Pruinosanones A-C, anti-inflammatory isoflavone derivatives from Caragana pruinosa

Pruinosanone A (1), a novel spirochromone, was isolated from the roots of Caragana pruinosa. Two biogenetically related isoflavone intermediates, pruinosanones B and C (2 and 3), were also isolated, together with five known analogs identified as 3-hydroxy-9-methoxypterocarpan (4), 7,2′-dihydroxy-4′-methoxyisoflavanol (5), retusin-8-methylether (6), 7,2′-dihydroxy-8,4′-dimethoxy isoflavone (7) and 7,3′-dihydroxy-8,4′-dimethoxy isoflavone (8). The structures of 1–3 were elucidated based on extensive spectroscopic methods. Notably, 1 is the first example of a spirochromone possessing an unprecedented pentacyclic skeleton containing a spiro[benzo[d][1,3]dioxole-2,3′-chroman]-4′-one motif, which was confirmed by X-ray diffraction analysis. A plausible biosynthetic pathway for 1 was also proposed. Compounds 1–8 were tested for their ability to inhibit nitric oxide (NO) production in LPS-induced RAW 264.7 macrophages, and compounds 1–3 were the most potent inhibitors of NO production, with IC50 values of 1.96, 1.93 and 1.58 μM, respectively. A structure-activity relationship analysis revealed that the fused 2-isopropenyl-2,3-dihydrofuran moiety plays a vital role in the potency of these compounds. Moreover, 1 was found to significantly inhibit inducible nitric oxide synthase (iNOS) protein expression, which accounts for the potent inhibition of NO production by this spirochromone.

Pruinosanone A (1), obtained from C. pruinosa as optically active, colorless needles ( α [ ] D 25 + 25°), possesses the molecular formula C 22 H 20 O 7 (13 degrees of unsaturation), which was deduced from HRESIMS analysis ([M + H] + at m/z 397.1269), 13 C NMR data, and DEPT spectra (see Supplementary Information). The 1 H NMR spectrum (Table 1)    Hz)] neighboring the quaternary carbon C-3 (δ C 106.0), with a large down-field shift in 1. C-3 was assigned to be an acetal carbon deduced from two more oxygen atoms that remained according to the molecular formula, which led to the formation of a spiro system between rings C and B. The HMBC experiment also indicated correlations between H-2/C-3, C-4, H-5′ /C-1′ , and H-6′ /C-2′ . Furthermore, the HMBC correlations ( Fig. 2) between H-2″ /C-7 and C-8 revealed that the oxygenated 1-isopentenyl group was attached at C-8. The downfield shifts of C-2″ and C-7 indicated that C-2″ and C-7 are linked through an oxygen bridge, thus forming a furan ring. In addition, the HMBC spectrum of compound 1 displayed long-range correlations of H-8′ , H-6′ /C-4′ and H-7′ , H-5′ /C-3′ , combined with a NOESY cross peak between H-8′ and H-5′ , indicating that one methoxy group was linked to C-3′ and the other to C-4′ . Based on these data, we hypothesized that compound 1 likely possesses an unprecedented pentacyclic skeleton containing a spiro[benzo[d] [1,3]dioxole-2,3′ -chroman]-4′ -one motif. The planar structure that was established is shown in Fig. 1, and this compound was named pruinosanone A. The relative stereochemistry of 1 was deduced on the basis of its NOESY spectroscopic data (Fig. 2). The NOESY correlations of H-2″ with H-1″ β indicated that these two protons are located on the same face. The configuration of 1 at C-2″ was thereby established, whereas no obvious NOESY correlation was detected for determining the relative configuration of another chiral carbon at C-3.
Fortunately, 1 was crystallized by slow evaporation from a solution in a mixture of CH 3 OH/CH 2 Cl 2 (1:1) over 7 days. The resulting crystals were suitable in size and quality for single-crystal X-ray analysis using anomalous dispersion with Cu Kα radiation, which revealed an absolute structure parameter (Flack's x) of 0.03(7) 17,18 , allowing unambiguous assignment of the complete absolute configuration of 1 as a 3S and 2″ S configuration (Fig. 3). The structure of pruinosanone A (1) was therefore defined as (2S, 8′ S) Compound 2, named pruinosanone B, was obtained as a yellow, amorphous powder. The positive HRESIMS data ([M + H] + at m/z 381.1343, calcd 381.1338) indicated the molecular formula of 2 to be C 22 H 20 O 6 . Its 1 H and 13 C NMR spectra ( Table 2) were characterized by data typical for an isoflavone skeleton and were analogous to those of 1, except for the major differences in the signals corresponding to C-2 and H-2 (δ H 8.24, s, 1H and δ C 154.6 in 2; δ H 4.63, 4.64, d, J = 12.6, 2H and δ C 70.5 in 1) and the appearance of several slightly shifted signals from the B-ring. These data indicated that 2 was an isoflavone derivative of 1, which was confirmed by detailed HMBC analysis, particularly the HMBC correlations from H-2 (δ H 8.24, s, 1H) to C-4 (δ C 175.3), C-1′ (δ C 113.9) and from H-6′ (δ H 6.90, d, J = 8.4, 1H) to C-3 (δ C 122.2) (Fig. 2). The signals for two pairs of ortho-aromatic protons in 2 were nearly the same as those in 1, revealing an identical substitution pattern in rings A and B for 2 and 1. The NOESY correlations were also similar to those of 1. The absolute configuration at C-2″ could be assigned by analogy to the configuration of 1 from a biosynthetic view and was confirmed by the similar signals for H-2″ [δ H 5.54 (dd, 9.6, 7.8) in 2; δ H 5.37 (dd, 10.2, 7.8) in 1] and C-2″ (δ C 87.8 in 2; δ C 88.1 in 1). The structure of pruinosanone B (2) was therefore defined as (2″ S)-8-(2-methylbut-1-en-4-yl)-7,2″ -epoxy-2′ -hydroxy-3′ ,4′ -dimethoxyisoflavone.   Table 2 and suggest that the compound has most of the same structural features as 2, except that compound 3 has one less degree of unsaturation than 2. Considering the molecular weights of 3 and 2, compound 3 was considered to be a 2,3-hydrogenation derivative of 2, which was further confirmed by detailed HMBC, COSY and NOESY examination. The equatorial orientation of the B-ring was verified by the NMR coupling constant between the trans-diaxial H-2β and H-3 of ca. 11 Hz 19 . Furthermore, circular dichroism (CD) was used to establish the absolute configuration of C-3 as 3R based on the positive cotton effect (CE) of the n → π * carbonyl absorption band at 329 nm 20 . Thus, pruinosanone C (3) was concluded to be (3R, 2″ S)-8-(2-methylbut-1-en-4-yl)-7,2″ -epoxy-2′ -hydroxy-3′ ,4′ -dimethoxyisoflavanone.
The biosynthetic pathways for 1-3 were proposed to start from a chalcone derivative (Fig. 4). This pathway involves two key epoxidation steps 21 that occur between C-2/C-3 and C-3/C-1′ , respectively, followed by an intermolecular carbonyl addition reaction to form a five-membered spiro-heterocycle between rings C and B.
Ability of the compounds to inhibit LPS-induced NO production. NO acts as a host defense mechanism by damaging pathogenic DNA and is also a regulatory molecule with homeostatic activities 22 . However, an excess production of NO in biological systems gives rise to various diseases, such as inflammation, cancer, and atherosclerosis 23 . Therefore, substances that inhibit NO release may be of therapeutic benefit in various disorders induced by pathological levels of NO 21 . Compounds 1-8 were tested for their ability to inhibit NO production in LPS-induced RAW 264.7 macrophages according to a previously described method 24,25 . The results (Table 3) indicated that compounds 1-3, which have a 2-isopropenyl-2,3-dihydrofuran moiety fused with ring A, possessed much better inhibitory activity, with IC 50 values ranging from 1.58 to 1.96 μ M, than compounds 4-8, which had IC 50 values > 100 μ M. This suggests that the presence of a fused 2-isopropenyl-2,3-dihydrofuran ring plays a vital role in the potency of these compounds. Pruinosanone A (1) was a potent inhibitor of NO production, remarkably suppressing NO release in a dose-dependent manner (Fig. 5A), with an IC 50 value of 1.96 μ M, which is much less than that of the positive control aminoguanidine (AG, IC 50 20.13 μ M).
Cell viability. Cell viability was determined using the CCK-8 method to evaluate whether the inhibition of NO production was due to the cytotoxicity of the tested compounds. It was found that none of the concentrations used in this experiment were cytotoxic (Fig. 5B). Thus, the inhibitory activity of these isoflavone derivatives was not due to their cytotoxic properties but to their ability to suppress NO production, which merited further study regarding the precise site and mechanism of action of these compounds.  Western blot analysis. Nitric oxide synthases (NOSs) play a very important role in catalyzing the production of NO from L-arginine. Inflammatory mediators, such as IL-1, TNF-α , and LPS, stimulate the expression of the inducible isoform of NOS (iNOS) in rodent macrophages, which leads to the prolonged production of large amounts of NO, a characteristic of many inflammatory diseases [26][27][28][29] . To elucidate the underlying mechanism of these compounds in the inhibition of NO, pruinosanone A (1) was selected to investigate the effect of the compound on iNOS protein expression in LPS-induced RAW 264.7 cells. According to the results, pruinosanone A was found to significantly down-regulate the expression of iNOS in a dose-dependent manner, which accounts for the potent inhibitory activity of compound 1 against NO production (Fig. 5C).
In conclusion, pruinosanone A (1) is the first pentacyclic spirochromone containing a spiro[benzo[d] [1,3] dioxole-2,3′ -chroman]-4′ -one motif, which has never been observed before for any chromone. Therefore, this structure represents a new carbon skeleton. The structure of 1 provides not only an interesting synthetic target but also a potent inhibitor of NO release. Further studies on this molecule will provide valuable insights into the development of anti-inflammatory drugs.

Methods
General experimental procedures. Optical rotations were acquired with a Perkin-Elmer 341 polarimeter.
The crystallographic data for the structure of pruinosanone A (1)  Inhibition of LPS-induced NO production. RAW 264.7 macrophages were seeded in 96-well plates (10 5 cells/well). The cells were co-incubated with the isolated compounds and LPS (1 μ g/mL) for 24 h. The amount of NO was assessed by determining the nitrite concentration in the cultured RAW 264.7 macrophage supernatants with the Griess reagent. Aliquots of the supernatants (100 μ L) were incubated in sequence with 50 μ L of 1% sulfanilamide and 50 μ L of 0.1% naphthylethylenediamine in a 2.5% phosphoric acid solution. The absorbance at 548 nm was read using a microplate reader (POLARstar).
Cell viability. Cell viability was assessed using the mitochondrial respiration-dependent MTT reduction method. After transferring the required supernatant to another plate for the Griess assay, the remaining supernatant was aspirated from the 96-well plates, and 100 μ L of fresh medium containing 2 mg/mL of MTT was added to each well. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO 2 . After incubating for 3 h, the medium was removed, and the violet crystals of formazan in the viable cells were dissolved in DMSO. The absorbance at 570 nm was measured using a microplate reader.
Western blot analysis. The murine RAW 264.7 cell line was seeded at an initial density of 2 × 10 6 cells/well in 6-well tissue culture plates and incubated overnight. The cells were exposed to Escherichia coli LPS (1 μ g/mL; Sigma) for 24 h in the presence or absence of the tested compounds. These compounds were dissolved in DMSO at an initial concentration of 10 mM and diluted to an appropriate concentration using culture medium; the final concentration of DMSO was adjusted to ≤ 0.01%. Beta-actin (β -actin) was used to ensure that the amounts of protein were equal in each lane. Protein samples were collected and prepared as described previously 30 , and the iNOS expression levels were investigated using western blot analysis. Briefly, samples containing equal quantities of protein (50 μ g) were subjected to SDS/20%-polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to nitrocellulose (NC) membranes. The resulting NC membranes were incubated with a blocking solution and probed with an antibody specific to the iNOS protein (1:1000 dilution; Cell Signaling). Then, the antibodies were visualized using an ECL detection kit (Western Lightning Chemiluminescence Reagent Plus, PerkinElmer).