Synthesis of novel flavone hydrazones: In-vitro evaluation of α-glucosidase inhibition, QSAR analysis and docking studies

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Graphical Abstract

Synthesis of Novel Flavone Hydrazones: In-Vitro Evaluation

of α-Glucosidase Inhibition, QSAR Analysis and Docking

Studies

a,b

a,b∗

c

Syahrul Imran , Muhammad Taha , Nor Hadiani Ismail , Syed Muhammad Kashif , Fazal

d

c

e

Rahim , Waqas Jamil , Maywan Hariono , Muhammad Yusuf , Habibah Wahab

a

Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA, Puncak Alam Campus,

b

4

2300, Selangor D.E., Malaysia, Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam,

e

0450, Selangor D.E., Malaysia, Institute of Advance Research Studies in Chemical Sciences, University of

d

Sindh, 76080, Jamshoro, Pakistan, Department of Chemistry, Hazara University, 21300, Mansehra, Pakistan,

e

Pharmaceutical Design and Simulation Laboratory, School of Pharmaceutical Sciences, Universiti Sains

Malaysia, 11800, Pulau Pinang, Malaysia

A new series of potent novel flavone derivatives 5-34 as α-glucosidase inhibitors was identified.

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Synthesis of Novel Flavone Hydrazones: In-Vitro Evaluation of α-Glucosidase Inhibition,

QSAR Analysis and Docking Studies

a,b

a,b∗

c

Syahrul Imran , Muhammad Taha , Nor Hadiani Ismail , Syed Muhammad Kashif , Fazal

d

c

e

Rahim , Waqas Jamil , Maywan Hariono , Muhammad Yusuf , Habibah Wahab

a

Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA, Puncak Alam Campus, 42300, Selangor D.E.,

Malaysia

b

c

d

e

Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam, 40450, Selangor D.E., Malaysia

Institute of Advance Research Studies in Chemical Sciences, University of Sindh, 76080, Jamshoro, Pakistan

Department of Chemistry, Hazara University, 21300, Mansehra, Pakistan

Pharmaceutical Design and Simulation Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800, Pulau Pinang,

Malaysia

ABSTRACT

Thirty derivatives of flavone hydrazone (5-34) had been synthesized through a five-step

reaction and screened for their α-glucosidase inhibition activity. Chalcone 1 was synthesized

through aldol condensation then subjected through oxidative cyclization, esterification, and

condensation reaction to afford the final products. The result for baker’s yeast α-glucosidase

(EC 3.2.1.20) inhibition assay showed that all compounds are active with reference to the IC₅₀

value of the acarbose (standard drug) except for compound 3. Increase in activity observed for

compounds 2 to 34 clearly highlights the importance of flavone, hydrazide and hydrazone

linkage in suppressing the activity of α-glucosidase. Additional functional group on N-

benzylidene moiety further enhances the activity significantly. Compound 5 (15.4 ± 0.22 µM),

a 2,4,6-trihydroxy substituted compound, is the most active compound in the series. Other

compounds which were found to be active are those having chlorine, fluorine, and nitro

substituents. Compounds with methoxy, pyridine, and methyl substituents are weakly active.

Further studies showed that they are not active in inhibiting histone deacetylase activity and do

not possess any cytotoxic properties. QSAR model was being developed to further identify the

structural requirements contributing to the activity. Using Discovery Studio (DS) 2.5, various

2

D descriptors were being used to develop the model. The QSAR model is able to predict the

pIC₅₀and could be used as a prediction tool for compounds having the same skeletal

framework. Molecular docking was done for all compounds using homology model of α-

glucosidase to identify important binding modes responsible for inhibition activity.

Keywords: Flavone, hydrazone, α-glucosidase, 2D-QSAR, docking.

∗

Correspondence and reprints

E-mail: taha_hej@yahoo.com and muhamm9000@puncakalam.uitm.edu.my Tel: 0060193098141 (M.T.);

norhadiani@puncakalam.uitm.edu.my, Tel: 0060132675756 (N.H.I.)

1

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1

. Introduction

Diabetes mellitus is a disease involving metabolic disorder resulting from defects in insulin

secretion and action. This result in increase of blood glucose level (hyperglycemia) and causes

dysfunction to important organs like blood vessels and nerves [1]. Study shows that the number

of diabetes mellitus patients increases each year. The trend displayed that patient with diabetes

increases from 153 million to 347 million between the year 1980 to 2008 [2]. Based on World

Health Organization (WHO) projection, diabetes is predicted to become the seventh leading

cause of death worldwide by 2030. One of the approaches, which can be taken to reduce

diabetes is to inhibit enzymes like α-glucosidase by controlling the postprandial glucose levels

and suppressing postprandial hyperglycemia [3].

Inhibitors for α-glucosidase function by reversibly inhibit digestive α-glucosidase that retards

glucose liberation from complex starch and carbohydrate. This results in delay for glucose to

be absorbed into bloodstream and, therefore, decreases plasma glucose level. Inhibition of a-

glucosidase has attracted plenty of interest by pharmaceutical industry as a treatment method of

diseases like diabetics, viral infections, hepatitis, and cancer [4-6]. Inhibitors for α-Glucosidase

are found to show antitumor, antidiabetic, antiviral, and immunoregulatory activities [7-9].

Research on α-glucosidase inhibitors such as castanospermine, N-butyl-deoxynojirimycin and

deoxynojirimycin showed that they are potent against HIV replication [10].

Currently, glycosidic based α-glucosidase inhibitors such as miglitol [11], voglibose [12],

acarbose [13] and nojirimycin [14] are used to control glucose level in blood of diabetic

patients. Despite being potent, these inhibitors have some limitations such as diarrhea,

abdominal distension, meteorism and flatulence [15]. Acarbose, deoxynojirimycin, miglitol,

and voglibose are among the key known candidates which are used extensively to inhibit α-

glucosidase activity. Due to side effects and absorptivity problems associated with these

inhibitors [16, 17], new potent α-glucosidase inhibitors are highly desired. Hence, a lot of

efforts have been put in to design and develop non-glycosidic based inhibitors for safer [18]

and effective α-glucosidase inhibitors [19].

α-Glucosidase (EC3.2.1.20) is an enzyme commonly found in small intestine. α-Glucosidase

hydrolyzes carbohydrates and produces α-d-glucose during food digestion. Carbohydrates and

α-d-glucose that has been produced are being absorbed into blood stream, thus increases

postprandial blood glucose level and leads to diabetes. Therefore, α-glucosidase inhibitors are

of significant importance to control diabetes since they could reduce carbohydrate digestion

2

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and monosaccharide absorption [20, 21]. Glucose concentration in blood is extremely critical

for diabetes mellitus management and it must be between an acceptable ranges (70-100 mg/dl)

[

22]. In addition to preventing diabetes, blood glucose level which is controlled may avoid

hyperlipoproteinemia, obesity, and hyperlipidemia [23].

α-Glucosidase also enables monosaccharide removal from viral glycoproteins, thus, its

inhibitors could alter cell-to-cell signaling, virus recognition by the cell and could be used in

the treatment of viral diseases, cancer, and immune-regulations [24-28].

Flavone is one of the class of flavonoids that is present in fruits and vegetables. Flavones are

being consumed in daily diet and they improve health without giving any major side effect

[

29]. In order to explore diverse roles of flavones, investigating various methods for their

synthesis and structural modification of flavones ring has now become important goals of

several research groups. Thus, naturally obtained flavone moiety having a variety of biological

activities can be taken as lead compound for the synthesis of semi- and purely synthetic flavone

derivatives with different functional groups at different positions of flavone skeleton.

Flavone scaffold is an important structure found in many pharmaceutically active compounds.

They are structurally diverse and possess a variety of biological activity. This reason has

increased the interest of medicinal chemists to further study flavones as lead molecules to treat

various diseases. Recently, researchers had focused a lot on flavonoids bioactivities like free

radicals scavenging ability and protection against peroxidation of lipid [30, 31]. Researchers

also showed interest in some flavonoids due to their ability to modulate NADPH oxidase

activity and endothelial nitric oxide metabolism [32-38]. Various research indicate that

flavonoids could reduce hyperglycemia, improve sensitivity and enhance secretion of insulin

[

39]. It has been established that flavonoids like kaempferol, luteolin, apigenin, chrysin and

baicalein have the capability of inhibiting α-glucosidase activity [40, 41], taking into

consideration of the importance of ring A, B, and C towards the inhibition of α-glucosidase

activity.

Benzohydrazides had been reported to possess various biological activities, which includes

antileishmanial [42], antioxidant [43], antiglycation [44], antibacterial [45], and α-glucosidase

inhibition [46] activities. In this paper, flavone derivatives possessing hydrazone linkage

synthesized and evaluated for their α-glucosidase inhibition activity. We attempted to

rationalize the effect of various substituents at different position on the inhibitory effect in

terms of how the molecules bind with α-glucosidase protein.

3

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2

. Result and discussion

2

.1. Chemistry

The title compounds were synthesized through scheme 1 and 2. Flavone hydrazones 5-34 were

synthesized through a single step reaction of 4-(4-oxo-4H-chromen-2-yl)benzohydrazide 4 with

appropriate aryl aldehydes, in the presence of catalytic amount of glacial acetic acid.

Compound 4 which is the key intermediate for the synthesis of target compounds 5-34 was

prepared through a four-step sequence. Initially 2’-hydroxyacetophenone undergoes Aldol

condensation with 4-formylbenzoic acid using aqueous KOH in ethanol to form (E)-4-(3-(2-

hydroxyphenyl)-3-oxoprop-1-en-1-yl)benzoic acid 1. Chalcone 1 was cyclized into flavone

through oxidative cyclization by using well-known I -DMSO mixture as the oxidizing agent.

2

Carboxylic acid of the resulting product 2 was converted into ester 3 before finally reacted it

with hydrazine to form compound 4.

Insert Scheme 1 here

Insert Scheme 2 here

The structure of newly synthesized compounds were confirmed by the various analytical

1

techniques, H-NMR, 3C-NMR, IR, MS and elemental analysis. All spectral data were in

1

accordance with assumed structures. In the H-NMR spectra of chalcone 1, olefinic protons H

α

and H (Figure-1) appeared as doublets at 7.86 ppm and 8.12 ppm respectively. The formation

β

of chalcone 1 having trans conformation was verified using the coupling constant values (15.6

Hz) of both vinyl hydrogens (H and H ). Oxidation of chalcone 1 leads to the formation of

α

β

flavone 2 which involves removal of hydrogen from hydroxyl group and H . This was being

β

1

confirmed through several changes to the H-NMR spectra. Splitting produced by vinylic

protons (H and H ) were no longer observed. This deduction was further confirmed by the

α

β

appearance of singlet proton at 7.13 ppm representing H on the chromone ring.

α

Insert Figure 1 here

Insert Table 1 here

4

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2

.2. In-vitro α-glucosidase inhibition

In continuation of our work on enzyme inhibition [47], we had synthesized compounds 1-34

and evaluate them for their α-glucosidase inhibition activity. The assay was carried out using

baker’s yeast α-glucosidase (EC 3.2.1.20). The result in table 2 showed, with the exception of

compound 3, the compounds synthesized in this study are more active than the standard drug.

Compounds 1 to 4, which are the intermediate compounds for synthesis of the final compounds

5

-34 were also evaluated for their inhibition activity. Comparison between results for

intermediates 1 and 2 showed that flavone 2 has better activity as compared to chalcone 1. This

result indicates that the chromone moiety is playing a significant role in inhibiting the enzyme

activity. Conversion of carboxylic acid to hydrazide 3 enhances the inhibition activity.

Converting hydrazide 4 into hydrazones 5-34 further improves the inhibition activity.

Comparison between results for intermediate compounds 1-4 with flavone-hydrazone

derivatives 5-34 showed a significant improvement in the inhibitory activity, which indicates

the importance of hydrazone linkage in inhibiting α-glucosidase activity. Compounds having

hydroxyl, chloro and fluoro substituent showed potent activity with IC ranging between 15.4

5

0

to 86.3 µM while the other compounds having methyl, methoxy, nitro and bromo substituent

are weakly active. Compounds 28, 29 and 30 that are having pyridine ring also showed weak

inhibitory activity against α-glucosidase. In order to understand further the effect of structural

features and mechanism of action, the compounds were subjected through 2D QSAR and

docking studies, which studies enable us to identify key structural features responsible for the

inhibition activity as well as rationalizing the best binding modes in the active site of α-

glucosidase. The compounds were not showing any activity against histone deacetylase

inhibition and cytotoxicity activity.

Insert Table 2 here

2

.3. 2D QSAR study

2

.3.1.QSAR model development and validation

Quantitative structural activity relationship (QSAR) for α-glucosidase inhibition activity was

performed to determine the factors/descriptors which correlates the bioassay results obtained

with flavone derivatives (5-34), and to determine structural features contributing towards the

inhibition activity. In this study, the compounds for training set and test set were selected using

"cluster analysis" protocol in Discovery Studio 2.5. Cluster selection was done by allowing

5

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cluster to have fixed number of compounds for each cluster. Compounds selection was done

using predefined set consisting of items such as AlogP, molecular weight, number of hydrogen

donors and acceptors. The training set consisting of 21 compounds from the newly synthesized

flavone hydrazones together with their measured pIC₅₀(-logIC ) was used to develop the

50

QSAR model. The molecular properties for the compounds in the training set were calculated

using “Calculate Molecular Properties” protocol. The protocol includes calculation for 2D

molecular properties, energies of highest occupied and lowest unoccupied molecular orbitals

(HOMO and LUMO) for compounds of the training set. In this study, 2D descriptors like

AlogP, molecular weight, number of hydrogen donor, and number of rotatable bonds were

utilized in developing the model.

Notably, AlogP is a measure of the hydrophobicity of the molecule and calculated in Discovery

Studio as the Log of the octanol-water partition coefficient, while Molecular Fractional Polar

Surface Area is the ratio of the polar surface area divided by the total surface area of the

molecule.

In this study, Multiple Linear Regression (MLR) analysis method was being used to obtain the

model. Descriptors were selected based on the results of the intercorrelation matrix between the

descriptors, which should intercorrelate more than 0.6. Descriptors that were selected for this

study have intercorrelation values lower than 0.5 (table 3a) to prevent model overfitting. These

value also showed that these descriptors are independent. Robustness of the established QSAR

2

models were verified using Leave-one-out (LOO) internal validation or cross-validation (q ),

2

where r (squared correlation coefficient value) equals 0.848, while the r (pred), which is

2

equivalent to q from a leave-one-out cross validation, is 0.705 (table 3b).

Insert Table 3 here

In addition, validation was done by measuring the residuals between the experimental and the

predicted activity of the training set (Table 4). A correlation plot representing the correlation

between predicted pIC against experimental pIC of test set (Fig. 2b) showed that the plot

5

0

50

2

has a regression correlation coefficient (r ) of 0.783, which is considered quite good. Despite

showing good correlation, the QSAR model can be further improved to be used applied for

prediction of more effective hits having the same skeletal framework.

6

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Insert Figure 2 here

Insert Table 4 here

2

.3.2. 2D QSAR model analysis

Equation 1 represents the model obtained using Multiple Linear Regression and it is

graphically visualized using a scatter plot of predicted pIC against experimental pIC values

5

0

50

for the training set and test set compounds as shown in Figure 2. Equation 1 describes that α-

glucosidase inhibition activity of flavone derivatives 5-34 are affected by four descriptors,

which are AlogP, molecular weight, number of hydrogen bond donor, and number of rotatable

bonds. Comparison between descriptors in the equation showed that AlogP, number of

hydrogen bond donor, and number of rotatable bonds are among those which contributed

significantly towards the high correlation as compared to molecular weight. Despite showing

some effect on the pIC , small molecular variation that only involves substitution on the

5

0

extended benzylidene ring has affected the molecular weight descriptor. Having the lowest

constant value, the equation suggest that small changes in molecular weight could slightly

affect the IC₅₀value as compared to the other descriptors. Based on the equation, it was

observed that increasing the number of rotatable bonds descriptor, which has the highest

constant value, in the molecule could significantly increase the pIC₅₀value and, therefore,

gives a lower IC . However, other descriptors in the equation, which are AlogP and molecular

5

0

weight, suggest that increasing the lipophilicity and molecular weight of the compounds will

only reduce the pIC value and gives a higher IC value. This observation supports the actual

5

0

50

activity which showed that adding rotatable and non-polar substituents methyl and methoxy

substituent to compounds could increase the lipophilicity and molecular weight of the

compounds and will significantly reduce the pIC value to give a higher IC value. On the

50

other hand, the equation also suggest that increasing the number of hydrogen bond donor could

increases the activity of the compounds by lowering the IC value, which also supports the

5

0

general observation results from in-vitro activity, which show that increasing hydroxyl group in

the structure could enhance the activity by giving lower IC value. Docking results further

5

0

validate this QSAR model by taking into accounts the effect of all descriptors used in this

model.

Equation 1. Equation representing the QSAR model;

-2

pIC = -16.827 - 0.509 (AlogP) - 5.819 x 10 (Molecular_Weight) + 0.213 (Num_H_Donors)

5

0

+

1.334 (Num_RotatableBonds)

7

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2

.4. Docking study

Molecular docking was performed on all flavones derivatives (5-34) to identify possible

binding mode which explains the reason for their potency. Since crystal structure for α-

glucosidase of S. cerevisiae is still not available, docking study was conducted using a

homology model for α-glucosidase. Preliminary results on sequence analysis of α-glucosidase

from S. cerevisiae showed that the most suitable template for homology modelling is

isomaltase (EC 3.2.1.10, oligo-1,6-glucosidase, MALX3)(PDB ID: 3A4A) from baker’s yeast

which shares 71% identity and 84% similarity with the target enzyme, α-glucosidase of S.

cerevisiae. Sequence analysis on the homology model’s sequence showed high sequence

homology with the target enzyme. The active site contains important and highly conserved

amino acids amino acids (Fig. 3) [48]. The final structure of α-glucosidase generated from

homology modeling was evaluated using PROCHECK. The Ramachandran plot obtained from

PROCHECK showed that 87.5% of residues of the final 3D structure lied in most favored

regions (Fig. 4).

Insert Figure 3 here

Insert Figure 4 here

Prior to docking and analysis of the binding mode of the most active compound 5, the docking

method was validated through control docking of native inhibitor. Acarbose was docked into α-

glucosidase from Sugar beet (PDB code: 3W37) and compared by superimposing with the

native ligand in the protein (Figure 5a). The rmsd value between docked and actual pose of

acarbose was found to be 0.65Å. Even though α-glucosidase from Sugar beet share relatively

low homology with baker’s yeast α-glucosidase (16% identity), the active site is highly

conserved and main interactions of the ligand remain the same [48]. Another control docking

was done on the target protein, isomaltase from Baker’s yeast, using α-D-glucopyranose which

is the native ligand located in the active site (Fig. 5b). The rmsd value for docked pose and

native ligand was found to be 0.93Å.

Insert Figure 5 here

All flavones were docked and aligned in the binding pocket. Based on docking results of the

aligned molecules (Fig. 6), the benzylidene moiety of the compounds are oriented towards the

core of the binding pocket. This enables the substituents on the benzylidene moiety to interact

8

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closely with important residues in the active site. Introduction of hydrophilic substituents with

H-bond donating properties like hydroxyl groups on the benzylidene moiety showed the most

interaction with active residues which led to extra interactions and increase of the activity.

Benzohydrazone linkage provides torsional degree of freedom (rotatability) that substantially

decreases the entropic penalty for the formation of enzyme-inhibitor complex. This feature

validates one of the descriptors used in the QSAR model, which suggest that increase in

number of rotatable bonds could enhance the inhibition activity. Four hydrogen bonds are

established between the phenolic and carbonyl oxygen of compound 5 and the side chains of

His239, His245, His279, and Thr275. Benzohydrazone moiety resides in close proximity to the

catalytic residues including Asp214, Glu276, His348, and Asp349, indicating that it can serve

as surrogate for the terminal glucose in the substrate.

Insert Figure 6 here

The main flavone scaffold of all derivatives (5-34) are better aligned when compared to the

benzylidene moiety. The flavone scaffold which is positioned in a hydrophobic pocket allows

formation of highly stable complexes. Several observations were made based on the interaction

of flavone moiety with the enzyme. The main interaction which played the most significant

role in stabilizing the enzyme-inhibitor complex is the interaction of benzopyrone ring of

flavone moiety with Phe311 and Lys155 through a Pi-Pi stacking and Pi-cation interaction,

respectively. Ring B of flavone forms additional Pi-Pi stacking with Phe311.

Two hydrogen bonds were observed for the most active compound, 5, in the series (Fig. 7a).

Besides forming pi-cation interaction, NH fragment of Lys155 also forms hydrogen bonding

with oxygen of the ether linkage on ring C which further stabilizes the enzyme inhibitor

complex. On the other hand, hydroxyl forms hydrogen bonding with catalytic residue His279,

which is believed to have some reducing effect in the enzyme’s activity. In figure 6b, nitrogen

of benzohydrazone backbone forms a Pi-cation interaction with another catalytic residue

His239.

Insert Figure 7 here

Attempt to validate the QSAR model using docking results showed a high degree of correlation

in terms of substitutent’s lipophilicity, ability to form hydrogen bonding, and rotatability.

9

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Docking studies showed that increase in number of hydrogen bond allows more interaction to

form between between active residues and the ligand. This observation satisfy the number of

hydrogen bond donor descriptor, which was observed to reduce the IC value when number of

5

0

hydroxyl substituent was increased. In a different observation, it is also clearly visualized that

increasing lipophilicity properties of the substituents by replacing hydroxyl with methoxy

substituents causes shifting of benzylidene ring and prevents optimal interaction from taking

place. Inhibition activity also decreases due to steric hindrance imposed by methoxy and

inability to function as a hydrogen bond donor. The effect from steric hindrance by methoxy is

visualized in figure 8c and 8d. Compounds 14 and 15, which contains both hydroxyl and

methoxy substituents, displayed better activity as compared to compounds 30 and 31 which are

single substituted with methoxy substituent. For compounds 14 and 15, the hydroxyl groups for

both compounds are oriented towards the hydrophilic pocket (Fig. 8a and 8b). It was observed

that substituents for both compounds 14 and 15 are not capable of interacting with any catalytic

residues due to steric hindrance imposed by methoxy substituent. For compound 14, methoxy

group forms hydrogen bonding with Thr217, while for compound 15, hydroxyl group interacts

with Asn241.

Insert Figure 8 here

Eventhough the activity for compound 14 and 15 are considered quite good, hydrogen bond

between methoxy of compound 14 and hydroxyl of compound 15 were not enough to increase

the activity in a significant way. Rather than adding more hydroxyl substituents, it seems more

important to place hydroxyl substituents on N-benzylidene ring at the correct position for the

compounds to be able to inhibit α-glucosidase activity.

3

. Conclusion

All flavone-hydrazones synthesized in this study are active in inhibiting α-glucosidase. The

result strongly suggest that chromone moiety and hydrazone linkage played important role in

suppressing the activity of α-glucosidase activity as the activity of all the compounds improve

significantly as compared to the starting material 4. 2D QSAR model established showed

correlation and contribution of various structural features in the activity. Descriptors such as

AlogP, number of H bond acceptor/donor, molecular weight, and number of rotatable bonds

are statistically important in this study. AlogP and number of rotatable bonds play an important

10

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role as compared to other descriptors like H-bond donors and molecular weight. QSAR

equation suggest that by increasing number of hydrogen bond donor, molecular weight, and

number of rotatable bonds, the inhibition activity could be improved. The validated QSAR

model is suitable to predict α-glucosidase inhibition activity for compounds having similar

skeletal structure in the future. Docking studies showed that benzopyrone ring of flavone

moiety that is located in hydrophobic pocket stabilizes the enzyme-inhibitor complex by

forming Pi-Pi stacking with Phe311 and Pi-cation interaction with Lys155. N-benzylidene

moiety resides in close proximity to catalytic residues like Asp214, Glu276, His348, and

Asp349 indicates that it can serve as a surrogate for terminal glucose in α-glucosidase. On the

other hand, benzohydrazone linkage provides torsional degree of freedom in terms of its

rotatablility, which had substantially decreased the entropic penalty for the formation of

enzyme-inhibitor complex. Phenolic and carbonyl oxygens of compound 5, which is the most

active derivative, forms 4 hydrogen bonds with side chains of His239, His245, His279, and

Thr275. Therefore, the compounds are most likely to be capable of inhibiting the catalytic

action of α-glucosidase by a tight binding in the active site through the multiple hydrogen bond

and hydrophobic interactions in a cooperative fashion. In addition to a-glucosidase inhibition

activity, the compounds were also tested for their cytotoxicity and ability to inhibit histone

deacetylase. It was found that they do not show any activity for histone deacetylase inhibition

and cytotoxicity activity.

4

. Experimental

.1. Chemistry

4

Melting points were determined using Sinosource SGW X-4 melting point apparatus with

microscope (Guangzhou, China). IR spectra obtained using PerkinElmer Spectrum 100 FTIR

Spectrometer (Waltham, MA, USA) equipped with a diamond crystal Attenuated Total

Reflectance (ATR) accessory by PIKE Technologies (Madison, WI, USA). NMR spectroscopy

was obtained using Bruker Ultra Shield FT NMR 500 MHz and Avance III 600 Ascend

spectrometer (Wissembourg, France). EI-MS spectroscopic analysis had been obtained using

Finnigan-MAT-311-A instrument (Bremen, Germany). Thin layer chromatography (TLC) was

performed using precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.20 mm).

4

.2. Synthesis of (E)-4-(3-(2-hydroxyphenyl)-3-oxoprop-1-en-1-yl)benzoic acid (1)

In a 250 ml flask, 2’-hydroxyacetophenone (30 mmole) was being mixed with 4-formylbenzoic

acid (30 mmole). The mixture was dissolved in 100 ml of 15% (w/v) sodium hydroxide in

11

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ethanol and stirred at room temperature. Reaction progress was monitored using TLC and upon

completion, diluted sulphuric acid was added to allow precipitation. The precipitate was

filtered and crystallized in ethanol to afford pure product. Yellow solid; yield 78 %; m.p. 250-

-1

1

2

52 °C; IR (ATR) cm : 2990, 2831, 1678, 1643, 1568, 1291; H-NMR (600 MHz, DMSO-d6):

δ 7.02-7.05 (m, 2H), 7.58 (t, J = 7.2 Hz, 1H), 7.86 (d, J = 15.6 Hz, 1H), 8.00-8.05 (m, 4H),

1

3

8

.13 (d, J = 15.6 Hz, 1H), 8.24 (d, J = 7.5 Hz, 1H), 11.15 (s, 1H), 12.36 (s, 1H); C-NMR (125

MHz, DMSO-d6): δ 117.7, 119.5, 120.9, 122.3, 126.3, 126.3, 128.6, 130.6, 130.6, 133.5,

1

35.5, 137.5, 143.3, 161.4, 168.5, 193.5; Anal. Calcd for C H O : C = 71.64, H = 4.51,

1

6

12

4

+

Found: C = 71.63, H = 4.52; EI MS m/z (% rel. abund.): 268.07 (M , 68.2 %).

4

.3. Synthesis of 4-(4-oxo-4H-chromen-2-yl)benzoic acid (2)

Compound 1 (22.4 mmole) was mixed with iodine (0.23 mmole). The mixture was dissolved in

0 mL of DMSO and refluxed at 170 °C. After 3 hours, sodium thiosulfate was added to the

5

reaction mixture followed by excess amount of water to allow precipitation. The product was

rinsed and allowed to dry at room temperature to afford pure product. Light yellow solid; yield

-1

9

0%; m.p. 298-300 °C; IR (ATR) cm : 3420, 3081, 2885, 1707, 1623, 1588, 1567, 1415,

1

242; H-NMR (500 MHz, DMSO-d6): δ 7.13 (s, 1H), 7.51(t, J = 7.5 Hz, 1H), 7.80 (d, J = 8.5

Hz, 1H), 7.84 (t, J = 7.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 8.09 (d, J = 8.5 Hz, 2H), 8.22 (d, J =

1

3

8

.5 Hz, 2H), 12.21 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 105.9, 118.4, 124.6, 124.6,

1

24.9, 125.7, 126.0, 128.9, 130.3, 130.3, 132.6, 134.4, 156.8, 163.7, 168.8, 178.0; Anal. Calcd

for C H O : C = 72.18, H = 3.79, Found: C = 72.19, H = 3.81; EI MS m/z (% rel. abund.):

1

6

10

4

+

2

66.06 (M , 73.6%)

4

.4. Synthesis of methyl 4-(4-oxo-4H-chromen-2-yl)benzoate (3)

Compound 2 (18.8 mmole) was mixed with 1 ml of concentrated sulphuric acid and dissolved

in 100 ml methanol. The reaction was refluxed for more than 16 hours and monitored using

TLC. Upon completion, methanol was being removed using rotavap. Residue was collected

and rinsed using excessive amount of water and crystallized in methanol to afford pure product.

-1

White solid; yield 95%; m.p. 268-270 °C; IR (ATR) cm : 3325, 3073, 2873, 1742, 1707, 1648,

1

556, 1412, 1262; H-NMR (500 MHz, DMSO-d6): δ 3.91 (s, 3H), 7.16 (s, 1H), 7.52(t, J = 7.0

Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.86 (t, J = 7.0 Hz,1H), 8.07(d, J = 8.0 Hz, 1H), 8.13(d, J =

1

3

8

1

.5 Hz, 2H), 8.27(d, J = 8.5 Hz, 2H); C-NMR (125 MHz, DMSO-d6): δ 55.6, 105.91, 118.2,

24.7, 125.1, 125.1, 125.7, 126.5, 128.2, 128.2, 131.0, 131.5, 133.2, 156.8, 163.7, 167.4, 178.0;

12

ACCEPTED MANUSCRIPT

Anal. Calcd for C H O : C = 72.85, H = 4.32, Found: C = 72.87, H = 4.35; EI MS m/z (% rel.

1

7

12

4

+

abund.): 280.07 (M , 76.3 %),

4

.5. Synthesis of 4-(4-oxo-4H-chromen-2-yl)benzohydrazide (4)

Compound 3 (17.7 mmole) was dissolved in 100 ml methanol containing 25 ml hydrazine

hydrate. The reaction mixture was refluxed for at least 6 hours. Reaction progress was

monitored using TLC. Methanol was removed using rotavap and product was rinsed with

plenty of water. The product was rinsed and allowed to dry at room temperature to afford pure

-1

product. White solid; yield 88%; m.p. 197-199 °C; IR (ATR) cm : 3327, 3139, 2997, 1630,

1

7

1

558, 1469, 1341, 1251; H-NMR (500 MHz, DMSO-d6): δ 4.51 (s, 2H), 6.91 (t, J = 7.5 Hz,

H), 6.96 (d, J = 7.5 Hz, 1H), 7.19 (t, J = 7.0 Hz, 1H), 7.33 (s, 1H), 7.74 (d, J = 7.5 Hz, 1H),

1

3

.918 (s, 4H), 9.82 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 105.8, 118.3, 124.3, 124.8,

25.2, 125.6, 125.6, 126.3, 126.3, 133.8, 137.3, 137., 157.2, 163.3, 167.4, 178.2; Anal. Calcd

for C H N O : C = 68.56, H = 4.32, N = 9.99, Found: C = 68.57, H = 4.34, N = 9.97; EI MS

1

6

12

2

3

+

m/z (% rel. abund.): 280.17 (M , 58.2%)

4

.6. General procedure for synthesis of flavone hydrazones (5-34)

Compound 4 (0.5 mmole) and substituted benzaldehydes (0.5 mmole) were being mixed with 1

ml of glacial acetic acid in a 50 mL round bottom flask. The mixture was being dissolved in 20

mL of methanol and refluxed. Progress monitored using TLC and upon completion, solvent

was being removed using rotary evaporator. Product was collected and rinsed with diethyl

ether to afford pure product.

4

.6.1.(E)-4-(4-oxo-4H-chromen-2-yl)-N'-(2,4,6-trihydroxybenzylidene)benzohydrazide (5)

°

-1

Light yellow solid; Yield 76 %; m.p. 283-285 C; IR (ATR) cm : 3353, 3139, 2994, 1657,

1

2

7

611, 1556, 1519, 1470, 1372, 1269, 1186, 760; H-NMR (500 MHz, DMSO-d6): δ 5.86 (s,

H), 6.93 (t, J = 7.5 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.93 (t, J = 7.0 Hz, 1H), 7.38 (s, 1H),

1

3

.75 (s, 1H), 7.99-8.04 (m, 4H), 8.83 (s, 1H), 9.85 (s, 1H), 11.13 (s, 2H), 11.94 (s, 1H); C-

NMR (125 MHz, DMSO-d6): δ 95.8, 95.8, 105.5, 106.8, 118.2, 125.1, 125.4, 125.4, 125.6,

26.2, 126.8, 126.8, 133.7, 137.2, 138.8, 144.6, 156.2, 161.7, 161.7, 163.2, 163.5, 164.3, 178.1;

Anal. Calcd for C H N O : C = 66.34, H = 3.87, N = 6.73, Found: C = 66.33, H = 3.88, N =

1

2

3

16

2

6

+

6

.71; EI MS m/z (% rel. abund.): 416.20 (M , 72.5%)

4

.6.2.(E)-4-(4-oxo-4H-chromen-2-yl)-N'-(2,3-dihydroxybenzylidene)benzohydrazide (6)

13

ACCEPTED MANUSCRIPT

°

-1

White solid; Yield 81 %; m.p. 288-290 C; IR (ATR) cm : 3373, 3159, 2835, 1609, 1539,

1

469, 1357, 1257, 1186, 754; H-NMR (500 MHz, DMSO-d6): δ 6.75 (t, J = 7.5 Hz, 1H), 6.87

(d, J = 7.0 Hz, 1H), 6.92 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 7.5 Hz, 2H), 7.20 (t, J = 7.0 Hz, 1H),

1

3

7

.76 (s, 1H), 8.01-8.04 (m, 5H), 8.63 (s, 1H), 9.25 (s, 1H), 11.14 (s, 1H), 12.19 (s, 1H); C-

NMR (125 MHz, DMSO-d6): δ 105.9, 118.2, 119.7, 119.9, 121.3, 122.0, 125.0, 125.2, 125.4,

25.4, 126.2, 126.9, 126.9, 133.9, 137.1, 138.7, 144.4, 146.4, 149.3, 155.9, 163.4, 164.5, 178.3;

Anal. Calcd for C H N O : C = 69.00, H = 4.03, N = 7.00, Found: C = 69.01, H = 4.05, N =

1

2

3

16

2

5

+

6

.98; EI MS m/z (% rel. abund.): 400.12 (M , 64.7%)

4

.6.3.(E)-4-(4-oxo-4H-chromen-2-yl)-N'-(2,4-dihydroxybenzylidene)benzohydrazide (7)

°

-1

Light yellow solid; Yield 88 %; m.p. 294-296 C; IR (ATR) cm : IR (ATR) cm : 3354, 3212,

1

3

021, 1626, 1606, 1495, 1466, 1259, 1166, 749; H-NMR (500 MHz, DMSO-d6): δ 6.34 (s,

H), 6.37 (d, J = 8.0 Hz, 1H), 6.91 (t, J = 7.5 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 7.20 (t, J= 7.5

1

Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.36 (s, 1H), 7.75 (d, J= 7.0 Hz, 1H), 8.00-8.03 (m, 4H),

1

3

8

1

6

4

.53 (s, 1H), 9.23 (s, 1H), 10.14 (s, 1H), 11.53 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ

03.0, 105.9, 109.2, 113.1, 118.2, 125.1, 125.4, 125.4, 125.4, 126.2, 126.9, 126.9, 130.8, 133.9,

37.1, 138.7, 151.1, 156.2, 160.5, 160.5, 163.4, 164.3, 178.1; Anal. Calcd for C H N O : C =

2

3

16

2

5

9.00; H = 4.03; N = 7.00; Found: C = 69.02; H = 4.04; N = 7.01; EI MS m/z (% rel. abund.):

+

00.25 (M , 69.2%)

4

.6.4.(E)-4-(4-oxo-4H-chromen-2-yl)-N'-(2,5-dihydroxybenzylidene)benzohydrazide (8)

°

-1

Light orange solid; Yield 74%; m.p. > 320 C; IR (ATR) cm : IR (ATR) cm : 3244, 3164,

1

3

6

1

029, 1647, 1580, 1545, 1487, 1272, 1256, 1141, 747; H-NMR (500 MHz, DMSO-d6): δ

.73-6.78 (m, 2H), 6.91 (t, J = 7.0 Hz, 1H), 6.97-6.99 (m, 2H), 7.20 (t, J= 8.5 Hz, 1H), 7.37 (s,

H), 7.75 (d, J= 7.5 Hz, 1H), 8.00-8.05 (m, 4H), 8.60 (s, 1H), 9.04 (s, 1H), 10.48 (s, 1H), 12.04

1

3

(

s, 1H); C-NMR (125 MHz, DMSO-d6): δ 105.9, 116.4, 118.1, 118.2, 121.6, 122.0, 125.0,

1

=

25.2, 125.4, 125.4, 126.2, 126.9, 126.9, 133.9, 137.1, 138.7, 149.6, 151.2, 152.7, 156.2, 163.4,

64.3, 178.1; Anal. Calcd for C H N O : C = 69.00, H = 4.03, N = 7.00; Found: C = 68.69, H

23

16

2

5

+

4.04, N = 6.99; EI MS m/z (% rel. abund.): 400.15 (M , 71.5%)

4

.6.5.(E)-4-(4-oxo-4H-chromen-2-yl)-N'-(3,4-dihydroxybenzylidene)benzohydrazide (9)

14

ACCEPTED MANUSCRIPT

°

-1

Yellowish white solid; Yield 92 %; m.p. 293-295 C; IR (ATR) cm : IR (ATR) cm : 3381,

1

3

062, 2898, 1613, 1590, 1558, 1470, 1445, 1277, 1180, 752; H-NMR (500 MHz, DMSO-d6):

δ 6.79 (d, J = 8.0 Hz, 1H), 6.91-6.98 (m, 4H), 7.19 (t, J = 7.5 Hz, 1H), 7.35 (s, 1H), 7.75 (d, J =

1

3

7

.0 Hz, 1H), 7.76 (s, 1H), 7.99 (m, 4H), 8.29 (s, 1H), 10.23 (s, 1H), 11.59 (s, 1H); C-NMR

125 MHz, DMSO-d6): δ 105.96, 115.90, 116.62, 118.24, 121.06, 125.0, 125.2, 125.44, 125.4,

26.9, 127.9, 127.9, 133.9, 137.1, 138.7, 145.5, 148.1, 148.6, 149.6, 156.2, 163.4, 164.3, 178.9;

Anal. Calcd for C H N O : C = 69.00, H = 4.03, N = 7.00, Found: C = 69.01, H = 4.02, N =

(

1

2

3

16

2

5

+

6

.99; EI MS m/z (% rel. abund.): 400.27 (M , 76.8%)

4

.6.6.(E)-4-(4-oxo-4H-chromen-2-yl)-N'-(2-hydroxybenzylidene)benzohydrazide (10)

°

-1

White solid; Yield 76 %; m.p. 305-306 C; IR (ATR) cm : 3329, 3210, 3021, 1636, 1606,

1

4

7

537, 1488, 1348, 1259, 1183, 1115, 746; H-NMR (500 MHz, DMSO-d6): δ 6.91-6.99 (m,

H), 7.20 (t, J = 7.5 Hz, 1H), 7.31 (t, J= 8.0 Hz, 1H), 7.37 (s, 1H), 7.56 (d, J= 7.5 Hz, 1H),

1

3

.75 (d, J= 7.0 Hz, 1H), 8.01-8.06 (m, 4H), 8.67 (s, 1H), 10.28 (s, 1H), 11.32 (s, 1H); C-

NMR (125 MHz, DMSO-d6): δ 105.9, 117.3, 118.2, 120.3, 121.1, 125.0, 125.2, 125.4, 125.4,

26.2, 126.9, 126.9, 128.5, 129.7, 133.9, 137.1, 138.7, 151.0, 156.2, 158.3, 163.6, 164.6, 178.1;

Anal. Calcd for C H N O : C = 71.87, H = 4.20, N = 7.29, Found: C = 71.88, H = 4.22, N =

1

2

3

16

2

4

+

7

.30; EI MS m/z (% rel. abund.): 384.32 (M , 67.4%)

4

.6.7.(E)-4-(4-oxo-4H-chromen-2-yl)-N'-(3-hydroxybenzylidene)benzohydrazide (11)

°

Light red solid; Yield 83 %; m.p. 299-301 C; IR (ATR) cm-1: 3385, 3212, 3021, 1626, 1606,

1

495, 1466, 1259, 1166, 756; H-NMR (500 MHz, DMSO-d6): δ 6.84 (d, J = 8.0 Hz, 1H), 6.92

(t, J = 7.5 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 7.5 Hz, 1H), 7.20-7.23 (m, 2H), 7.26

1

3

(

t, J = 7.5 Hz, 1H), 7.75 (s, 1H), 8.01 (m, 5H), 8.39 (s, 1H), 9.67 (s, 1H), 11.85 (s, 1H); C-

NMR (125 MHz, DMSO-d6): δ 105.95, 117.32, 118.20, 119.35, 120.08, 125.03, 125.21, 125.4,

1

4

25.4, 126.2, 126.9, 126.9, 130.6, 133.9, 136.8, 137.2, 138.7, 148.7, 156.2, 156.7, 163.4, 164.3,

78.1; Anal. Calcd for C H N O : C = 71.87, H = 4.20, N = 7.29; Found: C = 71.85, H =

23

16

2

4

+

.22, N = 7.28; EI MS m/z (% rel. abund.): 384.13 (M , 69.2%);

4

.6.8.(E)-4-(4-oxo-4H-chromen-2-yl)-N'-(4-hydroxybenzylidene)benzohydrazide (12)

°

-1

Brown solid; Yield 85 %; m.p. 292-294 C; IR (ATR) cm : 3429, 3254, 3062, 1607, 1587,

1

507, 1467, 1275, 1236, 1180, 755; H-NMR (500 MHz, DMSO-d6): δ 6.85 (d, J = 8.0 Hz,

15

ACCEPTED MANUSCRIPT

1

H), 6.91 (t, J = 7.5 Hz, 1H), 6.97 (d, J= 8.0 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.40 (s, 1H),

.58 (d, J= 8.5 Hz, 2H), 7.75 (s, 1H), 8.00 (m, 5H), 8.38 (s, 1H), 9.98 (s, 1H), 11.70 (s, 1H);

C-NMR (125 MHz, DMSO-d6): δ 105.9, 115.6, 115.6, 118.2, 124.3, 124.8, 125.4, 125.4,

25.9, 126.3, 126.8, 126.8, 129.7, 133.8, 137.1, 137.1, 138.6, 149.3, 156.3, 158.4, 163.4, 164.2,

78.0; Anal. Calcd for C H N O : C = 71.87, H = 4.20, N = 7.29, Found: C = 71.88, H =

7

1

3

1

4

23

16

2

4

+

.21, N = 7.27; EI MS m/z (% rel. abund.): 384.18 (M , 61.5%)

4

.6.9.(E)-N'-(2-hydroxy-4-methoxybenzylidene)-4-(4-oxo-4H-chromen-2-

yl)benzohydrazide (13)

°

-1

Dark yellow solid; Yield 94 %; m.p. 311-312 C; IR (ATR) cm : 3347, 3182, 3075, 2838,

1

633, 1603, 1489, 1465, 1260, 1184, 750; H-NMR (500 MHz, DMSO-d6): δ 3.79 (s, 3H),

.52 (s, 1H), 6.54 (d, J = 8.5 Hz, 1H), 6.92 (t, J = 7.5 Hz, 1H), 6.97 (d, J= 8.0 Hz, 1H), 7.20 (t,

6

J = 8.0 Hz, 1H), 7.44 (d, J = 8.5, 1H), 7.75 (s, 1H), 8.02 (m, 5H), 8.58 (s, 1H), 11.65 (s, 1H),

1

3

1

6

2.04 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 56.3, 105.9, 107.2, 113.9, 118.2, 124.9,

25.2, 125.4, 125.4, 126.2, 126.9, 126.9, 130.8, 133.9, 137.1, 138.7, 151.0, 156.0, 156.2, 161.2,

62.0, 163.4, 164.3, 178.1; Anal. Calcd for C H N O : C = 69.56, H = 4.38, N = 6.76, Found:

2

4

18

2

5

+

9.57, H = 4.39, N = 6.74; EI MS m/z (% rel. abund.): 414.03 (M , 86.9%)

4

.6.10. (E)-N'-(3-hydroxy-4-methoxybenzylidene)-4-(4-oxo-4H-chromen-2-yl)

benzohydrazide (14)

°

-1

Dark orange solid; Yield 87 %; m.p. 293-295 C; IR (ATR) cm : 3389, 3249, 3064, 2843,

1

603, 1584, 1561, 1520, 1470, 1440, 1276, 1246, 1171, 752; H-NMR (500 MHz, DMSO-d6):

δ 3.82 (s, 3H), 6.91 (t, J = 7.5 Hz, 1H), 6.97 (t, J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 1H), 7.20

(t, J = 8.0 Hz, 1H), 7.29 (s, 1H), 7.39 (s, 1H), 7.76 (s, 1H), 8.00 (s, 4H), 8.33 (s, 1H), 9.35 (s,

1

3

1

H), 11.72 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 56.4, 105.8, 115.0, 115.2, 118.2, 120.3,

25.0, 125.3, 125.5, 125.5, 126.2, 126.8, 126.8, 129.5, 133.8, 137.2, 138.6, 146.6, 148.5, 149.7,

56.8, 163.4, 164.4, 178.8; Anal. Calcd for C H N O : C = 69.56, H = 4.38, N = 6.76, Found:

2

4

18

2

5

+

C = 69.55, H = 4.39, N = 6.74; EI MS m/z (% rel. abund.): 414.24 (M , 65.0%)

4

.6.11. (E)-N'-(2-hydroxy-5-methoxybenzylidene)-4-(4-oxo-4H-chromen-2-yl)

benzohydrazide (15)

°

-1

Dark orange solid; Yield 68 %; m.p. 305-307 C; IR (ATR) cm : 3143, 2962, 2835, 1642,

1

614, 1583, 1490, 1462, 1266, 1164, 756; H-NMR (500 MHz, DMSO-d6): δ 3.75 (s, 3H),

16

ACCEPTED MANUSCRIPT

6

4

1

.87-6.99 (m, 4H), 7.14 (s, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.37 (s, 1H), 7.76 (s, 1H), 8.02 (m,

1

3

H), 8.66 (s, 1H), 10.71 (s, 1H), 12.02 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 56.3, 105.9,

12.8, 116.3, 116.5, 118.2, 121.7, 124.9, 125.1, 125.3, 125.3, 126.2, 126.9, 126.9, 133.9, 137.1,

38.7, 149.6, 154.2, 154.4, 156.2, 163.4, 164.3, 178.2; Anal. Calcd for C H N O : C = 69.56,

24

18

2

5

H = 4.38, N = 6.76, Found: C = 69.57, H = 4.36, N = 6.78; EI MS m/z (% rel. abund.): 414.19

+

(

M , 72.5%)

4

.6.12. (E)-N'-(3,5-dimethoxybenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide

16)

(

°

-1

Light orange solid; Yield 71 %; m.p. 232-234 C; IR (ATR) cm : 3394, 3296, 3002, 2837,

1

6

=

684, 1652, 1585, 1462, 1266, 1205, 1157, 736; H-NMR (500 MHz, DMSO-d6): δ 3.86 (s,

H), 6.57 (s, 1H), 6.95 (m, 2H), 7.09 (s, 2H), 7.21 (m, 2H), 7.74 (d, J = 7.5 Hz, 1H), 8.00 (d, J

1

3

8.0 Hz, 2H), 8.06 (d, J = 8.0 Hz, 2H), 8.30 (s, 1H), 11.97 (s, 1H); C-NMR (125 MHz,

DMSO-d6): δ 56.2, 56.2, 98.5, 105.4, 105.4, 105.9, 118.4, 124.9, 125.2, 125.5, 125.5, 126.2,

26.9, 126.9, 133.9, 137.1, 138.2, 138.7, 147.9, 156.3, 161.2, 161.2, 163.4, 164.3, 178.3; Anal.

Calcd for C H N O : C = 70.09, H = 4.71, N = 6.54, Found: C = 70.07, H = 4.70, N = 6.55;

1

2

5

20

2

5

+

EI MS m/z (% rel. abund.): 428.35 (M , 54.1%).

4

.6.13. (E)-N'-(3-bromo-4-hydroxybenzylidene)-4-(4-oxo-4H-chromen-2-

yl)benzohydrazide (17)

°

-1

Brown solid; Yield 79 %; m.p. 254-255 C; IR (ATR) cm : 3352, 3140, 3054, 2897, 1650,

1

591, 1496, 1282, 1205, 750; H-NMR (500 MHz, DMSO-d6): δ 6.91 (t, J = 7.5 Hz, 1H), 6.99

(s, 1H), 7.03 (d, J = 8.0 Hz, 1H), 7.20 (t, J= 8.5 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.73 (s, 1H),

1

3

7

.89 (s, 1H), 8.00 (m, 5H), 8.35 (s, 1H), 10.78 (s, 1H), 11.83 (s, 1H); C-NMR (125 MHz,

DMSO-d6): δ 105.9, 109.9, 116.3, 118.2, 125.1, 125.4, 125.4, 125.4, 125.7, 126.2, 126.9,

26.9, 128.2, 130.4, 133.9, 137.1, 138.7, 148., 153.7, 156.2, 163.4, 164.3, 178.1; Anal. Calcd

for C H BrN O : C = 59.63, H = 3.26, N = 6.05, Found: C = 59.64, H = 3.24, N = 6.06; EI

1

2

3

15

2

4

MS m/z (% rel. abund.): 462.02 (M+, 54.7%)

4

.6.14. (E)-N'-(2-methylbenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (18)

°

-1

White solid; Yield 85 %; m.p. 225-226 C; IR (ATR) cm : 3433, 3253, 3021, 2361, 1609,

1

546, 1466, 1277, 755; H-NMR (500 MHz, DMSO-d6): δ 2.47 (s, 3H), 6.91 (t, J = 7.5 Hz,

H), 6.98 (s, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.27-7.34 (m, 4H), 7.87 (d, J = 7.5 Hz, 1H), 8.02

1

3

(

m, 5H), 8.78 (s, 1H), 11.88 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 21.1, 105.9, 118.2,

17

ACCEPTED MANUSCRIPT

1

4

7

25.1, 125.3, 125.5, 125.5, 126.0, 126.3, 126.6, 126.9, 126.9, 128.2, 130.5, 133.5, 133.8, 135.4,

37.1, 138.6, 145.4, 156.3, 163.5, 164.3, 178.1; Anal. Calcd for C H N O : C = 75.38, H =

2

4

18

2

3

+

.74, N = 7.33, Found: C = 75.39, H = 4.75, N = 7.31; EI MS m/z (% rel. abund.): 382.29 (M ,

0.3%).

4

.6.15. (E)-N'-(3-methylbenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (19)

°

-1

White solid; Yield 93 %; m.p. 210-211 C; IR (ATR) cm : 3406, 3193, 3023, 1625, 1557,

1

309, 1256, 747; H-NMR (500 MHz, DMSO-d6): δ 2.37 (s, 3H), 6.91 (t, J = 7.0 Hz, 1H), 6.98

(d, J = 7.5 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 7.27(d, J = 7.5 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H),

7

1

.45 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.59 (s, 1H), 7.75 (s, 1H), 8.01 (m, 4H), 8.45 (s, 1H),

1

3

1.89 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 21.4, 105.9, 118.2, 123.5, 124.8, 125.2,

25.5, 125.5, 126.0, 126.8, 126.8, 128.2, 128.6, 129.7, 133.8, 136.6, 137.2, 137.9, 138.8, 148.7,

56.2, 163.4, 164.3, 178.3; Anal. Calcd for C H N O : C = 75.38, H = 4.74, N = 7.33, Found:

2

4

18

2

3

+

C = 75.39, H = 4.73, N = 7.31; EI MS m/z (% rel. abund.): 382.12 (M , 61.7%).

4

.6.16. (E)-N'-(4-methylbenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (20)

°

-1

Light yellow solid; Yield 74 %; m.p. 232-234 C; IR (ATR) cm : 3422, 3382, 3242, 3164,

1

609, 1464, 1270, 749; H-NMR (MeOD-d , 500 MHz): δ 2.41 (s, 3H), 6.94-6.98 (m, 2H),

4

7

.21-7.24 (m, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.75-7.77 (m, 3H), 7.99 (d, J = 7.5 Hz, 2H), 8.06

13

(

d, J = 7.5 Hz, 2H), 8.36 (s, 1H), 12.27 (s, 1H); C-HMR (125 MHz, DMSO-d6): δ 21.15,

1

7

3

05.9, 118.2, 125.0, 118.2, 125.0, 125.3, 125.3, 125.8, 126.1, 126.8, 126.8, 127.3, 129.2, 131.8,

33.9, 137.2, 138.2, 138.7, 149.4, 156.3, 163.5, 164.5, 178.0; Anal. Calcd for C H N O : C =

2

4

18

2

3

5.38, H = 4.74, N = 7.33, Found: C = 75.39, H = 4.76, N = 7.31; EI MS m/z (% rel. abund.):

+

82.07 (M , 62.5%).

4

.6.17. (E)-N'-(2-chlorobenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (21)

°

-1

White solid; Yield 78 %; m.p. 294-296 C; IR (ATR) cm : 3432, 3183, 3065, 1613, 1589,

1

543, 1467, 1280, 757; H-NMR (500 MHz, DMSO-d6): δ 1H-NMR (MeOD-d4, 500 MHz): δ

.94-6.98 (m, 2H), 7.21-7.25 (m, 2H), 7.42-7.46 (m, 2H), 7.48 (d, J = 8.0 Hz, 1H), 7.75 (dd, J

6

=

7.5 Hz, 1.5 Hz, 1H), 8.00 (d, J = 7.0 Hz, 2H), 8.08 (d, J = 7.5 Hz, 2H), 8.31 (dd, J = 7.5 Hz,

13

1

.5 Hz, 1H), 8.91 (s, 1H), 12.23 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 105.9, 118.2,

25.0, 125.2, 125.4, 125.4, 126.5, 126.9, 126.9, 127.3, 127.5, 128.6, 130.9, 131.3, 132.7, 133.9,

37.1, 138.7, 149.8, 156.2, 163.2, 164.3, 178.1; Anal. Calcd for C H ClN O : C = 68.58, H =

2

3

15

2

3

18

ACCEPTED MANUSCRIPT

3

7

.75, N = 6.95, Found: C = 68.59, H = 3.76, N = 6.93; EI MS m/z (% rel. abund.): 402.08 (M+,

4.2%), 404.08 (M+2, 13.8%).

4

.6.18. (E)-N'-(3-chlorobenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (22)

°

-1

Light yellow solid; Yield 69 %; m.p. 304-306 C; IR (ATR) cm : 3273, 3120, 2915, 1660,

1

542, 1469, 1253, 743; H-NMR (500 MHz, DMSO-d6): δ 6.89 (t, J = 7.5 Hz, 1H), 6.96 (d, J

=

8.0 Hz , 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.34 (s, 1H), 7.51 (d, J = 4.0 Hz, 2H), 7.72-7.75 (m,

1

3

2

H), 7.81 (s, 1H), 8.02 (s, 4H), 8.47 (s, 1H), 12.06 (s, 1H); C-NMR (125 MHz, DMSO-d6):

δ 105.9, 118.2, 124.4, 124.8, 125.2, 125.4, 125.4, 126.2, 126.9, 126.9, 127.6, 128.0, 130.2,

33.7, 134.0, 135.3, 137.1, 138.7, 148.6, 156.2, 163.4, 164.3, 178.2; Anal. Calcd for

C H ClN O : C = 68.58, H = 3.75, N = 6.95, Found: C = 68.57, H = 3.77, N, 6.97; EI MS m/z

1

23

15

2

3

(% rel. abund.): 402.08 (M+, 35.4%), 404.36 (M+2, 13.8%).

4

.6.19. (E)-N'-(4-chlorobenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (23)

°

-1

Yellow solid; Yield 91 %; m.p. 295-297 C; IR (ATR) cm : 3427, 3126, 2947, 1608, 1589,

1

466, 1273, 752; H-NMR (MeOD-d , 500 MHz): δ 6.94-6.98 (m, 2H), 7.21 (td, J = 8.5 Hz,

4

1

.0 Hz, 1H), 7.24 (s, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.75 (dd, J = 7.5 Hz, 1.5 Hz, 1H), 7.87 (d, J

=

8.5 Hz, 2H), 8.00 (d, J = 7.5 Hz, 2H), 8.02 (d, J = 8.0 Hz, 2H), 8.38 (s, 1H), 12.03 (s, 1H);

C-NMR (125 MHz, DMSO-d6): δ 105.9, 118.4, 125.1, 125.4, 125.4, 125.8, 126.3, 126.8,

26.8, 129.0, 129.0, 129.4, 129.4, 132.3, 134.1, 135.1, 137.1, 138.7, 149.4, 156.3, 163.3, 164.3,

78.3. Anal. Calcd for C H ClN O : C = 68.58, H = 3.75, N = 6.95, Found: C = 68.59, H =

1

3

1

3

2

3

15

2

3

.74, N = 6.94; EI MS m/z (% rel. abund.): 402.08 (M+, 64.7%), 404.21 (M+2, 13.8%).

4

.6.20. (E)-N'-(2-nitrobenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (24)

°

-1

Yellow solid; Yield 65 %; m.p. 305-306 C; IR (ATR) cm : 3431, 3255, 3153, 3024, 1623,

1

543, 1519, 1468, 1274, 755; H-NMR (MeOD-d , 500 MHz): δ 6.91 (t, J = 7.5 Hz, 1H), 6.98

4

(d, J = 7.5 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.37 (s, 1H), 7.69-7.93 (m, 3H), 8.03-8.18 (m,

1

3

6

1

H), 8.91 (s, 1H), 12.26 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 105.91, 118.28, 125.00,

25.1, 125.2, 125.4, 125.4, 126.2, 126.9, 126.9, 128.0, 129.3, 130.4, 132.6, 133.7, 137.1, 138.7,

42.9, 147.9, 156.9, 163.4, 164.3, 178.1; Anal. Calcd for C H N O : C = 66.83, H = 3.66, N

2

3

15

3

5

19

ACCEPTED MANUSCRIPT

+

=

10.17, Found: C = 66.84, H = 3.65, N = 10.19; EI MS m/z (% rel. abund.): 413.07 (M ,

4

2.7%).

4

.6.21. (E)-N'-(3-nitrobenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (25)

°

-1

Light orange solid; Yield 62 %; m.p. 312-314 C; IR (ATR) cm : 3312, 3179, 3143, 2886,

1

657, 1519, 1469, 1342, 1274, 1145, 732; H-NMR (500 MHz, DMSO-d6): δ 6.91 (t, J = 7.5

Hz, 1H), 6.98 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.37 (s, 1H), 7.75-7.77 (m, 2H),

8

1

.03 (m, 4H), 8.17 (d, J = 6.5 Hz, 1H), 8.27(d, J = 7.5 Hz, 1H), 8.57-8.60 (m, 3H), 12.16 (s,

1

3

H); C-NMR (125 MHz, DMSO-d6): δ 105.94, 118.22, 122.37, 124.18, 125.05, 125.24,

25.5, 125.5, 126.2, 126.9, 126.9, 129.9, 132.6, 133.9, 137.1, 137.7, 138.7, 147.41, 148.6,

56.2, 163.4, 164.3, 178.1; Anal. Calcd for C H N O : C = 66.83, H = 3.66, N = 10.17,

23

15

3

5

+

Found: C = 66.84, H = 3.65, N = 10.18; EI MS m/z (% rel. abund.): 413.15 (M , 52.6%).

4

.6.22. (E -(4-nitrobenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (26)

°

-1

Dark brown solid; Yield 88 %; m.p. 297-299 C; IR (ATR) cm : 3413, 3075, 2833, 1633,

1

8

514, 1466, 1341, 1269, 1113, 756; H-NMR (500 MHz, DMSO-d6): δ 6.92 (t, J = 7.5 Hz,

H), 6.98 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.37 (s, 1H), 7.75 (d, J = 7.0 Hz, 1H),

1

3

.03 (m, 6H), 8.32-8.33 (m, 2H), 8.59 (s, 1H), 12.22 (s, 1H); C-NMR (125 MHz, DMSO-d6):

δ 106.5, 118.2, 124.4, 124.4, 125.0, 125.2, 125.4, 125.4, 125.6, 126.2, 126.9, 126.9, 127.9,

27.9, 133.9, 137.1, 138.7, 148.0, 149.3, 156.2, 163.4, 164.3, 178.1; Anal. Calcd for

C H N O : C = 66.83, H = 3.66, N = 10.17, Found: C = 66.82, H = 3.67, N = 10.18; EI MS

1

23

15

3

5

+

m/z (% rel. abund.): 413.19 (M , 64.8%).

4

.6.23. (E)-N'-(2-fluorobenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (27)

°

-1

White solid; Yield 84 %; m.p. 257-259 C; IR (ATR) cm : 3434, 3232, 3019, 2861, 1660,

1

2

1

608, 1519, 1466, 1360, 1272, 1187, 752; H-NMR (MeOD-d , 500 MHz): δ 6.96-6.98 (m,

4

H), 7.19-7.24 (m, 3H), 7.28 (t, J = 7.5 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.74 (dd, J = 7.5 Hz,

.5 Hz, 1H), 8.00 (d, J = 8.5 Hz, 2H), 8.07 (d, J = 8.5 Hz, 2H), 8.24 (t, J = 7.5 Hz, 1H), 8.70

1

3

(

s, 1H), 12.11 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 105.9, 117.4, 118.3, 124.6, 125.1,

1

3

25.4, 125.4, 125.6, 125.8, 126.3, 126.9, 126.9, 129.4, 130.9, 133.9, 137.1, 138.7, 149.3, 156.1,

59.3 (d, J = 282.5 Hz), 163.4, 164.3, 178.3; Anal. Calcd for C H FN O : C = 71.50, H =

23

15

2

3

.91, F = 4.92, N = 7.25, Found: C = 71.51, H = 3.89, F = 4.91, N = 7.26; EI MS m/z (% rel.

+

abund.): 386.06 (M , 72.6%).

20

ACCEPTED MANUSCRIPT

4

.6.24. (E)-N'-(3-fluorobenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (28)

°

White solid; Yield 78 %; m.p. 254-256 C; IR (ATR) cm-1: 3119, 3026, 2828, 1658, 1539,

1

7

8

1

449, 1269, 745; H-NMR (MeOD-d , 500 MHz): δ 6.94-6.98 (m, 2H), 7.18-7.24 (m, 3H),

4

.46 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.72-7.76 (m, 2H), 8.00 (d, J = 7.5 Hz, 2H),

1

3

.06 (d, J = 8.0 Hz, 2H), 8.38 (s, 1H), 12.20 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 105.8,

14.4, 117.1, 118.4, 121.8, 124.7, 125.2, 125.7, 125.7, 126.2, 126.9, 126.9, 129.9, 133.9, 137.1,

38.2, 138.7, 148.6, 156.2, 161.6 (d, J = 193.75 Hz), 163.7, 164.4, 178.1; Anal. Calcd for

C H FN O : C = 71.50, H = 3.91, N = 7.25. Found: C = 71.51, H = 3.93, F = 4.91, N = 7.24;

23

15

2

3

+

EI MS m/z (% rel. abund.): 386.11 (M , 62.7%).

4

.6.25. (E)-N'-(4-fluorobenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (29)

°

Light yellow solid; Yield 95 %; m.p. 252-254 C; IR (ATR) cm-1: 3421, 3282, 3061, 2947,

1

8

1

633, 1609, 1505, 1467, 1233, 752; H-NMR (500 MHz, DMSO-d6): δ 6.92 (t, J = 7.0 Hz,

H), 6.98 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.30-7.36 (m, 3H), 7.75-7.82(m, 3H),

1

3

.02(m, 4H), 8.49(s, 1H), 11.92(s, 1H); C-NMR (125 MHz, DMSO-d6): δ 105.9, 115.3,

15.3, 115.6, 118.2, 124.6, 125.3, 125.7, 125.7, 126.2, 126.9, 126.9, 130.3, 130.8, 130.8, 133.8,

37.1, 138.7, 149.3, 156.2, 160.3 (d, J = 245.00 Hz), 163.3, 164.3, 178.2; Anal. Calcd for

C H FN O : C = 71.50, H = 3.91, N = 7.25, Found: C = 71.49, H = 3.93, N = 7.26; EI MS

23

15

2

3

+

m/z (% rel. abund.): 386.41 (M , 47.1%).

4

.6.26. (E)-N'-(3-methoxybenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (30)

°

Light brown solid; Yield 91 %; m.p. 231-233 C; IR (ATR) cm-1: 3539, 3252, 3051, 2835,

1

640, 1564, 1469, 1253, 1141, 747; H-NMR (MeOD-d , 500 MHz): δ 3.90 (s, 3H), 6.94-6.98

4

(m, 2H), 7.02 (d, J = 8.0 Hz, 1H), 7.21-7.25 (m, 2H), 7.33-7.39 (m, 2H), 7.62 (s, 1H), 7.75 (d, J

=

6.5 Hz, 1H), 8.00 (d, J = 7.0 Hz, 2H), 8.07 (d, J = 7.5 Hz, 2H), 8.36 (s, 1H), 12.08 (s, 1H);

C-NMR (125 MHz, DMSO-d6): δ 56.2, 105.9, 112.2, 114.8, 118.2, 121.2, 124.9, 125.2,

25.4, 125.4, 126.2, 126.9, 126.9, 129.2, 133.9, 136.9, 137.1, 138.7, 148.6, 156.3, 160.2, 163.2,

64.2, 178.3; Anal. Calcd for C H N O : C = 72.35; H = 4.55; N = 7.03, Found: C = 72.33; H

1

3

1

=

2

4

18

2

4

+

4.56; N = 7.01; EI MS m/z (% rel. abund.): 398.26 (M , 51.7%).

4

.6.27. (E)-N'-(4-methoxybenzylidene)-4-(4-oxo-4H-chromen-2-yl)benzohydrazide (31)

°

Yellow solid; Yield 89 %; m.p. 237-239 C; IR (ATR) cm-1: 3424, 3269, 3065, 2843, 1610,

1

507, 1466, 1356, 1244, 755; H-NMR (500 MHz, DMSO-d6): δ 3.83 (s, 3H), 6.92 (t, J = 7.0

Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 7.04 (d, J = 8.5 Hz, 2H), 7.20 (t, J = 7.0 Hz, 1H), 7.36 (s,

21

ACCEPTED MANUSCRIPT

1

3

1

H), 7.70-7.77 (m, 3H), 8.00 (s, 4H), 8.44 (s, 1H), 11.77 (s, 1H); C-NMR (125 MHz, DMSO-

d6): δ 56.0, 105.9, 114.3, 114.3, 118.2, 125.0, 125.2, 125.4, 125.4, 126.2, 126.6, 127.0, 127.0,

29.1, 129.1, 133.9, 137.1, 138.7, 149.3, 156.2, 160.1, 163.2, 164.6, 178.3. Anal. Calcd for

C H N O : C = 72.35, H = 4.55, N = 7.03, Found: C = 72.36, H = 4.53, N = 7.02; EI MS m/z

1

24

18

2

4

+

(

% rel. abund.): 398.17 (M , 69.4%).

4

.6.28. (E)-4-(4-oxo-4H-chromen-2-yl)-N'-(pyridin-2-ylmethylene)benzohydrazide (32)

°

Orange solid; Yield 81 %; m.p. 170-172 C; IR (ATR) cm-1: 3395, 3170, 3058, 2849, 1643,

1

2

564, 1465, 1255, 734; H-NMR (MeOD-d , 500 MHz): δ 6.95-6.98 (m, 2H), 7.21-7.26 (m,

4

H), 7.46 (d, J = 5.0 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.93 (t, J = 7.5 Hz, 1H), 8.01-8.02 (m,

1

3

H), 8.07-8.13 (m, 3H), 8.34 (d, J = 7.5 Hz, 1H), 8.45 (s, 1H), 11.78 (s, 1H); C-NMR (125

MHz, DMSO-d6): δ 105.9, 118.2, 119.0, 123.0, 125.0, 125.2, 125.4, 125.4, 126.3, 126.9,

26.9, 133.9, 137.1, 137.6, 138.7, 146.1, 147.9, 154.6, 156.2, 163.2, 164.5, 178.0. Anal. Calcd

for C H N O : C = 71.54, H = 4.09, N = 11.38, Found: C = 71.55, H = 4.10, N = 11.39; EI

1

2

15

3

+

MS m/z (% rel. abund.): 369.21 (M , 47.0%).

4

.6.29. (E)-4-(4-oxo-4H-chromen-2-yl)-N'-(pyridin-3-ylmethylene)benzohydrazide (33)

°

Orange solid; Yield 79 %; m.p. 210-212 C; IR (ATR) cm-1: 3417, 3037, 2837, 1641, 1556,

1

467, 1280, 755; H-NMR (MeOD-d , 500 MHz): δ 6.96 (m, 2H), 7.21-7.24 (m, 2H), 7.55 (d, J

4

=

5.0 Hz, 1H), 7.75 (d, J = 5.5 Hz, 1H), 8.00-8.07 (m, 4H), 8.43-8.45 (m, 2H), 8.61 (d, J = 3.5

1

3

Hz, 1H), 8.95 (s, 1H), 12.21 (s, 1H); C-NMR (125 MHz, DMSO-d6): δ 105.9, 118.2, 124.41,

1

25.0, 125.2, 125.4, 125.4, 126.2, 126.9, 126.9, 133.8, 134.6, 135.6, 137.1, 138.7, 145.5, 146.3,

49.8, 156.2, 163.4, 164.2, 178.1; Anal. Calcd for C H N O : C = 71.54, H = 4.09, N =

2

15

3

+

1.38, Found: C = 71.53, H = 4.08, N = 11.37; EI MS m/z (% rel. abund.): 369.15 (M , 67.0%).

4

.6.30. (E)-4-(4-oxo-4H-chromen-2-yl)-N'-(pyridin-4-ylmethylene)benzohydrazide (34)

°

Dark red solid; Yield 76 %; m.p. 198-200 C; IR (ATR) cm-1: 3289, 3172, 2850, 1657, 1544,

1

466, 1262, 1113, 754; H-NMR (MeOD-d , 500 MHz): δ 6.94-6.98 (m, 2H), 7.21 (t, J = 7.5

4

Hz, 1H), 7.25 (s, 1H), 7.74 (dd, J = 8.0 Hz, 1.5 Hz, 1H), 7.88 (d, J = 5.0 Hz, 2H), 8.01 (d, J =

8

.5 Hz, 2H), 8.08 (d, J = 8.0 Hz, 2H), 8.39 (s, 1H), 8.64 (d, J = 5.5 Hz, 2H), 12.76 (s, 1H);

C-NMR (125 MHz, DMSO-d6): δ 105.9, 118.2, 122.3, 122.3, 125.0, 125.28, 125.4, 125.4,

26.2, 126.9, 126.9, 133.9, 137.1, 138.7, 140.7, 149.3, 150.0, 150.0, 156.2, 163.4, 164.3, 178.1.

1

3

1

22

ACCEPTED MANUSCRIPT

Anal. Calcd for C H N O : C = 71.54, H = 4.09, N = 11.38, Found: C = 71.56, H = 4.11, N =

2

15

3

1

1.36; EI MS m/z (% rel. abund.): 369.07 (M+, 64.5%)

4

.4 α-Glucosidase Inhibition Assay

The α-glucosidase inhibition assay had been carried out using baker’s yeast α-glucosidase (EC

.2.1.20) and p-nitrophenyl-α-D-glucopyranoside [49]. The samples (5 µg/mL) were prepared

3

by dissolving the compounds 1-35 in DMSO. Test samples (10 µL) which had been prepared

were reconstituted in 100 µL of phosphate buffer (100 mM) at pH 6.8 in 96-well micro-plate

and incubated with 50 µL of baker’s yeast α-glucosidase for 5 minutes before 50 µL of p-

nitrophenyl-α-D-glucopyranoside (5 mM) was added. After incubating for 5 minutes, the

absorbance was measured at 405 nm using SpectraMax plus384 (Molecular Devices

Corporation, Sunnyvale, CA, USA). Blank in which the substrate was changed with 50 µL of

buffer were analysed to accurately determine the background absorbance. Control sample was

prepared to contain 10 µL DMSO instead of test samples. Percentage of enzyme inhibition was

measured using the following formula.

%

inhibition = [(A – B)/A] × 100

[

A] represents absorbance of control samples, and [B] corresponding to absorbance in presence

of test samples.

4

.5 In Vitro HDAC Inhibition Assay

This assay had been performed in accordance with method in Li et al. (2014) [50]. In this

assay, 10 µL of enzyme solution was mixed with 50 µL of tested compound at various

concentrations. The mixture was incubated at 37 °C for 5 minutes prior to addition of 40 µL

fluorogenic substrate Boc-Lys(acetyl)-AMC. After further incubation at 37 °C for 30 min, the

reaction was stopped by adding 100 µL of trypsin and TSA. Fluorescence intensity was

measured after 20 minutes using a microplate reader at excitation and emission wavelengths of

3

90 and 460 nm, respectively.

4

.6 Cytotoxicity

The Neutral Red cytotoxicity assay is based on the initial protocol described by Borenfreund et

al. (1998) [51] with some modifications. Briefly, the cells (1x104/well) were seeded in 96-well

23

ACCEPTED MANUSCRIPT

microtiter plates (Nunc) and allowed to grow for 24 hours before treatment. After 24 hours of

incubation, the cells were treated with six different concentrations (0.1-100 µg/mL) of test

compounds, in three replicates. The plates were incubated for 72 h at 37°C in a 5% CO₂

incubator. A stock solution was obtained by dissolving the test compounds in DMSO. Further

dilution to different tested concentrations were then carried out ensuring that the final

concentration of DMSO in the test and control wells was not in excess of 1% (v/v). No effect

due to the DMSO was observed. Doxorubicin was used as the positive control. The well

containing untreated cells was the negative control. At the end of the incubation period, the

media were replaced with medium containing 50 µg/mL of Neutral Red. The plates were

incubated for another 3 hours to allow for uptake of the vital dye into the lysosomes of viable

and injured cells. After the incubation period, the media were removed and cells were washed

with the neutral red washing solution. The dye was eluted from the cells by adding 200 µL of

Neutral Red resorb solution and incubated for 30 minutes at room temperature with rapid

agitation on a microtiter plate shaker. Dye absorbance was measured at 540 nm using a

spectrophotometer ELISA plate reader.

4

.7 2D QSAR

Training set and test set compounds were being selected using “Cluster Ligands” protocol in

Discovery Studio DS 2.5 software (Discovery Studio 2.5, Accelrys, Co. Ltd). Another module,

“Calculate Molecular Properties”, was used to calculate the 2D molecular properties as well as

energies of highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO) of

the training set compounds. All semi-empirical calculation and density functional theory was

carried out using software package VAMP and DMol3 in Discovery Studio 2.5. Different 2D

descriptors such as AlogP, molecular properties (molecular weight), molecular property counts

(Num_aromatic

Rings,

Num_-H_Acceptors,

Num_H_Donors,

Num_Rings

and

Num_Rotatable Bonds) and surface area and volume (Molecular_Fractional Polar Surface

Area), were utilized in our model. The model was validated using test set correlation and

Leave-one-out (LOO) cross validation.

4

.8 Docking studies

The structure of all compounds were prepared using Chem3D by CambridgeSoft. The

geometry and energy of the structures were being optimized using Steepest-Descent and Polak-

Ribiere algorithm in HyperChem. AutoDock 4.2 [52] was used to identify the binding modes

of flavone derivatives (5-34) responsible for the activity. Genetic Algorithm (GA) with default

24

ACCEPTED MANUSCRIPT

settings was employed for the studies. Protein sequence for Baker’s yeast α-glucosidase

(MAL12) was obtained from uniprot (http://www.uniprot.org). Homology model for

Saccharomyces cerevisiae α-glucosidase was built using crystal structure of α-D-glucose

bound isomaltase from Saccharomyces cerevisiae (PDB ID: 3A4A) which shares 72% identical

and 85% similar sequence with α-glucosidase. Sequence alignment and homology modelling

were performed using Swiss-Model, which is a fully automated homology modelling pipeline

SWISS-MODEL, managed by Swiss Institute of Bioinformatics [53-55]. The docking results

had been visualized using Discovery Studio visualizer 3.5 [56] and PyMol [57]. The homology

model was being evaluated by using PROCHECK [58, 59].

Acknowledgement

The authors would like to acknowledge Universiti Teknologi MARA for the financial support

through research grant, UiTM 100-RMI/MOA 16/6/2 (1/2013). The first author would like to

thank UiTM and Ministry of Higher Education Malaysia for the financial funding through

Dana Kecemerlangan Pendidikan UiTM and MyBrain15 scholarship.

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Article Doi

Synthesis of novel flavone hydrazones: In-vitro evaluation of α-glucosidase inhibition, QSAR analysis and docking studies

DOI: 10.1016/j.ejmech.2015.10.017

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Article abstract of DOI:10.1016/j.ejmech.2015.10.017

Full text of DOI:10.1016/j.ejmech.2015.10.017

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