Efficient green synthesis of

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668 RESEARCH PAPER

VOL. 39

NOVEMBER, 668–673

JOURNAL OF CHEMICAL RESEARCH 2015

Efficient green synthesis of N′-benzylidene-2-(2-fluorobiphenyl) propanehydrazides: crystal structure and anti-oxidant potential Muhammad Zaheera,b, Muhammad Zia-ur-Rehman∗a, Nadia Jamilb, Muhammad Nadeem Arshadc, Sabahat Zahra Siddiquid and Abdullah Mohammad Asiric Applied Chemistry Research Centre, PCSIR Laboratories Complex, Lahore-54600 Pakistan College of Earth & Environmental Sciences, University of the Punjab, Lahore-54590 Pakistan c Chemistry Department & Centre of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, Jeddah 21589, Saudi Arabia d Department of Chemistry, Government College University, Lahore-54600, Pakistan a

b

For the synthesis of N’-benzylidene-2-(2-fluorobiphenyl)propanehydrazides, 2-(2-fluorobiphenyl-4-yl)propanoic acid was esterified to methyl 2-(2-fluorobiphenyl-4-yl)propanoate under microwave irradiation followed by hydrazinolysis. This hydrazide was then reacted in an ultrasonic bath with different benzaldehydes to afford a series of N’-benzylidene-2-(2-fluorobiphenyl)propanehydrazides. Comparison of both the conventional and ultrasound assisted reactions indicates a significant reduction in reaction times and improvement in yields. Besides chemical characterisation and X-ray crystallography, all the synthesised compounds were evaluated for their anti-oxidant potential (total anti-oxidant activity, ferric reducing antioxidant power and total phenolic content) and most of them were found to have useful potential for pharmaceutical applications especially derivative bearing 2-hydroxy substituents. Keywords: efficient synthesis, ultrasonics, microwaves, carbohydrazide, NSAIDs Oxidation is a useful process in many organisms for the biological conversion of matter to energy. The process involves the production of free radicals which, if uncontrolled, may lead to a number of diseases such as rheumatoid arthritis, cancer and atherosclerosis.1 It may also lead to the degenerative processes associated with ageing.2 Oxidative stress may result in the damage of biopolymers including nucleic acids, proteins, polyunsaturated fatty acids and carbohydrates which play a significantly pathological role in human diseases such as cancer, emphysema, cirrhosis and atherosclerosis.3,4 The importance of anti-oxidants to prevent the progression of agerelated diseases and their role in enzymatic and non-enzymatic defense mechanisms in both the lipid and aqueous phases is well established.5 In order to minimise the free radical damage to the human body, a number of natural, as well as synthetic antioxidants such as ascorbic acid, vitamins A and E, glutathione and propanolol (Fig. 1), are used. However, unfortunately most of the commonly used synthetic anti-oxidants are suspected to cause liver damage and carcinogenesis making it essential to develop new anti-oxidant molecules and pharmacophores which can protect the human H

body from free radicals and retard the progress of many chronic diseases.6 Carbohydrazides, on the other hand, possess a diverse set of biological activities7 and are emerging as potential anticancer agents due to their anti-proliferative properties. They are also known to suppress tumor formation.8-11 Keeping in mind the potential activities of carbohydrazides, we attempted to create a carbohydrazide functionality on a well-known nonsteroidal anti-inflammatory, analgesic and anti-pyretic drug, flurbiprofen12 for further derivatisation to afford a series of novel N′-benzylidene-2-(2-fluorobiphenyl)propanehydrazides (Scheme 1). In the key step, ultrasonic waves are used to carry out the condensations, which have proved to be successful to enhance reaction rates and shorten the reaction times (Table 1).

Results and discussion Chemistry 2-(2-Fluorobiphenyl-4-yl)propanehydrazide (3), the key precursor, was synthesised by esterification of 2-(2-fluorobiphenyl-4-yl) propanoic acid (1) under microwave irradiation using sulfuric acid as catalyst followed by hydrazinolysis of the ester. Use of

OH OH

O

OH

O

CH3

CH3

OH

OMe

OH Vitamin A

OH

MeOH, H

O

Ascorbic acid

F H2N

+

COOH

Microwaves

(1)

(2)

Microwaves

HO

HN O

O SH

Vitamin E

CH3

O

NH

O

COOH Propanolol

F

N2H4.H2O

HN

O

O

Glutathione

Fig. 1 Structures of some known natural and synthetic anti-oxidants. * Correspondent. E-mail: [email protected]

CH3 N

R

(4a-s)

H N

NH2

O

+

RCHO/ H / EtOH Ultrasounds

F OH

H N

F (3)

Scheme 1 Synthesis of N´-benzylidene-2-(2-fluorobiphenyl)propanehydrazides.

JOURNAL OF CHEMICAL RESEARCH 2015 669 microwaves in the esterification step plays significant role in minimising the reaction time and increasing the yield. The reaction time was reduced to 3 hours to 15 minutes13 while the yield of the reaction was enhanced from 93% to 98.8%. In the second step, the ester was reacted with hydrazine hydrate (80%) in the presence of ethanol under microwave irradiation; this reaction could not be completed using a conventional reflux method. The use of microwave energy is becoming popular for a number of reactions due to low catalyst loadings, shorter reaction times and to limit the generation of unwanted side products;14 and the same procedure was adopted for the conversion of methyl 2-(2-fluorobiphenyl-4yl)propanoate (2) to 2-(2-fluorobiphenyl-4-yl)propanehydrazide (3) in good yield (96.2%). Both of the reactions were attempted in an ultrasonic bath but were unsuccessful. Condensation of 2-(2-fluorobiphenyl-4-yl)propanehydrazide (3) with different substituted aldehydes was carried out to obtain the title compounds in ethanol using glacial acetic acid as the catalyst. All the condensation reactions were attempted in an ultrasonic heating bath which was found to be more effective than using thermal conditions, as shown by the greater yields and shorter reaction times (Table 1). Ultrasound tends to create cavities within the reaction mixture and their collapse may release very high local temperatures and pressures which lead to an enhancement of reaction rates and product yield.15-17 All of the newly synthesised compounds were characterised using spectroscopic techniques (FTIR, 1H NMR, 13C NMR and mass spectrometry) along with their elemental analyses and were found in accordance with the calculated values. Stereochemistry and X-ray crystallography To explore the stereochemistry (E or Z configuration) of the C=N bond of the compounds under investigation (4a–s), a single crystal of N’-(2-chlorophenylmethylidene-2-(2-fluorobiphenyl4-yl)propanehydrazide (4b) was grown by recrystallising the compound from 90% ethanol and was studied by single crystal X-ray crystallography. It was found that C=N bond exhibits E configuration and the compound 4b crystallises in monoclinic space group P 21/a with Z = 4. The aromatic rings of biphenyl moiety are oriented at dihedral angle of 48.15(8) o. The root mean square deviation for the fitted atoms of aliphatic

backbone “propanehydrazide” is 0.1009(2)Å with maximum deviation from C14 = 0.1872(2)Å and C13 = 0.1295(2)Å. The chlorophenyl ring is oriented at dihedral angle of 28.95(1) o with respect to this backbone. On the other hand, the dihedral angle between this aliphatic hydrazide moiety and flourophenyl ring is 76.96(6) o and appears perpendicular to each other (Fig 2). There is a combination of classical and non-classical hydrogen bonding interaction which connects the molecules along the a axis to form infinite chains. These chains were further connected through symmetry related C–H…F interaction (Fig 3). Supplementary crystallographic data have been deposited with the CCDC number 986584. These data can be obtained free of charge from www.ccdc.cam.ac.uk/data_request/cif. Details of crystal data and structure refinement have given in Table 2. Anti-oxidant activity The total antioxidant capacity of the synthesised derivatives was measured using UV-Vis spectrophotometry at 695 nm by a literature procedure18 in which Mo (VI) is reduced to Mo (V) by the compounds under investigation. This change was detected by the formation of green phosphate Mo(V) compounds.

Fig. 2 ORTEP3 diagram of compound 4b with the numbering scheme. Displacement ellipsoids are drawn at the 50% probability level; H atoms are represented by circles of arbitrary radii.

Table 1 Synthesis of N’-benzylidene-2-(2-fluorobiphenyl)propanehydrazides under conventional and ultrasound conditions (4a–s) Conventional Ultrasound Entry Product R M.p./°C Time/min Yield/%a Time/min Yield/%a 1 4a Phenyl 187–188 70 90 04.5 98 2 4b 2-Chlorophenyl 201 110 87 09.0 95 3 4c 3-Chlorophenyl 200–202 90 85 07.5 93 4 4d 2,3-Dichlorophenyl 180–181 120 86 12.0 92 5 4e 2,4-Dichlorophenyl 194–195 110 83 10.5 94 6 4f 3,4-Dichlorophenyl 166 100 88 09.0 95 7 4g 2-Hydroxyphenyl 176–177 120 84 15.0 90 8 4h 4-Hydroxyphenyl 235–236 100 89 10.5 96 196–197 110 83 13.5 94 9 4i 4-N,N -Dimethylphenyl 10 4j 4-Hydroxy,3-methoxyphenyl 171–173 115 83 12.0 92 11 4k 9-Anthracenyl 218–219 110 86 09.0 95 12 4l 2-Furfuryl 197 90 88 07.5 96 13 4m 5-Methyl furfuryl 199–200 120 84 13.5 93 14 4n 1-Pentyl-2-propenyl benzene 104 120 83 15.0 92 15 4o 2-Methylphenyl 177–178 110 80 15.0 94 16 4p 3-Methyphenyl 184–185 120 79 15.0 91 17 4q 4-Methylphenyl 189–190 100 84 12.0 96 18 4r 2-Nitrophenyl 210–211 120 78 15.0 92 19 4s 3,4-Dimethoxyphenyl 170 135 77 18.0 90 a

Isolated yields based on 2-(2-fluorobiphenyl-4-yl)propanehydrazide (3).

670 JOURNAL OF CHEMICAL RESEARCH 2015 Table 2 Crystallographic parameters for compound 4b Structural formula C22H18ClFN2O Formula weight Crystal system Space group T/K a/Å; α b/Å; α c/Å; γ 2θ range for data collection

380.83 Monoclinic P21/a 569(2) 8.1561(4); 90 16.0334(13); 97.243(5) 14.8277(10); 90 8.16 to 155.18°

Fig. 3 Unit cell diagram of compound molecule 4b showing the hydrogen bonding interaction using dashed lines. Hydrogen atoms not involved in bonding have been omitted.

The results of the total antioxidant activity revealed that N’benzylidene-2-(2-fluorobiphenyl)propanehydrazides possess excellent anti-oxidant potentials especially, derivatives containing halo/ hydroxy substituents as compared to the standard butylated hydroxytoluene (BHT) of 0.46. The highest total anti-oxidant activity was exhibited by 2-hydroxy derivative 4g (1.998); the other incorporated substituents which were key contributors towards potent activity were in the order of: 2,3-dichloro 4d (1.032) > N,N-dimethyl 4i (0.988) > 4-hydroxy 4h (0.980), 3,4-dichloro 4f (0.975) which displayed excellent activity. Derivatives 4b, 4c, 4e, 4h, 4i and 4q also revealed good activities which were better than the standard. Derivatives 4a (0.455) and 4k (0.411) showed activities almost equal to the standard while 4j, 4l and 4n were found to be less active. The data of the synthesised derivatives are shown in Table 3. The anti-oxidant activity of the synthesised compounds was correlated with their total phenolic contents. Phenolic concentration is expressed as mg of gallic acid equivalents (GAEs) per g of the anti-oxidant under investigation.19 Results revealed that the compounds bearing hydroxyl groups were potent anti-oxidants. The highest activity was displayed by 2-hydroxy substituent 4g having a value of (381) followed by the 4-hydroxy-3-methoxy derivative 4j (315), 4-hydroxy derivative 4h (166), 3-chloro derivative 4c (137). It was observed that halogen substituted derivatives possess moderate to weak activities while compounds containing furfuryl 4l, 1-pentyl-2propenylbenzene 4n and 3-methylphenyl 4p groups were found

Cell volume

1923.5(2)

Z Calculated density Crystal size Reflections collected Independent reflections Data/restraints/parameters Goodness-of-fit on F2 F(000)

4 1.315 0.46 × 0.08 × 0.07 20314 4016 [R(int) = 0.0595] 4016/0/247 1.026 792.0

Table 3 Total antioxidant activity, total phenolic content and FRAP values of the synthesised compounds FRAP value Total antioxidant Total phenolic /TE µM mL-1 Entry Product activity content -1 ± SEM /GAE mg g ± SEM ± SEM) 1 4a 0.455±0.12 0.34±0.08 6.40±1.10 2 4b 0.923±0.09 0.21±0.07 0.23±0.08 3 4c 0.720±0.03 137±0.32 2.41±0.01 0.04+0.01 4 4d 1.032±0.01 0.21±0.09 5 4e 0.759±0.01 69.0±1.56 0.84±0.02 6 4f 0.975±0.02 113±1.08 1.00±0.10 7 4g 1.998±0.22 381±1.13 8.00±1.00 8 4h 0.980±0.08 166±1.03 10.8±0.09 151+0.98 21.2±0.49 9 4i 0.988±0.12 10 4j 0.385±0.03 315±0.86 NA 11 4k 0.411±0.29 0.22±0.07 0.03±0.06 12 4l 0.385±0.16 NA NA 13 4m 0.555±0.04 0.41±0.05 1.79±0.48 14 4n 0.382±0.06 NA 0.041±0.02 0.32+0.06 3.23±0.13 15 4o 0.610±0.18 0.038+0.01 16 4p 0.408±0.14 NA 0.28+0.04 2.87±0.76 17 4q 0.569±0.11 18 4r 0.745±0.07 0.41±0.09 6.45±0.84 0.23+0.03 6.27±0.29 19 4s 0.458±0.12 a 20 Control BHT (0.46±0.09) a NA, no activity observed. a Readings were calculated with reference to the calibration curves drawn with reference to Gallic acid and Trolox for TPC and FRAP respectively.

inactive. The activity was calculated as compared to GAEs per g of anti-oxidant by plotting a standard calibration curve having straight line equation (y = 0.0043x + 0.4348; R² = 0.9945). The reducing power of the synthesised compounds was checked using a ferric reducing antioxidant power (FRAP) assay20 which portrays anti-oxidants as reductants in a redoxlinked colorimetric method using an easily reduced oxidant system at low pH. It involves reduction of yellow coloured ferric tripyridyltriazine (FeIII TPTZ) complex to various shades of green and blue depending on reducing power of the derivatives under investigation. Any change in absorbance is directly related to the combined reducing power of the electron donating antioxidants present in the reaction mixture which is measured at 595 nm.21 The present study revealed that most of the compounds under study were active except compounds 4j and 4l. The highest activity was exhibited by N,N-dimethyl derivative 4i i.e., Trolox equivalents (TE) µM mL-1 (21.2) followed by 4-hydroxy derivative 4h (10.8) and 2-hydroxy 4g (8.00). In general, the antioxidant study revealed that the compounds containing halo, hydroxy and N,N-dimethyl moieties are potent antioxidants. The results were expressed in TE µM mL-1 by plotting a standard calibration curve with a straight line equation (y = 0.0008x + 0.29; R² = 0.9082).

JOURNAL OF CHEMICAL RESEARCH 2015 671

Experimental CAUTION: The use of a domestic microwave oven for chemical purposes can be hazardous so adequate safety precautions must be taken. All the chemicals were purchased from E. Merck, BDH or Fluka and used without purification; however, solvents were purified through distillation. 1H and 13C NMR spectra were recorded on Bruker AVANCEIII 600 MHz spectrophotometer and tetramethylsilane as an internal standard. FTIR spectra were recorded on a Bruker Tensor 27 spectrometer. Mass spectra were recorded on LCQ Advantage Max Thermo fisher instrument using ESI mode. Melting points were recorded on a Mettller Toledo FP 62 melting point apparatus and are uncorrected. Microwave assisted reactions were carried out in a domestic microwave oven (Orient eNNe781JF) equipped with inverter technology (generating fixed frequency throughout the required time) for realistic control of the microwaves operating at multiples of 100 W up to 1000 W generating 2450 MHz frequency. Ultrasonic mediated reactions were carried out in Clifton Ultrasonic Bath (2 x T2A, 300W, DU-4) made by Nickel Electro Ltd, Weston-S-Mare Somerset, England. The apparatus was modified for laboratory applications, equipped with magnetic stirrer and an external reflux condenser. Methyl 2-(2-fluorobiphenyl-4-yl)propionate (2): Prepared using a modified literature procedure.13 A mixture of 2-(2-fluorobiphenyl4-yl)propanoic acid (1) (4.50 g; 18.423 mmol), methanol (100 mL) and concentrated sulfuric acid (2–3 drops) was irradiated under microwaves (300 W) to reflux. After completion of the reaction (as indicated by TLC), excess methanol was distilled off and the resultant mixture was poured over ice cooled aqueous sodium bicarbonate solution (4%) to get the required product in the form of off-white hard flakes. Off white solid; m.p. 40˚C (lit.13 39 ˚C). IR (KBr): 2984, 1733, 1623 cm ; 1H NMR (CDCl3) δ 1.52(d, J = 6.8 Hz, 3H, CH3), 3.71(s, 3H, OCH3), 3.78 (q, J = 6.8, 1.0 Hz, 1H, CH), 7.39–7.54 (m, 8H, ArH). MS m/z [M+H]+ 259.28. 2-(2-Fluorobiphenyl-4-yl)propanehydrazide (3) was prepared using modified literature procedure14 with slight modification. A mixture of methyl 2-(2-fluorobiphenyl-4-yl)propanoate (2) (10.0 g; 38.716 mmol), hydrazine hydrate 80% (2.35 mL; 38.716 mmol) and ethanol (200 mL) was irradiated to reflux under microwaves (300 W). After completion of the reaction, excess ethanol was removed under reduced pressure and the resultant mixture was acidified with hydrochloric acid (5% v/v) to Congo red to get the product. Recrystallisation of the crude product afforded needle like crystalline material; m.p. 110˚C (lit.14 101˚C). IR (KBr): 3309, 2987, 1633 cm-1; 1H NMR (CDCl3) δ 1.53 (d, J = 6.8 Hz, 3H, CH3), 2.63 (s, 2H, NH2), 3.57 (q, J = 6.8, 1.0 Hz, 1H, CH), 7.09–7.54 (m, 8H, ArH), 8.49 (s, 1H, NH); MS m/z [M+H]+ 259.29 -1

Synthesis of N’-benzylidene-2-(2-fluorobiphenyl)propanehydrazides (4a-s); general procedure A mixture of 2-(2-fluorobiphenyl-4-yl)propanehydrazide (3) (200 mg; 0.774 mmoles), benzaldehyde (0.774 mmol), ethanol (25 mL) and glacial acetic acid (one or two drops) was reacted in an ultrasonic bath at 80oC until completion of the reaction (reaction times are shown in Table 1). After removal of excess ethanol under vacuum, contents were neutralised with ice-cooled aqueous sodium bicarbonate solution (4% w/w). Recrystallisation of the product gave the products which were then recrystallised in ethanol. 2-(2-Fluorobiphenyl-4-yl)-N’-phenylmethylidene propanehydrazide (4a): White powder; m.p. 187˚C. IR (KBr): 3028 (NH), 1644 (C=O), 1600 (C=N) cm-1; 1H NMR (CDCl3, 600 MHz) δ 1.56 (d, J = 6.8 Hz, 3H, CH3), 4.09 (q, J = 6.8, 1.0 Hz, 1H, CH), 7.08 (s, 1H, ArH), 7.15 (m, 4H, ArH), 7.42–7.46 (m, 2H, ArH), 7.50–7.56 (m, 2H, ArH), 7.63–7.66 (m, 3H, ArH), 7.90 (dd, J = 7.5, 5.6 Hz, 1H, ArH), 8.34 (s, 1H, N=CH), 10.03 (s, 1H, NH); 13C NMR δ 18. 3, 43.5, 114.7, 115.2, 123.9, 124.6, 126.4, 127.8, 128.7, 129.4, 130.1, 131.4, 132.2, 133.3, 134.6, 139.0, 143.6, 144.7, 158.1, 159.3, 169.9, 174.7. Anal. calcd for C22H19FN2O: C, 76.28; H, 5.53; N, 8.09; found: C, 76.22; H, 5.49; N, 8.12%; MS m/z [M+H]+ 347.4.

N’- (2- Chlorophenylmethylidene-2- (2-f luorobiphenyl- 4 -yl) propanehydrazide (4b): White crystalline powder; m.p. 201˚C. IR (KBr):

3033 (NH), 1650 (C=O), 1623 (C=N) cm-1; 1H NMR (CDCl3) δ 1.45 (d, J = 6.7 Hz, 3H, CH3), 3.76 (q, J = 6.8, 1.0 Hz, 1H, CH) 7.25 (s, 1H, ArH) 7.27–7.61 (m, 8H, ArH), 7.92 (dd, J = 7.5, 5.7 Hz, 1H, ArH), 7.99–8.01 (m, 2H, ArH), 8.33(s, 1H, N=CH), 11.56 (s, 1H, NH) . 13C NMR δ 18.3, 39.5, 43.6, 115.0, 124.0, 126.8, 127.8, 128.7, 129.9, 130.7, 131.5, 132.9, 133.1, 134.9, 139.1, 142.7, 158.0, 169.3, 174.5. Anal. calcd for C22H18ClFN2O: C, 69.38 H 4.76; N, 7.36; found: C, 69.31; H, 4.77; N, 7.38%; MS m/z: [M+H]+ 381.84, [M+H+2]+ 383.82

N’- (3- Chlorophenylmethylidene-2- (2-f luorobiphenyl- 4 -yl) propanehydrazide (4c): White crystalline powder; m.p. 200˚C. IR (KBr): 3032 (NH), 1651 (C=O), 1623 (C=N) cm-1; 1H NMR (CDCl3) δ 1.67 (d, J = 6.8 Hz, 3H, CH3), 4.05 (q, J = 6.7, 1.0 Hz, 1H, CH), 6.60 (s, 1H, ArH), 7.36–7.54 (m, 7H, ArH) 7.77 (t, J = 2.0 Hz, 1H, ArH), 7.78–7.87 (m, 2H, ArH), 8.41(s, 1H, N=CH), 9.98 (s, 1H, NH) . 13C NMR δ 18.3, 40.2, 43.2, 115.2, 124.2, 126.7, 127.3, 128.2, 129.9, 130.9, 131.4, 132.4, 133.4, 134.0, 139.2, 143.2, 158.0, 169.4, 174.7. Anal. calcd for

C22H18ClFN2O: C, 69.38; H, 4.76; N, 7.36; found: C, 69.27; H, 4.75; N, 7.39%; MS m/z: [M+H]+ 381.84, [M+H+2]+ 383.81 N’- (2,3-Dichlorophenylmethylidene-2- (2-fluorobiphenyl-4-yl) propanehydrazide (4d): White powder; m.p. 180˚C. IR (KBr): 2975 (NH), 1652 (C=O), 1559 (C=N) cm-1; 1H NMR (CDCl3) δ 1.67 (d, J = 6.8 Hz, 3H, CH3), 4.68 (q, J = 6.7, 1.0 Hz, 1H, CH), 6.62 (s, 1H, ArH), 7.27–7.55 (m, 9H, ArH), 7.96 (dd, J = 7.5, 5.7 Hz, 1H, ArH), 8.40 (s, 1H, N=CH), 10.45 (s, 1H, NH). 13C NMR δ ppm: 18.3, 44.5, 114.3, 115.4, 124.2, 127.2, 128.4, 129.7, 130.2, 133.4, 135.8, 136.3, 137.1, 158.3, 159.9, 161.3, 162.7, 169.3, 174.7. Anal. calcd for C22H17Cl2FN2O: C, 63.63; H, 4.13; N, 6.75; found: C, 63.56; H, 4.14; N, 6.76%; MS m/z: [M+H]+ 416.29, [M+H+4]+ 420.28

N’- (2,4-Dichlorophenylmethylidene-2- (2-fluorobiphenyl-4-yl) propanehydrazide (4e): Off-white crystalline powder; m.p. 194˚C. IR (KBr): 3051 (NH), 1663 (C=O), 1586 (C=N) cm-1; 1H NMR (CDCl3 δ 1.67 (d, J = 6.8 Hz, 3H, CH3), 4.67 (q, J = 6.7, 1.0 Hz, 1H, CH), 7.09 (s, 1H, ArH), 7.20–7.55 (m, 8H, ArH), 7.89 (d, J = 7.4 Hz, 1H, ArH), 8.04 (s, 1H, ArH), 8.43 (s, 1H, N=CH), 10.42 (s, 1H, NH). 13C NMR δ 18.3, 44.4, 114.2, 116.0, 124.5, 126.0, 127.7, 128.0, 130.7, 131.2, 132.1, 134.1, 136.8, 143.7, 157.3, 159.5, 160.0, 162.1, 174.4. Anal. calcd for C22H17Cl2FN2O: C, 63.63; H, 4.13; N, 6.75, Found: C, 63.68; H, 4.12; N, 6.76%; MS m/z: [M+H]+ 416.29, [M+H+4]+ 420.28

N’- (3,4-Dichlorophenyl) methylidene-2- (2-fluorobiphenyl-4-yl) propanehydrazide (4f): Off-white crystalline powder; m.p. 166˚C. IR (KBr): 3075 (NH), 1668 (C=O), 1606 (C=N) cm-1; 1H NMR (CDCl3) δ 1.67 (d, J = 6.7 Hz, 3H, CH3), 4.67(q, J = 6.8, 1.0 Hz, 1H, CH), 6.63 (s, 1H, ArH), 7.39–7.73 (m, 9H, ArH), 7.96–7.97 (m, 1H, ArH), 8.56(s, 1H, N=CH), 9.95(s, 1H, NH). 13C NMR δ 18.3, 44.5, 115.7, 116.0 124.9, 126.1, 127.1, 128.4, 129.6, 130.3, 131.4, 135.2, 136.7, 144.1, 158.4, 159.4, 160.3, 162.7, 174.0. Anal. calcd for C22H17Cl2FN2O: C, 63.63; H, 4.13; N, 6.75, Found: C, 63.78; H, 4.12; N, 6.73%; MS m/z: [M+H]+ 416.29, [M+H+4]+ 420.28

2- (2-Fluorobiphenyl-4-yl) -N’-[(2-hydroxyphenyl) methylidene] propanehydrazide (4g): Off-white crystalline powder; m.p. 176˚C. IR (KBr): 2983 (NH), 1659 (C=O), 1639 (C=N) cm ; 1H NMR δ 1.67 (d, J = 6.8 Hz, 3H, CH3), 4.70 (q, J = 6.8, 1.0 Hz, 1H, CH), 6.96–7.05 (m, 9H, ArH) 7.37 (dd , J = 7.5, 5.7 Hz, 1H,) 7.39 –7.42 (m, 3H, ArH), 8.65 (s, 1H, N=CH), 9.91 (s, 1H, NH) 11.02 (s, 1H, OH),. 13C NMR (CDCl3) δ -1

ppm: 18.1, 43.9, 115.2, 124.3, 126.1, 127.2, 128.8, 129.6, 131.0, 131.5, 133.2, 134.8, 139.2, 144.1, 157.1, 158.1, 159.5, 161.0, 169.3, 175.6. Anal. calcd for

C22H19FN2O2: C, 72.91; H, 5.28; N, 7.73; found: C, 72.83; H, 5.29; N, 7.75%; MS m/z [M+H]+ 363.40 2- (2-Fluorobiphenyl-4-yl) -N’-[(2-hydroxyphenyl) methylidene] propanehydrazide (4h): Off-white powder; m.p. 235˚C. IR (KBr): 3050 (NH), 1644 (C=O), 1596 (C=N) cm ; 1H NMR δ 1.64 (d, J = 6.8 Hz, 3H, CH3), 4.72 (q, J = 6.8, 1.0 Hz, 1H, CH), 6.87 (m, 1H, ArH), 7.08 (s, 1H, ArH), 7.22–7.43 (m, 7H, ArH) 7.55 (dd, J = 7.5, 5.7 Hz, 1H, ArH), 7.60 (s, 1H, ArH), 8.01 (s, 1H, ArH), 8.30 (s, 1H, N=CH), 9.87 (s, 1H, OH), 9.95 (s, 1H, NH),. 13C NMR δ 18.3, 43.8, 115.8, 124.2, 126.8, 127.6, 128.4, -1

129.1, 130.4, 131.5, 132.1, 134.0, 140.5, 144.2, 157.7, 158.7, 159.4, 160.8, 162.5, 175.0. Anal. calcd for C22H19FN2O2: C, 72.91; H, 5.28; N, 7.73; found: C, 73.02; H, 5. 29; N, 7.72%; MS m/z [M+H]+ 363.40.

672 JOURNAL OF CHEMICAL RESEARCH 2015 N’-4- (Dimethylamino) phenylmethylidene-2- (2-fluorobiphenyl) propanehydrazide (4i): Off-white powder; m.p. 196˚C. IR (KBr): 3031 (NH), 1643 (C=O), 1598 (C=N) cm-1; 1H NMR δ 1.63 (d, J = 6.8 Hz, 3H, CH3), 3.03 (s, 5H, N(CH3) 2 4.75 (q, J = 6.7, 1.0 Hz, 1H, CH), 6.71 (d, J = 6.8 Hz , 1H, CH), 7.25 –7.56 (m, 10H, ArH), 7.58 (s, 1H, ArH), 7.90 (s, 1H, ArH), 8.35 (s, 1H, N=CH), 10.03 (s, 1H, NH). 13C NMR δ 18.4, 43.1, 44.4, 113.6, 124.2, 126.2, 127.4, 128.3, 129.2, 130.5, 133.1, 137.6, 141.7, 148.3, 157.3, 158.6, 160.1, 161.3, 163.4, 175.5. Anal. calcd for C24H24FN3O: C, 74.01; H, 6.21; N, 10.79; found: C, 73.91; H, 6.24; N, 10.81%; MS m/z [M+H]+ 390.46 2- (2-Fluorobiphenyl- 4 -yl) -N’- [(4 - hydrox y-3- methox yphenyl) methylidene]propanehydrazide (4j): Off-white powder; m.p. 171˚C. IR (KBr): 2988 (NH), 1655 (C=O), 1637(C=N) cm-1; 1H NMR δ 1.64 (d, J = 6.8 Hz, 3H, CH3), 3.98 (s, 2H, OCH3), 4.68 (q, J = 6.7, 1.0 Hz, 1H, CH), 5.85 (s, 1H, OH) 7.26–7.54 (m, 10H, ArH), 7.57 (s, 1H, ArH), 7.95 (s, 1H, ArH), 8.53 (s, 1H, N=CH), 9.97 (s, 1H, NH). 13C NMR δ 18.3, 44.7, 55.4, 113.5, 114.2, 124.2, 127.1, 128.8, 130.1, 131.3, 132.2, 137.6, 140.8, 144.3, 148.5, 158.6, 160.2, 161.2, 162.1, 174.9. Anal. calcd for C23H21FN2O3: C, 70.40; H, 5.39; N, 7.14; found: C, 70.26 H 5.40; N, 7.16%; MS m/z [M+H]+ 393.42.

2- (2-Fluorobiphenyl- 4-yl) -N’- (anthracen-9-ylmethylidene) propanehydrazide (4k): Light yellowish powder; m.p. 218˚C. IR (KBr): 3006 (NH), 1668 (C=O), 1644 (C=N) cm-1; 1H NMR δ 1.43(d, J = 6.8 Hz, 3H, CH3), 4.83 (q, J = 6.8, 1.0 Hz, 1H, CH), 6.51 (d, J = 7.5 Hz, 1H, ArH),

6.87 (m, 1H, ArH), 7.27–7.60 (m, 6H, ArH), 7.65–7.66 (m, 2H, ArH), 7.73 (dd, J = 7.4, 5.7 Hz, 1H, ArH), 7.83–8.08 (m, 5H, ArH), 8.17 (s, 1H, ArH), 8.42 (s, 1H, N=CH), 10.15(s, 1H, NH). 13C NMR δ 18.3, 43.6, 114.4, 123.6, 124.3, 125.3, 126.1, 127.57, 128.3, 129.2, 130.2, 131.0, 131.5, 131.8, 132.6, 134.2, 135.6, 141.8, 143.3, 159.4, 160.4, 161.3, 162.2, 164.3, 174.9. Anal. calcd for C30H23FN2O: C, 80.70; H, 5.19; N, 6.27; found: C, 80. 89; H, 5.20; N, 6.29%; MS m/z [M+H]+ 447.51

2 - ( 2 - F l u o r o b i p h e n y l - 4 -y l ) - N’- ( f u r a n - 2 -y l m e t h y l i d e n e ) propanehydrazide (4l): Buff coloured powder; m.p. 197˚C. IR (KBr): 2988 (NH), 1645 (C=O), 1581(C=N) cm-1; 1H NMR δ 1.63 (d, J = 6.7 Hz, 3H, CH3), 4.72 (q, J = 6.8, 1.0 Hz, 1H, CH), 6.50 (dd, J = 7.4, 1.5 Hz, 1H,

ArH), 6.71 (d, J = 7.5, Hz, 1H, ArH), 6.76–7.54 (m, 8H, ArH), 7.57(s, 1H, ArH), 8.41 (s, 1H, N=CH), 10.34 (s, 1H, NH). 13C NMR δ 18.1, 44.1, 113.4, 114.6, 119.7, 123.3, 128.4, 129.3, 131.0, 134.4, 136.8, 143.3, 144.2, 150.8, 161.6, 162.1, 174.7. Anal. calcd for C20H17FN2O2: C, 71.42; H, 5.09; N, 8.33, Found: C, 71.48; H, 5. 11; N, 8.36%; MS m/z [M+H]+ 337.36

2- (2-Fluorobiphenyl-4-yl) -N’- (5-methylfuran-2-ylmethylidene) propanehydrazide (4m): Off-white powder; m.p. 199˚C. IR (KBr): 3050 (NH), 1651 (C=O), 1622 (C=N) cm-1; 1H NMR δ 1.63 (d, J = 6.8 Hz, 3H, CH3), 2.39 (s, 2H, CH3), 4.72 (q, J = 6.7, 1.0 Hz, 1H, CH), 6.06 (d, J=

7.5Hz 1H, ArH), 6.09 (dd, J = 6.8, 1.0 Hz, 1H, ArH), 6.58 (d, J = 7.5Hz, 1H, ArH), 7.28 (s, 1H, ArH), 7.29–7.54 (m, 7H, ArH), 8.34 (s, 1H, N=CH), 10.45 (s, 1H, NH). 13C NMR δ 14.7, 18.5, 43.3, 108.4, 114.4, 119.3, 123.9, 128.2, 129.0, 130.5, 134.5, 136.4, 144.0, 150.3, 158.1, 160.6, 162.5, 175.5. Anal. calcd for C21H19FN2O2: C, 71.98; H, 5.47; N, 8.00; found: C, 72.03; H, 5.49; N, 8.02%; MS m/z [M+H]+ 351.39 2-(2-Fluorobiphenyl)-4-yl-N’-[3-(2-pentylphenyl)prop-2-en-1-ylidene] propanehydrazide (4n): Off-white powder; m.p. 104˚C. IR (KBr): 2976 (NH), 1649 (C=O), 1577 (C=N) cm-1; 1H NMR δ 0.95 (t, J = 7.8 Hz, 3H, CH3), 1.35–1.59 (m, 5H, CH3), 2.57–2.63 (m, 4H, CH3) 4.61 (q, J = 6.8, 1.0 Hz, 1H, CH), 6.64 (s, 1H, CH3), 7.18–7.43 (m, 10H, ArH), 7.51–7.69 (m, 5H, ArH), 8.35 (s, 1H, N=CH), 10.67 (s, 1H, NH). 13C NMR δ 14.2, 18.6, 24.38, 30.1, 32.5, 44.1, 117.6, 118.3, 122.1, 125.2, 126.3, 128.2, 129.1, 130.4, 131.3, 132.1, 134.2, 138.2, 139.8, 140.8, 144.3, 159.3, 161.6, 162.8, 174.3. Anal. calcd for C29H31FN2O: C, 78.70; H, 7.06; N, 6.33; found: C, 78.87; H, 7.09; N, 6.35%; MS m/z [M+H]+ 443.56

2- (2-Fluorobiphenyl- 4-yl) -N’- [(2-methylphenyl) methylidene] propanehydrazide (4o): Off-white powder; m.p. 178˚C. IR (KBr): 2987 (NH), 1648 (C=O), 1594 (C=N) cm-1; 1H NMR (CDCl3, 600 MHz) δ 1.45 (d, J = 6.8 Hz, 3H, CH3), 2.35 (s, 3H, CH3), 4.73 (q, J = 6.8, 1.0 Hz, 1H, CH), 7.10–7.31 (m, 4H, ArH), 7.42–7.46 (m, 2H, ArH), 7.50–7.56 (m, 2H, ArH), 7.63–7.66 (m, 3H, ArH), 7.30 (dd, J = 7.5, 5.6 Hz, 1H, ArH), 8.42 (s, 1H, N=CH), 11.03 (s, 1H, NH), 13C NMR δ 18. 3, 21.2, 44.5, 113.7, 114.2, 124.9, 126.9 127.8, 128.7, 129.4, 130.1, 131.4, 132.2,

136.5, 137.1, 143.6, 148.4, 159.3, 162.9, 170.8. Anal. calcd for C23H21FN2O: C, 76.64; H, 5.87; N, 7.77; found: C, 76.62; H, 5.83; N, 7.73%; MS m/z [M+H]+ 361.42

2- (2-Fluorobiphenyl- 4-yl) -N’- [(3-methylphenyl) methylidene] propanehydrazide (4p): Off-white powder; m.p. 184˚C. IR (KBr): 2987 (NH), 1671 (C=O), 1650 (C=N) cm-1; 1H NMR (CDCl3, 600 MHz) δ 1.44 (d, J = 6.8 Hz, 3H, CH3), 2.35 (s, 3H, CH3), 4.76 (q, J = 6.8, 1.0 Hz, 1H, CH), 7.18–7.28 (m, 2H, ArH), 7.35–7.42 (m, 4H, ArH), 7.48–7.57 (m, 5H, ArH), 7.92 (s, 1H, ArH), 8.17 (s, 1H, N=CH), 11.36 (s, 1H, NH); 13 C NMR δ 18. 4, 22.0, 43.5, 112.7, 113.9, 125.9, 127.1, 127.9, 129.7, 130.0, 131.3, 131.8, 133.2, 134.5, 138.1, 144.6, 147.4, 160.3, 163.9, 171.1. Anal. calcd for C23H21FN2O: C, 76.64; H, 5.87; N, 7.77; found: C, 76.72; H, 5.85; N, 7.79%; MS m/z [M+H]+ 361.42.

2- (2-Fluorobiphenyl- 4-yl) -N’- [(4-methylphenyl) methylidene] propanehydrazide (4q): White crystalline powder; m.p. 190˚C. IR (KBr): 3027 (NH), 1647(C=O), 1606 (C=N) cm-1; 1H NMR (CDCl3, 600 MHz) δ 1.56 (d, J = 6.8 Hz, 3H, CH3), 2.39 (s, 3H, CH3), 3.75 (q, J = 6.8, 1.0 Hz, 1H, CH), 7.35–7.50 (m, 3H, ArH), 7.56–7.65 (m, 6H, ArH), 7.78–7.95(m, 3H, ArH), 8.35 (s, 1H, N=CH), 11.17 (s, 1H, NH); 13 C NMR δ 18. 1, 22.9, 44.0, 113.1, 113.8, 126.8, 127.1, 128.9, 130.0, 131.0, 131.2, 132.8, 133.2, 135.5, 135.1, 142.6, 149.4, 160.3, 162.5, 169.0. Anal. calcd for C23H21FN2O: C, 76.64; H, 5.87; N, 7.77; found: C, 76.62; H, 5.89; N, 7.80%; MS m/z [M+H]+ 361.42

2- (2-Fluorobiphenyl- 4-yl) -N’- [(2-nitrophenyl) methylidene] propanehydrazide (4r): Off-white powder; m.p. 211˚C. IR (KBr): 2987 (NH), 1654 (C=O), 1560 (C=N) cm-1; 1H NMR (CDCl3, 600 MHz) δ 1.42 (d, J = 6.8 Hz, 3H, CH3), 3.70 (q, J = 6.8, 1.0 Hz, 1H, CH), 7.25–7.43 (m, 3H, ArH), 7.46–7.69 (m, 4H, ArH), 7.82 (s, 1H, ArH), 8.0–8.11 (m, 4H, ArH), 8.64 (s, 1H, N=CH), 11.93 (s, 1H, NH); 13C NMR δ 18. 7, 43.9, 113.1, 114.1, 124.6, 127.4, 127.9, 128.9, 131.0, 131.4, 134.9, 143.5, 154.1, 158.3, 171.7. Anal. calcd for C22H18FN3O3: C, 67.51; H, 4.64; N, 10.74; found: C, 67.49; H, 4.69; N, 10.69%; MS m/z [M+H]+ 392.39.

2-(2-Fluorobiphenyl-4-yl)-N’-[(3,4-dimethoxyphenyl)methylidene] propanehydrazide (4s): Light yellow powder; m.p. 170˚C. IR (KBr): 2987 (NH), 1653 (C=O), 1600 (C=N) cm-1; 1H NMR (CDCl3, 600 MHz) δ 1.44 (d, J = 6.8 Hz, 3H, CH3), 3.72–3.82 (m, 6H, OCH3), 4.72 (q, J = 7.0, 1.0 Hz, 1H, CH), 7.0–7.18 (m, 3H, ArH), 7.24–7.47 (m, 5H, ArH), 7.50–7.57 (m, 2H, ArH), 7.86 (s, 1H, ArH), 8.34 (s, 1H, N=CH), 10.03 (s, 1H, NH); 13C NMR δ 18. 3, 43.5, 113.5, 114.1, 123.9, 125.1, 127.3, 128.7, 129.1, 130.4, 131.2, 132.3, 136.0, 144.1, 148.7, 150.2, 157.1, 160.1, 168.9, 171.6. Anal. calcd for C24H23FN2O3: C, 70.92; H, 5.70; N, 6.89; found: C, 70.98; H, 5.65; N, 6.97%; MS m/z [M+H]+ 407.44

X-ray data collection and structure determination Suitable crystals of N’-(2-chlorophenylmethylidene-2-(2-fluorobiphenyl-4-yl)propanehydrazide (4b) were selected and mounted on Agilent Super Nova (Dual source) Agilent Technologies Diffractometer for single crystal diffraction studies. The data were collected using CrysAlisPro software at 296 K. The structures solution was performed by direct methods using SHELXS–9722 and refined by full-matrix least-squares methods on F2 using SHELXL-97 in-built with X-Seed.23 All non-hydrogen atoms were refined anisotropically by full-matrix least squares methods.22 All hydrogen atoms were positioned geometrically and treated as riding atoms with C–H distances of 0.93 Å for aromatic, 0.96 Å for methyl, 0.98 Å for chiral and 0.86 Å for N-H hydrogen atoms. These were refined using a riding model with Uiso (H) = 1.5 Ueq (C) for methyl and Uiso (H) = 1.2 Ueq (C) for all other atoms. Supplementary crystallographic data have been deposited with the CCDC number 986584 at www.ccdc.cam.ac.uk/ data_request/cif. Details of crystal data and structure refinement have been provided in Table 2. Anti-oxidant assay Total antioxidant activity of all the compounds synthesised was evaluated by phosphomolybdenum complex formation method.24 Each compound (500 µg mL-1) was mixed with reagent solution (4 mL) which was prepared by mixing of sodium phosphate (5.32 g), ammonium molybdate (2.47 g) and conc. sulfuric acid (16.7 mL) in distilled water (500 mL). All the vials including blank were capped

JOURNAL OF CHEMICAL RESEARCH 2015 673 and incubated in water bath at 95oC for 90 minutes. After the samples were cooled to room temperature, absorbance of the mixtures was measured at 695 nm against blank. Antioxidant activity was expressed relative to BHT (1.218). All the assays were conducted in triplicate and mean values were calculated (Table 3). Ferric reducing anti-oxidant power assay FRAP assay was performed according to Benzie and Strain method25 with some modifications. Stock solutions of acetate buffer (300 mM), TPTZ (2, 4, 6-tripyridyl-s-triazine) solution (10 mM in hydrochloric acid) and ferric chloride hexahydrate solution (20 mM) were prepared from commercially available chemicals. Fresh working solution was prepared by mixing acetate buffer (25 mL), TPTZ solution (2.5 mL) and ferric chloride solution (2.5 mL) followed by warming at 37oC prior to use. Solutions of all the compounds and trolox were prepared in methanol (1 mg mL-1). 10 µL of each of the sample and BHT solution were taken in separate test tubes and 2990 µL of FRAP solution were added to it to make total volume up to 3 mL. The sample was allowed to react with FRAP solution in the dark for 30 min. Readings of the coloured product(ferrous tripyridyltriazine complex) were noted at 593 nm and FRAP values were determined as micromoles of trolox equivalents per mL of the sample by computing with standard calibration curve constructed for different concentrations of trolox. Results are expressed in TE µM mL-1 (Table 3). Total phenolic contents The total phenolic contents of the compounds under investigation were determined by the method of Makkar et al.26 Solutions of the test compounds (0.1 mL) were prepared by dissolving them in methanol (0.5 mg mL-1) and each sample solution was mixed with sodium carbonate (2.8 mL; 10%) and Folin-Ciocalteu reagent (0.1 mL; 2N). Absorbance of the test solution was measured after 40 min at 725 nm by UV-Vis spectrophotometer. Total phenolics were determined as mg of gallic acid equiv. per g of sample by computing with standard calibration curve drawn for different concentrations of gallic acid. Standard calibration curve was linear between 50 µg mL-1 to 400 µg mL-1 of gallic acid and the results are expressed in GAEs µg mL-1 (Table 3). Statistical analysis All the measurements were done in triplicate and statistical analysis was performed in MS Excel 2007. Results are presented as average ±SEM.

Conclusions Prompted by the well-established anti-oxidant properties of carbohydrazides, a series of novel N’-benzylidene-2-(2fluorobiphenyl)propanehydrazides (4a–s) has been synthesised emphasising the advantage of combining two chemically different but pharmacologically compatible moieties (flurbiprofen nucleus and 1-phenylethylidene carbohydrazides) in one frame. The title compounds were synthesised under ultrasonic waves and assayed in vitro for the evaluation of their anti-oxidant potential (total anti-oxidant activity, FRAP and total phenolic contents). The

compounds obtained by synergism are significantly anti-oxidant in nature and could be useful as a template for designing more potent biologically active compounds. Facilitation of the syntheses in an ultrasound bath was found to be useful to obtain higher yields and purity than those which were carried out by simple thermal condensation in methanol/ethanol and the duration of reactions was reduced considerably, from 70–135 to 4.5–18 min. The authors acknowledge PCSIR Laboratories Complex, Lahore, Pakistan for the provision of necessary facilities. Received 5 August 2015; accepted 8 October 2015 Paper 1503526 doi: 10.3184/174751915X14452514747565 Published online: 1 November 2015

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