*3.1. Conformational Analysis of Fagopyrins*

The structure of fagopyrin consists of a polycyclic system of eight rings. As shown in Scheme 2, each ring, named A–H, consists of six carbon atoms. Rings A–H are characterized by the presence of at least one substituent. In the A + B + C and F + G + H regions of the molecule, there are two hydroxyl groups and carbonyl oxygen. Such a close position of the substituents allows the formation of a hydrogen bond system in which hydroxyl groups are directed to the centrally located carbonyl oxygen. As shown for the hypericin molecule [13], such formation of strong hydrogen bonds is energetically preferred, and these hydrogen bonds are difficult to break. What is new in the structure of fagopyrin is the close position of piperidine and pyrrolidine substituents. These substituents can occur at positions named R1 and R2 (Scheme 2). The rings containing the substituents with nitrogen atoms give an additional possibility to form of intramolecular OHN hydrogen bond and possible breaking of the strong OHO hydrogen bonds. So far, no studies have been found

on the arrangement of these substituents in fagopyrin molecules. Another interesting part of the fagopyrin molecule is the "bay" region consisting of the A + D + F ring system. In hypericin molecule, the preferred arrangement of the substituents in the "bay" region is to form an OHO hydrogen bond between two hydroxyl groups. In the fagopyrin molecule, the addition of piperidine and pyrrolidine rings allows the interactions to be directed to the nitrogen atom forming new OHN interactions. Another part of fagopyrin molecule that may affect the overall structure is the presence of R3 and R4 substituents. Depending on the type of fagopyrin A–F molecule, these parts can be substituted by protons or methyl groups. As shown for hypericin, the close distance of two methyl groups may cause strain in the entire molecule and can strongly affect the planarity of the polycyclic system. Such a variety of substituents and possible strain effects from methyl groups make the structure and intramolecular interactions in fagopyrin molecules worth describing.

**Scheme 2.** Double anthrone polycyclic system of fagopyrin A–H.

Six low-energy conformers of fagopyrin A have been obtained, and structure 2 is the minimum energy conformer (Figure 1). The hydrogen bonds in the "peri" region of the molecule show alignment to the carbonyl oxygen. Two OHN hydrogen bonds in the "bay" region are preferred; however, breaking one OHN hydrogen bond in the "bay" region and forming OHO hydrogen bond between hydroxyl groups results in a total energy change of only 3.8 kcal·mol−<sup>1</sup> (structure 1). A similar change in the total energy of the system (Δ<sup>E</sup> ≈ 4.9 kcal·mol<sup>−</sup>1) is caused by breaking a strong OHO hydrogen bond in the "peri" region and the formation of an OHN bond with the pyrrolidine substituent (structure 3). Breaking the OHO hydrogen bond in the "peri" region without the formation of another interaction destabilizes the fagopyrin structure and raises its energy (structure 4, 6).

For fagopyrin B, six low-energy conformers (Figure 2) have been obtained. Structure 8 showing the lowest energy is characterized by the OHO hydrogen bond arrangement in the "peri" region typical for anthrones. In the "bay" region, the OHN hydrogen bonds linking the hydroxyl group and the nitrogen atom are formed. The energy differences between the structures 7, 8, and 9 show the energy difference up to 10.0 kcal·mol−1. The energy difference for these conformers is larger than the analogous difference for structures 1, 2, and 3 of fagopyrin A. Formation of OHN hydrogen bonds with the piperidine ring (fagopyrin A) shows larger energy differences than the formation of OHN interactions with the pyrrolidine ring (fagopyrin B). Additionally, it can be seen that the piperidine ring in the fagopyrin B prefers a "chair" conformation; however, interaction with the hydroxyl substituent in the "peri" and "bay" region can disrupt the chair conformation (structure 7–12). The presence of a free hydroxyl group (structure 10, 12) results in a significant increase in the energy of fagopyrin B. In contrast, the lack of the methyl groups brings the double anthrone system closer to planarity.

**Figure 2.** Conformers (**7**–**12**) of fagopyrin B.

Six conformers that were obtained for fagopyrin C (Figure 3) are characterized by low energy. The structure with the lowest energy (structure 14) favors the formation of

OHN hydrogen bonds and the breaking of the OHO hydrogen bonds in the "bay" region of the molecule. The piperidine ring shows a "chair" conformation for all the obtained structures (structure 13–18). Breaking of the OHN hydrogen bond located in the "bay" region results in leaving the piperidine ring free and increasing the energy of the molecule by 7.0 kcal·mol−<sup>1</sup> (structure 13). Breaking of the OHO hydrogen bond in the "bay" region together with the formation of the OHN hydrogen bond with a hydroxyl group located in the "peri" region (structure 15) is associated with an energy increase of 9.6 kcal·mol−1. Leaving the "free" hydroxyl group in the "peri" region results in a significant increase in the energy <sup>Δ</sup><sup>E</sup> ≈ 27.7 kcal·mol−<sup>1</sup> (structure 16) and <sup>Δ</sup><sup>E</sup> ≈ 65.8 kcal·mol−<sup>1</sup> (structure 18).

**Figure 3.** Conformers (**13**–**18**) of fagopyrin C.

Six low-energy conformers (Figure 4) were obtained for fagopyrin D. The lowest energy structure (structure 20) is characterized by the formation of an OHN hydrogen bond in the "bay" region. Structure 19 is characterized by a "hypericin-like" arrangement of the hydroxyl groups in the "bay", and the "peri" region differs in energy by 7.0 kcal·mol−<sup>1</sup> from the lowest energy structure. The "chair" conformation is preferred for both piperidine rings in fagopyrin D. Structure 21 is characterized by the breaking of the strong OHO hydrogen bond in the "peri" region and the formation of an OHN hydrogen bond to the piperidine ring. Such transfer of the hydrogen interaction results in the energy difference of 9.6 kcal·mol−<sup>1</sup> to the minimum energy structure (structure 20). As in the fagopyrin A–C structure, the "free" hydroxyl group (22, 24) increases the energy of the conformer; however, in such a polycyclic system, this may not be a direct expression of breaking the OHN hydrogen bond but also due to possible structural changes of the multi-ring molecule. The formation of the OHN hydrogen bond in the "peri" region stabilizes the fagopyrin D molecule.

**Figure 4.** Conformers (**19**–**24**) of fagopyrin D.

Six conformers (Figure 5) were obtained for fagopyrin E. The lowest-energy conformer (structure 26) shows hydrogen bonding in the "bay" region of the molecule. The OHN hydrogen bonds are formed by the hydroxyl groups to both nitrogen atoms in the piperidine and pyrrolidine substituent. In the minimum-energy conformer, the hydrogen bonds in the "peri" region are directed to the carbonyl oxygen. The "chair" conformation of the piperidine substituent is preferred. Conformer characterized by the "free" piperidine group (structure 25) differs in the energy of 7.5 kcal·mol<sup>−</sup>1. Additionally, breaking of OHO hydrogen bond in the "peri" region and transferring it to the "free" piperidine substituent (structure 27) raises the energy relative to conformer 25 by 2.1 kcal·mol−1. As in the fagopyrin structures described previously, breaking of a strong OHO hydrogen bond in the "peri" region and leaving the hydroxyl group unbound raises the total energy of the polycyclic system (structure 28 and structure 30).

For fagopyrin F, six low-energy conformers were obtained. The lowest-energy structure again is characterized by the formation of the OHN hydrogen bonds in the "bay" region (structure 32). The chair conformation of the piperidine substituents is preferred. The energetically similar conformers 31 and 33 are characterized by an energy difference of 7.5 and 9.7 kcal·mol−1, relatively to the minimum. As in conformers of fagopyrin E, it is possible to break the OHN hydrogen bond in the "bay" region and form an OHN hydrogen bond in the "peri" region. Breaking of the strong OHO hydrogen bond system in the "peri" region causes the deformation of the polycyclic system and deviates the molecule from planarity (36).

In general, the structure of fagopyrin tends to form OHN hydrogen bonds in the "bay" region. Energetically preferred formation of strong OHO hydrogen bonds to carbonyl oxygen in the "peri" region is evident in most conformers, and breaking of these interactions has the consequence of raising the energy of the system. Nevertheless, it is possible to break the strong OHO hydrogen bonds in the "peri" region in favor of the formation of an OHN hydrogen bond with the piperidine or pyrrolidine substituent. In summary, the introduction of piperidine and pyrrolidine substituents into the hypericin system provides an opportunity to form an OHN hydrogen bond instead of the strongest OHO.

**Figure 5.** Conformers (**25**–**30**) of fagopyrin E.
