Synthesis and crystal structure of (2S,4aR,8aR)-6-oxo-2,4a,6,8a-tetrahydropyrano[3,2-b]pyran-2-carboxamide

(2S,4aR,8aR)-6-Oxo-2,4a,6,8a-tetrahydropyrano[3,2-b]pyran-2-carboxamide, a potentially effective antibacterial agent, was prepared by a chemoselective hydration of the corresponding nitrile using a heterogeneous catalytic method based on copper(II) supported on molecular sieves, in the presence of acetaldoxime. It belongs to a new class of pyranopyrans that possess antibacterial and phytotoxic activity.


Chemical context
The phytotoxin diplopyrone 1 was isolated from the fungus Diplodia mutila and reported in 2003 (Evidente et al., 2003). This fungus is considered a causative agent of cork oak decline and diplopyrone is implicated as the main phytotoxin responsible for this disease, the economic and environmental impacts of which are well known (Giorgio et al., 2005). The proposed structure of diplopyrone contains a cis-fused pyranopyran core and four chirality centers, originally assigned as 9S,6R,8aS,4aS, but revised recently to 9R,6S,8aS,4aS (Fusè et al., 2019). In 2019, our laboratory published the synthesis and biological evaluation of pyranopyran analogs based on the structure of diplopyrone (Lazzara et al., 2019). These enantiomeric analogs showed antibacterial and phytotoxic activity, in one case exceeding the activity of a commercially used antibiotic that is used to treat bacterial diseases in pond-raised catfish, which is the largest segment of aquaculture in the United States. Pyranopyran nitrile 2 was approximately 100 times more potent in bioassay than florfenicol against Edwardsiella ictaluri, which causes enteric septicemia (ESC), a disease that can result in losses of tens of millions of dollars to the industry annually. Compound 2 was also phytotoxic in an assay using the aquatic plant Lemna paucicostata (L.) Hegelm. (duckweed). As part of our ongoing efforts to synthesize additional analogs of 1 for testing as new antibacterials and herbicides, we have recently prepared amide 3, by a heterogeneous catalytic method that uses copper(II) supported on molecular sieves, in the presence of acetaldoxime to carry out chemoselective hydration of 2 (Kiss & Hell, 2011).

Structural commentary
Pyranopyrans in which the two rings are cis-fused are relatively rare compared to trans-fused pyranopyrans (Giuliano, 2014). A consequence of the cis ring fusion is that the molecule has more of a bent shape than it would if trans-fused, which is demonstrated by the O1-C8A-C4A-O5 torsion angle of 72.95 (15) versus 177 for a comparable trans-fused pyranopyran (Yu et al., 2017). Both rings adopt half-chair conformations, placing the amide group in a near 1,3-diaxial interaction with H4A. These features are consistent with the results in the computational study reported (Evidente et al., 2003). The study suggests the hydroxyethyl side chain is involved in an intramolecular hydrogen bond between the hydroxyl group and the O5 ring oxygen. By contrast, the amide side chain in 3 does not exhibit a similar intramolecular hydrogen bond with its amino group in the solid state, as shown in Fig. 1. The overall structure of 3 is nearly identical to that of the pyranopyran nitrile 2 with obvious deviation at the side chain.
The NMR spectra of the pyranopyran amide 3 are similar to those of pyranopyran nitrile 2. The most obvious difference in the 13 C spectra is the presence of the additional (amide) carbonyl carbon in 3 at 174.1 ppm and the absence of the nitrile carbon that occurs at 114.9 in 2. The 1 H spectrum of 3 shows slight changes in the chemical shifts of most protons, for example there is a downfield shift of H4A from 4.45 ppm in the nitrile to 4.61 ppm in the amide. The vinyl hydrogen H4 is also further downfield in the amide ( 7.10 ppm vs 6.91 ppm). The torsion angle of 45.8 for H4A-C4A-C8A-H8A in 3 is consistent with the observed vicinal coupling constant of 4.5 Hz for H4A-H8A in the associated 1 H NMR spectrum.

Supramolecular features
The amino hydrogen atoms of 3 are involved in intermolecular hydrogen bonding with adjacent carbonyl oxygen atoms: H1A with O2 i and H1B with O3 ii (Fig. 3, Table 1, Symmetry codes: (i) x, y + 1, z; (ii) x + 1, y, z.). A packing diagram of 3 ( Fig. 2a) shows the N-HÁ Á ÁO hydrogen-bonding interactions forming molecular planes defined by the crystallographic a-and b-axes; packing of these hydrogen-bonded layers appears to be a function of solvent exclusion and van der Waals contact alone, lacking any hydrogen bonding.
The hydrogen-bonded network in the ab plane also presents an arrangement of C-HÁ Á ÁO and C-HÁ Á Á interactions that suggests two potential additional forces at play within the lattice of 3. Fig. 2b and Table 1 depict distances between H6 and O3 ii and C6 and O3 ii of adjacent copies of 3. These distances fall within parameters for C-HÁ Á ÁO hydrogen bonding as has been described in well-characterized membrane proteins and peptidomimetics (Senes et al., 2001;Giuliano et al., 2009); the -protons implicated in these systems are structurally analogous to the C6-H6 bond of 3. While we will not speculate on the energetic significance of this interaction, which can arise as a coincidence of crystal packing (Dunitz & Gavezzotti, 2005), we note that such interactions have been spectroscopically measured within the core of the dimeric membrane peptide glycophorin A (Arbely & Arkin, 2004). Further, solid-state NMR studies have observed that 1 H and 13 C NMR shifts change for anomeric C-H bonds in crystalline maltose samples, suggesting that such interactions as described in this study (the C6-H6 bond in 3 is pseudo-anomeric) are not consequences of an energetically dominant lattice arrangement and N-HÁ Á ÁO hydrogen bonding, but rather have some measurable, albeit weak, energetic contribution to intermolecular association (Yates et al., 2005).
Within the ab plane, H4A of one copy of 3 comes into close approach with its closest neighbor along the a axis. Fig. 3c depicts these distances, which place the centroid of the C7 C8 double bond within distance parameters similar to those calculated for aliphatic C-HÁ Á Á interactions (Karthikeyan et al., 2013). We investigated this further using a semiempirical protocol to generate partial charges for the atoms of 3. This only allows for qualitative comparison, and, as the color coding in Fig. 2c  Molecular structure of 3 with displacement ellipsoids at the 50% probability level. Table 1 Hydrogen-bond geometry (Å , ).
positive). Proper exploration of this would require more advanced QM/MM methods, however, the crystal packing of 3 is at least suggestive of a favorable geometry and electrostatic environment for C-HÁ Á Á interactions.

Database survey
A search of the Cambridge Structural Database (CSD Version 5.41, November 2019; Groom et al., 2016) using the core fused ring lactone in the search query revealed only three similar structures (Somarathne et al., 2019;Lazzara et al., 2019) in which the pyranopyran ring system is cis-fused and the two double bonds are in the same location as they are in 3. Among the total 40 structures that were found in the search, the pyranopyran core of several were trans-fused, for example, the bergenins and also truncated ladder ethers related to brevitoxin. Some compounds possessed aryl rings fused to the pyranopyran system while others had double bonds at alternate positions including the ring junction.

Refinement
Crystal data, data collection, and structure refinement details are summarized in Table 2. The absolute configuration was known from the synthetic route and assigned accordingly. The amino hydrogen atoms were found in the electron difference map and refined isotropically, while all other hydrogen atoms were treated as idealized contributions with C-H = 0.95-1.00 Å and U iso (H) = 1.2U eq (C).  (c) Measured distances and electrostatic coloring between H4A, C7, and C8 used to explore a potential C-HÁ Á Á interaction within the ab plane molecular layers. Partial charges were generated within UCSF Chimera (Pettersen et al., 2004) using the Amber ff14SB forcefield in Antechamber (Wang et al., 2006) with the semi-empirical AM1 À BCC method and color coded with pink for negative charges and light blue for positive charges.  Computer programs: APEX3 (Bruker, 2016), SAINT (Bruker, 2015), SIR92 (Altomare et al., 1994), SHELXL2014 (Sheldrick, 2015) and OLEX2

Special details
Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Refinement. 1. Fixed Uiso At 1.2 times of: All C(H) groups 2.a Ternary CH refined with riding coordinates: C4A(H4A), C6(H6), C8A(H8A) 2.b Aromatic/amide H refined with riding coordinates: C3(H3), C4(H4), C7(H7), C8(H8) 3. N1(H1A) and N1(H1B) located from the difference map with refined Uiso  (2)