Investigations on the antimalarial activity of alkoxylated and hydroxylated chalcones 3

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 su

SECTION SIX MODE OF ANTIMALARIAL ACTIVITY OF CHALCONES

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):

[14C] 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

6.2.1 Degradation of [ 14C] 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, 4oC) 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, 4oC to give supernatant containing crude

parasite extracts (lysate) Following a reported method 125, aliquots of the lysate (25

µl, estimated to contain 1-1.5 µg protein /µl) and test compound in DMSO (10 µl) was

added to 50 µl sodium acetate (0.1 M, pH 6.0) and sufficient distilled water to give a

final volume of 100 µl The final concentration of test compound was 100 µM After

1 h of incubation at 37oC, 5 µl of [14C] 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, 4oC before centrifugation at 13,000 g , 4oC

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 ([14C] methemoglobin and lysate) and

samples containing only [14C] 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

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 IC50 values from rates

versus concentration plots

Chalcones which inhibited falcipain-2 with IC50 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

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

µM) 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 [14C] 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

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 (IC50 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 µM

was used to distinguish between chalcones with inhibitory activity (IC50 < 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

dimethoxychalcones (2, 5) The 3-quinolinyl chalcone 2 7 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 (IC50 1.4 µM) which had weak in vitro antimalarial activity (IC50 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 IC50 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 µM (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

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

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

Table 6.1 Effect of antimalarial chalcones on the enzymatic activity of a crude P

falciparum K1 extract using [14C] methemoglobin as substrate, in vitro

falcipain inhibition and cell viability

KB3-1 cell viability (%) c(SD)

% inhibition of [14C] MetHb breakdown at

100 µ M (SD)

IC50 ( µ M) Falcipain inhibition

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)

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

N D

2.3 7.8

7.0 4.8 14.4 6.4

78.9(16.7) 82.9(15.2)

N D 121.6(13.3)

16.1 16.0 19.7 20.0 12.3

89.3(28.5)*

88.9(22.1)*

96.4(19.3)*

N D 100.7(32.0)*

N D

N D 9.4 (3.7)

16.3 18.4 17.7

112.3 (18.2)*

90.6 (23.3)*

84.8 (32.2)*

N D 40.7 (9.7) 39.0 (14.3)

N D 2.4 9.7

234 2-hydroxy 4-chloro 12.9 N D 14.7 (8.6) 2.3 Chloroquine

E64

Pepstatin A

0.27 4.0 80.0

104.1 (17.5)

N D

N D

N D 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 [3H] 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

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

Table 6.2 Effect of chalcones on Soret band absorbance of hematin

Number Ring B a Ring A a

IC50 ( µ M) for

In vitro

antimalarial activity

% Decrease in absorbance of hematin b (SD)

5.4 3.0 2.0 60.0 9.5

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)

7.0 4.8 43.0 6.4

<10

<10

<10 47.4 (1.0)

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)

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)

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

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 (IC50 < 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

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 pKa 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

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

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 1st 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 IC50 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 kw) 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 activity correlations reached in this study

The 2nd 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

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 IC50 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 humancervical 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

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

Appendix Table 1 Physical and analytical data of synthesized chalcones

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) 168.1-169.3c 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 CDClαH) 6.52-7.79 (m, 5’H, 6’H, 2H, 3H, 5H, 6H) 3.92-3.88 (t, OCH3, 7.67-7.61 (d, J=15.688Hz, βH) 7.29-7.24 (d, J=15.653Hz, 3 )

3.03 (s, N(CH 3 ) 2 ) 13 C-NMR: 191.324 (C=O)

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)

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)

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

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)

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)

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)

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)

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, βH), 7.46 (m, J = 15.82Hz, αH) 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)

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) 79.8-80d (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 CDClαH) 6.4-8.0 (m, 3’H, 5’H, 6’H, 3H, 5H, 6H) 3.90- 3.87 (d, OCH3, 7.99-7.94(d, J=15.825Hz, βH) 7.51-7.46(d, J=15.795Hz, 3 )