European Journal of Medicinal Chemistry xxx (2012) 1e5
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Short communication
Laurene-type sesquiterpenes from the Red Sea red alga Laurencia obtusa as potential antitumoreantimicrobial agents Walied M. Alarif a, *, Sultan S. Al-Lihaibi a, Seif-Eldin N. Ayyad b, Mohamed H. Abdel-Rhman c, Farid A. Badria d a
Department of Marine Chemistry, Faculty of Marine Sciences, King Abdulaziz University, PO. Box 80207, Jeddah 21589, Saudi Arabia Department of Chemistry, Faculty of Science, King Abdulaziz University, PO. Box 80203, Jeddah 21589, Saudi Arabia Department of Chemistry, Faculty of Science, Mansoura University, Mansoura, Egypt d Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 May 2012 Received in revised form 24 June 2012 Accepted 30 June 2012 Available online xxx
Three new laurene-type sesquiterpenes, 12-hydroxy isolaurene (1), 8,11-dihydro-12-hydroxy isolaurene (2) and isolauraldehyde (3) were isolated from the organic extract of the red alga Laurencia obtusa. The chemical structures of isolates were determined by interpretation of their spectral data 1D and 2D NMR, UV, IR and MS. The newly isolated compounds were tested for their antimicrobial and antitumor activities. Compounds (1e3) exhibited potent activity against the Gram-positive Bacillus subtilis and Staphylococcus aureus, where 3 proved to be the most active (MIC 35 and 27 mg/mL, respectively). Moreover, compound 3 exhibited a significant activity against Candida albicans (MIC of 70 mg/mL) and revealed to have very promising activity in an in vitro model of Ehrlich ascites Carcinoma. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Marine algae Terpenoids Gram-positive bacteria Candida albicans EAC
1. Introduction Bioactive metabolites originated from marine organisms exhibited different effects on many diseases other than that of the terrestrial counterparts, which may lead to the discovery of new efficient bioactive metabolites with different modes of action [1]. The different locations with diversity of atmosphere led to production of different active substances as well as structures [2]. Laurene-type sesquiterpenes are aryl cyclopentanes substituted with three methyl groups in 1, 2 and 3 fashion. In addition to laurenes, two closely similar sesquiterpene families; cuparenes and laurokamurenes, differ from each other only in the methylation pattern (1, 2, 2 and 2, 2, 3 for cuparenes and laurokamurenes, respectively). On the contrary to the laurenes, the biological activities of several members of the cuparenes family have been examined as antifungal, antibiotic, neurotrophic and antilipidperoxidation agents [3].
* Corresponding author. Tel.: þ966 2 6952383, þ966 56 0352034 (mobile); fax: þ966 2 6401747. E-mail address:
[email protected] (W.M. Alarif).
Among all marine algal flora, species of the genus Laurencia are always expected to be a source of novel secondary metabolites, especially sesquiterpenes, diterpenes and halogenated C15 acetogenins [4,5]. Several metabolites of Laurencia showed noticeable antibacterial [6,7] insecticidal [8] antifungal [9], antiviral activity [10], tyrosine inhibitor [11] and apoptosis inducing or suppressing activity [5], this tempted us to investigate some more chemical constituents of this organism. Laurene sesquiterpenes have been isolated from the red algae of the genus Laurencia (Rhodomelaceae, Ceramiales) [12,13]. These were first isolated from the sea hare Aplysia kurodai [14] which suggests that this sea hare may consume Laurencia sp. and concentrate these sesquiterpenoid compounds in its body [15]. Ehrlich Ascites Carcinoma (EAC) is a common tumor. It is an undifferentiated carcinoma with high transplantable capability, noregression, rapid proliferation, shorter life span, 100% malignancy and also does not have tumor-specific transplantation antigen (TSTA) [16]. Three methods are applied for cancer therapy; chemotherapy, radiotherapy and surgery. Recently, chemotherapy is the most widely used therapy [17]. The main principle of chemotherapy, which serves as a drug treatment in cancer, is to prevent the growth and progression of tumor cells or to destroy
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2012.06.060
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W.M. Alarif et al. / European Journal of Medicinal Chemistry xxx (2012) 1e5
them by the effect it has on tumor cells more than the normal cells of the patient without or with minimum side effects [18]. As a part of systematic endeavors to isolate bioactive compounds from the Saudi Red Sea organisms, we investigated constituents of Laurencia obtusa collected off the coast of Jeddah, KSA. We report the isolation and characterization of three new laurenes (1e3) along with one known chamigrene sesquiterpene (4), in addition to one known steroid, cholest-4-en-3-one (5) obtained from the pet-ether extract of the red alga L. obtusa. 2. Results and discussion 2.1. Chemistry The sequential use of separation techniques including column and preparative TLC of the petroleum ether extract of the red alga L. obtusa afforded three new sesquiterpenes 1, 2 and 3, in 0.005%, 0.0015% and 0.002% yield (based on dry weight of the algal material), respectively, in addition to known a-chamigrene (4) and a steroidal ketone compound (5) (Fig. 1). Compound 1 was obtained as an optically active colorless oil [a]D ¼ þ41.7 (CHCl3; c ¼ 0.01). The molecular formula of 1, C15H20O (corresponding to 6 degrees of unsaturation), was deduced from the HRESIMS analysis, m/z 215.1423 [M H]þ in negative-ion mode. The EIMS of 1 showed a molecular ion peak at m/z 216. The parent peak at m/z 201 corresponding to the formula C14H17Oþ arises from the expulsion of a methyl group, and the m/z 109, 91 and 77 peaks would be from an aromatic group. The existence of a substituted benzene ring was concluded from UV absorption spectrum, which showed maxima at 273 and 279 nm, supported by IR absorption at 1510 cm1. Moreover, the IR bands at nmax 3298 and 1637 cm1 were attributed to a hydroxyl and to an isolated double bond, respectively. The 1H, 13C and DEPT NMR spectra of 1 showed the presence of 15 carbon atoms (Table 1), including three methyl groups, three methylenes (one oxygenated at dH/dC 4.62/65.2), four sp2 methine
(dC 2 126.7 and 2 127.1) and five quaternary carbons (dC 54.8, 137.2, 132.1, 148.9 and 137.8). From the previous discussion, the structure suggested to be composed of a 1,4-disubstituted benzene ring, extra double bond, primary hydroxyl group and one more ring (to fulfill 6 degrees of unsaturation). HSQC spectrum indicates the presence of three tertiary methyl groups signals appeared at dH/dC: 1.36/24.2, 1.41/10.3 and 1.72/14.3, in addition to a singlet signal at dH 4.62 that was attributed to two protons linked to a carbon atom at dC 65.2. Furthermore, the 1He1H COSY spectrum supported the existence of 1,4-disubstituted benzene ring through the 1He1H spin system between H-7 (11) and H-8 (10) and the long range correlation between H-8 and H212. This allowed us to indicate the presence of 1,4-disubstituted benzene ring together with a 1,2,3-trimethylcyclopentenyl partial structure (Fig. 1). The spectral data of compound 1 resembles that was reported for isolaurene (6) [19], except the appearance of a hydroxyl function. The absence of any aromatic methyl group in compound 1 compared to isolaurene (6) and the presence of a hydroxy methyl group together with HMBC correlations between 2H-12 and C-9 in addition to C-8 and C-10 confirmed the location of the carbinol group as p-position to the other substitution. Moreover, the HMBC correlations between H3-13 (d 1.36) and C-1, C-2, C-5 and C-6 is a further confirmation of the structure of compound (The down field shift of the Me-13 dH 1.36, could be attributed to the anisotropic effect of the benzene ring). Thus, in view of the abovementioned data and discussion, compound 1 has been identified as 12-hydroxy isolaurene. Compound 2 was obtained as an optically active colorless oil [a]D ¼ þ11.5 (CHCl3; c ¼ 0.01). The molecular formula of 2, C15H22O, was deduced from the analysis of the HRESIMS m/z 217.1579 [M H]þ, in negative-ion mode. The EIMS of 2 showed a molecular ion peak at m/z 218. The parent peak at m/z 203 corresponding to the formula C14H19Oþ arises from the loss of a methyl group, and the m/z 185 corresponding to molecular formula C14Hþ 17 arises from the expulsion of water molecule from the Mþ CH3. The m/z 90 and 77
Fig. 1. Structures of compounds 1e6.
Please cite this article in press as: W.M. Alarif, et al., Laurene-type sesquiterpenes from the Red Sea red alga Laurencia obtusa as potential antitumoreantimicrobial agents, European Journal of Medicinal Chemistry (2012), http://dx.doi.org/10.1016/j.ejmech.2012.06.060
W.M. Alarif et al. / European Journal of Medicinal Chemistry xxx (2012) 1e5 Table 1 1 H [600 MHz] and Position
3
13
C NMR [150 MHz] NMR spectral data of 1e3.a,b 1
2
3
dH (J in Hz)
dCc
dH (J in Hz)
dC
dH (J in Hz)
dC
1 2 3 4
e e e Ha & Hb 2.30 (m)
54.8 137.2 132.1 35.8
e e e Ha & Hb 2.21 (m)
55.4 134.3 131.7 35.9
55.0 137.0 133.0 35.9
5
41.5
148.9 126.5 127.1
27.7
e 7.40 (d, 7.8) 7.8 (d, 7.8)
143.0 127.0 129.7
9 10 11
e 7.21 (d, 8.4 Hz) 7.27 (d, 8.4 Hz)
137.8 127.1 126.5
139.7 121.1 26.2
e 7.8 (d, 7.8) 7.40 (d, 7.8)
134.4 129.7 127.0
12 13 14 15
4.62 (s) 1.36 (brs) 1.41(brs) 1.72 (brs)
65.2 24.2 10.3 14.3
Ha 1.83 (ddd, 13.2, 7.8 & 5.4 Hz) Hb 1.52 (ddd, 13.2, 4.2 & 1.8 Hz) e 5.45 (m) 116.4 Ha 2.70 (m), Hb 2.66 (m) e 5.69 (m) Ha 2.62 (m), Hb 2.40 (m) 4.00 (s) 1.13 (brs) 1.36 (brs) 1.63 (brs)
36.2
6 7 8
Ha 1.92 (ddd, 13.2, 8.2 & 5.4 Hz) Hb 1.90 (ddd, 13.2, 6.6 & 4.8) e 7.27 (d, 8.4 Hz) 7.21 (d, 8.4 Hz)
e e e Ha 2.34 (m), Hb 2.30 (m) Ha 1.88 (m), Hb 1.95 (m)
66.8 23.8 10.0 14.3
9.98 1.36 1.45 1.72
192.2 24.0 10.3 14.1
a b c
135.9
(s) (brs) (s) (brs)
41.4
The solvent is CDCl3. All assignments are based on 1D and 2D measurements (HMBC, HSQC, COSY). Implied multiplicities were determined by DEPT (C ¼ s, CH ¼ d, CH2 ¼ t).
peaks should be arising from an aromatic group. Compound 2 is two mass units more compared to compound 1, which led us to believe that 2 is a partial saturated form of 1. The IR spectral data indicated the presence of hydroxyl and double bond vibration bands at 3324 and 1641 cm1, respectively. The 1H, 13C and DEPT NMR spectra of 2 showed the presence of 15 carbon atoms (Table 1), including three methyl groups, five methylenes (one oxygenated at dH/dC 4.00/66.8), two sp2 methines (dC 116.4 and 121.1) and five quaternary carbons (dC 55.4, 134.2, 131.7, 135.8 and 139.7). Since compound 2 has five degrees of unsaturation with six signals due to olefinic carbons (i.e. three double bonds); from the HSQC spectrum, a set of three tertiary methyl groups signals appeared at dH/dC: 1.13/23.9, 1.36/10.0 and 1.63/14.4, that are closely similar to that of compound 1 (i.e. a 1,2,3-trimethylcyclopentenyl partial structure); together with the absence of any absorptions due to benzene ring in both UV and IR spectra, hence, compound 2 should be bicyclic. The presence of a sixmembered ring was deduced by analysis of the 1He1H COSY spectrum in which the olefinic proton H-10 (dH 5.69) is correlated with 2H-12 and H-11; H-7 (dH 5.45) is correlated to H-8; and both H-7 and 2H-12 are correlated with H-8. As a result of absence of any absorption at about 260 nm in the UV spectrum (i.e. no double bond conjugation) [20], the two double bonds should constitute 1,4positions within the ring. Therefore, compound 2 is composed of cyclohex-1,4-dien ring linked to a 1,2,3-trimethylcyclopentenyl group. The structure connectivity’s and the location of the hydroxy methyl group were achieved by studying HMBC spectrum where
Fig. 2. Selected HMBC of compound 2.
2H-12 is correlated with C-9, C-8 and C-10; H-7 is correlated with C-6, C-1, C-8 and C-11; H-10 is correlated with C-11, C-6 and H-13 is correlated with C-1, C-6 and C-2 (Fig. 2). Thus, in view of the above-mentioned data, compound 2 can be identified as 8,11-dihydro-12-hydroxy isolaurene. Compound 3 was obtained as an optically active colorless oil [a]D ¼ þ11.5 (CHCl3; c ¼ 0.01). The molecular formula of 3, C15H18O, was deduced from the analysis of the HRESIMS m/z 213.1267 [M H]þ, in negative-ion mode, corresponding to 7 degrees of unsaturation. By analysis of the spectroscopic data of compound 3, including 1D (1H, 13C and DEPT) and 2D (COSY, HSQC and HMBC) NMR (Table 1), we can conclude that, 3 is the oxidized form of 1, that was supported by the appearance of an NMR signal at dH/dC 9.98/192.2, characteristic of aldehyde group (supported by an IR absorption band at 1728 cm1) and disappearance of hydroxyl methyl signal. Hence, compound 3 can be assigned the structure as isolaureldehyde (Fig. 1). 2.2. Pharmacology 2.2.1. Antifungal activity The antifungal activity of compounds 1e3 was assessed using the microtiter broth dilution method for yeast susceptibility testing. Compound 3 had significant activity against Candida albicans with a minimal inhibitory concentration (MIC) of 70 mg/mL, and showed medium activity against Aspergillus fumigatus and Aspergillus flavus with a MIC of 100 and 1000 mg/mL, respectively. Compound 2 had similar activity against C. albicans with MIC of 120 mg/mL and weaker activity against A. fumigatus and A. flavus with MIC of 200 and 1250 mg/mL, respectively. Moreover, compound 1 had weaker activity in all cases, with MIC of 2000, 2000 and 5000 against C. albicans, A. fumigatus and A. flavus, respectively. The positive control, ketoconazole, showed potent activity against C. albicans and A. fumigatus with a MIC of 40 mg/mL, and 625 mg/mL toward A. flavus. 2.2.2. Antibacterial activity Biological activity of the compounds 1e3, were screened for their antibacterial activity by disk-diffusion technique [21] against
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two Gram-positive (Bacillus subtilis ATCC6633 and Staphylococcus aureus ATCC 29213) and one Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27953), at 1 mg/mL concentration. Furacin was used as a standard compound. The MIC of all compounds was also measured. The data obtained showed that, all compounds 1e3 exhibited potent activity against the Gram-positive B. subtilis with MIC of 46, 39 and 35 mg/mL, respectively, and MIC of 52, 31 and 27 micro g/mL against S. aureus for compounds 1e3, respectively. On the other hand, all compounds showed non-significant activity against the Gram negative bacteria P. aeruginosa. 2.2.3. Antitumor activity using in vitro Ehrlich ascites assay Compounds 2 and 3, with highest antimicrobial activity were screened for their antitumor activity. The viability of the cells used in control experiments exceeded 95%. Compound 3 proved to have the highest cytotoxic activity (83.1%) followed by compound 2 (79.9%). 3. Conclusions Despite the huge number of secondary metabolites isolated from the red algae of the genus Laurencia, re-investigation of the petroleum ether extract of the red alga Laurencia obtusa afforded three new sesquiterpenes. The isolated compounds showed significant activity against gram positive bacteria, some yeast, in addition to antitumor activity in vitro Ehrlich ascites assay. While, the activity of the isolated compounds against Gram negative bacteria was insignificant. 4. Experimental 4.1. General Optical rotations were measured on ATAGO POLAX-L 2 polarimeter. EI/MS analyses were carried out on a Shimadzu-QP 2010. GC/MS analyses were carried out using RTX-1 column (30 m, 0.25 mm) was used. 1D and 2D NMR spectra were recorded on Bruker AVANCE III WM 600 MHz spectrometers and 13C NMR at 150 MHz. Chemical shifts are given in d (ppm) relative to TMS as internal standard. Chemical shifts are given in ppm relative to TMS as internal standard. Thin layer chromatography was performed on silica gel (Kieselgel 60, F254) of 0.25 mm layer thickness. Spots were detected by using ethanol/sulfuric acid as spray reagent. The red alga L. obtusa was collected in June 2011, off the Saudi Arabia Red Sea Coast at Jeddah. Voucher sample (JAD 03060) was deposited at the Marine Chemistry Department, King Abdulaziz University, Jeddah, Saudi Arabia. 4.2. Extraction and isolation Algal material was washed with water and dried in the shade at room temperature. The dried material of the red alga L. obtusa (200 g) was exhaustively extracted with equal volumes of petroleum ether/diethyl ether (2 6l, 24 h for each batch) at room temperature. The residue (6 g) was partitioned between ether and water, the organic layer (5 g) was chromatographed on NP (Merck, 60G) column chromatography employing n-hexane/diethyl ether mixtures with increasing polarity. Fractions of w50 mL were collected. TLC was carried out by employing silica-gel chromatoplates, appropriate solvent system and 50%-sulfuric acid in methanol as spraying reagent. Fractions containing a single compound were combined and further purified by preparative TLC of glass supported silica gel plates (20 cm 20 cm) of 250 mm thickness.
4.2.1. 12-Hydroxy isolaurene (1) Colorless oil (10 mg, 0.005% yield); Rf ¼ 0.45 (Si gel, nhexaneeEt2O, 8:2, violet appearance under UV, while yelloworange spot developed upon spraying with 50%-sulfuric acid in methanol); [a]D ¼ þ41.7 (CHCl3; c ¼ 0.01). UV (MeOH) 212, 273, 1 279 nm; IR (neat) n1 cm: 3298, 2925, 1637, 1580, 1510 cm ; EIMS m/z 216 (28), 201 (100), 171 (55), 143 (90), 109 (70), 91 (90), 77 (70) 41 (50); HRESI-MS data m/z 215.1423 [M]þ (calculated for C15H20O 215.1436); 1H, 13C NMR (Table 1 and Figs. S1 and S2). 4.2.2. 8,11-Dihydro-12-hydroxy isolaurene (2) Colorless oil (3 mg, 0.0015% yield); Rf ¼ 0.60 (Si gel, nhexaneeEt2O, 8:2, yellow-orange spot spraying with 50%-sulfuric acid in methanol); [a]D ¼ þ11.5 (CHCl3; c ¼ 0.02). IR (neat) n1 cm: 3324, 2925, 1641 cm1; EIMS m/z 218 (4), 203 (100), 185 (45), 91 (50), 41 (37); HRESI-MS data m/z 217.1579 [M H]þ (calculated for C15H21O 217.1592); 1H, 13C NMR (Table 1 and Figs. S3 and S4). 4.2.3. Isolauraldehyde (3) Colorless oil (4 mg, 0.002% yield); Rf ¼ 0.82 (Si gel, nhexaneeEt2O, 8:2, violet appearance under UV, while yelloweorange spot slowly developed upon spraying with 50%sulfuric acid in methanol); [a]D ¼ þ37.0 (CHCl3; c ¼ 0.02). UV (Et2O) 212, 262, 293 nm; IR (neat) n1 cm: 2892, 2780, 1728, 1638, 1580, 1508 cm1; EIMS m/z 214 (12), 199 (100), 171 (45), 157 (25), 105 (60), 77 (30), 41 (55); HRESI-MS data m/z 213.1267 [M]þ (calculated for C15H18O 213.1279); 1H, 13C NMR (Table 1 and Figs. S5 and S6). 4.2.4. 2,10-Dibromo-3-chloro-7-chamigrene (4) The fraction eluted with n-hexane, Rf ¼ 0.95 (10 mg, 0.008% yield) was purified by preparative TLC using the solvent system nhexane (100%), the violet color band with sulfuric acidemethanol was extracted to give colorless oil (10 mg). The spectral data of 4 (and Figs. S7 and S8) were identical to these reported for 2,10-Dibromo-3-chloro-7-chamigrene which was previously isolated from Laurencia nipponica [22]. 4.2.5. Cholest-4-en-3-one (5) White powder (8 mg, 0.004% yield); Rf ¼ 0.90 (Si gel, nhexaneeEt2O, 8:2, violet appearance under UV). This compound was identified as cholest-4-en-3-one, by analysis of its spectral data, and by comparison of its physical data with authentic standard material. 4.3. Biological activity 4.3.1. Antifungal activity Microtiter broth dilution method for yeast susceptibility testing: Sterile microtiter assay trays containing 96 round-bottom wells (Dynatech Laboratories, Inc., Alexandria, VA.) were employed. The stock solutions of antifungal agents were appropriately diluted in the various assay media to give the following working solution of ketoconazole (Janssen Pharmaceuticals, Piscataway, N.J.), starting from 20 to 1000 mg/mL. Two fold serial dilutions were performed with respective assay broths and dispensed into appropriate wells. Each vertical column of wells contained a single antifungal agent in progressive dilutions and was inoculated with a single clinical isolate. The first horizontal row of wells (A1eA12) contained no antifungal agent and served both as a growth and sterility control. The inoculation of 0.1 mL of yeast suspension created a final range of antifungal concentrations identical to that of the agar dilution plates. The final volume in each well was 0.2 mL. Inoculum: A. fumigatus WT (Af293), A. flavus WT (NRRL3357), and C. albicans were grown in YPD (1% yeast, 2% peptone and 2%
Please cite this article in press as: W.M. Alarif, et al., Laurene-type sesquiterpenes from the Red Sea red alga Laurencia obtusa as potential antitumoreantimicrobial agents, European Journal of Medicinal Chemistry (2012), http://dx.doi.org/10.1016/j.ejmech.2012.06.060
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dextrose) medium. After 24 h of incubation at 37 C in a 5% CO2 atmosphere, the isolates were diluted 1:100 in normal saline, followed by a 1:100 dilution in yeast peptone dextrose medium. Prepared in this manner, a volume of 0.1 mL of the final broth dilution contained approximately 1000 CFU of the previously mentioned fungi. Microtiter broth dilution plates were incubated at 37 C in 5% CO2 for 48 h. MIC was defined as that concentration of the antifungal agent contained in the microtiter well or in the agar dilution plate in which the absence of visual turbidity (colonies) was first observed.
Acknowledgments
4.3.2. Antibacterial activity The Antibacterial activity was evaluated by disk-diffusion technique [21].
Appendix A. Supplementary material
4.3.3. Minimum inhibitory concentration (MIC) The bacteriostatic activity of the isolated compounds of inhibition zone above 15 mm was then evaluated using the two fold serial dilution technique. 4.4. Ehrlich ascites in vitro assay [23] Different concentrations of the tested compounds were prepared (100, 50 and 25 mg/mL DMSO). Ascites fluid from the peritoneal cavity of the donor animal (contains Ehrlich cells) was aseptically aspirated. The cells were grown partly floating and partly attached in a suspension culture (RPMI 1640 medium, supplemented with 10% fetal bovine serum). They were maintained at 37 C in a humidified atmosphere with 5% CO2 for 2 h. The viability of the cells used in control experiments (without drug) exceeded 95% as determined by the microscopic examination using a hemocytometer and using trypan blue stain (stains only the dead cells). 4.4.1. Antitumor activity using Ehrlich ascites in vitro assay Different concentrations of the tested compounds were prepared (100, 50 and 25 ml from 1 mg/mL in DMSO (
