experimental/theoretical electrostatic properties of a styrylquinoline-type hiv-1 integrase...

Experimental/Theoretical Electrostatic Properties of a Styrylquinoline-Type HIV-1Integrase Inhibitor and Its Progenitors

Delphine Firley,† Blandine Courcot,† Jean-Michel Gillet,† Bernard Fraisse,† Fatima Zouhiri, ‡,§

Didier Desmae1le,‡ Jean d’Angelo,‡ and Nour Eddine Ghermani*,†,|

Laboratoire Structures, Proprie´tes 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,Facultede Pharmacie, UniVersiteParis-Sud XI, 5, rue Jean-Baptiste Cle´ment,92296 Chaˆ tenay-Malabry Cedex, France, BioAlliance Pharma S.A. 59, BouleVard du General Martial Valin,75015 Paris, France, and Laboratoire Physico-Chimie, Pharmacotechnie, Biopharmacie, UMR CNRS 8612,Facultede Pharmacie, UniVersiteParis-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-quinoli-necarboxylic acid (1) as a promising lead. Previous molecular dynamics simulations and docking procedureshave shown that the inhibitory activity involves one or two metal cations (Mg2+), which are present in thevicinity of the active center of the enzyme. However, such methods are generally based on a force-fieldapproach 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 electrondensity and electrostatic properties in the structure-activity relationships of this class of HIV-1 antiviraldrugs. The electron properties of the two chemical progenitors of1 were derived from both high-resolutionX-ray diffraction experiments and ab initio calculations. The twinning phenomenon and solvent disorderwere observed during the crystal structure determination of1. Molecule1 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 of1. The electron densities, electrostaticpotentials, and atomic charges of1 and its progenitors are here compared and analyzed. The experimentaland theoretical deformation density bond peaks are very comparable for the two progenitors. However, theexperimental electrostatic potential is strongly affected by the crystal field and cannot straightforwardly beused as a predictive index. The weak difference in the theoretical electron densities between1 and its progenitorsreveals that each component of1 conserves its intrinsic properties, an assumption reinforced by a13C NMRstudy. 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 of1 are discussed in relationwith the localization of possible metal chelation sites.

Introduction

Acquired immunodeficiency syndrome (AIDS) is one of thegreatest challenges to humankind. All oral agents licensed totreat HIV-1 diseases target two of the three essential, virallyencoded enzymes, reverse transcriptase (RT) and protease (PR).1

However, although the advent of combination therapy withHIV-1 RT and PR inhibitors has made it possible to suppressthe replication of the virus in infected persons to such an extentthat it becomes almost undetectable in the plasma for more than2 years, HIV-1 persists in sanctuaries such as peripheral bloodmononuclear cells or resting T-lymphocytes. This means that

AIDS can be temporarily controlled but not eradicated withcurrent treatments.1-3 Therefore, additional therapeutic ap-proaches are warranted.

One such approach is to target the third viral enzyme,integrase (IN), that inserts the viral DNA into the cellulargenome through a multistep process that includes two catalyticreactions, 3′-endonucleolytic processing of both ends of thenative viral DNA consisting of the excision of a dinucleotideadjacent 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 requiredboth for 3′-processing and strand transfer and for the assemblyof HIV-1 IN onto specific viral donor DNA to form a complexcompetent to carry out either function.5-7 Mg2+ is the likelycation cofactor in vivo. Recent molecular modeling wasperformed by docking various inhibitors into a structural modelof full-length HIV-1 IN dimer complexed with donor DNA,suggesting that two divalent cations are involved in the catalyticcycle.8 HIV-1 IN is essential for retroviral replication, and the

* 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,

UniversiteParis-Sud XI.§ BioAlliance Pharma.|| Laboratoire Physico-Chimie, Pharmacotechnie, Biopharmacie, UMR

CNRS 8612, Faculte´ de Pharmacie, Universite´ Paris-Sud XI.

537J. Phys. Chem. B2006,110,537-547

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

absence of a host-cell equivalent of IN means that IN inhibitorsdo not interfere with normal cellular processes and thereforehave a high therapeutic index. In this respect, we have reportedthat polyhydroxylated styrylquinolines (SQLs), exemplified by1 (Figure 1), are potent HIV-1 IN inhibitors in in vitroexperiments, block the replication of HIV-1 in cell culture, andare devoid of cytotoxicity.9-18

These properties make SQLs promising candidates for thedevelopment of therapeutic HIV-1 IN inhibitors. Although theexact mechanism by which drug1 and analogues exert theirinhibitory potency is still a matter of controversy, it recentlyhas been proposed that such compounds might act prior tointegration by preventing viral DNA-IN binding.19,20 Withinthe framework of SQLs, we have identified the 7-COOH and8-OH (salicylic acid moiety) of the quinoline ring and the 4′-OH (Figure 1) of the ancillary aromatic nucleus as criticalpharmacophores for antiviral activity.10 SQLs were originallydesigned to chelate the divalent metal cation(s) in the catalyticcore domain of HIV-1 IN. A modified docking protocol,consisting of coupling a grid search method with full energyminimization, has been specially formatted to study the interac-tion between SQLs and retroviral INs. The docking procedureshows that SQLs bind closely to the Mg2+ cation in the vicinityof the active site of the catalytic core of the protein and thatthe Mg2+-drug interaction most likely involves the salicylicacid moiety of the quinoline half of the inhibitor.11,21 Thisoutcome was recently strengthened by the work of Ma et al.22

Structurally, SQL drugs are formed by a quinoline moietyconnected to an ancillary aromatic nucleus by means of anethylenic spacer. It was, however, shown that the replacementof this linker fragment by a variety of spacers has an importantimpact on both inhibitory potency and toxicity of the drugs.16

With the aim to assign the respective role played by each ofthe three subunits of SQLs, the present investigation deals withthe characterization at the atomic level of drug1 and itsprogenitors that are quinoline half2 and benzaldehyde derivative3 (Figure 2).

The electron density distribution and the electrostatic proper-ties (charges, electrostatic potential, dipole moments) werecarefully analyzed. Such properties were derived from X-raydiffraction experiments at 100 K and compared to the resultsobtained from ab initio quantum mechanical calculations.Comparisons between intrinsic progenitor electrostatic propertiesand those of drug1 are discussed. As shown from the above-mentioned docking study,11,21,22these electrostatic properties are

of particular importance in relation with the biological activity.This work is a part of our ongoing research devoted to this typeof HIV-1 IN inhibitor.

Experimental and Theoretical Procedures

Crystallization, Data Collection, and Reduction. In thisstudy, all of the diffraction data were collected at 100.0(1) Kon a Bruker-SMART charge couple device (CCD) diffracto-meter using graphite monochromated Mo KR radiation (wave-lengthλ ) 0.71073 Å). Cooling to 100 K was achieved by anN2 gas stream device (Oxford Cryosystem). The area detectorsurface was placed at 4.02 cm from the crystal sample. Differentdata collection strategies were used for the three compoundsunder study as detailed in Table 1. The Lorentz-polarizationcorrection and the integration of the diffracted intensities wereperformed with the SAINT software package.23 An empiricalabsorption correction was applied using the SADABS computerprogram.23 Finally, the SORTAV program was used for sortingand averaging equivalent and redundant data of high-resolutiondiffraction experiments performed for progenitors2 and3.24

Figure 2 illustrates the chemical synthesis of1 from itsprogenitors. The (E)-8-hydroxy-2-[2-(4,5-dihydroxy-3-methoxy-phenyl)-ethenyl]-7-quinolinecarboxylic acid (drug1, hereaftercompound I ) crystals were obtained by cooling to roomtemperature a hot saturated solution in dimethyl sulfoxide(DMSO). Despite sustained efforts directed toward this crystal-lization, the large number of crystal samples that we haveexamined were invariably found to be of very small size, needle-shaped, and twinned. The crystal used in the present study wasmounted in a sealed glass capillary due to its instability in air.The diffraction data were collected asω-scans (∆ω ) 0.25°)at one detector position (2θ ) -28°) whereθ is the Bragg angle.An exceptional exposure time of 120 s per frame was used dueto the weak diffraction power of the sample. The maximumresolution reached for this experimental data set is (sinθ/λ)max

) H/2 ) 0.65 Å-1, whereH is the Bragg vector modulus. Atwin analysis of all reflections was carried out using the GEMINIprogram.25 This reveals a nonmerohedral twinning with twocrystal components and a twofold twin operation along thereciprocal axisc*. A total of 4310 unique reflections (I > 2σ-(I)) were used in the structure refinements,σ(I) being theestimated standard deviation of the diffracted intensityI.

The 8-hydroxy-2-methyl-quinoline-7-carboxylic acid (pro-genitor2, hereafter compoundII ) crystals were obtained by slowevaporation of water/acetic acid (1:4) solution at room temper-ature. The crystal was also placed in a sealed glass capillarydue to instability in air. The diffraction data were collected atdifferent detector positions: 2θ ) -23°, (45°, (60°, (75°,(85°. The data spots were recorded asω-scans (∆ω ) 0.20°)to reconstruct accurate three-dimensional diffracted intensityprofiles. According to theθ dependence of the diffractedintensities, the chosen exposure times were, respectively, 5, 10,15, 30, and 60 s per frame for the detector positions given above.

Figure 1. Chemical structure of styrylquinoline1.

Figure 2. Synthesis of1 from progenitors2 and3.

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

The maximum resolution reached for this data set is (sinθ/λ)max

) 1.18 Å-1. Exactly 11 596 unique reflections (I > 2σ(I)) wereused in the conventional refinement, and 10 859 reflections (I> 3σ(I)) for the electron density refinement.

The diffraction data for 3,4-dihydroxy-5-methoxybenzalde-hyde (progenitor3, hereafter compoundIII ) were collected atfour different detector positions: 2θ ) -28°, +45°, -60°,+75°. The data spots were recorded asω-scans (∆ω ) 0.20°).The chosen exposure times were, respectively, 15, 30, 60, and90 s per frame for the four detector positions. The maximumresolution was (sinθ/λ)max ) 1.10 Å-1. Exactly 5455 uniquereflections (I > 2σ(I)) were used in the structure refinement,and 4206 reflections (I > 3σ(I)) for the electron densityrefinement.

Structure and Electron Density Refinements.The threecrystal structures were solved and refined using the WINGXsoftware package.26 For compoundI , the statistical factor beforethe twin analysis remains high (R) 16.3%). When the twinningeffect was taken into account,R dropped to 8.66% (Table 1).The ratio of the two twin components refined to 0.313(2). Thecrystallographic structure revealed an s-trans zwitterionic formof the molecule in the solid state. The attached proton of thequinoline nitrogen atom was located by Fourier differencesynthesis. All other hydrogen atoms were positioned theoreti-cally with geometrically fixed distances, except those of thehydroxyl groups of the ancillary nucleus (Figure 3).

The crystal structures of compoundsII and III weredetermined unambiguously (Table 1). Figure 3 gives the atom-

numbering system for the three molecules isolated from thecrystal lattice. For compoundsII and III , conventional andelectron density refinements were carried out using the MOLLYprogram based on the Hansen-Coppens multipole model.27 Thefrozen core and valence spherical densities are calculated fromthe Hartree-Fock free atom wave functions.28 In this study,theêl exponents (in bohr-1) of the radial functions were chosento be equal to 3.0, 4.5, and 3.8 andnl ) 2, 2, and 3 up to theoctupole level (l ) 3) for C, O, and N atoms, respectively;êl

) 2.26 bohr-1, andnl ) 1 (dipole level,l ) 1) for the hydrogenatoms.29 All of the multipole parameters were obtained by theleast-squares fit to the observed X-ray diffraction structureamplitudesF. Before the electron density refinement, the atomicpositions and anisotropic thermal displacements for C, O, andN were estimated on the basis of high-order data (sinθ/λ g0.8 Å-1 ). Their attached hydrogen atoms were extended to theneutron diffraction distances (Caromatic-H ) 1.08 Å, Cmethyl-H) 1.07 Å, N-H ) 1.01 Å, Ohydroxyl-H ) 0.96 Å). All of thesestructural and thermal parameters were relaxed in the last cyclesof the refinements. Figure 4 displays the residual electron densitymaps obtained after the multipole refinements. In these maps,the absolute residues of the electron density do not exceed0.20eÅ-3 (this limit is observed for compoundII ), attesting thegood convergence of the refinements. For comparison, theexperimental errors in the electron density are⟨σ2(∆F)⟩1/2 )0.025eÅ-3 for II and 0.038eÅ-3 for III and ⟨σ2

res⟩1/2 )0.024eÅ-3 for II and 0.032eÅ-3 for III . 30,31

TABLE 1: Details of the Experimental Diffraction Data Collections and Refinements

I II III

chemical formula C19H15NO6‚H2O‚(C2H6SO)2 C11H9NO3.C2H4O2 C8H8O4

molecular weight 527.6 263.2 168.1crystal size (mm3) 0.25× 0.08× 0.08 1.0× 0.45× 0.45 0.75× 0.18× 0.04color, form red, needle yellow, prism white, prismcell setting, space group monoclinic,P21/c triclinic, P1h monoclinic,P21/ca (Å) 7.096(2) 6.9937(7) 6.2088(1)b (Å) 19.114(7) 9.4230(9) 14.2712(3)c (Å) 17.902(5) 9.7841(9) 8.4469(2)R (deg) 68.546(2)â (deg) 91.44(2) 85.286(2) 97.836(1)γ (deg) 77.742(2)V (Å3) 2427.5 (2) 581.9(2) 741.46(3)Z 4 2 4Dx (Mg m-3) 1.38 1.47 1.50radiation type Mo KR Mo KR Mo KRµ (mm-1) 0.270 0.114 0.122T (K) 100.0(1) 100.0(1) 100.0(1)(sin θ/λ)max (Å-1) 0.65 1.18 1.10diffractometer Bruker SMART CCD Bruker SMART CCD Bruker SMART CCDdata collection method ω-scans ω-scans ω-scansmeasured reflections 36 387 116 729 64 887unique reflections 6824 14 519 6953Rint (%) 1.87 2.09index ranges -9 f h f9 -15 f h f 16 -13 f h f 13

-24f k f 24 -22 f k f 22 -29 f k f 29-23 f l f 23 -22 f l f 22 -18 f l f 18

spherical refinementR(F2 > 2σ(F2)) (%) 8.66 4.00 3.52wR(F2) (%); S 26.8; 1.03 10.5; 0.98 9.4; 0.88reflections used (F2 > 2σ(F2)) 4310 11 596 5455number of parameters 340 224 142(∆/σ) max 0.0030 0.0020 -0.0020∆Fmax,∆Fmin (eÅ-3) 1.07,-1.40 0.68,-0.31 0.58,-0.29

multipole refinementreflections used (F2 > 3σ(F2)) 10 859 4206R[F] (%) 1.75 2.05Rw[F] (%) 1.90 1.78goodness of fit 0.89 0.80

SQL HIV-1 Integrase Inhibitors J. Phys. Chem. B, Vol. 110, No. 1, 2006539

Quantum Mechanical Calculations. Ab initio single-molecule calculations for all three molecules were performedat the Hartree-Fock (HF) level using the program Gaussian03.32 The atomic coordinates of moleculesII andIII are thoseobtained from the X-ray data refinements. For compoundI ,however, the missing hydrogen atoms of the hydroxyl groupsin the crystal structure determination were added manually, andtheir positions were optimized according to energy minimiza-tions. The theoretical calculations were performed in a vacuumusing for the HF level the standard molecular split valence6-31G++** basis set. DFT calculations were carried out usingthe B3LYP basis set.33,34

For comparison with experimental electron deformationdensity maps (∆F(r) ) Fmolecule(r) - Fpromolecule(r)), we developeda routine using the Mathematica 5.0 software.35 Each promol-ecule electron density (superimposition of spherically sym-metrical densities of isolated atoms) was computed from thesame basis set as for the calculation of the molecular density tominimize the errors due to the basis set dependency.

Electrostatic Potential. The electrostatic potentialV(r) is ameasure of the electrostatic energy experienced by a unit pointcharge approaching the chemical system.V(r) is the sum of thenegative contribution generated by the electron density and thepositive one due to the nuclear charges. The electrophilic andnucleophilic characteristics of a molecule can then be revealedby the electrostatic potential. In many cases, the interactionenergy of molecular systems is dominated by the electrostaticpart. This makesV(r) a predictive property of particularimportance for the quantification of the chemical reactivity ofmolecules. In the present study, once the electron density wasdetermined from the experiment and theory, the electrostaticpotential was generated on a three-dimensional (3D) grid aroundthe molecules using the ELECTROS program36 and the corre-sponding routine implemented in the Gaussian 03 package,32

respectively. The MOLEKEL graphic software has been usedfor visualizing and plotting the electrostatic potential.37

Results and Discussion

Structural Analysis. CompoundI crystallizes in theP21/cmonoclinic space group with one water molecule and twodisordered dimethyl sulfoxide (DMSO) molecules in the asym-metric unit. The carboxyl group was found to be ionized, andthe quinoline ring nitrogen atom was found to be protonated,emphasizing the zwitterionic form ofI in the solid state. Thecorresponding H1 atom (Figure 3) is engaged in a hydrogenbond with a water molecule characterized by a donor-acceptordistance D‚‚‚A ) 2.802(6) Å. A strong intramolecular bondconnects O2 and O3 atoms (O2‚‚‚O3 ) 2.488(5) Å). Thisbelongs to the type of resonance-assisted hydrogen bond(RAHB) described theoretically by Wojtulewski and Grabow-ski.38 The quinoline and ancillary aromatic rings of moleculeIare almost in the same plane, the torsion angle around the C10-C11 connecting bond being close to 180° (C1′-C11-C10-C2 ) 179.8(4)°).

MoleculeII also crystallizes in a zwitterionic state. The spacegroup isP1h, and the asymmetric unit contains one acetic acidsolvent molecule. The H1 atom of the NH+ group is involvedin a hydrogen bond with one solvent molecule [D‚‚‚A )2.7771(1) Å). Both carboxylic O1 and O2 atoms participate inintermolecular hydrogen bonds. As inI , the carboxylate andhydroxyl groups are also connected via a strong intramolecularhydrogen bond: O2‚‚‚O3 ) 2.4516(2) Å.

MoleculeIII crystallizes in theP21/c space group. Both H3and H4 atoms (Figure 3) are involved in two intramolecularhydrogen 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 anglesof the three molecules in their respective crystals. Although lessprecise, the geometrical features ofI are in good agreementwith those derived from multipole electron density refinementsof compoundsII and III . For example, the C-N and C-Obond distances have similar trends, emphasizing the zwitterioniccharacter ofI and II . In this case, the protonation of thequinoline nitrogen atom is also characterized by the increasingC-N-C angle value (C2-N-C8a ) 123.7(4)° in I and123.09(1)° in II ) as recently reported by Dobson and Gerkin39

and Okabe and Muranishi40 for protonated quinoline rings.

Figure 3. ORTEP view of compoundsI , II , and III with the atom-numbering systems.


Electron Deformation Density.Figure 5 depicts the experi-mental static electron deformation density maps of moleculesII and III (STATDENS program)36 on one hand and thoseobtained from ab initio Hartree-Fock calculations for isolatedmolecules on the other hand.32 These maps are comparativelysimilar in features of C-C, C-N, and C-H bonds, displayingan average accumulation of electron density around 0.6-0.7eÅ-3 as generally found in organic molecules. However,some discrepancies appear for the oxygen atom lone pairs andfor the C-O bonds in both compounds. The electron densityaround the oxygen atoms is more contracted in the experimentalmaps as shown for O1 and O2 of the carboxylate group inIIand also for O3 involved in the intramolecular hydrogen bond.In the latter case, the electron concentration is much morepronounced in the O3-H31 bond in the theoretical maps.Conversely, in the experimental map, the O3 lone pair is ratherpolarized toward the NH+ group for which H1 is involved in ahydrogen bond with the solvent molecule in the solid state.

When experimental and theoretical results are compared, sucheffects of crystal environment and of the pseudoatom modellimitations have also been reported by Volkov et al.41 andCoppens and Volkov.42 In moleculeIII , quantitative differencesexist for the hydroxyl electron densities. Indeed, the experi-mental electron deformation density peak heights in C3-O2,C4-O3, C5-O4, and O4-C8 do not exceed 0.3eÅ-3 comparedto 0.6eÅ-3 found in the theoretical maps. The same remarkshold for the oxygen lone pair electron density accumulations.

Difference Densities from Theoretical Population Ma-trices. The ab initio HF electron deformation density of isolatedmolecule I is shown in Figure 6. The theoretical electrondeformation densities of both quinoline and ancillary aromaticring parts resemble those shown in Figure 5. To investigatefurther the specificities of each chemical progenitorII andIIIwithin the final moleculeI , another type of difference densitywas performed, the purpose being to detect the differences inthe charge density forII andIII in a vacuum on one hand andwhen they are embedded inI on the other hand. For this purpose,an original procedure was adopted.

Given the fact that the geometries are weakly but somewhatsignificantly different, no straightforward charge density dif-ferences could be envisaged. However, HF theoretical chargedensities are calculated using a set of nuclei-centered atomiclikebasis functions{Φi} with

wherePij values are the elements of the matrix. Given theiratomiclike nature, basis set functions are rather localized andcross contributions (i not equal toj in eq 1), particularly thoseinvolving remote atoms, are expected to be quite weak. Somebasis set functions can then arbitrarily be separated into functionswell identified to one or the other end of moleculeI , therebybeing identical to those found inII and III .

Electron density variations can therefore be evaluated, notfrom the difference of charge distributions, but from differencesof populations submatrices obtained by a projection of the fullmatrices onto the subspace of basis functions common to thetwo molecules to be compared. As an example, let us considerthe case of isolated moleculeII and the behavior of its commonpart in I . Ab initio calculations of the latter yield a rather largepopulation matrixPij

I ; meanwhile calculations ofII provide us

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

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

I II III

bondO4′-C5′ 1.369(6) O4-C5 1.3624(4)O3′-C4′ 1.353(6) O3-C4 1.3510(4)O2′-C3′ 1.369(6) O2-C3 1.3584(4)C2-N 1.338(7) 1.3356(2)C8a-N 1.384(6) 1.3730(2)C9-O1 1.249(6) 1.2561(2)C9-O2 1.276(6) 1.2772(2)C8-O3 1.336(6) 1.3304(2)C2-C10 1.437(7) 1.4921(2)C7-C9 1.502(7) 1.4978(2)C11-C10 1.347(7)C1′-C11 1.464(7) C1-C7 1.4586(4)O4′-C7′ 1.412(7) O4-C8 1.4322(4)

angleC5′-O4′-C7′ 117.0(4) C5-O4-C8 116.24(2)C2-N-C8a 123.7(4) 123.09(1)N-C2-C10 117.8(4) 119.24(2)C2′-C1′-C11 123.1(4) C2-C1-C7 117.25(3)C7-C8-O3 123.9(4) 123.08(1)O1-C9-O2 123.8(4) 124.23(2)

torsion angleC1′-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.

F(r) ) ∑i,j

PijΦi(r)Φj(r) (1)


with a smaller one,PijII . It is clear that, since the geometries

slightly differ, few basis functions can rigorously be consideredidentical from the atomic positions point of view. However, allof the functions, for a given pair of equivalent atoms in bothmolecules, have exactly the same mathematical expressions andonly differ in the weak atomic position shifts experiencedbetween the two geometries. It was therefore believed that aquantity such as

where [I ∩ II ] means that basis functions on which thesubmatrices are expressed are those identified as “genuinemembers” of bothI and II is not only trustworthy butmeaningful as long as one concentrates on the region expected

to be the most chemically similar. Equation 2 implies anarbitrary choice (I or II ) for locating the basis functions,Φi.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 smallcontour values (0.02eÅ-3) were chosen. The regions situatedat the right of moleculeII and at the left ofIII correspond tospurious difference densities due to the truncation of thepopulation matrices and should therefore be discarded. Whatappears quite clearly in Figure 7 is the weak perturbationencountered in moleculeII . As it turns out,σ-type electronsshow little change with the exception of the C5-C6 bond andthe attached H5 and H6 atoms. A quite weak but clear changein the polarization of O1 and O3 lone pairs can also be observed.

Figure 5. Electron deformation density of moleculesII and III : top panel, from experiment; bottom panel, from theory. Contour intervals are0.10eÅ-3; negative contours are dashed.

Figure 6. Theoretical electron deformation density of molecule1. Contour intervals are 0.10eÅ-3; negative contours are dashed.

δFII-I(r) ) ∑i,j∈[I∩II ]

(PijII - Pij

I ) Φi(r)Φj(r) (2)


No significantπ-type electron contributions can be spotted inthe plane parallel at 0.5 Å above the quinoline ring (bottom ofFigure 7).

The situation in moleculeIII is rather different in that theσ-type electrons exhibit changes on most of the atoms of theextreme right such as O2, H3, O3, H4, C8, and its connectedH81, H82, and H83 atoms (Figure 7). A weak effect can benoted on the ring itself. Aπ-electron transfer is detected mostlyin the O4-C8 region (bottom of Figure 7). Again, this lack ofeffect on the ring is confirmed by the population analysis, andon the opposite, strong charge fluctuations are observed on theO4-C8 region as well as around O3. The features observed onO2 in the σ-plane are quite isolated. None of these densityfluctuations can be related to significant integrated atomic chargevariations and can therefore only be attributed to a mere atomicpolarization. These considerations strongly support the pictureof almost two independent fragments, and the activity ofmoleculeI seems to be mostly driven by its spatial conforma-tion.

Electrostatic Potential.Figures 8 and 9 display the molecularsurfaces of the investigated compounds colored in accordancewith the electrostatic potential. These surfaces correspond tothe isodensity value of 0.007eÅ-3 (0.001 a.u.) as initially definedby Bader et al.43 This representation is of particular interest tohighlight the noncovalent interactions occurring at the molecularsurface (electrostatic complementarities, van der Waals interac-tions, and so on). The most nucleophilic regions (negativeelectrostatic potential) are in red, and the most electrophilic

regions (positive electrostatic potential) are in blue. The gradientvector of the electrostatic potential is characterized by the widthof the consecutive colored stripes corresponding to intermediateisovalues. The molecular volumes and the extrema of experi-mental and theoretical electrostatic potentials for the threemolecules are reported in Table 3. Only HF calculation resultsare presented here since they are very similar to those derivedfrom the DFT method.

As expected, the experimental molecular volumes forII andIII are smaller than those calculated for isolated moleculesaccording to the contraction of the electron density in thecondensed state. For the sake of comparison, we have chosenas a reference the largest molecular surface derived from theHF calculations to be colored in concordance with the electro-static potential. On these surfaces, we note that both minimumand maximum absolute magnitudes are higher for the experi-mental electrostatic potential due to the polarization of themolecular electron density induced by the crystal field. Thiseffect is clearly shown in Figure 8 for both moleculesII andIII displaying a higher experimental electric field on themolecular surfaces. As also can be seen in Figure 8 forII , thenegative region of the electrostatic potential is extended aroundthe carboxylate and O3-H31 hydroxyl groups, with a minimumvalue of -0.389eÅ-1 found in the vicinity of the O1 atom(Figure 3). From theoretical calculations, however, the corre-sponding negative area is limited to the proximity of thecarboxylate oxygen atoms with a minimumVmin )-0.269eÅ-1 found also close to O1.

Figure 7. Theoretical electron difference densities of moleculesII andIII with their equivalent embedded intoI : 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.


For III , the differences in absolute magnitudes of the extremaof experimental and theoretical electrostatic potentials are lesspronounced than inII (Table 3). However, the narrow stripesof the molecular surface experimental electrostatic potentialisovalues reveal a high gradient in the crystalline environment.A large negative area of the experimental electrostatic potentialsurrounding the O2-H3 hydroxyl group extends to the centerof the aromatic ring, as can be seen in Figure 8. This was notobserved from the theoretical calculations that yield a nearlyflat electrostatic potential in this region. The minima reportedin Table 3 for moleculeIII 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 onthe molecular surface ofI . For quantitative comparison withIIand III , the same potential range limits were chosen corre-sponding to-0.13 and+0.13eÅ-1, respectively. The mostnucleophilic region (double minima of-0.281eÅ-1 found closeto both O1 and O2 in good agreement withVmin )-0.269eÅ-1 found close to O1 inII ) surrounds the carboxylategroup of the molecule, and the area corresponding to the cutoffof -0.13eÅ-1 is much more extended than inII . In the rightpart of I , a minimum of the electrostatic potential is observedin the vicinity of the O2′ atom (V ) -0.072eÅ-1).

The previous outcomes relate to the zwitterionic form ofmoleculeI as it stands in the solid state. In a further study, wehave carried out HF calculations (with the same basis set as forI ) for the nonzwitterionic form obtained by attaching a hydrogen

atom to O2 of the acid group, removing H1, and rotating H31to the right side toward N. The molecular surface electrostaticpotential is displayed on the bottom of the left column in Figure9. In this case, the extrema of the electrostatic potential areVmin

) -0.135eÅ-1 and Vmax ) +0.177eÅ-1, respectively. Theelectrostatic potential became rather flat on both centers of thequinoline and ancillary phenyl rings. The electrophilic regionis largely reduced in comparison to that displayed by themolecular electrostatic potential of zwitterionic moleculeI (topof the left column in Figure 9). The most nucleophilic areasare now located in the vicinity of O1, between O2 and O3, andaround O2′ (Figure 3), respectively. Besides the molecularsurface electrostatic features, the right column in Figure 9compares the zwitterionic and nonzwitterionic forms using theisopotential surfaces. In the former, a large negative electrostaticpotential isosurface surrounds the carboxylate group. In thenonzwitterionic form, however, the corresponding region is splitinto two parts on each side of the carboxylic O2 atom. Thispincerlike shape of positive and negative potential arisingbetween O2 and O3 and their attached hydrogen atoms wouldincrease the ability to chelate positive ions such as metals.

Atomic Charges and Dipole Moments.From the consid-erations given above, it follows that whenI is formed in itszwitterionic state, the left part corresponding toII (also azwitterion) conserves to some extent its own electrostaticproperties, especially the nucleophilic character of the salicylicacid moiety. It is not the case for the right part correspondingto III . As can be expected, the removal of the O1 atom from

Figure 8. Isodensity molecular surface (0.007eÅ-3) colored in accordance with the electrostatic potential ofII andIII : 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 sameas in Figure 3.


III should a priori perturb the electron distribution in the restof the molecule. These assumptions would be confirmed bycomparing the net atomic charges derived from the theoreticalelectrostatic potential for the three molecules. We used themethod “charges from electrostatic potential grid” (ChelpGcharges) developed by Breneman and Wiberg44 to retrieve theatomic charges. These charges are also able to estimate thedipole moments of the molecules as can be seen in Table 4. Asexpected, we can see that a large difference in dipole magnitudesappears between the zwitterionic and the nonzwitterionic formsof I . It is noteworthy that, for the most polar molecules

(compoundsII and I in zwitterionic form), HF and DFTmethods yield different values of dipole moments. Figure 10depicts the correlation between the charges of the equivalentatoms inI , II , andIII , respectively. The two connecting carbonatoms that are C10-methyl ofII (future C10-ethylenic centerof I ) and C7-carbonyl ofIII (future C11-ethylenic center ofI )are ignored in the present correlation. Surprisingly, the linearregression fits to the paired data reveal excellent statisticalfactors: R) 99.7%, root-mean-square deviation (rmsd)) 0.04,slope) 1.067 (I vs II ) andR ) 98.4%, rmsd) 0.07, slope)1.052 (I vs III ) for HF charges. The highest atomic chargediscrepancies were found for C1′/C1 (0.168e in I and-0.041ein 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 obtainedfrom the DFT method. Except couples C1′/C1 and C6′/C6, theweak local difference densities depicted in Figure 7 and

Figure 9. Electrostatic potential of1 obtained from HF calculations: top panel, zwitterionic form (I ); bottom panel, nonzwitterionic form. The leftcolumn 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 Vol-umes Corresponding to the Isodensity Surface of 0.007eÅ-3 a

I II III

molecular volume (Å3) exp 213.91 178.46HF 398.39 236.24 195.59

Vmax(eÅ-1) exp 0.492 0.241HF 0.213 0.228 0.178

Vmin (eÅ-1) exp -0.389 -0.156HF -0.281 -0.269 -0.128

a The maximum (Vmax) and minimum (Vmin) electrostatic potentialvalues 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 from ChelpG charges

compound HF DFT HF DFT

II 20.37 18.23 20.29 18.12III 3.96 4.55 3.88 4.46I (zwitterionic form) 25.69 24.23 25.55 24.04I (nonzwitterionic form) 8.09 7.96 8.07 7.91

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

carbon atom 1 2 4 5

2 160.8 160.8 160.2 159.03 121.5 125.0 121.8 122.34 139.6 140.9 138.3 138.04a 130.6 130.4 131.0 133.55 113.5 112.8 115.6 116.96 127.3 127.5 126.6 126.77 112.8 112.6 111.5 111.18 153.0 156.2 153.7 153.48a 135.8 135.0 137.4 137.29 170.9 170.7 171.6 171.7

a 13C NMR spectra were recorded on a Bruker AC 200 Pspectrometer (50 MHz for13C, 300 K).


commented in the previous section seem to have no incidenceon the net atomic charges derived from the electrostatic potential.Furthermore, assuming these atomic charges as partitioningindexes of the electron density, it can be argued that theethylenic linker blocks any charge transfer between the two partsof I .

13C NMR Study. To assess the last statement, a comparative13C NMR study was undertaken. The chemical shifts obtainedfor drug 110 (harboring a π-excessive ancillary aromaticnucleus), its analogues4 and510 (in which the ancillary nucleiare neutral andπ-deficient, respectively), and progenitor2, werecompared (Figure 11 and Table 5). Regarding the 10 carbonatoms of the quinolinecarboxylic acid moiety of these molecules,only very discrete chemical shift changes were observed. WithinSQL series (compounds1, 4, and 5), the following standarddeviations 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.(1.5ppm for carbon atoms 4a and 5. This clearly reveals nosignificant shielding/deshielding of the quinoline part. Since the13C chemical shift of heteroaromatic compounds correlates

almost linearly with the electron density at the individualcarbon,45 the hypothesis that a push-pull-type electron delo-calization relayed by the ethylenic linker might occur betweenthe ancillary aromatic nucleus and the quinoline half of the SQLsshould be discarded.

Conclusion

High-resolution X-ray diffraction experiments and ab initiotheoretical calculations have been used to derive the electrondensity and electrostatic properties of the progenitors of a potentHIV-1 IN inhibitor, (E)-8-hydroxy-2-[2-(4,5-dihydroxy-3-meth-oxyphenyl)-ethenyl]-7-quinolinecarboxylic acid (1). This gavethe opportunity to highlight the specific electron properties ofthe quinoline and ancillary nucleus parts embedded in1, inrelation with the putative activity of a pharmacophore. Althoughthe experimental and theoretical electron densities of progenitorswere similar in comparison, the experimental electrostaticpotential, strongly influenced by the crystal field and by thelimitations of the multipole model, did not agree as well withthe calculated one. However, the comparison of the theoreticalelectrostatic potential features of progenitors and those of drug1 has revealed the absence of any effect of electron delocal-ization through the ethylenic linker. Accordingly, the atomiccharge values derived from the electrostatic potential remainedalmost unchanged for corresponding atoms in both progenitorsand drug1. This observation was reinforced by the13C NMRstudy of a variety of SQLs, showing that the electron excess ordeficiency at the ancillary aromatic nucleus did not affect thequinoline part. This result is of particular significance for thefuture development of potent IN inhibitors based on SQLs.

HIV-1 IN belongs to a superfamily of polynucleotidyltranferases that all require Mg2+ ions as cofactors to achievephosphodiester bond cleavage of DNA. Three-dimensionalstructures 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, islocated near a cluster of aspartic/glutamic acid residues,suggesting an electrostatic interaction between the cation(s) andthe negatively charged carboxyl groups. These observationsreinforce the hypothesis that inhibition of HIV-1 IN by SQLsmight be due to the functional sequestration of the critical Mg2+

cofactor by their salicylic part. Accordingly, the most nucleo-philic regions of the electrostatic potential generated for bothzwitterionic and nonzwitterionic forms of drug1 were foundin the vicinity of the salicylic fragment of the quinoline moiety.However, recent studies of molecular dynamics51 and drugdocking in the active site of the enzyme52 suggest that SQLsmight inhibit IN at its interface with viral DNA and divalentmetal. To assess the validity of this model, further investigationsincluding the elaboration and crystallographic characterizationof SQL-divalent metal complexes are under way. Finally, itshould be pointed out that the outcomes of the present studyconstitute a significant advance in the rationalization/predictionof SQL structure-activity relationships that could be exploitedto design more potent and more selective HIV-1 IN inhibitors.

Figure 10. Correlation between ChelPG theoretical atomic chargesof the quinoline half and ancillary aromatic nucleus of moleculeI andthe corresponding progenitorsII and III .

Figure 11. Chemical structure of styrylquinolines4 and5.


Acknowledgment. The financial support of the CNRS, EcoleCentrale Paris, and Universite´ Paris XI is gratefully acknowl-edged. The authors thank Dr P. Roussel (Universite´ de Lille,France) for his help and advise in the data processing for thetwinned crystal.

Supporting Information Available: Fractional coordinates(multiplied by 105) and atomic thermal parameters (multipliedby 105) for C19H15NO6‚H2O‚(C2H6SO)2 (compoundI), fractionalcoordinates (multiplied by 105) and atomic thermal parameters(multiplied by 105) from the multipole refinements for C11H9-NO3.C2H4O2 (compoundII ), κ, κ′, Pval, and Plm multipoleparameters for C11H9NO3.C2H4O2 (compoundII ), fractionalcoordinates (multiplied by 105) and atomic thermal parameters(multiplied by 105) from the multipole refinements for C8H8O4

(compoundIII ), κ, κ′, Pval, andPlm multipole parameters forC8H8O4 (compoundIII ), and theoretical CHelPG atomic chargesin e units. This material is available free of charge via theInternet at http://pubs.acs.org.

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experimental/theoretical electrostatic properties of a styrylquinoline-type hiv-1 integrase...

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