Cytotoxic Sesterterpenes from Thai Marine Sponge Hyrtios erectus

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.

Compound 3 was obtained as a pale yellow solid, exhibiting similar ultraviolet (UV), infrared (IR), 1 H, and 13 C NMR spectra (Table 2) 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  Compound 2 was obtained as optically active ([α] 26 D −18.5), and its molecular formula was determined to be C 25 H 34 O 3 (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 1 H and 13 C 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 = 13.2Hz, 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 ([α] 26 D −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), 1 H, and 13 C NMR spectra (Table 2, 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 C 26 H 38 ClO 5 , 465.2413), consistent with the molecular formula C 26 H 38 O 5 .This difference of 14 amu compared to the molecular formula of 14, and the appearance of a signal of methoxy group in 1 H, and 13 C 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 (Figures 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 Sand R-MTPA esters 3a and 3b, respectively.The 1 H NMR chemical shifts were assigned by the analysis of the 1 H-1 H COSY NMR data for each MTPA ester (experimental section).The calculated ∆δ S−R values were positive for the H 2 -5 (+0.03 and +0.03), H-6 (+0.15),H 2 -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 ([α] 26 D −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) and 6Z-neomanoalide (13) suggested that compound 4 was also a neomanoalide-type sesterterpene [17].
The main differences in the 1 H 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   Compound 4 was obtained as an optically active ([α] 26 D −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) and 6Z-neomanoalide (13) suggested that compound 4 was also a neomanoalide-type sesterterpene [17].
The main differences in the 1 H 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 (δH 4. )} [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 ([α] 25 D +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 1 H and 13 C NMR spectra of 5 as shown in Table 3 suggests that Compound 4 was obtained as an optically active ([α] 26 D −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) and 6Z-neomanoalide (13) suggested that compound 4 was also a neomanoalide-type sesterterpene [17].
The main differences in the 1 H 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 (δH 4. )} [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 ([α] 25 D +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 1 H and 13 C NMR spectra of 5 as shown in Table 3 suggests that Compound 4 was obtained as an optically active ([α] 26 D −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) and 6Z-neomanoalide (13) suggested that compound 4 was also a neomanoalide-type sesterterpene [17].

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 CDCl 3 or C 6 D 6 solution containing 4 Si 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.

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.
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.

Figure 3 .
Figure 3. ΔδS-R values in ppm for S-and R-MTPA esters of compound 3 in CDCl3.

Figure 3 .
Figure 3. ∆δ S-R values in ppm for Sand R-MTPA esters of compound 3 in CDCl 3 .Compound 4 was obtained as an optically active ([α] 26 D −20.9), and its molecular formula was determined to be C 25 H 38 O 5 (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 (Table2, FiguresS17 and S18) and 6Z-neomanoalide(13) suggested that compound 4 was also a neomanoalide-type sesterterpene[17].The main differences in the 1 H 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, H 2 -22).Observation of a sp 2 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 13 C 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 1 H-13 C HMBC correlations (Figures 4 and S20) (H 2 -13, H 2 -16, H 2 -18, H 3 -21/22 to C-14, H 2 -17 to C-15, H 4.83 and 4.91, H 2 -22).Observation of a sp 2 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 13 C 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 1 H-13 C HMBC correlations (Figures 4 and S20 ) (H 2 -13, H 2 -16, H 2 -18, H 3 -21/22 to C-14, H 2 -17 to C-15, and H 2 -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 H 2 -13, and equatorial CH 3 -20 (δ H 0.96) and correlations among axial H 3 -21 (δ H 0.88), exocyclic methylene H 2 -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 H 2 -5 and H 2 -24 and between H 2 -9 and H 3 -23, respectively.Compound 4 exhibited a negative optical rotation {[α] 25 D −20.9 (c 1.64, CHCl 3 )}, similar to that of 6Z-neomanoalide (13) {[α] 25 D −28 (c 0.8, CH 2 Cl 2 )} [12]the relative stereochemistry at C-4 of 4 was then assigned to be R. Thus, the structure 4 was concluded as shown in Figure1and was named erectusolide D.

Figure 3 .
Figure 3. ΔδS-R values in ppm for S-and R-MTPA esters of compound 3 in CDCl3.

Figure 3 .
Figure 3. ΔδS-R values in ppm for S-and R-MTPA esters of compound 3 in CDCl3.

Figure 6 .
Figure 6.ΔδS-R values in ppm for S-and R-MTPA esters of compounds 5 and 6 in CDCl3.

Figure 6 .
Figure 6.ΔδS-R values in ppm for S-and R-MTPA esters of compounds 5 and 6 in CDCl3.

Figure 6 .
Figure 6.ΔδS-R values in ppm for S-and R-MTPA esters of compounds 5 and 6 in CDCl3.

Figure 6 .
Figure 6.ΔδS-R values in ppm for S-and R-MTPA esters of compounds 5 and 6 in CDCl3.

Figure 6 .
Figure 6.ΔδS-R values in ppm for S-and R-MTPA esters of compounds 5 and 6 in CDCl3.

Figure 6 .
Figure 6.∆δ S-R values in ppm for Sand R-MTPA esters of compounds 5 and 6 in CDCl 3 .