Next Article in Journal
Anti-Anxiety Effects of Essential Oil Microemulsion in Chronic Unpredictable Mild Stress-Induced Rats: Preparation, Characterization, and Mechanisms
Previous Article in Journal
Pressure Dependence of Structural Behavior in the Polymorphs of Fe(PM–BiA)2(NCS)2
Previous Article in Special Issue
Synthesis, Stability, and Biological Evaluation of Novel Aminoderivatives Incorporating the Aza-Acridine Scaffold
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 30
Issue 12
10.3390/molecules30122650
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Total Synthesis and Biological Evaluation of 22-Hydroxyacuminatine and the Related Natural Products Norketoyobyrine and Naucleficine

by
Shohta Mizuno
1,†,
Takashi Nishiyama
1,
Hana Bessho
1,
Tetsuya Nakamura
1,†,
Tomoki Oe
1,
Nanako Hayashi
1,
Yuhzo Hieda
1,
Toshio Motoyashiki
1,
Toshiyuki Hata
1,
Noriyuki Hatae
2,* and
Tominari Choshi
1,*
1
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Sanzo, Gakuen-cho, Fukuyama 729-0292, Hiroshima, Japan
2
Faculty of Pharmaceutical Sciences, Yokohama University of Pharmacy, 601 Matano, Totsuka-ku, Yokohama 245-0066, Kanagawa, Japan
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2025, 30(12), 2650; https://doi.org/10.3390/molecules30122650
Submission received: 26 May 2025 / Revised: 16 June 2025 / Accepted: 16 June 2025 / Published: 19 June 2025
(This article belongs to the Special Issue Exclusive Feature Papers in Synthetic Medicinal Chemistry)
Browse Figures
Versions Notes

Abstract

Aromathecin compounds—which contain the same indolizine core structure as camptothecin-like compounds—are expected to show anticancer activity. Among them, 22-hydroxyacuminatine—which has a substituent on the E-ring of the pentacyclic scaffold—exhibits topoisomerase 1 inhibitory activity; therefore, the development of efficient methods for its synthesis has been actively pursued. Herein, we report a versatile synthetic methodology for introducing various substituents on the E-ring, leading to the total synthesis of 22-hydroxyacuminatine as a model compound of the aromathecin family. The synthesis comprises the following key steps: the synthesis of an isoquinoline N-oxide via the thermal cyclization of 2-alkynylbenzaldehyde oxime, the subsequent Reissert–Henze-type reaction to yield an isoquinolone, and the construction of the indolizine moiety (CD-ring) through C–N bond formation via the Mitsunobu reaction. Consequently, a pentacyclic benz[6,7]indolizino[1,2-b]quinolin-11(13H)-one framework is obtained. Using this methodology, the total synthesis of the natural products norketoyobyrine and naucleficine and an intermediate of the latter, which are indoloquinolizidine-type alkaloids, was achieved, and their antiproliferative activity against HCT-116 human colon cancer cells and HepG2 human liver cancer cells was assessed. Naucleficine and its intermediate exhibited moderate antiproliferative activity against HCT-116 cells, with IC50 values of 55.58 and 41.40 μM, respectively.

Graphical Abstract

1. Introduction

Camptothecin (CPT: 1) was isolated from the Chinese tree Camptotheca acuminata by Wani et al. in 1966 and reportedly had topoisomerase I (Top1) inhibitory activity (Figure 1) [1]. Subsequently, 1 served as a lead compound for the development of pharmaceuticals, including irinotecan, which are now clinically used. Meanwhile, rosettacin (2), in which the lactone E-ring moiety of 1 is replaced by a benzene ring, was synthesized while developing the synthesis method for 1 [2]. Later, in 1992, 22-hydroxyacuminatine (4), having the skeleton of 2, was isolated along with 1 from the seeds of C. acuminata [3]. Compound 4 has reportedly displayed weak Top1 inhibitory activity. Compounds 2 and 4, as well as acuminatine (3), are the three members of the aromathecin family targeted for intense synthetic research [4,5]. In particular, the total synthesis of 4 has been reported by six research groups [6,7,8,9,10,11]. These derivatives and other compounds containing the indolizine scaffold have been the subject of drug discovery studies.
Norketoyobyrine (5), ketoyobyrine (6), and naucleficine (7) have a pentacyclic structure similar to that of aromathecins and belong to the indoloquinolizidine alkaloid family. They have been isolated from the Nauclea latifolia plant and used in folk medicine (Figure 1) [12]. These products have attracted considerable interest in synthesis owing to their structural features. Various synthetic approaches have been developed for norketoyobyrine (5) [11,13,14,15,16,17,18,19,20,21,22,23,24,25] and ketoyobyrine (6) [13,26,27]—a degradation product of yohimbine. Naucleficine (7) is isolated from the stems of Nauclea officinalis (Pierre ex Pitard) and has been used as an anti-inflammatory and antibacterial agent in Chinese folk medicine [28]. The total synthesis of 7 has been reported by three research groups so far [11,29,30]. These products 5–7 can be considered as derivatives of aromathecin that underwent a skeletal transformation of its B- and C-rings to conduct structure–activity relationship (SAR) studies. To this aim, performing the total synthesis of these compounds would be highly beneficial.
Our research group has been interested in the unique structure and pharmacological action of condensed heteroaromatic compounds and has been searching for highly active compounds based on these naturally occurring compounds and their derivatives. To date, we have achieved the total synthesis of β-carboline alkaloids (R)-(–)-pyridindolols [31,32] and 8-oxoprotoberberine alkaloids alangiumkaloids A and B [33] using a series of key steps, namely, the thermal cyclization of 2-alkynylaryl aldoxime to afford fused pyridine N-oxide, followed by the synthesis of fused pyridone via a Reissert–Henze-type reaction.
Furthermore, we have previously reported a synthetic route to 2 by applying a similar series of reactions as the key steps [34]. Herein, we describe the application of this method to the synthesis of 4 (Scheme 1). During the final step for constructing the pentacyclic scaffold by forming the C-ring, isoquinolone 8 was treated with H2SO4 in EtOH at 110 °C. As expected, a C-ring was formed; however, the 7-methoxymethoxymethyl group was converted to a methyl group, and 3 was obtained in 79% yield. This result, whose reason remains unclear, indicates that this methodology might not be applicable to the synthesis of derivatives with certain substituents on the E-ring.
Therefore, a versatile strategy is still required to synthesize the natural product 22-hydroxyacuminatine (4) and its derivatives bearing substituents on the E-ring. In this study, we improved the previously reported synthetic method and applied it to the synthesis of indoloquinolizidine-type alkaloids.

2. Results and Discussion

As a starting point of our investigation, we considered intramolecular cyclization under mild conditions to form a C-ring. The retrosynthetic analysis of the pentacyclic scaffolds of indolizine-type 2–4 and quinolizine-type 5–7 is shown in Scheme 2. The construction of the indolizine and quinolizine scaffolds would be achieved via a C-ring formation in the final stage. To this aim, we considered the Mitsunobu reaction between the alcohol moiety and the amide nitrogen of isoquinolone 9 to form a C–N bond. Precursor 9 would be synthesized via the Reissert–Henze-type reaction of isoquinoline N-oxide 10, formed by the thermal cyclization of 2-alkynylbenzaldehyde oxime 11. Therefore, we investigated the optimal substrate for converting 10 to 9.
As shown in Scheme 3, 2-arylethynylbenzaldehyde 14a was obtained in 86% yield via the Sonogashira reaction of 3-hydroxymethyl-2-iodoquinoline (12) [34] and 2-ethynylbenzaldehyde 13 [33] in the presence of CuI, iPr2NH and Pd2(dba)3. To investigate the optimal synthetic route, tert-butyldimethylsilyl (TBS) and acetyl (Ac) groups were selected as protecting groups for the hydroxyl group of 14a. Compounds 14b,c were obtained by introducing each protecting group to 14a under general conditions. Subsequently, the three 2-arylethynylbenzaldehydes 14a–c were treated with NH2OH to obtain oximes 15a–c, followed by heating in 1,2-dichlorobenzene (DCB) at 80 °C to obtain N-oxides 16a–c of the key precursor in moderate yield. Oxime 15b was unstable and thus was subjected to a thermal cyclization without purification to obtain N-oxide 16b in 63% yield over two steps.
Subsequently, we investigated the conversion of N-oxides 16a–c to the desired isoquinolones 17a–c in Ac2O at 80 °C with or without microwave (MW) irradiation. The Reissert–Henze-type reaction of N-oxides 16 produced 4-hydroxyisoquinolines 18 along with the desired isoquinolones 17. The product ratios are summarized in Table 1.
Initially, when unprotected N-oxide 16a was heated in Ac2O at 80 °C, many products were observed on thin-layer chromatography (TLC). Among them, isoquinolone 17a and 4-OH 18a were not obtained, and only small amounts of 17c and 18c, which are acetylated hydroxymethyl groups of 17a and 18a, were isolated (Table 1, entries 1 and 2). Substrate 16a did not afford isoquinolone 17c in the absence or presence of MW irradiation.
Moreover, when N-oxide 16b containing a TBS group was heated in Ac2O at 80 °C, 16b disappeared as revealed by TLC monitoring, but isoquinolone 17b was obtained in a low 20% yield (Table 1, entry 3). The yield of 17b could be improved to 36%, and the reaction time was considerably shortened by reacting 16b under MW irradiation (Table 1, entry 4). However, the yield was still not satisfactory for proceeding to the next step.
Furthermore, we investigated the conversion of O-Ac-N-oxide 16c to isoquinolone 17c. When heated at 80 °C, 17c and 18c were obtained in 20% and 27% yields, respectively (Table 1, entry 5). The product ratio of 17c improved when heating under MW irradiation, affording a yield of 50% (Table 1, entry 6). Considering these results, the Ac group is the most suitable protecting group for the hydroxymethyl group in N-oxide 16.
As a key precursor for the C-ring formation, alcohol 19 was obtained in 93% yield by treating 17c with 1 M NaOH in MeOH (Scheme 4). Further, 19 was subjected to the Mitsunobu reaction in the presence of diisopropyl azodicarboxylate (DIAD) and PPh3, which promoted the C-ring formation to obtain the desired pentacyclic compound 20. However, 20 was obtained as a mixture with the by-product triphenylphosphine oxide and could not be separated. Therefore, the crude 20 was treated with 6 M HCl and ethylene glycol in THF to deprotect the MOM group [11], affording the desired 4 in 60% yield over two steps. Thus, the total synthesis of 22-hydroxyacuminatine (4) as a model compound with a substituent on the E-ring was achieved via an eight-step sequence in 10.5% overall yield. The physical and spectroscopic data of synthesized 4 were consistent with previously reported values [6].
Having established an improved synthetic method for the benzo[6,7]indolizino[1,2-b]quinoline-11(13H)-one scaffold—the core structure of the aromathecin family—and achieved the total synthesis of 4, we addressed the synthesis of indoloquinolizidine-type alkaloids by applying the same methodology (Scheme 5). Methyl indolylacetate 21 [35] was treated with MOMCl and NaH in DMF to give N-MOM-indolylacetate 22 in 85% yield. Subsequently, the Sonogashira coupling of 22 and trimethylsilyl (TMS)-acetylene in the presence of CuI, Et3N, and PdCl2(PPh3)2 produced TMS-acetylene 23 in 78% yield. Further, the desilylation of 23 using TBAF provided 2-ethynylindole 24 in 92% yield. The Sonogashira coupling of 24 and 2-iodobenzaldehyde 25a in the presence of CuI, Et3N, and PdCl2(PPh3)2 produced 2-alkynylbenzaldehyde 26a in 80% yield. The treatment of 26a with NH2OH in EtOH furnished 2-alkynylbenzaldehyde oxime 27a in 88% yield. Subsequently, heating 27a in 1,2-DCB at 80 °C to give isoquinoline N-oxide 28a in 82% yield followed by heating 28a in Ac2O at 80 °C under MW irradiation furnished isoquinolone 29a and 4-OAc 31a in 60% and 18% yields, respectively.
Isoquinoline 29a was reduced with LiAlH4 to give alcohol 32a in 95% yield. The Mitsunobu reaction of 32a with DIAD and PPh3 formed the C-ring, giving indoloquinolizine 33a in 95% yield (Scheme 6). Finally, 33a was treated with 6 M HCl in the presence of ethylene glycol to remove the MOM group, giving 5 in 90% yield. Using the same synthetic route, we also attempted the total synthesis of naucleficine (7). In this case, when isoquinolone 29b was synthesized from N-oxide 28b via the Reissert–Henze-type reaction as shown in Scheme 5, 1-OAc-isoquinoline 30b was produced in 20% yield along with the desired 29b (38%) and 4-OAc 31b (32%). Subsequently, the reduction of 29b and 30b with LiAlH4 afforded alcohol 32b in 72% and 59% yields, respectively. Therefore, after obtaining 29b and 30b from N-oxide 28b, the mixture was reduced with LiAlH4, and alcohol 32b was afforded as a single product in 69% yield over two steps (Scheme 6). After the Mitsunobu reaction of 32b to obtain indoloquinolizine 33b in 90% yield, the two MOM groups of 33b were removed by treating with 6 M HCl, furnishing alcohol 34 in 90% yield. Finally, 34 was oxidized with MnO2 to produce 7 in 70% yield. Thus, we achieved the total synthesis of the indoloquinolizidine-type alkaloids norketoyobyrine (5: 10 steps, total yield 17.2%) and naucleficine (7: 11 steps, total yield 13.3%).
The physical and spectroscopic data of synthesized 5 and 7 were consistent with the previously reported values [11]. Furthermore, 1H NMR, 13C NMR, and mass spectroscopy characterization of all the synthesized compounds supported the identified structures (Supplementary Materials).
Furthermore, we compared the total synthesis yields of the three natural products that we synthesized with other methodologies that have already been published, and we summarized the results in Table 2.
On preliminary work, the indolizine derivatives 2, 3, and 4 were indicated weak antiproliferative activity against HCT-116 cells. The synthesized indoloquinolizine-type natural products 5 and 7 and intermediate 34 were assessed for their antiproliferative activity against HCT-116 human colon and HepG2 human liver cancer cells. Figure 2 summarizes the results of the tumor cell viability treatments, which were performed at compound concentrations of 10 and 100 μM for 72 h. Naucleficine (7) and intermediate 34 at a concentration of 100 μM showed moderate antiproliferative activities against HCT-116 cells, with IC50 values of 55.58 and 41.40 μM, respectively (Table 3). Meanwhile, tested compounds were indicated to have low or almost lost their activity against HepG2 cells.

3. Materials and Methods

3.1. Chemistry

All non-aqueous reactions were carried out under an atmosphere of nitrogen in dried glassware unless otherwise noted. Solvents were dried and distilled according to standard protocols. Analytical thin-layer chromatography was performed with Silica gel 60PF254 (Merck KGaA, Darmstadt, Germany). Silica gel column chromatography was performed with Silica gel 60 (70–230 mesh, Kanto Co. Lit., Tokyo, Japan). All melting points were determined on micro-melting point apparatus MP-500D (Yanaco Technical Science Co., Ltd., Tokyo, Japan) and are uncorrected. Proton nuclear magnetic resonance (1H-NMR) spectra were recorded on JEOL JNM-ECZ400S (JEOL Ltd., Tokyo, Japan). Chemical shifts are reported relative to Me4Si (δ 0.00). Multiplicity is indicated by one or more of the following: s (singlet); d (doublet); t (triplet); q (quartet); m (multiplet); and br (broad). Carbon nuclear magnetic resonance (13C-NMR) spectra were recorded on a JEOL JNM-ECZ400S at 100 MHz. Chemical shifts are reported relative to CDCl3 (δ 77.0) and DMSO-d6 (δ 39.7). Infrared spectra were recorded with ATR method using a Shimadzu FTIR-8000 spectrophotometer (Shimadzu corporation, Kyoto, Japan) and DuraScop (Sensir Technologies, NC, USA). Low and high-resolution mass spectra were recorded on JEOL JMS-700 spectrometers (JEOL Ltd., Tokyo, Japan) by direct inlet system. The microwave assisted reaction was carried out at 180 W and 2450 MHz with Discover (CEM corporation, NC, USA).

3.1.1. 2-(3-Hydroxymethylquinolin-2-yl)ethynyl-3-[(methoxymethoxy)methyl]benzaldehyde (14a)

To a solution of 2-iodoquinoline 12 (95 mg, 0.33 mmol), CuI (6 mg, 0.033 mmol), Pd2(dba)3 (13 mg, 0.013 mmol) and iPr2NH (0.3 mL, 2.31 mmol) in THF (1.5 mL), a solution of 2-ethynylbenzaldehyde 13 (81 mg, 0.40 mmol) in THF (1.5 mL) was added. The reaction mixture was stirred at 100 °C for 10 min. After cooling to ambient temperature, the reaction mixture was filtered through Celite pad, washed with EtOAc, and the filtrate was evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:3 v/v) to give the 2-alkynylbenzaldehyde 14a (124 mg, 86%) as a white solid. mp was 59–61 °C (EtOAc-hexane). IR (ATR) ν = 1685, 2190, and 3629 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.41 (s, 3H), 3.52 (br s, 1H), 4.79 (s, 2H), 5.00 (s, 2H), 5.06 (s, 2H), 7.54–7.60 (m, 2H), 7.74 (t, J = 7.8 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 7.8 Hz, 1H), 8.25 (s, 1H), and 10.70 (s, 1H). 13C-NMR (400 MHz, CDCl3) δ 55.6, 62.8, 67.1, 85.6, 95.9, 98.2, 123.7, 127.5 (2C), 127.8, 127.9, 129.1, 129.4, 130.1, 133.8, 135.0, 135.2, 136.8, 141.8, 142.0, 147.6, and 191.5. MS m/z: 361 (M+). HRMS (EI): calcd for C22H19NO4 361.1314; found 361.1332.

3.1.2. 2-{[3-(tert-Butyldimethylsilyloxy)methyl]quinolin-2-yl}ethynyl-3-[(methoxymethoxy)methyl]benzaldehyde (14b)

A solution of alcohol 14a (120 mg, 0.33 mmol) in CH2Cl2 (2 mL) was dropwise added to a suspension of imidazole (56 mg, 0.82 mmol) in CH2Cl2 (1 mL) under ice cooling. After stirring at same temperature for 15 min, TBSCl (123 mg, 0.82 mmol) in CH2Cl2 (1 mL) was added to the reaction mixture and then was stirred at rt for 12 h. After quenching with H2O, the reaction mixture was extracted with CH2Cl2. The organic layer was washed with brine, dried with Na2SO4, and evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:3 v/v) to give the silyl ether 14b (123 mg, 78%) as a yellow solid. mp was 74–75 °C (EtOAc-hexane). IR (ATR) ν = 1697, 2168 cm−1. 1H-NMR (400 MHz, CDCl3) δ 0.19 (s, 6H), 1.00 (s, 9H), 3.42 (s, 3H), 4.82 (s, 2H), 5.00 (s, 2H), 5.14 (s, 2H), 7.54–7.61 (m, 2H), 7.73 (t, J = 7.8 Hz, 1H), 7.84–7.88 (m, 2H), 7.95 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 7.8 Hz, 1H), 8.33 (s, 1H), and 10.77 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ -5.3 (2C), 18.4, 25.9 (3C), 55.6, 62.2, 67.0, 85.7, 96.3, 97.9, 123.7, 126.7, 127.6 (2C), 127.7, 129.1, 129.4, 129.6, 132.7, 133.0, 135.9, 136.7, 140.3, 142.0, 147.3, and 191.2. MS m/z: 475 (M+). HRMS (EI): calcd for C28H33NO4Si 475.2179; found 475.2181.

3.1.3. 2-[3-(Acetoxymethyl)quinolin-2-yl]ethynyl-3-[(methoxymethoxy)methyl]benzaldehyde (14c)

A solution of alcohol 14a (30 mg, 0.083 mmol) in CH2Cl2 (1 mL) was dropwise added to a suspension of DMAP (12 mg, 0.10 mmol) in CH2Cl2 (1 mL) under ice cooling. After stirring at same temperature for 15 min, Ac2O (9 μL, 0.10 mmol) was added to the reaction mixture and then was stirred at rt for 1 h. After quenching with H2O, the reaction mixture was extracted with CH2Cl2. The organic layer was washed with brine, dried with Na2SO4, and evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:3 v/v) to give the compound 14c (25 mg, 75%) as a yellow solid. mp was 110–111 °C (EtOAc-hexane). IR (ATR) ν = 1685, 1736, and 2198 cm−1. 1H-NMR (400 MHz, CDCl3) δ 2.17 (s, 3H), 3.43 (s, 3H), 4.84 (s, 2H), 5.01 (s, 2H), 5.53 (s, 2H), 7.57 (t, J = 7.8 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.85–7.87 (m, 2H), 7.96 (d, J = 7.8 Hz, 1H), 8.13 (d, J = 7.8 Hz, 1H), 8.27 (s, 1H), and 10.77 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 20.9, 55.6, 63.5, 67.0, 85.7, 96.4, 97.9, 123.5, 126.6, 127.2, 127.7, 128.0, 129.3, 129.5, 130.3, 130.5, 132.9, 136.2, 136.8, 142.0, 142.2, 147.9, 170.6, and 191.4. MS m/z: 403 (M+). HRMS (EI): calcd for C24H21NO5 403.1420; found 403.1411.

3.1.4. 2-(3-Hydroxymethylquinolin-2-yl)ethynyl-3-[(methoxymethoxy)methyl]benzaldehyde oxime (15a)

A mixture of benzaldehyde 14a (100 mg, 0.25 mmol), NH2OH·HCl (34 mg, 0.50 mmol), and AcONa (40 mg, 0.50 mmol) in EtOH (5 mL) was stirred at rt for 2 h. After removal of solvent, the residue was diluted with H2O and then filtered off to give the oxime 15a (65 mg, 63%) as a yellow solid. Mp was 220–221 °C (EtOAc-hexane). IR (ATR) ν = 1736, 2191, 3444, and 3610 cm−1. 1H-NMR (400 MHz, DMSO-d6) δ 4.76 (s, 2H), 4.85 (s, 2H), 4.91 (d, J = 5.5 Hz, 3H), 5.66 (t, J = 5.5 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 8.03–8.06 (m, 2H), 8.44 (s, 1H), 8.68 (s, 1H), and 11.72 (s, 1H). 13C-NMR (100 MHz, DMSO-d6) δ 55.0, 60.3, 67.0, 86.7, 95.8, 96.7, 119.1, 123.9, 127.2, 127.7, 127.9, 128.4, 128.7, 129.7, 129.8, 133.0, 134.8, 136.9, 140.9, 141.2, 146.0, and 146.6. MS m/z: 376 (M+). HRMS (EI): calcd for C22H20N2O4 376.1423; found 376.1441.

3.1.5. 2-[3-(Acetoxymethyl)quinolin-2-yl]ethynyl-3-[(methoxymethoxy)methyl]benzaldehyde oxime (15c)

The same procedure as above was carried out with 2-arylethynylbenzaldehyde 14c (96 mg, 0.23 mmol) to give the oxime 15c (78 mg, 81%) as a pink solid. mp was 138–139 °C (EtOAc-hexane). IR (ATR) ν = 1751, 2233, and 3629 cm−1. 1H-NMR (400 MHz, DMSO-d6) δ 2.12 (s, 3H), 3.29 (s, 3H), 4.74 (s, 2H), 4.84 (s, 2H), 5.47 (s, 2H), 7.53 (t, J = 7.8 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.82–7.87 (m, 2H), 8.05–8.08 (m, 2H), 8.49 (s, 1H), 8.65 (s, 1H), and 11.72 (s, 1H). 13C-NMR (100 MHz, DMSO-d6) δ 20.7, 55.0, 63.0, 67.0, 86.7, 95.7, 96.6, 118.8, 123.9, 126.8, 128.1 (2C), 128.5, 128.6, 129.9, 130.5, 130.8, 135.0, 136.1, 141.3, 141.8, 145.9, 147.1, and 170.3. MS m/z: 418 (M+). HRMS (EI): calcd for C24H22N2O5 418.1529; found 418.1533.

3.1.6. 3-(3-Hydroxymethylquinolin-2-yl)-5-[(methoxymethoxy)methyl]isoquinoline N-oxide (16a)

A solution of oxime 15a (65 mg, 0.16 mmol) in 1,2-dichlorobenzene (4 mL) was stirred at 80 °C for 20 h. After removal of the solvent, the residue was purified by column chromatography (EtOAc/hexane 1:1 v/v) to give the N-oxide 16a (43 mg, 72%) as a white solid. mp was 239–240 °C (EtOAc-hexane). IR (ATR) ν = 1230, 3610 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.36 (s, 3H), 4.57 (s, 2H), 4.71 (s, 2H), 5.02 (s, 2H), 5.61 (br s, 1H), 7.63–7.67 (m, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.27 (s, 1H), 8.40 (s, 1H), and 8.99 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 55.7, 63.7, 66.2, 95.8, 123.2, 125.5, 127.7, 127.8 (2C), 128.7, 129.4, 129.7, 130.0, 130.1 (2C), 133.8, 134.2, 137.8, 138.1, 146.4, 147.6, and 151.9 MS m/z: 376 (M+). HRMS (EI): calcd for C22H20N2O4 376.1423; found 376.1417.

3.1.7. 2-{3-[(tert-Butyldimethylsilyloxy)methyl]quinolin-2-yl}-5-[(methoxymethoxy)methyl]isoquinoline N-oxide (16b)

A mixture of benzaldehyde 15b (43 mg, 0.090 mmol), NH2OH·HCl (12 mg, 0.18 mmol), and AcONa (11 mg, 0.14 mmol) in EtOH (3 mL) was stirred at rt for 2 h. The resulting mixture was quenched with water and extracted with EtOAc. The organic layer was washed with water and brine, dried with Na2SO4, and evaporated in vacuo. Next, 1,2-dichlorobenzene (2 mL) was added to the residue, and the mixture was stirred at 80 °C for 12 h. After removal of the solvent, the residue was purified by column chromatography (EtOAc/hexane 1:1 v/v) to give the N-oxide 16b (28 mg, 63%) as a black oil. IR (ATR) ν = 1254 cm−1. 1H-NMR (400 MHz, CDCl3) δ –0.07 (s, 3H), 0.02 (s, 3H), 0.83 (s, 9H), 3.36 (s, 3H), 4.70 (s, 2H), 4.77 (d, J = 13.7 Hz, 1H), 4.95 (d, J = 11.9 Hz, 1H), 5.02 (d, J = 11.9 Hz, 1H), 5.11 (d, J = 13.7 Hz, 1H), 7.59–7.67 (m, 3H), 7.71–7.76 (m, 2H), 7.93 (d, J = 7.8 Hz, 1H), 8.13 (d, J = 7.8 Hz, 1H), 8.19 (s, 1H), 8.37 (s, 1H), and 8.90 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ -5.5 (2C), 18.2, 25.8 (3C), 55.6, 62.2, 66.2, 95.7, 122.6, 124.9, 127.4, 127.6 (2C), 128.3, 129.0, 129.3 (3C), 130.2, 133.9, 134.0, 135.3, 136.5, 146.5, 146.9, and 150.9. MS m/z: 490 (M+). HRMS (EI): calcd for C28H34N2O4Si 490.2288; found 490.2276.

3.1.8. 2-(3-Acetoxymethylquinolin-2-yl)-5-[(methoxymethoxy)methyl]isoquinoline N-oxide (16c)

The N-oxide 16c (yield 72%) was prepared according to a synthetic method for 16a as a black solid. mp was 169–171 °C (EtOAc-hexane). IR (ATR) ν = 1230, 1743 cm−1. 1H-NMR (400 MHz, CDCl3) δ 1.94 (s, 3H), 3.37 (s, 3H), 4.71 (s, 2H), 4.97 (d, J = 11.9 Hz, 1H), 5.05 (d, J = 11.9 Hz, 1H), 5.27 (d, J = 13.3 Hz, 1H), 5.45 (d, J = 13.3 Hz, 1H), 7.61–7.67 (m, 3H), 7.76 (t, J = 7.8 Hz, 2H), 7.93 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.26 (s, 1H), 8.33 (s, 1H), and 8.91 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 20.7, 55.6, 63.5, 66.2, 95.7, 122.9, 125.0 (2C), 127.6, 127.7, 127.9, 129.2, 129.4, 130.0 (2C), 130.4, 134.0, 135.6 (2C), 136.7, 146.0, 147.3, 151.4, and 170.3. MS m/z: 418 (M+). HRMS (EI): calcd for C24H22N2O5 418.1529; found 418.1535.

3.1.9. 3-{3-[(tert-Butyldimethylsilyloxy)methyl]quinolin-2-yl}-5-[(methoxymethoxy)methyl]isoquinolin-1-one (17b)

A solution of N-oxide 16b (25 mg, 0.051 mmol) in Ac2O (1 mL) was heated at 80 °C under microwave irradiation for 2 h. After removal of the solvent, the residue was purified by column chromatography (EtOAc/hexane 2:3 v/v) to give the isoquinolone 17b (9 mg, 36%) as a yellow solid. mp was 114–115 °C (EtOAc-hexane). IR (ATR) ν = 1651, 3305 cm−1. 1H-NMR (400 MHz, CDCl3) δ 0.21 (s, 6H), 1.00 (s, 9H), 3.40 (s, 3H), 4.73 (s, 2H), 4.97 (s, 2H), 5.20 (s, 2H), 7.43 (s, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.49 (d, J = 7.8 Hz, 2H), and 10.73 (br s, 1H). 13C-NMR (100 MHz, CDCl3) δ-5.1 (2C), 18.4, 25.9 (3C), 55.5, 63.1, 66.7, 95.2, 105.1, 127.2, 127.3, 127.5, 127.7 (2C), 127.8, 129.2, 130.3, 132.4, 133.3, 133.6, 136.2, 136.4, 136.6, 146.2, 148.3, and 162.6. MS m/z: 490 (M+). HRMS (EI): calcd for C28H34N2O4Si 490.2288; found 490.2284.

3.1.10. 3-(3-Acetoxymethylquinolin-2-yl)-5-[(methoxymethoxy)methyl]isoquinolin-1-one (17c) and 3-[3-(Acetoxymethyl)quinolin-2-yl]-4-hydroxy-3-[(methoxymethoxy)methyl]isoquinoline (18c)

A solution of N-oxide 16c (30 mg, 0.071 mmol) in Ac2O (2 mL) was heated at 80 °C under MW irradiation for 5 h. After removal of the solvent, the residue was purified by column chromatography (EtOAc/hexane 1:1 v/v) to give the isoquinolone 17c (15 mg, 50%) and the 4-acetoxyisoquinoline 18c (11 mg, 37%).
17c: yellow solid, mp 149–150 °C (EtOAc-hexane). IR (ATR) ν = 1643, 1743, and 3286 cm−1. 1H-NMR (400 MHz, CDCl3) δ 2.25 (s, 3H), 3.39 (s, 3H), 4.71 (s, 2H), 4.95 (s, 2H), 5.54 (s, 2H), 7.54 (t, J = 7.8 Hz, 1H), 7.57 (s, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.83 (t, J = 7.8 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 8.17 (d, J = 7.8 Hz, 1H), 8.45 (s, 1H), 8.50 (d, J = 7.8 Hz, 1H), and 10.36 (br s, 1H). 13C-NMR (100 MHz, CDCl3) δ 21.0, 55.5, 64.1, 66.6, 95.1, 105.3, 126.5, 127.2, 127.3, 127.4, 127.5, 127.8, 128.1, 129.2, 131.1, 133.4, 133.7, 135.3, 136.3, 140.7, 146.8, 149.7, 162.6, and 170.6. MS m/z: 418 (M+). HRMS (EI): calcd for C24H22N2O5 418.1529; found 418.1523.
18c: yellow solid, mp 108–109 °C (EtOAc-hexane). IR (ATR) ν = 1732 cm−1. 1H-NMR (400 MHz, CDCl3) δ 2.08 (s, 3H), 3.47 (s, 3H), 4.80 (s, 2H), 5.16 (s, 2H), 5.71 (s, 2H), 7.60 (t, J = 7.8 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 8.01 (d, J = 7.8 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.36 (s, 1H), 8.70 (s, 1H), and 9.36 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 21.0, 55.7, 64.3, 66.5, 95.9, 117.7, 127.0, 127.4 (2C), 127.5, 127.8, 128.3, 128.9, 129.4, 129.8, 130.4, 133.9, 135.0, 136.0, 147.2, 151.8, 152.2, 156.3, and 170.7. MS m/z: 418 (M+). HRMS (EI): calcd for C24H22N2O5 418.1529; found 418.1518.

3.1.11. 3-(3-Hydroxymethylquinolin-2-yl)-5-[(methoxymethoxy)methyl]isoquinolin-1-one (19)

To a solution of isoquinolone 17c (9 mg, 0.020 mmol) in MeOH (1 mL), 1 M NaOH aq. (0.1 mL) was added dropwise under ice cooling and was stirred at 70 °C for 15 min. After cooling at ambient temperature, the resulting mixture was quenched with water and extracted with EtOAc. The organic layer was washed with water and brine, dried with Na2SO4, and evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 3:7 v/v) to give the alcohol 19 (7 mg, 93%) as a white solid. mp was 199–200 °C (EtOAc-hexane). IR (ATR) ν = 1620, 3286, 3737 cm−1. 1H-NMR (400 MHz, DMSO-d6) δ 3.28 (s, 3H), 4.70 (s, 2H), 4.80 (d, J = 5.0 Hz, 2H), 4.85 (s, 2H), 5.78 (t, J = 5.0 Hz, 1H), 7.16 (s, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.77–7.83 (m, 2H), 8.08–8.12 (m, 2H), 8.26 (d, J = 7.8 Hz, 1H), 8.55 (s, 1H), and 11.48 (br s, 1H). 13C-NMR (100 MHz, DMSO-d6) δ 55.0, 60.5, 66.6, 95.5, 103.1, 126.2, 126.7, 126.8, 127.5 (2C), 127.6, 127.8, 128.8, 130.0, 133.0, 133.6, 133.9, 136.0, 138.0, 145.9, 151.3, and 161.9. MS m/z: 376 (M+). HRMS (EI): calcd for C22H20N2O4 376.1423; found 376.1433.

3.1.12. 22-Hydroxyacuminatine (4)

To a solution of alcohol 19 (8 mg, 0.021 mmol) and PPh3 (8 mg, 0.032 mmol) in THF (1 mL), DIAD (1.9 M in toluene, 0.017 mL, 0.032 mmol) was added dropwise under ice cooling, and was stirred at 0 °C for 1 h. The resulting mixture was quenched with water and extracted with EtOAc. The organic layer was washed with water and brine, dried with Na2SO4, and evaporated in vacuo. To a suspension of the residue and ethylene glycol (0.1 mL) in THF (0.5 mL), 6 M HCl (0.5 mL) was added dropwise under ice cooling and then stirred at 50 °C for 3 h. After cooling to ambient temperature, the residue was alkalified with 1 M aqueous NaOH. The resulting mixture was extracted with EtOAc. The organic layer was washed with water and brine, dried with Na2SO4, and evaporated in vacuo. The residue was filtered off to give 22-hydroxyacuminatine (4) (4 mg, 60%) as a white solid. mp 255–256 °C (EtOAc-hexane lit. [3] mp 258–260 °C). IR (ATR) ν = 1651, 3359 cm−1. 1H-NMR (400 MHz, DMSO-d6) δ 4.95 (d, J = 5.5 Hz, 2H), 5.36 (s, 2H), 5.50 (t, J = 5.5 Hz, 1H), 7.54–7.63 (m, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.73 (s, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.83–7.87 (m, 1H), 8.11 (d, J = 7.8 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.30 (d, J = 7.8 Hz, 1H), and 8.66 (s, 1H). 13C-NMR (100 MHz, DMSO-d6) δ 49.6, 61.2, 96.2, 125.9 (2C), 126.7, 127.3, 127.9, 128.5, 128.9, 129.7, 130.2, 131.1, 131.3, 135.3, 138.5, 140.1, 148.0, 153.4, and 159.9. MS m/z: 314 (M+). HRMS (EI): calcd for C20H14N2O2 314.1055; found 314.1061.

3.1.13. Methyl [2-Iodo-N-(methoxymethyl)indol-3-yl]acetate (22)

A solution of methyl indolylacetate 21 (470 mg, 1.49 mmol) in DMF (8 mL) was added dropwise to a suspension of NaH (71 mg, 1.79 mmol) in DMF (4 mL) under ice cooling. After stirring at same temperature for 30 min, MOMCl (0.13 mL, 1.79 mmol) was added to the reaction mixture and then was stirred at rt for 2 h. After quenching with H2O, the reaction mixture was extracted with EtOAc. The organic layer was washed with brine, dried with Na2SO4, and evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:3 v/v) to give the N-MOM-indolylacetate 22 (454 mg, 85%) as a yellow solid. mp was 83–85 °C (EtOAc-hexane). IR (ATR) ν = 1728 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.31 (s, 3H), 3.69 (s, 3H), 3.79 (s, 2H), 5.54 (s, 2H), 7.16 (t, J = 7.8 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), and 7.55 (d, J = 7.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ 33.6, 52.1, 56.0, 77.5, 88.0, 110.1, 116.1, 118.5, 120.7, 122.7, 128.4, 138.6, and 171.3. MS m/z: 359 (M+). HRMS (EI): calcd for C13H14INO3 359.0018; found 359.0022.

3.1.14. Methyl [2-Trimethylsilylethynyl-N-(methoxymethyl)lindol-3-yl]acetate (23)

A solution of 2-iodoindole 22 (200 mg, 0.56 mmol), CuI (11 mg, 0.056 mmol), PdCl2(PPh3)2 (23 mg, 0.022 mmol), and TMS-acetylene (0.091 mL, 0.66 mmol) in Et3N (5 mL, 35.6 mmol) was stirred at rt for 23 h. Afterward, the reaction mixture was filtered through Celite pad, washed with EtOAc, and the filtrate was evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:9 v/v) to give the TMS-acetylene 23 (143 mg, 78%) as a yellow oil. IR (ATR) ν = 1736, 2152 cm−1. 1H-NMR (400 MHz, CDCl3) δ 0.30 (s, 9H), 3.28 (s, 3H), 3.70 (s, 3H), 3.87 (s, 2H), 5.57 (s, 2H), 7.17 (t, J = 7.8 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), and 7.57 (d, J = 7.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ -0.14 (3C), 31.2, 52.0, 56.0, 75.1, 94.4, 105.3, 110.3, 115.1, 119.6, 121.0, 121.3, 124.1, 127.1, 136.6, and 171.6. MS m/z: 329 (M+). HRMS (EI): calcd for C18H23NO3Si 329.1447; found 329.1451.

3.1.15. Methyl [2-Ethynyl-N-(methoxymethyl)indol-3-yl]acetate (24)

A solution of TBAF (1.0 M in THF, 2.19 mL, 2.19 mmol) was added dropwise to a solution of TMS-acetylene 23 (600 mg, 1.82 mmol) in THF (20 mL) at 0 °C. After being stirred at rt for 10 min, the reaction mixture was quenched with water and then extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and evaporated in vacuo. The residue was purified by column chromatography using EtOAc-hexane (1:4, v/v) as an eluent to give the 2-ethynylindole 24 (430 mg, 92%) as a red solid. mp was 92–94 °C (EtOAc-hexane). IR (ATR) ν = 1732, 2102 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.29 (s, 3H), 3.68 (s, 1H), 3.70 (s, 3H), 3.90 (s, 2H), 5.59 (s, 2H), 7.19 (t, J = 7.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), and 7.60 (d, J = 7.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ 31.0, 52.1, 56.0, 73.9, 75.1, 86.9, 110.3, 115.8, 119.7, 120.1, 121.1, 124.3, 127.0, 136.7, and 171.4. MS m/z: 257 (M+). HRMS (EI): calcd for C15H15NO3 257.1052; found 257.1046.

3.1.16. Methyl {2-[2-(2-Formylphenyl)ethynyl]-N-(methoxymethyl)indol-3-yl}acetate (26a)

A solution of 2-ethynylindole 24 (81 mg, 0.32 mmol), CuI (2.7 mg, 0.014 mmol), PdCl2(PPh3)2 (6 mg, 0.0086 mmol), and 2-iodobenzaldehyde 25a (66 mg, 0.29 mmol) in Et3N (2.6 mL, 18 mmol) was stirred at 70 °C for 45 min. Afterward, the reaction mixture was filtered through Celite pad, washed with EtOAc, and the filtrate was evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:9 v/v) to give the 2-alkynylbenzaldehyde 26a (83 mg, 80%) as a yellow solid. mp was 75–77 °C (EtOAc-hexane). IR (ATR) ν = 1689, 1736, and 2202 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.35 (s, 3H), 3.71 (s, 3H), 3.98 (s, 2H), 5.70 (s, 2H), 7.22 (t, J = 7.8 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.61–7.66 (m, 2H), 7.71 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), and 10.59 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 31.3, 52.2, 56.2, 75.3, 86.1, 95.1, 110.3, 116.3, 119.8, 120.5, 121.2, 124.6, 125.2, 127.3, 128.6, 129.0, 133.3, 133.8, 135.6, 137.2, 171.3, and 191.1. MS m/z: 361 (M+). HRMS (EI): calcd for C22H19NO4 361.1314; found 361.1322.

3.1.17. Methyl {2-[2-(2-Formyl-3-[(methoxymethoxy)methyl]phenyl)ethynyl]-N-(methoxymethylindol-3-yl}acetate (26b)

A solution of 2-ethynylindole 24 (266 mg, 1.04 mmol), CuI (20 mg, 0.10 mmol), PdCl2(PPh3)2 (36 mg, 0.052 mmol), and 2-iodobenzaldehyde 25b (377 mg, 1.25 mmol) in iPr2NH (8 mL) was stirred at 100 °C for 3 h. Afterward, the reaction mixture was filtered through Celite pad, washed with EtOAc, and the filtrate was evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:9 v/v) to give the 2-alkynylbenzaldehyde 26b (410 mg, 91%) as a yellow solid. mp was 76–78 °C (EtOAc-hexane). IR (ATR) ν = 1693, 1724, 2198 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.34 (s, 3H), 3.42 (s, 3H), 3.71 (s, 3H), 4.00 (s, 2H), 4.80 (s, 2H), 4.98 (s, 2H), 5.71 (s, 2H), 7.21 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H), and 10.64 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 31.3, 52.2, 55.5, 56.0, 66.8, 75.3, 91.3, 92.2, 96.1, 110.3, 116.6, 119.8, 120.3, 121.2, 123.5, 124.7, 127.3, 127.7, 128.7, 132.9, 135.9, 137.4, 141.2, 171.2, and 191.2. MS m/z: 435 (M+). HRMS (EI): calcd for C25H25NO6 435.1682; found 435.1696.

3.1.18. Methyl {2-[22-Hydroxyiminophenyl)ethynyl]-N-(methoxymethyl)indol-3-yl}acetate (27a)

A mixture of benzaldehyde 26a (184 mg, 0.51 mmol), NH2OH·HCl (70 mg, 1.02 mmol), and AcONa (83 mg, 1.02 mmol) in EtOH (4 mL) was stirred at rt for 1.5 h. After removal of solvent, the residue was diluted with H2O and then filtered off to give the oxime 27a (170 mg, 88%) as a yellow solid. mp was 110–113 °C (EtOAc-hexane). IR (ATR) ν = 1701, 1732, 2218, and 3062 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.36 (s, 3H), 3.73 (s, 3H), 4.03 (s, 2H), 5.68 (s, 2H), 7.22 (t, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.38–7.40 (m, 2H), 7.49 (d, J = 7.8 Hz, 1H), 7.60–7.62 (m, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.70–7.72 (m, 1H), 8.46 (br s, 1H), and 8.59 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 31.5, 52.4, 56.2, 75.3, 84.4, 97.0, 110.2, 115.3, 119.8, 121.1, 121.2, 121.2, 124.3, 127.2, 127.4, 128.9, 129.3, 132.8, 133.0, 137.1, 148.8, and 172.3. MS m/z: 376 (M+). HRMS (EI): calcd for C22H20N2O4 376.1423; found 376.1427.

3.1.19. Methyl {2-[2-(2-Hydroxyimino-3-[(methoxymethoxy)methyl]phenyl)ethynyl]-N-methoxymethylindol-3-yl}acetate (27b)

The same procedure as above was carried out with 2-arylethynylbenzaldehyde 26b (450 mg, 1.03 mmol) to give the oxime 27b (414 mg, 89%) as a white solid. Mp was 94–95 °C (EtOAc-hexane). IR (ATR) ν = 1736, 2164, and 3302 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.35 (s, 3H), 3.42 (s, 3H), 3.71 (s, 3H), 4.03 (s, 2H), 4.78 (s, 2H), 4.92 (s, 2H), 5.68 (s, 2H), 7.21 (t, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.66–7.70 (m, 2H), 8.45 (s, 1H), and 8.68 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 31.4, 52.3, 55.4, 56.0, 67.4, 75.3, 89.8, 93.9, 95.9, 110.2, 115.6, 119.9 (2C), 121.0, 121.1, 124.4, 126.0, 127.4, 128.7, 128.7, 133.5, 137.3, 140.6, 149.0, and 172.0. MS m/z: 450 (M+). HRMS (EI): calcd for C25H26N2O6 450.1791; found 450.1799.

3.1.20. 3-[3-(2-Methoxy-2-oxoethyl)-1-(methoxymethyl)indol-2-yl]isoquinoline N-oxide (28a)

A solution of oxime 27a (45 mg, 0.12 mmol) in 1,2-dichlorobenzene (3 mL) was stirred at 120 °C for 17 h. After removal of the solvent, the residue was purified by column chromatography (EtOAc/hexane 1:1 v/v) to give the N-oxide 28a (37 mg, 82%) as a brown oil. IR (ATR) ν = 1315, 1736 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.09 (s, 3H), 3.55 (d, J = 15.6 Hz, 1H), 3.69 (s, 3H), 3.83 (d, J = 15.6 Hz, 1H), 5.35 (d, J = 11.4 Hz, 1H), 5.67 (d, J = 11.4 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.62–7.71 (m, 3H), 7.79 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 8.17 (s, 1H), and 8.94 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 30.9, 52.1, 56.0, 76.4, 110.4, 111.1, 119.7, 120.5, 123.8, 124.6, 127.2, 127.6, 128.5, 129.0, 129.2, 129.7, 129.8, 129.9, 137.0, 137.4, 138.2, and 172.1. MS m/z: 376 (M+). HRMS (EI): calcd for C22H20N2O4 376.1423; found 376.1441.

3.1.21. 3-[3-(2-Methoxy-2-oxoethyl)-1-(methoxymethyl)indol-2-yl)-5-[(methoxymethoxy)methyl]isoquinoline N-oxide (28b)

The same procedure as above was carried out with the oxime 27b (30 mg, 0.067 mmol) to give the N-oxide 28b (21 mg, 69%) as a brown oil. IR (ATR) ν = 1323, 1731 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.10 (s, 3H), 3.36 (s, 3H), 3.54 (d, J = 15.6 Hz, 1H), 3.70 (s, 3H), 3.84 (d, J = 15.6 Hz, 1H), 4.73 (s, 2H), 4.94 (d, J = 12.3 Hz, 1H), 5.05 (d, J = 12.3 Hz, 1H), 5.37 (d, J = 11.0 Hz, 1H), 5.67 (d, J = 11.0 Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.62–7.68 (m, 2H), 7.71 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 8.43 (s, 1H) and 8.94 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 31.1, 52.1, 55.6, 56.0, 66.3, 95.9, 110.5, 111.2, 119.8 (2C), 120.6, 123.9, 124.9, 126.1, 127.1, 127.7, 129.3, 129.7, 129.9, 130.4, 134.3, 137.2, 137.5, 138.1 and 172.0. MS m/z: 450 (M+). HRMS (EI): calcd for C25H26N2O6 450.1791; found 450.1795.

3.1.22. Methyl 2-[1-(methoxymethyl)-2-(1-oxo-1,2-dihydroisoquinolin-3-yl)indol-3-yl]acetate (29a) and Methyl 2-[2-(4-acetoxyisoquinolin-3-yl)-1-(methoxymethyl)indol-3-yl]acetate (31a)

A solution of N-oxide 28a (15 mg, 0.040 mmol) in Ac2O (1 mL) was heated at 80 °C under MW irradiation for 6 h. After removal of the solvent, the residue was purified by column chromatography (EtOAc/hexane 1:1 v/v) to give the isoquinolone 29a (9 mg, 60%) and the 4-acetoxyisoquinoline 31a (3 mg, 18%).
29a: White solid, mp was 150–151 °C (EtOAc-hexane). IR (ATR) ν = 1624, 1739, and 3649 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.46 (s, 3H), 3.79 (s, 3H), 3.86 (s, 2H), 5.48 (s, 2H), 6.90 (s, 1H), 7.26 (t, J = 7.8 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.51–7.57 (m, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.67–7.73 (m, 2H), 8.47 (d, J = 7.8 Hz, 1H), and 9.98 (br s, 1H). 13C-NMR (100 MHz, CDCl3) δ 30.9, 52.6, 56.5, 74.9, 109.2, 109.9, 110.5, 119.6, 121.3, 124.4, 125.9, 126.8, 127.4 (2C), 127.7, 129.6, 132.3, 132.9, 137.4, 137.6, 162.8, and 172.8. MS m/z: 376 (M+). HRMS (EI): calcd for C22H20N2O4 376.1423; found 376.1431.
31a: Brown oil. IR (ATR) ν = 1732, 1774 cm−1. 1H-NMR (400 MHz, CDCl3) δ 2.16 (s, 3H), 3.16 (s, 3H), 3.63 (s, 3H), 3.64–3.75 (m, 2H), 5.34 (d, J = 11.0 Hz, 1H), 5.56 (d, J = 11.0 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.74 (t, J = 7.8 Hz, 1H), 7.82 (t, J = 7.8 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), and 9.30 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 20.4, 30.7, 51.8, 55.9, 75.5, 109.7, 110.8, 119.6, 120.5, 121.1, 123.1, 127.8, 128.0, 128.5, 129.4, 130.5, 131.4, 132.8, 136.5, 137.7, 142.2, 150.3, 168.5, and 172.0. MS m/z: 418 (M+). HRMS (EI): calcd for C24H22N2O5 418.1529; found 418.1527.

3.1.23. Methyl 2-{2-[5-[(Methoxymethoxy)methyl]-1-oxo-1,2-dihydroisoquinolin-3-yl]-1-(methoxymethyl)indol-3-yl}acetate (29b), 1-Acetoxy-3-[3-methoxycarbonylmethyl-N-(methoxymethyl)indol-2-yl]-5-[(methoxymethoxy)methyl]isoquinoline (30b) and 4-Acetoxy-3-[3-methoxycarbonylmethyl-N-(methoxymethyl)indol-2-yl]-5-[(methoxymethoxy)methyl]isoquinoline (31b)

The same procedure as above was carried out with the oxime 28b (32 mg, 0.071 mmol) to give the isoquinolone 29b (12 mg, 38%), the 1-acetoxyisoquinoline 30b (7 mg, 20%), and the 4-acetoxyisoquinoline 31b (11 mg, 32%).
29b: Orange oil. IR (ATR) ν = 1643, 1736, and 3672 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.39 (s, 3H), 3.49 (s, 3H), 3.79 (s, 3H), 3.87 (s, 2H), 4.73 (s, 2H), 4.89 (s, 2H), 5.49 (s, 2H), 7.17 (s, 1H), 7.26 (t, J = 7.8 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.50–7.54 (m, 2H), 7.69 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 8.47 (d, J = 7.8 Hz, 1H), and 10.11 (br s, 1H). 13C-NMR (100 MHz, CDCl3) δ 30.9, 52.6, 55.5, 56.5, 66.8, 74.9, 95.8, 105.7, 109.9, 110.7, 119.7, 121.3, 124.4, 126.4, 126.9, 127.5, 127.8, 129.8, 132.5, 133.3, 133.4, 136.2, 137.7, 162.8, and 172.7. MS m/z: 450 (M+). HRMS (EI): calcd for C25H26N2O6 450.1791; found 450.1783.
30b: Orange oil. IR (ATR) ν = 1736, 1778 cm−1. 1H-NMR (400 MHz, CDCl3) δ 2.51 (s, 3H), 3.18 (s, 3H), 3.41 (s, 3H), 3.74 (s, 3H), 3.88 (s, 2H), 4.78 (s, 2H), 5.08 (s, 2H), 5.75 (s, 2H), 7.22 (t, J = 7.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), and 8.39 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 21.3, 31.3, 52.2, 55.6, 56.0, 66.6, 75.2, 96.0, 109.8, 110.6, 118.8, 119.7 (2C), 120.8, 123.6, 123.7, 127.9, 128.2, 131.4, 134.3, 136.5, 137.9, 137.9, 141.7, 155.7, 168.9, and 172.5. MS m/z: 492 (M+). HRMS (EI): calcd for C27H28N2O7 492.1897; found 492.1891.
31b: Yellow oil. IR (ATR) ν = 1736, 1774 cm−1. 1H-NMR (400 MHz, CDCl3) δ 1.99 (s, 3H), 3.28 (s, 3H), 3.36 (s, 3H), 3.63 (s, 3H), 3.64 (d, J = 16.5 Hz, 1H), 3.73 (d, J = 16.5 Hz, 1H), 4.61 (d, J = 6.9 Hz, 1H), 4.64 (d, J = 6.9 Hz, 1H), 5.02 (d, J = 13.3 Hz, 1H), 5.21 (d, J = 13.3 Hz, 1H), 5.26 (d, J = 11.0 Hz, 1H), 5.57 (d, J = 11.0 Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H), and 9.28 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 21.0, 30.8, 51.8, 55.4, 56.1, 67.7, 75.9, 94.7, 109.3, 111.0, 119.6 (2C), 120.4, 123.0, 128.0, 128.3, 129.5, 130.7, 132.0, 133.1, 133.2, 137.7, 138.8, 142.3, 150.8, 168.8, and 172.0. MS m/z: 492 (M+). HRMS (EI): calcd for C27H28N2O7 492.1897; found 492.1889.

3.1.24. 3-[3-(2-Hydroxyethyl)-N-(methoxymethyl)indol-2-yl]isoquinolin-1-one (32a)

A solution of the isoquinolone 29a (25 mg, 0.066 mmol) in THF (2 mL) was added dropwise to a suspension of LiAlH4 (4 mg, 0.10 mmol) in THF (2 mL) under ice cooling and then stirred at 70 °C for 30 min. After quenching with H2O, the reaction mixture was filtered through Celite pad and washed with H2O and EtOAc, and the filtrate was extracted with EtOAc. The organic layer was washed with brine, dried with Na2SO4, and evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:1 v/v) to give the alcohol 32a (22 mg, 95%) as a white solid. mp was 211–212 °C (EtOAc-hexane). IR (ATR) ν = 1732, 3351, 3629 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.12 (t, J = 5.5 Hz, 2H), 3.39 (s, 3H), 3.57 (br s, 1H), 4.12 (t, J = 5.5 Hz, 2H), 5.47 (s, 2H), 6.89 (s, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.48–7.54 (m, 2H), 7.61–7.63 (m, 2H), 7.69 (t, J = 7.8 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H), and 11.36 (br s, 1H). 13C-NMR (100 MHz, CDCl3) δ 27.1, 56.2, 61.7, 74.9, 108.4, 110.4, 114.4, 119.2, 120.9, 124.0, 125.5, 126.7, 127.0, 127.4, 127.5, 130.4, 132.5, 132.7, 137.7, 138.3, and 163.3. MS m/z: 348 (M+). HRMS (EI): calcd for C21H20N2O3 348.1474; found 348.1468.

3.1.25. 3-[3-(2-Hydroxyethyl)-N-(methoxymethyl)indol-2-yl]-5-[(methoxymethoxy)methyl]isoquinolin-1-one (32b)

A solution of N-oxide 28b (25 mg, 0.056 mmol) in Ac2O (1 mL) was heated at 80 °C under MW irradiation for 4.5 h. After removal of the solvent, the residue was purified by column chromatography (EtOAc/hexane 1:3 v/v) to give a mixture of isoquinolone 29b and the 1-acetoxyisoquinoline 30b. A solution of the mixture in THF (1 mL) was added dropwise to a suspension of LiAlH4 (4 mg, 0.11 mmol) in THF (1 mL) under ice cooling and then stirred at 0 °C for 1 h. After quenching with H2O, the reaction mixture was filtered through Celite pad and washed with H2O and EtOAc, and the filtrate was extracted with EtOAc. The organic layer was washed with brine, dried with Na2SO4, and evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:1 v/v) to give the alcohol 32b (16 mg, 69%) as a white solid. mp was 175–176 °C (EtOAc-hexane). IR (ATR) ν = 1639, 3309, and 3529 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.11 (t, J = 5.5 Hz, 2H), 3.39 (s, 3H), 3.42 (s, 3H), 4.12 (t, J = 5.5 Hz, 2H), 4.73 (s, 2H), 4.87 (s, 2H), 5.46 (s, 2H), 7.16 (s, 1H), 7.23 (t, J = 7.8 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 8.35 (d, J = 7.8 Hz, 1H), and 11.63 (br s, 1H). 13C-NMR (100 MHz, CDCl3) δ 27.1, 55.5, 56.2, 61.7, 67.0, 75.0, 95.8, 104.9, 110.3, 114.8, 119.3, 120.9, 124.0, 126.0, 126.4, 127.5, 127.7, 130.7, 132.6, 132.9, 133.3, 136.5, and 138.4, 163.4. MS m/z: 422 (M+). HRMS (EI): calcd for C24H26N2O5 422.1842; found 422.1844.

3.1.26. N-Methoxymethyl-3,14,15,16,17,18,19,20-octadehydroyohimban-21-one (33a)

To a solution of alcohol 32a (21 mg, 0.060 mmol) and PPh3 (47 mg, 0.18 mmol) in THF (3 mL), DIAD (1.9 M in toluene, 0.94 mL, 0.18 mmol) was added dropwise under ice cooling and was stirred at 60 °C for 3 h. After cooling at ambient temperature, the resulting mixture was quenched with water and extracted with EtOAc. The organic layer was washed with water and brine, dried with Na2SO4, and evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:3 v/v) to give the indoloquinolizine 33a (19 mg, 95%) as a white solid. mp was 163–166 °C (EtOAc-hexane). IR (ATR) ν = 1732 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.10 (t, J = 6.2 Hz, 2H), 3.59 (s, 3H), 4.50 (t, J = 6.2 Hz, 2H), 5.62 (s, 2H), 7.24 (t, J = 7.8 Hz, 1H), 7.31 (s, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.46 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.59–7.66 (m, 3H), and 8.45 (d, J = 7.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ 20.1, 40.4, 56.1, 75.1, 103.4, 109.9, 116.5, 119.4, 121.1, 124.5, 125.2, 125.4, 126.6, 128.0 (2C), 130.7, 131.1, 132.3, 136.4, 140.6, and 162.5. MS m/z: 330 (M+). HRMS (EI): calcd for C21H18N2O2 330.1368; found 330.1354.

3.1.27. N-Methoxymethyl-16-[(methoxymethoxy)methyl]-3,14,15,16,17,18,19,20-octadehydroyohimban-21-one (33b)

The same procedure as above was carried out with alcohol 32b (35 mg, 0.083 mmol) to give the indoloquinolizine 33b (30 mg, 90%) as a yellow solid. mp was 128–129 °C (EtOAc-hexane). IR (ATR) ν = 1736 cm−1. 1H-NMR (400 MHz, CDCl3) δ 3.10 (t, J = 6.4 Hz, 2H), 3.43 (s, 3H), 3.60 (s, 3H), 4.49 (t, J = 6.4 Hz, 2H), 4.79 (s, 2H), 4.90 (s, 2H), 5.63 (s, 2H), 7.24 (t, J = 7.8 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.58 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), and 8.46 (d, J = 7.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ 20.1, 40.4, 55.4, 56.1, 66.9, 75.3, 95.9, 99.9, 110.0, 116.6, 119.4, 121.2, 124.6, 125.4, 125.7, 126.1, 128.3, 130.9, 131.3, 132.8, 132.9, 135.2, 140.7, and 162.5. MS m/z: 404 (M+). HRMS (EI): calcd for C24H24N2O4 404.1736; found 404.1744.

3.1.28. Norketoyobyrine (5)

To a suspension of indoloquinolizine 33a (23 mg, 0.070 mmol) and ethylene glycol (0.78 mg, 0.014 mmol) in THF (5 mL), 6 M HCl (3 mL) was added dropwise under ice cooling and then stirred at 60 °C for 15 h. After cooling to ambient temperature, the solvent was evaporated in vacuo. The residue was diluted with water and then was alkalified with 1 M aqueous NaOH. The resulting mixture was extracted with EtOAc. The organic layer was washed with water and brine, dried with Na2SO4, and evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:1 v/v) to give norketoyobyrine (5; 18 mg, 90%) as a yellow solid. mp was 298–299 °C (EtOAc-hexane lit. [16] mp of 299–300 °C). IR (ATR) ν = 1732 cm−1. 1H-NMR (400 MHz, DMSO-d6) δ 3.08 (t, J = 6.6 Hz, 2H), 4.39 (t, J = 6.6 Hz, 2H), 7.06 (t, J = 7.8 Hz, 1H), 7.06 (s, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.55–7.63 (m, 2H), 7.70 (t, J = 7.8 Hz, 1H), 8.23 (d, J = 7.8 Hz, 1H), and 11.69 (br s, 1H). 13C-NMR (100 MHz, DMSO-d6) δ 19.3, 40.4, 99.1, 111.7, 112.5, 119.1, 119.5, 123.5, 124.5, 125.6, 126.0, 126.2, 127.5, 128.2, 132.4, 132.7, 136.2, 138.0, and 161.3. MS m/z: 286 (M+). HRMS (EI): calcd for C19H14N2O 286.1106; found 286.1114.

3.1.29. 16-Hydroxymethyl-3,14,15,16,17,18,19,20-octadehydroyohimban-21-one (34)

The same procedure as above was carried out with alcohol 33b (40 mg, 0.099 mmol) to give alcohol 34 (28 mg, 90%) as a white solid. Mp was 283–284 °C (EtOAc-hexane lit. [30] mp of 280–283 °C). IR (ATR) ν = 1747, 3317 cm−1. 1H-NMR (400 MHz, DMSO-d6) δ 3.08 (t, J = 6.6 Hz, 2H), 4.39 (t, J = 6.6 Hz, 2H), 4.87 (d, J = 5.5 Hz, 2H), 5.40 (t, J = 5.5 Hz, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.19–7.23 (m, 2H), 7.40–7.45 (m, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), and 11.72 (br s, 1H). 13C-NMR (100 MHz, DMSO-d6) δ 19.3, 40.4, 60.4, 95.6, 111.6, 112.5, 119.2, 119.5, 123.6, 124.7, 125.6 (2C), 126.2, 128.4, 130.2, 132.0, 133.6, 137.3, 138.0, and 161.4. MS m/z: 316 (M+). HRMS (EI): calcd for C20H16N2O2 316.1212; found 316.1208.

3.1.30. Naucleficine (7)

A suspension of alcohol 34 (30 mg, 0.095 mmol) and active MnO2 (165 mg, 1.9 mmol) in CH2Cl2 (2 mL) was stirred at reflux for 1 h. The reaction mixture was filtered through a Celite pad. The filtrate was evaporated in vacuo. The residue was purified by column chromatography (EtOAc/hexane 1:1, v/v) to give naucleficine (7; 21 mg, 70%) as an orange solid. mp was 276–277 °C (CHCl3 lit. [30] mp of 284–290 °C). IR (ATR) ν = 1592, 1643, and 3221 cm−1. 1H-NMR (400 MHz, DMSO-d6) δ 3.11 (t, J = 6.6 Hz, 2H), 4.41 (t, J = 6.6 Hz, 2H), 7.08 (t, J = 7.8 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.59–7.64 (m, 2H), 8.12 (s, 1H), 8.25 (d, J = 7.8 Hz, 1H), 8.53 (d, J = 7.8 Hz, 1H), 10.42 (s, 1H), 11.82 (br s, 1H). 13C-NMR (100 MHz, DMSO-d6) δ 19.2, 40.5, 94.9, 112.0, 114.1, 119.4, 119.7, 124.1, 125.4, 125.5 (2C), 127.9, 129.6, 133.8, 135.1, 135.6, 138.5, 138.6, 160.8, and 192.8. MS m/z: 314 (M+). HRMS (EI): calcd for C20H14N2O2 314.1055; found 314.1066.

3.2. Biochemistry

3.2.1. Cell Lines and Cell Cultures

For testing the antiproliferative cell activities, two types of cancer cell lines were used in this study: HCT-116 cells (human colon cancer) and HepG2 cells (human liver cancer), which were purchased from the American Type Culture Collection (Manassas, VA, USA) and the Japanese Collection of Research Bioresources (Osaka, Japan). The HCT-116 and HepG2 cells were maintained in a McCOY’s 5A medium (Sigma-Aldrich, MO, USA) with L-glutamine and 10% heat inactivated (55 °C for 30 min) fetal bovine serum (FBS) and in a Dulbecco’s Modified Eagle’s medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) with L-glutamine and 10% heat-inactivated FBS, respectively, at 37 °C in an atmosphere of 5% CO2.

3.2.2. Cell Viability Assays

The HCT-116 cells’ viability assay was conducted using the MTT method based on the procedure described by Mosmann [36]. Briefly, cells were placed in 96-well flat-bottomed tissue culture plates with 2.0 × 103 cells per well in a 100 μL culture medium. This was followed by incubation at 37 °C in an atmosphere of 5% CO2 for 24 h to allow the cells to attach to the wells. The cells were treated with the indicated concentrations of test agents in a culture medium without FBS. Following a further 72 h incubation, 10 μL of MTT (5 mg/mL in phosphate-buffered saline) was added per well, and the plate was incubated for 4 h to allow the MTT to metabolize by cellular mitochondrial dehydrogenases. The excess MTT was aspirated, and the produced formazan crystals were dissolved by adding 100 μL of dimethyl sulfoxide. The absorbance of the purple formazan was read at 570 nm using a microplate reader. The results following the test agents’ exposure were calculated as a percentage relative to untreated controls. The HepG2 cells’ viability assay was conducted using the WST-8 method based on the procedure described by Tominaga [37]. The cells were seeded in 96-well flat-bottomed tissue culture plates with 1.0 × 104 cells per well in 100 µL of the FBS-containing culture medium with the indicated concentrations of test agents. Following a further 72 h incubation, 10 µL of a mixture of WST-8 solution was added per well, and the plate was incubated for 2 h to allow the WST-8/1-methoxy PMS to metabolize by cellular mitochondrial dehydrogenases. The absorbance of the orange formazan was read at 450 nm using a microplate reader. The results following the test agents’ exposure were calculated as a percentage relative to untreated controls.

3.2.3. Statistical Calculation

The concentration cells’ viability curves were fitted to a four-parametric logistic equation using a nonlinear curve-fitting program that derived the IC50 values (Kaleida-graph ver. 3.6; Synergy Software, PA, USA). Wherever appropriate, the results were expressed as means ± sem, with n ¼ 3 or higher in at least one out of three similar experiments.

4. Conclusions

We developed a versatile synthetic methodology for introducing various substituents into the E-ring of the pentacyclic aromathecin scaffold, which allowed us to achieve the total synthesis of 22-hydroxyacuminatine (4) as a model compound of the aromathecin family.
The pentacyclic scaffold was obtained by constructing the indolizine moiety from isoquinolone 17 by C–N bond formation via the Mitsunobu reaction. Isoquinolone 17 was synthesized via the thermal cyclization of 2-alkynylbenzaldehyde oxime to obtain N-oxide 16, which was then subjected to the Reissert–Henze-type reaction. Consequently, 4 was obtained in 10.5% overall yield via an eight-step sequence. Using this methodology, we achieved the total synthesis of the indoloquinolizidine-type alkaloids norketoyobyrine (5: 10 steps, total yield 17.2%) and naucleficine (7: 11 steps, total yield 13.3%). Further, we assessed the antiproliferative activity of the synthesized 5, 7, and intermediate 34 against HCT-116 human colon cancer cells and HepG2 human liver cancer cells, concluding that 7 and 34 exhibited moderate antiproliferative activity against HCT-116 cells with IC50 values of 55.58 and 41.40 μM, respectively. Future research will focus on the synthesis of diverse pentacyclic compounds with promising therapeutic profiles by adopting this strategy.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules30122650/s1, copies of the 1H NMR and 13C NMR spectra for compounds 14–34, 22-hydroxyacuminatine (4), norketoyobyrine (5), and naucleficine (7).

Author Contributions

Conceptualization, T.C. and T.N. (Takashi Nishiyama); formal analysis, S.M., H.B., Y.H. and T.N. (Takashi Nishiyama); investigation, S.M., H.B., T.O., N.H. (Nanako Hayashi), Y.H. and T.N. (Takashi Nishiyama); resources, T.M., T.N. (Tetsuya Nakamura) and N.H. (Noriyuki Hatae); writing—original draft preparation, T.C. and T.N. (Takashi Nishiyama); writing—review and editing, T.C., T.N. (Takashi Nishiyama), T.N., T.M., T.H. and N.H. (Noriyuki Hatae). All authors have read and agreed to the published version of the manuscript.

Funding

This work was partly supported by Fukuyama University Grant for Academic Research Projects (Grant Number: GARP2022-223 (S.M.), GARP2024-224 (H.B.)).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
TBStert-butyldimethylsilyl
Acacetyl
1,2-DCB1,2-dichlorobenzene
MWmicrowave
TLCthin-layer chromatography
DIADdiisopropyl azodicarboxylate
MOMmethoxymethyl
TBAFtetrabutylammonium fluoride

References

  1. Wall, M.E.; Wani, M.C.; Cook, C.E.; Palmer, K.H.; McPhail, A.T.; Sim, G.A. Plant Antitumor Agents. I. The Isolation and Structure of Camptothecin, a Novel Alkaloidal Leukemia and Tumor Inhibitor from Camptotheca acuminata. J. Am. Chem. Soc. 1966, 88, 3888–8890. [Google Scholar] [CrossRef]
  2. Shamma, M.; Novak, L. Synthetic approaches to camptothecin. Tetrahedron 1969, 25, 2275–2279. [Google Scholar] [CrossRef]
  3. Lin, L.Z.; Cordell, G.A. Quinoline Alkaloids from Camptotheca acuminata. Phytochemistry 1989, 28, 1295–1297. [Google Scholar] [CrossRef]
  4. Tang, X.; Jiang, Y.; Song, L.; Van der Eycken, E.V. Recent Advances in the Synthesis of Rosettacin. Molecules 2024, 29, 2176. [Google Scholar] [CrossRef]
  5. Nishiyama, T.; Mizuno, S.; Hieda, Y.; Choshi, T. Progress on the Synthesis of the Aromathecin Family of Compounds: An Overview. Molecules 2024, 29, 2380. [Google Scholar] [CrossRef] [PubMed]
  6. Ma, Z.; Lee, D.Y.W. Total synthesis of the cytotoxic alkaloid 22-hydroxyacuminatine. Tetrahedron Lett. 2004, 45, 6721–6723. [Google Scholar] [CrossRef]
  7. Babjak, M.; Kanazawa, A.; Anderson, R.J.; Greene, A.E. Concise synthesis of 22-hydroxyacuminatine, cytotoxic camptothecinoid from Camptotheca acuminata, by pyridone benzannulation. Org. Biomol. Chem. 2006, 4, 407–409. [Google Scholar] [CrossRef]
  8. Xiao, X.; Antony, S.; Pommier, Y.; Cushman, M. Total Synthesis and Biological Evaluation of 22-Hydroxyacuminatine. J. Med. Chem. 2006, 49, 1408–1412. [Google Scholar] [CrossRef]
  9. Zhou, H.-B.; Liu, G.-S.; Yao, Z.-J. Short and Efficient Total Synthesis of Luotonin A and 22-Hydroxyacuminatine Using A Common Cascade Strategy. J. Org. Chem. 2007, 72, 6270–6272. [Google Scholar] [CrossRef]
  10. Grillet, F.; Baumlová, B.; Prévost, G.; Constant, J.-F.; Chaumeron, S.; Bigg, D.C.H.; Greene, A.E.; Kanazawa, A. Synthesis and bioevaluation of 22-hydroxyacuminatine analogs. Bioorg. Med. Chem. Lett. 2008, 18, 2143–2146. [Google Scholar] [CrossRef]
  11. Li, K.; Ou, J.; Gao, S. Total Synthesis of Camptothecin and Related Natural Products by a Flexible Strategy. Angew. Chem. Int. Ed. 2016, 55, 14778–14783. [Google Scholar] [CrossRef] [PubMed]
  12. Boucherle, B.; Haudecoeur, R.; Queiroz, E.F.; De Waard, M.; Wolfender, J.-L.; Robins, R.J.; Boumendjel, A. Nauclea latifolia: Biological activity and alkaloid phytochemistry of a West African tree. Nat. Prod. Rep. 2016, 33, 1034–1043. [Google Scholar] [CrossRef] [PubMed]
  13. Swan, G.A. The Constitution of Yohimbine and Related Alkaloids. Part III. The Structure of Sempervirine and Some Further Observations on the Structure of Ketoyobyrine. J. Chem. Soc. 1949, 1720–1724. [Google Scholar] [CrossRef]
  14. Ninomiya, I.; Takasugi, H.; Naito, T. Total Synthesis of Yohimbine-type Alkaloids. The Yohimbine Skeleton and Angustidine. J. Chem. Soc. Chem. Commun. 1973, 732. [Google Scholar] [CrossRef]
  15. Ninomiya, I.; Naito, T.; Takasugi, H. Photocyclization of enamides. Part X. Total syntheses of the yohimbine skeleton: Demethoxyc arbonyldihydrogambirtannine, angustidine, nauclétine, (±)-angustoline, and related compounds. J. Chem. Soc. Perkin Trans. 1 1976, 1865–1868. [Google Scholar]
  16. Grigg, R.; Sridharan, S.; Stevenson, P.; Worakun, T. Palladium(II) Catalyzed Construction of Tetrasubstituted Carbon Centres, and Spiro- and Bridged-ring Compounds from Enamides of 2-lodobenzoic Acids. J. Chem. Soc. Chem. Commun. 1986, 1697–1699. [Google Scholar] [CrossRef]
  17. Grigg, R.; Sridharan, S.; Stevenson, P.; Sukirthalingama, S.; Worakumb, T. The Synthesis of Fused Ring Nitrogen Heterocycles via Reriospecific Intramolecular Heck Reactions. Tetrahedron 1990, 46, 4003–4018. [Google Scholar] [CrossRef]
  18. Yamaguchi, R.; Otsuji, A.; Utimoto, K.; Kozima, S. 1,2-Addition of allyl and 2-oxo-2H-pyran-6-ylcarbonyl groups to cyclic carbon-nitrogen double bonds by means of organotin reagent for alkaloids synthesis; a facile synthesis of 8-oxoprotoberberine and norketoyobirine (demethoxycarbonyloxogambirtannine). Bull. Chem. Soc. Jpn. 1992, 65, 298–300. [Google Scholar] [CrossRef]
  19. Sánchez-Sancho, F.; Mann, E.; Herradón, B. Efficient Syntheses of Polyannular Heterocycles Featuring Microwave Accelerated Bischler-Napieralski Reaction, Stereoselective Heck Cyclization, and Claisen Rearrangement. Synlett 2000, 2000, 509–513. [Google Scholar] [CrossRef]
  20. Knölker, H.-J.; Cämmerer, S. Transition metal complexes in organic synthesis. Part 62: Total synthesis of (±)-demethoxycarbonyldihydrogambirtannine and norketoyobyrine by an iron-mediated [2+2+1] cycloaddition. Tetrahedron Lett. 2000, 41, 5035–5038. [Google Scholar] [CrossRef]
  21. Pan, X.; Bannister, T.D. Sequential Sonagashira and Larock Indole Synthesis Reactions in a General Strategy to Prepare Biologically Active β-Carboline-Containing Alkaloids. Org. Lett. 2014, 16, 6124–6127. [Google Scholar] [CrossRef] [PubMed]
  22. Bai, L.-G.; Zhou, Y.; Zhuang, X.; Zhang, L.; Xue, J.; Lin, X.-L.; Cai, T.; Luo, Q.-L. Base-promoted aerobic oxidation of N-alkyl iminium salts derived from isoquinolines and related heterocycles. Green Chem. 2020, 22, 197–203. [Google Scholar] [CrossRef]
  23. He, G.; List, B.; Christman, M. Unified Synthesis of Polycyclic Alkaloids by Complementary Carbonyl Activation. Angew. Chem. Int. Ed. 2021, 60, 13591–13596. [Google Scholar] [CrossRef] [PubMed]
  24. Li, S.; Nie, H.; Duan, M.; Wang, W.; Zhu, C.; Song, C. Construction of a Protoberberine Alkaloid Skeleton via the Palladium-Catalyzed α-Arylation−Amide Formation Cascade. Org. Lett. 2021, 23, 9631–9634. [Google Scholar] [CrossRef]
  25. Yan, X.; Zhenga, J.; Li, W.-D.Z. Concise total syntheses of berberine and its analogues enabled by trifluoroacetic anhydride-promoted decarbonylative-elimination reaction. Tetrahedron Lett. 2023, 132, 154826. [Google Scholar] [CrossRef]
  26. Woodward, R.B.; Witkop, B. The Structure of Ketoyobyrine. J. Am. Chem. Soc. 1948, 70, 2409–2412. [Google Scholar] [CrossRef]
  27. Levin, R.H.; Wesner, M.M.; Meinzer, E.M. The Synthesis of Ketoyobyrine. J. Am. Chem. Soc. 1948, 70, 2834–2835. [Google Scholar] [CrossRef]
  28. Mao, L.; Xin, L.; Dequan, Y. Alkaloids of Nauclea oficinalis. Planta Medica 1984, 459–461. [Google Scholar] [CrossRef] [PubMed]
  29. Naito, T.; Doi, E.; Miyata, O.; Ninomiya, I. Total synthesis of Nauclea alkaloid naucleficine. Heterocycles 1986, 24, 903–906. [Google Scholar]
  30. Naito, T.; Kuroda, E.; Miyata, O.; Ninomiya, I. Photocyclization of enamides. XXXIV. A practical total synthesis of aromatic yohimboid alkaloids, oxogambi rtannine and naucleficine. Chem. Pharm. Bull. 1991, 39, 2216–2218. [Google Scholar] [CrossRef]
  31. Kanekiyo, N.; Kuwada, T.; Choshi, T.; Nobuhiro, J.; Hibino, S. Total syntheses of β-carboline alkaloids, (R)-(−)-pyridindolol K1, (R)-(−)-pyridindolol K2, and (R)-(−)-pyridindolol. J. Org. Chem. 2001, 66, 8793–8798. [Google Scholar] [CrossRef] [PubMed]
  32. Kanekiyo, N.; Choshi, T.; Kuwada, T.; Sugino, E.; Hibino, S. The first total synthesis of (R)-(−)-pyridindolol K2 and its enantiomer. Heterocycles 2000, 53, 1877–1880. [Google Scholar]
  33. Nishiyama, T.; Hironaka, M.; Taketomi, M.; Taguchi, R.; Kotouge, R.; Shigemori, Y.; Hatae, N.; Ishikura, M.; Choshi, T. Total Synthesis of Two 8-Oxoprotoberberine Alkaloids: Alangiumkaloids A and B. Eur. J. Org. Chem. 2018, 2018, 673–678. [Google Scholar] [CrossRef]
  34. Mizuno, S.; Nishiyama, T.; Endo, M.; Sakoguchi, K.; Yoshiura, T.; Bessho, H.; Motoyashiki, T.; Hatae, N.; Choshi, T. Novel Approach to the Construction of Fused Indolizine Scaffolds: Synthesis of Rosettacin and the Aromathecin Family of Compounds. Molecules 2023, 28, 4059. [Google Scholar] [CrossRef]
  35. Soto, S.; Vaz, E.; Dell’Aversana, C.; Álvarez, R.; Altucci, R.; de Lera, Á.R. New synthetic approach to paullones and characterization of their SIRT1 inhibitory activity. Org. Biomol. Chem. 2012, 10, 2101–2112. [Google Scholar] [CrossRef]
  36. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
  37. Tominaga, H.; Ishiyama, M.; Ohseto, F.; Sasamoto, K.; Hamamoto, T.; Suzuki, K.; Watanabe, M. A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal. Commun. 1999, 36, 47–50. [Google Scholar] [CrossRef]
Molecules 30 02650 g001
Figure 1. Structures of natural products containing the indolizinone or quinolizinone scaffold.
Figure 1. Structures of natural products containing the indolizinone or quinolizinone scaffold.
Molecules 30 02650 g001
Molecules 30 02650 sch001
Scheme 1. Synthesis of acuminatine by constructing a C-ring.
Scheme 1. Synthesis of acuminatine by constructing a C-ring.
Molecules 30 02650 sch001
Molecules 30 02650 sch002
Scheme 2. Retrosynthetic analysis of indolizine-type and quinolizine-type pentacyclic scaffolds.
Scheme 2. Retrosynthetic analysis of indolizine-type and quinolizine-type pentacyclic scaffolds.
Molecules 30 02650 sch002
Molecules 30 02650 sch003
Scheme 3. Synthesis of isoquinoline N-oxides 16a–c.
Scheme 3. Synthesis of isoquinoline N-oxides 16a–c.
Molecules 30 02650 sch003
Molecules 30 02650 sch004
Scheme 4. Synthesis of 22-hydroxyacuminatine (4).
Scheme 4. Synthesis of 22-hydroxyacuminatine (4).
Molecules 30 02650 sch004
Molecules 30 02650 sch005
Scheme 5. Synthesis of isoquinolones 29a and 29b.
Scheme 5. Synthesis of isoquinolones 29a and 29b.
Molecules 30 02650 sch005
Molecules 30 02650 sch006
Scheme 6. Synthesis of indoloquinolizine-type alkaloids 5 and 7.
Scheme 6. Synthesis of indoloquinolizine-type alkaloids 5 and 7.
Molecules 30 02650 sch006
Molecules 30 02650 g002
Figure 2. Antiproliferative activities of indoloquinolizine-type natural products 5 and 7 and intermediate 34 against (A) HCT-116 and (B) HepG2 cells. The open and filled columns correspond to the treatment with compound concentrations of 10 and 100 μM, respectively.
Figure 2. Antiproliferative activities of indoloquinolizine-type natural products 5 and 7 and intermediate 34 against (A) HCT-116 and (B) HepG2 cells. The open and filled columns correspond to the treatment with compound concentrations of 10 and 100 μM, respectively.
Molecules 30 02650 g002
Table 1. Investigation of optimal isoquinoline N-oxides for synthesis of isoquinolones by Reissert–Henze-type reaction.
Table 1. Investigation of optimal isoquinoline N-oxides for synthesis of isoquinolones by Reissert–Henze-type reaction.
Molecules 30 02650 i001
EntryRCompd. No.Time (h) (1)MWYield (%)
Isoquinolone 17 (2)4-OH-isoquinoline 18 (3)
1Ha12−trace (4)trace (4)
2Ha5+trace (4)trace (4)
3TBSb24−20−
4TBSb2+36−
5Acc6−2027
6Acc5+5037
(1) Reissert–Henze-type reaction is carried out while monitoring the disappearance of N-oxides 16 by TLC. (2) Isoquinolones 17 are identified by observing the chemical shift in their 4-H at approximately 7.43 ppm as a singlet peak in 1H-NMR measurement. (3) 4-OH-isoquinolines 18 are identified by observing the chemical shift in their 1-H at approximately 9.36 ppm as a singlet peak in 1H-NMR measurement. (4) 17a and 18a are not obtained, and only small amounts of 17c and 18c, which are acetylated hydroxymethyl groups of 17a and 18a, are isolated.
Table 2. A summary on total synthesis of 22-hydroxyacuminatine (4), norketoyobyrine (5), and naucleficine (7).
Table 2. A summary on total synthesis of 22-hydroxyacuminatine (4), norketoyobyrine (5), and naucleficine (7).
Natural ProductYearAuthorsStepsTotal Yield (%)Reference
22-hydroxyacuminatine Our group810.5
2004Lee714.9[6]
2006, 2008Kanazawa721.9[7,10]
2006Cushman97.4[8]
2007Yao921.1[9]
2016Gao109.3[11]
norketoyobyrine Our group1017.2
1949Swan512.6[13]
1973, 1976Ninomiya231.0[14,15]
1986, 1990Grigg219.2[16,17]
1992Yamaguchi354.1[18]
2000Sánchez-Sancho538.1[19]
2000Knölker825.5[20]
2014Bannister445.1[21]
2016Gao1229.9[11]
2020Cai and Luo446.5[22]
2021Christman25.7[23]
2021Zhu and Song255.2[24]
naucleficine Our group1113.3
1986, 1991Naito523.0[29,30]
2016Gao1020.2[11]
Table 3. IC50 values of synthetic products on antiproliferative activities against HCT-116 and HepG2 cells.
Table 3. IC50 values of synthetic products on antiproliferative activities against HCT-116 and HepG2 cells.
Compd. No.IC50 Value (μM)
HCT-116 CellsHepG2 Cells
5>100>100
755.58 ± 3.79>100
3441.40 ± 1.28>100
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Mizuno, S.; Nishiyama, T.; Bessho, H.; Nakamura, T.; Oe, T.; Hayashi, N.; Hieda, Y.; Motoyashiki, T.; Hata, T.; Hatae, N.; et al. Total Synthesis and Biological Evaluation of 22-Hydroxyacuminatine and the Related Natural Products Norketoyobyrine and Naucleficine. Molecules 2025, 30, 2650. https://doi.org/10.3390/molecules30122650

AMA Style

Mizuno S, Nishiyama T, Bessho H, Nakamura T, Oe T, Hayashi N, Hieda Y, Motoyashiki T, Hata T, Hatae N, et al. Total Synthesis and Biological Evaluation of 22-Hydroxyacuminatine and the Related Natural Products Norketoyobyrine and Naucleficine. Molecules. 2025; 30(12):2650. https://doi.org/10.3390/molecules30122650

Chicago/Turabian Style

Mizuno, Shohta, Takashi Nishiyama, Hana Bessho, Tetsuya Nakamura, Tomoki Oe, Nanako Hayashi, Yuhzo Hieda, Toshio Motoyashiki, Toshiyuki Hata, Noriyuki Hatae, and et al. 2025. "Total Synthesis and Biological Evaluation of 22-Hydroxyacuminatine and the Related Natural Products Norketoyobyrine and Naucleficine" Molecules 30, no. 12: 2650. https://doi.org/10.3390/molecules30122650

APA Style

Mizuno, S., Nishiyama, T., Bessho, H., Nakamura, T., Oe, T., Hayashi, N., Hieda, Y., Motoyashiki, T., Hata, T., Hatae, N., & Choshi, T. (2025). Total Synthesis and Biological Evaluation of 22-Hydroxyacuminatine and the Related Natural Products Norketoyobyrine and Naucleficine. Molecules, 30(12), 2650. https://doi.org/10.3390/molecules30122650

Article Metrics

Back to TopTop