2,4-Dichlorophenoxyacetic Acid Derived Schiff Base and Its Lanthanide(III) Complexes: Synthesis, Characterization, Spectroscopic Studies, and Plant Growth Activity

2,4-Dichlorophenoxyacetic acid derived Schiff base (HL) and its lanthanide [La(III), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III), Dy(III), Y(III)] complexes were synthesized and characterized by various spectroscopic (H, C, DEPT and 2D HMQC NMR, FT-IR, �V-Vis, and mass) techni�ues and other analytical methods. HL exhibits �E� and ��� isomerism and was con�rmed by variable temperature HNMR studies. e spectral and analytical data reveals the bidentate coordination of HL to lanthanide(III) ion, through carboxylic acid group via deprotonation. Fluorescence spectrum of europium complex shows bands at 578, 592, and 612 nm assignable to D0 → F0, D0 → F1, and D0 → F2, respectively. Auxin activity ofHL and lanthanum(III) complex onwheat seeds (Triticum durum) was measured at different concentrations. e percentage germination, root length, and shoot length were recorded. An enhancement in the plant growth activity of the ligand was observed on complexation and the best activity was observed at 10−6 M concentration.


Introduction
Rare earth elements are being used in agriculture as micronutrients and fertilizers [1][2][3][4].ese are found to have nitrogen �xing capacity, to enhance activity of hydrolytic enzymes, to promote seed germination, to strengthen photosynthetic rate, and to reduce water loss in plants [5,6].Recent experiments indicate that lanthanide salts could accelerate the seed germination, increases chlorophyll content, and improve root growth [7,8].ese elements also found to have signi�cant effect on the physiological and biochemical reactions in plant growth and development [9,10].e effect of lanthanide(III) complexes on the growth of plants has been an important topic in agricultural �eld.ere are only few reports which explain the auxin effect of lanthanide complexes on plant growth [11,12].Indole-3 acetic acid, naphthalene acetic acid, and 2,4dichlorophenoxyacetic acid are the well known plant growth regulating hormones, widely used in agricultural �eld [13,14].e derivatives of these hormones were also found to have plant growth regulating and other biological activities [13,15,16].
Literature survey reveals that chelation of lanthanide metal ions with plant growth promoting auxins synergistically increases the plant growth [12].Keeping this in view, we have synthesized 2,4-dichlorophenoxy acetic acid derived Schiff base and its lanthanide(III) complexes and screened for their auxin activity on wheat seeds.e work is focused on the synthesis, characterization, spectroscopic investigation, thermal, �uorescent behavior, and plant growth promoting activity of a series of rare earth complexes of 2,4dichlorophenoxy acetic acid derived Schiff base (HL).

Experimental
2.1.Instrumentation and Materials.Elemental analyses (C, H, and N) were performed using Leco Model TruSpec CHNS analyzer.e percentage of metal content was determined according to the literature procedure [17] with xylenol orange as the indicator and EDTA as the chelating agent.SHIMADZU LCMS 2010A spectrometer is used to know the mass.Molar conductivities in DMSO (10 −3 M) at room temperature were measured using an Elico conductivity bridge having platinum electrode.Melting points were determined in an open capillary on a Gallenkamp melting point apparatus and are uncorrected.IR spectra were recorded in a KBr matrix using an Impact-410 Nicolet (USA) FT-IR spectrometer in 4000-400 cm −1 range.e 1 H, 13 C, DEPT, 2D HMQC NMR, and variable temperature 1 H NMR were recorded in DMSO-d 6 solvent on BRUKER AV-500 MHz High Resolution Multinuclear FT-NMR spectrometer using SiMe 4 as an internal standard at  = 0 ppm.ermogravimetry (TG) and differential thermal analysis (DTA) were run in nitrogen atmosphere in a temperature range of 20 ○ C to 1000 ○ C at a heating rate of 10 ○ C min −1 using a Perkin-Elmer (Pyris Diamond) analyzer.UV-Visible spectra were recorded on a CARY 50 Bio UV-Visible spectrophotometer in 200-1100 nm range in DMSO solvent.Fluorescence spectrum was measured on F-7000 FL spectrophotometer.Plant growth activity was performed in a dual chamber seed germinator at 25 ○ C temperature and 70 ± 5% humidity with proper illumination.
2,4-Dichlorophenoxyacetic acid and 2-formyl phenoxyacetic acid were obtained from Sigma Aldrich.Lanthanide(III) oxides were procured from Indian Rare Earths Ltd, India.Other chemicals were obtained from s.d.Fine Chemicals, India, and used as received.Solvents were puri�ed by standard methods [18].Lanthanide nitrates were prepared by dissolving the corresponding oxide (99.99%,) in 50% HNO 3 followed by evaporation of the excess acid.All the compounds were routinely checked by thin-layer chromatography (TLC) on aluminum-backed silica gel plates.e wheat seeds (Triticum durum) DWR-2006 were collected from University of Agricultural Sciences (UAS), Dharwad, India.

General Procedure for the Synthesis of Lanthanide
Complexes.e complexes were prepared by treating HL with freshly prepared lanthanide(III) nitrates in presence of triethyl amine according to literature procedure [19] with slight modi�cations.e mixture of HL (0.5 gm, 1.26 mmol) and triethyl amine (0.14 g, 1.38 mmol) was taken in THF (50 mL) and stirred till HL completely dissolves.To this solution, freshly prepared Ln(NO 3 ) 3 (0.42 mmol) (Ln = La, Pr, Nd, Sm, Eu, Gd, Dy, Y) was added slowly under stirring and was further re�uxed for 2 hrs.Solvent was evaporated under reduced pressure.e solid obtained was washed with water and THF several times.e product was dried under vacuum.Yield: 60-70%.

Protocol for the Plant Growth Activity on Wheat Seeds.
DWR-2006 (Triticum durum), a local variety of wheat seeds developed at University of Agricultural Sciences, Dharwad, India, was selected to investigate the growth activities of the synthesized compounds.e solutions of HL, lanthanum(III) complex, La(NO 3 ) 3 , and 2,4-dichlorophenoxyacetic acid were prepared by dissolving them in minimum quantity of DMSO (∼1 mL) and further diluted with distilled water to obtain the solutions of 1 × 10 −5 M, 1 × 10 −6 M and, 1 × 10 −7 M concentrations.Seed germination experiments were carried out according to the literature method [20] with slight modi�cations.Healthy wheat seeds were selected and washed with distilled water before soaking in test solutions.Hundred seeds were soaked in test solutions of particular concentration for 3 minutes and then arranged on the moistened specially prepared germination paper placed on polythene sheet.One more moistened germination paper was placed over the seeds and loosely rolled.Each roll was labeled clearly and kept upright in the seed germinator and maintained at 25 ○ C temperature and 70 ± 5% humidity, with proper illumination.e conditions were maintained for eight days.Aer eight days, percentage germination was calculated.Further, 10 seeds were randomly selected from each roll for root and shoot length measurements.e experiments were performed in triplicate.

Results and Discussions
e Schiff base (HL) was prepared as shown in Scheme 1. e structure of HL was con�rmed by IR, NMR, and mass spectral analysis.All the complexes were obtained in moderate to good yields (60-70%) by reacting HL and lanthanide(III) nitrates in THF in 3 : 1 (ligand to metal) molar ratio.e complexes are soluble in DMSO and DMF.e analytical results given in Table 1 agree with the suggested formula of complexes.Elemental analysis, FT-IR, 1 H, 13 C, 2D HMQC NMR, TGA/DSC, and UV-Visible spectroscopy were used to characterize the complexes.e elemental analyses indicate 3 : 1 (ligand to metal) stoichiometry.e lower molar conductance values of the complexes in DMSO at 10 −3 M concentration suggest their nonelectrolytic nature and are given in Table 1.

IR Spectral Studies. e characteristic IR bands of HL
and Ln(III) complexes are compiled in Table 2.In the IR spectrum of HL, the broad band observed at 3290 cm −1 was assigned to (OH) group of carboxylic acid.e appearance of this frequency at a slightly lower wave number is due to the involvement of OH group in intermolecular hydrogen bonding with oxygen atom of amide (C=O) functional group [21].e HL shows a very strong absorption band at 1736 cm −1 assigned to the (C=O) of the carboxylic acid.e bands at 1659 cm −1 and 1609 cm −1 were assigned to amide (C=O) and (C=N), respectively.e absence of band due to carboxylic OH group in the spectra of all lanthanide (III) complexes suggests the coordination to the metal ion via deprotonation.e band due to (C=O) of carboxylic acid of HL was absent in all the lanthanide(III) complexes, indicating the participation of carbonyl oxygen in coordination to the metal ion [22][23][24].Whereas the two new characteristic bands appeared on complexation in the ranges of 1590-1562 cm −1 and 1372-1346 cm −1 were assigned to asymmetric and symmetric stretching frequencies of carboxylate ion, respectively.e difference between  asy (COO − ) and  sy (COO − ) frequencies in all complexes were found to be less than the difference observed in the sodium salt of HL. is implies that carboxylic acid group of HL has coordinated to metal ion in bidentate fashion via deprotonation as suggested by Deacon [22,[25][26][27][28].
A strong band at 1659 cm −1 in the spectrum of uncoordinated ligand is assigned to amide (C=O) and has shied to higher wave number in all complexes.is increase in frequency of amide (C=O) functional group may be due to the breaking of hydrogen bond present in HL on complexation.Azomethine nitrogen (C=N) has not suffered any change on complexation indicating its noninvolvement in coordination.
e presence of a broad band in the region 3460-3421 cm −1 was attributed to the (O-H) of coordinated water molecules in all complexes [29].is was further con�rmed by the appearance of a weak nonligand band in the region 831-856 cm −1 , assignable to rocking mode of coordinated water molecule [30].e presence of coordinated water molecules was further con�rmed by thermal studies.

NMR Spectral Studies of HL. e NMR spectrum of HL
shows double set of signals for all the protons and carbons due to its existence in E and Z isomeric forms arising due to the restricted rotation along the (-HC=N-) functional group.From the 1 H NMR data, the ratio of E and Z isomers was approximately found to be 70 : 30 with E isomer as the predominant one over Z isomer.
e isomeric structure of HL and its numbering are presented in Figure 1.e 1 H NMR spectrum is given in Figure 2. e detailed assignments for both the isomers are given in the experimental section.e singlet observed at 13.16 ppm was assigned to OH proton of carboxylic acid.Two singlets observed at 11.72 and 11.80 ppm were assigned to amide NH and NH ′ protons, respectively.e C11H and C11 ′ H protons present adjacent to carbonyl functional group were observed at 4.80 and 5.31 ppm, respectively.e C2H and C2 ′ H protons present adjacent to carboxylic functional group were observed at 4.82 and 4.90 ppm, respectively.e azomethine protons C9H and C9 ′ H were observed at 8.42 and 8.65 ppm, respectively [31].e aromatic protons were observed between 6.99-7.90ppm.
1 H-NMR analysis is supported by the 13 C and DEPT NMR spectral analysis for the con�rmation of E and Z isomerism in HL. e 13 C spectrum of HL is given in Figure 3 and its DEPT NMR is presented at   All assignments were further studied by 2D HMQC NMR spectral analysis and the spectrum is given in Figure 5. e spectrum correlates the directly bonded 1 H and 13 C NMR resonances.e resonance observed in 1 H NMR spectrum It is well known that, in case of E and Z isomers, as the temperature increases, interconversion of two isomers also increases due to the increase in rotation along C8-C9 bond and one of them will become predominant over the other isomer at higher temperature [32].To study the interconvertion phenomenon, variable temperature 1 H NMR analyses were undertaken [33] in the temperature range from 298 K to 363 K and the spectrum is given in Figure 6.

NMR Spectral Studies of Lanthanum(III) Complex.
In the spectrum of the lanthanum(III) complex shown at Figure 7, only one set of signals was observed for each proton and carbon, indicating the presence of in only one form.e resonance at 13.16 ppm assigned to carboxylic acid proton in HL has completely disappeared in lanthanum(III) complex indicating the coordination of HL through carboxylic group via deprotonation [22].e carboxylic acid coordination was supported by down�eld shi of CH 2 protons adjacent to carboxylic acid.e C11H, C9H, and NH signals observed at 4.80, 8.42, and 11.72 ppm, respectively in the spectrum of HL have now been observed at 4.70, 8.41, and 11.64 ppm, respectively, in the NMR spectrum of lanthanum(III) complex.is observation con�rms that the carbonyl oxygen and azomethine nitrogen were not involved in coordination.Signi�cant changes were not observed in the chemical shi values of aromatic protons aer complexation. 13C NMR spectrum further supports the mode of coordination of HL. e spectrum is given in Figure 8. e carbonyl carbon C1 of carboxylic acid observed at 169.88 ppm in HL has shied down�eld to 176.46 ppm in the complex suggesting the involvement of carboxylic acid in coordination [22].It was further supported by down�eld shi of C2 carbon resonance from 64.62 ppm to 67.55 ppm on complexation.Small change was observed in the chemical shi values of carbonyl carbon C10 on complexation.is  indicates breakdown of hydrogen bond aer complexation [34].
e assignments were further studied by 2D HMQC NMR spectrum.e spectrum is given in Figure 10.e resonance observed at 11.64 ppm was devoid of any attached carbon signal con�rming their assignment to NH proton.

Complex
Temperature range (   loss corresponds to three coordinated water molecules.e second weight loss between 365.71 ○ C to 510.60 ○ C is due to the loss of three ligand molecules in all complexes.ird step weight losses correspond to the formation of residue.e observed weight losses match with calculated values for the metal content [35].

ESI Mass
Analysis.e ESI mass spectrum of HL is given in Figure 12. e molecular ion peak m/z = 419 corresponds to sodium adduct of HL. e mass spectra of complexes are in good agreement with the expected molecular weights.All the molecular weights are adducting with the proton.e representative spectrum of lanthanum complex is given in Figure 13.In the given mass spectrum of La(III) complex, the molecular ion peak m/z = 1358 due to [M-H] + ion.
3.5.Electronic Spectra.e electronic spectra of HL and its lanthanide(III) complexes were recorded in DMSO at room temperature.e two absorption maxima at 278 nm and 318 nm in the spectrum of HL were assigned to    * transitions of carbonyl oxygen and azomethine (-C=N-) moiety.No signi�cant changes were observed in these bands on complexation.
3.6.Emission Spectra.e emission spectrum of Eu(III) complex was recorded in the solid state in the range of 300-700 nm by selective excitation wavelength at 318 nm.e three emission peaks observed were at 578, 592, and 612 nm.e bands at 578, 592, and 612 nm were assigned to 5 D 0  7 F 0 , 5 D 0  7 F 1 , and 5 D 0  7 F 2 , respectively.e intensity of 612 nm band was found to be more intense than other two bands [36,37].From the spectral and analytical data, the general molecular formula for complexes is given as [Ln(L) 3 ⋅nH 2 O].e tentative structure for the complexes is given in Figure 14.

Plant Growth
Activity.e effect of HL and its La(III) complex at different concentrations in the growth of root and shoot of the germinated wheat seeds was analyzed with statistical analysis and data are compiled in Table 4.Among the three concentrations used, that is, 1 × 10 −5 , 1 × 10 −6 , and 1 × 10 −7 M, the growth of root and shoot is more at 1 × 10 −6 M concentration.e growth activity exhibited by HL (Group 5) and La(III) complex (Group 6) is compared with that of control (Group 1), solvent (Group 2), standard auxin (Group 3), and metal salt (Group 4).At 1 × 10 −5 M concentration, the percentage germination is less, while it is good and almost equal at 1 × 10 −6 and 1 × 10 −7 M concentrations.is indicates that, at 10 −6 M concentration, both HL and La(III) complex have more growth promoting activity than the standard auxin used.3.7.1.Shoot Length.At 1 × 10 −5 M concentration, surprisingly Groups 5 and 6 have shown a decreased activity while at 1 × 10 −7 M, a signi�cant change was observed.�ut at 1 × 10 −6 M, a signi�cant enhancement in shoot length is observed on complexation (Group 6).e graph is given in Figure 15.
3.7.2.Root Length.At 1 × 10 −5 M concentration, Group 4 has shown signi�cant increase in root length compared to Groups 3, 5, and 6.At 1 × 10 −6 M, Groups 5 and 6 have shown signi�cant enhancement in root length compared to even Group 3 (the commercial auxin).When compared among the Groups 5 and 6, the activity has enhanced on complexation (Group 6).A similar observation is made at 1 × 10 −7 M concentration, but the extent of enhancement is less compared to that at 1 × 10 −6 M. e comparative graph is given in Figure 16.

Conclusion
A series of lanthanide complexes were prepared by treating novel 2-{[2-(2, 4-dichloro-phenoxy)-acetyl]-hydrazono-methyl}-phenoxy)-acetic acid with lanthanide(III) nitrates.e structure of HL was con�rmed by various spectroscopic techniques.NMR spectral data of HL reveal its existence in E and Z isomeric forms in solution at room temperature.e coordination mode of ligand is well established from elemental analysis, molar conductivity, IR, NMR, mass, electronic spectral, and thermal studies.e results con�rm that ligand has coordinated in bidentate fashion through carboxylic acid group via deprotonation.Based on spectral and analytical results, tentative structure for complexes is given in Figure 14.e results obtained on plant growth activity indicate that germination percentage is more at 1 × 10 −6 M and 1 × 10 −7 M concentrations.When compared between the standard, HL and lanthanum(III) complex, an enhancement in the plant growth activity was observed on complexation and the best activity was observed at 10 −6 M concentration.

S 1 :
Synthetic route for the preparation of HL.

F 1 :F 2 : 1 H
Figure 4. DEPT NMR at 135 ○ pulse assisted the assignment of 13 C Isomeric structures of HL.NMR spectrum of HL.

F 6 :
Variable temperature 1 HNMR spectra of HL. at 13.16 ppm, 11.72, and 11.80 were assigned to OH, NH, and NH ′ protons, respectively, which are devoid of any attached carbon signals, and were con�rmed in 2D HMQC NMR spectral analysis.Isomeric Investigation of HL by Variable Temperature NMR.
At room temperature (298 K), the resonances for both E and Z isomers are well distinguishable.As the temperature is increased (308 K), the intensities of signals assigned to Z isomer have comparatively decreased.At 338 K, signals were reduced to more than half of their original intensity.It implies that increase in temperature causes the interconvertion of two isomers.At 348 K, the signals observed at 8.65 ppm (C9 ′ H), 7.61 ppm (C7 ′ H), and 5.31 ppm (C11 ′ H) for Z isomer have

F 8 :
13 C NMR spectrum of [La (L)3⋅2H2O].completely disappeared and at the same time, the remaining signals have also su�ered a signi�cant decrease in their intensities.At 363 K, almost a single set of signals was observed.

3. 3 .F 12 :
ermal Analysis.e thermal stability data of Ln(III) complexes (where, Ln = La, Pr, Nd, Sm, Eu, Gd, Dy, Y) are compiled in Table3.As a representative, the thermogram of La(III) complex is shown in Figure11.Under nitrogen atmosphere, the complex undergoes a three-step decomposition.e �rst weight loss found between 137.03 ○ C to 210.02 ○ C corresponds to the loss of coordinated water molecules.In La(III), Pr(III), Sm(III), Eu(III), and Y(III) complexes, the weight loss corresponds to two coordinated water molecules while in Nd(III), Gd(III), and Dy(III) complexes, the weight Mass spectrum of HL.
* e values in the parenthesis are calculated ones.* * * n.o: not observed.