Agelasine Diterpenoids and Cbl-b Inhibitory Ageliferins from the Coralline Demosponge Astrosclera willeyana

Abstract

An extract of the coralline demosponge Astrosclera willeyana inhibited the ubiquitin ligase activity of the immunomodulatory protein Cbl-b. The bioassay-guided separation of the extract provided ten active compounds, including three new N-methyladenine-containing diterpenoids, agelasines W–Y (1–3), a new bromopyrrole alkaloid, N(1)-methylisoageliferin (4), and six known ageliferin derivatives (5–10). The structures of the new compounds were elucidated from their spectroscopic and spectrometric data, including IR, HRESIMS, and NMR, and by comparison with spectroscopic data in the literature. While all of the isolated compounds showed Cbl-b inhibitory activities, ageliferins (4–10) were the most potent metabolites, with IC₅₀ values that ranged from 18 to 35 μM.

1. Introduction

The ubiquitin protein ligase (E3), referred to as Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), negatively regulates the costimulatory pathway in T cells, decreasing the immune response and setting the threshold for anergy in T cells [1]. Cbl-b is essential for the negative regulation of T-cell activation, and thus, it reduces the immune response to cancer cells [2,3]. In line with this function, cells that lack the cblb gene rejected tumors in various models and were resistant to rechallenge with tumors after initial tumor rejection in a variety of tumor models [4,5,6,7]. Thus, targeting Cbl-b may be an effective strategy to enhance antitumor immunity. As part of an ongoing effort to identify small molecule inhibitors of the Cbl-b function from natural products [8], an extract of the sponge Astrosclera willeyana was screened and showed a marked reduction of Cbl-b ligase activity.

The calcareous demosponge Astrosclera willeyana is often referred to as a “living fossil” that is representative of late Paleozoic and Mesozoic reef sponges, and it has provided unique secondary metabolites such as N-methylated ageliferin derivatives and manzacidin D [9,10]. These compounds contain pyrrole-2-carboxylic acid moieties, and their novel structures have been the focus of numerous synthetic efforts [11,12,13,14,15]. To date, little is known about the biological properties of these compounds, except for the reported cytotoxic and antibacterial activities of the nonmethylated form of ageliferin [16,17]. In our current study, bioassay-guided fractionation of the A. willeyana extract provided ten active compounds, including three new N-methyladenine-containing diterpeneoids named agelasines W–Y (1–3). The agelasines represent a family of diterpene–adenine conjugates that has only been described from sponges in the genus Agelas. In addition, a new bromopyrrole alkaloid, N(1)-methylisoageliferin (4), along with six known ageliferin derivatives (5–10) were isolated and identified. Herein, we describe the isolation, structure elucidation, and biological activities of the A. willeyana metabolites.

2. Results and Discussion

The organic solvent extract of the sponge Astrosclera willeyana was separated by bioassay-guided diol flash chromatography and C₁₈ HPLC to yield four new metabolites named agelasines W–Y (1–3) and N(1)-methylisoageliferin (4) (Figure 1), along with six known compounds: N(1′)-methylisoageliferin (5), N(1′)-methylageliferin (6), N(1),N(1′)-dimethylisoageliferin (7), N(1),N(1′)-dimethylageliferin (8), N(1′)-methyl-2-bromoageliferin (9), and N(1′)-methyl-2′-bromoageliferin (10).

Agelasine W (1) was obtained as a colorless oil. The molecular formula C₂₆H₄₀N₅⁺, with 10 degrees of unsaturation, was determined by HRESIMS measurements ([M]⁺ m/z 422.3285, calcd for C₂₆H₄₀N₅⁺, 422.3278). The ¹H and ¹³C NMR data (

Table 1. ¹H NMR (600 MHz) and ¹³C NMR (150 MHz) data for agelasines W–Y (1–3) in CD₃OD.

Position	1		2		3
	δH (J in Hz)	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)	δC, Type
1	5.36, t (4.0)	121.4, CH	5.36, t (4.0)	121.4, CH	2.01, m 1.83, m	18.8, CH2
2	2.04, m	24.1, CH2	2.04, m	24.1, CH2	2.15, m 2.01, m	25.0, CH2
3	1.37, m 1.13, m	34.2, CH2	1.37, m 1.13, m	34.2, CH2	5.28, br s	124.4, CH
4		32.4, C		32.4, C		141.0, C
5	1.69, m	44.8, CH	1.69, m	44.9, CH		38.0, C
6	1.59, m 1.30, m	24.8, CH2	1.59, m 1.30, m	24.8, CH2	2.03, m 1.09, m	38.8, CH2
7	2.02, m 1.37, m	30.2, CH2	2.02, m 1.37, m	30.2, CH2	1.25, m	29.9, CH2
8	1.55, m	40.6, CH	1.55, m	40.6, CH	1.48, m	38.6, CH
9		44.1, C		44.1, C		41.3, C
10		142.7, C		142.6, C	1.40, m	45.9, CH
11	2.10, m 1.26, m	38.6, CH2	2.10, m 1.26, m	38.7, CH2	1.65, m 1.37, m	37.6, CH2
12	2.00, m 1.81, m	35.5, CH2	2.00, m 1.81, m	35.5, CH2	2.03, m	33.9, CH2
13		147.4, C		147.7, C		147.7, C
14	5.45, t (6.9)	117.5, CH	5.45, t (6.9)	117.2, CH	5.50, t (7.0)	117.3, CH
15	5.12, br d (6.9)	46.6, CH2	5.13, br d (6.9)	46.7, CH2	5.15, br d (7.0)	46.7, CH2
16	1.84, s	17.0, CH3	1.84, s	17.0, CH3	1.86, s	17.0, CH3
17	0.83, d (6.4)	16.0, CH3	0.83, d (6.4)	16.0, CH3	0.80, d (6.4)	16.3, CH3
18	0.84, s	26.6, CH3	0.84, s	26.6, CH3	1.69, s	20.0, CH3
19	0.88, s	28.7, CH3	0.88, s	28.7, CH3	1.04, s	33.6, CH3
20	0.94, s	22.8, CH3	0.94, s	22.8, CH3	0.85, s	17.9, CH3
2′	8.57, s	149.5, CH	8.67, s	149.5, CH	8.67, s	149.5, CH
3′-NMe	4.04, s	36.6, CH3	4.06, s	36.6, CH3	4.05, s	36.6, CH3
4′		151.5, C		150.0, C		150.4, C
5′		112.4, C		113.1, C		113.1, C
6′		155.1, C		153.8, C		153.8, C
8′	8.44, s	148.0, CH	8.38, s	147.1, CH	8.39, s	147.1, CH
10′-NMe			3.27, s	29.3, CH3	3.27, s	29.3, CH3

) of compound 1 showed characteristic signals of an adenine moiety at δ_H/δ_C 8.44 (1H, s, H-8′)/148.0 (C-8′), 8.57 (1H, s, H-2′)/149.5 (C-2′), δ_C 112.4 (C-5′), 151.5 (C-4′), and 155.1 (C-6′), and an N-methyl group at δ_H/δ_C 4.04 (3H, s)/36.6, revealing the presence of an N-methyladeninium unit in (1). The remaining C₂₀H₃₃ portion was defined as a halimane diterpenoid moiety by comparison of its NMR spectroscopic data with those of related diterpenes [18,19,20]. The four methyl singlets at δ_H 0.84 (H₃-18), 0.88 (H₃-19), 0.94 (H₃-20), and 1.84 (H₃-16), and a doublet at δ_H 0.83 (3H, d, J = 6.4 Hz, H-17), were compatible with a bicyclic halimane ring system, while the ¹H NMR signals of δ_H 2.10 (1H, m, H-11a), 1.26 (1H, m, H-11b), 2.00 (1H, m, H-12a), 1.81 (1H, m, H-12b), 5.45 (1H, t, J = 6.9 Hz, H-14), 5.12 (2H, br d, J = 6.9 Hz, H₂-15), and 1.84 (3H, s, H₃-16) were assigned to a 3-methyl-2-pentenyl chain, according to their COSY and HMBC correlations (Figure 2). The HMBC correlations from H-1 to C-3 and C-5, and from H-8 and H₂-11 to C-10, established the location of a trisubstituted olefin at Δ¹⁻¹⁰, while the HMBC correlations from H₂-15 to C-5′ and C-8′ defined the attachment of C-15 to N-7′ of the adenine subunit. Moreover, the ¹H and ¹³C data of (1) were highly similar to those of agelasine C [21]. However, the N-methyl group in (1) showed HMBC correlations to C-2′ and C-4′, which revealed its location on N-3′, while agelasine C had a methyl group substituted at N-9′. The E configuration of the C-13/C-14 olefin was assigned from NOESY correlations between H-14/H-12b and H₂-15/H₃-16. Additional NOESY correlations of H-5/H-11a and H-8/H-11b suggested that H-5, H-8, and H₂-11 were on the same face of the molecule, while a correlation between H₃-17 and H₃-20 supported this assignment. The chemical shift of C-20 appeared at δ_H 0.94 ppm, which was consistent with the C-17 and C-20 methyl groups being cis, since it was reported that C-20 is more shielded in a cis than a trans orientation of these methyls [21]. The absolute configuration of agelasine W (1) is suggested as 5R, 8R, 9,S according to the positive optical rotation of (1) ([α]${}_{\mathrm{D}}^{25}$ + 17) compared to those for (+)-and (−)-agelasine C, +36.7 and −55.1, respectively [20,21].

Agelasine X (2) was obtained as a colorless oil, and the HRESIMS spectrum displayed a [M]⁺ ion at m/z 436.3452, corresponding to the molecular formula of C₂₇H₄₂N₅⁺ with 10 degrees of unsaturation. The ¹H and ¹³C NMR data of compound 2 were nearly identical to those of (1), except for the presence of an additional N-methyl group at δ_H/δ_C 3.27 (3H, s)/29.3. The location of the N-methyl group was assigned at N-10′ based on an HMBC correlation to C-6′ (δ_C 153.8). The relative and absolute configurations of agelasine X (2) were assigned the same as (1) based on their close spectroscopic similarities and its positive optical rotation ([α]${}_{\mathrm{D}}^{25}$ + 20).

Agelasine Y (3) was also isolated as a colorless oil, and HRESIMS ([M]⁺ m/z 436.3439, calcd for C₂₇H₄₂N₅⁺, 436.3435) established a molecular formula of C₂₇H₄₂N₅⁺ that was isomeric with compound 2. Agelasine Y (3) shared many similar NMR features with those of (2), except for differences in select signals in the bicyclic diterpene ring system (Table 1). The diterpene portion of (3) was assigned as a clerodane skeleton by NMR analysis and a comparison of its spectroscopic data with those of previously reported clerodane diterpenes [20,22,23,24]. The location of the trisubstituted C-3/C-4 olefin was defined by a COSY correlation between the H-2/H-3 and HMBC correlations from H₂-1 to C-3, H-10 to C-4, H₃-18 to C-3 and C-5, and from H₃-19 to C-4 (Figure 3). The relative configuration of the bicyclic ring system of (3) was deduced as a cis-clerodane from the characteristic deshielded carbon signals at δ_C 25.0 (CH₂, C-2) and 33.6 (CH₃, C-19) [22], in addition to a NOESY correlation between H-10 and H₃-19. Additional NOESY correlations of H-8/H-10, H-10/H-11a, and H₃-17/H₃-20 established the relative configurations at C-8 and C-9. Furthermore, a comparison of the ¹³C NMR data in CDCl₃ with four closely related clerodane diastereomers (cis-cis, cis-trans, trans-trans, and trans-cis) showed that the chemical shifts of (3) (Supplementary Materials) were in good agreement with those of neo-cis-cis-kolavenol [25], supporting the assigned configuration of the bicyclic scaffold. The E configuration of the side chain olefin was assigned from NOESY correlations of H-14/H-12b and H₂-15/H₃-16. The structure of (3) resembled that of agelasine Except for the position of the N-methyl groups, the rotation for (3) ([α]${}_{\mathrm{D}}^{25}$ + 15) and agelasine A ([α]${}_{\mathrm{D}}^{25}$ − 31.3) have opposite signs [20]. Therefore, the absolute configuration of agelasine Y (3) is suggested as shown.

N(1)-methylisoageliferin (4) was obtained as a pale-yellow glass. The molecular formula C₂₃H₂₆Br₂N₁₀O₂ with 15 degrees of unsaturation was determined by an HRESIMS of the doubly charged ion ([M + 2H]²⁺ m/z 317.0374, calcd for C₂₃H₂₈N₁₀O₂⁷⁹Br₂²⁺, 317.0376). The ¹H and ¹³C NMR data of (4) (

Table 2. ¹H NMR (600 MHz) and ¹³C (150 MHz) NMR data for N(1)-methylisoageliferin (4) in CD₃OD.

Position	δH (J in Hz)	δC, Type
2	6.91, d (1.5)	129.1, CH
2′		104.7, C
3		95.6, C
3′	6.14, d (4.0)	112.5, CH
4	6.84, d (1.5)	116.1, CH
4′	6.81, d (4.0)	113.5, CH
5		127.0, C
5′		128.4, C
6		163.9, C
6′		163.0, C
8	3.72, dd (14.8, 3.2); 3.43, dd (14.8, 4.3)	40.4, CH2
8′	3.63, dd (14.0, 2.7); 3.37, dd (14.0, 2.7)	42.5, CH2
9	2.17, m	43.8, CH
9′	2.25, m	37.2, CH
10	3.82, br d (8.5)	33.6, CH
10′	2.72, dd (16.3, 5.3); 2.47, ddd (16.3, 9.0, 2.9)	23.5, CH2
11		127.6, C
11′		122.8, C
13		149.3, C
13′		149.2, C
15	6.77, s	112.9, CH
15′		119.1, C
NMe	3.90, s	37.2, CH3

) closely resembled those of the known compound N(1′)-methylisoageliferin (5) [10], and the molecular formula of (4) was isomeric with (5). NMR signals characteristic of the 3-bromo-N-methylpyrrole 5-carbozamide and 2-bromopyrrole 5-carboxamide ring systems were apparent, as well as signals for a highly substituted cyclohexene and two amino imidazole rings. The HMBC correlations from H-4 and H₂-8 to C-6 (Figure 4) revealed that the 3-bromo-N-methylpyrrole 5-carboxamide ring was linked to C-8, and the 2-bromopyrrole 5-carboxamide ring was linked to C-8′ via the HMBC correlations from H-4′ and H₂-8′ to C-6′. Thus, the constitution of (4) only differed from (5) by the location of N-methyl substitution on the pyrrole ring. The configuration of N(1)-methylisoageliferin (4) was identical to that of (5), since its NMR data for the cyclohexene ring, including proton coupling constants and NOE enhancements, and its optical rotation, as well as its ECD data, were fully consistent with those of (5) [10].

The known compounds 5–10 were identified as N(1′)-methylisoageliferin (5), N(1′)-methylageliferin (6), N(1),N(1′)-dimethylisoageliferin (7), N(1),N(1′)-dimethylageliferin (8), N(1′)-methyl-2-bromoageliferin (9), and N(1′)-methyl-2′-bromoageliferin (10) by comparison of their spectroscopic data with the appropriate literature values [10].

Compounds 1–10 were tested for their ability to inhibit the in vitro enzymatic activity of the Cbl-b ubiquitin ligase (

Table 3. Cbl-b inhibitory activities of compounds 1–10 (IC₅₀ values in μM).

Compound	IC50	Compound	IC50
1	57	6	30
2	72	7	18
3	66	8	19
4	33	9	19
5	25	10	35

). The adenine–diterpenoid metabolites agelasines W–Y (1–3) showed weak inhibitory activities against Cbl-b (IC₅₀ > 50 µM), while the ageliferin derivatives (4–10) had more pronounced inhibitory effects (IC₅₀ = 18~35 µM). Compounds 7–9 were the most potent metabolites among the tested compounds, but definitive SAR conclusions were difficult to make. There was no clear pattern of N-methylation or bromine substitution of the two pyrrole rings that directly correlated with the observed Cbl-b inhibitory activity. While the ageliferins are rather modest inhibitors of Cbl-b, the ageliferin structural scaffold could serve as a starting point for the development of more potent analogs with enhanced inhibitory properties.

3. Materials and Methods

3.1. General Experimental Procedures

Flash chromatography was performed using a CombiFlash system (Teledyne Isco, Lincoln, NE, USA). High-performance liquid chromatography (HPLC) was performed using a Varian ProStar 215 solvent delivery module equipped with a Varian ProStar 340 UV-Vis detector, operating under Star 6.41 chromatography workstation software (Agilent Technologies, Santa Clara, CA, USA). HPLC fractions were subsequently dried on Explorer-220 (Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra were acquired with a Bruker Avance III NMR spectrometer equipped with a 3-mm cryogenic probe and operated at 600 MHz for ¹H and 150 MHz for ¹³C (Bruker, Billerica, MA, USA). Spectra were calibrated to their residual solvent signals at δ_H 3.31 and δ_C 49.0 for CD₃OD. LRESIMS studies were measured on an Agilent 6130 Quadrapole LC/MS system (Agilent Technologies, Santa Clara, CA, USA). HRESIMS data were carried out on an Agilent Technology 6530 Accurate-mass Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA). UV spectra were measured with a PerkinElmer Lambda 465 UV/Vis photodiode array spectrophotometer (PerkinElmer, Waltham, MA, USA). ECD spectra were obtained with a Jasco-1500 circular dichroism spectrophotometer (JASCO, Tokyo, Japan). Optical rotations were recorded on a Rudolph research analytical AUTOPOL IV spectropolarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). IR spectra were measured with a Bruker ALPHA II FT-IR spectrometer (Bruker, Billerica, MA, USA).

3.2. Animal Material

Specimens of the sponge Astrosclera willeyana were collected in Tonga in November 1997 and kept frozen until extraction. The collection was carried out by the Coral Reef Research Foundation under contract with the Natural Products Branch, U.S. National Cancer Institute. A voucher specimen (voucher ID # 0CDN5435) was deposited at the Smithsonian Institution, Washington, DC, USA.

3.3. Extraction and Isolation

Following the standard NCI protocol for marine samples [26], the frozen sponge sample (2127 g, wet weight) was ground and processed to provide 4.50 g of organic solvent (CH₂Cl₂-MeOH 1:1 and 100% MeOH) extract (NSC #C017821). A 3.10-g aliquot of the extract was loaded onto a cotton plug and then applied to a diol flash chromatography column (150 g) through a CombiFlash system, sequentially eluting with hexane, CH₂Cl₂, EtOAc/CH₂Cl₂ 1:1 (v/v), EtOAc, MeOH/EtOAc 1:4 (v/v), MeOH/EtOAc 1:1 (v/v), MeOH/EtOAc 4:1 (v/v), and MeOH to obtain nine fractions (Frac. A–I). The active Frac. E (eluted by MeOH/EtOAc 1:4, (v/v) 735 mg) was fractionated by semipreparative HPLC (Phenomenex Luna C18(2), 5 µM, 100 Å, 250 × 21.2 mm), using a linear gradient of CH₃CN/H₂O 3:7–1:0 with 0.1% trifluoroacetic acid (TFA) as the mobile phase to afford agelasine W (1, 7.0 mg), agelasine X (2, 18.9 mg), and agelasine Y (3, 4.7 mg). Another more active Frac. F (eluted by EtOAc/MeOH 1:1, 1787 mg) was separated by semipreparative HPLC in the same way as Frac. E to afford N(1)-methylisoageliferin (4, 10.7 mg), N(1′)-methylisoageliferin (5, 20.0 mg), N(1′)-methylageliferin (6, 26.3 mg), N(1),N(1′)-dimethylisoageliferin (7, 53.1 mg), N(1), N(1′)-dimethylageliferin (8, 6.9 mg), N(1′)-methyl-2-bromoageliferin (9, 26.9 mg), and N(1′)-methyl-2′-bromoageliferin (10, 14.7 mg).

Agelasine W (1): colorless oil; [α]${}_{\mathrm{D}}^{25}$ + 17 (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 280 (3.70) nm; IR (neat) ν_max 2951, 2357, 1659, 1434, 1196, 1133 cm⁻¹; ¹H and ¹³C NMR, Table 1; HRESIMS m/z 422.3285 [M]⁺ (calcd for C₂₆H₄₀N₅⁺, 422.3278).

Agelasine X (2): colorless oil; [α]${}_{\mathrm{D}}^{25}$ + 20 (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 285 (3.89); IR (neat) ν_max 2952, 2357, 1643, 1395, 1196, 1132 cm⁻¹; ¹H and ¹³C NMR, Table 1; HRESIMS m/z 436.3452 [M]⁺ (calcd for C₂₇H₄₂N₅⁺, 436.3435).

Agelasine Y (3): colorless oil; [α]${}_{\mathrm{D}}^{25}$ + 15 (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 285 (3.95) nm; IR (neat) ν_max 2944, 2357, 1643, 1394, 1196, 1132 cm⁻¹; ¹H and ¹³C NMR, Table 1; HRESIMS m/z 436.3439 [M]⁺ (calcd for C₂₇H₄₂N₅⁺, 436.3435).

N(1)-Methylisoageliferin (4): pale-yellow glass; [α]${}_{\mathrm{D}}^{25}$ + 50 (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 218 (4.30), 228 (4.25) and 270 (4.32) nm; ECD (c 7.12 × 10⁻³ M, MeOH) λ_max (Δε) 284 (−0.75), 250 (+0.96), 229 (−4.56), and 212 (+1.33) nm; IR (neat) ν_max 3172, 1678, 1200, 1139 cm⁻¹; ¹H and ¹³C NMR, Table 2; HRESIMS m/z 317.0374 [M + 2H]²⁺ (calcd for C₂₃H₂₈N₁₀O₂⁷⁹Br₂²⁺, 317.0376).

3.4. Cbl-b Biochemical Assay

An extract of Astrosclera willeyana was identified as an active source in a screening campaign of prefractionated natural product samples for the inhibition of Cbl-b ubiquitin ligase activity [8]. The bulk extract was fractionated as described above, and the resulting fractions were evaluated for activity in the Cbl-b bioassay, the details of which have already been reported [8]. In brief, dose response experiments with chromatography fractions and purified compounds were carried out in a Tris-HCl-based enzymatic assay buffer with the following final composition: 75-nM Cbl-b (N1/2 construct) [27], 50-nM Ube2d2 protein (E2) [28], 10-nM UBE1protein (E1), 50-nM biotinylated ubiquitin, and 0.5-µM unlabeled recombinant ubiquitin. The assay buffer also contained the following additives: 0.1-mM dithiothreitol, 0.5-mg/mL bovine gelatin (type B), 0.5-mM magnesium chloride, and 0.01% Triton X-100. Reactions were initiated by the addition of ATP to a final concentration of 100 µM. Following initiation, reactions were transferred to plates previously precoated overnight with 10 µg/mL of the polyubiquitin-binding domain of Cbl-b (UBA) [29]. The UBA domain of Cbl-b binds ubiquitin chains, which results in the capture and enrichment of autopolyubiquitinated Cbl-b. Following a reaction interval of 60 min, the reactions were quenched by the addition of zinc to a final concentration of 2.4 mM. Quenched reactions were incubated at room temperature overnight. Following this incubation period reaction, plates were processed as standard ELISA plates: washed thrice in Tris-buffered saline (with 0.1% Tween-20, 1X TBST), probed with avidin-conjugated horse radish peroxidase, which binds captured biotinylated ubiquitin, washed again in 1X TBST (3X), and then, an avidin-HRP-dependent fluorescent signal (indicating avidin-HRP/biotin-polyubiquitin complexes captured by the UBA coating on the plate) was detected (excitation 325 nm, emission 420 nm) using an Infinite M1000 (Tecan US, Inc., Raleigh, NC, USA) plate reader.

4. Conclusions

Two different classes of Cbl-b inhibitory metabolites, including three new adenine-diterpenoid conjugates (1–3) and seven ageliferin derivatives (4–10), were obtained from the marine sponge Astrosclera willeyana. Agelasines W–Y (1–3) have bicyclic terpenoid skeletons with a prenyl side chain that terminates with an N-methyladenine subunit. These alkaloids arise from a mixed biosynthetic process that incorporates both terpene and purine components, and their new structures enrich the known chemo-diversity of Astrosclera sponges. Diterpene alkaloids with an N-methyladenine moiety are generally methylated at the N-9′ position [30,31,32,33,34]; however, agelasines W–Y (1–3), along with the previously reported agelasimines [35,36], have methyl substituents at both the N-3′ and N-10′ positions. The new compound N(1)-methylisoageliferin (4) and six known analogs of ageliferin (5–10) exhibited significant Cbl-b inhibitory properties, and they could provide a structural framework for lead compound development.