Amides, Isoquinoline Alkaloids and Dipeptides from the Aerial Parts of Piper mullesua

Abstract One undescribed amide, pipermullesine A, two undescribed isoquinoline alkaloids, pipermullesines B and C, and six undescribed dipeptides, pipermullamides A–F, along with 28 known compounds, were isolated from the aerial parts of Piper mullesua. The structures of the undescribed compounds were elucidated based on the analysis of 1D and 2D NMR and MS data. Furthermore, the structures of pipermullesines A–C were confirmed by single crystal X-ray diffraction analysis. All isolates were evaluated for inhibitory activity against platelet aggregation induced by thrombin (IIa) or platelet-activating factor (PAF). (-)-Mangochinine, pellitorine, and (2E,4E)-N-isobutyl-2,4-dodecadienamide showed weak inhibitory activity against rabbit platelet aggregation induced by PAF, with IC50 values of 470.3 µg/mL, 614.9 µg/mL, and 579.7 µg/mL, respectively. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-018-0180-z) contains supplementary material, which is available to authorized users.

Pipermullesine A (1) had the molecular formula C 15 H 15 NO 4 based on 13 C NMR (Table 1) and HREIMS data. Its IR spectrum showed absorption peaks for a tertiary amide at 1643 cm −1 and a phenyl ring at 1595, 1513, and 1461 cm −1 . The 1 H NMR data (Table 1) [14], and two methoxy groups [δ H 3.92 (3H, s) and 3.91 (3H, s)]. The above NMR characteristic signals implied that compound 1 might be a cinnamamide derivative.
The crystals for pipermullesine B trifluoroacetate (2a) were obtained from methanol. The NMR data of 2a (Table 2) and the result of its single-crystal X-ray diffraction analysis (Fig. 3) further supported the structure elucidation of 2.
On the basis of 2D NMR correlations (Fig. 2), a 1-(4acetamidobutyl)-6-hydroxy-3,4-dihydroisoquinoline moiety was determined. One more ring is needed to meet the unsaturation, and the ring was deduced as an oxazole ring attached to C-7 and C-8 by comparison of the NMR data with those of benzoxazoles in the literature [15,16]. The additional 4-acetamidobutyl group was located at C-1′′ of the oxazole ring by the HMBC correlation from H-3′′ to C-1′′. Thus, the structure of 3 (pipermullesine C) was determined.
Fortunately, the crystals for pipermullesine C trifluoroacetate (3a) were also obtained from methanol. The NMR data of 3a (Table 3) and the result of its singlecrystal X-ray diffraction analysis (Fig. 3) confirmed the chemical structure of 3.
The molecular formula of pipermullamide A (4), C 18 H 28 N 2 O 3 , was determined by 13    . By comparing the NMR data of 4 with those of phenylalanine and leucine trimethylbetaine [17,18], compound 4 might comprise the two fragments, which was confirmed through its 1 H-1 H COSY and HMBC correlations (Fig. 2). The amino of phenylalanine was acylated by the carboxyl group of leucine trimethylbetaine according to the HMBC correlation from H-8′ to C-1. Natural amino acids generally have an L configuration. Compound 10 from the plant is also a derivative of Lphenylalanine. Accordingly, compound 4 (pipermullamide A) was elucidated as L-(N,N,N-trimethyl)leucyl-Lphenylalanine.
The molecular formulae of pipermullamides B to F (5-9) were determined as C 18

General Experimental Procedures
The instruments and materials for isolation and identification of compounds from the herb were presented in Supplementary Material.

Extraction and Isolation
The air-dried, powdered P. mullesua plant (1.8 kg) was exhaustively extracted with MeOH (4910 L) at room temperature. The MeOH extracts (92.5 g) were suspended in H 2 O and further partitioned with petroleum ether and CHCl 3 . The petroleum ether-soluble part (31.2 g) and CHCl 3 -soluble part (4.8 g) were combined (36.0 g, part B) according to the testing results of thin-layer chromatography. The water phase was partitioned by D101 resin column chromatography to obtain the water-eluted part (discarded) and 95% EtOH-eluted part (7.2 g, part A).

In vitro Platelet Aggregation Assay
The inhibitory effects of compounds against rabbit platelet aggregation induced by PAF or Thrombin (IIa) were evaluated according to the published methods [50][51][52][53]. The details were presented in Supplementary Material.

Conclusion
Thirty-seven compounds were isolated from the folk Chinese medicine Piper mullesua with the "Huayu" function associated with the antiplatelet therapies. The antiplatelet compounds, especially (-)-mangochinine, pellitorine, and (2E,4E)-N-isobutyl-2,4-dodecadienamide, might be scientific evidence to support the traditional use of the plant as folk medicine. In order to make better use of the folk medicine to serve for human health, further research needs to be conducted on bioguided isolation of compounds from the plant, based on both in vitro and in vivo bioassay testing.