New Monoterpenoid Indole Alkaloids from Tabernaemontana crassa Inhibit β-Amyloid42 Production and Phospho-Tau (Thr217)

Eleven monoterpenoid indole alkaloids, including three new ones, tabercrassines A–C (1–3), were isolated from the seeds of Tabernaemontana crassa. Tabercrassine A (1) is an ibogan–ibogan-type bisindole alkaloid which is formed by the polymerization of two classic ibogan-type monomers through a C3 unit aliphatic chain. Their structures were established by extensive analysis of HRESIMS, NMR, and ECD spectra. Cellular assays showed that alkaloids 1–3 all reduce Aβ42 production and inhibit phospho-tau (Thr217), a new biomarker of Alzheimer’s disease [AD] associated with BACE1-, NCSTN-, GSK3β-, and CDK5-mediated pathways, suggesting these alkaloids’ potential against AD.


Introduction
Alzheimer's disease (AD), the most common type of dementia, accounts for up to 70% of neurodegenerative diseases [1][2][3]. It is now one of the most common fatal diseases, and over 50 million individuals worldwide suffer from AD [4]. The main pathological hallmarks of AD are senile plaques and neurofibrillary tangles (NFTs) composed of amyloid beta (Aβ) and over-phosphorylated tau protein, respectively [3,[5][6][7]. However, as AD is a heterogeneous, polygenic, and complex disease, there are no efficacious disease-modifying therapeutics to date [7,8]. Hence, the discovery of a diversification of AD-druggable polytargets or therapeutic drug combination strategies might provide new drug discovery avenues [8]. Among these avenues, inhibitors of amyloid precursor protein (APP) protease, β-site cleaving enzyme 1 (BACE1), and cyclin-dependent kinase 5 (CDK5) contributing to tau phosphorylation have been suggested as appealing drug targets for AD [9][10][11][12]. Previous studies have shown that natural products exhibited biological activities against AD by inhibiting cyclin-dependent kinase 5 and tau phosphorylation and reducing Aβ42 and Aβ40 production toward the nonamyloidogenic pathway [13,14].
Monoterpenoid indole alkaloids (MIAs) are a category of natural products that are attractive due to their intriguing skeletons and promising bioactivities [15][16][17]. Among MIAs, reserpine, vincristine and its derivatives are outstanding representatives [18,19]. The genus Tabernaemontana (Apocynaceae family) comprises 99 species worldwide, some of which have been widely used as folk medicine for the treatment of hypertension, snake poisoning, and rheumatalgia [20]. Interestingly, ibogaine is the most abundant ibogantype MIA in the genus Tabernaemontana, and has been used as a psychopharmacological sacrament in the Bwiti religion (West Africa) for several centuries [21]. In recent years, various classes of MIAs have been isolated and identified from this genus and some of them possessed novel skeletons and intriguing biological activities [22][23][24][25]. Previously, we found that kopoffines A-C, which are MIA dimers, showed significant inhibition against cyclin-dependent kinase 5 and decreased the protein levels of phospho-tau (pTau217 and pTau396) without influencing Aβ production [13].
Monoterpenoid indole alkaloids (MIAs) are a category of natural products that are attractive due to their intriguing skeletons and promising bioactivities [15][16][17]. Among MIAs, reserpine, vincristine and its derivatives are outstanding representatives [18,19]. The genus Tabernaemontana (Apocynaceae family) comprises 99 species worldwide, some of which have been widely used as folk medicine for the treatment of hypertension, snake poisoning, and rheumatalgia [20]. Interestingly, ibogaine is the most abundant ibogan-type MIA in the genus Tabernaemontana, and has been used as a psychopharmacological sacrament in the Bwiti religion (West Africa) for several centuries [21]. In recent years, various classes of MIAs have been isolated and identified from this genus and some of them possessed novel skeletons and intriguing biological activities [22][23][24][25]. Previously, we found that kopoffines A-C, which are MIA dimers, showed significant inhibition against cyclin-dependent kinase 5 and decreased the protein levels of phospho-tau (pTau217 and pTau396) without influencing Aβ production [13].

Characterization of the New Isolates
The relative configuration of 1 was same as voacangine and tabervarine A, based on their identical ROESY correlations [26,34] Figure S17), which corresponded to amino, carbonyl, and aromatic functionalities. Analysis of the NMR data ( Table 2, Figures S10 and S11) suggested that the structure of 2 shared the same basic skeleton with that of voacangine [26], except for the presence of a C6 unit aliphatic chain group (δH 2.64, H-22a; δH 2.80, H-22b; δH 2.58, H-24a; . The signals are attributed to one downfield non-protonated carbon, two methylenes, one ketone carbonyl, and two methyl groups in a C6 unit, respectively. The presence of 1 H-1 H COSY cross-peaks of H-3 (δH 3.37) and H-22a and the HMBC correlations from H-3, H-22b, and H-24a to C-23, as well as H-3 to C-5 (δC 52.4), established that the C6 unit aliphatic chain was located at C-3. Further analysis of the 2D NMR (HSQC, HMBC, and 1 H-1 H COSY) spectra determined the planar structure of 2 (Figures 3A and S12-S14).
The relative configuration of 2 was deduced from the analysis of its ROESY spectrum ( Figure 3B), which was identical with that of voacangine. The ROESY correlation of H-3 with H-17a (δH 1.90) indicated that both protons were co-facial and were assigned arbitrarily as β-oriented ( Figure S15). Finally, time-dependent density functional theory (TDDFT) ECD calculation was applied to clarify the absolute configuration of 2. The calculated ECD data of (3R,14R,16S,20S,21S)-2 matched well with the experimental data (Figures 4, S18, and S28), confirming the absolute configuration of 2 as shown in Figure 1.  (Figure S17), which corresponded to amino, carbonyl, and aromatic functionalities. Analysis of the NMR data ( Table 2, Figures S10 and S11) suggested that the structure of 2 shared the same basic skeleton with that of voacangine [26] ; δ C 211.9, C-23; δ C 48.1, C-22; δ C 55.6, C-24; δ C 69.9, C-25; δ C 29.9, C-26; δ C 29.8, C-27). The signals are attributed to one downfield non-protonated carbon, two methylenes, one ketone carbonyl, and two methyl groups in a C6 unit, respectively. The presence of 1 H-1 H COSY cross-peaks of H-3 (δ H 3.37) and H-22a and the HMBC correlations from H-3, H-22b, and H-24a to C-23, as well as H-3 to C-5 (δ C 52.4), established that the C6 unit aliphatic chain was located at C-3. Further analysis of the 2D NMR (HSQC, HMBC, and 1 H-1 H COSY) spectra determined the planar structure of 2 ( Figure 3A and Figures S12-S14).
The relative configuration of 2 was deduced from the analysis of its ROESY spectrum ( Figure 3B), which was identical with that of voacangine. The ROESY correlation of H-3 with H-17a (δ H 1.90) indicated that both protons were co-facial and were assigned arbitrarily as β-oriented ( Figure S15). Finally, time-dependent density functional theory (TDDFT) ECD calculation was applied to clarify the absolute configuration of 2. The calculated ECD data of (3R,14R,16S,20S,21S)-2 matched well with the experimental data (    Tabercrassine C (3) was separated as a colorless oily substance, w MeOH), whose molecular formula was deduced as C21H24N2O4 based ion at m/z 369.1816 [M + H] + (calcd 369.1809) ( Figure S25), correspondin unsaturation. The IR spectrum showed bands at 3393, 1719, and 1623 aldehyde, and amide functions, respectively ( Figure S26). The 13 C NMR 2) showed a total of 21 separate carbon resonances, which were class thyls, four methylenes, five methines, and nine non-protonated carbon sis of its NMR data demonstrated that 3 was essentially similar to the a alkaloid jerantiphylline A [35], indicating both alkaloids had the same eton. The striking differences were the observation of chemical shifts ppm), C-10 (Δδ −18.   (Figure S25), corresponding to 11 degrees of unsaturation. The IR spectrum showed bands at 3393, 1719, and 1623 cm −1 due to NH, aldehyde, and amide functions, respectively ( Figure S26). The 13 C NMR spectrum (Table 2) showed a total of 21 separate carbon resonances, which were classified as three methyls, four methylenes, five methines, and nine non-protonated carbons. Detailed analysis of its NMR data demonstrated that 3 was essentially similar to the aspidosperma-type alkaloid jerantiphylline A [35], indicating both alkaloids had the same basic carbon skeleton. The striking differences were the observation of chemical shifts of C-9 (∆δ +13.6 ppm), C-10 (∆δ −18.2 ppm), C-11 (∆δ −17.5 ppm), and C-13 (∆δ +16.3 ppm) in 3, which were attributed to the absence of methoxy and hydroxyl moieties in the indole ring of 3. The 1 H-1 H COSY correlation of H-10 (δ H 6.87) and H-11 (δ H 7.19), and the key HMBC correlations of H-10 with C-8 (δ C 136.7) and C-12 (δ C 110.9), and of H-11 with C-9 (δ C 122.4) and C-13 (δ C 144.7), confirmed the above elucidation ( Figure 5A). The 2D NMR spectra ( Figures S19-S24, Supporting Information) confirmed that the other partial structures of the molecule were the same as jerantiphylline A. It is worth noting that tabercrassine C (3) represents the second example of a ring-D-seco-tabersonine alkaloid with the fracture at the C-14 and C-15 positions. This ring-opened alkaloid might be originated from a 3-oxotabersonine derivative such as melosine C [36], via a retro-aldol reaction.
The relative configuration of 3 was deduced from the analysis of its ROESY spectrum ( Figure 5B and Figure S24). The observed ROESY correlations of H-21 (δ H 4.05) and H-18 (δ H 0.73), and of H-21 and H-19b (δ H 1.81), indicated that the ring-D-seco-tabersonine alkaloid had a relative configuration that was identical to that of jerantiphylline A. In an attempt to assign the absolute configuration of 3, time-dependent density functional theory (TDDFT) ECD calculation was performed. The matched experimental and calculated ECD spectra finally confirmed the absolute configuration of 3 ( Figure 6, Figures S27 and S28).

Biological Activity of the New Isolates
The cytotoxic activities of the new alkaloids (1-3) were evaluat man cancer cell lines, HepG-2 (liver cancer), CNE-2 (nasophary HCT-116 (colon cancer), and MDA-MB-231 (triple-negative breast ca MTT method, according to our previous studies [23]. Unfortunately inactive (IC50 > 40 μM). Then, we conducted cellular analyses using h cells stably expressing the human APP mutant (APP-p. K670N/M671L a cellular AD model that was created in our previous studies [37][38][39] methyl sulfoxide) was used as the solvent and the control, gemfibroz US Food and Drug Administration, primarily for treating hyperlipi used as a positive control in cellular assays, as it could reduce Aβ p increase Aβ clearance [37][38][39]. At concentrations of 5 μM and 20 showed no apparent toxicity for U251-APP cells ( Figure 7A). We mea Aβ42 species, which play major synaptotoxic roles in AD [43][44][45]. A pernatants of U251-APP cells treated with alkaloids 1-3 showed a sig levels of Aβ42 (20 μM), as determined by the enzyme-linked imm (ELISA) ( Figure 7B). Interestingly, the effect of 1-3 on Aβ production that of gemfibrozil at a dose of 20 μM, suggesting that alkaloids 1-3 p of preventing Aβ production and its downstream consequence. Add ated anti-tau phosphorylation effects of alkaloids 1-3. We used Dinac

Biological Activity of the New Isolates
The cytotoxic activities of the new alkaloids (1-3) were evaluated against four human cancer cell lines, HepG-2 (liver cancer), CNE-2 (nasopharyngeal carcinoma), HCT-116 (colon cancer), and MDA-MB-231 (triple-negative breast cancer), by using the MTT method, according to our previous studies [23]. Unfortunately, all of them were inactive (IC 50 > 40 µM). Then, we conducted cellular analyses using human glioma U251 cells stably expressing the human APP mutant (APP-p. K670N/M671L) (U251-APP cells), a cellular AD model that was created in our previous studies [37][38][39]. While DMSO (dimethyl sulfoxide) was used as the solvent and the control, gemfibrozil approved by the US Food and Drug Administration, primarily for treating hyperlipidemia [40,41], was used as a positive control in cellular assays, as it could reduce Aβ production [42] and increase Aβ clearance [37][38][39]. At concentrations of 5 µM and 20 µM, alkaloids 1-3 showed no apparent toxicity for U251-APP cells ( Figure 7A). We measured the levels of Aβ42 species, which play major synaptotoxic roles in AD [43][44][45]. All of the culture supernatants of U251-APP cells treated with alkaloids 1-3 showed a significant decrease in levels of Aβ42 (20 µM), as determined by the enzyme-linked immunosorbent assay (ELISA) ( Figure 7B). Interestingly, the effect of 1-3 on Aβ production was comparable to that of gemfibrozil at a dose of 20 µM, suggesting that alkaloids 1-3 possess the property of preventing Aβ production and its downstream consequence. Additionally, we evaluated anti-tau phosphorylation effects of alkaloids 1-3. We used Dinaciclib, a CDK5 selective inhibitor [46] which can inhibit tau phosphorylation [13], as a positive treatment in this assay. The levels of phospho-tau (Thr217, a new biomarker of AD) [47][48][49], phospho-tau (Thr181, pTau181), and phospho-tau (Ser396, pTau396), which play major roles in the formation of NFT [50], were determined by ELISA. Unexpectedly, we found that alkaloids 1-3 all significantly decreased the level of pTau217, whereas the levels of pTau396 and pTau181 were not influenced ( Figure 7C-E). These results suggest that these alkaloids can inhibit the phosphorylation of tau and its downstream consequence.
tive inhibitor [46] which can inhibit tau phosphorylation [13], as a positive treatment in this assay. The levels of phospho-tau (Thr217, a new biomarker of AD) [47][48][49], phospho-tau (Thr181, pTau181), and phospho-tau (Ser396, pTau396), which play major roles in the formation of NFT [50], were determined by ELISA. Unexpectedly, we found that alkaloids 1-3 all significantly decreased the level of pTau217, whereas the levels of pTau396 and pTau181 were not influenced ( Figure 7C-E). These results suggest that these alkaloids can inhibit the phosphorylation of tau and its downstream consequence.  (R) A proposed potential role of 1 against AD by downregulating BACE1, NCSTN, CDK5, and GSK3β-mediated pathways, resulting in Aβ42 reduction and decreased pTau217. Data are presented as the means ± SD; ns, not significant; ***, p < 0.001; **, p < 0.01; and *, p < 0.05; one-way ANOVA with Bonferroni's post hoc test. We further measured CDK5 and GSK3β, which play important roles in Tau phosphorylation [51]. Western blot analyses showed that alkaloids 1-3 significantly decreased the protein level of GSK3β ( Figure 7F-K). Moreover, alkaloids 1-3 decreased the level of phospho-CDK5 (Tyr15) (pCDK5), an index of CDK5 enzyme activity [13,[52][53][54], although the CDK5 protein level was not changed ( Figure 7F-K). These results suggest the potential of alkaloids 1-3 to inhibit GSK3β and the CDK5-mediated pathway. In addition, we checked the protein levels of BACE1-the first protease that processes APP in the pathway, leading to the production of toxic Aβ and, therefore, playing a key role in the pathogenesis of AD [3,55]-and the components of γ-secretase, including NCSTN (nicastrin), γ-secretase subunit), PSEN1 (presenilin 1), and PSEN2 (presenilin 2). Western blot analyses showed that alkaloids 1-3 significantly decreased the protein levels of BACE1 and NCSTN, whereas the protein levels of PSEN1 and PSEN2 were not significantly changed ( Figure 7L-Q).
In this study, tabercrassines A-C (1-3), three MIAs with intriguing structures, were isolated and identified from the seeds of T. crassa. Moreover, alkaloids 1-3 possess potential bioactivity against AD by inhibiting Aβ production and tau phosphorylation at a site of Thr217 in cellular models ( Figure 7R), based on three lines of evidence: (1) alkaloids 1-3 decrease Aβ42 production in U251-APP cells; (2) alkaloids 1-3 decrease the protein levels of BACE1 and NCSTN; (3) alkaloids 1-3 inhibit the protein level of GSK3β and the activity of CDK5, and then inhibit the level of pTau217. Thus, it would be rewarding to perform further focused studies by testing whether alkaloids 1-3 would inhibit the production of Aβ42 and pTau217, thereby improving cognitive functions in AD animal models.

Plant Material
The seeds of T. crassa were collected in November 2016 from Ghana, Africa, and were identified by Dr.

Cytotoxic Activity
The cytotoxic activities of the new alkaloids (1-3) were evaluated against four human cancer cell lines by using the MTT method, according to our previous studies [23].
3.8. Enzyme Linked Immunosorbent Assay (ELISA) for Aβ42, pTau217, pTau396 and pTau181 The level of Aβ42 in the culture media of U251-APP cells was determined using commercial ELISA kit (Elabscience, E-EL-H0543c; Wuhan, China), as described in our previous study [14,38,39]. The levels of pTau217, pTau396, and pTau181 in cell lysates were determined using the commercial ELISA kit (RUIFAN, RF13027 to detect pTau217; Elabscience, E-EL-H5314c to detect pTau396; FineTest, EH4701 to detect pTau181), according to the manufacturer's instructions. We normalized the values of the control group with 3 biological replicates and the other treatment groups were compared with the control group.

Statistical Analysis
Data analyses were carried out by using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA) [38,39,57,58]. The results are expressed as means ± SD. Statistical analysis was performed using one-way ANOVA with Bonferroni's post hoc test, and differences of p < 0.05 were considered statistically significant. It was considered to be statistically significant if a p value < 0.05. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Conclusions
In conclusion, three new MIAs, tabercrassines A-C (1-3), were obtained from the seeds of T. crassa. Intriguingly, alkaloids 1 and 2 represent novel alkaloids within the ibogantype MIAs category, and 1-3 possess potential against AD by inhibiting Aβ production and tau phosphorylation in cellular models. Indeed, studies conducted on extracts or pure compounds of the Tabernaemontana genus have reported diverse pharmacological activities including anticancer, antimicrobial, antiviral activities, etc. However, few therapeutic agents from the genus Tabernaemontana have been reported for the treatment of neurodegenerative diseases. Our findings shed light on natural products that may provide novel therapeutic strategies for modulating AD from multiple aspects. Thus, it would be rewarding to perform further studies testing whether other ibogan-type MIAs possess the potential for treatment of AD, and whether alkaloids 1-3 would improve the cognitive function in AD animal models.

Conflicts of Interest:
The authors declare no conflict of interest. Glycogen synthase kinases-3β pCDK5