Extended and Clustered Conformers of Epothilone A - The Journal of

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Extended and Clustered Conformers of Epothilone A Danuta Rusinska-Roszak, Honorata Tatka, Rafal Pawlak, and Marek Lozynski* Institute of Chemical Technology and Engineering, Poznan University of Technology, Pl. M. Sklodowskiej-Curie 2, 60-965 Poznan, Poland

bS Supporting Information ABSTRACT: The conformational properties of epothilone A have been analyzed in detail using electronic structure calculations to better understand the effect of intramolecular hydrogen bonding on the conformational energies of this highly potent anticancer molecule. Single-point second-order Møller
Plesset calculations done in vacuo at the MP2/631þG(d,p)//B3LYP/6-31þG(d,p) level yielded data on the relative stability of conformers that were more distinct than data obtained from the standard DFT model, although the structural trends are in fair agreement. We studied torsional profiles of both hydroxyl groups and sampled energies of the side chain and thiazole moiety rotamers within the whole set of all experimentally accessible conformers. The aldol hydrogen bonds, though relatively weak, generally contribute to the conformational profile, while dipole
dipole interactions, ester group puckering, transannular repulsions between hydrogen atoms, steric effects, and syn-pentane effects have a limited influence. A salient result of our calculations is the determination that the energy of the clustered exo conformer P01 lays 9.3 kcal/mol below that of the extended, experimental conformer P11, apparently due to the unconstrained, near linear 3-OH hydrogen bond to thiazole. Another finding to be noted is the corroboration of the remarkable ability of 3-OH to form transannular hydrogen bonding with the epoxide, which releases the conformational strain of the macroring and thus leads to extra stabilization energy within the endo W subset. Finally, we found that the general trend of the conformer populations of epothilone A obtained from conformational energies resembles those derived from experiments and can be used to interpret values of NMR vicinal coupling constants. The calculated geometries and energies provide essential data for further discussion of the mechanism of biological activity of epothilone A and might be of importance in the explanation of its ADME properties.

’ INTRODUCTION Epothilones are antimitotic, naturally occurring compounds that are active against human cancer cell proliferation.1
3 These compounds stabilize microtubules by inhibiting their depolymerization through a “taxol-like” mode of action,4,5 though without any apparent structural similarity to taxol itself. The chemistry, synthesis, and structure
activity relationships of these compounds have been studied extensively in the last two decades.6
10 A number of reviews report on the effects of structural modification on the macrocyclic ring,11 side chains,12
14 and structural alterations of the macrolide scaffold.15 At present, a number of compounds are undergoing clinical efficacy evaluations.16 Epothilones are polyketides that are produced from the myxobacterium Sorangium cellulosum. Structurally, epothilones are lactones with a sixteen-membered ring. Due to their polyketide structure and as they are microbial polyketide synthase (PKS) products, these compounds are related to some macrolide antibiotics.17 Epothilone A (1) (Scheme 1) possesses a flexible macrolactone ring bearing a total of seven chiral centers, as shown by X-ray crystallographic analysis2 (Scheme 1). The heptaketide-derived framework of 1 is composed of three tact propionate and four acetate units on the end of the side chain, which is bound with a thiazole ring derived from cysteine.18 r 2011 American Chemical Society

One of two 4-methyl groups has a biosynthetic origin and is derived from S-adenosyl-methionine.18 Finally, epothilone A is a product of the epoxidation of a PKS end product by a monooxygenase, acting as a post-PKS modifying enzyme.19 Efforts are currently being made to understand the structure
activity relationships of antimitotic agents in anticancer activity. On the basis of the similarity of the mode of action of paclitaxel and epothilone, it would seem likely that both of these structurally different ligands occupy a common binding pocket in Rβ-tubulin.20 However, two electron crystallographic (EC) approaches have shown that the same compartment of this protein is used in a spatially selective manner.21
23 Also, a consistent model of association for epothilone that reasonably reproduces all structure
activity relationship (SAR) data has not yet been developed. The experimental difficulty in elucidating epothilone in its binding site is perhaps due to an unnatural β-tubulin structure (Rβ-tubulin polymerized on zinc-stabilized sheets (EC))24 or a β-tubulin monomer (solution-state NMR).25 These challenges were effectively overcome by the use of well-ordered tubules with a low molar Received: December 29, 2010 Revised: February 22, 2011 Published: March 15, 2011 3698

dx.doi.org/10.1021/jp112353p | J. Phys. Chem. B 2011, 115, 3698–3707

The Journal of Physical Chemistry B Scheme 1. Chemical Structure of Epothilone A (Atom Numbering As in Reference 25)

ratio of 13C-labeled epothilone to heterodimer of Rβ-tubulin.26 Solid-state NMR spectroscopy was applied, and the set of differences between the 13C chemical shifts observed for the ligand in the crystal state and the signals observed in the complex with Rβ-tubulin now constitutes a valid criterion for any molecular model of the epothilone binding site. To date, several calculations on the epothilone system have been reported in the literature.27
29 Very recently, Jimenez30 used density functional theory (DFT) calculations at B3LYP and X3LYP at the 6-31G(d) level to study the potential energy surface (PES) of epothilone A and to present a reduced model of the tubulin receptor. However, this computational assessment has been equivocal because of the relative flatness of the PES. Our aim was to provide more valuable information on the energy and conformational properties of epothilone A. We are interested in discovering whether and how hydrogen bonds and the rotation of the side chains would influence the total energy. The relative energy values have been used to probe different aspects of conformational equilibrium. The amounts of clustered and extended forms of epothilone A have been determined in vacuo directly from electron correlation calculations based on DFT geometries. The results of these studies are discussed in light of the effect of structural features on conformer stability. To gain information about the value of the obtained results, we compare the calculated average of the coupling constant with experimental data.

’ COMPUTATIONAL METHODOLOGY It is widely accepted that in protein
ligand complexes the bound-state conformers of small molecules are usually not in their global minimum energy state. Typically, the bound ligand conformation resembles a local potential energy minimum because the binding induces some degree of strain in the ligand. Also, the receptor gradually reorganizes to optimize the fit of the specific interactions of the binding site. In the case studied here, one cannot exclude two22,25 (or more30) experiment-dependent tubulin
epothilone models, and therefore, more than one bioactive conformation should be considered. Because epothilone can adopt multiple structural modes with different rotational (side chain, hydroxyl) groups and/or conformational (macrolide scaffold) preferences, detailed knowledge about the equilibrated, i.e., energetically accessible, unbound-state conformations within ca. 3 kcal/mol is strongly needed. Generally, in the case of strong hydrogen bonds or electrostatic effects, the accuracy of DFT, especially of B3LYP,31 BLYP,32 and XLYP33 methods (gradient-corrected functionals), is approximately the same as that of correlated MP2 calculations.34 However, in the case of weak (intramolecular) hydrogen bonds,

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repulsion of heteroatom lone pairs, transannular phenomena involving hydrogen atoms, and significant dispersion forces, the limits of DFT are evident.35 MP2 can describe geometries of molecules accurately enough;36 however, the computational resources required make this methodology impractical for use with large-sized molecules. Keeping in mind that DFT functionals cannot truly describe correlation effects like London forces and thus give reliable relative energies, we employed in vacuo MP2/6-31þG(d,p)//B3LYP/6-31þG(d,p), which is a compromise between the size of the system studied and the computational demand. We observed that the model chemistry used predicts the relative interaction energies spread over a much larger range than those calculated using the standard B3LYP/631þG(d,p)//B3LYP/6-31þG(d,p) optimization model.37 The default loose convergence criteria of the Gaussian program38 were applied for final DFT optimizations. A systematic conformational search for the epothilone A molecule becomes impractical; there are 1296 generated conformers for only one nonplanar macrolactone ring, i.e., scaffold (for 30° resolution and only four rotatable bonds: 3-OH, 7-OH, C15
C16, and C17
C18 of the side chain). Although full knowledge of its conformational surface at a high ab initio level cannot be obtained, we used systematic scanning for selected torsions to provide comprehensive information about the local conformational profile of a chosen part of the molecule. We anticipate that this reduced conformational analysis is representative for the defined structural feature for the molecule concerned, at least in its nearest environment. Initial molecular coordinates of the representative exo and endo forms were taken from geometries available experimentally from X-ray crystallography (H,2 P39), electron crystallography (S22), and NMR spectroscopy (M,25 T40) or on the basis of QSAR studies (W41). Coordinates of epothilone A bound to tubulin S (1TVK) or cytochrome P450epoK P (1Q5D) were extracted from Protein Data Bank (PDB) files.42

’ RESULTS AND DISCUSSION Survey on Macrolide Scaffold Conformations. The aim of this section is to conduct a short structural survey of experimentally developed, computationally optimized models of epothilones to assess possible contributing factors affecting the conformational energy level. On the basis of general conformational preferences, it is reasonable to assume that the ratio of preferred43,44 antiperiplanar and synclinal43,44 (ap and ( sc) and unstable synperiplanar and anticlinal ring torsions (sp and ( ac) may be decisive for the total energy of the conformer (Table 1; Supporting Information (SI) Table SI1). We estimate that the total stability of the conformer scaffold is also highly affected by a number of interactions, including intramolecular hydrogen bonding (Table 1), ester group puckering, dipole
dipole interactions (SI, Table SI3), and transannular effects (SI, Table SI4). The severe 1,3-diaxial interaction of the C4 and C6 methyl groups is permanent, but the syn-pentane effect (SI, Table SI5) seems to be less important. Usually zero or one syn-pentane-type interaction was found for both the exo and endo epothilone A conformers. The H conformer subset, with four syn-pentane interactions, is an interesting exception (SI, Table SI5). It was established that the tubulin-bound conformation M25 of epothilone A bears a resemblance to the X-ray conformer H2 (Figure 1). The similarity includes almost the entire macrocycle 3699

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The Journal of Physical Chemistry B

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Figure 1. Stick models of the most stable representatives of conformers of epothilone A (cyan, carbon; red, oxygen, blue, nitrogen; yellow, sulfur; white, hydrogen).

except for the region between the ester and carbonyl group. The heme-bound X-ray bioconformer P39 looks like the solution conformer T;40 only two torsions, O16
C1
C2
C3 in the ester and C10
C11
C12
C13 in the epoxide environments, are essentially different (SI, Table SI2). The N and S endo conformers22 are also similar, but noticeable differences appear on both sides of the ester functionality. Unexpectedly, the W41 conformer, which is different than its S endo counterpart, partially exhibits some closeness to conformer H. However, this conformer shows more particular similarity to the M conformer in C1
C11 and to N in the C12
C1 region of the macrolide. Sixteen atoms of “hybrid” W01 are being tested for congruity; the rms deviation of the macrocycle matched atoms of the C1
C11 fragment is 0.08 Å with the M16 conformer but only 0.04 Å through C12
C1 sequence with N01. The T and P conformers are the most stable among the exo and endo families of epothilone A conformers. Indeed, both conformer subsets have as many as 12 nonconflicting torsions. However, there is only one exact ac torsion and two where the torsional strain is elevated (SI, Table SI2). Moreover, both β-hydroxyoxo (aldol) units of the T and P conformers enjoy two intramolecular hydrogen bonds directed to the ketone oxygen (7-OH) and to the carbonyl ester oxygen (3-OH) (Table 1). It seems that some dipole
dipole interaction and three prominent transannular interactions (H2
H7, H3
H6, and H7
H10) (SI, Table SI4) are unable to change the high, net-positive output. Note that the representative P03 structure, which is 1.0 kcal/mol less stable than T01, has an equal number of ap and sc torsions, while the last one has seven ap and only five sc torsions. In addition, a negligible puckering (less than 3.0°) of the trans ester group does not destabilize the macrocycle. Subsets H and M reveal a less promising picture; these macrocycles include only five and seven optimum torsions, respectively (SI, Table SI2). Thus, the torsional strain is discharged through the macrocycle, partly engaging the ester group. The 7-hydroxyl proton donor does not have any acceptor in its environment with which it can form an intramolecular hydrogen bond (Table 1). We assume that hydrogen bonding of the 3-hydroxyl to the carbonyl ester oxygen is more stabilizing than

that if it bonded to the ring ester oxygen, mainly due to geometry and a slightly more negative NBO atomic charge. In fact, the H01 conformer is 0.7 kcal/mol more stable than M03. The crystal structure of epothilone A reveals a zigzag antiparallel arrangement in the C7
C12 segment.45 Our calculations confirm this feature for all H and M conformers. However, the two remaining exo subsets, P and T, widen the antiperiplanar alignment to the C5
C13 segment, which consisted of four ap torsions divided by two sc conformations in the middle of the segment. We also observed that some P and T conformers have more stable ring alternatives. The ap conformation “migrates” from regular (C10
C11
C12
C13) in P09 to (C8
C9
C10
C11) in P07, while in the case of T02 (C10
C11
C12
C13), the ap torsion is scattered around, causing T01 to be more stable than T02 by 0.5 kcal/mol (SI, Table SI2). Interestingly, the original P06 variant does not have a regular P counterpart. For the more compact endo subset, the accumulation of dipole
dipole moments is probably of great importance. In fact, the least stable N conformer has three such interactions, while the stable W conformer has only one. The W macrocyclic structure presents 4 trans and 7 gauche torsional forms and a minor amount of ester group puckering. This is not the case for the N01 conformer, where the ester puckering is 18.6° (SI, Table SI3), and there is only one ap and six sc conformations. We computed such restrained puckering for methyl acetate and found a loss of energy (1.2 kcal/mol). It is interesting that free access of the 3- and 7-hydroxyl groups to the carbonyl oxygen acceptors realizes such effective intramolecular hydrogen bonds, and the characteristically more stable S conformer forms the hydrogen bridge that is shorter by about 0.3 Å of this of N conformer (Table 1). Conformer W forms a hydrogen bond in a unique manner: the 3-hydroxyl interacts transannularly as a proton donor to the endo oxygen atom of the epoxide and is directed to the noncarbonyl ester oxygen.41 The last contact (2.56 Å, 101° for W01) seems to be an electrostatic interaction rather than a regular hydrogen bond. It is not quite clear whether such a bifurcated interaction may sufficiently explain the relative stability of the W conformer when the 7-OH group is inactive in hydrogen bonding. 3700

dx.doi.org/10.1021/jp112353p |J. Phys. Chem. B 2011, 115, 3698–3707

The Journal of Physical Chemistry B Conformers of epothilone A occur as a mixture of exo and endo epoxides, differing in their C10
C11
C12
O12 torsional angles. The most stable structures, P01, P02, and T01, and all of the H and M conformers have the O12 and C10 atoms as ap, but a part of the P and T subsets have this torsion as characteristically ac (Table 1). The endo stereoisomers of W have the O12 oxygen atom typically sp, but N and S partly decrease torsional strain, keeping this angle at about 30°. Side Chain and Thiazole Rotation. Systematic conformational scanning studies of epothilone based on the M conformer provided expected information about the side chain disposition toward the macrolide scaffold (Table 1). We found five C14
C15
C16
C17 side chain torsions; however, unexpectedly, none of them favored the stable trans or gauche position.43,44 The two most stable conformations have torsional angles of 88.7° and
153.5°, constrained by intramolecular hydrogen bonding of the 3-hydroxyl to the nitrogen atom of the side chain of the thiazole ring. Interestingly, the remaining three conformers, M08, M12, and M16, adopt (()ac and sc conformations with an energy gap of only 1.1 kcal/mol rather than the expected stable staggered positions. Further subtle differences appear in the arrangement of the thiazole ring. The obvious electron conjugation of the thiazole aromatic system and the carbon
carbon double bond prefers a coplanar disposition and therefore two distinct C16
C17
C18
C19 dihedral angles. The s-trans conformation of the carbon
carbon double bonds of the side chain (i.e., the olefinic C16
C17 and that of the thiazole ring) usually ensures coplanarity and therefore effective π-conjugation, and with one exception (P05), a deviation of torsional angle is seldom greater than five degrees. The alternative s-cis conformation is not so rigid: the torsional angles are instead between 12 and 20°. We tested the problem through all subsets of conformers. As expected, the energy differences are quite negligible, usually less than 0.5 kcal/mol (cf. H04 and H06, H08 and H09, H12 and H13, M14 and M15, S03 and S04, T02 and T03) or slightly larger, but still insignificant (cf. H01 and H02, H05 and H11, H07 and H14, M10 and M12, P08 and P10, S01 and S02, T05 and T06, W01 and W03). In some cases, the torsional angle of the thiazole plane is split into two levels around 0° (the value of a perfectly π-conjugated double bond
thiazole system), but the C16
C17
C18
C19 torsion angle differs by less than 40°. For example, the thiazole plane is partially distorted on both sides of the C16
C17 double bond (17.3° and
19.6° for M17 and M14, respectively) with a negligible energy difference of 0.4 kcal/mol. With an almost symmetrical tilt of the thiazole (15.6° and
19.0° for H12 and H10, respectively) and almost no difference in energy (0.07 kcal/ mol), these pairs of rotamers resemble “degenerate” energy states. Although “degenerate” states are not a rule, we see that the existence of subtle torsional differences of the functionalized macrocycle implies a complex stereoelectronic effect on the delocalized electronic system of the side chain. The calculated relative low-energy differences between side-chain rotamers within the macrocycle H and the M scaffold predict its remarkable sensitivity to demonstrate surface recognition properties of its double bond/thiazole conjugated system. In the case of exo structures, the side chain resides exclusively as a single, bulky substituent in the ring equatorial position (or pseudoequatorial for some conformers of the M subset). All endo conformers, although they still have a side chain spatially equatorial in respect to the macroring, cannot avoid steric hindrance from

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sharing the C13
C14
C15
C16 gauche torsion. Distinctly, conformers S03 and S04 are nearly identical in the C13
O16 region and have C4 and C17 methyl substituents at less than the methyl
methyl van der Waals contact distance46 of 4.0 Å (3.79 and 3.73 Å, respectively). It seems that such interaction is repulsive rather than attractive, a favorable interaction arising between the two alkyl groups. Indeed, the energy of the S03 and S04 conformers is 1.1 and 2.5 kcal/mol higher, respectively, than the alternative, sterically unhindered conformers (Table 1). Intramolecular Hydrogen Bonding. Three subsets of conformers under investigation, H, M, and W, are structurally unable to form intramolecular hydrogen bonds with the 7-hydroxyl, while for the remaining subsets both the 3- and 7-hydroxyl groups can potentially be active as intramolecular proton donors. Molecular determinants of the epothilone A structure limit hydrogen bonding of the 7-hydroxyl to the ketone at C5 (aldol moiety defines interaction as six-membered ring). Hydrogen bonding to the epoxide is excluded because of the consequent ap sequence of the C
C region and is excluded with the 3-hydroxyl because of the gem-4,4-dimethyl hindrance. The case of 3-hydroxyl is completely different; the proton donating OH can “choose” between two carbonyl oxygens, including the strong proton acceptor nearby at C1 and C5 and the noncarbonyl oxygen atom of the ester group to form six-membered rings. The endo conformation of the macrolactone ring allows it to, at least hypothetically, form an unconstrained, ten-membered, transannular interaction with the oxygen atom of the epoxide. A free rotation of two sigma bonds of the side chain can lead to hydrogen bonding contact with the nitrogen atom of thiazole, but the stereochemistry excludes even weak interactions with the thiazole sulfur atom. Importantly, the defined absolute configuration of the C3 chiral atom, the anisotropy of the sp2 and sp3 oxygen atoms of the ester group, and the spatial relationships of the ester functional group’s environment determine the ability to form patterns of intramolecular hydrogen bonding due to its two faces: Re and Si.43,44 In the case of the most stable representatives of subsets H and W, the Si face is preferred, but for the others, the Re face is the more approachable. We present below (Table 1) a few of the hydrogen bonds from each family of epothilone A conformers that will illustrate energy trends as a function of structure through the range of scaffolds covered by this study. For the H conformer, a rotation of the 7-OH group when 3-OH is held fixed leads to a change in energy of 0.3
0.4 kcal/mol for two triads of conformers, including H15, H16, and H17, where the 3-hydroxyl is engaged in hydrogen bonding with the ketone, and for H03, H05, and H07, where it does not. Interestingly, H15, the most stable conformer of the former group, is less stable than H03 by 1.6 kcal/mol, although the former forms a six-membered hydrogen bond with interaction parameters of 2.002 Å (142.8°). The H macroring scaffold provides the possibility of the 3-hydroxyl group hydrogen bonding to the sp2 hybridized oxygen atoms of the ketone and ester groups or avoids such interactions entirely. Likely, conformers H01 and H17 exhibit intramolecular hydrogen bonding to the ester group (Si face) or to the ketone, although the latter structure is 2.8 kcal/mol less stable than the former, in spite of the shorter length and more linear geometry of the hydrogen bond. We obtained a quite surprising result concerning the M conformer. Similar in shape, conformers M06, M07, and M08 differ only in that the 3-hydroxyl forms three isomers with the ester groups (Re face) that are energetically almost equivalent (in the range of 0.25 kcal/mol), but notwithstanding, these conformers use 3701

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The Journal of Physical Chemistry B

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Table 1. Selected Torsion Angle Valuesa, Hydrogen Bond Parametersb, and Correlated Energy Correction (MP2) Relative Energiesc for Stable DFT Conformations of Epothilone A at the MP2/6-31Gþ(d,p)//B3LYP/6-31Gþ(d,p) Level of Theory hydrogen bond distances and anglese

torsional angles conf.

10
11
12
O12

14
15
16
17

16
17
18
19

2
3
O3
H3

3-OHd

6
7
O7
H7

7-OHd

MP2 energiesc,f

H01

178.2


124.8

177.5

67.1


82.7

2.180 Å(127.4°)EC

noHB

6.75

H02

177.4


121.6


17.9

66.2


82.3

2.144 Å(128.3°)EC

noHB

7.34

H03 H04

173.2 178.8


125.0 93.0

176.2 179.1

163.9 67.8


170.2
82.4

noHB 2.198 Å(126.8°)EC

noHB noHB

7.60 7.61

H05

174.9


125.2

176.4

160.1


84.3

noHB

noHB

7.73

H06

179.1

99.4


14.4

67.9


82.5

2.204 Å(126.6°)EC

noHB

7.73

H07

173.4


125.1

176.4

164.9

76.6

noHB

noHB

7.89

H08

176.4

86.5

179.1

162.6


84.1

noHB

noHB

8.23 8.26

H09

176.2

92.6


19.6

163.5


83.4

noHB

noHB

H10

175.5

93.1


19.0

168.2

75.2

noHB

noHB

8.41

H11 H12

175.6 174.9


119.0 98.9


16.3 15.6

159.4 166.1


83.9 74.6

noHB noHB

noHB noHB

8.45 8.48

H13

175.5

87.4

179.4

166.6

76.2

noHB

noHB

8.50

H14

173.3


119.9


18.3

163.6

76.4

noHB

noHB

8.64

H15

172.1


125.0

174.9


103.6


170.8

2.002 Å(142.8°)C

noHB

9.21

H16

173.5


125.1

175.2


107.6

76.6

2.020 Å(142.8°)C

noHB

9.48

H17

174.4


124.2

174.7


102.9


86.3

2.041 Å(142.3°)C

noHB

9.59

M01


174.2

88.7

40.1

138.0


165.1

2.083 Å(160.7°)thia

noHB

2.94

M02 M03


175.2
177.3


153.5 72.6


83.1 179.8

128.1 69.7


165.9
164.4

2.046 Å(163.6°)thia 2.451 Å(119.1°)EE

noHB noHB

5.16 7.41

M04


178.2


115.6


13.5

41.0


85.2

1.988 Å(141.6°)EC

noHB

7.58

M05


179.0


129.5

177.2

60.9


164.9

2.099 Å(130.0°)EE

noHB

7.72

M06


179.2


118.5


12.5


49.1


166.0

3.105 Å(98.9°) EC

noHB

7.74

M07


179.5


115.1


12.9

41.9


164.7

1.998 Å(141.2°)EC

noHB

7.84

M08


174.9


119.9

18.8

59.4


164.5

2.045 Å(132.0°)EE

noHB

8.01

M09


177.7


113.5

20.2


48.7


85.9

3.076 Å(99.1°) EC

noHB

8.06

M10 M11


169.7
176.9

104.6
113.8


179.2 20.5

61.8
49.7


164.8 72.9

2.074 Å(130.6°)EE 3.111 Å(97.7°) EC

noHB noHB

8.08 8.38

M12


168.7

106.1


23.4

59.3


164.2

2.100 Å(130.4°)EE

noHB

8.60

M13


179.1


115.1


13.4

40.6

72.2

1.983 Å(141.7°)EC

noHB

8.66

M14

179.7

15.8


19.6

66.9


163.6

2.082 Å(127.8°)EE

noHB

8.89

M15

177.9

23.4

179.9

63.6


164.3

2.068 Å(129.1°)EE

noHB

8.93

M16


156.6


7.3


23.5

51.8


166.2

2.112 Å(133.6°)EE

noHB

9.17

M17

177.2

19.9

17.3

67.6


164.8

2.100 Å(126.9°)EE

noHB

9.27

M18 M19


155.9
176.3


9.1
120.3

177.5
16.2

50.9 152.2


166.0
166.1

2.111 Å(133.8°)EE noHB

noHB noHB

9.32 9.39

M20


174.9


120.6


16.9

148.7


87.7

noHB

noHB

9.60

M21


174.5


120.5


17.1

156.7

73.5

noHB

noHB

10.01

N01

37.9

114.2


174.7


70.3


59.4

2.236 Å(129.7°)C

2.159 Å(130.0°)C

N02

38.4

111.4

15.9


70.7


59.0

2.228 Å(130.0°)C

2.151 Å(130.2°)C

8.23

N03

38.5


11.2


20.1


69.8


59.1

2.257 Å(129.1°)C

2.158 Å(130.1°)C

9.66

N04

37.9


14.5

176.5


70.1


58.4

2.244 Å(129.5°)C

2.139 Å(130.8°)C

10.46

N05 N06

36.5 34.1

111.1 112.9

17.1
175.8

55.2 46.3


55.3
55.1

3.415 Å(116.2°)EC 3.199 Å(122.7°)EC

2.047 Å(133.9°)C 2.055 Å(133.8°)C

12.14 12.36

7.61

N07

36.4


11.6


21.4

47.5


55.2

3.330 Å (120.3°)EC

2.058 Å(133.7°)C

14.07

N08

36.0


14.5

176.8

50.0


54.9

3.359 Å (119.0°)EC

2.052 Å(134.0°)C

14.69

P01

179.4

90.8

40.9

129.5


43.9

2.048 Å(167.5°)thia

1.956 Å(139.4°)C

0.00

P02


178.9


141.3


134.9

104.7


44.4

2.110 Å(159.7°)thia

1.958 Å(139.2°)C

0.36

P03

176.6


122.4

175.3

33.0


47.3

2.235 Å(139.6°)EC

1.966 Å(138.0°)C

5.13

P04

124.0


125.5

178.9

70.1


43.4

2.292 Å(120.1°)EE

2.003 Å(138.5°)C

5.47

P05

125.3

115.8


169.7


44.8


45.4

3.161 Å(98.6°)EC

1.974 Å(138.5°)C

6.10

3702

dx.doi.org/10.1021/jp112353p |J. Phys. Chem. B 2011, 115, 3698–3707

The Journal of Physical Chemistry B

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Table 1. Continued hydrogen bond distances and anglese

torsional angles conf.

10
11
12
O12

14
15
16
17

P06 P07

175.9
146.8


111.7 14.7

16
17
18
19

2
3
O3
H3

6
7
O7
H7

21.1 176.7


44.5 156.4


45.0
45.5

3-OHd 3.158 Å(96.6°)EC noHB

7-OHd

MP2 energiesc,f

1.992 Å(138.3°)C 2.043 Å(136.8°)C

6.28 7.50

P08

121.6

7.4


22.9


51.6


45.2

noHB

1.970 Å(138.5°)C

8.02

P09

121.8

11.7

176.2

150.9


45.2

noHB

1.975 Å(138.4°)C

8.62

P10

121.0

5.5

177.6


51.2


45.5

noHB

1.970 Å(138.5°)C

8.72

P11

173.1

10.7

177.3


51.5


46.5

noHB

1.978 Å(138.0°)C

9.26

S01

29.0

77.2

178.0


81.6


60.1

1.931 Å(138.6°)C

2.051 Å(131.1°)C

7.17

S02

28.0

80.7

27.2


80.6


58.8

1.943 Å(138.0°)C

2.028 Å(132.3°)C

8.16

S03 S04

28.0 26.4


94.7
92.3

17.0 176.7


81.5
81.0


60.4
59.8

1.931 Å(138.4°)C 1.939 Å(138.1°)C

2.043 Å(131.1°)C 2.029 Å(131.8°)C

9.25 9.71

T01


178.1

81.8


21.1

37.1


48.5

2.120 Å(140.3°)EC

1.979 Å(137.3°)C

4.10

T02

123.3

78.3


22.9

33.6


46.1

2.118 Å(141.3°)EC

1.944 Å(138.8°)C

4.60

T03

120.4


119.6

174.6

29.7


46.6

2.127 Å(141.9°)EC

1.952 Å(138.4°)C

5.01

T04

122.1

75.6

179.7

32.9


46.3

2.108 Å(141.5°)EC

1.948 Å(138.6°)C

5.08

T05

119.0


105.8

177.9

32.8


46.3

2.073 Å(142.1°)EC

1.949 Å(138.7°)C

5.57

T06

119.9


114.5


13.0

29.0


46.5

2.109 Å(142.2°)EC

1.953 Å(138.4°)C

6.07

W01


1.4

115.1


175.3

90.1


166.1

2.171 Å(143.1°)epo 2.561 Å(101.1°)EE

noHB

4.39

W02


1.8


107.9

174.1

92.8


166.6

2.075 Å(145.6°)epo

noHB

5.83

noHB

5.86

noHB

6.20

noHB noHB

6.60 6.90

2.718 Å (97.7°)EE W03


3.4

104.8


18.5


165.3

92.5

2.108 Å(144.3°)epo 2.680 Å(98.4°)EE

W04


3.0


101.5


12.3


166.0

95.5

2.051 Å(147.2°)epo 2.756 Å (94.9°)EE

W05 W06

3.4 4.2


108.1
104.4

176.2 0.9

57.6 56.4

166.0
165.3

2.069 Å(132.9°) EE 2.050 Å(133.8°)EE

a deg. b Å, deg. c kcal mol
1. d Specific electrostatic interaction rather than hydrogen bonding are italic. e Hydrogen bonding to: C, O atom of ketone; EC, carbonyl O atom of ester; EE, noncarbonyl O atom of ester; epo, O atom of epoxide; thia, N atom of thiazole; noHB, lack of hydrogen bonding, f Absolute energies in Hartrees are given in SI, Table SI1.

the oxygen atoms of the ester group in three various ways (Table 1). The change in stability arises from the formation of an almost linear, eleven-membered intramolecular hydrogen bond to the nitrogen atom of the thiazole. In optimal mutual alignment within 3-hydroxyl and thiazole, the difference in energy between M01 and M06 conformers approaches 4.8 kcal/mol. The considerable difference in energy between M01 and M02, 2.2 kcal/mol, can be attributed to the lack of conjugation between these two groups (for M02 C16
C17
C18
C19 =
83°) with the still relatively high perturbation observed for M01 (C16
C17
C18
C19 = 40°). Note that in the case of conformers not involved in hydrogen bonding the side chain system remains nearly coplanar (for M06 C16
C17
C18
C19 =
12.5°). For the P subset, hydrogen bonding between the 7-hydroxyl and the 5-carbonyl is structurally determined, but the 3-OH group may realize hydrogen bonding in two different ways with either the carbonyl or noncarbonyl oxygen atoms of the ester group. The energy difference between the P03 and P04 conformers is only 0.3 kcal/mol, and it is not surprising that the conformer that bonds with the carbonyl atom is more stable, as observed for the M scaffold. Rotational reorientation of the 3-hydroxyl in P05 leads to disruption of its hydrogen bond with the sp2 oxygen atom of the ester and the subsequent formation of a relatively strong interaction to the thiazole (2.048 Å (167.5°) and 2.110 Å (159.7°) for P01 and P02, respectively). The

Figure 2. Ball (hydrophobic fragments) and stick models of the dihydrates of clustered conformers M01 (left) and P01 (right). Hydrogen bonds are shown as broken lines.

conformer P01 probably represents the global minimum of the PES of epothilone A at both MP2 and B3LYP levels, though this conformer is only 0.36 kcal/mol more stable than the competitive side-chain rotamer P02 in which the π-conjugation of the olefinic bond and thiazole is perhaps not so effective because the C16
C17
C18
C19 angle is slightly more obtuse. Conformers P01 and P11 (the conformer of crystallographic provenance)39 have very similar shapes of macrolide scaffolds and differ only in side-chain disposition. However, P01 is as much as 9.3 kcal/mol more stable than P11, and as the fully relaxed scaffold is the same as in the original, we presume that the hydrogen bond is the only extra impact on our energy result. For 3703

dx.doi.org/10.1021/jp112353p |J. Phys. Chem. B 2011, 115, 3698–3707

The Journal of Physical Chemistry B comparison, the M01 and M02 thiazoles that are involved in the intramolecular hydrogen bonding structures disperse conformational strain, relaxing all torsions of the macroring and additionally partly canceling the syn-pentane effect (SI, Table SI5). It is worth noting that the formation of the M and P clustered conformers occurs on account of the ester group puckering in the range of 12.7
19.5°, a value that is rather untypical for extended exo structures. Also, such a shape creates a large dipole
dipole repulsion of C1
O and C3
O, producing approximately collinear bonds. Clustered structures are formed as a result of the functional compensation of both hydrophilic nitrogen atoms of the thiazole and the 3-OH proton donor. As a consequence, the southern domain of epothilone A demonstrates pronounced hydrophobic character in striking contrast to the organization in the clustered ensemble of the nonpolar groups of paclitaxel.47 The hydrogen bond bridge is well shielded from the bulk solvent by the gemdimethyl group at C4, the C21 methyl, and the sulfur atom of the thiazole moiety. Furthermore, the carbon
carbon double bond and the C27 methyl hinder hydration (and hydrolysis) of the ester group (Figure 1). Indeed, such a conformation provides a rational guideline for considering the effects of limited amounts of water molecules on the stability of the crucial macroring side chain bridge. The presented structure of M01 dihydrate (Figure 2) exhibits remarkable shortening of the intramolecular hydrogen bond by ca. 0.06 Å (from 2.047 Å for M01 to 1.989 Å for M01 dihydrate), attributable to the cooperative binding of two water molecules, which could lead to strengthening of the bond. The covalent bond lengthening in the hydroxyl group that is involved in the bridge supports this conclusion (from 0.976 Å for M01 to 0.980 Å for M01 dihydrate; the unbound 3-OH in M19 has a bond length of 0.964 Å). For comparison, the analogous values are 1.968 Å and 170.0° for the hydrogen bond parameters of P01 dihydrate (Figure 2) and 0.982 Å for its 3-OH bond length. We assume that an environment rich in water may transform such compact structures into extended ones that are unconstrained by intramolecular hydrogen bonds. The endo subset N provides the unique possibility of analyzing a conformational effect that relies on the alteration of the weak electrostatic interaction of the 3-hydroxyl group directed to the O1 ester carbonyl oxygen by a hydrogen bond to the C5 ketone oxygen. Despite the fact that the ketone is used as a proton acceptor by the 7-hydroxyl, the formation of an additional intramolecular hydrogen bond and chelation of the ketone oxygen causes an essential increase in the stability of the system by 4.1
4.5 kcal/mol for four selected pairs of conformers (N01 and N05, N02 and N06, N03 and N07, and N04 and N08). Special attention should be paid to subset W, which forms the most stable endo scaffold. Here, for conformational reasons, the 7-hydroxyl is unable to establish a hydrogen bond with the ketone, while the 3-hydroxyl may operate as a proton donor with a proton acceptor in two fashions: in the plane of the ester group, employing both lone pairs of the sp3 oxygen, and more effectively on the Si face, placing it on the inside of the macrocycle ring. A three-center (bifurcated) hydrogen bond allows the realization of a ten-membered pattern with the length of 2.171 Å and an angle of 143.1° for the W01 conformer (or even shorter cf. 2.051 Å, 147.2° for W04). Moreover, a contact directed to the noncarbonyl ester oxygen at 2.561 Å is about 0.16 Å below the sum of the van der Waals radii.48 This causes it to elude rigorous hydrogen bond length definition but ignores the bond angle requirement. Such oriented dipole interactions, which do not meet the arbitrary

ARTICLE

criterion of the hydrogen bond, are common through the analyzed series of conformers of epothilone A (cf. Table 1) and probably help to stabilize conformers. The resulting energy gain, although not large, probably allows for the inclusion of W01 and some related conformers to the set of equilibrated entities in a polar environment. The formation of new bonds or the transformation of hydrogen bonds causes a change of only one or two torsional angles of the macroring while preserving the other ones. Usually, the torsion angle formed by C3
C4
C5
C6, being in the close surroundings of C3, is considerably affected. Also, the R bond to the ester function O16
C1
C2
C3 is sensitive to correction. Note that the dihedral angle that defines the ester group puckering keeps its value. Some examples of macrolactone conversion(s) related to hydrogen bonding are given below. The energetically favorable conformer H03 rotates around its free 3-hydroxyl (C2
C3
O3
H3 = 163.9°), and the resultant conformer H15, the C2
C3
O3
H3 =
103.6° conformation of the former, is trapped in a 1.6 kcal/mol higher-energy local minimum. Conformers of the M subset convert similarly; lacking a hydrogen bond, the high-energy conformer M21 forms intramolecular hydrogen bonds between the 3-OH group and the carbonyl oxygen of the ester, resulting in M13 (1.983 Å, 141.7°). The selective, structural macroring alterations of the C3
C4
C5
C6 dihedral range from
76.3° to
28.2°, and those of O16
C1
C2
C3 range from
47.5° to
127.9°, accompanying the M21 to M13 interconversion, respectively (SI, Table SI2). The hydrogen bonding pattern change occurs when a local internal conversion of the 3-OH in the ester region takes place: the transposition of the sp3 (P04) to sp2 oxygen atom as a proton acceptor (P03) (Table 1) induces a slight 11° correction of the C3
C4
C5
C6 angle and a considerable change of 73° of O16
C1
C2
C3 torsion. Moreover, the typical ap (of
166.4°) torsion of C10
C11
C12
C13 in P04 changes into
113.1° in P03, which is characteristic rather for P06, an alkyl variant value. The last conclusion strongly confirms the previous statement that the three torsional angles of the neighboring environment of functional groups of the macrolide are the most sensitive on structural conversion as follows: ester (O16
C1
C2
C3), ketone (C3
C4
C5
C6), and epoxide (C10
C11
C12
C13). Such judgment seems to be logical if we consider a complex electronic structure and conformational flexibility of functional groups, which can much better accommodate an additional torsion strain than an unfunctionalized aliphatic chain. Extended vs Clustered Epothilone. It is interesting to remember that no active epothilones are known without or with a reduced side chain.6,49 One cannot exclude the fact that the side chain is a moiety that provides critical structural change, enabling the molecule to achieve a compact shape and a hydrophobic character that is more relevant to the diffusion through the cell membrane rather than to the final binding on tubulin. On the basis of the available MP2 energy data, our conformational analysis suggests that the population of clustered conformations might be important, at least in apolar media. The hydrogen bond of the 3-hydroxyl to the thiazole ring, due to the presence of a nitrogen atom in the 20 -position of the thiazole moiety, does appear to be a driving force in the formation of such clustered conformations. Since the arrangement of the side-chain clustered conformation is so energetically favorable and contributes strongly to epothilone stability, we would like to investigate 3704

dx.doi.org/10.1021/jp112353p |J. Phys. Chem. B 2011, 115, 3698–3707

The Journal of Physical Chemistry B whether this conformation might coexist with a side-chain extended arrangement. Using the Boltzmann distribution in calculations to predict the epothilone populations leads us to believe that the vast majority of compact conformers are the P01, P02 (99.35%), and M01 (0.45%) conformers with traces of the T and W subsets. If we accept that clustered conformers are solvent dependent and in the solvolytic conditions, which disfavor the presence of intramolecular hydrogen bonds50 (i.e., 3OH
thiazole bond), open, and do not equilibrate, the extended structures dominate with the following population: T (62.56%), W (24.62%), and P (10.76%). The impact of the remaining subsets on the equilibrium does not exceed 1%. Next, the calculated51 weightedaverage coupling constant is JH6
H7 = 4.6 Hz. Note that the equilibrium population exists as a mixture of conformers of low value JH6
H7 (ca. 1.5 Hz for T and P) and high value JH6
H7 (ca. 14.5 Hz for W, M and H) (SI, Table SI3). Conformers N and S appear too unstable to exist in vacuo and in solution as free species. Experimental data confirmed this way of thinking. Also, the integral ratios of the NOE cross peaks H19TH27 and H19TH17 are sensitive to changing solvent polarity11,52 and may therefore reflect the relatively high concentration of the clustered P01 (and M01) syn-periplanar C16
C17
C18
C19 conformer(s) equilibrating with the anti-P02. However, the same experimental data using the NAMFIS protocol applied to polar solvents11 preclude W (near one-fourth of the total population (see above)) as the species having conformer torsional angles C10
C11
C12
C13 and C12
C13
C14
C15 of ca. 70° and
140°, respectively. This reveals that in polar solvents the apparent participation of H conformers (or clustered M conformers) is most likely what occurs. As seen in 2D NMR spectroscopy, there are at least two solution conformations of epothilone that exist in both DMSO/ D2O and CD2Cl2 solutions.40 The coupling constant data suggest the sensitivity of conformational equilibrium on the solvent polarity. When the solvent polarity decreases, the JH6
H7 changes from 9.5 to 4.8 Hz.40 This is due not only to a higher presence of any T and P subsets of conformers but also more importantly to higher concentrations of the related, but less polar, clustered P01 and P02. We concur with other groups23,53 that are skeptical of a literature claim that the “NMR tubulin conformation” is the binding site conformer. The finding of such relevant stability of clustered structures induces us to try to explain the appearance of the M subset of conformers. The transformation of any H conformer into an M scaffold template requires the interconversion of the C1
C5 polar macroring sequence and can probably be facilitated in a solvent-free environment. We anticipate that under solution-state NMR experimental conditions25 a promiscuous binding site of the β-tubulin monomer may mediate the rapid interconversion of an H conformer, possibly into stable, clustered M01 (or M02) conformer(s). Thus, we propose that after binding site desorption in polar surroundings that are rich in water the clustered conformation M01 undergoes slow (in the NMR time scale) opening and extending, and the solventexposed epothilone molecule is recognized by NMR as M19.25

’ CONCLUSIONS The hydroxyl group in the C3 position governs the intramolecular hydrogen bonding pattern of epothilone A. The key

ARTICLE

interaction of the PES of epothilone is the intramolecular hydrogen bond of the 3-OH group and a nitrogen atom of thiazole. Conformational change provides stable, clustered, and therefore hydrophobic conformations. In vacuo, and probably in a nonpolar environment, two structures, P01 and P02, exclusively dominate the conformational space of epothilone A. The third lowest-energy conformer, M01, is conceivably the only one that can compete within the range of conformational energy of 3 kcal/mol. In light of the apparent importance of the 3-OH hydrogen bonding, it is not surprising that its replacement with a cyano group reduces the rate of tubulin polymerization.54 However, it is obviously difficult to accept that 3-deoxy- and (E)-2,3-didehydroepothilone derivatives, which are comparably as potent as patupilone11 in the acceleration of tubulin polymerization, act according to the epothilone mechanism. Intramolecular hydrogen bonds within aldol fragments are not a determining factor in the stability control of conformers, and their energy impact does not exceed 3 kcal/mol. However, as an exception, the participation of both competing hydroxyl groups leads to a chelate hydrogen bond in the N subset with a surprising result of ca. 4
5 kcal/mol. For the endo W subset, the hydroxyl group at C3 can also act transannularly as an effective proton donor to make a hydrogen bond with the oxygen atom of epoxide.41 Rotation of the side chain and orientation of the π
π conjugated thiazole ring also has little effect on the total energy unless the proximity of the 3-OH and nitrogen thiazole causes a hydrogen bond to be formed. Our computational investigations attempted to interpret the results of solution NMR.25 We assume that in the polar solution a representative(s) of H and/or P, T populations undergoes catalytic conformational isomerization of the macroring, perhaps at the binding site of an unpolymerized β-tubulin, to the scaffold M and eventually forms the extended conformation M19.25 We speculate that in solution during standard tubulin dimer polymerization the prebound M19 finally transforms into the bioactive ligand, as observed by ssNMR spectroscopy.26 Similarly, these findings do not exclude the idea that equilibrating solution conformers might, in the binding-site-mediated process, transform first into M01 (the compact, clustered intermediate30) and then through internal rotation around the C16
C17 single bond form the related M19 extended conformation.25 This tentative mechanistic approach would be plausibly integrated into the molecular model by ab initio minireceptor studies. In the light of experimental11 and MP2/B3LYP calculations, the presented epothilone structure exhibits altered and labile drug shape and polarity characteristics. It is very important in the context of a still unclear insensibility of epothilones to P-glycoprotein,1 a cell membrane transport protein that usually acts as an efflux of lipophilic molecules. Our findings underlay the problem of potential cell membrane permeability of epothilones that would modulate their chemotherapeutic efficacy. These results show the importance of controlling (and/or exploiting) such features in the design and optimization of new antimitotic drugs. Further study of this subject will continue to add to our understanding of the effects of the solvent on the conformational equilibrium concentrations. We expect that both experimental and theoretical studies have led to a full, molecular picture of the bioactive conformation of epothilone. It is reasonable to anticipate that a successful design of new, improved therapeutic entities could be obtained through polyketide engineering or PKS genes biosynthesis,17 rather than a conventional organic synthesis approach. 3705

dx.doi.org/10.1021/jp112353p |J. Phys. Chem. B 2011, 115, 3698–3707

The Journal of Physical Chemistry B

’ ASSOCIATED CONTENT

bS

Supporting Information. Tables containing coordinates, energies of the optimized conformers of epothilone A, and other structural data. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Tel.: (þ48)61-665-3534. Fax: (þ48)61-665-3649.

’ ACKNOWLEDGMENT This study was supported by Poznan University of Technology/DS 32/045/2010. The authors are grateful to Professor R. E. Taylor (University of Notre Dame, Notre Dame, IN), Professor J. P. Snyder and Dr. M. Wang (Emory University, Atlanta, GA), and Professor J. Meiler (Vanderbilt University, Nashville, TN) for providing original coordinates of conformers to prepareinitial guesses for the generation of epothilone A representatives. We also acknowledge Poznanskie Centrum Superkomputerowo
Sieciowe, Poznan, Poland, for computational time. ’ REFERENCES (1) Bollag, D. M.; McQueney, P. A.; Zhu, J.; Hensens, O.; Koupal, L.; Liesch, J.; Goetz, M.; Lazarides, E.; Woods, C. M. Cancer Res. 1995, 55, 2325–2333. (2) H€ofle, G.; Bedorf, N.; Steinmetz, H.; Schomburg, D.; Gerth, K.; Reichenbach, H. Angew. Chem., Int. Ed. Engl. 1996, 35, 1567–1569. (3) Gerth, K.; Bedorf, N.; H€ofle, G.; Irschik, H.; Reichenbach, H. J. Antibiot. 1996, 49, 560–563. (4) Jordan, M. A. Curr. Med. Chem. Anti-Cancer Agents 2002, 2, 1–17. (5) Jordan, M. A.; Wilson, L. Nat. Rev. Cancer 2004, 4, 253–265. (6) Nicolaou, K. C.; Roschangar, F.; Vourloumis, D. Angew. Chem., Int. Ed. 1998, 37, 2015–2045. (7) Mulzer, J.; Altmann, K.-H.; H€ofle, G.; M€uller, R.; Prantz, K. C. R. Chim. 2008, 11, 1336–1368. (8) Altmann, K.-H.; Gertsch, J. Nat. Prod. Rep. 2007, 24, 327–357. (9) Nicolaou, K. C.; Sasmal, P. K.; Rassias, G.; Reddy, M. V.; Altmann, K.-H.; Wartmann, M.; O’Brate, A.; Giannakakou, P. Angew. Chem., Int. Ed. 2003, 42, 3515–3520. (10) Altmann, K.-H. Curr. Pharm. Des. 2005, 11, 1595–1613. (11) Erdelyi, M.; Pfeiffer, B.; Hauenstein, K.; Fohrer, J.; Gertsch, J.; Altmann, K.-H.; Carlomagno, T. J. Med. Chem. 2008, 51, 1469–1473. (12) Nicolaou, K. C.; Scarpelli, R.; Bollbuck, B.; Werschkun, B.; Pereira, M. M. A.; Wartmann, M.; Altmann, K.-H.; Zaharewitz, D.; Gussio, R.; Giannakakou, P. Chem. Biol. 2000, 7, 593–599. (13) Nicolaou, K. C.; Pratt, B. A.; Arseniyadis, S.; Wartmann, M.; O’Brate, A.; Giannakakou, P. ChemMedChem 2006, 1, 41–44. (14) Nicolaou, K. C.; Vourloumis, D.; Li, T.; Pastor, J.; Winssinger, N.; He, Y.; Ninkovic, S.; Sarabia, F.; Vallberg, H.; Roschangar, F.; King, N. P.; Finlay, M. R. V.; Giannakakou, P.; Verdier-Pinard, P.; Hamel, E. Angew. Chem., Int. Ed. Engl. 1997, 36, 2097–2103. (15) Nicolaou, K. C.; Sarabia, F.; Ninkovic, S.; Finlay, M. R. V.; Boddy, C. N. C. Angew. Chem., Int. Ed. Engl. 1998, 37, 81–87. (16) Harrison, M.; Swanton, C. Expert Opin. Invest. Drugs 2008, 17, 523–46. (17) McDaniel, R.; Welch, M.; Hutchinson, C. R. Chem. Rev. 2005, 105, 543–558. (18) Gerth, K.; Steinmetz, H.; H€ofle, G.; Reichenbach, H. J. Antibiot. 2001, 54, 144–148. (19) Gerth, K.; Steinmetz, H.; H€ofle, G.; Reichenbach, H. J. Antibiot. 2000, 53, 1373–1377.

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