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Synthesis, spectral characterization, molecular structure and pharmacological studies of N’-(1, 4-naphtho-quinone-2yl) isonicotinohyWdrazide
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 1156–1161
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Synthesis, spectral characterization, molecular structure and pharmacological studies of N’-(1, 4-naphtho-quinone-2yl) isonicotinohydrazide P.R. Kavitha Rani a,⇑, Annette Fernandez b, Annie George a, V.K. Remadevi a, M.R. Sudarsanakumar c, Shiny P. Laila b, Muhammed Arif d a
Dept. of Chemistry, Govt. College for Women, Thiruvananthapuram 695014, Kerala, India Dept. of Chemistry, College of Engineering, Trivandrum (CET) 695016, Kerala, India c Dept. of Chemistry, Mahatma Gandhi College, Thiruvananthapuram 695004, Kerala, India d Govt. Arts College, Thiruvananthapuram 695014, Kerala, India b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Sonochemical synthesis of organic
compound with anticancer properties. Single crystals X-ray analysis of N’-(1,4-naphtho-quinone-2yl) isonicotinohydrazide. DNA cleavage studies and bioactivity. Pharmacological studies of N’-(1, 4naphtho-quinone-2yl) isonicotinohydrazide.
a r t i c l e
i n f o
Article history: Received 27 May 2014 Received in revised form 10 July 2014 Accepted 29 July 2014 Available online 8 August 2014 Keywords: Ultrasound irradiation Lawsone Isonicotinoyl hydrazine and MTT assay
a b s t r a c t A simple and efficient procedure was employed for the synthesis of N’-(1,4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH) by the reaction of 2-hydroxy-1,4-naphthaquinone (lawsone) and isonicotinoyl hydrazine in methanol using ultrasonic irradiation. Lawsone is the principal dye, isolated from the leaves of henna (Lawsonia inermis). Structural modification was done on the molecule aiming to get a more active derivative. The structure of the parent compound and the derivative was characterized by elemental analyses, infrared, electronic, 1H, 13C NMR and GC-MS spectra. The fluorescence spectral investigation of the compound was studied in DMSO and ethanol. Single crystal X-ray diffraction studies reveal that NIH crystallizes in monoclinic space group. The DNA cleavage study was monitored by gel electrophoresis method. The synthesized compound was found to have significant antioxidant activity against DPPH radical (IC50 = 58 lM). The in vitro cytotoxic studies of the derivative against two human cancer cell lines MCF-7 (human breast cancer) and HCT-15 (human colon carcinoma cells) using MTT assay revealed that the compound exhibited higher cytotoxic activity with a lower IC50 value indicating its efficiency in killing the cancer cells even at low concentrations. These results suggest that the structural modifications performed on lawsone could be considered a good strategy to obtain a more active drug. Ó 2014 Published by Elsevier B.V.
⇑ Corresponding author. Tel.: +91 8547854534. E-mail address: [email protected] (P.R. Kavitha Rani). http://dx.doi.org/10.1016/j.saa.2014.07.092 1386-1425/Ó 2014 Published by Elsevier B.V.
P.R. Kavitha Rani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 1156–1161
Introduction Though numerous naturally occurring compounds with biological activity have been investigated, however many of them are not suitable for therapeutic use due to their toxic, carcinogenic and mutagenic properties. Nowadays, it is possible to make modifications of active chemical structures, in order to synthesize compounds with improved therapeutic activity and reduced toxicity. The quinones represent a large group of substances present in several families of plants, fungi, lichens and bacteria. Quinones are classified according to the basic aromatic system as benzoquinones, naphthaquinone and anthraquinones [1]. The most important naphthaquinone containing plant families are Bignoniaceae, Droseraceae, Ebenaceae, Juglandaceae, Plumbaginaceae and Lythraceac [2]. Lawsonia inermis (Lythraceac) is commonly called as henna and its synonym is Lawsonia alba Linn. The coloring matter in the leaves of henna is lawsone and identified as 2-hydroxy 1, 4-naphthaquinone (C10H6O3) which is present in dried leaves. Lawsone have long been recognized to possess anti-inflammatory, antioxidant, antiallergic, hepatoprotective, antiviral, and anticarcinogenic activities. The hydroxyquinones are typical phenolic compounds and, therefore, act as potent metal chelators and free radical scavengers. They are powerful chain-breaking antioxidants [3,4]. In spite of these promising pharmacological profiles, it has not been modified to be used as a drug. This work aims to modify lawsone to obtain an efficient drug. With this objective, a new derivative was synthetized by the reaction with isonicotinoyl hydrazine to obtain the corresponding hydrazide. Such derivative has been shown to improve not only the cytotoxic activity but also exhibit fluorescence. The fluorescent materials are of interest in many disciplines such as emitters for electroluminescence devices, molecular probes for biochemical research, in traditional textile and polymer fields, and fluorescent whitening agents [5]. This derivative, N’-(1,4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH) was synthesized by ultrasound accelerated technique. Ultrasonic-assisted organic synthesis is a powerful and green approach which is being used to accelerate synthesis of organic compounds. Increase in reaction rate and yield takes place on application of ultrasound wave [6]. The structure of the compound was characterized by elemental analyses, infrared, electronic 1H, 13 C NMR and GC-MS spectra. Single crystal diffraction studies reveal that NIH ligand crystallizes in monoclinic space group. To know the applicability of NIH as a drug, cytotoxicity studies were conducted in human breast adenocarcinoma (MCF-7) and colon cancer (HCT-15) cell lines using MTT assay. The derivative has good ability to cleave pET 20b Plasmid DNA. Experimental Materials and instrumentations Isolation and purification of lawsone was done as per literature [7,8]. Isonicotinoyl hydrazine (INH) (Merck), (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT), DPPH were purchased and used without purification. Solvents were purified and dried according to the standard procedure [9]. TLC analyses were performed on precoated aluminum plates of silica gel 60F 254 plates (0.25 mm, Merck). Column chromatography (CC) was performed using silica gel G (mesh 100–200). In vitro cytotoxicity assay was conducted in HCT-15 colon carcinoma and MCF-7 human breast adenocarcinoma cell lines using MTT assay. Melting points were obtained using a capillary melting point apparatus. IR spectra were recorded on a Shimadzu IR Prestige-21 FTIR spectrometer on KBr pellets. 1H and 13C NMR spectra were measured on a 500 MHz Bruker Advanced DPX spectrometer with CDCl3 as solvent. CHN analyses were carried out on an Elementar vario MICRO cube
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Elemental Analyzer. UV–visible spectrum was recorded on Shimadzu UV-3600 UV–vis–NIR spectrometer. The fluorescence emissions were measured by a Horiba FluoroLog Spectrophotometer. Mass spectra were recorded on a Shimadzu Gas Chromatographic Mass Spectrometer. X-ray diffraction studies were carried using a Bruker AXS Kappa Apex2 CCD diffractometer, with graphite monochromated Mo Ka (k = 0.7103 Å) radiation. Synthesis of N’-(1, 4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH) A solution of lawsone was prepared by dissolving 0.174 g (1 mmol) in 25 mL methanol and added to 0.137 g (1 mmol) of isonicotinoyl hydrazine in 25 mL methanol in presence of acetic acid. The mixture was sonicated for 30 min in an ultrasound sonictor and this mixture was kept at 4 °C for 3 h which then yielded a yellow colored precipitate (Scheme 1). The precipitate was purified by column chromatography and eluted with methanol–chloroform mixture (1:3v/v). Single crystal suitable for X-ray diffraction studies were grown by slow evaporation in methanol. Yield – 69% m.p. 205 °C. X-ray crystallography The crystallographic data and structure refinement parameters are given in Table 1. The unit cell dimensions and intensity data were recorded at 293 K. The program SAINT/XPREP was used for data reduction and APEX2/SAINT for cell refinement [10]. The structure was solved using SIR92 and refinement was carried out by full matrix least squares of F2 using SHELXL-97 [11,12]. All non-hydrogen atoms were refined with anisotropic thermal parameters. All hydrogen atoms with the exception of those on nitrogen atoms were geometrically fixed and refined using a riding model. Molecular graphics employed were MERCURY and PLATON [13]. In vitro pharmacology analyzes DPPH radical scavenging Free radical scavenging activity of NIH was studied using 2, 2-Diphenyl-2-picryl-hydrazyl (DPPH). For this, a solution of 0.1 mM DPPH was prepared. Different concentrations (20–100 lg/ mL) of the compound were prepared in methanol and each sample was made to 500 ll using methanol. The sample was made up to 0.5 mL with methanol. 5 mL of DPPH solution was added to each test tube and shaken well. The tubes were kept at (30 ± 1) °C in dark for 30 min. A control was prepared with 5 mL DPPH solution and 0.5 mL methanol. Ascorbic acid was used as a reference to antioxidant compounds. Absorbance was measured after 30 min at 517 nm in a UV–visible spectrophotometer. Percentage of radical scavenging activity was calculated using the formula, [14].
% of Radical scavenging activity ¼
Absorbance of control absorbance of sample 100 Absorbance of control
ð1Þ
DNA cleavage studies a. Preparation of culture media and DNA isolation of Escherichia coli microbial strains were done according to the reported procedure [15,16]. b. Agarose gel electrophoresis. Cleavage products were analyzed by agarose gel electrophoresis method. Test samples (1 mg/mL) were prepared in DMSO. 0.3 lg of isolated plasmid DNA pET20b was incubated with 50 lM of the
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Scheme 1. Synthesis of N’-(1, 4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH).
Results and discussion Table 1 Crystal data of NIH. Empirical formula
C16H11N3O3
Formula weight Crystal system Space group a (Å) b (Å) c (Å)
293.28 Monoclinic P21/c 4.6352(9) 13.573(3) 21.681(4) 90 95 90 1357.4 4 1.435 0.102 5, 16, 25 608 0.982 0.990 2397 0.1387 1.360 0.3166 R1 = 0.2122 wR2 = 0.4185 0.1425
a b
a Volume (A3) Z D cat (g cm3) l (m m1) h, k, l F (0 0 0) T min T max No: of reflections Rw Goodness of fit wR2 (reflections) R indices (all data) R factor
derivatives in 50 lM Tris buffer pH 8.0. The reaction mixture was incubated at 37 °C for 3 h. After incubation the samples were mixed with gel loading dye and a constant 50 V of electricity passed for around 30 min. Then it was analyzed by agarose gel electrophoresis along with untreated control plasmids and photographed to determine the extent of DNA cleavage. MTT assay In vitro cytotoxicity of the derivatives on HCT-15 colon carcinoma and MCF-7 human breast adenocarcinoma cell lines were determined using MTT assay. Cells grown in Dulbeccos Modified Eagles Medium containing 10% FBS were seeded in (10 103/well) 96-well plates and incubated at 37 °C in 5% CO2 and 95% air at a relative humidity of 100%. The test compounds were dissolved in DMSO and diluted in the respective serum free medium. After 24 h, 100 ll of the medium containing the test compounds with various concentrations (6.25 lM, 12.5 lM, 25 lM, 50 lM, 100 lM) were added to the cells. After 24 h incubation, MTT at a concentration of 5 mg/mL in PBS (pH 7.4) was added to each well (10% v/v) and the cells were further incubated for 4 h at 37 °C. After removing the unreacted medium the blue crystals were dissolved in 100 ll DMSO and optical density was measured at 570 nm. The medium containing no test compounds served as the control. Cell inhibition (%) related to control wells containing cell culture medium without treatment was calculated by% cell inhibition = 100-Abs (drug)/Abs (control) 100. All the measurements were done in triplicate [17].
Spectral studies of the extracted lawsone were carried out and the data were consistent with the reported data [18]. The structure of lawsone is as shown in (Scheme 1). CHN analysis Purity and composition of the synthesized compound was ascertained using CHN analysis. C16H11N3O3; Calculated C = 65.55%, H = 14.33%, N = 3.55%; Found C = 65.15%, H = 14.84%, N = 3.75%. The values obtained were in good agreement with the calculated values. UV–visible and fluorescence spectra The UV–visible spectrum of lawsone in ethanol shows two bands at 248 nm and 276 nm. Another band observed at 334 nm is responsible for the yellowish color of lawsone. The UV–visible absorption spectrum of NIH in ethanol exhibited absorption max at 474 nm which is attributed to n-p* transition due to the extended conjugation. The medium absorption at 256 and 380 nm respectively are due to p–p* transition. It is earlier reported that the UV–visible spectrum of lawsone with acidic proton removed, exhibited bands at 450 nm, support the spectrum of the NIH [18]. The fluorescence property of NIH was investigated in two different solvents with respect to polarity. The compound exhibited emission maxima at 442 nm (strong) and 600 nm (broad) in DMSO where as in ethanol it gave emission maxima at 458 nm when it is excited at 365 nm which is shown in Fig. 1. This strong emission is due to the intramolecular charge transfer (ICT) transition through the delocalized hydrazide group present in the derivative, whereas the weak one may be due to the n-p* transition of the pyridyl N-atom [19]. The weak emission band (600 nm) gets broadened in ethanol. The molecule was designed with unique combination of electron donating and electron accepting group. This leads to the formation of pushpull system [20]. The high sensitivity to solvent is due to a charge shift away from the amino group (electron donor) in the excited state, towards the electron acceptor. These results in a large dipole moment in the excited state. This dipole moment interact with the polar solvent molecules to reduce the energy of the excited state [21,5]. The fluorescence property of compound NIH was found to be dependent on solvent which either increase the intensity or enhancement of quenching property [19]. Hence these quinone derivatives can be used as effective fluorophore for sensor application [22]. FT-IR spectral analysis The parent compound, lawsone, exhibits a broad spectrum at 3400 cm1 due to hydrogen bonded OH. This band is absent in NIH indicating that substitution has occurred at the hydroxyl group [23]. The strong band at 3250 cm1 is attributed to NH stretching
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The protonated carbon atom at C9 shows more down field shift in the spectrum (d = 107 ppm), due to an increase in electron density resulting from the presence of p electron delocalization on the C8AC9AC10 bond. In the pyridine ring the C13 and C14 carbon atoms adjacent to more electronegative nitrogen atom are shifted further down field to d = 155 ppm. Gas chromatographic mass spectra of NIH Gas chromatographic mass spectrum (Fig. S3) provides a vital clue for elucidating the structure of the compound. The mass spectrum of NIH is dominated by the intense molecular ion peak M-1 at m/z = 292 which corresponds to [C16H10N3O3]+. The base peak is obtained at m/ z = 149 which corresponds to C9H7O2. The spectrum also exhibits the fragments at m/z 278, 250, 223 and 167 due to [C16H11N2O3]+, [C16H11N2O2]+, [C12H5N2O2]+ and [C10H5N2O2]+ respectively. Single crystal XRD studies of NIH Fig. 1. Fluorescent spectrum of NIH in DMSO and ethanol.
frequency. The strong bands at 1672 cm1 and 1639 cm1 are assigned to quinone frequencies of naphthaquinone. The strong band at 1697 cm1 is due to C@O stretching mode of amide. The NAN band in NIH is observed at 1032 cm1. The CAN stretch is observed at 1250 cm1 [24]. NMR spectral analysis 1
H NMR spectra In the 1HNMR spectrum (Fig. S1) of the compound NIH, the naphthalene ring system consists of two doublets at 7.62 ppm and two triplets at 7.86 ppm. The singlet C3 proton appears at 8.26 ppm. The two singlets observed at approximately 6.07 ppm are due to the N(1)H and N(2)H protons. This downfield shift is due to increase in electron density resulting from p electron delocalization. The four pyridyl protons can be easily assigned, since the two ortho protons with respect to nitrogen are largely deshielded and appear at about 8.82 ppm [18]. 13
C NMR spectrum The 13C NMR spectrum provides direct information about the carbon skeleton of NIH (Fig. S2). There are 16 unique carbon atoms in the molecule which give a total 16 different peaks in the 13C NMR spectrums. The quinonoid carbonyl C1 signal was observed at 186.23 ppm and C8 at 187.12 ppm. The peak due to C@O observed down field at d = 169 ppm is the resultant of conjugative effect of the AN1AN2ACOA isonicotinoyl hydrazine skeleton [25].
The derivative NIH crystallizes in monoclinic space group P21/c. The molecular structure of derivative is shown is Fig. 2. Crystallographic data has been summarized in Table 2. Selected bond length and bond angles of NIH are given in Table 3. The carbon oxygen double bonds distance C1AO1 is 1.200 Å and in C8AO2 as 1.220AÅ. The C@O bond length of C11AO3 is 1.210 Å indicates that the compound exist in keto form. C10AN1 bond length is 1.357 Å is greater than C@N bond length 1.28 Å. This confirms the formation of CAN bond rather than C@N bond. Examination of CAO and CAC bond lengths and CACAC and CACAO angles give evidence of the quinonoid nature of the C1AC10AC9AC8AC7AC2 ring in the planar 1,4 naphthaquinone moiety [26]. The selected values of bond angles C10AN1AN3 is 117.6 Å, N2AC11AC12 is 116.56 Å, C1AC10AN1 is 113.05 Å, C9A C10AN1 is 125.79 Å deviate from the ideal value of 120° characteristic to a sp2 hybridization of C10 and N1 atoms. The hydrazenic N atoms are approximately coplanar with atoms of isonicotinic acid ring, and this is a consequence of steric effect. In the crystal packing, molecules are linked via intermolecular NAH. . .O and NAH. . .N hydrogen bonds (Table 3) into extended one-dimension chains along the c axis (Fig. 3). Adjacent chains are further cross-linked via NAH. . .O interactions into a two dimensional network. Pharmacology results DPPH-free radical scavenging activity The damage to DNA, which is induced by free radicals, has been suggested to aging and various diseases including cancer and chronic inflammation. The hydroxyl free radical is by far the most
Fig. 2. The molecular structure of N’-(1,4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH) Ellipsoids are drawn with 50% probability.
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Table 2 Selected bond length and bond angles (Å) bond angles (°). C1AO1 C1AC10 C10AC9 C9AC8 C8AC7 C8AO2 C8AO2 C7AC2 C2AC3 C2AC3 C3AC4 C4AC5
1.204 1.486 1.349 1.438 1.496 1.229 1.229 1.392 1.364 1.364 1.364 1.375
C5AC6 C10AN1 N1AN2 N2AC11 C11AO3 C11AC12 C11AC12 C12AC13 C12AC13 C13AC14 N3AC15 C14AN3
1.361 1.364 1.391 1.327 1.202 1.496 1.496 1.381 1.381 1.368 1.326 1.320
Bond angles O1AC1AC10 CAC10AN1 C9AC10AN1 C10AN1AN2 N2AC11AC12 C14AN3AC15 O3AC11AN2
120.45 113.05 125.79 117.66 116.56 115.44 122.56
O2AC8AC9 O2AC8AC9 O2AC8AC7 C10AN1AC11 C9AC8AC7 C2AC1AC10 C7AC2AC1
120.45 120.44 120.17 124.21 118.61 122.19 120.31 Fig. 4. DPPH free radical scavenging of NIH using ascorbic acid as standard.
Table 3 Hydrogen bonds of NIH. DAH. . .A
(DAA) (Å)
(H. . .A) (Å)
(D. . .A) (Å)
DHA (°)
N1AH. . .N3 N2AHAO3
0.894 0.796
2.102 2.077
2.88 2.854
146.21 164.93
potent and therefore the most dangerous oxygen metabolite. Elimination of such radical is one of the major aims of antioxidant administration [27]. 2,2-Diphenyl-2-picryl-hydrazyl (DPPH) assay is widely used for assessing the ability of radical scavenging activity and it is measured in terms of IC50 values. Because of the presence of odd electron, DPPH shows a strong absorption band at 517 nm in the visible spectrum. As this electron becomes paired off in the presence of a free radical scavenger, this absorption vanishes, and the resulting de-colorization is stoichiometric with respect to the number of electrons taken up [15]. The DPPH assay of the tested compound is shown in Fig. 4, it is seen from the results that the derivative exhibited good activity compared to the standard ascorbic acid. The IC50 values of NIH (58 lM) indicates that it has antioxidant activity, which show the ability of a compound to scavenge free radicals and to promote the formation of the non-radical form DPPH-H by hydrogen-donating action [28]. DNA cleavage studies DNA cleavage activity of lawsone and its derivative at a 50 lM concentration were studied using pET20b plasmid DNA. In the control experiment using DNA alone (lane-1), no significant cleavage of DNA was observed even on longer exposure time. It is evident from Fig. 5, that the derivative and lawsone have the ability cleave
DNA. The new derivative was observed to cleave the DNA more efficiently than the parent lawsone. This observation suggests that the compound has ability to cleave DNA by non oxidative mechanism. Oxidative cleavage agents require the addition of external agent (e.g. light or H2O2) to initiate cleavage and thus limited to in vitro applications. Hydrolytic cleavage does not require co-reactants and therefore, may be more useful in drug design [29]. As the new derivative was observed to cleave the DNA, it can be concluded that the compound inhibits the growth of the pathogenic organism by cleavage the genome [30]. In vitro cytotoxicity studies The antiproliferative activity of the compound was screened by MTT assay against two human cancer cell lines, human breast adenocarcinoma (MCF-7) and colon carcinoma cells (HCT-15). It was observed that the compound was toxic to cell lines in a dose dependent manner. Comparing the results, the compound NIH was found to be more active than the parent lawsone. From the results, MCF7cell lines were found to be more sensitive which is shown in Fig. 6. The IC50 value of compound NIH in MCF-7 cells was lower (20.86 lM) where as in lawsone the value is 24.15 lM which is already reported [3]. The IC50 value of compound NIH in HCT-15 colon cancer cells was (28.15 lM) where as in lawsone the value is 35.15 lM. The higher cytotoxic activities with the lower IC50 values indicate their efficiency in killing the cancer cells even at low concentrations. These results suggest that the structural modifications performed on the compound could be considered a good strategy to obtain a more active drug.
Fig. 3. Unit cell packing diagram of NIH viewed along c-axis.
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performed on lawsone have resulted in a more active derivative which poses to have good therapeutical applications. Acknowledgements The authors would like to thank the Department of Chemistry, Govt. College for Women, Thiruvananthapuram, School of Chemistry and School of Biology (IISER), Thiruvananthapuram, and College of Engineering Trivandrum for providing the laboratory and instrumental facilities. 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.saa.2014.07.092. References
Fig. 5. Agarose gel showing result of electrophoresis of pET20b plasmid DNA, lane 1: DNA control, Lane2: DNA+NIH, Lane 3:DNA+Lawsone.
Fig. 6. Cytotoxic activity of NIH and lawsone using cell lines MCF-7 human breast adenocarcinoma cell lines and HCT-15 colon carcinoma cell lines.
Conclusion The derivative N’-(1,4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH) was synthesized from lawsone by ultrasound irradiation technique, which is a simple and efficient procedure and gives good yield. The new compound was characterized by various spectroscopic and single crystal XRD studies. Various pharmacological studies were carried out. The compound exhibits good antioxidant activity. DNA cleavage studies reveal that the synthesized derivative exhibits more cleavage property than the parent lawsone. The cytotoxic studies in two different cell lines MCF-7 (human breast cancer) and HCT-15 (human colon carcinoma cells) were done and it was observed that NIH exhibits potential bioactivity than lawsone. These results suggest that the structural modification
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Synthesis, spectral characterization, molecular structure and pharmacological studies of N’-(1, 4-naphtho-quinone-2yl) isonicotinohydrazide P.R. Kavitha Rani a,⇑, Annette Fernandez b, Annie George a, V.K. Remadevi a, M.R. Sudarsanakumar c, Shiny P. Laila b, Muhammed Arif d a
Dept. of Chemistry, Govt. College for Women, Thiruvananthapuram 695014, Kerala, India Dept. of Chemistry, College of Engineering, Trivandrum (CET) 695016, Kerala, India c Dept. of Chemistry, Mahatma Gandhi College, Thiruvananthapuram 695004, Kerala, India d Govt. Arts College, Thiruvananthapuram 695014, Kerala, India b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Sonochemical synthesis of organic
compound with anticancer properties. Single crystals X-ray analysis of N’-(1,4-naphtho-quinone-2yl) isonicotinohydrazide. DNA cleavage studies and bioactivity. Pharmacological studies of N’-(1, 4naphtho-quinone-2yl) isonicotinohydrazide.
a r t i c l e
i n f o
Article history: Received 27 May 2014 Received in revised form 10 July 2014 Accepted 29 July 2014 Available online 8 August 2014 Keywords: Ultrasound irradiation Lawsone Isonicotinoyl hydrazine and MTT assay
a b s t r a c t A simple and efficient procedure was employed for the synthesis of N’-(1,4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH) by the reaction of 2-hydroxy-1,4-naphthaquinone (lawsone) and isonicotinoyl hydrazine in methanol using ultrasonic irradiation. Lawsone is the principal dye, isolated from the leaves of henna (Lawsonia inermis). Structural modification was done on the molecule aiming to get a more active derivative. The structure of the parent compound and the derivative was characterized by elemental analyses, infrared, electronic, 1H, 13C NMR and GC-MS spectra. The fluorescence spectral investigation of the compound was studied in DMSO and ethanol. Single crystal X-ray diffraction studies reveal that NIH crystallizes in monoclinic space group. The DNA cleavage study was monitored by gel electrophoresis method. The synthesized compound was found to have significant antioxidant activity against DPPH radical (IC50 = 58 lM). The in vitro cytotoxic studies of the derivative against two human cancer cell lines MCF-7 (human breast cancer) and HCT-15 (human colon carcinoma cells) using MTT assay revealed that the compound exhibited higher cytotoxic activity with a lower IC50 value indicating its efficiency in killing the cancer cells even at low concentrations. These results suggest that the structural modifications performed on lawsone could be considered a good strategy to obtain a more active drug. Ó 2014 Published by Elsevier B.V.
⇑ Corresponding author. Tel.: +91 8547854534. E-mail address: [email protected] (P.R. Kavitha Rani). http://dx.doi.org/10.1016/j.saa.2014.07.092 1386-1425/Ó 2014 Published by Elsevier B.V.
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Introduction Though numerous naturally occurring compounds with biological activity have been investigated, however many of them are not suitable for therapeutic use due to their toxic, carcinogenic and mutagenic properties. Nowadays, it is possible to make modifications of active chemical structures, in order to synthesize compounds with improved therapeutic activity and reduced toxicity. The quinones represent a large group of substances present in several families of plants, fungi, lichens and bacteria. Quinones are classified according to the basic aromatic system as benzoquinones, naphthaquinone and anthraquinones [1]. The most important naphthaquinone containing plant families are Bignoniaceae, Droseraceae, Ebenaceae, Juglandaceae, Plumbaginaceae and Lythraceac [2]. Lawsonia inermis (Lythraceac) is commonly called as henna and its synonym is Lawsonia alba Linn. The coloring matter in the leaves of henna is lawsone and identified as 2-hydroxy 1, 4-naphthaquinone (C10H6O3) which is present in dried leaves. Lawsone have long been recognized to possess anti-inflammatory, antioxidant, antiallergic, hepatoprotective, antiviral, and anticarcinogenic activities. The hydroxyquinones are typical phenolic compounds and, therefore, act as potent metal chelators and free radical scavengers. They are powerful chain-breaking antioxidants [3,4]. In spite of these promising pharmacological profiles, it has not been modified to be used as a drug. This work aims to modify lawsone to obtain an efficient drug. With this objective, a new derivative was synthetized by the reaction with isonicotinoyl hydrazine to obtain the corresponding hydrazide. Such derivative has been shown to improve not only the cytotoxic activity but also exhibit fluorescence. The fluorescent materials are of interest in many disciplines such as emitters for electroluminescence devices, molecular probes for biochemical research, in traditional textile and polymer fields, and fluorescent whitening agents [5]. This derivative, N’-(1,4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH) was synthesized by ultrasound accelerated technique. Ultrasonic-assisted organic synthesis is a powerful and green approach which is being used to accelerate synthesis of organic compounds. Increase in reaction rate and yield takes place on application of ultrasound wave [6]. The structure of the compound was characterized by elemental analyses, infrared, electronic 1H, 13 C NMR and GC-MS spectra. Single crystal diffraction studies reveal that NIH ligand crystallizes in monoclinic space group. To know the applicability of NIH as a drug, cytotoxicity studies were conducted in human breast adenocarcinoma (MCF-7) and colon cancer (HCT-15) cell lines using MTT assay. The derivative has good ability to cleave pET 20b Plasmid DNA. Experimental Materials and instrumentations Isolation and purification of lawsone was done as per literature [7,8]. Isonicotinoyl hydrazine (INH) (Merck), (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT), DPPH were purchased and used without purification. Solvents were purified and dried according to the standard procedure [9]. TLC analyses were performed on precoated aluminum plates of silica gel 60F 254 plates (0.25 mm, Merck). Column chromatography (CC) was performed using silica gel G (mesh 100–200). In vitro cytotoxicity assay was conducted in HCT-15 colon carcinoma and MCF-7 human breast adenocarcinoma cell lines using MTT assay. Melting points were obtained using a capillary melting point apparatus. IR spectra were recorded on a Shimadzu IR Prestige-21 FTIR spectrometer on KBr pellets. 1H and 13C NMR spectra were measured on a 500 MHz Bruker Advanced DPX spectrometer with CDCl3 as solvent. CHN analyses were carried out on an Elementar vario MICRO cube
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Elemental Analyzer. UV–visible spectrum was recorded on Shimadzu UV-3600 UV–vis–NIR spectrometer. The fluorescence emissions were measured by a Horiba FluoroLog Spectrophotometer. Mass spectra were recorded on a Shimadzu Gas Chromatographic Mass Spectrometer. X-ray diffraction studies were carried using a Bruker AXS Kappa Apex2 CCD diffractometer, with graphite monochromated Mo Ka (k = 0.7103 Å) radiation. Synthesis of N’-(1, 4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH) A solution of lawsone was prepared by dissolving 0.174 g (1 mmol) in 25 mL methanol and added to 0.137 g (1 mmol) of isonicotinoyl hydrazine in 25 mL methanol in presence of acetic acid. The mixture was sonicated for 30 min in an ultrasound sonictor and this mixture was kept at 4 °C for 3 h which then yielded a yellow colored precipitate (Scheme 1). The precipitate was purified by column chromatography and eluted with methanol–chloroform mixture (1:3v/v). Single crystal suitable for X-ray diffraction studies were grown by slow evaporation in methanol. Yield – 69% m.p. 205 °C. X-ray crystallography The crystallographic data and structure refinement parameters are given in Table 1. The unit cell dimensions and intensity data were recorded at 293 K. The program SAINT/XPREP was used for data reduction and APEX2/SAINT for cell refinement [10]. The structure was solved using SIR92 and refinement was carried out by full matrix least squares of F2 using SHELXL-97 [11,12]. All non-hydrogen atoms were refined with anisotropic thermal parameters. All hydrogen atoms with the exception of those on nitrogen atoms were geometrically fixed and refined using a riding model. Molecular graphics employed were MERCURY and PLATON [13]. In vitro pharmacology analyzes DPPH radical scavenging Free radical scavenging activity of NIH was studied using 2, 2-Diphenyl-2-picryl-hydrazyl (DPPH). For this, a solution of 0.1 mM DPPH was prepared. Different concentrations (20–100 lg/ mL) of the compound were prepared in methanol and each sample was made to 500 ll using methanol. The sample was made up to 0.5 mL with methanol. 5 mL of DPPH solution was added to each test tube and shaken well. The tubes were kept at (30 ± 1) °C in dark for 30 min. A control was prepared with 5 mL DPPH solution and 0.5 mL methanol. Ascorbic acid was used as a reference to antioxidant compounds. Absorbance was measured after 30 min at 517 nm in a UV–visible spectrophotometer. Percentage of radical scavenging activity was calculated using the formula, [14].
% of Radical scavenging activity ¼
Absorbance of control absorbance of sample 100 Absorbance of control
ð1Þ
DNA cleavage studies a. Preparation of culture media and DNA isolation of Escherichia coli microbial strains were done according to the reported procedure [15,16]. b. Agarose gel electrophoresis. Cleavage products were analyzed by agarose gel electrophoresis method. Test samples (1 mg/mL) were prepared in DMSO. 0.3 lg of isolated plasmid DNA pET20b was incubated with 50 lM of the
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Scheme 1. Synthesis of N’-(1, 4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH).
Results and discussion Table 1 Crystal data of NIH. Empirical formula
C16H11N3O3
Formula weight Crystal system Space group a (Å) b (Å) c (Å)
293.28 Monoclinic P21/c 4.6352(9) 13.573(3) 21.681(4) 90 95 90 1357.4 4 1.435 0.102 5, 16, 25 608 0.982 0.990 2397 0.1387 1.360 0.3166 R1 = 0.2122 wR2 = 0.4185 0.1425
a b
a Volume (A3) Z D cat (g cm3) l (m m1) h, k, l F (0 0 0) T min T max No: of reflections Rw Goodness of fit wR2 (reflections) R indices (all data) R factor
derivatives in 50 lM Tris buffer pH 8.0. The reaction mixture was incubated at 37 °C for 3 h. After incubation the samples were mixed with gel loading dye and a constant 50 V of electricity passed for around 30 min. Then it was analyzed by agarose gel electrophoresis along with untreated control plasmids and photographed to determine the extent of DNA cleavage. MTT assay In vitro cytotoxicity of the derivatives on HCT-15 colon carcinoma and MCF-7 human breast adenocarcinoma cell lines were determined using MTT assay. Cells grown in Dulbeccos Modified Eagles Medium containing 10% FBS were seeded in (10 103/well) 96-well plates and incubated at 37 °C in 5% CO2 and 95% air at a relative humidity of 100%. The test compounds were dissolved in DMSO and diluted in the respective serum free medium. After 24 h, 100 ll of the medium containing the test compounds with various concentrations (6.25 lM, 12.5 lM, 25 lM, 50 lM, 100 lM) were added to the cells. After 24 h incubation, MTT at a concentration of 5 mg/mL in PBS (pH 7.4) was added to each well (10% v/v) and the cells were further incubated for 4 h at 37 °C. After removing the unreacted medium the blue crystals were dissolved in 100 ll DMSO and optical density was measured at 570 nm. The medium containing no test compounds served as the control. Cell inhibition (%) related to control wells containing cell culture medium without treatment was calculated by% cell inhibition = 100-Abs (drug)/Abs (control) 100. All the measurements were done in triplicate [17].
Spectral studies of the extracted lawsone were carried out and the data were consistent with the reported data [18]. The structure of lawsone is as shown in (Scheme 1). CHN analysis Purity and composition of the synthesized compound was ascertained using CHN analysis. C16H11N3O3; Calculated C = 65.55%, H = 14.33%, N = 3.55%; Found C = 65.15%, H = 14.84%, N = 3.75%. The values obtained were in good agreement with the calculated values. UV–visible and fluorescence spectra The UV–visible spectrum of lawsone in ethanol shows two bands at 248 nm and 276 nm. Another band observed at 334 nm is responsible for the yellowish color of lawsone. The UV–visible absorption spectrum of NIH in ethanol exhibited absorption max at 474 nm which is attributed to n-p* transition due to the extended conjugation. The medium absorption at 256 and 380 nm respectively are due to p–p* transition. It is earlier reported that the UV–visible spectrum of lawsone with acidic proton removed, exhibited bands at 450 nm, support the spectrum of the NIH [18]. The fluorescence property of NIH was investigated in two different solvents with respect to polarity. The compound exhibited emission maxima at 442 nm (strong) and 600 nm (broad) in DMSO where as in ethanol it gave emission maxima at 458 nm when it is excited at 365 nm which is shown in Fig. 1. This strong emission is due to the intramolecular charge transfer (ICT) transition through the delocalized hydrazide group present in the derivative, whereas the weak one may be due to the n-p* transition of the pyridyl N-atom [19]. The weak emission band (600 nm) gets broadened in ethanol. The molecule was designed with unique combination of electron donating and electron accepting group. This leads to the formation of pushpull system [20]. The high sensitivity to solvent is due to a charge shift away from the amino group (electron donor) in the excited state, towards the electron acceptor. These results in a large dipole moment in the excited state. This dipole moment interact with the polar solvent molecules to reduce the energy of the excited state [21,5]. The fluorescence property of compound NIH was found to be dependent on solvent which either increase the intensity or enhancement of quenching property [19]. Hence these quinone derivatives can be used as effective fluorophore for sensor application [22]. FT-IR spectral analysis The parent compound, lawsone, exhibits a broad spectrum at 3400 cm1 due to hydrogen bonded OH. This band is absent in NIH indicating that substitution has occurred at the hydroxyl group [23]. The strong band at 3250 cm1 is attributed to NH stretching
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The protonated carbon atom at C9 shows more down field shift in the spectrum (d = 107 ppm), due to an increase in electron density resulting from the presence of p electron delocalization on the C8AC9AC10 bond. In the pyridine ring the C13 and C14 carbon atoms adjacent to more electronegative nitrogen atom are shifted further down field to d = 155 ppm. Gas chromatographic mass spectra of NIH Gas chromatographic mass spectrum (Fig. S3) provides a vital clue for elucidating the structure of the compound. The mass spectrum of NIH is dominated by the intense molecular ion peak M-1 at m/z = 292 which corresponds to [C16H10N3O3]+. The base peak is obtained at m/ z = 149 which corresponds to C9H7O2. The spectrum also exhibits the fragments at m/z 278, 250, 223 and 167 due to [C16H11N2O3]+, [C16H11N2O2]+, [C12H5N2O2]+ and [C10H5N2O2]+ respectively. Single crystal XRD studies of NIH Fig. 1. Fluorescent spectrum of NIH in DMSO and ethanol.
frequency. The strong bands at 1672 cm1 and 1639 cm1 are assigned to quinone frequencies of naphthaquinone. The strong band at 1697 cm1 is due to C@O stretching mode of amide. The NAN band in NIH is observed at 1032 cm1. The CAN stretch is observed at 1250 cm1 [24]. NMR spectral analysis 1
H NMR spectra In the 1HNMR spectrum (Fig. S1) of the compound NIH, the naphthalene ring system consists of two doublets at 7.62 ppm and two triplets at 7.86 ppm. The singlet C3 proton appears at 8.26 ppm. The two singlets observed at approximately 6.07 ppm are due to the N(1)H and N(2)H protons. This downfield shift is due to increase in electron density resulting from p electron delocalization. The four pyridyl protons can be easily assigned, since the two ortho protons with respect to nitrogen are largely deshielded and appear at about 8.82 ppm [18]. 13
C NMR spectrum The 13C NMR spectrum provides direct information about the carbon skeleton of NIH (Fig. S2). There are 16 unique carbon atoms in the molecule which give a total 16 different peaks in the 13C NMR spectrums. The quinonoid carbonyl C1 signal was observed at 186.23 ppm and C8 at 187.12 ppm. The peak due to C@O observed down field at d = 169 ppm is the resultant of conjugative effect of the AN1AN2ACOA isonicotinoyl hydrazine skeleton [25].
The derivative NIH crystallizes in monoclinic space group P21/c. The molecular structure of derivative is shown is Fig. 2. Crystallographic data has been summarized in Table 2. Selected bond length and bond angles of NIH are given in Table 3. The carbon oxygen double bonds distance C1AO1 is 1.200 Å and in C8AO2 as 1.220AÅ. The C@O bond length of C11AO3 is 1.210 Å indicates that the compound exist in keto form. C10AN1 bond length is 1.357 Å is greater than C@N bond length 1.28 Å. This confirms the formation of CAN bond rather than C@N bond. Examination of CAO and CAC bond lengths and CACAC and CACAO angles give evidence of the quinonoid nature of the C1AC10AC9AC8AC7AC2 ring in the planar 1,4 naphthaquinone moiety [26]. The selected values of bond angles C10AN1AN3 is 117.6 Å, N2AC11AC12 is 116.56 Å, C1AC10AN1 is 113.05 Å, C9A C10AN1 is 125.79 Å deviate from the ideal value of 120° characteristic to a sp2 hybridization of C10 and N1 atoms. The hydrazenic N atoms are approximately coplanar with atoms of isonicotinic acid ring, and this is a consequence of steric effect. In the crystal packing, molecules are linked via intermolecular NAH. . .O and NAH. . .N hydrogen bonds (Table 3) into extended one-dimension chains along the c axis (Fig. 3). Adjacent chains are further cross-linked via NAH. . .O interactions into a two dimensional network. Pharmacology results DPPH-free radical scavenging activity The damage to DNA, which is induced by free radicals, has been suggested to aging and various diseases including cancer and chronic inflammation. The hydroxyl free radical is by far the most
Fig. 2. The molecular structure of N’-(1,4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH) Ellipsoids are drawn with 50% probability.
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Table 2 Selected bond length and bond angles (Å) bond angles (°). C1AO1 C1AC10 C10AC9 C9AC8 C8AC7 C8AO2 C8AO2 C7AC2 C2AC3 C2AC3 C3AC4 C4AC5
1.204 1.486 1.349 1.438 1.496 1.229 1.229 1.392 1.364 1.364 1.364 1.375
C5AC6 C10AN1 N1AN2 N2AC11 C11AO3 C11AC12 C11AC12 C12AC13 C12AC13 C13AC14 N3AC15 C14AN3
1.361 1.364 1.391 1.327 1.202 1.496 1.496 1.381 1.381 1.368 1.326 1.320
Bond angles O1AC1AC10 CAC10AN1 C9AC10AN1 C10AN1AN2 N2AC11AC12 C14AN3AC15 O3AC11AN2
120.45 113.05 125.79 117.66 116.56 115.44 122.56
O2AC8AC9 O2AC8AC9 O2AC8AC7 C10AN1AC11 C9AC8AC7 C2AC1AC10 C7AC2AC1
120.45 120.44 120.17 124.21 118.61 122.19 120.31 Fig. 4. DPPH free radical scavenging of NIH using ascorbic acid as standard.
Table 3 Hydrogen bonds of NIH. DAH. . .A
(DAA) (Å)
(H. . .A) (Å)
(D. . .A) (Å)
DHA (°)
N1AH. . .N3 N2AHAO3
0.894 0.796
2.102 2.077
2.88 2.854
146.21 164.93
potent and therefore the most dangerous oxygen metabolite. Elimination of such radical is one of the major aims of antioxidant administration [27]. 2,2-Diphenyl-2-picryl-hydrazyl (DPPH) assay is widely used for assessing the ability of radical scavenging activity and it is measured in terms of IC50 values. Because of the presence of odd electron, DPPH shows a strong absorption band at 517 nm in the visible spectrum. As this electron becomes paired off in the presence of a free radical scavenger, this absorption vanishes, and the resulting de-colorization is stoichiometric with respect to the number of electrons taken up [15]. The DPPH assay of the tested compound is shown in Fig. 4, it is seen from the results that the derivative exhibited good activity compared to the standard ascorbic acid. The IC50 values of NIH (58 lM) indicates that it has antioxidant activity, which show the ability of a compound to scavenge free radicals and to promote the formation of the non-radical form DPPH-H by hydrogen-donating action [28]. DNA cleavage studies DNA cleavage activity of lawsone and its derivative at a 50 lM concentration were studied using pET20b plasmid DNA. In the control experiment using DNA alone (lane-1), no significant cleavage of DNA was observed even on longer exposure time. It is evident from Fig. 5, that the derivative and lawsone have the ability cleave
DNA. The new derivative was observed to cleave the DNA more efficiently than the parent lawsone. This observation suggests that the compound has ability to cleave DNA by non oxidative mechanism. Oxidative cleavage agents require the addition of external agent (e.g. light or H2O2) to initiate cleavage and thus limited to in vitro applications. Hydrolytic cleavage does not require co-reactants and therefore, may be more useful in drug design [29]. As the new derivative was observed to cleave the DNA, it can be concluded that the compound inhibits the growth of the pathogenic organism by cleavage the genome [30]. In vitro cytotoxicity studies The antiproliferative activity of the compound was screened by MTT assay against two human cancer cell lines, human breast adenocarcinoma (MCF-7) and colon carcinoma cells (HCT-15). It was observed that the compound was toxic to cell lines in a dose dependent manner. Comparing the results, the compound NIH was found to be more active than the parent lawsone. From the results, MCF7cell lines were found to be more sensitive which is shown in Fig. 6. The IC50 value of compound NIH in MCF-7 cells was lower (20.86 lM) where as in lawsone the value is 24.15 lM which is already reported [3]. The IC50 value of compound NIH in HCT-15 colon cancer cells was (28.15 lM) where as in lawsone the value is 35.15 lM. The higher cytotoxic activities with the lower IC50 values indicate their efficiency in killing the cancer cells even at low concentrations. These results suggest that the structural modifications performed on the compound could be considered a good strategy to obtain a more active drug.
Fig. 3. Unit cell packing diagram of NIH viewed along c-axis.
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performed on lawsone have resulted in a more active derivative which poses to have good therapeutical applications. Acknowledgements The authors would like to thank the Department of Chemistry, Govt. College for Women, Thiruvananthapuram, School of Chemistry and School of Biology (IISER), Thiruvananthapuram, and College of Engineering Trivandrum for providing the laboratory and instrumental facilities. 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.saa.2014.07.092. References
Fig. 5. Agarose gel showing result of electrophoresis of pET20b plasmid DNA, lane 1: DNA control, Lane2: DNA+NIH, Lane 3:DNA+Lawsone.
Fig. 6. Cytotoxic activity of NIH and lawsone using cell lines MCF-7 human breast adenocarcinoma cell lines and HCT-15 colon carcinoma cell lines.
Conclusion The derivative N’-(1,4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH) was synthesized from lawsone by ultrasound irradiation technique, which is a simple and efficient procedure and gives good yield. The new compound was characterized by various spectroscopic and single crystal XRD studies. Various pharmacological studies were carried out. The compound exhibits good antioxidant activity. DNA cleavage studies reveal that the synthesized derivative exhibits more cleavage property than the parent lawsone. The cytotoxic studies in two different cell lines MCF-7 (human breast cancer) and HCT-15 (human colon carcinoma cells) were done and it was observed that NIH exhibits potential bioactivity than lawsone. These results suggest that the structural modification
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