Synthesis and Structure–Activity Relationship Correlations of Gnidimacrin Derivatives as Potent HIV-1 Inhibitors and HIV Latency Reversing Agents

Abstract

Currently, due to the HIV latency mechanism, the search continues for effective drugs to combat this issue and provide a cure for AIDS. Gnidimacrin activates latent HIV-1 replication and inhibits HIV-1 infection at picomolar concentrations. This natural diterpene was able to markedly reduce the latent HIV-1 DNA level and the frequency of latently infected cells. Therefore, gnidimacrin is an excellent lead compound, and its anti-HIV potential merits further investigation. Twenty-nine modified gnidimacrin derivatives were synthesized and evaluated in assays for HIV replication and latency activation to establish which molecular structures must be maintained and which can tolerate changes that may be needed for better pharmacological properties. The results indicated that hydroxyl substituents at C-5 and C-20 are essential, while derivatives modified at 3-OH with aromatic esters retain anti-HIV replication and latent activation activities. The half-lives of the potent GM derivatives are over 20 h, which implies that they are stable in the plasm even though they contain ester linkages. The established structure–activity relationship should be useful in the development of gnidimacrin or structurally related compounds as clinical trial candidates.

Graphical Abstract

INTRODUCTION

Natural products (NPs) and NP-derived compounds, such as gnidimacrin (GM, 1),¹ betulinic acid,^2,3 bevirimat,^4,5 bengamide A,⁶ and LAF-389,⁷ present major opportunities for the inhibition and elimination of the human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS).^8,9 Approaches to combat HIV are hindered by its ability to hide in an inactive state inside certain immune cells and avoid drug treatment. Viral rebound occurs if combination antiretroviral treatment is discontinued.^10,11 Thus, latent HIV reservoirs must be eliminated. “Shock-and-kill” strategies involve two steps: first, reactivating the latent virus hidden in immune cells using latency reversing agents and, second, targeting the reactivated cells for clearance by the immune system and cytopathic effects.^9,12,13 GM, a daphnane diterpenoid isolated from plants in the family Thymeleaceae, significantly reduces latently HIV-infected cells at picomolar concentrations.^1,8 Its potent activity is achieved through selective activation of protein kinase C βI and βII (PKC βI and βII).^5,14 Furthermore, GM reduces the frequency of HIV-1 latently infected cells at concentrations that do not cause global T cell activation or stimulate inflammatory cytokine production.¹

GM has a polyhydroxylated 5/7/6 tricyclic skeleton with a macrocyclic bridge connecting the aliphatic orthoester group with the C-1 position of ring A. Two (C-3 and C-18) of the hydroxyl groups are benzoylated. Several natural diterpenoids, such as tiglianes and ingenanes, with similar daphnane-type skeletons also inhibit HIV replication in lymphocytes^5,15,16 and display HIV-1 latency-reversing activity.^16,17 Studies have shown that such diterpenes can form hydrogen bonds through moieties at their C-20, C-4, and C-3 positions with residues Thr-242, Leu-251, and Gly-253, respectively, in the activator binding groove of PKC isoforms.^18,19

In the current study, a structure–activity relationship (SAR) study was performed on the extremely potent GM to determine which structural elements are adaptable for the generation of optimal dichotomous activity. This present study included novel modified GM derivatives with changes at the C-3, C-5, C-20, and C-2′ hydroxyl groups (Figure 1). All synthesized GM analogs were evaluated with a multicycle viral replication assay in MT4 cells. A selected compound was also evaluated together with GM in a mechanistic study to assure that GM and its modified derivatives have the same mechanism of action.

Figure 1.

Strategy for modification of anti-HIV lead gnidimacrin.

RESULTS

Design and Synthesis of GM Derivatives.

New derivatives were synthesized according to the procedures in Schemes 1-4. Compound 1 was acylated separately with acetic anhydride and benzoyl chloride in anhydrous pyridine to obtain the 5,20,2′-triacetate 2 and 20,2′-dibenzoate 3, respectively. Also, compound 1 was treated with potassium carbonate in dry MeOH to afford the 3,18-debenzylated 4 and 3-debenzylated 5. Reduction of the 15,16-double bond of 1 was achieved through catalytic hydrogenation (10% Pd/C) in anhydrous EtOAc to give the target molecule 6 (Scheme 1). The C-5 and C-20 hydroxyl moieties of 1 were protected as a 2,2-di-tert-butyldioxasilole so that the C-2′ hydroxyl could be modified selectively (Scheme 2). Oxidation of the protected 7 with Dess–Martin periodinane gave the 2′-oxo derivative 8a, while acylation of 7 with acid anhydrides or chlorides gave 2′-acylated derivatives 8b–8h. Desilyation of 8a–8h provided the target compounds 9a–9h. The C-3 modified derivatives were synthesized as shown in Scheme 3. The hydroxy groups at C-20 and C-2′ were selectively protected to afford the tert-butyldimethylsilyl (TBS) ether 10. Then, 3-debenzoylation (11) and subsequent re-esterification (12) followed by removal of the protecting group with pyridinium poly(hydrogen fluoride) provided the target compounds 13a–13e. As also shown in Scheme 3, the 5-benzoyloxy (15a) and 5-oxo (15b) derivatives of 1 were synthesized from 10 by treatment with benzoic acid, N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide (EDCI), and 4-dimethylaminopyridine (DMAP) or Dess–Martin periodinane, respectively, followed by removal of the silyl protecting groups from 14a and 14b. As shown in Scheme 4, the silyl ether intermediate 16 was acetylated with acetic anhydride in anhydrous pyridine to give 17. Deprotection gave the 5-acetoxy derivative 18. Also, the primary hydroxy group at C-20 in 1 was esterified with appropriate acid anhydrides in the presence of DMAP to give 19a–19e and 19g. The C-20 esters 19f and 19h were obtained by reaction of 1 with Et₃N and (1S)-camphanic and trans-4-methoxycinnamoyl chlorides, respectively. All reactions were monitored by LCMS, and the target compounds were purified by semipreparative RP-HPLC.

Scheme 1. Synthesis of 2–6^a.

^aReagents and conditions: (a) acetic anhydride, pyridine, 40 °C, overnight (60% yield); (b) benzyl chloride, pyridine, rt, overnight (100% yield); (c) K₂CO₃, MeOH, reflux (39% yield); (d) K₂CO₃, MeOH, rt (81%yield); (e) Pd/C, EtOAc, rt, 4 h (67% yield).

Scheme 4. Synthesis of 18 and 19a–19h^a.

^aReagents and conditions (a) TBDMSCl, DMAP, pyridine, rt, 36 h (74% yield) (b) acetic anhydride, DMAP, pyridine, rt, 4h (58% yield) (c) Py•HF, pyridine, rt, over two days (45% yield) (d) acetic anhydride/benzoic anhydride/succinic anhydride/2,2-dimethylsuccinic anhydride, pyridine, DMAP,12 h–72 h; (e) Et₃N, DCM, (1S)-camphanic chloride/trans-4-methoxycinnamoyl chloride, rt, 12 h (24–53% yield).

Scheme 2. Synthesis of GM Derivatives 9a–9h^a.

^aReagents and conditions: (a) bis(trifluoromethanesulfonic acid), pyridine, 0°C, 1 h (25% yield) (b) Dess-Martin periodinane, DCM, rt, 6 h/acetic/tiglic anhydride, DMAP, pyridine, 40°C, 4 h or 7 days/corresponding acid chloride, pyridine, rt, 12 h–48 h (50–97% yield); (c) Et₃N 3HF, THF, rt, 4 h (76–99% yield).

Scheme 3. Synthesis of 13a–13e^a.

^aReagents and conditions: (a) TBSOTf, pyridine, rt, 1 h (69% yield) (b) K₂CO₃, MeOH, rt (82% yield) (c) corresponding acid, EDCI, DMAP, DCM, rt (48–71% yields) (d) Py•HF, pyridine, rt, over two days (40–74% yield) (e) benzoic acid, EDCI, DMAP, DCM, rt, over 7 days/Dess-Martin periodinane, DCM, rt, overnight (48 and 80% yield) (f) Py•HF, pyridine, rt, over two days (80–94% yield).

Anti-HIV Activities of 1 and Its Derivatives.

Parent compound 1 and the 29 synthesized 1-derivatives were evaluated for anti-HIV activity with a multicycle viral replication assay in MT-4 cells. The results are summarized in Table 1. Inhibition of HIV-1 replication was used as an indicator for the potency of GM derivatives because both latency-reversing and replication-inhibiting activities stem from targeting protein kinase C.^1,10 Four compounds (1, 9a, 13e, and 15b) were also evaluated in an HIV latency activation assay. The corresponding EC₅₀ values were 0.19 ± 0.05, 0.60 ± 0.14, 0.55 ± 0.19, and >3.9 nM.

Table 1.

Anti-HIV Replication Activities of GM Derivatives

compd	R1	R2	R3	R4	R5	15,16	anti-HIV IC50 (nM)	cytotoxicity CC50 (nM)
1	OBz	OH	OH	OH	OBz	C═CH2	0.13 ± 0.04	>1
2	OBz	OAc	OAc	OAc	OBz	C═CH2	>1	>1
3	OBz	OH	OBz	OBz	OBz	C═CH2	>1	>1
4	OH	OH	OH	OH	OH	C═CH2	>1	>1
5	OH	OH	OH	OH	OBz	C═CH2	>1	>1
6	OBz	OH	OH	OH	OBz	CH─CH3	0.18 ± 0.05	>1
9a	OBz	OH	OH	═O	OBz	C═CH2	0.14 ± 0.08	>1
9b	OBz	OH	OH	OAc	OBz	C═CH2	0.09 ± 0.04	>1
9c	OBz	OH	OH	tigloxy	OBz	C═CH2	>1	>1
9d	OBz	OH	OH	OBz	OBz	C═CH2	>1	>1
9e	OBz	OH	OH	O-4–methoxybenzoyl	OBz	C═CH2	>1	>1
9f	OBz	OH	OH	O-4–nitrobenzoyl	OBz	C═CH2	>1	>1
9g	OBz	OH	OH	O-trans-cinnamoyl	OBz	C═CH2	>1	>1
9h	OBz	OH	OH	O-2-naphthoyl	OBz	C═CH2	>1	>1
13a	O-4-methoxybenzoyl	OH	OH	OH	OBz	C═CH2	0.17 ± 0.04	>1
13b	O-4-nitrobenzoyl	OH	OH	OH	OBz	C═CH2	0.22 ± 0.05	>1
13c	O-trans-cinnamoyl	OH	OH	OH	OBz	C═CH2	>1	>1
13d	O-2-naphthoyl	OH	OH	OH	OBz	C═CH2	0.15 ± 0.03	>1
13e	O-4-pyridinylcarbonyl	OH	OH	OH	OBz	C═CH2	0.36 ± 0.09	>1
15a	OBz	OBz	OH	OH	OBz	C═CH2	>1	>1
15b	OBz	═O	OH	OH	OBz	C═CH2	>1	>1
18	OBz	OAc	OH	OH	OBz	C═CH2	>1	>1
19a	OBz	OH	OAc	OH	OBz	C═CH2	>1	>1
19b	OBz	OH	OBz	OH	OBz	C═CH2	>1	>1
19c	OBz	OH	succinoxy	OH	OBz	C═CH2	>1	>1
19d	OBz	OH	2,2-dimethylsuccinoxy	OH	OBz	C═CH2	>1	>1
19e	OBz	OH	3,3-dimethylsuccinoxy	OH	OBz	C═CH2	>1	>1
19f	OBz	OH	O-camphanoyl	OH	OBz	C═CH2	>1	>1
19g	OBz	OH	O-2,2,5-trimethyl-1,3-dioxane-5-carboxyl	OH	OBz	C═CH2	>1	>1
19h	OBz	OH	O-trans-4-methoxycinnamoyl	OH	OBz	C═CH2	>1	>1

MOA Comparison Study.

As found previously, protein kinase C beta (PKC-β) is responsible for the effect of 1 on HIV-1 replication.¹ To determine whether 1 and its analogs share the same mechanism of action (MOA), the antiviral activities of 1 and 13e were determined in the presence of the selective PKC-β antagonist enzastaurin. Enzastaurin is expected to antagonize the anti-HIV activity of 1.¹ The concentrations of 1 and 13e used in this experiment were intended to inhibit 90% of HIV replication so that the antagonistic effects of enzastaurin can be clearly demonstrated. As shown in Figure 2, enzastaurin at 1 μM effectively antagonized GM’s HIV-1 inhibitory activity. Compound 1 at 0.6 nM inhibited the virus replication by approximately 90%. This inhibitory activity was largely abrogated in the presence of enzastaurin at 1 μM. The antiviral potency of 13e was approximately threefold weaker than 1 in this assay. Enzastaurin was able to abrogate the HIV-1 inhibitory activity of 13e at 2 nM to a degree comparable to that of 1. These results suggest that 1 and 13e share the same mechanism of action even though 13e is a weaker PKC agonist.

Figure 2.

Enzastaurin abrogated the anti-HIV-1 activity of GM (1) and 13e. The effects of 1 or 13e on HIV-1 NL4-3 NanoLuc-sec infection of MT4 cells were determined in the presence or absence of enzastaurin. The 100% control in the y axis denotes HIV-1 replication in the absence of any compounds. The assays were performed in the presence of 0.6 nM 1 (blue bars), 2 nM 13e (orange bars), or without any PKC agonists (NA, gray bars). Enz-0, Enz-1 μM, and Enz-1/3 μM in the x axis represent the experimental groups with enzastaurin at 0 μM, 1 μM, and 1/3 μM, respectively. The data shown in the figure were derived from a duplicated antiviral assay.

In Vitro Stability in Plasma.

Most of the synthesized GM derivatives are ester-linked compounds and could possibly be decomposed by esterase enzymes in plasma. Due to this concern, the plasma stabilities of GM, 9a, and 13d were determined. The protocol used to assess the in vitro stability in plasma was modified from that used in the study by Hartman et al.²⁰ The plasma stabilities of GM, 9a, and 13d were studied in Sprague Dawley rat plasma. After sample preparation, each sample was analyzed by LCMS. The results showed that the half-lives of GM, 9a, and 13d were 21.77 ± 3.85, 21.65 ± 0.63, and 26.17 ± 1.18 h, respectively, indicating that these ester-linked GM derivatives are stable in plasma.

DISCUSSION AND CONCLUSIONS

In the anti-HIV replication screen, seven new compounds, 6, 9a, 9b, 13a, 13b, 13d, and 13e, exhibited significant potency (IC₅₀ = 0.09–0.36 nM), generally equivalent to that of 1 (IC₅₀ = 0.13 nM), while the EC₅₀ values of the remaining compounds were greater than 1 nM. The compounds’ structures and anti-HIV potencies were compared to identify useful SAR correlations.

None of the derivatives (2, 3, and 19a–19h) with an esterified 20-OH, regardless of the type of ester or other modifications in the molecule, exhibited an IC₅₀ value less than 1 nM. Thus, the free 20-OH found in the parent compound 1 is optimal for activity in this compound set.

In derivatives 15a, 15b, and 18, the 5-OH in 1 was changed to benzoyloxy, oxo, and acetoxy groups, respectively. These modifications at C-5 raised the IC₅₀ value above 1 nM. Therefore, a free 5-OH is likely pivotal to the anti-HIV activity.

Compound 1 (IC₅₀ = 0.13 nM) was also distinctly more potent than derivatives 4 and 5 (IC₅₀ > 1 nM). In derivative 5, the benzoate ester at C-3 in 1 has been removed to leave a free hydroxy group, while the benzoates at both C-3 and C-18 are absent in 4. The results suggest that a benzoylated rather than free hydroxy group at C-3 is important for activity. The anti-HIV potency was generally maintained when the phenyl ring in the 3-benzoate contained a para-methoxy (13a, IC₅₀ = 0.17 nM) or -nitro (13b, IC₅₀ = 0.22 nM) group or was replaced by a pyridinyl (13d, IC₅₀ = 0.15 nM) or naphthyl (13e, IC₅₀ = 0.36 nM) ring. However, a cinnamoyl ester (13c) on the 3-OH led to a significant loss of potency (IC₅₀ > 1 nM). Thus, insertion of a double bond (CH═CH) between the phenyl ring and carbonyl was unfavorable.

Regarding the C-18 position, in a prior study, stelleralide A, which is identical to 1 at all positions except C-18 (acetoxy rather than benzyoyloxy), showed excellent anti-HIV activity (IC₉₀ = 0.40 nM).²¹ In the current study, only one derivative, 4, was modified at C-18 (hydroxy). While 4 showed significantly lower potency compared with 1, the compound also contained a free hydroxyl group rather than a benzoate ester at C-3, which could be responsible for the decreased potency.

The hydroxyl group on the 1α-position of the macrocyclic ring (2′-OH) in 1 was modified by oxidization and esterification. Oxidation or acetylation (9a and 9b, respectively) resulted in potent analogs (IC₅₀ = 0.14 and 0.09 nM). However, compounds containing larger ester groups (e.g., 9c–9h: tiglate, benzoate, cinnamate, naphthoate, etc.) exhibited EC₅₀ values greater than 1 nM. Thus, C-2′ accepts only certain changes.

Derivative 6 with an isopropyl side chain on C-13 and parent compound 1 with an isopropenyl side chain exhibited identical potency. Thus, the oxidation state of this group did not influence the anti-HIV activity.

In summary, the following SAR conclusions were identified in this study. The free 5- and 20-OH groups of 1 are likely essential, and an aromatic acyl group on the 3-OH at C-3 is important for enhanced activity. Minor modifications at the 2′-OH are acceptable, including oxidation and acetylation. Thus far, modifications at C-18 have been limited and could be further studied. Reduction of Δ15,16 did not affect anti-HIV activity. These results are summarized in Figure 3.

Figure 3.

SAR correlations of derivatives of 1.

Furthermore, because the parent compound 1 exhibits potent dichotomous activity against HIV-1,¹⁰ three selected derivatives, 9a, 13b, and 15b, were evaluated in parallel with 1 in an HIV latency activation assay. Compound 1 exhibited the highest potency (EC₅₀ = 0.19 nM) in this assay, and derivatives 9a and 13e were equipotent (EC₅₀ = 0.6 nM). All three compounds were also potent in the anti-HIV-1 replication assay (Table 1). Thus, 2′-oxidation (9a) and 3-benzoylation (13b) are acceptable for both types of anti-HIV activity. In contrast, 5-oxognidimacrin (15b) was less potent in both assays (latency EC₅₀ > 3.9 nM, replication IC₅₀ > 1 nM). Thus, the free 5-OH is important for dichotomous activity against HIV-1.

In summary, the SAR study on 1 has identified the C-5 and C-20 hydroxyl groups as critical moieties to be retained. Some modifications at C2′, C-18, and Δ15,16 are tolerated. Finally, the C-3 benzoate ester can be changed to other aromatic acyl groups, which offers an opportunity to optimize pharmacological properties.

EXPERIMENTAL SECTION

Instrumentation and Reagents.

Optical rotations were measured on a JASCO P-2200 polarimeter in a 0.5 dm cell. The UV spectra were obtained with a Shimadzu UV-160 spectrophotometer. The IR spectra were measured on a JASCO FT/IR-4100 Fourier transform infrared spectrometer by the KBr disk method. The ¹H and ¹³C NMR spectra were measured on a JEOL ECA-500 spectrometer with the deuterated solvent as the internal reference, and the chemical shifts are expressed in δ (ppm). HRFABMS and HRESITOFMS were conducted using a JEOL JMS-700 MStation and a JEOL JMST100LP AccuTOF LC-plus mass spectrometer, respectively. Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan) was used for column chromatography. RP-HPLC was performed on a Waters 515 HPLC pump equipped with a Shodex RI-101 differential refractometer detector and a JASCO UV-970 intelligent UV–vis detector. An RP-C18 silica gel column (YMC-Pack Pro C18, 150 × 20 mm) was used at a flow rate of 5.0 mL/min. Sep-Pak C18 and Sep-Pak silica cartridges were purchased from Waters (Milford, MA, U.S.A.). The purity of the tested compounds (>95%) was confirmed by HPLC-PDA analysis and ¹H-NMR spectroscopic analysis. LCMS analysis was conducted on a Shimadzu LCMS-8040 Triple Quadrupole LC/MS/MS mass spectrometer. Solvents for LCMS analysis and synthesis were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Isolation of Gnidimacrin (1).

The roots of Stellera chamaejasme L. were collected from Chele (alt. 3115 m) to Shyammochem (alt. 3755 m), upper Mustang District, Nepal. The plant materials were identified by Dr. Takashi Watanabe (School of Pharmacy, Kumamoto University). A specimen of the plant (TH-1015) is kept in the herbarium of the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Toho University, Japan. The roots (30 kg) were extracted with MeOH. The MeOH extract (840 g) was partitioned between EtOAc and H₂O. The EtOAc layer was evaporated under reduced pressure below 40 °C to give a residue (471 g), which was applied to an ODS column and eluted with a gradient of MeOH─H₂O to give eight fractions. Fraction 4 (54 g) was further chromatographed on a silica gel column eluted with a gradient of MeOH─CHCl₃, and purification of the fractions by repeated preparative HPLC afforded 1 (1.0 g).

Preparation of Multiacylated Derivatives 2 and 3. 5,20,2′-Triacetoxygnidimacin (2).

Compound 1 (5 mg, 6.5 μmol) in anhydrous pyridine (3 mL) was combined with acetic anhydride (15 μL) at rt for 24 h under argon. After removal of solvent in vacuo, the residue was purified by RP-HPLC (MeCN, 5 mL/min) to give 2 (3.5 mg, 60%, t_R = 21.0 min). 2: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ + 18.5 (c 0.15, CHCl₃); UV (MeCN) λ_max (log ε): 200 (4.80), 257 (4.50) nm; IR (KBr) max: 2929, 2858, 1749, 1719, 1452, 1371, 1272, 1237, 1072, 1027, 755, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.86 (1H, t, J = 12.0 Hz, H-1), 1.93 (1H, m, H-2), 4.71 (1H, d, J = 4.0 Hz, H-3), 5.50 (1H, s, H-5), 3.25 (1H, s, H-7), 3.18 (1H, d, J = 2.8 Hz, H-8), 3.15 (1H, d, J = 12.0 Hz, H-10), 2.75 (1H, ddd, J = 10.6, 7.7, 2.3 Hz, H-11), 2.32 (1H, d, J = 14.6 Hz, H-12a), 1.97 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.26 (1H, d, J = 2.8 Hz, H-14), 5.11 (1H, s, H-16a), 4.91 (1H, brs, H-16b), 1.79 (3H, s, CH₃-17), 5.07 (1H, dd, J = 10.6, 2.3 Hz, H-18a), 4.39 (1H, t, J = 10.6 Hz, H-18b), 1.11 (3H, d, J = 6.6 Hz, CH₃-19), 4.75 (1H, d, J = 12.0 Hz, H-20a), 3.43 (1H, d, J = 12.0 Hz, H-20b), 5.29 (1H, d, J = 7.7 Hz, H-2′), 1.81 (1H, o, H-3′a), 1.69 (1H, m, H-3′b), 1.58 (1H, m, H-4′a), 1.46 (1H, m, H-4′b), 1.41 (1H, m, H-5′a), 1.19 (1H, m, H-5′b), 1.40 (1H, m, H-6′a), 1.31 (1H, m, H-6′b), 1.58 (1H, m, H-7′a), 1.45 (1H, m, H-7′b), 1.68 (1H, m, H-8′a), 1.06 (1H, m, H-8′b), 2.35 (1H, m, H-9′), 1.13 (3H, d, J = 6.9 Hz, H-10′). 3-OBz: 8.08 (2H, dd, J = 8.3, 1.1 Hz, H-3, H-7), 7.45 (2H, dd, J = 8.3, 7.5 Hz, H-4, H-6), 7.58 (1H, tt, J = 7.5, 1.2 Hz, H-5). 18-OBz: 8.07 (2H, dd, J = 8.0, 1.4 Hz, H-3, H-7), 7.27 (2H, dd, J = 8.0, 7.5 Hz, H-4, H-6), 7.48 (1H, tt, J = 7.5, 1.4 Hz, H-5). 5-Acetyl: 2.25 (3H, s, 5-COCH₃). 20-Acetoxy: 2.07 (3H, s, 20-COCH₃). 2′-Acetoxy: 2.03 (3H, s, 2′-COCH₃); ¹³C NMR (CDCl₃, 125 MHz) δ 49.5 (C-1), 37.0 (C-2), 82.9 (C-3), 80.6 (C-4), 70.4 (C-5), 58.9 (C-6), 63.4 (C-7), 36.6 (C-8), 81.1 (C-9), 47.5 (C-10), 41.0 (C-11), 29.3 (C-12), 84.0 (C-13), 81.2 (C-14), 146.0 (C-15), 111.8 (C-16), 18.7 (C-17), 67.6 (C-18), 14.8 (C-19), 67.4 (C-20), 117.6 (C-1′), 70.3 (C-2′), 27.1 (C-3′), 24.5 (C-4′), 24.5 (C-5′), 23.6 (C-6′), 23.5 (C-7′), 23.0 (C-8′), 27.7 (C-9′), 18.2 (C-10′). 3-Bz: 168.5 (C-1), 130.6 (C-2), 129.8 (C-3, C-7), 128.5 (C-4, C-6), 133.0 (C-5). 18-OBz: 166.3 (C-1), 129.8 (C-2), 129.6 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5). 5-Acetoxy: 170.7 (5-COCH₃), 21.0 (5-COCH₃). 20-Acetyl: 170.4 (20-COCH₃), 20.7 (20-COCH₃). 2′-Acetoxy: 170.3 (2′-COCH₃), 20.9 (2′-COCH₃); positive-ion HRFABMS m/z 901.4018 [M + Na]⁺, (calcd for C₅₀H₆₀O₁₅Na, 910.4010).

20,2′-O-Dibenzoyl Gnidimacin (3).

Compound 1 (5.5 mg, 6.8 μmol) was treated similarly with benzoyl chloride (10 μL) at rt overnight. The residue was purified by RP-HPLC (MeCN, 5 mL/min) to give 3 (6.9 mg, 100%, t_R = 27.0 min). 3: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 10.2 (c 0.38, CHCl₃); IR (KBr) max: 2932, 1719, 1451, 1385, 1270, 1114, 1025, 759, 710 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.88 (1H, m, H-1), 1.88 (1H, m, H-2), 5.00 (1H, d, J = 4.9 Hz, H-3), 4.09 (1H, s, H-5), 3.32 (1H, s, H-7), 3.08 (1H, d, J = 2.8 Hz, H-8), 3.15 (1H, d, J = 12.0 Hz, H-10), 2.87 (1H, m, H-11), 2.35 (1H, d, J = 14.6 Hz, H-12a), 1.99 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.28 (1H, d, J = 2.6 Hz, H-14), 5.17 (1H, s, H-16a), 4.88 (1H, brs, H-16b), 1.73 (3H, s, CH₃-17), 5.12 (1H, dd, J = 10.6, 2.3 Hz, H-18a), 4.47 (1H, t, J = 10.6 Hz, H-18b), 1.16 (3H, d, J = 6.6 Hz, CH₃-19), 5.10 (1H, d, J = 12.1 Hz, H-20a), 3.93 (1H, d, J = 12.1, Hz, H-20b), 5.57 (1H, d, J = 7.2 Hz, H-2′), 1.95 (1H, m, H-3′a), 1.88 (1H, m, H-3′b), 1.60 (1H, m, H-4′a), 1.32 (1H, m, H-4′b), 1.46 (1H, m, H-5′a), 1.24 (1H, m, H-5′b), 1.58 (1H, m, H-6′a), 1.31 (1H, m, H-6′b), 1.58 (1H, m, H-7′a), 1.42 (1H, m, H-7′b), 1.70 (1H, m, H-8′a), 1.09 (1H, m, H-8′b), 2.35 (1H, m, H-9′), 1.15 (3H, d, J = 7.2 Hz, H-10′). 3-Bz: 8.13 (2H, o, H-3, H-7), 7.31 (2H, dd, J = 8.3, 7.5 Hz, H-4, H-6), 7.50 (1H, m, H-5). 18-OBz: 8.12 (2H, m, H-3, H-7), 7.47 (2H, m, H-4, H-6), 7.58 (1H, m, H-5). 20-OBz: 8.12 (2H, m, H-3, H-7), 7.47 (2H, m, H-4, H-6), 7.58 (1H, m, H-5). 2′-Bz: 8.08 (2H, m, H-3, H-7), 7.40 (2H, m, H-4, H-6), 7.52 (1H, m, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 49.8 (C-1), 36.4 (C-2), 82.9 (C-3), 79.7 (C-4), 71.6 (C-5), 59.6 (C-6), 63.5 (C-7), 36.5 (C-8), 81.5 (C-9), 47.9 (C-10), 41.2 (C-11), 29.6 (C-12), 84.1 (C-13), 81.3 (C-14), 145.8 (C-15), 111.7 (C-16), 18.9 (C-17), 67.8 (C-18), 14.7 (C-19), 68.1 (C-20), 117.7 (C-1′), 70.8 (C-2′), 27.5 (C-3′), 24.1 (C-4′), 24.4 (C-5′), 23.7 (C-6′), 23.7 (C-7′), 23.1 (C-8′), 27.9 (C-9′), 18.3 (C-10′). 3-OBz: 167.9 (C-1), 130.7 (C-2), 130.2 (C-3, C-7), 128.5 (C-4, C-6), 133.3 (C-5). 18-OBz: 166.7 (C-1), 130.2 (C-2), 130.0 (C-3, C-7), 128.4 (C-4, C-6), 133.2 (C-5). 20-Bz: 166.4 (C-1), 129.8 (C-2), 130.0 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5). 2′-Bz: 165.8 (C-1), 129.6 (C-2), 129.6 (C-3, C-7), 128.2 (C-4, C-6), 132.8 (C-5); positive-ion HRFABMS m/z 1005.4036 [M + Na]⁺, (calcd for C₅₈H₆₂O₁₄Na, 1005.4037).

Preparation of Debenzoylated Derivatives 4 and 5. 3,18-Debenzoyl Gnidimacrin (4).

A mixture of 1 (8 mg, 10 μmol) and K₂CO₃ (8 mg, 56 μmol) was refluxed in MeOH (10 mL) for 72 h under argon. MeOH was removed under reduced pressure, and the residue was purified by RP-HPLC (MeCN─H₂O, 35:65, 5 mL/min) to give 4 (2.2 mg, 39%, t_R = 21.2 min). 4: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ + 61.3 (c 0.10, CHCl₃); IR (KBr) max: 2930, 1452, 1387, 1287, 1171, 1013, 757 cm⁻¹; ¹H NMR (pyridine-d₅, 500 MHz) δ 2.60 (1H, t, J = 12.0 Hz, H-1), 1.67 (1H, m, H-2), 4.21 (1H, brs, H-3), 4.27 (1H, d, J = 8.3 Hz, H-5), 3.74 (1H, s, H-7), 3.42 (1H, d, J = 2.9 Hz, H-8), 3.28 (1H, d, J = 12.0 Hz, H-10), 3.12 (1H, ddd, J = 9.4, 7.7, 3.2 Hz, H-11), 3.04 (1H, dd, J = 14.3, 7.7, Hz, H-12a), 2.20 (1H, d, J = 14.3 Hz, H-12b), 4.50 (1H, d, J = 2.9 Hz, H-14), 5.47 (1H, s, H-16a), 4.92 (1H, t, J = 1.4 Hz, H-16b), 1.87 (3H, s, CH₃-17), 4.57 (1H, brd, J = 10.3 Hz, H₂-18a), 4.44 (1H, dd, J = 10.3, 9.4 Hz, H₂-18b), 1.17 (3H, d, J = 6.9 Hz, CH₃-19), 4.69 (1H, brd, J = 12.1 Hz, H-20a), 4.14 (1H, brd, J = 12.1 Hz, H-20b), 4.30 (1H, dd, J = 7.8, 4.6 Hz, H-2′), 2.08 (1H, t, J = 13.6 Hz, H-3′a), 1.90 (1H, m, H-3′b), 1.88 (1H, m, H-4′a), 1.32 (1H, m, H-4′b), 1.36 (1H, m, H-5′a), 1.29 (1H, m, H-5′b), 1.39 (1H, m, H-6′a), 1.30 (1H, m, H-6′b), 1.52 (1H, m, H-7′a), 1.30 (1H, m, H-7′b), 1.67 (1H, m, H-8′a), 0.96 (1H, m, H-8′b), 2.55 (1H, m, H-9′), 1.00 (3H, d, J = 7.4 Hz, H-10′); ¹³C NMR (pyridine-d₅, 125 MHz) δ 49.5 (C-1), 38.7 (C-2), 78.6 (C-3), 79.1 (C-4), 72.9 (C-5), 63.0 (C-6), 64.2 (C-7), 37.5 (C-8), 82.1 (C-9), 49.4 (C-10), 45.7 (C-11), 30.2 (C-12), 84.7 (C-13), 82.4 (C-14), 147.9 (C-15), 111.3 (C-16), 19.2 (C-17), 63.9 (C-18), 15.4 (C-19), 65.9 (C-20), 119.8 (C-1′), 71.1 (C-2′), 29.5 (C-3′), 26.1 (C-4′), 23.8 (C-5′), 24.6 (C-6′), 24.2 (C-7′), 23.3 (C-8′), 27.5 (C-9′), 18.8 (C-10′); positive-ion HRFABMS m/z 589.2995 [M + Na]⁺, (calcd for C₃₀H₄₆O₁₀Na, 589.2988).

Stelleramacrin (5).

K₂CO₃ (5.0 mg, 36 μmol) was added to a solution of 1 (10.0 mg, 12.9 μmol) in MeOH (10.0 mL), and the mixture was stirred at rt for 5 h under argon. After removal of MeOH in vacuo, the residue was purified by RP-HPLC (MeCN─H₂O, 60:40, 5 mL/min) to give 5 (7.0 mg, 81%, t_R = 17.5 min). 5: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ 16.0 (c 0.1, CHCl₃); IR (KBr) max: 2926, 1716, 1456, 1386, 1271, 1171, 1106, 1013, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.69 (1H, t, J = 12.3 Hz, H-1), 1.70 (1H, m, H-2), 3.79 (1H, m, H-3), 3.72 (1H, d, J = 10.4 Hz, H-5), 3.38 (1H, s, H-7), 3.04 (1H, d, J = 2.5 Hz, H-8), 2.96 (1H, d, J = 12.3 Hz, H-10), 2.74 (1H, ddd, J = 10.6, 7.4, 1.7 Hz, H-11), 2.33 (1H, d, J = 14.6 Hz, H-12a), 2.00 (1H, dd, J = 14.6, 7.4 Hz, H-12b), 4.40 (1H, d, J = 2.5 Hz, H-14), 5.19 (1H, s, H-16a), 4.95 (1H, t, J = 1.4 Hz, H-16b), 1.83 (3H, s, CH₃-17), 4.91 (1H, dd, J = 10.6, 1.7 Hz, H-18a), 4.39 (1H, t, J = 10.6 Hz, H-18b), 1.16 (3H, d, J = 6.6 Hz, CH₃-19), 3.91 (1H, d, J = 12.0 Hz, H-20a), 3.60 (1H, d, J = 12.0 Hz, H-20b), 3.88 (1H, d, J = 7.5 Hz, H-2′), 1.72 (1H, m, H-3′a), 1.53 (1H, m, H-3′b), 1.61 (1H, m, H-4′a), 1.26 (1H, m, H-4′b), 1.57 (1H, m, H-5′a), 1.31 (1H, m, H-5′b), 1.32 (1H, m, H-6′a), 1.22 (1H, m, H-6′b), 1.56 (1H, m, H-7′a), 1.36 (1H, m, H-7′b), 1.61 (1H, m, H-8′a), 0.99 (1H, m, H-8′b), 2.26 (1H, m, H-9′), 1.00 (3H, d, J = 7.4 Hz, H-10′). 18-OBz: 8.08 (2H, dd, J = 8.0, 1.2 Hz, H-3, H-7), 7.45 (2H, dd, J = 8.0, 7.5 Hz, H-4, H-6), 7.57 (1H, tt, J = 7.5, 1.2 Hz, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 48.2 (C-1), 37.6 (C-2), 79.4 (C-3), 78.5 (C-4), 72.1 (C-5), 61.3 (C-6), 63.1 (C-7), 36.6 (C-8), 81.1 (C-9), 47.9 (C-10), 40.9 (C-11), 29.6 (C-12), 84.4 (C-13), 81.4 (C-14), 145.5 (C-15), 111.9 (C-16), 19.0 (C-17), 68.3 (C-18), 14.6 (C-19), 65.2 (C-20), 118.5 (C-1′), 70.8 (C-2′), 28.6 (C-3′), 25.3 (C-4′), 24.2 (C-5′), 23.6 (C-6′), 23.1 (C-7′), 22.9 (C-8′), 27.3 (C-9′), 18.2 (C-10′). 18-OBz: 167.1 (C-1), 130.3 (C-2), 129.7 (C-3, C-7), 128.4 (C-4, C-6), 133.1 (C-5).ESI-MS (positive) m/z 671 [M + H]⁺; (negative) m/z 669 [M − H]⁻.

Preparation of 15,16-Reduced Derivative 6.

A mixture of 1 (3.5 mg, 4.6 μmol) and 10% Pd/C (1 mg, 9.4 μmol) in anhydrous EtOAc (3.0 mL) was stirred under a hydrogen atmosphere (balloon) at rt for 24 h. The mixture was filtered, and the solution was purified by HPLC chromatography (YMC Pack Pro C₁₈; MeCN─H₂O, 90:10; 5 mL/min) to give 6 (2.4 mg, 67%, t_R = 19.5 min).

15,16-Dihydrognidimacrin (6).

Colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 8.2 (c 0.20, CHCl₃); IR (KBr) max: 3487, 2930, 1717, 1452, 1385, 1286, 1217, 1112, 1008, 757, 711 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.86 (1H, t, J = 12.3 Hz, H-1), 1.85 (1H, m, H-2), 4.94 (1H, d, J = 5.2 Hz, H-3), 4.05 (1H, d, J = 3.1 Hz, H-5), 3.39 (1H, s, H-7), 2.93 (1H, d, J = 2.8 Hz, H-8), 2.98 (1H, d, J = 12.3 Hz, H-10), 2.75 (1H, ddd, J = 10.6, 7.6, 2.6 Hz, H-11), 2.18 (1H, d, J = 14.6 Hz, H-12a), 1.69 (1H, dd, J = 14.6, 7.6 Hz, H-12b), 4.35 (1H, d, J = 2.8 Hz, H-14), 1.95 (1H, sep, J = 6.9 Hz, H-15), 0.97 (1H, d, J = 6.9 Hz, CH₃-16), 0.95 (1H, d, J = 6.9 Hz, CH₃-17), 5.05 (1H, dd, J = 10.6, 2.6 Hz, H-18a), 4.37 (1H, t, J = 10.6 Hz, H-18b), 1.15 (3H, d, J = 6.6 Hz, CH₃-19), 3.84 (2H, d, J = 6.5 Hz, H-20), 3.82 (1H, dd, J = 7.1, 4.0 Hz, H-2′), 1.71 (1H, t, J = 13.5 Hz, H-3′a), 1.40–1.60 (1H, m, H-3′b), 1.58–1.62 (1H, m, H-4′a), 1.22–1.29 (1H, m, H-4′b), 1.58–1.62 (1H, m, H-5′a), 1.22–1.29 (1H, m, H-5′b), 1.22–1.29 (2H, m, H-6), 1.58–1.62 (1H, m, H-7′a), 1.22–1.29 (1H, m, H-7′b), 1.58–1.62 (1H, m, H-8′a), 1.00–1.02 (1H, m, H-8′b), 2.30–2.40 (1H, m, H-9′), 1.10 (3H, d, J = 7.5 Hz, CH₃-10′). 3-OBz: 8.15 (2H, dd, J = 8.3, 1.1 Hz, H-3, H-7), 7.34 (2H, dd, J = 8.3, 7.5 Hz, H-4, H-6), 7.53 (1H, tt, J = 7.5, 1.1 Hz, H-5). 18-OBz: 8.11 (2H, dd, J = 8.3, 1.2 Hz, H-3, H-7), 7.47 (2H, dd, J = 8.3, 7.5 Hz, H-4, H-6), 7.58 (1H, tt, J = 7.5, 1.2 Hz, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 49.4 (C-1), 37.4 (C-2), 82.7 (C-3), 79.7 (C-4), 73.8 (C-5), 60.6 (C-6), 63.8 (C-7), 36.9 (C-8), 81.3 (C-9), 48.2 (C-10), 41.3 (C-11), 28.1 (C-12), 86.1 (C-13) 78.3 (C-14), 34.8 (C-15), 17.1 (C-16), 17.4 (C-17), 67.8 (C-18), 14.6 (C-19), 65.9 (C-20), 118.0 (C-1′), 70.8 (C-2′), 28.6 (C-3′), 25.2 (C-4′), 24.0 (C-5′), 23.7 (C-6′), 23.0 (C-7′), 22.8 (C-8′), 27.3 (C-9′), 18.2 (C-10′). 3-OBz: 168.3 (C-1), 129.4 (C-2), 130.1 (C-3, C-7), 128.4 (C-4, C-6), 133.5 (C-5). 18-OBz: 166.4 (C-1), 130.7 (C-2), 129.6 (C-3, C-7), 128.5 (C-4, C-6), 132.9 (C-5); positive-ion HRFABMS m/z 777.3852 [M + H]⁺, (calcd for C₄₄H₅₇O₁₂, 776.3772); 799.3668 [M + Na]⁺, (calcd for C₄₄H₅₆O₁₂Na, 799.3668).

General Procedure for Preparation of 2′-Derivatives (9a-9h)).

Bis(trifluoromethanesulfonic acid) was added to a solution of 1 in anhydrous pyridine (5.0 mL). The mixture was stirred at 0 °C for 1 h under argon. Purification by HPLC (YMC Pack Pro C₁₈, MeOH, 5 mL/min) gave 7. Compound 8a was obtained by oxidation of 7 (4.5 mg) with Dess–Martin periodinane in anhydrous DCM (3.0 mL) at rt for 6 h under argon and subsequently purified by RP-HPLC. To give the 2′-acylated derivatives 8b–8h, the appropriate acid anhydride or chloride was added to the stirred solution of 7 in anhydrous pyridine (3.0 mL) under argon. After removal of solvent in vacuo, the residue was purified by RP-HPLC to give 8b–8h. The deprotection of the silyl group in 8a–8h was easily achieved using Et₃N·3HF (2 μL, 12.2 μmol) at rt for 4 h to give 9a–9h.

2′-Oxognidimacrin (9a).

Compound 9a was obtained from the deprotection of 8a. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 9a (4.0 mg, 96.0%, t_R = 25.8 min). 9a: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 23.7 (c 0.10, CHCl₃); IR (KBr) max: 2925, 1718, 1452, 1382, 1273, 1069, 1003, 760, 711 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.92 (1H, t, J = 12.3 Hz, H-1), 1.84 (1H, m, H-2), 4.95 (1H, d, J = 5.1 Hz, H-3), 4.09 (1H, d, J = 3.8 Hz, H-5), 3.41 (1H, s, H-7), 3.10 (1H, d, J = 2.8 Hz, H-8), 3.15 (1H, d, J = 12.3 Hz, H-10), 2.92 (1H, ddd, J = 10.3, 7.8, 2.2 Hz, H-11), 2.39 (1H, d, J = 14.6 Hz, H-12a), 2.00 (1H, d, J = 14.6, 7.8 Hz, H-12b), 4.44 (1H, d, J = 2.8 Hz, H-14), 5.01 (1H, brs, H-16a), 4.92 (1H, t, J = 1.1 Hz, H-16b), 1.75 (3H, s, CH₃-17), 5.10 (1H, dd, J = 10.6, 2.2 Hz, H-18a), 4.49 (1H, dd, J = 10.6, 10.3 Hz, H-18b), 1.17 (3H, d, J = 6.6 Hz, CH₃-19), 3.86 (1H, d, J = 12.3 Hz, H-20a), 3.82 (1H, d, J = 12.3 Hz, H-20b), 2.75 (1H, ddd, J = 16.4, 9.7, 2.3 Hz, H-3′a), 2.65 (1H, ddd, J = 16.4, 8.5, 2.5 Hz, H-3′b), 1.36 (1H, m, H-4′a), 1.23 (1H, m, H-4′b), 1.92 (1H, m, H-5′a), 1.62 (1H, m, H-5′b), 1.23 (2H, m, H-6′), 1.44 (1H, m, H-7′a), 1.32 (1H, m, H-7′b), 1.44 (1H, m, H-8′a), 1.04 (1H, m, H-8′b), 2.30 (1H, m, H-9′), 1.12 (1H, d, J = 7.2 Hz, H-10′). 3-OBz: 8.13 (2H, dd, J = 8.0, 1.2 Hz, H-3, H-7), 7.31 (2H, dd, J = 8.0, 7.7 Hz, H-4, H-6), 7.51 (1H, tt, J = 7.7, 1.2 Hz, H-5). 18-Bz: 8.11 (2H, dd, J = 8.0, 1.2 Hz, H-3, H-7), 7.47 (2H, dd, J = 8.0, 7.5 Hz, H-4, H-6), 7.59 (1H, tt, J = 7.5, 1.2 Hz, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 49.2 (C-1), 37.2 (C-2), 82.7 (C-3), 79.8 (C-4), 73.7 (C-5), 60.6 (C-6), 63.2 (C-7), 36.9 (C-8), 83.1 (C-9), 47.8 (C-10), 41.6 (C-11), 29.9 (C-12), 85.2 (C-13), 81.6 (C-14), 145.2 (C-15), 112.0 (C-16), 18.7 (C-17), 67.6 (C-18), 14.7 (C-19), 65.8 (C-20), 113.6 (C-1′), 197.6 (C-2′), 36.5 (C-3′), 23.1 (C-4′), 28.5 (C-5′), 24.9 (C-6′), 23.9 (C-7′), 25.7 (C-8′), 27.8 (C-9′), 18.7 (C-10′). 3-OBz: 168.4 (C-1), 130.6 (C-2), 130.1 (C-3,7), 128.5 (C-4,6), 133.0 (C-5). 18-OBz: 166.4 (C-1), 130.6 (C-2), 129.7 (C-3,7), 128.4 (C-4,6), 133.5 (C-5); positive-ion HRFABMS m/z 795.3356 [M + Na]⁺, (calcd for C₄₄H₅₆O₁₂Na, 795.3356).

2′-Acetoxygnidimacrin (9b).

Compound 9b was obtained from the deprotection of 8b. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 9b (2.8 mg, 93.7%, t_R = 22.5 min). 9b: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ + 5.8 (c 0.10, CHCl₃); IR (KBr) max: 2933, 1750, 1718, 1472, 1452, 1384, 1279, 1236, 1119, 1026, 828, 759, 726 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.88 (1H, t, J = 12.3 Hz, H-1), 1.87 (1H, m, H-2), 4.95 (1H, d, J = 4.8 Hz, H-3), 4.07 (1H, s, H-5), 3.34 (1H, s, H-7), 3.00 (1H, d, J = 2.8 Hz, H-8), 3.06 (1H, d, J = 12.3 Hz, H-10), 2.83 (1H, ddd, J = 10.3, 7.5, 2.0 Hz, H-11), 2.33 (1H, d, J = 14.6 Hz, H-12a), 1.97 (1H, d, J = 14.6, 7.5 Hz, H-12b), 4.27 (1H, d, J = 2.9 Hz, H-14), 5.13 (1H, brs, H-16a), 4.92 (1H, t, J = 1.4 Hz, H-16b), 1.80 (3H, s, CH₃-17), 5.08 (1H, dd, J = 10.6, 2.0 Hz, H-18a), 4.40 (1H, dd, J = 10.6, 10.3 Hz, H-18b), 1.17 (3H, d, J = 6.6 Hz, CH₃-19), 3.85 (1H, d, J = 12.3 Hz, H-20a), 3.79 (1H, d, J = 12.3 Hz, H-20b), 5.29 (1H, d, J = 7.4 Hz, H-2′), 1.83 (1H, m, H-3′a), 1.71 (1H, m, H-3′b), 1.38 (1H, m, H-4′a), 1.23 (1H, m, H-4′b), 1.41 (1H, m, H-5′a), 1.30 (1H, m, H-5′b), 1.40 (1H, m, H-6′a), 1.29 (1H, m, H-6′b), 1.61 (1H, m, H-7′a), 1.42 (1H, m, H-7′b), 1.64 (1H, m, H-8′a), 1.05 (1H, m, H-8′b), 2.39 (1H, m, H-9′), 1.83 (1H, d, J = 7.5 Hz, H-10′). 3-OBz: 8.14 (2H, dd, J = 8.0, 1.1 Hz, H-3, H-7), 7.34 (2H, dd, J = 8.0, 7.4 Hz, H-4, H-6), 7.52 (1H, tt, J = 7.4, 1.1 Hz, H-5). 18-OBz: 8.11 (2H, dd, J = 8.0, 1.4 Hz, H-3, H-7), 7.43 (2H, dd, J = 8.0, 7.4 Hz, H-4, H-6), 7.59 (1H, tt, J = 7.4, 1.4 Hz, H-5). 2′-Acetoxy: 2.06 (3H, s, COCH₃); ¹³C NMR (CDCl₃, 125 MHz) δ 49.4 (C-1), 37.4 (C-2), 82.8 (C-3), 79.7 (C-4), 73.7 (C-5), 60.5 (C-6), 63.3 (C-7), 36.5 (C-8), 81.6 (C-9), 47.9 (C-10), 41.2 (C-11), 29.3 (C-12), 82.8 (C-13), 81.5 (C-14), 146.0 (C-15), 111.7 (C-16), 18.7 (C-17), 67.7 (C-18), 14.7 (C-19), 65.7 (C-20), 117.5 (C-1′), 70.3 (C-2′), 27.1 (C-3′), 24.4 (C-4′), 23.9 (C-5′), 23.6 (C-6′), 23.5 (C-7′), 22.8 (C-8′), 27.7 (C-9′), 18.2 (C-10′). 3-OBz: 168.3 (C-1), 129.5 (C-2), 130.1 (C-3, C-7), 128.5 (C-4, C-6), 133.4 (C-5). 18-Bz: 166.4 (C-1), 130.7 (C-2), 129.6 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5). 2′-Acetoyl: 170.4 (C-1), 21.0 (C-2); positive-ion HRFABMS m/z 817.3799 [M + H]⁺, (calcd for C₄₆H₅₇O₁₃, 817.3799).

2′-Tigloxygnidimacrin (9c).

Compound 9c was obtained from the deprotection of 8c. The residue was purified by RP-HPLC (MeCN─H₂O, 95:5, 5 mL/min) to give 9c (2.0 mg, 86.2%, t_R = 22.5 min). 9c: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 7.2 (c 0.20, CHCl₃); IR (KBr) max: 3464, 2927, 2857, 1717, 1363, 1602, 1451, 1381, 1314, 1273, 1174, 1109, 1015, 711 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.87 (1H, dd, J = 12.3, 12.0 Hz, H-1), 1.87 (1H, m, H-2), 4.93 (1H, d, J = 5.2 Hz, H-3), 4.06 (1H, s, H-5), 3.33 (1H, s, H-7), 2.99 (1H, d, J = 2.8, Hz, H-8), 3.08 (1H, d, J = 12.3 Hz, H-10), 2.82 (1H, m, H-11), 2.30 (1H, d, J = 14.6, H-12a), 1.95 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.28 (1H, d, J = 2.8 Hz, H-14), 5.14 (1H, brs, H-16a), 4.89 (1H, t, J = 1.4 Hz, H-16b), 1.80 (3H, s, CH₃-17), 5.08 (1H, dd, J = 10.6, 2.0 Hz, H-18a), 4.42 (1H, dd, J = 10.6, 10.3 Hz, H-18b), 1.16 (3H, d, J = 6.5 Hz, CH₃-19), 3.83 (1H, d, J = 12.3 Hz, H-20a), 3.79 (1H, d, J = 12.3 Hz, H-20b), 5.38 (1H, d, J = 6.9 Hz, H-2′), 1.86 (1H, m, H-3′a), 1.75 (1H, m, H-3′b), 1.43 (1H, m, H-4′a), 1.20 (1H, m, H-4′b), 1.43 (1H, m, H-5′a), 1.20 (1H, m, H-5′b), 1.37 (1H, m, H-6′a), 1.32 (1H, m, H-6′b), 1.40 (1H, m, H-7′a), 1.29 (1H, m, H-7′b), 1.64 (1H, m, H-8′a), 1.06 (1H, m, H-8′b), 2.38 (1H, m, H-9′), 1.82 (1H, m, H-10′). 3-OBz: 8.13 (2H, dd, J = 8.4, 1.5 Hz, H-3, H-7), 7.32 (2H, dd, J = 8.4, 7.5 Hz, H-4, H-6), 7.41 (1H, tt, J = 7.4, 1.5 Hz, H-5). 18-OBz: 8.10 (2H, dd, J = 8.0, 1.4 Hz, H-3, H-7), 7.46 (2H, dd, J = 8.0, 7.4 Hz, H-4, H-6), 7.57 (1H, tt, J = 7.5, 1.4 Hz, H-5). 2′-Tigloxy: 6.92 (1H, qq, J = 7.2, 1.4 Hz, H-3), 1.75 (3H, dq, J = 7.2, 0.9 Hz, CH₃-4), 1.83 (3H, brs, CH₃-5); ¹³C NMR (CDCl₃, 125 MHz) δ 49.3 (C-1), 37.3 (C-2), 82.7 (C-3), 79.7 (C-4), 73.7 (C-5), 60.3 (C-6), 63.3 (C-7), 36.5 (C-8), 81.4 (C-9), 47.9 (C-10), 41.2 (C-11), 29.6 (C-12), 84.0 (C-13), 81.2 (C-14), 145.6 (C-15), 111.6 (C-16), 18.9 (C-17), 67.8 (C-18), 14.7 (C-19), 65.7 (C-20), 117.7 (C-1′), 70.1 (C-2′), 27.4 (C-3′), 24.2 (C-4′), 24.0 (C-5′), 23.7 (C-6′), 23.6 (C-7′), 23.0 (C-8′), 28.0 (C-9′), 18.3 (C-10′). 3-OBz: 168.3 (C-1), 129.5 (C-2), 130.1 (C-3, C-7), 128.4 (C-4, C-6), 133.4 (C-5). 18-OBz: 166.4 (C-1), 130.6 (C-2), 129.6 (C-3, C-7), 128.5 (C-4, C-6), 132.9 (C-5). 2′-Tigloxy: 167.3 (C-1), 128.3 (C-2), 137.8 (C-3), 14.4 (C-4), 12.1 (C-5); positive-ion HRFABMS m/z 879.3933 [M + Na]⁺, (calcd for C₄₆H₆₀O₁₃Na, 879.3932).

2′-O-Benzoylgnidimacrin (9d).

Compound 9d was obtained from the deprotection of 8d. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 9d (3.8 mg, 91.8%, t_R = 40 min). 9d: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 8.5 (c 0.30, CHCl₃); IR (KBr) max: 2930, 1717, 1461, 1383, 1271, 1115, 1026, 838, 779, 711 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.89 (1H, dd, J = 12.3, 12.1 Hz, H-1), 1.87 (1H, m, H-2), 4.96 (1H, d, J = 4.8 Hz, H-3), 4.08 (1H, s, H-5), 3.33 (1H, s, H-7), 3.00 (1H, d, J = 2.8, Hz, H-8), 3.10 (1H, d, J = 12.3 Hz, H-10), 2.82 (1H, ddd, J = 10.3, 7.7, 2.2 Hz, H-11), 2.32 (1H, d, J = 14.6, H-12a), 1.95 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.24 (1H, d, J = 2.8 Hz, H-14), 5.14 (1H, brs, H-16a), 4.86 (1H, t, J = 1.4 Hz, H-16b), 1.68 (3H, s, CH₃-17), 5.11 (1H, dd, J = 10.6, 2.2 Hz, H-18a), 4.45 (1H, dd, J = 10.6, 10.3 Hz, H-18b), 1.17 (3H, d, J = 6.9 Hz, CH₃-19), 3.84 (1H, d, J = 12.0 Hz, H-20a), 3.80 (1H, d, J = 12.0 Hz, H-20b), 5.56 (1H, d, J = 6.9 Hz, H-2′), 1.97 (1H, m, H-3′a), 1.89 (1H, m, H-3′b), 1.47 (1H, m, H-4′a), 1.30 (1H, m, H-4′b), 1.43 (1H, m, H-5′a), 1.39 (1H, m, H-5′b), 1.47 (1H, m, H-6′a), 1.39 (1H, m, H-6′b), 1.59 (1H, m, H-7′a), 1.44 (1H, m, H-7′b), 1.66 (1H, m, H-8′a), 1.06 (1H, m, H-8′b), 2.43 (1H, m, H-9′), 1.13 (3H, d, J = 7.5 Hz, H-10′). 3-OBz: 8.14 (2H, dd, J = 8.0, 1.0 Hz, H-3, H-7), 7.32 (2H, dd, J = 8.0, 7.5 Hz, H-4, H-6), 7.50 (1H, tt, J = 7.5, 1.0 Hz, H-5). 18-OBz: 8.10 (2H, dd, J = 8.3, 1.4 Hz, H-3, H-7), 7.46 (2H, dd, J = 8.3, 7.4 Hz, H-4, H-6), 7.50 (1H, tt, J = 7.5, 1.4 Hz, H-5). 2′-Bz: 8.08 (2H, dd, J = 8.3, 1.0 Hz, H-3, H-7), 7.40 (2H, dd, J = 8.2, 7.5 Hz, H-4, H-6), 7.50 (1H, tt, J = 7.5, 1.0 Hz, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 49.4 (C-1), 37.4 (C-2) 82.9 (C-3), 79.7 (C-4), 73.7 (C-5), 60.5 (C-6), 63.3 (C-7), 36.6 (C-8), 81.6 (C-9), 47.9 (C-10), 41.2 (C-11), 29.6 (C-12), 84.1 (C-13), 81.3 (C-14), 145.7 (C-15), 111.6 (C-16), 18.9 (C-17), 67.8 (C-18), 14.7 (C-19), 65.8 (C-20), 117.6 (C-1′), 70.8 (C-2′), 27.3 (C-3′), 24.3 (C-4′), 24.1 (C-5′), 23.7 (C-6′), 23.7 (C-7′), 23.0 (C-8′), 28.0 (C-9′), 18.3 (C-10′). 3-OBz: 168.3 (C-1), 129.5 (C-2), 130.1 (C-3, C-7), 128.5 (C-4, C-6), 133.4 (C-5). 18-Bz: 166.4 (C-1), 130.7 (C-2), 129.6 (C-3,7), 128.4 (C-4, C-6), 132.9 (C-5). 2′-OBz: 166.4 (C-1), 130.7 (C-2), 129.6 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5); positive-ion HRFABMS m/z 901.3777 [M + Na]⁺, (calcd for C₅₁H₅₈O₁₃Na, 901.3775).

2′-O-(4-Methoxybenzoyl)gnidimacrin (9e).

Compound 9e was obtained from the deprotection of 8e. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 9e (3.0 mg, 88.8%, t_R = 37.5 min). 9e: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 11.4 (c 0.30, CHCl₃); IR (KBr) max: 2932, 1717, 1605, 1510, 1452, 1271, 1167, 1101, 1027, 768, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.89 (1H, dd, J = 12.3, 12.0 Hz, H-1), 1.87 (1H, m, H-2), 4.96 (1H, d, J = 4.8 Hz, H-3), 4.06 (1H, s, H-5), 3.33 (1H, s, H-7), 2.99 (1H, d, J = 2.8, Hz, H-8) 3.10 (1H, d, J = 12.3 Hz, H-10), 2.82 (1H, ddd, J = 10.3, 7.6, 2.2 Hz, H-11), 2.32 (1H, d, J = 14.6 Hz, H-12a), 1.95 (1H, dd, J = 14.6, 7.6 Hz, H-12b), 4.24 (1H, d, J = 2.8 Hz, H-14), 5.14 (1H, brs, H-16a), 4.86 (1H, t, J = 1.4 Hz, H-16b), 1.68 (3H, s, CH₃-17), 5.12 (1H, dd, J = 10.6, 2.2 Hz, H-18a), 4.45 (1H, dd, J = 10.6, 10.3 Hz, H-18b), 1.14 (3H, d, J = 6.6 Hz, CH₃-19), 3.85 (1H, d, J = 12.0 Hz, H-20a), 3.80 (1H, d, J = 12.0 Hz, H-20b), 5.53 (1H, d, J = 6.9 Hz, H-2′), 1.90 (1H, m, H-3′a), 1.85 (1H, m, H-3′b), 1.46 (1H, m, H-4′a), 1.30 (1H, m, H-4′b), 1.44 (1H, m, H-5′a), 1.30 (1H, m, H-5′b), 1.45 (1H, m, H-6′a), 1.39 (1H, m, H-6′b), 1.59 (1H, m, H-7′a), 1.44 (1H, m, H-7′b), 1.66 (1H, m, H-8′a), 1.07 (1H, m, H-8′b), 2.42 (1H, m, H-9′), 1.14 (3H, d, J = 7.5 Hz, H-10′). 3-Bz: 8.14 (2H, dd, J = 8.3, 1.2 Hz, H-3, H-7), 7.32 (2H, dd, J = 8.3, 7.5 Hz, H-4, H-6), 7.51 (1H, tt, J = 7.5, 1.2 Hz, H-5). 18-OBz: 8.10 (2H, dd, J = 8.0, 1.2 Hz, H-3, H-7), 7.46 (2H, dd, J = 8.0, 7.5 Hz, H-4, H-6), 7.58 (1H, tt, J = 7.5, 1.2 Hz, H-5). 2′-O-(4-Methoxybenzoyl): 8.08 (2H, brd, J = 9.1 Hz, H-3, H-7), 7.40 (2H, brd, J = 9.1 Hz, H-4, H-6), 3.84 (3H, s, 5-OMe); ¹³C NMR (CDCl₃, 125 MHz) δ 49.4 (C-1), 37.4 (C-2), 82.8 (C-3), 79.7 (C-4), 73.8 (C-5), 60.4 (C-6), 63.4 (C-7), 36.5 (C-8), 81.6 (C-9), 47.9 (C-10), 41.3 (C-11), 29.6 (C-12), 84.1 (C-13), 81.3 (C-14), 145.8 (C-15), 111.6 (C-16), 18.9 (C-17), 67.8 (C-18), 14.7 (C-19), 65.8 (C-20), 117.7 (C-1′), 70.5 (C-2′), 27.4 (C-3′), 24.3 (C-4′), 24.1 (C-5′), 23.7 (C-6′), 23.7 (C-7′), 23.1 (C-8′), 28.0 (C-9′), 18.3 (C-10′). 3-OBz: 168.3 (C-1), 129.5 (C-2), 130.1 (C-3, C-7), 128.5 (C-4, C-6), 133.4 (C-5). 18-Bz: 166.4 (C-1), 130.7 (C-2), 129.6 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5). 2′-O-(4-Methoxybenzoyl): 165.9 (C-1), 122.5 (C-2), 132.0 (C-3, C-7), 113.5 (C-4, C-6), 163.3 (C-5), 55.4 (5-OMe); positive-ion HRFABMS m/z 931.3881 [M + Na]⁺, (calcd for C₅₂H₆₀O₁₄Na, 931.3881).

2′-O-(4-Nitrobenzoyl)gnidimacrin (9f).

Compound 9f was obtained from the deprotection of 8f at rt overnight. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 9f (3.0 mg, 77.9%, t_R = 38.0 min). 9f: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 11.4 (c 0.30, CHCl₃); IR (KBr) max: 2939, 1716, 1530, 1455, 1271, 1102, 1015, 714 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.88 (1H, dd, J = 12.3, 11.5 Hz, H-1), 1.88 (1H, m, H-2), 4.94 (1H, d, J = 4.8 Hz, H-3), 4.08 (1H, s, H-5), 3.32 (1H, s, H-7), 3.02 (1H, d, J = 2.6, Hz, H-8), 3.10 (1H, d, J = 12.3 Hz, H-10), 2.84 (1H, ddd, J = 10.3, 7.6, 2.2 Hz, H-11), 2.32 (1H, d, J = 14.6, H-12a), 1.98 (1H, dd, J = 14.6, 7.6 Hz, H-12b), 4.27 (1H, d, J = 2.6 Hz, H-14), 5.15 (1H, brs, H-16a), 4.89 (1H, t, J = 1.4 Hz, H-16b), 1.70 (3H, s, CH₃-17), 5.11 (1H, dd, J = 10.6, 2.2 Hz, H-18a), 4.45 (1H, dd, J = 10.6, 10.3 Hz, H-18b), 1.17 (3H, d, J = 6.6 Hz, CH₃-19) 3.84 (1H, d, J = 12.3 Hz, H-20a), 3.80 (1H, d, J = 12.3 Hz, H-20b), 5.55 (1H, dd, J = 7.4, 1.6 Hz, H-2′), 1.96 (1H, m, H-3′a), 1.87 (1H, m, H-3′b), 1.44 (1H, m, H-4′a), 1.41 (1H, m, H-4′b), 1.41 (1H, m, H-5′a), 1.30 (1H, m, H-5′b), 1.47 (1H, m, H-6′a), 1.31 (1H, m, H-6′b), 1.56 (1H, m, H-7′a), 1.47 (1H, m, H-7′b), 1.65 (1H, m, H-8′a), 1.08 (1H, m, H-8′b), 2.42 (1H, m, H-9′), 1.14 (3H, d, J = 7.5 Hz, H-10′). 3-OBz: 8.14 (2H, dd, J = 8.3, 1.1 Hz, H-3, H-7), 7.32 (2H, dd, J = 8.3, 7.5 Hz, H-4,6), 7.52 (1H, tt, J = 7.5, 1.1 Hz, H-5). 18-Bz: 8.11 (2H, dd, J = 8.3, 1.2 Hz, H-3,7), 7.48 (2H, dd, J = 8.3, 7.5 Hz, H-4, H-6), 7.59 (1H, tt, J = 7.5, 1.2 Hz, H-5). 2′-O-(4-Nitrobenzoyl): 8.24 (2H, brd, J = 9.1 Hz, H-3, H-7), 8.27 (2H, brd, J = 9.1 Hz, H-4, H-6); ¹³C NMR (CDCl₃, 125 MHz) δ 49.4 (C-1), 37.4 (C-2), 82.8 (C-3), 79.7 (C-4), 73.7 (C-5), 60.5 (C-6), 63.3 (C-7), 36.6 (C-8), 81.8 (C-9), 48.0 (C-10), 41.2 (C-11), 29.7 (C-12), 84.3 (C-13), 81.3 (C-14), 145.5 (C-15), 111.6 (C-16), 18.9 (C-17), 67.7 (C-18), 14.7 (C-19), 65.8 (C-20), 117.3 (C-1′), 71.9 (C-2′), 27.2 (C-3′), 24.2 (C-4′), 24.1 (C-5′), 23.7 (C-6′), 23.7 (C-7′), 23.1 (C-8′), 28.1 (C-9′), 18.3 (C-10′). 3-Bz: 168.4 (C-1), 129.4 (C-2), 130.1 (C-3, C-7), 128.6 (C-4, C-6), 133.5 (C-5). 18-OBz: 166.5 (C-1), 130.6 (C-2), 129.6 (C-3, C-7), 128.4 (C-4, C-6), 133.0 (C-5). 2′-O-(4-Nitrobenzoyl): 164.1 (C-1), 135.6 (C-2), 131.0 (c-3, C-7), 123.4 (C-4, C-6), 150.6 (C-5); positive-ion HRFABMS m/z 946.3625 [M + Na]⁺, (calcd for C₅₁H₅₇NO₁₅Na, 946.3625).

2′-O-(trans-Cinnamoyl)gnidimacrin (9g).

Compound 9g was obtained from the deprotection of 8g. The residue was purified by RP-HPLC (MeCN─H₂O, 95:5, 5 mL/min) to give 9g (2.8 mg, 75.9%, t_R = 18.5 min). 9g: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 7.2 (c 0.20, CHCl₃); IR (KBr) max: 2926, 1716, 1456, 1386, 1271, 1171, 1106, 1013, 768, 712 cm⁻¹; ¹H NMR (CDC1₃, 500 MHz) δ 2.82 (1H, dd, J = 12.3, 11.4 Hz, H-1), 1.85 (1H, m, H-2), 5.12 (1H, d, J = 5.2 Hz, H-3), 4.34 (1H, s, H-5), 3.20 (1H, s, H-7), 3.06 (1H, d, J = 2.8 Hz, H-8), 3.10 (1H, d, J = 12.3 Hz, H-10), 2.82 (1H, ddd, J = 10.3, 7.7, 2.0 Hz, H-11), 2.32 (1H, d, J = 14.6, H-12a), 1.94 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.26 (1H, d, J = 2.8 Hz, H-14), 5.11 (1H, brs, H-16a), 4.90 (1H, t, J = 1.2 Hz, H-16b), 1.77 (3H, s, CH₃-17), 5.11 (1H, dd, J = 10.6, 2.0 Hz, H-18a), 4.45 (1H, t, J = 10.6, 10.3 Hz, H-18b), 1.17 (3H, d, J = 6.9 Hz, CH₃-19), 3.84 (1H, d, J = 12.3 Hz, H-20a), 3.80 (1H, d, J = 12.3 Hz, H-20b), 5.43 (1H, dd, J = 7.5 Hz, H-2′), 1.87 (1H, m, H-3′a), 1.80 (1H, m, H-3′b), 1.48 (1H, m, H-4′a), 1.24 (1H, m, H-4′b), 1.40 (1H, m, H-5′a), 1.28 (1H, m, H-5′b), 1.47 (1H, m, H-6′a), 1.31 (1H, m, H-6′b), 1.63 (1H, m, H-7′a), 1.43 (1H, m, H-7′b), 1.66 (1H, m, H-8′a), 1.06 (1H, m, H-8′b), 2.40 (1H, m, H-9′), 1.13 (3H, d, J = 7.5 Hz, H-10′). 3-OBz: 8.14 (2H, dd, J = 8.1, 1.1 Hz, H-3, H-7), 7.32 (2H, dd, J = 8.1, 7.5 Hz, H-4, H-6), 7.41 (1H, tt, J = 7.5, 1.1 Hz, H-5). 18-OBz: 8.11 (2H, dd, J = 8.3, 1.3 Hz, H-3, H-7), 7.48 (2H, dd, J = 8.3, 7.5 Hz, H-4, H-6), 7.58 (1H, tt, J = 7.5, 1.3 Hz, H-5). 2′-O-(trans-Cinnamoyl): 6.40 (1H, d, J = 16.0 Hz, H-2), 7.72 (1H, d, J = 16.0 Hz, H-3), 7.48 (1H, dd, J = 6.6, 1.1 Hz, H-5, H-9), 7.35 (1H, m, H-6, H-8), 7.35 (1H, m, H-7); ¹³C NMR (CDCl₃, 125 MHz) δ 49.4 (C-1), 37.4 (C-2), 82.8 (C-3), 79.7 (C-4), 73.8 (C-5), 60.4 (C-6), 63.3 (C-7), 36.6 (C-8), 81.6 (C-9), 47.9 (C-10), 41.3 (C-11), 29.4 (C-12), 84.0 (C-13), 81.4 (C-14), 146.0 (C-15), 111.7 (C-16), 18.8 (C-17), 67.8 (C-18) 14.7 (C-19), 65.8 (C-20), 117.3 (C-1′), 70.4 (C-2′), 27.3 (C-3′), 24.5 (C-4′), 23.9 (C-5′), 23.7 (C-6′), 23.5 (C-7′), 22.8 (C-8′), 28.1 (C-9′), 18.2 (C-10′). 3-Bz: 168.3 (C-1), 129.5 (C-2), 130.1 (C-3, C-7), 128.5 (C-4, C-6), 133.4 (C-5). 18-OBz: 166.4 (C-1), 130.7 (C-2), 129.6 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5). 2′-O-(trans-Cinnamoyl): 166.3 (C-1), 118.1 (C-2), 145.2 (C-3), 134.6 (C-4), 128.1 (C-5,9), 128.9 (C-6, C-8), 130.0 (C-7); positive-ion HRFABMS m/z 927.3936 [M + Na]⁺, (calcd for C₅₃H₆₀O₁₃Na, 927.3932).

2′-O-(2-Naphthoyl)gnidimacrin (9h).

Compound 9h was obtained from the deprotection of 8h at rt overnight. The residue was purified by RP-HPLC (MeCN─H₂O, 95:5, 5 mL/min) to give 9h (3.1 mg, 99.1%, t_R = 21 min). 9h: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 21.1 (c 0.30, CHCl₃); IR (KBr) max: 2931, 1717, 1541, 1456, 1273, 1099, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.90 (1H, t, J = 12.3 Hz, H-1), 1.90 (1H, m, H-2), 4.97 (1H, d, J = 4.6 Hz, H-3), 4.10 (1H, s, H-5), 3.33 (1H, s, H-7), 3.00 (1H, d, J = 2.8, Hz, H-8), 3.12 (1H, d, J = 12.3 Hz, H-10), 2.83 (1H, ddd, J = 10.6, 7.7, 2.0 Hz, H-11), 2.31 (1H, d, J = 14.6 Hz, H-12a), 1.91 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.22 (1H, d, J = 2.8 Hz, H-14), 5.17 (1H, brs, H-16a), 4.85 (1H, brs, H-16b), 1.65 (3H, s, CH₃-17), 5.14 (1H, dd, J = 10.6, 2.0 Hz, H-18a), 4.47 (1H, t, J = 10.6 Hz, H-18b), 1.18 (3H, d, J = 7.4 Hz, CH₃-19), 3.85 (1H, d, J = 12.3 Hz, H-20a), 3.81 (1H, d, J = 12.3 Hz, H-20b), 5.62 (1H, dd, J = 7.4, 1.4 Hz, H-2′), 1.98 (1H, m, H-3′a), 1.91 (1H, m, H-3′b), 1.49 (1H, m, H-4′a), 1.31 (1H, m, H-4′b), 1.45 (1H, m, H-5′a), 1.35 (1H, m, H-5′b), 1.42 (1H, m, H-6′a), 1.32 (1H, m, H-6′b), 1.58 (1H, m, H-7′a), 1.42 (1H, m, H-7′b), 1.68 (1H, m, H-8′a), 1.09 (1H, m, H-8′b), 2.44 (1H, m, H-9′), 1.16 (3H, d, J = 7.5 Hz, H-10′). 3-Bz: 8.15 (2H, dd, J = 8.3, 1.1 Hz, H-3, H-7), 7.33 (2H, dd, J = 8.3, 7.5 Hz, H-4,6), 7.49 (1H, tt, J = 7.5, 1.1 Hz, H-5). 18-Bz: 8.15 (2H, dd, J = 8.3, 1.1 Hz, H-3, H-7), 7.33 (2H, dd, J = 8.3, 7.5 Hz, H-4, H-6), 7.49 (1H, tt, J = 7.5, 1.1 Hz, H-5). 2′-O-(2-Naphthoyl): 8.09 (1H, dd, J = 8.6, 1.6 Hz, H-3), 7.84 (1H, d, J = 8.6 Hz, H-4), 7.84 (1H, brd, J = 8.0 Hz, H-5), 7.53 (1H, ddd, J = 8.3, 6.9, 1.6 Hz, H-6), 7.47 (1H, ddd, J = 8.0, 6.9, 1.4 Hz, H-7), 7.89 (1H, brd, J = 8.0 Hz, H-8), 8.64 (1H, brs, H-9); ¹³C NMR (CDCl₃, 125 MHz) δ 49.5 (C-1), 37.4 (C-2), 82.8 (C-3), 79.7 (C-4), 73.8 (C-5), 60.4 (C-6), 63.4 (C-7), 36.6 (C-8), 81.7 (C-9), 47.9 (C-10), 41.2 (C-11), 29.5 (C-12), 84.2 (C-13), 81.2 (C-14), 145.7 (C-15), 111.7 (C-16), 18.9 (C-17), 67.8 (C-18), 14.7 (C-19), 65.9 (C-20), 117.6 (C-1′), 71.0 (C-2′), 27.4 (C-3′), 24.4 (C-4′), 24.0 (C-5′), 23.7 (C-6′), 23.6 (C-7′), 23.0 (C-8′), 27.9 (C-9′), 18.3 (C-10′). 3-Bz: 168.2 (C-1), 129.5 (C-2), 130.1 (C-3, C-7), 128.5 (C-4, C-6), 133.4 (C-5). 18-OBz: 166.4 (C-1), 130.7 (C-2), 129.7 (C-3,7), 128.4 (C-4,6), 132.9 (C-5). 2′-O-(2-Naphthoyl): 166.1 (C-1), 127.3 (C-2), 125.6 (C-3), 127.9 (C-4), 127.7 (C-5), 128.2 (C-6), 126.5 (C-7), 129.3 (C-8), 131.5 (C-9), 132.5 (C-10), 135.6 (C-11); positive-ion HRFABMS m/z 951.3933 [M + Na]⁺, (calcd for C₅₅H₆₀O₁₃Na, 951.3932).

General Procedure for Replacement of the 3-Benzoyl Group with Different Ester Groups (13a–13e).

tert-Butyldimethylsilyl trifluoromethanesulfonate was added to a solution of 1 in anhydrous pyridine (5.0 mL). The mixture was stirred at 0 °C for 1 h under argon. Purification by HPLC (YMC Pack Pro C₁₈, MeOH, 5 mL/min) gave intermediate 10. Intermediate 11 was obtained by adding K₂CO₃ to a stirred solution of 10 in MeOH (10.0 mL). To a stirred solution of 11 in anhydrous DCM (3.0 mL) was added the 4-methoxybenzoyl chloride or appropriate acid, EDCI, DMAP under argon. After removal of solvent in vacuo, the residue was purified by RP-HPLC to give 12a–12e. The deprotection of 13a–13e was easily achieved using Py·HF (2 equiv) at rt for 4 h.

3-O-(4-Methoxybenzoyl) Stelleramacrin (13a).

Compound 13a was obtained from the deprotection of 12a. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 13a (2.1 mg, 45.7%, t_R = 25.5 min). 13a: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ + 2.4 (c 0.20, CHCl₃); IR (KBr) max: 2925, 1717, 1457, 1376, 1272, 1169, 1105, 1015, 766, 713 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.86 (1H, t, J = 12.3 Hz, H-1), 1.84 (1H, m, H-2), 4.90 (1H, d, J = 4.9 Hz, H-3), 4.04 (1H, brs, H-5), 3.35 (1H, s, H-7), 3.02 (1H, d, J = 2.9, Hz, H-8), 2.98 (1H, d, J = 12.3 Hz, H-10), 2.82 (1H, ddd, J = 10.3, 7.5, 2.0 Hz, H-11), 2.32 (1H, d, J = 14.6, H-12a), 1.96 (1H, dd, J = 14.6, 7.5 Hz, H-12b), 4.36 (1H, d, J = 2.9 Hz, H-14), 5.16 (1H, brs, H-16a), 4.92 (1H, t, J = 1.5 Hz, H-16b), 1.80 (3H, s, CH₃-17), 5.11 (1H, dd, J = 10.6, 2.0 Hz, H-18a), 4.36 (1H, dd, J = 10.6, 10.3 Hz, H-18b), 1.14 (3H, d, J = 6.8 Hz, CH₃-19), 3.85 (1H, d, J = 12.3 Hz, H-20a), 3.81 (1H, d, J = 12.3 Hz, H-20b), 3.87 (1H, d, J = 7.7 Hz, H-2′), 1.73 (1H, t, J = 13.7 Hz, H-3′a), 1.53 (1H, m, H-3′b), 1.60 (1H, m, H-4′a), 1.27 (1H, m, H-4′b), 1.32 (1H, m, H-5′a), 1.22 (1H, m, H-5′b), 1.60 (1H, m, H-6′a), 1.34 (1H, m, H-6′b), 1.39 (1H, m, H-7′a), 1.28 (1H, m, H-7′b), 1.60 (1H, m, H-8′a), 1.04 (1H, m, H-8′b), 2.35 (1H, m, H-9′), 1.08 (3H, d, J = 7.5 Hz, H-10′). 3-O-(4-Methoxybenzoyl): 8.10 (2H, dt, J = 9.2, 2.2 Hz, H-3, H-7), 7.44 (2H, dt, J = 9.2, 2.2 Hz, H-4, H-6), 3.71 (3H, s, 4-OCH₃). 18-OBz: 8.10 (2H, dd, J = 8.3, 1.4 Hz, H-3, H-7), 7.46 (2H, dd, J = 8.3, 7.4 Hz, H-4, H-6), 7.57 (1H, tt, J = 7.4, 1.4 Hz, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 49.3 (C-1), 37.2 (C-2), 82.3 (C-3), 79.8 (C-4), 73.8 (C-5), 60.7 (C-6), 63.6 (C-7), 36.6 (C-8) 81.4 (C-9), 48.1 (C-10), 41.3 (C-11), 29.7 (C-12), 84.4 (C-13), 81.4 (C-14), 145.5 (C-15), 111.8 (C-16), 19.0 (C-17), 67.7 (C-18), 14.6 (C-19), 67.7 (C-20), 118.5 (C-1′), 70.7 (C-2′), 28.5 (C-3′), 25.2 (C-4′), 24.0 (C-5′), 23.7 (C-6′), 23.1 (C-7′), 22.6 (C-8′), 27.3 (C-9′), 18.1 (C-10′). 3-O-(4-Methoxybenzoyl): 168.1 (C-1), 129.5 (C-2), 132.2 (C-3,7), 113.9 (C-4,6), 163.9 (C-5), 55.3 (5-OMe). 18-OBz: 166.4 (C-1), 130.7 (C-2), 129.7 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5); positive-ion HRFABMS m/z 805.3797 [M + H]⁺, (calcd for C₄₅H₅₇O₁₃, 805.3799); 827.3619 [M + Na]⁺, (calcd for C₄₅H₅₆O₁₃Na, 827.3619).

3-O-(4-Nitrobenzoyl) Stelleramacrin (13b).

Compound 13b was obtained from the deprotection of 12b. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 13b (2.3 mg, 73.5%, t_R = 24.0 min). 13b: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ + 10.7 (c 0.20, CHCl₃); IR (KBr) max: 3447, 2924, 1719, 1458, 1273, 1104, 1014, 762, 713 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 3.00 (1H, t, J = 12.4, 12.0 Hz, H-1), 1.86 (1H, m, H-2), 4.95 (1H, d, J = 4.6 Hz, H-3), 4.09 (1H, brs, H-5), 3.34 (1H, s, H-7), 2.99 (1H, d, J = 2.9, Hz, H-8), 3.00 (1H, d, J = 12.4 Hz, H-10), 2.75 (1H, ddd, J = 10.6, 7.7, 2.9 Hz, H-11), 2.34 (1H, d, J = 14.6, H-12a), 1.93 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.35 (1H, d, J = 2.9 Hz, H-14), 5.17 (1H, s, H-16a), 4.93 (1H, t, J = 1.5 Hz, H-16b), 1.79 (3H, s, CH₃-17), 5.17 (1H, m, H-18a), 4.38 (1H, dd, J = 10.9, 10.6 Hz, H-18b), 1.20 (3H, d, J = 6.8 Hz, CH₃-19), 3.89 (1H, d, J = 12.4 Hz, H-20a), 3.77 (1H, d, J = 12.4 Hz, H-20b), 3.88 (1H, d, J = 7.4 Hz, H-2′), 1.73 (1H, dd, J = 14.0, 13.2 Hz, H-3′a), 1.53 (1H, m, H-3′b), 1.60 (1H, m, H-4′a), 1.25 (1H, m, H-4′b), 1.37 (1H, m, H-5′a), 1.24 (1H, m, H-5′b), 1.61 (1H, m, H-6′a), 1.37 (1H, m, H-6′b), 1.37 (1H, m, H-7′a), 1.25 (1H, m, H-7′b), 1.61 (1H, m, H-8′a), 1.03 (1H, m, H-8′b), 2.37 (1H, m, H-9′), 1.15 (3H, d, J = 7.5 Hz, H-10′). 3-O-(4-Nitrobenzoyl): 8.34 (2H, dt, J = 8.9, 2.3 Hz, H-3, H-7), 8.17 (2H, dd, J = 8.9, 2.3 Hz, H-4, H-6). 18-OBz: 8.10 (2H, dd, J = 8.3, 1.2 Hz, H-3, H-7), 7.42 (2H, dd, J = 8.3, 7.7 Hz, H-4, H-6), 7.55 (1H, tt, J = 7.7, 1.2 Hz, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 49.3 (C-1), 37.3 (C-2), 83.6 (C-3), 79.6 (C-4), 73.8 (C-5), 60.7 (C-6), 63.6 (C-7), 36.7 (C-8), 81.2 (C-9), 48.2 (C-10), 41.2 (C-11), 29.4 (C-12), 84.4 (C-13), 81.3 (C-14), 145.5 (C-15), 111.8 (C-16), 19.0 (C-17), 67.5 (C-18), 14.6 (C-19), 65.9 (C-20), 118.5 (C-1′), 70.6 (C-2′), 28.5 (C-3′), 25.1 (C-4′), 24.0 (C-5′), 23.7 (C-6′), 23.0 (C-7′), 22.5 (C-8′), 27.3 (C-9′), 18.1 (C-10′). 3-O-(4-Nitrobenzoyl): 166.4 (C-1), 134.9 (C-2), 131.3 (C-3, C-7), 123.6 (C-4, C-6), 150.8 (C-5). 18-OBz: 166.6 (C-1), 130.7 (C-2), 129.5 (C-3, C-7), 128.6 (C-4, C-6), 133.3 (C-5); positive-ion HRFABMS m/z 842.3363 [M + Na]⁺, (calcd for C₄₄H₅₃NO₁₄Na, 842.3364).

3-O-(trans-Cinnamoyl) Stelleramacrin (13c).

Compound 13c was obtained from the deprotection of 12c. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 13c (1.6 mg, 40.3%, t_R = 27.0 min). 13c: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ + 9.7 (c 0.20, CHCl₃); IR (KBr) max: 3472, 2926, 1718, 1458, 1386, 1272, 1170, 1105, 1024, 766, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.76 (1H, dd, J = 12.6, 12.0 Hz, H-1), 1.81 (1H, m, H-2), 4.84 (1H, d, J = 4.9 Hz, H-3), 4.01 (1H, brs, H-5), 3.36 (1H, s, H-7), 3.03 (1H, d, J = 3.0 Hz, H-8), 2.96 (1H, d, J = 12.6 Hz, H-10), 2.83 (1H, ddd, J = 10.3, 7.7, 2.6 Hz, H-11), 2.36 (1H, d, J = 14.6, H-12a), 2.00 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.38 (1H, d, J = 3.0 Hz, H-14), 5.16 (1H, s, H-16a), 4.92 (1H, t, J = 1.5 Hz, H-16b), 1.79 (3H, s, CH₃-17), 5.01 (1H, dd, J = 10.3, 2.6 Hz, H-18a), 4.38 (1H, t, J = 10.3 Hz, H-18b), 1.13 (3H, d, J = 6.6 Hz, CH₃-19), 3.83 (2H, brs, H₂-20), 3.87 (1H, d, J = 7.8 Hz, H-2′), 1.73 (1H, t, J = 13.8 Hz, H-3′a), 1.55 (1H, m, H-3′b), 1.61 (1H, m, H-4′a), 1.25 (1H, m, H-4′b), 1.34 (1H, m, H-5′a), 1.26 (1H, m, H-5′b), 1.58 (1H, m, H-6′a), 1.35 (1H, m, H-6′b), 1.37 (1H, m, H-7′a), 1.24 (1H, m, H-7′b), 1.59 (1H, m, H-8′a), 1.01 (1H, m, H-8′b), 2.37 (1H, m, H-9′), 1.08 (3H, d, J = 7.5 Hz, H-10′). 3-O-(trans-Cinnamoyl): 7.80 (1H, d, J = 16.1 Hz, H-2), 6.49 (1H, d, J = 16.1 Hz, H-3), 7.49 (2H, dd, J = 7.8, 1.5 Hz, H-5, H-9), 7.26 (2H, dd, J = 7.8, 7.5 Hz, H-6, H-8), 7.37 (1H, tt, J = 7.5, 1.5 Hz, H-7). 18-Bz: 8.06 (2H, dd, J = 8.3, 1.1 Hz, H-3, H-7), 7.40 (2H, dd, J = 8.3, 7.5 Hz, H-4, H-6), 7.54 (1H, tt, J = 7.5, 1.1 Hz, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 49.3 (C-1), 37.4 (C-2), 82.2 (C-3), 79.6 (C-4), 73.8 (C-5), 60.7 (C-6), 63.5 (C-7), 36.8 (C-8), 81.3 (C-9), 48.1 (C-10), 41.1 (C-11), 29.6 (C-12), 84.3 (C-13), 81.5 (C-14), 145.5 (C-15), 111.8 (C-16), 19.0 (C-17), 67.7 (C-18), 14.5 (C-19), 65.8 (C-20), 118.5 (C-1′), 70.7 (C-2′), 28.5 (C-3′), 25.2 (C-4′), 24.0 (C-5′), 23.7 (C-6′), 23.0 (C-7′), 22.5 (C-8′), 27.2 (C-9′), 18.1 (C-10′). 3-O-trans-Cinnamoyl: 168.6 (C-1), 117.3 (C-2), 146.4 (C-3), 134.2 (C-4), 128.4 (C-5,9), 128.8 (C-6,8), 130.6 (C-7). 18-OBz: 166.5 (C-1), 130.7 (C-2), 129.6 (C-3,7), 128.6 (C-4,6), 132.8 (C-5); positive-ion HRFABMS m/z 823.3668 [M + Na]⁺, (calcd for C₄₆H₅₆O₁₂Na, 823.3669).

3-O-(2-Naphthoyl) Stelleramacrin (13d).

Compound 13d was obtained from the deprotection of 12d. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 13d (4.4 mg, 72.0%, t_R = 36.0 min). 13d: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ + 22.1 (c 0.30, CHCl₃); IR (KBr) max: 2923, 1717, 1458, 1272, 1106, 767, 717 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.92 (1H, dd, J = 12.3, 12.0 Hz, H-1), 1.84 (1H, m, H-2), 4.90 (1H, d, J = 4.9 Hz, H-3), 4.09 (1H, brs, H-5) 3.36 (1H, s, H-7), 3.03 (1H, d, J = 2.9, Hz, H-8), 3.02 (1H, d, J = 12.0 Hz, H-10), 2.82 (1H, ddd, J = 10.3, 7.6, 2.3 Hz, H-11), 2.32 (1H, d, J = 14.6, H-12a), 1.94 (1H, dd, J = 14.6, 7.6 Hz, H-12b), 4.37 (1H, d, J = 2.6 Hz, H-14), 5.15 (1H, s, H-16a), 4.91 (1H, t, J = 1.5 Hz, H-16b), 1.79 (3H, s, CH₃-17), 5.15 (1H, m, H-18a), 4.38 (1H, dd, J = 10.6, 10.3 Hz, H-18b), 1.20 (3H, d, J = 6.8 Hz, CH₃-19), 3.85 (2H, brs H-20), 3.88 (1H, d, J = 7.4 Hz, H-2′), 1.73 (1H, dd, J = 12.6 Hz, H-3′a), 1.53 (1H, m, H-3′b), 1.60 (1H, m, H-4′a), 1.25 (1H, m, H-4′b), 1.35 (1H, m, H-5′a), 1.20 (1H, m, H-5′b), 1.60 (1H, m, H-6′a), 1.37 (1H, m, H-6′b), 1.35 (1H, m, H-7′a), 1.28 (1H, m, H-7′b), 1.62 (1H, m, H-8′a), 1.05 (1H, m, H-8′b), 2.35 (1H, m, H-9′), 1.15 (3H, d, J = 7.5 Hz, H-10′). 3-O-(2-Naphthoyl): 8.09 (1H, dd, J = 8.6, 1.5 Hz, H-3), 7.72 (1H, d, J = 8.6 Hz, H-4), 7.79 (1H, brd, J = 8.0 Hz, H-5), 7.52 (1H, ddd, J = 8.3, 6.0, 1.1 Hz, H-6), 7.43 (1H, m, H-7), 7.91 (1H, brd, J = 8.3 Hz, H-8), 8.70 (1H, brs, H-9). 18-OBz: 8.10 (2H, dd, J = 8.3, 1.2 Hz, H-3, H-7), 7.42 (2H, dd, J = 8.3, 7.7 Hz, H-4, H-6), 7.55 (1H, tt, J = 7.7, 1.2 Hz, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 49.6 (C-1), 37.5 (C-2), 82.8 (C-3), 79.7 (C-4), 73.8 (C-5), 60.7 (C-6), 63.6 (C-7), 36.8 (C-8), 81.4 (C-9), 48.2 (C-10), 41.1 (C-11), 29.4 (C-12), 84.3 (C-13), 81.3 (C-14), 145.5 (C-15), 111.8 (C-16), 19.0 (C-17), 67.7 (C-18), 14.7 (C-19), 65.8 (C-20), 118.5 (C-1′), 70.7 (C-2′), 28.5 (C-3′), 25.2 (C-4′), 24.0 (C-5′), 23.7 (C-6′), 23.0 (C-7′), 22.6 (C-8′), 27.3 (C-9′), 18.2 (C-10′). 3-O-(2-Naphthoyl): 168.5 (C-1), 126.6 (C-2), 125.3 (C-3), 128.4 (C-4), 127.6 (C-5), 128.5 (C-6), 126.6 (C-7), 129.8 (C-8), 131.9 (C-9), 132.5 (C-10), 135.8 (C-11). 18-OBz: 166.5 (C-1‴), 130.7 (C-2‴), 129.7 (C-3‴, C-7‴), 128.3 (C-4‴, C-6‴), 132.7 (C-5‴); positive-ion HRFABMS m/z 847.3668 [M + Na]⁺, (calcd for C₄₈H₅₆O₁₂Na, 847.3669).

3-O-(4-Pyridinylcarbonyl) Stelleramacrin (13e).

Compound 13e was obtained from the deprotection of 12e. The residue was purified by RP-HPLC (MeCN─H₂O, 85:15, 5 mL/min) to give 13e (3.4 mg, 50.1%, t_R = 19.5 min). 13e: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 13.7 (c 0.20, CHCl₃); IR (KBr) max: 2854, 1719, 1645, 1465, 1378, 1272, 1109, 1013, 714 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 3.00 (1H, m, H-1), 1.85 (1H, m, H-2), 4.95 (1H, d, J = 4.9 Hz, H-3), 4.08 (1H, brs, H-5), 3.34 (1H, s, H-7), 3.00 (1H, m, H-8), 3.00 (1H, m, H-10), 2.78 (1H, ddd, J = 10.9, 7.7, 2.6 Hz, H-11), 2.34 (1H, d, J = 14.6 Hz, H-12a), 1.93 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.35 (1H, d, J = 2.9 Hz, H-14), 5.16 (1H, s, H-16a), 4.93 (1H, t, J = 1.4 Hz, H-16b), 1.80 (3H, s, CH₃-17), 5.17 (1H, dd, J = 10.9, 2.6 Hz, H-18a), 4.38 (1H, t, J = 10.9 Hz, H₂-18b), 1.17 (3H, d, J = 6.6 Hz, CH₃-19), 3.89 (1H, d, J = 12.6 Hz, H-20a), 3.79 (1H, d, J = 12.6 Hz, H-20b), 3.87 (1H, d, J = 7.3 Hz, H-2′), 1.73 (1H, t, J = 13.6 Hz, H-3′a), 1.53 (1H, m, H-3′b), 1.60 (1H, m, H-4′a), 1.25 (1H, m, H-4′b), 1.30 (1H, m, H-5′a), 1.23 (1H, m, H-5′b), 1.60 (1H, m, H-6′a), 1.37 (1H, m, H-6′b), 1.37 (1H, m, H-7′a), 1.25 (1H, m, H-7′b), 1.61 (1H, m, H-8′a), 1.03 (1H, m, H-8′b), 2.35 (1H, m, H-9′), 1.12 (3H, d, J = 7.4 Hz, H-10′). 3-O-(4-Pyridinylcarbonyl): 8.01 (2H, dt, J = 8.9, 2.3 Hz, H-3, H-7), 8.70 (2H, dt, J = 8.9, 2.3 Hz, H-4, H-6). 18-Bz: 8.10 (2H, dd, J = 8.3, 1.1 Hz, H-3, H-7), 7.49 (2H, dd, J = 8.3, 7.5 Hz, H-4, H-6), 7.55 (1H, tt, J = 7.5, 1.1 Hz, H-5‴); ¹³C NMR (CDCl₃, 125 MHz) δ 49.3 (C-1), 37.4 (C-2), 83.6 (C-3), 79.6 (C-4), 73.3 (C-5), 60.7 (C-6), 63.5 (C-7), 36.7 (C-8), 81.3 (C-9), 48.2 (C-10), 41.1 (C-11), 29.3 (C-12), 84.4 (C-13), 81.4 (C-14), 145.5 (C-15), 111.9 (C-16), 19.0 (C-17), 67.5 (C-18), 14.6 (C-19), 65.9 (C-20), 118.5 (C-1′), 70.6 (C-2′), 28.5 (C-3′), 25.1 (C-4′), 24.0 (C-5′), 23.7 (C-6′), 23.0 (C-7′), 22.5 (C-8′), 27.3 (C-9′), 18.1 (C-10′). 3-O-(4-Pyridinylcarbonyl): 166.6 (C-1), 136.9 (C-2), 123.3 (C-3, C-7), 150.6 (C-4, C-6). 18-Bz: 166.8 (C-1), 130.5 (C-2), 129.5 (C-3, C-7), 128.6 (C-4, C-6), 133.2 (C-5); positive-ion HRFABMS m/z 776.3644 [M + H]⁺, (calcd for C₄₃H₅₄NO₁₂, 776.3646); 798.3465 [M + Na]⁺, (calcd for C₄₃H₅₃NO₁₂Na, 798.3465).

Preparation of 5-Derivatives (15a, 15b, and 18). 5-O-Benzoyl Gnidimacrin (15a).

To a stirred solution of 8 in anhydrous DCM (3.0 mL) was added benzoic acid (10 equiv) in the presence of EDCI and DMAP under argon. After removal of solvent in vacuo, the residue was purified by RP-HPLC to give 14a. Compound 15a was obtained from the deprotection of 14a using Py·HF (1.6 mL) in pyridine (1 mL) at rt for 4 days. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 15a (2.2 mg, 93.7%, t_R = 31.5 min). 15a: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 5.0 (c 0.20, CHCl₃); IR (KBr) max: 2926, 2855, 1718, 1452, 1383, 1271, 1106, 1069, 1025, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.94 (1H, dd, J = 12.3, 11.7 Hz, H-1), 2.01 (1H, m, H-2), 4.86 (1H, d, J = 4.3 Hz, H-3), 5.69 (1H, brs, H-5), 3.52 (1H, s, H-7), 3.25 (1H, d, J = 2.8 Hz, H-8), 3.15 (1H, d, J = 12.3 Hz, H-10), 2.78 (1H, ddd, J = 10.0, 7.3, 2.0 Hz, H-11), 2.31 (1H, d, J = 14.6, H₂-12a), 1.96 (1H, dd, J = 14.6, 7.3 Hz, H₂-12b), 4.40 (1H, d, J = 2.8 Hz, H-14), 5.17 (1H, brs, H-16a), 4.93 (1H, t, J = 1.4 Hz, H-16b), 1.81 (3H, s, CH₃-17), 5.10 (1H, dd, J = 10.9, 2.0 Hz, H-18a), 4.38 (1H, dd, J = 10.9, 10.0 Hz, H-18b), 1.13 (3H, d, J = 6.6 Hz, CH₃-19), 3.81 (1H, d, J = 12.3 Hz, H-20a), 3.60 (1H, d, J = 12.3 Hz, H₂-20b), 3.91 (1H, d, J = 7.4 Hz, H-2′), 1.78 (1H, t, J = 13.5 Hz, H-3′a), 1.55 (1H, m, H-3′b), 1.50–1.90 (1H, m, H-4′a), 1.25–1.40 (1H, m, H-4′b), 1.50–1.90 (1H, m, H-5′a), 1.25–1.40 (1H, m, H-5′b), 1.50–1.90 (1H, m, H-6′a), 1.25–1.40 (1H, m, H-6′b), 1.25–1.40 (1H, m, H-7), 1.50–1.90 (1H, m, H-8′a), 1.10–1.20 (1H, m, H-8′b), 2.31–2.38 (1H, m, H-9′), 1.13 (3H, d, J = 7.4 Hz, H-10′). 3-OBz: 7.99 (2H, dd, J = 8.6, 1.4 Hz, H-3, H-7), 7.20 (2H, dd, J = 8.6, 7.4 Hz, H-4, H-6), 7.42 (1H, tt, J = 7.4, 1.4 Hz, H-5). 5-OBz: 8.13 (2H, dd, J = 8.4, 1.1 Hz, H-3, H-7), 7.42 (2H, dd, J = 8.4, 7.5 Hz, H-4, H-6), 7.62 (1H, tt, J = 7.5, 1.1 Hz, H-5). 18-Bz: 8.05 (2H, dd, J = 8.4, 1.4 Hz, H-3, H-7), 7.49 (2H, dd, J = 8.0, 7.5 Hz, H-4, H-6), 7.56 (1H, tt, J = 7.5, 1.4 Hz, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 49.7 (C-1), 37.2 (C-2), 82.5 (C-3), 80.7 (C-4), 72.9 (C-5), 60.6 (C-6), 62.9 (C-7), 36.9 (C-8), 81.0 (C-9), 47.8 (C-10), 41.1 (C-11), 30.0 (C-12), 84.6 (C-13), 81.3 (C-14), 145.6 (C-15), 111.8 (C-16), 19.0 (C-17), 67.6 (C-18), 14.8 (C-19), 62.7 (C-20), 118.6 (C-1′), 70.8 (C-2′), 28.7 (C-3′), 25.3 (C-4′), 24.3 (C-5′), 23.7 (C-6′), 23.2 (C-7′), 22.9 (C-8′), 27.5 (C-9′), 18.2 (C-10′). 3-OBz: 168.3 (C-1), 130.6 (C-2), 130.0 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5). 5-OBz: 166.3 (C-1), 129.0 (C-2), 130.2 (C-3, C-7), 128.3 (C-4, C-6), 129.0 (C-5). 18-OBz: 166.3 (C-1), 130.8 (C-2), 129.7 (C-3, C-7), 128.6 (C-4, C-6), 133.0 (C-5); positive-ion HRFABMS m/z 901.3774 [M + Na]⁺, (calcd for C₅₁H₅₈O₁₃Na, 901.3775).

5-Oxognidimacrin (15b).

To a stirred solution of 8 in super-dehydrated DCM (3.0 mL) was added Dess–Martin periodinane under argon. After removal of solvent in vacuo, the residue was purified by RP-HPLC to give 14b. Compound 15b was obtained from the deprotection of 14b similar to 15a from that of 14a. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 15b (3.1 mg, 80.3%, t_R = 18.3 min). 15b: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 48.1 (c 0.30, CHCl₃); IR (KBr) max: 2925, 2853, 1719, 1458, 1271, 1114, 1068, 1024, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.76 (1H, t, J = 12.3 Hz, H-1), 2.43 (1H, m, H-2), 5.19 (1H, d, J = 10.1 Hz, H-3), 3.63 (1H, s, H-7), 3.16 (1H, d, J = 2.9 Hz, H-8), 3.04 (1H, d, J = 12.3 Hz, H-10), 2.75 (1H, ddd, J = 10.6, 7.7, 2.0 Hz, H-11), 2.31 (1H, d, J = 14.6, H-12a), 1.93 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.35 (1H, d, J = 2.9 Hz, H-14), 5.16 (1H, s, H-16a), 4.93 (1H, t, J = 1.3 Hz, H-16b), 1.80 (3H, s, CH₃-17), 4.93 (1H, dd, J = 10.9, 2.0 Hz, H-18a), 4.38 (1H, dd, J = 10.9, 10.6 Hz, H-18b), 1.30 (3H, d, J = 6.9 Hz, CH₃-19), 4.31 (1H, d, J = 12.8 Hz, H-20a), 3.81 (1H, d, J = 12.8 Hz, H-20b), 3.98 (1H, d, J = 8.0 Hz, H-2′), 1.83 (1H, o, H-3′a), 1.52 (1H, m, H-3′b), 1.66 (1H, m, H-4′a), 1.35 (1H, m, H-4′b), 1.63 (1H, m, H-5′a), 1.41 (1H, m, H-5′b), 1.50 (1H, m, H-6′a), 1.22 (1H, m, H-6′b), 1.51 (1H, m, H-7′a), 1.22 (1H, m, H-7′b), 1.83 (1H, m, H-8′a), 1.11 (1H, m, H-8′b), 2.25 (1H, m, H-9′), 0.97 (3H, d, J = 7.1 Hz, H-10′). 3-OBz: 8.01 (2H, dd, J = 8.6, 1.4 Hz, H-3, H-7), 7.44 (2H, dd, J = 8.6, 7.4 Hz, H-4, H-6), 7.58 (1H, tt, J = 7.4, 1.4 Hz, H-5). 18-OBz: 8.07 (2H, dd, J = 8.6, 1.4 Hz, H-3, H-7), 7.43 (2H, dd, J = 8.6, 7.5 Hz, H-4, H-6), 7.55 (1H, tt, J = 7.5, 1.4 Hz, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 51.0 (C-1), 33.6 (C-2), 75.0 (C-3), 81.4 (C-4), 208.9 (C-5), 65.9 (C-6), 60.8 (C-7), 36.7 (C-8), 84.6 (C-9), 46.5 (C-10), 40.9 (C-11), 30.5 (C-12), 85.4 (C-13), 79.9 (C-14), 144.8 (C-15), 112.2 (C-16), 19.0 (C-17), 67.9 (C-18), 18.9 (C-19), 60.2 (C-20), 118.6 (C-1′), 70.0 (C-2′), 28.4 (C-3′), 24.7 (C-4′), 24.1 (C-5′), 23.8 (C-6′), 22.5 (C-7′), 23.3 (C-8′), 29.6 (C-9′), 17.7 (C-10′). 3-OBz: 166.2 (C-1), 129.4 (C-2), 129.7 (C-3, C-7), 128.6 (C-4, C-6), 133.6 (C-5). 18-OBz: 166.4 (C-1), 130.5 (C-2), 129.5 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5); positive-ion HRFABMS m/z 773.3535 [M + H]⁺, (calcd for C₄₄H₅₄O₁₂, 773.3459); 795.3357 [M + Na]⁺, (calcd for C₄₄H₅₂O₁₂Na, 795.3357).

5-Acetoxygnidimacrin (18).

TBDMSCl (40.0 mg, 240 μmol) and DMAP (1.0 mg) were added to a solution of 1 (20.0 mg, 25.8 μmol) in anhydrous pyridine (3.0 mL). Then, the mixture was stirred at rt for 36 h under N₂. After removal of pyridine in vacuo, the residue was purified by HPLC chromatography (YMC Actus Triant C18, MeCN, 5 mL/min) to give intermediate 16 (17.0 mg, 73.6%, t_R = 22.8 min). Acetic anhydride and DMAP were added to a solution of 16 (10.0 mg, 11.3 μmol) in anhydrous pyridine (3.0 mL). The mixture was stirred at rt for 2 h under argon. After removal of pyridine in vacuo, the residue was purified by HPLC chromatography (YMC Pack Pro C18, MeCN, 5 mL/min) to obtain 17 (6.0 mg, 58%, t_R = 34.5 min). Et₃N·3HF (8.0 μL, 48.8 μmol) was added to a solution of 17 in THF (3.0 mL). The mixture was stirred at rt for 5 days under argon. Purification by HPLC chromatography (YMC Pack Sil, hexane-EtOAc, 35:65, 5 mL/min) gave 18 (2.4 mg, 45%, t_R = 25.5 min). Colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ + 12.4 (c 0.20, CHCl₃); IR (KBr) max: 2928, 1717, 1452, 1382, 1271, 1121, 1112, 1024, 754, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.92 (1H, dd, J = 12.2 Hz, H-1), 1.86 (1H, m, H-2), 4.98 (1H, d, J = 4.9 Hz, H-3), 5.41 (1H, s, H-5), 3.26 (1H, s, H-7), 3.04 (1H, d, J = 2.9 Hz, H-8), 3.01 (1H, d, J = 12.2 Hz, H-10), 2.75 (1H, ddd, J = 10.6, 7.7, 2.0 Hz, H-11), 2.33 (1H, d, J = 14.6, H-12a), 1.95 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.35 (1H, d, J = 2.9 Hz, H-14), 5.15 (1H, s, H-16a), 4.92 (1H, t, J = 1.4 Hz, H-16b), 1.80 (3H, s, CH₃-17), 5.09 (1H, dd, J = 10.6, 2.0 Hz, H-18a), 4.37 (1H, t, J = 10.6 Hz, H-18b), 1.17 (3H, d, J = 6.6 Hz, CH₃-19), 4.78 (1H, d, J = 12.0 Hz, H-20a), 3.82 (1H, d, J = 12.0 Hz, H-20b), 3.87 (1H, d, J = 7.4 Hz, H-2′), 1.73 (1H, t, J = 13.8 Hz, H-3′a), 1.56 (1H, m, H-3′b), 1.61 (1H, m, H-4′a), 1.31 (1H, m, H-4′b), 1.40 (1H, m, H-5′a), 1.20 (1H, m, H-5′b), 1.59 (1H, m, H-6′a), 1.33 (1H, m, H-6′b), 1.37 (1H, m, H-7′a), 1.26 (1H, m, H-7′b), 1.63 (1H, m, H-8′a), 1.03 (1H, m, H-8′b), 2.35 (1H, m, H-9′), 1.11 (3H, d, J = 7.4 Hz, H-10′). 3-OBz: 8.16 (2H, dd, J = 8.3, 1.2 Hz, H-3, H-7), 7.34 (2H, dd, J = 8.3, 7.4 Hz, H-4, H-6), 7.52 (1H, tt, J = 7.4, 1.2 Hz, H-5). 18-OBz: 8.11 (2H, dd, J = 8.3, 1.4 Hz, H-3, H-7), 7.46 (2H, dd, J = 8.3, 7.5 Hz, H-4, H-6), 7.58 (1H, tt, J = 7.5, 1.4 Hz, H-5). 5-Acetoxy: 2.13 (3H, s, COCH₃); ¹³C NMR (CDCl₃, 125 MHz) δ 49.4 (C-1), 37.5 (C-2), 82.8 (C-3), 80.0 (C-4), 71.8 (C-5), 60.6 (C-6), 63.6 (C-7), 36.7 (C-8), 81.2 (C-9), 48.0 (C-10), 41.1 (C-11), 29.5 (C-12), 84.3 (C-13), 81.4 (C-14), 145.6 (C-15), 111.9 (C-16), 19.0 (C-17), 67.6 (C-18), 14.6 (C-19), 67.0 (C-20), 118.6 (C-1′), 70.7 (C-2′), 28.6 (C-3′), 23.1 (C-4′), 25.2 (C-5′), 23.7 (C-6′), 23.0 (C-7′), 22.6 (C-8′), 27.3 (C-9′), 18.1 (C-10′). 3-OBz: 168.1 (C-1), 129.5 (C-2), 130.1 (C-3, C-7), 128.6 (C-4, C-6), 133.4 (C-5). 18-OBz: 166.4 (C-1), 130.6 (C-2), 129.7 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5). 5-Acetoxy: 170.8 (COCH₃), 20.9 (COCH₃); positive-ion HRFABMS m/z 817.3799 [M + H]⁺, (calcd for C₄₆H₅₇O₁₃, 817.3799).

General Procedure for Preparation of 20-Ester Derivatives 19a–19h.

To a stirred solution of 1 in anhydrous pyridine (3 mL) was added the corresponding anhydride or acid chloride under argon at rt. After removal of pyridine in vacuo, the residue was purified by HPLC chromatography (YMC Pack Pro C₁₈) to give 19a–19h.

20-Acetoxygnidimacrin (19a).

Compound 1 (6.0 mg, 7.8 μmol) was treated with acetic anhydride (8.0 μL, 88 μmol) and DMAP (1.0 mg) at rt for 14 h. The residue was purified by RP-HPLC (MeCN─H₂O, 90:10, 5 mL/min) to give 19a (3.4 mg, 53.4%, t_R = 15.0 min). 19a: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 8.5 (c 0.19, CHCl₃); IR (KBr) max: 3480, 2931, 1741, 1717, 1452, 1383, 1272, 1112, 1024, 756, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.91 (1H, t, J = 12.3 Hz, H-1), 1.90 (1H, m, H-2), 4.69 (1H, d, J = 4.3 Hz, H-3), 5.41 (1H, brs, H-5), 3.45 (1H, s, H-7), 3.16 (1H, d, J = 2.9 Hz, H-8), 3.04 (1H, d, J = 12.3 Hz, H-10), 2.75 (1H, ddd, J = 10.6, 7.7, 2.0 Hz, H-11), 2.31 (1H, d, J = 14.6, H-12a), 1.96 (1H, dd, J = 14.6, 7.7 Hz, H₂-12b), 4.36 (1H, d, J = 2.9 Hz, H-14), 5.15 (1H, s, H-16a), 4.92 (1H, t, J = 1.3 Hz, H-16b), 1.80 (3H, s, CH₃-17), 5.09 (1H, dd, J = 10.6, 2.0 Hz, H-18a), 4.38 (1H, t, J = 10.6 Hz, H-18b), 1.13 (3H, d, J = 6.6 Hz, CH₃-19), 4.78 (1H, d, J = 12.0 Hz, H-20a), 3.82 (1H, d, J = 12.0 Hz, H-20b), 3.89 (1H, d, J = 7.5 Hz, H-2′), 1.74 (1H, t, J = 13.8 Hz, H-3′a), 1.56 (1H, m, H-3′b), 1.62 (1H, m, H-4′a), 1.39 (1H, m, H-4′b), 1.37 (1H, m, H-5′a), 1.22 (1H, m, H-5′b), 1.59 (1H, m, H-6′a), 1.33 (1H, m, H-6′b), 1.36 (1H, m, H-7′a), 1.26 (1H, m, H-7′b), 1.61 (1H, m, H-8′a), 1.06 (1H, m, H-8′b), 2.35 (1H, m, H-9′), 1.12 (3H, d, J = 7.4 Hz, H-10′). 3-OBz: 8.10 (2H, dd, J = 8.2, 1.1 Hz, H-3, H-7), 7.27 (2H, dd, J = 8.2, 7.5 Hz, H-4, H-6), 7.45 (1H, tt, J = 7.5, 1.1 Hz, H-5). 18-OBz: 8.07 (2H, dd, J = 8.1, 1.2 Hz, H-3, H-7), 7.49 (2H, dd, J = 8.1, 7.5 Hz, H-4, H-6), 7.56 (1H, tt, J = 7.5, 1.2 Hz, H-5). 20-Acetoxy: 2.13 (s, COCH₃); ¹³C NMR (CDCl₃, 125 MHz) δ 49.5 (C-1), 37.1 (C-2), 82.2 (C-3), 80.5 (C-4), 72.1 (C-5), 60.3 (C-6), 62.8 (C-7), 36.8 (C-8), 81.0 (C-9), 47.7 (C-10), 41.1 (C-11), 29.6 (C-12), 84.5 (C-13), 81.3 (C-14), 145.5 (C-15), 111.8 (C-16), 19.0 (C-17), 67.5 (C-18), 14.8 (C-19), 62.7 (C-20), 118.6 (C-1′), 70.8 (C-2′), 28.6 (C-3′), 25.2 (C-4′), 24.2 (C-5′), 23.7 (C-6′), 23.1 (C-7′), 22.7 (C-8′), 27.4 (C-9′), 18.1 (C-10′). 3-OBz: 168.4 (C-1), 129.8 (C-2), 130.0 (C-3, C-7), 128.4 (C-4, C-6), 133.1 (C-5). 18-OBz: 166.3 (C-1), 130.6 (C-2), 129.7 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5). 20-Acetoxy: 170.8 (COCH₃), 20.9 (COCH₃); positive-ion HRFABMS m/z 817.3798 [M + H]⁺, (calcd for C₄₆H₅₇O₁₃Na, 817.3799).

20-O-Benzoyl Gnidimacrin (19b).

Compound 1 (5.0 mg, 6.3 μmol) was treated with benzyl anhydride (10.0 mg, 44 μmol) at rt for 14 h. The residue was purified by RP-HPLC (MeCN─H₂O, 95:5, 5 mL/min) to give 19b (2.7 mg, 48.8%, t_R = 30.0 min). 19b: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 6.8 (c 0.19, CHCl₃); IR (KBr) max: 2929, 1719, 1451, 1383, 1274, 1114, 1024, 711 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.90 (1H, dd, J = 12.6, 11.8 Hz, H-1), 1.87 (1H, m, H-2), 4.99 (1H, d, J = 4.9 Hz, H-3), 4.07 (1H, s, H-5), 3.32 (1H, s, H-7), 3.09 (1H, d, J = 2.6 Hz, H-8), 3.03 (1H, d, J = 12.6 Hz, H-10), 2.87 (1H, ddd, J = 10.6, 7.8, 2.0 Hz, H-11), 2.43 (1H, d, J = 14.6, H₂-12a), 1.97 (1H, dd, J = 14.6, 7.8 Hz, H₂-12b), 4.37 (1H, d, J = 2.6 Hz, H-14), 5.17 (1H, s, H-16a), 4.93 (1H, t, J = 1.4 Hz, H-16b), 1.81 (3H, s, H₃–17), 5.10 (1H, dd, J = 10.9, 2.0 Hz, H₂-18a), 4.47 (1H, dd, J = 10.9, 10.6 Hz, H₂-18b), 1.13 (3H, d, J = 6.6 Hz, H₃-19), 5.07 (1H, d, J = 12.0 Hz, H₂-20a), 4.08 (1H, d, J = 12.0 Hz, H₂-20b), 3.89 (1H, d, J = 7.4 Hz, H-2′), 1.74 (1H, t, J = 13.5 Hz, H-3′a), 1.52 (1H, m, H-3′b), 1.65 (1H, m, H-4′a), 1.26 (1H, m, H-4′b), 1.35 (1H, m, H-5′a), 1.24 (1H, m, H-5′b), 1.54 (1H, m, H-6′a), 1.38 (1H, m, H-6′b), 1.35 (1H, m, H-7′a), 1.27 (1H, m, H-7′b), 1.62 (1H, m, H-8′a), 1.05 (1H, m, H-8′b), 2.38 (1H, m, H-9′), 1.13 (3H, d, J = 7.4 Hz, H-10′). 3-Bz: 8.15 (2H, dd, J = 8.3, 1.2 Hz, H-3,7), 7.33 (2H, dd, J = 8.3, 7.5 Hz, H-4,6), 7.51 (1H, tt, J = 7.5, 1.2 Hz, H-5). 18-Bz: 8.11 (2H, dd, J = 8.3, 1.2 Hz, H-3,7), 7.47 (2H, dd, J = 8.3, 7.5 Hz, H-4,6), 7.58 (1H, tt, J = 7.5, 1.2 Hz, H-5). 20-Bz: 8.09 (2H, dd, J = 8.3, 1.2 Hz, H-3,7), 7.46 (2H, dd, J = 8.3, 7.4 Hz, H-4,6), 7.58 (1H, tt, J = 7.5, 1.2 Hz, H-5); ¹³C NMR (CDCl₃, 125 MHz) δ 49.5 (C-1), 37.5 (C-2), 82.8 (C-3), 79.6 (C-4), 70.7 (C-5), 59.7 (C-6), 63.6 (C-7), 36.7 (C-8), 81.2 (C-9), 48.0 (C-10), 41.2 (C-11), 29.6 (C-12), 84.3 (C-13), 81.4 (C-14), 145.6 (C-15), 111.9 (C-16), 19.1 (C-17), 67.6 (C-18), 14.6 (C-19), 67.5 (C-20), 118.6 (C-1′), 71.9 (C-2′), 28.6 (C-3′), 25.2 (C-4′), 24.4 (C-5′), 24.0 (C-6′), 23.7 (C-7′), 22.6 (C-8′), 27.3 (C-9′), 18.1 (C-10′). 3-Bz: 168.0 (C-1), 130.6 (C-2), 130.1 (C-3, C-7), 128.5 (C-4, C-6), 133.4 (C-5). 18-Bz: 166.7 (C-1), 129.8 (c-2), 130.0 (C-3, C-7), 128.4 (C-4, C-6), 133.2 (C-5). 20-Bz: 166.4 (C-1), 129.8 (C-2), 129.6 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5); positive-ion HRFABMS m/z 901.3650 [M + Na]⁺, (calcd for C₅₁H₅₈O₁₃Na, 901.3673).

20-Succinoxy Gnidimacrin (19c).

Compound 1 (5.0 mg, 6.3 μmol) was treated with 2,2-dimethylsuccinic anhydride (30.0 μL, 27 μmol) and DMAP (3.0 mg) at rt for 14 h. The residue was purified by RP-HPLC (MeCN─H₂O, 95:5, 5 mL/min) to give 19c (3.8 mg, 42.2%, recycle 5 times). 19c: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 3.8 (c 0.20, CHCl₃); IR (KBr) max: 3506, 2933, 2860, 1718, 1601, 1452, 1383, 1272, 1171, 1116, 1024, 756, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.87 (1H, t, J = 12.3 Hz, H-1), 1.82 (1H, m, H-2), 5.02 (1H, d, J = 4.6 Hz, H-3), 4.05 (1H, s, H-5), 3.26 (1H, d, J = 2.9 Hz, H-8), 3.00 (1H, d, J = 12.3 Hz, H-10), 2.88 (1H, m, H-11), 2.32 (1H, d, J = 14.6, H-12a), 1.97 (1H, dd, J = 14.6, 7.7 Hz, H-12b), 4.37 (1H, d, J = 2.9 Hz, H-14), 5.15 (1H, s, H-16a), 4.92 (1H, s, H-16b), 1.81 (3H, s, CH₃-17), 5.09 (1H, dd, J = 10.3, 1.7 Hz, H-18a), 4.37 (1H, t, J = 10.3 Hz, H-18b), 1.12 (3H, d, J = 6.6 Hz, CH₃-19), 4.93 (1H, d, J = 12.0 Hz, H-20a), 3.76 (1H, d, J = 12.0 Hz, H-20b), 3.86 (1H, d, J = 7.8 Hz, H-2′), 1.74 (1H, t, J = 13.8 Hz, H-3′a), 1.52 (1H, m, H-3′b), 1.60 (1H, m, H-4′a), 1.28 (1H, m, H-4′b), 1.34 (1H, m, H-5′a), 1.24 (1H, m, H-5′b), 1.58(1H, m, H-6′a), 1.34 (1H, m, H-6′b), 1.34 (1H, m, H-7′a), 1.28 (1H, m, H-7′b), 1.65 (1H, m, H-8′a), 1.03 (1H, m, H-8′b), 2.34 (1H, m, H-9′), 1.10 (3H, d, J = 7.2 Hz, H-10′). 3-Bz: 8.13 (2H, dd, J = 8.0, 1.4 Hz, H-3, H-7), 7.19 (2H, dd, J = 8.0, 7.7 Hz, H-4, H-6), 7.41 (1H, tt, J = 7.7, 1.4 Hz, H-5). 18-OBz: 8.09 (2H, dd, J = 8.0, 1.4 Hz, H-3, H-7), 7.45 (2H, dd, J = 8.0, 7.5 Hz, H-4, H-6), 7.57 (1H, tt, J = 7.5, 1.4 Hz, H-5). 20-Succinoxy: 2.68–2.71 (2H, m, H-2, H-3); ¹³C NMR (CDCl₃, 125 MHz) δ 49.6 (C-1), 37.4 (C-2), 82.9 (C-3), 79.6 (C-4), 71.2 (C-5), 59.7 (C-6), 63.1 (C-7), 36.6 (C-8), 81.2 (C-9), 47.7 (C-10), 41.1 (C-11), 29.6 (C-12), 84.4 (C-13), 81.3 (C-14), 145.6 (C-15), 111.8 (C-16), 18.1 (C-17), 67.6 (C-18), 14.7 (C-19), 67.5 (C-20), 118.5 (C-1′), 70.8 (C-2′), 28.7 (C-3′), 23.1 (C-4′), 25.2 (C-5′), 24.0 (C-6′), 23.7 (C-7′), 22.7 (C-8′), 27.4 (C-9′), 18.1 (C-10′). 3-OBz: 166.6 (C-1), 129.6 (C-2), 130.1 (C-3, C-7), 128.5 (C-4, C-6), 133.4 (C-5). 18-Bz: 166.4 (C-1), 130.6 (C-2), 129.6 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5). 20-Succinoxy: 172.0 (C-1), 29.3 (C-2,3), 176.0 (C-4); positive-ion HRFABMS m/z 897.3674 [M + Na]⁺, (calcd for C₄₈H₅₈O₁₅Na, 897.3673).

20-(2,2-Dimethylsuccinoxy) Gnidimacrin (19d) and 20-(3,3-Dimethylsuccinoxy) Gnidimacrin (19e).

2,2-Dimethylsuccinic anhydride (30.0 μL, 27 μmol) and DMAP (3.0 mg) were added to a solution of 1 (10.0 mg, 12.9 μmol) in anhydrous pyridine (3.0 mL). The mixture was stirred at 60 °C for 48 h under argon. Purification by HPLC chromatography (YMC Pack Pro C₁₈; MeCN─H₂O (0.1% HCOOH), 85:15; 5 mL/min) to give 19d (2.7 mg, 24.1%, recycle 13 times) and 19e (2.9 mg, 24.9%, recycle 13 times).

19d: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ + 11.7 (c 0.15, CHCl₃); IR (KBr) max: 2936, 1718, 1452, 1383, 1272, 1171, 1109, 1013, 761, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.85 (1H, m, H-1), 1.83 (1H, m, H-2), 5.03 (1H, d, J = 5.1 Hz, H-3), 3.97 (1H, s, H-5), 3.25 (1H, s, H-7), 3.08 (1H, d, J = 2.8 Hz, H-8), 3.00 (1H, d, J = 12.3 Hz, H-10), 2.85 (1H, o, H-11), 2.32 (1H, d, J = 14.6 Hz, H-12a), 2.00 (1H, dd, J = 14.6, 7.4 Hz, H-12b), 4.38 (1H, d, J = 2.8 Hz, H-14), 5.15 (1H, s, H-16a), 4.92 (1H, s, H-16b), 1.80 (3H, s, CH₃-17), 5.07 (1H, dd, J = 10.6, 2.0 Hz, H-18a), 4.39 (1H, t, J = 10.6 Hz, H-18b), 1.10 (3H, d, J = 6.5 Hz, CH₃-19), 5.05 (1H, d, J = 12.0 Hz, H-20a), 3.60 (1H, d, J = 12.0 Hz, H-20b), 3.86 (1H, d, J = 7.7 Hz, H-2′), 1.75 (1H, t, J = 13.8 Hz, H-3′a), 1.53 (1H, m, H-3′b), 1.50–1.70 (1H, m, H-4′a), 1.20–1.45 (1H, m, H-4′b), 1.50–1.70 (1H, m, H-5′a), 1.20–1.45 (1H, m, H-5′b), 1.20–1.45 (1H, m, H-6), 1.50–1.70 (1H, m, H-7′a), 1.20–1.45 (1H, m, H-7′b), 1.50–1.70 (1H, m, H-8′a), 1.00–1.02 (1H, m, H-8′b), 2.32–2.40 (1H, m, H-9′), 1.07 (3H, d, J = 7.4 Hz, H-10′). 3-OBz: 8.11 (2H, dd, J = 8.0, 1.2 Hz, H-3, H-7), 7.29 (2H, dd, J = 8.0, 7.5 Hz, H-4, H-6), 7.49 (1H, tt, J = 7.5, 1.2 Hz, H-5). 18-OBz: 8.09 (2H, dd, J = 8.0, 1.4 Hz, H-3, H-7), 7.45 (2H, dd, J = 8.0, 7.4 Hz, H-4, H-6), 7.58 (1H, tt, J = 7.4, 1.4 Hz, H-5). 20-(2,2-Dimethylsuccinoxy): 2.74 (1H, d, J = 16.6 Hz, H-2a), 2.66 (1H, d, J = 16.6 Hz, H-2b), 1.34 (3H, s, CH₃-5), 1.30 (3H, s, CH₃-6); ¹³C NMR (CDCl₃, 125 MHz) δ 49.7 (C-1), 37.2 (C-2), 83.0 (C-3), 79.8 (C-4), 71.0 (C-5), 60.0 (C-6), 63.1 (C-7), 36.6 (C-8), 81.2 (C-9), 47.5 (C-10), 41.2 (C-11), 29.9 (C-12), 84.4 (C-13), 81.3 (C-14), 145.6 (C-15), 111.8 (C-16), 19.0 (C-17), 67.9 (C-18), 15.0 (C-19), 68.0 (C-20), 118.6 (C-1′), 70.8 (C-2′), 28.6 (C-3′), 25.2 (C-4′), 24.2 (C-5′), 23.7 (C-6′), 23.2 (C-7′), 22.9 (C-8′), 27.6 (C-9′), 18.2 (C-10′). 3-OBz: 167.8 (C-1), 129.6 (C-2), 130.0 (C-3, C-7), 128.5 (C-4, C-6), 133.3 (C-5). 18-OBz: 166.4 (C-1), 130.6 (C-2), 129.6 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5). 20-(2,2-Dimethylsuccinoxy): 176.4 (C-1), 44.0 (C-2), 40.8 (C-3), 174.4 (C-4), 25.7 (C-5), 25.6 (C-6); positive-ion HRFABMS m/z 925.3984 [M + Na]⁺, (calcd for calcd for C₅₀H₆₂O₁₅Na, 925.3986).

19e: colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}$ − 5.4 (c 0.19, CHCl₃); IR (KBr) max: 3495, 2927, 2856, 1720, 1452, 1272, 1177, 1115, 1024, 761, 712 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 2.86 (1H, t, J = 12.3 Hz, H-1), 1.86 (1H, m, H-2), 5.04 (1H, d, J = 5.1 Hz, H-3), 4.06 (1H, s, H-5), 3.27 (1H, s, H-7), 3.04 (1H, d, J = 2.9 Hz, H-8), 3.01 (1H, d, J = 12.3 Hz, H-10), 2.82 (1H, ddd, J = 10.3, 7.5, 2.0 Hz, H-11), 2.32 (1H, d, J = 14.6 Hz, H₂-12a), 1.98 (1H, dd, J = 14.6, 7.5 Hz, H₂-12b), 4.40 (1H, d, J = 2.9 Hz, H-14), 5.15 (1H, s, H-16a), 4.92 (1H, s, H-16b), 1.80 (3H, s, H₃-17), 5.07 (1H, dd, J = 10.9, 2.0 Hz, H-18a), 4.92 (1H, dd, J = 10.6, 10.3 Hz, H-18b), 1.14 (3H, d, J = 6.5 Hz, H₃-19), 4.75 (1H, d, J = 12.0 Hz, H-20a), 3.87 (1H, d, J = 12.0 Hz, H-20b), 3.86 (1H, d, J = 6.9 Hz, H-2′), 1.74 (1H, t, J = 13.7 Hz, H-3′a), 1.58–1.62 (1H, m, H-3′b), 1.58–1.62 (1H, m, H-4′a), 1.22–1.32 (1H, m, H-4′b), 1.58–1.62 (1H, m, H-5′a), 1.22–1.32 (1H, m, H-5′b), 1.22–1.32 (1H, m, H-6′), 1.58–1.62 (1H, m, H-7′a), 1.22–1.32 (1H, m, H-7′b), 1.58–1.62 (1H, m, H-8′a), 1.00–1.02 (1H, m, H-8′b), 2.30–2.40 (1H, m, H-9′), 1.08 (3H, d, J = 7.5 Hz, H-10′). 3-OBz: 8.12 (2H, dd, J = 8.0, 1.1 Hz, H-3,7), 7.31 (2H, dd, J = 8.0, 7.5 Hz, H-4, H-6), 7.49 (1H, tt, J = 7.5, 1.1 Hz, H-5). 18-Bz: 8.10 (2H, dd, J = 8.3, 1.4 Hz, H-3,7), 7.46 (2H, dd, J = 8.3, 7.4 Hz, H-4, H-6), 7.57 (1H, tt, J = 7.4, 1.4 Hz, H-5). 20-(3,3-Dimethylsuccinoxy): 2.74 (1H, d, J = 15.5 Hz, H-2a), 2.57 (1H, d, J = 15.5 Hz, H-2b), 1.32 (3H, s, H-5), 1.31 (3H, s, H-6); ¹³C NMR (CDCl₃, 125 MHz) δ 49.6 (C-1), 37.2 (C-2), 83.0 (C-3), 79.8 (C-4), 71.6 (C-5), 59.4 (C-6), 63.0 (C-7), 36.6 (C-8), 81.3 (C-9), 47.7 (C-10), 41.2 (C-11), 29.8 (C-12), 84.4 (C-13), 81.3 (C-14), 145.5 (C-15), 111.9 (C-16), 19.0 (C-17), 67.8 (C-18), 15.0 (C-19), 66.9 (C-20), 118.5 (C-1′), 70.8 (C-2′), 28.8 (C-3′), 25.2 (C-4′), 24.2 (C-5′), 23.7 (C-6′), 23.2 (C-7′), 22.8 (C-8′), 27.6 (C-9′), 18.0 (C-10′). 3-Bz: 168.1 (C-1), 129.5 (C-2), 130.1 (C-3, C-7), 128.5 (C-4, C-6), 133.4 (C-5). 18-OBz: 166.4 (C-1), 130.6 (C-2), 129.6 (C-3, C-7), 128.4 (C-4, C-6), 132.9 (C-5). 20-(3,3-Dimethylsuccinoxy): 170.8 (C-1), 44.6 (C-2), 40.5 (C-3), 181.5 (C-4), 25.1 (C-5), 26.0 (C-6); positive-ion HRFABMS m/z 925.3986 [M + Na]⁺, (calcd for C₅₀H₆₂O₁₅Na, 925.3986).

19f: To a stirred solution of 1 (10.0 mg, 0.013 mmol) in CH₂Cl₂ (1.0 mL) were added Et₃N (1.9 mg, 0.019 mmol) and (1S)-camphanic chloride (3.8 mg, 0.019 mmol) sequentially at rt. The reaction mixture was stirred at the same temperature for 12 h. Then, water (1 mL) and 1 N HCl solution (0.1 mL) were added at 0 °C to quench the reaction. The aqueous layer was separated and extracted with CH₂Cl₂ (3 × 1.5 mL). The combined organic extracts were washed with brine, dried over MgSO₄, filtered, and concentrated to give the crude residue, which was purified by flash chromatography on silica gel with MeOH/CH₂Cl₂ to afford compound 19f (4.3 mg, 35% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 8.14 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H), 7.58–7.53 (m, 2H), 7.45 (t, J = 7.6 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 5.15 (s, 1H), 5.08–5.06 (m, 1H), 4.95–4.90 (m, 3H), 4.37–4.32 (m, 2H), 4.03 (d, J = 4.0 Hz, 1H), 3.94 (d, J = 12.0 Hz, 1H), 3.83 (d, J = 8.0 Hz, 1H), 3.66 (d, J = 4.4 Hz, 1H), 3.23 (s, 1H), 3.02 (d, J = 2.8 Hz, 1H), 3.00–2.94 (m, 1H), 2.90 (d, J = 10.8 Hz, 1H), 2.84 (s, 1H), 2.80–2.75 (m, 1H), 2.50–2.42 (m, 1H), 2.36–2.28 (m, 2H), 2.09–2.01 (m, 2H), 1.96–1.84 (m, 2H), 1.78 (s, 3H), 1.71–1.68 (m, 2H), 1.60–1.53 (m, 4H), 1.33–1.19 (m, 12H), 1.15 (d, J = 6.4 Hz, 2H), 1.10 (s, 3H), 1.08 (s, 3H), 0.99 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 175.8, 167.0, 158.3, 144.5, 139.8, 134.8, 134.0, 130.0, 129.5, 129.3, 128.4, 128.3, 124.2, 118.4, 111.7, 109.9, 97.8, 84.3, 82.4, 79.4, 73.0, 70.6, 67.5, 65.0, 63.0, 54.8, 48.0, 41.0, 37.3, 34.0, 29.6, 29.5, 28.5, 27.3, 26.6, 25.2, 23.9, 23.6, 20.6, 19.0, 18.0, 16.6, 14.5; ESI MS m/z calcd for C₅₄H₆₇O₁₅ 955.44 [M + H]⁺, found 955.30.

19g: To a stirred solution of 1 (10.0 mg, 0.013 mmol) and 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (2.0 mg, 0.019 mmol) in CH₂Cl₂ (1.0 mL) were added EDCI (3.0 mg, 0.019 mmol), hydroxybenzotriazole (HOBt) (2.7 mg, 0.002 mmol), and DMAP (2.5 mg, 0.002 mmol) at rt. The reaction mixture was stirred at the same temperature for 12 h, and then H₂O (2.0 mL) was added to quench the reaction. The aqueous layer was separated and extracted with CH₂Cl₂ (3 × 2 mL). The combined organic extracts were washed with brine, dried over MgSO₄, filtered, and concentrated to give the crude product, which was then purified by flash chromatography on silica gel with MeOH/CH₂Cl₂ to afford compound 19g (4.8 mg, 40% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 8.14–8.11 (m, 3H), 7.61–7.57 (m, 1H), 7.52–7.45 (m, 3H), 7.31 (t, J = 8.0 Hz, 2H), 5.18 (s, 1H), 5.11–5.09 (m, 1H), 5.04 (d, J = 4.8 Hz, 1H), 5.00 (d, J = 12.0 Hz, 1H), 4.94 (s, 1H), 4.42–4.39 (m, 1H), 4.37–4.36 (m, 1H), 4.32 (dd, J = 12.0, 2.0 Hz, 1H), 4.26 (dd, J = 11.6, 2.0 Hz, 1H), 4.16 (d, J = 5.6 Hz, 1H), 4.02 (d, J = 5.6 Hz, 1H), 3.87 (d, J = 8.4 Hz, 1H), 3.77 (d, J = 12.0 Hz, 1H), 3.68 (d, J = 11.6 Hz, 1H), 3.26 (s, 1H), 3.11 (d, J = 2.8 Hz, 1H), 3.00–2.97 (m, 1H), 2.93 (s, 1H), 2.91–2.88 (m, 1h), 2.86–2.83 (m, 1H), 2.39–2.32 (m, 2H), 2.00–1.97 (m, 2H), 1.82 (s, 3H), 1.65–1.55 (m, 8H), 1.47 (s, 3H), 1.42 (s, 3H), 1.39–1.30 (m, 7H), 1.25 (s, 3H), 1.17 (s, 3H), 1.15–1.10 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 173.8, 167.3, 166.3, 145.4, 133.0, 132.8, 130.5, 129.9, 129.5, 128.3, 128.3, 118.4, 111.8, 98.5, 96.5, 84.3, 82.4, 81.2, 81.0, 79.6, 71.3, 70.7, 68.2, 67.5, 66.6, 66.4, 62.8, 59.8, 49.5, 47.6, 42.4, 41.0, 37.3, 36.5, 29.6, 29.5, 28.5, 27.3, 26.6, 25.2, 23.9, 23.6, 20.6, 19.0, 18.0, 17.9, 14.7; ESI MS m/z calcd for C₅₂H₆₇O₁₅ 931.44 [M + H]⁺, found 931.40.

19h: To a stirred solution of 1 (10.0 mg, 0.013 mmol) in CH₂Cl₂ (1.0 mL) were added Et₃N (1.9 mg, 0.019 mmol) and trans-4-methoxycinnamoyl chloride (3.7 mg, 0.019 mmol) sequentially at rt. The reaction mixture was stirred at the same temperature for 12 h. Then, water (1 mL) and 1 N HCl solution (0.1 mL) were added to quench the reaction at 0 °C. The aqueous layer was separated and extracted with CH₂Cl₂ (3 × 1.5 mL). The combined organic extracts were washed with brine, dried over MgSO₄, filtered, and concentrated to give a crude residue, which was purified by flash chromatography on silical gel with MeOH/CH₂Cl₂ to afford compound 19h (4.8 mg, 40% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 8.14–8.08 (m, 4H), 7.69 (d, J = 15.6 Hz, 1H), 7.58–7.54 (m, 1H), 7.51–7.43 (m, 5H), 7.32–7.28 (m, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.39 (d, J = 15.6 Hz, 1H), 5.15 (s, 1H), 5.10–5.07 (m, 1H), 5.00 (d, J = 4.8 Hz, 1H), 4.96 (d, J = 12.0 Hz, 1H), 4.91 (s, 1H), 4.38–4.33 (m, 2H), 4.04–4.03 (m, 1H), 3.94–3.89 (m, 2H), 3.86–3.85 (m, 1H), 3.81 (s, 3H), 3.30 (s, 1H), 3.08 (d, J = 2.8 Hz, 1H), 2.91 (s, 1H), 2.87–2.79 (m, 2H), 2.36–2.29 (m, 2H), 2.01–1.94 (m, 1H), 1.87–1.83 (m, 1H), 1.79 (s, 3H), 1.72–1.68 (m, 1H), 1.63–1.49 (m, 6H), 1.35–1.19 (m, 5H), 1.12 (d, J = 6.4 Hz, 3H), 1.09 (d, J = 7.2 Hz, 3H), 0.87–0.80 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 167.8, 167.4, 166.3, 161.5, 145.5, 133.2, 132.9, 130.5, 130.0, 129.9, 129.6, 129.5, 128.4, 128.3, 127.0, 118.5, 114.7, 114.3, 111.8, 84.3, 82.7, 81.3, 81.1, 79.5, 71.4, 70.6, 67.5, 67.0, 63.7, 59.8, 55.3, 49.4, 47.8, 41.0, 37.4, 36.3, 29.5, 28.5, 27.2, 25.2, 23.9, 23.6, 23.0, 22.5, 19.0, 18.0, 14.6; ESI MS m/z calcd for C₅₄H₆₂O₁₄ 935.41 [M + H]⁺, found 935.35.

Multicycle Viral Replication in MT4 Cell Assay.

HIV-1 NL4-3 NanoLuc-sec at a dose of 50 TCID₅₀/well was used to infect MT4 cells (1 × 10⁵ cells/mL) in the presence of compounds at various concentrations in 96-well plates. On day 3 post infection, supernatant samples were harvested and assayed for luciferase activity using the Promega Nano-Glo Luciferase assay system. The antiviral potency is defined as the drug concentration that reduces the luciferase activity by 50% (IC₅₀).

Cytotoxicity Assay.

A CytoTox-Glo cytotoxicity assay (Promega) was used to determine the cytotoxicity of the tested compounds. MT4 cells were cultured in the presence of various concentrations of the compounds for three days. Cytotoxicity of the compounds was determined by following the protocol provided by the manufacturer. The 50% cytotoxic concentration (CC₅₀) was defined as the concentration that caused a 50% reduction of cell viability.

HIV Latency Activation Assay.

U1 cells were used as an HIV-1 latency model. U1 cells (2 × 10⁵ cells/mL) were incubated in the presence of various concentrations of compounds at 37 °C for 72 h. The culture supernatant was assayed for p24 with an HIV p24 ELISA kit (ZeptoMetrix) following the manufacturer’s protocol. The drug concentration that activated HIV-1 p24 production by 50% of the maximum is defined as the EC₅₀ of the compounds. EC₅₀ was determined with a nonlinear regression analysis using Biosoft software. The cytotoxicity against U937 cells, the parental cell line of U1, was measured by the same method as that for MT4 cells. U937 does not harbor HIV-1; therefore, analysis of cytotoxicity caused by the compounds is not complicated by HIV-1-associated cell killing or apoptosis.

In Vitro Stability in Plasma.

The protocol for this assay was modified from that used by Hartman et al.²⁰ The plasma stability of the test compounds was studied in Sprague Dawley rat plasma. The rat plasma in sodium heparin was purchased from Innovative Research, Inc. (catalog no. 50-642-312). The concentration of the test compounds in the stock solution was 5 mM in DMSO. The initial plasma solutions were incubated at 37 °C. After incubation for 10 min, the test compounds were added into the preheated plasma. The final tested concentration of test compounds was 0.05 mM. The sampling times were 0, 0.5, 1, 2, 4, 6, 10, and 24 h. Each sample was deproteinized with two times the volume of MeOH. The deproteinized samples were mixed for 1 min and then centrifuged at 4 °C for 15 min at 14,000 rpm. The clear supernatants were filtered through a 0.22 μm filter. Then, each sample was analyzed by LCMS. The values represent the mean of three independent experiments.

LCMS Analysis.

An LCMS-2020 single quadrupole liquid chromatograph–mass spectrometer was used for sample analysis. The chromatographic separation for all analytes was achieved on a reversed-phase Leapsil C18 column (150 × 2.1 mm, 2.7 μm; Dikma, Beijing, China) using isocratic elution with a mobile phase of 5% solvent A (5 mM ammonia acetate) and 95% solvent B (0.1% formic acid in acetonitrile). The column oven was maintained at 40 °C, and the autosampler temperature was set at 4 °C. The other LCMS parameters were set as follows: electrospray ionization interface, positive and negative ion modes; desolvation line temperature, 250 °C; heat block temperature, 400 °C; interface voltage, −3.5 kV; detector voltage, 1.10 kV; nebulizing gas flow, 1.5 L/min; drying gas flow, 15.0 L/min; nebulizing gas and drying gas, both nitrogen. Samples were quantified in the selective ion monitoring (SIM) mode. The SIM values were [M + H]⁺ m/z 775.9 for GM, [M − H]⁻ m/z 771.9 for 9a and [M − H]⁻ m/z 823.9 for 13d.

ABBREVIATIONS

AIDS

acquired immunodeficiency syndrome

CC₅₀

half-maximum cytotoxic concentration

DMAP

4-dimethylaminopyridine

EC₅₀

half maximal effective concentration

EDCI

N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide

GM

gnidimacrin

HIV

human immunodeficiency virus

IC₅₀

half-maximum inhibitory concentration

LCMS

liquid chromatography–mass spectrometry

NMR

nuclear magnetic resonance

NP

natural product

PKC

protein kinase C

SAR

structure–activity relationship

SIM

selective ion monitoring

TBS

tert-butyldimethylsilyl