Crystal structure of the cytotoxic macrocyclic trichothecene Isororidin A

The absolute configuration of Isororidin A, isolated from the fungus Myrothesium verrucaria, has been determined by X-ray crystallography.

Abstract

The highly cytotoxic macrocyclic trichothecene Isororidin A (C₂₉H₄₀O₉) was isolated from the fungus Myrothesium verrucaria endophytic on the wild medicinal plant ‘Datura’ (Datura stramonium L.) and was characterized by one- (1D) and two-dimensional (2D) NMR spectroscopy. The three-dimensional structure of Isororidin A has been confirmed by X-ray crystallography at 0.81 Å resolution from crystals grown in the ortho­rhom­bic space group P2₁2₁2₁, with one mol­ecule per asymmetric unit. Isororidin A is the epimer of previously described (by X-ray crystallography) Roridin A at position C-13′ of the macrocyclic ring.

Introduction

Macrocyclic trichothecenes (MTs) constitute the second major group (the other being the simple trichothecenes) of a class of highly functionalized sesquiterpenoid secondary metabolites, mainly of fungal origin, which are well known for their severe toxicity to both animals and humans (Grove, 2007 ▸; Shank et al., 2011 ▸; Wu et al., 2017 ▸). Most trichothecenes are at least tetra­cyclic, as they contain a spiro-epoxide group in the 12–13 position of the ‘trichothecane’ sesquiterpene skeleton. They also usually com­prise a double bond at C9—C10; thus, they are considered as 12–13 ep­oxy-trichothec-9-ene derivatives [see (a) in Scheme 1]. In MTs, an extra cyclic diester or triester ring is connected to the trichothecene core skeleton at C-4 and C-15, making them penta­cyclic macrolides. The presence of the spiro-ep­oxy group, the Δ^9,10 bond and the macrocyclic ring in the mol­ecule appear to be crucial for their biological properties, which include anti­fungal, anti­malarial, anti­virus and anti­cancer activity (de Carvalho et al., 2015 ▸; Jarvis & Mazzola, 1982 ▸; McCormick et al., 2011 ▸; Wu et al., 2017 ▸). The MTs are further classified into the subgroups of the Roridoids, to which Roridin A and Isororidin A belong [see (b) and (c) in Scheme 1, respectively], the Baccharinoids, the Verrucaroids and the Trichoverroids, which are considered the biosynthetic precursors of the three former subgroups of MTs (Bräse et al., 2009 ▸).

There has been a series of articles since the 1980s con­cer­ning the elucidation of the configuration of the stereogenic centres of the macrocyclic ring of the MTs, especially C-6′ and C-13′ in the Roridoids (Jarvis et al., 1982 ▸, 1987 ▸, 1996 ▸; Jarvis & Wang, 1999 ▸). The task was based mainly on NMR spectroscopy (despite the technical limitations of the method at that time), as well as chemical manipulations when there were adequate qu­anti­ties available, aided – in rare cases – by stereoselective synthesis and X-ray diffraction analyses. In 1982, Jarvis and co-workers isolated Roridin A and Isororidin A from a large-scale fermentation of Myrothesium verrucaria and resolved the relative configuration of Roridin A by X-ray crystallography. The absolute configuration of Roridin A was confirmed after oxidative cleavage of its hy­droxy­ethyl moiety, which produced Verrucarin A, an MT whose absolute con­fig­uration had already been established (Jarvis et al., 1982 ▸). The ¹H and ¹³C NMR spectra of Roridin A and Isororidin A in CDCl₃ were almost identical, except for carbon C-6′, which differed in the ¹³C NMR spectra by 1.1 ppm. The epimeric relation of the two fungal metabolites at C-13′ was deduced indirectly by the selective hydrogenation of Roridin A and Isororidin A to their respective tetra­hydro derivatives, and then oxidation of the C-6′ hy­droxy­ethyl group of these tetra­­hydro derivatives to an identical (in the ¹H NMR spectrum) corresponding methyl ketone (Jarvis et al., 1982 ▸). Even though Isororidin A was re-isolated a few times in subsequent years from different fungal strains and by different research groups, verification of its structure was performed only by com­parison of the NMR data in CDCl₃ with those reported in 1982, but without submitting the NMR data. Isororidin A is one of the most cytotoxic metabolites among all com­pounds containing C, H and O, and was on the shortlist of the National Cancer Institute (NCI) for the most promising anti­cancer agents in the 2000s (Amagata et al., 2003 ▸; de Carvalho et al., 2015 ▸; Sy-Cordero et al., 2010 ▸). The mechanism of action of the macrocyclic trichothecenes is still underexplored, possibly due to their severe general cytotoxicity. However, there is evi­dence that MTs show large variations in both activity and selectivity against different cancer cell lines induced by alterations in their mol­ecular structure. These findings indicate that MTs may still be considered as highly promising anti­tumour agents, as long as more detailed structure–activity relationship (SAR) and qu­anti­tative structure–activity relationship (QSAR) studies have been performed. For these studies, knowledge of the configuration and conformation of the MTs is undoubtedly critical (Wu et al., 2017 ▸; Zhu et al., 2020 ▸). In the current article, Isororidin A was isolated from the fun­gus Myrothesium verrucaria endophytic on the wild medicinal plant ‘Datura’ (Datura stramonium L.) and was characterized by 1D and 2D NMR spectroscopy. Its crystal structure is presented for the first time at 0.81 Å resolution.

Experimental

Isolation and crystallization

Isororidin A was isolated as a colourless solid (19.5 mg) after high-performance liquid chromatography (HPLC) using a semipreparative C18 column eluted with a linear gradient mixture of water and methanol. The gross structure of the com­pound was elucidated on the basis of a detailed analysis of its 1D/2D NMR and high-resolution mass spectroscopic (HRMS) data. The full 1D and 2D NMR data recorded in CD₃OD are reported for the first time (see the Analytical data section in the supporting information and Table 1 ▸). The relative configuration of its chiral centres was deduced from a combined study of nuclear Overhauser effect (NOE) correlations and ³J_HH coupling constants, and by com­parison with the NMR data of other Roridoids having similar structures (Amagata et al., 2003 ▸; Jarvis & Wang, 1999 ▸). The absolute configuration of all its chiral centres was confirmed by the X-ray crystallographic analysis of its colourless needle-like crystals that were obtained after the slow evaporation of a solution in methanol from an NMR tube. Most of the Isororidin A crystals had morphological defects that may have led to twinned spots on the diffraction pattern and potential issues at the stage of processing and deconvolution. Therefore, a small fragment of an Isororidin A crystal, with the least morphological defects, was isolated and mounted on a litho loop to minimize the background contribution when exposed to X-rays. The loop was placed on the goniometer head and diffraction data were collected at 0.81 Å resolution.

Table 1. NMR spectroscopic data for Isororidin A [400 (¹H) and 100 MHz (¹³C), δ ppm]^a.

Position (Scheme 1)	1H NMR (J in Hz)	13C NMR	COSY	HMBC	NOESY
2	3.74 (d, 5.1)	80.4	3b	4, 5, 12	3′, 13a
3	b: 2.14 (overlap by H-3′)	35.7	2, 4	2, 4
	a: 2.47 (dd, 8.2, 15.2)			2, 5, 12
4	5.84 (dd, 4.5, 8.2)	76.0	3	2, 3, 5, 6, 12, 11′	11
5	Cq	50.5
6	Cq	45.0
7	1.87 (m, 2H)	21.3	8	6, 8, 9, 11	13, 14
8	a: 1.93 (d, 8.0)	28.7	7	6,7, 9, 10
	b: 1.98 (m)
9	Cq	141.7
10	5.41 (d, 5.4)	119.7	11, 16	6, 8, 11, 16
11	3.72 (br d, 5.4)	68.5	10	7, 10, 15	4
12	Cq	66.4
13	2.86 (d, 4.0)	48.5		2, 5, 12	14
	3.05 (d, 4.0)
14	0.81 (s)	8.0	,	4, 5, 6, 12	2′, 3′, 15, 12′
15	4.32 (d, 12.2)	64.8	15	5, 6, 7, 1′	14
	4.46 (d, 12.2)			5, 6, 7, 11, 1′
16	1.72 (s)	23.3	10	8, 9, 10
1′	CO	175.6
2′	4.04 (d, 4.0)	76.7	3′	1′, 4′, 12′	14, 3′, 12′
3′	2.08 (m	37.7	2′, 12′	1′, 2′	14, 2′
4′	1.58 (m)	34.9	4′, 5′	3′, 5′, 12′
	1.73 (m)			2′, 3′, 5′
5′	3.50 (ddd, 5.2, 8.7, 9.1)	70.9	4′, 5′	3′, 4′, 6′
	3.58 (ddd, 5.2, 9.6, 9.8)
6′	3.82 (m)	84.6	7′, 13′	5′, 7′, 8′, 14′	8′, 14′
7′	6.17 (dd, 3.0, 15.4)	142.3	6′, 8′	6′, 8′, 9′	13′, 14′
8′	7.60 (ddt, 11.4, 15.4, 1.1)	126.8	7′, 9′	6′, 9′, 10′	14, 3′, 10′, 12′
9′	6.75 (t, 11.4)	145.5	8′, 10′	7′, 8′, 11′	7′
10′	5.76 (d, 11.2)	117.9	9′	8′, 9′, 11′	14
11′	CO	168.1
12′	1.09 (d, 6.8)	15.1	3′	2′, 3′, 4′	14, 2′, 3′, 8′
13′	3.69 (m)	71.0	6′, 14′	6′, 14′	7′, 8′, 14′
14′	1.16 (d, 6.4)	18.4	13′	6′, 13′	6′, 7′, 8′,13′

Note: (a) the assignments were based on ¹H–¹H COSY, HSQC–DEPT and HMBC experiments, and recorded in MeOD-d₄.

Refinement

Crystal data, data collection and structure refinement details are summarized in Table 2 ▸. Details of the geometry of the Isororidin A crystal structure regarding bond lengths (Å), bond angles (°), torsion angles (°) and the geometry of the hydrogen bonds [distances (Å) and angles (°)] are presented in the supporting information (Tables S1–S5).

Table 2. Experimental details.

Crystal data
Chemical formula	C29H40O9
M r	532.61
Crystal system, space group	Orthorhombic, P212121
Temperature (K)	100
a, b, c (Å)	9.2707 (4), 15.2236 (6), 20.0806 (8)
V (Å3)	2834.0 (2)
Z	4
Radiation type	Cu Kα
μ (mm−1)	0.76
Crystal size (mm)	0.08 × 0.06 × 0.04
Data collection
Diffractometer	Bruker APEXII
Absorption correction	Multi-scan (SADABS; Bruker, 2021 ▸)
Tmin, Tmax	0.673, 0.754
No. of measured, independent and observed [I > 2σ(I)] reflections	101717, 5548, 5338
R int	0.055
(sin θ/λ)max (Å−1)	0.617
Refinement
R[F2 > 2σ(F2)], wR(F2), S	0.041, 0.109, 1.07
No. of reflections	5548
No. of parameters	349
H-atom treatment	H-atom parameters constrained
Δρmax, Δρmin (e Å−3)	0.34, −0.22
Absolute structure	Flack x determined using 2264 quotients [(I+) − (I−)]/[(I+) + (I−)] (Parsons et al., 2013 ▸)
Absolute structure parameter	−0.02 (3)

Computer programs: APEX2 (Bruker, 2021 ▸), SAINT (Bruker, 2021 ▸), SORTAV (Blessing, 1995 ▸), SHELXT (Sheldrick, 2015a ▸) and SHELXL2018 (Sheldrick, 2015b ▸).

Results and discussion

Structural commentary

The crystal structure of Isororidin A, isolated from the ethyl acetate extract of the culture broth of the endophytic fungus M. verrucaria, after a series of chromatographic separations, is presented at 0.81 Å resolution and confirms the configuration at position C13′. Isororidin A crystallized in the ortho­rhom­bic space group P2₁2₁2₁ (No. 19). A data set was initially collected at room temperature at 0.81 Å resolution (Table S1 in the supporting information) and the calculated Flack parameter (Flack, 1983 ▸; Parsons et al., 2013 ▸) was 0.4 (4), which was not sufficient to assess the absolute configuration of Isororidin A. Therefore, a new data set was collected at 100 K. The crystal lattice and space group remained P2₁2₁2₁, with unit-cell dimensions a = 9.2707 (4), b = 15.2236 (6), c = 20.0806 (8) Å and α = β = γ = 90°, and the Flack parameter calculated for this structure was −0.02 (3), confirming the absolute configuration of Isororidin A. The experimental details are summarized in Table 2 ▸ and Tables S2–S6 of the supporting information. The two experiments reveal no temperature-dependent phase change, as the unit-cell param­eters are almost identical (Table 2 ▸ and Table S1). The measurement at 100 K resulted in an overall better data set with an improved R parameter and a higher precision Flack parameter. Therefore, the structure analysis that follows focuses on the structure determined at 100 K.

The packing of the mol­ecules is stabilized by two inter­molecular hydrogen-bond inter­actions between atom O7, which acts as a donor to symmetry-related O8ⁱ and O8, which acts as a donor to symmetry-related O2ⁱⁱ, as well as inter­molecular C—H⋯O inter­actions between C4 and O1ⁱⁱⁱ, C13 and O9^iv, and C7′ and O6^v (see Table 3 ▸ for symmetry codes). A schematic representation of the crystal structure, showing the stereoconfiguration of Isororidin A and its packing within the unit cell, is presented in Fig. 1 ▸.

Table 3. Hydrogen-bond geometry (Å, °).

D—H⋯A	D—H	H⋯A	D⋯A	D—H⋯A
O7—H7⋯O8i	0.84	1.94	2.761 (3)	167
O8—H8⋯O2ii	0.84	2.10	2.895 (3)	158
C4—H4⋯O1iii	1.00	2.55	3.467 (3)	153
C13—H13B⋯O9iv	0.99	2.65	3.490 (3)	143
C7′—H7′⋯O6v	0.95	2.62	3.473 (3)	150

Symmetry codes: (i) ; (ii) ; (iii) , ; (iv) ; (v) .

Figure 1.

(a) Schematic representation of the Isororidin A X-ray diffraction solution, drawn with 50% probability displacement ellipsoids. O atoms are shown in red, C atoms in light grey and H atoms in pale pink. The absolute configurations of C6′ (R) and C13′ (S) shown in Scheme 1 are indicated. (b) A view of the inter­molecular hydrogen-bond inter­actions formed between Isororidin A (shown in red) and its symmetry-related mol­ecules [colour code for symmetry codes: (x + , −y + , −z + 1) in lime, (−x + 1, y + , −z + ) in lavender, (x − , −y + , −z + 1) in orange, (x + 1, y, z) in tan and (−x + , −y + 1, z + ) in salmon], while the hydrogen bonding is indicated with blue dashed lines.

Superposition of the crystal structure of Isororidin A with the only available previously determined structure of Roridin A (CCDC deposition No. 1110357, CSD refcode BIDPIN10; Jarvis et al., 1982 ▸) showed that the overall structure is the same; more pro­nounced differences are observed in the macrocyclic ring, more specifically, in the vicinity of the C13′ atom [Figs. 1 ▸(a) and 2 ▸]. Both saturated pyran rings adopt dis­torted chair con­formations, with a torsion angle C5—C6—C11—O1 of −46.7 (2)° in Isororidin A versus −41.5° in Roridin A. The un­saturated cyclo­hexene rings adopt flattened half-chair con­formations, while the five-membered rings in both structures adopt envelope conformations, with the C atom at position C12 (C11 for the Roridin A structure) pointing out of the plane. The differences observed between the two structures relate to the hy­droxy­ethyl group and neighbouring atoms that include a significant rotation of the torsion angles O4—C6′—C13′—O8 and C7′—C6′—C13′—O8 by 106.7 and 104.6°, respectively. Additional differences are observed for torsion angles O7—C2′—C3′—C4′ by 18.4°, O7—C2′—C3′—C12′ by 17.2°, O9—C11′—C10′—C9′ by 17.7°, O5—C11′—C10′—C9′ by 16.2°, C7′—C6′—C13′—C14′ by 10.9°, O4—C6′—C13′—C14′ by 9.2° and O6—C1′—C2′—C3′ by 7.3°. The rest of the differences in the torsion angles ob­served in the 18-membered macrocyclic ring are less profound and in the range of 5°; for example, torsion angle O6—C1′—C2′—O7′ by 4.2° (Table S7 in the supporting information). These changes may be attributed to the inter­molecular inter­actions formed in Isororidin A com­pared to Roridin A [Fig. 1 ▸(b)].

Figure 2.

Superposition of the three-dimensional structures of determined Isororidin A (with an S configuration at C13′) and its stereoisomer (epimeric at C28 with an R configuration) Roridin A. Isororidin A is shown in red and Roridin A in grey.

Supra­molecular features

A schematic representation of the structure of Isororidin A and its packing with symmetry-related mol­ecules within the crystal is shown in Fig. 3 ▸. Isororidin A crystallized in the ortho­rhom­bic space group P2₁2₁2₁. The difference observed in the epimeric C atom seems to foster the inter­molecular inter­actions within the unit cell. Atom O8 is hydrogen bonded to O2 of a symmetry-related mol­ecule within the unit cell, while in the case of Roridin A, the same atom inter­acts with O1.

Figure 3.

Schematic representation of the supra­molecular structure of Isororidin A. The asymmetric unit is highlighted in black and the hydrogen bonds are indicated in blue. [Symmetry codes: (i) −x+, y − , −z + ; (ii) −x + , −y + 1, z + ; (iii) −x + 1, y + , −z + ; (iv) −x + , −y + , −z + 1; (v) −x + , −y + 1, z − .]

Database survey

One entry is available in the Cambridge Structural Database (CSD; Groom et al., 2016 ▸) for the structure of Roridin A (CCDC deposition No. 1110357, CSD refcode BIDPIN10; Jarvis et al., 1982 ▸) determined in the space group P2₁ with unit-cell dimensions a = 10.197 (3), b = 14.079 (4), c = 9.606 (2) Å, α = γ = 90° and β = 94.6 (1)°.

Supplementary Material

CCDC references: 2083096, 2279458

Funding Statement

This work was funded by Greek General Secretariat for Research and Innovation grant 5002550 to Evangelia Chrysina.