Two new indole alkaloids from Nauclea officinalis and their evaluation for cytotoxic activities

Abstract Two new indole alkaloids, naucleamide H (1) and (±)-19-O-butylangustoline (8), along with seven known alkaloids, 3,14-dihydroangustine (2), (-)-naucleofficine D (3a), (+)-naucleofficine D (3b), nauclefine (4), angustidine (5),19-O-ethylangustoline (6) and angustine (7) were isolated from the water extract of Nauclea officinalis. The structures of these compounds were established by spectroscopic analysis. Among them, the cytotoxicity of 1, 2, 6 and 8 were evaluated against six human cancer cell lines (HepG-2, SKOV3, HeLa, SGC 7901, MCF-7 and KB) in vitro for the first time with 5-fluorouracil as a positive control drug. The new compound 1 had a strong inhibitory effect on the proliferation of HepG-2 with an IC50 value of 19.59 μg/mL. The new compound 8 had a strong inhibitory effect on HepG-2, SKOV3, HeLa, MCF-7 and KB, IC50 value was 5.530, 23.11, 31.30, 32.42 and 37.26 μg/mL, respectively. GRAPHICAL ABSTRACT


Introduction
Nauclea officinalis is the branch and bark of the Nauclea officinalis Pierrc ex Pitard, which belongs to the genus Nauclea in family Rubiaceae.It is mainly distributed in Hainan, Guangdong, Guangxi and Hunan Province of China.As a traditional folk medicine, it is commonly used in colds, fever, acute tonsillitis, pharyngitis, bronchitis, pneumonia, urinary tract infection, enteritis, dysentery, cholecystitis and other diseases treatment (Liu et al. 2019;Zhou et al. 2021).It is reported that Nauclea officinalis was rich in indole alkaloids (Li et al. 2020;Rosales et al. 2020;Wang et al. 2022), and had many pharmacological activities such as anti-inflammatory (Zhai et al. 2016;Song et al. 2020), antimalarial (Sun et al. 2008), and anti-tumor (Wang et al. 2015;Liu et al. 2018;Omar et al. 2021).In order to further explore the active indole alkaloids in Nauclea officinalis and make full use of this rare medicinal resource, the study on the chemical constituents of Nauclea officinalis was carried out.As a result, two new indole alkaloids naucleamide H ( 1) and (±)-19-O-butylangustoline (8) with seven known alkaloids 3,14-dihydroangustine ( 2), (-)-naucleofficine D (3a), (+)-naucleofficine D (3b), nauclefine (4), angustidine (5), 19-O-ethylangustoline (6) and angustine (7) were isolated from the water extract of Nauclea officinalis (Figure 1).Among them, four compounds were evaluated for their antitumor activities for the first time.The results showed that the four compounds could inhibit the proliferation of multiple human cancer cells.The new compound 1 had a strong inhibitory effect on the proliferation of HepG-2, and new compound 8 had strong inhibitory effects on HepG-2, SKOV3, HeLa, MCF-7 and KB.In this paper, we described the isolation, structure identification and antitumor activities of these compounds.

Phytochemical investigation
Compound 1 was isolated as a white amorphous powder with the specific rotation of [α] 20D +91.1 (c 0.12, MeOH).The reaction of bismuth potassium iodide reagent was positive, suggesting that the compound was alkaloid.The uV spectrum of 1 showed absorption maxima at 224 and 280 nm, suggesting the characteristic absorption of the indole ring.2) and the presence of a trisubstituted olefin with a methyl group at C-19 (δ c 156.0) and an formly group at C-20 (δ c 145.2).The above data revealed that the structure of 1 was similar to that of naucleamide A. The only different between them was the presence of the oxymethylene group at C-20 in naucleamide A was replaced by formly group in 1.The NOESY correlations of H-15 to H-18 indicated that the geometry of the trisubstituted olefin was E. In the NOESY spectrum, the correlations of H-3 to H-14a, H-14a to H-16, and H-14b to H-15 indicated that these protons are cofacial.The relative configuration of H-3 and H-15 of 1 was different from that of naucleamide A by comparing the coupling constants and NOESY data.According to the report on naucleamide A in the literature (Shigemori et al. 2003), NOESY correlation of H-14a to H-16 and H-14b to H-15 and 1 H-1 H coupling constants (J 3, 14a =5.9 Hz, J 3, 14b =3.7 Hz, J 14a, 15 =11.1 Hz, J 15, 16 =10.6Hz) indicated a trans relationship between H-15 and H-16, α-orientation of H-3 and H-16, and a β-orientation of H-15.In fact, the 1 H-1 H coupling constants of 1 were J 3, 14a =4.9 Hz, J 3, 14b =12.9 Hz, J 14a, in Figure S1.The absolute configuration of 1 was determined by electronic circular dichroism (ECD) calculation.The calculated ECD curve was consistent with the experimentally measured curve (Figure S2).Therefore, the absolute configuration of the three chiral carbons of 1 is 3 R, 15S, 16S.After searching for Scifinder, compound 1 was inferred to be a new indole alkaloid.The structure is shown in Figure 1 and named as naucleamide H.
Compound 8 was obtained as yellow powder with the specific rotation of [α] 20D −2.4 (c 0.12, CH 3 OH).The reaction of bismuth potassium iodide reagent was positive, suggesting that 8 was alkaloid.The uV spectrum of 8 showed absorption maxima at 390, 372, 300, 252 and 217 nm, indicating the characteristic absorption of the indole ring.The molecular formula of C 24 H 25 N 3 O 2 was established by the positive ion HR-ESI-TOF-MS (m/z 388.2021 [M + H] + , calcd for 388.2020).The 1 H, 13 C NMR (Table S1) and HSQC spectra indicated the presence of several groups including eight aromatic carbons, one lactam carbonyl group, two methyls, one methyne and five methylenes.Four aromatic proton signals at δ H 7.63 (1H, d, J = 7.9 Hz, H-9), δ H 7.10 (1H, t.J = 7.5 Hz, H-10), δ H 7.26 (1H, d, J = 7.9 Hz, H-11), δ H 7.50 (1H, d, J = 8.2 Hz, H-12), two methylene groups δ H 4.33 (1H, ddd, J = 13.6,7.6, 6.3 Hz, H-5a), δ H 4.47 (1H, dt, J = 13.6,7.6, 6.3 Hz, H-5b) and δ H 3.12 (2H, m, H-6) indicated the presence of the tetrahydro β-carboline structure.A comparison of the 1 H and 13 C-NMR spectra of 8 with 19-O-methyl-3,14-angustoline (Xu et al. 2012) showed that they were very similar the only different between them was the presence of the double bond between C-3 and C-14 in 8 and oxymethyl in 19-O-methyl-3,14-angustoline was substituted by oxybutyl in 8.The 1 H-1 H COSY spectrum showed the correlations of C-18 to C-19, C-23 to C-26 , indicated that C-23 is connected to a propyl group.The chemical shift of C-19 was δ c 72.6, and the chemical shift of C-23 was δ c 68.0, indicating that both C-19 and C-23 are linked to oxygen atom.The structure of 8 was deduced to be a natural derivative of angustoline, based on the screen results in the crude extract of Nauclea officinalis by using uPLC-Q-TOF-MS/MS method (Wang et al. 2022).The molecular weight of 8 could be deduced as 387.19 (C 24 H 25 N 3 O 2 ) by [M + H] + at m/z 388.2024.In the positive MS/MS spectrum, the [M + H] + of 8 was 42 Da higher than that of 19-O-methylangustoline (Li et al. 2011)  The HMBC spectrum showed that correlation of H-18 was associated with C-19 (δ c 72.6) and C-20 (δ c 131.7), and H-19 was associated with C-15 (δ c 139.1) and C-20 (δ c 131.7), suggested that H-19 was related to the C-20, and H-23 was related to C-19 (δ c 72.6), further confirmed that the oxybutyl group was attached at position C-19.The relative configuration of 8 was deduced to be the same as that of angustoline based on the comparison of their NMR data, as well as its biogenetic relationship (Erdelmeier et al. 1992).Therefore, the structure of 8 could unambiguously be established, detailed 2D NMR correlation analysis (including HMBC and 1 H-1 H COSY spectra) inferred the planar structure of 8 as shown in Figure S3.The specific optical rotation of 8 was −2.4 (c 0.12, CH 3 OH), suggesting that 8 should be a partial racemate.After searching for Scifinder, compound 8 was inferred to be a new indole alkaloid, named as (±) −19-O-butylangustoline.

Cytotoxic activity
Six human cancer cell lines, HepG-2, SKOV3, HeLa, SGC 7901, MCF-7 and KB, were used to evaluate their cytotoxicity in vitro, 5-fluorouracil was used as a positive control drug.The cytotoxicity of four compounds in vitro were evaluated for the first time.The results showed that four compounds had different inhibitory effects on the proliferation of multiple kinds of cancer cells.Among them, new compound 1 had a strong inhibitory effect on the proliferation of HepG-2, and had a weak inhibitory effect on the proliferation of SKOV3 and SGC 7901.New compound 8 had a strong inhibitory effect on the proliferation of HepG-2, SKOV3, HeLa, MCF-7 and KB.The specific activity data was shown in Table S2.

Cytotoxicity assay
The cytotoxicity assays of compounds 1, 2, 6 and 8 were performed using the MTT method in 96-well microplates with 5-fluorouracil as the positive control.The suspension of cells (5 × 10 4 cells/mL) in the logarithmic phase was dispersed in three replicates in 96-well plates (200 μL/well).The cells in plates were cultured at 37 °C with 5% CO 2 saturated humidity incubator for 24 h.Culture medium containing drugs of a series of concentrations (1.5, 3, 6.25, 12.5, 25, 50 and 100 μg/mL) were added into the 96-well plates after removing the old medium, three replicates were performed for each concentration.At the same time, the positive (5-fluorouracil), negative (only cells, no drugs) and blank control groups (only culture medium) were arranged.Twenty microliter MTT reagent (5 mg/mL) was added into each well after removing the old medium after 48 h of incubation.Then, cells were further incubated for 4 h.Next, removing the culture medium and 150 μL dimethyl sulfoxide (DMSO) was added into 96-well plates, vibrating for 10 min, the concentration of formazan solubilized in DMSO was determined by wavelength detection at 492 nm with a microplate reader.The rate of cell inhibition was calculated as follows: Cell inhibition (%)= [A (negative)-A (test)]/[A (negative)-A (blank)] × 100%.The results of the IC 50 values (μg/mL) were calculated by SPSS 19.0 software.

Conclusion
In this study, the chemical investigation on N. officinalis was undertaken and had led to isolation and characterization of two new monoterpene indole alkaloids, naucleamide H (1) and (±)-19-O-butylangustoline (8), together with seven known alkaloids (2-7).The cytotoxicity of 1, 2, 6 and 8 were evaluated against six human cancer cell lines.In vitro cytotoxic activity screening of the new compounds 1 and 8 showed strong inhibitory activity against human liver cancer (HepG-2).Moreover, the cytotoxic activities of the new compounds 1 and 8 against HepG-2 were even stronger than that of the positive control drug 5-fluorouracil.These findings that the discovery of the new indole alkaloid with significant cytotoxic activity may be important for the development of novel antitumor drugs.