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[19] Pagani I, Liolios K, Jansson J, Chen IMA, Smirnova T, Nosrat B, et al. The genomes OnLine database (GOLD) v.4: Status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Research. 2012;**40**:D571-

*Computational Biology and Chemistry*

[20] Kolmogorov V. Blossom V: A new implementation of a minimum cost perfect matching algorithm. Mathematical Programming Computation. 2009;**1**:43-67

[21] Lu CL. An efficient algorithm for the contig ordering problem under algebraic rearrangement distance. Journal of Computational Biology. 2015;

[22] Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, et al. Versatile and open software for comparing large genomes. Genome Biology. 2004;**5**. Available from: https:// genomebiology.biomedcentral.com/ articles/10.1186/gb-2004-5-2-r12

[23] Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: Quality assessment

tool for genome assemblies. Bioinformatics. 2013;**29**:1072-1075

D579

**22**:975-987

**80**

**83**

**Chapter 5**

**Abstract**

**1. Introduction**

Biological Evaluation and

*Kodakkat Parambil Safna Hussan,* 

*and Karuvanthodi Muraleedharan*

Molecular Docking Studies of

*Mohamed Shahin Thayyil, Thaikadan Shameera Ahamed* 

The third-generation ionic liquids (ILs), which are being used to produce double active pharmaceutical ingredients (d-APIs) with tunable biological activity along with novel performance, enhancement, and delivery options, have been revolutionizing the area of drug discovery since the past few decades. Herein we report the in vitro antibacterial and anti-inflammatory activity of benzalkonium ibuprofenate (BaIb) that are being used as in-house d-API, with a particular focus on its interaction with respective protein target through molecular docking study. The evaluation of the biological activity of BaIb with the antibacterial and anti-inflammatory target at the molecular level revealed that the synthesized BaIb could be designed as a potential double active drug since it retains the antibacterial and anti-inflammatory activity of its parent drugs, benzalkonium chloride (BaCl) and sodium ibuprofenate (NaIb), respectively.

**Keywords:** benzalkonium ibuprofenate, double active pharmaceutical ingredient,

In the pharmaceutical field, ionic liquids (ILs) by salification of drugs were widely applied to improve the performance of drugs on its oral administration, especially, their solubility, bioavailability, and stability. The research on the antimicrobial activity of ILs is a growing field because of its unprecedented flexibility for chemical diversity in a severely drained arsenal of antimicrobial. The third-generation ionic liquids give us the freedom to tune the biological properties in addition to its physical and chemical properties. The proper selection of ions with synergetic effects may result in the formation of double active pharmaceutical ingredient (d-API). Thus the d-APIs are composed of asymmetric organic ions, which prevent the formation of the stable crystal lattice and are liquid at unusually low temperatures. Such d-APIs can be used for the ailment situations where the two activities are required. This strategy will reduce the excess in taking of unwanted chemicals and

Benzalkonium ibuprofenate (BaIb) is a double active pharmaceutical ingredient designed by combining benzalkonium cations with ibuprofen. Benzalkonium chloride

molecular docking studies, anti-inflammatory, antibacterial activities

will enhance the solubility and bioavailability [1, 2].

Benzalkonium Ibuprofenate

**Chapter 5**

## Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate

*Kodakkat Parambil Safna Hussan, Mohamed Shahin Thayyil, Thaikadan Shameera Ahamed and Karuvanthodi Muraleedharan*

### **Abstract**

The third-generation ionic liquids (ILs), which are being used to produce double active pharmaceutical ingredients (d-APIs) with tunable biological activity along with novel performance, enhancement, and delivery options, have been revolutionizing the area of drug discovery since the past few decades. Herein we report the in vitro antibacterial and anti-inflammatory activity of benzalkonium ibuprofenate (BaIb) that are being used as in-house d-API, with a particular focus on its interaction with respective protein target through molecular docking study. The evaluation of the biological activity of BaIb with the antibacterial and anti-inflammatory target at the molecular level revealed that the synthesized BaIb could be designed as a potential double active drug since it retains the antibacterial and anti-inflammatory activity of its parent drugs, benzalkonium chloride (BaCl) and sodium ibuprofenate (NaIb), respectively.

**Keywords:** benzalkonium ibuprofenate, double active pharmaceutical ingredient, molecular docking studies, anti-inflammatory, antibacterial activities

### **1. Introduction**

In the pharmaceutical field, ionic liquids (ILs) by salification of drugs were widely applied to improve the performance of drugs on its oral administration, especially, their solubility, bioavailability, and stability. The research on the antimicrobial activity of ILs is a growing field because of its unprecedented flexibility for chemical diversity in a severely drained arsenal of antimicrobial. The third-generation ionic liquids give us the freedom to tune the biological properties in addition to its physical and chemical properties. The proper selection of ions with synergetic effects may result in the formation of double active pharmaceutical ingredient (d-API). Thus the d-APIs are composed of asymmetric organic ions, which prevent the formation of the stable crystal lattice and are liquid at unusually low temperatures. Such d-APIs can be used for the ailment situations where the two activities are required. This strategy will reduce the excess in taking of unwanted chemicals and will enhance the solubility and bioavailability [1, 2].

Benzalkonium ibuprofenate (BaIb) is a double active pharmaceutical ingredient designed by combining benzalkonium cations with ibuprofen. Benzalkonium chloride (BaCl) is a potential antibacterial drug and sodium ibuprofen is a prospective antiinflammatory drug [3]. It is important to evaluate the pharmaceutical profiles of the d-API to confirm the retainity of the biological activities of the parent drugs. We have synthesized a d-API, benzalkonium ibuprofenate, carried out its quantum mechanical calculations using density functional theory, characterized different experimental techniques, and reported glass-forming ability in earlier works [4, 5]. Now, in this work, the biological evaluations, in particular, the antibacterial and anti-inflammatory activities, were performed and the results with the activity of parent drugs, BaCl and sodium ibuprofenate (NaIb), compared. The molecular docking of all the samples was done to get a better understanding of the mode of interaction between the drugs and respective targeted proteins and to trace their binding pores and cites.

### **2. Materials and method**

### **2.1 Materials**

The benzalkonium chloride and sodium ibuprofenate were purchased from Sigma-Aldrich (USA). Cell culture plastic flasks, test tubes, and culture plates were purchased from Borosil (India).

### **2.2 Experimental**

### *2.2.1 Synthesis*

The double active pharmaceutical ingredient, benzalkonium ibuprofenate, was synthesized using stoichiometric metathesis reaction. Solid (1 mmol) BaCl and NaIb were dissolved in 50 mL distilled water, each taken in two beakers and stirred separately with gentle heating (40–60°C). Then the two solutions were mixed together and again stirred for another 30 min with heating (around 80°C) and then cooled to room temperature. 60 mL of chloroform was added to separate the organic and inorganic part; then the chloroform phase was washed with cold distilled water until it removes the inorganic salt completely. AgNO3 test was used to confirm the absence of chloride anions in the product. This is followed by continuous washing of the chloroform phase with deionized (DI) water until the water washings tested negative for NaCl or NaBr via AgNO3 test. The chloroform was then evaporated using rotary evaporator, and the BaIb was dried under high vacuum for 12 h with gentle heating (50–60°C) [4, 6].

The double active pharmaceutical ingredient, BaIb was characterized well using Nuclear Magnetic Resonance using Bruker Avance III, 400MHzwith a 9.4 Tesla super-conducting magnet in an operating temperature at 309 K, Fourier transform infrared spectroscopy JASCO FTIR-4100 spectrophotometer, Fourier Transform-Raman spectroscopy and UV–visible spectroscopy using Jasco UV–Visible Spectrophotometer model V-550 (USA) and are reported in earlier works [4, 5].

### *2.2.2 Biological evaluation*

### *2.2.2.1 Antibacterial activity*

The synthesized double active pharmaceutical ingredient BaIb was screened against Gram-negative bacteria to confirm the retainity of the biological activity of its parent drug BaCl, which is a potential germicide. For this study, we have chosen *Pseudomonas aeruginosa* and *E. coli* as Gram-positive, while DMSO is considered as

**85**

*Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate*

inhibition, and the zone diameter was measured using a scale [4, 7]. *Formula for ABA*<sup>=</sup>*sample diameter* \_\_\_\_\_\_\_\_\_\_\_\_

%*inhibition* = *Abscontrol*− *Abstest* \_\_\_\_\_\_\_\_\_\_\_\_

was used for hemolysis at 560 nm and its percentage recorded [4, 8, 9].

of proteins were used for molecular docking studies.

Gram-negative. Paper disk method was used for the in vitro analysis. The experiment was started with the preparation of nutrient agar (28 g in 100 mL) and was sterilized by autoclaving. This nutrient agar media was cooled without solidifying and incubated overnight. *P. aeruginosa* and *E. coli* were prepared. 15–20 mL of this media was poured into sterilized Petri plate and allowed to solidify. Then sterilized disks were placed in this solidified agar plate; each plate contains three disks. 10 μL negative controls on to one disk, 10 μL standard parent drugs on to the second disk, and 10 μL samples on to the third disk were added and appropriately labeled. After incubating the Petri dishes in 310 K for 24 h, the plates were checked for the zone of

The anti-inflammatory activity of BaIb and NaIb was done using an egg albumin. For this, a reaction mixture of 5 mL was made with fresh hen's egg (0.2 mL) and phosphate buffer saline with pH = 6.4 of 2 mL with varying concentrations of extract for preparing the concentrations of 100, 200, 300, 400, and 500 μg/ mL. The same steps were repeated for the preparation of double distilled water, which served as control. Then the prepared mixtures were incubated in BOD incubator (Labline Technologies, India) at 210 ± 2 K for 15 min; after that, it is heated to 343 K and was hold for 5 min. The absorbance was measured after incubating using Shimadzu (Japan), UV 1800 at 660 nm. At the final concentration of 100, 200, 300, 400, and 500 μg/mL, acetyl salicylic acid was used as reference drug. The protein denaturation inhibition percentage was calculated using the following formulae:

*Abscontrol*

In addition, the synthesized drug BaIb was screened for its anti-inflammatory activity, and its efficiency with the parent drug NaIb and drug diclofenac was compared. For this, blood samples from a healthy donor (male) were collected and mixed with sterilized Alsever's solution before centrifuging it at 3000 rpm. The suspension of packed cells was made with isoline. This suspension with phosphate buffer, hyposaline, was mixed with diclofenac at varying concentration, where the distilled water is taken as control while diclofenac as standard. Then the mixtures were incubated for 30 min at 303 K and centrifuged. Spectrophotometric analysis

The input structures of BaCl (PubChem: 2330), NaIb (PubChem: 5338317), and BaIb (PubChem: 86612072) were taken from the PubChem database [9] and optimized using density functional theory with B3LYP level of theory and 631-G+(d,p) [10] basis sets using Gaussian software packages [11]. Further, the molecular docking was also conducted using Schrodinger Maestro software package [10]. The optimized structures and downloaded Protein Data Bank (PDB) files [12]

<sup>+</sup>*ve* <sup>c</sup>*ontrol* <sup>×</sup> 100 (1)

× 100 (2)

*DOI: http://dx.doi.org/10.5772/intechopen.90191*

*2.2.2.2 Anti-inflammatory activity*

*2.2.2.2.2 In human serum albumin*

**2.3 Computational**

*2.2.2.2.1 In chick albumin*

*Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate DOI: http://dx.doi.org/10.5772/intechopen.90191*

Gram-negative. Paper disk method was used for the in vitro analysis. The experiment was started with the preparation of nutrient agar (28 g in 100 mL) and was sterilized by autoclaving. This nutrient agar media was cooled without solidifying and incubated overnight. *P. aeruginosa* and *E. coli* were prepared. 15–20 mL of this media was poured into sterilized Petri plate and allowed to solidify. Then sterilized disks were placed in this solidified agar plate; each plate contains three disks. 10 μL negative controls on to one disk, 10 μL standard parent drugs on to the second disk, and 10 μL samples on to the third disk were added and appropriately labeled. After incubating the Petri dishes in 310 K for 24 h, the plates were checked for the zone of inhibition, and the zone diameter was measured using a scale [4, 7].

 *Formula for ABA*<sup>=</sup>*sample diameter* \_\_\_\_\_\_\_\_\_\_\_\_ <sup>+</sup>*ve* <sup>c</sup>*ontrol* <sup>×</sup> 100 (1)

### *2.2.2.2 Anti-inflammatory activity*

### *2.2.2.2.1 In chick albumin*

*Computational Biology and Chemistry*

**2. Materials and method**

purchased from Borosil (India).

12 h with gentle heating (50–60°C) [4, 6].

*2.2.2 Biological evaluation*

*2.2.2.1 Antibacterial activity*

**2.1 Materials**

**2.2 Experimental**

*2.2.1 Synthesis*

(BaCl) is a potential antibacterial drug and sodium ibuprofen is a prospective antiinflammatory drug [3]. It is important to evaluate the pharmaceutical profiles of the d-API to confirm the retainity of the biological activities of the parent drugs. We have synthesized a d-API, benzalkonium ibuprofenate, carried out its quantum mechanical calculations using density functional theory, characterized different experimental techniques, and reported glass-forming ability in earlier works [4, 5]. Now, in this work, the biological evaluations, in particular, the antibacterial and anti-inflammatory activities, were performed and the results with the activity of parent drugs, BaCl and sodium ibuprofenate (NaIb), compared. The molecular docking of all the samples was done to get a better understanding of the mode of interaction between the drugs

and respective targeted proteins and to trace their binding pores and cites.

The benzalkonium chloride and sodium ibuprofenate were purchased from Sigma-Aldrich (USA). Cell culture plastic flasks, test tubes, and culture plates were

The double active pharmaceutical ingredient, benzalkonium ibuprofenate, was synthesized using stoichiometric metathesis reaction. Solid (1 mmol) BaCl and NaIb were dissolved in 50 mL distilled water, each taken in two beakers and stirred separately with gentle heating (40–60°C). Then the two solutions were mixed together and again stirred for another 30 min with heating (around 80°C) and then cooled to room temperature. 60 mL of chloroform was added to separate the organic and inorganic part; then the chloroform phase was washed with cold distilled water until it removes the inorganic salt completely. AgNO3 test was used to confirm the absence of chloride anions in the product. This is followed by continuous washing of the chloroform phase with deionized (DI) water until the water washings tested negative for NaCl or NaBr via AgNO3 test. The chloroform was then evaporated using rotary evaporator, and the BaIb was dried under high vacuum for

The double active pharmaceutical ingredient, BaIb was characterized well using

Nuclear Magnetic Resonance using Bruker Avance III, 400MHzwith a 9.4 Tesla super-conducting magnet in an operating temperature at 309 K, Fourier transform infrared spectroscopy JASCO FTIR-4100 spectrophotometer, Fourier Transform-Raman spectroscopy and UV–visible spectroscopy using Jasco UV–Visible Spectrophotometer model V-550 (USA) and are reported in earlier works [4, 5].

The synthesized double active pharmaceutical ingredient BaIb was screened against Gram-negative bacteria to confirm the retainity of the biological activity of its parent drug BaCl, which is a potential germicide. For this study, we have chosen *Pseudomonas aeruginosa* and *E. coli* as Gram-positive, while DMSO is considered as

**84**

The anti-inflammatory activity of BaIb and NaIb was done using an egg albumin. For this, a reaction mixture of 5 mL was made with fresh hen's egg (0.2 mL) and phosphate buffer saline with pH = 6.4 of 2 mL with varying concentrations of extract for preparing the concentrations of 100, 200, 300, 400, and 500 μg/ mL. The same steps were repeated for the preparation of double distilled water, which served as control. Then the prepared mixtures were incubated in BOD incubator (Labline Technologies, India) at 210 ± 2 K for 15 min; after that, it is heated to 343 K and was hold for 5 min. The absorbance was measured after incubating using Shimadzu (Japan), UV 1800 at 660 nm. At the final concentration of 100, 200, 300, 400, and 500 μg/mL, acetyl salicylic acid was used as reference drug. The protein denaturation inhibition percentage was calculated using the following formulae:

$$\text{Non minimum premiumage was can enhance using } \text{uncology} \text{ returning norm.}$$

$$\text{@minability} = \frac{Abs\_{\text{control}} \cdot Abs\_{\text{test}}}{Abs\_{\text{control}}} \times 100\tag{2}$$

### *2.2.2.2.2 In human serum albumin*

In addition, the synthesized drug BaIb was screened for its anti-inflammatory activity, and its efficiency with the parent drug NaIb and drug diclofenac was compared. For this, blood samples from a healthy donor (male) were collected and mixed with sterilized Alsever's solution before centrifuging it at 3000 rpm. The suspension of packed cells was made with isoline. This suspension with phosphate buffer, hyposaline, was mixed with diclofenac at varying concentration, where the distilled water is taken as control while diclofenac as standard. Then the mixtures were incubated for 30 min at 303 K and centrifuged. Spectrophotometric analysis was used for hemolysis at 560 nm and its percentage recorded [4, 8, 9].

### **2.3 Computational**

The input structures of BaCl (PubChem: 2330), NaIb (PubChem: 5338317), and BaIb (PubChem: 86612072) were taken from the PubChem database [9] and optimized using density functional theory with B3LYP level of theory and 631-G+(d,p) [10] basis sets using Gaussian software packages [11]. Further, the molecular docking was also conducted using Schrodinger Maestro software package [10]. The optimized structures and downloaded Protein Data Bank (PDB) files [12] of proteins were used for molecular docking studies.

The Schrodinger's Glide module was used for docking analysis of the present work. Glide offers the full range of speed vs. accuracy options, from the HTVS (high-throughput virtual screening) mode for efficiently enriching million compound libraries, to the SP (standard precision) mode for reliably docking tens to hundreds of thousands of ligand with high accuracy, and to the extra precision (XP) mode where further elimination of false positives is accomplished by more extensive sampling and advanced scoring, resulting in even higher enrichment. Many researchers carried out extensive comparisons of several docking programs and scoring functions using an extensive data set of pharmaceutically attractive targets and active compounds [13–18]. All the study leads to the same result that Glide XP methodology was shown to yield enrichments superior to the alternative methods consistently. Glide SP scoring also shows improvement as compared to the scoring in GOLD and DOCK. The drawbacks of Glide come from the fact that it's increasing computational time. From computational efficiency, the CPU time required on average for Glide XP calculations (7.0 min per ligand) is larger than other methods except for the most accurate version of Goldscore (8.5 min per ligand). This extra cost for Glide XP is the trade-off for the higher enrichment factors obtained. Glide SP delivers the second best overall enrichment performance while providing a considerable speedup (0.42 min per ligand) as compared to all approaches except for the fast version of GOLD Chemscore setting.

### *2.3.1 Molecular docking studies*

The structure-based drug design always promotes the in silico method for molecular docking before going to lab screening. In silico methods can site the binding pores and predict the mechanism of protein-ligand interactions as well as target binding.

Moreover, the analysis and interpretation of the binding behavior play a crucial role in rational drug designs and in elucidating fundamentals of biochemical processes. The antibacterial activity of BaCl and BaIb was studied using LpxC enzymes since the enzyme LpxC places an important role in the lipid A biosynthesis. Lipid A acts as a hydrophobic membrane of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane of Gram-negative bacteria; however, the bacteria is with a defective lipid. A synthesis reduces its hydrophobicity and shows increased membrane permeability, which in turn increase the sensitivity to the antibiotics, and hence, it results in cell death. For this work, we have selected LpxC from *Escherichia coli* and *P. aeruginosa*. In the same way, the targeted protein for the anti-inflammatory activity of NaIb and BaIb were studied using human serum albumin (HSA).

Thus the structures of proteins used in this work were downloaded from the Protein Data Bank [12]. The detailed information of the selected proteins, their PDB IDs, inbuilt inhibitor, X-ray resolution, etc., were given in **Table 1**. Molecular docking study has been carried out by the Glide docking program [19–21] provided by Schrodinger suite. Protein preparation is done by using the Protein Preparation Wizard module of Glide [22, 23]. Initially, all the protein structures must be preprocessed to be used as a receptor for docking. Some of the typical operations in preprocessing include (i) addition of hydrogen atoms, (ii) assignment of atomic charges, and (iii) elimination of water molecules that are not involved in ligand binding. Missing chains and loops can also be added if necessary. Preprocessed protein was optimized with PROPKA and then minimized with OPSL3 force field function, which is followed by a convergence of heavy atoms of RMSD 0.3 Å.

Then, the Glide's receptor grid generation wizard was used to generate a threedimensional (3D) grid with a maximal size of 20 × 20 × 20 Å with 0.5 Å spacing.

**87**

dotted lines.

module.

**Table 1.**

**Sl. no.**

**3. Results and discussions**

*3.1.1 In vitro studies*

**3.1 Antibacterial activities of BaCl and BaIb**

*Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate*

**inhibitor**

3P3G 3p3 1.65 Å BaCl,

5U3B NVS 2.00 Å BaCl,

2BXG Ibuprofen 2.70 Å NaIb,

**X-ray resolution** **Ligands Activity**

Antibacterial

Antibacterial

Antiinflammatory

BaIb

BaIb

BaIb

**Protein Organism PDB-ID Inbuilt** 

There is enough option to apply any constraints such as precision constraints, H-bond constraint, etc., in the receptor grid generation wizard. At last, flexible docking was performed with extra precision docking mode in Glide docking

As we know, benzalkonium chloride is a prominent germicide widely used in medicinal chemistry [24]. It is mandatory to confirm the retainity of BaCl's antibacterial effect in the synthesized d-API, BaIb. The inhibition zone method using agar diffusion was used to screen the antibacterial activity of the prepared as well as parent drug against *P. aeruginosa* and *E. coli* bacterial strains [25]. The percentage of inhibition and its diameter are listed in **Table 2**. The results emphasized that BaIb retains the antibacterial activity of parent drug BaCl, though it had less inhibitory

Here, molecular docking of the parent and daughter drugs, BaCl and BaIb with *Escherichia coli* LpxC/LPC-009 complex, has been employed to trace its binding pore and binding affinity. The docking scores and binding free energies of lowest energy pose of its inbuilt inhibitor, 3p3 and the samples under study, BaCl, and BaIb in active sites on chain A of the LpxC/LPC-009 X-ray crystal structures have been computed after deleting the unwanted ligands and amino acids (So4 at 501, 502, 504, 505, 506; dimethyl sulfide (DMS) at 701; and UKW) using Schrodinger Glide module and are given in **Table 3**. The docking result points out that the drugs BaCl and BaIb show considerable binding affinity scores compared to the inbuilt ligand. However, interestingly the d-API BaIb exhibits high docking score compared to the parent drug BaCl, which emphasize that the interaction between the ligand and

**Figure 1** demonstrates the three-dimensional protein-ligand interaction of the three samples under study in the dynamic site of LpxC/LPC-009 obtained from graphical interface Maestro. All the ligands are found to be buried in the deep binding pocket of LpxC/LPC-009 in the same way. The d-API BaIb interacts with the active site's amino acids of the protein by H-bonding, which is depicted in red and

action against *E. coli* and *P. aeruginosa* than the parent drug [25].

protein increases on double active formation with ibuprofen.

*3.1.2 Molecular docking studies with LpxC (E. coli)*

*DOI: http://dx.doi.org/10.5772/intechopen.90191*

*coli*

*aeruginosa*

*sapiens*

*Detailed information regarding the proteins under study.*

1 LpxC *Escherichia* 

2 LpxC *Pseudomonas* 

3 HSA *Homo* 


**Table 1.**

*Computational Biology and Chemistry*

for the fast version of GOLD Chemscore setting.

*2.3.1 Molecular docking studies*

binding.

albumin (HSA).

The Schrodinger's Glide module was used for docking analysis of the present work. Glide offers the full range of speed vs. accuracy options, from the HTVS (high-throughput virtual screening) mode for efficiently enriching million compound libraries, to the SP (standard precision) mode for reliably docking tens to hundreds of thousands of ligand with high accuracy, and to the extra precision (XP) mode where further elimination of false positives is accomplished by more extensive sampling and advanced scoring, resulting in even higher enrichment. Many researchers carried out extensive comparisons of several docking programs and scoring functions using an extensive data set of pharmaceutically attractive targets and active compounds [13–18]. All the study leads to the same result that Glide XP methodology was shown to yield enrichments superior to the alternative methods consistently. Glide SP scoring also shows improvement as compared to the scoring in GOLD and DOCK. The drawbacks of Glide come from the fact that it's increasing computational time. From computational efficiency, the CPU time required on average for Glide XP calculations (7.0 min per ligand) is larger than other methods except for the most accurate version of Goldscore (8.5 min per ligand). This extra cost for Glide XP is the trade-off for the higher enrichment factors obtained. Glide SP delivers the second best overall enrichment performance while providing a considerable speedup (0.42 min per ligand) as compared to all approaches except

The structure-based drug design always promotes the in silico method for molecular docking before going to lab screening. In silico methods can site the binding pores and predict the mechanism of protein-ligand interactions as well as target

role in rational drug designs and in elucidating fundamentals of biochemical processes. The antibacterial activity of BaCl and BaIb was studied using LpxC enzymes since the enzyme LpxC places an important role in the lipid A biosynthesis. Lipid A acts as a hydrophobic membrane of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane of Gram-negative bacteria; however, the bacteria is with a defective lipid. A synthesis reduces its hydrophobicity and shows increased membrane permeability, which in turn increase the sensitivity to the antibiotics, and hence, it results in cell death. For this work, we have selected LpxC from *Escherichia coli* and *P. aeruginosa*. In the same way, the targeted protein for the anti-inflammatory activity of NaIb and BaIb were studied using human serum

Moreover, the analysis and interpretation of the binding behavior play a crucial

Thus the structures of proteins used in this work were downloaded from the Protein Data Bank [12]. The detailed information of the selected proteins, their PDB IDs, inbuilt inhibitor, X-ray resolution, etc., were given in **Table 1**. Molecular docking study has been carried out by the Glide docking program [19–21] provided by Schrodinger suite. Protein preparation is done by using the Protein Preparation Wizard module of Glide [22, 23]. Initially, all the protein structures must be preprocessed to be used as a receptor for docking. Some of the typical operations in preprocessing include (i) addition of hydrogen atoms, (ii) assignment of atomic charges, and (iii) elimination of water molecules that are not involved in ligand binding. Missing chains and loops can also be added if necessary. Preprocessed protein was optimized with PROPKA and then minimized with OPSL3 force field function, which is followed by a convergence of heavy atoms of RMSD 0.3 Å.

Then, the Glide's receptor grid generation wizard was used to generate a threedimensional (3D) grid with a maximal size of 20 × 20 × 20 Å with 0.5 Å spacing.

**86**

*Detailed information regarding the proteins under study.*

There is enough option to apply any constraints such as precision constraints, H-bond constraint, etc., in the receptor grid generation wizard. At last, flexible docking was performed with extra precision docking mode in Glide docking module.

### **3. Results and discussions**

### **3.1 Antibacterial activities of BaCl and BaIb**

### *3.1.1 In vitro studies*

As we know, benzalkonium chloride is a prominent germicide widely used in medicinal chemistry [24]. It is mandatory to confirm the retainity of BaCl's antibacterial effect in the synthesized d-API, BaIb. The inhibition zone method using agar diffusion was used to screen the antibacterial activity of the prepared as well as parent drug against *P. aeruginosa* and *E. coli* bacterial strains [25]. The percentage of inhibition and its diameter are listed in **Table 2**. The results emphasized that BaIb retains the antibacterial activity of parent drug BaCl, though it had less inhibitory action against *E. coli* and *P. aeruginosa* than the parent drug [25].

### *3.1.2 Molecular docking studies with LpxC (E. coli)*

Here, molecular docking of the parent and daughter drugs, BaCl and BaIb with *Escherichia coli* LpxC/LPC-009 complex, has been employed to trace its binding pore and binding affinity. The docking scores and binding free energies of lowest energy pose of its inbuilt inhibitor, 3p3 and the samples under study, BaCl, and BaIb in active sites on chain A of the LpxC/LPC-009 X-ray crystal structures have been computed after deleting the unwanted ligands and amino acids (So4 at 501, 502, 504, 505, 506; dimethyl sulfide (DMS) at 701; and UKW) using Schrodinger Glide module and are given in **Table 3**. The docking result points out that the drugs BaCl and BaIb show considerable binding affinity scores compared to the inbuilt ligand. However, interestingly the d-API BaIb exhibits high docking score compared to the parent drug BaCl, which emphasize that the interaction between the ligand and protein increases on double active formation with ibuprofen.

**Figure 1** demonstrates the three-dimensional protein-ligand interaction of the three samples under study in the dynamic site of LpxC/LPC-009 obtained from graphical interface Maestro. All the ligands are found to be buried in the deep binding pocket of LpxC/LPC-009 in the same way. The d-API BaIb interacts with the active site's amino acids of the protein by H-bonding, which is depicted in red and dotted lines.


### **Table 2.**

*Preliminary in vitro antibacterial screening activity of BaIb.*


### **Table 3.**

*Docking scores and binding free energies of inbuilt inhibitor 3P3, BaCl, and BaIb to the LpxC/LPC-009 using Schrodinger Maestro software.*

### **Figure 1.**

*Three-dimensional (3D) protein-ligand interactions diagram using Schrodinger software (I) with LpxC protein of* E. coli *using (a) inbuilt ligand 3P3, (b) parent ligand BaCl, and (c) double active pharmaceutical ingredient BaIb; (II) with LpxC (*P. aeruginosa*) using (a) inbuilt ligand 3P3, (b) parent ligand BaCl, and (c) double active pharmaceutical ingredient BaIb; and (III) with human serum albumin using (a) inbuilt ligand ibuprofen and (b) double active pharmaceutical ingredient BaIb.*

In addition to the 3D binding orientations of the ligands in the protein, the docking results provide further insights into selective interactions of the ligands with the *E. coli* LpxC in the 2D image, as shown in **Figure 2**. The ligands were encompassed by active site amino acids THR191, PHE192, SER211, PHE212, CYS214, LYS239, HIS238, HIS265, etc., of LpxC. The co-crystallized ligand 3P3 occupied the deep cavity by forming three hydrogen bonds and one π-π interaction with active site amino acid PHE212. Though the parent ligand BaCl occupied the deep cavity of

**89**

**Figure 2.**

*LpxC, (b) BaCl with LpxC, and (c) BaIb with LpxC.*

*Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate*

formed one hydrogen bond with the active amino acid LYS239.

*3.1.3 Molecular docking studies with LpxC (P. aeruginosa)*

LpxC with the support of salt bridges between them, the daughter ligand BaIb

Despite the binding energy and docking score difference between parent and daughter drugs, BaCl and BaIb were well occupied in the binding site of the LpxC protein as similar to the native ligand 3P3 by forming a hydrogen bond with active site amino acids. Besides, the binding energy and docking score emphasize that the d-API has a higher binding affinity with LpxC than the parent drug BaCl. This indicates that BaIb can be considered as a potential inhibitor of LpxC protein with

Here, molecular docking of the parent and daughter drugs, BaCl and BaIb with LpxC protein of *Pseudomonas aeruginosa*, has been employed to trace the nature of its binding interaction and binding affinity value. The docking scores and binding free energies of lowest energy pose of its inbuilt inhibitor, NVS-LpxC-01 and the samples under study, BaCl, and BaIb in active sites on chain B of the NVS-LpxC X-ray crystal structures, have been computed after deleting the unwanted ligands and amino acids and including the Zn2+ using Schrodinger Maestro software and are given in **Table 4**. The docking result points out that the drugs BaCl and BaIb show considerable binding affinity scores compared to the inbuilt ligand. However,

*Schematic representations of ligand-protein interaction and binding interaction using stick mode. (a) 3P3 with* 

*DOI: http://dx.doi.org/10.5772/intechopen.90191*

antibacterial activity.

### *Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate DOI: http://dx.doi.org/10.5772/intechopen.90191*

LpxC with the support of salt bridges between them, the daughter ligand BaIb formed one hydrogen bond with the active amino acid LYS239.

Despite the binding energy and docking score difference between parent and daughter drugs, BaCl and BaIb were well occupied in the binding site of the LpxC protein as similar to the native ligand 3P3 by forming a hydrogen bond with active site amino acids. Besides, the binding energy and docking score emphasize that the d-API has a higher binding affinity with LpxC than the parent drug BaCl. This indicates that BaIb can be considered as a potential inhibitor of LpxC protein with antibacterial activity.

### *3.1.3 Molecular docking studies with LpxC (P. aeruginosa)*

Here, molecular docking of the parent and daughter drugs, BaCl and BaIb with LpxC protein of *Pseudomonas aeruginosa*, has been employed to trace the nature of its binding interaction and binding affinity value. The docking scores and binding free energies of lowest energy pose of its inbuilt inhibitor, NVS-LpxC-01 and the samples under study, BaCl, and BaIb in active sites on chain B of the NVS-LpxC X-ray crystal structures, have been computed after deleting the unwanted ligands and amino acids and including the Zn2+ using Schrodinger Maestro software and are given in **Table 4**. The docking result points out that the drugs BaCl and BaIb show considerable binding affinity scores compared to the inbuilt ligand. However,

### **Figure 2.**

*Schematic representations of ligand-protein interaction and binding interaction using stick mode. (a) 3P3 with LpxC, (b) BaCl with LpxC, and (c) BaIb with LpxC.*

*Computational Biology and Chemistry*

**Compound Schrodinger software**

*Preliminary in vitro antibacterial screening activity of BaIb.*

**The diameter of zone of inhibition (mm) Percentage of inhibition (%)**

3P3 −13.682 −0.507 BaCl −3.234 −0.147 BaIb −6.315 −0.175

Standard BaCl 19 38 Standard BaCl 21.11 42.22 Sample BaIb 14 31 Sample BaIb 15.55 34.44 Negative (DMSO) 0 0 Negative (DMSO) 0 0

*Docking scores and binding free energies of inbuilt inhibitor 3P3, BaCl, and BaIb to the LpxC/LPC-009 using* 

**Glide docking score (kcal/mol) Glide ligand efficiency**

*E. coli P. aeruginosa E. coli P. aeruginosa*

**88**

**Figure 1.**

**Table 3.**

**Table 2.**

*Schrodinger Maestro software.*

In addition to the 3D binding orientations of the ligands in the protein, the docking results provide further insights into selective interactions of the ligands with the *E. coli* LpxC in the 2D image, as shown in **Figure 2**. The ligands were encompassed by active site amino acids THR191, PHE192, SER211, PHE212, CYS214, LYS239, HIS238, HIS265, etc., of LpxC. The co-crystallized ligand 3P3 occupied the deep cavity by forming three hydrogen bonds and one π-π interaction with active site amino acid PHE212. Though the parent ligand BaCl occupied the deep cavity of

*Three-dimensional (3D) protein-ligand interactions diagram using Schrodinger software (I) with LpxC protein of* E. coli *using (a) inbuilt ligand 3P3, (b) parent ligand BaCl, and (c) double active pharmaceutical ingredient BaIb; (II) with LpxC (*P. aeruginosa*) using (a) inbuilt ligand 3P3, (b) parent ligand BaCl, and (c) double active pharmaceutical ingredient BaIb; and (III) with human serum albumin using (a) inbuilt* 

*ligand ibuprofen and (b) double active pharmaceutical ingredient BaIb.*


**Table 4.**

*Docking scores and binding free energies of inbuilt inhibitor NVS, BaCl, and BaIb to the LpxC/LPC-009 using Schrodinger Maestro software.*

### **Figure 3.**

*Schematic representations of ligand-protein interaction and binding interaction using stick mode. (a) 3P3 with LpxC, (b) BaCl with LpxC, and (c) BaIb with LpxC using Schrodinger software.*

interestingly the d-API BaIb exhibits high docking score compared to the parent drug BaCl, which emphasizes that the interaction between the ligand and protein increased on double active formation with ibuprofen.

**Figure 1** demonstrates the three-dimensional protein-ligand interaction of three samples under study in the active site of LpxC in complex obtained from graphical interface Maestro. All the ligands are found to be buried in the deep binding pocket of LpxC in the complex in an indistinguishable way. The d-API BaIb interact with the active site's amino acids of the protein by H-bonding, which is depicted in red and dotted lines.

**91**

*Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate*

In addition to the 3D binding orientations of the ligands in the protein, the docking results provide further insights into selective interactions of the ligands with the *Pseudomonas aeruginosa* LpxC in the 2D image as shown in **Figure 3**. The ligands were surrounded by active site amino acids THR190, GLY192, PHE191, SER211, PHE193, MET194, ASP196, DLE197, LEU200, ARG201, VAL216, etc., of LpxC. The co-crystallized ligand NVS occupied the deep cavity by forming five hydrogen bonds in addition to its salt bridge. Though the parent ligand BaCl occupied the deep cavity of LpxC with the support of salt bridges between them, the daughter ligand BaIb formed one hydrogen bond with the active amino acid LYS238 and a

Despite the binding energy and docking score difference of parent as well as the daughter drugs, BaCl and BaIb were well occupied in the binding site of the LpxC protein as similar to the native ligand NVS with hydrogen bonding with active site amino acids. Also, the binding energy and docking score emphasize that the d-API has a higher binding affinity with LpxC than the parent drug BaCl. This indicates the BaIb can be considered as a potential inhibitor of LpxC protein with antibacte-

The anti-inflammatory properties of the synthesized drug BaIb and parent drug NaIb were studied in chick albumin membrane. **Figure 4** depicts the bar diagram of the percentage inhibition of inflammation against the concentration of NaIb and BaIb. Sample NaIb and BaIb have almost the same inhibitory activity in all concentration, which is around 95%. Thus from this analysis, one can confirm that BaIb

The anti-inflammatory properties of the synthesized drug BaIb, parent drug NaIb, and drug diclofenac as standard samples were given in **Table 5**. Among samples provided, diclofenac shows maximum inhibitory activity, whereas the NaIb and BaIb are less active than the reference compound, but still, its activity is significant as an inflammatory agent. This fact suggests that the anti-inflammatory activity of NaIb was retained in the double active pharmaceutical ingredient. Thus the in vitro study confirms that the synthesized BaIb is a double active pharmaceu-

tical ingredient by retaining the biological activities of the parent drugs.

protein is still functional on double active formation with ibuprofen.

Here, molecular docking of the parent and daughter drugs, NaIb and BaIb, respectively, with human serum albumin complex has been employed to analyze its binding mode and binding affinity value. The docking scores and binding free energies of lowest energy pose of its inbuilt inhibitor, ibuprofen and the BaIb in active sites on chain A of the 2BXG X-ray crystal structures, have been computed after deleting the unwanted ligands and amino acids using Schrodinger Maestro software and are given in **Table 6**. The docking result points out that the drugs ibuprofen and BaIb show considerable binding affinity scores compared to the inbuilt ligand. However, interestingly the d-API BaIb exhibited almost similar docking score to the parent drug Ib, which emphasizes that the interaction between the ligand and

*DOI: http://dx.doi.org/10.5772/intechopen.90191*

rial activity.

π-cation interaction with active site amino acid PHE191.

**3.2 Anti-inflammatory activities of NaIb and BaIb**

retains the anti-inflammatory activity of NaIb and states.

*3.2.3 Molecular docking studies of NaIb and BaIb with HSA*

*3.2.2 In vitro studies using human serum albumin*

*3.2.1 In vitro studies using chick albumin*

*Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate DOI: http://dx.doi.org/10.5772/intechopen.90191*

In addition to the 3D binding orientations of the ligands in the protein, the docking results provide further insights into selective interactions of the ligands with the *Pseudomonas aeruginosa* LpxC in the 2D image as shown in **Figure 3**. The ligands were surrounded by active site amino acids THR190, GLY192, PHE191, SER211, PHE193, MET194, ASP196, DLE197, LEU200, ARG201, VAL216, etc., of LpxC. The co-crystallized ligand NVS occupied the deep cavity by forming five hydrogen bonds in addition to its salt bridge. Though the parent ligand BaCl occupied the deep cavity of LpxC with the support of salt bridges between them, the daughter ligand BaIb formed one hydrogen bond with the active amino acid LYS238 and a π-cation interaction with active site amino acid PHE191.

Despite the binding energy and docking score difference of parent as well as the daughter drugs, BaCl and BaIb were well occupied in the binding site of the LpxC protein as similar to the native ligand NVS with hydrogen bonding with active site amino acids. Also, the binding energy and docking score emphasize that the d-API has a higher binding affinity with LpxC than the parent drug BaCl. This indicates the BaIb can be considered as a potential inhibitor of LpxC protein with antibacterial activity.

### **3.2 Anti-inflammatory activities of NaIb and BaIb**

### *3.2.1 In vitro studies using chick albumin*

*Computational Biology and Chemistry*

**Table 4.**

*Schrodinger Maestro software.*

**Compound Schrodinger software**

NVS −11.095 −0.482 BaCl −4.540 −0.206 BaIb −5.494 −0.153

*Docking scores and binding free energies of inbuilt inhibitor NVS, BaCl, and BaIb to the LpxC/LPC-009 using* 

**Glide docking score (kcal/mol) Glide ligand efficiency**

**90**

**Figure 3.**

and dotted lines.

interestingly the d-API BaIb exhibits high docking score compared to the parent drug BaCl, which emphasizes that the interaction between the ligand and protein

*Schematic representations of ligand-protein interaction and binding interaction using stick mode. (a) 3P3 with* 

**Figure 1** demonstrates the three-dimensional protein-ligand interaction of three samples under study in the active site of LpxC in complex obtained from graphical interface Maestro. All the ligands are found to be buried in the deep binding pocket of LpxC in the complex in an indistinguishable way. The d-API BaIb interact with the active site's amino acids of the protein by H-bonding, which is depicted in red

increased on double active formation with ibuprofen.

*LpxC, (b) BaCl with LpxC, and (c) BaIb with LpxC using Schrodinger software.*

The anti-inflammatory properties of the synthesized drug BaIb and parent drug NaIb were studied in chick albumin membrane. **Figure 4** depicts the bar diagram of the percentage inhibition of inflammation against the concentration of NaIb and BaIb. Sample NaIb and BaIb have almost the same inhibitory activity in all concentration, which is around 95%. Thus from this analysis, one can confirm that BaIb retains the anti-inflammatory activity of NaIb and states.

### *3.2.2 In vitro studies using human serum albumin*

The anti-inflammatory properties of the synthesized drug BaIb, parent drug NaIb, and drug diclofenac as standard samples were given in **Table 5**. Among samples provided, diclofenac shows maximum inhibitory activity, whereas the NaIb and BaIb are less active than the reference compound, but still, its activity is significant as an inflammatory agent. This fact suggests that the anti-inflammatory activity of NaIb was retained in the double active pharmaceutical ingredient. Thus the in vitro study confirms that the synthesized BaIb is a double active pharmaceutical ingredient by retaining the biological activities of the parent drugs.

### *3.2.3 Molecular docking studies of NaIb and BaIb with HSA*

Here, molecular docking of the parent and daughter drugs, NaIb and BaIb, respectively, with human serum albumin complex has been employed to analyze its binding mode and binding affinity value. The docking scores and binding free energies of lowest energy pose of its inbuilt inhibitor, ibuprofen and the BaIb in active sites on chain A of the 2BXG X-ray crystal structures, have been computed after deleting the unwanted ligands and amino acids using Schrodinger Maestro software and are given in **Table 6**. The docking result points out that the drugs ibuprofen and BaIb show considerable binding affinity scores compared to the inbuilt ligand. However, interestingly the d-API BaIb exhibited almost similar docking score to the parent drug Ib, which emphasizes that the interaction between the ligand and protein is still functional on double active formation with ibuprofen.

### **Figure 4.**

*Plot of percentage inhibition of inflammation against the concentration of NaIb and BaIb studied in chick albumin membrane.*


### **Table 5.**

*Preliminary in vitro anti-inflammatory properties of BaIb.*


### **Table 6.**

*Docking scores and binding free energies of inbuilt inhibitor ibuprofen and BaIb to the 2BXG using Schrodinger Maestro software.*

**Figure 1** demonstrates the three-dimensional protein-ligand interaction of three samples under study in the dynamic site of 2BXG obtained from graphical interface Maestro. All the ligands are found to be well occupied in the deep binding pocket of 2BXG in the same way. The d-API BaIb interact with the active site's amino acids of the protein by H-bonding, which is depicted in red and dotted lines. Interaction of amino acids at the active site of HAS with the studied compound is displayed in the 2D image (**Figure 5**). The ligands were encircled by amino acids like SER480, LBU481, VAL482, ASN483, PHE205, ARG209, ALA210, ALA213, etc., of HSA. The co-crystallized ligand ibuprofen occupies the deep cavity by forming three hydrogen bonds with active sites of amino acids SER480, LBU481, VAL482, and one π-anion interaction with active site amino acid LYS351. The daughter ligand BaIb forms only one hydrogen bond with the amino acid LYS239.

Despite the binding energy and docking score difference of parent as well as the daughter drug, the daughter drug, BaIb, was well occupied in the binding site of the

**93**

*Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate*

HSA protein as similar to the native ligand ibuprofen by forming a hydrogen bond

*Schematic representations of ligand-protein interaction and binding interaction using stick mode. (a)* 

*Ibuprofen with 2BXG and (b) BaIb with 2BXG using Schrodinger software.*

In this work, the biological evaluations of a synthesized double active pharmaceutical ingredient BaIb were done to confirm the retainity of the biological activities of its parent drugs and to elucidate information regarding its potential activities against their respective ailments. Further molecular docking studies were done to get a better understanding about the mode of interaction of the parent as well as daughter drugs with the targeted proteins and trace out the binding pore and cites

The in vitro studies revealed that the synthesized BaIb could be designed as a potential double active drug since it retained the antibacterial activity of its parent BaCl with considerable inhibitory action against *E. coli* and *P. aeruginosa* compared to the parent drug. The binding energy and docking score of BaCl and BaIb again confirm that the prepared d-API BaIb docks well into the LpxC proteins of *E. coli* and *P. aeruginosa* with high docking and Glide score compared to

Similarly, the results from both in vitro and in silico method emphasize that the prepared d-API retained the anti-inflammatory action of its parent NaIb and bound well to the deep pocket of the active site in the human serum albumin. Thus, in total, one can conclude that the prepared BaIb can be used as a potential double

The authors thankfully acknowledge the fruitful discussions they had with Dr. Deshpande. The authors also acknowledge the Central Sophisticated Instrumentation Facility (CSIF), University of Calicut, for the Schrodinger software support. KPSH acknowledges UGC-MANF for fellowship with sanction number MANF2017-18-KER-78598. KPSH and MST gratefully acknowledge

active drug with antibacterial and anti-inflammatory actions.

*DOI: http://dx.doi.org/10.5772/intechopen.90191*

with active site amino acid.

in the targeted proteins.

the parent drug BaCl.

**Acknowledgements**

**4. Conclusions**

**Figure 5.**

*Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate DOI: http://dx.doi.org/10.5772/intechopen.90191*

**Figure 5.**

*Computational Biology and Chemistry*

**Percentage of inhibition of hemolysis (%)**

*Preliminary in vitro anti-inflammatory properties of BaIb.*

Diclofenac 92.16 Standard sample NaIb 88.47 Sample BaIb 88.59

**Compound Schrodinger software**

Ibuprofen −5.435 −0.362 BaIb −3.554 −0.099

*Plot of percentage inhibition of inflammation against the concentration of NaIb and BaIb studied in chick* 

**Figure 1** demonstrates the three-dimensional protein-ligand interaction of three samples under study in the dynamic site of 2BXG obtained from graphical interface Maestro. All the ligands are found to be well occupied in the deep binding pocket of 2BXG in the same way. The d-API BaIb interact with the active site's amino acids of the protein by H-bonding, which is depicted in red and dotted lines. Interaction of amino acids at the active site of HAS with the studied compound is displayed in the 2D image (**Figure 5**). The ligands were encircled by amino acids like SER480, LBU481, VAL482, ASN483, PHE205, ARG209, ALA210, ALA213, etc., of HSA. The co-crystallized ligand ibuprofen occupies the deep cavity by forming three hydrogen bonds with active sites of amino acids SER480, LBU481, VAL482, and one π-anion interaction with active site amino acid LYS351. The daughter ligand BaIb

*Docking scores and binding free energies of inbuilt inhibitor ibuprofen and BaIb to the 2BXG using Schrodinger* 

**Glide docking score (kcal/mol) Glide ligand efficiency**

Despite the binding energy and docking score difference of parent as well as the daughter drug, the daughter drug, BaIb, was well occupied in the binding site of the

forms only one hydrogen bond with the amino acid LYS239.

**92**

**Table 5.**

**Table 6.**

*Maestro software.*

**Figure 4.**

*albumin membrane.*

*Schematic representations of ligand-protein interaction and binding interaction using stick mode. (a) Ibuprofen with 2BXG and (b) BaIb with 2BXG using Schrodinger software.*

HSA protein as similar to the native ligand ibuprofen by forming a hydrogen bond with active site amino acid.

### **4. Conclusions**

In this work, the biological evaluations of a synthesized double active pharmaceutical ingredient BaIb were done to confirm the retainity of the biological activities of its parent drugs and to elucidate information regarding its potential activities against their respective ailments. Further molecular docking studies were done to get a better understanding about the mode of interaction of the parent as well as daughter drugs with the targeted proteins and trace out the binding pore and cites in the targeted proteins.

The in vitro studies revealed that the synthesized BaIb could be designed as a potential double active drug since it retained the antibacterial activity of its parent BaCl with considerable inhibitory action against *E. coli* and *P. aeruginosa* compared to the parent drug. The binding energy and docking score of BaCl and BaIb again confirm that the prepared d-API BaIb docks well into the LpxC proteins of *E. coli* and *P. aeruginosa* with high docking and Glide score compared to the parent drug BaCl.

Similarly, the results from both in vitro and in silico method emphasize that the prepared d-API retained the anti-inflammatory action of its parent NaIb and bound well to the deep pocket of the active site in the human serum albumin. Thus, in total, one can conclude that the prepared BaIb can be used as a potential double active drug with antibacterial and anti-inflammatory actions.

### **Acknowledgements**

The authors thankfully acknowledge the fruitful discussions they had with Dr. Deshpande. The authors also acknowledge the Central Sophisticated Instrumentation Facility (CSIF), University of Calicut, for the Schrodinger software support. KPSH acknowledges UGC-MANF for fellowship with sanction number MANF2017-18-KER-78598. KPSH and MST gratefully acknowledge the collaborative research grant from UGC-DAE (No. UDCSR/MUM/AO/CRS-M-2I0/2015/501 dated 06/01/2015). MST further acknowledges the financial assistance from KSCSTE (SRS, SARD), UGC (MRP), and DST FIST.

### **Author details**

Kodakkat Parambil Safna Hussan1 , Mohamed Shahin Thayyil1 , Thaikadan Shameera Ahamed<sup>2</sup> and Karuvanthodi Muraleedharan<sup>2</sup> \*

1 Department of Physics, University of Calicut, Malappuram, Kerala, India

2 Department of Chemistry, University of Calicut, Malappuram, Kerala, India

\*Address all correspondence to: kmuralika@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**95**

*Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate*

Glycyrrhiza radix and its bioactive compounds. China Journal of Chinese

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Springer Publishers; 2011

Gaussian, Inc.; 1998

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[15] Warren GL, Andrews CW, Capelli A-M, Clarke B, LaLonde J, Lambert MH, et al. A critical assessment of docking programs and scoring functions.

[13] Zhou Z, Felts AK, Friesner RA, Levy RM. Comparative performance of several flexible docking programs and scoring functions: Enrichment studies for a diverse set of pharmaceutically relevant targets. Journal of Chemical

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Pharmaceutical and biotechnological

Rodriguez H, Swatloski RP, Spear SK, Daly DT, et al. The third evolution of ionic liquids: Active pharmaceutical ingredients. New Journal of Chemistry.

[3] Ferraz R, Branco LC, Prudêncio C, Noronha JP, Petrovski Ž. Ionic liquids as active pharmaceutical ingredients. ChemMedChem. 2011;**6**:975-985

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the collaborative research grant from UGC-DAE (No. UDCSR/MUM/AO/CRS-M-2I0/2015/501 dated 06/01/2015). MST further acknowledges the financial

, Mohamed Shahin Thayyil1

1 Department of Physics, University of Calicut, Malappuram, Kerala, India

\*Address all correspondence to: kmuralika@gmail.com

provided the original work is properly cited.

2 Department of Chemistry, University of Calicut, Malappuram, Kerala, India

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and Karuvanthodi Muraleedharan<sup>2</sup>

,

\*

assistance from KSCSTE (SRS, SARD), UGC (MRP), and DST FIST.

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Kodakkat Parambil Safna Hussan1

Thaikadan Shameera Ahamed<sup>2</sup>

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[19] Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, et al. Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. Journal of Medicinal Chemistry. 2004;**47**:1739-1749

[20] Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT, et al. Glide: A new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. Journal of Medicinal Chemistry. 2014;**47**:1750-1759

[21] Friesner R, Murphy R, Repasky M, Frye L, Greenwood J, Halgren T, et al. Extra precision glide: Docking and scoring incorporating a model of hydrophobic enclosure for proteinligand complexes. Journal of Medicinal Chemistry. 2006;**49**:6177-6196

[22] Sastry G, Adzhigirey M, Day T, Annabhimoju R, Sherman W. Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments. Journal of Computer-Aided Molecular Design. 2013;**27**:221-234

[23] Epik. Schrödinger Suite 2019-2 Protein Preparation Wizard; 2019

[24] Malizia WF, Gangarosa EJ, Goley AF. Benzalkonium chloride as a source of infection. New England Journal of Medicine. 2010;**263**:800-802

[25] Prasad NK. Synthesis, characterisation and biological activity of metal complexes of furoic acid. International Journal of Basic and Applied Chemical Sciences. 2015;**5**:52-57

**97**

**Figure 1.**

*General molecular structure of hydrazones.*

**Chapter 6**

**Abstract**

**1. Introduction**

Chemosensors

Hydrazone-Based Small-Molecule

The hydrazone functional group is widely applied in several fields. The versatility and large use of this chemotype are attributed to its easy and straightforward synthesis and unique structural characteristics which is useful for different chemical and biological purposes. Recently hydrazone scaffold has been widely adopted in the design of small-molecule fluorescent and colorimetric chemosensors for detecting metals and anions because of its corresponding non-covalent interactions. This chapter provides an overview of hydrazone-based fluorescent and colorimetric chemosensors for anions and metals of biological interest, with their representative rational designs in the last 15 years. We hope this chapter inspires the development of novel and powerful

*Thiago Moreira Pereira and Arthur Eugen Kümmerle*

fluorescent and colorimetric chemosensors for a broad range of applications.

**Keywords:** hydrazone, cyanide, acetate, fluoride, zinc, copper, aluminum, magnesium, mercury, coumarin, fluorescein, rhodamine, Schiff base

Hydrazone-based molecular structures are ubiquitous in many research fields, such as medicinal chemistry [1], organic synthesis [2], supramolecular chemistry [3], metal-organic coordination [4], dyes [5], fluorescent sensors, and molecular machines [6], besides others applications [7]. Over the last decades, the popularity of hydrazone group has increased due to its easy and direct-obtaining synthesis, stability toward hydrolysis in comparison with imines, modularity, and mainly, functional diversity of C═N▬N useful in several fields (**Figure 1**). In terms of structure, hydrazones are considered as azomethine compounds; however they are distinguished from imines and oximes, for example, by the presence of additional linked nitrogen atom [8]. Hydrazone backbone has an imine carbon that has an

### **Chapter 6**

*Computational Biology and Chemistry*

Journal of Medicinal Chemistry.

[16] Schulz-Gasch T, Stahl M. Binding site characteristics in structure-based virtual screening: Evaluation of current docking tools. Journal of Molecular

[24] Malizia WF, Gangarosa EJ, Goley AF. Benzalkonium chloride as a source of infection. New England Journal of Medicine. 2010;**263**:800-802

[25] Prasad NK. Synthesis,

characterisation and biological activity of metal complexes of furoic acid. International Journal of Basic and Applied Chemical Sciences. 2015;**5**:52-57

[17] Pagadala NS, Syed K, Tuszynski J. Software for molecular docking: A review. Biophysical Reviews.

[18] Castro-Alvarez A, Costa AM, Vilarrasa J. The performance of several docking programs at reproducing protein-macrolide-like crystal structures. Molecules. 2017;**22**:136

[19] Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, et al. Glide: A new approach for rapid,

accurate docking and scoring. 1. Method and assessment of docking accuracy. Journal of Medicinal Chemistry.

Pollard WT, et al. Glide: A new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. Journal of Medicinal Chemistry. 2014;**47**:1750-1759

[21] Friesner R, Murphy R, Repasky M, Frye L, Greenwood J, Halgren T, et al. Extra precision glide: Docking and scoring incorporating a model of hydrophobic enclosure for proteinligand complexes. Journal of Medicinal

Chemistry. 2006;**49**:6177-6196

[22] Sastry G, Adzhigirey M, Day T, Annabhimoju R, Sherman W. Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments. Journal of Computer-Aided Molecular Design.

[23] Epik. Schrödinger Suite 2019-2 Protein Preparation Wizard; 2019

2006;**49**:5912-5931

Modeling. 2003;**9**:47-57

2017;**9**:91-102

2004;**47**:1739-1749

[20] Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL,

**96**

2013;**27**:221-234

## Hydrazone-Based Small-Molecule Chemosensors

*Thiago Moreira Pereira and Arthur Eugen Kümmerle*

### **Abstract**

The hydrazone functional group is widely applied in several fields. The versatility and large use of this chemotype are attributed to its easy and straightforward synthesis and unique structural characteristics which is useful for different chemical and biological purposes. Recently hydrazone scaffold has been widely adopted in the design of small-molecule fluorescent and colorimetric chemosensors for detecting metals and anions because of its corresponding non-covalent interactions. This chapter provides an overview of hydrazone-based fluorescent and colorimetric chemosensors for anions and metals of biological interest, with their representative rational designs in the last 15 years. We hope this chapter inspires the development of novel and powerful fluorescent and colorimetric chemosensors for a broad range of applications.

**Keywords:** hydrazone, cyanide, acetate, fluoride, zinc, copper, aluminum, magnesium, mercury, coumarin, fluorescein, rhodamine, Schiff base

### **1. Introduction**

Hydrazone-based molecular structures are ubiquitous in many research fields, such as medicinal chemistry [1], organic synthesis [2], supramolecular chemistry [3], metal-organic coordination [4], dyes [5], fluorescent sensors, and molecular machines [6], besides others applications [7]. Over the last decades, the popularity of hydrazone group has increased due to its easy and direct-obtaining synthesis, stability toward hydrolysis in comparison with imines, modularity, and mainly, functional diversity of C═N▬N useful in several fields (**Figure 1**). In terms of structure, hydrazones are considered as azomethine compounds; however they are distinguished from imines and oximes, for example, by the presence of additional linked nitrogen atom [8]. Hydrazone backbone has an imine carbon that has an

**Figure 1.** *General molecular structure of hydrazones.*

electrophile character, two nucleophilic nitrogen in both imine and amine groups and a possible isomerization of C═N double bond typically from the conjugation of imine and the acid N▬H. These structural properties play a crucial role to determine the specificity of applications which hydrazone group can be involved [6, 9].

The main synthesis of hydrazones is carried out from acid-catalyzed condensation between hydrazines (R1NHNH2) and activated carbonyl aldehydes or ketones, generally in alcoholic media. Other forms to obtain hydrazones are from Japp-Klingemann reaction (i.e., aryl diazonium salts coupling with β-keto esters or acids) and coupling between aryl halides and non-substituted hydrazones [9].

### **1.1 Hydrazone-based compounds as fluorescent chemosensors**

Most hydrazone derivative fluorescent chemosensors were designed combining fluorophores or aromatic structures with this functional group. The wide range of chemical reactivity of hydrazones allows their application in the detection of anions, cations, and other species [10].

Some hydrazone-based chemosensors have weak fluorescence because of quenching effects such as E/Z double bond isomerization in the excited state; photoinduced electron transfer (PET) process (excited electron is transferred from donor to acceptor; generating a charge separation, i.e., redox reaction takes place in excited state); [11] excited state intramolecular proton transfer (ESIPT) process (photoexcited molecule relax their energy through tautomerization by transfer a proton); and others [12, 13]. The main objective for this class of chemosensors is inhibiting the quenching effects after interaction with some analytes promoting a fluorescence state. Other possible mechanisms are based on nucleophilic addition or induced N▬H and O▬H deprotonation. These mechanisms will be detailed after.

A quick literature survey using Scopus database has shown few reviews on the chemistry of hydrazones, most of them focusing on medicinal chemistry [14] or organic synthesis [15]. Only one review on hydrazone compounds describes some examples of hydrazone-based fluorescent chemosensor, which covered some results reported before 2014 [6].

This chapter book aims to present the progress of fluorescent and colorimetric chemosensor based on hydrazone scaffold, as reported in the literature in the period of 2006 until 2019. We hope that this chapter book helps in the design and development of new and selective fluorescent and colorimetric chemosensors for a broad range of applications.

### **2. Fluorescent chemosensors for anions**

Anions, such as cyanide (CN<sup>−</sup>), fluoride (F<sup>−</sup>), chlorine (Cl<sup>−</sup>), and acetate (AcO<sup>−</sup>), play an important role in many environmental, clinical, chemical, and biological processes. Due to these important roles, anion recognition is an area with growing interest in supramolecular chemistry, and considerable efforts has been focused on the design of receptors (compounds) that are able to recognize anions. The detection and quantification of anions is a challenge, especially in biological systems. Some aspects as microenvironmental sensitivity, specificity, basicity, and nucleophilicity are among the main complicating factors in the detection of anions. One way to solve these problems is to develop chemosensors with high specificity for individual anions [16, 17]. Among different types of anions, fluoride and cyanide aroused great interest. The optimum concentration of F<sup>−</sup> anions in the human body is a positive aspect to our health and can prevent dental caries and osteoporosis; however the excess of F<sup>−</sup> may cause dental or osseous fluorosis, thyroid and liver

**99**

**Figure 2.**

*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

× 10<sup>−</sup><sup>6</sup>

anions is of great importance.

*2.1.1 Nucleophilic addition of CN<sup>−</sup>*

damage, and bone diseases [18–20]. Additionally, F<sup>−</sup> is known as a test index for residues of some nerve agents, being also associated with certain drugs, Alzheimer disease, and drinking water. The level of F<sup>−</sup> recommended in potable water by the

Commonly involved in chemicals and industrial processes, cyanide is highly toxic and its exposure to live organisms and environment is extremely detrimental. Moreover, cyanide anion has a strong affinity with cytochrome a3 which can lead to cell death because of respiratory arrest [22]. According to the World Health Organization (WHO), the permissible level of cyanide in drinking water is 1.9

 M [18]. Therefore, considering the notorious toxicity of CN<sup>−</sup> and F<sup>−</sup>, the development of sensitive sensors for the accurate detection and quantification of

The main mechanisms of fluorescent sensing CN<sup>−</sup> in hydrazone derivatives are based on nucleophilic addition to polarized C═N [23, 24] and C═O [25] bonds, which leads to disruption of C═N and C═O double bond to C▬NH or C▬OH forms, deprotonation of NH or OH by means of acid-base reactions [26–28], and the displacement of fluorescent hydrazones from hydrazone-copper complexes.

Two highly selective CN<sup>−</sup> chemosensors **1**–**2** based on hydrazones functionalized with salicylaldehyde were described as capable of detecting this anion in aqueous solution at very low concentrations (**Figure 2A**). The ability of **1**–**2** complexing with several anions was tested by means of UV-vis absorption and fluorescence spectrometry. Among these anions, only CN<sup>−</sup> caused spectral changes due to its nucleophilic attack to the imine group (**Figure 2B**), and spectroscopy analysis (H1 NMR and MS studies) confirmed a 1:1 binding stoichiometry. In the presence of CN<sup>−</sup> (0–120 equivalents), a new intramolecular hydrogen bond network is formed, resulting in a turn-on fluorescence response for probe **1** and colorimetric naked-eye

*(A) Molecular structures of CN<sup>−</sup> chemosensors 1–3 with polarized C═N bond as sensing sites. (B) Proposed cyanide sensing mechanism of 1. (C) Proposed CN<sup>−</sup> sensing mechanism for 4 based on polarized C═O bond.*

US Environmental Protection Agency (EPA) is about 2 ppm [18–21].

**2.1 Hydrazone derivatives as chemosensors for CN<sup>−</sup>**

*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

*Computational Biology and Chemistry*

anions, cations, and other species [10].

reported before 2014 [6].

broad range of applications.

**2. Fluorescent chemosensors for anions**

electrophile character, two nucleophilic nitrogen in both imine and amine groups and a possible isomerization of C═N double bond typically from the conjugation of imine and the acid N▬H. These structural properties play a crucial role to determine

The main synthesis of hydrazones is carried out from acid-catalyzed condensation between hydrazines (R1NHNH2) and activated carbonyl aldehydes or ketones, generally in alcoholic media. Other forms to obtain hydrazones are from Japp-Klingemann reaction (i.e., aryl diazonium salts coupling with β-keto esters or acids)

Most hydrazone derivative fluorescent chemosensors were designed combining fluorophores or aromatic structures with this functional group. The wide range of chemical reactivity of hydrazones allows their application in the detection of

Some hydrazone-based chemosensors have weak fluorescence because of quenching effects such as E/Z double bond isomerization in the excited state; photoinduced electron transfer (PET) process (excited electron is transferred from donor to acceptor; generating a charge separation, i.e., redox reaction takes place in excited state); [11] excited state intramolecular proton transfer (ESIPT) process (photoexcited molecule relax their energy through tautomerization by transfer a proton); and others [12, 13]. The main objective for this class of chemosensors is inhibiting the quenching effects after interaction with some analytes promoting a fluorescence state. Other possible mechanisms are based on nucleophilic addition or induced N▬H and O▬H deprotonation. These mechanisms will be detailed after. A quick literature survey using Scopus database has shown few reviews on the chemistry of hydrazones, most of them focusing on medicinal chemistry [14] or organic synthesis [15]. Only one review on hydrazone compounds describes some examples of hydrazone-based fluorescent chemosensor, which covered some results

This chapter book aims to present the progress of fluorescent and colorimetric

chemosensor based on hydrazone scaffold, as reported in the literature in the period of 2006 until 2019. We hope that this chapter book helps in the design and development of new and selective fluorescent and colorimetric chemosensors for a

Anions, such as cyanide (CN<sup>−</sup>), fluoride (F<sup>−</sup>), chlorine (Cl<sup>−</sup>), and acetate (AcO<sup>−</sup>), play an important role in many environmental, clinical, chemical, and biological processes. Due to these important roles, anion recognition is an area with growing interest in supramolecular chemistry, and considerable efforts has been focused on the design of receptors (compounds) that are able to recognize anions. The detection and quantification of anions is a challenge, especially in biological systems. Some aspects as microenvironmental sensitivity, specificity, basicity, and nucleophilicity are among the main complicating factors in the detection of anions. One way to solve these problems is to develop chemosensors with high specificity for individual anions [16, 17]. Among different types of anions, fluoride and cyanide aroused great interest. The optimum concentration of F<sup>−</sup> anions in the human body is a positive aspect to our health and can prevent dental caries and osteoporosis; however the excess of F<sup>−</sup> may cause dental or osseous fluorosis, thyroid and liver

the specificity of applications which hydrazone group can be involved [6, 9].

and coupling between aryl halides and non-substituted hydrazones [9].

**1.1 Hydrazone-based compounds as fluorescent chemosensors**

**98**

damage, and bone diseases [18–20]. Additionally, F<sup>−</sup> is known as a test index for residues of some nerve agents, being also associated with certain drugs, Alzheimer disease, and drinking water. The level of F<sup>−</sup> recommended in potable water by the US Environmental Protection Agency (EPA) is about 2 ppm [18–21].

Commonly involved in chemicals and industrial processes, cyanide is highly toxic and its exposure to live organisms and environment is extremely detrimental. Moreover, cyanide anion has a strong affinity with cytochrome a3 which can lead to cell death because of respiratory arrest [22]. According to the World Health Organization (WHO), the permissible level of cyanide in drinking water is 1.9 × 10<sup>−</sup><sup>6</sup> M [18]. Therefore, considering the notorious toxicity of CN<sup>−</sup> and F<sup>−</sup>, the development of sensitive sensors for the accurate detection and quantification of anions is of great importance.

### **2.1 Hydrazone derivatives as chemosensors for CN<sup>−</sup>**

The main mechanisms of fluorescent sensing CN<sup>−</sup> in hydrazone derivatives are based on nucleophilic addition to polarized C═N [23, 24] and C═O [25] bonds, which leads to disruption of C═N and C═O double bond to C▬NH or C▬OH forms, deprotonation of NH or OH by means of acid-base reactions [26–28], and the displacement of fluorescent hydrazones from hydrazone-copper complexes.

### *2.1.1 Nucleophilic addition of CN<sup>−</sup>*

Two highly selective CN<sup>−</sup> chemosensors **1**–**2** based on hydrazones functionalized with salicylaldehyde were described as capable of detecting this anion in aqueous solution at very low concentrations (**Figure 2A**). The ability of **1**–**2** complexing with several anions was tested by means of UV-vis absorption and fluorescence spectrometry. Among these anions, only CN<sup>−</sup> caused spectral changes due to its nucleophilic attack to the imine group (**Figure 2B**), and spectroscopy analysis (H1 NMR and MS studies) confirmed a 1:1 binding stoichiometry. In the presence of CN<sup>−</sup> (0–120 equivalents), a new intramolecular hydrogen bond network is formed, resulting in a turn-on fluorescence response for probe **1** and colorimetric naked-eye

### **Figure 2.**

*(A) Molecular structures of CN<sup>−</sup> chemosensors 1–3 with polarized C═N bond as sensing sites. (B) Proposed cyanide sensing mechanism of 1. (C) Proposed CN<sup>−</sup> sensing mechanism for 4 based on polarized C═O bond.*

changes for probe **2** [23]. With the same sensing mechanism based on nucleophilic attack of CN<sup>−</sup> on the imine group, a remarkably and selective fluorescent and colorimetric chemosensor **3** was reported (**Figure 2A**). Among various anions, sensor **3** responded to only CN<sup>−</sup>, resulting in a color change from colorless to yellow. Moreover, a fluorescence analysis showed **3** has a weak fluorescence, and after addition of CN<sup>−</sup> (0–120 equivalents), the fluorescence emission has increased to a bright green fluorescence [24].

Exploring the same principle of nucleophilic attack, a highly selective and sensitive naphthalene-acylhydrazone chemosensor for CN<sup>−</sup> in aqueous media was designed. By this time, the mechanism proposed was the nucleophilic attack on the carbonyl group instead of the imine one, according to <sup>1</sup> H NMR, 13C NMR, ESI-MS, and DFT calculations data (**Figure 2C**). Among several anions tested, only CN<sup>−</sup> could induce a remarkable color change from colorless to yellow and increase of fluorescence emission in DMSO/H2O solution. Moreover, the detection limits were 5.0 × 10<sup>−</sup><sup>7</sup> M and 2.0 × 10<sup>−</sup><sup>9</sup> M of CN<sup>−</sup> for color and fluorescence changes respectively, far lower than the WHO guideline of 1.9 × 10<sup>−</sup><sup>6</sup> M [25].

### *2.1.2 Deprotonation mechanism*

Cyanide anion is a Lewis base and can form hydrogen bonds with hydrogen bond donors as hydroxyl and amines usually followed by deprotonation.

The highly selective and sensitive chemosensor (**5**) based on acyl hydrazone could detect CN<sup>−</sup> in aqueous solution with colorimetric and fluorimetric turn-on response (**Figure 3A**). The detection limit of CN<sup>−</sup> was 1.2 × 10<sup>−</sup><sup>9</sup> M, which is lower than the maximum level of 1.9 × 10<sup>−</sup><sup>6</sup> M for cyanide in drinking water according to WHO guidelines. Additionally, test strips based on **5** were fabricated and demonstrated that it could be used as an efficient CN<sup>−</sup> sensing in aqueous solution [26].

Another deprotonation mechanism was reported in the design of a two-dimensional carbazole-based chromophore **6** as chemosensor for the measurement of CN<sup>−</sup>. In terms of structure, this compound possesses two types of donor-π-acceptor (D-π-A) chromophores, where carbazole moiety is a donor for the two branches, each one with an accepting group (**Figure 3B**). Addition of CN<sup>−</sup> to the solution of **6** in DMSO/H2O (95/5, v/v) redshifted from 440 nm to 500 nm its absorption, changing its naked-eye observed color from yellow to violet. The sensing mechanism proposed was based on proton abstraction of ▬NH▬ when this group reacts with CN<sup>−</sup> resulting in the obvious color change [27].

Like compound **5**, a rhodamine B hydrazone derivative (**7**) was reported as highly selective chemosensor for CN<sup>−</sup> by means of a phenol deprotonation.

**Figure 3.** *Molecular structures of CN<sup>−</sup> chemosensors 5 (A), 6 (B), and 7 (C) and their proposed sensing mechanism.*

**101**

**Figure 4.**

*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

tion limit observed of 5.81 × 10<sup>−</sup><sup>8</sup>

detection [28].

intramolecular charge transfer (ICT), supported by <sup>1</sup>

**2.2 Hydrazone derivatives as chemosensors for F<sup>−</sup>**

donors. This coordination usually promotes deprotonation.

was successful, and fluoride solution exhibited color changes [29].

protons in a three-step process (**Figure 4B**) confirmed by <sup>1</sup>

from the observed peak of the FHF− at *δ* = 16.13 ppm [30].

*Molecular structure of F<sup>−</sup> chemosensor 8 (A) and 9 (B) and their proposed sensing mechanism.*

*2.2.1 Hydrogen bond interactions and deprotonation*

The complete sensing mechanism proposed was deprotonation followed by an

calculations (**Figure 3C**). This compound is a fluorescence and colorimetric sensor in DMSO/H2O (1:9) medium with a significant color change after addition of CN<sup>−</sup> to a chemosensor solution from colorless to pale yellow visible to the naked eye, and the fluorescence increased to strong green fluorescence. The good detec-

for cyanide according to WHO guidelines, leading this compound to be applied in the detection of CN<sup>−</sup> in germinated potatoes and also in tests strips for CN<sup>−</sup>

Fluoride is a weak Lewis base and can form hydrogen bonds with hydrogen bond

A Ru-bpy-based quinone hydrazone was designed as chromo-fluorogenic hybrid chemosensor (**8**) for F<sup>−</sup> (**Figure 4A**). This compound contains a quinone-hydrazone group that can be converted to azophenol tautomer in **8-F**<sup>−</sup> induced by the proton transfer from **8** to F<sup>−</sup> causing a color change from orange to blue-violet. Only F<sup>−</sup> was capable of inducing color change to **8** in MeCN, suggesting the high selectivity could be attributed to the strong intramolecular N▬H▬O hydrogen bond interaction in **8**, which means only the most electronegative anion could form an additional hydrogen bond. Generally, anion sensors based on hydrogen bond interactions cannot serve as good sensors in aqueous media due to hydrogen bond competition with water. To avoid this problem, a filter paper impregnated with acetonitrile solution of **8** and dried in air has been prepared. Immersing this paper into aqueous fluoride solution

A thiocarbonohydrazone anion chemosensor **9** was described and rationally designed based on previously reported anion chemosensors, where the presence of strong electron withdrawing ▬NO2 group enhanced the acidity of the thioamide protons and stabilized the negative deprotonated species (**Figure 4B**). After successive addition of F<sup>−</sup>, the UV-vis absorption band with maximum at 360 nm has decreased, whereas new peaks at 407 and 495 nm appeared. The absorption at 360 nm is attributed to the Ar▬CH═N▬NH conjugation moiety, and its bathochromic shift clearly indicated an interaction/reaction of fluoride with this portion. After addition of more than four equivalents of F<sup>−</sup>, a new absorption band at 600 nm appeared with a new isosbestic point at 535 nm. The significant changes in UV-vis spectra of chemosensor **9** was attributed to the deprotonation of thioamide

H NMR studies and DFT

H NMR titration analysis

M

is lower than the maximum level of 1.9 × 10<sup>−</sup><sup>6</sup>

*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

*Computational Biology and Chemistry*

green fluorescence [24].

M and 2.0 × 10<sup>−</sup><sup>9</sup>

than the maximum level of 1.9 × 10<sup>−</sup><sup>6</sup>

CN<sup>−</sup> resulting in the obvious color change [27].

*2.1.2 Deprotonation mechanism*

5.0 × 10<sup>−</sup><sup>7</sup>

changes for probe **2** [23]. With the same sensing mechanism based on nucleophilic attack of CN<sup>−</sup> on the imine group, a remarkably and selective fluorescent and colorimetric chemosensor **3** was reported (**Figure 2A**). Among various anions, sensor **3** responded to only CN<sup>−</sup>, resulting in a color change from colorless to yellow. Moreover, a fluorescence analysis showed **3** has a weak fluorescence, and after addition of CN<sup>−</sup> (0–120 equivalents), the fluorescence emission has increased to a bright

Exploring the same principle of nucleophilic attack, a highly selective and sensitive naphthalene-acylhydrazone chemosensor for CN<sup>−</sup> in aqueous media was designed. By this time, the mechanism proposed was the nucleophilic attack on the

and DFT calculations data (**Figure 2C**). Among several anions tested, only CN<sup>−</sup> could induce a remarkable color change from colorless to yellow and increase of fluorescence emission in DMSO/H2O solution. Moreover, the detection limits were

Cyanide anion is a Lewis base and can form hydrogen bonds with hydrogen bond

The highly selective and sensitive chemosensor (**5**) based on acyl hydrazone could detect CN<sup>−</sup> in aqueous solution with colorimetric and fluorimetric turn-on

WHO guidelines. Additionally, test strips based on **5** were fabricated and demonstrated that it could be used as an efficient CN<sup>−</sup> sensing in aqueous solution [26]. Another deprotonation mechanism was reported in the design of a two-dimensional carbazole-based chromophore **6** as chemosensor for the measurement of CN<sup>−</sup>. In terms of structure, this compound possesses two types of donor-π-acceptor (D-π-A) chromophores, where carbazole moiety is a donor for the two branches, each one with an accepting group (**Figure 3B**). Addition of CN<sup>−</sup> to the solution of **6** in DMSO/H2O (95/5, v/v) redshifted from 440 nm to 500 nm its absorption, changing its naked-eye observed color from yellow to violet. The sensing mechanism proposed was based on proton abstraction of ▬NH▬ when this group reacts with

Like compound **5**, a rhodamine B hydrazone derivative (**7**) was reported as highly selective chemosensor for CN<sup>−</sup> by means of a phenol deprotonation.

*Molecular structures of CN<sup>−</sup> chemosensors 5 (A), 6 (B), and 7 (C) and their proposed sensing mechanism.*

M of CN<sup>−</sup> for color and fluorescence changes respec-

M [25].

M for cyanide in drinking water according to

H NMR, 13C NMR, ESI-MS,

M, which is lower

carbonyl group instead of the imine one, according to <sup>1</sup>

tively, far lower than the WHO guideline of 1.9 × 10<sup>−</sup><sup>6</sup>

donors as hydroxyl and amines usually followed by deprotonation.

response (**Figure 3A**). The detection limit of CN<sup>−</sup> was 1.2 × 10<sup>−</sup><sup>9</sup>

**100**

**Figure 3.**

The complete sensing mechanism proposed was deprotonation followed by an intramolecular charge transfer (ICT), supported by <sup>1</sup> H NMR studies and DFT calculations (**Figure 3C**). This compound is a fluorescence and colorimetric sensor in DMSO/H2O (1:9) medium with a significant color change after addition of CN<sup>−</sup> to a chemosensor solution from colorless to pale yellow visible to the naked eye, and the fluorescence increased to strong green fluorescence. The good detection limit observed of 5.81 × 10<sup>−</sup><sup>8</sup> is lower than the maximum level of 1.9 × 10<sup>−</sup><sup>6</sup> M for cyanide according to WHO guidelines, leading this compound to be applied in the detection of CN<sup>−</sup> in germinated potatoes and also in tests strips for CN<sup>−</sup> detection [28].

### **2.2 Hydrazone derivatives as chemosensors for F<sup>−</sup>**

### *2.2.1 Hydrogen bond interactions and deprotonation*

Fluoride is a weak Lewis base and can form hydrogen bonds with hydrogen bond donors. This coordination usually promotes deprotonation.

A Ru-bpy-based quinone hydrazone was designed as chromo-fluorogenic hybrid chemosensor (**8**) for F<sup>−</sup> (**Figure 4A**). This compound contains a quinone-hydrazone group that can be converted to azophenol tautomer in **8-F**<sup>−</sup> induced by the proton transfer from **8** to F<sup>−</sup> causing a color change from orange to blue-violet. Only F<sup>−</sup> was capable of inducing color change to **8** in MeCN, suggesting the high selectivity could be attributed to the strong intramolecular N▬H▬O hydrogen bond interaction in **8**, which means only the most electronegative anion could form an additional hydrogen bond. Generally, anion sensors based on hydrogen bond interactions cannot serve as good sensors in aqueous media due to hydrogen bond competition with water. To avoid this problem, a filter paper impregnated with acetonitrile solution of **8** and dried in air has been prepared. Immersing this paper into aqueous fluoride solution was successful, and fluoride solution exhibited color changes [29].

A thiocarbonohydrazone anion chemosensor **9** was described and rationally designed based on previously reported anion chemosensors, where the presence of strong electron withdrawing ▬NO2 group enhanced the acidity of the thioamide protons and stabilized the negative deprotonated species (**Figure 4B**). After successive addition of F<sup>−</sup>, the UV-vis absorption band with maximum at 360 nm has decreased, whereas new peaks at 407 and 495 nm appeared. The absorption at 360 nm is attributed to the Ar▬CH═N▬NH conjugation moiety, and its bathochromic shift clearly indicated an interaction/reaction of fluoride with this portion. After addition of more than four equivalents of F<sup>−</sup>, a new absorption band at 600 nm appeared with a new isosbestic point at 535 nm. The significant changes in UV-vis spectra of chemosensor **9** was attributed to the deprotonation of thioamide protons in a three-step process (**Figure 4B**) confirmed by <sup>1</sup> H NMR titration analysis from the observed peak of the FHF− at *δ* = 16.13 ppm [30].

**Figure 4.** *Molecular structure of F<sup>−</sup> chemosensor 8 (A) and 9 (B) and their proposed sensing mechanism.*

Still exploring the N▬H acidity of hydrazones, a new series of diketopyrrolopyrrole (DPP) derivatives **10**–**14** bearing phenylhydrazone group was described presenting a ESIPT-PET fluoride sensing mechanism. The authors aimed to study the effect of nitro substituent of phenylhydrazone on their photophysical property and optical fluoride sensing. The anion sensing capabilities of **10**–**14** were evaluated in DMSO by the addition of several anions. The presence of nitro substituent at *ortho*-position of phenylhydrazone in **11** and **14** significantly altered the electronic properties through intramolecular hydrogen bonding and furnished an excited state intramolecular proton transfer (**Figure 5**). So, *o*-NO2-DPPH (**11**) has a weak fluorescence, partly attributed to the photoinduced electron transfer from the imine and similar situation was found in *o, p*-NO2-DPPH (**14**) (**Figure 5**).

The visual naked-eye color change was observed under natural light for DPPPH (**10**), *m*-NO2-DPPPH (**12**), *p*-NO2-DPPPH (**13**), and *o, p*-NO2-DPPPH (**14**) in the presence of F<sup>−</sup>. Among these designed chemoreceptors, *p*-NO2-DPPPH demonstrated selectivity for F<sup>−</sup> in UV-vis and fluorescence evaluations in DMSO solution. Additionally, experimental results of 1 H NMR and 19F NMR revealed that the spectral changes occur due to deprotonation of the hydrazone N▬H moiety by fluoride ion [31].

### **2.3 Hydrazone derivatives as chemosensor for AcO<sup>−</sup>**

Acetate (AcO<sup>−</sup>) and dicarboxylate are essential components in several metabolic processes in living organisms. Without them, many enzymes and antibodies are unable to function properly. In this sense, the synthesis of chemosensors that can recognize AcO<sup>−</sup>, mainly via hydrogen bond interaction, is of great importance for biological systems [32].

An interesting naked-eye selective colorimetric sensor for AcO<sup>−</sup> based on 1,10-phenanthroline-2,9-dicarboxyaldehyde-di-(p-nitrophenylhydrazone) (**15**) was described by Lin's group. The UV-vis absorption in DMSO showed a dramatic color change from yellow to green in the presence of AcO<sup>−</sup> with no changes for other anions. The presence of electron withdrawing groups increasing the hydrogen bond donor ability of N▬H framework was favorable for AcO<sup>−</sup> sensing. In addition, 1 H NMR studies showed that after interaction, AcO<sup>−</sup> lead to deprotonation of **15** [33].

Similarly a fluorescent and naked-eye colorimetric chemosensor (**16**) for AcO<sup>−</sup> based on thiosemicarbazone was evaluated by UV–vis spectroscopic titrations in dry DMSO solution, presenting high selectivity and affinity for AcO<sup>−</sup>. After addition of this anion, the absorption band at 373 nm decreased gradually, whereas a new band appeared at 457 nm, followed by color change in the solution from light yellow to orange. The complex formed between AcO<sup>−</sup> and **16**, through hydrogen bond interactions, caused intramolecular charge transfer between the electron-rich urea unit and the electron-deficient benzene moiety (**Figure 6**). In addition, the fluorescence spectroscopic titrations were carried out, and the **16** also displayed a switch-on reaction toward AcO<sup>−</sup>, which was attributed to the binding-induced

**103**

**Figure 7.**

further <sup>1</sup>

**Figure 6.**

H2PO4

*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

rigidity of the host molecule. The free receptor (**16**) had a flexible configuration and could rotate freely; however, after complexation with AcO<sup>−</sup>, the host molecule

In contrast to previously described, where receptors had a certain degree of selectivity toward single anion, several hydrazone-based chemosensors have been published exhibiting a response to more than one anion species, and some interest-

A tripodal benzaldehyde-phenylhydrazone (**17**) was developed as a colorimetric

UV-vis spectroscopic studies in DMSO were used to determine the binding mode of **17**. After addition of these anions, a naked-eye color change from yellow to purple and a significant bathochromic shift of 124 nm were observed. However, both the color and spectral changes were reverted by the addition of protic solvent such as H2O into the mentioned host-guest system, indicating that protic solvents could compete with anion binding sites. Considering these results, the authors proposed that a strong hydrogen bonding interaction was taking place between receptor

described in **Figure 7** was only attributed after assays indicating that stoichiometry of the host and specific guests was different depending on the anion. The receptor formed 1:1 complex with F<sup>−</sup> ion and 1:3 complexes with AcO<sup>−</sup>, OH<sup>−</sup>, and H2PO4

This was attributed to the smaller ionic radius of F<sup>−</sup> than that of other larger ones. A

to NH protons, exhibited a downfield to 12.27 ppm upon addition of 2 equivalents

A Schiff-base thiophene-based hydrazone (**18**) was described as visual anion chemosensor in aqueous media exhibiting sensing properties for F<sup>−</sup>, AcO<sup>−</sup>, and

<sup>−</sup>, among several anions tested with colorimetric response changes from

of F<sup>−</sup> ion confirming the formation of NH…F<sup>−</sup> hydrogen bonding [34].

*Molecular structures of multianalyte chemosensor 17 and their proposed sensing mechanism.*

H NMR investigation showed that resonance peak at 11.77 ppm, attributed

<sup>−</sup> by Lin and colleagues, and

<sup>−</sup>. The complete interaction mode

−.

was rigidified, and the fluorescence emission has increased [32].

*Molecular structures of acetate chemosensors 15–16 and the proposed sensing mechanism.*

**2.4 Hydrazone derivatives as chemosensors for multiple anions**

ing examples will be described below [34–36].

**17** and AcO<sup>−</sup> as well as F<sup>−</sup>, OH<sup>−</sup>, and H2PO4

naked-eye chemosensor for AcO<sup>−</sup>, F<sup>−</sup>, and H2PO4

**Figure 5.** *Molecular structures of F<sup>−</sup> chemosensors 10–14.*

*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

*Computational Biology and Chemistry*

Additionally, experimental results of 1

biological systems [32].

**2.3 Hydrazone derivatives as chemosensor for AcO<sup>−</sup>**

Still exploring the N▬H acidity of hydrazones, a new series of diketopyrrolopyrrole (DPP) derivatives **10**–**14** bearing phenylhydrazone group was described presenting a ESIPT-PET fluoride sensing mechanism. The authors aimed to study the effect of nitro substituent of phenylhydrazone on their photophysical property and optical fluoride sensing. The anion sensing capabilities of **10**–**14** were evaluated in DMSO by the addition of several anions. The presence of nitro substituent at *ortho*-position of phenylhydrazone in **11** and **14** significantly altered the electronic properties through intramolecular hydrogen bonding and furnished an excited state intramolecular proton transfer (**Figure 5**). So, *o*-NO2-DPPH (**11**) has a weak fluorescence, partly attributed to the photoinduced electron transfer from the imine

The visual naked-eye color change was observed under natural light for DPPPH (**10**), *m*-NO2-DPPPH (**12**), *p*-NO2-DPPPH (**13**), and *o, p*-NO2-DPPPH (**14**) in the presence of F<sup>−</sup>. Among these designed chemoreceptors, *p*-NO2-DPPPH demonstrated selectivity for F<sup>−</sup> in UV-vis and fluorescence evaluations in DMSO solution.

changes occur due to deprotonation of the hydrazone N▬H moiety by fluoride ion [31].

Acetate (AcO<sup>−</sup>) and dicarboxylate are essential components in several metabolic

processes in living organisms. Without them, many enzymes and antibodies are unable to function properly. In this sense, the synthesis of chemosensors that can recognize AcO<sup>−</sup>, mainly via hydrogen bond interaction, is of great importance for

An interesting naked-eye selective colorimetric sensor for AcO<sup>−</sup> based on 1,10-phenanthroline-2,9-dicarboxyaldehyde-di-(p-nitrophenylhydrazone) (**15**) was described by Lin's group. The UV-vis absorption in DMSO showed a dramatic color change from yellow to green in the presence of AcO<sup>−</sup> with no changes for other anions. The presence of electron withdrawing groups increasing the hydrogen bond donor ability of N▬H framework was favorable for AcO<sup>−</sup> sensing. In addition, 1

NMR studies showed that after interaction, AcO<sup>−</sup> lead to deprotonation of **15** [33]. Similarly a fluorescent and naked-eye colorimetric chemosensor (**16**) for AcO<sup>−</sup> based on thiosemicarbazone was evaluated by UV–vis spectroscopic titrations in dry DMSO solution, presenting high selectivity and affinity for AcO<sup>−</sup>. After addition of this anion, the absorption band at 373 nm decreased gradually, whereas a new band appeared at 457 nm, followed by color change in the solution from light yellow to orange. The complex formed between AcO<sup>−</sup> and **16**, through hydrogen bond interactions, caused intramolecular charge transfer between the electron-rich urea unit and the electron-deficient benzene moiety (**Figure 6**). In addition, the fluorescence spectroscopic titrations were carried out, and the **16** also displayed a switch-on reaction toward AcO<sup>−</sup>, which was attributed to the binding-induced

H NMR and 19F NMR revealed that the spectral

H

and similar situation was found in *o, p*-NO2-DPPH (**14**) (**Figure 5**).

**102**

**Figure 5.**

*Molecular structures of F<sup>−</sup> chemosensors 10–14.*

**Figure 6.** *Molecular structures of acetate chemosensors 15–16 and the proposed sensing mechanism.*

rigidity of the host molecule. The free receptor (**16**) had a flexible configuration and could rotate freely; however, after complexation with AcO<sup>−</sup>, the host molecule was rigidified, and the fluorescence emission has increased [32].

### **2.4 Hydrazone derivatives as chemosensors for multiple anions**

In contrast to previously described, where receptors had a certain degree of selectivity toward single anion, several hydrazone-based chemosensors have been published exhibiting a response to more than one anion species, and some interesting examples will be described below [34–36].

A tripodal benzaldehyde-phenylhydrazone (**17**) was developed as a colorimetric naked-eye chemosensor for AcO<sup>−</sup>, F<sup>−</sup>, and H2PO4 <sup>−</sup> by Lin and colleagues, and UV-vis spectroscopic studies in DMSO were used to determine the binding mode of **17**. After addition of these anions, a naked-eye color change from yellow to purple and a significant bathochromic shift of 124 nm were observed. However, both the color and spectral changes were reverted by the addition of protic solvent such as H2O into the mentioned host-guest system, indicating that protic solvents could compete with anion binding sites. Considering these results, the authors proposed that a strong hydrogen bonding interaction was taking place between receptor **17** and AcO<sup>−</sup> as well as F<sup>−</sup>, OH<sup>−</sup>, and H2PO4 <sup>−</sup>. The complete interaction mode described in **Figure 7** was only attributed after assays indicating that stoichiometry of the host and specific guests was different depending on the anion. The receptor formed 1:1 complex with F<sup>−</sup> ion and 1:3 complexes with AcO<sup>−</sup>, OH<sup>−</sup>, and H2PO4 −. This was attributed to the smaller ionic radius of F<sup>−</sup> than that of other larger ones. A further <sup>1</sup> H NMR investigation showed that resonance peak at 11.77 ppm, attributed to NH protons, exhibited a downfield to 12.27 ppm upon addition of 2 equivalents of F<sup>−</sup> ion confirming the formation of NH…F<sup>−</sup> hydrogen bonding [34].

A Schiff-base thiophene-based hydrazone (**18**) was described as visual anion chemosensor in aqueous media exhibiting sensing properties for F<sup>−</sup>, AcO<sup>−</sup>, and H2PO4 <sup>−</sup>, among several anions tested with colorimetric response changes from

**Figure 7.** *Molecular structures of multianalyte chemosensor 17 and their proposed sensing mechanism.*

**Figure 8.** *Molecular structure of multianalyte chemosensor 18 (A) and 19–22 (B) and mechanism of acetate detection for 18 (A).*

orange to violet at a micromolar level. The UV-vis analysis confirmed the nakedeye colorimetric changes and showed a decrease in band centered at 420 nm and increase in intensity of the band at 590 nm with the clear isosbestic point at 520 nm after addition of F<sup>−</sup>, AcO<sup>−</sup>, and H2PO4 <sup>−</sup> to **18** solution. The chemosensor **18** works by means of a N▬H deprotonation mechanism. 1 H NMR analysis confirmed this mechanism, showing that **18** presented signals at *δ* 11.79 and 8.91 corresponding to N▬H and imine protons, respectively. After addition of AcO<sup>−</sup> and F<sup>−</sup> ion, N▬H signal disappeared (deprotonation), while the signal corresponding to the imine and phenyl rings shifted to the upfield at the region of *δ* 8.59 and 8.54 (**Figure 8**). Additionally, a real sample qualitative estimation analysis of F<sup>−</sup> and AcO<sup>−</sup> in commercially available toothpaste and vinegar was successfully achieved by this simple and easy colorimetric method [35].

Four furan/thiophene-based fluorescent hydrazones **19**–**22** were described as CN<sup>−</sup> and F<sup>−</sup> sensors (**Figure 8**) and could detect these ions with naked-eye color changes from yellow to blue, while their fluorescence emission intensities were completely quenched. The presence of electron donating/withdrawing groups attached to furan ring as in **21** (▬NO2) and **22** (▬CH3) curiously resulted in increased selectivity for CN<sup>−</sup> ions compared to F<sup>−</sup> ones. 1 H NMR confirmed that the sensing mechanism goes through hydrogen bonding interaction between sensors and F<sup>−</sup>/CN<sup>−</sup>, followed by deprotonation, leading to elicited ICT. Job's plot afforded a stoichiometry of 2:1 binding ratio between **19** and **20** and F<sup>−</sup> ions. However, curiously **21** and **22** exhibited a 1:1 ratio with F<sup>−</sup> and CN<sup>−</sup> due to steric constraint. The limit of detection (LOD) analysis revealed that the four sensors displayed the LOD below 0.3 ppm for CN<sup>−</sup> and F<sup>−</sup> and a good selectivity. Competitive experiments revealed a negligible perturbation in the optical response which confirms a higher selectivity for F<sup>−</sup> and CN<sup>−</sup> than other competitor anions [36].

### **3. Fluorescent chemosensors for metal ions**

Metal ions such as Cu2+, Zn2+, Fe3+, Al3+, Hg2+, Mg2+, etc. play an important role in many biological and environmental processes, and excessive or insufficient amounts may lead to diseases [37]. As an example, copper (Cu2+) is the third most abundant transition metal in the human body and plays essential roles in several environmental, chemical, and physiological systems. In living organisms, Cu2+ acts as a key catalytic center in many enzymes and as cofactor in a variety of metalloproteins [38]. Its insufficient concentration may affect the development of bone and brain tissues as well as the nervous and immune system, whereas excessive intake may lead to serious problems including cirrhosis and neurological diseases such as Alzheimer's and Wilson's diseases and prion disorders [39]. The extreme toxicity of heavy metal ions such as Pb2+ and Hg2+, even in small amounts, remains a danger to

**105**

**Figure 9.**

be 6.4 × 105

such as K<sup>+</sup>

M<sup>−</sup><sup>1</sup>

, Ag+

*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

human health and the environment, but they have been widely used in industrial processes [40]. Therefore, the development of sensitive sensors for the accurate

Fluorescent and colorimetric hydrazone-based chemosensors for Cu2+ attract interest and are mainly based on coordination mechanism, often quenching the

Coumarins are widely associated with hydrazones for sensing Cu2+ and the on–off fluorescent chemosensor (**23**) was described for Cu2+ detection in aqueous media. This chemosensor showed very strong luminescence in H2O/DMSO (9:1, v/v) with quantum yield of 0.289, which was almost completely quenched after addition of copper (1 equivalent), decreasing the quantum yield to 0.024. This process was associated with the complexation of Cu2+ to the tautomeric enol-like form of **23** leading to **23-Cu2+** and the PET mechanism (**Figure 9**). Compound **20** showed detection limit of 0.1 μM for Cu2+, which is useful to sense Cu2+ in blood system, a 1:1 binding mode supported by a Job's plot, an association constant estimated to

cation. Only Cu2+ causes a significant fluorescence decrease, while other metals

did not cause any significant change. In addition, living cell experiments were successfully applied, and confocal image changes were observed, demonstrating its

Differently from turn-off PET mechanism, off-on (turn-on) chemosensors for detecting Cu2+ often present a FRET mechanism (described by energy transfer between two light-sensitive molecules or chromophores, where a donor chromophore in its electronic excited state may transfer energy to an acceptor chromophore through nonradiative dipole-dipole coupling). An example is the hybrid coumarin-rhodamine hydrazone chemosensor **24** based on metal ioninduced FRET. In this mechanism, coumarin nucleus acts as donor and rhodamine acts as acceptor of energy. Free ligand coumarin-rhodamine hydrazone (**24**) absorbs around 460 nm, which is attributed to coumarin chromophore. The absorbance remained unchanged upon addition of various metal ions except Cu2+. Upon addition of Cu2+, the solution color changes from yellow to bright red indicating metal complexation. It was confirmed analyzing the decrease in absorption band centered at 460 nm with a slowly redshifts to 475 nm, while a new peak at 556 nm arise from the rhodamine chromophore in the visible region.

*Molecular structures of Cu2+ chemosensors 23 (A) and 24 (B) and their proposed sensing mechanism.*

value in practical applications and biological systems [42].

, and a response time of 2 min upon addition of 1 equivalent of this

, Ca2+, Cd2+, Co2+, Cr3+, Fe2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+ and Zn2+

detection and quantification of these ions is of great importance.

**3.1 Hydrazone derivatives as chemosensors for Cu2+**

fluorescence emission due to PET mechanism [41].

*Computational Biology and Chemistry*

**Figure 8.**

*for 18 (A).*

after addition of F<sup>−</sup>, AcO<sup>−</sup>, and H2PO4

and easy colorimetric method [35].

by means of a N▬H deprotonation mechanism. 1

increased selectivity for CN<sup>−</sup> ions compared to F<sup>−</sup> ones. 1

selectivity for F<sup>−</sup> and CN<sup>−</sup> than other competitor anions [36].

**3. Fluorescent chemosensors for metal ions**

orange to violet at a micromolar level. The UV-vis analysis confirmed the nakedeye colorimetric changes and showed a decrease in band centered at 420 nm and increase in intensity of the band at 590 nm with the clear isosbestic point at 520 nm

*Molecular structure of multianalyte chemosensor 18 (A) and 19–22 (B) and mechanism of acetate detection* 

mechanism, showing that **18** presented signals at *δ* 11.79 and 8.91 corresponding to N▬H and imine protons, respectively. After addition of AcO<sup>−</sup> and F<sup>−</sup> ion, N▬H signal disappeared (deprotonation), while the signal corresponding to the imine and phenyl rings shifted to the upfield at the region of *δ* 8.59 and 8.54 (**Figure 8**). Additionally, a real sample qualitative estimation analysis of F<sup>−</sup> and AcO<sup>−</sup> in commercially available toothpaste and vinegar was successfully achieved by this simple

Four furan/thiophene-based fluorescent hydrazones **19**–**22** were described as CN<sup>−</sup> and F<sup>−</sup> sensors (**Figure 8**) and could detect these ions with naked-eye color changes from yellow to blue, while their fluorescence emission intensities were completely quenched. The presence of electron donating/withdrawing groups attached to furan ring as in **21** (▬NO2) and **22** (▬CH3) curiously resulted in

sensing mechanism goes through hydrogen bonding interaction between sensors and F<sup>−</sup>/CN<sup>−</sup>, followed by deprotonation, leading to elicited ICT. Job's plot afforded a stoichiometry of 2:1 binding ratio between **19** and **20** and F<sup>−</sup> ions. However, curiously **21** and **22** exhibited a 1:1 ratio with F<sup>−</sup> and CN<sup>−</sup> due to steric constraint. The limit of detection (LOD) analysis revealed that the four sensors displayed the LOD below 0.3 ppm for CN<sup>−</sup> and F<sup>−</sup> and a good selectivity. Competitive experiments revealed a negligible perturbation in the optical response which confirms a higher

Metal ions such as Cu2+, Zn2+, Fe3+, Al3+, Hg2+, Mg2+, etc. play an important role in many biological and environmental processes, and excessive or insufficient amounts may lead to diseases [37]. As an example, copper (Cu2+) is the third most abundant transition metal in the human body and plays essential roles in several environmental, chemical, and physiological systems. In living organisms, Cu2+ acts as a key catalytic center in many enzymes and as cofactor in a variety of metalloproteins [38]. Its insufficient concentration may affect the development of bone and brain tissues as well as the nervous and immune system, whereas excessive intake may lead to serious problems including cirrhosis and neurological diseases such as Alzheimer's and Wilson's diseases and prion disorders [39]. The extreme toxicity of heavy metal ions such as Pb2+ and Hg2+, even in small amounts, remains a danger to

<sup>−</sup> to **18** solution. The chemosensor **18** works

H NMR analysis confirmed this

H NMR confirmed that the

**104**

human health and the environment, but they have been widely used in industrial processes [40]. Therefore, the development of sensitive sensors for the accurate detection and quantification of these ions is of great importance.

### **3.1 Hydrazone derivatives as chemosensors for Cu2+**

Fluorescent and colorimetric hydrazone-based chemosensors for Cu2+ attract interest and are mainly based on coordination mechanism, often quenching the fluorescence emission due to PET mechanism [41].

Coumarins are widely associated with hydrazones for sensing Cu2+ and the on–off fluorescent chemosensor (**23**) was described for Cu2+ detection in aqueous media. This chemosensor showed very strong luminescence in H2O/DMSO (9:1, v/v) with quantum yield of 0.289, which was almost completely quenched after addition of copper (1 equivalent), decreasing the quantum yield to 0.024. This process was associated with the complexation of Cu2+ to the tautomeric enol-like form of **23** leading to **23-Cu2+** and the PET mechanism (**Figure 9**). Compound **20** showed detection limit of 0.1 μM for Cu2+, which is useful to sense Cu2+ in blood system, a 1:1 binding mode supported by a Job's plot, an association constant estimated to be 6.4 × 105 M<sup>−</sup><sup>1</sup> , and a response time of 2 min upon addition of 1 equivalent of this cation. Only Cu2+ causes a significant fluorescence decrease, while other metals such as K<sup>+</sup> , Ag+ , Ca2+, Cd2+, Co2+, Cr3+, Fe2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+ and Zn2+ did not cause any significant change. In addition, living cell experiments were successfully applied, and confocal image changes were observed, demonstrating its value in practical applications and biological systems [42].

Differently from turn-off PET mechanism, off-on (turn-on) chemosensors for detecting Cu2+ often present a FRET mechanism (described by energy transfer between two light-sensitive molecules or chromophores, where a donor chromophore in its electronic excited state may transfer energy to an acceptor chromophore through nonradiative dipole-dipole coupling). An example is the hybrid coumarin-rhodamine hydrazone chemosensor **24** based on metal ioninduced FRET. In this mechanism, coumarin nucleus acts as donor and rhodamine acts as acceptor of energy. Free ligand coumarin-rhodamine hydrazone (**24**) absorbs around 460 nm, which is attributed to coumarin chromophore. The absorbance remained unchanged upon addition of various metal ions except Cu2+. Upon addition of Cu2+, the solution color changes from yellow to bright red indicating metal complexation. It was confirmed analyzing the decrease in absorption band centered at 460 nm with a slowly redshifts to 475 nm, while a new peak at 556 nm arise from the rhodamine chromophore in the visible region.

**Figure 9.** *Molecular structures of Cu2+ chemosensors 23 (A) and 24 (B) and their proposed sensing mechanism.*

Free coumarin-rhodamine hydrazone (**24**) emits in the green fluorescence region with a fluorescence band centered at 524 nm which is attributed to *N,N*-diethylcoumarin moiety upon excitation at 480 nm. Upon sequential addition of Cu2+, coumarin emission band was observed at 524 nm, while an emission signal corresponding to ring-opened rhodamine appeared at 582 nm (**Figure 9**). This chromo-fluorogenic probe can detect concentrations below 20 μM of Cu2+ in aqueous buffer medium. The FRET mechanism is possible due to integral overlap between emission band of *N,N*-diethyl coumarin moiety and absorption band of ring-opened zwitterionic rhodamine unit in buffer medium. Additionally, probe **24** undergoes a 1:1 stoichiometric complexation with Cu2+ with the calculated association constants of 8.81 M<sup>−</sup><sup>1</sup> and was successfully employed as ratiometric biosensor for living cell imaging of Cu2+ [43].

A highly selective and sensitive naked-eye colorimetric chemosensor for Cu2+ in aqueous solution was designed and developed based on hydrazone framework. In the UV-vis spectroscopic studies, compound **25** exhibited a broad band at 336 nm, and after addition of copper, a new absorption band at 502 nm appeared, whereas the absorption band at 336 nm was gradually reduced. This gradually increasing absorption peak at 502 nm (after sequential addition of Cu2+) was attributed to the coordination of **25** with Cu2+.

Trying to understand its sensing mechanism, the stoichiometry of the **25-Cu2+** complexation was determined by the Job's plot analysis which indicates a 2:1 stoichiometric between **25** and Cu2+ and after confirmed by ESI/MS analysis. With stoichiometry on hands, complementary infrared (IR) and <sup>1</sup> H NMR spectroscopy were employed, and even Cu2+ which is a paramagnetic ion helped to describe the coordination mode. The <sup>1</sup> H NMR revealed that N▬H (**a**) proton almost completely disappeared upon addition of Cu2+ and proton **b** became broader after binding, indicating that Cu2+ binds with nitrogen of pyridine after an initial tautomerization with a deprotonation of O▬H.

The selectivity of **25** over other metals was investigated by adding several metal cations to the solution of **25** in THF/H2O (9:1, v/v), and there was no obvious change with any other metal. The colorless solution of the compound **25** became pink after addition of Cu2+ with the detection limit by the naked eye around 2 μM which is lower than the limit of copper in drinking water (~20 μM). Additionally, to evaluate the practical application of chemosensor **25**, competition experiments of Cu2+ mixed with other metal ions were carried out from UV-vis absorption spectra. The treatment of **25** solution with Cu2+ in the presence of the same concentration of other metal cations did not show any significant changes [44].

Using a strategy of intraligand charge transfer transition (ILCT) turn-on mechanism, a small chromo-fluorogenic chemosensor (**26**) for Cu2+, based on hydrazones, was described. This chemosensor exhibited a weak fluorescence in DMSO with a 44-fold increase in fluorescence emission intensity upon addition of Cu2+, being attributed to ILCT. This receptor showed a prominent colorimetric change from yellow to brown in the presence of Cu2+ with a detection limit in the order of 10−<sup>8</sup> M. In the presence of other environmentally significant metal cations such as Hg2+, Pb2+, Cd2+, Ni2+, Co2+, Fe2+, Fe3+, Mn2+, Zn2+, Al3+, and Cr3+, no significant spectral changes were observed. Interesting, this chemosensor was capable of extracting Cu2+ selectively from aqueous mixture of metal ions using dichloromethane as solvent with an efficiency of 94% at 6.5–11 pH range.

The binding sense mechanism was explained by <sup>1</sup> H NMR titration in DMSO, in which the peak attributed to acid O▬H proton of **26** gradually decreased upon addition of Cu2+, also indicating a 2:1 (**26-Cu2+**) stoichiometric binding (**Figure 10**). During the extraction process, the stoichiometry of ligand and Cu2+ was confirmed to be 2:1, and, in addition, the color changes and concentration of Cu2+ were

**107**

cence [48].

*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

chemical and biological systems [45].

**Figure 10.**

*and its sensing mechanism (B).*

these probes often still lack specificity.

**3.2 Hydrazone derivatives as chemosensors for Zn2+**

monitored by a readily usable smartphone as an analysis tool. Interesting, this receptor showed a good recyclability and reusability in Cu2+ extraction, being very useful for the detection and selective extraction of Cu2+ from aqueous media in

*Molecular structures of Cu2+ chemosensor 25 and its coordination mode (A) and structure of chemosensor 26*

Zinc (Zn2+) is the second most abundant transition metal (after Fe3+) in the human body and is considered essential for living organisms. Zn2+ exerts influence on many cellular processes, including proliferation, differentiation, apoptosis, transcription, neural signal transmission, and microtubule polymerization. Therefore, significant changes in Zn2+ concentration may be related to many diseases, including Alzheimer's and Parkinson's diseases, diabetes, and prostate cancer [46, 47]. Chemosensors for Zn2+ are mainly based on the coordination mechanism; however,

Aroylhydrazone derivatives (**27–28**) were described as fluorescent chemosensors for Zn2+ recognition. These ligands and their metal complexes (**27** (Zn2+, Cu2+), **28** (Zn2+, Co2+, Fe3+)) have been synthesized and characterized in terms of their crystal structures, elemental analysis, and spectroscopic properties. First, chemosensor **27** displayed high selectivity for Zn2+ over other transition metals compared to **28** in aqueous ethanol solution, which indicate that hydroxyl group exerts effect on the selectivity of the fluorescent chemosensor. The possible reason is that the presence of hydroxyl group gives the carbonyl a better binding ability for Zn2+ to form a 1:1 complex. The fluorescence response of **27** in solution increased approximately 25-fold upon addition of 10 equivalents of Zn2+ probably due to rigidity imposed by the complex formed, inhibiting isomerization (**Figure 11**). Otherwise, **28** exhibited only small increase in fluorescence (5-fold) when Zn2+ was introduced to the solution, indicating also the importance of *ortho*-hydroxyl group for fluores*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

### **Figure 10.**

*Computational Biology and Chemistry*

association constants of 8.81 M<sup>−</sup><sup>1</sup>

coordination of **25** with Cu2+.

coordination mode. The <sup>1</sup>

with a deprotonation of O▬H.

biosensor for living cell imaging of Cu2+ [43].

stoichiometry on hands, complementary infrared (IR) and <sup>1</sup>

other metal cations did not show any significant changes [44].

solvent with an efficiency of 94% at 6.5–11 pH range. The binding sense mechanism was explained by <sup>1</sup>

Free coumarin-rhodamine hydrazone (**24**) emits in the green fluorescence region with a fluorescence band centered at 524 nm which is attributed to *N,N*-diethylcoumarin moiety upon excitation at 480 nm. Upon sequential addition of Cu2+, coumarin emission band was observed at 524 nm, while an emission signal corresponding to ring-opened rhodamine appeared at 582 nm (**Figure 9**). This chromo-fluorogenic probe can detect concentrations below 20 μM of Cu2+ in aqueous buffer medium. The FRET mechanism is possible due to integral overlap between emission band of *N,N*-diethyl coumarin moiety and absorption band of ring-opened zwitterionic rhodamine unit in buffer medium. Additionally, probe **24** undergoes a 1:1 stoichiometric complexation with Cu2+ with the calculated

A highly selective and sensitive naked-eye colorimetric chemosensor for Cu2+ in aqueous solution was designed and developed based on hydrazone framework. In the UV-vis spectroscopic studies, compound **25** exhibited a broad band at 336 nm, and after addition of copper, a new absorption band at 502 nm appeared, whereas the absorption band at 336 nm was gradually reduced. This gradually increasing absorption peak at 502 nm (after sequential addition of Cu2+) was attributed to the

Trying to understand its sensing mechanism, the stoichiometry of the **25-Cu2+** complexation was determined by the Job's plot analysis which indicates a 2:1 stoichiometric between **25** and Cu2+ and after confirmed by ESI/MS analysis. With

were employed, and even Cu2+ which is a paramagnetic ion helped to describe the

disappeared upon addition of Cu2+ and proton **b** became broader after binding, indicating that Cu2+ binds with nitrogen of pyridine after an initial tautomerization

cations to the solution of **25** in THF/H2O (9:1, v/v), and there was no obvious change with any other metal. The colorless solution of the compound **25** became pink after addition of Cu2+ with the detection limit by the naked eye around 2 μM which is lower than the limit of copper in drinking water (~20 μM). Additionally, to evaluate the practical application of chemosensor **25**, competition experiments of Cu2+ mixed with other metal ions were carried out from UV-vis absorption spectra. The treatment of **25** solution with Cu2+ in the presence of the same concentration of

the presence of other environmentally significant metal cations such as Hg2+, Pb2+, Cd2+, Ni2+, Co2+, Fe2+, Fe3+, Mn2+, Zn2+, Al3+, and Cr3+, no significant spectral changes were observed. Interesting, this chemosensor was capable of extracting Cu2+ selectively from aqueous mixture of metal ions using dichloromethane as

in which the peak attributed to acid O▬H proton of **26** gradually decreased upon addition of Cu2+, also indicating a 2:1 (**26-Cu2+**) stoichiometric binding (**Figure 10**). During the extraction process, the stoichiometry of ligand and Cu2+ was confirmed to be 2:1, and, in addition, the color changes and concentration of Cu2+ were

The selectivity of **25** over other metals was investigated by adding several metal

Using a strategy of intraligand charge transfer transition (ILCT) turn-on mechanism, a small chromo-fluorogenic chemosensor (**26**) for Cu2+, based on hydrazones, was described. This chemosensor exhibited a weak fluorescence in DMSO with a 44-fold increase in fluorescence emission intensity upon addition of Cu2+, being attributed to ILCT. This receptor showed a prominent colorimetric change from yellow to brown in the presence of Cu2+ with a detection limit in the order of 10−<sup>8</sup>

and was successfully employed as ratiometric

H NMR revealed that N▬H (**a**) proton almost completely

H NMR spectroscopy

M. In

H NMR titration in DMSO,

**106**

*Molecular structures of Cu2+ chemosensor 25 and its coordination mode (A) and structure of chemosensor 26 and its sensing mechanism (B).*

monitored by a readily usable smartphone as an analysis tool. Interesting, this receptor showed a good recyclability and reusability in Cu2+ extraction, being very useful for the detection and selective extraction of Cu2+ from aqueous media in chemical and biological systems [45].

### **3.2 Hydrazone derivatives as chemosensors for Zn2+**

Zinc (Zn2+) is the second most abundant transition metal (after Fe3+) in the human body and is considered essential for living organisms. Zn2+ exerts influence on many cellular processes, including proliferation, differentiation, apoptosis, transcription, neural signal transmission, and microtubule polymerization. Therefore, significant changes in Zn2+ concentration may be related to many diseases, including Alzheimer's and Parkinson's diseases, diabetes, and prostate cancer [46, 47]. Chemosensors for Zn2+ are mainly based on the coordination mechanism; however, these probes often still lack specificity.

Aroylhydrazone derivatives (**27–28**) were described as fluorescent chemosensors for Zn2+ recognition. These ligands and their metal complexes (**27** (Zn2+, Cu2+), **28** (Zn2+, Co2+, Fe3+)) have been synthesized and characterized in terms of their crystal structures, elemental analysis, and spectroscopic properties. First, chemosensor **27** displayed high selectivity for Zn2+ over other transition metals compared to **28** in aqueous ethanol solution, which indicate that hydroxyl group exerts effect on the selectivity of the fluorescent chemosensor. The possible reason is that the presence of hydroxyl group gives the carbonyl a better binding ability for Zn2+ to form a 1:1 complex. The fluorescence response of **27** in solution increased approximately 25-fold upon addition of 10 equivalents of Zn2+ probably due to rigidity imposed by the complex formed, inhibiting isomerization (**Figure 11**). Otherwise, **28** exhibited only small increase in fluorescence (5-fold) when Zn2+ was introduced to the solution, indicating also the importance of *ortho*-hydroxyl group for fluorescence [48].

**Figure 11.**

*Molecular structures of Zn2+ chemosensors 27–28 and their sensing mechanism.*

A fluorescein-coumarin conjugate (**29**) was reported as turn-on fluorescent sensor for Zn2+ in aqueous medium. The mechanism involved in this chemosensor is related to spirolactam ring opening mediated selectively by Zn2+ over other earth and transition metal ions. The free chemosensor **29** showed almost no absorption characteristic of the fluorescein moiety which indicate the existence of the spirolactam form. However, upon Zn2+ addition in the sensing system, a new absorption band corresponding to fluorescein moiety increased indicating the generation of a ring-opening amide form (**29-Zn2+**). The absorbance ascended linearly as a function of Zn2+ concentration with a saturation at the ratio of 1:1 (**Figure 12**). The fluorescence emission intensity was increased 33-fold at 501 nm upon zinc addition, which is characteristic of fluorescein, confirming the spirocycle opening of **29** after coordination. In addition, this chemosensor was highly selective toward several metals such as Na+ , K+ , Ca2+, Mg2+, Cu2+, Fe3+, Co2+, Ni2+, Hg2+, Cd2+, and Cr3+ that showed none or little fluorescence intensity changes. The ability of chemosensor **29** works in aqueous medium and shows potential applications in environmental, biological, and medicinal areas [49].

The hydrazone-based fluorescent chemosensor **30** was reported as an interesting and selective sensor for Zn2+ over other biologically important metal ions (Na+ , K+ , Mg2+, Ca2+, Co2+, Ni2+, Cu2+, Cd2+, Ag+ , Pb2+, Hg2+, Al3+, Cr3+, Fe3+, and Zn2+) under completely physiological conditions (HEPES buffer medium (1 mM, pH = 7.4) containing 0.33% of DMSO), also demonstrating detection of zinc in live Hela cells by fluorescence imaging. The recognition of Zn2+ was investigated by absorption and emission spectroscopy, DFT calculations, ESI-MS experiment, and <sup>1</sup> H NMR titration.

The free ligand (**30**) exhibited two absorption peaks at 305 nm and 367 nm attributed to the π-π\* transitions, in which zinc addition (10 equivalents) promoted a prominent change where the absorption band at 305 nm decreased, whereas a new peak at 450 nm emerged. This absorption spectra change became minimum upon the addition of two equivalents of metal ions, which suggested a 1:2 ratio between **30** and Zn2+. Free receptor (**30**) has weak fluorescence with visible light excitability; however zinc caused drastic enhancement in the fluorescence emission with a prominent peak at 555 nm. The Zn2+ sensing mechanism was attributed to

**109**

concentration (3.6 × 10<sup>−</sup><sup>9</sup>

water (LOD = 10 nM) [54].

*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

(Φ = 0.16, 18-fold) of **30** (**Figure 12**) [50].

mechanism.

**3.3 Hydrazone derivatives as chemosensors for Hg2+**

indicating a 2:1 complex formation (**Figure 13**) [53].

the chelation-enhanced fluorescence (CHEF) and ICT processes. The low fluorescence state of **30** (Φ = 0.009) is may be due to free rotation of imine (▬C═N) bond (**Figure 12**). Upon Zn2+ addition, the coordination of this metal with imine nitrogen and the hydroxyl group inhibited the free rotation around the imine bond, leaving the system more rigid. Thus, the chelation of Zn2+ leads to the formation of binuclear zinc complex with drastic increase in conjugation, resulting in a CHEF effect. Furthermore, due to the binding of Zn2+, the ICT is facilitated over the π-system. These conjugated effects caused the improvement in the fluorescence emission

Mercuric ion (Hg2+) is considered highly dangerous, because it is known as one of the most toxic metal ions and is generated by many sources such as mercury lamps, gold production, electronic equipment, paints, and batteries [51]. Mercuric ion can cause serious detrimental effects to living organisms, resulting in hepatitis, uremia, digestive diseases, and fatal damage to the central nervous system, and its accumulation can lead to various cognitive and motor disorders, such as Minamata disease [52]. Due to its high toxicity, considerable attention has been devoted to the development of new sensors for Hg2+ detection. Hydrazonebased fluorescence chemosensors for Hg2+ are mainly based on coordination

One example is the 3,4-ethylenedioxythiophene (EDOT) rhodamine-hydrazinebased compound **31** which acts as colorimetric and turn-on fluorescent chemosensor for Hg2+ detection. Between several metals, only Hg2+ coordination promoted the turn-on effect. The sensing mechanism is quite similar to that observed to compound **29**, in which free receptor has a spirolactam moiety, which inhibits the intramolecular charge transfer between electron-acceptor moiety of xanthene and the electron donor of EDOT. Once Hg2+ complexation occurs, a ring opening takes place delivering the rhodamine B moiety, which is a well-known fluorophore. The spirolactam moiety of the rhodamine acts as a signal switcher, which is envisioned to turn on upon complexation with the cation. The spectroscopic parameters of Hg2+ in mixed ethanol and HEPES (10 mM, 1:1, v/v, pH = 7.2) solution demonstrated that only Hg2+ was capable to change the color of the sensing solution from colorless to red (570 nm) and to increase fluorescence emission (593 nm), also

The previous exposed FRET mechanism has been used in the design of selective turn-on fluorescent chemosensor for Hg2+ based on bis-hydrazone derivative from 2,5-furancarboxaldehyde. In this case, furan ring is the donor, and rhodamine B is the acceptor chromophore. Free ligand has the spirolactam moiety which in turn inhibits the charge transfer between these chromophores. When Hg2+ binds to **32**, a rapid naked-eye visual color change occurs from colorless to sharp pink, as well as a bright red fluorescent emission under UV lamp irradiation, which is attributed to the spirolactam ring opening (**Figure 13**). The FRET mechanism was confirmed by overlap of the emission band of 2,5-furan-dicarboxaldehyde (donor) and absorption spectra of rhodamine B (acceptor). After the binding of **32** to Hg2+, leading to **32-Hg2+**, furan moiety makes an energy transfer to induce the spirolactam opening which allowed the FRET process with increasing in the conjugation of the system (**Figure 13**). Additionally, the high increase in quantum yield of **32-Hg2+** (0.23) when they compared **32** (0.015) and its limit of the detection at very low ppb level

M) allowed applications for detecting Hg2+ in drinking

**Figure 12.** *Molecular structures of Zn2+ chemosensors 29 (A) and 30 (B) and their sensing mechanism.*

*Computational Biology and Chemistry*

metals such as Na+

**Figure 11.**

titration.

, K+

biological, and medicinal areas [49].

Mg2+, Ca2+, Co2+, Ni2+, Cu2+, Cd2+, Ag+

A fluorescein-coumarin conjugate (**29**) was reported as turn-on fluorescent sensor for Zn2+ in aqueous medium. The mechanism involved in this chemosensor is related to spirolactam ring opening mediated selectively by Zn2+ over other earth and transition metal ions. The free chemosensor **29** showed almost no absorption characteristic of the fluorescein moiety which indicate the existence of the spirolactam form. However, upon Zn2+ addition in the sensing system, a new absorption band corresponding to fluorescein moiety increased indicating the generation of a ring-opening amide form (**29-Zn2+**). The absorbance ascended linearly as a function of Zn2+ concentration with a saturation at the ratio of 1:1 (**Figure 12**). The fluorescence emission intensity was increased 33-fold at 501 nm upon zinc addition, which is characteristic of fluorescein, confirming the spirocycle opening of **29** after coordination. In addition, this chemosensor was highly selective toward several

*Molecular structures of Zn2+ chemosensors 27–28 and their sensing mechanism.*

showed none or little fluorescence intensity changes. The ability of chemosensor **29** works in aqueous medium and shows potential applications in environmental,

and selective sensor for Zn2+ over other biologically important metal ions (Na+

completely physiological conditions (HEPES buffer medium (1 mM, pH = 7.4) containing 0.33% of DMSO), also demonstrating detection of zinc in live Hela cells by fluorescence imaging. The recognition of Zn2+ was investigated by absorption

The free ligand (**30**) exhibited two absorption peaks at 305 nm and 367 nm attributed to the π-π\* transitions, in which zinc addition (10 equivalents) promoted a prominent change where the absorption band at 305 nm decreased, whereas a new peak at 450 nm emerged. This absorption spectra change became minimum upon the addition of two equivalents of metal ions, which suggested a 1:2 ratio between **30** and Zn2+. Free receptor (**30**) has weak fluorescence with visible light excitability; however zinc caused drastic enhancement in the fluorescence emission with a prominent peak at 555 nm. The Zn2+ sensing mechanism was attributed to

and emission spectroscopy, DFT calculations, ESI-MS experiment, and <sup>1</sup>

*Molecular structures of Zn2+ chemosensors 29 (A) and 30 (B) and their sensing mechanism.*

The hydrazone-based fluorescent chemosensor **30** was reported as an interesting

, Ca2+, Mg2+, Cu2+, Fe3+, Co2+, Ni2+, Hg2+, Cd2+, and Cr3+ that

, Pb2+, Hg2+, Al3+, Cr3+, Fe3+, and Zn2+) under

, K+ ,

H NMR

**108**

**Figure 12.**

the chelation-enhanced fluorescence (CHEF) and ICT processes. The low fluorescence state of **30** (Φ = 0.009) is may be due to free rotation of imine (▬C═N) bond (**Figure 12**). Upon Zn2+ addition, the coordination of this metal with imine nitrogen and the hydroxyl group inhibited the free rotation around the imine bond, leaving the system more rigid. Thus, the chelation of Zn2+ leads to the formation of binuclear zinc complex with drastic increase in conjugation, resulting in a CHEF effect. Furthermore, due to the binding of Zn2+, the ICT is facilitated over the π-system. These conjugated effects caused the improvement in the fluorescence emission (Φ = 0.16, 18-fold) of **30** (**Figure 12**) [50].

### **3.3 Hydrazone derivatives as chemosensors for Hg2+**

Mercuric ion (Hg2+) is considered highly dangerous, because it is known as one of the most toxic metal ions and is generated by many sources such as mercury lamps, gold production, electronic equipment, paints, and batteries [51]. Mercuric ion can cause serious detrimental effects to living organisms, resulting in hepatitis, uremia, digestive diseases, and fatal damage to the central nervous system, and its accumulation can lead to various cognitive and motor disorders, such as Minamata disease [52]. Due to its high toxicity, considerable attention has been devoted to the development of new sensors for Hg2+ detection. Hydrazonebased fluorescence chemosensors for Hg2+ are mainly based on coordination mechanism.

One example is the 3,4-ethylenedioxythiophene (EDOT) rhodamine-hydrazinebased compound **31** which acts as colorimetric and turn-on fluorescent chemosensor for Hg2+ detection. Between several metals, only Hg2+ coordination promoted the turn-on effect. The sensing mechanism is quite similar to that observed to compound **29**, in which free receptor has a spirolactam moiety, which inhibits the intramolecular charge transfer between electron-acceptor moiety of xanthene and the electron donor of EDOT. Once Hg2+ complexation occurs, a ring opening takes place delivering the rhodamine B moiety, which is a well-known fluorophore. The spirolactam moiety of the rhodamine acts as a signal switcher, which is envisioned to turn on upon complexation with the cation. The spectroscopic parameters of Hg2+ in mixed ethanol and HEPES (10 mM, 1:1, v/v, pH = 7.2) solution demonstrated that only Hg2+ was capable to change the color of the sensing solution from colorless to red (570 nm) and to increase fluorescence emission (593 nm), also indicating a 2:1 complex formation (**Figure 13**) [53].

The previous exposed FRET mechanism has been used in the design of selective turn-on fluorescent chemosensor for Hg2+ based on bis-hydrazone derivative from 2,5-furancarboxaldehyde. In this case, furan ring is the donor, and rhodamine B is the acceptor chromophore. Free ligand has the spirolactam moiety which in turn inhibits the charge transfer between these chromophores. When Hg2+ binds to **32**, a rapid naked-eye visual color change occurs from colorless to sharp pink, as well as a bright red fluorescent emission under UV lamp irradiation, which is attributed to the spirolactam ring opening (**Figure 13**). The FRET mechanism was confirmed by overlap of the emission band of 2,5-furan-dicarboxaldehyde (donor) and absorption spectra of rhodamine B (acceptor). After the binding of **32** to Hg2+, leading to **32-Hg2+**, furan moiety makes an energy transfer to induce the spirolactam opening which allowed the FRET process with increasing in the conjugation of the system (**Figure 13**). Additionally, the high increase in quantum yield of **32-Hg2+** (0.23) when they compared **32** (0.015) and its limit of the detection at very low ppb level concentration (3.6 × 10<sup>−</sup><sup>9</sup> M) allowed applications for detecting Hg2+ in drinking water (LOD = 10 nM) [54].

**Figure 13.** *Molecular structure of Zn2+ chemosensors 31 (A) and 32 (B) and their sensing mechanism.*

### **3.4 Hydrazone derivatives as chemosensors for Al3+**

Aluminum (Al3+) is the most abundant (8.3% by weight) metallic element and, after oxygen and silicon, is the third most abundant of all elements in the earth. Aluminum is widely used in the environment around us in modern society, such as in water treatment, food packing, medicines, etc. However, the excess of this metal can result in health problems such as Alzheimer's and Parkinson's diseases [55]. Moreover, it is believed that around 40% of the world's acid solid are caused by aluminum toxicity, which is harmful to plants' performance [56]. Thus, the detection of aluminum is essential in controlling its effects on environment and on human health. Hydrazone-based chemosensors for aluminum ion (Al3+) are mainly based on coordination with fluorescence turn-on response as a result of restricted molecular motion through inhibiting ESIPT or PET effects.

A Schiff-base 7-methoxychromone-3-carbaldehyde-(pyridylformyl) hydrazone was reported as turn-on fluorescent and colorimetric chemosensor for Al3+. This chemosensor (**33**) is colorless and nonfluorescent either in aqueous medium or organic solvents; however, in the presence of aluminum ions (Al3+), the development of a yellow-green color and yellow-green fluorescence occurred. The emission intensity of **33** is very low with low fluorescence quantum yield of 0.051 in ethanol, being attributed to a PET mechanism from the Schiff-base nitrogen free pair electrons. As exposed, PET process involves the deactivation of excited state of a fluorophore by the addition of an electron to one of its excited state frontier orbitals which leaves the fluorophore in a non-emissive state.

Metal complexation to Schiff base produces a less efficient electron donor character, interrupting the PET process and, in some cases, improving the fluorescence emission, which is known as CHEF effect [57]. The selective coordination between **33** and Al3+ on the carbonyl of chromone, nitrogen of imine (▬C═N),

**111**

**Figure 14.**

*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

(**Figure 14**) [58].

mental systems [55].

one metal ion.

and carbonyl of pyridylformyl hydrazone moiety suppressed the PET effect, restoring the fluorescence of the system with an increase of more than 800-fold

**3.5 Hydrazone derivatives as chemosensors for multiple metals**

*Molecular structures of Al3+ chemosensors 33 (A) and 34 (B) and their sensing mechanism.*

Although several chemosensors have relatively high degree of selectivity as previously exposed, some chemosensors have been reported to recognize more than

Following a previous described strategy, probes based on the opening of spirolactam ring upon metal coordination were designed as single molecule multianalyte (Cu2+ and Hg2+) chemosensors. Compound **35** was reported as colorless and nonfluorescent in aqueous or organic medium (**Figure 15**). The UV-vis spectroscopy indicated that this chemosensor is a good chromogenic probe for Cu2+ in ethanolwater (1:99, v/v), whereas other competitive cations failed. Upon the addition of Cu2+ to the solution of **35**, a strong absorption band centered at 530 nm appeared, with changes from colorless to pink, because of spirolactam opening (**35-Cu2+**). A significant increase in the fluorescence emission in ethanol was also observed in the presence of Hg2+ (ϕ = 0.335) and Cr3+ (ϕ = 0.445). However, small addition of water to the ethanol quenched the fluorescence produced by Cr3+, whereas the fluorescence intensity of **35-Hg2+** declined just a little. The rapid quenching of the **35-Cr3+** is justified due to strong coordination between Cr3+ and water which may lead to

The simple and selective fluorescent naphthalene-hydrazone chemosensor (**34**) for Al3+ is a good example of chemosensor based on the excited state intramolecular proton transfer mechanism, that is a process in which photoexcited molecules relax their energy through tautomerization by transfer of protons. Compound **34** has a characteristic UV-vis bands 325 nm and 366 nm which should be assigned to π-π\* transitions of the naphthalene. Only in the presence of Al3+ the spectra of **34** exhibited a peak at 432 nm, which remained constant even after more than 1 equivalent of aluminum addition, which indicates 1:1 binding stoichiometry between **34** and Al3+. The free receptor (**34**) exhibited no fluorescence emission with low fluorescence quantum yield (0.5%), justified by the electron transfer from nitrogen atom of imine to the naphthalene ring (PET) and also transfer of the hydroxyl proton to a neighboring imine nitrogen along with the formation of intramolecular hydrogen bond (OH<sup>⋯</sup>N) (ESIPT). Upon addition of several metal ions, only Al3+ could cause a significant enhancement in the fluorescence emission at 475 nm with a high fluorescence quantum yield (0.26%), due to PET and ESIPT process inhibition (**Figure 14**). Chemosensor **34** showed an interesting fluorogenic response to Al3+ in fully aqueous medium which allows its application in biological assays and environ*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

*Computational Biology and Chemistry*

**3.4 Hydrazone derivatives as chemosensors for Al3+**

*Molecular structure of Zn2+ chemosensors 31 (A) and 32 (B) and their sensing mechanism.*

which leaves the fluorophore in a non-emissive state.

Aluminum (Al3+) is the most abundant (8.3% by weight) metallic element and, after oxygen and silicon, is the third most abundant of all elements in the earth. Aluminum is widely used in the environment around us in modern society, such as in water treatment, food packing, medicines, etc. However, the excess of this metal can result in health problems such as Alzheimer's and Parkinson's diseases [55]. Moreover, it is believed that around 40% of the world's acid solid are caused by aluminum toxicity, which is harmful to plants' performance [56]. Thus, the detection of aluminum is essential in controlling its effects on environment and on human health. Hydrazone-based chemosensors for aluminum ion (Al3+) are mainly based on coordination with fluorescence turn-on response as a result of

A Schiff-base 7-methoxychromone-3-carbaldehyde-(pyridylformyl) hydrazone was reported as turn-on fluorescent and colorimetric chemosensor for Al3+. This chemosensor (**33**) is colorless and nonfluorescent either in aqueous medium or organic solvents; however, in the presence of aluminum ions (Al3+), the development of a yellow-green color and yellow-green fluorescence occurred. The emission intensity of **33** is very low with low fluorescence quantum yield of 0.051 in ethanol, being attributed to a PET mechanism from the Schiff-base nitrogen free pair electrons. As exposed, PET process involves the deactivation of excited state of a fluorophore by the addition of an electron to one of its excited state frontier orbitals

Metal complexation to Schiff base produces a less efficient electron donor character, interrupting the PET process and, in some cases, improving the fluorescence emission, which is known as CHEF effect [57]. The selective coordination between **33** and Al3+ on the carbonyl of chromone, nitrogen of imine (▬C═N),

restricted molecular motion through inhibiting ESIPT or PET effects.

**110**

**Figure 13.**

and carbonyl of pyridylformyl hydrazone moiety suppressed the PET effect, restoring the fluorescence of the system with an increase of more than 800-fold (**Figure 14**) [58].

The simple and selective fluorescent naphthalene-hydrazone chemosensor (**34**) for Al3+ is a good example of chemosensor based on the excited state intramolecular proton transfer mechanism, that is a process in which photoexcited molecules relax their energy through tautomerization by transfer of protons. Compound **34** has a characteristic UV-vis bands 325 nm and 366 nm which should be assigned to π-π\* transitions of the naphthalene. Only in the presence of Al3+ the spectra of **34** exhibited a peak at 432 nm, which remained constant even after more than 1 equivalent of aluminum addition, which indicates 1:1 binding stoichiometry between **34** and Al3+. The free receptor (**34**) exhibited no fluorescence emission with low fluorescence quantum yield (0.5%), justified by the electron transfer from nitrogen atom of imine to the naphthalene ring (PET) and also transfer of the hydroxyl proton to a neighboring imine nitrogen along with the formation of intramolecular hydrogen bond (OH<sup>⋯</sup>N) (ESIPT). Upon addition of several metal ions, only Al3+ could cause a significant enhancement in the fluorescence emission at 475 nm with a high fluorescence quantum yield (0.26%), due to PET and ESIPT process inhibition (**Figure 14**). Chemosensor **34** showed an interesting fluorogenic response to Al3+ in fully aqueous medium which allows its application in biological assays and environmental systems [55].

### **3.5 Hydrazone derivatives as chemosensors for multiple metals**

Although several chemosensors have relatively high degree of selectivity as previously exposed, some chemosensors have been reported to recognize more than one metal ion.

Following a previous described strategy, probes based on the opening of spirolactam ring upon metal coordination were designed as single molecule multianalyte (Cu2+ and Hg2+) chemosensors. Compound **35** was reported as colorless and nonfluorescent in aqueous or organic medium (**Figure 15**). The UV-vis spectroscopy indicated that this chemosensor is a good chromogenic probe for Cu2+ in ethanolwater (1:99, v/v), whereas other competitive cations failed. Upon the addition of Cu2+ to the solution of **35**, a strong absorption band centered at 530 nm appeared, with changes from colorless to pink, because of spirolactam opening (**35-Cu2+**). A significant increase in the fluorescence emission in ethanol was also observed in the presence of Hg2+ (ϕ = 0.335) and Cr3+ (ϕ = 0.445). However, small addition of water to the ethanol quenched the fluorescence produced by Cr3+, whereas the fluorescence intensity of **35-Hg2+** declined just a little. The rapid quenching of the **35-Cr3+** is justified due to strong coordination between Cr3+ and water which may lead to

**Figure 14.** *Molecular structures of Al3+ chemosensors 33 (A) and 34 (B) and their sensing mechanism.*

**Figure 15.**

*Molecular structures of Cu2+ and Hg2+ multianalyte chemosensor 35 (A) and of Cu2+ and Zn2+ multianalyte chemosensor 36 (B).*

hydrolysis of **35-Cr3+**, resulting in Cr(OH)3. In addition, the open ring form of **35** after binding with Cu2+ has no fluorescence, which is attributed to the quenching of the fluorescence by Cu2+, due to paramagnetic properties of the d9 Cu2+ system [59].

With a similar structure to compound **30**, a hydrazone-based chemosensor (**36**) with *off*-*on* fluorescence response to Cu2+ and Zn2+ ion in aqueous media was reported. The reaction of **36** with Cu2+ and Zn2+ formed their corresponding dimeric complexes, which were characterized by single X-ray analysis (**Figure 15**).

The UV-vis absorption and fluorescence spectroscopy (CH3CN/0.02 M HEPES buffer at pH 7.3) indicated the binding behavior of chemosensor **36** toward Zn2+ and Cu2+. The electronic spectra of **36** exhibit two sharp bands at 300 and 363 nm, and upon gradual addition of Cu2+ and Zn2+, new absorption bands appeared at 423 and 415 nm, attributed to charge transfer in complexes **36-Cu2+** and **36-Zn2+**. The occurrence of three well-defined isosbestic points demonstrated an equilibrium between **36** and **36-M2+**. The little fluorescence presented by **36** (at 493 nm) was almost quenched upon sequential addition of Cu2+, being ascribed to the reverse PET from the 4-methylphenyl moiety to the phenolic hydroxyl, and carbohydrazide nitrogen and oxygen atoms, arising from the decrease in electron density after copper ion binding (**Figure 15**). In contrast, Zn2+ ion caused the enhancement in the fluorescence emission (~4.1-fold) of **36** due to the filled d10 electronic configuration of the Zn2+ ion, which does not usually involve energy or electron transfer mechanisms for the deactivation of the excited state (**Figure 15**).

Finally, the sensing mechanism of **36-Cu<sup>2</sup>**<sup>+</sup> and **36-Zn2+** has been shown to be reversible in the presence of EDTA, in which the fluorescence of **36** was almost recovered immediately from both complexes, which suggests the high reversibility of the chemosensor and the potential application in real-time monitoring [60].

**113**

**Author details**

Thiago Moreira Pereira and Arthur Eugen Kümmerle\* Rural Federal University of Rio de Janeiro, Seropédica, Brazil

provided the original work is properly cited.

The authors declare no conflict of interest.

\*Address all correspondence to: akummerle@hotmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Hydrazone-Based Small-Molecule Chemosensors DOI: http://dx.doi.org/10.5772/intechopen.92144*

As exposed in this chapter, hydrazone derivatives have been extensively employed as fluorescent and colorimetric chemosensors targeting important biological analytes such as inorganic anions and metal cations. Thus, it is clear that hydrazone scaffold is of great importance in the design of optical sensors. Here we have demonstrated just some representative examples of hydrazone and their ability as chemosensor for CN<sup>−</sup>, F<sup>−</sup>, AcO<sup>−</sup>, multiple anions, Cu2+, Zn2+, Hg2+, Al3+, and multiple metals. Furthermore, we hope that this book chapter with discussions about the sensing mechanisms (PET, FRET, ESIPT, etc.) could be an important tool and contribute to the development of new rational research projects with the hydrazine scaffold for biological and environmental monitoring of metals and anions.

This work was supported by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico

**4. Conclusion**

**Acknowledgements**

**Conflict of interest**

(CNPq).

### **4. Conclusion**

*Computational Biology and Chemistry*

hydrolysis of **35-Cr3+**, resulting in Cr(OH)3. In addition, the open ring form of **35** after binding with Cu2+ has no fluorescence, which is attributed to the quenching of

*Molecular structures of Cu2+ and Hg2+ multianalyte chemosensor 35 (A) and of Cu2+ and Zn2+ multianalyte* 

With a similar structure to compound **30**, a hydrazone-based chemosensor (**36**) with *off*-*on* fluorescence response to Cu2+ and Zn2+ ion in aqueous media was reported. The reaction of **36** with Cu2+ and Zn2+ formed their corresponding dimeric complexes, which were characterized by single X-ray analysis

The UV-vis absorption and fluorescence spectroscopy (CH3CN/0.02 M HEPES buffer at pH 7.3) indicated the binding behavior of chemosensor **36** toward Zn2+ and Cu2+. The electronic spectra of **36** exhibit two sharp bands at 300 and 363 nm, and upon gradual addition of Cu2+ and Zn2+, new absorption bands appeared at 423 and 415 nm, attributed to charge transfer in complexes **36-Cu2+** and **36-Zn2+**. The occurrence of three well-defined isosbestic points demonstrated an equilibrium between **36** and **36-M2+**. The little fluorescence presented by **36** (at 493 nm) was almost quenched upon sequential addition of Cu2+, being ascribed to the reverse PET from the 4-methylphenyl moiety to the phenolic hydroxyl, and carbohydrazide nitrogen and oxygen atoms, arising from the decrease in electron density after copper ion binding (**Figure 15**). In contrast, Zn2+ ion caused the enhancement in the fluorescence emission (~4.1-fold) of **36** due to the filled d10 electronic configuration of the Zn2+ ion, which does not usually involve energy or electron transfer mechanisms for the deactivation

reversible in the presence of EDTA, in which the fluorescence of **36** was almost recovered immediately from both complexes, which suggests the high reversibility of the chemosensor and the potential application in real-time monitoring [60].

Cu2+ system [59].

and **36-Zn2+** has been shown to be

the fluorescence by Cu2+, due to paramagnetic properties of the d9

**112**

(**Figure 15**).

**Figure 15.**

*chemosensor 36 (B).*

of the excited state (**Figure 15**).

Finally, the sensing mechanism of **36-Cu<sup>2</sup>**<sup>+</sup>

As exposed in this chapter, hydrazone derivatives have been extensively employed as fluorescent and colorimetric chemosensors targeting important biological analytes such as inorganic anions and metal cations. Thus, it is clear that hydrazone scaffold is of great importance in the design of optical sensors. Here we have demonstrated just some representative examples of hydrazone and their ability as chemosensor for CN<sup>−</sup>, F<sup>−</sup>, AcO<sup>−</sup>, multiple anions, Cu2+, Zn2+, Hg2+, Al3+, and multiple metals. Furthermore, we hope that this book chapter with discussions about the sensing mechanisms (PET, FRET, ESIPT, etc.) could be an important tool and contribute to the development of new rational research projects with the hydrazine scaffold for biological and environmental monitoring of metals and anions.

### **Acknowledgements**

This work was supported by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Thiago Moreira Pereira and Arthur Eugen Kümmerle\* Rural Federal University of Rio de Janeiro, Seropédica, Brazil

\*Address all correspondence to: akummerle@hotmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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From Bioinformatics to

Computational Biology

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Section 5
