Modeling of Purine Derivatives Transport across Cell Membranes

Mercaptopurine (6-MP), thioguanine (6-TG), and azathioprine (AZA) are purine antimetabolites introduced as anticancer or immunosuppressive drugs decad...
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Brief Articles Modeling of Purine Derivatives Transport across Cell Membranes Based on Their Partition Coefficient Determination and Quantum Chemical Calculations Marcin Hoffmann,*,†,‡ Maria Chrzanowska,*,§ Tadeusz Hermann,§ and Jacek Rychlewski†,# Quantum Chemistry Group, Faculty of Chemistry, A. Mickiewicz University, 6 Grunwaldzka Street, 60-780 Poznan´ , Poland, BioInfoBank Institute, 24 a Limanowskiego Street, 60-744 Poznan´ , Poland, and Department of Physical Pharmacy and Pharmacokinetics, University of Medical Sciences, 6 SÄ wie¸ cickiego Street, 60-781 Poznan´ , Poland Received June 17, 2004

Mercaptopurine (6-MP), thioguanine (6-TG), and azathioprine (AZA) are purine antimetabolites introduced as anticancer or immunosuppressive drugs decades ago. Methylated AZA, called MAZA, is among the investigational drugs. The present study compares MAZA to the widely recognized drugs AZA, 6-MP, and 6-TG with respect to the ability of being transported across cell membranes. The obtained octanol/water phases partition coefficients and results of quantum chemical calculations predict the following sequence of hydrophobicity: MAZA > AZA > 6-TG > 6-MP. Introduction Mercaptopurine (6-MP), thioguanine (6-TG), and azathioprine (AZA) are substances of significant biological activities. Thus, these antimetabolites were introduced as anticancer and immunosuppressive drugs decades ago.1,2 They are used in the treatment of leukemia (6MP, 6-TG) and as immunosupressants (AZA).3,4 Although AZA is still often used in therapy,5 unwanted effects of AZA are remarkable, thus limiting its use in therapy. Therefore, the search for new compounds and modification of the already existing ones continue with the aim of obtaining agents with appropriate activity.6 A methylated AZA i.e., MAZA is among these investigational drugs whose synthesis is protected by a patent.7 (Chart 1 presents chemical formulas of AZA, MAZA, 6-MP, and 6-TG.) The kinetics of AZA and MAZA metabolism in human blood8 and their mercaptolysis in the presence of physiological thiols glutathione and cysteine9 were examined, showing similarities between AZA and MAZA. The results indicated that the mercaptolysis of MAZA is faster under the influence of the thiols than the mercaptolysis of AZA. The apparent firstorder rate constants for MAZA are approximately 1.8fold greater than those obtained for AZA. For MAZA and AZA, mercaptolysis proceeded slightly and yet significantly faster under the influence of cysteine than glutathione. Our previous studies allowed us to propose a mechanism for AZA bioactivation.10 From the studies on AZA and MAZA metabolism in human blood and their mercaptolysis, we can conclude that MAZA is bioactivated in the same fashion as AZA. * To whom correspondence should be addressed. For M.H.: phone, +48 61 829 12 89; fax, +48 61 865 80 08; e-mail, [email protected]. For M.C.: phone, +48 61 854 64 31; fax, +48 61 854 64 30; e-mail, [email protected]. † A. Mickiewicz University. ‡ BioInfoBank Institute. § University of Medical Sciences. # Professor Jacek Rychlewski died before this article was finalized.

Chart 1. Chemical Formulas of 6-MP, 6-Thioguanine 6-TG, AZA, MAZA

Therefore, the present study is aimed at comparing some other physical and chemical properties of MAZA, compared to recognized drugs AZA, 6-MP, and 6-TG. We decided to model the transport of these compounds across a cell membrane. Therefore, we measured the octanol/aqueous phases partition coefficients, which reflect to a certain extent processes of a passive drug transport in the body, and compared the experimental results with those yielded by the quantum chemical calculations. The obtained results shed new light on abilities of the studied compounds to be transported across cell membranes. During recent years quantum mechanical calculations taking into account solvation effects have been successfully employed in studies on various systems.11 For example, Lombardo et al. utilized solvation free energies in water and n-hexsadecane calculated with AMSOL to compute brain-blood partitioning of organic compounds12 and Giesen et al. used the SM5.4 model to calculate partitioning of nucleic acid bases between chloroform and water.13 In the case of 6-MP, thiocy-

10.1021/jm0495273 CCC: $30.25 © 2005 American Chemical Society Published on Web 05/27/2005

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Scheme 1. Thione (Left) and Thiole (Right) Tautomeric Forms of 6-Mercaptopurine (6-MP)

tosine, and similar compounds, solvation effects favor formation of thione tautomers over the thiole ones.14,15 Experimental Section Methods. The employed experimental procedure for noctanol/phosphate buffer partition coefficients determination is described in detail in ref 16. Briefly, the partition coefficients of the studied compounds were determined for n-octanol/ phosphate buffer, pH 5.7 and 7.4 at 37 °C. Samples of 4 mL of solutions of the studied purines in phosphate buffer of pH 5.7 or 7.4 at 3.525 × 10-5 or 1.766 × 10-5 M were placed in screwed-cap tubes. Then an amount of 4 mL of n-octanol was added. The tubes were placed in a shaking water bath at 37 °C. After 5 h of reaching equilibrium the tubes were centrifuged. The layers were separated, and the absorbances were read for the n-octanol and aqueous phases at a suitable λmax against n-octanol or the phosphate buffer of a corresponding pH. The partition coefficients were calculated as an average of 10 determinations each.16 Computational Details. Density functional theory (DFT) calculations with the hybrid Becke’s three-parameter functional17 and Lee-Yang-Parr exchange-correlation potential18 (B3LYP at 6-31+G(d) basis set19) were carried out to obtain equilibrium geometries of the lowest energy forms of AZA, MAZA, 6-TG, and 6-MP. Then the solvation model SM5.4 with AM1 and PM3 Hamiltonian implemented in the AMSOL package20 was utilized to calculate their solvation free energies in water, octanol, and n-hexadecane and to assess partition coefficients for individual solutes. Briefly, the solvation models (SMx) are semiempirical models that introduce into calculations the effects of solvents such as water,21 alkanes,22 chloroform,23 or others.24 In the SM5.4 terms responsible for cavity formation, dispersion, solvent structure, and local field polarization are present. The solvation energy is obtained via the usual approximation that solute treated at the quantum mechanical level is immersed in an isotropic, polarizable continuum representation of a solvent. In this work we used the SM5.4 model,21c in which the solute Hamiltonian is modeled using PM3 or AM1 molecular orbital theory with class IV atomic charges from the charge models.25 In this work we utilized the SM5.4 method to calculate partition coefficients between octanol and aqueous solutions according to

log Koctanol/water )

∆G°water - ∆G°octanol 2.303RT

(1)

where R is the universal gas constant and T is the temperature (298 K). ∆G is the weighted average of the values for individual tautomers.

Results and Discussion The only two stable conformers of AZA were identified previously26 and were used as starting conformers for geometry optimization at the B3LYP/6-31G+(d) level. Similar two conformers of MAZA were utilized for geometry optimization. In the case of 6-TG and 6-MP, several tautomeric structures were examined, including 6-thione ones that are observed in experiments27 and are of lowest energy in the calculations27c,28 (Scheme 1). To compare the electronic structure of the studied molecules, natural population analyses were performed. Table 1 presents point charges derived from such analyses for lowest energy structures of AZA and MAZA at the B3LYP/6-31+g(d) level. For the corresponding

Table 1. Point Charges Derived from Natural Population Analysis for AZA and MAZAa atom

cAZA

cMAZA

difference (cAZA - cMAZA)

N (1p) C (2p) N (3p) C (4p) C (5p) C (6p) N (7p) C (8p) N (9p) S N (1i) C (2i) N (3i) C (4i) C (5i) N (6i) O (NO2) O (NO2) C (7i) C (8i)

-0.5210 0.2411 -0.4918 0.3560 0.0199 0.0906 -0.4665 0.2194 -0.5855 0.4008 -0.3883 0.2038 -0.4472 0.2262 -0.0439 0.4797 -0.3921 -0.3546 -0.4760

-0.5220 0.2403 -0.4931 0.3550 0.0193 0.0871 -0.4667 0.2186 -0.5857 0.4091 -0.3884 0.4005 -0.4545 0.2274 -0.0440 0.4798 -0.3940 -0.3581 -0.4789 -0.7323

0.0009 0.0008 0.0014 0.0010 0.0006 0.0035 0.0002 0.0008 0.0002 -0.0082 0.0001 -0.1967 0.0073 -0.0013 0.0001 -0.0002 0.0018 0.0035 0.0029

a

Numbering scheme as in Chart 1.

Figure 1. Electrostatic potential around AZA (top) and MAZA (bottom) mapped on the surface of electron density of 0.0004 au is ranging from +0.06 (navy blue) to -0.06 (red).

atoms the differences in point charges between AZA and MAZA are small, ranging from -0.197 to 0.007. As expected, the largest difference (-0.197) is obtained for the C(2i) atom to which the additional methyl group is attached in MAZA. If this value is excluded, the differences in point charges between AZA and MAZA are in the range from -0.008 to +0.007 and the average unsigned difference equals 0.002, indicating that the charge distribution is very similar for the both molecules (see Figure 1 where electrostatic potentials around AZA and MAZA are presented.) For all the obtained structures of 6-TG, 6-MP, AZA, MAZA we performed solvation model calculations in water, octanol, and n-hexadecane, which allowed us to identify the predominant forms in solution for the molecules studied. In the case of 6-TG and 6-MP the thione forms are better solvated than the thiol forms by slightly over 10 kcal/mol in water, about 7 kcal/mol in octanol, and about 5 kcal/mol in n-hexadecane, indicating that the thione forms predominately in solution, which is in agreement with experimental findings in condensed media.27 In aqueous solution, the free energy of solvation of the lowest energy form of 6-TG is -35 kcal/mol if the geometry of the thione

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Table 2. Solvation Gibbs Free Energies for Aqueous (aq), Octanol (oc), and n-Hexadecane (hd) Solutions for 6-TG, 6-MP, AZA, and MAZA for Geometries Optimized in Vacuo Fixed and Relaxed in Solutions energy (kcal/mol) 6-TG

6-MP

AZA

MAZA

∆G°aq -34.6 -45.5 -29.1 -38.0 -24.3 -45.5 -23.8 -44.1 ∆G°oc -30.7 -41.5 -25.7 -34.7 -24.6 -46.2 -25.2 -45.8 ∆G°hd -19.0 -29.5 -16.4 -25.1 -17.9 -39.3 -18.6 -39.2

Table 3. Gibbs Free Energies Changes from Aqueous to Octanol (aq f oc) and Aqueous to n-Hexadecane (aq f hd) Solutions for 6-TG, 6-MP, AZA, and MAZA for Geometries Optimized in Vacuo Fixed and Relaxed in Solutions energy (kcal/mol) 6-MP

AZA

MAZA

fixed relaxed fixed relaxed fixed relaxed fixed relaxed ∆G°aqfoc 3.9 ∆G°aqfhd 15.6

4.0 16.0

3.4 12.7

3.3 12.9

-0.3 6.4

Table 4. Apparent Partition Coefficients for Octanol/Water Measured in pH 5.7 and 7.4 and Calculated Partition Coefficients for 6-TG, 6-MP, AZA, and MAZA log Koctanol/water compd exptl, pH 5.7 exptl, pH 7.4 calcd, SM5.4/P calcd, SM5.4/A

fixed relaxed fixed relaxed fixed relaxed fixed relaxed

6-TG

Brief Articles

-0.7 6.2

-1.4 5.2

-1.7 4.9

tautomer, optimized in gas phase, is fixed, and -45 kcal/ mol if the geometery of the molecule is allowed to relax in solution. For 6-MP the solvation free energies in aqueous solution is -29 kcal/mol for fixed geometry and -38 kcal/mol if the geometry from gas phase is allowed to relax. These values agree well with the values from previous SM1 and SM2 calculations.14a The lowest energy structure of AZA has solvation energy in water of -24 kcal/mol for the fixed geometry and -45 kcal/ mol for the relaxed geometry, while MAZA was solvated in water by about l kcal/mol worse than AZA. Free energies of solvation obtained with SM5.4/P method in aqueous, octanol, and n-hexadecane solutions are presented in Table 2. They differ only slightly from the results from the SM5.4/A method. Thus, we presented them in Supporting Information in Table S1 and based further discussion on the SM5.4/P results. In octanol solution solvation energies for fixed gasphase geometries are about -31, -26, -25, and -25 kcal/mol for 6-TG, 6-MP, AZA, and MAZA, respectively. Interestingly, 6-MP and 6-TG are better solvated in aqueous solution then in octanol, whereas AZA and MAZA are better solvated in octanol then water. For the n-hexadecane solvent and for fixed gas-phase geometries, solvation energies are in the range -16 to -19 kcal/mol. Table 3 presents Gibbs free energies changes upon going from aqueous to octanol and aqueous to n-hexadecane solutions. As is clear from Table 2 and even more from Table 3, the relaxation in solution of gas phase geometries from B3LYP/6-31G+G(d) calculations leads to an additional energy of solvation of constant value, independent of the solvent. Thus, the hypothesis of Lombardo et al. that “full relaxation of the geometry in the presence of solvent is expected to have only a minor influence on the geometry and energetics at considerably computational cost12” is only partially true. The full relaxation of gas phase geometry in solution introduces a constant difference in free energy of solvation. However, this allows us to select only one set of energies for further calculations of partitioning between different solvents. Here, we used free solvation energies obtained for geometries optimized at a relatively high level (B3LYP),

6-TG 6-MP AZA MAZA

-0.26 -0.17 0.18 0.27

-0.16 -0.37 -0.09 0.17

-2.90 -2.51 0.21 1.07

-3.71 -3.50 0.03 0.90

which takes into account dynamic electron correlation with a flexible basis set 6-31+G(d) that is augmented by diffuse and polarization functions. The changes in Gibbs free energies of solvation upon going from aqueous to octanol solution are about 4, 3.5, -0.5, and -1.5 kcal/mol for 6-TG, 6-MP, AZA, and MAZA, respectively. These data indicate that the hydrophilic character of the studied compounds increases in the order MAZA, AZA, 6-MP, 6-TG. Indeed, as seen in Table 3, partition coefficients for the studied compounds in pH 5.7 and apparent partition coefficients between octanol and aqueous solutions are increasing in the order 6-TG, 6-MP, AZA, MAZA, so the hydrophobicity of theses compounds is also increasing in this same way. Differences in the experimentally measured apparent partition coefficients were detected when different buffer solutions were used. As expected, at higher pH (7.4) all purines showed lower partition coefficients in comparison to the buffer solution with pH 5.7 because they undergo dissociation. The highest in the series is the partition coefficient for MAZA; thus, it is the most lipophilic compound among the purines studied. At pH 5.7, 6-MP has a higher partition coefficient (-0.17) than 6-TG (-0.26), while at pH 7.4 the apparent partition coefficients are -0.37 and -0.16, for 6-MP and 6-TG, respectively. The rationale for such a difference comes from different values of the acidic dissociation constant; the pKa for 6-TG is 8.52, while for 6-MP it is 7.72.16 It is clear from Table 4 that the calculated partition coefficients between octanol and water for the studied compounds predict the same trends in the hydrophobic character of the studied molecules. However, the actual numbers of the calculated and measured partition coefficients are far from each other. It is not very surprising. First, experimentally measured coefficients are apparent partition coefficients that take into account dissociated and undissociated species. Second, even small errors in calculating solvation energies are transferred, via eq 1, into large differences in concentrations in aqueous and octanol solutions. Last, in our calculations we neglected changes in conformational and tautomeric equilibria upon going from one solvent to another and the possibility of dimer formation in aqueous or organic solutions. It should be mentioned here that Cini et al.28 suggested that the observed dimer of 6-MP is relatively strong and its formation energy is comparable to guanine-cytosine Watson-Crick pairing energies. All in all, although the actual values of the calculated partitioning coefficients differ significantly from the apparent ones obtained from experimental measurements, the computationally inexpensive SM5.4 methods allow for fast assessment of the relative hydrophobicity/hydrophilicity of the studied compounds.

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Conclusions The calculated electrostatic potential around MAZA is very similar to that of AZA (Figure 1), indicating similarities between these molecules, which agrees very well with the reactivates of these compounds observed in experiments. However, the obtained results, allowing us to compare the hydrophilic and hydrophobic character of MAZA to that of the recognized drugs AZA, 6-MP, and 6-TG, identify the following sequence of hydrophobicity: MAZA > AZA > 6-TG > 6-MP. The recorded octanol/aqueous phases partition coefficients, reflecting to a certain extent processes of a passive drug transport in the body, suggest that MAZA is likely to cross cell membranes more easily than AZA. Therefore, the biological efficacy of MAZA is likely to be greater than that of AZA. These data indicate that MAZA is a good candidate as a drug, fully deserving further studies. Acknowledgment. The authors thank Professors D. G. Truhlar and C. J. Cramer for providing free academic license for AMSOL-6.5.3. The authors are grateful to Poznan´ Supercomputing and Networking Center for access to computer resources therein. Financial support from A. Mickiewicz University and University of Medical Sciences in Poznan´ Grant No. PU-II/9, the Foundation for Polish Science Grant No. 06/2003, and State Committee for Scientific Research (KBN) Grant No. T09 A18525 is gratefully acknowledged. Supporting Information Available: Comparison of the solvation free energies obtained with SM5.4/A and SM5.4P methods, results of measurements of 6-TG, 6-MP, AZA, and MAZA in n-octanol and aqueous phases, and Cartesian coordinates of the lowest energy structures for 6-MP, 6-TG, AZA, and MAZA after B3LPY/6-31+G(d) optimization. This material is available free of charge via the Internet at http:// pubs.acs.org.

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