Theoretical Electrostatic Properties of a Styrylquinoline

J. Phys. Chem. B 2006, 110, 537-547

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Experimental/Theoretical Electrostatic Properties of a Styrylquinoline-Type HIV-1 Integrase Inhibitor and Its Progenitors Delphine Firley,† Blandine Courcot,† Jean-Michel Gillet,† Bernard Fraisse,† Fatima Zouhiri,‡,§ Didier Desmae1 le,‡ Jean d’Angelo,‡ and Nour Eddine Ghermani*,†,| Laboratoire Structures, Proprie´ te´ s et Mode´ lisation des Solides, UMR CNRS 8580, Ecole Centrale Paris, Grande Voie des Vignes, 92295 Chaˆ tenay-Malabry Cedex, France, Laboratoire BIOCIS, UMR CNRS 8076, Faculte´ de Pharmacie, UniVersite´ Paris-Sud XI, 5, rue Jean-Baptiste Cle´ ment, 92296 Chaˆ tenay-Malabry Cedex, France, BioAlliance Pharma S.A. 59, BouleVard du Ge´ ne´ ral Martial Valin, 75015 Paris, France, and Laboratoire Physico-Chimie, Pharmacotechnie, Biopharmacie, UMR CNRS 8612, Faculte´ de Pharmacie, UniVersite´ Paris-Sud XI, 5, rue Jean-Baptiste Cle´ ment, 92296 Chaˆ tenay-Malabry Cedex, France ReceiVed: August 3, 2005

We have established that polyhydroxylated styrylquinolines are potent inhibitors of HIV-1 integrase (IN). Among them, we have identified (E)-8-hydroxy-2-[2-(4,5-dihydroxy-3-methoxyphenyl)-ethenyl]-7-quinolinecarboxylic acid (1) as a promising lead. Previous molecular dynamics simulations and docking procedures have shown that the inhibitory activity involves one or two metal cations (Mg2+), which are present in the vicinity of the active center of the enzyme. However, such methods are generally based on a force-field approach and still remain not as reliable as ab initio calculations with extended basis sets on the whole system. To go further in this area, the aim of the present study was to evaluate the predictive ability of the electron density and electrostatic properties in the structure-activity relationships of this class of HIV-1 antiviral drugs. The electron properties of the two chemical progenitors of 1 were derived from both high-resolution X-ray diffraction experiments and ab initio calculations. The twinning phenomenon and solvent disorder were observed during the crystal structure determination of 1. Molecule 1 exhibits a planar s-trans conformation, and a zwitterionic form in the crystalline state is obtained. This geometry was used for ab initio calculations, which were performed to characterize the electronic properties of 1. The electron densities, electrostatic potentials, and atomic charges of 1 and its progenitors are here compared and analyzed. The experimental and theoretical deformation density bond peaks are very comparable for the two progenitors. However, the experimental electrostatic potential is strongly affected by the crystal field and cannot straightforwardly be used as a predictive index. The weak difference in the theoretical electron densities between 1 and its progenitors reveals that each component of 1 conserves its intrinsic properties, an assumption reinforced by a 13C NMR study. This is also shown through an excellent correlation of the atomic charges for the common fragments. The electrostatic potential minima in zwitterionic and nonzwitterionic forms of 1 are discussed in relation with the localization of possible metal chelation sites.

Introduction Acquired immunodeficiency syndrome (AIDS) is one of the greatest challenges to humankind. All oral agents licensed to treat HIV-1 diseases target two of the three essential, virally encoded enzymes, reverse transcriptase (RT) and protease (PR).1 However, although the advent of combination therapy with HIV-1 RT and PR inhibitors has made it possible to suppress the replication of the virus in infected persons to such an extent that it becomes almost undetectable in the plasma for more than 2 years, HIV-1 persists in sanctuaries such as peripheral blood mononuclear cells or resting T-lymphocytes. This means that * Author to whom correspondence should be addressed. Phone: +33 (0)1 46 83 56 48. Fax: +33 (0)1 46 83 58 82. E-mail: [email protected]. † Ecole Centrale Paris. ‡ Laboratoire BIOCIS, UMR CNRS 8076, Faculte ´ de Pharmacie, Universite´ Paris-Sud XI. § BioAlliance Pharma. || Laboratoire Physico-Chimie, Pharmacotechnie, Biopharmacie, UMR CNRS 8612, Faculte´ de Pharmacie, Universite´ Paris-Sud XI.

AIDS can be temporarily controlled but not eradicated with current treatments.1-3 Therefore, additional therapeutic approaches are warranted. One such approach is to target the third viral enzyme, integrase (IN), that inserts the viral DNA into the cellular genome through a multistep process that includes two catalytic reactions, 3′-endonucleolytic processing of both ends of the native viral DNA consisting of the excision of a dinucleotide adjacent to a conserved CA sequence and joining of the 3′processed viral and host chromosomal DNAs (strand transfer).4 Divalent metal cations such as Mg2+ and Mn2+ are required both for 3′-processing and strand transfer and for the assembly of HIV-1 IN onto specific viral donor DNA to form a complex competent to carry out either function.5-7 Mg2+ is the likely cation cofactor in vivo. Recent molecular modeling was performed by docking various inhibitors into a structural model of full-length HIV-1 IN dimer complexed with donor DNA, suggesting that two divalent cations are involved in the catalytic cycle.8 HIV-1 IN is essential for retroviral replication, and the

10.1021/jp0582179 CCC: $33.50 © 2006 American Chemical Society Published on Web 12/10/2005

538 J. Phys. Chem. B, Vol. 110, No. 1, 2006

Firley et al. of particular importance in relation with the biological activity. This work is a part of our ongoing research devoted to this type of HIV-1 IN inhibitor. Experimental and Theoretical Procedures

Figure 1. Chemical structure of styrylquinoline 1.

absence of a host-cell equivalent of IN means that IN inhibitors do not interfere with normal cellular processes and therefore have a high therapeutic index. In this respect, we have reported that polyhydroxylated styrylquinolines (SQLs), exemplified by 1 (Figure 1), are potent HIV-1 IN inhibitors in in vitro experiments, block the replication of HIV-1 in cell culture, and are devoid of cytotoxicity.9-18 These properties make SQLs promising candidates for the development of therapeutic HIV-1 IN inhibitors. Although the exact mechanism by which drug 1 and analogues exert their inhibitory potency is still a matter of controversy, it recently has been proposed that such compounds might act prior to integration by preventing viral DNA-IN binding.19,20 Within the framework of SQLs, we have identified the 7-COOH and 8-OH (salicylic acid moiety) of the quinoline ring and the 4′OH (Figure 1) of the ancillary aromatic nucleus as critical pharmacophores for antiviral activity.10 SQLs were originally designed to chelate the divalent metal cation(s) in the catalytic core domain of HIV-1 IN. A modified docking protocol, consisting of coupling a grid search method with full energy minimization, has been specially formatted to study the interaction between SQLs and retroviral INs. The docking procedure shows that SQLs bind closely to the Mg2+ cation in the vicinity of the active site of the catalytic core of the protein and that the Mg2+-drug interaction most likely involves the salicylic acid moiety of the quinoline half of the inhibitor.11,21 This outcome was recently strengthened by the work of Ma et al.22 Structurally, SQL drugs are formed by a quinoline moiety connected to an ancillary aromatic nucleus by means of an ethylenic spacer. It was, however, shown that the replacement of this linker fragment by a variety of spacers has an important impact on both inhibitory potency and toxicity of the drugs.16 With the aim to assign the respective role played by each of the three subunits of SQLs, the present investigation deals with the characterization at the atomic level of drug 1 and its progenitors that are quinoline half 2 and benzaldehyde derivative 3 (Figure 2). The electron density distribution and the electrostatic properties (charges, electrostatic potential, dipole moments) were carefully analyzed. Such properties were derived from X-ray diffraction experiments at 100 K and compared to the results obtained from ab initio quantum mechanical calculations. Comparisons between intrinsic progenitor electrostatic properties and those of drug 1 are discussed. As shown from the abovementioned docking study,11,21,22 these electrostatic properties are

Figure 2. Synthesis of 1 from progenitors 2 and 3.

Crystallization, Data Collection, and Reduction. In this study, all of the diffraction data were collected at 100.0(1) K on a Bruker-SMART charge couple device (CCD) diffractometer using graphite monochromated Mo KR radiation (wavelength λ ) 0.71073 Å). Cooling to 100 K was achieved by an N2 gas stream device (Oxford Cryosystem). The area detector surface was placed at 4.02 cm from the crystal sample. Different data collection strategies were used for the three compounds under study as detailed in Table 1. The Lorentz-polarization correction and the integration of the diffracted intensities were performed with the SAINT software package.23 An empirical absorption correction was applied using the SADABS computer program.23 Finally, the SORTAV program was used for sorting and averaging equivalent and redundant data of high-resolution diffraction experiments performed for progenitors 2 and 3.24 Figure 2 illustrates the chemical synthesis of 1 from its progenitors. The (E)-8-hydroxy-2-[2-(4,5-dihydroxy-3-methoxyphenyl)-ethenyl]-7-quinolinecarboxylic acid (drug 1, hereafter compound I) crystals were obtained by cooling to room temperature a hot saturated solution in dimethyl sulfoxide (DMSO). Despite sustained efforts directed toward this crystallization, the large number of crystal samples that we have examined were invariably found to be of very small size, needleshaped, and twinned. The crystal used in the present study was mounted in a sealed glass capillary due to its instability in air. The diffraction data were collected as ω-scans (∆ω ) 0.25°) at one detector position (2θ ) -28°) where θ is the Bragg angle. An exceptional exposure time of 120 s per frame was used due to the weak diffraction power of the sample. The maximum resolution reached for this experimental data set is (sin θ/λ)max ) H/2 ) 0.65 Å-1, where H is the Bragg vector modulus. A twin analysis of all reflections was carried out using the GEMINI program.25 This reveals a nonmerohedral twinning with two crystal components and a twofold twin operation along the reciprocal axis c*. A total of 4310 unique reflections (I > 2σ(I)) were used in the structure refinements, σ(I) being the estimated standard deviation of the diffracted intensity I. The 8-hydroxy-2-methyl-quinoline-7-carboxylic acid (progenitor 2, hereafter compound II) crystals were obtained by slow evaporation of water/acetic acid (1:4) solution at room temperature. The crystal was also placed in a sealed glass capillary due to instability in air. The diffraction data were collected at different detector positions: 2θ ) -23°, (45°, (60°, (75°, (85°. The data spots were recorded as ω-scans (∆ω ) 0.20°) to reconstruct accurate three-dimensional diffracted intensity profiles. According to the θ dependence of the diffracted intensities, the chosen exposure times were, respectively, 5, 10, 15, 30, and 60 s per frame for the detector positions given above.

SQL HIV-1 Integrase Inhibitors

J. Phys. Chem. B, Vol. 110, No. 1, 2006 539

TABLE 1: Details of the Experimental Diffraction Data Collections and Refinements chemical formula molecular weight crystal size (mm3) color, form cell setting, space group a (Å) b (Å) c (Å) R (deg) β (deg) γ (deg) V (Å3) Z Dx (Mg m-3) radiation type µ (mm-1) T (K) (sin θ/λ)max (Å-1) diffractometer data collection method measured reflections unique reflections Rint (%) index ranges

R(F2 > 2σ(F2)) (%) wR(F2) (%); S reflections used (F2 > 2σ(F2)) number of parameters (∆/σ) max ∆Fmax, ∆Fmin (eÅ-3) reflections used (F2 > 3σ(F2)) R[F] (%) Rw[F] (%) goodness of fit

I

II

C19H15NO6‚H2O‚(C2H6SO)2 527.6 0.25 × 0.08 × 0.08 red, needle monoclinic, P21/c 7.096(2) 19.114(7) 17.902(5)

C11H9NO3.C2H4O2 263.2 1.0 × 0.45 × 0.45 yellow, prism triclinic, P1h 6.9937(7) 9.4230(9) 9.7841(9) 68.546(2) 85.286(2) 77.742(2) 581.9(2) 2 1.47 Mo KR 0.114 100.0(1) 1.18 Bruker SMART CCD ω-scans 116 729 14 519 1.87 -15 f h f 16 -22 f k f 22 -22 f l f 22

741.46(3) 4 1.50 Mo KR 0.122 100.0(1) 1.10 Bruker SMART CCD ω-scans 64 887 6953 2.09 -13 f h f 13 -29 f k f 29 -18 f l f 18

4.00 10.5; 0.98 11 596 224 0.0020 0.68, -0.31

3.52 9.4; 0.88 5455 142 -0.0020 0.58, -0.29

10 859 1.75 1.90 0.89

4206 2.05 1.78 0.80

91.44(2) 2427.5 (2) 4 1.38 Mo KR 0.270 100.0(1) 0.65 Bruker SMART CCD ω-scans 36 387 6824 -9 f h f9 -24f k f 24 -23 f l f 23

III C8H8O4 168.1 0.75 × 0.18 × 0.04 white, prism monoclinic, P21/c 6.2088(1) 14.2712(3) 8.4469(2) 97.836(1)

spherical refinement 8.66 26.8; 1.03 4310 340 0.0030 1.07, -1.40 multipole refinement

The maximum resolution reached for this data set is (sin θ/λ)max ) 1.18 Å-1. Exactly 11 596 unique reflections (I > 2σ(I)) were used in the conventional refinement, and 10 859 reflections (I > 3σ(I)) for the electron density refinement. The diffraction data for 3,4-dihydroxy-5-methoxybenzaldehyde (progenitor 3, hereafter compound III) were collected at four different detector positions: 2θ ) -28°, +45°, -60°, +75°. The data spots were recorded as ω-scans (∆ω ) 0.20°). The chosen exposure times were, respectively, 15, 30, 60, and 90 s per frame for the four detector positions. The maximum resolution was (sin θ/λ)max ) 1.10 Å-1. Exactly 5455 unique reflections (I > 2σ(I)) were used in the structure refinement, and 4206 reflections (I > 3σ(I)) for the electron density refinement. Structure and Electron Density Refinements. The three crystal structures were solved and refined using the WINGX software package.26 For compound I, the statistical factor before the twin analysis remains high (R ) 16.3%). When the twinning effect was taken into account, R dropped to 8.66% (Table 1). The ratio of the two twin components refined to 0.313(2). The crystallographic structure revealed an s-trans zwitterionic form of the molecule in the solid state. The attached proton of the quinoline nitrogen atom was located by Fourier difference synthesis. All other hydrogen atoms were positioned theoretically with geometrically fixed distances, except those of the hydroxyl groups of the ancillary nucleus (Figure 3). The crystal structures of compounds II and III were determined unambiguously (Table 1). Figure 3 gives the atom-

numbering system for the three molecules isolated from the crystal lattice. For compounds II and III, conventional and electron density refinements were carried out using the MOLLY program based on the Hansen-Coppens multipole model.27 The frozen core and valence spherical densities are calculated from the Hartree-Fock free atom wave functions.28 In this study, the ξl exponents (in bohr-1) of the radial functions were chosen to be equal to 3.0, 4.5, and 3.8 and nl ) 2, 2, and 3 up to the octupole level (l ) 3) for C, O, and N atoms, respectively; ξl ) 2.26 bohr-1, and nl ) 1 (dipole level, l ) 1) for the hydrogen atoms.29 All of the multipole parameters were obtained by the least-squares fit to the observed X-ray diffraction structure amplitudes F. Before the electron density refinement, the atomic positions and anisotropic thermal displacements for C, O, and N were estimated on the basis of high-order data (sin θ/λ g 0.8 Å-1 ). Their attached hydrogen atoms were extended to the neutron diffraction distances (Caromatic-H ) 1.08 Å, Cmethyl-H ) 1.07 Å, N-H ) 1.01 Å, Ohydroxyl-H ) 0.96 Å). All of these structural and thermal parameters were relaxed in the last cycles of the refinements. Figure 4 displays the residual electron density maps obtained after the multipole refinements. In these maps, the absolute residues of the electron density do not exceed 0.20eÅ-3 (this limit is observed for compound II), attesting the good convergence of the refinements. For comparison, the experimental errors in the electron density are 〈σ2(∆F)〉1/2 ) 0.025eÅ-3 for II and 0.038eÅ-3 for III and 〈σ2res〉1/2 ) 0.024eÅ-3 for II and 0.032eÅ-3 for III. 30,31


Firley et al. For comparison with experimental electron deformation density maps (∆F(r) ) Fmolecule(r) - Fpromolecule(r)), we developed a routine using the Mathematica 5.0 software.35 Each promolecule electron density (superimposition of spherically symmetrical densities of isolated atoms) was computed from the same basis set as for the calculation of the molecular density to minimize the errors due to the basis set dependency. Electrostatic Potential. The electrostatic potential V(r) is a measure of the electrostatic energy experienced by a unit point charge approaching the chemical system. V(r) is the sum of the negative contribution generated by the electron density and the positive one due to the nuclear charges. The electrophilic and nucleophilic characteristics of a molecule can then be revealed by the electrostatic potential. In many cases, the interaction energy of molecular systems is dominated by the electrostatic part. This makes V(r) a predictive property of particular importance for the quantification of the chemical reactivity of molecules. In the present study, once the electron density was determined from the experiment and theory, the electrostatic potential was generated on a three-dimensional (3D) grid around the molecules using the ELECTROS program36 and the corresponding routine implemented in the Gaussian 03 package,32 respectively. The MOLEKEL graphic software has been used for visualizing and plotting the electrostatic potential.37

Figure 3. ORTEP view of compounds I, II, and III with the atomnumbering systems.

Quantum Mechanical Calculations. Ab initio singlemolecule calculations for all three molecules were performed at the Hartree-Fock (HF) level using the program Gaussian 03.32 The atomic coordinates of molecules II and III are those obtained from the X-ray data refinements. For compound I, however, the missing hydrogen atoms of the hydroxyl groups in the crystal structure determination were added manually, and their positions were optimized according to energy minimizations. The theoretical calculations were performed in a vacuum using for the HF level the standard molecular split valence 6-31G++** basis set. DFT calculations were carried out using the B3LYP basis set.33,34

Results and Discussion Structural Analysis. Compound I crystallizes in the P21/c monoclinic space group with one water molecule and two disordered dimethyl sulfoxide (DMSO) molecules in the asymmetric unit. The carboxyl group was found to be ionized, and the quinoline ring nitrogen atom was found to be protonated, emphasizing the zwitterionic form of I in the solid state. The corresponding H1 atom (Figure 3) is engaged in a hydrogen bond with a water molecule characterized by a donor-acceptor distance D‚‚‚A ) 2.802(6) Å. A strong intramolecular bond connects O2 and O3 atoms (O2‚‚‚O3 ) 2.488(5) Å). This belongs to the type of resonance-assisted hydrogen bond (RAHB) described theoretically by Wojtulewski and Grabowski.38 The quinoline and ancillary aromatic rings of molecule I are almost in the same plane, the torsion angle around the C10C11 connecting bond being close to 180° (C1′-C11-C10C2 ) 179.8(4)°). Molecule II also crystallizes in a zwitterionic state. The space group is P1h, and the asymmetric unit contains one acetic acid solvent molecule. The H1 atom of the NH+ group is involved in a hydrogen bond with one solvent molecule [D‚‚‚A ) 2.7771(1) Å). Both carboxylic O1 and O2 atoms participate in intermolecular hydrogen bonds. As in I, the carboxylate and hydroxyl groups are also connected via a strong intramolecular hydrogen bond: O2‚‚‚O3 ) 2.4516(2) Å. Molecule III crystallizes in the P21/c space group. Both H3 and H4 atoms (Figure 3) are involved in two intramolecular hydrogen contacts of the hydroxyl groups (H3‚‚‚O3 ) 2.2432(3) and H4‚‚‚O4 ) 2.3483(2) Å). Table 2 compares the bond lengths, angles, and torsion angles of the three molecules in their respective crystals. Although less precise, the geometrical features of I are in good agreement with those derived from multipole electron density refinements of compounds II and III. For example, the C-N and C-O bond distances have similar trends, emphasizing the zwitterionic character of I and II. In this case, the protonation of the quinoline nitrogen atom is also characterized by the increasing C-N-C angle value (C2-N-C8a ) 123.7(4)° in I and 123.09(1)° in II) as recently reported by Dobson and Gerkin39 and Okabe and Muranishi40 for protonated quinoline rings.



Figure 4. Residual electron density of molecules II and III, contour intervals 0.10eÅ-3. Negative contours are dashed.

TABLE 2: Selected Geometric Parameters (Å and deg)a I

II

III

bond O4′-C5′ O3′-C4′ O2′-C3′ C2-N C8a-N C9-O1 C9-O2 C8-O3 C2-C10 C7-C9 C11-C10 C1′-C11 O4′-C7′

1.369(6) 1.353(6) 1.369(6) 1.338(7) 1.384(6) 1.249(6) 1.276(6) 1.336(6) 1.437(7) 1.502(7) 1.347(7) 1.464(7) 1.412(7)

C5′-O4′-C7′ C2-N-C8a N-C2-C10 C2′-C1′-C11 C7-C8-O3 O1-C9-O2

117.0(4) 123.7(4) 117.8(4) 123.1(4) 123.9(4) 123.8(4)

O4-C5 O3-C4 O2-C3

1.3624(4) 1.3510(4) 1.3584(4)

C1-C7 O4-C8

1.4586(4) 1.4322(4)

C5-O4-C8

116.24(2)

C2-C1-C7

117.25(3)

1.3356(2) 1.3730(2) 1.2561(2) 1.2772(2) 1.3304(2) 1.4921(2) 1.4978(2)

angle 123.09(1) 119.24(2) 123.08(1) 124.23(2)

torsion angle C1′-C11-C10-C2 179.8(4) C6-C7-C9-O2 178.9(4) 178.01(4) C4′-C5′-O4′-C7′ -174.0(4) C4-C5-O4-C8 -174.38(3) a

Standard deviations are given in parentheses.

Electron Deformation Density. Figure 5 depicts the experimental static electron deformation density maps of molecules II and III (STATDENS program)36 on one hand and those obtained from ab initio Hartree-Fock calculations for isolated molecules on the other hand.32 These maps are comparatively similar in features of C-C, C-N, and C-H bonds, displaying an average accumulation of electron density around 0.60.7eÅ-3 as generally found in organic molecules. However, some discrepancies appear for the oxygen atom lone pairs and for the C-O bonds in both compounds. The electron density around the oxygen atoms is more contracted in the experimental maps as shown for O1 and O2 of the carboxylate group in II and also for O3 involved in the intramolecular hydrogen bond. In the latter case, the electron concentration is much more pronounced in the O3-H31 bond in the theoretical maps. Conversely, in the experimental map, the O3 lone pair is rather polarized toward the NH+ group for which H1 is involved in a hydrogen bond with the solvent molecule in the solid state.

When experimental and theoretical results are compared, such effects of crystal environment and of the pseudoatom model limitations have also been reported by Volkov et al.41 and Coppens and Volkov.42 In molecule III, quantitative differences exist for the hydroxyl electron densities. Indeed, the experimental electron deformation density peak heights in C3-O2, C4-O3, C5-O4, and O4-C8 do not exceed 0.3eÅ-3 compared to 0.6eÅ-3 found in the theoretical maps. The same remarks hold for the oxygen lone pair electron density accumulations. Difference Densities from Theoretical Population Matrices. The ab initio HF electron deformation density of isolated molecule I is shown in Figure 6. The theoretical electron deformation densities of both quinoline and ancillary aromatic ring parts resemble those shown in Figure 5. To investigate further the specificities of each chemical progenitor II and III within the final molecule I, another type of difference density was performed, the purpose being to detect the differences in the charge density for II and III in a vacuum on one hand and when they are embedded in I on the other hand. For this purpose, an original procedure was adopted. Given the fact that the geometries are weakly but somewhat significantly different, no straightforward charge density differences could be envisaged. However, HF theoretical charge densities are calculated using a set of nuclei-centered atomiclike basis functions {Φi} with

F(r) )

PijΦi(r)Φj(r) ∑ i,j

(1)

where Pij values are the elements of the matrix. Given their atomiclike nature, basis set functions are rather localized and cross contributions (i not equal to j in eq 1), particularly those involving remote atoms, are expected to be quite weak. Some basis set functions can then arbitrarily be separated into functions well identified to one or the other end of molecule I, thereby being identical to those found in II and III. Electron density variations can therefore be evaluated, not from the difference of charge distributions, but from differences of populations submatrices obtained by a projection of the full matrices onto the subspace of basis functions common to the two molecules to be compared. As an example, let us consider the case of isolated molecule II and the behavior of its common part in I. Ab initio calculations of the latter yield a rather large population matrix PIij; meanwhile calculations of II provide us


Firley et al.

Figure 5. Electron deformation density of molecules II and III: top panel, from experiment; bottom panel, from theory. Contour intervals are 0.10eÅ-3; negative contours are dashed.

Figure 6. Theoretical electron deformation density of molecule 1. Contour intervals are 0.10eÅ-3; negative contours are dashed.

with a smaller one, PIIij . It is clear that, since the geometries slightly differ, few basis functions can rigorously be considered identical from the atomic positions point of view. However, all of the functions, for a given pair of equivalent atoms in both molecules, have exactly the same mathematical expressions and only differ in the weak atomic position shifts experienced between the two geometries. It was therefore believed that a quantity such as

δFII-I(r) )

∑

(PIIij - PIij) Φ ˜ i(r)Φ ˜ j(r)

(2)

i,j∈[I∩II]

where [I ∩ II] means that basis functions on which the submatrices are expressed are those identified as “genuine members” of both I and II is not only trustworthy but meaningful as long as one concentrates on the region expected

to be the most chemically similar. Equation 2 implies an arbitrary choice (I or II) for locating the basis functions, Φ ˜ i. We thereafter decided to use the geometry of the progenitor, and other choices proved to yield very similar results. Figure 7 displays the graphical results of these comparisons. Given the weakness of the “embedding effect”, rather small contour values (0.02eÅ-3) were chosen. The regions situated at the right of molecule II and at the left of III correspond to spurious difference densities due to the truncation of the population matrices and should therefore be discarded. What appears quite clearly in Figure 7 is the weak perturbation encountered in molecule II. As it turns out, σ-type electrons show little change with the exception of the C5-C6 bond and the attached H5 and H6 atoms. A quite weak but clear change in the polarization of O1 and O3 lone pairs can also be observed.



Figure 7. Theoretical electron difference densities of molecules II and III with their equivalent embedded into I: top panel, in the molecular plane; bottom panel, 0.5 Å above the molecular plane. Contour intervals are 0.02eÅ-3; negative contours are dashed.

No significant π-type electron contributions can be spotted in the plane parallel at 0.5 Å above the quinoline ring (bottom of Figure 7). The situation in molecule III is rather different in that the σ-type electrons exhibit changes on most of the atoms of the extreme right such as O2, H3, O3, H4, C8, and its connected H81, H82, and H83 atoms (Figure 7). A weak effect can be noted on the ring itself. A π-electron transfer is detected mostly in the O4-C8 region (bottom of Figure 7). Again, this lack of effect on the ring is confirmed by the population analysis, and on the opposite, strong charge fluctuations are observed on the O4-C8 region as well as around O3. The features observed on O2 in the σ-plane are quite isolated. None of these density fluctuations can be related to significant integrated atomic charge variations and can therefore only be attributed to a mere atomic polarization. These considerations strongly support the picture of almost two independent fragments, and the activity of molecule I seems to be mostly driven by its spatial conformation. Electrostatic Potential. Figures 8 and 9 display the molecular surfaces of the investigated compounds colored in accordance with the electrostatic potential. These surfaces correspond to the isodensity value of 0.007eÅ-3 (0.001 a.u.) as initially defined by Bader et al.43 This representation is of particular interest to highlight the noncovalent interactions occurring at the molecular surface (electrostatic complementarities, van der Waals interactions, and so on). The most nucleophilic regions (negative electrostatic potential) are in red, and the most electrophilic

regions (positive electrostatic potential) are in blue. The gradient vector of the electrostatic potential is characterized by the width of the consecutive colored stripes corresponding to intermediate isovalues. The molecular volumes and the extrema of experimental and theoretical electrostatic potentials for the three molecules are reported in Table 3. Only HF calculation results are presented here since they are very similar to those derived from the DFT method. As expected, the experimental molecular volumes for II and III are smaller than those calculated for isolated molecules according to the contraction of the electron density in the condensed state. For the sake of comparison, we have chosen as a reference the largest molecular surface derived from the HF calculations to be colored in concordance with the electrostatic potential. On these surfaces, we note that both minimum and maximum absolute magnitudes are higher for the experimental electrostatic potential due to the polarization of the molecular electron density induced by the crystal field. This effect is clearly shown in Figure 8 for both molecules II and III displaying a higher experimental electric field on the molecular surfaces. As also can be seen in Figure 8 for II, the negative region of the electrostatic potential is extended around the carboxylate and O3-H31 hydroxyl groups, with a minimum value of -0.389eÅ-1 found in the vicinity of the O1 atom (Figure 3). From theoretical calculations, however, the corresponding negative area is limited to the proximity of the carboxylate oxygen atoms with a minimum Vmin ) -0.269eÅ-1 found also close to O1.


Firley et al.

Figure 8. Isodensity molecular surface (0.007eÅ-3) colored in accordance with the electrostatic potential of II and III: top panel, from experiment; bottom panel, from theory. Blue and red areas correspond to +0.13 and -0.13eÅ-1, respectively. The orientations of the molecules are the same as in Figure 3.

For III, the differences in absolute magnitudes of the extrema of experimental and theoretical electrostatic potentials are less pronounced than in II (Table 3). However, the narrow stripes of the molecular surface experimental electrostatic potential isovalues reveal a high gradient in the crystalline environment. A large negative area of the experimental electrostatic potential surrounding the O2-H3 hydroxyl group extends to the center of the aromatic ring, as can be seen in Figure 8. This was not observed from the theoretical calculations that yield a nearly flat electrostatic potential in this region. The minima reported in Table 3 for molecule III are found in the vicinity of O2 (Vmin ) -0.156eÅ-1) from experiment and of O1 (Vmin ) -0.128eÅ-1) from theory, respectively. Figure 9 displays the HF electrostatic potential generated on the molecular surface of I. For quantitative comparison with II and III, the same potential range limits were chosen corresponding to -0.13 and +0.13eÅ-1, respectively. The most nucleophilic region (double minima of -0.281eÅ-1 found close to both O1 and O2 in good agreement with Vmin ) -0.269eÅ-1 found close to O1 in II) surrounds the carboxylate group of the molecule, and the area corresponding to the cutoff of -0.13eÅ-1 is much more extended than in II. In the right part of I, a minimum of the electrostatic potential is observed in the vicinity of the O2′ atom (V ) -0.072eÅ-1). The previous outcomes relate to the zwitterionic form of molecule I as it stands in the solid state. In a further study, we have carried out HF calculations (with the same basis set as for I) for the nonzwitterionic form obtained by attaching a hydrogen

atom to O2 of the acid group, removing H1, and rotating H31 to the right side toward N. The molecular surface electrostatic potential is displayed on the bottom of the left column in Figure 9. In this case, the extrema of the electrostatic potential are Vmin ) -0.135eÅ-1 and Vmax ) +0.177eÅ-1, respectively. The electrostatic potential became rather flat on both centers of the quinoline and ancillary phenyl rings. The electrophilic region is largely reduced in comparison to that displayed by the molecular electrostatic potential of zwitterionic molecule I (top of the left column in Figure 9). The most nucleophilic areas are now located in the vicinity of O1, between O2 and O3, and around O2′ (Figure 3), respectively. Besides the molecular surface electrostatic features, the right column in Figure 9 compares the zwitterionic and nonzwitterionic forms using the isopotential surfaces. In the former, a large negative electrostatic potential isosurface surrounds the carboxylate group. In the nonzwitterionic form, however, the corresponding region is split into two parts on each side of the carboxylic O2 atom. This pincerlike shape of positive and negative potential arising between O2 and O3 and their attached hydrogen atoms would increase the ability to chelate positive ions such as metals. Atomic Charges and Dipole Moments. From the considerations given above, it follows that when I is formed in its zwitterionic state, the left part corresponding to II (also a zwitterion) conserves to some extent its own electrostatic properties, especially the nucleophilic character of the salicylic acid moiety. It is not the case for the right part corresponding to III. As can be expected, the removal of the O1 atom from



Figure 9. Electrostatic potential of 1 obtained from HF calculations: top panel, zwitterionic form (I); bottom panel, nonzwitterionic form. The left column contains the isodensity molecular surface (0.007eÅ-3) colored in harmony with the electrostatic potential; blue and red areas correspond to +0.13 and -0.13eÅ-1, respectively (see color chart in Figure 8). The right column contains isopotential surfaces: gray, +0.30eÅ-1; red, -0.10eÅ-1. The orientation of the molecule is the same as in Figure 3.

TABLE 3: Experimental and Theoretical Molecular Volumes Corresponding to the Isodensity Surface of 0.007eÅ-3 a I molecular volume

(Å3

)

Vmax (eÅ-1) Vmin (eÅ-1)

exp HF exp HF exp HF

398.39 0.213 -0.281

II

III

carbon atom

1

2

4

5

213.91 236.24 0.492 0.228 -0.389 -0.269

178.46 195.59 0.241 0.178 -0.156 -0.128

2 3 4 4a 5 6 7 8 8a 9

160.8 121.5 139.6 130.6 113.5 127.3 112.8 153.0 135.8 170.9

160.8 125.0 140.9 130.4 112.8 127.5 112.6 156.2 135.0 170.7

160.2 121.8 138.3 131.0 115.6 126.6 111.5 153.7 137.4 171.6

159.0 122.3 138.0 133.5 116.9 126.7 111.1 153.4 137.2 171.7

a

The maximum (Vmax) and minimum (Vmin) electrostatic potential values are given on the HF surface chosen as a reference.

TABLE 4: Theoretical HF and DFT Dipole Moments (D) for the Three Compounds from total density

TABLE 5: 13C NMR Shifts (ppm) for Compounds 1, 2, 4, and 5 (in d6-DMSO)a

from ChelpG charges

compound

HF

DFT

HF

DFT

II III I (zwitterionic form) I (nonzwitterionic form)

20.37 3.96 25.69 8.09

18.23 4.55 24.23 7.96

20.29 3.88 25.55 8.07

18.12 4.46 24.04 7.91

III should a priori perturb the electron distribution in the rest of the molecule. These assumptions would be confirmed by comparing the net atomic charges derived from the theoretical electrostatic potential for the three molecules. We used the method “charges from electrostatic potential grid” (ChelpG charges) developed by Breneman and Wiberg44 to retrieve the atomic charges. These charges are also able to estimate the dipole moments of the molecules as can be seen in Table 4. As expected, we can see that a large difference in dipole magnitudes appears between the zwitterionic and the nonzwitterionic forms of I. It is noteworthy that, for the most polar molecules

a 13 C NMR spectra were recorded on a Bruker AC 200 P spectrometer (50 MHz for 13C, 300 K).

(compounds II and I in zwitterionic form), HF and DFT methods yield different values of dipole moments. Figure 10 depicts the correlation between the charges of the equivalent atoms in I, II, and III, respectively. The two connecting carbon atoms that are C10-methyl of II (future C10-ethylenic center of I) and C7-carbonyl of III (future C11-ethylenic center of I) are ignored in the present correlation. Surprisingly, the linear regression fits to the paired data reveal excellent statistical factors: R ) 99.7%, root-mean-square deviation (rmsd) ) 0.04, slope ) 1.067 (I vs II) and R ) 98.4%, rmsd ) 0.07, slope ) 1.052 (I vs III) for HF charges. The highest atomic charge discrepancies were found for C1′/C1 (0.168e in I and -0.041e in III from HF calculations) and C6′/C6 (-0.441e in I and -0.297e in III from HF calculations) as shown in Figure 10. The same remarks hold for the correlation of charges obtained from the DFT method. Except couples C1′/C1 and C6′/C6, the weak local difference densities depicted in Figure 7 and


Firley et al. almost linearly with the electron density at the individual carbon,45 the hypothesis that a push-pull-type electron delocalization relayed by the ethylenic linker might occur between the ancillary aromatic nucleus and the quinoline half of the SQLs should be discarded. Conclusion

Figure 10. Correlation between ChelPG theoretical atomic charges of the quinoline half and ancillary aromatic nucleus of molecule I and the corresponding progenitors II and III.

commented in the previous section seem to have no incidence on the net atomic charges derived from the electrostatic potential. Furthermore, assuming these atomic charges as partitioning indexes of the electron density, it can be argued that the ethylenic linker blocks any charge transfer between the two parts of I. 13C NMR Study. To assess the last statement, a comparative 13C NMR study was undertaken. The chemical shifts obtained for drug 110 (harboring a π-excessive ancillary aromatic nucleus), its analogues 4 and 510 (in which the ancillary nuclei are neutral and π-deficient, respectively), and progenitor 2, were compared (Figure 11 and Table 5). Regarding the 10 carbon atoms of the quinolinecarboxylic acid moiety of these molecules, only very discrete chemical shift changes were observed. Within SQL series (compounds 1, 4, and 5), the following standard deviations were found: ca. (0.4 ppm for carbon atoms 3, 6, 8, and 9; ca. (1 ppm for carbon atoms 2, 4, 7, and 8a; ca. (1.5 ppm for carbon atoms 4a and 5. This clearly reveals no significant shielding/deshielding of the quinoline part. Since the 13C chemical shift of heteroaromatic compounds correlates

Figure 11. Chemical structure of styrylquinolines 4 and 5.

High-resolution X-ray diffraction experiments and ab initio theoretical calculations have been used to derive the electron density and electrostatic properties of the progenitors of a potent HIV-1 IN inhibitor, (E)-8-hydroxy-2-[2-(4,5-dihydroxy-3-methoxyphenyl)-ethenyl]-7-quinolinecarboxylic acid (1). This gave the opportunity to highlight the specific electron properties of the quinoline and ancillary nucleus parts embedded in 1, in relation with the putative activity of a pharmacophore. Although the experimental and theoretical electron densities of progenitors were similar in comparison, the experimental electrostatic potential, strongly influenced by the crystal field and by the limitations of the multipole model, did not agree as well with the calculated one. However, the comparison of the theoretical electrostatic potential features of progenitors and those of drug 1 has revealed the absence of any effect of electron delocalization through the ethylenic linker. Accordingly, the atomic charge values derived from the electrostatic potential remained almost unchanged for corresponding atoms in both progenitors and drug 1. This observation was reinforced by the 13C NMR study of a variety of SQLs, showing that the electron excess or deficiency at the ancillary aromatic nucleus did not affect the quinoline part. This result is of particular significance for the future development of potent IN inhibitors based on SQLs. HIV-1 IN belongs to a superfamily of polynucleotidyl tranferases that all require Mg2+ ions as cofactors to achieve phosphodiester bond cleavage of DNA. Three-dimensional structures of a variety of polynucleotidyl tranferase-Mg2+ ion(s) complexes were determined by X-ray crystallography.46-50 In all cases, the Mg2+-binding site, essential for activity, is located near a cluster of aspartic/glutamic acid residues, suggesting an electrostatic interaction between the cation(s) and the negatively charged carboxyl groups. These observations reinforce the hypothesis that inhibition of HIV-1 IN by SQLs might be due to the functional sequestration of the critical Mg2+ cofactor by their salicylic part. Accordingly, the most nucleophilic regions of the electrostatic potential generated for both zwitterionic and nonzwitterionic forms of drug 1 were found in the vicinity of the salicylic fragment of the quinoline moiety. However, recent studies of molecular dynamics51 and drug docking in the active site of the enzyme52 suggest that SQLs might inhibit IN at its interface with viral DNA and divalent metal. To assess the validity of this model, further investigations including the elaboration and crystallographic characterization of SQL-divalent metal complexes are under way. Finally, it should be pointed out that the outcomes of the present study constitute a significant advance in the rationalization/prediction of SQL structure-activity relationships that could be exploited to design more potent and more selective HIV-1 IN inhibitors.

SQL HIV-1 Integrase Inhibitors Acknowledgment. The financial support of the CNRS, Ecole Centrale Paris, and Universite´ Paris XI is gratefully acknowledged. The authors thank Dr P. Roussel (Universite´ de Lille, France) for his help and advise in the data processing for the twinned crystal. Supporting Information Available: Fractional coordinates (multiplied by 105) and atomic thermal parameters (multiplied by 105) for C19H15NO6‚H2O‚(C2H6SO)2 (compound I), fractional coordinates (multiplied by 105) and atomic thermal parameters (multiplied by 105) from the multipole refinements for C11H9NO3.C2H4O2 (compound II), κ, κ′, Pval, and Plm multipole parameters for C11H9NO3.C2H4O2 (compound II), fractional coordinates (multiplied by 105) and atomic thermal parameters (multiplied by 105) from the multipole refinements for C8H8O4 (compound III), κ, κ′, Pval, and Plm multipole parameters for C8H8O4 (compound III), and theoretical CHelPG atomic charges in e units. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) De Clercq, E. Med. Res. ReV. 2002, 22, 531-565. (2) Zhang, L.; Ramratnam, B.; Tenner-Racz, K.; He, Y.; Vesanen, M.; Lewin, S.; Talal, A.; Rasz, P.; Perelson, A.; Korber, B.; Markowitz, M.; Ho, D. N. Engl. J. Med. 1999, 340, 1605-1613. (3) Furtado, M.; Callaway, D.; Phair, J.; Kunstman, K.; Stanton, J.; Macken, C.; Perelson, A.; Wolinsky, S. N. Engl. J. Med. 1999, 340, 16141622. (4) Brown, P. O. Curr. Top. Microbiol. Immunol. 1990, 157, 19-48. (5) Ellison, V.; Brown, P. O. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7316-7320. (6) Vink, C.; Lutzke, R. A. P.; Plasterk, R. H. A. Nucleic Acids Res. 1994, 22, 4103-4110. (7) Wolfe, A. L.; Felock, P. J.; Hastings, J. C.; Uncapher Blau, C.; Hazuda, D. J. J. Virol. 1996, 70, 1424-1432. (8) Marchand, C.; Johnson, A. A.; Karki, R. G.; Pais, G. C. G.; Zhang, X.; Cowansage, K.; Patel, T. A.; Nicklaus, M. C.; Burke, T. R., Jr.; Pommier, Y. Mol. Pharmacol. 2003, 64, 600-609. (9) Mekouar, K.; Mouscadet, J.-F.; Desmae¨le, D.; Subra, F.; Leh, H.; Savoure´, D.; Auclair, C.; d’Angelo, J. J. Med. Chem. 1998, 41, 28462857. (10) Zouhiri, F.; Mouscadet, J.-F.; Mekouar, K.; Desmae¨le, D.; Savoure´, D.; Leh, H.; Subra, F.; Le Bret, M.; Auclair, C.; d’Angelo, J. J. Med. Chem. 2000, 43, 1533-1540. (11) Ouali, M.; Laboulais, C.; Leh, H.; Gill, D.; Desmae¨le, D.; Mekouar, K.; Zouhiri, F.; d’Angelo, J.; Auclair, C.; Mouscadet, J.-F.; Le Bret, M. J. Med. Chem. 2000, 43, 1949-1957. (12) d’Angelo, J.; Mouscadet, J.-F.; Desmae¨le, D.; Zouhiri, F.; Leh, H. Pathol. Biol. 2001, 49, 237-246. (13) Burdujan, R.; d’Angelo, J.; Desmae¨le, D.; Zouhiri, F.; Tauc, P.; Brochon, J.-C.; Auclair, C.; Mouscadet, J.-F.; Pernot, P.; Tfibel, F.; Enescu, M.; Fontaine-Aupart, M.-P. Phys. Chem. Chem. Phys. 2001, 3, 3797-3804. (14) Zouhiri, F.; Desmae¨le, D.; d’Angelo, J.; Ourevitch, M.; Mouscadet, J.-F.; Leh, H.; Le Bret, M. Tetrahedron Lett. 2001, 42, 8189-8192. (15) Polanski, J.; Zouhiri, F.; Jeanson, L.; Desmae¨le, D.; d’Angelo, J.; Mouscadet, J.-F.; Gieleciak, R.; Gasteiger, J.; Le Bret, M. J. Med. Chem. 2002, 45, 4647-4654. (16) Beńard, C.; Zouhiri, F.; Normand-Bayle, M.; Danet, M.; Desmae¨le, D.; Leh, H.; Mouscadet, J.-F.; Mbemba, G.; Thomas, C.-M.; Bonnenfant, S.; Le Bret, M.; d’Angelo, J. Bioorg. Med. Chem. Lett. 2004, 14, 24732476. (17) Zouhiri, F.; Danet, M.; Beńard, C.; Normand-Bayle, M.; Mouscadet, J.-F.; Leh, H.; Thomas, C.-M.; Mbemba, G.; d’Angelo, J.; Desmae¨le, D. Tetrahedron Lett. 2005, 46, 2201-2205. (18) Normand-Bayle, M.; Beńard, C.; Zouhiri, F.; Mouscadet, J.-F.; Leh, H.; Thomas, C.-M.; Mbemba, G.; Desmae¨le, D.; d’Angelo, J. Bioorg. Med. Chem. Lett. 2005, 15, 4019-4022. (19) Deprez, E.; Barbe, S.; Kolaski, M.; Leh, H.; Zouhiri, F.; Auclair, C.; Brochon, J.-C.; Le Bret, M.; Mouscadet, J.-F. Mol. Pharmacol. 2004, 65, 85-98.

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Theoretical Electrostatic Properties of a Styrylquinoline

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