Unusual alkaloids of the highland species Astragalus cryptanthus Wedd. (Fabaceae)

Abstract Two unusual caprolactam alkaloids, 3-(dimethylamino)hexahydro-2H-azepin-2-one and 3-(methylamino)-hexahydro-2H-azepin-2-one, were isolated from the aerial parts of Astragalus cryptanthus Wedd.; their structures were unambiguously determined based on data from extensive 1D and 2D NMR, GC-MS and FT-IR spectroscopic analyses. This is the first report of this alkaloid type in the genus Astragalus.


Introduction
The genus Astragalus (Fabaceae) has a worldwide distribution with over 3000 accepted species (Osaloo et al. 2003). The most common use of Astragalus is as forage for livestock and wild animals, although a number of species have been shown to be toxic for livestock (Panter et al. 2012) and the toxins transferred to humans through meat and milk (Panter & James 1990). These are generically referred to as locoweeds from the Spanish 'loco' (crazy), based on the neurological disturbances they cause on animals. Two different kinds of toxic nitrogen compounds have been described in the genus Astragalus: indolizidine alkaloids such as swainsonine, and nitro compounds such as nitropropionic acid-glucose derivatives; the plants that cause locoweed poisoning contain swainsonine (Molyneux & James 1982). Among the toxicological effects described for swainsonine are the induction of high mountain disease in the animals that graze at high elevation (James et al. 1991). As far as we are aware, most phytochemical studies that isolated alkaloids have been performed in Astragalus species collected at relatively low altitudes (Pistelli 2002;Yang et al. 2013). Astragalus cryptanthus is a herb that grows in the high plateau ('altiplano') of the southcentral Andes at altitudes above 4500 masl, in places where wild camelids such as vicuña (Vicugna vicugna) and guanaco (Lama guanicoe) graze. Intensive conservation programmes for vicuña which have been implemented locally prompted us to undertake the study of alkaloids present in extracts of A. cryptanthus. We did not find swainsonine, but rather two unusual caprolactam alkaloids -one of them described for the first time from natural sources -which can be related biogenetically to swainsonine.

Results and discussion
Compounds 1 and 2 ( Figure 1) were presumably endowed with nitrogen atom or atoms on the basis of TlC examinations using Dragendorff's reagent. The molecular formulae of 1 and 2 were determined as C 8 H 16 N 2 O and C 7 H 14 N 2 O (both with an unsaturation degree of 2) based on their ESI-MS which showed M + ion peaks at m/z 156 (Calcd 156.13) and m/z 142 (Calcd 142.11), respectively. These even molecular masses indicated the likely presence of two nitrogen atoms; in fact, the IR spectra showed absorption bands assignable to amino and amide functionalities. The mass spectrum of 1 showed main ions at m/z 111, 98, 85, 84, 71, 70, 56, 44 and 42, while the mass spectrum of 2 showed main ions at m/z 99, 84, 71, 70, 56 and 42. Relative intensities of ions in the mass spectra of 1 and 2 are given in Figure S1 and a proposal for the fragmentation patterns leading to the main ions in Figure S2.
The analysis of the 1D and 2D NMR spectra of 1 and 2 ( Figure S3) led to the full assignment of the carbon and proton signals. Compounds 1 and 2 showed similar chemical shifts. However, in the 1 H NMR of 2 the unique signal at 3.26 ppm was attributed to the N-H proton of the N-monomethyl fragment, consistent with the presence of the singlet at 2.43 ppm integrating for a single methyl group and the singlet at 2.35 ppm in 1 integrating for two methyl groups. The use of 2D methods (COSY, HMQC, HMBC, and NOESY) allowed the assignment of all 13 C NMR resonances of these compounds. A comparison of the 13 C/DEPT NMR spectra of 1 and 2 showed that the main differences between them occurred in the 13 C NMR chemical shifts of the signals for the C3 and C2, the C atoms closest to the substituent differing between the two compounds. COSY data analysis confirmed the caprolactam system and the linkage of the carbonyl unit was established via HMBC correlations ( Figure S4).
Although caprolactam alkaloids are not common in nature, volatile extracts from Astragalus gombiformis Pomel collected in Tunisia have been shown to contain caprolactam (Teyeb et al. 2011), alkaloid 1 was found in the leaves and tubers of Ceropegia bulbosa (Apocynaceae) collected in the state of Rajasthan, India (Palawat & lodha 2014), while alkaloid 2 has not been previously reported from natural sources. Caprolactam alkaloids such as 1 and 2 can be biosynthesised from lysine via pipecolic acid, through several biosynthetic steps preceding the generation of indolizidinic alkaloids such as swaisonine.
Not all species of Astragalus contain swainsonine (Ibrahim et al. 2013), but this alkaloid occurs in all of those that typically have been known as locoweeds (James et al. 1992). Thus, absence of reports of high mountain disease in wild animals in the Andes altiplano which consume A. cryptanthus is consistent with the absence of swainsonine from extracts of A. cryptanthus and with eventual bioactivity of 1 and 2 being unrelated to this disease.

Experimental
Please refer to the Supplementary Material online for more on the "Experimental" section.

Conclusion
Two caprolactam alkaloids were isolated from the aerial parts of A. cryptanthus. These compounds are the first examples of caprolactam alkaloids in the genus Astragalus; one of them was isolated for the first time from natural sources.