70 Hz, Ha),
7
3
1
1
3
1
3
.99 (s, 6H, OCH3); C NMR (CDCl ) d 188.5 (C¼O),
3
0
53.1 (C6, C7), 147.8 (C3 ), 146.0 (C2), 138.6 (C8a),
0
38.9 (Cb), 133.2 (C1), 129.7 (C4 ), 125.7 (Ca), 120.7
0
0
0
(
5
C6 ), 117.8 (C8), 114.9 (C5 ), 112.9 (C2 ), 104.7 (C5),
+
6.6 (2 OCH ), 55.9 (OCH ). MS m/z: [MꢀH] 387,
3
3
+
Enzymatic assays
+
+
[
(
MꢀOCH ] 357, [Mꢀ29] 356, [Mꢀ77] 311. Anal.
3
C H ClNO ), C: calcd 63.91, found 63.89; H: calcd
5
Strains and culture conditions. The S. cerevisiae strain
used was MATa trpI ura3 leu2 his3 pep4::HIS3
nuc1::LEU2. Routine yeast growth (YEPD) was as
2
2
20
4
8
.88, found 4.90; N: calcd 3.39, found 3.35; Cl: calcd
.46, found 8.45.
3
5
described.
0 0
-(2 ,5 -Dimethoxyphenyl)-3-(2-chloro-6,7-dimethoxy-3-
quinolinyl)-2-propen-1-one (41). (0.40 g, 90%). IR (KBr)
1
Enzyme preparation. Cell extracts were obtained essen-
36
1
1
1
651 (C¼O), 1609 (C¼C). H NMR (CDCl ) d 8.30 (s,
tially as described previously. Early logarithmic phase
3
H, H4), 7.98 (d, 1H, J=16.0 Hz, Hb), 7.49 (d, 1H,
cells grown in 100 mL YEPD medium were collected,
washed once with 50 mM Tris–HCl pH 7.5, suspended
in 100 mL of the same buffer and broken with glass
beads in a FastPrep FP120 apparatus (Savant, BIO 101,
Inc.) (once a 15 s pulse at speed of 6.0). Broken material
was collected and cell debris was removed by low speed
0
J=15.70 Hz, Ha), 7.36 (s, 1H, H8), 7.26 (s, 1H, H6 ),
0
.02 (s 6H, OCH ), 3.86 (s, 6H, OCH ); C NMR
0
7
4
(
1
(
1
(
3
.12 (s, 1H, H5), 7.03 (dd, 2H, J=8.2, 2.2, H3 , H4 ),
1
3
3
3
0
53.2 (C2 , C7), 148.6 (C3 ), 14 (C2), 138.6 (C8a), 138.9
CDCl ) d 189.8 (C¼O), 155.2 (C6, C7), 152.0 (C5 , C6),
3
0
0
0
19.0 (C8), 105.1 (C5), 56.8 (OCH ), 56.3 (OCH ), 56.2
ꢁ
Cb), 134.5 (C1), 129.0 (C4a), 125.83 (Ca), 122.8 (C6 ),
centrifugation (5000ꢃg, 10 min at 4 C). The super-
ꢁ
natant was centrifuged at 18,000ꢃg for 30 min at 4 C
3
3
+
+
OCH ), 56.0 (OCH ). MS m/z: [Mꢀ1] 413, [Mꢀ29]
and the pellet was resuspended in 50 mM Tris–HCl pH
7.5 containing33% lgy cerol (at a concentration of
approximately 3 mgprotein per mL) and stored at
3
3
+
+
83, [Mꢀ31] 383, [Mꢀ138] 275. Anal. (C H ClNO ),
2
2
20
5
C: calcd 63.91; found 63.93; H: calcd 4.88, found 4.87; N:
calcd 3.39, found 3.38; Cl: calcd 8.46, found 8.47.
ꢁ
ꢀ80 C. Protein was quantified by the Bradford dye-
3
7
bindingprocedure usingthe Bio-Rad Protein Assay
Dye Reagent and bovine serum albumin as standard.
Biological Evaluation
ꢀ
(1,3)-Glucan synthase assay. b(1,3)-glucan synthase
3
6
Microorganisms and media
assay was essentially as described previously. The stan-
dard assay mixture contained 5 mL enzyme (15mgprotein),
in a total volume of 40mL. Two microliters of methanol or
the correspondingcompounds (kept in stock solution,
The microorganisms used for the fungistatic evaluation
were purchased from the American Type Culture

2012
S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
ꢁ
1
0 mg/mL in methanol at ꢀ20 C) were added to each
wish to thank Professor A. Duran (CSIC/Univ. de Sal-
´
ꢁ
reaction. The reaction was incubated for 30 min at 30 C
and stopped by addition of 1 mL 10% trichloroacetic
acid. All reactions were carried out in duplicate.
amanca) for helpingand supportingpart of this work.
This work is part of the Iberoamerican Project PIBEA-
FUN (Search and development of new antifungal
agents) from RIPRONAMED (Iberoamerican Network
on Medicinal Natural Products), part of the Iberoamer-
ican Program of Science and Technology for the Devel-
opment (CYTED). Collaboration from RIIDDMED
(Iberoamerican Network on Investigation and Develop-
ment of Medicines) of CYTED is gratefully acknowl-
edged. R.D.E. is a carrier researcher of CONICET.
The drugs Papulacandin B and Aculeacin A were gen-
erous gifts from K. Scheibli and P. Traxler (Novartis,
Basel, Switzerland) and K. Mizuno (Tokyo Jozo Co.
Ltd., Tagatagun, Shizuoka-ken, Japan), respectively.
The antibiotics were kept in stock solution (10 mg/mL
ꢁ
in methanol) at ꢀ20 C.
Chitin synthase-1 assay. Chitin synthase-1 assay was
3
8
performed as described previously.
The standard
References and Notes
assay mixture contained 10 mL enzyme (30 mgprotein),
in a total volume of 50 mL. Two microliters of methanol
or the correspondingcompounds (kept in stock solu-
1
. Barrett-Bee, K. J.; Ryder, N. S. In Emerging Targets in
Antibacterial and Antifungal Chemotherapy; Sutcliffe, J. M.,
Georgopapadakou, N. H., Eds.; Chapman and Hall: New
York, 1992; pp 410–436.
. Walsh, T. J. In Emerging Targets in Antibacterial and Anti-
fungal Chemotherapy; Sutcliffe J. M., Georgopapadakou, N.
ꢁ
tion, 10 mg/mL in methanol at ꢀ20 C) were added to
each reaction. Enzyme activation was performed by
partial proteolysis of the reaction mixture with 2 mL
2
ꢁ
trypsin (0.25 mg/mL) during15 min at 30 C and stopped
by the addition of 2 mL trypsin inhibitor (0.375 mg/mL).
The reaction was incubated for 90 min at 30 C and
H., Eds.; Chapman and Hall: New York, 1992; pp 349–373.
ꢁ
3. Caceres, A.; Lopez, B. R.; Giro
Ethnopharm. 1991, 31, 263.
. White, T. C.; Marr, K. A.; Bowdenn, R. A. Clin. Microbiol.
Rev. 1998, 11, 382.
. Selitrennikoff, C. P. Antifungal drugs: (1,3)-ꢀ-Glucan Syn-
thase Inhibitors; Springer: Heidelberg, 1995; pp 91–132.
´
n, M. A.; Logeman, H. J.
stopped by addition of 1 mL 10% trichloroacetic acid.
All reactions were carried out in duplicate.
4
5
Computational methods
6
5
7
. Georgopapadakou, N. H. Curr. Opin. Microbiol. 1998, 1,
47.
. Groll, A. H.; De Lucca, A. J.; Walsh, T. J. Trends Micro-
Gaussian 9839 has been used to perform RHF calcula-
tions in the compounds of interest. The calculations
have been carried out at both RHF/3-21G and RHF/6-
biol. 1998, 6, 117.
8. DiDomenico, B. Curr. Opin. Microbiol. 1999, 2, 509.
3
1G levels of theory.
9. Urbina, J. M.; Cortes, J. C. G.; Palma, A.; Lopez, S. N.;
´ ´
Zacchino, S. A.; Enriz, R. D.; Ribas, J. C.; Kouznetzov, V.
In a first step a systematic conformational search using
4
0ꢀ42
Bioorg . Med. Chem. 2000, 8, 691.
the GASCOS method developed by the San Luis
group was carried out. The compounds of interest were
1
0. Zacchino, S. A.; Lo
Santecchia, C. B.; Mun
A. M.; Enriz, R. D. J. Nat. Prod. 1999, 63, 1353.
1. Zacchino, S. A.; Rodrıguez, G. E.; Pezzenati, G.; Santec-
chia, C. B.; Giannini, F. A.; Enriz, R. D. J. Ethnopharm. 1998,
2, 35.
12. Zacchino, S. A.; Rodrı
´
pez, S. N.; Pezzenati, G.; Furla
´
n, R. L.;
˜
oz, L.; Giannini, F. A.; Rodrı
´
guez,
4
3,44
then modeled by the semi-empirical AM1 method.
After the structures were determined at RHF/3-21G
level of theory, partially relaxed scan calculations were
1
´
ꢁ
run for every 15 of rotation of the torsional angles f
6
and y. The minima obtained by the scan for each com-
pound were optimized at RHF/3-21G and RHF/6-31G
levels of theory. Frequency calculations performed at
the RHF/6-31G level ensured that the critical points
given as minima by the optimizations are indeed
minima on the potential energy surface.
´
guez, G. E.; Pezzenati, G.; Orel-
lana, G.; Enriz, R. D.; Gonzalez Sierra, M. J. Nat. Prod. 1997,
60, 659.
13. Rodrıguez, A. M.; Giannini, F. A.; Baldoni, H. A.; Suvire,
´
F. D.; Zacchino, S. A.; Sosa, C. P.; Enriz, R. D.; Csazar, P.;
Csizmadia, I. G. J. Mol. Struct. (TEOCHEM) 1999, 463, 283.
1
4. Tsuchiya, H.; Sato, M.; Akagiri, M.; Takagi, N.; Tanaka,
T.; Iinuma, M. Pharmazie 1994, 49, 756.
5. Sato, M.; Tsuchiya, H.; Akagiri, M.; Fujiwara, S.; Fujii,
T.; Takagi, N.; Matsuura, N.; Iinuma, M. Lett. Appl. Microb.
994, 18, 53.
AM1 and the RHF/3-21G optimized coordinates were
imported into PC Spartan.45 Single point calculations
were performed to generate the wave functions, using
PC Spartan, thus enablingthe display of LUMO and
MEP maps.
1
1
16. Gafner, S.; Wolfender, J. L.; Mavi, S.; Hostettman, K.
Planta Medica 1996, 62, 67.
1
1
7. Szajda, M.; Kedzia, B. Pharmazie 1989, 44, 190.
8. Cid, V. J.; Duran, A.; del Rey, F.; Snyder, M. P.; Nom-
nchez, M. Microbiol. Rev. 1995, 59, 345.
9. Klis, F. M. Yeast 1994, 10, 4381.
´
bela, C.; Sa
´
1
2
2
2
0. Wattanasin, S.; Murphy, W. S. Synthesis 1980, 647.
1. Meth-Cohn, O.; Narine, B. Tetrahedron Lett. 1978, 23, 2945.
2. Vogel, A. A Textbook of Practical Organic Chemistry, 3rd
Acknowledgements
ed.; Wiley: New York, 1956; Chapter IV, p 718.
3. Wachter-Jurcsak, N.; Zamani, H. J. J. Chem. Educ. 1998,
76, 653.
24. Dickinson, R.; Heilbron, I.; Irving, F. J. Chem. Soc 1927,
1888.
This work was supported by grants to SAZ (Agencia de
Promociones Cientı
´
2
ficas y Tecnolo
´
gicas de la Argentina
PICT # 06-06454); to SNL (Grantee IFS F/3114-1), to
RDE (Proyecto 81-01, Univ. Nac. San Luis). Authors

S. N. L o´ pez et al. / Bioorg. Med. Chem. 9 (2001) 1999–2013
38. Choi, W.; Cabib, E. Anal. Biochem. 1994, 219, 368.
2013
2
5. Bradsher, C. K.; Brow, F. C.; Blue, W. B. J. Am. Chem.
Soc. 1959, 71, 3570.
6. Iwata, S.; Nagata, N.; Omae, A.; Yamaguchi, S.; Okada,
Y.; Shibata, S.; Okuyama, T. Biol. Pharm. Bull. 1999, 22, 323.
7. Edwads, M. L.; Stemerick, D. M.; Sunkara, P. S. J. Med.
Chem. 1190, 33, 1948.
8. Herencia, F.; Ferra
Charris, J.; Lobo, G.; Alcara
39. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuresia,
G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.;
Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.;
Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.;
Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.;
Cammi, R.; Menucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.;
Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Mor-
okuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ciolowski, J.; Ortiz, J. V.; Baboul, A. G.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komar-
omi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-
Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.;
Challacombe, M.; Gill, P. M.; Johnson, B.; Chen, W.; Wong,
M. W.; Andres, J. L.; Gonzalez, C.; Head-Gordon, M.;
Replogle, E. S.; Pople, J. A. Gaussian 98, Revision A7; Gaus-
sian, Inc: Pittsburgh, PA, 1998.
2
2
2
´
ndiz, M.; Ubeda, A.; Domı
z, M. Bioorg. Med. Chem. Lett.
´
nguez, J.;
´
1
2
998, 8, 1169.
9. Devia, A. C.; Ferretti, F. H.; Ponce, C. A.; Tomas, F. J.
Mol. Struct. (TEOCHEM) 1999, 493, 187.
0. Lawrence, N. J.; McGown, A. T.; Ducki, S.; Hadfield,
J. A. Anti-Cancer Drug Des. 2000, 15, 135.
3
3
1. Rabinovich, D.; Shokked, Z. Acta Cryst. B 1974, 30, 2829.
2. Tkacz, J. In Emerging Targets in Antibacterial and
3
Antifungal Chemotherapy; Sutcliffe J. M., Georgopapada-
kou, N. H., Eds.; Chapman and Hall: New York, 1992; pp
4
3
95–523.
3. Cabib, E.; Kang, M. S.; Au-Young, J. In Methods in
40. Santagata, L. N.; Suvire, F. D.; Enriz, R. D.; Torday, V.;
Csizmadia, I. G. J. Mol. Struct. (TEOCHEM) 1999, 465, 33.
41. Santagata, L. N.; Suvire, F. D.; Enriz, R. D. J. Mol.
Struc. (TEOCHEM) 2000, 504, 35.
Enzymology; Abelson, J., Simmons, M., Eds.; Academic: San
Diego, 1990; Vol. 138, pp 643–669.
3
4. Wright, L.; Scott, E.; Gorman, S. J. Antimicrob. Che-
mother. 1983, 12, 317.
5. Alfa, C.; Fantes, P.; Hyams, J.; McLeod, M.; Warbrick,
42. Santagata, L. N.; Suvire, F. D.; Enriz, R. D. J. Mol.
Struc. (TEOCHEM) 2000, 536, 173.
43. Dewar, M. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. P.
J. Am. Chem. Soc. 1985, 107, 3902.
44. Stewart, J. J. In MOPAC 6.0 Manual: A Semiempirical
Molecular Orbital Program, 6th ed.; F.J. Seiler Research
Laboratory, Eds.; United States Air Force Academy: Boulder,
1990; pp 1–19.
45. PC Spartan Pro-User’s Guide; Wave Function Inc.: Irvine,
CA, 1999.
3
E. In Experiments with Fission Yeast: A Laboratory Course
Manual; Alfa, C., Fantes, P., Hyams, J., McLeod, M., War-
brick, Eds.; Cold SpringHarbor Laboratory: Cold Spring
Harbor, 1993.
3
6. Ishiguro, J.; Saitou, A.; Dura
iol. 1997, 179, 7653.
7. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
´
n, A.; Ribas, J. C. J. Bacter-
3