Microwave Assisted Synthesis and Evaluation of N-Cinnamoyl Aryl Hydrazones for Cytotoxic and Antioxidant Activities

Introduction

Acyl hydrazone  derivatives possessing an azometine -NHN=CH- proton  are emerging as a novel class  of compounds for new lead development ^1-4.They were reported to possess, antimicrobial^5-6, antitubercular⁷, antitumour^8-9, antioxidant^10-12, antiinflammatory¹³, analgesic¹⁴ ,antimalarial ¹⁵,  anti platelet activities^16-17 etc. It was reported that the presence of styryl ketone moiety in the compounds were found to be potent scavengers of free radicals. In view of these observations it was considered of interest to synthesise  a new class of acyl hydrazones  by  incorporating the styryl carbonyl moiety along with aryl hydrazone unit. The aim of present research  was to synthesize various N¹-(substituted benzylidene)-2-cyano-3-(4-dimethylamino ) phenyl acrylohydrazides  and to evaluate  for  their  in-vitro antioxidant and cytotoxic activities.

Materials and Methods

All the chemicals and solvents used in the present study were purchased from Merck, Hi media, S.D. fine Chemicals limited, Mumbai and Sigma Aldrich, USA. Melting points were determined in an open capillary tube in Thermonik precision melting point cum boiling point (C-PMB) apparatus and are uncorrected. Silica gel G coated on laboratory micro slides prepared by dipping method were used. IR spectra (KBr discs) were  confirmed by Shimadzu FT-IR spectrophotometer using KBr pellets technique, ¹H  NMR spectra were recorded on Bruker 300 MHz  NMR spectrometer  using DMSO as solvent. Mass spectra were recorded on Apex mass spectrophotometer.

Chemistry

General method of  synthesis of  compounds (1a-1i) 

To 0.01 mol of various substituted  benzaldehyde , 0.01 mol of cyanoacetohydrazide was added  in few ml of ethanol  followed  by few drops of  glacial acetic acid and irradiated in microoven for 1 -3 minutes at 140 watts .The reaction was monitored by TLC  and the solid formed was collected  and  recrystallized  from methanol .

General method of  synthesis of  compounds (2a-2i) 

To 0.01 mol of various N¹– substituted benzylidene-2-cyanoacetohydrazides  ,0.01mol of  N,N-di methylamino benzaldehyde  was added in few ml of ethanol  followed  by  few drops of  pyridine  and  irradiated  in  microoven for 1 -3 minutes at 140 watts . The reaction was monitored by TLC  and the solid formed is  collected  and recrystallized from methanol.

Scheme Click here to View Scheme

 

The physical data of compounds 2a-2i was tabulated in table-1 .

Table 1: Physical data  of  N¹-benzylidene-2-cyano-3-(4-dimethylamino)phenyl acrylohydrazides (2a-2i) 

Compound code	R1	R2 RRRRR2		R3	M.P(OC)		Yield (%)			Molecular  Formula
2a	H	H	H			161-162		80	C19H18N4 O
2b	OCH3	OCH3	H			173-176		85	C21H22N4 O3
2c	OCH3	OH	H			178-179		84	C20H18N4 O2
2d	OCH3	OCH3	OCH3			161-166		82	C22H24N4 O4
2e	H	OCH3	H			167-175		89	C20H20N4 O2
2f	H	OH	H			160-162		60	C19H18N4 O2
2g	H	4-CH3	H			178-179		50	C20H20N4 O
2h	H	N(CH3)2	H			158-160		49	C21H23N4 O
2i	3-NO2	H	H			178-179		76	C19H17N5 O3

N¹-benzylidene-2-cyano-3-(4-dimethylamino) phenyl acrylo hydrazide (2a) 

Mol. Formula C₁₉H₁₈ON₄,  Yield : 80 % ; m.p.: 161-162 ⁰ C ; IR (KBr) cm^-1: 3209 (N-H), 3075 (Ar-H), 2258 (C≡N), 1671(C=O), 1551 (C=C) . ¹H  NMR (300 MHz, DMSO-d₆); δ 3.1 (s,6H ,N(CH3)₂) , 6.8-7.77 (m,9H, Ar-H) , 8.0(s,IH,C=CH), 8.2(s,IH,N=CH),9.7(s 1H,-CONH);    Mass: m/z ( M±1) 318, (M+H)⁺ 319

N¹– (3,4-dimethoxy-benzylidene)-2-cyano-3-(4-dimethylamino) phenyl acrylohydrazide (2b)  

Mol. Formula C₂₁H₂₂N₄O₃, Yield : 95 %;  m.p.: 173-176⁰ C ; IR (KBr) cm^-1 : 3307 (N-H) , 3068 (Ar-H), 2253 (C≡N) , 1665(C=O), 1566 (C=C). ¹H NMR (300 MHz,DMSO- d₆) ; δ 3.0 (s,6H ,N(CH₃)₂) , 3.7-3.8 (s,6H,-OCH₃),  6.7-7.7 (m,7H, Ar-H), 7.9(s,IH,C=CH),  8.0 (s,IH,N=CH) , 9.6(s 1H,-CONH)

N¹-(3-methoxy,4-hydroxy-benzylidene)-2-cyano-3-(4-dimethylamino) phenyl acrylohydrazide( 2c) 

Mol. formula C₁₉H₁₈N₄O₂, Yield : 94%; m.p.:178-179⁰C ; IR (KBr) cm^-1 : 3442 (N-H) , 3078 (Ar-H), 2239 (C≡N), 1653(C=O), 1530 (C=C).  ¹H  NMR (300 MHz,DMSO- d₆) ; δ 3.0 (s,6H ,N(CH₃)₂), 3.7 (s,3H,OCH₃),  6.7-6.8 (m,7H,Ar-H) , 7.8 (s,IH,C=CH), 8.0(s,IH,N=CH), 9.6(s ,1H,-CON-H)

N¹– (3,4,5-trimethoxy -benzylidene)-2-cyano-3-(4-dimethylamino) phenyl acrylohydrazide (2d)

 Mol. Formula C₂₂H₂₄N₄O₄, Yield :92 %; m.p.161-166 ⁰C;IR-(KBr) cm^-1 : 3253 (N-H), 3068 (Ar-H), 2319 (C≡N),1660 (C=O) , 1521 (C=C) .    ¹H  NMR  (300 MHz,DMSO- d₆) ; δ 3.0  (s,6H ,N(CH3) ₂) , 3.7-3.8 (s,9H,OCH₃),  6.7-8.0(m,6H,Ar-H), 9.6(s, 1H,-CONH), MASS:m/z ( M±1) 408,  ( M+H  )⁺  409

N¹– (4-methoxy-benzylidene)-2-cyano-3-(4-dimethylamino) phenyl acrylohydrazide (2e)

Mol. formula  C₂₀H₂₀N₄O₂ , Yield :89 %; m.p.167-175 ⁰ C ; IR (KBr) cm^-1 : 3213 (N-H) , 3088 (Ar-H), 2320 (C≡N) , 1672 (C=O), 1564 (C=C) .¹H  NMR (300 MHz,DMSO- d₆) ; δ   3.0 (s,6H ,N(CH₃)₂), 3.7 (s,3H,-OCH₃),  6.7-7.6(m, Ar-H),  7.9(s,1H,C=CH) , 8.1(s,1H,N=CH) , 9.6(s, 1H,-CONH)

N¹– (4-hydroxy-benzylidene)-2-cyano-3-(4-dimethylamino) phenyl acrylohydrazide (2f)

Mol.  formula  C₁₉H₁₈N₄O₂ , Yield :60 %; m.p.160-162 ⁰ C ;  IR (KBr) cm^-1 : 3282 (N-H) , 3095 (Ar-H), 2232 (C≡N) , 1628(C=O), 1553 (C=C) . ¹H  NMR  (300 MHz,DMSO- d₆) ; δ  3.0 (s,6H ,N(CH₃)₂), 6.7-7.5( m,8H,Ar-H),  7.9(s,1H,C=CH) , 8.1(s,1H,N=CH) , 9.6(s ,1H,-CONH)

N¹– (4-methyl-benzylidene)-2-cyano-3-(4-dimethylamino) phenyl acrylohydrazide (2g)

Mol. formula C₂₀H₂₀N₄O,   Yield : 50 %; m.p.178-179 ⁰ C ;  IR (KBr) cm^-1 : 3260 (N-H) , 3100 (Ar-H),  2321 (C≡N) , 1688(C=O), 1511 (C=C) .¹H  NMR  (300 MHz,DMSO- d₆) ; δ 1.9 (s,1H,CH ₃ ) ,3.0 (s,6H ,N(CH₃)₂), 6.7-7.7( m,8H,Ar-H),  7.9(s,IH,C=CH) , 8.1(s,IH,N=CH) , 9.6(s ,1H,-CONH)

N¹– (4 – dimethylamino -benzylidene)-2-cyano-3-(4-dimethylamino) phenyl acrylohydrazide (2h)

Mol. formula C₂₁H₂₃N₄O, Yield :89 %; m.p.158-160 ⁰ C ; IR (KBr) cm^-1 : 3242(N-H) , 3076 (Ar-H), 2325 (C≡N) , 1683(C=O), 1543 (C=C) .¹H  NMR (300 MHz,DMSO- d₆)  ; δ 2.9-3.0  (s,12H ,N(CH₃)₂), 6.7-7.5(m,8H, Ar-H),  7.9(s,IH,C=CH) , 8.0(s,IH,N=CH) , 9.6(s ,1H,-CONH)

N¹– (3-nitro -benzylidene)-2-cyano-3-(4-dimethylamino) phenyl acrylohydrazide (2i)

Mol. formula  C₁₉H₁₇N₅O₃ ,  Yield :89 %; m.p.178-179⁰ C ;  IR (KBr) cm^-1 : 3222 (N-H) , 3086 (Ar-H),  2285 (C≡N) , 1628(C=O), 1513 (C=C) .¹H- NMR (300 MHz,DMSO- d₆) ; δ  3.0(s,6H ,N(CH₃)₂), 6.7-7.7( m,8H,Ar-H),  7.9(s,1H,C=CH) , 8.1(s,1H,N=CH) , 9.6(s ,1H,-CONH)

Figure Click here to View Figure

 

Cytotoxic activity

Brine shrimp lethality test¹⁸

Brine Shrimp (Artemia salina) nauplii were hatched in sterile brine solution (prepared using sea salt 38g/L and adjusted the pH to 8.5 using  1N NaOH) under constant aeration for  48 hr. After hatching,10 nauplii were placed in each vial and added various concentrations of drug solutions in a final volume of 5 mL, maintained at 37ºC for 24 h under the light of incandescent lamps and surviving larvae were counted . Each experiment was conducted along with control (vehicle treated), at various concentrations of the test substances. Percentage lethality was determined by comparing the mean surviving larvae of test and control tubes. ED₅₀ values were obtained by using Finney probed analysis software .The result for test compound was compared with the positive control podophyllotoxin.

Antioxidant activity

Determination of nitric oxide Scavenging Activity¹⁹ 

Nitric oxide scavenging activity of samples was determined by the following procedure. 2ml (10mM) of  sodium nitro prusside dissolved in 1.5ml phosphate buffer saline (PH-7.4) and 1 ml of different test samples corresponding to  100μM concentration was added in different test tubes respectively and incubated at 25^OC for about 150 min. From this 0.5ml was taken and 1ml sulphanilic acid reagent (33% in 20% glacial acetic acid) was added and incubated at room temperature for 5min.  1ml  of naphthyl ethylene diamine dihydro chloride (0.1% w/v) was added and again incubated at room temperature for 30min, then measured the absorbance at 540 nm in spectrophotometer.

Determination of the effect of samples on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) Radical ¹⁹

DPPH scavenging activity was assessed according to the reported method. Solutions of  various test samples at 100µM concentration were added to 100µM  DPPH  in 95 % ethanol and  tubes were kept at an ambient temperature for 20-30 min  and  absorbance was  measured at 517 nm. Ethanol was used as blank and DPPH  solution in ethanol served as the control. The effect of Ascorbic acid on DPPH was also assessed for comparison with that of samples.

Determination of Hydrogen Peroxide Scavenging  Activity²⁰

4mM solution of H₂O₂ was prepared in phosphate – buffered saline (PBS, pH 7.4). H₂O₂ concentration was determined spectrophotometrically from absorbance at 230 nm using molar absorptivity 81 M^-1 cm^-1 . 1 ml of different samples corresponding to 100μM concentration were added to 0.6ml hydrogen peroxide- PBS solution respectively and control without sample. Absorbance of  H₂O₂ at  230nm was determined 10 minutes later against a blank solution .

Results and Discussion

Chemistry

A series of N¹-benzylidene-2-cyano-3-(4-dimethylamino)phenyl acrylohydrazides (2a-2i) were  synthesized by two step procedure .In the first step various N¹-substituted benzylidene-2-cyanoacetohydrazides were synthesized by taking various substituted aromatic aldehydes  and cyanoacetohydrazide in few ml of ethanol by  adding a few drops of  glacial acetic acid and irradiated in microoven for 1 -3 minutes at 140 watts . The free amino group of cyanoacetohydrazide was  condensed  with carbonyl group of aldehyde to form schiffs linkage.  In the second step the various N¹– substituted benzylidene-2-cyanoacetohydrazides are condensed with N,N–dimethylamino benzaldehyde  at the electrophilic carbon of cyanoacetohydrazide . The structures of these compounds were established by means of their TLC, IR, ¹H NMR and Mass spectra  .The synthesized compounds were  evaluated for in-vitro antioxidant  activity.  Among the  nine  compounds synthesized six were evaluated for in-vitro cytotoxic activity.

 Antioxidant activity

The in-vitro antioxidant activity was evaluated by the reported  methods of  DPPH, nitric oxide and hydrogen peroxide . The results of antioxidant activities of the synthesized compounds were shown in table-2. The unsubstituted (2a ) and 4-methoxy derivatives (2e)  showed good scavenging activity  towards all the  three models at 100µM concentration , when compared with the standard  ascorbic acid. From the structure activity relationship studies of the compounds 2a-2i , it was observed that the nature of the substitution on the  benzylidene moiety affects  the  activity. Almost all the compounds showed good to moderate  percentage scavenging activity towards DPPH and NO  radicals . The moderately electron releasing methoxy derivatives showed good to moderate activity towards  H₂O₂  model. The electron releasing like 4-CH₃ and N,N-dimethylamino derivatives showed less scavenging activity towards hydrogen peroxide radical when compared with the standard.

Cytotoxic activity

 The brine shrimp lethality test was performed in order to evaluate the cytotoxic nature of the compounds. ED₅₀  values were calculated, based on the percentage of larvae survived at different concentrations of test and standard drugs. Compounds  2a (3.07 µg/ml) and 2e  (3.7 µg/ml) showed good  ED₅₀.The results are given in table-2. It was observed that the compounds with unsubstituted  and 4-methoxy derivatives  showed good activity .

Table 2: Antioxidant activity  and  Cyotoxic activity of  compounds (2a-2i) 

Compound	% Inhibition of  NO		% Inhibition of  H2O2	% Inhibition of  DPPH	ED50 (µg/ml)
2a	10		59	50	3.07
2b	8		31	36	5.28
2c	11		34	45	6.37
2d	8		39	38	10.50
2e	11		49	51	3.77
2f	8		20	31	–
2g	8		18	31	–
2h	7		20	25	4.07
2i	7		32	40	–
Ascorbic acid	22		71	52	–
Podopyllotoxin	–		–	–	1.64

Conclusion

In the present study we have described the synthesis , invitro cytotoxicity screening and antioxidant study of  various  N¹– (substituted benzylidene)-2-cyano-3-(4-dimethylamino) phenyl acrylohydrazides. From the results it was evident that the  further substitution  and modification on the  benzylidene moiety brings a new lead molecule.

Acknowledgements

The authors  are thankful to  the Siddhartha Academy of General and Technical Education for providing  necessary facilities to carry out  this research work. The authors are also thankful to laila impex ,vijayawada for providing spectral analysis and  laila neutraceuticals  for  screening of cytotoxic activity.

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