Ab Initio Conformational Study of the Phenylisoserine Side Chain of

J. Med. Chem. 1999, 42, 291-299

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Ab Initio Conformational Study of the Phenylisoserine Side Chain of Paclitaxel Marco Milanesio, Piero Ugliengo, and Davide Viterbo* Dipartimento di Chimica IFM, Universita` , Via P. Giuria 7, I-10125 Torino, Italy, and Dipartimento di Scienze e Tecnologie Avanzate, Universita` del Piemonte Orientale, “A. Avogadro”, Corso T. Borsalino 54, I-15100 Alessandria, Italy

Giovanni Appendino Dipartimento di Scienza e Tecnologia del Farmaco, Universita` , Via P. Giuria 9, I-10125 Torino, Italy Received September 16, 1998

Paclitaxel (Taxol) and related compounds are important antitumor drugs, currently used for the treatment of several types of cancer. The flexible amino acidic C13 side chain is a key element of the taxoid pharmacophore, and the identification of the bioactive conformation is a top priority for a better understanding of the mode of action of these anticancer agents. The conformational features of the side chain have been investigated by Hartree-Fock ab initio and semiempirical PM3 calculations. To gain a better understanding of solvent effects, different molecular models of paclitaxel were used in the calculations. The gas-phase calculations confirm that only one conformation, named ch1 (very similar to the one found in the crystal structure of docetaxel), is present in apolar environments. The preference for this conformer has been rationalized in terms of its L shape, which minimizes steric and Coulombic interactions, and of a favorable arrangement of the glycolate moiety. When a polar solvent was simulated by different methods, a greater conformational variability was found, with different conformations differing by less than 1.5 kcal/mol. Among these conformations, only one (ch5′, similar to molecule B of the crystal structure of paclitaxel) is particularly apt to interact with solvent molecules. In light of these data, it seems reasonable to assume that, when the drug is bound to the lipophilic pocket of the tubuline receptor, the C13 amino acidic side chain assumes a conformation close to ch1. Introduction

Chart 1

The anticancer drug paclitaxel (Taxol) (1) (Chart 1) has been the focus of considerable chemical modifications, spurred by the possibility of improving the pharmacological profile of the natural product and shedding light on its structure-activity relationships.1,2 These studies have highlighted the relevance of the amino acidic side chain at C13 for significant anticancer activity. The structure-activity relationships in this moiety are well-established, but their translation in conformational terms has lagged behind. Indeed, the side chain of paclitaxel is flexible and can adopt different conformations, referred to as the “polar” and the “apolar” forms. Similar results have been reported for the taxoid alkaloid taxine A3, which also contains a phenylisoserine side chain. From the NMR data alone, it was not possible to assess if the “polar” conformation of paclitaxel is a conformational extreme or, conversely, an averaged form, as is the analogous conformation of taxine A3. A better appreciation of the precise geometry of the “polar” conformation of paclitaxel and of factors underlying the conformational behavior of the amino acidic side chain of taxoids has great relevance for the study of how tubulin, the target of antitumor taxoids, discriminates between the various conformations of paclitaxel. To address these questions, X-ray data4-11 and molecular mechanics12-15 and semiempirical calculations16 * Corresponding author. Fax: +39-11-6707855. E-mail: viterbo@ ch.unito.it.

have been employed. However, no clear-cut conclusions could be drawn, since often a host of solvent molecules were present in the crystal, making it difficult to assess their relevance for the conformational studies in solution. The conformation of amino alcohols of the type ArCH(NR2)-CHOH-X has also been extensively investigated by NMR12-15 spectroscopy. The results showed that rotation around the sp3 carbons is restricted and that the conformation is determined not only by purely steric factors but also by the tendency to form a net of intramolecular hydrogen-bond interactions between the functional groups. In paclitaxel, hydrophobic interactions might also play a role, owing to the presence of lipophilic ester groups on the chain and on the terpenoid core.17 The major focus of the studies in solution was the conformation around the bond C2′-C3′. Simple esters of N-acylphenylisoserine adopt a staggered conformation both in apolar solvents and in the solid state. This conformation is also adopted by the amino acidic side chain of paclitaxel in apolar solvents (mostly CDCl3). A survey of the conformation in different solvents of various taxane derivatives of phenylisoserine shows that their conformation depends on several

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Chart 2

Figure 1. Phenylisoserine side chain with the most important degrees of freedom.

factors, the most important being the nature of the solvent, the hydrogen-bonding donor/acceptor properties of the chain, and the substituent at the amino acidic nitrogen. We report here an attempt to rationalize the conformational equilibria of the amino acidic side chain of paclitaxel and related compounds in light of the existing X-ray and NMR data and of new ab initio quantummechanical calculations on the isolated side chain. Details of the Calculations Ab initio Hartree-Fock (HF) SCF-MO calculations on the different models of the C13 side chain (2) of paclitaxel were performed using the Gaussian 92/DFT18a and Gaussian 9418b programs on a variety of workstations. The initial geometries where those derived from NMR and molecular mechanics calculations12 and kindly supplied by Dr. Swindell; they were checked and extended using the molecular mechanics approach, with the SYBYL19 force field as embedded in SPARTAN,20 as well as the ab initio Hartree-Fock method using MINI-1,21a 3-21G, and the polarized 6-31G(d,p)21b basis sets. The results obtained at various levels were also compared with the available crystal structures.4-11 The complete phenylisoserine chain 2 (Chart 2) with 39 atoms is too large for full geometry optimization with the extended 6-31G(d,p) basis set. For the calculations on isolated molecules (gas phase), we reduced the model by substituting the C5′ phenyl by a hydrogen (N-formylphenylisoserine methyl ester, 3), after checking by semiempirical PM322 calculations (SPARTAN) on both models 2 and 3 that this substitution did not affect the geometry nor the energy of the analyzed conformers. The importance of the C3′ substituent was investigated by comparing the calculations on model 3 with those on the even smaller model 4. To analyze the influence of a polar solvent on the side chain conformations, we first used the simple selfconsistent reaction field (SCRF23) model, as implemented in Gaussian 92/DFT and Gaussian 94, for modeling a continuum highly polar solvent, providing its dielectric constant . We then introduced two explicit water molecules (supermolecular approach) in order to account for the most relevant local effects. To make the supermolecular approach computationally feasible, we had to perform our calculations with a mixed basis set: 6-31G(d)21b for nonaromatic carbons, oxygens, nitrogens, and nonaromatic hydrogens not involved in hydrogen bonds, 6-31G(d,p)21b for hydrogens involved in hydrogen bonds, and MINI-1 with geometric constraints on the C-H distances for the aromatic rings. Finally the supermolecular assemblies were embedded in the continuum polar solvent simulated by the SCRF method. To verify some possible steric effects we have also performed, on much larger molecular fragments, both ab initio calculations with mixed basis sets and semiempirical calculations at the PM3 level. (Coordinates of all optimized geometries can be requested from the authors, e-mail: [email protected].) All the molecular models were inspected and analyzed using the molecular graphic tools of MOLDRAW24 and SPARTAN.

Results and Discussion Calculations on the Isolated Molecule of the C13 Paclitaxel Side Chain. 1. Choice of the Molecular

Table 1. Main Torsion Angles of the Nine Conformers of Model 3 after HF/6-31G(d,p) Complete Geometry Optimization conformer

ω1 (deg)a

ω2 (deg)a

ω3 (deg)a

ch1 ch2 ch3 ch4 ch5 ch6 ch7 ch8 ch9

-3 -171 0 172 53 -171 94 -124 116

-52 167 161 -50 49 -76 45 52 41

-139 -141 -133 -148 -86 48 30 -160 -164

a ω ) O -C1′-C2′-O ; ω ) O -C2′-C3′-N; ω ) C2′1 car ox 2 ox 3 C3′-N-C5′.

Model and Identification of the Possible Conformers. Since accurate ab initio calculations can only be performed on relatively small molecular moieties, our study was mainly performed on the phenylisoserine chain 2, which is essential for the activity and is the most flexible part of the paclitaxel molecule. In this chain both the amidic and the ester groups are planar and rigid, while the most important degrees of freedom are the three torsion angles around the three single bonds in the central part of the chain (bold lines in Figure 1). On the basis of NMR data and of molecular mechanics calculations, Williams and co-workers12 reported 12 conformers: 6 in apolar and 6 in polar solvents. The analysis of these conformers by HF/3-21G full geometry optimization showed that four of them were just small modifications of other conformers, while three others were not real minima. We therefore considered only the remaining five conformers, but we then undertook a complete conformational search. Calculations at the HF/ 3-21G//HF/3-21G level indicated that the torsion angle Ocar-C1′-C2′-Oox (ω1) can assume four possible mean values (0, (90, 180°), the torsion angle Oox-C2′-C3′-N (ω2) three values ((60, 180°), and C2′-C3′-N-C5′ (ω3) also three values ((60, -140°). The analysis of the resulting 36 conformations allowed us to confirm the validity of the five geometries derived from the literature and to add four new conformations. These nine conformations may be considered as a complete set of possible conformers, and their geometrical features, after complete optimization at the HF/6-31G(d,p) level, carried out on model 3, are summarized in Table 1 and Figure 2, where they have been labeled as ch# (chain no.). The relative energy values obtained from these calculations (Table 2) indicate that one conformation (named ch1) is the most stable, among the nine analyzed, by at least 2 kcal/mol, indicating that the population of ch1 is greater than 95%. 2. Factors Contributing to the Stability of ch1. As shown in Figure 2, the main chain of conformer ch1 has an L shape. The two weak intramolecular hydrogenbond interactions, Oox-H‚‚‚‚Ocar and N-H‚‚‚‚Oox, in-

Phenylisoserine Side Chain of Paclitaxel

Journal of Medicinal Chemistry, 1999, Vol. 42, No. 2 293 Table 2. Relative Stability at the HF/6-31G(d,p)//HF/ 6-31G(d,p) Level of the Nine Gas-Phase Conformers with Respect to ch1 conformer

∆E (kcal/mol)


0 2.1 2.5 3.2 3.6 4.0 4.7 5.9 6.7

Chart 3

Figure 2. Nine conformers of the phenylisoserine side chain optimized at the HF/6-31G(d,p) level.

voked by Williams and co-workers,12 Gueritte-Voegelein et al.,5 and Vander Velde et al.,17 are present, but they involve the formation of rather strained five-member rings in which the NH and OH groups cannot assume optimal positions for the interactions, as indicated by the long distances, H‚‚‚‚Ocar ) 2.06 Å, H‚‚‚‚Oox ) 2.44 Å, and small angles, Oox-H‚‚‚‚Ocar ) 116.2°, N-H‚‚‚‚ Oox ) 96.8°. Other conformations, such as ch5 and ch6, allow stronger hydrogen bonds, but the relatively strong repulsive interactions, due to the formation of approximately planar six- or seven-member rings, exceed their stabilizing effect.

The L shape itself is an important factor as it allows to minimize the number and type of repulsive contacts. This was confirmed by estimating24 the number and strength of all repulsive nonbonding interactions in the different conformers, which are minimal for ch1. The graphic inspection of the nine conformers (Figure 2) suggests that only in ch1 can the C3′ phenyl be accommodated in a favorable position with the π orbital as far as possible from the Ocar and Oam lone pairs and the two ortho hydrogens pointing toward the Oam and Oox lone pairs. To gain a more quantitative picture, we decided to analyze the possible stabilizing factors one at a time, by performing, for each conformation, ab initio calculations on the four smaller molecular fragments (Chart 3) sampling all possible weak interactions: Oox-H‚‚‚‚Ocar in 5, N-H‚‚‚‚Oox and Oox-H‚‚‚‚Oam in 6, N-H‚‚‚‚Ocar in 7, and N-H‚‚‚‚Oet in 8. It turned out that the only interaction contributing by more than 2 kcal/ mol is Oox-H‚‚‚‚Ocar, while the N-H‚‚‚‚Oox is a purely electrostatic dipole-dipole interaction. Therefore only the results obtained on the methyl glycolate fragment 5 are worth describing and are summarized in Table 3. Figure 3 shows that the conformation a with the OoxH‚‚‚‚Ocar interaction is the most stable, while rotation of 180° around the C-C or/and C-Oox bonds (b-d) destabilizes the molecule. Nevertheless we have seen that the stabilizing effect of the Oox-H‚‚‚‚Ocar interaction is due more to the shielding of the Oox‚‚‚‚Ocar Coulombic repulsion by the interposed hydrogen than to the formation of a hydrogen bond. Indeed the simple break of the Oox-H‚‚‚‚Ocar hydrogen bond by a +60° rotation of the C-C-Oox-H torsion angle (e, where the shielding effect is only weakened) only costs 2 kcal/mol, much less than the 5 kcal/mol needed to obtain the unshielded conformation by 180° rotation (b). The preferred ch1 conformation shows the glycolate group in its most stable geometry, while ch4, differing from ch1 only for a 180° rotation of Ocar-C1′-C2′-Oox (ω1) in order to form an Oox-H‚‚‚‚Oet interaction, is less stable by 3.2 kcal/mol. Conformer ch2 with the glycolate group in the same conformation as in ch4, but with

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Table 3. Geometric and Energetic Features of the Different Conformations (shown in Figure 3) of the Methyl Glycolate Fragmenta glycolate as in

ω1 (deg)b

ω4 (deg)b

contact

∆E (kcal/mol)

a: ch1, ch3 e c: ch2, ch4 b f: ch5, ch7, ch8, ch9 d

0 0 180 0 60 180

0 60 0 180 -60 180

Oox-Hr‚‚‚fOcar none Oox-Hr‚‚‚fOet Oox T Ocar Oet T H2′ Oox T Oet

0.0 2.0 2.2 5.0 5.2 5.3

a Continuous double arrows for repulsive interactions, dotted double arrows for attractive interactions. b ω ) O -C1′-C2′-O ; ω 1 car ox 4 ) C1′-C2′-Oox-H.

Figure 3. Conformations of the methyl glycolate moiety 5 optimized at the HF/6-31G(d,p) level (continuous double arrows for repulsive interactions, dotted double arrows for attractive interactions).

fewer repulsive interactions, is less stable than ch1 by only 2.2 kcal/mol, a value very close to that given in Table 3. Ch3 has the glycolate group in its most stable conformation but is destabilized by other repulsive interactions between the C3′ phenyl and the methoxy group. All other conformers, with much higher energy, have a nonplanar glycolate group. We may therefore conclude that the geometry of the glycolate fragment plays a central role in the stabilization of the ch1 conformer. The validity of these theoretical calculations was also checked by analyzing the experimental conformations found in 1228 crystal structures, retrieved from the Cambridge Structural Database (CSD),25 containing the glycolate fragment 5 without the methoxy group and with an sp3 hybridization of C2′. We have seen that 65% of the structures have values of the torsion angle Ocar-C1′-C2′-Oox (ω1) in the interval -10° to +10°, while 23% have values near 180°, thus confirming the results of the calculations indicating an overwhelming preference for planar conformations with a greater weight of the conformation with torsion angle close to 0°. Also for the C1′-C2′-Oox-H (ω4) torsion angle, there is a preference for a planar geometry, but with a greater weight of the 180° conformation with respect to Ocar-C1′-C2′-Oox. Values around (60° are also frequent, but these are found in compounds with the OH group involved in intermolecular hydrogen bonds; this is in agreement with our finding that such a rotation only costs 2 kcal/mol (conformation e in Table 3). We can conclude that the stability of form a of the glycolate group is due more to the minimization of the steric and

Coulombic repulsions than to the formation of an intramolecular hydrogen bond, Oox-H‚‚‚Ocar. Finally to investigate the role of the phenyl group in C3′, we carried out HF/6-31G(d,p) calculations on model 4 with the C3′ phenyl substituted by a hydrogen; for this very simple fragment the number of possible lowenergy conformers increases. Comparison of the gasphase calculations on models 3 and 4 indicates that the main effects of the C3′ substituent are (i) nonsignificant effects on the relative energies of ch1, ch2, and ch5 and (ii) destabilization of all other new conformers with H2′ and H3′ antiperiplanar. We can therefore assume that the presence of a phenyl in C3′ is an important factor in selecting ch1 as the most populated conformer, in agreement with the findings of William and coworkers.15 The stabilizing effects are mainly steric, and the C3′ substituent does not need to be necessarily a phenyl but can be any other aliphatic or aromatic bulky hydrophobic group; in apolar solvents, this group must be large enough to be able to screen the neighboring polar residues of the chain. These results are in keeping with the SAR indications that taxols with a cyclohexyl,26a isobutyl,26b or tert-butyl26c in the C3′ position show comparable activity as well as similar NMR behavior in apolar solvents. Calculations on the C13 Side Chain of Paclitaxel with Simulation of a Polar Solvent. NMR data and molecular mechanics calculations have indicated the great influence of solvent polarity in dictating the conformation of the phenylisoserine side chain. In polar solvents, the increase of the coupling constant JH2′-H3′ up to values between 5 and 8 Hz indicates a significantly greater weight of conformations such as ch5 and ch9 with H2′ and H3′ antiperiplanar (calculated JH2′-H3′ > 10-12 Hz). Our calculations on the isolated molecule are in agreement with the data in apolar solvents, but to model the behavior of the side chain in polar solvents (water in particular), one has to perform the calculations taking into account the solvent effects. Different approaches were followed. 1. Onsager Continuum Approach. The solvent is modeled as a continuum dielectric medium, with a given dielectric constant ( ) 80 for water), interacting with the molecular dipole of the solute. All specific interactions between solute and solvent are therefore neglected. The results of the calculations, in terms of the relative stability of the nine conformers using model 3, are shown in Table 4. The most relevant effect of the polar solvent is that four other conformations besides ch1 are stabilized, resulting in five conformers with energies differing by less than 1 kcal/mol. While ch1, with a relatively small dipole moment, remains almost unaffected, conformers ch2, ch3, ch4, and ch5, with a dipole moment larger than 5 D, are stabilized. It is worth


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Table 4. Gas-Phase Dipole Moment µ and Relative Stability at the HF/6-31G(d,p) Level of the Nine Conformers in a Continuum Polar Solvent (Onsager Model, ) 80) Compared with That in the Gas Phase ∆E (kcal/mol) conformer

µ (Debye)

Onsager

gas phase


5.38 1.84 5.69 5.57 5.78 3.55 6.36 2.31 2.60

-0.14 0 +0.14 +0.40 +0.80 +3.3 +3.8 +5.7 a

+2.1 0 +2.5 +3.2 +3.6 +4.0 +6.7 +5.9 +4.7

a Conformation ch7 is not an energy minimum and collapses to ch5.

noting that conformer ch7, when optimized in the presence of a polar solvent, is driven to the ch5 conformation with the largest dipole moment. The presence, among the five low-energy conformations, of conformer ch5, with H2′ and H3′ antiperiplanar, is in agreement with the increase of JH2′-H3′. Besides, the importance of the stabilization by the polar solvent of conformer ch5 is in keeping with the fact that in the solvated crystal structures of both molecules of 10deacetyl-7-epitaxol6 and of molecule B of paclitaxel the side chain is in a conformation close to it. 2. Supermolecular Approach. The Onsager approach is certainly inadequate to treat a model with five polar sites capable of interacting with water molecules. To take into account the possible local interactions, we decided to use a supermolecular approach in which specific water molecules are added to the model in appropriate positions. This approach is clearly more computer-time-consuming, and it is therefore necessary to limit the number of water molecules in the model by including only the ones which are most tightly bound. We have chosen to consider a microsolvation environment with at most two water molecules located in such a way to act both as hydrogen-bond donors and as acceptors. The number of possible geometries is thus drastically reduced, and the initial search for the best positions of the water molecules was performed using the MINI-1 minimal basis set. These calculations also indicated that the C5′ phenyl is important in determining the water binding geometry and cannot be omitted. The size of the system then becomes exceedingly large to be treated with a full 6-31G(d,p) basis set. The MINI-1 basis set was therefore used to treat the atoms belonging to the phenyl rings; however, because of the systematic tendency of MINI-1 to lengthen the C-H distances, they were constrained to the values computed with the 6-31G(d,p) basis set. The calculations were only performed on the six most significant conformers, namely: ch1′, ch2′, ch3′, ch4′, ch5′, and ch9′. Besides the five most stable conformations in polar continuum solvent (Onsager method), ch9′ was also taken into account because its extended geometry is similar to the conformation of molecule A in the crystal structure of paclitaxel. The relative energies of these six optimized assemblies (inclusive of the two water molecules) are reported in the second column of Table 5. Even though ch1′ remains the most stable conformation, ch5′ is now only ≈1 kcal/mol above the absolute minimum and its population is conse-

Table 5. Relative Stability of Some Selected Conformations of N-Formylphenylisoserine Methyl Ester (3)a ∆E (kcal/mol) conformer

SM

SMO

GP

SE (kcal/mol)

ch1′ ch5′ ch2′ ch4′ ch3′ ch9′

0 +1.0 +2.1 +6.6 +8.9 +10.2

0 +0.5 +1.2

0 +3.6 +2.1 +3.2 +2.5 +6.7

-18.6 -22.0 -18.6 -15.2 -12.2 -15.1

a SM refers to the microsolvated model; SMO refers to SM plus the continuum solvent; GP refers to the gas-phase model. The last column shows the solvation energies (SE) of each conformer with respect to the two water molecules.

quently relevant. On the other hand, the relative population of ch2′ remains the same as that in the gas phase (third column) and is substantially reduced with respect to the results of the Onsager approach. The remaining three conformers, ch3′, ch4′, and ch9′, have a much higher energy and are not populated. In the last column of Table 5 we have also reported the microsolvation energies (SE) of the six conformers with respect to the two water molecules, evaluated as

SE(i) ) ESM(i) - [EGP(i) + 2EGP(H2O)], i ) ch1, ‚‚‚, ch9 in which ESM(i) is the total energy of the assembly inclusive of the two water molecules, EGP(i) is the gasphase energy of conformer i without water molecules, and EGP(H2O) is the energy of an isolated water molecule. Their values show that ch5′ is the conformer most stabilized by solvation and is the most apt to interact with a polar solvent (see Figure 4b). This is due to the extended conformation of ch5′, in which the polar residues are easily accessible by the solvent molecules. The stabilization trend is further confirmed by the calculations performed only on the first three conformers of Table 5, in which the supermolecular assemblies with two water molecules are embedded in a continuum polar solvent simulated by the Onsager approach, which yield the values reported in the third column. Conformer ch1′, shown in Figure 4a, is less stabilized than ch5′ by water, as is clearly indicated by the values of the solvation energies in Table 5. Nevertheless ch1′ remains the absolute minimum, even though the energy difference reduces from 3.6 kcal/mol for the gas phase to 1.0 kcal/mol for the supermolecular assemblies and to only 0.5 kcal/mol when these assemblies are included in a continuum water solvent. It must be pointed out that the presence of two water molecules induces significant distortions on ch1′ with respect to ch1; in particular the polar solvent induces a rotation of the phenyl in C3′ and a narrowing of the angle of the L shape of ch1 due to the rotation of the C2′-C3′-NC5′ (ω3) torsion angle (Figure 5): as a result, the amide group gets closer to the other end of the chain (Figure 4a). It is worth noting that these distortions move ch1′ away from the conformation found in the crystal structures of 2-debenzoyl-acetylpaclitaxel,8 docetaxel (Taxotere),5 and the methyl ester of the paclitaxel side chain,11 which are very close to ch1, while similar distortions have been found in the structure of 7-mesylpaclitaxel,7 where several polar solvent molecules are present in the crystal.

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Figure 4. Geometry of the microsolvated conformers ch1′ (a) and ch5′ (b).

Figure 5. Superimposed skeletons of conformation ch1′ (full line) and ch1 (dashed line).

As further evidence of the different stabilizations induced by the solvent on conformers ch1′, ch2′, and ch5′, we have also analyzed the geometry of all the hydrogen-bond interactions of the chain atoms with the water molecules. Indeed ch5′ is involved in the largest number of fairly strong interactions with the two solvent molecules (five interactions, compared with four in ch1′ and three in ch2′). Calculations on the Complete Paclitaxel Mol-

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ecule. As already pointed out, the fact of neglecting the presence of the bulky diterpene core in our simplified model of the paclitaxel side chain may affect our results. For instance, in ch1′ the oxygen Oam gets rather close to the methyl C13 (Figure 4a), with distances just above the sum of the van der Waals radii. These contacts may became much tighter and repulsive when the methyl group of our simplified model is substituted by the bulky diterpene core and the two hydrogen atoms become carbons. To verify the consequences of these steric effects, semiempirical and ab initio calculations with mixed basis set on the complete paclitaxel molecule have been performed. They also allow to check all NOE effects resulting from the NMR analyses.12,15 Starting from the conformation of molecule B in the paclitaxel crystal structure,4 the initial models were obtained by modifying the C13 side chain in such a way as to obtain the conformations resulting from the ab initio calculations described in the previous sections. Because of the large size of our molecular models, only the three gas-phase conformers ch1, ch2, and ch5 and the corresponding supermolecular conformers ch1′, ch2′, and ch5′ were considered. All models were optimized by the PM3 semiempirical method, with geometric constraints on the amide group. The PM3 method, in comparison with other semiempirical procedures, turned out to be the most apt to reproduce the ab initio geometries. To be able to distinguish between the geometrical and energetic variations due to the simplified computational method and those due to actual steric effects, we have also carried out PM3 calculations on the two models used for the ab initio calculations: the complete methyl ester of the phenylisoserine side chain 2 and the same fragment without the C5′ phenyl ring (3). The energy values obtained by the PM3 method must be analyzed with some caution, because of the known difficulties to accurately evaluate the energy contribution from weak interactions. To that purpose comparison between the PM3 results on models 2 and 3 with the full HF/6-31G(d,p) ones shows that geometrical changes indeed occur, and they are ascribable to the weakness of the PM3 method. The calculations on the gas-phase conformers show that extension of the model fragments to the whole paclitaxel molecule causes only limited geometric rearrangements, which are mainly due to the limitations of the PM3 method: the relative stability of ch1 (0.0 kcal/ mol), ch2 (+4.1 kcal/mol), and ch5 (+3.0 kcal/mol) remains similar to that of the ab initio calculations on model 3. On the contrary, the steric effects due to the presence of the diterpene core became important in polar environment and induce some relevant geometrical deformations in the chain; indeed the phenyl at C3′ gets very close to the C18 methyl of the core. While conformers ch2′ and ch5′ are almost insensitive to the size of the model, the extension of model 2 for conformer ch1′ increases the destabilization energy by more than 6 kcal/mol. The largest deformation is found for the torsion angle C2′-C3′-N-C5′ (ω3) which varies from -92° to -121°. It is important to note that these deformations move the ab initio ch1′ conformation toward the ab initio ch1 geometry. Also the energetic


Figure 6. Two-pin plug: dotted van der Waals representation of paclitaxel with the C13 amino acidic side chain in conformation ch1 (optimized HF/mixed-base geometry).

results for the three bihydrated conformers ch1′ (+6.6 kcal/mol), ch2′ (+5.1 kcal/mol), and ch5′ (0.0 kcal/mol) confirm that ch5′ is the one which is most stabilized by the water molecules. Finally for the three gas-phase conformers, we have also carried out geometry optimization at the ab initio level on an almost complete paclitaxel molecule (pruned of the unessential for activity C10 acetyl1), using the same type of basis sets used for the supermolecular calculations on the side chain and the STO-3G21b minimal basis set for the remaining atoms. The choice of the STO-3G minimal basis set for the atoms of the diterpene core is justified by the stiffness of this moiety. The results confirm those obtained by the PM3 calculations, and the geometries obtained in this way will be used to analyze the possible NOE effects and compare them with the experimental NMR data.12,15 Comparison with Experimental Data. Our ab initio and semiempirical PM3 calculations confirm that ch1 is the predominant conformation of the paclitaxel side chain in apolar environments. Indeed in ch1 the two protons H2′ and H3′ are synclinal (torsion angle H2′-C2′-C3′-H3′ ) 72°) and the calculated value of JH2′-H3′ ) 2.64 Hz is close to the values (2-2.8) measured for paclitaxel,12 docetaxel,14 and the methyl ester of the paclitaxel side chain in chloroform.12 Moreover in most crystal structures of derivatives containing the phenylisoserine chain (2-debenzoylacetylpaclitaxel,8 docetaxel,5 and the methyl ester of the paclitaxel side chain11) this group adopts the ch1 conformation. Finally, the values of the proton-proton distances in the ab initio optimized geometries of the complete paclitaxel molecule indicate that only for ch1 there is a good agreement with the NOEs reported by Gueritte-Voegelein and co-workers.14 We can therefore conclude that, in apolar solvents, conformer ch1 (Figure 6) is the prevailing conformation and has no drive to conformational changes. On the contrary, it is more difficult to rationalize the conformational features of the solvated phenylisoserine chain using only the results of ab initio calculations.

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Indeed ch5′ is the conformation which is most stabilized by water, and this result does not seem to depend on the size of the model or on the computational method employed in order to consider the solvent effect. Furthermore, ch5′ is very similar to the side chain of molecule B in the crystal structure of paclitaxel.4 In this conformation the hydrogen atoms H2′ and H3′ are antiperiplanar, with the torsion angle H2′-C2′-C3′H3′ equal to 164.4°, giving a calculated coupling constant JH2′-H3′ ) 12.2 Hz. Its relevant population in water is in agreement with the already mentioned net increase of the coupling constant found experimentally in polar solvents.12 A significant population of a conformer with the two protons in the synclinal position, which probably adopts an atomic arrangement intermediate between ch1 and ch1′ and similar to the one found in 7-mesylpaclitaxel,7 is always present, with a weight depending on the amount of steric effects. Indeed the experimental value of JH2′-H3′ increases from about 5 Hz (fraction of antiperiplanar conformer ≈ 0.25) for the simple methyl ester of the paclitaxel side chain, where the steric effects destabilizing ch1′ are minimal, to about 8 Hz (fraction of antiperiplanar conformer ≈ 0.6) for the whole paclitaxel molecule. A small population of the other synclinal conformer ch2′ may not be excluded, but the values of the proton-proton distances in the PM3 optimized geometries, compared with the NOEs reported by Williams and co-workers,12 seem to exclude this conformer. As further evidence we may notice that, while the transition from ch1′ to ch5′ involves a rotation of about 90° of the torsion angle Oox-C2′-C3′-N (ω2) and smaller variations of the other torsion angles, the transition from ch1′ or ch5′ to ch2′ involves much larger changes including a rotation of almost 180° of the Ocar-C1′-C2′-Oox (ω1) angle in the glycolate group. Conclusions Our gas-phase calculations (Table 2) confirm the hypothesis of Williams and co-workers,12 based on NMR and molecular mechanics data, that ch1 is the predominant conformation of the paclitaxel side chain in apolar environments. The results of the semiempirical PM3 and mixed-base ab initio calculations on the whole paclitaxel molecule show that the indications obtained from the gas-phase ab initio calculations on the reduced model 3 are not significantly affected by the presence of the bulky diterpene core. Our detailed analysis shows that the relative stability of ch1 depends more on the minimization of the steric and Coulombic repulsions than on the formation of the intramolecular hydrogenbond interactions shown in Figure 2. The geometry of the glycolate fragment plays an important role in the stabilization of the ch1 conformer. Its L shape is the other relevant stabilizing factor, since it allows to minimize the intramolecular repulsions while maintaining a rather curled up geometry as expected for a molecule with polar groups in an apolar medium. An important role in stabilizing the ch1 conformation in apolar solvents is also played by the presence in C3′ of a phenyl or any other bulky hydrophobic group capable of screening the neighboring polar residues. Polar solvents induce large conformational changes in the side chain, and these changes may be hindered by the presence of the bulky diterpene core. The pres-

298


ence of five polar residues enshrined into a hydrophobic matrix (the C3′ and C5′ phenyls, the C2 benzoate, the C4 acetate) makes it difficult to rationalize the conformational features of the side chain in polar solvents. Nevertheless, even the rather crude continuum SCRF method is capable of accounting for the increase of JH2′-H3′ in polar solvents, indicating that nonlocal interactions play an important role in the chain rearrangement induced by the solvent. All our calculations simulating a polar solvent indicate that ch5′ is the conformation most stabilized by water, and this result does not seem to depend on the size of the model or on the adopted computational method. Its extended geometry (Figure 4b) makes this conformation, with the polar and apolar residues on opposite sides of the C1′-C2′C3′-N plane, the most apt to interact with a polar solvent; indeed, ch5′ forms the largest number of hydrogen bonds with the two water molecules. The gauche conformations ch1′ and ch2′, in which polar and apolar residues are not so well-separated, are less favored; other considerations seem to exclude ch2′. In conformer ch5′ the presence of water molecules does not induce rotation of the C3′ phenyl ring with respect to ch5, while the same ring is rotated by almost 35° in conformer ch1′ with respect to ch1. This rotation in ch1 reduces the screening effect of the phenyl on the neighboring polar groups, which become then more accessible to the molecules of a polar solvent. The C3′ phenyl ring can now be approached by water molecules either through proton-π interactions (molecule W1 in Figure 4a) or through C-H‚ ‚ ‚O interactions. The hydrophobic collapse invoked by Vander Velde et al.17 can therefore be associated to two changes induced by the polar solvent in the C13 side chain: a rearrangement of ch1 to ch1′ followed by a rotation around C2′C3′, which causes the conformational transition from ch1′ to ch5′, with overall translation of weak proton-π interactions into stronger H-bonds between heterotaoms (see Figure 4). It has been found that when the C3′ substituent is a bulky alkylic group (cyclohexyl,26a isobutyl,26b or tert-butyl26c) JH2′-H3′ does not increase in polar solvents. This fact deserves further analysis, but we suggest that this behavior might be related to the high hydrophobicity of these aliphatic substituents, which, unlike a phenyl group or other aryl groups (pyridyl or furyl),27 are precluded from any interaction with water. The “hydrophobic collapse” may indeed be triggered by water-phenyl (or in general water-aryl) interactions, in accordance with the findings that it is inhibited by hydrogenation of the C3′ phenyl, but not of the C2 benzoate.26a Recently Ojima et al.28 have proposed a new interpretation of the conformational changes in paclitaxel in polar solvents and have indicated the presence of a new conformer with an almost eclipsed arrangement of the substituents around the C2′-C3′ bond. We have no direct evidence of this unusual conformer, with Oox and N facing each other, but we have observed that the presence of two water molecules induces a narrowing of the N-C3′-C2′-Oox (ω2) torsion angle of up to 10° in ch5′. The validation by computational methods of the hypothesis of Ojima et al.28 is in progress. SAR studies1,2 have shown that hydrophobic groups at C3′ and C5′ on the phenylisoserine side chain, and

Milanesio et al.

at C2 on the terpenoid core, are essential for the antitumor activity of taxoids. This suggested that the paclitaxel recognition site is a hydrophobic pocket of the tubulin structure. The recent electron crystallography study of this protein at 3.7 Å29 has confirmed this hypothesis, since the majority of the amino acids in the sequences surrounding the host paclitaxel molecule have nonpolar residues. It may therefore be assumed that, when the drug is bound to the receptor, the C13 amino acidic side chain will be in a conformation very close to ch1, the preferred conformation in apolar media. This is corroborated by the fact that the L shape of ch1 puts the hydrophobic C3′ phenyl ring in an external position, where it is free to bind to the lipophilic pocket of the receptor. Indeed, the removal of this phenyl group is detrimental for the activity, which is instead retained or even increased by substitution with other bulky hydrophobic groups. Finally, the conformational variability in polar solvents, with an increase of the entropic gain, may improve solubility and affect the pharmacokinetics of taxoids. The role of the bulky diterpene core might be that of holding into a proper orientation the substituents entering the lipophilic pocket, acting as the handle of a two-pin plug (Figure 6), in which the pins are the lipophilic side chains at C13 and C2. Acknowledgment. We are grateful to MURST and CNR for their financial support, to Dr. C. S. Swindell (Bryn Mawr College, Department of Chemistry) for supplying the initial conformations of the paclitaxel side chain, and to Dr. D. G. Vander Velde (University of Kansas, Department of Medicinal Chemistry) for providing unpublished information on the conformation of butitaxel. We also thank one of the reviewers for helpful suggestions. References (1) Nicolaou, K. C.; Dai, W. M.; Guy R. K. Chemistry and Biology of Taxol. Angew. Chem., Int. Ed. Engl. 1994, 33, 15-44. (2) Chen, S. H.; Farina, V. The chemistry and pharmacology of Paclitaxel and its derivatives; Elsevier: Amsterdam, 1995. (3) Barboni, L.; Gariboldi, P.; Appendino, G.; Enriu`, R.; Gabetta, B.; Bombardelli, E. New Taxoids from Taxus baccata. Liebigs Ann. 1995, 345. (4) Mastropaolo, D.; Camerman, A.; Luo, Y.; Brayer, G. D.; Camerman, N. Crystal and Molecular structure of Paclitaxel (Taxol). Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6920-6924. (5) Gueritte-Voegelein, F.; Guenard, D.; Mangatal, L.; Potier, P.; Guilhem, J.; Cesario, M.; Pascard, C. Structure of a Synthetic Taxol Precursor: N-tert-Butoxycarbonyl-10-deacetyl-N-debenzoyltaxol. Acta Crystallogr. 1990, C46, 781-784. (6) Gao, Q.; Parker, W. L. The “Hydrophobic Collapse” Conformation of Paclitaxel (Taxol) Has Been Observed in a Nonaqueous Environment: Crystal Structure of 10-Deacetyl-7-epitaxol. Tetrahedron 1996, 52, 2291-2300. (7) Gao, Q.; Chen, S. H. An Unprecedented side Chain Conformation of Paclitaxel (Taxol): Crystal Structure of 7-Mesylpaclitaxel. Tetrahedron Lett. 1996, 37, 3425-3428. (8) Gao, Q.; Wei, J. M.; Chen, S. H. Crystal Structure of 2-Debenzoyl2-Acetoxy Paclitaxel (Taxol): Conformation of the Paclitaxel Side Chain. Pharm. Res. 1995, 12, 337-341. (9) Gao, Q.; Golik, J. 2′-Carbamate Taxol. Acta Crystallogr. 1995, C51, 295-298. (10) Miller, R. W.; Powell, R. G.; Smith, C. R., Jr.; Arnold, E.; Clardy, J. Antileukemic Alkaloids from Taxus wallichiana Zucc. J. Org. Chem. 1981, 46, 1469-1474. (11) Peterson, J. R.; Do, H. D.; Rogers, R. D. X-ray Structure and Crystal Lattice Interactions of the Taxol Side-Chain Methyl Ester. Pharm. Res. 1991, 8, 908-912. (12) Williams, H. J.; Scott, A. I.; Dieden, R. A.; Swindell, C. S.; Chirlian, L. E.; Francl, M. M.; Heerding, J. M.; Krauss, N. E. NMR and Molecular Modeling Study of the Conformations of Taxol and of its Side Chain Methyl Ester in Aqueous and NonAqueous Solution. Tetrahedron 1993, 49, 6545-6560.

Phenylisoserine Side Chain of Paclitaxel (13) Williams, H. J.; Moyna, G.; Scott, A. I.; Swindell, C. S.; Chirlian, L. E.; Heerding, J. M.; Williams, D. K. NMR and Molecular Modeling Study of the Conformations of Taxol 2′-Acetate in Chloroform and Aqueous Dimethyl Sulfoxide Solutions. J. Med. Chem. 1996, 39, 1555-1559. (14) Dubois, J.; Gue´nard, D.; Gueritte-Voegelein, F.; Guedira, N.; Potier, P.; Gillet, B.; Beloeil, J. C. Conformation of Taxotere and Analogues Determined by NMR Spectroscopy and Molecular Modeling Studies. Tetrahedron 1993, 49, 6533-6544. (15) Williams, H. J.; Scott, A. I.; Dieden, R. A.; Swindell, C. S.; Chirlian, L. E.; Francl, M. M.; Heerding, J. M.; Krauss, N. E. NMR and Molecular Modeling Study of Active and Inactive Taxol Analogues in Aqueous and Nonaqueous Solution. Can. J. Chem. 1994, 72, 252-260. (16) Łoz`yn`ski, M.; Rusin´ska-Roszak, D. PM3 Conformations of C-13 Taxol Side Chain Methyl Ester. Tetrahedron Lett. 1995, 36, 8849-8852. (17) Vander Velde, D. G.; Georg, G. I.; Grunewald, G. L.; Gunn, C. D.; Mitsher, L. A. “Hydrophobic Collapse” of Taxol and Taxotere Solution Conformations in Mixtures of Water and Organic Solvent. J. Am. Chem. Soc. 1993, 115, 11650-11651. (18) (a) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Gill, P. M. W.; Johnson, B. G.; Wong, M. W.; Foresman, J. B.; Robb, M. A.; Head-Gordon, M.; Replogle, E. S.; Gomperts, R.; Andres, J. L.; Raghavachari, K.; Binkley, J. S.; Gonzalez, C.; Martin, R. L.; Fox, D. J.; Defrees, D. J.; Baker, J.; Stewart, J. J. P.; Pople, J. A. Gaussian 92/DFT, revision G.3; Gaussian, Inc.: Pittsburgh, PA, 1993. (b) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Gill, P. M. W.; Johnson, B. G.; Robb, M. A.; Cheeseman, J. R.; Keith, T.; Petersson, G. A.; Montgomery, J. A.; Raghavachari, K.; AlLaham, M. A.; Zakrzewski, V. G.; Ortiz, J. V.; Foresman, J. B.; Cioslowski, J.; Stefanov, B. B.; Nanayakkara, A.; Challacombe, M.; Peng, C. Y.; Ayala, P. Y.; Chen, W.; Wong, M. W.; Andres, J. L.; Replogle, E. S.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Binkley, J. S.; Defrees, D. J.; Baker, J.; Stewart, J. J. P.; HeadGordon, M.; Gonzalez, C.; Pople, J. A. Gaussian 94, revision C.3; Gaussian, Inc.: Pittsburgh, PA, 1995. (19) Clark, M.; Cramer, R. D., III; Van Opdenbosch, N. Validation of the General Purpose Tripos 5.2 Force Field. J. Comput. Chem. 1989, 10, 982-1012. (20) Spartan IBM, version 5.0.2 X11 AIX 4.1.4; Wavefunction Inc., 1997.

Journal of Medicinal Chemistry, 1999, Vol. 42, No. 2 299 (21) (a) Tatewaki, H.; Huzinaga, S. A Systematic Preparation of New Contracted Gaussian-Type Orbital Sets. III. Second Row Atoms from Lithium trough Neon. J. Comput. Chem. 1980, 1, 205228. (b) Hehre, W. J.; Radom, L.; Schleyer, P. v. R.; Pople, J. A. Ab initio molecular orbital theory, 1st ed.; John Wiley & Sons: New York, 1986; Chapter 4. (22) (a) Stewart, J. J. P. Optimization of Parameters for Semiempirical Methods I. Method. J. Comput. Chem. 1989, 10, 209220. (b) Stewart, J. J. P. Optimization of Parameters for Semiempirical Methods II. Applications. J. Comput. Chem. 1989, 10, 221-264. (23) Onsager, L. Electric Moments of Molecules in Liquids. J. Am. Chem. Soc. 1936, 58, 1486-1493. (24) Ugliengo, P.; Viterbo, D.; Chiari, G. MOLDRAW: Molecular Graphics on a Personal Computer. Z. Kristallog. 1993, 207, 9-23. Available at http://www.ch.unito.it/ifm/fisica/moldraw/ moldraw.html. (25) Allen, F. H.; Kennard, O. 3D Search and Research Using the Cambridge Structure Database. Chem. Des. Automation News 1993, 8, 1, 31-37. (26) (a) Boge, T. C.; Himes, R. H.; Vander Velde, D. G.; Georg, G. I. The Effect of the Aromatic Rings of Taxol on Biological Activity and Solution Conformation: Synthesis and Evaluation of Saturated Taxol and Taxotere Analogues. J. Med. Chem. 1994, 37, 3337-3343. (b) Appendino, G. Unpublished data and indication by one of the reviewers. (c) Vander Velde, D. G. Private communication to G.A. and indication by one of the reviewers. (27) Georg, G. I.; Harriman, G. C. B.; Hepperle, M.; Clowers, J. S.; Vander Velde, D. G.; Himes, R. H. Synthesis, Conformational Analysis, and Biological Evaluation of Heteroaromatic Taxanes. J. Org. Chem. 1996, 61, 2664-2676. (28) Ojima, I.; Scott, D. K.; Chakravarty, S.; Ourevitch, M.; Begue´, J. P. A Novel Approach to the Study of Solution Structures and Dynamic Behavior of Paclitaxel and Docetaxel Using FluorineContaining Analogues as Probes. J. Am. Chem. Soc. 1997, 119, 5519-5527. (29) Nogales, E.; Wolf, S. G.; Downing, K. H. Structure of the Rβ Tubulin Dimer by Electron Crystallography. Nature 1998, 391, 199-203.

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Ab Initio Conformational Study of the Phenylisoserine Side Chain of

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