New alkaloids from Daphniphyllum himalense

Abstract

Nine new Daphniphyllum alkaloids (1–9) and 16 biosynthetically related known ones were obtained during the phytochemical investigation into a Nepalese plant, Daphniphyllum himalense. Structures were assigned to these compounds based on careful spectroscopic interpretation and NMR comparison with literature data. Deuteration of H₂-16 in CD₃OD due to quick keto-enol tautomerization was observed for both 2 and 3. The recently reported 3β-hydroxydaphniyunnine A was re-examined and its structure was revised to be 21-deoxymacropodumine D via X-ray diffraction analysis. All the isolates were screened preliminarily for their in vitro inhibitory effects against four kinases, PTP1B, aurora A, HDAC6 and IKK-β, and selective alkaloids exhibited weak activities at 20 μg mL⁻¹.

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Introduction

Plants of the genus Daphniphyllum (family Daphniphyllaceae) are widely known in the chemical community for their unique secondary metabolites called ‘Daphniphyllum alkaloids’.¹ This family of fascinating molecules has been studied a lot since the 1960s owing to their diverse and complex ring systems, and particularly, the first decade of this century has witnessed the most rapid development of the investigations into these structures from both natural products and synthetic chemists.¹

D. himalense, one of the 13 species of Daphniphyllum genus, only grows in a small area of Asia, i.e. Nepal, northeast of India and limited regions in southwest of China.² As this species has not been studied much^3–5 due to colleting difficulties, we have recently investigated the EtOH extract of its twigs and leaves and isolated a group of new hydroxylated calyciphylline A-type alkaloids from the more polar fractions.⁶ To accomplish the full chemical profiling of the alkaloidal constituents in this species, we have continued to work on the remaining fractions for less polar compounds. The current study has yielded nine new Daphniphyllum alkaloids (1–9) whose structures were characterized on the basis of careful examination of spectroscopic data and NMR comparison with known analogues. In addition to the new isolates, we have also obtained 16 known ones and identified them by comparison with authentic samples and/or literature data.

All the alkaloids were tested in a panel of kinase inhibitory assays [protein tyrosine phosphatase 1B (PTP1B), aurora kinase A, histone deacetylase 6 (HDAC6), and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKK-β)], and selective compounds showed weak inhibitory effects toward three kinases, PTP1B, aurora kinase A and IKK-β. We herein report the isolation, structure characterization and biological tests of these Daphniphyllum alkaloids.

Results and discussion

Alkaloid 1 was assigned a molecular formula of C₂₃H₂₉NO₃ by (+)-HRESIMS analysis at m/z 368.2230 ([M + H]⁺, calcd 368.2226), supportive of ten double bond equivalents (DBEs).

The NMR data (Table 1 and 2) of 1 revealed the presence of a tetrasubstituted (δ_C 144.6 & 133.6) and a trisubstituted (δ_C 134.5 & 109.6; δ_H 5.69) double bonds, a methoxyl (δ_C 51.4; δ_H 3.64), and two methyls (δ_C 29.4 & 19.7; δ_H 1.36 & 1.68). Other NMR resonances included those for seven methylenes, five methines, and two quaternary carbons, all being sp³ hybridized. The aforementioned observations accounted for four DBEs requiring a hexacyclic backbone for 1.

Table 1 ¹H NMR data for alkaloids 1–5

No.	1^a	2^b	3^b	4^a	5^c
a Measured in CDCl3.b Measured in C5D5N.c Measured in CD3OD.
2	2.84, m	2.16, brd (3.5)	2.20, brs	2.05, m	2.25, m
3a	2.12, ddd, (13.9, 3.1, 3.1)	1.94, m	1.97, dd (15.0, 4.4)	2.04, m	2.30, m
3b	1.98, ddd (13.9, 3.0, 2.6)	1.90, m	1.89, ddd (15.0, 5.0, 1.1)	1.93, m	2.11,ddd (15.1, 4.8, 1.3)
4	3.04, m	3.32, brd (4.3)	3.33, brd (4.1)	3.28, brd (4.8)	3.34, brd (4.8)
6	1.81, brdd (12.2, 6.8)	2.25, m	2.28, m	2.25, m	2.32, m
7α	2.83, d (12.2)	3.03, dd (12.6, 8.5)	2.99, dd (12.6, 8.5)	2.64, dd (11.6, 9.0)	2.95, dd (13.3, 9.9)
7β	3.33, dd (12.2, 6.8)	2.78, dd (8.5, 6.3)	2.80, dd (8.5, 6.1)	2.91, dd (9.0, 6.6)	2.80, dd (9.9, 6.4)
11α	2.05, m	2.19, m	2.19, m	2.04, m	1.87, m
11β	1.97, m	2.67, ddd (15.8, 4.6, 2.9)	2.67, ddd (16.2, 4.3, 2.9)	1.96, m	2.27, m
12α	1.68, m	1.70, m	1.69, m	1.65, m	1.83, m
12β	1.29, ddd (14.0, 3.3, 3.3)	1.55, m	1.55, m	1.92, m	1.61, m
13	2.22, dd (15.0, 9.6)	2.74, brd (18.4)	3.17, d (17.8)	2.20, m	2.35, dd (13.4, 5.8)
13	2.97, dd (15.0, 2.7)	3.27, brd (18.4)	3.67, brd (17.8)	2.73, m	2.56, brdd (13.4, 5.1)
14	2.89, ddd (10.2, 9.6, 2.7)	5.67, m		2.70, m	α 1.00, m
					β 1.98, m
15	3.57, m			3.45 (m)	2.75, m
16	α 1.93, m	a 2.91, brd (20.4)	3.28, s (2H)	1.90, m	α 2.14, dd (16.9, 4.0)
	β 1.52, m	b 2.96, brd (20.4)		1.27, m	β 2.59, dd (16.9, 5.9)
17	α 2.63, m			2.71, m
	β 2.40, brdd (15.4, 8.8)			2.33, brdd (15.3, 8.7)
18		2.91, m	2.93, m	2.84, m	2.76, m
19	5.69, q (1.3)	α 2.74, dd (14.6, 7.7)	α 2.75, dd (14.7, 7.8)	α 2.75, m	α 2.85, dd (14.4, 7.4)
		β 2.50, dd (14.6, 10.3)	β 2.50, dd (14.7, 10.2)	β 2.46, dd (13.2, 9.5)	β 2.54, dd (14.4, 10.5)
20	1.68, d (1.3)	0.84, d (6.8)	0.86, d (6.9)	0.97, d (6.8)	1.02, d (6.8)
21	1.36, s	1.02, s	1.07, s	1.32, s	1.27, s
OMe	3.64, s		3.72, s
OEt				1.24, t (7.1)
				4.13, m; 4.07, m

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Table 2 ¹³C NMR data for alkaloids 1–9 and calyciphylline Q

No.	1^a	2^b	3^b	4^a	5^c	6^c	7^c	8^a	9^a	Calyciphylline Q^a
a Measured in CDCl3.b Measured in C5D5N.c Measured in CD3OD.
1	209.4	213.4	213.1	217.0	214.8	219.9	216.4	201.5	218.5	206.5
2	43.4	45.0	44.8	44.4	45.4	45.3	43.4	50.0	44.3	43.1
3	23.5	21.0	20.9	20.7	21.5	20.4	19.7	18.4	20.7	23.6
4	64.9	64.9	65.0	66.6	66.2	65.9	91.3	67.3	65.8	63.4
5	54.6	50.1	50.3	52.1	49.5	53.9	51.7	52.5	53.7	54.9
6	57.9	52.0	51.9	51.5	52.4	51.3	47.6	52.1	49.3	56.8
7	61.6	54.2	53.9	56.9	55.1	55.0	73.0	61.4	53.8	61.4
8	56.5	65.1	64.7	62.2	66.7	68.6	64.5	143.8	71.5	55.9
9	144.6	179.9	177.7	141.4	184.5	187.7	51.0	124.3	140.5	148.2
10	133.6	140.0	145.6	138.9	142.9	38.3	186.5	176.0	205.5	151.0
11	33.5	18.9	19.4	25.7	18.2	25.2	28.1	32.3	37.1	33.7
12	32.1	24.7	24.1	27.3	25.6	21.1	24.1	32.9	19.5	31.2
13	43.7	46.3	44.9	40.3	39.2	40.5	31.9	136.2	33.8	46.6
14	41.9	124.9	126.0	42.1	28.5	25.4	25.4	118.3	36.2	115.1
15	56.9	144.7	153.7	53.4	49.6	152.6	51.7	124.4	152.1	171.5
16	27.5	36.1	37.4	28.3	43.2	206.6	213.8	24.2	29.6	25.9
17	40.9	205.3	203.9	41.7	210.8	50.3	132.9	69.4	62.7	40.8
18	109.6	34.1	34.3	33.8	33.1	33.2	33.5	31.0	32.6	110.5
19	134.5	50.0	49.8	49.7	50.6	50.6	67.7	54.2	50.0	134.7
20	19.7	19.0	18.9	18.8	19.4	19.6	19.8	18.8	19.0	19.9
21	29.4	22.7	22.7	24.8	24.1	22.4	27.7	34.8	22.2	27.1
22	175.1		165.2	174.9						166.5
OMe	51.4		52.0							51.3
OEt				14.6
				60.4
OAc									21.2
									171.0

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Further analysis of 2D NMR data (Fig. 1) confirmed a calyciphylline A-type skeleton¹ for 1 as described below. Examination of ¹H–¹H COSY spectrum revealed three spin systems of H-2 to H-4, H₂-7 via H-6 and H₂-12 to H₂-11, and H₂-13 to H₂-17. The HMBC correlations from H₂-13 to C-1, C-5 and C-8, from H₃-20 to C-2, C-18 and C-19, from H₃-21 to C-4, C-5, C-6 and C-8, from H-2 to C-1, and from H-19 to C-4 and C-7, established A, B and C rings as shown. Subsequent observation of correlations from H₂-11 to C-9 and C-10, from H₂-13 to C-9 and C-15, and from H₂-17 to C-9, C-10 and C-15, further constructed D, E and F rings to complete the core planar structure of 1. Finally, the HMBC correlations from H-14 and the methoxyl protons to C-22 (δ_C 175.1) fixed the methoxycarbonyl group at C-14. The relative configuration of 1 was assigned by analysis of NOESY data (Fig. 1). The strong NOESY correlations of H₃-21 with H-4, H-6 and H-13 at δ_H 2.97 suggested that they were cofacial and were determined to be β-oriented as those in other calyciphylline A-type alkaloids,¹ while the correlations of H-13β/H-3a, H-13α/H-14 and H-14/H-15 established the configurations of C-2, C-14 and C-15 as drawn. Alkaloid 1 was thus unequivocally characterized to be the 18,19-didehydro derivative of daphniyunnine A.⁷

Fig. 1 Key 2D NMR correlations for 1.

Alkaloid 2 exhibited a protonated molecular ion peak at m/z 324.1965 (calcd 324.1964) in the (+)-HRESIMS spectrum, corresponding to a molecular formula of C₂₁H₂₅NO₂ and suggestive of a didehydro congener of daphnilongeranin B.⁸ Analyses of the NMR data (Table 1 and 2) for 2 corroborated this deduction with the observation of diagnostic signals for an additional double bond [δ_C 124.9 (C-14) & 144.7 (C-15); δ_H 5.67 (H-14)] in place of those for the sp³ methylene [δ_C 32.6 (C-14)] and methine [δ_C 45.8 (C-15)] groups in daphnilongeranin B. Further examination of 2D NMR data (ESI Fig. S1†) confirmed the planar structure of 2 with an extended conjugation system to Δ¹⁴ as supported by the key COSY correlations of H₂-13/H-14 and HMBC correlations from H₂-16 to C-14 and C-15. The relative configurations at all chiral centers in 2 were assigned to be identical with their counterparts in daphnilongeranin B via diagnostic NOESY correlations (ESI Fig. S1†) of H₃-21/H-4, H-6 and H-13β, H-13β/H-3a, and H-3b/H₃-20. Alkaloid 2 was hence characterized to be the 14,15-didehydro analogue of daphnilongeranin B.⁸ Of note, quick enolation equilibration was observed for 2 in protonic solvent such as methanol; therefore, H₂-16 was not resolved in the ¹H NMR spectrum acquired in CD₃OD due to deuteration (ESI Table S1†). The occurrence of this quick enolation was attributable to the presence of the extended conjugation system in 2.

Alkaloid 3 gave a molecular formula of C₂₃H₂₇NO₄ as established by the (+)-HRESIMS ion at m/z 382.2014 ([M + H]⁺, calcd 382.2018). The NMR data (Table 1 and 2) for 3 revealed high similarities to those for 2 with the only difference being the presence of signals [δ_C 52.0 & 165.2 (C-22); δ_H 3.72] for a methoxycarbonyl functionality and the absence of that (δ_H 5.67) for an olefinic proton in the latter. The methoxycarbonyl group was attached to C-14 as further evidenced by the HMBC correlations (ESI Fig. S2†) from H₂-13 and H₂-16 to this quaternary carbon at δ_C 126.0. The relative configuration of 3 was in accord with that of 2 based on excellent NMR comparisons and ROESY data (ESI Fig. S24†). Deuteration of H₂-16 in CD₃OD was also observed for 3 (ESI Table S1†).

Alkaloid 4 displayed a quasi-molecular ion peak in (+)-HRESIMS analysis at m/z 384.2544 ([M + H]⁺, calcd 384.2539), consistent with a molecular formula of C₂₄H₃₃NO₃. The ¹H and ¹³C NMR spectra (ESI Fig. S28 & S29†) of 4 were nearly superimposable with those of daphniyunnine A⁷ except for resonances (δ_C 14.6 & 60.4; δ_H 4.07, 4.13 & 1.24) for an ethoxyl group instead of those (δ_C 52.0; δ_H 3.62) for a methoxyl in the latter. Further inspection of 2D NMR data (ESI Fig. S30–S32†) confirmed the structure of 4 as shown with the relative configuration being identical with that of daphniyunnine A⁷ based on excellent NMR comparisons and NOESY data. Considering the abundance of daphniyunnine A in the same species and the use of ethanol for extraction, alkaloid 4 could be a solvolysis artifact from the former.

Alkaloid 5 was assigned a molecular formula (C₂₁H₂₇NO₂) same as daphnilongeranin B⁸ by (+)-HRESIMS analysis at m/z 326.2114 ([M + H]⁺, calcd 326.2020), supportive of an isomer of the latter. The NMR data (Table 1 and 2) for 5 were highly comparable with those for daphnilongeranin B and the major differences were observed for signals around C-15 chiral center. Subsequent acquisition of 2D NMR data (Fig. 2) for 5 facilitated the construction of its planar structure, being the same as that of daphnilongeranin B. Analysis of NOESY data (Fig. 2) revealed that all stereocenters except C-15 in 5 possessed identical relative configurations with their counterparts in daphnilongeranin B based on the correlations of H₃-21/H-4, H-6 and H-13β, H-13α/H-3a, and H-3b/H₃-20. Finally, H-15 was considered to be β-directed via its NOESY correlations with H-13β and H₃-21. Alkaloid 5 was thereby identified to be the 15-epimer of daphnilongeranin B.⁸

Fig. 2 Key 2D NMR correlations for 5.

Alkaloid 6 gave a molecular formula of C₂₁H₂₇NO₂ as determined by the (+)-HRESIMS ion at m/z 326.2115 ([M + H]⁺, calcd 326.2120), suggestive of a deoxy congener of daphnipaxianine A.⁹ Analyses of the NMR data (Table 2 and 3) for 6 corroborated this hypothesis with diagnostic resonances (δ_C 75.1) for the oxygenated quaternary C-10 in daphnipaxianine A being replaced by a normal methine (δ_C 38.3; δ_H 3.63) in 6. Further examination of 2D NMR data (Fig. 3) revealed COSY correlations of H-10 with both H₂-11 and H₂-17, which supported the aforementioned conclusion and also confirmed the planar structure of 6 as drawn. While H-10 were determined to be α-positioned via the NOESY correlations of H-7α/H-11α and H-11α/H-10, the relative configurations at other stereocenters in 6 were consistent with their counterparts in daphnipaxianine A⁹ based on good NMR comparisons and similar NOESY data. Alkaloid 6 was thus characterized to be the 10-deoxy analogue of daphnipaxianine A.⁹

Table 3 ¹H NMR data for alkaloids 6–9 and calyciphylline Q

No.	6^a	7^a	8^b	9^b	Calyciphylline Q^b
a Measured in CD3OD.b Measured in CDCl3.
2	2.16, brd (4.5)	2.37, m	2.61, m	2.04, m	2.90, m
3a	2.35, m	2.59, brdd (16.1, 4.5)	2.33, m	2.46, m	2.19, ddd (14.0, 3.1, 3.1)
3b	2.11, ddd (15.4, 4.9, 1.5)	2.40, ddd (16.1, 4.6, 1.6)	2.14, brd (15.2)	2.34, m	1.99, ddd (14.0, 2.8, 2.8)
4	3.39, brd (4.9)	3.78, m	3.08, d (5.8)	3.56, brd (4.6)	3.06, m
6	2.13, m	3.05, m	2.35, m	2.32, m	1.75, brdd (12.1, 6.2)
7α	2.87, dd (12.9, 9.8)	3.10, dd (13.6, 11.8)	2.28, dd (11.7, 6.9)	2.86, m	2.84, d (12.2)
7β	2.75, dd (9.8, 6.3)	3.27, dd (11.8, 5.2)	3.59, dd (11.7, 9.4)	2.82, m	3.29, dd (12.2, 6.2)
9		4.14, brd (5.6)
10	3.63, m
11α	1.81, m	2.96, m	2.59, m	2.51, m	2.40, brd (19.5)
11β	1.52, brdd (15.1, 6.1)	3.03, m	2.68, brdd (18.5, 11.9)	2.35, m	2.03, m
12α	1.69, m	1.76, m	1.59, m	1.80, m	1.84, m
12β	1.58, m	2.04, m	2.25, m	1.93, m	1.28, ddd (14.4, 3.6, 3.6)
13	α 2.21, dd (13.4, 9.2)	α 2.02, m	α 1.78, m	α 3.32, brd (16.8)
13	β 3.01, ddd (13.4, 7.4, 2.0)	β 1.78, m		β 2.59, m	β 2.84, m
14	α 2.29, m	α 1.29, m	6.50, brs	2.49, m (2H)
	β 2.33, m	β 1.79, m
15		2.69, brdd (9.9, 5.6)
16			a 2.78, m	a 2.89, m	2.77, m (2H)
			b 2.75, m	b 2.48, m
17	α 2.97, dd (18.2, 6.2)	6.10, m	a 4.55, ddd (10.5, 4.9, 1.8)	a 4.19, m	a 2.84, m
	β 2.42, dd (18.2, 3.5)		b 4.08, ddd (13.7, 10.5, 4.8)	b 4.13, m	b 2.75, m
18	2.71, m	2.52, m	2.02, m	2.80, m
19	α 2.85, dd (14.3, 7.3)	α 3.61, dd (13.3, 7.2)	α 2.93, dd (15.6, 3.7)	α 2.80, m	5.70, q (1.3)
	β 2.55, dd (14.3, 10.5)	β 2.97, dd (13.3, 11.4)	β 2.57, m	β 2.54, m
20	1.02, d (6.8)	1.16, d (6.8)	1.30, d (7.1)	0.97, d (6.6)	1.71, d (1.3)
21	1.23, s	1.37, s	1.32, s	1.27, s	1.16, s
OMe					3.70, s
OAc				2.03, s

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Fig. 3 Key 2D NMR correlations for 6.

A molecular formula of C₂₁H₂₇NO₃ was assigned to alkaloid 7 based on the (+)-HRESIMS ion at m/z 342.2061 ([M + H]⁺, calcd 342.2069), indicative of an oxygenated congener of daphniyunnine C.⁷ The NMR data (Table 2 and 3) for 7, including proton couplings and number of carbon types, were comparable with those for daphniyunnine C, and the main differences occurred to CH-4, CH₂-7 and CH₂-19 whose NMR resonances were dramatically shifted to the downfield region. Further examination of 2D NMR data (ESI Fig. S3†) for 7 returned a completely identical structure, including relative configuration, with that of daphniyunnine C. The above-mentioned observations strongly supported an oxidation at the N-position of 7.¹⁰ Alkaloid 7 was hence elucidated to be the N-oxide of daphniyunnine C.⁷

(+)-HRESIMS analysis of alkaloid 8 revealed a protonated molecular ion at m/z 324.1955 (calcd 324.1964), in accord with a molecular formula of C₂₁H₂₅NO₂ suggesting a deoxy congener of daphnicyclidin G.¹¹ Analyses of the NMR data (Table 2 and 3) for 8 confirmed this deduction with diagnostic resonances (δ_C 74.3) for the oxygenated quaternary C-2 in daphnicyclidin G being replaced by those (δ_C 50.0; δ_H 2.61) for a routine methine in 8. Subsequent inspection of 2D NMR data (ESI Fig. S4†) revealed COSY correlations of H-2 with both H₂-3 and H-18, which supported the aforementioned conclusion and also corroborated the planar structure of 8 as shown. The relative configuration of 8 was established to be the same as that of daphnicyclidin G via the similar NOESY correlations (ESI Fig. S4†) of H-3a/H₃-20 and H₃-21/H-3b, H-4 and H-6. Alkaloid 8 was thus identified to be the 2-deoxy derivative of daphnicyclidin G.¹¹

Alkaloid 9 was assigned a molecular formula of C₂₃H₃₁NO₄ based on the (+)-HRESIMS ion at m/z 386.2325 ([M + H]⁺, calcd 386.2331), indicating an acetylated congener of daphniyunnine B.⁷ Compared to daphniyunnine B, the NMR data (Table 2 and 3) for 9 showed characteristic signals for an acetyl group (δ_C 21.2 & 171.0; δ_H 2.03) which was connected to C-17 via the HMBC correlations from H₂-17 to the acetyl carbonyl carbon. Other diagnostic observations included the downfield shifted H₂-17 signals (Δδ_H 0.41) caused by the acetylation effect. Careful examination of 2D NMR data (ESI Fig. S74 & S75†) confirmed the above-mentioned conclusion and the relative configuration of 9 was assigned to be identical with that of daphniyunnine B by excellent NMR comparisons and analysis of NOESY data (ESI Fig. S76†). Alkaloid 9 was then elucidated to be 17-O-acetyldaphniyunnine B.⁷

Alkaloid 10 was isolated from a more polar fraction in our recent report and was identified as 3β-hydroxydaphniyunnine A via spectroscopic data analysis.⁶ However, another portion of sample from a close fraction crystallized in CD₃OD in a NMR tube during long-term storage in the fridge. In order to confirm the previous structure assignment, we run an X-ray diffraction experiment which led to a revision of the structure for 10 to be 21-deoxymacropodumine D and also established the absolute configuration as shown [Fig. 4, absolute structure parameter: 0.19(11)].¹² The macropodumine D skeleton was only reported once by Guo and coworkers,¹³ and 10 represents the second example bearing this backbone.

Fig. 4 ORTEP drawing of 10 (with a methanol molecule).

Along with the new alkaloids, 16 known cometabolites were also isolated and structurally characterized on the basis of spectroscopic analyses and comparison with authentic samples.

They were identified to be calyciphylline Q,¹⁴ daphniyunnines A–E,⁷ daphnilongeranins A and B,⁸ daphlongamines E and F,¹⁵ daphnipaxianine A,⁹ daphnipaxinin,¹⁶ dehydroxymacropodumine A,¹⁷ deoxycalyciphylline B,¹⁰ deoxyisocalyciphylline B,¹⁰ and longistylumphylline A.¹⁸

All the alkaloids were tested in vitro for their inhibitory activities (ESI Tables S2–S5†) against four kinases, PTP1B, aurora A, HDAC6 and IKK-β at 20 μg mL⁻¹. While only 5, daphniyunnine C, and longistylumphylline A exhibited marginal activities toward aurora A with >30% inhibitory rates, more tested alkaloids (1, 2, 9, daphniyunnine D, daphnilongeranin A, daphnipaxianine A and longistylumphylline A) showed more than 30% inhibition against PTP1B. None of the compounds were active (<20% inhibition) against HDAC6 at the tested concentration, and only daphnipaxianine A weakly inhibited IKK-β with an inhibitory rate of 31%.

Experimental

General experimental procedures

Optical rotations were acquired on a Rudolph Autopol VI automatic polarimeter. UV data were obtained on a Shimadzu UV-2550 UV/visible spectrophotometer. IR spectra were recorded on a Perkin-Elmer 577 spectrometer. NMR experiments were carried out on a Bruker AM-500 spectrometer with a cryoprobe and data were referenced to deuterated solvent peaks [δ_H 7.26 and δ_C 77.23 for CDCl₃; δ_H 3.31 and δ_C 49.15 for CD₃OD; δ_H 7.58 (H-3) and δ_C 135.91 (C-3) for C₅D₅N). LR- and HR-ESIMS analyses were performed on a Bruker Daltonics esquire3000plus and a Waters LCT Premier XE spectrometers, respectively. Pre-coated silica gel GF₂₅₄ plates (Yantai Huiyou Silica Gel Exploitation Company, Ltd., China) were used for TLC analyses. Silica gel H (300–400 mesh, Qingdao Haiyang Chemical Plant, Ltd., China) and amino silica gel (20–45 μm, Fuji Silysia Chemical, Ltd., Japan) were used for normal column chromatography (CC). HPLC purifications were conducted on a Waters 1525 binary pump system equipped with a 2489 UV/Visible detector and a Waters X-bridge Prep C18 column (5 μm, 10 × 250 mm) or a YMC TriArt C18 column (5 μm, 10 × 250 mm). All solvents used for CC were of at least analytical grade (Shanghai Chemical Reagents Company, Ltd., China), and solvents used for UV, [α]_D, NMR measurements, and HPLC separations were of suitable chromatographic grades from Merck or Sigma-Aldrich.

Plant materials

The twigs and leaves of Daphniphyllum himalense (Benth.) Muell.-Arg. were collected in March 2012 at Shivapuri Nagarjun National Park, Nepal, and were identified via comparison with the herbarium specimen (voucher number: 12 682) deposited at the National Herbarium Laboratory, Department of Plant Resources, Godawari, Nepal. A voucher specimen has been deposited at Shanghai Institute of Materia Medica, Chinese Academy of Sciences (accession number: 2012-DH-Y1).

Extraction and isolation

The air-dried powder of the twigs and leaves (9.5 kg) of D. himalense were extracted three times (each for seven days) with 95% EtOH at room temperature. The solvent of the EtOH extraction was removed under reduced pressure to yield the final crude extract (1.0 kg) which was re-suspended in 4.0 L water. The water suspension was then acidified by 1.0 mol L⁻¹ H₂SO₄ to pH 2–3 and was partitioned with EtOAc (6 × 2.0 L) to remove the non-polar neutral constituents. The crude alkaloids (12.3 g) were obtained via subsequent basification with saturated Na₂CO₃ solution to pH 9–10 and extraction with CHCl₃ (4 × 2.0 L). The aforementioned crude alkaloids were subjected to a silica gel column eluted with petroleum ether-EtOAc (5 : 1 to 1 : 5 with 5% NHEt₂) to return 12 fractions A1–A12.

Fraction A1 (1.34 g) was first separated over silica gel eluted with CHCl₃–MeOH (200 : 1 to 15 : 1) to give eight elutions A1a–A1h. Elution A1a (114 mg) was re-chromatographed by a silica gel column (CHCl₃–MeOH, 400 : 1) to further return five subfractions and alkaloid 1 (3.2 mg), and calyciphylline Q (4.6 mg) were isolated from the third (15.6 mg) and fourth (25.3 mg) subfractions by HPLC [TriArt C18 column, 50–90% (in 18 min) and 40–70% (in 15 min) MeCN in H₂O for 1 and calyciphylline Q, respectively]. Elution A1c (45 mg) was purified by HPLC (80% MeOH in H₂O) to yield deoxycalyciphylline B (21.9 mg) and elution A1e (64 mg) was purified by HPLC (70–100% MeOH in H₂O in 15 min) to yield deoxyisocalyciphylline B (27.0 mg). A portion of elution A1h (75 mg) was fractionated by HPLC (78% MeCN in H₂O) to give daphniyunnine A (48.7 mg) and alkaloid 4 (3.1 mg). Fraction A4 (111 mg) was first fractionated by silica gel CC (CHCl₃–MeOH, 75 : 1 to 35 : 1) to afford four subfractions and daphnilongeranin A (16.8 mg) and longistylumphylline A (22.9 mg) were obtained from the fourth one (57 mg) by HPLC purification (40–50% MeCN in H₂O in 15 min). Fraction A5 (264 mg) was first separated on a silica gel column (CHCl₃–MeOH, 50 : 1 to 10 : 1) to furnish five subfractions A5a–A5e, and A5b (30 mg) was purified by HPLC (30–40% MeCN in H₂O in 15 min) to give alkaloid 3 (1.6 mg). Daphnilongeranin B (11.1 mg) was obtained from A5c (58 mg) via HPLC separation (20–80% MeCN in H₂O in 20 min), while dehydroxymacropodumine A (4.3 mg) and alkaloid 8 were isolated from A5e (25 mg) also via HPLC fractionation (30–45% MeCN in H₂O in 20 min). Fractionation of A6 (265 mg) over silica gel (CHCl₃–MeOH, 75 : 1 to 20 : 1) returned six subfractions A6a–A6f and alkaloid 5 (16.6 mg) was obtained via HPLC separation (45–70% MeOH in H₂O in 15 min) of A6d (50 mg). Subfraction A6c (88 mg) was separated by HPLC (40–70% MeOH in H₂O in 20 min) to sequentially afford alkaloid 7 (2.1 mg), an elution (4.5 mg) containing 2, and an elution (38 mg) containing 6 and daphniyunnine C. Alkaloid 2 (2.3 mg) was acquired by further HPLC purification on TriArt C18 column (36% MeCN in H₂O), while alkaloid 6 (17.4. mg) and daphniyunnine C (6.8 mg) were only separated by a chiral HPLC column (Diacel AD-H, 2.5 mL min⁻¹ EtOH).

Silica gel CC (CHCl₃–MeOH, 70 : 1 to 30 : 1) of fraction A7 (405 mg) afforded six subfractions and daphniyunnine D (13.8 mg), and daphlongamine E (11.1 mg) were isolated from the fifth one by HPLC fractionation (35–55% MeCN in H₂O in 15 min). Fraction A8 (417 mg) was fractionated by silica gel CC (CHCl₃–MeOH, 50 : 1 to 12 : 1) to furnish seven subfractions A8a–A8g. Daphnipaxianine A (14.0 mg) and alkaloid 9 (4.0 mg) were recovered via the same HPLC condition (30–45% MeCN in H₂O in 20 min) from A8c (99 mg) and A8d (25 mg), respectively, while HPLC fractionation (30% MeCN in H₂O) of A8e (34 mg) yielded daphlongamine F (17.4 mg). Subfraction A8g (134 mg) was first processed with amino silica gel (CHCl₃–MeOH, 100 : 1 to 50 : 1) and then purified by HPLC (30% MeCN in H₂O) to give daphniyunnine E (16.7 mg). Fraction A10 (468 mg) was separated by silica gel CC (CHCl₃–MeOH, 30 : 1 to 10 : 1) to give five subfractions and daphniyunnine B (18.8 mg) was acquired from the third one (29 mg) via HPLC purification (20–40% MeCN in H₂O in 15 min).

All HPLC purifications were performed on a Waters X-bridge C18 column at a flow rate of 3.5 mL min⁻¹ with 0.02% HNEt₂ as modifier, unless specified.

Characterization of new compounds

18,19-Didehydrodaphniyunnine A (1). White amorphous powder; [α]²⁵_D 36 (c 0.03, MeOH); UV (MeOH) λ_max (log ε) 208 (3.94) nm; IR (KBr) ν_max 2953, 2925, 1736, 1691, 1637, 1453, 1436, 1385, 1193, 1169 cm⁻¹; ¹H and ¹³C NMR (CDCl₃) see Table 1 and 2; (+)-ESIMS m/z 368.1 [M + H]⁺; (+)-HRESIMS m/z 368.2230 [M + H]⁺ (calcd for C₂₃H₃₀NO₃, 368.2226).

14,15-Didehydrodaphnilongeranin B (2). White amorphous powder; [α]²⁴_D −175 (c 0.22, MeOH); UV (MeOH) λ_max (log ε) 289 (4.09), 208 (3.77) nm; IR (KBr) ν_max 2956, 2922, 2867, 1698, 1636, 1441, 1384, 1301, 1224, 1061, 1034 cm⁻¹; ¹H and ¹³C NMR (C₅D₅N) see Table 1 and 2; (+)-ESIMS m/z 324.2 [M + H]⁺; (+)-HRESIMS m/z 324.1965 [M + H]⁺ (calcd for C₂₁H₂₆NO₂, 324.1964).

17-Oxolongistylumphylline A (3). White amorphous powder; [α]²⁵_D −93 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 297 (4.24), 216 (3.85) nm; IR (KBr) ν_max 2957, 2926, 1701, 1665, 1637, 1438, 1384, 1354, 1262, 1123, 1062 cm⁻¹; ¹H and ¹³C NMR (C₅D₅N) see Table 1 and 2; (+)-ESIMS m/z 382.3 [M + H]⁺; (+)-HRESIMS m/z 382.2014 [M + H]⁺ (calcd for C₂₃H₂₈NO₄, 382.2018).

Daphnilongeranin C ethyl ester (4). White amorphous powder; [α]²⁵_D −38 (c 0.24, MeOH); UV (MeOH) no obvious absorption maximum between 400–200 nm; IR (KBr) ν_max 2958, 2925, 2856, 1728, 1703, 1633, 1458, 1400, 1175 cm⁻¹; ¹H and ¹³C NMR (CDCl₃) see Table 1 and 2; (+)-ESIMS m/z 384.2 [M + H]⁺; (+)-HRESIMS m/z 384.2544 [M + H]⁺ (calcd for C₂₄H₃₄NO₃, 384.2539).

15-Epidaphnilongeranin B (5). White amorphous powder; [α]²⁵_D −17 (c 0.20, MeOH); UV (MeOH) λ_max (log ε) 251 (4.08) nm; IR (KBr) ν_max 2956, 2922, 2868, 1697, 1665, 1443, 1401, 1384, 1284, 1261, 1177, 1104, 1057 cm⁻¹; ¹H and ¹³C NMR (CD₃OD) see Table 1 and 2; (+)-ESIMS m/z 326.2 [M + H]⁺; (+)-HRESIMS m/z 326.2114 [M + H]⁺ (calcd for C₂₁H₂₈NO₂, 326.2020).

10-Deoxydaphnipaxianine A (6). White amorphous powder; [α]²⁵_D −111 (c 0.20, MeOH); UV (MeOH) λ_max (log ε) 248 (4.12) nm; IR (KBr) ν_max 2926, 2871, 1700, 1630, 1455, 1384, 1269, 1193, 1151, 1101, 1075 cm⁻¹; ¹H and ¹³C NMR (CD₃OD) see Table 2 and 3; (+)-ESIMS m/z 326.2 [M + H]⁺; (+)-HRESIMS m/z 326.2115 [M + H]⁺ (calcd for C₂₁H₂₈NO₂, 326.2120).

Daphniyunnine C N-oxide (7). White amorphous powder; [α]²⁵_D −125 (c 0.20, MeOH); UV (MeOH) λ_max (log ε) 239 (3.98) nm; IR (KBr) ν_max 2956, 2927, 1695, 1635, 1608, 1458, 1388, 1262, 1089, 988 cm⁻¹; ¹H and ¹³C NMR (CD₃OD) see Table 2 and 3; (+)-ESIMS m/z 342.1 [M + H]⁺, 683.3 [2M + H]⁺; (+)-HRESIMS m/z 342.2061 [M + H]⁺ (calcd for C₂₁H₂₈NO₃, 342.2069).

2-Deoxydaphnicyclidin G (8). White amorphous powder; [α]²⁶_D −77 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 335 (4.06) 275 (3.92) nm; IR (KBr) ν_max 2929, 1627, 1592, 1483, 1458, 1397, 1371, 1322, 1209, 1179, 1140, 938 cm⁻¹; ¹H and ¹³C NMR (CDCl₃) see Table 2 and 3; (+)-ESIMS m/z 324.3 [M + H]⁺; (+)-HRESIMS m/z 324.1955 [M + H]⁺ (calcd for C₂₁H₂₆NO₂, 324.1964).

17-O-Acetyldaphniyunnine B (9). White amorphous powder; [α]²⁵_D 36 (c 0.20, MeOH); UV (MeOH) λ_max (log ε) 243 (3.48) nm; IR (KBr) ν_max 2961, 2925, 1737, 1699, 1675, 1629, 1441, 1384, 1237, 1083, 1041 cm⁻¹; ¹H and ¹³C NMR (CDCl₃) see Table 2 and 3; (+)-ESIMS m/z 386.1 [M + H]⁺; (+)-HRESIMS m/z 386.2325 [M + H]⁺ (calcd for C₂₃H₃₂NO₄, 386.2331).

X-ray diffraction analysis

21-Deoxymacropodumine D was crystallized from CD₃OD in an NMR tube at 4 °C. The X-ray crystallographic data were obtained on a Bruker APEX-II CCD detector employing graphite monochromated Cu-Kα radiation (λ = 1.54178 Å) at 293.15 K, and operated in the ϕ–ω scan mode. The structure was solved by direct method using SHELXS-97 (Sheldrick 2008) and refined with full-matrix least-squares calculations on F² using SHELXL-97 (Sheldrick 2008). All non-hydrogen atoms were refined anisotropically. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms.

Bioassays

The PTP1B,¹⁹ aurora A,²⁰ HDAC6,²¹ and IKK-β²² inhibitory assays were performed as previously reported.

Acknowledgements

This project was financially supported by the National Natural Science Foundation (No. 81322045) of P. R. China.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: 1D & 2D NMR, IR and MS spectra for all new compounds, along with the key X-ray crystallographic parameters of 10. CCDC 1462825. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra06420e

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