Ochrocephalamine A, a new quinolizidine alkaloid from Oxytropis ochrocephala Bunge

Abstract

One dimeric matrine-type alkaloid, ochrocephalamine A (1), was isolated from the poisonous plant Oxytropis ochrocephala Bunge. Its structure was elucidated by spectroscopic data and single-crystal X-ray diffraction. The insecticidal and cytotoxic activities of 1 were evaluated.

Graphical Abstract

The plant Oxytropis ochrocephala Bunge is a common poisonous plant found in the western grasslands of China^1–2. After ingesting the plant, livestock gradually develop a chronic neurological disease, characterized by a staggering gait and muscular incoordination, giving rise to the vivid name ‘locoweed’ for this poisonous plant³. Previous phytochemical investigations on O. ochrocephala provided quinolizidine and indolizidine alkaloids, as well as flavonoids and saponins^4–10.

Quinolizidine alkaloids are important natural products exhibiting a broad array of pharmacological activities, such as antineoplastic, antibacterial, antiviral, and insecticidal properties¹¹. Recently, we reported the synthesis of sophoridine derivatives as potential antitumor agents¹². As part of our ongoing research program to identify bioactive constituents, particularly from this alkaloid class, we obtained a new quinolizidine alkaloid, ochrocephalamine A (1), from O. ochrocephala. Herein, we describe the isolation, structural elucidation and bioactivity of this compound.

The whole plant (20.0 kg) of O. ochrocephala was percolated three times with 95% EtOH to give a crude extract (3.0 kg). The extract was concentrated to dryness under reduced pressure, followed by partitioning between CH₂Cl₂ and 2% HCl. The aqueous phase was then adjusted to pH 11 with 3% NaOH and extracted with CH₂Cl₂ to give crude alkaloids (100 g). The crude alkaloids were subjected to a silica gel column chromatography eluted with CH₂Cl₂/MeOH (1:0 to 0:1) to obtain fractions A, B and C. Fraction B was chromatographed on silica gel [CH₂Cl₂/MeOH (10:1)] and then RP-18 (30% MeOH) columns to yield ochrocephalamine A (1, 20 mg).

Ochrocephalamine A (1)¹³ was obtained as a light-yellow solid (CH₂Cl₂), and its molecular formula was established as C₃₀H₄₂N₄O₂ by HR-EI-MS (m/z 490.3296 [M]⁺, calcd 490.3308), requiring 12 degrees of unsaturation. The ¹H NMR data of 1 (Table 1) showed two groups of signals characteristic for a matrine-type alkaloid at δ_H 4.29 (1H, dd, J = 12.8, 4.0 Hz) and 3.11 (1H, br t, J = 12.8 Hz) and at 4.09 (1H, dd, J = 12.8, 7.2 Hz) and 3.80 (1H, br t, J = 12.8 Hz)¹⁴. In addition, two coupled olefinic protons [δ_H 7.30 (1H, d, J = 7.6 Hz), 6.51 (1H, d, J = 7.6 Hz)] were distinguished by NMR analysis. The ¹³C-NMR data (Table 1) accounted for 30 carbon signals, classified as four sp² quaternary carbons including two lactam-CO signals at δ_C 170.9 and 164.6, two sp² and eight sp³ methines, and 16 methylenes. Six sp² carbons accounted for 4 out of the 12 degrees of unsaturation, and the remaining 8 degrees required that 1 is an octacyclic system.

Table 1.

¹H (400 MHz) and ¹³C NMR (100 MHz) Data of Ochrocephalamine A (1) in CD₃OD

No	δH (mult, J)	δC	no.	δH (mult, J)	δC
2	2.82 (m)a 2.07 (m) a	57.8	2′	2.82 (m) a 2.07 (m) a	58.3
3	1.56–1.65 (m) a	22.2	3′	1.56–1.65 (m) a	21.6
4	1.79 (m) a	27.8	4′	1.67–1.71 (m) a	28.7
5	2.25 (m)	32.9	5′	1.80 (m)	37.5
6	2.33 (t, 4)	61.5	6′	2.29 (br s)	65.6
7	3.03 (br s)	39.5	7′	1.89 (m)	42.6
8	2.15 (m)	27.5	8′	2.44–2.50(m)	28.4
9	1.56–1.65 (m) a	22.2	9′	1.56–1.65 (m) a	21.3
10	2.82 (m) a 2.07 (m) a	57.6	10′	2.82 (m) a 2.07 (m) a	58.2
11		147.7	11′	3.94 (m)	53.8
12	6.51 (d, 7.6)	106.6	12′	2.14 (m)	29.8
13	7.30 (d, 7.6)	135.7	13′	3.38 (m)	30.0
14		130.1	14′	2.62(dd, 16.0, 4.0) 2.46(dd, 16.0, 8.0)	37.5
15		164.6	15′		170.9
17	4.09 (dd, 12.8, 7.2) 3.80 (br t, 12.8)	45.5	17′	4.29 (dd, 12.8, 4.0) 3.11 (br t, 12.8)	43.7

^aoverlapped.

Comprehensive analysis of the 1D NMR data suggested that 1 contains two matrine-type alkaloid moieties, sophoramine and matrine¹⁵. Considering the octacyclic ring system of 1, this compound was likely a dimeric alkaloid of sophoramine and matrine connected through a C-C bond. Accordingly, the methine of sophoramine at C-14 (δ_C 116.4) and methylene of matrine at C-13 (δ_C 19.2)¹⁵ were replaced by the quaternary carbon (δ_C 130.1) and methine (δ_C 30.0), respectively, in 1. The HMBC correlations from H-13 to C-15, C-11 and C-13′, H₂-14′ to C-15′, C-13′ and C-14, H-13′ to C-15′ and C-14′, and H₂-12′ to C-7′, C-11′, C-13′ and C-14 confirmed the C-14–C-13′connection. Thus, the gross planar structure of ochrocephalamine A was established as shown (Figure 2a).

Figure 2.

a) key HMBC correlations of 1, b) X-ray crystal structure of 1.

Determinations of the stereo configurations of 1 were particularly challenging. The ROESY correlations of H-17′α to H-5′, H-6′ to H-7′ indicated H-5′, H-6′ and H-7′ were α configuration¹⁰. H-11α was assigned in β-configuration on the basis of the mutual ROESY between H-17′β and H-11′¹⁰. The relative configuration of H-13′ could not be determined by a ROESY experiment due to unobserved correlations between H-11′ and H-13′ as well as H-11′ and H-14′. Additionally, the relative configuration of H-5, H-6 and H-7 also can’t be determined by ROESY experiment due to unobserved correlations between one of them with another confirmed proton configuration. Thus, we attempted to prepare crystals of 1 for X-ray determination. Eventually, a single crystal of this compound was obtained from EtOH. The connection between C-14 and C-13′ was confirmed by X-ray crystallographic diffraction with CuKα radiation and the relative configurations of H-5, H-6, H-7 and H-13′ were α-configuration (Figure 2b)^13,16. The absolute configurations of 1 were also determined by the refined Hooft parameter value 0.01(6) for 1892 Bijovet pairs with a probability of 1.000 as shown in Figure 2b¹⁶.

From a biogenetic point of view, matrine could react with known sophoridine through a Michael addition reaction to give the intermediate B. Aromatization of this intermediate would then yield 1.

We evaluated the contact toxicity of 1 against Spodoptera litura third-instar larvae by the micro-drip method. One μL of 1 (3 mg/mL in EtOH) was dropped on the pronotum of larvae. After five days, 50% of larvae were killed. In cytotoxicity assays, compound 1 was not active against A549, KB, KB-VIN, and MDA-MB-231 cell lines (ED₅₀ > 20 μM).

O. ochrocephala is widely distributed in the grasslands of eastern and central Asia, Australia, and western North and South America, where it poses an extreme risk to grazing animals, resulting in a chronic neurological disease^1,3. The indolizidine alkaloid swainsonine is claimed as the constituent poisonous to livestock³. In view of the plant itself, poisonous secondary metabolites can improve the plant’s chances of survival and reproduction by deterring animals, such as sheep, cattle, and horses. In addition, quinolizidine alkaloids^{8–9, 17}, such as 1, play an important role in defense against insects. Biogenetically, indolizidine and quinolizidine alkaloids originate from the same precursor L-lysine¹⁸. The use of one biogenetic pathway to produce two different types of alkaloids involved in plant defense against both livestock and insects, respectively, is a smart and efficient strategy to combat the survival challenge.

Figure 1.

The structure of ochrocephalamine A (1)

Scheme 1.

Biogenetic pathway proposed of ochrocephalamine A (1)

• Ochrocephalamine A (1) was isolated from Oxytropis ochrocephala Bunge.

• The poisonous plant O. ochrocephala poses an extreme risk to grazing animals.

• Compound 1 is a dimeric matrine-type alkaloid.

• Its structure was solved from spectral data and single-crystal X-ray diffraction.

• Compound 1 showed contact toxicity against Spodoptera litura third-instar larvae.