Investigations on the antimalarial activity of alkoxylated and hydroxylated chalcones 3

SECTION SIX
MODE OF
ANTIMALARIAL ACTIVITY OF CHALCONES

99
6. MODE OF ANTIMALARIAL ACTIVITY OF CHALCONES
6.1 Introduction
The mode of action of chalcones in malaria remains uncertain. As described in
the Introduction (Section 1.3.1) the antimalarial activity of chalcones came to the fore,
in part, due to a database search of compounds that could fit the active site of the
malarial cysteine proteases (falcipains).
72
In silico simulations indicated that several
antimalarial chalcones have an excellent fit onto the enzyme active site.
72
Subsequent
investigations explored this connection but so far, the correlation between antimalarial
activity and inhibition of falcipains has not been convincing.
74
In this section, the
likelihood of chalcones acting on targets in the digestive vacuole of Plasmodium is
investigated. The decision to focus on events involving hemoglobin degradation in the
digestive vacuole is prompted largely by earlier findings that chalcones are potential
cysteine protease inhibitors. The aspects investigated are the effects of selected
chalcones on the enzymatic breakdown of radiolabelled methemoglobin by a crude
plasmodial extract, the hydrolysis of a fluorogenic substrate by recombinant
plasmodial cysteine protease (falcipain-2) and binding to hematin. The results
obtained from these experiments are presented and discussed in the following

paragraphs.

6.2 Materials and Methods
The following chemicals were purchased from Sigma Chem. Co. (MO, USA):
[
14
C] methemoglobin (30 mCi/g, 0.17 mg/ml), pepstatin A, L-trans-epoxysuccinyl-
leucylamido-(4-guanidino)-butane (E64), benzyloxycarbonyl-Phe-Arg-7-amino-4-
methylcourmarin (Z-Phe-Arg-AMC), porcine hematin (ferriprotoporphyrin IX
hydroxide), chloroquine diphosphate. Other reagents were of analytical grade.
100

6.2.1 Degradation of [
14
C] methemoglobin by extracts of P. falciparum (K1)
P. falciparum (K1)-infected erythrocytes were synchronized at least twice
using 5% sorbitol to yield cultures of trophozoites at approximately 20% parasitemia.
The infected cells were harvested, washed with PBS, treated with 0.1% (w/v) saponin
in PBS, washed 3 × with ice-cold PBS and centrifuged (1000 g, 10 min, 4
o
C). Water
was added to the resulting pellet to give a solution which was subjected to 2 freeze-
thaw cycles, centrifuged at 13,000 g, 10 min, 4
o
C to give supernatant containing crude
parasite extracts (lysate). Following a reported method
125
, aliquots of the lysate (25
µ
l, estimated to contain 1-1.5

µ
g protein /
µ
l) and test compound in DMSO (10
µ
l) was
added to 50 µl sodium acetate (0.1 M, pH 6.0) and sufficient distilled water to give a
final volume of 100
µ
l. The final concentration of test compound was 100
µ
M. After
1 h of incubation at 37
o
C, 5 µl of [
14
C] methemoglobin (30 mCi/g, 0.17 mg/ml) was
added. Incubation was continued for another 3 h after which additions of bovine serum
albumin (50 µl, 3 mg/ml) and 50% w/v trichloroacetic acid (100 µl) were made. The
samples were incubated for another 30 min, 4
o
C before centrifugation at 13,000 g, 4
o
C.
Aliquots (100 µl) of the supernatant were taken, added to 4 ml of scintillation fluid and
radioactivity was determined with a scintillation counter. Controls consisted of
samples processed without test compound ([
14
C] methemoglobin and lysate) and
samples containing only [

14
C] methemoglobin in the incubation mixture. The same
volume of DMSO used to deliver test compound was added in both controls.

6.2.2 Inhibition of falcipain-2
Experiments on the inhibition of falcipain-2 were not carried out by the
candidate but were tested in Dr Philip Rosenthal’s laboratory in the School of
Medicine, University of California, San Francisco, USA. Briefly, soluble parasite
101
extracts containing falcipain-2 were incubated with dithiothreitol (10 mM) and test
compound in sodium acetate buffer (0.1 M, pH 5.5) for 30 min at room temperature,
after which the fluorogenic substrate Z-Phe-Arg-AMC was added to give a final
concentration of 50 µM.
126
Cleavage of the substrate caused an increase in
fluorescence (due to the free coumarin) which was monitored for 30 min at excitation
and emission wavelengths of 380 nm and 460 nm respectively. An inhibitor of
falcipain will cause fluorescence to decrease. The rates of hydrolysis of the substrate
in the presence and absence of test compound were determined. Fluorescence from
control cuvettes containing only substrate or compound was also monitored. Test
compounds were monitored at different concentrations to give IC
50
values from rates
versus concentration plots.
Chalcones which inhibited falcipain-2 with IC
50
values of ≤ 10 µM were
further investigated for the appearance of an abnormal food vacuole in P. falciparum
trophozoites. Ring-stage parasites were incubated with the test compound for 24 h,
after which Giemsa-stained smears were prepared and evaluated microscopically for

abnormal morphology.


6.2.3 Effect on Soret band of hematin
The interaction of chalcones with porcine hematin was investigated by
monitoring changes in the Soret band of hematin, following a reported method.
25

Appropriate aliquots of hematin (2 mM stock solution in 0.1 M NaOH), test compound
(stock solutions of 2 mM or 0.2 mM in methanol) were added to a cuvette (1 ml)
containing 43% methanol in sodium acetate buffer (10 mM, pH 5.5), to give final
concentrations of 14 µM hematin and 2-128 µM test compound. The solution was
vortexed for 15 s and the spectrum was collected from 250 -650 nm. Under these
conditions, the Soret band of hematin was observed at 400 nm. Some of the test
102
compounds have strong absorbance in the range of 380-420 nm at concentrations >
100 µM. Thus correction for background absorbance was made using as blank, a
solution containing test compound in buffer with no hematin. Chloroquine (2-128
µ
M) was used as a positive control. The decrease in Soret band absorbance was
expressed as a % of the control absorbance obtained in the absence of test compound:
% Decrease in absorbance =
(Absorbance
Control
– Absorbance
test compound
) / Absorbance
Control
×
100

6.3 Results
6.3.1 Effects on the enzymatic activity of a crude plasmodial extract catalyzing the
breakdown of radiolabelled methemoglobin
A crude plasmodial extract was prepared by saponinizing P. falciparum infected
erythrocytes to release the intraerythrocytic parasites. The extract has enzymatic
activity and catalyzes the breakdown of [
14
C] methemoglobin, an oxidized and
denatured derivative of hemoglobin, into smaller radiolabelled peptide /amino acid
fragments that are recovered in the supernatant. Pepstatin A (a specific inhibitor of
aspartate protease) and E64 (a specific cysteine protease inhibitor) at 100 µM inhibited
the enzymatic activity of the extract to about the same extent (32-38%)
A search of the literature indicated that there were no reports of the inhibitory
activities of these compounds on a plasmodial extract prepared from P. falciparum for
comparison with the present results. However, Pandey and coworkers
127
had
investigated the effects of pepstatin A and E64 on the enzymatic activity of a crude P.
yoelii extract. They found that the breakdown of radiolabelled methemoglobin was
reduced by 71% and 26% in the presence of pepstatin and E64 (both at 100 µM)
respectively. Noting that P. yoelii is a murine strain, the inhibitory effects of pepstatin
103
A and E64 were investigated using extracts prepared in a similar manner from another
murine plasmodia (P. berghei ANKA). This time, the levels of inhibition (21%, 62 %
for 100 µM E64, pepstatin A respectively) were comparable to those obtained with P.
yoelii.
Fifteen of the twenty “actives” were tested for inhibitory activity on the
breakdown of methemoglobin at a fixed concentration of 100 µM. Less active

chalcones
41
and
125
were also included for comparison (
Table 6.1
). As seen from
Table 6.1, varying levels of inhibition are observed among the chalcones. No
inhibitory activity was noted for several active members (6, 8, 19, 113) but both the
inactives (41, 125) inhibited enzymatic activity by 26-38%. On the other hand,
maximum inhibition (45 %) was noted for 27 which had the highest in vitro
antimalarial activity (IC
50
2 µM) for the present series of chalcones. Overall, no
discernible trend is evident from the results.
Table 6.1

6.3.2 Effects on falcipain-2 and associated changes in the food vacuole on
incubation
The same chalcones that were tested for activity on the crude plasmodial
extract were investigated for their effects on falcipain-2-catalyzed hydrolysis of the
fluorogenic substrate Z-Phe-Arg-AMC. An arbitrary cut-off concentration of 10
µ
M
was used to distinguish between chalcones with inhibitory activity (IC
50
< 10 µM) and
those without activity. Based on these criteria, 8 chalcones comprising of two inactives
(41, 125) and six actives (2, 5, 211, 227, 228, 234) were found to be inhibitory (Table
6.1). Interestingly, the actives are derived from only two classes of ring B substituted

chalcones, namely the hydroxylated chalcones (211, 227, 228, 234) and the
104
dimethoxychalcones (2, 5). The 3-quinolinyl chalcone 27 which inhibited
methemoglobin breakdown to the greatest extent was not among the falcipain-2
inhibitors. Strongest falcipain-2 inhibitory activity was found in the ethoxychalcone
125 (IC
50
1.4 µM) which had weak in vitro antimalarial activity (IC
50
39 µM). It is
clear that there is no correlation between falcipain-2 inhibition, inhibition of
methemoglobin breakdown and in vitro antimalarial activity.
Inhibition of falcipain-2 is accompanied by visible changes in the digestive
vacuole of Plasmodium, namely the appearance of swollen vacuoles filled with
undegraded hemoglobin. These changes were not evident when the chalcones that
inhibited falcipain-2 with IC
50
values of < 10 µM were incubated with the ring-stage
parasites. A similar finding was reported for some antimalarial phenothiazines that
inhibited falcipain-2 but did not cause the expected changes in the food vacuole.
74
The
proffered explanation was that the phenothiazines were cytotoxic and had other effects
on parasite morphology (cytoplasmic vacuolization) that overshadowed changes in the
food vacuole. This may be true for the chalcones as well, but this would be difficult to
reconcile with the results of the MTT assay which showed that these chalcones were
essentially not cytotoxic at 20
µ
M (Section 4.3.3). One possibility is that these results
reflect the limitations on the transport of these chalcones into the food vacuole that

would be necessary before detection of morphological changes becomes evident. That
is, the chalcones may be able to inhibit the enzyme when it is isolated (as in an in vitro
assay) but may not be able to gain access into the food vacuole readily to cause the
anticipated morphological changes. The present series of chalcones are neutral or
weakly basic compounds and will not be trapped in the acidic food vacuole in the same
way as the more basic aminoquinolines like chloroquine (pKa values = 8.1, 10.2).

6.3.3 Binding to heme
105
Degradation of hemoglobin results in the formation of toxic heme, the disposal
of which has been the target of several antimalarial drugs.
4
In the food vacuole, heme
is formatted to non-toxic hemozoin. Compounds that interfere with this process are
characterized by binding to heme. In this investigation, the interaction of the
chalcones with hematin was investigated in an acetate buffer with an apparent pH 5.5
to mimic the acidic pH of the plasmodial food vacuole.
25
The buffer contained a high
proportion of methanol (43%) to keep heme in solution and in the monomeric state.
Under these conditions, hematin displays a Soret band at 400 nm with a shoulder at
360 nm. When a test compound binds to heme, a decrease in the Soret band
absorbance is observed. This is illustrated with chloroquine which was used in this
investigation as a positive control. The incubation of hematin with chloroquine (2-128
µM) caused a concentration-dependent fall in the Soret band absorbance (Figure 6.1),
similar to that reported by other investigators.
25
At the highest concentration (128 µM)
of chloroquine, the observed absorbance was approximately 48% of the control Soret
band absorbance. That is, chloroquine has reduced Soret band absorbance by 52% at

this concentration.
36 chalcones were screened for changes in Soret band absorbance. These
included 19 active chalcones (except 234) in Table 6.1 as well as 16 other less active
members. Table 6.2 lists the % decrease in the absorbance of the Soret band in the
presence of 128 µM test compound. A compound that binds to hematin should cause a
large drop in absorbance. Only compounds that decreased absorbance by more than
10% were considered to have an effect on the binding interaction with hematin. 14
such compounds were identified, but only five of these compounds are actives (7, 113,
207, 211, 228). The greatest reduction in the Soret band absorbance (64.5%) was
observed for the dihydroxychalcone 205. A concentration-dependent effect was also
106
evident for these compounds, that is increasing concentrations resulted in a greater
reduction in the Soret band absorbance.
The results show a clear structural trend among chalcones that bind to hematin.
Binding is observed mostly among the hydroxylated chalcones, in particular, the 2’-
hydroxychalcones. There is also a strong preference for naphthalene and pyridine
rings among the hematin-binding chalcones. In contrast, quinoline and the usual
benzenoid ring A are conspicuously under-represented. Since the structural features
that predispose towards binding to hematin are not the same as those associated with
good antimalarial activity, it is probable that heme binding does not contribute
significantly to antimalarial activity. However, heme binding may account to some
extent for the activity of the less active chalcones like the 2’-hydroxychalcones.
Figure 6.1
Table 6.2

107
Table 6.1 Effect of antimalarial chalcones on the enzymatic activity of a crude P.
falciparum K1 extract using [
14
C] methemoglobin as substrate, in vitro
falcipain inhibition and cell viability.
O
B
A RR'

Substitution on No.
Ring B Ring A
IC

50
(µM)
in vitro
antimalarial
activity
b


KB3-1 cell
viability (%)
c

(SD)
% inhibition of
[
14
C] MetHb
breakdown at
100 µM (SD)
IC
50
(µM)
Falcipain
inhibition
3
6
27
36

2,3,4-trimethoxy 2,4-dichloro

4-trifluoromethyl
3-quinolinyl
a
4-fluoro

5.4
3.0
2.0
9.5
88.8 (17.5)
80.8 (15.0)
77.8 (23.5)
126.1 (19.7)

10.8 (4.0)
0
45.1 (16.0)
25.8 (5.3)
>10
>10
>10
>10
2
5
7
8
29

2,4-dimethoxy 4-trifluoromethyl
2,4-difluoro

2,4-dimethoxy
4-ethyl
3-quinolinyl
a

5.9
6.2
2.1
2.4
2.2

99.6 (18.3)
107.8(7.5)
84.0 (20.5)
85.4 (8.2)
82.6 (17.1)

27.2 (12.4)
32.1 (1.9)
30.8 (16.2)
0
ND

2.3
7.8
>10
>10
ND
19
31

38
113

4-methoxy 4-hydroxy
3-quinolinyl
4-fluoro
2,4-dimethoxy
7.0
4.8
14.4
6.4
78.9(16.7)
82.9(15.2)
ND
121.6(13.3)
0
ND
ND
0

>10
ND
ND
>10
125
127
4-ethoxy 4-nitro
4-cyano

39.0

540.0
ND
ND
25.6 (12.8)
ND
1.4
>10
202
203
207
208
211
2,4-dihydroxy 3-quinolinyl
a
2,4-difluoro
2-pyridinyl
a
2-naphthalenyl
a
4-chloro

16.1
16.0
19.7
20.0
12.3
89.3(28.5)*
88.9(22.1)*
96.4(19.3)*
ND

100.7(32.0)*
ND
ND
9.4 (3.7)
ND
0
ND
ND
>10
ND
9.8
214
227
228
4-hydroxy 2-pyridinyl
a
3,4-dichloro
4-dimethylamino
16.3
18.4
17.7

112.3 (18.2)*
90.6 (23.3)*
84.8 (32.2)*
ND
40.7 (9.7)
39.0 (14.3)

ND

2.4
9.7
234 2-hydroxy 4-chloro

12.9 ND 14.7 (8.6) 2.3
Chloroquine
E64
Pepstatin A
0.27
4.0
80.0

104.1 (17.5)

ND
ND
ND
38.2
d
(16.3)
31.5
d
(5.3)
ND
ND
ND

ND = not done. SD is given in parentheses.
a. Phenyl ring A is substituted by heterocyclic or naphthalene ring.
b. Inhibition of [

3
H] hypoxanthine uptake into P. falciparum K1 infected erythrocytes.

c. Mean of 2 or more determinations. Compounds are tested at 20 µM except for chloroquine
(80 µM) and those marked with * (40 µM).

d. % inhibition of extracts prepared from P. berghei (ANKA) infected erythrocytes for E64
and pepstatin A were 24.3 (12.9) and 62.2 (18.7) respectively.
108
Figure 6.1 Spectra of heme with CQ at pH 5.5



Absorbance of heme at ( _____ ) 0 µM, ( _____ ) 2 µM, ( _____ ) 4 µM, ( _____ ) 8
µM, ( _____ ) 16 µM, ( _____ ) 32 µM, ( _____ ) 64 µM, and ( _____ ) 128 µM CQ.




109
Table 6.2 Effect of chalcones on Soret band absorbance of hematin

Number


Ring B
a

Ring A
a

IC
50
(µM) for
In vitro
antimalarial
activity
% Decrease in
absorbance of
hematin
b
(SD)
3
6
27
28
36
2,3,4-trimethoxy 2,4-dichloro
4-trifluoromethyl
3-quinolinyl
4-quinolinyl
4-fluoro
5.4
3.0
2.0
60.0
9.5
< 10
<10
<10

<10
<10
2
5
7
8
29
30
110
2,4-dimethoxy 4-trifluoromethyl
2,4-difluoro
2,4-dimethoxy
4-ethyl
3-quinolinyl
4-quinolinyl
1-naphthalenyl
5.9
6.2
2.1
2.4
2.2
27.0
320.0
<10
<10
42.0 (2.3)
<10
<10
13.5 (2.2)
52.6 (3.3)

19
31
32
113
4-methoxy 4-hydroxy
3-quinolinyl
4-quinolinyl
2,4-dimethoxy
7.0
4.8
43.0
6.4
<10
<10
<10
47.4 (1.0)
33
34
4-ethoxy 3-quinolinyl
4-quinolinyl
24.9
100.0
<10
<10
202
203
205
207
208
210

211
2,4-dihydroxy 3-quinolinyl
2,4-difluoro
1-naphthalenyl
2-pyridinyl
2-naphthalenyl
4-quinolinyl
4-chloro
16.1
16.0
24.8
19.7
20.0
92.8
12.3
<10
<10
64.5 (4.5)
32.4 (4.4)
44.7 (11.7)
<10
14.4 (1.8)
212
213
214
215
216
227
228
4-hydroxy 1-naphthalenyl

3-quinolinyl
2-pyridinyl
4-quinolinyl
2-naphthalenyl
3,4-dichloro
4-dimethylamino
39.9
41.0
16.3
51.0
27.5
18.4
17.7
48.0 (3.4)
<10
<10
<10
26.0 (2.5)
<10
15.8 (3.4)
233
241
243
244
2-hydroxy 3-quinolinyl
2-pyridinyl
2-naphthalenyl
4-quinolinyl
28.0
31.0

29.5
Not Done
31.8 (7.9)
16.5 (0.4)
59.1 (7.2)
63.0 (5.4)
chloroquine 0.27 52.4 (0.6)

a. Structure of chalcones is given in Table 6.1.
b. Monitored at 400 nm, pH 5.5. Compounds are tested at 128 µM, except for 228 (64 µM).
Mean (SD) for 3 or more determinations.



110
6.4 Discussion
The present investigations reveal that chalcones do interfere with the various
stages associated with the degradation of hemoglobin. However, their activities are
unlikely to contribute significantly to antimalarial activity. This can be seen from the
following evidences. Firstly, inhibition of the enzymatic activity of the plasmodial
extract was observed at a relatively high concentration (100 µM) of the chalcone, and
even then, the maximum level of inhibition detected was modest (45% inhibition).
More importantly, correlation between inhibition and in vitro antimalarial activity is
lacking. Secondly, moderate inhibition (IC
50
< 10 µM) of falcipain-2 was observed for
several chalcones including the weakly active members (e.g. 125), but the expected
morphological changes in the parasite which accompanies such inhibition (swollen
food vacuoles filled with undegraded hemoglobin) were not evident. This could be
due to problems associated with the diffusion / retention of the chalcones within the

food vacuole. Thus the chalcone template may predispose chalcones to inhibition of
falcipain-2, as predicted from in silico studies and observed in this and other in vitro
studies, but falcipain-2 inhibition alone is unlikely to account for the antimalarial
activity of the chalcones. Finally, there is little evidence of significant binding to
hematin among the active chalcones. Decreases in the Soret band of hematin were
observed mainly for 2’-hydroxychalcones that have naphthalene and pyridine rings,
and to a lesser extent among alkoxylated chalcones and chalcones that have other types
of Ring A. The planarity of naphthalene and pyridine rings may favor π-π interactions
between the electron clouds of these rings and the porphyrin ring of hematin, although
the structurally related quinoline ring is noticeable in its exclusion from similar
interactions. Earlier studies
135
have shown that only quinolines with amino functions
(like 2 and 4-aminoquinolines) bind with strong affinity to heme. This would explain
111
the results observed with the quinolinyl chalcones which are essentially weak bases
(pKa ≈ 4-5) but does not explain why the non-basic naphthalenyl chalcones or the
pyridinyl chalcones which are also weak bases (pKa ≈ 4-5) should behave otherwise.
Since these structural features (2-hydroxy on Ring B, naphthalene and pyridine as Ring
A) are not associated with chalcones having good antimalarial activity, the lack of
correlation between in vitro antimalarial activity and heme binding is to be expected.

6.5 Conclusion
The results of this study have shown that chalcones do interfere with critical
processes that affect the growth of the intraerythrocytic plasmodia, namely enzymatic
degradation of hemoglobin and binding to heme. But it is unlikely that interference
with these processes is solely responsible for the in vitro antimalarial activity of
chalcones. The targets considered here are localized in the digestive vacuole of
Plasmodium which would mean that the chalcones must gain access into this
compartment before they can interfere with hemoglobin breakdown. Access may be

gained by diffusion across the vacuole membrane, which is highly probable as the
chalcones are lipophilic compounds. However, most of the chalcones are not basic
molecules. Even those that have basic nitrogen atoms (Ring A = quinoline, pyridine or
carry an aromatic amino substituent) are only weakly basic with pK
a
values of around
4-5. In the acidic food vacuole (pH 5 -5.2), there would be approximately equal
amounts of protonated and non-protonated species, and the latter can readily diffuse
out of the food vacuole. Thus, the “ion-trapping” or “weak-base” mechanism widely
ascribed to explain the accumulation of chloroquine in the food vacuole is unlikely to
be applicable to the chalcones.
112
It is possible that chalcones may interfere with targets outside the food vacuole.
Ginsburg and coworkers
128
have proposed that monomeric heme exits the food
vacuole and is subsequently degraded by reaction with glutathione in the parasite
cytosol. Drugs like chloroquine
128, 129
and clotrimazole
130
are reported to form
complexes with heme, thus interfering with its degradation by glutathione. The
resulting drug-heme complex is toxic and contributes to cell death. Chalcones may
also interfere with transport pathways present in infected erythrocytes. Several
flavonoids have been reported to inhibit the passage of essential solutes via parasite-
induced pathways on the host erythrocyte.
51, 52
Chalcones which are biosynthetic
precursors of flavonoids may have a similar effect.

SECTION SEVEN
CONCLUSION

113
7. CONCLUSIONS
It is appropriate at this juncture to consider the original hypotheses which have
been the driving force of this investigation and to evaluate how well the questions can
now be answered as a result of the work presented in this thesis.
The 1
st
hypothesis is that oxygenated chalcones exhibits antimalarial activity
and that an optimal substitution pattern exists which will favor activity. 102 chalcones
have been synthesized and evaluated for in vitro antimalarial activity. Of these, only
13 members have IC
50
values of ≥ 100 µM. Therefore, about 13% of the synthesized
compounds can be classified as inactive. This is a relatively low attrition rate and it is
appropriate to conclude that the presence of oxygenated groups like hydroxyl and
alkoxy predisposes chalcones to acceptable antimalarial activity.
As to whether there is an optimal substitution pattern for activity, the structure
activity studies which have been carried out imply that such a pattern exists.
Multivariate analysis identifies size and partitioning (log k
w
) characteristics to be
important parameters of active chalcones. Input from multiple linear regression and
CoMFA suggests that this should be a large-size ring B (di or tri substituted) and a
polar ring A substituted with electron withdrawing groups. Suitably designed
compounds may be synthesized in the near future to test the reliability of the structure-
activity correlations reached in this study.

The 2
nd
hypothesis proposes that similar structural requirements exist for
antimalarial and antileishmanial activities. The results of the present investigation
suggest that this is not true. Good antimalarial activity is mostly associated with
alkoxylated chalcones, unlike antileishmanial activity which is found predominately
among the hydroxylated chalcones. QSAR studies propose that the size of ring B and
the polarity of ring A contribute to antimalarial activity. In contrast, ring A appears to
114
have a higher profile than ring B in antileishmanial activity. In addition, the size rather
than electrostatic nature of ring A appears to be more important. Not withstanding
these conclusions, the present study has identified two chalcones (alkoxylated
derivatives
8
and
19
) that combine good antimalarial and antileishmanial activities.
8

is of particular interest as it is one of two chalcones that is capable of increasing the
survivability of P. berghei ANKA infected mice.
The last hypothesis states that oxygenated chalcones would act on multiple
targets in Plasmodium. In this study, only the processes involved in the breakdown of
hemoglobin in the Plasmodium food vacuole were investigated. The results do support
the view that particular chalcones interfere with several processes (heme binding,
falcipain-2 inhibition, methemoglobin degradation) in hemoglobin degradation, but at
concentrations that do not match their antimalarial IC
50
values. In addition, there are
several confounding examples of compounds with poor antimalarial activity but good

falcipain-2 inhibitory activity or heme binding properties. This has led to the
conclusion that hemoglobin breakdown is an unlikely target of oxygenated chalcones.
Not withstanding their interference at various stages of hemoglobin breakdown, the
chalcones appear to be selective in their antiplasmodial activity at the concentrations
employed. The MTT assay using a human cervical carcinoma epithelial cell line
(KB3-1) showed that chalcones were not cytotoxic at the concentrations used for in
vitro antimalarial tests. Thus, the chalcones specifically target Plasmodium at the
concentrations used.
In conclusion, the present work has pointed to new directions that could be
pursued to unravel the yet unanswered question of how chalcones exert their
antiplasmodial activity. For example, a careful electron microscope-based study of
chalcone-treated cells may point to sites of damage evoked by these compounds.
115
Alternatively, a careful analysis of the stage-dependency of killing, or an examination
of the synergistic or antagonistic effects of anti-oxidants or other drugs may be useful.
The effects of chalcones on other plasmodial targets such as transport processes or the
biosynthesis of critical macromolecules in the infected erythrocytes may also be
helpful.


APPENDICES

A-1

Appendix Table 1 Physical and analytical data of synthesized chalcones

Compd
No.
Melting point (°C)
(Recryst. solvent)
a


Yield
(%)

Elemental Analysis
Accurate Mass
(M+1)
IR (cm
-1
)
1
H-NMR (δ ppm)
3
105.1-107.6 (A)
108-109
b

93.1
C: calcd, 59.01 found, 58.78

H: calcd, 4.41 found, 4.29
Cl: calcd, 19.11 found, 19.48
366.0443
(C
18
H
16
O
4
Cl
2

= 366.0426)
1658.48
(υC=O)
In CDCl
3
, 8.02-7.97 (d, J=15.858Hz, βH) 7.50-7.45 (d, J=15.693Hz,
αH) 6.8-8.02 (m, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.94- 3.92 (t, OCH
3
)
13
C-NMR: 190.164 (C=O)

4
95.9-99.7 (B)
168.1-169.3
c

30.0

C: calcd, 70.35 found, 70.06
H: calcd, 6.79 found, 6.64
N: calcd, 4.11 found, 4.10
341.1602
(C
20
H
23
O
4
N =
341.1627)
1642.09
(υC=O)

In CDCl
3
, 7.67-7.61 (d, J=15.688Hz, βH) 7.29-7.24 (d, J=15.653Hz,
αH) 6.52-7.79 (m, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.92-3.88 (t, OCH
3
)
3.03 (s, N(CH
3
)
2
)
13
C-NMR: 191.324 (C=O)
6
e

82.8-86.0 (A) 30.8
C: calcd, 62.28 found, 61.79
H: calcd, 4.68 found, 4.57
F: calcd, 15.57 found, 15.74
366.1071
(C
19
H
17
O
4
F
3
=
366.1079)
1657.52
(υC=O)
In CDCl
3
, 7.72-7.66 (d, J=16.044Hz, βH) 7.61-7.56 (d, J=15.824Hz,
αH) 6.76-7.73 (m, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.94- 3.93(d, OCH
3
)
13
C-NMR: 190.199 (C=O)
11
e
81.2-84.4 (A) 45.0

C: calcd, 67.01 found, 66.87

H: calcd, 6.19 found, 6.01
358.1428
(C
20
H
22
O
6
=
358.1416)
1655.59
(υC=O)
In CDCl
3
, 7.97-7.92(d, J=15.908Hz, βH) 7.46-7.40 (d, J=15.985Hz,
αH) 6.46-7.97 (m, 5’H, 6’H, 3H, 5H, 6H) 3.92-3.84 (m, OCH
3
)
13
C-
NMR: 191.748 (C=O)
12
e
106.3-111.2 (A) 70.8
C: calcd, 73.05 found, 72.81
H: calcd, 6.46 found, 6.50

312.1352
(C
19

H
20
O
4
=
312.1362)
1658.48
(υC=O)
In CDCl
3
, 7.69-7.64 (d, J=15.852Hz, βH) 7.48-7.42 (d, J=15.819Hz,
αH) 6.74-7.70 (m, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.91-3.93(t, OCH
3
)
2.38 (s, CH
3
)
13
C-NMR: 191.022 (C=O)
13
e
66.6-69.6 (A) 86.7

C: calcd, 73.59 found, 73.84
H: calcd, 6.80 found, 6.66
326.1538
(C
20
H
22

O
4
=
326.1518)
1598.70
(υC=O)
In CDCl
3
, 7.70-7.65 (d, J=15.825Hz, βH) 7.48-7.43 (d, J=15.817Hz,
αH) 6.7-7.7 (m, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.98-3.87(t, OCH
3
)
2.72-2.64 (q, CH
2
) 1.28-1.23 (t, CH
3
)
13
C-NMR: 191.054 (C=O)
27
e
135.0-136.5 (A) 37.7
C: calcd, 72.18 found, 72.30
H: calcd, 5.49 found, 5.25
N: calcd, 4.01 found, 4.07
349.1330
(C
21
H
19

O
4
N =
349.1314)

In CDCl
3
, 9.20 (s, 2H) 7.88-7.83(d, J=16.2Hz, βH) 7.62-7.56 (d,
J=16.2Hz, αH) 8.30-6.78 (m, 4H, 5H, 6H, 7H, 8H, 5’H, 6’H) 3.96-
3.94 (t, OCH
3
)
13
C-NMR: 190.016 (C=O)
28
e

195.0-197.0
(A)
10.0
C: calcd, 72.18 found, 71.93
H: calcd, 5.49 found, 5.33
N: calcd, 4.01 found, 4.05
349.1336
(C
21
H
19
O
4

N =
349.1314)

In DMSO, 9.19-9.17 (d, 2H) 8.35-8.30(d, J=15.45Hz, βH) 7.91-7.86
(d, J=15.44Hz, αH) 8.53-7.00 (m, 3H, 5H, 6H, 7H, 8H, 2’H, 3’H,
5’H, 6’H) 3.91-3.87 (t, OCH
3
)
13
C-NMR: 188.647 (C=O)
35
e
97.2-101.8 (A) 63.2

C: calcd, 69.48 found, 69.43
H: calcd, 6.14 found, 6.09
328.1292
(C
19
H
20
O
5
=
328.1311)
1649.80
(υ
C=O
)
In CDCl

3
, 7.68-7.63 (d, J=15.83Hz, βH) 7.39-7.34 (d, J=15.45Hz,
αH) 6.74-7.68 (m, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.99-3.85 (m, OCH
3
)
13
C-NMR: 191.030 (C=O)
A-2

36
e
87.5-94.2 (A) 41.0
C: calcd, 68.33 found, 68.31
H: calcd, 5.42 found, 5.60
F: calcd, 6.01 found, 5.90
316.1132
(C
18
H
17
O
4
F =
316.1111)
1663.30
(υ
C=O
)
In CDCl
3

, 7.68-7.63 (d, J=15.83Hz, βH) 7.46-7.41 (d, J=15.82Hz,
αH) 6.75-7.86 (m, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.93-3.92 (m, OCH
3
)
13
C-NMR: 190.605 (C=O)
40
e
116.0-118.0 (A) 74.6
C: calcd, 76.97 found, 76.89
H: calcd, 5.93 found, 5.88

374.1503
(C
24
H
22
O
4
=
374.1518)

In CDCl
3
, 7.76-7.71 (d, J=15.83Hz, βH) 7.49-7.44 (d, J=15.83Hz,
αH) 6.75-7.76 (m, 5’H, 6’H, 2H, 3H, 5H, 6H, 2’’H, 3’’H, 4’’H,
5’’H, 6’’H) 3.93 (m, OCH
3
)
13

C-NMR: 190.293 (C=O)
128
e
78.0-80.2 (A) 38.2
C: calcd, 64.65 found, 64.43
H: calcd, 4.83 found, 4.66
F: calcd, 11.37 found, 11.22
334.1017
(C
18
H
16
O
4
F
2
=
334.1017)

In CDCl
3
, 7.57-7.52 (d, J=16.17Hz, βH) 7.46-7.44 (d, J=16.06Hz,
αH) 7.83-6.45 (m, 5’H, 6’H, 3H, 5H, 6H) 3.93-3.91 (t, OCH
3
)
13
C-NMR: 189.682 (C=O)
129
e
155.7-158.0 (C) 55.5

C: calcd, 62.95 found, 62.90
H: calcd, 4.99 found, 4.73
N: calcd, 4.08 found, 4.46
343.1066
(C
18
H
17
O
6
N =
343.1055)
1663.30
(υ
C=O
)
In CDCl
3
, 7.62 (m, J = 15.83Hz, βH), 7.57 (m, J = 14.69Hz, αH)
6.77-8.28 (m, 2H, 3H, 5H, 6H, 5’H, 6’H) 3.93-3.95 (t, OCH
3
)
13
C-NMR: 189.494 (C=O)
130
e
97.9-98.9 (C) 47.3
C: calcd, 59.01 found, 58.95
H: calcd, 4.41 found, 4.33
Cl: calcd, 19.11 found, 20.57

366.0427
(C
18
H
16
O
4
Cl
2
=
366.0425)
1656.55
(υ
C=O
)
In CDCl
3
, 7.50 (m, J = 15.44Hz, βH), 7.44 (m, J = 15.45Hz, αH)
6.75-7.69 (m, 2H, 5H, 6H, 5’H, 6’H), 3.92-3.94 (t, OCH
3
)
13
C-NMR: 189.745 (C=O)
131
e
100.6-102.0 (A) 39.0
C: calcd, 65.04 found, 64.82
H: calcd, 5.16 found, 5.13
Cl: calcd, 10.53 found, 10.62
332.0817

(C
18
H
17
O
4
Cl =
332.0815)
1661.37
(υ
C=O
)
In CDCl
3
, 7.56 (m, J = 15.45Hz,
β
H), 7.46 (m, J = 15.82Hz,
α
H)
6.75-7.66 (m, 2H, 3H, 5H, 6H, 5’H, 6’H), 3.91-3.93 (t, OCH
3
)
13
C-NMR: 189.837 (C=O)
132
e
87.9-89.4 (A) 33.7
C: calcd, 65.04 found, 65.04
H: calcd, 5.16 found, 5.17
Cl: calcd, 10.53 found, 10.87

332.0808
(C
18
H
17
O
4
Cl =
332.0815)
1660.41
(υ
C=O
)
In CDCl
3
, 8.04 (d, J = 15.82Hz, βH), 7.45 (m, J = 15.82Hz, αH)
6.75-8.10 (m, 3H, 4H, 5H, 6H, 5’H, 6’H), 3.92-3.93 (t, OCH
3
)
13
C-NMR: 189.514 (C=O)
133
e
177.3-178.8 (A) 26.0
C: calcd, 65.04 found, 65.12
H: calcd, 5.16 found, 5.20
Cl: calcd, 10.53 found, 10.67
332.0816
(C
18

H
17
O
4
Cl =
332.0815)
1651.73
(υ
C=O
)
In CDCl
3
, 7.43 (m, J = 15.07Hz, βH), 7.31 (m, J = 15.07Hz, αH)
6.72-7.82 (m, 2H, 4H, 5H, 6H, 5’H, 6’H), 3.80-3.89 (m, OCH
3
)
13
C-NMR: 186.881 (C=O)
134 69.2-70.2 (A) 30.9

C: calcd, 72.45 found, 72.25
H: calcd, 6.09 found, 6.17
298.1199
(C
18
H
18
O
4
=

298.1025)
1650.77
(υ
C=O
)
In CDCl
3
, 7.66 (d, J = 15.83Hz, βH), 7.48 (d, J = 15.83Hz, αH)
6.75-7.62 (m, 2H, 3H, 4H, 5H, 6H, 5’H, 6’H), 3.92-3.93 (d, OCH
3
)
13
C-NMR: 189.976 (C=O)

41
69.8-71.2 (A)
79.8-80
d
(B)
50.1

C: calcd, 74.08 found, 74.15
H: calcd, 7.11 found, 7.16
340.1680
(C
21
H
24
O
4

=
340.1675)

In CDCl
3
, 8.09-8.04 (d, J=15.45Hz, βH) 7.58-7.53 (d, J=15.83Hz,
αH) 6.48-8.09 (m, 2’H, 3’H, 5’H, 6’H, 3H, 5H, 6H), 4.06-3.88(t, -
OCH
2
-) 3.92-3.85(m, OCH
3
) 1.82-0.98(m, -(CH
2
)
2
, -CH
3
)
1
155.7-158.5 (C)
152-153
b
(A/C=1:1)
83.0
C: calcd, 60.71 found, 59.51
H: calcd, 4.20 found, 4.26
Cl: calcd, 20.81 found, 21.05
336.0346
(C
17

H
14
O
3
Cl
2

= 336.0320)
1650.77
(υC=O)
In CDCl
3
, 7.99-7.94(d, J=15.825Hz, βH) 7.51-7.46(d, J=15.795Hz,
αH) 6.4-8.0 (m, 3’H, 5’H, 6’H, 3H, 5H, 6H) 3.90- 3.87 (d, OCH
3
)
A-3

2
110.9-115.1 (A)
115-116
b

79.8
C: calcd, 64.27 found, 64.05
H: calcd, 4.50 found, 4.29
F: calcd, 16.96 found, 17.56
336.0990
(C
18

H
15
O
3
F
3
=
336.0973)
1662.34
(υC=O)
In CDCl
3
, 7.71-7.66 (d, J=15.83Hz,βH) 7.62-7.57 (d, J=15.82Hz,
αH) 6.3-7.8 (m, 3’H, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.92- 3.88 (d,
OCH
3
)
5
115.7-118.5 (A)
107-108
b
(A/C/DMF=2:2:1)
75.3
C: calcd, 67.09 found, 66.85
H: calcd, 4.64 found, 4.30
F: calcd, 12.50 found, 12.42
304.0918
(C
17
H

14
O
3
F
2
=
304.0911)
1642.09
(υC=O)
In CDCl
3
, 7.64-7.59(d, J=15.431Hz, βH) 7.60-7.55(d, J=15.54Hz,
αH) 6.3-7.8 (m, 3’H, 5’H, 6’H, 3H, 5H, 6H) 3.95- 3.92(d, OCH
3
)

7 129.3-132.9 (A) 56.8

C: calcd, 69.48 found, 69.34
H: calcd, 6.14 found, 6.03
328.1332
(C
19
H
20
O
5
=
328.1311)
1644.02

(υC=O)
In CDCl
3
, 7.94-7.89 (d, J=15.926Hz, βH) 7.46-7.41(d, J=15.86Hz,
αH) 6.34-7.94 (m, 3’H, 5’H, 6’H, 3H, 5H, 6H) 3.86-3.82 (m, CH
3
)
8
e
<62 (C) 69.0

C: calcd, 76.99 found, 77.37
H: calcd, 6.81 found, 6.72
296.1421
(C
19
H
20
O
3
=
296.1412)
1655.59
(υC=O)
In CDCl
3
, 7.53-7.48 (d, J=15.799Hz,
β
H) 7.33-7.28(d, J=15.79Hz,
αH) 6.24-7.6 (m, 3’H, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.70-3.97 (d,

OCH
3
) 2.55-2.41 (q, CH
2
) 1.49-1.01 (t, -CH
3
)
29
e
140-147 (A) 42.3
C: calcd, 75.21 found, 75.00
H: calcd, 5.37 found, 5.35
N: calcd, 4.39 found, 4.23
319.1220
(C
20
H
17
O
3
N =
319.1208)

In CDCl
3
, 9.20 (s, 2H) 7.77-7.71(d, J=15.45Hz,
β
H) 7.60-7.55 (d,
J=15.07Hz, αH) 8.26-6.52 (m, 4H, 5H, 6H, 7H, 8H, 3’H, 5’H, 6’H)
3.94-3.89 (d, OCH

3
)
30
e

200.0-202.0
(A)
10.0
C: calcd, 75.21 found, 74.93
H: calcd, 5.37 found, 5.23
N: calcd, 4.39 found, 4.28
319.1214
(C
20
H
17
O
3
N =
319.1208)

In DMSO, 9.19-9.18 (d, 2H) 8.32-8.27(d, J=15.45Hz,
β
H) 7.98-7.93
(d, J=15.44Hz, αH) 8.53-6.69 (m, 3H, 5H, 6H, 7H, 8H, 2’H, 3’H,
5’H, 6’H) 3.91-3.85 (d, OCH
3
)
101 112 (A) 38.4
(C

18
H
18
O
3
0.2 H
2
O)
C: calcd, 76.56 found, 76.30
H: calcd, 6.43 found, 6.77
282.1248
(C
18
H
18
O
3
=
282.1256)
1648.84
(ν C=O)
In CDCl
3
, 7.75(d, J=8.57Hz, 1H, 6'-H), 7.66(d, J=15.8Hz, 1H,β -H),
7.51-7.44(m, 3H, 2-H, 6-H,α-H), 7.20(d, J=8.0Hz, 2H), 6.57(d,
J=2.3Hz and 8.6Hz, 1H, 5’-H), 6.51(d, J=2.3Hz, 1H, 3’-H), 3.91(s,
3H, OCH
3
), 3.87(s, 3H, OCH
3

), 2.39(s, 3H, CH
3
)
102 83 (A) 15.9

(C
18
H
18
O
4.
0.15H
2
O)
C: calcd, 72.45 found, 72.26
H: calcd, 6.09 found, 6.23
298.1229
(C
18
H
18
O
4
=298.1205)
1647.87
(
ν
C=O)
In CDCl
3

, 7.73(d, J=8.7Hz, 1H, 6'-H), 7.64(d, J=15.8Hz, 1H,β -H),
7.54(d, J=8.7Hz, 2H, 2-H, 6-H), 7.38(d, J=15.8Hz, 1H, α-H), 6.91(d,
J=9.1Hz, 2H, 3-H, 5-H), 6.55(d, J=2.3Hz and 8.7Hz, 1H, 5’-H), 6.49
(d, J=2.3Hz, 1H, 3’-H), 3.92(s, 3H, OCH
3
), 3.82(s, 3H, OCH
3
),
3.80(s, 3H, OCH
3
)
103 89-91 (B) 10.5

(C
19
H
21
NO
3.
0.75H
2
O)
C: calcd, 73.28 found, 73.50
H: calcd, 6.80 found, 6.87
N: calcd, 4.50 found, 3.61
311.1530
(C
19
H
21

O
3
N =
311.1521)
1636.30
(νC=O)
In CDCl
3
, 7.70(d, J=8.5Hz, 1H, 6'-H), 7.63(d, J=15.7Hz, 1H, β-H),
7.50(d, J=8.9Hz, 2H), 7.28(d, J=15.6Hz, 1H, α-H), 6.68(d, J=8.9Hz,
2H), 6.55(d, J=2.3Hz and 8.5Hz, 1H, 5’-H), 6.50(d, J=2.2Hz, 1H, 3’-
H), 3.89(s, 3H, OCH
3
), 3.87(s, 3H, OCH
3
), 3.03(s, 6H, N(CH
3
)
2
)
A-4

104 90-92 (A) 16.5
(C
17
H
15
FO
3
0.02H

2
O)
C: calcd, 71.30 found, 71.50
H: calcd, 5.28 found, 5.20
F: calcd, 6.64 found, 6.87
286.0997
(C
17
H
15
O
3
F
=286.1005)
1663.30
(νC=O)
In CDCl
3
, 7.81(d, J=8.7Hz, 1H, 6'-H), 7.69(d, J=15.8Hz, 1H, β-H),
7.64-7.60(m, 2H), 7.49(d, J=15.8Hz, α-H), 7.15-7.09(m, 2H),
6.61(d, J=2.3Hz and 8.7Hz, 1H, 5’-H), 6.54(d, J=2.3Hz, 1H, 3’-H),
3.95(s, 3H, OCH
3
), 3.92(s, 3H, OCH
3
)
105 115.3-115.6 (A) 36.4
(C
17
H

15
ClO
3
0.15H
2
O)
C: calcd, 67.53 found, 67.78
H: calcd, 5.01 found, 5.18
Cl: calcd, 11.58 found, 11.79
302.0706
(C
17
H
15
O
3
Cl
=302.0710)
1660.41
(νC=O)
In CDCl
3
, 7.80(d, J=8.7Hz, 1H, 6'-H), 7.66(d, J=15.8Hz, 1H, β-H),
7.57-7.50(m, 3H), 7.39(d, J=8.7Hz, 2H), 6.60(d, J=2.3Hz and 8.7Hz,
1H, 5'-H), 6.53(d, J=2.3Hz, 1H, 3'-H), 3.95(s, 3H, OCH
3
), 3.91(s,
3H, OCH
3
)

106 121.5-121.8 (A) 72.1
(C
17
H
15
O
3
0.18H
2
0)
C: calcd, 58.96 found, 59.00
H: calcd, 4.37 found, 4.36
Br: calcd, 22.81 found, 22.66
346.0217
(C
17
H
15
O
3
Br
=346.0205)
1660.41
(νC=O)
In CDCl
3
, 7.80(d, J=8.7Hz, 1H, 6'-H), 7.64(d, J=15.8Hz, 1H, β-H),
7.57-7.47(m, 5H), 6.60(d, J=2.3Hz and 8.7Hz, 1H, 5'-H), 6.55(d,
J=2.3Hz, 1H, 3'-H), 3.94(s, 3H, OCH
3

), 3.91(s, 3H, OCH
3
)
107
e
112-113 (A) 53.0
C: calcd, 63.74 found, 63.90
H: calcd, 4.41, found, 4.87
F: calcd, 5.94, found, 4.35
Cl: calcd, 10.93, found, 10.95
320.0597
(C
17
H
14
O
3
FCl
=320.0615)
1650.77
(νC=O)
In CDCl
3
, 7.99(d, J=15.8Hz, 1H, β-H), 7.78(d, J=8.7Hz, 1H), 7.74-
7.64(m, 1H), 7.38(d, J=15.8Hz, 1H, α-H), 7.17(d, J=2.6Hz and
8.3Hz, 1H), 7.05-6.95(m, 1H), 6.57(d, J=2.3Hz and 8.7Hz, 1H, 5'-
H), 6.49(d, J=2.3Hz, 1H, 3'-H), 3.90(s, 3H, OCH
3
), 3.88(s, 3H,
OCH

3
)
108
e
130-131 (C) 53.6
(C
17
H
14
O
3
Cl
2
.0.05H
2
0)
C: calcd, 60.71 found, 60.57
H: calcd, 41.99 found, 41.85
Cl: calcd, 20.81 found, 22.66
336.0306
(C
17
H
14
O
3
Cl
2

=336.0320)

1665.23
(νC=O)
In CDCl
3
, 7.78(d, J=8.7Hz, 1H), 7.65(d, J=1.9Hz, 1H), 7.52(d,
J=5.7Hz, 2H), 7.47-7.38(m, 2H), 6.57(d, J=2.3Hz and 8.7Hz, 1H, 5'-
H), 6.49(d, J=2.3Hz, 1H, 3'-H), 3.92, (s, 3H, OCH
3
), 3.88(s, 3H,
OCH
3
)
109 191-192 (C) 74.3
(C
17
H
15
O
5
N. 0.25H
2
O)
C: calcd, 65.16 found, 65.32
H: calcd, 4.83 found, 4.84
N: calcd, 4.47 found, 4.53
313.0940
(C
17
H
15

O
5
N
=313.0950)
1662.34
(νC=O)
In CDCl
3
, 8.28(d, J=9.0Hz, 2H), 7.85(d, J=8.7Hz, 1H), 7.77-7.70(m,
4H), 6.61(d, J=2.3Hz and 8.7Hz, 1H, 5'-H), 6.53(d, J=2.3Hz, 1H, 3'-
H), 3.93(s, 3H, OCH
3
), 3.90(s, 3H, OCH
3
)
110 73-74 (A) 12.1

C: calcd, 79.21 found, 79.43
H: calcd, 5.70 found, 5.71
318.1269
(C
21
H
18
O
3

=318.1256)
1649.80
(νC=O)

In CDCl
3
, 8.51(d, J=15.8Hz, 1H, β-H), 8.28(d, J=8.3Hz, 1H), 7.90-
7.82(m, 4H), 7.63-7.47(m, 4H), 6.59(d, J=2.3Hz and 8.7Hz, 1H, 5'-
H), 6.51(d, J=2.3Hz, 1H, 3'-H), 3.94(s, 3H, OCH
3
), 3.88(s, 3H,
OCH
3
)
19 181.2-184 (A) 61.6

C: calcd, 75.56 found, 75.44
H: calcd, 5.55 found, 5.52
254.0933
(C
16
H
14
O
3
=
254.0943)

3158.83
(υ
OH
)
1644.02
(υ

C=O
)
In CDCl
3
, 9.54 (OH), 7.75-7.70 (d, J=15.82Hz, βH) 7.44-7.39 (d,
J=15.83Hz, αH) 6.87-8.04 (m, 2’H, 3’H, 5’H, 6’H, 2H, 3H, 5H, 6H)
3.89 (s, OCH
3
)

A-5

22
e
101.5-102.9 (A) 72.6
C: calcd, 70.05 found, 69.70
H: calcd, 4.41 found, 4.07
F: calcd, 13.86 found, 13.77
274.0802
(C
16
H
12
O
2
F
2
=
274.0805)

In CDCl
3
, 7.81-7.76 (d, J=15.535Hz, βH) 7.46-7.40 (d, J=15.648Hz,
αH) 6.91-8.06 (m, 2’H, 3’H, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.89-3.85
(d, OCH
3
)
23
133.4-137.0 (A)
101
f

83.7
C: calcd, 76.09 found, 75.68
H: calcd, 6.01 found, 5.80
268.1122
(C
17
H
16
O
3
=
268.1099)

In CDCl
3
, 7.85-7.80 (d, J=15.853Hz, βH) 7.63-7.57 (d, J=15.826Hz,
αH) 6.85-8.06 (m, 2’H, 3’H, 5’H, 6’H, 3H, 5H, 6H) 3.89 (s, OCH
3

)

31
e
137.2-144.4 (A) 92.1
C: calcd, 78.86 found, 78.56
H: calcd, 5.23 found, 5.55
N: calcd, 4.84 found, 4.53
289.1103
(C
19
H
15
O
2
N =
289.1103)

In CDCl
3
, 9.21- 9.20 (d, 2’H) 7.81-7.75(d, J=15.83Hz, βH) 7.63-
7.57 (d, J=15.83Hz, αH) 8.35-7.00 (m, 4’H, 5’H, 6’H, 7’H, 8’H, 2H,
3H, 5H, 6H) 3.88 (s, OCH
3
)
32
e

215.0-217.0
(A)

15.0
C: calcd, 78.86 found, 78.65
H: calcd, 5.23 found, 5.15
N: calcd, 4.84 found, 4.69
289.1109
(C
19
H
15
O
2
N =
289.1103)

In DMSO, 9.28-9.27 (d, 2H) 8.51-8.46(d, J=15.44Hz, βH) 8.40-8.35
(d, J=15.44Hz, αH) 8.58-7.00 (m, 3H, 5H, 6H, 7H, 8H, 2’H, 3’H,
5’H, 6’H) 3.90 (s, OCH
3
)
38
112.0-114.0
(A)
44.4
C: calcd, 74.97 found, 74.76
H: calcd, 5.12 found, 5.09
F: calcd, 7.42 found, 7.26
256.0876
(C
16
H

13
O
2
F =
256.0900)

In CDCl
3
, 7.79-7.74 (d, J=15.45Hz, βH) 7.50-7.44 (d, J=16.2Hz,
αH) 6.96-8.06 (m, 2’H, 3’H, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.89 (s,
CH
3
)
111 132-136 (A) 41.6
C: calcd, 62.74 found, 62.58
H: calcd, 3.95 found, 3.87
Cl: calcd, 22.85 found, 23.33
306.0190
(C
16
H
12
O
2
Cl
2

= 306.0214)
1657.52
(υC=O)

In CDCl
3
, 8.12 (d, J=15.72Hz, βH) 7.52 (d, J=15.72Hz, αH) 7.00-
8.15 (m, 2’H, 3’H, 5’H, 6’H, 3H, 5H, 6H) 3.89 (s, OCH
3
)
112
e
135-137 (A) 46.7
C: calcd, 66.65 found, 66.48
H: calcd, 4.28 found, 4.17
F: calcd, 18.62 found, 18.74
306.0868
(C
17
H
13
O
2
F
3

= 306.0848)
1660.41
(υC=O)
In CDCl
3
, 7.79 (d, J=15.82Hz, βH) 7.60 (d, J=15.83Hz, αH) 6.98-
8.07 (m, 2’H, 3’H, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.91 (s, OCH

3
)
113 84.5-87.2 (A) 20.8
C: calcd, 72.45 found, 72.56
H: calcd, 6.09 found, 6.18

298.1202
(C
18
H
18
O
4

= 298.1205)
1642.09
(υC=O)

In CDCl
3
, 8.04 (d, J=16.20Hz,
β
H) 7.56 (d, J=15.82Hz,
α
H) 6.47-
8.07 (m, 2’H, 3’H, 5’H, 6’H, 3H, 5H, 6H) 3.88 (t, 3 OCH
3
)
114 125.3-127 (A) 37.3
C: calcd, 80.92 found, 81.14

H: calcd, 6.40 found, 6.57
252.1165
(C
17
H
16
O
2
= 252.1150)
1653.66
(υC=O)
In CDCl
3
, 7.79 (d, J=15.82Hz, βH) 7.51 (d, J=15.45Hz, αH) 6.96-
8.05 (m, 2’H, 3’H, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.90 (s, OCH
3
), 2.39
(S, CH
3
)
115 170-174 (A) 13.9
C: calcd, 67.82 found, 68.00
H: calcd, 4.62 found, 4.50
N: calcd, 4.95 found, 5.37
283.0849
(C
16
H
13
O

4
N
= 283.0845)
1658.48
(υC=O)

In CDCl
3
, 7.79 (d, J=15.82Hz, βH) 7.65 (d, J=15.83Hz, αH) 7.00-
8.26 (m, 2’H, 3’H, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.91 (s, OCH
3
)
116 125-128 (C) 24.4
C: calcd, 76.83 found, 76.56
H: calcd, 6.81 found, 6.92
N: calcd, 4.98
281.1430
(C
18
H
19
O
2
N
= 281.1416)
1598.70
(υC=O)

In CDCl
3

, 7.79 (d, J=15.83Hz, βH) 7.59 (d, J=15.80Hz, αH) 7.01-
8.06 (m, 2’H, 3’H, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.03 (s, -N-(CH
3
)
2
)
3.91 (s, OCH
3
)