Sinularamides A–G, Terpenoid-Derived Spermidine and Spermine Conjugates with Casitas B-Lineage Lymphoma Proto-Oncogene B (Cbl-b) Inhibitory Activities from a Sinularia sp. Soft Coral

Abstract

An extract of a Sinularia sp. soft coral showed inhibitory activity against the E3-ubiquitin ligase casitas B-lineage lymphoma proto-oncogene B (Cbl-b). Subsequent bioassay-guided separation of the extract provided a series of terpenoid-derived spermidine and spermine amides that were named sinularamides A–G (1–7). Compounds 1–7 represent new natural products; however, sinularamide A (1) was previously reported as a synthetic end product. The structures of sinularamides A–G (1–7) were elucidated by analysis of spectroscopic and spectrometric data from NMR, IR, and HRESIMS experiments and by comparison with literature data. All of the isolated compounds showed Cbl-b inhibitory activities with IC₅₀ values that ranged from approximately 6.5 to 33 μM.

Graphical Abstract

In many cases, tumor growth and lack of anticancer immunity can be attributed to insufficient T cell stimulation by tumor cells or the induction of tolerance in the tumor-reactive T cell population.^1,2 Casitas B-lineage lymphoma proto-oncogene B (Cbl-b), a ubiquitin ligase (E3), acts as a negative regulator of both T cell and B cell activation.³ In the absence of Cbl-b, T cells are hyperproliferative and able to be fully activated even without CD28 costimulatory signaling, and T cells that lack Cbl-b have increased antitumor activity.^3–5 In addition, natural killer (NK) cells in mice lacking Cbl-b exhibit enhanced antitumor activity.⁶ Thus, inhibition of Cbl-b activity could enhance the adaptive and innate immune system and help facilitate antitumor immunotherapies.⁷ As part of a search for small molecule inhibitors of Cbl-b from natural sources,⁸ a prefractionated sample from the organic solvent extract of a Sinularia sp. soft coral was found to inhibit the ubiquitin ligase activity of Cbl-b.

Sinularia is a prominent genus of soft coral in the family Alcyoniidae with 166 described species, and it has proven to be a rich source of secondary metabolites, particularly cembranoid diterpenes and steroids.⁹ Spermidine- or spermine-based aliphatic amines are widely distributed polycationic molecules, which have two or more amine groups in linear hydrocarbon chains.¹⁰ They play important roles in a variety of physiological processes such as signal transduction, gene expression, cell proliferation, chromatin organization, and cell death.¹⁰ Some of the bioactivities have been related to their cationic nature, because they can interact with negatively charged macromolecules such as DNA, RNA, and proteins. It has also been shown that they can bind to specific positions or structural motifs in macromolecules via multiple noncovalent interactions, thereby affecting the structure and function of their macromolecular targets.^10–12 Among the numerous compounds isolated from Sinularia soft corals, spermidine or spermine derivatives are rare.^13–16 These primarily consist of fatty acid substituted derivatives, and only one compound previously reported from Sinularia contained polyprenylated spermidine or spermine moieties. This compound was named sinulamide, and it was initially assigned structure 1 by the Fusetani group.¹⁶ Their subsequent synthetic studies provided compound 1, but this revealed that the natural product sinulamide was actually a double bond isomer where the C-2/C-3 olefin had Z geometry. Herein, we describe the isolation and assignment of a new natural product we are calling sinularamide A (1), which has the same structure as the previously reported synthetic product 1,¹⁶ and six new metabolites named sinularamides B–G (2–7) from a Palau collection of a Sinularia sp. soft coral. The Cbl-b inhibitory activities of all the isolated metabolites were also evaluated.

RESULTS AND DISCUSSION

Bioassay-guided separation of the Sinularia extract on diol solid-phase extraction (SPE) cartridges followed by C₁₈ HPLC yield seven prenylated spermidine or spermine derivatives. These comprised a new natural product named sinularamide A (1), which was previously generated via a synthetic effort,¹⁶ along with the six related compounds sinularamides B–G (2–7). All of these soft coral metabolites showed significant Cbl-b inhibitory activities with IC₅₀ values that ranged from 6.5 to 33 μM.

Sinularamide A (1) was isolated as a colorless oil. Its molecular formula of C₃₄H₆₆N₄O²⁺ was established from HRESIMS measurements of its doubly charged [M]²⁺ molecular ion with m/z 273.2612. The molecular formula of 1 was isomeric with that previously reported for sinulamide.¹⁶ The ¹H and ¹³C NMR data for 1 and sinulamide were also highly similar. The only differences in the NMR data were resonances associated with the C-2/C-3 olefin, which was definitively established as Z in sinulamide and assigned as E in sinularamide A (1) based on NOE correlations between H-2/H-4 and H-2/NH-21. The NMR data that we recorded for sinularamide A (1) exactly matched the data reported for synthetic 1,¹⁶ which thus confirmed the assigned structure.

Sinularamide B (2) was obtained as a colorless oil, and its molecular formula of C₃₁H₅₈N₃O⁺, with five degrees of unsaturation, was determined by HRESIMS measurements ([M]⁺ m/z 488.4593, calcd for C₃₁H₅₈N₃O⁺, 488.4574). The IR spectrum displayed absorption bands at 3420 cm⁻¹ for amine and 1685 cm⁻¹ for conjugated amide functionalities. A linear geranylgeranylic acid diterpenoid moiety with all E olefins like that seen in 1 was recognized from characteristic ¹H and ¹³C NMR data (Tables 1 and 2), as well as COSY, NOESY, and HMBC data (Figure 1). The remaining signals in the ¹H and ¹³C NMR spectra were largely attributed to a spermidine unit, including one exchangeable proton δ_H 7.99 (1H, t, J = 5.8 Hz, NH-21), four deshielded N-methyls [δ_H 2.99 (6H, s) and 2.77 (6H, s); δc 50.1 (2C) and 42.2 (2C)], four nitrogen bearing methylenes (δc 62.3, 61.6, 55.9, and 35.4), and three aliphatic methylenes (δc 22.6, 20.8, and 19.1). HMBC correlations from the N-methyl groups H₃-31, H₃-32 to C-24, and C-26, as well as H₃-33 and H₃-34 to C-29, led to the assignment of the N²⁵,N²⁵,N³⁰,N³⁰-tetramethylspermidine moiety. The deshielded chemical shifts of C-24, C-26, C-31, and C-32 were consistent with the presence of an N-25 quaternary amine group. The linkage between the diterpenoid and spermidine subunits was confirmed by HMBC correlations from H-22 and H-2 to C-1 (δc 166.5) and from NH-21 to C-1 and C-22; thus, the structure of sinularamide B (2) was defined as shown (Figure 1).

Table 1.

¹³C NMR Spectroscopic Data (150 MHz) for Compounds 2–7 in DMSO-d₆ (δ_C, Type)

positiona	2	3	4	5	6	7
1/1′	166.5, C	166.4, C	166.4, C	166.4, C	166.8, C	166.4, C
2/2′	118.5, CH	118.4, CH	118.4, CH	118.4, CH	118.8, CH	118.4, CH
3/3′	152.3, C	152.3, C	152.2, C	152.2, C	152.7, C	152.2, C
4/4′	40.3, CH2	40.3, CH2	40.2, CH2	40.2, CH2	40.7, CH2	40.1, CH2
5/5′	25.8, CH2	25.8, CH2	25.8, CH2	25.8, CH2	26.2, CH2	25.8, CH2
6/6′	123.4, CH	123.3, CH	123.3, CH	123.3, CH	123.8, CH	123.8, CH
7/7′	135.2, C	135.1, C	135.1, C	135.1, C	135.5, C	134.3, C
8/8′	39.3, CH2	39.3, CH2	39.4, CH2	39.4, CH2	39.5, CH2	35.0, CH2
9/9′	26.3, CH2	26.2, CH2	26.2, CH2	26.2, CH2	26.5, CH2	30.8, CH2
10/10′	124.0, CH	124.2, CH	124.1, CH	124.1, CH	124.8, CH	75.3, CH
11/11′	134.5, C	134.5, C	134.4, C	134.4, C	134.1, C	147.2, C
12/12′	39.3, CH2	39.3, CH2	39.4, CH2	39.4, CH2	35.3, CH2	31.6, CH2
13/13′	26.1, CH2	26.1, CH2	26.1, CH2	26.1, CH2	30.8, CH2	25.9, CH2
14/14′	124.2, CH	123.9, CH	123.9, CH	123.9, CH	76.4, CH	123.9, CH
15/15′	130.8, C	130.7, C	130.7, C	130.7, C	143.6, C	131.2, C
16/16′	25.6, CH3	25.6, CH3	25.5, CH3	25.5, CH3	18.4, CH3	25.6, CH3
17/17′	17.8, CH3	17.6, CH3	17.6, CH3	17.6, CH3	112.8, CH2	17.6, CH3
18/18′	15.8, CH3	15.8, CH3	15.8, CH3	15.8, CH3	16.2, CH3	111.0, CH2
19/19′	15.9, CH3	15.8, CH3	15.8, CH3	15.8, CH3	16.2, CH3	15.7, CH3
20/20′	17.6, CH3	17.8, CH3	17.7, CH3	17.7, CH3	18.2, CH3	17.8, CH3
22	35.4, CH2	35.4, CH2	35.4, CH2	35.4, CH2	35.8, CH2	35.4, CH2
23	22.6, CH2	22.7, CH2	22.5, CH2	22.5, CH2	23.0, CH2	22.7, CH2
24	61.6, CH2	61.6, CH2	61.6, CH2	61.6, CH2	62.0, CH2	61.6, CH2
26	62.3, CH2	62.3, CH2	62.2, CH2	62.2, CH2	62.7, CH2	62.3, CH2
27	19.1, CH2	19.0, CH2	19.0, CH2	19.0, CH2	19.4, CH2	19.0, CH2
28	20.8, CH2	19.0, CH2	19.0, CH2	19.0, CH2	19.4, CH2	19.0, CH2
29	55.9, CH2	62.3, CH2	62.2, CH2	62.2, CH2	63.1, CH2	62.7, CH2
31/32	50.1, CH3	50.1, CH3	50.1, CH3	50.1, CH3	50.5, CH3	50.1, CH3
33/34	42.2, CH3	50.1, CH3	50.1, CH3	50.1, CH3	50.5, CH3	50.1, CH3
35		61.6, CH2	61.2, CH2	61.2, CH2	60.7, CH2	60.3, CH2
36		22.7, CH2	22.6, CH2	22.6, CH2	20.9, CH2	20.5, CH2
37		35.4, CH2	34.2, CH2	35.6, CH2	36.6, CH2	36.2, CH2
39			161.5, C	169.6, C
40				22.6, CH3
14/10-OCOCH3					170.1, C	169.7, C
14/10-OCOCH3					21.3, CH3	20.9, CH3

^aPrime numbers are for compound 3.

Table 2.

¹H NMR Spectroscopic Data (600 MHz) for Compounds 2–7 in DMSO-d₆ [δ_H, Multiplet (mult.) (J in Hz)]

positiona	2	3	4	5	6	7
2/2′	5.63, s	5.62, s	5.63, s	5.63, s	5.62, s	5.62, s
4/4′	2.04, m	2.05, m	2.04, m	2.04, m	2.03, m	2.03, m
5/5′	2.09, m	2.10, m	2.09, m	2.09, m	2.10, m	2.10, m
6/6′	5.08, t (6.7)	5.09, t (6.7)	5.09, t (6.7)	5.09, t (6.7)	5.09, t (6.7)	5.07, t (6.7)
8/8′	1.93, m	1.94, m	1.94, m	1.92, m	1.92, m	1.90, m
9/9′	2.02, m	2.03, m	2.02, m	2.02, m	2.03, m	1.65, m
10/10′	5.06, t (6.7)	5.07, t (6.7)	5.07, t (6.7)	5.06, t (6.7)	5.06, t (6.7)	5.03, t (6.7)
12/12′	1.91, m	1.92, m	1.92, m	1.92, m	1.90, m	1.98, m
13/13′	2.00, m	2.01, m	2.01, m	2.01, m	1.63, m	2.02, m
14/14′	5.06, t (6.7)	5.07, t (6.7)	5.07, t (6.7)	5.06, t (6.7)	5.00, t (6.7)	5.09, t (6.7)
16/16′	1.62, s	1.63, s	1.63, s	1.63, s	1.66, s	1.63, s
17/17′	1.54, s	1.55, s	1.55, s	1.55, s	4.87, br s
4.86, br s	1.57, s
18/18′	1.54, s	1.55, s	1.55, s	1.55, s	1.55, s	4.95, br s
4.88, br s
19/19′	1.56, s	1.57, s	1.57, s	1.57, s	1.57, s	1.57, s
20/20′	2.07, s	2.08, s	2.08, s	2.08, s	2.08, s	2.08, s
21-NH	7.99, t (5.8)	7.95, t (5.8)	7.98, t (5.8)	7.95, t (5.8)	8.00, t (5.8)	7.97, t (5.8)
22	3.12, dt (6.2, 5.8)	3.12, dt (6.2, 5.8)	3.12, dt (6.2, 5.8)	3.12, dt (6.2, 5.8)	3.12, dt (6.2, 5.8)	3.13, dt (6.2, 5.8)
23	1.82, m	1.82, m	1.83, m	1.83, m	1.83, m	1.82, m
24	3.24, m	3.25, m	3.25, m	3.25, m	3.27, m	3.26, m
26	3.27, m	3.27, m	3.27, m	3.27, m	3.29, m	3.27, m
27	1.68, m	1.66, m	1.67, m	1.67, m	1.69, m	1.68, m
28	1.64, m	1.66, m	1.67, m	1.67, m	1.69, m	1.68, m
29	3.07, m	3.27, m	3.27, m	3.27, m	3.29, m	3.30, m
31/32	2.99, s	3.00, s	3.01, s	3.01, s	3.00, s	3.00, s
33/34	2.77, s	3.00, s	3.01, s	3.01, s	3.00, s	3.00, s
35		3.25, m	3.25, m	3.25, m	3.35, m	3.33, m
36		1.82, m	1.83, m	1.83, m	2.00, m	1.97, m
37		3.12, dt (6.2, 5.8)	3.15, m	3.08, dt (6.3, 5.5)	2.87, m	2.86, m
38-NH		7.95, t (5.8)	8.22, t (5.8)	8.03, t (5.5)	8.11, 2H, br s	8.06, 2H, br s
39			8.05, s
40				1.82, s
14/10-OCOCH3					2.01, s	2.01, s

^aPrime numbers are for compound 3.

Figure 1.

Key 2D correlations for sinularamides B (2) and C (3).

Sinularamide C (3) with a molecular formula of C₅₄H₉₆N₄O₂²⁺, with 10 degrees of unsaturation, was established from a doubly charged molecular ion in the HRESIMS ([M]²⁺ m/z 416.3767, calcd for C₅₄H₉₆N₄O₂²⁺, 416.3761). The isotope distribution pattern for the molecular ion (adjacent peaks differ by 0.5 amu) indicated that it carried two positive charges. The ¹H and ¹³C NMR spectra for 3 showed only half as many signals as predicted by the molecular formula; therefore, it was a symmetrical molecule. The NMR data (Tables 1 and 2) revealed characteristic signals that could be assigned to one geranylgeranylic acid subunit, but the remaining signals defined only half of an N,N-tetramethyl spermine moiety. Thus, it was apparent that sinularamide C (3) is comprised of an N,N-tetramethyl spermine unit, in which the two primary amines were each acylated with a geranylgeranylic acid diterpenoid chain. This assignment was supported by HMBC correlations from NH-21 (δ_H 7.95, t, J = 5.8 Hz) to C-1 (δc 166.4) and C-22 (δ_C 35.4) and from H-2 (δ_H 5.62, s) to C-1. An HMBC correlation observed between the spectroscopically equivalent C-27 and C-28 methylene groups (H₂-27/C-28 and H₂-28/C-27) supported the symmetrical, dimeric nature of 3, while diagnostic NOESY correlations confirmed an E configuration for the C-2/C-3 olefin (Figure 1).

Sinulamide D (4) was obtained as a colorless oil. The molecular formula C₃₅H₆₆N₄O₂²⁺ with six degrees of unsaturation was determined by HRESIMS measurements of its doubly charged molecular ion ([M]²⁺ m/z 287.2585, calcd for C₃₅H₆₆N₄O₂²⁺, 287.2587). The NMR data of 4 (Tables 1 and 2) were very similar to those recorded for 1; however, the ¹H NMR spectrum of 4 contained an additional deshielded proton signal at δ_H 8.05 (1H, s, H-39) that correlated with a new carbonyl carbon at δ_C 161.5 (C-39) in the HSQC spectrum. HMBC correlations between H-39 and C-37 (δ_C 34.2), in conjunction with correlations from NH-38 (δ_H 8.22, t, J = 5.8 Hz) and H₂-37 (δ_H 3.15, m) to C-39, revealed the presence of a formamide group attached to C-37 (Figure 2). The olefin configurations were assigned from ¹³C and NOESY data, which established the structure of sinularamide D (4) as the N-formyl derivative of sinularamide A (1).

Figure 2.

Key 2D correlations for sinularamides D–G (4–7).

Sinularamide E (5) was also a colorless oil with a molecular formula of C₃₆H₆₈N₄O₂²⁺ that was determined by HRESIMS measurements ([M]²⁺ m/z 294.2663, calcd for C₃₆H₆₈N₄O₂²⁺, 294.2665). The molecular formula of 5 represented an addition of CH₂ to the formula of compound 4. The ¹H and ¹³C NMR data recorded for 5 closely paralleled those of 4, except for the loss of the signals attributed to the formamide group and the appearance of an additional methyl at δ_H 1.82 (3H, s, H-40) in 5. HMBC correlations (Figure 2) between these methyl protons and a carbonyl carbon at δ_C 169.6 (C-39), along with correlations from both NH-38 (δ_H 8.03, t, J = 5.5 Hz) and H₂-37 (δ_H 3.08, m) to C-39, confirmed the presence of a terminal acetamide group, which completed the structural assignment of sinularamide E (5).

Sinularamide F (6) was a colorless oil with a molecular formula C₃₆H₆₈N₄O₃²⁺ determined by HRESIMS ([M]²⁺ m/z 302.2653, calcd for C₃₆H₆₈N₄O₃²⁺, 302.2640) which had an additional oxygen relative to the molecular formula of sinularamide E (5). The ¹H and ¹³C NMR spectra of 6 contained signals that closely matched the spermine portion of sinularamide A (1), but distinct changes in the diterpenoid moiety were apparent. The appearance of an additional methyl group at δ_H 2.01 (3H, s)/δ_C 21.3 and ester carbonyl δ_C 170.1 suggested the presence of an acetate group in 6. An oxymethine was revealed from the deshielded H-14 signal (δ_H 5.00, 1H, t, J = 6.7 Hz) and the chemical shift of C-14 (δ_C 76.4). HMBC correlations (Figure 2) from H-14 and the acetate methyl to the ester carbonyl established an O-acetyl group substituted at C-14. Broadened olefinic singlets at δ_H 4.87 (H-17a) and 4.86 (H-17b) correlated with the same sp² carbon (112.8) in the HSQC spectrum and showed HMBC correlations with C-14 (δ_C 76.4), C-15 (δ_C 143.6), and C-16 (δ_C 18.4), indicative of a 1,1-disubstituted olefin involving carbons 15 and 17. The remaining NMR signals of sinularamide F (6), including NOESY correlations around the C-2/C-3, C-6/C-7, and C-10/C-11 olefins, were virtually identical to those of 1, which allowed assignment of the planar structure, but the absolute configuration at C-14 was not assigned.

Sinularamide G (7), had a molecular formula of C₃₆H₆₈N₄O₃²⁺ that was isomeric with 6 ([M]²⁺ m/z 302.2648, calcd for C₃₆H₆₈N₄O₃²⁺, 302.2640), and the ¹H and ¹³C NMR data for 7 were also very similar to those of 6. The position of the acetyl group and the 1,1-disubstituted double bond (δ_C 111.0 and 147.2) in 7 were deduced by HMBC correlations (Figure 2) from H₂-18 (δ_H 4.95, 4.88, each br s) to C-10 (δ_C 75.3) and C-12 (δ_C 31.6) and HMBC correlations from both H-10 (δ_H 5.03, 1H, t, J = 6.7 Hz) and the acetate methyl signal at δ_H 2.01 to the carbonyl carbon at δ_C 169.7. Diagnostic NOEs defined E configurations for the C-2/C-3 and C-6/C-7 double bonds. Thus, the structure of sinularamide G (7) was defined, but the absolute configuration at C-10 was not assigned.

Sinularamides A–G (1–7) were tested in vitro to assess their ability to inhibit Cbl-b dependent autoubiquitination.⁸ Compounds 1–7 exhibited moderate inhibitory activities with IC₅₀ values that ranged from approximately 6.5 to 33 μM (Figure 3 and Supporting Information). Sinularamide C (3) was the most potent inhibitor with an IC₅₀ of 6.5 μM. Cbl-b is a RING finger E3 ligase that regulates tyrosine kinase signaling, and it has been implicated in various oncogenic processes and as a regulator of immune cell function.^3,4,17 Loss of the E3 function of Cbl-b results in activation of NK immune cells and T cells and renders them capable of rejecting tumors.^4–6,18 Thus, compounds such as the sinularamides, which can inhibit Cbl-b activity in vitro, may provide a template to develop agents targeting Cbl-b that can be used to boost immune activation against cancer cells. However, the cationic nature of polyamines such as the sinularamides may limit their chemotherapeutic potential due to issues related to cellular permeability and nonspecific electrostatic interactions with other macromolecules.

Figure 3.

Cbl-b inhibitory activity of sinularamide C (3).

EXPERIMENTAL SECTION

General Experimental Procedures.

Optical rotations were measured on a Rudolph research analytical AUTOPOL IV spectropolarimeter. IR spectra were recorded with a Bruker ALPHA II FT-IR spectrometer. NMR spectra were obtained 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. NMR spectra were calibrated to the residual DMSO-d₆ solvent signals at δ_H 2.50 and δ_C 39.5. The (+)-HRESIMS data were acquired on an Agilent Technology 6530 Accurate-mass Q-TOF LC/MS. 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.

Animal Material.

Specimens of the soft coral Sinularia sp. were collected in Palau, in November 1996, 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 no. 0CDN4378) was deposited at the Smithsonian Institution, Washington, DC, and a high-resolution in situ photo of the coral is available in the Supporting Information. The cream colored Sinularia sp. colonies were low and encrusting with numerous small knobs and ridges. Confirmation of the Sinularia sp. taxonomy was provided by octocoral specialists Andrea Quattrini and Catherine McFadden. The animal material (624.4 g, wet weight) was ground and processed using the standard NCI method for marine samples to provide 9.24 g of organic solvent (CH₂Cl₂–MeOH, 1:1) extract (NSC no. C016809).¹⁹

Isolation.

A portion of the crude extract (500.0 mg) was applied on SPE-diol cartridges (2 g) eluted with hexane–CH₂Cl₂ 9:1 (v/v), CH₂Cl₂–EtOAc 20:1 (v/v), EtOAc, EtOAc–MeOH 5:1 (v/v), and MeOH in a stepwise manner. The active hydrophilic fraction (eluted by MeOH, 295.0 mg) was separated by semipreparative HPLC (Phenomenex Luna C18, 5 μ, 100 Å, 250 mm × 21.2 mm), using a linear gradient of MeOH–H₂O 1:9 to 1:0 [v/v, containing 0.1% trifluoroacetic acid (TFA)] as the mobile phase to yield 9 fractions (Frs. A–I); Fr. H was pure sinularamide A (1, 69.6 mg). The other fractions were further purified on the former HPLC column, with a linear gradient elution of CH₃CN–H₂O (v/v, containing 0.1% TFA). Fr .B (10.0 mg) was eluted with a linear gradient (4:6 to 9:1) to afford sinularamide E (5, 1.3 mg). Fr. D (14.4 mg) was eluted with a linear gradient (3:7 to 9:1) to obtain sinularamide F (6, 3.8 mg) and sinularamide G (7, 1.6 mg). Fr. F (17.2 mg) was eluted with a linear gradient (4:6 to 9:1) to yield sinularamide D (4, 3.8 mg). Fr. G (20.8 mg) and Fr. I (50.0 mg) were eluted with the same linear gradient (6:4 to 1:0) to yield sinularamide B (2, 11.9 mg) and sinularamide C (3, 1.2 mg), respectively.

Sinularamide B (2).

This compound is a colorless oil; IR (neat) ν_max 3420, 1685, 1640, 1201, 1176, 1127, 830, 720 cm⁻¹; ¹H NMR data, see Table 1; ¹³C NMR data, see Table 2; HRESIMS m/z 488.4594 [M]⁺ (calcd for C₃₁H₅₈N₃O⁺, 488.4574).

Sinularamide C (3).

This compound is a colorless oil; IR (neat) ν_max 3420, 1684, 1202, 1177, 1128, 668 cm⁻¹; ¹H NMR data, see Table 1; ¹³C NMR data, see Table 2; HRESIMS m/z 416.3767 [M]²⁺ (calcd for C₅₄H₉₆N₄O₂²⁺, 416.3761).

Sinularamide D (4).

This compound is a colorless oil; IR (neat) ν_max 3420, 1671, 1201, 1176, 1127, 829, 801, 720 cm⁻¹; ¹H NMR data, see Table 1; ¹³C NMR data, see Table 2; HRESIMS m/z 287.2585 [M]²⁺ (calcd for C₃₅H₆₆N₄O₂²⁺, 287.2587).

Sinularamide E (5).

This compound is a colorless oil; IR (neat) ν_max 3420, 1683, 1202, 1178, 1128, 831, 802, 720 cm⁻¹; ¹H NMR data, see Table 1; ¹³C NMR data, see Table 2; HRESIMS m/z 294.2663 [M]²⁺ (calcd for C₃₆H₆₈N₄O₂²⁺, 294.2665).

Sinularamide F (6).

This compound is a colorless oil; ${\left[\alpha \right]}_{\text{D}}^{\text{20}}+8.0\left(c0.06,\text{\hspace{0.17em}MeOH}\right)$; IR (neat) ν_max 3421, 1685, 1202, 1128, 831, 802, 721 cm⁻¹; ¹H NMR data, see Table 1; ¹³C NMR data, see Table 2; HRESIMS m/z 302.2653 [M]²⁺ (calcd for C₃₆H₆₈N₄O₃²⁺, 302.2640).

Sinularamide G (7).

This compound is a colorless oil; ${\left[\alpha \right]}_{\text{D}}^{\text{20}}+8.9\left(c0.03,\text{\hspace{0.17em}MeOH}\right)$; IR (neat) ν_max 3421, 1685, 1202, 1178, 1128, 831, 802, 720 cm⁻¹; ¹H NMR data, see Table 1; ¹³C NMR data, see Table 2; HRESIMS m/z 302.2648 [M]²⁺ (calcd for C₃₆H₆₈N₄O₃²⁺, 302.2640).

Cbl-b Biochemical Assay.

Chromatography fractions and pure compounds were evaluated for activity in a Cbl-b bioassay, details of which have already been reported.⁸ In brief, dose response experiments with the purified compounds were carried out in Tris–HCl buffer (pH 7.5) that contained 15 nM E1 protein (UBE1), 75 nM E2 protein (Ube2d2),²⁰ 112 nM Cbl-b protein (N1/2 Construct),²¹ 75 nM biotinylated ubiquitin, 750 nM ubiquitin, 0.1 mM dithiothreitol, 0.5 mg/mL bovine gelatin type B, 0.5 mM magnesium chloride, and 0.01% Triton X-100. Addition of ATP into the enzyme solution initiated the enzymatic reaction cascade. Initiated reactions were then transferred to plates that had been precoated overnight with 10 μg/mL polyubiquitin binding portion of Cbl-b (UBA)²² which allowed for the binding and specific enrichment of autopolyubiquitinated Cbl-b. After 1 h, the reactions were quenched, and the reaction plates were sealed and incubated overnight at room temperature. The following day, reaction plates were probed with avidin-conjugated horse radish peroxidase and washed three times; then, an avidin-HRP dependent fluorescent signal (indicating the presence of avidin-HRP/biotin-polyubiquitin complexes bound by the UBA coated plate) was detected (excitation 325 nm, emission 420 nm) using a Tecan Infinite M1000 plate reader.