Conformational flexibility of antifungal atropisomeric strobilurin analogues: a quantum mechanical investigation

Journal of Molecular Structure: THEOCHEM 719 (2005) 69–74 www.elsevier.com/locate/theochem

Conformational flexibility of antifungal atropisomeric strobilurin analogues: a quantum mechanical investigation Ya-Jun Zheng*, Daniel A. Kleier** DuPont Crop Protection, Stine-Haskell Research Center, P.O. Box 30, Newark, Delaware 19714, USA Received 6 December 2004; revised 26 January 2005; accepted 27 January 2005

Abstract Quantum mechanical calculations were performed on novel N-phenyl-triazolone inhibitors of the mitochondrial bc1 complex. Both conformational flexibility and rotational barrier were examined. For all of the triazolones investigated, there is generally a large inter-ring torsion angle. The rotational barrier varies considerably among these analogues. For example, when a 6-methyl group is substituted on the phenyl ring at a position ortho to the triazolone, the calculated barrier is large enough to allow atropisomers to be stable even at room temperature. The calculated results are in agreement with experimental studies. q 2005 Elsevier B.V. All rights reserved. Keywords: Fungicide; bc1 complex; Inhibitor; Molecular modeling

1. Introduction Ubiquinol-cytochrome c oxidoreductase (bc1 complex), an essential component of the eukaryotic or bacterial respiratory chain and of the photosynthetic apparatus in purple bacteria [1], has been validated as an antifungal target [2]. Crystallographic studies of bc1 complexes from several sources have been reported [3–7]; binding sites of several classes of inhibitors have been identified. There are two distinct ubiquinone (or ubiquinol) binding sites: ubiquinol oxidation site (Qo site) and ubiquinol reduction site (Qi site). Two general approaches were employed in the discovery of bc1 complex inhibitors. First, procurement and screening of novel compounds have resulted in commercialization of famoxadone (3-anilino-5-methyl-5(4-phenoxyphenyl)-1,3oxazolidine-2,4-dione) fungicide under the trade name Famoxatew [8]. A second approach involving systematic modification of the naturally occurring inhibitors has proven * Corresponding authors. Tel.: C1 302 451 4604; fax: C1 302 366 5738. ** Address: Department of Chemistry, Drexel University, Philadelphia, PA 19104, USA. E-mail addresses: [email protected] (Y.-J. Zheng), [email protected] (D.A. Kleier). 0166-1280/$ - see front matter q 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2005.01.025

to be another extremely successful strategy [2]. The availability of high resolution crystal structures of the bc1 complex will certainly usher in a complementary approach that involves structure-based antifungal design. Strobilurins such as strobilurin A (Scheme 1) are naturally occurring b-methoxyacrylates with fungicidal activity [9]. The biological target of strobilurins is the mitochondrial respiratory chain. The binding of strobilurin to the ubiquinol oxidation center of cytochrome b disrupts the electron transfer from ubiquinol to cytochrome c. However, these natural products have some undesirable properties such as low light stability, which make them less attractive fungicides. Numerous attempts were made to design synthetic strobilurin analogues with improved light stability and systemic properties over the natural strobilurins [10]. Scheme 1 lists several classes of synthetic strobilurins. Synthetic strobilurins such as BAS 490F and ICI A5504 are broad-spectrum fungicides that control a number of major plant pathogens on various crops. The resemblance of those strobilurin analogs to the natural one is still very obvious. Similarly, studies at DuPont have resulted in a class of novel triazolone strobilurin analogues (Scheme 2) [11]. As part of our effort to understand the structure-activity relationship of these triazolones, conformational analysis was performed on several model compounds.

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Y.-J. Zheng, D.A. Kleier / Journal of Molecular Structure: THEOCHEM 719 (2005) 69–74

O O

O

O

CH3 N

H3C

H3C

OH N

O

OMe

N N

kresoxim-methyl (BAS 490F)

O

OMe

N N

1

H N

OMe

N N

2

3

N O H N

O O

N

N

O

O

O

Strobilurin A

N

H3C

O

O

O

N

O

O

OH N

O

N N

metominostrobin (SSF 126)

4

O

azoxystrobin (ICI A 5504)

H

H3C

OMe

N

O

OMe

H3C

OMe

O

N N

OMe N N N

5

Scheme 1.

SMe

6

Scheme 3.

3. Results and discussions R2

R1

N

O N

3.1. Inter-ring torsion angle

R3 N

Scheme 2.

2. Theoretical procedure There are a number of theoretical methods one could use to perform conformational analysis [12]. These methods range from high level ab initio and semiempirical quantum mechanical approaches to molecular mechanics. The theoretical method used for these calculations was chosen in such a fashion to give reasonable geometries and energetics without requiring too much computational resource. The geometry of each compound was initially energy minimized using ab initio molecular orbital theory at the HF/3–21G level of theory; rotational transition state was located and characterized at the same level by starting with a structure with the corresponding inter-ring torsion angle. Stationary points were identified as either a minimum (no imaginary frequency) or a transition state (one imaginary frequency); for transition state, the vector corresponding to the imaginary frequency was inspected visually to confirm that it indeed corresponds to the inter-ring torsion. The calculations were then repeated at both the HF/6–31G(d) level of theory and a hybrid density functional theory (DFT at the B3LYP/ 6–31G(d) levels of theory). The latter method has been demonstrated to be reliable in dealing with similar systems (rotational barrier of chiral biphenyls) [13]. The rotational barrier is calculated as the calculated electronic energy difference between the minimum energy conformation and the nearly planar rotational transition state. Six compounds were investigated as shown in Scheme 3. The calculations were carried out using the Gaussian 03 program [14].

Like the biphenyls [15], the N-phenyl-triazolone strobilurin analogs (as shown in Scheme 2) are twisted and the inter-ring torsion angle is determined by the nature of the nearby ortho substituents on the phenyl ring. The lowest energy conformation for all six compounds is twisted with an absolute inter-ring torsion angle of less than 908 as shown in Tables 1 and 2. Since the rings in these compounds are neither coplanar nor orthogonal in the lowest energy conformation, the energy profile in these compounds is complex. Transition states have inter-ring torsion angles Table 1 The calculated relative energies (in kcal/mol) at the B3LYP/6–31G(d) level of theory for two conformers and transition states at 0 and 1808 Compound

Conformer 1

Conformer 2

08 TS

1808 TS

1 2 3 4 5 6

0 0 0 0 0 0

0 4.2 0.7 5.5 0.8 1.2

34.7 26.4 15.4 7.1 35.0 33.6

34.7 32.8 16.3 13.4 37.3 43.6

Table 2 The calculated torsion angles (in degrees) Compound

HF/3–21G

HF/6–31G(d)

B3LYP/6–31G(d)

1 2 3 4 5 6

68.5 59.4 64.8 42.6 71.1 72.9

81.2 56.6 73.1 50.6 80.3 85.5

70.1 51.5 64.5 44.4 71.8 75.9

Torsion angle is defined as the C 0 –N–C–C0(R) where C 0 is the carbonyl carbon and C0(R) is the ortho carbon attached to the following R groups (RZMe in 1, OH in 2, H in 3, OH in 4, Me in 5 and 6).

Y.-J. Zheng, D.A. Kleier / Journal of Molecular Structure: THEOCHEM 719 (2005) 69–74

180° TS 0° TS

15.6 kcal/mol 15.4 kcal/mol

90 °TS 1.1

0.4 min2

min1 Scheme 4.

near 0, 90 and 1808 with the maximum at 08 (or 1808) being much higher in energy. For our purposes, only the rotational transition state with near 08 inter-ring torsion angle is relevant since passage through this transition state changes the chirality while passage through the near orthogonal transition state does not. A schematic potential energy curve for compound 3 at the B3LYP/6–31G(d) level of theory is depicted in the following scheme (Scheme 4). The optimized geometries corresponding to the minima in Scheme 4 are shown in Figs. 1 and 2. Compounds 2–6 exist in four stable conformations, an enantiomeric pair with absolute twist angle less than 908 (see Fig. 1 for one member of the pair) and a second pair with absolute twist angle

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larger than 908 (see Fig. 2) respectively. All stable conformations were calculated, but in what follows only the most stable conformers are discussed. As shown in Table 1, in general, the energy difference between these two conformers is rather small except in cases such as 2 and 4 where when intramolecular hydrogen bond is present, the energy difference becomes large. For compound 1, owing to the symmetry of the phenyl ring, those two minima are identical in energy with a negligibly small interconversion barrier of 0.6 kcal/mol at the B3LYP/6–31G(d) level of theory. The calculated torsion angles are very similar at the HF/3–21G and B3LYP/6–31G(d) levels of theory while the calculated values at the HF/6–31G(d) level of theory are generally w108 higher with the exception of compounds 2 and 4. All three methods predict the torsion angles for the six compounds to increase in the following order: 4!2! 3!1w5!6. The lower inter-ring torsion angles in compounds 2 and 4 are clearly due to the presence of an intramolecular hydrogen bond between the carbonyl oxygen and the phenolic HO. The smaller angles at the HF/3–21G and B3LYP/6–31G(d) levels compared to the HF/6–31G(d) values seem to suggest a stronger intramolecular hydrogen bond at the HF/3–21G and B3LYP/6–31G(d) levels. The HF/6–31G(d) level of theory is known to give reasonable hydrogen bonding energy while HF/3–21G tends to overestimate the hydrogen bonding interaction [16]. Thus, it is likely that the intramolecular hydrogen bond is

Fig. 1. The optimized global minimum geometries for 1–6.

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Fig. 2. The isomeric rotamers of compounds 2–4. These rotamers correspond to local minima that are slightly higher in energy relative to the corresponding rotamers illustrated in Fig. 1.

overestimated by HF/3–21G and B3LYP/6–31G(d) in the present system. Furthermore, the potential energy surface around the 908 torsion angle is expected to be rather flat and the inter-ring torsion angle can be easily perturbed. Indeed, the recently reported crystal structure for racemic compound 2 consists of dimers of the two atropisomeric enantiomers [11a]. The torsion angle is about 94.88 the dimer is held together by two inter-molecular hydrogen bonds formed between the phenolic HO of one enantiomer and the carbonyl oxygen of the other. However, crystal structures formed from enantiomerically pure compound contain extended hydrogen bonded chains in the crystalline state where two conformers with inter-ring torsion angles of 67.8 and 103.08 are present. Similarly, NMR studies also demonstrated that racemic and enantiomerically pure compound behaves differently in aprotic solvents. Even though there are no experimental data to compare for compounds 1, 5, and 6, X-ray crystal structures have been reported for related systems such as 1-phenylpyrimidine-2-thiones and 3-phenylthiazoline-2-thiones [17]. Those compounds and their carbonyl analogs do exhibit atropisomerism and the inter-ring torsion angles between the heterocycle and the phenyl group were determined by X-ray crystallography to be 81.5 and 84.28, respectively [17]. These values are similar to those calculated torsion angles reported here for N-phenyltriazolones at the HF/6– 31G(d) level of theory.

asymmetrical, each enantiomer could interact with the target differently. As it is often the case, only one enantiomer is bioactive. Unlike typical chiral compounds, chirality resulting from restricted rotation is inherently prone to spontaneous racemization. The rate of racemization depends on the rotational barrier. For instance, for a barrier of 20 kcal/mol, the half-life time of an atropiosmeric enantiomer is only about one minute at 25 8C. Therefore, it is important to know the barrier. The calculated barriers are summarized in Table 3. When there is only one ortho substituent present in the phenyl moiety (in 3 and 4), the rotational barrier is relatively small; in the transition state as shown in Fig. 3 for compound 4, both rings remain close to be planar with slight distortion. However, when both position 2 and 6 of the phenyl moiety are substituted (as in 1, 2, 5, and 6), the barrier increase considerably and significant distortion of the phenyl and triazolone rings is shown (Fig. 3) in the transition state. It is interesting to note that the HF and DFT (B3LYP/6–31G(d)) barriers agree with each other very well when there is little or no distortion in the transition state, but significant differences occur when there is large distortion in the transition state. In the transition states, the inter-ring C–N ˚ with respect to the bond lengthens by 0.02–0.06 A corresponding ground state. According to our calculated barriers, compounds 2, 5, and 6 should be resolvable at

3.2. Rotational barrier

Table 3 The calculated barriers (in kcal/mol) at three different levels of theory for compounds 1–6

Since these strobilurins are non-planar, they should in principle exhibit atropisomerism. Whether they are chiral or not depends on the barrier of racemization. If the rotational barrier is small, they exist as a racemic mixture. However, if the barrier is large with respect to thermal energy at room temperature, the strobilurin analogues could be resolved as pure enantiomers. Since the biologic target of strobilurin is

Compound

HF/3–21G

HF/6–31G(d)

B3LYP/6–31G(d)

1 2 3 4 5 6

41.8 31.4 16.5 6.8 41.7 38.2

42.3 34.5 20.9 11.7 41.9 41.7

34.7 26.4 15.4 7.1 35.0 33.6

Y.-J. Zheng, D.A. Kleier / Journal of Molecular Structure: THEOCHEM 719 (2005) 69–74

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Fig. 3. Calculated rotational transition states for 1, 2, and 4 in two different perspectives.

room temperature, whereas 3 and 4 may only be resolvable at lower temperature. The effective barrier for compounds 2 and 4 could be much larger in solution as well as in crystalline state owing to the presence of inter-molecular hydrogen bonding interactions that are likely to stabilize the twisted minimum conformations more than the near planar transition states. It should be noted that compound 1 cannot be resolved due to the inherent symmetry of the phenyl ring. However, slight perturbation of the symmetry of the phenyl (e.g. replacing one CH3 by CD3) should result in a resolvable chiral compound. Compound 2 has indeed been resolved and enantiomerically pure sample made [11a]. As mentioned previously, 1-phenylpyrimidine-2-thione and 3-phenylthiazoline-2-thione and their carbonyl analogs have been investigated and thermal racemization processes examined. The former racemizes through a ring opening/ closing reaction whereas the latter undergoes a rotation around the interring C–N bond [17]. The racemization barriers for 3-phenylthiazoline-2-thione and its carbonyl analogs with only one ortho substituent on the phenyl ring are around 30 kcal/mol. Our analysis suggests that the lower barriers in 2 and 4 again are due to intra-molecular hydrogen bonding that differentially stabilizes the near planar transition states compared with the twisted minimum energy structures.

4. Conclusions Quantum mechanical methods were used to investigate the conformational flexibility of a number of novel 4phenyl-triazolone inhibitors of the mitochondrial bc1 complex. Our calculations predicted that these compounds are twisted with a large inter-ring torsion angle comparable

to those seen for chiral biphenyls. Our calculations also indicate that the torsion angle can be reduced by the presence of intramolecular hydrogen bonding interactions between ortho hydroxyls on the phenyl ring and the carbonyl oxygen of the triazolone. The findings have been confirmed by subsequent X-ray crystallographic studies [11a]. Our calculations also suggested that the rotational barriers are very significant in compounds 1, 2, 5, and 6. The barrier is large enough to allow atropisomers to be resolved. Again, this is consistent with experimental observations. Now with a better understanding of the structure and conformational flexibility of these triazolone compounds and the availability of crystal structures of mitochondria bc1 complexes from different organisms, one may begin to understand how bc1 complex differentially binds the atropisomers.

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