Cytotoxic Sesterterpenes from Thai Marine Sponge Hyrtios erectus

Abstract

Four sesterterpenes, erectusolides B, C, D, and seco-manoalide-25-methyl ether, two 2-furanone derivatives, erectusfuranones A and B, together with thirteen known sesterterpenes, (6Z)-neomanoalide-24-acetate, two diastereomers of 24-O-methylmanoalide, luffariolide B, manoalide, (6E)- and (6Z)-neomanoalide, seco-manoalide, scalarafuran, 12-acetylscalarolide, 12-epi-O-deacetyl-19-deoxyscalarin, 12-epi-scalarin, and 12-O-deacetyl-12-epi-scalarin, three indole alkaloids, 5-hydroxy-1H-indole-3-carbaldehyde, hyrtiosine A, and variabine B, and one norterpene, cavernosine were isolated from the marine sponge Hyrtios erectus. Their structures were determined by means of spectroscopic methods and the absolute configurations of the asymmetric centers were determined using the modified Mosher’s method. The cytotoxic activities for the isolated compounds have been reported.

1. Introduction

Marine organisms have always been an attractive source of natural products with novel and exotic structures and useful biological activities [1,2,3,4,5,6,7,8,9,10,11]. Marine sponges of the genus Hyrtios (order Dictyoceratida family Thorectidae) have yielded scalarane [6,8,12,13,14] and manoalide [12] types sesterterpenes, which are important groups of active secondary metabolites. They have been reported to possess many biological activities, such as cytotoxic [3,6,15,16], antibacterial [9,11], antibiotic [17] activities, inhibition of the DNA-relaxing activity of mouse DNA topoisomerase I [12], and enhancement nerve growth factor synthesis in cultured astroglial cells [13]. As a part of our ongoing research program focused on the discovery of cytotoxic compounds from Thai marine sponges [18,19,20], drawing from the previous report, one sesterterpene, erectusolide A, six phenolic alkenes, erectuseneols A–F, together with nine known compounds, were isolated from the EtOAc soluble extract of Hyrtios erectus (Chulabhorn Research Institute (CRI) 588) [3]. Of these, some sesterterpenes exhibited significant cytotoxic activities against the MOLT-3 cell line with an IC₅₀ values of 3.79–5.82 μM [3]. Accordingly, further investigation of the EtOAc soluble extract was carried out, leading to the isolation of four new sesterterpenes, erectusolides B (1), C (2), D (4), and seco-manoalide-25-methyl ether (3), two new 2-furanone derivatives, erectusfuranones A and B (5 and 6), together with seventeen known sesterterpenes, which were identified as (6Z)-neomanoalide-24-acetate (7) [9], two diastereomers of 24-O-methylmanoalide (8 and 9) [10], luffariolide B (10) [15], manoalide (11) [10,12,17], (6E)- and (6Z)-neomanoalides (12 and 13) [12,15,17], seco-manoalide (14) [12,17] scalarafuran (15) [8,16], 12-acetylscalarolide (17) [21], 12-epi-O-deacetyl-19-deoxyscalarin (18) [8,16], 12-epi-scalarin (19) [13,16], and 12-O-deacetyl-12-epi-scalarin (20) [13,14,16], three indole alkaloids, 5-hydroxy-1H-indole- 3-carbaldehyde (21) [14,22], hyrtiosine A (22) [14,22], and variabine B (23) [7], and one norterpene, cavernosine (16) [3,23]. All of the known compounds (Figure 1) were readily identified by extensive study of their spectral data, including high resolution atmospheric pressure chemical ionization mass spectrometry (HRAPCIMS) or high resolution electrospray ionization mass spectrometry (HRESIMS), 1D and 2D nuclear magnetic resonance (NMR) data, as well as by comparison with those reported in the literatures.

2. Results

Two separate collections of H. erectus were studied; and they were collected in February 2011 from different locations of the Similan Island, Phangnga province, Thailand. The MeOH extract of the Thai marine sponge H. erectus from collection CRI 588 was dissolved in H₂O and partitioned with EtOAc. The EtOAc soluble extract was fractionated by column chromatography over silica gel, Sephadex LH-20, and reversed-phase high performance liquid chromatography (HPLC) to afford three new sesterterpenes, erectusolides B (1), C (2), and seco-manoalide-25-methyl ether (3), two new 2-furanone derivatives, erectusfuranones A (5) and B (6) together with eight known sesterterpenes, 7–14. One additional new sesterterpene, erectusolide D (4), and ten known compounds (13, 15–23) were obtained from the H. erectus collection CRI 572. All new compounds showed considerable structural similarity with the co-occurring known sesterterpenes.

Compound 1 was obtained as a weak optical rotation value (${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{26}$ −1.3), and its molecular formula was determined to be C₂₅H₃₆O₃ (eight degrees of unsaturation) by HRAPCIMS. Infrared (IR) absorption bands of compound 1 suggested the presence of β-substituted α,β-unsaturated γ-lactone (β-substituted butenolide) at 1779 and 1746 cm⁻¹ and hydroxyl group at 3443 cm⁻¹. Four of the eight degrees of unsaturation implied by the molecular formula of 1 were taken up in one carbon–oxygen double bonds and three carbon–carbon double bonds, thus indicating the tetracyclic nature of the molecule. The ¹³C and (distortionless enhancement by polarization transfer (DEPT) NMR spectra of 1 showed 25 carbons including, 4 tertiary methyls, 7 methylenes, 2 oxymethylenes, 3 methines, 2 olefinic methines, and 7 quaternary carbons. The ¹H and ¹³C NMR spectral data of 1 (

Table 1. ¹H (600 MHz) and ¹³C (150 MHz) NMR data (^a in CDCl₃ ^b in C₆D₆) of compounds 1 and 2.

Position	1			2
	a δC (Type)	a δH, Mult. (J in Hz)	b δH, Mult. (J in Hz)	a δC (Type)	a δH, Mult. (J in Hz)	b δH, Mult. (J in Hz)
1	174.0 (C)	-	-	173.3 (C)	-	-
2	115.3 (CH)	5.86, brd (1.5)	5.66, q (1.6)	116.0 (CH)	5.93, brd (1.6)	5.46, brs
3	169.7 (C)	-	-	168.1 (C)	-	-
4	51.0 (CH)	2.74, d (10.0)	2.24, d (10.1)	50.8 (CH)	2.85, d (9.9)	1.93, d (10.5)
5	33.3 (CH)	2.85, brt (9.0)	2.60–2.66, m	34.5 (CH)	3.14, brt (8.8)	2.40, brt (10.5)
6	122.1 (CH)	5.67, brd (0.9)	5.48, brd (1.4)	149.1 (CH)	6.74, t (3.0)	5.85, brt (3.1)
7	140.9 (C)	-	-	143.7 (C)	-	-
8	24.0 (CH2)	1.90–2.00, o 2.09, dt (16.4, 3.4)	1.76, brt (13.0) 1.90–1.98, o	19.4 (CH2)	1.86–1.93, o 2.65, dt (16.8, 3.6)	1.51–1.62, o 2.60, dt (13.3, 3.1)
9	22.8 (CH2)	1.65, qd (12.2, 4.2) 1.82–2.00, o	1.42, qd (12.3, 4.1) 1.57–1.67, m	22.3 (CH2)	1.50–1.58, o 1.88–1.99, o	0.88–0.96, m 1.27–1.37, 0
10	37.0 (CH)	2.25, dt (12.2, 6.9)	2.12, ddd (12.8, 7.9, 6.8)	38.0 (CH)	2.37, dt (11.7, 6.7)	1.82–1.88, o
11	43.0 (C)	-	-	43.7 (C)	-	-
12	36.6 (CH2)	1.08–1.20, m 1.49–1.61, o	1.28–1.36, m 1.58–1.66, o	36.6 (CH2)	1.15–1.18, o 1.55–1.58, o	1.10–1.17, m 1.35–1.40, o
13	22.4 (CH2)	1.90–2.00, o	1.94–2.06, o	22.2 (CH2)	1.88–1.99, o	1.23–1.40, o 1.78–1.84, o
14	136.3 (C)	-	-	136.0 (C)	-	-
15	127.5 (C)	-	-	127.7 (C)	-	-
16	32.7 (CH2)	1.89–1.98, o	1.92–2.06, o	32.7 (CH2)	1.85–1.99, o	1.78–1.89, o
17	19.4 (CH2)	1.53–1.62, o	1.60–1.68, o	19.4 (CH2)	1.52–1.62, o	1.49–1.58, o
18	39.7 (CH2)	1.40–1.44, m	1.49–1.52, m	39.7 (CH2)	1.40–1.44, m	1.37–1.42, o
19	35.1 (C)	-	-	35.1 (C)	-	-
20	28.71 (CH3)	0.96 *, s	1.14, s	28.74 (CH3)	0.96, s	0.99, s
21	28.72 (CH3)	0.97 *, s	1.11, s	28.7 (CH3)	0.98, s	1.02, s
22	19.8 (CH3)	1.55, s	1.66, s	19.8 (CH3)	1.56, s	1.52, s
23	21.5 (CH3)	1.13, s	0.88, s	21.5 (CH3)	1.16, s	0.63, s
24	66.8 (CH2)	4.03, d (13.2) 4.06, d (13.2)	3.88, s	192.9 (CH)	9.45, s	9.37, s
25	73.2 (CH2)	4.62, dd (17.4, 1.6) 4.65, dd (17.4, 1.0)	4.02, dd (17.1, 0.8) 4.16, dd (17.1, 1.7)	72.9 (CH2)	4.64, dd (17.3, 1.8) 4.68, dd (17.3, 1.1)	3.85, dd (17.2) 4.00, dd (17.2)

* Interchangeable; o = overlapped signals; Assignments are based on COSY, HSQC (Figures S3, S9 and S10), and HMBC experiments.

, Figures S1 and S2) were comparable to those of luffariolides A (Figure 1) and B (10) and implied that all compounds possessed identical two terminal units, which included a polyalkylated-cyclohexene (C₁₂–C₂₂) and β-substituted butenolide moieties. The major differences were found in the C₄–C₁₁, and C-23. In the ¹H–¹H COSY correlation of 1 (Figure 2), H-6 (δ_H 5.67) showed allylic coupling to H_ax-8 (1H, δ_H 1.90–2.00) and H₂-24 (2H, δ_H 4.03/4.06) and H-5 was coupled to H-4, H-6, and H-10. From its ¹H–¹H correlation spectroscopy (COSY) spectrum of 1 (Figure 2), typical allylic coupling of the olefinic methines at δ_H 5.86 (H-2) and 5.67 (H-6) with the respective methine signal at δ_H 2.74 (H-4) and oxymethylene at δ_H 4.03/4.06 (H₂-24) were discernable. Both the COSY and heteronuclear multiple bond correlation (HMBC) data indicated that the double bonds were not conjugated to each other. The ¹H, ¹³C, and COSY NMR data confirmed the presence of a 3-hydroxymethylbicyclo [4.2.0] oct-2-ene (C₄–C₁₁ and C-24) in the molecule. The HMBC correlations of 1 (Figure 2 and Figure S4), the proton signal at δ_H 5.86 (H-2) correlated to methine carbon at δ_C 51.0 (C-4), H-4 at δ_H 2.74 correlated to quaternary carbon at δ_C 169.7 (C-3), methine carbon at δ_C 115.3 (C-2), and methylene carbon at δ_C 73.2 (C-25) allowed the connectivity of the butenolide with the bicyclic ring through the linkage of C-3 and C-4. The linkage of the polyalkylated-cyclohexene and CH₃-23 at quaternary C-11 on the molecule were deduced from the HMBC correlations of H₃-23 (δ_H 1.13) with C-4, C-10 (δ 37.0), C-11 (δ 43.0), and C-12 (δ 36.6) and the correlations of H₂-12 (δ_H 1.28–1.36 and 1.58–1.66) with C-4, C-10, C-14 (δ 136.3), and C-23 (δ 21.5). The large trans-diaxial coupling constant between H_ax-9 and H_ax-10 (J = 12.2 Hz) on the cyclohexene ring suggested the β-configuration of H_ax-10. The relative stereochemistry of 1 was established by analysis of the (nuclear overhauser effect spectroscopy (NOESY) spectrum (in CDCl₃ and C₆D₆; Figure 2). NOESY cross-peaks resonance of H-4, H_α-9, and H₃-23 and H-5, H-10, H-12, and H-2 implied a cis-junction for rings B/C and H-5, H_ax-10, β-substituted butenolide moiety, and polyalkylated-cyclohexene (C₁₂–C₂₂) are on the same face while the methyl group (CH₃-23) and H-4 on the opposite face of the molecule. Structure 1 with relative stereochemistry as shown was thus assigned to erectusolide B, which contained fused cyclohexene and cyclobutane rings. It could derive from luffariolide A (Figure 1) [11], through a biogenetic pathway involving a 2 + 2 cycloaddition of double bonds at C-4/C-5 and C-10/C-11 as suggested by Lin, H.W. and co-workers [4]. Furthermore, compounds 1 and 2 represent two important compounds which lend further support for the proposed biosynthetic pathway of Lin et. al. for the cyclobutane formation [4]. This substance has an optical rotation near zero (${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{26}$ −1.3); attempting to separate the substance using various chiral columns was found to be inseparable.

Compound 2 was obtained as optically active (${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{26}$ −18.5), and its molecular formula was determined to be C₂₅H₃₄O₃ (nine degrees of unsaturation) by HRAPCIMS. Infrared (IR) absorption bands of compound 2 suggested the presence of β-substituted α,β-unsaturated γ-lactone at 1779 and 1746 cm⁻¹ and α,β-unsaturated carbonyl group at 1682 cm⁻¹. The ¹H and ¹³C NMR spectroscopic data of 2 (Table 1, Figures S4–S8) indicated that it was essentially identical to compound 1, except for the presence of an aldehyde group (δ_H 9.45, s; δ_C 192.9) for 2 in place of the hydroxylmethyl group (δ_H 4.03/4.06, each d, J = 13.2 Hz, δ_C 66.8) for 1. This was further confirmed by the HMBC correlations (Figures S11 and S12) between olefinic H-6 (δ_H 6.74) and aldehydic carbon (δ_C 192.9) and between proton aldehyde (δ_H 9.45) and C-7 (δ_C 143.7) and C-8 (δ_C 19.4). The NOESY spectrum of 2 was similar to that observed of 1 indicating the same relative stereochemistry. Thus, compound 2 was suggested to be the formaldehyde analog of 1, and named erectusolide C. This compound has low optical rotation (${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{26}$ −18.5); attempting to separate the compound using various chiral columns was unsuccessful.

Compound 3 was obtained as a pale yellow solid, exhibiting similar ultraviolet (UV), infrared (IR), ¹H, and ¹³C NMR spectra (

Table 2. ¹H (600 MHz) and ¹³C (150 MHz) NMR data (CDCl₃) of compounds 3 and 4.

Position	3		4
	δC (Type)	δH, Mult. (J in Hz)	δC (Type)	δH, Mult. (J in Hz)
1	169.2 (C)	-	172.7 (C)	-
2	119.3 (CH)	6.09, brt (1.3)	116.0 (CH)	6.04, brd (1.4)
3	166.3 (C)	-	171.7 (C)	-
4	66.9 (CH)	4.73, brdd (6.7, 4.1)	81.8 (CH)	5.08, m
5	34.7 (CH2)	2.78, dt (15.7, 7.5) 2.92, ddd (15.7, 7.1, 4.1)	30.2 (CH2)	2.57, ddd (15.0, 7.3, 5.5) 2.78, dt (15.0, 7.0)
6	147.0 (CH)	6.54, t (7.1)	119.9 (CH)	5.19, t (7.5)
7	146.1 (C)	-	143.0 (C)	-
8	24.6 (CH2)	2.32, t (7.4)	35.4 (CH2)	2.10–2.22, m
9	26.7 (CH2)	2.09, q (7.4)	26.3 (CH2)	2.07–2.17, m
10	122.4 (CH)	5.13, td (7.3, 1.0)	123.6 (CH)	5.07, m
11	137.5 (C)	-	136.6 (C)
12	40.2 (CH2)	1.95–2.07, m	33.7 (CH2)	1.68, td (13.5, 3.5) 1.86–1.96, m
13	27.8 (CH2)	1.97–2.07, m	31.0 (CH2)	1.49–1.60, m 1.88, td (11.7, 4.3)
14	137.0 (C)	-	80.4 (C)	-
15	127.0 (C)	-	150.5 (C)	-
16	32.8 (CH2)	1.90, t (6.2)	34.0 (CH2)	1.97, td (13.0, 5.7) 2.32, brd (13.0)
17	19.5 (CH2)	1.54–1.59, m	22.7 (CH2)	1.43–1.65, m
18	39.9 (CH2)	1.39–1.43, m	38.0 (CH2)	1.37, brd (13.7) 1.63, td (13.7, 4.5)
19	35.0 (C)	-	39.7 (C)	-
20	28.6 (CH3)	0.99, s	24.1 (CH3)	0.96, s
21	28.6 (CH3)	0.99, s	22.1 (CH3)	0.88, s
22	19.8 (CH3)	1.59, s	108.3 (CH2)	4.83, brs 4.91, brs
23	16.0 (CH3)	1.61, s	16.3 (CH3)	1.60, s
24	194.2 (CH)	9.43, s	60.3 (CH2)	4.10, d (12.1) 4.13, d (12.1)
25	103.3 (CH)	5.84, s	58.6 (CH2)	4.44, d (16.9) 4.52, d (16.9)
OMe-25	57.9 (CH3)	3.65, s

Assignments are based on COSY, HSQC (Figure S15) or HMQC (Figure S19), and HMBC experiments.

, Figures S13 and S14) as (6E) seco-manoalide (14) [17]. Accurate mass measurement by HRAPCIMS of 3 indicated a pseudo molecular ion peak at m/z 465.2414 [M + Cl]⁻ (calcd for C₂₆H₃₈ClO₅, 465.2413), consistent with the molecular formula C₂₆H₃₈O₅. This difference of 14 amu compared to the molecular formula of 14, and the appearance of a signal of methoxy group in ¹H, and ¹³C NMR spectra of 3 at δ_H 3.65 and δ_C 57.9. It is suggested that the hydroxyl group at C-25 was replaced by a methoxyl group. This was further confirmed by the HMBC correlations (Figure S16) between the methoxyl protons (δ_H 3.65) and hemiacetal carbon (δ_C 103.3) and between H-25 (δ_H 5.84) and methoxyl carbon (δ_C 57.9). The absolute stereochemistry at C-4 of 3 was determined by the modified Mosher’s method [24]. The hydroxyl group of 3 was converted into both the S- and R-MTPA esters 3a and 3b, respectively. The ¹H NMR chemical shifts were assigned by the analysis of the ¹H–¹H COSY NMR data for each MTPA ester (experimental section). The calculated Δδ_S−R values were positive for the H₂-5 (+0.03 and +0.03), H-6 (+0.15), H₂-8 (+0.04), and H-24 (+0.11) and negative for the H-2 (−0.13), 25-OMe (−0.004), and H-25 (−0.02) (Figure 3), implying that the absolute configuration of C-4 was R. Thus, compound 3 was characterized as (4R,6E) seco-manoalide-25-methyl ether.

Compound 4 was obtained as an optically active (${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{26}$ −20.9), and its molecular formula was determined to be C₂₅H₃₈O₅ (seven degrees of unsaturation) by HRAPCIMS. Five of the seven degrees of unsaturation implied by the molecular formula of 4 were taken up in one carbon–oxygen double bonds and four carbon–carbon double bonds, thus indicating the bicyclic nature of the molecule. The IR spectrum exhibited absorption bands corresponding to a hydroxyl group (3424 cm⁻¹), an ester carbonyl (1739 cm⁻¹), and an exomethylene substituent (898 cm⁻¹). Similarities in the NMR spectra between compounds 4 (Table 2, Figures S17 and S18) and 6Z-neomanoalide (13) suggested that compound 4 was also a neomanoalide-type sesterterpene [17].

The main differences in the ¹H NMR spectra of 4 and compound 13 were the absence of one olefinic methyl group resonance in compound 4 and the appearance of a resonance attributable to an exomethylene moiety (δ_H 4.83 and 4.91, H₂-22). Observation of a sp² methylene carbon resonance (δ_C 108.3) and a corresponding quaternary carbon (δ_C 150.5) further supported the presence of a exocyclic methylene functionality in the structure of 4. An oxygenated quaternary carbon (δ_C 80.4, C-14) was observed by ¹³C NMR and DEPT experiments. The assignment and placement of the hydroxyl substituent at C-14 and the exocyclic methylene at C-15 were deduced by observation of long range ¹H–¹³C HMBC correlations (Figure 4 and Figure S20) (H₂-13, H₂-16, H₂-18, H₃-21/22 to C-14, H₂-17 to C-15, and H₂-16 to C-22). The relative stereochemistry of the methylenecyclohexane unit was elucidated mainly on the basis of NOESY correlations (Figure 4). The correlations among axial H_α-18 (δ_H 1.63, td, J = 13.0, 5.7 Hz), axial methylene H₂-13, and equatorial CH₃-20 (δ_H 0.96) and correlations among axial H₃-21 (δ_H 0.88), exocyclic methylene H₂-22, and equatorial H_β-18 (δ_H 1.37, brd, J = 13.7 Hz) that were observed in 4 indicated that the equatorial hydroxyl group at C-14 occupied the β-face. The geometry of the olifinic bonds was assigned as 6Z,10E on the basis of NOESY correlations between H₂-5 and H₂-24 and between H₂-9 and H₃-23, respectively. Compound 4 exhibited a negative optical rotation {${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −20.9 (c 1.64, CHCl₃)}, similar to that of 6Z-neomanoalide (13) {${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −28 (c 0.8, CH₂Cl₂)} [12] the relative stereochemistry at C-4 of 4 was then assigned to be R. Thus, the structure 4 was concluded as shown in Figure 1 and was named erectusolide D.

Compound 5 was obtained as optically active (${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +1.3), and its molecular formula was determined to be C₂₃H₄₂O₃ (three degrees of unsaturation) by HRESIMS. The IR absorption bands data of compound 5 suggested the presence of β-substituted α, β-unsaturated γ-lactone (β-substituted butenolide for three degrees of unsaturation in the structure at 1777 and 1739 cm⁻¹ in addition to the hydroxyl group at 3442 cm⁻¹. The ¹H and ¹³C NMR spectra of 5 as shown in

Table 3. ¹H (600 MHz) and ¹³C (150 MHz) NMR data (CDCl₃) of compounds 5 and 6.

Position	5			6
	δC (Type)		δH, Mult. (J in Hz)	δC (Type)		δH, Mult. (J in Hz)
2	173.8 (C)		-	173.9 (C)		-
3	117.3 (CH)		5.93, s	117.2 (CH)		5.93, s
4	167.3 (C)		-	167.4 (C)		-
5	73.8 (CH2)		4.85, s	73.8 (CH2)		4.85, s
1′	36.3 (CH2)		2.52, dd (14.8, 8.2) 2.64, dd (14.8, 3.2)	36.5 (CH2)		2.52, dd (15.2, 8.2) 2.64, dd (15.2, 3.4)
2′	70.2 (CH)		3.84–3.90, m	70.2 (CH)		3.84–3.89, m
3′	37.7 (CH2)		1.49–1.54, m	37.7 (CH2)		1.49–1.55, m
4′	25.5 (CH2)		1.28–1.38, m 1.39–1.46, m	25.5 (CH2)		1.30–1.47, m
		(CH2)-5′– (CH2)-14′ 30.0, 29.68, 29.64, 29.63, 29.61, 29.57, 29.49, 29.49, 29.48, 27.1 (C-14′)	1.22–1.50, m		(CH2)-5′– (CH2)-14′ 29.65, 29.65, 29.65, 29.63, 29.61, 29.61, 29.58, 29.50, 29.50, 29.42	1.22–1.38, m
15′	36.6 (CH2)		1.07–1.17, m 1.23–1.37, m	29.3 (CH2)		1.22–1.38, m
16′	34.4 (CH)		1.23–1.37, m	31.9 (CH2)		1.22–1.31, m
17′	29.42 (CH2)		1.22–1.50, m	22.6 (CH2)		1.22–1.38, m
18′	11.3 (CH3)		0.85, t (7.2)	14.0 (CH3)		0.88, t (7.0)
19′	19.2 (CH3)		0.84, d (6.0)	-		-

Assignments are based on COSY, HSQC (Figures S23 and S27), and HMBC (Figures S24 and S28) experiments.

(Figures S11 and S22) suggests that compound 5 possess β-substituted butenolide moiety to which a saturated long chain hydrocarbon group is attached. The ¹H NMR signals at δ_H 5.93 (1H) and 4.85 (2H) assignable to an olefinic proton and methylene protons at the α- and γ-positions of the α,β-unsaturated γ-lactone, respectively, reveals that the long chain hydrocarbon group is attached at the β-position (C-4). The 2-hydroxy-16-methyloctadecane group (long chain hydrocarbon) was assigned by a combination of HRESIMS, ¹H and ¹³C NMR, DEPT, COSY, HSQC (Figure S23), and HMBC analyses. The HMBC spectrum of 5 (Figure 5 and Figure S24) showed the correlations from two of the methyl protons at δ_H 0.85 (H₃-18′, t, J = 7.2 Hz) and δ_H 0.84 (H₃-19′, d, J = 6.0 Hz) to the methine carbon at δ_C 34.4 (C-16′) and methylene carbon at δ_C 29.42 (C-17′) suggesting that the position of the second methyl group was attached at C-16′. In the HMBC spectrum of 5 also showed the correlations between methylene H₂-1′ (δ_H 2.52/2.64) and C-3 (δ_C 117.3), C-2′ (δ_C 70.2), C-3′ (δ_C 37.7), and C-5 (δ_C 73.8) and between H-2′ (δ_H 3.84–3.90) and C-4 (δ_C 167.3), C-4′ (δ_C 25.5) indicating clearly that the methylene carbon (C-1′) of 2-hydroxy-16-methyloctadecyl attached to the C-4 of the α,β-unsaturated γ-lactone. The absolute stereochemistry at C-2′ of 5 was determined by the modified Mosher’s method [24]. The hydroxyl group of 5 was converted into both the S- and R-MTPA esters 5a and 5b, respectively, each of which was a single diastereoisomer by ¹H and COSY NMR experiments. The calculated Δδ_S-R values were positive for the H₂-3′ (+0.04) and H₂-4′ (+0.06), while Δδ_S-R values were negative for H₂-1′ (−0.07 and −0.06), H-3 (−0.09), and H₂-5 (−0.13 and −0.27) (Figure 6), which implied the absolute configuration of C-2′ was R. Thus, compound 5 was characterized as 4-((2R)-2-hydroxy-16-methyloctadecyl)furan-2(5H)-one and was named erectusfuranone A.

Compound 6 was obtained as optically active (${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{26}$ −4.9), and its molecular formula was determined to be C₂₂H₄₀O₃ (three degrees of unsaturation) by HRESIMS. The UV, IR, ¹H and ¹³C NMR (Figures S25 and S26) spectroscopic data of 6 indicated that it was essentially identical to compound 5. The signals of a methine moiety (C-16′; δ_C 34.3, δ_H 1.23–1.37) and a methyl group (C-19′; δ_C 19.2, δ_H 0.84) were not evident in 6, instead a methylene group (C-16′; δ_C 31.9, δ_H 1.22–1.31) was observed. The absolute stereochemistry at C-2′ of 6 was determined by the modified Mosher’s method [24]. The hydroxyl group of 6 was converted into both the S- and R-MTPA esters 6a and 6b, respectively, each of which was a single diastereoisomer judged by ¹H and COSY NMR experiments. The Δδ_S-R (Figure 6) values observed in the ¹H NMR spectra were calculated and the resulting positive Δδ values for H₂-6 (+0.04 and +0.04) and H₂-7 (+0.06), and negative Δδ values for H₂-1′ (−0.06 and −0.06), H-3 (−0.09), and H₂-5 (−0.12 and −0.26) were consistent with the 2′R configuration. Thus, compound 6 was characterized as (R)-4-(2-hydroxyoctadecyl) furan-2(5H)-one and was named erectusfuranone B.

Cytotoxicity of the isolated compounds 3, 5, 7–20 and 23 were evaluated against several cancer cell lines such as (

Table 4. Cytotoxicity of compounds 3, 5, 7–20 and 23 (IC₅₀, μM).

Compound	MOLT-3	HepG2	HeLa	HuCCA-1	A549	H69AR	KB	T47D	MDA-MB-231	MRC-5
3	15.84	27.91	20.02	24.72	41.12	- a	- a	- a	40.09	- a
5	31.75	107.46	28.79	65.57	57.24	- a	- a	- a	89.26	- a
7	40.11	I	87.95	I	I	- a	- a	- a	I	- a
8	1.77	53.49	8.42	11.02	16.77	- a	- a	- a	39.53	13.00
9	1.30	27.91	1.51	8.60	12.00	- a	- a	- a	18.60	10.84
10	35.45	I	78.71	I	I	- a	- a	- a	I	- a
11	17.38	I	87.04	84.52	I	- a	- a	- a	I	- a
12	37.81	I	79.20	119.35	I	- a	- a	- a	I	- a
13	34.10	88.73	59.30	94.65	114.30	- a	- a	- a	92.04	- a
14	6.68	101.75	21.80	47.38	58.13	- a	- a	- a	84.13	17.48
15	- a	- a	63.08	49.07	- a	51.40	58.41	28.04	14.02	- a
16	124.32	I	77.54	I	I	- a	- a	- a	I	- a
17	- a	- a	63.08	60.75	- a	107.48	63.08	42.06	60.75	- a
18	- a	- a	23.32	41.45	- a	56.99	6.99	5.18	5.96	- a
19	13.83	75.45	65.32	60.81	60.81	- a	- a	- a	- a	- a
20	7.64	27.36	- a	87.06	73.38	- a	- a	- a	- a	70.97
23	77.98	I	I	I	I	- a	- a	- a	- a	- a
Doxorubicin	- a	0.36 ± 0.05	0.19 ± 0.00	0.40 ± 0.03	0.35 ± 0.02		1.64 ± 0.15		1.64 ± 0.17	3.16 ± 0.10
Etoposide	0.04 ± 0.01	- a	- a	- a	- a		- a		- a	- a

I = inactive at IC₅₀ > 50 µg/mL; ^a not determined; etoposide and doxorubicin were used as the reference compounds.

), MOLT-3 (acute lymphoblastic leukemia), HepG2 (hepatocarcinoma), HeLa (human cervical carcinoma), HuCCA-1 (human chlolangiocarcinoma), A549 (non-small-cell lung cancer), H69AR (multidrug resistance small-cell lung cancer), KB (human epidermoid carcinoma in the mouth), T47D (hormone dependent breast cancer), MDA-MB-231 (hormone independent breast cancer), and MRC-5 (normal embryonic lung cell). Compounds 8, 9, 14 and 20 showed good cytotoxic activity against MOLT-3 cell line with IC₅₀ values of 1.77, 1.30, 6.68, and 7.64 μM, respectively, compounds 8 and 9 also showed cytotoxic activity against HeLa, HuCCA-1, and A549 cell lines with IC₅₀ values of 1.51–16.77 μM. In addition, compounds 8 and 20 selectively exhibited cytotoxic activity toward the MOLT-3 (IC₅₀ 1.77 and 7.64 µM, respectively) cancer cell line with the selectivity index (SI) value of 7 (IC₅₀ 13.00 µM for normal cell line, MRC-5) and 9 (IC₅₀ 70.97 µM for MRC-5), respectively. The SI value is the ratio of IC₅₀ of normal cell (MRC-5) and IC₅₀ of cancer cell line. Compound 9 selectively exhibited cytotoxic activity against MOLT-3 (IC₅₀ 1.30 µM) and HeLa (IC₅₀ 1.51 µM) cell lines with respective SI values of 8 and 7 (IC₅₀ 10.84 µM for MRC-5). Manoalides 8 and 9, the acetal derivatives of the hemiacetal 11, showed higher activity than 11, suggesting that the presence of 24-O-methyl of manoalides 8 and 9 were important for cytotoxic activity (Table 4). Manoalide 11 was previously reported to possess good cytotoxicity against L1210 (mouse lymphocytic leukemia) and KB (mouth epidermal carcinoma) cell lines with the IC₅₀ value of 0.053 and 0.63 µM, respectively [12]. Compound 20 (12-O-deacetyl-12-epi-scalarin) exhibited cytotoxic activity against A549 cells with an IC₅₀ of 73.38 µM (Table 4), while its IC₅₀ have been reported in the literature to be 36.82 μM [16]. Luffariolide B (10), (6E)- and (6Z)-neomanoalides (12 and 13) showed weak cytotoxicity against the MOLT-3 cell line with IC₅₀ of 35.45, 37.81, and 34.10 μM, respectively, but they exhibited cytotoxicity against the L1210 cell line with IC₅₀ of 3.23, 24.38, and 13.93 μM, respectively [15].

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were recorded on a JASCO DIP 1020 polarimeter using cylindrical glass cell (10 mm inner diameter (I.D.) × 10 mm). UV spectra were measured with a UV-1700 Pharma Spec (Shimadzu, Kyoto, Japan) spectrophotometer. Fourier transform infrared (FTIR) spectra were obtained using a universal attenuated total reflectance attached on Perkin Elmer Spectrum One spectrometer (PerkinElmer, Waltham, MA, USA). Nuclear magnetic resonance (NMR) spectra were recorded in a CDCl₃ or C₆D₆ solution containing Me₄Si as internal standard on Bruker AM400 or AVANCE600 spectrometer (Bruker Corporation, Billerica, MA, USA). HR–MS was performed on a Bruker (Micro ToF, Bruker Corporation, Billerica, MA, USA) spectrometer. HPLC was carried out on a Waters 600 system (Warers Corporation, Milford, MA, USA) equipped with a Waters Delta 600 pump, a Waters 600 Controller, a Waters 2998 photodiode array detector, and Waters Empower 2 software. Sephadex^™ LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was used for a column gel filtration. All commercial grade solvents were distilled prior to use and spectral grade solvents were used for spectroscopic measurements.

3.2. Sponge Material

Sponges (Hyrtios erectus) CRI 572 and CRI 588 were collected by hand using scuba at a depth of 30–40 feet in the Similan Island at the Andaman Sea (Phangnga province, Thailand) on 22 and 23 February 2011, respectively. The sponges were identified by Dr. Sumaitt Putchakarn, Head of Marine Biodiversity Research, Unit Curator of Porifera and Echinodermata, Institute of Marine Science, Burapha University, Bangsaen, Chonburi, Thailand. The voucher specimens (CRI 572 and CRI 588) were presently deposited at the Laboratory of Natural Products, Chulabhorn Research Institute, Bangkok, Thailand.

3.3. Extraction and Isolation

A frozen sample (2.6 kg) of H. erectus collection CRI 572 was cut into small pieces and extracted exhaustively with MeOH. The extract was filtered through cotton, and then evaporated under reduced pressure to give an aqueous residue, which was partitioned with EtOAc. The organic layer was concentrated to give a dark brown solid (7.52 g). The EtOAc–soluble fraction was subjected to vacuum liquid chromatography on silica gel and eluted with an EtOAc–hexane gradient (0→100% of EtOAc). Nine fractions (F1–F9) were obtained. F2 (340.5 mg) was subjected to column chromatography on Sephadex LH-20, using CH₂Cl₂–MeOH (1:1) to give compound 15 (16 mg). F4 (293.3 mg) was repeatedly fractionated by Sephadex LH-20, using CH₂Cl₂–MeOH (1:1) to give a mixture compounds (96.8 mg) that was separated using on a HPLC column (Hichrome C18, 5 µm, 21.2 mm × 250 mm with MeOH–H₂O (gradient, 77→97% MeOH for 40 min), flowrate 12 mL/min, λ 220, 265 nm) to afford compounds 16 (28 mg, at 12.7 min), 17 (4.9 mg, at 32.5 min). F5 (574.4 mg) was subjected to column chromatography on Sephadex LH-20, using 100% MeOH to obtain a mixtures compound (9.3 mg) that was separated using on a HPLC column (Sunfire C18, 5 µm, 10 mm × 250 mm, with 80% CH₃CN–H₂O, flowrate 3 mL/min, λ 220 nm) to afford 18 (4.0 mg, at 25.7 min). F6 (976.4 mg) was subjected to column chromatography on Sephadex LH-20, using CH₂Cl₂–MeOH (1:1) to give three fractions (f1–f3). Fraction f1 was subjected to repeated chromatography on Sephadex LH-20, using 100% MeOH to give a mixtures compound (73.5 mg) that was separated using a HPLC column (Hichrome C18, 5 µm, 21.2 mm × 250 mm, with MeOH–H₂O (gradient, 75→100% MeOH for 50 min), flowrate 12 mL/min, λ 220 nm) to afford compound 19 (2.3 mg, at 35.2 min). Fraction f2 was subjected to column chromatography on Sephadex LH-20, using 100% MeOH to give a mixtures compound (45.6 mg) that was separated using on a HPLC column (Sunfire C18, 5 µm, 19 mm × 250 mm, with 88% MeOH–H₂O, flowrate 12 mL/min, λ 220 nm) to afford 20 (20 mg, at 16.7 min). Fraction f3 was subjected to repeated chromatography on Sephadex LH-20, using 100% MeOH to give a mixtures compound (6.9 mg) that was separated using on a preparative thin layer chromatography (PTLC) [hexane–CH₂Cl₂–acetone (2:2:1) as eluent] to afford 21 (1.4 mg). F7 (508.1 mg) was subjected to column chromatography on Sephadex LH-20, using 100% MeOH to give a mixtures compound (12.1 mg) that was separated using on a HPLC column (Sunfire C18, 5 µm, 19 mm × 250 mm, with 20% MeOH–H₂O, flowrate 12 mL/min, λ 254 nm) to afford compound 22 (7.5 mg, at 12.8 min). F8 (298.1 mg) was repeatedly fractionated by Sephadex LH-20, using CH₂Cl₂–MeOH (1:1) to give a mixture compound (127.6 mg) that was separated using a HPLC column (Hichrome C18, 5 µm, 21.2 mm × 250 mm with 75% MeOH–H₂O, flowrate 12 mL/min, λ 222 nm) to afford compounds 4 (16.4 mg, at 11.3 min), and 13 (16.1 mg, at 33.5 min). F9 (249.2 mg) was subjected to repeated chromatography on Sephadex LH-20, using CH₂Cl₂–MeOH (1:1) to give a mixtures compound (20.3 mg) that was separated using a HPLC column (Hichrome C18, 5 µm, 21.2 mm × 250 mm, with MeOH–H₂O (45–55% MeOH for 20 min), flowrate 12 mL/min, λ 234 nm) to afford compound 23 (5.4 mg, at 14.7 min). The flesh sponge Hyrtios erectus collection CRI 588 (14.0 kg) was cut into small pieces and extracted repeatedly with MeOH (20 L) three times (3 × 20 L). After the evaporation of the solvent, the concentrated MeOH extracts was partitioned between EtOAc and water and the EtOAc fraction was chromatographed on a silica gel column with CH₂Cl₂–hexane (1:1) containing increasing proportions of acetone as eluent, to give 7 fractions (A–G). Fraction C (4.0 g) was fractionated by Sephadex LH-20 with MeOH–CH₂Cl₂ (1:1) as eluent, to give 2 fractions (C1–C2). C2 (230.3 mg) was repeatedly chromatography on Sephadex LH-20, using CH₂Cl₂–MeOH (4:1) to give a mixtures compound (200 mg) that was separated using a HPLC column (Sunfire Prep C18, 5 µm, 10 mm × 250 mm) with MeOH–H₂O (93:7), flowrate 2.5 mL/min, λ 224 nm, to afford compounds 5 (4.1 mg at 45.5 min), and 6 (7.2 mg at 48.2 min). Fraction D (9.0 g) was further fractionated on a Sephadex LH-20 column chromatography with MeOH–CH₂Cl₂ (1:1) to yield 4 fractions (D1–D4). D4 (400 mg) was further purified by HPLC column (Sunfire C18, 5 µm, 19 mm × 250 mm) with CH₃CN–H₂O (80:20), flowrate 8 mL/min, λ 224, 236 nm, to afford compounds 1 (5.9 mg, at 23.0 min), 2 (1.6 mg, at 29.6 min), and 3 (7.2 mg, at 35.6 min). Fraction E (25.0 g) was further fractionated by vacuum liquid chromatography eluted with CH₂Cl₂–hexane (1:1) containing increasing proportions of acetone as eluent, to give 4 fractions (E1–E4). Fraction E1 (2 g) was further purified by a HPLC column (Sunfire C18, 5 µm, 19 mm × 250 mm) with CH₃CN–H₂O (85:15), flowrate 8 mL/min, λ 224, 236 nm, to afford compounds 7 (74 mg, at 32.3 min), 8 (52 mg, at 47.9 min), and 9 (229 mg, 52.8 min). Fraction E3 (3 g) was further purified by a HPLC column (Sunfire C18, 5 µm, 19 mm × 250 mm) with CH₃CN–H₂O (80:20), flowrate 8 mL/min, λ 224, 236 nm, to afford compounds 10 (27 mg, at 19.9 min), and 11 (56 mg, at 32.5 min). Fraction E4 (1.4 g) was further purified by HPLC column (Sunfire C18, 5 µm, 19 mm × 250 mm) with CH₃CN–H₂O (82:18), flowrate 8 mL/min, λ 224, 236 nm, to afford compounds 13 (72 mg, at 27.35 min) and 14 (33 mg, at 29.3 min). Fraction G (4.0 g) was further filtrated on a Sephadex LH-20 column chromatography with MeOH to give a residue (655 mg), which was further purified by HPLC column (Cosmosil, 5 μ C18-MS-II, 20 mm × 250 mm) with CH₃CN–H₂O (60:40), flowrate 8 mL/min, λ 224, 236 nm, to afford compound 12 (70.0 mg, at 64.2 min).

Preparation of (S)- and (R)-MTPA Esters of compound 3: To a solution of 3 (3.1 mg) in pyridine (0.9 mL) was added (R)-MTPA-chloride (17 µL). The mixture was stirred at room temperature for 3 h, checked with thin layer chromatography (TLC) to make sure that the reaction was complete, quenched by the addition of 2 mL of H₂O, and the mixture was subsequently extracted with CH₂Cl₂ (2 mL) three times (3 × 2 mL). The CH₂Cl₂ soluble layers were combined, dried over anhydrous MgSO₄, and evaporated. The residue was subjected to short silica gel column chromatography using hexane–EtOAc (4:1) to give the (S)-MTPA ester 3a (1.6 mg).

Selected ¹H NMR (CDCl₃ 600 MHz) of 3a: δ_H 9.32, s, H-24; 6.28, t (J = 7.1 Hz), H-6; 5.86, brs, H-2; 5.79, s, H-25; 5.74, dd (J = 7.2, 4.2 Hz), H-4; 5.09, t (J = 7.1 Hz), H-10; 3.03, ddd (J = 16.1, 6.6, 4.2 Hz), Ha-5; 2.90, dt (J = 16.1, 8.0 Hz), Hb-5; 2.24, t (J = 7.6 Hz), H₂-8; 2.02, m, H₂-9.

The same procedure was used to prepare the (R)-MTPA ester 3b (2.5 mg from 3.9 mg of 3) with (S)-MTPA chloride.

Selected ¹H NMR (CDCl₃ 600 MHz) of 3b: δ_H 9.21, s, H-24; 6.13, t (J = 7.1 Hz), H-6; 6.00, brs, H-2; 5.81, s, H-25; 5.74, dd (J = 7.0, 4.2 Hz), H-4; 5.07, t (J = 6.9 Hz), H-10; 3.00, ddd (J = 16.2, 6.8, 4.3 Hz), Ha-5; 2.87, dt (J = 16.2, 7.5 Hz), Hb-5; 2.20, t (J = 7.7 Hz), H₂-8; 1.99, m, H₂-9

(S)-MTPA esters 5a (1.3 mg from 1.4 mg of 5) and 6a (1.8 mg from 1.4 mg of 6) and (R)-MTPA esters 5b (1.5 mg from 1.4 mg of 5), 6b (1.7 mg from 1.2 mg of 6) were prepared by the method mentioned above.

Selected ¹H NMR (CDCl₃, 400 MHz) of 5a: δ_H 5.77, brs, H-3; 2.72, dd (J = 15.7, 6.9 Hz), Ha-1′; 2.62, dd (J = 15.7, 3.1 Hz), Hb-1′; 5.28, m, H-2′; 1.67, m, H₂-3′; 4.56, dd (J = 17.8, 1.8 Hz), Ha-5; 4.25, brd (J = 17.6 Hz), Hb-5.

Selected ¹H NMR (CDCl₃, 400 MHz) of 5b: δ_H 5.86, s, H-3; 2.78, dd (J = 15.7, 7.3 Hz), Ha-1′; 2.69, dd (J = 15.7, 3.1 Hz), Hb-1′; 5.29, m, H-2′; 1.63, m, H₂-3′; 4.69, brd (J = 17.4 Hz), Ha-5; 4.52, brd (J = 17.4 Hz), Hb-5.

Selected ¹H NMR (CDCl₃, 600 MHz) of 6a: δ_H 5.77, brs, H-3; 2.72, dd (J = 15.6, 7.1 Hz), Ha-1′; 2.63, dd (J = 15.6, 3.4 Hz), Hb-1′; 5.28, m, H-2′; 1.71, m, Ha-3′; 1.59, m, Hb-3′; 1.29, m, H₂-4′; 4.57, dd (J = 17.8, 1.6 Hz), Ha-5; 4.26, brd (J = 16.7 Hz), Hb-5.

Selected ¹H NMR (CDCl₃, 600 MHz) of 6b: δ_H 5.86, brs, H-2; 2.78, dd (J = 15.4, 6.58 Hz), Ha-1′; 2.69, dd (J = 15.4, 3.6 Hz), Hb-1′; 5.29, m, H-2′; 1.67, m, Ha-3′; 1.55, m, Hb-3′; 1.23, m, H₂-4′; 4.69, d (J = 17.5 Hz), Ha-5; 4.52, d (J = 17.5 Hz), Hb-5.

Erectusolide B (1): Colorless powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{26}$ −1.3 (c 0.56, CHCl₃); UV λ_max (MeOH) nm (log ε) 202 (3.9), 224 (3.4), IR (ATR) ν_max 3443 (br), 2927, 2861, 1779, 1746, 1629, 1456, 1379, 1266, 1143, 1041, 888, 851, 735, 702 cm⁻¹; ¹H and ¹³C NMR data see Table 1; HRAPCIMS m/z 385.2727 [M + H]⁺ (calcd for C₂₅H₃₇O₃, 385.2737).

Erectusolide C (2): Colorless powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{26}$ −18.5 (c 0.16, CHCl₃); UV λ_max (MeOH) nm (log ε) 234 (4.3); IR (ATR) ν_max 2924, 2855, 1779, 1748, 1682, 1632, 1458, 1378, 1261, 1142, 1040, 887 cm⁻¹; ¹H and ¹³C NMR data see Table 1; HRESIMS m/z 405.2390 [M + Na]⁺ (calcd for C₂₅H₃₄NaO₃, 405.2400).

seco-Manoalide-25-methyl ether (3): Pale yellow solid; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{24}$ −22.2 (c 1.86, CHCl₃); UV λ_max (MeOH) nm (log ε) 203.5 (4.3), 232 (sh); IR (ATR) ν_max 3446 (br), 2930, 2863, 1762, 1687, 1457, 1371, 1203, 1120, 1070, 960, 898, 870, 735, 702 cm⁻¹; ¹H and ¹³C NMR data see Table 2; HRAPCIMS m/z 465.2414 [M + Cl]⁻ (calcd for C₂₆H₃₈ClO₅, 465.2413).

Erectusolide D (4): Pale yellow gum; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −20.9 (c 1.64, CHCl₃); UV λ_max (MeOH) nm (log ε) 202 (4.3), IR (ATR) ν_max 3424 (br), 2928, 2857, 1739, 1642, 1456, 1382, 1258, 1172, 1063, 898 cm⁻¹; ¹H and ¹³C NMR data see Table 2; HRAPCIMS m/z 453.2418 [M + Cl]⁻ (calcd for C₂₅H₃₈ClO₅, 453.2413).

erectusfuranone A (5): Colorless powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +1.3 (c 0.32, CHCl₃); UV λ_max (MeOH) nm (log ε) 206 (3.9); IR (ATR) ν_max 3442 (br), 2923, 2853, 1777, 1739, 1637, 1457, 1378, 1175, 1027, 888, 720 cm⁻¹; ¹H and ¹³C NMR data see Table 3; HRAPCIMS m/z 367.3213 [M + H]⁺ (calcd for C₂₃H₄₃O₃, 367.3207).

erectusfuranone B (6): Colorless powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{26}$ −4.9 (c 0.61, CHCl₃); UV λ_max (MeOH) nm (log ε) 206 (3.9); IR (ATR) ν_max 3402 (br), 2916, 2851, 1779, 1756, 1736, 1634, 1469, 1025, 892, 720 cm⁻¹; ¹H and ¹³C NMR data see Table 3; HRESIMS m/z 353.3053 [M + H]⁺ (calcd for C₂₂H₄₁O₃, 353.3050).

3.4. Cytotoxicity Assay

Cytotoxic activity for adhesive cell lines including HepG2, HeLa, HuCCA-1, A549, H69AR, KB, T47D, MDA-MB-231, and MRC-5 cell lines were evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay [25,26]. For the non-adhesive MOLT-3 cell line, the cytotoxicity was assessed using the 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl-(2H)-tetrazolium-5-carboxanilide (XTT) assay [27]. Etoposide and doxorubicin were used as positive controls (Table 4)

4. Conclusions

Two separate collections of H. erectus were studied. Three new sesterterpenes, erectusolides B (1), C (2), and seco-manoalide-25-methyl ether (3), two new 2-furanone derivatives, erectusfuranones A (5) and B (6) together with eight known sesterterpenes, 7–14 were isolated from H. erectus collection CRI 588. One additional new sesterterpene, erectusolide D (4), and ten known compounds (13, 15–23) were also obtained from the H. erectus collection CRI 572. Three sesterterpenes 8, 9, and 20 showed good cytotoxic activity against the MOLT-3 cell line with IC₅₀ values of 1.30–7.64 μM and with SI (selectivity index) values 7–9.