An Insight into Prototropism and Supramolecular Motifs in Solid-State

Article pubs.acs.org/JPCB

An Insight into Prototropism and Supramolecular Motifs in SolidState Structures of Allopurinol, Hypoxanthine, Xanthine, and Uric Acid. A 1H−14N NQDR Spectroscopy, Hybrid DFT/QTAIM, and Hirshfeld Surface-Based Study Jolanta Natalia Latosińska,*,† Magdalena Latosińska,† Janez Seliger,‡,§ Veselko Ž agar,‡ and Zygmunt Kazimierczuk∥ †

Faculty of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznań, Poland “Jozef Stefan” Institute, Jamova 39, 1000 Ljubljana, Slovenia § Faculty of Mathematics and Physics, University of Ljubljana, Jadranska 19, 1000 Ljubljana, Slovenia ∥ Institute of Chemistry, Warsaw University of Life Sciences, 159C Nowoursynowska St., 02-787 Warsaw, Poland ‡

ABSTRACT: Allopurinol (1,5-dihydro-4H-pyrazolo [3,4-d]pyrimidin-4-one), the active pharmaceutical ingredient (API) of the drugs applied for the treatment of gout and tumor lysis syndrome, recently discovered to have multifaceted therapeutic potential, and hypoxanthine which is a naturally occurring purine have been studied experimentally in the solid state by 1H−14N NMR-NQR double resonance. Twelve 14 N resonance frequencies have been detected at 295 K and assigned to two pairs of two kinds of nitrogen sites (N and NH) in each compound. The experimental results are supported by and interpreted with the help of quantum theory of atoms in molecules (QTAIM)/density functional theory (DFT) calculations. The factors, such as the substituent effect, in particular the shift of nitrogen from position 7 (as in hypoxanthine) to position 8 (as in allopurinol), hybridization, possible prototropic tautomerism, and the pattern of intermolecular bonding, have been taken into account in 1H−14N NMR-NQR spectra interpretation. This study demonstrates the advantages of combining NQR, DFT/QTAIM, and Hirshfeld surface analysis to extract detailed information on electron density distribution and complex H-bonding networks in crystals of purinic type heterocycles, relevant in pharmacological processes. In the absence of X-ray data for xanthine, the NQR parameters supported by DFT/QTAIM calculations and Hirshfeld surface analysis were proved to be valuable tools for clarifying the details of crystalline packing and predicting an unsolved crystalline structure of xanthine. The influence of a decrease in purine ring conjugation level upon oxidation on the biological activity of allopurinol, a xanthine oxidase (XO) enzyme inhibitor, which blocks the conversion of hypoxanthine to xanthine and subsequently xanthine to uric acid, is also discussed.

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INTRODUCTION Development of a new drug can take 10 years and more and costs over a billion dollars. The old drugs and drug-like compounds awoke growing interests for new applications. The idea of repositioning and repurposing is one of the most popular in the drug business. For this reason, detailed structural investigation of old drugs could be inspiring for novel applications on the basis of molecular recognitions of new targets. In this study, we describe molecular properties of allopurinol (ALP)a potent xanthine oxidase inhibitor1,2 and three ketopurineshypoxanthine (HPX), xanthine (XAN), and uric acid (UCA)which are substrates and products of purine catabolism, catalyzed by xanthine oxidase (XO). The choice of allopurinol was not coincidental because, besides the long-known therapeutical profile, numerous new activities of this substance have been discovered.3−6 Allopurinol (ALP, 1,5-dihydro-4H-pyrazolo [3,4-d]pyrimidin-4-one, trade name Zyloprim) is a structural isomer © 2014 American Chemical Society

of the natural purine base hypoxanthine (HPX), Figure 1, invented by G. B. Elion and G. H. Hitchings in 19567 during the search for antimetabolite antineoplastic agents. ALP inhibits xanthine oxidase (E.C. 1.2.3.2, XO) enzyme, which catalyzes the conversion of hypoxanthine to xanthine and subsequently xanthine to uric acid, Figure 1. Overactivity of XO causes overproduction of uric acid which results in hyperuricemia in primary or secondary gout. ALP inhibits this enzyme, blocks the production of uric acid, and thus reduces high levels of uric acid in blood and serum.8 The fundament of the mechanism of its activity is that ALP, similarly to hypoxanthine or xanthine, can bind to XO, but unlike hypoxanthine and xanthine it cannot be oxidized, because N(7) is shifted to position 8. In addition to preventing uric acid production, inhibition of XO Received: May 17, 2014 Revised: July 26, 2014 Published: July 31, 2014 10837

dx.doi.org/10.1021/jp504871y | J. Phys. Chem. B 2014, 118, 10837−10853

The Journal of Physical Chemistry B

Article

Figure 1. Chemical structure of allopurinol, hypoxanthine, xanthine, and uric acid. The applied and canonical (purine type) numbering of atoms in a molecule is indicated.

and nonpolar media27 accompanied by the presence of nine different canonical tautomeric forms resulting from the prototropic (pyrazole moiety) and lactam−lactim (pyrimidine moiety) tautomerism clearly indicates the significance of proton transfer, which is a very important factor in biochemical and pharmacological research. In the gas phase, among the lowenergy isomers of allopurinol, the oxo-N(1)H,N(8)H is higher in energy by at least 14.8 kJ/mol than any other tautomer.28 On the other hand, in DMSO as a polar aprotic solvent, the coexistence of oxo-N(1)H,N(9)H and oxo-N(1)H,N(8)H tautomers of ALP has been suggested by 13C NMR (nuclear magnetic resonance) spectra.29 Hence, the thermal population of all hydroxy tautomers is very low and these forms would not be detectable by the 1H−14N NQDR; thus, they can be ruled out from further analysis. While the predominance of the ketonic form in solids was evident from the Raman and IR spectra, revealing an intense band attributable to the carbonyl stretching mode at ν− ≥ ν0 were calculated from45

and solid effect dips, there is also a strong dip at ν = 600 kHz (ν = 3νH) in the spectrum measured at νH = 200 kHz. The analysis of the solid effect spectra shows the presence of six 14N NQR frequencies690, 790, 840, 1060, 1630, and 1750 kHzwhich can be combined into two triplets (ν+, ν−, ν0): N(1) (1750, 1060, 690 kHz) and N(9) (1630, 840, 790 kHz). The same procedure was used to obtain the allopurinol spectrum shown in Figure 6.

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DFT CALCULATIONS NQR Spectra Simulation. Quantum chemical calculations were carried out within the Gaussian 09 code41 run on the CRAY supercomputer at the Poznan Supercomputer and Network Centre (PCSS) in Poznan, Poland. All calculations were performed within the density functional theory (DFT) with the exchange-correlation hybrid functional B3LYP (threeparameter exchange functional of Becke B342 combined with the Lee−Yang−Parr correlation functional LYP43) using the extended basis sets with polarization and diffuse functions 6-311++G(d,p). The calculations were carried out under the assumption of the crystallographic as well as the partially optimized geometry made using the Berny algorithm in which only the positions of the hydrogen atoms were allowed to relax while those of all other atoms remained frozen. High electron density between the proton and heavy atoms as well as libration

ν+ =

e 2qQ (3 + η) 4h

ν− =

e 2qQ (3 − η) 4h

ν0 = ν+ − ν− = 2

e 2qQ η 2h

(2)

2

where e Qq/h = e Qqzz/h is the nuclear quadrupole coupling constant and η = (qxx − qyy)/qzz is the asymmetry parameter. The quadrupole coupling constant e2qQ/h, the asymmetry parameter η for the 14N nucleus, and the principal values of the nuclear quadrupole coupling qii tensor are related to these frequencies by the formulas e 2qQ 2 = (ν+ + ν−) h 3 η= 10840

3(ν+ − ν−) ν+ + ν−

(3)


a

3.31

3.20

N(3)H

N(7)

10841

1.87

3.527 3.060 2.877 2.877

N(9)H

N(1)H N(3)H N(7)H N(9)H

N(1)H

0.556 0.523 0.629 0.553

0.771

0.45

0.26

0.706 0.419 0.797 0.988 0.737 0.758 0.151 0.264 0.261 0.959 0.996 0.658

η

2.895 2.695 2.610 2.555

1.763

2.760

2.698

1.81a 2.65a 3.145a 2.735a 1.75a 1.76b 2.863a 2.587a 2.59 1.630a 1.64 2.460

ν+ (MHz)

1.990 1.895 1.705 1.760

1.042

2.040

2.267

1.12a 2.00a 1.825a 1.38a 1.060a 1.05 2.588a 2.168a 2.18 0.840a 0.82 1.575

ν− (MHz)

H−14N NQDR

1

0.905 0.800 0.905 0.795

0.721

0.720

0.430

0.69a 0.65a 1.32a 1.355a 0.69a 0.71 0.275a 0.419a 0.41 0.790a 0.82 0.885

ν0 (MHz)

4.149 4.759 4.852 4.302

3.919

5.037

5.023

4.097

3.813

3.837 5.214

3.233 4.215 5.309 4.149 2.851

−1

e Qqh (MHz)

2

0.191 0.229 0.074 0.347

0.160

0.130

0.059

0.201

0.128

0.312 0.108

0.449 0.223 0.665 0.369 0.145

η

3.310 3.842 3.729 3.600

3.096

3.941

3.841

3.279

2.982

3.177 4.051

2.788 3.396 4.864 3.494 2.242

ν+ (MHz)

2.914 3.297 3.549 2.853

2.782

3.614

3.693

2.867

2.738

2.578 3.770

2.062 2.926 3.099 2.729 2.035

ν− (MHz)

0.396 0.545 0.180 0.746

0.314

0.327

0.148

0.412

0.244

0.599 0.282

0.726 0.470 1.765 0.765 0.207

ν0 (MHz)

DFT (monomer) −1

3.27 3.183 4.202 4.496 3.55 4.949 2.036 2.725 3.767 3.300 3.222 3.204

2.047

4.047 3.680

2.225 3.361 4.506 3.184 2.067

e Qqh (MHz)

2

0.533 0.569 0.108 0.078 0.368 0.069 0.723 0.644 0.616 0.478 0.862 0.568

0.616

0.143 0.174

0.711 0.374 0.748 0.931 0.774

η

2.888 2.840 3.265 3.460 2.989 3.797 1.895 2.482 3.405 2.869 3.111 2.858

1.85

3.18 2.92

2.064 2.835 4.222 3.129 1.95

ν+ (MHz)

2.017 1.934 3.038 3.284 2.336 3.626 1.159 1.605 2.245 2.081 1.722 1.948

1.22

2.89 2.6

1.273 2.206 2.537 1.647 1.15

ν− (MHz)

0.871 0.906 0.227 0.175 0.653 0.171 0.736 0.877 1.160 0.787 1.389 0.910

0.64

0.29 0.32

0.791 0.629 1.685 1.482 0.81

ν0 (MHz)

DFT (cluster) cluster type

1 layer, 7 molecules

1 layer, 4 molecules, putative structures I and IId



This paper, measured at 295 K. bCalculated ν+ − ν− = ν0. cOpposite assignment to those given in refs 55 and 56. dPutative structures I (hypoxanthine type) and II (ureic acid type)

uric acid

xanthine

N(3) N(7)

hypoxanthinec

N(9)H

1.955 3.100 3.315 2.745 1.873 1.87 3.634 3.170 3.18 1.647 1.64 2.69

e Qqh (MHz)

N(1)H N(3) N(8) N(9)H N(1)H

site

−1

allopurinol

compound

2

Table 1. Experimental and Calculated NQR Parameters for Allopurinol, Hypoxanthine, Xanthine, and Uric Acid

The Journal of Physical Chemistry B Article



Article

Figure 7. Chemical structures of possible tautomeric forms: (a) allopurinol; (b) hypoxanthine; (c) xanthine; (d) uric acid.

qZZ =

e 2qQ h

qYY = −

q e 2qQ (1 + η) = − ZZ (1 + η) 2h 2

qXX = −

q e 2qQ (1 − η) = − ZZ (1 − η) 2h 2

local maximum of electron density, BCP (bond critical point) minimum in the direction of the nucleus but maximum in another main direction, RCP (ring critical point) - minimum in two principal axes, and CCP (cage critical point) - local minimum of electron density, respectively. The kind of extreme was determined with the help of a Hessian matrix composed of nine second-order derivatives of ρ(r). The Poincare−Hopf relationship46 was used as a consistency check. At each extreme point the topological parameters, the electron density, and its Laplacian were calculated. In addition, the ellipticity of the bond, ε, the total electron energy density at BCP (HBCP) and its components, the local kinetic energy density (GBCP), the local potential energy density (VBCP), and the hydrogen bonding energy EE according to Espinosa (EE = (1/2)VBCP),47 were calculated at each BCP. The theoretical reactivity indices the absolute electronegativity [χ = −(ELUMO + EHOMO)/2; eV]; absolute hardness [η = ELUMO − EHOMO; eV)]; electrophilicity index (reactivity) [ω = χ 2 /2η; eV]; and softness

(4)

Quantum Theory of Atoms in Molecules. The topological analysis of the electron density was performed within Bader’s quantum theory of atoms in molecules (QTAIM).46 Within this approach, the electron density ρ(r) of a molecule treated as a scalar field was examined by analysis of its gradient vector field. Depending on the nature of the extremes (maxima, saddle points, or minima in the electron density), they are named as core, bond, ring, and cage critical points and denoted as NACP (nuclear attractor critical point) 10842



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unfortunately, the full spectra have been recorded only for a few of them and the assignment of frequencies is not always accurate. Nonetheless, the assignment of each set of lines to particular nitrogen sites requires additional data, because theoretically any two of four nitrogen atoms can be protonated. According to the available X-ray data,30 the crystalline lattice of allopurinol is made of a layered structure of hydrogenbonded (NH···N and CH···O) molecules. The mean stacking distance between molecules from adjacent layers is quite short and is equal to 3.301 Å. Because light atoms are somewhat more difficult to localize than heavy nuclei by X-ray, the presence of the “ordered” and “disordered” hydrogen bonds (H-bonds) in ALP could not be simply excluded. Fortunately, in the solid state, the H-bonding pattern, which makes the proton migration channels available, restricts the possible ketonic tautomeric forms in ALP, Figure 7a, to oxo-N(1)H,N(9)H (A1) or oxo-N(3)H,N(8)H (A2). The predicted higher stability of oxo-N(1)H,N(9)H than any other form well agrees with the general observation that the requirement of a small number of the transferred protons is an obstacle for the prototropy in the chains of infinitive length in solids.51 Additionally, only one set of resonance lines detected experimentally by 14N NQDR also suggests that only one tautomeric form contributes to the NQR spectra. Comparison of the experimental 14N NQR spectrum of ALP with that theoretically predicted at the DFT (B3LYP/6-311++G(d,p)) level for the two most abundant isomers (monomers and cluster built of seven molecules) is presented in Table 1. The evident discrepancy between the experiment and results of DFT calculations performed upon monomer assumption, Table 1, confirms the validity and necessity of the supramolecular approach, which was proposed earlier for cytosine52 and a few other molecular systems.34,35 A good agreement between the patterns of experimental and theoretical spectra supports the conclusion that the oxo-N(1)H,N(9)H is adopted by ALP in solid, similarly as in the gas phase. It also suggests that the possible presence of the “disordered” H-bonds in crystalline ALP can result from the scattering of proton position rather than the formal migrations of proton. The final assignment of the 14N NQR frequencies to the particular N and  NH sites in ALP, made on the basis of NQDR and verified and supplemented by DFT, is given in Table 1 and shown in Figure 8. A more detailed analysis allows concluding which effects (substituent, hydrogen bonding, atomic contacts) and to which extent contribute to NQR parameters at particular sites. According to X-ray30 data, each molecule is surrounded by six molecules in the plane and forms the following hydrogen bonds: N(9)−H···N(3) of 2.880 Å, N(8)···HN(1) of 2.875 Å, and O(6)···HC(2) of 3.208 Å (much weaker than the two former) with only five of them and the overlapping molecules participating in four kinds of contacts: O(6)···C(6) of 3.323 Å, N(1)···O(6) of 3.430 Å, N(9)···N(8) of 3.445 Å, and N(8)··· C(7) of 3.448 Å. To detect weak interactions in the crystalline structure, including atomic contacts, which are often omitted in standard X-ray data analysis, and to get further insights into the nature of the above-mentioned intermolecular interactions, ρ(r), Δρ(r), ε, and HBCP and its components GBCP and VBCP were calculated for the charge distribution at BCP and RCP using the QTAIM approach, describing their molecular topology. The results are presented in Table 2. The QTAIM analysis confirmed the presence of N(9)−H···N(3) of −33.87 kJ/mol, N(8)···H−N(1) of −34.00 kJ/mol, and O(6)···H−

[S = 1/2η; 1/eV] were calculated using the Par and Pearson HOMO (highest occupied molecular orbital)−LUMO (lowest unoccupied molecular orbital) approach.48

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RESULTS AND DISCUSSION Allopurinol (ALP). The 1H−14N NQDR spectrum of ALP as obtained by the solid-effect technique at 295 K and νL = 100 kHz is presented in Figure 6. Strong dips observed at 300 kHz, 200 kHz, and below correspond to direct saturation of the proton signal. Two strong triplets centered at 1.810 and 2.740 MHz correspond to the ν+ transitions (ν+ − νL, ν+, ν+ + νL) at the NH nitrogen positions N(1) and N(9), respectively. The doublets (0.690 MHz, 0.790 MHz) and (1.120 MHz, 1.220 MHz) represent the level crossing signals (0.690 MHz, 1.120 MHz) and the solid effect signals from the nitrogen position N(1) corresponding to the 14N NQR transitions ν0(1) = 0.690 MHz and ν−(1) = 1.120 MHz. A similar pair of doublets from the nitrogen position N(9) merges into a single slightly broader doublet centered at 1.370 MHz. Thus, the NQR frequencies ν0(9) and ν−(9) are nearly the same (ν0(9) ≈ ν−(9) ≈ ν+(9)/2 = 1.370 MHz). The solid effect signals from the nonprotonated nitrogen positions N(3) and N(8) are expected to be weak, and the level crossing signals are in this case often not observed. The dip centered at 3.245 MHz is the solid effect dip at the frequency ν+(8) + νL (ν+(8) = 3.145 MHz). The dips associated with this dip at the frequencies ν0(8) = 1.320 MHz and ν−(8) = 1.825 MHz are masked by much stronger dips from the NH nitrogen positions. The frequency ν0(8) is observed in the cross relaxation spectrum. From among the frequencies assigned to nitrogen N(3), only the dip at ν−(3) + νL = 2.000 MHz + 0.100 MHz = 2.100 MHz is observed in the solid effect spectrum. Two other frequencies, ν+(3) = 2.650 MHz and ν0(3) = 0.650 MHz, were roughly determined from the cross relaxation spectrum and later refined by the twofrequency irradiation technique. The experimental NQR frequencies are collected in Table 1. The number of lines in the 1H−14N NQR spectrum, 12 in total, suggests that there are no crystallographically inequivalent molecules in the unit cell. The latter is consistent with the X-ray data30 according to which ALP crystallizes in the monoclinic P21/c space group with a = 3.683(1), b = 14.685(3), c = 10.318(2) Å, and β = 97.47°. The assignment of one set of NQR frequencies to particular nitrogen sites is not straightforward; however, the third equation of set 2, which enables grouping of resonance lines in a set of three for each site, is much helpful. Another observation that facilitates the assignment of NQR frequencies to particular nitrogen sites in the ALP molecule is the appearance of level crossing lines and strong solid effect lines from the protonated nitrogen positions N(1) and N(9). Additionally, the 14N quadrupole coupling constants e2qQ/h = 1.955 and 2.745 MHz are within the range expected for the NH nitrogen sites, while e2qQ/h = 3.100 and 3.315 MHz are within the range expected for the N nitrogen sites in purines; however, in fact, all of them are significantly lower in comparison with those known for pyrimidine (4.439 and 4.434 MHz49) or pyrazole (3.783, 3.756, 2.496, and 2.442 MHz50), respectively. On the other hand, all the values of asymmetry parameters 0.707 and 0.797 for NH and 0.419 and 0.988 for N sites, however quite typical of purines, are relatively high in comparison with those known for pyrimidine (0.386 and 0.385) or pyrazole (0.793, 0.802, 0.750, and 0.818). A comparison of NQR parameters with those for other purine compounds could be helpful; 10843



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the pure closed-shell (CS region I). Inside region I (pure CS), the bond degree parameter HBCP/ρBCP, Table 2, indicates a softening degree (SD) per electron-density unit at BCP; the higher the HBCP/ρBCP, the weaker the interaction and the greater the SD magnitude. It is worth noting that for N−H···N bonds the H···N distance is 2.065 Å, for C−H···O bonds the H···O distance is 2.194 Å, and only one is much shorter than 2.2 Å, Table 2; thus, within the classification proposed by Jeffrey,54 the first two are considered to be moderate and partially covalent. For C−H···O and C−H···H−C bonds, the H···O and H···H distances are 2.552 and 2.802 Å, respectively; thus, within the classification proposed by Jeffrey, these bonds are weak and mainly electrostatic. The Laplacian, ΔρBCP, recovers the shell structure of atoms and allows tracing the effects of chemical bonding in the total charge density. The relief map of the Laplacian of electron density in the plane of intermolecular H-bonds for ALP, Figure 9a, clearly exhibits maxima in the negative Laplacian on either side of the oxygen and nitrogen atoms, corresponding to the lone pair model. Moreover, it shows the polarization of the oxygen/nitrogen lone pair electrons toward hydrogen and differences in polarization of the oxygen/nitrogen lone pairs. The N−H···N interactions linking N(9) with N(3) and N(8) with N(1) sites proved to be the strongest in terms of the total energy density, and thus dominant in the interactions within the neighboring molecules in the crystal lattice and to force the layered structure in ALP crystal. Therefore, we could not expect that diversity of the NQR parameters observed at nitrogen sites is a net result of the substituent/hybridization effect. Actually, the resonance withdrawing effect of oxygen (in keto form) decreases the electron density on the ring (ρRCP, Table 2) and subsequently increases e2Qqh−1 at the N(1) site at ortho and to a lower degree at N(3) at para, is marginal at N(8) and N(9) sites. The close-packing of N(8) and N(9) atoms in adjacent positions in a five-membered ring is similar to that observed in pyrazole; thus, it justifies the observed e2qQ/h and η values but does not explain the noticeably higher e2qQ/h and lower η at the N(3) site. In general, the asymmetry parameters of the EFG tensor at N(1) and N(3) sites smaller than those at N(8) and N(9) sites can be justified by the twice smaller electron density at RCP, ρRCP, located on a six-membered ring than on a five-membered ring (0.02577 versus 0.05591) and in addition by low symmetry and localization of electron density in the neighborhood of nitrogen atoms N(8) an N(9), as revealed by ΔρRCP, Figure 9a. Incorporation of almost equivalent in strength N−H···N hydrogen bonds linking N(1) with N(8) and N(9) with N(3) to a great extent explains the observed diversity at NQR parameters; however, some discrepancy at N(8) and N(3) being proton acceptors is still noticeable, Table 1. An additional source of the discrepancy in NQR parameters can be generally weak but differing in strength intermolecular contacts: N(1)···O(6), N(9)···N(8), and N(8)···C(7) in which N(1), N(8), and N(9) only participate. Indeed, the lack of such a contact for N(3) and the presence of two for N(8) is well reflected by NQR parameters, Table 1. Hypoxanthine (HPX). Hypoxanthine, a naturally occurring purine and a metabolic intermediate of nucleic acid occasionally found in the anticodon of tRNA, has an important property of binding via weak hydrogen bonds to any of the four natural DNA bases. HPX differs from ALP (its isomer) in the presence of nitrogen in position 7 instead of 8, Figure 1. Unfortunately, for HPX, only seven 14N NQR resonance lines have been measured and the assignment of the frequencies to particular

Figure 8. (a) Correlation between experimental and calculated NQR frequencies; solid line - linear fit. (b) Correlation between the components of EFG tensor; solid lines - linear fit.

C(2) of −11.55 kJ/mol hydrogen bonds but also revealed an additional weak C(7)−H···O(6) hydrogen bond of 3.530 Å and −5.91 kJ/mol and C(7)−H···H−C(7′) dihydrogen (bifurcated) bond of 4.312 Å and −1.18 kJ/mol. The presence of these two bonds explains the experimentally observed high nonlinearity (∠O(6)HC(2) = 131°) of the C(2)−H···O(6) hydrogen bond. The AIM calculations yielded a value of electron densities, ρBCP, of 0.008−0.035 au (it falls within a certain range of values, typically between 0.001 and 0.035 au) markedly lower than for the covalent bonds. The corresponding Laplacian values ΔρBCP are positive and amount to 0.024− 0.098 au (typically between 0.006 and 0.130 au), which is indicative of the closed-shell interaction. The ρBCP and ΔρBCP are extremely low for dihydrogen bonds (0.019 and 0.074 au, respectively) and take limit values. The Koch and Popelier53 topological criteria required for H-bonds allow their classification as hydrogen bonds. Recently, Espinosa47 proposed a classification of three kinds of atomic interactions: pure closed shell [CS, region I, ΔρBCP > 0, HBCP > 0], pure shared shell [SS, region III, ΔρBCP < 0, HBCP < 0], and transit closed shell [CS, region II, ΔρBCP > 0, HBCP < 0]. Very small values of ρ(r), small and positive values of Laplacian, relatively high values of ε, nearly zero values of HBCP, and values of |VBCP|/GBCP > 1 permit classification of the N−H···N interactions as the transit closed-shell (CS region II) while C−H···O and C−H···H−C as 10844


10845

N(9)−H···N(3) N(1)−H···N(8) C(2)−H···O(6) C(7)−H···O(6) C(7)−H···H−C(7) six-membered ring five-membered ring N(1)−H···O(6) N(7)−H···N(9) C(8)−H···O(6) C(2)−H···N(3) C(2)−H···H−C(8) N(1)−H···H−C(8) six-membered ring five-membered ring N(7)−H···O(6) N(1)−H···N(9) C(8)−H···O(3) six-membered ring five-membered ring N(9)−H···O(5) N(3)−H···O(3) six-membered ring five-membered ring N(9)−H···O(6) N(1)−H···O(2) N(3)−H···O(8) N(7)−H···O(8) six-membered ring five-membered ring

RCP RCP BCP BCP BCP BCP BCP BCP RCP RCP BCP BCP BCP RCP RCP BCP BCP RCP RCP BCP BCP BCP BCP RCP RCP

bond/ring

BCP BCP BCP BCP BCP

critical point type

2.728 2.831 2.806 2.795

2.728 2.830

2.834 2.921 3.132

2.785 2.821 3.193 3.376 4.858 3.575

2.880 2.875 3.208 3.530 4.312

R(X−H···Y) (Å)

0.768 1.006 0.918 1.122

1.015 1.014

1.033 1.032 0.891

1.041 1.029 1.079 1.081 1.088 1.279

1.032 1.034 1.086 1.083 1.083 1.082

R(N−H) (Å)

1.997 1.826 1.909 1.734

1.769 1.817

1.724 1.902 2.276

1.746 1.799 2.369 2.513 2.261 2.291

1.864 1.872 2.195 2.552 2.798

R(Y···H) (Å)

0.0258 0.0559 0.0406 0.0417 0.0115 0.0099 0.0008 0.0008 0.0249 0.0582 0.0406 0.0321 0.0138 0.0219 0.0604 0.0370 0.0336 0.0221 0.0552 0.0208 0.0328 0.0269 0.0410 0.0218 0.0523

0.0356 0.0354 0.0145 0.0082 0.0019

ρBCP(r) (au)

0.1766 0.3787 0.1256 0.1056 0.0400 0.0299 0.0035 0.0033 0.1816 0.4071 0.1365 0.0920 0.0477 0.1633 0.4209 0.1347 0.1159 0.1658 0.3981 0.1076 0.1160 0.1110 0.1179 0.1653 0.3885

0.0956 0.0985 0.0555 0.0240 0.0074

ΔρBCP(r) (au)

0.0505 0.0444 0.0421 0.0385

0.0206 0.0436

0.0221 0.0633 0.0608

0.0338 0.0527 0.0942 0.0603 0.2841 0.3462

0.0529 0.0623 0.0248 0.0298 0.6066

ε

GBCP (au) 0.0248 0.0253 0.0113 0.0052 0.0014 0.0381 0.0925 0.0335 0.0307 0.0085 0.0063 0.0006 0.0006 0.0376 0.0967 0.0357 0.0234 0.0102 0.0329 0.1006 0.0333 0.0284 0.033 0.092 0.0217 0.0281 0.0249 0.0324 0.0333 0.0895

VBCP (au) −0.0258 −0.0259 −0.0088 −0.0045 −0.0009 −0.0320 −0.0903 −0.0355 −0.0350 −0.0071 −0.0050 −0.0003 −0.0003 −0.0298 −0.0916 −0.0372 −0.0238 −0.0084 −0.0250 −0.0960 −0.0329 −0.0279 −0.025 −0.085 −0.0165 −0.0272 −0.0220 −0.0353 −0.0254 −0.0818

0.0061 0.0022 −0.0020 −0.0043 0.0014 0.0012 0.0003 0.0003 0.0078 0.0051 −0.0015 −0.0004 0.0018 0.0079 0.0046 −0.0003 −0.0005 0.0079 0.0071 −0.0052 −0.0009 −0.0029 0.0029 0.0080 −0.0076

−0.0010 −0.0006 0.0025 0.0007 0.0005

HBCP (au)

0.8399 0.9762 1.0597 1.1401 0.8353 0.7937 0.5000 0.5000 0.7926 0.9473 1.0420 1.0171 0.8235 0.7599 0.9543 0.0370 0.0336 0.7610 0.9230 0.7604 0.9680 0.8835 1.0895 0.7628 0.9140

1.0403 1.0237 0.7788 0.8654 0.6429

|VBCP|/GBCP

−21.66 −35.71 −28.88 −46.34

−43.19 −36.63

−48.83 −31.24 −11.03

−46.60 −45.95 −9.32 −6.56 −0.39 −0.39

−33.87 −34.00 −11.55 −5.91 −1.18

EE (kJ/mol)

a

The hydrogen bond length R(XH···Y); the proton−donor distance R(NH); the proton−acceptor distance R(H···Y), where Y = N or O; the electron density at BCP (ρBCP(r)) and its Laplacian Δ (ρBCP); the potential electron energy density (VBCP); the kinetic electron energy density (GBCP); the total electron energy density (HBCP); and the estimated hydrogen bonding energy according to Espinosa EE calculated at the B3LYP/6-311++G(d,p) level of theory. bPutative structures I (hypoxanthine type) and II (ureic acid type).

uric acid

xanthine (putative IIb)

xanthine (putative Ib)

hypoxanthine

allopurinol

compound

Table 2. Topological Parametersa of ρ(r) for Selectedb Hydrogen Bonds in Allopurinol, Xanthine, Hypoxanthine, and Uric Acid

The Journal of Physical Chemistry B Article



Article

Figure 9. Laplacian contour and surface for (a) allopurinol, (b) hypoxanthine, (c) xanthine (putative structure I), (d) xanthine (putative structure II), and (e) uric acid.

sites given by Edmonds, Speight55 or Garcia, Smith56 in the light of the results for ALP raises some doubts, reinforced by the knowledge of the X-ray data.57 Therefore, we decided to perform a NQR experiment for hypoxanthine. The results are collected in Table 1. The 14N quadrupole coupling constant e2qQ/h = 3.18 MHz is within the range expected for the N nitrogen sites, while e2qQ/h = 1.87 and 1.64 MHz are within the range expected for the NH nitrogen sites in purines or benzimidazole. The values of asymmetry parameters, 0.261 for NH and 0.758 and 0.996 for N sites, are quite typical of purines. In fact, all of them are much smaller than those for ALP, which is surprising because the difference between both isomers is limited to two sites and it would be reasonable to expect noticeable changes limited only to the close neighborhood of these two sites (N(8) and N(9)). The DFT/QTAIM analysis should shed some light on the source of these differences and help with the assignment of the 14N NQR frequencies. The same methodology as for ALP was applied. HPX, similarly to ALP, exhibits a layered structure, but in

contradiction to ALP, it is built of two types of hydrogen bonds: NH···O and NH···N. The mean stacking distance between molecules of adjacent layers is 3.25 Å, i.e., shorter than that in ALP. It is also smaller than the sum of van der Waals radii of two carbon atoms in an aromatic ring (3.4 Å) but a bit larger than the sum of van der Waals radii of two nitrogen atoms (3.1 Å). According to X-ray data,57 each of two inequivalent molecules in a crystal unit is involved in six hydrogen bonds, two in the bond type N−H···O of 2.779 Å, two in the bond type N−H···N of 2.786 Å, C−H···O of 3.193 Å, and C−H···N of 3.376 Å. As suggested by the IR spectra of isolated molecules, in the gas phase,58 the keto form N(1)H/N(9)H (H1), Figure 3b, predominates, similarly as in ALP. According to 13C NMR, in DMSO solution, the N(7)H (H6) form is more favored,59 while, in aqueous solution, UV spectroscopic studies suggest the presence of two tautomers N(7)H and N(9) H of which N(9)H dominates. 10846



Article

In low temperature matrix60 and crystal,51 HPX occurs as the N(9)H tautomeric form. In general, the H-bonding pattern, which makes the proton migration channels available, restricts the possible tautomeric forms in crystalline HPX to ketonic, H1 and H6, and hydroxy, H3 and H4, Figure 7b. Only one set of resonance lines detected experimentally by 14N NQDR suggests that only one tautomeric form contributes to the NQR spectra. A good agreement between the patterns of experimental and theoretical spectra supports the conclusion that H1 is adopted by HPX in solid, similarly as in the gas phase. The calculations performed for a single molecule show the dominant influence of the substituent effect on NQR parameters, but such an estimation of NQR parameters is rough. The assumption of a supramolecular cluster consisting of five molecules gives results in much better agreement with the experimental data, Table 1, Figure 8. A good agreement between the patterns of experimental and theoretical NQR spectra supports the conclusion that oxo-N(1)H,N(9)H is adopted by HPX in solid. Thus, the final assignment of the 14N NQR frequencies, to the particular N and NH sites given in Table 1, was partially made on the basis of NQDR and then verified by DFT. This assignment is different than those given earlier by Edmonds, Speight55 or Garcia, Smith;56 however, 9 of 12 frequencies are not far from 9 of the previously reported ones. To detect all of the possible weak interactions including atomic contacts and to get further insights into their nature and strength, ρ(r), Δρ(r), ε, and HBCP and its components GBCP and VBCP were calculated for the charge distribution at BCPs and RCPs using the QTAIM approach. The results are presented in Table 2. The N(1)−H···O(6) bonds of −46.60 kJ/mol and N(9)−H···N(7) of −45.95 kJ/mol which are stronger and C(8)−H···O(6) of −9.32 kJ/mol which are weaker than their equivalents in ALP and C(2)−H···N(3) of −6.56 kJ/mol were revealed. Similarly to ALP, additional weak C(2)−H···H−C(8′) and C(2)−H···H−N(1′) dihydrogen bonds of 4.857 and 4.836 Å and both of −0.39 kJ/mol were found. Very close to zero values of ρ(r), small and positive values of Laplacian, nearly zero values of total energy density HBCP (0.0008 au), and values of |VBCP|/GBCP < 1 classify this bond as a border case between pure and transit closed shell (according to Espinosa47 classification). Generally, very small values of ρ(r), small and positive values of Laplacian, relatively high values of ε, and nearly zero values of HBCP and values of | VBCP|/GBCP classify N(1)−H···O(6) and N(9)−H···N(7) interactions as the CS II, while the remaining as CS I. Similarly to ALP, the resonance withdrawing effect of oxygen, which decreases the electron density on the ring and subsequently e2qQ/h at the N(1) site at ortho and to a lower degree at N(3) at para, is marginal at N(8) and N(9) sites. The lack of close-packing of nitrogens in the five-membered ring explains to a great extent the noticeably lower e2qQ/h and lower η at both sites; thus, the NQR parameters for HPX resemble those for benzimidazoles. This indicates that the diversity of the NQR parameters observed at nitrogen sites is mainly caused by the pure substituent/hybridization effect. In general, the asymmetry parameters of the EFG tensor at N(1) and N(3) sites smaller than those at N(8) and N(9) sites can be justified by the twice smaller electron density at RCP, ρRCP, located on a six-membered ring than on a five-membered ring (0.02495 versus 0.05828) and in addition by low symmetry and localization of electron density in the neighborhood of nitrogen atoms N(7) an N(9), as revealed by ΔρRCP, Figure 9b. Incorporation of almost equivalent in strength N−H···N

hydrogen bonds linking N(1) with N(8) and N(9) with N(3) to a great extent explains the observed diversity at NQR parameters; however, some discrepancy at N(8) and N(3) being proton acceptors is still noticeable, Table 1. Xanthine (XAN). Xanthine, a naturally occurring purine base found in most human body tissues and fluids, resembling hypoxanthine, differs from it by the presence of an additional oxygen atom, Figure 1. However, the 14N NQR data for XAN are known, but neither X-ray nor neutron scattering data which could reveal the intermolecular bond pattern are to our knowledge available. XAN is poorly soluble; thus, its crystallization is highly difficult. Our attempts in this field have failed. The 14N quadrupole coupling constants e2qQ/h are 3.31, 1.87, and 2.69 MHz, which is within the range expected for the NH nitrogen sites, while e2qQ/h = 3.2 MHz, which is within the range expected for the N nitrogen sites in the abovementioned purines, but in fact all of them are significantly lower than those known for pyrimidine. The values of asymmetry parameters 0.658 and 0.445 for NH and 0.771 for N sites are quite typical of purines and close to those known for pyrimidine or benzimidazole.35 The order of the NQR parameters and additionally a poor quality of their reproduction by DFT upon monomer assignment suggests that each nitrogen site participates in strong intermolecular contact. In the absence of the real crystallographic data, we assumed that, like in the other purines, the molecule of xanthine in the crystalline structure is bonded to four associate molecules and except for N(3), C(4), and C(5) all the remaining atoms participate in intermolecular interactions. Two putative structures I and II, Figure 10, were proposed. Structure I was deduced using

Figure 10. Two putative structures of xanthine: (a) structure I deduced using hypoxanthine; (b) structure II deduced using uric acid. Both structures were optimized under assumption of periodic boundary conditions (PBC). Then, the cluster (tetramer) shown in the figure was extracted.

hypoxanthine, while structure II was deduced using uric acid. Both structures were optimized under the assumption of periodic boundary conditions (PBC). Then, the cluster (tetramer) was extracted. The calculations of NQR parameters have been performed upon this assumption (clusters, Table 1). The reproduction of NQR parameters with the use of putative structure I is much better than assuming putative structure II; the scattering of the NQR parameters, Figure 8a, is small. 10847



Article

Figure 11. Hirshfeld surface with the normalized contact distance dnorm and 2D molecular fingerprint for each purine studied: (a) allopurinol; (b) hypoxanthine; (c) xanthine; (d) uric acid.

were found. Generally, very small values of ρ(r), small and positive values of Laplacian, relatively high values of ε, and nearly zero values of HBCP and values of |VBCP|/GBCP classify N(7)−H···O(6) and N(1)−H···N(9) interactions as CS II, while C(8)−H···O(3) as CS I. The calculations performed for a single molecule give a rough estimation of the pure substituent effect, but the NQR parameters obtained assuming the putative structure, Table 1, Figure 8, are in good agreement with the

The validity of this structural concept was checked by DFT calculations. Two N−H···N hydrogen bonds of 2.921 Å and −31.24 kJ/mol, which link N(9) (hydrogen acceptor) and N(1) (proton donor), two N−H···O hydrogen bonds of 2.834 Å and −48.83 kJ/mol which link O(6) (hydrogen acceptor) and N(7) (proton donor), and two C−H···O hydrogen bonds of 3.132 Å and −11.03 kJ/mol which link O(3) (hydrogen acceptor) and C(8) (proton donor), as shown in Figure 9c, 10848



Article

Table 3. A Comparison of % of the Hirshfeld Surface, Total Hirshfeld Surface, and Volume Characterizing Allopurinol, Hypoxanthine, Xanthine, and Uric Acid twist angle (%)

N···O (%)

O···H (%)

N···H (%)

O···All (%)

N···All (%)

surface (Å2)

volume (Å3)

globularity

asphericity

0.24 0.49

2.2 2.8

22.2 22.6

31.2 26.6

134.18 136.94

0.856 0.853

0.091 0.110

64.11

8.3

31.5

14.5

158.98

146.38

xanthine (putative structure II) uric acid

64.11 64.74

8.3 9.7

31.6 51.2

14.6 4

25 24.4 (40.1)a 18.3 (31.4)a 31.4a 11

148.10 150.67

xanthine (putative structure I)

14.2 14.6 (31.2)a 26.4 (49.1)a 49a 38.2

158.98 160.94

146.38 146.31

0.834

0.128

compound allopurinol hypoxanthine

a

Reciprocal contacts included.

Moreover, upon oxidation, the involvement of nitrogen atoms in binding decreases, while that of oxygen atoms increases and hydrogen bonds change from N−H···N type to much stronger N−H···O type. Thus, hydrogen bonds play a key role in packing context of purines. The Hirshfeld surface-based approach64,65 provides the distribution of interactions of the molecule with its environment and thus is very convenient for comparison of different molecular systems. The Hirshfeld surface with the normalized contact distance dnorm and 2D molecular fingerprint plot for each purine (Figure 11) reveal some additional details on the hydrogen bond pattern. The hydrogen bonds visualized as red areas on the Hirshfeld surfaces form sharp distinct spikes in the 2D molecular fingerprint plot, Figure 11. The top left and bottom right of the 2D fingerprint plot reveal characteristic “wings”, which can be identified as a result of hydrogen bond X−H···Y (X = N or C and Y = N or O) interactions. The differences between 2D molecular fingerprint plots for ALP, HPX, and UCA are evident. The pair of wings at the top left, di < de, can be assigned to the surface around the donors (N−H and C−H bonds in ALP and HPX), whereas those at the bottom right, de > di, correspond to the surface around the acceptor (N in ALP and HPX). In UCA, the only one wing at the top left, di < de, can be assigned to the surface around the donor (N−H bond), whereas that at the bottom right, de > di, corresponds to the surface around the acceptor (O). A small but discernible splitting of spikes assigned to hydrogen bonds in ALP and HPX is a clear indication of the presence of a mixture of two H-bond types. The lack of such a splitting in UCA reflects the equivalency of all hydrogen bonds in UCA crystal. Indeed, according to the X-ray and QTAIM study of the crystalline structure of ALP and UCA, all nitrogen atoms participate only in one kind of hydrogen bonds, N−H···N and N−H···O, respectively, while in HPX and XAN the two kinds of bonds, N−H···N and N−H···O, exist. Therefore, although the same number of nitrogen atoms is in all three compounds, the percentage of the Hirshfeld surface of all bonds/contacts in which nitrogen atoms are involved decreased from 25 and 24.4 to 11% (for ALL, HPX, and UCA, respectively), Table 3. This feature reflects the increase in the number of oxygen atoms in a molecule (molecular volume/surface/asphericity). The contribution of bonds/contacts in which oxygen atoms are involved is relatively small in ALP and HPX (about 14%) but increases significantly with an increasing number of oxygen atoms (38.2% in UCA), Table 3. The equilibration of the contributions of bonds/contacts in which oxygen and nitrogen participate forces different crystalline packing for different purines.

experimental data and suggest that such a structure is highly probable. A high asymmetry parameter at −N(1)H− and −N(9)H− sites evidently results from the coexistence of substituent and hydrogen bonding effects. Uric Acid (UCA). Uric acid, the end product of purine catabolism in humans, formed by a series of oxidations of hypoxanthine and of xanthine, catalyzed by xanthine oxidase, structurally resembles hypoxanthine but differs from it by the presence of two additional oxygen atoms, Figure 1. The 14N NQR resonance lines for all nitrogen sites in uric acid were detected.61 The 14N quadrupole coupling constants e2qQ/h = 3.257, 3.063, 2.877, and 2.77 MHz and asymmetry parameters 0.556, 0.523, 0.629, and 0.553 are noticeably less differentiated than in the other purines but within the range expected for  NH nitrogen sites. The assignment of the frequencies to nitrogen sites, all of NH type, is not straightforward, and a simple comparison with the similar compounds can lead to an erroneous conclusion; thus, it should be verified using DFT. In comparison to HPX or ALP, all values of e2qQ/h are noticeably higher, but the asymmetry parameters are smaller, which suggests a different pattern of intermolecular interactions. Indeed, according to X-ray data,62 UCA alone among purines exhibits a unique structure of intermolecular H-bonds, which are exclusively NH···O type and engage all hydrogen atoms, Figure 9d. Each molecule in UCA is bonded to six neighboring molecules via the hydrogen bonds in which NH is a proton donor and O is a proton acceptor: N(1)H···O(2) of 2.831 Å, N(3)H···O(8) of 2.806 Å, N(7)H···O(8) of 2.792 Å, and N(9)H···O(6) of 2.728 Å. The fully keto form of uric acid is found to be most stable in the gas phase, aqueous medium,63 as well as in solid.62 The small scattering of NQR parameters on nitrogen atoms, Table 2, Figure 8, confirms the adoption of such a tautomeric form. To detect other weak interactions, including atomic contacts, ρ(r), Δρ(r), ε, and HBCP and its components GBCP and VBCP were calculated for the charge distribution at BCP and RCP using the QTAIM approach. The results are collected in Table 2 and shown in Figure 9d. The QTAIM analysis confirmed the presence of four strong hydrogen bonds. Generally, very small values of ρ(r), small and positive values of Laplacian, relatively high values of ε, and nearly zero values of HBCP and values of |VBCP|/GBCP classify these interactions as the CS II type, except for N(7) H···O(8). The presence of four hydrogen bonds of the same kind, close in length and strength, explains the small diversity of NQR parameters. Hirshfeld Surfaces. As follows from our studies, the NQR spectra (and derived parameters) for ALP and HPX differ considerably from those of XAN and UCA because the less oxidized purine ring is conjugated to a greater degree. 10849



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Table 4. Chemical Reactivity Descriptors According to Parr Definitiona compound

EHOMO (eV)

ELUMO (eV)

η (eV)

χ (eV)

ω (eV)

S (1/eV)

allopurinol hypoxanthine xanthine 9H xanthine 7H uric acid oxipurinol (pyrazolo[3,4-d]pyrimidine-4,6-dione)

−9.274 −8.679 −9.097 −9.246 −8.953 −11.916

0.722 0.784 0.362 0.688 0.562 −1.463

4.998 4.732 4.729 4.967 4.757 5.226

4.276 3.947 4.367 4.279 4.196 6.689

1.829 1.646 2.016 1.843 1.850 4.281

0.200 0.211 0.211 0.201 0.210 0.191

a The highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), chemical potential (χ), absolute hardness (η), electrophilicity index (ω), and chemical softness (S) for allopurinol, hypoxanthine, xanthine, uric acid, and oxipurinol calculated by the B3LYP and HF combined approach.

Reactivity. Some preliminary findings important for biological activity prediction come from the analysis of HOMO, LUMO, HOMO−LUMO gaps, and the chemical reactivity natural descriptors, i.e., the electrophilicity ω, electronegativity χ, and hardness η, Table 4. The widest HOMO−LUMO gap for ALP (and its metabolite alloxanthine called oxypurinol OXY) and the narrowest for XAN mean that the structures of ALP (and OXY) and XAN (9H) are the most and least stable, respectively. The ALP reduction potential is lower than that of HPX but higher than that of XAN and UCA. However, ALP has an unusually low lying HOMO (highest occupied molecular orbital) and thus a higher oxidation potential than HPX, XAN, and UCA, Figure 12. Therefore,

conversion, ALP may act in a noncompetitive way; thus, it suggests either a different character of inhibition in each case HPX and XAN or the influence of proton migration (intermolecular hydrogen bonding). A comparison of geometries of single molecules in all above-mentioned purines in crystalline states leads to a conclusion that all the CO bonds are relatively short (C(6)O(6) of 1.2275 Å in ALP, 1.2352 Å in HPX, and 1.2334 Å in UCA; C(2)O(2) in UCA is even shortened 1.2234 Å, while C(8)O(8) is elongated 1.2413 Å). The 2D relief maps and 3D isosurfaces of Laplacian for ALP reveal additional details, important from the point of view of further comparison with other purines, Figures 9 and 13. Three valence shell charge concentrations (VSCCs) oriented toward their bonding partners and consistent with the sp2 hybridization are found at the oxygen atom. One of the VSCCs at the oxygen atom points toward the carbon atom and one VSCC at the carbon points toward the oxygen atom. This suggests that the CO bond is strongly polarized. The VSCCs of carbon C(7) directed toward the N(8) nitrogen atom is shifted to the nitrogen basin. Nevertheless, the NC, NN, and NH bonds show features typical of covalent interactions. The search for minima in the Laplacian around nitrogen atoms of each type N and NH of the ring gives three VSCCs, oriented toward their bonding partners and consistent with the sp2 hybridization. The isosurface representation of the Laplacian around N reveals the expected VSCC of the H-bonddirected in-plane of the lone pair with a well pronounced maximum; by contrast, the donor nitrogen atom NH exhibits a more symmetrical distribution of the Laplacian and VSCC directed toward H with an even better pronounced (higher) maximum. The electron depletion regions at each carbon atom represent possible sites for nucleophilic attack, but the space above and below the carbon bonded to oxygen is more widely open for a potential nucleophilic attack than any other, while the claws made of the nitrogen atom provide additional shielding. Irrespective of the presence of the atom type in positions 7 and 8 of the ring (i.e., nitrogen or carbon), the covalent bond linking atoms in positions 7 and 8 of the ring is longer than those linking atoms in positions 8 and 9. However, the electron density at BCP of the bond linking atoms in positions 7 and 8 is higher than that of the bond linking atoms in 8 and 9 for ALP and UCA, while the opposite effect is observed for HPX and XAN (0.3522 versus 0.3388 for ALP, 0.3564 versus 0.3235 for HPX, 0.3259 versus 0.3125 for UCA, and 0.339 versus 0.3533 for XAN). The asymmetry ε describing the curvature of ρ(r) at BCP of the bond linking atoms in 7 and 8 is higher than that of the bond linking atoms in 8 and 9 (0.2288 versus 0.1124 for ALL, 0.2275 versus 0.1407 for HPX, and 0.2459 versus 0.1845 for UCA). The shift of a nitrogen atom from N(8) to N(7) results in a decrease in the

Figure 12. Comparison of HOMO and LUMO levels.

its oxidation which involves the attachment of oxygen at C(2), i.e., removal of electron from the HOMO orbital, is the easiest among this set. Thanks to the low-lying HOMO, ALP can be easily oxidized to its metabolite OXY, which is the actual inhibitor of XO in vivo. OXY binds tightly (covalently) to reduced molybdenum in the center of the enzyme.66 Strongly electrophilic reagents are known to lead to a low substrate selectivity, which for XO is a crucial factor. A reliable measure of the electrophilic capabilities of molecules is the electrophilicity, ω. It can be derived directly from HOMO−LUMO and is known to be almost insensitive to solvent effects. The electrophilicity is the highest for XAN(9H) but the lowest for HPX and ALP, Table 4. An increase in electrophilicity after nitrogen shift from position 7 (as in HPX) to position 8 (as in ALP) or C(2) oxidation (XAN) is remarkable, Table 4. The smaller ω value for HPX than ALP is surprising, because ALP is in vitro competitive to both HPX and XAN. After metabolic 10850



Article

Figure 13. 3D distributions of the Laplacian of electron density calculated by DFT for the compounds studied (isocontour ±0.35 au), with the regions of negative Laplacian in red and the regions of positive Laplacian in blue: (a) allopurinol; (b) hypoxanthine; (c) xanthine (putative II); (d) uric acid.

which is a very good indicator of hydrogen bond strength, suggests a higher proton affinity (PA) of N(9) than N(1). However, the stronger HB is expected at those sites for which PA is higher; thus, the N(8) proton affinity should be higher than that of N(9), but the difference between the H-bond strength is small. Since it is known that N(8) in ALP cannot be oxidized by the enzyme, it may be involved in the main chain at an active site. Such a hypothesis was confirmed recently by the crystalline structure of reduced bovine milk XO bound with oxipurinol (metabolite of ALP, which similarly to ALP has a low lying HOMO, Table 4) in which N(8) binds to molybdenum, while N(9) binds to glutamate 1261.66−68 Thus, NQR parameters permit characterization of a dehydrogenation site, i.e., the factor determining the biological activity of ALP and making it a potent inhibitor of XO.

electron density at RCP of the six-membered ring (from 0.0258 in ALP to 0.0249 in HPX; 0.0219 in XAN and 0.0218 in UCA) and an increase in RCP of the five-membered ring (from 0.0559 in ALP to 0.0582 in HPX; 0.0604 in XAN but 0.0523 in UCA), which means that it generates in the heterocyclic rings an effect similar to that of HPX oxidation to XAN. In the fivemembered ring, this effect is the opposite to that generated by XAN oxidation to the final product of metabolism, i.e., UCA, because the density in RCP of the five-membered ring ALP is lower than that in HPX and XAN but greater than that in UCA. The effect of electron withdrawing from the ring by each subsequent oxygen atom substituted to a six-membered ring is clearly pronounced, while for the five-membered ring this effect in XAN is disturbed by the competitive interaction of both oxygen atoms. The X-ray and NQR data for most purines studied, apart from XAN, suggest that the keto N(1)HN(9)H tautomer predominates in solid. From the biological activity point of view, the fact that oxo-N(1)H,N(9)H is adopted by ALP in solid gives a credible answer to the important question of the site of proton removal, which was suggested to be N(9) instead of N(8), required for the explanation of the substrates for the malarial and Leishmanial HGPRTs.31 The shift of the nitrogen from the 7 to 8 position results in a decrease in proton affinity (PA) of N(9) and facilitates deprotonation of this site. Thus, it can be expected that N(9) takes part in a stronger HB, which is also reflected by both NQR parameters (e2Qqh−1 and η for HPX versus ALP), Table 1. The NQR asymmetry parameter η,

■

CONCLUSIONS (1) Twelve 14N resonance frequencies have been detected and assigned to two pairs of two kinds of nitrogen sites (N and NH) in allopurinol and hypoxanthine. (2) As follows from the multiplicity of NQR spectra and the X-ray data for most purinic type heterocycles relevant in pharmacological processes, the keto N(1)HN(9)H tautomer predominates in solid. Upon oxidation, the purine ring is less conjugated and the involvement of nitrogen atoms in binding decreases, while that of oxygen atoms increases and hydrogen bonds change from N−H···N type to a much stronger N−H··· 10851



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Effect of Initial Management of Hyperleukocytosis on Early Complications and Outcome of Children with Acute Lymphoblastic Leukemia. J. Clin. Oncol. 1988, 6, 1425−1432. (12) Holdsworth, M. T.; Nguyen, P. Role of I.V. Allopurinol and Rasburicase in Tumor Lysis Syndrome. Am. J. Health-Syst. Pharm. 2003, 60, 2213−22. (13) Nakamura, K.; Natsugoe, S.; Kumanohoso, T.; Shinkawa, T.; Kariyazono, H.; Yamada, K.; Baba, M.; Yoshinaka, H.; Fukumoto, T.; Aikou, T. Prophylactic Action of Allopurinol Against ChemotherapyInduced Stomatitis-Inhibition of Superoxide Dismutase and Proteases. Anticancer Drugs 1996, 7, 235−239. (14) Saadeh, C. E. Chemotherapy- and Radiotherapy-Induced Oral Mucositis: Review of Preventive Strategies and Treatment. Pharmacotherapy 2005, 25, 540−554. (15) Krakoff, I. H. Use of Allopurinol in Preventing Hyperuricemia in Leukemia and Lymphoma. Cancer 1966, 19, 1489−1496. (16) Langer, J. C.; Sohal, S. S.; Blennerhassett, P. Mucosal Permeability After Subclinical Intestinal Ischemia-Reperfusion Injury: An Exploration of Possible Mechanisms. J. Pediatr. Surg. 1995, 30, 568−572. (17) Coghlan, J. G.; Flitter, W. D.; Clutton, S. M.; Panda, R.; Daly, R.; Wright, G.; Ilsley, C. D.; Slater, T. F. Allopurinol Pretreatment Improves Postoperative Recovery and Reduces Lipid Peroxidation in Patients Undergoing Coronary Artery Bypass Grafting. J. Thorac. Cardiovasc. Surg. 1994, 107, 248−256. (18) Godin, D. V.; Bhimji, S. Effects of Allopurinol on Myocardial Ischemic Injury Induced by Coronary Artery Ligation and Reperfusion. Biochem. Pharmacol. 1987, 36, 2101−2107. (19) Guan, W.; Osanai, T.; Kamada, T.; Hanada, H.; Ishizaka, H.; Onodera, H.; Iwasa, A.; Fujita, N.; Kudo, S.; Ohkubo, T.; et al. Effect of Allopurinol Pretreatment on Free Radical Generation After Primary Coronary Angioplasty for Acute Myocardial Infarction. J. Cardiovasc. Pharmacol. 2003, 41, 699−705. (20) Kelkar, A.; Kuo, A.; Frishman, W. H. Allopurinol As a Cardiovascular Drug. Cardiol. Rev. 2011, 19, 265−271. (21) Stewart, J. R.; Crute, S. L.; Loughlin, V.; Hess, M. L.; Greenfield, L. J. Prevention of Free Radical-Induced Myocardial Reperfusion Injury with Allopurinol. J. Thorac. Cardiovasc. Surg. 1985, 90, 68−72. (22) Hopson, S. B.; Lust, R. M.; Sun, Y. S.; Zeri, R. S.; Morrison, R. F.; Otaki, M.; Chitwood, W. R., Jr. Allopurinol Improves Myocardial Reperfusion Injury in a Xanthine Oxidase-Free Model. J. Natl. Med. Assoc. 1995, 87, 480−484. (23) Lara, D. R.; Cruz, M. R.; Xavier, F.; Souza, D. O.; Moriguchi, E. H. Allopurinol for the Treatment of Aggressive Behaviour in Patients with Dementia. Int. Clin. Psychopharmacol. 2003, 18, 53−55. (24) Fan, A.; Berg, A.; Bresee, C.; Glassman, L. H.; Rapaport, M. H. Allopurinol Augmentation in the Outpatient Treatment of Bipolar Mania: A Pilot Study. Bipolar Disord. 2012, 14, 206−210. (25) Buie, L. W.; Oertel, M. D.; Cala, S. O. Allopurinol as Adjuvant Therapy in Poorly Responsive or Treatment Refractory Schizophrenia. Ann. Pharmacother. 2006, 40, 2200−2204. (26) Watts, R. W. Allopurinol In the Therapy of Neoplasia and Blood Diseases. Metabolic Aspects. Ann. Rheum. Dis. 1966, 25, 657−659. (27) Samy, E. M.; Hassan, M. A.; Tous, S. S.; Rhodes, C. T. Improvement of Availability of Allopurinol from Pharmaceutical Dosage Forms I - Suppositories. Eur. J. Pharm. Biopharm. 2000, 49, 119−127. (28) Hussain, A.; Rytting, J. H. Prodrug Approach to Enhancement of Rate of Dissolution of Allopurinol. J. Pharm. Sci. 1974, 63, 798−799. (29) Babushkina, T. A.; Leonova, T. S.; Chernyshev, A. I.; Yashunskii, V. G. Study of Tautomerism in Allopurinol and its Methyl Derivatives by 13 C Spectroscopy. Chem. Heterocycl. Compd. 1979, 15, 1240−1243. (30) Prusiner, P.; Sundaralingam, M. Stereochemistry of Nucleic Acids and Their Constituents. XXIX. Crystal and Molecular Structure of Allopurinol, A Potent Inhibitor of Xanthine Oxidase. Acta Crystallogr., Sect. B 1972, 28, 2148−2152. (31) Shivashankar, K.; Sujay Subbayya, I. N.; Balaram, H. Development of a Bacterial Screen for Novel Hypoxanthine-Guanine

O type. NQR spectra (and derived parameters) for ALP and HPX differ considerably from those of XAN and UCA. (3) NQR parameters obtained assuming the putative XAN structure (tetramer) are in good agreement with the experimental data and suggest that such a structure is highly probable. (4) The shift of nitrogen from the 8 to 7 position of the ring is strongly manifested in the values of NQR parameters at all 14 N atoms, relatively small at the −N(1)H site and significant at the −N(9)H− site. A small difference in the strength of Hbonds linking N(8)···H−N(1) and N(9)−H···N(3) is important for binding capabilities of reduced bovine milk XO with oxypurinol (metabolite of ALP). N(8) binds to molybdenum, while N(9) binds to glutamate 1261.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +48-61-8295277. E-mail: Jolanta.Latosinska@amu. edu.pl. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The study was supported by the Foundation for Development Diagnostics and Therapy, Warsaw, Poland (J.N.L. and M.L.). Generous allotment of computer time from the Poznań Supercomputing and Networking Center (PCSS) in Poznań, Poland is gratefully acknowledged.

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REFERENCES

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An Insight into Prototropism and Supramolecular Motifs in Solid-State

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