Carbohydrazones as new class of carbonic anhydrase inhibitors: Synthesis, kinetics, and ligand docking studies

Bioorganic Chemistry 72 (2017) 89–101

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Carbohydrazones as new class of carbonic anhydrase inhibitors: Synthesis, kinetics, and ligand docking studies Sarosh Iqbal a,f, Muhammad Saleem a,g, M. Kamran Azim a, Muhammad Taha b,c, Uzma Salar a, Khalid Mohammed Khan a,⇑, Shahnaz Perveen d, M. Iqbal Choudhary a,e,⇑ a

H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA, Puncak Alam Campus, 42300, Malaysia Faculty of Applied Science, Universiti Teknologi MARA, Shah Alam, 40450 Selangor D. E., Malaysia d PCSIR Laboratories Complex Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi 75280, Pakistan e Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 214412, Saudi Arabia f Department of Chemistry, Government College University Faisalabad, 38000, Pakistan g Department of Chemistry, University of Education Lahore, Campus Dera Ghazi Khan, Punjab, Pakistan b c

a r t i c l e

i n f o

Article history: Received 13 February 2017 Revised 28 March 2017 Accepted 28 March 2017 Available online 30 March 2017 Keywords: Carbohydrazones bis-Schiff bases Carbonic anhydrase II inhibitors Ligand docking Kinetic studies Cheminformatic analysis

a b s t r a c t Discovery and development of carbonic anhydrase inhibitors is crucial for their clinical use as antiepileptic, diurectic and antiglaucoma agents. Keeping this in mind, we have synthesized carbohydrazones 1–27 and evaluated them for their in vitro carbonic anhydrase inhibitory potential. Out of twenty-seven compounds, compounds 1 (IC50 = 1.33 ± 0.01 mM), 2 (IC50 = 1.85 ± 0.24 mM), 3 (IC50 = 1.37 ± 0.06 mM), and 9 (IC50 = 1.46 ± 0.12 mM) have showed carbonic anhydrase inhibition better than the standard drug zonisamide (IC50 = 1.86 ± 0.03 mM). Moreover, compounds 4 (IC50 = 2.32 ± 0.04 mM), 5 (IC50 = 3.96 ± 0.35 mM), 7 (IC50 = 2.33 ± 0.02 mM), and 8 (IC50 = 2.67 ± 0.01 mM) showed good inhibitory activity. Cheminformatic analysis has shown that compounds 1 and 2 possess lead-like properties. In addition, kinetic and molecular docking studies were also performed to investigate the binding interaction between carbohydrazones and carbonic anhydrase enzyme. This study has identified a novel and potent class of carbonic anhydrase inhibitors with the potential to be investigated further. Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction During the last decade, carbohydrazones or bis-Schiff bases of carbohydrazide have received major attention of medicinal chemists due to the presence of multiple potential donor sites to form mononuclear, dinuclear and even tetranuclear molecular square grid complexes [1–4], thereby providing unique supramolecular assemblies. In last few years, several reports describing the transition metal complexes of these ligands have been appeared in literature [5–11]. Moreover, these compounds also exhibit antimicrobial activity towards bacteria and fungi [12,13]. Carbohydrazide has structural sequence of urea and semicarbazide [14] therefore, can react with carbonyl compounds from both ends to afford bis-Schiff bases. Hydrazones are not only used as biologically active compounds but also as analytical reagents. ⇑ Corresponding authors at: H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan (M.I. Choudhary). E-mail addresses: [email protected] (K.M. Khan), [email protected] (M.I. Choudhary). http://dx.doi.org/10.1016/j.bioorg.2017.03.014 0045-2068/Ó 2017 Elsevier Inc. All rights reserved.

Hydrazones play vital role in the treatment of diseases such as leishmania, diabetes, tumor, tuberculosis, leprosy and mental disorder [15–18]. Hydrazones also find applications in detection, determination and isolation of compounds having carbonyl moiety. More recently, they have been extensively used in the detection of several metals [19]. Carbonic anhydrases (EC 4.2.1.1) are metalloproteins, found in mammals. They are divided into four major subgroups, further comprised of several isoforms. Carbonic anhydrase (CA-II) contains a tightly bound Zn2+ at the active site. The Zn2+ cation is bound with one water and three histidine molecules. The function of Zn2+ cation, a strong Lewis acid, is to bind and activate substrate water molecule to catalyze the reversible hydration reaction of carbon dioxide into bicarbonate as shown in Fig. 1. In the absence of CA enzymes, hydration of carbon dioxide does not proceed at a considerable rate under physiological conditions [20]. CA enzymes play a pivotal role in diverse processes, such as physiological pH control, gas balance, calcification, respiration and secretion of electrolytes. Carbonic anhydrase inhibitors are known to serve as antiepileptic, diurectic and antiglaucoma agents [21–25].

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Fig. 1. Mechanism of action of carbonic anhydrase.

Carbonic anhydrase inhibitors have been used in the treatment of glaucoma for past three decades. CA inhibition has a crucial role in cancer treatment through reducing the provision of bicarbonate for the synthesis of nucleotides and other cell components, such as membrane lipids [26]. Currently, carbonic anhydrase inhibitors are administered systemically, and include acetazolamide, dichlorophenamide, ethoxzolamide and methazolamide [27–29]. A number of carbonic anhydrase inhibitors has been reported to lower the intraocular pressure when instilled topically in animals. Intraocular pressure is decreased by reduction in humor formation, stemming from the inhibition of carbonic anhydrase present in the ciliary epithelium [30,31]. To the best of our knowledge, this is the first report describing the carbonic anhydrase inhibitiory activity of carbohydrazones. To date, all known inhibitors of carbonic anhydrase, i.e.; acetazolamide, dichlorophenamide, ethoxzolamide, and methazolamide, belong to sulphonamide class of compounds [32]. As several side effects are associated with sulphonamides along with their involvement in the inhibition of other enzymes for instance, CelecoxibÒ, a non-selective carbonic anhydrase inhibitor, involved in the inhibition of COX-1, COX-2, and phosphodiesterease-5 enzyme. Therefore, there is an urgent need to explore other classes of compounds having selective carbonic anhydrase inhibitory activity without cytotoxicity. Keeping this in mind, we have synthesized several classes of small molecules which may act as selective carbonic anhydrase inhibitors [11,17,18,33]. In current study, we choose carbohydrazone class of compounds particularly based on mechanism of action of carbonic anhydrase depicting in Fig. 1. Our assumption was that a compound with sterically unhindered carbonyl moiety may act as carbonic anhydrase inhibitor. To test our hypothesis, we synthesized a variety of carbohydrazones (1–27) and screened them for carbonic anhydrase inhibitory activity. A proposed mechanism of action of carbohydrazones in carbonic anhydrase inhibition is shown in Fig. 2.

anhydrase II by carbohydrazone derivatives of the general structure, shown in Table 1, is reported. Carbohydrazones 1–27 were synthesized by reacting commercially available carbohydrazide and a range of aromatic aldehydes (Scheme 1). In a typical reaction, few drops of acetic acid were added to a stirred mixture of substituted aromatic aldehyde (4.0 mmol) and carbohydrazide (2.0 mmol) in anhydrous ethanol. The reaction mixture was refluxed and the reaction progress was monitored by TLC. After completion, the reaction mixture was allowed to cool to room temperature; the crude product obtained was filtered and washed with hexanes. After crystallization from ethanol, the pure carbohydrazones 1–27 were obtained in excellent yields. The structures of all the synthetic compounds were deduced by using spectroscopic techniques (1H NMR, EI-MS, HREI-MS, TOF-MS, and HRTOF-MS). Elemental analyses were found to be in agreement with the calculated values. In addition, 13C NMR and IR spectroscopy was performed on new compounds 11, 17, 24, and 27.

2. Results and discussion

sented CH-2/CH-6, CH-3/CH-5 and methylene carbon (@CH),

2.1. Chemistry Synthesis of lead molecules against well-defined biological targets is an ongoing research of our group. In recent years, we have reported Schiff bases exhibiting diverse biological activities [11,17,18,33]. In the light of these findings, we designed our current project. In this report, in vitro inhibition of bovine carbonic

2.2. General structure elucidation of compounds by spectroscopic techniques The structures of all synthetic compounds were deduced by spectroscopic techniques. The 1H NMR spectrum of representative compound 6 was recorded in deuterated DMSO. Since, there is symmetry in the molecule so only half of the molecule appeared in 1H and 13C NMR spectra. A singlet at d 10.78 showed the presence of benzilidene proton while a broad singlet at d 8.16 indicated the presence of an amide proton. The phenyl ring C-2/C-6 protons coupled with C-3/C-5 protons and appeared as doublet at d 7.78 (J2,3 = J6,5 = 8.4 Hz). Likewise, C-3/C-5 protons appeared as doublet at d 7.50 (J3,2 = J5,6 = 8.4 Hz) (Fig. 3). The 13C NMR spectrum of compound 6 was recorded in deuterated DMSO. Signals appeared at d 128.73, 128.40, and 141.8 reprerespectively. Quaternary C-1, C-4 and C@O appeared at d 133.6, 133.8 and 151.9, respectively (Fig. 4). Geometrical isomerism was confirmed by NOE analysis in which signal enhancement was typically observed for amidic protons by irradiating benzilidine protons, likewise signal enhancement was also observed for benzilidine protons, on irradiating the amidic protons indicating that they are located on the same side.

Fig. 2. A proposed mechanism of inhibition of carbonic anhydrase with carbohydrazone.

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S. Iqbal et al. / Bioorganic Chemistry 72 (2017) 89–101 Table 1 Structures of synthetic carbohydrazones 1-27 and their IC50 values for carbonic anhydrase II inhibition. Comp. no.

Structure and IC50 ± SEMa (mM)

Comp. no.

Structure and IC50 ± SEMa (mM)

2

1

1.33 ± 0.01 µM 3

1.85 ± 0.24 µM 4

2.32 ± 0.04 µM

1.37 ± 0.06 µM 5

6

No Inhibition

3.96 ± 0.35 µM 7

8

2.67 ± 0.50 µM

2.33 ± 0.02 µM 9

10

No Inhibition

1.46 ± 0.12 µM 11

12

No Inhibition

No Inhibition 13

14

15

16

No Inhibition 17

No Inhibition 18

No Inhibition

No Inhibition (continued on next page)

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Table 1 (continued) Comp. no.

Structure and IC50 ± SEMa (mM)

Comp. no.

Structure and IC50 ± SEMa (mM)

20

19

No Inhibition

No Inhibition

21

22

No Inhibition

No Inhibition

23

24

No Inhibition

No Inhibition

25

26

No Inhibition

No Inhibition 27

28

No Inhibition

a

1.86 ± 0.03 µM Zonisamide, Std. Carbonic anhydrase II inhibitor

SEM is standard error of mean.

Scheme 1. Synthesis of carbohydrazones (1–27).

Fig. 3. 1H NMR chemical shifts of compound 6.

Fig. 4.

13

C NMR chemical shifts of compound 6.

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The structure of compound 6 was also deduced by EI-MS spectrum having a molecular ion peak at m/z 334 which correspond to molecular ion peak (M+) of C15H12Cl2N4O. Isotopic peaks M++2 and M++4 appeared at m/z 336 and m/z 338 due to the presence of two chlorine atoms. Ions at m/z 111 and 124 were due to the chlorophenyl and methylene chlorophenyl groups, respectively (Fig. 5). 2.3. Structure-activity relationship All the synthetic compounds 1–27 were screened for their in vitro carbonic anhydrase II inhibitory activities. Out of twentyseven (27), eight (8) compounds showed a varying degree of inhibition with IC50 values ranging between 1.33 and 3.96 mM. Compounds 1 (IC50 = 1.33 ± 0.01 mM), 3 (IC50 = 1.37 ± 0.06 mM), and 9 (IC50 = 1.46 ± 0.12 mM), showed potent activity, better than standard drug zonisamide (IC50 = 1.86 ± 0.03 mM). Moreover, compound 2 (IC50 = 1.85 ± 0.24 mM) also exhibited excellent activity, comparable to standard. Likewise, compounds 4 (IC50 = 2.32 ± 0.04 mM), 5 (IC50 = 3.96 ± 0.35 mM), 7 (IC50 = 2.33 ± 0.02 mM), and 8 (IC50 = 2.67 ± 0.01 mM) have also demonstrated a good tendency to inhibit carbonic anhydrase II. However, rest of the compounds was considered as inactive due to less than 50% inhibition at 0.2 mM concentration. Activities of these compounds revealed that specific substituent at distinct position of phenyl group found to be responsible for altering the activity of compound. Fig. 5. Key mass fragmentation pattern of compound 6.

O2N

NO2

O N

N H

4

N H

NO2 N

N

IC50 = 1.33 ± 0.01 µM

N H

5

N H

N

IC50 = 1.85 ± 0.24 µM

Compound 1, having a nitro group at para position of phenyl ring, showed the strongest inhibition of the enzyme among the series with an IC50 value of 1.33 ± 0.01 mM. However, molecule 2 having a nitro group at ortho- position of phenyl ring showed slightly less inhibitory activity with an IC50 value of 1.85 ± 0.24 mM found to be more active than 1. Likewise compound 4, with 4-bromo substituent at phenyl ring showed more inhibitory potential with an IC50 value of 2.32 ± 0.04 mM as compared to its 2-bromo analogue having an IC50 value of 3.96 ± 0.35 mM.

Br N H

4

In contrast, in case of chloro substituents at phenyl ring, such as compound 6, having chloro group at para position was found to be completely inactive. Compounds 7, 8, and 9 having 2-chloro (IC50 = 2.33 ± 0.02 mM), 2,4-dichloro (IC50 = 2.67 ± 0.01 mM), and 2,6-dichloro substituents (IC50 = 1.46 ± 0.12 mM), respectively, have shown carbonic anhydrase inhibitory potential in such a fashion that inhibitory tendency is increased when both ortho-positions of phenyl ring (2,6-dichloro) are occupied with chloro substituent, but decreased if an ortho and a para position (2,4-dichloro) is

Br

O N

O2N

O

N H

N

IC50 = 2.32 ± 0.04 µM

Br

Br

O N

N H

5

N H

N

IC50 = 3.96 ± 0.35 µM

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occupied with chloro substituent as compared to singly ortho substituted compound 7.

Cl N

N H

6

Cl

Cl

O N H

N

N

Cl

O N

Cl

N H

8

N H

N H

7

N H

N

IC50 = 2.33 ± 0.02 µM

No Inhibition

Cl

Cl

O

Cl

Cl

N

N Cl

IC50 = 1.46 ± 0.12 µM

N H

8

Cl

Cl

O N H

N

IC50 = 2.67 ± 0.01 µM

2-nitro, 2-chloro-5-nitro, 2-chloro, 2,4-dichloro, 2,6-dichloro, 2bromo, 4-bromo), is more electron deficient as compared to carbohydrazone derivatives, having electron donating substituents at phenyl ring (2-hydroxy, 3-hydroxy 4-hydroxy, 2,3-dihydroxy, 2,5dihydroxy, 2,4,6-trihydroxy, 2,3,4-trihydroxy, 2-methoxy, 3-methoxy, 4-methoxy, 2-dihydroxy-5-methoxy, 2-dihydroxy-3methoxy, 2-dihydroxy-3-ethoxy, and 2,3-dimethoxy), therefore, it is more susceptible for nucleophilic attack by hydroxyl group present at the active site of carbonic anhydrase-II enzyme.

Limited SAR results indicate that both nature and position of substituents at phenyl ring play an important role in determining the inhibitory potential of the series. However, only those compounds, having electron-withdrawing substituents at phenyl ring (4-nitro, 2-nitro, 2-chloro-5-nitro, 2-chloro, 2,4-dichloro, 2,6-dichloro, 2bromo, 4-bromo) showed inhibitory activity. These results further supported our proposed mechanism of inhibition of carbonic anhydrase (Fig. 2). As carbonyl moiety of carbohydrazone derivatives, having electron withdrawing substituents at phenyl ring (4-nitro,

Table 2 Cheminformatic analysis of carbohydrazones having carbonic anhydrase II inhibitory potential.

a

Compound no.

IC50 ± SEMa (mM)

Log P

PSA

MSA

Obey Lipinski rule of five

Lead likeness (Obey rule of three)

1 2 3 4 5 7 8 9

1.33 ± 0.01 1.85 ± 0.24 1.37 ± 0.06 2.32 ± 0.04 3.96 ± 0.35 2.33 ± 0.02 2.67 ± 0.50 1.46 ± 0.12

2.79 2.79 4.00 4.45 4.45 4.12 5.33 5.33

152.13 152.13 152.13 65.85 65.85 65.85 65.85 65.85

444.65 444.63 476.55 409.84 408.21 400.49 432.42 430.25

Yes Yes Yes Yes Yes Yes No No

Yes Yes No No No No No No

SEM is standard error of mean.

Table 3 Kinetic data of carbohydrazones against CA-II. Comp. no.

Vmax, (mM/min)
1

Km, mM

Vmax (app), (mM/min)
1

Km (app), mM

Ki, mM

Type of inhibition

1 2 3 4 7 9

78.32 81.96 69.60 72.56 98.03 68.47

3.25 2.98 3.52 3.54 3.50 3.43

35.23 15.60 24.97 25.78 28.65 21.56

1.45 1.95 2.36 1.56 2.41 2.56

2.35 1.81 3.58 2.52 4.12 1.78

Mixed type Mixed type Mixed- type Mixed type Mixed type Mixed type

(a) A plot for 1/Vmax vs 1/Substrate (4-nitrophenyl acetate) in the presence of different concentrations of mixed-type inhibitor. (b) Secondary re-plot from reciprocal plot for Ki which is a plot for slope vs [I]). (c) Dixon plot which further confirms the Ki and type of inhibition which is mixed-type for compound 7 against CA-II.

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I = 5 µM

0.6

Slope = Km/Vmax

I = 0.625 µM

0.4

1/Vmax µM/min

0.12

I = 1.25 µM I = 0.0 µM

0.2 0 -0.2

0.08 0.04 0

Slope = Km/Vmax.Ki

-0.04 -0.08

-2

0

2

4

-8

6

-6

-4

-2

0

2

4

6

8

10

[I] µM

1/Substrate(p-nitrophenyl acetate)

(b)

(a) 0.6 0.4 0.2

1/Vmax

0 -0.2 -0.4 -6

-4

-2

0

2

4

6

[I] µM

(c) Fig. 6. Steady State Inhibition by the compound 7.

Consequently, compounds 1, 2, 3, 4, 5, 7, 8, and 9, identified as better carbonic anhydrase inhibitors. All the synthetic compounds were also screened for their activity against urease and a-chymotrypsin and found to be inactive. Therefore it is assumed that they are target specific inhibitors. 2.4. Cheminformatic analysis Cheminformatic analysis of the compounds which showed carbonic anhydrase-II inhibition was carried out. Log P, polar surface area (PSA), and molecular surface area (MSA) was calculated as shown in Table 2 [34]. The PSA parameter provides an indication of drug transport properties by correlating the presence of polar atoms with membrane permeability. Additionally, reported ADMET (Absorption, distribution, metabolism, excretion, toxicity) rules of thumb indicates that neutral molecules with calculated Log P  4 and molecular weight  400 are likely to exhibit average value for a variety of indicators, including solubility, bioavailability and plasma protein binding, consequently provide good library start points [35]. Table 2 shows that out of eight compounds having carbonic anhydrase-II inhibitory potential, six compounds 1–5, and 7 followed Lipinski’s rule of five. Out of these six, two compounds 1 and 2 have lead like properties. Therefore, further research on these two molecules may lead to development of novel drug for the treatment of epilepsy and glaucoma. 2.5. Kinetic studies Kinetic studies on the most active compounds 1–4, 7, and 9 were performed in order to determine the inhibition mechanism

of these compounds. In this study, different concentrations of test compounds were used. These compounds inhibited bovine erythrocyte carbonic anhydrase-II in a concentration-dependent manner with Ki values between 1.78 and 4.12 mM. Kinetic studies shown all compounds as mixed-type inhibitors (Table 3). The types of inhibition of carbonic anhydrase-II were determined by Lineweavere Burk plots. The reciprocal of the rate of the reaction were plotted against the reciprocal of substrate concentration to monitor the effect of inhibitor on both km and Vmax values. The Ki values were calculated by plotting the slope of each line in the Lineweavere Burk plots against the different concentrations of compounds. The Ki values were confirmed from Dixon plot by plotting the reciprocal of the rate of reaction against different concentrations of compounds. In mixed-type of inhibition, both km and Vmax are affected. The high km and low Vmax of these compounds indicated a mixed type of inhibition (Fig. 6).

2.6. Ligand docking Molecular docking studies were conducted to predict binding mode of carbohydrazones in the active site of carbonic anhydrase II. Crystal structure of bovine carbonic anhydrase II complexed with zinc atom (PDB id: 1V9E) [36] was used for the docking of compounds 1–5 and 7–9 by using AutoDock Vina program [37]. These compounds inhibited carbonic anhydrase II with IC50 values in low micromolar range. AutoDock Vina offers multi-core capability, high performance, enhanced accuracy and ease of use. It significantly improves the average accuracy of the binding mode predictions as compared to AutoDock 4 [38]. The active site of carbonic anhydrase II lies at the bottom of a deep cleft, where a zinc atom is bound. Nitrogen atoms of 3 highly conserved histidines

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pound 1 with two residues (His93 and Thr198), involved in zinc atom binding and catalysis. The His93 formed p-p interactions with the phenyl ring as well as the hydrazide group, whereas side chain of Thr198 interacted with the carbonyl oxygen atom of hydrazide group. On the other hand, in binding mode 2, compounds are docked in an extended conformation (Fig. 7b). In this binding mode, compounds did not interact with zinc-binding residues. Interestingly, in both binding modes, the solvent exposed Phe129 residue is predicted to form p-p interactions with the phenyl ring of the ligands. Three-dimensional structural analysis of the predicted binding modes of carbohydrazone compounds provided clue of carbonic anhydrase inhibition from a structural point of view. Different substitutions and heterocyclic extensions may lead to the discovery of novel compounds with better potency and selectivity towards carbonic anhydrases for therapeutic intervention.

3. General experimental

Fig. 7. Docking of carbohydrazone derivatives in the active site cleft of bovine carbonic anhydrase (PDB id 1V9E). (a) Molecular surface representation of carbonic anhydrase complexed with (a) compound 1 and (b) compound 9. The Carbohydrazone compounds are shown in stick representation.

(numbered 93, 95 and 118) directly coordinate with the zinc ligand. The amino acid residues Thr98 and Glu105 interact indirectly through the bound water. Three-dimensional structural analysis of the predicted binding modes of carbohydrazone derivatives provided information related to carbonic anhydrase inhibition from structural stand point. The five top binders of carbohydrazones were modeled into the active site of carbonic anhydrase II to examine interactions with protein residues. Analysis of Autodock Vina docking solutions predicted binding of ligands (i.e. compounds 1–5 and 7–9) at the active site cleft (Fig. 7). The ligands were predicted to make an extensive network of interactions with side chains of active site cleft amino acids. Ligand docking of compounds 1–5 and 7–9 predicted the placement of compounds near the opening of the deep cleft (Fig. 7). The eight carbohydrazones (i.e. compounds 1–5 and 7–9) docked in two slightly different binding fashions. Compounds 1, 4, 7, and 8 docked with same binding mode (binding mode 1), whereas, compounds 2, 3, 5, and 9 docked with different binding fashion (binding mode 2) (Fig. 7a and b). In binding mode 1, compounds are docked in U-shaped conformation with hydrazide moiety pointing towards the bottom of the active site and the phenyl rings are solvent exposed. Fig. 8a shows the 2D map of interactions of compound 1 (a representative ligand docked with binding mode 1) with the active site residues. Docking predicted interaction of com-

NMR experiments were performed on Avance Bruker AM 300, 400 MHz instruments. Chemical shifts were recorded in parts per million (ppm) and coupling constants were recorded to the nearest 0.1 Hz. Multiplicities are reported as singlet (s), doublet (d), triplet (t), doublet of doublets (dd), doublet of triplets (dt), quartet (q) or multiplet (m). 13C NMR spectra were recorded on Avance Bruker AM 75 MHz. Chemical shifts were recorded in parts per million (ppm). Assignment of 1H and 13C NMR spectra were made by using COSY (correlation spectroscopy), HSQC (heteronuclear single quantum coherence), and HMBC (heteronuclear multiple bond coherence). Geometrical isomerism was determined by NOE measurement. Electron impact mass spectra (EI-MS) were recorded on a Finnigan MAT-311A, Germany. Thin layer chromatography (TLC) was performed on pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany). UV light (wavelength, 365 and 254 nm) was used to visualize chromatograms.

3.1. General procedure for the synthesis of carbohydrazones (1–27) Carbohydrazone derivatives 1–27 were synthesized by refluxing a mixture of carbohydrazide (2 mmol) and a variety of substituted benzaldehydes (4 mmol) in the presence of 3–4 drops of acetic acid using ethanol as solvent. Completion of reaction was monitored by TLC (hexane: acetone). After completion of reaction, the reaction mixture was allowed to cool at room temperature. The precipitate obtained was filtered and washed with hexane and dried to obtain pure compounds 1–27 in high yields. Crystallization from ethanol afforded pure products. The structures of synthetic compounds were confirmed by 1H NMR, EI-MS, HREIMS, TOF-MS, HRTOF-MS, and elemental analyses. However, 13C NMR and IR spectroscopy was performed on new compounds such as 11, 17, 24 and 27.

3.1.1. N00 ,N000 -Bis[(E)-(4-nitrophenyl)methylidene]carbonic dihydrazide (1) Solid; yield: 93%; m.p. 284–286 °C; Rf: 0.59 (acetone/hexanes, 3:7); 1H NMR (300 MHz, DMSO-d6): d 11.19 (s, 2H, 2  NH), 8.29 (d, J30 ,20 = J50 ,60 = 9 Hz, 6H, 2  @CH, 2  H-30 ,50 ), 8.03 (d, J20 ,30 = J60 ,50 = 8.4 Hz, 4H, 2  H-20 ,60 ); EI-MS: m/z (rel. abund. %) 356 (M+, absent), 298 (2.9), 191 (2.7), 176 (5.0), 165 (100), 149 (12.9), 135 (15.2); TOF-MS: 357 (M++H+); HRTOF-MS: calcd for C15H13N6O5: m/z = 357.0947, Found: 357.0956; Anal. Calcd for C15H12N6O5: C, 50.57; H, 3.39; N, 23.59; Found: C, 50.59; H, 3.38; N, 23.53.

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Fig. 8. 2D Diagrammatic representation of (a) compound 1 and (b) compound 9 and active site cleft residues of bovine carbonic anhydrase generated by Discovery Studio 3 Visualizer. Hydrogen bonds and p-p interactions are represented by dashed lines and thin lines, respectively.

3.1.2. N00 ,N000 -Bis[(E)-(2-nitrophenyl)methylidene]carbonic dihydrazide (2) Solid; yield: 93%; m.p. 246–248 °C; Rf: 0.59 (acetone/hexanes, 3:7); 1H NMR (300 MHz, DMSO-d6): d 11.18 (s, 2H, 2  NH), 8.57 (s, 2H, 2  @CH), 8.28 (d, J30 ,40 = 8.1 Hz, 2H, 2  H-30 ), 8.05 (dd, J60 ,50 = 8.1 Hz, J60 ,40 = 1.2 Hz, 2H, 2  H-60 ), 7.82 (br.t, J50 (40 ,60 ) = 7.8 Hz, 2H, 2  H-50 ), 7.66 (dt, J40 (30 ,50 ) = 8.7 Hz, J40 ,60 = 1.5 Hz, 2H, 2  H-40 ); EI-MS: m/z (rel. abund. %) 356 (M+, 2.8%), 221 (4.5), 192 (62.6), 135 (92.2), 118 (100); HREI-MS: calcd for C15H12N6O5: m/z = 356.0869, Found: 356.0875; Anal. Calcd for C15H12N6O5: C, 50.57; H, 3.39; N, 23.59; Found: C, 50.50; H, 3.37; N, 23.56. 00

000

3.1.3. N ,N -Bis[(E)-(2-chloro-5-nitrophenyl)methylidene]carbonic dihydrazide (3) Solid; yield: 92%; m.p. 266–268 °C; Rf: 0.59 (acetone/hexanes, 3:7); 1H NMR (400 MHz, DMSO-d6): d 11.49 (s, 2H, 2  NH), 8.86

(s, 2H, 2  @CH), 8.63 (br.s, 2H, 2  H-60 ), 8.22 (dd, J40 ,30 = 8.8 Hz, J40 ,60 = 2.8 Hz, 2H, 2  H-40 ), 7.84 (d, J30 ,40 = 8.8 Hz, 2H, 2  H-30 ); EI-MS: m/z (rel. abund. %) 425 (M+, absent), 424 (1.9), 226 (15.4), 169 (46.2), 123 (100); TOF-MS 425 (M+); HRTOF-MS: calcd for C15H11Cl2N6O5: m/z = 425.0167, Found: 425.0149; Anal. Calcd for C15H10Cl2N6O5: C, 42.37; H, 2.37; N, 19.77; Found: C, 42.39; H, 2.30; N, 19.79. 3.1.4. N00 ,N000 -Bis[(E)-(4-bromophenyl)methylidene]carbonic dihydrazide (4) Solid; yield: 89%; m.p. 246–248 °C; Rf: 0.59 (acetone/hexanes, 3:7); 1H NMR (400 MHz, DMSO-d6): d 10.79 (s, 2H, 2  NH), 8.13 (br.s, 2H, 2  @CH), 7.70 (d, J20 ,30 = J60 ,50 = 8.4 Hz, 4H, 2  H-20 , 60 ), 7.63 (d, J30 ,20 = J50 ,60 = 8.8 Hz, 4H, 2  H-30 , 50 ); EI-MS: m/z (rel. abund. %) 424 (M+, 5.0), 426 (M++2, 10.1), 428 (M++4, 4.8), 241 (4.1), 226 (12.3), 198 (100), 183 (36.4), 156 (6.2); HREI-MS: calcd

98

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for C15H12Br2N4O1: m/z = 421.9378, Found: 421.9355; Anal. Calcd for C15H12Br2N4O: C, 42.48; H, 2.85; N, 13.21; Found: C, 42.44; H, 2.87; N, 13.20.

3.1.10. N00 ,N000 -Bis[(E)-(4-fluorophenyl)methylidene]carbonic dihydrazide (10) Solid; yield: 89%; m.p. 243–244 °C; Rf: 0.57 (acetone/hexanes, 5:5); 1H NMR (300 MHz, DMSO-d6): d 10.70 (s, 2H, 2  NH), 8.15

3.1.5. N00 ,N000 -Bis[(E)-(2-bromophenyl)methylidene]carbonic dihydrazide (5) Solid; yield: 88%; m.p. 238–240 °C; Rf: 0.61 (acetone/hexanes, 5:5); 1H NMR (400 MHz, DMSO-d6): d 10.68 (s, 2H, 2  NH), 8.16 (s, 2H, 2  @CH), 7.81 (dd, J30 ,40 = J60 ,50 = 7.6 Hz J30 ,50 = J60 ,40 = 1.6 Hz, 4H, 2  H-30 ,60 ), 7.28 (t, J30 ,20 = J50 ,60 = 8.8 Hz, 4H, 2  H-30 ,50 ); EIMS: m/z (rel. abund. %) 424 (M+, absent), 198 (8.4), 183 (15.7), 119 (78.7), 103 (12.7), 89 (100); TOF-MS 425 (M++H+); HRTOFMS: calcd for C15H13Br2N4O: m/z = 422.9456, Found: 422.9463; Anal. Calcd for C15H12Br2N4O: C, 42.48; H, 2.85; N, 13.21; Found: C, 42.40; H, 2.89; N, 13.25. 00

000

3.1.6. N ,N -Bis[(E)-(4-chlorophenyl)methylidene]carbonic dihydrazide (6) Solid; yield: 88%; m.p. 247–248 °C; Rf: 0.46 (acetone/hexanes, 4:6); 1H NMR (300 MHz, DMSO-d6): d 10.78 (s, 2H, 2  NH), 8.16 (s, 2H, 2  @CH), 7.78 (d, J20 ,30 = J60 ,50 = 8.4 Hz, 4H, 2  H-20 ,60 ), 7.50 (d, J30 ,20 = J50 ,60 = 8.4 Hz, 4H, 2  H-30 ,50 ); 13C NMR (75 MHz, DMSOd6): d 151.89 (C@O), 141.76 (@CH), 133.80 (C-4), 133.55 (C-1), 128.73 (CH-2, CH-6), 128.40 (CH-3, CH-5); EI-MS: m/z (rel. abund. %) 334 (M+, 28.5), 336 (M++2, 13.1),181 (72.3), 154 (100), 137 (56.9), 124 (47.2), 111 (43); HREI-MS: calcd for C15H12Cl2N4O1: m/z = 334.0388, Found: 334.0386; Anal. Calcd for C15H12Cl2N4O: C, 53.75; H, 3.61; N, 16.74; Found: C, 53.70; H, 3.56; N, 16.78. 3.1.7. N00 ,N000 -Bis[(E)-(2-chlorophenyl)methylidene]carbonic dihydrazide (7) Solid; yield: 87%; m.p. 228–230 °C; Rf: 0.61 (acetone/hexanes, 5:5); 1H NMR (300 MHz, DMSO-d6): d 11.05 (s, 2H, 2  NH), 8.57 (s, 2H, 2  @CH), 8.15 (br.s, 2H, 2  H-60 ), 7.51 (m, 2H, 2  H-50 ), 7.44 (m, 4H, 2  H-40 ,30 ); EI-MS: m/z (rel. abund. %) 335 (M+, 2.0), 181 (77.1), 154 (88.2), 119 (100), 89 (67.0); HREI-MS: calcd for C15H12Cl2N4O1: m/z = 334.0388, Found: 334.0398; Anal. Calcd for C15H12Cl2N4O: C, 50.57; H, 3.39; N, 23.59; Found: C, 50.53; H, 3.36; N, 23.50. 3.1.8. N00 ,N000 -Bis[(E)-(2,4-dichlorophenyl)methylidene]carbonic dihydrazide (8) Solid; yield: 89%; m.p. 244–246 °C; Rf: 0.42 (acetone/hexanes, 3:7); 1H NMR (300 MHz, DMSO-d6): d 11.12 (s, 2H, 2  NH), 8.52 (s, 2H, 2  @CH), 8.17 (d, J60 ,50 = 8.4 Hz, 2H, 2  H-60 ), 7.68 (d, J30 ,50 = 2.1 Hz, 2H, 2  H-30 ), 7.52 (dd, J50 ,60 = 8.7 Hz, J50 ,30 = 2.1 Hz, 2H, 2  H-50 ); EI-MS: m/z (rel. abund. %) 404 (M+, 21.6), 406 (M++2, 9.8), 215 (85.2), 188 (94.2), 153 (100), 123 (51.6); HREIMS: calcd for C15H10Cl4N4O1: m/z = 401.9609, Found: 401.9617; Anal. Calcd for C15H10Cl4N4O: C, 44.59; H, 2.49; N, 13.87; Found: C, 44.56; H, 2.53; N, 13.90. 3.1.9. N00 ,N000 -Bis[(E)-(2,6-dichlorophenyl)methylidene]carbonic dihydrazide (9) Solid; yield: 87%; m.p. 305–306 °C; Rf: 0.59 (acetone/hexanes, 3:7); 1H NMR (300 MHz, DMSO-d6): d 10.98 (s, 2H, 2  NH), 8.28 (s, 2H, 2  @CH), 7.55 (d, J30 ,20 = J50 ,60 = 8.4 Hz, 4H, 2  H-30 , 50 ), 7.43 (t, J40 (30 ,50 ) = 7.2 Hz, 2H, 2  H-40 ); EI-MS: m/z (rel. abund. %) 404 (M+, absent), 402 (1.1), 231 (19.0), 188 (53.3), 173 (70.1), 146 (13.5); TOF-MS 405 (M++H+); HRTOF-MS: calcd for C15H11 Cl4N4O: m/z = 402.9686, Found: 402.9693; Anal. Calcd for C15H10Cl4N4O: C, 44.59; H, 2.49; N, 13.87; Found: C, 44.55; H, 2.47; N, 13.83.

(s, 2H, 2  @CH), 7.81 (t, J20 (30 ,F) = J60 (50 ,F) = 7.8 Hz, 4H, 2  H-20 ,60 ), 7.29 (t, J30 (20 ,F) = J50 (60 ,F) = 8.7 Hz, 4H, 2  H-30 ,50 ); EI-MS: m/z (rel. abund. %) 302 (M+, 5.5), 165 (29.4), 138 (100), 122 (41.7), 108 (46.5), 95 (95.8); HREI-MS: calcd for C15H12F2N4O1: m/z = 302.0979, Found: 302.0972; Anal. Calcd for C15H12F2N4O: C, 59.60; H, 4.00; N, 18.53; Found: C, 59.65; H, 4.03; N, 18.57. 3.1.11. N00 ,N000 -Bis[(E)-(2-fluorophenyl)methylidene]carbonic dihydrazide (11) Solid; yield: 89%; m.p. 206–208 °C; Rf: 0.57 (acetone/hexanes, 5:5); 1H NMR (400 MHz, DMSO-d6): d 10.93 (s, 2H, 2  NH), 8.41 (br.s, 2H, 2  @CH), 8.09 (br.s, 2H, 2  H-30 ), 7.45 (m, 2H, 2  H-60 ), 7.29 (m, 4H, 2  H-40 ,50 ); 13C NMR (75 MHz, DMSO-d6): d 162.14 (C-2), 158.84 (C-1), 151.78 (C@O), 135.68 (@CH), 131.34 (CH-4), 126.42 (CH-6), 124.72 (CH-5), 115.96 (CH-3); EI-MS: m/z (rel. abund. %) 302 (M+, 30.4), 181 (16.5), 165 (100), 138 (100), 122 (43.8), 108 (40.6), 95 (34.8); HREI-MS: calcd for C15H12F2N4O1: m/z = 302.0979, Found: 302.0971; Anal. Calcd for C15H12F2N4O: C, 59.60; H, 4.00; N, 18.53; Found: C, 59.57; H, 4.04; N, 18.56; IR (KBr, cm
1): 3335 (N-H), 1718 (C@O), 1660 (C@N), 1611 (C@C). 3.1.12. N00 ,N000 -Bis[(E)-phenylmethylidene]carbonic dihydrazide (12) Solid; yield: 89%; m.p. 217–218 °C; Rf: 0.52 (acetone/hexanes, 5:5); 1H NMR (400 MHz, DMSO-d6): d 10.68 (s, 2H, 2  NH), 8.17 (s, 2H, 2  @CH), 7.74 (d, J20 ,30 = J60 ,50 = 7.2 Hz, 4H, 2  H-20 , 60 ), 7.44 (m, 6H, 2  H-30 ,40 ,50 ); EI-MS: m/z (rel. abund. %) 266 (M+, 42.5), 162 (16.8), 147 (78.7), 120 (100), 104 (65.6), 77 (78.1); HREI-MS: calcd for C15H14N4O1: m/z = 266.1168, Found: 266.1169; Anal. Calcd for C15H14N4O: C, 67.65; H, 5.30; N, 21.04; Found: C, 67.60; H, 5.33; N, 21.09. 3.1.13. N00 ,N000 -Bis[(E)-(2-hydroxyphenyl)methylidene]carbonic dihydrazide (13) Solid; yield: 83%; m.p. 231–233 °C; Rf: 0.51 (acetone/hexanes, 5:5); 1H NMR (400 MHz, DMSO-d6): d 10.83 (s, 2H, 2  NH), 8.41 (s, 2H, 2  @CH), 7.68 (br.s, 2H, 2  H-60 ), 7.25 (dt, J40 (30 ,50 ) = 8.0 Hz, J40 ,60 = 1.6 Hz, 2H, 2  H-40 ), 6.89 (m, 4H, 2  H-30 ,50 ); EI-MS: m/z (rel. abund. %) 298 (M+, 5.0), 240 (80.8), 162 (41.6), 147 (23.4), 136 (100), 120 (58.2), 106 (25.7), 93 (20.2); TOF-MS 299 (M++H+); HRTOF-MS: calcd for C15H15N4O3: m/z = 299.1144, Found: 299.1124; Anal. Calcd for C15H14N4O3: C, 60.40; H, 4.73; N, 18.78; Found: C, 60.44; H, 4.72; N, 18.70. 3.1.14. N00 ,N000 -Bis[(E)-(3-hydroxyphenyl)methylidene]carbonic dihydrazide (14) Solid; yield: 81%; m.p. 246–248 °C; Rf: 0.45 (acetone/hexanes, 6:4); 1H NMR (300 MHz, DMSO-d6): d 10.59 (s, 2H, 2  NH), 9.54 (s, 2H, 2  OH), 8.06 (s, 2H, 2  @CH), 7.23 (m, 6H, 2  H-20 ,50 ,60 ), 6.79 (dd, J40 ,50 = 8 Hz, J40 ,60 = 1.6 Hz, 2H, H-40 ); EI-MS: m/z (rel. abund. %) 298 (M+, absent), 240 (100), 163 (9.7), 147 (96.6), 120 (54.5), 106 (10.9), 93 (25.0); TOF-MS 299 (M++H+); HRTOF-MS: calcd for C15H15N4O3: m/z = 299.1144, Found: 299.1145; Anal. Calcd for C15H14N4O3: C, 60.40; H, 4.73; N, 18.78; Found: C, 60.41; H, 4.79; N, 18.69. 3.1.15. N00 ,N000 -Bis[(E)-(4-hydroxyphenyl)methylidene]carbonic dihydrazide (15) Solid; yield: 83%; m.p. 254–258 °C; Rf: 0.46 (acetone/hexanes, 6:4); 1H NMR (400 MHz, DMSO-d6): d 10.35 (s, 2H, 2  NH), 9.87

S. Iqbal et al. / Bioorganic Chemistry 72 (2017) 89–101

(s, 2H, 2  OH), 8.03 (s, 2H, 2  @CH), 7.55 (d, J20 ,30 = J60 ,50 = 8.8 Hz, 4H, 2  H-20 , 60 ), 6.80 (d, J30 ,20 = J50 ,60 = 8.4 Hz, 4H, 2  H-30 ,50 ); EIMS: m/z (rel. abund. %) 298 (M+, absent), 240 (79.3), 147 (89.6), 136 (100), 120 (40.2), 106 (34.1); TOF-MS 299 (M++H+); HRTOFMS: calcd for C15H15N4O3: m/z = 299.1144, Found: 299.1124; Anal. Calcd for C15H14N4O3: C, 60.40; H, 4.73; N, 18.78; Found: C, 60.41; H, 4.75; N, 18.79. 00

000

3.1.16. N ,N -Bis[(E)-(2,3-dihydroxyphenyl)methylidene]carbonic dihydrazide (16) Solid; yield: 82%; m.p. 240–243 °C; Rf: 0.56 (acetone/hexanes, 1

6:4); H NMR (300 MHz, DMSO-d6): d 10.80 (s, 2H, 2  NH), 9.24 0

99

(br.s, 2H, 2  H-60 ), 6.36 (d, J50 ,60 = 8.4 Hz, 2H, 2  H-50 ); EI-MS: m/ z (rel. abund. %) 362 (M+, absent), 360 (2.0), 304 (100), 194 (15.9), 168 (29.7), 152 (36.0), 138 (11.2); TOF-MS 363 (M++H+); HRTOF-MS: calcd for C15H15N4O7: m/z = 363.0940, Found: 363.0946; Anal. Calcd for C15H14N4O7: C, 49.73; H, 3.89; N, 15.46; Found: C, 49.70; H, 3.92; N, 15.46. 3.1.21. N00 ,N000 -Bis[(E)-(2-methoxyphenyl)methylidene]carbonic dihydrazide (21) Solid; yield: 83%; m.p. 210–211 °C; Rf: 0.51 (acetone/hexanes, 5:5); 1H NMR (400 MHz, DMSO-d6): d 10.68 (s, 2H, 2  NH), 8.49 (s, 2H, 2  @CH), 7.99 (br.s, 2H, 2  H-60 ), 7.38 (dt, J40 (30 ,50 ) = 8.4 Hz, J40 ,60 = 1.6 Hz, 2H, H-40 ), 7.08 (d, J30 ,40 = 8.4 Hz, 2H,

(s, 2H, 2  OH), 8.39 (s, 2H, 2  @CH), 7.10 (br.s, 2H, 2  H-6 ), 6.81, (dd, J40 ,50 = 1.2 Hz, J40 ,60 = 7.5 Hz, 2H, 2  H-40 ), 6.71 (t, J50 (40 ,60 ) = 7.8 Hz, 2H, 2  H-50 ); EI-MS: m/z (rel. abund. %) 330 (M+, 21.7), 330 (M+, 21.7), 272 (100), 178 (86.9), 152 (100), 108 (32.2); HREI-MS: calcd for C15H14N4O5: m/z = 330.0964, Found: 330.0976; Anal. Calcd for C15H14N4O5: C, 54.55; H, 4.27; N, 16.96; Found: C, 54.60; H, 4.29; N, 16.92.

2  H-30 ) 7.01 (t, J5ꞌ(4ꞌ,6ꞌ) = 7.2 Hz, 2H, H-50 ), 3.84 (s, 6H, OCH3); EI-MS: m/z (rel. abund. %) 326 (M+, 5.8), 177 (29.3), 150 (36.6), 134 (20.2), 119 (58.3), 107 (18.9), 91 (100); HREI-MS: calcd for C17H18N4O3: m/z = 326.1379, Found: 326.1382; Anal. Calcd for C17H18N4O3: C, 62.57; H, 5.56; N, 17.17; Found: C, 62.59; H, 5.60; N, 17.19.

3.1.17. N00 ,N000 -Bis[(E)-(2,5-dihydroxyphenyl)methylidene]carbonic dihydrazide (17) Solid; yield: 81%; m.p. 276–279 °C; Rf: 0.59 (acetone/hexanes,

3.1.22. N00 ,N000 -Bis[(E)-(3-methoxyphenyl)methylidene]carbonic dihydrazide (22) Solid; yield: 85%; m.p. 191–193 °C; Rf: 0.48 (acetone/hexanes,

3:7); 1H NMR (300 MHz, DMSO-d6): d 10.74 (s, 2H, 2  NH), 9.90 (br.s 2H, 2  OH), 8.86 (s, 2H, 2  OH), 8.31 (s, 2H, 2  @CH), 7.06 (br.s, 2H, 2  H-50 ), 6.71 (m, 4H, 2  H-30 ,40 ); 13C NMR (75 MHz, DMSO-d6): d 151.89 (C@O), 149.79 (C-2), 149.45 (C-5), 142.44 (@CH), 119.91 (C-1), 118.02 (CH-3), 116.74 (CH-4), 113.18 (CH-6); EI-MS: m/z (rel. abund. %) 330 (M+, absent), 272 (100), 255 (80.7), 178 (11.7), 163 (16.5), 152 (13.4), 136 (79.3); TOF-MS 331 (M++H+); HRTOF-MS: calcd for C15H15N4O5: m/z = 331.1042, Found: 331.1045; Anal. Calcd for C15H14N4O5: C, 54.55; H, 4.27; N, 16.96; Found: C, 54.50; H, 4.29; N, 16.99; IR (KBr, cm
1): 3469 (OH), 3335 (N-H), 1717 (C@O), 1662 (C@N), 1614 (C@C). 3.1.18. N00 ,N000 -Bis[(E)-(5-bromo-2-hydroxyphenyl) methylidene]carbonic dihydrazide (18) Solid; yield: 82%; m.p. 286–290 °C; Rf: 0.53 (acetone/hexanes, 5:5); 1H NMR (300 MHz, DMSO-d6): d 11.01 (s, 2H, 2  NH), 8.37 (s, 2H, 2  @CH), 7.94 (br.s, 2H, 2  H-60 ), 7.37 (dd, J40 ,30 = 8.7 Hz, J40 ,60 = 2.7 Hz, 2H, 2  H-40 ), 6.86 (d, J30 ,40 = 8.7 Hz, 2H, 2  H-30 ); EI-MS: m/z (rel. abund. %) 454 (M+, 11.7), 456 (M++2, 22.5), (M++4, 11.2), 257 (3.4), 242 (59.7), 214 (100), 199 (59.3), 185 (74.0); HREI-MS: calcd for C15H12Br2N4O3: m/z = 453.9276, Found: 453.9274; Anal. Calcd for C15H12Br2N4O3: C, 39.50; H, 2.65; N, 12.28; Found: C, 39.54; H, 2.67; N, 12.21. 3.1.19. N00 ,N000 -Bis[(E)-(2,4,6-trihydroxyphenyl) methylidene]carbonicdihydrazide (19) Solid; yield: 77%; m.p. 248–250 °C; %; Rf: 0.59 (acetone/hexanes,

5:5); 1H NMR (300 MHz, DMSO-d6): d 10.72 (s, 2H, 2  NH), 8.14 (s, 2H, 2  @CH), 7.36 (m, 6H, 2  H-20 ,50 ,60 ), 6.98 (dd, J40 ,50 = 8.1 Hz, J40 ,60 = 2.4 Hz, 2H, H-40 ), 3.80 (s, 6H, OCH3); EI-MS: m/z (rel. abund. %) 326 (M+, 49.0), 268 (16.2), 192 (5.7), 150 (100), 134 (36.8), 120 (54.0); HREI-MS: calcd for C17H18N4O3: m/z = 326.1379, Found: 326.1384; Anal. Calcd for C17H18N4O3: C, 62.57; H, 5.56; N, 17.17; Found: C, 62.60; H, 5.53; N, 17.16. 3.1.23. N00 ,N000 -Bis[(E)-(4-methoxyphenyl)methylidene]carbonic dihydrazide (23) Solid; yield: 84%; m.p. 210–211 °C; Rf: 0.40 (acetone/hexanes, 5:5); 1H NMR (400 MHz, DMSO-d6): d 10.47 (s, 2H, 2  NH), 8.09 (s, 2H, 2  @CH), 7.68 (d, J20 ,30 = J60 ,50 = 8.8 Hz, 4H, 2  H-20 ,60 ), 6.99 (d, J30 ,20 = J50 ,60 = 8.8 Hz, 4H, 2  H-30 ,50 ), 3.79(s, 6H, 2  OCH3); EIMS: m/z (rel. abund. %) 326 (M+, 6.1), 177 (19.3), 150 (100), 120 (28.6); HREI-MS: calcd for C17H18N4O3: m/z = 326.1379, Found: 326.1382; Anal. Calcd for C17H18N4O3: C, 62.57; H, 5.56; N, 17.17; Found: C, 62.52; H, 5.57; N, 17.17. 3.1.24. N00 ,N000 -Bis[(E)-(2-hydroxy-5-methoxyphenyl) methylidene]carbonic dihydrazide (24) Solid; yield: 82% m.p. 227–228 °C; Rf: 0.59 (acetone/hexanes, 3:7);

1

H NMR (300 MHz, DMSO-d6): d 10.87 (s, 2H, 2  NH),

10.23 (br.s 2H, 2  OH), 8.37 (s, 2H, 2  @CH), 7.26 (br.s, 2H, 2  H-60 ), 6.86 (m, 4H, 2  H-30 ,40 ), 3.73 (s, 6H, 2  OCH3);

13

C

NMR (75 MHz, DMSO-d6): d 152.17 (C@O), 151.91 (C-2), 150.68

3:7); 1H NMR (400 MHz, DMSO-d6): d 10.54 (s, 6H, 6  OH), 9.69 (s,

(C-5), 142.30 (@CH), 119.94 (C-1), 117.12 (CH-4), 116.94 (CH-3),

2H, 2  NH), 8.46 (s, 2H, 2  @CH), 5.80 (s, 4H, 2  H-30 ,50 ); EI-MS: m/z (rel. abund. %)362 (M+, absent) 226 (7.3), 194 (31), 168 (100), 152 (48.9), 138 (15.6), 108 (16.5); TOF-MS 363 (M++H+); HRTOFMS: calcd for C15H15N4O7: m/z = 363.0940, Found: 363.0950; Anal. Calcd for C15H14N4O7: C, 49.73; H, 3.89; N, 15.46; Found: C, 49.77; H, 3.84; N, 15.49.

111.69 (CH-6), 55.53 (OCH3); EI-MS: m/z (rel. abund. %) 358 (M+, 5.2), 300 (69.3), 192 (17.9), 166 (41.7), 150 (52.2), 136 (100); HREI-MS: calcd for C17H18N4O5: m/z = 358.1277, Found: 358.1271; Anal. Calcd for C17H18N4O5: C, 56.98; H, 5.06; N, 15.63; Found: C, 56.95; H, 5.02; N, 15.67; IR (KBr, cm
1): 3465 (OH), 3330 (N-H), 1711 (C@O), 1665 (C@N), 1615 (C@C).

3.1.20. N00 ,N000 -Bis[(E)-(2,3,4-trihydroxyphenyl)methylidene]carbonic dihydrazide (20) Solid; yield: 78%; m.p. 258–260 °C; Rf: 0.59 (acetone/hexanes,

3.1.25. N00 ,N000 -Bis[(E)-(2-hydroxy-3-methoxyphenyl) methylidene]carbonic dihydrazide (25) Solid; yield: 81%; m.p. 207–209 °C; Rf: 0.59 (acetone/hexanes,

3:7); 1H NMR (400 MHz, DMSO-d6): d 10.57 (s, 2H, 2  NH), 9.37

3:7); 1H NMR (300 MHz, DMSO-d6): d 10.83 (s, 2H, 2  NH), 8.42

(s, 2H, 2  OH), 8.40 (s, 2H, 2  OH), 8.23 (s, 2H, 2  @CH), 6.89

(s, 2H, 2  @CH), 7.28 (br.s, 2H, 2  H-60 ), 6.98 (d, J50 ,60 = 6.6 Hz,

100

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2H, 2  H-40 ), 6.84 (t, J50 (60 ,40 ) = 8.1 Hz, 2H, 2  H-50 ), 3.80 (s, 6H, 2  OCH3); EI-MS: m/z (rel. abund. %) 358 (M+, 3.7), 300 (27.4), 166 (43.1), 150 (48.7), 136 (22.6), 124 (3.6), 106 (77.0); HREIMS: calcd for C17H18N4O5: m/z = 358.1277, Found: 358.1263; Anal. Calcd for C17H18N4O5: C, 56.98; H, 5.06; N, 15.63; Found: C, 56.97; H, 5.02; N, 15.63. 3.1.26. N00 ,N000 -Bis[(E)-(3-ethoxy-2-hydroxyphenyl) methylidene]carbonic dihydrazide (26) Solid; yield: 80%; m.p. 189–190 °C; Rf: 0.48 (acetone/hexanes, 5:5); 1H NMR (300 MHz, DMSO-d6): d 10.83 (s, 2H, 2  NH), 8.42 0

(s, 2H, 2  @CH), 7.27 (br.s, 2H, 2  H-6 ), 6.97 (dd, J40 ,50 = 8.1 Hz, J40 ,60 = 1.2 Hz, 2H, 2  H-40 ), 6.82 (t, J50 (40 ,60 ) = 7.8 Hz, 2H, 2  H-50 ), 4.08 (q, J = 6.9 Hz, J = 13.8 Hz, 4H, 2  OCH2CH3), 1.36 (t, J = 6.9 Hz, 6H, 2  OCH2CH3); EI-MS: m/z (rel. abund. %) 386 (M+, 2.1), 328 (44.9), 206 (52.2), 180 (100), 164 (66.3); HREI-MS: calcd for C19H22N4O5: m/z = 386.1590, Found: 386.1584; Anal. Calcd for C19H22N4O5: C, 59.06; H, 5.74; N, 14.50; Found: C, 59.09; H, 5.78; N, 14.55. 3.1.27. N00 ,N000 -Bis[(E)-(3,4-dimethoxyphenyl)methylidene]carbonic dihydrazide (27) Solid; yield: 88%; m.p. 171–172 °C; Rf: 0.59 (acetone/hexanes, 3:7); 1H NMR (300 MHz, DMSO-d6): d 10.55 (s, 2H, 2  NH), 8.07 (s, 2H, 2  @CH), 7.39 (s, 2H, 2  H-20 ), 7.17 (dd J60 ,50 = 8.1 Hz, J60 ,40 = 1.5 Hz, 2H, 2  H-60 ), 6.99 (d, J50 ,60 = 8.1 Hz, 2H, 2  H-50 ), 3.82 (s, 6H, 2  OCH3), 3.78 (s, 6H, 2  OCH3);

13

C NMR (75 MHz,

DMSO-d6): d 152.10 (C@O), 150.19 (C-4), 149.03 (C-3), 143.07 (@CH), 127.41 (C-1), 121.12 (CH-6), 111.45 (CH-2), 108.55 (CH5), 55.57 (OCH3), 55.53 (OCH3); EI-MS: m/z (rel. abund. %) 386 (M+, 2.8), 328 (100), 207 (19.8), 191 (49.2), 180 (82.5), 150 (22.7); HREI-MS: calcd for C19H22N4O5: m/z = 386.1590, Found: 386.1570; Anal. Calcd for C19H22N4O5: C, 59.06; H, 5.74; N, 14.50; Found: C, 59.09; H, 5.71; N, 14.55; IR (KBr, cm
1): 3333 (N-H), 1711 (C@O), 1663 (C@N), 1608 (C@C). 3.2. Assay for carbonic anhydrase II In this assay, 4-nitrophenyl acetate (4-NPA), a colorless compound, was hydrolyzed to 4-nitrophenol and CO2. The reaction was followed by measuring the formation of 4-nitrophenol, a yellow colored compound. The reaction was performed at 25 °C in buffer containing HEPES and Tris-HCl at a total concentration of 20 mM and pH of 7.4 for each sample. The reaction mixture contained 140 lL of the HEPES-tris solution, 20 lL of freshly prepared aqueous solution of purified bovine erythrocyte CA-II (0.1 mg/mL of deionized water for 96-well), 20 lL of test compound in DMSO (10% final concentration), 20 lL of substrate 4-NPA at a concentration of 0.7 mM diluted in ethanol. The reaction was initiated by addition of 4-NPA after 15 min incubation of test compound, and each compound was tested 3times at different concentrations. In this assay, the reaction was performed using 96-well plates. The plate was placed in a spectrophotometer and the amount of product formed was monitored at a 1 min interval for 30 min at 400 nm [39].

bottom plate in ELISA reader (Spectra max 384, Molecular Devices USA) after addition of substrate for a period of 30 min at 25 °C with 1 min interval. The Km value was obtained by constructing a Lineweavere Burk-plot between 1/S vs 1/Vmax. For Ki determination, the double reciprocal plot was constructed which was plotted between inhibitor concentration [I] and slope (Slope = Km/Vmax). The Dixon plot was also constructed between inhibitor concentrations [I] and 1/Vmax which further confirm the Ki value and type of inhibition [40]. 3.4. Statistical analysis Grafit 7.0 version was used to determine the kinetics parameter. The software was purchased from Erithacus Software Ltd. Wilmington House, High Street, East Grinstead, West Sussex RH19 3AU, UK. 3.5. Ligand docking Single crystal X-ray diffraction, structural coordinates of bovine carbonic anhydrase (PDB id; 1V9E) [36] was used for docking of carbohydrazone compounds. Ligand docking was carried out by using AutoDock Vina program [37]. The structure was checked for missing atoms, bonds and contacts. The B chain of protein and hetero-atoms were removed from original PDB file. Hydrogen atoms were added to the enzyme structure by using MGLTools (http://autodock.scripps.edu/). The 3D models of the carbohydrazones were constructed in SYBYL mol2 format. After each docking run, 10 top ranking docking solutions were saved and considered for detailed analysis. For its inputs and outputs, AutoDock Vina uses the same PDBQT molecular structure file format used by AutoDock. PDBQT files were generated interactively and viewed using MGLTools (http:// autodock.scripps.edu). A configuration file was made with following parameters; docking site was defined as all atoms within 10 Å of a specified centroid; number of points in x-dimension =
25.2, number of points in y-dimension =
26.9, number of points in z-dimension = 0.88; Center Grid Box (size_x = 22, size_y = 24, size_z = 28); exhaustness = 8. AutoDock Vina ligand docking was carried out allowing full flexibility for the ligands, while keeping the protein fixed. All single bonds were treated as rotatable. The docking calculations were performed on IBM Intellistation workstation running under SUSE 11.0 Linux operating system. Acknowledgement This work was supported by the Higher Education Commission (HEC) Pakistan, Project No. 20-1910 under the National Research Program for Universities. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bioorg.2017.03. 014. References

3.3. Kinetic studies Kinetic studies were performed by using different concentration of inhibitors over different concentrations of substrate (4-NPA) such as 0.175, 0.35, 0.70 and 1.40 mM. The enzyme 0.2 mg/mL concentration for each well was used after dissolving in de-ionized water. HEPES-tris ammonia was used as buffer at pH of 7.4. The change in absorbance was measured by keeping the 96-well flat

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