Synthesis, characterization and antitubercular activities of novel pyrrolyl hydrazones and their Cu-complexes

Article abstract of DOI:10.1016/j.ejmech.2016.05.025

Novel pyrrolyl hydrazones and their copper complexes have been synthesized and characterized using analytical and spectral techniques to show the tetrahedral geometry for Cu(II) complexes. Biological activities of hydrazones have been assessed to understand the role of metal ion on their biological activity and the effect of pyrrolyl hydrazones. In?vitro antitubercular activity against Mycobacterium tuberculosis of the metal complexes (13b and 13r) exhibited the highest antitubercular activity that are quite close to rifampicin (0.4?μg/mL), giving a MIC of 0.8?μg/mL. All other compounds showed good activity with the MIC values ranging from 1.6 to 100?μg/mL. A comparative study of inhibition values of the ligands and their complexes showed higher antimicrobial activity of the complexes than the ligands. Some compounds have a good activity against InhA and in particular, compounds 12r, 13b and 13r exhibited more than 60% binding with the enzyme even at 5?μM (exhibited good IC50 upto 2.4?μM). Most of the active molecules have a very less cytotoxicity against the human lung cancer cell-line A549. The docking and 3D-QSAR studies have been carried out to provide some insights into the mechanism of action for this class of compounds.

Full text of DOI:10.1016/j.ejmech.2016.05.025

Accepted Manuscript
Synthesis, characterization and antitubercular activities of novel pyrrolyl hydrazones
and their Cu-complexes
Shrinivas D. Joshi, Devendra Kumar, Sheshagiri R. Dixit, Nageshwar Tigadi, Uttam A.
More, Christian Lherbet, Tejraj M. Aminabhavi, Kap Seung Yang
PII:
S0223-5234(16)30413-5
10.1016/j.ejmech.2016.05.025
EJMECH 8617
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 January 2016
Revised Date: 7 May 2016
Accepted Date: 9 May 2016
Please cite this article as: S.D. Joshi, D. Kumar, S.R. Dixit, N. Tigadi, U.A. More, C. Lherbet, T.M.
Aminabhavi, K.S. Yang, Synthesis, characterization and antitubercular activities of novel pyrrolyl
hydrazones and their Cu-complexes, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.05.025.
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GRAPHICAL ABSTRACT
Synthesis, characterization and antitubercular activities of novel pyrrolyl hydrazones
and their Cu-complexes
Shrinivas D. Joshi, Devendra Kumar, Sheshagiri R. Dixit, Nageshwar Tigadi, Uttam A.
More, Christian Lherbet, Tejraj M. Aminabhavi, Kap Seung Yang
Synthetic route for the synthesis of biologically active metal complexes as enoyl ACP
reductase inhibitors
1

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Synthesis, characterization and antitubercular activities of novel pyrrolyl hydrazones
and their Cu-complexes
a
a
a
a
Shrinivas D. Joshi* , Devendra Kumar , Sheshagiri R. Dixit , Nageshwar Tigadi ,
a
b,c
a
d
Uttam A. More , Christian Lherbet , Tejraj M. Aminabhavi , Kap Seung Yang
a
Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry,
S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-580 002, India.
b
Universite de Toulouse, UPS, Laboratoire de Synthese et Physico-chimie de Molecules
d’Interet Biologique, LSPCMIB, 118 Roote de Narbonne, F-31062, Toulouse Cedex 9,
France.
c
ITAV-USR3505, Université de Toulouse, CNRS, UPS, F-31106 Toulouse, France.
d
Department of Polymer and Fiber System Engineering, Chonnam National University, 300
Yongbong-Dong, Bukgu, Gwangju, 500 757, Korea.
Corresponding Author
Shrinivas D. Joshi, Novel Drug Design and Discovery Laboratory, Department of
Pharmaceutical Chemistry, S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-
5
80 002, India.
E-mail: shrinivasdj@rediffmail.com.
Tel.: +91 9986151953; Fax; +91 836 2467190
1

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Abstract
Novel pyrrolyl hydrazones and their copper complexes have been synthesized and
characterized using analytical and spectral techniques to show the tetrahedral geometry for
Cu(II) complexes. Biological activities of hydrazones have been assessed to understand the
role of metal ion on their biological activity and the effect of pyrrolyl hydrazones. In vitro
antitubercular activity against Mycobacterium tuberculosis of the metal complexes (13b and
1
3r) exhibited the highest antitubercular activity that are quite close to rifampicin (0.4
µg/mL), giving a MIC of 0.8 µg/mL. All other compounds showed good activity with the
MIC values ranging from 1.6-100 µg/mL. A comparative study of inhibition values of the
ligands and their complexes showed higher antimicrobial activity of the complexes than the
ligands. Some compounds have a good activity against InhA and in particular, compounds
1
2r, 13b and 13r exhibited more than 60% binding with the enzyme even at 5 µM (exhibited
good IC50 upto 2.4 µM). Most of the active molecules have a very less cytotoxicity against
the human lung cancer cell-line A549. The docking and 3D-QSAR studies have been carried
out to provide some insights into the mechanism of action for this class of compounds.
2

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Keywords: Surflex docking, Pyrrolyl Schiff bases, Pyrrolyl Cu-complexes, Anti-tubercular
activity, Cytotoxicity activity, Enzyme inhibition studies.
3

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1
. Introduction
Tuberculosis (TB) is the leading airborne contagious disease caused by Mycobacterium
tuberculosis (MTB) and is the second leading cause of death worldwide [1]. TB is known to
be one of the major causes of death in HIV patients. As per World Health Organization
(
WHO) 2012 global report on TB, an estimated 3.7% (range 2.1-5.2%) of new cases and 20%
range 13-26%) of the previously treated cases show multidrug-resistant TB (MDR-TB)
(
features. In Eastern Europe and central Asia, nearly 9-32% of new cases and more than 50%
of the previously treated patients acquired MDR-TB [2]. Global increase in drug resistance,
mainly MDR-TB, reflects at least in part, improper use of anti-TB drugs during the treatment
course of TB patients with drug susceptible strains [3]. Additional factors such as sex, age,
HIV infection and socio-economic factors have also shown to be responsible for increased
prevalence of MDR-TB [4]. Nearly 50% of global MDR-TB cases have been reported in
China and India [5].
Currently, TB treatment using DOTS (directly observed therapy short-course) requires a
combination of three to four drugs, which includes isoniazid, pyrazinamide, rifampin, and
ethambutol for over a period of 6-12 months. The extended treatment schedule for TB is due
to the presence of non-replicating persistent MTB phenotype and therefore, novel drugs are
urgently required for the treatment of this deadly disease.
Isoniazid (INH), a frontline antitubercular agent, is a pro-drug that requires activation to form
active metabolite (INH-NAD adduct), which exerts its lethal action on intracellular target [6,
7
]. The INH-NAD adduct inhibits mycolic acid biosynthesis in MTB by affecting InhA, a 2-
trans-enoyl-acyl carrier protein reductase enzyme of the type II fatty acid synthesis (FAS-II)
system, which catalyses the last step of fatty acid elongation cycle [8]. Among them, fatty
acid biosynthesis-I (FabI) constitutes a single isoform in major pathogens such as
Staphylococcus aureus [9], Escherichia coli [10], and MTB [11]. The clinical success of
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InhA inhibitor isoniazid [12] and numerous reports of FabI inhibitors [13] involving
diazaborines [14], 4-pyridones [15], naphthyridinones [16], triclosan and its analogues [17,
1
8] have validated this target as one of the most attractive of the FAS-II pathways. This
enzyme is recognized and validated as an important drug target in MTB, since its homolog in
human is absent.
Several azole compounds containing imidazole, pyrrole, toluidine or methanamine group
were tested for anti-TB activity against drug resistant and intramacrophagic mycobacteria
[
19]. Amongst these compounds, pyrrole derivative BM212 appeared to be particularly
potent due to its selective antimycobacterial properties [20]. Recently, spontaneous mutants
resistant to BM212 and SQ109, compounds with anti-TB activities, were shown to contain
mutations mapping to mmpL3 (for mycobacterium large membrane protein) [21]. The mmpL3
was found to be essential in mycobacteria and conditional depletion of mmpL3 in
Mycobacterium smegmatis resulted in a loss of cell wall mycolylation [22].
Realizing the widespread application of transition metal complexes in living systems [23-25]
and due to their favorable biological activity [26], the complexes of tetradentate Schiff base
ligands with transition metals (manganese, copper and nickel) have been shown to exhibit
antibacterial, antifungal and cytotoxicity activities [27-29]. In our earlier studies, we have
developed a new series of compounds that have shown promising antimycobacterial activities
[
30, 31]. However, keeping in view of the biological and medicinal properties of pyrroles and
the potentiality of transition metal chemistry, it was felt necessary to design metal complexes
with organic ligands such as pyrrolyl hydrazone having biological activity that could help to
treat against the resistant bacteria species. As per Fig. 1, some marketed drugs that were
considered in the synthesis of new derivatives following the Paal-Knorr pyrrole synthesis,
wherein we have designed novel concepts to describe the framework to understand their
mechanisms by combining molecular docking and other parameters for their structural
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characteristics that affect the binding of pyrrolyl hydrazones and copper complexes with
enoyl ACP reductase receptor. Molecular docking studies have been used to correlate in
silico results with in vitro analysis to find ENR as a potential target for these derivatives.
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2
. Molecular modeling/docking studies
The 3D structures were generated using SYBYL package (Tripos Associates, St. Louis, MO,
USA) [32]. By using standard bond lengths and bond angles, the geometry optimization was
carried out with the help of standard Tripos Force Field [33] with a distance dependent-
dielectric function, energy gradient of 0.001 kcal/mol and Gasteiger-Huckel as the
electrostatics. Conformational analysis of all the ligands was performed using the repeated
molecular dynamics-based simulated annealing approach as implemented in Sybyl-X 2.0. All
the conformations were minimized with Gasteiger-Huckel charges.
2
.1. Data set and structures
The in vitro antitubercular activity (expressed as MIC) was converted into pMIC (log MIC)
values, which were used to construct 3D-QSAR models. Antitubercular activity results of the
synthesized compounds are summarized in Table 1. The 3D-QSAR models were generated
using a training set of 19 molecules, and predictive power of the resulting models was
evaluated using a test set of six molecules. Test compounds were selected randomly by
diversity method such that the data set included diverse structures and a wide range of
activity.
2
.2. Alignment rule
A common substructure-based alignment was adopted, wherein molecules were aligned to the
template molecule on a common backbone. For database alignment of the inhibitors, the
structure of compound 12b was used as a templet.
2
.3. CoMFA and CoMSIA settings
In order to understand the contributions of electrostatic and steric fields in the binding affinity
and potency of pyrrole scaffold, as well as to build the predictive 3D-QSAR models, the
CoMFA studies were performed on the ligands based on molecular alignment. The
contributions of steric and electrostatic fields were calculated using the Lennard-Jones and
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Coulombic potentials, respectively [34]. The aligned training set of molecules was then
placed in a 3D grid box to include the entire set. The CoMFA steric and electrostatic fields
3
were generated at each grid point with Tripos force field using sp carbon atom with +1
charge.
The CoMSIA was used in which similarity indices (steric, electrostatic, hydrophobic and H-
bond donor and H-bond acceptors) were calculated at different points on a regularly spaced
grid for pre-aligned molecules with a radius of 1.0 Å and a default value of 0.3 as the
attenuation factor (α), a grid spacing of 2.0 Å in x, y, and z directions used for both CoMFA
2
and CoMSIA. The q resulted in an optimum number of components and the lowest standard
error of prediction.
2
.4. Partial least squares (PLS)
PLS analysis provides a correlation between anti-TB activity of the compounds with the
predictive values of CoMFA and CoMSIA containing a magnitude of steric, electrostatic,
hydrophobic potentials, H-bond donor, and acceptor. This regression method was used to
analyze the training set by correlating dependent variable values (anti-TB activity) with
variations in their independent variables (CoMFA/CoMSIA interaction fields). All the latent
variable path models in PLS consist of three sets of relationships: (a) the inner model, which
specifies the relationship between latent variables (LVs), (b) the outer model, which specifies
the relationship between LVs and their association observed or manifest variables (MVs), and
(c) the weight relations upon which the case values for LVs can be estimated. Without loss of
generality, it can be assumed that LVs and MVs are scaled to zero means and unit variance
such that location parameter (i.e., constant parameter terms) can be eliminated [35].
2
2
.5. Predictive ability of CoMFA and CoMSIA models (r pred
)
2
The value of r is a measure of % of data that can be satisfactorily explained by the regression
analysis [36]. Predictive ability of each analysis was determined from the test set molecules
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that were not included in the training set. These molecules were aligned and their activities
were predicted by each PLS analysis.
2
By PLS analysis, r predicted for CoMFA and CoMSIA were found to be 0.86 and 0.69,
respectively. Cross validated and non-cross validated statistical parameters of CoMFA and
CoMSIA models are summarized in Table 2.
3
. RESULTS AND DISCUSSION
3
.1. Chemistry
Studies were undertaken to synthesize novel pyrrole ring bearing hydrazone derivatives and
their copper complexes to investigate their anti-TB effects. Compounds 6a-y were
synthesized as per Scheme 1. Various carboxamides were synthesized by reacting the
commercially available acids viz., pyruvic acid (1) / lavulanic acid (2) in the presence of
thionyl chloride or HBTU [(N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uraniumhexa
fluorophosphate] /DIEA (N,N-diisopropylethylamine). Pyrrole synthesis is achieved via Paal-
Knorr mechanism by the condensation of 2,5-dimethoxytetrahydrofuran or 1,4-diketone (2,5-
hexanedione) and ethyl 4-amino benzoate (7) in the presence of dry acetic acid to get 4-(1H-
pyrrol-1-yl)benzoate (8) or 4-(2,5-dimethyl-1H-pyrrol-1-yl)benzoate (9) in good yield. Thus
obtained esters were converted to their corresponding hydrazides (10, 11) by hydrazinolysis
using hydrazine hydrate (Scheme 2). The NH in hydrazides (10, 11) is more nucleophilic
2
than NH, which reacts preferentially with the more reactive carbonyl group (6a-y), leading to
the formation of hydrazone derivative (12a-y) (Scheme 3) as the major products, especially if
the reaction is carried out in the presence of a catalytic amount of acetic acid or TFAA upon
heating or without heating.
The structures of hydrazones (12a-y) were established by spectral analysis. The compound
-1
-1
1
2r showed characteristic absorption bands at 1701 cm and 1687 cm for carbonyl groups
-1
-1
and the absence of NH stretching band around 3335 cm . A sharp peak at 1577 cm
2
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1
indicates the presence of C=N group. The H NMR showed two singlets at 9.88 and 10.54 δ
ppm corresponding to two -NH protons. A singlet at δ 1.98 ppm, which, was accounted for
three protons of methyl group and a multiplet at δ 2.51-2.56 ppm representing the four
protons of pentamide. All other 12 protons of aromatic rings resonated in the expected region
between δ 6.29-8.23 ppm. The number of protons calculated from the integration curves and
those obtained from the values of the expected CHN analyses agree with each other.
3
.2. Synthesis and characterization of copper complexes
The synthesis of copper complexes was carried out as per Scheme 4. Hydrazones (12a-y)
were reacted with copper acetate in a refluxing ethanol medium to furnish the targeted copper
complexes (13a-y), which were readily soluble in DMSO and DMF solvents. They are non-
hygroscopic and stable in both solid and solution phases. The elemental analyses data for Cu
(
II) complexes are in good agreement with their molecular formulae and all metal complexes
are green in color.
.3. FTIR spectra
3
FTIR spectra of the metal complexes are compared with that of the ligand itself to find the
coordination sites involved in the bond formation. There are some guide peaks in the
spectrum of the ligand, which enabled us to understand this aspect. These peaks change either
in their positions and/or their intensities upon chelation, but some peaks disappeared after
chelation.
-1
The carbonyl stretching bands found in the region of 1603-1714 cm (12a-y), which have
disappeared, indicating the involvement of carbonyl group in bond formation with metal,
while two -C=N peaks are observed in FTIR spectrum of the metal complex. From these
observations, it can be concluded that two carbonyl groups of the ligand have undergone
-
mesomerism, since the mesomeric form containing -C=N-N-O type of arrangement might be
involved in the bond formation with the central metal. Further, this type of mesomerism was
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-1
proved by the appearance of two -C=N- stretching bands in the region of 1690-1579 cm
(
medium band). From the FTIR spectra, it was found that hydrazide nitrogen might have been
involved in coordinate bond formation with the central metal ion (13a-y).
.4. UV-Visible Spectroscopy
3
UV (ultraviolet)-visible spectroscopy was used at >300 nm to analyze the geometry of metal
complexes, which confirmed tetrahedral geometry.
3
.5. Thermogravimetric analysis
Water of hydration is related with the central metal ion complex formation around the
coordination sphere, which generally takes place around the temperature range of 50-140°C,
indicating no loss of any fragment around the water molecule. This further confirmes that
there is no water molecule attached outside the coordination sphere. The coordinated water
molecules directly bonded to metal ion are generally knocked off at higher temperature of
1
50-250°C [35-37]. However, mass spectrometry suggests that complexes may not contain
water molecule either inside or outside the coordination sphere, which further confirms the
results of TGA.
Metal complexes decompose at 195-285°C in two steps via the formation of unstable
intermediates ending at 490-750°C (oxides formation). The % metal complexes were
calculated from the % residual metal oxide formed in the final step, and these are in good
agreement with the results obtained by MacDonald wet combustion method [40]. TGA data
of copper complexes are summarized in Table 3. On the basis of the above observations,
following general scheme for thermal decomposition can be proposed for metal complexes in
the ratio of 1:2 (M:L)
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3
.6. Mass spectrometry
Mass spectroscopy was used to determine the ratio of ligands involved in the complex
formation with the central metal ion as well as to estimate the mole reactants used in the
synthesis. It also gives appropriate information about the involvement of water or chlorine
molecule during the complexation step. In this study, neither water nor chlorine was involved
in the formation of the coordination sphere. Molecular ion peak for ligand (12r) is observed
at 392 m/z, whereas its copper complex 13r shows molecular ion peak at 844 m/z, which
confirms the molecular weight of 12r and 13r.
3
.7. Cyclic voltammetry
Cyclic voltammetry of the complexes were performed on 0.025M solutions in DMSO solvent
containing 0.2M [NBu ][ClO ] in acetonitrile as the supporting electrolyte in the range of +1
4
4
to -0.4 V. All solutions were purged with nitrogen gas for 5 min before measurement and
working electrode was polished before each experiment with 0.5 µ particle size α-alumina
slurry. The procedure was performed at room temperature and nitrogen atmosphere was
maintained over the solution during measurements. In the positive range of +1 to 0 V the
oxidation of Cu(III) to Cu(II) was observed.
3
.8. Stability of complexes in solution
The stability of complexes in aqueous solution was studied by observing UV-VIS spectra.
The compounds were prepared in DMSO solvent and for experiments freshly diluted in
phosphate buffer system. UV-VIS spectra and molar conductivities were measured at
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different time intervals. It can be seen from the results show that there are no change in
absorption bands, indicating that these complexes are quite stable in phosphate buffer.
3
.9. Molecular modeling
In order to investigate detailed intermolecular interactions between the ligand and the target
protein, a program Surflex-Dock was used. The 3D structure information on target protein
was taken from PDB entry 2AQI [41] (Crystal structure of isoniazid-resistant I47T enoyl
ACP (CoA) reductase mutant enzyme from MTB in complex with NADH). Processing of the
protein included the removal of ligand and solvent molecules as well as addition of essential
hydrogen atoms. All 50 compounds were docked into active site of the enzyme and predicted
binding energies of the compounds are listed in Tables 4 and 5.
As depicted in Fig. 2, fluorine atom attached to aromatic ring of compound 12r makes two H-
bond interaction (F-----H-PHE41, ASP42) i.e., hydrogen of amide linkage attached to 4-
fluoro phenyl ring makes one bonding interaction with SER94 (NH-CO-----O-SER94) and
oxygen of another amide linkage attached to bridging phenyl makes one H-bonding
interaction (CONH------H-THR196). As depicted in Fig. 3 (A and B), the hydrogen of
hydrazide of compound 13b makes bonding interaction with amino acid residue (NH----H-
GLY96).
It was found that hydrophobic region of mutant ENR enzyme overlap with the hydrophobic
region of copper complex (Fig 3B) and due to such type of overlapping as well as
involvement of different forces such as van der Waals, and π-π interaction along with H-
bonding are strong base for such interaction between copper complex and mutant ENR
enzyme.
3
.10. CoMFA and CoMSIA models
.10.1. Contour maps
3
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Steric and Electrostatic field. In 3D-QSAR analysis, a very less difference between the actual
pMIC and predicted pMIC was observed (Table 6). From the 3D-CoMFA analysis, it can be
confirmed that if the steric bulk is increased on pyrrole ring, biological activity of the
compound will be diminished (Fig 4A) as was also supported by CoMSIA analysis (Fig. 5A).
In CoMSIA, the blue counter map on pyrrole confirms that this is the sterically disfavored
region. This is one of the causes of decrease in biological activity of some of the derivatives
nd
th
having the methyl group on their 2 and 5 positions with ligands such as 12v (exceptional),
2w and 12x that suffer from an increase in MIC values up to 12.50, 100 and 25 µg/ml,
1
respectively. In docking analysis, it was found that all these methyl containing pyrrole
ligands have less binding affinity with the mutant ENR enzyme. The ligands 12v, 12w and
1
2x have the binding scores of 4.52, 4.47 and 3.97, respectively along with a slightly high
crash score. Benzene ring that is directly attached to the pyrrole contains a big section of blue
contour map, due to the bulky group of unfavorable contour. Blue contour map on anilide
benzene ring reveals that substitution on this ring is less bulky, but cyan contour map in
CoMFA and CoMSIA over this ring is a clear indication of the electronegative substitution
(Fig. 4B and Fig. 5B).
Hydrogen-bond-acceptor field. In Fig. 5C, it can be observed that in the highly active
template molecule 12b, the green contour in the region of hydrazone bridge favors its
activity.
Hydrogen-bond-donor field. In Fig. 5D, pink contour on NH of anilide and also on hydrazone
bridge of molecule 12b favor the activity.
Hydrophobicity field. According to CoMSIA model, as shown in Fig. 5E, hydrophobicity is
favored near the anilide ring (purple contour).
3
.11. Antimycobacterial studies and InhA enzyme inhibition studies
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The MIC values were determined for hydrazones (12a-y) and their copper complexes (13a-y)
against M. tuberculosis strain H37Rv using the Microplate Alamar Blue assay (MABA)
method using ethambutol and rifampicin as the standard drugs. The tested compounds
showed activities against mycobacteria with the MIC values ranging from 0.8 to 100 µg/mL
(Table 1). In most of the cases, higher activity was exhibited by the copper complexes than
the ligands. Compounds 13b, 13h, 13o and 13r inhibited mycobacterial growth very
effectively compared to other tested compounds with the MIC values ranging from 0.8 and
1
.6 µg/mL.
By considering in vitro antimycobacterial studies, we have selected ten compounds for in
vitro enzyme inhibition activity against InhA from M. tuberculosis at 50 µM (and/or at 5 µM)
by utilizing the commonly used approach. [42] (Table 7). Triclosan was used as reference and
showed a complete inhibition of InhA enzyme at 50 µM.
th
Ligand 12r having fluorine at the 4 position exhibited 100% binding with the enzyme with
IC at 7.7 µM. The corresponding copper complex (13r) showed the same activity profile
5
0
with IC at 2.4 µM. This increase in biological activity is due to high electro-negativity and
5
0
th
small size of the substituent on the 4 position of anilide ring. The molecule 12b also showed
remarkable value of 35% binding and its copper complex (13b) had extraordinary binding
affinity with the enzyme viz., 100% at 50 µM (IC = 2.4 µM). The molecule 12o and its
50
copper complex 13o also passes the notable binding affinity with enzyme.
.12. Cytotoxicity
3
Among the 10 compounds utilized for enzyme assay, seven most promising compounds were
taken forward for cytotoxicity study against the human lung cancer cell-line A549 (Table 8).
It was concluded from cytotoxic activity that the most active ligand molecules are very much
safe towards human lung cell-line. Compounds 12b, 12o and 12r showed IC that are almost
50
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overlapping with the standard compound (INH). The cytotoxic activity of copper complexes
is in the acceptable range of 281-305 µM concentration.
3
.13. Structure-activity relationship
From the results of CoMFA, CoMSIA, and docking analysis, it was concluded that all those
th
molecules having electronegative substitution on the 4 position of anilide ring might have
favored the biological activity, as in case of ligands containing 4-F, 4-Br or 4-Cl that have
shown the best anti-TB activities along with a high binding interaction with the mutant ENR
enzyme. Compounds 12b and 12r (MIC=3.12 µg/mL) exhibited the best docking pose with
the mutant ENR enzyme having total docking scores of 7.96 and 8.01, respectively. The
copper complexes of these ligands have shown even better scores with the mutant ENR
enzyme. Copper complexes 13b and 13r (MIC 0.8 µg/mL) have shown the total docking
scores of 11.96 and 12.71 against the mutant ENR. Ligands 12h, 12k and 12o having 4-F, 4-
Br and 4-Cl substitutions have shown the MIC values of 6.25, 6.25 and 3.12 µg/mL
respectively. All these observation suggest that copper complexes of these ligands have a
much better biological activity; 13h, 13k and 13o have the MIC value of 1.60, 3.12 and 1.60
µg/ml, respectively. Requirement of different substitution pattern on the ligand molecule to
get the desirable biological activity are depicted in Fig 6.
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4
. EXPERIMENTAL SECTION
Kinetic studies were performed on a Cary Bio 100. All chemicals were obtained from Aldrich
and used without further purification. Melting points were determined using Shital-digital
programmable apparatus and are uncorrected. FTIR spectra in KBr pellets were recorded on a
1
13
Bruker FTIR spectrophotometer. The H and C NMR spectra were recorded on a Bruker
AVANCE II at 400 and 100/75 MHz, respectively; chemical shifts are expressed in parts per
million (ppm) relative to TMS. The abbreviations used to describe the peak patterns are: (b)
broad, (s) singlet, (d) doublet, (t) triplet, (q) quartet, and (m) multiplet. Since copper (II)
complexes are paramagnetic in nature hence, their NMR spectra could not be obtained.
Mass spectra (MS) were recorded on a JEOL GCMATE II GC-Mass spectrometer and
Schimadzu QP 20105 GC-Mass spectrometer. Elemental analysis data (performed on Leco
Tru Spec CHNS Analyzer) for C, H, and N fall within 0.4% of the theoretical data. Thin-
layer chromatography (TLC) was performed on pre-coated TLC sheets of silica gel 60 F254
(Merck, Darmstadt, Germany) visualized by long- and short-wavelength UV lamps.
Chromatographic purifications were done on Merck aluminum oxide (70-230 mesh) and
Merck silica gel (70-230 mesh). UV-spectra were recorded on a Shimadzu
spectrophotometer. Electrochemical studies were carried out on a CHI-1103a electrochemical
Analyzer (CH Instruments Ltd. Co., USA, version 12.01) electrode system involving a GCE
(3 mm diameter) as the working electrode, a platinum wire as a counter electrode and an
Ag/AgCl as reference electrode, standardized for the redox couple ferricinium/ferrocene
(
E =+0.400 V, ꢀE =60 mV).
1/2 p
4
.1. General procedure for the synthesis of 3-oxo-N-substituted phenylpentamides or 2-oxo-
N- substituted phenylpropamides 6(a-y)
Compounds [6(a-f), 6(k-p), 6v, 6w, 6y] were prepared by heating a mixture of levulinic acid
(1 mol) or pyruvic acid 2 (1 mol) and thionyl chloride (2.5 mol) at 70 ºC for 30 min. The
1
17

ACCEPTED MANUSCRIPT
reaction was monitored using TLC. After cooling the reaction mixture, excess of thionyl
chloride was distilled off to get compounds 2-oxopropanoyl chloride 3 and 4-oxopentanoyl
chloride 4. To this crude product, a solution of substituted anilines in diethyl ether was added
and continued heating for another 30 min. and the reaction was monitored using TLC. After
cooling to ambient temperature, the separated solid was filtered, washed with diethyl ether
and acetonitrile, and purified by column chromatography on silica gel with a mixture of
chloroform: ethyl acetate (6:4).
For preparation of compounds [6(g-j), 6(q-t), 6u, 6x], compounds 1 and 2 (0.0019 mol) and
different anilines (0.0018 mol) were dissolved in dry DMF solvent (58 mL). HBTU (0.87g,
o
0
.0023 mol) and DIEA (0.93 mL, 0.0053 mol) were added and stirred for 42-48 h at 23 C.
The reaction was monitored using TLC, and after completion of the reaction, mixture was
poured onto distilled water. The resulting mixture was extracted with a mixture of
ethylacetate:methanol (9:1). The organic layer was dried over anhydrous sodium sulphate.
The residue left out after concentrating the organic layer in vacuum was purified by column
chromatography on silica gel with a mixture of chloroform: ethyl acetate (6:4).
4
.2.1. Synthesis of 4-(1H-pyrrol-1-yl)benzoic acid hydrazide 10
Ethyl 4-(1H-pyrrol-1-yl)benzoate 8 (15 mmol) was refluxed with hydrazine hydrate (10 mL)
in absolute ethanol (10 mL) for 3 h. The reaction mixture was cooled and the crystalline mass
obtained was further recrystallized from ethanol [45].
4
.2.2. Synthesis of 4-(2,5-dimethylpyrrol-1-yl)benzoic acid hydrazide 11
Compound 11 was synthesized by refluxing a mixture of ethyl 4-(2,5-dimethylpyrrol-1-
yl)benzoate 9 (3.64 g, 15 mmol) with hydrazine hydrate (10 mL) in absolute ethanol (10 mL)
for 3 h (monitored by TLC). The cooled mixture was poured gradually onto the crushed ice
with stirring. The mixture was allowed to stand, solid separated, filtered, and washed
18

ACCEPTED MANUSCRIPT
thoroughly with cold water, then dried and recrystallized from ethanol (yield 80%). mp 170-
0
1
72 C [45].
4
.2.General procedure for synthesis of pyrrolyl hydrazones (12a-y)
An ethanolic solution of N-substituted phenylalkylamides 6(a-y) (0.005 mol) was added to a
hot ethanolic solution of 4-(1H-pyrrol-1-yl)benzoic acid hydrazide 10 (0.005 mol) or 4-(2,5-
dimethylpyrrol-1-yl)benzoic acid hydrazide 11 (0.005 mol) in the presence of catalytic
amount of acetic acid. The mixture was heated under reflux for 72-78 h and cooled to
ambient temperature. The crude product was filtered, washed with ethanol, dried and purified
using column chromatography on silica gel with a mixture of chloroform:ethyl acetate (6:4)
to afford the final compounds 12(a-y).
4
.3.1. N-(3-Bromophenyl)-2-{2-[4-(1H-pyrrol-1-yl)benzoyl]hydrazinylidene}propanamide
12a).
(
-1
(
Yield 68%). mp 230-232 °C; FTIR (KBr) cm : 3255 (2° amine), 2922 (Ar-H), 1693, 1666
1
(
C=O), 1585 (C=N); H NMR (400 MHz, DMSO) δ ppm: 2.24 (s, 3H, -CH ), 6.30 (t, 2H,
3
pyrrole-C , C -H), 7.18-7.36 (m, 5H, pyrrole-C , C -H and bromophenyl-C , C , C -H), 7.64
3
4
2
5
4
5
6
(
t, 3H, bridging phenyl-C C and bromophenyl-C -H), 8.01 (d, 2H, bridging phenyl-C C -
2, 6 2 3, 5
1
3
H), 10.39 (s, 1H, hydrazinylidene-NH), 11.07 (s, 1H, propanamide-NH); C NMR (75 MHz,
DMSO) δ ppm: 11.5, 111.1, 118.3, 118.4, 118.6, 121.5, 122.2, 126, 129.1, 130, 130.1, 139.7,
+
+
1
42.6, 162.5; MS (ESI): m/z found 425.00, 426.14 [M M (1:1)]; calcd. 425.28. Anal.
,
Calcd. For C H N O Br: C, 56.48; H, 4.03; N, 13.17. Found: C, 56.52; H, 4.00; N, 13.10;
2
0
17
4
2
UV-Visible λmax: 320 nm.
.3.2. N-(4-Bromophenyl)-2-{2-[4-(1H-pyrrol-1-yl)benzoyl]hydrazinylidene}propanamide
12b)
4
(
-1
Yield 72%; mp 240-242°C; FTIR (KBr) cm : 3247 (2° amine), 3102, (Ar-H), 1663, 1603
1
(
C=O), 1591 (C=N); H NMR (400 MHz, DMSO) δ ppm: 2.28 (s, 3H, -CH ), 6.45 (t, 2H,
3
19

ACCEPTED MANUSCRIPT
pyrrole-C , C -H), 7.21 (t, 2H, pyrrole-C , C -H), 7.49 (t, 2H, bromophenyl-C , C -H), 7.55-
3
4
2
5
3
5
7
.58 (m, 4H, bromophenyl-C , C -H and bridging phenyl-C , C -H), 7.96-7.98 (dd, 2H,
2 6 2 6
bridging phenyl-C , C -H), 9.0 (s, 1H, hydrazinylidene-NH), 10.09 (s, 1H, propanamide-
3
5
1
3
NH); C NMR (75 MHz, DMSO) δ ppm: 11.5, 118.3, 118.4, 121.5, 126, 129.1, 130.1, 139.7,
+
+2
1
42.6, 162.5; MS (ESI): m/z found 425.26, 427.14 [M , M (1:1)]; calcd. 425.28. Anal.
Calcd. For C H N O Br: C, 56.48; H, 4.03; N, 13.17. Found: C, 55.98; H, 4.00; N, 13.15;
2
0
17
4
2
UV-Visible λmax: 325 nm.
4
.3.3. N-(2,6-Dichlorophenyl)-2-{2-[4-(1H-pyrrol-1-yl)benzoyl]hydrazinylidene}
propanamide (12c)
-1
Yield 57%; mp 211-213°C; FTIR (KBr) cm : 3287 (2° amine), 2923 (Ar-H), 1670, 1604
1
(
C=O) 1579 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.98 (s, 3H, propanamide-H), 6.29
(
t, 2H, pyrrole C , C -H) 7.39 (t, 3H, pyrrole-C C -H and chlorophenyl-C -H), 7.62 (dd, 2H,
3
4
2,
5
4
chlorophenyl-C , C -H), 7.94 (dd, 2H, bridging phenyl-C , C -H), 8.23 (s, 2H, bridging
3
5
2
6
1
3
phenyl-C , C -H), 9.80 (s, 1H, hydrazinylidene-NH), 11.01 (s, 1H, propanamide-NH); C
3
5
NMR (75 MHz, DMSO) δ ppm: 11.2, 111.5, 118.4, 118.7, 118.8, 121.2, 121.7, 126.4, 129.7,
+
1
30.8, 130.4, 139.1, 142.4, 162.8; MS (ESI): m/z found found 414.41, 416.24, 418.56 [M ,
+
2
+4
M , M (9:6:1)]; calcd. 414.07. Anal. Calcd. For C H N O Cl : C, 57.37; H, 3.84; N,
2
0
16
4
2
2
1
3.47. Found: C, 57.84; H, 3.88; N, 13.49; UV-Visible λmax: 267 nm.
.3.4. N-(3,5-Dichlorophenyl)-2-{2-[4-(1H-pyrrol-1-yl)benzoyl]hydrazinylidene}
propanamide (12d)
4
-1
Yield 62%; mp 183-185°C; FTIR (KBr) cm : 3235 (2° amine), 2904 (Ar-H), 1701, 1684
1
(
C=O), 1599 (C=N); H NMR (400 MHz, DMSO) δ ppm: 2.53 (m, 3H, propanamide-H),
6
.31 (t, 2H, pyrrole C , C -H) 7.41 (t, 3H, pyrrole-C C -H and chlorophenyl-C -H), 7.68 (d,
3 4 2, 5 4
2
H, chlorophenyl-C , C -H), 7.98 (d, 2H, bridging phenyl-C , C -H), 8.20 (s, 2H, bridging
2
6
2
6
1
3
phenyl-C , C -H), 9.80 (s, 1H, hydrazinylidene-NH), 11.01 (s, 1H, propanamide-NH); C
3
5
20

ACCEPTED MANUSCRIPT
NMR (75 MHz, DMSO) δ ppm: 11.5, 111.4, 118.2, 118.6, 118.7, 121.7, 121.1, 126.4, 129.2,
+
1
30.4, 130.7, 139.2, 142.4, 162.8; MS (ESI): m/z found 414.42 [M ]; calcd. 414.07. Anal.
Calcd. For C H N O Cl : C, 57.70; H, 3.86; N, 13.48.Found: C, 57.84; H, 3.88; N, 13.49;
2
0
16
4
2
2
UV-Visible λmax: 314 nm.
.3.5. N'-(1-(4-Methoxyphenylcarbamoyl)ethylidene)-4-(1H-pyrrol-1-yl)benzohydrazide
12e)
4
(
-1
Yield 80%; mp 108-112 °C; FTIR (KBr) cm : 3210 (2° amine), 2930 (Ar-H), 1680, 1653
1
(
C=O), 1588 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.19 (s, 3H, -CH ), 3.63 (s, 3H, -
3
OCH ), 6.39 (t, 2H, pyrrole-C , C -H), 7.23 (m, 4H, pyrrole C , C -H and methoxyphenyl-C
3,
3
3
4
2
5
C -H), 7.60 (d, 2H, methoxyphenyl-C C -H), 7.73-7.76 (m, 2H, bridging phenyl C , C -H),
5
2,
6
2
6
7
.80-7.89 (m, 2H, bridging phenyl C , C -H), 9.12 (s, 1H, hydrazinylidene-NH), 10.00 (s, 1H,
3 5
1
3
propanamide-NH); C NMR (75 MHz, DMSO) δ ppm: 11.2, 50.1, 111.3, 118.6, 118.1,
1
18.5, 121.6, 121.1, 126.3, 129, 130.6, 130.4, 139, 142.3, 153, 156, 162.5; MS (ESI): m/z
+
found 376.86 [M ]; calcd. 376.15. Anal. Calcd. For C H N O : C, 67.01; H, 5.36; N, 14.88.
2
1
20
4
3
Found: C, 67.10; H, 5.33; N, 14.86; UV-Visible λmax: 283 nm.
.3.6. N'-(1-(3-Methoxyphenylcarbamoyl)ethylidene)-4-(1H-pyrrol-1-yl)benzohydrazide
12f)
4
(
-1
Yield 68%; mp 98-100 °C; FTIR (KBr) cm : 3248 (2° amine), 2937 (Ar-H), 1688, 1650
1
(
C=O), 1590 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.99 (s, 3H, -CH ), 3.66 (s, 3H, -
3
OCH ), 6.34 (t, 2H, pyrrole-C , C -H), 6.80 (t, 1H, methoxyphenyl-C -H), 7.20-7.38 (m, 5H,
3
3
4
4
pyrrole-C , C -H and methoxyphenyl-C C , C -H), 7.48-7.52 (m, 2H, bridging phenyl-C ,
2
5
2,
5
6
2
C -H), 7.64-7.70 (m, 2H, bridging phenyl-C , C -H), 9.10 (s, 1H, hydrazinylidene-NH),
6
4
5
+
1
0.03 (s, 1H, propanamide-NH); MS (ESI): m/z found 376.50 [M ]; calcd. 376.15. Anal.
Calcd. For C H N O : C, 67.01; H, 5.36; N, 14.88. Found: C, 67.17; H, 5.30; N, 14.89; UV-
2
1
20
4
3
Visible λmax: 317 nm.
21

ACCEPTED MANUSCRIPT
4
.3.7. N'-(1-(3-Fluorophenylcarbamoyl)ethylidene)-4-(1H-pyrrol-1-yl)benzohydrazide (12g)
-1
Yield 60%; mp 114-117°C; FTIR (KBr) cm : 3241 (2° amine), 2932 (Ar-H), 1696, 1670
1
(
C=O), 1601 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.84 (s, 3H, -CH ), 6.24 (t, 2H,
3
pyrrole-C , C -H), 7.18 (t, 1H, fluorophenyl-C -H), 7.26 (t, 2H, pyrrole-C , C -H), 7.35 (t,
3
4
4
2
5
1
H, fluoropheyl-C -H), 7.64 (t, 1H, fluoropheyl-C -H), 7.87 (t, 1H, fluorophenyl C , H),
5 6 2
7
.93-7.96 (m, 2H, bridging phenyl-C , C -H), 7.99 (d, 2H, bridging phenyl-C , C -H), 10.34
2
6
3
5
(s, 1H, hydrazinylidene-NH), 11.04 (s, 1H, propanamide-NH); MS (ESI): m/z found 364.04
+
[
M ]; calcd. 364.13. Anal. Calcd. For C H N O F: C, 65.93; H, 4.70; N, 15.38. Found: C,
2
0
17
4
2
6
4
5.70; H, 4.68; N, 15.31; UV-Visible λmax: 291 nm.
.3.8. N'-(1-(4-Fluorophenylcarbamoyl)ethylidene)-4-(1H-pyrrol-1-yl)benzohydrazide (12h)
-1
Yield 71%; mp 87-89°C; FTIR (KBr) cm : 3261 (2° amine), 2940 (Ar-H), 1680, 1674
1
(
C=O), 1610 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.86 (s, 3H, -CH ), 6.25 (t, 2H,
3
pyrrole-C , C -H), 7.18 (t, 2H, fluoropheyl-C C -H), 7.38 (t, 2H, pyrrole-C , C -H), 7.94-
3
4
3,
5
2
5
7
.98 (m, 4H, fluorophenyl-C , C -H and bridging phenyl-C , C -H), 8.21 (d, 2H, bridging
2 6 2 6
1
3
phenyl-C , C -H), 10.36 (s, 1H, hydrazinylidene-NH), 11.06 (s, 1H, propanamide-NH); C
3
5
NMR (75 MHz, DMSO) δ ppm: 11.5, 111.1, 118.3, 118.4, 118.6, 121.5, 122.2, 126, 129.1,
+
1
30, 130.1, 139.7, 142.6, 153.8, 162.5; MS (ESI): m/z found 364.65 [M ]; calcd. 364.13.
Anal. Calcd. For C H N O F: C, 65.93; H, 4.70; N, 15.38. Found: C, 65.72; H, 4.64; N,
2
0
17
4
2
1
5.34; UV-Visible λmax: 287 nm.
4
.3.9. N'-(1-(2-Fluorophenylcarbamoyl)ethylidene)-4-(1H-pyrrol-1-yl)benzohydrazide (12i)
-1
Yield 54%; mp 107-109°C; FTIR (KBr) cm : 3345 (2° amine), 2934 (Ar-H), 1694, 1677
1
(
C=O); H NMR (400 MHz, DMSO) δ ppm: 1.81 (s, 3H, -CH ), 6.20 (t, 2H, pyrrole C , C ,
3
3
4
H), 7.10 (t, 1H, fluorophenyl-C -H), 7.33 (m, 4H, fluorophenyl-C C and pyrrole-C , C -H),
4
3,
5
2
5
7
.84 (d, 2H, bridging phenyl-C , C -H), 7.90 (t, 1H, fluorophenyl-C -H), 7.96 (d, 2H,
2 6 6
bridging phenyl-C , C -H), 10.32 (s, 1H, hydrazinylidene-NH), 11.00 (s, 1H, propanamide-
3
5
22

ACCEPTED MANUSCRIPT
+
NH); MS (ESI): m/z found 364.36 [M ]; calcd. 364.13. Anal. Calcd. For C H N O F: C,
20
17
4
2
6
5.93; H, 4.70; N, 15.38. Found: C, 65.80; H, 4.71; N, 15.36; UV-Visible λmax: 331 nm.
.3.10. N-(3,5-Dibromophenyl)-2-{2-[4-(1H-pyrrol-1-yl)benzoyl]hydrazinylidene}
propanamide (12j)
4
-1
Yield 54%; mp 170-174°C; FTIR (KBr) cm : 3245 (2° amine), 2900 (Ar-H), 1714, 1680
1
(
C=O), 1579 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.84 (s, 3H, -CH ), 6.20 (t, 2H,
3
pyrrole-C , C -H), 6.91 (t, 2H, pyrrole-C , C -H), 7.54-7.60 (m, 5H, bridging phenyl-C , C -
3
4
2
5
2
6
H and bromophenyl-C , C , C -H), 7.84 (d, 2H, bridging phenyl-C , C -H), 7.94 (d, 2H,
2
4
6
2
6
bridging phenyl-C , C -H), 9.10 (s, 1H, hydrazinylidene -NH), 11.12 (s, 1H, propanamide -
3
5
1
3
NH); C NMR (75 MHz, DMSO) δ ppm: 11.5, 111.4, 118.2, 118.6, 118.7, 121.7, 121.1,
1
5
3
4
26.4, 129.2, 130.4, 130.7, 139.2, 142.4, 153.5, 162.8; MS (ESI): m/z found 504.14, 506.12,
+
+2
+4
08.24 [M , M , M (1:2:1)]; calcd. 504.17. Anal. Calcd. For C H N O Br : C, 47.64; H,
2
0
16
4
2
2
.20; N, 11.11.Found: C, 47.60; H, 3.22; N, 11.10; UV-Visible λmax: 279 nm.
.3.11. N-(4-Bromophenyl)-4-{2-[4-(1H-pyrrol-1-yl)benzoyl]hydrazinylidene}pentanamide
12k)
(
-1
Yield 74%; mp 242-246°C; FTIR (KBr) cm : 3278 (2° amine), 2923 (Ar-H), 1684, 1644
1
(
C=O), 1610 (C=O); H NMR (400 MHz, DMSO) δ ppm: 1.24 (s, 3H, -CH ), 2.53 (t, 4H,
3
pentamide-H), 6.28-6.30 (m, 2H, pyrrole-C , C -H), 7.36 (d, 2H, pyrrole-C , C -H), 7.60-7.66
3
4
2
5
(
q, 4H, bromophenyl-C C C , C -H), 7.94 (d, 2H, bridging phenyl-C , C -H), 8.01-8.19 (m,
2, 3, 5 6 2 6
2
H, bridging phenyl-C , C -H), 9.79 (s, 1H, hydrazinylidene -NH), 10.05 (s, 1H,
3 5
1
3
pentanamide-NH); C NMR (75 MHz, DMSO) δ ppm: 11.5, 30.1, 110.9, 111, 118.4, 118.7,
+
1
18.8, 128.5, 129.1, 129.5, 141.7, 159.6, 165.1; MS (ESI): m/z found 452.02, 454.17 [M ,
+
2
M
(1:1)]; calcd. 452.08. Anal. Calcd. For C H N O Br: C, 58.29; H, 4.67; N, 12.36.
22 21 4 2
Found: C, 57.97; H, 4.62; N, 12.34; UV-Visible λmax: 330 nm.
23

ACCEPTED MANUSCRIPT
4
.3.12. N-(3-Bromophenyl)-4-{2-[4-(1H-pyrrol-1-yl)benzoyl]hydrazinylidene}pentanamide
(12l)
-1
Yield 72%; mp 234-236°C; FTIR (KBr) cm : 3241 (2° amine), 2924 (Ar-H), 1693, 1660
1
(
C=O), 1591 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.30 (s, 3H, -CH ), 2.54-2.55 (m,
3
4
H, pentamide-H), 6.28-6.31 (m, 2H, pyrrole-C , C -H), 7.28-7.32 (m, 5H, pyrrole-C ,C -H
3 4 2 5
and bromophenyl-C , C -H), 7.53-7.59 (m, 1H, bromophenyl-C -H), 7.93-8.19 (m, 5H,
5
6
4
bridging phenyl-C C , C , C , H and bromophenyl C -H), 9.78 (s, 1H, hydrazinylidene-NH),
2
,
3
5
6
2
1
3
9
.99 (s, 1H, pentanamide-NH); C NMR (75 MHz, DMSO) δ ppm: 20, 30.8, 110.9, 111,
+
+2
1
18.3, 118.4, 118.7, 128.5, 129.1, 141.7; MS (ESI): m/z found 452.18, 454.14 [M , M
(
1:1)]; calcd. 452.08. Anal. Calcd. For C H N O Br: C, 58.14; H, 4.64; N, 12.30. Found: C,
22 21 4 2
5
4
8.29; H, 4.67; N, 12.36; UV-Visible λmax: 295 nm.
.3.13. N-(2-Chlorophenyl)-4-{2-[4-(1H-pyrrol-1-yl)benzoyl]hydrazinylidene}pentanamide
(12m)
-1
Yield 68%; mp 244-248°C; FTIR (KBr) cm : 3241 (2° amine), 2923 (Ar-H), 1694, 1644
1
(
C=O), 1588 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.94 (s, 3H, -CH ), 2.50-2.52 (m,
3
4
H, pentamide-H), 6.28-6.30 (m, 2H, pyrrole C , C , H), 7.40-7.44 (m, 4H, chlorophenyl-C ,
3 4 4
C -H and pyrrole-C ,C -H), 7.62-7.71 (m, 1H, chlorophenyl-C -H), 7.91-7.98 (q, 2H,
5
2
5
3
bridging phenyl-C C -H), 8.25 (s, 3H, chlorophenyl-C -H and bridging phenyl-C , C -H),
2
,
6
6
3
5
9
4
5
4
.88 (s, 1H, hydrazinylidene-NH), 10.30 (s, 1H, pentanamide-NH); MS (ESI): m/z found
+
+2
08.06, 410.14 [M , M (3:1)]; calcd. 408.14. Anal. Calcd. For C H N O Cl: C, 64.62; H,
2
2
21
4
2
.18; N, 13.70. Found: C, 64.47; H, 5.16; N, 13.72; UV-Visible λmax: 280 nm.
.3.14. N-(3-Chlorophenyl)-4-{2-[4-(1H-pyrrol-1-yl)benzoyl]hydrazinylidene}pentanamide
(12n)
-1
Yield 67%; mp 249-251°C; FTIR (KBr) cm : 3245 (2° amine), 2924 (Ar-H), 1692, 1644
1
(
C=O), 1590 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.94 (s, 3H, -CH ), 2.51-2.52 (m,
3
24

ACCEPTED MANUSCRIPT
4
H, pentamide-H), 6.29-6.31 (m, 2H, pyrrole-C , C -H), 7.41-7.45 (m, 3H, pyrrole-C ,C and
3
4
2
5
chlorophenyl-C -H), 7.65-8.23 (m, 7H, chlorophenyl-C , C , C -H and bridging phenyl-C ,
4
2
5
6
2
C , C , C -H), 9.88 (s, 1H, hydrazinylidene-NH), 10.30 (s, 1H, pentanamide-NH); MS (ESI):
3
5
6
+
+2
m/z found 408.14, 410.18 [M , M (3:1)]; calcd. 408.14. Anal. Calcd. For C H N O Cl: C,
2
2
21
4
2
6
4.62; H, 5.18; N, 13.70. Found: C, 64.39; H, 5.14; N, 13.71; UV-Visible λmax: 310 nm.
4
.3.15. N-(4-Chlorophenyl)-4-{2-[4-(1H-pyrrol-1-yl)benzoyl]hydrazinylidene}pentanamide
(12o)
-1
Yield 67%; mp 250-252°C; FTIR (KBr) cm : 3234 (2° amine), 3035 (Ar-H), 1693, 1644
1
(
C=O), 1590 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.98 (s, 3H, -CH ), 2.51-2.56 (m,
3
4
H, pentamide-H), 6.29 (t, 2H, pyrrole-C , C -H), 7.43 (t, 2H, pyrrole-C , C -H), 7.72 (dd,
3 4 2 5
2
H, chlorophenyl-C C -H), 7.96-8.11 (m, 4H, chlorophenyl-C , C and bridging phenyl-C ,
3
,
5
2
6
2
C -H), 8.23 (s, 2H, bridging phenyl-C , C -H), 9.94 (s, 1H, hydrazinylidene-NH), 10.40 (s,
6
3
5
+
+2
1
H, pentanamide-NH); MS (ESI): m/z found 408.47, 410.25 [M , M (3:1)]; calcd. 408.14.
Anal. Calcd. For C H N O Cl: C, 64.62; H, 5.18; N, 13.70. Found: C, 64.67; H, 5.15; N,
2
2
21
4
2
1
3.72; UV-Visible λmax: 287 nm.
4
.3.16. N-(3,5-Dichlorophenyl)-4-{2-[4-(1H-pyrrol-1-yl)benzoyl]hydrazinylidene}
pentanamide (12p)
-1
Yield 58%; mp 201-203°C; FTIR (KBr) cm : 3325 (2° amine), 3015 (Ar-H), 1697, 1667
1
(
C=O), 1618 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.98 (s, 3H, -CH ), 2.51-2.56 (m,
3
4
H, pentamide-H), 6.30 (t, 2H, pyrrole-C , C -H), 7.20 (t, 2H, pyrrole-C , C -H), 7.41 (t, 1H,
3 4 2 5
chlorophenyl-C -H), 7.72 (m, 2H, chlorophenyl-C , C and bridging phenyl-C , C -H), 7.96
4
2
6
2
6
(
d, 2H, bridging phenyl-C , C -H), 9.88 (s, 1H, hydrazinylidene-NH), 10.54 (s, 1H,
3 5
+1
pentanamide-NH); MS (ESI): m/z found 444 [M ]; calcd. 443.33. Anal. Calcd. For
C H N O Cl : C, 59.60; H, 4.55; N, 12.64. Found: C, 60.67; H, 5.15; N, 13.72; UV-Visible
22
20
4
2
2
λmax: 286 nm.
25

ACCEPTED MANUSCRIPT
4
.3.17. N'-(4-(2-Methoxyphenylcarbamoyl)butan-2-ylidene)-4-(1H-pyrrol-1-yl)
benzohydrazide (12q)
-1
Yield 61%; mp 88-90°C; FTIR (KBr) cm : 3314 (2° amine), 3014 (Ar-H), 1702, 1687
1
(
C=O), 1608 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1. 84 (s, 3H, -CH ), 2.02-2.08 (m,
3
4
H, pentamide-H), 3.22 (s, 3H, -OCH ), 6.29 (t, 2H, pyrrole-C , C -H), 6.89-7.22 (m, 5H,
3 3 4
methoxyphenyl-C , C , C and pyrrole-C , C -H), 7.72 (m, 3H, methoxyphenyl-C and
3
4
5
2
5
6
bridging phenyl-C , C -H), 8.11 (d, 2H, bridging phenyl-C , C -H), 9.69 (s, 1H,
2
6
3
5
hydrazinylidene-NH), 10.49 (s, 1H, pentanamide-NH); UV-Visible λmax: 284 nm.
.3.18. N'-(4-(4-Fluorophenylcarbamoyl)butan-2-ylidene)-4-(1H-pyrrol-1-yl)benzohydrazide
12r)
4
(
-1
Yield 71%; mp 140-142°C; FTIR (KBr) cm : 3274 (2° amine), 2924 (Ar-H), 1701, 1687
1
(
C=O), 1577 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.98 (s, 3H, -CH ), 2.51-2.56 (m,
3
4
H, pentamide-H), 6.29 (t, 2H, pyrrole-C , C -H), 7.28-7.32 (m, 4H, pyrrole-C , C and
3 4 2 5
fluorophenyl-C , C -H), 7.72 (d, 2H, fluorophenyl-C C -H), 7.96-8.01 (m, 2H, bridging
3
5
2,
6
phenyl-C , C -H), 8.23 (s, 2H, bridging phenyl-C , C -H), 9.88 (s, 1H, hydrazinylidene-NH),
2
6
3
5
+
1
0.54 (s, 1H, pentanamide-NH); MS (ESI): m/z found 392.64 [M ]; calcd. 392.16 Anal.
Calcd. For C H N O F: C, 67.37; H, 5.35; N, 14.24. Found: C, 67.27; H, 5.37; N, 14.26;
2
2
21
4
2
UV-Visible λmax: 282 nm.
.3.19. N'-(4-(3-Fluorophenylcarbamoyl)butan-2-ylidene)-4-(1H-pyrrol-1-yl)benzohydrazide
12s)
4
(
-1
Yield 64%; mp 120-122°C; FTIR (KBr) cm : 3241 (2° amine), 2942 (Ar-H), 1707, 1685
1
(
C=O), 1579 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.74 (s, 3H, -CH ), 2.63-2.69 (m,
3
4
H, pentamide-H), 6.35 (t, 2H, pyrrole-C , C -H), 7.38 (t, 3H, pyrrole-C , C and fluoro
3 4 2 5
phenyl-C -H), 7.47 (t, 2H, fluorophenyl-C C -H), 7.73 (t, 3H, flurophenyl-C and bridging
4
5,
6
2
phenyl-C , C -H), 8.07 (t, 2H, bridging phenyl-C , C -H), 10.43 (s, 1H, hydrazinylidene-NH),
2
6
3
5
26

ACCEPTED MANUSCRIPT
+
1
1.15 (s, 1H, pentanamide-NH); MS (ESI): m/z found 392.64 [M ]; calcd. 392.16 Anal.
Calcd. For C H N O F: C, 67.37; H, 5.35; N, 14.24. Found: C, 67.27; H, 5.37; N, 14.26;
2
2
21
4
2
UV-Visible λmax: 285 nm.
.3.20. N'-(4-(2-Fluorophenylcarbamoyl)butan-2-ylidene)-4-(1H-pyrrol-1-yl)benzohydrazide
12t)
4
(
-1
Yield 52%; mp 89-91°C; FTIR (KBr) cm : 3285 (2° amine), 2961 (Ar-H), 1692, 1670
1
(
C=O), 1611 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.78 (s, 3H, -CH ), 2.34 (m, 4H,
3
pentamide-H), 6.31 (t, 2H, pyrrole-C , C -H), 6.90-7.26 (m, 5H, pyrrole-C , C -H and
3
4
2
5
fluorophenyl-C , C , C -H), 7.82 (t, 2H, bridging phenyl-C , C -H), 8.00 (t, 1H, flurophenyl-
3
4
5
2
6
C -H), 8.12 (d, 2H, bridging phenyl-C , C -H), 10.31 (s, 1H, hydrazinylidene-NH), 11.04 (s,
6
3
5
+
1
H, pentanamide-NH); MS (ESI): m/z found 392.60 [M ]; calcd. 392.16 Anal. Calcd. For
C H N O F: C, 67.40; H, 5.36; N, 14.25. Found: C, 67.27; H, 5.37; N, 14.26; UV-Visible
22
21
4
2
λmax: 275 nm.
.3.21. N'-(4-(phenylcarbamoyl)butan-2-ylidene)-4-(1H-pyrrol-1-yl)benzohydrazide (12u)
4
-1
Yield 78%; mp 93-95 °C; FTIR (KBr) cm : 3276 (2° amine), 2940 (Ar-H), 1680, 1674
1
(
C=O), 1579 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.72 (s, 3H, -CH ), 2.53-2.54 (m,
3
4
H, pentamide-H), 6.24 (t, 2H, pyrrole C , C4, H), 7.20 (t, 3H, pyrrole-C , C and phenyl-C -
3 2 5 4
H), 7.34 (d, 2H, phenyl-C , C -H), 7.48 (d, 2H, phenyl-C , C -H), 7.50 (t, 2H, bridging
3
5
2
6
phenyl-C C -H), 7.71 (t, 2H, bridging phenyl-C C -H), 10.25 (s, 1H, hydrazinylidene-NH),
2
,
6
3,
5
+
1
0.51 (s, 1H, pentanamide-NH); MS (ESI): m/z found 374.47 [M ]; calcd. 374.17 Anal.
Calcd. For C H N O : C, 70.57; H, 5.92; N, 14.96;. Found: C, 70.50; H, 5.90; N, 14.94;
2
2
22
4
2
UV-Visible λmax: 327 nm.
4
.3.22. N'-(1-(4-Bromophenylcarbamoyl)ethylidene)-4-(2,5-dimethyl-1H-pyrrol-1-yl)
benzohydrazide (12v)
27

ACCEPTED MANUSCRIPT
-1
Yield 70%; mp 128-130°C; FTIR (KBr) cm : 3274 (2° amine), 3074 (Ar-H), 1692, 1665
1
(
C=O), 1590 (C=N); H NMR (400 MHz, DMSO) δ ppm: 2.10 (s, 6H, 2-CH ), 2.31 (s, 3H, -
3
CH ), 5.98 (s, 2H, pyrrole-C , C -H), 7.28-8.00 (m, 8H, bridging phenyl-C , C C , C and
3
3
4
2
3,
5
6
bromophenyl- C , C C , C -H), 9.00 (s, 1H, hydrazinylidene-NH), 9.42 (s, 1H,
2
3,
5
6
1
3
propanamide-NH); C NMR (75 MHz, DMSO) δ ppm: 12.7, 22.7, 106.8, 114.1. 116.9,
+
+2
1
21.6, 128.5, 131.9, 135.3, 143, 160, 175; MS (ESI): m/z found 437.23, 439.35 [M , M
(
1:1)]; calcd. 437.07. Anal. Calcd. For C H BrN O : C, 60.28; H, 4.60; N, 9.59; Found: C,
2
2
20
3
2
6
4
0.24; H, 4.64; N, 9.57; UV-Visible λmax: 320 nm.
.3.23. N-(2,6-Dichlorophenyl)-2-{2-[4-(2,5-dimethyl-1H-pyrrol-1yl)benzoyl]
hydrazinylidene}propanamide (12w)
-1
Yield 61%; mp 180-182°C; FTIR (KBr) cm : 3254 (2° amine), 2923 (Ar-H), 1662, 1646
1
(
C=O), 1594 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.99 (s, 6H, 2-CH ), 2.53 (t, 3H, -
3
CH ), 5.80 (s, 2H, pyrrole C , C , H), 6.70 (s, 1H, chlorophenyl-C -H), 7.01 (d, 2H,
3
3
4
4
chlorophenyl-C , C -H), 7.30 (d, 2H, bridging phenyl-C , C -H), 7.97 (d, 2H, bridging
3
5
2
6
phenyl-C , C -H), 9.88 (s, 1H, hydrazinylidene-NH), 10.28 (s, 1H, propanamide-NH); MS
3
5
+
+2
+4
(
ESI): m/z found 442.01, 444.15, 446.25 [M , M , M (9:6:1)]; calcd. 442.21. Anal. Calcd.
For C H N O Cl : C, 59.51; H, 4.54; N, 12.65. Found: C, 59.60; H, 4.55; N, 12.64; UV-
2
2
20
4
2
2
Visible λmax: 254 nm.
4
.3.24. N'-(1-(4-Methoxyphenylcarbamoyl)ethylidene)-4-(2,5-dimethyl-1H-pyrrol-1-yl)
benzohydrazide (12x)
-1
Yield 72%; mp 94-96°C; FTIR (KBr) cm : 3281 (2° amine), 2953 (Ar-H), 1692, 1666
1
(
C=O), 1608 (C=N); H NMR (400 MHz, DMSO) δ ppm: 1.19 (s, 6H, -CH ), 2.53 (s, 3H, -
3
CH ), 3.63 (s, 3H, -OCH ), 5.82 (t, 2H, pyrrole-C , C -H), 7.23 (t, 2H, methoxyphenyl-C ,
3
3
3
4
3
C -H), 7.40 (t, 2H, methoxyphenyl-C C -H), 7.53-7.56 (m, 2H, bridging phenyl-C , C -H),
5
2,
6
2
6
7
.70-7.79 (m, 2H, bridging phenyl-C , C -H), 9.12 (s, 1H, hydrazinylidene-NH), 10.00 (s,
3 5
28