Design, synthesis and 2D QSAR study of novel pyridine and quinolone hydrazone derivatives as potential antimicrobial and antitubercular agents

ACCEPTED MANUSCRIPT

Graphical abstract

Compounds 6o, 6p, and 6r displayed superior antimycobacterial (MIC = 0.39 µg/mL) and

antimicrobial (MICs = 0.49- 3.9 µg/mL) activities. Compounds 6e, 6f, 6l and 6n showed very

good antimycobacterial activities with MIC 0.78 µg/mL and good antimicrobial activity MICs =

0

.49- 15.63 µg/mL.

OCH₃

O

N

- electronic effect

different positions

N

R

N

H

-

H

N

H CO

3

N

Cl= 3-or 4-Cl

Cl

6p

MIC _a_n_t_i_b_a_c_t_.₌0.49, 0.98 µg/mL

MIC _M_._T_u_b_e_r_c_u_l_o_s_i_s= 0.39 µg/mL

ACCEPTED MANUSCRIPT

1

. Introduction

Tuberculosis (TB) could not be merely considered a disease, but rather a prevalent

epidemic, which according to the World Health Organization (WHO), is considered as the world’s

deadliest communicable disease [1]. As stated in the latest WHO report, there were an estimated

10.4 million new (incident) TB cases in 2015 worldwide, of which HIV patients accounted for 1.2

million (11%) of all new TB cases, [2]. There was also an estimation of 1.4 million TB deaths in

the same year, with an additional of 0.4 million deaths among HIV infected people. Although the

number of TB deaths fell by 22% between 2000 and 2015, TB remained one of the top 10 causes

of death worldwide in 2015 [2-4]. The development of drug resistant strains including multidrug-

resistant (MDR), extremely drug resistance (XDR) and the recently cases of totally drug resistance

(TDR), profoundly increase the challenges to eradicate TB worldwide [4,5]. In 2015, there were an

estimated 480 000 new cases of multidrug-resistant TB (MDR-TB) [2]. Moreover, WHO estimates

that one third of the world population are infected by latent TB, where treatment is still unavailable

due to the lack of new drugs.

In the same context, the emergence of resistance to the major classes of antibacterial agents

constitute a serious public health threat. Particularly, in recent years, much attention has been

directed towards the multi-drug resistant bacteria and fungi, resulting mainly from the widespread

use and misuse of classical antimicrobial drugs [6]. On the report of WHO, multidrug-resistant

bacteria are responsible for approximately 25000 deaths in Europe each year [7,8]. In fact, bacterial

infection has evolved once again as a global serious threat.

Consequently, one of the fundamental challenges in medicinal chemistry has become the

development of new antimycobacterial and antibacterial agents with potent activity against these

drug-resistant microorganisms [9]. Regarding development of novel antitubercular and

antibacterial agents, quinolone derivatives and isonicotinyl hyrazone analoges arose much interest

due to their well established activities and clinical applications [10-19].

Quinolones are one of the most useful and versatile antibacterial agents, where several candidates

are already in clinical use such as ciprofloxacin I, norfloxacin II, it is also a core pharmacophore in

the recently developed antitubercular drugs such as Bedaquiline III, mefloquine IV, moxifloxacin

V, (Figure 1) [20-23]. In addition to novel synthesized compounds that with promising

antitubercular and antibacterial agents such as VI [24], (Figure 1).

Moreover, several INH derivatives have also shown interesting anti-TB activities such as, saluzide

VII, salizide VIII, verazide IX, ftivazide X, INHA-17 XI, (Figure 1) [25-33].

1

ACCEPTED MANUSCRIPT

It is also interesting to note that, substituted carbohyrazone moiety has been reported as a well-

known pharmacophoric group for antitubercular and antibacterial activity. [34-41]

Therefore, the target compounds 1,4-dihydroquinoline-3-carbohydrazide 12a-l and substituted

benzylidene-2-methylnicotinohydrazide 6a-r, were rationalized so as to comprise these hydrazone

pharmacophores. With the aim of extending the antibacterial spectrum, structural modifications on

the benzenoid and the pyridine rings of quinolone, as well as the nicotinohydrazone scaffold were

carried out. Furthermore, 2D-QSAR models for the novel compounds were generated in order to

correlate these activities to their physically relevant descriptors.

2. Results and Discussion

2.1. Chemistry

The synthetic pathways adopted to prepare the new targeted compounds are depicted in

schemes 1-2. In scheme 1, ethyl 2-methyl-6-arylnicotinate 3a,b was obtained via the reaction of

enaminones 2a,b with ethyl acetoacetate and ammonium acetate in refluxing acetic acid [42]. The

nicotinic acid hydrazides 4a,b were obtained in 70-90% yield, through hydrazinolysis of the ester

derivatives 3a,b (Scheme 1).

IR spectra of the hydrazides 4a,b showed absorption bands due to the carbonyl group in the

-1

region 1643, 1660 cm , in addition to bands in the region from 3282, 3321 and 3322, 3195 cm ,

1

assigned to the NH and NH groups. The H NMR spectra of 4a,b showed two singlet D O-

2

exchangeable signals attributed to NH and NH protons in the region δ 9.63 and 4.54 ppm,

2

respectively, while the methyl (-CH ) protons appeared as singlets around δ 2.50, 2.61 ppm.

3

1

3

Moreover, C NMR spectra of 4a,b showed signals resonating around δ 166 ppm attributable to

the carbons of carbonyl groups, while the carbons of the methyl groups appeared in δ 23.04, 23.56

ppm. Consequently, these hydrazide derivatives 4a and 4b were reacted with different aldehydes

5a-j, in ethanol in the presence of a catalytic amount of glacial acetic acid to furnish the target

derivatives 6a-r (Scheme 1).

IR spectra of the synthetic compounds 6a-r showed absorption bands due to the NH groups in

-1

1

the region 3163-3228 cm , in addition to a carbonyl band in the region 1643-1665 cm . The H

NMR spectra of 6a-r showed two singlet D O-exchangeable signals attributable to NH protons of

2

the hydrazine function (=N-NH-) in the region δ 10.70- 12.36 ppm, respectively, and the methyl (-

CH ) protons appeared as singlet signals around δ 2.51- 2.67 ppm, while the (CH=N) protons

3

1

3

appeared as a singlet around δ 7.97-8.67 ppm. Furthermore, C NMR spectra of 6a-r showed

2

ACCEPTED MANUSCRIPT

signals resonating in the range δ 164.15- 164.40 ppm attributable for the carbons of carbonyl

groups, while the carbons of the methyl groups appeared in the range δ 21.43-23.44 ppm.

It has been reported that compounds containing arylidene–hydrazide structure may exist as E/Z

geometrical isomers about –C=N– double bond and cis/trans amide conformers around amide

function (-CONH-). These compounds were present in higher percentage in DMSO-d6 in the form

of geometrical E isomer about –C=N– bond [43,44]. The presence of cis/trans conformers can be

established by observing the signal of the amide group (–CONH–) which appeared as two singlets.

Consequently, in all cases throughout the work, two signals of azamethine of E isomer proton (–

N=CH–) appeared at δ ≈ 7.97-8.10 and 8.21-8.43 ppm [45,46] due to the formation of cis/trans

conformers. In the present study, it could be suggested that the signals of Z isomer (which are the

up-field signals [47,48] have not appeared, whereas the cis/trans conformers of E isomer were

clearly observed.

Scheme 1

On the other hand, ethyl anilinomethylenemalonate 8a,b was obtained via reaction of substituted

aniline 7a,b with diethyl ethoxy methylene malonate. Cyclization of the 8a,b in diphenyl ether

gave the substituted 1, 4-dihydro-4 oxoquinoline-3-carboxylic acid ethyl ester, 9a,b. The 1,4-

dihydro-4-oxo-quinoline-3-carbohydrazide derivatives 10a,b were obtained through the

hydrazinolysis of the ester derivatives 9a,b with refluxing hydrazine hydrate with 70-80% yield.

The target compounds 12a-l were obtained through reaction of the hydrazides 10a,b with different

aldehydes in DMF (Scheme 2).

IR spectra of compounds 12a-l showed absorption bands due to the carbonyl group in the region

-1

1

647-1680 cm , in addition to bands in the region from 3201 to 3425 cm , which were assigned to

1

the NH groups. The H NMR spectra of 12a-l showed two singlets D O-exchangeable singlets

2

attributed to –CONH and NH protons in the region δ 12.65-13.16 and 13.03 -13.28 ppm,

1

3

respectively, while the (CH=N) protons appeared as a singlet around δ 8.31-8.67 ppm. C NMR

spectra of 12a-l showed signals resonating around δ 161 ppm attributable to the carbons of

carbonyl groups.

Scheme 2

3

ACCEPTED MANUSCRIPT

2

.2. Biological Evaluation

.2. Anti-Microbial Activity

2

Antibacterial and antifungal activities were performed at the Regional Center for Mycology and

Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. Target compounds 6a–r, 12a-l and

reference drugs were evaluated in vitro for their antibacterial, antifungal and anti-mycobacterial

activities, by inhibition zone technique and minimum inhibitory concentration (MIC), using two

fungi: Aspergillus fumigatus (RCMB 02568, Af) and Candida albicans (RCMB 05036, Ca), two

gram-positive bacteria: Streptococus pneumoniae (RCMB 010010, Sp), and Staphylococcus

aureus (RCMB 010028, Sa), two gram-negative bacteria: Pseudomonas aeruginosa (RCMB

0

10043, Pa), Escherichia coli (RCMB 010052, Ec), and Mycobaterium tuberculosis (RCMB

10126).

2

.2.1. Anti-Fungal Activity

The results of the tested compounds against Aspergillus fumigatus and Candida albicans were

presented in tables 1 and 2, compounds 6f, and 6p, 2-methylnicotinohydrazide exhibited superior

antifungal activities over all the synthesized target compounds. This compound 6f demonstrated

two-folds the activity against Aspergillus fumigatus and Candida albicans with concentrations 0.98

and 0.49 µg/mL, respectively, while compound 6p showed two-folds of activity against

Aspergillus fumigatus and the same against Candida albicans with concentrations 0.98 and 0.98

µg/mL, respectively, compared to concentration of amphotericin B (0.98 and 1.95 µg/mL,

respectively).

Table 1.

Table 2.

These results of antifungal activities were greatly appreciated as it is well demonstrated that

Aspergillus fumigatus cause invasive aspergillosis on immune comprised patients [49]. In addition

to the well-known resistance of Candida albicans to antifungal agents [50]. Similarly, the

trimethoxy phenyl counterpart 6l and 2,5-dimethoxy phenyl 6r showed significant antifungal

activities against Aspergillus fumigatus with a concentration half the standard amphotericin B (0.98

and 1.95 µg/mL, respectively), good activity was also demonstrated against Candida albicans, with

MIC= 1.95 µg/mL.

Compound 6a, bearing 4-Chloro benzylidene and 2-hydroxy3-methoxy benzyl 6o was equipotent

to amphotericin B against Aspergillus fumigatus (MIC = 1.95 µg/mL) and showing moderate

activity against Candida albicans. It is worth mentioning also that 4-Cl-substitution showed better

4

ACCEPTED MANUSCRIPT

activity than the 2,4-dichloro benzylidine derivative 6b. Moderate activities was also displayed by

compound 12b against both antifungal strains, and 12e against Aspergillus fumigatus. The

remarkable antifungal activity of 6f, points out the significance of addition of trimethoxy phenyl

carbohydrazide moiety and demonstrated as well better activity conferred by changing the chloro

group from 4-to 3-postion.

2.2.2. Antibacterial Activity

As indicated in Tables 1 and 2, compounds 6a, 6f, 6l, 6p and 12b displayed significant

antibacterial activity against the tested gram-positive and gram-negative bacteria as compared

with ampicillin and ciprofloxacin, respectively.

As regards to the antibacterial activity, compounds 6f and 6p comprising 3,4,5-

trimethoxybenzylidene or 2,5-dimethoxy moieties elicited significant activities against both tested

gram-positive and gram-negative organisms, and even showing 2-fold increased inhibition against

Streptococus pneumonia with (MIC = 0.49 µg/mL), compared to ampicillin (MIC= 0.98 µg/mL).

Besides compounds 6o, 6r and 12b exhibited equipotent activity to ampicillin against

Streptococus pneumonia (MIC = 0.98 µg/mL), while compounds to 6l and 6p demonstrated the

same activity as ampicillin against Staphylococcus aureus with MIC= 0.98 µg/mL. Amongst all

the novel methylnicotinohydrazone series, it was observed that, compounds bearing electron-

donating groups showed better activities than the electron withdrawing ones.

Comparing compound 12b with its counterpart, 12a, it could be assumed that the 2,4-

dichlorobenzylidene shows better activity than, the 4-chlorobenzylidene-3-quinolino

carbohydrazide substitution (MIC = 0.98, 15.63 µg/mL, respectively). Whilst, changing position

of the chloro group from 7- to 6-position resulted in a substantial loss of activity from 0.98 to 7.81

µg/mL, against Streptococus pneumonia. Other compounds also showed moderate to good

activities against Streptococus pneumonia and Staphylococcus aureus at different concentrations

levels from 1.95 to 3.9 µg/mL.

In general, methylnicotinohydrazone analogs demonstrated better activities over the quinolinyl

derivatives which was also greatly enhanced by incorporating electron-donating groups into the

benzylidene moiety for example compounds 6l, 6m, 6o, 6p and 6r, whereas this activity

decreased by the incorporation of electron-withdrawing groups in the following order from 2,5-

(

OCH ) >3,4,5-(OCH ) > 4-Cl > 4-OCH >2,4-(Cl) > F.

3 2 3 2 3 2

5

ACCEPTED MANUSCRIPT

Regarding gram negative bacteria, remarkable activities was elicited by the derivatives 6a, 6f, 6p

and 12b against Escherichia coli showing two fold the potency of the standard, ciprofloxacin (MIC

=

0.49, 0.98 µg/mL respectively).

In addition, compound 6r showed very good activity against Escherichia coli with MIC = 0.98 as

the standard ciprofloxacin, alongside good to moderate activities were also demonstrated by

compounds 6e, 6h, 6l and 12e, finally, it is worth mentioning that substitution on the benzylideine

with 4-Floro or 4-methyl abolished the activity.

Unfortunately, all the synthesized compounds were inactive against Pseudomonas aeruginosa.

Whereas, the results obtained by compounds 6a, 6f, 6p, and 12b against Escherichia coli, were

greatly appreciated due to the resistances of gram-negative bacteria [51].

2.2.3. Antimycobacterial Activity

The activity of the target synthesized compounds 6a-l and 12a-l were evaluated against M.

tuberculosis (RCMB 010126) using the microplate Alamar blue assay (MABA) [52]. Isoniazide

was used as reference drug. The results of the in vitro antimycobacterial screening, as percent

inhibition and minimum inhibitory concentration (MIC), are present in Table 3.

Table 3.

The results revealed that compounds 6o, 6p, and 6r possessed superior activities against M.

tuberculosis with MIC = 0.39 µg/mL, 2-folds the activity of isoniazide (MIC = 0.75 µg/mL).

Interestingly, these derivatives had electron-donating groups on the benzylidene moiety, 2,5-

(

OCH ) as in compounds 6p and 6r or 2-OH-3-OCH as compound 6o. In addition, compounds

3

2

3

6e, 6f, 6l and 6n also showed very good antimycobaterial activities with MIC values 0.78 µg/mL,

equipotent to the standard isoniazide (MIC = 0.75 g/mL)..

Other methylnicotinohydrazone derivatives possessed antimycobacterial activity better than INH or

equal to it such as 6c, 6d, 6k, 6m and 6q at dose levels ranging from 1.56 to 3.12 µg/mL. These

results once again, demonstrates the marked effect of electron-donating groups, mostly represented

by –OCH groups, over the other substituents. Moreover, there was no substantial impact of

3

changing the position of chloro neither in the methylnicotinohydrazone nor in the quinolone series.

In general, all the 1,4-dihydroquinoline-3-carbohydrazide derivatives were inactive against M.

tuberculosis.

In conclusion, the 6-(4-Chlorophenyl)-N'-(2,5-dimethoxybenzylidene)-2-methylnicotinohydrazide

6p emerged as the most potent derivative amongst all the synthesized compounds, exhibiting

6

ACCEPTED MANUSCRIPT

promising results over a range of antifungal, antibacterial and antimycobacterial organisms with

significant dose levels 0.39 – 0.98 µg/mL, compared to the reference drugs.

2.2.4. In vitro cytotoxic activity:

In addition to the antimycobacterial evaluation, the most active compounds 6d, 6e, 6f, 6k,

l, 6m, 6n, 6o, 6p ,6q and 6r were evaluated for cytotoxic activities against human lung fibroblast

6

normal cells (WI-38), in order to determine the selectivity of hydrazones towards M.tuberculosis.

Results presented in (Table 4) indicate that all the tested hydrazones displayed poor or no

cytotoxic activity at 0-500 µg/mL concentrations. Thus compounds 6d, 6e, 6f, 6k, 6l, 6m, 6n, 6o,

6

p ,6q and 6r showed high selectivity towards M.tuberculosis.

Table 4.

2

.3. 2D QSAR study:

.3.1. Development of QSAR models:

With the aim to assess the structural basis for antifungal, antibacterial and antimycobacterial

inhibitory activity of the novel derivatives (6a-r and 12a-l), 2D-QSAR analysis was carried out.

This analysis was run by means of the DS 4.0 software (Discovery Studio 4.0, Accelrys, Co. Ltd)

[

53].

A set of the newly synthesized hydrazones (6a-r and 12a-l) was used as training set with their

measured pMIC against Aspergillus fumigatus, Streptococus pneumoniae and Mycobacterium

tuberculosis for QSAR modeling, compounds 6f, 12g and 12h were chosen as statistical outliers

while building model for Aspergillus fumigatus, and Streptococus pneumoniae whereas,

compounds 6f, 6j, 12g and 12h were chosen for the Mycobacterium tuberculosis model.

“Calculate Molecular Properties” module was used for calculating the molecular properties.

Different molecular descriptors were utilized in the models generation representing

thermodynamic, electronic, spatial, structural, thermodynamic, geometric, topological and quantum

mechanical properties. “Genetic function approximation” (GFA) protocol was applied in order to

choose the best descriptors that characterizes the activity. “Multiple linear regression” (MLR)

protocol was then employed to search for optimal QSAR models with the best statistical validation

measures and capable of correlating bioactivity variation across the used training set collection.

QSAR model was validated employing leave one-out cross-validation by setting the folds to a

2

number much larger than the number of samples , r (squared correlation coefficient value) and r

prediction (predictive squared correlation coefficient value), residuals between the predicted and

experimental activity of the test set and training set.

7

ACCEPTED MANUSCRIPT

2

.3.2. QSAR study results

Equation 1 Represents the best performing QSAR model for the activity against Aspergillus

fumigatus;

-logMIC= 0.1402 + 0.4747 Kappa_3_AM - 4.272 Shadow_YZfrac

Equation 2 Represents the best performing QSAR model for the activity against Streptococus

pneumoniae;

-logMIC= 3.992 + 0.2114 ALogP - 8.107 Shadow_YZfrac

Equation 3 Represents the best performing QSAR model for M.tuberculosis;

logMIC= 9.377 + 0.8318 ALogP - 59.44 Jurs_RPCG + 0.01429 Jurs_WNSA_2

-

According to the former equations these QSAR models were represented graphically by scattering

plots of the experimental versus the predicted bioactivity values –logMIC for the training set

compounds as shown in Figures 2- 4.

The three MLR models exhibited good correlation coefficient r2 of 0.784, 0.776 and 0.884, r2

(adj) = 0.754, 0.744 and 0.862, respectively, r2 (pred) = 0.672, 0.673 and 0.803, Least-Squared

error = 0.2196, 0.2562 and 0.2201 respectively, where r2 (adj) is r2 adjusted for the number of

terms in the model; r2 (pred) is the prediction r2, equivalent to q2 from a leave-1-out cross-

validation [54].

Equation 1 suggested that the antifungal activity of the synthesized compounds was positively

affected by Kappa_3_AM, which is a topological molecular property describing the molecular

shape index. The antifungal and antibacterial activities were also affected by the high and negative

value of Shadow_YZfrac. Shadow indices are a set of geometric descriptors that characterize the

shape of the molecules. They are calculated by projecting the model surface on three mutually

perpendicular planes: xy, yz, and xz. These descriptors depend not only on conformation, but also

on the orientation of the model. In order to calculate them, the models are first rotated to align the

principal moments of inertia with the x-, y-, and z-axes. Shadow_YZ is area of the molecular

shadow in the yz plane [55].

Whereas, equations 2 and 3 showed that, ALogP contributed positively to the antibacterial and

antimycobacterial activity. ALogP is a descriptor that computes the lipophilicity of the molecule

and is calculated in Discovery Studio as the Log of the octanol water partition coefficient using

Ghose and Crippen's method [56]. Interestingly lipophilicity is one of the fundamental molecular

properties controlling the antimycobacterial activity [18,57].

8

ACCEPTED MANUSCRIPT

Equation 3 showed that the antimycobacterial activities were also affected by jurs descriptors.

Jurs descriptors are a group of geometric descriptors, that combine both shape and electronic

information which may characterize the molecules [56]. These descriptors are calculated by

mapping atomic partial charges on solvent-accessible surface areas of individual atoms. In

particular, Jurs_RPCG which contributes inversely to the activity, represents the relative positive

charge on the molecule. This indicates that less positive charge on the molecule gives better

activity.

2.3.3. QSAR validation

The established QSAR models (1, 2 and 3) were verified by applying; Leave-one-out (LOO)

internal validation (r2 = 0.784, 0.776 and 0.884, respectively). Cross-validation was also employed

where q2, which is equivalent to r2 (pred), was 0.672, 0.673 and 0.803, respectively. In addition,

validation was employed by measuring the residuals between the experimental and the predicted

activities of the training set listed in (Table 4 and Table 5). Interestingly, the predicted activities of

the QSAR models were found very close to those experimentally observed, Furthermore, to

evaluate the predictive ability of the developed models Aspergillus fumigatus, and Streptococus

pneumoniae, compounds 6f, 12g and 12h were applied as test compounds, where they were not

included in model generation, the same was also applied for the Mycobacterium tuberculosis

model using compounds 6f, 6j, 12g and 12h.

Table 5.

Table 6.

2.4. ADME study

The ADME of the biologically active hybrids (6a-r and 12a-l) was predicted via a theoretical

kinetic study that is performed by means of Discovery Studio software (Table 6). Both, AlogP98

and PSA_2D descriptors were calculated to evaluate the lipophilicity and polar surface area. Also,

solubility, absorption and CYP2D inhibition levels were predicted. Active members of the first

series 6a-r and compound 12h were found to have good lipophilicity, ALogP values ranging from

2.64 to 5.75, this lipophilicity was found to play an important role in antimycobacterial activity

[

58]. All series of quinolone compounds 12a-l showed low lipophilicity levels except 12h. Whilst,

all the examined derivatives seemed to possess good absorption levels and predicted to be CYP2D

non-inhibitors, generally all the molecules passed the Lipinski's rule of five.

Table 7.

9

ACCEPTED MANUSCRIPT

3

. Conclusion

In this work two series of nicotinc acid hydrazone derivatives 6a-r and quinolone hydrazone

2a-l were synthesized. The antimycobacterial and antimicrobial activity of the newly synthesized

1

target compounds were evaluated. The results manifested that compounds 6p and 6f show broad

spectrum of activity against antifungal, gram positive and gram negative bacteria. In addition, the

methylnicotinohydrazone derivatives 6o, 6p and 6r showed the most promising

antimmycobacterial activity against M. tuberculosis with MIC = 0.39 µg/mL. Moreover,

compounds 6e, 6f, 6l and 6n also showed good antimycobaterial activities with MIC values 0.78

µg/mL. These results manifested that 6-(4-chlorophenyl)-N'-(2,5-dimethoxybenzylidene)-2-

methylnicotinohydrazide 6p could be further investigation as apromising candidate for a potential

antimicrobial agent. Finally, the generated QSAR models, performed to explore the structural

requirements controlling the observed antibacterial properties, indicated that the antibacterial and

antimmycobacterial were affected by lipophilicity and that these generated models had good

prediction ability.

4

. Experimental

.1. Chemistry

.1.1. General

Melting points were measured with a Stuart melting point apparatus and were uncorrected. The

NMR spectra were recorded by Varian Gemini-400BB 400 MHz FT-NMR spectrometers (Varian

1

13

Inc., Palo Alto, CA). H and C spectra were run at 400 and 100 MHz, respectively, in deuterated

dimethylsulphoxide (DMSO-d ). Chemical shifts (δ ) are reported relative to TMS as internal

6

H

standard. All coupling constant (J) values are given in hertz. Chemical shifts (δ ) are reported

C

relative to DMSO-d as internal standards. The abbreviations used are as follows: s, singlet; d,

6

doublet; m, multiplet. IR spectra were recorded with a Bruker FT-IR spectrophotometer. Reaction

courses and product mixtures were routinely monitored by thin layer chromatography (TLC) on

silica gel precoated F₂₅₄Merck plates. Unless otherwise noted, all solvents and reagents were

commercially available and used without further purification.

4.1.2. Ethyl 2-methyl-6-arylnicotinate 3a,b.

To a solution of the appropriate enaminone 2a,b (5 mmol) in glacial acetic acid (15 mL), ethyl

acetoacetate (5.5 mmol) and ammonium acetate (40 mmol) were added. The reaction mixture was

heated under reflux for 5 h. After cooling and pouring into ice-water, the residue obtained was

filtered and washed with petroleum ether then with water and finally crystallized from ethanol [59].

1

0

ACCEPTED MANUSCRIPT

.1.2.1. 6-(3-Chlorophenyl)-2-methylnicotinate ester (3a).

4

-1

1

White crystals (yield 80%), m.p. 62-65°C; IR (KBr, ν cm ): 1716 (C=O); H NMR (DMSO-d )

δ

6

ppm: 1.34 (t, 3H, CH ), 2.50 (s, 3H, CH pyridine), 4.31 (q, 2H, CH –CH ), 7.54 (d, 2H, H-4, H-6,

3

2

3

1

2

1

-Cl-C H ), 7.79 (d, 1H, J = 8.0 Hz, H-4 pyridine,), 8.09-8.11 (m, 1H,H-5, 3-Cl-C H ), 8.19 (s,

6

4

6

4

1

3

H, H-2, 3-Cl-C H ), 8.24 (d, 1H, J = 8.0 Hz, H-5 pyridine) ; C NMR (DMSO-d )

δ

ppm: 14.51,

6

4

6

5.10, 61.50 (CH ), 118.32, 124.71, 126.03, 127.06, 130.11, 131.21, 134.28, 139.89, 140.01,

3

56.41, 159.07, 166.24.

4.1.2.2. 6-(4-Chlorophenyl)-2-methylnicotinate ester (3b).

White crystals (yield 75%), m.p. 42-47°C, as reported [60].

4.1.3. 6-Aryl-2-methylnicotinohydrazide 4a,b.

A mixture of the appropriate ester 3a,b (5 mmol) and 99% hydrazine hydrate (2 mL) was

refluxed for 6 h. The solid product obtained upon cooling was filtered off and recrystallized from

dioxan to afford the corresponding 6-aryl-2-methylnicotinohydrazides 4a,b. [59].

4.1.3.1. 6-(3-Chlorophenyl)-2-methylnicotinohydrazide (4a).

-1

White crystals (yield 90%), m.p. 208-210 °C; IR (KBr, ν cm ): 3282, 3321 (NH, NH ) and 1660

2

1

(

C=O); H NMR (DMSO-d6)

δ

ppm: 2.61 (s, 3H, CH pyridine), 4.54 (s, 2H, NH , D O

3

2

exchangeable), 7.50-7.55 (m, 2H, H-4, H-6, 3-Cl-C H ), 7.78 (d, 1H, J = 8.0 Hz, H-4 pyridine,),

6

4

7

.92 (d, 1H, J = 8.0 Hz, H-5 pyridine), 8.06-8.09 (m, 1H, H-5, 3-Cl-C H ), 8.16 (s, 1H, H-2, 3-Cl-

6

4

1

3

C H ), 9.63 (s, 1H, NH, D O exchangeable); C NMR (DMSO-d )

δ

ppm: 23.04, (CH ), 117.87,

6

4

2

6

3

125.69, 126.73, 129.57, 130.29, 131.16, 134.21, 137.02, 140.54, 154.50, 156.22, 167.43.

4.1.3.2. 6-(4-Chlorophenyl)-2-methylnicotinohydrazide (4b).

White crystals (yield 75%), m.p. 215-220 °C, as reported [60].

4.1.4.1. Preparation of 6-(3-Chlorophenyl)-N'-(4-chlorobenzylidene)-2-methylnicotinohydrazide

(6a).

Different aldehydes 5a-j, (1 mmol) was added to a suspension of 6-aryl-2-methylnicotinohydrazide

derivatives 4a, b (1 mmol) in ethanol (10 mL) and catalytic amount of glacial acetic acid. The

reaction mixture was refluxed for 4 h. The precipitate formed was collected by filtration while hot,

washed with hot ethanol, dried and crystallized from ethanol to furnish compounds 6a-r.

1

-1

1

ACCEPTED MANUSCRIPT

White crystals (yield 90%), m.p. 215-220 °C; IR (KBr, ν cm ): 3174 (NH) and 1651 (C=O); H

NMR (DMSO-d₆) ppm: 2.50, 2.63 (s, 3H, CH ), 7.40- 7.42 (m, 2H, H-3, H-5, 4-Cl-C H ), 7.51-

.55 (m, 2H, H-4, H-6, 3-Cl-C H ), 7.74 (d, 2H, J = 8.0 Hz, H-2, H-6, 4-Cl-C H ), 7.82 (d, J = 8.0

δ

3

6

4

7

6

4

6

4

Hz, 1H, H-4 pyridine), 7.93 (d, 1H, J = 8.0 Hz, H-5 pyridine), 8.07-8.11 (m, 1H, H-5, 3-Cl-C H ),

6

4

8

.17 (s, 1H, H-2, 3-Cl-C H ), 8.28, 8.00 (s, 1H, CH=N), 11.98, 12.04 (s, 1H, –CONH, D O

6

4

2

1

3

exchangeable); C NMR (DMSO-d )

δ

ppm: 23.37, 23.43, (CH ), 117.63, 117.96, 125.74, 125,83,

6

3

1

6

26.81, 126.84, 128.72, 129.27, 129.43, 129.59, 129.75, 129.85, 130.68, 131.20, 133.25, 133.52,

34.26, 134.81, 135.15, 137.40, 137.74, 140.44, 140.58, 143.72, 147.09, 154.29, 155.03, 156.51,

64.34, 170.35; Anal. calcd. for C H Cl N O (384.26): C, 62.51; H, 3.93; N, 10.94. Found C,

2

0

15

2

3

3.04; H, 4.03; N, 11.35.

.1.4.2. 6-(3-Chlorophenyl)-N'-(2,4-dichlorobenzylidene)-2-methylnicotinohydrazide (6b).

4

-1

1

White crystal (yield 80%), m.p. 245-250 °C; IR (KBr, ν cm ): 3170 (NH) and 1662 (C=O); H

NMR (DMSO-d₆) ppm: 2.51, 2.65 (s, 3H, CH ), 7.52- 7.55 (m, 2H, H-4, H-6, 3-Cl-C H ), 7.64,

δ

3

6

4

7

.71 (s, 1H, H-3, 2,4(Cl) -C H ), 7.84, 7.38 (d, 1H, J = 8.4 Hz, H-4 pyridine), 7.92, 7.34 (d, 1H, J

2 6 3

=

8.0 Hz, H-5 pyridine), 7.97- 8.01 (m, 2H, H-5, 3-Cl-C H , and H-5, 2,4(Cl) -C H ), 8.09 (d, 1H,

6 4 2 6 3

H-6, 2,4(Cl) -C H ), 8.18, 8.04 (s, 1H, H-2, 3-Cl-C H ), 8.43, 8.66 (s, 1H, CH=N), 12.15, 12.36 (s,

2

6

3

6

4

1

H, –CONH, D O exchangeable); Anal. calcd. for C H Cl N O (418.70): C, 57.37; H, 3.37; N,

2

20 14

3

10.04. Found C, 57.71; H, 3.39; N, 10.57.

4.1.4.3. 6-(3-Chlorophenyl)-N'-(4-florobenzylidene)-2-methylnicotinohydrazide (6c).

-1

1

White crystals (yield 60%), m.p. 180-183 °C; IR (KBr, ν cm ): 3178 (NH) and 1643 (C=O); H

NMR (DMSO-d₆) ppm: 2.50, 2.63 (s, 3H, CH ), 7.27, 7.14 (t, 2H, J = 8.8 Hz, H-3, H-5, 4-F-

C H ), 7.50-7.55 (m, 2H, H-4, H-6, 3-Cl-C H ), 7.77-7.80 (dd, 2H, J = 6.0 Hz, J = 8.8 Hz, H-2 and

δ

3

6

4

6

4

H-6, 4-F-C H ), 7.82, 7.99 (d, 1H, J = 8.0 Hz, H-4 pyridine), 7.93 (d, 1H, J = 8.0 Hz, H-5

6

4

pyridine), 8.08- 8.10 (m, 1H, H-5, 3-Cl-C H ), 8.17 (s, 1H, H-2, 3-Cl-C H ) 8.29 (s, 1H, CH=N),

6

4

6

4

+

1

1.91, 11.97 (s, 1H, –CONH, D O exchangeable); MS m/z %: 369.06 (M +2, 0.97), 367.09 (M ,

2

.30), 230.05 (100%); Anal. calcd. for C H ClFN O (367.81): C, 65.31; H, 4.11; N, 11.42. Found

2

0

14

3

C, 65.48; H, 4.18; N, 11.80.

4.1.4.4. 6-(3-Chlorophenyl)-N'-(4-methylbenzylidene)-2-methylnicotinohydrazide (6d).

-1

1

White crystals (yield 70%), m.p. 210-213 °C; IR (KBr, ν cm ): 3213 (NH) and 1651 (C=O); H

NMR (DMSO-d₆) ppm: 2.24, 2.33 (s, 3H, CH C H ), 2.50, 2.63 (s, 3H, CH ), 7.12, 7.25 (d, 2H,

J = 8.0 Hz, H-3, H-5, 4-CH -C H ), 7.50-7.53 (m, 2H, H-4, H-6, 3-Cl-C H ), 7.55, 7.60 (d, 2H, J =

δ

3

6

5

3

6

4

6

5

8

.0 Hz, H-2, H-6, 4-CH3-C H ), 7.82, 7.97 (d, 1H, J = 8.4 Hz, H-4 pyridine), 7.93, 7.95 (d, 1H, J =

6 4

1

2

ACCEPTED MANUSCRIPT

.6 Hz, H-5 pyridine), 8.08-8.10 (m, 1H, H-5, 3-Cl-C H ), 8.17, (s, 1H, H-2, 3-Cl-C H ), 8.25,

6 4 6 4

5

1

3

8

.05 (s, 1H, CH=N), 11.84, 11.90 (s, 1H, –CONH, D O exchangeable); C NMR (DMSO-d )

δ

2

6

ppm: 21.43, 21.50, 23.41, (CH ), 117.61, 117.95, 125.74, 125,81, 126.83, 127.08, 127.62, 129.56,

3

1

2

6

29.72, 129.87, 129.94, 130.04, 131.16, 131.20, 134.25, 137.36, 137.46, 140.47, 140.57, 145.01,

+

48.44, 154.93, 156.46, 164.20, 170.17; MS m/z %: 365.12 (M +2, 1.48), 363.10 (M , 3.79),

30.06 (100%); Anal. calcd. for C H ClN O (363.85): C, 69.32; H, 4.99; N, 11.55. Found C,

2

1

18

3

9.55; H, 5.04; N, 11.78.

.1.4.5. 6-(3-Chlorophenyl)-N'-(3,4-dimethoxybenzylidene)-2-methylnicotinohydrazide (6e).

4

-1

1

White crystal (yield 75%), m.p. 190-192 °C; IR (KBr, ν cm ): 3221 (NH) and 1651 (C=O); H

NMR (DMSO-d₆) ppm: 2.51, 2.63 (s, 3H, CH ), 3.56, 3.79 (s, 3H, OCH -C H ), 3.70, 3.81 (s,

H, OCH -C H ), 7.00, 6.89 (d, 1H, J = 8.0 Hz, H-5, 3,4-(OCH ) -C H ), 7.18, 6.96 (d, 1H, J = 8.4

δ

3

6

3

6

3

3 2

6

3

Hz, H-6, 3,4-(OCH ) -C H ), 7.33, 6.92 (s, 1H, H-2, 3,4-(OCH ) -C H ), 7.52-7.55 (m, 2H, H-4,

3

2

6

3

3 2

6

3

H-6, 3-Cl-C H ), 7.82 (d, 1H, H-4 pyridine), 7.93 (d, 1H, J = 8.0 Hz, H-5 pyridine), 8.06- 8.09 (m,

6

5

1

H, H-5, 3-Cl-C H ), 8.17 (s, 1H, H-2, 3-Cl-C H ), 8.21, 7.98 (s, 1H, CH=N), 11.78, 12.07 (s, 1H,

6 4 6 5

1

3

–

CONH, D O exchangeable); C NMR (DMSO-d ) δ ppm: 23.39, (CH ), 55.91, 56.03, (OCH ),

2 6 3 3

108.71, 111.93, 117.96, 122.46, 125.80, 126.82, 127.22, 129.70, 130.15, 131.20, 134.25, 137.33,

1

40.48, 148.55, 149.53, 154.90, 156.41, 164.09; Anal. calcd. for C H ClN O (409.87): C, 64.47;

2

20

3

H, 4.92; N, 10.25. Found C, 64.73; H, 4.97; N, 10.49.

4.1.4.6. 6-(3-Chlorophenyl)-N'-(3,4,5-trimethoxybenzylidene)-2-methylnicotinohydrazide (6f).

-1

1

White crystal (yield 80%), m.p. 200-203 °C; IR (KBr, ν cm ): 3224 (NH) and 1651 (C=O); H

NMR (DMSO-d₆) ppm: 2.51, 2.64 (s, 3H, CH ), 3.61 (s, 3H, OCH -C H ), 3.70 (s, 3H, OCH -

C H ), 3.82 (s, 3H, OCH -C H ), 7.02, 6.7 (s, 2H, H-2, H-6, 3,4,5-(OCH ) -C H ), 7.51- 7.55 (m,

δ

3

6

3

6

3

6

3

3 3

6

2

1

–

H, H-4, H-6, 3-Cl-C H ), 7.83 (d, 1H, H-4 pyridine), 7.92 (d, 1H, H-5 pyridine), 8.04- 8.09 (m,

6 4

H, H-5, 3-Cl C H ), 8.17 (s, 1H, H-2, 3-Cl C H ), 8.22, 8.13 (s, 1H, CH=N), 11.87, 12.00 (s, 1H,

6

4

6

4

1

3

CONH, D O exchangeable); C NMR (DMSO-d )

δ ppm: 23.39, (CH ), 56.04, 56.42, 60.57,

3

2

6

(

OCH ), 104.84, 117.99, 125.82, 126.83, 129.72, 130.04, 130.06, 131.21, 134.26, 137.38, 139.77,

3

1

40.47, 148.35, 153.65, 154.97, 156.41, 164.25; Anal. calcd. for C H ClN O (439.90): C, 62.80;

23 22 3 4

H, 5.04; N, 9.55. Found C, 62.80; H, 5.08; N, 9.95.

4.1.4.7. 6-(4-Chlorophenyl)-N'-(4-chlorobenzylidene)-2-methylnicotinohydrazide (6g).

-1

1

White crystals (yield 90%), m.p. 215-220 °C; IR (KBr, ν cm ): 3228 (NH) and 1654 (C=O); H

NMR (DMSO-d₆)

δ

ppm: 2.94, 2.62 (s, 3H, CH ), 7.40- 7.42 (m, 2H, H-3, H-5, 4-Cl-C H (ald)),

3

6

4

7

.51- 7.85 (m, 2H, , H-3, H-5, 4-Cl C H ), 7.74 (d, 2H, J = 8.0 Hz, H-2, H-6, 4-Cl C H (ald)),

6 4 6 4

1

3

ACCEPTED MANUSCRIPT

7

2

.81, 7.93 (d, 1H, J = 8 Hz, H-4 pyridine), 7.89, 7.95 (d, 1H, J = 8 Hz, H-5 pyridine), 7.95-7.97 (m,

H, H-2 and H-6, 4-Cl-C H ), 8.27, 8.07 (s, 1H, CH=N), 11.98, 12.05 (s, 1H, –CONH, D O

6

4

2

1

3

exchangeable); C NMR (DMSO-d₆)

δ

ppm: 23.44, (CH ), 117.59, 123.85, 128.70, 128.98,

3

1

3

29.26, 129.30, 129.42, 129.48, 133.51, 134.84, 137.13, 137.35, 147.04, 155.42, 156.43, 164.40,

68.14; Anal. calcd. for C H Cl N O (384.26): C, 62.51; H, 3.93; N, 10.94. Found C, 62.74; H,

20

15

2

3

.97; N, 11.47.

.1.4.8. 6-(4-Chlorophenyl)-N'-(2,4-dichlorobenzylidene)-2-methylnicotinohydrazide (6h).

4

-1

1

White crystal (yield 80%), m.p. 245-250 °C; IR (KBr, ν cm ): 3170 (NH) and 1665 (C=O); H

NMR (DMSO-d₆) ppm: 2.50, 2.64 (s, 3H, CH ), 7.54 (d, 2H, J = 8.8 Hz, H-3, H-5, 4-Cl-C H ),

.64, 7.71 (s, 1H, H-3, 2,4(Cl) -C H ), 7.83, 7.38 (d, 1H, H-4 pyridine), 7.88, 7.34 (d, 1H, H-5

δ

3

6

4

7

2

6

3

pyridine), 7.93 (d, 1H, J = 8.4 Hz, H-6, 2,4(Cl) -C H ), 7.99- 8.03 (m, 1H, H-5, 2,4(Cl) -C H ),

2

6

3

2

6

3

8

.15 (d, 2H, J = 8.8 Hz, H-2, H-6, 4-Cl-C H ), 8.43, 8.66 (s, 1H, CH=N), 12.14, 12.17 (s, 1H, –

6 4

CONH, D O exchangeable); Anal. calcd. for C H Cl N O (418.70): C, 57.37; H, 3.37; N, 10.04.

2

20 14

3

Found C, 57.98; H, 3.39; N, 10.35.

4.1.4.9. 6-(4-Chlorophenyl)-N'-(4-florobenzylidene)-2-methylnicotinohydrazide (6i).

-1

1

White crystals (yield 60%), m.p. 180-183 °C; IR (KBr, ν cm ): 3167 (NH) and 1643 (C=O); H

NMR (DMSO-d₆) ppm: 2.50, 2.63 (s, 3H, CH ), 7.26, 7.14 (t, 2H, H-3, H-5, 4-F-C H ), 7.54 (d,

H, J = 8.5 Hz, H-3, H-5, 4-Cl-C H ), 7.76, 7.42 (dd, 2H, J = 5.6 Hz, J = 8.8 Hz, H-2 and H-6, 4-

δ

3

6

4

2

6

4

F-C H ), 7.81, 7.91 (d, 1H, J = 8.4 Hz, H-4 pyridine), 7.88, 7.94 (d, 1H, J = 8.0 Hz, H-5 pyridine),

6

4

8

.14 (d, J = 8.4 Hz, 2H, H-2, H-6, 4-Cl-C H ), 8.30, 8.08 (s, 1H, CH=N), 11.87, 11.94 (s, 1H, –

6 4

CONH, D O exchangeable); Anal. calcd. for C H ClFN O (367.81): C, 65.31; H, 4.11; N, 11.42.

2

20 14

3

Found C, 65.59; H, 4.18; N, 11.93.

4.1.4.10. 6-(4-Chlorophenyl)-N'-(4-methylbenzylidene)-2-methylnicotinohydrazide (6j).

-1

1

White crystals (yield 70%), m.p. 210-213 °C; IR (KBr, ν cm ): 3163 (NH) and 1643 (C=O); H

NMR (DMSO-d₆) ppm: 2.24, 2.33 (s, 3H, CH -C H ), 2.49, 2.63 (s, 3H, CH ), 7.12, 7.25 (d, 2H,

J = 8.0 Hz, H-3, H-5 of 4-CH -C H ), 7.54 (d, 2H, H-3, H-5, 4-Cl-C H ), 7.60 (d, 2H, J = 8.0 Hz,

δ

3

6

4

3

6

4

6

4

H-2, H-6 of 4-CH -C H ), 7.80, 7.88 (d, 1H, J = 7.6 Hz, H-4 pyridine), 7.91, 7.93 (d, J = 8.4 Hz,

3

6

4

1

H, H-5 pyridine), 8.14 (d, 2H, H-2, H-6, 4-Cl-C H ), 8.25, 8.05 (s, 1H, CH=N), 11.81, 11.88 (s,

6 4

1

3

H, –CONH, D O exchangeable); C NMR (DMSO-d₆)

δ ppm: 21.50, 23.43, (CH ), 117.58,

3

2

27.08, 127.60, 128.92, 128.97, 129.26, 129.30, 129.87, 129.94, 131.85, 137.17, 137.33, 140.55,

48.39, 155.34, 156.40, 164.25; Anal. calcd. for C H ClN O (363.85): C, 69.32; H, 4.99; N,

21

18

3

1.55. Found C, 69.45; H, 5.09; N, 11.97.

1

4

ACCEPTED MANUSCRIPT

.1.4.11. 6-(4-Chlorophenyl)-N'-(3,4-dimethoxybenzylidene)-2-methylnicotinohydrazide (6k).

4

-1

1

White crystal (yield 75%), m.p. 190-192 °C; IR (KBr, ν cm ): 3190 (NH) and 1651 (C=O); H

NMR (DMSO-d₆) ppm: 2.50, 2.62 (s, 3H, CH ), 3.56, 3.79 (s, 3H, OCH -C H ), 3.70, 3.81 (s,

H, OCH -C H ), 7.00, 6.89 (d, 1H, J = 8.0 Hz, H-5, 3,4-(OCH ) -C H ), 7.18, 6.96 (d, 1H, J = 8.4

δ

3

6

3

6

3

3 2

6

3

Hz, H-6, 3,4-(OCH ) -C H ), 7.33, 6.92 (s, 1H, H-2, 3,4-(OCH ) -C H ), 7.54 (d, 2H, J = 8.8 Hz,

3

2

6

3

3 2

6

3

H-3, H-5, 4-Cl-C H ), 7.81 (d, 1H, J = 8.0 Hz, H-4 pyridine), 7.87 (d, 1H, J = 8.0 Hz, H-5

6

5

pyridine), 8.14 (d, 2H, J = 8.8 Hz, H-2, H-6, 4-Cl-C H ), 8.21, 7.97 (s, 1H, CH=N), 11.75, 11.86

6

4

1

3

(

s, 1H, –CONH, D O exchangeable); C NMR (DMSO-d ) δ ppm: 23.41, (CH ), 55.91, 56.03,

2 6 3

(

OCH ), 108.71, 111.93, 117.60, 122.44, 127.23, 128.82, 128.97, 129.30, 129.80, 134.80, 137.20,

3

+

1

37.29, 148.51, 149.53, 151.32, 155.30, 156.35, 164.15; MS m/z %: 411.09 (M +2, 7.49), 409.10

+

(

M , 20.34), 230.05 (100%); Anal. calcd. for C H ClN O (409.87): C, 64.47; H, 4.92; N,

2

20

3

1

0.25. Found C, 64.72; H, 4.92; N, 10.64.

.1.4.12. 6-(4-Chlorophenyl)-N'-(3,4,5-trimethoxybenzylidene)-2-methylnicotinohydrazide (6l).

4

-1

1

White crystal (yield 80%), m.p. 200-203 °C; IR (KBr, ν cm ): 3221 (NH) and 1651 (C=O); H

NMR (DMSO-d₆) ppm: 2.50, 2.63 (s, 3H, CH ), 3.61, 3.74 (s, 3H, OCH C H ), 3.69, 3.72 (s,

H, OCH -C H ), 3.82, 3.84 (s, 3H, OCH -C H ), 6.70, 7.02 (s, 2H, H-2, H-6, 3,4,5-(OCH ) -

δ

3

6

3

6

3

6

3

3 3

C H ), 7.54 (d, 2H, J = 8.8 Hz, H-3, H-5, 4-Cl-C H ) 7.82 (d, 1H, J = 8.0 Hz, H-4 pyridine), 7.87

6

2

6

5

(

d, 1H, J = 8.0 Hz, H-5 pyridine), 8.14 (d, 2H, J = 8.8 Hz, H-2, H-6, 4-Cl-C H ), 8.10, 8.21 (s, 1H,

6 4

1

3

CH=N), 11.87, 12.00 (s, 1H, –CONH, D O exchangeable); C NMR (DMSO-d )

δ

ppm: 23.41,

2

6

(

CH ), 56.04, 56.42, 56.52, 60.57, (OCH ), 104.83, 117.62, 128.98, 129.31, 129.71, 130.04,

3

+

1

4

9

34.83, 137.18, 137.34, 140.32, 153.65, 155.38, 156.35, 164.31; MS m/z %: 441.16 (M +2, 15.42),

+

39.17 (M , 48.65), 230.06 (100%); Anal. calcd. for C H ClN O (439.90): C, 62.80; H, 5.04; N,

2

3

22

3

4

.55. Found C, 62.98; H, 5.18; N, 9.95.

.1.4.13. 6-(4-Chlorophenyl)-N'-(4-hydroxybenzylidene)-2-methylnicotinohydrazide (6m).

4

-1

1

White crystals (yield 90%), m.p. 210-215 °C; IR (KBr, ν cm ): 3170 (NH) and 1662 (C=O); H

NMR (DMSO-d₆) ppm: 2.51, 2.66 (s, 3H, CH ), 6.73, 6.86 (d, 2H, J = 8.0 Hz, H-3, H-5, 4-OH-

C H ), 7.26, 7.94 (d, 2H, J = 8.4 Hz, H-2, H-6, 4-OH C H ), 7.56 (d, 2H, J = 8.0 Hz, H-3 and H-5,

δ

3

6

4

6

4

-Cl-C H ), 8.16 (d, 2H, J = 8.0 Hz, H-2 and H-6, 4-Cl-C H ), 7.83, 7.96 (d, 1H, J = 8.0 Hz, H-4

6 4 6 4

pyridine), 7.89 (d, 1H, H-5 pyridine), 8.03, 8.27 (s, 1H, CH=N), 9.95 (s, 1H, OH, D O

2

exchangeable), 11.73, 11.80 (s, 1H, –CONH, D O exchangeable); Anal. calcd. for C H ClN O

2

20 16

3

(365.82): C, 65.67; H, 4.41; N, 11.49. Found C, 62.77; H, 4.55; N, 11.67.

1

5

ACCEPTED MANUSCRIPT

.1.4.14. 6-(4-Chlorophenyl)-N'-(4-methoxybenzylidene)-2-methylnicotinohydrazide (6n).

4

-1

1

White crystals (yield 80%), m.p. 205-210 °C; IR (KBr, ν cm ): 3228 (NH) and 1651 (C=O); H

NMR (DMSO-d₆) ppm: 2.51, 2.66 (s, 3H, CH ), 3.73, 3.87 (s, 3H, CH -OCH ), 6.90, 7.03 (d,

δ

3

2

7

H, J = 8.4 Hz, H-3, H-5, 4-OCH - C H ), 7.57 (d, 2H, J = 8.4 Hz, H-3, H-5, 4-Cl C H ), 7.69,

3 6 4 6 4

.35 (d, 2H, J = 8.4 Hz, H-2, H-6, 4-OCH - C H ), 7.83, 7.96 (d, 1H, J = 8.0 Hz, H-4 pyridine),

3

6

4

.89, 7.93 (d, 1H, H-5 pyridine), 8.16 (d, 2H, J = 8.4 Hz, H-2, H-6, 4-Cl-C H ), 8.06, 8.27 (s, 1H,

6

4

CH=N), 11.80, 11.87 (s, 1H, –CONH, D O exchangeable); Anal. calcd. for C H ClN O

2

21 18

3

2

(397.84): C, 66.40; H, 4.78; N, 11.06. Found C, 66.65; H, 4.90; N, 11.14.

4.1.4.15. 6-(4-Chlorophenyl)-N'-(2-hydroxy-3-methoxybenzylidene)-2-methylnicotinohydrazide

(6o).

-1

1

White crystals (yield 75%), m.p. 200-205 °C; IR (KBr, ν cm ): 3228 (NH) and 1654 (C=O); H

NMR (DMSO-d₆) ppm: 2.48, 2.65 (s, 3H, CH ), 3.72, 3.80 (s, 3H, CH -OCH ), 6.67, 6.83 ( t,

δ

3

1H, H-5, 2-OH-3-OCH -C H ), 6.89 (dd, 1H, J = 1.2 Hz, J = 8.0 Hz, H-4, 2-OH-3-OCH -C H ),

3 6 3 3 6 3

7

.55, 7.02 (d, 1H, J = 8.0 Hz, H-6, 2-OH-3-OCH -C H ), 7.55 (d, 2H, J = 8.4 Hz, H-3, H-5, 4-Cl-

3

6

3

C H ), 7.82, 7.99 (d, 1H, J = 8.0 Hz, H-4 pyridine), 7.90, 7.93 (d, 1H, J = 8.4 Hz, H-5 pyridine),

6

4

8

.15 (d, 2H, J = 8.4 Hz, H-2, H-6 of 4-Cl C H ), 8.36, 8.52 (s, 1H, CH=N), 10.70 (s, 1H, OH, D O

6

4

2

exchangeable), 12.06 (s, 1H, –CONH, D O exchangeable); Anal. calcd. for C H ClN O

2

21 18

3

(395.84): C, 63.72; H, 4.58; N, 10.62. Found C, 63.95; H, 4.76; N, 10.85.

4.1.4.16. 6-(4-Chlorophenyl)-N'-(2,5-dimethoxybenzylidene)-2-methylnicotinohydrazide (6p).

-1

1

White crystals (yield 85%), m.p. 220-225 °C; IR (KBr, ν cm ): 3178 (NH) and 1604 (C=O); H

NMR (DMSO-d₆) ppm: 2.51, 2.67 (s, 3H, CH ), 3.78, 3.81 (s, 6H, CH -OCH ), 6.90, 7.07 (m,

H, H-3, H-4, 2,5-(OCH ) -C H ), 7.41, 6.81 (s, 1H, H-6, 2,5-(OCH ) -C H ), 7.56 (d, 1H, J = 8.0

δ

3

2

3

2

6

3

3 2

6

3

Hz, H-3, H-5, 4-Cl-C H ), 7.84, 7.99 (d, 1H, J = 8.0 Hz, H-4 pyridine), 7.90, 7.93 (d, 1H, J = 8.0

6

4

Hz, H-5 pyridine), 8.17 (d, 1H, J = 8.0 Hz, H-2, H-6, 4-Cl-C H ), 8.39, 8.67 (s, 1H, CH=N), 11.93,

6

4

1

3

1

5

1

1.98 (s, 1H, –CONH, D O exchangeable); C NMR (DMSO-d ) δ ppm: 23.44, (CH ), 55.45,

2 6 3

5.94, 56.63. 56.72, (OCH ), 109.60, 109.96, 113.71, 113.93, 117.00. 117.10, 117.55, 118.39,

3

23.15, 128.84, 128.98, 129.29, 129.50, 130.55, 134.64, 134.85, 137.19, 137.36, 139.86, 143.81,

52.52, 152.85, 153.43, 153.76, 154.67, 155.07, 155.40, 156.57, 164.25, 170.36; Anal. calcd. for

C H ClN O (409.87): C, 64.47; H, 4.92; N, 10.25. Found C, 64.72; H, 4.92; N, 10.64.

22

20

3

1

6

ACCEPTED MANUSCRIPT

.1.4.17. 6-(3-Chlorophenyl)-N'-(4-methoxybenzylidene)-2-methylnicotinohydrazide (6q).

4

-1

1

White crystals (yield 90%), m.p. 203-208 °C; IR (KBr, ν cm ): 3178 (NH) and 1604 (C=O); H

NMR (DMSO-d₆) ppm: 2.51, 2.67 (s, 3H, CH ), 3.74, 3.83 (s, 3H, CH -OCH ), 6.90, 7.03 (d,

δ

3

2

H, J = 8.0 Hz, H-3, H-5, 4-OCH -C H ), 7.55- 7.58 (m, 2H, H-4, H-6, 3-Cl C H ), 7.69, 7.36 (d,

3 6 4 6 4

H, H-2, H-6, 4-OCH -C H ), 7.85 (d, 1H, H-4 pyridine), 7.95 (d, 1H, H-5 pyridine), 8.12- 8.22

3

6

4

(

m, 2H, H-2, H-5, 3-Cl C H ), 8.07, 8.28 (s, 1H, CH=N), 11.83, 11.86 (s, 1H, –CONH, D O

6

4

2

exchangeable); Anal. calcd. for C H ClN O (379.84): C, 66.40; H, 4.78; N, 11.06. Found C,

2

1

18

3

2

6

6.65; H, 4.90; N, 11.14.

.1.4.18. 6-(3-Chlorophenyl)-N'-(2,5-dimethoxybenzylidene)-2-methylnicotinohydrazide (6r).

4

-1

1

White crystals (yield 90%), m.p. 220-225 °C; IR (KBr, ν cm ): 3170 (NH) and 1662 (C=O); H

NMR (DMSO-d₆) ppm: 2.51, 2.68 (s, 3H, CH ), 3.78, 3.81 (s, 6H, CH -OCH ), 6.91- 7.08 (m,

H, H-3, H-4, 2,5-(OCH ) -C H ), 7.42, 6.81 (s, 1H, H-6, 2,5-(OCH ) -C H ), 7.54- 7.76 (m, 1H,

δ

3

2

3

2

6

3

3 2

6

3

H-4, H-6, 3-Cl C H ), 7.86 (d, 1H, H-4 pyridine), 7.95 (d, 1H, H-5 pyridine), 8.00- 8.21 (m, 2H,

6

4

H-2, H-5, 3-Cl C H ), 8.39, 8.67 (s, 1H, CH=N), 11.93, 11.96 (s, 1H, –CONH, D O exchangeable);

6

4

2

1

3

C NMR (DMSO-d₆)

δ

ppm: 23.44, (CH ), 55.45, 55.95, 56.64. 56.73, (OCH ), 109.59, 109.98,

3

1

13.72, 113.94, 117.00. 117.49, 117.92, 118.41, 123.14, 125.67, 125.83, 126.76, 126.87, 129.53,

29.72, 129.86, 130.93, 131.17, 134.28, 137.41, 139.92, 140.49, 140.68, 143.85, 152.53, 152.86,

53.43, 153.76, 154.27, 155.07, 155.12, 156.62, 164.19, 170.30; Anal. calcd. for C H ClN O

3

2

20

3

(409.87): C, 64.47; H, 4.92; N, 10.25. Found C, 64.72; H, 4.92; N, 10.64.

4.1.5. Preparation of 1,4-dihydro-4-oxo-quinoline-3-carbohydrazide derivatives 10a,b

Substituted aniline 7a,b (0.01 mol) and diethyl ethoxy methylene malonate (0.01 mol) were mixed

0

and heated at 125-130 C for 3 h. Ethanol was evaporated off from the resulting mixture of ethyl

anilinomethylene malonate. The crude solid was filtered on sintered funnel, dried. The malonate

8a,b (0.01 mol) was refluxed with diphenylether (50 mL) for 2 h to give 1,4-dihydro-4-

oxoquinoline-3-carboxylic acid ethyl ester 9a,b. After 1 h the solution was cooled and the resulting

precipitate was filtered off and dried.

Substituted-1,4-dihydro-4-oxoquinoline- 3-carboxylic acid ethyl ester 9a,b (0.01 mol) was refluxed

for 12 h with hydrazine hydrate (0.01 mol) in absolute ethanol ( 9 mL) to give substituted- 4-oxo-

1,4 dihydroquinoline-3-carbohydrazide 10a,b. The excess solvent was evaporated off and the

resulting mixture was poured into crushed ice. The solid separated was filter on sintered funnel,

washed with water and dried.

1

7

ACCEPTED MANUSCRIPT

The physical properties and spectral data of 10a,b were being identical with those were reported

[

61].

4.1.5.1.

Preparation

of

7-Chloro-N'-(4-chlorobenzylidene)-4-oxo-1,4-dihydroquinoline-3-

carbohydrazide (12a).

1,4-dihydro-4-oxo-quinoline-3-carbohydrazide derivatives 10a,b (1 mmol) reacted with different

aldehydes 11a-f, (1 mmol) in DMF (10 mL). The reaction mixture was refluxed for 4 h. Then, the

precipitate formed was collected by filtration while hot, washed with hot ethanol, dried and

crystallized from ethanol\DMF to furnish compounds 12a-l.

-1

White crystal (yield 86%), m.p. above 300 °C; IR (KBr, ν cm ): 3251 (NH, NH) and 1666, 1670

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 7.51-7.56 (m, 3H, H-3, H-5, 4-Cl-C H and H-6 quinoline),

6

4

7

.76- 7.79, (m, 3H, H-2, H-6, 4-Cl-C H and H-8 quinoline), 8.26 (d, 1H, J = 8.8 Hz, H-5

6 4

quinoline), 8.44 (s, 1H, CH=N), 8.90 (s, 1H, H-2 quinoline), 13.16 (s, 1H, –CONH, D O

2

exchangeable), 13.19 (s, 1H, NH, D O exchangeable); Anal. calcd. for C H Cl N O (360.18): C,

2

17 11

2

3

2

56.69; H, 3.08; N, 11.67. Found C, 56.69; H, 3.12; N, 11.67.

4.1.5.2.

7-Chloro-N'-(2,4-dichlorobenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide

(12b).

-1

White crystal (yield 85%), m.p. above 300 °C; IR (KBr, ν cm ): 3402 (NH, NH) and 1651, 1660

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 7.45- 7.47 (m, 2H, H-5, 2,4(Cl) -C H and H-6 quinoline),

6

2

6

3

7

.60 (s,1H, H-8 quinoline), 7.68 (s, 1H, H-3, 2,4(Cl) -C H ), 7.91 (d, 1H, H-6 2,4(Cl) -C H ), 8.17

2 6 3 2 6 3

(d, 1H, J = 8.0 Hz, H-5 quinoline), 8.52 (s, 1H, CH=N), 8.81 (s, 1H, H-2 quinoline), 13.20 (s, 1H,

1

3

–

1

2

5

CONH, D O exchangeable), 13.23 (s, 1H, NH, D O exchangeable); C NMR (DMSO-d )

δ

ppm:

2

6

10.87, 118.80, 126.17, 128.12, 128.41, 128.63, 129.82, 134.31, 138.56, 142.82, 143.73, 145.79,

+

51.26, 152.39, 159.94, 161.81, 163.16; MS m/z %: 441.16 (M +2, 15.42), 439.17 (M , 48.65),

30.06 (100%);Anal. calcd. for C H Cl N O (394.64): C, 51.74; H, 2.55; N, 10.65. Found C,

1

7

10

3

2

1.82; H, 2.51; N, 10.65.

.1.5.3. 7-Chloro-N'-(4-florobenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (12c).

4

-1

White crystal (yield 79%), m.p. above 300 °C; IR (KBr, ν cm ): 3248 (NH, NH) and 1662, 1680

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 7.25 (t, 2H, J = 8.8 Hz, H-2, H-6, 4-F-C H ), 7.52- 7.55 (m,

6

4

1H, H-6 quinoline), 7.79 (s, 1H, , H-8 quinoline), 7.88 (dd, 2H, J = 8.8 Hz, J = 5.4 Hz, H-3, H-5, 4-

F-C H ), 8.29- 8.31 (m, 2H, H-5 quinoline and CH=N), 8.93 (s, 1H, H-2 quinoline), 12.65 (s, 1H, –

6

4

+

CONH, D O exchangeable), 13.11 (s, 1H, NH, D O exchangeable); MS m/z %: 345.11 (M +2,

2

1

8

+

ACCEPTED MANUSCRIPT

.39), 343.09 (M , 4.16), 206.04 (100%); Anal. calcd. for C H ClFN O (343.74): C, 59.40; H,

17 11 3 2

1

3.23; N, 12.22. Found C, 59.64; H, 3.28; N, 12.22.

4.1.5.4. 7-Chloro-N'-(4-methylbenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (12d).

-1

White crystal (yield 83%), m.p. above 300 °C; IR (KBr, ν cm ): 3201 (NH, NH) and 1651, 1660

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 2.50 (s, 3H, CH ), 7.26 (d, J = 7.6 Hz, 2H, H-3, H-5, 4- CH -

6

3

C H ), 7.53 (d, J = 8.4 Hz, 1H, H-6 quinoline), 7.64, (d, J = 7.6 Hz, 2H, H-2, H-6, 4-CH - C H ),

6

4

3

6

4

7

1

.79 (s, 1H, H-8 quinoline), 8.26 (d, J = 8.8 Hz, 1H, H-5 quinoline), 8.37 (s, 1H, CH=N), 8.90 (s,

H, H-2 quinoline), 12.91 (s, 1H, –CONH, D O exchangeable), 13.10 (s, 1H, NH, D O

2

1

3

exchangeable); C NMR (DMSO-d )

δ

ppm: 111.18, 118.92, 119.24, 125.03, 126.05, 127.65,

6

128.18, 128.94, 129.83, 132.13, 137.95, 140.31, 140.40, 145.57, 148.27, 161.37, 162.77; MS m/z

+

%

: 441.16 (M +2, 15.42), 439.17 (M , 48.65), 230.06 (100%);Anal. calcd. for C H Cl N O

17 10 3 3 2

(394.64): C, 51.74; H, 2.55; N, 10.65. Found C, 51.82; H, 2.51; N, 10.65; MS m/z %: 341.12

+

(

M +2, 1.96), 339.09 (M , 2.41), 230.00 (100%); Anal. calcd. for C H ClN O (339.78): C,

18 14 3 2

63.63; H, 4.15; N, 12.37. Found C, 63.90; H, 4.23; N, 12.37.

4.1.5.5. 7-Chloro-N'-(3,4-dimethoxybenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide

(12e).

-1

White crystal (yield 80%), m.p. above 300 °C; IR (KBr, ν cm ): 3201 (NH, NH) and 1670, 1680

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 3.81 (s, 3H, OCH ), 3.83 (s, 3H, OCH ), 7.03 (d, 1H, J = 8.0

6

3

Hz, H-5, 3,4-(OCH ) -C H ), 7.26 (d, 1H, H-6 quinoline), 7.37 (s, 1H, H-2, 3,4-(OCH ) -C H ),

3

2

6

3

3 2

6

3

7

.53 (d, 2H, J = 8.4 Hz, 1H, H-6, 3,4-(OCH ) -C H ), 7.80 (s, 1H, H-8 quinoline), 8.27 (d, J = 8.4

3 2 6 3

Hz, 1H, H-5 quinoline), 8.34 (s, 1H, CH=N), 8.89 (s, 1H, H-2 quinoline), 13.13 (s, 1H, –CONH,

1

3

D O exchangeable), 13.16 (s, 1H, NH, D O exchangeable); C NMR (DMSO-d )

δ ppm: 103.44,

2

6

1

09.03, 111.05, 111.90, 122.49, 125.25, 126.05, 128.22, 128.25, 137.91, 141.88, 142.23, 143.16,

45.64, 149.44, 151.29, 161.37; Anal. calcd. for C H ClN O (385.80): C, 59.15; H, 4.18; N,

1

9

16

3

4

0.89. Found C, 59.34; H, 4.25; N, 10.89.

.1.5.6. 7-Chloro-N'-(3,4,5-trimethoxybenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide

4

(12f).

-1

White crystal (yield 82%), m.p. above 300 °C; IR (KBr, ν cm ): 3255 (NH, NH) and 1651, 1670

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 3.71 (s, 3H, OCH ), 3.84 (s, 6H, OCH ), 7.09 (s, 2H, H-2, H-

6

3

6

, 3,4,5-(OCH ) -C H ), 7.53- 7.55 (m, 1H, H-6 quinoline), 7.80 (s, 1H, H-8 quinoline), 8.27 (d, J

3 3 6 2

=

8.8 Hz, 1H, H-5 quinoline), 8.35 (s, 1H, CH=N), 8.89 (s, 1H, H-2 quinoline), 12.91 (s, 1H, –

1

9

ACCEPTED MANUSCRIPT

CONH, D O exchangeable), 13.16 (s, 1H, NH, D O exchangeable); Anal. calcd. for C H ClN O

5

2

20 18

3

(415.83): C, 57.77; H, 4.36; N, 10.11. Found C, 57.89; H, 4.39; N, 10.11.

4.1.5.7. 6-Chloro-N'-(4-chlorobenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (12g).

-1

Yellow crystal (yield 81%), m.p. above 300 °C; IR (KBr, ν cm ): 3255 (NH, NH) and 1647, 1660

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 7.52 (d, 2H, J = 7.2 Hz, H-3, H-5, 4-Cl-C H ), 7.77, (d, 2H, J

6

4

=

7.2 Hz, H-2, H-6, 4-Cl-C H ), 7.80-7.86 (m, 2H, H-7, H-8 quinoline), 8.20 (s, 1H, H-5

6 4

quinoline), 8.45 (s, 1H, CH=N), 8.90 (s, 1H, H-2 quinoline), 13.10 (s, 1H, –CONH, D O

2

exchangeable), 13.16 (s, 1H, NH, D O exchangeable); Anal. calcd. for C H Cl N O (360.18): C,

2

17 11

2

3

2

56.69; H, 3.08; N, 11.67. Found C, 56.69; H, 3.12; N, 11.67.

4.1.5.8.

6-Chloro-N'-(2,4-dichlorobenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide

(12h).

-1

Yellow crystal (yield 82%), m.p. above 300 °C; IR (KBr, ν cm ): 3255 (NH, NH) and 1651, 1660

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 7.52 (d, 1H, J = 8.0 Hz, H-5, 2,4(Cl) -C H ), 7.69 (s,1H, H-3,

6

2

6

3

2

,4(Cl) -C H ), 7.76-7.95 (m, 2H, H-7, H-8 quinoline), 8.00 (d, 1H, J = 8.4 Hz, H-6, 2,4(Cl) -

2

6

3

2

C H ), 8.22 (s, 1H, H-5 quinoline), 8.67 (s, 1H, CH=N), 8.81 (s, 1H, H-2 quinoline), 13.12 (s, 1H,

6

3

1

3

–

1

2

CONH, D O exchangeable), 13.28 (s, 1H, NH, D O exchangeable); C NMR (DMSO-d ) δ ppm:

2 2 6

10.60, 124.82, 127.20, 128.40, 128.78, 129.79, 130.54, 131.09, 133.59, 134.30, 135.56, 136.92,

38.21, 143.31, 145.26, 161.98, 163.35; Anal. calcd. for C H Cl N O (394.64): C, 51.74; H,

1

7

10

3

2

.55; N, 10.65. Found C, 51.97; H, 2.53; N, 10.68.

.1.5.9. 6-Chloro-N'-(4-florobenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (12i).

4

-1

Yellow crystal (yield 78%), m.p. above 300 °C; IR (KBr, ν cm ): 3317 (NH, NH) and 1651, 1670

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 7.26 (t, 2H, J = 8.8 Hz, H-2, H-6, 4-F-C H ), 7.76 (dd, 2H, J

6

4

=

5

8.8 Hz, H-3, H-5, 4-F-C H ), 7.80- 7.84 (m, 2H, H-7, H-8 quinoline), 8.18 (s, 1H, J = 2.8 Hz, H-

6 4

quinoline), 8.43 (s, 1H, CH=N), 8.87 (s, 1H, H-2 quinoline), 13.10 (s, 2H, NH, –CONH, D O

2

exchangeable); Anal. calcd. for C H ClFN O (343.74): C, 59.40; H, 3.23; N, 12.22. Found C,

1

7

11

3

2

5

9.74; H, 3.21; N, 12.22.

.1.5.10. 6-Chloro-N'-(4-methylbenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (12j).

4

-1

Yellow crystal (yield 86%), m.p. above 300 °C; IR (KBr, ν cm ): 3255 (NH, NH) and 1654, 1660

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 2.32 (s, 3H, CH ) 7.23 (d, 2H, J = 8.0 Hz, H-3, H-5, 4- CH -

6

3

C H ), 7.61 (d, 2H, J = 8.0 Hz, H-2, H-6, 4-CH - C H ), 7.74- 7.82 (m, 2H, H-7, H-8 quinoline),

6

4

3

6

4

8.17 (s, 1H, H-5 quinoline), 8.35 (s, 1H, CH=N), 8.86 (s, 1H, H-2 quinoline), 13.04 (s, 2H, NH, –

2

0

ACCEPTED MANUSCRIPT

CONH, D O exchangeable); Anal. calcd. for C H ClN O (339.78): C, 63.63; H, 4.15; N, 12.37.

2

18 14

3

2

Found C, 63.90; H, 4.23; N, 12.37.

4.1.5.11. 6-Chloro-N'-(3,4-dimethoxybenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide

(12k).

-1

Yellow crystal (yield 80%), m.p. above 300 °C; IR (KBr, ν cm ): 3255 (NH, NH) and 1658, 1670

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 3.79 (s, 3H, OCH ), 3.80 (s, 3H, OCH ), 7.01 (d, 1H, J = 8.4

6

3

Hz, H-5, 3,4-(OCH ) -C H ), 7.24 (d, 1H, J = 8.4 Hz, H-6, 3,4-(OCH ) -C H ), 7.35 (s, 1H, H-2,

3

2

6

3

3 2

6

3

3,4-(OCH ) -C H ), 7.76- 7.84 (m, 2H, H-7, H-8 quinoline), 8.33 (s, 1H, H-5 quinoline), 8.34 (s,

3 2 6 3

1

3

1

H, CH=N), 8.86 (s, 1H, H-2 quinoline), 13.03 (s, 2H, NH, –CONH, D O exchangeable); C

2

NMR (DMSO-d₆)

δ

ppm: 109.05, 110.97, 111.98, 122.42, 124.81, 126.87, 127.08, 127.49, 128.49,

+

1

3

1

30.36, 133.52, 138.34, 145.12, 148.50, 149.47, 151.21, 161.98; MS m/z %: 387.29 (M +2, 7.78),

+

86.21 (M , 22.27), 81.08 (100%); Anal. calcd. for C H ClN O (385.80): C, 59.15; H, 4.18; N,

19

16

3

4

0.89. Found C, 59.39; H, 4.12; N, 10.89.

.1.5.12. 6-Chloro-N'-(3,4,5-trimethoxybenzylidene)-4-oxo-1,4-dihydroquinoline-3-

4

carbohydrazide (12l).

-1

Yellow crystal (yield 85%), m.p. above 300 °C; IR (KBr, ν cm ): 3425 (NH, NH) and 1651, 1660

1

(

C=O); H NMR (DMSO-d )

δ

ppm: 3.71 (s, 3H, OCH ), 3.84 (s, 6H, OCH ), 7.09 (s, 2H, H-2, H-

6

3

6

1

, 3,4,5-(OCH ) -C H ), 7.75- 7.85 (m, 2H, H-7, H-8 quinoline), 8.20 (s, H-5 quinoline), 8.35 (s,

3 3 6 2

H, CH=N), 8.89 (s, 1H, H-2 quinoline), 13.06 (s, 1H, –CONH, D O exchangeable), 13.13 (s, 1H,

2

+

NH, D O exchangeable); MS m/z %: 417.14 (M +2, 4.69), 415.12 (M , 9.56), 193.10 (100%);

2

Anal. calcd. for C H ClN O (415.83): C, 57.77; H, 4.36; N, 10.11. Found C, 57.98; H, 4.43; N,

2

0

18

3

5

10.37.

4

.2 Biological evaluation

.2.1. Antimicrobial activity

All strains were provided from culture collection of the Regional Center for Mycology and

Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. Antibacterial and antifungal activities

were expressed as the diameter of inhibition zones; agar well diffusion method was used. Holes (1

cm diameter) were digger in the agar using sterile cork borer in sterile malt agar plates for fungi

and sterile nutrient agar plates for bacteria, which had previously been uniformly seeded with

tested microorganisms. The holes were filled by fungal filtrates (100 µl). Plates were left in a

cooled incubator at 4 °C for one hour for diffusion and then incubated at 37°C for tested bacteria

and 28°C for tested fungi. Inhibition zones developed due to active antimicrobial metabolites were

2

1

ACCEPTED MANUSCRIPT

measured after 24 hour of incubation for bacteria and 48 hour of incubation for fungi.

Amphotericin B and ciprofloxacin were used as antifungal and antibacterial positive control;

respectively. The experiment was performed in triplicate and the average zone of inhibition was

calculated.

4.2.2. Minimum inhibitory concentration

MIC was performed by a serial dilution technique described by Irobi et al., [62], starting with

00 mmol concentration of all compounds dissolved in 1 mL DMSO and then reduced by

1

successive twofold dilutions of stock solution using a calibrated micropipette. Amphotericin B and

ciprofloxacin were used as the reference compounds for fungi and bacteria, respectively. The final

solutions concentrations were 125, 62.50, 31.25, 15.63, 7.81, 3.90, 1.95, 0.98, 0.49, 0.24 and 0.12

µmol/mL. The microtiter plates were incubated at 37°C for tested bacteria and 28°C for tested

fungi and were readied using microplate reader after 24 h for bacteria and after 48 h for fungi. In

each case, triplicate tests were performed and the average was taken as final reading. MIC was

expressed as the lowest concentration inhibiting test organism's growth [63].

4.2.3. Antimycobacterial activity

The M. tuberculosis (RCMB 010126) strain was provided from culture collection clinically

isolated of the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University

(Cairo, Egypt). Isoniazide was used as reference drugs. Antimycobacterial activity of the

synthesized compounds was evaluated using the microplate Alamar blue assay (MABA). Briefly,

the inoculum was prepared from fresh LJ medium re-suspended in 7H9-S medium (7H9 broth,

0.1% casitone, 0.5% glycerol, supplemented oleic acid, albumin, dextrose, and catalase [OADC]),

adjusted to a McFarland tube No. 1, and diluted 1:20; 100 µl was used as inoculum. Each

compound and Isoniazide stock solution was thawed and diluted in 7H9-S at four-fold the final

highest concentration tested. Serial two-fold dilutions of each compound and Isoniazide were

prepared directly in a sterile 96-well microtiter plate using 100 µl 7H9-S. A growth control

containing no antibiotic and a sterile control were also prepared on each plate. Sterile water was

added to all perimeter wells to avoid evaporation during the incubation. The plate was covered,

o

sealed in plastic bags and incubated at 37 C in normal atmosphere. After 7 days incubation, 30 ml

of alamar blue solution was added to each well, and the plate was re-incubated overnight. A change

in colour from blue (oxidised state) to pink (reduced) indicated the growth of bacteria, Percent

inhibition was defined as: 1 − (mean of test well/mean of B wells) × 100. the MIC was defined as

the lowest concentration of drug that prevented this change in colour [64].

2

ACCEPTED MANUSCRIPT

.2.4. In vitro cytotoxic activity

2

Mammalian cell lines: WI-38 cells (human lung fibroblast normal cell line), were obtained from

VACSERA Tissue Culture Unit.

Chemicals Used: Dimethyl sulfoxide (DMSO), crystal violet and trypan blue dye were purchased

from Sigma (St. Louis, Mo., USA).

Fetal Bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin and

0

.25% Trypsin-EDTA were purchased from Lonza.

Crystal violet stain (1%): It composed of 0.5% (w/v) crystal violet and 50% methanol then made

up to volume with ddH2O and filtered through a Whatmann No.1 filter paper.

Cell line Propagation:

The cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented

with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer and 50µg/ml

gentamycin. All cells were maintained at 37ºC in a humidified atmosphere with 5% CO2 and were

subcultured two times a week.

Cytotoxicity evaluation using viability assay: For cytotoxicity assay, the cells were seeded

in 96-well plate at a cell concentration of 1×104 cells per well in 100µl of growth medium. Fresh

medium containing different concentrations of the test sample was added after 24 h of seeding.

Serial two-fold dilutions of the tested chemical compound were added to confluent cell monolayers

dispensed into 96-well, flat-bottomed microtiter plates (Falcon, NJ, USA) using a multichannel

pipette. The microtiter plates were incubated at 37ºC in a humidified incubator with 5% CO2 for a

period of 48 h. Three wells were used for each concentration of the test sample. Control cells were

incubated without test sample and with or without DMSO. After incubation of the cells for at 37°C,

various concentrations of sample were added, and the incubation was continued for 24 h and viable

cells yield was determined by a colorimetric method.

In brief, after the end of the incubation period, media were aspirated and the crystal violet

solution (1%) was added to each well for at least 30 minutes. The stain was removed and the plates

were rinsed using tap water until all excess stain is removed. Glacial acetic acid (30%) was then

added to all wells and mixed thoroughly, and then the absorbance of the plates were measured after

gently shaken on Microplate reader (TECAN, Inc.), using a test wavelength of 490 nm. All results

were corrected for background absorbance detected in wells without added stain. Treated samples

were compared with the cell control in the absence of the tested compounds. All experiments were

carried out in triplicate. The cell cytotoxic effect of each tested compound was calculated. The

optical density was measured with the microplate reader (SunRise, TECAN, Inc, USA) to

determine the number of viable cells and the percentage of viability was calculated as [1-

(ODt/ODc)]x100% where ODt is the mean optical density of wells treated with the tested sample

and ODc is the mean optical density of untreated cells. The relation between surviving cells and

drug concentration is plotted to get the survival curve of each tumor cell line after treatment with

the specified compound. The 50% inhibitory concentration (IC50), the concentration required to

cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response

curve for each conc. using Graphpad Prism software (San Diego, CA. USA) [65,66].

2

3

ACCEPTED MANUSCRIPT

4

.3. 2D QSAR study:

All title compounds were Sketched in 2D and minimized by the protocol “Prepared Ligands” in

the DS 4.0 software (Discovery Studio 4.0, Accelrys, Co. Ltd). The antifungal, antibacterial and

antimycobacterial activities of the compounds MIC were converted into the logarithmic scale

PMIC (PMIC= -log MIC) and then proceed to the QSAR analysis as the response variables. The

data set was divided into the two subsets (Training and test set). Training set for antifungal and

antibacterial was 17 compounds and test set were 3 compounds. While for antimycobacterial it was

20 compounds and test set, 4 compounds. The test set compounds were selected manually

considering the distribution of biological data and structural diversity. The training set was used to

build a regression model, and the test set was used to evaluate the predictive ability of the model

obtained [67].

In this study, sixty eight molecular descriptors representing thermodynamic, electronic, spatial,

structural, thermodynamic, geometric, topological and quantum mechanical properties were

calculated using “Calculate Molecular Properties” protocol of the Discovery Studio 4.0. where all

the descriptor values for the molecules were considered as independent variables while, the

inhibitory concentration results (pMIC) were taken as dependent variables.

Then, by applying the “Genetic Function Approximation” module, certain descriptors were

selected then used to build a significant QSAR model by one of the best methods “Multiple Linear

Regression” (MLR) [68].

Acknowledgements

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,

Cairo, Egypt, is highly appreciated for supporting this research.

2

4

ACCEPTED MANUSCRIPT

References

[

1] Chaudhari, K., Surana, S., Jain, P. and Patel, H.M, Mycobacterium Tuberculosis (MTB) GyrB

inhibitors: An attractive approach for developing novel drugs against TB, Eur. J. Med.

Chem. 124 (2016) 160-185.

[

2] World

Health

Organization.

Global

Tuberculosis

Report

2015,

http://www.who.int/tb/publications/global_report/en.

3] Saxena, S., Samala, G., Renuka, J., Sridevi, J.P., Yogeeswari, P. and Sriram, D, Development

of 2-amino-5-phenylthiophene-3-carboxamide derivatives as novel inhibitors of

Mycobacterium tuberculosis DNA GyrB domain, Bioorg. Med. Chem. 23 (2015) 1402-1412.

4] Dye, C., Global epidemiology of tuberculosis. Lancet. 367 (2006) 938-940.

[

5] WHO, Multidrug-resistant Tuberculosis (MDR-TB), Update, World Health Organization,

2013.

[

6] Bielenica, A., Stefańska, J., Stępień, K., Napiórkowska, A., Augustynowicz-Kopeć, E., Sanna,

G., Madeddu, S., Boi, S., Giliberti, G., Wrzosek, M. and Struga, M, Synthesis, cytotoxicity and

antimicrobial activity of thiourea derivatives incorporating 3-(trifluoromethyl) phenyl

moiety, Eur. J. Med. Chem.101 (2015) 111-125.

[

7] Roca, I., Akova, M., Baquero, F., Carlet, J., Cavaleri, M., Coenen, S., Cohen, J., Findlay, D.,

Gyssens, I., Heure, O.E. and Kahlmeter, G, The global threat of antimicrobial resistance:

science for intervention, New microbes and new infections, 6 (2015) 22-29.

8] Janeczko, M., Demchuk, O.M., Strzelecka, D., Kubiński, K. and Masłyk, M, New family of

antimicrobial agents derived from 1,4-naphthoquinone, Eur. J. Med. Chem. 124 (2016) 1019-

1025.

9] Ruddarraju, R.R., Murugulla, A.C., Kotla, R., Tirumalasetty, M.C.B., Wudayagiri, R.,

Donthabakthuni, S., Maroju, R., Baburao, K. and Parasa, L.S, Design, synthesis, anticancer,

antimicrobial activities and molecular docking studies of theophylline containing acetylenes

and theophylline containing 1,2,3-triazoles with variant nucleoside derivatives, Eur. J. Med.

Chem. 123 (2016) 379-396.

[

10] Mitscher, L.A, Bacterial topoisomerase inhibitors: quinolone and pyridone antibacterial

agents, Chem. Rev. 105 (2005) 559-592.

11] Suresh, N., Nagesh, H.N., Renuka, J., Rajput, V., Sharma, R., Khan, I.A. and Gowri,

C.S.K.V, Synthesis and evaluation of 1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-7-(4-(2-(4-

substitutedpiperazin-1-yl) acetyl) piperazin-1-yl) quinoline-3-carboxylic acid derivatives as

anti-tubercular and antibacterial agents, Eur. J. Med. Chem. 71 (2014) 324-332.

12] Musiol, R., Serda, M., Hensel-Bielowka, S. and Polanski, J, Quinoline-based

antifungals, Curr. Med. Chem. 17 (2010) 1960-1973.

[

2

5

ACCEPTED MANUSCRIPT

13] Patel, S.R., Gangwal, R., Sangamwar, A.T. and Jain, R, Synthesis, biological evaluation

[

and 3D-QSAR study of hydrazide, semicarbazide and thiosemicarbazide derivatives of 4-

(adamantan-1-yl) quinoline as anti-tuberculosis agents, Eur. J. Med. Chem. 85 (2014) 255-267.

[

14] Kumar, S., Bawa, S. and Gupta, H, Biological activities of quinoline derivatives, Mini.

Rev. Med. Chem. 9 (2009) 1648-1654.

15] Daneshtalab, M. and Ahmed, A, Nonclassical biological activities of quinolone

derivatives, J. Pharm. Pharma. Sci. 15 (2011) 52-72.

16] Al-Amiery, A.A., Al-Bayati, R.I., Saour, K.Y. and Radi, M.F, Cytotoxicity, antioxidant,

and antimicrobial activities of novel 2-quinolone derivatives derived from coumarin, Res.

Chem. Intermed. 38 (2012) 559-569.

[

17] Rachakonda, V., Alla, M., Kotipalli, S.S. and Ummani, R, Design, diversity-oriented

synthesis and structure activity relationship studies of quinolinyl heterocycles as

antimycobacterial agents, Eur. J. Med. Chem. 70 (2013) 536-547.

[

18] Jardosh, H.H. and Patel, M.P, Design and synthesis of biquinolone–isoniazid hybrids as a

new class of antitubercular and antimicrobial agents, Eur. J. Med. Chem. 65 (2013) 348-359.

19] Oliveri, V. and Vecchio, G, 8-Hydroxyquinolines in medicinal chemistry: A structural

perspective, Eur. J. Med. Chem. 120 (2016) 252-274.

20] Matviiuk, T., Madacki, J., Mori, G., Orena, B.S., Menendez, C., Kysil, A., André-Barrès,

C., Rodriguez, F., Korduláková, J., Mallet-Ladeira, S. and Voitenko, Z, Pyrrolidinone and

pyrrolidine derivatives: Evaluation as inhibitors of InhA and Mycobacterium tuberculosis, Eur.

J. Med. Chem. 123 (2016) 462-475.

[

21] Jayaprakash, S., Iso, Y., Wan, B., Franzblau, S.G. and Kozikowski, A.P, Design, Synthesis,

and SAR Studies of Mefloquine‐ Based Ligands as Potential Antituberculosis Agents, Chem.

Med. Chem. 1 (2006) 593-597.

[

22] Keri, R.S. and Patil, S.A, Quinoline: a promising antitubercular target, Biomed.

Pharmacother. 68 (2014) 1161-1175.

23] Eswaran, S., Adhikari, A.V., Pal, N.K. and Chowdhury, I.H, Design and synthesis of some

new quinoline-3-carbohydrazone derivatives as potential antimycobacterial agents, Bioorg.

Med. Chem. Lett. 20 (2010) 1040-1044.

[

24] Thomas, K.D., Adhikari, A.V., Telkar, S., Chowdhury, I.H., Mahmood, R., Pal, N.K., Row,

G. and Sumesh, E, Design, synthesis and docking studies of new quinoline-3-carbohydrazide

derivatives as antitubercular agents, Eur. J. Med. Chem. 46 (2011) 5283-5292.

25] Manetti, F., Corelli, F., Biava, M., Fioravanti, R., Porretta, G.C. and Botta, M, Building a

pharmacophore model for a novel class of antitubercular compounds, Il Farmaco. 55 (2000)

484-491.

2

6

ACCEPTED MANUSCRIPT

26] Da Silva Lourenço, M.C., de Lima Ferreira, M., de Souza, M.V.N., Peralta, M.A.,

[

Vasconcelos, T.R.A. and Maria das Graças, M.O, Synthesis and anti-mycobacterial activity of

(E)-N′-(monosubstituted-benzylidene) isonicotinohydrazide derivatives, Eur. J. Med. Chem. 43

(2008) 1344-1347.

[

27] Abdel-Aziz, M. and Abdel-Rahman, H.M, Synthesis and anti-mycobacterial evaluation of

some pyrazine-2-carboxylic acid hydrazide derivatives, Eur. J. Med. Chem. 45 (2010) 3384-

3388.

28] Maccari, R., Ottana, R. and Vigorita, M.G, In vitro advanced antimycobacterial screening

of isoniazid-related hydrazones, hydrazides and cyanoboranes, Bioorg. Med. Chem. Lett. 15

(2005) 2509-2513.

29] Silva, F.P., Ellena, J., de Lima Ferreira, M., Mascarenhas, Y.P., de Souza, M.V.,

Vasconcelos, T.R., Wardell, J.L. and Wardell, S.M, Experimental and theoretical structure

characterization of two isoniazid derivatives: 2,4-Difluoro-N′-isonicotinoylbenzohydrazide and

2,4-dichloro-N′-isonicotinoylbenzohydrazide hydrochloride, J. Mol. Struct. 788 (2006) 63-71.

[

30] Carvalho, S.A., da Silva, E.F., de Souza, M.V., Lourenço, M.C. and Vicente, F.R,

Synthesis and antimycobacterial evaluation of new trans-cinnamic acid hydrazide

derivatives, Bioorg. Med. Chem. Lett. 18 (2008) 538-541.

31] Sriram, D., Yogeeswari, P. and Madhu, K, Synthesis and in vitro and in vivo

antimycobacterial activity of isonicotinoyl hydrazones, Bioorg. Med. Chem. Lett. 15 (2005)

4502-4505.

32] Navarrete-Vázquez, G., Marı, G., Duarte-Fajardo, Z.V., Vargas-Villarreal, J., Estrada-Soto,

S., González-Salazar, F., Hernández-Núñez, E. and Said-Fernández, S, Synthesis and

antimycobacterial activity of 4-(5-substituted-1,3,4-oxadiazol-2-yl) pyridines, Bioorg. Med.

Chem. 15 (2007) 5502-5508.

[

33] Bartzatt, R., LG Cirillo, S. and D Cirillo, J, Small molecule hydrazide agents to inhibit

growth and proliferation of Mycobacterium tuberculosis, Med. Chem. 8 (2012) 273-280.

34] Zhang, L., Addla, D., Ponmani, J., Wang, A., Xie, D., Wang, Y.N., Zhang, S.L., Geng,

R.X., Cai, G.X., Li, S. and Zhou, C.H, Discovery of membrane active benzimidazole

quinolones-based topoisomerase inhibitors as potential DNA-binding antimicrobial agents, Eur.

J. Med. Chem. 111 (2016) 160-182.

[

35] Raju, B., Mortell, K., Anandan, S., O'Dowd, H., Gao, H., Gomez, M., Hackbarth, C., Wu,

C., Wang, W., Yuan, Z. and White, R, N-and C-terminal modifications of negamycin, Bioorg.

Med. Chem. Lett 13 (2003) 2413-2418.

2

7

ACCEPTED MANUSCRIPT

[

36] Imramovský, A., Polanc, S., Vinšová, J., Kočevar, M., Jampílek, J., Rečková, Z. and

Kaustová, J, A new modification of anti-tubercular active molecules, Bioorg. Med. Chem. 15

(

2007) 2551-2559.

37] Nayyar, A., Malde, A., Coutinho, E. and Jain, R, Synthesis, anti-tuberculosis activity, and

D-QSAR study of ring-substituted-2/4-quinolinecarbaldehyde derivatives, Bioorg. Med.

3

Chem. 14 (2006) 7302-7310.

38] Sriram, D., Yogeeswari, P. and Devakaram, R.V, Synthesis, in vitro and in vivo

antimycobacterial activities of diclofenac acid hydrazones and amides, Bioorg. Med. Chem. 14

(2006) 3113-3118.

39] Sinha, N., Jain, S., Tilekar, A., Upadhayaya, R.S., Kishore, N., Jana, G.H. and Arora, S.K,

Synthesis of isonicotinic acid N′-arylidene-N-[2-oxo-2-(4-aryl-piperazin-1-yl)-ethyl]-

hydrazides as antituberculosis agents, Bioorg. Med. Chem. Lett. 15 (2005) 1573-1576.

40] Kathiravan, A., Sundaravel, K., Jaccob, M., Dhinagaran, G., Rameshkumar, A., Arul

Ananth, D. and Sivasudha, T, Pyrene schiff base: photophysics, aggregation induced emission,

and antimicrobial properties, J. Phys. Chem. B. 118 (2014) 13573-13581.

41] Liao, Z.Q., Dong, C., Carlson, K.E., Srinivasan, S., Nwachukwu, J.C., Chesnut, R.W.,

Sharma, A., Nettles, K.W., Katzenellenbogen, J.A. and Zhou, H.B, Triaryl-substituted Schiff

bases are high-affinity subtype-selective ligands for the estrogen receptor, J. Med. Chem. 57

(2014) 3532-3545.

[

42] Eldehna, W.M., Fares, M., Abdel-Aziz, M.M. and Abdel-Aziz, H.A, Design, synthesis and

antitubercular activity of certain nicotinic acid hydrazides, Molecules. 20 (2015) 8800-8815.

43] Demirbas, N., Karaoglu, S.A., Demirbas, A. and Sancak, K, Synthesis and antimicrobial

activities of some new 1-(5-phenylamino-[1,3,4] thiadiazol-2-yl) methyl-5-oxo-[1,2,4] triazole

and 1-(4-phenyl-5-thioxo-[1,2,4] triazol-3-yl) methyl-5-oxo-[1,2,4] triazole derivatives, Eur. J.

Med. Chem. 39 (2004) 793-804.

[

44] Abdel-Aziz, H.A., Ghabbour, H.A., Eldehna, W.M., Qabeel, M.M. and Fun, H.K.,

Synthesis, Crystal Structure, and Biological Activity of cis/trans Amide Rotomers of (Z)-N′-(2-

Oxoindolin-3-ylidene) formohydrazide, J. Chem. 2014.

45] Dos Santos Filho, J.M., Moreira, D.R.M., de Simone, C.A., Ferreira, R.S., McKerrow, J.H.,

Meira, C.S., Guimarães, E.T. and Soares, M.B.P, Optimization of anti-Trypanosoma cruzi

oxadiazoles leads to identification of compounds with efficacy in infected mice, Bioorg. Med.

Chem. 20 (2012) 6423-6433.

[

46] Dos Santos Filho, J.M, Mild, Stereoselective, and Highly Efficient Synthesis of

N‐ Acylhydrazones Mediated by CeCl3· 7H2O in a Broad Range of Solvents, Eur. J. Org.

Chem. 2014 (2014) 6411-6417.

2

8

Article Doi

Design, synthesis and 2D QSAR study of novel pyridine and quinolone hydrazone derivatives as potential antimicrobial and antitubercular agents

DOI: 10.1016/j.ejmech.2017.07.004

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2017.07.004

Full text of DOI:10.1016/j.ejmech.2017.07.004

Products guided by the article

R&D Labs maybe for 108715-11-3

Relevant to this article

Hot Product