Accepted Manuscript XRD, vibrational spectra and quantum chemical studies of an anticancer drug: 6-Mercaptopurine S. Suresh Kumar, S. Athimoolam, B. Sridhar PII: DOI: Reference:

S1386-1425(15)00278-4 http://dx.doi.org/10.1016/j.saa.2015.02.104 SAA 13406

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

14 June 2014 20 February 2015 24 February 2015

Please cite this article as: S. Suresh Kumar, S. Athimoolam, B. Sridhar, XRD, vibrational spectra and quantum chemical studies of an anticancer drug: 6-Mercaptopurine, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.02.104

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XRD, vibrational spectra and quantum chemical studies of an anticancer drug: 6-Mercaptopurine S. Suresh Kumara, S. Athimoolama*, B. Sridharb a

Department of Physics, University College of Engineering Nagercoil, Anna University, Nagercoil 629 004, India. b Laboratory of X-ray Crystallography, Indian Institute of Chemical Technology, 500 007, Hyderabad, India *E-mail: [email protected] Abstract The single crystal of the hydrated anticancer drug, 6-Mercaptopurine (6-MP), has been grown by slow evaporation technique under room temperature. The structure was determined by single crystal X-ray diffraction. The vibrational spectral analysis was carried out using Laser Raman and FT-IR spectroscopy in the range of 3300-100 and 4000–400 cm-1. The single crystal X-ray studies shows that the crystal packing is dominated by N–H...O and O–H...N classical hydrogen bonds leading to a hydrogen bonded ensemble. This classical hydrogen bonds were further connected through O–H...S hydrogen bond to form two primary ring R44(16) and R44(12) motifs. These two primary ring motifs are interlinked with each other to build a ladder like structure. These Ladders are connected through N–H...N hydrogen bond along c-axis of the unit cell through chain C(5) motifs. Further, the strength of the hydrogen bonds is studied through vibrational spectral measurements. The shifting of bands due to the intermolecular interactions was also analyzed in the solid crystalline state. Geometrical optimizations of the drug molecule were done by Density Functional Theory (DFT) using the B3LYP function and Hartree-Fock (HF) level with 6-311++G(d,p) basis set. The optimized molecular geometry and computed vibrational spectra are compared with experimental results which show significant agreement. The natural bond orbital (NBO) analysis was carried out to interpret hyperconjucative interaction and Intramolecular Charge Transfer (ICT). The chemical hardness, electro-negativity and chemical potential of the molecule are carried out by HOMO - LUMO plot. In which, the frontier orbitals has lower band gap value indicating the possible pharmaceutical activity of the molecule. Keywords: Single Crystal XRD, FT-IR, Laser Raman, DFT, NBO and HOMO - LUMO

1

Introduction Cancer is a high proportion death disease in many countries. About two-thirds of them are arising in developing countries. The cancer occurs after normal cells have been transformed into neoplastic cells through alteration of their genetic material and the abnormal expression of certain genes. The cancer can be treated with surgery, radiotherapy and chemotherapy. In which chemotherapy is the best acceptable and unavoidable method for treating cancer, especially, leukemia, lymphomas and testicular cancer. The anticancer drugs play essential role in the development of chemotherapy. 6-mercaptopurine is an antineoplastics agent which has the additional application as immuno-suppressive [1] and anti-inflammatory [2] agent. 6mercaptopurine is used in the treatment of tumors and leukemia [3-5]. It is also used in the treatment of inflammatory bowel problem such as Crohn's disease [6]. The chemotherapeutic activity of 6-mercaptopurine in cancer cells is due to its ability of transform the donor sites into the respective ribosides [7]. Also, the intense interest on the antitumor properties of the molecule is arised due to its coordination behavior through the nitrogen and sulfur donor sites. To understand the mechanism of drug molecule in human cells and tissues, the knowledge about their structure is vital. Mainly the problem involved in the tautomerism of its structure which has four tautomeric form.

Scheme 1 Representation of possible tautomerism in 6-mercaptopurine molecule The above scheme shows the possible proton transfer between the nitrogen atoms in the ring. The existence of tautomerism in the ring is due to the re-arrangement of π-bond in the system. 6-mercaptopurine is also focused for their potent acid-base properties which offer a variety of metal coordination [8]. Metal complexes of 6-mercaptopurine have more anticancer activity than the free ligands [9]. The necessary mechanisms of anti-inflammatory action of 6-mercaptopurine has been explained by the formation of the copper chelate [10]. The single crystal of anhydrous 6-mercaptopurine [11, 12], 6-mercaptopurine

2

monohydrate [13-16], 6-mercaptopurine

hydrochloride [15, 17] and few metal complexes of Copper, cadmium [18], Sodium and Platinum [19] were already reported. The present work was carried out as the re-determination of the molecular structure with single crystal XRD studies with the focus on hydrogen bonding network. Further, theoretical optimization and vibrational analyses were also attempted. From the thorough survey of literature, it is to be identified that the detail discussion about the hydrogen bonding interactions of the drug, complete vibrational assignment and molecular orbital analyses are not found. The vibrational analyses of the molecule are crucial for understanding the strength of the intermolecular forces and delocalization of electron density inside the molecule. The quantum chemical calculations using Hartree Fock (HF) and Density Functional Theory (DFT) methods have been performed to identify various vibrational modes with their wavenumbers and have been correlated with experimental data. The Chemical hardness, Chemical Potential and Electronegativity were calculated by Frontier Molecular Orbitals (FMO) analyses. As the stabilization energy is playing an important role in the biological field, it is calculated using the Natural Bond Orbitals (NBO) analyses. Experimental The single crystals of 6-Mercaptopurine monohydrate (6-MP) have been grown by slow evaporation method under room temperature. The good X-ray quality crystals were obtained within two weeks. The grown crystals are shown in the Fig 1. The density of the crystals were measured by sink and swim method (flotation technique) using a liquid mixture of xylene and bromoform.

The observed densities of the crystals were found to be 1.55 (1) Mg.m-3

respectively. The preliminary crystallographic calculations, i.e., the unit cell parameters of 6-MP and full data collection were done from single-crystal X-ray diffraction done by Bruker SMART APEX CCD area detector diffractometer [20] (graphite- monochromated, MoKα = 0.71073 Å). Crystallographic data, details of data collection and refinement statistics are given in Table 1. The structure was solved by direct methods using SHELXTL/PC [21]. All the non-H atoms were refined anisotropically. All the H atoms were located in difference fourier map and refined iostropically. A Jasco FT-IR spectrometer of model 410 with resolution of 4 cm-1 and scanning speed of 2 mm/sec was used for IR spectral measurements. The samples were prepared using pellet technique with KBr and the spectra were recorded over the range 4000–400 cm-1. The Laser Raman spectrum was recorded in the frequency range of 100-3300 cm-1 using a Renishaw 3

Invia Laser Raman Microscope module. The He-Ne Laser source was operated at 633 nm for the excitation with the power output of 18 mW. Computational Details The geometries, electronic structure for anhydrate 6-Mercaptopurine were carried out theoretically by the 6-311++G(d,p) method on a Intel Core i5/ 3.20 GHz computer using Gaussian 09W [22] program package without any constraint on the geometry optimization [23]. Initial geometry was taken from the single crystal X-ray studies without water molecule and it was minimized (optimized) by Hartee−Fock (HF) method using the 6-311++G(d,p) basis set. These molecular geometries have also been optimized by the Density Functional Theory (DFT) using the Becker's three - parameters exchange functional (B3) [24] in combination with the Lee - Yang - Parr correlation functional (LYP) [25]. It is accepted as a cost-effective approach for the computation of molecular structure, vibrational frequencies and energies of optimized structures. The optimized structural parameters were used in the vibrational frequency calculations at the same level to characterize all stationary points as minima. Then vibrationally averaged nuclear positions of the structure were used for harmonic vibrational frequency calculations resulting in IR and Raman frequencies. Finally, the calculated normal mode vibrational frequencies provide thermodynamic properties also through the principle of statistical mechanics. By combing the results of the GAUSSVIEW program [26] with symmetry considerations, vibrational frequency assignments were made with a high degree of accuracy. There is always some ambiguity in defining internal coordination. However, the defined coordinate from complete set matches quite well with the motions observed using the GAUSSVIEW program. Also the natural Bonding orbital (NBO) calculation was carried out using HF and DFT method with the 6-311++G(d,p) basis set to get more detailed information about the chemical bonds and hydrogen bonding interaction within the molecule. The frontier molecular orbitals (FMO) have been computed and analyzed by HF and DFT method with the 6-311++G(d,p) basis set. Results and Discussion Molecular Structure Analysis Single crystal XRD The molecular structure of 6-MP, a 6-Mercaptopurine and a lattice water molecule in the asymmetric unit, is shown in Fig. 2. The 6-MP was crystallized in monoclinic crystal system with C2/c space group. The unit cell parameters are a=15.360 (2) Å, b = 7.746 (9) Å, 4

c = 12.376 (14) Å and β = 101.526 (3) ̊. The cell parameters are in good agreement with reported data [13, 14]. Though the overall comparison of bond lengths and bond angles are appreciably matching with the reported literature, the crystal structure of the present molecule has better R-factor (2.8% instead of previous 3.8% and 3.4 %) than the previous reports. In imidazole ring, the hydrogen atom attached with the N10 atom rather than the N9 atom which is due to electron transfer around the N10 atom to attain the equilibrium. This result shows the possible tautomerism of the molecule between the two nitrogen atoms (N10↔N9) in the imidazole ring, which is due to the re-arrangement of the π- bond around C5 atom between these two nitrogens.

Optimized molecular geometry The optimized geometry of 6-MP molecule was obtained by both HF and DFT calculations with 6-311++G(d,p) basis set. The optimized structures of 6-MP molecule are shown in Fig. 2 and compared with experimental structure. The optimized structures obtained from DFT and single XRD methods are appreciably matching with the tautomeric form IV (scheme 1). But, in the case of HF method, it corresponds to tautomeric form III. This results shows that the 6-MP has two different protonation sites in the gaseous phase which is due to the intra molecular proton transfer of two nitrogen atoms (N7↔ N8) in the pyrimidine ring. The optimized bond lengths and bond angles of 6-MP are listed in Table 2 with the experimental data. From the table, it is observed that the most of the optimized bond lengths and bond angles are slightly deviated from the experimental counterparts. Because, theoretical calculations were carried out on molecule in gaseous phase whereas the experimental results correspond to molecule in solid crystalline state. However, the overall bond angles and bond distances of B3LYP method correlated well when compared with experimental results. The optimized bond length of C–C in pyrimidine ring fall in the range 1.400 to 1.412 Å for B3LYP/6-311++G(d,p) method, which is in good agreement with the experimental value. The bond length of C4–C6 is 1.400 Å in the DFT method whereas in the HF method the bond length is 1.359 Å which is more deviated (0.038 Å) from the experimental data. The above result shows that the electron delocalization occurs between the two carbon atoms in the aromatic ring. This type of electron delocalization or charge transfer is occurred to make the stable structure. The two C–N bonds of the pyrimidine ring are observed to be 1.372/1.276 Å and 1.296/1.352Å for DFT/HF methods respectively. The calculated bond length of C3-N7 bond is 1.276 Å in the HF method. It shows the negative deviation (~-0.079 Å) from the experimental result. This result 5

confirms that C–N is double bond due to more charge transfer from the carbon atom to nitrogen atom. Another C3–N8 bond has positive deviation (~0.043 Å) from the experimental value at HF method. It is illustrated that electron delocalization is occurred between carbon and nitrogen atoms. This charge transfer was obtained between nitrogen to carbon because nitrogen (N7) has more electronegativity than the carbon atom in HF method. Hence, the present study features two possible tautomers of the 6-MP. This large number of electron delocalization or charge transfer property around the ring stimulates the more bioactivity of the molecule.

Hydrogen bonding features Intermolecular forces play an important role in the formation of Bio-molecular systems like, DNA, RNA etc, through hydrogen bonds. Studies of hydrogen bond behaviour of a drug molecule give the significance awareness for the pharmaceutical activity. The present molecule has nitrogen and oxygen sites as Donor (or) acceptors in the crystalline state. The crystal structure is stabilized through a three dimensional hydrogen bonding network formed through N–H...O, N–H...N, O–H..N and O–H...S hydrogen bonds [27]. The packing diagram of 6-MP shows the intricate three dimensional hydrogen bonding network with different hydrophilic layers (Fig.3). These layers are formed by the intermolecular hydrogen bond between 6-Mercaptopurine and lattice water molecule. The water molecule is making exclusive hydrogen bonding interactions with the drug molecules. The water O atom act as the acceptor for the hydrogen bond with N atom of the pyrimidine ring and donor for the hydrogen bonds with N atoms of the imidazole ring. These interaction makes an infinite chain C22(8) motif running along the b-axis of the unit cell. Also, the N atom of the pyrimidine ring and the N atom of the imidazole ring were connected via., N–H...N bonds form another chain C(5) motif, which is extending along the c-axis of the unit cell. The combination of these two primary chain motifs form a ring R88(31) motif (Fig. 4 (a)). The molecular aggregation formed through chain C22(8) motif construct two parallel layers in the unit cell. These two layers are cross-linked with O–H...S hydrogen bonds, form a ring R44(16) motif. This ring motif leads to a cavity with the approximate dimension of 8.566 × 3.654 × 4.377 Å3 (Fig. 4(b)). Another centrosymmetric ring R44(12) motif is observed with the N–H...O and O–H...S hydrogen bonds (Fig. 4(c)). These two secondary ring motifs generate a ladder-like structure extending along the diagonal of the ab-axis. This ladder is connected through the linear N–H...N hydrogen bonds, to produce another ladder resembling 6

structure. These adjacent ladders in the crystal structure are connected by 21 screw axes parallel to the c-axis and inclined at an angle of 61.9 (1)° to each other (Fig. 4(d)). These ladders are parallel to the (2 2 1) and (2 2 1) plane of the unit cell which give strong x-ray diffraction peaks.

Mulliken atomic charge analysis The charge distribution on the molecule has an important role in the application of quantum chemical calculation of molecular system. The charge distribution of a molecule has significant influence on dipole moment, polarizability, electronic structure and vibrational modes [28]. The calculated Mulliken charges at the HF and DFT levels with the 6-311++G(d,p) atomic basis sets are given in Table 4 and the corresponding comparison diagram is shown in Fig 5. The charge distribution of drug molecule shows that the carbon atoms with hydrogen (C3 and C5) are positive, because those atoms are surrounded by two electronegative nitrogen atoms. These two electronegative atoms are withdrawing the electron charges from the carbon and become negative. The remaining carbon atoms (C2 and C4) are more negative charge, which is due to the more electron delocalization around the aromatic ring. All the hydrogen atoms are positive charge. Moreover, it is observed shows that the H1 (0.333 e and 0.365 e for B3LYP and HF method respectively) and H14 (0.323 e and 0.371 e for B3LYP and HF method respectively) atoms have higher positive charges than the other hydrogen atoms. This is due to the presence of attached electronegative nitrogen atoms (N7 and N10). Further, these hydrogens are playing crucial role in the formation of hydrogen bonding network in crystalline state.

Vibrational analyze of 6-Mercaptopurine (6-MP) The single crystal analysis of the 6-MP obviously predicts the molecular structure, crystal symmetry, atomic vibrations (via thermal displacement parameters) and different types of bonds. But, the vibrational spectroscopy can only give valuable information regarding the bonding forces and strength of intermolecular bonds in addition to symmetry of the individual molecule. The effect of hydrogen bond on vibration modes is vital in crystal environment [29]. The strong, normal and weak hydrogen bonds will cause downshifting of stretching mode of vibrations and up shifting of deformation modes with different numerical values of the wave numbers depends upon their strength (strong/normal/weak). Normally, the shifts in stretching modes are greater 7

than the deformation modes which indicate the more linear distortion than the angular distortion. The assignment of the vibrational spectra was carried out on the basis of a set of locally symmetrized vibrational modes that can be easily correlated with characteristic group wavenumbers. Excluding the lattice water molecule, 6-MP has 14 atoms and 36 normal modes of vibrations. The experimental FTIR and Laser Raman spectra of the compound are shown in the Figs.6 and 7 respectively. The calculated harmonic vibrational frequencies by DFT/B3LYP and HF have been compared with experimental results. The assignments are shown in Table 4. The 6-MP molecule consists of two rings viz., pyrimidine and imidazole. These two rings have functional groups such as C-H, N-H, C-C, C=S and C=N. The N-H stretching vibrations of aromatic ring always occur in the wavenumber region 3450-3250 cm-1, which is the region for identification of the structure. The present structure has one N-H band of the pyrimidine ring, which is observed as very strong peak at 3432 cm-1 in the IR spectra and 3541 cm-1 in Raman spectra. This corresponding stretching vibration is computed at 3576 cm-1 in B3LYP method and 3869 cm-1 in HF level (mode 35). This result shows that HF level more deviated (~ 437 cm-1) from the experimental value due to the shifting of N-H band from ortho position to para position. The N-H stretching vibration of the imidazole ring is observed at 3623 cm-1 in B3LYP level and 3874 cm-1in HF level (mode 36), it is not present in experimental results. The C-H stretching vibration in the heterocyclic aromatic pyrimidine ring is observed in the region 3100 - 3000 cm-1. Generally in this region the bands are not affected substantially by the nature of the substituent. The medium band at 3001 cm-1 in IR spectra and the corresponding Raman spectra at 3104 cm-1 are observed due to the C-H stretching vibration of the pyrimidine ring. This same vibration is computed at 3180 cm-1 in B3LYP level. More deviation (~345 cm-1) occurs in HF level due to the electron delocalization of the aromatic ring. The medium band appearing at 3091 cm-1 in the FTIR spectrum is assigned to C-H stretching vibration of the imidazole ring. The same vibration predicted at 3245 cm-1 in B3LYP level and 3411 cm-1 in HF level (mode 34). A broad peak is observed in the wavenumber region 3000-2600 cm-1 in IR spectra, which is due to the O-H stretching vibration of the water molecule. This stretching vibration is not observed in the theoretical calculation, because theoretical calculation was carried out without lattice water molecule. In the medium wave number region (2000-1000 cm-1), most of the bands are assigned to the in-plane N-H and C-H bending vibrations. The strong peak observed around 1612 cm-1 in IR spectra and 1540 cm-1 in Raman,

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are due to the in-plane N-H and C-H bending vibrations. This vibration is computed as very strong peaks at 1542 cm-1 in B3LYP method and 1663 cm-1 in HF method. This strong peak occurred due to the more electron delocalization around the aromatic ring. The C-H wagging vibration is computed at 1395 cm-1 in B3LYP method, 1509 cm-1 in HF level and is not observed experimentally. The lower wavenumber region (1000 cm-1 - 200 cm-1) was allotted to the N-H and C-H out-of-plane bending vibrations. In this region, the experimental and theoretical results are well correlated to each other.

Natural bond orbital The NBO analysis of Weinhold et.al [30, 31] is a helpful tool for understanding the hydrogen bonding interactions and delocalization of electron density from occupied donor and unoccupied acceptor within the molecule. It is also allow us to estimate the energy of the molecule with the same geometry in the absence of electronic delocalization. The stabilization of orbital interaction is proportional to the energy difference between interacting orbital and it plays an important role in the biological field. The stabilization energy E(2) associated with i (donor) → j(acceptor) delocalization is estimated by the following equation, E(2) = qi

,

(1)

 

where qi is the donor orbital occupancy, εj and εi are diagonal elements orbital energies and F(i,j) is the off diagonal NBO Fock matrix element. NBO analysis has been performed in order to determine the intermolecular interaction, rehypridization and hyberconjucation of electron density within the molecule using second order perturbation theory, which is presented in Table 5. The percentages of s and p orbitals on each natural atomic hybrid of the NBO are tabulated in Table 6. The large stabilization energy shows more intensive interaction between the donors and acceptors. The hyperconjugative interactions are formed by the orbital overlap between the π (C3-N8) bonding orbital to π*(C4-C6) anti-bonding orbital, this interaction can be increased in electron density in antibonding orbital. This interaction leads to the enormous stabilization energy 20.3 kJ/mol. Another strong electron delocalization was occurred in the interaction between the bonding orbital π (C4-C6) and anti bonding orbital π*(C2-S11), which leading the larger stabilization 37.34 kJ/mol. This interactions are formed by orbital overlap between π (C4-C6) and π*(C2-S11) bond orbital in aromatic ring results intra molecular charge

9

transfer causing stabilization of the system. This strong stabilization energy denotes the larger delocalization.

HOMO and LUMO band gap The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbitals are named as frontier molecular orbitals (FMOs). FMOs play important role in the intramolecular charge transfer between molecules as well as in electronic spectra of molecule [32]. The energy gap between HOMO and LUMO determines the chemical hardness, electronegativity and chemical potential of a molecule, as present in Table 7. The chemical hardness is a good indicator of the chemical stability. The chemical hardness value of 6-MP is small in DFT calculation, when compared with HF level. It shows that 6-MP is a soft molecule and also have small energy gap. The small energy gap is associated with a high chemical reactivity and low kinetic stability. The frontier orbital of HOMO and LUMO plots for the 6-MP is shown in the Fig. 8. The calculated energy values of HOMO and LUMO in DFT/ HF levels are -0.232 au/ -0.298 au and -0.077 au/-0.041 au, the energy gap value between the frontier molecular orbitals are 0.155 au/0.257 au. The DFT calculation shows that 6-MP has the lower value of frontier orbital energy gap. The lower value of HOMO and LUMO energy gap explains that large number of intramolecular charge transfer takes place within the molecule. This large charge transfer influences the biological activity of the molecule. Conclusion In the present study, the molecular structure of the crystallized material has been identified by the single crystal X-ray diffraction. This studies shows that the crystal packing is dominated by N–H...O, N–H...N, O–H...N and O–H...S classical hydrogen bonds leading to a hydrogen bonded ensemble. The intricate three dimensional hydrogen bonding interactions connect the drug molecules in a ladder-like fashion through lattice water molecule. The theoretical study was attempted to predict the optimized geometry and computed vibrational spectra by HF and DFT levels with 6-311++G(d,p) basis set. The shifting of vibrational bands due to the intermolecular hydrogen bonds was analyzed with experimental and theoretically spectra. The comparison of experimental and theoretical molecular geometries reiterates the two tautomeric forms of the drug. Natural bond orbital analysis indicates the possible intermolecular interactions which is in 10

agreement with the experimental intermolecular hydrogen bonding interactions. Natural bond orbital analysis of the molecule confirms that the intra molecular charge transfer caused by π electron cloud movement from donor to acceptor must be responsible for bioactivity of the molecule. The value of the energy gap between the HOMO and LUMO give the information about chemical hardness, electronegativity and chemical potential of the molecule. Acknowledgements: The authors grateful to Department of Science and Technology, SERB for the financial support of this work in the form of Fast track Research Project scheme.

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Hratchian, A.F.Izmaylov, J.Bloino, G. Zheng, J.L.Sonnenberg, M. Hada, M.Ehara, K. Toyota, R.Fukuda, J.Hasegawa, M.Ishida, T.Nakajima, Y.Honda, O.Kitao, H.Nakai, T. Vreven, J.A.Montgomery, Jr. JE.Peralta., F.Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N.Staroverov, R.Kobayashi, J.Normand, K.Raghavachari, A.Rendell, J.C. Burant, S.S.Iyengar, J. Tomasi, M. Cossi, N.Rega, J.M.Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C.Adamo, J.Jaramillo, R. Gomperts, R.E. Stratmann, O.Yazyev, A.J. Austin, R. Cammi, C.Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma,V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S.Dapprich, A.D. Daniels,O. Farkas, J.B.Foresman, J.V.Ortiz, J.Cioslowski, D.J.Fox. Gaussian 09, Revision A.1, Gaussian, Inc., Wallingford CT, (2013). [23] H.B. Schlegel, J. Comput. Chem. 3 (1982), 214-218. [24] P. Hohenberg, W.Kohn, Phys.rev. B 136 (1964), 864-871. [25] A.D. Becke. J.Chem. Phys. 98 (1993), 5648-5653. [26] R. Dennington, T.Keith and J. Millam, J. Semichem Inc., Shawnee Mission KS, (2009). [27] L. L. Xu, J.M. Chen, Y. Yan, and T.B. Lu, Cryst. Growth Des. (2012), 6004-6011. [28] I. Sidir, Y.G. Sidir, M. Kumalar, E. Tasal, J. Mol. Struct. 964 (2010), 134–138. [29] G. Ploug-Sørensen, E.K. Andersen. Acta Cryst. C39 (1983), 112–114. [30] F. Weinhold, C.R. Landis, Valency and Bonding: A Natural Bond Orbital Donor– Acceptor Perspective, Cambridge University Press, Cambridge, (2005). [31] F. Weinhold, Natural bond orbital methods, in: P.V.R. Schleyer, N.L. Allinger, T.Clark, J. Gasteiger, P.A. Kollman, H.F. Schaefer III, P.R. Schreiner (Eds.),Encyclopedia of Computational Chemistry, John Wiley & Sons, Chichester, UK, (1998). [32] I. Fleming, Frontier Orbitals and Organic Chemical Reactions, John Wiley & Sons, New York, (1976).

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Table 1 Crystallographic data and Structure refinement Parameters for the 6-MP Empirical formula

C5 H6 N4 O S

Formula weight

170.2

Temperature

293(2) K

Wavelength

0.71073 Å

Crystal system, space group

Monoclinic, C2/c

Unit cell dimensions

a = 15.360 (2) Å b = 7.746 (9) Å beta = 101.526 (3) ̊ c = 12.376 (1) Å

Volume

1442.9 (3) Å3

Z, Calculated density

8, 1.567 Mg/m3

Absorption coefficient

0.390 mm-1

F(000)

704

Crystal size

0.21 x 0.23 x 0.25 mm

Theta range for data collection

2.71 to 25.00 ̊

Limiting indices

-18 ≤ h ≤ 18, -9 ≤ k ≤ 9, -14 ≤ 1 ≤ 14

Reflections collected / unique

6374 / 1276 [R(int) = 0.0384]

Completeness to theta = 25.00

100%

Refinement method

Full-matrix least-squares on F2

Data / restraints / parameters

1276 / 0 / 124

Goodness-of-fit on F2

1.068

Final R indices [I>2sigma(I)]

R1 = 0.0289, wR2 = 0.0818

R indices (all data)

R1 = 0.0299, wR2 = 0.0828

Largest diff. peak and hole

0.247 and -0.150 e.Å-3

CCDC No

CCDC 998661

14

Table 2 Comparison of Optimized molecular Geometrical parameters of 6-MP

Parameter

HF/6Experimental 311++G(d,p)

DFT/6311++G(d,p)

Bond Length (Å) C2-N7

1.379 (2)

1.383

1.393

C2-C6

1.409 (2)

1.421

1.412

C2-S11

1.678 (2)

1.653

1.669

C3-N8

1.308 (2)

1.352

1.296

C3-N7

1.353 (2)

1.276

1.372

C3-H12

0.939 (2)

1.076

1.084

C4-N8

1.368 (2)

1.361

1.364

C4-N9

1.370 (2)

1.347

1.368

C4-C6

1.395 (2)

1.359

1.400

C5-N9

1.328 (2)

1.305

1.316

C5-N10

1.350 (2)

1.336

1.367

C5-H4

0.915 (1)

1.070

1.079

C6-N4

1.373 (2)

1.374

1.369

N7-H1

0.840 (2)

-

1.012

N10-H14

0.850 (2)

0.995

1.010

O1W-H1W

0.850 (3)

-

-

O1W-H2W

0.830 (3)

-

-

Bond Angle ( ͦ ) N7-C2-C6

110.45 (1)

113.68

109.15

N7-C2-S11

122.55 (1)

123.11

123.05

C6-C2-S11

126.99 (1)

123.20

127.79

N8-C3-N7

125.37 (1)

126.24

125.16

N8-C3-H12

120.30 (1)

115.22

119.85

N7-C3-H12

114.30 (1)

118.55

114.98

N8-C4-N9

125.76 (1)

128.51

126.36

N8-C4-C6

124.04 (1)

118.94

123.19

N9-C4-C6

110.20 (1)

112.53

110.44

N9-C5-N4

113.57 (1)

113.52

113.29

15

N9-C5-H13

123.60 (1)

124.12

124.95

N10-C5-H13

122.90 (1)

122.35

121.75

N10-C5-C3

105.69 (1)

103.98

105.38

N10-C6-C2

132.39 (1)

132.85

131.29

C4-C6-C2

121.91 (1)

123.16

123.32

C2-N7-C3

125.23 (1)

121.30

125.22

C3-N7-H14

115.80 (1)

-

119.14

C2-N7-H14

119.00 (1)

-

115.62

C3-N8-C4

112.98 (1)

116.65

113.93

C5-N9-C4

104.23 (1)

103.45

104.61

C5-N10-C6

106.30 (1)

106.5

106.27

C5-N10-H14

127.00 (1)

128.15

128.40

C6-N10-H14

126.70 (1)

125.33

125.32

Table 3 Hydrogen Bonding geometry of 6-MP (D-H) (Å)

(H...A) (Å)

(D...A) (Å)

(D–H...A) (°°)

N7– H14...O1W

0.84 (2)

1.94 (2)

2.76 (2)

172 (2)

N10– H14...N8 (i)

0.85 (2)

2.07 (1)

2.92 (2)

173 (2)

O1W – H1W...N9 (ii)

0.85 (2)

1.98 (3)

2.81 (18)

169 (1)

O1W – H2W...S11 (iii)

0.83 (2)

2.58 (3)

3.38 (16)

164 (2)

D-H...A (Å,°°)

Equivalent Positions: i. ii. iii.

x,-y+2, z-1/2 x+1/2, y-1/2, +z –x+2, -y+1, -z+1

16

Table 4 Mulliken atomic charges for optimized geometry of 6-MP

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

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

H1

0.365

0.333

C2

-0.376

-0.032

C3

0.322

0.175

C4

-0.099

-0.739

C5

0.287

0.261

C6

0.736

0.435

N7

-0.122

-0.081

N8

-0.524

-0.145

N9

-0.289

-0.134

N10

-0.402

-0.202

S11

-0.573

-0.629

H12

0.157

0.218

H13

0.147

0.218

H14

0.371

0.323

Atom label

17

Table 5 Experimental, HF and B3LYP levels computed vibrational frequencies (cm-1) obtained for 6-MP Mode Number

Experimental FT-IR

Raman

HF/6-311++G(d,p) νcal

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

a IR

b Raman

I

νcal

I

a IR

b Raman

I

Assignment

I

1

117

1.32

0.16

130

11.77

0.04

Forbidden

2

201

0.8

0.12

198

9.4

0.01

Forbidden

3

234

5.6

5.44

211

10.02

6.31

ω (N-H)

4

322

30.89

0.04

269

6.17

0.28

Forbidden

467

20.02

9.1

433

4.35

13.77

β (N-H)+β (C-H)

510

34.8

0.94

508

6.15

1.44

β (N-H)+β (C-H)

7

521

17.22

0.61

541

34.52

1.79

γ (N-H)+ γ (C-H)

8

593

91.97

0.26

565

45.65

0.37

γ (N-H)+ γ (C-H)

610

93.95

0.34

587

2.11

11.34

Planar ring distortion

10

622

0.13

5.06

597

28.29

0.9

γ (N-H)+ γ (C-H)

11

694

16.39

0.81

670

1.39

0.29

γ (N-H)+ γ (C-H)

724

3.18

17.28

682

0.69

17.07

Planar ring distortion

13

735

1.33

0.56

691

60.74

0.67

γ (N-H)+β (C-H)

14

823

5.46

2.26

776

0.26

0.84

Forbidden

953

52.95

2.8

866

10.35

0.52

γ (C-H)+ Planar distortion

16

1028

4.19

1.59

872

37.39

2.86

Planar ring distortion + γ (C-H)

17

1037

25.22

11.61

926

4.59

0.45

Planar ring distortion + γ (C-H)

1084

0.8

0.05

959

3.39

9.76

Planar ring distortion + γ (C-H)

1090

66.74

11.03

1022

60.01

12.24

Planar ring distortion

1154

196.43

15.48

1089

32.64

4.2

5 6

9

12

15

436 m 499 m

586 m

647 m

677s

867 m

18

933 m

19

1006 s

20

588 m

1032 w

18

β (N-H)+β (C-H)

Mode Number

Experimental FT-IR

Raman

HF/6-311++G(d,p) νcal

a IR

b Raman

I

I

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

b Raman

I

νcal

Assignment

I

21

1218

37.54

13.68

1104

12.1

5.68

ω C-H

22

1300

166.68

3.23

1191

153.44

5.92

β (N-H)+β (C-H)

1278 vs

1329

131.71

21.39

1230

5.69

24.87

β (N-H)+β (C-H)

24

1352 s

1408

71.49

17.39

1310

13.2

31.71

β (N-H)+β (C-H)

25

1376 m

1452

106.01

85.75

1355

39.76

31.21

β (N-H)+β (C-H)

1509

136.46

146.68

1395

141.9

178.37

ω C-H

1535

168.23

48.94

1410

3.43

14.26

β (N-H)+β (C-H)

28

1597

81.21

9.83

1440

42.12

12.81

β (N-H)+β (C-H)

29

1617

120.56

38.84

1476

29.35

11.97

Planar ring distortion

1540 m

1663

18.75

118.09

1542

58.58

56.17

β (N-H)+β (C-H)

1594 m

1771

157.93

26.97

1582

249.83

66.87

β (N-H)+β (C-H)

1801

869.06

67.44

1640

262.38

3.23

β (N-H)+β (C-H)

3346

13.86

126.54

3180

4.61

138.72

ν C-H (pyrimidine)

23

1212 s

26 27

30

1404 vs

1612 s

31

1409 m

32

1847 m

33

3001 m

34

3091 m

3411

2.04

94.81

3245

2.33

125.45

ν C-H (imidazole)

35

3432 vs

3869

203.6

111.14

3576

60.05

83.66

ν N-H (pyrimidine)

3874

142.55

30.35

3623

86.69

80.46

ν N-H (imidazole)

36

3104 s

w-weak; vs - very strong; m- medium; ν- Stretching;β- in plane bending; γ- out -of- plane bending; ω-Wagging

19

Table 6 Second order perturbation theory analysis of Fock Matrix in NBO for 6-MP

Donor (i)

Type

ED (e)

E (2)a

E (j)-E (i)b

F (i,j)c

(kJ/mol)

(a.u)

(a.u)

2.48

1.52

0.055

1.03

1.04

0.029

4.84

1.28

0.071

C4-N9

1.2

1.23

0.034

C5-N10

1.03

1.19

0.031

C6-N10

2.46

1.17

0.048

H1-N7

2.63

1.63

0.058

C2-C6

0.74

1.37

0.029

1.79

1.29

0.043

C3-H12

0.55

1.48

0.025

C6-N10

4.56

1.28

0.068

C2-C6

2.17

1.21

0.046

C2-N7

0.52

1.11

0.022

Acceptor (j)

Type

ED (e)

H1-N7 C2-S11 C2-C6

C2-N7

C2-S11

C3-N7

σ

σ

1.977

1.985

C4-C6

C3-N7

σ*

0.046

0.068

σ*

σ

1.982

C3-N7

σ*

0.013

3.17

1.14

0.054

π

1.969

C4-C6

π*

0.504

7.78

0.29

0.047

H1-N7

1.93

1.63

0.05

C2-N7

2.13

1.26

0.047

3.17

1.15

0.054

0.81

1.41

0.03

σ

1.987

C2-S11

0.044

σ*

C3-N8 20

C3-N8

σ

1.983

C3-H12

0.51

1.49

0.025

H1-N7

0.77

1.63

0.032

C3-N7

0.78

1.3

0.029

2.01

1.49

0.049

0.79

1.34

0.029

5.18

1.34

0.075

C4-C6

20.3

0.34

0.08

H1-N7

0.63

1.4

0.027

C2-N7

5.31

1.03

0.067

1.73

1.18

0.04

C4-N8

3.89

1.11

0.059

C2 -C6

4.75

1.23

0.068

C2-S11

4.46

1.01

0.06

1.68

1.19

0.04

C4-N9

1.1

1.19

0.032

C6-N10

0.86

1.14

0.028

N10-H14

3.2

1.52

0.062

37.34

0.21

0.082

C3-N8

11.35

0.26

0.05

C5-N9

13.79

0.26

0.054

C2-C6

2.92

1.31

0.056

C3-N8

1.38

1.34

0.039

C3-H12

0.009

σ*

C4-N8 π

C3-H12

σ

σ

1.840

1.980

1.973

C4-C6

π

1.624

C4-N9

C3-N8

C4-N8

C2-S11

0.276

π*

0.018

σ*

0.045

σ*

0.424

π*

21

C4-N9

C5-N9

σ

σ

1.982

1.981

C4-N8

1.45

1.27

0.038

C 5-N10

1.02

1.24

0.032

C5-H13

3.33

1.48

0.063

C6-N10

0.77

1.22

0.027

C4-N8

6.99

1.29

0.085

2.05

1.51

0.05

1.83

1.62

0.049

23.78

0.32

0.084

C2-C6

5.07

1.35

0.074

C4-N8

0.84

1.31

0.03

0.79

1.53

0.031

C6-N10

2.01

1.26

0.045

N10-H14

2.12

1.64

0.053

C4-N9

2.35

1.12

0.046

1.61

1.17

0.039

C6-N10

2.49

1.07

0.046

C2-C6

2.91

1.34

0.056

C2-N7

0.87

1.23

0.03

C4-C6

1.38

1.36

0.039

C4-N8

3.39

1.3

0.059

0.57

1.35

0.025

1.52

1.27

0.039

C5-H13

0.026

σ*

0.007

σ*

N10-H14 π

C5-N10

C5-H13

C6-N10

σ

σ

σ

1.826

1.986

1.985

1.982

C4-C6

C5-H13

C5-N9

C5-N9

0.344

π*

0.031

σ*

0.015

σ*

0.024

σ*

C5-N10

22

N10-H14

a

σ

1.988

C5-H13

2.08

1.51

0.05

N10-H14

2.66

1.62

0.059

C4 -C6

1.15

1.35

0.035

1.22

1.34

0.036

C5-N0

1.14

1.26

0.034

C6-N10

2.00

1.24

0.045

C5-N9

0.012

σ*

E(2) means energy of hyperconjugative interactions

b

Energy difference between donor and acceptor i and j NBO orbitals

c

F(i,j) is the Fock matrix element between i and j NBO orbitals

23

Table 7 Percentage of s and p character on each natural atomic hybrid of the NBO

Bond (A-B)

Energy (KJ/mol)

EDA (%)

EDB (%)

S (%)

P (%)

H1-N7

-0.813

31.33

68.67

99.98

0.02

31.07

68.88

35.99

63.95

38.59

61.39

27.73

72.15

35.7

64.27

36.16

63.73

18.85

80.51

0.01

99.84

0.02

99.76

30.29

69.62

33.07

66.89

34.95

64.98

34.01

65.89

0.01

99.85

0.01

99.83

34.75

65.19

99.94

0.06

35.91

64.04

33.23

66.73

0.02

99.95

0.02

99.97

32.61

67.31

33.53

66.38

31.05

68.86

32.41

67.49

33.05

66.86

31.63

68.26

C2-C6 C2-N7 C2-S11 C2-S11 C3-N7 C3-N8 C3-N8 C3-H12 C4-C6 C4-C6 C4-N8 C4-N9 C5-N9

-0.763 -0.870 -0.719 -0.299 -0.875 -0.879 -0.350 -0.647 -0.731 -0.296 -0.835 -0.812 -0.835

48.58 37.34 59.55 32.22 37.18 42.03 39.63 58.13 48.01 45.16 41.96 42.29 42.34

24

51.42 62.66 40.45 67.78 62.82 57.97 60.37 41.87 51.99 54.84 58.04 57.71 57.66

Bond (A-B)

Energy (KJ/mol)

EDA (%)

EDB (%)

S (%)

P (%)

C5-N9

-0.333

40.03

59.97

0.01

99.82

0.01

99.82

29.25

70.65

32.24

67.72

37.6

62.35

99.94

0.00

28.00

71.92

32.32

67.65

35.26

64.69

99.98

0.00

C5-N10

-0.856

C5-H13

-0.662

C6-N10

-0.840

N10-H14

-0.832

37.15 58.05 38.88 69.11

62.85 41.95 61.12 30.89

Table 8 Calculated energy values of 6MP HF/6311++G(d,p)

DFT/6311++G(d,p)

EHOMO

-0.298

-0.232

ELUMO

-0.041

-0.077

∆(ELUMO- EHOMO)

0.257

0.155

Chemical Hardness (h)

0.128

0.077

Electronegativity (χ)

-0.169

-0.155

Chemical Potential (µ)

0.169

0.155

Parameters (au)

25

Figure Caption Fig. 1. Grown crystal of 6-MP Fig. 2. (a) The molecular structure of the 6-MP with atom numbering scheme and 50% probability displacement ellipsoids and (b) Optimized Structure with atomic numbering for HF (c) Optimized Structure with atomic numbering for DFT Fig. 3. Packing diagram for 6-MP viewed along the c axis. H-bonds are shown as dashed lines. Fig. 4 (a). Molecular aggregations formed through 6MP and water molecule showing ring and chain motifs. Hydrogen bonds are shown dashed lines. Fig. 4 (b). O1W–H1W...N3 and O–H2W...S1 hydrogen bond formed R44(16) motif with a cavity

of approximate area 8.566 × 3.654 × 4.377 Å3 Fig. 4 (c). Centrosymmetric ring R44(12) ring motif of N1-H1...O1W and O1W–H2W...S1 hydrogen

bonds Fig. 4 (d). Ladder like structure formed by alternating R44(16) and R44(12) ring motifs Fig. 5. The atomic charges of the optimized molecular structures for 6MP by (a) HF and (b) B3LYP levels Fig.6. Comparative representations of FT-IR spectra for 6MP Fig. 7. Comparative representations of Raman spectra for 6MP Fig. 8. (a) Molecular orbitals and energies for the HOMO and LUMO of 6MP in DFT and (b) Molecular orbitals and energies for the HOMO and LUMO of 6MP in HF

26

28

HIGHLIGHTS •

Single crystal of the hydrated anticancer drug, 6-Mercaptopurine (6-MP), has been grown by slow evaporation technique.

•

The crystal and molecular structure identified by single crystal XRD

•

Hydrogen bonding interactions connect the drug molecules in a ladder-like fashion through lattice water molecule

•

Theoretical calculation was attempted by the HF and DFT method

•

Experimental and theoretical molecular geometries reiterate the two tautomeric forms of the drug

•

The chemical hardness, electro-negativity and chemical potential of the molecule were carried out by HOMO - LUMO plot

27

29