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Spectrochimica Acta Part A 78 (2011) 1058–1067

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journa l homepage: www.e lsev ier .com/ locate /saa

xperimental and theoretical investigation of the molecular and electronictructure of anticancer drug camptothecin

. Subramaniana,b, N. Sundaraganesanc,∗, S. Sudhac, V. Aroulmojid, G.D. Sockalingame, M. Bergamind

Govt. college of Education, Komarapalayam 638 183, Tamil Nadu, IndiaDepartment of Physics, Dravidian University, Kuppam, A.P., IndiaDepartment of Physics (Engg.), Annamalai University, Annamalai Nagar 608 002, Chidambaram, IndiaPROTOS Research Institute, Via Flavia 23/1, c/o Incubatori FVG, 34148 Trieste, ItalyMéDIAN-Médicaments: Dynamique Intracellulaire et Architecture, Nucléaire CNRS-URCA, Unversité de Reims Champagne-Ardenne, France

r t i c l e i n f o

rticle history:eceived 15 May 2010eceived in revised form 5 December 2010ccepted 14 December 2010

eywords:

a b s t r a c t

The Fourier Transform Infrared spectrum of (S)-4 ethyl-4-hydroxy-1H-pyrano [3′,4′:6,7]-indolizino-[1,2-b-quinoline-3,14-(4H,12H)-dione] [camptothecin] was recorded in the region 4000–400 cm−1. TheFourier Transform Raman spectrum of camptothecin (CPT) was also recorded in the region 3500–50 cm−1.Quantum chemical calculations of geometrical structural parameters and vibrational frequencies of CPTwere carried out by MP2/6-31G(d,p) and density functional theory DFT/B3LYP/6-311++G(d,p) methods.

T-IRT-RamanMRP2FTamptothecin

The assignment of each normal mode has been made using the observed and calculated frequencies, theirIR and Raman intensities. The harmonic vibrational frequencies were calculated and the scaled valueshave been compared with experimental FT-IR and FT-Raman spectra. Most of the computed frequencieswere found to be in good agreement with the experimental observations. The isotropic chemical shiftscomputed by 13C and 1H NMR analysis also show good agreement with experimental observations. Com-parison of calculated spectra with the experimental spectra provides important information about the

etho

ability of computational m

. Introduction

Camptothecin (CPT), (S)-4 ethyl-4-hydroxy-1H-pyrano3′,4′:6,7]-indolizino-[1,2-b-quinoline-3,14-(4H,12H)-dione] aatural plant alkaloid extracted from Camptotheca Acuminata, haseen shown to be a potent anticancer drug targeting intracellularopoisomerase I [1–3]. This enzyme plays an important role duringhe DNA replication, unwinding the double strand and allowing the

etabolic process to occur. Its inhibition completely blocks the cellroliferation and hence the cancer growth. CPT and its derivativesave shown their anticancer activity against a wide spectrumf human malignancies including human lung, prostate, breast,olon, stomach, ovarian carcinomas, melanoma, lymphomas andarcomas [4–6]. It is a poorly water soluble substance and existsn two forms depending on the pH value. The lactone form at pHelow 5 shows high biological activity and, on the contrary, the

pen ring (carboxylate form) is the inactive one [7]. CPT havingromising antitumor efficacy has not been successfully used inancer therapy because of its intrinsic water insolubility and lac-one ring instability [8]. It has been reported that the lactone ring

∗ Corresponding author. Tel.: +91 9442068405.E-mail address: sundaraganesan n2003@yahoo.co.in (N. Sundaraganesan).

386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2010.12.049

d to describe the vibrational modes of large sized organic molecule.© 2010 Elsevier B.V. All rights reserved.

in CPT in the blood stream could be rapidly hydrolyzed yieldingto the less active and highly toxic open form (carboxylate form)[9–11]. Stability studies have demonstrated that the active lactoneform of CPT analogs is readily hydrolyzed under physiologicalcondition (pH 7.4, 37 ◦C), and the hydrolysis rates were alsoaccelerated in the presence of human serum albumin. Because ofthese inadequencies, many efforts have been made to design newdrug carrier systems that are able to improve the drug’s solubilityand stability in vivo, while retaining its anticancer therapeuticefficiency.

In the previous work, the Raman spectra of CPT derivativeswere obtained in dimethyl sulfoxide (DMSO) and FT-Raman spec-tra of CPT derivatives were obtained in aqueous buffer. The effectsof lactone hydrolysis and of substitutions on CPT molecule werestudied by SERS together with other complementary vibrationalspectroscopic techniques: Raman and FT-Raman [12]. UV–Vis spec-troscopic data of camptothecin in solvents with different polaritiesand hydrogen-bonding abilities have been reported. Also, theabsorption and fluorescence spectra of CPT have been analyzed in

different solvents such as dichloromethane, acetonitrile and iso-propanol [13]. Two usual base pairs GC, AT (G – guanine, C –cytosine, A – adenine and T – thymine) and mismatch base pairsGG, CC, AA, TT and their respective drug interacting complexeshave been optimized using the B3LYP method of density functional

N. Subramanian et al. / Spectrochimica Acta Part A 78 (2011) 1058–1067 1059

trum

ttgTtt

sNicla

2

Sopiutacl

optimization [16]. The geometry of the title compound, togetherwith that of tetramethylsilane (TMS) is fully optimized. 1H and 13CNMR chemical shifts are calculated with GIAO approach [17,18]

1 13

Fig. 1. FT-IR spec

heory with the 6-31+G* basis set. The optimized geometries ofhe usual and mismatch base pairs are almost planar whereas theeometries of drug interacting complexes deviate from planarity.he presence of hydrogen bonds has been identified and studiedhrough the interaction energy and many body interaction energy,opological and NBO analysis [14].

In this study, we present the results of a detailed investigation oftructural characterization of camptothecin using FT-IR, FT-Raman,MR and quantum chemical methods. GIAO 1H and 13C NMR chem-

cal shifts of the title compound in the ground state have beenalculated using DFT/B3LYP with 6-31G (d) as basis set. These calcu-ations are valuable for providing insight into molecular parametersnd the vibration and NMR spectra.

. Experimental

The camptothecin sample was purchased from theigma–Aldrich Chemical Company (USA) with a stated purityf greater then 99% and it was used as such without any furtherurification. The FTIR spectrum of this compound was recorded

n region of 4000–400 cm−1 on an IFS 66 V spectrophotometersing KBr pellet technique. The spectrum was recorded at room

emperature with a scanning speed of 10 cm−1 per minute andt the spectral resolution of 2.0 cm−1. The FT-Raman spectrum ofamptothecin has been recorded using 1064 nm line of Nd:YAGaser as excitation wavelength in the region 3500–50 cm−1 on

20000

15000

10000

5000

0

500 1000 1500 2000 2500

Wavenumber (cm-1)

Ram

an

In

ten

sity

(arb

un

its)

Fig. 2. FT-Raman spectrum of camptothecin.

of camptothecin.

a Brucker model IFS 66 V spectrophotometer equipped with anFRA 106 FT-Raman module accessory. The observed experimentalFT-IR and FT-Raman spectra are shown in Figs. 1 and 2. The theo-retically predicted IR spectra at HF and DFT/B3LYP/6-311++G(d,p)level calculations is shown in Fig. 3, along with the experimentalspectrum. For NMR spectra the samples were prepared in (2H6)dimethyl sulfoxide (DMSO) solution. NMR experiments werecarried out on a Varian INOVA 500 MHz NMR spectrometer at aconstant temperature of 25 ◦C. The measured 13C and 1H NMRspectra are shown in Figs. 4–5. Chemical shifts were referencedto internal TMS. The spectral measurements were carried out atCentral Electro Chemical Research Institute (CECRI), Karaikudi,Tamil Nadu, India.

3. Computational details

Ab initio molecular calculations were performed using theGaussian 03W [15] program package, invoking gradient geometry

by applying B3LYP method. The theoretical H and C chemical

IR I

nte

nsi

ty (

Km

/mol)

4000 3500 3000 2500 2000 1500 1000 500 0

Wavenumber (cm-1)

B3LYP/6-311++G(d,p)

Fig. 3. Theoretical FT-IR spectrum of camptothecin.

1060 N. Subramanian et al. / Spectrochimica Acta Part A 78 (2011) 1058–1067

ectrum

s[aT

Fig. 4. 13C NMR sp

hift values were obtained by subtracting the GIAO calculation19,20]. 13C and 1H NMR isotropic magnetic shielding (IMS) ofny X carbon atom were made according to the value 13C IMS ofMS. CSx = IMSTMS− IMSx. The geometrical parameter and vibra-

Fig. 5. 1H NMR spectrum

of camptothecin.

tional frequencies of CPT were carried out by MP2/6-31G(d,p)and B3LYP/6-311++G(d,p) methods respectively. The optimizedstructural parameters were used in the vibrational frequency calcu-lations at DFT level to characterize all stationary points as minima.

of camptothecin.

N. Subramanian et al. / Spectrochimica Acta Part A 78 (2011) 1058–1067 1061

ing sch

Atc

4

4

fpcctmaroB

sbbaBstalsCowaC

4

‘ect

Fig. 6. Molecular structure and atom number

n empirical scaling factor of 0.9572 was used to offset the sys-ematic error caused by neglecting anharmonicity and electronorrelation [21].

. Results and discussion

.1. Molecular structure

The molecular structure and atomic numbering scheme adoptedor the present study is shown in Fig. 6. The first task for the com-utational work was to determine the optimized geometry of theamptothecin. The calculated relevant geometric parameters foramptothecin are summarized in Table 1. The geometries of camp-othecin was optimized in the gas phase at the MP2/6-31G(d,p)

ethod. Our optimized bond lengths, bond angles and dihedralngles of CPT by MP2/6-31G(d,p) method were compared with theeported X-ray crystal data results obtained for related moleculef camptothecin iodoacetate [22] and theoretical calculations of3LYP/6-311++G(d,p) method [23].

The CPT molecule consists of five rings out of which four areix-membered ring and one is five-membered ring. Both N–Conds of 2nd have identical length of 1.340 A [24–26]. The N–Conds is about 1.34 A for both 2-amino-5-methyl pyridine and 2-mino-6-methyl pyridine by B3LYP/6-311++G(d,p) method [27].ut in our title molecule, the N1–C2 and N1–C10 bond length ofix-membered 2nd ring calculated to be 1.325 A and 1.363 A respec-ively by MP2/6-31G(d,p) method. In the 1st ring, C–C bond length isbout 1.396 A [28], our calculations by MP2/6-31G(d,p) were simi-ar to this value, for example the optimized bond length of C–C in 1stix-membered ring fall in the range 1.379–1.417 A (C5–C6 = 1.417 A,6–C7 = 1.380 A, C7–C8 = 1.415 A, C8–C9 = 1.379 A). All the ringsf CPT are coplanar except the 5th six-membered ring,hich is nonplanar as evident from Table 1 of torsional

ngles of C14–C15–C19–C20 = 41.0◦, C18–C21–C20–C19 = 8.7◦,15–C19–C20–O22 = 137.0◦ and C15–C14–C18–O21 =−32.7◦.

.2. Vibrational assignments

It is well known, that the calculated HF and DFT ‘raw’ ornon-scale’ harmonic frequencies could significantly overestimatexperimental values due to lack of electron correlation, insuffi-ient basis sets and anharmonicity. Much effort has been devotedo accurately reproducing experimental frequencies in theoretical

eme adopted in this study for camptothecin.

calculations. The Hartree–Fock calculated results are usually moreover-estimated than the corresponding DFT ones [29]. Frequencycalculations at the B3LYP level of theory revealed no imaginary fre-quencies, indicating that an optimal geometry at these levels ofapproximation was found for the title compound. To make com-parison with experiment, we present correlation graphics in Fig. 7based on the calculations. As we can see from correlation graphicin Fig. 7 experimental fundamentals are in better agreement withthe scaled fundamentals and found to have a better correlation forB3LYP.

The molecule of camptothecin consists of 42 atoms. The 120normal modes of vibration of camptothecin, which span in theirreducible representations as 120A under the C1 point group sym-metry, have been assigned according to the detailed motion ofthe individual atoms. The vibrational frequency and the approxi-mate description of each normal modes obtained using DFT/B3LYPmethod with 6-311++G(d,p) basis set is given in Table 2. The solidphase FT-IR and FT-Raman spectra recorded in this study was usedas an experimental result which is also given in Table 2. The com-parison of theoretical and experimental IR spectra indicates that theintense vibrations in the experimental spectrum are also intensivein theoretical spectrum.

4.2.1. C O group vibrationsThe structural unit of C O has an excellent group frequency,

which is described as a stretching vibration. Since the C O groupis a terminal group, only the carbon is involved in a second chem-ical bond this reduces the number of force constants determiningthe spectral position of the vibration. The C O stretching vibrationusually appears in a frequency range that relatively free of othervibrations. For example, in many carbonyl compounds the doublebond of the C O has a force constant different from those of suchstructural units such as C C, C–C, C–H etc., only structural units ofC C have force constants of magnitudes similar to that of the C Ogroup. The C C vibration could interact with C O if it was the samespecies, but generally it is not. Almost all carbonyl compounds havea very intense and narrow peak in the range of 1800–1600 cm−1

[30,31] or in other words the carbonyl stretching frequency has

been most extensively studied by infrared spectroscopy [32]. Themultiple bonded group is highly polar and therefore gives rise toan intense infrared absorption band in the region 1700–1800 cm−1.The carbon–oxygen double bond is formed by P�–P� bondingbetween carbon and oxygen. Because of the different electroneg-

1062 N. Subramanian et al. / Spectrochimica Acta Part A 78 (2011) 1058–1067

Table 1Geometrical parameters optimized in camptothecin, bond length (A), bond angle (◦) and dihedral angle (◦).

Parameters aExperimental aB3LYP/6-311++G(d,p)

MP2/6-31G(d,p) (inthis study)

Parameters aExperimental aB3LYP/6-311++G(d,p)

MP2/6-31G(d,p) (inthis study)

Bond length (A)N1–C2 1.31 1.311 1.325 C13–C14 1.46 1.450 1.446N1–C10 1.38 1.363 1.369 C13–023 1.20 1.231 1.242C2–C3 1.44 1.424 1.422 C14–C15 1.33 1.373 1.379C2–C17 1.47 1.466 1.459 C14–C18 1.54 1.495 1.493C3–C4 1.29 1.367 1.373 C15–C16 1.39 1.418 1.412C3–C11 1.53 1.503 1.501 C15–C19 1.52 1.519 1.507C4–C5 1.42 1.421 1.419 C16–C17 1.41 1.366 1.373C4–H27 1.085 C16–H34 1.079C5–C6 1.40 1.418 1.417 C18–O21 1.452C5–C10 1.42 1.437 1.439 C18–H35 1.088C6–C7 1.39 1.374 1.380 C18–H36 1.093C6–H28 1.084 C19–C20 1.56 1.542 1.529C7–C8 1.43 1.415 1.415 C19–024 1.42 1.414 1.415C7–H29 1.083 C19–C25 1.60 1.560 1.543C8–C9 1.37 1.374 1.379 C20–O21 1.31 1.336 1.346C8–H30 1.083 C20–O22 1.19 1.207 1.222C9–C10 1.34 1.419 1.417 O24–H37 2.046 0.973C9–H31 1.082 C25–C26 1.52 1.529 1.523C11–N12 1.49 1.473 1.469 C25–H38 1.092C11–H32 1.092 C25–H39 1.092C11–H33 1.092 C26–H40 1.089N12–C13 1.40 1.396 1.395 C26–H41 1.088N12–C17 1.31 1.377 1.377 C26–H42 1.090

Bond angle (◦)C2–N1–C10 115.0 116.3 115.1 C7–C6–H28 120.5N1–C2–C3 126.4 C6–C7–C8 123.0 120.4 120.5N1–C2–C17 126.0 126.1 125.3 C6–C7–H29 119.8C3–C2–C17 107.0 108.1 108.3 C8–C7–H29 119.6C2–C3–C4 118.0 118.7 118.8 C7–C8–C9 120.5C2–C3–C11 109.0 109.4 109.4 C7–C8–H30 119.6C4–C3–C11 133.0 131.9 131.8 C9–C8–H30 119.9C3–C4–C5 117.7 C8–C9–C10 120.0 120.5 120.4C3–C4–H27 122.4 C8–C9–H31 122.0C5–C4–H27 119.9 C10–C9–H31 117.6C4–C5–C6 119.0 122.6 122.3 N1–C10–C5 123.2C4–C5–C10 118.8 N1–C10–C9 117.6C6–C5–C10 118.9 C5–C10–C9 119.2C5–C6–C7 120.5 C3–C11–N12 100.0 102.1 101.9C5–C6–H28 119.0 C3–C11–H32 114.0

Bond angle (◦)C3–C11–H33 114.0 O21–C18–H36 108.2N12–C11–H32 109.5 H35–C18–H36 108.0N12–C11–H33 109.5 C15–C19–C20 110.0 108.9 108.6H32–C11–H33 107.7 C15–C19–O24 110.6C11–N12–C13 117.0 121.8 120.7 C15–C19–C25 110.3C11–N12–C17 114.0 113.3 113.5 C20–C19–O24 109.0 108.4 108.4C13–N12–C17 128.0 124.9 125.8 C20–C19–C25 113.0 107.7 109.9N12–C13–C14 112.2 O24–C19–C25 109.1N12–C13–O23 123.0 120.8 121.0 C19–C20–O21 118.5C14–C13–O23 126.8 C19–C20–O22 120.8C13–C14–C15 122.5 O21–C20–O22 122.0 121.0 120.7C13–C14–C18 112.0 117.5 117.5 C18–O21–C20 121.0 120.8 119.6C15–C14–C18 120.0 C19–O24–H37 104.7C14–C15–C16 121.8 C19–C25–C26 112.0 113.5 112.9C14–C15–C19 116.6 C19–C25–H38 109.8C16–C15–C19 121.6 C19–C25–H39 105.5C15–C16–C17 117.0 117.3 116.7 C26–C25–H38 110.8C15–C16–H34 122.3 C26–C25–H39 110.4C17–C16–H34 121.1 H38–C25–H39 107.0C2–C17–N12 106.9 C25–C26–H40 110.3C2–C17–C16 132.1 C25–C26–H41 110.1N12–C17–C16 119.0 121.2 121.0 C25–C26–H42 111.6C14–C18–O21 112.7 H40–C26–H41 108.4C14–C18–H35 109.9 H40–C26–H42 108.0C14–C18–H36 112.3 H41–C26–H42 108.4O21–C18–H35 105.4

Dihedral angle (◦)C10–N1–C2-C17 180.0 C11–C12–C13–O23 −0.6C2–N1–C10-C9 −179.9 O23–C13–C14–C15 −179.7C14–C15–C19–C20 41.0 C15–C14–C18–H35 −150.0C18–C21–C20–C19 8.7 C14–C15–C19–O24 159.9C15–C19–C20–O22 137.0 H34–C16–C17–N12 178.8C15–C14–C18–O21 −32.7 H35–C18–O21–C20 150.0

N. Subramanian et al. / Spectrochimica Acta Part A 78 (2011) 1058–1067 1063

Table 1 (Continued)

Parameters aExperimental aB3LYP/6-311++G(d,p)

MP2/6-31G(d,p) (inthis study)

Parameters aExperimental aB3LYP/6-311++G(d,p)

MP2/6-31G(d,p) (inthis study)

C19–C20–O21–C18 8.7 O24–C19–C20–O21 −164.7C

aaigd

tbip(siWFR1bma

4

nvtcp3otasmbatmaC

Ft

O22–C20–O21–C18 −172.8C19–C25–C26–H40 174.6

a Taken from Refs. [22,23].

tivities of the carbon and oxygen atoms, the bonding electronsre not equally distributed between the two atoms. The follow-ng two resonance forms contribute to the bonding of the carbonylroup >C O←→C+–O−. The lone pair of electrons on oxygen alsoetermines nature of the carbonyl group [33].

In our present study, we can expect two C O stretching vibra-ion (i.e. C13 O23 and C20 O22), the conjugation of C20 O22ond C13 O23 may increase its single bond character resulting

n lowered values of carbonyl stretching wavenumbers. The com-uted wavenumber by B3LYP/6-311++G (d,p) method at 1711 cm−1

mode no. 17) is assigned to C20 O22 stretching vibrations. Theharp intense band in FT-IR spectrum at 1741 cm−1 and weak bandn FT-Raman at 1732 cm−1 is assigned to C20 C22 stretching mode.

hile in the case of C13 O23 stretching mode, the shoulder band inT-IR spectrum at 1651 cm−1 and 1654 cm−1 as a strong band in FT-aman are in moderate agreement with computed wavenumber at631 cm−1 (mode no. 18). The lowering of wavenumber ∼24 cm−1

etween the recorded as well as computed for C13 O23 stretchay be due to formation of intramolecular hydrogen bonding with

djacent CH2 moiety.

.2.2. Aromatic C–H vibrationsThe existence of one or more aromatic ring in a structure is

ormally readily determined from the C–H and C C–C relatedibrations. The C–H stretching occurs above 3000 cm−1 and isypically exhibited as a multiplicity of weak to moderate bands,ompared with the aliphatic C–H stretch [33]. The aromatic com-ounds show the presence of C–H stretching vibrations around100–3000 cm−1 range. In amino quinoline, these modes werebserved at 3103, 3060 and 3037 cm−1 [34]. For our title moleculehe band predicted by B3LYP method at 3062, 3052, 3038, 3029nd 3026 cm−1 (mode nos. 3–7) have been designated as C–Htretching vibrations of quinoline ring of camptothecin. The experi-ental observations of FT-IR spectrum at 3116 cm−1 as a moderate

and show excellent agreement with our theoretical values as wells the literature data [35,36]. The C–H in-plane bending vibra-

ions are observed in the region 1350–950 cm−1 and are usually

edium to weak. The C–H out-of plane bending modes are usu-lly of weak intensity arises in the region 600–950 cm−1. All these–H in-plane and out-of plane bending modes of this compound

B3LYP/6-311++G(d,p)

y = 0.995x + 3.2439

R2 = 0.9989

0

500

1000

1500

2000

2500

3000

3500

4000

40003000200010000

Experimental

Calc

ula

ted

ig. 7. Correlation graphics of calculated and experimental frequencies of camp-othecin.

15–C19–C25–C26 177.68.7

are also assigned within the said region and are represented inTable 2.

4.2.3. C–C vibrationsThe carbon–carbon stretching modes of the phenyl group are

expected in the region from 1650 to 1200 cm−1. The actual posi-tion of these modes are determined not so much by the nature ofthe substituents but by the form of substitution around the ring[37], although heavy halogens cause the frequency to diminishundoubtedly [38]. In amino methyl quinoline [36] under Cs sym-metry the carbon–carbon stretching bands appeared in the infraredspectrum at 1617, 1570, 1518, 1470, 1415, 1241 and 1129 cm−1

and were assigned to skeletal C–C bonds while the bands at 1593and 1371 cm−1 had been assigned to the ring C–N stretching vibra-tions. The recorded FT-IR spectrum at 1570, 1500 cm−1 and 1622,1503, 1394, 1383, 1364 cm−1 in FT-Raman spectrum are assigned toC–C stretching vibrations for our title molecule. The C–C stretchingmodes predicted by B3LYP/6-311++G (d,p) method at 1595, 1585,1510, 1471, 1398, 1376, 1355 and 1353 cm−1 (mode nos. 19, 20,23, 24, 31, 32, 34 and 35). The C N and C–N stretching vibrationspredicted by same B3LYP/6-311++G(d,p) method are at 1575 and1204 cm−1 (mode nos. 21 and 44). These two C N and C–N stretch-ing vibrations are exactly correlate with experimental FT-Ramanrecorded spectrum at 1564 and 1210 cm−1 as well as the literaturedata [36].

The bands occurring at 979 and 509 cm−1 in Raman spectrumwere assigned to the skeletal CCC and CCN in-plane bending modesof amino methyl quinoline respectively. The out-of-plane bendingring vibrations under Cs symmetry are assigned to the bands at 648,619, 538, 449 and 105 cm−1. Normal coordinate analysis shows thatsignificant mixing of skeletal in-plane bending with C–H in-planebending and vice versa occurs. In accordance with above conclu-sion, the bands predicted by B3LYP/6-311++G(d,p) method at 861,781, 775 and 748 cm−1 (mode nos. 70, 73, 74 and 77) are assignedto CCC/CCN in-plane bending modes of CPT show good agreementwith recorded FT-IR band at 789, 777, 750 cm−1 and 773, 750 cm−1

in FT-Raman spectrum respectively.

4.2.4. O–H vibrationsThe O-H group gives rise to three vibrations (stretching, in-

plane bending and out-of-plane bending vibrations). The hydroxylstretching vibrations are generally observed in the region around3500 cm−1 [39]. The O–H group vibrations are likely to be the mostsensitive to the environment, so they show pronounced shifts inthe spectra of the hydrogen bonded species [40]. IR spectrum inthe high wavenumber region shows a broad band at 3433 cm−1 isattributed to hydrogen bonded OH stretching vibration. The the-oretically computed value at 3550 cm−1 by B3LYP/6-311++G(d,p)(mode no. 1) method shows a deviation about 117 cm−1 with theexperimental data. The peak is broader and its intensity is higherthan that of a free O–H vibration which indicates involvement inan intramolecular hydrogen bond.

The in-plane O–H deformation vibration usually appears inthe region 1440–1260 cm−1 in IR spectrum. In our title molecule,the O–H in-plane bending vibrations present in the region1400–1350 cm−1. Also, the band predicted by B3LYP method at1376, 1353 and 1331 cm−1 (mode nos. 32, 35 and 36) have been

1064 N. Subramanian et al. / Spectrochimica Acta Part A 78 (2011) 1058–1067

Table 2Vibrational wavenumbers obtained for camptothecin at B3LYP/6-311++G(d,p) method.

Mode nos. Experimental wavenumber (cm−1) Theoretical wavenumber (cm−1) Vibrational assignments

FT-IR FT-Raman B3LYP/6-311++G(d,p)

Freq. (unscaled) Freq. (scaled) IR int

1 3433br 3709 3550 122.37 �O–H2 3261br 3243 3104 8.14 �C16–H3 3116m 3199 3062 10.07 �C–H4 3188 3052 16.37 �C–H5 3174 3038 8.72 �C–H6 3164 3029 11.15 �C–H7 3161 3026 0.25 �C–H8 3127 2993 1.07 �asymC18H2

9 2974m 2984vw 3112 2979 18.97 �asymCH3

10 2963w 3097 2964 23.36 �asymCH3

11 2943w 3076 2944 3.09 �asymC11H2

12 2938m 3073 2941 7.30 �asymC25H2

13 2920w 2925w 3047 2917 7.56 �symC18H2

14 3038 2908 18.47 �symCH3

15 3036 2906 28.07 �symC25H2

16 3032 2902 21.99 �symC18H2

17 1741ms 1732w 1788 1711 403.88 �C20 O22 + �C13 O2318 1651m 1654s 1704 1631 535.23 �C13 O2319 1622vs 1666 1595 140.22 �C–C20 1656 1585 66.12 �C–C21 1570sh 1564vs 1645 1575 83.78 �C–N/�C–N22 1605 1536 8.84 �C–C/�C–N/�C–H23 1500m 1503m 1578 1510 125.59 �C–C + �C18H2

24 1473m 1537 1471 28.91 �C–C25 1455ms 1509 1444 5.30 CH3 asym. deform26 1441ms 1507 1443 12.23 �C18H2

27 1439m 1502 1438 4.34 CH3 asym. deform28 1498 1434 5.27 �C25H2

29 1496 1432 11.04 �C25H2 + �C–H30 1478 1415 3.20 �C11H2

31 1400w 1394vw 1461 1398 28.72 �C–C + �C11H2/�C18H2

32 1383s 1438 1376 6.95 �C–C + �C11H2 + �C–H + �O–H33 1370w 1424 1363 61.53 CH3 sym. deform34 1364m 1416 1355 1.96 �C–C + �C–H35 1349w 1413 1353 1.01 �C–C + �C–N + �O–H36 1391 1331 42.16 �O–H + �CH2

37 1384 1325 26.21 Ring deform38 1323w 1322vw 1380 1321 14.22 �C–C + �C–H + �C11H2

39 1294vs 1349 1291 19.82 �C25H2

40 1344 1286 6.72 �C–H41 1252w 1312 1256 1.65 tC25H2

42 1240vw 1305 1249 4.31 tC25H2 + �C11H2

43 1234w 1225w 1277 1222 0.82 �C–H44 1210vw 1258 1204 47.40 �C–N + tCH2

45 1254 1200 75.79 �C11H2 + �C–H46 1197w 1187s 1243 1190 28.22 tC18H2

47 1221 1169 2.27 tC18H2 + �C–H48 1160w 1208 1156 20.60 �C–H49 1157m 1154w 1206 1154 9.91 �C–C + �C–H50 1197 1146 1.08 tC11H2

51 1138w 1184 1133 15.50 �C–H52 1171 1121 109.00 �C O/�C18H2

53 1114w 1165 1115 104.96 �C–H54 1103vw 1109w 1158 1108 12.30 �C–H55 1132 1084 14.17 �C25H2

56 1071vw 1056m 1124 1076 3.67 �C11H2 + �C–H57 1042vw 1043vw 1083 1037 25.96 �C19–C2558 1019vw 1071 1025 13.29 �C–CH3/�C25–C2659 1005m 1005vw 1050 1005 99.66 �C18–O2160 1038 994 3.42 �C–H61 970vw 1013 970 14.83 �C18H2

62 967vw 1007 964 0.32 �C11H2 + �C–H63 998 955 7.06 �C18H2

64 998 955 0.13 �C–H65 981 939 4.20 �C–C–C + �C–H66 934w 933vw 975 933 2.31 �C–H67 917ms 919w 942 902 3.91 �C–C–C68 886w 927 887 6.27 �C–H in benzene69 871w 916 877 12.86 �C–H + �C11H2

70 899 861 8.36 �C–C–C71 840m 874 837 0.66 �C–C–C72 829s 829vw 870 833 29.77 �C–C–C

N. Subramanian et al. / Spectrochimica Acta Part A 78 (2011) 1058–1067 1065

Table 2 (Continued)

Mode nos. Experimental wavenumber (cm−1) Theoretical wavenumber (cm−1) Vibrational assignments

FT-IR FT-Raman B3LYP/6-311++G(d,p)

Freq. (unscaled) Freq. (scaled) IR int

73 789w 816 781 14.61 �C–C–N + �C–N–C74 777w 773vs 810 775 4.18 �C–C–C + �C–C–O75 764w 795 761 8.64 �C–H76 789 755 5.53 �CH3

77 750vw 750w 781 748 2.01 �C–C–C + �C–N–C78 739ms 771 738 41.30 �C–H79 724w 728vw 758 726 1.85 �C–C–C + �C–N–C80 746 714 3.59 �C–C–C81 707vw 707m 736 704 15.10 �C–C–C + �C–C–N82 730 699 1.74 �C–C–O83 659vw 708 678 8.45 �C–C–O84 642s 671 642 5.95 �C–C–C + �C–N–C85 621w 619vw 652 624 1.09 �C–C–C + �N–C–C86 606w 632 605 5.38 �C–C–C + �C–C–O87 588vw 618 592 1.53 �C–C–C + �C–C–N88 565m 603 577 13.15 �C–C–C + �N–C–C89 553m 571 547 1.67 �C–O–C + �C–C–C90 564 540 0.97 �C–C–C + �C–C–N91 522vw 538 515 3.80 �C–C–C + �C–N–C92 500vw 528 505 2.56 �C–C–C + �C–N–C93 482vw 500 479 7.87 �C19–C2594 474vw 485 464 5.13 �C–C–C + �C–N–C95 460s 465 445 12.19 �O–H96 444vw 463 443 56.86 �C–C–C97 425vw 440 421 7.01 �O–H98 372w 409 391 0.05 �C–C–C + �C–N–C99 353w 370 354 5.93 �C–C–C + �C–N–C

100 340w 357 342 15.13 �O–H + �C–C–C101 325m 334 320 5.75 �C–C–C + �C–N–C102 302w 321 307 0.84 �C–C–C103 301 288 6.07 �C O + �C–C–C + �N–C–C104 297 284 4.26 �C–C–C + �C–N–C105 278 266 0.82 �C–C–C + �C–N–C106 260 249 0.66 �C–C–C + �C–N–C107 255 244 3.61 �C–C–C + �C–N–C108 243 233 1.93 tCH3 + tC18H2

109 220 211 4.92 tCH3

110 199 190 1.73 tCH3

111 177 169 5.24 tC18H2

112 165 158 0.71 CH3

113 148 142 6.95 tCH2

114 122 117 0.16 �C O115 101 97 0.39 �CH3

116 93 89 1.69 �C–C–N117 79 76 0.18 CH3–CH2

118 62 59 0.03 CH3–CH2

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Rint – IR intensity (K mmol−1); w – weak; vw – very weak; s – strong; vs – very stroasy – asymmetric stretching; � – in plane bending; � – out-of-plane bending; � –

onformed as O–H bending vibrations of CPT. The experimentalata of FT-IR spectrum at 1349 cm−1 and 1383 cm−1 in FT-Ramanhows an excellent agreement with computed wavenumber. Thisibration mixes with C–N and C–C stretch and C–H in-plane bend-ng vibrations as shown in Table 2.

The O–H out-of-plane deformation vibration for phenol liesn the region 290–320 cm−1 for free OH and in the region17–710 cm−1 for associated O–H [41]. The bands at 445 and21 cm−1 (mode nos. 95 and 97) by B3LYP/6-311++G(d,p) methodhow excellent correlation with recorded FT-Raman band at 460,25 cm−1.

.2.5. Methyl group vibrations

The title molecule under consideration possesses one CH3 unit

hich lies in the terminal group of molecule. For the assignmentsf CH3 group frequencies one can expect nine fundamentals can bessociated to each CH3 group. Methyl group vibrations are generallyeferred to as electron-donating substituent in the aromatic rings

48 0.45 Ring torsion31 0.54 Ring bending

– medium; br, sh – broad, shoulder; � – stretching; �sym – symmetric stretching;ng; t – twisting; � – scissoring; – torsion.

system, the antisymmetric C–H stretching mode of CH3 is expectedaround 2980 cm−1 and CH3 symmetric stretching is expected at2870 cm−1 [42,43]. The first of these results from the antisymmetricstretching of CH3 mode in which the two C–H bonds of the methylgroup are expanding while the third one is contracting. The sec-ond arises from the symmetric stretching, in which all the threeC–H bonds expand and contract in phase. The antisymmetric andsymmetric stretching vibrations are observed in the 2970–3010and 2900–2940 cm−1 regions respectively. For benzene deriva-tives containing a CH3 group two bands which are antisymmetricaland symmetrical stretching occur at about 2900 and 2850 cm−1

respectively [44]. In our present work, the antisymmetric stretch-ing vibrations of CH3 group predicted by B3LYP/6-311++G (d,p)

method at 2979 and 2964 cm−1 (mode nos. 9 and 10). The sym-metric stretching vibration of CH3 group is predicated theoreticallyat 2908 cm−1 by B3LYP/6-311++G(d,p) method (mode no. 14). Twobending modes can occur within a methyl group. The first of these,the symmetric bending vibration, involves the in-phase bending

1 imica Acta Part A 78 (2011) 1058–1067

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Table 3Experimental and theoretical chemical shifts (13C, 1H) of camptothecin by B3LYP/6-31G(d) method (ppm)].

Atom position B3LYP Atom position B3LYP

Expt. 6-31G(d) Expt. 6-31G(d)

N1 −39.70 O22 −116.81C2 152.43 155.15 O23 42.00C3 129.70 126.5 O24 289.80C4 131.70 123.65 C25 29.90 39.42C5 127.80 118.62 C26 7.65 15.05C6 128.40 147.31 H27 8.70 8.43C7 127.52 133.02 H28 8.14 8.26C8 130.20 137.06 H29 7.72 7.42C9 128.90 125.66 H30 7.87 7.57C10 147.90 140.98 H31 8.18 8.06C11 49.90 56.98 H32 5.29 4.22N12 H33 5.29 4.22C13 156.69 145.20 H34 7.36 6.74C14 118.94 124.51 H35 5.44 4.76C15 149.86 146.37 H36 5.44 4.74C16 96.40 99.14 H37 6.53 0.67C17 145.34 144.06 H38 1.88 2.64C18 64.90 72.77 H39 1.88 2.39

066 N. Subramanian et al. / Spectroch

f C–H bonds. The second, the antisymmetrical bending vibration,nvolves out-of-phase bending of the C–H bonds. The antisym-

etric deformations are expected in the region 1400–1485cm−1.he antisymmetric and symmetric bending deformations are pre-icted by B3LYP/6-311++G(d,p) method at 1444, 1438 cm−1 and363 cm−1 (mode nos. 25, 27 and 33) respectively.

The weak intense bands in IR spectrum at 764 cm−1 arettributed to the CH3 rocking mode. The methyl twisting mode ofibration coupled with methylene group. The other CH3 torsionalode and CH3 out-of-plane bending vibrations are also predicted

heoretically at 211, 190 and 97 cm−1. In the FT-Raman spectrum,he C–CH3 in-plane bending vibration is observed at 1019 cm−1.

.2.6. CH2 group vibrationsFor the assignments of CH2 group frequencies, basically six

undamentals can be associated to each CH2 group namely, CH2ym-symmetric stretch, CH2-asym, antisymmetric stretch, CH2cis, scissoring and CH2 rock, rocking modes which belong to in-lane vibrations of A’ species. In addition to that CH2 wag, waggingnd CH2 twist, twisting modes of CH2 group would be expected toe for out-of-plane bending vibrations of A” species.

In our title molecule there are three CH2 groups (C11H2, C18H2nd C25H2). The C–H stretching vibrations of the methylene groupre at lower frequencies than those of the aromatic C–H ringtretching. The antisymmetric CH2 stretching vibration generallybserved in the region 3000–2900 cm−1, while the CH2 sym-etric stretch will appear between 2900–2800 cm−1 [45,46]. The

H2 antisymmetric and symmetric stretching vibrations computedy B3LYP/6-311++(d,p) method at 2944, 2941, 2917, 2906 and902 cm−1 (mode nos. 11, 12, 13, 15 and 16). The FT-IR band at 2938,920 cm−1 and 2925 cm−1 in FT-Raman spectrum are assigned to11H2, C18H2 and C25H2 antisymmetric and symmetric stretchingibrations respectively.

In the present study, the CH2 bending modes follow, inecreasing wavenumber, the general order CH2 deformation > CH2agging > CH2 twist > CH2 rock. Since the bending modes involv-

ng hydrogen atom attached to the central carbon falls into the450–875 cm−1 range, there is extensive vibrational coupling ofhese modes with CH2 deformations particularly with the CH2wist. It is notable that both CH2 scissoring and CH2 rocking wereensitive to the molecular conformation. For cyclohexane, the CH2cissoring mode has been assigned to the medium intensity IR bandt about 1450 cm−1 [46]. In our title molecule the scaled vibra-ional frequencies computed by B3LYP/6-311++G(d,p) method at443, 1434, 1432 and 1415 cm−1 (mode nos. 26, 28, 29 and 30)re assigned to CH2 scissoring modes of C18H2, C25H2 and C11H2nits show good correlation with recorded FT-Raman spectrum at441 cm−1.

The computed wavenumber by B3LYP/6-311++G(d,p) methodt 1398, 1376, 1331, 1321, 1291, 1249, and 1200 cm−1 (mode nos.1, 32, 36, 38, 39, 42 and 45) are assigned to CH2 wagging vibra-ion of C18H2, C11H2 and C25H2 units shows good agreement withecorded FT-IR and FT-Raman bands as shown in Table 2. The CH2wisting vibrations are observed as a weak to shoulder band inT-IR spectrum at 1252, 1197 cm−1 and 1210, 1187 cm−1 in FT-aman spectrum as a weak band are assigned to CH2 twistingibrations.

.3. 13C and 1H NMR spectral analysis

The isotropic chemical shifts are frequently used as an aid in

dentification of reactive organic as well as ionic species. It isecognized that accurate predictions of molecular geometries aressential for reliable calculations of magnetic properties. There-ore, full geometry optimization of CPT were performed by using3LYP/6-311++G (d,p) method. Then, Gauge-Including Atomic

C19 72.25 72.77 H40 0.89 1.44C20 172.34 172.49 H41 0.89 2.18O21 192.46 H42 0.89 1.28

Orbital (GIAO) 1H and 13C chemical shift calculations of the com-pound has been made by same method.

Application of the GIAO [17] approach to molecular systems wassignificantly improved by an efficient application of the methodto the ab initio SCF calculations, using techniques borrowed fromanalytic derivative methodologies. GIAO procedure is somewhatsuperior since it exhibits a faster convergence of the calculatedproperties upon extension of the basis set used. Taking intoaccount the computational cost and the effectiveness of calcula-tion, the GIAO method seems to be preferable from many aspectsat the present state of this subject. On the other hand, the den-sity functional methodologies offer an effective alternative to theconventional correlated methods, due to their significantly lowercomputational cost.

The molecular structure of camptothecin is optimized by B3LYPmethod with 6-311++G (d,p). Then, gauge-including atomic orbital(GIAO) 13C and 1H chemical shift calculation of title compoundis made by using B3LYP method. The 1H and 13C chemical shiftswere measured in a less polar (CDCl3) solvent. The result in Table 3shows that the range 13C NMR chemical shift of the typical organicmolecule usually is >100 ppm [47,48], the accuracy ensures reliableinterpretation of spectroscopic parameters. It is true from the aboveliterature value, in our present study, the title molecule camp-tothecin also falls with the above literature data except with themethylene carbon atoms (C11, C18 and C25) olefinic carbon (C16),carbonyl carbon (C20) and methyl carbon atom (C26). In the presentpaper, the signal observed at 29.90 ppm in 13C NMR spectrum isassigned to C25 carbon atom correlates with theoretically predictedvalue at 39.42 ppm. The signals for aromatic carbons were observedat 127.52–152.43 ppm in 13C NMR spectrum for the title molecule,since those carbon atoms which belong to quinoline also exactlycorrelate with theoretically predicted value at 118.62–155.15 ppm.The signals of the aromatic proton were observed at 7.72–8.70 ppm.The calculated proton NMR chemical shifts show moderate agree-ment with the experimental values except with aliphatic protonH34. The H atom is the smallest of all atoms and mostly localized onthe periphery of molecules. Therefore their chemical shifts would

be more susceptible to intermolecular interactions in the aque-ous solution as compared to that for other heavier atoms. Anotherimportant aspect is that, hydrogen attached or nearby electron-withdrawing atom or group can decrease the shielding and moves

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he resonance of attached proton towards to a higher frequency.y contrast electron donating atom or group increases the shield-

ng and moves the resonance towards to a lower frequency. In thistudy, the chemical shifts obtained and calculated for the hydro-en atoms of methyl groups are quite low. All values are ≤3 ppm49] due to shielding effect. It is true from above literature datan our present study the methyl protons at C26 appears as singlet

ith three proton integral at 0.89 ppm shows good agreement withomputed chemical shift values are shown in Table 3. The com-uted methylene H atom chemical shifts by B3LYP method are inood agreement with experimental ppm value as shown in Table 3.

.4. Conclusion

The FT-IR and FT-Raman spectra of camptothecin were recordednd analyzed. The vibrational frequencies are calculated theoreti-ally using Gaussian 03 software package. The normal modes ofhe camptothecin molecule are assigned by comparing experimen-al and theoretically calculated wavenumbers. For the geometricarameters as well as vibrational wavenumbers, the result of B3LYPethod has shown a better fit to experimental ones in evaluating

eometrical parameters and vibrational wavenumbers. The com-arisons of theoretical and experimental IR spectra indicate thathe intense vibrations in the experimental spectrum are also inten-ive in theoretical spectrum. The NMR chemical shifts, infraredntensities and Raman activities are reported. The calculated NMRhemical shift values are comparable with measured NMR values.

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